U.S. patent application number 17/087765 was filed with the patent office on 2021-05-20 for antibodies targeting quiescin sulfhydryl oxidase (qsox1) and uses of same.
This patent application is currently assigned to Yeda Research and Development Co. Ltd.. The applicant listed for this patent is Yeda Research and Development Co. Ltd.. Invention is credited to Deborah FASS, Iris GROSSMAN, Tal ILANI.
Application Number | 20210147575 17/087765 |
Document ID | / |
Family ID | 1000005360728 |
Filed Date | 2021-05-20 |
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United States Patent
Application |
20210147575 |
Kind Code |
A1 |
FASS; Deborah ; et
al. |
May 20, 2021 |
ANTIBODIES TARGETING QUIESCIN SULFHYDRYL OXIDASE (QSOX1) AND USES
OF SAME
Abstract
An antibody comprising an antigen recognition domain exhibiting
species cross reactivity to human QSOX1 and murine QSOX1 is
disclosed. Methods of producing the antibody, pharmaceutical
compositions comprising the antibody and methods of using the
antibody for treating medical conditions are also disclosed.
Inventors: |
FASS; Deborah; (Rehovot,
IL) ; GROSSMAN; Iris; (Rehovot, IL) ; ILANI;
Tal; (Rehovot, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Yeda Research and Development Co. Ltd. |
Rehovot |
|
IL |
|
|
Assignee: |
Yeda Research and Development Co.
Ltd.
Rehovot
IL
|
Family ID: |
1000005360728 |
Appl. No.: |
17/087765 |
Filed: |
February 7, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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15961928 |
Apr 25, 2018 |
10829561 |
|
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17087765 |
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PCT/IL2016/051147 |
Oct 25, 2016 |
|
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15961928 |
|
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|
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62246076 |
Oct 25, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 39/395 20130101;
C07K 2317/33 20130101; C07K 2317/92 20130101; C07K 16/40 20130101;
C07K 2317/73 20130101; C07K 2317/622 20130101; C07K 2317/565
20130101; C07K 2317/76 20130101; A01K 2227/105 20130101; A01K
2267/0306 20130101; A61P 35/00 20180101; C07K 16/18 20130101; A61K
2039/505 20130101 |
International
Class: |
C07K 16/40 20060101
C07K016/40; A61K 39/395 20060101 A61K039/395; C07K 16/18 20060101
C07K016/18 |
Claims
1. An antibody comprising an antigen recognition domain exhibiting
species cross reactivity to human QSOX1 and murine QSOX1, said
antigen recognition domain comprising complementarity determining
regions (CDRs) as set forth in SEQ ID NOs: 26-31.
2. The antibody of claim 1, wherein said antibody is an antibody
fragment, a single chain antibody or a monoclonal antibody.
3. The antibody of claim 2, wherein said monoclonal antibody is
Mab316.1 and comprises CDRs SEQ ID NOs: 26-31.
4. The antibody of claim 2, wherein said single chain antibody is
Mab316.1 and comprises CDRs SEQ ID NOs: 26-31.
5. A pharmaceutical composition comprising as an active ingredient
the monoclonal antibody of claim 1 and a pharmaceutically
acceptable carrier.
6. A method for preventing or treating a laminin-associated disease
or condition in a subject in need thereof, the method comprising
administering to the subject a therapeutically effective amount of
the pharmaceutical composition of claim 5.
Description
RELATED APPLICATIONS
[0001] This application is a Division of U.S. patent application
Ser. No. 15/961,928 filed on Apr. 25, 2018, which is a Continuation
of PCT Patent Application No. PCT/IL2016/051147 having
International Filing date of Oct. 25, 2016, which claims the
benefit of priority of U.S. Provisional Patent Application No.
62/246,076 filed on Oct. 25, 2015.
[0002] The contents of the above applications are all incorporated
by reference as if fully set forth herein in their entirety.
SEQUENCE LISTING STATEMENT
[0003] The ASCII file, entitled 84654SequenceListing.txt, created
on Nov. 3, 2020, comprising 77,398 bytes, submitted concurrently
with the filing of this application is incorporated herein by
reference. The sequence listing submitted herewith is identical to
the sequence listing forming part of the international
application.
FIELD AND BACKGROUND OF THE INVENTION
[0004] The present invention, in some embodiments thereof, relates
to antibodies targeting QSOX1 including cross-species-specific
antibodies, and, more particularly, but not exclusively, to the
generation and use of same.
[0005] Monoclonal antibody therapy has become an integral part of
cancer diagnostics and treatment. The success of antibody-based
therapy stems from the high specificity and affinity that
antibodies offer compared to other anti-tumor agents. In addition,
tumors express on their cell-surfaces many potential targets for
antibody therapeutics. Antibodies supplied extracellularly can both
neutralize the function of their cell-surface antigen and recruit
the immune system for a more extensive anti-tumor response.
Recently, the realization that tumor stroma has a major role in
supporting tumor development and metastasis inspired antibody-based
cancer therapies targeting extracellular matrix (ECM) components in
addition to targeting tumor cells directly. Examples for such
agents are antibodies that affect the extracellular glycoprotein
tenascin or fibroblast activation protein, found in stromal
fibroblasts of most human carcinomas.
[0006] ECM proteins are good candidates for antibody therapy
because they are both accessible and abundant in most tissues,
making the same ECM components a target in various cancers. A major
component of the ECM that is over-expressed, reorganized, and
cross-linked in tumorigenesis is collagen. Collagen cross-linking
by the enzyme lysyl oxidase (LOX) is increased in several cancers
and contributes to matrix stiffening, thereby promoting cell
adhesion and migration. Inhibitors of LOX activity, including
monoclonal antibodies, significantly inhibited tumor growth and
metastasis in gastric carcinoma. Laminin is another abundant
scaffolding ECM protein that interacts with integrins to mediate
cell adhesion and migration, a requirement for metastasis. Indeed,
laminin is over-expressed in various cancers, and its chain
isotypes serve as tumor biomarkers. Like collagen cross-linking and
integrin blocking, laminin incorporation into the matrix may serve
as a complementary target for antibody-based cancer
therapeutics.
[0007] Fass D. and co-workers have recently shown that laminin
incorporation into the ECM is affected by the disulfide catalyst
Quiescin sulfhydryl oxidase 1 (QSOX1) [Ilani, T. et al. (2013)
Science 341: 74-76]. The enzyme QSOX1 is a fusion of two
thioredoxin (Trx) domains and an Erv-fold sulfhydryl oxidase module
(FIG. 1A). QSOX1 contains two CXXC motifs as redox-active sites
that cooperate to relay electrons from reduced thiols of substrate
proteins to molecular oxygen. Mechanistically, after oxidizing the
substrate, the Trx active site transfers two electrons to the Erv
CXXC motif through an inter-domain disulfide intermediate (FIG.
1C). The electrons proceed to the adjacent flavin adenine
dinucleotide cofactor, which in turn reduces oxygen to hydrogen
peroxide, leaving QSOX1 oxidized and ready for another catalytic
cycle. Unlike other disulfide catalysts, QSOX1 is localized
downstream of the endoplasmic reticulum (ER). It is found in the
Golgi apparatus and secreted from quiescent fibroblasts into the
ECM, where it affects ECM composition and especially laminin
incorporation [Ilani T. et al. (2013), supra]. Specifically, QSOX1
affects the incorporation of laminin isoforms that contain an
.alpha.4 chain [Ilani T. et al. (2013), supra], a known marker for
tumor progression. Together with the over-production of QSOX1 in
various adenocarcinomas and associated stroma, these findings point
to a possible role of QSOX1 in stimulating tumor cell migration via
laminin incorporation.
[0008] The multi-step catalytic cycle of QSOX1 implies that
obscuring any one of several sites on the protein by interaction
with antibody may accomplish inhibition. Fass D. and co-workers
have developed an inhibitory monoclonal antibody, MAb492.1, which
blocks substrate access to the Trx CXXC redox-active site of human
QSOX1 (HsQSOX1) (FIG. 1B) [Grossman I. et al. (2013)1 Mol. Biol.
425: 4366-4378]. MAb492.1 efficiently inhibited HsQSOX1 activity,
and consequently inhibited adhesion and migration of cancer cells
to and through fibroblasts from corresponding tissues [Ilani T. et
al. (2013), supra]. Accordingly, MAb492.1 may serve as an
anti-metastatic drug in antibody-based cancer therapy.
[0009] In certain cases an antibody targeting a human protein is
sufficient for pre-clinical trials in animal models due to the
availability of xenograft and tumor graft models. However, a major
disadvantage of these models is the artificial interaction of the
tumor with surrounding tissues. Moreover, eventual recruitment of
mouse fibroblasts will create an inhomogeneous tumor
microenvironment composed of both mouse and human constituents.
Hence, when targeting a secreted protein such as QSOX1, mouse
models with a natural organization of murine cancer cells and
stroma, such as genetically modified mice that develop spontaneous
tumors, may be preferable for studying metastasis development. In
such configurations, when targeting an endogenous animal protein,
the lead compound e.g., antibody which recognizes the human
protein, is often irrelevant.
[0010] U.S. Patent Application no. 20140141015 (to Lake and
Katchman) discloses tumor treatment by administering an inhibitor
of QSOX1, e.g., an antibody. Specifically, U.S. 20140141015
discloses the use of QSOX1 inhibitors for the treatment of tumors
that over-express QSOX1, pancreatic ductal adenocarcinoma (PDA) and
breast adenocarcinoma.
[0011] Additional background art includes PCT publication nos. WO
2013/132495 (to Fass et al.), WO 2010/077921 (to Lake et al.) and
WO 2010/071787 (to Lake et al.).
SUMMARY OF THE INVENTION
[0012] According to an aspect of some embodiments of the present
invention there is provided an antibody comprising an antigen
recognition domain exhibiting species cross reactivity to human
QSOX1 and murine QSOX1.
[0013] According to an aspect of some embodiments of the present
invention there is provided an antibody comprising an antigen
recognition domain exhibiting species cross reactivity to human
QSOX1 and murine QSOX1, the antigen recognition domain comprising
complementarity determining regions (CDRs) as set forth in SEQ ID
NOs: 46-51.
[0014] According to an aspect of some embodiments of the present
invention there is provided a pharmaceutical composition comprising
as an active ingredient the antibody of some embodiments of the
invention and a pharmaceutically acceptable carrier.
[0015] According to an aspect of some embodiments of the present
invention there is to provided a method for preventing or treating
a laminin-associated disease or condition in a subject in need
thereof, the method comprising administering to the subject a
therapeutically effective amount of the pharmaceutical composition
of some embodiments of the invention.
[0016] According to an aspect of some embodiments of the present
invention there is provided a use of the antibody of some
embodiments of the invention for the manufacture of a medicament
identified for preventing or treating a laminin-associated disease
or condition in a subject in need thereof.
[0017] According to an aspect of some embodiments of the present
invention there is provided an article of manufacture comprising
the antibody of some embodiments of the invention being packaged in
a packaging material and identified in print, in or on the
packaging material for use in the treatment of a laminin-associated
disease or condition.
[0018] According to an aspect of some embodiments of the present
invention there is provided an antibody comprising an antigen
recognition domain comprising CDRs as set forth in SEQ ID NOs:
26-31, wherein the antibody specifically binds murine QSOX1.
[0019] According to an aspect of some embodiments of the present
invention there is provided a method for in vivo determining the
efficiency of an antibody in reducing a laminin-associated disease
or condition in a murine animal, the method comprising
administering to the murine animal the antibody of some embodiments
of the invention and monitoring progression of a laminin-associated
disease or condition in the murine animal, thereby determining the
efficiency of the antibody.
[0020] According to an aspect of some embodiments of the present
invention there is provided an isolated polynucleotide comprising a
nucleic acid sequence encoding the antibody of some embodiments of
the invention.
[0021] According to an aspect of some embodiments of the present
invention there is provided an isolated polynucleotide comprising a
nucleic acid sequence encoding an antibody comprising the CDRs set
forth in SEQ ID NOs: 46-51.
[0022] According to an aspect of some embodiments of the present
invention there is provided an isolated polynucleotide comprising a
nucleic acid sequence encoding an antibody comprising the CDRs set
forth in SEQ ID NOs: 26-31.
[0023] According to an aspect of some embodiments of the present
invention there is provided a method of producing an antibody which
comprises an antigen recognition domain exhibiting species cross
reactivity to QSOX1, the method comprising: (a) introducing at
least one point mutation in the antigen binding domain of an
antibody which binds QSOX1 of a first species, which the at least
one point mutation increases the water-mediated hydrogen bonding in
the interaction interface between the antigen binding domain of the
antibody and the QSOX1 of the first species, and alternatively or
additionally the at least one point mutation reduces aromatic
interactions between the antigen binding domain of the antibody and
the QSOX1 of the first species, wherein the at least one point
mutation does not substantially affect affinity of the antibody to
the QSOX1 of the first species; and (b) testing binding of the
antibody having the at least one point mutation to QSOX1 of a
second species, wherein when the antibody having the at least one
point mutation binds the QSOX1 of the first species and the second
species with substantially the same affinity the antibody is
considered having cross reactivity to QSOX1.
[0024] According to some embodiments of the invention, the antibody
comprises a lower content of aromatic amino acids in CDRs of the
antigen recognition domain as compared to that of a
species-specific antibody to the human QSOX1.
[0025] According to some embodiments of the invention, the aromatic
amino acids comprise tyrosine.
[0026] According to some embodiments of the invention, the antibody
comprises at least one charged amino acid in CDR3 of a variable
heavy chain of the antibody.
[0027] According to some embodiments of the invention, the charged
amino acid comprises aspartic acid.
[0028] According to some embodiments of the invention, the antibody
inhibits QSOX1 activity in mediating laminin incorporation in the
basement membrane.
[0029] According to some embodiments of the invention, the activity
is assayed by at least one of an immunofluorescence (IF) staining
assay of the extracellular matrix or western blot assay detecting
for soluble laminin.
[0030] According to some embodiments of the invention, the antibody
is an antibody fragment.
[0031] According to some embodiments of the invention, the antibody
is selected from the group consisting of a Fab fragment, an Fv
fragment, a single chain antibody and a single domain antibody.
[0032] According to some embodiments of the invention, the antibody
is a monoclonal antibody.
[0033] According to some embodiments of the invention, the
monoclonal antibody is MAb492gen and comprises CDRs SEQ ID NOs:
46-51.
[0034] According to some embodiments of the invention, the antibody
is a single chain antibody.
[0035] According to some embodiments of the invention, the single
chain antibody is seFV492gen and comprises CDRs SEQ ID NOs:
46-51.
[0036] According to some embodiments of the invention, the antibody
is humanized.
[0037] According to some embodiments of the invention, the antibody
is a chimeric antibody.
[0038] According to some embodiments of the invention, the antibody
is immobilized to a solid support.
[0039] According to some embodiments of the invention, the antibody
is attached to a detectable moiety.
[0040] According to some embodiments of the invention, the antibody
comprises an amino acid sequence as set forth in SEQ ID NOs: 44 and
45.
[0041] According to some embodiments of the invention, the method
further comprises administering to the subject a chemotherapeutic
agent.
[0042] According to some embodiments of the invention, the use
further comprises the use of a chemotherapeutic agent.
[0043] According to some embodiments of the invention, the article
of manufacture further comprises a chemotherapeutic agent.
[0044] According to some embodiments of the invention, the
laminin-associated disease or condition is a tumor.
[0045] According to some embodiments of the invention, the tumor is
a metastasizing solid tumor.
[0046] According to some embodiments of the invention, the tumor is
an adenocarcinoma.
[0047] According to some embodiments of the invention, the tumor is
a cancer selected from the group consisting of a prostate cancer, a
lung cancer, a breast cancer, a cervical cancer, an urachus cancer,
a vaginal cancer, a colon cancer, an esophagus cancer, a pancreatic
cancer, a throat cancer, a stomach cancer and a myeloid
leukemia.
[0048] According to some embodiments of the invention, the
laminin-associated disease or condition is associated with
fibrosis.
[0049] According to some embodiments of the invention, the amino
acid sequence is as set forth in SEQ ID NOs: 9 and 10.
[0050] According to some embodiments of the invention, the antibody
is a monoclonal antibody.
[0051] According to some embodiments of the invention, the at least
one point mutation is in a CDR sequence of the antibody.
[0052] According to some embodiments of the invention, the at least
one point mutation which reduces aromatic interactions is in a
tyrosine.
[0053] According to some embodiments of the invention, the at least
one point mutation which reduces aromatic interactions comprises an
amino acid comprising a flexible side chain.
[0054] According to some embodiments of the invention, the at least
one point mutation which increases the water-mediated hydrogen
bonding comprises at least one charged amino acid.
[0055] According to some embodiments of the invention, the at least
one point mutation which increases the water-mediated hydrogen
bonding is an aspartic acid.
[0056] Unless otherwise defined, all technical and/or scientific
terms used herein have the same meaning as commonly understood by
one of ordinary skill in the art to which the invention pertains.
Although methods and materials similar or equivalent to those
described herein can be used in the practice or testing of
embodiments of the invention, exemplary methods and/or materials
are described below. In case of conflict, the patent specification,
including definitions, will control. In addition, the materials,
methods, and examples are illustrative only and are not intended to
be necessarily limiting.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0057] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0058] Some embodiments of the invention are herein described, by
way of example only, with reference to the accompanying drawings.
With specific reference now to the drawings in detail, it is
stressed that the particulars shown are by way of example and for
purposes of illustrative discussion of embodiments of the
invention. In this regard, the description taken with the drawings
makes apparent to those skilled in the art how embodiments of the
invention may be practiced.
[0059] In the drawings:
[0060] FIGS. 1A-1B are schematic illustrations of QSOX1 domain
organization and inhibition by MAb492.1. FIG. 1A shows that the two
modules of mammalian QSOX1 enzymes are linked by a flexible linker
(black). The amino--terminal module, QSOX1.sub.Trx, is composed of
two Trx-fold domains, the first of which has a redox-active CXXC
motif (yellow balls). A second redox-active CXXC motif is located
in the carboxy-terminal Erv domain. This domain binds an FAD
cofactor (fused hexagons). The domain labeled .psi.Erv is a
degenerate Erv domain lacking active-site cysteines and a FAD
cofactor; and FIG. 1B shows the surface presentation of a complex
of a Fab fragment of Mab492.1 (Fab492.1) and HsQSOX1.sub.Trx (PDB
code: 4IJ3) showing that Fab492.1 inhibits HsQSOX1 by burying the
Trx CXXC active site (yellow spheres).
[0061] FIG. 1C is a schematic illustration of the catalytic cycle
of QSOX1 oxidizing two free thiols in a substrate molecule to a
disulfide bond, and reducing oxygen to hydrogen peroxide. Different
chemical enzyme states in the cycle are designated with numbers
1-5. In state 3, an inter-domain disulfide requires a
conformational change that leads to physical proximity of the two
CXXC motifs. Fused yellow balls represent disulfide bonds.
Separated yellow bills indicate reduced cysteines. Orange FAD
hexagons represent oxidized FAD, whereas yellow hexagons indicate
reduced FAD.
[0062] FIGS. 2A-2D illustrate that MAb492.1 is species specific.
FIG. 2A shows the turnover numbers of various mammalian QSOX1
enzymes in the absence and presence of 250 nM or 1 .mu.M MAb492.1.
Activity was evaluated using an oxygen consumption assay; FIG. 2B
shows the sequence alignment of HsQSOX1 and other mammalian QSOX1
orthologs in the region bound by MAb492.1. Residues involved in
interactions with MAb492.1 are indicated in bold. Residues from
MmQSOX1, RnQSOX1, and CpQSOX1 that differ from the corresponding
HsQSOX1 residues are colored red; FIG. 2C shows superposition of
the Fab492.1-HsQSOX1.sub.Trx complex structure (PDB code: 4IJ3) and
the structure of MmQSOX1.sub.Trx (PDB code: 5D8I), The two chains
of MmQSOX1.sub.Trx from the crystal asymmetric unit are light
(chain B) and dark (chain A) gray, and HsQSOX1.sub.Trx is pink.
Right, close-up of the expected clash between CDR H3 and residues
TLPG(138-141) from MmQSOX1. Left, close-up of the expected clash
between CDR L3 and Asn117 from MmQSOX1. Only chain B of
MmQSOX1.sub.Trx is shown for simplicity; and FIG. 2D shows the
percent activity for different MmQSOX1 mutants in the presence of
MAb492.1. Oxygen consumption measurements were conducted with 100
nM enzyme, 250 nM MAb492.1, and 200 .mu.M DTT. Percent activity was
calculated according to the results of the same measurements in the
absence of antibody. Error bars represent standard deviations from
an average of three measurements.
[0063] FIGS. 3A-3I illustrate the evaluation of binding and
inhibition constants of antibodies targeting MmQSOX1. FIG. 3A shows
histograms of fluorescence (APC-conjugated streptavidin) reporting
MmQSOX1.sub.Trx binding to the surface of yeast displaying
scFv492.1 or a scFv492.1 mutant (mutant d, Table 3). Each histogram
represents 50,000 yeast cells. The scFv492.1 mutant shows an
increase in APC fluorescence compared to wild-type scFv492.1; FIG.
3B shows a dose-response curve of MAb492gen to 150 or 25 nM
MmQSOX1, based on results from a colorimetric assay quantifying
RNase A oxidation. The inhibitory activity is expressed as
absorbance at 412 nm, representing free thiols that reacted with
5,5'-dithiobis-(2-nitrobenzoic acid). Error bars represent standard
deviations from an average of three measurements. The IC.sub.50
values were determined by nonlinear regression analysis and yielded
values close to the MmQSOX1 concentration; FIG. 3C shows the
inhibition curve of MAb492gen to 50 nM MmQSOX1, based on results
from oxygen electrode assays at a range of MAb492gen
concentrations. Inhibitory activity is expressed as the ratio of
the inhibited rate to the uninhibited rate (v.sub.i/v.sub.0). Error
bars represent standard deviations from an average of three
measurements. The K.sub.i value was determined by nonlinear
regression analysis; FIG. 3D shows the same as FIG. 3C only for
MAb316.1; FIGS. 3E-3H show representative images of laminin
immunostaining in cultures treated with no antibody (FIG. 3E),
MAb492.1 (FIG. 3F), MAb316.1 (FIG. 3H), or MAb492gen (FIG. 3G).
Scale bar is 20 .mu.m, Additional images are presented in FIGS.
4B-4I; and FIG. 3I shows quantification of adhesion of
fluorescently labeled epithelial cells to a mouse fibroblast
monolayer grown in the absence or presence of MAb492.1, MAb316.1,
or MAb492gen. Error bars are standard error of the mean. Sample
images are shown in FIGS. 4J-O.
[0064] FIG. 3J illustrates a sequence alignment of MAb492gen and
MAb492.1 variable regions. Mutated residues are colored red.
[0065] FIGS. 3K-3L illustrate histograms of red fluorescence (APC
bound to streptavidin) representing MmQSOX1.sub.Trx binding at
various concentrations to the surface of yeast displaying
scFv492gen (FIG. 3K). Each histogram represents 50,000 yeast cells;
and FIG. 3L shows a quantification of FIG. 3K. For each histogram
in FIG. 3K, the mean APC fluorescence was measured and plotted as
function of MmQSOX1.sub.Trx concentration. Data were fit to a
binding isotherm, yielding an apparent K.sub.d of 65.+-.10 nM for
MmQSOX1.sub.Trx binding on the surface of yeast.
[0066] FIG. 4A illustrates a MmQSOX1 immunoblot with a rabbit
polyclonal antibody (dilution 1:1000). Lane 1, recombinant MmQSOX1.
Lane 2, the supernatant of confluent mouse fibroblasts, showing
that MmQSOX1 is secreted. Recombinant MmQSOX1 is truncated at the
carboxy-terminus and so migrates faster than endogenous secreted
MmQSOX1. Endogenous MmQSOX1 appears as two bands due to its two
splice variants.
[0067] FIGS. 4B-4I illustrate additional representative images of
laminin immunostaining, as shown in FIGS. 3E-I. Scale bar is 40
.mu.m.
[0068] FIGS. 4J-4O illustrate representative images of adherent
epithelial cells to a MEF monolayer. Fifteen such fields were
quantified in each treatment (FIG. 3I). Scale bar is 100 .mu.m.
[0069] FIGS. 5A-5E are schematic illustrations of the structure of
the Fab492gen-MmQSOX1Trx complex. FIG. 5A shows a surface
presentation of the Fab492gen-MmQSOX1Trx complex (PDB code: 5D96)
showing that Fab492gen binds MmQSOX1 in the same mode as Fab492.1
with respect to HsQSOX1Trx, by burying the Trx CXXC active site
(yellow spheres); FIG. 5B shows the top view of MAb492.1 and
MAb492gen CDRs. Coloring of Fab492gen chains is as in FIG. 5A. The
Fab492gen-MmQSOX1Trx complex was overlaid on the
Fab492.1-HsQSOX1Trx complex by aligning MmQSOX1Trx and HsQSOX1Trx;
FIG. 5C shows a cartoon presentation of CDRs L2 and H3 in
Fab492gen, showing their close interaction. Residues in stick
presentation were mutated in the development of Fab492gen from
Fab492.1. The dashed black line represents a hydrogen bond. FIGS.
5D-5E show a comparison of the CDR L1-QSOX1Trx interaction in
Fab492gen-MmQSOX1Trx and Fab492.1-HsQSOX1Trx complexes. Coloring is
as in FIG. 5A and. FIG. 5B. One hydrogen bond is possible between
Fab492.1 and HsQSOX1Trx, and none between Fab492.1 and MmQSOX1Trx
(FIG. 5D). Two hydrogen bonds provide interactions in the
Fab492gen- MmQSOX1Trx complex (FIG. 5E).
[0070] FIG. 5F is a schematic illustration showing a comparison of
CDR H3 between Fab492.1 and Fab492gen. In addition to the shift in
backbone, mutation of Tyr to Met prevents a clash between CDR H3
and MmQSOX1Trx. The position of CDR H3 in Fab492gen enables binding
of both MmQSOX1 and HsQOX1.
[0071] FIGS. 6A-6C illustrate that MAb316.1 binds the Trx domain of
MmQSOX1. FIG. 6A shows elution profiles of MAb316.1 and MmQSOX1. or
its fragments from analytical size-exclusion chromatography.
MAb316.1 binds the Trx module; FIG. 6B shows the surface
presentation of the Fab316.1-HsQSOX1.sub.Trx complex (PDB code:
5D93) showing that Fab316.1 does not bury the CXXC motif,
accessible at the complex surface (yellow); and FIG. 6C shows
superposition of the Fab31.6.1-HsQSOX1.sub.Trx, complex (PDB code:
5D93) and MmQSOX1 C76A/C455S (PDB code: 3T58). Only the variable
region of the light chain is shown (gray) from Fab316.1. Yellow
spheres represent the interdomain disulfide between the Trx and Erv
active-site cysteines. MmQSOX1.sub.Erv clashes with the Fab316.1
light chain (black circle), suggesting that Fab316.1 inhibits
MmQSOX1 by interfering with formation of the inter-domain
electron-transfer intermediate.
[0072] FIGS. 7A-7D illustrate HsQSOX1 inhibition by MAb316.1 and
MAb492gen. FIG. 7A shows turnover numbers of HsQSOX1 in the absence
and presence of 250 nM or 1 .mu.M MAb316.1. Activity was evaluated
using an oxygen consumption assay. Error bars represent standard
deviations from an average of three measurements; FIG. 7B shows the
inhibition curve of MAb492gen calculated from oxygen electrode
assays of 50 nM HsQSOX1 in the presence of various MAb492gen
concentrations. Inhibitory activity is expressed as the ratio of
the inhibited rate to the uninhibited rate (v.sub.i/v.sub.0). Error
bars represent standard deviations from an average of three
measurements. The K.sub.i value was determined by nonlinear
regression analysis; FIG. 7C shows the superposition of
HsQSOX1.sub.Trx (PDB code: 4IJ3, chain a) and
Fab316.1-MmQSOX1.sub.Trx complex (PDB code: 5D93). Residues that
differ in sequence between the two QSOX1.sub.Trx orthologs, namely
the VFPV(135-138) loop from HsQSOX1 and the corresponding
TLPG(138-141) loop from MmQSOX1, are in stick presentation. FIG. 7D
shows the percent activity of MmQSOX1, HsQSOX1, and its mutant
VFPV(135-138)TLPG, designated TLPG, in the presence of MAb316.1.
Measurements were conducted in an oxygen consumption assay with 100
nM enzyme, 250 nM MAb492.1, and 200 .mu.M DTT. Percent activity was
calculated according to the results of the same measurements in the
absence of antibody. Error bars represent standard deviations from
an average of three measurements.
[0073] FIG. 8 illustrates in vivo that treatment with MAb316.1
slows the progression of an aggressive breast cancer in mice. Mice
were treated with 15 mg/kg or 30 mg/kg of MAb316.1 and/or with
doxorubicin (as described in Table 1B, hereinbelow). Average tumor
volumes of the five groups of mice injected with 4T1 cells was
monitored. Tumor dimensions were measured twice a week with a
caliper. Tumor volume was calculated as: x*y*z*6/.pi.. Error bars
represent SEM.
DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION
[0074] The present invention, in some embodiments thereof, relates
to antibodies targeting QSOX1 including cross-species-specific
antibodies, and, more particularly, but not exclusively, to the
generation and use of same.
[0075] The principles and operation of the present invention may be
better understood with reference to the drawings and accompanying
descriptions.
[0076] Before explaining at least one embodiment of the invention
in detail, it is to be understood that the invention is not
necessarily limited in its application to the details set forth in
the following description or exemplified by the Examples. The
invention is capable of other embodiments or of being practiced or
carried out in various ways. Also, it is to be understood that the
phraseology and terminology employed herein is for the purpose of
description and should not be regarded as limiting.
[0077] The secreted disulfide catalyst Quiescin sulfhydryl oxidase
1 (QSOX1) affects extracellular matrix (ECM) composition and is
over-expressed in various adenocarcinomas and associated stroma.
Specifically, QSOX1 is found in the Golgi apparatus and is secreted
from quiescent fibroblasts into the ECM, where it affects ECM
composition and especially laminin incorporation into the basement
membrane (BM), a layer of the ECM at the interface between body
cavities or blood vessels and underlying stromal fibroblasts.
[0078] Inhibition of extracellular human QSOX1 by a monoclonal
antibody has been previously suggested to decrease tumor cell
migration, However, the species-specificity of the QSOX1 monoclonal
antibody has been a setback in assessing its utility as an
anti-metastatic agent in vivo, a common problem in the
antibody-therapy industry.
[0079] The introduction of desired properties into monoclonal
antibodies is often challenging as such properties may be
accomplished on the expense of other properties. The present
inventors demonstrated herein the importance of using complex
structural information for identifying the specificity determinants
in broadening of the antibody specificity. Thus, although human and
mouse QSOX1 have a high sequence identity (79%) structural
differences are noted between HsQSOX1 and MmQSOX1 which affect
species specificity of the antibody. In order to broaden antibody
species specificity, the present inventors found that relieving
steric clashes between the anti-QSOX1 antibody and its target is
necessary but not sufficient to extend binding to a distinct
ortholog of the target human QSOX1 and that additional favorable
interactions have to be introduced. These interactions are
accomplished by altering the association between the heavy and
light antibody chains, which recruits CDRs L1 and L2 into the
antibody-MmQSOX1 interface. Moreover, dual specificity is enabled
by substituting the constrained rings of aromatic residues with
more flexible side chains, including those that can participate in
water-bridged polar interactions. CDRs that recognize structurally
conserved elements are left in their natural state, preserving
specificity to QSOX1 antigens.
[0080] Thus, while reducing the present invention to practice, the
present inventors have performed a structure-guided modification of
the MAb492.1 antibody (which binds human QSOX1) to introduce point
mutations rendering the antibody (termed MAb492gen) capable of
binding the mouse QSOX1 ortholog. The present inventors have also
generated a new anti-QSOX1 inhibitory monoclonal antibody targeting
murine QSOX1. These antibodies can be used in in vivo pre-clinical
trials overcoming the barriers of species cross-reactivity as well
as for various biotechnological applications.
[0081] As is shown herein below and in the Examples section which
follows, the present inventors have used structure-guided
engineering to expand the antibody species specificity, improving
its affinity towards mouse QSOX1 by at least four orders of
magnitude (Tables 3 and 4, FIGS. 3A-3L). A crystal structure of the
re--engineered variant, complexed with its mouse antigen, revealed
that the antibody accomplishes dual-species targeting through
altered contacts between its heavy and light chains, plus
replacement of bulky aromatics by flexible side chains and
versatile water-bridged polar interactions (FIGS. 5A-5F). The to
present inventors have also produced an antibody targeting mouse
QSOX1, termed MAb316.1, which exhibits a new QSOX1 inhibition mode
(Table 2, FIGS. 6A-6C). Thus, while MAb492.1 and MAb492gen inhibit
QSOX1 by blocking substrate access to the Trx CXXC redox-active
site (FIG. 1B), Fab316.1 envelopes the helix containing the Trx
redox-active site at its amino terminus but does not block access
to the CXXC motif itself (FIG. 6B). Accordingly, MAb316.1 inhibits
QSOX1 by physically preventing formation of the inter-domain
electron-transfer intermediate of MmQSOX1 (FIG. 6C), thus
interrupting a different step in the catalytic cycle than MAb492.1
and MAb492gen (transition from state 2 to 3, rather than 1 to 2 in
FIG. 1C).
[0082] These discoveries provide the framework for generating
cross-species-specific antibodies capable of binding and inhibiting
mouse and human QSOX1 for pre-clinical trials for better qualifying
antibodies for future therapeutic applications.
[0083] Thus, according to one aspect of the present invention there
is provided an antibody comprising an antigen recognition domain
exhibiting species cross reactivity to human QSOX1 and murine
QSOX1.
[0084] As used herein, the term "QSOX1" relates to the Quiescin
Sulfhydryl Oxidase 1. The protein accession number for the long
variant of human QSOX1 on the NCBI database is NP_002817 (SEQ ID
NO: 3), and the accession number for the short form of human QSOX1
is NP_001004128 (SEQ ID NO: 4). The protein accession number for
the long variant of mouse QSOX1 on the NCBI database is
NM_001024945.1 (SEQ ID NO: 34), and the accession number for the
short form of mouse QSOX1 is NM_023268.2 (SEQ ID NO: 35).
[0085] The antibody of the present invention exhibits species cross
reactivity to human QSOX1 and murine QSOX1 (e.g., mouse QSOX1).
[0086] The term "species cross reactivity" as used herein refers to
binding of the antigen recognition domain described herein to the
same target molecule in humans and mice (i.e. QSOX1) with
substantially the same affinity (as determined by a specific
affinity assay e.g., BiaCore, ELISA). According to one embodiment,
the binding of the antigen recognition domain described herein to
the same target molecule in humans and mice (i.e. QSOX1) results in
the same activity (e.g., inhibition of QSOX1 activity).
[0087] As used herein "substantially the same affinity" refers to a
binding affinity which is the same or within one order of magnitude
difference, as determined in the same, binding assay.
[0088] According to one embodiment, the affinity of the antibody to
human QSOX1 is characterized by a K.sub.D of 0.1-100 nM, K.sub.D of
0.1-50 nM, K.sub.D of 0.1-10 nM, K.sub.D of 1-10 nM, K.sub.D of
0.1-5 nM, K.sub.D of 1-5 nM, K.sub.D of 1-3 nM, K.sub.D of 1.5-2
nM, or K.sub.D of 1.6 nM.
[0089] According to one embodiment, the affinity of the antibody to
mouse QSOX1 is characterized by a K.sub.D of 0.1-100 nM, K.sub.D of
0.1-50 nM, K.sub.D of 0.1-10 nM, K.sub.D of 1-10 nM, K.sub.D of
0.1-5 nM, K.sub.D of 1-5 nM, K.sub.D of 2-3 nM, K.sub.D of 2-2.5
nM, or K.sub.D of 2.2 nM.
[0090] Thus, "species cross reactivity" is to be understood as an
interspecies reactivity to the same molecule (i.e. QSOX1) of
different species, but not to a molecule other than QSOX1.
[0091] Species cross reactivity of an antibody recognizing human
QSOX1 and mouse QSOX1 can be determined, for example, by FACS
analysis. The FACS analysis is carried out in a way that the
respective antibody is tested for binding to the human and mouse
QSOX1 proteins. For example, a yeast-surface display may be used
for expression of single-chain variable fragments (scFv) of the
antibody tested. Binding of scFv displayed on cells to soluble
QSOX1 (e.g., human or mouse) can be monitored using FACS analysis,
e.g., utilizing streptavidin-APC fluorescence. Alternatively,
species cross reactivity may be determined by any other method
known in the art, e.g., by ELISA.
[0092] The term "antibody" as used in this invention includes
intact molecules as well as functional fragments thereof (such as
Fab, F(ab')2, Fv, scFv, dsFv, or single domain molecules such as VH
and VL) that are capable of binding to an epitope of an
antigen.
[0093] The term "isolated" refers to at least partially separated
from the natural environment e.g., from a hybridoma cell.
[0094] Suitable antibody fragments for practicing some embodiments
of the invention include a complementarity-determining region (CDR)
of an immunoglobulin light chain (referred to herein as "light
chain"), a complementarity-determining region of an immunoglobulin
heavy chain (referred to herein as "heavy chain"), a variable
region of a light chain, a variable region of a heavy chain, a
light chain, a heavy chain, an Fd fragment, and antibody fragments
comprising essentially whole variable regions of both light and
heavy chains such as an Fv, a single chain Fv (scFv), a
disulfide-stabilized Fv (dsFv), an Fab, an Fab', and an
F(ab')2.
[0095] As used herein, the terms "complementarily-determining
region" or "CDR" are used to interchangeably to refer to the
antigen binding regions found within the variable region of the
heavy and light chain polypeptides. Generally, antibodies comprise
three CDRs in each of the VH (CDR HI or HI; CDR H2 or H2; and CDR
H3 or H3) and three in each of the VL (CDR LI or LI; CDR L2 or L2;
and CDR L3 or L3).
[0096] The identity of the amino acid residues in a particular
antibody that make up a variable region or a CDR can be determined
using methods well known in the art and include methods such as
sequence variability as defined by Kabat et al. (See, e.g., Kabat
et al., 1992, Sequences of Proteins of immunological interest, 5th
ed., Public Health Service, NIH, Washington D.C.), location of the
structural loop regions as defined by Chothia et al. (see, e.g.,
Chothia et al., Nature 342:877-883, 1989.), a compromise between
Kabat and Chothia using Oxford Molecular's AbM antibody modeling
software (now Accelrys.RTM., see, Martin et al., 1989, Proc. Natl
Acad Sci USA. 86:9268; and world wide web site
www(dot)bioinf-org(dot)uk/abs), available complex crystal
structures as defined by the contact definition (see MacCallum et
al., J. Mol. Biol. 262:732-745, 1996) and the "conformational
definition" (see, e.g., Makabe et al., Journal of Biological
Chemistry, 283:1156-1166, 2008).
[0097] As used herein, the "variable regions" and "CDRs" may refer
to variable regions and CDRs defined by any approach known in the
art, including combinations of approaches.
[0098] Functional antibody fragments comprising whole or
essentially whole variable regions of both light and heavy chains
are defined as follows:
[0099] (i) Fv, defined as a genetically engineered fragment
consisting of the variable region of the light chain (VL) and the
variable region of the heavy chain (VH) expressed as two
chains;
[0100] (ii) single chain Fv ("seFv"), a genetically engineered
single chain molecule including the variable region of the light
chain and the variable region of the heavy chain, linked by a
suitable polypeptide linker as a genetically fused single chain
molecule;
[0101] (iii) disulfide-stabilized Fv ("dsFv"), a genetically
engineered antibody including the variable region of the light
chain and the variable region of the heavy chain, linked by a
genetically engineered disulfide bond;
[0102] (iv) Fab, a fragment of an antibody molecule containing a
monovalent antigen-binding portion of an antibody molecule which
can be obtained by treating whole antibody with the enzyme papain
to yield the intact light chain and the Fd fragment of the heavy
chain which consists of the variable and CH1 domains thereof;
[0103] (v) Fab', a fragment of an antibody molecule containing a
monovalent antigen-binding portion of an antibody molecule which
can be obtained by treating whole antibody with the enzyme pepsin,
followed by reduction (two Fab' fragments are obtained per antibody
molecule);
[0104] (vi) F(ab')2, a fragment of an antibody molecule containing
a monovalent antigen-binding portion of an antibody molecule which
can be obtained by treating whole antibody with the enzyme pepsin
(i.e., a dimer of Fab' fragments held together by two disulfide
bonds); and
[0105] (vii) Single domain antibodies or nanobodies are composed of
a single VH or VL domains which exhibit sufficient affinity to the
antigen.
[0106] Methods of producing polyclonal and monoclonal antibodies as
well as fragments thereof are well known in the art (See for
example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold
Spring Harbor Laboratory, New York, 1988, incorporated herein by
reference).
[0107] Antibody fragments according to some embodiments of the
invention can be prepared by proteolytic hydrolysis of the antibody
or by expression in E. coli or mammalian cells (e.g., Chinese
hamster ovary cell culture or other protein expression systems) of
DNA encoding the fragment. Antibody fragments can be obtained by
pepsin or papain digestion of whole antibodies by conventional
methods. For example, antibody fragments can be produced by
enzymatic cleavage of antibodies with pepsin to provide a 5S
fragment denoted F(ab')2. This fragment can be further cleaved
using a thiol reducing agent, and optionally a blocking group for
the sulfhydryl groups resulting from cleavage of disulfide
linkages, to produce 3.5S Fab' monovalent fragments. Alternatively,
an enzymatic cleavage using pepsin produces two monovalent Fab'
fragments and an Fc fragment directly. These methods are described,
for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647,
and references contained therein, which patents are hereby
incorporated by reference in their entirety. See also Porter, R. R.
[Biochem. J. 73: 119-126 (1959)], Other methods of cleaving
antibodies, such as separation of heavy chains to form monovalent
light-heavy chain fragments, further cleavage of fragments, or
other enzymatic, chemical, or genetic techniques may also be used,
so long as the fragments bind to the antigen that is recognized by
the intact antibody.
[0108] Fv fragments comprise an association of VH and VL chains.
This association may be noncovalent, as described in Inbar et al,
[Proc. Nat'l Acad. Sci. USA 69:2659-62 (19720]. Alternatively, the
variable chains can be linked by an intermolecular disulfide bond
or cross-linked by chemicals such as glutaraldehyde. Preferably,
the Fv fragments comprise VH and VL chains connected by a peptide
linker. These single-chain antigen binding proteins (sFv) are
prepared by constructing a structural gene comprising DNA sequences
encoding the VH and VL domains connected by an oligonucleotide. The
structural gene is inserted into an expression vector, which is
subsequently introduced into a host cell such as E. coli. The
recombinant host cells synthesize a single polypeptide chain with a
linker peptide bridging the two V domains. Methods for producing
sFvs are described, for example, by [Whitlow and Filpula, Methods
2: 97-105 (1991); Bird et al., Science 242:423-426 (1988); Pack et
al., Bio/Technology 11:1271-77 (1993); and U.S. Pat. No. 4,946,778,
which is hereby incorporated by reference in its entirety.
[0109] Another form of an antibody fragment is a peptide coding for
a single complementarity-determining region (CDR). CDR peptides
("minimal recognition units") can be obtained by constructing genes
encoding the CDR of an antibody of interest. Such genes are
prepared, for example, by using the polymerase chain reaction to
synthesize the variable region from RNA of antibody-producing
cells. See, for example, Larrick and Fry [Methods, 2:106-10
(1991)].
[0110] Humanized forms of non-human (e.g., murine) antibodies are
chimeric molecules of immunoglobulins, immunoglobulin chains or
fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other
antigen-binding subsequences of antibodies) which contain minimal
sequence derived from non-human immunoglobulin. Humanized
antibodies include human immunoglobulins (recipient antibody) in
which residues form a complementary determining region (CDR) of the
recipient are replaced by residues from a CDR of a non-human
species (donor antibody) such as mouse, rat or rabbit having the
desired specificity, affinity and capacity. In some instances, Fv
framework residues of the human immunoglobulin are replaced by
corresponding non-human residues. Humanized antibodies may also
comprise residues which are found neither in the recipient antibody
nor in the imported CDR or framework sequences. In general, the
humanized antibody will comprise substantially all of at least one,
and typically two, variable domains, in which all or substantially
all of the CDR regions correspond to (hose of a non-human
immunoglobulin and all or substantially all of the FR regions are
those of a human immunoglobulin consensus sequence. The humanized
antibody optimally also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann
et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct.
Biol., 2:593-596 (1992)].
[0111] Methods for humanizing non-human antibodies are well known
in the art. Generally, a humanized antibody has one or more amino
acid residues introduced into it from a source which is non-human.
These non-human amino acid residues are often referred to as import
residues, which are typically taken from an import variable domain.
Humanization can be essentially performed following the method of
Winter and co-workers [Jones et al., Nature, 321:522-525 (1986);
Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al.,
Science, 239:1534-1536 (1988)], by substituting rodent CDRs or CDR
sequences for the corresponding sequences of a human antibody.
Accordingly, such humanized antibodies are chimeric antibodies
(U.S. Pat. .No. 4,816,567), wherein substantially less than an
intact human variable domain has been substituted by the
corresponding sequence from a non-human species. In practice,
humanized antibodies are typically human antibodies in which some
CDR residues and possibly some FR residues are substituted by
residues from analogous sites in rodent antibodies.
[0112] Human antibodies can also be produced using various
techniques known in the art, including phage display libraries
[Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et
al., J. Mol. Biol., 222:581 (1991)]. The techniques of Cole et al.
and Boerner et al. are also available for the preparation of human
monoclonal antibodies [Cole et al., Monoclonal Antibodies and
Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boemer et al., J.
Immunol., 147(1):86-95 (1991)]. Similarly, human antibodies can be
made by introduction of human immunoglobulin loci into transgenic
animals, e.g., mice in which the endogenous immunoglobulin genes
have been partially or completely inactivated. Upon challenge,
human antibody production is observed, which closely resembles that
seen in humans in all respects, including gene rearrangement,
assembly, and antibody repertoire. This approach is described, for
example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825;
5,625,126; 5,633,425; 5,661,016, and in the following scientific
publications: Marks et al., Bio/Technology 10,: 779-783 (1992);
Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368
812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51
(1996); Neuberger, Nature Biotechnology 14: 826 (1996); and Lonberg
and Huszar, Intern. Rev. Immunol. 13, 65-93 (1995).
[0113] According to one embodiment, there is provided a method of
producing an antibody which comprises an antigen recognition domain
exhibiting species cross reactivity to QSOX1, the method
comprising: (a) introducing at least one point mutation in the
antigen binding domain of an antibody which binds QSOX1 of a first
species, which at least one point mutation increases the
water-mediated hydrogen bonding in the interaction interface
between the antigen binding domain of the antibody and QSOX1 of the
first species, and alternatively or additionally the at least one
point mutation reduces aromatic interactions between the antigen
binding domain of the antibody and QSOX1 of the first species,
wherein the at least one point mutation does not substantially
affect affinity of the antibody to QSOX1 of the first species; and
(b) testing binding of the antibody having the at least one point
mutation to QSOX1 of a second species, wherein when the antibody
having the at least one point mutation binds QSOX1 of the first
species and the second species with substantially the same affinity
the antibody is considered having cross reactivity to QSOX1.
[0114] Accordingly, any antibody which binds a first species (e.g.,
human) QSOX1 may be used. Such antibodies may be obtained
commercially (e.g., from Merck Millipore, Atlas Antibodies, MBL
International or Thermo Scientific Pierce Antibodies), or may be
produced by any method known in the art, e.g., as described below,
provided that their coding sequence can be elucidated.
[0115] According to one embodiment, anti-QSOX1 monoclonal
antibodies may be produced e.g., in murine animals, such as in
mice, by first immunizing with an emulsion of recombinant human
QSOX1 and Complete Freund's adjuvant (e.g., obtained from
DifcoLboratories). For instance, mice may be immunized three, four,
or five times at intervals of two or three weeks. QSOX1 peptides
used for immunization may comprise between 50-100 amino acids,
between 50-150 amino acids, between 50-200 amino acids, between
50-232 amino acids, between 100-200 amino acids or between 150-232
amino acids.
[0116] According to one embodiment, immunization can be done using
a fragment spanning 33 to 546 of Homo sapiens QSOX1, which
comprises all the functional domains of the enzyme.
[0117] According to one embodiment, immunization can be done using
a fragment spanning residues 33 to 272 of Homo sapiens QSOX1.
[0118] According to one embodiment, immunization can be done using
the Trx module of QSOX1 (e.g., two Trx domains) or using the Erv
module of QSOX1 (e.g., two Erv domains).
[0119] According to one embodiment, immunization can be done using
the full-length QSOX1.
[0120] Next, spleen cells from selected mice are fused with myeloma
cells (e.g., NSO myeloma cells) using polyethylene glycol.
Hybridoma cells are then selected by a selection medium (e.g., HAT
medium) and supernatants of the cells (i.e. comprising the
antibodies) are screened for specific binding to human QSOX1 and/or
inhibition thereof.
[0121] In order to generate antibodies with species cross
reactivity (e.g., to human and mouse QSOX1), at least one amino
acid sequence of the antibody is modified (e.g., by point
mutations, deletions and/or insertions), wherein the modification
enables functional flexibility, i.e., flexibility in the
interaction interface of the antibody with an antigen, enabling
interaction with more than one antigen (e.g., QSOX1 of different
species).
[0122] As detailed in the Examples section which follows, the amino
acid sequence of the antibody may be modified to comprise a lower
content of aromatic amino acids (e.g., tyrosines) as compared to
that of a species-specific antibody to the human QSOX1. The
aromatic amino acids may be replaced by more flexible amino acids
(i.e. amino acids comprising flexible side chains). Furthermore,
the amino acid of the antibody may be modified to include at least
one charged amino acid (i.e., amino acid which comprises a charged
residue). It will be appreciated that charged amino acids comprise
energetically favorable contact h water and accordingly can
participate in water-bridged polar interactions between the
antibody and QSOX1.
[0123] Thus, according to one embodiment, in order to generate
antibodies with species cross reactivity an antibody recognizing
human QSOX1 is modified in at least one amino acid residue.
[0124] According to one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12 or more point mutations are generated in the amino acid
sequence of the antibody.
[0125] According to one embodiment, the point mutation is generated
in the heavy chain of the antibody.
[0126] According to one embodiment, the point mutation is generated
in the light chain of the antibody. According to an embodiment, the
point mutation may be a plurality of point mutations in the heavy
chain and in the light chain of the antibody.
[0127] According to one embodiment, the at least one point mutation
is at the antigen binding domain of the antibody.
[0128] According to one embodiment, the at least one point mutation
is in a CDR sequence of the antibody.
[0129] According to one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12 or more point mutations are generated in CDR sequences of
the antibody.
[0130] According to a specific embodiment, a point mutation is
generated in the heavy chain variable region of the antibody, e.g.,
in CDR1 CDR2 and/or CDR3.
[0131] According to specific embodiment, a point mutation is
generated in the light chain variable region of the antibody, e.g.,
in CDR1, CDR2 and/or CDR3.
[0132] According to one embodiment, the at least one point mutation
which reduces aromatic interactions is in an aromatic amino acid
(e.g. tyrosine). Accordingly, aromatic amino acids within the amino
acid sequence of the antibody may be replaced by more flexible
amino acids (as discussed in detail below).
[0133] According to one embodiment, the at least one point mutation
which reduces aromatic interactions comprises an amino acid
comprising a flexible side chain.
[0134] According to one embodiment, the amino acid sequence of the
antibody comprises a lower content of aromatic amino acids (e.g.,
tyrosines) as compared to that of a species-specific antibody to
the human QSOX1.
[0135] According to one embodiment, the amino acid sequence of the
antibody comprises a lower content of aromatic amino acids (e.g.,
tyrosines) in CDRs of the antigen recognition domain as compared to
that of a species-specific antibody to the human QSOX1.
[0136] A lower content of aromatic amino acids may include 1, 2, 3,
4, or 5 fewer aromatic amino acids (e.g., tyrosines) in the amino
acid sequence of the antibody as compared to that of a
species-specific antibody to the human QSOX1. (e.g., mAb492.1).
[0137] Aromatic amino acids include, for example, tyrosine,
phenylalanine, histidine and tryptophan.
[0138] According to a specific embodiment, the amino acid sequence
of the antibody comprises a lower content of tyrosines as compared
to that of a species-specific antibody to the human QSOX1.
[0139] According to a specific embodiment, the amino acid sequence
of the antibody comprises a lower content of tyrosines in CDRs of
the antigen recognition domain as compared to that of a
species-specific antibody to the human QSOX1.
[0140] A lower content of tyrosines may include 1, 2, 3, 4, or 5
fewer tyrosines in the amino acid sequence of the antibody as
compared to that of a species-specific antibody to the human QSOX1
(e.g., mAb492.1).
[0141] According to another specific embodiment, the amino acid
sequence of the antibody comprises a lower content of aromatic
amino acids (e.g., tyrosine) in CDR3 of the variable heavy chain of
the antibody (as compared to that of a species-specific antibody to
the human QSOX1). Additionally or alternatively, the amino acid
sequence of the antibody comprises a lower content of aromatic
amino acids (e.g., tyrosine) in CDR2 of the variable light chain of
the antibody (as compared to that of a species-specific antibody to
the human QSOX1).
[0142] According to another specific embodiment, tyrosine at
position 99 of the variable heavy chain of the antibody as set
forth in SEQ ID NO: 37 is replaced by a point mutation.
[0143] Additionally or alternatively, tyrosine at position 100 of
the variable heavy chain of the antibody as set forth in SEQ ID NO:
37 is replaced by a point mutation. Additionally or alternatively,
tyrosine at position 53 of the variable light chain of the antibody
as set forth in SEQ ID NO: 36 is replaced by a point mutation.
[0144] According to another specific embodiment, aromatic amino
acids (e.g., tyrosine) are replaced by more flexible amino acids
(i.e. amino acids comprising flexible side chains), including, but
not limited to glutamine, methionine, arginine, lysine, aspartate,
glutamate, and serine.
[0145] According to another specific embodiment, tyrosine at
position 99 of the variable heavy chain of the antibody as set
forth in SEQ ID NO: 37 is replaced by a serine. Additionally or
alternatively, tyrosine at position 100 of the variable heavy chain
of the antibody as set forth in SEQ ID NO: 37 is replaced by a
methionine. Additionally or alternatively, tyrosine at position 53
of the variable light chain of the antibody as set forth in SEQ ID
NO: 36 is replaced by a glutamine.
[0146] As discussed above, in order to improve antibody binding to
murine QSOX1, the antibody of the invention may alternatively or
additionally be modified to include at least one charged amino acid
residue.
[0147] According to one embodiment, the at least one point mutation
which increases the water-mediated hydrogen bonding comprises at
least one charged or polar (e.g. non-charged polar) amino acid.
[0148] According to one embodiment, the amino acid sequence of the
antibody comprises at least 1, 2, 3 or more charged or polar amino
acids in the amino acid sequence of the antibody as compared to
that of a species-specific antibody to human QSOX1 (e.g.,
mAb492.1).
[0149] According to one embodiment, the amino acid sequence of the
antibody comprises at least one charged or polar amino acid in a
CDR of the antigen recognition domain of the antibody.
[0150] According to one embodiment, the amino acid sequence of the
antibody comprises at least one charged or polar amino acid in CDR3
of a variable heavy chain of the antibody.
[0151] Charged amino acids (i.e. amino acids which comprise a
charged residue) include, but are not limited to, lysine, arginine,
aspartic acid, histidine and glutamic acid.
[0152] Polar amino acids include, but are not limited to, serine,
threonine, asparagine, glutamine, histidine and tyrosine.
[0153] According to one embodiment, the at least one point mutation
which increases the water-mediated hydrogen bonding is an aspartic
acid.
[0154] According to a specific embodiment, the amino acid sequence
of the antibody comprises aspartic acid in CDR3 of a variable heavy
chain of the antibody (e.g., at position 101 of a variable heavy
chain amino acid sequence of the antibody as set forth in SEQ ID
NO: 45).
[0155] According to one embodiment, the amino acid sequence of the
antibody comprises a serine, methionine, aspartic acid, proline
(i.e. SMDP) sequence in CDR3 of the variable heavy chain of the
antibody (e.g., at positions 99-102 of a variable heavy chain amino
acid sequence of the antibody as set forth in SEQ ID NO: 45).
[0156] The modification to the antibody's amino acid sequence is
typically carried out using the minimal number of amino acid
alteration in order to increase binding affinity to and optimally
inhibit mouse QSOX1 without substantially losing binding affinity
to human QSOX1. (i.e. binding affinity which is the same or within
one order of magnitude difference, as determined in the same
binding assay).
[0157] In order to select an antibody with species cross
reactivity, the antibody generated according to the teachings of
some embodiments of the invention is tested for binding of the
antibody having the at least one point mutation to QSOX1 of a
second species, wherein when the antibody having the at least one
point mutation binds QSOX1 of the first species and the second
species with substantially the same affinity the antibody is
considered having cross reactivity to QSOX1 .
[0158] Selecting antibodies which bind to a first species (e.g.,
human) QSOX1 and a second species (e.g., mouse) QSOX1. (i.e.
species cross reactivity) can be carried out using any method known
in the art (e.g., by a specific affinity assay using, for example,
BiaCore, ELISA or FACS analyses as described in detail hereinabove)
as long as the same assay and conditions are used for both species
human and murine) QSOX1.
[0159] Large scale antibodies may then be produced using, for
example, a miniPERM bioreactor (Sarstedt) in serum--free medium
(DCCM).
[0160] Thus, the teachings of the present invention provide for an
antibody comprising an antigen recognition domain which binds to
human QSOX1 and murine QSOX1.
[0161] According to one aspect of the invention, there is provided
an antibody comprising an antigen recognition domain exhibiting
species cross reactivity to human QSOX1 and murine QSOX1, the
antigen recognition domain comprising complementarity determining
regions (CDR) as set forth in SEQ ID NOs: 46-51.
[0162] According to a specific embodiment, the antibody of the
present invention is a monoclonal antibody (MAb).
[0163] According to a specific embodiment, CDRs 1-3 (SEQ ID NOs:
46-48 respectively) are located on the light chain of the MAb
antibody.
[0164] According to another specific embodiment, CDRs 1-3 (SEQ ID
NOs: 49-51, respectively) are located on the heavy chain of the MAb
antibody.
[0165] According to a specific embodiment the MAb antibody is
MAb492gen.
[0166] According to another embodiment, the antibody of the present
invention is a single chain antibody.
[0167] According to another specific embodiment, CDRs 1-3 (SEQ ID
NOs: 46-48, respectively) are located on the light chain of the
single chain antibody.
[0168] According to another specific embodiment, CDRs 1-3 (SEQ ID
NOs: 49-51, respectively) are located on the heavy chain of the
single chain antibody.
[0169] An exemplary single chain antibody which may be used in
accordance with the present teachings is scFv492gen.
[0170] According to another embodiment scFv492gen comprises CDRs
set forth SEQ ID NOs: 46-51.
[0171] According to another embodiment, the antibody of the present
invention is a fragment antigen-binding (Fab) antibody.
[0172] According to another specific embodiment, CDRs 1-3 (SEQ ID
NOs: 46-48, respectively) are located on the :light chain of the
Fab antibody.
[0173] According to another specific embodiment, CDRs 1-3 (SEQ ID
NOs: 49-51, respectively) are located on the heavy chain of the Fab
antibody.
[0174] An exemplary Fab antibody which may be used in accordance
with the present teachings is Fab492gen.
[0175] According to anther embodiment Fab492gen comprises CDRs set
forth in SEQ ID NOs: 46-51.
[0176] According to anther embodiment, an isolated antibody of the
present invention comprises the amino acid sequence as set forth in
SEQ ID NOs: 44 and 45.
[0177] According to some embodiments of the invention, the amino
acid sequence comprises an amino acid sequence having at least 80%,
at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology or
identity to the peptide set forth in SEQ ID NOs: 44 and 45, wherein
the antibody is capable of binding but an QSOX1 and mouse QSOX1
according to the cross reactivity definition.
[0178] Homology (e.g., percent homology, identity+similarity) can
be determined using any homology comparison software, including for
example, the BlastP or TBLASTN software of the National Center of
Biotechnology Information (NCBI) such as by using default
parameters, when starting from a polypeptide sequence; or the
tBLASTX algorithm (available via the NCBI) such as by using default
parameters, which compares the six-frame conceptual translation
products of a nucleotide query sequence (both strands) against a
protein sequence database.
[0179] For example, default parameters for tBLASTX include: Max
target sequences: 100; Expected threshold: 10; Word size: 3; Max
matches in a query range: 0; Scoring parameters: Matrix--BLOSUM62;
filters and masking: Filter--low complexity regions.
[0180] The teachings of the present invention also provide for an
antibody capable of binding mouse QSOX1.
[0181] According to one aspect of the invention, there is provided
an antibody comprising an antigen recognition domain comprising
CDRs as set forth in SEQ ID NOs: 26-31, wherein the antibody
specifically binds murine QSOX1.
[0182] According to a specific embodiment, the antibody capable of
binding murine QSOX1. of the present invention is a monoclonal
antibody (MAb).
[0183] According to a specific embodiment, CDRs 1-3 (SEQ ID NOs:
26-28, respectively) are located on the light chain of the MAb
antibody.
[0184] According to a specific embodiment CDRs 1-3 (SEQ ID NOs:
29-31, respectively) are located on the heavy chain of the MAb
antibody.
[0185] An exemplary monoclonal antibody which may be used in
accordance with the present teachings is MAb316.1.
[0186] According to a specific embodiment, MAb316.1 comprises CDRs
set forth in SEQ ID NOs: 26-31.
[0187] According to another embodiment, an isolated antibody of the
present invention comprises the amino acid sequence as set forth in
SEQ ID NOs: 9 and 10.
[0188] According to some embodiments of the invention, the amino
acid sequence comprises an amino acid sequence having at least 80%,
at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology
or identity to the peptide set forth in SEQ ID NOs: 9 and 10.
wherein the antibody is capable of binding mouse QSOX1.
[0189] The antibodies and antibody fragments generated according to
the teachings of the present invention serve as inhibitors of
QSOX1.
[0190] According to one embodiment of the invention, the antibodies
or antibody fragments inhibit QSOX1 activity in mediating laminin
incorporation in the basement membrane, e.g., laminin assembly that
supports cell migration.
[0191] As used herein, the term "laminin" refers to a human laminin
protein. Typically laminins are trimeric proteins that contain an
.alpha.-chain, a .beta.-chain, and a .gamma.-chain (found in five,
four, and three genetic variants, respectively). Thus, the term
laminin as used herein encompasses any type of human laminin,
including any of the different chain combinations or any individual
subunits of laminin. The different chains and trimer molecules
differ with respect to their tissue distribution apparently
reflecting diverse functions in vivo. Exemplary laminin subunits of
the present invention include, but are not limited to, LAMA1,
LAMA2, LAMA3, LAMA4, LAMA5, LAMB1, LAMB2, LAMB3, LAMB4, LAMC1,
LAMC2 and LAMC3.
[0192] According to an embodiment of the present invention, the
laminin comprises an alpha 4 chain.
[0193] According to a specific embodiment, the laminin is
laminin-411 or laminin- 421.
[0194] The term "laminin assembly" refers to the incorporation of
laminin proteins into the basal lamina (i.e. one of the layers of
the basement membrane). Typically, laminin is secreted from cells
fibroblasts, epithelial cells, tumor cells) and is incorporated
into cell-associated extracellular matrices where they form
independent networks and are associated with type IV collagen
networks via entactin, fibronectin and perlecan.
[0195] The term "basement membrane" or "laminin-comprising basement
membrane" refers to the thin layer of fibers which anchors and
supports the epithelium and endothelium and comprises the basal
lamina (i.e. comprising laminin).
[0196] The phrase "inhibiting or preventing laminin assembly"
refers to reducing, reversing, attenuating, minimizing, suppressing
or halting laminin assembly in a basement membrane. According to
one embodiment, inhibiting or preventing laminin assembly is by at
least about 10%, by at least about 20%, by at least about 30%, by
at least about 40%, by at least about 50%, by at least about 60%,
by at least about 70%, by at least about 80%, by at least about 90%
or by about 100%, as compared to laminin assembly in the absence of
the anti-QSOX1 antibody or antibody fragment (as described
hereinabove). Thus, according to an embodiment of the invention
laminin is not incorporated into the basement membrane.
[0197] Laminin which is not incorporated into the basal membrane
can be found in soluble form (e.g., in the culture medium of in
vitro cultured cells). Thus, monitoring reduction in laminin
assembly can be monitored by e.g., immunofluorescence (IF) staining
of the extracellular matrix or by Western blotting of the soluble
laminin (i.e, that which was not incorporated into the basal
membrane).
[0198] According to an embodiment of the invention, the activity of
the antibody in inhibiting QSOX1 activity is assayed by at least
one of an immunofluorescence (IF) staining assay of the
extracellular matrix or Western blot assay for soluble laminin
(i.e. that which is not incorporated into the basal membrane, as
further described in the Examples section which follows).
[0199] It will be appreciated that inhibiting or preventing laminin
assembly may be advantageous in situations in which excess
connective tissue is produced in a non-structured manner in an
organ or tissue in a reparative or reactive process, such as
fibrosis. Thus, while further reducing the present invention to
practice, inhibition of QSOX1 and subsequently generation of
soluble laminin may be therapeutic for fibrotic processes.
[0200] It will be appreciated that laminins are an important
biologically active part of the basal lamina and basal membrane
influencing cell adhesion, signaling, migration, phenotype,
differentiation and survival. An exemplary cell migration of the
present invention comprises tumor cell migration leading to
metastasis.
[0201] Accordingly, inhibiting or preventing laminin assembly may
be advantageous in situations in which inhibition of cell migration
is warranted. The cell may comprise, for example, a brain cell, a
neuron, a cardiac cell, a muscle cell, a skin cell, a bone cell, a
pancreatic cell, a liver cell, a kidney cell, an intestinal cell, a
spleen cell, a respiratory cell, a lung cell, a lymphocyte or a
monocyte. The cell of the present invention may comprise a healthy
cell or may alternately comprise a mutated cell (e.g., a tumor
cell).
[0202] According to one embodiment, inhibiting or preventing cell
migration refers to reducing, reversing, attenuating, minimizing,
suppressing or halting migration of a cell (e.g., tumor cell) via a
laminin-comprising basement membrane.
[0203] According to one embodiment, inhibiting or preventing cell
migration is by at least about 10%, by at least about 20%, by at
least about 30%, by at least about 40%, by at least about 50 by at
least about 60%, by at least about 70%, by at least about 80%, by
at least about 90% or by at least about 100%, as compared to cell
migration via a laminin-comprising basement membrane in the absence
of the anti-QSOX1 antibody or antibody fragment (as described
hereinabove). Thus, according to an embodiment of the invention
cell migration is completely inhibited through the basement
membrane.
[0204] The methods of the present invention (e.g., inhibiting cell
migration) may be effected in vitro, in vivo or ex vivo.
[0205] As mentioned, the ability to modulate cell migration can be
used as a therapeutic modality.
[0206] Accordingly, one specific use for the antibodies of the
present invention is for preventing or treating a
laminin-associated disease or condition in a subject in need
thereof.
[0207] The phrase "preventing or treating" refers to inhibiting or
arresting the development of a disease, disorder or condition
and/or causing the reduction, remission, or regression of a
disease, disorder or condition or keeping a disease, disorder or
medical condition from occurring in a subject who may be at risk
for the disease disorder or condition, but has not yet been
diagnosed as having the disease disorder or condition. Those of
skill in the art will understand that various methodologies and
assays can be used to assess the development of a disease, disorder
or condition, and similarly, various methodologies and assays may
be used to assess the reduction, remission or regression of a
disease, disorder or condition.
[0208] As used herein, the term "subject" refers to an animal,
preferably a mammal, most preferably a human being, including both
young and old human beings of both genders who suffer from or are
predisposed to a laminin-associated disease or condition.
[0209] As used herein, the term "laminin-associated disease or
condition" refers to a disease or condition in which laminin
function is associated with the onset or progression of a
disease.
[0210] According to one embodiment, e laminin-associated disease or
condition is a tumor.
[0211] Examples of tumors include, but are not limited to,
carcinoma, blastoma and sarcoma. Particular examples of cancerous
diseases but are not limited to: myeloproliferative diseases, such
as solid tumors benign meningioma, mixed tumors of salivary gland,
colonic adenomas; adenocarcinomas, such as small cell lung cancer,
kidney, uterus, prostate, bladder, ovary, colon, sarcomas,
liposarcoma, myxoid, synovial sarcoma, rhabdomyosarcoma (alveolar),
extraskeletal myxoid chonodrosarcoma, Ewing's tumor; other include
testicular and ovarian dysgerminoma, retinoblastoma, Wilms' tumor,
neuroblastoma, malignant melanoma, mesotheliorna, breast, skin,
prostate, and ovarian.
[0212] According to an embodiment, the tumor is a metastasizing
solid tumor (e.g., formed by metastatic cancer cells).
[0213] According to an embodiment, the tumor is an
adenocarcinoma.
[0214] According to one embodiment the tumor is a cancer.
[0215] Types of cancerous diseases amenable to treatment by the
methods of some embodiments of the invention include benign tumors,
warts, polyps, pre-cancers, and malignant tumors/cancers.
[0216] Specific examples of cancerous diseases which can be treated
using the methods of the present invention include, but are not
limited to, tumors of the gastrointestinal tract (colon carcinoma,
rectal carcinoma, colorectal carcinoma, colorectal cancer,
colorectal adenoma, hereditary nonpolyposis type 1, hereditary
nonpolyposis type 2, hereditary nonpolyposis type 3, hereditary
nonpolyposis type 6; colorectal cancer, hereditary nonpolyposis
type 7, small and/or large bowel carcinoma, esophageal carcinoma,
tylosis with esophageal cancer, stomach carcinoma, pancreatic
carcinoma, pancreatic endocrine tumors), endometrial carcinoma,
dermatofibrosarcoma protuberans, gallbladder carcinoma, Biliary
tract tumors, prostate cancer, prostate adenocarcinoma, renal
cancer (e.g., Wilms' tumor type 2. or type 1), liver cancer (e.g.,
hepatoblastoma, hepatocellular carcinoma, hepatocellular cancer),
bladder cancer, embryonal rhabdomyosarcoma, germ cell tumor,
trophoblastic tumor, testicular germ cells tumor, immature teratoma
of ovary, uterine, epithelial ovarian, sacrococcygeal tumor,
choriocarcinoma, placental site trophoblastic tumor, epithelial
adult tumor, ovarian carcinoma, serous ovarian cancer, ovarian sex
cord tumors, cervical carcinoma, uterine cervix carcinoma,
small-cell and non-small cell lung carcinoma, nasopharyngeal,
breast carcinoma (e.g., ductal breast cancer, invasive intraductal
breast cancer, sporadic; breast cancer, susceptibility to breast
cancer, type 4 breast cancer, breast cancer-1, breast cancer-3;
breast-ovarian cancer), squamous cell carcinoma (e.g., in head and
neck), neurogenic tumor, astrocytoma, ganglioblastoma,
neuroblastoma, lymphomas (e.g., Hodgkin's disease, non-Hodgkin's
lymphoma, B cell, Burkitt, cutaneous T cell, histiocytic,
lymphoblastic, T cell, thymic), gliomas, adenocarcinoma, adrenal
tumor, hereditary adrenocortical carcinoma, brain malignancy
(tumor), various other carcinomas (e.g., bronchogenic large cell,
ductal, Ehrlich-Lettre ascites, epidermoid, large cell, Lewis lung,
medullary, mucoepidermoid, oat cell, small cell, spindle cell,
spinocellular, transitional cell, undifferentiated, carcinosarcoma,
choriocarcinoma, cystadenocarcinoma), ependimoblastoma,
epithelioma, erythroleukemia (e.g., Friend, lymphoblast),
fibrosarcoma, giant cell tumor, glial tumor, glioblastoma (e.g.,
multiforme, astrocytorna), glioma hepatoma, heterohybridoma,
heteromyeloma, histiocytoma, hybridoma (e.g., B cell),
hypernephroma, insulinoma, islet tumor, keratoma,
leioniyoblastorna, leiomyosarcoma, leukemia (e.g., acute lymphatic,
acute lymphoblastic, acute lymphoblastic pre-B cell, acute
lymphoblastic T cell leukemia, acute- megakaryoblastic, monocytic,
acute myelogenous, acute myeloid, acute myeloid with eosinophilia,
B cell, basophilic, chronic myeloid, chronic, B cell, eosinophilic,
Friend, granulocytic or myelocytic, hairy cell, lymphocytic,
megakaryoblastic, monocytic, monocytic-macrophage, myeloblastic,
myeloid, myelomonocytic, plasma cell, pre-B cell, promyelocytic,
subacute, T cell, lymphoid neoplasm, predisposition to myeloid
malignancy, acute nonlymphocytic leukemia), lymphosarcoma,
melanoma, mammary tumor, mastocytoma, medulloblastoma,
mesothelioma, metastatic tumor, monocyte tumor, multiple myeloma,
myelodysplastic syndrome, myeloma, nephroblastoma, nervous tissue
glial tumor, nervous tissue neuronal tumor, neurinoma,
neuroblastoma, oligodendroglioma, osteochondroma, osteomyeloma,
osteosarcoma Ewing's), papilloma, transitional cell,
pheochromocytoma, pituitary tumor (invasive), plasmacytoma,
retinoblastoma, rhabdomyosarcoma, sarcoma (e.g., Ewing's,
histiocytic cell, Jensen, osteogenic, reticulum cell), schwannoma,
subcutaneous tumor, teratocarcinoma (e.g., pluripotent), teratoma,
testicular tumor, thymoma and trichoepithelioma, gastric cancer,
fibrosarcoma, glioblastoma multiforme; multiple glomus tumors,
Li-Fraumeni syndrome, liposarcoma, lynch cancer family syndrome II,
male germ cell tumor, mast cell leukemia, medullary thyroid,
multiple meningioma, endocrine neoplasia myxosarcoma,
paraganglioma, familial nonchromaffin, pilomatricoma, papillary,
familial and sporadic, rhabdoid predisposition syndrome, familial,
rhabdoid tumors, soft tissue sarcoma, and Turcot syndrome with
glioblastoma.
[0217] According to a specific embodiment of this aspect of the
present invention, the cancers which may be treated in accordance
with the present teachings, include but are not limited to,
prostate cancer, lung cancer, breast cancer, cervical cancer,
urachus cancer, vaginal cancer, colon cancer, esophagus cancer,
pancreatic cancer, throat cancer, stomach cancer and myeloid
leukemia.
[0218] According to one embodiment, the laminin-associated disease
or condition is associated with fibrosis.
[0219] The term "fibrosis" refers to a formation or a presence of
excess connective tissue in an organ or tissue. It may occur as a
repair or replacement response to a stimulus such as tissue injury
or inflammation.
[0220] Examples of disorders involving fibrosis include, but are
not limited to, liver fibrosis, pulmonary fibrosis, renal fibrosis,
pancreatic fibrosis, scleroderma, connective tissue diseases,
scarring, skin fibrosis, cardiac fibrosis, organ transplant,
vascular stenosis, restenosis, arterial fibrosis, arthrofibrosis,
breast fibrosis, muscle fibrosis, retroperitoneal fibrosis, thyroid
fibrosis, lymph node fibrosis, bladder fibrosis, pleural fibrosis
and COPD.
[0221] According to one embodiment, the laminin-associated disease
or condition is a bacterial disease, a viral disease or a parasitic
disease.
[0222] An exemplary parasitic disease which may be treated by the
teachings of the present invention includes African
trypanosomiasis.
[0223] According to the present teachings, in order to treat the
laminin-associated disease or condition, the subject is
administered with the anti-QSOX1 antibody (or antibody fragment) of
some embodiments of the invention, as further detailed
hereinabove.
[0224] Each of the antibody or antibody fragments described
hereinabove can be administered to the individual per se or as part
of a pharmaceutical composition which also includes a
physiologically acceptable carrier. The purpose of a pharmaceutical
composition is to facilitate administration of the active
ingredient to an organism.
[0225] As used herein a "pharmaceutical composition" refers to a
preparation of one or more of the active ingredients described
herein with other chemical components such as physiologically
suitable carriers and excipients. The purpose of a pharmaceutical
composition is to facilitate administration of a compound to an
organism.
[0226] Herein the term "active ingredient" refers to the anti-QSOX1
antibody or fragment thereof accountable for the biological
effect.
[0227] Hereinafter, the phrases "physiologically acceptable
carrier" and "pharmaceutically acceptable carrier" which may be
interchangeably used refer to a carrier or a diluent that does not
cause significant irritation to an organism and does not abrogate
the biological activity and properties of the administered
compound. An adjuvant is included under these phrases.
[0228] Herein the term "excipient" refers to an inert substance
added to a pharmaceutical composition to further facilitate
administration of an active ingredient. Examples, without
limitation, of excipients include calcium carbonate, calcium
phosphate, various sugars and types of starch, cellulose
derivatives, gelatin, vegetable oils and polyethylene glycols.
[0229] Techniques for formulation and administration of drugs may
be found in "Remington's Pharmaceutical Sciences," Mack Publishing
Co., Easton, Pa., latest edition, which is incorporated herein by
reference.
[0230] Suitable routes of administration may, for example, include
oral, rectal, transmucosal , especially transnasal, intestinal or
parenteral delivery, including intramuscular, subcutaneous and
intramedullary injections as well as intrathecal, direct
intraventricular, intracardiac, e.g., into the right or left
ventricular cavity, into the common coronary artery, intravenous,
intraperitoneal, intranasal, or intraocular injections.
[0231] Conventional approaches for drug delivery to the central
nervous system (CNS) include: neurosurgical strategies
intracerebral injection or intracerebroventricular infusion);
molecular manipulation of the agent (e.g., production of a chimeric
fusion protein that comprises a transport peptide that has an
affinity for an endothelial cell surface molecule in combination
with an agent that is itself incapable of crossing the BBB) in an
attempt to exploit one of the endogenous transport pathways of the
BBB; pharmacological strategies designed to increase the lipid
solubility of an agent (e.g., conjugation of water-soluble agents
to lipid or cholesterol carriers); and the transitory disruption of
the integrity of the BBB by hyperosmotic disruption (resulting from
the infusion of a mannitol solution into the carotid artery or the
use of a biologically active agent such as an angiotensin
peptide).
[0232] Alternately, one may administer the pharmaceutical
composition in a local rather than systemic manner, for example,
via injection of the pharmaceutical composition directly into a
tissue region of a patient.
[0233] Pharmaceutical compositions of some embodiments of the
invention may be manufactured by processes well known in the art,
e.g., by means of conventional mixing, dissolving, granulating,
dragee-making, levigating, emulsifying, encapsulating, entrapping
or lyophilizing processes.
[0234] Pharmaceutical compositions for use in accordance with some
embodiments of the invention thus may be formulated in conventional
manner using one or more physiologically acceptable carriers
comprising excipients and auxiliaries, which facilitate processing
of the active ingredients into preparations which, can be used
pharmaceutically. Proper formulation is dependent upon the route of
administration chosen.
[0235] For injection, the active ingredients of the pharmaceutical
composition may be formulated in aqueous solutions, preferably in
physiologically compatible buffers such as Hank's solution,
Ringer's solution, or physiological salt buffer. For transmucosal
administration, penetrants appropriate to the barrier to be
permeated are used in the formulation. Such penetrants are
generally known in the art.
[0236] For oral administration, the pharmaceutical composition can
be formulated readily by combining the active compounds with
pharmaceutically acceptable carriers well known in the art. Such
carriers enable the pharmaceutical composition to be formulated as
tablets, pills, dragees, capsules, liquids, gels, syrups, slurries,
suspensions, and the like, for oral ingestion by a patient.
Pharmacological preparations for oral use can be made using a solid
excipient, optionally grinding the resulting mixture, and
processing the mixture of granules, after adding suitable
auxiliaries if desired, to obtain tablets or dragee cores. Suitable
excipients are, in particular, fillers such as sugars, including
lactose, sucrose, mannitol, or sorbitol; cellulose preparations
such as, for example, maize starch, wheat starch, rice starch,
potato starch, gelatin, gum tragacanth, methyl cellulose,
hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or
physiologically acceptable polymers such as polyvinylpyrrolidone
(PVP). If desired, disintegrating agents may be added, such as
cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt
thereof such as sodium alginate.
[0237] Dragee cores are provided with suitable coatings. For this
purpose, concentrated sugar solutions may be used which may
optionally contain gum arabic, talc, polyvinyl pyrrolidone,
carbopol gel, polyethylene glycol, titanium dioxide, lacquer
solutions and suitable organic solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee
coatings for identification or to characterize different
combinations of active compound doses.
[0238] Pharmaceutical compositions which can be used orally,
include push-fit capsules made of gelatin as well as soft, sealed
capsules made of gelatin and a plasticizer, such as glycerol or
sorbitol. The push-fit capsules may contain the active ingredients
in admixture with filler such as lactose, binders such as starches,
lubricants such as talc or magnesium stearate and, optionally,
stabilizers, In soft capsules, the active ingredients may be
dissolved or suspended in suitable liquids, such as fatty oils,
liquid paraffin, or liquid polyethylene glycols. In addition,
stabilizers may be added. All formulations for oral administration
should be in dosages suitable for the chosen route of
administration.
[0239] For buccal administration, the compositions may take the
form of tablets or lozenges formulated in conventional manner.
[0240] For administration by nasal inhalation, the active
ingredients for use according to some embodiments of the invention
are conveniently delivered in the form of an aerosol spray
presentation from a pressurized pack or a nebulizer with the use of
a suitable propellant, e.g., dichlorodifluoromethane,
trichlorofluoromethane, dichlorotetrafluoroethane or carbon
dioxide. In the case of a pressurized aerosol, the dosage unit may
be determined by providing a valve to deliver a metered amount.
Capsules and cartridges of, e.g., gelatin for use in a dispenser
may be formulated containing a powder mix of the compound and a
suitable powder base such as lactose or starch.
[0241] The pharmaceutical composition described herein may be
formulated for parenteral administration, e.g., by bolus injection
or continuous infusion. Formulations for injection may be presented
in unit dosage form, e.g., in ampoules or in multidose containers
with optionally, an added preservative. The compositions may be
suspensions, solutions or emulsions in oily or aqueous vehicles,
and may contain formulatory agents such as suspending, stabilizing
and/or dispersing agents.
[0242] Pharmaceutical compositions for parenteral administration
include aqueous solutions of the active preparation in
water-soluble form. Additionally, suspensions of the active
ingredients may be prepared as appropriate oily or water based
injection suspensions. Suitable lipophilic solvents or vehicles
include fatty oils such as sesame oil, or synthetic fatty acids
esters such as ethyl oleate, triglycerides or liposomes. Aqueous
injection suspensions may contain substances, which increase the
viscosity of the suspension, such as sodium carboxymethyl
cellulose, sorbitol or dextran. Optionally, the suspension may also
contain suitable stabilizers or agents which increase the
solubility of the active ingredients to allow for the preparation
of highly concentrated solutions.
[0243] Alternatively, the active ingredient may be in powder form
for constitution with a suitable vehicle, e.g., sterile,
pyrogen-free water based solution, before use.
[0244] The pharmaceutical composition of some embodiments of the
invention may also be formulated in rectal compositions such as
suppositories or retention enemas, using, e.g., conventional
suppository bases such as cocoa butter or other glycerides.
[0245] Pharmaceutical compositions suitable for use in context of
some embodiments of the invention include compositions wherein the
active ingredients are contained in an amount effective to achieve
the intended purpose. More specifically, a therapeutically
effective amount means an amount of active ingredients (anti-QSOX1
antibody or fragment thereof) effective to prevent, alleviate or
ameliorate symptoms of a disorder (e.g., laminin-associated disease
or condition) or prolong the survival of the subject being
treated.
[0246] Determination of a therapeutically effective amount is well
within the capability of those skilled in the art, especially in
light of the detailed disclosure provided herein.
[0247] For any preparation used in the methods of the invention,
the therapeutically effective amount or dose can be estimated
initially from in vitro and cell culture assays. For example, a
dose can be formulated in animal models to achieve a desired
concentration or titer. Such information can be used to more
accurately determine useful doses in humans.
[0248] Animal models for laminin-associated diseases include, for
example, the murine animal model for liver fibrosis [see e.g.,
review paper by Hiromitsu Hayashi and Takao Sakail , Amer Journal
Physiol-GI(2011)300(5):G729-G738] and the murine animal model for
metastatic breast cancer [Anna Fantozzi and Gerhard Christofori,
Breast Cancer Research (2006) 8:212].
[0249] Toxicity and therapeutic efficacy of the active ingredients
described herein can be determined by standard pharmaceutical
procedures in vitro, in cell cultures or experimental animals. The
data obtained from these in vitro and cell culture assays and
animal studies can be used in formulating a range of dosage for use
in human. The dosage may vary depending upon the dosage form
employed and the route of administration utilized. The exact
formulation, route of administration and dosage can be chosen by
the individual physician in view of the patient's condition. (See
e.g., Fingl, et al., 1975, in "The Pharmacological Basis of
Therapeutics", Ch. 1 p.1).
[0250] Dosage amount and interval may be adjusted individually to
provide the active ingredient at a sufficient amount to induce or
suppress the biological effect (minimal effective concentration,
MEC). The MEC will vary for each preparation, but can be estimated
from in vitro data. Dosages necessary to achieve the MEC will
depend on individual characteristics and route of administration.
Detection assays can be used to determine plasma
concentrations.
[0251] Depending on the severity and responsiveness of the
condition to be treated, dosing can be of a single or a plurality
of administrations, with course of treatment lasting from several
days to several weeks or until cure is effected or diminution of
the disease state is achieved.
[0252] It will be appreciated that the antibodies of some
embodiments of the invention exhibiting cross-species reactivity
(e.g., recognizing both human and mouse QSOX1) are particularly
useful for preclinical trials to determine the therapeutic
effective amount, toxicity and the efficacy of the antibodies for
treatment. Furthermore, the anti-mouse QSOX1 antibodies of some
embodiments of the invention may also be used for preclinical
trials to determine the therapeutic effective amount, toxicity and
the efficacy of the antibodies for treatment.
[0253] The amount of a composition to be administered will, of
course, be dependent on the subject being treated, the severity of
the affliction, the manner of administration, the judgment of the
prescribing physician, etc.
[0254] Compositions of some embodiments of the invention may, if
desired, be presented in a pack or dispenser device, such as an FDA
approved kit, which may contain one or more unit dosage forms
containing the active ingredient. The pack may, for example,
comprise metal or plastic foil, such as a blister pack. The pack or
dispenser device may be accompanied by instructions for
administration. The pack or dispenser may also be accommodated by a
notice associated with the container in a form prescribed by a
governmental agency regulating the manufacture, use or sale of
pharmaceuticals, which notice is reflective of approval by the
agency of the form of the compositions or human or veterinary
administration. Such notice, for example, may be of labeling
approved by the U.S. Food and Drug Administration for prescription
drugs or of an approved product insert. Compositions comprising a
preparation of the invention formulated in a compatible
pharmaceutical carrier may also be prepared, placed in an
appropriate container, and labeled for treatment of an indicated
condition, as is further detailed above.
[0255] According to one embodiment, the antibody of some
embodiments of the invention is used in conjunction with another
agent capable of treating a laminin-associated disease or condition
in a subject (e.g. tumor). In such cases, the antibody may be
administered to the subject prior to, concomitantly with, or
following said other agent (e.g. within a time frame of 30 minutes,
1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48
hours, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 14 days, 21
days, 30 days, 60 days, 90 days or 120 days of each other).
[0256] Exemplary agents include, but are not limited to,
chemotherapeutic agents (e.g. cytotoxic drugs), hormonal
therapeutic agents, radiotherapeutic agents, anti-proliferative
agents, and combinations thereof.
[0257] Non-limiting examples of chemotherapeutic agents include,
but are not limited to, platinum-based drugs (e.g., oxaliplatin,
cisplatin, carboplatin, spiroplatin, iproplatin, satraplatin,
etc.), alkylating agents (e.g., cyclophosphamide, ifosfamide,
chlorambucil, busulfan, melphalan, mechlorethamine, uramustine,
thiotepa, nitrosoureas, etc.), anti-metabolites (e.g.,
5-fluorouracil, azathioprine, 6-mercaptopurine, methotrexate,
leucovorin, capecitabine, cytarabine, floxuridine, fludarabine,
gemcitabine (Gemzar.RTM.), pemetrexed (ALIMTA.RTM.), raltitrexed,
etc.), plant alkaloids (e.g., vincristine, vinblastine,
vinorelbine, vindesine, podophyllotoxin, paclitaxel (Taxol.RTM.),
docetaxel (Taxotere.RTM.), etc.), topoisomerase inhibitors (e.g.,
irinotecan, topotecan, amsacrine, etoposide (VP16), etoposide
phosphate, teniposide, etc.), antitumor antibiotics (e.g.,
doxorubicin, adriamycin, daunorubicin, epirubicin, actinomycin,
Neomycin, mitomycin, mitoxantrone, plicamycin, etc.),
pharmaceutically acceptable salts thereof, stereoisomers thereof,
derivatives thereof, analogs thereof, and combinations thereof.
[0258] According to a specific embodiment the chemotherapeutic
agent is Doxorubicin.
[0259] It will be appreciated that the antibody may allow lower
doses of chemotherapeutic agents to be used (e.g. doses which are
less than the current gold standard), thus minimizing adverse
toxicity typically associated with the use of such treatments.
[0260] Examples of hormonal therapeutic agents include, but are not
limited to, aromatase inhibitors (e.g., aminoglutethimide,
anastrozole (Arimidex.RTM.), letrozole (Femora.RTM.), vorozole,
exemestane (Aromasin.RTM.), 4-androstene-3,6,17-trione (6-OXO),
1,4,6-androstatrien-3,17-dione (ATD), formestane (Lentaron.RTM.),
etc.), selective estrogen receptor modulators (e.g., bazedoxifene,
clomifene, fulvestrant, lasofoxifene, raloxifene, tamoxifen,
toremifene, etc.), steroids (e.g., dexamethasone), finasteride, and
gonadotropin-releasing hormone agonists (GnRH) such as goserelin,
pharmaceutically acceptable salts thereof, stereoisomers thereof,
derivatives thereof, analogs thereof, and combinations thereof.
[0261] Radiation therapy includes, but is not limited to,
fractionated radiotherapy, non-fractionated radiotherapy and
hyper-fractionated radiotherapy, and combination radiation and
chemotherapy. Types of radiation also include ionizing (gamma)
radiation, particle radiation, low energy transmission (LET), high
energy transmission (HET), ultraviolet radiation, infrared
radiation, visible light, and photosensitizing radiation.
[0262] Exemplary anti-proliferative agents include mTOR inhibitors
such as sirolimus (rapamycin), temsirolimus (CCI-779), and
everolimus (RAD001); Akt inhibitors such as
IL6-hydroxymethyl-chiro-inositol-2-(R)-2-O-methyl-3-O-octadecyl-sn-glycer
ocarbonate, 9-methoxy-2-methylellipticinium acetate,
1,3-dihydro-1-(1-((4-(6-phenyl-1H-imidazo [4,5-g]quinoxalin-7-yl)
phenyl)me-thyl)-4-piperidinyl)-2H-benzimidazol-2-one,10-(4'-(N-diethylami-
no)butyl) -2-chlorophenoxazine, 3-formylchromone thiosemicarbazone
(Cu(II)Cl.sub.2 complex), API-2, a 15-mer peptide derived from
amino acids 10-24 of the proto-oncogene TCL1 (Hiromura et al., J.
Biol. Chem., 279:53407-53418 (2004), KP372-1, and the compounds
described in Kozikowski et al., J. Am. Chem. Soc., 125:1144-1145
(2003) and Kau et al., Cancer Cell, 4:463-476 (2003); and
combinations thereof.
[0263] It will be appreciated that since the anti-QSOX1 antibody or
antibody fragment of the present invention is capable of
specifically binding human QSOX1 and mouse QSOX1, and since QSOX1
levels are elevated in medical conditions associated with laminin
(e.g., tumors or fibrosis), such an antibody can be used in
assessing the efficiency of treatment (e.g., in preclinical trials
prior to human therapeutics).
[0264] Thus, according to an aspect of the present invention there
is provided a method for in vivo determining the efficiency of an
antibody in reducing a laminin-associated disease or condition in a
murine animal, the method comprising administering to the murine
animal the antibody of some embodiments of the invention and
monitoring progression of a laminin-associated disease or condition
in the murine animal, thereby determining the efficiency of the
antibody.
[0265] As used herein, the term "in vivo" refers to a process
occurring within a living organism.
[0266] According to one embodiment, the organism is a murine animal
including a mouse or a rat. Alternatively, other animal models
known in the art, including e.g., guinea pigs, hamsters, rabbits or
gerbils, can be used in accordance with the present teachings.
[0267] According to a specific embodiment, the murine animal is a
mouse.
[0268] According to one embodiment, the murine animal (e.g., mouse)
can lack all or part of a functional immune system (e.g., SCID
mouse) or a full or part of a gene (e.g., knockout mouse or
transgenic mouse). The murine animal may also be a xenograft animal
model (e.g., wherein a graft from another organism e.g., human
graft, such as a patient-derived tumor, is engrafted into
immunodeficient mice) or and tumor graft animal model (e.g.,
wherein a tumor, from any source, is engrafted into the animal).
The murine animal can also be a model in which the disease
spontaneously occurs or wherein a tumor is induced by a carcinogen
(for further details see e.g.,
www.emice(dot)nci(dot)nih(dot)gov/aam/mouse/carcinogen-induced
-and-spontaneous-mouse-models).
[0269] As used herein the phrase "determining the efficiency"
refers to monitoring disease progression using the antibody of the
invention as compared to disease progression without the use of the
antibody.
[0270] For example, when the disease is a tumor or metastasizing
tumor, efficiency of treatment is determined when a reduction of
about 10%, about 20%, about 30%, about 40 %, about 50%, about 60%,
about 70%, about 80%, about 90% or about 100% is observed in tumor
size, in the number of metastatic lesions (e.g., one lesion),
and/or in the number of areas of metastatic lesions (e.g., in the
bone, brain, liver, lymph nodes, etc) as compared to disease
progression without the use of the antibody.
[0271] For example, when the disease is a fibrosis, efficiency of
treatment is determined when a reduction of about 10%, about 20%,
about 30%, about 40%, about 50%, about 60%, about 70%, about 80%,
about 90% or about 100% is observed in excess connective tissue (in
an organ or tissue) as compared to excess connective tissue without
the use of the antibody.
[0272] In order to determine the efficiency of the antibody or
antibody fragment of some embodiments of the invention, the
antibody is administered to the animal (e.g., at different doses
and intervals) and disease progression is monitored using any
method known in the art, for example, by surgery (e.g., dissecting
the animal after a predetermined time), by ultrasound, CT, PET,
MRI, by blood test, etc.
[0273] Alternatively, a biological sample may be obtained from the
animal and analyzed for disease parameters (e.g., for expression
levels of liver peptides, pulmonary peptides, renal peptides,
pancreatic peptides, tumor peptides, etc.).
[0274] A biological sample may refer to a sample of tissue or fluid
isolated from a subject, including but not limited to, cells (e.g.,
liver cells, pulmonary cells, renal cells, etc.), tissues, organs,
various tumors (e.g., tumor biopsy sample) and fluids such as
blood, serum, plasma, lymph, bile fluid, urine, saliva, sputum,
synovial fluid, semen, tears, cerebrospinal fluid, bronchioalveolar
large fluid, ascites fluid, pus, conditioned medium, and also
samples of in vivo cell culture constituents.
[0275] It will be appreciated that, the antibody of some
embodiments of the present invention may be attached to a
detectable moiety in order to enable detection (e.g., in vivo
detection) of the antibody.
[0276] Various types of detectable or reporter moieties may be
conjugated to the antibody of the invention. These include, but not
are limited to, a radioactive isotope (such as .sup.[125]iodine), a
phosphorescent chemical, a chemiluminescent chemical, a fluorescent
chemical (fluorophore), an enzyme, a fluorescent polypeptide, an
affinity tag, and molecules (contrast agents) detectable by
Positron Emission Tomography (PET) or Magnetic Resonance Imaging
(MRI).
[0277] Examples of suitable fluorophores include, but are not
limited to, phycoerythrin (PE), fluorescein isothiocyanate (FITC),
Cy-chrome, rhodamine, green fluorescent protein (GFP), blue
fluorescent protein (BFP), Texas red, PE-Cy5, and the like. For
additional guidance regarding fluorophore selection, methods of
linking fluorophores to various types of molecules see Richard P.
Haugland, "Molecular Probes: Handbook of Fluorescent Probes and
Research Chemicals 1992-4994", 5th ed., Molecular Probes, Inc.
(1994); U.S. Pat. No. 6,037,137 to Oncoimmunin Inc.; Hennanson,
"Bioconjugate Techniques", Academic Press New York, N.Y. (1995);
Kay M. et al., 1995. Biochemistry 34:293; Stubbs et al., 1996.
Biochemistry 35:937; Gakamsky D. et al., "Evaluating Receptor
Stoichiometry by Fluorescence Resonance Energy Transfer," in
"Receptors: A Practical Approach," 2nd ed., Stanford C. and Horton
R. (eds.), Oxford University Press, UK. (2001); U.S. Pat. No.
6,350,466 to Targesome, Inc.]. Fluorescence detection methods which
can be used to detect the antibody when conjugated to a fluorescent
detectable moiety include, for example, fluorescence activated flow
cytometry (FACS), immunofluorescence confocal microscopy,
fluorescence in-situ hybridization (FISH) and fluorescence
resonance energy transfer (FRET).
[0278] Numerous types of enzymes may be attached to the antibody of
the invention [e.g., horseradish peroxidase (HPR),
beta-galactosidase, and alkaline phosphatase (AP)] and detection of
enzyme-conjugated antibodies can be performed using ELISA (e.g., in
solution), enzyme-linked immunohistochemical assay (e.g., in a
fixed tissue), enzyme-linked chemiluminescence assay (e.g.,, in an
electrophoretically separated protein mixture) or other methods
known in the art [see Khatkhatay M I. and Desai M., 1999. J
Immunoassay 20:151-83; Wisdom G B., 1994. Methods Mol Biol.
32:433-40; Ishikawa E. et al., 1983. J Immunoassay 4:209-327;
Oellerich M., 1980. J Clin Chem Clin Biochem. 18:197-208; Schuurs A
H. and van Weemen B K., 1980. J Immunoassay 1:229-49).
[0279] The affinity tag (or a member of a binding pair) can be an
antigen identifiable by a corresponding antibody [e.g., digoxigenin
(DIG) which is identified by an anti-DIG antibody) or a molecule
having a high affinity towards the tag [e.g., streptavidin and
biotin]. The antibody or the molecule which binds the affinity tag
can be fluorescently labeled or conjugated to enzyme as described
above.
[0280] Various methods, widely practiced in the art, may be
employed to attach a streptavidin or biotin molecule to the
antibody of the invention. For example, a biotin molecule may be
attached to the antibody of the invention via the recognition
sequence of a biotin protein ligase (e.g., BirA) as described in
the Examples section which follows and in Denkberg, G. et al.,
2000. Eur. J. Immnunol. 30:3522-3532. Alternatively, a streptavidin
molecule may be attached to an antibody fragment, such as a single
chain Fv, essentially as described in Cloutier S M. et al., 2000.
Molecular Immunology 37:1067-1077; Dubel S. et al., 1995. J Immunol
Methods 178:201; Huston J S. et al., 1991. Methods in Enzymology
203:46; Kipriyanov S M. et al., 1995. Hum Antibodies Hybridomas
6:93; Kipriyanov S M. et al., 1996. Protein Engineering 9:203;
Pearce L A. et al., 1997. Biochem Molec Biol Intl
42:1179-1188).
[0281] Functional moieties, such as fluorophores, conjugated to
streptavidin are commercially available from essentially all major
suppliers of immunofluorescence flow cytometry reagents (for
example, Pharmingen or Becton-Dickinson).
[0282] According to some embodiments of the invention, biotin
conjugated antibodies are bound to a streptavidin molecule to form
a multivalent composition (e.g., a dimer or tetramer form of the
antibody).
[0283] Table 1 A provides non-limiting examples of identifiable
moieties which can be conjugated to the antibody of the
invention.
TABLE-US-00001 TABLE 1A Amino Acid Nucleic Acid Identifiable
sequence (GenBank sequence (GenBank Moiety Accession No.) Accession
No.) Green Fluorescent AAL33912 AF435427 protein Alkaline AAK73766
AY042185 phosphatase Peroxidase CAA00083 A00740 Histidine tag Amino
acids 264-269 Nucleotides 790-807 of GenBank Accession of GenBank
Accession No. AAK09208 No. AF329457 Myc tag Amino acids 273-283
Nucleotides 817-849 of GenBank Accession of GenBank Accession No.
AAK09208 No. AF329457 Biotin ligase tag LHHILDAQKMVWNHR orange
fluorescent AAL33917 AF435432 protein Beta galactosidase ACH42114
EU626139 Streptavidin AAM49066 AF283893
[0284] According to one embodiment, the antibody of some
embodiments of the invention can be used for in vitro or ex vivo
applications e.g., for detection of QSOX1 levels in biological
samples).
[0285] According to one embodiment, the antibody of the invention
may be immobilized on to a solid support (e.g., for formation of an
immunocomplex between the antibody and QSOX1 proteins in ex vivo or
in vitro settings). As used herein the phrase "solid support"
refers to a non-aqueous matrix to which a reagent of interest
(e.g., the antibody of this aspect of the present invention) can
adhere. Examples of solid supports include, but are not limited to,
solid supports formed partially or entirely of glass (e.g.,
controlled pore glass), polysaccharides (e.g., agarose),
polyacrylamides, polystyrene, polyvinyl alcohol and silicones. In
certain embodiments, depending on the context, the solid support
can comprise the well of an assay plate; in others it is a
purification column (e.g., an affinity chromatography column). This
term also includes a discontinuous solid phase of discrete
particles, such as those described in U.S. Pat. No. 4,275,149.
[0286] The agents described hereinabove may be included in a
diagnostic kit/article of manufacture preferably along with
appropriate instructions for use and labels indicating FDA approval
for use in diagnosing and/or assessing efficiency of treatment of a
laminin-associated disease.
[0287] According to another aspect of the present invention, there
is provided a kit for detecting a level of QSOX1 in a biological
sample.
[0288] Such a kit can include, for example, at least one container
including at least one of the above described diagnostic agents
(e.g., antibodies comprising an antigen recognition domain to
QSOX1) and an imaging reagent packed in another container (e.g.,
enzymes, secondary antibodies, buffers, chromogenic substrates,
fluorogenic material). The kit may also include appropriate buffers
and preservatives for improving the shelf-life of the kit.
[0289] According to another aspect of the present invention, there
is provided a kit for preventing or treating a laminin-associated
disease or condition.
[0290] Such a kit can include, for example, at least one container
including at least one of the above described antibodies comprising
an antigen recognition domain to QSOX1 and an additional
therapeutic agent packed in another container (e.g.,
chemotherapeutic agents, hormonal therapeutic agents,
radiotherapeutic agents, anti--proliferative agents). According to
another embodiment, the therapeutic agent (e.g., antibody
comprising an antigen recognition domain to QSOX1) and the
additional therapeutic agent are packed in the same container. The
kit may also include appropriate buffers and preservatives for
improving the shelf-life of the kit.
[0291] As used herein the term "about" refers to .+-.10%.
[0292] The terms "comprises", "comprising", "includes",
"including", "having" and their conjugates mean "including but not
limited to".
[0293] The term "consisting of" means "including and limited
to".
[0294] The term "consisting essentially of" means that the
composition, method or structure may include additional
ingredients, steps and/or parts, but only if the additional
ingredients, steps and/or parts do not materially alter the basic
and novel characteristics of the claimed composition, method or
structure.
[0295] As used herein, the singular form "a", "an" and "the"
include plural references unless the context clearly dictates
otherwise. For example, the term "a compound" or "at least one
compound" may include a plurality of compounds, including mixtures
thereof.
[0296] Throughout this application, various embodiments of this
invention may be presented in a range format. It should be
understood that the description in range format is merely for
convenience and brevity and should not be construed as an
inflexible limitation on the scope of the invention. Accordingly,
the description of a range should be considered to have
specifically disclosed all the possible subranges as well as
individual numerical values within that range. For example,
description of a range such as from 1 to 6 should be considered to
have specifically disclosed subranges such as from 1 to 3, from 1
to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as
well as individual numbers within that range, for example, 1, 2, 3,
4, 5, and 6. This applies regardless of the breadth of the
range.
[0297] Whenever a numerical range is indicated herein, it is meant
to include any cited numeral (fractional or integral) within the
indicated range. The phrases "ranging/ranges between" a first
indicate number and a second indicate number and "ranging/ranges
from" a first indicate number "to" a second indicate number are
used herein interchangeably and are meant to include the first and
second indicated numbers and all the fractional and integral
numerals therebetween.
[0298] As used herein the term "method" refers to manners, means,
techniques and procedures for accomplishing a given task including,
but not limited to, those manners, means, techniques and procedures
either known to, or readily developed from known manners, means,
techniques and procedures by practitioners of the chemical,
pharmacological, biological, biochemical and medical arts.
[0299] It is appreciated that certain features of the invention,
which are, for clarity, described in the context of separate
embodiments, may also be provided in combination in a single
embodiment. Conversely, various features of the invention, which
are, for brevity, described in the context of a single embodiment,
may also be provided separately or in any suitable subcombination
or as suitable in any other described embodiment of the invention.
Certain features described in the context of various embodiments
are not to be considered essential features of those embodiments,
unless the embodiment is inoperative without those elements.
[0300] Various embodiments and aspects of the present invention as
delineated hereinabove and as claimed in the claims section below
find experimental support in the following examples.
EXAMPLES
[0301] Reference is now made to the following examples, which,
together with the above descriptions, illustrate the invention in a
non limiting fashion.
[0302] Generally, the nomenclature used herein and the laboratory
procedures utilized in the present invention include molecular,
biochemical, microbiological and recombinant DNA techniques. Such
techniques are thoroughly explained in the literature. See, for
example, "Molecular Cloning: A laboratory Manual" Sambrook et al.,
(1989); "Current Protocols in Molecular Biology" Volumes I-III
Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in
Molecular Biology", John Wiley and Sons, Baltimore, Md. (1989);
Perbal, "A Practical Guide to Molecular Cloning", John Wiley &
Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific
American Books, New York; Birren et al. (eds) "Genome Analysis: A
Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory
Press, New York (1998); methodologies as set forth in U.S. Pat.
Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057;
"Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E.,
ed. (1994); "Current Protocols in Immunology" Volumes I-III Coligan
J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical
Immunology" (8th Edition), Appleton & Lange, Norwalk, CT
(1994); Mishell and Shiigi (eds), "Selected Methods in Cellular
Immunology", W. H. Freeman and Co., New York (1980); available
immunoassays are extensively described in the patent and scientific
literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;
3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;
3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;
5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J.,
ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins
S. J., eds. (1985); "Transcription and Translation" Hames, B. D.,
and Higgins S. J., Eds. (1984); "Animal Cell Culture" Freshney, R.
I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986);
"A Practical Guide to Molecular Cloning" Perbal, B., (1984) and
"Methods in Enzymology" Vol. 1-317Academic Press; "PCR Protocols: A
Guide To Methods And Applications", Academic Press, San Diego,
Calif. (1990); Marshak et al., "Strategies for Protein Purification
and Characterization --A Laboratory Course Manual" CSHL Press
(1996); all of which are incorporated by reference as if fully set
forth herein. Other general references are provided throughout this
document. The procedures therein are believed to be well known in
the art and are provided for the convenience of the reader. All the
information contained therein is incorporated herein by
reference.
GENERAL MATERIALS AND EXPERIMENTAL PROCEDURES
Plasmid Construction
[0303] HsQSOX1 and MmQSOX1 mutants were made by restriction-free
cloning based on the published HsQSOX1 [Grossman I. et al. (2013)
J. Mol. Biol. 425: 4366-4378] and MmQSOX1 [Alon A. et al. (2012)
Nature 488: 414-418] expression plasmids. MmQSOX1.sub.Trx and
MmQSOX1.sub.Erv span residues 36-275 and 289-550, respectively, of
Mus musculus QSOX1. The RnQSOX1 construct was previously described
[Gat Y. et al. (2014) Protein Sci. 23: 1102-1112]. The CpQSOX1
protein spans residues 34-547 of Cavia porcellus QSOX1. A synthetic
gene (Genescript) codon-optimized for CpQSOX1 production in E. coil
was cloned between the NdeI and BamHI sites of the pET-15b vector
(Novagen). For production of biotinylated MmQSOX1.sub.Trx an AviTag
[Kay B et al. (2009) Methods Mol. Biol. 498: 185-196] was added at
the carboxy terminus.
QSOX1 Expression and Purification
[0304] Recombinant HsQSOX1 [Grossman L et al. (2013), supra] and
RnQSOX1. [Gat Y. et al. (2014), supra] were expressed and purified
as described. MmQSOX1, MmQSOX1.sub.Erv, and CpQSOX1 were prepared
as for HsQSOX1. MmQSOX1.sub.Trx used for crystallization was
produced in the BL21 (DE3) E. coli strain. Cells were grown in LB
media to OD.sub.600 nm 0.5 at 37.degree. C.
Isopropyl-1-thio-.beta.-D-galactopyranoside (IPTG) was added to a
final concentration of 0.5 mM, and the cultures were grown for a
further 40 hours at 15.degree. C. Cells were lysed in 20 mM Tris
buffer, pH 8.5, 500 mM NaCl, 20 mM imidazole, sonicated, and
centrifuged for 1 hour at 40,000 x g. The supernatant was applied
to a Ni-NTA column (GE healthcare), and protein was eluted in 20 mM
Tris buffer, pH 8.5, 500 mM. NaCl, and a gradient of imidazole (20
to 500 mM). Eluted protein was further purified by size exclusion
chromatography on a Superdex 75 16/60 column in 10 mM Tris buffer,
pH 8, 100 mM NaCl. Purified MmQSOX1.sub.Trx was concentrated to 12
mg/ml, and immediately before crystallization was mixed with
thrombin (3 units/mg MmQSOX1.sub.Trx). Biotinylated MmQSOX1.sub.Trx
for yeast-surface display screening was co-expressed with an
expression plasmid for biotin ligase. Upon induction of protein
expression with 500 .mu.M IPTG, biotin was added to the growth
medium at a concentration of 50 .mu.M, and the cultures were grown
for a further 24 hours at 20.degree. C. Purification of
biotinylated MmQSOX1.sub.Trx, was as for MmQSOX1.sub.Trx.
Biotinylation was verified by ELISA.
Oxygen Consumption Assays for Testing QSOX1 Enzymes Activity and
Inhibition
[0305] Recombinant mammalian QSOX1 enzymes (100 nM) were assayed
with 200 .mu.M DTT and various MAb492.1 concentrations in a
Clarke-type oxygen electrode (Hansatech Instruments) as previously
described [Grossman I. et al. (2013), supra]. Reactions were
initiated by DTT injection, and oxygen consumption rates were
obtained from initial slopes. For testing inhibition of MmQSOX1 and
HsQSOX1 mutants by MAb492.1 and MAb316.1, respectively, 50 nM
enzyme was assayed with 200 .mu.M DTT, with and without 250 nM
antibody. Measurements were performed 3 times, and resulting rates
were averaged. For each mutant, the rate in the presence of
inhibitory antibody was divided by the rate in the absence of
antibody to get percent activity.
Generation and Selection of MAb316.1
[0306] Hybridomas were generated, and supernatants of approximately
1000 clones were screened for MmQSOX1 binding as previously
described [Ilani T. et al. (2013) Science 341: 74-76]. Twenty top
binders were tested for MmQSOX1 inhibition in the in vitro oxygen
consumption assay, in which hybridoma supernatants were mixed with
100 nM MrnQSOX1 and 200 .mu.m DTT. Two inhibitory clones in
addition to three strong binders were chosen for sub-cloning. Each
of the sub-clones was tested for binding by ELISA. Approximately 40
sub-clones were chosen for inhibition assays. Supernatants of
sub-clones 316.1 and 947.3 inhibited MmQSOX1 activity repeatedly,
and so were chosen for further studies. MAb316.1 used for
inhibition assays was produced in a miniPERM bioreactor (Sarstedt)
in serum-free medium (DCCM) and was purified as previously
described [Ilani T. et al. (2013), supra]. Variable region
sequencing of both monoclonal antibodies was performed as
previously described [Grossman I. et al. (2013), supra]. Briefly,
the variable region was reverse transcribed from hybridomal mRNA
and amplified by PCR using sets of degenerate primers [described in
Benhar I. and Reiter Y. (2002) In Curr. Protoc. Immunol. Chapter
10: Unit 10.19B; and Zhou, H. et al. (1994) Nucleic Acids Res. 22:
888-889]. Amplified fragments of the heavy chain and light chain
were then cloned into the pGEM-T Easy vector, sequenced, and
analyzed in the ImMunoGeneTics database [Lefranc M. P. et al.
(2005) Nucleic Acids Res. 33: 593-597]. A productively rearranged
sequence was confirmed for each fragment and verified on the
protein level by liquid chromatography-tandem mass spectrometry of
purified MAb316.1 (Table 2, below).
Screening of seFv492.1 Mutants with Yeast-Surface Display
[0307] The scFv492.1 construct, without the His-tag and thrombin
cleavage site [Grossman I. et al, (2013), supra], was cloned
between the Ndel and BamHI sites in the yeast display plasmid
pETCON [Fleishman S. J. et al. (2011) Science 332: 816-821].
Mutants of scFv492.1 were prepared on the basis of this plasmid by
in vivo recombination in EBY100 yeast using the LiAc method. Yeast
growth and induction of scFv expression were carried out as
previously described [Chao G. et al. (2006) Nat. Protoc.
1:755-768], except that for induction 10 g raffinose per liter were
added to the media and the cultures were grown for 16 hours at
20.degree. C. Yeast were labeled with anti-cMyc (Santa Cruz
Biotechnology) at a dilution 1:50 to monitor scFv expression, and
with biotinylated MmQSOX1.sub.Trx (for concentrations see Table 3,
below) for 45 minutes. After washing, secondary labeling was
performed with streptavidin-allophycocyanin (streptavidin-APC) from
Jackson Immunoresearch Laboratories, Inc. at a 1:50 dilution and
with goat anti-mouse IgG1 secondary antibody Alexa fluor 488
conjugate (Life technologies) at a 1:100 dilution, for the
anti-cMyc labeling. Alternatively, in the first rounds of screening
(Table 3, below), yeast were labeled with anti-cMyc, washed, and
then labeled with goat anti-mouse IgG1 secondary antibody Alexa
fluor 488 conjugate and with biotinylated MmQSOX1.sub.Trx ,
pre-loaded with streptavidin-APC in a 1:4 molar ratio [Chao G. et
al. (2006), supra]. Display of scFv clones and MmQSOX1.sub.Trx
binding was monitored by Alexa 488 and APC fluorescence,
respectively, using an Accuri C6 flow cytometer. Anti-fluorescein
scFv (PDB code: 1X9Q) was used as a positive control for scFv
display. Yeast displaying scFv492.1 labeled with biotinylated
HsQSOX1.sub.Trx were used as a positive control for binding.
[0308] Small scFv libraries were constructed by fully randomizing
specific residue positions (Table 3, below) using the NNS codon.
Oligonucleotides with NNS codons in desired positions and wild-type
flanking regions were ordered from Sigma. NdeI and BamHI sites were
deleted from the N- and C-termini of the scFv construct in the
pETCON plasmid and inserted flanking the desired region for
randomization. The plasmid was restricted, and the oligonucleotide
pool was inserted through in vivo recombination using the LiAc
method. In all libraries constructed, the number of colonies
obtained was at least an order of magnitude larger than the
potential size of the library (20.sup.number of randomized
positions). Libraries were induced and labeled as described above.
Cells were sorted using a FACSAria III Cell Sorter in 3 iterative
rounds of enrichment. In the first sorting round, the top 5% cells
found within the green and red fluorescence area were collected
into growth media. In the following sorting rounds, the top 1-4%
cells were collected. Plasmids from the last cycle of FAGS
enrichment were sequenced. Eighteen colonies enriched from the
library constructed in CDR H3 were sequenced, yielding two distinct
sequences. Each of the two sequences was tested separately for
MmQSOX1.sub.Trx binding, and the better binder (Table 3, below) was
chosen for further study. Enrichment of the library constructed in
L1 yielded a single clone, verified by sequencing 12 colonies.
Enrichment of the library constructed in L2 yielded eight clones,
which were tested for MmQSOX1.sub.Trx binding separately. The
enriched L1 sequence was incorporated to the top binder. After
verifying that the combined clone showed increased MmQSOX1.sub.Trx
binding compared to the clones enriched directly from the L1 and L2
libraries, this clone was subjected to epPCR on the entire scFv
sequence using the Agilent GeneMorph II Random Mutagenesis kit.
Recombination in vivo was performed by electroporation, yielding a
library of size 5*10.sup.7. The library was subjected to 3 rounds
of selection as described above, recovering scFv492gen.
Recombinant MAb492gen Production
[0309] The light chain and heavy chain variable regions of
scFv492gen were amplified and cloned separately into mammalian
expression vectors for human IgG1 antibodies [Tiller T. et al.
(2008) J. Immunol. Methods 329: 112-124]. MAb492gen was expressed
by transient co-transfection of the two plasmids in human embryonic
kidney (HEK) 293T cells using polyethylenimine. Cell supernatants
were collected and replaced with fresh medium every 4 days.
Expression and secretion to the medium was verified by western
blot. MAb492gen was purified from the media using protein G [Ilani
T. et al. (2013), supra]. Purified MAb492gen with human constant
regions was used for inhibition assays.
Inhibitory Constant Determination
[0310] Colorimetric assays of RNase A oxidation were performed as
described previously to obtain IC.sub.50 values [Grossman I. et al.
(2013), supra]. K.sub.i values were obtained in oxygen consumption
assays as previously described [Grossman I. et al. (2013),
supra].
Laminin Staining and Cell Adhesion Assay
[0311] The assays were conducted essentially as previously
described [Ilani T. et al. (2013), supra]. Instead of human
fibroblasts, mouse fibroblasts were grown for three days in the
presence of various antibodies. HEK 293T cells were used as
adhering cells.
Analytical Size-Exclusion Chromatography
[0312] MsQSOX1, its fragments, MAb316.1, or MAb947.3 were loaded
onto a Superdex 200 column (GE HealthCare) equilibrated with
phosphate buffered saline (PBS) at a flow rate of 0.8 mL/min.
MsQSOX1-antibody complexes were injected after a 30 min
co-incubation at 4.degree. C. Elution of proteins was monitored by
absorbance at 280 nm.
Fab-MmQSOX1.sub.Trx Complex Formation
[0313] MAb492.1 light chain and heavy chain were amplified from
hybridomal cDNA [Grossman I. et al. (2013), supra] and cloned into
the MAb492gen expression vectors, separately. Variable regions of
scFv492gen were amplified and cloned into these vectors instead of
the 492,1 variable regions. A stop codon was inserted after the CH1
segment of the heavy chain, to obtain a coding sequence for a Fab
fragment. Fab492gen was expressed and purified as was MAb492gen.
The heavy and light variable regions of MAb316.1 were amplified
from hybridomal cDNA and cloned separately into the above mentioned
MAb492gen expression vectors. MAb316.1 was expressed and purified
as for MAb492gen. Fab316.1 was prepared by papain digestion as
previously described [Grossman I. et al. (2013), supra]. Purified
Fab492gen or Fab316.1 was mixed with purified MmQSOX1.sub.Trx at a
1:2 ratio for 30 minutes at 4.degree. C. to form complexes, which
were purified from excess MmQSOX1.sub.Trx by size-exclusion
chromatography. Prior to crystallization the complexes were
concentrated to 10 mg/ml.
[0314] Protein Crystallization
[0315] Crystals were grown by hanging-drop vapor diffusion at 293
K. MmQSOX1.sub.Trx crystals used for seeding were grown over a well
solution containing 0.2 M ammonium sulfate, 0.1 M sodium acetate,
pH 4.6, 21% w/v polyethylene glycol (PEG) 4 kD. These crystals were
crushed and seeded into drops of MmQSOX1.sub.Trx grown over a well
solution containing 5% w/v dimethyl suboxide, 0.1 M sodium acetate,
pH 4.6, 7 w/v PEG monomethyl ether 2 kD. Crystals were transferred
to a solution containing 0.1 M sodium acetate, pH 4.6, 15% w/v PEG
monomethyl ether 2 kD, 25% glycerol and flash frozen in a nitrogen
stream at 100 K. Crystals of the MmQSOX1.sub.Trx-Fab31.6.1 complex
were grown over a well solution containing 50 mM ammonium sulfate,
0.1 M bis-tris methane buffer, pH 5.5, 22% w/v PEG 3.35 kD.
Crystals were transferred to a solution with the same content and
25% w/v glycerol for freezing. Crystals of the
MmQSOX1.sub.Trx-Fab492gen complex were grown over a well solution
containing 50 mM CaCl.sub.2, 0.1 M 2-(N-morpholino)ethanesulfonic
acid buffer, pH 6, 22.5% w/v PEG 6 kD, and were transferred to the
same solution containing 25% w/v glycerol prior to freezing.
Data Collection
[0316] Diffraction data for MmQSOX1.sub.Trx,
MmQSOX1.sub.Trx-Fab316.1 complex, and MmQSOX1.sub.Trx-Fab492gen
complex were collected at 100 K on a RU-H3R generator (Rigaku)
equipped with a RaxisIV++ image plate system and Osmic mirrors. For
MmQSOX1.sub.Trx, data were collected to 2.05 .ANG. resolution from
a crystal of space group P2.sub.1. MmQSOX1.sub.Trx-Fab316.1 complex
data were collected to 2.2 .ANG. resolution from a crystal of space
group P2.sub.12.sub.12.sub.1. Diffraction data for
MmQSOX1.sub.Trx-Fab492gen were collected to 2.3 .ANG. resolution
from a crystal of space group P2.sub.1. All data sets were
processed and scaled using DENZO and SCALEPACK [Broennimann Ch. et
al. (2006) J. Synchrotron Radiat. 13: 120-130].
Structure Solution
[0317] All three structures were determined by molecular
replacement using Phaser [McCoy A. J. et al. (2007) J. Appl.
Crystallogr. 40: 658-674]. The HsQSOX1.sub.Trx structure (PDB code:
3Q6O) was used as a search model for MmQSOX1.sub.Trx, and model
rebuilding and mutagenesis was done in Coot [Emsley P. and Cowtan
K. (2004) Acta Crystallogr. D. Biol. Crystallogr. 60: 2126-2132].
The resulting MmQSOX1.sub.Trx structure (PDB code: 5D8I) was used
as a search model for both MmQSOX1.sub.Trx-Fab complexes. After
translation and rotation functions were found for MmQSOX1.sub.Trx,
a search for the constant region of Fab316.1 was performed using an
identical constant region (PDB code: 3D85, chain A spanning
residues 108-213 and chain B spanning residues 116-217). The
variable region of Fab316.1 heavy chain was searched with a model
having 87% identity (PDB code: 4Q0X, chain H spanning residues
1-118 without CDR H3), and the light chain variable region was
searched with a 94% identical model (PDB code: 3AB0, chain C
spanning residues 1-106). For Fab492gen, three search models from
Fab492.1 (PDB code: 4IJ3, chains B and C) were used: the constant
region, the light chain variable region without the mutated
residues in Fab492gen, and the heavy chain variable region without
CDRs H1 and H3. For both complexes addition of CDR loops and model
rebuilding were done using Coot [Emsley P. and Cowtan K. (2004),
supra]. Refinement was performed using Phenix [Afonine P. V. et al.
(2005) CCP4 Newsletter 42, contribution 8]. Validation was done
using MolProbity [Lovell S. C. et al. (2003) Proteins 50, 437-450],
according to which there are no Ramachandran outliers in the
structures reported herein.
Treatment Regime in Murine 4T1 Breast Cancer Model
[0318] 4T1 mCherry cells were suspended in Hanks' Balanced Salt
Solution at a concentration of 2.5.times.10.sup.6 cells/ml and
mixed 1:1 with Cultrex.RTM. Basement Membrane Extract. 250,000
cells were injected into the mammary fat pad of 45 BALB/c female
mice. Animals were divided into 5 groups containing 9 animals each
(as depicted in Table 1B, below). Treatments were administered by
intraperitoneal injection, beginning 3 days after cell injection.
Tumor dimensions (x,y,z) were measured with a caliper twice a week.
Tumor volumes were calculated according to: x*y*z*6/.pi.. Animals
were weighed once a week. Twenty days post cell injection all
animals were sacrificed by CO.sub.2 asphyxiation. Tumors and lungs
were removed into 4% formaldehyde solution and prepared for
histology.
TABLE-US-00002 TABLE 1B Treatment regime in murine 4T1 breast
cancer model Frequency of Group Treatment administration Vehicle 1
50 mg/kg control IgG antibody twice a week PBS 2 30 mg/kg MAb316.1
twice a week PBS 3 8 mg/kg doxorubicin once a week PBS 4 8 mg/kg
doxorubicin once a week PBS 30 mg/kg MAb316.1 twice a week 5 8
mg/kg doxorubicin once a week PBS 15 mg/kg MAb316.1 twice a
week
Example 1
The QS0X1 Inhibitor MAb492.1 is Species-Specific
[0319] MAb492.1. which inhibits HsQSOX1 activity in vitro and in
cell culture at a near-stoichiometric concentration [Grossman I. et
al. (2013) supra], was tested on other mammalian QSOX1 enzymes to
find a suitable model for assessing QSOX1 inhibition in vivo. Three
mammalian QSOX1 enzymes, from common experimental organisms and
having 78-79% sequence identity with HsQSOX1, were chosen: Mus
musculus QSOX1 (MmQSOX1), Rattus norvegicus QSOX1 (RnQSOX1), and
Cavia porcellus (CpQSOX1). Enzyme activity was assessed in an
oxygen consumption assay on the model substrate dithiothreitol
(DTT). According to the assay, MAb492.1 had no effect on MmQSOX1,
RnQSOX1, or CpQSOX1, even at 1 .mu.M, a 10:1 molar ratio of
antibody to enzyme (FIG. 2A).
[0320] To understand the molecular basis for MAb492.1 species
restriction, the epitope on HsQSOX1 was compared with the
corresponding regions of RnQSOX1, CpQSOX1, and particularly MmQSOX1
by crystallizing and solving the structure of the Trx module of
MmQSOX1 (MmQSOX1.sub.Trx) to 2.05 .ANG. resolution (Table 1C,
below). Two MmQSOX1.sub.Trx molecules were present in the crystal
asymmetric unit. The atomic coordinates of these two molecules were
overlaid on the previously solved structure of a complex between
HsQSOX1.sub.Trx and a Fab fragment of MAb492.1 (Fab492.1) [Grossman
I. et al. (2013) supra]. Although the regions near the CXXC
redox-active site are identical in sequence among QSOX1 orthologs,
a few other positions of contact between Fab492.1 and HsQSOX1
differ (FIG. 2B). In particular, HsQSOX1 P116, which fits well into
a cleft between hydrophobic complementary determining regions
(CDRs) L3, H2, and H3 of Fab492.1, is replaced with alanine in
other mammalian QSOX1 enzymes. The MmQSOX1.sub.Trx structure showed
that this alanine residue cannot fill the hydrophobic cleft in a
hypothetical complex between MmQSOX1. and Fab492.1. Moreover, the
replacement of praline by alanine affects the position of the
backbone nearby, such that a clash would form between MmQSOX1 N117
and CDR L3 (FIG. 2C, left). Another region that is not conserved
among the QSOX1 enzymes examined is VFPV(135-138) from HsQSOX1. The
corresponding TLPG(138-141) loop in one of the MmQSOX1 molecules is
displaced from the antibody CDRs L1 and L2 and is found closer to
CDR H3 than the VFPV(135-138) loop of HsQSOX1 (FIG. 2C, right). As
a result, MAb492.1 CDR H3 would clash sterically with this loop,
eliminating potential interactions of MmQSOX1 with CDRs L1 and
L2.
TABLE-US-00003 TABLE 1C Summary of crystallographic data collection
and refinement statistics Fab492gen- MmQSOX1.sub.Trx Fab316.1-
MmQSOX1.sub.Trx MmQSOX1.sub.Trx complex complex Date collection
Space group P2.sub.1 P2.sub.1 P2.sub.12.sub.12.sub.1 Cell
dimensions a, b, c (.ANG.) 42.5, 116.4, 50.0 78.8, 204.8, 44.7
65.5, 112.7, 193.4 .alpha., .beta., .gamma. (.degree.) 90, 103, 90
90, 90, 90 90, 90, 90 Copies in asymmetric unit 2 2 2 Resolution
(.ANG.) 50-2.05 (2.05-2.09) 50-2.30 (2.30-2.34) 50-2.20 (2.20-2.24)
Measured reflections 104,982 190,891 358,728 Unique reflections
28,528 (1197) 62,461 (3167) 72,630 (3564) Completeness (%) 95.8
(79.2) 99.7 (100) 98.9 (98.8) Redundancy 3.7 (2.9) 3.1 (3.0) 4.9
(3.8) <I/.sigma.I> 11.3 (2.2) 8.3 (2.2) 7.6 (2.0) Rsym 0.063
(0.318) 0.084 (0.418) 0.088 (0.408) Refinement Resolution (.ANG.)
28.5-2.05 27.4-2.3 23.2-2.2 Number of reflections 28,485 62,422
72,573 in working set Number of reflections 1957 2002 3588 in test
set Rwork/Rfree 0.201/0.252 0.167/0.228 0.174/0.236 Number of
protein atoms 3774 10180 10156 Number of water 288 425 751
molecules Mean B-factor 31.50 34.98 32.65 Root mean square
deviations Bond length (.ANG.) 0.006 0.004 0.008 Bond angle
(.degree.) 1.275 1.013 1.113 Ramachandran plot Favored regions (%)
96.0 98.1 96.6 Additional allowed regions (%) 4.0 1.9 3.4
Disallowed regions (%) 0 0 0 Values in parentheses are for the
highest-resolution shell.
[0321] To confirm that the structural differences noted between
HsQSOX1 and MmQSOX1 are the cause of MAb492.1 species specificity,
three MmQSOX1 mutants that mimic HsQSOX1 in distinct positions were
constructed. MAb492.1 inhibition was tested on these mutants to
identify the residues that interfere with MAb492.1-MmQSOX1 complex
formation. The first mutant, A116P, was inhibited by about 60%
(FIG. 2D), and the second mutant, TLPG(138-141)VFPV, by about 50%.
The third mutant, which comprises both the above mutations, was
inhibited by MAb492.1. to the same extent as HsQSOX1 (100%),
confirming the identity of residues that determine the specificity
of MAb492.1 towards HsQSOX1.
Example 2
Generation and Characterization of a Murine Antibody Inhibitor
Targeting MmQSOX1
[0322] The species-specificity exhibited by MAb492.1 is a common
feature shared by natural monoclonal antibodies. Although
differences in antigen structure might be small among orthologs,
they are exploited by the immune system to bind the foreign antigen
and avoid self-reactivity. In the large antigen surface area buried
by antibodies, minor differences between orthologs can cause steric
clashes that cannot be remedied by a simple corresponding change in
the antibody. QSOX1 knock-out (QSOX1.-KO) mice produced in the
laboratory were then exploited to generate surrogate antibodies
against MmQSOX1, which is a foreign antigen for these animals.
Hybridoma supernatants were screened for binding of MmQSOX1 using a
standard enzyme-linked immunosorbent assay (ELISA). Top hinders
were tested for MmQSOX1 inhibition, and five were chosen for
sub-cloning (described in the materials and experimental procedures
section above). Antibody sub-clones derived from one particular
clone inhibited MmQSOX1 and one sub-clone, designated MAb316.1 and
classified to be of the IgG1 isotype, was selected for further
study. The sequences of the MAb316.1 variable regions (described in
the materials and experimental procedures section above) are
presented in Table 2, below.
TABLE-US-00004 TABLE 2 MAb316.1 variable region amino acid
sequences Chain Sequence light
QIVLTQSPAIMSASPGEKVTISCSASSSVSYMYWYHQKPGSSPKPWIYRTSNLASGVPARFSG
SGSGTSYSLTISSMEAEDAATYYCQQYHSYPLTFGAGTKLELK (SEQ ID NO: 9) heavy
QVQLQQSGPELVKPGASVKISCKASGYSFTSYYIHWVKQRPGQGLEWIGWIYPGSYNTEYSEK
FKGKATLTADTSSSTAYMQLSSLTSEDSAVYYCARSEDWFAYWGQGTLVTVS (SEQ ID NO:
10) MW (calc) MW (exp) peptide enzyme light 1855.9729 1855.9713
QIVLTQSPAIMSASPGEK (SEQ ID NO: 11) trypsin 2936.3419 2936.3473
VTISCSASSSVSYMYWYHQKPGSSPK (SEQ ID NO: 12) 1071.5671 1071.5673
TSNLASGVRAR (SEQ ID NO: 13) 3389.4864 3389.4856
TISSMEAEDAATYYCQQYHSYPLTEGAGTK (SEQ ID NO: 14) 1650.8829 1650.8842
IYRTSNLASGVPARF (SEQ ID NO: 15) chymotrypsin/ 1374.7352 1374.7368
RTSNLASGVPARF (SEQ ID NO: 16) Asp-N 1586.7319 1586.7325
ASGVPARFSGSGSGTSY (SEQ ID NO; 17) 793.4351 793.4334 TFGAGTKLEL (SEQ
ID NO: 18) heavy 1992.0977 1992.1004 QVQLQQSGPELVKPGASVK (SEQ ID
NO: 19) trypsin 1807.8521 1807.8570 ASGYSFTSYYIHWVK (SEQ ID NO: 20)
2957.3959 2957.3984 QRPGQGLEWIGWIYPGSYNTEYSEK (SEQ ID NO: 21)
2079.9242 2079.9241 MQLSSLTSEDSAVYYCAR (SEQ ID NO: 22) 2390.2278
2390.2264 QQSGPELVKPGASVKISCKASGY chymotrypsin/ (SEQ ID NO: 23)
Asp-N 1652.8761 1652.8787 VKQRPGQGLEWIGW (SEQ ID NO: 24) 1420.6153
1420.6147 PGSYNTEYSEKF (SEQ ID NO: 25) The table displays
representative peptides detected by liquid chromatography-tandem
mass spectrometry.
Example 3
Generation of a MAb492.1 Variant Targeting MmQOX1
[0323] In parallel to obtaining an antibody targeting MmQSOX1 T
from hybridoma clones, a variant of MAb492.1 that inhibits MmQSOX1
was developed. Though occasionally a single point mutation may
modulate antibody species specificity to some extent, substantial
re-engineering is often required to obtain the desired
target-recognition properties. According to the observation that
mutating four residues of MmQSOX1 was sufficient to achieve
inhibition by MAb492.1 (FIG. 2D), inventors reasoned that
inhibiting MmQSOX1 would be possible by making a set of mutations
in the MAb492.1 CDRs or surrounding regions. However, in contrast
to modifying MmQSOX1. to mimic HsQSOX1, modification of the
antibody had to be made without a guiding structure. Furthermore,
the four QSOX1 residues controlling reactivity with MAb492.1
affected the position of the QSOX1 polypeptide backbone (FIG. 2C),
hinting that substantial compensating mutations affecting CDR loop
position and/or structure would be needed to convert MAb492.1 into
a cross-reactive reagent. Candidate residues for mutation to
improve MmQSOX1.sub.Trx binding were identified based on the
structures of MmQSOX1.sub.Trx and the Fab492.1-HsQSOX1.sub.Trx
complex (Table 3, below). Specific point mutations were tested and
small libraries were constructed, varying up to four CDR residues
in specific locations, from which binding mutants were enriched.
This careful approach was taken to increase the likelihood of
gaining functionality against MmQSOX1 without losing binding to the
targeted epitope.
[0324] Screening was performed using yeast-surface display of
single-chain variable fragments (scFv) (described in the materials
and experimental procedures section above) [Chao G. et al. (2006)
Nat. Protoc. 1:755-768]. A scFv variant of MAb492.1. (scFv492.1)
had been constructed, expressed in E. coli, and shown to inhibit
HsQSOX1 successfully [Grossman I. et al. (2013), supra], laying the
groundwork for using yeast-surface display to select for MmQSOX1
inhibitors. A scFv variant carrying ten mutations in the CDRs
achieved by specific rational mutations and consecutive rounds of
library sorting and enrichment (Table 3, below) bound
MmQSOX1.sub.Trx on the surface of yeast (FIG. 3A). Since mutations
in other CDR residues did not improve MmQSOX1.sub.Trx binding
further, one round of affinity maturation was performed on the
scFv492.1 mutant, for fine-tuning, To this end a gene library
(total size of 5*10.sup.7 clones) was constructed by error-prone
polymerase chain reaction (epPCR), with an average of three
mutations per gene, and subjected the library to three rounds of
flow-cytometry selection for MmQSOX1.sub.Trx binding. A mutant with
a total of 12 mutations on scFv492.1, designated scFv492gen (FIG.
3J), was isolated and characterized, yielding an apparent K.sub.d
of 65 nM on the surface of yeast (FIGS. 3K-3L). Based on the
scFv492gen sequence, a recombinant full-length antibody, MAb492gen,
was produced in mammalian cells.
TABLE-US-00005 TABLE 3 MAb492.1 residues mutated to generate
MAb492gen MmQSOX1.sub.Trx concentration Template Template for used
for Positive name mutations Residue.sup.a Mutation Strategy binding
tests mutations.sup.b wt scFv492.1 Y92 (light chain) N/D 25 of the
possible combinatorial 500 nM pre-loaded S93A S93 (light chain) G/A
mutants involving these with streptavidin-APC Y100 (heavy chain)
D/N/G/S three residues were tested a scFv492.1 Y99-S102 All
possible Library in potential size of 250 nM pre-loaded Y99S S93A
(heavy chain) amino acids 20.sup.4 clones subjected with
streptavidin- APC Y100K to three rounds of selection G101D S102P b
scFv492.1 Y92-P95 All possible Library in potential size of 250 nM
pre-loaded -- S93A (light chain) amino acids 20.sup.4 clones
subjected with streptavidin- APC Y99S to three rounds of selection
Y100K T30-Y32 All possible Library in potential size of 250 nM
pre-loaded -- G101D (heavy chain) amino acids 20.sup.3 clones
subjected with streptavidin- APC S102P to three rounds of selection
W52-D54, R56 All possible Library in potential size of 250 nM
pre-loaded -- (heavy chain) amino acids 20.sup.4 clones subjected
with streptavidin- APC to three rounds of selection D58 E/Q Tested
point mutations for 250 nM pre-loaded D58E (heavy chain)
MmQSOX1.sub.Trx binding with streptavidin- APC c scFv492.1 S30-T31
All possible Library in potential size of 250 nM pre-loaded T31G
S93A (light chain) amino acids 20.sup.2 clones subjected with
streptavidin- APC Y99S to three rounds of selection Y100K H49-S50,
Y52 All possible Library in potential size of 250 nM pre-loaded
H49S G101D (light chain) amino acids 20.sup.3 clones subjected with
streptavidin- APC S50M S102P to three rounds of selection Y52Q D58E
d scFv492.1 All All Library was created by epPCR 250 nM G33S S93A
and its size was evaluated as (not pre-loaded) N35I Y99S 5*10.sup.7
clones. S100M Y100K Library was subjected to three (heavy chain)
G101D rounds of selection S102P D58E T31G H49S S50M Y52Q
.sup.aNumbering is according to structure of
HsQSOX1.sub.Trx-Fab492.1 complex (PDB code: 4IJ3). .sup.bMutations
that improved MmQSOX1.sub.Trx binding.
Example 4
Comparison of Inhibition Constants
[0325] MmQSOX1 inhibition by MAb492gen was next quantified and
compared to inhibition by the natural antibody MAb316.1. In one
assay, MmQSOX1 activity was measured by the number of free thiols
remaining at the end of the oxidation reaction of denatured and
reduced RNase A. Various MAb492gen concentrations were scanned
against two MmQSOX1 concentrations. In both cases the IC.sub.50
values were similar to the MmQSOX1 concentration used in the assay
(FIG. 3B), indicating tight-binding inhibition. In a second assay
MmQSOX1 catalytic activity on DTT was measured using the oxygen
consumption assay, providing data that were fitted to a model of
tight-binding inhibition [Bieth, J. G. (1995) Methods Enzymol. 248:
59-84] to yield an inhibitory constant (K.sub.i) of 2.2.+-.0.5 nM
(FIG. 3C and Table 4, below). Like MAb492gen, MAb316.1 exhibited
tight-binding inhibition of MmQSOX1, but with a slightly larger
K.sub.i value of 16.+-.2 nM (FIG. 3D and Table 4, below).
TABLE-US-00006 TABLE 4 Inhibition constants for antibodies
targeting QSOX1 Antibody/enzyme HsQSOXl MmQSOXl MAb492.1 0.9 .+-.
0.1 nM No inhibition at 1 .mu.M MAb492gen 1.6 .+-. 0.6 nM 2.2 .+-.
0.5 nM MAb316.1 No inhibition 16 .+-. 2 nM at 1 .mu.M
Example 5
Antibodies Inhibit MmQSOX1 Activity in Cell Culture
[0326] MmQSOX1 inhibition by MAb316.1 and MAb492gen was examined in
cell culture using two readouts of QSOX1 activity [Macri T. et al.
(2013), supra]. QSOX1 and laminin are secreted front quiescent
fibroblasts into the ECM, where QSOX1 promotes laminin
incorporation, and consequently the adherence of epithelial cells
to the fibroblast monolayer. Thus, intact laminin networks in the
ECM and cell adherence indicate functional QSOX1 activity in the
ECM [Ilani T. et al. (2013), supra]. Inventors first verified that
MmQSOX1 is secreted from confluent mouse embryonic fibroblasts
(MEFs) (FIG. 4A). Next, extracellular laminin secreted by MEFs
grown tor three days in the presence of MAb492.1 MAb316.1, or
MAb492gen was stained. As in human fibroblast cultures [Ilani T. et
al. (2013), supra], an extensive laminin network was observed only
in ECM produced with active QSOX1 (FIGS. 3E-3H and FIGS. 4B-4I). A
quantitative assay of epithelial cell adhesion to MEF monolayers
was performed next. Fluorescently labeled epithelial cells were
added to MEFs grown in the absence or presence of the antibodies,
and adherent cells were quantified. As expected, more epithelial
cells adhered to MEFs grown in the absence of inhibitory antibody
compared to MEFs grown in the presence of either 250 nM or 1 .mu.M
antibody targeting MmQSOX1 (MAb316.1 and MAb492gen). MAb492.1,
which inhibits HsQSOX1 but not MmQSOX1, did not affect the laminin
network or epithelial cell adherence in the mouse fibroblast
cultures (FIGS. 3E-3I and FIGS. 4J-4O).
Example 6
MAb492gen Binds MmQSOX1 Due to Alterations at the Interface of the
Heavy and Light Chains
[0327] After attaining a MAb492.1 mutant that inhibits MmQSOX1,
X-ray crystallography was used to examine the structural effects of
mutations that enabled antigen recognition. A complex between a Fab
fragment of MAb492gen (Fab492gen) and MmQSOX1.sub.Trx was formed,
and its structure was solved to 2.3 .ANG. resolution (Table 1C).
Two complexes were found in the asymmetric unit, deviating from one
another mainly in the angle between the constant and the variable
regions of the Fab fragment. As expected, the MAb492gen epitope on
MmQSOX1 corresponds to the HsQSOX1 region bound by MAb492.1 (FIG.
5A). MmQSOX1.sub.Trx bound by MAb492gen takes the conformation of
chain B from the unbound MmQSOX1.sub.Trx, in which the
TLPG(138-141) loop approaches the CXXC motif. MAb492gen differs
from MAb492.1 primarily around this QSOX1.sub.Trx region (FIG. 5B).
The complex structure revealed how a combination of relieving
steric clashes and introducing new favorable interactions enabled
recognition of MmQSOX1.
[0328] Based on the Fab492gen-MmQSOX.sub.Trx structure, inventors
suspected that mutations introduced into CDR L3 and H3 prevented
collisions between MmQSOX1 and the antibody. CDR H3 is the most
variable CDR in sequence and structure among antibodies and is
usually responsible for most of the paratope [Xu J. L. and Davis M.
M. (2000) Immunity 13: 37-45]. The YYGS-to-SMDP mutation of CDR H3
in Fab492gen both eliminated a clashing tyrosine and caused a
rearrangement of the backbone conformation (FIGS. 5B and 5F),
resulting in the first significant improvement in binding. Changing
the structure of the CDR H3 loop also allowed closer approach of
CDR L2, together with the rest of the light chain, toward the heavy
chain (FIG. 5B). This movement appears to have helped relieve the
expected clash between CDR L3 and N117 in MmQSOX1 (FIG. 2C).
[0329] A second effect of the slight change in the relative
orientations of the heavy and light chain variable regions observed
in the Fab492gen-MmQSOX1.sub.Trx structure was the introduction of
new interactions. Some interactions were generated between the
heavy and light chains, and some with the target. Mutating H49 in
CDR L2 to serine enabled an interaction with the new CDR H3 SMDP
loop (FIG. 5C), potentially contributing to pre-organization of the
paratope. Mutating T31 in CDR L1 to glycine enabled formation of
hydrogen bonds between CDR L1 and the MmQSOX1 TLPG(138-141) loop
that could not form between MAb492.1 and MmQSOX1 (FIGS. 5D and
5E).
Example 7
MAb316.1 Exhibits a New Inhibition Mode of QSOX1
[0330] MAb492.1 and MAb492gen bind the N-terminal QSOX1 Trx domain.
Since MAb316.1 was generated in mice injected with full-length
MmQSOX1, inventors sought to localize its epitope. Size exclusion
chromatography was used to compare the elution profiles of MmQSOX1,
or each of its constituent modules, to their elution profiles when
mixed with MAb316.1. In size exclusion chromatography, this
antibody shifted the elution of both full-length MmQSOX1 and the
Trx module to higher apparent molecular weights (FIG. 6A). In
contrast, migration of the MmQSOX1 Erv module, responsible for the
sulthydryl oxidase activity, was not affected, pointing at the Trx
module as the MAb316.1 binding region in MmQSOX1.
[0331] To localize the MAb316.1 epitope to specific residues in
MmQSOX1.sub.Trx, a Fab fragment of MAb316.1 (Fab316.1) was
co-crystallized with MnQSOX1.sub.Trx and the structure was solved
to 2.2 .ANG. resolution (see Table 1C, above). Two
MmQSOX1.sub.Trx-Fab316.1 complexes were present in the crystal
asymmetric unit. The complex structure revealed a distinct QSOX1
inhibition mode from the one displayed by MAb492.1 and MAb492gen.
Fab316.1 envelopes the helix containing the Trx redox-active site
at its amino terminus but does not block access to the CXXC motif
itself (FIG. 6B). It seems that a small substrate like DTT might be
able to reduce the Trx active site even in the presence of
MAb316.1. Nevertheless, MAb316.1 would physically prevent formation
of the inter-domain electron-transfer intermediate of MmQSOX1 (FIG.
6C), thus interrupting a different step in the catalytic cycle than
MAb492.I and MAb492gen (transition from state 2 to 3, rather than 1
to 2 in FIG. 1C).
Example 8
MAb492gen is the only QSOX1 Inhibitory Antibody Cross-Reactive with
Mouse and Human Orthologs
[0332] A QSOX1- targeting antibody with dual species specificity,
i.e , that inhibits HsQSOX1 as well as MmQSOX1, would be a valuable
tool that could be used in animal models, and in turn, with minimal
engineering, in clinical trials. Inventors therefore tested for
MA6492gen and MAb316.1 inhibition of HsQSOX1. MAb316.1 up to a
concentration of 1 .mu.M did not inhibit HsQSOX1 (FIG. 7A). In
contrast, though no selection for binding of HsQSOX1 was performed
during its development, MAb492gen retained tight-binding activity
against HsQSOX1, having a Ki value of 1.6.+-.0.6 nM for HsQSOX1
(FIG. 7B and Table 4, above) compared to 0.9.+-.0.1 nM previously
measured for MA6492.1 [Grossman I. et al. (2013), supra].
[0333] A comparison of the three available QSOX1-antibody complex
structures suggests features that prevent or contribute to target
species specificity. Lack of binding of HsQSOX1.sub.Trx by
MAb316.1. can be explained once again by the VFPV(135-138) loop,
which was a major specificity determinant for MAb492.1. In the
Fab31.6.1-MmQSOX1.sub.Trx complex, however, the corresponding
TLPG(138-141) loop is not directly in the interface, but rather
affects the position of the conserved downstream residues
AGA(139-142). These downstream residues in HsQSOX1 are expected to
clash sterically with CDR H2 of MAb316.1 (FIG. 7C). The
constellation of aromatic residues (light chain: Y31, Y90, Y93;
heavy chain: Y33, W50, Y52) that mediates interaction between
Fab31.6.1 and MmQSOX1 presumably does not accommodate minor
differences in backbone position between the mouse and human QSOX1
orthologs. Mutating HsQSOX1 to mimic MmQSOX1 by converting
TLPG(138-141) to VFPV(138-141) made MAb316.1 inhibition of HsQSOX1
possible, confirming this region as the block to cross-reactivity
(FIG. 7D).
[0334] As opposed to MAb316.1 and MAb492.1, MAb492gen accommodates
both the TLPG(138-141) loop from MmQSOX1 and presumably the
corresponding VFPV(135-138) from HsQSOX1 between its H3, L1, and L2
CDRs, resulting in dual specificity (FIG. 5F). One key difference
between MAb492gen and the species-specific antibodies (MAb492.1 and
MAb316.1) is its lower content of tyrosines and other aromatic
residues in the CDRs. Tyrosines are highly prevalent and effective
in antibody CDRs, where they can make van der Wools, hydrogen
bonding, and cation-.pi. interactions. In MAb492gen, however, three
tyrosines were replaced with more flexible residues. Another
difference is the introduction of a charged residue, D101, into CDR
H3, which becomes buried at the QSOX1-antibody interface. D101
forms water-mediated interactions with the MmQSOX1 TLPG (138-141)
loop, while also hydrogen bonding to the light chain (FIG. 5C). The
bulky aromatic residues within the corresponding YYGS sequence of
MAb492.1 H3 were apparently unable to accommodate the deviation of
MmQSOX1 TLPG(138-141) from HsQSOX1 VFPV(135-138). Though the
structural variability of this QSOX1 loop likely positions backbone
hydrogen bond donor and acceptor groups differently in space, the
water network through which Asp101 interacts with its target in the
Fab492gen-MmQSOX.sub.Trx complex suggests that MAb492gen can
accommodate these differences through a re-organized water
structure. Through features offering greater flexibility, MAb492gen
acquired expanded capabilities, but it presumably retained its
specificity towards QSOX1 antigens by maintaining CDRs that
interact with the CXXC motif region (CDRs H1 and H2), the universal
catalytic element in QSOX1 Trx domains.
[0335] Although the invention has been described in conjunction
with specific embodiments thereof, it, is evident that many
alternatives, modifications and variations will be apparent to
those skilled in the art. Accordingly, it is intended to embrace
all such alternatives, modifications and variations that fall
within the spirit and broad scope of the appended claims.
[0336] It is the intent of the applicant(s) that all publications,
patents and patent applications referred to in this specification
are to be incorporated in their entirety by reference into the
specification, as if each individual publication, patent or patent
application was specifically and individually noted when referenced
that it is to be incorporated herein by reference. In addition,
citation or identification of any reference in this application
shall not be construed as an admission that such reference is
available as prior art to the present invention. To the extent that
section headings are used, they should not be construed as
necessarily limiting. In addition, any priority document(s) of this
application is/are hereby incorporated herein by reference in
its/their entirety.
Example 9
MAb316.1 is Therapeutic in Vivo in a Breast Cancer Murine Model
[0337] The ability of an antibody inhibiting MmQSOX1 to control
tumor growth and metastasis was evaluated in vivo in the 4T1 murine
mammary carcinoma breast cancer model previously described [Pulaski
and Ostrand-Rosenberg (2001) Mouse 4T1 breast tumor model. Curr.
Protoc. Immunol. Chapter 20, Unit 20.2.]. Mice were injected with
4T1 cells together with matrigel, which aided in formation of a
localized tumor, and then divided into five groups (as described in
Table 1B, above). Each group received a different treatment: 50
mg/kg control IgG antibody twice a week, 8 mg/kg doxorubicin (a
common chemotherapeutic agent) once a week, 30 mg/kg MAb316.1 twice
a week, a combination of 8 mg/kg doxorubicin and 30 mg/kg MAb316.1,
or a combination of 8 mg/kg doxorubicin and 15 mg/kg MAb316.1.
Tumor volumes were measured twice a week. Animals were sacrificed
after 20 days, and their tumors and lungs were removed for
evaluation.
[0338] During the entire experiment, mice treated with doxorubicin
alone had smaller tumors than control mice, as previously reported
[Gao et al. (2011) J. Control Release 152: 84-89]. Interestingly,
treatment with the higher concentration of MAb316.1 used (30 mg/kg)
reduced tumor volumes, and treatment with a combination of
doxorubicin and 30 mg/kg MAb316.1 decreased tumor volumes compared
to treatment with doxorubicin alone (FIG. 8). Treatment with the
lower MAb316.1 concentration and doxorubicin resulted in tumors
with a comparable size to tumors in the group treated with only
doxorubicin. These results show that treatment with 30 mg/kg
MAb316.1 twice a week slows the progression of an aggressive breast
cancer in mice.
Sequence CWU 1
1
5113316DNAHomo sapiens 1ggaggcaggc ggtgccgcgg cgccgggacc cgactcatcc
ggtgcttgcg tgtggtggtg 60agcgcagcgc cgaggatgag gaggtgcaac agcggctccg
ggccgccgcc gtcgctgctg 120ctgctgctgc tgtggctgct cgcggttccc
ggcgctaacg cggccccgcg gtcggcgctc 180tattcgcctt ccgacccgct
gacgctgctg caggcggaca cggtgcgcgg cgcggtgctg 240ggctcccgca
gcgcctgggc cgtggagttc ttcgcctcct ggtgcggcca ctgcatcgcc
300ttcgccccga cgtggaaggc gctggccgaa gacgtcaaag cctggaggcc
ggccctgtat 360ctcgccgccc tggactgtgc tgaggagacc aacagtgcag
tctgcagaga cttcaacatc 420cctggcttcc cgactgtgag gttcttcaag
gcctttacca agaacggctc gggagcagta 480tttccagtgg ctggtgctga
cgtgcagaca ctgcgggaga ggctcattga cgccctggag 540tcccatcatg
acacgtggcc cccagcctgt cccccactgg agcctgccaa gctggaggag
600attgatggat tctttgcgag aaataacgaa gagtacctgg ctctgatctt
tgaaaaggga 660ggctcctacc tgggtagaga ggtggctctg gacctgtccc
agcacaaagg cgtggcggtg 720cgcagggtgc tgaacacaga ggccaatgtg
gtgagaaagt ttggtgtcac cgacttcccc 780tcttgctacc tgctgttccg
gaatggctct gtctcccgag tccccgtgct catggaatcc 840aggtccttct
ataccgctta cctgcagaga ctctctgggc tcaccaggga ggctgcccag
900accacagttg caccaaccac tgctaacaag atagctccca ctgtttggaa
attggcagat 960cgctccaaga tctacatggc tgacctggaa tctgcactgc
actacatcct gcggatagaa 1020gtgggcaggt tcccggtcct ggaagggcag
cgcctggtgg ccctgaaaaa gtttgtggca 1080gtgctggcca agtatttccc
tggccggccc ttagtccaga acttcctgca ctccgtgaat 1140gaatggctca
agaggcagaa gagaaataaa attccctaca gtttctttaa aactgccctg
1200gacgacagga aagagggtgc cgttcttgcc aagaaggtga actggattgg
ctgccagggg 1260agtgagccgc atttccgggg ctttccctgc tccctgtggg
tcctcttcca cttcttgact 1320gtgcaggcag ctcggcaaaa tgtagaccac
tcacaggaag cagccaaggc caaggaggtc 1380ctcccagcca tccgaggcta
cgtgcactac ttcttcggct gccgagactg cgctagccac 1440ttcgagcaga
tggctgctgc ctccatgcac cgggtgggga gtcccaacgc cgctgtcctc
1500tggctctggt ctagccacaa cagggtcaat gctcgccttg caggtgcccc
cagcgaggac 1560ccccagttcc ccaaggtgca gtggccaccc cgtgaacttt
gttctgcctg ccacaatgaa 1620cgcctggatg tgcccgtgtg ggacgtggaa
gccaccctca acttcctcaa ggcccacttc 1680tccccaagca acatcatcct
ggacttccct gcagctgggt cagctgcccg gagggatgtg 1740cagaatgtgg
cagccgcccc agagctggcg atgggagccc tggagctgga aagccggaat
1800tcaactctgg accctgggaa gcctgagatg atgaagtccc ccacaaacac
caccccacat 1860gtgccggctg agggacctga ggcaagtcga cccccgaagc
tgcaccctgg cctcagagct 1920gcaccaggcc aggagcctcc tgagcacatg
gcagagcttc agaggaatga gcaggagcag 1980ccgcttgggc agtggcactt
gagcaagcga gacacagggg ctgcattgct ggctgagtcc 2040agggctgaga
agaaccgcct ctggggccct ttggaggtca ggcgcgtggg ccgcagctcc
2100aagcagctgg tcgacatccc tgagggccag ctggaggccc gagctggacg
gggccgaggc 2160cagtggctgc aggtgctggg agggggcttc tcttacctgg
acatcagcct ctgtgtgggg 2220ctctattccc tgtccttcat gggcctgctg
gccatgtaca cctacttcca ggccaagata 2280agggccctga agggccatgc
tggccaccct gcagcctgaa ccacctgggg aggaggcggg 2340agagggagct
gccatctcta ggcacctcaa gccccctgac cccattccct cccctcccac
2400cccttgctcc ttgtctggcc tagaagtgtg ggaaattcag gaaaacgagt
tgctccagtg 2460aagcttcttg gggttgctag gacagagagc tcctttgaca
caaaagacag gagcagggtc 2520caggttcccc tgctgtgcag ggagggcagc
cccgggcagt gggcataggg cagctcagtc 2580cctggcctct tagcaccaca
ttcctgtttt tcagcttatt tgaagtcctg cctcattctc 2640actggagcct
cagtctctcc tgcttggtct tggccctcaa ctggggcaag tgaagccaga
2700ggagggtccc ccagctgggt gggctggaat ggaactcctc actagctgct
ggggctccgc 2760ccaccctgct cccttccgga caatgaagaa gcctttgcac
cctgggagga aggaccaccc 2820cgggccctct atgcctggcc agcctccagc
tcctcagacc tcctgggtgg ggtttggctt 2880cagggtgggg tttggaagct
tctggaagtc gtgctggtct cccaggtgag gcaagccatg 2940gttgctgggc
tgtagggtga gtggcttgct tggtgggacc tgacgagttg gtggcatggg
3000aaggatgtgg gtctctagtg ccttgccctg gcttagctgc aggagaagat
ggctgctttc 3060acttcccccc attgagctct gctccctctg agcctggtct
tttgtccttt tttattttgg 3120tctccaagat gaatgctcat ctttggaggg
tgccaggtag aagctaggga ggggagtgtc 3180ttctctctcc aggtttcacc
ttccagtgtg cagaagttag aagggtctgg cgggggcagt 3240gccttacaca
tgcttgattc ccacgctacc ccctgccttg ggaggtgtgt ggaataaatt
3300atttttgtta aggcaa 331622583DNAHomo sapiens 2ggaggcaggc
ggtgccgcgg cgccgggacc cgactcatcc ggtgcttgcg tgtggtggtg 60agcgcagcgc
cgaggatgag gaggtgcaac agcggctccg ggccgccgcc gtcgctgctg
120ctgctgctgc tgtggctgct cgcggttccc ggcgctaacg cggccccgcg
gtcggcgctc 180tattcgcctt ccgacccgct gacgctgctg caggcggaca
cggtgcgcgg cgcggtgctg 240ggctcccgca gcgcctgggc cgtggagttc
ttcgcctcct ggtgcggcca ctgcatcgcc 300ttcgccccga cgtggaaggc
gctggccgaa gacgtcaaag cctggaggcc ggccctgtat 360ctcgccgccc
tggactgtgc tgaggagacc aacagtgcag tctgcagaga cttcaacatc
420cctggcttcc cgactgtgag gttcttcaag gcctttacca agaacggctc
gggagcagta 480tttccagtgg ctggtgctga cgtgcagaca ctgcgggaga
ggctcattga cgccctggag 540tcccatcatg acacgtggcc cccagcctgt
cccccactgg agcctgccaa gctggaggag 600attgatggat tctttgcgag
aaataacgaa gagtacctgg ctctgatctt tgaaaaggga 660ggctcctacc
tgggtagaga ggtggctctg gacctgtccc agcacaaagg cgtggcggtg
720cgcagggtgc tgaacacaga ggccaatgtg gtgagaaagt ttggtgtcac
cgacttcccc 780tcttgctacc tgctgttccg gaatggctct gtctcccgag
tccccgtgct catggaatcc 840aggtccttct ataccgctta cctgcagaga
ctctctgggc tcaccaggga ggctgcccag 900accacagttg caccaaccac
tgctaacaag atagctccca ctgtttggaa attggcagat 960cgctccaaga
tctacatggc tgacctggaa tctgcactgc actacatcct gcggatagaa
1020gtgggcaggt tcccggtcct ggaagggcag cgcctggtgg ccctgaaaaa
gtttgtggca 1080gtgctggcca agtatttccc tggccggccc ttagtccaga
acttcctgca ctccgtgaat 1140gaatggctca agaggcagaa gagaaataaa
attccctaca gtttctttaa aactgccctg 1200gacgacagga aagagggtgc
cgttcttgcc aagaaggtga actggattgg ctgccagggg 1260agtgagccgc
atttccgggg ctttccctgc tccctgtggg tcctcttcca cttcttgact
1320gtgcaggcag ctcggcaaaa tgtagaccac tcacaggaag cagccaaggc
caaggaggtc 1380ctcccagcca tccgaggcta cgtgcactac ttcttcggct
gccgagactg cgctagccac 1440ttcgagcaga tggctgctgc ctccatgcac
cgggtgggga gtcccaacgc cgctgtcctc 1500tggctctggt ctagccacaa
cagggtcaat gctcgccttg caggtgcccc cagcgaggac 1560ccccagttcc
ccaaggtgca gtggccaccc cgtgaacttt gttctgcctg ccacaatgaa
1620cgcctggatg tgcccgtgtg ggacgtggaa gccaccctca acttcctcaa
ggcccacttc 1680tccccaagca acatcatcct ggacttccct gcagctgggt
cagctgcccg gagggatgtg 1740cagaatgtgg cagccgcccc agagctggcg
atgggagccc tggagctgga aagccggaat 1800tcaactctgg accctgggaa
gcctgagatg atgaagtccc ccacaaacac caccccacat 1860gtgccggctg
agggacctga gcttatttga agtcctgcct cattctcact ggagcctcag
1920tctctcctgc ttggtcttgg ccctcaactg gggcaagtga agccagagga
gggtccccca 1980gctgggtggg ctggaatgga actcctcact agctgctggg
gctccgccca ccctgctccc 2040ttccggacaa tgaagaagcc tttgcaccct
gggaggaagg accaccccgg gccctctatg 2100cctggccagc ctccagctcc
tcagacctcc tgggtggggt ttggcttcag ggtggggttt 2160ggaagcttct
ggaagtcgtg ctggtctccc aggtgaggca agccatggtt gctgggctgt
2220agggtgagtg gcttgcttgg tgggacctga cgagttggtg gcatgggaag
gatgtgggtc 2280tctagtgcct tgccctggct tagctgcagg agaagatggc
tgctttcact tccccccatt 2340gagctctgct ccctctgagc ctggtctttt
gtcctttttt attttggtct ccaagatgaa 2400tgctcatctt tggagggtgc
caggtagaag ctagggaggg gagtgtcttc tctctccagg 2460tttcaccttc
cagtgtgcag aagttagaag ggtctggcgg gggcagtgcc ttacacatgc
2520ttgattccca cgctaccccc tgccttggga ggtgtgtgga ataaattatt
tttgttaagg 2580caa 25833747PRTHomo sapiens 3Met Arg Arg Cys Asn Ser
Gly Ser Gly Pro Pro Pro Ser Leu Leu Leu1 5 10 15Leu Leu Leu Trp Leu
Leu Ala Val Pro Gly Ala Asn Ala Ala Pro Arg 20 25 30Ser Ala Leu Tyr
Ser Pro Ser Asp Pro Leu Thr Leu Leu Gln Ala Asp 35 40 45Thr Val Arg
Gly Ala Val Leu Gly Ser Arg Ser Ala Trp Ala Val Glu 50 55 60Phe Phe
Ala Ser Trp Cys Gly His Cys Ile Ala Phe Ala Pro Thr Trp65 70 75
80Lys Ala Leu Ala Glu Asp Val Lys Ala Trp Arg Pro Ala Leu Tyr Leu
85 90 95Ala Ala Leu Asp Cys Ala Glu Glu Thr Asn Ser Ala Val Cys Arg
Asp 100 105 110Phe Asn Ile Pro Gly Phe Pro Thr Val Arg Phe Phe Lys
Ala Phe Thr 115 120 125Lys Asn Gly Ser Gly Ala Val Phe Pro Val Ala
Gly Ala Asp Val Gln 130 135 140Thr Leu Arg Glu Arg Leu Ile Asp Ala
Leu Glu Ser His His Asp Thr145 150 155 160Trp Pro Pro Ala Cys Pro
Pro Leu Glu Pro Ala Lys Leu Glu Glu Ile 165 170 175Asp Gly Phe Phe
Ala Arg Asn Asn Glu Glu Tyr Leu Ala Leu Ile Phe 180 185 190Glu Lys
Gly Gly Ser Tyr Leu Gly Arg Glu Val Ala Leu Asp Leu Ser 195 200
205Gln His Lys Gly Val Ala Val Arg Arg Val Leu Asn Thr Glu Ala Asn
210 215 220Val Val Arg Lys Phe Gly Val Thr Asp Phe Pro Ser Cys Tyr
Leu Leu225 230 235 240Phe Arg Asn Gly Ser Val Ser Arg Val Pro Val
Leu Met Glu Ser Arg 245 250 255Ser Phe Tyr Thr Ala Tyr Leu Gln Arg
Leu Ser Gly Leu Thr Arg Glu 260 265 270Ala Ala Gln Thr Thr Val Ala
Pro Thr Thr Ala Asn Lys Ile Ala Pro 275 280 285Thr Val Trp Lys Leu
Ala Asp Arg Ser Lys Ile Tyr Met Ala Asp Leu 290 295 300Glu Ser Ala
Leu His Tyr Ile Leu Arg Ile Glu Val Gly Arg Phe Pro305 310 315
320Val Leu Glu Gly Gln Arg Leu Val Ala Leu Lys Lys Phe Val Ala Val
325 330 335Leu Ala Lys Tyr Phe Pro Gly Arg Pro Leu Val Gln Asn Phe
Leu His 340 345 350Ser Val Asn Glu Trp Leu Lys Arg Gln Lys Arg Asn
Lys Ile Pro Tyr 355 360 365Ser Phe Phe Lys Thr Ala Leu Asp Asp Arg
Lys Glu Gly Ala Val Leu 370 375 380Ala Lys Lys Val Asn Trp Ile Gly
Cys Gln Gly Ser Glu Pro His Phe385 390 395 400Arg Gly Phe Pro Cys
Ser Leu Trp Val Leu Phe His Phe Leu Thr Val 405 410 415Gln Ala Ala
Arg Gln Asn Val Asp His Ser Gln Glu Ala Ala Lys Ala 420 425 430Lys
Glu Val Leu Pro Ala Ile Arg Gly Tyr Val His Tyr Phe Phe Gly 435 440
445Cys Arg Asp Cys Ala Ser His Phe Glu Gln Met Ala Ala Ala Ser Met
450 455 460His Arg Val Gly Ser Pro Asn Ala Ala Val Leu Trp Leu Trp
Ser Ser465 470 475 480His Asn Arg Val Asn Ala Arg Leu Ala Gly Ala
Pro Ser Glu Asp Pro 485 490 495Gln Phe Pro Lys Val Gln Trp Pro Pro
Arg Glu Leu Cys Ser Ala Cys 500 505 510His Asn Glu Arg Leu Asp Val
Pro Val Trp Asp Val Glu Ala Thr Leu 515 520 525Asn Phe Leu Lys Ala
His Phe Ser Pro Ser Asn Ile Ile Leu Asp Phe 530 535 540Pro Ala Ala
Gly Ser Ala Ala Arg Arg Asp Val Gln Asn Val Ala Ala545 550 555
560Ala Pro Glu Leu Ala Met Gly Ala Leu Glu Leu Glu Ser Arg Asn Ser
565 570 575Thr Leu Asp Pro Gly Lys Pro Glu Met Met Lys Ser Pro Thr
Asn Thr 580 585 590Thr Pro His Val Pro Ala Glu Gly Pro Glu Ala Ser
Arg Pro Pro Lys 595 600 605Leu His Pro Gly Leu Arg Ala Ala Pro Gly
Gln Glu Pro Pro Glu His 610 615 620Met Ala Glu Leu Gln Arg Asn Glu
Gln Glu Gln Pro Leu Gly Gln Trp625 630 635 640His Leu Ser Lys Arg
Asp Thr Gly Ala Ala Leu Leu Ala Glu Ser Arg 645 650 655Ala Glu Lys
Asn Arg Leu Trp Gly Pro Leu Glu Val Arg Arg Val Gly 660 665 670Arg
Ser Ser Lys Gln Leu Val Asp Ile Pro Glu Gly Gln Leu Glu Ala 675 680
685Arg Ala Gly Arg Gly Arg Gly Gln Trp Leu Gln Val Leu Gly Gly Gly
690 695 700Phe Ser Tyr Leu Asp Ile Ser Leu Cys Val Gly Leu Tyr Ser
Leu Ser705 710 715 720Phe Met Gly Leu Leu Ala Met Tyr Thr Tyr Phe
Gln Ala Lys Ile Arg 725 730 735Ala Leu Lys Gly His Ala Gly His Pro
Ala Ala 740 7454604PRTHomo sapiens 4Met Arg Arg Cys Asn Ser Gly Ser
Gly Pro Pro Pro Ser Leu Leu Leu1 5 10 15Leu Leu Leu Trp Leu Leu Ala
Val Pro Gly Ala Asn Ala Ala Pro Arg 20 25 30Ser Ala Leu Tyr Ser Pro
Ser Asp Pro Leu Thr Leu Leu Gln Ala Asp 35 40 45Thr Val Arg Gly Ala
Val Leu Gly Ser Arg Ser Ala Trp Ala Val Glu 50 55 60Phe Phe Ala Ser
Trp Cys Gly His Cys Ile Ala Phe Ala Pro Thr Trp65 70 75 80Lys Ala
Leu Ala Glu Asp Val Lys Ala Trp Arg Pro Ala Leu Tyr Leu 85 90 95Ala
Ala Leu Asp Cys Ala Glu Glu Thr Asn Ser Ala Val Cys Arg Asp 100 105
110Phe Asn Ile Pro Gly Phe Pro Thr Val Arg Phe Phe Lys Ala Phe Thr
115 120 125Lys Asn Gly Ser Gly Ala Val Phe Pro Val Ala Gly Ala Asp
Val Gln 130 135 140Thr Leu Arg Glu Arg Leu Ile Asp Ala Leu Glu Ser
His His Asp Thr145 150 155 160Trp Pro Pro Ala Cys Pro Pro Leu Glu
Pro Ala Lys Leu Glu Glu Ile 165 170 175Asp Gly Phe Phe Ala Arg Asn
Asn Glu Glu Tyr Leu Ala Leu Ile Phe 180 185 190Glu Lys Gly Gly Ser
Tyr Leu Gly Arg Glu Val Ala Leu Asp Leu Ser 195 200 205Gln His Lys
Gly Val Ala Val Arg Arg Val Leu Asn Thr Glu Ala Asn 210 215 220Val
Val Arg Lys Phe Gly Val Thr Asp Phe Pro Ser Cys Tyr Leu Leu225 230
235 240Phe Arg Asn Gly Ser Val Ser Arg Val Pro Val Leu Met Glu Ser
Arg 245 250 255Ser Phe Tyr Thr Ala Tyr Leu Gln Arg Leu Ser Gly Leu
Thr Arg Glu 260 265 270Ala Ala Gln Thr Thr Val Ala Pro Thr Thr Ala
Asn Lys Ile Ala Pro 275 280 285Thr Val Trp Lys Leu Ala Asp Arg Ser
Lys Ile Tyr Met Ala Asp Leu 290 295 300Glu Ser Ala Leu His Tyr Ile
Leu Arg Ile Glu Val Gly Arg Phe Pro305 310 315 320Val Leu Glu Gly
Gln Arg Leu Val Ala Leu Lys Lys Phe Val Ala Val 325 330 335Leu Ala
Lys Tyr Phe Pro Gly Arg Pro Leu Val Gln Asn Phe Leu His 340 345
350Ser Val Asn Glu Trp Leu Lys Arg Gln Lys Arg Asn Lys Ile Pro Tyr
355 360 365Ser Phe Phe Lys Thr Ala Leu Asp Asp Arg Lys Glu Gly Ala
Val Leu 370 375 380Ala Lys Lys Val Asn Trp Ile Gly Cys Gln Gly Ser
Glu Pro His Phe385 390 395 400Arg Gly Phe Pro Cys Ser Leu Trp Val
Leu Phe His Phe Leu Thr Val 405 410 415Gln Ala Ala Arg Gln Asn Val
Asp His Ser Gln Glu Ala Ala Lys Ala 420 425 430Lys Glu Val Leu Pro
Ala Ile Arg Gly Tyr Val His Tyr Phe Phe Gly 435 440 445Cys Arg Asp
Cys Ala Ser His Phe Glu Gln Met Ala Ala Ala Ser Met 450 455 460His
Arg Val Gly Ser Pro Asn Ala Ala Val Leu Trp Leu Trp Ser Ser465 470
475 480His Asn Arg Val Asn Ala Arg Leu Ala Gly Ala Pro Ser Glu Asp
Pro 485 490 495Gln Phe Pro Lys Val Gln Trp Pro Pro Arg Glu Leu Cys
Ser Ala Cys 500 505 510His Asn Glu Arg Leu Asp Val Pro Val Trp Asp
Val Glu Ala Thr Leu 515 520 525Asn Phe Leu Lys Ala His Phe Ser Pro
Ser Asn Ile Ile Leu Asp Phe 530 535 540Pro Ala Ala Gly Ser Ala Ala
Arg Arg Asp Val Gln Asn Val Ala Ala545 550 555 560Ala Pro Glu Leu
Ala Met Gly Ala Leu Glu Leu Glu Ser Arg Asn Ser 565 570 575Thr Leu
Asp Pro Gly Lys Pro Glu Met Met Lys Ser Pro Thr Asn Thr 580 585
590Thr Pro His Val Pro Ala Glu Gly Pro Glu Leu Ile 595
6005604PRTHomo sapiens 5Met Arg Arg Cys Asn Ser Gly Ser Gly Pro Pro
Pro Ser Leu Leu Leu1 5 10 15Leu Leu Leu Trp Leu Leu Ala Val Pro Gly
Ala Asn Ala Ala Pro Arg 20 25 30Ser Ala Leu Tyr Ser Pro Ser Asp Pro
Leu Thr Leu Leu Gln Ala Asp 35 40 45Thr Val Arg Gly Ala Val Leu Gly
Ser Arg Ser Ala Trp Ala Val Glu 50 55 60Phe Phe Ala Ser Trp Cys Gly
His Cys Ile Ala Phe Ala Pro Thr Trp65 70 75 80Lys Ala Leu Ala Glu
Asp Val Lys Ala Trp Arg Pro Ala Leu Tyr Leu 85 90 95Ala Ala Leu Asp
Cys Ala Glu Glu Thr Asn Ser Ala Val Cys Arg Asp 100 105 110Phe Asn
Ile Pro Gly Phe Pro Thr Val Arg Phe Phe Lys Ala Phe Thr
115 120 125Lys Asn Gly Ser Gly Ala Val Phe Pro Val Ala Gly Ala Asp
Val Gln 130 135 140Thr Leu Arg Glu Arg Leu Ile Asp Ala Leu Glu Ser
His His Asp Thr145 150 155 160Trp Pro Pro Ala Cys Pro Pro Leu Glu
Pro Ala Lys Leu Glu Glu Ile 165 170 175Asp Gly Phe Phe Ala Arg Asn
Asn Glu Glu Tyr Leu Ala Leu Ile Phe 180 185 190Glu Lys Gly Gly Ser
Tyr Leu Gly Arg Glu Val Ala Leu Asp Leu Ser 195 200 205Gln His Lys
Gly Val Ala Val Arg Arg Val Leu Asn Thr Glu Ala Asn 210 215 220Val
Val Arg Lys Phe Gly Val Thr Asp Phe Pro Ser Cys Tyr Leu Leu225 230
235 240Phe Arg Asn Gly Ser Val Ser Arg Val Pro Val Leu Met Glu Ser
Arg 245 250 255Ser Phe Tyr Thr Ala Tyr Leu Gln Arg Leu Ser Gly Leu
Thr Arg Glu 260 265 270Ala Ala Gln Thr Thr Val Ala Pro Thr Thr Ala
Asn Lys Ile Ala Pro 275 280 285Thr Val Trp Lys Leu Ala Asp Arg Ser
Lys Ile Tyr Met Ala Asp Leu 290 295 300Glu Ser Ala Leu His Tyr Ile
Leu Arg Ile Glu Val Gly Arg Phe Pro305 310 315 320Val Leu Glu Gly
Gln Arg Leu Val Ala Leu Lys Lys Phe Val Ala Val 325 330 335Leu Ala
Lys Tyr Phe Pro Gly Arg Pro Leu Val Gln Asn Phe Leu His 340 345
350Ser Val Asn Glu Trp Leu Lys Arg Gln Lys Arg Asn Lys Ile Pro Tyr
355 360 365Ser Phe Phe Lys Thr Ala Leu Asp Asp Arg Lys Glu Gly Ala
Val Leu 370 375 380Ala Lys Lys Val Asn Trp Ile Gly Cys Gln Gly Ser
Glu Pro His Phe385 390 395 400Arg Gly Phe Pro Cys Ser Leu Trp Val
Leu Phe His Phe Leu Thr Val 405 410 415Gln Ala Ala Arg Gln Asn Val
Asp His Ser Gln Glu Ala Ala Lys Ala 420 425 430Lys Glu Val Leu Pro
Ala Ile Arg Gly Tyr Val His Tyr Phe Phe Gly 435 440 445Cys Arg Asp
Cys Ala Ser His Phe Glu Gln Met Ala Ala Ala Ser Met 450 455 460His
Arg Val Gly Ser Pro Asn Ala Ala Val Leu Trp Leu Trp Ser Ser465 470
475 480His Asn Arg Val Asn Ala Arg Leu Ala Gly Ala Pro Ser Glu Asp
Pro 485 490 495Gln Phe Pro Lys Val Gln Trp Pro Pro Arg Glu Leu Cys
Ser Ala Cys 500 505 510His Asn Glu Arg Leu Asp Val Pro Val Trp Asp
Val Glu Ala Thr Leu 515 520 525Asn Phe Leu Lys Ala His Phe Ser Pro
Ser Asn Ile Ile Leu Asp Phe 530 535 540Pro Ala Ala Gly Ser Ala Ala
Arg Arg Asp Val Gln Asn Val Ala Ala545 550 555 560Ala Pro Glu Leu
Ala Met Gly Ala Leu Glu Leu Glu Ser Arg Asn Ser 565 570 575Thr Leu
Asp Pro Gly Lys Pro Glu Met Met Lys Ser Pro Thr Asn Thr 580 585
590Thr Pro His Val Pro Ala Glu Gly Pro Glu Leu Ile 595
6006748PRTMus musculus 6Met Arg Arg Cys Gly Arg Leu Ser Gly Pro Pro
Ser Leu Leu Leu Leu1 5 10 15Leu Leu Leu Leu Ser Pro Leu Leu Phe Ser
Gly Pro Gly Ala Tyr Ala 20 25 30Ala Arg Leu Ser Val Leu Tyr Ser Ser
Ser Asp Pro Leu Thr Leu Leu 35 40 45Asp Ala Asp Ser Val Arg Pro Thr
Val Leu Gly Ser Ser Ser Ala Trp 50 55 60Ala Val Glu Phe Phe Ala Ser
Trp Cys Gly His Cys Ile Ala Phe Ala65 70 75 80Pro Thr Trp Lys Glu
Leu Ala Asn Asp Val Lys Asp Trp Arg Pro Ala 85 90 95Leu Asn Leu Ala
Val Leu Asp Cys Ala Glu Glu Thr Asn Ser Ala Val 100 105 110Cys Arg
Glu Phe Asn Ile Ala Gly Phe Pro Thr Val Arg Phe Phe Gln 115 120
125Ala Phe Thr Lys Asn Gly Ser Gly Ala Thr Leu Pro Gly Ala Gly Ala
130 135 140Asn Val Gln Thr Leu Arg Met Arg Leu Ile Asp Ala Leu Glu
Ser His145 150 155 160Arg Asp Thr Trp Pro Pro Ala Cys Pro Pro Leu
Glu Pro Ala Lys Leu 165 170 175Asn Asp Ile Asp Gly Phe Phe Thr Arg
Asn Lys Ala Asp Tyr Leu Ala 180 185 190Leu Val Phe Glu Arg Glu Asp
Ser Tyr Leu Gly Arg Glu Val Thr Leu 195 200 205Asp Leu Ser Gln Tyr
His Ala Val Ala Val Arg Arg Val Leu Asn Thr 210 215 220Glu Ser Asp
Leu Val Asn Lys Phe Gly Val Thr Asp Phe Pro Ser Cys225 230 235
240Tyr Leu Leu Leu Arg Asn Gly Ser Val Ser Arg Val Pro Val Leu Val
245 250 255Glu Ser Arg Ser Phe Tyr Thr Ser Tyr Leu Arg Gly Leu Pro
Gly Leu 260 265 270Thr Arg Asp Ala Pro Pro Thr Thr Ala Thr Pro Val
Thr Ala Asp Lys 275 280 285Ile Ala Pro Thr Val Trp Lys Phe Ala Asp
Arg Ser Lys Ile Tyr Met 290 295 300Ala Asp Leu Glu Ser Ala Leu His
Tyr Ile Leu Arg Val Glu Val Gly305 310 315 320Lys Phe Ser Val Leu
Glu Gly Gln Arg Leu Val Ala Leu Lys Lys Phe 325 330 335Val Ala Val
Leu Ala Lys Tyr Phe Pro Gly Gln Pro Leu Val Gln Asn 340 345 350Phe
Leu His Ser Ile Asn Asp Trp Leu Gln Lys Gln Gln Lys Lys Arg 355 360
365Ile Pro Tyr Ser Phe Phe Lys Ala Ala Leu Asp Ser Arg Lys Glu Asp
370 375 380Ala Val Leu Thr Glu Lys Val Asn Trp Val Gly Cys Gln Gly
Ser Glu385 390 395 400Pro His Phe Arg Gly Phe Pro Cys Ser Leu Trp
Val Leu Phe His Phe 405 410 415Leu Thr Val Gln Ala Asn Arg Tyr Ser
Glu Ala His Pro Gln Glu Pro 420 425 430Ala Asp Gly Gln Glu Val Leu
Gln Ala Met Arg Ser Tyr Val Gln Phe 435 440 445Phe Phe Gly Cys Arg
Asp Cys Ala Asp His Phe Glu Gln Met Ala Ala 450 455 460Ala Ser Met
His Gln Val Arg Ser Pro Ser Asn Ala Ile Leu Trp Leu465 470 475
480Trp Thr Ser His Asn Arg Val Asn Ala Arg Leu Ser Gly Ala Leu Ser
485 490 495Glu Asp Pro His Phe Pro Lys Val Gln Trp Pro Pro Arg Glu
Leu Cys 500 505 510Ser Ala Cys His Asn Glu Leu Asn Gly Gln Val Pro
Leu Trp Asp Leu 515 520 525Gly Ala Thr Leu Asn Phe Leu Lys Ala His
Phe Ser Pro Ala Asn Ile 530 535 540Val Ile Asp Ser Ser Ala Ser Arg
His Thr Gly Arg Arg Gly Ser Pro545 550 555 560Glu Ala Thr Pro Glu
Leu Val Met Asp Thr Leu Lys Leu Glu Ser Arg 565 570 575Asn Ser Val
Leu Gly His Glu Gln Ala Ala Ser Ala Glu Ser Pro Gly 580 585 590Ala
Thr Ala Leu Asp Val Pro Ala Glu Lys Pro Glu Ala Ser Gly Pro 595 600
605Gln Glu Leu Tyr Thr Gly Leu Arg Met Gly Gly Ala Ser Pro Gly Gln
610 615 620Gly Pro Pro Glu Arg Met Glu Asp His Gln Arg Asp Met Gln
Glu Asn625 630 635 640Ala Pro Gly Gln Gln His Leu Ser Lys Arg Asp
Thr Glu Ala Leu Phe 645 650 655Leu Pro Glu Val Asn His Leu Gln Gly
Pro Leu Glu Leu Arg Arg Gly 660 665 670Gly Arg Ser Pro Lys Gln Leu
Ala Pro Ile Leu Glu Glu Glu Pro Glu 675 680 685Ala Leu Ala Ile Gln
Gly Gln Gly Gln Trp Leu Gln Val Leu Gly Gly 690 695 700Gly Ile Ser
His Leu Asp Ile Ser Leu Cys Val Gly Leu Tyr Ser Val705 710 715
720Ser Phe Met Gly Leu Leu Ala Met Tyr Thr Tyr Phe Arg Ala Arg Leu
725 730 735Arg Thr Pro Lys Gly His Ala Ser Tyr Pro Thr Ala 740
7457750PRTRattus norvegicus 7Met Arg Arg Cys Gly Arg His Ser Gly
Pro Pro Ser Leu Leu Leu Leu1 5 10 15Leu Leu Leu Leu Pro Pro Leu Leu
Leu Ser Val Pro Gly Ala Tyr Ala 20 25 30Ala Arg Leu Ser Val Leu Tyr
Ser Ser Ser Asp Pro Leu Thr Leu Leu 35 40 45Asp Ala Asp Thr Val Arg
Pro Ala Val Leu Gly Ser Ser Ser Ala Trp 50 55 60Ala Val Glu Phe Phe
Ala Ser Trp Cys Gly His Cys Ile Ala Phe Ala65 70 75 80Pro Thr Trp
Lys Glu Leu Ala Asn Asp Val Lys Asp Trp Arg Pro Ala 85 90 95Leu Asn
Leu Ala Val Leu Asp Cys Ala Asp Glu Thr Asn Ser Ala Val 100 105
110Cys Arg Glu Phe Asn Ile Ala Gly Phe Pro Thr Val Arg Phe Phe Lys
115 120 125Ala Phe Ser Lys Asn Gly Thr Gly Thr Ala Leu Pro Ala Ala
Gly Ala 130 135 140Asn Val Gln Thr Leu Arg Met Arg Leu Ile Asp Ala
Leu Glu Ser His145 150 155 160Arg Asp Thr Trp Pro Pro Ala Cys Pro
Pro Leu Glu Pro Ala Lys Leu 165 170 175Lys Asp Ile Asn Glu Phe Phe
Thr Arg Ser Lys Ala Glu Tyr Leu Ala 180 185 190Leu Ile Phe Glu Arg
Glu Asp Ser Tyr Leu Gly Arg Glu Val Thr Leu 195 200 205Asp Leu Ser
Gln Phe His Ala Val Ala Val Arg Arg Val Leu Asn Ser 210 215 220Glu
Ser Asp Val Val Ser Lys Phe Ala Val Thr Asp Phe Pro Ser Cys225 230
235 240Tyr Leu Leu Leu Arg Asn Gly Ser Val Ser Arg Val Pro Val Leu
Val 245 250 255Glu Ser Arg Pro Phe Tyr Thr Ser Tyr Leu Arg Gly Leu
Pro Gly Leu 260 265 270Thr Arg Glu Ala Pro Pro Thr Thr Ala Ala Pro
Val Thr Pro Asp Lys 275 280 285Ile Ala Pro Thr Val Trp Lys Phe Ala
Asp Arg Ser Lys Ile Tyr Met 290 295 300Ala Asp Leu Glu Ser Ala Leu
His Tyr Ile Leu Arg Val Glu Val Gly305 310 315 320Lys Phe Ser Val
Leu Glu Gly Gln Arg Leu Val Ala Leu Lys Lys Phe 325 330 335Val Ala
Val Leu Ala Lys Tyr Phe Pro Gly Gln Pro Leu Val Gln Asn 340 345
350Phe Leu His Ser Ile Asn Asp Trp Leu Gln Lys Gln Gln Lys Lys Lys
355 360 365Ile Pro Tyr Ser Tyr Phe Lys Ala Ala Leu Asp Ser Arg Lys
Glu Asn 370 375 380Ala Val Leu Ala Glu Lys Val Asn Trp Ile Gly Cys
Gln Gly Ser Glu385 390 395 400Pro His Phe Arg Gly Phe Pro Cys Ser
Leu Trp Val Leu Phe His Phe 405 410 415Leu Thr Val Gln Ala His Arg
Tyr Ser Glu Ala His Pro Gln Glu Pro 420 425 430Ala Asp Gly Gln Glu
Val Leu Gln Ala Met Arg Ser Tyr Val Gln Ser 435 440 445Phe Phe Gly
Cys Arg Asp Cys Ala Asn His Phe Glu Gln Met Ala Ala 450 455 460Ala
Ser Met His Gln Val Lys Ser Pro Ser Asn Ala Val Leu Trp Leu465 470
475 480Trp Thr Ser His Asn Arg Val Asn Ala Arg Leu Ser Gly Ala Leu
Ser 485 490 495Glu Asp Pro Gln Phe Pro Lys Val Gln Trp Pro Pro Arg
Glu Leu Cys 500 505 510Ser Ala Cys His Asn Glu Val Asn Gly Gln Val
Pro Leu Trp Asp Leu 515 520 525Gly Ala Thr Leu Asn Phe Leu Lys Ala
His Phe Ser Pro Ala Asn Ile 530 535 540Val Arg Asp Pro Pro Ala Pro
Gly Pro Ala Ser Arg Arg Gly Thr Gln545 550 555 560Asp Pro Glu Ala
Ser Pro Asn Leu Val Met Asp Thr Leu Lys Leu Glu 565 570 575Thr Gly
Asn Ser Val Leu Gly His Glu Gln Ala Ala Ser Ala Ala Ser 580 585
590Pro Gly Ala Thr Ala Leu Asp Val Pro Ala Gly Lys Pro Glu Ala Ser
595 600 605Gly Pro Gln Glu Leu Asn Ala Gly Leu Ser Met Gly Gly Ala
Ser Pro 610 615 620Gly Gln Gly Pro Pro Glu His Thr Glu Glu Leu Leu
Arg Asp Val Gln625 630 635 640Glu Asn Ala Gln Gly Gln Gln His Leu
Ser Lys Arg Asp Thr Glu Ala 645 650 655Leu Leu Leu Pro Glu Val Asn
His Leu Gln Gly Pro Leu Ala Pro Arg 660 665 670Arg Gly Gly His Ser
Pro Lys Gln Leu Ala Ser Ile Leu Glu Gly Glu 675 680 685Pro Glu Ala
Leu Ala Ile Gln Gly Arg Arg Gln Trp Leu Gln Val Leu 690 695 700Gly
Gly Gly Val Ser Phe Leu Asp Ile Ser Leu Cys Val Gly Leu Tyr705 710
715 720Ser Val Ser Phe Met Gly Leu Leu Ala Met Tyr Thr Tyr Phe Arg
Ala 725 730 735Arg Met Arg Thr Pro Lys Gly His Val Ser Tyr Pro Thr
Ala 740 745 7508613PRTCavia porcellus 8Met Thr Gly Cys Gly Arg Arg
Ser Gly Trp Leu Pro Pro Leu Arg Leu1 5 10 15Leu Leu Leu Pro Leu Leu
Leu Gly Gly Pro Gly Val Gly Ala Ala Gln 20 25 30Leu Ala Ala Leu Tyr
Ser Ala Ser Asp Pro Leu Thr Leu Leu Gln Ala 35 40 45Asp Thr Val Arg
Ser Thr Val Leu Asn Ser Pro Ser Ala Trp Ala Val 50 55 60Glu Phe Phe
Ala Ser Trp Cys Gly His Cys Ile Ala Phe Ala Pro Thr65 70 75 80Trp
Lys Ala Leu Ala Lys Asp Ile Lys Asp Trp Arg Pro Ala Leu Asn 85 90
95Leu Ala Ala Leu Asn Cys Ala Asp Glu Thr Asn Asn Ala Val Cys Arg
100 105 110Asp Phe Asn Ile Ala Gly Phe Pro Ser Val Arg Phe Phe Lys
Ala Phe 115 120 125Ser Lys Asn Ser Thr Gly Thr Thr Leu Pro Val Ala
Gly Ala Asn Val 130 135 140Gln Met Leu Arg Glu Arg Leu Ile Asp Ala
Leu Glu Ser His His Asp145 150 155 160Thr Trp Pro Ser Ala Cys Pro
Pro Leu Glu Pro Val Lys Pro Lys Glu 165 170 175Ile Asp Thr Phe Phe
Ala Arg Asn Asn Gln Glu Tyr Leu Val Leu Ile 180 185 190Phe Glu Gln
Glu Asn Ser Tyr Leu Gly Arg Glu Val Thr Leu Asp Leu 195 200 205Ser
Gln His His Asp Leu Val Val Arg Arg Val Leu Ser Thr Glu Ala 210 215
220Asn Val Val Arg Lys Phe Gly Val Ala Asp Phe Pro Ser Cys Tyr
Leu225 230 235 240Leu Phe Arg Asn Gly Ser Val Ser Arg Val Pro Val
Leu Val Glu Ser 245 250 255Arg Arg Phe Tyr Thr Ala Tyr Leu Gln Arg
Leu Ser Glu Val Thr Arg 260 265 270Glu Gly Thr Pro Thr Pro Ala Val
Pro Thr Ile Ser Asp Gln Ile Ala 275 280 285Pro Thr Val Trp Lys Phe
Ala Asp Arg Ser Lys Ile Tyr Met Ala Asp 290 295 300Leu Glu Ser Ala
Leu His Tyr Ile Leu Arg Val Glu Val Gly Arg Phe305 310 315 320Ser
Val Leu Glu Gly Gln Arg Leu Met Ala Leu Lys Lys Phe Val Thr 325 330
335Val Leu Thr Lys Tyr Phe Pro Gly Gln Pro Leu Val Arg Asn Phe Leu
340 345 350Gln Ser Thr Asn Glu Trp Leu Lys Arg Gln His Lys Lys Lys
Met Pro 355 360 365Tyr Ser Phe Phe Lys Thr Ala Met Asp Ser Arg Asn
Glu Glu Ala Val 370 375 380Ile Thr Lys Glu Val Asn Trp Val Gly Cys
Gln Gly Ser Glu Ser His385 390 395 400Phe Arg Gly Phe Pro Cys Ser
Leu Trp Ile Leu Phe His Phe Leu Thr 405 410 415Val Gln Ala Ser Gln
Lys Asn Ala Glu Ser Ser Gln Lys Pro Ala Asn 420 425 430Gly Gln Glu
Val Leu Gln Ala Ile Arg Asn Tyr Val Arg Phe Phe Phe 435 440 445Gly
Cys Arg Asp Cys Ala Asn His Phe Glu Gln Met Ala Ala Gly Ser 450 455
460Met His Arg Val Lys Ser Pro Asn Asp Ala Val Leu Trp Leu Trp
Thr465 470 475 480Ser His Asn Arg Val Asn Ala Arg Leu Ala
Gly Ala Pro Ser Glu Asp 485 490 495Pro Gln Phe Pro Lys Val Gln Trp
Pro Pro Pro Glu Leu Cys Ser Ala 500 505 510Cys His Asn Glu Leu Ser
Gly Glu Pro Val Trp Asp Val Asp Ala Thr 515 520 525Leu Arg Phe Leu
Lys Thr His Phe Ser Pro Ser Asn Ile Val Leu Asn 530 535 540Phe Pro
Pro Ala Glu Pro Ala Ser Arg Ser Ser Val His Ser Trp Gly545 550 555
560Ala Thr Pro His Leu Glu Leu Asp Ala Leu Gly Leu Val Thr Arg Asn
565 570 575Ser Ala Leu Ala Leu Glu Arg Ala Glu Ile Ser Glu Ser Pro
Gly Ser 580 585 590Asn Ala Met Pro Asn Ile Pro Ala Glu Arg Pro Glu
Leu Phe Glu Ala 595 600 605Leu Ser His Ser Arg 6109106PRTArtificial
SequenceMAb316.1 variable region - light chain 9Gln Ile Val Leu Thr
Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly1 5 10 15Glu Lys Val Thr
Ile Ser Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30Tyr Trp Tyr
His Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr 35 40 45Arg Thr
Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60Gly
Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu65 70 75
80Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Tyr His Ser Tyr Pro Leu Thr
85 90 95Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100
10510115PRTArtificial SequenceMAb316.1 variable region - heavy
chain 10Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly
Ala1 5 10 15Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr
Ser Tyr 20 25 30Tyr Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu
Glu Trp Ile 35 40 45Gly Trp Ile Tyr Pro Gly Ser Tyr Asn Thr Glu Tyr
Ser Glu Lys Phe 50 55 60Lys Gly Lys Ala Thr Leu Thr Ala Asp Thr Ser
Ser Ser Thr Ala Tyr65 70 75 80Met Gln Leu Ser Ser Leu Thr Ser Glu
Asp Ser Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ser Glu Asp Trp Phe Ala
Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110Thr Val Ser
1151118PRTArtificial SequenceMAb316.1 variable region partial
sequences (light chain) 11Gln Ile Val Leu Thr Gln Ser Pro Ala Ile
Met Ser Ala Ser Pro Gly1 5 10 15Glu Lys1226PRTArtificial
SequenceMAb316.1 variable region partial sequences (light chain)
12Val Thr Ile Ser Cys Ser Ala Ser Ser Ser Val Ser Tyr Met Tyr Trp1
5 10 15Tyr His Gln Lys Pro Gly Ser Ser Pro Lys 20
251311PRTArtificial SequenceMAb316.1 variable region partial
sequences (light chain) 13Thr Ser Asn Leu Ala Ser Gly Val Pro Ala
Arg1 5 101430PRTArtificial SequenceMAb316.1 variable region partial
sequences (light chain) 14Thr Ile Ser Ser Met Glu Ala Glu Asp Ala
Ala Thr Tyr Tyr Cys Gln1 5 10 15Gln Tyr His Ser Tyr Pro Leu Thr Phe
Gly Ala Gly Thr Lys 20 25 301515PRTArtificial SequenceMAb316.1
variable region partial sequences (light chain) 15Ile Tyr Arg Thr
Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe1 5 10
151613PRTArtificial SequenceMAb316.1 variable region partial
sequences (light chain) 16Arg Thr Ser Asn Leu Ala Ser Gly Val Pro
Ala Arg Phe1 5 101717PRTArtificial SequenceMAb316.1 variable region
partial sequences (light chain) 17Ala Ser Gly Val Pro Ala Arg Phe
Ser Gly Ser Gly Ser Gly Thr Ser1 5 10 15Tyr1810PRTArtificial
SequenceMAb316.1 variable region partial sequences (light chain)
18Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu1 5 101919PRTArtificial
SequenceMAb316.1 variable region partial sequences (heavy chain)
19Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala1
5 10 15Ser Val Lys2015PRTArtificial SequenceMAb316.1 variable
region partial sequences (heavy chain) 20Ala Ser Gly Tyr Ser Phe
Thr Ser Tyr Tyr Ile His Trp Val Lys1 5 10 152125PRTArtificial
SequenceMAb316.1 variable region partial sequences (heavy chain)
21Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Trp Ile Tyr Pro Gly1
5 10 15Ser Tyr Asn Thr Glu Tyr Ser Glu Lys 20 252218PRTArtificial
SequenceMAb316.1 variable region partial sequences (heavy chain)
22Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys1
5 10 15Ala Arg2323PRTArtificial SequenceMAb316.1 variable region
partial sequences (heavy chain) 23Gln Gln Ser Gly Pro Glu Leu Val
Lys Pro Gly Ala Ser Val Lys Ile1 5 10 15Ser Cys Lys Ala Ser Gly Tyr
202414PRTArtificial SequenceMAb316.1 variable region partial
sequences (heavy chain) 24Val Lys Gln Arg Pro Gly Gln Gly Leu Glu
Trp Ile Gly Trp1 5 102512PRTArtificial SequenceMAb316.1 variable
region partial sequences (heavy chain) 25Pro Gly Ser Tyr Asn Thr
Glu Tyr Ser Glu Lys Phe1 5 10265PRTArtificial SequenceMAb316.1
light chain CDR1 amino acid sequence 26Ser Ser Val Ser Tyr1
5273PRTArtificial SequenceMAb316.1 light chain CDR2 amino acid
sequence 27Arg Thr Ser1289PRTArtificial SequenceMAb316.1 light
chain CDR3 amino acid sequence 28Gln Gln Tyr His Ser Tyr Pro Leu
Thr1 5298PRTArtificial SequenceMAb316.1 heavy chain CDR1 amino acid
sequence 29Gly Tyr Ser Phe Thr Ser Tyr Tyr1 5308PRTArtificial
SequenceMAb316.1 heavy chain CDR2 amino acid sequence 30Ile Tyr Pro
Gly Ser Tyr Asn Thr1 5319PRTArtificial SequenceMAb316.1 heavy chain
CDR3 amino acid sequence 31Ala Arg Ser Glu Asp Trp Phe Ala Tyr1
532748PRTHomo sapiens 32Met Arg Arg Cys Gly Arg Leu Ser Gly Pro Pro
Ser Leu Leu Leu Leu1 5 10 15Leu Leu Leu Leu Ser Pro Leu Leu Phe Ser
Gly Pro Gly Ala Tyr Ala 20 25 30Ala Arg Leu Ser Val Leu Tyr Ser Ser
Ser Asp Pro Leu Thr Leu Leu 35 40 45Asp Ala Asp Ser Val Arg Pro Thr
Val Leu Gly Ser Ser Ser Ala Trp 50 55 60Ala Val Glu Phe Phe Ala Ser
Trp Cys Gly His Cys Ile Ala Phe Ala65 70 75 80Pro Thr Trp Lys Glu
Leu Ala Asn Asp Val Lys Asp Trp Arg Pro Ala 85 90 95Leu Asn Leu Ala
Val Leu Asp Cys Ala Glu Glu Thr Asn Ser Ala Val 100 105 110Cys Arg
Glu Phe Asn Ile Ala Gly Phe Pro Thr Val Arg Phe Phe Gln 115 120
125Ala Phe Thr Lys Asn Gly Ser Gly Ala Thr Leu Pro Gly Ala Gly Ala
130 135 140Asn Val Gln Thr Leu Arg Met Arg Leu Ile Asp Ala Leu Glu
Ser His145 150 155 160Arg Asp Thr Trp Pro Pro Ala Cys Pro Pro Leu
Glu Pro Ala Lys Leu 165 170 175Asn Asp Ile Asp Gly Phe Phe Thr Arg
Asn Lys Ala Asp Tyr Leu Ala 180 185 190Leu Val Phe Glu Arg Glu Asp
Ser Tyr Leu Gly Arg Glu Val Thr Leu 195 200 205Asp Leu Ser Gln Tyr
His Ala Val Ala Val Arg Arg Val Leu Asn Thr 210 215 220Glu Ser Asp
Leu Val Asn Lys Phe Gly Val Thr Asp Phe Pro Ser Cys225 230 235
240Tyr Leu Leu Leu Arg Asn Gly Ser Val Ser Arg Val Pro Val Leu Val
245 250 255Glu Ser Arg Ser Phe Tyr Thr Ser Tyr Leu Arg Gly Leu Pro
Gly Leu 260 265 270Thr Arg Asp Ala Pro Pro Thr Thr Ala Thr Pro Val
Thr Ala Asp Lys 275 280 285Ile Ala Pro Thr Val Trp Lys Phe Ala Asp
Arg Ser Lys Ile Tyr Met 290 295 300Ala Asp Leu Glu Ser Ala Leu His
Tyr Ile Leu Arg Val Glu Val Gly305 310 315 320Lys Phe Ser Val Leu
Glu Gly Gln Arg Leu Val Ala Leu Lys Lys Phe 325 330 335Val Ala Val
Leu Ala Lys Tyr Phe Pro Gly Gln Pro Leu Val Gln Asn 340 345 350Phe
Leu His Ser Ile Asn Asp Trp Leu Gln Lys Gln Gln Lys Lys Arg 355 360
365Ile Pro Tyr Ser Phe Phe Lys Ala Ala Leu Asp Ser Arg Lys Glu Asp
370 375 380Ala Val Leu Thr Glu Lys Val Asn Trp Val Gly Cys Gln Gly
Ser Glu385 390 395 400Pro His Phe Arg Gly Phe Pro Cys Ser Leu Trp
Val Leu Phe His Phe 405 410 415Leu Thr Val Gln Ala Asn Arg Tyr Ser
Glu Ala His Pro Gln Glu Pro 420 425 430Ala Asp Gly Gln Glu Val Leu
Gln Ala Met Arg Ser Tyr Val Gln Phe 435 440 445Phe Phe Gly Cys Arg
Asp Cys Ala Asp His Phe Glu Gln Met Ala Ala 450 455 460Ala Ser Met
His Gln Val Arg Ser Pro Ser Asn Ala Ile Leu Trp Leu465 470 475
480Trp Thr Ser His Asn Arg Val Asn Ala Arg Leu Ser Gly Ala Leu Ser
485 490 495Glu Asp Pro His Phe Pro Lys Val Gln Trp Pro Pro Arg Glu
Leu Cys 500 505 510Ser Ala Cys His Asn Glu Leu Asn Gly Gln Val Pro
Leu Trp Asp Leu 515 520 525Gly Ala Thr Leu Asn Phe Leu Lys Ala His
Phe Ser Pro Ala Asn Ile 530 535 540Val Ile Asp Ser Ser Ala Ser Arg
His Thr Gly Arg Arg Gly Ser Pro545 550 555 560Glu Ala Thr Pro Glu
Leu Val Met Asp Thr Leu Lys Leu Glu Ser Arg 565 570 575Asn Ser Val
Leu Gly His Glu Gln Ala Ala Ser Ala Glu Ser Pro Gly 580 585 590Ala
Thr Ala Leu Asp Val Pro Ala Glu Lys Pro Glu Ala Ser Gly Pro 595 600
605Gln Glu Leu Tyr Thr Gly Leu Arg Met Gly Gly Ala Ser Pro Gly Gln
610 615 620Gly Pro Pro Glu Arg Met Glu Asp His Gln Arg Asp Met Gln
Glu Asn625 630 635 640Ala Pro Gly Gln Gln His Leu Ser Lys Arg Asp
Thr Glu Ala Leu Phe 645 650 655Leu Pro Glu Val Asn His Leu Gln Gly
Pro Leu Glu Leu Arg Arg Gly 660 665 670Gly Arg Ser Pro Lys Gln Leu
Ala Pro Ile Leu Glu Glu Glu Pro Glu 675 680 685Ala Leu Ala Ile Gln
Gly Gln Gly Gln Trp Leu Gln Val Leu Gly Gly 690 695 700Gly Ile Ser
His Leu Asp Ile Ser Leu Cys Val Gly Leu Tyr Ser Val705 710 715
720Ser Phe Met Gly Leu Leu Ala Met Tyr Thr Tyr Phe Arg Ala Arg Leu
725 730 735Arg Thr Pro Lys Gly His Ala Ser Tyr Pro Thr Ala 740
74533568PRTHomo sapiens 33Met Arg Arg Cys Gly Arg Leu Ser Gly Pro
Pro Ser Leu Leu Leu Leu1 5 10 15Leu Leu Leu Leu Ser Pro Leu Leu Phe
Ser Gly Pro Gly Ala Tyr Ala 20 25 30Ala Arg Leu Ser Val Leu Tyr Ser
Ser Ser Asp Pro Leu Thr Leu Leu 35 40 45Asp Ala Asp Ser Val Arg Pro
Thr Val Leu Gly Ser Ser Ser Ala Trp 50 55 60Ala Val Glu Phe Phe Ala
Ser Trp Cys Gly His Cys Ile Ala Phe Ala65 70 75 80Pro Thr Trp Lys
Glu Leu Ala Asn Asp Val Lys Asp Trp Arg Pro Ala 85 90 95Leu Asn Leu
Ala Val Leu Asp Cys Ala Glu Glu Thr Asn Ser Ala Val 100 105 110Cys
Arg Glu Phe Asn Ile Ala Gly Phe Pro Thr Val Arg Phe Phe Gln 115 120
125Ala Phe Thr Lys Asn Gly Ser Gly Ala Thr Leu Pro Gly Ala Gly Ala
130 135 140Asn Val Gln Thr Leu Arg Met Arg Leu Ile Asp Ala Leu Glu
Ser His145 150 155 160Arg Asp Thr Trp Pro Pro Ala Cys Pro Pro Leu
Glu Pro Ala Lys Leu 165 170 175Asn Asp Ile Asp Gly Phe Phe Thr Arg
Asn Lys Ala Asp Tyr Leu Ala 180 185 190Leu Val Phe Glu Arg Glu Asp
Ser Tyr Leu Gly Arg Glu Val Thr Leu 195 200 205Asp Leu Ser Gln Tyr
His Ala Val Ala Val Arg Arg Val Leu Asn Thr 210 215 220Glu Ser Asp
Leu Val Asn Lys Phe Gly Val Thr Asp Phe Pro Ser Cys225 230 235
240Tyr Leu Leu Leu Arg Asn Gly Ser Val Ser Arg Val Pro Val Leu Val
245 250 255Glu Ser Arg Ser Phe Tyr Thr Ser Tyr Leu Arg Gly Leu Pro
Gly Leu 260 265 270Thr Arg Asp Ala Pro Pro Thr Thr Ala Thr Pro Val
Thr Ala Asp Lys 275 280 285Ile Ala Pro Thr Val Trp Lys Phe Ala Asp
Arg Ser Lys Ile Tyr Met 290 295 300Ala Asp Leu Glu Ser Ala Leu His
Tyr Ile Leu Arg Val Glu Val Gly305 310 315 320Lys Phe Ser Val Leu
Glu Gly Gln Arg Leu Val Ala Leu Lys Lys Phe 325 330 335Val Ala Val
Leu Ala Lys Tyr Phe Pro Gly Gln Pro Leu Val Gln Asn 340 345 350Phe
Leu His Ser Ile Asn Asp Trp Leu Gln Lys Gln Gln Lys Lys Arg 355 360
365Ile Pro Tyr Ser Phe Phe Lys Ala Ala Leu Asp Ser Arg Lys Glu Asp
370 375 380Ala Val Leu Thr Glu Lys Val Asn Trp Val Gly Cys Gln Gly
Ser Glu385 390 395 400Pro His Phe Arg Gly Phe Pro Cys Ser Leu Trp
Val Leu Phe His Phe 405 410 415Leu Thr Val Gln Ala Asn Arg Tyr Ser
Glu Ala His Pro Gln Glu Pro 420 425 430Ala Asp Gly Gln Glu Val Leu
Gln Ala Met Arg Ser Tyr Val Gln Phe 435 440 445Phe Phe Gly Cys Arg
Asp Cys Ala Asp His Phe Glu Gln Met Ala Ala 450 455 460Ala Ser Met
His Gln Val Arg Ser Pro Ser Asn Ala Ile Leu Trp Leu465 470 475
480Trp Thr Ser His Asn Arg Val Asn Ala Arg Leu Ser Gly Ala Leu Ser
485 490 495Glu Asp Pro His Phe Pro Lys Val Gln Trp Pro Pro Arg Glu
Leu Cys 500 505 510Ser Ala Cys His Asn Glu Leu Asn Gly Gln Val Pro
Leu Trp Asp Leu 515 520 525Gly Ala Thr Leu Asn Phe Leu Lys Ala His
Phe Ser Pro Ala Asn Ile 530 535 540Val Ile Asp Ser Ser Ala Ser Arg
His Thr Gly Arg Arg Gly Ser Pro545 550 555 560Glu Ala Thr Pro Glu
Leu Leu Leu 565343399DNAHomo sapiens 34ctcctcctcc tcctccgggg
cggaggctgt tggtgcgcgg caggctccgg atactgacta 60gtcacaaact tgaaggaggt
ggacattcaa gccgcctagg atgaggaggt gcggccgcct 120ctcggggccg
ccatcgctgc tgctactact gctgctgctc tcgcctctgc tcttctcggg
180gcccggcgct tacgcggccc ggctctcagt gctctactcg tcctctgacc
cgctgacgct 240gctggatgct gattcggtgc gtcccactgt gctcggctcc
agcagcgcct gggcggtgga 300gttcttcgcc tcctggtgtg gccactgcat
cgccttcgcc ccgacgtgga aggagcttgc 360taacgacgtg aaagactgga
ggccagcact caatcttgct gtcctggact gtgctgagga 420gaccaacagt
gctgtctgca gagagttcaa catcgctggc ttcccgactg tgaggttttt
480tcaggccttt accaagaacg gttctggagc gacactgcca ggtgctggcg
ctaatgtgca 540gactctgcgt atgaggctca tcgatgctct ggagtcccac
cgtgacacat ggcccccagc 600ctgtccacct ctggaacctg ccaagctgaa
tgatatcgac ggattcttta caagaaataa 660agcagattac ctggccctgg
tctttgaaag ggaagactcc tacctgggta gagaggtaac 720tctggacctg
tcccagtacc atgctgtggc agtgcgcagg gtcttgaata cagagagtga
780cctggtgaac aagtttggcg tcactgactt cccatcttgt tacctgctgc
ttcggaatgg 840ctctgtctcc cgagtgcctg tgctggtgga gtccaggtct
ttctatacat cctatcttcg 900ggggctacct ggactgacca gggatgctcc
cccaaccaca gccaccccag tcactgctga 960taagatagca cccacagtgt
ggaagtttgc agaccgctcc aagatctaca tggccgacct 1020ggagtccgca
ctccactaca tcttgcgtgt agaagtgggg aagttctcag tgctggaggg
1080acagcgcctg gtggccctga aaaagtttgt ggcagtattg gccaagtact
tccctgggca 1140gcctttggtc cagaacttct tgcattccat aaacgactgg
cttcagaagc agcagaagaa 1200gaggatcccc tacagtttct tcaaagctgc
tctggacagc aggaaggagg atgctgtcct 1260tactgagaag gtgaactggg
tcggctgcca gggcagtgag ccacacttcc gggggtttcc 1320ctgctcactg
tgggtcctct tccacttcct gacggtgcag gcaaaccgat atagtgaggc
1380ccacccacag gaaccagctg atggccagga ggtcctccaa
gccatgagga gctatgttca 1440gttcttcttt ggctgtcgtg actgtgcgga
ccattttgag cagatggctg cagcgtccat 1500gcaccaagtg agaagtccca
gtaatgcgat tctttggctc tggactagcc acaacagggt 1560taacgctcgc
ctctcaggtg ctctgagtga ggacccccac ttccccaagg tgcagtggcc
1620tccccgtgag ctgtgttctg cctgccataa tgaactcaac ggacaggtgc
ctttgtggga 1680ccttggtgcc accctcaact ttctcaaggc tcacttctcc
ccagcaaaca tcgtcataga 1740ctcttctgca tctagacaca caggccggag
agggagtcca gaagctaccc ccgagctggt 1800aatggataca ttaaaactgg
agagcagaaa ttcagtgttg ggccatgagc aggctgcttc 1860tgcagagtcc
cctggagcca ctgccctaga tgtaccagct gagaagcctg aagcaagtgg
1920cccccaagaa ctatacacag gcctcagaat gggtggagct tcaccagggc
agggccctcc 1980tgagcgcatg gaagaccacc agagggatat gcaggagaat
gcccccgggc agcaacactt 2040gagcaagaga gacactgaag ccttatttct
gcctgaggtg aaccacctcc aaggcccttt 2100agagctcagg cgagggggcc
gcagccccaa gcaactagcc cccatacttg aagaggaacc 2160agaggcccta
gctatacagg gccaaggcca gtggctgcag gttctaggag ggggcatttc
2220ccacctggac attagcctct gtgtggggct ctactccgtg tccttcatgg
gcttactggc 2280catgtacacc tactttcggg ccaggctgag aaccccaaag
ggccatgcta gttaccccac 2340agcctgaact gcctcggcag aggacagaca
aggagctgct gatgtctggg gctttgggtt 2400tttttttttt ttttttggcc
cactggcccc cttgcttcct ttctacccct tgttctgttg 2460tctagcttag
gagagtggca agtccaagaa agtgagttgt ttcagtgaac cctggagtct
2520actgtgagag gattccctag acaaaacaga atcagggtgc cagtctcgcc
tgaccagcat 2580ggggtaggag cagcctgacg caggggctca ggaggcttct
gggaggctca ggccctggct 2640tcagcaccaa attctgtttt tcagctcttg
tgaagtcctg ccccattcct gctgaagtct 2700caatgtgtcc tgcttggtct
tggccttaga ttgaggcaga caagtccaga gtttccaagg 2760tttctcatcc
agaggagtgt atgtgggccg ggtgggctgg agtggacctc ctcactgcat
2820tctgagactg tctactcccc tccctcacga catgaagaaa aagcatccct
ccggccttct 2880ctggcctggc cagcttcagc acctcaggag tgggtggggc
ttagcttcaa gaatgggtct 2940gggagcctca gaaaatgatt ctggtcaccc
aggttcaaac tggggtttct gctgcaagag 3000tgtggtttgg caggctggga
gggtgacagt tgctgcactg tagggtgtag tgcttgcttg 3060gtggtagggg
caagggggtg ggtgatgctg gggtctgtag tgccttagtc ctgggctagc
3120tgggagaggg cagactcctc tctgagcggg ggtgcttgtc tcttactttg
gtctctaaaa 3180tcaatgctaa ctttggggtg tggaaggtgc tgggtttagg
gcagtgggca gagccctctc 3240tctcccgctt gggtccactg tgggcaggag
ggggtggaga gaccagccca catttccttc 3300ccctgctcct gtggcttggg
agggatgtgg aataaaatta tttttgttaa gtcacgcaga 3360aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaa 3399352533DNAHomo sapiens
35ctcctcctcc tcctccgggg cggaggctgt tggtgcgcgg caggctccgg atactgacta
60gtcacaaact tgaaggaggt ggacattcaa gccgcctagg atgaggaggt gcggccgcct
120ctcggggccg ccatcgctgc tgctactact gctgctgctc tcgcctctgc
tcttctcggg 180gcccggcgct tacgcggccc ggctctcagt gctctactcg
tcctctgacc cgctgacgct 240gctggatgct gattcggtgc gtcccactgt
gctcggctcc agcagcgcct gggcggtgga 300gttcttcgcc tcctggtgtg
gccactgcat cgccttcgcc ccgacgtgga aggagcttgc 360taacgacgtg
aaagactgga ggccagcact caatcttgct gtcctggact gtgctgagga
420gaccaacagt gctgtctgca gagagttcaa catcgctggc ttcccgactg
tgaggttttt 480tcaggccttt accaagaacg gttctggagc gacactgcca
ggtgctggcg ctaatgtgca 540gactctgcgt atgaggctca tcgatgctct
ggagtcccac cgtgacacat ggcccccagc 600ctgtccacct ctggaacctg
ccaagctgaa tgatatcgac ggattcttta caagaaataa 660agcagattac
ctggccctgg tctttgaaag ggaagactcc tacctgggta gagaggtaac
720tctggacctg tcccagtacc atgctgtggc agtgcgcagg gtcttgaata
cagagagtga 780cctggtgaac aagtttggcg tcactgactt cccatcttgt
tacctgctgc ttcggaatgg 840ctctgtctcc cgagtgcctg tgctggtgga
gtccaggtct ttctatacat cctatcttcg 900ggggctacct ggactgacca
gggatgctcc cccaaccaca gccaccccag tcactgctga 960taagatagca
cccacagtgt ggaagtttgc agaccgctcc aagatctaca tggccgacct
1020ggagtccgca ctccactaca tcttgcgtgt agaagtgggg aagttctcag
tgctggaggg 1080acagcgcctg gtggccctga aaaagtttgt ggcagtattg
gccaagtact tccctgggca 1140gcctttggtc cagaacttct tgcattccat
aaacgactgg cttcagaagc agcagaagaa 1200gaggatcccc tacagtttct
tcaaagctgc tctggacagc aggaaggagg atgctgtcct 1260tactgagaag
gtgaactggg tcggctgcca gggcagtgag ccacacttcc gggggtttcc
1320ctgctcactg tgggtcctct tccacttcct gacggtgcag gcaaaccgat
atagtgaggc 1380ccacccacag gaaccagctg atggccagga ggtcctccaa
gccatgagga gctatgttca 1440gttcttcttt ggctgtcgtg actgtgcgga
ccattttgag cagatggctg cagcgtccat 1500gcaccaagtg agaagtccca
gtaatgcgat tctttggctc tggactagcc acaacagggt 1560taacgctcgc
ctctcaggtg ctctgagtga ggacccccac ttccccaagg tgcagtggcc
1620tccccgtgag ctgtgttctg cctgccataa tgaactcaac ggacaggtgc
ctttgtggga 1680ccttggtgcc accctcaact ttctcaaggc tcacttctcc
ccagcaaaca tcgtcataga 1740ctcttctgca tctagacaca caggccggag
agggagtcca gaagctaccc ccgagctgct 1800cttgtgaagt cctgccccat
tcctgctgaa gtctcaatgt gtcctgcttg gtcttggcct 1860tagattgagg
cagacaagtc cagagtttcc aaggtttctc atccagagga gtgtatgtgg
1920gccgggtggg ctggagtgga cctcctcact gcattctgag actgtctact
cccctccctc 1980acgacatgaa gaaaaagcat ccctccggcc ttctctggcc
tggccagctt cagcacctca 2040ggagtgggtg gggcttagct tcaagaatgg
gtctgggagc ctcagaaaat gattctggtc 2100acccaggttc aaactggggt
ttctgctgca agagtgtggt ttggcaggct gggagggtga 2160cagttgctgc
actgtagggt gtagtgcttg cttggtggta ggggcaaggg ggtgggtgat
2220gctggggtct gtagtgcctt agtcctgggc tagctgggag agggcagact
cctctctgag 2280cgggggtgct tgtctcttac tttggtctct aaaatcaatg
ctaactttgg ggtgtggaag 2340gtgctgggtt tagggcagtg ggcagagccc
tctctctccc gcttgggtcc actgtgggca 2400ggagggggtg gagagaccag
cccacatttc cttcccctgc tcctgtggct tgggagggat 2460gtggaataaa
attatttttg ttaagtcacg cagaaaaaaa aaaaaaaaaa aaaaaaaaaa
2520aaaaaaaaaa aaa 253336107PRTArtificial SequenceMAb492.1 variable
light amino acid sequence 36Asp Val Val Met Thr Gln Thr His Lys Phe
Met Ser Thr Ser Val Gly1 5 10 15Asp Arg Val Ser Ile Thr Cys Lys Ala
Ser Gln Asp Val Ser Thr Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Ser
Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45His Ser Ala Ser Tyr Arg Tyr
Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Phe Thr Ile Ser Ser Val Gln Ala65 70 75 80Glu Asp Leu Ala
Val Tyr Tyr Cys Gln Gln His Tyr Ser Ile Pro Leu 85 90 95Thr Phe Gly
Ala Gly Thr Lys Leu Glu Leu Lys 100 10537118PRTArtificial
SequenceMAb492.1 variable heavy amino acid sequence 37Gln Val Gln
Leu Lys Gln Ser Gly Pro Gly Leu Val Ala Pro Ser Gln1 5 10 15Ser Leu
Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Gly Tyr 20 25 30Gly
Val Asn Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu 35 40
45Gly Met Ile Trp Gly Asp Gly Arg Thr Asp Tyr Lys Ser Ala Leu Lys
50 55 60Ser Arg Leu Ser Ile Thr Lys Asp Asn Ser Lys Ser Gln Val Phe
Leu65 70 75 80Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Arg Tyr
Phe Cys Ala 85 90 95Ser Asp Tyr Tyr Gly Ser Gly Ser Phe Ala Tyr Trp
Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ala
115386PRTArtificial SequenceMAb492.1 light chain CDR1 amino acid
sequence 38Gln Asp Val Ser Thr Ala1 5393PRTArtificial
SequenceMAb492.1 light chain CDR2 amino acid sequence 39Ser Ala
Ser1409PRTArtificial SequenceMAb492.1 light chain CDR3 amino acid
sequence 40Gln Gln His Tyr Ser Ile Pro Leu Thr1 5418PRTArtificial
SequenceMAb492.1 heavy chain CDR1 amino acid sequence 41Gly Phe Ser
Leu Thr Gly Tyr Gly1 5427PRTArtificial SequenceMAb492.1 heavy chain
CDR2 amino acid sequence 42Ile Trp Gly Asp Gly Arg Thr1
54312PRTArtificial SequenceMAb492.1 heavy chain CDR3 amino acid
sequence 43Ala Ser Asp Tyr Tyr Gly Ser Gly Ser Phe Ala Tyr1 5
1044107PRTArtificial SequenceMAb492.1gen variable light AA sequence
44Asp Val Val Met Thr Gln Thr His Lys Phe Met Ser Thr Ser Val Gly1
5 10 15Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Gly
Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Ser Gly Gln Ser Pro Lys Leu
Leu Ile 35 40 45Ser Met Ala Ser Gln Arg Tyr Thr Gly Val Pro Asp Arg
Phe Thr Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser
Ser Val Gln Ala65 70 75 80Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln
His Tyr Ala Ile Pro Leu 85 90 95Thr Phe Gly Ala Gly Thr Lys Leu Glu
Leu Lys 100 10545118PRTArtificial SequenceMAb492.1gen variable
heavy AA sequence 45Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val
Ala Pro Ser Gln1 5 10 15Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe
Ser Leu Thr Gly Tyr 20 25 30Ser Val Ile Trp Val Arg Gln Ser Pro Gly
Lys Gly Leu Glu Trp Leu 35 40 45Gly Met Ile Trp Gly Asp Gly Arg Thr
Glu Tyr Lys Ser Ala Leu Lys 50 55 60Ser Arg Leu Ser Ile Thr Lys Asp
Asn Ser Lys Ser Gln Val Phe Leu65 70 75 80Lys Met Asn Ser Leu Gln
Thr Asp Asp Thr Ala Arg Tyr Phe Cys Ala 85 90 95Ser Asp Ser Met Asp
Pro Gly Ser Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr
Val Ser Ala 115466PRTArtificial SequenceMAb492.1gen light chain
CDR1 AA sequence 46Gln Asp Val Ser Gly Ala1 5473PRTArtificial
SequenceMAb492.1gen light chain CDR2 AA sequence 47Met Ala
Ser1489PRTArtificial SequenceMAb492.1gen light chain CDR3 AA
sequence 48Gln Gln His Tyr Ala Ile Pro Leu Thr1 5498PRTArtificial
SequenceMAb492.1gen heavy chain CDR1 AA sequence 49Gly Phe Ser Leu
Thr Gly Tyr Ser1 5507PRTArtificial SequenceMAb492.1gen heavy chain
CDR2 AA sequence 50Ile Trp Gly Asp Gly Arg Thr1 55112PRTArtificial
SequenceMAb492.1gen heavy chain CDR3 AA sequence 51Ala Ser Asp Ser
Met Asp Pro Gly Ser Phe Ala Tyr1 5 10
* * * * *
References