U.S. patent application number 16/105651 was filed with the patent office on 2019-03-14 for novel anti-claudin antibodies and methods of use.
This patent application is currently assigned to ABBVIE STEMCENTRX LLC. The applicant listed for this patent is ABBVIE STEMCENTRX LLC. Invention is credited to Monette AUJAY, Paul Anthony ESCARPE, Orit FOORD, David LIU, Marianne SANTAGUIDA, Laura SAUNDERS, Robert A. STULL.
Application Number | 20190077876 16/105651 |
Document ID | / |
Family ID | 53042314 |
Filed Date | 2019-03-14 |
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United States Patent
Application |
20190077876 |
Kind Code |
A1 |
SANTAGUIDA; Marianne ; et
al. |
March 14, 2019 |
NOVEL ANTI-CLAUDIN ANTIBODIES AND METHODS OF USE
Abstract
Provided herein are novel anti-CLDN antibodies and antibody drug
conjugates (ADC), including derivatives thereof, and methods of
using the same to treat proliferative disorders.
Inventors: |
SANTAGUIDA; Marianne;
(Belmont, CA) ; AUJAY; Monette; (San Francisco,
CA) ; SAUNDERS; Laura; (San Francisco, CA) ;
LIU; David; (San Francisco, CA) ; FOORD; Orit;
(Foster City, CA) ; STULL; Robert A.; (Alameda,
CA) ; ESCARPE; Paul Anthony; (San Bruno, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ABBVIE STEMCENTRX LLC |
NORTH CHICAGO |
IL |
US |
|
|
Assignee: |
ABBVIE STEMCENTRX LLC
NORTH CHICAGO
IL
|
Family ID: |
53042314 |
Appl. No.: |
16/105651 |
Filed: |
August 20, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15034992 |
May 6, 2016 |
10053511 |
|
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PCT/US2014/064165 |
Nov 5, 2014 |
|
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16105651 |
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61900916 |
Nov 6, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 47/6869 20170801;
C07K 2317/56 20130101; C07K 16/3069 20130101; A61K 47/6851
20170801; C07K 16/30 20130101; C07K 2317/53 20130101; C07K 2317/92
20130101; C07K 2317/34 20130101; C07K 2317/77 20130101; C07K
2317/33 20130101; C07K 16/28 20130101; C07K 2317/73 20130101; C07K
2317/24 20130101; A61K 47/6857 20170801; C07K 2317/522 20130101;
A61P 35/00 20180101; C07K 16/3023 20130101; C12N 5/0093
20130101 |
International
Class: |
C07K 16/30 20060101
C07K016/30; A61K 47/68 20170101 A61K047/68; C12N 5/00 20060101
C12N005/00; C07K 16/28 20060101 C07K016/28 |
Claims
1.-23. (canceled)
24. A method of treating ovarian cancer comprising administering to
a subject in need thereof an antibody drug conjugate comprising a
monoclonal antibody conjugated, linked, or otherwise associated
with a cytotoxic agent, wherein the monoclonal antibody binds to a
human CLDN6 protein; and wherein the monoclonal antibody comprises
three complementarity determining regions of a light chain variable
region comprising an amino acid sequence set forth as SEQ ID NO:
73, and three complementarity determining regions of a heavy chain
variable region comprising an amino acid sequence set forth as SEQ
ID NO: 87.
25.-32. (canceled)
33. The method of claim 24, wherein the monoclonal antibody is an
internalizing antibody.
34. The method of claim 24, wherein the monoclonal antibody is
selected from the group consisting of a chimeric antibody,
humanized antibody, and CDR grafted antibody.
35. The antibody of claim 24, wherein the monoclonal antibody
comprises: (a) residues 24-34 of SEQ ID NO: 73 for CDR-L1, residues
50-56 of SEQ ID NO: 73 for CDR-L2, residues 89-97 of SEQ ID NO: 73
for CDR-L3, residues 31-35 of SEQ ID NO: 87 for CDR-H1, residues
50-65 of SEQ ID NO: 87 for CDR-H2 and residues 95-102 of SEQ ID NO:
87 for CDR-H3, wherein the residues are numbered according to
Kabat; (b) residues 24-34 of SEQ ID NO: 73 for CDR-L1, residues
50-56 of SEQ ID NO: 73 for CDR-L2, residues 89-97 of SEQ ID NO: 73
for CDR-L3, residues 26-32 of SEQ ID NO: 87 for CDR-H1, residues
52-56 of SEQ ID NO: 87 for CDR-H2 and residues 95-102 of SEQ ID NO:
87 for CDR-H3, wherein the residues are numbered according to
Chothia; or (c) residues 30-36 of SEQ ID NO: 73 for CDR-L1,
residues 46-55 of SEQ ID NO: 73 for CDR-L2, residues 89-96 of SEQ
ID NO: 73 for CDR-L3, residues 30-35 of SEQ ID NO: 87 for CDR-H1,
residues 47-58 of SEQ ID NO: 87 for CDR-H2 and residues 93-101 of
SEQ ID NO: 87 for CDR-H3, wherein the residues are numbered
according to MacCallum.
36. The method of claim 24, wherein the monoclonal antibody
comprises a light chain variable region comprising an amino acid
sequence set forth as SEQ ID NO: 73 and a heavy chain variable
region comprising an amino acid sequence set forth as SEQ ID NO:
87.
37. The method of claim 36, wherein the monoclonal antibody is an
IgG1 antibody comprising: (i) a cysteine residue at heavy chain
position 220 and a deletion of a cysteine residue at light chain
position 214, (ii) a cysteine residue at heavy chain position 220
and a substitution of a cysteine residue at light chain position
214, (iii) a cysteine residue at light chain position 214 and a
deletion of a cysteine residue at heavy chain position 220, or (iv)
a cysteine residue at light chain position 214 and a substitution
of a cysteine residue at heavy chain position 220; and wherein the
engineered antibody comprises native cysteine residues at heavy
chain positions 226 and 229.
38. The method of claim 37, wherein the monoclonal antibody
comprises a cysteine residue at light chain position 214.
39. The method of claim 24, wherein the cytotoxic agent is a
pyrrolobenzodiazepine, an auristatin, a maytansinoid, a
calicheamicin, or a radioisotope.
40. The method of claim 24, wherein the cytotoxic agent is a
pyrrolobenzodiazepine.
41. The method of claim 24, wherein the antibody drug conjugate
comprises the formula M-[L-D]n wherein: M comprises the monoclonal
antibody; L comprises an optional linker; D comprises a drug, which
is the cytotoxic agent; and n is an integer from 1 to 20.
42. The method of claim 24, wherein the monoclonal antibody is
conjugated to the cytotoxic agent via a linker.
43. The method of claim 42, wherein the linker comprises a
cleavable linker.
44. The method of claim 24, wherein the antibody drug conjugate has
a drug loading of 2.
45. The method of claim 24, wherein the monoclonal antibody
comprises a light chain variable region comprising an amino acid
sequence set forth as SEQ ID NO: 73 and a heavy chain variable
region comprising an amino acid sequence set forth as SEQ ID NO:
87, and wherein the cytotoxic agent is a pyrrolobenzodiazepine.
46. The method of claim 24, wherein the cancer is refractory,
relapsed, or resistant to a platinum based agent.
47. A method of treating ovarian cancer comprising administering to
a subject in need thereof an antibody drug conjugate comprising a
monoclonal antibody conjugated, linked, or otherwise associated
with a cytotoxic agent, wherein the monoclonal antibody binds to a
human CLDN6 protein and comprises a light chain variable region
comprising an amino acid sequence set forth as SEQ ID NO: 73 and a
heavy chain variable region comprising an amino acid sequence set
forth as SEQ ID NO: 87; wherein the cytotoxic agent is a
pyrrolobenzodiazepine; and wherein the antibody drug conjugate has
a drug loading of 2.
48. A method of treating cancer comprising administering to a
subject in need thereof an antibody drug conjugate comprising a
monoclonal antibody conjugated, linked, or otherwise associated
with a cytotoxic agent, wherein the monoclonal antibody comprises
three complementarity determining regions of a light chain variable
region comprising an amino acid sequence set forth as SEQ ID NO: 73
and three complementarity determining regions of a heavy chain
variable region comprising an amino acid sequence set forth as SEQ
ID NO: 87.
49. The method of claim 48, wherein the monoclonal antibody
comprises a light chain variable region comprising an amino acid
sequence set forth as SEQ ID NO: 73 and a heavy chain variable
region comprising an amino acid sequence set forth as SEQ ID NO:
87, and wherein the cytotoxic agent is a pyrrolobenzodiazepine.
50. The method of claim 49, wherein the cancer is lung
adenocarcinoma.
51. The method of claim 49, wherein the cancer is endometrial
cancer.
Description
CROSS REFERENCED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/900,916 filed on Nov. 6, 2013, which is
incorporated herein by reference in its entirety.
SEQUENCE LISTING
[0002] This application contains a sequence listing which has been
submitted in ASCII format via EFS-Web and is hereby incorporated by
reference in its entirety. Said ASCII copy, created on Nov. 5,
2014, is named sc2701pct_S69697_1040 WO_SEQL_110514.txt and is
229,316 bytes in size.
FIELD OF THE INVENTION
[0003] This application generally relates to novel anti-CLDN
antibodies or immunoreactive fragments thereof and compositions,
including antibody drug conjugates, comprising the same for the
treatment, diagnosis or prophylaxis of cancer and any recurrence or
metastasis thereof. Selected embodiments of the invention provide
for the use of such anti-CLDN antibodies or antibody drug
conjugates for the treatment of cancer comprising a reduction in
tumorigenic cell frequency.
BACKGROUND OF THE INVENTION
[0004] Differentiation and proliferation of stem cells and
progenitor cells are normal ongoing processes that act in concert
to support tissue growth during organogenesis, cell repair and cell
replacement. The system is tightly regulated to ensure that only
appropriate signals are generated based on the needs of the
organism. Cell proliferation and differentiation normally occur
only as necessary for the replacement of damaged or dying cells or
for growth. However, disruption of these processes can be triggered
by many factors including the under- or overabundance of various
signaling chemicals, the presence of altered microenvironments,
genetic mutations or a combination thereof. Disruption of normal
cellular proliferation and/or differentiation can lead to various
disorders including proliferative diseases such as cancer.
[0005] Conventional therapeutic treatments for cancer include
chemotherapy, radiotherapy and immunotherapy. Often these
treatments are ineffective and surgical resection may not provide a
viable clinical alternative. Limitations in the current standard of
care are particularly evident in those cases where patients undergo
first line treatments and subsequently relapse. In such cases
refractory tumors, often aggressive and incurable, frequently
arise. The overall survival rates for many solid tumors have
remained largely unchanged over the years due, at least in part, to
the failure of existing therapies to prevent relapse, tumor
recurrence and metastasis. There remains therefore a great need to
develop more targeted and potent therapies for proliferative
disorders. The current invention addresses this need.
SUMMARY OF THE INVENTION
[0006] The invention is broadly directed to antibodies and antibody
drug conjugates (ADC) that bind to at least one member of the
claudin (CLDN) family of proteins.
[0007] In selected embodiments the invention comprises an antibody
that binds to cancer stem cells expressing at least one protein of
the CLDN family. In another embodiment the antibodies of the
invention bind specifically to CLND6 or specifically to CLDN6 and
CLDN9. In another embodiment, the antibodies of the invention bind
to CLDN6 and CLDN9 with substantially the same apparent binding
affinity. Any of the anti-CLDN antibodies of the invention may be
internalizing antibodies.
[0008] In one embodiment the antibody of the invention binds to at
least one member of the CLDN family and competes for binding with
an antibody comprising: a light chain variable region (VL) of SEQ
ID NO: 21 and a heavy chain variable region (VH) of SEQ ID NO: 23;
or a VL of SEQ ID NO: 25 and a VH of SEQ ID NO: 27; or a VL of SEQ
ID NO: 29 and a VH of SEQ ID NO: 31; or a VL of SEQ ID NO: 33 and a
VH of SEQ ID NO: 35; or a VL of SEQ ID NO: 37 and a VH of SEQ ID
NO: 39; or a VL of SEQ ID NO: 41 and a VH of SEQ ID NO: 43; or a VL
of SEQ ID NO: 45 and a VH of SEQ ID NO: 47; or a VL of SEQ ID NO:
49 and a VH of SEQ ID NO: 51; or a VL of SEQ ID NO: 53 and a VH of
SEQ ID NO: 55; or a VL of SEQ ID NO: 57 and a VH of SEQ ID NO:
59.
[0009] In another embodiment the antibody of the invention binds
specifically to CLND6; or binds specifically to CLDN6 and CLDN9 and
competes for binding with an antibody comprising: a light chain
variable region (VL) of SEQ ID NO: 21 and a heavy chain variable
region (VH) of SEQ ID NO: 23; or a VL of SEQ ID NO: 25 and a VH of
SEQ ID NO: 27; or a VL of SEQ ID NO: 29 and a VH of SEQ ID NO: 31;
or a VL of SEQ ID NO: 33 and a VH of SEQ ID NO: 35; or a VL of SEQ
ID NO: 37 and a VH of SEQ ID NO: 39; or a VL of SEQ ID NO: 41 and a
VH of SEQ ID NO: 43; or a VL of SEQ ID NO: 45 and a VH of SEQ ID
NO: 47; or a VL of SEQ ID NO: 49 and a VH of SEQ ID NO: 51; or a VL
of SEQ ID NO: 53 and a VH of SEQ ID NO: 55; or a VL of SEQ ID NO:
57 and a VH of SEQ ID NO: 59.
[0010] Any of the anti-CLDN antibodies disclosed herein may be a
chimeric, CDR grafted, humanized or recombinant antibody, or a
fragment thereof.
[0011] In a particular embodiment the invention comprises a
humanized antibody that binds to at least one protein of the CLDN
family and competes for binding with an antibody comprising three
variable light chain CDRs (CDRL) as set forth in SEQ ID NO: 61; and
three variable heavy chain CDRs (CDRH) as set forth in SEQ ID NO:
63; or three CDRL as set forth in SEQ ID NO: 65 and three CDRH as
set forth in SEQ ID NO: 67; or three CDRL as set forth in SEQ ID
NO: 69 and three CDRH as set forth in SEQ ID NO: 71; three CDRL as
set forth in SEQ ID NO: 73 and three CDRH as set forth in SEQ ID
NO: 75.
[0012] In a further embodiment the invention comprises a humanized
antibody that binds to at least one protein of the CLDN family and
competes for binding with an antibody comprising a VH and VL,
wherein the VL has three CDRL comprising a CDRL1 of SEQ ID NO: 151,
a CDRL2 of SEQ ID NO: 152 and a CDRL3 of SEQ ID NO: 153; or a VL
having three CDRLs comprising a CDRL1 of SEQ ID NO: 157, a CDRL2 of
SEQ ID NO: 158 and a CDRL3 of SEQ ID NO: 159; or a VL having three
CDRLs comprising a CDRL1 of SEQ ID NO: 163, a CDRL2 of SEQ ID NO:
164 and a CDRL3 of SEQ ID NO: 165; or a VL having three CDRLs
comprising a CDRL1 of SEQ ID NO: 169, a CDRL2 of SEQ ID NO: 170 and
a CDRL3 of SEQ ID NO: 171.
[0013] In a further embodiment the invention comprises a humanized
antibody that binds to at least one protein of the CLDN family and
competes for binding with an antibody comprising a VL and a VH,
wherein the VH has three CDRs (CDRH) comprising a CDRH1 of SEQ ID
NO: 154, a CDRH2 of SEQ ID NO: 155 and a CDRH3 of SEQ ID NO: 156;
or the VH has three CDRHs comprising a CDRH1 of SEQ ID NO: 160, a
CDRH2 of SEQ ID NO: 161 and a CDRH3 of SEQ ID NO: 162; or the VH
has three CDRHs comprising a CDRH1 of SEQ ID NO: 166, a CDRH2 of
SEQ ID NO: 167 and a CDRH3 of SEQ ID NO: 168; or the VH has three
CDRHs comprising a CDRH1 of SEQ ID NO: 172, a CDRH2 of SEQ ID NO:
173 and a CDRH3 of SEQ ID NO: 174.
[0014] In a further embodiment the invention comprises a humanized
antibody that binds to at least one protein of the CLDN family and
competes for binding with an antibody comprising a VL and VH
wherein the VL has three CDRLs comprising a CDRL1 of SEQ ID NO:
151, a CDRL2 of SEQ ID NO: 152 and a CDRL3 of SEQ ID NO: 153 and
the VH has three CDRHs comprising a CDRH1 of SEQ ID NO: 154, a
CDRH2 of SEQ ID NO: 155 and a CDRH3 of SEQ ID NO: 156; or an
antibody comprising a VL and VH wherein the VL has three CDRLs
comprising a CDRL1 of SEQ ID NO: 157, a CDRL2 of SEQ ID NO: 158 and
a CDRL3 of SEQ ID NO: 159 and the VH has three CDRHs comprising a
CDRH1 of SEQ ID NO: 160, a CDRH2 of SEQ ID NO: 161 and a CDRH3 of
SEQ ID NO: 162; or an antibody comprising a VL and VH wherein the
VL has three CDRLs comprising a CDRL1 of SEQ ID NO: 163, a CDRL2 of
SEQ ID NO: 164 and a CDRL3 of SEQ ID NO: 165 and the VH has three
CDRHs comprising a CDRH1 of SEQ ID NO: 166, a CDRH2 of SEQ ID NO:
167 and a CDRH3 of SEQ ID NO: 168; or an antibody comprising a VL
and VH wherein the VL has three CDRLs comprising a CDRL1 of SEQ ID
NO: 169, a CDRL2 of SEQ ID NO: 170 and a CDRL3 of SEQ ID NO: 171
and the VH has three CDRHs comprising a CDRH1 of SEQ ID NO: 172, a
CDRH2 of SEQ ID NO: 173 and a CDRH3 of SEQ ID NO: 174.
[0015] In one embodiment the invention comprises a humanized
antibody that binds to at least one protein of the CLDN family
comprising a full length light chain set forth as SEQ ID NO: 114
and a full length heavy chain set forth as SEQ ID NO: 115; or a
full length light chain set forth as SEQ ID NO: 116 and a full
length heavy chain set forth as SEQ ID NO: 117; or a full length
light chain set forth as SEQ ID NO: 118 and a full length heavy
chain set forth as SEQ ID NO: 119; or a full length light chain set
forth as SEQ ID NO: 120 and a full length heavy chain set forth as
SEQ ID NO: 121.
[0016] In one embodiment the invention comprises an antibody drug
conjugate (ADC) comprising any anti-CLDN antibody disclosed herein,
wherein the antibody is conjugated to a payload. In another
embodiment the invention comprises a pharmaceutical composition
comprising an ADC, wherein the ADC comprises an anti-CLDN antibody
of the invention conjugated to a payload.
[0017] In another embodiment the invention comprises a nucleic acid
encoding the antibody of any of the anti-CLDN antibodies disclosed
herein. In a related embodiment, the invention comprises a vector
comprising one or more of the nucleic acids encoding an anti-CLDN
antibody disclosed herein or a host cell comprising said
vector.
[0018] In a preferred embodiment, the invention comprises an
antibody drug conjugate (ADC) comprising a chimeric, CDR grafted,
humanized or recombinant human antibody, or a fragment thereof,
which binds to cancer stem cells expressing at least one protein of
the CLDN family, wherein the antibody is conjugated to a cytotoxic
agent.
[0019] In another embodiment the invention comprises an ADC of the
formula Ab-[L-D]n, wherein Ab is any one of the anti-CLDN
antibodies disclosed herein; L is an optional linker; D is a drug;
and n is an integer from about 1 to about 20.
[0020] In one embodiment the invention comprises a method of
treating cancer comprising administering to a subject in need
thereof a pharmaceutical composition comprising an ADC, wherein the
ADC comprises an anti-CLDN antibody of the invention conjugated to
a payload.
[0021] In some embodiments the invention comprises a method of
treating cancer comprising administering to a subject in need
thereof a pharmaceutical composition comprising an anti-CLDN ADC,
wherein the cancer is selected from ovarian cancer, lung cancer,
e.g. lung adenocarcinoma, breast cancer and pancreatic cancer.
[0022] In one embodiment the invention comprises a method of
treating cancer comprising administering to a subject in need
thereof a pharmaceutical composition comprising an anti-CLDN ADC
and at least one additional therapeutic moiety.
[0023] In one embodiment the invention comprises a method of
reducing cancer stem cells in a tumor cell population, wherein the
method comprises contacting a tumor cell population comprising
cancer stem cells and tumor cells other than cancer stem cells with
an anti-CLDN ADC; whereby the frequency of cancer stem cells is
reduced, for example, such contacting can be performed in vivo or
in vitro.
[0024] In one embodiment the invention comprises a method of
delivering a cytotoxin to a cell comprising contacting the cell
with an ADC comprising any anti-CLDN antibody disclosed herein.
BRIEF DESCRIPTION OF THE FIGURES
[0025] FIG. 1 shows the relative mRNA expression levels of CDLN4,
CLDN6, and CLDN9 determined by whole transcriptome (SOLiD)
sequencing in selected patient-derived xenograft (PDX) tumors.
Tumor types are denoted according the abbreviations listed in Table
4;
[0026] FIG. 2A shows the relative mRNA expression of CLDN4, CLDN6
and CLDN9 in cancer stem cells (CSCs; grey bar), compared to
non-tumorigenic (NTG; white bar) cells and in matched normal tissue
(black bar), as determined by qRT-PCR;
[0027] FIGS. 2B-2D show relative mRNA expression levels of CLDN4,
CLDN6 and CLDN9, respectively, in PDX tumors determined by
qRT-PCR;
[0028] FIGS. 2E-2G show the relative levels of mRNA expression of
CLDN4 (FIG. 2E), CLDN6 (FIG. 2F) or CLDN9 (FIG. 2G) as measured by
qRT-PCR in whole tumor specimens (black dot) or matched normal
adjacent tissue (white dot) from patients with one of eighteen
different tumor types;
[0029] FIGS. 3A and 3B show relative mRNA expression of CLDN6 (FIG.
3A) and CLDN9 (FIG. 3B) across a large number of tumor and normal
tissues derived from a public database;
[0030] FIG. 3C shows the relative mRNA expression of CLDN6 (x-axis)
versus that of CLDN9 (y-axis) for individual tumor samples for five
tumor types derived from a public database;
[0031] FIG. 3D shows a plot of the centroids (center of mass) of
the scatter graphs for the five different tumor types shown in FIG.
3C, as well as a best fit regression line for the plotted
centroids;
[0032] FIG. 4A is a dendrogram showing the relative degree of
similarity between the 30 CLDN proteins encoded by the 23 human
CLDN genes;
[0033] FIG. 4B shows a tabular representation of the percent
identity of amino acid residues in extracellular domain (ECD) 1 or
ECD2 in CLDN4, CLDN6 and CLDN9;
[0034] FIG. 4C shows a tabular representation of the percent
identity of amino acid residues in the ECD1 and ECD2 loops amongst
the 16 proteins comprising the set of human, rat, mouse and
cynomolgus monkey orthologs of CLDN4, CLDN6 and CLDN9;
[0035] FIGS. 5A-5H provide amino acid and nucleic acid sequences of
mouse and humanized anti-CLDN antibodies. FIGS. 5A and 5B show
light chain (FIG. 5A) and heavy chain (FIG. 5B) variable region
amino acid sequences of exemplary mouse and humanized anti-CLDN
antibodies (SEQ ID NOS: 21-75, odd numbers) and variants of
hSC27.22, hSC27.108 and hSC27.204. FIG. 5C shows the nucleic acid
sequences of the same light and heavy chain variable regions of
such exemplary mouse and humanized anti-CLDN antibodies (SEQ ID
NOS: 20-74, even numbers) and variants of hSC27.22, hSC27.108 and
hSC27.204. FIG. 5D shows the amino acid sequences of the full
length light and heavy chains of humanized antibodies hSC27.1 and
hSC27.22, thirteen variants of hSC27.22, one variant of hSC27.108
and fifteen variants of hSC27.204. FIGS. 5E-5H show annotated amino
acid sequences (numbered as per Kabat et al.) of the light and
heavy chain variable regions of the humanized anti-CLDN antibodies,
hSC27.1 (FIG. 5E), hSC27.22 (FIG. 5F), hSC27.108 (FIG. 5G), and
hSC27.204 (FIG. 5H), wherein the CDRs are derived using Kabat,
Chothia, ABM and Contact methodology;
[0036] FIG. 6A shows the ability of anti-CLDN antibodies SC27.1 and
SC27.22 to bind HEK-293T cells overexpressing human CLDN4, CLDN6
and CLDN9 as detected by flow cytometry, where results are shown as
change in mean fluorescence intensity (.DELTA.MFI) and a histogram,
with the solid black line indicating the binding of the indicated
antibody to cells overexpressing the indicated CLDN protein
compared to fluorescence minus one (FMO) isotype-control
(gray-fill);
[0037] FIG. 6B shows the ability of anti-CLDN antibodies to bind
HEK-293T cells overexpressing CLDN4, CLDN6 and CLDN9 as detected by
flow cytometry, where the results are shown as mean fluorescence
intensity (MFI) for each antibody binding to each cell line;
[0038] FIG. 6C shows the apparent binding affinity of an exemplary
anti-CLDN antibody for CLDN6 and CLDN9 as determined by a titration
of the amount of antibody versus a fixed number of cells expressing
the antigen of interest;
[0039] FIG. 7 shows expression of CLDN4, CLDN6, and CLDN9 proteins
in cell populations derived from liver, lung, ovarian and
pancreatic PDX tumors (solid black line) compared to fluorescence
minus one (FMO) isotype-control (gray-fill);
[0040] FIG. 8A shows expression of CLDN4, CLDN6, and CLDN9 proteins
in human CSC (solid black line) compared to non-tumorigenic (dashed
line) ovarian, pancreatic and lung tumor cell populations and FMO
isotype controls (gray-fill);
[0041] FIG. 8B shows the growth of tumors in mice transplanted with
CLDN.sup.+ (closed circles) or CLDN.sup.- (open circles) ovarian
tumor cells where CLDN.sup.+ tumor cells exhibit enhanced
tumorigenicity compared to CLDN.sup.- ovarian tumor cells;
[0042] FIGS. 9A and 9B show that anti-CLDN antibodies SC27.1 and
SC27.22 are able to internalize into cells overexpressing human
CLDN4, CLDN6 and CLDN9 and mediate the delivery of saporin
cytotoxin;
[0043] FIG. 10A shows expression of CLND6 in various PDX lung,
breast and ovarian tumor cells using immunohistochemistry; and
[0044] FIG. 10B shows expression of CLND6 in various primary
ovarian tumors using immunohistochemistry.
DETAILED DESCRIPTION OF THE INVENTION
[0045] The invention may be embodied in many different forms.
Disclosed herein are non-limiting, illustrative embodiments of the
invention that exemplify the principles thereof. Any section
headings used herein are for organizational purposes only and are
not to be construed as limiting the subject matter described. For
the purposes of the instant disclosure all identifying sequence
accession numbers may be found in the NCBI Reference Sequence
(RefSeq) database and/or the NCBI GenBank.RTM. archival sequence
database unless otherwise noted.
[0046] CLDN has surprisingly been found to be a biological marker
of a number of tumor types and this association may be exploited
for the treatment of such tumors. It has also unexpectedly been
found that CLDN is associated with tumorigenic cells and may be
effectively exploited to inhibit or eliminate them. Tumorigenic
cells, which will be described in more detail below, are known to
exhibit resistance to many conventional treatments. In contrast to
the teachings of the prior art, the disclosed compounds and methods
effectively overcome this inherent resistance.
[0047] The invention provides anti-CLDN antibodies (including
antibody drug conjugates) and their use in the prognosis,
diagnosis, theragnosis, treatment and/or prevention of a variety of
CLDN-associated cancers regardless of any particular mechanism of
action or specifically targeted cellular or molecular
component.
I CLAUDIN (CLDN) PHYSIOLOGY
[0048] Claudins are integral membrane proteins comprising a major
structural protein of tight junctions, the most apical cell-cell
adhesion junction in polarized cell types such as those found in
epithelial or endothelial cell sheets. Tight junctions are composed
of strands of networked proteins that form continuous seals around
cells to provide a physical but modulatable barrier to the
transport of solutes and water in the paracellular space. The
claudin family of proteins in humans is comprised of at least 23
members, ranging in size from 22-34 kDa. All claudins possess a
tetraspanin topology in which both protein termini are located on
the intracellular face of the membrane, resulting in the formation
of two extracellular (EC) loops, EC1 and EC2. The EC loops mediate
head-to-head homophilic, and for certain combinations of claudins,
heterophilic interactions that lead to formation of tight
junctions. The specific claudin-claudin interactions and claudin EC
sequences are a key determinant of ion selectivity and tight
junction strength (for example, see Nakano et al., 2009, PMID:
19696885). Typically, EC1 is about 50-60 amino acids in size,
contains a conserved disulfide bond within a larger
W-X(17-22)-W-X(2)-C-X(8-10)-C motif, and numerous charged residues
that participate in ion channel formation (Turksen and Troy, 2004,
PMID: 15159449). EC2 is smaller than EC1, being approximately 25
amino acids. Due to its helix-turn-helix conformation, it has been
suggested that EC2 contributes to dimer or multimer formation of
claudins on opposing cell membranes, although mutations in both
loops may perturb complex formation. Claudin-claudin complexes in
vitro may range in size from dimers to hexamers, depending upon the
specific claudins involved (Krause et al., 2008, PMID: 18036336).
Individual claudins show a range of tissue specific expression
patterns, as well as developmentally regulated expression as
determined by PCR analyses (Krause et al., 2008, PMID:18036336;
Turksen, 2011, PMID:21526417).
[0049] Sequence analysis can be used to construct phylogenetic
trees for the claudin family members, indicating the relationship
and degrees of relatedness of the protein sequences (FIG. 4A). For
instance, it can be seen that the CLDN6 and CLDN9 proteins are
closely related which, given the adjacent head-to-head location of
their genes at the chromosomal location 16p3.3, is suggestive of an
ancestral gene duplication. These similarities likely translate to
an ability of these family members to interact heterotypically.
Similarly, the CLDN3 and CLDN4 proteins are closely related by
sequence analysis, and their genes can be found in tandem at the
chromosomal location 7r11.23. High homology in the EC1 or EC2 loops
between certain family members (e.g. FIG. 4B) provides opportunity
to develop antibodies that are multi-reactive with various claudin
family members.
[0050] CLDN6, also known as skullin, is a developmentally regulated
claudin. Representative CLDN6 protein orthologs include, but are
not limited to, human (NP_067018), chimpanzee (XP_523276), rhesus
monkey (NP_001180762), mouse (NP_061247), and rat (NP_001095834).
In humans, the CLDN6 gene consists of 2 exons spanning
approximately 3.5 kBp at the chromosomal location 16p13.3.
Transcription of the CLDN6 locus yields a mature 1.4 kB mRNA
transcript (NM_021195), encoding a 219 amino acid protein
(NP_061247). CLDN6 is expressed in ES cell derivatives committed to
an epithelial fate (Turksen and Troy, 2001, PMID: 11668606), in the
periderm (Morita et al., 2002, PMID: 12060405), and in the
suprabasal level of the epidermis (Turkson and Troy, 2002, PMID:
11923212). It is also expressed in developing mouse kidney (Abuazza
et al., 2006, PMID: 16774906), although expression is not detected
in adult kidney (Reyes et al., 2002, PMID: 12110008). CLDN6 is also
a coreceptor for hepatitis C virus, along with CLDN1 and CLDN9
(Zheng et al., 2007, PMID: 17804490).
[0051] CLDN9 is the most closely related family member to CLDN6.
Representative CLDN9 protein orthologs include, but are not limited
to, human (NP_066192), chimpanzee (XP_003314989), rhesus monkey
(NP_001180758), mouse (NP_064689), and rat (NP_001011889). In
humans, the CLDN9 gene consists of a single exon spanning
approximately 2.1 kBp at the chromosomal locus 16p13.3.
Transcription of the intronless CLDN9 locus yields a 2.1 kB mRNA
transcript (NM_020982), encoding a 217 amino acid protein
(NP_0066192). CLDN9 is expressed in various structures of the inner
ear (Kitarjiri et al., 2004, PMID:14698084; Nankano et al., 2009,
PMID: 19696885), the cornea (Ban et al., 2003, PMID:12742348), the
liver (Zheng et al., 2007, PMID:17804490) and developing kidney
(Abuazza et al., 2006, PMID:16774906). Consistent with its
expression in the cochlea, animals expressing a CLDN9 protein with
a missense mutation show defects in hearing likely due to altered
paracellular K.sup.+ permeability with consequent perturbation of
ion currents critical for depolarization of hair cells involved in
sound detection. Expression of CLDN9 in cells of the inner ear is
specifically localized to a subdomain underneath more apical
tight-junction strands formed by other claudins, indicating that
not all claudins in normal tissues are found in the most apical and
accessible tight junctions (Nankano et al., 2009, PMID: 19696885).
In contrast to the results in the cochlea, mice expressing missense
CLDN9 showed no signs of hepatic or renal defects (Nankano et al.,
2009, PMID: 19696885).
[0052] CLDN4 is also known as the Clostridium perfringens
enterotoxin receptor, due to its high affinity binding of this
toxin responsible for food poisoning and other gastrointestinal
illnesses. Representative CLDN4 protein orthologs include, but are
not limited to, human (NP_001296), chimpanzee (XP_519142), rhesus
monkey (NP_001181493), mouse (NP_034033), and rat (NP_001012022).
In humans, the intronless CLDN4 gene spans approximately 1.82 kBp
at the chromosomal location 17q11.23. Transcription of the CLDN4
locus yields a 1.82 kB mRNA transcript (NM_001305), encoding a 209
amino acid protein (NP_001296). Consistent with the ability of
CLDN4 to bind a toxin produced by a gastrointestinal pathogen,
CDLN4 expression can be detected throughout the GI tract as well as
in prostate, bladder, breast, and lung (Rahner et al., 2001,
PMID:11159882; Tamagawa et al., 2003, PMID:12861044; Wang et al.,
2003, PMID:12600828; Nichols et al., 2004, PMID:14983936).
[0053] Although claudins are important in the function and
homeostasis of normal tissues, tumor cells frequently exhibit
abnormal tight junction function. This may be linked to
disregulated expression and/or localization of claudins as a
consequence of the dedifferentiation of tumor cells, or the
requirement of rapidly growing cancerous tissues to efficiently
absorb nutrients within a tumor mass with abnormal vascularization
(Morin, 2005, PMID: 16266975). Individual claudin family members
may be up-regulated in certain cancer types, yet down-regulated in
others. For example, CLDN3 and CLDN4 expression is elevated in
certain pancreatic, breast and ovarian cancers, yet may be lower in
other breast (e.g., "claudin-low") carcinomas. Claudin proteins may
be particularly good targets for antibody drug conjugates (ADCs)
since it is known that claudins undergo endocytosis, turnover time
of some claudins is short relative to other membrane proteins (Van
Itallie et al., 2004, PMID: 15366421), claudin expression is
disregulated in cancer cells and tight junctions structures among
tumor cells are disrupted in cancer cells. These properties may
afford more opportunities for antibodies to bind claudin proteins
in neoplastic but not in normal tissues. Although antibodies
specific to individual claudins may be useful, it is also possible
that polyreactive claudin antibodies would be more likely to
facilitate the delivery of payloads to a broader patient
population. Specifically, polyreactive claudin antibodies may
permit more efficient targeting of cells expressing multiple
claudin proteins due to higher aggregate antigen density, reduce
the likelihood of escape of tumor cells with low levels of antigen
expression of any individual claudin, and as can be seen in the
expression examples below, expand the number of therapeutic
indications for a single ADC.
II CANCER STEM CELLS
[0054] According to the current models, a tumor comprises
non-tumorigenic cells and tumorigenic cells. Non-tumorigenic cells
do not have the capacity to self-renew and are incapable of
reproducibly forming tumors, even when transplanted into
immunocompromised mice in excess cell numbers. Tumorigenic cells,
also referred to herein as "tumor initiating cells" (TICs), which
make up 0.1-40% of a tumor's cell population, have the ability to
form tumors. Tumorigenic cells encompass both tumor perpetuating
cells (TPCs), referred to interchangeably as cancer stem cells
(CSCs) and tumor progenitor cells (TProgs).
[0055] CSCs, like normal stem cells that support cellular
hierarchies in normal tissue, are able to self-replicate
indefinitely while maintaining the capacity for multilineage
differentiation. CSCs are able to generate both tumorigenic progeny
and non-tumorigenic progeny and are able to completely recapitulate
the heterogeneous cellular composition of the parental tumor as
demonstrated by serial isolation and transplantation of low numbers
of isolated CSCs into immunocompromised mice.
[0056] TProgs, like CSCs have the ability to fuel tumor growth in a
primary transplant. However, unlike CSCs, they are not able to
recapitulate the cellular heterogeneity of the parental tumor and
are less efficient at reinitiating tumorigenesis in subsequent
transplants because TProgs are typically only capable of a finite
number of cell divisions as demonstrated by serial transplantation
of low numbers of highly purified TProg into immunocompromised
mice. TProgs may further be divided into early TProgs and late
TProgs, which may be distinguished by phenotype (e.g., cell surface
markers) and their different capacities to recapitulate tumor cell
architecture. While neither can recapitulate a tumor to the same
extent as CSCs, early TProgs have a greater capacity to
recapitulate the parental tumor's characteristics than late TProgs.
Notwithstanding the foregoing distinctions, it has been shown that
some TProg populations can, on rare occasion, gain self-renewal
capabilities normally attributed to CSCs and can themselves become
CSCs.
[0057] CSCs exhibit higher tumorigenicity and are relatively more
quiescent than: (i) TProgs (both early and late TProgs); and (ii)
non-tumorigenic cells such as tumor-infiltrating cells, for
example, fibroblasts/stroma, endothelial and hematopoietic cells
that may be derived from CSCs and typically comprise the bulk of a
tumor. Given that conventional therapies and regimens have, in
large part, been designed to debulk tumors and attack rapidly
proliferating cells, CSCs are more resistant to conventional
therapies and regimens than the faster proliferating TProgs and
other bulk tumor cell populations such as non-tumorigenic cells.
Other characteristics that may make CSCs relatively chemoresistant
to conventional therapies are increased expression of multi-drug
resistance transporters, enhanced DNA repair mechanisms and
anti-apoptotic gene expression. These properties in CSCs constitute
a key reason for the failure of standard oncology treatment
regimens to ensure long-term benefit for most patients with
advanced stage neoplasia because standard chemotherapy does not
target the CSCs that actually fuel continued tumor growth and
recurrence.
[0058] It has surprisingly been discovered that CLDN expression is
associated with various tumorigenic cell subpopulations. The
invention provides anti-CLDN antibodies that may be particularly
useful for targeting tumorigenic cells and may be used to silence,
sensitize, neutralize, reduce the frequency, block, abrogate,
interfere with, decrease, hinder, restrain, control, deplete,
moderate, mediate, diminish, reprogram, eliminate, or otherwise
inhibit (collectively, "inhibit") tumorigenic cells, thereby
facilitating the treatment, management and/or prevention of
proliferative disorders (e.g. cancer). Advantageously, the novel
anti-CLDN antibodies of the invention may be selected so they
preferably reduce the frequency or tumorigenicity of tumorigenic
cells upon administration to a subject regardless of the form of
the CLDN determinant (e.g., phenotypic or genotypic). The reduction
in tumorigenic cell frequency may occur as a result of (i)
inhibition or eradication of tumorigenic cells; (ii) controlling
the growth, expansion or recurrence of tumorigenic cells; (iii)
interrupting the initiation, propagation, maintenance, or
proliferation of tumorigenic cells; or (iv) by otherwise hindering
the survival, regeneration and/or metastasis of the tumorigenic
cells. In some embodiments, the inhibition of tumorigenic cells may
occur as a result of a change in one or more physiological
pathways. The change in the pathway, whether by inhibition of the
tumorigenic cells, modification of their potential (for example, by
induced differentiation or niche disruption) or otherwise
interfering with the ability of tumorigenic cells to influence the
tumor environment or other cells, allows for the more effective
treatment of CLDN associated disorders by inhibiting tumorigenesis,
tumor maintenance and/or metastasis and recurrence.
[0059] Methods that can be used to assess the reduction in the
frequency of tumorigenic cells, include but are not limited to,
cytometric or immunohistochemical analysis, preferably by in vitro
or in vivo limiting dilution analysis (Dylla et al. 2008, PMID:
PMC2413402 and Hoey et al. 2009, PMID: 19664991).
[0060] In vitro limiting dilution analysis may be performed by
culturing fractionated or unfractionated tumor cells (e.g. from
treated and untreated tumors, respectively) on solid medium that
fosters colony formation and counting and characterizing the
colonies that grow. Alternatively, the tumor cells can be serially
diluted onto plates with wells containing liquid medium and each
well can be scored as either positive or negative for colony
formation at any time after inoculation but preferably more than 10
days after inoculation.
[0061] In vivo limiting dilution is performed by transplanting
tumor cells, from either untreated controls or from tumors exposed
to selected therapeutic agents, into immunocompromised mice in
serial dilutions and subsequently scoring each mouse as either
positive or negative for tumor formation. The scoring may occur at
any time after the implanted tumors are detectable but is
preferably done 60 or more days after the transplant. The analysis
of the results of limiting dilution experiments to determine the
frequency of tumorigenic cells is preferably done using Poisson
distribution statistics or assessing the frequency of predefined
definitive events such as the ability to generate tumors in vivo or
not (Fazekas et al., 1982, PMID: 7040548).
[0062] Flow cytometry and immunohistochemistry may also be used to
determine tumorigenic cell frequency. Both techniques employ one or
more antibodies or reagents that bind art recognized cell surface
proteins or markers known to enrich for tumorigenic cells (see WO
2012/031280). As known in the art, flow cytometry (e.g. florescence
activated cell sorting (FACS)) can also be used to characterize,
isolate, purify, enrich or sort for various cell populations
including tumorigenic cells. Flow cytometry measures tumorigenic
cell levels by passing a stream of fluid, in which a mixed
population of cells is suspended, through an electronic detection
apparatus which is able to measure the physical and/or chemical
characteristics of up to thousands of particles per second.
Immunohistochemistry provides additional information in that it
enables visualization of tumorigenic cells in situ (e.g., in a
tissue section) by staining the tissue sample with labeled
antibodies or reagents which bind to tumorigenic cell markers.
[0063] The antibodies of the invention may be useful for
identifying, characterizing, monitoring, isolating, sectioning or
enriching populations or subpopulations of tumorigenic cells
through methods such as, for example, flow cytometry, magnetic
activated cell sorting (MACS), laser mediated sectioning or FACS.
FACS is a reliable method used to isolate cell subpopulations at
more than 99.5% purity based on specific cell surface markers.
Other compatible techniques for the characterization and
manipulation of tumorigenic cells including CSCs can be seen, for
example, in U.S. patent Ser. Nos. 12/686,359, 12/669,136 and
12/757,649.
[0064] Listed below are markers that have been associated with CSC
populations and have been used to isolate or characterize CSCs:
ABCA1, ABCA3, ABCG2, ADAM9, ADCY9, ADORA2A, AFP, AXIN1, B7H3, BCL9,
Bmi-1, BMP-4, C20orf52, C4.4A, carboxypeptidase M, CAV1, CAV2,
CD105, CD133, CD14, CD16, CD166, CD16a, CD16b, CD2, CD20, CD24,
CD29, CD3, CD31, CD324, CD325, CD34, CD38, CD44, CD45, CD46, CD49b,
CD49f, CD56, CD64, CD74, CD9, CD90, CEACAM6, CELSR1, CPD, CRIM1,
CX3CL1, CXCR4, DAF, decorin, easyh1, easyh2, EDG3, eed, EGFR,
ENPP1, EPCAM, EPHA1, EPHA2, FLJ10052, FLVCR, FZD1, FZD10, FZD2,
FZD3, FZD4, FZD6, FZD7, FZD8, FZD9, GD2, GJA1, GLI1, GL12, GPNMB,
GPR54, GPRC5B, IL1R1, IL1RAP, JAM3, Lgr5, Lgr6, LRP3, LY6E, MCP,
mf2, mIIt3, MPZL1, MUC1, MUC16, MYC, N33, Nanog, NB84, nestin,
NID2, NMA, NPC1, oncostatin M, OCT4, OPN3, PCDH7, PCDHA10, PCDHB2,
PPAP2C, PTPN3, PTS, RARRES1, SEMA4B, SLC19A2, SLC1A1, SLC39A1,
SLC4A11, SLC6A14, SLC7A8, smarcA3, smarcD3, smarcE1, smarcA5, Sox1,
STAT3, STEAP, TCF4, TEM8, TGFBR3, TMEPAI, TMPRSS4, transferrin
receptor, TrkA, WNT10B, WNT16, WNT2, WNT2B, WNT3, WNT5A, YY1 and
.beta.-catenin. See, for example, Schulenburg et al., 2010, PMID:
20185329, U.S. Pat. No. 7,632,678 and U.S.P.N.s. 2007/0292414,
2008/0175870, 2010/0275280, 2010/0162416 and 2011/0020221.
[0065] Similarly, non-limiting examples of cell surface phenotypes
associated with CSCs of certain tumor types include
CD44.sup.hiCD24.sup.low, ALDH.sup.+, CD133.sup.+, CD123.sup.+,
CD34.sup.+CD38.sup.-, CD44.sup.+CD24.sup.-,
CD46.sup.hiCD324.sup.+CD66c.sup.-,
CD133.sup.+CD34.sup.+CD10.sup.-CD19.sup.-,
CD138.sup.-CD34.sup.-CD19.sup.+, CD133.sup.+RC2.sup.+,
CD44.sup.+.alpha..sub.2.beta..sub.1.sup.hiCD133.sup.+,
CD44.sup.+CD24.sup.+ESA.sup.+, CD271.sup.+, ABCB5.sup.+ as well as
other CSC surface phenotypes that are known in the art. See, for
example, Schulenburg et al., 2010, supra, Visvader et al., 2008,
PMID: 18784658 and U.S.P.N. 2008/0138313. Of particular interest
with respect to the instant invention are CSC preparations
comprising CD46.sup.hiCD324.sup.+ phenotypes. "Positive," "low" and
"negative" expression levels as they apply to markers or marker
phenotypes are defined as follows. Cells with negative expression
(i.e. "-") are herein defined as those cells expressing less than,
or equal to, the 95th percentile of expression observed with an
isotype control antibody in the channel of fluorescence in the
presence of the complete antibody staining cocktail labeling for
other proteins of interest in additional channels of fluorescence
emission. Those skilled in the art will appreciate that this
procedure for defining negative events is referred to as
"fluorescence minus one", or "FMO", staining. Cells with expression
greater than the 95th percentile of expression observed with an
isotype control antibody using the FMO staining procedure described
above are herein defined as "positive" (i.e. "+"). As defined
herein there are various populations of cells broadly defined as
"positive." A cell is defined as positive if the mean observed
expression of the antigen is above the 95th percentile determined
using FMO staining with an isotype control antibody as described
above. The positive cells may be termed cells with low expression
(i.e. "10") if the mean observed expression is above the 95.sup.th
percentile determined by FMO staining and is within one standard
deviation of the 95.sup.th percentile. Alternatively, the positive
cells may be termed cells with high expression (i.e. "hi") if the
mean observed expression is above the 95.sup.th percentile
determined by FMO staining and greater than one standard deviation
above the 95.sup.th percentile. In other embodiments the 99th
percentile may preferably be used as a demarcation point between
negative and positive FMO staining and in particularly preferred
embodiments the percentile may be greater than 99%.
[0066] The CD46.sup.hiCD324.sup.+ marker phenotype and those
exemplified immediately above may be used in conjunction with
standard flow cytometric analysis and cell sorting techniques to
characterize, isolate, purify or enrich TIC and/or TPC cells or
cell populations for further analysis.
[0067] The ability of the antibodies of the current invention to
reduce the frequency of tumorigenic cells can therefore be
determined using the techniques and markers described above. In
some instances, the anti-CLDN antibodies may reduce the frequency
of tumorigenic cells by 10%, 15%, 20%, 25%, 30% or even by 35%. In
other embodiments, the reduction in frequency of tumorigenic cells
may be in the order of 40%, 45%, 50%, 55%, 60% or 65%. In certain
embodiments, the disclosed compounds my reduce the frequency of
tumorigenic cells by 70%, 75%, 80%, 85%, 90% or even 95%. It will
be appreciated that any reduction of the frequency of tumorigenic
cells is likely to result in a corresponding reduction in the
tumorigenicity, persistence, recurrence and aggressiveness of the
neoplasia.
III ANTIBODIES
[0068] A. Antibody Structure
[0069] Antibodies and variants and derivatives thereof, including
accepted nomenclature and numbering systems, have been extensively
described, for example, in Abbas et al. (2010), Cellular and
Molecular Immunology (6.sup.th Ed.), W.B. Saunders Company; or
Murphey et al. (2011), Janeway's Immunobiology (8.sup.1h Ed.),
Garland Science.
[0070] As used herein an "antibody" or "intact antibody" typically
refers to a Y-shaped tetrameric protein comprising two heavy (H)
and two light (L) polypeptide chains held together by covalent
disulfide bonds and non-covalent interactions. Human light chains
are classified as kappa or lambda light chains. Each light chain is
composed of one variable domain (VL) and one constant domain (CL).
Each heavy chain comprises one variable domain (VH) and a constant
region, which in the case of IgG, IgA, and IgD, comprises three
domains termed CH1, CH2, and CH3 (IgM and IgE have a fourth domain,
CH4). In IgG, IgA, and IgD classes the CH1 and CH2 domains are
separated by a flexible hinge region, which is a proline and
cysteine rich segment of variable length (generally from about 10
to about 60 amino acids in IgG). The variable domains in both the
light and heavy chains are joined to the constant domains by a "J"
region of about 12 or more amino acids and the heavy chain also has
a "D" region of about 10 additional amino acids. Each class of
antibody further comprises inter-chain and intra-chain disulfide
bonds formed by paired cysteine residues.
[0071] As used herein the term "antibody" includes polyclonal
antibodies, multiclonal antibodies, monoclonal antibodies, chimeric
antibodies, humanized and primatized antibodies, CDR grafted
antibodies, human antibodies, recombinantly produced antibodies,
intrabodies, multispecific antibodies, bispecific antibodies,
monovalent antibodies, multivalent antibodies, anti-idiotypic
antibodies, synthetic antibodies, including muteins and variants
thereof, immunospecific antibody fragments such as Fd, Fab,
F(ab').sub.2, F(ab') fragments, single-chain fragments (e.g. ScFv
and ScFvFc); and derivatives thereof including Fc fusions and other
modifications, and any other immunoreactive molecule so long as it
exhibits preferential association or binding with a determinant.
Moreover, unless dictated otherwise by contextual constraints the
term further comprises all classes of antibodies (i.e. IgA, IgD,
IgE, IgG, and IgM) and all subclasses (i.e., IgG1, IgG2, IgG3,
IgG4, IgA1, and IgA2). Heavy-chain constant domains that correspond
to the different classes of antibodies are typically denoted by the
corresponding lower case Greek letter .alpha., .delta., .epsilon.,
.gamma., and .mu., respectively. Light chains of the antibodies
from any vertebrate species can be assigned to one of two clearly
distinct types, called kappa (.kappa.) and lambda (.lamda.), based
on the amino acid sequences of their constant domains.
[0072] The variable domains of antibodies show considerable
variation in amino acid composition from one antibody to another
and are primarily responsible for antigen recognition and binding.
Variable regions of each light/heavy chain pair form the antibody
binding site such that an intact IgG antibody has two binding sites
(i.e. it is bivalent). V.sub.H and V.sub.L domains comprise three
regions of extreme variability, which are termed hypervariable
regions, or more commonly, complementarity-determining regions
(CDRs), framed and separated by four less variable regions known as
framework regions (FRs). The non-covalent association between the
V.sub.H and the V.sub.L region forms the Fv fragment (for "fragment
variable") which contains one of the two antigen-binding sites of
the antibody. ScFv fragments (for single chain fragment variable),
which can be obtained by genetic engineering, associates in a
single polypeptide chain, the V.sub.H and the V.sub.L region of an
antibody, separated by a peptide linker.
[0073] As used herein, the assignment of amino acids to each
domain, framework region and CDR may be in accordance with one of
the numbering schemes provided by Kabat et al. (1991) Sequences of
Proteins of Immunological Interest (5.sup.th Ed.), US Dept. of
Health and Human Services, PHS, NIH, NIH Publication no. 91-3242;
Chothia et al., 1987, PMID: 3681981; Chothia et al., 1989, PMID:
2687698; MacCallum et al., 1996, PMID: 8876650; or Dubel, Ed.
(2007) Handbook of Therapeutic Antibodies, 3.sup.rd Ed., Wily-VCH
Verlag GmbH and Co. unless otherwise noted. The amino acid residues
which comprise CDRs as defined by Kabat, Chothia and MacCallum (or
"Contact") as obtained from the Abysis website database (infra.)
are set out below.
TABLE-US-00001 TABLE 1 Kabat Chothia MacCallum VH CDR1 31-35 26-32
30-35 VH CDR2 50-65 52-56 47-58 VH CDR3 95-102 95-102 93-101 VL
CDR1 24-34 24-34 30-36 VL CDR2 50-56 50-56 46-55 VL CDR3 89-97
89-97 89-96
[0074] Variable regions and CDRs in an antibody sequence can be
identified according to general rules that have been developed in
the art (as set out above, such as, for example, the Kabat et al.
numbering system) or by aligning the sequences against a database
of known variable regions. Methods for identifying these regions
are described in Kontermann and Dubel, eds., Antibody Engineering,
Springer, New York, N.Y., 2001 and Dinarello et al., Current
Protocols in Immunology, John Wiley and Sons Inc., Hoboken, N.J.,
2000. Exemplary databases of antibody sequences are described in,
and can be accessed through, the "Abysis" website at
www.bioinf.org.uk/abs (maintained by A. C. Martin in the Department
of Biochemistry & Molecular Biology University College London,
London, England) and the VBASE2 website at www.vbase2.org, as
described in Retter et al., Nucl. Acids Res., 33 (Database issue):
D671-D674 (2005). Preferably the sequences are analyzed using the
Abysis database, which integrates sequence data from Kabat et al.,
IMGT and the Protein Data Bank (PDB) with structural data from the
PDB. See Dr. Andrew C. R. Martin's book chapter Protein Sequence
and Structure Analysis of Antibody Variable Domains. In: Antibody
Engineering Lab Manual (Ed.: Duebel, S. and Kontermann, R.,
Springer-Verlag, Heidelberg, ISBN-13: 978-3540413547, also
available on the website bioinforg.uk/abs). The Abysis database
website further includes general rules that have been developed for
identifying CDRs which can be used in accordance with the teachings
herein. Unless otherwise indicated, all CDRs set forth herein are
derived according to the Abysis database website as per Kabat et
al.
[0075] For heavy chain constant region amino acid positions
discussed in the invention, numbering is according to the Eu index
first described in Edelman et al., 1969, Proc. Natl. Acad. Sci. USA
63(1): 78-85 describing the amino acid sequence of myeloma protein
Eu, which reportedly was the first human IgG1 sequenced. The EU
index of Edelman is also set forth in Kabat et al., 1991 (supra.).
Thus, the terms "EU index as set forth in Kabat" or "EU index of
Kabat" or "EU numbering" in the context of the heavy chain refers
to the residue numbering system based on the human IgG1 Eu antibody
of Edelman et al. as set forth in Kabat et al., 1991 (supra.) The
numbering system used for the light chain constant region amino
acid sequence is similarly set forth in Kabat et al., (supra.) An
exemplary kappa light chain constant region amino acid sequence
compatible with the present invention is set forth immediately
below:
TABLE-US-00002 (SEQ ID NO: 1)
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG
NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC.
Similarly, an exemplary IgG1 heavy chain constant region amino acid
sequence compatible with the present invention is set forth
immediately below:
TABLE-US-00003 (SEQ ID NO: 2)
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP
KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC
LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPG.
[0076] The disclosed constant region sequences, or variations or
derivatives thereof, may be operably associated with the disclosed
heavy and light chain variable regions using standard molecular
biology techniques to provide full-length antibodies that may be
used as such or incorporated in the anti-CLDN ADCs of the
invention.
[0077] More generally the antibodies or immunoglobulins of the
invention may be generated from any antibody that specifically
recognizes or associates with the relevant determinant. As used
herein "determinant" or "target" means any detectable trait,
property, marker or factor that is identifiably associated with, or
specifically found in or on a particular cell, cell population or
tissue. Determinants or targets may be morphological, functional or
biochemical in nature and are preferably phenotypic. In certain
preferred embodiments a determinant is a protein that is
differentially expressed (over- or under-expressed) by specific
cell types or by cells under certain conditions (e.g., during
specific points of the cell cycle or cells in a particular niche).
For the purposes of the instant invention a determinant preferably
is differentially expressed on aberrant cancer cells and may
comprise a CLDN protein, or any of its splice variants, isoforms or
family members, or specific domains, regions or epitopes thereof.
An "antigen", "immunogenic determinant", "antigenic determinant" or
"immunogen" means any protein or any fragment, region or domain
thereof that can stimulate an immune response when introduced into
an immunocompetent animal and is recognized by the antibodies
produced from the immune response. The presence or absence of the
determinants contemplated herein may be used to identify a cell,
cell subpopulation or tissue (e.g., tumors, tumorigenic cells or
CSCs).
[0078] There are two types of disulfide bridges or bonds in
immunoglobulin molecules: interchain and intrachain disulfide
bonds. As is well known in the art the location and number of
interchain disulfide bonds vary according to the immunoglobulin
class and species. While the invention is not limited to any
particular class or subclass of antibody, the IgG1 immunoglobulin
shall be used throughout the instant disclosure for illustrative
purposes. In wild-type IgG1 molecules there are twelve intrachain
disulfide bonds (four on each heavy chain and two on each light
chain) and four interchain disulfide bonds. Intrachain disulfide
bonds are generally somewhat protected and relatively less
susceptible to reduction than interchain bonds. Conversely,
interchain disulfide bonds are located on the surface of the
immunoglobulin, are accessible to solvent and are usually
relatively easy to reduce. Two interchain disulfide bonds exist
between the heavy chains and one from each heavy chain to its
respective light chain. It has been demonstrated that interchain
disulfide bonds are not essential for chain association. The IgG1
hinge region contain the cysteines in the heavy chain that form the
interchain disulfide bonds, which provide structural support along
with the flexibility that facilitates Fab movement. The heavy/heavy
IgG1 interchain disulfide bonds are located at residues C226 and
C229 (Eu numbering) while the IgG1 interchain disulfide bond
between the light and heavy chain of IgG1 (heavy/light) are formed
between C214 of the kappa or lambda light chain and C220 in the
upper hinge region of the heavy chain.
[0079] B. Antibody Generation and Production
[0080] Antibodies of the invention can be produced using a variety
of methods known in the art.
[0081] 1. Generation of Polyclonal Antibodies in Host Animals
[0082] The production of polyclonal antibodies in various host
animals is well known in the art (see for example, Harlow and Lane
(Eds.) (1988) Antibodies: A Laboratory Manual, CSH Press; and
Harlow et al. (1989) Antibodies, NY, Cold Spring Harbor Press). In
order to generate polyclonal antibodies, an immunocompetent animal
is immunized with an antigenic protein or cells or preparations
comprising an antigenic protein. After a period of time, polyclonal
antibody-containing serum is obtained by bleeding or sacrificing
the animal. The serum may be used in the form obtained from the
animal or the antibodies may be partially or fully purified to
provide immunoglobulin fractions or isolated antibody
preparations.
[0083] Any form of antigen, or cells or preparations containing the
antigen, can be used to generate an antibody that is specific for a
determinant. The term "antigen" is used in a broad sense and may
comprise any immunogenic fragment or determinant of the selected
target including a single epitope, multiple epitopes, single or
multiple domains or the entire extracellular domain (ECD). The
antigen may be an isolated full-length protein, a cell surface
protein (e.g., immunizing with cells expressing at least a portion
of the antigen on their surface), or a soluble protein (e.g.,
immunizing with only the ECD portion of the protein). The antigen
may be produced in a genetically modified cell. Any of the
aforementioned antigens may be used alone or in combination with
one or more immunogenicity enhancing adjuvants known in the art.
The DNA encoding the antigen may be genomic or non-genomic (e.g.,
cDNA) and may encode at least a portion of the ECD, sufficient to
elicit an immunogenic response. Any vectors may be employed to
transform the cells in which the antigen is expressed, including
but not limited to adenoviral vectors, lentiviral vectors,
plasmids, and non-viral vectors, such as cationic lipids.
[0084] 2. Monoclonal Antibodies
[0085] In selected embodiments, the invention contemplates use of
monoclonal antibodies. The term "monoclonal antibody" or "mAb"
refers to an antibody obtained from a population of substantially
homogeneous antibodies, i.e., the individual antibodies comprising
the population are identical except for possible mutations (e.g.,
naturally occurring mutations), that may be present in minor
amounts.
[0086] Monoclonal antibodies can be prepared using a wide variety
of techniques including hybridoma techniques, recombinant
techniques, phage display technologies, transgenic animals (e.g., a
XenoMouse.RTM.) or some combination thereof. For example, in
preferred embodiments monoclonal antibodies can be produced using
hybridoma and biochemical and genetic engineering techniques such
as described in more detail in An, Zhigiang (ed.) Therapeutic
Monoclonal Antibodies: From Bench to Clinic, John Wiley and Sons,
1.sup.st ed. 2009; Shire et. al. (eds.) Current Trends in
Monoclonal Antibody Development and Manufacturing, Springer
Science+Business Media LLC, 1.sup.st ed. 2010; Harlow et al.,
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory
Press, 2nd ed. 1988; Hammerling, et al., in: Monoclonal Antibodies
and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981). Following
generation of a number of monoclonal antibodies that bind
specifically to a determinant, particularly suitable antibodies may
be selected through various screening processes, based on, for
example, affinity for the determinant or rate of internalization.
In particularly preferred embodiments monoclonal antibodies
produced as described herein may be used as source antibodies and
further modified to, for example, to improve affinity for the
target, improve its production in cell culture, reduce
immunogenicity in vivo, create multispecific constructs, etc. A
more detailed description of monoclonal antibody production and
screening is set out below and in the appended Examples.
[0087] 3. Human Antibodies
[0088] The antibodies may comprise fully human antibodies. The term
"human antibody" refers to an antibody (preferably a monoclonal
antibody) which possesses an amino acid sequence that corresponds
to that of an antibody produced by a human and/or has been made
using any of the techniques for making human antibodies described
below.
[0089] In one embodiment, recombinant human antibodies may be
isolated by screening a recombinant combinatorial antibody library
prepared using phage display. In one embodiment, the library is a
scFv phage or yeast display library, generated using human VL and
VH cDNAs prepared from mRNA isolated from B-cells.
[0090] Human antibodies can also be made by introducing human
immunoglobulin loci into transgenic animals, e.g., mice in which
the endogenous immunoglobulin genes have been partially or
completely inactivated and human immunoglobulin genes have been
introduced. Upon challenge antibody generation is observed which
closely resembles that seen in humans in all respects, including
gene rearrangement, assembly and fully human 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 6,075,181 and 6,150,584 regarding XenoMouse.RTM. technology;
and Lonberg and Huszar, 1995, PMID: 7494109). Alternatively, a
human antibody may be prepared via immortalization of human B
lymphocytes producing an antibody directed against a target antigen
(such B lymphocytes may be recovered from an individual suffering
from a neoplastic disorder or may have been immunized in vitro).
See, e.g., Cole et al., Monoclonal Antibodies and Cancer Therapy,
Alan R. Liss, p. 77 (1985); Boerner et al., 1991, PMID: 2051030;
and U.S. Pat. No. 5,750,373.
[0091] 4. Derived Antibodies:
[0092] Once the source antibodies have been generated, selected and
isolated as described above they may be further altered to provide
anti-CLDN antibodies having improved pharmaceutical
characteristics. Preferably the source antibodies are modified or
altered using known molecular engineering techniques to provide
derived antibodies having the desired therapeutic properties.
[0093] 4.1 Chimeric and Humanized Antibodies
[0094] Selected embodiments of the invention comprise murine
antibodies that immunospecifically bind to CLDN and, for the
purposes of the instant disclosure, may be considered "source"
antibodies. In selected embodiments, antibodies compatible with the
invention can be derived from such "source" antibodies through
optional modification of the constant region and/or the antigen
binding amino acid sequences of the source antibody. In certain
embodiments an antibody is "derived" from a source antibody if
selected amino acids in the source antibody are altered through
deletion, mutation, substitution, integration or combination. In
another embodiment, a "derived" antibody is one in which fragments
of the source antibody (e.g., one or more CDRs or the entire heavy
and light chain variable regions) are combined with or incorporated
into an acceptor antibody sequence to provide the derivative
antibody (e.g. chimeric or humanized antibodies). These "derived"
antibodies can be generated using standard molecular biological
techniques as described below, such as, for example, to improve
affinity for the determinant; to improve antibody stability; to
improve production and yield in cell culture; to reduce
immunogenicity in vivo; to reduce toxicity; to facilitate
conjugation of an active moiety; or to create a multispecific
antibody. Such antibodies may also be derived from source
antibodies through modification of the mature molecule (e.g.,
glycosylation patterns or pegylation) by chemical means or
post-translational modification.
[0095] In one embodiment, the chimeric antibodies of the invention
comprise chimeric antibodies that are derived from protein segments
from at least two different species or class of antibodies that
have been covalently joined. The term "chimeric" antibody is
directed to constructs in which a portion of the heavy and/or light
chain is identical or homologous to corresponding sequences in
antibodies from a particular species or belonging to a particular
antibody class or subclass, while the remainder of the chain(s) is
identical or homologous to corresponding sequences in antibodies
from another species or belonging to another antibody class or
subclass, as well as fragments of such antibodies (U.S. Pat. No.
4,816,567; Morrison et al., 1984, PMID: 6436822). In some preferred
embodiments chimeric antibodies of the instant invention may
comprise all or most of the selected murine heavy and light chain
variable regions operably linked to human light and heavy chain
constant regions. In other particularly preferred embodiments,
anti-CLDN antibodies may be "derived" from the mouse antibodies
disclosed herein.
[0096] In other embodiments, the chimeric antibodies of the
invention are "CDR grafted" antibodies, where the CDRs (as defined
using Kabat, Chothia, McCallum, etc.) are derived from a particular
species or belonging to a particular antibody class or subclass,
while the remainder of the antibody is derived from an antibody
from another species or belonging to another antibody class or
subclass. For use in humans, one or more selected rodent CDRs
(e.g., mouse CDRs) may be grafted into a human acceptor antibody,
replacing one or more of the naturally occurring CDRs of the human
antibody. These constructs generally have the advantages of
providing full strength human antibody functions, e.g., complement
dependent cytotoxicity (CDC) and antibody-dependent cell-mediated
cytotoxicity (ADCC) while reducing unwanted immune responses to the
antibody by the subject. In particularly preferred embodiments the
CDR grafted antibodies will comprise one or more CDRs obtained from
a mouse incorporated in a human framework sequence.
[0097] Similar to the CDR-grafted antibody is a "humanized"
antibody. As used herein, a "humanized" antibody is a human
antibody (acceptor antibody) comprising one or more amino acid
sequences (e.g. CDR sequences) derived from one or more non-human
antibodies (donor or source antibody). In certain embodiments,
"back mutations" can be introduced into the humanized antibody, in
which residues in one or more FRs of the variable region of the
recipient human antibody are replaced by corresponding residues
from the non-human species donor antibody. Such back mutations may
to help maintain the appropriate three-dimensional configuration of
the grafted CDR(s) and thereby improve affinity and antibody
stability. Antibodies from various donor species may be used
including, without limitation, mouse, rat, rabbit, or non-human
primate. Furthermore, humanized antibodies may comprise new
residues that are not found in the recipient antibody or in the
donor antibody to, for example, further refine antibody
performance. CDR grafted and humanized antibodies compatible with
the instant invention are provided as set forth in Example 7
below.
[0098] Various art recognized techniques can be used to determine
which human sequences to use as acceptor antibodies to provide
humanized constructs in accordance with the instant invention.
Compilations of compatible human germline sequences and methods of
determining their suitability as acceptor sequences are disclosed,
for example, in Tomlinson, I. A. et al. (1992) J. Mol. Biol.
227:776-798; Cook, G. P. et al. (1995) Immunol. Today 16: 237-242;
Chothia, D. et al. (1992) J. Mol. Biol. 227:799-817; and Tomlinson
et al. (1995) EMBO J 14:4628-4638 each of which is incorporated
herein in its entirety. The V-BASE directory (VBASE2--Retter et
al., Nucleic Acid Res. 33; 671-674, 2005) which provides a
comprehensive directory of human immunoglobulin variable region
sequences (compiled by Tomlinson, I. A. et al. MRC Centre for
Protein Engineering, Cambridge, UK) may also be used to identify
compatible acceptor sequences. Additionally, consensus human
framework sequences described, for example, in U.S. Pat. No.
6,300,064 may also prove to be compatible acceptor sequences are
can be used in accordance with the instant teachings. In general,
human framework acceptor sequences are selected based on homology
with the murine source framework sequences along with an analysis
of the CDR canonical structures of the source and acceptor
antibodies. The derived sequences of the heavy and light chain
variable regions of the derived antibody may then be synthesized
using art recognized techniques.
[0099] By way of example CDR grafted and humanized antibodies, and
associated methods, are described in U.S. Pat. Nos. 6,180,370 and
5,693,762. For further details, see, e.g., Jones et al., 1986,
PMID: 3713831); and U.S. Pat. Nos. 6,982,321 and 7,087,409.
[0100] The sequence identity or homology of the CDR grafted or
humanized antibody variable region to the human acceptor variable
region may be determined as discussed herein and, when measured as
such, will preferably share at least 60% or 65% sequence identity,
more preferably at least 70%, 75%, 80%, 85%, or 90% sequence
identity, even more preferably at least 93%, 95%, 98% or 99%
sequence identity. Preferably, residue positions which are not
identical differ by conservative amino acid substitutions. A
"conservative amino acid substitution" is one in which an amino
acid residue is substituted by another amino acid residue having a
side chain (R group) with similar chemical properties (e.g., charge
or hydrophobicity). In general, a conservative amino acid
substitution will not substantially change the functional
properties of a protein. In cases where two or more amino acid
sequences differ from each other by conservative substitutions, the
percent sequence identity or degree of similarity may be adjusted
upwards to correct for the conservative nature of the
substitution.
[0101] It will be appreciated that the annotated CDRs and framework
sequences as provided in the appended Figures are defined as per
Kabat et al. using a proprietary Abysis database. However, as
discussed herein one skilled in the art could readily identify the
CDRs in accordance with the numbering schemes provided by Chothia
et al. or MacCallum et al.
[0102] 4.2 Site-Specific Antibodies
[0103] The antibodies of the instant invention may be engineered to
facilitate conjugation to a cytotoxin or other anti-cancer agent
(as discussed in more detail below). It is advantageous for the
antibody drug conjugate (ADC) preparation to comprise a homogenous
population of ADC molecules in terms of the position of the
cytotoxin on the antibody and the drug to antibody ratio (DAR).
Based on the instant disclosure one skilled in the art could
readily fabricate site-specific engineered constructs as described
herein. As used herein a "site-specific antibody" or "site-specific
construct" means an antibody, or immunoreactive fragment thereof,
wherein at least one amino acid in either the heavy or light chain
is deleted, altered or substituted (preferably with another amino
acid) to provide at least one free cysteine. Similarly, a
"site-specific conjugate" shall be held to mean an ADC comprising a
site-specific antibody and at least one cytotoxin or other compound
conjugated to the unpaired cysteine(s). In certain embodiments the
unpaired cysteine residue will comprise an unpaired intrachain
residue. In other preferred embodiments the free cysteine residue
will comprise an unpaired interchain cysteine residue. The
engineered antibody can be of various isotypes, for example, IgG,
IgE, IgA or IgD; and within those classes the antibody can be of
various subclasses, for example, IgG1, IgG2, IgG3 or IgG4. For IgG
constructs the light chain of the antibody can comprise either a
kappa or lambda isotype each incorporating a C214 that, in
preferred embodiments, may be unpaired due to a lack of a C220
residue in the IgG1 heavy chain.
[0104] In one embodiment the engineered antibody comprises at least
one amino acid deletion or substitution of an intrachain or
interchain cysteine residue. As used herein "interchain cysteine
residue" means a cysteine residue that is involved in a native
disulfide bond either between the light and heavy chain of an
antibody or between the two heavy chains of an antibody while an
"intrachain cysteine residue" is one naturally paired with another
cysteine in the same heavy or light chain. In one embodiment the
deleted or substituted interchain cysteine residue is involved in
the formation of a disulfide bond between the light and heavy
chain. In another embodiment the deleted or substituted cysteine
residue is involved in a disulfide bond between the two heavy
chains. In a typical embodiment, due to the complementary structure
of an antibody, in which the light chain is paired with the VH and
C.sub.H1 domains of the heavy chain and wherein the CH2 and
C.sub.H3 domains of one heavy chain are paired with the CH2 and CH3
domains of the complementary heavy chain, a mutation or deletion of
a single cysteine in either the light chain or in the heavy chain
would result in two unpaired cysteine residues in the engineered
antibody.
[0105] In some embodiments an interchain cysteine residue is
deleted. In other embodiments an interchain cysteine is substituted
for another amino acid (e.g., a naturally occurring amino acid).
For example, the amino acid substitution can result in the
replacement of an interchain cysteine with a neutral (e.g. serine,
threonine or glycine) or hydrophilic (e.g. methionine, alanine,
valine, leucine or isoleucine) residue. In one particularly
preferred embodiment an interchain cysteine is replaced with a
serine.
[0106] In some embodiments contemplated by the invention the
deleted or substituted cysteine residue is on the light chain
(either kappa or lambda) thereby leaving a free cysteine on the
heavy chain. In other embodiments the deleted or substituted
cysteine residue is on the heavy chain leaving the free cysteine on
the light chain constant region. Upon assembly it will be
appreciated that deletion or substitution of a single cysteine in
either the light or heavy chain of an intact antibody results in a
site-specific antibody having two unpaired cysteine residues.
[0107] In one particularly preferred embodiment the cysteine at
position 214 (C214) of the IgG light chain (kappa or lambda) is
deleted or substituted. In another preferred embodiment the
cysteine at position 220 (C220) on the IgG heavy chain is deleted
or substituted. In further embodiments the cysteine at position 226
or position 229 on the heavy chain is deleted or substituted. In
one embodiment C220 on the heavy chain is substituted with serine
(C220S) to provide the desired free cysteine in the light chain. In
another embodiment C214 in the light chain is substituted with
serine (C214S) to provide the desired free cysteine in the heavy
chain. Such site-specific constructs provided in Example 8. A
summary of these preferred constructs is shown in Table 2
immediately below where all numbering is according to the EU index
as set forth in Kabat and WT stands for "wild-type" or native
constant region sequences without alterations and delta (4)
designates the deletion of an amino acid residue (e.g., C2144
indicates that the cysteine at position 214 has been deleted).
TABLE-US-00004 TABLE 2 Antibody Designation Component Alteration
ss1 Heavy Chain C220S Light Chain WT ss2 Heavy Chain C220.DELTA.
Light Chain WT ss3 Heavy Chain WT Light Chain C214.DELTA. ss4 Heavy
Chain WT Light Chain C214S
[0108] In an analogous manner preferred embodiments may comprise
site-specific IgG4 antibodies where the C127 residue of the heavy
chain is altered or eliminated to provide a free cysteine at the
220 position of the light chain. As set forth in the Examples
below, such embodiments may exhibit improved stability and reduced
toxicity.
[0109] The strategy for generating antibody-drug conjugates with
defined sites and stoichiometries of drug loading, as disclosed
herein, is broadly applicable to all anti-CLDN antibodies as it
primarily involves engineering of the conserved constant domains of
the antibody. As the amino acid sequences and native disulfide
bridges of each class and subclass of antibody are well documented,
one skilled in the art could readily fabricate engineered
constructs of various antibodies without undue experimentation and,
accordingly, such constructs are expressly contemplated as being
within the scope of the instant invention.
[0110] 4.3 Constant Region Modifications and Altered
Glycosylation
[0111] Selected embodiments of the present invention may also
comprise substitutions or modifications of the constant region
(i.e. the Fc region), including without limitation, amino acid
residue substitutions, mutations and/or modifications, which result
in a compound with preferred characteristics including, but not
limited to: altered pharmacokinetics, increased serum half-life,
increase binding affinity, reduced immunogenicity, increased
production, altered Fc ligand binding to an Fc receptor (FcR),
enhanced or reduced ADCC or CDC, altered glycosylation and/or
disulfide bonds and modified binding specificity.
[0112] Compounds with improved Fc effector functions can be
generated, for example, through changes in amino acid residues
involved in the interaction between the Fc domain and an Fc
receptor (e.g., Fc.gamma.RI, Fc.gamma.RIIA and B, Fc.gamma.RIII and
FcRn), which may lead to increased cytotoxicity and/or altered
pharmacokinetics, such as increased serum half-life (see, for
example, Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991);
Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J.
Lab. Clin. Med. 126:330-41 (1995).
[0113] In selected embodiments, antibodies with increased in vivo
half-lives can be generated by modifying (e.g., substituting,
deleting or adding) amino acid residues identified as involved in
the interaction between the Fc domain and the FcRn receptor (see,
e.g., International Publication Nos. WO 97/34631; WO 04/029207;
U.S. Pat. No. 6,737,056 and U.S.P.N. 2003/0190311). With regard to
such embodiments, Fc variants may provide half-lives in a mammal,
preferably a human, of greater than 5 days, greater than 10 days,
greater than 15 days, preferably greater than 20 days, greater than
25 days, greater than 30 days, greater than 35 days, greater than
40 days, greater than 45 days, greater than 2 months, greater than
3 months, greater than 4 months, or greater than 5 months. The
increased half-life results in a higher serum titer which thus
reduces the frequency of the administration of the antibodies
and/or reduces the concentration of the antibodies to be
administered. Binding to human FcRn in vivo and serum half-life of
human FcRn high affinity binding polypeptides can be assayed, e.g.,
in transgenic mice or transfected human cell lines expressing human
FcRn, or in primates to which the polypeptides with a variant Fc
region are administered. WO 2000/42072 describes antibody variants
with improved or diminished binding to FcRns. See also, e.g.,
Shields et al. J. Biol. Chem. 9(2):6591-6604 (2001).
[0114] In other embodiments, Fc alterations may lead to enhanced or
reduced ADCC or CDC activity. As in known in the art, CDC refers to
the lysing of a target cell in the presence of complement, and ADCC
refers to a form of cytotoxicity in which secreted Ig bound onto
FcRs present on certain cytotoxic cells (e.g., Natural Killer
cells, neutrophils, and macrophages) enables these cytotoxic
effector cells to bind specifically to an antigen-bearing target
cell and subsequently kill the target cell with cytotoxins. In the
context of the instant invention antibody variants are provided
with "altered" FcR binding affinity, which is either enhanced or
diminished binding as compared to a parent or unmodified antibody
or to an antibody comprising a native sequence FcR. Such variants
which display decreased binding may possess little or no
appreciable binding, e.g., 0-20% binding to the FcR compared to a
native sequence, e.g. as determined by techniques well known in the
art. In other embodiments the variant will exhibit enhanced binding
as compared to the native immunoglobulin Fc domain. It will be
appreciated that these types of Fc variants may advantageously be
used to enhance the effective anti-neoplastic properties of the
disclosed antibodies. In yet other embodiments, such alterations
lead to increased binding affinity, reduced immunogenicity,
increased production, altered glycosylation and/or disulfide bonds
(e.g., for conjugation sites), modified binding specificity,
increased phagocytosis; and/or down regulation of cell surface
receptors (e.g. B cell receptor; BCR), etc.
[0115] Still other embodiments comprise one or more engineered
glycoforms, e.g., a site-specific antibody comprising an altered
glycosylation pattern or altered carbohydrate composition that is
covalently attached to the protein (e.g., in the Fc domain). See,
for example, Shields, R. L. et al. (2002) J. Biol. Chem.
277:26733-26740. Engineered glycoforms may be useful for a variety
of purposes, including but not limited to enhancing or reducing
effector function, increasing the affinity of the antibody for a
target or facilitating production of the antibody. In certain
embodiments where reduced effector function is desired, the
molecule may be engineered to express an aglycosylated form.
Substitutions that may result in elimination of one or more
variable region framework glycosylation sites to thereby eliminate
glycosylation at that site are well known (see e.g. U.S. Pat. Nos.
5,714,350 and 6,350,861). Conversely, enhanced effector functions
or improved binding may be imparted to the Fc containing molecule
by engineering in one or more additional glycosylation sites.
[0116] Other embodiments include an Fc variant that has an altered
glycosylation composition, such as a hypofucosylated antibody
having reduced amounts of fucosyl residues or an antibody having
increased bisecting GlcNAc structures. Such altered glycosylation
patterns have been demonstrated to increase the ADCC ability of
antibodies. Engineered glycoforms may be generated by any method
known to one skilled in the art, for example by using engineered or
variant expression strains, by co-expression with one or more
enzymes (for example N-acetylglucosaminyltransferase III (GnTIII)),
by expressing a molecule comprising an Fc region in various
organisms or cell lines from various organisms or by modifying
carbohydrate(s) after the molecule comprising Fc region has been
expressed (see, for example, WO 2012/117002).
[0117] 4.4 Fragments
[0118] Regardless of which form of antibody (e.g. chimeric,
humanized, etc.) is selected to practice the invention it will be
appreciated that immunoreactive fragments, either by themselves or
as part of an antibody drug conjugate, of the same may be used in
accordance with the teachings herein. An "antibody fragment"
comprises at least a portion of an intact antibody. As used herein,
the term "fragment" of an antibody molecule includes
antigen-binding fragments of antibodies, and the term
"antigen-binding fragment" refers to a polypeptide fragment of an
immunoglobulin or antibody that immunospecifically binds or reacts
with a selected antigen or immunogenic determinant thereof or
competes with the intact antibody from which the fragments were
derived for specific antigen binding.
[0119] Exemplary site-specific fragments include: variable light
chain fragments (VL), an variable heavy chain fragments (VH), scFv,
F(ab')2 fragment, Fab fragment, Fd fragment, Fv fragment, single
domain antibody fragments, diabodies, linear antibodies,
single-chain antibody molecules and multispecific antibodies formed
from antibody fragments. In addition, an active site-specific
fragment comprises a portion of the antibody that retains its
ability to interact with the antigen/substrates or receptors and
modify them in a manner similar to that of an intact antibody
(though maybe with somewhat less efficiency). Such antibody
fragments may further be engineered to comprise one or more free
cysteines.
[0120] In other embodiments, an antibody fragment is one that
comprises the Fc region and that retains at least one of the
biological functions normally associated with the Fc region when
present in an intact antibody, such as FcRn binding, antibody
half-life modulation, ADCC function and complement binding. In one
embodiment, an antibody fragment is a monovalent antibody that has
an in vivo half-life substantially similar to an intact antibody.
For example, such an antibody fragment may comprise an antigen
binding arm linked to an Fc sequence comprising at least one free
cysteine capable of conferring in vivo stability to the
fragment.
[0121] As would be well recognized by those skilled in the art,
fragments can be obtained by molecular engineering or via chemical
or enzymatic treatment (such as papain or pepsin) of an intact or
complete antibody or antibody chain or by recombinant means. See,
e.g., Fundamental Immunology, W. E. Paul, ed., Raven Press, N.Y.
(1999), for a more detailed description of antibody fragments.
[0122] 4.5 Multivalent Constructs
[0123] In other embodiments, the antibodies and conjugates of the
invention may be monovalent or multivalent (e.g., bivalent,
trivalent, etc.). As used herein, the term "valency" refers to the
number of potential target binding sites associated with an
antibody. Each target binding site specifically binds one target
molecule or specific position or locus on a target molecule. When
an antibody is monovalent, each binding site of the molecule will
specifically bind to a single antigen position or epitope. When an
antibody comprises more than one target binding site (multivalent),
each target binding site may specifically bind the same or
different molecules (e.g., may bind to different ligands or
different antigens, or different epitopes or positions on the same
antigen). See, for example, U.S.P.N. 2009/0130105.
[0124] In one embodiment, the antibodies are bispecific antibodies
in which the two chains have different specificities, as described
in Millstein et al., 1983, Nature, 305:537-539. Other embodiments
include antibodies with additional specificities such as
trispecific antibodies. Other more sophisticated compatible
multispecific constructs and methods of their fabrication are set
forth in U.S.P.N. 2009/0155255, as well as WO 94/04690; Suresh et
al., 1986, Methods in Enzymology, 121:210; and WO96/27011.
[0125] Multivalent antibodies may immunospecifically bind to
different epitopes of the desired target molecule or may
immunospecifically bind to both the target molecule as well as a
heterologous epitope, such as a heterologous polypeptide or solid
support material. While preferred embodiments only bind two
antigens (i.e. bispecific antibodies), antibodies with additional
specificities such as trispecific antibodies are also encompassed
by the instant invention. Bispecific antibodies also include
cross-linked or "heteroconjugate" antibodies. For example, one of
the antibodies in the heteroconjugate can be coupled to avidin, the
other to biotin. Such antibodies have, for example, been proposed
to target immune system cells to unwanted cells (U.S. Pat. No.
4,676,980), and for treatment of HIV infection (WO 91/00360, WO
92/200373, and EP 03089). Heteroconjugate antibodies may be made
using any convenient cross-linking methods. Suitable cross-linking
agents are well known in the art, and are disclosed in U.S. Pat.
No. 4,676,980, along with a number of cross-linking techniques.
[0126] In certain preferred embodiments the antibodies of the
invention may be utilized in adoptive immunity gene therapy to
treat tumors. In one embodiment the antibodies of the invention
(e.g. ScFv fragments) may be used to generate a chimeric antigen
receptor (CAR). A "CAR" is a fused protein made up of an ECD
comprising the anti-CLDN antibodies of the invention or
immunoreactive fragments thereof (e.g. ScFv fragments), a
transmembrane domain, and at least one intracellular domain. In one
embodiment, T-cells, natural killer cells or dendritic cells that
have been genetically engineered to express CARs can be introduced
into a subject suffering from cancer in order to stimulate the
immune system of the subject to specifically target tumor cells
expressing CLDN. In preferred embodiments the CARs of the invention
will comprise an intracellular domain that initiates a primary
cytoplasmic signaling sequence, that is, a sequence for initiating
antigen-dependent primary activation via a T-cell receptor complex,
for example, intracellular domains derived from CD3.zeta.,
FcR.gamma., FcR.beta., CD3.gamma., CD3.delta., CD3.epsilon., CD5,
CD22, CD79a, CD79b, and CD66d. In other preferred embodiments, the
CARs of the invention will comprise an intracellular domain that
initiates a secondary or co-stimulating signal, for example,
intracellular domains derived from CD2, CD4, CD5, CD8.alpha.,
CD8.beta., CD28, CD134, CD137, ICOS, CD154, 4-1BB and
glucocorticoid-induced tumor necrosis factor receptor (see U.S.P.N.
US/2014/0242701).
[0127] In yet other embodiments, antibody variable domains with the
desired binding specificities (antibody-antigen combining sites)
are fused to immunoglobulin constant domain sequences, such as an
immunoglobulin heavy chain constant domain comprising at least part
of the hinge, CH2, and/or CH3 regions, using methods well known to
those of ordinary skill in the art.
[0128] 5. Recombinant Production of Antibodies
[0129] Antibodies and fragments thereof may be produced or modified
using genetic material obtained from antibody producing cells and
recombinant technology (see, for example, Berger and Kimmel, Guide
to Molecular Cloning Techniques, Methods in Enzymology vol. 152
Academic Press, Inc., San Diego, Calif.; Sambrook and Russell
(Eds.) (2000) Molecular Cloning: A Laboratory Manual (3.sup.rd
Ed.), NY, Cold Spring Harbor Laboratory Press; Ausubel et al.
(2002) Short Protocols in Molecular Biology: A Compendium of
Methods from Current Protocols in Molecular Biology, Wiley, John
& Sons, Inc.; and U.S. Pat. No. 7,709,611).
[0130] Another aspect of the invention pertains to nucleic acid
molecules that encode the antibodies of the invention. The nucleic
acids may be present in whole cells, in a cell lysate, or in a
partially purified or substantially pure form. A nucleic acid is
"isolated" or rendered substantially pure when separated from other
cellular components or other contaminants, e.g., other cellular
nucleic acids or proteins, by standard techniques, including
alkaline/SDS treatment, CsCl banding, column chromatography,
agarose gel electrophoresis and others well known in the art. A
nucleic acid of the invention can be, for example, DNA (e.g.
genomic DNA, cDNA), RNA and artificial variants thereof (e.g.,
peptide nucleic acids), whether single-stranded or double-stranded
or RNA, RNA and may or may not contain introns. In a preferred
embodiment, the nucleic acid is a cDNA molecule.
[0131] Nucleic acids of the invention can be obtained using
standard molecular biology techniques. For antibodies expressed by
hybridomas (e.g., hybridomas prepared as set forth in the Examples
below), cDNAs encoding the light and heavy chains of the antibody
can be obtained by standard PCR amplification or cDNA cloning
techniques. For antibodies obtained from an immunoglobulin gene
library (e.g., using phage display techniques), nucleic acid
encoding the antibody can be recovered from the library.
[0132] DNA fragments encoding VH and VL segments can be further
manipulated by standard recombinant DNA techniques, for example to
convert the variable region genes to full-length antibody chain
genes, to Fab fragment genes or to a scFv gene. In these
manipulations, a VL- or VH-encoding DNA fragment is operatively
linked to another DNA fragment encoding another protein, such as an
antibody constant region or a flexible linker. The term
"operatively linked", as used in this context, means that the two
DNA fragments are joined such that the amino acid sequences encoded
by the two DNA fragments remain in-frame.
[0133] The isolated DNA encoding the VH region can be converted to
a full-length heavy chain gene by operatively linking the
VH-encoding DNA to another DNA molecule encoding heavy chain
constant regions (CH1, CH2 and CH3). The sequences of human heavy
chain constant region genes are known in the art (see e.g., Kabat,
et al. (1991) (supra)) and DNA fragments encompassing these regions
can be obtained by standard PCR amplification. The heavy chain
constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or
IgD constant region, but most preferably is an IgG1 or IgG4
constant region. An exemplary IgG1 constant region is set forth in
SEQ ID NO: 2. For a Fab fragment heavy chain gene, the VH-encoding
DNA can be operatively linked to another DNA molecule encoding only
the heavy chain CH1 constant region.
[0134] The isolated DNA encoding the VL region can be converted to
a full-length light chain gene (as well as a Fab light chain gene)
by operatively linking the VL-encoding DNA to another DNA molecule
encoding the light chain constant region, CL. The sequences of
human light chain constant region genes are known in the art (see
e.g., Kabat, et al. (1991) (supra)) and DNA fragments encompassing
these regions can be obtained by standard PCR amplification. The
light chain constant region can be a kappa or lambda constant
region, but most preferably is a kappa constant region. In this
respect an exemplary compatible kappa light chain constant region
is set forth in SEQ ID NO: 1.
[0135] Contemplated herein are certain polypeptides (e.g. antigens
or antibodies) that exhibit "sequence identity", sequence
similarity" or "sequence homology" to the polypeptides of the
invention. A "homologous" polypeptide may exhibit 65%, 70%, 75%,
80%, 85%, or 90% sequence identity. In other embodiments a
"homologous" polypeptides may exhibit 93%, 95% or 98% sequence
identity. As used herein, the percent homology between two amino
acid sequences is equivalent to the percent identity between the
two sequences. The percent identity between the two sequences is a
function of the number of identical positions shared by the
sequences (i.e., % homology=# of identical positions/total # of
positions.times.100), taking into account the number of gaps, and
the length of each gap, which need to be introduced for optimal
alignment of the two sequences. The comparison of sequences and
determination of percent identity between two sequences can be
accomplished using a mathematical algorithm, as described in the
non-limiting examples below.
[0136] The percent identity between two amino acid sequences can be
determined using the algorithm of E. Meyers and W. Miller (Comput.
Appl. Biosci., 4:11-17 (1988)) which has been incorporated into the
ALIGN program (version 2.0), using a PAM120 weight residue table, a
gap length penalty of 12 and a gap penalty of 4. In addition, the
percent identity between two amino acid sequences can be determined
using the Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970))
algorithm which has been incorporated into the GAP program in the
GCG software package (available at www.gcg.com), using either a
Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14,
12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
[0137] Additionally or alternatively, the protein sequences of the
present invention can further be used as a "query sequence" to
perform a search against public databases to, for example, identify
related sequences. Such searches can be performed using the XBLAST
program (version 2.0) of Altschul, et al. (1990) J. Mol. Biol.
215:403-10. BLAST protein searches can be performed with the XBLAST
program, score=50, wordlength=3 to obtain amino acid sequences
homologous to the antibody molecules of the invention. To obtain
gapped alignments for comparison purposes, Gapped BLAST can be
utilized as described in Altschul et al., (1997) Nucleic Acids Res.
25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs,
the default parameters of the respective programs (e.g., XBLAST and
NBLAST) can be used.
[0138] Residue positions which are not identical may differ by
conservative amino acid substitutions or by non-conservative amino
acid substitutions. A "conservative amino acid substitution" is one
in which an amino acid residue is substituted by another amino acid
residue having a side chain with similar chemical properties (e.g.,
charge or hydrophobicity). In general, a conservative amino acid
substitution will not substantially change the functional
properties of a protein. In cases where two or more amino acid
sequences differ from each other by conservative substitutions, the
percent sequence identity or degree of similarity may be adjusted
upwards to correct for the conservative nature of the substitution.
In cases where there is a substitution with a non-conservative
amino acid, in preferred embodiments the polypeptide exhibiting
sequence identity will retain the desired function or activity of
the polypeptide of the invention (e.g., antibody.)
[0139] Also contemplated herein are nucleic acids that that exhibit
"sequence identity", sequence similarity" or "sequence homology" to
the nucleic acids of the invention. A "homologous sequence" means a
sequence of nucleic acid molecules exhibiting at least about 65%,
70%, 75%, 80%, 85%, or 90% sequence identity. In other embodiments,
a "homologous sequence" of nucleic acids may exhibit 93%, 95% or
98% sequence identity to the reference nucleic acid.
[0140] The instant invention also provides vectors comprising such
nucleic acids described above, which may be operably linked to a
promoter (see, e.g., WO 86/05807; WO 89/01036; and U.S. Pat. No.
5,122,464); and other transcriptional regulatory and processing
control elements of the eukaryotic secretory pathway. The invention
also provides host cells harboring those vectors and
host-expression systems.
[0141] As used herein, the term "host-expression system" includes
any kind of cellular system that can be engineered to generate
either the nucleic acids or the polypeptides and antibodies of the
invention. Such host-expression systems include, but are not
limited to microorganisms (e.g., E. coli or B. subtilis)
transformed or transfected with recombinant bacteriophage DNA or
plasmid DNA; yeast (e.g., Saccharomyces) transfected with
recombinant yeast expression vectors; or mammalian cells (e.g.,
COS, CHO-S, HEK-293T, 3T3 cells) harboring recombinant expression
constructs containing promoters derived from the genome of
mammalian cells or viruses (e.g., the adenovirus late promoter).
The host cell may be co-transfected with two expression vectors,
for example, the first vector encoding a heavy chain derived
polypeptide and the second vector encoding a light chain derived
polypeptide.
[0142] Methods of transforming mammalian cells are well known in
the art. See, for example, U.S. Pat. Nos. 4,399,216, 4,912,040,
4,740,461, and 4,959,455. The host cell may also be engineered to
allow the production of an antigen binding molecule with various
characteristics (e.g. modified glycoforms or proteins having GnTIII
activity).
[0143] For long-term, high-yield production of recombinant proteins
stable expression is preferred. Accordingly, cell lines that stably
express the selected antibody may be engineered using standard art
recognized techniques and form part of the invention. Rather than
using expression vectors that contain viral origins of replication,
host cells can be transformed with DNA controlled by appropriate
expression control elements (e.g., promoter or enhancer sequences,
transcription terminators, polyadenylation sites, etc.), and a
selectable marker. Any of the selection systems well known in the
art may be used, including the glutamine synthetase gene expression
system (the GS system) which provides an efficient approach for
enhancing expression under certain conditions. The GS system is
discussed in whole or part in connection with EP 0 216 846, EP 0
256 055, EP 0 323 997 and EP 0 338 841 and U.S. Pat. Nos. 5,591,639
and 5,879,936. Another preferred expression system for the
development of stable cell lines is the Freedom.TM. CHO-S Kit (Life
Technologies).
[0144] Once an antibody of the invention has been produced by
recombinant expression or any other of the disclosed techniques, it
may be purified or isolated by methods known in the art, meaning
that it is identified and separated and/or recovered from its
natural environment and separated from contaminants that would
interfere with diagnostic or therapeutic uses for the antibody.
Isolated antibodies include antibodies in situ within recombinant
cells.
[0145] These isolated preparations may be purified using various
art recognized techniques, such as, for example, ion exchange and
size exclusion chromatography, dialysis, diafiltration, and
affinity chromatography, particularly Protein A or Protein G
affinity chromatography.
[0146] 6. Post-Production Selection
[0147] No matter how obtained, antibody-producing cells (e.g.,
hybridomas, yeast colonies, etc.) may be selected, cloned and
further screened for desirable characteristics including, for
example, robust growth, high antibody production and desirable
antibody characteristics such as high affinity for the antigen of
interest. Hybridomas can be expanded in vitro in cell culture or in
vivo in syngeneic immunocompromised animals. Methods of selecting,
cloning and expanding hybridomas and/or colonies are well known to
those of ordinary skill in the art. Once the desired antibodies are
identified the relevant genetic material may be isolated,
manipulated and expressed using common, art-recognized molecular
biology and biochemical techniques.
[0148] The antibodies produced by naive libraries (either natural
or synthetic) may be of moderate affinity (K.sub.a of about
10.sup.6 to 10.sup.7 M.sup.-1). To enhance affinity, affinity
maturation may be mimicked in vitro by constructing antibody
libraries (e.g., by introducing random mutations in vitro by using
error-prone polymerase) and reselecting antibodies with high
affinity for the antigen from those secondary libraries (e.g. by
using phage or yeast display). WO 9607754 describes a method for
inducing mutagenesis in a CDR of an immunoglobulin light chain to
create a library of light chain genes.
[0149] Various techniques can be used to select antibodies,
including but not limited to, phage or yeast display in which a
library of human combinatorial antibodies or scFv fragments is
synthesized on phages or yeast, the library is screened with the
antigen of interest or an antibody-binding portion thereof, and the
phage or yeast that binds the antigen is isolated, from which one
may obtain the antibodies or immunoreactive fragments (Vaughan et
al., 1996, PMID: 9630891; Sheets et al., 1998, PMID: 9600934; Boder
et al., 1997, PMID: 9181578; Pepper et al., 2008, PMID: 18336206).
Kits for generating phage or yeast display libraries are
commercially available. There also are other methods and reagents
that can be used in generating and screening antibody display
libraries (see U.S. Pat. No. 5,223,409; WO 92/18619, WO 91/17271,
WO 92/20791, WO 92/15679, WO 93/01288, WO 92/01047, WO 92/09690;
and Barbas et al., 1991, PMID: 1896445). Such techniques
advantageously allow for the screening of large numbers of
candidate antibodies and provide for relatively easy manipulation
of sequences (e.g., by recombinant shuffling).
IV CHARACTERISTICS OF ANTIBODIES
[0150] In selected embodiments, antibody-producing cells (e.g.,
hybridomas or yeast colonies) may be selected, cloned and further
screened for favorable properties including, for example, robust
growth, high antibody production and, as discussed in more detail
below, desirable site-specific antibody characteristics. In other
cases characteristics of the antibody may be imparted by selecting
a particular antigen (e.g., a specific CLDN isoform) or
immunoreactive fragment of the target antigen for inoculation of
the animal. In still other embodiments the selected antibodies may
be engineered as described above to enhance or refine
immunochemical characteristics such as affinity or
pharmacokinetics.
[0151] A. Neutralizing Antibodies
[0152] In selected embodiments the antibodies of the invention may
be "antagonists" or "neutralizing" antibodies, meaning that the
antibody may associate with a determinant and block or inhibit the
activities of said determinant either directly or by preventing
association of the determinant with a binding partner such as a
ligand or a receptor, thereby interrupting the biological response
that otherwise would result from the interaction of the molecules.
A neutralizing or antagonist antibody will substantially inhibit
binding of the determinant to its ligand or substrate when an
excess of antibody reduces the quantity of binding partner bound to
the determinant by at least about 20%, 30%, 40%, 50%, 60%, 70%,
80%, 85%, 90%, 95%, 97%, 99% or more as measured, for example, by
target molecule activity or in an in vitro competitive binding
assay. It will be appreciated that the modified activity may be
measured directly using art recognized techniques or may be
measured by the impact the altered activity has downstream (e.g.,
oncogenesis or cell survival).
[0153] B. Internalizing Antibodies
[0154] There is evidence that a substantial portion of expressed
CLDN protein remains associated with the tumorigenic cell surface,
thereby allowing for localization and internalization of the
disclosed antibodies or ADCs. In preferred embodiments such
antibodies will be associated with, or conjugated to, one or more
drugs that kill the cell upon internalization. In particularly
preferred embodiments the ADCs of the instant invention will
comprise an internalizing site-specific ADC.
[0155] As used herein, an antibody that "internalizes" is one that
is taken up (along with any cytotoxin) by the cell upon binding to
an associated antigen or receptor. For therapeutic applications,
internalization will preferably occur in vivo in a subject in need
thereof. The number of ADCs internalized may be sufficient to kill
an antigen-expressing cell, especially an antigen-expressing cancer
stem cell. Depending on the potency of the cytotoxin or ADC as a
whole, in some instances, the uptake of a single antibody molecule
into the cell is sufficient to kill the target cell to which the
antibody binds. For example, certain drugs are so highly potent
that the internalization of a few molecules of the toxin conjugated
to the antibody is sufficient to kill the tumor cell. Whether an
antibody internalizes upon binding to a mammalian cell can be
determined by various art-recognized assays including those
described in the Examples below. Methods of detecting whether an
antibody internalizes into a cell are also described in U.S. Pat.
No. 7,619,068.
[0156] C. Depleting Antibodies
[0157] In other embodiments the antibodies of the invention are
depleting antibodies. The term "depleting" antibody refers to an
antibody that preferably binds to an antigen on or near the cell
surface and induces, promotes or causes the death of the cell
(e.g., by CDC, ADCC or introduction of a cytotoxic agent). In
preferred embodiments, the selected depleting antibodies will be
conjugated to a cytotoxin. Preferably a depleting antibody will be
able to kill at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%,
95%, 97%, or 99% of CLDN-expressing cells in a defined cell
population. The term "apparent 1050", as used herein, refers to the
concentration at which a primary antibody linked to a toxin kills
50 percent of the cells expressing the antigen(s) recognized by the
primary antibody. The toxin can be directly conjugated to the
primary antibody, or can be associated with the primary antibody
via a secondary antibody or antibody fragment that recognizes the
primary antibody, and which secondary antibody or antibody fragment
is directly conjugated to a toxin. Preferably a depleting antibody
will have an IC50 of less than 5 .mu.M. less than 1 .mu.M, less
than 100 nM, less than 50 nM, less than 30 nM, less than 20 nM,
less than 10 nM, less than 5 nM, less than 2 nM or less than 1 nM.
In some embodiments the cell population may comprise enriched,
sectioned, purified or isolated tumorigenic cells, including cancer
stem cells. In other embodiments the cell population may comprise
whole tumor samples or heterogeneous tumor extracts that comprise
cancer stem cells. Standard biochemical techniques may be used to
monitor and quantify the depletion of tumorigenic cells in
accordance with the teachings herein.
[0158] D. Binding Affinity
[0159] Disclosed herein are antibodies that have a high binding
affinity for a specific determinant e.g. CLDN. The term "K.sub.D"
refers to the dissociation constant of a particular
antibody-antigen interaction. An antibody of the invention can
immunospecifically bind its target antigen when the dissociation
constant K.sub.D (k.sub.off/k.sub.on) is .ltoreq.10.sup.-7 M. The
antibody specifically binds antigen with high affinity when the
K.sub.D is .ltoreq.5.times.10.sup.-9 M, and with very high affinity
when the K.sub.D is .ltoreq.5.times.10.sup.-10 M. In one embodiment
of the invention, the antibody has a K.sub.D of .ltoreq.10.sup.-9 M
and an off-rate of about 1.times.10.sup.-4/sec. In one embodiment
of the invention, the off-rate is <1.times.10.sup.-5/sec. In
other embodiments of the invention, the antibodies will bind to a
determinant with a K.sub.D of between about 10.sup.-7 M and
10.sup.-10 M, and in yet another embodiment it will bind with a
K.sub.D.ltoreq.2.times.10.sup.-10 M. Still other selected
embodiments of the invention comprise antibodies that have a
K.sub.D (k.sub.off/k.sub.on) of less than 10.sup.-6 M, less than
5.times.10.sup.-6 M, less than 10.sup.-7 M, less than
5.times.10.sup.-7 M, less than 10.sup.-8 M, less than
5.times.10.sup.-8 M, less than 10.sup.-9 M, less than
5.times.10.sup.-9 M, less than 10.sup.-10 M, less than
5.times.10.sup.-10 M, less than 10.sup.-11 M, less than
5.times.10.sup.-11 M, less than 10.sup.-12 M, less than
5.times.10.sup.-12 M, less than 10.sup.-13 M, less than
5.times.10.sup.-13 M, less than 10.sup.-14 M, less than
5.times.10.sup.-14 M, less than 10.sup.-15 M or less than
5.times.10.sup.-15 M.
[0160] In certain embodiments, an antibody of the invention that
immunospecifically binds to a determinant e.g. CLDN may have an
association rate constant or k.sub.on (or k.sub.a) rate
(antibody+antigen (Ag).sup.k.sub.on.rarw.antibody-Ag) of at least
10.sup.5 M.sup.-ls.sup.-l, at least 2.times.10.sup.5
M.sup.-ls.sup.-l, at least 5.times.10.sup.5 M.sup.-ls.sup.-l, at
least 10.sup.6 M.sup.-ls.sup.-l, at least 5.times.10.sup.6
M.sup.-ls.sup.-l, at least 10.sup.7 M.sup.-ls.sup.-l, at least
5.times.10.sup.7 M.sup.-ls.sup.-l, or at least 10.sup.8
M.sup.-ls.sup.-l.
[0161] In another embodiment, an antibody of the invention that
immunospecifically binds to a determinant e.g. CLDN may have a
disassociation rate constant or k.sub.off (or k.sub.d) rate
(antibody+antigen (Ag).sup.k.sub.off.rarw.antibody-Ag) of less than
10.sup.-l s.sup.-l, less than 5.times.10.sup.-ls.sup.-l, less than
10.sup.-2 s.sup.-l, less than 5.times.10.sup.-2 s.sup.-l, less than
10.sup.-3 s.sup.-l, less than 5.times.10.sup.-3 s.sup.-l, less than
10.sup.-4 s.sup.-l, less than 5.times.10.sup.4 s.sup.-l, less than
10.sup.-5 s.sup.-l, less than 5.times.10.sup.-5 s.sup.-l, less than
10.sup.-6 s.sup.-l, less than 5.times.10.sup.-6 s.sup.-l less than
10.sup.-7 s.sup.-l, less than 5.times.10.sup.-7 s.sup.-l, less than
10.sup.-8 s.sup.-l, less than 5.times.10.sup.-8 s.sup.-l, less than
10.sup.-9 s.sup.-l, less than 5.times.10.sup.-9 s.sup.-l or less
than 10.sup.-10 s.sup.-l.
[0162] Binding affinity may be determined using various techniques
known in the art, for example, surface plasmon resonance, bio-layer
interferometry, dual polarization interferometry, static light
scattering, dynamic light scattering, isothermal titration
calorimetry, ELISA, analytical ultracentrifugation, and flow
cytometry.
[0163] The term "apparent binding affinity" as used herein, refers
to the apparent binding of an antibody to its target antigen when
the antigen is overexpressed on the surface of a cell. The apparent
binding affinity of an antibody for an antigen is described herein
as an "apparent EC50", which is the concentration of antibody at
which 50% maximal binding to cells overexpressing the antigen
occurs. In one embodiment, two antibodies can be said to have
"substantially the same" apparent binding affinity for an antigen,
with >99% confidence, if they have apparent EC50 values that do
not differ from one another by more than 45%, by more than 40%, by
more than 35%, by more than 30%, by more than 25%, by more than
20%, by more than 10% or by more than 5%. In another embodiment an
antibody that binds multiple target antigens, e.g. is multireactive
towards one or more CLDN proteins, can be said to have
"substantially the same" apparent binding affinity for the multiple
antigens, with >99% confidence, if the apparent EC50 values of
the antibody for each of the antigens do not differ from one
another by more than 45%, by more than 40%, by more than 35%, by
more than 30%, by more than 25%, by more than 20%, by more than 10%
or by more than 5%. Since the assays used to determine the apparent
binding affinity of an antibody for an antigen typically utilize
cells overexpressing the antigen and which are exposed to
antibodies under presumed equilibrium or near equilibrium
conditions, the apparent EC50 value is reflective of the avidity,
or combined or accumulated strength of multiple apparent binding
affinities. Thus, in a related embodiment two antibodies will share
substantially the same avidity for a target cell line expressing
the antigen, with >99% confidence, if their apparent binding
affinities for the cell line, expressed as apparent EC50 values, do
not differ from one another by more than 45%, by more than 40%, by
more than 35%, by more than 30%, by more than 25%, by more than
20%, by more than 10% or by more than 5%. Similarly an antibody
that binds multiple target antigens, e.g. is multireactive towards
one or more CLDN proteins, can be said to have substantially the
same avidity for multiple antigens, with >99% confidence, if the
apparent EC50 values for each of the antigens do not differ from
one another by more than 45%, by more than 40%, by more than 35%,
by more than 30%, by more than 25%, by more than 20%, by more than
10% or by more than 5%.
[0164] E. Binning and Epitope Mapping
[0165] As used herein, the term "binning" refers to methods used to
group antibodies into "bins" based on their antigen binding
characteristics and whether they compete with each other. The
initial determination of bins may be further refined and confirmed
by epitope mapping and other techniques as described herein.
However it will be appreciated that empirical assignment of
antibodies to individual bins provides information that may be
indicative of the therapeutic potential of the disclosed
antibodies.
[0166] More specifically, one can determine whether a selected
reference antibody (or fragment thereof) competes for binding with
a second test antibody (i.e., is in the same bin) by using methods
known in the art and set forth in the Examples herein. In one
embodiment, a reference antibody is associated with CLDN antigen
under saturating conditions and then the ability of a secondary or
test antibody to bind to CLDN is determined using standard
immunochemical techniques. If the test antibody is able to
substantially bind to CLDN at the same time as the reference
anti-CLDN antibody, then the secondary or test antibody binds to a
different epitope than the primary or reference antibody. However,
if the test antibody is not able to substantially bind to CLDN at
the same time, then the test antibody binds to the same epitope, an
overlapping epitope, or an epitope that is in close proximity (at
least sterically) to the epitope bound by the primary antibody.
That is, the test antibody competes for antigen binding and is in
the same bin as the reference antibody.
[0167] The term "compete" or "competing antibody" when used in the
context of the disclosed antibodies means competition between
antibodies as determined by an assay in which a test antibody or
immunologically functional fragment being tested inhibits specific
binding of a reference antibody to a common antigen. Typically,
such an assay involves the use of purified antigen (e.g., CLDN or a
domain or fragment thereof) bound to a solid surface or cells, an
unlabeled test antibody and a labeled reference antibody.
Competitive inhibition is measured by determining the amount of
label bound to the solid surface or cells in the presence of the
test antibody. Usually the test antibody is present in excess
and/or allowed to bind first. Additional details regarding methods
for determining competitive binding are provided in the Examples
herein. Usually, when a competing antibody is present in excess, it
will inhibit specific binding of a reference antibody to a common
antigen by at least 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75%.
In some instance, binding is inhibited by at least 80%, 85%, 90%,
95%, or 97% or more.
[0168] Conversely, when the reference antibody is bound it will
preferably inhibit binding of a subsequently added test antibody
(i.e., an anti-CLDN antibody) by at least 30%, 40%, 45%, 50%, 55%,
60%, 65%, 70% or 75%. In some instance, binding of the test
antibody is inhibited by at least 80%, 85%, 90%, 95%, or 97% or
more.
[0169] Generally binning or competitive binding may be determined
using various art-recognized techniques, such as, for example,
immunoassays such as western blots, radioimmunoassays, enzyme
linked immunosorbent assay (ELISA), "sandwich" immunoassays,
immunoprecipitation assays, precipitin reactions, gel diffusion
precipitin reactions, immunodiffusion assays, agglutination assays,
complement-fixation assays, immunoradiometric assays, fluorescent
immunoassays and protein A immunoassays. Such immunoassays are
routine and well known in the art (see, Ausubel et al, eds, (1994)
Current Protocols in Molecular Biology, Vol. 1, John Wiley &
Sons, Inc., New York). Additionally, cross-blocking assays may be
used (see, for example, WO 2003/48731; and Harlow et al. (1988)
Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed
Harlow and David Lane).
[0170] Other technologies used to determine competitive inhibition
(and hence "bins"), include: surface plasmon resonance using, for
example, the BIAcore.TM. 2000 system (GE Healthcare); bio-layer
interferometry using, for example, a ForteBio.RTM. Octet RED
(ForteBio); or flow cytometry bead arrays using, for example, a
FACSCanto II (BD Biosciences) or a multiplex LUMINEX.TM. detection
assay (Luminex).
[0171] Luminex is a bead-based immunoassay platform that enables
large scale multiplexed antibody pairing. The assay compares the
simultaneous binding patterns of antibody pairs to the target
antigen. One antibody of the pair (capture mAb) is bound to Luminex
beads, wherein each capture mAb is bound to a bead of a different
color. The other antibody (detector mAb) is bound to a fluorescent
signal (e.g. phycoerythrin (PE)). The assay analyzes the
simultaneous binding (pairing) of antibodies to an antigen and
groups together antibodies with similar pairing profiles. Similar
profiles of a detector mAb and a capture mAb indicates that the two
antibodies bind to the same or closely related epitopes. In one
embodiment, pairing profiles can be determined using Pearson
correlation coefficients to identify the antibodies which most
closely correlate to any particular antibody on the panel of
antibodies that are tested. In preferred embodiments a
test/detector mAb will be determined to be in the same bin as a
reference/capture mAb if the Pearson's correlation coefficient of
the antibody pair is at least 0.9. In other embodiments the
Pearson's correlation coefficient is at least 0.8, 0.85, 0.87 or
0.89. In further embodiments, the Pearson's correlation coefficient
is at least 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99 or
1. Other methods of analyzing the data obtained from the Luminex
assay are described in U.S. Pat. No. 8,568,992. The ability of
Luminex to analyze 100 different types of beads (or more)
simultaneously provides almost unlimited antigen and/or antibody
surfaces, resulting in improved throughput and resolution in
antibody epitope profiling over a biosensor assay (Miller, et al.,
2011, PMID: 21223970).
[0172] "Surface plasmon resonance," refers to an optical phenomenon
that allows for the analysis of real-time specific interactions by
detection of alterations in protein concentrations within a
biosensor matrix.
[0173] In other embodiments, a technique that can be used to
determine whether a test antibody "competes" for binding with a
reference antibody is "bio-layer interferometry", an optical
analytical technique that analyzes the interference pattern of
white light reflected from two surfaces: a layer of immobilized
protein on a biosensor tip, and an internal reference layer. Any
change in the number of molecules bound to the biosensor tip causes
a shift in the interference pattern that can be measured in
real-time. Such biolayer interferometry assays may be conducted
using a ForteBio.RTM. Octet RED machine as follows. A reference
antibody (Ab1) is captured onto an anti-mouse capture chip, a high
concentration of non-binding antibody is then used to block the
chip and a baseline is collected. Monomeric, recombinant target
protein is then captured by the specific antibody (Ab1) and the tip
is dipped into a well with either the same antibody (Ab1) as a
control or into a well with a different test antibody (Ab2). If no
further binding occurs, as determined by comparing binding levels
with the control Ab1, then Ab1 and Ab2 are determined to be
"competing" antibodies. If additional binding is observed with Ab2,
then Ab1 and Ab2 are determined not to compete with each other.
This process can be expanded to screen large libraries of unique
antibodies using a full row of antibodies in a 96-well plate
representing unique bins. In preferred embodiments a test antibody
will compete with a reference antibody if the reference antibody
inhibits specific binding of the test antibody to a common antigen
by at least 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75%. In other
embodiments, binding is inhibited by at least 80%, 85%, 90%, 95%,
or 97% or more.
[0174] Once a bin, encompassing a group of competing antibodies,
has been defined further characterization can be carried out to
determine the specific domain or epitope on the antigen to which
the antibodies in a bin bind. Domain-level epitope mapping may be
performed using a modification of the protocol described by Cochran
et al., 2004, PMID: 15099763. Fine epitope mapping is the process
of determining the specific amino acids on the antigen that
comprise the epitope of a determinant to which the antibody binds.
The term "epitope" is used in its common biochemical sense and
refers to that portion of the target antigen capable of being
recognized and specifically bound by a particular antibody. In
certain embodiments, epitopes or immunogenic determinants include
chemically active surface groupings of molecules such as amino
acids, sugar side chains, phosphoryl groups, or sulfonyl groups,
and, in certain embodiments, may have specific three-dimensional
structural characteristics, and/or specific charge characteristics.
In certain embodiments, an antibody is said to specifically bind an
antigen when it preferentially recognizes its target antigen in a
complex mixture of proteins and/or macromolecules.
[0175] When the antigen is a polypeptide such as CLDN, epitopes may
generally be formed from both contiguous amino acids and
noncontiguous amino acids juxtaposed by tertiary folding of a
protein ("conformational epitopes"). In such conformational
epitopes the points of interaction occur across amino acid residues
on the protein that are linearly separated from one another.
Epitopes formed from contiguous amino acids (sometimes referred to
as "linear" or "continuous" epitopes) are typically retained upon
protein denaturing, whereas epitopes formed by tertiary folding are
typically lost upon protein denaturing. An antibody epitope
typically includes at least 3, and more usually, at least 5 or 8-10
amino acids in a unique spatial conformation. Methods of epitope
determination or "epitope mapping" are well known in the art and
may be used in conjunction with the instant disclosure to identify
epitopes on CLDN bound by the disclosed antibodies.
[0176] Compatible epitope mapping techniques include alanine
scanning mutants, peptide blots (Reineke (2004) Methods Mol Biol
248:443-63), or peptide cleavage analysis. In addition, methods
such as epitope excision, epitope extraction and chemical
modification of antigens can be employed (Tomer (2000) Protein
Science 9: 487-496). In other embodiments Modification-Assisted
Profiling (MAP), also known as Antigen Structure-based Antibody
Profiling (ASAP) provides a method that categorizes large numbers
of monoclonal antibodies directed against the same antigen
according to the similarities of the binding profile of each
antibody to chemically or enzymatically modified antigen surfaces
(U.S.P.N. 2004/0101920). This technology allows rapid filtering of
genetically identical antibodies, such that characterization can be
focused on genetically distinct antibodies. It will be appreciated
that MAP may be used to sort the anti-CLDN antibodies of the
invention into groups of antibodies binding different epitopes
[0177] Once a desired epitope on an antigen is determined, it is
possible to generate antibodies to that epitope, e.g., by
immunizing with a peptide comprising the epitope using techniques
described in the present invention. Alternatively, during the
discovery process, the generation and characterization of
antibodies may elucidate information about desirable epitopes
located in specific domains or motifs. From this information, it is
then possible to competitively screen antibodies for binding to the
same epitope. An approach to achieve this is to conduct competition
studies to find antibodies that compete for binding to the antigen.
A high throughput process for binning antibodies based upon their
cross-competition is described in WO 03/48731. Other methods of
binning or domain level or epitope mapping comprising antibody
competition or antigen fragment expression on yeast are well known
in the art.
V ANTIBODY CONJUGATES
[0178] In certain preferred embodiments the antibodies of the
invention may be conjugated with pharmaceutically active moiety or
diagnostic moieties to form an "antibody drug conjugate" (ADC) or
"antibody conjugate". The term "conjugate" is used broadly and
means the covalent or non-covalent association of any
pharmaceutically active moiety or diagnostic moiety with an
antibody of the instant invention regardless of the method of
association. In certain embodiments the association is effected
through a lysine or cysteine residue of the antibody. In
particularly preferred embodiments the pharmaceutically active or
diagnostic moieties may be conjugated to the antibody via one or
more site-specific free cysteine(s). The disclosed ADCs may be used
for therapeutic and diagnostic purposes.
[0179] The ADCs of the instant invention may be used to deliver
cytotoxins or other payloads to the target location (e.g.,
tumorigenic cells and/or cells expressing CLDN). As used herein the
terms "drug" or "warhead" may be used interchangeably and will mean
a biologically active or detectable molecule or compound, including
anti-cancer agents as described below. A "payload" may comprise a
drug or warhead in combination with an optional linker compound.
The warhead on the conjugate may comprise peptides, proteins,
prodrugs which are metabolized to an active agent in vivo,
polymers, nucleic acid molecules, small molecules, binding agents,
mimetic agents, synthetic drugs, inorganic molecules, organic
molecules and radioisotopes. In an advantageous embodiment, the
disclosed ADCs will direct the bound payload to the target site in
a relatively unreactive, non-toxic state before releasing and
activating the payload. This targeted release of the payload is
preferably achieved through stable conjugation of the payloads via
one or more cysteines or lysines and the relatively homogeneous
composition of the ADC preparations which minimize over-conjugated
toxic species. Coupled with drug linkers that are designed to
largely release the drug once it has been delivered to the tumor
site, the conjugates of the instant invention can substantially
reduce undesirable non-specific toxicity. This advantageously
provides for relatively high levels of the active cytotoxin at the
tumor site while minimizing exposure of non-targeted cells and
tissue thereby providing an enhanced therapeutic index.
[0180] It will be appreciated that, while preferred embodiments of
the invention comprise payloads of therapeutic moieties (e.g.,
cytotoxins), other payloads such as diagnostic agents and
biocompatible modifiers may benefit from the targeted release
provided by the disclosed conjugates. Accordingly, any disclosure
directed to exemplary therapeutic payloads is also applicable to
payloads comprising diagnostic agents or biocompatible modifiers as
discussed herein unless otherwise dictated by context. The selected
payload may be covalently or non-covalently linked to, the antibody
and exhibit various stoichiometric molar ratios depending, at least
in part, on the method used to effect the conjugation. The
conjugates of the instant invention may be represented by the
formula:
[0181] Ab-[L-D]n or a pharmaceutically acceptable salt thereof
wherein [0182] a) Ab comprises an anti-CLDN antibody; [0183] b) L
comprises an optional linker; [0184] c) D comprises a drug; and
[0185] d) n is an integer from about 1 to about 20.
[0186] Those of skill in the art will appreciate that conjugates
according to the aforementioned formula may be fabricated using a
number of different linkers and drugs and that conjugation
methodology will vary depending on the selection of components. As
such, any drug or drug linker compound that associates with a
reactive residue (e.g., cysteine or lysine) of the disclosed
antibodies are compatible with the teachings herein. Similarly, any
reaction conditions that allow for conjugation (e.g., site-specific
conjugation) of the selected drug to an antibody are within the
scope of the present invention. Notwithstanding the foregoing,
particularly preferred embodiments of the instant invention
comprise selective conjugation of the drug or drug linker to free
cysteines using stabilization agents in combination with mild
reducing agents as described herein. Such reaction conditions tend
to provide more homogeneous preparations with less non-specific
conjugation and contaminants and correspondingly less toxicity.
[0187] Exemplary payloads compatible with the teachings herein are
set forth below:
[0188] A. Therapeutic Agents
[0189] The antibodies of the invention may be conjugated, linked or
fused to or otherwise associated with a pharmaceutically active
moiety which is a therapeutic moiety or a drug such as an
anti-cancer agent including, but not limited to, cytotoxic agents,
cytostatic agents, anti-angiogenic agents, debulking agents,
chemotherapeutic agents, radiotherapeutic agents, targeted
anti-cancer agents, biological response modifiers, cancer vaccines,
cytokines, hormone therapies, anti-metastatic agents and
immunotherapeutic agents.
[0190] Preferred exemplary anti-cancer agents (including homologs
and derivatives thereof) comprise 1-dehydrotestosterone,
anthramycins, actinomycin D, bleomycin, calicheamicin, colchicin,
cyclophosphamide, cytochalasin B, dactinomycin (formerly
actinomycin), dihydroxy anthracin, dione, emetine, epirubicin,
ethidium bromide, etoposide, glucocorticoids, gramicidin D,
lidocaine, maytansinoids such as DM-1 and DM-4 (Immunogen),
mithramycin, mitomycin, mitoxantrone, paclitaxel, procaine,
propranolol, puromycin, tenoposide, tetracaine and pharmaceutically
acceptable salts or solvates, acids or derivatives of any of the
above.
[0191] Additional compatible cytotoxins comprise dolastatins and
auristatins, including monomethyl auristatin E (MMAE) and
monomethyl auristatin F (MMAF) (Seattle Genetics), amanitins such
as alpha-amanitin, beta-amanitin, gamma-amanitin or
epsilon-amanitin (Heidelberg Pharma), DNA minor groove binding
agents such as duocarmycin derivatives (Syntarga), alkylating
agents such as modified or dimeric pyrrolobenzodiazepines (PBD),
mechlorethamine, thioepa, chlorambucil, melphalan, carmustine
(BCNU), lomustine (CCNU), cyclothosphamide, busulfan,
dibromomannitol, streptozotocin, mitomycin C and cisdichlorodiamine
platinum (II) (DDP) cisplatin, splicing inhibitors such as
meayamycin analogs or derivatives (e.g., FR901464 as set forth in
U.S. Pat. No. 7,825,267), tubular binding agents such as epothilone
analogs and paclitaxel and DNA damaging agents such as
calicheamicins and esperamicins, antimetabolites such as
methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, and
5-fluorouracil decarbazine, anti-mitotic agents such as vinblastine
and vincristine and anthracyclines such as daunorubicin (formerly
daunomycin) and doxorubicin and pharmaceutically acceptable salts
or solvates, acids or derivatives of any of the above.
[0192] In one embodiment the antibodies of the instant invention
may be associated with anti-CD3 binding molecules to recruit
cytotoxic T-cells and have them target tumorigenic cells (BiTE
technology; see e.g., Fuhrmann et. al. (2010) Annual Meeting of
AACR Abstract No. 5625).
[0193] In further embodiments ADCs of the invention may comprise
therapeutic radioisotopes conjugated using appropriate linkers.
Exemplary radioisotopes that may be compatible with such
embodiments include, but are not limited to, iodine (.sup.131I,
.sup.125I, .sup.123I, .sup.121I), carbon (.sup.14C), copper
(.sup.62Cu, .sup.64Cu, .sup.67Cu), sulfur (.sup.35S), tritium
(.sup.3H), indium (.sup.115In, .sup.113In, .sup.112In, .sup.111In),
bismuth (.sup.212Bi, .sup.213Bi), technetium (.sup.99Tc), thallium
(.sup.201Ti), gallium (.sup.68Ga, .sup.67Ga), palladium
(.sup.103Pd), molybdenum (.sup.99Mo), xenon (.sup.133Xe), fluorine
(.sup.18F), .sup.153Sm, .sup.177Lu, .sup.159Gd, .sup.149Pm,
.sup.140La, .sup.175Yb, .sup.166Ho, .sup.90Y, .sup.47Sc,
.sup.186Re, .sup.188Re, .sup.142Pr, .sup.105Rh, .sup.97Ru,
.sup.68Ge, .sup.57Co, .sup.65Zn, .sup.85Sr, .sup.32P, .sup.153Gd,
.sup.169Yb, .sup.51Cr, .sup.54Mn, .sup.75Se, .sup.113Sn,
.sup.117Sn, .sup.225Ac, .sup.76Br, and .sup.211At. Other
radionuclides are also available as diagnostic and therapeutic
agents, especially those in the energy range of 60 to 4,000
keV.
[0194] In certain preferred embodiments, the ADCs of the invention
may comprise pyrrolobenzodiazepines (PBDs) as a cytotoxic agent and
pharmaceutically acceptable salts or solvates, acids or derivatives
thereof. PBDs are alkylating agents that exert antitumor activity
by covalently binding to DNA in the minor groove and inhibiting
nucleic acid synthesis. PBDs have been shown to have potent
antitumor properties while exhibiting minimal bone marrow
depression. PBDs compatible with the invention may be linked to an
antibody using several types of linkers (e.g., a peptidyl linker
comprising a maleimido moiety with a free sulfhydryl), and in
certain embodiments are dimeric in form (i.e., PBD dimers).
Compatible PBDs (and optional linkers) that may be conjugated to
the disclosed antibodies are described, for example, in U.S. Pat.
Nos. 6,362,331, 7,049,311, 7,189,710, 7,429,658, 7,407,951,
7,741,319, 7,557,099, 8,034,808, 8,163,736, 2011/0256157,
WO2011/130613, WO2011/128650, WO2011/130616 and WO2014/057074.
[0195] Antibodies of the present invention may also be conjugated
to biological response modifiers. For example, in particularly
preferred embodiments the drug moiety can be a polypeptide
possessing a desired biological activity. Such proteins may
include, for example, a toxin such as abrin, ricin A, Onconase (or
another cytotoxic RNase), pseudomonas exotoxin, cholera toxin,
diphtheria toxin; an apoptotic agent such as tumor necrosis factor
e.g. TNF-.alpha. or TNF-.beta., .alpha.-interferon,
.beta.-interferon, nerve growth factor, platelet derived growth
factor, tissue plasminogen activator, AIM I (WO 97/33899), AIM II
(WO 97/34911), Fas Ligand (Takahashi et al., 1994, PMID: 7826947),
and VEGI (WO 99/23105), a thrombotic agent, an anti-angiogenic
agent, e.g., angiostatin or endostatin, a lymphokine, for example,
interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6),
granulocyte macrophage colony stimulating factor (GM-CSF), and
granulocyte colony stimulating factor (G-CSF), or a growth factor
e.g., growth hormone (GH).
[0196] B. Diagnostic or Detection Agents
[0197] In other preferred embodiments, the antibodies of the
invention, or fragments or derivatives thereof, are conjugated to a
diagnostic or detectable agent, marker or reporter which may be,
for example, a biological molecule (e.g., a peptide or nucleotide),
a small molecule, fluorophore, or radioisotope. Labeled antibodies
can be useful for monitoring the development or progression of a
hyperproliferative disorder or as part of a clinical testing
procedure to determine the efficacy of a particular therapy
including the disclosed antibodies (i.e. theragnostics) or to
determine a future course of treatment. Such markers or reporters
may also be useful in purifying the selected antibody, for use in
antibody analytics (e.g., epitope binding or antibody binning),
separating or isolating tumorigenic cells or in preclinical
procedures or toxicology studies.
[0198] Such diagnosis, analysis and/or detection can be
accomplished by coupling the antibody to detectable substances
including, but not limited to, various enzymes comprising for
example horseradish peroxidase, alkaline phosphatase,
beta-galactosidase, or acetylcholinesterase; prosthetic groups,
such as but not limited to streptavidinlbiotin and avidin/biotin;
fluorescent materials, such as but not limited to, umbelliferone,
fluorescein, fluorescein isothiocynate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; luminescent materials, such as but not limited to,
luminol; bioluminescent materials, such as but not limited to,
luciferase, luciferin, and aequorin; radioactive materials, such as
but not limited to iodine (.sup.131I, .sup.125I, .sup.123I,
.sup.121I), carbon (.sup.14C), sulfur (.sup.35S), tritium
(.sup.3H), indium (.sup.115In, .sup.113In, .sup.112In, .sup.111In),
and technetium (.sup.99Tc), thallium (.sup.201Ti), gallium
(.sup.68Ga, .sup.67Ga), palladium (.sup.103Pd), molybdenum
(.sup.99Mo), xenon (.sup.133Xe), fluorine (.sup.18F), .sup.153Sm,
.sup.177Lu, .sup.159Gd, .sup.149Pm, .sup.140La, .sup.175Yb,
.sup.166Ho, .sup.90Y, .sup.47Sc, .sup.186Re, .sup.188Re,
.sup.142Pr, .sup.105Rh, .sup.97Ru, .sup.68Ge, .sup.57Co, .sup.65Zn,
.sup.85Sr, .sup.32P, .sup.153Gd, .sup.169Yb, .sup.51Cr, .sup.54Mn,
.sup.75Se, .sup.113Sn, and .sup.117Tin; positron emitting metals
using various positron emission tomographies, non-radioactive
paramagnetic metal ions, and molecules that are radiolabeled or
conjugated to specific radioisotopes. In such embodiments
appropriate detection methodology is well known in the art and
readily available from numerous commercial sources.
[0199] In other embodiments the antibodies or fragments thereof can
be fused or conjugated to marker sequences or compounds, such as a
peptide or fluorophore to facilitate purification or diagnostic or
analytic procedures such as immunohistochemistry, bio-layer
interferometry, surface plasmon resonance, flow cytometry,
competitive ELISA, FACs, etc. In preferred embodiments, the marker
comprises a histidine tag such as that provided by the pQE vector
(Qiagen), among others, many of which are commercially available.
Other peptide tags useful for purification include, but are not
limited to, the hemagglutinin "HA" tag, which corresponds to an
epitope derived from the influenza hemagglutinin protein (Wilson et
al., 1984, Cell 37:767) and the "flag" tag (U.S. Pat. No.
4,703,004).
[0200] C. Biocompatible Modifiers
[0201] In selected embodiments the antibodies of the invention may
be conjugated with biocompatible modifiers that may be used to
adjust, alter, improve or moderate antibody characteristics as
desired. For example, antibodies or fusion constructs with
increased in vivo half-lives can be generated by attaching
relatively high molecular weight polymer molecules such as
commercially available polyethylene glycol (PEG) or similar
biocompatible polymers. Those skilled in the art will appreciate
that PEG may be obtained in many different molecular weights and
molecular configurations that can be selected to impart specific
properties to the antibody (e.g. the half-life may be tailored).
PEG can be attached to antibodies or antibody fragments or
derivatives with or without a multifunctional linker either through
conjugation of the PEG to the N- or C-terminus of said antibodies
or antibody fragments or via epsilon-amino groups present on lysine
residues. Linear or branched polymer derivatization that results in
minimal loss of biological activity may be used. The degree of
conjugation can be closely monitored by SDS-PAGE and mass
spectrometry to ensure optimal conjugation of PEG molecules to
antibody molecules. Unreacted PEG can be separated from
antibody-PEG conjugates by, e.g., size exclusion or ion-exchange
chromatography. In a similar manner, the disclosed antibodies can
be conjugated to albumin in order to make the antibody or antibody
fragment more stable in vivo or have a longer half-life in vivo.
The techniques are well known in the art, see e.g., WO 93/15199, WO
93/15200, and WO 01/77137; and EP 0 413, 622. Other biocompatible
conjugates are evident to those of ordinary skill and may readily
be identified in accordance with the teachings herein.
[0202] D. Linker Compounds
[0203] Numerous linker compounds can be used to conjugate the
antibodies of the invention to the relevant drug. Preferably
linkers will covalently bind with the reactive residue (preferably
a cysteine or lysine) and the selected drug compound. Accordingly,
any linker that reacts with the selected antibody residue and may
be used to provide the relatively stable conjugates (site-specific
or otherwise) of the instant invention is compatible with the
teachings herein.
[0204] Numerous compatible linkers can advantageously bind to
reduced cysteines and lysines, which are nucleophilic. Conjugation
reactions involving reduced cysteines and lysines include, but are
not limited to, thiol-maleimide, thiol-halogeno (acyl halide),
thiol-ene, thiol-yne, thiol-vinylsulfone, thiol-bisulfone,
thiol-thiosulfonate, thiol-pyridyl disulfide and thiol-parafluoro
reactions. As further discussed herein, thiol-maleimide
bioconjugation is one of the most widely used approaches due to its
fast reaction rates and mild conjugation conditions. One issue with
this approach is the possibility of the retro-Michael reaction and
loss or transfer of the maleimido-linked payload from the antibody
to other proteins in the plasma, such as, for example, human serum
albumin. However, in preferred embodiments the use of selective
reduction and site-specific antibodies as set forth herein in
Example 15 may be used to stabilize the conjugate and reduce this
undesired transfer. Thiol-acyl halide reactions provide
bioconjugates that cannot undergo retro-Michael reaction and
therefore are more stable. However, the thiol-halide reactions in
general have slower reaction rates compared to maleimide-based
conjugations and are thus not as efficient. Thiol-pyridyl disulfide
reaction is another popular bioconjugation route. The pyridyl
disulfide undergoes fast exchange with free thiol resulting in the
mixed disulfide and release of pyridine-2-thione. Mixed disulfides
can be cleaved in the reductive cell environment releasing the
payload. Other approaches gaining more attention in bioconjugation
are thiol-vinylsulfone and thiol-bisulfone reactions, each of which
are compatible with the teachings herein and expressly included
within the scope of the invention.
[0205] In preferred embodiments compatible linkers will confer
stability on the ADCs in the extracellular environment, prevent
aggregation of the ADC molecules and keep the ADC freely soluble in
aqueous media and in a monomeric state. Before transport or
delivery into a cell, the ADC is preferably stable and remains
intact, i.e. the antibody remains linked to the drug moiety. While
the linkers are stable outside the target cell they are designed to
be cleaved or degraded at some efficacious rate inside the cell.
Accordingly an effective linker will: (i) maintain the specific
binding properties of the antibody; (ii) allow intracellular
delivery of the conjugate or drug moiety; (iii) remain stable and
intact, i.e. not cleaved or degraded, until the conjugate has been
delivered or transported to its targeted site; and (iv) maintain a
cytotoxic, cell-killing effect or a cytostatic effect of the drug
moiety. The stability of the ADC may be measured by standard
analytical techniques such as mass spectroscopy, hydrophobic
interaction chromatography (HIC), HPLC, and the separation/analysis
technique LC/MS. As set forth above covalent attachment of the
antibody and the drug moiety requires the linker to have two
reactive functional groups, i.e. bivalency in a reactive sense.
Bivalent linker reagents which are useful to attach two or more
functional or biologically active moieties, such as MMAE and
site-specific antibodies are known, and methods have been described
to provide their resulting conjugates.
[0206] Linkers compatible with the present invention may broadly be
classified as cleavable and non-cleavable linkers. Cleavable
linkers, which may include acid-labile linkers, protease cleavable
linkers and disulfide linkers, are internalized into the target
cell and are cleaved in the endosomal-lysosomal pathway inside the
cell. Release and activation of the cytotoxin relies on
endosome/lysosome acidic compartments that facilitate cleavage of
acid-labile chemical linkages such as hydrazone or oxime. If a
lysosomal-specific protease cleavage site is engineered into the
linker the cytotoxins will be released in proximity to their
intracellular targets. Alternatively, linkers containing mixed
disulfides provide an approach by which cytotoxic payloads are
released intracellularly as they are selectively cleaved in the
reducing environment of the cell, but not in the oxygen-rich
environment in the bloodstream. By way of contrast, compatible
non-cleavable linkers containing amide linked polyethyleneglycol or
alkyl spacers liberate toxic payloads during lysosomal degradation
of the ADC within the target cell. In some respects the selection
of linker will depend on the particular drug used in the
conjugate.
[0207] Accordingly, certain embodiments of the invention comprise a
linker that is cleavable by a cleaving agent that is present in the
intracellular environment (e.g., within a lysosome or endosome or
caveolae). The linker can be, for example, a peptidyl linker that
is cleaved by an intracellular peptidase or protease enzyme,
including, but not limited to, a lysosomal or endosomal protease.
In some embodiments, the peptidyl linker is at least two amino
acids long or at least three amino acids long. Cleaving agents can
include cathepsins B and D and plasmin, each of which is known to
hydrolyze dipeptide drug derivatives resulting in the release of
active drug inside target cells. Exemplary peptidyl linkers that
are cleavable by the thiol-dependent protease Cathepsin-B are
peptides comprising Phe-Leu since cathepsin-B has been found to be
highly expressed in cancerous tissue. Other examples of such
linkers are described, for example, in U.S. Pat. No. 6,214,345. In
a specific preferred embodiment, the peptidyl linker cleavable by
an intracellular protease is a Val-Cit linker, a Val-Ala linker or
a Phe-Lys linker such as is described in U.S. Pat. No. 6,214,345.
One advantage of using intracellular proteolytic release of the
therapeutic agent is that the agent is typically attenuated when
conjugated and the serum stabilities of the conjugates are
typically high.
[0208] In other embodiments, the cleavable linker is pH-sensitive.
Typically, the pH-sensitive linker will be hydrolyzable under
acidic conditions. For example, an acid-labile linker that is
hydrolyzable in the lysosome (e.g., a hydrazone, oxime,
semicarbazone, thiosemicarbazone, cis-aconitic amide, orthoester,
acetal, ketal, or the like) can be used (See, e.g., U.S. Pat. Nos.
5,122,368; 5,824,805; 5,622,929). Such linkers are relatively
stable under neutral pH conditions, such as those in the blood, but
are unstable at below pH 5.5 or 5.0, the approximate pH of the
lysosome.
[0209] In yet other embodiments, the linker is cleavable under
reducing conditions (e.g., a disulfide linker). A variety of
disulfide linkers are known in the art, including, for example,
those that can be formed using SATA
(N-succinimidyl-S-acetylthioacetate), SPDP
(N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB
(N-succinimidyl-3-(2-pyridyldithio) butyrate) and SMPT
(N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene)-
. In yet other specific embodiments, the linker is a malonate
linker (Johnson et al., 1995, Anticancer Res. 15:1387-93), a
maleimidobenzoyl linker (Lau et al., 1995, Bioorg-Med-Chem.
3(10):1299-1304), or a 3'-N-amide analog (Lau et al., 1995,
Bioorg-Med-Chem. 3(10):1305-12).
[0210] In particularly preferred embodiments (set forth in U.S.P.N.
2011/0256157) compatible peptidyl linkers will comprise:
##STR00001##
[0211] where the asterisk indicates the point of attachment to the
drug, CBA is the anti-CLDN antibody, L.sup.1 is a linker, A is a
connecting group connecting L.sup.1 to a reactive residue on the
antibody, L.sup.2 is a covalent bond or together with
--OC(.dbd.O)-- forms a self-immolative linker, and L.sup.1 or
L.sup.2 is a cleavable linker.
[0212] L.sup.1 is preferably the cleavable linker, and may be
referred to as a trigger for activation of the linker for
cleavage.
[0213] The nature of L.sup.1 and L.sup.2, where present, can vary
widely. These groups are chosen on the basis of their cleavage
characteristics, which may be dictated by the conditions at the
site to which the conjugate is delivered. Those linkers that are
cleaved by the action of enzymes are preferred, although linkers
that are cleavable by changes in pH (e.g. acid or base labile),
temperature or upon irradiation (e.g. photolabile) may also be
used. Linkers that are cleavable under reducing or oxidising
conditions may also find use in the present invention.
[0214] L.sup.1 may comprise a contiguous sequence of amino acids.
The amino acid sequence may be the target substrate for enzymatic
cleavage, thereby allowing release of the drug.
[0215] In one embodiment, L.sup.1 is cleavable by the action of an
enzyme. In one embodiment, the enzyme is an esterase or a
peptidase.
[0216] In one embodiment, L.sup.1 comprises a dipeptide. The
dipeptide may be represented as --NH--X.sub.1-X.sub.2--CO--, where
--NH-- and --CO-- represent the N- and C-terminals of the amino
acid groups X.sub.1 and X.sub.2 respectively. The amino acids in
the dipeptide may be any combination of natural amino acids. Where
the linker is a cathepsin labile linker, the dipeptide may be the
site of action for cathepsin-mediated cleavage.
[0217] Additionally, for those amino acids groups having carboxyl
or amino side chain functionality, for example Glu and Lys
respectively, CO and NH may represent that side chain
functionality.
[0218] In one embodiment, the group --X.sub.1-X.sub.2-- in
dipeptide, --NH--X.sub.1-X.sub.2--CO--, is selected from:
-Phe-Lys-, -Val-Ala-, -Val-Lys-, -Ala-Lys-, -Val-Cit-, -Phe-Cit-,
-Leu-Cit-, -Ile-Cit-, -Phe-Arg- and -Trp-Cit- where Cit is
citrulline.
[0219] Preferably, the group --X.sub.1-X.sub.2-- in dipeptide,
--NH--X.sub.1-X.sub.2--CO--, is selected from: -Phe-Lys-,
-Val-Ala-, -Val-Lys-, -Ala-Lys-, and -Val-Cit-.
[0220] Most preferably, the group --X.sub.1-X.sub.2-- in dipeptide,
--NH--X.sub.1-X.sub.2--CO--, is -Phe-Lys- or -Val-Ala-.
[0221] In one embodiment, L.sup.2 is present and together with
--C(.dbd.O)O-- forms a self-immolative linker. In one embodiment,
L.sup.2 is a substrate for enzymatic activity, thereby allowing
release of the drug.
[0222] In one embodiment, where L.sup.1 is cleavable by the action
of an enzyme and L.sup.2 is present, the enzyme cleaves the bond
between L.sup.1 and L.sup.2.
[0223] L.sup.1 and L.sup.2, where present, may be connected by a
bond selected from: --C(.dbd.O)NH--, --C(.dbd.O)O--,
--NHC(.dbd.O)--, --OC(.dbd.O)--, --OC(.dbd.O)O--, --NHC(.dbd.O)O--,
--OC(.dbd.O)NH--, and --NHC(.dbd.O)NH--.
[0224] An amino group of L.sup.1 that connects to L.sup.2 may be
the N-terminus of an amino acid or may be derived from an amino
group of an amino acid side chain, for example a lysine amino acid
side chain.
[0225] A carboxyl group of L.sup.1 that connects to L.sup.2 may be
the C-terminus of an amino acid or may be derived from a carboxyl
group of an amino acid side chain, for example a glutamic acid
amino acid side chain.
[0226] A hydroxyl group of L.sup.1 that connects to L.sup.2 may be
derived from a hydroxyl group of an amino acid side chain, for
example a serine amino acid side chain.
[0227] The term "amino acid side chain" includes those groups found
in: (i) naturally occurring amino acids such as alanine, arginine,
asparagine, aspartic acid, cysteine, glutamine, glutamic acid,
glycine, histidine, isoleucine, leucine, lysine, methionine,
phenylalanine, proline, serine, threonine, tryptophan, tyrosine,
and valine; (ii) minor amino acids such as ornithine and
citrulline; (iii) unnatural amino acids, beta-amino acids,
synthetic analogs and derivatives of naturally occurring amino
acids; and (iv) all enantiomers, diastereomers, isomerically
enriched, isotopically labelled (e.g. .sup.2H, .sup.3H, .sup.14C,
.sup.15N), protected forms, and racemic mixtures thereof.
[0228] In one embodiment, --C(.dbd.O)O-- and L.sup.2 together form
the group:
##STR00002##
[0229] where the asterisk indicates the point of attachment to the
drug or cytotoxic agent position, the wavy line indicates the point
of attachment to the linker L.sup.1, Y is --N(H)--, --O--,
--C(.dbd.O)N(H)-- or --C(.dbd.O)O--, and n is 0 to 3. The phenylene
ring is optionally substituted with one, two or three substituents
as described herein. In one embodiment, the phenylene group is
optionally substituted with halo, NO.sub.2, R or OR.
[0230] In one embodiment, Y is NH.
[0231] In one embodiment, n is 0 or 1. Preferably, n is 0.
[0232] Where Y is NH and n is 0, the self-immolative linker may be
referred to as a p-aminobenzylcarbonyl linker (PABC).
[0233] In another particularly preferred embodiments the linker may
include a self-immolative linker and the dipeptide together form
the group --NH-Val-Ala-CO--NH-PABC-, which is illustrated
below:
##STR00003##
[0234] where the asterisk indicates the point of attachment to the
selected cytotoxic moiety, and the wavy line indicates the point of
attachment to the remaining portion of the linker (e.g., the
spacer-antibody binding segments) which may be conjugated to the
antibody. Upon enzymatic cleavage of the dipeptide the
self-immolative linker will allow for clean release of the
protected compound (i.e., the cytotoxin) when a remote site is
activated, proceeding along the lines shown below:
##STR00004##
[0235] where L* is the activated form of the remaining portion of
the linker comprising the now cleaved peptidyl unit. The clean
release of the drug ensures they will maintain the desired toxic
activity.
[0236] In one embodiment, A is a covalent bond. Thus, L.sup.1 and
the antibody are directly connected. For example, where L.sup.1
comprises a contiguous amino acid sequence, the N-terminus of the
sequence may connect directly to the antibody residue.
[0237] In another embodiment, A is a spacer group. Thus, L.sup.1
and the antibody are indirectly connected.
[0238] L.sup.1 and A may be connected by a bond selected from:
--C(.dbd.O)NH--, --C(.dbd.O)O--, --NHC(.dbd.O)--, --OC(.dbd.O)--,
--OC(.dbd.O)O--, --NHC(.dbd.O)O--, --OC(.dbd.O)NH--, and
--NHC(.dbd.O)NH--.
[0239] As will be discussed in more detail below the drug linkers
of the instant invention will preferably be linked to reactive
thiol nucleophiles on cysteines, including free cysteines. To this
end the cysteines of the antibodies may be made reactive for
conjugation with linker reagents by treatment with various reducing
agent such as DTT or TCEP or mild reducing agents as set forth
herein. In other embodiments the drug linkers of the instant
invention will preferably be linked to a lysine.
[0240] Preferably, the linker contains an electrophilic functional
group for reaction with a nucleophilic functional group on the
antibody. Nucleophilic groups on antibodies include, but are not
limited to: (i) N-terminal amine groups, (ii) side chain amine
groups, e.g. lysine, (iii) side chain thiol groups, e.g. cysteine,
and (iv) sugar hydroxyl or amino groups where the antibody is
glycosylated. Amine, thiol, and hydroxyl groups are nucleophilic
and capable of reacting to form covalent bonds with electrophilic
groups on linker moieties and linker reagents including: (i)
maleimide groups (ii) activated disulfides, (iii) active esters
such as NHS (N-hydroxysuccinimide) esters, HOBt
(N-hydroxybenzotriazole) esters, haloformates, and acid halides;
(iv) alkyl and benzyl halides such as haloacetamides; and (v)
aldehydes, ketones, carboxyl, and, some of which are exemplified as
follows:
##STR00005##
[0241] In particularly preferred embodiments the connection between
a site-specific antibody and the drug-linker moiety is through a
thiol residue of a free cysteine of the site specific antibody and
a terminal maleimide group of present on the linker. In such
embodiments, the connection between the antibody and the
drug-linker is:
##STR00006##
[0242] where the asterisk indicates the point of attachment to the
remaining portion of drug-linker and the wavy line indicates the
point of attachment to the remaining portion of the antibody. In
this embodiment, the S atom is preferably derived from a
site-specific free cysteine. With regard to other compatible
linkers the binding moiety comprises a terminal iodoacetamide that
may be reacted with activated residues to provide the desired
conjugate. In any event one skilled in the art could readily
conjugate each of the disclosed drug-linker compounds with a
compatible anti-CLDN site-specific antibody in view of the instant
disclosure.
[0243] E. Conjugation
[0244] It will be appreciated that a number of well known different
reactions may be used to attach the drug moiety and/or linker to
the selected antibody. For example, various reactions exploiting
sulfhydryl groups of cysteines may be employed to conjugate the
desired moiety. Particularly preferred embodiments will comprise
conjugation of antibodies comprising one or more free cysteines as
discussed in detail below. In other embodiments ADCs of the instant
invention may be generated through conjugation of drugs to
solvent-exposed amino groups of lysine residues present in the
selected antibody. Still other embodiments comprise activation of
the N-terminal threonine and serine residues which may then be used
to attach the disclosed payloads to the antibody. The selected
conjugation methodology will preferably be tailored to optimize the
number of drugs attached to the antibody and provide a relatively
high therapeutic index.
[0245] Various methods are known in the art for conjugating a
therapeutic compound to a cysteine residue and will be apparent to
the skilled artisan. Under basic conditions the cysteine residues
will be deprotonated to generate a thiolate nucleophile which may
be reacted with soft electrophiles, such as maleimides and
iodoacetamides. Generally reagents for such conjugations may react
directly with a cysteine thiol of a cysteine to form the conjugated
protein or with a linker-drug to form a linker-drug intermediate.
In the case of a linker, several routes, employing organic
chemistry reactions, conditions, and reagents are known to those
skilled in the art, including: (1) reaction of a cysteine group of
the protein of the invention with a linker reagent, to form a
protein-linker intermediate, via a covalent bond, followed by
reaction with an activated compound; and (2) reaction of a
nucleophilic group of a compound with a linker reagent, to form a
drug-linker intermediate, via a covalent bond, followed by reaction
with a cysteine group of a protein of the invention. As will be
apparent to the skilled artisan from the foregoing, bifunctional
linkers are useful in the present invention. For example, the
bifunctional linker may comprise a thiol modification group for
covalent linkage to the cysteine residue(s) and at least one
attachment moiety (e.g., a second thiol modification moiety) for
covalent or non-covalent linkage to the compound.
[0246] Prior to conjugation, antibodies may be made reactive for
conjugation with linker reagents by treatment with a reducing agent
such as dithiothreitol (DTT) or (tris(2-carboxyethyl)phosphine
(TCEP). In other embodiments additional nucleophilic groups can be
introduced into antibodies through the reaction of lysines with
reagents, including but not limited to, 2-iminothiolane (Traut's
reagent), SATA, SATP or SAT(PEG)4, resulting in conversion of an
amine into a thiol.
[0247] With regard to such conjugations cysteine thiol or lysine
amino groups are nucleophilic and capable of reacting to form
covalent bonds with electrophilic groups on linker reagents or
compound-linker intermediates or drugs including: (i) active esters
such as NHS esters, HOBt esters, haloformates, and acid halides;
(ii) alkyl and benzyl halides, such as haloacetamides; (iii)
aldehydes, ketones, carboxyl, and maleimide groups; and (iv)
disulfides, including pyridyl disulfides, via sulfide exchange.
Nucleophilic groups on a compound or linker include, but are not
limited to amine, thiol, hydroxyl, hydrazide, oxime, hydrazine,
thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groups
capable of reacting to form covalent bonds with electrophilic
groups on linker moieties and linker reagents.
[0248] Preferred labelling reagents include maleimide, haloacetyl,
iodoacetamide succinimidyl ester, isothiocyanate, sulfonyl
chloride, 2,6-dichlorotriazinyl, pentafluorophenyl ester, and
phosphoramidite, although other functional groups can also be used.
In certain embodiments methods include, for example, the use of
maleimides, iodoacetimides or haloacetyl/alkyl halides, aziridne,
acryloyl derivatives to react with the thiol of a cysteine to
produce a thioether that is reactive with a compound. Disulphide
exchange of a free thiol with an activated piridyldisulphide is
also useful for producing a conjugate (e.g., use of
5-thio-2-nitrobenzoic (TNB) acid). Preferably, a maleimide is
used.
[0249] As indicated above, lysine may also be used as a reactive
residue to effect conjugation as set forth herein. The nucleophilic
lysine residue is commonly targeted through amine-reactive
succinimidylesters. To obtain an optimal number of deprotonated
lysine residues, the pH of the aqueous solution must be below the
pKa of the lysine ammonium group, which is around 10.5, so the
typical pH of the reaction is about 8 and 9. The common reagent for
the coupling reaction is NHS-ester which reacts with nucleophilic
lysine through a lysine acylation mechanism. Other compatible
reagents that undergo similar reactions comprise isocyanates and
isothiocyanates which also may be used in conjunction with the
teachings herein to provide ADCs. Once the lysines have been
activated, many of the aforementioned linking groups may be used to
covalently bind the warhead to the antibody.
[0250] Methods are also known in the art for conjugating a compound
to a threonine or serine residue (preferably a N-terminal residue).
For example methods have been described in which carbonyl
precursors are derived from the 1,2-aminoalcohols of serine or
threonine, which can be selectively and rapidly converted to
aldehyde form by periodate oxidation. Reaction of the aldehyde with
a 1,2-aminothiol of cysteine in a compound to be attached to a
protein of the invention forms a stable thiazolidine product. This
method is particularly useful for labelling proteins at N-terminal
serine or threonine residues.
[0251] In particularly preferred embodiments reactive thiol groups
may be introduced into the selected antibody (or fragment thereof)
by introducing one, two, three, four, or more free cysteine
residues (e.g., preparing antibodies comprising one or more free
non-native cysteine amino acid residues). Such site-specific
antibodies or engineered antibodies, allow for conjugate
preparations that exhibit enhanced stability and substantial
homogeneity due, at least in part, to the provision of engineered
free cysteine site(s) and/or the novel conjugation procedures set
forth herein. Unlike conventional conjugation methodology that
fully or partially reduces each of the intrachain or interchain
antibody disulfide bonds to provide conjugation sites (and is fully
compatible with the instant invention), the present invention
additionally provides for the selective reduction of certain
prepared free cysteine sites and direction of the drug-linker to
the same. The conjugation specificity promoted by the engineered
sites and the selective reduction allows for a high percentage of
site directed conjugation at the desired positions. Significantly
some of these conjugation sites, such as those present in the
terminal region of the light chain constant region, are typically
difficult to conjugate effectively as they cross-react with other
free cysteines. However, through molecular engineering and
selective reduction of the resulting free cysteines efficient
conjugation rates may be obtained which considerably reduces
unwanted high-DAR contaminants and non-specific toxicity. More
generally the engineered constructs and disclosed novel conjugation
methods comprising selective reduction provide ADC preparations
having improved pharmacokinetics and/or pharmacodynamics and,
potentially, an improved therapeutic index.
[0252] The site-specific constructs present free cysteine(s), which
when reduced comprise thiol groups that are nucleophilic and
capable of reacting to form covalent bonds with electrophilic
groups on linker moieties such as those disclosed above. Preferred
antibodies of the instant invention will have reducible unpaired
interchain or intrachain cysteines, i.e. cysteines providing such
nucleophilic groups. Thus, in certain embodiments the reaction of
free sulfhydryl groups of the reduced unpaired cysteines and the
terminal maleimido or haloacetamide groups of the disclosed
drug-linkers will provide the desired conjugation. In such cases
the free cysteines of the antibodies may be made reactive for
conjugation with linker reagents by treatment with a reducing agent
such as dithiothreitol (DTT) or (tris (2-carboxyethyl)phosphine
(TCEP). Each free cysteine will thus present, theoretically, a
reactive thiol nucleophile. While such reagents are compatible it
will be appreciated that conjugation of the site-specific
antibodies may be effected using various reactions, conditions and
reagents known to those skilled in the art.
[0253] In addition it has been found that the free cysteines of
engineered antibodies may be selectively reduced to provide
enhanced site-directed conjugation and a reduction in unwanted,
potentially toxic contaminants. More specifically "stabilizing
agents" such as arginine have been found to modulate intra- and
inter-molecular interactions in proteins and may be used, in
conjunction with selected reducing agents (preferably relatively
mild), to selectively reduce the free cysteines and to facilitate
site-specific conjugation as set forth herein. As used herein the
terms "selective reduction" or "selectively reducing" may be used
interchangeably and shall mean the reduction of free cysteine(s)
without substantially disrupting native disulfide bonds present in
the engineered antibody. In selected embodiments this may be
affected by certain reducing agents. In other preferred embodiments
selective reduction of an engineered construct will comprise the
use of stabilization agents in combination with reducing agents
(including mild reducing agents). It will be appreciated that the
term "selective conjugation" shall mean the conjugation of an
engineered antibody that has been selectively reduced with a
cytotoxin as described herein. In this respect the use of such
stabilizing agents in combination with reducing agents can markedly
improve the efficiency of site-specific conjugation as determined
by extent of conjugation on the heavy and light antibody chains and
DAR distribution of the preparation.
[0254] While not wishing to be bound by any particular theory, such
stabilizing agents may act to modulate the electrostatic
microenvironment and/or modulate conformational changes at the
desired conjugation site, thereby allowing relatively mild reducing
agents (which do not materially reduce intact native disulfide
bonds) to facilitate conjugation at the desired free cysteine site.
Such agents (e.g., certain amino acids) are known to form salt
bridges (via hydrogen bonding and electrostatic interactions) and
may modulate protein-protein interactions in such a way as to
impart a stabilizing effect that may cause favorable conformation
changes and/or may reduce unfavorable protein-protein interactions.
Moreover, such agents may act to inhibit the formation of undesired
intramolecular (and intermolecular) cysteine-cysteine bonds after
reduction thus facilitating the desired conjugation reaction
wherein the engineered site-specific cysteine is bound to the drug
(preferably via a linker). Since the reaction conditions do not
provide for the significant reduction of intact native disulfide
bonds the conjugation reaction is naturally driven to the
relatively few reactive thiols on the free cysteines (e.g.,
preferably 2 free thiols). As previously alluded to this
considerably reduces the levels of non-specific conjugation and
corresponding impurities in conjugate preparations fabricated as
set forth herein.
[0255] In selected embodiments stabilizing agents compatible with
the present invention will generally comprise compounds with at
least one amine moiety having a basic pKa. In certain embodiments
the amine moiety will comprise a primary amine while in other
preferred embodiments the amine moiety will comprise a secondary
amine. In still other preferred embodiments the amine moiety will
comprise a tertiary amine. In other selected embodiments the amine
moiety will comprise an amino acid while in other compatible
embodiments the amine moiety will comprise an amino acid side
chain. In yet other embodiments the amine moiety will comprise a
proteinogenic amino acid. In still other embodiments the amine
moiety comprises a non-proteinogenic amino acid. In particularly
preferred embodiments, compatible stabilizing agents may comprise
arginine, lysine, proline and cysteine. In addition compatible
stabilizing agents may include guanidine and nitrogen containing
heterocycles with basic pKa.
[0256] In certain embodiments compatible stabilizing agents
comprise compounds with at least one amine moiety having a pKa of
greater than about 7.5, in other embodiments the subject amine
moiety will have a pKa of greater than about 8.0, in yet other
embodiments the amine moiety will have a pKa greater than about 8.5
and in still other embodiments the stabilizing agent will comprise
an amine moiety having a pKa of greater than about 9.0. Other
preferred embodiments will comprise stabilizing agents where the
amine moiety will have a pKa of greater than about 9.5 while
certain other embodiments will comprise stabilizing agents
exhibiting at least one amine moiety having a pKa of greater than
about 10.0. In still other preferred embodiments the stabilizing
agent will comprise a compound having the amine moiety with a pKa
of greater than about 10.5, in other embodiments the stabilizing
agent will comprise a compound having a amine moiety with a pKa
greater than about 11.0, while in still other embodiments the
stabilizing agent will comprise a amine moiety with a pKa greater
than about 11.5. In yet other embodiments the stabilizing agent
will comprise a compound having an amine moiety with a pKa greater
than about 12.0, while in still other embodiments the stabilizing
agent will comprise an amine moiety with a pKa greater than about
12.5. Those of skill in the art will understand that relevant pKa's
may readily be calculated or determined using standard techniques
and used to determine the applicability of using a selected
compound as a stabilizing agent.
[0257] The disclosed stabilizing agents are shown to be
particularly effective at targeting conjugation to free
site-specific cysteines when combined with certain reducing agents.
For the purposes of the instant invention, compatible reducing
agents may include any compound that produces a reduced free
site-specific cysteine for conjugation without significantly
disrupting the engineered antibody native disulfide bonds. Under
such conditions, provided by the combination of selected
stabilizing and reducing agents, the activated drug linker is
largely limited to binding to the desired free site-specific
cysteine site. Relatively mild reducing agents or reducing agents
used at relatively low concentrations to provide mild conditions
are particularly preferred. As used herein the terms "mild reducing
agent" or "mild reducing conditions" shall be held to mean any
agent or state brought about by a reducing agent (optionally in the
presence of stabilizing agents) that provides thiols at the free
cysteine site(s) without substantially disrupting native disulfide
bonds present in the engineered antibody. That is, mild reducing
agents or conditions are able to effectively reduce free
cysteine(s) (provide a thiol) without significantly disrupting the
protein's native disulfide bonds. The desired reducing conditions
may be provided by a number of sulfhydryl-based compounds that
establish the appropriate environment for selective conjugation. In
preferred embodiments mild reducing agents may comprise compounds
having one or more free thiols while in particularly preferred
embodiments mild reducing agents will comprise compounds having a
single free thiol. Non-limiting examples of reducing agents
compatible with the instant invention comprise glutathione,
n-acetyl cysteine, cysteine, 2-aminoethane-1-thiol and
2-hydroxyethane-1-thiol.
[0258] It will be appreciated that selective reduction process set
forth above is particularly effective at targeted conjugation to
the free cysteine. In this respect the extent of conjugation to the
desired target site (defined here as "conjugation efficiency") in
site-specific antibodies may be determined by various art-accepted
techniques. The efficiency of the site-specific conjugation of a
drug to an antibody may be determined by assessing the percentage
of conjugation on the target conjugation site (in this invention
the free cysteine on the c-terminus of the light chain) relative to
all other conjugated sites. In certain embodiments, the method
herein provides for efficiently conjugating a drug to an antibody
comprising free cysteines. In some embodiments, the conjugation
efficiency is at least 5%, at least 10%, at least 15%, at least
20%, at least 25%, at least 30%, at least 35%, at least 40%, at
least 45%, at least 50%, at least 55%, at least 60%, at least 70%,
at least 75%, at least 80%, at least 85%, at least 90%, at least
95%, at least 98% or more as measured by the percentage of target
conjugation relative to all other conjugation sites.
[0259] It will further be appreciated that engineered antibodies
capable of conjugation may contain free cysteine residues that
comprise sulfhydryl groups that are blocked or capped as the
antibody is produced or stored. Such caps include proteins,
peptides, ions and other materials that interact with the
sulfhydryl group and prevent or inhibit conjugate formation. In
some cases the unconjugated engineered antibody may comprise free
cysteines that bind other free cysteines on the same or different
antibodies. As discussed herein such cross-reactivity may lead to
various contaminants during the fabrication procedure. In some
embodiments, the engineered antibodies may require uncapping prior
to a conjugation reaction. In specific embodiments, antibodies
herein are uncapped and display a free sulfhydryl group capable of
conjugation. In specific embodiments, antibodies herein are
subjected to an uncapping reaction that does not disturb or
rearrange the naturally occurring disulfide bonds. It will be
appreciated that in most cases the uncapping reactions will occur
during the normal reduction reactions (reduction or selective
reduction).
[0260] F. DAR Distribution and Purification
[0261] One of the advantages of conjugation with site specific
antibodies of the present invention is the ability to generate
relatively homogeneous ADC preparations comprising a narrow DAR
distribution. In this regard the disclosed constructs and/or
selective conjugation provides for homogeneity of the ADC species
within a sample in terms of the stoichiometric ratio between the
drug and the engineered antibody. As briefly discussed above the
term "drug to antibody ratio" or "DAR" refers to the molar ratio of
drug to antibody. In some embodiments a conjugate preparation may
be substantially homogeneous with respect to its DAR distribution,
meaning that within the preparation is a predominant species of
site-specific ADC with a particular DAR (e.g., a DAR of 2 or 4)
that is also uniform with respect to the site of loading (i.e., on
the free cysteines). In certain embodiments of the invention it is
possible to achieve the desired homogeneity through the use of
site-specific antibodies or selective combination. In other
preferred embodiments the desired homogeneity may be achieved
through the use of site-specific constructs in combination with
selective reduction. In yet other particularly preferred
embodiments the preparations may be further purified using
analytical or preparative chromatography techniques. In each of
these embodiments the homogeneity of the ADC sample can be analyzed
using various techniques known in the art including but not limited
to SDS-PAGE, HPLC (e.g. size exclusion HPLC, RP-HPLC, HIC-HPLC
etc.) or capillary electrophoresis.
[0262] With regard to the purification of ADC preparations it will
be appreciated that standard pharmaceutical preparative methods may
be employed to obtain the desired purity. As discussed herein
liquid chromatography methods such as reverse phase (RP) and
hydrophobic interaction chromatography (HIC) may separate compounds
in the mixture by drug loading value. In some cases, mixed-mode
chromatography (MMC) may also be used to isolate species with a
specific drug load. More generally, once insoluble contaminants are
removed the antibody preparation may be further purified using
standard techniques such as, for example, hydroxylapatite
chromatography, gel electrophoresis, dialysis, and affinity
chromatography, with affinity chromatography of particular
interest. In this regard protein A can be used to purify antibodies
that are based on human IgG1, IgG2 or IgG4 heavy chains while
protein G is recommended for all mouse isotypes and for human IgG3.
Other techniques for protein purification such as fractionation on
an ion-exchange column, ethanol precipitation, chromatography on
silica, chromatography on heparin, sepharose chromatography on an
anion or cation exchange resin (such as a polyaspartic acid
column), chromatofocusing, SDS-PAGE and ammonium sulfate
precipitation are also available depending on the antibody or
conjugate to be recovered.
[0263] The disclosed ADCs and preparations thereof may comprise
drug and antibody moieties in various stoichiometric molar ratios
depending on the configuration of the antibody (e.g., an engineered
construct) and, at least in part, on the method used to effect
conjugation. In certain embodiments the drug loading per ADC may
comprise from 1-20 warheads (i.e., n is 1-20). Other selected
embodiments may comprise ADCs with a drug loading of from 1 to 15
warheads. In still other embodiments the ADCs may comprise from
1-12 warheads or, more preferably, from 1-10 warheads. In certain
preferred embodiments the ADCs will comprise from 1 to 8
warheads.
[0264] With regard to site-specific conjugates. depending on how
many and which interchain and intrachain disulfide bonds are
disrupted theoretical drug loading may be relatively high though
practical limitations such as free cysteine cross reactivity would
limit the generation of homogeneous preparations comprising such
DAR due to aggregates and other contaminants. That is, higher drug
loading, e.g. >6, may cause aggregation, insolubility, toxicity,
or loss of cellular permeability of certain antibody-drug
conjugates. In view of such concerns practical drug loading
provided by the instant invention preferably ranges from 1 to 8
drugs per conjugate, i.e. where 1, 2, 3, 4, 5, 6, 7, or 8 drugs are
covalently attached to each antibody (e.g., for IgG1, other
antibodies may have different loading capacity depending the number
of disulfide bonds). Preferably the DAR of compositions of the
instant invention will be approximately 2, 4 or 6 and in
particularly preferred embodiments the DAR will comprise
approximately 2.
[0265] Despite the relatively high level of homogeneity provided by
the instant invention the disclosed compositions actually comprise
a mixture of conjugates with a range of drug loads (e.g., from 1 to
8 drugs per IgG1 antibody) at various concentrations (along with
certain reaction contaminants primarily caused by free cysteine
cross reactivity); and which comprise drug moieties that are
attached to the antibody by various thiol groups. Using selective
reduction and post-fabrication purification the conjugate
compositions may be driven to the point where they largely contain
a single predominant desired ADC species (e.g., with a drug loading
of 2) with relatively low levels of other ADC species (e.g., with a
drug loading of 1, 4, 6, etc.). The average DAR value represents
the weighted average of drug loading for the composition as a whole
(i.e., all the ADC species taken together). Due to inherent
uncertainty in the quantification methodology employed and the
difficulty in completely removing the non-predominant ADC species
in a commercial setting, acceptable DAR values or specifications
are often presented as an average, a range or distribution (i.e.,
an average DAR of 2+/-0.5). Preferably compositions comprising a
measured average DAR within the range (i.e., 1.5 to 2.5) would be
used in a pharmaceutical setting.
[0266] Thus, in certain preferred embodiments the present invention
will comprise compositions having an average DAR of 1, 2, 3, 4, 5,
6, 7 or 8 each +/-0.5. In other preferred embodiments the present
invention will comprise an average DAR of 2, 4, 6 or 8+/-0.5.
Finally, in selected preferred embodiments the present invention
will comprise an average DAR of 2+/-0.5. It will be appreciated
that the range or deviation may be less than 0.4 in certain
preferred embodiments. Thus, in other embodiments the compositions
will comprise an average DAR of 1, 2, 3, 4, 5, 6, 7 or 8 each
+/-0.3, an average DAR of 2, 4, 6 or 8+/-0.3, even more preferably
an average DAR of 2 or 4+/-0.3 or even an average DAR of 2+/-0.3.
In other embodiments IgG1 conjugate compositions will preferably
comprise a composition with an average DAR of 1, 2, 3, 4, 5, 6, 7
or 8 each +/-0.4 and relatively low levels (i.e., less than 30%) of
non-predominant ADC species. In other preferred embodiments the ADC
composition will comprise an average DAR of 2, 4, 6 or 8 each
+/-0.4 with relatively low levels (<30%) of non-predominant ADC
species. In particularly preferred embodiments the ADC composition
will comprise an average DAR of 2+/-0.4 with relatively low levels
(<30%) of non-predominant ADC species. In yet other embodiments
the predominant ADC species (e.g., DAR of 2) will be present at a
concentration of greater than 70%, a concentration of greater than
75%, a concentration of greater that 80%, a concentration of
greater than 85%, a concentration of greater than 90%, a
concentration of greater than 93%, a concentration of greater than
95% or even a concentration of greater than 97% when measured
against other DAR species.
[0267] As detailed in the Examples below the distribution of drugs
per antibody in preparations of ADC from conjugation reactions may
be characterized by conventional means such as UV-Vis
spectrophotometry, reverse phase HPLC, HIC, mass spectroscopy,
ELISA, and electrophoresis. The quantitative distribution of ADC in
terms of drugs per antibody may also be determined. By ELISA, the
averaged value of the drugs per antibody in a particular
preparation of ADC may be determined. However, the distribution of
drug per antibody values is not discernible by the antibody-antigen
binding and detection limitation of ELISA. Also, ELISA assay for
detection of antibody-drug conjugates does not determine where the
drug moieties are attached to the antibody, such as the heavy chain
or light chain fragments, or the particular amino acid
residues.
VI DIAGNOSTICS AND SCREENING
[0268] A. Diagnostics
[0269] The invention provides in vitro and in vivo methods for
detecting, diagnosing or monitoring proliferative disorders and
methods of screening cells from a patient to identify tumor cells
including tumorigenic cells. Such methods include identifying an
individual having cancer for treatment or monitoring progression of
a cancer, comprising contacting the patient or a sample obtained
from a patient (either in vivo or in vitro) with an antibody as
described herein and detecting presence or absence, or level of
association, of the antibody to bound or free target molecules in
the sample. In some embodiments the antibody will comprise a
detectable label or reporter molecule as described herein.
[0270] In some embodiments, the association of the antibody with
particular cells in the sample can denote that the sample may
contain tumorigenic cells, thereby indicating that the individual
having cancer may be effectively treated with an antibody as
described herein.
[0271] Samples can be analyzed by numerous assays, for example,
radioimmunoassays, enzyme immunoassays (e.g. ELISA),
competitive-binding assays, fluorescent immunoassays, immunoblot
assays, Western Blot analysis and flow cytometry assays. Compatible
in vivo theragnostic or diagnostic assays can comprise art
recognized imaging or monitoring techniques, for example, magnetic
resonance imaging, computerized tomography (e.g. CAT scan),
positron tomography (e.g., PET scan), radiography, ultrasound,
etc.
[0272] In a particularly preferred embodiment the antibodies of the
instant invention may be used to detect and quantify levels of a
particular determinant (e.g., CLDN) in a patient sample (e.g.,
plasma or blood) which may, in turn, be used to detect, diagnose or
monitor proliferative disorders that are associated with the
relevant determinant. In related embodiments the antibodies of the
instant invention may be used to detect, monitor and/or quantify
circulating tumor cells either in vivo or in vitro (WO
2012/0128801). In still other embodiments the circulating tumor
cells may comprise tumorigenic cells.
[0273] In certain embodiments of the invention, the tumorigenic
cells in a subject or a sample from a subject may be assessed or
characterized using the disclosed antibodies prior to therapy or
regimen to establish a baseline. In other examples, the tumorigenic
cells can be assessed from a sample that is derived from a subject
that was treated.
[0274] B. Screening
[0275] In certain embodiments, the antibodies can be used to screen
samples in order to identify compounds or agents (e.g., antibodies
or ADCs) that alter a function or activity of tumor cells by
interacting with a determinant. In one embodiment, tumor cells are
put in contact with an antibody or ADC and the antibody or ADC can
be used to screen the tumor for cells expressing a certain target
(e.g. CLDN) in order to identify such cells for purposes, including
but not limited to, diagnostic purposes, to monitor such cells to
determine treatment efficacy or to enrich a cell population for
such target-expressing cells.
[0276] In yet another embodiment, a method includes contacting,
directly or indirectly, tumor cells with a test agent or compound
and determining if the test agent or compound modulates an activity
or function of the determinant-associated tumor cells for example,
changes in cell morphology or viability, expression of a marker,
differentiation or de-differentiation, cell respiration,
mitochondrial activity, membrane integrity, maturation,
proliferation, viability, apoptosis or cell death. One example of a
direct interaction is physical interaction, while an indirect
interaction includes, for example, the action of a composition upon
an intermediary molecule that, in turn, acts upon the referenced
entity (e.g., cell or cell culture).
[0277] Screening methods include high throughput screening, which
can include arrays of cells (e.g., microarrays) positioned or
placed, optionally at pre-determined locations, for example, on a
culture dish, tube, flask, roller bottle or plate. High-throughput
robotic or manual handling methods can probe chemical interactions
and determine levels of expression of many genes in a short period
of time. Techniques have been developed that utilize molecular
signals, for example via fluorophores or microarrays (Mocellin and
Rossi, 2007, PMID: 17265713) and automated analyses that process
information at a very rapid rate (see, e.g., Pinhasov et al., 2004,
PMID: 15032660). Libraries that can be screened include, for
example, small molecule libraries, phage display libraries, fully
human antibody yeast display libraries (Adimab), siRNA libraries,
and adenoviral transfection vectors.
VII PHARMACEUTICAL PREPARATIONS AND THERAPEUTIC USES
[0278] A. Formulations and Routes of Administration
[0279] The antibodies or ADCs of the invention can be formulated in
various ways using art recognized techniques. In some embodiments,
the therapeutic compositions of the invention can be administered
neat or with a minimum of additional components while others may
optionally be formulated to contain suitable pharmaceutically
acceptable carriers. As used herein, "pharmaceutically acceptable
carriers" comprise excipients, vehicles, adjuvants and diluents
that are well known in the art and can be available from commercial
sources for use in pharmaceutical preparation (see, e.g., Gennaro
(2003) Remington: The Science and Practice of Pharmacy with Facts
and Comparisons: Drugfacts Plus, 20th ed., Mack Publishing; Ansel
et al. (2004) Pharmaceutical Dosage Forms and Drug Delivery
Systems, 7.sup.th ed., Lippencott Williams and Wilkins; Kibbe et
al. (2000) Handbook of Pharmaceutical Excipients, 3.sup.rd ed.,
Pharmaceutical Press.)
[0280] Suitable pharmaceutically acceptable carriers comprise
substances that are relatively inert and can facilitate
administration of the antibody or can aid processing of the active
compounds into preparations that are pharmaceutically optimized for
delivery to the site of action.
[0281] Such pharmaceutically acceptable carriers include agents
that can alter the form, consistency, viscosity, pH, tonicity,
stability, osmolarity, pharmacokinetics, protein aggregation or
solubility of the formulation and include buffering agents, wetting
agents, emulsifying agents, diluents, encapsulating agents and skin
penetration enhancers. Certain non-limiting examples of carriers
include saline, buffered saline, dextrose, arginine, sucrose,
water, glycerol, ethanol, sorbitol, dextran, sodium carboxymethyl
cellulose and combinations thereof. Antibodies for systemic
administration may be formulated for enteral, parenteral or topical
administration. Indeed, all three types of formulation may be used
simultaneously to achieve systemic administration of the active
ingredient. Excipients as well as formulations for parenteral and
nonparenteral drug delivery are set forth in Remington: The Science
and Practice of Pharmacy (2000) 20th Ed. Mack Publishing.
[0282] Suitable formulations for enteral administration include
hard or soft gelatin capsules, pills, tablets, including coated
tablets, elixirs, suspensions, syrups or inhalations and controlled
release forms thereof.
[0283] Formulations suitable for parenteral administration (e.g.,
by injection), include aqueous or non-aqueous, isotonic,
pyrogen-free, sterile liquids (e.g., solutions, suspensions), in
which the active ingredient is dissolved, suspended, or otherwise
provided (e.g., in a liposome or other microparticulate). Such
liquids may additionally contain other pharmaceutically acceptable
carriers, such as anti-oxidants, buffers, preservatives,
stabilizers, bacteriostats, suspending agents, thickening agents,
and solutes that render the formulation isotonic with the blood (or
other relevant bodily fluid) of the intended recipient. Examples of
excipients include, for example, water, alcohols, polyols,
glycerol, vegetable oils, and the like. Examples of suitable
isotonic pharmaceutically acceptable carriers for use in such
formulations include Sodium Chloride Injection, Ringer's Solution,
or Lactated Ringer's Injection.
[0284] Compatible formulations for parenteral administration (e.g.,
intravenous injection) may comprise ADC or antibody concentrations
of from about 10 .mu.g/mL to about 100 mg/mL. In certain selected
embodiments antibody or ADC concentrations will comprise 20
.mu.g/mL, 40 .mu.g/mL, 60 .mu.g/mL, 80 .mu.g/mL, 100 .mu.g/mL, 200
.mu.g/mL, 300, .mu.g/mL, 400 .mu.g/mL, 500 .mu.g/mL, 600 .mu.g/mL,
700 .mu.g/mL, 800 .mu.g/mL, 900 .mu.g/mL or 1 mg/mL. In other
preferred embodiments ADC concentrations will comprise 2 mg/mL, 3
mg/mL, 4 mg/mL, 5 mg/mL, 6 mg/mL, 8 mg/mL, 10 mg/mL, 12 mg/mL, 14
mg/mL, 16 mg/mL, 18 mg/mL, 20 mg/mL, 25 mg/mL, 30 mg/mL, 35 mg/mL,
40 mg/mL, 45 mg/mL, 50 mg/mL, 60 mg/mL, 70 mg/mL, 80 mg/mL, 90
mg/mL or 100 mg/mL.
[0285] The compounds and compositions of the invention may be
administered in vivo, to a subject in need thereof, by various
routes, including, but not limited to, oral, intravenous,
intra-arterial, subcutaneous, parenteral, intranasal,
intramuscular, intracardiac, intraventricular, intratracheal,
buccal, rectal, intraperitoneal, intradermal, topical, transdermal,
and intrathecal, or otherwise by implantation or inhalation. The
subject compositions may be formulated into preparations in solid,
semi-solid, liquid, or gaseous forms; including, but not limited
to, tablets, capsules, powders, granules, ointments, solutions,
suppositories, enemas, injections, inhalants, and aerosols. The
appropriate formulation and route of administration may be selected
according to the intended application and therapeutic regimen.
[0286] B. Dosages
[0287] The particular dosage regimen, i.e., dose, timing and
repetition, will depend on the particular individual, as well as
empirical considerations such as pharmacokinetics (e.g., half-life,
clearance rate, etc.). Determination of the frequency of
administration may be made by persons skilled in the art, such as
an attending physician based on considerations of the condition and
severity of the condition being treated, age and general state of
health of the subject being treated and the like. Frequency of
administration may be adjusted over the course of therapy based on
assessment of the efficacy of the selected composition and the
dosing regimen. Such assessment can be made on the basis of markers
of the specific disease, disorder or condition. In embodiments
where the individual has cancer, these include direct measurements
of tumor size via palpation or visual observation; indirect
measurement of tumor size by x-ray or other imaging techniques; an
improvement as assessed by direct tumor biopsy and microscopic
examination of a tumor sample; the measurement of an indirect tumor
marker (e.g., PSA for prostate cancer) or an antigen identified
according to the methods described herein; reduction in the number
of proliferative or tumorigenic cells, maintenance of the reduction
of such neoplastic cells; reduction of the proliferation of
neoplastic cells; or delay in the development of metastasis.
[0288] The CLDN antibodies or ADCs of the invention may be
administered in various ranges. These include about 5 .mu.g/kg body
weight to about 100 mg/kg body weight per dose; about 50 .mu.g/kg
body weight to about 5 mg/kg body weight per dose; about 100
.mu.g/kg body weight to about 10 mg/kg body weight per dose. Other
ranges include about 100 .mu.g/kg body weight to about 20 mg/kg
body weight per dose and about 0.5 mg/kg body weight to about 20
mg/kg body weight per dose. In certain embodiments, the dosage is
at least about 100 .mu.g/kg body weight, at least about 250
.mu.g/kg body weight, at least about 750 .mu.g/kg body weight, at
least about 3 mg/kg body weight, at least about 5 mg/kg body
weight, at least about 10 mg/kg body weight.
[0289] In selected embodiments the CLDN antibodies or ADCs will be
administered (preferably intravenously) at approximately 10, 20,
30, 40, 50, 60, 70, 80, 90 or 100 .mu.g/kg body weight per dose.
Other embodiments may comprise the administration of ADCs at about
200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300,
1400, 1500, 1600, 1700, 1800, 1900 or 2000 .mu.g/kg body weight per
dose. In other preferred embodiments the disclosed conjugates will
be administered at 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.58, 9
or 10 mg/kg. In still other embodiments the conjugates may be
administered at 12, 14, 16, 18 or 20 mg/kg body weight per dose. In
yet other embodiments the conjugates may be administered at 25, 30,
35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90 or 100 mg/kg body weight
per dose. With the teachings herein one of skill in the art could
readily determine appropriate dosages for various CLDN antibodies
or ADCs based on preclinical animal studies, clinical observations
and standard medical and biochemical techniques and
measurements.
[0290] Other dosing regimens may be predicated on Body Surface Area
(BSA) calculations as disclosed in U.S. Pat. No. 7,744,877. As is
well known, the BSA is calculated using the patient's height and
weight and provides a measure of a subject's size as represented by
the surface area of his or her body. In certain embodiments, the
conjugates may be administered in dosages from 1 mg/m.sup.2 to 800
mg/m.sup.2, from 50 mg/m.sup.2 to 500 mg/m.sup.2 and at dosages of
100 mg/m.sup.2, 150 mg/m.sup.2, 200 mg/m.sup.2, 250 mg/m.sup.2, 300
mg/m.sup.2, 350 mg/m.sup.2, 400 mg/m.sup.2 or 450 mg/m.sup.2. It
will also be appreciated that art recognized and empirical
techniques may be used to determine appropriate dosage.
[0291] Anti-CLDN antibodies or ADCs may be administered on a
specific schedule. Generally, an effective dose of the CLDN
conjugate is administered to a subject one or more times. More
particularly, an effective dose of the ADC is administered to the
subject once a month, more than once a month, or less than once a
month. In certain embodiments, the effective dose of the CLDN
antibody or ADC may be administered multiple times, including for
periods of at least a month, at least six months, at least a year,
at least two years or a period of several years. In yet other
embodiments, several days (2, 3, 4, 5, 6 or 7), several weeks (1,
2, 3, 4, 5, 6, 7 or 8) or several months (1, 2, 3, 4, 5, 6, 7 or 8)
or even a year or several years may lapse between administration of
the disclosed antibodies or ADCs.
[0292] In certain preferred embodiments the course of treatment
involving conjugated antibodies will comprise multiple doses of the
selected drug product over a period of weeks or months. More
specifically, antibodies or ADCs of the instant invention may
administered once every day, every two days, every four days, every
week, every ten days, every two weeks, every three weeks, every
month, every six weeks, every two months, every ten weeks or every
three months. In this regard it will be appreciated that the
dosages may be altered or the interval may be adjusted based on
patient response and clinical practices.
[0293] Dosages and regimens may also be determined empirically for
the disclosed therapeutic compositions in individuals who have been
given one or more administration(s). For example, individuals may
be given incremental dosages of a therapeutic composition produced
as described herein. In selected embodiments the dosage may be
gradually increased or reduced or attenuated based respectively on
empirically determined or observed side effects or toxicity. To
assess efficacy of the selected composition, a marker of the
specific disease, disorder or condition can be followed as
described previously. For cancer, these include direct measurements
of tumor size via palpation or visual observation, indirect
measurement of tumor size by x-ray or other imaging techniques; an
improvement as assessed by direct tumor biopsy and microscopic
examination of the tumor sample; the measurement of an indirect
tumor marker (e.g., PSA for prostate cancer) or a tumorigenic
antigen identified according to the methods described herein, a
decrease in pain or paralysis; improved speech, vision, breathing
or other disability associated with the tumor; increased appetite;
or an increase in quality of life as measured by accepted tests or
prolongation of survival. It will be apparent to one of skill in
the art that the dosage will vary depending on the individual, the
type of neoplastic condition, the stage of neoplastic condition,
whether the neoplastic condition has begun to metastasize to other
location in the individual, and the past and concurrent treatments
being used.
[0294] C. Combination Therapies
[0295] The CLDN proteins are expressed in the tight junctions of
epithelial cells where they are thought to establish the
paracellular barrier that controls the flow of molecules in the
intercellular space between epithelial cells. The use of an
anti-CLDN antibodies may result in the disruption of the tight
junctions of epithelial cells and thus improve access of
therapeutics that otherwise would not be able to penetrate cancer
cells. Thus, the use of various therapies in combination with the
anti-CLDN antibodies and ADCs of the invention may be useful in
preventing or treating cancer and in preventing metastasis or
recurrence of cancer. "Combination therapy", as used herein, means
the administration of a combination comprising at least one
anti-CLDN antibody or ADC and at least one therapeutic moiety
(e.g., anti-cancer agent) wherein the combination preferably has
therapeutic synergy or improves the measurable therapeutic effects
in the treatment of cancer over (i) the anti-CLDN antibody or ADC
used alone, or (ii) the therapeutic moiety used alone, or (iii) the
use of the therapeutic moiety in combination with another
therapeutic moiety without the addition of an anti-CLDN antibody or
ADC. The term "therapeutic synergy", as used herein, means the
combination of an anti-CLDN antibody or ADC and one or more
therapeutic moiety(ies) having a therapeutic effect greater than
the additive effect of the combination of the anti-CLDN antibody or
ADC and the one or more therapeutic moiety(ies).
[0296] Desired outcomes of the disclosed combinations are
quantified by comparison to a control or baseline measurement. As
used herein, relative terms such as "improve," "increase," or
"reduce" indicate values relative to a control, such as a
measurement in the same individual prior to initiation of treatment
described herein, or a measurement in a control individual (or
multiple control individuals) in the absence of the anti-CLDN
antibodies or ADCs described herein but in the presence of other
therapeutic moiety(ies) such as standard of care treatment. A
representative control individual is an individual afflicted with
the same form of cancer as the individual being treated, who is
about the same age as the individual being treated (to ensure that
the stages of the disease in the treated individual and the control
individual are comparable.)
[0297] Changes or improvements in response to therapy are generally
statistically significant. As used herein, the term "significance"
or "significant" relates to a statistical analysis of the
probability that there is a non-random association between two or
more entities. To determine whether or not a relationship is
"significant" or has "significance," a "p-value" can be calculated.
P-values that fall below a user-defined cut-off point are regarded
as significant. A p-value less than or equal to 0.1, less than
0.05, less than 0.01, less than 0.005, or less than 0.001 may be
regarded as significant.
[0298] A synergistic therapeutic effect may be an effect of at
least about two-fold greater than the therapeutic effect elicited
by a single therapeutic moiety or anti-CLDN antibody or ADC, or the
sum of the therapeutic effects elicited by the anti-CLDN antibody
or ADC or the single therapeutic moiety(ies) of a given
combination, or at least about five-fold greater, or at least about
ten-fold greater, or at least about twenty-fold greater, or at
least about fifty-fold greater, or at least about one hundred-fold
greater. A synergistic therapeutic effect may also be observed as
an increase in therapeutic effect of at least 10% compared to the
therapeutic effect elicited by a single therapeutic moiety or
anti-CLDN antibody or ADC, or the sum of the therapeutic effects
elicited by the anti-CLDN antibody or ADC or the single therapeutic
moiety(ies) of a given combination, or at least 20%, or at least
30%, or at least 40%, or at least 50%, or at least 60%, or at least
70%, or at least 80%, or at least 90%, or at least 100%, or more. A
synergistic effect is also an effect that permits reduced dosing of
therapeutic agents when they are used in combination.
[0299] In practicing combination therapy, the anti-CLDN antibody or
ADC and therapeutic moiety(ies) may be administered to the subject
simultaneously, either in a single composition, or as two or more
distinct compositions using the same or different administration
routes. Alternatively, treatment with the anti-CLDN antibody or ADC
may precede or follow the therapeutic moiety treatment by, e.g.,
intervals ranging from minutes to weeks. In one embodiment, both
the therapeutic moiety and the antibody or ADC are administered
within about 5 minutes to about two weeks of each other. In yet
other embodiments, several days (2, 3, 4, 5, 6 or 7), several weeks
(1, 2, 3, 4, 5, 6, 7 or 8) or several months (1, 2, 3, 4, 5, 6, 7
or 8) may lapse between administration of the antibody and the
therapeutic moiety.
[0300] The combination therapy can be administered until the
condition is treated, palliated or cured on various schedules such
as once, twice or three times daily, once every two days, once
every three days, once weekly, once every two weeks, once every
month, once every two months, once every three months, once every
six months, or may be administered continuously. The antibody and
therapeutic moiety(ies) may be administered on alternate days or
weeks; or a sequence of anti-CLDN antibody or ADC treatments may be
given, followed by one or more treatments with the additional
therapeutic moiety. In one embodiment an anti-CLDN antibody or ADC
is administered in combination with one or more therapeutic
moiety(ies) for short treatment cycles. In other embodiments the
combination treatment is administered for long treatment cycles.
The combination therapy can be administered via any route.
[0301] In some embodiments the anti-CLDN antibodies or ADCs may be
used in combination with various first line cancer treatments. In
one embodiment the combination therapy comprises the use of an
anti-CLDN antibody or ADC and a platinum analog (e.g. ifosfamide,
mytomycin C, vindesine, vinblastine, etoposide, ironitecan,
gemcitabine, taxanes, vinorelbine, methotrexate, and pemetrexed)
and optionally one or more other therapeutic moiety(ies).
[0302] In another embodiment the combination therapy comprises the
use of an anti-CLDN antibody or ADC and a platinum-based drug (e.g.
carboplatin or cisplatin) and optionally one or more other
therapeutic moiety(ies) (e.g. vinorelbine; gemcitabine; a taxane
such as, for example, docetaxel or paclitaxel; irinotican; or
pemetrexed).
[0303] In another embodiment combination therapy for the treatment
of EGFR-positive NSCLC comprises the use of an anti-CLDN antibody
or ADC and afatinib and optionally one or more other therapeutic
moiety(ies) (e.g. erlotinib and/or bevacizumab).
[0304] In another embodiment combination therapy for the treatment
of EGFR-positive NSCLC comprises the use of an anti-CLDN antibody
or ADC and erlotinib and optionally one or more other therapeutic
moiety(ies) (e.g. bevacizumab).
[0305] In another embodiment combination therapy for the treatment
of ALK-positive NSCLC comprises the use of an anti-CLDN antibody or
ADC and ceritinib and optionally one or more other therapeutic
moiety(ies).
[0306] In another embodiment combination therapy for the treatment
of ALK-positive NSCLC comprises the use of an anti-CLDN antibody or
ADC and crizotinib and optionally one or more other therapeutic
moiety(ies).
[0307] In another embodiment the combination therapy comprises the
use of an anti-CLDN antibody or ADC and bevacizumab and optionally
one or more other therapeutic moiety(ies) (e.g. a taxane such as,
for example, docetaxel or paclitaxel; and/or a platinum
analog).
[0308] In another embodiment the combination therapy comprises the
use of an anti-CLDN antibody or ADC and bevacizumab and optionally
one or more other therapeutic moiety(ies) (e.g. gemcitabine and/or
a platinum analog).
[0309] In one embodiment the combination therapy comprises the use
of an anti-CLDN antibody or ADC and platinum-based drug (e.g.
carboplatin or cisplatin) analog and optionally one or more other
therapeutic moiety(ies) (e.g. a taxane such as, for example,
docetaxel and paclitaxel).
[0310] In one embodiment the combination therapy comprises the use
of an anti-CLDN antibody or ADC and platinum-based drug (e.g.
carboplatin or cisplatin) analog and optionally one or more other
therapeutic moiety(ies) (e.g. a taxane such, for example, docetaxel
and paclitaxel and/or gemcitabine and/or doxorubicin).
[0311] In a particular embodiment the combination therapy for the
treatment of platinum-resistant tumors comprises the use of an
anti-CLDN antibody or ADC and doxorubicin and/or etoposide and/or
gemcitabine and/or vinorelbine and/or ifosfamide and/or
leucovorin-modulated 5-fluoroucil and/or bevacizumab and/or
tamoxifen; and optionally one or more other therapeutic
moiety(ies).
[0312] In another embodiment the combination therapy comprises the
use of an anti-CLDN antibody or ADC and a PARP inhibitor and
optionally one or more other therapeutic moiety(ies).
[0313] In another embodiment the combination therapy comprises the
use of an anti-CLDN antibody or ADC and bevacizumab and optionally
cyclophosphamide.
[0314] The invention also provides for the combination of anti-CLDN
antibodies or ADCs with radiotherapy. The term "radiotherapy", as
used herein, means, any mechanism for inducing DNA damage locally
within tumor cells such as gamma-irradiation, X-rays,
UV-irradiation, microwaves, electronic emissions and the like.
Combination therapy using the directed delivery of radioisotopes to
tumor cells is also contemplated, and may be used in combination or
as a conjugate of the anti-CLDN antibodies disclosed herein.
Typically, radiation therapy is administered in pulses over a
period of time from about 1 to about 2 weeks. Optionally, the
radiation therapy may be administered as a single dose or as
multiple, sequential doses.
[0315] In other embodiments an anti-CLDN antibody or ADC may be
used in combination with one or more of the anti-cancer agents
described below.
[0316] D. Anti-Cancer Agents
[0317] The term "anti-cancer agent" or "chemotherapeutic agent" as
used herein is one subset of "therapeutic moieties", which in turn
is a subset of the agents described as "pharmaceutically active
moieties". More particularly "anti-cancer agent" means any agent
that can be used to treat a cell proliferative disorder such as
cancer, and includes, but is not limited to, cytotoxic agents,
cytostatic agents, anti-angiogenic agents, debulking agents,
chemotherapeutic agents, radiotherapy and radiotherapeutic agents,
targeted anti-cancer agents, biological response modifiers,
therapeutic antibodies, cancer vaccines, cytokines, hormone
therapy, anti-metastatic agents and immunotherapeutic agents. It
will be appreciated that in selected embodiments as discussed
above, such anti-cancer agents may comprise conjugates and may be
associated with antibodies prior to administration. In certain
embodiments the disclosed anti-cancer agent will be linked to an
antibody to provide an ADC as disclosed herein.
[0318] The term "cytotoxic agent", which can also be an anti-cancer
agent means a substance that is toxic to the cells and decreases or
inhibits the function of cells and/or causes destruction of cells.
Typically, the substance is a naturally occurring molecule derived
from a living organism (or a synthetically prepared natural
product). Examples of cytotoxic agents include, but are not limited
to, small molecule toxins or enzymatically active toxins of
bacteria (e.g., Diptheria toxin, Pseudomonas endotoxin and
exotoxin, Staphylococcal enterotoxin A), fungal (e.g.,
.alpha.-sarcin, restrictocin), plants (e.g., abrin, ricin,
modeccin, viscumin, pokeweed anti-viral protein, saporin, gelonin,
momoridin, trichosanthin, barley toxin, Aleurites fordii proteins,
dianthin proteins, Phytolacca mericana proteins (PAPI, PAPII, and
PAP-S), Momordica charantia inhibitor, curcin, crotin, saponaria
officinalis inhibitor, mitegellin, restrictocin, phenomycin,
neomycin, and the tricothecenes) or animals, (e.g., cytotoxic
RNases, such as extracellular pancreatic RNases; DNase I, including
fragments and/or variants thereof).
[0319] An anti-cancer agent can include any chemical agent that
inhibits, or is designed to inhibit, a cancerous cell or a cell
likely to become cancerous or generate tumorigenic progeny (e.g.,
tumorigenic cells). Such chemical agents are often directed to
intracellular processes necessary for cell growth or division, and
are thus particularly effective against cancerous cells, which
generally grow and divide rapidly. For example, vincristine
depolymerizes microtubules, and thus inhibits cells from entering
mitosis. Such agents are often administered, and are often most
effective, in combination, e.g., in the formulation CHOP. Again, in
selected embodiments such anti-cancer agents may be conjugated to
the disclosed antibodies.
[0320] Examples of anti-cancer agents that may be used in
combination with (or conjugated to) the antibodies of the invention
include, but are not limited to, alkylating agents, alkyl
sulfonates, anastrozole, amanitins, aziridines, ethylenimines and
methylamelamines, acetogenins, a camptothecin, BEZ-235, bortezomib,
bryostatin, callystatin, CC-1065, ceritinib, crizotinib,
cryptophycins, dolastatin, duocarmycin, eleutherobin, erlotinib,
pancratistatin, a sarcodictyin, spongistatin, nitrogen mustards,
antibiotics, enediyne dynemicin, bisphosphonates, esperamicin,
chromoprotein enediyne antiobiotic chromophores, aclacinomysins,
actinomycin, authramycin, azaserine, bleomycins, cactinomycin,
canfosfamide, carabicin, carminomycin, carzinophilin,
chromomycinis, cyclosphosphamide, dactinomycin, daunorubicin,
detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin,
esorubicin, exemestane, fluorouracil, fulvestrant, gefitinib,
idarubicin, lapatinib, letrozole, lonafarnib, marcellomycin,
megestrol acetate, mitomycins, mycophenolic acid, nogalamycin,
olivomycins, pazopanib, peplomycin, potfiromycin, puromycin,
quelamycin, rapamycin, rodorubicin, sorafenib, streptonigrin,
streptozocin, tamoxifen, tamoxifen citrate, temozolomide, tepodina,
tipifarnib, tubercidin, ubenimex, vandetanib, vorozole, XL-147,
zinostatin, zorubicin; anti-metabolites, folic acid analogues,
purine analogs, androgens, anti-adrenals, folic acid replenisher
such as frolinic acid, aceglatone, aldophosphamide glycoside,
aminolevulinic acid, eniluracil, amsacrine, bestrabucil,
bisantrene, edatraxate, defofamine, demecolcine, diaziquone,
elfornithine, elliptinium acetate, epothilone, etoglucid, gallium
nitrate, hydroxyurea, lentinan, lonidainine, maytansinoids,
mitoguazone, mitoxantrone, mopidanmol, nitraerine, pentostatin,
phenamet, pirarubicin, losoxantrone, podophyllinic acid,
2-ethylhydrazide, procarbazine, polysaccharide complex, razoxane;
rhizoxin; SF-1126, sizofiran; spirogermanium; tenuazonic acid;
triaziquone; 2,2',2''-trichlorotriethylamine; trichothecenes (T-2
toxin, verracurin A, roridin A and anguidine); urethan; vindesine;
dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;
gacytosine; arabinoside; cyclophosphamide; thiotepa; taxoids,
chloranbucil; gemcitabine; 6-thioguanine; mercaptopurine;
methotrexate; platinum analogs, vinblastine; platinum; etoposide;
ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone;
teniposide; edatrexate; daunomycin; aminopterin; xeloda;
ibandronate; irinotecan, topoisomerase inhibitor RFS 2000;
difluorometlhylornithine; retinoids; capecitabine; combretastatin;
leucovorin; oxaliplatin; XL518, inhibitors of PKC-alpha, Raf,
H-Ras, EGFR and VEGF-A that reduce cell proliferation and
pharmaceutically acceptable salts or solvates, acids or derivatives
of any of the above. Also included in this definition are
anti-hormonal agents that act to regulate or inhibit hormone action
on tumors such as anti-estrogens and selective estrogen receptor
antibodies, aromatase inhibitors that inhibit the enzyme aromatase,
which regulates estrogen production in the adrenal glands, and
anti-androgens; as well as troxacitabine (a 1,3-dioxolane
nucleoside cytosine analog); antisense oligonucleotides, ribozymes
such as a VEGF expression inhibitor and a HER2 expression
inhibitor; vaccines, PROLEUKIN.RTM. rIL-2; LURTOTECAN.RTM.
topoisomerase 1 inhibitor; ABARELIX.RTM. rmRH; Vinorelbine and
Esperamicins and pharmaceutically acceptable salts or solvates,
acids or derivatives of any of the above.
[0321] Particularly preferred anti-cancer agents comprise
commercially or clinically available compounds such as erlotinib
(TARCEVA.RTM., Genentech/OSI Pharm.), docetaxel (TAXOTERE.RTM.,
Sanofi-Aventis), 5-FU (fluorouracil, 5-fluorouracil, CAS No.
51-21-8), gemcitabine (GEMZAR.RTM., Lilly), PD-0325901 (CAS No.
391210-10-9, Pfizer), cisplatin (cis-diamine, dichloroplatinum(II),
CAS No. 15663-27-1), carboplatin (CAS No. 41575-94-4), paclitaxel
(TAXOL.RTM., Bristol-Myers Squibb Oncology, Princeton, N.J.),
trastuzumab (HERCEPTIN.RTM., Genentech), temozolomide
(4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo [4.3.0]
nona-2,7,9-triene-9-carboxamide, CAS No. 85622-93-1, TEMODAR.RTM.,
TEMODAL.RTM., Schering Plough), tamoxifen
((Z)-2-[4-(1,2-diphenylbut-1-enyl)phenoxy]-N,N-dimethylethanamine,
NOLVADEX.RTM., ISTUBAL.RTM., VALODEX.RTM.), and doxorubicin
(ADRIAMYCIN.RTM.). Additional commercially or clinically available
anti-cancer agents comprise oxaliplatin (ELOXATIN.RTM., Sanofi),
bortezomib (VELCADE.RTM., Millennium Pharm.), sutent
(SUNITINIB.RTM., SU11248, Pfizer), letrozole (FEMARA.RTM.,
Novartis), imatinib mesylate (GLEEVEC.RTM., Novartis), XL-518 (Mek
inhibitor, Exelixis, WO 2007/044515), ARRY-886 (Mek inhibitor,
AZD6244, Array BioPharma, Astra Zeneca), SF-1126 (PI3K inhibitor,
Semafore Pharmaceuticals), BEZ-235 (PI3K inhibitor, Novartis),
XL-147 (PI3K inhibitor, Exelixis), PTK787/ZK 222584 (Novartis),
fulvestrant (FASLODEX.RTM., AstraZeneca), leucovorin (folinic
acid), rapamycin (sirolimus, RAPAMUNE.RTM., Wyeth), lapatinib
(TYKERB.RTM., GSK572016, Glaxo Smith Kline), lonafarnib
(SARASAR.TM., SCH 66336, Schering Plough), sorafenib (NEXAVAR.RTM.,
BAY43-9006, Bayer Labs), gefitinib (IRESSA.RTM., AstraZeneca),
irinotecan (CAMPTOSAR.RTM., CPT-11, Pfizer), tipifarnib
(ZARNESTRA.TM., Johnson & Johnson), ABRAXANE.TM.
(Cremophor-free), albumin-engineered nanoparticle formulations of
paclitaxel (American Pharmaceutical Partners, Schaumberg, II),
vandetanib (rINN, ZD6474, ZACTIMA.RTM., AstraZeneca),
chloranmbucil, AG1478, AG1571 (SU 5271; Sugen), temsirolimus
(TORISEL.RTM., Wyeth), pazopanib (GlaxoSmithKline), canfosfamide
(TELCYTA.RTM., Telik), thiotepa and cyclosphosphamide
(CYTOXAN.RTM., NEOSAR.RTM.); vinorelbine (NAVELBINE.RTM.);
capecitabine (XELODA.RTM., Roche), tamoxifen (including
NOLVADEX.RTM.; tamoxifen citrate, FARESTON.RTM. (toremifine
citrate) MEGASE.RTM. (megestrol acetate), AROMASIN.RTM.
(exemestane; Pfizer), formestanie, fadrozole, RIVISOR.RTM.
(vorozole), FEMARA.RTM. (letrozole; Novartis), and ARIMIDEX.RTM.
(anastrozole; AstraZeneca).
[0322] The term "pharmaceutically acceptable salt" or "salt" means
organic or inorganic salts of a molecule or macromolecule. Acid
addition salts can be formed with amino groups. Exemplary salts
include, but are not limited, to sulfate, citrate, acetate,
oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate,
acid phosphate, isonicotinate, lactate, salicylate, acid citrate,
tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate,
succinate, maleate, gentisinate, fumarate, gluconate, glucuronate,
saccharate, formate, benzoate, glutamate, methanesulfonate,
ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate
(i.e., 1,1' methylene bis-(2-hydroxy 3-naphthoate)) salts. A
pharmaceutically acceptable salt may involve the inclusion of
another molecule such as an acetate ion, a succinate ion or other
counterion. The counterion may be any organic or inorganic moiety
that stabilizes the charge on the parent compound. Furthermore, a
pharmaceutically acceptable salt may have more than one charged
atom in its structure. Where multiple charged atoms are part of the
pharmaceutically acceptable salt, the salt can have multiple
counter ions. Hence, a pharmaceutically acceptable salt can have
one or more charged atoms and/or one or more counterion.
[0323] "Pharmaceutically acceptable solvate" or "solvate" refers to
an association of one or more solvent molecules and a molecule or
macromolecule. Examples of solvents that form pharmaceutically
acceptable solvates include, but are not limited to, water,
isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid,
and ethanolamine.
[0324] In other embodiments the antibodies or ADCs of the instant
invention may be used in combination with any one of a number of
antibodies (or immunotherapeutic agents) presently in clinical
trials or commercially available. The disclosed antibodies may be
used in combination with an antibody selected from the group
consisting of abagovomab, adecatumumab, afutuzumab, alemtuzumab,
altumomab, amatuximab, anatumomab, arcitumomab, bavituximab,
bectumomab, bevacizumab, bivatuzumab, blinatumomab, brentuximab,
cantuzumab, catumaxomab, cetuximab, citatuzumab, cixutumumab,
clivatuzumab, conatumumab, daratumumab, drozitumab, duligotumab,
dusigitumab, detumomab, dacetuzumab, dalotuzumab, ecromeximab,
elotuzumab, ensituximab, ertumaxomab, etaracizumab, farletuzumab,
ficlatuzumab, figitumumab, flanvotumab, futuximab, ganitumab,
gemtuzumab, girentuximab, glembatumumab, ibritumomab, igovomab,
imgatuzumab, indatuximab, inotuzumab, intetumumab, ipilimumab,
iratumumab, labetuzumab, lambrolizumab, lexatumumab, lintuzumab,
lorvotuzumab, lucatumumab, mapatumumab, matuzumab, milatuzumab,
minretumomab, mitumomab, moxetumomab, narnatumab, naptumomab,
necitumumab, nimotuzumab, nivolumab, nofetumomabn, obinutuzumab,
ocaratuzumab, ofatumumab, olaratumab, olaparib, onartuzumab,
oportuzumab, oregovomab, panitumumab, parsatuzumab, patritumab,
pemtumomab, pertuzumab, pidilizumab, pintumomab, pritumumab,
racotumomab, radretumab, ramucirumab, rilotumumab, rituximab,
robatumumab, satumomab, selumetinib, sibrotuzumab, siltuximab,
simtuzumab, solitomab, tacatuzumab, taplitumomab, tenatumomab,
teprotumumab, tigatuzumab, tositumomab, trastuzumab, tucotuzumab,
ublituximab, veltuzumab, vorsetuzumab, votumumab, zalutumumab,
CC49, 3F8, MDX-1105 and MED14736 and combinations thereof.
[0325] Other particularly preferred embodiments comprise the use of
antibodies approved for cancer therapy including, but not limited
to, rituximab, gemtuzumab ozogamcin, alemtuzumab, ibritumomab
tiuxetan, tositumomab, bevacizumab, cetuximab, patitumumab,
ofatumumab, ipilimumab and brentuximab vedotin. Those skilled in
the art will be able to readily identify additional anti-cancer
agents that are compatible with the teachings herein.
[0326] E. Radiotherapy
[0327] The present invention also provides for the combination of
antibodies or ADCs with radiotherapy (i.e., any mechanism for
inducing DNA damage locally within tumor cells such as
gamma-irradiation, X-rays, UV-irradiation, microwaves, electronic
emissions and the like). Combination therapy using the directed
delivery of radioisotopes to tumor cells is also contemplated, and
the disclosed antibodies or ADCs may be used in connection with a
targeted anti-cancer agent or other targeting means. Typically,
radiation therapy is administered in pulses over a period of time
from about 1 to about 2 weeks. The radiation therapy may be
administered to subjects having head and neck cancer for about 6 to
7 weeks. Optionally, the radiation therapy may be administered as a
single dose or as multiple, sequential doses.
VIII INDICATIONS
[0328] The invention provides for the use of antibodies and ADCs of
the invention for the diagnosis, theragnosis, treatment and/or
prophylaxis of various disorders including neoplastic,
inflammatory, angiogenic and immunologic disorders and disorders
caused by pathogens. Particularly, key targets for treatment are
neoplastic conditions comprising solid tumors, although hematologic
malignancies are within the scope of the invention. In certain
embodiments the antibodies of the invention will be used to treat
tumors or tumorigenic cells expressing a particular determinant
(e.g. CLDN). Preferably the "subject" or "patient" to be treated
will be human although, as used herein, the terms are expressly
held to comprise any mammalian species.
[0329] Neoplastic conditions subject to treatment in accordance
with the instant invention may be benign or malignant; solid tumors
or other blood neoplasia; and may be selected from the group
including, but not limited to: adrenal gland tumors,
AIDS-associated cancers, alveolar soft part sarcoma, astrocytic
tumors, autonomic ganglia tumors, bladder cancer (squamous cell
carcinoma and transitional cell carcinoma), blastocoelic disorders,
bone cancer (adamantinoma, aneurismal bone cysts, osteochondroma,
osteosarcoma), brain and spinal cord cancers, metastatic brain
tumors, breast cancer, carotid body tumors, cervical cancer,
chondrosarcoma, chordoma, chromophobe renal cell carcinoma, clear
cell carcinoma, colon cancer, colorectal cancer, cutaneous benign
fibrous histiocytomas, desmoplastic small round cell tumors,
ependymomas, epithelial disorders, Ewing's tumors, extraskeletal
myxoid chondrosarcoma, fibrogenesis imperfecta ossium, fibrous
dysplasia of the bone, gallbladder and bile duct cancers, gastric
cancer, gastrointestinal, gestational trophoblastic disease, germ
cell tumors, glandular disorders, head and neck cancers,
hypothalamic, intestinal cancer, islet cell tumors, Kaposi's
Sarcoma, kidney cancer (nephroblastoma, papillary renal cell
carcinoma), leukemias, lipoma/benign lipomatous tumors,
liposarcoma/malignant lipomatous tumors, liver cancer
(hepatoblastoma, hepatocellular carcinoma), lymphomas, lung cancers
(small cell carcinoma, adenocarcinoma, squamous cell carcinoma,
large cell carcinoma etc.), macrophagal disorders, medulloblastoma,
melanoma, meningiomas, multiple endocrine neoplasia, multiple
myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine
tumors, ovarian cancer, pancreatic cancers, papillary thyroid
carcinomas, parathyroid tumors, pediatric cancers, peripheral nerve
sheath tumors, phaeochromocytoma, pituitary tumors, prostate
cancer, posterious unveal melanoma, rare hematologic disorders,
renal metastatic cancer, rhabdoid tumor, rhabdomysarcoma, sarcomas,
skin cancer, soft-tissue sarcomas, squamous cell cancer, stomach
cancer, stromal disorders, synovial sarcoma, testicular cancer,
thymic carcinoma, thymoma, thyroid metastatic cancer, and uterine
cancers (carcinoma of the cervix, endometrial carcinoma, and
leiomyoma).
[0330] In other preferred embodiments, the disclosed antibodies and
ADCs are especially effective at treating lung cancer, including
the following subtypes: small cell lung cancer and non-small cell
lung cancer (e.g. squamous cell non-small cell lung cancer or
squamous cell small cell lung cancer). In selected embodiments the
antibodies and ADCs can be administered to patients exhibiting
limited stage disease or extensive stage disease. In other
preferred embodiments the disclosed conjugated antibodies will be
administered to refractory patients (i.e., those whose disease
recurs during or shortly after completing a course of initial
therapy); sensitive patients (i.e., those whose relapse is longer
than 2-3 months after primary therapy); or patients exhibiting
resistance to a platinum based agent (e.g. carboplatin, cisplatin,
oxaliplatin) and/or a taxane (e.g. docetaxel, paclitaxel, larotaxel
or cabazitaxel).
[0331] The invention also provides for a preventative or
prophylactic treatment of subjects who present with benign or
precancerous tumors. No particular type of tumor or proliferative
disorder is excluded from treatment using the antibodies of the
invention.
IX ARTICLES OF MANUFACTURE
[0332] The invention includes pharmaceutical packs and kits
comprising one or more containers, wherein a container can comprise
one or more doses of an antibody or ADC of the invention. In
certain embodiments, the pack or kit contains a unit dosage,
meaning a predetermined amount of a composition comprising, for
example, an antibody or ADC of the invention, with or without one
or more additional agents and optionally, one or more anti-cancer
agents.
[0333] The kit of the invention will generally contain in a
suitable container a pharmaceutically acceptable formulation of the
antibody or ADC of the invention and, optionally, one or more
anti-cancer agents in the same or different containers. The kits
may also contain other pharmaceutically acceptable formulations or
devices, either for diagnosis or combination therapy. Examples of
diagnostic devices or instruments include those that can be used to
detect, monitor, quantify or profile cells or markers associated
with proliferative disorders (for a full list of such markers, see
above). In particularly preferred embodiments the devices may be
used to detect, monitor and/or quantify circulating tumor cells
either in vivo or in vitro (see, for example, WO 2012/0128801). In
still other preferred embodiments the circulating tumor cells may
comprise tumorigenic cells. The kits contemplated by the invention
can also contain appropriate reagents to combine the antibody or
ADC of the invention with an anti-cancer agent or diagnostic agent
(e.g., see U.S. Pat. No. 7,422,739).
[0334] When the components of the kit are provided in one or more
liquid solutions, the liquid solution can be non-aqueous, however,
an aqueous solution is preferred, with a sterile aqueous solution
being particularly preferred. The formulation in the kit can also
be provided as dried powder(s) or in lyophilized form that can be
reconstituted upon addition of an appropriate liquid. The liquid
used for reconstitution can be contained in a separate container.
Such liquids can comprise sterile, pharmaceutically acceptable
buffer(s) or other diluent(s) such as bacteriostatic water for
injection, phosphate-buffered saline, Ringer's solution or dextrose
solution. Where the kit comprises the antibody or ADC of the
invention in combination with additional therapeutics or agents,
the solution may be pre-mixed, either in a molar equivalent
combination, or with one component in excess of the other.
Alternatively, the antibody or ADC of the invention and any
optional anti-cancer agent or other agent can be maintained
separately within distinct containers prior to administration to a
patient.
[0335] The kit can comprise one or multiple containers and a label
or package insert in, on or associated with the container(s),
indicating that the enclosed composition is used for diagnosing or
treating the disease condition of choice. Suitable containers
include, for example, bottles, vials, syringes, etc. The containers
can be formed from a variety of materials such as glass or plastic.
The container(s) can comprise a sterile access port, for example,
the container may be an intravenous solution bag or a vial having a
stopper that can be pierced by a hypodermic injection needle.
[0336] In some embodiments the kit can contain a means by which to
administer the antibody and any optional components to a patient,
e.g., one or more needles or syringes (pre-filled or empty), an eye
dropper, pipette, or other such like apparatus, from which the
formulation may be injected or introduced into the subject or
applied to a diseased area of the body. The kits of the invention
will also typically include a means for containing the vials, or
such like, and other components in close confinement for commercial
sale, such as, e.g., blow-molded plastic containers into which the
desired vials and other apparatus are placed and retained.
X MISCELLANEOUS
[0337] Unless otherwise defined herein, scientific and technical
terms used in connection with the invention shall have the meanings
that are commonly understood by those of ordinary skill in the art.
Further, unless otherwise required by context, singular terms shall
include pluralities and plural terms shall include the singular. In
addition, ranges provided in the specification and appended claims
include both end points and all points between the end points.
Therefore, a range of 2.0 to 3.0 includes 2.0, 3.0, and all points
between 2.0 and 3.0.
[0338] Generally, techniques of cell and tissue culture, molecular
biology, immunology, microbiology, genetics and chemistry described
herein are those well known and commonly used in the art. The
nomenclature used herein, in association with such techniques, is
also commonly used in the art. The methods and techniques of the
invention are generally performed according to conventional methods
well known in the art and as described in various references that
are cited throughout the present specification unless otherwise
indicated.
XI REFERENCES
[0339] The complete disclosure of all patents, patent applications,
and publications, and electronically available material (including,
for example, nucleotide sequence submissions in, e.g., GenBank and
RefSeq, and amino acid sequence submissions in, e.g., SwissProt,
PIR, PRF, PBD, and translations from annotated coding regions in
GenBank and RefSeq) cited herein are incorporated by reference,
regardless of whether the phrase "incorporated by reference" is or
is not used in relation to the particular reference. The foregoing
detailed description and the examples that follow have been given
for clarity of understanding only. No unnecessary limitations are
to be understood therefrom. The invention is not limited to the
exact details shown and described. Variations obvious to one
skilled in the art are included in the invention defined by the
claims. Any section headings used herein are for organizational
purposes only and are not to be construed as limiting the subject
matter described.
XII SEQUENCE LISTING SUMMARY
[0340] Appended to the instant application is a sequence listing
comprising a number of nucleic acid and amino acid sequences. The
following Table 3 provides a summary of the included sequences.
TABLE-US-00005 TABLE 3 SEQ ID NO Description 1 Kappa light chain
(LC) constant region protein 2 IgGI heavy chain (HC) constant
region protein 3-19 Reserved 20 SC27.1 VL DNA 21 SC27.1 VL protein
22 SC27.1 VH DNA 23 SC27.1 VH protein 24-59 Additional mouse clones
as in SEQ ID NOs: 20-23 60 hSC27.1 VL DNA 61 hSC27.1 VL protein 62
hSC27.1 VH DNA 63 hSC27.1 VH protein 64-75 Additional humanized
clones as in SEQ ID NOs: 60-63 76-77 hSC27.108v1 VL DNA and protein
78-79 hSC27.22-VH1-8 VH DNA and protein 80-81 hSC27.22-VH1-46 VH
DNA and protein 82-83 hSC27.22-VH1-69 VH DNA and protein 84-85
hSC27.204v1 DNA and protein 86-87 hSC27.204v2 DNA and protein 88-89
hSC27.204v3 DNA and protein 90-91 hSC27.204v4 DNA and protein 92-93
hSC27.204v5 DNA and protein 94-95 hSC27.204v6 DNA and protein 96-97
hSC27.204v7 DNA and protein 98-99 hSC27.204v8 DNA and protein
100-101 hSC27.204v9 DNA and protein 102-103 hSC27.204v10 DNA and
protein 104-105 hSC27.204v11 DNA and protein 106-107 hSC27.204v12
DNA and protein 108-109 hSC27.204v13 DNA and protein 110-111
hSC27.204v14 DNA and protein 112-113 hSC27.204v15 DNA and protein
114-115 hSC27.1 full length LC and HC protein 116-117 hSC27.22 full
length LC and HC protein 118-119 hSC27.108 full length LC and HC
protein 120-121 hSC27.204 full length LC and HC protein 122
hSC27.22ss1 full length HC protein 123 hSC27.22-VH1-8 full length
HC protein 124 hSC27.22-VH1-46 full length HC protein 125
hSC27.22-VH1-69 full length HC protein 126 hSC27.22 IgG2 full
length HC protein 127 hSC27.22 IgG4 R409K full length HC protein
128 hSC27.22 IgG4 S228P full length HC protein 129 hSC27.22 IgG4
S228P K370E R409K full length HC protein 130 hSC27.22 IgG4 K370E
full length HC protein 131 hSC27.22 IgG4 S228P K370E full length HC
protein 132 hSC27.22 IgG4 C127S S228P full length HC protein 133
hSC27.22 IgG4 C127S K370E full length HC protein 134 hSC27.22 IgG4
C127S S228P K370E full length HC protein 135 hSC27.108v1 full
length LC protein 136 hSC27.204v1 full length HC protein 137
hSC27.204v2 full length HC protein 138 hSC27.204v3 full length HC
protein 139 hSC27.204v4 full length HC protein 140 hSC27.204v5 full
length HC protein 141 hSC27.204v6 full length HC protein 142
hSC27.204v7 full length HC protein 143 hSC27.204v8 full length HC
protein 144 hSC27.204v9 full length HC protein 145 hSC27.204v10
full length HC protein 146 hSC27.204v11 full length HC protein 147
hSC27.204v12 full length HC protein 148 hSC27.204v13 full length HC
protein 149 hSC27.204v14 full length HC protein 150 hSC27.204v15
full length HC protein 151-156 hSC27.1 CDRL1; CDRL2; CDRL3, CDRH1;
CDRH2; CDRH3 157-162 hSC27.22 CDRL1; CDRL2; CDRL3, CDRH1; CDRH2;
CDRH3 163-168 hSC27.108 CDRL1; CDRL2; CDRL3, CDRH1; CDRH2; CDRH3
169-174 hSC27.204 CDRL1; CDRL2; CDRL3, CDRH1; CDRH2; CDRH3 175
CDRH2 of hSC27.204v1; hSC27.204v5 and hSC27.405v13 176 CDRH2 of
hSC27.204v2; hSC27.204v6 and hSC27.405v14 177 CDRH2 of hSC27.204v3;
hSC27.204v7 and hSC27.405v15 178 Codon optimized hSC27.22ss1 full
length HC DNA
XIII EXAMPLES
[0341] The invention, thus generally described above, will be
understood more readily by reference to the following examples,
which are provided by way of illustration and are not intended to
be limiting of the instant invention. The examples are not intended
to represent that the experiments below are all or the only
experiments performed. Unless indicated otherwise, parts are parts
by weight, molecular weight is weight average molecular weight,
temperature is in degrees Centigrade, and pressure is at or near
atmospheric.
[0342] PDX tumor cell types are denoted by an abbreviation followed
by a number, which indicates the particular tumor cell line. The
passage number of the tested sample is indicated by p0-p# appended
to the sample designation where p0 is indicative of an unpassaged
sample obtained directly from a patient tumor and p# is indicative
of the number of times the tumor has been passaged through a mouse
prior to testing. As used herein, the abbreviations of the tumor
types and subtypes are shown in Table 4 as follows:
TABLE-US-00006 TABLE 4 Abbre- Tumor Type viation Tumor subtype
Abbreviation Bladder BL Breast BR basal-like BR-Basal Like estrogen
receptor positive BR-ERPR and/or progesterone receptor positive
ERBB2/Neu positive BR-ERBB2/Neu HER2 positive BR-HER2
triple-negative TNBC luminal A BR-lumA claudin subtype of triple-
TNBC-CL negative claudin low BR-CLDN-Low Cervical CER Colorectal CR
Endometrial EM Gastric GA diffuse adenocarcinoma GA-Ad-Dif/Muc
intestinal adenocarcinoma GA-Ad-Int stromal tumors GA-GIST
Glioblastoma GB Head and HN neck Kidney KDY clear renal cell
carcinoma KDY-CC papillary renal cell KDY-PAP carcinoma
transitional cell or KDY-URO urothelial carcinoma unknown KDY-UNK
Liver LIV hepatocellular carcinoma LIV-HCC cholangiocarcinoma
LIV-CHOL Lymphoma LN Lung LU adenocarcinoma LU-Ad carcinoid LU-CAR
large cell neuroendocrine LU-LCC non-small cell NSCLC squamous cell
LU-SCC small cell SCLC spindle cell LU-SPC Ovarian OV clear cell
OV-CC endometroid OV-END mixed subtype OV-MIX malignant mixed
OV-MMMT mesodermal mucinous OV-MUC neuroendocrine OV-NET papillary
serous OV-PS serous OV-S small cell OV-SC transitional cell
carcinoma OV-TCC Pancreatic PA acinar cell carcinoma PA-ACC
duodenal carcinoma PA-DC mucinous adenocarcinoma PA-MAD
neuroendocrine PA-NET adenocarcinoma PA-PAC adenocarcinoma exocrine
PA-PACe type ductal adenocarcinoma PA-PDAC ampullary adenocarcinoma
PA-AAC Prostate PR Skin SK melanoma MEL squamous cell carcinomas
SK-SCC uveal melanoma UVM Testicular TES Thyroid THY
Example 1
Identification of CLDN4, CLDN6 and CLDN9 Expression Using Whole
Transcriptome Sequencing
[0343] To characterize the cellular heterogeneity of solid tumors
as they exist in cancer patients, aid in the identification of CSCs
using particular phenotypic markers and identify clinically
relevant therapeutic targets, a large PDX tumor bank was developed
and maintained using art recognized techniques. The PDX tumor bank,
comprising a large number of discrete tumor cell lines, was
propagated in immunocompromised mice through multiple passages of
heterogeneous tumor cells originally obtained from numerous cancer
patients afflicted by a variety of solid tumor malignancies. The
continued availability of a large number of discrete early passage
PDX tumor cell lines having well defined lineages greatly
facilitates the identification and isolation of CSCs as the PDX
tumors allow for the reproducible and repeated characterization of
CSCs. The use of minimally passaged PDX tumor cell lines simplifies
in vivo experimentation and provides readily verifiable results.
Moreover, early passage PDX tumors respond to therapeutic agents
such as irinotecan (i.e. Camptosar.RTM.), which provides clinically
relevant insight into underlying mechanisms driving tumor growth,
resistance to current therapies and tumor recurrence.
[0344] To generate RNA from the PDX tumor cell lines, tumors were
resected from mice after they reached 800-2,000 mm.sup.3 and the
tumors were dissociated into single cell suspensions using
art-recognized enzymatic digestion techniques (see, for example,
U.S.P.N. 2007/0292414). Select dissociated PDX tumor cell
preparations were depleted of mouse cells and sorted based on their
expression of CD46.sup.hi and/or CD324, markers of CSC
subpopulations (see U.S.P.N 2013/0260385 for the definition of
CD46.sup.hi). Cells that expressed human EpCAM, CD46.sup.hi and/or
CD324 (i.e. CSC) or EpCAM but not CD46.sup.hi and/or CD324 (i.e.
NTG cells), were isolated by FACS using a BD FACSAria cell sorter
and lysed in RLTplus RNA lysis buffer (Qiagen) per the
manufacturer's instructions. The lysates were then stored at
-80.degree. C. and thawed for RNA extraction. Upon thawing, total
RNA was extracted using an RNeasy isolation kit (Qiagen, GmbH)
following the vendor's instructions and then quantified using a
Nanodrop spectrophotometer (Thermo Scientific) and/or a Bioanalyzer
2100 (Agilent Technologies), again using the manufacturer's
protocols and recommended instrument settings. The resulting total
RNA preparations were assessed by genetic sequencing and gene
expression analyses.
[0345] Whole transcriptome sequencing of qualified, high quality
RNA was performed using an Applied Biosystems (ABI) Sequencing by
Oligo Ligation/Detection (SOLiD) 4.5 or SOLiD 5500xl next
generation sequencing system (Life Technologies). cDNA was
generated from 1 ng total RNA samples using either a modified whole
transcriptome protocol from ABI designed for low input total RNA or
the Ovation RNA-Seq System V2.TM. (NuGEN Technologies). The
resulting cDNA library was fragmented and barcode adapters were
added to allow pooling of fragment libraries from different samples
during sequencing runs. Data generated by the SOLiD platform mapped
to 34,609 genes as annotated by RefSeq version 47 using NCBI
version hg19.2 of the published human genome and provided
verifiable measurements of RNA levels in most samples. Sequencing
data from the SOLiD platform is nominally represented as a
transcript expression value using the metrics RPM (reads per
million) or RPKM (read per kilobase per million) mapped to exon
regions of genes, enabling basic gene expression analysis to be
normalized and enumerated as RPM_Transcript or RPKM_Transcript.
[0346] The results of whole transcriptome sequencing using SOLiD
showed elevated expression of CLDN4 mRNA in sorted CSC compared to
NTG in the following PDX cell lines: BR13, BR22, OV100, PA20 and
PA3, as well as high expression in additional CSC populations
including BR36, OV106MET, OV72MET, and OV91MET (FIG. 1). CLDN6 mRNA
was elevated in sorted CSC populations including BR36, OV106MET,
OV72MET, and OV91MET (FIG. 1). Unlike the case for CLDN4 or CLDN6,
the related family member, CLDN9, was observed to have low
expression in all sorted tumor populations. In contrast to the
tumor samples, normal ovary and pancreas tissues showed no or very
low mRNA expression of all three family members, CLDN4, CLDN6 and
CLDN9.
[0347] The identification of elevated CLDN4 and CLDN6 mRNA
expression in different types of human tumor indicated these
antigens merited further evaluation as a potential diagnostic
and/or immunotherapeutic targets.
Example 2
Expression of CLDN4, CLDN6 and CLDN9 mRNA in Tumors Using
qRT-PCR
[0348] To confirm expression of CLDN4, CLDN6 and further delineate
CLDN9 expression in tumor cell subpopulations, qRT-PCR was run on
RNA samples obtained from sorted CSC and NTG populations (as
described in Example 1) from various PDX models. qRT-PCR was
performed using the Fluidigm BioMark.TM. HD System according to
industry standard protocols. 1 ng of RNA, prepared as described in
Example 1, was converted to cDNA using the High Capacity cDNA
Archive kit (Life Technologies) according to the manufacturer's
instructions. cDNA material, pre-amplified using CLDN4, CLDN6 and
CLDN9 specific Taqman assays, was then used for subsequent qRT-PCR
experiments.
[0349] As shown in FIG. 2A, CLDN4 exhibited elevated expression in
CSC subpopulations of BR31 (TNBC), LU86 (SCLC) and PA14 compared to
NTG populations from the same PDX tumor lines. Compared to
expression in normal lung and pancreas, sorted CSC populations
showed 10-100 fold higher expression of CLDN4, suggesting a
therapeutic window and potential benefit for targeting the CSC of
these PDX models. CLDN6 was also found to exhibit elevated
expression in CSC subpopulations of BR13 (TNBC), CR81 and PA3 when
compared to NTG populations derived from the same PDX tumor lines,
with CSC expression of CLDN6 approximately 10,000 fold greater than
CLDN6 expression in normal pancreas and colon (FIG. 2A). Finally,
CLDN9 was also found to show elevated expression in CSC populations
of BR13 (TNBC), CR81, OV27MET (OV-S) and OV44 (OV-S) as compared
with NTG populations obtained from the same PDX tumor lines, with
expression of CLDN9 approximately 10,000 fold higher than that
found in normal colon cells. These findings validate the results
obtained from whole transcriptome sequencing of similar populations
with regard to CLDN4 and CLDN6 and clearly show that, in various
sorted CSC populations, CDLN4, CLDN6 and/or CLDN9 are highly
overexpressed.
[0350] To further determine the expression levels of CLDN4, CLDN6
and CLDN9 in additional tumor specimens, mRNA expression of the
relevant CLDN family members in various bulk (non-sorted) PDX tumor
cell lines was compared to mRNA expression in normal tissues that
might be of concern for intolerable toxicity when using ADC
therapeutics (NormTox: colon, stomach, small intestine, lung and
pancreas) and normal tissues of less concern in terms of toxicity
(Norm: breast, ovary). CLDN4 mRNA expression was found to be
elevated in many bulk BR, CR, LU-SCC and PA PDX lines, with highest
expression seen in BR-TNBC, CR, LU-SCC, PA (FIG. 2B). CLDN6 mRNA
was highly overexpressed in OV-S and OV-PS (FIG. 2C), while CLDN6
mRNA levels were also elevated in BR-TNBC and in subsets of LU-Ad
and CR PDX lines (FIG. 2C). Finally, CLDN9 mRNA was found to be
elevated in certain PDX cell lines including BR-TNBC, CR, LU-Ad, OV
and PA PDX lines (FIG. 2D) when compared with the levels found in
normal tissues. These results demonstrate that CLDN4, CLDN6 and
CLDN9 expression is elevated across different PDX tumor models,
indicating that antibodies against CLDN4, CLDN6 and CLDN9 will
allow for comprehensive targeting of multiple cancer indications.
These findings further suggest that, in certain selected
embodiments multireactive antibodies (i.e. those that
immunospecifically bind to more than one of the CLDN4, CLDN6 and
CLDN9 antigens) may be particularly effective in reducing or
eliminating tumorigenic cells.
[0351] To further expand the analysis to a larger group of primary
human tumor samples as well as normal tissue samples, a qRT-PCR
assay was performed using CLDN4, CLDN6 or CLDN9-specific Taqman
assays on a TissueScan.TM. qPCR (Origene Technologies) 384-well
array according to the manufacturer's instructions. This array
enables comparison of gene expression across 18 different tumor
types, with multiple patient derived samples for each tumor type.
Significantly, the Origene assay also enables the comparison of
expression from normal tissue versus tumor tissue of the same
tissue types. FIGS. 2E, 2F and 2G show the expression levels of
CLDN4, CLDN6 and CLDN9, respectively, in various whole tumors
specimens (black dots) normalized against .beta.-actin and plotted
relative to expression in the matched normal tissue (white dots)
for each tumor type analyzed. Specimens not amplified were assigned
a cycle count value (Ct) of 45, which represents the last cycle of
amplification in the experimental protocol. Each dot represents a
single tissue specimen, with the mean geometric value of the
samples for an indicated tumor or matched normal tissue type
represented as a black line.
[0352] Overexpression of CLDN4 relative to matched normal tissue
was seen in cervical, endometrial, and ovarian tumors, and in a
subset of esophageal, liver, gastric, lung, testicular and bladder
tumors (FIG. 2E). This includes 2/2 cholangiocarcinomas of the
liver, 1/3 adenomas of the liver and 3/12 hepatocellular
carcinomas. The gastric tumor subsets with overexpression of CLDN4
included 7/7 adenocarcinomas and 1/1 villous adenomas (data not
shown). Overexpression of CLDN6 relative to matched normal tissue
was seen in endometrial, ovarian and testicular tumors, as well as
subsets of adrenal, breast, esophageal, liver, lung, lymphoma and
gastric tumors (FIG. 2F). This includes 2/2 cholangiocarcinomas of
the liver, 2/3 adenomas of the liver and 7/12 hepatocellular
carcinomas (data not shown). Overexpression of CLDN9 relative to
matched normal tissue was seen in adrenal, endometrial, esophageal,
and ovarian tumors, and a subset of breast, lung and bladder tumors
(FIG. 2G). These data suggest that abnormal CLDN4, CLDN6 and CLDN9
expression may be indicative of and/or implicated in tumorigenesis
and/or tumor progression in the above-mentioned tumors, and again
demonstrate that antibodies directed to these claudin proteins may
be used to effectively treat various tumors.
Example 3
CLDN Expression Profiles in Primary Tumors from the Cancer Genome
Atlas
[0353] Overexpression of mRNA of CLDN6 and CLDN9 family members was
confirmed in various tumors using a large, publically available
dataset of tumors and normal samples known as The Cancer Genome
Atlas (TCGA, National Cancer Institute). Exon level 3 expression
data from the IlluminaHiSeq_RNASeqV2 platform was downloaded from
the TCGA Data Portal
(https://tcga-data.nci.nih.qov/tcga/tcgaDownload.jsp) and parsed to
aggregate the reads from the individual exons of each single gene
to generate a single value read per kilobase of exon per million
mapped reads (RPKM) for each gene in each sample. The rolled up
data was then displayed using Tableau software. The parsed data for
CLDN6 and CLDN9 are shown in FIGS. 3A and 3B, respectively, in
which each sample is represented as a single dot, and the black
horizontal lines represent the quartile boundaries for the setoff
data points within a given normal tissue or tumor subtype. FIG. 3A
shows that CLDN6 expression is elevated in OV tumors, which were
subtyped as ovarian serous cystadenocarcinomas, compared to all
other normal tissues. In addition, CLDN6 is elevated in a large
number of LU-Ad samples compared to normal lung samples, and a
substantial number of breast invasive carcinoma tumors (BRCA).
Similar overexpression patterns can be see for CLDN9 as those
observed for CLDN6 (FIG. 3B). Again, these data indicate that CLDN6
and CLDN9 expression levels are indicative of tumorigenesis in
various tumors and reinforce their selection as potential
therapeutic targets.
[0354] In five selected tumor types found in the TOGA data: OV,
LU-Ad, LU-SC (squamous cell carcinoma), BR, and SK, the relative
expression of CLDN6 versus CLDN9, for samples showing non-zero RPKM
expression values for both genes, was plotted (FIG. 3C). These
tumor types were selected to encompass a range of relative
expression levels, from high (OV) to low (SK). FIG. 3C shows a
progressive shift in the co-expression of both genes, from high
(upper right quadrant in scatter plots) to low (lower left
quadrant) across the indications, suggesting the expression of
these genes may be linked. This can be more easily visualized by
plotting the centroids (center of mass) from each of the
scatterplots (FIG. 3D). The centroids show a very tight correlation
of high significance for the CLDN6 and CLDN9 genes (r.sup.2=0.0996;
p=0.0001), which are proximal to one another (head to head on
chromosome 16). In these same five indications, there was no
significant correlation between: the expression of CLDN6 and a
different claudin gene, CLDN1 (r.sup.2=0.14); between CLDN6 and
another tetraspanin, CD81 (r.sup.2=0.02); or between CLDN6 and its
other neighboring gene on chromosome 16, TNFRSF12A (r.sup.2=0.47)
(data not shown). A more modest yet significant correlation was
seen in the co-expression of CLDN6 and CLDN4 genes (r.sup.2=0.80;
p<0.05) (data not shown). This rather surprisingly strong
correlation between CLDN6 and CLDN9 is likely the result of the
proximity of these two genes to one another, but is also suggestive
of a functional link or functional co-disregulation of these genes.
The tight co-expression pattern between CLDN6 and CLDN9 in several
cancer indications provides a rationale for targeting multiple CLDN
proteins as a therapeutic strategy.
Example 4
Cloning and Expression of Recombinant CLDN Proteins and Engineering
of Cell Lines Overexpressing Cell Surface CLDN Proteins
[0355] In order to deduce the relationship between claudin protein
sequences, the AlignX program of the Vector NTI software package
was used to align 30 claudin protein sequences from 23 human CLDN
genes. The results of this alignment are depicted as a dendrogram
in FIG. 4A. A review of the figure shows that CLDN6 and CLDN9 are
very closely related in sequence, appearing adjacent to one another
on the same branch of the dendrogram. FIG. 4A also shows that CLDN4
is the next most closely related family member to CLDN6. A more
detailed review of the data shows that the human CLDN6 protein is
very closely related to the human CLDN9 protein sequence in the
extracellular domains (ECD), with >98% identity in ECD1 and
>91% identity in ECD2 (FIG. 4B). Human CLDN4 was also found to
be closely related to human CLDN6 in the ECD sequences, with
>84% identity in ECD1 and >78% identity in ECD2 (FIG. 4B).
Based upon these protein sequence relationships, it was
hypothesized that immunization with a human CLDN6 antigen would
yield antibodies recognizing human CLDN6 that will also be
cross-reactive with human CLDN9, and perhaps also cross-reactive
with human CLDN4.
[0356] In order to determine which species orthologs of CLDN6,
CLDN9 and CLDN4 would be required for screening these multireactive
claudin antibodies, ECD sequences of CLDN4, CLDN6 and CLDN9 were
analyzed from each of the following species: human, cynomolgus
monkey, mouse and rat. The analysis was performed using AlignX and
NCBI database protein sequences when available (NP accession
numbers of human, mouse and rat proteins are indicated in FIG. 4C).
Alternatively, protein sequences were deduced from translation of
the cynomolgus monkey CLDN genes assembled by BLAST of human CLDN
open reading frame sequences versus cynomolgus monkey whole genome
shotgun sequencing contigs. Inspection of these alignments reveals
that: (1) deduced cynomolgus monkey protein ECD sequences for
CLDN4, CLDN6, and CLDN9 proteins are 100% identical to the
respective human ECD sequences; (2) mouse and rat CLDN9 ECD
sequences are 100% identical to the human ortholog sequence; (3)
and mouse and rat CLDN4 and CLDN6 ECD sequences differ from one
another and from the respective human orthologs. Therefore,
generation of a set of seven constructs comprising human CLDN4,
human CLDN6, human CLDN9, mouse CLDN4, mouse CLDN6, rat CLDN4 and
rat CLDN6 should enable determination of cross-reactivity for any
antibody with all possible 12 orthologs.
[0357] DNA Fragments Encoding Human CLDN6, CLDN4, and CLDN9
Proteins.
[0358] To generate all molecular and cellular materials required in
the present invention pertaining to the human CLDN6 (hCLDN6)
protein, a codon-optimized DNA fragment encoding a protein
identical to NCBI protein accession NP_067018 was synthesized
(IDT). This DNA clone was used for all subsequent engineering of
constructs expressing the mature hCLDN6 protein or fragments
thereof. Similarly, codon-optimized DNA fragments encoding proteins
identical to NCBI protein accession NP_001296 for human CLDN4
(hCLDN4), or NCBI protein accession NP_066192 for human CLDN9
(hCLDN9) were purchased and used for all subsequent engineering of
constructs expressing the hCLDN4 or hCLDN9 proteins or fragments
thereof.
[0359] DNA Fragments Encoding Mouse CLDN6 and CLDN4 Proteins.
[0360] To generate all molecular and cellular materials required in
the present invention pertaining to the mouse CLDN6 (mCLDN6)
protein, a codon-optimized DNA fragment encoding a protein
identical to NCBI protein accession NP_061247 was synthesized
(IDT). This DNA clone was used for all subsequent engineering of
constructs expressing the mature mCLDN6 protein or fragments
thereof. Similarly, a codon-optimized DNA fragment encoding a
protein identical to NCBI protein accession NP_034033 for mouse
CLDN4 (mCLDN4) was purchased and used for all subsequent
engineering of constructs expressing the mature mCLDN4 protein or
fragments thereof.
[0361] DNA Fragments Encoding Rat CLDN6 and CLDN4 Proteins.
[0362] To generate all molecular and cellular materials required in
the present invention pertaining to the rat CLDN6 (rCLDN6) protein,
a codon-optimized DNA fragment encoding a protein identical to NCBI
protein accession NP_001095834 was synthesized (IDT). This DNA
clone was used for all subsequent engineering of constructs
expressing the mature rCLDN6 protein or fragments thereof.
Similarly, a codon-optimized DNA fragment encoding a protein
identical to NCBI protein accession NP_001012022 for rat CLDN4
(rCLDN4) was purchased and used for all subsequent engineering of
constructs expressing the mature rCLDN4 protein or fragments
thereof.
[0363] Cell Line Engineering
[0364] Engineered cell lines overexpressing the various CLDN
proteins listed above were constructed using lentiviral vectors to
transduce HEK-293T or 3T3 cell lines using art recognized
techniques. First, PCR was used to amplify the DNA fragments
encoding the protein of interest (e.g., hCLDN6, mCLDN6, rCLDN6,
hCLDN9, hCLDN4, mCLDN4, or rCLDN4) using the commercially
synthesized DNA fragments described above as templates. Then, the
individual PCR products were subcloned into the multiple cloning
site (MCS) of the lentiviral expression vector,
pCDH-EF1-MCS-T2A-GFP (System Biosciences), to generate a suite of
lentiviral vectors. The T2A sequence in resultant
pCDH-EF1-CLDN-T2A-GFP vectors promotes ribosomal skipping of a
peptide bond condensation, resulting in expression of two
independent proteins: high level expression of the specific CLDN
protein encoded upstream of the T2A peptide, with co-expression of
the GFP marker protein encoded downstream of the T2A peptide. This
suite of lentiviral vectors was used to create separate stable
HEK-293T or 3T3 cell lines overexpressing individual CLDN proteins
using standard lentiviral transduction techniques well known to
those skilled in the art. CLDN-positive cells were selected with
FACS using high-expressing HEK-293T subclones, which were also
strongly positive for GFP.
Example 5
Generation of Anti-CLDN Antibodies
[0365] Because CLDN6 is most homologous to CLDN4 and CLDN9 (see
FIG. 4A and analysis as described in Example 4, above), CLDN6 was
used as the immunogen with which to generate multireactive
anti-CLDN antibodies. Mice were inoculated with HEK-293T cells or
3T3 cells overexpressing hCLDN6 (generated as described in Example
4) in order to produce antibody-generating hybridomas. Six mice
(two each of the following strains: Balb/c, CD-1, FVB) were
inoculated with 1 million hCLDN6-HEK-293T cells emulsified with an
equal volume of TiterMax.RTM. adjuvant. A second, separate
inoculation of six mice (two each of the following strains: Balb/c,
CD-1, FVB) was performed using 3T3 cells overexpressing CLDN6.
Following the initial inoculation the mice were injected twice
weekly for 4 weeks with cells overexpressing CLDN6 emulsified with
an equal volume of alum adjuvant.
[0366] Mice were sacrificed and draining lymph nodes (popliteal,
inguinal, and medial iliac) were dissected and used as a source for
antibody producing cells. A single cell suspension of B cells
(305.times.10.sup.6 cells) were fused with non-secreting
P3.times.63Ag8.653 myeloma cells (ATCC #CRL-1580) at a ratio of 1:1
by electro cell fusion using a model BTX Hybrimmune System (BTX
Harvard Apparatus). Cells were resuspended in hybridoma selection
medium: DMEM medium (Cellgro) supplemented with azaserine (Sigma),
15% fetal clone I serum (Hyclone), 10% BM condimed (Roche Applied
Sciences), 1 mM sodium pyruvate, 4 mM L-glutamine, 100 IU
penicillin-streptomycin, 50 .mu.M 2-mercaptoethanol, and 100 .mu.M
hypoxanthine, and cultured in three T225 flasks in 90 mL selection
medium per flask. The flasks were placed in a humidified 37.degree.
C. incubator containing 5% CO.sub.2 and 95% air for 6 days. The
library was frozen down in 6 vials of CryoStor CS10 buffer (BioLife
Solutions), with approximately 15.times.10.sup.6 viable cells per
vial, and stored in liquid nitrogen.
[0367] One vial from the library was thawed at 37.degree. C. and
the frozen hybridoma cells were added to 90 mL hybridoma selection
medium, described above, and placed in a T150 flask. The cells were
cultured overnight in a humidified 37.degree. C. incubator with 5%
CO.sub.2 and 95% air. The following day hybridoma cells were
collected from the flask and plated at one cell per well (using a
FACSAria I cell sorter) in 200 .mu.L of supplemented hybridoma
selection medium into 48 Falcon 96-well U-bottom plates. The
hybridomas were cultured for 10 days and the supernatants were
screened for antibodies specific to hCLDN6, hCLDN4 or hCLDN9
proteins using flow cytometry. Flow cytometry was performed as
follows: 1.times.10.sup.5 per well of HEK-293T cells, stably
transduced with lentiviral vectors encoding hCLDN6, hCLDN4 or
hCLDN9, were incubated for 30 mins. with 100 .mu.L hybridoma
supernatent. Cells were washed with PBS/2% FCS and then incubated
with 50 .mu.L per sample DyeLight 649 labeled goat-anti-mouse IgG,
Fc fragment specific secondary antibody diluted 1:200 in PBS/2%
FCS. After a 15 min. incubation cells were washed twice with PBS/2%
FCS and re-suspended in PBS/2% FCS with DAPI (to detect dead cells)
and analyzed by flow cytometry for fluorescence exceeding that of
cells stained with an isotype control antibody. Selected hybridomas
that tested positive for antibodies directed to one or more of the
CLDN antigens were set aside for further characterization.
Remaining, unused hybridoma library cells were frozen in liquid
nitrogen for future library testing and screening.
Example 6
Sequencing of Anti-CLDN Antibodies
[0368] Anti-CLDN antibodies were generated as described above and
then sequenced as follows. Total RNA was purified from selected
hybridoma cells using the RNeasy Miniprep Kit (Qiagen) according to
the manufacturer's instructions. Between 10.sup.4 and 10.sup.5
cells were used per sample. Isolated RNA samples were stored at
-80.degree. C. until used. The variable region of the Ig heavy
chain of each hybridoma was amplified using two 5' primer mixes
comprising 86 mouse specific leader sequence primers designed to
target the complete mouse VH repertoire in combination with a 3'
mouse C.gamma. primer specific for all mouse Ig isotypes.
Similarly, two primer mixes containing 64 5' VK leader sequences
designed to amplify each of the VK mouse families was used in
combination with a single reverse primer specific to the mouse
kappa constant region in order to amplify and sequence the kappa
light chain. The VH and VL transcripts were amplified from 100 ng
total RNA using the Qiagen One Step RT-PCR kit as follows. A total
of four RT-PCR reactions were run for each hybridoma, two for the
VK light chain and two for the VH heavy chain. PCR reaction
mixtures included 1.5 .mu.L of RNA, 0.4 .mu.L of 100 .mu.M of
either heavy chain or kappa light chain primers (custom synthesized
by IDT), 5 .mu.L of 5.times.RT-PCR buffer, 1 .mu.L dNTPs, and 0.6
.mu.L of enzyme mix containing reverse transcriptase and DNA
polymerase. The thermal cycler program included the following
steps: RT step 50.degree. C. for 60 min., 95.degree. C. for 15 min.
followed by 35 cycles of (94.5.degree. C. for 30 seconds,
57.degree. C. for 30 seconds, 72.degree. C. for 1 min.), and a
final incubation at 72.degree. C. for 10 min. The extracted PCR
products were sequenced using the same specific variable region
primers as described above. PCR products were sent to an external
sequencing vendor (MCLAB) for PCR purification and sequencing
services.
[0369] FIG. 5A depicts the contiguous amino acid sequences of
numerous novel mouse light chain variable regions from anti-CLDN
antibodies (SEQ ID NOS: 21-57, odd numbers). FIG. 5B depicts the
contiguous amino acid sequences of novel mouse heavy chain variable
regions from the same anti-CLDN antibodies (SEQ ID NOS: 23-59, odd
numbers). Mouse light and heavy chain variable region nucleic acid
sequences are provided in FIG. 5C (SEQ ID NOS: 20-58, even
numbers). Taken together FIGS. 5A and 5B provide the annotated
sequences of 10 mouse anti-CLDN antibodies, termed SC27.1, SC27.22,
SC27.103, SC27.104, SC27.105, SC27.106, SC27.108 (identical to
SC27.109), SC27.201, SC27.203 and SC27.204. The amino acid
sequences are annotated to identify the framework regions (i.e.
FR1-FR4) and the complementarity determining regions (i.e.
CDRL1-CDRL3 in FIG. 5A or CDRH1-CDRH3 in FIG. 5B) defined as per
Kabat. The variable region sequences were analyzed using a
proprietary version of the Abysis database to provide the CDR and
FR designations. Though the CDRs are numbered according to Kabat
those skilled in art will appreciate that the CDR and FR
designations can also be defined according to Chothia, McCallum or
any other accepted nomenclature system.
[0370] The SEQ ID NOS of each particular antibody are sequential
odd numbers. Thus the monoclonal anti-CLDN antibody, SC27.1,
comprises amino acid SEQ ID NOS: 21 and 23 for the VL and VH,
respectively; and SC27.22 comprises SEQ ID NOS: 25 and 27 etc. The
corresponding nucleic acid sequence for each antibody amino acid
sequence is included in FIG. 5C and has the SEQ ID NO immediately
preceding the corresponding amino acid SEQ ID NO. Thus, for
example, the SEQ ID NOS of the nucleic acid sequences of the VL and
VH of the SC27.1 antibody are SEQ ID NOS: 20 and 22,
respectively.
Example 7
Generation of Chimeric and Humanized Anti-CLDN Antibodies
[0371] Chimeric anti-CLDN antibodies were generated using
art-recognized techniques as follows. Total RNA was extracted from
the anti-CLDN antibody-producing hybridomas using the method
described in Example 6 and the RNA was PCR amplified. Data
regarding V, D and J gene segments of the VH and VL chains of the
mouse antibodies were obtained from the nucleic acid sequences of
the anti-CLDN antibodies of the invention (see FIG. 5C for nucleic
acid sequences). Primer sets specific to the framework sequence of
the VH and VL chain of the antibodies were designed using the
following restriction sites: AgeI and XhoI for the VH fragments,
and XmaI and DraIII for the VL fragments. PCR products were
purified with a Qiaquick PCR purification kit (Qiagen), followed by
digestion with restriction enzymes AgeI and XhoI for the VH
fragments and XmaI and DraIII for the VL fragments. The VH and VL
digested PCR products were purified and ligated into IgH or IgK
expression vectors, respectively. Ligation reactions were performed
in a total volume of 10 .mu.L with 200 U T4-DNA Ligase (New England
Biolabs), 7.5 .mu.L of digested and purified gene-specific PCR
product and 25 ng linearized vector DNA. Competent E. coli DH10B
bacteria (Life Technologies) were transformed via heat shock at
42.degree. C. with 3 .mu.L ligation product and plated onto
ampicillin plates at a concentration of 100 .mu.g/mL. Following
purification and digestion of the amplified ligation products, the
VH fragment was cloned into the AgeI-XhoI restriction sites of the
pEE6.4 expression vector (Lonza) comprising HuIgG1 (pEE6.4HuIgG1)
and the VL fragment was cloned into the XmaI-DraIII restriction
sites of the pEE12.4 expression vector (Lonza) comprising a human
kappa light constant region (pEE12.4Hu-Kappa).
[0372] Chimeric antibodies were expressed by co-transfection of
either HEK-293T or CHO-S cells with pEE6.4HuIgG1 and
pEE12.4Hu-Kappa expression vectors. Prior to transfection the
HEK-293T cells were cultured in 150 mm plates under standard
conditions in Dulbecco's Modified Eagle's Medium (DMEM)
supplemented with 10% heat inactivated FCS, 100 .mu.g/mL
streptomycin and 100 U/mL penicillin G. For transient transfections
cells were grown to 80% confluency. 2.5 .mu.g each of pEE6.4HuIgG1
and pEE12.4Hu-Kappa vector DNA were added to 10 .mu.L HEK-293T
transfection reagent in 1.5 mL Opti-MEM. The mix was incubated for
30 min. at room temperature and added to cells. Supernatants were
harvested three to six days after transfection. For CHO-S cells,
2.5 .mu.g each of pEE6.4HuIgG1 and pEE12.4Hu-Kappa vector DNA were
added to 15 .mu.g PEI transfection reagent in 400 .mu.L Opti-MEM.
The mix was incubated for 10 min. at room temperature and added to
cells. Supernatants were harvested three to six days after
transfection. Culture supernatants containing recombinant chimeric
antibodies were cleared from cell debris by centrifugation at
800.times.g for 10 min. and stored at 4.degree. C. Recombinant
chimeric antibodies were purified with Protein A beads
[0373] Mouse anti-CLDN antibodies were humanized using a
proprietary computer-aided CDR-grafting method (Abysis Database,
UCL Business) and standard molecular engineering techniques as
follows. Human framework regions of the variable regions were
designed based on the highest homology between the framework
sequences and CDR canonical structures of human germline antibody
sequences, and the framework sequences and CDRs of the relevant
mouse antibodies. For the purpose of the analysis the assignment of
amino acids to each of the CDR domains was done in accordance with
Kabat numbering. Once the variable regions were selected, they were
generated from synthetic gene segments (Integrated DNA
Technologies). Humanized antibodies were cloned and expressed using
the molecular methods described above for chimeric antibodies.
[0374] The VL and VH amino acid sequences of the humanized
antibodies were derived from the VL and VH sequences of the
corresponding mouse antibody (e.g. hSC27.1 is derived from mouse
SC27.1). There were no framework changes or back mutations made in
the light or heavy chain variable regions of the four humanized
antibodies generated: hSC27.1, hSC27.22, hSC17.108 and
hSC27.204.
[0375] To address stability concerns, three variants of hSC27.22
were produced using different VH frameworks in the same VH1 family.
The variants were termed hSC27.22-VH1-8; hSC27.22-VH1-46;
hSC27.22-VH1-69. In addition, one variant of hSC27.108 was
constructed, termed hSC27.108v1, which shares the same heavy chain
as hSC27.108 (SEQ ID NO: 119) but differs in light chain compared
to hSC27.108. In addition, several variants of hSC27.204 were
generated, termed hSC27.204v1 through hSC27.204v15, all of which
share the same light chain (SEQ ID NO: 120) but differ in the heavy
chain. The heavy chains of hSC27.204 and hSC27.204v4 differ in a
single framework region mutation, T28D. hSC27.204v1 through
hSC27.204v3 and hSC27.204v5 through hSC27.204v7 incorporate
conservative mutations in the CDRs to address stability concerns.
Specifically, hSC27.204v1, hSC27.204v2, and hSC27.204v3 contain the
modifications N58K, N58Q, and T60N, respectively, on the hSC27.204
heavy chain background. Similarly, hSC27.204v5, hSC27.204v6, and
hSC27.204v7 contain the modifications N58K, N58Q, and T60N,
respectively on the hSC27.204v4 background. Lastly, variants
hSC27.204v8 and hSC27.204v9 do not include a back mutation at
position 93 of the heavy chain in order to minimize immunogenicity.
Specifically, variants hSC27.204v8, hSC27.204v9, hSC27.204v10,
hSC27.204v11, hSC27.204v12, hSC27.204v13, hSC27.204v14, and
hSC27.204v15 correspond to variants hSC27.204, hSC27.204v1,
hSC27.204v2, hSC27.204v3, hSC27.204v4, hSC27.204v5, hSC27.204 6,
and hSC27.204v7, respectively, except that variants 8-15 lack the
A93T back mutation.
[0376] In addition, 9 variants of the hSC27.22 humanized antibody
constant region were constructed. The first variant, hSC27.22ss1 is
a site specific variant and is described in more detail in Example
8 below. The other variants were constructed by substituting the
IgG isotype with either IgG2 (termed, "hSC27.22 IgG2") or mutated
forms of IgG4 (termed, "hSC27.22 IgG4 R409K"; "hSC27.22 IgG4
S228P"; "hSC27.22 IgG4 S228P K370E R409K"; "hSC27.22 IgG4 K370E";
"hSC27.22 IgG4 S228P K370E"; "hSC27.22 IgG4 C127S S228P"; "hSC27.22
IgG4 C127S K370E"; and "hSC27.22 IgG4 C127S S228P K370E"). Table 5
below shows a summary of the humanized anti CLDN antibodies and
their variants, numbered according to Kabat et al.
[0377] In each case, the binding affinity of the humanized antibody
was checked to ensure that it was substantially equivalent to the
corresponding mouse antibody. FIG. 5A depicts the contiguous amino
acid sequences of the VL of exemplary humanized antibodies and
their variants. FIG. 5B depicts the contiguous amino acid sequences
of the VH of exemplary humanized antibodies and their variants. The
nucleic acid sequences of the light and heavy chain variable
regions of the anti-CLDN humanized antibodies are provided in FIG.
5C.
[0378] FIG. 5D shows the full length sequences of the light and
heavy chains of exemplary humanized antibodies and their variants.
hSC27.1 (SEQ ID NOS: 75 and 76) and hSC27.22 (SEQ ID NOS: 77 and
78).
[0379] FIGS. 5E to 5H comprise annotated amino acid sequences
(numbered as per Kabat et al.) of the light and heavy chain
variable regions of hSC27.1 (FIG. 5E); hSC27.22 (FIG. 5F);
hSC27.108 (FIG. 5G); and hSC27.204 (FIG. 5H) humanized antibodies
showing CDRs as determined using Kabat, Chothia, ABM and Contact
methodology.
TABLE-US-00007 TABLE 5 human VH FR VH CDR human VK FR VK CDR mAb
Isotype human VH JH changes Changes human VK JK changes Changes
hSC27.1 IgG1 IGHV1-3*01 JH1 None None IGKV1-12*01 JK2 None None
hSC27.22 IgG1 IGHV1-3*01 JH6 None None IGKV4-1*01 JK2 None None
hSC27.22ss1 IgG1 C220S IGHV1-8*01 JH6 None None IGKV4-1*01 JK2 None
None hSC27.22-VH1-8 IgG1 IGHV1-8*01 JH6 None None IGKV4-1*01 JK2
None None hSC27.22 IgG1 IGHV1-46*01 JH6 None None IGKV4-1*01 JK2
None None VH1-46 hSC27.22 IgG1 IGHV1-69*01 JH6 None None IGKV4-1*01
JK2 None None VH1-69 hSC27.22 IgG2 IgG2 IGHV1-3*01 JH6 None None
IGKV4-1*01 JK2 None None hSC27.22 IgG4 IgG4 R409K IGHV1-3*01 JH6
None None IGKV4-1*01 JK2 None None R409K hSC27.22 IgG4 IgG4 S228P
IGHV1-3*01 JH6 None None IGKV4-1*01 JK2 None None S228P hSC27.22
IgG4 IgG4 S228P R409K IGHV1-3*01 JH6 None None IGKV4-1*01 JK2 None
None S228P K370E R409K K370E hSC27.22 IgG4 IgG4 K370E IGHV1-3*01
JH6 None None IGKV4-1*01 JK2 None None K370E hSC27.22 IgG4 IgG4
S228P K370E IGHV1-3*01 JH6 None None IGKV4-1*01 JK2 None None S228P
K370E hSC27.22 IgG4 IgG4 C127S S228P IGHV1-3*01 JH6 None None
IGKV4-1*01 JK2 None None C127S S228P hSC27.22 IgG4 IgG4 C127S K370E
IGHV1-3*01 JH6 None None IGKV4-1*01 JK2 None None C127S K370E
hSC27.22 IgG4 IgG4 C127S S228P IGHV1-3*01 JH6 None None IGKV4-1*01
JK2 None None C127S S228P K370E K370E hSC27.108 IgG1 IGHV1-18*01
JH1 None None IGKV3-11*01 JK4 None None hSC27.108 v1 IgG1
IGHV1-18*01 JH1 None None IGKV6-21*01 JK4 L47W K49Y None hSC27.204
IgG1 IGHV3-23*01 JH1 A93T K94G None IGKV1-16*01 JK4 None None
hSC27.204 v1 IgG1 IGHV3-23*01 JH1 A93T K94G N58K IGKV1-16*01 JK4
None None hSC27.204v2 IgG1 IGHV3-23*01 JH1 A93T K94G N58Q
IGKV1-16*01 JK4 None None hSC27.204v3 IgG1 IGHV3-23*01 JH1 A93T
K94G T60N IGKV1-16*01 JK4 None None hSC27.204v4 IgG1 IGHV3-23*01
JH1 T28D, A93T None IGKV1-16*01 JK4 None None K94G hSC27.204v5 IgG1
IGHV3-23*01 JH1 T28D, A93T N58K IGKV1-16*01 JK4 None None K94G
hSC27.204v6 IgG1 IGHV3-23*01 JH1 T28D, A93T N58Q IGKV1-16*01 JK4
None None K94G hSC27.204v7 IgG1 IGHV3-23*01 JH1 T28D, A93T T60N
IGKV1-16*01 JK4 None None K94G hSC27.204v8 IgG1 IGHV3-23*01 JH1
K94G None IGKV1-16*01 JK4 None None hSC27.204v9 IgG1 IGHV3-23*01
JH1 K94G N58K IGKV1-16*01 JK4 None None hSC27.204v10 IgG1
IGHV3-23*01 JH1 K94G N58Q IGKV1-16*01 JK4 None None hSC27.204v11
IgG1 IGHV3-23*01 JH1 K94G T60N IGKV1-16*01 JK4 None None
hSC27.204v12 IgG1 IGHV3-23*01 JH1 T28D, K94G None IGKV1-16*01 JK4
None None hSC27.204v13 IgG1 IGHV3-23*01 JH1 T28D, K94G N58K
IGKV1-16*01 JK4 None None hSC27.204v14 IgG1 IGHV3-23*01 JH1 T28D,
K94G N58Q IGKV1-16*01 JK4 None None hSC27.204v15 IgG1 IGHV3-23*01
JH1 T28D, K94G T60N IGKV1-16*01 JK4 None None
Example 8
Generation of Site-Specific Anti-CLDN Antibodies
[0380] An engineered human IgG1/kappa anti-CLDN site-specific
antibody was constructed comprising a native light chain (LC)
constant region and mutated heavy chain (HC) constant region,
wherein cysteine 220 (C220) in the upper hinge region of the HC,
which forms an interchain disulfide bond with cysteine 214 (C214)
in the LC, was substituted with serine (C220S). When assembled, the
HCs and LCs form an antibody comprising two free cysteines that are
suitable for conjugation to a therapeutic agent. Unless otherwise
noted, all numbering of constant region residues is in accordance
with the EU numbering scheme as set forth in Kabat et al.
[0381] The engineered antibody was generated as follows. The
nucleic acid sequence of the HC of the hSC27.22 antibody (SEQ ID
NO: 67) was codon optimized by DNA2.0 (Menlo Park, Calif.) to
generate the following nucleic acid sequence:
TABLE-US-00008 (SEQ ID NO: 178)
CAAGTGCAGCTCGTCCAGTCCGGTGCCGAAGTCAAGAAGCCGGGCGCATC
AGTGAAAGTGTCGTGCAAAGCCTCCGGGTACACCTTCACCTCATACTGGA
TGAACTGGGTCCGCCAAGCCCCGGGACAGAGACTGGAGTGGATGGGCATG
ATTCACCCATCCGATTCCGAGATCCGGCTGAACCAGAAGTTCAAGGACCG
CGTGACCATCACCCGGGACACCAGCGCCAGCACTGCCTACATGGAATTGA
GCTCGCTGCGGTCCGAGGATACCGCTGTGTACTATTGCGCGAGGATCGAC
TCCTACTACGGCTACCTTTTCTACTTCGACTACTGGGGACAAGGGACGAC
CGTGACTGTGTCGAGC.
[0382] The optimized nucleic acid was cloned onto an expression
vector containing the C220S mutation in the constant region of the
HC. The vector encoding the mutant C220S HC of hSC27.22 was
co-transfected in CHO-S cells with a vector encoding the native
IgG1 kappa LC of hSC27.22, and expressed using a mammalian
transient expression system. The engineered anti-CLDN site-specific
antibody containing the C220S mutant was termed hSC27.22ss1. The
amino acid sequence of the full length HC of the hSC27.22ss1 site
specific antibody is shown in FIG. 5D (SEQ ID NO: 122). The amino
acid sequence of the LC of hSC27.22ss1 is identical to that of
hSC27.22 (SEQ ID NO: 123).
[0383] Engineered human IgG4/kappa anti-CLDN site-specific
antibodies were also constructed comprising a native LC constant
region and mutated HC constant region, wherein cysteine 127 (C127)
in the CH1 of the IgG4 heavy chain, which forms an interchain
disulfide bond with cysteine 220 (C220) in the LC, was substituted
with serine (C127S). When assembled the HCs and LCs form an
antibody comprising two free cysteines that are suitable for
conjugation to a therapeutic agent. This modification was made
using the Quikchange Site Directed Mutagenesis Kit (Agilent)
according to the manufacturer's protocols using the IgG4 expression
vector as a template.
[0384] The engineered antibodies were generated as follows. The
codon optimized nucleic acid sequence of hSC27.22 (SEQ ID NO: 178),
was cloned onto an expression vector containing the C127S mutation
in the constant region of the HC. The vector encoding the mutant
C127S HC of hSC27.22 was co-transfected in CHO-S cells with a
vector encoding the native IgG1 kappa LC of hSC27.22, and expressed
using a mammalian transient expression system. The C127S
modification was applied to the various modified IgG4 constructs
generated as described in Example 7 above. The resulting IgG4 site
specific constructs are shown in Table 5 above and FIG. 5D and are
termed: hSC27.22 IgG4 S228P; hSC27.22 IgG4 R409K; hSC27.22 IgG4
S228P K370E R409K; hSC27.22 IgG4 K370E; hSC27.22 IgG4 S228P K370E;
hSC27.22 IgG4 C127S S228P; hSC27.22 IgG4 C127S K370E; and hSC27.22
IgG4 C127S S228P K370E.
[0385] The engineered anti-CLDN site specific antibodies were
characterized by SDS-PAGE to confirm that the correct mutants had
been generated. SDS-PAGE was conducted on a pre-cast 10%
Tris-Glycine mini gel from Life Technologies in the presence and
absence of a reducing agent such as DTT (dithiothreitol). Following
electrophoresis, the gels were stained with a colloidal coomassie
solution (data not shown). Under reducing conditions, two bands
corresponding to the free LCs and free HCs, were observed. This
pattern is typical of IgG molecules in reducing conditions. Under
non-reducing conditions, the band patterns were different from
native IgG molecules, indicative of the absence of a disulfide bond
between the HC and LC. A band around 98 kD corresponding to the
HC-HC dimer was observed. In addition, a faint band corresponding
to the free LC and a predominant band around 48 kD that
corresponded to a LC-LC dimer was observed. The formation of some
amount of LC-LC species is expected due to the free cysteines on
the c-terminus of each LC.
Example 9
Specificity of Anti-CLDN Antibodies
[0386] The mouse antibodies generated as described in Example 5,
were characterized to determine whether they cross reacted with
CLDN family members and orthologs of CLDN family members.
[0387] Flow cytometry analyses were performed as follows: HEK-293T
cells were stably transduced with (i) lentiviral vectors encoding
hCLDN6, mCLDN6, and rCLDN6; (ii) hCLDN9; or (iii) hCLDN4, mCLDN4
and rCLDN4, made as described in Example 4 above. 1.times.10.sup.5
HEK-293T cells stably transduced with the aforementioned expression
constructs were incubated at 4.degree. C. for 30 mins. with either
hSC27.1 or hSC27.22 antibodies, diluted to 10 .mu.g/ml into a final
volume of 50 .mu.l PBS/2% FCS. Following incubation, cells were
washed with 200 .mu.L PBS/2% FCS, pelleted by centrifugation,
supernatant was discarded, and cell pellets were resuspended in 50
.mu.L per sample DyeLight 649 labeled goat-anti-mouse IgG, Fc
fragment specific secondary antibody diluted 1:200 in PBS/2% FCS.
After a 15 min. incubation at 4.degree. C. cells were washed and
pelleted twice with PBS/2% FCS as previously described and
resuspended in 100 .mu.L PBS/2% FCS with 2 .mu.g/mL
4',6-diamidino-2-phenylindole dihydrochloride (DAPI). Samples were
analyzed by flow cytometry and live cells were assessed with
DyeLight 649 for fluorescence exceeding that of cells stained with
an isotype control antibody.
[0388] The flow cytometry assay described above resulted in the
identification of numerous anti-CLDN antibodies. Cross reactivity
was determined based on the change in geometric mean fluorescence
intensity (.DELTA.MFI) for the binding of the antibody to the cell
lines specifically overexpressing the indicated CLDN family member
versus the signal determined using a fluorescence minus one (FMO)
isotype-control (gray-fill) (FIG. 6A). Thus, the two hCLDN6-binding
antibodies SC27.1 and SC27.22 can be described as claudin
multireactive antibodies since they cross react in this assay with
three members of the human CLDN family: hCLDN6, hCLDN4 and hCLDN9.
SC27.1 and SC27.22 antibodies also bound to mouse and rat orthologs
of CLDN4 and CLDN9 (data not shown).
[0389] To test the ability of various additional mouse antibodies
to bind to CLDN family members, flow cytometry was performed using
cell lines overexpressing human CLDN4, CLDN6 or CLND9 that had been
incubated with 10 .mu.g/mL of purified primary anti-CLDN antibody,
or a mouse IgG2b control antibody, followed by incubation with an
Alexa 647 anti-mouse secondary antibody. As shown in FIG. 6B, all
the antibodies bound to CLDN6, whereas some were CLDN6-specific
(e.g. SC27.102, SC27.105, and SC27.108), and others were
multireactive and bound to both CLDN6 and CLDN9 (e.g., SC27.103 and
SC27.204), or to CLDN6 and CLDN4 (e.g., SC27.104). Thus a wide
range of multireactive binding profiles was obtained for the
antibodies of the invention.
[0390] To compare the apparent binding affinity of the
multireactive anti-CLDN antibodies for CLDN6 and CLDN9, flow
cytometry was performed with a serial dilution of humanized
anti-CLDN antibody hSC27.22. The antibody was serially diluted to
concentrations ranging from 50 .mu.g/ml to 100 .mu.g/ml and was
added to a 96 well plate containing HEK-293T cells overexpressing
CLDN6 or CLDN9, and kept on ice for one hour. A secondary
anti-human antibody (Jackson ImmunoResearch Cat. #109-605-098) was
added and incubated for one hour in the dark. The cells were washed
twice in PBS after which Fixable Viability Dye (eBioscience Cat
#65-0863-14) was added for 10 mins. Following additional washing
with PBS, cells were fixed with paraformaldehyde (PFA) and read on
a BD FACS Canto II flow cytometer in accordance with the
manufacturer's instructions. MFI values were normalized using
fluorescent microspheres (Bangs Laboratories) according to
manufacturer's instructions. Normalized maximal MFI values observed
for the binding of the antibody to either CLDN6 or CLDN9 expressing
cells were used to transform the data into fraction maximal binding
for each overexpressing cell, using the equation: fraction maximal
binding=(observed normalized MFI/maximal normalized MFI). Apparent
EC50 values for the binding of hSC27.22 to each cell line were then
calculated using a four parameter variable slope curve fitting for
a log (inhibitor) vs. response model supplied in the GraphPad Prism
software package (La Jolla, Calif.). FIG. 6C shows that the
humanized multireactive anti-CLDN6 antibody, hSC27.22, has an
apparent EC50 for CLDN6 which is substantially the same as that for
CLDN9. (apparent EC50 CLDN6-3.45 .mu.g/mL (r.sup.2 for goodness of
fit=0.9987, 99% confidence bounds: 2.51-4.75 .mu.g/mL); apparent
EC50 CLDN9-4.66 .mu.g/mL (r.sup.2 for goodness of fit=0.9998, 99%
confidence bounds: 4.09-5.31 .mu.g/mL)).
Example 10
Detection of CLDN Protein Expression on PDX Tumors Using Flow
Cytometry
[0391] Flow cytometry was used to assess the ability of the two
representative hCLDN6-binding antibodies hSC27.1 and hSC27.22 to
specifically detect the presence of hCLDN proteins on the surface
of PDX tumor cells. Isotype-stained and fluorescence minus one
(FMO) controls were employed to confirm staining specificity.
[0392] Art-recognized enzymatic tissue digestion techniques were
used to obtain single cell suspensions of PDX tumor cells (see, for
example, U.S.P.N. 2007/0292414). PDX tumors were harvested,
dissociated, and co-stained with commercially available anti-mouse
CD45 and H-2kD antibodies (to demarcate mouse cells) and anti-human
EpCAM and anti-CLDN antibodies. The anti-hCLDN antibody, hSC27.1,
demonstrated positive staining on a subset of human (i.e. mCD45 and
H-2kD negative) EpCAM-positive tumor cells, including OV-S (e.g.,
OV44, OV54), OV-PS (e.g. OV63MET), PA, LU-SCC (e.g., LU22), LU-Ad
(e.g., LU134, LU135), and LIV (FIG. 7). Isotype control antibodies
and FMO controls were employed to confirm staining specificity as
is standard practice in the art. Flow cytometry was performed using
a BD FACS Canto II flow cytometer in accordance with the
manufacturer's instructions.
[0393] The levels of hCLDN staining varied across different PDX
tumor cell lines, where some tumor cells did not stain at all (data
not shown) while other tumor cell lines exhibited nearly uniformly
positive staining of the human tumor cells (e.g. OV63MET) compared
to isotype controls (FIG. 7). These data suggest that hCLDNs are
expressed on the surface of a subpopulation of human tumor
subtypes, which may be amenable to treatment using anti-CLDN
antibodies or ADCs of the invention.
Example 11
Enrichment of CLDN Expression in Cancer Stem Cell Populations
[0394] Tumor cells can be divided broadly into two types of cell
subpopulations: non-tumorigenic cells (NTG) and tumor initiating
cells or tumorigenic cells. Tumorigenic cells have the ability to
form tumors when implanted into immunocompromised mice, whereas
non-tumorigenic cells do not. Cancer stem cells (CSCs) are a subset
of tumorigenic cells and are able to self-replicate indefinitely
while maintaining the capacity for multilineage
differentiation.
[0395] To confirm the observations in Examples 1 and 2, which
showed overexpression of CLDN4, CLDN6 and CLDN9 in CSC
subpopulations of various tumors, and to determine whether the
anti-CLDN antibodies of the invention are able to detect
tumorigenic CSC populations, PDX tumors were dissociated into
single cell suspensions as described in Example 10 above and
selective markers, CD46.sup.hiCD324.sup.+, were used to enrich for
CSC tumor cell subpopulations (see WO 2012/031280) as follows.
[0396] PDX tumor single cell suspensions were incubated with the
following antibodies: anti-CLDN SC27.1; anti-human EPCAM;
anti-human CD46; anti-human CD324; and anti-mouse CD45 and H-2kD
antibodies. The tumor cells were then assessed for staining by flow
cytometry using a BD FACS Canto II flow cytometer. The human
EPCAM.sup.+CD46.sup.hiCD324.sup.+ CSC tumor cell subpopulations of
OV-S(e.g., OV44 and OV54MET), OV-PS (e.g. OV91MET), PA, LU-Ad
(e.g., LU135), and LU-Sq (e.g., LU22) PDX tumors demonstrated
positive staining with the anti-CLDN SC27.1 antibody, whereas NTG
cells (CD46.sup.lo/- and/or CD324.sup.-) demonstrated significantly
less staining with anti-CLDN antibodies (FIG. 8A). Isotype control
antibodies and FMO controls were employed to confirm staining
specificity as is standard practice in the art. A table summarizing
the differential staining of anti-CLDN antibodies observed on the
surface of CSC and NTG cells is shown in FIG. 8A, with expression
enumerated as the change in geometric mean fluorescence intensity
(.DELTA.MFI) between the indicated anti-CLDN antibody and the
isotype control for the respective tumor cell subpopulations. These
data confirm the expression of hCLDN proteins on CSCs and again
suggest anti-CLDN antibodies may be effective for the treatment of
cancer.
[0397] To determine whether CLDN expression in tumors could be
correlated with enhanced tumorigenicity, the following study was
conducted. Human OV PDX tumor samples (OV91MET) were grown in
immunocompromised mice and were resected after the tumor reached
800-2,000 mm.sup.3. The tumors were dissociated into single cell
suspensions using art-recognized enzymatic digestion techniques
(see, for example, U.S.P.N. 2007/0292414). Human OV PDX tumor cells
were stained with mouse anti-CD45 or anti-H2kD antibodies, and with
anti-ESA antibodies to differentiate between human tumor cells and
mouse cells. The tumors were also stained with anti-CLDN antibody
(SC27.22) and then sorted using a FACSAria.TM. Flow Cytometer (BD
Biosciences). The human OV PDX tumor cells were separated into
CLDN.sup.+ and CLDN.sup.- subpopulations. Five female NOD/SCID
immunocompromised mice were injected subcutaneously with 200
CLDN.sup.+ OV tumor cells; and five mice were injected with 200
CLDN.sup.- OV tumor cells. Tumor volumes were measured on a weekly
basis for four months.
[0398] FIG. 8B shows that CLDN.sup.+ (closed circles) tumor cells
were able to functionally reconstitute tumors in vivo, whereas
CLDN.sup.- tumors (open circles) were not. Thus, tumor cells
expressing CLDN were much more tumorigenic than those tumor cells
that did not express CLDN, suggesting that the CLDN protein can
functionally define a tumorigenic subpopulation within human
tumors, and supporting the concept that selected anti-CLDN ADCs can
be used to target a tumorigenic subpopulation of tumor cells, which
could result in significant tumor regression and prevention of
tumor recurrence.
Example 12
Anti-CLDN Antibodies Facilitate Delivery of Cytotoxic Agents In
Vitro
[0399] To determine whether anti-CLDN antibodies are able to
internalize and mediate the delivery of cytotoxic agents to live
tumor cells, an in vitro cell killing assay was performed using
selected anti-CLDN antibodies and saporin linked to a secondary
anti-mouse antibody FAB fragment. Saporin is a plant toxin that
deactivates ribosomes, thereby inhibiting protein synthesis and
resulting in the death of the cell. Saporin is only cytotoxic
inside the cell where it has access to ribosomes, but is unable to
internalize on its own. Therefore, saporin-mediated cellular
cytotoxicity in these assays is indicative of the ability of the
anti-mouse FAB-saporin conjugate to internalize into the target
cell only upon binding and internalization of anti-CLDN
antibodies.
[0400] Single cell suspensions of HEK-293T cells and HEK-293T cells
overexpressing hCLDN6, hCLDN4, or hCLDN9 were plated at 500 cells
per well into BD Tissue Culture plates (BD Biosciences). One day
later, 250 pM of purified SC27.1, SC27.22, or isotype control
(mIgG1) antibodies and a fixed concentration of 2 nM anti-Mouse IgG
FAB-saporin conjugate (Advanced Targeting Systems) were added to
the culture. The HEK-293T cells were incubated for 72 hours post
antibody treatment. After the incubation, viable cells were
enumerated using CellTiter-Glo.RTM. (Promega) as per the
manufacturer's instructions. Raw luminescence counts using cultures
containing cells incubated only with the secondary FAB-saporin
conjugate were set as 100% reference values and all other counts
calculated accordingly. Both of the anti-CLDN antibodies, SC27.1
and SC27.22, at a concentration of 250 pM effectively killed
HEK-293T cells overexpressing hCLDN6 and hCLDN9 (FIG. 9A), whereas
the mouse IgG1 isotype control antibody (mIgG1) at the same
concentration did not. Naive HEK-293T cells were not effectively
killed by the treatment whereas HEK-293T cells overexpressing
hCLDN4 were effectively killed by SC27.1 but were not killed by
SC27.22 treatment at the dose tested. The dashed horizontal line
represents the level at which no cytotoxicity was observed.
[0401] In order to determine the apparent IC50 of additional
antibodies for CLDN4, CLDN6 or CLDN9, the experiment described in
the paragraph above was repeated with titrations of antibodies,
across a concentration range of 0.15 nM to 1000 nM (FIG. 9B). The
percentage of cell killing observed at each antibody concentration
was enumerated by CellTiter-Glo.RTM. as described above, and a
curve was fitted to the resulting data in order to calculate an
apparent IC50 for the killing activity of antibody on each cell
line. Antibodies which had an apparent IC50 of >2000 nM were
deemed not to kill a particular cell line and are denoted as "NK"
in FIG. 9B. A control mouse IgG1 antibody also did not kill any of
the cell lines tested. Although this cytotoxicity assay measures
the ability of various antibodies to mediate delivery of a
cytotoxin via internalization of bound antigen rather than
providing a direct measure of antibody binding affinity, the
apparent IC50 of the antibodies shown in FIG. 9B in general
correlates well with the single point flow cytometry data presented
in FIG. 6B. For example, in both experiments SC27.108 is shown to
be CLDN6-specific (apparent IC50=100 nM). Similarly, by flow
cytometry SC27.103 shows strong binding to CLDN6 and moderate
binding to CLDN9, which correlates with an apparent IC50 value of
58 nM for CLDN6 and 466 nM for CLDN9. However, it is also clear
that detectable binding above background does not always result in
detectable killing (e.g., SC27.104 binds to CLDN9 (see FIG. 6B) but
is not able to effectively internalize and kill
CLDN9-overexpressing cells (see FIG. 9B); whereas SC27.201 binds
CLDN9 (see FIG. 6B) and is able to internalize into cells
expressing CLDN9 and kill those cells (see FIG. 9B)).
[0402] Together, the above results demonstrate the ability of
multireactive anti-CLDN antibodies to mediate internalization and
their ability to deliver cytotoxic payloads, supporting the
hypothesis that anti-CLDN antibodies may have therapeutic utility
as the targeting moiety for an ADC.
Example 13
Detection of CLDN6 on the Surface of Tumors Using
Immunohistochemistry
[0403] To assess the extent of CLDN6 protein expression in tumors,
immunohistochemistry (IHC) was performed on formalin fixed paraffin
embedded (FFPE) PDX tumors and tissue microarrays (TMAs) of primary
ovarian tumors (Oklahoma University).
[0404] Planar sections of cell pellet blocks were cut and mounted
on glass microscope slides. After xylene de-paraffinization 5 .mu.m
sections were pre-treated with Antigen Retrieval Solution (Dako)
for 20 min. at 99.degree. C., cooled to 75.degree. C. and then
treated with 0.3% hydrogen peroxide in PBS followed by treatment
with Avidin/Biotin Blocking Solution (Vector Laboratories). FFPE
slides were then blocked with 10% donkey serum in 3% BSA in PBS
buffer and incubated with a primary anti-CLDN6 rabbit polyclonal
antibody purchased from IBL America (Catalog #18865), diluted to 10
.mu.g/ml in 3% BSA/PBS, for 30 min. at room temperature. FFPE
slides were incubated with biotin-conjugated donkey anti-rabbit
antibody (Vector Laboratories), diluted to 2.5 .mu.g/ml in 3%
BSA/PBS, for 30 min. at room temperature followed by incubation
with streptavidin-HRP (ABC Elite Kit; Vector Laboratories).
Chromogenic detection was developed with 3,3'-diaminobenzidine
(Thermo Scientific) for 5 min. at room temperature and tissues were
counterstained with Meyer's hematoxylin (IHC World), washed with
alcohol and immersed in xylene.
[0405] To confirm the specificity of the primary anti-CLDN6
antibody, IHC was performed on FFPE slides of HEK-293T cells
overexpressing hCLDN6, hCLDN4 or hCLND9. The anti-CLDN6 polyclonal
antibody specifically stained hCLDN6-overexpressing HEK-293T cell
pellets but did not stain overexpressing cell lines of hCLDN4 and
hCLDN9 (data not shown).
[0406] FIG. 10A shows a summary overview of hCLDN6 expression in
OV, BR, and LU PDX tumors, as determined by IHC. Staining intensity
was scored from no staining (-) to high staining intensity (+++).
The percentage of tumor cells that expressed CLDN6 is also noted.
CLDN6 expression was observed in LU, BR and OV tumors with many PDX
lines showing expression in 90% of the cells on the FFPE slide.
[0407] To determine the penetrance of hCLDN6 expression in ovarian
cancer patients, IHC was also performed on FFPE slides generated
from TMAs made from 125 primary ovarian tumors resected from cancer
patients (Oklahoma University). The H-Score was generated utilizing
the Leica Biosystems Tissue IA software on digitally scanned images
of the TMAs. Briefly, staining preferences specific to hCLDN6 were
assigned in the Measure Stained Cells Algorithm under the Tissue IA
Optimiser. TMA cores were then individually annotated so that the
image analysis would only analyze tumor cells and not other tissue
constituents such as stroma. The TMA was analyzed using the hCLDN6
Membrane Stain algorithm which produced an H-score. The H-score
algorithm was calculated for membrane staining of tumor cells using
the following formula; H-Score=(% staining intensity at 0)*0+(%
staining intensity at 1+)*1+(% staining intensity at 2+)*2+(%
staining intensity at 3+)*3. Thus, this score produces a continuous
variable that ranges from 0 to 300. The results in the table in
FIG. 10B show level of expression of CLDN6 in the 125 tumor samples
of the TMA, with 70% of the tumors expressing some level of
CLDN6.
[0408] Together, these IHC data demonstrate that CLDN6 is expressed
on the cell surface of ovarian, breast and lung tumors as well as
in primary human tumors, reconfirming that claudins are relevant
targets for the development of antibody and ADC therapeutics for
treatment of a significant number of cancer patients. Anti-CLDN6
may have diagnostic utility in these and possibly additional cancer
indications.
Example 14
Preparation of Anti-CLDN6 Antibody-Drug Conjugates
[0409] Anti-CLDN antibody drug conjugates (ADCs) are prepared
having the Ab-[L-D] structure, where Ab refers to the anti-CLDN
antibody, L refers to an optional linker (e.g. a linker comprising
a terminal maleimido moiety with a free sulfhydryl group) and D
refers to a drug or cytotoxin (e.g. auristatins, calicheamicin
etc.). Each ADC comprises an anti-CLDN antibody covalently linked
to a linker-drug. ADCs are synthesized and purified using
techniques known in the art, for example, essentially as follows.
The cystine bonds of anti-CLDN antibodies are partially reduced
with a pre-determined molar addition of mol
tris(2-carboxyethyl)-phosphine (TCEP) per mol antibody for 90 min.
at 20.degree. C. in phosphate buffered saline (PBS) with 5 mM EDTA.
The linker-drug, dissolved in dimethyl acetamide (DMA), is added at
a ratio of 3 mol/mol anti-CLDN antibody. The reaction is allowed to
proceed for 30 min. Using a 10 mM stock solution of N-acetyl
cysteine (NAC) prepared in water, the reaction is quenched with the
addition of excess NAC to linker-drug. After a minimum quench time
of 20 mins., the pH is adjusted to 6.0 with the addition of 0.5 M
acetic acid and buffer exchanged by diafiltration into
diafiltration buffer using a 30 kDa membrane. The dialfiltered
anti-CLDN ADC is then formulated with sucrose and polysorbate-20 to
the target final concentration. The resulting anti-CLDN ADCs are
analyzed for protein concentration (by measuring UV), aggregation
(SEC), drug to antibody ratio (DAR) by reverse-phase HPLC (RP-HPLC)
and in vitro cytotoxicity.
Example 15
Conjugation of Site Specific Anti-CLDN Antibodies Using a Selective
Reduction Process
[0410] Anti-CLDN antibody drug conjugates (ADCs) are prepared
having the Ab-[L-D] structure as described in Example 14 above,
wherein the Ab moiety is a site specific antibody, for example,
hSC27.22ss1, generated as set forth in Example 8 above. The desired
product is an ADC that is maximally conjugated on the unpaired
cysteine (C214 in the case of IgG1 site specific antibodies or C127
on IgG4 site specific antibodies) on each LC constant region and
that minimizes ADCs having a drug to antibody ratio (DAR) which is
greater than 2 (DAR>2) or less than 2 (DAR<2) while
maximizing ADCs having a DAR of 2 (DAR=2).
[0411] In order to further improve the specificity of the
conjugation and homogeneity of the final site-specific ADC, the
site specific antibody (e.g. "hSC27.22ss1" or "hSC27.22 IgG4 C127S
S228P") is selectively reduced using, for example, a process
comprising a stabilizing agent (e.g. L-arginine) and a mild
reducing agent (e.g. glutathione) prior to conjugation with the
linker-drug, followed by preparative hydrophobic interaction
chromatography (HIC) that is used to separate the different DAR
species. The above procedures are conducted, for example,
essentially as described below.
[0412] A preparation of the site specific antibody is partially
reduced in a buffer containing 1M L-arginine/5 mM glutathione,
reduced (GSH)/5 mM EDTA, pH 8.0 for a minimum of one hour at room
temperature. All preparations are then buffer exchanged into a 20
mM Tris/3.2 mM EDTA, pH 8.2 buffer using a 30 kDa membrane
(Millipore Amicon Ultra) to remove the reducing buffer. The
resulting partially reduced preparations are then conjugated to a
cytotoxin (e.g. auristatin, calicheamicin etc.) via a linker (e.g.
maleimide linker) for a minimum of 30 mins. at room temperature.
The reaction is then quenched with the addition of excess NAC to
linker-drug using a 10 mM stock solution of NAC prepared in water.
After a minimum quench time of 20 mins., the pH is adjusted to 6.0
with the addition of 0.5 M acetic acid. The site specific ADC is
buffer exchanged into diafiltration buffer using a 30 kDa membrane.
The site specific ADC preparation is then diluted with a high salt
buffer to increase the conductivity to promote binding onto the
resin, and then loaded on a Butyl HP resin chromatography column
(GE Life Sciences). A decreasing salt gradient is then employed to
separate the different DAR species based on hydrophobicity, where
DAR=0 species elute first, followed by DAR=1, DAR=2, and then
higher DAR species.
[0413] The final ADC "HIC purified DAR=2" preparation is analyzed
using RP-HPLC to determine the percent conjugation on the HCs and
LCs and the DAR distribution. The samples are also analyzed using
analytical HIC to determine the amount of DAR=2 species relative to
the unwanted DAR>2 and DAR<2 species.
Example 16
Humanized Anti-CLDN Antibody Drug Conjugates Suppress Tumor Growth
In Vivo
[0414] The anti-CLDN ADCs, generated, for example, as described in
Examples 14 and 15 above, are tested using art-recognized
techniques, essentially as described below, to demonstrate their
ability to suppress ovarian tumor growth in immunodeficient
mice.
[0415] PDX tumor lines expressing CLDN and control tumor lines
which do not express CLDN are grown subcutaneously in the flanks of
female NOD/SCID mice using art-recognized techniques. Tumor volumes
and mouse weights are monitored once or twice per week. When tumor
volumes reach 150-250 mm.sup.3, mice are randomly assigned to
treatment groups and injected intraperitoneally with a single dose
of 1 or 2 mg/kg humanized anti-CLDN ADC, a single dose of 2 mg/kg
anti-hapten control human IgG ADC or vehicle control, for example,
0.9% saline or 5% glucose. Following treatment, tumor volumes and
mouse weights are monitored until tumors exceed 800 mm.sup.3 or the
mice become sick. Mice treated with humanized anti-CLDN ADC that do
not exhibit any adverse health effects beyond those typically seen
in immunodeficient, tumor-bearing NOD/SCID mice and that
effectively reduce tumor volume compared to control IgG ADC and
vehicle are selected for further analysis including toxicity
studies.
Example 17
Reduction of Cancer Stem Cell Frequency by Anti-CLDN Antibody-Drug
Conjugates
[0416] As demonstrated in Example 11 CLDN expression is associated
with cancer stem cells. Accordingly, to demonstrate that treatment
with anti-CLDN ADCs reduces the frequency of cancer stem cells
(CSC) that are known to be drug resistant and to fuel tumor
recurrence and metastasis, in vivo limiting dilution assays (LDA)
are performed, for example, essentially as described below.
[0417] PDX tumors (e.g. melanoma or ovarian) are grown
subcutaneously in immunodeficient mice. When tumor volumes average
150 mm.sup.3-250 mm.sup.3 in size, the mice are randomly segregated
into two groups. One group is injected intraperitoneally with a
human IgG1 conjugated to a drug as a negative control; and the
other group is injected intraperitoneally with an anti-CLDN ADC
(e.g., as prepared in Examples 14 and 15). One week following
dosing, two representative mice from each group are euthanized and
their tumors are harvested and dispersed to single-cell
suspensions. The tumor cells from each treatment group are then
harvested, pooled and disaggregated as previously described in
Example 1. The cells are labeled with FITC conjugated anti-mouse
H2kD and anti-mouse CD45 antibodies to detect mouse cells; EpCAM to
detect human cells; and DAPI to detect dead cells. The resulting
suspension is then sorted by FACS using a BD FACS Canto II flow
cytometer and live human tumor cells are isolated and
collected.
[0418] Four cohorts of mice are injected with either 1250, 375, 115
or 35 sorted live, human cells from tumors treated with anti-CLDN
ADC. As a negative control four cohorts of mice are transplanted
with either 1000, 300, 100 or 30 sorted live, human cells from
tumors treated with the control IgG1 ADC. Tumors in recipient mice
are measured weekly, and individual mice are euthanized before
tumors reach 1500 mm.sup.3. Recipient mice are scored as having
positive or negative tumor growth. Positive tumor growth is defined
as growth of a tumor exceeding 100 mm.sup.3.
[0419] Poisson distribution statistics (L-Calc software, Stemcell
Technologies) is used to calculate the frequency of CSCs in each
population.
Sequence CWU 1
1
1781107PRTHomo sapiensMISC_FEATUREKappa light chain (LC) constant
region protein 1Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro
Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys
Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys
Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 2329PRTHomo
sapiensMISC_FEATUREIgGI heavy chain (HC) constant region protein
2Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1
5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys
Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135
140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260
265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro
Gly 325 38PRTMus musculus 3Arg Glu Ser Glu Arg Val Glu Asp 1 5
48PRTMus musculus 4Arg Glu Ser Glu Arg Val Glu Asp 1 5 58PRTMus
musculus 5Arg Glu Ser Glu Arg Val Glu Asp 1 5 68PRTMus musculus
6Arg Glu Ser Glu Arg Val Glu Asp 1 5 78PRTMus musculus 7Arg Glu Ser
Glu Arg Val Glu Asp 1 5 88PRTMus musculus 8Arg Glu Ser Glu Arg Val
Glu Asp 1 5 98PRTMus musculus 9Arg Glu Ser Glu Arg Val Glu Asp 1 5
108PRTMus musculus 10Arg Glu Ser Glu Arg Val Glu Asp 1 5 118PRTMus
musculus 11Arg Glu Ser Glu Arg Val Glu Asp 1 5 128PRTMus musculus
12Arg Glu Ser Glu Arg Val Glu Asp 1 5 138PRTMus musculus 13Arg Glu
Ser Glu Arg Val Glu Asp 1 5 148PRTMus musculus 14Arg Glu Ser Glu
Arg Val Glu Asp 1 5 158PRTMus musculus 15Arg Glu Ser Glu Arg Val
Glu Asp 1 5 168PRTMus musculus 16Arg Glu Ser Glu Arg Val Glu Asp 1
5 178PRTMus musculus 17Arg Glu Ser Glu Arg Val Glu Asp 1 5
188PRTMus musculus 18Arg Glu Ser Glu Arg Val Glu Asp 1 5 198PRTMus
musculus 19Arg Glu Ser Glu Arg Val Glu Asp 1 5 20321DNAMus
musculusmisc_featureSC27.1 Light Chain Variable Region 20gacatccaga
tgacacaatc ttcatcctcc ttttctgtat ctctaggaga cagagtcacc 60attacttgca
aggcaagtga agacatatat aatcggttag cctggtatca gcagaaacca
120ggaaatgctc ccaggctctt aatatctggt gcaaccagtt tggaaactgg
gactccttca 180agattcagtg gcagtggatc tggaaaggat tacactctca
gtattaccag tcttcggact 240gaagatgctg ctacttatta ctgtcaacaa
tattggagta ctccactcac gttcggtact 300gggaccaagc tggagctgaa a
32121107PRTMus musculusMISC_FEATURESC27.1 Light Chain Variable
Region 21Asp Ile Gln Met Thr Gln Ser Ser Ser Ser Phe Ser Val Ser
Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Glu Asp
Ile Tyr Asn Arg 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Asn
Ala Pro Arg Leu Leu Ile 35 40 45 Ser Gly Ala Thr Ser Leu Glu Thr
Gly Thr Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Lys Asp
Tyr Thr Leu Ser Ile Thr Ser Leu Arg Thr 65 70 75 80 Glu Asp Ala Ala
Thr Tyr Tyr Cys Gln Gln Tyr Trp Ser Thr Pro Leu 85 90 95 Thr Phe
Gly Thr Gly Thr Lys Leu Glu Leu Lys 100 105 22357DNAMus
musculusmisc_featureSC27.1 Heavy Chain Variable Region 22gaggtccagc
tgcaagagtc tagacctgag ctggtgaagc ctggggcttc agtgaagata 60tcctgcaaga
cttctggata cacattcact gaatacacct tgcactgggt gaagcagagt
120catggaaaga gccttgagtg gattggaggt attaatccta acaatggtga
tactatctac 180aaccagaaat tcaagggcaa ggccacattg actgtagaca
agtcctccag cacagcctac 240atggagctcc gcagcctgac atctgaatat
tctgcagtct attactgtgc aagaagggcg 300attacggtct atgctatgga
ctactggggt caaggtacct cagtcaccgt ctcctca 35723119PRTMus
musculusMISC_FEATURESC27.1 Heavy Chain Variable Region 23Glu Val
Gln Leu Gln Glu Ser Arg Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15
Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr 20
25 30 Thr Leu His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp
Ile 35 40 45 Gly Gly Ile Asn Pro Asn Asn Gly Asp Thr Ile Tyr Asn
Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser
Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Thr Ser Glu
Tyr Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Ala Ile Thr Val
Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Ser Val Thr Val
Ser Ser 115 24333DNAMus musculusmisc_featureSC27.22 Light Chain
Variable Region 24gacattgtgc tgacacagtc tcctgcttcc ttagctgtat
ctctggggca gagggccacc 60atctcatgca gggccagcca gactgtcagt acatctagct
atagttatat gcactggttc 120caacagaaac caggacagcc acccaaactc
ctcatcaagt ttgcatccaa cgtagaatct 180ggggtccctg ccagattcag
tggcagtggg tctgggacag acttcaccct caacatccat 240cctgtggagg
aggaggatat ttcaacatat tactgtcagc acagttggga gattccgtgg
300acgttcggtg gaggcaccaa gctggaaatc aaa 33325111PRTMus
musculusMISC_FEATURESC27.22 Light Chain Variable Region 25Asp Ile
Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15
Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Thr Val Ser Thr Ser 20
25 30 Ser Tyr Ser Tyr Met His Trp Phe Gln Gln Lys Pro Gly Gln Pro
Pro 35 40 45 Lys Leu Leu Ile Lys Phe Ala Ser Asn Val Glu Ser Gly
Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Glu Glu Asp Ile Ser Thr
Tyr Tyr Cys Gln His Ser Trp 85 90 95 Glu Ile Pro Trp Thr Phe Gly
Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 26366DNAMus
musculusmisc_featureSC27.22 Heavy Chain Variable Region
26caggtccaac tgcagcagcc tggggctgag ctggtgaggc ctggagcttc agtgaagctg
60tcctgcaagg cttctggcta caccttcacc agctactgga tgaactgggt gaagcagagg
120cctggacaag gccttgaatg gattgccatg attcatcctt ccgatagtga
aattaggtta 180aatcagaagt tcaaggacaa ggccacattg actgtagaca
gatcctccag cacagcctac 240atgcaactca gcagcccgac atctgaggac
tctgcggtct attactgtgc aagaattgat 300agttattatg gttacctgtt
ttactttgac tactggggcc aaggcaccac tctcacagtc 360tcctca
36627122PRTMus musculusMISC_FEATURESC27.22 Heavy Chain Variable
Region 27Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro
Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr
Phe Thr Ser Tyr 20 25 30 Trp Met Asn Trp Val Lys Gln Arg Pro Gly
Gln Gly Leu Glu Trp Ile 35 40 45 Ala Met Ile His Pro Ser Asp Ser
Glu Ile Arg Leu Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu
Thr Val Asp Arg Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser
Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Ile Asp Ser Tyr Tyr Gly Tyr Leu Phe Tyr Phe Asp Tyr Trp 100 105 110
Gly Gln Gly Thr Thr Leu Thr Val Ser Ser 115 120 28324DNAMus
musculusmisc_featureSC27.103 Light Chain Variable Region
28caaattgttc tcacccagtc tccagcaatc atgtctgcat ctctagggga acgggtcacc
60atgacctgca ctgccagctc aagtgtaagt tccagttact tgcactggta ccagcagaag
120ccaggatcct cccccacact ctggatttat aggacatccg acctggcttc
tggagtccca 180gctcgcttca gtggcagtgg atctgggacc tcttactctc
tcacaatcag cagcatggag 240gctgaagatg ctgccactta ttactgccac
cagtatcatc gttccccgtg gacgttcggt 300ggaggcacca ggctggaaat caaa
32429108PRTMus musculusMISC_FEATURESC27.103 Light Chain Variable
Region 29Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser
Leu Gly 1 5 10 15 Glu Arg Val Thr Met Thr Cys Thr Ala Ser Ser Ser
Val Ser Ser Ser 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly
Ser Ser Pro Thr Leu Trp 35 40 45 Ile Tyr Arg Thr Ser Asp Leu Ala
Ser Gly Val Pro Ala Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr
Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu 65 70 75 80 Ala Glu Asp Ala
Ala Thr Tyr Tyr Cys His Gln Tyr His Arg Ser Pro 85 90 95 Trp Thr
Phe Gly Gly Gly Thr Arg Leu Glu Ile Lys 100 105 30354DNAMus
musculusmisc_featureSC27.103 Heavy Chain Variable Region
30gaggtccacc tgcaacagtc tggacctgag ctagtgaagc ctggaggttc aatgaagata
60tcctgcaagg cttctggtta ctcattcact ggctacacca tgaactgggt gaagcagagc
120catggaaaga accttgagtg gattggactt tttaatcctt acaatggtgg
tactagttat 180aaccagaagt tcaagggcaa ggccacatta actgtagaca
agtcatccag cacagcctac 240atggagctcc tcagtctgac atctgaggac
tctgcagtct attactgtgc aagatgctat 300aggtacgacg gtcttgacta
ctggggccaa ggcaccactc tcacagtctc ctca 35431118PRTMus
musculusMISC_FEATURESC27.103 Heavy Chain Variable Region 31Glu Val
His Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Gly 1 5 10 15
Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20
25 30 Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp
Ile 35 40 45 Gly Leu Phe Asn Pro Tyr Asn Gly Gly Thr Ser Tyr Asn
Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser
Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Leu Ser Leu Thr Ser Glu
Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Cys Tyr Arg Tyr Asp
Gly Leu Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Leu Thr Val Ser
Ser 115 32321DNAMus musculusmisc_featureSC27.104 Light Chain
Variable Region 32gacatccaga tgacacaatc ttcatcctcc ttttctgtat
ctctaggaga cagagtcacc 60attacttgca aggcaagtga ggacatatat aatcggttag
cctggtatca gcagaaacca 120ggaaatgctc ccaggctctt aatatctggt
gcaaccagtt tggaaactgg ggttccttca 180agattcagtg gcagtggatc
tggaaaggat tacactctca gcattaccag tcttcagact 240gaagatgttg
ctacttatta ctgtcaacag tattggagta atcctccgac gttcggtgga
300ggcaccaagc tggaaatcaa a 32133107PRTMus
musculusMISC_FEATURESC27.104 Light Chain Variable Region 33Asp Ile
Gln Met Thr Gln Ser Ser Ser Ser Phe Ser Val Ser Leu Gly 1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Glu Asp Ile Tyr Asn Arg 20
25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Asn Ala Pro Arg Leu Leu
Ile 35 40 45 Ser Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60 Ser Gly Ser Gly Lys Asp Tyr Thr Leu Ser Ile
Thr Ser Leu Gln Thr 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln
Gln Tyr Trp Ser Asn Pro Pro 85 90 95 Thr Phe Gly Gly Gly Thr Lys
Leu Glu Ile Lys 100 105 34354DNAMus musculusmisc_featureSC27.104
Heavy Chain Variable Region 34gaggtccagc tgcaacagtc tggacctgag
ctggtgaagc ctggggcttc agtgaagata 60tcctgcaaga cttctggata cacattcact
gaatacaccg tgcactgggt gaagcagagc 120catggaaaga gccttgagtg
gattggaggt gtttatccta agaatggtga tactacctac 180aaccagaagt
tcaggggcaa ggccacattg actgtagaca agtcctccaa cacagcctat
240atggaactcc gcagcctgac atctgaggat tctgcagtct attactgtac
aggaaaggat 300gggtacgacg ggtttgctta ctggggccaa gggactctgg
tcactgtctc tgca 35435118PRTMus musculusMISC_FEATURESC27.104 Heavy
Chain Variable Region 35Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu
Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Thr Ser
Gly Tyr Thr Phe Thr Glu Tyr 20 25 30 Thr Val His Trp Val Lys Gln
Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Gly Val Tyr Pro
Lys Asn Gly Asp Thr Thr Tyr Asn Gln Lys Phe 50 55 60 Arg Gly Lys
Ala Thr Leu Thr Val Asp Lys Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met
Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90
95 Thr Gly Lys Asp Gly Tyr Asp Gly Phe Ala Tyr Trp Gly Gln Gly Thr
100 105 110 Leu Val Thr Val Ser Ala 115 36318DNAMus
musculusmisc_featureSC27.105 Light Chain Variable Region
36gatgttcaaa tgacccagtc tccatcctcc ctgtctgcat ctttgggaga gagagtctcc
60ctgacttgcc aggcaagtca gagtgttagc aataatttaa actggtatca gcaaacacca
120gggaaagctc ctaggctctt gatctatggt gcaagcaaat tggaagatgg
ggtctcttca 180aggttcagtg gcactggata tgggacagat ttcactttca
ccatcagcag cctggaggaa 240gaagatgtgg caacttattt ttgtctacag
cataggtatc tgtggacgtt cggtggaggc 300accaagctgg aaatcaaa
31837106PRTMus musculusMISC_FEATURESC27.105 Light Chain Variable
Region 37Asp Val Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Leu Gly 1 5 10 15 Glu Arg Val Ser Leu Thr Cys Gln Ala Ser Gln Ser
Val Ser Asn Asn 20 25 30 Leu Asn Trp Tyr Gln Gln Thr Pro Gly Lys
Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Lys Leu Glu Asp
Gly Val Ser Ser Arg Phe Ser Gly 50 55 60 Thr Gly Tyr Gly Thr Asp
Phe Thr Phe Thr Ile Ser Ser Leu Glu Glu 65 70 75
80 Glu Asp Val Ala Thr Tyr Phe Cys Leu Gln His Arg Tyr Leu Trp Thr
85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105
38369DNAMus musculusmisc_featureSC27.105 Heavy Chain Variable
Region 38gaggtccagc tgcagcagtc tggacctgag ttggtgaagc ctggggcttc
agtgaagata 60tcctgcaagg cttctggtta ctcattcact ggctactaca tgaactgggt
gaagcaaagt 120cctgaaaaga gccttgagtg gattggagag attaatccta
gcactggtag tactacttac 180aaccagaagt tcaaggccaa ggccacattg
actgtagaca aatcctccag cacagcctac 240atgcagctca agagcctgac
atctgaggac tctgcagtct attactgtgc aagaagggat 300tattactacg
gtagtggttt ctatgctatg gactactggg gtcaaggaac ctcagtcacc 360gtctcctca
36939123PRTMus musculusMISC_FEATURESC27.105 Heavy Chain Variable
Region 39Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro
Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser
Phe Thr Gly Tyr 20 25 30 Tyr Met Asn Trp Val Lys Gln Ser Pro Glu
Lys Ser Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asn Pro Ser Thr Gly
Ser Thr Thr Tyr Asn Gln Lys Phe 50 55 60 Lys Ala Lys Ala Thr Leu
Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Lys
Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Arg Asp Tyr Tyr Tyr Gly Ser Gly Phe Tyr Ala Met Asp Tyr 100 105 110
Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 115 120 40321DNAMus
musculusmisc_featureSC27.106 Light Chain Variable Region
40gacatccaga tgacacaatc ttcatcctcc ttttctgtat ctctaggaga cagagtcacc
60attacttgca aggcaagtga ggacatatat aatcggttag cctggtatca gcagaaacca
120ggaaatgctc ctaggctctt aatatgtggt gcaaccagtt tggaaactgg
ggttccttca 180agattcagtg gcagtggatc tggaaaggat tacactctca
gcattaccag tcttcagact 240gaagatgttg ctacttatta ctgtcaacag
tattggagta ctccgctcac gttcggtgct 300gggaccaaac tggagctgaa a
32141107PRTMus musculusMISC_FEATURESC27.106 Light Chain Variable
Region 41Asp Ile Gln Met Thr Gln Ser Ser Ser Ser Phe Ser Val Ser
Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Glu Asp
Ile Tyr Asn Arg 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Asn
Ala Pro Arg Leu Leu Ile 35 40 45 Cys Gly Ala Thr Ser Leu Glu Thr
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Lys Asp
Tyr Thr Leu Ser Ile Thr Ser Leu Gln Thr 65 70 75 80 Glu Asp Val Ala
Thr Tyr Tyr Cys Gln Gln Tyr Trp Ser Thr Pro Leu 85 90 95 Thr Phe
Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 42357DNAMus
musculusmisc_featureSC27.106 Heavy Chain Variable Region
42gaggtccagc tgcaacagtc tggacctgag ctggtgaagc ctggggcttc agtgaagata
60tcctgcaaga cttctggata cacattcact gaatacacca tgcactgggt gaagcagagc
120catggaaaga gccttgagtg gattggaggt attaatccta acaatggtgg
tactaactac 180aaccagaagt tcaagggcaa ggccacattg actgttgaca
agtcctccag cacagcctac 240atggagctcc gcagcctgac atctgaggat
tctgcagtct attactgtgc aagaaggctt 300attacttact atgctatgga
ctactggggt caaggaacct cagtcaccgt ctcctca 35743119PRTMus
musculusMISC_FEATURESC27.106 Heavy Chain Variable Region 43Glu Val
Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15
Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr 20
25 30 Thr Met His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp
Ile 35 40 45 Gly Gly Ile Asn Pro Asn Asn Gly Gly Thr Asn Tyr Asn
Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser
Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Thr Ser Glu
Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Leu Ile Thr Tyr
Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Ser Val Thr Val
Ser Ser 115 44324DNAMus musculusmisc_featureSC27.108 Light Chain
Variable Region 44gaaattgtgc tcacccagtc tccagcactc atggctgcat
ctccagggga gaaggtcacc 60atcacctgca gtgtcagctc aagtataagt tccagcaact
tgcactggta ccagcagaag 120tcaggaacct cccccaaact ctggatttat
ggcacatcca acctggcttc tggagtccct 180gttcgcttca gtggcagtgg
atctgggacc tcttattctc tcacaatcag caacatggag 240gctgaagatg
ctgccactta ttactgtcaa cagtggagta gttacccaca cacgttcgga
300ggggggacca agctggaaat aaaa 32445108PRTMus
musculusMISC_FEATURESC27.108 Light Chain Variable Region 45Glu Ile
Val Leu Thr Gln Ser Pro Ala Leu Met Ala Ala Ser Pro Gly 1 5 10 15
Glu Lys Val Thr Ile Thr Cys Ser Val Ser Ser Ser Ile Ser Ser Ser 20
25 30 Asn Leu His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Leu
Trp 35 40 45 Ile Tyr Gly Thr Ser Asn Leu Ala Ser Gly Val Pro Val
Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr
Ile Ser Asn Met Glu 65 70 75 80 Ala Glu Asp Ala Ala Thr Tyr Tyr Cys
Gln Gln Trp Ser Ser Tyr Pro 85 90 95 His Thr Phe Gly Gly Gly Thr
Lys Leu Glu Ile Lys 100 105 46378DNAMus
musculusmisc_featureSC27.108 Heavy Chain Variable Region
46caggtccaaa tgcagcagtc tggagctgag ctggtaaggc ctgggacttc agtgaaggtg
60tcctgcaagg cttctggata cgccttcact aattacttga tagagtgggt aaagcagagg
120cctggacagg gccttgagtg gattggactg attaatcctg gaagtggtgg
tactaattac 180aatgagaagt tcaagggcaa ggcaacactg actgcagaca
aatcctccac cactgcctac 240atgcagctca gcagcctgac atctgatgac
tctgcggttt atttctgtgc aagacggtcc 300cctctaggga gttggatcta
ctatgcttac gacggtgttg cttactgggg ccaagggact 360ctggtcactg tctctgca
37847126PRTMus musculusMISC_FEATURESC27.108 Heavy Chain Variable
Region 47Gln Val Gln Met Gln Gln Ser Gly Ala Glu Leu Val Arg Pro
Gly Thr 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala
Phe Thr Asn Tyr 20 25 30 Leu Ile Glu Trp Val Lys Gln Arg Pro Gly
Gln Gly Leu Glu Trp Ile 35 40 45 Gly Leu Ile Asn Pro Gly Ser Gly
Gly Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu
Thr Ala Asp Lys Ser Ser Thr Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser
Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg
Arg Ser Pro Leu Gly Ser Trp Ile Tyr Tyr Ala Tyr Asp Gly 100 105 110
Val Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala 115 120 125
48321DNAMus musculusmisc_featureSC27.201 Light Chain Variable
Region 48gacatccaga tgacacaatc ttcatcctcc ttttctgtct ctctgggaga
cagagtcact 60attacttgca aggcaagtga ggacatctat aatcggttag cctggtatca
acagaaacca 120ggaaatgctc ctaggctctt aatatctggt gcaaccagtt
tggaagctgg ggttccttca 180ggattcagtg gcagtggatc tggaaaggat
tacactctca gcattaccag tcttcagact 240gaagatgttg ctacttatta
ctgtcaacag tattggagta ctcctccgac gttcggtgga 300ggcaccaagc
tggaactcaa g 32149107PRTMus musculusMISC_FEATURESC27.201 Light
Chain Variable Region 49Asp Ile Gln Met Thr Gln Ser Ser Ser Ser Phe
Ser Val Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala
Ser Glu Asp Ile Tyr Asn Arg 20 25 30 Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Asn Ala Pro Arg Leu Leu Ile 35 40 45 Ser Gly Ala Thr Ser
Leu Glu Ala Gly Val Pro Ser Gly Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Lys Asp Tyr Thr Leu Ser Ile Thr Ser Leu Gln Thr 65 70 75 80 Glu
Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Ser Thr Pro Pro 85 90
95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys 100 105 50360DNAMus
musculusmisc_featureSC27.201 Heavy Chain Variable Region
50gaggtccagc tgcaacagtc tggacctgaa ctggtgaagc ctggggcttc agtgaagata
60tcctgcaaga cttctggata cacattcact gaaaacatca gacactgggt gaagcagagc
120cgaggaaaga gccttgagtg gattggtact attaatccta ataatggtga
gactaggtac 180aatcagaagt tcaagggcaa ggccacattg actgtagaca
agtcctccag cacagcctac 240atggagctcc gcagcctgac atctgaggat
tctgcagtct attactgtac aagggggatt 300acaaagtccc cttatggtat
ggactactgg ggtcaaggaa cctcaatcac cgtctcctca 36051120PRTMus
musculusMISC_FEATURESC27.201 Heavy Chain Variable Region 51Glu Val
Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15
Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Asn 20
25 30 Ile Arg His Trp Val Lys Gln Ser Arg Gly Lys Ser Leu Glu Trp
Ile 35 40 45 Gly Thr Ile Asn Pro Asn Asn Gly Glu Thr Arg Tyr Asn
Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser
Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Thr Ser Glu
Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Gly Ile Thr Lys Ser
Pro Tyr Gly Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Ile Thr
Val Ser Ser 115 120 52321DNAMus musculusmisc_featureSC27.203 Light
Chain Variable Region 52gacatccaga tgacacaatc ttcatcctcc ttttctgtat
ctctaggaga cagagtcacc 60atcacttgca aggcaagtga ggacatatat aatcggttag
cctggtatca gcagaatcca 120ggaaatactc ctaggctctt aatgtctggt
gcaaccagtt tggaaactgg ggttccttca 180agattcagtg gcagtggatc
tggaaaggat tacactctca gcattaccag tcttcagatt 240gaagatgttt
ctacttatta ctgtcaacaa tattggagta ctcctccgac gttcggtgga
300ggcaccaggc tggaaatcaa a 32153107PRTMus
musculusMISC_FEATURESC27.203 Light Chain Variable Region 53Asp Ile
Gln Met Thr Gln Ser Ser Ser Ser Phe Ser Val Ser Leu Gly 1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Glu Asp Ile Tyr Asn Arg 20
25 30 Leu Ala Trp Tyr Gln Gln Asn Pro Gly Asn Thr Pro Arg Leu Leu
Met 35 40 45 Ser Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60 Ser Gly Ser Gly Lys Asp Tyr Thr Leu Ser Ile
Thr Ser Leu Gln Ile 65 70 75 80 Glu Asp Val Ser Thr Tyr Tyr Cys Gln
Gln Tyr Trp Ser Thr Pro Pro 85 90 95 Thr Phe Gly Gly Gly Thr Arg
Leu Glu Ile Lys 100 105 54360DNAMus musculusmisc_featureSC27.203
Heavy Chain Variable Region 54gaggtccagc tgcaacagtc tggacctgag
ctggtgaagc ctggggcttc agtgaagata 60tcctgcaaga cttctggata cacattcact
gaaaacatca tacactgggt gaagcagagc 120catggaaaga gccttgagtg
gattggaggt attaatccta tcaatggtgg tactagctac 180aaccagaagt
tcaagggcaa ggccacattg actgtagaca agtcctccag cacagcctac
240atggagctcc gtagcctgac atctgaggat tctgcagtct attactgtgc
aagggggatt 300actacgtccc cttatgctat ggactactgg ggtcaaggaa
cctcagtcac cgtctcctca 36055120PRTMus musculusMISC_FEATURESC27.203
Heavy Chain Variable Region 55Glu Val Gln Leu Gln Gln Ser Gly Pro
Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys
Thr Ser Gly Tyr Thr Phe Thr Glu Asn 20 25 30 Ile Ile His Trp Val
Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Gly Ile
Asn Pro Ile Asn Gly Gly Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys
Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70
75 80 Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr
Cys 85 90 95 Ala Arg Gly Ile Thr Thr Ser Pro Tyr Ala Met Asp Tyr
Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser 115 120
56321DNAMus musculusmisc_featureSC27.204 Light Chain Variable
Region 56gacattgtga tgacccagtc tcaaaaattc atgtccacat cagtaggaga
cagggtcagc 60gtcgcctgca aggccggtca gaatgtgggt actagtgtag cctggtatca
acagaaacca 120ggacattctc ctaaatcact gatttactcg gcatcctacc
ggtacagtgg agtccctaat 180cgcttcacag gcagtggatc tgggacagat
ttcactctca ccatcagcaa tgtgcagtct 240gaagacttgg cagactattt
ctgtcagcaa tatatcacct atccgtacac gttcggaggg 300gggaccaagc
tggaaataat a 32157107PRTMus musculusMISC_FEATURESC27.204 Light
Chain Variable Region 57Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met
Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Val Ala Cys Lys Ala
Gly Gln Asn Val Gly Thr Ser 20 25 30 Val Ala Trp Tyr Gln Gln Lys
Pro Gly His Ser Pro Lys Ser Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr
Arg Tyr Ser Gly Val Pro Asn Arg Phe Thr Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser 65 70 75 80 Glu
Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Ile Thr Tyr Pro Tyr 85 90
95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Ile 100 105 58336DNAMus
musculusmisc_featureSC27.204 Heavy Chain Variable Region
58gaggtgaagg ttctcgagtc tggaggtggc ctggtgcagc ctggaggatc cctgaaactc
60tcctgtgcag cctcaggatt cgattttagt agatactgga tgagttgggt ccggcaggct
120ccagggaaag gcctagaatg gattggagaa attaatccag atagcagtac
gataaactat 180acgccatctc taaaggctaa attcatcatc tccagagaca
acgccaaaaa tacgctgtac 240ctgcaaatga gcaaagtgag atctgaggac
acagcccttt attactgtac aggaccagct 300tactggggcc aagggactct
ggtcactgtc tctgca 33659112PRTMus musculusMISC_FEATURESC27.204 Heavy
Chain Variable Region 59Glu Val Lys Val Leu Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser
Gly Phe Asp Phe Ser Arg Tyr 20 25 30 Trp Met Ser Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asn Pro
Asp Ser Ser Thr Ile Asn Tyr Thr Pro Ser Leu 50 55 60 Lys Ala Lys
Phe Ile Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu
Gln Met Ser Lys Val Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90
95 Thr Gly Pro Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala
100 105 110 60321DNAArtificial SequencehSC27.1 Light Chain Variable
Region 60gacatccaga tgacccagtc tccatcttcc gtgtctgcat ctgtaggaga
cagagtcacc 60atcacttgta aggcgagtga ggatatttac aaccggttag cctggtatca
gcagaaacca 120gggaaagccc ctaagctcct gatctatggt gcaaccagtt
tggaaactgg ggtcccatca 180aggttcagcg gcagtggatc tgggacagat
tacactctca ccatcagcag cctgcagcct 240gaagattttg caacttacta
ttgtcaacag tattggagta ctccactcac gttcggtcaa 300gggaccaagc
tggagattaa a 32161107PRTArtificial SequencehSC27.1 Light Chain
Variable Region 61Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser
Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser
Glu Asp Ile Tyr Asn Arg 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Thr Ser Leu
Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly
Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp
Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Ser Thr Pro Leu 85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105
62357DNAArtificial SequencehSC27.1 Heavy Chain Variable Region
62caggtccagc ttgtgcagtc
tggggctgag gtgaagaagc ctggggcctc agtgaaggtt 60tcctgcaagg cttctggata
caccttcact gagtatactc tgcattgggt gcgccaggcc 120cccggacaaa
ggcttgagtg gatgggaggg atcaacccta acaatggtga cacaatatat
180aaccagaagt tcaagggcag agtcaccatt accagggaca catccgcgag
cacagcctac 240atggagctga gcagcctgag atctgaagac acggctgtgt
attactgtgc gagaagggcg 300attacggtct atgctatgga ctactggggt
caaggtaccc tagtcaccgt ctcgagc 35763119PRTArtificial SequencehSC27.1
Heavy Chain Variable Region 63Gln Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Glu Tyr 20 25 30 Thr Leu His Trp Val
Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Gly Ile
Asn Pro Asn Asn Gly Asp Thr Ile Tyr Asn Gln Lys Phe 50 55 60 Lys
Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr 65 70
75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95 Ala Arg Arg Ala Ile Thr Val Tyr Ala Met Asp Tyr Trp
Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
64333DNAArtificial SequencehSC27.22 Light Chain Variable Region
64gacattgtca tgacccagtc ccctgacagt ttggccgtta gcttggggga gcgtgccacc
60atcaactgta gggctagtca aactgtttct acatcctcct actcttacat gcattggtat
120cagcagaaac ctggtcagcc tccaaaactg ctgatttatt tcgcatctaa
cgtcgagtcc 180ggagttcctg accggttcag cggatcagga agcggtacag
attttacact taccatctca 240tctctgcaag cagaagatgt ggccgtgtac
tattgtcagc attcctggga gatcccctgg 300accttcgggc agggaaccaa
gctcgagatt aaa 33365111PRTArtificial SequencehSC27.22 Light Chain
Variable Region 65Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala
Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser
Gln Thr Val Ser Thr Ser 20 25 30 Ser Tyr Ser Tyr Met His Trp Tyr
Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Phe
Ala Ser Asn Val Glu Ser Gly Val Pro Asp 50 55 60 Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu
Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln His Ser Trp 85 90 95
Glu Ile Pro Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105
110 66366DNAArtificial SequencehSC27.22 Heavy Chain Variable Region
66caggtgcagt tggtgcagag cggcgccgaa gtcaagaaac caggagcttc tgtcaaagtc
60tcctgtaaag cctccggata taccttcacc agctactgga tgaattgggt aagacaggcc
120cccggacaga ggcttgagtg gatgggaatg atccatccct ctgacagcga
gattcggctc 180aaccagaagt ttaaagaccg agtgactatc acacgcgata
ccagtgctag cacagcctac 240atggagttga gttctcttcg tagcgaggac
actgccgtgt attattgcgc ccgcatcgac 300tcatattatg gttatctgtt
ctacttcgac tattggggcc aggggaccac cgtgactgtg 360tcttcc
36667122PRTArtificial SequencehSC27.22 Heavy Chain Variable Region
67Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1
5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser
Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Arg Leu
Glu Trp Met 35 40 45 Gly Met Ile His Pro Ser Asp Ser Glu Ile Arg
Leu Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Ile Thr Arg Asp
Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ile Asp Ser
Tyr Tyr Gly Tyr Leu Phe Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly
Thr Thr Val Thr Val Ser Ser 115 120 68324DNAArtificial
SequencehSC27.108 Light Chain Variable Region 68gaaatcgtgc
ttacacaatc ccctgccact ctgagccttt ctccaggcga gcgagcaacc 60ctttcctgca
gtgtttcctc ttcaatcagt tccagcaatt tgcactggta ccagcagaag
120cctggtcagg caccccgatt gttgatctat ggcacatcta acctggccag
cggcatccct 180gctcggttca gtggatctgg ctccggaaca gatttcactc
tcactatcag ctcccttgag 240cctgaagatt ttgccgtgta ctactgtcag
caatggagtt cctaccccca cacctttggc 300ggcgggacaa aggtcgagat aaaa
32469108PRTArtificial SequencehSC27.108 Light Chain Variable Region
69Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1
5 10 15 Glu Arg Ala Thr Leu Ser Cys Ser Val Ser Ser Ser Ile Ser Ser
Ser 20 25 30 Asn Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
Arg Leu Leu 35 40 45 Ile Tyr Gly Thr Ser Asn Leu Ala Ser Gly Ile
Pro Ala Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Ser Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr
Tyr Cys Gln Gln Trp Ser Ser Tyr Pro 85 90 95 His Thr Phe Gly Gly
Gly Thr Lys Val Glu Ile Lys 100 105 70378DNAArtificial
SequencehSC27.108 Heavy Chain Variable Region 70caggtacagc
tggtccagtc cggcgctgag gttaagaagc ccggtgcctc cgtgaaggta 60tcttgtaagg
cctcaggtta cacctttaca aattatctga tcgaatgggt gagacaggcc
120ccaggtcagg gtctggaatg gatgggactc atcaaccctg ggagtggcgg
gaccaactac 180aacgaaaagt ttaaggggag agtgacaatg accacagata
ccagtacctc caccgcatat 240atggagctgc gaagcttgag gtccgatgac
actgctgtgt actattgcgc ccgtagaagc 300ccactcgggt cttggatcta
ttacgcatac gatggtgtgg cctattgggg ccagggcacc 360ctggtgacag tcagctcc
37871126PRTArtificial SequencehSC27.108 Heavy Chain Variable Region
71Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1
5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn
Tyr 20 25 30 Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Met 35 40 45 Gly Leu Ile Asn Pro Gly Ser Gly Gly Thr Asn
Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Thr Asp
Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg
Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Ser Pro
Leu Gly Ser Trp Ile Tyr Tyr Ala Tyr Asp Gly 100 105 110 Val Ala Tyr
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125
72321DNAArtificial SequencehSC27.204 Light Chain Variable Region
72gacatccaga tgacccagtc cccctccagc ctgtctgctt ccgtgggcga cagagtgacc
60atcacatgca aggccggcca gaacgtgggc acctctgtgg cctggttcca gcagaagcct
120ggcaaggccc ccaagtccct gatctactcc gcctcctaca gatactccgg
cgtgccctcc 180agattctccg gctctggctc tggcaccgac tttaccctga
ccatcagctc cctgcagccc 240gaggacttcg ccacctacta ctgccagcag
tacatcacct acccctacac cttcggcgga 300ggcaccaagg tggaaatcaa g
32173107PRTArtificial SequencehSC27.204 Light Chain Variable Region
73Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1
5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Gly Gln Asn Val Gly Thr
Ser 20 25 30 Val Ala Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys
Ser Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln Tyr Ile Thr Tyr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly
Thr Lys Val Glu Ile Lys 100 105 74336DNAArtificial
SequencehSC27.204 Heavy Chain Variable Region 74gaagtgcagc
tgctggaatc tggcggcgga ctggtgcagc ctggcggatc tctgagactg 60tcttgtgccg
cctccggctt caccttctcc cggtactgga tgtcctgggt gcgacaggct
120cctggcaagg gcctggaatg ggtgtccgag atcaaccccg actcctccac
catcaactac 180acccccagcc tgaaggcccg gttcaccatc tctcgggaca
actccaagaa caccctgtac 240ctgcagatga actccctgcg ggccgaggac
accgccgtgt actactgtac cggccctgct 300tattggggcc agggcaccct
cgtgaccgtg tcctct 33675112PRTArtificial SequencehSC27.204 Heavy
Chain Variable Region 75Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Arg Tyr 20 25 30 Trp Met Ser Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Glu Ile Asn Pro
Asp Ser Ser Thr Ile Asn Tyr Thr Pro Ser Leu 50 55 60 Lys Ala Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Thr Gly Pro Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
100 105 110 76324DNAArtificial SequencehSC27.108v1 Light Chain
Variable Region 76gaaatcgtgc tgactcagtc tcccgatttc cagtccgtca
cacccaagga gaaagtcacc 60atcacttgtt ctgtctcctc aagcatctct tctagtaacc
tgcactggta tcagcagaag 120cctgatcagt cccctaagct ttggatatac
ggcacctcaa acctcgcctc cggagttcct 180tcaaggtttt caggtagtgg
atctggaacc gatttcaccc ttacaataaa cagtcttgag 240gccgaggacg
ccgccactta ctactgccag cagtggagtt cttaccccca cacatttggg
300ggcggcacca aagtggagat caaa 32477108PRTArtificial
SequencehSC27.108v1 Light Chain Variable Region 77Glu Ile Val Leu
Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys 1 5 10 15 Glu Lys
Val Thr Ile Thr Cys Ser Val Ser Ser Ser Ile Ser Ser Ser 20 25 30
Asn Leu His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Trp 35
40 45 Ile Tyr Gly Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe
Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn
Ser Leu Glu 65 70 75 80 Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln
Trp Ser Ser Tyr Pro 85 90 95 His Thr Phe Gly Gly Gly Thr Lys Val
Glu Ile Lys 100 105 78366DNAArtificial SequencehSC27.22-VH1-8 Heavy
Chain Variable Region 78caggtgcagc tggtgcagtc tggcgccgaa gtgaagaaac
ctggcgcctc cgtgaaggtg 60tcctgcaagg cctccggcta cacctttacc agctactgga
tgaactgggt gcgacaggct 120accggccagg gcctggaatg gatgggcatg
atccacccct ccgactccga gatccggctg 180aaccagaaat tcaaggacag
agtgaccatg acccggaaca cctccatctc caccgcctac 240atggaactgt
cctccctgcg gagcgaggac accgccgtgt actactgcgc ccggatcgac
300tcctactacg gctacctgtt ctacttcgac tactggggcc agggcaccac
cgtgaccgtg 360tcatct 36679122PRTArtificial SequencehSC27.22-VH1-8
Heavy Chain Variable Region 79Gln Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met Asn Trp Val
Arg Gln Ala Thr Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Met Ile
His Pro Ser Asp Ser Glu Ile Arg Leu Asn Gln Lys Phe 50 55 60 Lys
Asp Arg Val Thr Met Thr Arg Asn Thr Ser Ile Ser Thr Ala Tyr 65 70
75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95 Ala Arg Ile Asp Ser Tyr Tyr Gly Tyr Leu Phe Tyr Phe
Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115
120 80366DNAArtificial SequencehSC27.22-VH1-46 Heavy Chain Variable
Region 80caggtgcagc tggtgcagtc tggcgccgaa gtgaagaaac ctggcgcctc
cgtgaaggtg 60tcctgcaagg cctccggcta cacctttacc agctactgga tgaactgggt
gcgacaggcc 120cctggacagg gcctggaatg gatgggcatg atccacccct
ccgactccga gatccggctg 180aaccagaaat tcaaggaccg cgtgaccatg
accagagaca cctccaccag caccgtgtac 240atggaactgt cctccctgcg
gagcgaggac accgccgtgt actactgcgc ccggatcgac 300tcctactacg
gctacctgtt ctacttcgac tactggggcc agggcaccac cgtgaccgtg 360tcatct
36681122PRTArtificial SequencehSC27.22-VH1-46 Heavy Chain Variable
Region 81Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr
Phe Thr Ser Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45 Gly Met Ile His Pro Ser Asp Ser
Glu Ile Arg Leu Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Met
Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Ile Asp Ser Tyr Tyr Gly Tyr Leu Phe Tyr Phe Asp Tyr Trp 100 105 110
Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 82366DNAArtificial
SequencehSC27.22-VH1-69 Heavy Chain Variable Region 82caggtgcagc
tggtgcagtc tggcgccgaa gtgaagaaac ccggctcctc cgtgaaggtg 60tcctgcaagg
cttccggcgg caccttctcc agctactgga tgaactgggt gcgacaggcc
120cctggacagg gcctggaatg gatgggcatg atccacccct ccgactccga
gatccggctg 180aaccagaaat tcaaggacag agtgaccatc accgccgacg
agtccacctc caccgcctac 240atggaactgt cctccctgcg gagcgaggac
accgccgtgt actactgcgc ccggatcgac 300tcctactacg gctacctgtt
ctacttcgac tactggggcc agggcaccac cgtgaccgtg 360tcatct
36683122PRTArtificial SequencehSC27.22-VH1-69 Heavy Chain Variable
Region 83Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr
Phe Ser Ser Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45 Gly Met Ile His Pro Ser Asp Ser
Glu Ile Arg Leu Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Ile
Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Ile Asp Ser Tyr Tyr Gly Tyr Leu Phe Tyr Phe Asp Tyr Trp 100 105 110
Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 84336DNAArtificial
SequencehSC27.204v1 Heavy Chain Variable Region 84gaagtgcagc
tgctggaatc tggcggcgga ctggtgcagc ctggcggatc tctgagactg 60tcttgtgccg
cctccggctt caccttctcc cggtactgga tgtcctgggt gcgacaggct
120cctggcaagg gcctggaatg ggtgtccgag atcaaccccg actcctccac
catcaagtac 180acccccagcc tgaaggcccg gttcaccatc tctcgggaca
actccaagaa caccctgtac 240ctgcagatga actccctgcg ggccgaggac
accgccgtgt actactgtac cggccctgct 300tattggggcc agggcaccct
cgtgaccgtg tcctct 33685112PRTArtificial SequencehSC27.204v1 Heavy
Chain Variable Region 85Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Arg Tyr 20 25 30 Trp Met Ser Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Glu Ile Asn Pro
Asp Ser Ser Thr Ile Lys Tyr Thr Pro Ser Leu 50 55 60 Lys Ala Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Thr Gly Pro Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
Ser
100 105 110 86336DNAArtificial SequencehSC27.204v2 Heavy Chain
Variable Region 86gaagtgcagc tgctggaatc tggcggcgga ctggtgcagc
ctggcggatc tctgagactg 60tcttgtgccg cctccggctt caccttctcc cggtactgga
tgtcctgggt gcgacaggct 120cctggcaagg gcctggaatg ggtgtccgag
atcaaccccg actcctccac catccagtac 180acccccagcc tgaaggcccg
gttcaccatc tctcgggaca actccaagaa caccctgtac 240ctgcagatga
actccctgcg ggccgaggac accgccgtgt actactgtac cggccctgct
300tattggggcc agggcaccct cgtgaccgtg tcctct 33687112PRTArtificial
SequencehSC27.204v2 Heavy Chain Variable Region 87Glu Val Gln Leu
Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30
Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ser Glu Ile Asn Pro Asp Ser Ser Thr Ile Gln Tyr Thr Pro Ser
Leu 50 55 60 Lys Ala Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Thr Gly Pro Ala Tyr Trp Gly Gln Gly
Thr Leu Val Thr Val Ser Ser 100 105 110 88336DNAArtificial
SequencehSC27.204v3 Heavy Chain Variable Region 88gaagtgcagc
tgctggaatc tggcggcgga ctggtgcagc ctggcggatc tctgagactg 60tcttgtgccg
cctccggctt caccttctcc cggtactgga tgtcctgggt gcgacaggct
120cctggcaagg gcctggaatg ggtgtccgag atcaaccccg actcctccac
catcaactac 180aaccccagcc tgaaggcccg gttcaccatc tctcgggaca
actccaagaa caccctgtac 240ctgcagatga actccctgcg ggccgaggac
accgccgtgt actactgtac cggccctgct 300tattggggcc agggcaccct
cgtgaccgtg tcctct 33689112PRTArtificial SequencehSC27.204v3 Heavy
Chain Variable Region 89Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Arg Tyr 20 25 30 Trp Met Ser Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Glu Ile Asn Pro
Asp Ser Ser Thr Ile Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Ala Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Thr Gly Pro Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
100 105 110 90336DNAArtificial SequencehSC27.204v4 Heavy Chain
Variable Region 90gaagtgcagc tgctggaatc tggcggcgga ctggtgcagc
ctggcggatc tctgagactg 60tcttgtgccg cctccggctt cgacttctcc cggtactgga
tgtcctgggt gcgacaggct 120cctggcaagg gcctggaatg ggtgtccgag
atcaaccccg actcctccac catcaactac 180acccccagcc tgaaggcccg
gttcaccatc tctcgggaca actccaagaa caccctgtac 240ctgcagatga
actccctgcg ggccgaggac accgccgtgt actactgtac cggccctgct
300tattggggcc agggcaccct cgtgaccgtg tcctct 33691112PRTArtificial
SequencehSC27.204v4 Heavy Chain Variable Region 91Glu Val Gln Leu
Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Ser Arg Tyr 20 25 30
Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ser Glu Ile Asn Pro Asp Ser Ser Thr Ile Asn Tyr Thr Pro Ser
Leu 50 55 60 Lys Ala Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Thr Gly Pro Ala Tyr Trp Gly Gln Gly
Thr Leu Val Thr Val Ser Ser 100 105 110 92336DNAArtificial
SequencehSC27.204v5 Heavy Chain Variable Region 92gaagtgcagc
tgctggaatc tggcggcgga ctggtgcagc ctggcggatc tctgagactg 60tcttgtgccg
cctccggctt cgacttctcc cggtactgga tgtcctgggt gcgacaggct
120cctggcaagg gcctggaatg ggtgtccgag atcaaccccg actcctccac
catcaagtac 180acccccagcc tgaaggcccg gttcaccatc tctcgggaca
actccaagaa caccctgtac 240ctgcagatga actccctgcg ggccgaggac
accgccgtgt actactgtac cggccctgct 300tattggggcc agggcaccct
cgtgaccgtg tcctct 33693112PRTArtificial SequencehSC27.204v5 Heavy
Chain Variable Region 93Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Asp Phe Ser Arg Tyr 20 25 30 Trp Met Ser Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Glu Ile Asn Pro
Asp Ser Ser Thr Ile Lys Tyr Thr Pro Ser Leu 50 55 60 Lys Ala Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Thr Gly Pro Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
100 105 110 94336DNAArtificial SequencehSC27.204v6 Heavy Chain
Variable Region 94gaagtgcagc tgctggaatc tggcggcgga ctggtgcagc
ctggcggatc tctgagactg 60tcttgtgccg cctccggctt cgacttctcc cggtactgga
tgtcctgggt gcgacaggct 120cctggcaagg gcctggaatg ggtgtccgag
atcaaccccg actcctccac catccagtac 180acccccagcc tgaaggcccg
gttcaccatc tctcgggaca actccaagaa caccctgtac 240ctgcagatga
actccctgcg ggccgaggac accgccgtgt actactgtac cggccctgct
300tattggggcc agggcaccct cgtgaccgtg tcctct 33695112PRTArtificial
SequencehSC27.204v6 Heavy Chain Variable Region 95Glu Val Gln Leu
Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Ser Arg Tyr 20 25 30
Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ser Glu Ile Asn Pro Asp Ser Ser Thr Ile Gln Tyr Thr Pro Ser
Leu 50 55 60 Lys Ala Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Thr Gly Pro Ala Tyr Trp Gly Gln Gly
Thr Leu Val Thr Val Ser Ser 100 105 110 96336DNAArtificial
SequencehSC27.204v7 Heavy Chain Variable Region 96gaagtgcagc
tgctggaatc tggcggcgga ctggtgcagc ctggcggatc tctgagactg 60tcttgtgccg
cctccggctt cgacttctcc cggtactgga tgtcctgggt gcgacaggct
120cctggcaagg gcctggaatg ggtgtccgag atcaaccccg actcctccac
catcaactac 180aaccccagcc tgaaggcccg gttcaccatc tctcgggaca
actccaagaa caccctgtac 240ctgcagatga actccctgcg ggccgaggac
accgccgtgt actactgtac cggccctgct 300tattggggcc agggcaccct
cgtgaccgtg tcctct 33697112PRTArtificial SequencehSC27.204v7 Heavy
Chain Variable Region 97Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Asp Phe Ser Arg Tyr 20 25 30 Trp Met Ser Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Glu Ile Asn Pro
Asp Ser Ser Thr Ile Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Ala Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Thr Gly Pro Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
100 105 110 98336DNAArtificial SequencehSC27.204v8 Heavy Chain
Variable Region 98gaagtgcagc tgctggaatc tggcggcgga ctggtgcagc
ctggcggatc tctgagactg 60tcttgtgccg cctccggctt caccttctcc cggtactgga
tgtcctgggt gcgacaggct 120cctggcaagg gcctggaatg ggtgtccgag
atcaaccccg actcctccac catcaactac 180acccccagcc tgaaggcccg
gttcaccatc tctcgggaca actccaagaa caccctgtac 240ctgcagatga
actccctgcg ggccgaggac accgccgtgt actactgtgc cggccctgct
300tattggggcc agggcaccct cgtgaccgtg tcctct 33699112PRTArtificial
SequencehSC27.204v8 Heavy Chain Variable Region 99Glu Val Gln Leu
Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30
Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ser Glu Ile Asn Pro Asp Ser Ser Thr Ile Asn Tyr Thr Pro Ser
Leu 50 55 60 Lys Ala Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Gly Pro Ala Tyr Trp Gly Gln Gly
Thr Leu Val Thr Val Ser Ser 100 105 110 100336DNAArtificial
SequencehSC27.204v9 Heavy Chain Variable Region 100gaagtgcagc
tgctggaatc tggcggcgga ctggtgcagc ctggcggatc tctgagactg 60tcttgtgccg
cctccggctt caccttctcc cggtactgga tgtcctgggt gcgacaggct
120cctggcaagg gcctggaatg ggtgtccgag atcaaccccg actcctccac
catcaagtac 180acccccagcc tgaaggcccg gttcaccatc tctcgggaca
actccaagaa caccctgtac 240ctgcagatga actccctgcg ggccgaggac
accgccgtgt actactgtgc cggccctgct 300tattggggcc agggcaccct
cgtgaccgtg tcctct 336101112PRTArtificial SequencehSC27.204v9 Heavy
Chain Variable Region 101Glu Val Gln Leu Leu Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30 Trp Met Ser Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Glu Ile Asn
Pro Asp Ser Ser Thr Ile Lys Tyr Thr Pro Ser Leu 50 55 60 Lys Ala
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Gly Pro Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
Ser 100 105 110 102336DNAArtificial SequencehSC27.204v10 Heavy
Chain Variable Region 102gaagtgcagc tgctggaatc tggcggcgga
ctggtgcagc ctggcggatc tctgagactg 60tcttgtgccg cctccggctt caccttctcc
cggtactgga tgtcctgggt gcgacaggct 120cctggcaagg gcctggaatg
ggtgtccgag atcaaccccg actcctccac catccagtac 180acccccagcc
tgaaggcccg gttcaccatc tctcgggaca actccaagaa caccctgtac
240ctgcagatga actccctgcg ggccgaggac accgccgtgt actactgtgc
cggccctgct 300tattggggcc agggcaccct cgtgaccgtg tcctct
336103112PRTArtificial SequencehSC27.204v10 Heavy Chain Variable
Region 103Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Arg Tyr 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ser Glu Ile Asn Pro Asp Ser Ser
Thr Ile Gln Tyr Thr Pro Ser Leu 50 55 60 Lys Ala Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Gly
Pro Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 100 105 110
104336DNAArtificial SequencehSC27.204v11 Heavy Chain Variable
Region 104gaagtgcagc tgctggaatc tggcggcgga ctggtgcagc ctggcggatc
tctgagactg 60tcttgtgccg cctccggctt caccttctcc cggtactgga tgtcctgggt
gcgacaggct 120cctggcaagg gcctggaatg ggtgtccgag atcaaccccg
actcctccac catcaactac 180aaccccagcc tgaaggcccg gttcaccatc
tctcgggaca actccaagaa caccctgtac 240ctgcagatga actccctgcg
ggccgaggac accgccgtgt actactgtgc cggccctgct 300tattggggcc
agggcaccct cgtgaccgtg tcctct 336105112PRTArtificial
SequencehSC27.204v11 Heavy Chain Variable Region 105Glu Val Gln Leu
Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30
Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ser Glu Ile Asn Pro Asp Ser Ser Thr Ile Asn Tyr Asn Pro Ser
Leu 50 55 60 Lys Ala Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Gly Pro Ala Tyr Trp Gly Gln Gly
Thr Leu Val Thr Val Ser Ser 100 105 110 106336DNAArtificial
SequencehSC27.204v12 Heavy Chain Variable Region 106gaagtgcagc
tgctggaatc tggcggcgga ctggtgcagc ctggcggatc tctgagactg 60tcttgtgccg
cctccggctt cgacttctcc cggtactgga tgtcctgggt gcgacaggct
120cctggcaagg gcctggaatg ggtgtccgag atcaaccccg actcctccac
catcaactac 180acccccagcc tgaaggcccg gttcaccatc tctcgggaca
actccaagaa caccctgtac 240ctgcagatga actccctgcg ggccgaggac
accgccgtgt actactgtgc cggccctgct 300tattggggcc agggcaccct
cgtgaccgtg tcctct 336107112PRTArtificial SequencehSC27.204v12 Heavy
Chain Variable Region 107Glu Val Gln Leu Leu Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Asp Phe Ser Arg Tyr 20 25 30 Trp Met Ser Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Glu Ile Asn
Pro Asp Ser Ser Thr Ile Asn Tyr Thr Pro Ser Leu 50 55 60 Lys Ala
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Gly Pro Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
Ser 100 105 110 108336DNAArtificial SequencehSC27.204v13 Heavy
Chain Variable Region 108gaagtgcagc tgctggaatc tggcggcgga
ctggtgcagc ctggcggatc tctgagactg 60tcttgtgccg cctccggctt cgacttctcc
cggtactgga tgtcctgggt gcgacaggct 120cctggcaagg gcctggaatg
ggtgtccgag atcaaccccg actcctccac catcaagtac 180acccccagcc
tgaaggcccg gttcaccatc tctcgggaca actccaagaa caccctgtac
240ctgcagatga actccctgcg ggccgaggac accgccgtgt actactgtgc
cggccctgct 300tattggggcc agggcaccct cgtgaccgtg tcctct
336109112PRTArtificial SequencehSC27.204v13 Heavy Chain Variable
Region 109Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp
Phe Ser Arg Tyr 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ser Glu Ile Asn Pro Asp Ser Ser
Thr Ile Lys Tyr Thr Pro Ser Leu 50 55 60 Lys Ala Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Gly
Pro Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 100 105 110
110336DNAArtificial SequencehSC27.204v14 Heavy Chain Variable
Region Variable Region 110gaagtgcagc tgctggaatc tggcggcgga
ctggtgcagc ctggcggatc tctgagactg 60tcttgtgccg cctccggctt cgacttctcc
cggtactgga tgtcctgggt gcgacaggct 120cctggcaagg gcctggaatg
ggtgtccgag atcaaccccg actcctccac catccagtac 180acccccagcc
tgaaggcccg gttcaccatc tctcgggaca actccaagaa caccctgtac
240ctgcagatga actccctgcg ggccgaggac accgccgtgt
actactgtgc cggccctgct 300tattggggcc agggcaccct cgtgaccgtg tcctct
336111112PRTArtificial SequencehSC27.204v14 Heavy Chain Variable
Region 111Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp
Phe Ser Arg Tyr 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ser Glu Ile Asn Pro Asp Ser Ser
Thr Ile Gln Tyr Thr Pro Ser Leu 50 55 60 Lys Ala Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Gly
Pro Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 100 105 110
112336DNAArtificial SequencehSC27.204v15 Heavy Chain Variable
Region 112gaagtgcagc tgctggaatc tggcggcgga ctggtgcagc ctggcggatc
tctgagactg 60tcttgtgccg cctccggctt cgacttctcc cggtactgga tgtcctgggt
gcgacaggct 120cctggcaagg gcctggaatg ggtgtccgag atcaaccccg
actcctccac catcaactac 180aaccccagcc tgaaggcccg gttcaccatc
tctcgggaca actccaagaa caccctgtac 240ctgcagatga actccctgcg
ggccgaggac accgccgtgt actactgtgc cggccctgct 300tattggggcc
agggcaccct cgtgaccgtg tcctct 336113112PRTArtificial
SequencehSC27.204v15 Heavy Chain Variable Region 113Glu Val Gln Leu
Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Ser Arg Tyr 20 25 30
Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ser Glu Ile Asn Pro Asp Ser Ser Thr Ile Asn Tyr Asn Pro Ser
Leu 50 55 60 Lys Ala Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Gly Pro Ala Tyr Trp Gly Gln Gly
Thr Leu Val Thr Val Ser Ser 100 105 110 114214PRTArtificial
SequencehSC27.1 Light Chain 114Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Val Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys
Lys Ala Ser Glu Asp Ile Tyr Asn Arg 20 25 30 Leu Ala Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala
Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70
75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Ser Thr Pro
Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr
Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn
Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp
Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr
Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr
Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195
200 205 Phe Asn Arg Gly Glu Cys 210 115448PRTArtificial
SequencehSC27.1 Heavy Chain 115Gln Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Glu Tyr 20 25 30 Thr Leu His Trp Val
Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Gly Ile
Asn Pro Asn Asn Gly Asp Thr Ile Tyr Asn Gln Lys Phe 50 55 60 Lys
Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr 65 70
75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95 Ala Arg Arg Ala Ile Thr Val Tyr Ala Met Asp Tyr Trp
Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195
200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Ser Asp
Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315
320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440
445 116218PRTArtificial SequencehSC27.22 Light Chain 116Asp Ile Val
Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu
Arg Ala Thr Ile Asn Cys Arg Ala Ser Gln Thr Val Ser Thr Ser 20 25
30 Ser Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45 Lys Leu Leu Ile Tyr Phe Ala Ser Asn Val Glu Ser Gly Val
Pro Asp 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Ala Glu Asp Val Ala Val Tyr
Tyr Cys Gln His Ser Trp 85 90 95 Glu Ile Pro Trp Thr Phe Gly Gln
Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg
Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155
160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly
Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215 117451PRTArtificial SequencehSC27.22 Heavy Chain 117Gln Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20
25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp
Met 35 40 45 Gly Met Ile His Pro Ser Asp Ser Glu Ile Arg Leu Asn
Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Ile Thr Arg Asp Thr Ser
Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ile Asp Ser Tyr Tyr
Gly Tyr Leu Phe Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Thr
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150
155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr
Ile Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp
Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys
Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275
280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395
400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu 435 440 445 Ser Pro Gly 450 118215PRTArtificial
SequencehSC27.108 Light Chain 118Glu Ile Val Leu Thr Gln Ser Pro
Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser
Cys Ser Val Ser Ser Ser Ile Ser Ser Ser 20 25 30 Asn Leu His Trp
Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr
Gly Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser 50 55 60
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu 65
70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Ser Ser
Tyr Pro 85 90 95 His Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu
Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys
Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185
190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys
195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215 119455PRTArtificial
SequencehSC27.108 Heavy Chain 119Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys
Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Leu Ile Glu Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Leu
Ile Asn Pro Gly Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe 50 55 60
Lys Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65
70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Arg Ser Pro Leu Gly Ser Trp Ile Tyr Tyr
Ala Tyr Asp Gly 100 105 110 Val Ala Tyr Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser Ala Ser 115 120 125 Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro Ser Ser Lys Ser Thr 130 135 140 Ser Gly Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro 145 150 155 160 Glu Pro Val
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val 165 170 175 His
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser 180 185
190 Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
195 200 205 Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
Lys Val 210 215 220 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala 225 230 235 240 Pro Glu Leu Leu Gly Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro 245 250 255 Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val 260 265 270 Val Asp Val Ser His
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 275 280 285 Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 290 295 300 Tyr
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 305 310
315 320 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala 325 330 335 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro 340 345 350 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
Arg Asp Glu Leu Thr 355 360 365 Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser 370 375 380 Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr 385 390 395
400 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
405 410 415 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe 420 425 430 Ser Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr Gln Lys 435 440 445 Ser Leu Ser Leu Ser Pro Gly 450 455
120214PRTArtificial SequencehSC27.204 Light Chain 120Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp
Arg Val Thr Ile Thr Cys Lys Ala Gly Gln Asn Val Gly Thr Ser 20 25
30 Val Ala Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Ser Leu Ile
35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Tyr Ile Thr Tyr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val
Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe
Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val
Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val
Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155
160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210
121441PRTArtificial SequencehSC27.204 Heavy Chain 121Glu Val Gln
Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25
30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ser Glu Ile Asn Pro Asp Ser Ser Thr Ile Asn Tyr Thr Pro
Ser Leu 50 55 60 Lys Ala Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Gly Pro Ala Tyr Trp Gly Gln
Gly Thr Leu Val Thr Val Ser Ser 100 105 110 Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 115 120 125 Ser Thr Ser Gly
Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 130 135 140 Phe Pro
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 145 150 155
160 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
165 170 175 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
Gln Thr 180 185 190 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
Lys Val Asp Lys 195 200 205 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
His Thr Cys Pro Pro Cys 210 215 220 Pro Ala Pro Glu Leu Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro 225 230 235 240 Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 245 250 255 Val Val Val
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 260 265 270 Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 275 280
285 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
290 295 300 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn 305 310 315 320 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly 325 330 335 Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg Asp Glu 340 345 350 Leu Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr 355 360 365 Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 370 375 380 Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 385 390 395 400
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 405
410 415 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr 420 425 430 Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440
122451PRTArtificial SequencehSC27.22ss1 Heavy Chain 122Gln Val Gln
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25
30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met
35 40 45 Gly Met Ile His Pro Ser Asp Ser Glu Ile Arg Leu Asn Gln
Lys Phe 50 55 60 Lys Asp Arg Val Thr Ile Thr Arg Asp Thr Ser Ala
Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ile Asp Ser Tyr Tyr Gly
Tyr Leu Phe Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Thr Val
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155
160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val Asn 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys
Lys Val Glu Pro Lys Ser 210 215 220 Ser Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280
285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser
Arg Asp Glu Leu Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405
410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu 435 440 445 Ser Pro Gly 450 123451PRTArtificial
SequencehSC27.22-VH1-8 Heavy Chain 123Gln Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser
Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met Asn
Trp Val Arg Gln Ala Thr Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly
Met Ile His Pro Ser Asp Ser Glu Ile Arg Leu Asn Gln Lys Phe 50 55
60 Lys Asp Arg Val Thr Met Thr Arg Asn Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Ile Asp Ser Tyr Tyr Gly Tyr Leu Phe Tyr
Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185
190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro
Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310
315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435
440 445 Ser Pro Gly 450 124451PRTArtificial SequencehSC27.22-VH1-46
Heavy Chain 124Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys
Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
Thr Phe Thr Ser Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Met Ile His Pro Ser Asp
Ser Glu Ile Arg Leu Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr
Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu
Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala
Arg Ile Asp Ser Tyr Tyr Gly Tyr Leu Phe Tyr Phe Asp Tyr Trp 100 105
110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly
Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His Lys Pro
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220 Cys
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230
235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350
Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 355
360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Pro Gly 450
125451PRTArtificial SequencehSC27.22-VH1-69 Heavy Chain 125Gln Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20
25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Met 35 40 45 Gly Met Ile His Pro Ser Asp Ser Glu Ile Arg Leu Asn
Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Ile Thr Ala Asp Glu Ser
Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ile Asp Ser Tyr Tyr
Gly Tyr Leu Phe Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Thr
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150
155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val Thr 180 185 190 Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205 His
Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215
220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340
345 350 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Pro
Gly 450 126447PRTArtificial SequencehSC27.22 IgG2 Heavy Chain
126Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr
Ser Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Arg
Leu Glu Trp Met 35 40 45 Gly Met Ile His Pro Ser Asp Ser Glu Ile
Arg Leu Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Ile Thr Arg
Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu
Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ile Asp
Ser Tyr Tyr Gly Tyr Leu Phe Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln
Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125
Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr 130
135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Asn Phe Gly Thr
Gln Thr Tyr Thr Cys Asn Val Asp 195 200 205 His Lys Pro Ser Asn Thr
Lys Val Asp Lys Thr Val Glu Arg Lys Cys 210 215 220 Cys Val Glu Cys
Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser 225 230 235 240 Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250
255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270 Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr
Phe Arg Val Val 290 295 300 Ser Val Leu Thr Val Val His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys
Gly Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Thr Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser
Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375
380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp
385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro Gly 435 440 445 127448PRTArtificial
SequencehSC27.22 IgG4 R409K Heavy Chain 127Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met
Asn Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45
Gly Met Ile His Pro Ser Asp Ser Glu Ile Arg Leu Asn Gln Lys Phe 50
55 60 Lys Asp Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala
Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Ile Asp Ser Tyr Tyr Gly Tyr Leu Phe
Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro
Cys Ser Arg Ser Thr Ser Glu Ser Thr 130 135 140 Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180
185 190 Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val
Asp 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu
Ser Lys Tyr 210 215 220 Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu
Phe Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser Gln Glu Asp 260 265 270 Pro Glu Val Gln
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys
Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val 290 295 300
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305
310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile
Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Gln Glu Glu Met Thr Lys
Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser
Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425
430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
435 440 445 128448PRTArtificial SequencehSC27.22 IgG4 S228P Heavy
Chain 128Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr
Phe Thr Ser Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly
Gln Arg Leu Glu Trp Met 35 40 45 Gly Met Ile His Pro Ser Asp Ser
Glu Ile Arg Leu Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Ile
Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Ile Asp Ser Tyr Tyr Gly Tyr Leu Phe Tyr Phe Asp Tyr Trp 100 105 110
Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115
120 125 Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser
Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu
Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp 195 200 205 His Lys Pro Ser
Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr 210 215 220 Gly Pro
Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro 225 230 235
240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln
Glu Asp 260 265 270 Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys 325 330 335 Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu
Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360
365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg
Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Glu Gly Asn Val Phe
Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Leu Gly 435 440 445 129448PRTArtificial
SequencehSC27.22 IgG4 S228P K370E R409K Heavy Chain 129Gln Val Gln
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25
30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met
35 40 45 Gly Met Ile His Pro Ser Asp Ser Glu Ile Arg Leu Asn Gln
Lys Phe 50 55 60 Lys Asp Arg Val Thr Ile Thr Arg Asp Thr Ser Ala
Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ile Asp Ser Tyr Tyr Gly
Tyr Leu Phe Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Thr Val
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro
Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr 130 135 140 Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155
160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr
Cys Asn Val Asp 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys
Arg Val Glu Ser Lys Tyr 210 215 220 Gly Pro Pro Cys Pro Pro Cys Pro
Ala Pro Glu Phe Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp 260 265 270 Pro
Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280
285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val
290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
Ser Ser Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Gln Glu Glu
Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Glu Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405
410 415 Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His
Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Leu Gly 435 440 445 130448PRTArtificial SequencehSC27.22 IgG4
K370E Heavy Chain 130Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln
Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Met Ile His Pro
Ser Asp Ser Glu Ile Arg Leu Asn Gln Lys Phe 50 55 60 Lys Asp Arg
Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Ile Asp Ser Tyr Tyr Gly Tyr Leu Phe Tyr Phe Asp Tyr Trp
100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr
Ser Glu Ser Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser
Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp 195 200 205 His
Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu
Ser Lys Tyr 210 215 220 Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu
Phe Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser Gln Glu Asp 260 265 270 Pro Glu Val Gln
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys
Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val 290 295 300
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305
310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile
Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Gln Glu Glu Met Thr Lys
Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Glu Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys 405 410 415 Ser
Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425
430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
435 440 445 131448PRTArtificial SequencehSC27.22 IgG4 S228P K370E
Heavy Chain 131Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys
Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
Thr Phe Thr Ser Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro
Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Met Ile His Pro Ser Asp
Ser Glu Ile Arg Leu Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr
Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu
Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala
Arg Ile Asp Ser Tyr Tyr Gly Tyr Leu Phe Tyr Phe Asp Tyr Trp 100 105
110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
115 120 125 Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu
Ser Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser
Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp 195 200 205 His Lys Pro
Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr 210 215 220 Gly
Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro 225 230
235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
Gln Glu Asp 260 265 270 Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln
Phe Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys
Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys 325 330 335 Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350
Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355
360 365 Cys Leu Val Glu Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Arg Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Glu Gly Asn Val
Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 435 440 445
132448PRTArtificial SequencehSC27.22 IgG4 C127S S228P Heavy Chain
132Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr
Ser Tyr 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Arg
Leu Glu Trp Met 35 40 45 Gly Met Ile His Pro Ser Asp Ser Glu Ile
Arg Leu Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Ile Thr Arg
Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu
Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ile Asp
Ser Tyr Tyr Gly Tyr Leu Phe Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln
Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125
Ser Val Phe Pro Leu Ala Pro Ser Ser Arg Ser Thr Ser Glu Ser Thr 130
135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr
Lys Thr Tyr Thr Cys Asn Val Asp 195 200 205 His Lys Pro Ser Asn Thr
Lys Val Asp Lys Arg Val Glu Ser Lys Tyr 210 215 220 Gly Pro Pro Cys
Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro 225 230 235 240 Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250
255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp
260 265 270 Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val
His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn
Lys Gly Leu Pro Ser Ser Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro
Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375
380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr
Val Asp Lys 405 410 415 Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys
Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser Leu Gly 435 440 445 133448PRTArtificial
SequencehSC27.22 IgG4 C127S K370E Heavy Chain 133Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30
Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35
40 45 Gly Met Ile His Pro Ser Asp Ser Glu Ile Arg Leu Asn Gln Lys
Phe 50 55 60 Lys Asp Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser
Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ile Asp Ser Tyr Tyr Gly Tyr
Leu Phe Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Thr Val Thr
Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu
Ala Pro Ser Ser Arg Ser Thr Ser Glu Ser Thr 130 135 140 Ala Ala Leu
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165
170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys
Asn Val Asp 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg
Val Glu Ser Lys Tyr 210 215 220 Gly Pro Pro Cys Pro Ser Cys Pro Ala
Pro Glu Phe Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp 260 265 270 Pro Glu
Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val 290
295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
Ser Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Gln Glu Glu Met
Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Glu Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys 405 410
415 Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Leu Gly 435 440 445 134448PRTArtificial SequencehSC27.22 IgG4 C127S
S228P K370E Heavy Chain 134Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met Asn Trp Val Arg
Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Met Ile His
Pro Ser Asp Ser Glu Ile Arg Leu Asn Gln Lys Phe 50 55 60 Lys Asp
Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Ile Asp Ser Tyr Tyr Gly Tyr Leu Phe Tyr Phe Asp Tyr
Trp 100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser Ser Arg Ser
Thr Ser Glu Ser Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro
Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp 195 200 205
His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr 210
215 220 Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly
Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser Gln Glu Asp 260 265 270 Pro Glu Val Gln Phe Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg
Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr
Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys 325 330
335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350 Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser
Leu Thr 355 360 365 Cys Leu Val Glu Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Arg Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Glu
Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 435 440 445
135215PRTArtificial SequencehSC27.108v1 Heavy Chain 135Glu Ile Val
Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys 1 5 10 15 Glu
Lys Val Thr Ile Thr Cys Ser Val Ser Ser Ser Ile Ser Ser Ser 20 25
30 Asn Leu His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Trp
35 40 45 Ile Tyr Gly Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg
Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Asn Ser Leu Glu 65 70 75 80 Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln
Gln Trp Ser Ser Tyr Pro 85 90 95 His Thr Phe Gly Gly Gly Thr Lys
Val Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser
Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155
160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His
Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215
136441PRTArtificial SequencehSC27.204v1 Heavy Chain 136Glu Val Gln
Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30 Trp Met Ser Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Glu Ile
Asn Pro Asp Ser Ser Thr Ile Lys Tyr Thr Pro Ser Leu 50 55 60 Lys
Ala Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70
75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95 Thr Gly Pro Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ser 100 105 110 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Ser Ser Lys 115 120 125 Ser Thr Ser Gly Gly Thr Ala Ala Leu
Gly Cys Leu Val Lys Asp Tyr 130 135 140 Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser 145 150 155 160 Gly Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 165 170 175 Leu Ser
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 180 185 190
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 195
200 205 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro
Cys 210 215 220 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro 225 230 235 240 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
Thr Pro Glu Val Thr Cys 245 250 255 Val Val Val Asp Val Ser His Glu
Asp Pro Glu Val Lys Phe Asn Trp 260 265 270 Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu 275 280 285 Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 290 295 300 His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 305 310 315
320 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
325 330 335 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Asp Glu 340 345 350 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr 355 360 365 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn 370 375 380 Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe 385 390 395 400 Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 405 410 415 Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 420 425 430 Gln
Lys Ser Leu Ser Leu Ser Pro Gly 435 440 137441PRTArtificial
SequencehSC27.204v2 Heavy Chain 137Glu Val Gln Leu Leu Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30 Trp Met Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Glu
Ile Asn Pro Asp Ser Ser Thr Ile Gln Tyr Thr Pro Ser Leu 50 55 60
Lys Ala Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65
70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Thr Gly Pro Ala Tyr Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser 100 105 110 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro Ser Ser Lys 115 120 125 Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr 130 135 140 Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 145 150 155 160 Gly Val His
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 165 170 175 Leu
Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 180 185
190 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
195 200 205 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro
Pro Cys 210 215 220 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro 225 230 235 240 Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys 245 250 255 Val Val Val Asp Val Ser His
Glu Asp Pro Glu Val Lys Phe Asn Trp 260 265 270 Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 275 280 285 Glu Gln Tyr
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 290 295 300 His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 305 310
315 320 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly 325 330 335 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
Arg Asp Glu 340 345 350 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr 355 360 365 Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn 370 375 380 Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe 385 390 395 400 Leu Tyr Ser Lys
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 405 410 415 Val Phe
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 420 425 430
Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 138441PRTArtificial
SequencehSC27.204v3 Heavy Chain 138Glu Val Gln Leu Leu Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30 Trp Met Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Glu
Ile Asn Pro Asp Ser Ser Thr Ile Asn Tyr Asn Pro Ser Leu 50 55 60
Lys Ala Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65
70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Thr Gly Pro Ala Tyr Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser 100 105 110 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro Ser Ser Lys 115 120 125 Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr 130 135 140 Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 145 150 155 160 Gly Val His
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 165 170 175 Leu
Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 180 185
190 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
195 200 205 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro
Pro Cys 210 215 220 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro 225 230 235 240 Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys 245 250 255 Val Val Val Asp Val Ser His
Glu Asp Pro Glu Val Lys Phe Asn Trp 260 265 270 Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 275 280 285 Glu Gln Tyr
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 290 295 300 His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 305 310
315 320 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly 325 330 335 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
Arg Asp Glu 340 345 350 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr 355 360 365 Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn 370 375 380 Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe 385 390 395 400 Leu Tyr Ser Lys
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 405 410 415 Val Phe
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 420 425 430
Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 139441PRTArtificial
SequencehSC27.204v4 Heavy Chain 139Glu Val Gln Leu Leu Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Asp Phe Ser Arg Tyr 20 25 30 Trp Met Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Glu
Ile Asn Pro Asp Ser Ser Thr Ile Asn Tyr Thr Pro Ser Leu 50 55 60
Lys Ala Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65
70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Thr Gly Pro Ala Tyr Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser 100 105 110 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro Ser Ser Lys 115 120 125 Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr 130 135 140 Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 145 150 155 160 Gly Val His
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 165 170 175 Leu
Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 180 185
190 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
195 200 205 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro
Pro Cys 210 215 220 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro 225 230 235 240 Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys 245 250 255 Val Val Val Asp Val Ser His
Glu Asp Pro Glu Val Lys Phe Asn Trp 260 265 270 Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 275 280 285 Glu Gln Tyr
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 290 295 300 His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 305 310
315 320 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly 325 330 335 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
Arg Asp Glu 340 345 350 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr 355 360 365 Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn 370 375 380 Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe 385 390 395 400 Leu Tyr Ser Lys
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 405 410 415 Val Phe
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 420 425 430
Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 140441PRTArtificial
SequencehSC27.204v5 Heavy Chain 140Glu Val Gln Leu Leu Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Asp Phe Ser Arg Tyr 20 25 30 Trp Met Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Glu
Ile Asn Pro Asp Ser Ser Thr Ile Lys Tyr Thr Pro Ser Leu 50 55 60
Lys Ala Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65
70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Thr Gly Pro Ala Tyr Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser 100 105 110 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro Ser Ser Lys 115 120 125 Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr 130 135 140 Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 145 150 155 160 Gly Val His
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 165 170 175 Leu
Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 180 185
190 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
195 200 205 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro
Pro Cys 210 215 220 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro 225 230 235 240 Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys 245 250 255 Val Val Val Asp Val Ser His
Glu Asp Pro Glu Val Lys Phe Asn Trp 260 265 270 Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 275 280 285 Glu Gln Tyr
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 290 295 300 His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 305 310
315 320 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly 325 330 335 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
Arg Asp Glu 340 345 350 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr 355 360 365 Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn 370 375 380 Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe 385 390 395 400 Leu Tyr Ser Lys
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 405 410 415 Val Phe
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 420 425 430
Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 141441PRTArtificial
SequencehSC27.204v6 Heavy Chain 141Glu Val Gln Leu Leu Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Asp Phe Ser Arg Tyr 20 25 30 Trp Met Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Glu
Ile Asn Pro Asp Ser Ser Thr Ile Gln Tyr Thr Pro Ser Leu 50 55 60
Lys Ala Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65
70
75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95 Thr Gly Pro Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ser 100 105 110 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Ser Ser Lys 115 120 125 Ser Thr Ser Gly Gly Thr Ala Ala Leu
Gly Cys Leu Val Lys Asp Tyr 130 135 140 Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser 145 150 155 160 Gly Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 165 170 175 Leu Ser
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 180 185 190
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 195
200 205 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro
Cys 210 215 220 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro 225 230 235 240 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
Thr Pro Glu Val Thr Cys 245 250 255 Val Val Val Asp Val Ser His Glu
Asp Pro Glu Val Lys Phe Asn Trp 260 265 270 Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu 275 280 285 Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 290 295 300 His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 305 310 315
320 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
325 330 335 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Asp Glu 340 345 350 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr 355 360 365 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn 370 375 380 Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe 385 390 395 400 Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 405 410 415 Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 420 425 430 Gln
Lys Ser Leu Ser Leu Ser Pro Gly 435 440 142441PRTArtificial
SequencehSC27.204v7 Heavy Chain 142Glu Val Gln Leu Leu Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Asp Phe Ser Arg Tyr 20 25 30 Trp Met Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Glu
Ile Asn Pro Asp Ser Ser Thr Ile Asn Tyr Asn Pro Ser Leu 50 55 60
Lys Ala Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65
70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Thr Gly Pro Ala Tyr Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser 100 105 110 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro Ser Ser Lys 115 120 125 Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr 130 135 140 Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 145 150 155 160 Gly Val His
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 165 170 175 Leu
Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 180 185
190 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
195 200 205 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro
Pro Cys 210 215 220 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro 225 230 235 240 Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys 245 250 255 Val Val Val Asp Val Ser His
Glu Asp Pro Glu Val Lys Phe Asn Trp 260 265 270 Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 275 280 285 Glu Gln Tyr
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 290 295 300 His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 305 310
315 320 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly 325 330 335 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
Arg Asp Glu 340 345 350 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr 355 360 365 Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn 370 375 380 Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe 385 390 395 400 Leu Tyr Ser Lys
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 405 410 415 Val Phe
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 420 425 430
Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 143441PRTArtificial
SequencehSC27.204v8 Heavy Chain 143Glu Val Gln Leu Leu Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30 Trp Met Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Glu
Ile Asn Pro Asp Ser Ser Thr Ile Asn Tyr Thr Pro Ser Leu 50 55 60
Lys Ala Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65
70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Gly Pro Ala Tyr Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser 100 105 110 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro Ser Ser Lys 115 120 125 Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr 130 135 140 Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 145 150 155 160 Gly Val His
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 165 170 175 Leu
Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 180 185
190 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
195 200 205 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro
Pro Cys 210 215 220 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro 225 230 235 240 Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys 245 250 255 Val Val Val Asp Val Ser His
Glu Asp Pro Glu Val Lys Phe Asn Trp 260 265 270 Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 275 280 285 Glu Gln Tyr
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 290 295 300 His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 305 310
315 320 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly 325 330 335 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
Arg Asp Glu 340 345 350 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr 355 360 365 Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn 370 375 380 Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe 385 390 395 400 Leu Tyr Ser Lys
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 405 410 415 Val Phe
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 420 425 430
Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 144441PRTArtificial
SequencehSC27.204v9 Heavy Chain 144Glu Val Gln Leu Leu Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30 Trp Met Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Glu
Ile Asn Pro Asp Ser Ser Thr Ile Lys Tyr Thr Pro Ser Leu 50 55 60
Lys Ala Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65
70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Gly Pro Ala Tyr Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser 100 105 110 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro Ser Ser Lys 115 120 125 Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr 130 135 140 Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 145 150 155 160 Gly Val His
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 165 170 175 Leu
Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 180 185
190 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
195 200 205 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro
Pro Cys 210 215 220 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro 225 230 235 240 Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys 245 250 255 Val Val Val Asp Val Ser His
Glu Asp Pro Glu Val Lys Phe Asn Trp 260 265 270 Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 275 280 285 Glu Gln Tyr
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 290 295 300 His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 305 310
315 320 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly 325 330 335 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
Arg Asp Glu 340 345 350 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr 355 360 365 Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn 370 375 380 Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe 385 390 395 400 Leu Tyr Ser Lys
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 405 410 415 Val Phe
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 420 425 430
Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 145441PRTArtificial
SequencehSC27.204v10 Heavy Chain 145Glu Val Gln Leu Leu Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30 Trp Met Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Glu
Ile Asn Pro Asp Ser Ser Thr Ile Gln Tyr Thr Pro Ser Leu 50 55 60
Lys Ala Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65
70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Gly Pro Ala Tyr Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser 100 105 110 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro Ser Ser Lys 115 120 125 Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr 130 135 140 Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 145 150 155 160 Gly Val His
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 165 170 175 Leu
Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 180 185
190 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
195 200 205 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro
Pro Cys 210 215 220 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro 225 230 235 240 Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys 245 250 255 Val Val Val Asp Val Ser His
Glu Asp Pro Glu Val Lys Phe Asn Trp 260 265 270 Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 275 280 285 Glu Gln Tyr
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 290 295 300 His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 305 310
315 320 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly 325 330 335 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
Arg Asp Glu 340 345 350 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr 355 360 365 Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn 370 375 380 Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe 385 390 395 400 Leu Tyr Ser Lys
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 405 410 415 Val Phe
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 420 425 430
Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 146441PRTArtificial
SequencehSC27.204v11 Heavy Chain 146Glu Val Gln Leu Leu Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30 Trp Met Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Glu
Ile Asn Pro Asp Ser Ser Thr Ile Asn Tyr Asn Pro Ser Leu 50 55 60
Lys Ala Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65
70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Gly Pro Ala Tyr Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser 100 105 110 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro Ser Ser Lys 115 120 125 Ser Thr Ser Gly Gly Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 130 135 140 Phe Pro Glu Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 145 150 155 160 Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 165 170
175 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
180 185 190 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
Asp Lys 195 200 205 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
Cys Pro Pro Cys 210 215 220 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro 225 230 235 240 Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys 245 250 255 Val Val Val Asp Val
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 260 265 270 Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 275 280 285 Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 290 295
300 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
305 310 315 320 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly 325 330 335 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Asp Glu 340 345 350 Leu Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr 355 360 365 Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn 370 375 380 Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 385 390 395 400 Leu Tyr
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 405 410 415
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 420
425 430 Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440
147441PRTArtificial SequencehSC27.204v12 Heavy Chain 147Glu Val Gln
Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Ser Arg Tyr 20 25
30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ser Glu Ile Asn Pro Asp Ser Ser Thr Ile Asn Tyr Thr Pro
Ser Leu 50 55 60 Lys Ala Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Gly Pro Ala Tyr Trp Gly Gln
Gly Thr Leu Val Thr Val Ser Ser 100 105 110 Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 115 120 125 Ser Thr Ser Gly
Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 130 135 140 Phe Pro
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 145 150 155
160 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
165 170 175 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
Gln Thr 180 185 190 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
Lys Val Asp Lys 195 200 205 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
His Thr Cys Pro Pro Cys 210 215 220 Pro Ala Pro Glu Leu Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro 225 230 235 240 Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 245 250 255 Val Val Val
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 260 265 270 Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 275 280
285 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
290 295 300 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn 305 310 315 320 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly 325 330 335 Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg Asp Glu 340 345 350 Leu Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr 355 360 365 Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 370 375 380 Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 385 390 395 400
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 405
410 415 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr 420 425 430 Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440
148441PRTArtificial SequencehSC27.204v13 Heavy Chain 148Glu Val Gln
Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Ser Arg Tyr 20 25
30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ser Glu Ile Asn Pro Asp Ser Ser Thr Ile Lys Tyr Thr Pro
Ser Leu 50 55 60 Lys Ala Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Gly Pro Ala Tyr Trp Gly Gln
Gly Thr Leu Val Thr Val Ser Ser 100 105 110 Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 115 120 125 Ser Thr Ser Gly
Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 130 135 140 Phe Pro
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 145 150 155
160 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
165 170 175 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
Gln Thr 180 185 190 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
Lys Val Asp Lys 195 200 205 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
His Thr Cys Pro Pro Cys 210 215 220 Pro Ala Pro Glu Leu Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro 225 230 235 240 Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 245 250 255 Val Val Val
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 260 265 270 Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 275 280
285 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
290 295 300 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn 305 310 315 320 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly 325 330 335 Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg Asp Glu 340 345 350 Leu Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr 355 360 365 Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 370 375 380 Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 385 390 395 400
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 405
410 415 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr 420 425 430 Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440
149441PRTArtificial SequencehSC27.204v14 Heavy Chain 149Glu Val Gln
Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Ser Arg Tyr 20 25
30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ser Glu Ile Asn Pro Asp Ser Ser Thr Ile Gln Tyr Thr Pro
Ser Leu 50 55 60 Lys Ala Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Gly Pro Ala Tyr Trp Gly Gln
Gly Thr Leu Val Thr Val Ser Ser 100 105 110 Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 115 120 125 Ser Thr Ser Gly
Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 130 135 140 Phe Pro
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 145 150 155
160 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
165 170 175 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
Gln Thr 180 185 190 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
Lys Val Asp Lys 195 200 205 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
His Thr Cys Pro Pro Cys 210 215 220 Pro Ala Pro Glu Leu Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro 225 230 235 240 Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 245 250 255 Val Val Val
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 260 265 270 Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 275 280
285 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
290 295 300 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn 305 310 315 320 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly 325 330 335 Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg Asp Glu 340 345 350 Leu Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr 355 360 365 Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 370 375 380 Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 385 390 395 400
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 405
410 415 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr 420 425 430 Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440
150441PRTArtificial SequencehSC27.204v15 Heavy Chain 150Glu Val Gln
Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Ser Arg Tyr 20 25
30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ser Glu Ile Asn Pro Asp Ser Ser Thr Ile Asn Tyr Asn Pro
Ser Leu 50 55 60 Lys Ala Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Gly Pro Ala Tyr Trp Gly Gln
Gly Thr Leu Val Thr Val Ser Ser 100 105 110 Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 115 120 125 Ser Thr Ser Gly
Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 130 135 140 Phe Pro
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 145 150 155
160 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
165 170 175 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
Gln Thr 180 185 190 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
Lys Val Asp Lys 195 200 205 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
His Thr Cys Pro Pro Cys 210 215 220 Pro Ala Pro Glu Leu Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro 225 230 235 240 Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 245 250 255 Val Val Val
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 260 265 270 Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 275 280
285 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
290 295 300 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn 305 310 315 320 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly 325 330 335 Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg Asp Glu 340 345 350 Leu Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr 355 360 365 Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 370 375 380 Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 385 390 395 400
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 405
410 415 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr 420 425 430 Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440
15111PRTArtificial SequencehSC27.1 CDRL1 151Lys Ala Ser Glu Asp Ile
Tyr Asn Arg Leu Ala 1 5 10 1527PRTArtificial SequencehSC27.1 CDRL2
152Gly Ala Thr Ser Leu Glu Thr 1 5 1539PRTArtificial
SequencehSC27.1 CDRL3 153Gln Gln Tyr Trp Ser Thr Pro Leu Thr 1 5
1545PRTArtificial SequencehSC27.1 CDRH1 154Glu Tyr Thr Leu His 1 5
15517PRTArtificial SequencehSC27.1 CDRH2 155Gly Ile Asn Pro Asn Asn
Gly Asp Thr Ile Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly
15610PRTArtificial SequencehSC27.1 CDRH3 156Arg Ala Ile Thr Val Tyr
Ala Met Asp Tyr 1 5 10 15715PRTArtificial SequencehSC27.22 CDRL1
157Arg Ala Ser Gln Thr Val Ser Thr Ser Ser Tyr Ser Tyr Met His 1 5
10 15 1587PRTArtificial SequencehSC27.22 CDRL2 158Phe Ala Ser Asn
Val Glu Ser 1 5 1599PRTArtificial SequencehSC27.22 CDRL3 159Gln His
Ser Trp Glu Ile Pro Trp Thr 1 5 1605PRTArtificial SequencehSC27.22
CDRH1 160Ser Tyr Trp Met Asn 1 5 16117PRTArtificial
SequencehSC27.22 CDRH2 161Met Ile His Pro Ser Asp Ser Glu Ile Arg
Leu Asn Gln Lys Phe Lys 1 5 10 15 Asp 16213PRTArtificial
SequencehSC27.22 CDRH3 162Ile Asp Ser Tyr Tyr Gly Tyr Leu Phe Tyr
Phe Asp Tyr 1 5
10 16312PRTArtificial SequencehSC27.108 CDRL1 163Ser Val Ser Ser
Ser Ile Ser Ser Ser Asn Leu His 1 5 10 1647PRTArtificial
SequencehSC27.108 CDRL2 164Gly Thr Ser Asn Leu Ala Ser 1 5
1659PRTArtificial SequencehSC27.108 CDRL3 165Gln Gln Trp Ser Ser
Tyr Pro His Thr 1 5 1665PRTArtificial SequencehSC27.108 CDRH1
166Asn Tyr Leu Ile Glu 1 5 16717PRTArtificial SequencehSC27.108
CDRH2 167Leu Ile Asn Pro Gly Ser Gly Gly Thr Asn Tyr Asn Glu Lys
Phe Lys 1 5 10 15 Gly 16817PRTArtificial SequencehSC27.108 CDRH3
168Arg Ser Pro Leu Gly Ser Trp Ile Tyr Tyr Ala Tyr Asp Gly Val Ala
1 5 10 15 Tyr 16911PRTArtificial SequencehSC27.204 CDRL1 169Lys Ala
Gly Gln Asn Val Gly Thr Ser Val Ala 1 5 10 1707PRTArtificial
SequencehSC27.204 CDRL2 170Ser Ala Ser Tyr Arg Tyr Ser 1 5
1719PRTArtificial SequencehSC27.204 CDRL3 171Gln Gln Tyr Ile Thr
Tyr Pro Tyr Thr 1 5 1725PRTArtificial SequencehSC27.204 CDRH1
172Arg Tyr Trp Met Ser 1 5 17317PRTArtificial SequencehSC27.204
CDRH2 173Glu Ile Asn Pro Asp Ser Ser Thr Ile Asn Tyr Thr Pro Ser
Leu Lys 1 5 10 15 Ala 1743PRTArtificial SequencehSC27.204 CDRH3
174Pro Ala Tyr 1 17517PRTArtificial SequencehSC27.204v1,
hSC27.204v5 and hSC27.405v13 CDRH2 175Glu Ile Asn Pro Asp Ser Ser
Thr Ile Lys Tyr Thr Pro Ser Leu Lys 1 5 10 15 Ala
17617PRTArtificial SequencehSC27.204v2, hSC27.204v6 and
hSC27.405v14 CDRH2 176Glu Ile Asn Pro Asp Ser Ser Thr Ile Gln Tyr
Thr Pro Ser Leu Lys 1 5 10 15 Ala 17717PRTArtificial
SequencehSC27.204v3, hSC27.204v7 and hSC27.405v15 CDRH2 177Glu Ile
Asn Pro Asp Ser Ser Thr Ile Asn Tyr Asn Pro Ser Leu Lys 1 5 10 15
Ala 178366DNAArtificial SequenceCodon optimized hSC27.22ss1 full
length HC DNA 178caagtgcagc tcgtccagtc cggtgccgaa gtcaagaagc
cgggcgcatc agtgaaagtg 60tcgtgcaaag cctccgggta caccttcacc tcatactgga
tgaactgggt ccgccaagcc 120ccgggacaga gactggagtg gatgggcatg
attcacccat ccgattccga gatccggctg 180aaccagaagt tcaaggaccg
cgtgaccatc acccgggaca ccagcgccag cactgcctac 240atggaattga
gctcgctgcg gtccgaggat accgctgtgt actattgcgc gaggatcgac
300tcctactacg gctacctttt ctacttcgac tactggggac aagggacgac
cgtgactgtg 360tcgagc 366
* * * * *
References