U.S. patent application number 16/813167 was filed with the patent office on 2020-09-03 for antibodies.
The applicant listed for this patent is GENMAB A/S. Invention is credited to Esther C. W. BREIJ, Bart E. C. G. DE GOEIJ, Patrick ENGELBERTS, Sjeng HORBACH, Kristel KEMPER, Paul PARREN, Rik RADEMAKER, David SATIJN, Edward N. VAN DEN BRINK, Dennis VERZIJL.
Application Number | 20200277397 16/813167 |
Document ID | / |
Family ID | 1000004867447 |
Filed Date | 2020-09-03 |
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United States Patent
Application |
20200277397 |
Kind Code |
A1 |
SATIJN; David ; et
al. |
September 3, 2020 |
ANTIBODIES
Abstract
The present invention relates to antibodies binding to 5T4,
including bispecific antibodies binding to 5T4 and CD3. The
invention further provides pharmaceutical compositions comprising
the antibodies and use of the antibodies for therapeutic and
diagnostic procedures, in particular in cancer therapy.
Inventors: |
SATIJN; David; (Utrecht,
NL) ; BREIJ; Esther C. W.; (Utrecht, NL) ; DE
GOEIJ; Bart E. C. G.; (Utrecht, NL) ; KEMPER;
Kristel; (Utrecht, NL) ; ENGELBERTS; Patrick;
(Copenhagen V, DK) ; VAN DEN BRINK; Edward N.;
(Halfweg, NL) ; RADEMAKER; Rik; (Utrecht, NL)
; VERZIJL; Dennis; (Amstelveen, NL) ; HORBACH;
Sjeng; (Oss, NL) ; PARREN; Paul; (Odijk,
NL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GENMAB A/S |
Copenhagen V |
|
DK |
|
|
Family ID: |
1000004867447 |
Appl. No.: |
16/813167 |
Filed: |
March 9, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
PCT/EP2019/056197 |
Mar 12, 2019 |
|
|
|
16813167 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/30 20130101;
C07K 2317/31 20130101; A61K 2039/505 20130101; C07K 2317/565
20130101; C12N 15/79 20130101; A61P 35/00 20180101; C07K 2317/51
20130101 |
International
Class: |
C07K 16/30 20060101
C07K016/30; C12N 15/79 20060101 C12N015/79; A61P 35/00 20060101
A61P035/00 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 12, 2018 |
EP |
18161293.8 |
May 31, 2018 |
EP |
18175347.6 |
Claims
1-133. (canceled)
134. An antibody which binds to human 5T4 and comprises a heavy
chain variable (VH) region and a light chain variable (VL) region
selected from the group consisting of: (a) a VH region comprising
the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 6, 7,
and 8, respectively, and a VL region comprising the CDR1, CDR2, and
CDR3 sequences set forth in SEQ ID NO: 10, the sequence AAS, and
SEQ ID NO: 11, respectively, (b) a VH region comprising the CDR1,
CDR2, and CDR3 sequences set forth in SEQ ID NOs: 13, 14, and 15,
respectively; and a VL region comprising the CDR1, CDR2, and CDR3
sequences set forth in SEQ ID NO: 17, the sequence DAS, and SEQ ID
NO:18, respectively, (c) a VH region comprising the CDR1, CDR2, and
CDR3 sequences set forth in SEQ ID NOs: 20, 21, and 22,
respectively; and a VL region comprising the CDR1, CDR2, and CDR3
sequences set forth in SEQ ID NO: 24, the sequence DAS, and SEQ ID
NO: 25, respectively, (d) a VH region comprising the CDR1, CDR2,
and CDR3 sequences set forth in SEQ ID NOs: 27, 28, and 29,
respectively; and a VL region comprising the CDR1, CDR2, and CDR3
sequences set forth in SEQ ID NO: 31, the sequence DVS, and SEQ ID
NO: 32, respectively, (e) a VH region comprising the CDR1, CDR2,
and CDR3 sequences set forth in SEQ ID NOs: 34, 35, and 36,
respectively; and a VL region comprising the CDR1, CDR2, and CDR3
sequences set forth in SEQ ID NO: 38, the sequence DAS, and SEQ ID
NO: 39, respectively, (f) a VH region comprising the CDR1, CDR2,
and CDR3 sequences set forth in SEQ ID NOs: 41, 42, and 43,
respectively, and a VL region comprising the CDR1, CDR2, and CDR3
sequences set forth in SEQ ID NO: 45, the sequence DAS, and SEQ ID
NO: 46, respectively, (g) a VH region comprising the CDR1, CDR2,
and CDR3 sequences set forth in SEQ ID NOs: 48, 49, and 50,
respectively, and a VL region comprising the CDR1, CDR2, and CDR3
sequences set forth in SEQ ID NO: 52, the sequence DAS, and SEQ ID
NO: 53, respectively; and (h) a VH region and a VL region, each
region comprising CDR1, CDR2 and CDR3 sequences, said CDR1, CDR2,
and CDR3 sequences comprising in total at most 1, 2, 3, 4, 5, 6, 7,
8, 9, or 10 amino acid substitutions relative to the CDR1, CDR2,
and CDR3 sequences defined in any one of (a) to (g).
135. An antibody which binds to human 5T4 and comprises a heavy
chain variable (VH) region and a light chain variable (VL) region
selected from the group consisting of: a) a VH region comprising a
sequence having at least 90% identity to the sequence of SEQ ID NO:
5, and a VL region comprising a sequence having at least 90%
identity to the sequence of SEQ ID NO: 9, b) a VH region comprising
a sequence having at least 90% identity to the sequence of SEQ ID
NO: 12, and a VL region comprising a sequence having at least 90%
identity to the sequence of SEQ ID NO: 16, c) a VH region
comprising a sequence having at least 90% identity to the sequence
of SEQ ID NO: 19, and a VL region comprising a sequence having at
least 90% identity to the sequence of SEQ ID NO: 23, d) a VH region
comprising a sequence having at least 90% identity to the sequence
of SEQ ID NO: 26, and a VL region comprising a sequence having at
least 90% identity to the sequence of SEQ ID NO: 30, e) a VH region
comprising a sequence having at least 90% identity to the sequence
of SEQ ID NO: 33, and a VL region comprising a sequence having at
least 90% identity to the sequence of SEQ ID NO: 37, f) a VH region
comprising a sequence having at least 90% identity to the sequence
of SEQ ID NO: 40, and a VL region comprising a sequence having at
least 90% identity to the sequence of SEQ ID NO: 44, and g) a VH
region comprising a sequence having at least 90% identity to the
sequence of SEQ ID NO: 47, and a VL region comprising a sequence
having at least 90% identity to the sequence of SEQ ID NO: 51.
136. The antibody of claim 134, which is a full length
antibody.
137. The antibody of claim 134, which is a monovalent antibody.
138. The antibody of claim 134, which is a bivalent antibody.
139. The antibody of claim 134, wherein the antibody comprises a
kappa light chain or lambda light chain.
140. The antibody of claim 134, which is a monospecific
antibody.
141. A bispecific antibody comprising an antigen-binding region of
the antibody of claim 134 and a second antigen-binding region.
142. The bispecific antibody of claim 141, wherein the second
antigen-binding region binds to human CD3.
143. The bispecific antibody of claim 142, wherein the
antigen-binding region that binds to human CD3 comprises a VH
region comprising the sequence set forth in SEQ ID NO: 57, except
for an amino acid substitution at a position selected from the
group consisting of T31, N57, H101, G105, S110, and Y114, the
positions being numbered according to the sequence of SEQ ID NO:
57; and a VL region comprising the CDR1, CDR2, and CDR3 sequences
set forth in SEQ ID NO: 58, the sequence GTN, and SEQ ID NO: 59,
respectively.
144. The bispecific antibody of claim 142, wherein the
antigen-binding region that binds to CD3 comprises: (a) a VH region
comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID
NOs: 54, 55 and 56, respectively, and a VL region comprising the
CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 58, the
sequence GTN, and SEQ ID NO: 59, respectively; (b) a VH region
comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID
NOs: 61, 55, and 56, respectively, and a VL region comprising the
CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 58, the
sequence GTN, and SEQ ID NO: 59, respectively; (c) a VH region
comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID
NOs: 63, 55, and 56, respectively, and a VL region comprising the
CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 58, the
sequence GTN, and SEQ ID NO: 59, respectively; (d) a VH region
comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID
NOs: 54, 65, and 56, respectively, and a VL region comprising the
CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 58, the
sequence GTN, and SEQ ID NO: 59, respectively; (e) a VH region
comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID
NOs: 54, 55, and 67, respectively, and a VL region comprising the
CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 58, the
sequence GTN, and SEQ ID NO: 59, respectively; (f) a VH region
comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID
NOs: 54, 55, and 69, respectively, and a VL region comprising the
CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 58, the
sequence GTN, and SEQ ID NO: 59, respectively; (g) a VH region
comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID
NOs: 54, 55, and 71, respectively, and a VL region comprising the
CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 58, the
sequence GTN, and SEQ ID NO: 59, respectively; (h) a VH region
comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID
NOs: 54, 55, and 73, respectively, and a VL region comprising the
CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 58, the
sequence GTN, and SEQ ID NO: 59, respectively; (i) a VH region
comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID
NOs: 54, 55, and 75, respectively, and a VL region comprising the
CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 58, the
sequence GTN, and SEQ ID NO: 59, respectively; (j) a VH region
comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID
NOs: 54, 55, and 77, respectively, and a VL region comprising the
CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 58, the
sequence GTN, and SEQ ID NO: 59, respectively; (k) a VH region
comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID
NOs: 54, 55, and 79, respectively, and a VL region comprising the
CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 58, the
sequence GTN, and SEQ ID NO: 59, respectively; or (l) a VH region
comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID
NOs: 54, 55, and 81, respectively, and a VL region comprising the
CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 58, the
sequence GTN, and SEQ ID NO: 59, respectively.
145. The bispecific antibody of claim 142, wherein: (a) the
antigen-binding region that binds to human 5T4 comprises a VH
region comprising the CDR1, CDR2, and CDR3 sequences set forth in
SEQ ID NOs: 41, 42, and 43, respectively, and a VL region
comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID
NO: 45, the sequence DAS, and SEQ ID NO: 46, respectively; and the
antigen-binding region that binds to human CD3 comprises a VH
region comprising the CDR1, CDR2, and CDR3 sequences set forth in
SEQ ID NOs: 54, 55, and 67, respectively, and a VL region
comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID
NO: 58, the sequence GTN, and SEQ ID NO: 59, respectively; (b) the
antigen-binding region that binds to 5T4 comprises a VH region
comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID
NOs: 41, 42, and 43, respectively, and a VL region comprising the
CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 45, the
sequence DAS, and SEQ ID NO: 46, respectively; and the
antigen-binding region that binds to human CD3 comprises a VH
region comprising the CDR1, CDR2, and CDR3 sequences set forth in
SEQ ID NOs: 54, 55, and 67, respectively, and a VL region
comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID
NO: 58, the sequence GTN, and SEQ ID NO: 59, respectively; or (c)
the antigen-binding region that binds to 5T4 comprises a VH region
comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID
NOs: 48, 49, and 50, respectively, and a VL region comprising the
CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 52, the
sequence DAS, and SEQ ID NO: 53, respectively; and the
antigen-binding region that binds to human CD3 comprises a VH
region comprising the CDR1, CDR2, and CDR3 sequences set forth in
SEQ ID NOs: 54, 55, and 67, respectively, and a VL region
comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID
NO: 58, the sequence GTN, and SEQ ID NO: 59, respectively.
146. The bispecific antibody of claim 142, wherein the
antigen-binding region that binds to human CD3 comprises: (a) VH
and VL sequences set forth in SEQ ID NOs: 62 and 60, respectively;
(b) VH and VL sequences set forth in SEQ ID NOs: 64 and 60,
respectively; (c) VH and VL sequences set forth in SEQ ID NOs: 66
and 60, respectively; (d) VH and VL sequences set forth in SEQ ID
NOs: 68 and 60, respectively; (e) VH and VL sequences set forth in
SEQ ID NOs: 70 and 60, respectively; (f) VH and VL sequences set
forth in SEQ ID NOs: 72 and 60, respectively; (g) VH and VL
sequences set forth in SEQ ID NOs: 74 and 60, respectively; (h) VH
and VL sequences set forth in SEQ ID NOs: 76 and 60, respectively;
(i) VH and VL sequences set forth in SEQ ID NOs: 78 and 60,
respectively; (j) VH and VL sequences set forth in SEQ ID NOs: 80
and 60, respectively; or (k) VH and VL sequences set forth in SEQ
ID NOs: 82 and 60, respectively;
147. The bispecific antibody of claim 142, wherein: (a) the
antigen-binding region that binds to 5T4 comprises a VH region
comprising an amino acid sequence having at least 90% identity to
the sequence set forth in SEQ ID NO: 5; and the antigen-binding
region that binds to human CD3 comprises a VH region comprising the
sequence set forth in SEQ ID NO: 68 and a VL region comprising the
sequence set forth in SEQ ID NO: 60; (b) the antigen-binding region
that binds to 5T4 comprises a VH region comprising an amino acid
sequence having at least 90% identity to the sequence set forth in
SEQ ID NO: 40; and the antigen-binding region that binds to human
CD3 comprises a VH region comprising the sequence set forth in SEQ
ID NO: 68 and a VL region comprising the sequence set forth in SEQ
ID NO: 60; (c) the antigen-binding region that binds to 5T4
comprises a VH region comprising an amino acid sequence having at
least 90% identity to the sequence set forth in SEQ ID NO: 47; and
the antigen-binding region that binds to human CD3 comprises a VH
region comprising the sequence set forth in SEQ ID NO: 68 and a VL
region comprising the sequence set forth in SEQ ID NO: 60; (d) the
antigen-binding region that binds to 5T4 comprises a VH region
comprising an amino acid sequence having at least 90% identity to
the sequence set forth in SEQ ID NO: 5, and a VL region comprising
an amino acid sequence having at least 90% identity to the sequence
set forth in SEQ ID NO: 9; and the antigen-binding region that
binds to human CD3 comprises a VH region comprising the sequence
set forth in SEQ ID NO: 68 and a VL region comprising the sequence
set forth in SEQ ID NO: 60; (e) the antigen-binding region that
binds to 5T4 comprises a VH region comprising an amino acid
sequence having at least 90% identity to the sequence set forth in
SEQ ID NO: 40, and a VL region comprising an amino acid sequence
having at least 90% identity to the sequence set forth in SEQ ID
NO: 44; and the antigen-binding region that binds to human CD3
comprises a VH region comprising the sequence set forth in SEQ ID
NO: 68 and a VL region comprising the sequence set forth in SEQ ID
NO: 60; or (f) the antigen-binding region that binds to 5T4
comprises a VH region comprising an amino acid sequence having at
least 90% identity to the sequence set forth in SEQ ID NO: 47, and
a VL region comprising an amino acid sequence having at least 90%
identity to the sequence set forth in SEQ ID NO: 51; and the
antigen-binding region that binds to human CD3 comprises a VH
region comprising the sequence set forth in SEQ ID NO: 68 and a VL
region comprising the sequence set forth in SEQ ID NO: 60.
148. The bispecific antibody of claim 142, wherein the antibody
comprises a first heavy chain and a second heavy chain, each of
said first and second heavy chains comprising at least a hinge
region, a CH2 region, and a CH3 region, wherein in said first heavy
chain at least one position corresponding to a position selected
from the group consisting of T366, L368, K370, D399, F405, Y407,
and K409 in a human IgG1 heavy chain has been substituted, and in
said second heavy chain at least one position corresponding to a
position selected from the group consisting of T366, L368, K370,
D399, F405, Y407, and K409 in a human IgG1 heavy chain has been
substituted, wherein said substitutions of said first and said
second heavy chains are not in the same positions, and wherein the
amino acid positions are numbered according to EU numbering.
149. The bispecific antibody of claim 142, which comprises a first
heavy chain and a second heavy chain, and wherein in both the first
heavy chain and second heavy chain: (a) amino acid residues at
positions corresponding to L234 and L235 in a human IgG1 heavy
chain according to EU numbering are F and E, respectively; (b) the
amino acid residue at the position corresponding to position D265
in a human IgG1 heavy chain according to EU numbering is A; or (c)
amino acid residues at positions corresponding to L234, L235, and
D265 in a human IgG1 heavy chain according to EU numbering are F,
E, and A, respectively.
150. The bispecific antibody of claim 142, wherein the antibody
comprises a kappa light chain or lambda light chain.
151. An immunoconjugate comprising the antibody according to claim
134, and a therapeutic moiety, such as a cytotoxic agent, a
chemotherapeutic drug, a cytokine, an immunosuppressant,
antibiotic, or a radioisotope.
152. A nucleic acid construct comprising a nucleic acid sequence
encoding the VH region and/or VL region of the antibody of claim
134, optionally wherein the nucleic acid construct further
comprises a nucleic acid sequence encoding the VH region and/or VL
region of an antibody that binds to CD3, wherein the antibody that
binds to CD3 comprises: (a) a VH region comprising the CDR1, CDR2,
and CDR3 sequences set forth in SEQ ID NOs: 54, 55 and 56,
respectively, and a VL region comprising the CDR1, CDR2, and CDR3
sequences set forth in SEQ ID NO: 58, the sequence GTN, and SEQ ID
NO: 59, respectively; (b) a VH region comprising the CDR1, CDR2,
and CDR3 sequences set forth in SEQ ID NOs: 61, 55, and 56,
respectively, and a VL region comprising the CDR1, CDR2, and CDR3
sequences set forth in SEQ ID NO: 58, the sequence GTN, and SEQ ID
NO: 59, respectively; (c) a VH region comprising the CDR1, CDR2,
and CDR3 sequences set forth in SEQ ID NOs: 63, 55, and 56,
respectively, and a VL region comprising the CDR1, CDR2, and CDR3
sequences set forth in SEQ ID NO: 58, the sequence GTN, and SEQ ID
NO: 59, respectively; (d) a VH region comprising the CDR1, CDR2,
and CDR3 sequences set forth in SEQ ID NOs: 54, 65, and 56,
respectively, and a VL region comprising the CDR1, CDR2, and CDR3
sequences set forth in SEQ ID NO: 58, the sequence GTN, and SEQ ID
NO: 59, respectively; (e) a VH region comprising the CDR1, CDR2,
and CDR3 sequences set forth in SEQ ID NOs: 54, 55, and 67,
respectively, and a VL region comprising the CDR1, CDR2, and CDR3
sequences set forth in SEQ ID NO: 58, the sequence GTN, and SEQ ID
NO: 59, respectively; (f) a VH region comprising the CDR1, CDR2,
and CDR3 sequences set forth in SEQ ID NOs: 54, 55, and 69,
respectively, and a VL region comprising the CDR1, CDR2, and CDR3
sequences set forth in SEQ ID NO: 58, the sequence GTN, and SEQ ID
NO: 59, respectively; (g) a VH region comprising the CDR1, CDR2,
and CDR3 sequences set forth in SEQ ID NOs: 54, 55, and 71,
respectively, and a VL region comprising the CDR1, CDR2, and CDR3
sequences set forth in SEQ ID NO: 58, the sequence GTN, and SEQ ID
NO: 59, respectively; (h) a VH region comprising the CDR1, CDR2,
and CDR3 sequences set forth in SEQ ID NOs: 54, 55, and 73,
respectively, and a VL region comprising the CDR1, CDR2, and CDR3
sequences set forth in SEQ ID NO: 58, the sequence GTN, and SEQ ID
NO: 59, respectively; (i) a VH region comprising the CDR1, CDR2,
and CDR3 sequences set forth in SEQ ID NOs: 54, 55, and 75,
respectively, and a VL region comprising the CDR1, CDR2, and CDR3
sequences set forth in SEQ ID NO: 58, the sequence GTN, and SEQ ID
NO: 59, respectively; (j) a VH region comprising the CDR1, CDR2,
and CDR3 sequences set forth in SEQ ID NOs: 54, 55, and 77,
respectively, and a VL region comprising the CDR1, CDR2, and CDR3
sequences set forth in SEQ ID NO: 58, the sequence GTN, and SEQ ID
NO: 59, respectively; (k) a VH region comprising the CDR1, CDR2,
and CDR3 sequences set forth in SEQ ID NOs: 54, 55, and 79,
respectively, and a VL region comprising the CDR1, CDR2, and CDR3
sequences set forth in SEQ ID NO: 58, the sequence GTN, and SEQ ID
NO: 59, respectively; or (l) a VH region comprising the CDR1, CDR2,
and CDR3 sequences set forth in SEQ ID NOs: 54, 55, and 81,
respectively, and a VL region comprising the CDR1, CDR2, and CDR3
sequences set forth in SEQ ID NO: 58, the sequence GTN, and SEQ ID
NO: 59, respectively.
153. An expression vector comprising the nucleic acid construct of
claim 152.
154. A cell comprising the nucleic acid construct of claim 152.
155. A composition comprising the antibody of claim 134.
156. A composition comprising the bispecific antibody of claim
142.
157. A kit comprising the antibody of claim 134.
158. A method of treating cancer comprising administering to a
subject in need thereof a therapeutically effective amount of the
antibody of claim 134, or a composition comprising the
antibody.
159. The method of claim 158, wherein the cancer is selected from
the group consisting of kidney/renal cancer, breast cancer,
colorectal cancer, prostate cancer, ovarian cancer, bladder cancer,
uterine/endometrial/cervical cancer, lung cancer, gastro-intestinal
cancer, stomach cancer, pancreatic cancer, thyroid cancer, head and
neck cancer, lymphoma, and acute myeloid leukemia.
160. A method of treating cancer comprising administering to a
subject in need thereof a therapeutically effective amount of the
bispecific antibody of claim 142, or a composition comprising the
bispecific antibody.
161. The method of claim 160, wherein the cancer is selected from
the group consisting of kidney/renal cancer, breast cancer,
colorectal cancer, prostate cancer, ovarian cancer, bladder cancer,
uterine/endometrial/cervical cancer, lung cancer, gastro-intestinal
cancer, stomach cancer, pancreatic cancer, thyroid cancer, head and
neck cancer, lymphoma, and acute myeloid leukemia.
162. A method of producing an antibody that binds to human 5T4 or a
bispecific antibody that binds to both human 5T4 and human CD3
comprising the steps of: a) culturing a host cell comprising the
expression vector of claim 153 in culture medium; and b) purifying
the antibody from the culture medium.
163. An anti-idiotypic antibody which binds to the antigen-binding
region of the antibody of claim 134.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of International
Application No. PCT/EP2019/056197, filed Mar. 12, 2019, which
claims priority to European Patent Application Nos. 18161293.8 and
18175347.6, filed on Mar. 12, 2018, and May 31, 2018, respectively.
The contents of the aforementioned applications are hereby
incorporated by reference.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Mar. 31, 2020, is named GMI_190PCCN_Sequence_Listing.txt and is
90,340 bytes in size.
FIELD OF INVENTION
[0003] The present invention relates to antibodies binding to 5T4,
including bispecific antibodies binding to 5T4 and CD3. The
invention further provides pharmaceutical compositions comprising
the antibodies and use of the antibodies for therapeutic and
diagnostic procedures, in particular in cancer therapy.
BACKGROUND
[0004] 5T4 (also known as trophoblast glycoprotein [TPBG] or
Wnt-activated inhibitory factor 1 [WAIF1]) is a 72 kDa, single-pass
transmembrane protein that contains 8 leucine-rich repeats (LRR)
and 7 potential N-glycosylation sites (Zhao et al., 2014 Structure
22, 612-620).
[0005] 5T4 expression is limited in normal adult tissues, except
for placenta (Southall et al., 1990 Br J Cancer 61, 89-95). 5T4 is
expressed in many human cancers, including renal, cervical,
ovarian, lung, prostate and colon cancer (Stern and Harrop, 2017
Cancer Immunol Immunother 66, 415-426; Southall et al., 1990 Br J
Cancer 61, 89-95). 5T4 expression in tumor cells drives tumor
development by 1) facilitating epithelial-to-mesenchymal transition
(Damelin et al., 2011 Cancer Res 71, 4236-4246; Carsberg et al.,
1996 Int J Cancer 68, 84-92), and 2) inhibition of the canonical
Wnt/beta-catenin signaling pathway and activation of the
non-canonical Wnt pathway (Kagermeier-Schenk et al., 2011 Dev Cell
21, 1129-1143).
[0006] 5T4-targeting antibodies and 5T4-targeting therapies have
clinical activity in several cancers known to express 5T4
(including colorectal, lung and renal cancer). For example,
naptumomab estafenatox is a recombinant fusion protein that consist
of the 5T4-Fab moiety genetically fused to the engineered
superantigen variant SEA/E-120. It is currently in clinical trials
as an immunotherapy for non-small cell lung cancer (NSCLC), renal
cell (RCC) and pancreatic cancer (see e.g. Eisen, et al., 2014 Curr
Oncol Rep 16, 370). Furthermore, TroVax.RTM. is a modified vaccinia
Ankara that expresses 5T4 constructs (MVA-5T4), which shows
clinical benefit in colorectal, prostate and renal cancer (see e.g.
Stern and Harrop, 2017 Cancer Immunol Immunother 66, 415-426; Scurr
et al., 2017 JAMA Oncol 12, 10). Further anti-5T4 antibodies have
been described in WO2007106744, WO03038098, WO2011048369,
WO2013041687, WO2017072207.
[0007] While significant progress has been made on eradication of
cancer, there is still a need for further improvement of
antibody-based cancer therapy.
SUMMARY OF INVENTION
[0008] It is an object of the present invention to provide an
antibody comprising at least one antigen-binding region capable of
binding to 5T4 (Trophoblast glycoprotein), wherein the antibody is
able to block binding to 5T4 of an antibody comprising a variable
heavy chain (VH) region comprising the sequence set forth in SEQ ID
NO: 5, and a variable light chain (VL) region comprising the
sequence set forth in SEQ ID NO: 9 [059].
[0009] The antibody may in particular be a bispecific antibody and
may further comprise an antigen binding region of an antibody that
binds to CD3, such as human CD3E (epsilon), such as human CD3E
(epsilon) as specified in SEQ ID NO: 4.
[0010] In another aspect, the present invention relates to a
nucleic acid construct comprising [0011] a) a nucleic acid sequence
encoding a heavy chain sequence of an antibody comprising an
antigen-binding region capable of binding to 5T4 as defined herein,
and/or [0012] b) a nucleic acid sequence encoding a light chain
sequence of an antibody comprising an antigen-binding region
capable of binding to 5T4 as defined herein.
[0013] In another aspect, the present invention relates to an
expression vector comprising [0014] a) a nucleic acid sequence
encoding a heavy chain sequence of an antibody comprising an
antigen-binding region capable of binding to 5T4 as defined herein,
and/or [0015] b) a nucleic acid sequence encoding a light chain
sequence of an antibody comprising an antigen-binding region
capable of binding to 5T4 as defined herein.
[0016] In another aspect, the present invention relates to a cell
comprising a nucleic acid construct or an expression vector as
defined herein.
[0017] In another aspect, the present invention relates to a
composition comprising an antibody according to the invention.
[0018] In another aspect, the present invention relates to a
pharmaceutical composition comprising an antibody as defined herein
and a pharmaceutically acceptable carrier.
[0019] In another aspect, the present invention relates to an
antibody as defined herein for use as a medicament, such as for use
in the treatment of a disease.
[0020] In another aspect, the present invention relates to a method
of treating a disease or disorder, the method comprising
administering an antibody, a composition or pharmaceutical
composition as defined herein, to a subject in need thereof.
[0021] In another aspect, the present invention relates to methods
for producing an antibody as defined herein.
[0022] In another aspect, the present invention relates to a
kit-of-parts, comprising an antibody as defined herein; and
instructions for use of said kit.
[0023] In another aspect, the present invention relates to an
anti-idiotypic antibody, which binds to the antigen-binding region
capable of binding to 5T4 of the antibody as defined herein.
BRIEF DESCRIPTION OF FIGURES
[0024] FIGS. 1A-1C: Antibody displacement of IgG1-5T4-059-FEAR,
IgG1-5T4-207-FEAR and IgG1-5T4-226-FEAR in combination with
IgG1-5T4-A3-F405L. Antibody displacement was determined by biolayer
interferometry on an Octet HTX instrument (ForteBio).
IgG1-5T4-A3-F405L was immobilized on the biosensor and loaded with
human 5T4ECDHis (mature protein of SEQ ID NO. 99). Subsequently,
the loaded biosensors were exposed to IgG1-5T4-A3-F405L,
IgG1-5T4-H8-FEAR, IgG1-5T4-059-FEAR, IgG1-5T4-207-FEAR or
IgG1-5T4-226-FEAR. The figure shows the association responses (500
s) upon exposure to the second antibodies. FIGS. 1A-1C.
IgG1-5T4-A3-F405L showed no binding to the immobilized
IgG1-5T4-A3-F405L-5T4ECDHis complex, indicating cross-block
(self-block) with IgG1-5T4-A3-F405L. IgG1-5T4-H8-FEAR antibodies
showed an increase in mass (indicating binding to the immobilized
IgG1-5T4-A3-F405L-5T4ECDHis complex) and hence no cross-block with
IgG1-5T4-A3-F405L. FIG. 1A. IgG1-5T4-059-FEAR, FIG. 1B.
IgG1-5T4-207-FEAR and FIG. 1C. IgG1-5T4-226-FEAR all showed an
initial increase in mass (indicating binding of the antibodies to
the immobilized IgG1-5T4-A3-F405L-5T4ECDHis complex) followed by a
rapid decrease in mass. This behavior of the antibodies is
indicative of antibody displacement (Abdiche Y N, et al. (2017)
Antibodies Targeting Closely Adjacent or Minimally Overlapping
Epitopes Can Displace One Another. PLoS ONE 12(1): e0169535.
doi:10.1371/journal.pone.0169535).
[0025] FIG. 2: Simultaneous binding of 5T4 antibodies to
membrane-bound 5T4 measured with flow cytometry. 5T4 antibodies
IgG1-5T4-H8-FEAR, IgG1-5T4-207-FEAR and IgG1-5T4-226-FEAR were
conjugated to fluorescein isothiocyanate (FITC) and added at a
concentration of 2 .mu.g/mL to 5T4-expressing SK-OV-3 cells in
presence of 10 .mu.g/mL unconjugated IgG1-5T4-H8-FEAR,
IgG1-5T4-A1-F405L, IgG1-5T4-A3-F405L, IgG1-b12, IgG1-5T4-207-FEAR
or IgG1-5T4-226-FEAR. Percentage binding of FITC-labeled antibodies
was calculated and depicted as mean percentage binding.+-.standard
deviation (SD).
[0026] FIGS. 3A and 3B: Binding of 5T4 antibodies to HEK-293 cells
transfected with full length human and chicken 5T4. HEK-293 cells
transiently transfected with full length human 5T4 (SEQ ID NO: 1)
(FIG. 3A) or chicken 5T4 (SEQ ID NO: 3) (FIG. 3B) were incubated
with various concentrations of IgG1-5T4-A3-F405L,
IgG1-5T4-059-FEAR, IgG1-5T4-207-FEAR or IgG1-5T4-226-FEAR
antibodies. After incubation with R-Phycoerythrin (PE)-conjugated
goat-anti-human IgG F(ab')2, the mean fluorescence intensity (MFI)
was determined by flow cytometry. As negative control,
IgG1-b12-K409R (10 .mu.g/mL) was included.
[0027] FIGS. 4A and 4B: Internalization capacity of monovalent 5T4
antibodies. Bispecific, toxin-conjugated antibodies that recognize
5T4 with one Fab-arm while recognizing an irrelevant antigen (HIV-1
gp120, which is not expressed on tumor cells) with the second
Fab-arm, were generated by controlled Fab-arm exchange of
unconjugated 5T4 antibodies with (HIV-1 gp120-specific) IgG1-b12
antibodies that had been conjugated with one Duostatin-3 molecule
per antibody. MDA-MB-468 (FIG. 4A) and HCC1954 (FIG. 4B) cells were
incubated with increasing concentrations of antibodies, as
indicated. Cell viability was measured after 5 days. Data are
presented as mean percentage viable cells of three replicate
experiments. As negative control, monospecific, bivalent IgG1-b12
conjugated with Duostatin-3 (IgG1-b12-vcDuo3) was included.
[0028] FIGS. 5A-5D: Binding of CD3x5T4 bispecific antibodies to
full length human and cynomolgus monkey 5T4 transfected into
HEK-293 cells. Binding of monovalent and bivalent 5T4 antibodies
was analysed using HEK-293 cells transiently transfected with full
length human (left panels) or cynomolgus monkey 5T4 (right panels).
Cells were incubated with increasing concentrations of antibodies,
as indicated. After secondary labelling with FITC conjugated
goat-anti-human IgG F(ab')2, binding was analysed by flow
cytometry. As negative control antibody, IgG1-b12-K409R (3
.mu.g/mL) was included. Data are presented as mean fluorescence
intensity (MFI) values of two technical replicates.+-.SD. FIG. 5A.
Binding of bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR and
IgG1-5T4-207-FEAR. FIG. 5B. Binding of
bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR and IgG1-5T4-226-FEAR. FIG.
5C. Binding of bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR and
IgG1-5T4-059-FEAR. FIG. 5D. Binding of
bsIgG1-huCD3-H101G-FEALx5T4-H8-FEAR and IgG1-5T4-H8-FEAR.
[0029] FIGS. 5E-5M: Binding of bispecific CD3x5T4 antibodies to
cynomolgus monkey and human 5T4 transfected into HEK-293 cells.
Mono- and bivalent binding of 5T4 antibodies was analysed using
HEK-293 cells transiently transfected with human 5T4 (left panels)
or with cynomolgus monkey 5T4 (right panels). Cells were incubated
with increasing concentrations of antibodies, as indicated. After
secondary labelling with phycoerythrin (PE)-conjugated
goat-anti-human IgG F(ab')2, binding was analysed by flow
cytometry. FIG. 5E. Binding of bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR
and IgG1-5T4-207-FEAR; FIG. 5F. Binding of
bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR and IgG1-5T4-226-FEAR; FIG.
5G. Binding of bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR and
IgG1-5T4-059-FEAR; FIG. 5H. Binding of
bsIgG1-huCD3-H101G-FEALx5T4-106-FEAR and IgG1-5T4-106-FEAR; FIG.
5I. Binding of bsIgG1-huCD3-H101G-FEALx5T4-076-FEAR and
IgG1-5T4-076-FEAR; FIG. 5J. Binding of
bsIgG1-huCD3-H101G-FEALx5T4-085-FEAR and IgG1-5T4-085-FEAR; FIG.
5K. Binding of bsIgG1-huCD3-H101G-FEALx5T4-127-FEAR and
IgG1-5T4-127-FEAR; FIG. 5L. Binding of
bsIgG1-huCD3-H101G-FEALx5T4-A1-FEAR and IgG1-5T4-A1-FEAR; FIG. 5M.
Binding of bsIgG1-huCD3-H101G-FEALx5T4-A3-FEAR and
IgG1-5T4-A3-FEAR.
[0030] FIGS. 6A-6C: Binding of CD3x5T4 bispecific and 5T4
monospecific antibodies to 5T4-positive human tumor cells. Mono-
and bivalent binding of 5T4 antibodies to HeLa cells (left panels)
or MDA-MB-231 cells (right panels) was determined by flow
cytometry. Cells were incubated with increasing concentrations of
antibodies. After secondary labelling with FITC-conjugated
goat-anti-human IgG F(ab')2, the MFI was determined by flow
cytometry. FIG. 6A. Binding of bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR
and IgG1-5T4-207-FEAR antibodies to HeLa cells (left panel) or
MDA-MB-231 cells (right panel). FIG. 6B. Binding of
bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR and IgG1-5T4-059-FEAR
antibodies to HeLa cells (left panel) or MDA-MB-231 cells (right
panel). FIG. 6C. Binding of bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR
and IgG1-5T4-226-FEAR antibodies to HeLa cells (left panel) or
MDA-MB-231 cells (right panel). IgG1-b12-K409R (3 .mu.g/mL) was
included as negative control (open circles).
[0031] FIGS. 6D-6K: Binding of CD3x5T4 bispecific and 5T4
monospecific antibodies to HeLa cells. Mono- and bivalent binding
of 5T4 antibodies to HeLa cells was determined by flow cytometry.
Cells were incubated with increasing concentrations of antibodies.
After secondary labelling with Phycoerythrin (PE)-conjugated
goat-anti-human IgG F(ab')2, the mean fluorescence intensity (MFI)
was determined by flow cytometry. FIG. 6D. Binding of
bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR and IgG1-5T4-207-FEAR; FIG.
6E. Binding of bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR and
IgG1-5T4-226-FEAR; FIG. 6F. Binding of
bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR and IgG1-5T4-059-FEAR; FIG.
6G. Binding of bsIgG1-huCD3-H101G-FEALx5T4-106-FEAR and
IgG1-5T4-106-FEAR; FIG. 6H. Binding of
bsIgG1-huCD3-H101G-FEALx5T4-085-FEAR and IgG1-5T4-085-FEAR; FIG.
6I. Binding of bsIgG1-huCD3-H101G-FEALx5T4-127-FEAR and
IgG1-5T4-127-FEAR; FIG. 6J. Binding of
bsIgG1-huCD3-H101G-FEALx5T4-A1-FEAR and IgG1-5T4-A1-FEAR; FIG. 6K.
Binding of bsIgG1-huCD3-H101G-FEALx5T4-A3-FEAR and
IgG1-5T4-A3-FEAR
[0032] FIGS. 6L-6S: Binding of CD3x5T4 bispecific and 5T4
monospecific antibodies to MDA-MB-231 cells. Mono- and bivalent
binding of 5T4 antibodies to MDA-MB-231 cells was determined by
flow cytometry. Cells were incubated with increasing concentrations
of antibodies. After secondary labelling with PE-conjugated
goat-anti-human IgG F(ab')2, the mean fluorescence intensity (MFI)
was determined by flow cytometry. FIG. 6L. Binding of
bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR and IgG1-5T4-207-FEAR; FIG.
6M. Binding of bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR and
IgG1-5T4-226-FEAR; FIG. 6N. Binding of
bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR and IgG1-5T4-059-FEAR; FIG.
6O. Binding of bsIgG1-huCD3-H101G-FEALx5T4-106-FEAR and
IgG1-5T4-106-FEAR; FIG. 6P. Binding of
bsIgG1-huCD3-H101G-FEALx5T4-085-FEAR and IgG1-5T4-085-FEAR; FIG.
6Q. Binding of bsIgG1-huCD3-H101G-FEALx5T4-127-FEAR and
IgG1-5T4-127-FEAR; FIG. 6R. Binding of
bsIgG1-huCD3-H101G-FEALx5T4-A1-FEAR and IgG1-5T4-A1-FEAR; FIG. 6S.
Binding of bsIgG1-huCD3-H101G-FEALx5T4-A3-FEAR and
IgG1-5T4-A3-FEAR.
[0033] FIGS. 7A-7C: Induction of cytotoxicity in vitro by CD3x5T4
bispecific antibodies in MDA-MB-231 cells using purified T cells as
effector cells. MDA-MB-231 cells were incubated with increasing
concentrations of CD3x5T4 bispecific antibodies or monospecific,
bivalent 5T4 antibodies and isolated T cells as effector cells in
an Effector:Target cell (E:T) ratio of 8:1. Purified T cells
obtained from two different donors were used for this experiment,
donor A (left panels) and donor B (right panels). Cytotoxicity was
determined by measuring the percentage of viable MDA-MB-231 cells
after 72 hrs of incubation (% viable cells=[absorbance
sample-absorbance staurosporine-treated target cells]/[absorbance
untreated target cells-absorbance staurosporine-treated target
cells].times.100). FIG. 7A. Cytotoxicity induced in the presence of
bsIgG1-huCD3-FEALx5T4-207-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR and IgG1-5T4-207-FEAR; FIG.
7B. Cytotoxicity induced in the presence of
bsIgG1-huCD3-FEALx5T4-226-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR and IgG1-5T4-226-FEAR; FIG.
7C. Cytotoxicity induced in the presence of
bsIgG1-huCD3-FEALx5T4-059-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR and IgG1-5T4-059-FEAR.
[0034] FIG. 7D: IC50 values of cytotoxicity induced in vitro by
CD3x5T4 bispecific antibodies in MDA-MB-231 cells using purified T
cells as effector cells. IC50 values of the T-cell mediated
cytotoxicity induced by bsIgG1-huCD3-FEALx5T4-207-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR,
bsIgG1-huCD3-FEALx5T4-226-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR,
bsIgG1-huCD3-FEALx5T4-059-FEAR or
bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR in MDA-MB-231 cells were
analyzed using GraphPad Prism V7.02 software. Data are presented as
mean IC50 values of two different donors.+-.SD.
[0035] FIGS. 8A-8F: Induction of cytotoxicity by CD3x5T4 bispecific
antibodies in MDA-MB-231 cells using T cells as effector cells in
vitro. MDA-MB-231 cells were incubated with increasing
concentrations of CD3x5T4 bispecific antibodies or 5T4 homodimers
and isolated T cells as effector cells in an E:T ratio of 8:1.
Three different donors were used for this experiment. Data shown
are mean % survival.+-.standard error of the mean (SEM) of three
donors tested. FIG. 8A. T-cell-mediated cytotoxicity (decrease in
survival) induced in the presence of
bsIgG1-huCD3-FEALx5T4-207-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR and IgG1-5T4-207-FEAR; FIG.
8B. T-cell-mediated cytotoxicity induced in the presence of
bsIgG1-huCD3-FEALx5T4-226-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR and IgG1-5T4-226-FEAR; FIG.
8C. T-cell-mediated cytotoxicity induced in the presence of
bsIgG1-huCD3-FEALx5T4-059-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR and IgG1-5T4-059-FEAR; FIG.
8D. T-cell-mediated cytotoxicity induced in the presence of
bsIgG1-huCD3-FEALx5T4-106-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-106-FEAR and IgG1-5T4-106-FEAR; FIG.
8E. T-cell-mediated cytotoxicity induced in the presence of
bsIgG1-huCD3-FEALx5T4-A1-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-A1-FEAR
and IgG1-5T4-A1-FEAR; FIG. 8F. T-cell-mediated cytotoxicity induced
in the presence of bsIgG1-huCD3-FEALx5T4-A3-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-A3-FEAR and IgG1-5T4-A3-FEAR.
[0036] FIGS. 8G-8H: IC50 values of cytotoxicity induced by CD3x5T4
bispecific antibodies in MDA-MB-231 cells using T cells as effector
cells in vitro. IC50 values of the T-cell-mediated cytotoxicity
induced CD3x5T4 bispecific antibodies in MDA-MB-231 cells were
analyzed using GraphPad Prism V7.02 software. Data are presented as
mean IC50 values of three different donors.+-.SD. FIG. 8G. IC50
values of the T-cell-mediated cytotoxicity induced by
bsIgG1-huCD3-FEALx5T4-207-FEAR, bsIgG1-huCD3-FEALx5T4-226-FEAR,
bsIgG1-huCD3-FEALx5T4-059-FEAR, bsIgG1-huCD3-FEALx5T4-106-FEAR,
bsIgG1-huCD3-FEALx5T4-A1-FEAR and bsIgG1-huCD3-FEALx5T4-A3-FEAR;
FIG. 8H. IC50 values of the T-cell-mediated cytotoxicity induced by
bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-106-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-A1-FEAR and
bsIgG1-huCD3-H101G-FEALx5T4-A3-FEAR.
[0037] FIGS. 9A-9C: In vitro T-cell activation by CD3x5T4
bispecific antibodies in the presence of MDA-MB-231 cells.
MDA-MB-231 cells were incubated with increasing concentrations of
CD3x5T4 bispecific antibodies and monospecific, bivalent 5T4
antibodies, as indicated, and isolated T cells as effector cells in
an E:T ratio of 8:1. The expression of three T cell activation
markers (PD1 [upper panels], CD25 [middle panels] and CD69 [lower
panels]) was analyzed by flow cytometry. Two different donors were
used for this experiment, donor A (closed symbols) and donor B
(open symbols). FIG. 9A. T-cell activation induced in the presence
of bsIgG1-huCD3-FEALx5T4-207-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR and IgG1-5T4-207-FEAR; FIG.
9B. T-cell activation induced in the presence of
bsIgG1-huCD3-FEALx5T4-226-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR and IgG1-5T4-226-FEAR; FIG.
9C. T-cell activation induced in the presence of
bsIgG1-huCD3-FEALx5T4-059-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR and IgG1-5T4-059-FEAR.
[0038] FIG. 9D: EC50 values of in vitro T-cell activation by
CD3x5T4 bispecific antibodies in the presence of MDA-MB-231 cells.
EC50 values of in vitro T-cell activation markers (PD1, CD25 and
CD69) induced by bsIgG1-huCD3-FEALx5T4-207-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR,
bsIgG1-huCD3-FEALx5T4-226-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR,
bsIgG1-huCD3-FEALx5T4-059-FEAR or
bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR in the presence of MDA-MB-231
cells were analyzed using GraphPad Prism V7.02 software. Data are
presented as mean of two different donors.+-.SD.
[0039] FIGS. 10A-10F: In vitro T-cell activation by CD3x5T4
bispecific antibodies in the presence of MDA-MB-231 cells.
MDA-MB-231 cells were incubated with increasing concentrations of
CD3x5T4 bispecific antibodies and 5T4 homodimers and isolated T
cells as effector cells in an E:T ratio of 8:1. T-cell activation
was measured by an increase in % CD69+ cells within the CD4+ (left
panels) and CD8+ (right panels) T cell populations. Three different
donors were used for this experiment; data shown are mean % CD69
upregulation.+-.SEM of three donors tested. FIG. 10A. T-cell
activation induced in the presence of
bsIgG1-huCD3-FEALx5T4-207-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR and IgG1-5T4-207-FEAR; FIG.
10B. T-cell activation induced in the presence of
bsIgG1-huCD3-FEALx5T4-226-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR and IgG1-5T4-226-FEAR; FIG.
10C. T-cell activation induced in the presence of
bsIgG1-huCD3-FEALx5T4-059-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR and IgG1-5T4-059-FEAR; FIG.
10D. T-cell activation induced in the presence of
bsIgG1-huCD3-FEALx5T4-106-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-106-FEAR and IgG1-5T4-106-FEAR; FIG.
10E. T-cell activation induced in the presence of
bsIgG1-huCD3-FEALx5T4-A1-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-A1-FEAR
and IgG1-5T4-A1-FEAR; FIG. 10F. T-cell activation induced in the
presence of bsIgG1-huCD3-FEALx5T4-A3-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-A3-FEAR and IgG1-5T4-A3-FEAR.
[0040] FIGS. 10G-10L: EC.sub.50 values of in vitro T-cell
activation by CD3x5T4 bispecific antibodies in the presence of
MDA-MB-231 cells. EC.sub.50 values of T-cell activation markers
(increase in % of CD69.sup.+ [FIGS. 10G-10H], CD25.sup.+ [FIGS.
10I-10J] and PD1.sup.+ [FIGS. 10K-10L], CD25 and CD69 cells within
the CD4.sup.+ and CD8.sup.+ T cell populations) induced in vitro by
CD3x5T4 bispecific antibodies in the presence of MDA-MB-231 cells
were analyzed using GraphPad Prism V7.02 software. Data are
presented as mean of three different donors.+-.SD. FIG. 10G.
EC.sub.50 values of the CD69 upregulation induced by
bsIgG1-huCD3-FEALx5T4-207-FEAR, bsIgG1-huCD3-FEALx5T4-226-FEAR,
bsIgG1-huCD3-FEALx5T4-059-FEAR, bsIgG1-huCD3-FEALx5T4-106-FEAR,
bsIgG1-huCD3-FEALx5T4-A1-FEAR and bsIgG1-huCD3-FEALx5T4-A3-FEAR;
FIG. 10H. EC.sub.50 values of the CD69 upregulation induced by
bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-106-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-A1-FEAR and
bsIgG1-huCD3-H101G-FEALx5T4-A3-FEAR. FIG. 10I. EC.sub.50 values of
the CD25 upregulation induced by bsIgG1-huCD3-FEALx5T4-207-FEAR,
bsIgG1-huCD3-FEALx5T4-226-FEAR, bsIgG1-huCD3-FEALx5T4-059-FEAR,
bsIgG1-huCD3-FEALx5T4-106-FEAR, bsIgG1-huCD3-FEALx5T4-A1-FEAR and
bsIgG1-huCD3-FEALx5T4-A3-FEAR; FIG. 10J. EC.sub.50 values of the
CD25 upregulation induced by bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-106-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-A1-FEAR and
bsIgG1-huCD3-H101G-FEALx5T4-A3-FEAR. FIG. 10K. EC.sub.50 values of
the PD1 upregulation induced by bsIgG1-huCD3-FEALx5T4-207-FEAR,
bsIgG1-huCD3-FEALx5T4-226-FEAR, bsIgG1-huCD3-FEALx5T4-059-FEAR,
bsIgG1-huCD3-FEALx5T4-106-FEAR, bsIgG1-huCD3-FEALx5T4-A1-FEAR and
bsIgG1-huCD3-FEALx5T4-A3-FEAR; FIG. 10L. EC.sub.50 values of the
PD1 upregulation induced by bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-106-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-A1-FEAR and
bsIgG1-huCD3-H101G-FEALx5T4-A3-FEAR.
[0041] FIGS. 11A and 11B: T cell cytokine release induced by
CD3x5T4 bispecific antibodies in the presence of 5T4-positive tumor
cells. MDA-MB-231 cells were incubated with 0.2 .mu.g/mL CD3x5T4
bispecific antibodies (bsIgG1-huCD3-FEALx5T4-207-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR,
bsIgG1-huCD3-FEALx5T4-226-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR,
bsIgG1-huCD3-FEALx5T4-059-FEAR or
bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR) and 5T4 monospecific
antibodies (IgG1-5T4-207-FEAR, IgG1-5T4-226-FEAR or
IgG1-5T4-059-FEAR) and isolated T cells as effector cells in an E:T
ratio of 8:1. Release of cytokines was analyzed by U-PLEX assay.
FIG. 11A. Concentration of IL-10, IL-13 and TNF in the supernatant
of T cell (derived from donor A)-tumor cell co-cultures, after 72 h
of incubation with CD3x5T4 bispecific antibodies or 5T4
monospecific antibodies. FIG. 11B. Concentration of IL-10, IL-13
and TNF in the supernatant of T cell (derived from donor B)-tumor
cell co-cultures, after 72 h of incubation with CD3x5T4 bispecific
antibodies or 5T4 monospecific antibodies.
[0042] FIGS. 12A and 12B: Induction of cytotoxicity in vitro by
CD3x5T4 bispecific antibodies in SK-OV-3 cells using PBMCs as
effector cells at varying E:T ratios. SK-OV-3 cells were incubated
with increasing concentrations of bsIgG1-huCD3-FEALx5T4-207-FEAR
(left panels) or bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR (right
panels) and PBMCs as effector cells in an E:T ratio of 1:2, 1:1,
2:1, 4:1, 8:1 and 12:1. Cytotoxicity was determined by measuring
the percentage of viable SK-OV-3 cells after 72 h of incubation (%
viable cells=[absorbance sample-absorbance staurosporine-treated
target cells]/[absorbance untreated target cells-absorbance
staurosporine-treated target cells].times.100). PBMCs from two
different donors were used for this experiment: FIG. 12A. donor C
and FIG. 12B. donor D.
[0043] FIGS. 13A and 13B: Induction of cytotoxicity in SK-OV-3
cells in vitro by CD3x5T4 bispecific antibodies using T cells as
effector cells at varying E:T ratios. SK-OV-3 cells were incubated
with increasing concentrations of bsIgG1-huCD3-FEALx5T4-207-FEAR
(left panels) or bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR (right
panels) and isolated T cells as effector cells in an E:T ratio of
1:2, 1:1, 2:1, 4:1 and 8:1. The efficiency of cytotoxicity was
determined by measuring the percentage of viable SK-OV-3 cells
after 72 h of incubation (% viable cells=[absorbance
sample-absorbance staurosporine-treated target cells]/[absorbance
untreated target cells-absorbance staurosporine-treated target
cells].times.100). T cells from two different donors were used for
this experiment: FIG. 13A. donor E and FIG. 13B. donor F.
[0044] FIGS. 14A and 14B: Anti-tumor activity of CD3x5T4 bispecific
antibodies in a MDA-MB-231 xenograft model in NSG-HIS mice. FIG.
14A. Average tumor size in the MDA-MB-231 xenograft model in
NSG-HIS mice after treatment with PBS (vehicle control), 0.5 mg/kg
bsIgG1-huCD3-FEALx5T4-207-FEAR or 0.5 mg/kg
bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR. Tumor size was assessed by
caliper measurement. Error bars indicate SEM. FIG. 14B. Percentage
of NSG-HIS mice injected with MDA-MB-231 cells with a tumor size
<500 mm.sup.3 after treatment with PBS,
bsIgG1-huCD3-FEALx5T4-207-FEAR or
bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR.
[0045] FIGS. 15A-15D: Binding of directly FITC-labeled 5T4-specific
antibodies to human 5T4 variants with single alanine mutations at
positions 32 to 355 of human 5T4 ECD, as determined by flow
cytometry. Binding was expressed as Z-score (fold change), as a
measure for change in binding compared to a non-cross blocking
5T4-specific control antibody (bsIgG1-5T4-A1-F405Lxb12-FEAR-FITC)
used for normalization. The number on the x-axis refers to the
amino acid positions in human 5T4 (SEQ ID: 1). Residues where the
Z-score in binding was lower than -1.5 (indicated by the dotted
line) were considered `loss of binding mutants`. Residues with a
positive Z-score in binding are loss of binding residues for the
non-cross blocking 5T4 specific control antibody
(bsIgG1-5T4-A1-67F-F405Lxb12-FEAR-FITC). Residues on aa position
38, 45, 49, 51, 54, 62, 64, 66, 68, 71, 72, 77, 91, 104, 108, 110,
112, 118, 121, 122, 135, 137, 155, 161, 167, 171, 201, 202, 205,
208, 218, 231, 269, 279, 298, 300, 303, 323, 324, 340 and 344 were
not evaluated, as these positions contained either endogenous
alanines or cysteines. Data shown are Z-scores for binding of (FIG.
15A) bsIgG1-b12-FEALx5T4-059-FEAR-FITC, (FIG. 15B)
bsIgG1-b12-FEALx5T4-207-FEAR-FITC, (FIG. 15C)
bsIgG1-b12-FEALx5T4-226-FEAR-FITC, and (FIG. 15D)
bsIgG1-5T4-A3-F405Lxb12-FEAR-FITC. Buried residues with a Z-score
just below -1.5 that were predicted to be spatially separated from
the majority of surface-exposed loss of binding residues were
excluded (for bsIgG1-b12-FEALx5T4-207-FEAR-FITC: L281 [Z-score:
-1.57] and P326 [Z-score: -1.54]; and for
bsIgG1-b12-FEALx5T4-226-FEAR-FITC: L273 [Z-score: -1.58], L281
[Z-score: -1.65], N294 [Z-score: -1.57], L309 [Z-score: -1.63] and
P326 [Z-score: -1.67]).
[0046] FIGS. 16A and 16B: Induction of cytotoxicity in vitro by
CD3x5T4 bispecific antibodies in tumor cells of different
indications using T cells as effector cells. Tumor cells were
incubated with increasing concentrations of
bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR or control antibodies
(bsIgG1-huCD3-H101G-FEALxb12-FEAR, bsIgG1-b12-FEALx5T4-207-FEAR)
and isolated T cells as effector cells in an E:T ratio of 4:1.
Cytotoxicity (decrease in survival) was determined by measuring the
percentage of viable tumor cells after 72 h of incubation. Data
shown are mean % survival.+-.SEM of duplicate wells from one
representative donor out of at least three donors tested. FIG. 16A.
Cytotoxicity (decrease in survival) induced in pancreas cancer cell
lines; FIG. 16B. Cytotoxicity (decrease in survival) induced in
cervical cancer cell lines.
[0047] FIG. 16C: IC50 values of cytotoxicity induced in vitro by
CD3x5T4 bispecific antibodies in tumor cell lines of different
indications using T cells as effector cells. IC50 values of the
T-cell-mediated cytotoxicity induced by
bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR in tumor cells of the
indicated indications were analyzed using GraphPad Prism V7.02
software. Data are presented as mean IC50 values of at least three
different donors (see Table 10).+-.SD.
[0048] FIGS. 17A-17D: In vitro T-cell activation by CD3x5T4
bispecific antibodies in the presence of tumor cells of different
indications. Tumor cells were incubated with increasing
concentrations of bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR or control
antibodies (bsIgG1-huCD3-H101G-FEALxb12-FEAR,
bsIgG1-b12-FEALx5T4-207-FEAR and isolated T cells as effector cells
in an E:T ratio of 4:1 for 72 h. T-cell activation was measured by
the upregulation of CD69 (% of CD69+ cells) within CD4.sup.+ (left
panels) and CD8.sup.+ (right panels) T-cell populations. Data shown
are mean % CD69+ cells.+-.SD of duplicate wells from one
representative donor out of at least three donors tested. FIG. 17A.
T-cell activation induced by CD3x5T4 bispecific antibodies in the
presence of pancreas cancer cell line BxPc-3; FIG. 17B. T-cell
activation induced by CD3x5T4 bispecific antibodies in the presence
of pancreas cancer cell line PANC-1; FIG. 17C. T-cell activation
induced by CD3x5T4 bispecific antibodies in the presence of
cervical cancer cell line SiHa; FIG. 17D. T-cell activation induced
by CD3x5T4 bispecific antibodies in the presence of cervical cancer
cell line Ca Ski.
[0049] FIGS. 17E-17F: EC50 values of in vitro T-cell activation by
CD3x5T4 bispecific antibodies in with the presence of tumor cell
lines of different indications. EC50 values of the T-cell
activation (% of CD69+ cells within CD4.sup.+ and CD8.sup.+ T-cell
populations) induced by bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR in
co-culture with tumor cell lines of the different indications were
analyzed using GraphPad Prism V7.02 software. Data are presented as
mean EC50 values of at least three different donors (see Table
10).+-.SD. FIG. 17E. EC50 values of CD4+ T-cell activation induced
by bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR in the presence of the
indicated tumor cell lines; FIG. 17F. EC50 values of CD8+ T-cell
activation induced by bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR in the
presence of the indicated tumor cell lines.
DETAILED DESCRIPTION
Definitions
[0050] The term "antibody" as used herein is intended to refer to
an immunoglobulin molecule, a fragment of an immunoglobulin
molecule, or a derivative of either thereof, which has the ability
to specifically bind to an antigen under typical physiological
and/or tumor-specific conditions with a half-life of significant
periods of time, such as at least about 30 minutes, at least about
45 minutes, at least about one hour, at least about two hours, at
least about four hours, at least about 8 hours, at least about 12
hours, at least about 24 hours or more, at least about 48 hours or
more, at least about 3, 4, 5, 6, 7 or more days, etc., or any other
relevant functionally-defined period (such as a time sufficient to
induce, promote, enhance, and/or modulate a physiological response
associated with antibody binding to the antigen and/or time
sufficient for the antibody to be internalized). The binding region
(or binding domain which may be used herein, both having the same
meaning) which interacts with an antigen, comprises variable
regions of both the heavy and light chains of the immunoglobulin
molecule. The constant regions of the antibodies (Abs) may mediate
the binding of the immunoglobulin to host tissues or factors,
including various cells of the immune system (such as effector
cells) and components of the complement system such as C1q, the
first component in the classical pathway of complement
activation.
[0051] In the context of the present invention, the term "antibody"
includes a monoclonal antibody (mAb), an antibody-like polypeptide,
such as a chimeric antibody and a humanized antibody, as well as an
`antibody fragment` or a `fragment thereof` retaining the ability
to specifically bind to the antigen (antigen-binding fragment)
provided by any known technique, such as enzymatic cleavage,
peptide synthesis, and recombinant techniques, and retaining the
ability to be conjugated to a toxin. An antibody as defined
according to the invention can possess any isotype unless the
disclosure herein is otherwise limited.
[0052] As indicated above, the term antibody as used herein, unless
otherwise stated or clearly contradicted by context, includes
fragments of an antibody that retain the ability to specifically
interact, such as bind, to the antigen. It has been shown that the
antigen-binding function of an antibody may be performed by
fragments of a full-length antibody. Examples of binding fragments
encompassed within the term "antibody" include (i) a Fab' or Fab
fragment, a monovalent fragment consisting of the light chain
variable domain (VL), heavy chain variable domain (VH), light chain
constant region (CL) and heavy chain constant region domain 1 (CH1)
domains, or a monovalent antibody as described in WO 2007/059782;
(ii) F(ab').sub.2 fragments, bivalent fragments comprising two Fab
fragments linked by a disulfide bridge at the hinge region; (iii)
an Fd fragment consisting essentially of the VH and CH1 domains;
(iv) an Fv fragment consisting essentially of the VL and VH domains
of a single arm of an antibody, (v) a dAb fragment Ward et al.,
Nature 341, 544-546 (1989), which consists essentially of a VH
domain and is also called domain antibody Holt et al; Trends
Biotechnol. 2003 November; 21(11):484-90; (vi) camelid or
nanobodies Revets et al; Expert Opin Biol Ther. 2005 January;
5(1):111-24 and (vii) an isolated complementarity determining
region (CDR). Furthermore, although the two domains of the Fv
fragment, VL and VH, are coded for by separate genes, they may be
joined, using recombinant methods, by a synthetic linker that
enables them to be made as a single protein chain in which the VL
and VH regions pair to form monovalent molecules (known as single
chain antibodies or single chain Fv (scFv), see for instance Revets
et al; Expert Opin Biol Ther. 2005 January; 5(1):111-24 and Bird et
al., Science 242, 423-426 (1988). Such single chain antibodies are
encompassed within the term antibody unless otherwise noted or
clearly indicated by context. Although such fragments are generally
included within the meaning of antibody, they collectively and each
independently are unique features of the present invention,
exhibiting different biological properties and utility. These and
other useful antibody fragments in the context of the present
invention are discussed further herein.
[0053] An antibody can be produced in and collected from different
in vitro or ex vivo expression or production systems, for example
from recombinantly modified host cells, from hybridomas or systems
that use cellular extracts supporting in vitro transcription and/or
translation of nucleic acid sequences encoding the antibody. It is
to be understood that a multitude of different antibodies, the
antibodies being as defined in the context of the present
invention, is one that can be provided by producing each antibody
separately in a production system as mentioned above and thereafter
mixing the antibodies, or by producing several antibodies in the
same production system.
[0054] The term "immunoglobulin heavy chain" or "heavy chain of an
immunoglobulin" as used herein is intended to refer to one of the
heavy chains of an immunoglobulin. A heavy chain is typically
comprised of a heavy chain variable region (abbreviated herein as
VH) and a heavy chain constant region (abbreviated herein as CH)
which defines the isotype of the immunoglobulin. The heavy chain
constant region typically is comprised of three domains, CH1, CH2,
and CH3. The term "immunoglobulin" as used herein is intended to
refer to a class of structurally related glycoproteins consisting
of two pairs of polypeptide chains, one pair of light (L) low
molecular weight chains and one pair of heavy (H) chains, all four
potentially inter-connected by disulfide bonds. The structure of
immunoglobulins has been well characterized (see for instance
Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press,
N.Y. (1989)). Within the structure of the immunoglobulin, the two
heavy chains are inter-connected via disulfide bonds in the
so-called "hinge region". Equally to the heavy chains, each light
chain is typically comprised of several regions; a light chain
variable region (abbreviated herein as VL) and a light chain
constant region. The light chain constant region typically is
comprised of one domain, CL. Furthermore, the VH and VL regions may
be further subdivided into regions of hypervariability (or
hypervariable regions which may be hypervariable in sequence and/or
form of structurally defined loops), also termed complementarity
determining regions (CDRs), interspersed with regions that are more
conserved, termed framework regions (FRs). Each VH and VL is
typically composed of three CDRs and four FRs, arranged from
amino-terminus to carboxy-terminus in the following order: FR1,
CDR1, FR2, CDR2, FR3, CDR3, FR4. CDR sequences are defined
according to IMGT (see Lefranc M P. et al., Nucleic Acids Research,
27, 209-212, 1999] and Brochet X. Nucl. Acids Res. 36, W503-508
(2008)).
[0055] When used herein, the terms "half molecule", "Fab-arm" and
"arm" refer to one heavy chain-light chain pair. When a bispecific
antibody is described to comprise a half-molecule antibody "derived
from" a first antibody, and a half-molecule antibody "derived from"
a second antibody, the term "derived from" indicates that the
bispecific antibody was generated by recombining, by any known
method, said half-molecules from each of said first and second
antibodies into the resulting bispecific antibody. In this context,
"recombining" is not intended to be limited by any particular
method of recombining and thus includes all of the methods for
producing bispecific antibodies described herein below, including
for example recombining by half-molecule exchange, as well as
recombining at nucleic acid level and/or through co-expression of
two half-molecules in the same cells.
[0056] The term "antigen-binding region" or "binding region" as
used herein, refers to a region of an antibody which is capable of
binding to the antigen. The antigen can be any molecule, such as a
polypeptide, e.g. present on a cell, bacterium, or virion. The
terms "antigen" and "target" may, unless contradicted by the
context, be used interchangeably in the context of the present
invention. The terms "antigen-binding region" and "antigen-binding
site" may, unless contradicted by the context, be used
interchangeably in the context of the present invention.
[0057] The term "blocks binding" or "blocking the binding of an
antibody" or "cross-blocking binding" or "cross-blocks binding"
refers to the situation where one antibody bound to a specific
antigen prevents binding of the second antibody to the same antigen
and vice versa. In the absence of the other antibody, each antibody
has the ability to bind to the antigen as determined by a
significant binding response, whereas one of the antibodies lacks a
binding response when the other antibody is present. The ability of
one antibody to block the binding of another antibody may be
determined by biolayer interferometry in a classical sandwich
epitope binning assay format, for instance as described in Example
3 in the present application and by Abdiche et al. (Abdiche Y N,
Malashock D S, Pinkerton A, Pons J. Exploring blocking assays using
Octet, ProteOn, and Biacore biosensors. Anal Biochem. 2009; 386(2):
172-180). Briefly, in a sandwich epitope binning assay, an antibody
in solution is tested for binding to its specific antigen that is
first captured via an immobilized antibody. In the context of the
present invention, one antibody does not block the binding of
another antibody if it is capable of "displacing" the other
antibody, according to the definition of "displacement" below. The
terms "blocks binding" and "blocking the binding of an antibody"
and "cross-blocking binding" and "cross-blocks binding" may, unless
contradicted by the context, be used interchangeably in the context
of the present invention. Preferably, the ability of one antibody
to block the binding of another antibody is determined using
full-length antibodies.
[0058] The term "displacement" or "ability to displace" or
"displacing" refers to the situation wherein two antibodies perturb
one another's binding to an antigen by kinetically altering one
another's binding to their specific antigen via the formation of a
transient trimolecular complex, which rapidly collapses by
retaining one antibody to the antigen and displacing the other.
Antibody displacement is defined in Abdiche et al., 2017 (Abdiche Y
N, Yeung A Y, Ni I, Stone D, Miles A, Morishige W, et al. (2017)
Antibodies Targeting Closely Adjacent or Minimally Overlapping
Epitopes Can Displace One Another. PLoS ONE 12(1): e0169535.
doi:10.1371/journal.pone.0169535). Antibody displacement may be
determined by biolayer interferometry using real-time label-free
biosensors in a classical sandwich assay format as described in
Abdiche et al. 2017 and Example 4 in the present application.
Preferably, antibody displacement is determined using antibodies
which are in the IgG format.
[0059] The term "binding" as used herein refers to the binding of
an antibody to a predetermined antigen or target, typically with a
binding affinity corresponding to a K.sub.D of 1E.sup.-6 M or less,
e.g. 5E.sup.-7 M or less, 1E.sup.-7 M or less, such as 5E.sup.-8 M
or less, such as 1E.sup.-8 M or less, such as 5E.sup.-9 M or less,
or such as 1E.sup.-9 M or less, when determined by biolayer
interferometry using the antibody as the ligand and the antigen as
the analyte and binds to the predetermined antigen with an affinity
corresponding to a K.sub.D that is at least ten-fold lower, such as
at least 100-fold lower, for instance at least 1,000-fold lower,
such as at least 10,000-fold lower, for instance at least
100,000-fold lower than its affinity for binding to a non-specific
antigen (e.g., BSA, casein) other than the predetermined antigen or
a closely-related antigen.
[0060] The term "K.sub.D" (M), as used herein, refers to the
dissociation equilibrium constant of a particular antibody-antigen
interaction, and is obtained by dividing k.sub.d by k.sub.a.
[0061] The term "k.sub.d" (sec.sup.-1), as used herein, refers to
the dissociation rate constant of a particular antibody-antigen
interaction. Said value is also referred to as the k.sub.off value
or off-rate.
[0062] The term "k.sub.a" (M.sup.-1.times.sec.sup.-1), as used
herein, refers to the association rate constant of a particular
antibody-antigen interaction. Said value is also referred to as the
k.sub.on value or on-rate.
[0063] The term "5T4" as used herein, refers to the protein
entitled 5T4, which is also referred to as trophoblast
glycoprotein, 5T4 oncofetal antigen, 5T4 oncofetal trophoblast
glycoprotein, TPBG, WAIF1 and M6P1. It is 72-80 kDa transmembrane
protein with an extensively N-linked glycosylated core. In humans
(Homo sapiens), the 5T4 protein has the amino acid sequence shown
in SEQ ID NO: 1 (Human Trophoblast glycoprotein: Uniprot accession
no. Q13641). In the amino acid sequence shown in SEQ ID NO: 1,
amino acid residues 1-31 are a signal peptide, and amino acid
residues 32-420 are the mature polypeptide. In cynomolgus monkey
(Macaca fascicularis), the 5T4 protein has the amino acid sequence
shown in SEQ ID NO: 2 (Uniprot accession no. Q4R8Y9). In the amino
acid sequence shown in SEQ ID NO: 2, amino acid residues 1-34 are a
signal peptide, and amino acid residues 35-420 are the mature
polypeptide. In chicken (Gallus gallus), the 5T4 protein has the
amino acid sequence shown in SEQ ID NO: 3 (Uniprot accession no.
R4GM46). In the sequence shown in SEQ ID NO: 3, amino acid residues
1-27 are a signal peptide, and amino acid residues 28-379 are the
mature polypeptide.
[0064] The term "CD3" as used herein, refers to the human Cluster
of Differentiation 3 protein which is part of the T-cell
co-receptor protein complex and is composed of four distinct
chains. CD3 is also found in other species, and thus, the term
"CD3" is not limited to human CD3 unless contradicted by context.
In mammals, the complex contains a CD3.gamma. (gamma) chain (human
CD3.gamma. chain UniProtKB/Swiss-Prot No P09693, or cynomolgus
monkey CD3.gamma. UniProtKB/Swiss-Prot No Q95LI7), a CD3.delta.
(delta) chain (human CD3.delta. UniProtKB/Swiss-Prot No P04234, or
cynomolgus monkey CD3.delta. UniProtKB/Swiss-Prot No Q95LI8), two
CD3.epsilon. (epsilon) chains (human CD3.epsilon.
UniProtKB/Swiss-Prot No P07766; amino acid residues 1-22 is a
signal peptide and amino acid residues 23-207 is the mature CD3E
polypeptide, which is identified herein as SEQ ID NO: 4; cynomolgus
monkey CD3.epsilon. UniProtKB/Swiss-Prot No Q95LI5; or rhesus
monkey CD3.epsilon. UniProtKB/Swiss-Prot No G7NCB9), and a
CD3-chain (zeta) chain (human CD3.zeta. UniProtKB/Swiss-Prot No
P20963, cynomolgus monkey CD3.zeta. UniProtKB/Swiss-Prot No
Q09TK0). These chains associate with a molecule known as the T-cell
receptor (TCR) and generate an activation signal in T lymphocytes.
The TCR and CD3 molecules together comprise the TCR complex.
[0065] The term "antibody binding region" refers to a region of the
antigen, which comprises the epitope to which the antibody binds.
An antibody binding region may be determined by epitope binning
using biolayer interferometry, by alanine scan, or by shuffle
assays (using antigen constructs in which regions of the antigen
are exchanged with that of another species and determining whether
the antibody still binds to the antigen or not). The amino acids
within the antibody binding region that are involved in the
interaction with the antibody may be determined by
hydrogen/deuterium exchange mass spectrometry and by
crystallography of the antibody bound to its antigen.
[0066] The term "epitope" means an antigenic determinant which is
specifically bound by an antibody. Epitopes usually consist of
surface groupings of molecules such as amino acids, sugar side
chains or a combination thereof and usually have specific three
dimensional structural characteristics, as well as specific charge
characteristics. Conformational and non-conformational epitopes are
distinguished in that the binding to the former but not the latter
is lost in the presence of denaturing solvents. The epitope may
comprise amino acid residues which are directly involved in the
binding, and other amino acid residues, which are not directly
involved in the binding, such as amino acid residues which are
effectively blocked or covered by the antibody when it is bound to
the antigen (in other words, the amino acid residue is within or
closely adjacent to the footprint of the specific antibody).
[0067] The terms "monoclonal antibody", "monoclonal Ab",
"monoclonal antibody composition", "mAb", or the like, as used
herein refer to a preparation of antibody molecules of single
molecular composition. A monoclonal antibody composition displays a
single binding specificity and affinity for a particular epitope.
Accordingly, the term "human monoclonal antibody" refers to
antibodies displaying a single binding specificity which have
variable and constant regions derived from human germline
immunoglobulin sequences. The human monoclonal antibodies may be
produced by a hybridoma which includes a B cell obtained from a
transgenic or transchromosomal non-human animal, such as a
transgenic mouse, having a genome comprising a human heavy chain
transgene and a light chain transgene, fused to an immortalized
cell. Monoclonal antibodies may also be produced from recombinantly
modified host cells, or systems that use cellular extracts
supporting in vitro transcription and/or translation of nucleic
acid sequences encoding the antibody.
[0068] The term "isotype" as used herein refers to the
immunoglobulin class (for instance IgG1, IgG2, IgG3, IgG4, IgD,
IgA, IgE, or IgM) or any allotypes thereof, such as IgG1m(za) and
IgG1m(f)) that is encoded by heavy chain constant region genes.
Further, each heavy chain isotype can be combined with either a
kappa (.kappa.) or lambda (.lamda.) light chain.
[0069] The term "full-length antibody" when used herein, refers to
an antibody (e.g., a parent or variant antibody) comprising one or
two pairs of heavy and light chains, each containing all heavy and
light chain constant and variable domains that are normally found
in a heavy chain-light chain pair of a wild-type antibody of that
isotype. In a full length variant antibody, the heavy and light
chain constant and variable domains may in particular contain amino
acid substitutions that improve the functional properties of the
antibody when compared to the full length parent or wild type
antibody. A full-length antibody according to the present invention
may be produced by a method comprising the steps of (i) cloning the
CDR sequences into a suitable vector comprising complete heavy
chain sequences and complete light chain sequence, and (ii)
expressing the complete heavy and light chain sequences in suitable
expression systems. It is within the knowledge of the skilled
person to produce a full-length antibody when starting out from
either CDR sequences or full variable region sequences. Thus, the
skilled person would know how to generate a full-length antibody
according to the present invention.
[0070] The term "human antibody", as used herein, is intended to
include antibodies having variable and framework regions derived
from human germline immunoglobulin sequences and a human
immunoglobulin constant domain. The human antibodies of the
invention may include amino acid residues not encoded by human
germline immunoglobulin sequences (e.g., mutations, insertions or
deletions introduced by random or site-specific mutagenesis in
vitro or by somatic mutation in vivo). However, the term "human
antibody", as used herein, is not intended to include antibodies in
which CDR sequences derived from the germline of another non-human
species, such as a mouse, have been grafted onto human framework
sequences.
[0071] The term "humanized antibody" as used herein, refers to a
genetically engineered non-human antibody, which contains human
antibody constant domains and non-human variable domains modified
to contain a high level of sequence homology to human variable
domains. This can be achieved by grafting of the six non-human
antibody complementarity-determining regions (CDRs), which together
form the antigen binding site, onto a homologous human acceptor
framework region (FR) (see WO92/22653 and EP0629240). In order to
fully reconstitute the binding affinity and specificity of the
parental antibody, the substitution of framework residues from the
parental antibody (i.e. the non-human antibody) into the human
framework regions (back-mutations) may be required. Structural
homology modeling may help to identify the amino acid residues in
the framework regions that are important for the binding properties
of the antibody. Thus, a humanized antibody may comprise non-human
CDR sequences, primarily human framework regions optionally
comprising one or more amino acid back-mutations to the non-human
amino acid sequence, and fully human constant regions. Optionally,
additional amino acid modifications, which are not necessarily
back-mutations, may be applied to obtain a humanized antibody with
preferred characteristics, such as affinity and biochemical
properties.
[0072] The term "Fc region" as used herein, refers to a region
comprising, in the direction from the N- to C-terminal end of the
antibody, at least a hinge region, a CH2 region and a CH3 region.
An Fc region of the antibody may mediate the binding of the
immunoglobulin to host tissues or factors, including various cells
of the immune system (such as effector cells) and components of the
complement system.
[0073] The term "hinge region" as used herein refers to the hinge
region of an immunoglobulin heavy chain. Thus, for example the
hinge region of a human IgG1 antibody corresponds to amino acids
216-230 according to the Eu numbering as set forth in Kabat Kabat,
E. A. et al., Sequences of proteins of immunological interest. 5th
Edition--US Department of Health and Human Services, NIH
publication No. 91-3242, pp 662,680,689 (1991). However, the hinge
region may also be any of the other subtypes as described
herein.
[0074] The term "CH1 region" or "CH1 domain" as used herein refers
to the CH1 region of an immunoglobulin heavy chain. Thus, for
example the CH1 region of a human IgG1 antibody corresponds to
amino acids 118-215 according to the Eu numbering as set forth in
Kabat (ibid). However, the CH1 region may also be any of the other
subtypes as described herein.
[0075] The term "CH2 region" or "CH2 domain" as used herein refers
to the CH2 region of an immunoglobulin heavy chain. Thus, for
example the CH2 region of a human IgG1 antibody corresponds to
amino acids 231-340 according to the Eu numbering as set forth in
Kabat (ibid). However, the CH2 region may also be any of the other
subtypes as described herein.
[0076] The term "CH3 region" or "CH3 domain" as used herein refers
to the CH3 region of an immunoglobulin heavy chain. Thus for
example the CH3 region of a human IgG1 antibody corresponds to
amino acids 341-447 according to the Eu numbering as set forth in
Kabat (ibid). However, the CH3 region may also be any of the other
subtypes as described herein.
[0077] The term "Fc-mediated effector functions," as used herein,
is intended to refer to functions that are a consequence of binding
a polypeptide or antibody to its target or antigen on a cell
membrane wherein the Fc-mediated effector function is attributable
to the Fc region of the polypeptide or antibody. Examples of
Fc-mediated effector functions include (i) C1q binding, (ii)
complement activation, (iii) complement-dependent cytotoxicity
(CDC), (iv) antibody-dependent cell-mediated cytotoxity (ADCC), (v)
Fc-gamma receptor (FcgR)-binding, (vi) antibody-dependent,
Fc.gamma.R-mediated antigen crosslinking, (vii) antibody-dependent
cellular phagocytosis (ADCP), (viii) complement-dependent cellular
cytotoxicity (CDCC), (ix) complement-enhanced cytotoxicity, (x)
binding to complement receptor of an opsonized antibody mediated by
the antibody, (xi) opsonisation, and (xii) a combination of any of
(i) to (xi).
[0078] The term "inertness", "inert" or "non-activating" as used
herein, refers to an Fc region which is at least not able to bind
any Fc.gamma.R, induce Fc-mediated cross-linking of Fc.gamma.Rs, or
induce Fc.gamma.R-mediated cross-linking of target antigens via two
Fc regions of individual antibodies, or is not able to bind C1q.
The inertness of an Fc region of an antibody, may be tested using
the antibody in a monospecific or bispecific format.
[0079] The term "full-length" when used in the context of an
antibody indicates that the antibody is not a fragment, but
contains all of the domains of the particular isotype normally
found for that isotype in nature, e.g. the VH, CH1, CH2, CH3,
hinge, VL and CL domains for an IgG1 antibody.
[0080] The term "monovalent antibody", in the context of the
present invention, refers to an antibody molecule that can interact
with a specific epitope on an antigen, with only one antigen
binding domain (e.g. one Fab arm). In the context of a bispecific
antibody, "monovalent antibody binding" refers to the binding of
the bispecific antibody to one specific epitope on an antigen with
only one antigen binding domain (e.g. one Fab arm).
[0081] The term "monospecific antibody" in the context of the
present invention, refers to an antibody that has binding
specificity to one epitope only. The antibody may be a
monospecific, monovalent antibody (i.e. carrying only one antigen
binding region) or a monospecific, bivalent antibody (i.e. an
antibody with two identical antigen binding regions).
[0082] The term "bispecific antibody" refers to an antibody having
two non-identical antigen binding domains, e.g. two non-identical
Fab-arms or two Fab-arms with non-identical CDR regions. In the
context of this invention, bispecific antibodies have specificity
for at least two different epitopes. Such epitopes may be on the
same or different antigens or targets. If the epitopes are on
different antigens, such antigens may be on the same cell or
different cells, cell types or structures, such as extracellular
matrix or vesicles and soluble protein. A bispecific antibody may
thus be capable of crosslinking multiple antigens, e.g. two
different cells.
[0083] The term "bivalent antibody" refers to an antibody that has
two antigen binding regions, which bind to epitopes on one or two
targets or antigens or binds to one or two epitopes on the same
antigen. Hence, a bivalent antibody may be a monospecific, bivalent
antibody or a bispecific, bivalent antibody.
[0084] The term "amino acid" and "amino acid residue" may herein be
used interchangeably, and are not to be understood limiting. Amino
acids are organic compounds containing amine (--NH.sub.2) and
carboxyl (--COOH) functional groups, along with a side chain (R
group) specific to each amino acid. In the context of the present
invention, amino acids may be classified based on structure and
chemical characteristics. Thus, classes of amino acids may be
reflected in one or both of the following tables:
[0085] Main classification based on structure and general chemical
characterization of R group
TABLE-US-00001 Class Amino acid Acidic Residues D and E Basic
Residues K, R, and H Hydrophilic Uncharged Residues S, T, N, and Q
Aliphatic Uncharged Residues G, A, V, L, and I Non-polar Uncharged
Residues C, M, and P Aromatic Residues F, Y, and W
[0086] Alternative Physical and Functional Classifications of Amino
Acid Residues
TABLE-US-00002 Class Amino acid Hydroxyl group containing residues
S and T Aliphatic residues I, L, V, and M Cycloalkenyl-associated
residues F, H, W, and Y Hydrophobic residues A, C, F, G, H, I, L,
M, R, T, V, W, and Y Negatively charged residues D and E Polar
residues C, D, E, H, K, N, Q, R, S, and T Positively charged
residues H, K, and R Small residues A, C, D, G, N, P, S, T, and V
Very small residues A, G, and S Residues involved in turn formation
A, C, D, E, G, H, K, N, Q, R, S, P, and T Flexible residues Q, T,
K, S, G, P, D, E, and R
[0087] Substitution of one amino acid for another may be classified
as a conservative or non-conservative substitution. In the context
of the invention, a "conservative substitution" is a substitution
of one amino acid with another amino acid having similar structural
and/or chemical characteristics, such substitution of one amino
acid residue for another amino acid residue of the same class as
defined in any of the two tables above: for example, leucine may be
substituted with isoleucine as they are both aliphatic, branched
hydrophobes. Similarly, aspartic acid may be substituted with
glutamic acid since they are both small, negatively charged
residues.
[0088] In the context of the present invention, a substitution in
an antibody is indicated as: [0089] Original amino
acid-position-substituted amino acid;
[0090] Referring to the well-recognized nomenclature for amino
acids, the three letter code, or one letter code, is used,
including the codes "Xaa" or "X" to indicate any amino acid
residue. Thus, Xaa or X may typically represent any of the 20
naturally occurring amino acids. The term "naturally occurring" as
used herein refers to any one of the following amino acid residues;
glycine, alanine, valine, leucine, isoleucine, serine, threonine,
lysine, arginine, histidine, aspartic acid, asparagine, glutamic
acid, glutamine, proline, tryptophan, phenylalanine, tyrosine,
methionine, and cysteine. Accordingly, the notation "K409R" or
"Lys409Arg" means, that the antibody comprises a substitution of
Lysine with Arginine in amino acid position 409.
[0091] Substitution of an amino acid at a given position to any
other amino acid is referred to as:
[0092] Original amino acid-position; or e.g. "K409"
[0093] For a modification where the original amino acid(s) and/or
substituted amino acid(s) may comprise more than one, but not all
amino acid(s), the more than one amino acid may be separated by ","
or "/". E.g. the substitution of Lysine with Arginine, Alanine, or
Phenylalanine in position 409 is:
[0094] "Lys409Arg,Ala,Phe" or "Lys409Arg/Ala/Phe" or "K409R,A,F" or
"K409R/A/F" or "K409 to R, A, or F".
[0095] Such designation may be used interchangeably in the context
of the invention but have the same meaning and purpose.
[0096] Furthermore, the term "a substitution" embraces a
substitution into any one or the other nineteen natural amino
acids, or into other amino acids, such as non-natural amino acids.
For example, a substitution of amino acid K in position 409
includes each of the following substitutions: 409A, 409C, 409D,
409E, 409F, 409G, 409H, 409I, 409L, 409M, 409N, 409Q, 409R, 409S,
409T, 409V, 409W, 409P, and 409Y. This is, by the way, equivalent
to the designation 409X, wherein the X designates any amino acid
other than the original amino acid. These substitutions may also be
designated K409A, K409C, etc. or K409A,C, etc. or K409A/C/etc. The
same applies by analogy to each and every position mentioned
herein, to specifically include herein any one of such
substitutions.
[0097] The antibody according to the invention may also comprise a
deletion of an amino acid residue. Such deletion may be denoted
"del", and includes, e.g., writing as K409del. Thus, in such
embodiments, the Lysine in position 409 has been deleted from the
amino acid sequence.
[0098] The term "host cell", as used herein, is intended to refer
to a cell into which an expression vector has been introduced. It
should be understood that such terms are intended to refer not only
to the particular subject cell, but also to the progeny of such a
cell. Because certain modifications may occur in succeeding
generations due to either mutation or environmental influences,
such progeny may not, in fact, be identical to the parent cell, but
are still included within the scope of the term "host cell" as used
herein. Recombinant host cells include, for example, transfectomas,
such as CHO cells, HEK-293 cells, Expi293F cells, PER.C6 cells, NS0
cells, and lymphocytic cells, and prokaryotic cells such as E. coli
and other eukaryotic hosts such as plant cells and fungi.
[0099] The term "transfectoma", as used herein, includes
recombinant eukaryotic host cells expressing the antibody or a
target antigen, such as CHO cells, PER.C6 cells, NS0 cells, HEK-293
cells, Expi293F cells, plant cells, or fungi, including yeast
cells.
[0100] For purposes of the present invention, the sequence identity
between two amino acid sequences is determined using the
Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol.
Biol. 48: 443-453) as implemented in the Needle program of the
EMBOSS package (EMBOSS: The European Molecular Biology Open
Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277),
preferably version 5.0.0 or later. The parameters used are gap open
penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62
(EMBOSS version of BLOSUM62) substitution matrix. The output of
Needle labeled "longest identity" (obtained using the -nobrief
option) is used as the percent identity and is calculated as
follows:
(Identical Residues.times.100)/(Length of Alignment-Total Number of
Gaps in Alignment).
[0101] The retention of similar residues may also or alternatively
be measured by a similarity score, as determined by use of a BLAST
program (e.g., BLAST 2.2.8 available through the NCBI using
standard settings BLOSUM62, Open Gap=11 and Extended Gap=1).
Suitable variants typically exhibit at least about 45%, such as at
least about 55%, at least about 65%, at least about 75%, at least
about 85%, at least about 90%, at least about 95%, or more (e.g.,
about 99%) similarity to the parent sequence.
[0102] The term "internalized" or "internalization" as used herein,
refers to a biological process in which molecules such as the
antibody according to the present invention, are engulfed by the
cell membrane and drawn into the interior of the cell.
Internalization may also be referred to as "endocytosis".
Antibodies
[0103] In a first aspect, the present invention provides an
antibody comprising at least one antigen-binding region capable of
binding to 5T4 (Trophoblast glycoprotein), wherein the antibody is
able to block binding to 5T4 of an antibody selected from the group
consisting of: [0104] a) an antibody comprising a VH region
comprising the sequence set forth in SEQ ID NO: 5 and a VL region
comprising the sequence set forth in SEQ ID NO: 9 [059], [0105] b)
an antibody comprising a VH region comprising the sequence set
forth in SEQ ID NO: 12 and a VL region comprising the sequence set
forth in SEQ ID NO: 16 [076], [0106] c) an antibody comprising a VH
region comprising the sequence set forth in SEQ ID NO: 19 and a VL
region comprising the sequence set forth in SEQ ID NO: 23 [085],
[0107] d) an antibody comprising a VH region comprising the
sequence set forth in SEQ ID NO: 26 and a VL region comprising the
sequence set forth in SEQ ID NO: 30 [106], [0108] e) an antibody
comprising a VH region comprising the sequence set forth in SEQ ID
NO: 33 and a VL region comprising the sequence set forth in SEQ ID
NO: 37 [127], [0109] f) an antibody comprising a VH region
comprising the sequence set forth in SEQ ID NO: 40 and a VL region
comprising the sequence set forth in SEQ ID NO: 44 [207]; and
[0110] g) an antibody comprising a VH region comprising the
sequence set forth in SEQ ID NO: 47 and a VL region comprising the
sequence set forth in SEQ ID NO: 51 [226].
[0111] In particular, the invention provides an antibody comprising
at least one antigen-binding region capable of binding to 5T4
(Trophoblast glycoprotein), wherein the antibody is able to block
binding to 5T4 of an antibody comprising a variable heavy chain
(VH) region comprising the sequence set forth in SEQ ID NO: 5, and
a variable light chain (VL) region comprising the sequence set
forth in SEQ ID NO: 9 [059].
[0112] The antibody may in particular be able to block binding to
5T4 of an antibody selected from the group consisting of: [0113] a)
an antibody comprising a variable heavy chain (VH) region
comprising the sequence set forth in SEQ ID NO: 40 and a variable
light chain (VL) region comprising the sequence set forth in SEQ ID
NO: 44 [207], [0114] b) an antibody comprising a variable heavy
chain (VH) region comprising the sequence set forth in SEQ ID NO:
47 and a variable light chain (VL) region comprising the sequence
set forth in SEQ ID NO: 51 [226]; and an antibody comprising a
variable heavy chain (VH) region comprising the sequence set forth
in SEQ ID NO: 5 and a variable light chain (VL) region comprising
the sequence set forth in SEQ ID NO: 9 [059].
[0115] In particular embodiments of the invention, the antibody is
able to block binding to 5T4 of an antibody selected from the group
consisting of: [0116] a) an antibody comprising a variable heavy
chain (VH) region comprising the sequence set forth in SEQ ID NO:
40 and a variable light chain (VL) region comprising the sequence
set forth in SEQ ID NO: 44 [207]; and [0117] b) an antibody
comprising a variable heavy chain (VH) region comprising the
sequence set forth in SEQ ID NO: 47 and a variable light chain (VL)
region comprising the sequence set forth in SEQ ID NO: 51 [226]
[0118] The antibodies according to the invention are characterized
by having specificity for or having the ability to bind human (Homo
sapiens) 5T4. Hence, 5T4 as referred to herein may in particular be
human 5T4, such as the mature polypeptide of SEQ ID NO: 1.
[0119] In further embodiments, the antibodies of the invention are
characterized by having specificity for or having the ability to
bind to cynomolgus monkey (Macaca fascicularis) 5T4, such as
specificity for or the ability to bind to both human and cynomolgus
monkey 5T4. Cynomolgus monkey 5T4 may in particular be the mature
polypeptide of SEQ ID NO: 2.
[0120] In still further embodiments, the antibodies according to
the invention have specificity for or have the ability to bind to
chicken (Gallus gallus) 5T4, such as specificity for or the ability
to bind to human 5T4 and chicken 5T4 or such as specificity for or
the ability to bind to human, cynomolgus monkey and chicken 5T4,
wherein chicken 5T4 in particular may have the amino acid sequence
of the mature polypeptide of SEQ ID NO: 3.
[0121] Accordingly, the antibodies of the invention may have
specificity for or be able to bind to human 5T4 such as the mature
polypeptide of SEQ ID NO: 1 and cynomologus monkey 5T4, such as the
mature polypeptide of SEQ ID NO: 2.
[0122] Further, the antibodies according to the invention may have
specificity for or be able to bind to human 5T4, such as the mature
polypeptide of SEQ ID NO: 1, cynomologus monkey 5T4 such as the
mature polypeptide of SEQ ID NO: 2 and chicken 5T4, such as the
mature polypeptide of SEQ ID NO: 3.
[0123] The antibodies according to the invention may be able to
bind human 5T4, cynomolgus monkey and/or chicken 5T4, with a
binding affinity that corresponds to a K.sub.D value of 1E-7 M or
less, such as a K.sub.D value of about 1E-7 M or less, 5E-8 M or
less, about 5E-8 M or less, 1E-8 M or less, about 1E-8 M or less,
5E-9 M or less, about 5E-9 M or less, such as 1E-9 M or less or
such as about 1E-9 M or less, such as with a binding affinity
corresponding to a K.sub.D value which is within the range of 1E-7
to 5E-10 M, such as within the range of about 1E-7 to about 5E-10
M, such as 1E-7 to 1E-9 M, such as about 1E-7 to about 1E-9 M, such
as 5E-8 to 5E-10 M, such as about 5E-8 to about 5E-10 M, such as
5E-8 to 1E-9 M, such as about 5E-8 to about 1E-9 M, such as 1E-8 to
5E-10 M, such as about 1E-8 to about 5E-10 M, such as 1E-8 to 1E-9
M, such as about 1E-8 to about 1E-9 M, such as 1E-8 to 5E-9 M or
such as about 1E-8 to about 5E-9 M.
[0124] While it is within the capacity of the skilled person to
determine the affinity of an antibody for binding to its target,
the binding affinity of the antibodies according to the invention
for 5T4 may in particular be determined by biolayer interferometry,
optionally as set forth in Example 2 herein.
[0125] More specifically, the binding affinity of an antibody
according to the invention may determined using a procedure, such
as a biolayer interferometry procedure, comprising the steps of:
[0126] I) Immobilizing the antibody at an amount of 1 .mu.g/mL for
600 seconds on an anti-human IgG Fc Capture biosensor; [0127] II)
Determining association over a time period of 200 seconds and
dissociation over a time period of 1000 seconds of 5T4ECDHis
(mature protein of SEQ ID NO: 99) or cynomolgus monkey 5T4 (mature
protein of SEQ ID NO: 2, or recombinant cynomolgus monkey 5T4
protein (Cusabio; cat. no. CSB-MP024093MOV), using 2-fold dilution
series ranging from 100 nM to 1.56 nM. [0128] III) Referencing the
data to a buffer control (0 nM).
[0129] The binding affinity of an antibody according to the
invention may in particular be determined using an antibody as
defined in any one of the preceding claims, which is a
monospecific, bivalent antibody, such as an antibody which is a
full length IgG1.
[0130] In further embodiments of the invention, the antibody
recognizes or binds to an epitope or antibody binding region or
binding site on 5T4, said binding site or epitope or antibody
binding region being recognized by any one of the antibodies
selected from the group consisting of: [0131] a) an antibody
comprising a VH region comprising the sequence set forth in SEQ ID
NO: 5 and a VL region comprising the sequence set forth in SEQ ID
NO: 9 [059], [0132] b) an antibody comprising a VH region
comprising the sequence set forth in SEQ ID NO: 12 and a VL region
comprising the sequence set forth in SEQ ID NO: 16 [076], [0133] c)
an antibody comprising a VH region comprising the sequence set
forth in SEQ ID NO: 19 and a VL region comprising the sequence set
forth in SEQ ID NO: 23 [085], [0134] d) an antibody comprising a VH
region comprising the sequence set forth in SEQ ID NO: 26 and a VL
region comprising the sequence set forth in SEQ ID NO: 30 [106],
[0135] e) an antibody comprising a VH region comprising the
sequence set forth in SEQ ID NO: 33 and a VL region comprising the
sequence set forth in SEQ ID NO: 37 [127], [0136] f) an antibody
comprising a VH region comprising the sequence set forth in SEQ ID
NO: 40 and a VL region comprising the sequence set forth in SEQ ID
NO: 44 [207]; and [0137] g) an antibody comprising a VH region
comprising the sequence set forth in SEQ ID NO: 47 and a VL region
comprising the sequence set forth in SEQ ID NO: 51 [226].
[0138] In still further embodiments, the antibody according to the
invention recognizes or binds to an antibody binding region, a
binding site or epitope on 5T4, which is not an antibody binding
region, a binding site or epitope bound by, or is different from an
antibody binding region, a binding site or epitope bound by, an
antibody selected from the group consisting of: [0139] a) an
antibody comprising a VH region comprising the sequence set forth
in SEQ ID NO: 87 and a VL region comprising the sequence set forth
in SEQ ID NO: 88 [H8], [0140] b) an antibody comprising a VH region
comprising the sequence set forth in SEQ ID NO: 83 and a VL region
comprising the sequence set forth in SEQ ID NO: 84 [A1]; and [0141]
c) An antibody comprising a VH region comprising the sequence set
forth in SEQ ID NO: 85 and a VL region comprising the sequence set
forth in SEQ ID NO: 86 [A3].
[0142] In other embodiments, the binding of the antibody according
to the invention to 5T4 is blocked by binding to 5T4 of an antibody
comprising a variable heavy chain (VH) region comprising the
sequence set forth in SEQ ID NO: 85 and a variable light chain (VL)
region comprising the sequence set forth in SEQ ID NO: 86 [A3]. An
antibody comprising the VH and VL sequences set forth in SEQ ID NOs
85 and 86 respectively, is antibody A3, one of three murine 5T4
antibodies disclosed in WO2007106744. Rephrase: antibody A3 with a
single aa substitution. In the CDR sequences?
[0143] In still other embodiments, the antibody according to the
invention shows displacement of an antibody bound to 5T4 or to
His-tagged extracellular domain of 5T4 (e.g. 5T4ECDHis/mature
protein of SEQ ID NO: 99), said antibody bound to 5T4 comprising a
variable heavy chain (VH) region comprising the sequence set forth
in SEQ ID NO: 85 and a variable light chain (VL) region comprising
the sequence set forth in SEQ ID NO: 86 [A3]. This displacement
behavior indicates that the antibody of the invention binds to an
epitope, which is different from the epitope bound by antibody A3,
but may be adjacent to or even overlapping with the epitope bound
by A3.
[0144] "Displacement" or the ability to displace a bound antibody
may be determined in a biolayer interferometry assay, such as in an
assay performed as described in Example 4 of the present
application.
[0145] "Cross-blocking", or the ability of an antibody as defined
according to the invention to block binding of another antibody to
5T4 may be determined by the use of a fluorescence-activated cell
sorting (FACS) assay, such as in an assay performed as described in
Example 5.
[0146] In particular, "cross-blocking", or the ability of an
antibody according to the invention to block binding of another
antibody to 5T4, is determined as the ability of an unconjugated
antibody to block binding of a conjugated antibody, and is
optionally determined in a procedure comprising the steps of:
[0147] i) Providing a set of samples, each sample comprising a
mixture of human ovary adenocarcinoma SK-OV-3 cells, an antibody
which binds to 5T4 and is conjugated to fluorescein isothiocyanate
(FITC), and an excess of unconjugated antibody targeting 5T4,
[0148] ii) Incubating the samples for 30 minutes at 4.degree. C.,
and thereafter subjecting the samples to centrifugation, [0149]
iii) Removing the supernatant from each sample and resuspending the
cells in buffer and determining mean fluorescence intensity (MFI)
of FITC using a flow cytometer; and [0150] iv) Calculating the
percentage of binding as following: [0151] The difference in MFI
between cells incubated with a mixture of FITC-conjugated
antibodies and unconjugated antibodies and cells incubated without
FITC-conjugated or unconjugated antibodies, multiplied by 100, and
subsequently divided by the difference in MFI between cells
incubated with a mixture of FITC-conjugated antibodies and IgG-b12
antibodies and cells incubated without FITC-conjugated or
unconjugated antibodies.
[0152] While the skilled person will be familiar with suitable
technologies for determining the ability of an antibody to block
the binding of another antibody to its target, or to displace
another antibody bound to its target, the present application
discloses procedures suitable for determining blocking of binding
and displacement. Hence, in some embodiments, the ability of an
antibody according to the invention to block binding of another
antibody to 5T4 or to displace another antibody bound to 5T4, may
be determined using biolayer interferometry, such as in biolayer
interferometry performed as described in Example 3.
[0153] In particular, the ability of an antibody according to the
invention to block binding of another antibody to 5T4, or to
displace another antibody bound to 5T4 is determined using biolayer
interferometry may be determined in a procedure comprising the
steps of: [0154] i) Immobilizing the antibody according to the
invention, at an amount of 20 .mu.g/mL in 10 mM sodium acetate
buffer to an activated Amine-Reactive 2.sup.nd Generation
biosensor, [0155] ii) Quenching the biosensor with the immobilized
antibody in ethanolamine pH 8.5, [0156] iii) Immersing the
biosensor with the immobilized antibody in a composition comprising
3.6 .mu.g/mL (100 nM) of human 5T4ECDHis (mature protein of SEQ ID
NO: 99) for a time period of 500 seconds, and then [0157] iv)
Immersing the biosensor with the immobilized antibody and 5T4ECDHis
in a composition comprising 10 .mu.g/mL of the other antibody
targeting 5T4 and determining the association response over a time
period of 500 seconds; [0158] wherein steps i)-iv) are performed at
a temperature of 30.degree. C. and with shaking at 1000 rpm.
[0159] The antibodies provided herein may bind to an epitope or
antibody binding region on human 5T4 comprising the amino acid
residues R73, Y92 and R94; the numbering of each amino acid residue
referring to its position in SEQ ID NO: 1.
[0160] Also provided herein are antibodies, which bind to an
epitope or antibody binding region on human 5T4 comprising the
amino acid residues S69, R73, Y92 and R94; the numbering of each
amino acid residue referring to its position in SEQ ID NO: 1.
[0161] Further provided herein are antibodies, which bind to an
epitope or antibody binding region on human 5T4 comprising the
amino acid residues R73, T74, Y92, R94 and N95; the numbering of
each amino acid residue referring to its position in SEQ ID NO:
1.
[0162] Based on the results provided in Example 16 herein it is
hypothesized, without any wish to be bound by theory, that any one
or more of these amino acid residues (i.e. S69, R73, T74, Y92, R94
and N95) is/are directly involved in binding of the antibody, such
as by way of non-covalent interactions; e.g with amino acid
residues within the CDR sequences of the antibody. The hypothesis
is supported by the fact that these residues were identified as
being surface-exposed on the structure of 5T4 (4cnm; provided in
the RCSB PDB Protein Data Bank; DOI: 10.2210/pdb4CNM/pdb) as
published in Zhao, Y., Malinauskas, T., Harlos, K., & Jones, E.
Y. (2014). Structural insights into the inhibition of Wnt signaling
by cancer antigen 5T4/Wnt-activated inhibitory factor 1. Structure,
22(4), 612-620.
[0163] One or more of the following additional amino acid residues
may be involved binding of the antibody, such as indirectly
involved in binding, e.g. by impacting protein folding and/or
positioning of one or more amino acid residues directly involved in
binding of the antibody: L89, F111, L117, F138, L144, D148, N152;
the numbering of each amino acid residue referring to its position
in SEQ ID NO: 1. In particular, L89, F111, L117, F138, L144 have
been identified as part of a hydrophobic core within 5T4 as
described by Zhao et al., Structure, 22(4), 612-620.
[0164] Further, the antibody disclosed herein may to an epitope or
antibody binding region on human 5T4 within which amino acid
residues R73, Y92 and R94 are directly involved in binding the
antibody, and wherein one or more of amino acid residues F111,
F138, L144 and D148 are indirectly involved in said binding; the
numbering of each amino acid residue referring to its position in
SEQ ID NO: 1.
[0165] The antibody provided herein may bind to an epitope or
antibody binding region on human 5T4 within which amino acid
residues S69, R73, Y92 and R94 are directly involved in binding the
antibody, and wherein one or more of amino acid residues F111,
F138, and D148 are indirectly involved in said binding; the
numbering of each amino acid residue referring to its position in
SEQ ID NO: 1.
[0166] Also, the present disclosure provides antibodies which bind
to an epitope or antibody binding region on human 5T4 within which
amino acid residues R73, T74, Y92, R94 and N95 are directly
involved in binding the antibody, and wherein amino acid residue
F138 is indirectly involved in said binding; the numbering of each
amino acid residue referring to its position in SEQ ID NO: 1.
[0167] The amino acid residues comprised by said epitope or
antibody binding region and optionally the one or more additional
amino acid residues which are indirectly involved in binding may be
identified by alanine scanning of human 5T4 having the amino acid
sequence set forth in SEQ ID NO: 1 or the mature polypeptide
sequence of SEQ ID NO: 1, or by alanine scanning of a polypeptide
comprising amino acid residues 32-355 of SEQ ID NO: 1.
[0168] The alanine scanning may in particular be performed as set
forth or essentially as set forth in Example 16 herein.
[0169] Further, the alanine scanning may be performed by a
procedure comprising the steps of: [0170] i) Expressing mutant
human 5T4 polypeptides in which all amino acid residues in the
extracellular domain of human 5T4 (corresponding to amino acid
residues 32-355 of SEQ ID NO: 1), except cysteines and alanines,
are individually substituted with alanine, and wild type 5T4
polypeptides (amino acid residues 32-355 of SEQ ID NO: 1)
individually in human embryonic kidney cells, e.g. HEK 293 cells,
such that for each mutant or wild type 5T4 a sample comprising
70-90.000 cells, such as 80.000 cells is provided, [0171] ii)
Incubating the cells in each sample with 20 .mu.L of said antibody
conjugated to fluorescein isothiocyanate (FITC)-conjugated antibody
(3 .mu.g/mL; in FACS buffer) for 40 minutes at room temperature,
and subsequently washing each sample twice in 150-180 .mu.L FACS
buffer (phosphate-buffered saline [PBS; Lonza, cat. no.
BE17-517]+0.1% [w/v] BSA [Roche, cat. no. 10735086001]+0.02% [w/v]
sodium azide [NaN.sub.3; EMELCA Bioscience, cat. no. 41920044-3])
and resuspending the cells in each sample in 30 .mu.L FACS buffer,
[0172] iii) Determining, for each sample, the average amount of
antibody bound per cell as the geometric mean of the fluorescence
intensity (gMFI) for the viable, single cell population in said
sample and normalizing the data for each test antibody against the
binding intensity of a non-cross blocking 5T4-specific control
antibody using the equation:
[0172] Normalized gMFI aa position = Log 10 ( gMFI Test Ab gMFI
Control Ab ) ##EQU00001## [0173] wherein `aa position` refers to
the position that was mutated into an alanine, [0174] wherein the
Z-score is calculated to express loss or gain of binding of the
antibody, according to the calculation:
[0174] Z - score ( fold change ) = Normalized gMFI aa position -
.mu. .sigma. ##EQU00002## [0175] wherein .mu. and .sigma. are the
mean and standard deviation, respectively, of the Normalized gMFI
calculated from all mutants, [0176] wherein data is excluded from
the analysis if the gMFI of the control antibody for a particular
5T4 mutant is lower than the mean gMFI.sub.Control Ab-2.5.times.SD
of the mean gMFI.sub.Control Ab (from all mutants); and optionally
[0177] wherein data is excluded from the analysis if a residue
binds with a Z-score just below -1.5 (e.g. between -1.5 and -1.8,
such as between -1.5 and -1.7 or such as between -1.5 and -1.6) and
that residue is predicted to be buried and spatially separated from
the majority of residues, which are predicted to be surface-exposed
and for which loss of binding or reduced binding is determined.
[0178] A suitable non-cross blocking 5T4-specific control antibody
to be used in step iii) is a bispecific antibody comprising [0179]
an antigen-binding region, which comprises a VH sequence as set
forth in SEQ ID NO: 83 and a VL sequence as set forth in SEQ ID NO:
84 [A1]; and [0180] an antigen binding region, which comprises a VH
sequence as set forth in SEQ ID NO: 97 and a VL sequence as set
forth in SEQ ID NO: 98 [B12].
[0181] The present invention provides antibodies which bind to 5T4
such that there is loss of binding or binding is reduced if any one
or more of the amino acid residues R73, Y92 and R94 is/are
substituted with alanine; the numbering of each amino acid residue
referring to its position in SEQ ID NO: 1.
[0182] In particular, the antibodies may bind to 5T4 such that
there is loss of binding or binding is reduced if any one or more
of the amino acid residues S69, R73, Y92 and R94 is/are substituted
with alanine; the numbering of each amino acid residue referring to
its position in SEQ ID NO: 1.
[0183] Further, the antibodies may bind to 5T4 such that there is
loss of binding or binding is reduced if any one or more of the
amino acid residues R73, T74, Y92, R94 and N95 is/are substituted
with alanine; the numbering of each amino acid residue referring to
its position in SEQ ID NO: 1.
[0184] Also, the antibodies disclosed herein may bind to 5T4 such
that there is loss of binding or binding is reduced if any one or
more of the amino acid residues: L89, F111, L117, F138, L144, D148,
N152 is/are substituted with alanine; the numbering of each amino
acid residue referring to its position in SEQ ID NO: 1.
[0185] Further, the antibodies may bind to 5T4 such that there is
loss of binding or binding is reduced if any one or more of the
amino acid residues R73, Y92, R94, F111, F138, L144 and D148 is/are
substituted with alanine; the numbering of each amino acid residue
referring to its position in SEQ ID NO: 1.
[0186] The antibodies may bind to 5T4 such that there is loss of
binding or binding is reduced if any one or more of the amino acid
residues S69, R73, Y92, R94, F111, F138, and D148 is/are
substituted with alanine; the numbering of each amino acid residue
referring to its position in SEQ ID NO: 1.
[0187] In other embodiments, the antibodies of the invention may
bind to 5T4 such that there is loss of binding or binding is
reduced if any one or more of the amino acid residues R73, T74,
Y92, R94, N95 and F138 is/are substituted with alanine; the
numbering of each amino acid residue referring to its position in
SEQ ID NO: 1.
[0188] The effect of any of the alanine substitutions provided
above may be determined by alanine scanning of a polypeptide
comprising amino acid residues 32-355 of SEQ ID NO: 1.
[0189] In particular, the effect of the alanine substitutions may
be determined by a procedure as set forth or essentially as set
forth in Example 16 herein.
[0190] Loss of binding may be defined as a Z-score in binding being
lower than 1.5; the Z-score optionally being calculated as set
forth or essentially as set forth in Example 16 herein.
[0191] The effect of any of the alanine substitutions may be
determined by a procedure comprising the steps of: [0192] i)
Expressing mutant human 5T4 polypeptides in which all amino acid
residues in the extracellular domain of human 5T4 (corresponding to
amino acid residues 32-355 of SEQ ID NO: 1), except cysteines and
alanines, are individually substituted with alanine, and wild type
5T4 polypeptides individually in human embryonic kidney cells, e.g.
HEK 293 cells, such that for each mutant or wild type 5T4 a sample
comprising 70-90.000 cells, such as 80.000 cells is provided,
[0193] ii) Incubating the cells in each sample with 20 .mu.L of
said antibody conjugated to fluorescein isothiocyanate
(FITC)-conjugated antibody (3 .mu.g/mL; in FACS buffer) for 40
minutes at room temperature, and subsequently washing each sample
twice in 150-180 .mu.L FACS buffer (phosphate-buffered saline [PBS;
Lonza, cat. no. BE17-517]+0.1% [w/v] BSA [Roche, cat. no.
10735086001]+0.02% [w/v] sodium azide [NaN.sub.3; EMELCA
Bioscience, cat. no. 41920044-3]) and resuspending the cells in
each sample in 30 .mu.L FACS buffer, [0194] iii) Determining, for
each sample, the average amount of antibody bound per cell as the
geometric mean of the fluorescence intensity (gMFI) for the viable,
single cell population in said sample and normalizing the data for
each test antibody against the binding intensity of a non-cross
blocking 5T4-specific control antibody using the equation:
[0194] Normalized gMFI aa position = Log 10 ( gMFI Test Ab gMFI
Control Ab ) ##EQU00003## [0195] wherein `aa position` refers to
the position that was mutated into an alanine, [0196] wherein the
Z-score is calculated to express loss or gain of binding of the
antibody, according to the calculation:
[0196] Z - score ( fold change ) = Normalized gMFI aa position -
.mu. .sigma. ##EQU00004## [0197] wherein .mu. and .sigma. are the
mean and standard deviation, respectively, of the Normalized gMFI
calculated from all mutants, [0198] wherein data is excluded from
the analysis if the gMFI of the control antibody for a particular
5T4 mutant is lower than the mean gMFI.sub.Control Ab-2.5.times.SD
of the mean gMFI.sub.Control Ab (from all mutants); and optionally
[0199] wherein data is excluded from the analysis if a residue
binds with a Z-score just below -1.5 (e.g. between -1.5 and -1.8,
such as between -1.5 and -1.7 or such as between -1.5 and -1.6) and
that residue is predicted to be buried and spatially separated from
the majority of residues, which are predicted to be surface-exposed
and for which loss of binding or reduced binding is determined.
[0200] A suitable non-cross blocking 5T4-specific control antibody
in step iii) of the procedure above is a bispecific antibody
comprising [0201] an antigen-binding region, which comprises a VH
sequence as set forth in SEQ ID NO: 83 and a VL sequence as set
forth in SEQ ID NO: 84 [A1]; and [0202] an antigen binding region,
which comprising a VH sequence as set forth in SEQ ID NO: 97 and a
VL sequence as set forth in SEQ ID NO: 98 [B12].
[0203] The antibody according to the invention may be characterized
by having reduced internalization capacity as shown by reduced
cytotoxicity when conjugated to a cytotoxic moiety as compared to a
likewise conjugated antibody comprising a variable heavy chain (VH)
region comprising the sequence set forth in SEQ ID NO: 87 and a
variable light chain (VL) region comprising the sequence set forth
in SEQ ID NO: 88 [H8]. An antibody comprising the VH and VL
sequences set forth in SEQ ID Nos: 87 and 88 respectively, may be
murine 5T4 antibody mAb5T4, also called the H8 antibody, (Shaw et
al. (2002), Biochem. J. 363: 137-45, WO98/55607). Various chimeric
or humanized versions of antibody H8 are disclosed in
WO06/031653.
[0204] Cytotoxicity or internalization of 5T4 antibodies that
monovalently bind 5T4 may be determined using a procedure as set
forth in Example 7 in the present application. In particular,
cytotoxicity may be determined in an assay comprising the steps of:
[0205] i) Providing an toxin-conjugated bispecific antibody that
monovalently binds 5T4, comprising a first-Fab arm of an antibody
as defined in any one of the preceding claims and a second Fab arm
capable of binding to HIV viral protein gp120 (HIV-1 gp120),
wherein the HIV-1 gp120-specific Fab-arm is conjugated to
Duostatin-3, [0206] ii) Incubating 5T4-positive breast cancer cells
MDA-MB-468 (ATCC clone HTB-132) or HCC1954 (ATCC clone CRL-1338)
with said bispecific antibody that monovalently binds 5T4 for 5
days at 37.degree. C.; and [0207] iii) Determining the viability of
the cells.
[0208] IgG-b12 is a HIV-1 gp120 specific antibody (Barbas, C F. J
Mol Biol. 1993 Apr. 5; 230(3):812-23). Sequences of the heavy chain
(VH) and light chain variable regions (VL) are set forth in SEQ ID
NOs: 97 and 98, respectively.
[0209] In certain embodiments, the antibody of the invention is
one, wherein said antigen-binding region, which is capable of
binding to 5T4 comprises a heavy chain variable region (VH)
selected from the group consisting of: [0210] a) a heavy chain
variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences
of SEQ ID NOs.: 6, 7 and 8 [059], [0211] b) a heavy chain variable
region (VH) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID
NOs.: 13, 14 and 15 [076], [0212] c) a heavy chain variable region
(VH) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs.:
20, 21 and 22 [085], [0213] d) a heavy chain variable region (VH)
comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs.: 27,
28 and 29 [106], [0214] e) a heavy chain variable region (VH)
comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs.: 34,
35 and 36 [127], [0215] f) a heavy chain variable region (VH)
comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs.: 41,
42 and 43 [207], [0216] g) a heavy chain variable region (VH)
comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs.: 48,
49 and 50 [226]; and [0217] h) a heavy chain variable region (VH)
comprising CDR1, CDR2 and CDR3 sequences, said CDR1, CDR2 and CDR3
sequences comprising in total, at the most 1, 2, 3, 4, 5, 6, 7, 8,
9 or at the most 10 amino acid substitutions, when compared to the
CDR1, CDR2 and CDR3 sequences defined in any one of a) to g).
[0218] In other embodiments, the antibody according to the
invention is one, wherein said antigen-binding region capable of
binding to 5T4 comprises a heavy chain variable region (VH)
selected from the group consisting of: [0219] a) a heavy chain
variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences
of SEQ ID NOs.: 6, 7 and 8 [059], [0220] b) a heavy chain variable
region (VH) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID
NOs.: 41, 42 and 43 [207]; [0221] c) a heavy chain variable region
(VH) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs.:
48, 49 and 50 [226]; and [0222] d) a heavy chain variable region
(VH) comprising CDR1, CDR2 and CDR3 sequences, said CDR1, CDR2 and
CDR3 sequences comprising in total, at the most 1, 2, 3, 4, 5, 6,
7, 8, 9 or at the most 10 amino acid substitutions, when compared
to the CDR1, CDR2 and CDR3 sequences defined in any one of a) to
c).
[0223] In particular, the antibody according to the invention may
be one, wherein said antigen-binding region capable of binding to
5T4 comprises a heavy chain variable region (VH) comprising CDR1,
CDR2, and CDR3 sequences of SEQ ID NOs.: 6, 7 and 8 [059].
[0224] Alternatively, the antibody according to the invention may
be one, wherein said antigen-binding region capable of binding to
5T4 comprises a heavy chain variable region (VH) selected from the
group consisting of: a heavy chain variable region (VH) comprising
CDR1, CDR2, and CDR3 sequences of SEQ ID NOs.: 41, 42 and 43
[207].
[0225] Also, the antibody according to the invention may be one,
wherein said antigen-binding region capable of binding to 5T4
comprises a heavy chain variable region (VH) selected from the
group consisting of: a heavy chain variable region (VH) comprising
CDR1, CDR2, and CDR3 sequences of SEQ ID NOs.: 48, 49 and 50
[226].
[0226] In other embodiments, the antibody according to the
invention is one, wherein said antigen-binding region capable of
binding to 5T4 comprises a heavy chain variable region (VH) and a
light chain variable region (VL) selected from the group consisting
of: [0227] a) a heavy chain variable region (VH) comprising the
CDR1, CDR2, and CDR3 sequences of SEQ ID NOs.: 6, 7 and 8,
respectively, and a light chain variable region (VL) comprising the
CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 10, AAS and SEQ ID NO:
11, respectively [059], [0228] b) a heavy chain variable region
(VH) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs.:
13, 14 and 15, respectively; and a light chain variable region (VL)
comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 17, DAS
and SEQ ID NO:18, respectively [076], [0229] c) a heavy chain
variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences
of SEQ ID NOs.: 20, 21 and 22, respectively; and a light chain
variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences
of SEQ ID NO: 24, DAS and SEQ ID NO: 25, respectively [085], [0230]
d) a heavy chain variable region (VH) comprising the CDR1, CDR2,
and CDR3 sequences of SEQ ID NOs.: 27, 28 and 29, respectively; and
a light chain variable region (VL) comprising the CDR1, CDR2, and
CDR3 sequences of SEQ ID NO: 31, DVS and SEQ ID NO: 32,
respectively [106], [0231] e) a heavy chain variable region (VH)
comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs.: 34,
35 and 36, respectively; and a light chain variable region (VL)
comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 38, DAS
and SEQ ID NO: 39, respectively [127], [0232] f) a heavy chain
variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences
of SEQ ID NOs.: 41, 42 and 43, respectively, and a light chain
variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences
of SEQ ID NO: 45, DAS and SEQ ID NO: 46, respectively [207], [0233]
g) a heavy chain variable region (VH) comprising the CDR1, CDR2,
and CDR3 sequences of SEQ ID NOs.: 48, 49 and 50, respectively, and
a light chain variable region (VL) region comprising the CDR1,
CDR2, and CDR3 sequences of SEQ ID NO: 52, DAS and SEQ ID NO: 53,
respectively [226]; and [0234] h) a heavy chain variable region
(VH) comprising CDR1, CDR2 and CDR3 sequences, said CDR1, CDR2 and
CDR3 sequences comprising in total, at the most 1, 2, 3, 4, 5, 6,
7, 8, 9 or at the most 10 amino acid substitutions, when compared
to the CDR1, CDR2 and CDR3 sequences defined in any one of a) to
g).
[0235] The antibody according to the invention may be an antibody,
wherein the six complementarity-determining regions (CDRs) of the
antigen binding region(s) capable of binding to 5T4 comprise, in
total, at the most 1, 2, 3, 4, 5, 6, 7, 8, 9 or at the most 10
amino acid substitutions, when compared to [0236] i) the CDR
sequences of SEQ ID NOs: 6, 7, 8, 10, AAS and SEQ ID NO: 11 [059],
[0237] ii) the CDR sequences of SEQ ID NOs.: 41, 42, 43, 45, DAS
and SEQ ID NO: 46 [207]; or [0238] iii) the CDR sequences of SEQ ID
NOs.: 48, 49, 50, 52, DAS and SEQ ID NO: 53 [226].
[0239] Preferably 1, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the
said amino acid substitutions is/are conservative amino acid
substitution(s).
[0240] The antibody may in particular comprise one or two heavy
chain variable regions in which the complementarity-determining
region 3 (CDR3) comprises six consecutive amino acid residues of
the sequence set forth in SEQ ID NO: 102 (YYGMDV) [059, 207, 226].
These six consecutive amino acid residues may be the most
C-terminal amino acid residues within the CDR3.
[0241] The antibody according to the invention may be an antibody,
wherein said antigen-binding region capable of binding to 5T4
comprises one or two heavy chain variable region(s) (VH) comprising
the CDR1 sequence of SEQ ID NO: 41 (GGSFSGYY), the CDR2 sequence of
SEQ ID NO: 103 (IDHSX.sub.1ST), and the CDR3 sequence of SEQ ID NO:
104 (AX.sub.2WFGELX.sub.3X.sub.4YYYGMDV), and a light chain
variable region (VL) comprising the CDR1 sequence of SEQ ID NO: 105
(QSVSSX.sub.5), the CDR2 sequence DAS, and the CDR3 sequence of SEQ
ID NO: 46 (QQRSNWPLT), and wherein X.sub.1 is G or E, X.sub.2 is A
or G, X.sub.3 is W or Y, X.sub.4 is D or Hand X.sub.5 is Y or F
[207, 226].
[0242] The antibody according to the invention may be one, wherein
said antigen-binding region capable of binding to 5T4 comprises a
heavy chain variable region (VH) comprising the CDR1, CDR2, and
CDR3 sequences of SEQ ID NOs.: 6, 7, and 8, respectively, and a
light chain variable region (VL) comprising the CDR1, CDR2, and
CDR3 sequences of SEQ ID NO: 10, AAS and SEQ ID NO: 11,
respectively [059].
[0243] Alternatively, the antibody according to the invention may
be one, wherein said antigen-binding region capable of binding to
5T4 comprises a heavy chain variable region (VH) comprising the
CDR1, CDR2, and CDR3 sequences of SEQ ID NOs.: 41, 42 and 43,
respectively, and a light chain variable region (VL) comprising the
CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 45, DAS and SEQ ID NO:
46, respectively [207].
[0244] Additionally, the antibody according to the invention may be
one, wherein said antigen-binding region capable of binding to 5T4
comprises a heavy chain variable region (VH) comprising the CDR1,
CDR2, and CDR3 sequences of SEQ ID NOs.: 48, 49 and 50,
respectively, and a light chain variable region (VL) comprising the
CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 52, DAS and 53,
respectively [226].
[0245] In some embodiments, the antibody according to the invention
is an antibody, wherein said antigen-binding region capable of
binding to 5T4 comprises a heavy chain variable region (VH)
selected from the group consisting of: [0246] a) a heavy chain
variable region (VH) comprising the sequence of SEQ ID NO: 5 or a
sequence having at least 90%, at least 95%, at least 97%, or at
least 99% amino acid sequence identity to the sequence of SEQ ID
NO: 5 [059], [0247] b) a heavy chain variable region (VH)
comprising the sequence of SEQ ID NO: 12 or a sequence having at
least 90%, at least 95%, at least 97%, or at least 99% amino acid
sequence identity to the sequence of SEQ ID NO: 12 [076], [0248] c)
a heavy chain variable region (VH) comprising the sequence of SEQ
ID NO: 19 or a sequence having at least 90%, at least 95%, at least
97%, or at least 99% amino acid sequence identity to the sequence
of SEQ ID NO: 19 [085], [0249] d) a heavy chain variable region
(VH) comprising the sequence of SEQ ID NO: 26 or a sequence having
at least 90%, at least 95%, at least 97%, or at least 99% amino
acid sequence identity to the sequence of SEQ ID NO: 26 [106],
[0250] e) a heavy chain variable region (VH) comprising the
sequence of SEQ ID NO: 33 or a sequence having at least 90%, at
least 95%, at least 97%, or at least 99% amino acid sequence
identity to the sequence of SEQ ID NO: 33 [127], [0251] f) a heavy
chain variable region (VH) comprising the sequence of SEQ ID NO: 40
or a sequence having at least 90%, at least 95%, at least 97%, or
at least 99% amino acid sequence identity to the sequence of SEQ ID
NO: 40 [207]; and [0252] g) a heavy chain variable region (VH)
comprising the sequence of SEQ ID NO: 47 or a sequence having at
least 90%, at least 95%, at least 97%, or at least 99% amino acid
sequence identity to the sequence of SEQ ID NO: 47 [226].
[0253] The antibody according to the invention may in particular be
an antibody, wherein said antigen-binding region capable of binding
to 5T4 comprises a heavy chain variable region (VH) comprising the
sequence of SEQ ID NO: 5 or a sequence having at least 90%, at
least 95%, at least 97%, or at least 99% amino acid sequence
identity to the sequence of SEQ ID NO: 5 [059].
[0254] Also, the antibody according to the invention may be one,
wherein said antigen-binding region capable of binding to 5T4
comprises a heavy chain variable region (VH) comprising the
sequence of SEQ ID NO: 40 or a sequence having at least 90%, at
least 95%, at least 97%, or at least 99% amino acid sequence
identity to the sequence of SEQ ID NO: 40 [207].
[0255] Additionally, the antibody according to the invention may be
an antibody, wherein said antigen-binding region capable of binding
to 5T4 comprises a heavy chain variable region (VH) comprising the
sequence of SEQ ID NO: 47 or a sequence having at least 90%, at
least 95%, at least 97%, or at least 99% amino acid sequence
identity to the sequence of SEQ ID NO: 47 [226].
[0256] In other embodiments, the antibody according to the
invention is an antibody, wherein said antigen-binding region
capable of binding to 5T4 comprises a heavy chain variable region
(VH) and a light chain variable region (VL) selected from the group
consisting of: [0257] a) a heavy chain variable region (VH)
comprising the sequence of SEQ ID NO: 5 or a sequence having at
least 90%, at least 95%, at least 97%, or at least 99% amino acid
sequence identity to the sequence of SEQ ID NO: 5, and a light
chain variable region (VL) comprising the sequence of SEQ ID NO: 9
or a sequence having at least 90%, at least 95%, at least 97%, or
at least 99% amino acid sequence identity to the sequence of SEQ ID
NO: 9 [059], [0258] b) a heavy chain variable region (VH)
comprising the sequence of SEQ ID NO: 12 or a sequence having at
least 90%, at least 95%, at least 97%, or at least 99% amino acid
sequence identity to the sequence of SEQ ID NO: 12, and a light
chain variable region (VL) comprising the sequence of SEQ ID NO: 16
or a sequence having at least 90%, at least 95%, at least 97%, or
at least 99% amino acid sequence identity to the sequence of SEQ ID
NO: 16 [076], [0259] c) a heavy chain variable region (VH)
comprising the sequence of SEQ ID NO: 19 or a sequence having at
least 90%, at least 95%, at least 97%, or at least 99% amino acid
sequence identity to the sequence of SEQ ID NO: 19, and a light
chain variable region (VL) comprising the sequence of SEQ ID NO: 23
or a sequence having at least 90%, at least 95%, at least 97%, or
at least 99% amino acid sequence identity to the sequence of SEQ ID
NO: 23 [085], [0260] d) a heavy chain variable region (VH)
comprising the sequence of SEQ ID NO: 26 or a sequence having at
least 90%, at least 95%, at least 97%, or at least 99% amino acid
sequence identity to the sequence of SEQ ID NO: 26, and a light
chain variable region (VL) comprising the sequence of SEQ ID NO: 30
or a sequence having at least 90%, at least 95%, at least 97%, or
at least 99% amino acid sequence identity to the sequence of SEQ ID
NO: 30 [106], [0261] e) a heavy chain variable region (VH)
comprising the sequence of SEQ ID NO: 33 or a sequence having at
least 90%, at least 95%, at least 97%, or at least 99% amino acid
sequence identity to the sequence of SEQ ID NO: 33, and a light
chain variable region (VL) comprising the sequence of SEQ ID NO: 37
or a sequence having at least 90%, at least 95%, at least 97%, or
at least 99% amino acid sequence identity to the sequence of SEQ ID
NO: 37 [127], [0262] f) a heavy chain variable region (VH)
comprising the sequence of SEQ ID NO: 40 or a sequence having at
least 90%, at least 95%, at least 97%, or at least 99% amino acid
sequence identity to the sequence of SEQ ID NO: 40, and a light
chain variable region (VL) comprising the sequence of SEQ ID NO: 44
or a sequence having at least 90%, at least 95%, at least 97%, or
at least 99% amino acid sequence identity to the sequence of SEQ ID
NO: 44 [207], [0263] g) a heavy chain variable region (VH)
comprising the sequence of SEQ ID NO: 47 or a sequence having at
least 90%, at least 95%, at least 97%, or at least 99% amino acid
sequence identity to the sequence of SEQ ID NO: 47, and a light
chain variable region (VL) comprising the sequence of SEQ ID NO: 51
or a sequence having at least 90%, at least 95%, at least 97%, or
at least 99% amino acid sequence identity to the sequence of SEQ ID
NO: 51 [226].
[0264] In one embodiment, the at least one binding region comprises
a variable heavy chain (VH) region and a variable light chain (VL)
region having at most 10 mutations or substitutions, at most 5
mutations or substitutions, such as at most 4 mutations or
substitutions, such as at most 3 mutations or substitutions, such
as at most 2 mutations or substitutions, such as at most 1 mutation
or substitution, across said heavy chain variable region (VH) and
light chain variable region (VL) region selected from the group
consisting of: [0265] a) a heavy chain variable region (VH)
comprising or consisting of the sequence of SEQ ID NO: 5, and a
light chain variable region (VL) comprising or consisting of the
sequence of SEQ ID NO: 9 [059], [0266] b) a heavy chain variable
region (VH) comprising or consisting of the sequence of SEQ ID NO:
12, and a light chain variable region (VL) comprising or consisting
of the sequence of SEQ ID NO: 16 [076], [0267] c) a heavy chain
variable region (VH) comprising or consisting of the sequence of
SEQ ID NO: 19, and a light chain variable region (VL) comprising or
consisting of the sequence of SEQ ID NO: 23 [085], [0268] d) a
heavy chain variable region (VH) comprising or consisting of the
sequence of SEQ ID NO: 26, and a light chain variable region (VL)
comprising or consisting of the sequence of SEQ ID NO: 30 [106],
[0269] e) a heavy chain variable region (VH) comprising or
consisting of the sequence of SEQ ID NO: 33, and a light chain
variable region (VL) comprising or consisting of the sequence of
SEQ ID NO: 37 [127], [0270] f) a heavy chain variable region (VH)
comprising or consisting of the sequence of SEQ ID NO: 40, and a
light chain variable region (VL) comprising or consisting of the
sequence of SEQ ID NO: 44 [207], [0271] g) a heavy chain variable
region (VH) comprising or consisting of the sequence of SEQ ID NO:
47, and a light chain variable region (VL) comprising or consisting
of the sequence of SEQ ID NO: 51 [226].
[0272] In some embodiments of the present disclosure, the at most
10 mutations or substitutions, at most 5 mutations or
substitutions, such as at most 4 mutations or substitutions, such
as at most 3 mutations or substitutions, such as at most 2
mutations or substitutions, such as at most 1 mutation or
substitution are allowed across the full length of the variable
heavy chain and the entire variable light chain. In other
embodiments, the at most 10 mutations or substitutions, at most 5
mutations or substitutions, such as at most 4 mutations or
substitutions, such as at most 3 mutations or substitutions, such
as at most 2 mutations or substitutions, such as at most 1 mutation
or substitution may not be within any of the 6 CDR sequences in the
said variable heavy chain and the variable light chain.
[0273] The up to 10 mutations or substitutions may be distributed
across the full length of the variable heavy chain and the variable
light chain of each binding region. Some or all of the mutations or
substitutions may be conservative substitutions in which one amino
acid residue is substituted with an amino acid residue of the same
class as indicated under the definition "amino acid" herein above;
for instance an acidic amino acid being substituted for another
acidic amino acid residue, and an aromatic residue may be
substituted for another aromatic residue. It may be preferred that
35% or more, 50% or more, 60% or more, 70% or more, 75% or more,
80% or more, 85% or more, 90% or more, 92% or more, 93% or more or
94% or more of the substitutions in the variant are conservative
amino acid residue replacements.
[0274] In particular, some or all of the mutations or substitutions
may be with amino acid residue(s) each having the same physical or
functional properties as the respective amino acid residue which
they substitute. Amino acid residues sharing physical and
functional properties are provided under the definition "amino
acid" herein above; for instance a under the definition "amino
acid" herein above; for instance a hydrophobic residue may be
substituted for another hydrophobic amino acid residue or a
cycloalkenyl-associated residue may be substituted for another
cycloalkenyl-associated residue.
[0275] Antibodies comprising substitutions or mutations as
disclosed above may in particular be functional variants of the VL
regions, VH regions, or one or more CDRs defined above with
reference to sequences identifiers. A functional variant of a VL,
VH, or CDR used in the context of the antibodies of the present
invention still allows the antibody to retain at least a
substantial proportion (at least about 50%, 60%, 70%, 80%, 90%,
95%, 99% or more) of the affinity and/or the
specificity/selectivity of the parent antibody, and in some cases
such an 5T4 antibody may even be associated with greater affinity,
selectivity and/or specificity than the parent antibody.
[0276] In further embodiments of the invention, the antibody is
one, wherein said antigen-binding region capable of binding to 5T4
comprises a heavy chain variable region (VH) and a light chain
variable region (VL) selected from the group consisting of: [0277]
a) a heavy chain variable region (VH) comprising or consisting of
the sequence of SEQ ID NO: 5, and a light chain variable region
(VL) comprising or consisting of the sequence of SEQ ID NO: 9
[059], [0278] b) a heavy chain variable region (VH) comprising or
consisting of the sequence of SEQ ID NO: 12, and a light chain
variable region (VL) comprising or consisting of the sequence of
SEQ ID NO: 16 [076], [0279] c) a heavy chain variable region (VH)
comprising or consisting of the sequence of SEQ ID NO: 19, and a
light chain variable region (VL) comprising or consisting of the
sequence of SEQ ID NO: 23 [085], [0280] d) a heavy chain variable
region (VH) comprising or consisting of the sequence of SEQ ID NO:
26, and a light chain variable region (VL) comprising or consisting
of the sequence of SEQ ID NO: 30 [106], [0281] e) a heavy chain
variable region (VH) comprising or consisting of the sequence of
SEQ ID NO: 33, and a light chain variable region (VL) comprising or
consisting of the sequence of SEQ ID NO: 37 [127], [0282] f) a
heavy chain variable region (VH) comprising or consisting of the
sequence of SEQ ID NO: 40, and a light chain variable region (VL)
comprising or consisting of the sequence of SEQ ID NO: 44 [207];
and [0283] g) a heavy chain variable region (VH) comprising or
consisting of the sequence of SEQ ID NO: 47, and a light chain
variable region (VL) comprising or consisting of the sequence of
SEQ ID NO: 51 [226].
[0284] The antibody of the invention may be a full-length antibody,
such as a full length IgG1 antibody.
[0285] Further, the antibody of the invention may be a monovalent
antibody. Alternatively, the antibody according to the invention
may be a bivalent antibody.
[0286] In other embodiments, the antibody provided according to the
present invention is a monospecific antibody.
[0287] Alternatively, the antibody according to the present
disclosure may be a bispecific antibody.
[0288] It is further within the scope of the present disclosure to
provide an antibody as defined above, the antibody comprising an
antigen binding region of an antibody that binds to CD3, such as
human CD3E (epsilon), such as human CD3E (epsilon) as specified in
SEQ ID NO: 4.
[0289] In particular, the present disclosure provides a bispecific
antibody comprising a first antigen binding region of an antibody
as disclosed above, and a second binding region which binds to CD3,
such as human CD3 as defined above.
[0290] Examples of bispecific antibody molecules which may be used
in the present invention include but are not limited to (i) a
single antibody that has two arms comprising different
antigen-binding regions, (ii) a single chain antibody that has
specificity to two different epitopes, e.g., via two scFvs linked
in tandem by an extra peptide linker; (iii) a dual-variable-domain
antibody (DVD-Ig.TM.), where each light chain and heavy chain
contains two variable domains in tandem through a short peptide
linkage Wu et al., Generation and Characterization of a Dual
Variable Domain Immunoglobulin (DVD-Ig.TM.) Molecule, In: Antibody
Engineering, Springer Berlin Heidelberg (2010); (iv) a
chemically-linked bispecific (Fab')2 fragment; (v) a Tandab.RTM.,
which is a fusion of two single chain diabodies resulting in a
tetravalent bispecific antibody that has two binding sites for each
of the target antigens; (vi) a flexibody, which is a combination of
scFvs with a diabody resulting in a multivalent molecule; (vii) a
so called "dock and lock" molecule (Dock-and-Lock.RTM.), based on
the "dimerization and docking domain" in Protein Kinase A, which,
when applied to Fabs, can yield a trivalent bispecific binding
protein consisting of two identical Fab fragments linked to a
different Fab fragment; (viii) a so-called Scorpion molecule,
comprising, e.g., two scFvs fused to both termini of a human
Fab-arm; and (ix) a diabody.
[0291] In one embodiment, the bispecific antibody of the present
invention is a diabody, a cross-body, such as CrossMabs, or a
bispecific antibody obtained via a controlled Fab arm exchange
(such as described in WO 2011/131746).
[0292] Examples of different classes of bispecific antibodies
include but are not limited to (i) IgG-like molecules with
complementary CH3 domains to force heterodimerization; (ii)
recombinant IgG-like dual targeting molecules, wherein the two
sides of the molecule each contain the Fab fragment or part of the
Fab fragment of at least two different antibodies; (iii) IgG fusion
molecules, wherein full length IgG antibodies are fused to extra
Fab fragment or parts of Fab fragment; (iv) Fc fusion molecules,
wherein single chain Fv molecules or stabilized diabodies are fused
to heavy-chain constant-domains, Fc-regions or parts thereof; (v)
Fab fusion molecules, wherein different Fab-fragments are fused
together, fused to heavy-chain constant-domains, Fc-regions or
parts thereof; and (vi) ScFv- and diabody-based and heavy chain
antibodies (e.g., domain antibodies, Nanobodies.RTM.) wherein
different single chain Fv molecules or different diabodies or
different heavy-chain antibodies (e.g. domain antibodies,
Nanobodies.RTM.) are fused to each other or to another protein or
carrier molecule fused to heavy-chain constant-domains, Fc-regions
or parts thereof.
[0293] Examples of IgG-like molecules with complementary CH3
domains molecules include but are not limited to the Triomab.RTM.
(Trion Pharma/Fresenius Biotech, WO/2002/020039), the
Knobs-into-Holes (Genentech, WO9850431), CrossMAbs (Roche,
WO2011117329) and the electrostatically-matched (Amgen, EP1870459
and WO2009089004; Chugai, US201000155133; Oncomed, WO2010129304),
the LUZ-Y (Genentech), DIG-body and PIG-body (Pharmabcine), the
Strand Exchange Engineered Domain body (SEEDbody)(EMD Serono,
WO2007110205), the Biclonics (Merus), Fc.DELTA.Adp (Regeneron, WO
2010/015792), bispecific IgG1 and IgG2 (Pfizer/Rinat, WO11143545),
Azymetric scaffold (Zymeworks/Merck, WO2012058768), mAb-Fv (Xencor,
WO2011028952), bivalent bispecific antibodies (Roche WO
2009/080254) and DuoBody.RTM. molecules (Genmab A/S, WO
2011/131746).
[0294] Examples of recombinant IgG-like dual targeting molecules
include but are not limited to Dual Targeting (DT)-Ig
(GSK/Domantis), Two-in-one Antibody (Genentech), Cross-linked Mabs
(Karmanos Cancer Center), mAb2 (F-Star, WO2008003116), Zybodies.TM.
(Zyngenia), approaches with common light chain (Crucell/Merus, U.S.
Pat. No. 7,262,028), kABodies (NovImmune) and CovX-body
(CovX/Pfizer).
[0295] Examples of IgG fusion molecules include but are not limited
to Dual Variable Domain (DVD)-Ig.TM. (Abbott, U.S. Pat. No.
7,612,181), Dual domain double head antibodies (Unilever; Sanofi
Aventis, WO20100226923), IgG-like Bispecific (ImClone/Eli Lilly),
Ts2Ab (Medlmmune/AZ) and BsAb (Zymogenetics), HERCULES (Biogen
Idec, US007951918), scFv fusion (Novartis), scFv fusion (Changzhou
Adam Biotech Inc, CN 102250246) and TvAb (Roche, WO2012025525,
WO2012025530).
[0296] Examples of Fc fusion molecules include but are not limited
to ScFv/Fc Fusions (Academic Institution), SCORPION (Emergent
BioSolutions/Trubion, Zymogenetics/BMS), Dual Affinity Retargeting
Technology (Fc-DART.TM.) (MacroGenics, WO2008157379, WO2010/080538)
and Dual(ScFv)2-Fab (National Research Center for Antibody
Medicine--China).
[0297] Examples of Fab fusion bispecific antibodies include but are
not limited to F(ab)2 (Medarex/AMGEN), Dual-Action or Bis-Fab
(Genentech), Dock-and-Lock.RTM. (DNL) (ImmunoMedics), Bivalent
Bispecific (Biotecnol) and Fab-Fv (UCB-Celltech).
[0298] Examples of scFv-, diabody-based and domain antibodies
include but are not limited to Bispecific T Cell Engager
(BiTE.RTM.) (Micromet, Tandem Diabody (Tandab.TM.) (Affimed), Dual
Affinity Retargeting Technology (DART) (MacroGenics), Single-chain
Diabody (Academic), TCR-like Antibodies (AIT, ReceptorLogics),
Human Serum Albumin ScFv Fusion (Merrimack) and COMBODY (Epigen
Biotech), dual targeting Nanobodies.RTM. (Ablynx), dual targeting
heavy chain only domain antibodies.
[0299] The antibody according to the present disclosure may in
particular be an antibody, wherein the antigen binding region that
binds to CD3 comprises [0300] a heavy chain variable region (VH)
comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs.: 54,
55 and 56, respectively; [huCD3-H1L1] (WO2015001085 (Genmab A/S));
[0301] and, optionally [0302] a light chain variable region (VL)
comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 58, GTN
and 59, respectively [huCD3-H1L1].
[0303] Also disclosed are antibodies wherein the antigen binding
region that binds to CD3 comprises [0304] a heavy chain variable
region (VH) comprising the sequence of SEQ ID NO: 57, or a sequence
having at least 90%, at least 95%, at least 97%, or at least 99%
amino acid sequence identity to the sequence of SEQ ID NO: 57
[huCD3-H1L1]; [0305] and, optionally [0306] a light chain variable
region (VL) comprising the sequence of SEQ ID NO: 60 or a sequence
having at least 90%, at least 95%, at least 97%, or at least 99%
amino acid sequence identity to the sequence of SEQ ID NO: 60,
[huCD3-H1L1].
[0307] The present disclosure further provides an antibody, wherein
[0308] the antigen-binding region capable of binding to 5T4
comprises a heavy chain variable region (VH) comprising CDR1, CDR2,
and CDR3 sequences of SEQ ID NOs.: 6, 7 and 8, respectively, and a
light chain variable region (VL) comprising the CDR1, CDR2, and
CDR3 sequences of SEQ ID NO: 10, AAS and 11, respectively [059];
[0309] and [0310] the antigen-binding region capable of binding to
CD3 comprises a heavy chain variable region (VH) comprising CDR1,
CDR2, and CDR3 having the sequences as set forth in SEQ ID NOs: 54,
55 and 56, respectively, and a light chain variable region (VL)
comprising CDR1, CDR2, and CDR3 having the sequences as set forth
in SEQ ID NO: 58, the sequence GTN, and the sequence as set forth
in SEQ ID NO: 59 [huCD3-H1L1], respectively.
[0311] Also, the disclosure provides an antibody, wherein [0312]
the antigen-binding region capable of binding to 5T4 comprises a
heavy chain variable region (VH) comprising the CDR1, CDR2, and
CDR3 sequences of SEQ ID NOs.: 41, 42 and 43, respectively, and a
light chain variable region (VL) comprising the CDR1, CDR2, and
CDR3 sequences of SEQ ID NOs.: 45, DAS and 46, respectively [207];
[0313] and [0314] the antigen-binding region capable of binding to
CD3 comprises a heavy chain variable region (VH) comprising CDR1,
CDR2, and CDR3 having the sequences as set forth in SEQ ID NOs: 54,
55 and 56, respectively, and a light chain variable region (VL)
comprising CDR1, CDR2, and CDR3 having the sequences as set forth
in SEQ ID NO: 58, the sequence GTN, and the sequence as set forth
in SEQ ID NO: 59 [huCD3-H1L1], respectively.
[0315] Also, disclosure provides an antibody, wherein [0316] the
antigen-binding region capable of binding to 5T4 comprises a heavy
chain variable region (VH) comprising the CDR1, CDR2, and CDR3
sequences of SEQ ID NOs.: 48, 49 and 50, respectively, and a light
chain variable region (VL) comprising the CDR1, CDR2, and CDR3
sequences of SEQ ID NO: 52, DAS and SEQ ID NO: 53, respectively
[226-VH+VL CDR1, -2 and -3 sequences]; [0317] and [0318] the
antigen-binding region capable of binding to CD3 comprises a heavy
chain variable region (VH) comprising CDR1, CDR2, and CDR3 having
the sequences as set forth in SEQ ID NOs: 54, 55 and 56,
respectively, and a light chain variable region (VL) comprising
CDR1, CDR2, and CDR3 having the sequences as set forth in SEQ ID
NO: 58, the sequence GTN, and the sequence as set forth in SEQ ID
NO: 59 [huCD3-H1L1], respectively.
[0319] The antigen binding region that binds to CD3, may bind with
an equilibrium dissociation constant K.sub.D within the range of
200-1000 nM, such as within the range of 300-1000 nM, within the
range of 400-1000 nM, within the range of 500-1000 nM, within the
range of 300-900 nM within the range of 400-900 nM, within the
range of 400-700 nM, within the range of 500-900 nM, within the
range of 500-800 nM, within the range of 500-700 nM, within the
range of 600-1000 nM, within the range of 600-900 nM, within the
range of 600-800 nM, or such as within the range of 600-700 nM.
[0320] In further embodiments, the antibody disclosed herein has a
lower human CD3E binding affinity than an antibody having an
antigen-binding region comprising a VH sequence as set forth in SEQ
ID NO: 57, and a VL sequence as set forth in SEQ ID NO: 60
[huCD3-H1L1], preferably wherein said affinity is at least 2-fold
lower, e.g. at least 5-fold lower, such as at least 10-fold lower,
e.g. at least 20-fold lower, at least 30-fold lower, at least
40-fold lower, at least 45-fold lower, at least 50-fold lower, at
least 55-fold lower, or such as at least 60-fold lower.
[0321] In particular, the antigen binding region that binds to CD3
may bind with an equilibrium dissociation constant K.sub.D within
the range of 1-100 nM, such as within the range of 5-100 nM, within
the range of 10-100 nM, within the range of 1-80 nM, within the
range of 1-60 nM within the range of 1-40 nM, within the range of
1-20 nM, within the range of 5-80 nM, within the range of 5-60 nM,
within the range of 5-40 nM, within the range of 5-20 nM, within
the range of 10-80 nM, within the range of 10-60 nM, within the
range of 10-40 nM, or such as within the range of 10-20 nM.
[0322] The affinity with which the antibody according to the
invention bind to CD3 may be determined by biolayer interferometry,
using a modification of the procedure described above or as set
forth in Example 2 herein, in which the antibody is immobilized on
a human IgG Fc Capture biosensor and association and dissociation
of the CD3E27-GSKa (mature protein of SEQ ID NO: 101) to the
immobilize antibody is determined. Further, the affinity with which
the antibody according to the invention bind to CD3 may be
determined by biolayer interferometry as provided in Example 9
herein.
[0323] Antibodies binding CD3, in particular human CD3, with
reduced affinity are provided in WO 2017/009442, and it is to be
understood that any of these antibodies may serve as the basis for
generating antibodies according to the present invention which in
addition to the ability to bind 5T4 also have the ability to bind
CD3 with reduced affinity. Hence, in further embodiments, the
antibody according to the invention is an antibody, wherein [0324]
the antigen binding region that binds to CD3 comprises a heavy
chain variable (VH) region comprising a CDR1 sequence, a CDR2
sequence and a CDR3 sequence, [0325] the heavy chain variable (VH)
region, when compared to a heavy chain variable (VH) region
comprising the sequence set forth in SEQ ID NO: 57, has an amino
acid substitution in one of the CDR sequences, the substitution
being at a position selected from the group consisting of: T31,
N57, H101, G105, S110 and Y114, the positions being numbered
according to the sequence of SEQ ID NO: 57; and [0326] the wild
type light chain variable (VL) region comprises the CDR1, CDR2 and
CDR3 sequences set forth in SEQ ID NOs: 58, GTN and SEQ ID NO: 59,
respectively.
[0327] It is preferred that the CDR1, CDR2 and CDR3 sequences of
the heavy chain variable (VH) region of the antigen binding region
that binds to CD3 comprise, in total, at the most 1, 2, 3, 4 or 5
amino acid substitutions, when compared to the sequence set forth
in SEQ ID NO: 57.
[0328] The amino acid sequences of the CDR1, CDR2 and CDR3 of the
heavy chain variable (VH) region of the antigen binding region that
binds to CD3 may have at least 95% sequence identity, such as at
least 96% sequence identity, at least 97% sequence identity, at
least 98% sequence identity or at least 99% sequence identity to
the amino acid sequences of the CDR1, CDR2 and CDR3 of the wild
type heavy chain variable (VH) region, sequence identity being
calculated based on an aligning an amino acid sequence consisting
of the sequences of the CDR1, CDR2 and CDR3 of the heavy chain
variable (VH) region of the antigen binding region that binds to
CD3 with an amino acid sequence comprising the sequences of the
CDR1, CDR2 and CDR3 of the wild type heavy chain variable (VH)
region.
[0329] In particular, the antigen binding region that binds to CD3
may comprise a mutation selected from the group consisting of:
T31M, T31P, N57E, H101G, H101N, G105P, S110A, S110G, Y114M, Y114R,
Y114V, the positions being numbered according to the reference
sequence of SEQ ID NO: 57.
[0330] In certain embodiments, the antibody according to the
invention is an antibody, wherein when said antibody is a
bispecific antibody, which is devoid of, or has reduced Fc-mediated
effector function ("inert" antibody), and comprises an antigen
binding region of an antibody that binds to CD3, then the antibody:
[0331] a) is capable of mediating concentration-dependent
cytotoxicity of SK-OV-3 cells, when using purified peripheral blood
mononuclear cells (PBMCs) or T cells as effector cells e.g. when
assayed as described in Example 14 herein, [0332] b) is capable of
mediating concentration-dependent cytotoxicity of MDA-MB-231 cells,
when using purified T cells as effector cells e.g. when assayed as
described in Example 13 herein, [0333] c) is capable of activating
T cells in vitro in the presence of MDA-MB-231 tumor cells; e.g.
when assayed as described in Example 13 (II) herein [0334] d) is
capable of activating T-cells in vitro in the presence of BxPC-3,
PANC-1, Ca Ski and/or SiHa tumor cells; e.g. when assayed as
described in Example 17 herein, [0335] e) is capable of inducing
cytotoxicity of BxPC-3, PANC-1, Ca Ski and/or SiHa tumor cells when
using purified T cells as effector cells e.g. when assayed as
described in Example 17 herein; and/or [0336] f) shows anti-tumor
activity, such as inhibition of tumor growth or delayed tumor
outgrowth, in a humanized immune hematopoietic stem cell
reconstitution mouse xenograft model, such as NOD.Cg-Prkdc.sup.scid
ll2rg.sup.tm1Wjl/SzJ inoculated with human MDA-MB-231 tumor cells;
e.g. when determined as described in Example 15; and
[0337] Further, the antibody according to the invention is an
antibody that, when assessed by flow cytometry or ELISA, does not
bind leukocyte Fc.gamma.Rs, and does not induce CD3-antibody
dependent, Fc.gamma.R-mediated CD3-crosslinking in absence of
target (5T4)-specific tumor cells by binding to C1q.
[0338] A more detailed disclosure of antibodies with reduced or no
Fc-mediated effector function ("inert" antibodies) can be found
herein below.
[0339] The ability of the antibody to mediate
concentration-dependent cytotoxicity of SK-OV-3 cells is determined
in an in vitro cytotoxicity assay comprising the steps of: [0340]
i) isolating PBMCs or T cells from healthy human donor buffy coats,
[0341] ii) providing [0342] a first set of samples, wherein each
sample comprises PBMCs and human ovary adenocarcinoma SK-OV-3
cells, and wherein the ratios PBMCs:SK-OV-3 cells in said samples
are 1:2, 1:1, 2:1, 4:1, 8:1, and 12:1; and [0343] a second set of
samples, wherein each sample comprises T cells and human ovary
adenocarcinoma SK-OV-3 cells and wherein the ratios of T
cells:SK-OV-3 cells in said samples are 1:2, 1:1, 2:1, 4:1 and 8:1
[0344] iii) adding the antibody to each set of samples at
concentrations ranging from 0.0128 ng/mL to 1000 ng/mL and
incubating the samples for 72 hours at 37.degree. C.; and then
[0345] iv) assessing the viability of the SK-OV-3 cells using
Resazurin (7-Hydroxy-3H-phenoxazin-3-one 10-oxide).
[0346] The ability to activate T cells in vitro in the presence of
MDA-MB-231 tumor cells may be determined in an assay comprising the
steps of: [0347] i) Isolating T cells from healthy human donor
buffy coats, [0348] ii) Providing a set of samples, wherein each
sample comprises T-cells and human breast adenocarcinoma MDA-MB-231
cells and wherein the ratio of T-cells:MDA-MB-231 cells in said
samples is 8:1, [0349] iii) adding the antibody to the set of
samples at concentrations ranging from 0.0128 ng/mL to 1000 ng/mL
and incubating the samples for 72 h at 37.degree. C., [0350] iv)
staining the Tcells with fluorescent-labeled antibodies against
T-cell activation markers, such as CD69-APC, CD25-PE-Cy7 and
CD279/PD 1-BV 604 antibodies, by incubation with said antibodies
for 30 min at 4.degree. C.; and [0351] v) analyzing the T cells by
flow cytometry.
[0352] APC anti-human CD69 (CD69-APC) antibodies are commercially
available, for instance from BioLegend (Cat. # s 310909 and
310910). CD25 Monoclonal Antibody, PE-Cyanine7 (CD25-PE-Cy7) is
also commercially available, for instance from ThermoFisher
Scientific (Cat. #25-0259-42) and from BD Biosciences (Cat.
#557741). Finally, CD279/PD 1-BV 604 antibodies may be obtained
commercially from Genscript (Cat. # A01828).
[0353] The activation of T cells in vitro in the presence of
BxPC-3, PANC-1, Ca Ski and/or SiHa tumor cells may be determined in
an procedure comprising the steps of: [0354] i) Providing T cells
isolated from healthy human donor buffy coats, [0355] ii) Providing
a set of samples, wherein each sample comprises said T cells and
BxPC-3, PANC-1, Ca Ski or SiHa tumor cells and wherein the ratio of
T cells:tumor cells in said samples is 4:1, [0356] iii) adding the
antibody to the set of samples at concentrations ranging from
0.0128 ng/mL to 5000 ng/mL (such as 5-fold dilutions) and
incubating the samples for 72 hours at 37.degree. C., [0357] iv)
collecting from each sample 110 .mu.L supernatant containing T
cells and staining the T cells with fluorescent-labeled antibodies
against T-cell markers, such as CD3-eFluor450, CD4-APC-eFluor780,
DC8-AF700, and with antibodies against T-cell markers, such as
69-APC, CD25-PE-Cy7 and CD279/PD1-BV604 antibodies, by incubation
with said antibodies for 30 minutes at 4.degree. C.; and [0358] v)
analyzing the samples by flow cytometry.
[0359] The ability to induce cytotoxicity of BxPC-3, PANC-1, Ca Ski
and/or SiHa tumor cells may be determined in a procedure comprising
the steps of [0360] i) Providing T cells isolated from healthy
human donor buffy coats, [0361] ii) Providing a set of test samples
and control samples, wherein each sample comprises said T-cells and
BxPC-3, PANC-1, Ca Ski or SiHa tumor cells which have been allowed
to adhere to the bottom of a 96-well tissue culture plate and
wherein the ratio of T-cells:tumor cells in said samples is 4:1,
[0362] iii) adding the antibody to the set of test samples at
concentrations ranging from 0.0128 ng/mL to 5000 ng/mL (such as
5-fold dilutions), while the control samples remain untreated or
are incubated with 5 .mu.M staurosporin, and incubating all samples
for 72 hours at 37.degree. C., [0363] iv) Incubating the adherent
cells in 10% (w/w) 7-hydroxy-3H-phenoxazin-3-one 10-oxide
(Resazurin) in RPMI-1640 medium supplemented with 10% (w/w) donor
bovine serum with iron and penicillin/streptomycin at 37.degree. C.
for 4 hours, [0364] v) Measuring the absorbance of the cells;
setting the absorbance of the cells incubated with staurosporin as
0% viability and the untreated cells as 100% viability and
calculating the percentage viable cells as
[0364] .times. 100 % viable cells = ( [ absorbance sample -
absorbance staurosporine treated cells ] [ absorbance untreated
cells - absorbance staurosporine treated cells ] ) ##EQU00005##
[0365] The antibody of the invention may in particular be an
antibody, wherein the antigen-binding region capable of binding to
CD3 comprises: [0366] a) a heavy chain variable region (VH)
comprising CDR1, CDR2, and CDR3 having the sequences as set forth
in SEQ ID NOs: 61, 55, and 56 [VH CDR1-T31P+Wild type VH CDRs 2,3],
respectively, and a light chain variable region (VL) comprising
CDR1, CDR2, and CDR3 having the sequences as set forth in SEQ ID
NO: 58, the sequence GTN, and the sequence as set forth in SEQ ID
NO: 59, respectively [Wild type VL CDRs 1,2,3], or [0367] b) a
heavy chain variable region (VH) comprising CDR1, CDR2, and CDR3
having the sequences as set forth in SEQ ID NOs: 63, 55, and 56 [VH
CDR1-T31M+Wild type VH CDRs 2,3], respectively, and a light chain
variable region (VL) comprising CDR1, CDR2, and CDR3 having the
sequences as set forth in SEQ ID NO: 58, the sequence GTN, and the
sequence as set forth in SEQ ID NO: 59, respectively [Wild type VL
CDRs 1,2,3], respectively, or [0368] c) a heavy chain variable
region (VH) comprising CDR1, CDR2, and CDR3 having the sequences as
set forth in SEQ ID NOs: 54, 65, and 56 [VH CDR-N57E+Wild type VH
CDRs 1,3], respectively, and a light chain variable region (VL)
comprising CDR1, CDR2, and CDR3 having the sequences as set forth
in SEQ ID NO: 58, the sequence GTN, and the sequence as set forth
in SEQ ID NO: 59, respectively [Wild type VL CDRs 1,2,3],
respectively, or [0369] d) a heavy chain variable region (VH)
comprising CDR1, CDR2, and CDR3 having the sequences as set forth
in SEQ ID NOs: 54, 55, and 67 [Wild type VH CDRs 1,2+VH
CDR3-H101G], respectively, and a light chain variable region (VL)
comprising CDR1, CDR2, and CDR3 having the sequences as set forth
in SEQ ID NO: 58, the sequence GTN, and the sequence as set forth
in SEQ ID NO: 59, respectively [Wild type VL CDRs 1,2,3],
respectively. [0370] e) a heavy chain variable region (VH)
comprising CDR1, CDR2, and CDR3 having the sequences as set forth
in SEQ ID NOs: 54, 55, and 69 [Wild type VH CDRs 1,2+VH
CDR3-H101N], respectively, and a light chain variable region (VL)
comprising CDR1, CDR2, and CDR3 having the sequences as set forth
in SEQ ID NO: 58, the sequence GTN, and the sequence as set forth
in SEQ ID NO: 59, respectively [Wild type VL CDRs 1,2,3],
respectively; [0371] f) a heavy chain variable region (VH)
comprising CDR1, CDR2, and CDR3 having the sequences as set forth
in SEQ ID NOs: 54, 55, and 71 [Wild type VH CDRs 1,2+VH
CDR3-G105P], respectively, and a light chain variable region (VL)
comprising CDR1, CDR2, and CDR3 having the sequences as set forth
in SEQ ID NO: 58, the sequence GTN, and the sequence as set forth
in SEQ ID NO: 59, respectively [Wild type VL CDRs 1,2,3],
respectively; [0372] g) a heavy chain variable region (VH)
comprising CDR1, CDR2, and CDR3 having the sequences as set forth
in SEQ ID NOs: 54, 55, and 73 [Wild type VH CDRs 1,2+VH
CDR3-S110A], respectively, and a light chain variable region (VL)
comprising CDR1, CDR2, and CDR3 having the sequences as set forth
in SEQ ID NO: 58, the sequence GTN, and the sequence as set forth
in SEQ ID NO: 59, respectively [Wild type VL CDRs 1,2,3],
respectively, or [0373] h) a heavy chain variable region (VH)
comprising CDR1, CDR2, and CDR3 having the sequences as set forth
in SEQ ID NOs: 54, 55, and 75 [Wild type VH CDRs 1,2+VH
CDR3-S110G], respectively, and a light chain variable region (VL)
comprising CDR1, CDR2, and CDR3 having the sequences as set forth
in SEQ ID NO: 58, the sequence GTN, and the sequence as set forth
in SEQ ID NO: 59, respectively [Wild type VL CDRs 1,2,3],
respectively, [0374] i) a heavy chain variable region (VH)
comprising CDR1, CDR2, and CDR3 having the sequences as set forth
in SEQ ID NOs: 54, 55, and 77 [Wild type VH CDRs 1,2+VH
CDR3-Y114V], respectively, and a light chain variable region (VL)
comprising CDR1, CDR2, and CDR3 having the sequences as set forth
in SEQ ID NO: 58, the sequence GTN, and the sequence as set forth
in SEQ ID NO: 59, respectively [Wild type VL CDRs 1,2,3],
respectively, or [0375] j) a heavy chain variable region (VH)
comprising CDR1, CDR2, and CDR3 having the sequences as set forth
in SEQ ID NOs: 54, 55, and 79 [Wild type VH CDRs 1,2+VH
CDR3-Y114M], respectively, and a light chain variable region (VL)
comprising CDR1, CDR2, and CDR3 having the sequences as set forth
in SEQ ID NO: 58, the sequence GTN, and the sequence as set forth
in SEQ ID NO: 59, respectively [Wild type VL CDRs 1,2,3],
respectively, or [0376] k) a heavy chain variable region (VH)
comprising CDR1, CDR2, and CDR3 having the sequences as set forth
in SEQ ID NOs: 54, 55, and 81 [Wild type VH CDRs 1,2+VH
CDR3-Y114R], respectively, and a light chain variable region (VL)
comprising CDR1, CDR2, and CDR3 having the sequences as set forth
in SEQ ID NO: 58, the sequence GTN, and the sequence as set forth
in SEQ ID NO: 59, respectively [Wild type VL CDRs 1,2,3],
respectively.
[0377] In certain embodiments, the antigen-binding region capable
of binding to CD3 a heavy chain variable region (VH) comprising
CDR1, CDR2, and CDR3 having the sequences as set forth in SEQ ID
NOs: 54, 55, and 67 [Wild type VH CDRs 1,2+VH CDR3-H101G],
respectively, and a light chain variable region (VL) comprising
CDR1, CDR2, and CDR3 having the sequences as set forth in SEQ ID
NO: 58, the sequence GTN, and the sequence as set forth in SEQ ID
NO: 59, respectively [Wild type VL CDRs 1,2,3], respectively.
[0378] Further, the present invention provides an antibody as
defined above, wherein [0379] the antigen-binding region capable of
binding to 5T4 comprises a heavy chain variable region (VH)
comprising CDR1, CDR2, and CDR3 sequences of SEQ ID NOs.: 6, 7 and
8, respectively, and a light chain variable region (VL) comprising
the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 10, AAS and SEQ ID
NO: 11, respectively [059]; [0380] and [0381] the antigen-binding
region capable of binding to CD3 comprises a heavy chain variable
region (VH) comprising CDR1, CDR2, and CDR3 having the sequences as
set forth in SEQ ID NOs: 54, 55, and 67 [Wild type VH CDRs 1,2+VH
CDR3-H101G], respectively, and a light chain variable region (VL)
comprising CDR1, CDR2, and CDR3 having the sequences as set forth
in SEQ ID NO: 58, the sequence GTN, and the sequence as set forth
in SEQ ID NO: 59, respectively [Wild type VL CDRs 1,2,3],
respectively.
[0382] Also, the invention provides an antibody as defined above,
wherein [0383] the antigen-binding region capable of binding to 5T4
comprises a heavy chain variable region (VH) comprising the CDR1,
CDR2, and CDR3 sequences of SEQ ID NOs.: 41, 42 and 43,
respectively, and a light chain variable region (VL) comprising the
CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 45, DAS and 46,
respectively [207]; [0384] and [0385] the antigen-binding region
capable of binding to CD3 comprises a heavy chain variable region
(VH) comprising CDR1, CDR2, and CDR3 having the sequences as set
forth in SEQ ID NOs: 54, 55, and 67 [Wild type VH CDRs 1,2+VH
CDR3-H101G], respectively, and a light chain variable region (VL)
comprising CDR1, CDR2, and CDR3 having the sequences as set forth
in SEQ ID NO: 58, the sequence GTN, and the sequence as set forth
in SEQ ID NO: 59, respectively [Wild type VL CDRs 1,2,3],
respectively.
[0386] Further, the invention provides an antibody as defined
above, wherein [0387] the antigen-binding region capable of binding
to 5T4 comprises a heavy chain variable region (VH) comprising the
CDR1, CDR2, and CDR3 sequences of SEQ ID NOs.: 48, 49 and 50,
respectively, and a light chain variable region (VL) comprising the
CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 52, DAS and 53,
respectively [226]; [0388] and [0389] the antigen-binding region
capable of binding to CD3 comprises a heavy chain variable region
(VH) comprising CDR1, CDR2, and CDR3 having the sequences as set
forth in SEQ ID NOs: 54, 55, and 67 [Wild type VH CDRs 1,2+VH
CDR3-H101G], respectively, and a light chain variable region (VL)
comprising CDR1, CDR2, and CDR3 having the sequences as set forth
in SEQ ID NO: 58, the sequence GTN, and the sequence as set forth
in SEQ ID NO: 59, respectively [Wild type VL CDRs 1,2,3],
respectively.
[0390] In the antibody according to the invention, the
antigen-binding region capable of binding to human CD3 may comprise
a VH sequence and a VL sequence selected from the group consisting
of: [0391] a) a VH sequence as set forth in SEQ ID NO: 62 [VH T31P]
and a VL sequence as set forth in SEQ ID NO: 60, [0392] b) a VH
sequence as set forth in SEQ ID NO: 64 [VH T31M] and a VL sequence
as set forth in SEQ ID NO: 60, [0393] c) a VH sequence as set forth
in SEQ ID NO: 66 [VH N57E] and a VL sequence as set forth in SEQ ID
NO: 60, [0394] d) a VH sequence as set forth in SEQ ID NO: 68 [VH
H101G] and a VL sequence as set forth in SEQ ID NO: 60, [0395] e) a
VH sequence as set forth in SEQ ID NO: 70 [VH H101N] and a VL
sequence as set forth in SEQ ID NO: 60, [0396] f) a VH sequence as
set forth in SEQ ID NO: 72 [VH G105P] and a VL sequence as set
forth in SEQ ID NO: 60, [0397] g) a VH sequence as set forth in SEQ
ID NO: 74 [VH S110A] and a VL sequence as set forth in SEQ ID NO:
60, [0398] h) a VH sequence as set forth in SEQ ID NO: 76 [VH
S110G] and a VL sequence as set forth in SEQ ID NO: 60, [0399] i) a
VH sequence as set forth in SEQ ID NO: 78 [VH Y114V] and a VL
sequence as set forth in SEQ ID NO: 60, [0400] j) a VH sequence as
set forth in SEQ ID NO: 80 [VH Y114M] and a VL sequence as set
forth in SEQ ID NO: 60; and [0401] k) a VH sequence as set forth in
SEQ ID NO: 82 [VH Y114R] and a VL sequence as set forth in SEQ ID
NO: 60.
[0402] In particular, the antibody according to the invention may
be an antibody, wherein the antigen-binding region capable of
binding to human CD3 comprises a VH sequence as set forth in SEQ ID
NO: 68 [VH H101G] and a VL sequence as set forth in SEQ ID NO:
60.
[0403] In some embodiments, the antibody according to the invention
is one, wherein [0404] the antigen-binding region capable of
binding to 5T4 comprises a heavy chain variable region (VH)
comprising the sequence of SEQ ID NO: 5 or a sequence having at
least 90%, at least 95%, at least 97%, or at least 99% amino acid
sequence identity to the sequence of SEQ ID NO: 5 [059]; [0405] and
[0406] the antigen-binding region capable of binding to human CD3
comprises a VH sequence as set forth in SEQ ID NO: 68 [VH H101G]
and a VL sequence as set forth in SEQ ID NO: 60.
[0407] In other embodiments, the antibody according to the
invention is one, wherein [0408] the antigen-binding region capable
of binding to 5T4 comprises a heavy chain variable region (VH)
comprising the sequence of SEQ ID NO: 40 or a sequence having at
least 90%, at least 95%, at least 97%, or at least 99% amino acid
sequence identity to the sequence of SEQ ID NO: 40 [207]; [0409]
and [0410] the antigen-binding region capable of binding to human
CD3 comprises a VH sequence as set forth in SEQ ID NO: 68 [VH
H101G] and a VL sequence as set forth in SEQ ID NO: 60.
[0411] In still other embodiments, the antibody according to the
invention is one, wherein [0412] the antigen-binding region capable
of binding to 5T4 comprises a heavy chain variable region (VH)
comprising the sequence of SEQ ID NO: 47 or a sequence having at
least 90%, at least 95%, at least 97%, or at least 99% amino acid
sequence identity to the sequence of SEQ ID NO: 47 [226]; [0413]
and [0414] the antigen-binding region capable of binding to human
CD3 comprises a VH sequence as set forth in SEQ ID NO: 68 [VH
H101G] and a VL sequence as set forth in SEQ ID NO: 60.
[0415] As will be well-known to the skilled person, each
antigen-binding region of an antibody generally comprises a heavy
chain variable region (VH) and a light chain variable region (VL),
and each of the variable regions comprises three CDR sequences,
CDR1, CDR2 and CDR3, respectively, and four framework sequences,
FR1, FR2, FR3 and FR4, respectively. This structure may also be
found in the antibodies according to the present invention.
Further, the antibodies according to the invention may comprise two
heavy chain constant regions (CH), and two light chain constant
regions (CL).
[0416] In particular embodiments, the antibody according to the
invention comprises a first and a second heavy chain, such as a
first and second heavy chain each comprising at least a hinge
region, a CH2 and CH3 region. Stable, heterodimeric antibodies can
be obtained at high yield for instance by so-called Fab-arm
exchange as provided in WO 2008/119353 and WO 2011/131746, on the
basis of two homodimeric starting proteins containing only a few,
asymmetrical mutations in the CH3 regions. Hence, in some
embodiments of the invention, the antibody a first heavy chain
wherein at least one of the amino acids at the positions
corresponding to positions selected from the group consisting of
T366, L368, K370, D399, F405, Y407 and K409 in a human IgG1 heavy
chain has been substituted, and a second heavy chain wherein at
least one of the amino acids in the positions corresponding to a
position selected from the group consisting of T366, L368, K370,
D399, F405, Y407, and K409 in a human IgG1 heavy chain has been
substituted, wherein said substitutions of said first and said
second heavy chains are not in the same positions, and wherein the
amino acid positions are numbered according to EU numbering.
[0417] In particular embodiments, the invention provides an
antibody, wherein the amino acid in the position corresponding to
K409 in a human IgG1 heavy chain is R in said first heavy chain,
and the amino acid in the position corresponding to F405 in a human
IgG1 heavy chain is L in said second heavy chain, or vice
versa.
[0418] In some embodiments, the antibody according to the present
invention comprises, in addition to the antigen-binding regions, an
Fc region consisting of the Fc sequences of the two heavy chains.
The first and second Fc sequence may each be of any isotype,
including any human isotype, such as an IgG1, IgG2, IgG3, IgG4,
IgE, IgD, IgM, or IgA isotype or a mixed isotype. Preferably, the
Fc region is a human IgG1, IgG2, IgG3, IgG4 isotype or a mixed
isotype, such as a human IgG1 isotype.
[0419] Antibodies according to the present invention may comprise
modifications in the Fc region to render the antibody an inert, or
non-activating, antibody. Hence, in the antibodies disclosed
herein, one or both heavy chains may be modified so that the
antibody induces Fc-mediated effector function to a lesser extent
relative to an antibody which is identical, except for comprising
non-modified first and second heavy chains. The Fc-mediated
effector function may be measured by determining Fc-mediated CD69
expression on T cells (i.e. CD69 expression as a result of CD3
antibody-mediated, Fc.gamma. receptor-dependent CD3 crosslinking),
by binding to Fc.gamma. receptors, by binding to C1q, or by
induction of Fc-mediated cross-linking of Fc.gamma.Rs. In
particular, the heavy chain constant sequences may be modified so
that the Fc-mediated CD69 expression is reduced by at least 50%, at
least 60%, at least 70%, at least 80%, at least 90%, at least 99%
or 100% when compared to a wild-type (unmodified) antibody, wherein
said Fc-mediated CD69 expression is determined in a PBMC-based
functional assay, e.g. as described in Example 3 of WO2015001085.
Modifications of the heavy and light chain constant sequences may
also result in reduced binding of C1q to said antibody. As compared
to an unmodified antibody the reduction may be by at least 70%, at
least 80%, at least 90%, at least 95%, at least 97%, or 100% and
the C1q binding may be determined by ELISA. Further, the Fc region
which may be modified so that said antibody mediates reduced
Fc-mediated T-cell proliferation compared to an unmodified antibody
by at least 50%, at least 60%, at least 70%, at least 80%, at least
90%, at least 99% or 100%, wherein said T-cell proliferation is
measured in a PBMC-based functional assay.
[0420] Examples of amino acid positions that may be modified, e.g.
in an IgG1 isotype antibody, include positions L234 and L235.
Hence, the antibody according to the invention may comprises a
first and a second heavy chain, and wherein in both the first and
the second heavy chain, the amino acid residues at the positions
corresponding to positions L234 and L235 in a human IgG1 heavy
chain according to EU numbering are F and E, respectively.
[0421] In addition, a D265A amino acid substitution can decrease
binding to all Fc.gamma. receptors and prevent ADCC (Shields et
al., 2001, J. Biol. Chem. (276):6591-604). Therefore, the antibody
according to the invention may comprise a first and a second heavy
chain, wherein in both the first and the second heavy chain, the
amino acid residue at the position corresponding to position D265
in a human IgG1 heavy chain according to EU numbering is A. Further
embodiments of the invention provide antibodies wherein, in at
least one, such as in both, of said first and second heavy chains
the amino acids in the positions corresponding to positions L234,
L235, and D265 in a human IgG1 heavy chain, are F, E, and A,
respectively. In the present application antibodies, which have the
combination of three amino acid substitutions L234F, L235E and
D265A and in addition the K409R or the F405L mutation disclosed
herein above are termed with the suffix "FEAR" or "FEAL",
respectively.
[0422] The amino acid sequence of the wild type IgG1 heavy chain
constant region is identified herein as SEQ ID NO: 89. Consistent
with the embodiments disclosed above, the antibody of the invention
may comprise an IgG1 heavy chain constant region carrying the F405L
substitution and having the amino acid sequence set forth in SEQ ID
NO: 90 and/or an IgG1 heavy chain constant region carrying the
K409R substitution and having the amino acid sequence set forth in
SEQ ID NO: 94.
[0423] The amino acid sequence of an IgG1 heavy chain constant
region carrying the L234F, L235E and D265A substitutions is
identified herein as SEQ ID NO: 91. The amino acid sequence of an
IgG1 heavy chain constant region carrying the L234F, L235E, D265A
and F405L substitutions is identified herein as SEQ ID NO: 92. The
amino acid sequence of an IgG1 heavy chain constant region carrying
the L234F, L235E, D265A and K409R substitutions is identified
herein as SEQ ID NO: 93.
[0424] The present invention further provides an antibody, wherein
[0425] a) the antigen-binding region(s) capable of binding to 5T4
is/are humanized, and/or [0426] b) the antigen-binding region
capable of binding to CD3, if present, is humanized.
[0427] Also, the invention provides an antibody, wherein [0428] a)
the antigen-binding region(s) capable of binding to 5T4 is/are
human, and/or [0429] b) the antigen-binding region capable of
binding to CD3, if present, is human.
[0430] Further, the invention provides an antibody, wherein [0431]
a) the antigen-binding region(s) capable of binding to 5T4 is/are
chimeric, and/or [0432] b) the antigen-binding region capable of
binding to CD3, if present, is chimeric.
[0433] In some embodiments of the invention, the antibody comprises
a kappa (.kappa.) light chain. The sequence of in particular
embodiments of the invention concerning bispecific antibodies, the
kappa light chain comprises the CDR1, -2 and -3 sequences of a 5T4
antibody light chain as disclosed above.
[0434] In further embodiments of the invention, the antibody
according to any one of the preceding claims, wherein said antibody
comprises a lambda (A) light chain. In particular embodiments of
the invention concerning bispecific antibodies, the lambda light
chain comprises the CDR1, -2 and -3 sequences of a CD3 antibody
light chain as disclosed above, in particular a the CDR1, -2 and -3
sequences of a CD3 antibody having reduced affinity for CD3 as
disclosed above. The amino acid sequence of a kappa light chain
constant region is included herein as SEQ ID NO: 95 and the amino
acid sequence of a lambda light chain constant region is included
herein as SEQ ID NO: 96.
[0435] In particular embodiments, the antibody comprises a lambda
(.lamda.) light chain and a kappa (.kappa.) light chain; e.g. an
antibody with a heavy chain and a lambda light chain which comprise
the binding region capable of binding to CD3, and a heavy chain and
a kappa light chain which comprise the binding region capable of
binding to 5T4.
Immunoconjugates
[0436] In another aspect, the invention provides an immunoconjugate
or antibody-drug conjugate (ADC) comprising the antibody defined
above, and a therapeutic moiety, such as a cytotoxic agent, a
chemotherapeutic drug, a cytokine, an immunosuppressant,
antibiotic, or a radioisotope. In general, the skilled person will
have at his disposition numerous cytotoxic agents, chemotherapeutic
drugs, cytokines, immunosuppressants, antibiotics and
radioisotopes, the optimal choice of therapeutic moiety depending
on the desired application of the immunoconjugate. For certain
applications the preferred cytotoxic agent may be a
microtubule-disrupting agent, such as a duostatin, e.g.
Duostatin-3.
Nucleic Acid Constructs
[0437] A further aspect of the invention provides nucleic acid
construct comprising [0438] a) a nucleic acid sequence encoding a
heavy chain sequence of an antibody comprising an antigen-binding
region capable of binding to 5T4 as defined herein before, and/or
[0439] b) a nucleic acid sequence encoding a light chain sequence
of an antibody comprising an antigen-binding region capable of
binding to 5T4 as defined herein before.
[0440] The nucleic acid construct may further comprise [0441] a) a
nucleic acid sequence encoding a heavy chain sequence of an
antibody comprising an antigen-binding region capable of binding to
CD3 as defined herein before; and/or [0442] b) a nucleic acid
sequence encoding a light chain sequence of an antibody comprising
an antigen-binding region capable of binding to CD3 as defined
herein before.
Expression Vectors
[0443] Another aspect of the invention provides an expression
vector comprising nucleic acid sequences encoding heavy and/or
light chain sequences of an antibody according to the invention. In
particular, the expression vector may comprise: [0444] a) a nucleic
acid sequence encoding a heavy chain sequence of an antibody
comprising an antigen-binding region capable of binding to 5T4 as
defined herein before, and/or [0445] b) a nucleic acid sequence
encoding a light chain sequence of an antibody comprising an
antigen-binding region capable of binding to 5T4 as defined herein
before.
[0446] The expression vector may further comprise: [0447] a) a
nucleic acid sequence encoding a heavy chain sequence of an
antibody comprising an antigen-binding region capable of binding to
CD3 as defined herein before; and/or [0448] b) a nucleic acid
sequence encoding a light chain sequence of an antibody comprising
an antigen-binding region capable of binding to CD3 as defined
herein before.
[0449] In a further embodiment, the expression vector further
comprises a nucleic acid sequence encoding the constant region of a
light chain, a heavy chain or both light and heavy chains of an
antibody, e.g. a human IgG1,.kappa. monoclonal antibody.
[0450] An expression vector in the context of the present invention
may be any suitable vector, including chromosomal, non-chromosomal,
and synthetic nucleic acid vectors (a nucleic acid sequence
comprising a suitable set of expression control elements). Examples
of such vectors include derivatives of SV40, bacterial plasmids,
phage DNA, baculovirus, yeast plasmids, vectors derived from
combinations of plasmids and phage DNA, and viral nucleic acid (RNA
or DNA) vectors. In one embodiment, an anti-5T4 antibody-encoding
nucleic acid is comprised in a naked DNA or RNA vector, including,
for example, a linear expression element (as described in for
instance Sykes and Johnston, Nat Biotech 17, 355-59 (1997)), a
compacted nucleic acid vector (as described in for instance U.S.
Pat. No. 6,077,835 and/or WO 00/70087), a plasmid vector such as
pBR322, pUC 19/18, or pUC 118/119, a "midge" minimally-sized
nucleic acid vector (as described in for instance Schakowski et
al., Mol Ther 3, 793-800 (2001)), or as a precipitated nucleic acid
vector construct, such as a CaP04-precipitated construct (as
described in for instance WO 00/46147, Benvenisty and Reshef, PNAS
USA 83, 9551-55 (1986), Wigler et al., Cell 14, 725 (1978), and
Coraro and Pearson, Somatic Cell Genetics 7, 603 (1981)). Such
nucleic acid vectors and the usage thereof are well known in the
art (see for instance U.S. Pat. Nos. 5,589,466 and 5,973,972).
[0451] In one embodiment, the vector is suitable for expression of
the anti-5T4 antibody in a bacterial cell. Examples of such vectors
include expression vectors such as BlueScript (Stratagene), pIN
vectors Van Heeke & Schuster, J Biol Chem 264, 5503 5509
(1989), pET vectors (Novagen, Madison Wis.) and the like).
[0452] An expression vector may also or alternatively be a vector
suitable for expression in a yeast system. Any vector suitable for
expression in a yeast system may be employed. Suitable vectors
include, for example, vectors comprising constitutive or inducible
promoters such as alpha factor, alcohol oxidase and PGH (reviewed
in: F. Ausubel et al., ed. Current Protocols in Molecular Biology,
Greene Publishing and Wiley InterScience New York (1987), and Grant
et al., Methods in Enzymol 153, 516 544 (1987)).
[0453] A nucleic acid construct and/or vector may also comprises a
nucleic acid sequence encoding a secretion/localization sequence,
which can target a polypeptide, such as a nascent polypeptide
chain, to the periplasmic space or into cell culture media. Such
sequences are known in the art, and include secretion leader or
signal peptides, organelle targeting sequences (e. g., nuclear
localization sequences, ER retention signals, mitochondrial transit
sequences, chloroplast transit sequences), membrane
localization/anchor sequences (e. g., stop transfer sequences, GPI
anchor sequences), and the like.
[0454] In an expression vector of the invention, anti-5T4
antibody-encoding nucleic acids may comprise or be associated with
any suitable promoter, enhancer, and other expression-facilitating
elements. Examples of such elements include strong expression
promoters (e.g., human CMV IE promoter/enhancer as well as RSV,
SV40, SL3-3, MMTV, and HIV LTR promoters), effective poly (A)
termination sequences, an origin of replication for plasmid product
in E. coli, an antibiotic resistance gene as selectable marker,
and/or a convenient cloning site (e.g., a polylinker). Nucleic
acids may also comprise an inducible promoter as opposed to a
constitutive promoter such as CMV IE (the skilled artisan will
recognize that such terms are actually descriptors of a degree of
gene expression under certain conditions).
[0455] In one embodiment, the anti-5T4-antibody-encoding expression
vector may be positioned in and/or delivered to a host cell or host
animal via a viral vector.
Cells and Host Cells
[0456] In a further aspect, the invention provides a cell
comprising a nucleic acid construct as defined herein above, or an
expression vector as defined herein above. It is to be understood
that the cell may have been obtained by transfecting a host cell
with said nucleic acid construct or expression vector, such as a
recombinant host cell.
[0457] The host cell may be of human origin, such as a human
embryonic kidney (HEK) cell, such as a HEK/Expi cell.
Alternatively, it may be of rodent origin, such as a Chinese
hamster ovary cell, such as a CHO/N50 cell. Further, the host cell
may be of bacterial origin.
[0458] The cell may comprise a nucleic acid sequence encoding an
antibody of the invention or parts thereof stably integrated into
the cellular genome. Alternatively, the cell may comprise a
non-integrated nucleic acid, such as a plasmid, cosmid, phagemid,
or linear expression element, which comprises a sequence coding for
expression of an anti-5T4 antibody of the invention or a part
thereof. In particular, the host cell may comprise a non-integrated
nucleic acid, such as a plasmid, cosmid, phagemid, or linear
expression element, which comprises a sequence coding for
expression of an anti-5T4 antibody or a part thereof.
Compositions
[0459] A still further aspect of the invention provides a
composition comprising an antibody; e.g. a bispecific antibody or
an immunoconjugate as defined in the above. The composition may be
a pharmaceutical composition comprising the antibody, bispecific
antibody or immunoconjugate and a pharmaceutically acceptable
carrier.
[0460] The pharmaceutical compositions may be formulated with the
carrier, excipient and/or diluent as well as any other components
suitable for pharmaceutical compositions, including known
adjuvants, in accordance with conventional techniques such as those
disclosed in Remington: The Science and Practice of Pharmacy, 19th
Edition, Gennaro, Ed., Mack Publishing Co., Easton, Pa., 1995. The
pharmaceutically acceptable carriers or diluents as well as any
known adjuvants and excipients should be suitable for the antibody
or antibody conjugate of the present invention and the chosen mode
of administration. Suitability for carriers and other components of
pharmaceutical compositions is determined based on the lack of
significant negative impact on the desired biological properties of
the chosen compound or pharmaceutical composition of the present
invention (e.g., less than a substantial impact [10% or less
relative inhibition, 5% or less relative inhibition, etc.] upon
antigen binding).
[0461] A pharmaceutical composition of the present invention may
include diluents, fillers, salts, buffers, detergents (e. g., a
nonionic detergent, such as Tween-20 or Tween-80), stabilizers
(e.g., sugars or protein-free amino acids), preservatives, tissue
fixatives, solubilizers, and/or other materials suitable for
inclusion in a pharmaceutical composition.
[0462] The actual dosage levels of the active ingredients in the
pharmaceutical compositions of the present invention may be varied
so as to obtain an amount of the active ingredient which is
effective to achieve the desired therapeutic response for a
particular patient, composition, and mode of administration,
without being toxic to the patient. The selected dosage level will
depend upon a variety of pharmacokinetic factors including the
activity of the particular compositions of the present invention
employed, or the amide thereof, the route of administration, the
time of administration, the rate of excretion of the particular
compound being employed, the duration of the treatment, other
drugs, compounds and/or materials used in combination with the
particular compositions employed, the age, sex, weight, condition,
general health and prior medical history of the patient being
treated, and like factors well known in the medical arts.
[0463] Pharmaceutically acceptable carriers include any and all
suitable solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonicity agents, antioxidants and
absorption-delaying agents, and the like that are physiologically
compatible with a compound of the present invention.
[0464] Examples of suitable aqueous and non-aqueous carriers which
may be employed in the pharmaceutical compositions of the present
invention include water, saline, phosphate buffered saline,
ethanol, dextrose, polyols (such as glycerol, propylene glycol,
polyethylene glycol, and the like), and suitable mixtures thereof,
vegetable oils, such as olive oil, corn oil, peanut oil, cottonseed
oil, and sesame oil, carboxymethyl cellulose colloidal solutions,
tragacanth gum and injectable organic esters, such as ethyl oleate,
and/or various buffers. Other carriers are well known in the
pharmaceutical arts.
[0465] Pharmaceutically acceptable carriers include sterile aqueous
solutions or dispersions and sterile powders for the extemporaneous
preparation of sterile injectable solutions or dispersion. The use
of such media and agents for pharmaceutically active substances is
known in the art. Except insofar as any conventional media or agent
is incompatible with the active compound, use thereof in the
pharmaceutical compositions of the present invention is
contemplated.
[0466] Pharmaceutical compositions of the present invention may
also comprise pharmaceutically acceptable antioxidants for instance
(1) water-soluble antioxidants, such as ascorbic acid, cysteine
hydrochloride, sodium bisulfate, sodium metabisulfite, sodium
sulfite and the like; (2) oil-soluble antioxidants, such as
ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated
hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol,
and the like; and (3) metal-chelating agents, such as citric acid,
ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid,
phosphoric acid, and the like.
[0467] Pharmaceutical compositions of the present invention may
also comprise isotonicity agents, such as sugars, polyalcohols,
such as mannitol, sorbitol, glycerol or sodium chloride in the
compositions.
[0468] The pharmaceutical compositions of the present invention may
also contain one or more adjuvants appropriate for the chosen route
of administration such as preservatives, wetting agents,
emulsifying agents, dispersing agents, preservatives or buffers,
which may enhance the shelf life or effectiveness of the
pharmaceutical composition. The compounds of the present invention
may be prepared with carriers that will protect the compound
against rapid release, such as a controlled release formulation,
including implants, transdermal patches, and micro-encapsulated
delivery systems. Such carriers may include gelatin, glyceryl
monostearate, glyceryl distearate, biodegradable, biocompatible
polymers such as ethylene vinyl acetate, polyanhydrides,
polyglycolic acid, collagen, poly-ortho esters, and polylactic acid
alone or with a wax, or other materials well known in the art.
Methods for the preparation of such formulations are generally
known to those skilled in the art, see e.g. Sustained and
Controlled Release Drug Delivery Systems, J. R. Robinson, ed.,
Marcel Dekker, Inc., New York, 1978.
[0469] In one embodiment, the compounds of the present invention
may be formulated to ensure proper distribution in vivo.
Pharmaceutically acceptable carriers for parenteral administration
include sterile aqueous solutions or dispersions and sterile
powders for the extemporaneous preparation of sterile injectable
solutions or dispersion. The use of such media and agents for
pharmaceutically active substances is known in the art. Except in
so far as any conventional media or agent is incompatible with the
active compound, use thereof in the pharmaceutical compositions of
the present invention is contemplated. Other active or therapeutic
compounds may also be incorporated into the compositions.
[0470] Pharmaceutical compositions for injection must typically be
sterile and stable under the conditions of manufacture and storage.
The composition may be formulated as a solution, micro-emulsion,
liposome, or other ordered structure suitable to high drug
concentration. The carrier may be an aqueous or a non-aqueous
solvent or dispersion medium containing for instance water,
ethanol, polyols (such as glycerol, propylene glycol, polyethylene
glycol, and the like), and suitable mixtures thereof, vegetable
oils, such as olive oil, and injectable organic esters, such as
ethyl oleate. The proper fluidity may be maintained, for example,
by the use of a coating such as lecithin, by the maintenance of the
required particle size in the case of dispersion and by the use of
surfactants. In many cases, it will be preferable to include
isotonic agents, for example, sugars, polyalcohols such as
glycerol, mannitol, sorbitol, or sodium chloride in the
composition. Prolonged absorption of the injectable compositions
may be brought about by including in the composition an agent that
delays absorption, for example, monostearate salts and gelatin.
Sterile injectable solutions may be prepared by incorporating the
active compound in the required amount in an appropriate solvent
with one or a combination of ingredients e.g. as enumerated above,
as required, followed by sterilization microfiltration. Generally,
dispersions are prepared by incorporating the active compound into
a sterile vehicle that contains a basic dispersion medium and the
required other ingredients e.g. from those enumerated above. In the
case of sterile powders for the preparation of sterile injectable
solutions, examples of methods of preparation are vacuum drying and
freeze-drying (lyophilization) that yield a powder of the active
ingredient plus any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0471] Sterile injectable solutions may be prepared by
incorporating the active compound in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by sterilization
microfiltration. Generally, dispersions are prepared by
incorporating the active compound into a sterile vehicle that
contains a basic dispersion medium and the required other
ingredients from those enumerated above. In the case of sterile
powders for the preparation of sterile injectable solutions,
examples of methods of preparation are vacuum-drying and
freeze-drying (lyophilization) that yield a powder of the active
ingredient plus any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0472] The pharmaceutical composition of the present invention may
contain one antibody, bispecific antibody or antibody-drug
conjugate (ADC) of the present invention, a combination of an
antibody, a bispecific antibody or ADC according to the invention
with another therapeutic compound, or a combination of compounds of
the present invention.
[0473] The pharmaceutical composition may be administered by any
suitable route and mode. Suitable routes of administering a
compound of the present invention in vivo and in vitro are well
known in the art and may be selected by those of ordinary skill in
the art.
[0474] In one embodiment, the pharmaceutical composition of the
present invention is administered parenterally; i.e. by a mode of
administration other than enteral and topical administration;
usually by injection, and include epidermal, intravenous,
intramuscular, intra-arterial, intrathecal, intracapsular,
intra-orbital, intracardiac, intradermal, intraperitoneal,
intratendinous, transtracheal, subcutaneous, subcuticular,
intra-articular, subcapsular, subarachnoid, intraspinal,
intracranial, intrathoracic, epidural and intrasternal injection
and infusion. In particular, the pharmaceutical composition of the
present invention may be administered by intravenous or
subcutaneous injection or infusion.
Uses and Therapeutical Applications
[0475] The present invention further provides an antibody, such as
a bispecific antibody, or an immunoconjugate or antibody-drug
conjugate (ADC) as defined herein for use as a medicament. The
anti-5T4 antibodies or immunoconjugates of the present invention
can be used in the treatment or prevention of a disease or disorder
involving cells expressing 5T4. In particular, the bispecific
antibodies according to the invention; i.e. antibodies which
comprise antigen binding regions capable of binding 5T4 and CD3 may
be useful in therapeutic settings in which specific targeting and T
cell-mediated killing of cells that express 5T4 is desired, and
they may be more efficient compared to a regular anti-5T4 antibody
in certain such indications and settings.
[0476] In one embodiment, the antibody, such as the bispecific
antibody, or immunoconjugate or antibody-drug conjugate (ADC) of
the present invention is disclosed herein for use in the treatment
of cancer. The antibody, such as the bispecific antibody, or the
immunoconjugate or antibody-drug conjugate (ADC) may in particular
be use in treatment of a cancer, wherein the cancer is
characterized by expression of 5T4 in at least some of the tumor
cells.
[0477] The cancer may in particular be selected from the group
consisting of kidney/renal cancer, breast cancer, colorectal
cancer, prostate cancer, ovarian cancer, bladder cancer,
uterine/endometrial/cervical cancer, lung cancer, gastro-intestinal
cancer, stomach cancer, pancreatic cancer, thyroid cancer, head and
neck cancer, lymphoma, acute myeloid leukemia.
[0478] Additionally, the invention relates to the use of an
antibody according to the invention for the manufacture of a
medicament, such as a medicament for the treatment of cancer, e.g.
a cancer selected from the group consisting of kidney/renal cancer,
breast cancer, colorectal cancer, prostate cancer, ovarian cancer,
bladder cancer, uterine/endometrial/cervical cancer, lung cancer,
gastro-intestinal cancer, stomach cancer, pancreatic cancer,
thyroid cancer, head and neck cancer, lymphoma, acute myeloid
leukemia.
[0479] In a further aspect, the invention provides method of
treating a disease, the method comprising administering an
antibody, an immunoconjugate, a composition, such as a
pharmaceutical composition or antibody-drug conjugate (ADC)
according to the invention to a subject in need thereof.
[0480] In particular embodiments of the invention, said method is
for treatment of a cancer. The method of the invention may in
particular comprise the steps of: [0481] a) selecting a subject
suffering from a cancer comprising tumor cells expressing 5T4
and/or a cancer known to express 5T4; and [0482] b) administering
to the subject the antibody, such as the bispecific antibody, the
pharmaceutical composition or the antibody-drug conjugate (ADC) of
the present invention.
[0483] The cancer may in particular be selected from the group
consisting of kidney/renal cancer, breast cancer, colorectal
cancer, prostate cancer, ovarian cancer, bladder cancer,
uterine/endometrial/cervical cancer, lung cancer, gastro-intestinal
cancer, stomach cancer, pancreatic cancer, thyroid cancer, head and
neck cancer, lymphoma, acute myeloid leukemia.
[0484] Dosage regimens in the above methods of treatment and uses
are adjusted to provide the optimum desired response (e.g., a
therapeutic response). For example, a single bolus may be
administered, several divided doses may be administered over time
or the dose may be proportionally reduced or increased as indicated
by the exigencies of the therapeutic situation. Parenteral
compositions may be formulated in dosage unit form for ease of
administration and uniformity of dosage.
[0485] The efficient dosages and the dosage regimens for the
antibodies depend on the disease or condition to be treated and may
be determined by the persons skilled in the art. An exemplary,
non-limiting range for a therapeutically effective amount of a
compound of the present invention is about 0.001-10 mg/kg, such as
about 0.001-5 mg/kg, for example about 0.001-2 mg/kg, such as about
0.001-1 mg/kg, for instance about 0.001, about 0.01, about 0.1,
about 1 or about 10 mg/kg. Another exemplary, non-limiting range
for a therapeutically effective amount of an antibody of the
present invention is about 0.1-100 mg/kg, such as about 0.1-50
mg/kg, for example about 0.1-20 mg/kg, such as about 0.1-10 mg/kg,
for instance about 0.5, about such as 0.3, about 1, about 3, about
5, or about 8 mg/kg.
[0486] A physician having ordinary skill in the art may readily
determine and prescribe the effective amount of the pharmaceutical
composition required. For example, the physician or veterinarian
could start doses of the antibody employed in the pharmaceutical
composition at levels lower than that required to achieve the
desired therapeutic effect and gradually increase the dosage until
the desired effect is achieved. In general, a suitable daily dose
of an antibody of the present invention will be that amount of the
compound which is the lowest dose effective to produce a
therapeutic effect. Administration may e.g. be parenteral, such as
intravenous, intramuscular or subcutaneous. In one embodiment, the
antibodies may be administered by infusion in a weekly dosage of
calculated by mg/m2. Such dosages can, for example, be based on the
mg/kg dosages provided above according to the following: dose
(mg/kg).times.70:1.8. Such administration may be repeated, e.g., 1
to 8 times, such as 3 to 5 times. The administration may be
performed by continuous infusion over a period of from 2 to 24
hours, such as from 2 to 12 hours. In one embodiment, the
antibodies may be administered by slow continuous infusion over a
long period, such as more than 24 hours, to reduce toxic side
effects.
[0487] In one embodiment, the antibodies may be administered in a
weekly dosage of calculated as a fixed dose for up to 8 times, such
as from 4 to 6 times when given once a week. Such regimen may be
repeated one or more times as necessary, for example, after 6
months or 12 months. Such fixed dosages can, for example, be based
on the mg/kg dosages provided above, with a body weight estimate of
70 kg. The dosage may be determined or adjusted by measuring the
amount of antibody of the present invention in the blood upon
administration by for instance taking out a biological sample and
using anti-idiotypic antibodies which target the 5T4 antigen
antigen-binding region of the antibodies of the present
invention.
[0488] In one embodiment, the antibodies may be administered as
maintenance therapy, such as, e.g., once a week for a period of 6
months or more.
[0489] An antibody may also be administered prophylactically to
reduce the risk of developing cancer, delay the onset of the
occurrence of an event in cancer progression, and/or reduce the
risk of recurrence when a cancer is in remission.
[0490] The antibodies of the invention may also be administered in
combination therapy, i.e., combined with other therapeutic agents
relevant for the disease or condition to be treated. Accordingly,
in one embodiment, the antibody-containing medicament is for
combination with one or more further therapeutic agents, such as a
cytotoxic, chemotherapeutic or anti-angiogenic agent.
Antibody Production
[0491] Also provided herein is a method for producing the antibody,
such as the bispecific antibody of the invention. In particular,
there is provided a method for producing the antibody of the
invention, comprising the steps of [0492] a) culturing a host cell
comprising an expression vector as defined herein; and [0493] b)
and purifying said antibody from the culture medium.
[0494] In embodiments of the invention, wherein the antibody
comprises a binding region capable of binding to 5T4 and a binding
region capable of binding to CD3, the antibody may be produced
using a method comprising the steps of [0495] a) Providing an
antibody capable of binding to 5T4 by culturing a host cell
comprising an expression vector as defined herein under conditions
allowing expression of the antibody capable of binding to 5T4, and
purifying the antibody capable of binding to 5T4 from the culture
medium; [0496] b) Providing an antibody capable of binding to CD3
by culturing a host cell comprising an expression vector comprising
[0497] I) a nucleic acid sequence encoding a heavy chain sequence
of an antibody comprising an antigen-binding region capable of
binding to CD3 as defined herein above; and [0498] II) a nucleic
acid sequence encoding a light chain sequence of an antibody
comprising an antigen-binding region capable of binding to CD3 as
defined herein above; [0499] under conditions allowing expression
of the antibody capable of binding to CD3, and purifying the
antibody capable of binding to CD3 from the culture medium; [0500]
c) incubating said antibody capable of binding to 5T4 together with
said antibody capable of binding to CD3 under reducing conditions
sufficient to allow cysteines in the hinge region to undergo
disulfide-bond isomerization, and [0501] d) obtaining said
antibody.
Kits
[0502] The invention further provides a kit-of-parts comprising an
antibody as disclosed above, such as a kit for use as a companion
diagnostic/for identifying within a population of patients, those
patients which have a propensity to respond to treatment with an
antibody as defined herein above or an immunoconjugate or
antibody-drug conjugate (ADC) as defined herein above, or for
predicting efficacy or anti-tumor activity of said antibody or
immunoconjugate or ADC when used in treatment of a patient, the kit
comprising an antibody as defined above; and instructions for use
of said kit.
Anti-Idiotypic Antibodies
[0503] In a further aspect, the invention relates to an
anti-idiotypic antibody which binds to an antibody comprising at
least one antigen-binding region capable of binding to 5T4, i.e. an
antibody according to the invention as described herein. In
particular embodiments, the anti-idiotypic antibody binds to the
antigen-binding region capable of binding to 5T4.
[0504] An anti-idiotypic (Id) antibody is an antibody which
recognizes unique determinants generally associated with the
antigen-binding site of an antibody. An anti-Id antibody may be
prepared by immunizing an animal of the same species and genetic
type as the source of an anti-5T4 monoclonal antibody with the
monoclonal antibody against which an anti-Id is being prepared. The
immunized animal typically can recognize and respond to the
idiotypic determinants of the immunizing antibody by producing an
antibody to these idiotypic determinants (the anti-Id antibody).
Such antibodies are described in for instance U.S. Pat. No.
4,699,880. Such antibodies are further features of the present
invention.
[0505] An anti-Id antibody may also be used as an "immunogen" to
induce an immune response in yet another animal, producing a
so-called anti-anti-Id antibody. An anti-anti-Id antibody may be
epitopically identical to the original monoclonal antibody, which
induced the anti-Id antibody. Thus, by using antibodies to the
idiotypic determinants of a monoclonal antibody, it is possible to
identify other clones expressing antibodies of identical
specificity. Anti-Id antibodies may be varied (thereby producing
anti-Id antibody variants) and/or derivatized by any suitable
technique, such as those described elsewhere herein with respect to
5T4-specific antibodies of the present invention. For example, a
monoclonal anti-Id antibody may be coupled to a carrier such as
keyhole limpet hemocyanin (KLH) and used to immunize BALB/c mice.
Sera from these mice typically will contain anti-anti-Id antibodies
that have the binding properties similar, if not identical, to an
original/parental anti-5T4 antibody.
TABLE-US-00003 Sequences SEQ ID NO: Name Domain Sequence 1 Human
5T4 ORF MPGGCSRGPAAGDGRLRLARLALVLLGWVSSSSPTSS
ASSFSSSAPFLASAVSAQPPLPDQCPALCECSEAART
VKCVNRNLTEVPTDLPAYVRNLFLTGNQLAVLPAGAF
ARRPPLAELAALNLSGSRLDEVRAGAFEHLPSLRQLD
LSHNPLADLSPFAFSGSNASVSAPSPLVELILNHIVP
PEDERQNRSFEGMVVAALLAGRALQGLRRLELASNHF
LYLPRDVLAQLPSLRHLDLSNNSLVSLTYVSFRNLTH
LESLHLEDNALKVLHNGTLAELQGLPHIRVFLDNNPW
VCDCHMADMVTWLKETEVVQGKDRLTCAYPEKMRNRV
LLELNSADLDCDPILPPSLQTSYVFLGIVLALIGAIF
LLVLYLNRKGIKKWMHNIRDACRDHMEGYHYRYEINA DPRLTNLSSNSDV 2 Cynomolgus
ORF MPGGCSRGPAAGDGRLRLARLALVLLGWVSSSSSTSSA monkey 5T4
SSSSSSAPFLASAASAQPPLPDQCPALCECSEAARTVK
CVNRNLTEVPTDLPLYVRNLFLTGNQLAVLPAGAFARR
PPLAELAALNLSGSRLDEVRGGAFEHLPSLRQLDLSHN
PLAYLSPFAFSGSNASISAPSPLVELILNHIVPPDDKR
QNRSFEGMVAAALVAGRALQGLHLLELASNHFLYLPRD
VLAQLPSLRYLDLSNNSLVSLTYVSFRNLTHLESLHLE
DNALKVLHNGTLAELQGLPHVRVFLDNNPWVCDCHMAD
MVTWLKQTGVVQGKDRLTCAFPEKMRNRVLLELNSADL
DCDPILPPSLQTSYVFLGIVLALIGAIFLLVLYLNRKG
IKKWMHNIRDACRDHMEGYHYRYEINADPRLTNLSSNS DV 3 Chicken 5T4 ORF
MPGREAERRGALCLGLLLHALLGCGSAQPPAACPAPCE
CSEAAKTVKCVNKNLTEVPPDLPPYVRNLFITGNRLGR
LPAGALSAPRLAELGSLNLSGNHLRAVEAGALAALPAL
RQLDLGGNPLAELSPLAFGRASPLEELALRGALREQGA
LLGLADLLQAGALRNLSRLELADNGLLLLPTGMLGALP
ALRHLDLSNNSLVGLRNVSFQGLVRLQSLNLSDNSLGV
LRNGTLAQWRGLPALRRISLSHNTWVCDCAIEDMVAWL
KESDQVEGKEALSCAFPEKMAGRALLKLNTSELNCSAP
VDVPSQLQTSYVFLGIVLALIGAIFLLVLYLNRKGIKK
WMHNIRDACRDHMEGYHYRYEINADPRLTNLSSNSDV 4 Mature Human Mature
QDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILW CD3.epsilon. (epsilon)
protein QHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYV
CYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVI
VDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQR
GQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI 5 HC_5T4-059 VH
QVQLVESGGGVVQPGRSLRLSCAVSGFTFSSYDMNWV
RQAPGKGLEWVTFISYDGSNKYNADSVKGRFTISRDN
SKNTLYLQMNSLRAEDTAVYYCARDSYSRSWYGDYYG MDVWGQGTTVTVSS 6 HC_5T4-059
VH_CDR1 GFTFSSYD 7 HC_5T4-059 VH_CDR2 ISYDGSNK 8 HC_5T4-059
VH_CD_R3 ARDSYSRSWYGDYYGMDV 9 LC_5T4-059 VL
DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQK
PEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSL
QPEDFATYYCQQYNSYPLTFGGGTKVEIK 10 LC_5T4-059 VL_CDR1 QGISSW
LC_5T4-059 VL_CDR2 AAS 11 LC_5T4-059 VL_CDR3 QQYNSYPLT 12
HC_5T4-076 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVR
QAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTST
RTAYMELRSLRSDDTAVYYCARDPGYFDWLYGDYWGQG TLVTVSS 13 HC_5T4-076
VH_CDR1 GYTFTSYG 14 HC_5T4-076 VH_CDR2 ISAYNGNT 15 HC_5T4-076
VH_CDR3 ARDPGYFDWLYGDY 16 LC_5T4-076 VL
AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQK
PGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSL
QPEDFATYYCQQFNSYPRTFGQGTKVEIK 17 LC_5T4-076 VL_CDR1 QGISSA
LC_5T4-076 VL_CDR2 DAS 18 LC_5T4-076 VL_CDR3 QQFNSYPRT 19
HC_5T4-085 VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVR
QAPGKGLEWVSAISGSGGSTYNADSVKGRFTIFRDNSK
NTLYLHMNSLRAEDTAVYYCARDPGYNNVEYLDHWGQG TLVTVSS 20 HC_5T4-085
VH_CDR1 GFTFSSYA 21 HC_5T4-085 VH_CDR2 ISGSGGST 22 HC_5T4-085
VH_CDR3 ARDPGYNNVEYLDH 23 LC_5T4-085 VL
AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQK
PGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSL
QPEDFATYYCQQFNSYPLTFGGGTKVEIK 24 LC_5T4-085 VL_CDR1 QGISSA
LC_5T4-085 VL_CDR2 DAS 25 LC_5T4-085 VL_CDR3 QQFNSYPLT 26
HC_5T4-106 VH EVQLVQSGAEVKKPGESLKISCKGSGYRFTSYWIGWVR
QMPGKGLEWMGIIYPGDSDARYSPSFQGQVTISADKSI
STAYLQWSSLKASDTGMYYCARSVLFDYWGQGTLVTVS S 27 HC_5T4-106 VH_CDR1
GYRFTSYW 28 HC_5T4-106 VH_CDR2 IYPGDSDA 29 HC_5T4-106 VH_CDR3
ARSVLFDY 30 LC_5T4-106 VL AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQK
PGKAPKLLIYDVSNLESGVPSRFSGSGSGTDFTLTISSL
QPEDFATYYCQQFNSYPHTFGQGTKLEIK 31 LC_5T4-106 VL_CDR1 QGISSA
LC_5T4-106 VL_CDR2 DVS 32 LC_5T4-106 VL_CDR3 QQFNSYPHT 33
HC_5T4-127 VH EVQLLESRGGLVQPGGSLRLSCAASGFTFSSYAMSWVR
QAPGKGLEWVSTISGSGGSTYYADSVKGRFTISRDNSK
KTLYLQMNSLRAEDTAVYYCAKDWGSGSYPAEYFQHWG QGTLVTVSS 34 HC_5T4-127
VH_CDR1 GFTFSSYA 35 HC_5T4-127 VH_CDR2 ISGSGGST 36 HC_5T4-127
VH_CDR3 AKDWGSGSYPAEYFQH 37 LC_5T4-127 VL
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQK
PGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSL
EPEDFAVYYCQQRSNWLMYTFGQGTKLEIK 38 LC_5T4-127 VL_CDR1 QSVSSY
LC_5T4-127 VL_CDR2 DAS 39 LC_5T4-127 VL_CDR3 QQRSNWLMYT 40
HC_5T4-207 VH QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWTWIRQ
PPGKGLEWIGEIDHSESTNYNPSLKSRVTISVDTSKNQF
SLKLSSVTAADTAVYYCAGWFGELYHYYYGMDVWGQGTT VTVSS 41 HC_5T4-207 VH_CDR1
GGSFSGYY 42 HC_5T4-207 VH_CDR2 IDHSEST 43 HC_5T4-207 VH_CDR3
AGWFGELYHYYYGMDV 44 LC_5T4-207 VL
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQK
PGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSL
EPEDFAVYYCQQRSNWPLTFGGGTKVEIK 45 LC_5T4-207 VL_CDR1 QSVSSY
LC_5T4-207 VL_CDR2 DAS 46 LC_5T4-207 VL_CDR3 QQRSNWPLT 47
HC_5T4-226 VH QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWI
RQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTS
KNQFSLKLSSVTAADTAVYYCAAWFGELWDYYYGMDV WGQGTTVTVSS 48 HC_5T4-226
VH_CDR1 GGSFSGYY 49 HC_5T4-226 VH_CDR2 IDHSGST 50 HC_5T4-226
VH_CDR3 AAWFGELWDYYYGMDV 51 LC_5T4-226 VL
EIVLTQSPATLSLSPGERATLSCRASQSVSSFLAWYQQK
PGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSL
EPEDFAVYYCQQRSNWPLTFGQGTRLEIK 52 LC_5T4-226 VL_CDR1 QSVSSF
LC_5T4-226 VL_CDR2 DAS 53 LC_5T4-226 VL_CDR3 QQRSNWPLT 54 VH_huCD3-
VH_CDR1 GFTFNTYA H1L1_CDR1 55 VH_huCD3- VH_CDR2 IRSKYNNYAT
H1L1_CDR2 56 VH_huCD3- VH_CDR3 VRHGNFGNSYVSWFAY H1L1_CDR3 57
VH_huCD3-H1L1 VH EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWV
RQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISR
DDSKSSLYLQMNNLKTEDTAMYYCVRHGNFGNSYVSW FAYWGQGTLVTVSS 58 VL_huCD3-
VL_CDR1 TGAVTTSNY H1L1_CDR1 VL_huCD3- VL_CDR2 GTN H1L1_CDR2 59
VL_huCD3- VL_CDR3 ALWYSNLWV H1L1_CDR3 60 VL_huCD3-H1L1 VL
QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANW
VQQTPGQAFRGLIGGTNKRAPGVPARFSGSLIGDKAA
LTITGAQADDESIYFCALWYSNLWVFGGGTKLTVL 61 VH CDR1-T31P VH_CDR1
GFTFNPYA HC_T31P CDR1 62 VH T31P full VH
EVKLVESGGGLVQPGGSLRLSCAASGFTFNPYAMNWV length sequence
RQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISR HC_T31P
DDSKSSLYLQMNNLKTEDTAMYYCVRHGNFGNSYVSW FAYWGQGTLVTVSS 63 VH
CDR1-T31M VH_CDR1 GFTFNMYA HC_T31M CDR1 64 VH T31M full VH
EVKLVESGGGLVQPGGSLRLSCAASGFTFNMYAMNW length sequence
VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI HC_T31M
SRDDSKSSLYLQMNNLKTEDTAMYYCVRHGNFGNSY VSWFAYWGQGTLVTVSS 65 VH
CDR2-N57E VH_CDR2 IRSKYNEYAT HC_N57E CDR2 66 VH N57E full VH
EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWV length sequence
RQAPGKGLEWVARIRSKYNEYATYYADSVKDRFTISR HC_N57E
DDSKSSLYLQMNNLKTEDTAMYYCVRHGNFGNSYVSW FAYWGQGTLVTVSS 67 VH_huCD3-
VH_CDR3 VRGGNFGNSYVSWFAY H1L1- H101G_CDR3 HC_H101G CDR3 68
VH_huCD3- VH EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWV H1L1-H101G
RQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISR HC_H101G
DDSKSSLYLQMNNLKTEDTAMYYCVRGGNFGNSYVSW FAYWGQGTLVTVSS 69 VH
CDR3-H101N VH_CDR3 VRNGNFGNSYVSWFAY HC_H101N CDR3
70 VH H101N full VH EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWV length
sequence RQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISR HC_H101N
DDSKSSLYLQMNNLKTEDTAMYYCVRNGNFGNSYVSW FAYWGQGTLVTVSS 71 VH
CDR3-G105P VH_CDR3 VRHGNFPNSYVSWFAY HC_G105P CDR3 72 VH G105P full
VH EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWV length sequence
RQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISR HC_G105P
DDSKSSLYLQMNNLKTEDTAMYYCVRHGNFPNSYVSW FAYWGQGTLVTVSS 73 VH
CDR3-S110A VH_CDR3 VRHGNFGNSYVAWFAY HC_S110A CDR3 74 VH S110A full
VH EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWV length sequence
RQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISR HC_S110A
DDSKSSLYLQMNNLKTEDTAMYYCVRHGNFGNSYVAW FAYWGQGTLVTVSS 75 VH
CDR3-S110G VH_CDR3 VRHGNFGNSYVGWFAY HC_S110G CDR3 76 VH S110G full
VH EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWV length sequence
RQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISR HC_S110G
DDSKSSLYLQMNNLKTEDTAMYYCVRHGNFGNSYVGW FAYWGQGTLVTVSS 77 VH
CDR3-Y114V VH_CDR3 VRHGNFGNSYVSWFAV HC_Y114V CDR3 78 VH Y114V full
VH EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWV length sequence
RQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISR HC_Y114V
DDSKSSLYLQMNNLKTEDTAMYYCVRHGNFGNSYVSW FAYWGQGTLVTVSS 79 VH
CDR3-Y114M VH_CDR3 VRHGNFGNSYVSWFAM HC_Y114M CDR3 80 VH Y114M full
VH EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWV length sequence
RQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISR HC_Y114M
DDSKSSLYLQMNNLKTEDTAMYYCVRHGNFGNSYVSW FAMWGQGTLVTVSS 81 VH
CDR3-Y114R VH_CDR3 VRHGNFGNSYVSWFAR HC_Y114R CDR3 82 VH Y114R full
VH EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWV length sequence
RQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISR HC_Y114R
DDSKSSLYLQMNNLKTEDTAMYYCVRHGNFGNSYVSW FARWGQGTLVTVSS 83 HC_5T4-A1
VH QIQLVQSGPELKKPGETVKISCKASGYTFTNFGMNWVK
QGPGEGLKWMGWINTNTGEPRYAEEFKGRFAFSLETTA
STAYLQINNLKNEDTATYFCARDWDGAYFFDYWGQGTT LTVSS 84 LC_5T4-A1 VL
SIVMTQTPKFLLVSAGDRVTITCKASQSVSNDVAWYQ
QKPGQSPKLLINFATNRYTGVPNRFTGSGYGTDFTFT
ISTVQAEDLALYFCQQDYSSPWTFGGGTKLEIK 85 HC_5T4-A3 VH
EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWV
RQAPGKGLEWVARIRSKSNNYATYYADSVKDRFTISRD
DSQSMLYLQMNNLKTEDTAMYYCVRQWDYDVRAM NYWGQGTSVTVSS 86 LC_5T4-A3 VL
DIVMTQSHIFMSTSVGDRVSITCKASQDVDTAVAWYQ
QKPGQSPKLLIYWASTRLTGVPDRFTGSGSGTDFTLT
ISNVQSEDLADYFCQQYSSYPYTFGGGTKLEIK 87 HC_5T4-H8 VH
EVQLQQSGPDLVKPGASVKISCKASGYSFTGYYMHWV
KQSHGKSLEWIGRINPNNGVTLYNQKFKDKAILTVDS
STTAYMELRSLTSEDSAVYYCARSTMITNYVMDYWGQ VTSVTVSS 88 LC_5T4-H8 VL
SIVMTQTPTFLLVSAGDRVTITCKASQSVSNDVAWYQ
QKPGQSPTLLISYTSSRYAGVPDRFIGSGYGTDFTFT
ISTLQAEDLAVYFCQQDYNSPPTFGGGTKLEIK 89 IgG1-Fc Constant
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV
SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
QTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEL
LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD
WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL
PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK 90
IgG1-Fc_F405L Constant ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV
SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
QTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEL
LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD
WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL
PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFLLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK 91
IgG1-Fc_FEA Constant ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV
SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
QTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEF
EGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEV
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD
WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL
PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK 92
IgG1-Fc_FEAL Constant ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV
SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
QTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEF
EGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEV
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD
WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL
PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFLLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK 93
IgG1-Fc_FEAR Constant ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV
SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
QTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEF
EGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEV
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD
WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL
PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK 94
IgG1-Fc_K409R Constant ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV
SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
QTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEL
LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD
WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL
PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK 95 Kappa
Constant RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK
VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK
ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 96 Lambda Constant
GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAV
TVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTP
EQWKSHRSYSCQVTHEGSTVEKTVAPTECS 97 b12_VH VH
QVQLVQSGAEVKKPGASVKVSCQASGYRFSNFVIHWV
RQAPGQRFEWMGWINPYNGNKEFSAKFQDRVTFTADT
SAMTAYMELRSLRSADTAVYYCARVGPYSWDDSPQDN YYMDVWGKGTTVIVSS 98 b12_VL VL
EIVLTQSPGTLSLSPGERATFSCRSSHSIRSRRVAWY
QHKPGQAPRLVIHGVSNRASGISDRFSGSGSGTDFTL
TITRVEPEDFALYYCQVYGASSYTFGQGTKLERK 99 5T4ECDHis ORF
MPGGCSRGPAAGDGRLRLARLALVLLGWVSSSSPTSSA
SSFSSSAPFLASAVSAQPPLPDQCPALCECSEAARTVK
CVNRNLTEVPTDLPAYVRNLFLTGNQLAVLPAGAFARR
PPLAELAALNLSGSRLDEVRAGAFEHLPSLQLDLSHNP
LADLSPFAFSGSNASVSAPSPLVELILNHIVPPEDERQ
NRSFEGMVVAALLAGRALQGLRRLELASNHFLYLPRDV
LAQLPSLRHLDLSNNSLVSLTYVSFRNLTHLESLHLED
NALKVLHNGTLAELQGLPHIRVFLDNNPWVCDCHMADM
VTWLKETEVVQGKDRLTCAYPEKMRNRVLLELNSADLD CDPILPPSLQTSHHHHHHHH 100
5T4ECD91- ORF MPGGCSRGPAAGDGRLRLARLALVLLGWVSSSSPTSSAS FcRbHis
SFSSSAPFLASAVSAQPPLPDQCPALCECSEAARTVKCV
NRNLTEVPTDLPAAPSTCSKPTCPPPELLGGPSVFIFPP
KPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQV
RTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKV
HNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRS
VSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSD
GSYFLYSKLSVPTSEWQRGDVFTCSVMHEALTHNHYTQK SISRSPGKHHHHHHHH 101
CD3E27-GSKa ORF MWWRLWWLLLLLLLLWPMVWAQDGNEEMGGITQTPYKVS
ISGTTVILTGGGGSGGGGSGGGGSEIVLTQSPATLSLSP
GERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNR
ATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSN
WPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASV
VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS
TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGE 102 HC_574-059 VH-CDR3
YYGMDV HC_5T4-207 C-term HC_5T4-226 103 HC_5T4-207 VH-CDR-2
IDHSX.sub.1ST; X.sub.1 is G or E HC_5T4-226 104 HC_5T4-207 VH-CDR-3
AX.sub.2WFGELX.sub.3X.sub.4YYYGMDV; X.sub.2 is A or G, HC_5T4-226
X.sub.3 is W or Y, X.sub.4 is D or H 105 HC_5T4-207 VL-CDR-1
QSVSSX.sub.5; X.sub.5 is Y or F HC_5T4-226
[0506] The present invention is further illustrated by the
following examples which should not be construed as further
limiting.
EXAMPLES
Example 1--Generation of 5T4 Antibodies and Screenings
Materials
Expression Constructs for 5T4
[0507] The following codon-optimized constructs for expression of
various full length 5T4 variants were generated: human (Homo
sapiens) 5T4 (Uniprot accession no. Q13641), cynomolgus monkey
(Macaca fascicularis) 5T4 (Uniprot accession no. Q4R8Y9), and
chicken (Gallus gallus) 5T4 (Uniprot accession no. R4GM46). In
addition, the following codon-optimized constructs for various 5T4
extracellular domain (ECD) variants were generated: the ECD of
human 5T4 (aa 1-355 from Uniprot accession no. Q13641) with a
C-terminal His tag (5T4ECDHis)(SEQ ID NO: 99), and the ECD of human
5T4 (aa 1-91) fused to rabbit Fc domain and C-terminal His-tag
(5T4ECD91-FcRbHis). In SEQ ID NO: 99, amino acid residues 1-31 are
a signal peptide; hence the mature 5T4ECDHis protein corresponds to
amino acid residues 32-363 of SEQ ID NO: 99. Likewise, amino acid
residues 1-31 of SEQ ID NO: 100 are a signal peptide and the mature
5T4ECD91-FcRbHis protein corresponds to amino acid residues 32-327
of SEQ ID NO: 100.
[0508] The constructs contained suitable restriction sites for
cloning and an optimal Kozak (GCCGCCACC) sequence (Kozak, M., Gene
1999; 234(2):187-208). The full length human 5T4 and cynomolgus
monkey 5T4 codon-optimized constructs were cloned in the mammalian
expression vector pcDNA3.3 (Invitrogen). The full length chicken
5T4 codon-optimized constructs was cloned in pSB, a mammalian
expression vector containing Sleeping Beauty inverter terminal
repeats flanking an expression cassette consisting of a CMV
promoter and HSV-TK polyA signal.
Generation of HEK-293F Cell Lines Transiently Expressing Full
Length Human, Cynomolgus or Chicken 5T4
[0509] Freestyle.TM. 293-F (a HEK-293 subclone adapted to
suspension growth and chemically defined Freestyle medium
[HEK-293F]) cells were obtained from Invitrogen (cat. no. R790-07)
and transfected with the codon-optimized constructs described
supra, using 293fectin (Invitrogen, cat. no. 12347-019) according
to the manufacturer's instructions.
Purification of His-Tagged 5T4
[0510] 5T4ECDHis (mature protein of SEQ ID NO: 99) was expressed in
HEK-293F cells as described supra. 5T4ECD91-FcRbHis was expressed
using the Expi293F expression platform (Thermo Fisher Scientific,
Waltham, Mass., USA, cat. no. A14527) essentially as described by
the manufacturer.
[0511] The His-tag enables purification with immobilized metal
affinity chromatography. In this process, a chelator fixed onto the
chromatographic resin is charged with Co.sup.2+ cations.
Supernatants containing the His-tagged protein were incubated with
the resin in batch mode (i.e. solution). The His-tagged protein
binds strongly to the resin beads, while other proteins present in
the culture supernatant do not bind or bind weakly compared to the
His-tagged proteins. After incubation, the beads were retrieved
from the supernatant and packed into a column. The column was
washed in order to remove weakly bound proteins. The strongly bound
His-tagged proteins were then eluted with a buffer containing
imidazole, which competes with the binding of His to Co.sup.2+. The
eluent was removed by buffer exchange on a desalting column.
Immunization
[0512] For generation of antibodies IgG1-5T4-207 and IgG1-5T4-226,
HCo17-BalbC transgenic mice (Bristol-Myers Squibb, New York, N.Y.,
USA) were immunized alternatingly intraperitoneally (IP) and
subcutaneously (SC) with 20 .mu.g of the 5T4ECDHis protein in Sigma
adjuvant system (Sigma-Aldrich, St. Louis, Mo., USA, cat. no.
56322) with an interval of 14 days. In total 8 immunizations were
performed: 4 IP and 4 SC.
[0513] For generation of antibodies IgG1-5T4-076 and IgG1-5T4-059,
HCo12-BalbC (IgG1-5T4-076) and HCo20-BalbC (IgG1-5T4-059)
transgenic mice (Bristol-Myers Squibb) were immunized alternatingly
IP and SC with 20 .mu.g of the 5T4ECDHis protein in Sigma adjuvant
system with an interval of 14 days. In total 8 immunizations were
performed: 4 IP and 4 SC.
[0514] For generation of antibody IgG1-5T4-085, HCo17-BalbC
transgenic mice were immunized alternatingly IP and SC with 20
.mu.g of the 5T4ECDHis protein and 20 .mu.g of the 5T4ECD91-FcRbHis
mature protein in Sigma adjuvant system with an interval of 14
days. In total 8 immunizations were performed: 4 IP and 4 SC.
[0515] For generation of antibodies IgG1-5T4-106 and IgG1-5T4-127,
HCo12-BalbC (IgG1-5T4-106) and HCo17-BalbC (IgG1-5T4-127)
transgenic mice were immunized alternatingly IP and SC with 20
.mu.g of the 5T4ECD91-FcRbHis mature protein in Sigma adjuvant
system with an interval of 14 days. In total 8 immunizations were
performed: 4 IP and 4 SC.
[0516] Mice with at least two sequential 5T4 specific antibody
titers in the antigen specific screening Fluorometric Micro volume
Assay Technology (FMAT) as described below, were boosted with 10
.mu.g of 5T4ECDHis or 10 .mu.g 5T4ECD91-FcRbHis (in PBS injected
intravenously) and splenocytes and lymph node cells of these mice
were fused 3-4 days later.
Homogeneous Antigen Specific Screening Assay
[0517] The presence of 5T4 antibodies in sera of immunized mice or
HuMAb (human monoclonal antibody) hybridoma or transfectoma culture
supernatant was determined by homogeneous antigen specific
screening assays using FMAT (Applied Biosystems, Foster City,
Calif., USA). For this, a combination of 4 cell based assays was
used.
[0518] Sera from immunized mice, or hybridoma or transfectoma
culture supernatant samples were analyzed for binding of human
antibodies to HEK-293F cells transiently expressing human 5T4,
HEK-293F cells transiently expressing cynomolgus monkey 5T4,
streptavidin-coated polystyrene particles (0.5% w/v; 6.7 urn;
Spherotech, Lake Forest, Ill., USA, cat. no. SVP-60-5) coated with
5T4ECD91-FcRBHis, and HEK-293 wild-type cells (negative
control).
[0519] Samples were added to the cells to allow binding to 5T4.
Subsequently, binding of HuMAb was detected using a fluorescent
conjugate (AffiniPure Goat Anti-Human IgG Fc gamma-Alexa Fluor.RTM.
647; Jackson ImmunoResearch, cat no. 109-605-098).
IgG1-5T4-H8-F405L was used as a positive control and ChromPure
Human IgG, whole molecule (Jackson ImmunoResearch, cat no.
009-000-003) was used as negative control. The samples were scanned
using an ImageXpress Velos (Molecular devices, LLC, Sunnyvale,
Calif., USA) and total fluorescence was used as read-out. Samples
were stated positive when counts were higher than 50 and counts x
fluorescence was at least three times higher than the negative
control.
HuMAb Hybridoma Generation
[0520] HuMAb mice with sufficient antigen-specific titer
development (described above) were sacrificed and the spleen and
lymph nodes flanking the abdominal aorta and vena cava were
collected. Fusion of splenocytes and lymph node cells to a mouse
myeloma cell line (SP2.0 cells) was done by electrofusion using a
CytoPulse CEEF 50 Electrofusion System (Cellectis, Paris, France),
essentially according to the manufacturer's instructions. Next, the
antigen-positive primary wells were sub-cloned using the ClonePix
system (Genetix, Hampshire, UK). To this end, specific primary well
hybridomas were seeded in semisolid medium made from 40% CloneMedia
(Genetix, Hampshire, UK) and 60% HyQ 2.times. complete media
(Hyclone, Waltham, USA). The subclones were retested for 5T4
binding according to the antigen-specific binding assay as
described above and scanned using the IsoCyte system (Molecular
Devices). IgG levels were measured using an Octet system (Fortebio,
Menlo Park, USA) in order to select the best producing clone per
primary well for further expansion. Further expansion and culturing
of the resulting HuMAb hybridomas were done based upon standard
protocols (e.g. as described in Coligan J. E., Bierer, B. E.,
Margulies, D. H., Shevach, E. M. and Strober, W., eds. Current
Protocols in Immunology, John Wiley & Sons, Inc., 2006).
Sequence Analysis of the 5T4 Antibody Variable Domains and Cloning
in Expression Vectors
[0521] Total RNA was prepared from 2 to 5.times.10.sup.6 hybridoma
cells and 5'-RACE-complementary DNA (cDNA) was prepared from 100 ng
total RNA, using the SMART RACE cDNA Amplification kit (Clontech),
according to the manufacturer's instructions. VH and VL coding
regions were amplified by PCR and cloned directly, in frame, in the
p33G1f and p33Kappa expression vectors (pcDNA3.3 based vectors with
codon optimized human IgG1m(f) and Kappa constant domains,
respectively), by ligation independent cloning (Aslanidis, C. and
P. J. de Jong, Nucleic Acids Res 1990; 18(20): 6069-74). The
variable domains from these expression vectors were sequenced and
CDRs were annotated according to IMGT definitions (Lefranc M P. et
al., Nucleic Acids Research, 27, 209-212, 1999 and Brochet X. Nucl.
Acids Res. 36, W503-508 (2008)). Clones with a correct Open Reading
Frame (ORF) were expressed and tested for binding to the antigen. A
lead panel was ordered as codon optimized sequences (GeneArt,
Thermo Fisher Scientific) and produced with the Expi293 expression
system according to manufacturer's instructions (Thermo Fisher
Scientific). The antibodies in these supernatants were purified and
used for functional characterization. The sequences of the
resulting lead clones are shown in the table above.
5T4 Control Antibodies
[0522] In some of the Examples comparison antibodies against 5T4
were used (IgG1-5T4-H8, IgG1-5T4-A3 and IgG1-5T4-A1) that have been
previously described in WO2007/106744. The codon optimized antibody
encoding sequences were synthesized and cloned in pCDNA3.3
expression vectors (Thermo Fisher Scientific).
IgG1-b12 Antibody
[0523] In some of the Examples the antibody b12, an HIV-1 gp120
specific antibody (Barbas, C F. J Mol Biol. 1993 Apr. 5;
230(3):812-23) was used as a negative control. The codon optimized
antibody encoding sequences for this control antibody were
synthesized and cloned into pCDNA3.3 expression vectors (Thermo
Fisher Scientific). The sequence of the variable heavy chain (VH)
region and the sequence of the variable light chain (VL) region are
included herein as SEQ ID NOs.: 97 and 98, respectively.
Example 2--Determination of the Binding Affinities of 5T4 Specific
Antibodies Using Biolayer Interferometry
[0524] Affinities of the 5T4 antibodies for recombinant 5T4 protein
were determined using label-free biolayer interferometry on an
Octet HTX instrument (ForteBio, Portsmouth, UK). 5T4 antibodies (1
.mu.g/mL) were immobilized for 600 seconds on anti-human IgG Fc
Capture biosensors (ForteBio). After a baseline measurement (100
s), the association (200 s) and dissociation (1000 s) of human
5T4ECDHis (mature protein of SEQ ID NO: 99) or recombinant
cynomolgus monkey 5T4 protein (Cusabio; cat. no. CSB-MP024093MOV)
in Sample Diluent (ForteBio) was determined using a 2-fold dilution
series (ranging from 100 nM to 1.56 nM) starting at 3.58 .mu.g/mL
(100 nM) human 5T4ECDHis or 3.99 .mu.g/mL (100 nM) cynomolgus 5T4,
while shaking at 1000 rpm at 30.degree. C. Data were analyzed with
Data Analysis Software v9.0.0.12 (ForteBio). Values of reference
wells containing only Sample Diluent during the association and
dissociation steps were subtracted from values of wells containing
antigen, for each antibody separately. The Y-axis was aligned to
the last 10 s of the baseline and Interstep Correction alignment to
dissociation as well as Savitzky-Golay filtering was applied.
Responses <0.05 nm were excluded from analysis. The data were
fitted using the 1:1 model and a global full fit with 200 s
association time and 1000 s or 50 s dissociation time as Window of
Interest. The fit with the full dissociation time (1000 s) as
Window of Interest was used by default. Based on the R.sup.2 value
and visual inspection of the fit, a dissociation time of 50s was
used as Window of Interest for IgG1-5T4-127-FEAR.
[0525] Table 1 shows the association rate constant k.sub.a (1/Ms),
dissociation rate constant k.sub.d (1/s) and equilibrium
dissociation constant K.sub.D (M) of the 5T4 antibodies for human
5T4ECDHis determined by biolayer interferometry. A range of
affinities of the antibodies to human 5T4 was measured ranging from
1.3.times.10.sup.-9-2.7.times.10.sup.-8 M. The response of
IgG1-5T4-085-FEAR was lower than 0.05 nm, which prevented proper
fitting of the data (low R.sup.2 values for these fits).
Furthermore, the response of IgG1-5T4-076-FEAR could not be fitted
properly. These data are shown in italics.
[0526] Table 2 shows the association rate constant k.sub.a (1/Ms),
dissociation rate constant k.sub.d (1/s) and equilibrium
dissociation constant K.sub.D (M) for cynomolgus monkey 5T4
determined with biolayer interferometry. A range of affinities of
the antibodies to cynomolgus monkey 5T4 was measured ranging from
1.1.times.10.sup.-9-4.1.times.10.sup.-8 M. The responses of
IgG1-5T4-085-FEAR, IgG1-5T4-106-FEAR and IgG1-5T4-H8-FEAR were
lower than 0.05 nm, which prevented proper fitting of the data (low
R.sup.2 values for these fits). Furthermore, the response of
IgG1-5T4-076-FEAR could not be fitted properly. These data are
shown in italics.
TABLE-US-00004 TABLE 1 Binding affinities of monospecific, bivalent
5T4 antibodies to human 5T4 extracellular domain as determined by
label-free biolayer interferometry. On-rate Off-rate Antibody
k.sub.a (1/Ms) k.sub.d (1/s) K.sub.D (M) IgG1-5T4-059-FEAR 2.1E+05
3.2E-04 1.5E-09 IgG1-5T4-076-FEAR No fit IgG1-5T4-085-FEAR Response
<0.05 nm IgG1-5T4-106-FEAR 2.1E+05 1.2E-03 5.5E-09
IgG1-5T4-127-FEAR 5.8E+05 1.6E-02 2.7E-08 IgG1-5T4-207-FEAR 2.7E+05
6.8E-04 2.6E-09 IgG1-5T4-226-FEAR 3.3E+05 8.1E-04 2.5E-09
IgG1-5T4-H8-FEAR 2.2E+05 2.9E-04 1.3E-09
TABLE-US-00005 TABLE 2 Binding affinities of monospecific, bivalent
5T4 antibodies to cynomolgus monkey 5T4 extracellular domain as
determined by label-free biolayer interferometry. On-rate Off-rate
Antibody k.sub.a (1/Ms) k.sub.d (1/s) K.sub.D (M) IgG1-5T4-059-FEAR
1.6E+05 2.8E-04 1.8E-09 IgG1-5T4-076-FEAR No fit IgG1-5T4-085-FEAR
Response <0.05 nm IgG1-5T4-106-FEAR Response <0.05 nm
IgG1-5T4-127-FEAR 3.7E+05 1.5E-02 4.1E-08 IgG1-5T4-207-FEAR 1.4E+05
8.7E-04 6.3E-09 IgG1-5T4-226-FEAR 1.4E+05 1.5E-03 1.1E-08
IgG1-5T4-H8-FEAR Response <0.05 nm
Example 3--Cross-Block of 5T4 Antibodies Determined by Biolayer
Interferometry
[0527] Antibody cross-block analysis (epitope binning) was
performed using biolayer interferometry on an Octet HTX instrument
(ForteBio). 5T4 antibodies (20 .mu.g/mL in 10 mM sodium acetate
buffer pH 6.0, ForteBio) were immobilized on Amine-Reactive 2nd
Generation (AR2G) biosensors (ForteBio) according to the
manufacturer's instructions. After a baseline measurement (100 s)
in Sample Diluent (ForteBio), biosensors containing immobilized
antibodies were loaded for 500 s with human 5T4ECDHis (mature
protein of SEQ ID NO: 99) 100 nM (3.6 .mu.g/mL). Next, the
association response of a second 5T4 antibody (10 .mu.g/mL) was
determined for 500 s. Biosensors were regenerated by 3 times 5 s
exposure to 10 mM glycine pH 2.5 followed by Sample Diluent, and
the measurement was repeated with a new set of second 5T4
antibodies starting from the baseline step. Each biosensor was used
four times. Measurements were performed at 30.degree. C. using a
shaker speed of 1000 rpm. Data were analyzed using Data Analysis
Software v9.0.0.12 (ForteBio). The Y-axis was aligned to the
association step and Savitzky-Golay filtering was applied. The
response of Sample Diluent during the association step was
subtracted from the association response of the second antibody in
order to correct for the dissociation of 5T4ECDHis from the
immobilized antibody. The corrected association responses were
plotted in a matrix format. In general, responses >0.1 nm were
considered non-blocking antibody pairs (white), while responses
between -0.1 and 0.1 nm were considered to be blocking antibody
pairs (dark grey). For some antibody pairs the second antibody
showed an initial positive response, followed by a decrease in
signal. This was considered to be antibody displacement (light
grey), i.e. the second antibody displacing the interaction between
the first antibody and the antigen (Abdiche Y N, Yeung A Y, Ni I,
Stone D, Miles A, Morishige W, et al. (2017) Antibodies Targeting
Closely Adjacent or Minimally Overlapping Epitopes Can Displace One
Another. PLoS ONE 12(1): e0169535.
doi:10.1371/journal.pone.0169535). In some cases, the data curves
needed visual inspection by an expert to assign blocking,
non-blocking or displacement properties to antibody pairs.
[0528] Cross-block experiments were performed for antibodies
IgG1-5T4-059-FEAR, IgG1-5T4-076-FEAR, IgG1-5T4-085-FEAR,
IgG1-5T4-106-FEAR, IgG1-5T4-127-FEAR, IgG1-5T4-207-FEAR,
IgG1-5T4-226-FEAR, and prior art antibodies IgG1-5T4-H8-FEAR,
IgG1-5T4-A1-F405L and IgG1-5T4-A3-F405L. The results are summarized
in Table 3.
[0529] None of the antibodies (except IgG1-5T4-A1-F405L itself)
blocked binding of IgG1-5T4-A1-F405L to 5T4ECDHis. Antibodies
IgG1-5T4-076-FEAR, IgG1-5T4-085-FEAR, IgG1-5T4-127-FEAR,
IgG1-5T4-106-FEAR, IgG1-5T4-059-FEAR, IgG1-5T4-207-FEAR and
IgG1-5T4-226-FEAR (as well as IgG1-5T4-H8-FEAR itself) blocked
binding of IgG1-5T4-H8-FEAR to 5T4ECDHis. Antibodies
IgG1-5T4-076-FEAR, IgG1-5T4-085-FEAR, and IgG1-5T4-127-FEAR (as
well as IgG1-5T4-A3-F405L itself) also blocked binding of
IgG1-5T4-A3-F405L to 5T4ECDHis, while antibodies IgG1-5T4-106-FEAR
and IgG1-5T4-H8-FEAR did not block binding of IgG1-5T4-A3-F405L to
5T4ECDHis. Antibodies IgG1-5T4-059-FEAR, IgG1-5T4-207-FEAR and
IgG1-5T4-226-FEAR showed antibody displacement in combination with
IgG1-5T4-A3-F405L, which is described in more detail in Example
4.
TABLE-US-00006 TABLE 3 Antibody cross-block as determined by
biolayer interferometry. A1 A3 076 085 127 106 H8 059 207 226 A1
-0.01 0.76 0.36 0.72 0.87 0.85 0.89 0.91 0.86 0.86 A3 0.69 0.01
0.00 0.00 0.01 0.57 0.50 * * * 076 0.04 0.00 -0.01 -0.02 -0.02
-0.02 0.00 -0.02 0.05 0.05 085 0.07 -0.01 -0.01 -0.01 0.00 -0.01
-0.01 -0.04 0.08 0.07 127 0.15 -0.01 -0.02 -0.01 -0.01 -0.01 -0.02
-0.05 0.16 0.16 106 0.79 0.56 -0.03 -0.04 -0.02 -0.02 -0.02 -0.03
-0.03 -0.02 H8 0.64 0.49 -0.02 -0.02 -0.01 -0.01 0.00 -0.02 -0.01
-0.01 059 0.96 * 0.00 -0.02 -0.10 0.01 0.01 0.01 0.02 0.01 207 1.29
* 1.22 1.03 1.29 -0.01 -0.01 -0.02 -0.02 -0.02 226 1.56 * 1.47 1.35
1.51 -0.02 -0.01 -0.02 -0.02 -0.02
[0530] The first column shows the immobilized antibodies and the
first row shows the antibodies in solution. Corrected association
responses of the antibodies in solution are shown. Cross-block of
antibodies is indicated by italics and underlining, displacing
antibody combinations are indicated by an asterisk. Non-blocking
antibody combinations are unmarked.
Example 4--Antibody Displacement of IgG1-5T4-059-FEAR,
IgG1-5T4-207-FEAR and IgG1-5T4-226-FEAR in Combination with
IgG1-5T4-A3-F405L
[0531] Antibody displacement was demonstrated using biolayer
interferometry on an Octet HTX instrument (ForteBio).
IgG1-5T4-A3-F405L (20 .mu.g/mL in 10 mM sodium acetate buffer pH
6.0, ForteBio) was immobilized on Amine-Reactive 2nd Generation
(AR2G) biosensors (ForteBio) according to the manufacturer's
instructions. After a baseline measurement (100 s) in Sample
Diluent (ForteBio), biosensors containing immobilized
IgG1-5T4-A3-F405L antibodies were loaded for 500 s with human
5T4ECDHis (mature protein of SEQ ID NO: 99) 100 nM (3.6 .mu.g/mL).
Next, the association response of a second 5T4 antibody
(IgG1-5T4-059-FEAR, IgG1-5T4-207-FEAR or IgG1-5T4-226-FEAR; 10
.mu.g/mL) or Sample Diluent (buffer control) was determined for 500
s. The experiment was performed at 30.degree. C. using a shaker
speed of 1000 rpm. Data was analyzed using Data Analysis Software
v9.0.0.12 (ForteBio). The buffer control response was subtracted
from the responses of the second antibodies to correct for the
dissociation of human 5T4ECDHis from the immobilized
IgG1-5T4-A3-F405L, the Y-axis was aligned to the association step
and Savitzky-Golay filtering was applied.
[0532] As shown in FIG. 1, IgG1-5T4-A3-F405L did not show binding,
indicating cross-block (self-block) with IgG1-5T4-A3-F405L.
IgG1-5T4-H8-FEAR showed binding to 5T4ECDHis and hence no
cross-block with IgG1-5T4-A3-F405L. IgG1-5T4-059-FEAR,
IgG1-5T4-207-FEAR and IgG1-5T4-226-FEAR initially showed a positive
response (indicating binding to the IgG1-5T4-A3-F405L-5T4ECDHis
complex instead of cross-blocking with IgG1-5T4-A3-F405L), followed
by a decrease in response that dropped below the self-block
response of IgG1-5T4-A3-F405L. This demonstrates loss of mass from
the IgG1-5T4-A3-F405L-5T4ECDHis complex, indicating that
IgG1-5T4-059-FEAR, IgG1-5T4-207-FEAR and IgG1-5T4-226-FEAR induce
dissociation of human 5T4ECDHis from IgG1-5T4-A3-F405L upon binding
to the complex. This phenomenon has been described as antibody
displacement and indicates that the epitopes are closely adjacent
or minimally overlapping (Abdiche Y N, Yeung A Y, Ni I, Stone D,
Miles A, Morishige W, et al. (2017) Antibodies Targeting Closely
Adjacent or Minimally Overlapping Epitopes Can Displace One
Another. PLoS ONE 12(1): e0169535.
doi:10.1371/journal.pone.0169535)). This indicates that antibodies
IgG1-5T4-059-FEAR, IgG1-5T4-207-FEAR and IgG1-5T4-226-FEAR bind to
a distinct epitope on 5T4 as compared to IgG1-5T4-A3-F405L.
Example 5--Simultaneous Binding of 5T4 Antibodies to Membrane-Bound
5T4 Measured with Flow Cytometry
[0533] Binding of IgG1-5T4-207-FEAR and IgG1-5T4-226-FEAR
antibodies to membrane-bound 5T4 in the presence of
IgG1-5T4-A1-F405L and IgG1-5T4-A3-F405L was assessed by flow
cytometry. IgG1-5T4-H8-FEAR, IgG1-5T4-207-FEAR and
IgG1-5T4-226-FEAR were conjugated to fluorescein isothiocyanate
(FITC, Thermo Fisher Scientific) according to manufacturer's
instructions. SK-OV-3 cells (50,000 cells per condition), which
express approximately 20,000 5T4 molecules/cell, were incubated
with mixtures of 10 .mu.g/mL unconjugated 5T4 antibodies
(IgG1-5T4-H8-FEAR, IgG1-5T4-A1-F405L, IgG1-5T4-A3-F405L, IgG1-b12,
IgG1-5T4-207-FEAR or IgG1-5T4-226-FEAR) and 2 .mu.g/mL
FITC-conjugated 5T4 antibodies (IgG1-5T4-H8-FEAR-FITC,
IgG1-5T4-207-FEAR-FITC and IgG1-5T4-226-FEAR-FITC). Table 4 shows
an overview of the tested combinations. After 30 min incubation at
4.degree. C., cells were centrifuged at 1200 RPM for 5 min, and the
supernatant was discarded. The cells were resuspended in 100 .mu.L
FACS-buffer supplemented with 1:4000 Topro-3-iodine (Molecular
Probes). Mean fluorescence intensity (MFI) of the FITC signal was
measured using a flow cytometer (FACS Fortessa, BD Biosciences).
Percentage of binding was calculated using the following
formula:
( [ MFI of cells with Ab - FITC and uncojugated AB - MFI of cells
without Ab - FITC or unconjugated Ab ] * 100 ) ( MFI of cells with
Ab - FITC and isotype control - MFI of cells without Ab - FITC or
unconjugated Ab ) ##EQU00006##
[0534] FIG. 2 shows that binding of IgG1-5T4-H8-FEAR-FITC,
IgG1-5T4-207-FEAR-FITC and IgG1-5T4-226-FEAR-FITC was blocked in
presence of their unconjugated counterpart. However, binding of
IgG1-5T4-207-FEAR-FITC and IgG1-5T4-226-FEAR-FITC to membrane-bound
5T4 was still observed in the presence of unconjugated
IgG1-5T4-A1-F405L, IgG1-5T4-A3-F405L or IgG1-b12, and was
comparable to binding of IgG1-5T4-H8-FEAR-FITC to membrane-bound
5T4 in the presence of unconjugated IgG1-5T4-A1-F405L,
IgG1-5T4-A3-F405L or IgG1-b12. This demonstrates that antibodies
IgG1-5T4-H8-FEAR, IgG1-5T4-207-FEAR and IgG1-5T4-226-FEAR bind to a
distinct epitope on 5T4 as compared to antibodies IgG1-5T4-A1-F405L
and IgG1-5T4-A3-F405L.
TABLE-US-00007 TABLE 4 Overview of antibody combinations used in
flow cytometry experiment. FITC-labeled antibody Unconjugated
antibody (2 .mu.g/mL) (10 .mu.g/mL) 1 IgG1-5T4-H8-FEAR-FITC
IgG1-5T4-H8-FEAR 2 IgG1-5T4-H8-FEAR-FITC IgG1-5T4-A3-F405L 3
IgG1-5T4-H8-FEAR-FITC IgG1-5T4-207-FEAR 4 IgG1-5T4-H8-FEAR-FITC
IgG1-5T4-226-FEAR 5 IgG1-5T4-H8-FEAR-FITC IgG1-5T4-A1-F405L 6
IgG1-5T4-H8-FEAR-FITC IgG1-b12 7 IgG1-5T4-207-FEAR-FITC
IgG1-5T4-H8-FEAR 8 IgG1-5T4-207-FEAR-FITC IgG1-5T4-A3-F405L 9
IgG1-5T4-207-FEAR-FITC IgG1-5T4-207-FEAR 10 IgG1-5T4-207-FEAR-FITC
IgG1-5T4-226-FEAR 11 IgG1-5T4-207-FEAR-FITC IgG1-5T4-A1-F405L 12
IgG1-5T4-207-FEAR-FITC IgG1-b12 13 IgG1-5T4-226-FEAR-FITC
IgG1-5T4-H8-FEAR 14 IgG1-5T4-226-FEAR-FITC IgG1-5T4-A3-F405L 15
IgG1-5T4-226-FEAR-FITC IgG1-5T4-207-FEAR 16 IgG1-5T4-226-FEAR-FITC
IgG1-5T4-226-FEAR 17 IgG1-5T4-226-FEAR-FITC IgG1-5T4-A1-F405L 18
IgG1-5T4-226-FEAR-FITC IgG1-b12
Example 6--Binding of 5T4 Antibodies to HEK-293 Cells Transfected
with Human or Chicken 5T4
[0535] Binding of 5T4 antibodies to HEK-293 cells transiently
transfected with full length human or chicken 5T4 (generated as
described in Example 1) was analyzed by flow cytometry. Cells
(5.times.10.sup.4 cells/well) were incubated in polystyrene 96-well
round-bottom plates (Greiner bio-one, cat. no. 650180) with serial
dilutions of 5T4 antibodies (range 0.01 to 10 .mu.g/mL in 3-fold
dilution steps) in 50 .mu.L PBS/0.1% BSA/0.02% azide (staining
buffer) at 4.degree. C. for 30 min. After washing twice in staining
buffer, cells were incubated in 50 .mu.L R-Phycoerythrin
(PE)-conjugated goat-anti-human IgG F(ab').sub.2 (1:500 in staining
buffer; Jackson ImmunoResearch Laboratories, Inc., West Grove, Pa.,
cat. no. 109-116-098) at 4.degree. C. for 30 min. Cells were washed
twice in staining buffer, re-suspended in 20 .mu.L staining buffer
and analyzed on an iQue screener (Intellicyt Corporation, USA).
Binding curves were analyzed by non-linear regression (sigmoidal
dose-response with variable slope) using GraphPad Prism V7.02
software (Graph Pad Software, San Diego, Calif., USA).
[0536] FIG. 3A shows dose-dependent binding of IgG1-5T4-207-FEAR,
IgG1-5T4-226-FEAR, IgG1-5T4-059-FEAR and IgG1-5T4-A3-F405L to
HEK-293 cells transfected with full length human 5T4. FIG. 3B shows
that while dose-dependent binding of IgG1-5T4-207-FEAR,
IgG1-5T4-226-FEAR and IgG1-5T4-059-FEAR to HEK-293 cells
transfected with full length chicken 5T4 was observed,
IgG1-5T4-A3-F405L showed minimal binding to HEK-293 cells
transfected with full length chicken 5T4. The negative control
antibody, IgG1-b12-K409R, did not show binding to HEK-293 cells
transfected with full length human or chicken 5T4 at a
concentration of 10 .mu.g/m L.
Example 7--Internalization Capacity of 5T4 Antibodies in Tumor
Cells
[0537] Experiments were performed to characterize the
internalization capacity of monovalent 5T4 antibodies.
Intracellular payload delivery and resulting cytotoxicity were used
as a read out for internalization of the 5T4 antibodies upon target
binding. Bispecific, toxin-conjugated antibodies that recognize 5T4
with one Fab-arm while recognizing an irrelevant antigen (HIV-1
gp120, which is not expressed on tumor cells) with the second
Fab-arm, were generated by controlled Fab-arm exchange of
unconjugated 5T4 antibodies with (HIV-1 gp120-specific) IgG1-b12
antibodies that had been conjugated with the microtubule-disrupting
agent Duostatin-3. The resulting bispecific Duostatin-3 conjugated
antibodies carry 1 toxin molecule per antibody (drug-antibody ratio
1). Serial dilutions (0.00152-10 .mu.g/mL, 3-fold) of Duostatin-3
conjugated bispecific antibodies that monovalently bind 5T4, were
added to MDA-MB-468 (mammary cancer cell line, ATCC, clone HTB-132)
or HCC1954 (mammary cancer cell line, ATCC, clone CRL-2338) cells
seeded in flat-bottom 96-well tissue culture plates (5,000
cells/well; Greiner-bio-one, The Netherlands, cat. no. 655180). The
cells were incubated for 5 days at 37.degree. C., after which cell
viability was assessed using a CellTiter-Glo Luminescent Cell
Viability Assay (Promega, USA, cat. no. G7570) according to
manufacturer's instructions. Cytotoxicity curves were analyzed
using non-linear regression (sigmoidal dose-response with variable
slope) using GraphPad Prism V7.02 software (GraphPad Software, San
Diego, Calif., USA).
[0538] FIG. 4 shows the cytotoxic capacity of Duostatin-3
conjugated bispecific antibodies that monovalently bind 5T4 in
MDA-MB-468 (A) or HCC1954 cells (B). BsIgG1-5T4-H8-FEARxb12-vcDuo3
was highly capable of inducing cytotoxicity, indicative of an
effective internalization capacity of the antibody. In contrast,
bsIgG1-5T4-076-FEARxb12-vcDuo3, bsIgG1-5T4-085-FEARxb12-vcDuo3 and
bsIgG1-5T4-127-FEARxb12-vcDuo3 did not induce any cytotoxicity;
dose response curves were similar to that of the non-binding
IgG1-b12-vcDuo3 control antibody. This indicates poor
internalization of those antibodies upon binding to membrane-bound
5T4. BsIgG1-5T4-059-FEARxb12-vcDuo3,
bsIgG1-5T4-106-FEARxb12-vcDuo3, bsIgG1-5T4-207-FEARxb12-vcDuo3, and
bsIgG1-5T4-226-FEARxb12-vcDuo3 induced intermediate cytotoxicity in
both tested cell lines, indicating that these monovalent 5T4
antibodies induced internalization but to a lesser extent than
bsIgG1-5T4-H8-FEARxb12-vcDuo3.
Example 8--Humanized CD3 Antibodies for the Generation of CD3x5T4
Bispecific Antibodies
[0539] The generation of humanized antibody IgG1-huCD3-H1L1 is
described in Example 1 of WO2015/001085. IgG1-huCD3-H1L1 is
referred to herein as `IgG1-huCD3`. Antibody IgG1-huCD3-H1L1-FEAL
is a variant hereof with amino acid substitutions in the Fc domain
that prevent interactions with IgG Fc receptors (Fc gamma receptors
[Fc.gamma.R]) and complement, in addition to a mutation that allows
the generation of bispecific antibodies through controlled Fab-arm
exchange: L234F, L235E, D265A and F405L, as described herein above.
It has previously been demonstrated that these mutation have no
effect on target binding of the antibodies in which they are
introduced (see e.g. US 2015/0337049)
[0540] The generation of humanized antibody IgG1-huCD3-H1L1-H101G
is described in Example 2 of WO2017/009442. IgG1-huCD3-H1L1-H101G
will be referred to as `IgG1-huCD3-H101G`. Antibody
IgG1-huCD3-H101G-FEAL is a variant hereof with amino acid
substitutions L234F, L235E, D265A and F405L, as described herein
above.
Example 9--CD3 Binding Affinity Determination Using Biolayer
Interferometry
[0541] Binding affinities of selected CD3 antibodies, including
IgG1-huCD3 and IgG1-huCD3-H101G, were determined as described in
Example 7 of WO2017/009442.
[0542] In short, binding affinities of selected CD3 antibodies in
an IgG1-huCD3-FEAL format to for recombinant soluble CD3.epsilon.
(CD3E27-GSKa) (mature protein of SEQ ID NO: 101) were determined
using biolayer interferometry on a ForteBio Octet HTX (ForteBio).
Anti-human Fc capture biosensors (ForteBio, cat. no. 18-5060) were
loaded for 600 s with hIgG (1 mg/mL). After a baseline measurement
(200 s), the association (1000 s) and dissociation (2000 s) of
CD3E27-GSKa was determined, using a CD3E27-GSKa concentration range
of 27.11 .mu.g/mL-0.04 .mu.g/mL (1000 nM-1.4 nM) with three-fold
dilution steps (sample diluent, ForteBio, cat. no. 18-5028). For
calculations, the theoretical molecular mass of CD3E27-GSKa based
on the amino acid sequence was used, i.e. 27.11 kDa. Experiments
were carried out while shaking at 1000 rpm and at 30.degree. C.
Each antibody was tested in at least two independent experiments.
Data was analyzed with ForteBio Data Analysis Software v8.1, using
the 1:1 model and a global full fit with 1000 s association time
and 100 s dissociation time. Data traces were corrected by
subtraction of a reference curve (antibody on biosensor,
measurement with sample diluent only), the Y-axis was aligned to
the last 10 s of the baseline, and interstep correction as well as
Savitzky-Golay filtering was applied. Data traces with a response
<0.05 nm were excluded from analysis.
[0543] Table 5 shows the association rate constant k.sub.a (1/Ms),
dissociation rate constant k.sub.d (1/s) and equilibrium
dissociation constant K.sub.D (M) for recombinant CD3E determined
by biolayer interferometry. IgG1-huCD3-FEAL showed a relatively
high (K.sub.D: 15 nM) binding affinity to recombinant CD3E compared
to IgG1-huCD3-H101G-FEAL (K.sub.D: 638 nM).
TABLE-US-00008 TABLE 5 Binding affinities of monospecific, bivalent
CD3 antibodies to recombinant CD3.epsilon. as determined by
label-free biolayer interferometry On-rate Off-rate Antibody
k.sub.a (1/Ms) k.sub.d (1/s) K.sub.D (nM) IgG1-huCD3-FEAL 2.7E+05
4.0E-03 15 IgG1-huCD3-H101G-FEAL 3.0E+04 2.0E-02 683
Example 10--Generation of Bispecific Antibodies by 2-MEA-Induced
Fab-Arm Exchange
[0544] Bispecific antibodies were generated in vitro using the
DuoBody.RTM. platform technology, i.e. 2-MEA-induced Fab-arm
exchange as described in WO2011147986, WO2011131746 and
WO2013060867 (Genmab) and Labrijn et al. (Labrijn et al., PNAS
2013, 110: 5145-50; Gramer et al., MAbs 2013, 5: 962-973). To
enable the production of bispecific antibodies by this method, IgG1
molecules carrying a single mutation in the CH3 domain were
generated: in one parental IgG1 antibody the F405L mutation (i.e.
the CD3 antibodies), in the other parental IgG1 antibody the K409R
mutation (i.e. the 5T4 or control, HIV-1 gp120-specific,
antibodies). In addition to these mutations, the parental IgG1
antibodies included substitutions that result in a Fc domain that
is unable to interact with IgG Fc receptors (Fc gamma receptors)
and complement: L234F, L235E, D265A (FEA).
[0545] To generate bispecific antibodies, the two parental
antibodies were mixed in equal mass amounts in PBS buffer
(Phosphate Buffered Saline; 8.7 mM HPO.sub.4.sup.2-, 1.8 mM
H.sub.2PO.sub.4.sup.-, 163.9 mM Na.sup.+, 140.3 mM Cl.sup.-, pH
7.4). 2-mercaptoethylamine-HCl (2-MEA) was added to a final
concentration of 75 mM and the reaction mixture was incubated at
31.degree. C. for 5 h. The 2-MEA was removed by dialysis into PBS
buffer using 10 kDa molecular-weight cutoff Slide-A-Lyzer carriages
(Thermo Fisher Scientific) according to the manufacturer's protocol
in order to allow re-oxidation of the inter-chain disulfide bonds
and formation of intact bispecific antibodies.
[0546] The following antibodies were used in the examples:
CD3 Antibodies
[0547] IgG1-huCD3-FEAL (having the VH and VL sequences set forth in
SEQ ID NO: 57 and SEQ ID NO: 60).
[0548] IgG1-huCD3-H101G-FEAL (having the VH and VL sequences set
forth in SEQ ID NO: 68 and SEQ ID NO: 60)
5T4 Antibodies
[0549] IgG1-5T4-207-FEAR (having the VH and VL sequences set forth
in SEQ ID NO: 40 and SEQ ID NO: 44)
[0550] IgG1-5T4-226-FEAR (having the VH and VL sequences set forth
in SEQ ID NO: 47 and SEQ ID NO: 51)
[0551] IgG1-5T4-059-FEAR (having the VH and VL sequences set forth
in SEQ ID NO: 5 and SEQ ID NO: 9)
[0552] IgG1-5T4-076-FEAR (having the VH and VL sequences set forth
in SEQ ID NO: 12 and SEQ ID NO: 16)
[0553] IgG1-5T4-085-FEAR (having the VH and VL sequences set forth
in SEQ ID NO: 19 and SEQ ID NO: 23)
[0554] IgG1-5T4-106-FEAR (having the VH and VL sequences set forth
in SEQ ID NO: 26 and SEQ ID NO: 30)
[0555] IgG1-5T4-127-FEAR (having the VH and VL sequences set forth
in SEQ ID NO: 33 and SEQ ID NO: 37)
[0556] IgG1-5T4-H8-FEAR (based on 5T4 antibody H8 from Wyeth (WO
2007/106744 and US2010/0173382); having the VH and VL sequences set
forth in SEQ ID NO: 87 and SEQ ID NO: 88)
[0557] IgG1-5T4-A1-F405L (based on 5T4 antibody A1 from Wyeth (WO
2007/106744 and U.S. Pat. No. 8,044,178); having the VH and VL
sequences set forth in SEQ ID NO: 83 and SEQ ID NO: 84)
[0558] IgG1-5T4-A1-FEAR (based on 5T4 antibody A1 from Wyeth (WO
2007/106744 and U.S. Pat. No. 8,044,178); having the VH and VL
sequences set forth in SEQ ID NO: 83 and SEQ ID NO: 84)
[0559] IgG1-5T4-A3-F405L (based on 5T4 antibody A3 from Wyeth (WO
2007/106744 and U.S. Pat. No. 8,759,495); having the VH and VL
sequences set forth in SEQ ID NO: 85 and SEQ ID NO: 86)
[0560] IgG1-5T4-A3-FEAR (based on 5T4 antibody A3 from Wyeth (WO
2007/106744 and U.S. Pat. No. 8,759,495); having the VH and VL
sequences set forth in SEQ ID NO: 85 and SEQ ID NO: 86)
Bispecific Antibodies
[0561] bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR
[0562] bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR
[0563] bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR
[0564] bsIgG1-huCD3-H101G-FEALx5T4-106-FEAR
[0565] bsIgG1-huCD3-H101G-FEALx5T4-076-FEAR
[0566] bsIgG1-huCD3-H101G-FEALx5T4-085-FEAR
[0567] bsIgG1-huCD3-H101G-FEALx5T4-127-FEAR
[0568] bsIgG1-huCD3-H101G-FEALx5T4-A1-FEAR
[0569] bsIgG1-huCD3-H101G-FEALx5T4-A3-FEAR
[0570] bsIgG1-huCD3-H101G-FEALx5T4-H8-FEAR
[0571] bsIgG1-huCD3-H101G-FEALxb12-FEAR
[0572] bsIgG1-huCD3-FEALx5T4-207-FEAR
[0573] bsIgG1-huCD3-FEALx5T4-226-FEAR
[0574] bsIgG1-huCD3-FEALx5T4-059-FEAR
[0575] bsIgG1-huCD3-FEALx5T4-106-FEAR
[0576] bsIgG1-huCD3-FEALx5T4-H8-FEAR
[0577] bsIgG1-huCD3-FEALx5T4-A1-FEAR
[0578] bsIgG1-huCD3-FEALx5T4-A3-FEAR
[0579] bsIgG1-b12-FEALx5T4-207-FEAR
Fluorescein Isothiocyanate (FITC)-Labeled Bispecific Antibodies
[0580] bsIgG1-b12-FEALx5T4-059-FEAR-FITC
[0581] bsIgG1-b12-FEALx5T4-207-FEAR-FITC
[0582] bsIgG1-b12-FEALx5T4-226-FEAR-FITC
[0583] bsIgG1-5T4-A1-F405Lxb12-FEAR-FITC
[0584] bsIgG1-5T4-A3-F405Lxb12-FEAR-FITC
Duostatin-3 Conjugated Bispecific Antibodies
[0585] BsIgG1-5T4-H8-FEARxb12-vcDuo3
[0586] bsIgG1-5T4-076-FEARxb12-vcDuo3
[0587] bsIgG1-5T4-085-FEARxb12-vcDuo3
[0588] bsIgG1-5T4-127-FEARxb12-vcDuo3
[0589] BsIgG1-5T4-059-FEARxb12-vcDuo3
[0590] bsIgG1-5T4-106-FEARxb12-vcDuo3
[0591] bsIgG1-5T4-207-FEARxb12-vcDuo3
[0592] bsIgG1-5T4-226-FEARxb12-vcDuo3.
Non-Binding Control Antibodies
[0593] IgG-b12 is a HIV-1 gp120 specific antibody (Barbas, C F. J
Mol Biol. 1993 Apr. 5; 230(3):812-23) that is used in some of the
examples as negative, non-binding, control second arm for
bispecific antibodies.
[0594] IgG1-b12-F405L is a variant hereof with the substitution
F405L.
[0595] IgG1-b12-FEAL is a variant hereof with substitutions that
result in a Fc domain that is unable to interact with IgG Fc
receptors (Fc gamma receptors) and complement, in addition to a
mutation that allows the generation of bispecific antibodies
through controlled Fab-arm exchange: L234F, L235E, D265A and
F405L.
[0596] IgG1-b12-K409R is a variant hereof with the substitution
K409R.
[0597] IgG1-b12-FEAR is a variant hereof with substitutions that
result in a Fc domain that is unable to interact with IgG Fc
receptors (Fc gamma receptors) and complement, in addition to a
mutation that allows the generation of bispecific antibodies
through controlled Fab-arm exchange: L234F, L235E, D265A and
K409R.
Example 11--Binding of CD3x5T4 Bispecific Antibodies to Cynomolgus
Monkey and Human 5T4 Expressed in HEK-293 Cells
[0598] Binding of bispecific, monovalent CD3x5T4 antibodies and
monospecific, bivalent 5T4 antibodies to the plasma membrane of
HEK-293 cells transiently transfected with human 5T4 or with
cynomolgus monkey (Macaca fascicularis) 5T4 (generated as described
in Example 1) was analyzed by flow cytometry.
[0599] Cells (3.times.10.sup.4 cells/well) were incubated in
polystyrene 96-well round-bottom plates (Greiner bio-one, cat. no.
650180) with serial dilutions of antibodies (ranging from 0.0137 to
10 .mu.g/mL in 3-fold dilution steps) in 100 .mu.L PBS/0.1%
BSA/0.02% azide (staining buffer) at 4.degree. C. for 30 min.
Experiments were performed in technical duplicate. After washing
twice in staining buffer, cells were incubated in 50 .mu.L
secondary antibody at 4.degree. C. for 30 min. As a secondary
antibody, FITC-conjugated goat-anti-human IgG F(ab').sub.2
(Southern Biotech, USA, cat. no. 2043-02) diluted 1:200 in staining
buffer, was used in all experiments. Cells were washed twice in
staining buffer, re-suspended in 30 .mu.L staining buffer and
analyzed on an iQue Screener (Intellicyt Corporation, USA). Binding
curves were analyzed using non-linear regression (sigmoidal
dose-response with variable slope) using GraphPad Prism V7.02
software (Graph Pad Software, San Diego, Calif., USA).
[0600] FIGS. 5A-5D (left panels) show that bispecific antibodies
bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR (FIG. 5A),
bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR (FIG. 5B),
bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR (FIG. 5C) and
bsIgG1-huCD3-H101G-FEALx5T4-H8-FEAR (FIG. 5D), that monovalently
bind 5T4, display dose-dependent binding to HEK-293 cells
transfected with human 5T4, which was comparable to binding of
monospecific, bivalent 5T4 antibodies IgG1-5T4-207-FEAR,
IgG1-5T4-226-FEAR, IgG1-5T4-059-FEAR and IgG1-5T4-H8-FEAR,
respectively.
[0601] FIGS. 5A-5D (right panels) show that bispecific antibodies
bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR (FIG. 5A),
bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR (FIG. 5B), and
bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR (FIG. 5C), that monovalently
bind 5T4, display dose-dependent binding to HEK-293 cells
transfected with cynomolgus monkey 5T4, which was comparable to
binding of monospecific, bivalent 5T4 antibodies IgG1-5T4-207-FEAR,
IgG1-5T4-226-FEAR and IgG1-5T4-059-FEAR, respectively.
BsIgG1-huCD3-H101G-FEALx5T4-H8-FEAR and IgG1-5T4-H8-FEAR show poor
binding to cynomolgus monkey 5T4, which is in line with Example 2
and experiments described in WO2007/106744. As negative control,
IgG1-b12-K409R (3 .mu.g/mL) was included in these experiments,
which showed no binding to HEK-293 cells transfected with either
human or cynomolgus monkey 5T4.
[0602] In a second experiment, the staining was performed as
described above with minor adjustments. The cells were incubated
with serial dilutions of antibodies ranging from 0.000128 to 10
.mu.g/mL, in 5-fold dilution steps. As a secondary antibody,
Phycoerythrin (PE)-conjugated goat-anti-human IgG F(ab')2 (Jackson
Immunoresearch, UK, cat. no. 109-116-098) diluted 1:200 in staining
buffer, was used.
[0603] FIGS. 5E-5M show that antibodies
bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR and IgG1-5T4-207-FEAR (FIG.
5E), bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR and IgG1-5T4-226-FEAR
(FIG. 5F), bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR and
IgG1-5T4-059-FEAR (FIG. 5G), bsIgG1-huCD3-H101G-FEALx5T4-106-FEAR
and IgG1-5T4-106-FEAR (FIG. 5H),
bsIgG1-huCD3-H101G-FEALx5T4-076-FEAR and IgG1-5T4-076-FEAR (FIG.
5I), bsIgG1-huCD3-H101G-FEALx5T4-085-FEAR and IgG1-5T4-085-FEAR
(FIG. 5J), bsIgG1-huCD3-H101G-FEALx5T4-127-FEAR and
IgG1-5T4-127-FEAR (FIG. 5K), bsIgG1-huCD3-H101G-FEALx5T4-A1-FEAR
and IgG1-5T4-A1-FEAR (FIG. 5L), bsIgG1-huCD3-H101G-FEALx5T4-A3-FEAR
and IgG1-5T4-A3-FEAR (FIG. 5M) display dose-dependent binding to
HEK-293 cells transfected with human 5T4 (left panels) as well as
HEK-293 cells with cynomolgus monkey 5T4 (right panels). Again, the
binding curves of the bivalent, monospecific and bispecific,
monovalent antibodies display a similar trend between human and
cynomolgus 5T4.
Example 12--Binding of CD3x5T4 Bispecific Antibodies to
5T4-Positive Human Tumor Cells
[0604] Binding of CD3x5T4 bispecific antibodies to the
5T4-expressing human tumor cell lines HeLa (cervix adenocarcinoma;
ATCC, cat. no. CCL-2) and MDA-MB-231 (breast adenocarcinoma; ATCC,
cat. no. HTB-26) cell line was analyzed by flow cytometry. Neither
HeLa nor MDA-MB-231 cells express CD3.
[0605] Cells (3.times.10.sup.4 cells/well) were incubated in
polystyrene 96-well round-bottom plates (Greiner bio-one, cat. no.
650180) with serial dilutions of antibodies (range 0.000152 to 3
.mu.g/mL in 3-fold dilution steps) in 100 .mu.L PBS/0.1% BSA/0.02%
azide (staining buffer) at 4.degree. C. for 30 min. After washing
twice in staining buffer, cells were incubated in 50 .mu.L
secondary antibody at 4.degree. C. for 30 min. As a secondary
antibody, Fluorescein isothiocyanate (FITC)-conjugated
goat-anti-human IgG F(ab').sub.2 (Southern Biotech, USA, cat. no.
2043-02) diluted 1:400 in staining buffer, was used for the first
experiment. Next, cells were washed twice in staining buffer,
re-suspended in 120 .mu.L staining buffer and analyzed on a BD
LSRFortessa FACS (BD Biosciences, USA). Binding curves were
analyzed using non-linear regression (sigmoidal dose-response with
variable slope) using GraphPad Prism V7.02 software (GraphPad
Software, San Diego, Calif., USA).
[0606] FIGS. 6A-6C (left panels) show that the CD3x5T4 bispecific
antibodies bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR (FIG. 6A) and
bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR (FIG. 6B) display
dose-dependent binding to HeLa cells, with higher maximum binding
than the monospecific, bivalent 5T4 antibodies IgG1-5T4-207-FEAR
and IgG1-5T4-059-FEAR. For bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR
(FIG. 6C) the maximum binding was similar to that of the
monospecific, bivalent 5T4 antibody IgG1-5T4-226-FEAR on HeLa
cells.
[0607] FIGS. 6A-6C (right panels) show that the CD3x5T4 bispecific
antibodies bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR (FIG. 6A),
bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR (FIG. 6B) and
bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR (FIG. 6C) display
dose-dependent binding to MDA-MB-231 cells, with higher maximum
binding than the monospecific, bivalent 5T4 antibodies
IgG1-5T4-207-FEAR, IgG1-5T4-226-FEAR and IgG1-5T4-059-FEAR. The
negative control antibody that was included in these experiments,
IgG1-b12-K409R (3 .mu.g/mL), did not show binding to HeLa and
MDA-MB-231 cells.
[0608] In a second experiment, the staining was performed as
described above with minor adjustments. The cells were incubated
with serial dilutions of antibodies, ranging from 0.000128 to 10
.mu.g/mL, in 5-fold dilution steps. As a secondary antibody,
Phycoerythrin (PE)-conjugated goat-anti-human IgG F(ab')2 (Jackson
Immunoresearch, UK, cat. no. 109-116-098) diluted 1:200 in staining
buffer, was used.
[0609] FIGS. 6D-6K and 6L-6S show that antibodies
bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR and IgG1-5T4-207-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR and IgG1-5T4-226-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR and IgG1-5T4-059-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-106-FEAR and IgG1-5T4-106-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-085-FEAR and IgG1-5T4-085-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-127-FEAR and IgG1-5T4-127-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-A1-FEAR and IgG1-5T4-A1-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-A3-FEAR and IgG1-5T4-A3-FEAR display
dose-dependent binding to HeLa and MDA-MB-231 tumor cells. In
general, bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-106-FEAR, IgG1-5T4-207-FEAR,
IgG1-5T4-226-FEAR, IgG1-5T4-059-FEAR, IgG1-5T4-106-FEAR,
IgG1-5T4-085-FEAR and IgG1-5T4-127-FEAR display binding at lower
antibody concentrations compared to
bsIgG1-huCD3-H101G-FEALx5T4-A1-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-A3-FEAR, IgG1-5T4-A1-FEAR and
IgG1-5T4-A3-FEAR.
Example 13--Induction of T-Cell Activation, Cytokine Release and
Cytotoxicity In Vitro by CD3x5T4 Bispecific Antibodies Using
Purified T Cells as Effector Cells
[0610] CD3x5T4 bispecific antibodies were tested in an in vitro
cytotoxicity assay using 5T4-positive tumor cell lines as target
cells and purified T cells as effector cells. T cells were derived
from healthy human donor buffy coats (Sanquin, Amsterdam, The
Netherlands) and isolated using the RosetteSep human T cell
enrichment cocktail (Stemcell Technologies, France, cat. no. 15061)
according to the manufacturer's instructions. To determine the
percentage of viable T cells after isolation (either total T cells,
CD4.sup.+ T cells or CD8.sup.+ T cells), a sample of the isolated T
cells (2.5.times.10.sup.5 cells per condition) was stained for 30
min at 4.degree. C. in a U-well 96-well plate (Cellstar, cat. no.
650180) using the following antibodies: Pacific Blue-anti-CD3
(eBiosciences, clone OKT3), APC-Cy-anti-CD4 (eBiosciences, clone
OKT4), AF700-anti-CD8 (Biolegend, clone RPA-T8) and viability
marker FVS 510 (BD Biosciences) in 100 .mu.L PBS/0.1% BSA/0.02%
azide (staining buffer). Next, cells were washed twice in staining
buffer, re-suspended in 120 .mu.L staining buffer and analyzed on a
BD LSRFortessa FACS (BD Biosciences, USA). The percentages of
CD3.sup.+, CD3.sup.+CD4.sup.+ and CD3.sup.+CD8.sup.+ T cells for
each of the donors used in the cytotoxicity experiment are
described in Table 6.
TABLE-US-00009 TABLE 6 Ratio CD3.sup.+, CD4.sup.+ and CD8.sup.+ T
cells per donor % CD4+ % CD8+ % CD3+ of within CD3+ withinCD3+
Donor viable cells cells cells A 91.2 84.2 11.8 B 77.8 78.3 18 C
97.6 78.1 19.6 D 92.6 77.3 15.5 E 99.2 78.4 20.3
[0611] MDA-MB-231 cells (16,000 cells/well) were seeded into flat
bottom 96-well plates (Greiner-bio-one, The Netherlands, cat. no.
655180) and left to adhere for 4 hours at 37.degree. C. T cells
were added to tumor cells at an E:T ratio=8:1. Serial dilutions of
bispecific CD3x5T4 antibodies or monospecific, bivalent 5T4
antibodies were added (final concentration ranging from 1000 to
0.0128 ng/mL; 5-fold dilutions) and plates were incubated for 72
hours at 37.degree. C. Next, 110 .mu.L supernatants containing T
cells were transferred to U-bottom 96 Well culture plates
(CellStar, cat. no. 650180). Plates were centrifuged (300.times.g)
for 3 min at 4.degree. C., after which 75 .mu.L of supernatant was
transferred to a new plate for cytokine production measurement, and
T cells were kept to assess T cell activation markers (described
below). Cytokine production induced by 0.2 .mu.g/mL CD3x5T4
bispecific antibodies was analyzed by a multiplex U-plex assay
(MeSo Scale Discovery, USA, cat. no. K15049K) according to
manufacturer's instructions.
[0612] T cells were stained for T-cell markers CD3 (1:200;
eBioscience, clone OKT3, conjugated to eFluor450), CD4 (1:50;
eBioscience, clone OKT4, conjugated to APC-eFluor780), CD8 (1:100;
Biolegend, clone RPA-T8, conjugated to AF700) and T-cell activation
markers CD69 (1:50; BD Biosciences, clone AB2439, conjugated to
APC), CD25 (1:50; eBioscience, clone BC96, conjugated to PE-Cy7)
and CD279/PD1 (1:50; Biolegend, clone EH12.2H7, conjugated to
BV605). Single stained samples with Ultracomp beads (5 .mu.L;
Invitrogen, cat. no. 01-2222-42) were used for compensation
adjustments of the flow cytometer. After 30 min of incubation at
4.degree. C., plates were washed three times with PBS/0.1%
BSA/0.02% azide (staining buffer). Cells were resuspended in 120
.mu.L staining buffer and analyzed using a FACS Fortessa (BD
Biosciences). Data were processed using FlowJo (BD
Biosciences).
[0613] In parallel, the viability of the tumor cells was assessed
using Resazurin (7-Hydroxy-3H-phenoxazin-3-one 10-oxide). The
adherent tumor cells were washed twice with PBS and incubated with
10% Resazurin (150 .mu.L; Life Technologies, The Netherlands, cat.
no. DAL1100) in RPMI-1640 (Lonza, Switzerland, cat. no. BE12-115F)
medium containing 10% donor bovine serum with iron (Life
Technologies, The Netherlands, cat. no. 10371-029) and pen/strep
(Lonza, cat. no. DE17-603E) for 4 h at 37.degree. C. The absorbance
was measured with an Envision multilabel plate reader (PerkinElmer,
US). The absorbance of staurosporine-treated (Sigma-Aldrich, US,
cat. no. 56942) tumor cell samples wasset as 0% viability and the
absorbance of untreated tumor cell samples was set as 100%
viability. The `percentage viable cells` was calculated as
follows:
% viable cells=([absorbance sample-absorbance staurosporine-treated
target cells]/[absorbance untreated target cells-absorbance
staurosporine treated target cells]).times.100.
[0614] Dose-response curves, EC50 and IC50 values were analyzed
using non-linear regression (sigmoidal dose-response with variable
slope) using GraphPad Prism V7.02 software (GraphPad Software, San
Diego, Calif., USA).
[0615] FIGS. 7A-7C show that bsIgG1-huCD3-FEALx5T4-207-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR,
bsIgG1-huCD3-FEALx5T4-226-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR,
bsIgG1-huCD3-FEALx5T4-059-FEAR and
bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR induced dose-dependent
cytotoxicity (shown as decrease in % viable cells) in the
5T4-positive tumor cell line MDA-MB-231. Donor-to-donor variation
was observed, but T cells of both donors induced maximum kill in
the presence of 1 .mu.g/mL CD3x5T4 bispecific antibody.
Monospecific, bivalent antibodies IgG1-5T4-207-FEAR,
IgG1-5T4-226-FEAR and IgG1-5T4-059-FEAR did not induce
cytotoxicity. IC.sub.50 values calculated from the graphs are
presented in FIG. 7D. The IC.sub.50 value of
bsIgG1-huCD3-FEALx5T4-207-FEAR and bsIgG1-huCD3-FEALx5T4-059-FEAR
were lower compared to bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR and
bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR, respectively. In contrast,
the IC.sub.50 value of bsIgG1-huCD3-FEALx5T4-226-FEAR was
comparable to bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR.
[0616] FIGS. 8A-8F show that bsIgG1-huCD3-FEALx5T4-207-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR,
bsIgG1-huCD3-FEALx5T4-226-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR,
bsIgG1-huCD3-FEALx5T4-059-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR,
bsIgG1-huCD3-FEALx5T4-106-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-106-FEAR,
bsIgG1-huCD3-FEALx5T4-A1-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-A1-FEAR,
bsIgG1-huCD3-FEALx5T4-A3-FEAR and
bsIgG1-huCD3-H101G-FEALx5T4-A3-FEAR induced T-cell mediated
cytotoxicity (shown as decrease in tumor cell survival) in
MDA-MB-231 cell line. Bivalent, monospecific antibodies
IgG1-5T4-207-FEAR, IgG1-5T4-226-FEAR, IgG1-5T4-059-FEAR,
IgG1-5T4-106-FEAR, IgG1-5T4-A1-FEAR and IgG1-5T4-A3-FEAR did not
induce T-cell-mediated cytotoxicity. IC50 values calculated from
the graphs are presented in FIGS. 8G-8H. IC50 values of the T-cell
mediated cytotoxicity induced by bsIgG1-huCD3-FEALx5T4-207-FEAR,
bsIgG1-huCD3-FEALx5T4-226-FEAR, bsIgG1-huCD3-FEALx5T4-059-FEAR and
bsIgG1-huCD3-FEALx5T4-106-FEAR are lower than the IC50 values of
bsIgG1-huCD3-FEALx5T4-A1-FEAR and bsIgG1-huCD3-FEALx5T4-A3-FEAR.
Also, IC50 values of the T-cell mediated cytotoxicity induced by
bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR and
bsIgG1-huCD3-H101G-FEALx5T4-106-FEAR are lower than the IC50 values
of bsIgG1-huCD3-H101G-FEALx5T4-A1-FEAR and
bsIgG1-huCD3-H101G-FEALx5T4-A3-FEAR.
[0617] T-cell activation was determined by flow cytometry through
staining for activation markers PD1, CD25 and CD69 (FIGS. 9A-9C).
Monospecific, bivalent antibodies IgG1-5T4-207-FEAR,
IgG1-5T4-226-FEAR and IgG1-5T4-059-FEAR did not induce upregulation
of these T-cell activation markers, while bispecific antibodies
bsIgG1-huCD3-FEALx5T4-207-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR,
bsIgG1-huCD3-FEALx5T4-226-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR,
bsIgG1-huCD3-FEALx5T4-059-FEAR and
bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR induced dose-dependent
upregulation of PD1, CD25 and CD69. EC.sub.50 values calculated
from the graphs are represented in FIG. 9D. The EC.sub.50 values
for upregulation of PD1, CD25 and CD69 by
bsIgG1-huCD3-FEALx5T4-207-FEAR and bsIgG1-huCD3-FEALx5T4-059-FEAR
were lower compared to bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR and
bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR, respectively. The EC.sub.50
values for upregulation of CD25 and CD69 by
bsIgG1-huCD3-FEALx5T4-226-FEAR were lower compared to
bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR, while the EC.sub.50 value for
PD1 upregulation was comparable between
bsIgG1-huCD3-FEALx5T4-226-FEAR and
bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR.
[0618] FIGS. 10A-10F show that bsIgG1-huCD3-FEALx5T4-207-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR,
bsIgG1-huCD3-FEALx5T4-226-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR,
bsIgG1-huCD3-FEALx5T4-059-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR,
bsIgG1-huCD3-FEALx5T4-106-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-106-FEAR,
bsIgG1-huCD3-FEALx5T4-A1-FEAR, bsIgG1-huCD3-H101G-FEALx5T4-A1-FEAR,
bsIgG1-huCD3-FEALx5T4-A3-FEAR and
bsIgG1-huCD3-H101G-FEALx5T4-A3-FEAR induced T-cell activation
(exemplified in FIGS. 10A-10F by increase in % CD69.sup.+ T cells
within the CD4.sup.+ and CD8.sup.+ T cell populations) when
incubated with the MDA-MB-231 cell line, while the bivalent,
monospecific antibodies IgG1-5T4-207-FEAR, IgG1-5T4-226-FEAR,
IgG1-5T4-059-FEAR, IgG1-5T4-106-FEAR, IgG1-5T4-A1-FEAR and
IgG1-5T4-A3-FEAR did not induce T-cell activation. EC50 values of
three
[0619] T-cell activation markers are shown in FIGS. 10G-10L. In
general, the EC50 values of the T-cell activation (increase in %
CD69.sup.+, CD25.sup.+ and PD1+ cells within the CD4.sup.+ and
CD8.sup.+ T cell populations) induced by
bsIgG1-huCD3-FEALx5T4-207-FEAR, bsIgG1-huCD3-FEALx5T4-226-FEAR,
bsIgG1-huCD3-FEALx5T4-059-FEAR and bsIgG1-huCD3-FEALx5T4-106-FEAR
are lower than the EC50 values of bsIgG1-huCD3-FEALx5T4-A1-FEAR and
bsIgG1-huCD3-FEALx5T4-A3-FEAR. Also, EC50 values of T-cell
activation (increase in % of CD69.sup.+, CD25.sup.+ and PD1.sup.+ T
cells within the CD4.sup.+ and CD8.sup.+ T cell populations)
induced by bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR and
bsIgG1-huCD3-H101G-FEALx5T4-106-FEAR are lower than the EC50 values
of bsIgG1-huCD3-H101G-FEALx5T4-A1-FEAR and
bsIgG1-huCD3-H101G-FEALx5T4-A3-FEAR.
[0620] Production of the cytokines IL-10, IL-13 and TNF after
exposure of co-cultures of T cells and MDA-MB-231 cells to 0.2
.mu.g/mL CD3x5T4 bispecific antibodies was measured in culture
supernatant, by multiplex U-plex assay. FIG. 11 shows the cytokine
levels in the supernatant of T cell-tumor cell co-cultures, after
incubation with bispecific antibodies. Experiments were performed
using T cells from two different healthy donors; FIG. 11A shows the
results from co-cultures with T cells derived from donor A, FIG.
11B shows the results from co-cultures with T cells derived donor
B. Bispecific antibodies bsIgG1-huCD3-FEALx5T4-207-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR,
bsIgG1-huCD3-FEALx5T4-226-FEAR,
bsIgG1-huCD3-H101G-FEALx5T4-226-FEAR,
bsIgG1-huCD3-FEALx5T4-059-FEAR and
bsIgG1-huCD3-H101G-FEALx5T4-059-FEAR all induced cytokine release,
although the cytokine levels in T cell-tumor cell co-cultures
incubated with CD3x5T4 bispecific antibodies containing a
IgG1-huCD3-H101G-FEAL-derived CD3-specific Fab-arm were lower than
cytokine levels in co-cultures that had been incubated with
bispecific antibodies containing a IgG1-huCD3-FEAL-derived
CD3-specific Fab-arm. The monospecific antibodies
IgG1-5T4-207-FEAR, IgG1-5T4-226-FEAR and IgG1-5T4-059-FEAR did not
induce any cytokine release.
Example 14--Induction of Cytotoxicity In Vitro by CD3x5T4
Bispecific Antibodies Using PBMCs or Purified T Cells as Effector
Cells at Varying Effector to Target Ratios
[0621] To determine the efficiency of the T-cell-mediated kill of
bispecific antibodies bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR and
bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR in more detail, a cytotoxicity
assay was performed as described in Example 13, with varying
effector to target cell (E:T) ratios. In addition, either
peripheral blood mononuclear cells (PBMCs) or isolated T cells were
used as effector cells. The ovarian cancer cell line SK-OV-3 (9,000
cells/well, ATCC, cat. no. HTB-77) was used as target cell line.
PBMCs were isolated from 40 mL of buffy coat of human blood
(Sanquin) using a Ficoll gradient (Lonza; lymphocyte separation
medium, cat. no. 17-829E) according to the manufacturer's
instructions. T cells were isolated as described in Example 13. For
PBMCs, the following E:T ratios were used: 1:2, 1:1, 2:1, 4:1, 8:1
and 12:1. For isolated T cells, the following E:T ratios were used:
1:2, 1:1, 2:1, 4:1 and 8:1. In each experiment, effector cells from
two separate donors were used. Table 7 provides an overview of the
percentage of CD3.sup.+, CD3.sup.+CD4.sup.+ and CD3.sup.+CD8.sup.+
T cells in the PMBC or T-cell isolates for each of the donors
(determined as described in Example 13).
TABLE-US-00010 TABLE 7 Ratio CD3.sup.+, CD4.sup.+ and CD8.sup.+ T
cells per donor. % CD3 with % CD4.sup.+ % CD8.sup.+ viable cell
within CD3.sup.+ within CD3.sup.+ Donor population cells cells C
(PBMCs) 75 56.8 28.9 D (PBMCs) 60 63.2 32 E (T cells) 98.3 59.6
31.6 F (T cells) 97.2 70 26.4
[0622] As shown in FIG. 12, using effector cells from two different
donors, E:T ratios from 4:1 to 12:1 resulted in efficient
PBMC-mediated kill of the SK-OV-3 cells in the presence of
bsIgG1-huCD3-FEALx5T4-207-FEAR or
bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR. At E:T ratios of 2:1 and
lower, maximum kill of the SK-OV-3 cells was not achieved at the
highest antibody concentration used (1000 ng/mL). A similar result
was observed when isolated T cells were used as effector cells
(FIG. 13). Using effector cells from two different donors, an E:T
ratio of 4:1 and 8:1 resulted in maximum T-cell-mediated kill of
the SK-OV-3 cells in the presence of bsIgG1-huCD3-FEALx5T4-207-FEAR
or bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR at the highest antibody
concentration used (1000 ng/mL), whereas lower E:T ratios were not
sufficient to induce maximum kill. The efficacy of the
T-cell-mediated kill induced by bsIgG1-huCD3-FEALx5T4-207-FEAR and
bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR is thus dependent on a
sufficiently high E:T ratio.
Example 15--Anti-Tumor Activity of CD3x5T4 Bispecific Antibodies in
a Humanized Immune System Mouse Xenograft Model
[0623] The in vivo anti-tumor efficacy of the CD3x5T4 bispecific
antibodies bsIgG1-huCD3-FEALx5T4-207-FEAR and
bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR was evaluated in humanized
(tail vein injected CD34+ hematopoietic stem cells [HSC] at an age
of 3-4 weeks) NOD.Cg-Prkdc.sup.scid ll2rg.sup.tm1Wjl/SzJ (NSG-HIS)
mice (obtained from The Jackson Laboratory) that were inoculated
subcutaneously with human MDA-MB-231 tumor cells. Humanization of
the immune system of NSG-HIS mice was confirmed 16 weeks
post-engraftment by flow cytometry. Subsequently, NSG-HIS mice were
randomized in three groups (8 mice per group), based on HSC donor
(#5239 or #2328) and the percentage of human CD3.sup.+ T cells
within the human CD45.sup.+ population in peripheral blood (mean %
hCD45.sup.+ and % hCD3.sup.+ cells respectively; 42% hCD45.sup.+
and 39% hCD3.sup.+ for the PBS group, 34% hCD45.sup.+ and 25%
hCD3.sup.+ for the bsIgG1-huCD3-FEALx5T4-207-FEAR group, and 36%
hCD45.sup.+ and 29% hCD3.sup.+ for the
bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR). 5.times.10.sup.6 MDA-MB-231
cells (in 100 .mu.L PBS) were injected subcutaneously (SC) in the
flank of the mice; this was indicated as day 0 in the study. At day
14, 18, 21 and 25, the mice were injected intravenously (IV) with
either 0.5 mg/kg antibody or PBS. Treatment groups are shown in
Table 8. Tumor growth was evaluated twice per week (starting at day
14) using a caliper. Tumor volumes (mm.sup.3) were calculated from
caliper measurements as
0.52.times.(length).times.(width).sup.2.
[0624] The results are shown in FIG. 14. FIG. 14A shows that both
bsIgG1-huCD3-FEALx5T4-207-FEAR (p<0.01) and
bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR (p<0.05) efficiently
inhibited tumor growth based on Mann-Whitney statistical analysis
at day 43 compared to the control group. Furthermore, statistical
analysis of the tumor-free survival curves (Kaplan Meier plot,
using a tumor size <500 mm.sup.3 as a cut-off) using a Mantel
Cox test demonstrated that the difference in tumor-free survival
was statistically different, showing increased tumor-free survival
in animals treated with bsIgG1-huCD3-FEALx5T4-207-FEAR (p<0.001)
or bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR (p<0.001) compared to
the untreated animals (FIG. 14B).
TABLE-US-00011 TABLE 8 Treatment groups. Treatment Animals Antibody
Dose days per group PBS -- 14, 18, 21, 25 8 bsIgG1-huCD3- 0.5 mg/kg
14, 18, 21, 25 8 FEALx5T4-207-FEAR bsIgG1-huCD3- 0.5 mg/kg 14, 18,
21, 25 8 H101G-FEALx5T4-207-FEAR
Example 16. Determination of the Contribution of 5T4 Amino Acid
Residues to Antibody Binding Using Alanine Scanning
Library Design
[0625] A human 5T4 (Uniprot ID Q13641) single residue alanine
library was synthesized (GeneArt, Thermo Fisher Scientific), in
which all amino acid residues in the extracellular domain of human
5T4 were individually mutated to alanine, except for positions
already containing an alanine or cysteine. To minimize the chance
of structural disruption of the antigen, cysteines were not
mutated. The library was cloned in the pMAC expression vector
containing a CMV/TK-polyA expression cassette, an Ampicillin
resistance gene and a pBR322 replication origin.
Library Production and Screening
[0626] The wild type 5T4 and alanine mutants were expressed
individually in FreeStyle HEK293 cells according to the
manufacturer's instructions (Thermo Fisher Scientific, cat. no.
12347-019). One day post transfection, the cells were harvested.
Approximately 80,000 cells were incubated with 20 .mu.L
FITC-conjugated antibody (3 .mu.g/mL; in FACS buffer (PBS [Lonza,
cat. no. BE17-517]+0.1% [w/v] BSA [Roche, cat. no.
10735086001]+0.02% [w/v] sodium azide [NaN.sub.3; EMELCA
Bioscience, cat. no. 41920044-3]); Table 9) at room temperature for
40 min. Subsequently, cells were washed twice by centrifugation
using 150-180 .mu.L FACS buffer. Cells were resuspended in 30 .mu.L
FACS buffer and stored at 4.degree. C. until analysis by flow
cytometry using an iQue screener (Intellicyt Corporation).
[0627] The entire experiment was performed twice yielding duplicate
measurements.
TABLE-US-00012 TABLE 9 Antibodies used in determination of the
contribution of 5T4 amino acid residues in antibody binding using
alanine scanning. Antibodies monovalently binding to 5T4 were
labeled with FITC (Thermo Fisher Scientific, cat. no. 46425), prior
to performing the experiment. IgG1-5T4-A1-F405L and
IgG1-5T4-A3-F405L are surrogate A1 and A3 antibodies, respectively,
that were cloned into the human IgG1 backbone containing the F405L
mutations. Hence, the surrogate A1 antibody has a variable region
identical to that of the A1 antibody disclosed in WO2007106744.
Likewise, the A3 surrogate antibody has a variable region identical
to that of the A3 antibody disclosed in WO2007106744. In both
antibodies, the Fc domain carries the F405L substitution. Antibody
Test or control antibody bsIgG1-b12-FEALx5T4-059-FEAR-FITC Test
antibody bsIgG1-b12-FEALx5T4-207-FEAR-FITC Test antibody
bsIgG1-b12-FEALx5T4-226-FEAR-FITC Test antibody
bsIgG1-5T4-A3-F405Lxb12-FEAR-FITC Test antibody
bsIgG1-5T4-A1-F405Lxb12-FEAR-FITC Control antibody used for
normalization
Data Analysis
[0628] For every sample, the average amount of antibody bound per
cell was determined as the geometric mean of the fluorescence
intensity (gMFI) for the viable, single cell population. The gMFI
is influenced by the affinity of the antibody for the 5T4 mutant
and the expression level of the 5T4 mutant per cell. Since specific
alanine mutations can impact the surface expression level of the
mutant 5T4, and to correct for expression differences for each 5T4
mutant in general, data for each test antibody were normalized
against the binding intensity of a non-cross blocking 5T4-specific
control antibody, using the following equation:
Normalized gMFI aa position = Log 10 ( gMFI Test Ab gMFI Control Ab
) ##EQU00007##
[0629] In which `aa position` refers to the position that was
mutated into an alanine; and the Z-score was calculated to express
loss or gain of binding of the antibodies, according to the
following calculation:
Z - score ( fold change ) = Normalized gMFI aa position - .mu.
.sigma. ##EQU00008##
[0630] Where .mu. and .sigma. are the mean and standard deviation
of the Normalized gMFI calculated from all mutants.
[0631] If the gMFI of the control antibody for a particular 5T4
mutant was lower than the mean gMFIControl Ab-2.5.times.SD of the
mean gMFIControl Ab (from all mutants), data were excluded from
analysis (it was assumed that expression levels for those 5T4
mutants were not sufficient to draw conclusions). This was the case
for amino acid W at position 296 (SEQ ID NO: 1).
Results
[0632] FIG. 15 shows the binding results of the tested antibodies
to human 5T4 variants with single alanine mutations in the ECD:
positions 32 to 355 (according to SEQ ID NO: 1). The results
indicate that antibody bsIgG1-b12-FEALx5T4-059-FEAR-FITC showed
loss of binding when aa R at position 73, T at position 74, Y at
position 92, R at position 94, N at position 95 or F at position
138 of human 5T4 were mutated to an alanine. This suggests that
binding of antibody IgG1-5T4-059-04-FEAR is at least dependent on
aa R73, T74, Y92, R94, N95, F138 of human 5T4 (SEQ ID NO: 1),
antibody bsIgG1-b12-FEALx5T4-207-FEAR-FITC showed loss of binding
when aa S at position 69, R at position 73, Y at position 92, R at
position 94, F at position 111, F at position 138, D at position
148 of human 5T4 were mutated to an alanine. This suggests that
binding of antibody IgG1-5T4-207-FEAR is at least dependent on aa
S69, R73, Y92, R94, F111, F138 and D148 of human 5T4 (SEQ ID NO:
1),antibody bsIgG1-b12-FEALx5T4-226-FEAR-FITC showed loss of
binding when aa R at position 73, Y at position 92, R at position
94, F at position 111, F at position 138, L at position 144 or D at
position 148 of human 5T4 were mutated to an alanine. This suggests
that binding of antibody IgG1-5T4-226-FEAR is at least dependent on
aa R73, Y92, R94, F111, F138, L144 and D148 of human 5T4 (SEQ ID
NO: 1), antibody bsIgG1-5T4-A3-F405Lxb12-FEAR-FITC showed loss of
binding when aa D at position 60, Q at position 61, D at position
88, L at position 89, Y at position 92, F at position 111, P at
position 115, L at position 117, F at position 138, D at position
148 or N at position 152 of human 5T4 were mutated to an alanine.
This suggests that binding of antibody IgG1-5T4-A3-FEAR is at least
dependent on aa D60, Q61, D88, L89, Y92, F111, P115, L117, F138,
D148 and N152 of human 5T4 (SEQ ID NO: 1).
[0633] Some amino acids might be indirectly involved in binding.
For example, mutating a hydrophobic residue to alanine might impact
the local folding and affect the positioning of directly
interacting residues (Zhao et al., 2014 Structure 22, 612-620).
Based on structural data (human 5T4 crystal structure 4cnm; RCSB
protein databank) the following residues are buried and therefore
expected to indirectly contribute to binding to: [0634] antibody
bsIgG1-b12-FEALx5T4-059-04-FEAR-FITC: F138, [0635] antibody
bsIgG1-b12-FEALx5T4-207-FEAR-FITC: F111, F138, D148, [0636]
antibody bsIgG1-b12-FEALx5T4-226-FEAR-FITC: F111, F138, L144, D148,
[0637] antibody bsIgG1-5T4-A3-F405Lxb12-FEAR-FITC: L89, F111, L117,
F138, D148, N152.
[0638] Since only surface-exposed residues can directly interact
with the antibody, the following residues are expected to directly
interact with: [0639] antibody bsIgG1-b12-FEALx5T4-059-FEAR-FITC:
R73, T74, Y92, R94 and N95, [0640] antibody
bsIgG1-b12-FEALx5T4-207-FEAR-FITC: S69, R73, Y92 and R94, [0641]
antibody bsIgG1-b12-FEALx5T4-226-FEAR-FITC: R73, Y92 and R94,
[0642] antibody bsIgG1-5T4-A3-F405Lxb12-FEAR-FITC: D60, Q61, D88,
Y92 and P115.
[0643] Together, these results propose that antibodies
IgG1-5T4-059, IgG1-5T4-207 and IgG1-5T4-226 all bind by direct
interaction with amino acid residues R73, Y92 and R94. The results
also indicate that antibodies IgG1-5T4-059, IgG1-5T4-207 and
IgG1-5T4-226 each bind to a epitope which is different from but
partially overlapping with the epitope bound by IgG1-5T4-A3. This
is in line with the displacement behavior described in Example 3
and 4.
Example 17: Induction of T-Cell Activation and Cytotoxicity by
CD3x5T4 Bispecific Antibodies in Cell Lines of Different
Indications In Vitro
[0644] CD3x5T4 bispecific antibodies were tested in an in vitro
cytotoxicity assay using tumor cell lines of pancreas and cervical
cancer as target cells and purified T cells as effector cells. For
each indication (pancreas cancer and cervical cancer) two
representative cell lines were selected. The tumor cell lines used
in the in vitro cytotoxicity assay are summarized in Table 10. T
cells were derived from human donor buffy coats (Sanquin,
Amsterdam, The Netherlands) and isolated using the RosetteSep human
T cell enrichment cocktail (Stemcell Technologies, France, cat. no.
15061) according to manufacturer's instructions. For each cell
line, at least three different donors were tested in the in vitro
cytotoxicity assay and T-cell activation analysis, as summarized in
Table 10.
TABLE-US-00013 TABLE 10 Tumor cell lines used for in vitro
cytotoxicity assay Tumor ATCC cytotox T-cell activation cell line
Indication clone no. (n) (n) BxPC-3 Pancreas CRL-1687 3 3 PANC-1
Pancreas CRL-1469 9 4 Ca Ski Cervical CRL-1550 5 3 SiHa Cervical
HTB-35 3 3
[0645] Tumor cells (16,000 cells/well) were seeded into flat-bottom
96-well plates (Greiner Bio-One, The Netherlands, cat. no. 655180)
and left to adhere at 37.degree. C. for 4 h. T cells were added to
tumor cells at an E:T ratio=4:1. Serial dilutions of
bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR or control antibodies
(bsIgG1-huCD3-H101G-FEALxb12-FEAR, bsIgG1-b12-FEALx5T4-207-FEAR)
were added (final concentration ranging from 5000 to 0.0128 ng/mL;
5-fold dilutions) and plates were incubated at 37.degree. C. for 72
h. Next, 110 .mu.L supernatants containing T cells were transferred
to round-bottom 96-well culture plates (CellStar, cat. no. 650180)
and centrifuged (300.times.g) at 4.degree. C. for 3 min. T cells
were stained for T-cell markers by incubation with CD3-eFluor450
(1:200; eBioscience, clone OKT3), CD4-APC-eFluor780 (1:50;
eBioscience, clone OKT4), CD8-AF700 (1:100; Biolegend, clone
RPA-T8) and T-cell activation markers CD69-APC (1:50; BD
Biosciences, clone AB2439), CD25-PE-Cy7 (1:50; eBioscience, clone
BC96) and CD279/PD1-BV605 (1:50; Biolegend, clone EH12.2H7) diluted
in 50 .mu.L PBS/0.1% BSA/0.02% azide (staining buffer). Single
stained samples with Ultracomp beads (5 .mu.L; Invitrogen, cat. no.
01-2222-42) were used for compensation adjustments of the flow
cytometer. After 30 min of incubation at 4.degree. C., plates were
washed three times with staining buffer. Cells were resuspended in
120 .mu.L staining buffer and analyzed using a FACS Fortessa (BD
Biosciences). Data were processed using FlowJo (version 10, BD
Biosciences).
[0646] In parallel, the viability of the tumor cells was assessed
using Resazurin (7-Hydroxy-3H-phenoxazin-3-one 10-oxide). The
adherent tumor cells were washed twice with PBS and incubated with
10% Resazurin (150 .mu.L; Life Technologies, The Netherlands, cat.
no. DAL1100) in RPMI-1640 medium (Lonza, Switzerland, cat. no.
BE12-115F) supplemented with 10% donor bovine serum with iron (Life
Technologies, The Netherlands, cat. no. 10371-029) and pen/strep
(Lonza, cat. no. DE17-603E) at 37.degree. C. for 4 h. The
absorbance was measured with an Envision multilabel plate reader
(PerkinElmer, US). The absorbance of staurosporine-treated
(Sigma-Aldrich, US, cat. no. 56942) cells were set as 0% viability
and the absorbance of untreated cells were set as 100% viability.
The `percentage viable cells` was calculated as follows:
% viable cells=([absorbance sample-absorbance staurosporine-treated
target cells]/[absorbance untreated target cells-absorbance
staurosporine treated target cells]).times.100.
[0647] Cytotoxicity curves, T-cell activation curves, IC50
(cytotoxicity) and EC50 (T-cell activation) values were analyzed
using non-linear regression (sigmoidal dose-response with variable
slope) using GraphPad Prism V7.02 software (GraphPad Software, San
Diego, Calif., USA).
[0648] FIGS. 16A-16B show that bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR
induced cytotoxicity in a range of cell lines of different
indications, while the control bispecific antibodies
(bsIgG1-huCD3-H101G-FEALxb12-FEAR, bsIgG1-b12-FEALx5T4-207-FEAR)
targeting only the tumor cells or the T cells did not show any
cytotoxicity. FIG. 16C shows the mean IC.sub.50 values for each of
the cell lines tested with different donors (at least n=3). FIGS.
17A-17D show the T-cell activation induced by
bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR in a range of cell lines of
different indications as measured by the upregulation of CD69 on
CD4.sup.+ and CD8.sup.+ T cells (% of CD69.sup.+ cells within the
CD4.sup.+ or CD8.sup.+ population). The control bispecific
antibodies (bsIgG1-huCD3-H101G-FEALxb12-FEAR,
bsIgG1-b12-FEALx5T4-207-FEAR) targeting only the tumor cells or the
T cells, did not induce any T-cell activation. FIGS. 17E-17F show
the mean EC.sub.50 values for each of the cell lines tested with
different donors (at least n=3).
[0649] These data indicate that
bsIgG1-huCD3-H101G-FEALx5T4-207-FEAR can specifically induce T-cell
mediated cytotoxicity and T-cell activation in pancreas and
cervical cancer, while control bispecific antibodies
bsIgG1-huCD3-H101G-FEALxb12-FEAR and bsIgG1-b12-FEALx5T4-207-FEAR
do not induce T-cell activation and T-cell mediated
cytotoxicity.
REFERENCES
[0650] 1. Zhao et al., 2014 Structure 22, 612-620 [0651] 2.
Southall et al., 1990 Br J Cancer 61, 89-95 [0652] 3. Stern and
Harrop, 2017 Cancer Immunol Immunother 66, 415-426; Southall et
al., 1990 Br J
[0653] Cancer 61, 89-95 [0654] 4. Damelin et al., 2011 Cancer Res
71, 4236-4246; Carsberg et al., 1996 Int J Cancer 68, 84-92 [0655]
5. Carsberg et al., 1996 Int J Cancer 68, 84-92 [0656] 6.
Kagermeier-Schenk et al., 2011 Dev Cell 21, 1129-1143 [0657] 7.
Eisen, et al., 2014 Curr Oncol Rep 16, 370 [0658] 8. Stern and
Harrop, 2017 Cancer Immunol Immunother 66, 415-426; Scurr et al.,
2017 JAMA Oncol 12, 10 [0659] 9. Scurr et al., 2017 JAMA Oncol 12,
10) [0660] 10. WO2007106744 [0661] 11. WO03038098 [0662] 12.
WO2011048369 [0663] 13. WO2013041687 [0664] 14. WO2017072207 [0665]
15. Abdiche Y N, Yeung A Y, Ni I, Stone D, Miles A, Morishige W, et
al. (2017) Antibodies Targeting Closely Adjacent or Minimally
Overlapping Epitopes Can Displace One Another. PLoS ONE 12(1):
e0169535. doi:10.1371/journal.pone.0169535) [0666] 16. WO
2007/059782; Genmab A/S [0667] 17. Ward et al., Nature 341, 544-546
(1989) [0668] 18. Holt et al; Trends Biotechnol. 2003 November;
21(11):484-90 [0669] 19. Revets et al; Expert Opin Biol Ther. 2005
January; 5(1):111-24 [0670] 20. Bird et al., Science 242, 423-426
(1988) [0671] 21. Huston et al., PNAS USA 85, 5879-5883 (1988)
[0672] 22. Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed.
Raven Press, N.Y. (1989) [0673] 23. Lefranc M P. et al., Nucleic
Acids Research, 27, 209-212, 1999 [0674] 24. Brochet X. Nucl. Acids
Res. 36, W503-508 (2008) [0675] 25. Abdiche Y N, Malashock D S,
Pinkerton A, Pons J. Exploring blocking assays using Octet,
ProteOn, and Biacore biosensors. Anal Biochem. 2009; 386(2):
172-180) [0676] 26. WO92/22653 [0677] 27. EP0629240 [0678] 28.
Kabat, E. A. et al., Sequences of proteins of immunological
interest. 5th Edition--US Department of Health and Human Services,
NIH publication No. 91-3242, pp 662,680,689 (1991) [0679] 29. Rice
et al., 2000, Trends Genet. 16: 276-277 [0680] 30. WO2007106744
[0681] 31. Shaw et al. (2002), Biochem. J. 363: 137-45, WO98/55607
[0682] 32. WO06/031653 [0683] 33. Wu et al., Generation and
Characterization of a Dual Variable Domain Immunoglobulin
(DVD-Ig.TM.) Molecule, In: Antibody Engineering, Springer Berlin
Heidelberg (2010) [0684] 34. WO 2011/131746; Genmab A/S [0685] 35.
WO/2002/020039; Trion Pharma/Fresenius Biotech [0686] 36.
WO9850431; Genetech [0687] 37. WO2011117329; Roche [0688] 38.
EP1870459; Amgen [0689] 39. WO2009089004; Amgen [0690] 40.
US201000155133; Chugai [0691] 41. WO2010129304; Oncomed [0692] 42.
WO2007110205; EMD Serono [0693] 43. WO 2010/015792; Regeneron
[0694] 44. WO11143545; Pfizer/Rinat [0695] 45. WO2012058768:
Zymeworks/Merck [0696] 46. WO2011028952; Xencor [0697] 47. WO
2009/080254; Roche [0698] 48. WO2008003116; F-5tar [0699] 49. U.S.
Pat. No. 7,262,028; Crucell/Merus [0700] 50. U.S. Pat. No.
7,612,181; Abbott [0701] 51. WO20100226923; Unilever, Sanofi
Aventis [0702] 52. US007951918; Biogen Idec [0703] 53. CN
102250246; Changzhou Adam Biotech Inc [0704] 54. WO2012025525;
Roche [0705] 55. WO2012025530; Roche [0706] 56. WO2008157379;
Macrogenics [0707] 57. WO2010/080538; Macrogenics [0708] 58. WO
2008/119353 [0709] 59. WO 2011/131746 [0710] 60. WO2015001085
[0711] 61. Shields et al., 2001, J. Biol. Chem. (276):6591-604)
[0712] 62. Sykes and Johnston, Nat Biotech 17, 355-59 (1997) [0713]
63. U.S. Pat. No. 6,077,835 [0714] 64. WO 00/70087 [0715] 65.
Schakowski et al., Mol Ther 3, 793-800 (2001) [0716] 66. WO
00/46147 [0717] 67. Benvenisty and Reshef, PNAS USA 83, 9551-55
(1986) [0718] 68. Wigler et al., Cell 14, 725 (1978) [0719] 69.
Coraro and Pearson, Somatic Cell Genetics 7, 603 (1981) [0720] 70.
U.S. Pat. No. 5,589,466 [0721] 71. U.S. Pat. No. 5,973,972 [0722]
72. Van Heeke & Schuster, J Biol Chem 264, 5503-5509 (1989)
[0723] 73. Remington: The Science and Practice of Pharmacy, 19th
Edition, Gennaro, Ed., Mack Publishing Co., Easton, Pa., 1995
[0724] 74. Sustained and Controlled Release Drug Delivery Systems,
J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978 [0725] 75.
U.S. Pat. No. 4,699,880 [0726] 76. Kozak, M., Gene 1999;
234(2):187-208) [0727] 77. Coligan J. E., Bierer, B. E., Margulies,
D. H., Shevach, E. M. and Strober, W., eds. Current Protocols in
Immunology, John Wiley & Sons, Inc., 2006) [0728] 78.
Aslanidis, C. and P. J. de Jong, Nucleic Acids Res 1990; 18(20):
6069-74 [0729] 79. Lefranc M P. et al., Nucleic Acids Research, 27,
209-212, 1999 [0730] 80. Brochet X. Nucl. Acids Res. 36, W503-508
(2008) [0731] 81. WO2007/106744 [0732] 82. WO2015/001085 [0733] 83.
WO2017/009442 [0734] 84. WO2011147986, [0735] 85. WO2011131746
[0736] 86. WO2013060867 [0737] 87. Labrijn et al., PNAS 2013, 110:
5145-50 [0738] 88. Gramer et al., MAbs 2013, 5: 962-973 [0739] 89.
Barbas, C F. J Mol Biol. 1993 Apr. 5; 230(3):812-23 [0740] 90.
WO2007/106744 [0741] 91. US 2015/0337049
Sequence CWU 1
1
1051420PRTHomo Sapiens 1Met Pro Gly Gly Cys Ser Arg Gly Pro Ala Ala
Gly Asp Gly Arg Leu1 5 10 15Arg Leu Ala Arg Leu Ala Leu Val Leu Leu
Gly Trp Val Ser Ser Ser 20 25 30Ser Pro Thr Ser Ser Ala Ser Ser Phe
Ser Ser Ser Ala Pro Phe Leu 35 40 45Ala Ser Ala Val Ser Ala Gln Pro
Pro Leu Pro Asp Gln Cys Pro Ala 50 55 60Leu Cys Glu Cys Ser Glu Ala
Ala Arg Thr Val Lys Cys Val Asn Arg65 70 75 80Asn Leu Thr Glu Val
Pro Thr Asp Leu Pro Ala Tyr Val Arg Asn Leu 85 90 95Phe Leu Thr Gly
Asn Gln Leu Ala Val Leu Pro Ala Gly Ala Phe Ala 100 105 110Arg Arg
Pro Pro Leu Ala Glu Leu Ala Ala Leu Asn Leu Ser Gly Ser 115 120
125Arg Leu Asp Glu Val Arg Ala Gly Ala Phe Glu His Leu Pro Ser Leu
130 135 140Arg Gln Leu Asp Leu Ser His Asn Pro Leu Ala Asp Leu Ser
Pro Phe145 150 155 160Ala Phe Ser Gly Ser Asn Ala Ser Val Ser Ala
Pro Ser Pro Leu Val 165 170 175Glu Leu Ile Leu Asn His Ile Val Pro
Pro Glu Asp Glu Arg Gln Asn 180 185 190Arg Ser Phe Glu Gly Met Val
Val Ala Ala Leu Leu Ala Gly Arg Ala 195 200 205Leu Gln Gly Leu Arg
Arg Leu Glu Leu Ala Ser Asn His Phe Leu Tyr 210 215 220Leu Pro Arg
Asp Val Leu Ala Gln Leu Pro Ser Leu Arg His Leu Asp225 230 235
240Leu Ser Asn Asn Ser Leu Val Ser Leu Thr Tyr Val Ser Phe Arg Asn
245 250 255Leu Thr His Leu Glu Ser Leu His Leu Glu Asp Asn Ala Leu
Lys Val 260 265 270Leu His Asn Gly Thr Leu Ala Glu Leu Gln Gly Leu
Pro His Ile Arg 275 280 285Val Phe Leu Asp Asn Asn Pro Trp Val Cys
Asp Cys His Met Ala Asp 290 295 300Met Val Thr Trp Leu Lys Glu Thr
Glu Val Val Gln Gly Lys Asp Arg305 310 315 320Leu Thr Cys Ala Tyr
Pro Glu Lys Met Arg Asn Arg Val Leu Leu Glu 325 330 335Leu Asn Ser
Ala Asp Leu Asp Cys Asp Pro Ile Leu Pro Pro Ser Leu 340 345 350Gln
Thr Ser Tyr Val Phe Leu Gly Ile Val Leu Ala Leu Ile Gly Ala 355 360
365Ile Phe Leu Leu Val Leu Tyr Leu Asn Arg Lys Gly Ile Lys Lys Trp
370 375 380Met His Asn Ile Arg Asp Ala Cys Arg Asp His Met Glu Gly
Tyr His385 390 395 400Tyr Arg Tyr Glu Ile Asn Ala Asp Pro Arg Leu
Thr Asn Leu Ser Ser 405 410 415Asn Ser Asp Val 4202420PRTMacaca
Fascicularis 2Met Pro Gly Gly Cys Ser Arg Gly Pro Ala Ala Gly Asp
Gly Arg Leu1 5 10 15Arg Leu Ala Arg Leu Ala Leu Val Leu Leu Gly Trp
Val Ser Ser Ser 20 25 30Ser Ser Thr Ser Ser Ala Ser Ser Ser Ser Ser
Ser Ala Pro Phe Leu 35 40 45Ala Ser Ala Ala Ser Ala Gln Pro Pro Leu
Pro Asp Gln Cys Pro Ala 50 55 60Leu Cys Glu Cys Ser Glu Ala Ala Arg
Thr Val Lys Cys Val Asn Arg65 70 75 80Asn Leu Thr Glu Val Pro Thr
Asp Leu Pro Leu Tyr Val Arg Asn Leu 85 90 95Phe Leu Thr Gly Asn Gln
Leu Ala Val Leu Pro Ala Gly Ala Phe Ala 100 105 110Arg Arg Pro Pro
Leu Ala Glu Leu Ala Ala Leu Asn Leu Ser Gly Ser 115 120 125Arg Leu
Asp Glu Val Arg Gly Gly Ala Phe Glu His Leu Pro Ser Leu 130 135
140Arg Gln Leu Asp Leu Ser His Asn Pro Leu Ala Tyr Leu Ser Pro
Phe145 150 155 160Ala Phe Ser Gly Ser Asn Ala Ser Ile Ser Ala Pro
Ser Pro Leu Val 165 170 175Glu Leu Ile Leu Asn His Ile Val Pro Pro
Asp Asp Lys Arg Gln Asn 180 185 190Arg Ser Phe Glu Gly Met Val Ala
Ala Ala Leu Val Ala Gly Arg Ala 195 200 205Leu Gln Gly Leu His Leu
Leu Glu Leu Ala Ser Asn His Phe Leu Tyr 210 215 220Leu Pro Arg Asp
Val Leu Ala Gln Leu Pro Ser Leu Arg Tyr Leu Asp225 230 235 240Leu
Ser Asn Asn Ser Leu Val Ser Leu Thr Tyr Val Ser Phe Arg Asn 245 250
255Leu Thr His Leu Glu Ser Leu His Leu Glu Asp Asn Ala Leu Lys Val
260 265 270Leu His Asn Gly Thr Leu Ala Glu Leu Gln Gly Leu Pro His
Val Arg 275 280 285Val Phe Leu Asp Asn Asn Pro Trp Val Cys Asp Cys
His Met Ala Asp 290 295 300Met Val Thr Trp Leu Lys Gln Thr Gly Val
Val Gln Gly Lys Asp Arg305 310 315 320Leu Thr Cys Ala Phe Pro Glu
Lys Met Arg Asn Arg Val Leu Leu Glu 325 330 335Leu Asn Ser Ala Asp
Leu Asp Cys Asp Pro Ile Leu Pro Pro Ser Leu 340 345 350Gln Thr Ser
Tyr Val Phe Leu Gly Ile Val Leu Ala Leu Ile Gly Ala 355 360 365Ile
Phe Leu Leu Val Leu Tyr Leu Asn Arg Lys Gly Ile Lys Lys Trp 370 375
380Met His Asn Ile Arg Asp Ala Cys Arg Asp His Met Glu Gly Tyr
His385 390 395 400Tyr Arg Tyr Glu Ile Asn Ala Asp Pro Arg Leu Thr
Asn Leu Ser Ser 405 410 415Asn Ser Asp Val 4203379PRTGallus Gallus
3Met Pro Gly Arg Glu Ala Glu Arg Arg Gly Ala Leu Cys Leu Gly Leu1 5
10 15Leu Leu His Ala Leu Leu Gly Cys Gly Ser Ala Gln Pro Pro Ala
Ala 20 25 30Cys Pro Ala Pro Cys Glu Cys Ser Glu Ala Ala Lys Thr Val
Lys Cys 35 40 45Val Asn Lys Asn Leu Thr Glu Val Pro Pro Asp Leu Pro
Pro Tyr Val 50 55 60Arg Asn Leu Phe Ile Thr Gly Asn Arg Leu Gly Arg
Leu Pro Ala Gly65 70 75 80Ala Leu Ser Ala Pro Arg Leu Ala Glu Leu
Gly Ser Leu Asn Leu Ser 85 90 95Gly Asn His Leu Arg Ala Val Glu Ala
Gly Ala Leu Ala Ala Leu Pro 100 105 110Ala Leu Arg Gln Leu Asp Leu
Gly Gly Asn Pro Leu Ala Glu Leu Ser 115 120 125Pro Leu Ala Phe Gly
Arg Ala Ser Pro Leu Glu Glu Leu Ala Leu Arg 130 135 140Gly Ala Leu
Arg Glu Gln Gly Ala Leu Leu Gly Leu Ala Asp Leu Leu145 150 155
160Gln Ala Gly Ala Leu Arg Asn Leu Ser Arg Leu Glu Leu Ala Asp Asn
165 170 175Gly Leu Leu Leu Leu Pro Thr Gly Met Leu Gly Ala Leu Pro
Ala Leu 180 185 190Arg His Leu Asp Leu Ser Asn Asn Ser Leu Val Gly
Leu Arg Asn Val 195 200 205Ser Phe Gln Gly Leu Val Arg Leu Gln Ser
Leu Asn Leu Ser Asp Asn 210 215 220Ser Leu Gly Val Leu Arg Asn Gly
Thr Leu Ala Gln Trp Arg Gly Leu225 230 235 240Pro Ala Leu Arg Arg
Ile Ser Leu Ser His Asn Thr Trp Val Cys Asp 245 250 255Cys Ala Ile
Glu Asp Met Val Ala Trp Leu Lys Glu Ser Asp Gln Val 260 265 270Glu
Gly Lys Glu Ala Leu Ser Cys Ala Phe Pro Glu Lys Met Ala Gly 275 280
285Arg Ala Leu Leu Lys Leu Asn Thr Ser Glu Leu Asn Cys Ser Ala Pro
290 295 300Val Asp Val Pro Ser Gln Leu Gln Thr Ser Tyr Val Phe Leu
Gly Ile305 310 315 320Val Leu Ala Leu Ile Gly Ala Ile Phe Leu Leu
Val Leu Tyr Leu Asn 325 330 335Arg Lys Gly Ile Lys Lys Trp Met His
Asn Ile Arg Asp Ala Cys Arg 340 345 350Asp His Met Glu Gly Tyr His
Tyr Arg Tyr Glu Ile Asn Ala Asp Pro 355 360 365Arg Leu Thr Asn Leu
Ser Ser Asn Ser Asp Val 370 3754186PRTHomo Sapiens 4Gln Asp Gly Asn
Glu Glu Met Gly Gly Ile Thr Gln Thr Pro Tyr Lys1 5 10 15Val Ser Ile
Ser Gly Thr Thr Val Ile Leu Thr Cys Pro Gln Tyr Pro 20 25 30Gly Ser
Glu Ile Leu Trp Gln His Asn Asp Lys Asn Ile Gly Gly Asp 35 40 45Glu
Asp Asp Lys Asn Ile Gly Ser Asp Glu Asp His Leu Ser Leu Lys 50 55
60Glu Phe Ser Glu Leu Glu Gln Ser Gly Tyr Tyr Val Cys Tyr Pro Arg65
70 75 80Gly Ser Lys Pro Glu Asp Ala Asn Phe Tyr Leu Tyr Leu Arg Ala
Arg 85 90 95Val Cys Glu Asn Cys Met Glu Met Asp Val Met Ser Val Ala
Thr Ile 100 105 110Val Ile Val Asp Ile Cys Ile Thr Gly Gly Leu Leu
Leu Leu Val Tyr 115 120 125Tyr Trp Ser Lys Asn Arg Lys Ala Lys Ala
Lys Pro Val Thr Arg Gly 130 135 140Ala Gly Ala Gly Gly Arg Gln Arg
Gly Gln Asn Lys Glu Arg Pro Pro145 150 155 160Pro Val Pro Asn Pro
Asp Tyr Glu Pro Ile Arg Lys Gly Gln Arg Asp 165 170 175Leu Tyr Ser
Gly Leu Asn Gln Arg Arg Ile 180 1855125PRTArtificial
sequenceAntibody variable domain 5Gln Val Gln Leu Val Glu Ser Gly
Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala
Val Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Asp Met Asn Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Thr Phe Ile Ser Tyr
Asp Gly Ser Asn Lys Tyr Asn Ala Asp Ser Val 50 55 60Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Asp Ser Tyr Ser Arg Ser Trp Tyr Gly Asp Tyr Tyr Gly Met 100 105
110Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120
12568PRTArtificial sequenceCDR sequence 6Gly Phe Thr Phe Ser Ser
Tyr Asp1 578PRTArtificial sequenceCDR sequence 7Ile Ser Tyr Asp Gly
Ser Asn Lys1 5818PRTArtificial sequenceCDR sequence 8Ala Arg Asp
Ser Tyr Ser Arg Ser Trp Tyr Gly Asp Tyr Tyr Gly Met1 5 10 15Asp
Val9107PRTArtificial sequenceAntibody variable domain 9Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30Leu
Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile 35 40
45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser
Tyr Pro Leu 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100
105106PRTArtificial sequenceCDR sequence 10Gln Gly Ile Ser Ser Trp1
5119PRTArtificial sequenceCDR sequence 11Gln Gln Tyr Asn Ser Tyr
Pro Leu Thr1 512121PRTArtificial sequenceAntibody variable domain
12Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser
Tyr 20 25 30Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala
Gln Lys Leu 50 55 60Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr
Arg Thr Ala Tyr65 70 75 80Met Glu Leu Arg Ser Leu Arg Ser Asp Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Pro Gly Tyr Phe Asp Trp
Leu Tyr Gly Asp Tyr Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val
Ser Ser 115 120138PRTArtificial sequenceCDR sequence 13Gly Tyr Thr
Phe Thr Ser Tyr Gly1 5148PRTArtificial sequenceCDR sequence 14Ile
Ser Ala Tyr Asn Gly Asn Thr1 51514PRTArtificial SequenceSynthetic
15Ala Arg Asp Pro Gly Tyr Phe Asp Trp Leu Tyr Gly Asp Tyr1 5
1016107PRTArtificial sequenceAntibody variable region 16Ala Ile Gln
Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ala 20 25 30Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40
45Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser
Tyr Pro Arg 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
105176PRTArtificial sequenceCDR sequence 17Gln Gly Ile Ser Ser Ala1
5189PRTArtificial sequenceCDR sequence 18Gln Gln Phe Asn Ser Tyr
Pro Arg Thr1 519121PRTArtificial sequenceAntibody variable region
19Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Asn Ala
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Phe Arg Asp Asn Ser Lys
Asn Thr Leu Tyr65 70 75 80Leu His Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Pro Gly Tyr Asn Asn Val
Glu Tyr Leu Asp His Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val
Ser Ser 115 120208PRTArtificial sequenceCDR sequence 20Gly Phe Thr
Phe Ser Ser Tyr Ala1 5218PRTArtificial sequenceCDR sequence 21Ile
Ser Gly Ser Gly Gly Ser Thr1 52214PRTArtificial sequenceCDR
sequence 22Ala Arg Asp Pro Gly Tyr Asn Asn Val Glu Tyr Leu Asp His1
5 1023107PRTArtificial sequenceAntibody variable region 23Ala Ile
Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ala 20 25
30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn
Ser Tyr Pro Leu 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105246PRTArtificial sequenceCDR sequence 24Gln Gly Ile Ser Ser
Ala1 5259PRTArtificial sequenceCDR sequence 25Gln Gln Phe Asn Ser
Tyr Pro Leu Thr1 526115PRTArtificial sequenceAntibody variable
region 26Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Glu1 5 10 15Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Arg Phe
Thr Ser Tyr 20 25 30Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly
Leu Glu Trp Met 35 40 45Gly Ile Ile Tyr Pro Gly Asp Ser Asp Ala Arg
Tyr Ser Pro Ser Phe 50 55 60Gln Gly Gln Val Thr Ile Ser Ala Asp Lys
Ser Ile Ser Thr Ala Tyr65 70 75 80Leu Gln Trp Ser Ser Leu Lys Ala
Ser Asp Thr Gly Met Tyr Tyr Cys 85 90 95Ala Arg Ser Val Leu Phe Asp
Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110Val Ser Ser
115278PRTArtificial
sequenceCDR sequence 27Gly Tyr Arg Phe Thr Ser Tyr Trp1
5288PRTArtificial sequenceAntibody variable region 28Ile Tyr Pro
Gly Asp Ser Asp Ala1 5298PRTArtificial sequenceCDR sequence 29Ala
Arg Ser Val Leu Phe Asp Tyr1 530107PRTArtificial sequenceAntibody
variable region 30Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser
Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Gly Ile Ser Ser Ala 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile 35 40 45Tyr Asp Val Ser Asn Leu Glu Ser Gly
Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln Phe Asn Ser Tyr Pro His 85 90 95Thr Phe Gly Gln Gly
Thr Lys Leu Glu Ile Lys 100 105316PRTArtificial sequenceCDR
sequence 31Gln Gly Ile Ser Ser Ala1 5329PRTArtificial sequenceCDR
sequence 32Gln Gln Phe Asn Ser Tyr Pro His Thr1 533123PRTArtificial
sequenceAntibody variable region 33Glu Val Gln Leu Leu Glu Ser Arg
Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala Met Ser Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Thr Ile Ser Gly
Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asn Ser Lys Lys Thr Leu Tyr65 70 75 80Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Lys Asp Trp Gly Ser Gly Ser Tyr Pro Ala Glu Tyr Phe Gln His 100 105
110Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
120348PRTArtificial sequenceCDR sequence 34Gly Phe Thr Phe Ser Ser
Tyr Ala1 5358PRTArtificial sequenceCDR sequence 35Ile Ser Gly Ser
Gly Gly Ser Thr1 53616PRTArtificial sequenceCDR sequence 36Ala Lys
Asp Trp Gly Ser Gly Ser Tyr Pro Ala Glu Tyr Phe Gln His1 5 10
1537108PRTArtificial sequenceAntibody variable region 37Glu Ile Val
Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg
Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40
45Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu
Pro65 70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn
Trp Leu Met 85 90 95Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105386PRTArtificial sequenceCDR sequence 38Gln Ser Val Ser Ser
Tyr1 53910PRTArtificial sequenceSynthetic 39Gln Gln Arg Ser Asn Trp
Leu Met Tyr Thr1 5 1040122PRTArtificial SequenceSynthetic 40Gln Val
Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu1 5 10 15Thr
Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr 20 25
30Tyr Trp Thr Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45Gly Glu Ile Asp His Ser Glu Ser Thr Asn Tyr Asn Pro Ser Leu
Lys 50 55 60Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe
Ser Leu65 70 75 80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val
Tyr Tyr Cys Ala 85 90 95Gly Trp Phe Gly Glu Leu Tyr His Tyr Tyr Tyr
Gly Met Asp Val Trp 100 105 110Gly Gln Gly Thr Thr Val Thr Val Ser
Ser 115 120418PRTArtificial SequenceSynthetic 41Gly Gly Ser Phe Ser
Gly Tyr Tyr1 5427PRTArtificial SequenceSynthetic 42Ile Asp His Ser
Glu Ser Thr1 54316PRTArtificial SequenceSynthetic 43Ala Gly Trp Phe
Gly Glu Leu Tyr His Tyr Tyr Tyr Gly Met Asp Val1 5 10
1544107PRTArtificial SequenceSynthetic 44Glu Ile Val Leu Thr Gln
Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu
Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30Leu Ala Trp Tyr
Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Asp Ala
Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro65 70 75
80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Leu
85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100
105456PRTArtificial SequenceSynthetic 45Gln Ser Val Ser Ser Tyr1
5469PRTArtificial sequenceCDR sequence 46Gln Gln Arg Ser Asn Trp
Pro Leu Thr1 547122PRTArtificial sequenceAntibody variable region
47Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu1
5 10 15Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly
Tyr 20 25 30Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu
Trp Ile 35 40 45Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro
Ser Leu Lys 50 55 60Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn
Gln Phe Ser Leu65 70 75 80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr
Ala Val Tyr Tyr Cys Ala 85 90 95Ala Trp Phe Gly Glu Leu Trp Asp Tyr
Tyr Tyr Gly Met Asp Val Trp 100 105 110Gly Gln Gly Thr Thr Val Thr
Val Ser Ser 115 120488PRTArtificial sequenceCDR sequence 48Gly Gly
Ser Phe Ser Gly Tyr Tyr1 5497PRTArtificial sequenceCDR sequence
49Ile Asp His Ser Gly Ser Thr1 55016PRTArtificial SequenceSynthetic
50Ala Ala Trp Phe Gly Glu Leu Trp Asp Tyr Tyr Tyr Gly Met Asp Val1
5 10 1551107PRTArtificial sequenceAntibody variable region 51Glu
Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10
15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Phe
20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
Ile 35 40 45Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe
Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Glu Pro65 70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg
Ser Asn Trp Pro Leu 85 90 95Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile
Lys 100 105526PRTArtificial SequenceSynthetic 52Gln Ser Val Ser Ser
Phe1 5539PRTArtificial SequenceSynthetic 53Gln Gln Arg Ser Asn Trp
Pro Leu Thr1 5548PRTArtificial sequenceCDR sequence 54Gly Phe Thr
Phe Asn Thr Tyr Ala1 55510PRTArtificial sequenceCDR sequence 55Ile
Arg Ser Lys Tyr Asn Asn Tyr Ala Thr1 5 105616PRTArtificial
SequenceSynthetic 56Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser
Trp Phe Ala Tyr1 5 10 1557125PRTArtificial SequenceSynthetic 57Glu
Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr
20 25 30Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr
Ala Asp 50 55 60Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser
Lys Ser Ser65 70 75 80Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu
Asp Thr Ala Met Tyr 85 90 95Tyr Cys Val Arg His Gly Asn Phe Gly Asn
Ser Tyr Val Ser Trp Phe 100 105 110Ala Tyr Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser 115 120 125589PRTArtificial sequenceCDR
sequence 58Thr Gly Ala Val Thr Thr Ser Asn Tyr1 5599PRTArtificial
sequenceCDR sequence 59Ala Leu Trp Tyr Ser Asn Leu Trp Val1
560109PRTArtificial sequenceAntibody variable region 60Gln Ala Val
Val Thr Gln Glu Pro Ser Phe Ser Val Ser Pro Gly Gly1 5 10 15Thr Val
Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30Asn
Tyr Ala Asn Trp Val Gln Gln Thr Pro Gly Gln Ala Phe Arg Gly 35 40
45Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro Ala Arg Phe
50 55 60Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly
Ala65 70 75 80Gln Ala Asp Asp Glu Ser Ile Tyr Phe Cys Ala Leu Trp
Tyr Ser Asn 85 90 95Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val
Leu 100 105618PRTArtificial SequenceSynthetic 61Gly Phe Thr Phe Asn
Pro Tyr Ala1 562125PRTArtificial SequenceSynthetic 62Glu Val Lys
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Pro Tyr 20 25 30Ala
Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp
50 55 60Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser
Ser65 70 75 80Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr
Ala Met Tyr 85 90 95Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr
Val Ser Trp Phe 100 105 110Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ser 115 120 125638PRTArtificial SequenceSynthetic 63Gly Phe
Thr Phe Asn Met Tyr Ala1 564125PRTArtificial SequenceSynthetic
64Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Met
Tyr 20 25 30Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr
Tyr Ala Asp 50 55 60Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp
Ser Lys Ser Ser65 70 75 80Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr
Glu Asp Thr Ala Met Tyr 85 90 95Tyr Cys Val Arg His Gly Asn Phe Gly
Asn Ser Tyr Val Ser Trp Phe 100 105 110Ala Tyr Trp Gly Gln Gly Thr
Leu Val Thr Val Ser Ser 115 120 1256510PRTArtificial
SequenceSynthetic 65Ile Arg Ser Lys Tyr Asn Glu Tyr Ala Thr1 5
1066125PRTArtificial sequenceAntibody variable region 66Glu Val Lys
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30Ala
Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ala Arg Ile Arg Ser Lys Tyr Asn Glu Tyr Ala Thr Tyr Tyr Ala Asp
50 55 60Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser
Ser65 70 75 80Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr
Ala Met Tyr 85 90 95Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr
Val Ser Trp Phe 100 105 110Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ser 115 120 1256716PRTArtificial SequenceSynthetic 67Val
Arg Gly Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr1 5 10
1568125PRTArtificial SequenceSynthetic 68Glu Val Lys Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30Ala Met Asn Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Arg Ile
Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60Ser Val
Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser65 70 75
80Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr
85 90 95Tyr Cys Val Arg Gly Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp
Phe 100 105 110Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 1256916PRTArtificial SequenceSynthetic 69Val Arg Asn Gly
Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr1 5 10
1570125PRTArtificial SequenceSynthetic 70Glu Val Lys Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30Ala Met Asn Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Arg Ile
Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60Ser Val
Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser65 70 75
80Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr
85 90 95Tyr Cys Val Arg Asn Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp
Phe 100 105 110Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 1257116PRTArtificial SequenceSynthetic 71Val Arg His Gly
Asn Phe Pro Asn Ser Tyr Val Ser Trp Phe Ala Tyr1 5 10
1572125PRTArtificial SequenceSynthetic 72Glu Val Lys Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30Ala Met Asn Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Arg Ile
Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60Ser Val
Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser65 70 75
80Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr
85 90 95Tyr Cys Val Arg His Gly Asn Phe Pro Asn Ser Tyr Val Ser Trp
Phe 100 105 110Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 1257316PRTArtificial SequenceSynthetic 73Val Arg His Gly
Asn Phe Gly Asn Ser Tyr Val Ala Trp Phe Ala Tyr1 5 10
1574125PRTArtificial SequenceSynthetic 74Glu Val Lys Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30Ala Met Asn Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Arg Ile
Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60Ser Val
Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser65 70 75
80Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr
85 90 95Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ala Trp
Phe 100 105 110Ala Tyr Trp Gly Gln Gly Thr
Leu Val Thr Val Ser Ser 115 120 1257516PRTArtificial
SequenceSynthetic 75Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Gly
Trp Phe Ala Tyr1 5 10 1576125PRTArtificial SequenceSynthetic 76Glu
Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr
20 25 30Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr
Ala Asp 50 55 60Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser
Lys Ser Ser65 70 75 80Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu
Asp Thr Ala Met Tyr 85 90 95Tyr Cys Val Arg His Gly Asn Phe Gly Asn
Ser Tyr Val Gly Trp Phe 100 105 110Ala Tyr Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser 115 120 1257716PRTArtificial SequenceSynthetic
77Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Val1
5 10 1578125PRTArtificial SequenceSynthetic 78Glu Val Lys Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30Ala Met Asn
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Arg
Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60Ser
Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser65 70 75
80Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr
85 90 95Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp
Phe 100 105 110Ala Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 1257916PRTArtificial SequenceSynthetic 79Val Arg His Gly
Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Met1 5 10
1580125PRTArtificial SequenceSynthetic 80Glu Val Lys Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30Ala Met Asn Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Arg Ile
Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60Ser Val
Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser65 70 75
80Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr
85 90 95Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp
Phe 100 105 110Ala Met Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 1258116PRTArtificial SequenceSynthetic 81Val Arg His Gly
Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Arg1 5 10
1582125PRTArtificial SequenceSynthetic 82Glu Val Lys Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30Ala Met Asn Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Arg Ile
Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60Ser Val
Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser65 70 75
80Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr
85 90 95Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp
Phe 100 105 110Ala Arg Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 12583119PRTHomo Sapiens 83Gln Ile Gln Leu Val Gln Ser Gly
Pro Glu Leu Lys Lys Pro Gly Glu1 5 10 15Thr Val Lys Ile Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Asn Phe 20 25 30Gly Met Asn Trp Val Lys
Gln Gly Pro Gly Glu Gly Leu Lys Trp Met 35 40 45Gly Trp Ile Asn Thr
Asn Thr Gly Glu Pro Arg Tyr Ala Glu Glu Phe 50 55 60Lys Gly Arg Phe
Ala Phe Ser Leu Glu Thr Thr Ala Ser Thr Ala Tyr65 70 75 80Leu Gln
Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95Ala
Arg Asp Trp Asp Gly Ala Tyr Phe Phe Asp Tyr Trp Gly Gln Gly 100 105
110Thr Thr Leu Thr Val Ser Ser 11584107PRTHomo Sapiens 84Ser Ile
Val Met Thr Gln Thr Pro Lys Phe Leu Leu Val Ser Ala Gly1 5 10 15Asp
Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Ser Asn Asp 20 25
30Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile
35 40 45Asn Phe Ala Thr Asn Arg Tyr Thr Gly Val Pro Asn Arg Phe Thr
Gly 50 55 60Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser Thr Val
Gln Ala65 70 75 80Glu Asp Leu Ala Leu Tyr Phe Cys Gln Gln Asp Tyr
Ser Ser Pro Trp 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 10585122PRTHomo Sapiens 85Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Lys Gly1 5 10 15Ser Leu Lys Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30Ala Met Asn Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Arg Ile Arg Ser Lys
Ser Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60Ser Val Lys Asp Arg
Phe Thr Ile Ser Arg Asp Asp Ser Gln Ser Met65 70 75 80Leu Tyr Leu
Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95Tyr Cys
Val Arg Gln Trp Asp Tyr Asp Val Arg Ala Met Asn Tyr Trp 100 105
110Gly Gln Gly Thr Ser Val Thr Val Ser Ser 115 12086107PRTHomo
Sapiens 86Asp Ile Val Met Thr Gln Ser His Ile Phe Met Ser Thr Ser
Val Gly1 5 10 15Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val
Asp Thr Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro
Lys Leu Leu Ile 35 40 45Tyr Trp Ala Ser Thr Arg Leu Thr Gly Val Pro
Asp Arg Phe Thr Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Asn Val Gln Ser65 70 75 80Glu Asp Leu Ala Asp Tyr Phe Cys
Gln Gln Tyr Ser Ser Tyr Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys
Leu Glu Ile Lys 100 10587120PRTHomo Sapiens 87Glu Val Gln Leu Gln
Gln Ser Gly Pro Asp Leu Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Ile
Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30Tyr Met His
Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45Gly Arg
Ile Asn Pro Asn Asn Gly Val Thr Leu Tyr Asn Gln Lys Phe 50 55 60Lys
Asp Lys Ala Ile Leu Thr Val Asp Lys Ser Ser Thr Thr Ala Tyr65 70 75
80Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95Ala Arg Ser Thr Met Ile Thr Asn Tyr Val Met Asp Tyr Trp Gly
Gln 100 105 110Val Thr Ser Val Thr Val Ser Ser 115 12088107PRTHomo
Sapiens 88Ser Ile Val Met Thr Gln Thr Pro Thr Phe Leu Leu Val Ser
Ala Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val
Ser Asn Asp 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro
Thr Leu Leu Ile 35 40 45Ser Tyr Thr Ser Ser Arg Tyr Ala Gly Val Pro
Asp Arg Phe Ile Gly 50 55 60Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr
Ile Ser Thr Leu Gln Ala65 70 75 80Glu Asp Leu Ala Val Tyr Phe Cys
Gln Gln Asp Tyr Asn Ser Pro Pro 85 90 95Thr Phe Gly Gly Gly Thr Lys
Leu Glu Ile Lys 100 10589330PRTHomo Sapiens 89Ala Ser Thr Lys Gly
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly
Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu
Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75
80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro
Cys 100 105 110Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys 130 135 140Val Val Val Asp Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200
205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Glu Glu225 230 235 240Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310 315
320Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325
33090330PRTArtificial SequenceSynthetic 90Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75
80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro
Cys 100 105 110Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys 130 135 140Val Val Val Asp Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200
205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Glu Glu225 230 235 240Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Leu 275 280 285Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310 315
320Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325
33091330PRTArtificial SequenceSynthetic 91Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75
80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro
Cys 100 105 110Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys 130 135 140Val Val Val Ala Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200
205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Glu Glu225 230 235 240Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310 315
320Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325
33092330PRTArtificial SequenceSynthetic 92Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75
80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro
Cys 100 105 110Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys 130 135 140Val Val Val Ala Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn
Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu225 230 235 240Met
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250
255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Leu 275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
Gln Gln Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met His Glu Ala
Leu His Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser
Pro Gly Lys 325 33093330PRTArtificial SequenceSynthetic 93Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25
30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
Lys Val Asp Lys 85 90 95Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His
Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Phe Glu Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Val Val Val Ala Val
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170
175Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Glu Glu225 230 235 240Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Leu
Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295
300Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325
33094330PRTArtificial SequenceSynthetic 94Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75
80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro
Cys 100 105 110Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys 130 135 140Val Val Val Asp Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200
205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Glu Glu225 230 235 240Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Leu Tyr Ser Arg Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310 315
320Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 33095107PRTHomo
Sapiens 95Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp Glu1 5 10 15Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu
Asn Asn Phe 20 25 30Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp
Asn Ala Leu Gln 35 40 45Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser 50 55 60Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu65 70 75 80Lys His Lys Val Tyr Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser 85 90 95Pro Val Thr Lys Ser Phe Asn
Arg Gly Glu Cys 100 10596106PRTHomo Sapiens 96Gly Gln Pro Lys Ala
Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser1 5 10 15Glu Glu Leu Gln
Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp 20 25 30Phe Tyr Pro
Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro 35 40 45Val Lys
Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn 50 55 60Lys
Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys65 70 75
80Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val
85 90 95Glu Lys Thr Val Ala Pro Thr Glu Cys Ser 100 10597127PRTHomo
Sapiens 97Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Gln Ala Ser Gly Tyr Arg Phe
Ser Asn Phe 20 25 30Val Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg
Phe Glu Trp Met 35 40 45Gly Trp Ile Asn Pro Tyr Asn Gly Asn Lys Glu
Phe Ser Ala Lys Phe 50 55 60Gln Asp Arg Val Thr Phe Thr Ala Asp Thr
Ser Ala Asn Thr Ala Tyr65 70 75 80Met Glu Leu Arg Ser Leu Arg Ser
Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Val Gly Pro Tyr Ser
Trp Asp Asp Ser Pro Gln Asp Asn Tyr 100 105 110Tyr Met Asp Val Trp
Gly Lys Gly Thr Thr Val Ile Val Ser Ser 115 120 12598108PRTHomo
Sapiens 98Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser
Pro Gly1 5 10 15Glu Arg Ala Thr Phe Ser Cys Arg Ser Ser His Ser Ile
Arg Ser Arg 20 25 30Arg Val Ala Trp Tyr Gln His Lys Pro Gly Gln Ala
Pro Arg Leu Val 35 40 45Ile His Gly Val Ser Asn Arg Ala Ser Gly Ile
Ser Asp Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Thr Arg Val Glu65 70 75 80Pro Glu Asp Phe Ala Leu Tyr Tyr
Cys Gln Val Tyr Gly Ala Ser Ser 85 90 95Tyr Thr Phe Gly Gln Gly Thr
Lys Leu Glu Arg Lys 100 10599363PRTArtificial SequenceSynthetic
99Met Pro Gly Gly Cys Ser Arg Gly Pro Ala Ala Gly Asp Gly Arg Leu1
5 10 15Arg Leu Ala Arg Leu Ala Leu Val Leu Leu Gly Trp Val Ser Ser
Ser 20 25 30Ser Pro Thr Ser Ser Ala Ser Ser Phe Ser Ser Ser Ala Pro
Phe Leu 35 40 45Ala Ser Ala Val Ser Ala Gln Pro Pro Leu Pro Asp Gln
Cys Pro Ala 50 55 60Leu Cys Glu Cys Ser Glu Ala Ala Arg Thr Val Lys
Cys Val Asn Arg65 70 75 80Asn Leu Thr Glu Val Pro Thr Asp Leu Pro
Ala Tyr Val Arg Asn Leu 85 90 95Phe Leu Thr Gly Asn Gln Leu Ala Val
Leu Pro Ala Gly Ala Phe Ala 100 105 110Arg Arg Pro Pro Leu Ala Glu
Leu Ala Ala Leu Asn Leu Ser Gly Ser 115 120 125Arg Leu Asp Glu Val
Arg Ala Gly Ala Phe Glu His Leu Pro Ser Leu 130 135 140Arg Gln Leu
Asp Leu Ser His Asn Pro Leu Ala Asp Leu Ser Pro Phe145 150 155
160Ala Phe Ser Gly Ser Asn Ala Ser Val Ser Ala Pro Ser Pro Leu Val
165 170 175Glu Leu Ile Leu Asn His Ile Val Pro Pro Glu Asp Glu Arg
Gln Asn 180 185 190Arg Ser Phe Glu Gly Met Val Val Ala Ala Leu Leu
Ala Gly Arg Ala 195 200 205Leu Gln Gly Leu Arg Arg Leu Glu Leu Ala
Ser Asn His Phe Leu Tyr 210 215 220Leu Pro Arg Asp Val Leu Ala Gln
Leu Pro Ser Leu Arg His Leu Asp225 230 235 240Leu Ser Asn Asn Ser
Leu Val Ser Leu Thr Tyr Val Ser Phe Arg Asn 245 250 255Leu Thr His
Leu Glu Ser Leu His Leu Glu Asp Asn Ala Leu Lys Val 260 265 270Leu
His Asn Gly Thr Leu Ala Glu Leu Gln Gly Leu Pro His Ile Arg 275 280
285Val Phe Leu Asp Asn Asn Pro Trp Val Cys Asp Cys His Met Ala Asp
290 295 300Met Val Thr Trp Leu Lys Glu Thr Glu Val Val Gln Gly Lys
Asp Arg305 310 315 320Leu Thr Cys Ala Tyr Pro Glu Lys Met Arg Asn
Arg Val Leu Leu Glu 325 330 335Leu Asn Ser Ala Asp Leu Asp Cys Asp
Pro Ile Leu Pro Pro Ser Leu 340 345 350Gln Thr Ser His His His His
His His His His 355 360100327PRTArtificial SequenceSynthetic 100Met
Pro Gly Gly Cys Ser Arg Gly Pro Ala Ala Gly Asp Gly Arg Leu1 5 10
15Arg Leu Ala Arg Leu Ala Leu Val Leu Leu Gly Trp Val Ser Ser Ser
20 25 30Ser Pro Thr Ser Ser Ala Ser Ser Phe Ser Ser Ser Ala Pro Phe
Leu 35 40 45Ala Ser Ala Val Ser Ala Gln Pro Pro Leu Pro Asp Gln Cys
Pro Ala 50 55 60Leu Cys Glu Cys Ser Glu Ala Ala Arg Thr Val Lys Cys
Val Asn Arg65 70 75 80Asn Leu Thr Glu Val Pro Thr Asp Leu Pro Ala
Ala Pro Ser Thr Cys 85 90 95Ser Lys Pro Thr Cys Pro Pro Pro Glu Leu
Leu Gly Gly Pro Ser Val 100 105 110Phe Ile Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr 115 120 125Pro Glu Val Thr Cys Val
Val Val Asp Val Ser Gln Asp Asp Pro Glu 130 135 140Val Gln Phe Thr
Trp Tyr Ile Asn Asn Glu Gln Val Arg Thr Ala Arg145 150 155 160Pro
Pro Leu Arg Glu Gln Gln Phe Asn Ser Thr Ile Arg Val Val Ser 165 170
175Thr Leu Pro Ile Ala His Gln Asp Trp Leu Arg Gly Lys Glu Phe Lys
180 185 190Cys Lys Val His Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile 195 200 205Ser Lys Ala Arg Gly Gln Pro Leu Glu Pro Lys Val
Tyr Thr Met Gly 210 215 220Pro Pro Arg Glu Glu Leu Ser Ser Arg Ser
Val Ser Leu Thr Cys Met225 230 235 240Ile Asn Gly Phe Tyr Pro Ser
Asp Ile Ser Val Glu Trp Glu Lys Asn 245 250 255Gly Lys Ala Glu Asp
Asn Tyr Lys Thr Thr Pro Ala Val Leu Asp Ser 260 265 270Asp Gly Ser
Tyr Phe Leu Tyr Ser Lys Leu Ser Val Pro Thr Ser Glu 275 280 285Trp
Gln Arg Gly Asp Val Phe Thr Cys Ser Val Met His Glu Ala Leu 290 295
300His Asn His Tyr Thr Gln Lys Ser Ile Ser Arg Ser Pro Gly Lys
His305 310 315 320His His His His His His His
325101276PRTArtificial SequenceSynthetic 101Met Trp Trp Arg Leu Trp
Trp Leu Leu Leu Leu Leu Leu Leu Leu Trp1 5 10 15Pro Met Val Trp Ala
Gln Asp Gly Asn Glu Glu Met Gly Gly Ile Thr 20 25 30Gln Thr Pro Tyr
Lys Val Ser Ile Ser Gly Thr Thr Val Ile Leu Thr 35 40 45Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu 50 55 60Ile Val
Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu65 70 75
80Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu
85 90 95Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
Tyr 100 105 110Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe
Ser Gly Ser 115 120 125Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Glu Pro Glu 130 135 140Asp Phe Ala Val Tyr Tyr Cys Gln Gln
Arg Ser Asn Trp Pro Ile Thr145 150 155 160Phe Gly Gln Gly Thr Arg
Leu Glu Ile Lys Arg Thr Val Ala Ala Pro 165 170 175Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 180 185 190Ala Ser
Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 195 200
205Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
210 215 220Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
Ser Ser225 230 235 240Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
His Lys Val Tyr Ala 245 250 255Cys Glu Val Thr His Gln Gly Leu Ser
Ser Pro Val Thr Lys Ser Phe 260 265 270Asn Arg Gly Glu
2751026PRTArtificial sequenceCDR consensus sequence 102Tyr Tyr Gly
Met Asp Val1 51037PRTArtificial sequenceCDR consensus
sequenceXaa(5)..(5)Xaa is G or E 103Ile Asp His Ser Xaa Ser Thr1
510416PRTArtificial sequenceCDR consensus sequenceXaa(2)..(2)Xaa is
A or GXaa(8)..(8)Xaa is W or YXaa(9)..(9)Xaa is D or H 104Ala Xaa
Trp Phe Gly Glu Leu Xaa Xaa Tyr Tyr Tyr Gly Met Asp Val1 5 10
151056PRTArtificial sequenceCDR consensus sequenceXaa(6)..(6)Xaa is
Y or F 105Gln Ser Val Ser Ser Xaa1 5
* * * * *