Rational design of monoclonal antibody inhibitors targeting human cathepsin B

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Content
Rational Design of Monoclonal Antibody Inhibitors
Targeting Human Cathepsin B
by
Zhefu (Jeff) Dai

This thesis is presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(PHARMACEUTICAL SCIENCES)

August 2016

Copy right 2016 Zhefu (Jeff). Dai
2

Table of Contents

Acknowledgements 3
List of Figures 4
Abbreviations 5
Abstract 7
Introduction 8
Methods 14
Results 21
Discussion 32
References 35

3

Acknowledgements
I hereby would like to express my gratitude for Dr. Yong (Tiger) Zhang for his being a
great mentor giving insightful advices from time to time.

Also, I would like to thank my committee members: Dr. Ian Haworth and Dr. Curtis
Okamoto for their valuable support on my thesis project.

Finally, I appreciate the kind help from all members of Dr. Zhang’s laboratory: Jingwen
(Julianna) Chen, Menglu Han, Albert Lam, Yanran Lu, Xiaojing Shi and Dr. Xiao-Nan
Zhang

4

List of Figures
Figure 1: The x-ray crystal structure of active human CTSB 11
Figure 2: The x-ray crystal structure of Cystatin A in complex with CTSB 17
Figure 3: The design of Cystatin A-Herceptin fusion antibody Fab V1 24
Figure 4: SDS-PAGE gel of the purified Cystatin A-Herceptin fusion Fab V1 25
Figure 5: The fusion Fab V1inhibited CTSB activity in a dose-dependent manner 26
Figure 6: Non-linear regression fitting of inhibition curves of fusion Fab V1---NO.1 27
Figure 7: Non-linear regression fitting of inhibition curves of fusion Fab V1---NO.2 28
Figure 8: Non-linear regression fitting of inhibition curves of fusion Fab V1---NO.3 29
Figure 9: SDS-PAGE gel of the purified Cystatin A-Herceptin fusion Fab V2 30
Figure 10: Inhibition assay of the fusion Fab V2 against CTSB 31

5

Abbreviations
Arg: Arginine
Asn: Asparagine
CDRs: Complementarity Determining Regions
CTS: Cathepsins
CTSB: Cathepsin B
CTSD: Cathepsin D
CTSK: Cathepsin K
CTSS: Cathepsin S
Cys: Cysteine
DTT: 1,4-Dithiothreitol
ECM: Extracellular Matrix
E.coli: Escherichia coli
Fab: Fragment antigen-binding
HER-2: human epidermal growth factor receptor-2
His: Histidine
[I]: Inhibitor concentration
𝐾 𝑖 : Inhibition constant
𝐾 𝑚 : Michaelis constant
MHC: Major histocompatibility complex
6

PBS: Phosphate buffered saline
Phe: Phenylalanine
[S]: Substrate concentration
Thr: Threonine
𝑉 0
: Initial reaction velocity in the absence of inhibitor
𝑉 0
′
: Initial reaction velocity in the presence of inhibitor

7

Abstract
Cathepsin B (CTSB) is one of the earliest identified cysteine proteases, which is highly
abundant in lysosomes. Mounting evidence showed that CTSB is secreted by cells into
extracellular regions for various physiological functions such as bone remodeling.
Moreover, overexpressed CTSB and its accumulation in extracellular regions are closely
related to the deterioration of joint tissue damage in rheumatoid arthritis and the
aggressiveness of invasive solid tumors in cancers. Thus, inhibitors targeting CTSB can
provide innovative therapeutic approaches. Currently, selective CTSB inhibitors have yet
been available for clinical applications. Small-molecule CTSB inhibitors are often
plagued by severe toxicity due to significant off-target effects. In addition,
peptide-based inhibitors are likely to suffer from short serum half-lives. In this study,
through rational design, two human monoclonal antibody-based CTSB inhibitors were
generated and examined for inhibition activity towards CTSB. One of the resulting
antibodies displayed potent inhibition activity at nanomolar levels. Further engineering
study will be performed to generate antibody-based therapeutics with prolonged plasma
half-lives and improved safety profiles.

8

Introduction:
Cathepsins (CTS) are a family of proteases in animals involved in various physiological
activities as well as disease conditions. Among 15 identified CTS proteases, 11 CTS are
cysteine proteases that utilize thiol groups of cysteine side chains at active sites as
nucleophiles to catalyze the cleavage of peptide bonds (Mitchel et al., 1970; Rossi et al.,
2004). It was proposed that those 11 cysteine CTS (also commonly known as papain-like
cysteine proteases) were all originated from a single ancestor gene through translocation
and duplication during evolution (Turk et al., 2000). Discovered in the early 20
th
century,
cysteine protease-based CTS are characterized by their high stability and active
proteolytic activity under reducing conditions and slightly acidic pH (pH 5.0 to 6.5)
(Willstätter and Bamann, 1929). Since cysteine CTS were reported to be unstable under
physiological pH (pH 7.4), it was once believed that cysteine CTS were merely
lysosomal proteases inside living cells, and perhaps, as executioner proteases during
apoptosis (Roberts et al., 1997). However, the discovery of new tissue specific cysteine
CTS in 1990s, such as Cathepsin K (CTSK) expressed nearly exclusively in osteoclasts,
proved the “lysosomal scavengers” theory too narrow in its scope (Drake et al., 1996).
Specifically, cysteine protease-based CTS were found to be transported outside
lysosomes to convert prorenin to renin (Wang et al., 1991). Moreover, Cathepsin B
(CTSB) and Cathepsin S (CTSS) were known to be essential for the processing of major
histocompatibility complex (MHC) class II for antigen presentation (Chapman et al.,
9

1997; Riese et al., 1998).

In addition to their intracellular functions outside of lysosomes, CTS were found to be
secreted into extracellular regions to degrade extracellular matrix (ECM) under various
physiological and pathological conditions. It was known for decades that secreted
Cathepsin D (CTSD) participated in the remodeling of cartilage (Poole et al., 1974).
Moreover, secreted CTSK was identified as the major player for degrading ECM in bone
reabsorption (Asagiri and Takayanagi, 2007). Under pathological conditions such as
rheumatoid arthritis, patients typically have excess amount of Cathepsin B, L and S
(CTSB, CTSL and CTSB) in their synovial fluid relative to healthy people (Schurigt,
2013). Further substrate specificity studies on CTS revealed that elastins, collagens and
proteoglycans in ECM are all recognized as favorable substrates of CTS (Lutgens et al.,
2007). Thus, inhibition of CTS-mediated ECM remodeling associated with rheumatoid
arthritis, atherosclerosis, and tumor metastasis is likely to lead to novel approaches to
cure those diseases (Heidtmann et al., 1992; Lutgens et al., 2007).

10

Human Cathepsin B (CTSB):
Human CTSB is a 254-amino acid cysteine protease with both endopeptidase and
peptidyl-dipeptidase activities (Chan et al., 1986; Erdos et al., 1977). Similar to other
members in the CTS family, CTSB preferentially cleaves the peptide bonds right after
two consecutive arginine (Arg) residues (Kirschke et al., 1976). As one of the first
discovered members of CTS, CTSB was once considered a promiscuous protease only
involved in the protein recycling because of its abundance in lysosomes (Barrett and
Kirschke, 1981). However, as reviewed by Bromme and Wilson (2011), CTSB was found
existed in the extracellular regions as well and was capable of degrading a wide variety of
proteins in extracellular matrix (ECM) (Brömme and Wilson, 2011). Furthermore, both
the expression levels and proteolytic activities of extracellular CTSB were demonstrated
to be correlated with the invasiveness of solid tumors (Brömme and Wilson, 2011;
Emmert-Buck et al., 1994; Yan et al., 1998). As being shown in previous studies, CTSB
was overexpressed in colon cancer cells and subsequently led to increased ECM
degradation at tumor sites (Cavallo-Medved et al., 2009; GUZINŃSKA-USTYMOWICZ
et al., 2004). In addition, tumor tissue imaging analysis indicated abnormally high levels
of active CTSB (bound to annexin II on tumor cell membrane) on the boundary of
invasive tumors (Emmert-Buck et al., 1994; Mai et al., 2000). Beside colon cancer, high
levels of CTSB activity were also shown to contribute to poor prognosis of patients with
breast cancer and lung cancer (Bröker et al., 2004; Thorpe et al., 1989).

11

Figure 1. The x-ray crystal structure of active human CTSB. The cysteine (Cys 29) and
histidine (His 199) at the catalytic site of CTSB were marked in stick mode in yellow and
blue, respectively. The flexible loop in purple is the occluding loop that partially restricts
the substrate access to the catalytic site (Musil et al., 1991).

12

Aims of the thesis project:
At present, except the remarkable success of utilizing CTSK inhibitors (such as
Odanacatib) in treating arthritis, nearly all commercially available CTS inhibitors are
hindered from clinical applications due to low selectivity, undesired inhibition of
intracellular CTS, and short plasma half-lives (Lampe and Gondi, 2014; Schenker et al.,
2008; Stoch et al., 2009). The two most promising CTSB specific inhibitors with
potential for clinical applications are CA074 and ZRLR, which are both derived from
peptides with limited cell membrane permeability due to their negative charges under
physiological conditions (Buttle et al., 1992; Wieczerzak et al., 2007). Unfortunately, due
to short serum half-lives of CA074 and ZRLR, it is unlikely for them to achieve
prolonged inhibition of CTSB in synovial fluid of joints or at metastatic tumor sites
(Withana et al., 2012).

Hereby, we aimed to develop humanized monoclonal antibody-based CTSB inhibitors
with improved potency, selectivity, and extended plasma half-lives. The generated highly
selective antibody-based inhibitors could be utilized in treating rheumatoid arthritis and
invasive solid tumors. Comparing to conventional small-molecule CTSB inhibitors,
antibody-based inhibitors are unlikely to readily cross cell membranes and cause
undesired inhibition of intracellular CTSB. Moreover, our antibody-based inhibitors are
expected to have markedly increased serum half-lives, because human antibodies are
13

more resistant to renal filtration and proteolysis (Haraldsson et al., 2008).
14

Methods
Rationale:
The 101-amino acid Cystatin A is a potent, human-derived broad spectrum inhibitor to
cysteine proteases, including CTSB (Lenarčič et al., 1996). The key structural elements
for Cystatin A to inhibit cysteine proteases include its free N-terminal residues and two
flexible loops on the top of two β-strands, as shown in Figure 2 (Bode et al., 1988).

According to the x-ray crystal structure of Cystatin A, three flexible inhibitory loops of
Cystatin A are arranged in conformation similar to that of Complementary Determination
Regions (CDRs) of human immunoglobulins. To design antibodies targeting CTSB,
trastuzumab (commercially known as Herceptin), a humanized anti-HER2 receptor
monoclonal antibody, was chosen as the major immunoglobulin scaffold for inhibitor
designs (Cho et al., 2003). It was hypothesized that genetically grafting the inhibitory
loops from Cystatin A into the CDRs region of humanized antibodies would generate
potent anti-CTSB inhibitors with improved specificity and pharmacological activities
(Amit et al., 1986). Furthermore, the affinity and selectivity of the resulting fusion
antibodies can be improved through modifying residues in the grafted loops from
Cystatin A.

15

The design for Cystatin A-Herceptin Fusion Fab V1:
The N-terminal segment of Cystatin A, NH
2
-IPGGL-COOH, was reversed into
NH
2
-LGGPI- COOH (DNA sequence: 5
′
-GCTGAATCTCTGGGAGGTCCGATT-3
′
)
and grafted into light chain CDR 3 of Herceptin between Histidine 91 (His91) and
Threonine 93 (Thr93). The loop NH
2
-VVAGT-COOH (DNA sequence:
5-GTCGTAGCGGGTACT-3
′
) of Cystatin A was inserted into the light chain of CDR1 of
Herceptin between Asparagine 30 (Asn30) and Threonine 31 (Thr31). The major
inhibition loop region of Cystatin A: NH
2
-KSLPGQNEDL-COOH was extended by
adding flexible GGS linkers at both N- and C- termini followed by insertion of the loop
NH
2
-GGSKSLPGQNEDLGGS-COOH (DNA sequence:
5
′
-GGGGGCTCTAAAAGCCTCCCTGGGCAGAACGAAGATCTGAGCGGGGGT-3
′
)
into light chain CDR 2 of Herceptin between Tyrosine49 (Tyr49) and Phenylalanine 53
(Phe53).

16

The design for Cystatin A-Herceptin Fusion Fab V2:
Based on the inhibition result of the V1 Fab, the new antibody inhibitor was designed to
further extend the grafted CDR loops from Cystatin A for improved potency. This
strategy is expected to minimize unfavorable interactions with CTSB contributed by
residues from other CDR loops on Herceptin. To this end, three more amino acid residues
from Cystatin A were appended onto each terminus of the original inserts in V1 Fab,
except for the insert NH
2
-LGGPI-COOH of V1 Fab, in which only three more amino
acid residues were added to the N-terminus. Specifically, the NH
2
-IPGGLSEA-COOH of
Cystatin A was reversed into NH
2
-LGGPIAES-COOH (DNA sequence:
5-GCTGAATCTCTGGGAGGTCCGATT-3) and grafted into light Chain CDR3 of
Herceptin between Histidine 91 (His91) and Threonine 93 (Thr93).
NH
2
-KTQVVAGTNYY-COOH (DNA
sequence:5
′
-AAAACCCAGGTCGTAGCGGGTACTAACTACTAC-3
′
)
of Cystatin A was inserted into light chain CDR 1 of Herceptin between Asparagine 30
(Asn30) and Threonine 31 (Thr31). The major inhibition loop
NH
2
-KVFKSLPGQNEDLVLT-COOH extended by flexible GGS linkers was inserted
into light chain CDR2 of Herceptin between Tyrosine49 (Tyr49) and Phenylalanine 53
(Phe53)

17

Figure 2. The x-ray crystal structure of Cystatin A in complex with CTSB. Three major
flexible loops from Cystatin A at the interface of Cystatin A and CTSB are essential for
the inhibition (Renko et al., 2010).

18

Molecular cloning of the expression construct
In order to efficiently express fusion antibodies in Escherichia coli (E.coli), all fusion
antibodies were produced as Fragment antigen binding (Fab) fragments without fragment
crystallizable region. The pBAD vector expressing wild-type Herceptin Fab was
amplified by AccuPrime Pfx DNA polymerase (Invitrogen, CA). The gene fragments
encoding three loop regions of Cystatin A were incorporated into six designed primers
synthesized by IDT (Coralville, IA) for overlap extension PCR with wild type Herceptin
Fragment Antigen-Binding (Fab) DNA as the template. A series of overlap extension
PCRs were performed to acquire the DNA fragments encoding Herceptin Fab-Cystatin A
fusion proteins. The acquired fusion antibody DNA fragments and pBAD vector were
then digested by NheI-HF and SaiI-HF restriction enzymes (New England Biolabs, MA)
at 37 ℃ for three hours. Then, digested fusion antibody DNA fragments and pBAD
vector were purified by DNA gel extraction kit (Zymogen, CA). Bacterial expression
vectors of the Herceptin Fab-Cystatin A fusion proteins were acquired through in-frame
ligation of the fusion gene and pBAD backbone vector using T4 DNA ligase (New
England Biolabs, MA), followed by electroporation with DH10B electro-competent cells..
The resulting pBAD expression vectors for the chimeric Herceptin Fab fusion proteins
were confirmed by DNA sequencing.

19

Expression and purification of Cystatin A-Herceptin fusion antibody Fabs
The fusion proteins were expressed in DH10B E.coli transformed with pBAD expression
vectors by electro-poration. DH10B E.coli cells were cultured in Miller’s LB broth under
37℃ in 1 liter shaker flasks (180rpm). The Herceptin Fab-Cystatin A fusion protein
expression was induced at 25℃ by 0.02% L-arabinose. Fusion antibodies were secreted
into the periplasm of E.coli. After 24 hours of induction, cells were harvested by
centrifugation. The fusion antibodies in periplasm were released with lysis buffer for 1
hour (20% sucrose, 30 mM Tris at pH 8, 1 mM EDTA and 0.2 g L
-1
lysozyme). The
expressed fusion proteins in periplasmic lysates were clarified by centrifugation and then
purified by Protein G affinity chromatography (Thermo Fisher Scientific, IL). The
purified antibodies were dialyzed against PBS buffer (pH 7.4), followed by concentration
to over 5 mg mL
-1
for storage at -80 ℃.

20

In vitro Cathepsin B activity assay
The assays were performed in 96-well plates with 2.64 µM (for fusion Fab V1) or 30
µM(for fusion Fab V2) of Z-Phe-Arg-AMC (R&D, MN), 0.4 nM of recombinant human
procathepsin B (Novoprotein, NJ), and varied concentrations of fusion antibodies. The
total volume for the reaction was 200 uL. Procathepsin B was first incubated for 5
minutes in 100 uL activation buffer (340 mM sodium acetate, 60 mM acetic acid, 4 mM
EDTA, pH 5) for full activation, followed by addition of fluorescent substrate
Z-Phe-Arg-AMC in activation buffer and varied concentrations of fusion antibodies. The
activity of Cathepsin B were measured through monitoring the increase in fluorescence
intensity at 460 nm (with excitation at 380 nm), resulting from the cleavage of amide
bond in Z-Phe-Arg-AMC by Cathepsin B. The assay was performed at room temperature
(25℃) for 20 minutes.The initial reaction velocities were measured on the basis of the
slope of the curves. Inhibitor constant (K
i
) of the fusion antibodies were calculated
through nonlinear regression fit with the function:, (V
0
′
/V
0
) = ( 𝐾 𝑚 + [S])/( 𝐾 𝑚 + [S] +
𝐾 𝑚 [I]/ 𝐾 𝑖 ), where V
0
′
is the initial velocity in the presence of inhibitor; V
0
is the slope of
fluorescence intensity vs time curve initial velocity in the absence of inhibitor; 𝐾 𝑚 is the
Michaelis constant of Cathepsin B, which was estimated to be 6.5µM in a preliminary
experiment; [S] is the concentration of substrate used in the reaction, which was 2.64
uM, and the [I] is the inhibitor concentration.

21

Results:
The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was utilized
to examine purified Cystatain A-Herceptin fusion antibody Fab V1 with Herceptin Fab as
a control. Since streptococcal protein G utilized in Protein G based chromatography was
known to bind to human immunoglobulin G (IgG) with high specificity and the purity of
acquired proteins were reasonably good (no apparent undesired bands in Figure 3 and 8),
western blot was not performed (Björck and Kronvall, 1984). The molecular weight of
the purified fusion antibody Fab V1 is estimated to be 50 kDa, which is 2 kDa larger than
that of Herceptin Fab (about 48 kDa).

Similarly, the purity of Cystatain A-Herceptin fusion antibody Fab V2 was also examined
by SDS-PAGE using Herceptin Fab as a control (shown in Figure 8). With further
extended inserts, the theoretical molecular weight of fusion antibody Fab V2 (51 kDa) is
slightly larger than that of fusion antibody Fab V1 (50 kDa).

22

To test the potency of the purified Cystatin A-Herceptin fusion antibody V1 and V2, the
assays were carried out in 96-well plates with the total volume of 200 uL in each well as
described in the Methods section. In each assay, Cystatin A-Herceptin fusion proteins at
varied concentrations were used for examination of the inhibition effects. The detected
fluorescence intensities were plotted against reaction time. The slopes of curves (initial
reaction velocity of CTSB activity) were determined and used for the calculation of
inhibition constant ( 𝐾 𝑖 ). Reactions without inhibitors were labelled as control groups. The
standard curves for the competitive inhibition assays were generated by the nonlinear
regression model in the software GraphPad Prism 6.

Four assay results are presented in this thesis: NO.1 (Figure 4, 5), NO.2 (Figure 6), NO.3
(Figure 7) for evaluating the 𝐾 𝑖 of fusion Fab V1 and NO.4 (Figure 9) for determining
the inhibition effect of fusion Fab V2.

The assay results indicated that the inhibitory effect observed for Cystatin A-Herceptin
Fusion Antibody Fab V1 is attributed to the three grafted loops from Cystatin A, because
Herceptin Fab revealed no significant inhibition activity as shown in Figure 4. The
determined 𝐾 𝑖 values of Cystatin A-Herceptin fusion Fab V1 varied in the three
independent assays, which ranged from 186.7 nM (Figure 6) to 401.7 nM (Figure 7).
Additional assays will be conducted to further evaluate the potency of the fusion Fab V1.

23

Since no significant inhibition effect was observed in assay NO.4 (Figure 9) and
additional assays (data not shown) with fusion Fab V2, the nonlinear regression fitting for
estimating its 𝐾 𝑖 was not performed. It is possible that the extended inserts into CDRs of
Herceptin may impede three grafted loops from Cystatin A acting as an effective inhibitor
due to their excessive flexibility, which is further discussed in the Discussion section.

24

Figure 3. The design of Cystatin A-Herceptin fusion antibody Fab V1. The structure on
the left is the x-ray crystal structure of Herceptin light chain variable region with three of
its CDR loops labeled. The structure on the right for fusion antibody Fab V1 light chain
variable region was modeled through SWISS-MODEL using solved Herceptin x-ray
crystal structure as the template. The regions marked in orange are three grafts from
Cystatin A, which were believed to be essential for inhibiting CTSB proteolytic activity.

25

Figure 4. SDS-PAGE gel of the purified Cystatin A-Herceptin fusion Fab V1. Lanes
marked as “V1 1” through “V1 6” are eluted fractions collected from protein G
chromatography. “Her Fab” is the purified Herceptin Fab, which is about 2 kDa lower
than the fusion Fab V1. The last two lanes in the SDS-PAGE are reduced fusion Fab V1
and reduced Herceptin Fab.

Fusion
Fab
1
Fusion
Fab
2
Fusion
Fab 1
3
Fusion
Fab
4
Herceptin
Fab
Herceptin
Fab
reduced
Fusion
Fab
reduced
Fusion
Fab
5
Fusion
Fab
6
26

Figure 5. The fusion Fab V1 inhibited CTSB activities in a dose-dependent manner in
assay NO.1. Herceptin alone (500 nM concentration) revealed no significant inhibitory
effect against CTSB.

27

Figure 6. Non-linear regression fitting of inhibition curves of fusion Fab V1---NO.1.
Extrapolated from raw data shown in Figure 4, in assay NO.1, the K
i
of fusion antibody
Fab V1 was calculated as 277 nM ± 104 nM and the R square was -3.99.

28

Figure 7. Non-linear regression fitting of inhibition curves of fusion Fab V1---NO.2.
According to assay NO. 2, the Ki of fusion antibody Fab V1 was 186.7 nM ± 58.28 nM
and the R square was 0.5611.

29

Figure 8. Non-linear regression fitting of inhibition curves of fusion Fab V1---NO.3. In
assay NO.3, the Ki of fusion antibody Fab V1 was 401.7 nM ± 60.87 nM and the R
square was 0.9143.

30

Figure 9. SDS-PAGE gel of the purified Cystatin A-Herceptin fusion Fab V2. Those
bands in the gel labeled “V2 1” through “V2 5” were eluted fractions of fusion Fab V2,
and “Her” was purified Herceptin used as control. The last two lanes labeled as “V2
reduce” and “Her reduce” were fusion Fab V2 and Herceptin Fab reduced with DTT.
Given its larger inserts in CDR loops relative to V1 Fab, the protein band of V2 Fab
migrated at a higher position. The molecular weight for Herceptin Fab was estimated to
be 48 kDa, while the Cystatin A-Herceptin fusion antibody Fab V2 was roughly 51 kDa.
V2
1
V2
2
V2
3
V2
4
V2
5
Her
V2
Reduced
Her
Reduced
31

Figure 10. Inhibition assay of the fusion Fab V2 against CTSB. Compared with V1Fab,
the Cystatin A-Herceptin fusion antibody Fab V2 revealed less inhibition effect against
CTSB (the adding of inhibitor did not cause the CTSB enzymatic to significantly
decrease), even at concentration of 4 uM.

32

Discussion:
Cathepsin B was shown to be involved in the development and progression of various
human diseases, including rheumatoid arthritis and cancer. Currently, selective and
noncell-permeable inhibitors of CTSB with good pharmacokinetic profiles have yet
progressed into the clinical applications. Conventional small molecule-based or
peptide-based inhibitors possess high toxicity and short plasma half-lives, which have
hindered them from being translated into therapeutics. To tackle these deficiencies of
current CTSB inhibitors, we aimed to develop highly specific large antibody-based
inhibitors for CTSB through combining humanized monoclonal antibodies and the
naturally occurring cysteine protease inhibitors. The monoclonal antibody Herceptin was
chosen as a human immunoglobulin scaffold for rational design of the fusion proteins to
bear three inhibition loop regions from Cystatin A (essential for inhibiting CTSB) which
were grafted into the CDR loops of Herceptin light chain hyper variable region. Since the
conformation and arrangements of those three essential loops in Cystatin A are similar to
those of CDR loops in antibodies, we expect that the fusion antibody would act as potent
CTSB inhibitors.

In this study, two fusion antibody Fabs were designed and generated and their efficacy in
inhibiting human CTSB were evaluated. On the basis of inhibition results, the Cystatin
A-Herceptin fusion antibody Fab V1 revealed potent activity towards CTSB with a K
i
around 500 nM, while the Cystatin A-Herceptin fusion antibody Fab V2 with further
33

extended inserts grafted from Cystatin A was shown to have less inhibition effect on
CTSB. The lack of inhibition for Cystatin A-Herceptin Fusion Antibody V2 is likely to be
caused by the disrupted conformation of the grafted loops comparing to their native
forms in Cystatin A. In Cystatin A-Herceptin Fusion Antibody Fab V1, loops from
Cystatin were grafted right above the β-strand stalks that originally support CDRs loops
in Herceptin, which is similar to how these loops are originally supported in Cystatin A.
However, on the contrary, those extra amino acid residues in Fab V2 may be unable to
form partial β-strand like structure, and instead give rise to longer and more flexible loop
conformations. Moreover, the insertion site on Herceptin CDR loop 1 were located in the
middle of an extended flexible. It is even more difficult for the extra-long insert into CDR
loop 1 to retain any constrained conformation.

Cystatin A was reported to be a highly potent 11 kDa protein-based inhibitor against
CTSB with a inhibition constant K
i
of 2 nM (Lenarčič et al., 1996). The K
i
of Cystatin
A-Herceptin fusion antibody Fab V1 is much higher than Cystatin, indicating further
engineering work need to be done for improved affinity.

Future experiments will also include determination of the inhibition specificity for the
fusion antibodies. Importantly, antibody engineer work will be first concentrated on
improving the affinity of the designed antibodies.

34

To further improve the affinity of Cystatin A-Herceptin Fusion Antibody Fab V1, total
deletion will be performed for the flexible loops on the top of β-strand stalks in the CDR
regions of Herceptin light chain, which are likely to act as a sturdy platform to constrain
the conformation of inserts from Cystatin A (for inserts in Fab V2). Moreover, according
to the solved x-ray crystal structure of Cystatin A in complex with CTSB, the loop at the
N terminus of Cystatin A form limited interaction with the protease. Thus, fusion
antibodies with only two inhibition loops will be generated. Finally, rearrangements of
the insertion sites for those grafted loops from Cystatin A will be performed.

35

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Asset Metadata

Creator Dai, Zhefu (Jeff) (author)

Core Title Rational design of monoclonal antibody inhibitors targeting human cathepsin B

Contributor Electronically uploaded by the author (provenance)

School School of Pharmacy

Degree Master of Science

Degree Program Pharmaceutical Sciences

Publication Date 07/27/2016

Defense Date 06/15/2016

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

Tag cathepsin B,monoclonal antibody based inhibitors,oai:digitallibrary.usc.edu:usctheses,OAI-PMH Harvest,rational design

Format application/pdf (imt)

Language English

Advisor Zhang, Yong (committee chair), Haworth, Ian (committee member), Okamoto, Curtis (committee member)

Creator Email jeffdai1992@gmail.com,zhefudai@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c40-282749

Unique identifier UC11281288

Identifier etd-DaiZhefuJe-4645.pdf (filename),usctheses-c40-282749 (legacy record id)

Legacy Identifier etd-DaiZhefuJe-4645-1.pdf

Dmrecord 282749

Document Type Thesis

Format application/pdf (imt)

Rights Dai, Zhefu (Jeff)

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 a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA

Abstract (if available)

Abstract Cathepsin B (CTSB) is one of the earliest identified cysteine proteases, which is highly abundant in lysosomes. Mounting evidence showed that CTSB is secreted by cells into extracellular regions for various physiological functions such as bone remodeling. Moreover, overexpressed CTSB and its accumulation in extracellular regions are closely related to the deterioration of joint tissue damage in rheumatoid arthritis and the aggressiveness of invasive solid tumors in cancers. Thus, inhibitors targeting CTSB can provide innovative therapeutic approaches. Currently, selective CTSB inhibitors have yet been available for clinical applications. Small-molecule CTSB inhibitors are often plagued by severe toxicity due to significant off-target effects. In addition, peptide-based inhibitors are likely to suffer from short serum half-lives. In this study, through rational design, two human monoclonal antibody-based CTSB inhibitors were generated and examined for inhibition activity towards CTSB. One of the resulting antibodies displayed potent inhibition activity at nanomolar levels. Further engineering study will be performed to generate antibody-based therapeutics with prolonged plasma half-lives and improved safety profiles.