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Imaging and prodrug -activating derivatives of chTNT-3 (tumor necrosis therapy) monoclonal antibody

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Imaging and prodrug -activating derivatives of chTNT-3 (tumor necrosis therapy) monoclonal antibody

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Content INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy subm itted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand comer and continuing from left to right in equal sections with small overlaps. Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6' x 9’ black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. ProQuest Information and Learning 300 North Zeeb Road, Ann Arbor, Ml 48106-1346 USA 800-521-0600 R e p ro d u c e d with p e rm issio n of the copyright ow ner. F u rth e r reprod u ctio n prohibited w ithout perm issio n . Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. IMAGING AND PRODRUG-ACTTVATING DERIVATIVES OF chTNT-3 (TUM OR NECROSIS THERAPY) MONOCLONAL ANTIBODY by Barbara H. Biela, M.D. A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (PATHOBIOLOGY) January 2001 Copyright 2001 Barbara H. Biela R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . UMI Number: 3041436 Copyright 2001 by Biela, Barbara Helena All rights reserved. ___ ® UMI UMI Microform 3041436 Copyright 2002 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm ission. UNIVERSITY OF SOUTHERN CALIFORNIA THE GRADUATE SCHOOL UNIVERS1TY PARK LOS ANGELES. CALIFORNIA 90007 This dissertation, written by bm .ihm .fi. tf, M/mjul. under the direction of h tr. Dissertation Committee, and approved by all its members, has been presented to and accepted by The Graduate School, in partial fulfillment of re quirements for the degree of DOCTOR OF PHILOSOPHY Dean o f Graduate Studies Date DISSERTATION COMMITTEE V R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth er reproduction prohibited w ithout perm issio n . Barbara H. Biela Alan L. Epstein ABSTRACT “GENETICALLY ENGINEERED IMAGING AND PRODRUG-ACTIVATING DERIVATIVES OF CHIMETIC TNT-3 (TUM OR NECROSIS THERAPY) M ONOCLONAL ANTIBODY” The prim ary objective o f our laboratory is to develop successful m ethods o f cancer im aging and therapy using monoclonal antibodies (M Abs). A novel approach to tum or targeting utilizes M Abs directed against com m on, abundant in tracellu lar antigens accessible only in necrotic areas o f tum ors. D esignated T um or N ecrosis Therapy (TN T), this targeting idea circum vents m any o f the lim itations o f M Ab therapy directed against surface antigens. In order to develop an imaging agent against solid tum ors, genetic engineering m ethods were used to produce faster clearing single chain (scFv). diabodv. Fab. F(ab'):. and triabody antibody derivatives o f chTNT-3 antibody directed against single-stranded DNA. By genetically engineering derivatives from the sam e MAb. it was possible to com pare the im aging capabilities o f each construct in order to identify the best agent for cancer detection. The best results were obtained using F(ab'): fragment in respect o f tum or uptake but also tum or imaging. F(ab'); fragm ent produced clear im ages as early 1 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . as 24 hr after the injection. Pretreatm ent o f the m ice w ith antibody IL-2 fusion m olecule further im proved the im ages produced by F (ab'h both in their clarity and early appearance. Additional studies presented in this dissertation evaluate combination o f a TNT- type antibody with the w ell-know n concept o f tum or treatm ent called Antibody- D irected Enzyme Prodrug Therapy (ADEPT). In the A D EPT concept, the conjugate betw een antibody and enzym e is used to target the enzyme to the tumor. Once in place, the enzym e conjugate activates a systematically adm inistered prodrug into active form. The generation o f fusion proteins consisting o f either Fab. or F(ab'); or single chain (scFv) fragm ents derived from chim eric TNT-3 MAb plus either modified bacterial enzym e cytosine deam inase or hum an [3-glucuronidase is presented. In vitro characterization as well as in vivo studies (clearance, biodistribution, determination of enzym e activity, and tum or treatm ent studies) were perform ed to evaluate this targeting regim en. These experiments provide preliminary data to support the notion o f superior perform ance o f TN T -type antibody as a carrier w hen applied to ADEPT-based treatm ent of solid tumors. R e p ro d u c e d with p e rm ission of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm ission. ACKNOWLEDGMENTS The art o f thanking is a difficult one. Not to m iss anyone, not to offend one... If anyone will think him /herself forgotten, it is my oversight and no one else. My deepest thanks go to my mentor. Dr. Alan L. Epstein. Without his initial trust and later patience, this Ph.D. would have never got done. Any mistakes in this m anuscripts are mine, any wonderfully worded sentences - his. He is the greatest boss, the one who knows when to push but also knows when to prize. His personality makes the work in his laboratory a great pleasure. And when the going gets tough, there is alw ays his great vision reaching beyond ordinary work in the laboratory. My heartfelt thanks also go to all: Dr. Peisheng Hu - the best one to help solve problems. Dr. Leslie A. Khawli - the one to go to talk to about every thing (whether or not it is related to science). R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . my fellow students in the lab: Jason H om ick (long gone to achieve greatness on a East Coast and w hose Nobel Prize award cerem ony I hope to atten d ) - for many great advises. Myra M izokam i - for nice lunches (including introduction to J Town) and help anytime. Jahangir Sharifi — for being a fun guy around and helpful as well (hoping that he will not forget me w hen he finally goes to become a part o f big government in W ashington. D.C.). Jiali Li - for cheerfully trying to pick up w here we left... labmates: Thom as Bai for being extremely helpful alw ays, even at the short notice. M aggie Y un for providing pleasant atm osphere. Gina R u o ff for showing what a blonde can do (and the best of luck to her in a m edical school). dissertation com m ittee mem bers: Dr. Z oltan Tokes and Dr. Florence Hofman for advises and trust, all this despite my infrequent reports about any progress. R e p ro d u c e d with p e rm ission of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm ission. others: M ihaela Velicescu for being a friend as soulful as only Romanian can be. Drs. Valda and A m is Richters for the help in difficult m om ents. Lisa Doum ak for answ ering ALL my questions. significant other: Mark A. Hanning-Lee. most delightful husband - for putting up with all this, for being there cheerful and not letting me w ork on weekends (even if it m aybe added an extra year to my P h .D .!). and for proof-reading my writings and useful suggestions (but he is not proof-reading this part). the rest o f my family and friends: my Mother, my Aunt, and many other friends o f their in Poland, who adopted me as a "niece”, for being so proud o f me and putting trust in me so great that I had no choice but succeed... R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . TABLE OF CONTENTS ACKNOWLEDGMENTS TABLE OF CONTENTS IV LIST OF FIGURES VIII LIST OF TABLES XI CHAPTER 1 . INTRODUCTION R EFER EN C ES.................................. 17 CHAPTER 2: GENETICALLY ENGINEERED FAB AND F(AB')2 FRAGMENTS OF THE chTNT-3 ANTIBODY AS ALTERNATIVES TO ENZYMATIC DIGESTION OF ANTIBODIES: CONSTRUCTION, EXPRESSION, MODIFICATIONS FOR GREATER STABILITY, AND CHARACTERIZATION IN VITRO AND IN VIVO AS POTENTIAL IMAGING AGENTS.......................................23 A B STR A C T .........................................................................................................................23 IN TRO D U CTIO N .............................................................................................................. 25 M ATERIALS AND M ETH O D S....................................................................................28 Reagents.............................................................................................................................2S Antibodies and cell lines................................................................................................2 > S Construction o f expression vectors.............................................................................29 Expression o f chTNT-3fragments................................................................................30 Purification o f chTNT-3 fragm ents............................................................................. 31 Radiolabelling o f fragm ents.........................................................................................32 Determination o f avidity............................................................................................... 33 Pharmacokinetic and biodistribution studies...........................................................34 Imaging studies................................................................................................................35 RESULTS In vitro characterization o f F fab'): variants. Determination o f avidity................................... 36 36 40 IV R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . Pharmacokinetic studies................................................................................................ 42 Biodistribution studies....................................................................................................43 Imaging studies.................................................................................................................4~ DISCUSSION.................................................................................................................. 51 REFERENCES................................................................................................................. 61 CHAPTER 3 . SM ALL FRAGMENTS OF chTNT-3 BASED ON A SINGLE CHAIN CONCEPT............................................................................69 ABSTRACT......................................................................................................................69 INTRODUCTION........................................................................................................... 71 MATERIALS AND METHODS................................................................................. 73 Reagents, and antibodies, and cell lines................................................................... ~3 Construction o f expression vectors............................................................................ ~4 Expression a n d purification o f chTXT-3fragm ents............................................... ~6 Determination o f avidity.............................................................................................. HPLC and im m unoassays............................................................................................ Radiolabelling o f fusions.............................................................................................. Pharmacokinetic and biodistrihution studies.......................................................... “S ’ RESULTS.......................................................................................................................... 78 Expression, purification, and characterization o f chTXT-3 fragm ents........... "S’ Pharmacokinetic, biodistrihution. and targeting studies ofchT X T -3 fragm ents............................................................................................................................ R5 Imaging studies.................................................................................................................6’9 DISCUSSION.................................................................................................................. 93 REFERENCES............................................................................................................... 100 CHAPTER 4 . FUSION PROTEINS BETWEEN CHIMERIC ANTIBODY chTNT-3 DIRECTED AGAINST ssDNA AND BACTERIAL ENZYME CYTOSINE DEAMINASE (CDASE) FOR THE TREATM ENT OF SOLID TUMORS ...............................................107 ABSTRACT....................................................................................................................107 INTRODUCTION......................................................................................................... 109 MATERIALS AND METHODS............................................................................. 1 14 Reagents, antibodies, and cel! lines......................................................................... 114 Construction o f expression vectors.......................................................................... 115 Expression a n d purification o f chTXT-3/CDase fu sio n products................... 116 Determination o f avidity............................................................................................116 Radiolabelling o f fu sio n proteins............................................................................ 11~ In vitro cytotoxicity studies........................................................................................ II Pharmacokinetic and biodistrihution studies........................................................ 119 \ R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . M easurem ent o f cytosine deaminase activity in vivo........................................... 119 In vivo m urine m odel treatment studies................................................................... 120 RESULTS.......................................................................................................................122 In vitro characterization o f fusion proteins.......................................................... 122 D eterm ination o f avidity............................................................................................ 124 In vitro cytotoxicity studies......................................................................................... 12~ Pharm acokinetic studies............................................................................................ 129 Biodistrihution studies................................................................................................131 M easurem ent o f Cytosine Deaminase activity in tum ors..................................134 In vivo m urine m odel treatment studies................................................................. 136 DISCUSSION.............................................................................................................. 138 REFERENCES............................................................................................................. 143 CHAPTER 5 . FUSION PROTEINS BETW EEN chTNT-3 DIRECTED AGAINST ss-DNA AND HUMAN p-GLUCURONIDASE ENZYME AS POTENTIALLY IDEAL TARGETING VEHICLES FOR ANTIBODY- DIRECTED ENZYME PRODRUG THERAPY (A D E P T )............................ 149 ABSTRACT..................................................................................................................149 INTRODUCTION....................................................................................................... 151 MATERIALS AND METHODS............................................................................. 155 Reagents, antibodies, and cell lines........................................................................ 155 Construction o f expression vectors......................................................................... 156 Expression a n d purification o f the chTNT-3/(3-glucuronidase fu sio n p ro ducts............................................................................................................... I4~ D eterm ination o f avidity............................................................................................ 15~ Radiolabeling o f fusions............................................................................................ 15S M easurem ent o f /3-glucuronidase activity in vitro.............................................. 1 5 > S ' Visualization o f /3-glucuronidase activity in vitro............................................... 159 In vitro cytotoxicity studies....................................................................................... 159 M odifications o f hum an /3-glucuronidase.............................................................. 160 Pharm acokinetic and biodistrihution studies....................................................... 161 M easurem ent o f /3-glucuronidase activity in vivo............................................... 162 In vivo m urine m odel treatment studies................................................................. /62 RESULTS.......................................................................................................................164 Characterization o f enzyme/antibody co n stru cts...............................................164 D eterm ination o f avidity............................................................................................ I6~ M easurem ent o f /3-glucuronidase activity in vitro............................................../ ~0 In vitro cytotoxicity studies....................................................................................... I~2 M odifications o f human /3-glucuronidase..............................................................l~4 Pharm acokinetic studies............................................................................................ 1 6 Biodistrihution studies................................................................................................ I~9 vi R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth er reproduction prohibited w ithout perm issio n . Clearance and biodistrihution o f enzymatically and chemically m odified chTNT-3 F(ah')2/'/3-glucuronidase fusion proteins.............................IS2 M easurement of/3-glucuronidase activity in vivo..................................................A S 7 5 In vivo murine m odel treatment studies.................................................................. M W D ISC U SSIO N ................................................................................................................190 REFER EN C ES..................................................................................................................199 CHAPTER 6 . SUM M ARY.......................................................................................206 BIBLIOGRAPHY.........................................................................................................212 vii R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . LIST OF FIGURES Figure 1-1. Tissue localization o f chT N T -3/B .................................................................5 Figure 1-2 Schematic representation o f chimeric MAb TNT-3 and its fragm ents.......................................................................................................................8 Figure 1-3. Generation o f a cytotoxic drug by A D E P T ................................................13 Figure 2-1. SDS-PAGE gel o f antibody fragm ents...................................................... 39 Figure 2-2. Affinity constants o f Fab' and F(ab'h antibody fragm ents.................... 41 Figure 2-3. The % o f injected dose/gram o f tissue at different time points for all F(ab') 2 antibody v arian ts..................................................................... 44 Figure 2-4. The tumor/organ ratio for all Ffab'h antibody v ariants .........................45 Figure 2-5. Post injection: 6 and 24 hr: Images o f Madison 109 tum or in Balb/C mice obtained using intact antibody and fragments ..........................4S Figure 2-6. Twenty four hours post injection: ''"'i-images o f M adison 109 tumor in Balb/C mice obtained using intact antibody and F(ab') 2 antibody fragments administered after pretreatment with IL-2 conjugate........................................................................................................50 Figure 3-1. Diabody and triab o d y .....................................................................................72 Figure 3-2. SDS-PAGE gel o f single chain derivatives.............................................. 80 Figure 3-3. HPLC chart o f single chain derivatives..................................................... 82 Figure 3-4. Affinity constants for single chain antibody fragm ents.........................84 Figure 3-5. The % o f injected dose/gram of tissue at different time points for all single chain derivatives at 6. 12. and 24 hours...............................87 Figure 3-6. The tumor/organ ratio at different time points for all single chain derivatives at 6. 12. and 24 hr ............................................................ 88 Figure 3-7. ljlI-images o f M adison 109 tumor in Balb/C mice obtained using intact antibody and antibody fragments (6 and 24 hrs post injection) .......................................................................................90 \ iii R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth er reproduction prohibited w ithout perm issio n . Figure 3-8. wTc-images o f LS174T tum or in nude Balb/C mice obtained using intact and m utant antibody, and single chain fragm ent (6 and 18 hrs)...................................................................................................92 Figure 4-1. Comassie blue stained SD S-PA G E gel..................................................123 Figure 4-2. Affinity Constants for antibody fragment/cytosine deam inase conjugates................................................................................................... 125 Figure 4-3. In vitro cytotoxic effect o f the 5-FC converted to 5-FU by antibody/CD2 conjugates .......................................................................... 128 Figure 4-4. Tissue biodistribution and tum or uptake (% o f injected dose/gram o f tissue) o f chTN T-3 fragments/bacterial cytosine deaminase conjugates in m urine Colon26 colorectal (panel A) and M adison 109 lung (panel B) carcinom a tumor-bearing Balb/C mice in selected organs at 24 and 48 h r ....................................................132 Figure 4-5. Tissue biodistribution and tum or uptake (tumor/organ ratio) o f chTNT-3 fragm ents/bacterial cytosine deaminase conjugates in murine Colon26 colorectal (panel A) and Madison 109 lung (panel B) carcinoma tum or-bearing Balb/C mice in selected organs at 24 and 48 hr................................................................................................... 133 Figure 4-6. Activity o f cytosine deam inase part o f fusion protein in a tumor at different tim e p o in ts.............................................................................135 Figure 4-7. In vivo treatem ent study using Colon 26 murine tum or model in Balb/C m ice................................................................................................... 137 Figure 5-1. SDS-PAGE gel doubly stained with Comassie blue (protein) and pararosanilin (enzym e activ ity )....................................................... 166 Figure 5-2. Binding affinities o f chTN T-3 antibody fragments/enzyme fusion m o le c u les........................................................................169 Figure 5-3. pH dependent activity o f P-glucuronidase part o f fusion protein.................................................................................................................. 1 71 Figure 5-4. In vitro cytotoxic effect o f the activated prodrug o f doxorubicin................................................................................................................ 173 ix R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . Figure 5-5. Tissue biodistribution and tum or uptake (% of injected dose/gram o f tissue) o f chTNT-3 fragm ents/hum an P-glucuronidase conjugates in murine M adison 109 lung carcinom a tum or-bearing Balb/C mice in selected organs at 12 and 24 h r ..........................................................................~........................................ 180 Figure 5-6. Tissue biodistribution and tum or uptake (tumor/organ ratio) o f chTNT-3 fragm ents/hum an P-glucuronidase conjugates in murine M adison 109 lung carcinom a tum or-bearing Balb/C mice in selected organs at 12 and 24 h r................................................................. 181 Figure 5-7. Tissue biodistribution and tum or uptake o f chTNT-3 F( ah’ (^/human P-glucuronidase fusion proteins non-m odified. chemically, and enzym atically modified in murine M adison 109 lung carcinoma tum or-bearing Balb/C mice in selected organs at 24 and 48 hr: A) % injected dose per gram o f tissue. B) tum or to organ ratio....................... 1 85 Figure 5-8. Activity o f P-glucuronidase part o f fusion protein in selected tissues at different time p o in ts................................................................. 187 Figure 5-9. Treatm ent o f murine lung carcinom a Madison 109 using com bination o f the antibody fragm ent/hum an p-glucuronidase fusion and prodrug o f doxorubicin.......................................................................... 1 89 \ R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . LIST OF TABLES Table 2-1. Affinity constants o f different F(ab');> variants...........................................40 Table 2-2. Half-lives o f different F(ab'): variants as determ ined by clearance o f radiolabeled proteins from Balb/C m ic e .....................................42 Table 3-1. Affinity constants o f different single chain variants................................. 83 Table 3-2. Half-lives o f different chTNT-3 derivatives as determ ined by clearance o f radiolabeled proteins from Balb/C m ic e .....................................85 Table 4-1. Groups for the therapy study........................................................................ 120 Table 4-2. Affinity constants o f different antibody/CDase constructs....................125 Table 4-3. Half-lives o f different antibody fragments'cytosine deaminase fusion m olecules as determined by clearance o f radiolabeled proteins from Balb/C m ic e .............................................................130 Table 5-1. Groups for the therapy study........................................................................ 163 Table 5-2. Avidity constants o f different antibody fragments and human P-glucuronidase fusion proteines................................................................ 168 Table 5-3. Half-lives o f antibody fragment/human p-glucuronidase fusion proteins as determ ined by clearance o f radiolabeled proteins from Balb/C m ic e ..........................................................................................177 \i R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . CHAPTER I: INTRODUCTION The monoclonal antibody concept encom passes m ethods to generate therapeutic m olecules with specific targeting properties 1 * -. By binding specifically to tum or-associated antigens, antibodies can either direct an immune response to the tum or, or target toxins, chem otherapeutic agents, radioactive isotopes, or genes^-6. or sim ply provide an effective diagnostic tool^. When the concept o f monoclonal antibodies (M Abs) was first presented to the scientific and lay com m unities K multiple hopes and expectations were bom . Now after so many years, the hopes and expectations are still there but som ehow tempered. M onoclonal antibodies after all did not show them selves to be the "magic bullet" able to wipe out all diseases, but then ... nothing else did. However w ith a judicious and skilful use. M A bs have proved extrem ely useful in the therapy o f selected malignancies and other diseases, in imaging tumors, and in detecting many clinical modalities^- 7-11. The problem underlying the difficulties o f applying the M A bs in oncology lies within the molecules themselves. Immunoglobulins are designed to recognize specific antigens^- *0’ *2 In tumors, however, there are no truly specific antigens and even these closely associated with the developm ent and progress o f cancer, often are expressed in a heterogenous manner, and are downregulated or shed 13. Therefore. 1 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . proper targeting using monoclonal antibodies is not always p o s sib le ^ . These findings highlight the need to find novel antigens and tum or targeting regimes^. These and other reasons such as the immunological response o f humans to murine M A bs *6- 18, and physiological barriers impeding antibody distribution within tum ors, such as high interstitial pressure or distances betw een blood vessels and tum or cells 19- 20 explain w hy the overall results o f antibody therapy in clinical settings have been largely disappointing. In an attem pt to circumvent the problem s associated with conventional antibody-based targeting strategies, our laboratory developed a novel approach that exploits the presence o f a high proportion o f degenerating and necrotic cells, which accumulate within solid tumors. This strategy designated Tumor Necrosis Therapy (TN T) targets the necrotic regions o f solid tum ors. The TNT approach instead o f using tum or-associated cell surface antigens deploys monoclonal antibodies directed against intracellular antigens exposed in abnormally permeable cells. The knowledge that rapidly grow ing tumors contain a proportion o f dying or dead cells in addition to numerous proliferating cells has long been recognized, but with attention focused upon attempts to kill the dividing cells, the degenerating component has largely been ignored. Calculation o f tum or cell 1oss21-23 has revealed that in malignant tum ors, roughly 50- 90% o f the progeny o f tumor cell divisions shortly undergoes degeneration and cell death. In tum ors, the imperfect vasculature and impaired phagocytic response appear ~ > R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth er reproduction prohibited w ithout perm issio n . to perm it the accum ulation of degenerating cells, often with the formation o f large areas o f n e c r o s i s 2 4 . Thus, the accumulation w ithin tum ors o f dying or dead cells can be regarded as a m ajor distinction between malignant tum ors and normal tissues, in which cell death occurs in the latter at a relatively low rate and is accompanied by a rapid and orderly removal o f necrotic elements. The TN T approach takes advantage o f this particular distinction between normal and diseased tissues. It, therefore, can be used to target different tum or ty p es including lung, colon, breast, prostate, brain, sarcoma, and liver tum ors— . In the T N T concept, the monoclonal antibody raised in mice recognizes some o f the elements present w ithin the necrotic areas. The initial TN T MAb. designated TN T-1. was directed against nucleosomal determinants (nucleohistones)-^. This antibody provided the p ro o f o f concept and showed promise in tum or imaging and treatm ent-^- -7. despite relatively low tumor uptake. Since absolute tum or accretion o f MAb is a critical factor o f antitum or efficacy in radioimaging and radioim m unotherapv. our laboratory- sought to identify new antinuclear antibodies that displayed even higher tum or localization properties. Another murine antinuclear antibody. muTNT-3. was developed and then chimerized into human-murine construct (chimeric TN T-3) which exhibited a 3-fold higher tumor uptake than T N T -1-8. ELISA studies using a series o f nuclear antigens confirmed that chTNT-3 is directed against single-stranded DNA and cytoplasm ic RNA, and does not cross-react with TNT-I. Tissue localization o f the R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . biotinyiated form o f the MAb, chTN T-3/B, was examined by storage phosphor screen autoradiography. This m ethod compares hematoxylin and eosin (H&E) staining to autoradiographic images o f the same tissue sections. As illustrated in Figure l-l A. I:'I- chTNT-3/B localizes to areas o f necrosis within the tum or xenografts. In contrast, there is no significant signal in sections o f liver (Figure 1-1 B). kidney, or spleen (Figure 1- IC). This confirms the specificity o f chTNT-3/B binding to degenerating and necrotic cells and illustrates the ability o f chTNT-3/B to penetrate deep within solid tumors. Imaging studies with the intact chTNT-1 and chTN T-3 antibodies, have shown excellent localization o f the antibodies in tumor tissue as well as their prolonged presence at the tum or s i t e ^ . Even after several days. TN T antibodies are still visible in necrotic centers o f tumors due to their binding o f accessible nucleic acids present in degenerating cells. R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . Figure 1-1: Tissue Localization of chTNT-3/B. The upper panel consists o f H&E-stained sections o f tumor and selected organs harvested from LS174T human colon carcinoma tumor-bearing athymic nude mice injected 3 days earlier with l25I-labeled chTNT-3/B. The lower panel consists of autoradiographic images o f the same slide sections produced using a storage phosphor screen. The hot spots in the tumor correspond to regions o f necrosis (lighter stain in H&E due to cell necrosis) to which chTNT-3/B localizes. Tissue sections include (A) tumor, (B) liver, (C) kidney and spleen. 5 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth er reproduction prohibited w ithout perm issio n . N ot only has the lack o f specific targets for monoclonal antibody-based cancer therapies and poor tum or uptake slowed the developm ent o f such therapeutic m odalities, but the nature o f the m olecules themselves has proved a m ajor limitation o f their clinical application. Since the original M Abs were murine in origin, the human immune system recognizes them as foreign proteins and produces an undesirable hum an anti-m ouse antibody (H A M A ) r e s p o n s e s 'll. ^ ere genetic engineering provided a solution. Using techniques o f molecular biology, murine M Abs have been modified to resemble human antibodies to varying degrees. Combining variable regions o f the murine antibodies with human constant regions generated so-called chimeric antibodies containing 65% o f hum an sequences and retaining 35% o f murine ones^O. Further replacem ent o f murine sequences by those o f human origin by preservation o f the DR regions (the smallest portion recognizing the antigen) o f the original murine antibody and grafting them onto human framework genes allowed for the "hum anization" o f murine antibodies, making them approx. 80% human in their sequence^ I . M ore recently, however, "phage display" techniques have been used to obtain fully human antibodies reactive with antigenic regions identified by mouse m onoclonal an tib o d ies^ . 33 A nother limiting factor in tum or targeting with M Abs is the fact that the uptake o f antibodies into tum or sites in vivo is very low. often restricted to the areas near the vessels, while the majority o f the dose disperses throughout the body^^. Although 6 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm ission. biodistribution studies in animal models showed a range o f uptake from 1 to 20% o f injected dose o f the radiolabeled antibody accumulated in the tum or mass, clinical studies in humans show tum or uptake to be only 0 . 0 5 % to 0 . 1 % 3 5 . Because o f these findings different methods have been employed to increase tum or accretion o f M Abs. including modification o f antibody size while still preserving its binding abilities. The pharmacokinetic properties o f antibodies have been modified by the p r o t e o l y t i c 3 6 and, more recently, recombinant generation o f antibody-binding fragments with differing molecular masses ( 2 5 to 1 2 5 kDa) and valency (monomeric, dimeric, and m ore)8' 37 This approach to redesigning the antibody molecule first requires an understanding o f the functional components of the wild type molecule. The immunoglobulin molecule consists of two heavy and two light chains connected by disulfide bonds (Figure 1 - 2 ) . The heavy and light chains can be divided into a variable and a constant domain. The constant domain amino acid sequence is relatively conserved among immunoglobulins o f a specific class (e.g.. IgA. IgG. IgM). whereas the variable dom ains o f an antibody population are highly heterogeneous. The variable domain gives the antibody its binding specificity and affinity. R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . Figure 1-2: Schematic Representation of Chimeric MAbTNT-3 and Its Fragments. From^O chTNT-3 - s s - chTNT-3/Fab /Anticsn 3 S'- * A & l C H £ S ' CH ss chTNT-3 (Fab'}, I V H k • n e - ' - V L chT Vr-.VOiahody cli INT-.V S d \ R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . Because the practical application o f a recombinant antibody requires it to maintain its specificity and affinity, the structure o f the variable regions must be preserved. At the same time, chimeric antibodies (chM Ab) can be engineered by transferring the murine variable regions o f a given antibody onto the constant regions o f human IgG to generate reagents, which can be used in patients. The developm ent o f chimeric and hum anized antibodies has enabled clinicians to adm inister multiple doses o f antibodies to patients w ithout evoking an immune response to the p r o d u c t ^ . 3 9 One o f the smallest forms o f the antibody, which still retains antigen binding capacity, is called a single chain (scFv) construct (Figure 1-2). In this derivative, fragments o f variable light (VL) and variable heavy (VH) domains are joined together by a flexible peptide linker in length o f approximately 15 A A ^ l. T he scFv molecule has one antigen binding site and with a size o f 30 kDa. its clearance tim e is significantly faster than whole antibody and its distribution throughout the tum or is more uniform and deeply penetrating into the tum or core42. However a sm aller size and fast clearance often means lower total tum or accumulation due to the short half-life o f the molecule. This and other reasons (e.g. desire to create bifunctional or even multivalent molecules) led to the developm ent o f other molecules constructed from the single chain. The m ost recent variations, described within the last few years include diabody and triabody m olecules (Figure 3-1). 9 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . D i a b o d i e s 4 3 consist o f single chain molecule where VL and VH portion are joined by a very short linker (5 AA). This short linker prevents the solitary diabody molecule from forming a single chain-like binding site since its structure is too rigid to perm it interactions between variable light and heavy portions present on the same chain. Active binding sites form in situ. when two molecules pair to form a crossed homodimer44. This concept has been advanced further in the construction o f the triabody. For this reagent, the linker between variable heavy and variable light portions is com pletely absent and this prevents dimerization. Because o f that fact, the triabody m onom er is forced to trimerize to be able to exercise its antigen-binding abilities'^. The divalent diabody with a molecular weight o f approxim ately 60 kDa. and the trivalent triabody with molecular weight o f approxim ately 90 kDa have significantly different clearing times compared to the whole intact antibody. While the single chain antibody fragment and its derivatives could only be obtained by implementing methods o f genetic engineering, other antibody fragments were produced earlier using more traditional methods such as enzymatic digestion. Desirable characteristics such as a faster clearance time and the relative ease with which they can be produced from the intact antibody make the Fab and F(ab’): fragments useful for targeting o f radioisotopes for the imaging or therapy o f tumors^- 36. 46. 47 1 0 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . The Fab fragment consists o f the portions o f variable heavy and light regions and a small part o f the constant region, while the F(ab'): fragment also contains part o f the hinge region and therefore forms dimers via disulfide bridges (Figure 1-2). Fab has one antigen binding site and a molecular weight o f 45 kDa. while F(ab'): has tw o antigen binding sites and a molecular weight o f 1 10 kDa. The F(ab'); fragment can be reduced to sm aller Fab’ fragment (with one binding site and only slightly bigger in size than Fab). Due to their diminished sizes and lack o f the constant (Fc) portion, both fragments clear faster than whole antibody from the circulation while showing better penetration into tum or sites-^. However, the predominant renal clearance o f monovalent fragments results in the accumulation o f signal in the kidneys and bladder^S. 49 which can obscure tumors in the abdomen and pelvis, while for the F(ab’)2 fragment, accumulation o f the signal in the liver presents another obstacle for tumor detection. M oreover, the substantially reduced total tum or accretion o f antibody fragments com pared to intact antibody- ^ . 50. 51 secondary to their rapid clearance can decrease their sensitivity for detecting tum or sites. Clearly one has to balance advantages and disadvantages o f each particular molecule in view o f its projected use. Creation o f many different molecules derived from the same m onoclonal parent antibody as described in Chapters 2 and 3 has led to obtaining a panel o f molecules with different properties thus making possible to choose the most appropriate agent for the im aging or therapy o f solid tumors. 1 1 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . As mentioned earlier, monoclonal antibodies can be also used to guide other molecules directed against particular targets ^ . In 1987. Bagshaw e^- used the concept o f a monoclonal antibody as a carrier molecule to suggest a w ay to decrease the toxicity o f chem otherapy in patients with solid cancers, thereby increasing the therapeutic range o f chem otherapeutic drugs. In his approach, termed A ntibody-D irected Prodrug Therapy (A D EPT), specific enzym es are pre-targeted to human tumors using monoclonal antibodies as delivery vehicles. In this first stage, antibody-directed conjugate localizes prim arily to the tum or mass, generating a high concentration o f the enzym e in the tum or microenvironment. A fter unbound conjugate clears from the circulation and other organs, a relatively non toxic prodrug is adm inistered in the second stage o f the treatm ent. This prodrug is later converted to a highly reactive metabolite upon the action o f the pre-targeted enzym e. Due to its catalytic abilities, one molecule o f the enzyme can generate hundreds o f active molecules o f the drug. In turn, the active drug diffuses into surrounding areas o f the tumor, reaching cells, w hich did not bind the conjugate (Figure 1-3). Several studies suggest that M Ab-enzym e conjugates can activate drugs intratumorally and that their adm inistration can lead to better therapeutic effects compared to system ic drug th e ra p y ^ - 54 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . Figure 1-3: Generation of a Cytotoxic Drug by ADEPT. From^S. Ab Fragment l v yn R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . Many different enzymes have been used so far in ADEPT therapy such as oxidases and P-glucosidase, alkaline phosphatase, penicillin amidase. carboxypeptidase. P-glucuronidase, and P-lactamase (see reviews by^5-57y Some o f those enzym es are o f bacterial and some o f human origin. In the past, the preference was for using bacterial enzym es in ADEPT studies due to their relative abundance, well studied structure and know n DNA sequence, small size, and ease of production in bacteria in great quantities. Additionally, being o f non-human origin, most prokaryotic enzymes have no human analogs, thus circumventing the problem o f extratumoral prodrug activation. Since the main drawback o f bacterial enzym es lies with their ability to evoke a vigorous immune response in h u m a n s^ , their usage should be weighted carefully against potential benefit provided by their uniqueness in a sense o f not having a human analog. Interestingly, there are some studies suggesting that in case o f using murine antibodies there may be an advantage in the development o f anti-idiotype antibodies as these antibodies could reactivate the host immune response to the targeted tum or cells by generating anti-anti- idiotypic an tib o d ies^ . 60 The same might be true while using foreign bacterial enzym es that may serve as superantigens. Human enzymes have an advantage o f being immunologicallv inert. On the other hand, they are also found in normal tissues and therefore present an increased risk o f unwanted prodrug activation at the wrong site. This problem can be partially circumvented by using an intracellular enzyme, which is unlikely to be found outside the 14 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth er reproduction prohibited w ithout perm issio n . cell. The other disadvantage o f at least some human enzym es is their high molecular weight and the fact that they tend to form complexes. Now that m ore is known about human enzym es and more gene sequences for different enzym es arc available, it is possible to choose a human enzym e o f a relatively small size (e.g. (3-glucosidase). However, at this point, the availability o f a suitable prodrug becom es an issue. For all its possible advantages, the ADEPT concept has not been dev eloping as rapidly in recent years as predicted. This is mostly due to the fact that its practical applications revealed some unexpected difficulties. One o f them w as related to the use o f murine antibodies and bacterial enzym es, which inevitably led to the developm ent o f an immune response in patients^K therefore limiting treatment to ju st a few (sometimes only one) rounds o f antibody conjugate administration. This problem has been now overcome by the usage o f chimeric antibodies and human enzym es such as e.g. (5- glucuronidase62 and carboxypeptidase A^6. The other problem that hinders the development o f A DEPT therapy lies in the antibody itself. For the ADEPT therapy, the antibody must localize precisely at the tum or site and stay there long enough to permit the clearance o f the unbound conjugate from the circulation. In most cases this proves to be difficult, as the localized antibody is internalized or shed at the tum or site before its level in the blood declines low- enough to allow the adm inistration o f the prodrug. This problem led to the developm ent o f the tw'o-step A D EPT therapy. Here, a "clearing antibody" is 15 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm ission. introduced as an extra step, in which it binds to the circulating conjugate and facilitates its removal from the blood*^. N eedless to say. this also leads to the diminished localization o f the conjugate at the tum or site. Polvethyleneglvcol ( PEG-vlation) attached to fusion proteins, has also been em ployed as a way to improve clearance o f the fusion proteins from the circulation^. This, however, increases the size of the molecule thus diminishing tum or penetration. Clearly, ways to circumvent these problem s need to be further investigated, especially the use o f antibody fragments to direct the enzym e to the tum or site. Such conjugates being smaller in size, should clear faster from the circulation. A nother avenue is to use an antibody with particular characteristics such as high binding affinity towards antigen and prolonged presence in the tumor. O ur laboratory believes that the unique attributes o f TNT antibodies as described above make them exceptionally well suited for the application in ADEPT therapy. Since the TNT antibody, once targeted to the tum or site, can stay there for a very long period o f time, this enables sufficient clearance period without the need o f either antibody modification or the introduction o f an extra clearing step. Also, once present at the tum or site, TN T antibody is neither internalized nor shed, thus allowing for adm inistrations of the prodrug. In short, the com bination o f TNT and ADEPT may catalyze the revival o f A D EPT therapy. Prelim inary data provided in chapters 4 and 5 support this suggestion. Additionally we are certain that the fairly specific but at the 16 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . same tim e muitispectral binding pattern o f the TNT-based antibodies will make them very useful for imaging and therapy o f many different solid tumors. Again, the data presented in chapters 2 and 3 provide basis for such assumption. REFERENCES: 1. Kohler. G.. and C. M ilstein. 1975. Continuous cultures o f fused cells secreting antibody o f predefined specificity. Nature. 256:495-497. 2. Wright. A.. Shin. S-U.. M orrison. S.L... 1992. Genetically engineered antibodies: progress and prospects. Critical Reviews in Immunology. 12:125. 3. Taylor. C.R. 1994. The current role of im m unohistochem istrv in diagnostic pathology. Advances in Pathology and Laboratory M edicine. 7:59-105. 4. Stigbrand. T.. A. Ullen. P. Sandstrom. H. M irzaie-Joniani. B. Sundstrom . B. Nillson. L. Arlestig. R.R. Norrlund. K.R. Ahlstrom. and S. Ilietala. 1996. Tw enty years with monoclonal antibodies: state of the ait— where do we go? 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M erchant. 1997. Engineering antibodies for cancer imaging and therapy. Current Oppinion in Biotechnology. 8:449-454. 33. Sands. H., and P.L. Jones. 1990. Physiology o f monoclonal antibody accretion by tumors. Cancer Treatment and Research. 51:97-122. 34. Buchsbaum. D.J. 1995. Experimental approaches to increase radiolabeled antibody localization in tumors. Cancer Research (Supplement). 55:5729s- 5732s. 35. Covell. D.G.. Barbet. J.. Holton O.D.. Black. V.. Parker R.J.. Weinstein J.N... 1986. Pharmacokinetics o f monoclonal immunoglobulin G! F(ab'), and Fab' in mice. Cancer Research. 46:3969. 36. King, D.J.. M ountain, A.. Adair. J.R.. Owens. R.J.. Harvey. A.. Yarraton, G.T... 1992. Tum or localization o f engineered antibody fragments. Antibody Immunoconjugates & Radiopharmacology. 5:159. 37. Kuus-Reichel. K.. L.S. Grauer. L.M. Karavodin. C. Knott. M . Krusemeier, and N.E. Kay. 1994. Will immunogenicity limit the use. efficacy, and future developm ent o f therapeutic monoclonal antibodies? Clinical and Diagnostic Laboratory Immunology'. 1:365-372. 38. Gilliland, L.K.. L.A. Walsh. M.R. Frewin. M.P. Wise. M. Tone. G. Hale, and H. Waldmann. 1999. Elimination o f the immunogenecity o f therapuetic antibodies. Journal o f Immunology. 162:3663-3671. 39. Huston. J.S.. M .N. M argolies. and E. Haber. 1996. Antibody binding sites. Advances in protein chemistry. 49:329. 40. Raag, R.. and M. W hitlow. 1995. Single-chain Fvs. FASEB Journal. 9:73-80. 41. Yokota, T.. M ilenic. D.E., Whitlow. M.. Seldom. J. 1992. Rapid tum or penetration o f single-chain Fv and comparision with other immunoglobulin forms. Cancer Research. 52:3402. 20 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . 42. Holliger. P., Prospero, T„ Winter. G... 1990. "Diabodies": small bivalent and bispecific antibody fragments. Proceedings o f Nationla Academy o f Sciences U.S.A. 90:6444. 43. Perisic, O.. Webb. P.A., Hollinger. P.. Winter. G.. W illiams. R.L... 1994. Crystal structure o f a diabody, a bivalent antibody fragment. Structure. 2 :1 2 1 S. 44. Illiades, P., Kortt. A.A.. Hudson. P.J... 1997. Triabodies: single chain Fv fragments w ithout a linker form trivalent trimers. FEBS Letters. 409:437. 45. Wahl. R.L.. C.W. Parker, and G.W. Philpott. 19X3. Improved radioimaging and tum or localization with monoclonal F(ab').- .Journal of Nuclear Medicine. 24:316-325. 46. Kojima. S.. N. Suzuki. N. Shimura. A. Kubodera. K. Kubota. T. Yamaguchi, T. Takahashi, and H. Oyamada. 1993. Com parative study of intact A7 M oAb and F(ab');> fragments for radioimmunoimaging o f human colon cancer in nude mice. Nuclear M edical Biology. 20:243. 47. Behr. T.. W. Becker. E. Hannappel. D.M. Goldenberg. and F. Wolf. 1995. Targeting o f liver metastases o f colorectal cancer with IgG. F(ab'),. and Fab' anti- carcinoembryonic antigen antibodies labeled with Tc: the role o f metabolism and kinetics. Cancer Research (Supplement). 55:5777s-5785s. 48. Podoloff. D.A.. Y.Z. Patt. S.A. Curley. E.E. Kim. Y.A. Bhadkamkar. and R.E. Smith. 1993. Imaging o f colorectal carcinoma with technetium-99m radiolabeled Fab’ fragments. Seminars in Nuclear Medicine. 23:89-98. 49. Buist, M.R., P. Kenemans. W. den Hollander. J.B. Yermorken. C.J..V1. M olthoff. C.W. Burger. T.J.M . Helmerhorst. J.P.A. Baak. and J.C. Roos. 1993. Kinetics and tissue distribution o f the radiolabeled chimeric monoclonal antibody M O vl8 IgG and F(ab’), fragments in ovarian carcinoma patients. Cancer Research. 53:5413-5418. 50. Behr, T.M ., W.S. Becker. H.-J. Bair. M.W. Klein. C.Y1. Stiihler. K.P. Cidlinsky, C.W. W ittekind. J.R. Scheele. and F.G. Wolf. 1995. Comparison o f com plete versus fragmented technetium-99m-labeled anti-CEA monoclonal antibodies for immunoscintigraphy in colorectal cancer. Journal o f Nuclear Medicine. 36:430-441. R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth er reproduction prohibited w ithout perm issio n . 51. Bagshawe. K.D. 1987. A ntibody directed enzym es revive anti-cancer prodrug concept. British Journal o f Cancer. 56:531. 52. Connors. T.A.. Knox. R.J. 1995. Prodrugs in cancer chem otherapy. Steam Cells. 13:501. 53. Eccles. S.A.. Court. W.J.. Box. G.A. 1994. Regression o f the established breast carconoma xenografts with antibody-directed enzym e prodrug therapy against c-erbB2 p 185. Cancer Research. 54:5171. 54. Bagshawe. K.D.. Sharima. S.K.. Springer. C.J.. Rogers. G.T. 1994. A ntibody directed enzym e prodrug therapy (ADEPT): A review of som e theoretical, experimental and clinical aspects. Annals o f Oncology. 5:879. 55. Melton. R.G.. and R.F. Sherwood. 1996. A ntibodv-enzym e conjugates for cancer therapy. Journal o f the National Cancer Institute. 88:153 -165. 56. Sharma. S.K. 1996. Immune response in ADEPT. Advances in Drug Delivery Review. 22:369. 57. Bosslet. K.. J. Czech. P. Lorenz. H.H. Sedlacek. M. Schuermann. and G. Seemann. 1992. Molecular and functional characterization o f fusion protein suited for tum our specific prodrug activation. British Journal o f Cancer. 65:234-238. 58. Kerr. D.E.. Garrigues. U.S.. Wallace. P.M.. Hellstrom . K.E.. Uellstrom. I.. Senter. P.D. 1993. Application o f monoclonal antibodies against cytosine deaminase for the in vivo clearance o f cytosine deaminase immunoconjugate. Bioconjugate Chem istiy. 4:353. 59. Cheng. T.-L., Chen. B.-M .. Chan. L.-Y.. Wu. P.-Y.. Chern. J.-YY.. Roffler. S.R. 1997. Poly(ethylene glycol) modification o f (3-glucuronidase-antibody conjugates for solid-tum or therapy by targeted activation o f glucuronide prodrugs. Cancer Immunology and Immunotherapy. 44:305. R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . CHAPTER 2: GENETICALLY ENGINEERED Fab AND F(ab’)2 FRAGMENTS OF THE chTNT-3 ANTIBODY AS ALTERNATIVES TO ENZYMATIC DIGESTION OF ANTIBODIES: CONSTRUCTION. EXPRESSION. MODIFICATIONS FOR GREATER STABILITY, AND CHARACTERIZATION IN VITRO AND IN VIVO AS POTENTIAL IMAGING AGENTS ABSTRACT The ability to produce large quantities o f monoclonal antibodies has facilitated the clinical use o f these products. However, for som e applications, antibody fragments are preferable. The m ost common process for obtaining Fab and F(ab'): fragments involves their digestion using specific enzym es, e.g. papain, pepsin, trypsin, or tlcin. The resulting product consists of the m ixture o f various fragments (Fab. F fab'K whole antibody) thus demanding further purification to obtain homogeneous fractions. This lengthy procedure can lead to the loss o f protein and its biological activity. As an alternative, attem pts to genetically engineer and produce antibody fragments have met with varying degrees o f success. In particular, expression is a critical step while attempting to produce molecules with more than one subunit. While m onovalent Fab fragments can be relatively easily expressed and purified from bacterial cultures, the R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . m ore biologically relevant bivalent F(ab')2 fragments are produced in low quantities and often found as Fab' due to the reducing action o f the bacterial environment. Our laboratory has previously described a human-mouse chimeric TN T-3 (chTN T-3) monoclonal antibody directed against nucleic acids that targets necrotic regions common to the majority o f human and veterinary cancers. In order to develop a faster-clearing derivative which can be used to image tum ors and m onitor the effectiveness o f therapeutic modalities, the F(ab’)2 fragment o f chimeric TNT-3 parent antibody has been constructed. Here, for the first time, a method to express fully stable F(ab')2 in m am m alian system is presented as well as modifications for improving its stability described. Additional studies comparing pharmacokinetic and imaging abilities o f the F(ab')2 fragm ent with those o f the parent antibody are also presented. R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . 1. INTRODUCTION Monoclonal antibodies have been deployed in medicine for many purposes, among them for cancer im aging and therapy, due to their ability to bind selectively to a specific target*"6. W hile intact antibodies are still the most com m only used tools, they are not necessarily the m ost efficient or desired forms o f antibody molecule to utilize. Antibody fragments, either bivalent or monovalent, have different phannacokinetic characteristics than their parent, and in some clinical applications, they have been proven to be the molecule o f choice. Because o f their faster clearance characteristics and the relative ease with which they can be produced from the intact antibody, the Fab and Ffab')? fragments have been often used for targeting o f the radioisotopes to tumors, either for the im aging or the therapy?" 10. Future clinical uses o f antibody fragments will undoubtedly require large quantities o f homogenous antibody fragment preparations. For the Fab and F(ab'): fragments o f the antibody, the most common method still involves digestion o f the intact whole antibody to smaller fragments with naturally occurring peptidases by removing the Fc portion o f the antibody* **13. Pepsin digestion has been routinely used to produce F(ab' ) 2 fragments in the past, but its use does not always yield immunoreactive fragments. Because pepsin's action is strongly pi 1-dependent, the optimum conditions for the digest are not always the ones favoring preservation o f the 25 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . antibody's activity. A dditionally, pepsin's efficiency can vary betw een antibodies, starting from the inability to digest the antibody and going all the way to reducing antibodies to small inactive fragm ents 11- 13- *4. Papain can also be used to obtain F (ab ')2 fragments but the preparation process is often inconsistent and the resulting product unstable 15. Bromelain^ and f ic in ^ have been used w ith better results and are now often enzym es o f choice for the digestion. N evertheless, the enzymatic generation o f antibody fragm ents often leads to low yield o f pure fragments, generates a mixture o f different fractions (Fab'. F(ab')2. and whole an tib o d y )* -, and causes dim inished binding affinity *0. The alternative method is to em ploy genetic engineering to create antibody fragments. Several review papers have been published * N-20 describing the production o f these antibody fragments in different expression system s, most commonly in bacteria or yeast. Even so. molecular engineering is chiefly used to obtain single chain fragments and their derivatives 1^- 21. Recent advances in molecular biology, however, have enabled the generation of Fab and F(ab')2 fragments via genetic engineering, thus sim plifying the whole process and generating uniform constructs* • 22-24. Unfortunately, m ost attem pts have also described several problems encountered during the expression, as discussed later. The obvious solution for most o f these problems would be to express antibody fragments in mammalian expression system . Up until recently this w ould not be possible due to the lack o f suitable 26 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . mam m alian expression system. However several such system s have been introduced a short while ago. One o f the mammalian system s developed takes advantage o f the Glutam ine Synthase (GS) enzym e as a selective and amplificationa! marker. First described by Bebbington25, this system uses a GS-detlcient murine m yeloma cell line NSO as the expression cell line. A fter introducing a vector containing the GS gene together with the gene for the protein o f interest, the transfected cell line is grow n in the absence o f glutamine, thereby assuring the continuous expression o f not only Glutam ine Synthase which is required for glutamine production and cell survival, but also the expression o f the desired protein. The protein o f interest is modified accordingly as to assure its secretion into the medium from which it is subsequently purified. Our laboratory has previously described a human-mouse chTN T-3 VIA b directed against nucleic acids, which are always present inside the cells but are accessible to the antibody only in dead and dying cells with permeable m em branes-^. and therefore able to target many solid tum ors with areas o f necrosis-"'. In the current study, we sought to engineer a stable chTNT-3 F(ab'): fragment, expressed in a high level in a mammalian GS system , then perform murine pharm acokinetics and biodistribution studies as prelim inary but necessary steps to evaluate the potential o f this antibody fragment as a diagnostic agent for cancer detection in humans. 27 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . 2. MATERIALS AND METHODS Reagents The plasmids pEE6hCM V-B and pEE12 were purchased with the Glutamine Synthetase Gene Amplification System from Lonza Biologies (Slough. UK). Restriction endonucleases. T4 DNA ligase. and other molecular biology reagents were purchased from New England Biolabs (Beverly. MA) or Boehringcr Mannheim (Indianapolis. IN). Dialyzed fetal bovine serum, single-stranded DNA from calf thymus, sodium chloride. ABTS (2 ,2 ’-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt), and other chemicals were purchased from Sigma Chemical Co. (St. Louis. MO). Iodine-125 was obtained as sodium iodide in 0.1N sodium hydroxide from DuPont/New England Nuclear (North Billerica. VIA). Balh C mice were purchased from Harlan Sprague Dawley (Indianapolis. IN). Antibodies and Cell Lines The chimeric MAb TNT-3 (chTNT-3. IgGi) was produced as described previously^. The NSO murine myeloma cell line was obtained from Lonza Biologies. The Raji cell line, derived from an African Burkitt's lym phom a-^, was obtained from the American Type Culture Collection (Rockville. MD). The M adison 109 2S R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm ission. (M AD 109) cell line derived from murine lung cancer line-5^ was obtained from the National C ancer Institute (Frederick. MD). Construction of Expression Vectors For the purpose of generating F(ab'); fragment o f the chTNT-3 antibody, the PCR reaction was performed to yield heavy chain F(ab'): fragment. For the 5' end. the TNT-3 VH leader sequence prim er with extended Kozak sequence^ I and X bal cloning site 5' - A G C T C TA G A G C C G C C A C C A TG G G A TG G A G C G G G A TC TTT - 3' was used. For the 3 ' end o f the Fab construct, the prim er overlapping with the 3'end o f the Fab (end part o f CH| region) and appending Not I cloning site: 5 ’ - G G A G TC G A A T TC T C A A G C G G C C G C TT TC T T G TC C A C C 'TT G G T G T T - 3 ’ was used. For the 3' end o f the F(ab'): construct, three different 3' prim ers were used, each containing hinge region with varying sequences coding for no (not underlined: in brackets), one (underlined; in brackets), or two (underlined twice: in brackets) cysteines present additionally to the original one and N otf cloning site: 5’ - C TC G A G TG C G G C C G C (T G G A G G A C A )(G G G T G G G C A )(C'GGTGGGCA) T G T G TG A G T - 3'. 29 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . The final assembled PCR fragment was then inserted into the X h a l and X o tl sites o f p E E l2 vector modified to contain for purification purposes six histidine tag (His-Tag) or streptavidin-affinity tag (Strep-TagI) at 3' end. resulting in the expression vectors: 12/chTNT-3/Fab/His-Tag, 12/chTN T-3/F(ab'): 1 Cvs/I lis-Tag. IZ ch T N T - 3/F(ab')2 /2C ys/H is-Tag. 12/chTNT-3/F(ab')2 /3C vs/Strep-Tagl. and 12. chT N T - 3/F(ab')2 /3C ys/H is-Tag respectively. The expression vector for the chimeric T N T -3 light chain. pEE6/chTN T-3 LC. was constructed as described previously-'1 '. Expression o f chTNT-3 Fragments The chTN T-3 based constructs were expressed from NSO murine m yelom a cells according to the manufacturer's protocol. Fragments of M A b-containing supernatants w ere initially identified by indirect ELISA screening using m icrotiter plates coated with crude DNA or single-stranded DNA from calf thymus, as described previously^. For production rate assays. 10r ’ cells w ere plated in 1 ml o f medium and allowed to incubate for 24 hr. Supernatants were serially diluted and applied to wells o f m icrotiter plates coated as above and then detected using goat anti-human kappa H PRO-conjugated antibody (CalTag. So. San Francisco. CA). Dilutions o f a control chimeric M Ab were used to generate a standard curve using 4-param eter fit by an autom ated ELISA reader (Bio-Tek Instrum ents. Inc.. W inooski. YT). from which concentrations o f unknowns were interpolated. Rates o f production were expressed as 30 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm ission. jig/ml/106 cells/24 hr and com pared among different clones to identify the highest producers. The chosen clones w ere frozen as stocks, while at the sam e time, they were also further subcloned to isolate pure subclones. Purification of chTNT-3 Fragments Each o f the highest producing clones was incubated in a 3 liter stir-flask vessel, and the chTNT-3 fragments were purified stepwise from cell culture medium by Ni- N TA (Q1AGEN Inc. Valencia, CA) or Streptactin (Sigma-Genosys. Woodland. TX) affinity chromatography according to the manufacturer's protocols. For the purification using Ni-NTA column, the cell supernatant was spun dow n at 2000 rpm at 4 °C to pellet cells, then filtered using 2-micron filter to remove cell fragments. The supernatant was equilibrated using lOx binding buffer (4.M NaC'l. 0.25M Tris. 0.5M N atTPC^, pH 7.5). The 2 ml Ni-NTA colum n was run overnight at 4 °C under gravity. Subsequently the column was washed twice with 10 ml o f Ix binding buffer. The fragments were eluted using 0.5M imidazole in binding buffer. For the Streptactin (modified streptavidin provided by Genosys) affinity purification, the m anufacturer’s protocol was slightly modified. In short, the supernatant was spun and filtered as above, than equilibrated with 25.x Tris buffer (2.5M Tris, 0.025M EDTA. pH 8.0). Additionally avidin was added (from 10 mg ml stock solution) to the final concentration o f 10 |.ig/ml to bind any free biotin present in R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . the solution, which may otherwise irreversibly couple with the streptavidin and inactivate resin. The column containing 2 ml o f Streptactin was run overnight at 4° C under gravity. In next step the column was washed with 2x10 ml o f Ix Tris buffer. The fragments were eluted using 10 mM desthiobiotin in Tris buffer and subsequently 1 V I Tris, pH 3.5. The purity o f the fragments obtained was examined both by SDS-PAGE and by High Performance Liquid Chrom atography (HPLC). using a Beckman 11PLC Gold System (Beckm an Instruments. Inc.. Fullerton. CA) equipped with two I 10B solvent pum ps, a 210A valve injector, a 166 programmable UV detector, and a 406 analog interface module. Size exclusion chrom atography was performed on a G4000SW column (TosoHaas: Montgomeryvilie. PA) with 0.1 M PBS. pH 7.2 as the solvent system , eluting at a flow rate o f 1 ml/'min. The UV absorbance o f the HPLC eluate was detected at 280 nm. Radiolabeling of Fragments The l25I-Iabeled MAb fragments were prepared using a modified chloram ine-T m ethod as described previously^-. Briefly. 1 mCi o f i:'I and 20 pi o f an aqueous solution o f chloramine-T (2 mg/ml) were added to a 5 ml test tube containing 100 pg M Ab in 100 pi PBS. The solution was quenched after 2 min with 20 pi o f an aqueous solution o f sodium metabisulfite. Each reaction mixture was purified using a Sephadex 32 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . G-25 column and typically recovered 90-95% yield. The radiolabeled antibodies were diluted with PBS for injection, stored at 4 °C. and adm inistered within 2 h after labeling. Radioiodinated antibodies were analyzed using an analytical ITLC system consisting o f silica gel impregnated glass fiber (Gelman Sciences. Ann A rbor. M I). Strips (2 x 20 cm ) were activated by heating at 110 °C for 15 min prior to use. sp otted with 1 pi o f sam ple, air dried and eluted with m ethanoI/H20 (80:20) for approxim ately 10 cm. again air dried, cut in half, and counted to determ ine protein bound and free radioiodine. Determination of Avidity The avidity constant o f the chTNT-3 fragm ents was determined by a fixed cell REA using the m ethod o f Frankel and G erhard-^ as described previously. Briefly. Raji lym phom a cells fixed in 2% paraformaldehyde (which causes disruption o f the cell membrane) were incubated with increasing amounts o f i:''I-labeled MAh for I hr with constant mixing. The cells were then washed and the radioactivity measured in a gamma counter. The am ount o f MAb bound was determ ined by the remaining cell- bound radioactivity (cpm ) in each tube and the specific activity (cpm ng) o f the radiolabeled antibody. Scatchard analysis was perform ed to obtain the slope. The equilibrium or avidity constant Ka was calculated by the equation K = -(slope /;). where n is the valence o f the antibody (1 for Fab’. 2 for F(ab'): ). - * JO R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . Pharmacokinetics and Biodistribution Studies It has previously been demonstrated that half-life values o f IgG clearance from Balb/C mice determ ined by whole-body dosim etry are statistically indistinguishable from those calculated by blood sam pling-^. Therefore, w hole-body dosimetry' was performed for this pharm acokinetic study. Six-week-old Balb/C mice were used to determine the pharm acokinetic clearance o f the chTN T-3 constructs. A group of mice (/7=5) previously fed potassium iodide in drinking w ater for I week to block thyroid uptake o f radioiodine were administered i.v. injections o f I_'I-labeled MAb (1.1-1.5 MBq/mouse). The w hole-body activity immediately post-injection and at selected times thereafter was m easured with a CRC-7 m icrodosim eter (C'apintec. Inc.. Pittsburgh. PA). The data was analyzed and half-life determined as described previously-^. To determine the tissue biodistribution o f the chTN T-3 constructs, six-week- old female Balb/C mice were injected with a 0.2 ml inoculum containing 2 x 1 0 o f MAD 109 murine lung carcinom a cells s.c. in the left thigh. The tum ors were grown for 10-14 days, until they reached close to 1 cm in diameter. W ithin each group (//=5). individual mice were injected i.v. with aO. I ml inoculum containing 3.7 MBq 10 _g o f i:!5 I-Iabeled MAb. Anim als were sacrificed by sodium pentobarbital overdose 6. 12. and 24 hr post-injection, and tissues were removed, lightly blotted to remove excess o f 34 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth er reproduction prohibited w ithout perm issio n . blood, than weighed, and their radioactivity measured in a gamma counter. For each mouse, data were expressed as percent injected dose/gram (% ID/g) and tumor organ ratio (cpm per gram tum or/cpm per gram organ). Significance levels were determined using the Wilcox rank sum test. Imaging Studies Six-week-old female Balb/C mice were injected with M AD 109 cells s.c. in the left thigh and the tumors were grown as described above. G roups o f mice (//=5) w ere injected i.v. with a 0.1 ml inoculum containing 7.4 MBq/10 _g o f 1' 1 1-labeled chTNT-3 or chTNT-3 constructs. At 6. 12, and 24 hr post-injection, the mice were anesthetized with a s.c. injection o f 0.8 mg sodium pentobarbital. The immobilized mice were then imaged in a prone position with a Spectrum 91 gamma camera equipped with a pinhole collim ator (Raytheon M edical Systems. Melrose Park. IL) set to record 10.000 counts using the Nuclear MAX Plus image analysis software package (MLiDX Inc.. Wood Dale. IL). For the pretreatm ent studies, the mice were first injected with chTNT-3 IL-2 fusion protein or chLym -l/IL-2 (irrelevant control antibody). Both antibodies were injected i.v. at the dose o f 20 pg/mouse. 2 hr before the injection o f radiolabeled intact chTNT-3 or its fragments. Subsequent imaging was performed as described above. 35 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm ission. 3. RESULTS In vitro Characterization of F(ab')2 Variants Several attempts were made to express the Fab antibody fragment. Despite repeated transfections, its expression level was extremely low. At the end the Fab' fragment resulting from the dissociation o f the least stable o f the F (ab'): variants (coded as 12/chTN T-3/F(ab')2/lCys/His-Tag) was used in further experiments in lieu o f the Fab. Based on previous publications--5- -4- the F(ab'): fragment was designed as three different variants containing from one to three cysteine residues at the end o f the hinge region. Expression levels o f the F(ab'): subclones ranged from 5 to 15 pg/m l/106 cells/24 hr. Since removal o f the Fc portion o f the antibody precludes the use o f the protein A for the purification, two different affinity tags were added at the C end o f the molecule. One o f the tags consisted o f six consecutive histidines, while the other was composed o f nine different amino acids (SAW RH PQ FG ) shown to be able to bind to the streptavidin in specific manner-^. Fragments with the six-histidine tag at the end appeared to be expressed at higher levels than the ones with streptavidin- affinity tag (personal observations). Streptavidin tag on the other hand allowed for easy screening of supernatants for positive clones by em ploying one-step detection with HPRO-conjugated streptavidin. Additionally the purification using Streptactin 36 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth er reproduction prohibited w ithout perm issio n . column allowed obtaining cleaner product in a single step and with no need for further purification steps to get rid o f impurities. Purification o f six histidine-tagged proteins also resulted in uniform final product but the conditions o f purification (m ainly the com position o f the binding buffer) required several adjustm ents to the m anufacturer's protocol before the one-step purification goal was achieved. Upon purification, it has been noted that the Fab' variant with a single cysteine at the hinge region coded as 12/chTN T-3/F(ab')vlC ys/H is-T ag. had little tendency to form F(ab')2 in vivo, with less than 20% o f total purified fraction initially forming dimeric structures. This percentage would further decrease during storage with less than 5% o f the original fragment retaining its dimeric form after 30 days. This variant was later em ployed in animal studies in lieu o f Fab fragment. For the variant possessing two cysteines at the hinge region, the stability was influenced by the ty p e o f the purification tag at the end o f the molecule. O nly approxim ately half o f the tw o- cysteines variant with six histidines tag coded 12 chTNT-3 F(ab'): 2Cys H is-Tag formed dimeric structures, while for the one with the Streptavidin-affinity tag coded l2/chTNT-3/F(ab')2/3Cys/Strep-TagI. approxim ately 90% o f the expressed protein formed F(ab')2 - The last variant possessing three cysteines at the end o f the hinge region (coded 12/chTNT-3/F(ab')2/3Cys/His-Tag) exhibited the greatest stability o f all F(ab') 2 variants. Regardless o f the tag at the end o f the protein. 100% o f the expressed protein was present as a bivalent form. 37 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . All the F(ab' ) 2 variants were analyzed on the SDS-PAGE gelj7 as presented in Figure 2-1. Reducing SDS-PAGE revealed two discrete bands at approxim ately 55 and 25 kDa. corresponding to the predicted molecular weights o f chimeric immunoglobulin heavy and light chains (data not shown). SDS-PAGE under non-reducing conditions revealed bands corresponding to either Fab* or F(ab")2 constm cts with varying degrees o f stability as described above. HPLC analysis (data not shown) demonstrated a retention time o f 12.92 minutes for fully assembled F (ab')2. and 15.07 minutes for the chTNT-3 Fab. with each protein eluding as a single peak. The F(ab'): fragment with two cysteines, which formed 50-50 mixture o f Fab’ and F(ab’)2 fragments, eluded as two distinctive peaks, one with the retention tim e o f 14.03 minutes, and the other o f 12.73 minutes which closely resembled the retention times o f pure Fab' and F (ab'): fragments. By- com parison. the intact chTNT-3 antibody retention time is 12 minutes. 3N R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . Figure 2-1: SDS-PAGE Gel of Antibody Fragments. 97.4 66 45 31 lane 1 lane 2 lane 3 lane 4 lane 5 lane 6 lane 1 & 6: low molecular weight marker lane 2: F(ab')2 fragment with one cystein lane 3: F(ab')2 fragment with two cysteins lane 4: F(ab')2 fragment with three cysteins lanes 2-4 have six-histidine affinity tag lane 5: F(ab')2 fragment - two cysteins and Strep-Tag I affinity tag R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . Determination o f Avidity The avidity constant o f the chTNT-3 fragments was determined by a fixed cell radioim m unoassay (R.IA) using Raji cells. The avidity constant Ka is calculated from the equation K = -(slope/n). where n is the valence o f the antibody (1 for Fab. 2 for F(ab’)2). Table 2-1: Avidity Constants of Different F(ab’)2 Variants. Fragment # of binding sites % Binding avidity constant Ka Fab’ one 60.1 0.48 x 10'’ NT' F(ab’)2 His-tag tw o 65.7 0.54 x 101 ’ VT1 F(ab’)2 Strep-TagI tw o 65.8 0.34 x 101 ’ NT1 chTN T-3 tw o 89.0 1.43 x I0 1 ' M '1 These studies showed that the avidity constants for different F(ab'); fragments are com parable to the parent antibody within one decimal place (Table 2-1. Figure 2- 2). Between them selves, the fragments varied slightly in their avidity, with the most stable construct F(ab')2 with three cysteines and His-Tag having the highest value. The affinity o f the equally stable F(ab’ ) 2 w ith two cysteines and Strep-TagI was som ew hat lower, how ever in biodistribution studies this fragment perform s just as well as F(ab’): with H is-Tag. 40 R e p ro d u c e d with p e rm ission of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm ission. Figure 2-2: Affinity Constants of Fab’ and F(ab’)2 Antibody Fragments. A f f in it y o f D i f f e r e n t V a r i a n t s o f F a b a n d F ( a b f)2 Fab' (One Cysteine & His-Tag) F(ab')2 (Three Cysteines & His-Ta F(ab')2 (Two Cysteines & Strep-Ta 0.75 0.5 0.25 30 20 25 10 40 bound (ng) R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm ission. Pharmacokinetics Studies The half-life values for F(ab')2 fragments clearance from Balb/C mice were determined by whole-body dosimetry. Six-week-old Balb/C mice were used to determine the pharmacokinetic clearance o f the chTNT-3 constructs. The data was analyzed and half-life determined as described previously-**. Table 2-2: Half-lives of Different F(ab’)2 Variants as Determined by Clearance of Radiolabeled Proteins From Balb/c Mice. Fragment Size (kDa) T„2 Fab’ (1 cysteine & His-tag) 55 kDa 6.3 0.77 hr F(ab')2 (3 cysteines & His-tag) 120 kDa 8.1 — 0.48 hr F (ab')2 (3 cysteines & Strep-TagI) 120 kDa 7.7 + - 0.33 hr chTN T-3 150 kDa 134.2 - -4 .0 hr The analysis revealed that half-lives o f all F(ab'); fragments varied between themselves only slightly and in accordance with the am ount o f stability shown by each o f the variants (Table 2-2). This finding is in agreement with other studies of the antibody fragments. Similar findings in respect o f clearance values have been reported previously for Fab’ and F(ab')2 fragments generated by enzym atic digestion** - 3* ' 1 and genetically engineered domain-deleted chimeric antibodies^. 40 por tjlc Fab' obtained 42 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth er reproduction prohibited w ithout perm issio n . via enzym atic digestion the clearance time was approx. 5 hr-5^. while for the F(ab’): values ranged o f 8 h r ^ to 14 hr^l to 30 hr^S. For CFT domain-deleted M Abs the half- lives w ere shown to be 7.8 hr^- and 12 hr-^9. Biodistribution Studies Since chTN T-3 binds to dead present in tum or o f any origin. M adison 109 murine lung carcinom a grown in immunocompetent Balb/C mice was utilized. The tumor and normal tissue biodistribution o f '^I-labeled chTNT-3 and chTN T-3 fragments was evaluated at 6. 12. and 24 hr post-injection. The obtained data w as expressed as overall uptake (%injected dose/gram o f tissue: Figure 2-3). as well as the ratio between tum or and normal organs (tumor/organ ratio: Figure 2-4). R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . Figure 2-3: The % of Injected Dose/Gram of Tissue at Different Time Points for All F(ab’)2 Antibody Variants. S3 L . ec z j \ r . C 5 . 5 8 7.5 7 6.5 6 5.5 5 4.5 4 3.5 3 2.5 ■ > i .5 1 0.5 0 L § hlood ec u 8.5 8 7.5 7 6.5 6 5.5 5 4.5 4 3.5 2.5 - > 1.5 H 1 0.5 0 liver kidney organ 12 hr ♦ ♦ ♦ ♦ ±± I blood liver kidney organ ec " c ; ■ o ' S i O ' 8.5 5 - 7.5 - 7 6.5 6 5.5 5 4.5 4 3 5 3 2.5 -I 2 1.5 I 0.5 0 24 hr i 0 F a b ’ 1 Cys. His-tag HD F la b ’12 3 Cys. His-Tag H F ( a b ’ 12 2 Cys, Strep-TagI tumor □ Fab I Cys. His-tag no F la b ’>2 3Cys. His-tag F (a h ’ 12 2Cys. Strep-tagI l □ Fab I Cys. His-tag IE F < a h ’ 12 3Cys. His-tag H F (a b ’) 2 2Cys. Strep-tagI hlood liver kidney organ 44 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth er reproduction prohibited w ithout perm issio n . Figure 2-4: The Tumor to Organ Ratio for All F(ab’)2 Variants. 5 4.5 - 4 - 3.5 - a ■ ? - ec ~ 2 . 5 - 6 hr s 1.5 - 1 - 0.5 - o -L» 5 3 ♦ ♦ i f e E Fab 1 Cys. His-tag IQ F (a b ' (2 3 Cys. His-tag H F( ah' )2 2 Cys. Strep-tagI hlood liver organ kidnev 4.5 - 4 - = 3 . 5 - F* 3 2.5 12 hr T T ♦ ♦ 4 ♦ ♦ 4 ♦ ♦ 4 ♦♦4 4 4 4 T I I t± ± hlood kidnev □ Fab ICys. His-tag IQ F (a h ') 2 3Cys. His-tag H F (a b ' 12 2Cys. Strep-tagI organ 24 hr i i i 1 i blood organ tver kidne\ B Fab ICys. His-tag OH F la b 112 3Cys. His-tag H Fab 2Cys. Strep-ta R e p ro d u c e d with p e rm ission of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm ission. The effect o f the rapid clearance was properly reflected in biodistribution o f the Fab and F (ab ')2 fragments as compared to the whole chTNT-3 antibody in M adison 109 lung tumor-bearing Balb/C mice. The overall tum or uptake o f the antibody fragm ents was significantly lower than that o f the intact antibody (Figure 2- 3). For the Fab, the 24 hr uptake was 1.4% injected dose/gram o f tissue, while for F(ab’)2 fragm ent is varied from 2.93% o f injected dose/gram o f tissue for F (ab')2 with six histidines tag to 3.19% o f injected dose/gram o f tissue for F (ab ')2 with the Streptavidin-affinity Tag. This is in agreement with other studies describing tumor uptake o f the antibody fragments. B ehr's experimental s tu d ie s ^ showed tumor uptake o f 1.76% ID/gram for the Fab' and 3% ID/gram for F (ab')2 fragment at the same tim e point. Generally these values are much lower than the ones for the intact antibody but on the other hand the tumor to organ ratios are much more favorable for the fragments as compared to the whole antibody (Figure 2-4). Interestingly the Fab fragment show ed the lowest tumor/organ ratio for the kidneys o f 1.62 at 24 hr time point as opposite to that o f 2.87 and 3.22 for variants o f F(ab')2 with different tags, and o f 2.81 for the chTNT-3. This finding is in agreement with other observations reporting renal retention of monovalent antibody derivatives^- - * - 1- 46 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm ission. Im aging Studies Im m unoscintigraphy was perform ed to examine the difference between tum or targeting with chTNT-3 and its F(ab'> 2 derivatives. Mice bearing MAD 109 tum ors w ere imaged at 6. 12. and 24 hr post-injection. Images obtained from representativ e m ice are presented in Figure 2-5. Imaging studies further confirmed the preserv ation o f the binding abilities o f antibody derivatives but also highlighted differences in their im aging capabilities, which were not obviously noted in biodistribution data. The Fab' fragment showed poor tum or localization most likely due to its low er binding affinity value, rapid elimination and sequestration in kidneys. At 6 hr tim e point m ost o f the label and presum ably most o f the protein was distributed ev enly betw een blood pool in and around the heart and the urinary' tract. By 12 hr the labeling localized m ostly within the urinary system . All this accounted for the fact that Fab' w as poorly visualized throughout the time course o f the study, with just traces o f tum or targeting appearing at 24 hr time point. Im m unoscintigraphy studies obtained by using F(ab')2 show ed superior images as com pared to the Fab' fragment in respect to tum or localization and to intact chTN T-3 antibody in respect o f the presence o f the background radioactivity (Figure 2-5). 4 7 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth er reproduction prohibited w ithout perm issio n . Figure 2-5: Post Injection: 6 and 24 hr; 1-131 Images of Madison 109 Tumor in Balb/C Mice Obtained Using Intact Antibody and Fragments. 6 l i a r 24 lur Fab ChTNT-3 48 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm ission. Additional im aging studies have been performed after pretreatm ent o f tum ors with antibody/InterIeukin-2 (IL-2) fusion molecule. It has been dem onstrated that pretreatment with IL-2 significantly increases the uptake o f the radiolabeled agent into tumors by increasing blood vessels permeability^3. Since in earlier imaging studies F(ab')2 em erged as the best candidate for the im m unoscintigraphy o f tumors. IL-2 pretreatment studies were only performed using F(ab'): m olecule. MAD 109 lung tumor-bearing Balb/C mice were first injected i.v. with either irrelevant control or chTNT-3/IL-2 fusion molecule. Two hours after the injection, radiolabeled F(ab'): fragment was adm inistered intravenously. The subsequent images taken at 6. 12 and 24 hr after the injection o f the antibody fragment show ed its rapid localization into tumor with equally swift clearance from other organs (Figure 2-6). In addition the tumor accretion o f the radiolabeled product was superior to that with no pretreatment. Tumors present in anim al pretreated with irrelevant antibody showed no significant changes in tum or im aging. 49 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . Figure 2-6 : Twenty Four Hours Post Injection; 1 3 1 I Images of Madison 109 Tumor In Balb/C Mice Obtained Using Intact Antibody and F(ab’)2 Fragments Administered After Pretreatment with IL-2 Conjugate. 6 hr 12 hr 24 hr no protreatmont IL-2 protroatmont 5 0 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . 4. DISCUSSION Classically, m any different enzym es, such as pepsin, papain, brom elain, or ficin have been used to generate sm aller fragments from the intact antibody 17. However, none o f these enzym es provides a straightforward m ethod for the production o f immunoreactive fragments. Each enzym e has its advantages and disadvantages but nevertheless, enzymatic reactions do not result in uniform preparations consisting o f single products. The end result is always a m ixture o f intact antibody. F(ab'):- Fab and Fc fragments thus requiring further purification steps. Genetic engineering offers an effective and attractive way to obtain many different proteins with desired m odifications and in large quantities-* - ® . These newer m ethods have already been used to produce Fab and F(ab'): antibody fragm ents--3- -° . but the results so far are not encouraging. Sim ple m onomer-based antibody fragments such as single chain, diabodies. or triabodies have been expressed s u c c e s s f u l l y -^ -^ but more complicated m ultim eric proteins seem to encounter difficulties while being e x p r e s s e d ^ 0 - 5 2 One o f the main reasons is the fact that the expression takes place in bacterial systems. Expressing antibody fragments in bacterial system has the advantages o f producing large quantities o f the desired molecules-®, mostly due to the fast grow th o f E. coli. and the ease o f E. coli transformation with engineered DNA-®. and additionally results in savings in cost and time due to the same reasons. 5 1 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . However, bacterial system s cannot glycosylate proteins and if glycosylation is vital to their function, the protein products will be inactive. If this occurs, other expression systems (e.g. yeast) can be utilized. Additionally, the expression o f recom binant antibody fragments in the reducing environment o f the bacterial cytoplasm leads to the formation o f insoluble inclusion bodies. In this case, there is a further need for the solubilization o f unfolded proteins from inclusion bodies, w hich often m eans purification under denaturing co nditions^- 51. 53 W hile protocols for the refolding o f proteins purified under these harsh denaturing conditions are well established, still more often than not. the expressed protein becomes partially inactive. In short, the production o f each m olecule manufactured in bacteria needs its own purification protocol for the best results. M any molecules expressed in bacteria easily assum e the correct conform ation, but some antibody fragments seem to adopt a preferred conform ation which is not that o f desired antigen binding m o le c u le ^ . 33. It has been dem onstrated that functional m onovalent antibody fragments (Fab or single chain) can be readily produced in bacteria^ I • ^6. Attempts to produce bivalent antibody fragments w ith enhanced antigen binding in E. coli. however, have met with significant difficulties. W hile som e research groups were able to obtain bivalent antibody fragments directly from E. c o li--- 57. the yield o f the fully formed F(ab’); was found to be low and variable--- --3- JZ). Specifically, reducing conditions often 52 R e p ro d u c e d with p e rm ission of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm ission. encountered by proteins within the bacterial cytoplasm (especially traces o f thiol) can severely dam age disulfide bridges in F(ab')2 and lead to the reduction o f F(ab'): to Fab' fragments. One w ay to overcome this obstacle is to recover Fab’ fragments from bacteria as Fab’-Sf-P^ and then chemically couple them in vitro directly--- -9 . 60 or by cr0ss- linking^l. A nother way is to m odify the type o f bridges connecting two Fab’ molecules to improve the stability. For example, thioether bonds, unlike disulfide bonds, are resistant to the action o f thiols. Therefore, thioether-bridged F(ab'): molecules m ay therefore be more stable in bacteria than disulfide-bridged F(ab'): A -. Since the choice of covalent linkage between Fab" fragments can severely impact future stability o f the Ffab’E construct, often several expression trials are needed before finding the right method. Also the engineering of the hinge region, m ostly by introducing additional cysteine residues at the carboxv-terminal o f the molecule--’, but also by adding new short p e p tid e s ^ , can lead to improved stability o f antibody fragments. However usage o f additional peptides for dimerization may lead to elevated im m unogenicity of the resulting product and can also affect the level o f production in bacteria in adverse manner. Logically, the solution to these problem s would be to express antibody fragments in a m am m alian system. Expression of the above proteins in a mammalian system has several advantages. Mammalian cells themselves strongly mimic the conditions found within 53 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . the hum an body thus ensuring proper processing o f the engineered protein. M oreover, since post-translational m odifications often are crucial to the protein folding and activity, this is a m ajor issue w hen choosing an expression system. Previously, early m am m alian systems were unable to provide desirable levels o f protein expression. However, with the introduction o f different expression system s, especially those containing additional prom oters, enhancers, and combining expression with am plification, this drawback has largely been overcom e-^. High levels o f chim eric antibodies, above the levels o f production in parental murine hvbridom as. have been reported-^. M eanwhile additional advantages o f using mammalian system s to express engineered proteins lie in the fact that the protein processing and folding is done under gentler conditions, less rich in reducing agents, than in bacterial systems thus resulting in a greater preservation o f the intact and active molecules. Also, using a m am m alian system eliminates the risk o f contam ination o f the preparation with bacterial endotoxins. Now that there are several m am m alian expression system s available, m any proteins especially o f hum an origins are more likely to be expressed in this manner. The data shown here strongly dem onstrates the importance o f the environm ent w here the expression o f antibody fragm ents takes place. In a harsh bacterial environm ent, the F(ab')2 often falls apart to Fab' fragments. To express it as a dim er. com plicated modifications to its C terminus^-5- 64 neecj tQ be introduced by genetic engineering or final coupling has to be perform ed after the purification--- 59- 61 54 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth er reproduction prohibited w ithout perm issio n . Regardless o f the modifications, w hen genetically engineered F(ab'); fragments are expressed in bacteria, the end result is always a mixture o f F(ab'); and Fab' molecules. The experiments presented in this chapter indicate that in the case o f the F(ab') 2 fragment, even mild mammalian environmental conditions do not protect it fully from dissociation into Fab' monomers. The molecule with only one cysteine residue (thus only one potential disulfide bridge) fell apart nearly com pletely. Introduction of one more cysteine residue increased the F(ab'): stability, and the molecule with three cysteine residues has shown itself to be com pletely immune from the reducing influences o f the environment. These results are superior to those described by others. Even the best F(ab'):. modified extensively to retain its stability, when expressed in * > bacteria, was present as a dimeric molecule only up to 70% according to Rodrigues— \ w hile the other group attempting to produce Ffab’h in bacteria was only able to obtain less than 30% o f the molecule expressed in dimeric form-4. 35 Additionally, the use o f tw o different tags for the F(ab'): purification highlights the im portance o f the tag itself on protein stability. The F(ab'): fragment with the six histidine tag at the end. needed three disulfide bridges before it became fully stable once expressed. If only two disulfide bridges were present, approximately half o f molecules were in form o f F(ab')2 , while the other h alf fell apart to Fab' fragments. For the F(ab'): fragm ent with the streptavidin-affinity tag. the stability increased significantly and just two disulfide bridges were sufficient to hold it together. The most likely explanation is 55 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . that more disulfide bridges are needed to offset the effect o f repelling charges o f tw o strands o f six histidines parallel to each other in case o f His-tag. This is not the case for streptavidin-affm ity tag. when all nine amino acids are different and two bridges formed by cysteines are sufficient for m olecule's stability. W hile in many cases the expression level in a mammalian system is still below the one achieved in bacteria, in our hands the GS system allows for very good production r a t e s ^ S , 65 [t js worth mentioning that the expression rates were the highest in case o f the intact antibody. Since intact antibody is the preferred expressed form o f the antibody in myeloma cells, it is possible that F(ah’); fragments are considered “dam aged” by a cell and destroyed before their secretion. This could explain the fact that during parallel expression o f the intact antibody and its antibody fragment in GS system , the levels o f expression for the antibody fragments were consistently several fold low er than these for the intact antibody (personal observations). However, in case o f the F(ab’): antibody fragment production, potential lower levels o f production as compared to the intact antibody expressed in mammalian system , or often higher levels o f bacterial production, are more than offset by the ease o f purification in a single step. Other than the stability issue, which has been readily overcome by proper engineering o f the C-term inus o f the molecule and choice o f the purification tag, the mammalian-produced F(ab'): seems comparable to its parent antibody in respect o f antigen binding and tum or targeting. Additionally obtaining the 56 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . F(ab’)2 fragment not by the enzymatic digestion but by the genetic engineering, could make it more suitable for further studies in vivo. It has been suggested that the main reason for chim eric antibody fragments instability' shown in vivo in some studies lies in the process o f preparation which weakens bonds betw een two Fab' monomers and in the fact that the resulting F(ab’)2 molecule has low er num bers o f disulfide bridges between the M A b’s heavy chains as compared to the intact antibody**. Here, the F(ab')2 has been genetically engineered without the need for enzymatic digestion and the number o f its disulfide bridges has been increased. Further characterization o f F(ab')2 m olecules with respect o f their binding affinity showed them to be able to bind to the antigen (single-stranded DNA) within the same decimal range as parental antibody. Biodistribution studies showed that the tum or uptake was diminished for all antibody fragments: 2.93% o f injected dose/gram o f tissue for F(ab'): with the six histidine tag and 3.19% o f injected dose/gram o f tissue for F(ab'); with the streptavidin-affinity tag at 24 hr time point as com pared with 11.5% o f injected dose/gram o f tissue in case o f the intact antibody. For the Fab. the 24 hr uptake was approximately half o f that o f the F(ab')2. i.e. 1.4% injected dose/gram of tissue. Since the binding affinity o f the Fab was nearly equal to that o f F (ab '): using in vitro tests (Table 2-1), this result could only be explained by the difference in size affecting the clearance pattern. Low tum or uptake due to significantly faster clearing time is a well 57 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . known finding in the case o f smaller antibody fragm ents66-68 However, recent work by Homick69 jn our laboratory also emphasized the im portance o f the intact Fc region for the antibody clearance and subsequently for its tum or uptake. In this work, a mutant antibody variant o f chTNT-3 was constructed. This m utant varied from the intact antibody by a single amino acid change in the Fc region, sufficient to abolish its binding to the Fc receptor. Despite the similar molecular weight, the mutant chTNT-3 cleared significantly faster (half-life o f 33.8 hr com pared to 133 hr) than the intact antibody and its tum or uptake was diminished as com pared to the intact antibody: only 2.25% o f injected dose/gram localized to the tum or at 24 hr. As one may note, this value is even low er than that o f the F(ab'): fragment, albeit as the studies were done in a different tum or model, proper comparison is not possible. Low tum or uptakes for the Fab and F(ab'); fragments were, however, more than offset by the excellent tum or to normal organ ratios, which in case o f blood pool varied from 1.21 and 1.58 for the F(ab'): with the six histidine and the strcptavidin- affinity tags, respectively, and 3.1 for Fab at 24 hr time point. Since tumor organ ratios reflect normal organ uptake/binding and provide an indication o f possible toxicity due to the action o f the radioactive compound attached to the protein in case o f radioimmunotherapy^O, as well as the assessment o f the possible background noise in case o f im m unoscintigraphy? 1. these results bode w'ell for the use o f chTNT-3 fragments in such studies. 58 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . This fact was further reinforced in imaging studies, where the F (ab'): fragments proved its superiority over all other fragments derived from the parental chTNT-3 antibody. Here, once again, the im portance o f the size o f the antibody fragment for its tim e and route o f clearance was highlighted, just as has been already emphasized in several studies-^. 66 p ue to lower uptake into the tum or as noted in biodistribution studies but also due to its fast and predominantly renal pattern o f clearance. Fab provided poor images with m ost o f the label present in kidneys and urinary bladder even at early time points (Figure 2-6). The Ffab'L with its size exceeding the renal threshold presented much clearer images. This finding is also in accordance with other studies using enzymatically generated F (ab'): fragments for the immunoscintigraphv o f tum ors 10' 41. 68 Since it has been dem onstrated that pretreatment with IL-2 significantly increases the uptake o f the radiolabeled agents into tumors45. 72 similar studies were perform ed here using the F(ab‘) ;> derivative. As expected, significant improvement in tum or imaging with the respect to the am ount o f the radioactivity present in the tumor, as well as its absence in other organs, was observed. Thus, the combination o f antibody-targeted pretreatm ent w ith IL-2 in combination with F(ab'); imaging is most likely to be employed in clinical studies in the future. R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . The w ork presented in this chapter demonstrates for the first time the successful and stable expression o f the F(ab'); antibody fragment in an mammalian expression system . The F (ab')2 fragment is fully stable and produced in quantities sufficient for further experiments. Additionally, it can be purified in one sim ple step as a single species, ready to use. This represents a viable alternative to standard enzym atic digestion o f the intact antibody and as explained earlier, most likely produces a superior molecule. Biodistribution studies showed this fragment to be capable o f binding the antigen recognized by the original parent antibody and imaging studies showed its superiority as compared with parent antibody and other antibody fragments with respect to low background and rapid imaging. The chTNT-3 F(ab'): fragment emerged as the best candidate for future imaging needs. Its pharmacokinetic behavior is intermediate between that o f intact MAbs and other derived fragments. Thus, the decreased tum or accretion o f the fragment is less than the decrease in tumor uptake observed for single-chain-based (Chapter 3) or Fab' fragments. M oreover, the lack o f accumulation o f signal in the kidneys in case o f the chTNT-3 F(ab'): fragment. in contrast to the predom inant renal clearance of the other smaller fragm ents'^- should result in improved discrimination of abdominal tum ors with radiolabeled chTNT-3 F(ab')2 fragment. R e p ro d u c e d with p e rm ission of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm ission. REFERENCES 1. Wright A. Shin. S-U.. M orrison. S.L... Genetically engineered antibodies: progress and prospects. Critical Review s in Immunology 1992: 12:125. 2. Taylor CR. The current role o f immunohistochemistry in diagnostic pathology. Advances in Pathology and Laboratory M edicine 1994; 7:59-105. 3. Stigbrand T. Ullen A. Sandstrom P. et al. 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Monoclonal antibody production: problems and solutions. 74th Forum in Immunology 1998:542-547. 51. Peterson NC. Considerations for in vitro monoclonal antibody production. 74th Forum in Immunology 1998:553-557. 6 5 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm ission. 52. Frenken LGJ. Hessing JGM . van der Hondel CA M JJ. V'errips CT. Recent advances in the large-scale production o f antibody using lower eukaryotic m icroorganism s. 74th Forum in Immunology 1998:589-599. 53. Nilsson J, Stahl S. Lunderberg J.. Uhlen M. Nygren P-A. Affinity fusion strategies for detection, purification and immobilization o f recombinant antibodies. Protein Expression and Purification 1997; 11:1-16. 54. H uston JS, Margolies M N. Haber E. Antibody binding sites. Advances in protein chem istry 1996; 49:329. 55. Wall JG. Pluckthun A. The hierarchy o f mutations influencing the folding o f antibody domains in Escherichia coli. Protein Engineering 1999; 12:605-61 1. 56. Gill DS, Wong YW. Margolies MN. Differences in sequence-specific expression o f two anti-arsonic Fabs in E. coli. Biotechnology Progress 1997; 13:692- 694. 57. Better M. Bernhard SL. Lei S-P. et al. Potent anti-CD5 ricin A chain immunoconjugates from bacterially produced Fab' and F(ab'): . Proceedings o f the National Academy o f Sciences 1993; 90:457. 58. Pluckthun A. M ono- and bivalent antibody fragments produced in Escherichia coli: engineering, folding and antigen binding. Immunology Reviews 1992: 130:151. 59. Shalaby MR. Shepard HM. Presta L. Rodrigues. M.I Beverley PCL. Feldm ann M. Carter P. Developm ent o f humanized bispecific antibodies reactive with cytotoxic lym phocytes and tum or cells overexpressing HER2 protooncogene. Journal o f Experimental Medicine 1992: 175:217. 60. Rodrigues ML. Shalaby MR. W ether W. Presta L. Carter P. Engineering a humianized bispecific F(ab')2 fragment for improved binding to T cells. International Journal o f Cancer Supplement 1992; 7:45. 61. King DJ, Turner A. Farnsworth APH. et al. Improved tum or targeting with chemically cross-linked recombinant antibody fragments. Cancer Research 1994: 54:6176. 6 6 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth er reproduction prohibited w ithout perm issio n . 62. Glennie M J, M cBride HM. Worth AT. Stevenson GT. Preparation and performance o f bispecific F(ab'y), antibody containing thioethcr-Iinkcd Fab'y fragments. Journal o f Immunology 1987; 139:2367. 63. Pack P. Pluckthun A. Miniantibodies: use o f amphiphatic helices to produce functional, flexibly linked dimeric Fv fragments with high avidity in Escherichia coli. Biochemistry 1992:31:1579. 64. Speck RR, Couto JR. Godwin SG. et al. Inverted Fab2s (IFab2s): engineering and expression o f novel, dimeric molecules with the molecular weight o f 100.000. Molecular Im m unology 1996; 33:1095-1102. 65. H om ick JL. IChawli. L.A.. Hu. P.. Lynch. M.. Anderson. P.M.. Epstein. A.L... Chimeric CLL-1 antibody fusion proteins containing Granulocyte M acrophage Colony-Stimulating Factor or Interleukin-1 with specificity for B-cell malignancies exhibit enhanced effector functions while retaining tum or targeting properties. Blood 1997; 89:4437. 66. Behr TM . Becker WS. Bair H-J. et al. Comparison o f complete versus fragmented technetium-99m-labeled anti-CEA monoclonal antibodies for im m unoscintigraphy in colorectal cancer. Journal o f Nuclear Medicine 1995; 36:430- 441. 67. M offat FL, Pinsky CM. Hammershaimb L. et al. Clinical utility o f external im m unoscintigraphy with the IM M U-4 technctium-99m Fab' antibody fragment in patients undergoing surgery for carcinoma o f the colon and rectum: results o f a pivotal, phase III trial. Journal o f Clinical Oncology 1996; 14:2295-2305. 68. Breitz HB, Tyler A. Bjom MJ. Lesley T. W eiden PL. Clinical experience w ith Tc-99m nofetumomab merpentan (Verluma) radioimmunoscintigraphy. Clinical Nuclear M edicine 1997; 22:615-620. 69. Hom ick JL, Sharifi, J.. Khawli, L.A.. Hu. P.. Bao W.G.. Allaudin. M .M .. Mizokami M.M., and Epstein. A.L... Single amino acid substitution in the Fc region o f chimeric TNT-3 antibody accelerates clearance and improves immunoscintigraphy o f solid tumors. Journal o f Nuclear Medicine 2000; 4 1 :355-362. R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . 70. Breitz HB. D urham JS. Fisher DR. Weiden PL. Radiation-absorbed dose estim ates to normal organs following intraperitoneal 1 ^R e-lab cled monoclonal antibody: methods and results. Cancer Research (Supplem ent) 1995: 55:58 1 7s-5N22s. 71. Steis RG. Carrasquillo JA. M cCabe R. et al. Toxicity, immunogcnicity. and tum or radioimmunodetecting ability o f two human monoclonal antibodies in patients with metastatic colorectal carcinom a. Journal o f Clinical Oncology 1990: 8:476-490. 72. Khawli LA, Epstein AL. Exploration o f novel strategies to enhance monoclonal antibodies targeting. Q uarterly Journal o f Nuclear M edicine 1997: 41:25-35. R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . CHAPTER 3: SMALL FRAGMENTS OF chTNT-3 BASED ON A SINGLE CHAIN CONCEPT ABSTRACT A novel approach to tum or targeting has been developed which utilizes monoclonal antibodies (M abs) directed against common and abundant intracellular antigens accessible only in necrotic areas o f tumors. Designated Tum or Necrosis Therapy (TNT), this targeting approach circumvents many o f the lim itations o f MAb therapy directed against surface antigens. To date, three M Abs have been chimerized in our laboratory, which specifically target animal and human tum ors but do not localize to normal tissues. In order to develop an imaging agent against solid tumors, genetic engineering m ethods were used to produce faster clearing single chain (scFv). diabody, and triabody antibody derivatives o f chTNT-3 antibody directed against single-stranded DNA. Each o f the constructs was m ass-produced in NSO myeloma cells using the glutamine synthase expression system and purified with streptavidin- affm ity columns. 6 9 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . First, pharmacokinetics clearance data were obtained, then using these results, biodistribution and imaging studies were performed in M adison 109 (M A D 109) lung carcinoma tumor-bearing Balb/C mice. By genetically engineering derivatives from the same M Ab, it was possible to compare the imaging capabilities o f each construct in order to identify the best agent for cancer detection. The results showed that derivatives based on single chain were cleared rapidly with little tumor uptake at 6. 12. and 24 hr. The main reason for this fact appears to lie mainly in the partial loss o f the antigen binding potency as compared to the intact antibody and perhaps in the stability and the accessibility o f antigen binding sites o f the newlv-created molecules. Overall, the findings were disappointing but since some o f the tum or models ( L S 174T versus MAD109) and particular labeling agents (technetium versus '"'i) imaged better, this suggests that single chain derivatives could still be used in imaging but only in a way tailored to a particular tumor. Further studies are planned to address these issues. 7 0 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth er reproduction prohibited w ithout perm issio n . I. INTRODUCTION To date, the optim al antibody-based construct for targeted diagnostic and therapeutic strategies involving monoclonal antibodies, has not been identified. Whole immunoglobulins (IgG) have the advantage o f strong and divalent binding, but they penetrate the tum or poorly due to their large size and interact with host effector elements via their Fc dom ain*. Smaller molecules are often preferable to IgG and could be generated using methods o f molecular biology. Resulting derivatives have the advantages o f small size, thus enabling a rapid tum or uptake, faster blood clearance. more homogeneous tum or penetration^-, and reduced immunogenicity. However, they suffer from the excess retention in kidneys and often altered binding characteristics-’ ’'^ . One o f the smallest forms o f the antibody which still retains antigen binding capacity is called a single chain (scFv) construct. The final outcome o f the scFv tum or binding lies in a fine balance between its ability to penetrate tum or tissues due to the small size and its fast clearance from the body by the kidneys. To tilt the balance further tow ards tum or binding, other variations on a single chain them e have been recently described. These include diabody. triabody^. and even (just recently developed) tetrabody m olecular derivatives (see Chapter 1 for detailed description). R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . Figure 3-1: Diabody & Triabody. 7 2 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n So far m ost o f the work on diabodies and triabodies has involved generation o f multivalent molecules with binding sites directed tow ards different molecules^- 1-. This approach is very useful w hen it deals, for example, with directing different cells o r molecules tow ards each o t h e r ^ ’ ^ Little work, however, has been done on creating multivalent molecules directed towards the sam e antigens and exploring how this will impact binding avidity. In this study, the m onovalent single chain, the divalent diabody, and the trivalent triabody were constructed with the hope that they would have significantly different clearing times as compared to the whole antibody, while m ost likely displaying similar or improved (especially in comparison to Fab and F(ab')2 fragments) binding abilities due to their multiple antigen binding sites. Studies from other laboratories on scFv fragments comparing them to other antibody fragments have found that the relative affinity constants o f the bivalent forms (IgG and F(ab'): ) were 7 to 10-fold greater than those for the monovalent forms (Fab' and scFv)^- 2. MATERIALS AND METHODS Reagents, Antibodies, and Cell Lines The source o f reagents and the origin o f antibody and cell lines used have been described in Chapter 2 in details. 73 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth er reproduction prohibited w ithout perm issio n . Construction of Expression Vectors Fragments com posed o f variable heavy and light chains portions joined by different length linkers^ were produced by PCR amplification o f the particular regions o f interest. The expression vectors: pEE12/chTNT-3 HC for a heavy chain, and pEE6/chTN T-3 LC for a light chain 16. were used as the tem plates. For the purpose o f generating the single chain, diabody. and triabody constructs, the prim ary PCR w as perform ed to yield variable portions o f heavy and light chains overlapping at the linker portion and joined by a linker varied in length (0. 5. or 15 AA in length). For the single chain variable light fragment, the 5' TNT-3 VL leader sequence prim er with extended Kozak sequence 17 and X b a l cloning site 5’ - G CTCTA G A G CCG CCA CCA TG G TA TCCA CA G CTCA G TTC - 3’ and the 3' prim er with partially overlapping 15 AA linker sequence 5'-A C C A G A T C C T G G T T T G C C G C T A C C G G A A G T A G A G C C T T T T T C T A TTTC C A G C TTG C T - 3’ was used, and for the variable heavy fragment, the 5' primer w ithout leader sequence and with partially overlapping 15 AA linker sequence 5' - G G CA A A C C A G G A TC TG G TG A A G G C TC A A G A A A A G A A C A G G TCC A A C TG CA GCA G - 3’ and the 3' end prim er containing Natl cloning site 5' - TA G TG CG G CCG CTG CA G A GA CA G TG A CCA G - 3' was used. 74 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . The 15 AA linker for a single chain is not a classical (G ly)4(Ser)| three times repeater 19 but a different linker nam ed 218 This linker was chosen to provide at the same tim e maximum flexibility and resistance to proteolytic action o f intracellular proteases, and it is suggested to be superior to the com m only used ones. The 5’ TNrT-3 VL leader sequence prim er and the last prim er containing S o il cloning site were also used for diabody and triabody constructions. For diabody variable light fragment construction, the 5' TNT-3 VL leader sequence prim er and the 3' prim er w ith partially overlapping 5 AA linker sequence 5' - G G A G G C G G TG G A A G TC A G G TC CA A C TG C A G CA G - 3' w as used, and for the variable heavy diabody fragment, the 5' prim er w ithout leader sequence and with partially overlapping 5 AA linker sequence 5’ - A C TTC C A C C G C C TC C T TTTA TTTC C A G C TTG G T - 3’ and the 3' end prim er containing N o tl cloning site and described above, were used. Sim ple 5 AA linker contained (Gly)4(Ser)i sequence. For triabody variable light fragment construction, the 5' TNT-3 VL leader sequence prim er and the 3' prim er partially overlapping variable heavy portion and with no linker sequence 5' - C TG C T G C A G T TG G A C C TG TTTTA TTTC C A G C TTG G T - 3’ R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . were used, and for the variable heavy triabody fragment, the 5' prim er partially overlapping with variable heavy sequence and w ithout leader sequence, and with no linker 5’ - A CCA A G C TG G A A A TA A A A C A G G TC C A A C TG C A G C A G - 3' and the 3' end prim er containing N otl cloning (as described earlier) site were used. These prim er pairs generated two PCR products per molecule. A secondary PCR assem bly was perform ed using the overlapping prim ary PCR fragments and the outer prim ers (5' with Xbcil site and 3' with N otl site) from the pairs listed above. The final assembled PCR fragments had their size confirmed by gel electrophoresis, and their sequences by sequencing by the M icrochemical Core facility o f Norris Com prehensive Cancer Center at USC School o f Medicine. Correct fragments were inserted into the Xbcil and N otl sites o f pEE12 which at the 3’ end contained an additional sequence coding for nine amino acids tail with affinity for streptavidin added for purification purposes. The following resulted in the expression vectors: 12/chTNT-3/scFv. 12/T3D. and 12/T3T encoding respectively the chimeric TN T-3 single chain, diabody, and triabody. Expression and Purification of chTNT-3 Fragments The chTNT-3 single chain based constructs' expression and purification m ethods are sim ilar to these described in details in C hapter 2. 76 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . Determination of Avidity The avidity constant o f the chTNT-3 fragments was determined by a fixed cell RIA as described previously (Chapter 2). The equilibrium or avidity constant Ka was calculated by the equation K . = -(slope/«). where n was the valence o f the antibody (1 for scFv. 2 for diabody. 3 for triabody). HPLC and Immunoassays The m ethodology used for in vitro characterization o f chTNT-3 based constructs has been described in details in Chapter 2. Radiolabeling of Fusions '"I-labeled single chain derivatives were prepared using a modified chloraminc- T method as described earlier (Chapter 2). with a yield o f 60-67°» radiolabeled product. The wTc-labeled MAbs were prepared using a modified direct labeling method essentially as described previously by A llau d in -l. In short, the MAbs were diluted to 0.4 mg/ml in a volume o f 500 |il using 0.1 M sodium borate buffer. pH 9.3. deoxygenated by bubbling N? gas through the solution, and sealed under nitrogen. " n,Tc sodium pertechnetate (111 MBq in 300 pi) was injected into the MAb solution. 77 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth er reproduction prohibited w ithout perm issio n . Subsequently, 40 |al o f stannous chloride/glucoheptanoic acid solution (containing 10 mg glucoheptanoic acid and 1 mg stannous chloride in 1 ml o f deo\vgenated w ater) were added to the above solution. The reaction mixture was incubated for 2 hr at 37° C before it was purified using a PD-IO column (Pharmacia Biotech. Piseataw ay. \ J ) . with a yield o f 60-67% o f product. Pharmacokinetics, Biodistribution, and Imaging Studies The protocols were described in details in Chapter 2. 3. RESULTS Expression, Purification, and Characterization of chT \T -3 Fragments The chTNT-3 based constructs were expressed using the NSO murine myeloma cell system . All antibody fragments were secreted into the medium and thus easily obtained from the culture supernatants. The production levels varied w ithin the range from 3 to 5 |ag/ml/106 cells/24 hr. The affinity tag added to the C-end o f each molecule for the purification purposes also provided an efficient screening system for the identification o f positive clones by em ploying one-step detection with HPRO - conjugated streptavidin. Simple purification process resulted in a clean product 78 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . recovered in a single-step procedure. The SDS-polvacrylam ide gel electrophoresis — o f the affinity-purified fragments showed that the eluted protein was > 95° < > pure and m igrated with the mobility consistent at the expected molecular weight. It was 29 kDa for diabody and 27 kDa for the triabody constructs, since both are slightly sm aller than the chTN T-3/scFv (30 kDa) due to shorter or absent linker ( Figure 3-2). R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . Figure 3-2: SDS-PAGE Gel of Single Chain Derivatives. m 97.4 66 45 31 21.5 lane 1 lane 2 lane 3 lane 4 lane 1: single chain (scFv) construct lane 2: diabody construct lane 3: triabody construct lane 4: low weight molecular marker R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . Since all the single chain derivatives are not held by any covalent bonds, they fall apart regardless o f the native or denaturing nature o f SDS-PAGE gel. Additionally the single chain and its derivatives are known to have a tendency to form multimers 1 19 at least when expressed and later purified from bacteria. To confinn the size o f the fragments, investigate their stability in solution, and tendency to the formations o f aggregates, size exclusion HPLC was perform ed. It showed (Figure 3-3) that the single chain (30 kDa) eluted with the retention tim e o f 15.01 minutes as a mostly single peak but with some tendency to form dimers. The diabody's retention time was 14.15 m inutes (58 kDa), and triabody - 13.66 m inutes (81 kDa). They both eluted as single peaks thus affirming their purity and stability. By comparison, the intact chTN T-3 antibody retention tim e was 12 minutes. 8 1 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . Figure 3-3: HPLC Chart of Single Chain Derivatives. b a i <■ ■ t i tm m m m , „ - J f t J f l d l 10*m M ■ ^ ^ T T r W T T T T r ’ . ....... . . . . . . . . . . I . I i % f t i / / S in g le C h ain Diabody I * 4 v / A a if r* «t rww T riabody I ' ■ i ■' <— ' ■ I ■ > ’H.ltlt1 gaw+ r.~ « + " I - » ■ ' « « « I w ^ .1 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . Affinity m easurem ents using RIA allowed determ ination o f the affinity constant (Table 3-1, Figure 3-4). The chTNT-3 fragm ents were found to have dim inished avidity constants (Table 3-1) as com pared to that o f 1.4 x 10" M '1 for chTNT-3 As expected from the literature*^ 23-25^ diabody showed an im provem ent in binding affinity as compared to single chain, but contrary to som e other rep o rts^ 15, 24^ the generation o f the triabody did not result in further im provem ent o f K.a. N evertheless this study dem onstrates that the fragments retained the binding properties o f chTNT-3 albeit at the low er values. Table 3-1: Avidity Constants of Different Single Chain Variants. F ra g m e n t # o f binding sites % B inding avidity co n stan t K a Single Chain one 67.9 0.25 x 10" v r 1 Diabody tw o 68.5 0.40 x 10" M '1 Triabody three 68.1 0.31 x 10" M"1 chTN T-3 two 89.0 1.43 x io" v r 1 8 3 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth er reproduction prohibited w ithout perm issio n . Figure 3-4: Affinity Constants for Single Chain Antibody Fragments. S in g le C h a i n D e r iv a tiv e s A f f in ity Single Chain Diabod v Triabodv 0.5 - 10 bound <ng) R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . Pharmacokinetic, Biodistribution, and Targeting Studies o f ch T \T -3 Fragments Clearance studies in Balb/C mice were performed to establish differences in pharmacokinetics characteristics between the parental chTN T-3 and the chTN T-3 fragments. It has previously been show n that chTNT-3 clears slowly with a whole- body half-life o f 134.2 +/- 4.0 hr in Balb/C mice, as typical o f a chimeric M A b containing human IgG| constant re g io n s^ . As depicted in Table 3-2. all chTN T-3 fragments showed increase in clearance time according to their size. Table 3-2: Half-lives of Different chTNT-3 Derivatives as Determined by the Clearance of Radiolabeled Proteins From Balb/c Mice. Fragment Size (kDa) T./2 Single Chain 30 kDa 4.9 - - 0.9S hr Diabody 60 kDa 5.1 - - 0.46 hr Triabody 90 kDa 9.9 - - 1.25 hr ChTNT-3 150 kDa 134.2 — 4.0 hr The analysis revealed that half-lives o f all single chain-derived fragments varied betw een themselves in accordance with the molecular size predicted for each o f the variants (Table 3-2) with the exception o f the diabody which seemed to clear faster X5 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . than expected for its size. Similar clearance values have been reported prev iously for several single chains and som e diabody fragments obtained by genetic engineering-^. For example, the single chain clearance times were shown to be in a range from 1.5 hr'"*’ to 2.8 hr27, and 3.5 hr^S. Interestingly, for the diabody. other investigators also reported surprisingly short half-life ranging from 2.89 hr-^ to 3.3 h r-^ . and 3.5 h r ^ with no real explanation as to reasons behind its short serum persistence. As for the recently described triabody. no anim al studies have been reported at this time. Biodistribution and tum or targeting for the chTNT-3 single chain, diabody. and triabody were evaluated in Balb/C mice bearing murine lung carcinom a Madison 109 tum ors. Since chTNT-3 binds to dead and dying cells present in the tum or, the results obtained with this animal tum or m odel should be representative. The biodistribution studies showed that despite their rapid elimination. chTNT-3 fragm ents localized efficiently to the tum or site, as depicted in Figure 3-5. For these studies, the tum or and normal tissue biodistributions o f l2'I-labeled chTNT-3 and chTNT-3 fragm ents w ere evaluated at 6, 12. and 24 hr post-injection. All fragments showed localization to the tum or site and the actual % o f dose/gram (Figure 3-5) as well as tum or organ ratio for each (Figure 3-6) varied depending on their size and the time point o f the measurement. No significant im provem ent in tum or uptake was noted for newly generated di- and triabody. 86 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . Figure 3-5: The % of Injected Dose/Gram of Tissue at Different Time Points For All Single Chain Derivatives at 6, 12, and 24 Hours. 7 - 6 - 5 - E e 3 - 1 - 0 - hlood liver kidney tumor organ m Single Chain Q Diahody T riahndy hlood liver kidney tumor organ < S 7 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . Figure 3-6: The Tumor/Organ Ratio at Different Time Points For All Single Chain Derivatives at 6, 12, and 24 Hours. 51 Single Chain Diabody Triabody kidnev blood nvcr organ 12 hr 888 mm m 1 1 sc u c hlood liver organ kidnev 24 hr hlood liver kidne\ Single Chain K3 Diabody E8 Triabody H Single Chain H Diabody 0 Triabodv organ N S R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . Imaging Studies Im m unoscintigraphy was performed to examine the difference between tum or targeting with chTN T-3 and its derivatives. M ice bearing MAD 109 tum ors were imaged at 6. 12, and 24 hr post-injection. Images obtained from representative mice are presented in Figure 3-7. These studies showed disappointing images obtained by using all single chain-derived antibody fragments with respect to tumor localization. The single chain construct showed no tum or localization at 6 hr time point and the labeling was present mostly in the urinary system . By 12 hr (data not show n), traces o f the single chain appeared within the tum or but the majority o f label w as still distributed betw een the heart and kidneys. No im provem ent in the image was obtained at 24 hr tim e point. Similarly, diabody and triabody failed to provide any tum or images at 6 hr and were present mainly within the blood pool. At 12 hr (data not included), the diabody showed some localization in tum or but still the majority o f the label in case o f diabody and practically all the label in case o f triabody was divided between the heart and kidneys. At 24 hr time point, both molecules exhibited faint traces o f tum or uptake, and the majority o f the label was still present in the blood pool and kidneys, contributing to the significant background. 8 9 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . Figure 3-7: I31I Images o f Madison 109 Tum or in Balb/C Mice Obtained Using Intact Antibody and Antibody Fragments (6 and 24 Hrs Post Injection). y ■ , 1 * ; - t v * 9 9 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth e r rep roduction prohibited w ithout p erm ission . Additional im aging studies were performed using the single chain and a different tum or model, nam ely hum an colon carcinoma LS174T in nude Balb/C mice. Tum or-bearing Balb/C mice were injected i.v. with chTNT-3/scFv molecule labeled with technetium as described by Allaudin. The subsequent images taken at 6 and 18 hr after the injection o f the antibody fragment showed its rapid localization into tum or with equally swift clearance from other organs (Figure 3-8). Additionally, the tum or accretion o f the radiolabeled product was superior to that obtained using whole chTNT-3 antibody and its m utant version with significantly shorter clearing time due to point m utation in Fc regional. Several investigations^- 32. 33 have shown that pairing the antibody fragm ent with the right radioactive com pound such as technetium can significantly improve imaging capability o f the re a g e n t^ - 35 This also appears to be a case for the chTNT-3/scFv construct. Obviously these initial studies using technetium will require further investigation with all the fragments under study. 91 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . Figure 3-8: wTc Images o f LS174T Tumor in Nude Balb/C Mice Obtained Using Intact and Mutant Antibody, and Single Chain Fragment (6 and 18 Hrs). £ n 6 hr 18 hr R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . 4. DISCUSSION Recently monoclonal antibodies are becoming accepted into the clinic not only as a diagnostic tool in p a t h o l o g y - ^ but also in other applications, including the detection and treatment o f c a n c e r^ ^ O . However, the current generation o f the antibodies entering the clinic is still handicapped by their physiological characteristics. First, immunoglobulins are design to recognized specific antigens^ 1 bin they are not alw ays able to find truly specific antigens. This could be circum vented by the judicious choice o f the antigen, just as it is in case o f Tum or Necrosis Therapy (T N T) approach (see C hapter 1). Secondly, the large size o f the monoclonal antibodies and presence o f Fc receptor causes them to remain in the circulation for prolonged periods o f tim e"^ and limits their ability to diffuse into tum ors. In order to address these lim itations, smaller antibody fragments were developed, first using proteolytic digestion-^ for the production o f F(ab')2 and Fab m o l e c u l e s ^ and. more recently, by the recombinant generation o f antigen-binding fragments with differing molecular m asses (25 to 125 kDa) and valency (monomeric, dimeric, and more)47-50 por these investigations, genetic engineering has provided us the opportunity to generate a fam ily o f molecular derivatives which can be directly com pared in appropriate animal models. 93 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . One o f the sm allest form s o f the antibody which still retains antigen binding capacity is the single chain construct^*. For practical reasons, it is the smallest antibody fragment developed to date with potential clinical applications. Several modification strategies have been em ployed to improve the localization o f scF\-based molecules in tum ors and to slow down their clearance from the body. Recently invented single chain-based derivatives include the diabody**. triab o d y --. and tetrabody24 molecules. In the past most genetic engineering approaches to the construction o f single chain, diabodies. triabodies, and Fab and F(ab'); derivatives o f the antibody involved the generation o f ju st one particular m olecule^ or sometimes bivalent molecules directed to two different targets* *. Work presented in this chapter and the previous one describes the construction o f the monovalent single chain and Fab' molecules, the divalent diabody and Ffab'): molecules, and trivalent triabody molecule from the same parent antibody with the hope that they will have substantially different pharmacokinetic characteristics as compared to the whole antibody, while most likely- displaying similar or im proved binding abilities due to their m ultiple antigen binding sites. It was hoped that the evaluation o f their properties would prov ide an assessment o f their potential as diagnostic agents for cancer detection. 94 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . In this study, different fragments based on a single chain of the chimeric T N T -3 antibody were produced by PCR assembly. The Glutamine Synthetase Gene Am plification System was used to produce the chTNT-3 derivatives in mammalian cells, so all necessary post-translational modifications could be preserved. While single chain reagents and their derivatives are in most cases produced in bacteria, in our hands the recom binant antibody fragments were produced using the GS system in sufficient quantities for pre-clinical studies. Antibody fragments produced in mammalian systems are generally easier to purify since they are secreted into the supernatant and can be recovered under native conditions. This bypasses the denaturation and refolding steps often necessary when expressing is perform ed in bacteria--. The single-chain, diabody and triabody fragments were expressed with strcptav idin-affinity tags attached to the C-end o f the protein thus making it possible to use modified streptavidin (Streptactin)-affinity column for p u r i f i c a t i o n ^ . Xhis method proved to be efficient and an easy single-step procedure. The chTNT-3 fragments retained only a part o f the high affinity o f chTN T-3. as dem onstrated by radioimmunoassay and Scatchard analysis. The F(ab'): fragment, however, possessed an affinity closest to that o f a parental antibody (see C hapter 2). While loss o f binding abilities happens relatively often when engineering single chain fragment from the intact antibody^5. 56 recent studies showed that modifying scFv to form a diabody^’ 23-25 ancj triabody^- ^4 usually results in new molecules 95 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . possessing superior binding affinities. Why only the diabody showed a relatively high affinity it is not clear at this time. Possibly since each engineered antibody is different, in this particular case the conformation o f the new ly-constructed fragments prevents them from utilizing additional binding sites. A recent paper by Le Gall-"* showed in vitro that in the case o f a particular single chain and its diabody. triabody and tetrabody fragments, only the diabody showed a real improv em ent in v alency and in antigen binding over the single chain. Triabody and tetrabody showed some improvement but not as great as expected. This was due to the fact that their configuration prevented them from utilizing all binding sites, thus in fact reducing their valency to that o f the diabody. The discussion above can also apply to the biodistribution results found in the MAD 109 tum or-bearing mice, shown in Figure 3-5. For the single chain, the °< i o f injected dose/gram o f tissue for the tumor at 6 hr time point is 4.4% . for the diabody it is 2%. for the triabody is 3.5%. In this case there is no detectable improv ement over the original single chain derivative. Only at the 24 hr tim e point all derivatives become more evenly distributed with the single chain showing an uptake o f 1.46% o f dose per gram o f tum or tissue, the diabody with 0.65% o f dose, and the triabody with 1.023% o f dose per gram. This change at the 24 hr time point is understandable since the single chain after that time is clearing from the body, while the much larger triabody still persists in the circulation. Further support for this conclusion com es from the fact that 96 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth er reproduction prohibited w ithout perm issio n . the tum or to normal organ ratio at the 24 hr time point is 2.25 in the blood for single chain, 9.34 for diabody. and only 1.468 for the triabody. Interestingly and disappointingly, diabody behaves very much like a single chain, clearing quickly from the circulation and showing low uptake in tum or but with a high tumor/organ ratio. This com es as a surprise in lieu o f its improved avidity to antigen as shown by in vitro studies. Possibly the im provem ent in binding affinity is not sufficient to offset its faster clearance. A lternatively the flaw lies within the molecule itself, perhaps in its lower stability. However the size exclusion studies and stability assessm ent (data not shown) do not support this suggestion. Several have reported a significant improvement in tum or uptake for diabodies. including those by Adam s^b with a 6.5-fold higher tum or uptake at 24 hr time point by V iti^? with a 6-fold increase, by B e r e s f o r d ^ S with a 4-fold increase by W u-5 with a 3-fold increase. O th e rs ^ contradict this finding, suggesting that the main prediction for the binding and targeting abilities o f the derivatives o f a single chain, still lies within the single chain itself. This m eans that uptake o f the antibody fragment into the tumor can be improved but only to som e degree over that o f the original single chain, or rather more precisely, over that o f the original parental antibody. In fact, m ost o f the papers reviewed above utilize monoclonal antibodies with very high tum or uptake in murine tum or models, approaching, for example 38% ID/gram for the intact antibody and 4.29% ID/gram for the scFv dim er 58 These data are not relevant to the human studies 97 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . where uptake rarely exceeds 1%^* ^9 As for the chTNT-3 and its single chain derivatives, the starting point is low since the uptake o f the parent antibody into the M adison 109 tum or is 14.6% ID/g at 24 hr time point. By comparison, the uptake o f the single chain in tum or (1.46% ID/g) is only I/ 10th o f the value for the intact parental antibody. Theoretically this is the same l/l0 th described by Beresford but again, the starting point is lower which may greatly affect tum or imaging later on. Still some o f the papers mentioned earlier, present data showing real improvement o f the antibody fragm ent's uptake into tumor when using the diabody as compared to the single chain26. 57 why this is not the case for the diabody and triabody derived from the chTNT-3 single chain most likely is due to the low binding affinity o f the single chain itself and subsequent lack o f its improvement upon generation o f the derivatives. In support o f this, several recent papers em phasized the importance o f the increased affinity o f the antibody fragment for selective minor binding and higher uptake-7- The immunoscintigraphic results presented in this report evaluated the ability o f the chTNT-3 and its fragments to target solid tumors. Previous publications used chTN T-3, fusion protein containing chTNT-3 and interleukin-2, and mutant chTN T- 331, 61 for tum or imaging and the results were encouraging. Here the imaging data are at best ambiguous. While the immunoscintigraphy using M adison 109 tumor model fails totally in respect o f single chain-derived fragments, it seems to work well for the F(ab' ) 2 fragment. In contrast, the LS174T human carcinoma/nude mice model showed 9S R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . excellent imaging results using single chain, thus demonstrating the need for additional studies, using perhaps different tum or models as well as different labeling reagents to define the optim al parameters. In conclusion. chTNT-3/F(ab')2 fragment proved to be the best imaging agent with l3II as the radionuclide (see C hapter 2). The rapid clearance o f the chTN T-3 F(ab): fragm ent from the blood and normal tissues resulted in decreased tum or levels o f radiolabeled M Ab. although the decrease in tum or levels was less than that seen in many normal tissues, which yielded higher tumor/organ ratios for many tissues. Even for the other antibody fragments which showed less tum or uptake than F(ab'): . the tumor/organ ratios were often higher than seen for the intact antibody. While single chain derivatives performed in a disappointing manner, they still may warrant further investigation. The fact that technetium-labeled single chain produced good images in the LS174T tum or model suggests that either the single chain itself or other derivativ es may be still useful for tum or imaging. These results point to the need for further investigations into the used labeling technique and the choice o f appropriate labeling agent. Additionally, further modification o f the single chain to improv e its binding affinity could be performed. Several papers suggest point mutations within antigen- recognizing dom ains as a good way to improve antigen binding^- 57. 60 anc] jn turn tum or retention o f the antibody. Recent publications also suggest that single chain derivatives with additional disulfide bridges introduced by genetic engineering can form 90 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . covalent multim ers which display greater stability and perform better after these modifications^ 58 y h e possibility o f producing a tetrameric molecule based on a single chain should also be investigated. Several papers describing such modifications professed the increased avidity o f new molecules, which could prove advantageous to the imaging and im m unotherapy o f tumors*-* -4- 63. 64 W hatever the outcom e o f the further investigations, one very prom ising imaging agent has already been described in Chapter 2. Indeed, due to the specificity o f TN'T- based antibodies for a universal nuclear antigen exposed in degenerating and necrotic cells, it is anticipated that products derived from this type o f M Abs will tind applications in targeting the m ajority o f human solid tumors. Most likely im aging with the chTNT-3 F(ab'):- fragm ent may find utility not only in the diagnosis and staging o f cancers, but in monitoring the progress o f various therapeutic modalities by imaging new areas o f degeneration and necrosis resulting from cancer treatments. REFERENCES 1. Foon K.A. Biological response modifiers: the new im m unotherapy. Cancer Research 1989; 49:1621. 2. Y okotaT , M ilenic, D.E.. Whitlow. M.. Schlom. J. Rapid tum or penetration o f single-chain Fv and com parision with other immunoglobulin forms. Cancer Research 1992; 52:3402. 100 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . 3. Larson SM. Radiolabeled monoclonal anti-tum or antibodies in diagnosis and therapy. Journal o f N uclear M edicine 1985: 26:538. 4. Podoloff DA, Patt YZ, Curley SA. Kim EE. Bhadkam kar VA. Smith RE. Imaging o f colorectal carcinoma with technetium-99m radiolabeled Fab’ fragments. 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W hitlow M. Bell. B.A.. Feng, S-L.. Filpula. D.. Hardman. K.D.. Hubert. S.L.. Rollence, M .L.. Wood. J.F.. Schott, M .E.. Milenic. D.E.. Yokota. T.. Seldom. J. An improved linker for single-chain Fv with reduced aggregation and enhanced proteolytic stability. Protein Engineering 1993; 6:989. 19. Turner DJ. Ritter. M.A.. George. A.T.J... Importance o f the linker in the expression o f single-chain Fv antibody fragments: optimisation o f peptide sequence using phage display technology. Journal o f Immunological M ethods 1997: 205:43. 20. Schm idt TGM. Skerra A... The random peptide library-assisted engineering of a C-term inal affinity peptide, useful for the detection and purification o f a finctional Ig Fv fragment. Protein Engineering 1993; 6:109-122. 21. Alauddin MM, Khawli LA. Epstein AL. An im proved method o f direct labeling monoclonal antibodies with technetium-99m. Nucl. Med. Biol. 1992: 19:445 22. Laemmli UK. 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In vivo tum or targeting o f a recombinant single-chain antigen-binding protein. Journal o f the National Cancer Institute 1990: 82:1191-1197. 28. Willuda J, Honegger A. Waibel R. et al. High thennal stability is essebtial for tumor targeting o f antibody fragments: engineering o f a humanized anti-epithelial Glycoprotein-2 (Epithelial Cell Adhesion Molecule) single-chain Fv fragment. Cancer Research 1999: 59:5758-5767. 29. Wu AM . al. e. Tum or localization o f anti-CEA single chain F\ s: Improved targeting by non-covalnet dimers. Imm unotechnology 1996: 2:21-36. 30. Adams GP, Schier R. M cCall AM . et al. Prolonged in vivo retention o f a human diabody targeting extracellular domain o f human HER2 rieu. British Journal o f Cancer 1999; 77:1405-1412. 31. Homick JL, Sharifi, J.. K.hawli. L.A.. Hu. P.. Bao W.G.. Allaudin. VI.VI.. Mizokami M .M.. and Epstein. A.L... Single amino acid substitution in the Fc region o f chimeric TNT-3 antibody accelerates clearance and improves im m unoscintigraphy o f solid tumors. Journal o f Nuclear M edicine 2000: 41:355-362. 32. Bruland OS. Cancer therapy with radiolabeled antibodies. An overview. Acta Oncologica 1995; 34:1085-1094. 1 0 3 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth er reproduction prohibited w ithout perm issio n . 33. Behr TM, Becker WS, Bair H-J. et al. Comparison o f com plete versus fragmented technetium-99m-labeled anti-CEA monoclonal antibodies for immunoscintigraphy in colorectal cancer. Journal o f Nuclear M edicine 1995; 36:430. 34. Chengazi VU. Feneley MR. Ellison D. et al. Imaging prostate cancer with technetium -99m -7El 1-C5.3 (CYT-351). Journal o f Nuclear M edicine 1997; 38:675. 35. Colcher D. Pavlikova G. Beresford G. Booth B.M .J.. Choudhury A.. Batra S.K. Pharmakinetics and biodistribution o f genetically-engineered antibodies. 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Radioimmunoscintigraphy with In-111-labeled capromab pendetide predicts prostate cancer response to salvage radiotherapy after failed radical prostatectom y. Journal of Clinical Oncology 1998: 16:284-289. 41. Roselli M, Guadagni F, Buonomo O. et al. Tum or markers as targets for selective diagnostic and therapeutic procedures. Anticancer Research 1996: 16:2187- 2192. 42. von Mehren M, W einer LM. Monoclonal antibody-based therapy. Current Opinion in Oncology 1996; 8:493-498. 104 R e p ro d u c e d with p e rm issio n of th e copyright ow ner. F u rth e r repro d u ctio n prohibited w ithout perm issio n . 43. Zhu H, Baxter LT, Jain RK. Potential and limitations o f radioimmunodetection and radioim m unotherapy with monoclonal antibodies. Journal o f Nuclear Medicine 1997: 38:731-741. 44. Ghetie V, Popov, S.. Borvak. J... FcRn: the MHC class I-rcIated receptor that is more than an IgG transporter. Immunology Today 1997; 18:592-598. 45. Covell DG. Barbet. J.. Holton O.D.. Black. V.. Parker R.J.. Weinstein J.N... Pharmacokinetics o f monoclonal immunoglobulin G j F fab 'b and FalV in mice. Cancer Research 1986:46:3969. 46. Khawli LA. M ilkie BS. Homick JL. et al. Production o f immunoreactive F(ab')2 fragments in high yield from murine IgGl monoclonal antibodies. 1JBC 1996; 2:89-99. 47. King DJ, M ountain. A.. Adair. J.R.. Owens. R.J.. Harvey. A.. Yarraton. Ci.T... Tum or localization o f engineered antibody fragments. A ntibody Immunoconjugates & Radiopharm acology 1992; 5:159. 48. Wright A. Shin, S-U., Morrison. S.L... Genetically engineered antibodies: progress and prospects. Critical Reviews in Immunology 1992: 12:125. 49. Stigbrand T, Ullen A. Sandstrom P. et al. Tw enty years with monoclonal antibodies: state o f the art— where do we go? Acta Oncologica 1996: 35:259-265. 50. Sensel M G, Coloma J, Harvill ET. Shin S-U. Smith RIF. M orrison SL. Engineering novel antibody molecules. Chemical Immunology 1997: 65:129-158. 51. Raag R, W hitlow M. Single-chain Fvs. FASEB Journal 1995: 9:73-80. 52. Illiades P. Kortt. A.A., Hudson. P.J... Triabodies: single chain Fv fragments without a linker form trivalent trimers. FEBS Letters 1997: 409:437. 53. Verma R, Boleti E, George AJT. A ntibody engineering: Comparison of bacterial, yeast, insect and mammalian expression system s. Journal o f Immunological M ethods 1998; 216:165-181. 54. Schmidt TM G , Skerra, A... One-step affinity purification o f a bacteriallv produced protein by means o f the "Strep-Tag" and immobilized core straptavidin. Journal o f C hrom atography A 1994; 676:337. 105 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm ission. 55. Huston JS. Levinson D. M udgett HM. et al. Protein engineering o f antibody binding sites: recovery o f specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Preceedings o f the National Academ y o f Sciences 19X8: 85:5879-5883. 56. Adams GP. Schier R. Generating improved single-chain Fv molecules for tum or targeting. Journal o f Immunological Metohds 1999; 231:249-260. 57. Viti F. Tarli L. Giovannoni L. Zardi L. Neri D. Increased binding affinity and valence o f recombinant antibody fragments lead to improved targeting o f tum or angiogenesis. Cancer Research 1999: 59:347-352. 58. Beresford GW . Pavlinkova G. Booth BJM. Batra SK. C'olcher D. Binding characteristics and tum or targeting o f a covalently linked divalent CC'49 single-chain antibody. International Journal o f Cancer 1999; 81:911-917. 59. Buist MR, Kenemans P. den Hollander V V . et al. Kinetics and tissue distribution o f the radiolabeled chimeric monoclonal antibody M OvlX IgG and F(ab’), fragments in ovarian carcinoma patients. Cancer Research 1993: 53:5413-541 S. 60. Adams GP. Schier R. Marshall K. W olf EJ. M cCall AM. Marks JD. Increased affinity leads to improved selective tumor delivery o f single-chain Fv antibodies. Cancer Research 1998; 58:485-490. 61. Homick JL, Khawli LA. Hu P. Epstein AL. Pretreatm ent with a monoclonal antibody/interleukin-2 fusion protein directed against DNA enhances the delivery o f therapeutic molecules to solid tumors. Clinical Cancer Research 1999: 5:51 -60. 62. FitzGerald K. HoIIiger P. W inter G. Improved tum or targeting by sulphide stabilized diabodies expressed in Pichia pastoris. Protein Engineering 1997: 10:1221- 1225. 63. Pack P, M uller K. Zahn R, Pluckthun A. Tetravalent miniantibodies with high avidity assembling in Escherichia coli. Journal o f M olecular Biology 1995: 246:28-34. 64. Rheinnecker M, Hardt C, Hag LL. et al. M ultivalent antibody fragments with high functional affinity for tumor-associated carbohydrate antigen. Journal o f Immunology 1996; 157:2989-2997. 106 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . CHAPTER 4: FUSION PROTEIN BETWEEN CHIMERIC ANTIBODY chTNT-3 DIRECTED AGAINST ssDNA AND BACTERIAL ENZYME CYTOSINE DEAMINASE (CDase) FOR THE TREATMENT OF SOLID TUMORS ABSTRACT The prim ary objective o f our laboratory is to develop successful m ethods o f cancer imaging and therapy using monoclonal antibodies. In support o f that goal, the study presented in this chapter evaluates a combination o f a novel monoclonal antibody-based regime developed in our laboratory for the therapy o f cancer in com bination with the well-known concept o f tumor treatment called A ntibody- Directed Enzym e Prodrug Therapy (A D EPT). A novel tumor targeting approach developed in our laboratory and designated Tumor Necrosis Treatment (TNT) uses monoclonal antibodies to target highly conserved and abundant intracellular antigens expressed in necrotic regions o f human tum ors. One o f the most interesting aspects o f the TN T approach is the fact, that once localized to the tumor, the antibody remains there for a prolonged time without being shed or internalized, or otherwise destroyed. In the A D EPT concept, the conjugate betw een antibody and enzyme is used to target the enzym e to the tumor. Once in place, the enzym e conjugate activates a 107 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . system atically adm inistered prodrug into active form to treat the tum or sparing normal tissues. For this very reason, it is im portant that the antibody is present at the tum or site for prolonged periods o f time. Since this is one o f the main characteristics o f T N T antibodies, we believe that combination o f both concepts will be a unique delivery m ethod for the A D EPT therapy. Here we present the first generation o f fusion proteins consisting o f either F(ab')2 or single chain (scFv) fragments derived from chim eric TNT-3 M Ab and the modified bacterial enzyme cytosine deaminase (pC D 2) in order to evaluate and choose the reagent with best phannacokinetics and tum or uptake. Additional experiments assessed in vitro and in vivo properties o f these genetically engineered TNT-3 antibody fusion products, as a delivery vehicle for the A D EPT therapy. The results o f these studies are expected to broaden the potential uses o f TNT and lay the foundation for future clinical trials o f this promising new approach. 10S R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . 1. INTRODUCTION Chem otherapy is based upon the presumption that there are differences between normal and neoplastic cells which can be exploited in the process o f designing novel approaches to cancer t herapy^Thi s presum ption, however, has only been partially true. There are differences between malignant and normal tissues but they are slight and inconsistent^- 4. For these reasons, the achievement o f a useful therapeutic index o f tumor cell killing versus toxicity against normal cells, has proven difficult. In general, the traditional therapeutic modalities o f surgery, radiation, and chem otherapy are non-specific, in that they remove or destroy normal cells along with cancer cells-. O ther factors that limit the effectiveness o f therapeutic strategies include distant metastases. cellular heterogeneity o f the cancer, and resistance to dings and radiation^. All this in turn has led to development o f the strategies combining therapeutic effects o f multiple cytotoxic drugs, radiotherapy and to a lesser degree, immunotherapy- - ^ . Significant im provem ents in survival have resulted for some tumors but m any malignant neoplasms remain refractory to therapy^ with an overall five year survival o f 50% (for all tum ors, all types, all stages), and 15% or less for advanced, deep-seated can cers^. Realistically, the therapy o f cancer must still be regarded as both empirical and experimental. 109 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . New research approaches aimed tow ard a more profound understanding o f the biology o f each cancer com bined with fresh and creative use o f acquired knowledge are needed to direct the search for clinically useful treatm ent strategies. One such relatively new and creative idea uses monoclonal antibodies, molecules with highly selective recognition abilities *® to target cytotoxic agents to tum ors. These cytotoxic agents could be conventional cytotoxic drugs (e.g. doxorubicin)**, radioisotopes*-, toxins derived from plant (ricin) and bacteria ( D i p h t h e r i a ) * * " * ■ . or enzym es (e.g. 1 3 - lactamase, (3-glucuronidase) *5. Although the system ic cytotoxicity is reduced due to specific targeting, the therapeutic results have often been poor, chiefly because o f an inadequate uptake o f the conjugate by the tum or cells and the consequent failure to adm inister a dose sufficiently damaging to the tumor. So far only system s capable o f acting at a distance on other cells (radioisotopes, activated diffusing prodrugs) or amplifying their actions (enzym es) showed more encouraging results. The A ntibody- Directed Enzyme Prodrug Therapy *6 represents such an approach, combining both o f these characteristics (action at a distance and amplification). In this concept specific enzym es are pre-targeted to tum ors using monoclonal antibodies as delivery vehicles and later used to activate the administered prodrug within the tum or site (for detailed description see C hapter 1). Two main requirements, however, have to be satisfied for this system to work properly. The first requirem ent is that the prodrug m ust be much less toxic than the drug, thus allowing its administration in higher doses. The second 1 10 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth er reproduction prohibited w ithout perm issio n . requirem ent is that the conjugate m ust be allowed to clear from the circulation before the administration o f the prodrug, while still preserving its presence and activity at the tum or site - this is an indispensable prerequisite to avoid prodrug activation in normal tissues and subsequent toxic side effects. This second condition is especially difficult to satisfy as often during the time necessary for the antibodv-enzym e construct to disappear from the circulation, the fusion product already bound to the tum or is being internalized or shed, thus removing the enzym e from the desired prodrug activation site. Interestingly enough the problems limiting the usage o f ADEPT therapy in cancer treatm ent are the very same problem s our laboratory has set to overcome by developing a new concept o f tum or targeting. The TNT antibodies developed in our laboratory target necrotic areas o f tum ors and are, therefore, applicable to a broad spectrum o f human cancers, while at the same time circumventing some o f the limitations o f MAb therapy such as tum or heterogeneity and antigenic modulations. It therefore seemed possible that combining T N T -type antibodies with ADEPT approach to cancer treatm ent could create an excellent combination. These particular characteristics provided the reason for choosing chimeric TNT-3 derivatives (see Chapters 2 and 3) as partners in the construction o f the delivery vehicles in the ADEPT studies. M any different enzym es have been used so far in ADEPT therapy, some o f them o f bacterial and some o f human origin. Among the bacterial enzym es, cytosine 1 1 I R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth er reproduction prohibited w ithout perm issio n . deaminase (CDase, CD2) is. after (3-Iactamase. the second most common bacterial enzyme used in ADEPT treatment. The CDase catalyzes the deamination o f eytosine to uracil. This very same reaction allows for the use o f the well-characterized antifungal drug 5-fluorocytosine (5-FC) as a prodrug for ADEPT. Under the action of cytosine deaminase, the 5-FC is converted to the highly toxic and well known antineoplastic drug 5-fluorouracil (5-FU) used against a wide range o f human malignancies. Despite the bacterial origin o f the enzym e, which may elicit an immune response from the p a tie n ts ^ , this possibility is more than offset by the fact that CDase is not present in any mammalian tissue. Also its prodrug 5-FC is widely available and well studied thus offering good pharm acokinetic characterization and definition o f the maximum tolerated dose (M TD). Also 5-FC can be administered in many different ways including intravenous injection, intraperitoneal injections or even oral administration. The CDase is also used in gene therapy, for example in VDEPT (Virally-Directed Enzyme Prodrug Therapy)*^- where it seems to be effective. Interesting as this concept is. gene therapy has not advanced sufficiently to enable direct targeting. Currently used methods o f transfecting cells with the vector in vitro and then transferring them in vivo to established tumors, have at least provided proof o.' concept but these methods are totally impractical in clinical settings. For gene therapy, targeting issues need to be solved before it is widely accepted. Its use. therefore, is limited to treatm ent o f genetic deficiencies and not cancer. Additionally. 1 12 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm ission. the first round o f treatm ent has a tendency to eradicate mainly the cells infected with the vector, since they are the ones possessing the majority o f the enzym e-^- - I. This inevitably results in a decrease in product at each consecutive round o f the treatm ent unless more cells are transfected. M eanwhile, monoclonal antibodies, which have proven themselves as excellent targeting agents, are already used in the clinic and do not possess these d ra w b ac k s^ . 22 therefore they could provide ideal carriers for CDase enzym es in ADEPT. To date, som e but rather sparse work has been done using antibody conjugates. In vitro studies with H2981 lung adenocarcinoma cells have dem onstrated that the 5-FC prodrug is non-toxic at up to 200 mM. whereas 5-FU has an I C 5 0 o f 20 |lM . In vivo experiments have shown that the combination o f specific immunoconjugate and 5-FC could result in cytotoxic effect similar to that o f 5-FU alone 23-25. Even if the bacterial enzym e is certain to evoke an immunological response upon the adm inistration, this problem could be overcome. In studies aimed to diminishing the immune response against foreign proteins, it has been shown that poly(ethylene glycolj-modification (PEG-ylation) o f proteins reduces their immunogenicity26, while use o f cyclosporin A27 or deoxvspergualin2N delays the human immune response against foreign proteins. Using these methods it may be possible to adm inister at least several rounds o f antibody-bacterial enzym e fusion protein before the immunological response alters the course o f treatm ent. Additionally . R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . if indeed the bacterial enzym e could w ork as a superantigcn. as it has been s u g g e s t e d ^ . 30 for the anti-idiotype antibodies in mice, this ouls provide an unexpected but most w elcom e bonus. Since TNT-3 antibody shows particularly favorable characteristics for its use in A D EPT, two derivatives o f this antibody were chosen as carriers for the fusion molecules between chTN T-3 and bacterial enzyme cytosine deaminase. This chapter outlines prelim inary studies assessing the usefulness o f such fusion proteins in the treatm ent o f solid tumors. MATERIALS AND METHODS Reagents, Antibodies, and Cell Lines The source o f reagents and the origin o f antibody and cell lines used have been described in Chapter 2 in details. Additionally used murine adenocarcinoma cell line Colon 26^ I • 32 was obtained from the National Cancer Institute DCTD Tum or R epository (Friderick, MD). 1 14 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . Construction o f Expression Vectors A ntibody fragments, namely F(ab')2 - and single chain, have been already successfully generated from the parent chimeric TNT-3 (chTN T-3) antibody using PCR and m olecular cloning techniques (see Chapters 2 and 3). The cDN'A encoding for bacterial cytosine deaminase has been obtained from ATCC as so-called pCD2. This is a genetically modified version o f cytosine deaminase where 88 base pairs prior to the start codon have been removed and the start codon itself has been mutated from CiTG to ATG — all this done to insure good expression in eukaryotic cells. This cDNA was PCR-am plified as two fragments. The first fragment was generated using 5' primer: 5 ' - G A C C G TA TTG C G G CC G C A G TG TC G A A TA A CG C TTTA C A A - 3* which introduced N oll cloning site at the 5' end o f the enzyme, and the 3' primer: 5' - CAGG CG TG A G G TA TACG C -3" incorporating A ccI restriction site already present in the enzyme. Second fragment has been generated using 5’ end primer: 5' - TATA ACG G G G CG TA TA CC - 3' also containing the same AccI restriction site and overlapping with the portion o f the enzym e cDNA and 3' end primer: 5 ' - G TC A TC G A TC TC G A G A CG TTTG TA A TCG A TG G C TTC - 3 ‘ with stop codon removed and X h o l cloning site incorporated. A fter PCR amplification. 1 15 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . both fragm ent were cloned end to end into Bluescript vector, and then in one final step the cD N A was transferred, after S o li and X h o l digestion, into S o il and X Im l sites at the 3' end o f p E E l2 vector containing either F(ab'): . or single chain cDNA. Cloning into these two restriction sites allowed enzym e to be aligned in frame with the antibody fragments and preserved at the C-end already present purification tags (either six histidines or nine amino acids Streptavidin-affm ity tagJ J ). The final constructs were obtained in pEE12 expression vector for use with the Glutamine Synthase (GS) expression system (Lonzo Ltd.. Slough. U.K.). A t each step, the assembled PCR fragments had their size continued by gel electrophoresis, and in final step, their sequences were verified by sequencing by the M icrochemical Core facility at USC School o f Medicine. Expression and Purification of the chTNT-3/CDase Fusion Products The protocol for the expression and purification o f the chTN T-3 fragment/enzym e constructs is based on the methodology described in detail in C hapter 2. Determination of Avidity The avidity constants o f the chTNT-3 fragments/CDase fusion proteins were determ ined by a fixed cell RIA as described previously (Chapter 2). The equilibrium or 1 16 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . avidity constant Ka was calculated by the equation K = -(slope.//), where // was the valence o f the antibody (1 for scFv/CDase. 2 for F(ab')v'CD ase). Radiolabeling of Fusion Proteins l2‘ I-labeled M Ab/CDase fusions were prepared using a modified chloraminc-T m ethod as described earlier (C hapter 2). with a yield o f 60-67% radiolabeled product. The radiolabeled antibodies were diluted with PBS for injection, stored at -UC. and adm inistered within 2 h after labeling. In Vitro Cytotoxicity Studies O ur laboratory has previously employed M adison 109 lung carcinoma cell line as a tum or model in im m unocom petent mice. However, since 5-FU is widely used to treat colon cancer, the 5-FC/CDase system seemed worth investigating in murine colon cancer tum or model and for this purpose murine adenocarcinoma Colon 26 was added to the stu d y ^ -. Both, murine lung carcinoma M adison 109 and Colon 26 murine adenocarcinom a cell lines were used in cytotoxicity studies. The prodrug used in these studies is 5-fluorocytosine. a well characterized antifungal drug available commercially (Sigma Chemical Co.. St. Louis. MO), which the action o f CDase converts to the chem otherapeutic drug - 5-fluorouracil. 1 17 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm ission. The sensitivity o f ceils was determined using the modified colorimetric assay originally used for the assessm ent o f TNF cytotoxicity-3 4 *. In short, cells were seeded in quadruplicate on 96 well plates at the density o f 2 x 104 well in an appropriate medium and allowed to attach overnight. On the next day the fusion proteins and the prodrug were added to the wells in varied concentrations, ranging from 0.1 mg ml to 0.01 mg/ml for fusion proteins and from 40 mM to 0.001 mtVI for 5-fluorocytosine. Appropriate controls consisting o f a drug (5-fluorouracil) on its own as well as an irrelevant antibody in com bination with a prodrug were included. The plates were incubated for 24 hours at 37°C, after which they were spun in the centrifuge at 1000 rpm for 10 min and supernatants discarded. Subsequently plates were w ashed tw ice with 200 pi o f saline. C rystal violet solution (0.2% w/v in methanol) was added as 50 pl/wcll and afterwards the plates were ineubated at room tem perature for 15 min. In the next steps plates w ere w ashed under the cold tap w ater three times and after shaking o ff the excess o f w ater, left to dry overnight. On the next day methanol was added as 100 pl/well. The color development w'as read at 550 nm. Data w as reported as mean ± standard deviation (SD), and differences between cell lines were analyzed by unpaired Student’s /-test. IC5 0 was determined as described in literature-0 . 1 IS R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . Pharmacokinetics and Biodistribution Studies The protocol for the pharmacokinetics and biodistribution studies were described in details in Chapter 2. For biodistribution studies two murine carcinoma cell lines M adison 109 and Colon 26 were used to establish tumors in Balb C' mice. M easurement of Cytosine Deaminase Activity In Vivo The TN T-based antibodies bind to any necrosis, therefore the murine tumor model was employed, using the Balb/C female mice injected with initial dose o f the 2 x 107 M adison 109 (M A D 109) lung carcinoma cells s.c. in the left thigh. The tumors were grown for 7-10 days, until they reached close to 1 cm in diameter. Within each group (/i=3). individual mice were injected i.v. with 100 pg o f MAh fusion. Animals were sacrificed by sodium pentobarbital overdose 5 and 7 days post-injection, and tissues were removed, weighed, and homogenized in 7 ml 0.02 M Tris-I ICI. pH 7 . 5 . 0.075 M NaCl for 30 sec. Subsequently, homogenates were centrifuged and than were assessed for their enzym atic activity. Activity o f the enzym atic part o f fusion protein was measured as described by W allace^. Briefly 50 pi o f a sam ple with the protein concentration adjusted to the sim ilar level was incubated with 950 ml o f 5-FC (3 mM in PBS) at 37 ''C. At various time intervals. 50 pi aliquots were remov ed and quenched with 1 ml o f 0.1N HC1. The concentrations o f 5-FC and 5-FU were determined I 19 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm ission. spectrophotom etrically at wave lengths o f 255 nm and 290 nm. Activity was subsequently calculated using the equations: 5-FC [ntM] = 0.119x Ay)n — 0.025 x A 5-FU [mM] = 0.185 x A:ss - 0.049 x A:w In Vivo Murine Model Treatment Studies The murine colon carcinoma cell line. Colon 26. was employed to produce tum ors in Balb/C mice. Tum ors were injected subcutaneously into the thigh, and allowed to grow for approx. one week. When they achieved a mean diameter o f approxim ately 1 cm. mice were randomly divided into the several groups with 5 mice/group (Table 4-1) and injected according to the schedule. All injections o f fusion proteins were done intravenously into the lateral tail vein while prodrug was injected intraperitoneally. Table 4-1: Groups for the Therapy Study. GROUP REGIMEN Control no treatm ent F(ab’);./CD2 plus 5-FC fusion: lO m g'm l 5-FC: S00 mg/kg Diabody plus 5-FC fusion: 10 mg/kg 5-FC: 800 mg'kg 1 2 0 R e p ro d u c e d with p e rm ission of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm ission. Pretreatm ent with chTN T-3/IL-2. than F(ab’)2/CD2 plus 5-FC chTNT-3/IL-2: 0.2 m g'kg. fusion: 10 mg/kg 5-FC: 800 mg'kg 5-FC only 5-FC: 800 mg'kg 5-FU only 5-FU: 12 mg'kg The literature readings and in vitro cytotoxicity studies suggested using the dose o f 5-FC o f 800 mg/kg (approx. 16 mg/mouse) per single dose, further increase o f the dose being limited not by toxicity but by solubility. Fusion protein was administered as a single dose o f 10 mg/kg (approx. 0.2 m gm ouse) at approxim ately 7 and 14 days after tum or inoculation. The prodrug was administered approx. 9 and 16 days after inoculation - 48 hours after administration o f the fusion construct. There was approxim ately a two w eek "w indow " for the treatment before Balb C mice started developing immune response to foreign proteins. Tum or growth was m easured at 2 day intervals, starting on the day o f first injections of fusion protein. T um or m ass was calculated from tum or volume (assuming a tissue density o f lg/cnr5 ), which was determined by caliper measurem ents. The formula: mass = 1 g /c n r x 0.5 x L x W : was used (L and W - the longest dimension and its perpendicular in cm.). The mice were sacrificed when the tum or size reached over approx. 5 cm3 or when they exhibited signs o f morbidity. The final data was expressed as a tum or volume change. 121 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm ission. 3. RESULTS In Vitro Characterization of Fusion Proteins The fusion proteins w ere expressed using the Glutamine Synthetase Gene Am plification System from Lonza Biologies (Slough. UK). Rates o f production varied from 2 fig/m I/106 cells/24 hr to 5 (ig/ml/106 cells/24 hr and were high enough to allow purification of sufficient am ount o f material for further experiments. The highest producing clones were incubated in a 3 liter stir-flask. and the fusions were purified stepw ise from cell culture medium by Ni-NTA or modified Streptavidin (Streptactin) affinity chromatography as described earlier. The purity o f the sam ples was determ ined using both SDS-PAGE (Figure 4-1) and HPLC chrom atography. SDS-PAGE gel showed proper assem bly o f the fusion proteins with single chain running at the size o f 82 kD a and F(ab'): with the size o f 214 kDa. A part from m ajor bands present at the appropriate points corresponding to their molecular weight, several fainter bands with much higher molecular weight were seen. This m ay suggest the possibility that cytosine deaminase fusion proteins form conjugates due to the influence o f their enzymatic part. Reducing SDS-PAGE gel produces one major single band for each o f the fusion molecules, with a molecular weight identical to that expected o f the reduced single chain or F(ab‘): fusion molecule. R e p ro d u c e d with p e rm ission of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm ission. Figure 4-1. Comm ass ie Blue Stained SDS-PAGE Gel. Lines A, B, and C show chTNT-3/scFv/pCD2 construct and chTNT-3/scFv fragment under non-reducing conditions. Lines a, b, and c show chTNT- 3/F(ab')2/pCD2 fusion protein and chTNT-3/F(ab')2 fragment under reducing conditions. A B C D a b C L M W M ark er Figurel: SDS-PAge gel of CD2 fusions. Lanes A and a: Fab' and F(ab')2 non-reduced and reduced respectively Lanes B and b: chTNT-3/ScFv/pCD2 non-reduced and reduced Lanes C and c: chTNT-3/F(ab')2/pCD2 non-reduced and reduced Lane D: high molecular marker LMW: low molecular marker 123 R e p ro d u c e d with p e rm ission of th e copyright ow ner. F u rth er reproduction prohibited w ithout perm ission. To confirm the size o f the fusion proteins and further investigate the possibility o f m ultim ers’ formation, size exclusion HPLC was performed. It show ed (data not presented) that the single chain/CDase fusion (82 kDa) eluted w ith the retention time o f 12.9 minutes, while the F(ab’)2/C D ase fusion's retention tim e was 7.4 m inutes which is expected for a protein w ith a size o f approx. 214 kDa. B y com parison, the intact chTNT-3 antibody’s retention time was 10 m inutes. The F(ab’)2/CD ase fusion eluted as a single peak, while the single chain showed one large peak corresponding to the fusion protein's expected size, followed by few more much sm aller peaks eluding as proteins with much higher mass. This could be explained as a result o f forming multimers. Whether these multim ers are due to well-known tendency o f single chains towards aggregation^. 37 or are the result o f multimers formed by the enzym e as reported by o th e rs ^ , 39 bas not been clarified. Determination of avidity The avidity constants o f the chTN T-3/enzym e fusions were detenuined by a fixed cell RIA method as described previously and are presented below (Table 4-2. Figure 4-2). 124 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth er reproduction prohibited w ithout perm issio n . Table 4-2: Avidity Constants o f Different Antibody/CDase Constructs. Fragment binding sites % Binding avidity constant Ka chTNT-3/scFv/pCD2 one 68.5 0.92 x 109 v r 1 chTNT-3/F(ab')2 /pCD2 two 66.8 i.oi x io9 v r 1 chTNT-3 two 89.0 1.43 x io9 v r 1 These studies show ed that the avidity constants for different antibody fragments/enzyme fusions were similar to within one decimal place (Figure 4-2). Interestingly, the binding affinities for scFv and F(ab'); fusions (0.92 x 109 M ' 1 and 1.0 x 109 M ' 1 respectively) were im proved comparing to scFv and F(ab’); (0.25 x IO9 VI' 1 and 0.54 x 109 M ' 1 respectively) themselves, thus approaching the value for the intact antibody. This could be explained by the influence o f the enzym e portion of the fusion molecule forcing the m onovalent scFv and divalent F (ab '): fragments into fonning multim ers, thus improving their binding to antigen. There have been reports by others ^ o suggesting that bacterial cytosine deaminase forms either tetram ersJt> or even pentam ers 39 1 2 5 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth er reproduction prohibited w ithout perm issio n . Figure 4-2: Afflniy Constants for Antibody Fragment/Cytosine DeaminaseConj ugates. p C D 2 C o n j u g a t e s A f f in ity B i n d in g .75 .25 - 0.75 - 0.5 - 0.25 20 25 30 40 1 0 hound (niz) 1 2 6 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . In vitro Cytotoxicity Studies The sensitivity o f cells to the prodrug (5-FC) and the drug (5-FU) was determined using a modified colorimetric assay used for the assessm ent of TN F cytotoxicity. The concentrations used for fusion proteins varied from 0.16 mgml to 0.01 mg/ml. For the prodrug, the concentration ranged from 20 m.M to 0.01 mVI. Results are presented in a Figure 4-3. Our prelim inary studies have shown that the prodrug 5-fluorocvtosine is non toxic to murine cancer cell lines tested (M adison 109 and Colon 26) at the concentrations o f over 20 mM. Compared to the cytotoxic effect o f the 5-fluorouraciI. the prodrug is approx. 8,000-fold less toxic than the actual chem otherapeutic drug. For 5-FU, its IC50 for both cell lines (M AD 109 and Colon 26) was approx. 2.5 jig. The action o f the F (ab')2/C D 2 fusion was able to shift the IC50 for 5-FC dow nwards and the IC50 for 5-FC decreased to 2 mM. This was due to the action o f just 10 L t g m l o f fusion protein. Further studies (data not shown) have revealed that using higher concentrations o f fusion proteins, shifted the IC50 even more dow nw ards, all the w ay to 0.5 mM when used together with 160 pg/ml o f F(ab'):/'CD2. Such concentrations o f the enzyme conjugates are not easily attainable in tumors, but it appears that prodrug activation also may take place with much lower concentrations o f enzyme conjugate. 1 2 7 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm ission. % of cell killing Figure 4-3: In Vitro Cytotoxic Effect o f the 5-FC Converted to 5-FU by i oo 9 0 - 80 - 70 - AO - 50 - 40 - 30 - 2 0 - I 0 - S i S i I I □ El 0 0 scFv/pCD 2 - Colon-26 scFv/pCD 2 - M A I) 109 F (ab')2/pC D 2 - Colon-26 F (a b ’)2/pCD2 - M V DI09 0.5 5-FC (m M ) Antibody/pCD2 Conjugates. 1 2 8 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . The shift in I C 5 0 values described here is not so significant as reported by other investigators. Using Enzyme/Prodrug Gene Therapy system and transfected human colorectal cell line WiDr. Trinh et a l . . 4 0 reported a change in l C 5 n for 5-flurocvtosine from 26 mM to 27 jlM . K u r i y a m a ^ l reported a shift in sensitivity from 420 ug ml to 3.5 jug ml for murine hepatocellular carcinoma. While the change in IC?,, brought upon by the action o f chTNT-3 fragment/CDase fusion proteins, is not as im pressive, it is there nevertheless proving the preservation o f activity o f the enzymatic part o f fusion molecule. Also, the ability o f the chem otherapeutic drug to kill tum or cells varies significantly between different cell lines. Therefore some other cells lines should also be investigated in the future. Pharm acokinetics Studies Clearance studies in Balb/C mice were perform ed to establish differences in the pharm acokinetics between different antibody-enzvm e conjugate as determ ined by w hole-body dosim etry. 129 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm ission. Table 4-3: Half-lives of Different Antibody-Bacterial Cytosine Deam inase (pCD2) Conjugates as Determined by Clearance of Radiolabeled Proteins from Balb/C Mice Fusion S ize T ,< : chTN T-3/scFv/pC D 2 82 kDa 6 . 8 - - 0.5 hr chT N T -3/F (abV pC D 2 214 kDa 8 . 1 — - 1 . 0 hr ChTNT-3 140 kDa 134.2 - - 4.0 hr It has previously been shown42 that the parenta chTNT-3 monoclonal antibody clears slowly with a whole-body half-life o f 134.2 hr in Balb C mice. As depicted in Table 4-3, chTNT-3 fragments/enzyme conjugates were eliminated significantly more rapidly and in similar time frames. This happened despite the size o f the conjugates approaching (as in case o f single chain construct) and well exceeding (for the F(ab')2 construct) the size o f the parental antibody. The results can be explained by the lack o f the Fc portion o f the antibody. As shown in several s tu d ie s^ - 44. the Fc portion significantly influences the clearance o f the antibody from the circulation and antibodies with missing (F(ab');<). deleted, or mutated to abolish binding45 parts o f FC region are cleared in much faster manner regardless o f their size. As antibody/enzyme conjugates often clear too slow ly from the circulation and require the use o f an extra clearing step, the short half-life o f chTNT-3 antibody fragm ent CD2 conjugate may prove beneficial. 130 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . Biodistribution Studies The biodistribution o f the chTN T-3/enzym e constructs was assessed in tumor models using Balb/C mice im planted s.c. with either M adison 109 murine lung carcinoma or Colon 26 murine adenocarcinoma. This was done to determ ine which o f these tw o cell lines were more suitable for subsequent treatm ent studies in animals. Biodistribution data were taken at several time points as shown in Figures 4-4 and 4-5. D espite their rapid elim ination, the conjugates retained the ability to localize to tum ors. Tum or uptake o f chTNT-3 fragment/enzyme conjugate at 1st and 2nd day post-injection varied approxim ately from 1.27 to 2 % ID/g at 24 hr time point to 0.77 to 1.69 % ID/g for the 48 hr time point (Figure 4-4). Usually the higher tum or uptake values were associated with F(ab' ) 2 antibody fragment o f the fusion molecule and lower w ith single chain construct. The difference was approxim ately tw o-fold, most likely due to the difference in number o f affinity binding sites. At the same time, the rapid clearance o f conjugates produced several-fold higher tum or 'normal organ ratios for many normal tissues (Figure 4-5). For example blood to tum or ratio was approx. 5 at 24 hr and then raised to nearly 10-fold high values at 48 hr. The F (ab'): construct had slightly lower ratios at 24 hr. as compared to the single chain-based molecule, which probably is due to the differences in molecular weight o f two conjugates. 31 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . Figure 4-4: Tissue Biodistribution and Tumor Uptake (% of injected Dose/Gram o f Tissue) of chTNT-3 Fragments/Bacterial C ytosine Deam inase Conjugates in Murine Colon26 Colorectal (panel A) and M adisonl09 Lung (panel B) Carcinoma Tumor-Bearing Balb/C Mice in Selected Organs at 24 and 48hr. £3 scFv/CD2 - 24 h r 0 F(ab')2/CD 2 - 24hr □ SC Fv/CD2 - 4 8 h r e a F(ab')2/CD 2 - 4 8 h r 2.5 u z t 0.5 - hlood tumor organ u z t B .5 - 1 - 0.5 - I i X T I hlood tumor organ scFv/CD2 - 24 h r 0 F(ab')2/CD2 - 24 hr □ scFv/CD2 - 48 h r F(ab')2/CD 2 - 48 hr 1 3 2 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm ission. Figure 4-5: Tissue Biodistribution and Tumor Uptake (Tumor/Organ Ratio) of chTNT-3 Fragments/Bacterial Cytosine Deaminase Conjugates in Murine Colon26 Colorectal (panel A) and Madison 109 Lung (panel B) Carcinoma Tumor-Bearing Balb/C Mice in Selected Organs at 24 and 48hr. A blood i ver organ scFv/CD2 - 24 h r □ F(ab')2/CD 2 - 24hr □ scFv/CD2 - 4 8 h r 0 F (ah’)2/CD2 - 48hr kiilnev B 14- I 2 ■ I 0 X - ec 4 - X i blood X iT liver o rgan T r kidnev 03 scFv/CD2 - 24lir 0 F(ab')2/CD 2 - 24hr □ scFv/CD2 - 48h r F(ab')2/CD 2 - 48hr R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . On a whole, the biodistribution data dem onstrated the specificity o f tum or targeting with chTNT-3 fragment/enzyme conjugates and. at the same time, their rapid elimination from blood and normal organs. Measurement of Cytosine Deaminase Activity in Tumors The activity o f the enzymatic portion o f the chTNT-3 enzym e constructs was assessed in Madison 109 murine lung carcinoma/Balb C tum or model system. A ctivity o f the enzyme was measured in tumor homogenates as described earlier on a day 5 and 7 post injection. Figure 4-6 shows that the enzym atic activity o f the bacterial cytosine deam inase portion o f the antibody/enzyme fusion is present in tumors for a prolonged time (5 and 7 days) after the administration o f the conjugate. Additionally, the enzym e m aintains its activity for the entire period o f assay duration (up to 48 hr). Since this is one o f the major prerequirements for a successful A DEPT therapy, these findings are encouraging. 134 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm ission. Figure 4-6: Activity of Cytosine Deaminase Part of Fusion Protein in a Tumor at Different Time Points 0.45 0.4 - 0.35 - 0.3 0.25 0.2 0 . 1 5 -t 0 . 1 0.05 666 & + * 1 * co n tro l T u m o r- 168hr post injection T u m o r - 120lir post injection 10 r 15 20 25 30 35 D uration of Assay (hr) 1 3 5 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth er reproduction prohibited w ithout perm issio n . In Vivo Murine Model Treatment Studies The murine colon carcinoma cell line. Colon 26. was em ployed to produce tumors in Balb/C mice. When tumors achieved a mean diam eter o f approximately 1 cm. mice were random ly divided into the several groups and injected according to the defined schedule. The study was term inated on day 20 due to animal welfare concerns. The obtained data indicate that the 5-FC is converted to its active form by the enzym e conjugate but at a very low rate. Increasing o f the am ount o f pretargeted enzyme (by pretreatm ent with IL-2 vasoactive conjugate) produced better results. The lack o f prom ising results in this study may be at least partially due to the several factors, m ainly relatively large size o f tumors and low solubility o f the 5-FC at high doses. It is worth m entioning that even the groups with unsupressed tum or growth still had lower m ortality rate than the group treated with 5-FU. which, again, proved to he a very toxic chemotherapeutic. 136 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . Figure 4-7: In Vivo Treatment Study Using Colon 26 Murine Tumor Model Balb/C Mice 1 0 co n tro l 9 T3D + 5-FC F(ab')2/C D 2 + 5-FC 8 5-FC onlv 7 5-FU only U chT N T -3/IL -2 pretxt. than F(ab')2/CD 2 + 5-FC 0 7 I 4 4 9 0 day R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . 5 4. DISCUSSION M onoclonal antibodies can be used in m any different ways. When aimed at tum or-associated antigens, they can. for example, direct an immune response to the tumor, or target toxins, chemotherapeutic agents, radioactive isotopes, or genes'*^. Unfortunately, as mentioned earlier, the process o f identifying tumor-specific or tum or-enhanced markers has been difficult, as malignant and nonnal tissues differ only slightly in their antigenic properties. This is one o f the m ain reasons while the A D EPT therapy has not progress much beyond the experim ental s t a g e d Targeting enzym es to the tum ors requires antibodies with very specific characteristics. Notably, these requirem ents that the antibody should stay bound w ithin the tumor and not undergo shedding or internalization until the adm inistration o f the prodrug are just the characteristics bestowed upon the TN T-type antibodies developed in our laboratory. It has been therefore decided to create by the m eans o f m olecular biology, the fusions molecules between chimeric monoclonal antibody chTNT-3 and enzyme suitable for A D EPT. First the fusion proteins were produced in a mammalian expression system and characterized in vitro. SDS-PAGE gel showed proper assem bly o f the fusion proteins. Binding affinity studies confirmed the preservation (or even improvement) o f their binding abilities. This is a not uncommon phenom enon when fusion proteins are 13cS R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth er reproduction prohibited w ithout perm issio n . produced incorporating multimeric proteins. As for the cytosine deaminase, data from the literature are som ehow conflicting, suggesting the possibility o f fonning multimers. perhaps tetra-38 or even pentamers-^^. but no clear evidence has emerged from these readings. This particular characteristic o f the enzym e could have a significant impact on its activity and on the final size o f the fusion protein. G enerally our in vitro characterization seems to support the possibility o f multimer formation. Additional bands on a SDS-PAGE gel and peaks in HPLC as well as improv em ent o f binding ability o f a monomeric antibody fragment to its antigen, suggest the kind o f effect o f the enzym e portion which is usually associated with multimeric enzym es (see C hapter 5). Cytotoxicity studies showed that both cell lines used in the assays (MAD 109 and Colon-26) were equally sensitive to the cytotoxic effects o f the 5-fluorouracil and very resistant to the action o f 5-fluorocytosine. The IC5 n for 5-FU was 2.5 u.VI and for 5-FC was 20 mM - a difference o f approxim ately 8.000 fold. The enzym atic action o f the CDase fusion proteins converted inactive 5-FC to an active 5-FU. albeit to a lower degree then previously reported by other investigators. The IC?0 for 5-FC shifted from over 20 mM to less than 2 mM (tenfold difference) when using low concentration o f the fusion proteins and 0.5 mM with more enzym e used. Perhaps the ideal conditions for the cytotoxicity assay should be investigated in detail. It seems that the am ount o f enzym e plays a significant role, but another im portant variable is time. This particular 139 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm ission. assay was perform ed for 72 hr but longer incubation periods (5 or 7 days) have also been re p o rte d ^ . As a prelim inary step for the treatm ent studies, the pharmacokinetic and biodistribution studies were done in Balb/C immunocompetent mice. They showed that both single chain and F(ab')2 fusion proteins cleared rapidly from the circulation despite the m olecular size closely resembling (for scFv) or exceeding (for F(ab’): ) that o f the intact antibody. There are only a few reports employing antibody enzyme conjugates, leaving little to compare our data with. In a publication describing the conjugate between the whole antibody and C D ase-^. this conjugate cleared very slow ly from circulation, so slowly in fact, that the additional step involving a secondary antibody was needed before the treatment with prodrug could commence. Similar findings were described for the conjugates between the whole antibody and bacterial enzym e carboxypeptidase G2, where the half-life o f the conjugate was around 4 days48. Surprisingly, the conjugate between the same enzym e and F(ab'); antibody fragment also resulted in a product with prolonged lifetime in v iv o ^ . As for the fast clearance o f TNT fragments/CDase. the most likely explanation lies in the antibody portion o f the fusion protein. As it has been showed in Chapter 2. the removal of the Fc portion o f the antibody significantly decreases its persistence in the serum. N evertheless, despite the short half-life, the fusion proteins were able to localize to the transplanted tumors with 1.5-2% o f the injected dose seen in the tumor. Since faster 140 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . clearance times also mean better tum or to organ ratios, this was indeed the case for the CD ase fusions proteins. At the 24 hr tim e point, the blood to tum or ratio was 5. while at 48 hr. the difference was 9-fold. Fast clearance but with sufficient tum or uptake means that in the course o f ADEPT one will be able to adm inister the prodrug sooner after the injection o f the enzyme conjugate. Perhaps even significantly sooner than is has been done in studies up to date, where the interval often approached several days unless a clearing antibody was adm inistered-^- 49 Still the half-life o f S-9 hours for fusion proteins allows sufficient time for the conjugate to bind to the tumor as shown by Sharma^O. Both cell lines MAD 109 and Colon 26 showed similar levels o f the conjugate uptake in tumors. It was observed, however, that Colon 26 grew at a much slow er rate than MAD 109. so this would be a recommended cell line for animal treatm ent studies. The uptake into tum or approached nearly 2° o o f injected dose at 48 hr. Several studies from VDEPT approach investigated the issue o f how many cells have to be infected with the virus for the therapy to w'ork. It seems that as little as 2"» o f cells with the enzyme was sufficient for hindering tum or growth"*^, while the 5"o o f transduced cells often sufficed to eradicate the tum oral . In another study by PerroiP *. cocultures o f antigen positive cells with antigen negative showed that I'\) expression gave approx. 30 % growth inhibition, while 5% resulted in approx. 50°■> inhibition. These finding should also be applicable to our studies. The single preliminary' study- using Colon 26 tumor model showed that the conjugate w'as able to activate 5-FC to its 141 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . toxic form and slow tum or growth. However, this happened only in ease o f pretreatm ent o f the animals with vasoactive molecule (IL-2) before the administration o f the conjugate. It has been shown in other studies (see C hapter 2). that pretreatm ent with a vasoactive molecule before the adm inistration o f the fusion protein leads to a further increase in tum or u p ta k e ^ . This is most likely the case in this instance as well. Since the am ount o f the enzyme in a tum or is crucial for the prodrug activation, this explains the positive result obtained in that particular study. These animal studies are not as encouraging as some treatm ent studies done by other groups but m ay be worth further investigation. This minimal success may be partially due to the fact that tum or were large in size when the treatm ent started and that the dosage o f the prodrug was limited by its low solubility. After solving these technical problem s, the results o f next study may be more promising. It is worth noticing that 5-FU regimen managed to slow tum or growth but at the cost o f high m ortality among animals. At the same time. 5-FC showed good slowing o f tum or grow th in the group with IL-2 pretreatm ent while the m ortality in that group was much lower. No m atter how promising preliminary data is. one should remember that cytosine deaminase is still a bacterial enzyme. So the immune response after its application is inevitable making a “window o f opportunity" questionable. Studies describing the use o f CDase/5-FC system reported some cures * ^ but there were also 142 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . incidences o f tum or rebound after discontinuation o f the treatm ent Additionally, only recently new reports surfaced claiming the superiority o f the yeast cytosine deam inase over the bacterial enzym e-^- 53 W hether it is indeed the case, remains to be seen once more data become available. C ytosine deaminase from veast seems to have higher affinity to 5-FC than the bacterial enzym e. On the other hand, it has shown itself to be less stabile in serum and prone to the loss o f activity upon protracted tum or localization-^, suggesting that further studies are warranted. Obviously more work is needed comparing both enzym es. At the end. w hether it will be bacterial or yeast cytosine deam inase as a viable candidate, remains to be seen. The av ailability of the well-characterized prodrug is som ething to consider, especially that is can be given in much higher doses than the drug., and it could outweigh the side effects associated with using an immunogenic protein. REFERENCES 1. Old LJ. Cancer immunology: the search for specificity. Cancer Research 19X1: 41:361. 2. Klein G. Immune and non-im mune control o f neoplastic developm ent: contrasting effect o f host and tum or evolution. Cancer 1980: 45:2486. 3. Emmelot P. Biochemical properties o f normal and neoplastic cell surfaces: a review. European Journal o f Cancer 1973; 9:319. 143 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . 4. W eber G. Biochemical strategy o f cancer cells and the design o f chcmotherpay. Cancer Research 1983; 43:3466. 5. Calabresi P. W elch. A.D... 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Verhaar-Langereis MJ. Zonnenberg BA. de Klerk JMH, Blijham GH. Radioimmunodiagnosis and therapy. Cancer Treatment Reviews 2000; 26:3-10. 13. Dillman RO. Antibodies as cytotoxic therapy. Journal o f Clinical Oncology 1994: 12:1497-1515. 14. Kreitman RJ. Immunotoxins in cancer therapy. Current Opinion in Immunology 1999; 11:570-578. 15. Syrigos KN, Epenetos AA. Antibody Directed Enzyme Prodrug Therapy (ADEPT): a review o f experimental and clinical considerations. Anticancer Research 1999; 19:605-614. 144 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . 16. Bagshawe KD. Antibody directed enzymes revive anti-cancer prodrug concept. British Journal o f Cancer 1987; 56:531. 17. Sharma SK. Bagshawe KD, . . Melton RG. Sherwood RF. Human im m une response to m onoclonal antibody-enzym e conjugates in A D EPT pilot clinical trial. Cell Biophysiology 1992; 21:109-120. 18. Consalvo M, M ullen CA, Modesti. A.. . Musiani P. et al. 5-fluorocvtosine- induced eradication o f murine carcinomas engineered to express the cytosine deam inase suicide gene requires host immune competence and leaves an efficient memory'. Journal o f Im m unology 1995:5302-5312. 19. Gnant M FX, Puhlm ann M, Alexander Jr. HR. Bartlett DL. .. Systemic administration o f a recombinant Vaccinia Virus expressing the cytosine deam inase gene and subsequent treatm ent with 5-fluorocytosine leads to tum or-specific gene expression and prolongation o f survival in mice. Cancer Research 1999: 59:3396-3403. 20. Connors TA. The choice o f prodrugs for the gene directed enzyme prodrug therapy o f cancer. G ene Therapy 1995; 2:702-709. 21. Davis BM. Koc ON, Keunmyoung L. Gerson S. C urrent progress in the gene therapy o f cancer. Current Opinion in Oncology 1996: 8:499-508. 22. Trail PA, Bianchi AB. Monoclonal antibody daig conjugates in the treatm ent o f cancer. Current O pinion in Immunology 1999; 1 1:5S4-588. 23. Senter PD. Activation o f prodrugs by antibody-enzym e conjugates: a new approach to cancer therapy. FASEB Journal 1990: 4:188-193. 24. Senter PD, W allace PM. Svensson HP. Kerr DE. Hellstrom I. Hellstrom KE. Activation o f prodrugs by antibody enzyme conjugates. A dvances in Experim ental Medical Biology 1991: 303:97-105. 25. W allace PM. M acM aster JF, Smith VF. Kerr DE. Senter P.D.. Cosand WL. Intratumoral generation o f 5-fluorouracil mediated by an antibodv-cytosine deam inase conjugate in com bination with 5-fluorocytosine. Cancer Research 1994: 54:2719-2723. 26. Delgado C, Francis G.E., Fisher D. The uses and properties o f PEG-linked. also the intravenous adm inistration o f PEG-linked proteins. Crit Rev Ther Drug C arrier Syst 1992; 9:249-304. 145 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . 27. Sharma SK. Immune response in ADEPT. Advances in Drug Delivery Review 1996; 22:369. 28. Dhingra K, Fritsche H. Murray JL. et al. Phase I clinical and pharmacological study o f suppression o f human antim ouse response to monoclonal antibody L6 by deoxyspergualin. Cancer Research 1995; 55:3060-3067. 29. Kosma C. Epenetos AA. Courtnay-Luck NS. Patients receiving murine m onoclonal antibody therapy for m alignancy develop T cells, that proliferate in vitro in response to the antibodies as antigen. British Journal o f Cancer 1991; 64:494-500. 30. Schultes BC, Yang R. Agopsowicz K.. et al. Anti-idiotvpic induction therapy for ovarian cancer: immune response in patients injected with Ovarex™ M ab-B43-13. Preceedings o f ASCO 1999:1399. 31. Corbett TH. Grisw old PD. Roberts BJ. Peckham JC. Schabcl KM. Tum or induction relationship in developm ent o f transplantable cancer o f the colon in mice for chem otherpay assays, with note on carcinogen structure. Cancer Research 1975: 35:2434-2439. 32. Tsuruo T. Yamorei T. Naganuma K. et al. M etastasis after intravenous inoculation o f highly metastatic variants o f mouse tumors and the effects o f several antitum or drugs on the tumors. Gann 1984: 75:193-19S. 33. Schmidt TGM . Skerra A... The random peptide library-assisted engineering o f a C-terminal affinity peptide, useful for the detection and purification o f a functional Ig Fv fragment. Protein Engineering 1993; 6:109-122. 34. Fransen L. van der Heyden. Ruyschaert R. Fiers W. Recombinant tum or necrosis factor: Its effect and its synergism with interferon-yon variety o f normal and transformed human cell lines. European Jom al o f Cancer and Clinican Oncology 1986: 22:419-426. 35. Duch DS, Banks S, Dev IK, et al. Biochemical and cellular phaimacologv o f 1843U89, a novel benzoquinazoline inhibitor o f thym idylate synthase. Cancer Research 1993; 53:810. 1 4 6 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . 36. W hitlow M. Bell. B.A.. Feng, S-L.. Filpula. D.. Hardman. K.D.. Hubert. S.L.. Rollence, M.L., W ood, J.F., Schott. M.E.. Milenic. D.E.. Yokota. T.. Schlom. J. An improved linker for single-chain Fv with reduced aggregation and enhanced proteolytic stability. Protein Engineering 1993: 6:989. 37. Turner DJ. Ritter. M.A.. George. A.T.J... Importance o f the linker in the expression o f single-chain Fv antibody fragments: optimisation o f peptide sequence using phage display technology. Journal o f Immunological Methods 1997; 205:43. 38. Danielsen S. Kilstrup M. Barilla K. Jochimsen B. Neuhard J. Characterization o f the Escherichia coli codBA operon encoding cytosine permease and cytosine deaminase. M olecular M icrobiology 1992: 6:1335-1344. 39. Kievit E. Bershad E. Ng E. et al. Superiority o f yeast over bacterial cytosine deaminase for enzym e/prodrug gene therapy in colon cancer xenografts. Cancer Research 1999; 59:1417-1421. 40. Trinh QT. Austin EA. M urray DM. Knick VC. Huber BE. Enzyme Prodrug Gene Therapy: com parison o f Cytosine Deaminase/5-fluorocvtosine versus Thymidine Kinase/gangciclovir enzyme/prodrug systems in human colorectal carcinoma cell line. Cancer Research 1995: 55:4808-4812. 41. Kuriyama S. K ikukawa M. Masui K. et al. Cytosine deam inase 5- fluorocytosine gene therapy can induce efficient anti-tumor effects and protective immunity in im m unocom petent mice but not in athymic mice. International Journal of Cancer 1999: 81:592-597. 42. Homick JL. Sharifi. J.. Khawli, L.A.. Biela. B.H.. Yun. A.. Taylor. C.R.. Epstein. A.L... A new chem ically modified chimeric monoclonal antibody directed against DNA for the im m unotherapy o f solid tumors. Cancer Biotherapy & Biopharmacology 1998: 13:255. 43. Covell DG. Barbet. J.. Holton O.D.. Black. V.. Parker R.J.. W einstein J.N... Pharmacokinetics o f monoclonal immunoglobulin G | F(ab'), and Fab' in mice. Cancer Research 1986:46:3969. 1 4 7 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth er reproduction prohibited w ithout perm issio n . 44. Mueller BM. Reisfeld RA, Gillies SD. Serum half-life and tum or localization o f a chimeric antibody deleted o f the C\{2 domain and directed against the disialoganglioside GD2. Proceedings o f the National Academy o f Sciences USA 1990: 87:5702-5705. 45. Homick JL. Sharifi. J.. Khawli. L.A.. Hu. P.. Bao W .G.. A liaudin. M.M.. M izokami M.M.. and Epstein. A.L.,. Single amino acid substitution in the Fc region o f chim eric TNT-3 antibody accelerates clearance and improves immunoscintigraphy o f solid tumors. Journal o f Nuclear Medicine 2000: 41:355-362. 46. von M ehren M. W einer LM. M onoclonal antibodv-based therapy. Current O pinion in Oncology 1996; 8:493-498. 47. Melton RG. Sherwood RF. A ntibody-enzym e conjugates for cancer therapy. Journal o f the National Cancer Institute 1996; 88:153-165. 48. Eccles SA. Court. W.J.. Box. G.A. Regression o f the established breast carconom a xenografts with antibody-directed enzym e prodrug therapy against c-erbB2 p 185. Cancer Research 1994; 54:5171. 49. Sharma SK. Bagshawe KD. Burke PJ. et al. Galactosvlated antibodies and antibody-enzym e conjugates in Antibody-Directed Enzyme Prodrug Therapy. Cancer Suppl.. 1993: 73:1114-1120. 50. Sharma SK. Bagshawe KD. Burke PJ. Boden RW. Rogers GT. Inactivation and clearance o f an anti-CEA carboxypeptidase G2 conjugate in blood after localization in a xenograft model. British Journal o f Cancer 1990; 61:659-662. 51. Perron P-J. Page. M... Activation o f m ethotrcxate-phenvlalanine by monoclonal antibody-carboxypeptidase A conjugate for the specific treatment of ovarian cancer in vitro. British Journal o f Cancer 1996: 73: 281-287. 52. Homick JL. Khawli LA. Hu P. Epstein AL. Pretreatment with a monoclonal antibody/interleukin-2 fusion protein directed against DNA enhances the delivery' o f therapeutic molecules to solid tumors. Clinical Cancer Research 1999; 5:51 -60. 53. Hamstra DA. Rice DJ. Fahmy S. Ross BD. Rehemtula A. Enzyme Prodrug therapy for head and neck cancer using catalitically superior cytosine deaminase. Human Gene Therapy 1999; 10:1993-2003. 148 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . CHAPTER 5: FUSION PROTEINS BETWEEN chTNT-3 DIRECTED AGAINST ssDNA AND HUMAN [3-GLUCURONIDASE ENZYME AS POTENTIALLY IDEAL TARGETING VEHICLES FOR ANTIBODY-DIRECTED ENZYME PRODRUG THERAPY (ADEPT) ABSTRACT Tum or Necrosis Therapy (TN T) uses a monoclonal antibody (M A b). directed against antigens accessible only in degenerating cells present in necrotic areas o f tum ors as a targeting molecule for the delivery o f various agents to the tum or site. Biodistribution studies perform ed using either murine or chimeric M Ab TN T-3 have show n excellent localization and persistence in tumor. Since the antigen-antibodv complex is neither shed, nor internalized, these particular properties o f the T N T -tv p e antibody would make it a perfect candidate for use in A ntibody-D irected Enzym e Prodrug Therapy (ADEPT). A D EPT is a method which pretargets an enzym e to the tum or site to convert an intravenously adm inistered prodrug to its active form, thereby- enhancing the therapeutic value o f the cytotoxic reagent. 149 Reproduced with p e rm issio n of th e copyright ow ner. F u rth e r reprod u ctio n prohibited w ithout perm issio n . For these studies non-im munogenic fusion proteins between single chain (scFv). Fab. and F(ab' ) 2 fragments o f chimeric TNT-3 and the human 3-glucuronidase enzym e w ere constructed and expressed in myeloma NSO cell line using the Glutam ine Synthase Am plification System , then purified via affinity columns. Functional testing was perform ed to assess the specificity and avidity o f the constructed conjugates as com pared to that o f the parent antibody and the single chain. Fab. and F(ab' ) 2 derivatives. Furtherm ore, the enzym atic activity o f the 3-glucuronidase present in a fusion protein was determined. In vitro cytotoxicity studies using murine lung carcinoma cell line M adison 109 (M A D 109) were perform ed to determine killing abilities o f the glucuronide prodrug o f doxorubicin when combined together with the 3 - glucuronidase construct. Enzymatic and chemical modifications o f enzym e were perform ed to improve enzym e localization into tumors. Animal studies were performed in Balb/C mice using the M adison 109 lung tum or model. Clearance, biodistribution, and determ ination o f enzym e activity over the tim e course studies were perform ed to evaluate this targeting m ethodology for the treatm ent o f solid tum ors. These studies provide prelim inary database to prove the usefulness o f this targeting m ethodology and its superior perform ance as applied to ADEPT-based treatm ent o f solid tumors. 1 5 0 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth er reproduction prohibited w ithout perm issio n . 1. INTRODUCTION Cancer chemotherapy has been based on the principle that it is possible to develop drugs that are selectively more toxic to tum or cells than to the host. Unfortunately, this premise has not been proven to be true for most solid tumors. While chemotherapeutic agents used in treatm ent regiments do kill neoplastic cells, they also affect rapidly growing cells present in high numbers in the bone marrow, gastrointestinal tract, and skin I. This frequently leads to side effects and patient m orbidity and also results in the termination o f the treatment before com plete tum or eradication. In addition, the sensitivity o f tum or cells to chemotherapeutic drugs varies widely and often clinicians encounter tum ors which are partially or totally drug- a resistant2 . A major objective o f cancer therapy is therefore to develop treatm ent modalities that decrease side effects and deliver higher levels of cytotoxic drug to the tumor site w ithout further com prom ising the host^- 4. It may be possible to decrease these effects using an interesting new approach to cancer therapy introduced by Bagshawe in the late eighties-. Designated A ntibody- Directed Enzyme Prodrug Therapy (A D EPT), this novel approach targets an enzym e to the tum or using a monoclonal antibody as a carrier molecule. Later, the tumor- localized enzym e activates the administered prodrug (for detailed discussion, see Chapters 1 and 4). In order to achieve this therapeutic effect, the antibody em ployed 151 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth er reproduction prohibited w ithout perm issio n . in making the enzyme construct should possess certain characteristics. M o st im portantly, it has to localize to the tum or prom ptly and be available there for a prolonged period o f time. Since clearing o f the antibody/enzvm e construct from the circulation is critical to avoiding extra-tumoral activation o f the prodrug and unw anted side effects, the importance o f this particular characteristic cannot be overem phasized. O ur laboratory believes that the TN T antibodies (see Chapter 1) developed here may act as an ideal carrier molecule for the ADEPT therapy since their characteristics match precisely requirem ents outlined above. The advantage o f using chTN T-3 in ADEPT is that chTNT-3 can potentially target all types o f cancer and provide a ‘self-perpetuating' treatm ent regimen. For antibody enzym e fusions described in this chapter, monoclonal antibody chTNT-3 was used as the tem plate. The antibody-enzym e constructs em ployed in ADEPT studies w ere usually com posed o f murine antibodies and bacterial enzymes. At that time, those were the m ost readily available com ponents. H ow ever both o f them showed them selves to be strongly immunogenic when used in hum ans^. While it is possible to devise m odifications rendering them less im m unogenic^ or suppress the immune response o f a patient by using additional drugs^. the m ost obvious path is to substitute both com ponents with their human analogs. R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . One human enzyme already used in AD EPT, human P-glucuronidase (pG ). looks very prom ising. Human p-glucuronidase is a tetrameric glycoprotein com posed o f four identical (77 kDa) subunits. A m ultim eric form plus glycosvlation arc essential for preserving the enzymatic activity^. The enzym e is a lysosomal acid hydrolase and plays a major role in the degradation o f glucuronic acid-containing glycosaminoglvcans. Lack o f this enzyme causes one o f the lysosomal storage diseases, m ucopolysaccharidosis VII. and therefore the enzym e and its properties have been already studied in detail *0. Because o f its lysosomal and microsomal localization* *. pG has very low concentration in human serum *-, thus minimizing the am ount o f unw anted activation o f the prodrug in the circulation. In addition. pG. despite being a lysosom al enzym e, still exhibits its activity at physiological pH* A A dditionally, it has been reported that the pH in tum ors often falls below the physiological range as a result o f poor blood flow and hypoxia *4. Extracellular pH in tumors can reach values below pH 6.3*5. If this is indeed the case, then using a lysosomal enzym e with an activity optim um in a range o f pH 5 m ay be particularly advantageous. The fact that glucuronide conjugates are already present in the body as naturally occurring metabolites o f m any substances *6 ' *7. indicates that glucuronide-based prodrugs should be stable in the blood and not prem aturely activated by endogenous P- glucuronidase. Additionally, the glucuronide prodrugs have an increased solubility 153 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . which may lead to an improved formulation compared to anti-neoplastic agents which are often insoluble^, ^ while at the same time they are still hydrophobic enough to prevent them for entering cells s p o n t a n e o u s l y - ® . Studies o f the natural com pound called epirubicin-glucuronide. found in patients undergoing therapy with epirubicin. revealed that this prodrug which is produced in the liver as a metabolite o f epirubicin and excreted via the kidneys is 1 0 0 - to 1 0 0 0 -fold less toxic (depending on a cell line used) than its active precursor. At the same time, the action of bacterial [3- glucuronidase from E.coli was able to restore its ftill activity Levels o f the endogenous (3-glucuronidase have been also reported to be elevated up to 6 -fold in 0 1 O O human breast tumors com pared to normal tissu e s-1, and lung tum ors— . thus suggesting an added benefit o f the enzym e available already at the tumor site. As a human enzyme, human P-glucuronidase should not evoke an immune response like the bacterial enzym es^. Early studies o f PG usage in ADEPT were done utilizing chemical conjugates o f a human enzym e and antibody-®. The major disadvantages o f such conjugates, however, were that they were heterogeneous mixture o f different components and that often after the conjugation, the activity o f a resulting molecule diminished. Recently, attempts have been made to engineer fusion molecules combining antibody and human enzyme together as a single fusion p r o t e i n ^ . -■*. 154 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth er reproduction prohibited w ithout perm issio n . In the previous chapter (Chapter 4), the design and characterization o f a fusion protein between the bacterial enzyme, cytosine deaminase and antibody fragments of chTN T-3 were described. Promising as these results were, one could immediately anticipated problem s related to the use o f a bacterial enzyme, sure to ev oke immune response. When the prodrug for the human enzyme p-glucuronidase became available through the collaboration effort with another laboratory, it was decided that the conjugate between human p-glucuronidase and a monoclonal antibody should be developed. This chapter describes in details the construction o f several fusion products betw een fragments o f chTNT-3 and human P-glucuronidase. and the subsequent evaluation o f their properties as reagents for ADEPT. 2. MATERIALS AND METHODS Reagents, Antibodies, and Cell Lines The source o f reagents and the origin o f antibody and cell lines used have been described in Chapter 2 in details. 155 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth er reproduction prohibited w ithout perm issio n . Construction of Expression Vectors Antibody fragments, nam ely Fab, Ffab'):- and single chain, have been already successfully generated from the parent chimeric TNT-3 (chTN T-3) antibody using PCR and molecular cloning techniques (see Chapters 2 and 3). The cDNA encoding for hum an P - g l u c u r o n i d a s e ^ ^ has been obtained from the ATCC and was PCR amplified as tw o fragments. The first fragment was generated using 5' end primer: 5 ' - G A TA A TA G C TC G A G A C TG C A G G G C G G G A TG C TG TA C - 3* which removed propeptide sequence from the 5’ end o f the enzym e and introduced XI7of cloning site, and the 3’ end primer: 5' - G C TA TC G A A TTC TG A C G TC TG T G C A G TC A G C TG - 3 ' partially identical the bases 425-445 in the enzym e's cDNA where the Aar! restriction site is located plus additional E coR I site for cloning this part I o f P-glucuronidase into Bluescript vector. Second fragment has been generated using 5’ end primer: 5' - A C TG C A C A G A C G TC A C TG G G G C C T - 3* identical the part o f PG cDNA containing Acitl restriction site and 3' end primer: 5 ' - C A TA G C A A G C TTC TC G A G A G TA A A C G G G C TG TTTTC - 3* removing the stop codon from a cD N A sequence and introducing two cloning sites: X h o l and Hindi I I necessary for further genetic manipulations. 156 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . After PCR am plification, first fragm ent I was cloned into X h o l and EcoRI sites in a Bluescript vector. In a second step, this vector with part I insert w as digested with A a tl and H indlll restriction enzym es and into this cut. the second part o f (3 - glucuronidase was cloned. Both fragments aligned in frame next to each other. In the last final step, the full length o f p-G was transferred after the X h o l digestion, into Xhol site at the 3' end o f pEE 12 vector containing either Fab, F(ab'):. or single chain cDNA (see Chapters 2 and 3) plus a cassette with Glutamine Synthase (GS) expression system (Lonzo Ltd.. Slough, U.K.). Cloning into X hol site allowed enzym e to be aligned in frame with the antibody fragments and preserved at the C-end already present purification tags; either six histidines tag (His-tag) or nine amino acids streptavidin-affinity tag (Strep-TagI) 26). Expression and Purification o f the chTNT-3/P-Glucuronidase Fusion Products The protocol for the expression and purification o f the chTNT-3 fragment/enzyme constructs is based on the methodology described in detail in C hapter 2. Determination of Avidity The avidity constants o f the chTNT-3 fragments/fusion proteins were determ ined by a fixed cell RIA as described previously (Chapter 2). The equilibrium or 157 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . avidity constant Ka was calculated by the equation K = -(slope//!), w here n was the valence o f the antibody (2 for scFv and Fab/enzyme fusion proteins, and 4 for F(ab'); enzym e fusions). Radiolabeling of Fusions l2 5 I-labeled Mab/(3-glucuronidase fusions were prepared using a modified chloramine-T method as described earlier (Chapter 2). with a yield o f 60-67% radiolabeled product. The radiolabeled antibodies were diluted with PBS for injection, stored at 4°C, and administered w ithin 2 h after labeling. Measurement of [3-Glucuronidase Activity' In Vitro Activity o f the enzym atic part o f fusion protein was measured using commercially available kit from Sigma (cat # 325-A). according to the manufacturer instructions. Briefly: 40 |il o f supernatant was incubated with 40 jul o f the substrate (phenolphthalein mono-[3-glucuronic acid) with addition o f 1 2 0 jil o f acetate buffer (pH 4.5) for an hour at 56°C. A fter the incubation. 1 ml o f 2-am ino-2-m ethvl-l- propanol (AMP) buffer was added and the color development assessed at wavelength o f 550 nm. Enzyme activity was m easured in so called "Sigma units" (approxim ately 4 times Fishman units) and calculated from the standard curve o f phenolphthalein concentration versus color developm ent, then converted using equation: 158 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth er reproduction prohibited w ithout perm issio n . glucuronidase (m odified Signui units ml) = phenolphthalein concentration (fig/ml) x 30 (dilution factor) For the pH dependent activity study. 40 pi o f the fusion protein (1 mg ml) was incubated with 40 p i o f the substrate in 120 pi o f acetate buffer o f varying pH values. The assay developm ent proceeded further as described above. Visualization of the P-Glucuronidase Activity Activity o f the enzym atic part o f fusion protein was visualized using protocol developed by M urray^?. Briefly a mixture was prepared containing 2 . 8 mg o f naphthol AS-BI P-D-glucuronide in 10 ml o f 0.2 N acetate buffer (pH 5.0). 10 ml o f distilled water and 0.6 ml o f freshly prepared hexazotized pararosanilin. To prepare hexazotized pararosanilin 0.8 ml o f 4% pararosaniline hydrochloride was mixed with 0.8 ml o f 4% sodium nitrite, then pH was adjusted using IN NaOH (approx. 0 . 6 ml). This mixture was prepared within minutes o f its use. The pH o f final solution was cheeked one more time and adjusted to 5.0 if necessary with 1 N NaOH. The SDS- PAGE gel was than incubated in a solution for 5 hr at 56°C w ater bath. In Vitro Cytotoxicity Studies The m urine lung carcinoma cell line Madison 109 (MAD 109) was used in these studies. The prodrug used in these ADEPT studies is the glucuronide derivative o f 159 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth er reproduction prohibited w ithout perm issio n . doxorubicin. This glucuronide is several fold less toxic than parent drug, while the action by the (3-glucuronidase restores its toxicity. Q uantities o f the prodrug sufficient for the prelim inary studies were a generous gift from Dr. Phil Thorpe (M aine Medical Center Research Institute, South Portland. ME). The sensitivity o f cells were determined using the modified colorimetric assay used norm ally for the assessm ent o f TNF cytotoxicity-^. as described in details earlier (Chapter 4). C oncentrations varied from 1 mguril to 0.1 m giul for fusion proteins and from 3 mM to 0.1 mM for prodrug. A ppropriate controls included a drug (doxorubicin) on its own as well as irrelevant antibody in combination with a prodrug. Modifications of Human [3-Glucuronidase Based upon the literature, fusion proteins were modified either by chemical modification with sodium periodate or by enzym atic digestion with |3-N- acetyloglucosaminidase. For both modifications, the results o f either enzym atic or chemical deglycosylation were later checked by SD S-PA G E gel and the degrees o f the activity preservation o f the antibody and the enzym e portions of the fusion protein were confirm ed by ELISA and enzym e activity assay respectively. For the chemical digestion, the periodate treatm ent protocol- 9 modified by Houba-30 was used. In short fusion protein (1 mgTnl) was first dialyzcd into 50 mM sodium acetate, pH 4.5 and then N aI0 4 was added to the final concentration o f 40 160 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . mM . The m ix was incubated for 2 hr at 4°C. A fter that time the reaction was stopped by adding an excess o f ethylene glycol (added to the final concentration o f 1.63 M) and sam ple was dialyzed overnight in large volume o f PBS. pH 7.5. In the next step N aBH 4 was added to the final concentration o f 40 mM and the mix incubated at 37”C for 3 min and than at 4°C for 1 hr. Again the sam ple was dialyzed overnight in large volum e o f PBS. pH 7.5. For the enzym atic digestion, the P-N-acetyloglucosaminidase (also known as jack bean oc-mannosidase: EC. 3.2.1.30) was used as described by Glaser-’ *. Briefly, the fusion protein (concentration 1 mg/ml) was dialyzed into 0.1 M citrate buffer. pH 5 with 0.2 M NaCl and 0.02% (w/v) Bovine Serum Albumin (BSA) overnight. In a next step the enzym e was added (25 units total; 0.1 unit/ml in citrate buffer) and the digestion preceeded for 1 day at room tem perature. Finally the reaction was stopped by the addition in excess o f the 0.2M borate buffer. pH 9.8 and digested fusion w as dialyzed back into PBS. Pharmacokinetics and Biodistribution Studies The protocol for the pharmacokinetics and biodistribution studies were described in details in Chapter 2. For biodistribution studies murine carcinoma cell line M AD 109 was used. 161 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm ission. Measurement o f (3-Glucuronidase Activity In Vivo The TN T-based antibodies bind to any necrosis, therefore the murine tum or model was employed, using the Balb/C female mice injected with initial dose o f the 2 x 107 Madison 109 (M AD 109) lung carcinoma cells s.c. in the left thigh. After tum ors reached close to 1 cm in diameter, individual mice within each group (n=3) were injected i.v. with a 100 pg o f MAb fusion. Animals were sacrificed by sodium pentobarbital overdose 24. 48. 96. 120. 16S. and 216 hr post-injection, and tissues were removed, weighed, and processed according to the protocol by F rankeP - w ith slight modifications. Briefly: removed tissues were homogenized in 7 ml 0.02 M Tris- HC1. pH 7.5. 0.075 M NaCl for 30 sec. This homogenate was then diluted further (1:5) in above buffer with addition o f bovine serum albumin (5 mg ml), and 0.2” ■ > sodium deoxycholate. Endogenous murine (3-glucuronidase was inactivated by incubation o f the diluted crude homogenate in 64°C water bath for 180 min. Subsequently homogenates were assessed for their enzymatic activity using commercially available kit from Sigma, according to the manufacturer instructions as described above. In Vivo Murine Model Treatment Studies The murine lung carcinoma cell line. M adison 109. was em ployed to produce tum ors in Balb/C mice in a manner similar to one described earlier. When tum ors 162 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm ission. achieved a mean diameter o f approximately I cm. mice were random ly divided into the several groups with 5 mice/group (Table 5-1) and injected according to the schedule. Table 5-1: Groups for the Therapy Study. GROUP REGIMEN Control no treatment Fab/P-glucuronidase plus prodrug fusion: 1 0 mg/ml prodrug: 1 0 0 mg/kg Fab/p-glucuronidase plus prodrug fusion: 1 0 m gkg prodrug: 2 0 0 m g kg Fab/P-glucuronidase plus saline fusion: 1 0 mg'kg Prodrug only prodrug: 2 0 0 m gkg Doxorubicin only 2 0 mg/kg The literature r e a d i n g s ^ . 34 as w eu as /„ r / 7 / * < ? cytotoxicity studies suggested using two different doses o f the prodrug: 1 0 0 mg'kg (approx. 2 mg mouse) and 2 0 0 mg/kg (approx. 4 mg/mouse) per single dose. Fusion protein was adm inistered as a single dose o f 10 mg/kg (approx. 0.2 m g/m ouse) at approximately 10 and 15 days after tum or inoculation. The prodrug was adm inistered approx. 12 and 17 days after inoculation - 48 hours after adm inistration o f the fusion construct. There was 163 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . approxim ately a two week "window" for the treatm ent before Balb/C mice started developing immune response to foreign proteins. Tum or growth was measured at 2 day intervals, starting on the day o f first injections o f fusion protein. Tum or mass was calculated from tum or volume (assuming a tissue density o f Ig/cnT). which was determined by caliper measurements. The formula: mass = 1 g/cnT x 0.5 x L x W~ was used (L and W - the longest dimension and its perpendicular in cm.). The mice were sacrificed when the tum or size reached over approx. 5 cnT or when they exhibited signs o f morbidity. The final data was expressed as a tum or mass growth curve. Additionally the surv ival time was also monitored. 3. RESULTS Characterization o f Enzyme/Antibody Constructs The fusion proteins were expressed in a mammalian expression system since the proper glycosylation o f a human [3-glucuronidase is im portant to its subsequent form ation o f tetram ers and full enzymatic activity^. Rates o f production varied from 2 jlg/ml/106 cells/24 hr to 5 jlg/ml/106 cells/24 hr. which is high enough to allow the purification o f sufficient amounts o f material for further experimentation. The highest producing clones were incubated in a 3 liter stir-flask, and the fusions were purified 164 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . stepw ise from cell culture medium by Ni-NTA or modified streptavidin (Streptactin) affinity chrom atography as described above. The purity o f the obtained fragments was examined both by SDS-PAGE and HPLC chrom atography. SDS-PAGE gel showed proper assem bly o f the fusions with marked tendency to form dimers and tetramers (Figure 5-1). Interestingly enough, single chain and Fab fusion proteins had preference for forming dimers, while F(ab' ) 2 fusion protein formed mostly tetramers. The most likely explanation for this finding is that F(ab' ) 2 being already a homodimer itself, encourages the enzyme part of the fusion to form tetramers. Single chain fusion protein also exhibited a tendency to fall apart into monomers upon prolonged storage. While its antigen binding abilities stayed intact, the breakdown to monomers abolished seFv fusion's enzym e activity as shown on SDS-PAGE gel stained for enzymatic activity. 1 6 5 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth er reproduction prohibited w ithout perm issio n . Figure 5-1. The SDS-PAGE Gel Double Stained with Commassie Blue (Protein) and Pararosanilin (Enzyme Activity). 200 kDa 116 kDa 97.4 kDa 66 kDa | 45 kDa? B D lane A: high molecular weight marker laneB: single chain/fl-glucuronidase fusion molecule lane C: Fab/p-glucuronidase fusion lane D: F(ab')2/{i-glucuronidase fusion; non-modified. Lines E and F represent enzymatically and chemically m odified ^-glucuronidase fusions, respectively. Lane G: Fab' and F(ab')2 mixture. To confirm the size o f the fragments and investigate their stability in solution, size exclusion HPLC was performed. It showed that all antibody fragment/enzyme fusion proteins presented the same pattern o f elution which consisted of multiple peaks o f constantly shifting arrangement (L.A. Khwali; personal communication). This 165 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . suggests that the chTN T-3/hum an |3-glucuronidase fusion proteins have a tendency to change between mono-, di-. and tetram ers states in a continuous and fluent manner w ith prevalent forms being dim ers and tetram ers. W ork done by others reveals that m ature [ 3 -glucuronidase is ordinarily fully active as a hom otetradim er but homodimers can also be activ e^ 35 C onstant variability between different configuration states as observed by HPLC can explain transient differences in the activity o f the enzym atic part o f fusion protein as som etim e observed in experim ents. Determination of Avidity The avidity constants o f the chTN T-3/enzym e fusion proteins arc presented below in Table and Figure 5-2. The n value (# o f binding sites) has been in this case assigned in rather arbitrary fashion since the fusion protein could assume different configurations (mono-, di-, and tetramer). However each shift in configuration and therefore in n value is accompanied by an adjustm ent in molecular weight, so these changes cancel each other out in the equation. 1 6 7 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . Table 5-2: Avidity Constants o f Different Antibody Fragments and Human (3-GIucuronidase Fusion Proteins. F ragment Binding Sites % Binding Avidity Constant Ka chTNT-3/scFv/pG one 60.4 0.88 x 10* M' 1 chTNT-3/Fab/pG two 72.5 1.05 x 10* M' 1 chTNT-3/F(ab’)2 /pG two 62.8 1.11 x 10* v r 1 chTNT-3 two 89.0 1.43 x 10* M-' R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . Figure 5-2: Binding Affinities of chTNT-3 Antibody Fragments/Enzyme Fusion Molecules. H u m a n ( 3 - G lu c u r o n id a s e F u s io n P r o t e i n s B in d in g C o n s t a n t D a t a 4 scFv/[}-gl u c u r o n id a s e F a h /[3 -"lu c u ro n id a se F (a b ' H /p -g lu c u ro n id a s e w o 40 I 0 20 30 bound (ng) 169 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . These studies show ed that the avidity constants for different antibody fragm ents/enzym e fusion proteins were sim ilar to within one decimal place to that o f a parent antibody. Interestingly, the binding affinities for scFv. Fab and F(ab'); fusion proteins were im proved comparing to scFv and Fab and F(ab'): them selves, thus approaching the value for the intact antibody. This could be explained by the influence o f the enzym e portion o f the fusion protein forcing the monovalent scFv and Fab. and divalent F(ab’)i fragments into forming dimers and tetramers. thus improving their binding to antigen. This phenom enon has been reported by o th ers1 - 3- — \ Measurement of (3-Glucuronidase Activity In Vitro Activity o f the enzym atic part o f fusion protein was measured using a commercially available kit from Sigma, and modified to include the whole range o f pH values. Enzyme activity was measured in "Sigma units" (approximately 4 times Fishman units) and calculated from the standard curve o f phenolphthalein concentration versus color developm ent, then converted using the equation: (5-glucuronidase (modified Sigm a units/ml) = phenolphthalein concentration tu g m l) x 30 (dilution factor) 1 7 0 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth er reproduction prohibited w ithout perm issio n . modified Sigma units Figure 5-3: pH-Dependent Activity of p-GIucuronidase Part of Fusion Proteins. 360 sc F v /[K ; - lOOn 340 - F a b / p o - 1 0 0 n s 320 - F (a b ' )2 /p C - 100ns 300 - 280 - 260 - 240 - 220 - 200 - 80 - 60 - 40 - I 2 0 - 00 - 80 - 60 - 40 - 20 - 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 S. 5 V 4.5 pH 171 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth er reproduction prohibited w ithout perm issio n . Activity o f the fusion constructs proved to be similar to the native enzym e (Figure 5-3) as reported by others 36 jn respect o f the shape o f the activity's curve in relation to pH. The maximum catalytic rate was observed at low pH o f 4.5-5 as it should be expected from a lysosomal enzyme. However approx. 20% o f the enzym e activity remained at pH 7-7.3. a pH value found within tum ors 1 _ *. which should be sufficient for the activation o f a prodrug within the tum or microenvironment. In vitro Cytotoxicity Studies The sensitivity o f cells to doxorubicin and the prodrug was determined using a modified colorimetric assay used for the assessment o f TNF cytotoxicity-^. The commonly used enzym e-release cytotoxicity assay ^ did not work properly due to color interference from doxorubicin and its prodrug contributing to the high background. The concentrations used for fusion proteins ranged from 0.5 mg ml to 0 . 1 mg/ml. The 0 .1 mg/ml level o f the fusion protein has been reported as an achievable concentration in patients after a 1 0 0 mg dose and 0 .0 1 % dose gram tumor uptake For the prodrug, the concentration ranged from 3 mM to 0.1 mM. 1 7 2 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . of cell k illin g Figure 5-4: In Vitro Cytotoxic Effect of the Activated Prodrug of Doxorubicin. lOO-i 90 - 8 0 - 7 0 - 60 - 5 0 - 30 - I 0 - 0 -J <>> < V , ► ►< 4 V ■ 4 1 ► - V ► A V A X ► ' A V A > a a ; ■ ► A V ► < V , ■ < : ■ ► , •4 % M <v> i 1.5 0.5 m g/m l o f scF v /en zy m c c o n ju g a te 0.5 m g/m l of F ab /en zy m e co n ju g ate ESI 0.125 m g/m l of scF v /en zy m e c o n ju g a te H 0.125 m g/m l of F ab /en zy m e c o n ju g a te A A M <5M <S.M <*.M < > ► < <v,H < v< <>► 4> ► <S> < V <V'-, “ i 0.75 p ro d ru g only A A A A A A S ' 1 A 5 • < > , m 5 • < > . ► < * - 4 M Z A v > 4 ?4W4 Z A • M 5 ■ < V H 2 • < > H 2 ■ < > H ? ■ • < X , M ? .4 v H ? -4 M Z A v M 2 < > . ► < ? -4 > , M Z A X , M Z A • • > 4 Z A X , M 2 < > . ► < 2 ’ < X ► < 2 « > , H 2 • < > . M 2 ■ < s.M | : s " . p i * I 0.375 h v 7 \ :: : : : f 7 \ * r f I Z k 4$> a y ► 4*> n ► 4 ► 4 4 >4 ' M ’ > - 4 ‘ ► 4 T4 A >< ► 4 4 > 4 4 ► 4 ► 4 ► 4 " 1 - 4 ♦4 4 a?L i 0.1 88 I 0.094 p ro d ru g co n c en tratio n (mM) i 7 3 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . In these studies (Figure 5-4) the prodrug was shown to be approxim ately 6000 tim es less active then the doxorubicin against MAD 109 m urine lung carcinoma cell line (IC 50 o f 0.25JJ.M com pared to 1.5 mM). The action o f the enzym e p an o f the fusion was able to restore prodrug activity to approx. 2 0 -fold closer to that o f the doxorubicin. The IC50 for the combination o f the prodrug and 0.125 mg ml o f fusion protein was 94 [J.M. O ther studies often reported the reversal o f the IC5,, o f the prodrug to that o f original drug. Haisma -4 reported activation o f the prodrug by the scFv/pG construct and shifting o f the IC50 for Daudi cells from 2 1 1 .V I to 0.3 li.M. approaching very closely the IC50 o f doxorubicin. The same group made another scFv fusion protein and reported that its action converted the doxorubicin prodrug to an active drug, thus inhibiting the growth o f OVCAR-3 cells to the same extent as the doxorubicin itself 23. in our studies, the action o f the fusion protein improv ed the IC50 o f the prodrug o f doxorubicin, but not to that o f original doxorubicin. It could be that the conditions for the assay need to be optim ized. Also the cell line used. M adison 109. seem s to be particularly sensitive to d o x o r u b ic in ^ . Modifications of Human (3-Glucuronidase Enzymatic and chemical modification were only perform ed on one o f the fusion protein, namely the Ffab'VP-glucuronidase fragment as a representative for all the other constructs. Based upon prior literature, fusion protein has been modified either 174 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth e r repro d u ctio n prohibited w ithout perm issio n . by chemical modification with sodium m etaperiodate followed by a reduction w ith sodium borohydride 39 or by enzymatic digestion with (3-N-acetyloglucosaminidase 31. Human P-glucuronidase tends to clear quickly from the circulation and accumulates in the liver due to the preferential uptake by the reticuloendothelial system present in this organ which recognizes exposed N-acetylglucosamine residues39-42 ^as been reported by other investigators^- 39. 4o t|lat periodate treatment o f the hum an P-glucuronidase abolishes this feature. This generally leads to a prolonged life o f the infused enzyme and better targeting into organs. At the same time, this treatm ent causes only slight loss o f enzym atic activity where it is reported to be a 11% loss according to Archord39 and less than 5% as reported by I louba3^. In our experiments, we observed that both the chemical and the enzym atic treatments o f the fusion protein slightly diminished the activity o f the enzymatic part o f the fusion protein. After treatment with m etaperiodate, a minimal loss o f activity was detected and incubation o f the fusion protein with jack bean o.-mannosidase resulted in the preservation o f approximately 80 % o f the enzyme activity. Equally important, the antigen binding abilities o f the fusion proteins were fully preserved (data not shown). On SDS-PAGE gel (Figure 5-1) several hands were visible. corresponding to monomers, dimers and tetram ers o f the enzyme. There was a slight but noticeable shift towards dimer and m onomer formation for the enzym atically- 175 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . modified enzym e. In the case o f the chemical modification, howev er, it seems that the resulting molecule formed only tetramers. Since the ability to form tetramers. and to smaller extent dimers, is essential for the preservation o f the enzymatic activ ity o f 1 3 - glucuronidase. partial reduction to the monomers by either treatment can explain the loss o f activity. At the same time even monovalent antibody fragments can still bind to the antigen and since it seemed that neither chemical nor enzymatic treatment modified the antigen binding site itself, no change was detected in the binding o f the c h T \T - 3/enzym e fusion(s) to its antigen. Additionally, the SDS-PAGE gel was also stained for the enzymatic activity- using the visualization method described by M urray-^. This particular gel showed that upon prolonged storage, the single chain construct became unstable and formed exclusively monomers. It is clearly visible that although the fusion protein was still there forming m onomers, the enzyme activity vvas not present for the single chain but was associated with all the other constructs that formed dimers and tetramers. Pharmacokinetics Studies W hole-body clearance studies in Balb/C mice were performed to establish differences in the pharmacokinetics between different antibody-enzym e fusion proteins as determined. The data were analyzed and half-life values calculated as described p re v io u sly ^ . 176 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n. Table 5-3: Half-lives o f Different Antibody/Human (3-GIucuronidase Fusion Proteins as Determined by Clearance of Radiolabeled Proteins from Balb/C Mice. Fusion size T ,2 chTN T-3/scFv/p-glucuronidase 115. 230. 460 kDa 9.9 - - 1.3 hr chTN T-3/Fab/p-glucuronidase 120, 240. 480 kDa 9.5 0.6 hr chTN T-3/F(ab')2 /P-glucuronidase non-modified 125. 250. 500 kDa 8.5 a - 1.6 hr chTNT-3/F(ab')2/P-glucuronidase chemically modified 125. 250. 500 kDa 7.5 - - 1.4 hr chTN T-3/F(ab')2 /P-glticuronidase enzym atically modified 125. 250. 500 kDa 7.8 + ■ '- 0.8 hr chTN T-3 140 kDa 134.2 4.0 hr It has previously been s h o w n ^ that parent chTNT-3 clears slow ly with a whole-body half-life o f 134.2 +/- 4.0 hr in Balb/C m ic e ^ . As depicted in Table 5-3. chTNT-3 fragm ent/enzym e fusion proteins were eliminated significantly more rapidly and with only slight variations between them selves. This occurred despite the sizes o f the fusion proteins approaching (as m onom ers) and well exceeding (as dimers and tetramers) the size o f the parental antibody. These findings are. however, in agreement with other published data where it has been found that conjugates containing enzym e 177 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . [3-glucuronidase clear significantly faster most likely due to the selective uptake o f the enzym e by the reticuloendothelial system in the liver*-. Murine antibody (3G conjugate used by C h e n g ^ cleared from the blood with a T, : of 17.7 hr., which is remarkably fast for the whole antibody construct. (PEG )-vlation o f the molecule extended its half-life and diminished liver and spleen uptake, albeit at the cost o f lower enzym e activity and decreased binding affinity. Other studies by Houba-^*. while using a murine antibody conjugated to (3G. showed that it cleared from the circulation w ithin minutes ( T |2 = 24 min) while the whole antibody needed 32 hr to clear. Chemical modification o f the conjugate resulted in an increased retention time o f S.6 hr. In our experiments fusion molecules behaved in a manner resembling already modified conjugates between antibody and (3G. even when no such modifications were introduced. Additionally, the question was raised about the stability o f the enzym e in mouse and human sera. Some groups claimed that the enzym e loses the activity upon prolonged incubation *9 while others found that the activity increased-* 0 . For these reasons, stability studies were performed. Fusion proteins were incubated for prolonged periods o f tim e (up to 12 days) in human serum at 37 "C before performing binding and enzym atic activity measurements. No changes were observed in enzym e activity under these conditions (data not shown). 17S R e p ro d u c e d with p e rm issio n of th e copyright ow ner. F u rth e r reprod u ctio n prohibited w ithout perm issio n . Biodistribution Studies The biodistribution o f the chTNT-3/enzyme constructs was assessed in MAD 109 murine lung tum or model system using Balb/C mice with implanted s.c. tumors. Biodistribution data were taken at 6. 12. 24. and occasionally at 4S hr time points (Figures 5-5 and 5-6). All fusions retained the ability to localize to tumors, as depicted in Figure 5-5. Tum or uptake o f chTNT-3 fragments enzyme fusion proteins at 1 day post-injection varied approxim ately from 2.3% ID/g (scFv/(3G) to 2.5% ID'g (Fab (iG) to 3% ID g (F(ab');>/pG). At the same time, the rapid clearance o f the fusion proteins produced several-fold higher tumor/normal organ ratios for many normal tissues (Figure 5-6). illustrating the specificity o f tum or targeting with chTNT-3 fragments-enzyme fusion proteins and their rapid elimination from the blood and normal organs. One o f the organs where the uptake was high was the liver possibly due to recognition o f the [3- glucuronidase portion by the reticular system 42 Up to 24 hr. tum or to organ ratios for liver was around 1.0 for all constructs. To investigate this issue further, chemical and enzymatic modification o f fusion protein were performed (described later). 1 7 9 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . Figure 5-5: Tissue Biodistribution and Tumor Uptake (% Injected Dose per Gram of Tissue) o f chTNT-3 Fragment/Human P-GIucuronidase Conjugates in Murine M adisonl09 Lung Carcinoma Tumor-Bearing Balb/C Mice in Selected Organs at 12 and 24 hr. 12 h r 5 4.5 4 ; 3 . 5 • 3 ■ :2.5 - •> _ 1.5 - I ■ 0.5 0 !5 fk T , B M B B B M M ■■■ B M M B M M B M B B B M M B M M B M M B M M B M B B BM B M H B H H i B M M BM E3 s c F v /p G H F ab /P G 0 F( a h ' »2/pG blood liver kidnev o r g a n 24 h r blood 0 F (a b '» 2 /p G s c F v /p G F a h /p c kidnev tumor IS O o r g a n R e p ro d u c e d with p erm issio n o f th e copyright ow ner. F u rth e r rep roduction prohibited w ithout perm ission. Figure 5-6: Tissue Biodistribution and Tumor Uptake (Tumor to Organ Ratio) of chTNT-3 Fragment/Human P-GIucuronidase Conjugates in M urine M adisonl09 Lung Carcinoma Tumor-Bearing Balb/C Mice in Selected Organs at 12 and 24 hr. 12 hr 0 £ u E a s hlood liver kidnev o r g a n 4.5 - 4 - 3.5 - s - I 2.5 u - > C c s 1.5 0.5 0 24 hr hlood s i X. r liver if # • ■ $ > ■ $ ■ • « sr: ir: IT ! i kidnev scFv/pC» Fah/pc; F ( a b ’)2/PG se F v /p c ; F ab /p G F(ab*)2/pC; o r g a n 1 S I R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth e r repro d u ctio n prohibited w ithout perm issio n . Rapid elim ination o f the fusion proteins resulted in good tum or organ ratios for all the genetically engineered constructs (Figure 5-6). At 24 hrs time point, the tumor/organ values varied from 1-1.5 times (blood, liver, spleen, stom ach) to 3.3 times (kidney) and 5-6 times (bone, muscle). This is a significant im provem ent over results obtained by other investigators. For example Houba^O reported tum or organ ratio for blood as 0.6 at 1 day. going up to over 2 only after 7 days. The F (ab ')2 /carboxypeptidase studies as presented by Stribbling showed tum or to organ ratio 1.3 for blood at 48 h r ^ . For other conjugates, the results were even less encouraging. Eccles^? reported ratios o f 1.5 in the blood after II days, whereas Sharma^S measured a blood/tum or ratio o f 2.9 at 72 hr post injection. Clearance and Biodistribution of Enzymatically and Chemically Modified chTNT-3 F(ab')2 /P-Glucuronidase Fusion Proteins The F(ab')2 fragment was chosen as a representative for all fusion proteins and was subjected to enzymatic and chemical modifications as described earlier. Studies of the clearance time for chemically and enzymatically modified fragment revealed no significant differences in clearance patterns between all proteins. While unmodified F(ab’)2/enzym e fusion cleared with a half-life of 8.5 hr. chemically modified derivatives had a half-life o f 7.3 hr and enzym atically modified proteins had a half-life o f 7.5 hr (Table 5-3). This is a different finding that the one reported by I lo u b a ^ where the 182 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth er reproduction prohibited w ithout perm issio n . conjugate prepared with native (3-glucuronidase cleared from a circulation within m inutes (tt 2 = 24 min). while the chemical modification significantly increased clearance time (t|,; = 8.6 hr). The lack o f differences in clearing tim e between modified and unmodified fusion proteins may be due to fact that in our experiments, even unmodified fusion proteins betw een chTNT-3 fragments and human (3-glucuronidase cleared from circulation in a relatively slow fashion, showing values more similar to Houba's modified antibody/enzym e conjugate. Moreover, it is possible that a genetic fusion protein composed o f the antibody and (3-glucuronidase in contrast to one prepared by chemical, provekes changes to the conformation o f the enzym e sufficient to render its recognition by the reticuloendothelial system impossible. Alternatively, the purification tag placed at the C-end o f the fusion protein changes the pattern of the protein termination. Since the protein no longer ends with mannose or N- acetylglucosamine, receptor recognition may have been affected therefore resulting in abolishing o f the rapid uptake o f the enzyme by the liver. Biodistribution performed using Balb/C mice implanted s.c. with M adison 109 murine lung carcinoma revealed findings similar in scale to those seen in pharmacokinetics studies (Figure 5-7). There was no statistically significant differences in the uptake in the liver or in the tum or between differently-m odified F(ab'): subspecies. Since the clearance data showed no differences, the biodistribution data are 183 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . as expected. Interestingly, the chemically modified F lab '^/en zy m e fusion protein at the 48 hr tim e point showed the best characteristics with the tum or uptake (4.9°o) and best tum or to organ ratios for blood, liver and kidney. This points to it being a likely candidate for further studies. 1 8 4 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . Figure 5-7: Tissue Biodistribution and Tumor Uptake of chTNT-3 F(ab’)2 /Human (3-GIucuronidase Fusion Proteins Non-modified, Chemically, and Enzymatically Modified in Murine Madison 109 Lung Carcinoma Tumor- Bearing Balb/C Mice in Selected Organs at 24, and 48 hr. A) % Injected Dose per Gram of Tissue B) Tumor to Organ Ratio C 5 f a . ec i tumor organs E 3 non-modified - 24 hr EJ chemically modified - 24 hr S3 enzymatically modified - 24 hr Hi non-modified - 48 hr E 8 3 chemically modified - 48 hr enzymatically modified - 48 hr blood organ E3 a 52 0 kidncv 185 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . Measurement of (3-glucuronidase activity in vivo The activity o f the enzym atic portion o f the chTN T-3/enzym e constructs was assessed in murine lung tum or model system using Balb/C mice im planted s.c. with Madison 109 murine lung carcinom a as described earlier. Human (3-glucuronidase activity was assessed in tissue homogenates after the inactivation o f murine [3-glucuronidase. It was noted that the inactivation step to delete the activity o f the endogenous murine (3-glucuronidase by incubation at 65° C for 90 min as recommended by F ran k eP -. was insufficient to abolish the activity o f murine enzyme to the significant degree. A fter additional experiments to establish the best time length for the procedure, the incubation time was increased to 180 min without any detrimental effect on the activity o f the human enzyme. Figure 5-8 clearly shows that the enzymatic activity o f the human (3- glucuronidase component o f the antibody/enzym e fusion protein is present for a very- long time after the administration o f the conjugate and it is preserved well and at a steady level. The differences present am ong the different fusion proteins such as a high liver uptake in the case o f the single-chain/(3-g!ucuronidase fusion protein are consistent with the data shown in the biodistribution experiments. 1 8 6 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm ission. modified Sigma units modified Sigma units modified Sigma u n i t s Figure 5-8: Activity of P-GIucuronidase Part of Fusion Protein in Selected Tissues at Different Time Points. Blood I 60 140 - 1 2 0 - 1 0 0 - 80 - 60 - 4 0 - 20 - 0 T 24 hr r 48 hr 0 scFv/PG 0 Fab/PG s F (ab’)2/PG 72 hr I 20 hr tim e — I---------------------- 1 ------ 168 hr 216 hr Liver 1 6 0 - 4 0 - <xm 24 hr 48 hr 72 hr 120 hr time B1 scFv/PG [U Fab/PG H F (ab’)2/pG I T 168 hr 216 hr MAD109 Tumor 160 140 - 120 - 100 - 8 0 - 6 0 - 4 0 - 2 0 - 0 T 24 hr < $ • < ! ■ & < ■ t 48 hr SB scFv/PG E l Fab/PG HI F(ab’)2/PG 72 hr 120 hr time 68 hr ! 16 hr I 8 7 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth er reproduction prohibited w ithout perm issio n . Additionally, sim ilar studies m easuring the presence o f human [3G in the blood and muscles at com parable tim e points, showed no traces o f the fusion protein (data not shown). This supports the biodistribution findings where the fusion proteins were found to localize specifically to the tum or and clear rapidly from the blood. The enzym atic activity assay, how ever, offers also the p ro o f o f prolonged preservation o f the enzym e activity associated with the fusion protein. Since the loss o f activity in fusion proteins upon protracted tum or localization has been reported for [3- glucuronidase^G and also for yeast cytosine d eam in ase^ , this is a valuable finding. In Vivo Murine Model Treatment Studies The murine lung carcinoma cell line. M adison 109. was employed to produce tumors in Balb/C mice. W hen tumors achieved a mean diam eter of approxim ately 1 cm. mice were randomly divided into the several groups and injected according to the defined schedule. The study was terminated on day 35 due to animal welfare concerns. The obtained data indicate that the prodrug is activated to its toxic form and slow s the growth o f the tum ors but only in a transient manner. The surv ival rate also appears to be affected. IKS R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . Figure 5-9: Treatment of Murine Lung Carcinoma Madisonl09 Using Combination of the Antibody/Fragment/Human-GIucuronidase Fusions and Prodrug of Doxorubicin. control doxorubicin: 20mg/kg prodrug only: 200 mg/kg 4.5 - chTN T-3/Fab/pG:10 mg/kg + prodrug: 200 mg/kg Z J 4 - u C chT N T -3/Fab/pG : 10 mg/kg + prodrug: 200 mg/kg . ■ — > 0.5 8 9 10 11 12 13 14 15 IA 17 IS 10 20 2 1 2 2 days I 8 9 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . 4. DISCUSSION With the establishm ent o f methods to produce monoclonal antibodies-0 and techniques o f genetic engineering allowing for their m o d ificatio n s^, the possibility o f creating new carrier molecules for the delivery o f radiolabeled and cytotoxic compounds to tumors in vivo moved one step closer tow ards becoming a reality. As targeting agents, monoclonal antibodies are ideal since they are homogeneous in nature, recognize specific antigenic determinants, can be massproduced. and used both in vitro and in i’ /vo52 Because o f their complex size, structure, and composition o f M A bs. many different methods have been devised to link radiodiagnostic and therapeutic reagents to them for clinical e v a l u a t i o n ^ . One o f the m ethods uses genetic engineering to create fusion proteins between the antibody and another protein, thus bypassing cumbersome chemical steps'^ 54 ancj producing homogenous product. Such fusion proteins have been developed for use in Antibody-Directed Enzyme Prodrug T h e r a p y 2 3 . 3 3 However their further investigation revealed specific requirement for the carrier antibody as outlined earlier. The TNT antibodies developed in our laboratory seemed to fit perfectly into that description Therefore it has been postulated that the combination o f TN T and ADEPT could significantly improve the results in ADEPT therapy. This chapter describes the creation o f fusion proteins 190 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . between fragments o f the chimeric monoclonal antibody chTN T-3 and human enzym e (3-glucuronidase. First the fusions proteins were produced in a mammalian expression system at the levels o f expression sufficient to allow further characterization in vitro and later in vivo. The use o f mammalian expression system ensured that the (3-glucuronidase was properly processed posttranslationally. Such processing, nam ely glvcosylation. has been proven to be critical for its enzym atic activity. The SD S-PA G E gel showed proper assem bly o f the fusion proteins. In order to reach its full activity. [3G needs to form either dimers or tetramers. This has been confirmed in several studies^- - 1- and also by the visualization o f the enzym e activity on a SDS-PAGE presented in this chapter (Figure 5-1). The in vitro staining o f the gel for enzym atic activity corroborated the fact that this activity is only present in dimers and tetram ers. making these the most desirable conformations. This could present a serious drawback with the respect to tum or penetration due to the large size o f the resulting fusion protein. The use o f antibody fragments allows for creating reagents w ith smaller molecular w eight and preserved binding capabilities. Still the need for dimer and tetramer formation would results in a large molecule, no matter what antibody fragment is used. How serious an impact this fact will have upon the tum or penetration remains to he seen. The observed improvement o f the binding affinities as show n by the avidity studies may contribute to better tum or uptake, however. Several studies using 191 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . antibody fragm ents for tumor targeting show ed the importance o f enhanced affinity in improving tum or u p ta k e ^ , 56 Since the affinities o f generated fusion proteins arc slightly better than for corresponding antibody fragments, this may offset the negative effect o f the increased molecular size. The increase in affinity may help to explain the results obtained in the biodistribution studies. First o f all. despite using the molecules with the size o f approx. 500 kDa. all fusion proteins cleared rapidly from the circulation. Their half- lives varied between 7 and 9 hr, as com pared to approx. 140 hr for the parental VIA b chTN T-3. These clearance times are very similar to these for the antibody fragments them selves (see Chapters 2 and 3). W hile part o f this effect could be explained by the active uptake o f the enzym e by the liver as described previously for [3- glucuronidase^' 42 biodistribution data (%ID/g) do not offer significant su p p o rt for this conclusion. The liver uptake for the antibody fragment'(3-glucuronidase fusion proteins are 3.7-2.3-3.3% ID/g, resulting in tum or to organ ratios o f 0.7-1.05-1.7 (scFv-Fab-F(ab’ ) 2 respectively) at the 24 hr time point, which are better than the values reported for other antibody/(3-glucuronidase fusion proteins-'(). An additional argum ent for the diminished liver uptake o f the enzyme fusion protein com es from the fact that the chem ical and enzymatic m odification o f the F(ab'):/[3-gIucuronidase fusion do not significantly alter their clearance tim es and biodistribution patterns. In other studies, such modifications greatly increased the half-life of the enzym e and the half- 192 R e p ro d u c e d with p e rm ission of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm ission. life o f antibody/enzym e conjugates^. The main explanation for this could be that in previous w ork by Houba, the fast-clearing antibody/enzym e conjugate was obtained by chemical means. Chemical conjugation often results in a mixed product where tw o parts are joined together randomly. Proteins presented in this chapter have been constructed by genetic engineering, thus enabling the production o f a single species o f conjugate. Additionally, placement o f the antibody fragment at the N-term inus o f enzyme molecule could conceivably alter enzyme conformation in a way as to make it unrecognizable for the liver receptor responsible for its active uptake. A lternatively, the placem ent o f the purification tag at the C-term inus o f the enzyme may prevent the enzyme from displaying mannose or N-acetylglucosamine. thus again abolishing recognition by the liver receptor. T t therefore is likely that generation o f the [3- glucuronidase fusion proteins by molecular biology may diminish preferential uptake o f the conjugate by the liver. This could explain our results and those reported by B o s s l e t ^ , w here a fusion protein between Fab and [3G was produced that had no preferential uptake by the liver. It would be interesting to see whether these finding will hold for single chain-PG fusion proteins described by Haisma--’ ’- which have only been characterized in vitro. The biodistribution studies presented and discussed in this chapter are promising since the observed tumor uptake was not that different from the values obtained for ju st antibody fragments. At the 24 hr time point, the tumor uptake for the 193 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm ission. antibody fragment/(3G fusion proteins varied from 2.3% ID/g for scFv'PG . to 2.5% ID/g for Fab/PG. and to 3% ID/g for F(ab'):/pG . Considering the size o f fusion proteins, these values are rem arkable. Apart from the influence o f the increased affinity as discussed earlier, the unique attributes o f the T N T -tvpe antibodies may allow for good tumor accumulation o f even large particles. Since the fusion proteins have to get into the necrosis present in the middle of the tum or before its binds to the antigen, it m ay be aided by the presence o f leaky vasculature often associated with necrotic centers of the tumor. A dditionally, in studies with antibodies directed towards surface antigens, it has been show n that they bind first to the antigens located in the vicinity o f tum or vessels and this im pedes further antigen p e n e t r a t i o n ^ - This result m ay be further exacerbated in case o f large molecules which penetrate poorly at the outset. In contrast, TNT antibodies have to pass through viable zones to encounter their antigen in necrotic regions. The question remains w hether this level o f tum or uptake is adequate to allow sufficient conversion o f the prodrug at the tum or site. As discussed in details in Chapter 4. several studies asking this question were done in gene therapy experiments where the fraction o f gene expression in cells is the crucial factor. It seem s that as little as 1-2% o f cells with the enzyme expression is sufficient for hindering tum or g ro w th ^ 60 w hile 5% o f transduced cells often suffices to eradicate the tum or 6! 194 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth er reproduction prohibited w ithout perm issio n . For these and other reasons, our treatm ent therapy studies show encouraging results. Shortage o f the prodrug in this prelim inary trial forced abbrev iated design of the whole therapy regim en. Nevertheless, despite the animal groups being too small in size to be statistically significant, and despite only two injections o f the prodrug, positive results w ere obtained showing decreased tum or growth. Control groups where only the prodrug or the fusion protein were adm inistered produced no such outcome. Especially in the period o f time between days 11 and 13. a slight but visible decrease in tum or size in all fusion/prodrug groups was noticeable. This trend is also present but to a lesser degree following the second administration o f the fusion protein (around day 17). It could be postulated that more frequent adm inistrations o f the fusion protein followed by injections o f the prodrug would produce better results. Ev en with only two adm inistration o f fusion protein followed by the adm inistration o f the prodrug, the tum or growth was tem porarily arrested and the growth curves for the fusion - prodrug groups ran parallel to that o f the doxorubicin-treated group. Additionally, a difference in survival time was noted. As reported by others ~ - ’,s untreated mice carrying M AD 109 tum ors usually died within 35 days as is common for MAD I 09 tumor-bearing animals. Indeed, there were no survivors in the control groups. In contrast, in groups receiving the combination o f the fusion protein and prodrug. 60°.. o f mice were still alive after 35 days in the group receiving the higher dose of the prodrug and 40% were alive in a group receiving the lower dose. The survival rate for 195 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth e r rep roduction prohibited w ithout perm ission. the group treated w ith doxorubicin was 60%. The group receiving or.iy the prodrug had a survival rate o f 20% . Last result may have been due to the presence o f endogenous 1 3 - glucuronidase w ithin tum ors, which activated the prodrug as has been reported by other investigators 2 L 22 Comparing all the constructs, it seems that the F(ab' ) 2 is the best fragment to use in combination with the enzym e. Its fusion with [3G forms mostly tetramers. thus assuring peak enzym e activity. It displays the best stability upon prolonged storage and in serum (data not shown). In biodistribution studies it localizes to the liver to a lesser extent than other fragment constructs and to tum ors more than others. O f course, the large final size o f the active antibody/[3G fusion molecule poses the question w hether it is not better to use just the whole antibody in its targeting part. Using the intact antibody would result in only a small change o f molecular weight o f the resulting fusion protein and most likely would provide much improved tum or uptake since com pared to all generated antibody fragments, the whole antibody’s tum or uptake is still about 5-fold better. However, there may be the reasons to use antibody fragments rather than intact antibody. Our main rationale is that the clearance tim e o f the fusion proteins is still significantly shorter than that o f the whole antibody despite their size being nearly a quadruple o f the antibody. The scFv. Fab. and F(ab’): fusion proteins cleared from Balb/C mice with the half-life o f 8-9 hours. Despite the 196 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . fast clearance, biodistribution studies showed the fusion proteins to be capable o f localizing to the tumor. At the same time, due to their short half-life, the tum or organ ratios are high within first 24 hours and even better at 48 hr. In the context o f A D EPT therapy, this m eans that the prodrug can be administered shortly after the injection o f the fusion as opposed to the usual 5 to 7 day wait as described in some protocols- 34. 45 Shorter time intervals mean that m ore courses o f the therapy can he given. This presents an additional bonus for TN T-based therapy since each round o f treatm ent will generate more dead cells and therefore more targets for the TNT antibody. Using the whole antibody for the carrier part o f the fusion protein would most likely result in an increased clearance time. This may not pose a significant problem for the TN T antibody which remains inert in the tumor. Additionally, the results discussed earlier showed that the enzym e part o f the fusion protein preserves its activity well. However, the prolonged half-life o f the fusion protein would mean a longer interval between adm inistration of the construct and the prodrug, as discussed earlier. M eanwhile the tum or would continue to grow. W hether this could be offset by better tum or targeting o f the fusion protein and therefore improved impact later due to increased prodrug activation, can be determined only by experimental procedures. Is it better to deliver lower but more frequent doses (as it would be the ease when using antibody fragments) or larger doses but sparsely (as is the case when using intact 1 9 7 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . antibody)? Interestingly enough in case o f radiotherapy and chem otherapy, this question has m ostly been answered in favor o f the first alternative I • -- 62-64. In sum m ary, the use o f the monoclonal antibodies imprints high selectivity in delivering exogenous enzymes to tumors and forms the base for the A D EPT concept. H ow ever, it is still impossible to ensure the delivery to all tum or cells, since not all o f them express targeting antigen. These cells also may not be accessible to large antibody-enzym es conjugates with limited diffusion. Finally, the bound antibodies may not be protected from internalization or shedding. Still, these drawback can be substantially overcom e by the catalytic capability o f the enzyme, the proper design o f prodrug, and the use o f a particular type o f the antibody. Tum or Necrosis Treatm ent (TN T) antibody seems to be just such a candidate. Binding to antigens present in necrotic areas o f tum ors, it can target m any different types o f malignancies. Once localized to the tum or, it is present there for a protracted period o f time. As suggested by our data, its properties may allow it to guide particularly large molecules to the tum or and ensure their localization in sufficient levels. All this suggests that the com bination o f the TN T antibody and the A D EPT therapy can result in superior perform ance which may revitalize A D EPT concept. Further exploration, especially- including in vivo animal treatment studies, is needed to fully answ er this question. 1 9 8 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth er reproduction prohibited w ithout perm issio n . REFERENCES 1. Haskell CM . Principles o f cancer chem otherapy. In: Cancer Treatm ent. C'.M. Haskell (Ed.). Saunders: Philadelphia 1985:21. 2. Young RC. The clinical problems. In: Drug Resistance in Cancer Therapy. R.F. Ozols (Ed.), K luw er Academic Publishers: Boston 1989:1. 3. Calabresi P. W elch. A.D... Cytotoxic drugs, hormones and radioactiv e isotopes. In: The pharmacological basis o f therapeutics. Goodman L.S.. Gilman A., (eds) 1994: M acmillan. New York: 1345. 4. Hudson P. Recombinant antibody constructs in cancer therapu. Current O pinion in Im m unology 1999; 11:548-557. 5. Bagshawe K.D. Antibody directed enzym es revive anti-cancer prodm g concept. British Journal o f Cancer 1987; 56:531. 6. Sharma SK. Bagshawe KD, . . M elton RG. Sherwood RF. Human immune response to monoclonal antibody-enzym e conjugates in ADEPT pilot clinical trial. Cell Biophysiology 1992: 21:109-120. 7. Delgado C. Francis G.E.. Fisher D. The uses and properties o f PEG-linked. also the intravenous administration of PEG-linked proteins. Crit Rev Ther Drug Carrier Syst 1992; 9:249-304. 8. Sharma SK. Immune response in ADEPT. Advances in Drug Delivery Review 1996: 22:369 9. Shipley JM . Grubb J.H.. Sly. W.S. 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Improved characteristics o f a human (3- glucuronidase-antibody cinjugate after deglycosylation for use in A ntibody-D irected Enzyme Prodrug Therapy. Bioconjugate Chem istry 1996; 7:606-61 I. 31. G laser JH. Roozen KJ, Brot FE. Sly S.W. M ultiple isoelectric and recognition forms o f hum an (3-glucuronidase activity. Archives o f Biochem istry and B iophysics 1975:536-542. 32. Frankel HA. G laser JH. Sly WS. Human b-glucuronidase. I. Recognition and uptake by animal fibroblasts suggests animal models for enzym e replacement studies. Pediatric Research 1977; 11:811-822. 33. B osslet K. Czech J, Hoffmann D. Tum or-selective prodrug activation by fusion protein-m ediated catalysis. Cancer Research 1994: 54:2152-2159. 34. Chen B-M . Chan L-Y. Wang S-M. Wu M-F. Chem J-W . Roffler SR. Cure o f malignant ascites and generation o f protective immunity by monoclonal antibody- targeted activation o f glucuronide prodrug in rats. International Journal o f Cancer 1 997: 73:392-402. 35. G ehrm ann MC. O pper M. Sedlacek HH. Bosslet K. Czech .1 . Journal o f Biochem istry 1994; 301: 821-828. 36. Islam MR, Tom atsu S. Shab GN. Grubb JH. Jains S.. Sly WS. Active site residues o f human P-glucuronidase. The Journal o f Biological Chem istry 1999; 274:23451-23455. 37. Korzeniewski C, Callewaert DM. An enzvm e-release assay for natural cytotoxicity. Journal o f Immunological M ethods 1983: 64:313-320. 38. M arks TA. W oodman RJ. Geran RI. Billups LIT. M adison R.V1. Characterization and responsiveness o f the M adison 109 lung carcinoma to various antitum or agents. Cancer Treatm ent Reports 1977; 61:1459-1470. 39. A chord DT. Brot FE. Bell CE, Sly WS. Human P-glucuronidase: In vivo clearance and in vitro uptake by a glycoprotein recognition system on reticuloendothelial cells. Cell 1978; 15:269-278. 40. Lagunoff D. Nicol DM. Pritzl P. Uptake o f P-glucuronidase by deficient human fibroblasts. Laboratory Investigations 1973: 29:449. 2 0 2 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm ission. 41. Lunney J, Ashwell G. A hepatic receptor o f avian origin binding specifically modified glycoproteins. PNAS 1976: 73:341. 42. Schlesinger PH. Rodman JS. Doebber TW . et al. The role o f extra-hepatic tissues in the receptor mediated plasma clearance o f glycoproteins terminated by mannose or N-acetylglucosamine. Biochemistry Journal 1980: 192:597-606. 43. Hickman S. Sapiro LJ. Naufeld EF. A recognition is required for uptake o f a lysosomal enzyme by cultured fibroblasts. Biochem. Biophvs. Research Commun. 1974; 57: 55. 44. Homick JL. Sharifi. J.. Khawli. L.A.. Biela. B.H.. Yun. A.. Taylor. C.R.. Epstein. A.L... A new chemically modified chimeric monoclonal antibody directed against DNA for the im m unotherapy o f solid tumors. Cancer Biotherapv & Biopharmacology 1998; 13:255. 45. Cheng T-L. Chen. B.-M .. Chan. L.-Y.. Wu. P.-Y.. Chern. J.-W .. Roffler. S.R. Poly(ethylene glycol) modification o f p-glucuronidase-antibody conjugates for solid- tum or therapy by targeted activation o f glucuronide prodrugs. Cancer Immunology and Im m unotherapy 1997; 44:305. 46. Stribbling SM. M artin J, Pedley RB. Boden JA. Sharma SK. Springer C'J. Biodistribution o f an antibody-enzym e conjugate for antibody-directed enzym e prodrug therapy in nude mice bearing a human colon adenocarcinoma xenograft. Cancer Chem otherapy and Pharmacology 1997: 40: 277-284. 47 Ecclcs SA. Court. W.J.. Box. G.A. Regression o f the established breast carconom a xenografts with antibody-directed enzyme prodaig therapy against c-erbB2 p 185. Cancer Research 1994; 54:5171. 48. Sharma SK. Bagshawe KD, Burke PJ. Boden RW. Rogers GT. Inactivation and clearance o f an anti-CEA carboxypeptidase G2 conjugate in blood after localization in a xenograft model. British Journal o f Cancer 1990: 61:659-662. 49. Kievit E. Bershad E. Ng E. et al. Superiority o f yeast over bacterial cytosine deaminase for enzym e/prodrug gene therapy in colon cancer xenografts. Cancer Research 1999; 59:1417-1421. 50. K ohler G, Milstein C. Continuous cultures o f fused cells secreting antibody o f predefined specificity. Nature 1975: 256:495-497. 203 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . 51. Sensel M G, Coloma J, Harvill ET. Shin S-U. Smith RIF. Morrison SL. Engineering novel antibody molecules. Chemical Immunology 1997: 65:129-158. 52. W aldm annT A . Monoclonal antibodies in diagnosis and therapy. Science 1991: 252:1657-1662. 53. von M ehren M, Weiner LM. M onoclonal antibody-based therapy. Current O pinion in Oncology 1996: 8:493-498. 54. Owens RJ. Young RJ. The genetic engineering o f monoclonal antibodies. Journal o f Immunological Methods 1994: 168:149-165. 55. Viti F. Tarli L. Giovannoni L. Zardi L. Neri D. Increased binding affinity and valence o f recombinant antibody fragments lead to im proved targeting o f tum or angiogenesis. Cancer Research 1999; 59:347-352. 56. Adams GP. Schier R. Marshall K. W olf EJ. McCall AM. Marks JD. Increased affinity leads to improved selective tum or delivery o f single-chain Fv antibodies. Cancer Research 1998; 58:485-490. 57. Buist MR. Kenemans P. den Hollander W. et al. Kinetics and tissue distribution o f the radiolabeled chimeric monoclonal antibody M O vlS IgG and F(ab’), fragments in ovarian carcinoma patients. Cancer Research 1993: 53:5413-541 S. 58. Reilly RM. Sandhu J.. Alvarez-Diez. T.M .. Gallinger. S.. Kirsh. J.. Stem. II... Problems o f delivery o f monoclonal antibodies. Pharmaceutical and pharmacokinetic solutions. Clinical Pharmacokinetics 1995: 28:126. 59. Trinh QT. Austin EA. Murray DM. Knick VC. Huber BE. Enzyme Prodrug Gene Therapy: comparison of Cytosine Deam inase/5-fluorocytosine versus Thym idine Kinase/gangciclovir enzyme/prodrug system s in human colorectal carcinoma cell line. Cancer Research 1995; 55:4808-4812. 60. Perron P-J. Page, M... Activation o f methotrexatc-phenylalanine by monoclonal antibody-carboxypeptidase A conjugate for the specific treatment o f ovarian cancer in vitro. British Journal o f Cancer 1996: 73: 281-287. 61. Kuriyama S. Kikukawa M. Masui K. et al. Cytosine deaminase 5- fluorocytosine gene therapy can induce efficient anti-tum or effects and protective imm unity in immunocom petent mice but not in athvmic mice. International Journal o f Cancer 1999; 81:592-597. 204 R e p ro d u c e d with p e rm ission of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm ission. 62. Bruland OS. Cancer therapy with radiolabeled antibodies. An overv iew. Acta Oncologica 1995;34:1085-1094. 63. Kairemo KJ. Radioim m unotherapy o f solid cancers: a review. Acta Oncologica 1996: 35:343-355. 64. Verhaar-Langereis MJ, Zonnenberg BA. de Klerk JMH. Blijham G1I. Radioimmunodiagnosis and therapy. Cancer Treatm ent Reviews 2000; 26:3-10. R e p ro d u c e d with p e rm issio n of the copyright ow ner. F u rth e r reprod u ctio n prohibited w ithout perm issio n . CHAPTER 6: CONCLUSIONS T argeted therapy, in w hich a therapeutic agent localizes selectively in the tum or thus m inim izing toxic effects on other normal tissues, is an attractive treatment prospect. O f course the selectivity o f this targeting approach is w hat m akes it so attractive but also so difficult to achieve. Despite the constant search, it seem s that the differences betw een norm al and m alignant cells are scant. It should not com e as a surprise since cancerous cells are after all derived from norm al cells, thus any differences, if present at all. are most often o f the quantitative than qualitative nature. This also explains w hy m onoclonal antibodies - proteins with properties enabling them to recognize in a selective m anner particular m arkers - have not becom e a "m agic bullet", as was hoped when they were first described. O ur laboratory believes that the approach developed here for targeting tumors offers m uch prom ise. This novel approach called "Tum or N ecrosis Therapy" (TNT) takes advantage o f the pathological change often associated with neoplastic disease, nam ely the developm ent o f necrosis. Monoclonal antibodies based upon this principle, and developed in our laboratory, target abundant intracellular antigens which are norm ally inaccessible in living cells due to the cellular membrane barrier. When this barrier breaks down as happens in dying and dead cells, the TNT antibodies can bind to its target. In healthy tissues w hen a cell dies, scavenging m acrophages quickly rem ove its rem nants. M alignant tum ors seem to lack this m echanism and dead cells 206 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . keep on accum ulating until large areas o f necrosis are formed. So while necrosis is not a tum or specific phenom enon, it is strongly associated with cancer, m aking TN T antibodies excellent targeting reagents for the m ajority o f solid tumors. A dditionally, once TNT-Iike antibodies reach their target, they remain for prolonged periods o f time and are not subjected to shedding or antigenic downregulation. Once TN T antibodies are coupled with toxic agents, the action o f these conjugates creates m ore dead cells, providing more target for the next round o f treatment. This represents an interesting exam ple o f the amplification system not found with any other antibody's action. Being in a possession o f a very promising antibody for tumor targeting, our laboratory set on course to improve and identify novel ideas o f cancer detection and therapy. Like m ost monoclonal antibodies, TNT monoclonal antibodies w ere originally- developed in mice. It has been shown that the use o f m urine antibodies in hum ans quickly leads to the developm ent o f an im m une response, thus lim iting or even precluding further therapy. To avoid this problem , m olecular biology m ethods w ere em ployed to generate a chimeric TN T antibody, where only parts essential to antigen binding are still murine in origin, while the rest o f the molecule is hum an-derived. Because o f their large m olecular w eight, m onoclonal antibodies show a relatively slow blood clearance. As demonstrated by biodistribution studies, antibodies radiolabeled for tum or targeting give rise to unfavorable tum or organ distribution ratios and radiotoxicity to norm al tissues. It is this radiotoxicity w hich lim its the am ount o f the antibody which can be delivered safely. Their large m olecular weight is also partially responsible for the poor tum or penetration o f antibodies, where only 207 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . 0.001-0.01% o f an injected dose localizes in a gram o f tum or in patients. V arious attem pts have been made to overcom e these deficiencies. These include increasing the dose o f the antibody, increasing vascular perm eability at the tumor site. e.g. with the pretreatm ent with vasoactive conjugates, and also increasing the duration o f antibody retention in a tum or for exam ple by increasing its affinity or upregulating the am ount o f the antigen present within the tum or by using e.g. hyperthermia, gene transfer or cytokines. Finally, efforts have also been directed towards the developm ent o f sm aller antibody fragm ents which show better tum or penetration and improved target/non target ratios. Chapters 2 and 3 sum m arize our laboratory's efforts to develop an im proved im aging agent using fragm ent derivatives o f chTNT-3 antibody. For these studies, a panel o f sm aller antibody fragments was engineered, produced, and evaluated. Single chain-based fragments (single-chain, diabody. and triabody) did not live up to the expectations but offered som e valuable insights into tum or imaging, m ainly on the im portance o f the affinity for the antigen for achieving good results. Still som e data (im ages obtained with Technetium -labeled single chain) are encouraging enough to w arrant further investigations, aim ed at looking at additional tum or m odels and labeling agents. The F(ab' ) 2 fragm ent proved to be the most promising am ong all the fra. nents produced and further studies will be perform ed to determ ine the proper dot ge and timing for imaging. Also, the effect o f various pretreatments (for exam ple with vasoactive conjugates) will be investigated. W orth noting is the fact that this F(ab' ) 2 m olecule represents the first effort to generate a fully active, stable bivalent 208 R e p ro d u c e d with perm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm issio n . antibody fragm ent in m am m alian expression system . T his attem pt, described in Chapter 2. was totally successful and represents a viable alternative to enzym atic digestion or bacterial expression previously used to obtain F (ab'); fragments. As for the m olecule itself, it is hoped that at the end it will enter clinical trials and finally become available to clinicians as a good and efficient im aging agent applicable to a variety o f tumors. M ore than tw enty years after first introducing the concept o f m onoclonal antibodies, a few m onoclonal antibodies have made their w ay into the clinic m ostly in the field o f oncology. M Abs are now a part o f everyday diagnosis and treatm ent. Other possible applications o f m onoclonal antibodies are still very much subjects o f intensive investigation and research. One such concept is A ntibody-D irected Enzym e Prodrug Therapy (A D EPT). Since the selective delivery o f toxic agents exclusively to the tum or site had proven to be elusive, attempts have been m ade to generate an active drug from an inactive precursor. The concept o f the prodrug is central to this idea: it is a m olecule not active itself, but easily converted to a cytotoxic agent by enzym atic activation in vivo. Ideally, the activation of the prodrug should be restricted to the site of the tumor. Up to now. few prodrugs have been described which can he activated by tumor cells them selves. This led to the concept o f using m onoclonal antibodies as carriers o f the enzym es to the tum or sites called AD EPT. Here antibody-enzvm e conjugates are delivered to the tum or and allow ed to clear from norm al tissues. Subsequently, prodrug is adm inistered and converted by the enzym e into its active cytotoxic form. The interval betw een these two steps is optim ized to achieve m inim al 209 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth er reproduction prohibited w ithout perm issio n . system ic toxicity by accom plishing satisfactory clearance o f the conjugate from the blood and normal tissues. In addition, as active drug diffuses throughout the tumor, it provides a bystander effect, killing cells which did not bind the conjugate initially. The optim al antigen for ADEPT has to be relatively abundant, easily accessible, and it should not be down-regulated or shed. O ur laboratory noted that these particular characteristics desirable for ADEPT are exactly the sam e displayed by TN T antibodies. For this reason, we believe that TN T -based A DEPT therapy m ay prove superior to other constructs employed in this tum or treatm ent regim ent. C hapters 4 and 5 present the genetic construction, the expression, and purification o f several fusion products betw een chTNT-3 antibody fragm ents and two different enzym es: bacterial C ytosine D eam inase (CD2) and hum an (3-glucuronidase. C onstructs based on both enzym es showed prom ise in prelim inary in vitro characterization studies. These fusion products between antibody fragm ents and enzymes have preserved antigen binding abilities and still display the activity o f their enzymatic com ponents. During studies o f the cytotoxic effects o f these fusion m olecules upon tum or cells, they have shown them selves able to convert inactive prodrug into fully active drug (5-fluorocytosine into 5-fluorouraciI in case o f cytosine deam inase and doxorubicin glucoronide into doxorubicin in case o f (3- glucuronidase), both capable o f significant tum or killing. Prelim inary studies o f their pharm acokinetic behavior and tum or targeting show ed that these enzym e fusion m olecules cleared rapidly from the circulation, thus enabling shorter periods between adm inistration o f the enzyme fusion protein and prodrug than has been done before. 210 R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth e r reproduction prohibited w ithout perm ission. but at the same tim e w ere able to be efficiently localized into the center o f tumors. Additional studies investigating the most appropriate dosage o f a prodrug and time intervals between adm inistration o f the fusion proteins and the prodrug arc needed to achieve not only slow ing down o f tum or growth but perhaps its complete eradication. Here, the pretreatm ent w ith IL-2 fusion as described for antibody fragm ent imaging, could also proved valuable in increasing the payload o f the MAb enzym e fusion localizing into the tumor. It is hoped that the w ork presented in this dissertation will enlarge on the already num erous applications o f TNT antibodies. Each o f the chapters have led to interesting and even exciting conclusions which dem and m ore experim ents and follow-up. R e p ro d u c e d with p erm issio n of th e copyright ow ner. F u rth er reproduction prohibited w ithout perm issio n . 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Creator Biela, Barbara Helena (author)

Core Title Imaging and prodrug -activating derivatives of chTNT-3 (tumor necrosis therapy) monoclonal antibody

Contributor Digitized by ProQuest (provenance)

School Graduate School

Degree Doctor of Philosophy

Degree Program Pathobiology

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag health sciences, immunology,health sciences, oncology,health sciences, pathology,OAI-PMH Harvest

Language English

Advisor Epstein, Alan L. (committee chair), Hofman, Florence (committee member), Khawli, Leslie A. (committee member), Tokes, Zoltan A. (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c16-118342

Unique identifier UC11338119

Identifier 3041436.pdf (filename),usctheses-c16-118342 (legacy record id)

Legacy Identifier 3041436.pdf

Dmrecord 118342

Document Type Dissertation

Rights Biela, Barbara Helena

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

Repository Name University of Southern California Digital Library

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