U.S. patent application number 17/377924 was filed with the patent office on 2022-01-27 for antibodies specific for immunoglobulin-like transcript 3 (ilt3) and uses thereof.
This patent application is currently assigned to Merck Sharp & Dohme Corp.. The applicant listed for this patent is Merck Sharp & Dohme Corp.. Invention is credited to Philip E. Brandish, Laurence Fayadat-Dilman, Veronica Juan, Michael A. Meehl, Carl Mieczkowski, Latika Singh.
Application Number | 20220025042 17/377924 |
Document ID | / |
Family ID | |
Filed Date | 2022-01-27 |
United States Patent
Application |
20220025042 |
Kind Code |
A1 |
Meehl; Michael A. ; et
al. |
January 27, 2022 |
ANTIBODIES SPECIFIC FOR IMMUNOGLOBULIN-LIKE TRANSCRIPT 3 (ILT3) AND
USES THEREOF
Abstract
Humanized, non-promiscuous monoclonal antibodies specific for
immunoglobulin-like transcript 3 (ILT3), also known as Leukocyte
immunoglobulin-like receptor subfamily B member 4 (LILRB4), are
described.
Inventors: |
Meehl; Michael A.;
(Northborough, MA) ; Brandish; Philip E.;
(Needham, MA) ; Fayadat-Dilman; Laurence;
(Sunnyvale, CA) ; Juan; Veronica; (Redwood City,
CA) ; Mieczkowski; Carl; (Hercules, CA) ;
Singh; Latika; (Belmont, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Merck Sharp & Dohme Corp. |
Rahway |
NJ |
US |
|
|
Assignee: |
Merck Sharp & Dohme
Corp.
Rahway
NJ
|
Appl. No.: |
17/377924 |
Filed: |
July 16, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16191485 |
Nov 15, 2018 |
11111297 |
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17377924 |
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62587604 |
Nov 17, 2017 |
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International
Class: |
C07K 16/28 20060101
C07K016/28; A61P 35/00 20060101 A61P035/00 |
Claims
1.-26. (canceled)
27. A nucleic acid molecule encoding an antibody or antigen
fragment that binds the extracellular domain of human
immunoglobulin-like transcript 3 (ILT3) having amino acids 1-238 of
SEQ ID NO: 1, wherein the antibody or antigen binding fragment
comprises: (a) a heavy chain complementarity determining region 1
(HC-CDR1), wherein the HC-CDR1 has the amino acid sequence set
forth in SEQ ID NO: 17; an HC-CDR2, wherein the HC-CDR2 has the
amino acid sequence set forth in SEQ ID NO: 19, 20, or 21; an
HC-CDR3, wherein the HC-CDR3 has the amino acid sequence set forth
in SEQ ID NO: 23; and (b) a light chain complementarity determining
region 1 (LC-CDR1), wherein the LC-CDR1 has the amino acid sequence
set forth in SEQ ID NO: 34, 35, 36, 37, 38, 39, 40, 41, or 42; an
LC-CDR2, wherein the LC-CDR2 has the amino acid sequence set forth
in SEQ ID NO: 43; and an LC-CDR3, wherein the LC-CDR3 has the amino
acid sequence set forth in SEQ ID NO: 44.
28. A vector comprising the nucleic acid molecule of claim 27.
29. A host cell comprising the vector of claim 28.
30. A method for producing an antibody or antigen binding fragment
that binds the extracellular domain of human immunoglobulin-like
transcript 3 (ILT3) having amino acids 1-238 of SEQ ID NO: 1
comprising: (a) providing a host cell comprising the vector of
claim 28; (b) culturing the host cell in culture medium under
conditions to enable expression of the antibody or antigen binding
fragment encoded by the vector; and, (c) obtaining the antibody or
antigen binding fragment from the culture medium to provide the
antibody or antigen binding fragment.
31. The nucleic acid molecule of claim 27, wherein (a) the HC-CDR1
has the amino acid sequence set forth in SEQ ID NO: 17; the HC-CDR2
has the amino acid sequence set forth in SEQ ID NO:20; and the
HC-CDR3 has the amino acid sequence set forth in SEQ ID NO: 23; and
(b) the LC-CDR1 has the amino acid sequence set forth in SEQ ID NO:
41; the LC-CDR2 has the amino acid sequence set forth in SEQ ID
NO:43; and, the LC-CDR3 has the amino acid sequence set forth in
SEQ ID NO: 44
32. The nucleic acid molecule of claim 27, wherein the V.sub.H
comprises a framework selected from the group consisting of human
V.sub.H1, V.sub.H2, V.sub.H3, V.sub.H4, V.sub.HS, and V.sub.H6, and
variants thereof having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid
substitutions, additions, deletions, or combinations thereof; and,
the V.sub.L comprises a framework selected from the group
consisting of human V.sub..kappa.1, V.sub..kappa.2, V.sub..kappa.3,
V.sub..kappa.4, V.sub.KS, V.sub..kappa.6, V.sub..lamda.1,
V.sub..lamda.2, V.sub..lamda.3, V.sub..lamda.4, V.sub..lamda.5,
V.sub..lamda.6, V.sub..lamda.7, V.sub..lamda.8, V.sub..lamda.9, and
V.sub..lamda.10, and variants thereof having 1, 2, 3, 4, 5, 6, 7,
8, 9, or 10 amino acid substitutions, additions, deletions, or
combinations thereof.
33. The nucleic acid molecule of claim 27, wherein the antibody
comprises an HC having a human IgG1, IgG2, IgG3, or IgG4 HC
constant domain or variant thereof having 1, 2, 3, 4, 5, 6, 7, 8,
9, or 10 amino acid substitutions, additions, deletions, or
combinations thereof compared to the amino acid sequence of the
native IgG1, IgG2, IgG3, or IgG4 isotype constant domain.
34. The nucleic acid molecule of claim 33, wherein the antibody
comprises an LC having a human kappa or lambda LC constant domain
or variant thereof comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10
amino acid substitutions, additions, deletions, or combinations
thereof compared to the amino acid sequence of the native human
kappa or lambda light chain constant domain.
35. The nucleic acid molecule of claim 31, wherein the antibody
comprises: (i) a V.sub.H having a framework selected from the group
consisting of human V.sub.H1, V.sub.H2, V.sub.H3, V.sub.H4,
V.sub.HS, and V.sub.H6 and a human IgG1 or IgG4 HC constant domain
or variant thereof comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10
amino acid substitutions, additions, deletions, or combinations
thereof compared to the amino acid sequence of the native IgG1 or
IgG4 isotype HC constant domain; and, (ii) a V.sub.L having a
framework selected from the group consisting of human
V.sub..kappa.1, V.sub..kappa.2, V.sub..kappa.3, V.sub..kappa.4,
V.sub.KS, V.sub..kappa.6, V.sub..lamda.1, V.sub..lamda.2,
V.sub..lamda.3, V.sub..lamda.4, V.sub..lamda.5, V.sub..lamda.6,
V.sub..lamda.7, V.sub..lamda.8, V.sub..lamda.9, and V.sub..kappa.10
and a human kappa or lambda LC constant domain or variant thereof
comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid
substitutions, additions, deletions, or combinations thereof
compared to the amino acid sequence of the native human kappa or
lambda LC constant domain.
36. The nucleic acid molecule of claim 32, wherein the antibody or
antigen binding fragment comprises a V.sub.H and a V.sub.L having
the amino acid sequences set forth in SEQ ID NO: 15 and SEQ ID NO:
16, respectively; SEQ ID NO:45 and SEQ ID NO: 46, respectively; SEQ
ID NO: 53 and SEQ ID NO: 54, respectively; SEQ ID NO: 61 and SEQ ID
NO: 62, respectively; SEQ ID NO: 69 and SEQ ID NO: 70,
respectively; SEQ ID NO: 77 and SEQ ID NO: 78, respectively; SEQ ID
NO: 85 and SEQ ID NO: 86, respectively; SEQ ID NO: 93 and SEQ ID
NO: 94, respectively; or SEQ ID NO:101 and SEQ ID NO: 102,
respectively.
37. The nucleic acid molecule of claim 32, wherein the antibody or
antigen binding fragment comprises a V.sub.H having the amino acid
sequence set forth in SEQ ID NO: 117, 118, 119, 123, 124, or 125
and a V.sub.L having the amino acid sequence set forth in SEQ ID
NO: 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137,
138, 139, 140, or 141.
38. The nucleic acid molecule of claim 37, wherein the antibody or
antigen binding fragment comprises a V.sub.H having the amino acid
sequence set forth in SEQ ID NO: 118 and a V.sub.L having the amino
acid sequence set forth in SEQ ID NO: 140.
39. The nucleic acid molecule of claim 35, wherein the antibody
comprises a heavy chain (HC) constant domain comprising the amino
acid sequence set forth in SEQ ID NO: 9, 10, 11, 12, or 13.
40. The nucleic acid molecule of claim 35, wherein the antibody
comprises a light chain (LC) constant domain comprising the amino
acid sequence set forth in SEQ ID NO: 14.
41. The nucleic acid molecule of claim 35, wherein the antibody
comprises a heavy chain (HC) comprising the amino acid sequence set
forth in SEQ ID NO: 142, 143, 144, 148, 149, 150, 167, 168, 169,
170, 174, 175, 176, 177, 178, 182, 183, 184, 185, 186, 187, 191,
192, or 193.
42. The nucleic acid molecule of claim 35, wherein the antibody
comprises a light chain (LC) comprising the amino acid sequence set
forth in SEQ ID NO: 151, 152, 153, 154, 155, 156, 157, 158, 159,
160, 161, 162, 163, 164, 165, or 166.
43. The nucleic acid molecule of claim 35, wherein the antibody
comprises a heavy chain (HC) comprising the amino acid sequence set
forth in SEQ ID NO: 143 and a light chain (LC) comprising the amino
acid sequence set forth in SEQ ID NO: 165, and variants thereof
wherein the HC lacks a C-terminal Lysine residue or a C-terminal
glycine-lysine.
44. A nucleic acid molecule encoding an antibody or antigen binding
fragment that binds the extracellular domain of human
immunoglobulin-like transcript 3 (ILT3) having amino acids 1-238 of
SEQ ID NO: 1, wherein the antibody or antigen binding fragment
comprises: (a) a heavy chain complementarity determining region 1
(HC-CDR1), wherein the HC-CDR1 has the amino acid sequence set
forth in SEQ ID NO: 17; an HC-CDR2, wherein the HC-CDR2 has the
amino acid sequence set forth in SEQ ID NO: 20; an HC-CDR3, wherein
the HC-CDR3 has the amino acid sequence set forth in SEQ ID NO: 23;
and (b) a light chain complementarity determining region 1
(LC-CDR1), wherein the LC-CDR1 has the amino acid sequence set
forth in SEQ ID NO: 41; an LC-CDR2, wherein the LC-CDR2 has the
amino acid sequence set forth in SEQ ID NO: 43; and an LC-CDR3,
wherein the LC-CDR3 has the amino acid sequence set forth in SEQ ID
NO: 44.
45. The nucleic acid molecule of claim 44, wherein the antibody or
antigen binding fragment comprises a V.sub.H and a V.sub.L having
the amino acid sequences set forth in SEQ ID NO: 118 and SEQ ID NO:
140, respectively.
46. The nucleic acid molecule of claim 45, wherein the antibody
comprises a heavy chain (HC) comprising the amino acid sequence set
forth in SEQ ID NO: 143 and a light chain (LC) comprising the amino
acid sequence set forth in SEQ ID NO: 165, and variants thereof
wherein the HC lacks a C-terminal Lysine residue or a C-terminal
glycine-lysine.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a divisional application of U.S.
Pat. No. 11,111,297 issued Sep. 7, 2021, which claims benefit of
U.S. Provisional Patent Application No. 62/587,604 filed Nov. 17,
2017, each of which is herein incorporated by reference in its
entirety.
REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY
[0002] The sequence listing of the present application is submitted
electronically via EFS-Web as an ASCII formatted sequence listing
with a file name "24530 US_NP_SEQTXT_05NOVEMBER2018.txt", creation
date of Nov. 5, 2018, and a size of 376 Kb. This sequence listing
submitted via EFS-Web is part of the specification and is herein
incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
(1) Field of the Invention
[0003] The present invention provides non-promiscuous monoclonal
antibodies specific for immunoglobulin-like transcript 3 (ILT3), an
inhibitory receptor expressed on the surface of myeloid immune
cells.
(2) Description of Related Art
[0004] Immunoglobulin-like transcript 3 (ILT3), designated CD85k
and also known as Leukocyte Immunoglobulin-Like Receptor subfamily
B member 4 (LILRB4) and Leukocyte Immunoglobulin-like Receptor 5
(LIR-5), is a type I membrane protein that contains cytoplasmic
immunoreceptor tyrosine-based inhibition motif (ITIM) motifs and is
involved in the down-regulation of immune responses (Cella et al.,
J Exp Med. 185 (10): 1743-51 (1997); Samaridis et al., Eur J
Immunol. 27 (3): 660-665 (1997). Expression of ILT3 is up-regulated
on tolerogenic dendritic cells. This gene is a member of the
leukocyte immunoglobulin-like receptor (LIR) family, which is found
in a gene cluster at chromosomal region 19q13.4. The encoded
protein belongs to the subfamily B class of LIR receptors, which
contain two or four extracellular immunoglobulin domains, a
transmembrane domain, and two to four ITIMs. ILT3 is selectively
expressed by myeloid antigen presenting cells (APCs) such as
monocytes, macrophages, and dendritic cells, e.g., monocyte-derived
dendritic cells differentiated in the presence of IL-10 or vitamin
D.sub.3. ILT3 consists of 447 amino acids with a predicted
molecular mass of about 47 kD. The amino terminal portion of ILT3
begins with a hydrophobic signal peptide of 23 amino acids followed
by an extracellular domain composed of two C.sub.2 type
immunoglobulin superfamily domains and having the amino acid
sequence set forth in SEQ ID NO: 1 less the C-terminal His Tag.
(The Rhesus monkey ILT3 extracellular domain has the amino acid
sequence set forth in SEQ ID NO: 2). The putative transmembrane
domain of ILT3 consists of 21 amino acids, followed by a long
cytoplasmic region of 167 amino acids, which is characterized by
the presence of motifs spaced by 26 amino acid residues and are
reminiscent of the ITIM motifs identified in KIRs (natural-killer
cell Ig receptors) as binding sites for protein tyrosine
phosphatase SHP-1. ILT3 is expressed on immune cells where it binds
to MHC class I molecules on antigen-presenting cells and transduces
a negative signal that inhibits stimulation of an immune response.
The receptor can also function in antigen capture and presentation.
ILT3 is thought to control inflammatory responses and cytotoxicity
to help focus the immune response and to limit auto-reactivity.
Multiple transcript variants encoding different isoforms of ILT3
have been identified.
[0005] Patent publications that disclose use of an antibody for
modulating ILT3 activity with applications for inhibiting
transplant rejection or for use in treatments for cancer or
infectious diseases include U.S. Pub. Nos. 20090202544,
20150110714, 20150139986, and 20170267759; and, Intl. Pub. Nos.
WO2013043569, WO2013181438, WO2014116846, WO2016049641,
WO2016127427, WO2018089300, and WO2018148494. Of interest is Intl.
Pub. No. WO2017015227, which discloses CD166, also known as
lymphocyte cell adhesion molecule (ALCAM), as a ligand for ILT3 and
provides methods for treating cancer comprising in some embodiments
an antibody against CD166 or ALCAM. Also of interest are U.S. Pat.
Nos. 7,777,008 and 8,901,281, which disclose monoclonal antibody
9B11 for use in various treatments where it is desirable to
upregulate the immune system for anti-cancer treatments and to
downregulate the immune system for inhibiting transplant
rejection.
[0006] While the patent publications disclose anti-ILT3 antibodies,
in some instances no specific antibody is disclosed or specific
antibodies are disclosed, which in some cases are shown to be
promiscuous and cross-react with one or more ILT3-related receptors
such as LILRA6 and ILT8. Promiscuous anti-ILT3 antibodies may have
off-target effects, which may have undesirable effects that
contraindicate its use for therapeutic applications. Therefore
there is a need for antibodies and antigen binding fragments that
specifically bind ILT3 and have no measurable promiscuity towards
other related receptors.
BRIEF SUMMARY OF THE INVENTION
[0007] The present invention provides monoclonal antibodies and
antigen binding fragments that bind specifically to
immunoglobulin-like transcript 3 (ILT3) with no measurable binding
to closely related proteins (e.g., ILT5, ILT7, ILT8, or ILT11) as
determined by (i) a cell ELISA using 10 .mu.g/mL antibody or
antigen binding fragment or (ii) Biacore using 10 .mu.g/mL antibody
or antigen binding fragment. In particular embodiments, the
antibodies and antigen binding fragments specifically bind to both
human ILT3 and Rhesus monkey ILT3. These antibodies and antigen
binding fragments are capable of antagonizing ILT3 activity thereby
enhancing dendritic cell activation and T cell priming. Tolerized
dendritic cells and myeloid-derived suppressor cells (MDSCs) are
also responsive to these antibodies. Furthermore, in vivo studies
of these antibodies in humanized NSG.TM. mouse model systems (The
Jackson Laboratories, Bar Harbor, Me.) show that these antibodies
may have the ability to reduce tumor burden and shift cellular
phenotypes to a more activated state.
[0008] In clinical trial samples, ILT3 expression, like PD-L1,
LAG3, and the GEP signature, was found to be associated with
responsiveness to the anti-PD-1 antibody, pembrolizumab. Soluble
ILT3 in circulation is also increased in certain cancer types.
Taken together, the anti-ILT3 antibodies of the present invention
may be useful for treating particular cancers either as a
monotherapy treatment or in combination with an anti-PD-1 and/or
anti-PD-L1 antibody to enhance responsiveness to the anti-PD-1 or
anti-PD-L1 antibody, particularly in cancer treatments in which the
cancer is non-responsive to anti-PD-1 or anti-PD-L1 monotherapies.
In particular embodiments, the present invention provides chimeric
or humanized anti-ILT3 antibodies. In certain embodiments, the
antibodies may be fully human antibodies that compete with the
antibodies disclosed herein for binding to the ILT3 epitope
disclosed herein.
[0009] The present invention provides an antibody or antigen
binding fragment comprising one, two, or three complementarity
determining regions (CDRs) of a heavy chain variable V.sub.H domain
having heavy chain complementarity determining region (HC-CDR) 1,
2, and 3 and one, two, or three CDRs of a light chain variable
domain V.sub.L having LC-CDR1, 2, and 3, wherein the antibody or
antigen binding fragment is capable of specifically binding human
ILT3 wherein the the binding of the antibody or antigen binding
fragment may be determined by cell ELISA or Biacore.
[0010] In a further embodiment, the antibody or antigen binding
fragment binds to an epitope on the human ILT3 or competes with an
antibody disclosed for binding to an epitope on the human ILT3,
wherein the epitope comprises at least one amino acid within one or
more of the amino acid sequences set forth in the group consisting
of SEQ ID NOs:3, 4, 5, 6, 7, and 8. In further embodiments, the
antibody or antigen binding fragment binds to an epitope on the
human ILT3 or competes with an antibody disclosed for binding to an
epitope on the human ILT3, wherein the epitope comprises one or
more of the amino acid sequences set forth in the group consisting
of SEQ ID NOs:3, 4, 5, 6, 7, and 8. In further embodiments, the
antibody or antigen binding fragment binds to an epitope on the
human ILT3 or competes with an antibody disclosed for binding to an
epitope on the human ILT3, wherein the epitope comprises the amino
acid sequences set forth in the group consisting of SEQ ID NOs:3,
4, 5, 6, 7, and 8. In particular embodiments, the epitope is
determined by hydrogen deuterium exchange mass spectrometry
(HDX-MS) analysis.
[0011] The present invention further provides an antibody or
antigen binding fragment that binds human ILT3 comprising a heavy
chain (HC) wherein the heavy chain variable domain (V.sub.H)
comprises a heavy chain complementarity determining region (HC-CDR)
3 having an amino acid sequence selected from the group consisting
of SEQ ID NO: 22, 49, 57, 65, 73, 81, 89, 97, and 105, or having an
amino acid sequence that has 3, 2, or 1 differences with an amino
acid sequence selected from the group consisting of SEQ ID NO: 22,
49, 57, 65, 73, 81, 89, 97, and 105. In some embodiments the amino
acid sequence differences are conservative changes/substitutions.
In particular embodiments, the antibody or antigen binding fragment
that binds human ILT3 comprises a heavy chain (HC) wherein the
heavy chain variable domain (V.sub.H) comprises a heavy chain
complementarity determining region (HC-CDR) 3 having an amino acid
sequence selected from the group consisting of SEQ ID NO: 23, 49,
57, 65, 73, 81, 89, 97, and 105, or having an amino acid sequence
that has 3, 2, or 1 differences with an amino acid sequence
selected from the group consisting of SEQ ID NO: 23, 49, 57, 65,
73, 81, 89, 97, and 105. In particular embodiments the amino acid
sequence differences are conservative changes/substitutions.
[0012] In a further embodiment, the antibody or antigen binding
fragment binds to an epitope on the human ILT3 or competes with an
antibody disclosed for binding to an epitope on the human ILT3,
wherein the epitope comprises at least one amino acid from one or
more of the amino acid sequences set forth in in the group
consisting of SEQ ID NO: 3, 4, 5, 6, 7, and 8. In further
embodiments, the antibody or antigen binding fragment binds to an
epitope on the human ILT3 or competes with an antibody disclosed
for binding to an epitope on the human ILT3, wherein the epitope
comprises one or more of the amino acid sequences set forth in SEQ
ID NOs:3, 4, 5, 6, 7, and 8. In further embodiments, the antibody
or antigen binding fragment binds to an epitope on the human ILT3
or competes with an antibody disclosed for binding to an epitope on
the human ILT3, wherein the epitope comprises the amino acid
sequences set forth in SEQ ID NOs:3, 4, 5, 6, 7, and 8. In
particular embodiments, the epitope is determined by hydrogen
deuterium exchange mass spectrometry (HDX-MS) analysis.
[0013] The present invention further provides an antibody or
antigen binding fragment that binds human ILT3 comprising (a) an HC
having a variable domain (V.sub.H) comprising a variable domain
complementarity determining region (HC-CDR) 1 having the amino acid
sequence set forth in SEQ ID NO: 17, 47, 55, 63, 71, 79, 87, 95, or
103; an HC-CDR2 having the amino acid sequence set forth in SEQ ID
NO: 18, 48, 56, 64, 72, 80, 88, 96, or 104; and an HC-CDR3 having
the amino acid sequence set forth in SEQ ID NO: 23, 49, 57, 65, 73,
81, 89, 97, or 105; and, variants thereof wherein one or more of
the HC-CDRs has one, two, or three amino acid substitutions,
additions, deletions, or combinations thereof; and (b) a light
chain (LC) having variable domain (V.sub.L) comprising a variable
domain complementarity determining region (LC-CDR) 1 having the
amino acid sequence set forth in SEQ ID NO: 27, 50, 58, 66, 74, 82,
90, 98, or 106; an LC-CDR2 having the amino acid sequence set forth
in SEQ ID NO: 43, 51, 59, 67, 75, 83, 91, 99, or 107; and an
LC-CDR3 having the amino acid sequence set forth in SEQ ID NO: 44,
60, 68, 76, 84, 92, 100, or 108; and, variants thereof wherein one
or more of the LC-CDRs has one, two, or three amino acid
substitutions, additions, deletions, or combinations thereof. In
particular embodiments the amino acid sequence differences are
conservative changes/substitutions.
[0014] In a further embodiment of the antibody or antigen binding
fragment, HC-CDR1 has the amino acid sequence set forth in SEQ ID
NO:17; HC-CDR2 has the amino acid sequence set forth in SEQ ID NO:
19, 20, or 21; HC-CDR3 has the amino acid sequence set forth in SEQ
ID NO: 23; and LC-CDR1 has the amino acid sequence set forth in SEQ
ID NO: 34, 35, 36, 37, 38, 39, 40, 41, or 42; LC-CDR2 has the amino
acid sequence set forth in SEQ ID NO: 43; and, LC-CDR3 has the
amino acid sequence set forth in SEQ ID NO:44; and, variants
thereof wherein one or more of the HC-CDRs and LC-CDRs has one,
two, or three amino acid substitutions, additions, deletions, or
combinations thereof. In particular embodiments the amino acid
sequence differences are conservative changes/substitutions.
[0015] In a further embodiment of the antibody or antigen binding
fragment, HC-CDR1 has the amino acid sequence set forth in SEQ ID
NO: 17; HC-CDR2 has the amino acid sequence set forth in SEQ ID NO:
20; and HC-CDR3 has the amino acid sequence set forth in SEQ ID NO:
23; and LC-CDR1 having the amino acid sequence set forth in SEQ ID
NO: 41; LC-CDR2 having the amino acid sequence set forth in SEQ ID
NO: 43; and, LC-CDR3 having the amino acid sequence set forth in
SEQ ID NO: 44; and, variants thereof wherein one or more of the
HC-CDRs and LC-CDRs has one, two, or three amino acid
substitutions, additions, deletions, or combinations thereof. In
particular embodiments the amino acid sequence differences are
conservative changes/substitutions.
[0016] In a further embodiment of the antibody or antigen binding
fragment, the antibody or antigen binding fragment comprises (a) a
V.sub.H having a framework selected from the group consisting of
human V.sub.H1, V.sub.H2, V.sub.H3, V.sub.H4, V.sub.H5, and
V.sub.H6 family and variants thereof having 1, 2, 3, 4, 5, 6, 7, 8,
9, or 10 amino acid substitutions, additions, deletions, or
combinations thereof; and, (b) a V.sub.L having a framework
selected from the group consisting of human V.sub..kappa.1,
V.sub..kappa.2, V.sub..kappa.3, V.sub..kappa.4, V.sub.KS,
V.sub..kappa.6, V.sub..lamda.1, V.sub..lamda.2, V.sub..lamda.3,
V.sub..lamda.4, V.sub..lamda.5, V.sub..lamda.6, V.sub..lamda.7,
V.sub..lamda.8, V.sub..lamda.9, and V.sub..lamda.10 family and
variants thereof having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid
substitutions, additions, deletions, or combinations thereof. In
particular embodiments the amino acid sequence differences are
conservative changes/substitutions.
[0017] In particular embodiments, the antibody or antigen binding
fragment comprises (a) a V.sub.H having a human V.sub.H1 family
framework or variant thereof having 1, 2, 3, 4, 5, 6, 7, 8, 9, or
10 amino acid substitutions, additions, deletions, or combinations
thereof; and, (b) a V.sub.L having a human V.sub..kappa.5 family
framework or variant thereof having 1, 2, 3, 4, 5, 6, 7, 8, 9, or
10 amino acid substitutions, additions, deletions, or combinations
thereof. In particular embodiments the amino acid sequence
differences are conservative changes/substitutions.
[0018] In a further embodiment of the antibody, the antibody
comprises a human IgG1, IgG2, IgG3, or IgG4 HC constant domain or
variant thereof having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid
substitutions, additions, deletions, or combinations thereof
compared to the amino acid sequence of the native human IgG1, IgG2,
IgG3, or IgG4 isotype HC constant domain. In particular aspects,
the constant domain may comprise a C-terminal lysine or may lack a
C-terminal lysine or a C-terminal glycine-lysine dipeptide.
[0019] In particular embodiments, the heavy chain constant domain
is of the human IgG1 isotype, which has been modified to have
reduced or minimal effector function. In further aspects, the
minimal effector function results from an effector-less Fc
mutation, which may comprise or consist of the mutation N297A or
D265A/N297A as identified using Kabat numbering in which case the
minimal effector function results from aglycosylation (see for
example, the amino acid sequence shown in SEQ ID NO: 211 wherein
the N297A mutation corresponds to amino acid position 180; a D265A
mutation, if present, would correspond to amino acid position 148).
In particular aspects, the IgG1 has been modified to comprise or
consist of an L234A, an L235A, and a D265S mutation as identified
using Kabat numbering to render the Fc effector-less (see for
example the amino acid sequence shown in SEQ ID NO: 12 or 13
wherein the L234A, L235A, and D265S mutations correspond to amino
acid positions 117, 118, and 148, respectively).
[0020] In a further aspect, the HC constant domain is of the human
IgG4 isotype and which isotype further includes a substitution of
the serine residue at position 228 (EU numbering) with proline,
which corresponds to position 108 of SEQ ID NO: 9 or 10 (Serine at
position 108).
[0021] In a further embodiment of the antibody or antigen binding
fragment, the antibody comprises a human kappa or lambda LC
constant domain or variant thereof comprising 1, 2, 3, 4, 5, 6, 7,
8, 9, or 10 amino acid substitutions, additions, deletions, or
combinations thereof compared to the amino acid sequence of the
native human kappa or lambda LC constant domain. In particular
embodiments the amino acid sequence differences are conservative
changes/substitutions.
[0022] In a further embodiment of the antibody or antigen binding
fragment, the antibody comprises (i) a V.sub.H having a framework
selected from the human V.sub.H1, V.sub.H2, V.sub.H3, V.sub.H4,
V.sub.H5, and V.sub.H6 family and a human IgG1 or IgG4 HC constant
domain or variant thereof comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or
10 amino acid substitutions, additions, deletions, or combinations
thereof compared to the amino acid sequence of the native human
IgG1 or IgG4 isotype HC constant domain; and, (ii) and a V.sub.L
having a framework selected from the human V.sub.K1,
V.sub..kappa.2, V.sub..kappa.3, V.sub..kappa.4, V.sub.KS,
V.sub..kappa.6, V.sub..lamda.1, V.sub..lamda.2, V.sub..lamda.3,
V.sub..lamda.4, V.sub..lamda.5, V.sub..lamda.6, V.sub..lamda.7,
V.sub..lamda.8, V.sub..lamda.9, and V.sub..lamda.10 family and a
human kappa or lambda LC constant domain or variant thereof
comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid
substitutions, additions, deletions, or combinations thereof
compared to the amino acid sequence of the native human kappa or
lambda LC constant domain. In particular embodiments the amino acid
sequence differences are conservative changes/substitutions.
[0023] In a further embodiment of the antibody or antigen binding
fragment, the antibody comprises (i) a V.sub.H having a human
V.sub.H2 family framework and a V.sub.L having a human
V.sub..kappa.5 family framework; (ii) a human IgG1 or IgG4 HC
constant domain or variant thereof comprising 1, 2, 3, 4, 5, 6, 7,
8, 9, or 10 amino acid substitutions, additions, deletions, or
combinations thereof compared to the amino acid sequence of the
native human IgG1 or IgG4 isotype HC constant domain; and, (iii) a
human kappa LC constant domain or variant thereof comprising 1, 2,
3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions,
deletions, or combinations thereof compared to the amino acid
sequence of the native human kappa LC constant domain. In
particular embodiments the amino acid sequence differences are
conservative changes/substitutions.
[0024] In a further embodiment of the antibody or antigen binding
fragment, the antibody comprises (i) a V.sub.H having a human
V.sub.H1 family framework and a human V.sub.L having a human
V.sub..kappa.5 family framework; (ii) a human IgG4 HC constant
domain or variant thereof comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or
10 amino acid substitutions, additions, deletions, or combinations
thereof compared to the amino acid sequence of the native human
IgG4 isotype HC constant domain; and, (iii) a human kappa LC
constant domain or variant thereof comprising 1, 2, 3, 4, 5, 6, 7,
8, 9, or 10 amino acid substitutions, additions, deletions, or
combinations thereof compared to the amino acid sequence of the
native human kappa LC constant domain. In particular embodiments
the amino acid sequence differences are conservative
changes/substitutions.
[0025] In a further embodiment of the antibody or antigen binding
fragment, the antibody or antigen binding fragment comprises a
V.sub.H and a V.sub.L having the amino acid sequences set forth in
SEQ ID NO: 15 and SEQ ID NO: 16, respectively; SEQ ID NO: 45 and
SEQ ID NO: 46, respectively; SEQ ID NO: 53 and SEQ ID NO: 54,
respectively; SEQ ID NO: 61 and SEQ ID NO: 62, respectively; SEQ ID
NO: 69 and SEQ ID NO: 70, respectively; SEQ ID NO: 77 and SEQ ID
NO: 78, respectively; SEQ ID NO: 85 and SEQ ID NO: 86,
respectively; SEQ ID NO: 93 and SEQ ID NO: 94, respectively; or SEQ
ID NO: 101 and SEQ ID NO: 102, respectively.
[0026] In a further embodiment of the antibody or antigen binding
fragment, the antibody or antigen binding fragment comprises a
V.sub.H having the amino acid sequence set forth in SEQ ID NO: 117,
118, 119, 123, 124, or 125 and a V.sub.L having the amino acid
sequence set forth in SEQ ID NO: 126, 127, 128, 129, 130, 131, 132,
133, 134, 135, 136, 137, 138, 139, 140, or 141.
[0027] In a further embodiment of the antibody or antigen binding
fragment, the antibody or antigen binding fragment comprises a
V.sub.H having the amino acid sequence set forth in SEQ ID NO: 118
and a V.sub.L having the amino acid sequence set forth in SEQ ID
NO: 140.
[0028] In a further embodiment of the antibody, the antibody
comprises an HC constant domain comprising the amino acid sequence
set forth in SEQ ID NO: 9, 10, 11, 12, or 13. In particular
aspects, the HC constant domain comprising the amino acid sequence
set forth in SEQ ID NOs: 9, 11, 12, or 13 may lack a C-terminal
lysine or a C-terminal glycine-lysine dipeptide. In particular
embodiments, the HC constant domain comprises the amino acid
sequence set forth in SEQ ID NO: 10.
[0029] In a further embodiment of the antibody, the antibody
comprises an LC constant domain comprising the amino acid sequence
set forth in SEQ ID NO: 14.
[0030] In a further embodiment of the antibody, the antibody
comprises an HC comprising the amino acid sequence of SEQ ID NO:
142, 143, 144, 148, 149, 150, 167, 168, 169, 170, 174, 175, 176,
177, 178, 182, 183, 184, 185, 186, 187, 191, 192, or 193. In
particular aspects, the HC comprising the amino acid sequence set
forth in SEQ ID NOs: 142, 143, 144, 148, 149, 150, 167, 168, 169,
170, 174, or 175, may lack a C-terminal lysine or a C-terminal
glycine-lysine dipeptide. In particular embodiments, the HC
comprises the amino acid sequence set forth in SEQ ID NO: 143 or
177. In particular embodiments, the HC set forth in SEQ ID NO: 177
further lacks a C-terminal glycine.
[0031] In a further embodiment of the antibody, the antibody
comprises an LC comprising the amino acid sequence set forth in SEQ
ID NO: 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162,
163, 164, 165, or 166. In particular embodiments, the LC comprises
the amino acid set forth in SEQ ID NO: 165.
[0032] In a further embodiment of the antibody, the antibody
comprises an HC having the amino acid sequence set forth in SEQ ID
NO:143 and an LC comprising the amino acid sequence set forth in
SEQ ID NO:165. In particular aspects, the HC comprising the amino
acid sequence set forth in SEQ ID NO: 143 lacks a C-terminal lysine
or a C-terminal glycine-lysine dipeptide.
[0033] The present invention further provides a chimeric,
humanized, or recombinant human antibody or antigen binding
fragment that binds to an epitope on a human ILT3, wherein the
epitope comprises at least one amino acid within the amino acid
sequences set forth in the group consisting of SEQ ID NOs:3, 4, 5,
6, 7, and 8. In a further embodiment, the chimeric, humanized, or
recombinant human antibody or antigen binding fragment binds to an
epitope on a human ILT3 comprising the amino acid sequences set
forth in SEQ ID NOs: 3, 4, 5, 6, 7, and 8. In these embodiments,
the epitope is determined by hydrogen deuterium exchange mass
spectrometry (HDX-MS) analysis.
[0034] The present invention further provides a chimeric,
humanized, or recombinant human antibody or antigen binding
fragment that binds ILT3 wherein the binding cross-blocks or
competes with the binding of an antibody comprising a heavy chain
having the amino acid sequence set forth in SEQ ID NO: 15 and a
light chain having the amino acid sequence shown in SEQ ID NO: 16.
In a further embodiment, the chimeric, humanized, or recombinant
human antibody or antigen binding fragment that cross-blocks or
competes with an antibody comprising a heavy chain having the amino
acid sequence set forth in SEQ ID NO: 15 and a light chain having
the amino acid sequence shown in SEQ ID NO: 16 binds an epitope on
ILT3 that comprises the amino acid sequences set forth in SEQ ID
NOS: 3, 4, 5, 6, 7, and 8.
[0035] The present invention further provides a composition
comprising one or more of any one of the antibody or antigen
binding fragment disclosed or claimed herein and a pharmaceutically
acceptable carrier.
[0036] The present invention further provides a method for treating
a cancer in a subject comprising administering to the subject an
effective amount of an antibody or antigen binding fragment
disclosed or claimed herein sufficient to treat the cancer in the
subject.
[0037] In a further embodiment, the cancer is pancreatic cancer,
melanomas, breast cancer, lung cancer, head and neck cancer,
bronchus cancer, colorectal cancer, prostate cancer, pancreatic
cancer, stomach cancer, ovarian cancer, urinary bladder cancer,
brain or central nervous system cancer, peripheral nervous system
cancer, esophageal cancer, cervical cancer, uterine or endometrial
cancer, cancer of the oral cavity or pharynx, liver cancer, kidney
cancer, testicular cancer, biliary tract cancer, small bowel or
appendix cancer, salivary gland cancer, thyroid gland cancer,
adrenal gland cancer, osteosarcoma, chondrosarcoma, or cancer of
hematological tissues.
[0038] The present invention further provides a method for
treatment of a cancer in a subject comprising administering to the
subject concurrently or consecutively an antibody or antigen
binding fragment disclosed herein in combination with one or more
inhibitors or antagonists of PD-1, PD-L1 and/or PD-L2. In one
embodiment, the antagonist of PD-1 is an antibody or antigen
binding fragment thereof that binds to human PD-1 and blocks the
binding of PD1 to human PD-L1 and PD-L2. In one embodiment, the
antagonist of PD-L1 or PD-L2 is an antibody or antigen binding
fragment thereof that binds to human PD-L1 or PD-L2 and blocks the
binding of human PD-L1 or PD-L2 PD1.
[0039] In a further embodiment, the anti PD1 antagonist is an
anti-PD-1 antibody is nivolumab, pembrolizumab, cemiplimab, or
pidilizumab and the PD-L1 inhibitor is durvalumab, atezolizumab,
avelumab, YW243.55.S70, MPDL3280A, MEDI-4736, MSB-0010718C, or
MDX-1105.
[0040] The present invention further provides an antibody or
antigen binding fragment disclosed or claimed herein for treatment
of cancer in a subject.
[0041] In a further embodiment, the cancer is pancreatic cancer,
melanomas, breast cancer, lung cancer, head and neck cancer,
bronchus cancer, colorectal cancer, prostate cancer, pancreatic
cancer, stomach cancer, ovarian cancer, urinary bladder cancer,
brain or central nervous system cancer, peripheral nervous system
cancer, esophageal cancer, cervical cancer, uterine or endometrial
cancer, cancer of the oral cavity or pharynx, liver cancer, kidney
cancer, testicular cancer, biliary tract cancer, small bowel or
appendix cancer, salivary gland cancer, thyroid gland cancer,
adrenal gland cancer, osteosarcoma, chondrosarcoma, or cancer of
hematological tissues.
[0042] The present invention further provides an antibody or
antigen binding fragment disclosed or claimed herein for treatment
of a cancer in a subject wherein the treatment further comprises
one or more inhibitors or antagonists of PD-1, PD-L1 and/or
PD-L2.
[0043] In one embodiment, the antagonist of PD-1 is an antibody or
antigen binding fragment thereof that binds to human PD-1 and
blocks the binding of PD1 to PD-L1 and PD-L2.
[0044] In one embodiment, the antagonist of PD-L1 or PD-L2 is an
antibody or antigen binding fragment thereof that binds to human
PD-L1 or PD-L2 and blocks the binding of human PD-L1 or PD-L2
PD1.
[0045] In a further embodiment, the anti-PD-1 antibody is
nivolumab, pembrolizumab, cemiplimab, or pidilizumab and the PD-L1
inhibitor is durvalumab, atezolizumab, avelumab, YW243.55.S70,
MPDL3280A, MEDI-4736, MSB-0010718C, or MDX-1105.
[0046] The present invention further provides for use of an
antibody or antigen binding fragment disclosed or claimed herein
for the treatment of a cancer.
[0047] The present invention further provides for use of an
antibody or antigen binding fragment disclosed or claimed herein
for the manufacture of a medicament for the treatment of a
cancer.
[0048] In a further embodiment, the cancer is pancreatic cancer,
melanomas, breast cancer, lung cancer, head and neck cancer,
bronchus cancer, colorectal cancer, prostate cancer, pancreatic
cancer, stomach cancer, ovarian cancer, urinary bladder cancer,
brain or central nervous system cancer, peripheral nervous system
cancer, esophageal cancer, cervical cancer, uterine or endometrial
cancer, cancer of the oral cavity or pharynx, liver cancer, kidney
cancer, testicular cancer, biliary tract cancer, small bowel or
appendix cancer, salivary gland cancer, thyroid gland cancer,
adrenal gland cancer, osteosarcoma, chondrosarcoma, or cancer of
hematological tissues.
[0049] The present invention further provides a composition
comprising any one of the aforementioned antibodies or antigen
binding fragments and a pharmaceutically acceptable carrier. In
particular embodiments, the composition comprises a mixture of
antibodies comprising a heavy chain having a C-terminal lysine and
antibodies comprising a heavy chain lacking a C-terminal lysine. In
particular embodiments, the composition comprises an antibody
disclosed herein wherein the predominant antibody form comprises a
heavy chain having a C-terminal lysine. In particular embodiments,
the composition comprises an antibody disclosed herein wherein the
predominant antibody form comprises a heavy chain lacking a
C-terminal lysine. In particular embodiments, the composition
comprises an antibody disclosed herein wherein about 100% of the
antibodies in the composition comprise a heavy chain lacking a
C-terminal lysine.
BRIEF DESCRIPTION OF THE DRAWINGS
[0050] FIG. 1A, FIG. 1B, FIG. 1C, FIG. 1D, FIG. 1E, and FIG. 1F
show a comparison of the selectivity of several of the anti-ILT3
antibodies disclosed herein to monoclonal antibody 9B11 and mouse
IgG1 (mIGgG1) using a cell-based ELISA format. CHO-K1 cells
expressing human ILT3 (FIG. 1A), Rhesus monkey ILT3 (FIG. 1B),
human ILT5 (FIG. 1C), human ILT7 (FIG. 1D), human ILT8 (FIG. 1E),
or human ILT11 (FIG. 1F) were each tested with monoclonal antibody
p40B5 (LB179.40B5.1A1), p49C6 (LB181.49C6.1A1), and p52B8
1b181.52B8.1B1); antibody 9B11 (U.S. Pat. No. 7,777,008 as having
the amino acid sequences of SEQ ID NO: 33 (light chain) and SEQ ID
NO: 34 (heavy chain)), and mouse IgG1.
[0051] FIG. 2A shows data characteristics on binding affinity,
isoelectric point, purity of monomer species, and thermal stability
measurements for variants of mAb 10. Terms: "huILT3" refers to
human ILT3; "rhILT3" refers to Rhesus monkey ILT3; "pI" refers to
isoelectric point; "Tm" refers to temperature mid-point of a
thermal unfolding curve; "Tagg" refers to mid-point of a thermal
aggregation curve; "SEC" refers size-exclusion ultra-high
performance liquid chromatography).
[0052] FIG. 2B shows the relationship of SEC purity and melting
temperature of humanized light chain variants of mAb 10 (M64V VH1
IgG4). VL1-VL8 refer to variants having the amino acid sequence set
forth in SEQ ID NOs: 126-133, respectively.
[0053] FIG. 3A shows a deuterium labeling difference heatmap of the
human ILT3 extracellular domain amino acid residues that are bound
by Chimeric Anti-ILT3 52B8 mouse 52B8 VH parental/human IgG4
(S228P): mouse 52B8 parental VL/human Kappa antibody ("c58B2"; mAb
73). These six peptide domains, which comprise the epitope bound by
the antibody (residues 18-23 (ISWGNS; SEQ ID NO: 3), residues 64-69
(IPSMTE; SEQ ID NO: 4), residues 96-101 (MTGAYS; SEQ ID NO: 5),
residues 124-131 (QSRSPMDT; SEQ ID NO: 6), residues 152-159
(AQQHQAEF; SEQ ID NO: 7) and residues 184-187 (LLSH; SEQ ID NO:
8)), are located near the border of the D1 and D2 domains of the
ILT3 extracellular domain. The amino acid sequence of human
extracellular domain with C-terminal His Tag is set forth in SEQ ID
NO: 1.
[0054] FIG. 3B shows a first view and a second view of a surface
structure model of the extracellular domain of human ILT3. The dark
region of the model shows the location of the six peptide domains
comprising the human ILT3-His epitope bound by c58B8 (mAb 73).
[0055] FIG. 3C is a ribbon diagram showing the placement of the
epitope on the ILT3 extracellular domain: ISWGNS (SEQ ID NO: 3),
IPSMTE (SEQ ID NO: 4), MTGAYS (SEQ ID NO: 5), QSRSPMDT (SEQ ID NO:
6), AQQHQAEF (SEQ ID NO: 7) and LLSH (SEQ ID NO: 8).
[0056] FIG. 3D shows a deuterium labeling difference heatmap of the
human ILT3 extracellular domain amino acid residues that are bound
by antibody ZM4.1.
[0057] FIG. 3E shows a deuterium labeling difference heatmap of the
human ILT3 extracellular domain amino acid residues that are bound
by antibody DX446.
[0058] FIG. 3F shows a deuterium labeling difference heatmap of the
human ILT3 extracellular domain amino acid residues that are bound
by antibody DX439.
[0059] FIG. 3G shows a deuterium labeling difference heatmap of the
human ILT3 extracellular domain amino acid residues that are bound
by antibody 9B11.
[0060] FIG. 4 shows free c52B8 (mAb 73) concentrations in blood
after multiple doses in humanized tumor models (Panc08.13 and
SK-MEL-5). Free c52B8 concentrations are expressed by circles and
squares. Dashed lines indicate simulated historical antibody levels
after IV bolus administration of 1, 3, 10, or 30 mg/kg of humanized
IgG4 in C57BL/6J mice.
[0061] FIG. 5A shows a human dendritic cell (DC) functional assay
demonstrating anti-ILT3 antibody chimeric antibodies in which the
V.sub.H and V.sub.L from p52B8 fused to IgG4 Fc (c52B8; mAb 73),
IgG1 Fc (mAb 78), or IgG1 (N297A) Fc (mAb 76) had comparable
ability to activate dendritic cells (DCs). Human immature DCs were
prepared and differentiated into CD11c+ dendritic cells with GM-CSF
(1000 U/mL) and IL-4 (1000 U/mL) over 5 days. These cells were
treated with IL-10, LPS (a gram negative bacterial cell wall
component and a TLR4 ligand (Raetz et al. Ann. Rev. Biochem. 71:
635-700 (2002)), and varying concentrations of the indicated
antibodies for 42 hours. The data shown are mean and s.d. of two
technical replicates. This experiment is representative of four
independent studies. Control IgGs had no effect (not shown).
[0062] FIG. 5B and FIG. 5C show that humanized 52B8 (lot 26AVY; mAb
46) is indistinguishable from c52B8 (mAb 73) in the human DC
functional assay using DCs from two different healthy human donors.
The data shown are mean and s.d. of two technical replicates. The
data shown are representative of three independent studies using
these two donors.
[0063] FIG. 6A and FIG. 6B show that anti-ILT3 antibody c52B8 (mAb
73) and humanized anti-ILT3 antibody 52B8 (mAb 46; lot 26AVY)
reduce suppressive capacity of myeloid-derived suppressor cells
(MDSCs). The T cell suppression assay was conducted with a T cell
to MDSC ratio of 4:1. The data shown are means and s.d. of three
technical replicates at the level of the T cell assay step. The
experiment shown is representative of two independent studies using
PBMCs from the same two donors with qualitatively similar
results.
[0064] FIG. 7 shows c52B8 inhibits growth of SK-MEL-5 tumors in
SK-MEL-5 human-NSG mice bearing SK-MEL-5 subcutaneous tumors.
Animals were randomized to treatment on the basis of tumor volume
on day 21 post-implantation and dosed s.c. with 20 mg/kg of c52B8
or isotype control once weekly beginning on day 21. Data shown in
the top panel are means and std. error (nine per group). Individual
animal tumor growth curves are shown in the middle and bottom
panels. Body weight decreased to a similar degree in both control
and 52B8 groups. This study is representative of three independent
studies.
[0065] FIG. 8A, FIG. 8B, FIG. 8C, and FIG. 8D show the effect of
c52B8 in tumor growth and immune activation in SK-MEL-5 hu-NSG
model. FIG. 8A shows a tumor growth curve; FIG. 8B shows CyTOF
quantification of TILs collected 7 days after the 2.sup.nd dose: %
CD4.sup.+ T regulatory cells and CD69 expression levels on
CD4.sup.+ T cells; FIG. 8C shows sHLA-G levels in blood plasma
harvested at the end of the study; FIG. 8D shows IHC analysis of
human CD3.sup.+ T cells infiltration in the tumor, 4 tumors in each
group.
[0066] FIG. 9A, FIG. 9B, FIG. 9C, and FIG. 9D show the effect of a
c52B8 and pembrolizumab combination in Panc 08.13 human-NSG mice.
FIG. 9A shows a tumor growth curve; FIG. 9B shows CyTOF
quantification of % Tregs and CD69 expression levels on CD4+ T
cells from tumors harvested at the end of the study; FIG. 9C shows
plasma sHLA-G levels in terminal blood samples; FIG. 9D shows
plasma IFN.gamma. and IL-8 levels in terminal blood samples
quantitated using 10 plex MSD (Meso Scale Discovery).
[0067] FIG. 10 shows that humanized anti-ILT3 antibody 52B8 (mAb
46) reduces the suppressive capacity of MDSCs to an extent
comparable to chimeric anti-ILT3 antibody c52B8 (mAb 73) in an
MDSC/T cell suppression assay at a 4:1 ratio of T cell to MDSC.
[0068] FIG. 11 shows the effect of the humanized anti-ILT3 antibody
52B8 (mAb 46) and pembrolizumab combination in an MDSC/T cell
suppression assay at either a 4:1 or 8:1 ratio of T cell to MDSC
using MDSC cells obtained from human donor D001003835.
[0069] FIG. 12 shows the effect of the humanized anti-ILT3 antibody
52B8 (mAb 46) and pembrolizumab combination in an MDSC/T cell
suppression assay at an 8:1 ratio of T cell to MDSC using MDSC
cells obtained from human donor D001003180.
[0070] FIG. 13 shows the effect of the humanized anti-ILT3 antibody
52B8 (mAb 46) and pembrolizumab combination in an MDSC/T cell
suppression assay at an 4:1 ratio of T cell to MDSC using MDSC
cells obtained from human donor D001003507.
[0071] FIG. 14 shows the effect of the humnized anti-ILT3 antibody
52B8 (mAb 46) and pembrolizumab combination in an MDSC/T cell
suppression assay at an 8:1 ratio of T cell to MDSC using MDSC
cells obtained from human donor D001003428.
[0072] FIG. 15 shows the effect humanized anti-ILT3 antibody 52B8
(mAb 46) and pembrolizumab combination in a mixed lymphocyte
reaction of IL-10-polarized human monocyte-derived dendritic cells
and allogenic CD8+ T cells, incubated for four days followed by
measurement of interferon gamma (IFN.gamma.) in the culture
supernatant as a read out of T cell activation.
DETAILED DESCRIPTION OF THE INVENTION
[0073] The present invention provides non-promiscuous monoclonal
antibodies specific for human immunoglobulin-like transcript 3
(ILT3), an inhibitory receptor expressed on the surface of myeloid
immune cells.
Definitions
[0074] The term "immunoglobulin-like transcript 3" (abbreviated
herein as "ILT3", and also known as LIR-5, LILRB4, or CD85k), as
used herein and unless otherwise indicated, refers to the human
member of the ILT3 family, which is selectively expressed by
myeloid antigen presenting cells (APCs) such as monocytes,
macrophages, and dendritic cells, e.g., monocyte-derived dendritic
cells differentiated in the presence of IL-10 or vitamin
D.sub.3.
[0075] As used herein, "antibody" refers to an entire
immunoglobulin, including recombinantly produced forms and includes
any form of antibody that exhibits the desired biological activity.
Thus, it is used in the broadest sense and specifically covers, but
is not limited to, monoclonal antibodies (including full length
monoclonal antibodies), polyclonal antibodies, multispecific
antibodies (e.g., bispecific antibodies), humanized antibodies,
fully human antibodies, biparatopic antibodies, humanized camelid
heavy chain antibodies, and non-human/human chimeric antibodies.
"Parental antibodies" are antibodies obtained by exposure of an
immune system to an antigen prior to modification of the antibodies
for an intended use, such as humanization of a non-human antibody
for use as a human therapeutic antibody.
[0076] An "antibody" refers, in one embodiment, to a glycoprotein
comprising at least two heavy chains (HCs) and two light chains
(LCs) inter-connected by disulfide bonds, or an antigen binding
portion thereof. Each heavy chain is comprised of a heavy chain
variable region or domain (abbreviated herein as V.sub.H) and a
heavy chain constant region or domain. In certain naturally
occurring IgG, IgD and IgA antibodies, the heavy chain constant
region is comprised of three domains, C.sub.H1, C.sub.H2 and
C.sub.H3. In certain naturally occurring antibodies, each light
chain is comprised of a light chain variable region or domain
(abbreviated herein as V.sub.L) and a light chain constant region
or domain. The light chain constant region is comprised of one
domain, CL. The human V.sub.H includes six family members:
V.sub.H1, V.sub.H2, V.sub.H3, V.sub.H4, V.sub.HS, and V.sub.H6 and
the human V.sub.L family includes 16 family members: V.sub.K1,
V.sub..kappa.2, V.sub..kappa.3, V.sub..kappa.4, V.sub.KS,
V.sub..kappa.6, V.sub..lamda.1, V.sub..lamda.2, V.sub..lamda.3,
V.sub..lamda.4, V.sub..lamda.5, V.sub..lamda.6, V.sub..lamda.7,
V.sub..lamda.8, V.sub..lamda.9, and V.sub..lamda.10. Each of these
family members can be further divided into particular subtypes.
[0077] The V.sub.H and V.sub.L regions can be further subdivided
into regions of hypervariability, termed complementarity
determining regions (CDR), interspersed with regions that are more
conserved, termed framework regions (FR). Each V.sub.H and V.sub.L
is composed of three CDR regions and four FR regions, arranged from
amino-terminus to carboxy-terminus in the following order: FR1,
CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy
and light chains contain a binding domain that interacts with an
antigen. The constant regions of the antibodies may mediate the
binding of the immunoglobulin to host tissues or factors, including
various cells of the immune system (e.g., effector cells) and the
first component (C1q) of the classical complement system. The
assignment of amino acids to each domain is, generally, in
accordance with the definitions of Sequences of Proteins of
Immunological Interest, Kabat, et al.; National Institutes of
Health, Bethesda, Md.; 5.sup.th ed.; NIH Publ. No. 91-3242 (1991);
Kabat (1978) Adv. Prot. Chem. 32:1-75; Kabat, et al., (1977) J.
Biol. Chem. 252:6609-6616; Chothia, et al., (1987) J Mol. Biol.
196:901-917 or Chothia, et al., (1989) Nature 342:878-883.
[0078] In general, while an antibody comprises six CDRs, three on
the V.sub.H and three on the V.sub.L, the state of the art
recognizes that in most cases, the CDR3 region of the heavy chain
is the primary determinant of antibody specificity, and examples of
specific antibody generation based on CDR3 of the heavy chain alone
are known in the art (e.g., Beiboer et al., J. Mol. Biol. 296:
833-849 (2000); Klimka et al., British J. Cancer 83: 252-260
(2000); Rader et al., Proc. Natl. Acad. Sci. USA 95: 8910-8915
(1998); Xu et al., Immunity 13: 37-45 (2000). See Kabat et al.
(1991) Sequences of Proteins of Immunological Interest, 5th Ed.
Public Health Service, National Institutes of Health, Bethesda, Md.
(defining the CDR regions of an antibody by sequence); see also
Chothia and Lesk (1987) J Mol. Biol. 196: 901-917 (defining the CDR
regions of an antibody by structure).
[0079] The following general rules shown in Table 1 may be used to
identify the CDRs in an antibody sequence. There are rare examples
where these virtually constant features do not occur; however, the
Cys residues are the most conserved feature.
TABLE-US-00001 TABLE 1 Light chain CDR1 Start About amino acid
residue 24 Residue before Usually a Cys Residue after Usually a
Trp. Typically Trp-Tyr-Gln, but also, Trp- Leu-Gln, Trp-Phe-Gln, or
Trp-Tyr-Leu Length 10 to 17 amino acid residues Light chain CDR2
Start Usually 16 amino acid residues after the end of CDR1 Residues
before Generally Ile-Tyr, but also, Val-Tyr, Ile-Lys, or Ile-Phe
Length Usually seven amino acid residues Light chain CDR3 Start
Usually 33 amino acid residues after end of CDR2 Residue before
Usually Cys Residues after Usually Phe-Gly-Xaa-Gly (SEQ ID NO: 221)
Length Seven to 11 amino acid residues Heavy chain CDR1 Start About
amino acid residue 26 (usually four amino acid residues after a
Cys) [Chothia / AbM definition]; Kabat definition starts five amino
acid residues later Residues before Usually Cys-Xaa-Xaa-Xaa (SEQ ID
NO: 222) Residues after Usually a Trp. Typically Trp-Val, but also,
Trp-Ile or Trp-Ala Length 10 to 12 amino acid residues [AbM
definition]; Chothia definition excludes the last four amino acid
residues Heavy chain CDR2 Start Usually 15 amino acid residues
after the end of Kabat / AbM definition) of heavy chain CDR1
Residues before Typically Leu-Glu-Trp-Ile-Gly (SEQ ID NO: 223), but
a number of variations Residues after
Lys/Arg-Leu/Ile/Val/Phe/Thr/Ala-Thr/Ser/Ile/Ala Length Kabat
definition 16 to 19 amino acid residues; AbM (and recent Chothia)
definition ends seven amino acid residues earlier Heavy chain CDR3
Start Usually 33 amino acid residues after end of heavy chain CDR2
(usually two amino acid residues after a Cys) Residues before
Usually Cys-Xaa-Xaa (typically Cys-Ala-Arg) Residues after Usually
Trp-Gly-Xaa-Gly (SEQ ID NO: 224) Length Three to 25 amino acid
residues
[0080] In general, the basic antibody structural unit comprises a
tetramer. Each tetramer includes two identical pairs of polypeptide
chains, each pair having one "light" chain (about 25 kDa) and one
"heavy" chain (about 50-70 kDa). The amino-terminal portion of each
chain includes a variable region of about 100 to 110 or more amino
acids primarily responsible for antigen recognition. The
carboxy-terminal portion of the heavy chain may define a constant
region primarily responsible for effector function of the antibody.
Typically, human light chains are classified as kappa and lambda
light chains. Furthermore, human heavy chains are typically
classified as mu, delta, gamma, alpha, or epsilon, and define the
antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
Within light and heavy chains, the variable and constant regions
are joined by a "J" region of about 12 or more amino acids, with
the heavy chain also including a "D" region of about 10 more amino
acids. See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed.,
2nd ed. Raven Press, N.Y. (1989).
[0081] The heavy chain of an antibody may or may not contain a
terminal lysine (K) residue, or terminal glycine and lysine (GK)
residues. Thus, in particular embodiments of the anti-ILT3
antibodies herein comprising a heavy chain constant region amino
acid sequence shown herein lacking a terminal lysine but
terminating with a glycine residue further include embodiments in
which the terminal glycine residue is also lacking. This is because
the terminal lysine and sometimes glycine and lysine together may
be cleaved during expression of the antibody or cleaved off when
introduced into the human body with no apparent adverse effect on
antibody efficacy, stability, or immunogenicity. In some cases
cases, the nucleic acid molecule encoding the heavy chain may
purposely omit the codons encoding the terminal lysine or the
codons for the terminal lysine and glycine.
[0082] As used herein, "antigen binding fragment" refers to
fragments of antibodies, i.e. antibody fragments that retain the
ability to bind specifically to the antigen bound by the
full-length antibody, e.g. fragments that retain one or more CDR
regions. Examples of antibody binding fragments include, but are
not limited to, Fab, Fab', F(ab').sub.2, and Fv fragments;
diabodies; single-chain antibody molecules, e.g., scFv; nanobodies
and multispecific antibodies formed from antibody fragments.
[0083] As used herein, a "Fab fragment" is comprised of one light
chain and the C.sub.H1 and variable regions of one heavy chain. The
heavy chain of a Fab molecule cannot form a disulfide bond with
another heavy chain molecule. A "Fab fragment" can be the product
of papain cleavage of an antibody.
[0084] As used herein, a "Fab' fragment" contains one light chain
and a portion or fragment of one heavy chain that contains the
V.sub.H domain and the C.sub.H1 domain and also the region between
the C.sub.H1 and C.sub.H2 domains, such that an interchain
disulfide bond can be formed between the two heavy chains of two
Fab' fragments to form a F(ab').sub.2 molecule.
[0085] As used herein, a "F(ab').sub.2 fragment" contains two light
chains and two heavy chains containing the VH domain and a portion
of the constant region between the C.sub.H1 and C.sub.H2 domains,
such that an interchain disulfide bond is formed between the two
heavy chains. An F(ab').sub.2 fragment thus is composed of two Fab'
fragments that are held together by a disulfide bond between the
two heavy chains. An "F(ab').sub.2 fragment" can be the product of
pepsin cleavage of an antibody.
[0086] As used herein, an "Fv region" comprises the variable
regions from both the heavy and light chains, but lacks the
constant regions.
[0087] These and other potential constructs are described in Chan
& Carter (2010) Nat. Rev. Immunol. 10:301. These antibody
fragments are obtained using conventional techniques known to those
with skill in the art, and the fragments are screened for utility
in the same manner as are intact antibodies. Antigen-binding
portions can be produced by recombinant DNA techniques, or by
enzymatic or chemical cleavage of intact immunoglobulins.
[0088] As used herein, an "Fc" region contains two heavy chain
fragments comprising the C.sub.H2 and C.sub.H3 domains of an
antibody. The two heavy chain fragments are held together by two or
more disulfide bonds and by hydrophobic interactions of the
C.sub.H3 domains.
[0089] As used herein, a "diabody" refers to a small antibody
fragment with two antigen-binding sites, which fragments comprise a
heavy chain variable domain (V.sub.H) connected to a light chain
variable domain (V.sub.L) in the same polypeptide chain
(V.sub.H--V.sub.L or V.sub.L--V.sub.H). By using a linker that is
too short to allow pairing between the two domains on the same
chain, the domains are forced to pair with the complementarity
domains of another chain and create two antigen-binding sites.
Diabodies are described more fully in, e.g., EP 404,097; WO
93/11161; and Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90:
6444-6448. For a review of engineered antibody variants generally
see Holliger and Hudson (2005) Nat. Biotechnol. 23:1126-1136.
[0090] As used herein, a "bispecific antibody" is an artificial
hybrid antibody having two different heavy/light chain pairs and
thus two different binding sites. For example, a bispecific
antibody may comprise a first heavy/light chain pair comprising one
heavy and one light chain of a first antibody comprising at least
the six CDRs of an anti-ILT3 antibody disclosed herein or
embodiments wherein one or more of the six CDRs has one, two, or
three amino acid substitutions, additions, deletions, or
combinations thereof along with a second heavy/light chain pair
comprising one heavy and one light chain of a second antibody
having specificity for an antigen of interest other than ILT3.
Bispecific antibodies can be produced by a variety of methods
including fusion of hybridomas or linking of Fab' fragments. See,
e.g., Songsivilai, et al., (1990) Clin. Exp. Immunol. 79: 315-321,
Kostelny, et al., (1992) J Immunol. 148:1547-1553. In addition,
bispecific antibodies may be formed as "diabodies" (Holliger, et
al., (1993) PNAS USA 90:6444-6448) or as "Janusins" (Traunecker, et
al., (1991) EMBO J. 10:3655-3659 and Traunecker, et al., (1992)
Int. J. Cancer Suppl. 7:51-52).
[0091] As used herein, "isolated" antibodies or antigen-binding
fragments thereof are at least partially free of other biological
molecules from the cells or cell cultures in which they are
produced. Such biological molecules include nucleic acids,
proteins, lipids, carbohydrates, or other material such as cellular
debris and growth medium. An isolated antibody or antigen-binding
fragment may further be at least partially free of expression
system components such as biological molecules from a host cell or
of the growth medium thereof. Generally, the term "isolated" is not
intended to refer to a complete absence of such biological
molecules or to an absence of water, buffers, or salts or to
components of a pharmaceutical formulation that includes the
antibodies or fragments.
[0092] As used herein, a "monoclonal antibody" refers to a
population of substantially homogeneous antibodies, i.e., the
antibody molecules comprising the population are identical in amino
acid sequence except for possible naturally occurring mutations
that may be present in minor amounts. In contrast, conventional
(polyclonal) antibody preparations typically include a multitude of
different antibodies having different amino acid sequences in their
variable domains that are often specific for different epitopes.
The modifier "monoclonal" indicates the character of the antibody
as being obtained from a substantially homogeneous population of
antibodies, and is not to be construed as requiring production of
the antibody by any particular method. For example, the monoclonal
antibodies to be used in accordance with the present invention may
be made by the hybridoma method first described by Kohler et al.
(1975) Nature 256: 495, or may be made by recombinant DNA methods
(see, e.g., U.S. Pat. No. 4,816,567). The "monoclonal antibodies"
may also be isolated from phage antibody libraries using the
techniques described in Clackson et al. (1991) Nature 352: 624-628
and Marks et al. (1991) J. Mol. Biol. 222: 581-597, for example.
See also Presta (2005) J. Allergy Clin. Immunol. 116:731.
[0093] As used herein, a "chimeric antibody" is an antibody having
the variable domain from a first antibody and the constant domain
from a second antibody wherein (i) the first and second antibodies
are from different species (U.S. Pat. No. 4,816,567; and Morrison
et al., (1984) Proc. Natl. Acad. Sci. USA 81: 6851-6855) or (ii)
the first and second antibodies are from different isotypes, e.g.,
variable domain from an IgG1 antibody and the constant domains from
an IgG4 antibody). In one aspect, the variable domains are obtained
from a non-human antibody such as a mouse antibody (the "parental
antibody"), and the constant domain sequences are obtained from a
human antibody. In a further aspect, the variable domains are
humanized variable domains from a mouse antibody and the constant
domains of a human antibody.
[0094] As used herein, a "humanized antibody" refers to forms of
antibodies that contain sequences from both human and non-human
(e.g., murine, rat) antibodies. In general, the humanized antibody
will comprise all of at least one, and typically two, variable
domains, in which the hypervariable loops correspond to those of a
non-human immunoglobulin, and all or substantially all of the
framework (FR) regions are those of a human immunoglobulin
sequence. The humanized antibody may optionally comprise at least a
portion of a human immunoglobulin constant region (Fc).
[0095] "Humanization" (also called Reshaping or CDR-grafting) is
now a well-established technique for reducing the immunogenicity of
monoclonal antibodies (mAbs) from xenogeneic sources (commonly
rodent) and for improving the effector functions (ADCC, complement
activation, C1q binding). The engineered mAb is engineered using
the techniques of molecular biology, however simple CDR-grafting of
the rodent complementarity-determining regions (CDRs) into human
frameworks often results in loss of binding affinity and/or
specificity of the original mAb. In order to humanize an antibody,
the design of the humanized antibody includes variations such as
conservative amino acid substitutions in residues of the CDRs, and
back substitution of residues from the rodent mAb into the human
framework regions (back mutations). The positions can be discerned
or identified by sequence comparison for structural analysis or by
analysis of a homology model of the variable regions' 3D structure.
The process of affinity maturation has most recently used phage
libraries to vary the amino acids at chosen positions. Similarly,
many approaches have been used to choose the most appropriate human
frameworks in which to graft the rodent CDRs. As the datasets of
known parameters for antibody structures increases, so does the
sophistication and refinement of these techniques. Consensus or
germline sequences from a single antibody or fragments of the
framework sequences within each light or heavy chain variable
region from several different human mAbs can be used. Another
approach to humanization is to modify only surface residues of the
rodent sequence with the most common residues found in human mAbs
and has been termed "resurfacing" or "veneering." Often, the human
or humanized antibody is substantially non-immunogenic in
humans.
[0096] As used herein, "non-human amino acid sequences" with
respect to antibodies or immunoglobulins refers to an amino acid
sequence that is characteristic of the amino acid sequence of a
non-human mammal. The term does not include amino acid sequences of
antibodies or immunoglobulins obtained from a fully human antibody
library where diversity in the library is generated in silico (See
for example, U.S. Pat. No. 8,877,688 or 8,691,730).
[0097] As used herein, "effector functions" refer to those
biological activities attributable to the Fc region of an antibody,
which vary with the antibody isotype. Examples of antibody effector
functions include: C1q binding and complement dependent
cytotoxicity (CDC); Fc receptor binding; antibody-dependent
cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of
cell surface receptors (e.g. B cell receptor); and B cell
activation.
[0098] As used herein, "conservatively modified variants" or
"conservative substitution" refers to substitutions of amino acids
with other amino acids having similar characteristics (e.g. charge,
side-chain size, hydrophobicity/hydrophilicity, backbone
conformation and rigidity, etc.), such that the changes can
frequently be made without altering the biological activity of the
protein. Those of skill in this art recognize that, in general,
single amino acid substitutions in non-essential regions of a
polypeptide do not substantially alter biological activity (see,
e.g., Watson et al. (1987) Molecular Biology of the Gene, The
Benjamin/Cummings Pub. Co., p. 224 (4th Ed.)). In addition,
substitutions of structurally or functionally similar amino acids
are less likely to disrupt biological activity. Exemplary
conservative substitutions are set forth in Table 2.
TABLE-US-00002 TABLE 2 Original Conservative Original Conservative
residue substitution residue substitution Ala (A) Gly; Ser Leu (L)
Ile; Val Arg (R) Lys; His Lys (K) Arg; His Asn (N) Gln; His Met (M)
Leu; Ile; Tyr Asp (D) Glu; Asn Phe (F) Tyr; Met; Leu Cys (C) Ser;
Ala Pro (P) Ala Gln (Q) Asn Ser (S) Thr Glu (E) Asp; Gln Thr (T)
Ser Gly (G) Ala Trp (W) Tyr; Phe His (H) Asn; Gln Tyr (Y) Trp; Phe
Ile (I) Leu; Val Val (V) Ile; Leu
[0099] As used herein, the term "epitope" or "antigenic
determinant" refers to a site on an antigen (e.g., ILT3) to which
an immunoglobulin or antibody specifically binds. Epitopes within
protein antigens can be formed both from contiguous amino acids
(usually a linear epitope) or noncontiguous amino acids juxtaposed
by tertiary folding of the protein (usually a conformational
epitope). Epitopes formed from contiguous amino acids are
typically, but not always, retained on exposure to denaturing
solvents, whereas epitopes formed by tertiary folding are typically
lost on treatment with denaturing solvents. A contiguous linear
epitope comprises a peptide domain on an antigen comprising at
least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids. A
noncontiguous conformational epitope comprises one or more peptide
domains or regions on an antigen bound by an antibody interspersed
by one or more amino acids or peptide domains not bound by the
antibody, each domain independently comprises at least 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids. Methods for
determining what epitopes are bound by a given antibody (i.e.,
epitope mapping) are well known in the art and include, for
example, immunoblotting and immunoprecipitation assays, wherein
overlapping or contiguous peptides (e.g., from ILT3) are tested for
reactivity with a given antibody (e.g., anti-ILT3 antibody).
Methods of determining spatial conformation of epitopes include
techniques in the art and those described herein, for example,
x-ray crystallography, two-dimensional nuclear magnetic resonance,
and HDX-MS (see, e.g., Epitope Mapping Protocols in Methods in
Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996)).
[0100] The term "epitope mapping" refers to the process of
identifying the molecular determinants on the antigen involved in
antibody-antigen recognition using techniques in the art and those
described herein, for example, x-ray crystallography,
two-dimensional nuclear magnetic resonance, and
Hydrogen-Deuterium-Exchange-with-Mass-Spectroscopy (HDX-MS).
[0101] The term "binds to the same epitope" with reference to two
or more antibodies means that the antibodies bind to the same
segment of amino acid residues or combinations of segments of amino
acids, as determined by a given method. Techniques for determining
whether antibodies bind to the "same epitope on ILT3" with the
antibodies described herein include, for example, epitope mapping
methods, such as, x-ray analyses of crystals of antigen:antibody
complexes, which provides atomic resolution of the epitope, and
HDX-MS. Other methods that monitor the binding of the antibody to
antigen fragments (e.g. proteolytic fragments) or to mutated
variations of the antigen where loss of binding due to a
modification of an amino acid residue within the antigen sequence
is often considered an indication of an epitope component (e.g.
alanine scanning mutagenesis--Cunningham & Wells (1985) Science
244:1081). In addition, computational combinatorial methods for
epitope mapping can also be used. These methods rely on the ability
of the antibody of interest to affinity isolate specific short
peptides from combinatorial phage display peptide libraries.
[0102] Antibodies that "compete with another antibody for binding
to a target such as ILT3" refer to antibodies that inhibit
(partially or completely) the binding of the other antibody to the
target, i.e., ILT3. Whether two antibodies compete with each other
for binding to a target, i.e., whether and to what extent one
antibody inhibits the binding of the other antibody to a target,
may be determined using known competition experiments. In certain
embodiments, an antibody competes with, and inhibits binding of
another antibody to a target by at least 10%, 20%, 30%, 40%, 50%,
60%, 70%, 80%, 90% or 100%. The level of inhibition or competition
may be different depending on which antibody is the "blocking
antibody" (i.e., the cold antibody that is incubated first with the
target). Competition assays can be conducted as described, for
example, in Ed Harlow and David Lane, Cold Spring Harb Protoc;
2006; doi:10.1101/pdb.prot4277 or in Chapter 11 of "Using
Antibodies" by Ed Harlow and David Lane, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., USA 1999. Competing
antibodies bind to the same epitope, an overlapping epitope or to
adjacent epitopes (e.g., as evidenced by steric hindrance).
[0103] Other competitive binding assays include: solid phase direct
or indirect radioimmunoassay (MA), solid phase direct or indirect
enzyme immunoassay (EIA), sandwich competition assay (see Stahli et
al., Methods in Enzymology 9:242 (1983)); solid phase direct
biotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614
(1986)); solid phase direct labeled assay, solid phase direct
labeled sandwich assay (see Harlow and Lane, Antibodies: A
Laboratory Manual, Cold Spring Harbor Press (1988)); solid phase
direct label MA using 1-125 label (see Morel et al., Mol. Immunol.
25(1):7 (1988)); solid phase direct biotin-avidin EIA (Cheung et
al., Virology 176:546 (1990)); and direct labeled MA. (Moldenhauer
et al., Scand. J. Immunol. 32:77 (1990)).
[0104] As used herein, "specifically binds" refers, with respect to
an antigen or molecule such as human ILT3, to the preferential
association of an antibody or other ligand, in whole or part, with
human ILT3 and not to other molecules, particularly molecules found
in human blood or serum. Antibodies typically bind specifically to
their cognate antigen with high affinity, reflected by a
dissociation constant (K.sub.D) of 10.sup.-7 to 10.sup.-11 M or
less. Any K.sub.D greater than about 10.sup.-6 M is generally
considered to indicate nonspecific binding. As used herein, an
antibody that "specifically binds" or "binds specifically" to human
ILT3 refers to an antibody that binds to the human ILT3 with high
affinity, which means having a K.sub.D of 10.sup.-7 M or less, in
particular embodiments a K.sub.D of 10.sup.-8 M or less, or
5.times.10.sup.-9 M or less, or between 10.sup.-8 M and 10.sup.-11
M or less, but does not bind with measurable binding to closely
related proteins such as human ILT5, human ILT7, human ILT8, and
human ILT11 as determined in a cell ELISA or Biacore assay using 10
.mu.g/mL antibody.
[0105] As used herein, an antigen is "substantially identical" to a
given antigen if it exhibits a high degree of amino acid sequence
identity to the given antigen, for example, if it exhibits at least
80%, at least 90%, at least 95%, at least 97%, or at least 99% or
greater amino acid sequence identity to the amino acid sequence of
the given antigen. By way of example, an antibody that binds
specifically to human ILT3 may also cross-react with ILT3 from
certain non-human primate species (e.g., rhesus monkey or
cynomolgus monkey).
[0106] As used herein, "isolated nucleic acid molecule" means a DNA
or RNA of genomic, mRNA, cDNA, or synthetic origin or some
combination thereof which is not associated with all or a portion
of a polynucleotide in which the isolated polynucleotide is found
in nature, or is linked to a polynucleotide to which it is not
linked in nature. For purposes of this disclosure, it should be
understood that "a nucleic acid molecule comprising" a particular
nucleotide sequence does not encompass intact chromosomes. Isolated
nucleic acid molecules "comprising" specified nucleic acid
sequences may include, in addition to the specified sequences,
coding sequences for up to ten or even up to twenty or more other
proteins or portions or fragments thereof, or may include operably
linked regulatory sequences that control expression of the coding
region of the recited nucleic acid sequences, and/or may include
vector sequences.
[0107] As used herein, "treat" or "treating" means to administer a
therapeutic agent, such as a composition containing any of the
antibodies or antigen binding fragments thereof of the present
invention, internally or externally to a subject or patient having
one or more disease symptoms, or being suspected of having a
disease, for which the agent has therapeutic activity or
prophylactic activity. Typically, the agent is administered in an
amount effective to alleviate one or more disease symptoms in the
treated subject or population, whether by inducing the regression
of or inhibiting the progression of such symptom(s) by any
clinically measurable degree. The amount of a therapeutic agent
that is effective to alleviate any particular disease symptom may
vary according to factors such as the disease state, age, and
weight of the patient, and the ability of the drug to elicit a
desired response in the subject. Whether a disease symptom has been
alleviated can be assessed by any clinical measurement typically
used by physicians or other skilled healthcare providers to assess
the severity or progression status of that symptom. The term
further includes a postponement of development of the symptoms
associated with a disorder and/or a reduction in the severity of
the symptoms of such disorder. The terms further include
ameliorating existing uncontrolled or unwanted symptoms, preventing
additional symptoms, and ameliorating or preventing the underlying
causes of such symptoms. Thus, the terms denote that a beneficial
result has been conferred on a human or animal subject with a
disorder, disease or symptom, or with the potential to develop such
a disorder, disease or symptom.
[0108] As used herein, "treatment," as it applies to a human or
veterinary subject, refers to therapeutic treatment, as well as
diagnostic applications. "Treatment" as it applies to a human or
veterinary subject, encompasses contact of the antibodies or
antigen binding fragments of the present invention to a human or
animal subject.
[0109] As used herein, "therapeutically effective amount" refers to
a quantity of a specific substance sufficient to achieve a desired
effect in a subject being treated. For instance, this may be the
amount necessary to inhibit activation of ILT3 or the amount
necessary for enhanced pembrolizumab responsiveness when
co-administered with pembrolizumab.
[0110] As used herein the term "PD-1" refers to the programmed
Death 1 (PD-1) protein, an inhibitory member of the extended
CD28/CTLA-4 family of T cell regulators (Okazaki et al. (2002) Curr
Opin Immunol 14: 391779-82; Bennett et al. (2003) J. Immunol.
170:711-8). Other members of the CD28 family include CD28, CTLA-4,
ICOS and BTLA. The PD-1 gene encodes a 55 kDa type I transmembrane
protein (Agata et al. (1996) Int Immunol. 8:765-72). Two ligands
for PD-1 have been identified, PD-L1 (B7-H1) and PD-L2 (B7-DC),
that have been shown to downregulate T cell activation upon binding
to PD-1 (Freeman et al. (2000) J. Exp. Med. 192:1027-34; Carter et
al. (2002) Eur. J. Immunol. 32:634-43). PD-1 is known as an
immunoinhibitory protein that negatively regulates TCR signals
(Ishida, Y. et al. (1992) EMBO J. 11:3887-3895; Blank, C. et al.
(Epub 2006 Dec. 29) Immunol. Immunother. 56(5):739-745). The
interaction between PD-1 and PD-L1 can act as an immune checkpoint,
which can lead to, e.g., a decrease in tumor infiltrating
lymphocytes, a decrease in T-cell receptor mediated proliferation,
and/or immune evasion by cancerous cells (Dong et al. (2003) J.
Mol. Med. 81:281-7; Blank et al. (2005) Cancer Immunol. Immunother.
54:307-314; Konishi et al. (2004) Clin. Cancer Res. 10:5094-100).
Immune suppression can be reversed by inhibiting the local
interaction of PD-1 with PD-L1 or PD-L2; the effect is additive
when the interaction of PD-1 with PD-L2 is blocked as well (Iwai et
al. (2002) Proc. Nat'l. Acad. Sci. USA 99:12293-7; Brown et al.
(2003) J. Immunol. 170:1257-66).
Antibodies and Antigen Binding Fragments
[0111] The present invention provides isolated chimeric, humanized,
and human antibodies and antigen binding fragments that
specifically bind ILT3 and have no measurable binding to closely
related proteins (e.g., ILT5, ILT7, ILT8, and ILT11) as determined
in a cell ELISA or Biacore assay using 10 .mu.g/mL antibody. The
anti-ILT3 antibodies increase activity of antigen presenting cells
and dendritic cells, reduce activity of monocyte repressors, and
increase priming of T-cells. Thus, the present invention further
includes the use of the anti-ILT3 antibodies in monotherapies for
the treatment of cancers and for use in combination with anti-PD-1
or anti-PD-L1 antibodies, for either in a first line, second line,
or third line therapy for the treatment of cancer.
[0112] An anti-ILT3 antibody includes any antibody disclosed herein
by amino acid sequence and includes any antibody that comprises (i)
at least one, two, three, four, five, or six CDRs of an antibody
disclosed herein by amino acid sequence or (ii) has no CDR amino
acid sequence disclosed herein but which binds the same epitope on
ILT3 as an antibody disclosed herein by amino acid sequence and
which may modulate ILT3 receptor signaling such that the antibody
increases activity of antigen presenting cells and dendritic cells,
reduces activity of monocyte repressors, and increases priming of
T-cells. In particular aspects, the antibody has no measurable
binding to human ILT5, human ILT7, human ILT8, and human ILT11 as
determined in a cell ELISA or in a Biacore assay using 10 .mu.g/mL
of the antibody. The term specifically excludes antibodies
comprising at least one CDR of antibody ZM4.1 or antibody 9B11 or
any of the other antibodies disclosed in U.S. Pat. Nos. 7,777,008
and 8,901,281 or in U.S. Pub. Nos. 20090202544, 20150110714,
20150139986, and 20170267759; and, Intl. Pub. Nos. WO2013043569,
WO2013181438, WO2014116846, WO2016049641, WO2016127427,
WO2018089300, and WO2018148494.
[0113] An anti-ILT3 antigen binding fragment and the like includes
any protein or peptide containing molecule that comprises (i) at
least a portion of an anti-ILT3 antibody disclosed herein by amino
acid sequence, (ii) at least one, two, three, four, five, or six
CDRs of an antibody disclosed herein by sequence, or (iii) has no
CDR amino acid sequence disclosed herein but which binds the same
epitope on ILT3 as an anti-ILT3 antibody disclosed herein by amino
acid sequence, and which may modulate ILT3 receptor signaling such
that the antigen binding fragment increases activity of antigen
presenting cells and dendritic cells, reduces activity of monocyte
repressors, and increases priming of T-cells. In particular
aspects, the antigen binding fragment has no measurable binding to
human ILT5, human ILT7, human ILT8, and human ILT11 as determined
in a cell ELISA or in a Biacore assay using 10 .mu.g/mL of the
anti-ILT3 antigen binding fragment. The term specifically excludes
antigen binding fragments comprising at least one CDR of antibody
ZM4.1 or antibody 9B11 or any of the other antibodies disclosed in
U.S. Pat. Nos. 7,777,008 and 8,901,281 or U.S. Pub. Nos.
20090202544, 20150110714, 20150139986, and 20170267759; and, Intl.
Pub. Nos. WO2013043569, WO2013181438, WO2014116846, WO2016049641,
WO2016127427, WO2018089300, and WO2018148494.
[0114] In a further embodiment, an anti-ILT3 antibody includes any
antibody that comprises (i) at least HC-CDR3 of an antibody
disclosed herein by amino acid sequence or (ii) has no H3-CDR3
amino acid sequence disclosed herein but which binds the same
epitope on ILT3 as an antibody disclosed herein by amino acid
sequence and which may modulate ILT3 receptor signaling such that
the antibody increases activity of antigen presenting cells and
dendritic cells, reduces activity of monocyte repressors, and
increases priming of T-cells. In particular aspects, the antibody
has no measurable binding to human ILT5, human ILT7, human ILT8,
and human ILT11 as determined in a cell ELISA or in a Biacore assay
using 10 .mu.g/mL of the antibody. The term specifically excludes
antibodies comprising at least one CDR of antibody ZM4.1 or
antibody 9B11 or any of the other antibodies disclosed in U.S. Pat.
Nos. 7,777,008 and 8,901,281 or in U.S. Pub. Nos. 20090202544,
20150110714, 20150139986, and 20170267759; and, Intl. Pub. Nos.
WO2013043569, WO2013181438, WO2014116846, WO2016049641,
WO2016127427, WO2018089300, and WO2018148494.
[0115] An anti-ILT3 antigen binding fragment and the like includes
any protein or peptide containing molecule that comprises (i) at
least a portion of an anti-ILT3 antibody disclosed herein by amino
acid sequence, (ii) at least the HC-CDR3 of an antibody disclosed
herein by sequence, or (iii) has no HC-CDR3 amino acid sequence
disclosed herein but which binds the same epitope on ILT3 as an
anti-ILT3 antibody disclosed herein by amino acid sequence, and
which may modulate ILT3 receptor signaling such that the antigen
binding fragment increases activity of antigen presenting cells and
dendritic cells, reduces activity of monocyte repressors, and
increases priming of T-cells. In particular aspects, the antigen
binding fragment has no measurable binding to human ILT5, human
ILT7, human ILT8, and human ILT11 as determined in a cell ELISA or
in a Biacore assay using 10 .mu.g/mL of the anti-ILT3 antigen
binding fragment. The term specifically excludes antigen binding
fragments comprising at least one CDR of antibody ZM4.1 or antibody
9B11 or any of the other antibodies disclosed in U.S. Pat. Nos.
7,777,008 and 8,901,281 or U.S. Pub. Nos. 20090202544, 20150110714,
20150139986, and 20170267759; and, Intl. Pub. Nos. WO2013043569,
WO2013181438, WO2014116846, WO2016049641, WO2016127427,
WO2018089300, and WO2018148494.
[0116] In particular embodiments, the anti-ILT3 antibody is a human
or humanized anti-ILT3 antibody or antigen binding fragment or a
chimeric anti-ILT3 antibody or antigen binding fragment that
comprises HC-CDR3 of an anti-ILT3 antibody molecule disclosed
herein or an H3-CDR3 shown in Table 3.
[0117] In particular embodiments, the anti-ILT3 antibody is a human
or humanized anti-ILT3 antibody or antigen binding fragment or a
chimeric anti-ILT3 antibody or antigen binding fragment that
comprises HC-CDR1, HC-CDR2, HC-CDR3, LC-CDR1, LC-CDR2, and LC-CDR3
of an anti-ILT3 antibody molecule disclosed herein or in Table
3.
TABLE-US-00003 TABLE 3 Seq Seq Seq mAb No. No. No. HC-CDR1 HC-CDR2
HC-CDR3 52B8 NYGMS 17 TISGGGDYTMYPDSVRG 20 RLWFRSLYYAMDY 23 40A6
SYSIN 47 RFWYDEGIAYNLTLES 48 DRDTVGITGWFAY 49 16B1 NYCVN 55
RFWFDEGKAYNLTLES 56 DRDTVGITGWFAY 57 11D1 TYWIE 63
EILPGNGNTHFNENFKD 64 RRLGRGPFDF 65 17H12 NFDMA 71 SITYDGGSTSYRDSVKG
72 VESIATISTYFDY 73 37C8 SYCVN 79 RFWYDEGKVYNLTLES 80 DRDTMGITGWFAY
81 1G12 TYWIQ 87 EILPGSGTTNYNENFKG 88 RLGRGPFDY 89 20E4 SYSVN 95
RFWYDGGTAYNSTLES 96 DRDTMGITGWFAY 97 24A4 SYCVN 103
RFWYDEGKVYNLTLES 104 DRDTLGITGWFAY 105 LC-CDR1 LC-CDR2 LC-CDR3 52B8
RASEKVDSFGQSFMH 41 LTSNLDS 43 QQNNEDPYT 44 40A6 KASQSVGVNVD 50
GSANRHT 51 LQYGSVPYT 52 16B1 KASQSVGINVD 58 GSANRHT 59 LQYGSVPYT 60
11D1 KASQDINEYIG 66 YTSTLQS 67 LQYANPLPT 68 17H12 RASQSVSMSRYDLIH
74 RASDLAS 75 QQTRKSPPT 76 37C8 KASQSVGINVD 82 GSANRHT 83 LQYGSVPYT
84 1G12 EASQDINKHID 90 YASILQP 91 LQYDNLLPT 92 20E4 KASQSVGVNVD 98
GSANRHT 99 LQYGSVPYT 100 24A4 KASQSVGINVD 106 GSANRHT 107 LQYGSVPYT
108
[0118] In particular embodiments, the anti-ILT3 antibody is a human
or humanized anti-ILT3 antibody or antigen binding fragment or a
chimeric anti-ILT3 antibody or antigen binding fragment, in each
case comprising a heavy chain variable domain (V.sub.H) having a
heavy chain complementarity determining region (HC-CDR) 3
comprising an amino acid sequence selected from the group
consisting of SEQ ID NO: 22, 49, 57, 65, 73, 81, 89, 97, and 105,
or an amino acid sequence that has 3, 2, or 1 differences with an
amino acid sequence selected from the group consisting of SEQ ID
NO: 22, 49, 57, 65, 73, 81, 89, 97, and 105. In a further
embodiment, the antibody or antigen binding fragment binds to an
epitope on the human ILT3, wherein the epitope comprises at least
one amino acid from one or more of the amino acid sequences set
forth in in the group consisting of SEQ ID NO: 3, 4, 5, 6, 7, and
8. In a further embodiment, the antibody or antigen binding
fragment binds to an epitope on the human ILT3, wherein the epitope
comprises the amino acid sequences set forth in in the group
consisting of SEQ ID NO: 3, 4, 5, 6, 7, and 8. In particular
embodiments the amino acid sequence differences are conservative
changes/substitutions.
[0119] In particular embodiments, the anti-ILT3 antibody is a
humanized or chimeric anti-ILT3 antibody disclosed herein. In
particular embodiments, the anti-ILT3 antibody is a human or
humanized anti-ILT3 antibody or antigen binding fragment or a
chimeric anti-ILT3 antibody or antigen binding fragment that binds
the same epitope bound by an anti-ILT3 antibody disclosed herein or
competes with the binding of an anti-ILT3 antibody disclosed herein
and the antibody comprises less than three or none of the CDRs of
an anti-ILT3 antibody disclosed herein.
[0120] The present invention further provides an antibody or
antigen binding fragment comprising (i) at least the six
complementary determining regions (CDRs) of an
anti-immunoglobulin-like transcript 3 (ILT3) antibody or (ii) at
least the six CDRs of an anti-ILT3 antibody wherein one or more of
the six CDRs has one, two, or three amino acid substitutions,
additions, deletions, or combinations; wherein the six CDRs of the
anti-ILT3 antibody comprise a heavy chain (HC)-CDR1 having the
amino acid sequence set forth in SEQ ID NO: 17, 47, 55, 63, 71, 79,
87, 95, or 103; an HC-CDR2 having the amino acid sequence set forth
in SEQ ID NO: 18, 48, 56, 64, 72, 80, 88, 96, or 104; an HC-CDR3
having the amino acid sequence set forth in SEQ ID NO: 22, 49, 57,
65, 73, 81, 89, 97, or 105; a light chain (LC)-CDR1 having the
amino acid sequence set forth in SEQ ID NO: 27, 50, 58, 66, 74, 82,
90, 98, or 106; an LC-CDR2 having the amino acid sequence set forth
in SEQ ID NO: 43, 51, 59, 67, 75, 83, 91, 99, or 107; and an
LC-CDR3 having the amino acid sequence set forth in SEQ ID NO: 44,
60, 68, 76, 84, 92, 100, or 108; and, wherein the antibody or
antigen binding fragment specifically binds human or rhesus ILT3 or
both human and rhesus ILT3. In particular embodiments the amino
acid sequence differences are conservative
changes/substitutions.
[0121] In particular embodiments, the present invention provides an
antibody or antigen binding fragment comprising the six CDRs of the
anti-ILT3 antibody comprise a heavy chain (HC)-CDR1 having the
amino acid sequence set forth in SEQ ID NO:17; an HC-CDR2 having
the amino acid sequence set forth in SEQ ID NO:19, 20, or 21; an
HC-CDR3 having the amino acid sequence set forth in SEQ ID NO: 23,
24, 25, or 26; a light chain (LC)-CDR1 having the amino acid
sequence set forth in SEQ ID NO: 34, 35, 36, 37, 38, 39, 40, 41, or
42; an LC-CDR2 having the amino acid sequence set forth in SEQ ID
NO: 43; and an LC-CDR3 having the amino acid sequence set forth in
SEQ ID NO: 44.
[0122] In particular embodiments, the present invention provides an
antibody or antigen binding fragment comprising the six CDRs of the
anti-ILT3 antibody having a heavy chain (HC)-CDR1 having the amino
acid sequence set forth in SEQ ID NO: 17; an HC-CDR2 having the
amino acid sequence set forth in SEQ ID NO: 20; an HC-CDR3 having
the amino acid sequence set forth in SEQ ID NO: 23; a light chain
(LC)-CDR1 having the amino acid sequence set forth in SEQ ID NO:
41; an LC-CDR2 having the amino acid sequence set forth in SEQ ID
NO: 43; and an LC-CDR3 having the amino acid sequence set forth in
SEQ ID NO: 44.
[0123] In particular embodiments, the present invention provides
the above antibody or antigen binding fragment wherein the antibody
or antigen binding fragment comprises a heavy chain variable domain
(V.sub.H) having a framework selected from the human V.sub.H1,
V.sub.H2, V.sub.H3, V.sub.H4, V.sub.H5, and V.sub.H6 family and
variants thereof having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid
substitutions, additions, deletions, or combinations thereof; and,
(b) a light chain variable domain (V.sub.L) having a framework
selected from the human V.sub..kappa.1, V.sub..kappa.2,
V.sub..kappa.3, V.sub..kappa.4, V.sub.KS, V.sub..kappa.6,
V.sub..lamda.1, V.sub..lamda.2, V.sub..lamda.3, V.sub..lamda.4,
V.sub..lamda.5, V.sub..lamda.6, V.sub..lamda.7, V.sub..lamda.8,
V.sub..lamda.9, and V.sub..kappa.10 family and variants thereof
having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions,
additions, deletions, or combinations thereof.
[0124] In particular embodiments, the present invention provides
the above antibody or antigen binding fragment wherein the antibody
comprises a human IgG1, IgG2, IgG3, or IgG4 heavy chain (HC)
constant domain or variant thereof comprising 1, 2, 3, 4, 5, 6, 7,
8, 9, or 10 amino acid substitutions, additions, deletions, or
combinations thereof compared to the amino acid sequence of the
native IgG1, IgG2, IgG3, or IgG4 isotype.
[0125] In particular embodiments, the present invention provides
the above antibody or antigen binding fragment wherein the antibody
comprises a human kappa or lambda light chain constant domain or
variant thereof comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino
acid substitutions, additions, deletions, or combinations thereof
compared to the amino acid sequence of the native human kappa or
lambda light chain domain.
[0126] In particular embodiments, the present invention provides
the above antibody or antigen binding fragment wherein the antibody
comprises (i) a human heavy chain variable domain (V.sub.H) having
a framework selected from the human V.sub.H3 family and a human
light chain variable domain (V.sub.L) having a framework selected
from the human V.sub..kappa.1, V.sub..kappa.3, and V.sub..kappa.4
family; (ii) a human IgG1 or IgG4 heavy chain (HC) constant domain
or variant thereof comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10
amino acid substitutions, additions, deletions, or combinations
thereof compared to the amino acid sequence of the native IgG1 or
IgG4 isotype; and, (iii) a human kappa or lambda light chain
constant domain or variant thereof comprising 1, 2, 3, 4, 5, 6, 7,
8, 9, or 10 amino acid substitutions, additions, deletions, or
combinations thereof compared to the amino acid sequence of the
native human kappa or lambda light chain domain. In particular
embodiments the amino acid sequence differences are conservative
changes/substitutions.
[0127] In particular embodiments, the present invention provides
the above antibody or antigen binding fragment wherein the antibody
or antigen binding fragment comprises a heavy chain variable domain
(V.sub.H) and a light chain variable domain (V.sub.L) having the
amino acid sequences set forth in SEQ ID NO: 15 and SEQ ID NO: 16,
respectively; SEQ ID NO: 45 and SEQ ID NO: 46, respectively; SEQ ID
NO: 53 and SEQ ID NO: 54, respectively; SEQ ID NO:61 and SEQ ID NO:
62, respectively; SEQ ID NO: 69 and SEQ ID NO: 70, respectively;
SEQ ID NO:77 and SEQ ID NO: 78, respectively; SEQ ID NO: 85 and SEQ
ID NO: 86, respectively; SEQ ID NO: 93 and SEQ ID NO: 94,
respectively; or SEQ ID NO: 101 and SEQ ID NO: 102,
respectively.
[0128] In particular embodiments, the present invention provides
the above antibody or antigen binding fragment wherein the antibody
or antigen binding fragment comprises a heavy chain variable domain
(V.sub.H) having the amino acid sequence set forth in SEQ ID NO:
117, 118, 119, 120, 121, 122, 123, 124, or 125 and a light chain
variable domain (V.sub.L) having the amino acid sequence set forth
in SEQ ID NO: 126, 127, 128, 129, 130, 131, 132, 133, 134, 135,
136, 137, 138, 139, 140, or 141.
[0129] In particular embodiments, the present invention provides
the above antibody or antigen binding fragment wherein the antibody
or antigen binding fragment comprises a heavy chain variable domain
(V.sub.H) having the amino acid sequence set forth in SEQ ID NO:
118 and a light chain variable domain (V.sub.L) having the amino
acid sequence set forth in SEQ ID NO: 140.
[0130] In particular embodiments, the present invention provides
the above antibody or antigen binding fragment wherein, the
antibody comprises a heavy chain (HC) constant domain comprising
the amino acid sequence set forth in SEQ ID NO: 9, 10, 11, 12, or
13 and variants of SEQ ID NO: 9, 11, 12, or 13 in which the HC
lacks a C-terminal Lysine or glycine-lysine.
[0131] In particular embodiments, the present invention provides
the above antibody or antigen binding fragment wherein, the
antibody comprises a light chain (LC) constant domain comprising
the amino acid sequence set forth in SEQ ID NO: 14.
[0132] In particular embodiments, the present invention provides
the above antibody or antigen binding fragment wherein, the
antibody comprises a heavy chain (HC) comprising the amino acid
sequence of SEQ ID NO: 142, 143, 144, 148, 149, 150, 167, 168, 169,
170, 174, 175, 176, 177, 178, 182, 183, 184, 185, 186, 187, 191,
192, or 193 and variants of an HC comprising the amino acid
sequence of SEQ ID NO: 143, 144, 148, 149, 150, 167, 168, 169, 170,
174, or 175 in which the HC lacks a C-terminal Lysine or
glycine-lysine.
[0133] In particular embodiments, the present invention provides
the above antibody or antigen binding fragment wherein, the
antibody comprises a light chain (LC) comprising the amino acid
sequence set forth in SEQ ID NO: 151, 152, 153, 154, 155, 156, 157,
158, 159, 160, 161, 162, 163, 164, 165, or 166.
[0134] In particular embodiments, the present invention provides
the above antibody or antigen binding fragment wherein, the
antibody comprises a heavy chain (HC) comprising the amino acid
sequence of SEQ ID NO: 142, 143, 144, 148, 149, 150, 167, 168, 169,
170, 174, 175, 176, 177, 178, 182, 183, 184, 185, 186, 187, 191,
192, or 193 and a light chain (LC) comprising the amino acid
sequence set forth in SEQ ID NO: 151, 152, 153, 154, 155, 156, 157,
158, 159, 160, 161, 162, 163, 164, 165, or 166, and variants of an
HC comprising the amino acid sequence of SEQ ID NO: 143, 144, 148,
149, 150, 167, 168, 169, 170, 174, or 175 in which the HC lacks a
C-terminal Lysine or glycine-lysine.
[0135] In particular embodiments, the present invention provides an
antibody selected from the antibodies presented in Table 4.
[0136] In particular embodiments, the present invention provides
the above antibody or antigen binding fragment wherein, the
antibody comprises a heavy chain (HC) having the amino acid
sequence set forth in SEQ ID NO: 143 and a light chain (LC)
comprising the amino acid sequence set forth in SEQ ID NO: 165 and
variants in which the HC lacks a C-terminal Lysine or
glycine-lysine.
[0137] In particular embodiments, the present invention provides
the above antibody or antigen binding fragment wherein the antibody
comprises a human IgG1, IgG2, IgG3, or IgG4 heavy chain (HC)
constant domain or variant thereof comprising 1, 2, 3, 4, 5, 6, 7,
8, 9, or 10 amino acid substitutions, additions, deletions, or
combinations thereof compared to the amino acid sequence of the
native IgG1, IgG2, IgG3, or IgG4 isotype, and variants thereof in
which the HC lacks a C-terminal Lysine or glycine-lysine.
[0138] In some embodiments, different constant domains may be fused
to a V.sub.L and V.sub.H regions comprising the CDRs provided
herein. In particular embodiments, the V.sub.H regions comprising
the CDRs provided herein may be fused to a human IgG1, IgG2, IgG3,
or IgG4 heavy chain (HC) constant domain or variant thereof
comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid
substitutions, additions, deletions, or combinations thereof
compared to the amino acid sequence of the native or wild-type
IgG1, IgG2, IgG3, or IgG4 isotype, and variants thereof in which
the HC lacks a C-terminal Lysine or glycine-lysine.
[0139] In particular embodiments, the anti-ILT3 antibody (or
antigen binding fragment) has an altered effector function and may
comprise a heavy chain constant domain other than native
(wild-type) human IgG1, for example a human IgG1 that has mutations
that abrogate or minimize one or more effector functions, including
ability to bind complement, human IgG4, or a hybrid human
IgG1/human IgG4, and variants thereof in which the HC lacks a
C-terminal Lysine or glycine-lysine.
[0140] Although native human IgG1 antibodies provide for long
half-life and for effector functions, such as complement activation
and antibody-dependent cellular cytotoxicity, such activities may
not be desirable for all uses of an antibody. Thus, in particular
embodiments, it is desirable that the heavy chain constant domain
or Fc have minimal or reduced effector function ("effector-less").
In those instances, the anti-ILT3 HC variable domain may be fused
to a human IgG4 constant domain, which is generally known to be
effector-less, or an IgG1 constant domain that has been mutated to
be rendered effecter-less. These effector-less molecules have
minimal or reduced binding to human Fc.gamma.RIIIA, and
Fc.gamma.RIIA, and Fc.gamma..RI compared to the polypeptide
comprising the wildtype IgG Fc region, wherein the affinity to each
of human Fc.gamma.RIIIA, and Fc.gamma.RIIA, and Fc.gamma.RI is
reduced by 1.15-fold to 100-fold compared to the polypeptide
comprising the wildtype IgG constant domain, and wherein the
antibody-dependent cell-mediated cytotoxicity (ADCC) induced by
said molecule is 0-20% of the ADCC induced by the polypeptide
comprising the wild-type human IgG1 constant domain.
[0141] Therefore in particular embodiments, the present invention
includes chimeric or humanized anti-ILT3 antibodies and
antigen-binding fragments thereof that comprise a human IgG4
constant domain. In a further embodiment, the human IgG4 constant
domain may be modified to differ from the native (wild-type) human
IgG4 constant domain (Swiss-Prot Accession No. P01861.1) at a
position corresponding to position 228 in the EU system and
position 241 in the Kabat system in which the native serine at
position 108 (Ser108) of the HC constant domain is replaced with
proline (Pro), see for example SEQ ID NO: 9. This modification
prevents formation of a potential inter-chain disulfide bond
between the cysteine at position 106 (Cys106) and the cysteine at
position 109 (Cys109), which correspond to positions Cys226 and
Cys229 in the EU system and positions Cys239 and Cys242 in the
Kabat system, which may interfere with proper intra-chain disulfide
bond formation. See Angal et al. Mol. Imunol. 30:105 (1993); see
also (Schuurman et. al., Mol. Immunol. 38: 1-8, (2001); SEQ ID NOs:
14 and 41). In particular embodiments, the human IgG4 constant
domain may further include in addition to the S228P substitution an
L235E substitution.
[0142] In another embodiment, the chimeric or humanized anti-ILT3
antibody may be fused to a modified human IgG1 constant domain,
which has been modified to be effector-less. In one embodiment, the
human IgG1 HC may include substitutions of human IgG2 HC residues
at positions 233-236 and IgG4 HC residues at positions 327, 330,
and 331 to greatly reduce ADCC and CDC (Armour et al., Eur J
Immunol. 29(8):2613-24 (1999); Shields et al., J Biol Chem.
276(9):6591-604(2001)). In particular embodiments, the antibody
comprises a human IgG1 heavy chain (HC) constant domain or variant
thereof comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid
substitutions, additions, deletions, or combinations thereof
compared to the amino acid sequence of the native IgG, which
provides an antibody having reduced or minimal effector function.
In particular aspects, the IgG1 has been modified to comprise or
consist of an L234A, an L235A, and a D265S mutation to render the
Fc effector-less. Other mutations that may be used to render an
IgG1 Fc effector-less may be found in U.S. Pat. No. 8,969,526.
[0143] In another embodiment, the human IgG1 HC is modified to lack
N-glycosylation of the asparagine (Asn) residue at around position
297 of the HC. The consensus sequence for N-glycosylation is
Asn-Xaa-Ser/Thr (wherein Xaa is any amino acid except Pro); in IgG1
the N-glycosylation consensus sequence is Asn-Ser-Thr. The
modification may be achieved by replacing the codon for the Asn at
position 297 in the nucleic acid molecule encoding the HC with a
codon for another amino acid, for example Gln. Alternatively, the
codon for Ser may be replaced with the codon for Pro or the codon
for Thr may be replaced with any codon except the codon for Ser,
e.g. N297A or N297D. Such modified IgG1 molecules have little or no
detectable effector function. Alternatively, all three codons are
modified.
[0144] In another embodiment, the human IgG1 constant domain is
modified to include one or more amino acid substitutions selected
from E233P, L234A, L235A, L235E, N297A, N297D, D265S, and P331S,
wherein the residues are numbered according to the EU index of
Kabat, and wherein said polypeptide exhibits a reduced affinity to
the human Fc.gamma.RIIIA and/or Fc.gamma.RIIA and/or Fc.gamma.RI
compared to a polypeptide comprising the wildtype IgG constant
domain region. In particular embodiments, the human IgG constant
domain comprises substitutions of L234A, L235A, and D265S as
illustrated by SEQ ID NO: 4, for example. In particular
embodiments, the human IgG1 constant domain comprises an amino acid
substitution at position Pro329 and at least one further amino acid
substitution E233P, L234A, L235A, L235E, N297A, N297D, D265S, and
P331S. These and other substitutions are disclosed in WO9428027;
WO2004099249; WO20121300831, U.S. Pat. Nos. 9,708,406; 8,969,526;
9,296,815; Sondermann et al. Nature 406, 267-273 (20 Jul.
2000)).
[0145] In an embodiment of the invention, the anti-ILT3 antibodies
or antigen binding fragments thereof include embodiments in which
one or more of the six CDRs has one, two, or three amino acid
substitutions, additions, deletions, or combinations thereof
comprise a full tetrameric structure having two light chains and
two heavy chains, including constant regions. The variable regions
of each light chain/heavy chain pair form the antibody binding
site. Thus, in general, an intact antibody has two binding sites.
Except in bispecific antibodies, the two binding sites are, in
general, the same.
[0146] In specific embodiments, the present invention provides the
anti-ILT3 antibodies shown in the Table 4. With the exception of
those antibodies comprising a replacement of the tryptophan residue
at position 101 of the V.sub.H, the antibodies disclosed herein
bind the human ILT3.
TABLE-US-00004 TABLE 4 SEQ ID NO: mAb Heavy Light No. Description
Chain Chain 1 Humanized anti-ILT3 mAb (52B8 VH1 / VL1) 142 151 IgG4
S228P / Kappa 2 Humanized anti-ILT3 mAb (52B8 VH1 / VL2) 142 152
IgG4 S228P / Kappa 3 Humanized anti-ILT3 mAb (52B8 VH1 / VL3) 142
153 IgG4 S228P / Kappa 4 Humanized anti-ILT3 mAb (52B8 VH1 / VL4)
142 154 IgG4 S228P / Kappa 5 Humanized anti-ILT3 mAb (52B8 VH2 /
VL1) 148 151 IgG4 S228P / Kappa 6 Humanized anti-ILT3 mAb (52B8 VH2
/ VL2) 148 152 IgG4 S228P / Kappa 7 Humanized anti-ILT3 mAb (52B8
VH2 / VL3) 148 153 IgG4 S228P / Kappa 8 Humanized anti-ILT3 mAb
(52B8 VH2 / VL4) 148 154 IgG4 S228P / Kappa 9 Humanized anti-ILT3
mAb (52B8 VH1 M64V / 143 151 VL1) IgG4 S228P / Kappa 10 Humanized
anti-ILT3 mAb (52B8 VH1 M64V / 143 152 VL2) IgG4 S228P / Kappa 11
Humanized anti-ILT3 mAb (52B8 VH1 M64V / 143 153 VL3) IgG4 S228P /
Kappa 12 Humanized anti-ILT3 mAb (52B8 VH1 M64V / 143 154 VL4) IgG4
S228P / Kappa 13 Humanized anti-ILT3 mAb (52B8 VH2 M64V / 149 151
VL1) IgG4 S228P / Kappa 14 Humanized anti-ILT3 mAb (52B8 VH2 M64V /
149 152 VL2) IgG4 S228P / Kappa 15 Humanized anti-ILT3 mAb (52B8
VH2 M64V / 149 153 VL3) IgG4 S228P / Kappa 16 Humanized anti-ILT3
mAb (52B8 VH2 M64V / 149 154 VL4) IgG4 S228P / Kappa 17 Humanized
anti-ILT3 mAb (52B8 VH1 M64L / 144 151 VL1) IgG4 S228P / Kappa 18
Humanized anti-ILT3 mAb (52B8 VH1 M64L / 144 152 VL2) IgG4 S228P /
Kappa 19 Humanized anti-ILT3 mAb (52B8 VH1 M64L / 144 153 VL3) IgG4
S228P / Kappa 20 Humanized anti-ILT3 mAb (52B8 VH1 M64L / 144 155
VL4) IgG4 S228P / Kappa 21 Humanized anti-ILT3 mAb (52B8 VH2 M64L /
150 151 VL1) IgG4 S228P / Kappa 22 Humanized anti-ILT3 mAb (52B8
VH2 M64L / 150 152 VL2) IgG4 S228P / Kappa 23 Humanized anti-ILT3
mAb (52B8 VH2 M64L / 150 153 VL3) IgG4 S228P / Kappa 24 Humanized
anti-ILT3 mAb (52B8 VH2 M64L / 150 154 VL4) IgG4 S228P / Kappa 25
Humanized anti-ILT3 mAb ((52B8 VH1 M64V / 169 152 VL2) L234A L235A
D265S) IgG1 / Kappa 26 Humanized anti-ILT3 mAb ((52B8 VH1 M64V /
169 155 VL5) L234A L235A D265S) IgG1 / Kappa 27 Humanized anti-ILT3
mAb ((52B8 VH1 M64V / 169 156 VL6) L234A L235A D265S) IgG1 / Kappa
28 Humanized anti-ILT3 mAb ((52B8 VH1 M64V / 169 157 VL7) L234A
L235A D265S) IgG1 / Kappa 29 Humanized anti-ILT3 mAb ((52B8 VH1
M64V / 169 158 VL8) L234A L235A D265S) IgG1 / Kappa 30 Humanized
anti-ILT3 mAb (52B8 VH1 M64V / 143 155 VL5) IgG4 S228P / Kappa 31
Humanized anti-ILT3 mAb (52B8 VH1 M64V / 143 156 VL6) IgG4 S228P /
Kappa 32 Humanized anti-ILT3 mAb (52B8 VH1 M64V / 143 157 VL7) IgG4
S228P / Kappa 33 Humanized anti-ILT3 mAb (52B8 VH1 M64V / 143 158
VL8) IgG4 S228P / Kappa 34 Humanized anti-ILT3 mAb (52B8 VH1 M64V
145 152 W101F / VL2) IgG4 S228P / Kappa 35 Humanized anti-ILT3 mAb
(52B8 VH1 M64V 146 152 W101Y / VL2) IgG4 S228P / Kappa 36 Humanized
anti-ILT3 mAb (52B8 VH1 M64V 147 152 W101Q / VL2) IgG4 S228P /
Kappa 37 Humanized anti-ILT3 mAb ((52B8 VH1 M64V 145 152 W101F /
VL2) L234A L235A D265S) IgG1 / Kappa 38 Humanized anti-ILT3 mAb
((52B8 VH1 M64V 146 152 W101Y / VL2) L234A L235A D265S) IgG1 /
Kappa 39 Humanized anti-ILT3 mAb ((52B8 VH1 M64V 147 152 W101Q /
VL2) L234A L235A D265S) IgG1 / Kappa 40 Humanized anti-ILT3 mAb
(52B8 VH1 M64V / 143 159 VL2 S35A) IgG4 S228P / Kappa 41 Humanized
anti-ILT3 mAb (52B8 VH1 M64V / 143 160 VL2 S35N) IgG4 S228P / Kappa
42 Humanized anti-ILT3 mAb (52B8 VH1 M64V / 143 161 VL2 N34Q) IgG4
S228P / Kappa 43 Humanized anti-ILT3 mAb (52B8 VH1 M64V / 143 162
VL2 N34D) IgG4 S228P / Kappa 44 Humanized anti-ILT3 mAb (52B8 VH1
M64V / 143 163 VL5 S35A) IgG4 S228P / Kappa 45 Humanized anti-ILT3
mAb (52B8 VH1 M64V / 143 164 VL5 S35N) IgG4 S228P / Kappa 46
Humanized anti-ILT3 mAb (52B8 VH1 M64V / 143 165 VL5 N34Q) IgG4
S228P / Kappa 47 Humanized anti-ILT3 mAb (52B8 VH1 M64V / 143 166
VL5 N34D) IgG4 S228P / Kappa 48 Humanized anti-ILT3 mAb (52B8 VH1
M64V 145 155 W101F / VL5) IgG4 S228P / Kappa 49 Humanized anti-ILT3
mAb (52B8 VH1 M64V 146 155 W101Y / VL5) IgG4 S228P / Kappa 50
Humanized anti-ILT3 mAb (52B8 VH1 M64V 147 155 W101Q / VL5) IgG4
S228P / Kappa 51 Humanized anti-ILT3 mAb (52B8 VH1 M64V 145 163
W101F / VL5 S35A) IgG4 S228P / Kappa 52 Humanized anti-ILT3 mAb
(52B8 VH1 M64V 145 164 W101F / VL5 S35N) IgG4 S228P / Kappa 53
Humanized anti-ILT3 mAb (52B8 VH1 M64V 145 165 W101F / VL5 N34Q)
IgG4 S228P / Kappa 54 Humanized anti-ILT3 mAb (52B8 VH1 M64V 145
166 W101F / VL5 N34D) IgG4 S228P / Kappa 55 Humanized anti-ILT3 mAb
(52B8 VH1 M64V 146 163 W101Y / VL5 S35A) IgG4 S228P / Kappa 56
Humanized anti-ILT3 mAb (52B8 VH1 M64V 146 164 W101Y / VL5 S35N)
IgG4 S228P / Kappa 57 Humanized anti-ILT3 mAb (52B8 VH1 M64V 146
165 W101Y / VL5 N34Q) IgG4 S228P / Kappa 58 Humanized anti-ILT3 mAb
(52B8 VH1 M64V 146 166 W101Y / VL5 N34D) IgG4 S228P / Kappa 59
Humanized anti-ILT3 mAb (52B8 VH1 M64V 147 163 W101Q / VL5 S35A)
IgG4 S228P / Kappa 60 Humanized anti-ILT3 mAb (52B8 VH1 M64V 147
164 W101Q / VL5 S35N) IgG4 S228P / Kappa 61 Humanized anti-ILT3 mAb
(52B8 VH1 M64V 147 165 W101Q / VL5 N34Q) IgG4 S228P / Kappa 62
Humanized anti-ILT3 mAb (52B8 VH1 M64V 147 166 W101Q / VL5 N34D)
IgG4 S228P / Kappa 63 Humanized anti-ILT3 mAb (52B8 VH1 M64V / 210
126 VL1 N34Q) IgG1 N297A / Kappa 64 Humanized anti-ILT3 mAb (52B8
VH1 M64V / 210 127 VL2 IgG1 N297A / Kappa 65 Humanized anti-ILT3
mAb (52B8 VH1 M64V / 210 161 VL2 N34Q) IgG1 N297A / Kappa 66
Humanized anti-ILT3 mAb (52B8 VH1 M64V / 210 128 VL3 N34Q) IgG1
N297A / Kappa 67 Humanized anti-ILT3 mAb (52B8 VH1 M64V / 210 129
VL4 N34Q) IgG1 N297A / Kappa 68 Humanized anti-ILT3 mAb (52B8 VH1
M64V / 210 130 VL5 IgG1 N297A / Kappa 69 Humanized anti-ILT3 mAb
(52B8 VH1 M64V / 210 165 VL5 N34Q) IgG1 N297A / Kappa 70 Humanized
anti-ILT3 mAb (52B8 VH1 M64V / 210 131 VL6 N34Q) IgG1 N297A / Kappa
71 Humanized anti-ILT3 mAb (52B8 VH1 M64V / 210 132 VL7 N34Q) IgG1
N297A / Kappa 72 Humanized anti-ILT3 mAb (52B8 VH1 M64V / 210 133
VL8 N34Q) IgG1 N297A / Kappa 73 Chimeric anti-ILT3 52B8 mouse
VH/human IgG4 113 116 (S228P): mouse VL/human Kappa 74 Chimeric
anti-ILT3 52B8 mouse VH M64V/ 114 116 human IgG4 (S228P): mouse
VL/human Kappa 75 Chimeric anti-ILT3 52B8 mouse VH M64L/ 115 116
human IgG4 (S228P): mouse VL/human Kappa 76 Chimeric anti-ILT3 52B8
mouse VH/human IgG1 Residues 116 (N297A): mouse VL/human Kappa
1-122 of SEQ ID NO: 113 And SEQ ID NO: 211 77 Chimeric anti-ILT3
52B8 mouse VH M64V/ Residues 116 human IgG1 (N297A): mouse VL/human
Kappa 1-122 of SEQ ID NO: 114 And SEQ ID NO: 211 78 Chimeric
anti-ILT3 52B8 mouse VH/human IgG1: Residues 116 mouse VL/human
Kappa 1-122 of SEQ ID NO: 113 And SEQ ID NO: 11 79 Chimeric
anti-ILT3 52B8 mouse VH M64V/human Residues 116 IgG1: mouse
VL/human Kappa 1-122 of SEQ ID NO: 114 And SEQ ID NO: 11 80
Chimeric anti-ILT3 40A6 rat VH /human IgG4 194 195 (S228P): rat
VL/human Kappa 81 Chimeric anti-ILT3 16B1 rat VH /human IgG4 196
197 (S228P): rat VL/human Kappa 82 Chimeric anti-ILT3 11D1 mouse VH
/human IgG4 198 199 (S228P): mouse VL/human Kappa 83 Chimeric
anti-ILT3 17H12 rat VH /human IgG4 200 201 (S228P): rat VL/human
Kappa 84 Chimeric anti-ILT3 37C8 rat VH /human IgG4 202 203
(S228P): rat VL/human Kappa 85 Chimeric anti-ILT3 1G12 mouse VH
/human IgG4 203 205 (S228P): mouse VL/human Kappa 86 Chimeric
anti-ILT3 20E4 rat VH /human IgG4 206 207 (S228P): rat VL/human
Kappa 87 Chimeric anti-ILT3 24A4 rat VH /human IgG4 208 209
(S228P): rat VL/human Kappa 88 Chimeric anti-ILT3 40A6 rat VH
/human IgG1 212 195 (N297A): rat VL/human Kappa 89 Chimeric
anti-ILT3 16B1 rat VH /human IgG1 213 197 (N297A): rat VL/human
Kappa 90 Chimeric anti-ILT3 11D1 mouse VH /human IgG1 214 199
(N297A): mouse VL/human Kappa 91 Chimeric anti-ILT3 17H12 rat VH
/human IgG1 215 201 (N297A): rat VL/human Kappa 92 Chimeric
anti-ILT3 37C8 rat VH /human IgG1 216 203 (N297A): rat VL/human
Kappa 93 Chimeric anti-ILT3 1G12 mouse VH /human IgG1 217 205
(N297A): mouse VL/human Kappa 94 Chimeric anti-ILT3 20E4 rat VH
/human IgG1 218 207 (N297A): rat VL/human Kappa 95 Chimeric
anti-ILT3 24A4 rat VH /human IgG1 219 209 (N297A): rat VL/human
Kappa 96 Chimeric anti-ILT3 40A6 rat VH /human IgG1 220 195
(N297A): rat VL/human Kappa
[0147] Epitope mapping by hydrogen-deuterium exchange mass
spectrometry (HDX-MS) as described in Example 4 shows that the
anti-ILT3 antibodies disclosed herein bind to an epitope on the
extracellular domain near the border between the D1 and D2 domains
of the extracellular domain of ILT3. The epitope identified using
HDX-MS indicates that the epitope bound by the anti-ILT3 antibodies
disclosed herein comprises or consists of at least one amino acid
within one or more of the peptide domain amino acid sequences
selected from the group consisting of SEQ ID NOs: 3, 4, 5, 6, 7,
and 8. In a further embodiment, the epitope comprises or consists
of one or more of the peptide domain amino acid sequences selected
from the group consisting of SEQ ID NOs: 3, 4, 5, 6, 7, and 8. In
certain embodiments, the epitope comprises or consists of at least
one amino acid in each of the peptide domain amino acid sequences
selected from the group consisting of SEQ ID NOs: 3, 4, 5, 6, 7,
and 8 and identified in the HDX-MS. In particular embodiments, the
epitope comprises or consists of one or more of the peptide domain
amino acid sequences selected from the group consisting of SEQ ID
NOs: 3, 4, 5, 6, 7, and 8. In particular embodiments, the epitope
comprises or consists of the peptide domains shown in SEQ ID Nos:
3, 4, 5, 6, 7, and 8.
[0148] Thus, the present invention further provides a chimeric,
humanized, or human antibody or antigen binding fragment that binds
to an epitope on ILT3 wherein the epitope comprises or consists of
at least one amino acid within one or more of the peptide domains
comprising amino acid sequences shown by the amino acid sequences
set forth in SEQ ID NOs: 3, 4, 5, 6, 7, and 8 as determined by
hydrogen deuterium exchange mass spectrometry (HDX-MS)
analysis.
[0149] In a further embodiment, the present invention further
provides a chimeric, humanized, or human antibody or antigen
binding fragment that binds to an epitope on ILT3 wherein the
epitope comprises or consists of amino acids within the peptide
domains shown in one or more of SEQ ID Nos: 3, 4, 5, 6, 7, and 8.
In certain embodiments, the epitope comprises or consists of at
least one amino acid in each of the peptide domains identified in
the heat map determined by HDX-MS and shown in FIG. 3A.
[0150] The present invention further provides a chimeric,
humanized, or human antibody or antigen binding fragment that
cross-blocks the binding of an antibody comprising a heavy chain
variable domain having the amino acid sequence set forth in SEQ ID
NO: 15 and a light chain variable domain having the amino acid
sequence shown in SEQ ID NO: 16 to an epitope on ILT3. In a further
embodiment, the epitope comprises or consists of at least one amino
acid within one or more of the peptide domains comprising or
consisting of amino acid sequences shown by the amino acid
sequences set forth in SEQ ID NOs: 3, 4, 5, 6, 7, and 8 as
determined by hydrogen deuterium exchange mass spectrometry
(HDX-MS) analysis. In a further embodiment, the epitope comprises
or consists of amino acids within the peptide domains shown in one
or more of SEQ ID NOs: 3, 4, 5, 6, 7, and 8. In certain
embodiments, the epitope comprises or consists of at least one
amino acid in each of the peptide domains identified in the
HDX-MS.
[0151] The present invention further provides bispecific antibodies
and antigen-binding fragments comprising a first antibody or
antigen binding fragment that binds ILT3 and a second antibody or
antigen binding fragment that binds a molecule other than ILT3,
wherein the first antibody or antigen binding fragment comprises at
least the amino acid sequence of an HC-CDR3 having an amino acid
sequence selected from the group consisting of SEQ ID NO: 22, 49,
57, 65, 73, 81, 89, 97, and 105, or having an amino acid sequence
that has 3, 2, or 1 differences with an amino acid sequence
selected from the group consisting of SEQ ID NO: 22, 49, 57, 65,
73, 81, 89, 97, and 105 and wherein the first antibody binds an
ILT3 epitope comprising amino acids within the sequences of SEQ ID
Nos: 3, 4, 5, 6, 7, and 8 and the second antibody binds a molecule
other than ILT3, and methods of use thereof.
[0152] The present invention further provides bispecific antibodies
and antigen-binding fragments comprising a first antibody or
antigen binding fragment that binds ILT3 and a second antibody or
antigen binding fragment that binds a molecule other than ILT3,
wherein the first antibody or antigen binding fragment comprising
at least the six CDRs of an anti-ILT3 antibody or embodiments
thereof wherein one or more of the CDRs has one, two, or three
amino acid substitutions, additions, deletions, or combinations
thereof and wherein the first antibody binds an ILT3 epitope
comprising amino acids within the sequences of SEQ ID NOs: 3, 4, 5,
6, 7, and 8 and the second antibody binds a molecule other than
ILT3, and methods of use thereof.
[0153] The present invention further provides biparatopic
antibodies (antibodies having binding specificity for different
epitopes on the same antigen) having a first heavy/light chain pair
of a first antibody that comprises at least an HC-CDR3 having an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 22, 49, 57, 65, 73, 81, 89, 97, and 105, or having an amino
acid sequence that has 3, 2, or 1 differences with an amino acid
sequence selected from the group consisting of SEQ ID NOs: 22, 49,
57, 65, 73, 81, 89, 97, and 105, wherein the first heavy/light
chain pair binds an ILT3 epitope comprising amino acids within the
sequences of SEQ ID NOs: 3, 4, 5, 6, 7, and 8 and the second
antibody binds a molecule other than ILT3 and a second heavy/light
chain pair of a second antibody having specificity for an anti-ILT3
epitope that is different from the epitope recognized by the first
heavy/light chain pair.
[0154] The present invention further provides biparatopic
antibodies (antibodies having binding specificity for different
epitopes on the same antigen) having first heavy/light chain pair
of a first antibody that comprises at least the six CDRs of an
anti-ILT3 antibody or embodiments thereof wherein one or more of
the CDRs has one, two, or three amino acid substitutions,
additions, deletions, or combinations thereof wherein the first
antibody binds an ILT3 epitope comprising amino acids within the
sequences of SEQ ID NOs: 3, 4, 5, 6, 7, and 8, wherein the first
heavy/light chain pair binds an ILT3 epitope comprising amino acids
within the sequences of SEQ ID NOs: 3, 4, 5, 6, 7, and 8 and the
second antibody binds a molecule other than ILT3 and a second
heavy/light chain pair of a second antibody having specificity for
an anti-ILT3 epitope that is different from the epitope recognized
by the first heavy/light chain pair.
Pharmaceutical Compositions and Administration
[0155] To prepare pharmaceutical or sterile compositions of the
anti-ILT3 antibodies or antigen binding fragments thereof, the
antibody or antigen binding fragments thereof is admixed with a
pharmaceutically acceptable carrier or excipient. See, e.g.,
Remington's Pharmaceutical Sciences and U.S. Pharmacopeia: National
Formulary, Mack Publishing Company, Easton, Pa. (1984) and
continuously updated on the Internet by the U.S. Pharmacopeial
Convention (USP) 12601 Twinbrook Parkway, Rockville, Md.
20852-1790, USA.
[0156] Formulations of therapeutic and diagnostic agents may be
prepared by mixing with acceptable carriers, excipients, or
stabilizers in the form of, e.g., lyophilized powders, slurries,
aqueous solutions or suspensions (see, e.g., Hardman, et al. (2001)
Goodman and Gilman's The Pharmacological Basis of Therapeutics,
McGraw-Hill, New York, N.Y.; Gennaro (2000) Remington: The Science
and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New
York, N.Y.; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms:
Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.)
(1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY;
Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms:
Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000)
Excipient Toxicity and Safety, Marcel Dekker, Inc., New York,
N.Y.).
[0157] In a further embodiment, a composition comprising an
antibody or antibody fragment disclosed herein is administered to a
subject in accordance with the Physicians' Desk Reference 2017
(Thomson Healthcare; 75st edition (Nov. 1, 2002)). Methods of
administering antibody molecules are known in the art and are
described below. Suitable dosages of the molecules used will depend
on the age and weight of the subject and the particular drug used.
Dosages and therapeutic regimens of the anti-ILT3 antibody or
antigen binding fragment can be determined by a skilled artisan. In
certain embodiments, the anti-ILT3 antibody or antigen binding
fragment is administered by injection (e.g., subcutaneously or
intravenously) at a dose of about 1 to 30 mg/kg, e.g., about 5 to
25 mg/kg, about 10 to 20 mg/kg, about 1 to 5 mg/kg, or about 3
mg/kg. In some embodiments, the anti-ILT3 antibody or antigen
binding fragment is administered at a dose of about 1 mg/kg, about
3 mg/kg, or 10 mg/kg, about 20 mg/kg, about 30 mg/kg, or about 40
mg/kg. In some embodiments, the anti-ILT3 antibody or antigen
binding fragment is administered at a dose of about 1-3 mg/kg, or
about 3-10 mg/kg. In some embodiments, the anti-ILT3 antibody or
antigen binding fragment is administered at a dose of about 0.5-2,
2-4, 2-5, 5-15, or 5-20 mg/kg. The dosing schedule can vary from
e.g., once a week to once every 2, 3, or 4 weeks. In one
embodiment, the anti-ILT3 antibody or antigen binding fragment is
administered at a dose from about 10 to 20 mg/kg every other
week.
[0158] The mode of administration can vary. Suitable routes of
administration is preferably parenteral or subcutaneous. Other
routes of administration may include oral, transmucosal,
intradermal, direct intraventricular, intravenous, intranasal,
inhalation, insufflation, or intra-arterial.
[0159] In particular embodiments, the anti-ILT3 antibodies or
antigen binding fragments thereof can be administered by an
invasive route such as by injection. In further embodiments of the
invention, the anti-ILT3 antibodies or antigen binding fragments
thereof, or pharmaceutical composition thereof, may be administered
intravenously, subcutaneously, intraarterially, or by inhalation,
aerosol delivery. Administration by non-invasive routes (e.g.,
orally; for example, in a pill, capsule or tablet) is also within
the scope of the present invention.
[0160] Compositions can be administered with medical devices known
in the art. For example, a pharmaceutical composition of the
invention can be administered by injection with a hypodermic
needle, including, e.g., a prefilled syringe or autoinjector.
[0161] The pharmaceutical compositions disclosed herein may also be
administered with a needleless hypodermic injection device; such as
the devices disclosed in U.S. Pat. Nos. 6,620,135; 6,096,002;
5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824 or
4,596,556.
[0162] The pharmaceutical compositions disclosed herein may also be
administered by infusion. Examples of well-known implants and
modules form administering pharmaceutical compositions include:
U.S. Pat. No. 4,487,603, which discloses an implantable
micro-infusion pump for dispensing medication at a controlled rate;
U.S. Pat. No. 4,447,233, which discloses a medication infusion pump
for delivering medication at a precise infusion rate; U.S. Pat. No.
4,447,224, which discloses a variable flow implantable infusion
apparatus for continuous drug delivery; U.S. Pat. No. 4,439,196,
which discloses an osmotic drug delivery system having
multi-chamber compartments. Many other such implants, delivery
systems, and modules are well known to those skilled in the
art.
[0163] The administration regimen depends on several factors,
including the serum or tissue turnover rate of the therapeutic
antibody, the level of symptoms, the immunogenicity of the
therapeutic antibody, and the accessibility of the target cells in
the biological matrix. Preferably, the administration regimen
delivers sufficient therapeutic antibody to effect improvement in
the target disease state, while simultaneously minimizing undesired
side effects. Accordingly, the amount of biologic delivered depends
in part on the particular therapeutic antibody and the severity of
the condition being treated. Guidance in selecting appropriate
doses of therapeutic antibodies is available (see, e.g.,
Wawrzynczak (1996) Antibody Therapy, Bios Scientific Pub. Ltd,
Oxfordshire, UK; Kresina (ed.) (1991) Monoclonal Antibodies,
Cytokines and Arthritis, Marcel Dekker, New York, N.Y.; Bach (ed.)
(1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune
Diseases, Marcel Dekker, New York, N.Y.; Baert, et al. (2003) New
Engl. J. Med. 348:601-608; Milgrom et al. (1999) New Engl. J. Med.
341:1966-1973; Slamon et al. (2001) New Engl. J. Med. 344:783-792;
Beniaminovitz et al. (2000) New Engl. J. Med. 342:613-619; Ghosh et
al. (2003) New Engl. J. Med. 348:24-32; Lipsky et al. (2000) New
Engl. J. Med 343:1594-1602).
[0164] Dosage regimens 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. It is especially advantageous to formulate parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the
subjects to be treated; each unit contains a predetermined quantity
of active compound calculated to produce the desired therapeutic
effect in association with the required pharmaceutical carrier. The
specification for the dosage unit forms described herein are
dictated by and directly dependent on (a) the unique
characteristics of the antibody or antibody binding fragment and
the particular therapeutic effect to be achieved, and (b) the
limitations inherent in the art of compounding such an active
molecules for the treatment of sensitivity in individuals. (see,
e.g., Yang, et al. (2003) New Engl. J. Med. 349:427-434; Herold, et
al. (2002) New Engl. J. Med. 346:1692-1698; Liu, et al. (1999) J.
Neurol. Neurosurg. Psych. 67:451-456; Portielji, et al. (20003)
Cancer Immunol. Immunother. 52:133-144).
Use of the Anti-ILT3 Antibodies or Antigen Binding Fragments
Disclosed Herein
[0165] The anti-ILT3 antibodies and antigen binding fragments
disclosed herein being non-promiscuous for related ILTs may be used
to specifically detect human ILT3 (e.g., in a biological sample,
such as serum or plasma), using a conventional immunoassay, such as
an enzyme linked immunosorbent assays (ELISA), an radioimmunoassay
(MA) or tissue immunohistochemistry. The invention thus provides a
method for detecting human ILT3 in a biological sample comprising
contacting a biological sample with an anti-ILT3 antibody or
antigen binding fragment and detecting either the anti-ILT3
antibody or antigen binding fragment bound to human ILT3 or unbound
anti-ILT3 antibody or antigen binding fragment disclosed herein, to
thereby detect human ILT3 in the biological sample. The anti-ILT3
antibody or antigen binding fragment is directly or indirectly
labeled with a detectable substance to facilitate detection of the
bound or unbound anti-ILT3 antibody or antigen binding fragment
disclosed herein. Suitable detectable substances include various
enzymes, prosthetic groups, fluorescent materials, luminescent
materials and radioactive materials. Examples of suitable enzymes
include horseradish peroxidase, alkaline phosphatase,
.beta.-galactosidase, or acetylcholinesterase; examples of suitable
prosthetic group complexes include streptavidin/biotin and
avidin/biotin; examples of suitable fluorescent materials include
umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; and examples of suitable radioactive material include
.sup.125I, .sup.131I, .sup.35S, and .sup.3H.
[0166] Alternative to labeling the anti-ILT3 antibody or antigen
binding fragment, human ILT3 can be assayed in biological fluids by
a competition immunoassay utilizing ILT3 standards labeled with a
detectable substance and an unlabeled anti-human ILT3 anti-ILT3
antibody or antigen binding fragment disclosed herein. In this
assay, the biological sample, the labeled ILT3 standards and the
anti-ILT3 antibody or antigen binding fragment are combined and the
amount of labeled ILT3 standard bound to the unlabeled anti-ILT3
antibody or antigen binding fragment disclosed herein is
determined. The amount of human ILT3 in the biological sample is
inversely proportional to the amount of labeled ILT3 standard bound
to the anti-ILT3 antibody or antigen binding fragment.
[0167] An anti-ILT3 antibody or antigen binding fragment disclosed
herein may also be used to detect ILT3 from a species other than
humans, in particular ILT3 from primates (e.g., cynomolgus monkey
or rhesus monkey).
Methods of Upmodulating Immune Responses In Vivo
[0168] The anti-ILT3 antibodies or antigen binding fragments
disclosed herein may be used as immunostimulatory compositions,
e.g., alone or as part of a vaccine or combination therapy, to
promote B cell, and/or T cell activation, e.g., either Th1 or Th2
cell activation, in a subject. That is, the anti-ILT3 antibody or
antigen binding fragment disclosed herein may serve as adjuvants
used in combination with an antigen of interest to enhance an
immune response to that antigen of interest in vivo. For example,
to stimulate an antibody or cellular immune response to an antigen
of interest (e.g., for vaccination purposes), the antigen and
anti-ILT3 antibody or antigen binding fragment disclosed herein may
be co-administered (e.g., co-administered at the same time in the
same or separate compositions, or sequentially in time such that an
enhanced immune response occurs). The antigen of interest and the
anti-ILT3 antibody or antigen binding fragment disclosed herein may
be formulated together into a single pharmaceutical composition or
in separate compositions. In one embodiment, the antigen of
interest and the anti-ILT3 antibody or antigen binding fragment
disclosed herein are administered simultaneously to the subject.
Alternatively, in certain situations it may be desirable to
administer the antigen first and then the anti-ILT3 antibody or
antigen binding fragment disclosed herein or vice versa (for
example, in the case of an antigen that naturally evokes a Th1
response, it may be beneficial to first administer the antigen
alone to stimulate a Th1 response and then administer an anti-ILT3
antibody or antigen binding fragment disclosed herein, alone or
together with a boost of antigen, to shift the immune response to a
Th2 response). In preferred embodiments, an anti-ILT3 antibody or
antigen binding fragment disclosed herein is administered at the
time of priming with antigen, i.e., at the time of the first
administration of antigen. For example, day -3, -2, -1, 0, +1, +2,
+3. A particularly preferred day of administration of an anti-ILT3
antibody or antigen binding fragment disclosed herein is day
-1.
[0169] In one embodiment, an anti-ILT3 antibody or antigen binding
fragment disclosed herein is administered with an antigen of
interest. An antigen of interest is one to which an immune response
is desired. For example, an antigen of interest is an antigen
capable of stimulating immune protection in a subject against
challenge by an infectious agent from which the antigen was
derived. Further contemplated is administration of an anti-ILT3
antibody or antigen binding fragment disclosed herein to increase
immune responses without having to administer an antigen.
[0170] Exemplary antigens of interest therefore include those
derived from infectious agents, wherein an immune response directed
against the antigen serves to prevent or treat disease caused by
the agent. Such antigens include, but are not limited to, viral,
bacterial, fungal or parasite proteins and any other proteins,
glycoproteins, lipoprotein, glycolipids, and the like. Antigens of
interest also include those which provide benefit to a subject
which is at risk for acquiring or which is diagnosed as having a
tumor. The subject is preferably a mammal and most preferably, is a
human.
[0171] Typical antigens of interest may be classified as follows:
protein antigens, such as ceruloplasmin and serum albumin;
bacterial antigens, such as teichoic acids, flagellar antigens,
capsular polysaccharides, and extra-cellular bacterial products and
toxins; glycoproteins and glycolipids; viruses, such as animal,
plant, and bacterial viruses; conjugated and synthetic antigens,
such as protein/hapten conjugates, molecules expressed
preferentially by tumors, compared to normal tissue; synthetic
polypeptides; and nucleic acids, such as ribonucleic acid and
deoxyribonucleic acid. The term "infectious agent," as used herein,
includes any agent which expresses an antigen, which elicits a host
cellular immune response. Non-limiting examples of viral antigens
which may be considered useful as include, but are not limited to,
the nucleoprotein (NP) of influenza virus and the Gag proteins of
HIV. Other heterologous antigens include, but are not limited to,
HIV Env protein or its component parts gp120 and gp41, HIV Nef
protein, and the HIV Pol proteins, reverse transcriptase and
protease. In addition, other viral antigens such as Ebola virus
(EBOV) antigens, such as, for example, EBOV NP or glycoprotein
(GP), either full-length or GP deleted in the mucin region of the
molecule (Yang et al., Nat Med 6:886 (2000), small pox antigens,
hepatitis A, B or C virus, human rhinovirus such as type 2 or type
14, herpes simplex virus, poliovirus type 2 or 3, foot-and-mouth
disease virus (FMDV), rabies virus, rotavirus, influenza virus,
coxsackie virus, human papilloma virus (HPV), for example the type
16 papilloma virus, the E7 protein thereof, and fragments
containing the E7 protein or its epitopes; and simian
immunodeficiency virus (SIV) may be used. The antigens of interest
need not be limited to antigens of viral origin. Parasitic
antigens, such as, for example, malarial antigens are included, as
are fungal antigens, bacterial antigens and tumor antigens.
Examples of antigens derived from bacteria are those derived from
Bordetella pertussis (e.g., P69 protein and filamentous
haemagglutinin (FHA) antigens), Vibrio cholerae, Bacillus
anthracis, and E. coli antigens such as E. coli heat labile toxin B
subunit (LT-B), E. coli K88 antigens, and enterotoxigenic E. coli
antigens. Other examples of antigens include Schistosoma mansoni
P28 glutathione S-transferase antigens (P28 antigens) and antigens
of flukes, mycoplasma, roundworms, tapeworms, Chlamydia
trachomatis, and malaria parasites, e.g., parasites of the genus
plasmodium or babesia, for example Plasmodium falciparum, and
peptides encoding immunogenic epitopes from the aforementioned
antigens.
[0172] By the term "tumor-related antigen," as used herein, is
meant an antigen which affects tumor growth or metastasis in a host
organism. The tumor-related antigen may be an antigen expressed by
a tumor cell, or it may be an antigen that is expressed by a
non-tumor cell but when so expressed, promotes the growth or
metastasis of tumor cells. The types of tumor antigens and
tumor-related antigens include any known or heretofore unknown
tumor antigen, including, without limitation, the bcr/abl antigen
in leukemia, HPVE6 and E7 antigens of the oncogenic virus
associated with cervical cancer, the MAGE1 and MZ2-E antigens in or
associated with melanoma, and the MVC-1 and HER-2 antigens in or
associated with breast cancer.
[0173] An infection, disease or disorder which may be treated or
prevented by the administration of a composition comprising an
anti-ILT3 antibody or antigen binding fragment disclosed herein
includes any infection, disease or disorder wherein a host immune
response acts to prevent the infection, disease or disorder.
Diseases, disorders, or infection which may be treated or prevented
by the administration of a composition comprising an anti-ILT3
antibody or antigen binding fragment disclosed herein include, but
are not limited to, any infection, disease or disorder caused by or
related to a fungus, parasite, virus, or bacteria, diseases,
disorders or infections caused by or related to various agents used
in bioterrorism, listeriosis, Ebola virus, SARS, small pox,
hepatitis A, hepatitis B, hepatitis C, diseases and disorders
caused by human rhinovirus, HIV and AIDS, Herpes, polio,
foot-and-mouth disease, rabies, diseases or disorders caused by or
related to: rotavirus, influenza, coxsackie virus, human papilloma
virus, SIV, malaria, cancer, e.g., tumors, and diseases or
disorders caused by or related to infection by Bordetella
pertussis, Vibrio cholerae, Bacillus anthracis, E. coli, flukes,
mycoplasma, roundworms, tapeworms, Chlamydia trachomatis, and
malaria parasites, etc.
Immune Responses to Tumor Cells
[0174] Regulatory T cells play an important role in the maintenance
of immunological self-tolerance by suppressing immune responses
against autoimmune diseases and cancer. Accordingly, in one
embodiment, upmodulating an immune response would be beneficial for
enhancing an immune response in cancer. Therefore, the anti-ILT3
antibodies or antigen binding fragments disclosed herein may be
used in the treatment of malignancies, to inhibit tumor growth or
metastasis. The anti-ILT3 antibodies or antigen binding fragments
disclosed herein may be administered systemically or locally to the
tumor site.
[0175] In one embodiment, modulation of human ILT3 function may be
useful in the induction of tumor immunity. An ILT3 binding molecule
may be administered to a patient having tumor cells (e.g., sarcoma,
melanoma, lymphoma, leukemia, neuroblastoma, carcinoma) to overcome
tumor-specific tolerance in the subject.
[0176] As used herein, the term "neoplastic disease" is
characterized by malignant tumor growth or in disease states
characterized by benign hyperproliferative and hyperplastic cells.
The common medical meaning of the term "neoplasia" refers to "new
cell growth" that results as a loss of responsiveness to normal
growth controls, e.g., neoplastic cell growth.
[0177] As used herein, the terms "hyperproliferative",
"hyperplastic", malignant" and "neoplastic" are used
interchangeably, and refer to those cells in an abnormal state or
condition characterized by rapid proliferation or neoplasia. The
terms are meant to include all types of hyperproliferative growth,
hyperplastic growth, cancerous growths or oncogenic processes,
metastatic tissues or malignantly transformed cells, tissues, or
organs, irrespective of histopathologic type or stage of
invasiveness. A "hyperplasia" refers to cells undergoing an
abnormally high rate of growth. However, as used herein, the terms
neoplasia and hyperplasia can be used interchangeably, as their
context will reveal, referring generally to cells experiencing
abnormal cell growth rates. Neoplasias and hyperplasias include
"tumors," which may be either benign, premalignant or
malignant.
[0178] The terms "neoplasia," "hyperplasia," and "tumor" are often
commonly referred to as "cancer," which is a general name for more
than 100 disease that are characterized by uncontrolled, abnormal
growth of cells. Examples of cancer include, but are not limited
to: breast; colon; non-small cell lung, head and neck; colorectal;
lung; prostate; ovary; renal; melanoma; and gastrointestinal (e.g.,
pancreatic and stomach) cancer; and osteogenic sarcoma.
[0179] In one embodiment, the cancer is selected from the group
consisting of: pancreatic cancer, melanomas, breast cancer, lung
cancer, head and neck cancer, bronchus cancer, colorectal cancer,
prostate cancer, pancreatic cancer, stomach cancer, ovarian cancer,
urinary bladder cancer, brain or central nervous system cancer
(e.g., gliobastoma), peripheral nervous system cancer, esophageal
cancer, cervical cancer, uterine or endometrial cancer, cancer of
the oral cavity or pharynx, liver cancer, kidney cancer, testicular
cancer, biliary tract cancer, small bowel or appendix cancer,
salivary gland cancer, thyroid gland cancer, adrenal gland cancer,
osteosarcoma, chondrosarcoma, and cancer of hematological
tissues.
Immune Responses to Infectious Agents
[0180] Upregulation of immune responses may be in the form of
enhancing an existing immune response or eliciting an initial
immune response. For example, enhancing an immune response by
modulation of ILT3 may be useful in cases of viral infection. As
the anti-ILT3 antibodies or antigen binding fragments disclosed
herein may act to enhance immune responses, they would be
therapeutically useful in situations where more rapid or thorough
clearance of pathogenic agents, e.g., bacteria and viruses would be
beneficial.
[0181] As used herein, the term "viral infection" includes
infections with organisms including, but not limited to, HIV (e.g.,
HIV-1 and HIV-2), human herpes viruses, cytomegalovirus (esp.
Human), Rotavirus, Epstein-Barr virus, Varicella Zoster Virus,
hepatitis viruses, such as hepatitis B virus, hepatitis A virus,
hepatitis C virus and hepatitis E virus, paramyxoviruses:
Respiratory Syncytial virus, parainfluenza virus, measles virus,
mumps virus, human papilloma viruses (for example HPV6, 11, 16, 18
and the like), flaviviruses (e.g. Yellow Fever Virus, Dengue Virus,
Tick-borne encephalitis virus, Japanese Encephalitis Virus) or
influenza virus.
[0182] As used herein, the term "bacterial infections" include
infections with a variety of bacterial organisms, including
gram-positive and gram-negative bacteria. Examples include, but are
not limited to, Neisseria spp, including N. gonorrhea and N.
meningitidis, Streptococcus spp, including S. pneumoniae, S.
pyogenes, S. agalactiae, S. mutans; Haemophilus spp, including H.
influenzae type B, non typeable H. influenzae, H. ducreyi;
Moraxella spp, including M. catarrhalis, also known as Branhamella
catarrhalis; Bordetella spp, including B. pertussis, B.
parapertussis and B. bronchiseptica; Mycobacterium spp., including
M. tuberculosis, M. bovis, M leprae, M avium, M. paratuberculosis,
M smegmatis; Legionella spp, including L. pneumophila; Escherichia
spp, including enterotoxic E. coli, enterohemorragic E. coli,
enteropathogenic E. coli; Vibrio spp, including V. cholera,
Shigella spp, including S. sonnei, S. dysenteriae, S. flexnerii;
Yersinia spp, including Y. enterocolitica, Y. pestis, Y.
pseudotuberculosis, Campylobacter spp, including C. jejuni and C.
coli; Salmonella spp, including S. typhi, S. paratyphi, S.
choleraesuis, S. enteritidis; Listeria spp., including L.
monocytogenes; Helicobacter spp, including H. pylori; Pseudomonas
spp, including P. aeruginosa, Staphylococcus spp., including S.
aureus, S. epidermidis; Enterococcus spp., including E. faecalis,
E. faecium; Clostridium spp., including C. tetani, C. botulinum, C.
difficile; Bacillus spp., including B. anthracis; Corynebacterium
spp., including C. diphtherias; Borrelia spp., including B.
burgdorferi, B. garinii, B. afzelii, B. andersonii, B. hermsii;
Ehrlichia spp., including E. equi and the agent of the Human
Granulocytic Ehrlichiosis; Rickettsia spp, including R. rickettsii;
Chlamydia spp., including C. trachomatis, C. neumoniae, C.
psittaci; Leptsira spp., including L. interrogans; Treponema spp.,
including T. pallidum, T. denticola, T. hyodysenteriae. Preferred
bacteria include, but are not limited to, Listeria, mycobacteria,
mycobacteria (e.g., tuberculosis), Anthrax, Salmonella and Listeria
monocytogenes.
[0183] In another embodiment, T cells can be removed from a
patient, and contacted in vitro with an anti-ILT3 antibody or
antigen binding fragment disclosed herein, optionally with an
activating signal (e.g., antigen plus APCs or a polyclonal
antibody) and reintroduced into the patient.
[0184] The anti-ILT3 antibodies or antigen binding fragments
disclosed herein may also be used prophylactically in vaccines
against various pathogens. Immunity against a pathogen, e.g., a
virus, could be induced by vaccinating with a viral protein along
with an anti-ILT3 antibody or antigen binding fragment disclosed
herein. Alternately, an expression vector that encodes genes for
both a pathogenic antigen and anti-ILT3 antibody or antigen binding
fragment disclosed herein, e.g., a vaccinia virus expression vector
engineered to express a nucleic acid encoding a viral protein and a
nucleic acid encoding an anti-ILT3 antibody or antigen binding
fragment disclosed herein, may be used for vaccination. Pathogens
for which vaccines may be useful include, for example, hepatitis B,
hepatitis C, Epstein-Barr virus, cytomegalovirus, HIV-1, HIV-2,
tuberculosis, malaria and schistosomiasis.
[0185] The present invention further encompasses an anti-ILT3
antibody or antigen binding fragment disclosed herein conjugated to
a diagnostic or therapeutic agent. The anti-ILT3 antibody or
antigen binding fragment disclosed herein can be used
diagnostically to, for example, monitor the development or
progression of a tumor as part of a clinical testing procedure to,
e.g., determine the efficacy of a given treatment regimen.
Detection may be facilitated by coupling the antibody to a
detectable substance. Examples of detectable substances include
various enzymes, prosthetic groups, fluorescent materials,
luminescent materials, bioluminescent materials, radioactive
materials, positron emitting metals using various positron emission
tomographies, and nonradioactive paramagnetic metal ions. The
detectable substance may be coupled or conjugated either directly
to the binding molecule or indirectly, through an intermediate
(such as, for example, a linker known in the art) using techniques
known in the art. U.S. Pat. No. 4,741,900 discloses metal ions that
may be conjugated to binding molecules. Examples of suitable
enzymes include horseradish peroxidase, alkaline phosphatase,
beta-galactosidase, or acetylcholinesterase; examples of suitable
prosthetic group complexes include streptavidin/biotin and
avidin/biotin; examples of suitable fluorescent materials include
umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and aequorin; and examples of suitable radioactive
materials are .sup.125I, .sup.131I, and .sup.99Tc.
[0186] Further, an anti-ILT3 antibody or antigen binding fragment
disclosed herein may be conjugated to a therapeutic moiety such as
a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic
agent or a radioactive metal ion, e.g., alpha-emitters such as, for
example, .sup.213Bi. A cytotoxin or cytotoxic agent includes any
agent that is detrimental to cells. Examples include paclitaxol,
cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,
etoposide, tenoposide, vincristine, vinblastine, colchicin,
doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,
mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,
procaine, tetracaine, lidocaine, propranolol, and puromycin and
analogs or homologs thereof. Therapeutic agents include, but are
not limited to, antimetabolites (e.g., methotrexate,
6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil
decarbazine), alkylating agents (e.g., mechlorethamine, thioepa
chlorambucil, melphalan, camustine (BSNU) and lomustine (CCNU),
cyclothosphamide, busulfan, dibromomannitol, streptozotocin,
mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)
cisplatin), anthracyclines (e.g., daunorubicin (formerly
daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin
(formerly actinomycin), bleomycin, mithramycin, and anthramycin
(AMC)), and anti-mitotic agents (e.g., vincristine and
vinblastine).
[0187] The present invention is further directed to therapies that
involve administering an anti-ILT3 antibody or antigen binding
fragment disclosed herein to an animal, preferably a mammal, and
most preferably a human, patient for treating, detecting, and/or
preventing one or more of the diseases, disorders, or conditions
disclosed herein. Therapeutic compounds of the invention include,
but are not limited to, anti-ILT3 antibody or antigen binding
fragment disclosed herein. The anti-ILT3 antibody or antigen
binding fragment disclosed herein may be used to treat, diagnose,
inhibit or prevent diseases, disorders or conditions associated
with aberrant activity of ILT3, including, but not limited to, any
one or more of the diseases, disorders, or conditions described
herein.
[0188] The anti-ILT3 antibody or antigen binding fragment disclosed
herein may be advantageously utilized in combination with other
monoclonal or chimeric binding molecules, or with lymphokines or
hematopoietic growth factors (such as, e.g., IL-2, IL-3 and IL-7),
for example, which serve to increase the number or activity of
effector cells which interact with the binding molecules.
[0189] The anti-ILT3 antibody or antigen binding fragment disclosed
herein may be administered alone or in combination with other types
of treatments, e.g., immunostimulatory treatments or treatments
designed to control the proliferation of a target of activated
immune cells (e.g., cancer cells or pathogens). Exemplary therapies
include e.g., radiation therapy, chemotherapy, hormonal therapy,
immunotherapy and anti-tumor agents, antibiotics, and
immunoglobulin.
[0190] An anti-ILT3 antibody or antigen binding fragment disclosed
herein may be administered to a human subject for therapeutic
purposes. Moreover, an anti-ILT3 antibody or antigen binding
fragment disclosed herein may be administered to a non-human mammal
expressing ILT3 with which the binding molecule cross-reacts (e.g.,
a primate) for veterinary purposes or as an animal model of human
disease.
Combinations
[0191] The anti-ILT3 antibodies or antigen binding fragments herein
may be used in unconjugated forms or conjugated to a second agent,
e.g., a cytotoxic drug, radioisotope, or a protein, e.g., a protein
toxin or a viral protein. This method includes: administering the
anti-ILT3 antibodies or antigen binding fragments herein, alone or
conjugated to a cytotoxic drug, to a subject requiring such
treatment. The anti-ILT3 antibodies or antigen binding fragments
herein may be used to deliver a variety of therapeutic agents,
e.g., a cytotoxic moiety, e.g., a therapeutic drug, a radioisotope,
molecules of plant, fungal, or bacterial origin, or biological
proteins (e.g., protein toxins) or particles (e.g., a recombinant
viral particles, e.g.; via a viral coat protein), or mixtures
thereof.
Additional Combination Therapies
[0192] The anti-ILT3 antibodies or antigen binding fragments herein
may be used in combination with other therapies. For example, the
combination therapy may include a composition comprising an
anti-ILT3 antibody or antigen binding fragment co-formulated with,
and/or co-administered with, one or more additional therapeutic
agents, e.g., one or more anti-cancer agents, cytotoxic or
cytostatic agents, hormone treatment, vaccines, and/or other
immunotherapies. In other embodiments, the anti-ILT3 antibody or
antigen binding fragment is administered in combination with other
therapeutic treatment modalities, including surgery, radiation,
cryosurgery, and/or thermotherapy. Such combination therapies may
advantageously utilize lower dosages of the administered
therapeutic agents, thus avoiding possible toxicities or
complications associated with the various monotherapies.
[0193] By "in combination with," it is not intended to imply that
the therapy or the therapeutic agents must be administered at the
same time and/or formulated for delivery together, although these
methods of delivery are within the scope described herein. The
anti-ILT3 antibody or antigen binding fragment may be administered
concurrently with, prior to, or subsequent to, one or more other
additional therapies or therapeutic agents. The anti-ILT3 antibody
or antigen binding fragment and the other agent or therapeutic
protocol may be administered in any order. In general, each agent
will be administered at a dose and/or on a time schedule determined
for that agent. In will further be appreciated that the additional
therapeutic agent utilized in this combination may be administered
together in a single composition or administered separately in
different compositions. In general, it is expected that additional
therapeutic agents utilized in combination be utilized at levels
that do not exceed the levels at which they are utilized
individually. In some embodiments, the levels utilized in
combination will be lower than those utilized individually.
[0194] In certain embodiments, an anti-ILT3 antibody or antigen
binding fragment described herein is administered in combination
with one or more check point inhibitors or antagonists of
programmed death receptor 1 (PD-1) or its ligand PD-L1 and PD-L2.
The inhibitor or antagonist may be an antibody, an antigen binding
fragment, an immunoadhesin, a fusion protein, or oligopeptide. In
some embodiments, the anti-PD-1 antibody is chosen from nivolumab
(OPDIVO.RTM., Bristol Myers Squibb, New York, N.Y.), pembrolizumab
(KEYTRUDA.RTM., Merck Sharp & Dohme Corp, Kenilworth, N.J.
USA), cetiplimab (Regeneron, Tarrytown, N.Y.) or pidilizumab
(CT-011). In some embodiments, the PD-1 inhibitor is an
immunoadhesin (e.g., an immunoadhesin comprising an extracellular
or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant
region (e.g., an Fc region of an immunoglobulin sequence)). In some
embodiments, the PD-1 inhibitor is AMP-224. In some embodiments,
the PD-L1 inhibitor is anti-PD-L1 antibody such durvalumab
(IMFINZI.RTM., Astrazeneca, Wilmingon, Del.), atezolizumab
(TECENTRIQ.RTM., Roche, Zurich, CH), or avelumab (BAVENCIO.RTM.,
EMD Serono, Billerica, Mass.). In some embodiments, the anti-PD-L1
binding antagonist is chosen from YW243.55.S70, MPDL3280A,
MEDI-4736, MSB-0010718C, or MDX-1105.
[0195] MDX-1105, also known as BMS-936559, is an anti-PD-L1
antibody described in WO2007/005874. Antibody YW243.55.S70 is an
anti-PD-L1 described in WO 2010/077634 (heavy and light chain
variable region sequences shown in SEQ ID NOs. 20 and 21,
respectively).
[0196] Nivolumab, also known as OPDIVO.RTM., MDX-1106-04, ONO-4538,
or BMS-936558, is a fully human IgG4 anti-PD-1 antibody described
in WO2006/121168 and U.S. Pat. No. 8,008,449.
[0197] Pembrolizumab, also known as KEYTRUDA.RTM., lambrolizumab,
MK-3475 or SCH-900475, is a humanized anti-PD-1 antibody described
in U.S. Pat. No. 8,354,509 and WO2009/114335 and disclosed, e.g.,
in Hamid, et al., New England J. Med. 369 (2): 134-144 (2013). The
heavy and light chains for prembrolizumab are shown by the amino
acid sequences set forth in SEQ ID Nos: 225 and 226,
respectively.
[0198] Pidilizumab, also known as CT-011 (Cure Tech) is a humanized
IgG1 monoclonal antibody that binds to PD-1. Pidilizumab and other
humanized anti-PD-1 monoclonal antibodies are disclosed in
WO2009/101611. Other anti-PD-1 antibodies include AMP 514
(Amplimmune), among others, e.g., anti-PD-1 antibodies disclosed in
U.S. Pat. No. 8,609,089; U.S Publication No. 2010028330; and U.S
Publication No. 20120114649.
[0199] AMP-224 (B7-DCIg; Amplimmune; e.g., disclosed in
WO2010/027827 and WO2011/066342), is a PD-L2 Fc fusion soluble
receptor that blocks the interaction between PD-1 and B7-H1.
[0200] MDPL3280A (Genentech/Roche) is a human Fc optimized IgG1
monoclonal antibody that binds to PD-L1. MDPL3280A and other human
monoclonal antibodies to PD-L1 are disclosed in U.S. Pat. No.
7,943,743 and U.S Publication No. 20120039906.
[0201] Other anti-PD-L1 binding agents include YW243.55.570 (heavy
and light chain variable regions are shown in SEQ ID NOs 20 and 21
in WO2010/077634) and MDX-1105 (also referred to as BMS-936559). It
and other anti-PD-L1 binding agents are disclosed in
WO2007/005874).
Kits
[0202] Further provided are kits comprising one or more components
that include, but are not limited to, the anti-ILT3 antibodies or
antigen binding fragments thereof, as discussed herein in
association with one or more additional components including, but
not limited to, a further therapeutic agent, as discussed herein.
The antibody or fragment and/or the therapeutic agent can be
formulated as a pure composition or in combination with a
pharmaceutically acceptable carrier, in a pharmaceutical
composition.
[0203] In one embodiment, the kit includes the anti-ILT3 antibodies
or antigen binding fragments thereof or a pharmaceutical
composition thereof in one container (e.g., in a sterile glass or
plastic vial) and a further therapeutic agent in another container
(e.g., in a sterile glass or plastic vial).
[0204] In another embodiment, the kit comprises a combination of
the anti-ILT3 antibodies or antigen binding fragments thereof or
pharmaceutical composition thereof in combination with one or more
therapeutic agents formulated together, optionally, in a
pharmaceutical composition, in a single, common container.
[0205] If the kit includes a pharmaceutical composition for
parenteral administration to a subject, the kit can include a
device for performing such administration. For example, the kit can
include one or more hypodermic needles or other injection devices
as discussed above. Thus, the present invention includes a kit
comprising an injection device and t the anti-ILT3 antibodies or
antigen binding fragments thereof, e.g., wherein the injection
device includes the antibody or fragment or wherein the antibody or
fragment is in a separate vessel.
[0206] The kit can include a package insert including information
concerning the pharmaceutical compositions and dosage forms in the
kit. Generally, such information aids patients and physicians in
using the enclosed pharmaceutical compositions and dosage forms
effectively and safely. For example, the following information
regarding a combination of the invention may be supplied in the
insert: pharmacokinetics, pharmacodynamics, clinical studies,
efficacy parameters, indications and usage, contraindications,
warnings, precautions, adverse reactions, overdosage, proper dosage
and administration, how supplied, proper storage conditions,
references, manufacturer/distributor information and patent
information.
Methods of Making Antibodies and Antigen Binding Fragments
Thereof
[0207] The anti-ILT3 antibodies or antigen binding fragments
thereof disclosed herein may also be produced recombinantly. In
this embodiment, nucleic acid molecules encoding the antibody
molecules may be inserted into a vector (plasmid or viral) and
transfected or transformed into a host cell where it may be
expressed and secreted from the host cell. There are several
methods by which to produce recombinant antibodies which are known
in the art.
[0208] In particular aspects, the present invention provides
nucleic acid molecules encoding an HC and an LC wherein the HC
comprises at least the HC-CDR3 of an anti-ILT3 antibody disclosed
herein or embodiment thereof wherein the HC-CDR3 has one, two, or
three amino acid substitutions, additions, deletions, or
combinations thereof. In further embodiments, the HC and/or LC
variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9,
or 10 amino acid substitutions, additions, deletions, or
combinations thereof.
[0209] In particular aspects, the present invention provides
nucleic acid molecules encoding an HC and an LC wherein the HC
comprises the HC-CDR1, 2, and 3 of an anti-ILT3 antibody disclosed
herein or embodiment thereof wherein one or more of HC-CDR1, 2, and
3 has one, two, or three amino acid substitutions, additions,
deletions, or combinations thereof and wherein the LC comprises the
LC-CDR1, 2, and 3 of an anti-ILT3 antibody disclosed herein or
embodiment thereof wherein one or more of HC-CDR1, 2, and 3 has
one, two, or three amino acid substitutions, additions, deletions,
or combinations thereof. In further embodiments, the HC and/or LC
variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9,
or 10 amino acid substitutions, additions, deletions, or
combinations thereof.
[0210] In particular aspects, the present invention provides a
first expression vector comprising a nucleic acid molecule encoding
an HC comprising at least the HC CDRs of an anti-ILT3 antibody
disclosed herein or embodiment thereof wherein one or more of the
three HC CDRs has one, two, or three amino acid substitutions,
additions, deletions, or combinations thereof and/or wherein the HC
variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9,
or 10 amino acid substitutions, additions, deletions, or
combinations thereof and a second expression vector comprising a
nucleic acid molecule encoding an LC comprising at least the LC
CDRs of an anti-ILT3 antibody disclosed herein or embodiment
thereof wherein one or more of the three LC CDRs has one, two, or
three amino acid substitutions, additions, deletions, or
combinations thereof and/or wherein the LC variable region
framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid
substitutions, additions, deletions, or combinations thereof.
[0211] In particular aspects, the present invention provides
nucleic acid molecules encoding a V.sub.H and a V.sub.L wherein the
V.sub.H comprises at least the HC-CDR3 of an anti-ILT3 antibody
disclosed herein or embodiment thereof wherein the HC-CDR3 has one,
two, or three amino acid substitutions, additions, deletions, or
combinations thereof. In further embodiments, the V.sub.H and/or
V.sub.L variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7,
8, 9, or 10 amino acid substitutions, additions, deletions, or
combinations thereof.
[0212] In particular aspects, the present invention provides
nucleic acid molecules encoding a V.sub.H and a V.sub.L wherein the
HC comprises the HC-CDR1, 2, and 3 of an anti-ILT3 antibody
disclosed herein or embodiment thereof wherein one or more of
HC-CDR1, 2, and 3 has one, two, or three amino acid substitutions,
additions, deletions, or combinations thereof and wherein the
V.sub.L comprises the LC-CDR1, 2, and 3 of an anti-ILT3 antibody
disclosed herein or embodiment thereof wherein one or more of
HC-CDR1, 2, and 3 has one, two, or three amino acid substitutions,
additions, deletions, or combinations thereof. In further
embodiments, the V.sub.H and/or V.sub.L variable region framework
comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid
substitutions, additions, deletions, or combinations thereof.
[0213] In particular aspects, the present invention provides
nucleic acid molecules encoding a V.sub.H comprising at least the
HC CDRs of an anti-ILT3 disclosed herein or embodiment thereof
wherein one or more of the three HC CDRs has one, two, or three
amino acid substitutions, additions, deletions, or combinations
thereof and/or wherein the V.sub.H and/or V.sub.L variable region
framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid
substitutions, additions, deletions, or combinations thereof and
nucleic acid molecules encoding a V.sub.L comprising at least the
LC CDRs of an anti-ILT3 antibody disclosed herein or embodiment
thereof wherein one or more of the three LC CDRs has one, two, or
three amino acid substitutions, additions, deletions, or
combinations thereof and/or wherein the V.sub.H and/or V.sub.L
variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9,
or 10 amino acid substitutions, additions, deletions, or
combinations thereof.
[0214] Mammalian cell lines available as hosts for expression of
the antibodies or fragments disclosed herein are well known in the
art and include many immortalized cell lines available from the
American Type Culture Collection (ATCC). These include, inter alia,
Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby
hamster kidney (BHK) cells, monkey kidney cells (COS), human
hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3
cells, human embryo kidney 293 (HEK-293) cells and a number of
other cell lines. Cell lines of particular preference are selected
through determining which cell lines have high expression levels.
Other cell lines that may be used are insect cell lines, such as SD
cells, amphibian cells, bacterial cells, plant cells, filamentous
fungus cells (e.g. Trichoderma reesei), and yeast cells (e.g.,
Saccharomyces cerevisiae or Pichia pastoris). In particular
aspects, the host cell may be a prokaryote host cell such as E.
coli.
[0215] When recombinant expression vectors comprising a nucleic
acid molecule encoding the heavy chain or antigen-binding portion
or fragment, the light chain and/or antigen-binding fragment are
introduced into host cells, the antibodies are produced by
culturing the host cells under conditions and for a period of time
sufficient to allow for expression of the antibody in the host
cells or, more preferably, secretion of the antibody into the
culture medium in which the host cells are grown. The antibodies
may be recovered from the culture medium and further purified or
processed to produce the antibodies of the invention.
[0216] In particular aspects, the host cells are transfected with
an expression vector comprising nucleic acid molecules encoding an
HC and an LC wherein the HC comprises at least the HC-CDR3 of an
anti-ILT3 antibody or embodiment thereof wherein the HC-CDR3 has
one, two, or three amino acid substitutions, additions, deletions,
or combinations thereof. In further embodiments, the HC and/or LC
variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9,
or 10 amino acid substitutions, additions, deletions, or
combinations thereof.
[0217] In particular aspects, the host cells are transfected with
an expression vector comprising nucleic acid molecules encoding an
HC and an LC wherein the HC comprises the HC-CDR1, 2, and 3 of an
anti-ILT3 antibody disclosed herein or embodiment thereof wherein
one or more of HC-CDR1, 2, and 3 has one, two, or three amino acid
substitutions, additions, deletions, or combinations thereof and
wherein the LC comprises the LC-CDR1, 2, and 3 of an anti-ILT3
antibody disclosed herein or embodiment thereof wherein one or more
of HC-CDR1, 2, and 3 has one, two, or three amino acid
substitutions, additions, deletions, or combinations thereof. In
further embodiments, the HC and/or LC variable region framework
comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid
substitutions, additions, deletions, or combinations thereof.
[0218] In particular aspects, the host cells are transfected with a
first expression vector comprising a nucleic acid molecule encoding
an HC comprising at least the HC CDRs of an anti-ILT3 antibody
disclosed herein or embodiment thereof wherein one or more of the
three HC CDRs has one, two, or three amino acid substitutions,
additions, deletions, or combinations thereof and/or wherein the HC
variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9,
or 10 amino acid substitutions, additions, deletions, or
combinations thereof and a second expression vector comprising a
nucleic acid molecule encoding an LC comprising at least the LC
CDRs of an antibody disclosed herein or embodiment thereof wherein
one or more of the three LC CDRs has one, two, or three amino acid
s substitutions, additions, deletions, or combinations thereof
and/or wherein the LC variable region framework comprises 0, 1, 2,
3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions,
deletions, or combinations thereof.
[0219] In particular aspects, the host cells are transfected with
an expression vector comprising nucleic acid molecules encoding a
V.sub.H and a V.sub.L wherein the V.sub.H comprises at least the
HC-CDR3 of an anti-ILT3 antibody disclosed herein or embodiment
thereof wherein the HC-CDR3 has one, two, or three amino acid
substitutions, additions, deletions, or combinations thereof. In
further embodiments, the V.sub.H and/or V.sub.L variable region
framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid
substitutions, additions, deletions, or combinations thereof.
[0220] In particular aspects, the host cells are transfected with
an expression vector comprising nucleic acid molecules encoding a
V.sub.H and a V.sub.L wherein the V.sub.H comprises the HC-CDR1, 2,
and 3 of an anti-ILT3 antibody disclosed herein or embodiment
thereof wherein one or more of HC-CDR1, 2, and 3 has one, two, or
three amino acid substitutions, additions, deletions, or
combinations thereof and wherein the V.sub.L comprises the LC-CDR1,
2, and 3 of an anti-ILT3 antibody disclosed herein or embodiment
thereof wherein one or more of HC-CDR1, 2, and 3 has one, two, or
three amino acid substitutions, additions, deletions, or
combinations thereof. In further embodiments, the V.sub.H and/or
V.sub.L variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7,
8, 9, or 10 amino acid substitutions, additions, deletions, or
combinations thereof.
[0221] In particular aspects, the host cells are transfected with a
first expression vector comprising a nucleic acid molecule encoding
a V.sub.H comprising at least the HC CDRs of an anti-ILT3 antibody
disclosed herein or embodiment thereof wherein one or more of the
three HC CDRs has one, two, or three amino acid substitutions,
additions, deletions, or combinations thereof and/or wherein the
V.sub.H variable region framework comprises 0, 1, 2, 3, 4, 5, 6, 7,
8, 9, or 10 amino acid substitutions, additions, deletions, or
combinations thereof and a second expression vector comprising a
nucleic acid molecule encoding a V.sub.L comprising at least the LC
CDRs of an anti-ILT3 antibody disclosed herein or embodiment
thereof wherein one or more of the three LC CDRs has one, two, or
three amino acid s substitutions, additions, deletions, or
combinations thereof and/or wherein the V.sub.L variable region
framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid
substitutions, additions, deletions, or combinations thereof.
[0222] In particular embodiments, the HC and LC or V.sub.H and
V.sub.L are expressed as a fusion protein in which the N-terminus
of the HC and the LC are fused to a leader sequence to facilitate
the transport of the antibody through the secretory pathway.
Examples of leader sequences that may be used include
MSVPTQVLGLLLLWLTDARC (SEQ ID NO: 12) or MEWSWVFLFFLSVTTGVHS (SEQ ID
NO: 11).
[0223] The present invention further provides a plasmid or viral
vector comprising a nucleic acid molecule encoding an anti-ILT3
antibody disclosed herein or antigen binding fragment thereof. The
present invention further provides a plasmid or viral vector
comprising a nucleic acid molecule encoding the HC of an anti-ILT3
antibody disclosed herein or antigen binding fragment thereof or
embodiment of the antibody or antigen binding fragment thereof
wherein one or more of the three CDRs has one, two, or three amino
acid substitutions, additions, deletions, or combinations thereof
and/or wherein the HC variable region framework comprises 0, 1, 2,
3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions,
deletions, or combinations thereof and a nucleic acid molecule
encoding the LC of an anti-ILT3 antibody disclosed herein or
antigen binding fragment thereof or embodiment of the antibody or
antigen binding fragment thereof wherein one or more of the three
CDRs has one, two, or three amino acid substitutions, additions,
deletions, or combinations thereof and/or wherein the LC variable
region framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10
amino acid substitutions, additions, deletions, or combinations
thereof.
[0224] The present invention further provides a plasmid or viral
vector comprising a nucleic acid molecule encoding the HC of an
anti-ILT3 antibody disclosed herein or antigen binding fragment
thereof and a plasmid or viral vector comprising a nucleic acid
molecule encoding the LC of an anti-ILT3 antibody disclosed herein
or antigen binding fragment thereof.
[0225] The present invention further provides a host cell
comprising a plasmid or viral vector comprising a nucleic acid
molecule encoding the HC of an anti-ILT3 antibody disclosed herein
or antigen binding fragment thereof or embodiment of an anti-ILT3
antibody disclosed herein or antigen binding fragment thereof
wherein one or more of the three CDRs has one, two, or three amino
acid substitutions, additions, deletions, or combinations thereof
and/or wherein the HC variable region framework comprises 0, 1, 2,
3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions,
deletions, or combinations thereof and a plasmid or viral vector
comprising a nucleic acid molecule encoding the LC of an anti-ILT3
antibody disclosed herein or antigen binding fragment thereof or
embodiment of an anti-ILT3 antibody disclosed herein or antigen
binding fragment thereof wherein one or more of the three CDRs has
one, two, or three amino acid substitutions, additions, deletions,
or combinations thereof and/or wherein the LC variable region
framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid
substitutions, additions, deletions, or combinations thereof. In
particular embodiments, the host cell is a CHO or HEK-293 host
cell.
[0226] The present invention further provides a plasmid or viral
vector comprising a nucleic acid molecule encoding an anti-ILT3
antibody disclosed herein or antigen binding fragment thereof. The
present invention further provides a plasmid or viral vector
comprising a nucleic acid molecule encoding the V.sub.H of an
anti-ILT3 antibody disclosed herein or antigen binding fragment
thereof or embodiment of the antibody or antigen binding fragment
thereof wherein one or more of the three CDRs has one, two, or
three amino acid substitutions, additions, deletions, or
combinations thereof and/or wherein the V.sub.H framework comprises
0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions,
additions, deletions, or combinations thereof and a nucleic acid
molecule encoding the V.sub.L of an anti-ILT3 antibody disclosed
herein or antigen binding fragment thereof or embodiment of the
antibody or antigen binding fragment thereof wherein one or more of
the three CDRs has one, two, or three amino acid substitutions,
additions, deletions, or combinations thereof and/or wherein the LC
framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid
substitutions, additions, deletions, or combinations thereof.
[0227] The present invention further provides a plasmid or viral
vector comprising a nucleic acid molecule encoding the V.sub.H of
an anti-ILT3 antibody disclosed herein or antigen binding fragment
thereof and a plasmid or viral vector comprising a nucleic acid
molecule encoding the V.sub.L of an anti-ILT3 antibody disclosed
herein or antigen binding fragment thereof.
[0228] The present invention further provides a host cell
comprising a plasmid or viral vector comprising a nucleic acid
molecule encoding the V.sub.H of an anti-ILT3 antibody disclosed
herein or antigen binding fragment thereof or embodiment of an
anti-ILT3 antibody disclosed herein or antigen binding fragment
thereof wherein one or more of the three CDRs has one, two, or
three amino acid substitutions, additions, deletions, or
combinations thereof and/or wherein the V.sub.H framework comprises
0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions,
additions, deletions, or combinations thereof and a plasmid or
viral vector comprising a nucleic acid molecule encoding the
V.sub.L of an anti-ILT3 antibody disclosed herein or antigen
binding fragment thereof or embodiment of an anti-ILT3 antibody
disclosed herein or antigen binding fragment thereof wherein one or
more of the three CDRs has one, two, or three amino acid
substitutions, additions, deletions, or combinations thereof and/or
wherein the V.sub.L framework comprises 0, 1, 2, 3, 4, 5, 6, 7, 8,
9, or 10 amino acid substitutions, additions, deletions, or
combinations thereof. In particular embodiments, the host cell is a
CHO or HEK-293 host cell.
[0229] The anti-ILT3 antibodies or antigen binding fragments
thereof can be recovered from the culture medium using standard
protein purification methods. Further, expression of antibodies of
the invention (or other moieties therefrom) from production cell
lines can be enhanced using a number of known techniques. For
example, the glutamine synthetase gene expression system (the GS
system) is a common approach for enhancing expression under certain
conditions.
[0230] In general, glycoproteins produced in a particular cell line
or transgenic animal will have a glycosylation pattern that is
characteristic for glycoproteins produced in the cell line or
transgenic animal (See for example, Croset et al., J. Biotechnol.
161: 336-348 (2012)). Therefore, the particular glycosylation
pattern of an antibody will depend on the particular cell line or
transgenic animal used to produce the antibody. However, all
antibodies encoded by the nucleic acid molecules provided herein,
or comprising the amino acid sequences provided herein, comprise
the instant invention, independent of the glycosylation pattern the
antibodies may have.
[0231] The following examples are intended to promote a further
understanding of the present invention.
General Methods
[0232] Standard methods in molecular biology are described
Sambrook, Fritsch and Maniatis (1982 & 1989 2nd Edition, 2001
3rd Edition) Molecular Cloning, A Laboratory Manual, Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Sambrook and
Russell (2001) Molecular Cloning, 3rd ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.; Wu (1993) Recombinant
DNA, Vol. 217, Academic Press, San Diego, Calif.). Standard methods
also appear in Ausbel, et al. (2001) Current Protocols in Molecular
Biology, Vols. 1-4, John Wiley and Sons, Inc. New York, N.Y., which
describes cloning in bacterial cells and DNA mutagenesis (Vol. 1),
cloning in mammalian cells and yeast (Vol. 2), glycoconjugates and
protein expression (Vol. 3), and bioinformatics (Vol. 4).
[0233] Methods for protein purification including
immunoprecipitation, chromatography, electrophoresis,
centrifugation, and crystallization are described (Coligan, et al.
(2000) Current Protocols in Protein Science, Vol. 1, John Wiley and
Sons, Inc., New York). Chemical analysis, chemical modification,
post-translational modification, production of fusion proteins,
glycosylation of proteins are described (see, e.g., Coligan, et al.
(2000) Current Protocols in Protein Science, Vol. 2, John Wiley and
Sons, Inc., New York; Ausubel, et al. (2001) Current Protocols in
Molecular Biology, Vol. 3, John Wiley and Sons, Inc., NY, NY, pp.
16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for Life
Science Research, St. Louis, Mo.; pp. 45-89; Amersham Pharmacia
Biotech (2001) BioDirectory, Piscataway, N.J., pp. 384-391).
Production, purification, and fragmentation of polyclonal and
monoclonal antibodies are described (Coligan, et al. (2001) Current
Protcols in Immunology, Vol. 1, John Wiley and Sons, Inc., New
York; Harlow and Lane (1999) Using Antibodies, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.; Harlow and Lane,
supra). Standard techniques for characterizing ligand/receptor
interactions are available (see, e.g., Coligan, et al. (2001)
Current Protocols in Immunology, Vol. 4, John Wiley, Inc., New
York).
[0234] Monoclonal, polyclonal, and humanized antibodies can be
prepared (see, e.g., Sheperd and Dean (eds.) (2000) Monoclonal
Antibodies, Oxford Univ. Press, New York, N.Y.; Kontermann and
Dubel (eds.) (2001) Antibody Engineering, Springer-Verlag, New
York; Harlow and Lane (1988) Antibodies A Laboratory Manual, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp.
139-243; Carpenter, et al. (2000) J. Immunol. 165:6205; He, et al.
(1998) J. Immunol. 160:1029; Tang et al. (1999) J. Biol. Chem.
274:27371-27378; Baca et al. (1997) J. Biol. Chem. 272:10678-10684;
Chothia et al. (1989) Nature 342:877-883; Foote and Winter (1992)
J. Mol. Biol. 224:487-499; U.S. Pat. No. 6,329,511).
[0235] An alternative to humanization is to use human antibody
libraries displayed on phage or human antibody libraries in
transgenic mice (Vaughan et al. (1996) Nature Biotechnol.
14:309-314; Barbas (1995) Nature Medicine 1:837-839; Mendez et al.
(1997) Nature Genetics 15:146-156; Hoogenboom and Chames (2000)
Immunol. Today 21:371-377; Barbas et al. (2001) Phage Display: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.; Kay et al. (1996) Phage Display of Peptides and
Proteins: A Laboratory Manual, Academic Press, San Diego, Calif.;
de Bruin et al. (1999) Nature Biotechnol. 17:397-399). Antibodies
can be conjugated, e.g., to small drug molecules, enzymes,
liposomes, polyethylene glycol (PEG). Antibodies are useful for
therapeutic, diagnostic, kit or other purposes, and include
antibodies coupled, e.g., to dyes, radioisotopes, enzymes, or
metals, e.g., colloidal gold (see, e.g., Le Doussal et al. (1991)
J. Immunol. 146:169-175; Gibellini et al. (1998) J. Immunol.
160:3891-3898; Hsing and Bishop (1999) J. Immunol. 162:2804-2811;
Everts et al. (2002) J. Immunol. 168:883-889).
[0236] Methods for flow cytometry, including fluorescence activated
cell sorting (FACS), are available (see, e.g., Owens, et al. (1994)
Flow Cytometry Principles for Clinical Laboratory Practice, John
Wiley and Sons, Hoboken, N.J.; Givan (2001) Flow Cytometry, 2nd
ed.; Wiley-Liss, Hoboken, N.J.; Shapiro (2003) Practical Flow
Cytometry, John Wiley and Sons, Hoboken, N.J.). Fluorescent
reagents suitable for modifying nucleic acids, including nucleic
acid primers and probes, polypeptides, and antibodies, for use,
e.g., as diagnostic reagents, are available (Molecular Probes
(2003) Catalogue, Molecular Probes, Inc., Eugene, Oreg.;
Sigma-Aldrich (2003) Catalogue, St. Louis, Mo.).
[0237] Standard methods of histology of the immune system are
described (see, e.g., Muller-Harmelink (ed.) (1986) Human Thymus:
Histopathology and Pathology, Springer Verlag, New York, N.Y.;
Hiatt, et al. (2000) Color Atlas of Histology, Lippincott,
Williams, and Wilkins, Phila, Pa.; Louis, et al. (2002) Basic
Histology: Text and Atlas, McGraw-Hill, New York, N.Y.). Software
packages and databases for determining, e.g., antigenic fragments,
leader sequences, protein folding, functional domains,
glycosylation sites, and sequence alignments, are available (see,
e.g., GenBank, VECTOR NTI.RTM. Suite (Informax, Inc, Bethesda,
Md.); GCG Wisconsin Package (Accelrys, Inc., San Diego, Calif.);
DECYPHER.RTM. (TimeLogic Corp., Crystal Bay, Nevada); Menne, et al.
(2000) Bioinformatics 16: 741-742; Menne, et al. (2000)
Bioinformatics Applications Note 16:741-742; Wren, et al. (2002)
Comput. Methods Programs Biomed. 68:177-181; von Heijne (1983) Eur.
J. Biochem. 133:17-21; von Heijne (1986) Nucleic Acids Res.
14:4683-4690).
[0238] Purity determinations: Size-exclusion ultra-high performance
liquid chromatography (SE-UPLC) or (SEC) was carried out on an
ACQUITY.RTM. UPLC.RTM. H-Class system. Column used was an
ACQUITY.RTM. UPLC.RTM. Protein BEH SEC column (Part No. 186005225,
1.7 .mu.m, 200.LAMBDA., 4.6 mm.times.150 mm) from Waters (Milford,
Mass.). Column temperature used was 25 C and 10 .mu.l sample at 1
mg/mL was injected using a system flow rate of 0.5 ml/min. Mobile
phase used was 100 mM sodium phosphate, 200 mM sodium chloride and
0.02% sodium azide, pH 7.0. Data was quantified at both 214 and 280
nm and analyzed using Empower 3 software. A BEH200 SEC Protein
Standard Mix (Part No. 186006518) from Waters (Milford, Mass.) was
utilized and injected at 10 ug and USP Resolution, Theoretical
plates, and Tailing was measured.
[0239] NANO-DSF.TM. (tradename for modified differential scanning
fluorimetry method to determine protein stability employing
intrinsic tryptophan or tyrosin fluorescence): the temperature
mid-point of a thermal unfolding curve, Tm, and mid-point of a
thermal aggregation curve, Tagg, were determined by NANO-DSF.TM.
using a PROMETHEUS.TM. NT.48 Differential Scanning Fluorimeter
(Nanotemper Technologies) controlled by PR THERMCONTROL.TM. v2.0.4
software. Excitation power was 40% and temperature was increased
from 20.degree. C. to 95.degree. C. at a rate of 1 C/minute. Tm and
Tagg were automatically measured. Samples were prepared by diluting
to 1 mg/mL in 20 mM sodium acetate pH 5.5 buffer and drawn by
capillary action into a PROMETHEUS.TM. glass capillary
(PR-L002).
[0240] Capillary Isoelectric Focusing (cIEF): cIEF was conducted on
a iCE3.TM. system from Protein Simple (San Jose, Calif.) using iCE
CFR.TM. software 4.1.1 for instrument control and data analysis.
cIEF Cartridge used was Fc-coated (Protein Simple, 101701) and
prepared according to manufacturer's instruction. A 200 .mu.L
sample consisting of 40 .mu.g of analyte and 1% v/v 3-10
PHARMALYTE.RTM., 0.5% v/v 8-10.5 PHARMALYTE.RTM., 0.5% v/v 5-8
PHARMALYTE.RTM. (GE Healthcare), 37.5% v/v 8.0 M Urea
(Sigma-Aldrich), 35% v/v 1% methyl cellulose and 1 .mu.L each of
5.85 and 9.22 pI markers (Protein Simple), was prepared. Samples
were injected for 60 seconds. Isoelectric focusing parameters were
1500 V for 1 minute and 3000 V for 8 minutes. pI was automatically
measured using the internal pI markers serving as a two-point
calibration standard. Calibrated data was further analyzed and
quantified by conversion to Empower format and analyzed using
Empower 3.
Example 1
[0241] Hybridoma clone 52B8 was identified via standard mouse and
rat immunization and hybridoma selections. In general, Balb/C mice
or rats were immunized with human ILT3-HIS recombinant protein in a
standard four week footpad immunization to generate a hyperimmune
response. Electrofusion of bulk lymphocytes from draining lymph
nodes with the P3 myeloma fusion partner produced immortalized
hybridomas. Hybridoma supernatant fluid was screened in a primary
cell-based ELISA binding assay on human CHO-human ILT3 cells. A
secondary screen on CHO parental, CHO-ILT3 SNP, CHO-rhesus ILT3,
CHO-ILT5, CHO-ILT8, and CHO-ILT11 cells was performed in a
cell-based ELISA format (See Example 2). Subcloning by limited
dilution was performed on the ILT3 specific and rhesus positive
hybridoma cells. Subclones were expanded to generate purified
protein to enable additional tests of Biacore analyses and
functional screening. Table 5 shows 10 hybridoma clones that
produced antibodies that binned together and had high affinity for
human ILT3 as shown by CELISA and Biacore preformed as disclosed in
Examples 2 and 4, respectively.
TABLE-US-00005 TABLE 5 cELISA- human cELISA- ILT3 rhesus ILT3
Biacore Kd Parental EC50 EC50 Biacore Kd (M)- Clone species (ng/mL)
(ng/mL) (M)-ILT3_H ILT3_MM LB181.52A8.1A1 Mouse 18.4 25 8.55
.times. 10.sup.-10 1.3 .times. 10.sup.-8 LB181.52B8.1B1 Mouse 15.5
23.2 6.58 .times. 10.sup.-10 2.44 .times. 10.sup.-8 LB182.11D1.1A1
Mouse 50.5 No Binding 1.41 .times. 10.sup.-08 No binding
LB182.1G12.1B1 Mouse 39.2 No Binding 1.69 .times. 10.sup.-08 No
binding LB184.16B1.1D2 Rat 64.9 67.9 9.57 .times. 10.sup.-11 2.59
.times. 10.sup.-10 LB184.20E4.1E1.1D1 Rat 2 18 6.99 .times.
10.sup.-9 1.8 .times. 10.sup.-8 LB184.24A4.1A1 Rat 21.4 23.1 2.05
.times. 10.sup.-11 1.26 .times. 10.sup.-10 LB184.37C8.1A3.1B1 Rat
7.7 9.5 1.18 .times. 10.sup.-11 1.5 .times. 10.sup.-10
LB184.40A6.1C1 Rat 17.9 25.9 1.79 .times. 10.sup.-09 9.46 .times.
10.sup.-10 LB190.17H12.1A1 Rat 139.2 No Binding 5.92 .times.
10.sup.-10 No binding H = human MM = rhesus monkey (Macaca
mulatta)
Table 6 shows the amino acid sequences for the heavy chain and
light chain variable domains for the mAbs obtained from the above
clones.
TABLE-US-00006 TABLE 6 SEQ ID NO: Heavy Chain Light Chain mAb
Variable Variable No. Description domain Domain p52B8 Mouse
anti-ILT3 mAb 52B8 IgG2a / 15 16 Kappa p40A6 Rat anti-ILT3 mAb 40A6
IgG2a / 45 46 Kappa p16B1 Rat anti-ILT3 mAb 16B1 IgG2a / 53 54
Kappa p49C6 Mouse anti-ILT3 mAb 49C6 IgG2a / Not Not Kappa
sequenced sequenced p11D1 Mouse anti-ILT3 mAb 11D1 IgG2b / 61 62
Kappa p17H12 Rat anti-ILT3 mAb 17H12 IgG1 / 69 70 Kappa p37C8 Rat
anti-ILT3 mAb 37C8 IgG2a / 77 78 Kappa p1G12 Mouse anti-ILT3 mAb
1G12 IgG2a / 85 86 Kappa p20E4 Rat anti-ILT3 mAb 20E4 IgG2a / 93 94
Kappa p24A4 Rat anti-ILT3 mAb 24A4 IgG2a / 101 102 Kappa
[0242] To ultimately guide the selection of a lead antibody,
antibodies were further analyzed and re-evaluated in a set of
bio-functional, biophysical, and physicochemical assays. Finally,
antibodies were tested in an in vivo, proof of biology tumor
regression study using human SKMEL5 melanoma-challenged humanized
mice.
Example 2
Selectivity of Various Anti-ILT3 Antibodies
[0243] Cell-based ELISA (cELISA) was used to show the selectivity
of the various parental anti-ILT3 antibodies shown in Table 5 and
humanized anti-ILT3 monoclonal antibody 9B11 disclosed in U.S. Pat.
No. 7,777,008 as having the amino acid sequences of SEQ ID NO: 33
(light chain) and SEQ ID NO: 34 (heavy chain).
[0244] Mouse anti-human ILT3 antibodies were tested for binding to
human ILT3, and cross-reactivity to Rhesus monkey ILT3, human ILT5,
human ILT7, human ILT8, and human ILT11 expressing CHO-K1 cells
using a cell-based ELISA format. CHO-K1 cells were plated in
96-well tissue-culture plates in 50 .mu.L of DMEM/F12, 10% BCS and
gentamycin (CHO-K1 media). Cells were plated at either
2.times.10.sup.4 cells/well two days prior to the assay or
4.times.10.sup.4 cells/well one day prior to the assay. Media was
removed from the wells prior to adding the test samples. Purified
antibody was serially-diluted in CHO-K1 media and added to the
CHO-K1 plates. The samples were incubated at room temperature for
30-60 minutes and plates were washed three times with PBS/05%
Tween-20 using the cell wash program on the Biotek EL405x Select CW
plate washer. Binding was detected using an HRP-conjugated goat
anti-mouse IgG (Southern Biotech cat #1031-05) secondary antibody
added at a 1:2000 dilution in CHO-K1 media and incubated at room
temperature for 30-60 minutes. Assay plates were washed as above
and developed with TMB and stopped with TMB stop solution (KPL cat
#50-85-06). The absorbance at 450 nm-620 nm was determined. Mouse
IgG1 (MIgG1) served as a control
[0245] The results are shown in FIGS. 1A, 1B, 1C, 1D, and 1E. The
figures show that representative antibodies from clones p40B5,
p49C6, and p52B8 were specific for ILT3 and did not cross-react
with or bind ILT5, ILT7, ILT8, and ILT11. Antibodies from clones
p49C6 and p52B8 as were the antibodies from the other clones were
capable of binding Rhesus monkey ILT3. The p52B8 clone was chosen
for in vivo characterization based on (1) its high affinity to
human ILT3, (2) lack of binding to other ILT family members, and
(3) cross-reactivity to rhesus ILT3.
Example 3
[0246] Parental mouse 52B8 heavy chain (VH) and light chain (VL)
variable domain sequences were compared to human germline
sequences. Human framework sequences closely homologous to the
framework of the mouse antibody were chosen.
[0247] The mouse V.sub.H domain of mouse anti-human ILT3 mAb 52B8
clone scored highly against human heavy chain germline 3-07 in
subgroup III and JH4 for the J region. Based on structural
considerations, two framework substitutions (R87K and A97G) were
incorporated to maintain binding equivalent to the parental
antibody. The mouse V.sub.L domain of the antibody clone scored
highly against human light chain germline 1-02 in kappa subgroup I.
Mouse 52B8 CDRs were engineered onto the variable light chain
sequence of 1-02 and JK2 for the J region. Based on structural
considerations, three framework substitutions (M4L, S64A, and G72R)
were incorporated.
[0248] To generate humanized variants, the humanized V.sub.H
sequence was cloned into a vector encoding human IgG4 S228P heavy
chain constant domain and the humanized V.sub.L domain was cloned
into a vector encoding for a kappa light chain constant domain. A
total of two humanized V.sub.H (VH1 and VH2) and 8 humanized
V.sub.L were designed. In silico sequence and structural analysis
of mouse 52B8 revealed six potential "hot spots" on the molecule:
two potential oxidation sites in VH-CDR2 (M64) and in VH-CDR3
(W101), one potential isomerization site in VH-CDR2 (D62), one
potential deamidation site in VL-CDR1 (N34), two potential
isomerization sites in VL-CDR1 (D30) and VL-CDR2 (D59). M64 was
modified to V64 or L64, which maintained favorable physicochemical
attributes and binding/functionality.
[0249] FIG. 2A provides a table showing data characteristics on
binding affinity, isoelectric point, purity of monomer species, and
thermal stability measurements for humanized variants that were
designed. Biacore was used to measure binding affinity, cIEF was
used to measure pI, purity was determined by SE-UPLC, Tm and Tgg
was determined by NANO-DSF.TM.. FIG. 2B shows the relationship of
SEC purity and melting temperature of various humanized light chain
variants. Data is plotted as values obtained from each of the eight
humanized light chain variants demonstrating that VL5 has both the
highest purity and thermal stability. Based on the data in FIG. 2A
and FIG. 2B, VL5 was selected for the light chain.
[0250] Initial studies were performed on the humanized VH1 M64V/VL5
produced in transient CHO cells. Forced deamidation conditions
employing both 50.degree. C. incubation and high pH stress
performed on unformulated humanized 52B8 VH1 M64V/VL5 revealed
deamidation of LC N34 in VL-CDR1 (4.0 and 7.2%, respectively) and
W101 oxidation in HC-CDR3 with 1.times. light stress exposure was
15.4%. Substitution of N34 to Q34 maintained binding affinity to
human and rhesus ILT3 assessed by a Biacore SPR assay and
functional activity assessed by a DC TNF.alpha. production assay;
however, substitution of the W101 residue resulted in significant
loss in binding as determined by a Biacore SPR assay.
[0251] In summary, the humanized 52B8 was anti-ILT3 mAb (52B8 VH1
M64V/VL5 N34Q IgG4 S228P/Kappa), contains one framework
substitution in V.sub.L (M4L) and one framework substitution in
V.sub.H (A97G).
Example 4
[0252] Binding Kinetics and Affinities for the Anti-Human ILT3
Antibodies to Recombinant Human or Rhesus ILT3
[0253] The binding kinetics and affinities of anti-human ILT3
clones for human or rhesus ILT3-His tagged recombinant protein were
measured by surface plasmon resonance using a Biacore T200 system
(GE Healthcare, Piscataway, N.J.). HBS-EP+ buffer (BR-1006-69) was
used as the running buffer. Anti-human Fc antibody (Human Fc
Capture Kit, BR100839, GE Healthcare) was immobilized via amine
coupling chemistry in all four flow cells on a Series S CMS sensor
chip (BR100530 or 29149603, GE Healthcare) following manufacturer
instructions. Flow cell 1 was used as reference for background
subtraction and was not used for capture. Anti-human ILT3
antibodies listed above (diluted to 1 .mu.g/mL in HBS-EP+ buffer)
were injected over the anti-human Fc capture surfaces in flow cells
2, 3 and 4 at 10 .mu.L/mL for 10 seconds which resulted in antibody
capture levels in the range of 60-70 RU Six-point, two-fold
dilution series of human or rhesus ILT3-His protein ranging from 20
nM to 0.31 nM and two zeros (HBS-EP+) were injected at 50 .mu.L/mL
over the reference and captured antibody surfaces for 180 seconds
of association followed by 600 seconds of dissociation. Following
each injection cycle, all four flow cells were regenerated using 30
second injection of 3M MgCl.sub.2 solution at a flow rate of 10
.mu.L/minute. Reference subtracted sensorgrams were fit to a 1:1
Langmuir Binding Model in the Biacore T200 Evaluation Software
(Version 2.0) to determine the association (ka) and dissociation
(kd) rate constants and the equilibrium dissociation constant KD
(=kd/ka).
[0254] Table 7 summarizes the binding kinetics and affinities for
the anti-human ILT3 antibodies to recombinant human or rhesus
ILT3.
TABLE-US-00007 TABLE 7 cELISA cELISA Biacore Biacore (human (rhesus
KD KD Purity ILT3- ILT3- (human (rhesus by SEC CHO) CHO) ILT3-
ILT3- (% mAb EC50 EC50 His) His) main No. Description (.mu.g/mL)
(.mu.g/mL) (nM) (nM) peak) PI 63 Chimeric anti- 0.064 0.091 0.46
9.5 95.9 n.d. ILT3 52B8 mouse VH/human IgG4 (S228P): mouse VL/human
Kappa 64 Chimeric anti- 0.075 0.096 0.44 9.2 95.3 n.d. ILT3 52B8
mouse VH M64V/human IgG4 (S228P): mouse VL/human Kappa 65 Chimeric
anti- 0.086 0.137 0.41 9.3 93.5 n.d. ILT3 52B8 mouse VH M64L/human
IgG4 (S228P): mouse VL/human Kappa 1 Humanized anti- n.d. n.d. 0.99
25 93.1 n.d. ILT3 mAb (52B8 VH1 / VL1) IgG4 S228P / Kappa 2
Humanized anti- 0.7 0.109 1.1 20 96.2 n.d. ILT3 mAb (52B8 VH1 /
VL2) IgG4 S228P / Kappa 3 Humanized anti- n.d. n.d. 1.1 26 90 n.d.
ILT3 mAb (52B8 VH1 / VL3) IgG4 S228P / Kappa 4 Humanized anti- n.d.
n.d. 1.4 29 93.3 n.d. ILT3 mAb (52B8 VH1 / VL4) IgG4 S228P / Kappa
5 Humanized anti- n.d. n.d. 0.94 25 93.1 n.d. ILT3 mAb (52B8 VH2 /
VL1) IgG4 S228P / Kappa 6 Humanized anti- 0.1 0.118 1.1 21 96.6
n.d. ILT3 mAb (52B8 VH2 / VL2) IgG4 S228P / Kappa 7 Humanized anti-
n.d. n.d. 0.96 26 89.6 6.33 ILT3 mAb (52B8 VH2 / VL3) IgG4 S228P /
Kappa 8 Humanized anti- n.d. n.d. 1.3 27 92.8 n.d. ILT3 mAb (52B8
VH2 / VL4) IgG4 S228P / Kappa 9 Humanized anti- n.d. n.d. 0.94 26
92.1 n.d. ILT3 mAb (52B8 VH1 M64V / VL1) IgG4 S228P / Kappa 10
Humanized anti- 0.085 0.148 1.1 22 95.1 n.d. ILT3 mAb (52B8 VH1
M64V / VL2) IgG4 S228P / Kappa 11 Humanized anti- n.d. n.d. 1.1 27
89.6 n.d. ILT3 mAb (52B8 VH1 M64V / VL3) IgG4 S228P / Kappa 12
Humanized anti- n.d. n.d. 1.5 29 92.4 n.d. ILT3 mAb (52B8 VH1 M64V
/ VL4) IgG4 S228P / Kappa 13 Humanized anti- n.d. n.d. 0.94 25 85.9
n.d. ILT3 mAb (52B8 VH2 M64V / VL1) IgG4 S228P / Kappa 14 Humanized
anti- 0.077 0.126 1 22 92.8 n.d. ILT3 mAb (52B8 VH2 M64V / VL2)
IgG4 S228P / Kappa 15 Humanized anti- n.d. n.d. 1 26 88.7 n.d. ILT3
mAb (52B8 VH2 M64V / VL3) IgG4 S228P / Kappa 16 Humanized anti-
n.d. n.d. 1.4 29 93 n.d. ILT3 mAb (52B8 VH2 M64V / VL4) IgG4 S228P
/ Kappa 17 Humanized anti- n.d. n.d. 0.87 24 90.2 n.d. ILT3 mAb
(52B8 VH1 M64L / VL1) IgG4 S228P / Kappa 18 Humanized anti- 0.079
0.137 1 22 92.2 n.d. ILT3 mAb (52B8 VH1 M64L / VL2) IgG4 S228P /
Kappa 19 Humanized anti- n.d. n.d. 0.99 26 87.4 n.d. ILT3 mAb (52B8
VH1 M64L / VL3) IgG4 S228P / Kappa 20 Humanized anti- n.d. n.d. 1.3
29 90.8 n.d. ILT3 mAb (52B8 VH1 M64L / VL4) IgG4 S228P / Kappa 21
Humanized anti- 0.079 0.112 0.88 27 91.2 n.d. ILT3 mAb (52B8 VH2
M64L / VL1) IgG4 S228P / Kappa 22 Humanized anti- 0.057 0.081 0.97
21 96.8 n.d. ILT3 mAb (52B8 VH2 M64L / VL2) IgG4 S228P / Kappa 23
Humanized anti- n.d. n.d. 0.96 24 88.5 n.d. ILT3 mAb (52B8 VH2 M64L
/ VL3) IgG4 S228P / Kappa 24 Humanized anti- n.d. n.d. 1.2 27 91.9
n.d. ILT3 mAb (52B8 VH2 M64L / VL4) IgG4 S228P / Kappa 25 Humanized
anti- n.d. n.d. 0.74 8.7 94.9 7.76 ILT3 mAb ((52B8 VH1 M64V / VL2)
L234A L235A D265S) IgG1 / Kappa 26 Humanized anti- n.d. n.d. 0.61
4.9 96.05 8.62 ILT3 mAb ((52B8 VH1 M64V / VL5) L234A L235A D265S)
IgG1 / Kappa 27 Humanized anti- n.d. n.d. 0.92 10 90.17 8.84 ILT3
mAb ((52B8 VH1 M64V / VL6) L234A L235A D265S) IgG1 / Kappa 28
Humanized anti- n.d. n.d. 0.57 5.6 94.4 8.8 ILT3 mAb ((52B8 VH1
M64V / VL7) L234A L235A D265S) IgG1 / Kappa 29 Humanized anti- n.d.
n.d. 0.56 5.7 94.14 8.85 ILT3 mAb ((52B8 VH1 M64V / VL8) L234A
L235A D265S) IgG1 / Kappa 30 Humanized anti- n.d. n.d. 0.60 4.8
98.22 7.21 ILT3 mAb (52B8 VH1 M64V / VL5) IgG4 S228P / Kappa 31
Humanized anti- n.d. n.d. 0.88 10 91.74 7.45 ILT3 mAb (52B8 VH1
M64V / VL6) IgG4 S228P / Kappa 32 Humanized anti- n.d. n.d. 0.53
5.6 97.79 7.45 ILT3 mAb (52B8 VH1 M64V / VL7) IgG4 S228P / Kappa 33
Humanized anti- n.d. n.d. 0.54 5.6 97.29 7.45 ILT3 mAb (52B8 VH1
M64V / VL8) IgG4 S228P / Kappa 34 Humanized anti- n.d. n.d. n.d.
n.d. n.d. n.d. ILT3 mAb (52B8 VH1 M64V W101F / VL2) IgG4 S228P /
Kappa 35 Humanized anti- n.d. n.d. n.d. n.d. n.d. n.d. ILT3 mAb
(52B8 VH1 M64V W101Y / VL2) IgG4 S228P / Kappa 36 Humanized anti-
n.d. n.d. n.d. n.d. n.d. n.d. ILT3 mAb (52B8 VH1 M64V W101Q / VL2)
IgG4 S228P / Kappa 37 Humanized anti- n.d. n.d. n.d. n.d. n.d. n.d.
ILT3 mAb ((52B8 VH1 M64V W101F / VL2) L234A L235A D265S) IgG1 /
Kappa 38 Humanized anti- n.d. n.d. n.d. n.d. n.d. n.d. ILT3 mAb
((52B8 VH1 M64V W101Y / VL2) L234A L235A D265S) IgG1 / Kappa 39
Humanized anti- n.d. n.d. n.d. n.d. n.d. n.d. ILT3 mAb ((52B8 VH1
M64V W101Q / VL2) L234A L235A D265S) IgG1 / Kappa 40 Humanized
anti- n.d. n.d. n.d. n.d. n.d. n.d. ILT3 mAb (52B8 VH1 M64V /
VL2 S35A) IgG4 S228P / Kappa 41 Humanized anti- n.d. n.d. n.d. n.d.
n.d. n.d. ILT3 mAb (52B8 VH1 M64V / VL2 S35N) IgG4 S228P / Kappa 42
Humanized anti- n.d. n.d. n.d. n.d. n.d. n.d. ILT3 mAb (52B8 VH1
M64V / VL2 N34Q) IgG4 S228P / Kappa 43 Humanized anti- n.d. n.d.
n.d. n.d. n.d. n.d. ILT3 mAb (52B8 VH1 M64V / VL2 N34D) IgG4 S228P
/ Kappa 44 Humanized anti- n.d. n.d. 2.6 34 n.d. n.d. ILT3 mAb
(52B8 VH1 M64V / VL5 S35A) IgG4 S228P / Kappa 45 Humanized anti-
n.d. n.d. 4.7 NB n.d. n.d. ILT3 mAb (52B8 (No VH1 M64V / Binding)
VL5 S35N) IgG4 S228P / Kappa 46 Humanized anti- 0.088 0.12 0.77 15
97.9 7.1 ILT3 mAb (52B8 VH1 M64V / VL5 N34Q) IgG4 S228P / Kappa 47
Humanized anti- n.d. n.d. 3.8 115 n.d. n.d. ILT3 mAb (52B8 VH1 M64V
/ VL5 N34D) IgG4 S228P / Kappa 48 Humanized anti- n.d. n.d. n.d.
n.d. n.d. n.d. ILT3 mAb (52B8 VH1 M64V W101F / VL5) IgG4 S228P /
Kappa 49 Humanized anti- n.d. n.d. n.d. n.d. n.d. n.d. ILT3 mAb
(52B8 VH1 M64V W101Y / VL5) IgG4 S228P / Kappa 50 Humanized anti-
n.d. n.d. n.d. n.d. n.d. n.d. ILT3 mAb (52B8 VH1 M64V W101Q / VL5)
IgG4 S228P / Kappa 51 Humanize anti- n.d. n.d. NB NB n.d. n.d. ILT3
mAb (52B8 (No (No VH1 M64V Binding) Binding) W101F / VL5 S35A) IgG4
S228P / Kappa 52 Humanized anti- n.d. n.d. NB NB n.d. n.d. ILT3 mAb
(52B8 (No (No VH1 M64V Binding) Binding) W101F / VL5 S35N) IgG4
S228P / Kappa 53 Humanized anti- n.d. n.d. 35 NB n.d. n.d. ILT3 mAb
(52B8 (No VH1 M64V Binding) W101F / VL5 N34Q) IgG4 S228P / Kappa 54
Humanized anti- n.d. n.d. NB NB n.d. n.d. ILT3 mAb (52B8 (No (No
VH1 M64V Binding) Binding) W101F / VL5 N34D) IgG4 S228P / Kappa 55
Humanized anti- n.d. n.d. NB NB n.d. n.d. ILT3 mAb (52B8 (No (No
VH1 M64V Binding) Binding) W101Y / VL5 S35A) IgG4 S228P / Kappa 56
Humanized anti- n.d. n.d. NB NB n.d. n.d. ILT3 mAb (52B8 (No (No
VH1 M64V Binding) Binding) W101Y / VL5 S35N) IgG4 S228P / Kappa 57
Humanized anti- n.d. n.d. NB NB n.d. n.d. ILT3 mAb (52B8 (No (No
VH1 M64V Binding) Binding) W101Y / VL5 N34Q) IgG4 S228P / Kappa 58
Humanized anti- n.d. n.d. NB NB n.d. n.d. ILT3 mAb (52B8 (No (No
VH1 M64V Binding) Binding) W101Y/VL5 N34D) IgG4 S228P / Kappa 59
Humanized anti- n.d. n.d. NB NB n.d. n.d. ILT3 mAb (52B8 (No (No
VH1 M64V Binding) Binding) W101Q / VL5 S35A) IgG4 S228P / Kappa 60
Humanized anti- n.d. n.d. NB NB n.d. n.d. ILT3 mAb (52B8 (No (No
VH1 M64V Binding) Binding) W101Q / VL5 S35N) IgG4 S228P / Kappa 61
Humanized anti- n.d. n.d. NB NB n.d. n.d. ILT3 mAb (52B8 (No (No
VH1 M64V Binding) Binding) W101Q / VL5 N34Q) IgG4 S228P / Kappa 62
Humanized anti- n.d. n.d. NB NB n.d. n.d. ILT3 mAb (52B8 (No (No
VH1 M64V Binding) Binding) W101Q / VL5 N34D) IgG4 S228P / Kappa
p52B8 Clone 52B8 15.5 23.2 0.658 24.4 98 n.d. Hybridoma extract
p40A6 Clone 40A6 17.9 25.9 0.713 0.995 n.d. n.d. Hybridoma extract
p16B1 Clone 16B1 n.d. n.d. 0.096 0.259 98.1 Hybridoma n.d. extract
p49C6 Clone 49C6 13.8 19.8 n.d. n.d. n.d. n.d. Hybridoma extract
(not sequenced) p11D1 Clone 11D1 50.46 2028 n.d. n.d. n.d. n.d.
Hybridoma extract p17H12 Clone 17H12 139.2 NB n.d. n.d. 95.7 n.d.
Hybridoma extract p37C8 Clone 37C8 7.719 9.478 0.012 0.145 98.4
n.d. Hybridoma extract p1G12 Clone 1G12 39.2 NB n.d. n.d. n.d. n.d.
Hybridoma extract p20E4 Clone 20E4 1.992 18.04 6.99 18.2 98.5 n.d.
Hybridoma extract p24A4 Clone 24A4 21.4 21.3 0.021 0.126 n.d. n.d.
Hybridoma extract
Example 5
[0255] Epitope Mapping of a chimeric anti-ILT3 52B8 mouse VH/human
IgG4 (S228P):mouse VL/human Kappa ("c58B8"; mAb 73) binding to
human ILT3 by Hydrogen Deuterium Exchange (HDX) Mass
Spectrometry
[0256] Contact areas of the antibody to human ILT3 extracellular
domain were determined by use of hydrogen deuterium exchange mass
spectrometry (HDX-MS) analysis. HDX-MS measures the incorporation
of deuterium into the amide backbone of the protein and changes in
this incorporation are influenced by the hydrogen's solvent
exposure. A comparison of the deuterium exchange levels in
antigen-alone samples and antibody-bound samples was done to
identify regions on the ILT3 extracellular domain that may be in
contact with the antibody. Human ILT3 extracellular domain with a
C-terminal His tag (human ILT3-His) has the amino acid sequence
shown in SEQ ID NO: 1.
[0257] His-tagged human ILT3-His extracellular domain was
pre-incubated with antibody c58B8 (mAb 73), a chimeric anti-ILT3
52B8 mouse VH M64V/human IgG4 (S228P):mouse VL/human Kappa
comprising a HC having the amino acid sequence of SEQ ID NO: 113
and a LC having the amino acid sequence shown in SEQ ID NO: 116,
before incubation in a deuterium buffer. Human ILT3-His and the
antibody were buffer exchanged to PBS pH 7.4 using 3k MWCO spin
columns. Human ILT3-His (80 pmol/.mu.L) was mixed with an equal
volume of the antibody (40 pmol/.mu.L) or, as the unbound control,
PBS pH 7.4. The antibody bound samples and the unbound control were
incubated at room temperature for one hour before beginning the
labeling experiment.
[0258] To deuterium label the samples, 2 .mu.L of sample was mixed
with 25 .mu.L of PBS in deuterium oxide pH 7.6. Labeling time
points were 30, 300, 3000, 6000 or 12000 seconds. After the set
time, 25 .mu.L of the labeling mixture was added to 30 .mu.L of
cold quench buffer (8M Urea, 150 mM TCEP). The quenched sample was
incubated at 1.5.degree. C. for 2 minutes. 53 .mu.L was then
injected into the column cooling chamber where the sample was
passed over the pepsin/protease XIII column and the resulting
peptides loaded onto the trapping column. After three minutes, the
analytical gradient and the mass spectrometer were started. A fully
deuterated sample was generated by incubating 2 .mu.L of human
ILT3-His with 108 .mu.L of deuterated denaturing buffer (4M Urea,
150 mM TCEP in 99.5% deuterium oxide). The sample was incubated at
37.degree. C. overnight. Then 55 .mu.L was directly injected into
the column chamber and the data acquired.
[0259] LC-MS/MS data was acquired of an unlabeled sample and
searched before deuterium labeling to verify successful digestion
of the proteins and to generate a list of peptides. Data was
database searched using Proteome Discoverer 1.4 and the SEQUEST HT
search algorithm (ThermoFisher Scientific). The protein database
used was the human ILT3-His sequence concatenated to the yeast
Saccharomycese cerevisiae database.
[0260] Following labeling, 55 .mu.L sample aliquotes were applied
to a NovaBioAssays Pepsin/Protease XIII column followed by
chromatography on Waters CSH C18 Guard column and Waters CSH C18
1.times.50 mm Analytical column in a loading buffer containing 2%
Acetonitrile, 0.1% TFA. Deuterium incorporation into the human
ILT3-His extracellular domain was measured by mass spectrometry.
Quench: 8M Urea, 150 mM TCEP; Labeling buffer: PBS, pH 7.6; Blank
buffer: PBS, pH 7.4. The mass spectrometer was a Thermo Scientific
ORBITRAP-ELITE.TM.. For the measurement of deuterium labeled
samples, the mass spectrometer was set to acquire one full scan MS
data in the orbitrap at 120,000 resolving power, a target ion count
of 1E6 and a maximum ion injection time of 500 millisecond. For the
acquisition of MS/MS data for peptide identifications, the mass
spectrometer was set to acquire one full scan spectrum at 120,000
resolving power followed by ten data-dependent MS/MS spectra in the
ion trap.
[0261] The liquid chromatography system used was a Waters
NANOACQUITY.RTM. for the analytical column gradient and a Waters
515 isocratic pump for the sample digestion and loading. For sample
digestion and loading, the buffer used was 2% acetonitrile and 0.1%
trifluoroacetic acid at a flow rate of 100 .mu.L/min. For the
analytical gradient, the buffers were Buffer A) 0.1% formic acid in
water and Buffer B) 0.1% formic acid in acetonitrile. The gradient
was at 40 .mu.L/min from 2% B to 36% B in 10 minutes, followed by a
wash of 80% B for 1.5 minute and a re-equilibration at 2% B for 3
minutes. The column was then washed by cycling the gradient between
2% and 80% B, three times with 1 minute at each step, followed by a
final equilibration at 2% B for 5 minutes. The trapping column was
a Waters VANGUARD.TM. C18 BEH 1.7 .mu.m Guard Column and the
analytical column was a Waters C18 BEH300, 1.7 .mu.m 1.times.50 mm
column.
[0262] Sample handling for the deuterium labeling was done by a
Leaptec H/D-X PAL.TM. system. The labeling sample tray was set to a
temperature of 25.degree. C., the quenching tray was set to 1.5 C
and the trap and analytical column chamber was set to 1.5.degree.
C. The immobilized pepsin column (Pepsin/Protease XIII column
NBA2014002, 2.1.times.30 mm, NovaBioAssay) was kept outside the
column chamber at room temperature.
[0263] A deuterium labeling difference heatmap of the human
ILT3-His amino acid residues bound by the antibody is shown in FIG.
3A. The HDX mass spectrometry shows that the antibody and the other
antibody families disclosed herein that cross-compete with the
antibody bind an epitope comprising or consisting of at least one
amino acid in one or more of amino acid residues 18-23 (ISWGNS; SEQ
ID NO: 3), 64-69 (IPSMTE; SEQ ID NO: 4), 96-101 (MTGAYS; SEQ ID NO:
5), 124-131 (QSRSPMDT; SEQ ID NO: 6), 152-159 (AQQHQAEF; SEQ ID NO:
7) and 184-187 (LLSH; SEQ ID NO: 8) of ILT3. FIG. 3B shows a
first-view and a second view of a three-dimensional surface
structure model of the human ILT3 extracellular domain with the
protected amino acid residues shown. These protected amino acid
residues comprise a split or non-contiguous epitope that spans the
border between the D1 and D2 domains of the extracellular domain.
FIG. 3C is a ribbon diagram showing the placement of the epitope on
the human ILT3 extracellular domain. Residues in black were
protected from labeling by the antibody. Residues in white showed
no changes in labeling and residues in dark gray did not have data
acquired for them. The deuterium labeling difference for each
residue was averaged and mapped onto a crystal structure of ILT3
(Cheng et al., "Crystal structure of leukocyte Ig-like receptor
LILRB4 (ILT3/LIR-5/CD85k): a myeloid inhibitory receptor involved
in immune tolerance." J Biol Chem 286:18013-25 (2011)).
[0264] Similar HDX mapping experiments were preformed using
antibodies ZM4.1, DX439, DX446, and 9B11. Antibody ZM4.1 is
commercially available from ThermoFisher Scientific, Carlsbad,
Calif. or BioLegend, San Diego, Calif. Antibodies DX439 and DX446
have been disclosed in WO2018089300 and Antibody 9B11 has been
disclosed in U.S. Pat. No. 7,777,008. Of these antibodies, only
antibody ZM4.1 was observed to bind an epitope that partially
overlapped with the epitope bound by the antibodies of the present
invention; however, binning studies showed that antibody ZM4.1 did
not cross block binding of the antibodies of the present invention.
FIGS. 3D, 3E, 3F, and 3G show heatmaps of the binding of antibodies
ZM4.1, DX439, DX446, and 9B11 to human ILT3.
Example 6
[0265] Pharmacokinetics of Chimeric Anti-ILT3 52B8 Mouse VH/Human
IGg4 (S228P):Mouse VL/Human Kappa ("c58B8"; mAb 73) in NSG Mice
[0266] The pharmacokinetics of chimeric anti-ILT3 52B8 mouse
VH/human IgG4 (S228P):mouse VL/human Kappa (c85B8; mAb 73) was
evaluated in Panc08.13 human-NSG mice model and SK-MEL-5 human
CD34+-NSG mice model.
[0267] SK-MEL-5 is a human melanoma-derived line that can grow as a
subcutaneous tumor. Panc 08.13 is a human pancreatic
carcinoma-derived tumor line. Panc 08.13 human-NSG model has been
shown to be sensitive to pembrolizumab and ipilimumab treatment.
SK-MEL-5 model has a robust and diverse myeloid infiltrate in the
tumor compared to Panc 08.13 model. Both models show increased ILT3
expression on human CD14+ myeloid cells in the tumor and
spleen.
[0268] An ECL-based target capture immunoassay was used to quantify
the antibody in humanized mice plasma. The assay was established
with biotinylated recombinant ILT3 as capture reagent, and sulfoTAG
labeled mouse anti-huIgG (Fc specific) from Southern Biotech (cat
#9190-01) for detection reagent. Both calibrators and QCs were
prepared in neat C57BL/6 plasma and diluted 100 times when testing
in plate. This assay has been qualified and the LLOQ of the assay
was determined to be 40 ng/mL with an MRD of 100.
[0269] In Panc08.13 hu-NSG mice model, 20 mg/kg of antibody was
administered with and without pembrolizumab (5 mg/kg) via IP weekly
for the first three doses and two weeks after the 3rd dose for the
4th dose. Blood samples were collected before the third dose
(Ctrough) and 24 hours after the third dose (Cmax). Terminal blood
samples on day 5 and 6 after the fourth dose were also collected.
In SK-MEL-5 huCD34+-NSG mice model, the antibody was administered
at 2 and 20 mg/kg via IP weekly. Blood samples were collected
before the third dose (Ctrough) and 24 h after the third dose
(Cmax). Terminal blood samples on day 3 and 7 after the third dose
were also collected. The free (unbound) antibody concentrations
were determined by an antigen-capture assay.
[0270] Pharmacokinetic parameters are generated from historical
IgG4 antibody data (IV bolus administration of 1, 3, 10, 30 mg/kg
of humanized IgG4 antibody in C57BL/6J mice) with Phoenix NLME. PK
profiles at the studied dose of the antibody were simulated based
on the generated pharmacokinetic parameters.
[0271] PK analysis of historical IgG4 antibody data showed a linear
relationship between AUC and studied dose (See FIG. 4). With the
assumptions including linear PK across different tested doses of
c52B8, no PK difference among different mouse strains, rapid
absorption and 100% bioavailable after IP administration of the
antibody, PK profiles at the studied dose of c52B8 were simulated
based on historical IgG4 antibody data. The results showed that the
simulated profile at 20 mg/kg in both Panc08.13 human-NSG model and
SK-MEL-5 huCD34+-NSG model follow the observed c52B8
concentrations.
Example 7
[0272] Anti-ILT3 Monoclonal Antibodies Activate Dendritic Cells and
Reduces Suppressive Capacity of Myeloid-Derived Suppressor Cells
(MDSCs)
[0273] Human PBMCs isolated from fresh leukopacs were frozen,
thawed and CD14+ monocytes were purified by negative selection. The
purified cells were cultured for 5 days with GM-CSF (1000 U/mL) and
IL4 (1000 U/mL). These immature DCs were then further cultured for
42 hours with addition of IL-10 (50 ng/mL) and LPS (1 ug/mL) with
or without anti-ILT3 antibody. TNF.alpha. is measured in the
culture supernatant.
[0274] Titration experiments showed that c52B8 caused a
dose-dependent increase in TNF.alpha. secretion in the culture
medium when added during the polarization step, whereas a control
IgG4 did not (the control is an variant of a commercial antibody
against RSV, trade name Synagis) (FIG. 5A). The concentration of
antibody required to produce half of the maximal increase in
TNF.alpha. levels (EC50) was approximately 1.9 ng/mL. This was not
different for chimeric variants in which V.sub.H and V.sub.L of
p58B8 were fused to Fc with a human IgG1 framework (mAb 78) or a
N297A mutated human IgG1 framework (mAb 76). These data indicate
that in this assay Fc receptor binding does not play any role in
the functional activity. The independence from Fc receptor binding
controls for the possibility that the mechanism of activation in
this assay is DCs becoming activated through recognition of other
DCs in the culture being decorated with antibody which would be a
mechanism unrelated to ILT3.
[0275] FIGS. 5B and 5C show there was no significant difference in
functional activity between c52B8 (mAb 73) and humanized anti-ILT3
mAb 52B8 VH1 M64V/VL5 N34Q) IgG4 S228P/Kappa (mAb 46) in two
donors. As shown, with antibody c52B8 added during polarization of
the DCs, but not during T cell priming, DCs were better able to
activate T cells to proliferate, similar to DCs not tolerized with
IL10. When antibody c52B8 was added during T cell priming but not
during DC polarization, T cells were better able to respond to
subsequent re-stimulation. Following humanization, variants that
retained binding comparable to the chimera were tested in this same
assay and found to be active, with no meaningful differences in
potency among them. These data indicate that data generated with
c52B8 is representative of what the data would be if humanized mAb
46 had been used.
Example 8
[0276] Anti-ILT3 Antibodies Reduce Suppressive Capacity of
Myeloid-Derived Suppressor Cells (MDSCs)
[0277] Without ascribing to any particular theory or hypothesis, we
hypothesize that a productive T cell response to tumor can be
limited in some cases by the presence of immature and suppressive
myeloid cells. These cells express ILT3 and we hypothesize that
ILT3 functions as an inhibitory manner to maintain an immature
state characterized by low HLA-DR expression, IL-10 production, and
effective suppression of T cell activation and proliferation.
Establishment of a model based on co-culture of human PBMCs with
SKMEL5 tumor cells in vitro, followed by purification of MDSCs and
testing of their ability to suppress proliferation of autologous
CD8+ T cells enabled exploration of this aspect of ILT3 biology.
This example shows that c52B8 and humanized 52B8 (mAb 46) are able
to impair the acquisition (or maintenance) of a T cell-suppressive
phenotype.
[0278] To generate MDSCs, healthy human PBMCs were cultured with
SKMEL5 cells and 20 ng/mL GM-CSF for 7 days. CD33+ cells were
collected by positive antibody-based magnetic bead selection and
then co-cultured at the indicated ratios with purified autologous
CD8+ T cells for 3 days in the presence of a polyclonal stimulus.
Cultures included c52B8 (mAb 73), humanized 52B8 (mAb 46), or
isotype control antibody (1 .mu.g/mL) in both the co-culture and T
cell suppression steps. The T cell suppression assay was conducted
with a T cell to MDSC ratio of 4:1 and measuring the amount of
interferon gamma (INF.gamma.) produced.
[0279] FIG. 6A and FIG. 6B exemplifies the activity of both
humanized 52B8 and c52B8 in the MDSC model at a ratio of T cells to
MDSCs where the effect of these antibodies was most evident show
that the antibodies reduce the suppressive capacity of MDSCs in a
comparable manner. These data further indicate that data generated
with c52B8 is representative of what would be found with humanized
mAb 46.
Example 9
[0280] Anti-ILT3 Antibody cC52B8 Inhibits Growth of SK-MEL-5 Tumors
in SK-MEL-5 Hu-NSG Mice Bearing SK-MEL-5 Subcutaneous Tumors
[0281] Systemic administration of c52B8 once weekly to mice bearing
established subcutaneous tumors afforded inhibition of tumor growth
(FIG. 7). Animals were randomized to treatment on the basis of
tumor volume on day 21 post-implantation and dosed s.c. with 20
mg/kg of c52B8 (mAb 73) or isotype control once weekly beginning on
day 21. Data shown in the left panel are means and std. error (nine
per group). Individual animal tumor growth curves are shown at
right. Body weight decreased to a similar degree in both control
and 52B8 groups. This study is representative of three independent
studies.
[0282] The degree of inhibition of tumor growth was consistent and
similar in three separate studies and was very similar to the
effect of anti-ILT4. None of the other mechanisms tested to date
(e.g. anti-PD-1, anti-ILT4, anti-CD27, anti-GITR) have afforded
regressions leading us to speculate that tumor stasis may represent
a floor for this model. This is clearly different from the mouse
syngenic models commonly used for preclinical efficacy assays.
Example 10
[0283] Immune Activation in SK-MEL-5 Hu-NSG after c52B8
Treatment
[0284] To understand immune mechanism that mediates the tumor
efficacy, tumor infiltrating immune cells were profiled and
measured sHLA-G levels were measured in the blood. Mice were
treated with c52B8 (2 and 20 mg/kg i.p. QW). Antibody doses were
selected based on C.sub.max and C.sub.trough levels detected in a
mini-PK and simulations using historical studies. Blood samples
were collected for PK, sHLA-G, and cytokine analyses. TILs
profiling was performed using CyTOF to detect 36 markers
simultaneously. Terminal tumor samples were fixed and used for
human CD3+ T cell IHC analysis. Thirty percent tumor growth
inhibition was observed in mice treated with 20 mpk 52B8. However,
no statistical significant difference was detected due to big
variability associated with the humanized tumor model. 52B8 modest
tumor efficacy was associated with a modest decrease in tumor
CD4+CD127-CD25+T suppressor cells (21% vs. 14%) and blood sHLA-G
levels and an increase in activation of T cells (CD69 intensity, 14
vs. 23) in the tumor. No cytokine change was detected with c52B8
treatment as seen in FIG. 8.
Example 11
[0285] Effect of Anti-ILT3 Antibody c52B8 in Combination with
Pembrolizumab in Panc 08.13 Hu-NSG Model: Tumor Efficacy and Immune
Activation
[0286] Anti-ILT3 antibody c52B8 was evaluated in Panc 08.13 hu-NSG
model. 52B8 used as a single agent showed minimum effect on tumor
growth inhibition. When 52B8 was used in combination with
pembrolizumab, one in five cohorts (five different human donors) of
humanized mice had 50% tumor growth inhibition (TGI) and the TGI
was associated with increased T cell activation and IFN.gamma.
production and decreased blood sHLA-G level as seen in FIG. 9A,
FIG. 9B, FIG. 9C, and FIG. 9D.
Example 12
[0287] Effect of Anti-ILT3 Antibody 52B8 in Combination with
Pembrolizumab in an MDSC/T Cell Suppression Assay
[0288] Humanized anti-ILT3 antibody 52B8 (mAb 46) with and without
pembrolizumab effected an increase T-Cell activity in MDSC/T-cell
suppression assays. The effect was additive when mAb 46 was used in
combination with pembrolizumab.
[0289] To generate MDSCs, healthy human PBMCs from a particular
donor were cultured with SKMEL5 cells and 20 ng/mL GM-CSF for seven
days. Cultures were treated with 52B8 (1 .mu.g/mL) or isotype
control antibody (1 .mu.g/mL). CD33+ cells were collected anti-CD33
magnetic microbeads and LS column separation (Miltenyi Biotec,
Germany) and then co-cultured at the indicated ratios with purified
autologous CD8+ T cells for 3 days in the presence of a polyclonal
stimulus. Autologous CD8+ T cells were isolated from healthy human
PBMCs using negative antibody-based magnetic bead selection (Stem
Cell Technologies, Canada) then co-cultured in 96 well plates with
CD33+ myeloid cells at the ratio of 8:1 (Tcell:MDSC) for 2 days.
Cultures included humanized 52B8 (mAb 46) or isotype control
antibody (IgG4) (1 .mu.g/mL) alone or in combination with
pembrolizumab (2 .mu.g/mL) in both the co-culture and T cell
suppression steps. Total antibody concentration in each treatment
is adjusted to 3 ug/mL with isotype control antibody. T cell
proliferation was induced by a polyclonal stimulus anti-CD3/CD28
beads and IL2. IFN.gamma. levels were determined in culture
supernatants using MSD ELISA (Mesoscale Discovery, Md.). The T cell
suppression assay was conducted with a T cell to MDSC ratio of 4:1
or 8:1 and measuring the amount of interferon gamma (INF.gamma.)
produced. The results are shown in FIGS. 10-14.
[0290] FIG. 10 shows that humanized anti-ILT3 antibody 52B8 (mAb
46) reduces the suppressive capacity of MDSCs to an extent
comparable to chimeric anti-ILT3 antibody c52B8 (mAb 73; lot 26AVY)
in MDSC/T-cell suppression assays using MDSCs obtained from PBMCs
from two different human donors (D00100385 and D001003507,
respectively).
[0291] As shown in FIGS. 11-14 humanized anti-ILT3 antibody 52B8
(mAb 46) in combination with pembrolizumab reduced MDSC inhibition
of T cell activation at a higher level compared to either alone in
an MDSC/T cell suppression assay (a) at either a 4:1 or 8:1 ratio
of T cell to MDSC using MDSCs obtained from PBMCs from human donor
D001003835 (FIG. 11); (b) at either a 4:1 or 8:1 ratio of MDSC to T
cell using MDSCs obtained from PBMCs from human donor D001003180
(FIG. 12); (c) at a 4:1 or 8:1 ratio of ratio of T cell to MDSC
using MDSCs obtained from PBMCs from human donor D001003507 (FIG.
13); and an 8:1 ratio of ratio of T cell to MDSC using MDSCs
obtained from PBMCs from human donor (FIG. 14). The results are
summarized in Tables 8 and 9. As shown in FIGS. 10-13 and Tables 8
and 9, combining an anti-ILT3 antibody 52B8 with pembrolizumab
resulted in an additive effect of increasing the activation of T
cells over that achievable using pembrolizumab or 52B8 alone. As
shown, increases in IFN.gamma. for the combination relative to the
other treatments ranged from 41% to 74%. These results indicate
that the combination of pembrolizumab with 52B8 does not result in
an excessive or uncontrolled escalation of T cell activation.
TABLE-US-00008 TABLE 8 Summary of the humanized anti-ILT3 antibody
52B8 and pembrolizumab combination data Mean Avg .+-. SD T cell +
MDSC T Cell: hIgG4 + 52B8 + MDSC T Cell hIgG4 + Pembro- hIgG4 +
Pembro- Donor ratio only hIgG4 lizumab 52B8 lizumab D001003835 4:1
19439 .+-. 3667 .+-. 4676 .+-. 6380 .+-. 10438 .+-. 4191 795 1162
1187 1132 8:1 32644 .+-. 17386 .+-. 20556 .+-. 28280 .+-. 38163
.+-. 4146 1628 5028 4643 7817 D001003180 4:1 38166 .+-. 1482 .+-.
1781 .+-. 3983 .+-. 3606 .+-. 7574 646 295 1528 1864 8:1 33250 .+-.
6823 .+-. 6768 .+-. 9532 .+-. 14896 .+-. 6021 2107 1287 3025 2932
D001003507 4:1 56836 .+-. 7364 .+-. 8111 .+-. 12202 .+-. 18422 .+-.
5777 2977 5220 3221 4135 8:1 55376 .+-. 23417 .+-. 23981 .+-. 26204
.+-. 36992 .+-. 6310 8640 3135 3075 1856 D001003428 8:1 159127 .+-.
81071 .+-. 87413 .+-. 98902 .+-. 123920 .+-. 10552 13458 15061 8994
22448
TABLE-US-00009 TABLE 9 52B8 Antibody + Pembrolizumab Combination- T
cell:MDSC ratio (8:1) Ratios of Condition GM 95% CI P-value (52B8 +
pembrolizumab) / 1.84 1.35, 2.53 0.0043 IgG4 (52B8 + pembrolizumab)
/ 1.73 1.26, 2.36 0.0057 pembrolizumab (52B8 + pembrolizumab) /
1.39 1.20, 1.61 0.0028 52B8 The p-values are from one-sided paired
t-tests comparing the 52B8 + pembrolizumab combination to each of
the other groups, using logs of IFN.gamma. values. GM = geometric
mean
Example 13
[0292] Effect of Anti-ILT3 Antibody 52B8 in Combination with
Pembrolizumab in Mixed Lymphocyte Reaction of Polarized IL-10 DCs
and Allogenic CD8+ T Cells
[0293] In this example, a mixed lymphocyte reaction of
IL-10-polarized human monocyte-derived dendritic cells and
allogenic CD8+ T cells, incubated for four days followed by
measurement of interferon gamma (IFN.gamma.) in the culture
supernatant as a read out of T cell activation. In this experiment,
the activities of pembrolizumab, 52B8, or the combination of the
two were compared to isotype control antibody (IgG4 in both cases),
in nine allogenic donor pairs.
[0294] Monocyte derived dendritic cells (DCs)-IL10 DCs from three
CD14+ monocyte donors were differentiated for seven days
(Granulocyte-macrophage colony-stimulating factor (GMCSF) and IL4
for five days and then two days with IL10, with and without IgG4
(lot 92ASJ), with and without 52B8 (Lot 41BAB) at 1 .mu.g/mL) to
produce DC129, DC226, and DC196. CD8+ cells from three donors were
isolated and mixed leukocyte reactions (MLR) were established at
1:5 DC:T cell ratio from the three donors in a 96 well format (30 k
DC vs 150 k CD8+ T cells) where cells were treated with and without
IgG4 (lot 92 ASJ); with and without Pembrolizumab (lot 42ASN) at 2
.mu.g/mL. IgG4 or 52B8 was also added back in the MLR at 1
.mu.g/mL. Wound up with nine MLR pairs of IL10 DCs:CD8+ T
cells:
[0295] DC129 vs T30, T3788 and T3259
[0296] DC226 vs T30, T3788 and T3259
[0297] DC196 vs T30, T3788 and T3259
IFN.gamma. supernatant was collected at day four and quantified
using Meso Scale Discovery (MSD). Additional supernatant fraction
was collected at day five and cells were collected and stained for
PD1 and PDL1 expression. Dendritic Cell Staining on Day seven of
differentiation (just prior to MLR setup). T cell Staining of CD8+
T cells at day five of MLR assay.
[0298] FIG. 15 shows the results for all donor pairs combined into
one figure (each mark is a donor pairing). As shown, 52B8 in
combination with pembrolizumab effected a reverse of T cell
tolerization, resulting in a statistically significant increase in
activation of CD8+ T cells.
TABLE-US-00010 Table of Sequences SEQ ID NO: Description Sequence 1
Human ILT3 QAGPLPKPTLWAEPGSVISWGNSVTIWCQGTLEAREYRLDK (LILRB4)
EESPAPWDRQNPLEPKNKARFSIPSMTEDYAGRYRCYYRSP extracellular
VGWSQPSDPLELVMTGAYSKPTLSALPSPLVTSGKSVTLLC domain with C-
QSRSPMDTFLLIKERAAHPLLHLRSEHGAQQHQAEFPMSPV terminal His Tag;
TSVHGGTYRCFSSHGFSHYLLSHPSDPLELIVSGSLEDPRPSP epitope domains
TRSVSTAAGPEDQPLMPTGSVPHSGLRRHWEHHHHHHHH identified by bold- face
type 2 Macaca mulatta QAGPLPKPTIWAEPGSVISWGSPVTIWCQGTLDAQEYYLDKE
(Rhesus) ILT3 GSPAPWDTQNPLEPRNKAKFSIPSMTQHYAGRYRCYYHSHP (LILRB4)
DWSEDSDPLDLVMTGAYSKPILSVLPSPLVTSGESVTLLCQS extracellular
QSPMDTFLLFKEGAAHPLPRLRSQHGAQLHWAEFPMGPVTS domain
VHGGTYRCISSRSFSHYLLSRPSDPVELTVLGSLESPSPSPTRSI (sequence obtained
SAAGPEDQSLMPTGSDPQSGLRRHWE from GenBank NP_001035766) 3 Human ILT3
ISWGNS peptide A 4 Human ILT3 IPSMTE peptide B 5 Human ILT3 MTGAYS
peptide C 6 Human ILT3 QSRSPMDT peptide D 7 Human ILT3 AQQHQAEF
peptide E 8 Human ILT3 LLSH peptide F 9 Human IgG4 HC
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA Constant domain
LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSN (S228P; shown in
TKVDKRVESKYGPPCP CPAPEFLGGPSVFLFPPKPKDTLMISR bold-face type)
TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNS
TYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQP
REPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP
ENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEA LHNHYTQKSLSLSLGK 10
Human IgG4 HC ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA
Constant domain LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSN
(S228P; shown in TKVDKRVESKYGPPCP CPAPEFLGGPSVFLFPPKPKDTLMISR
bold-face type) TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNS (lacks
C-terminal K TYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQP (herein
referred to REPQVYTLPPSQEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP as "K-"))
ENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEA LHNHYTQKSLSLSLG 11
Human IgG1 HC ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA
constant domain LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN
TKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL
MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA
KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK
12 Human IgG1 HC ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA
Constant domain LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN
(L234A, L235A, TKVDKKVEPKSCDKTHTCPPCPAPE GGPSVFLFPPKPKDTL D265S;
shown in MISRTPEVTCVVV VSHEDPEVKFNWYVDGVEVHNAKTKPREEQ bold-face
type) YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESN
GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK 13
Human IgG1 HC ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA
Constant domain LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN (K-)
(L234A, TKVDKKVEPKSCDKTHTCPPCPAPE GGPSVFLFPPKPKDTL L235A, D265S;
MISRTPEVTCVVV VSHEDPEVKFNWYVDGVEVHNAKTKPREEQ shown in bold-face
YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK type)
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESN
GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPG 14
Human LC Kappa RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD
Constant domain NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV
THQGLSSPVTKSFNRGEC 15 Anti-ILT3 52B8
EVQLVESGGDLVKPGGSLKLSCAASGFTFSNYGMSWVRQTP parental HC
DRRLEWVATISGGGDYTNYPDSMRGRFTISRDNAKNTLYLQ variable domain
MSSLKSEDTAMYYCGRRLWFRSLYYAMDYWGQGTSVTVSS 16 Anti-ILT3 52B8
NIVLTQSPASLAVSLGQRATISCRASEKVDSFGNSFMHWYQQ parental LC
KPGQPPKLLIYLTSNLDSGVPARFSGSGSRTDFALTIDPVEAD variable domain
DAATYYCQQNNEDPYTFGGGTKLEIK 17 52B8 HC-CDR1 NYGMS 18 52B8 HC-CDR2
TISGGGDYTNYPDSXRG (Wherein Xaa15 is M, V, or L) 19 52B8 HC-CDR2 M
TISGGGDYTNYPDSMRG 20 52B8 HC-CDR2 V TISGGGDYTNYPDSVRG 21 52B8
HC-CDR2 L TISGGGDYTNYPDSLRG 22 52B8 HC-CDR3 RLXFRSLYYAMDY (Wherein
Xaa3 is W, Y, Q, or F) 23 52B8 HC-CDR3 RLWFRSLYYAMDY 24 52B8
HC-CDR3 RLYFRSLYYAMDY 25 52B8 HC-CDR3 RLQFRSLYYAMDY 26 52B8 HC-CDR3
RLFFRSLYYAMDY 27 52B8 LC-CDR1 RASEKVDSFGXXFMH (Wherein Xaa11 is N,
D, or Q and Xaa12 is S, N, or A) 28 52B8 LC-CDR1 N RASEKVDSFGNXFMH
(Wherein Xaa12 is S, N, or A) 29 52B8 LC-CDR1 D RASEKVDSFGDXFMH
(Wherein Xaa12 is S, N, or A) 30 52B8 LC-CDR1 Q RASEKVDSFGQXFMH
(Wherein Xaa12 is S, N, or A) 31 52B8 LC-CDR1 S RASEKVDSFGXSFMH
(Wherein Xaa11 is N, D, or Q) 32 52B8 LC-CDR1 N RASEKVDSFGXNFMH
(Wherein Xaa11 is N, D, or Q) 33 52B8 LC-CDR1 A RASEKVDSFGXAFMH
(Wherein Xaa11 is N, D, or Q) 34 52B8 LC-CDR1 RASEKVDSFGNNFMH (NN)
35 52B8 LC-CDR1 RASEKVDSFGDNFMH (DN) 36 52B8 LC-CDR1
RASEKVDSFGQNFMH (QN) 37 52B8 LC-CDR1 RASEKVDSFGNSFMH (NS) 38 52B8
LC-CDR1 RASEKVDSFGDSFMH (DS) 39 52B8 LC-CDR1 RASEKVDSFGNAFMH (NA)
40 52B8 LC-CDR1 RASEKVDSFGDAFMH (DA) 41 52B8 LC-CDR1
RASEKVDSFGQSFMH (QS) 42 52B8 LC-CDR1 RASEKVDSFGQAFMH (AF) 43 52B8
LC-CDR2 LTSNLDS 44 52B8 LC-CDR3 QQNNEDPYT 45 Anti-ILT3 40A6
QVQLKESGPGLVQASETLSLTCTVSGFSLTSYSINWVRQSSG parental HC
KGPEWMGRFWYDEGIAYNLTLESRLSISGDTSKNQVFLKMN variable domain
SLRTGDTGTYYCTRDRDTVGITGWFAYWGQGTLVTVSS 46 Anti-ILT3 40A6
ETVMTQSPTSLSASIGERVTLNCKASQSVGVNVDWYQQTPG parental LC
QSPKLLIYGSANRHTGVPDRFTGSGFGSDFTLTISDVEPEDLG variable domain
VYYCLQYGSVPYTFGAGTKLELK 47 40A6 HC-CDR1 SYSIN 48 40A6 HC-CDR2
RFWYDEGIAYNLTLES 49 40A6 HC-CDR3 DRDTVGITGWFAY 50 40A6 LC-CDR1
KASQSVGVNVD 51 40A6 LC-CDR2 GSANRHT 52 40A6 LC-CDR3 LQYGSVPYT 53
Anti-ILT3 16B1 QVQLKESGPGLVQASETLSLTCTVSGFSLTNYCVNWVRQPS parental
HC GKGPEWLGRFWFDEGKAYNLTLESRLSISGDTSKNQVFLRM variable domain
NSLRADDTGTYYCTRDRDTVGITGWFAYWGQGTLVTVSS 54 Anti-ILT3 16B1
ETVMTQSPTSLSASIGERVTLNCKASQSVGINVDWYQQTPGQ parental LC
SPKLLIYGSANRHTGVPDRFTGSGFGSDFTLTISNVEPEDLGV variable domain
YYCLQYGSVPYTFGPGTKLELK 55 16B1 HC-CDR1 NYCVN 56 16B1 HC-CDR2
RFWFDEGKAYNLTLES 57 16B1 HC-CDR3 DRDTVGITGWFAY 58 16B1 LC-CDR1
KASQSVGINVD 59 16B1 LC-CDR2 GSANRHT 60 16B1 LC-CDR3 LQYGSVPYT 61
Anti-ILT3 11D1 QVQLQQSGAELMKPGASVKISCKATGYTFRTYWIEWVKQRP parental
HC GHGLEWIGEILPGNGNTHFNENFKDKATFTADTSSNAAYMQ variable domain
LSSLTSEDSAVYYCVRRLGRGPFDFWGQGTTLTVSS 62 Anti-ILT3 11D1
DIQMTQSPSSLSVSLGGKVTITCKASQDINEYIGWYQRKPGK parental LC
GPRLLIHYTSTLQSGIPSRFSGSGSGRDYSLSISNLEPEDIATYY variable domain
CLQYANPLPTFGGGTKLEIK 63 11D1 HC-CDR1 TYWIE 64 11D1 HC-CDR2
EILPGNGNTHFNENFKD 65 11D1 HC-CDR3 RRLGRGPFDF 66 11D1 LC-CDR1
KASQDINEYIG 67 11D1 LC-CDR2 YTSTLQS 68 11D1 LC-CDR3 LQYANPLPT 69
Anti-ILT3 17H12 EVQLVESGGGLVQPGRSMKLSCAASGFTFSNFDMAWVRQA parental
HC PTRGLEWVSSITYDGGSTSYRDSVKGRFTISRDNAKGTLYLQ variable domain
MDSLRSEDTATYYCTTVESIATISTYFDYWGQGVMVTVSS 70 Anti-ILT3 17H12
DIVLTQSPALAVSLGQRATISCRASQSVSMSRYDLIHWYQQK parental LC
PGQQPKLLIFRASDLASGIPARFSGSGSGTDFTLTINPVQADDI
variable domain ATYYCQQTRKSPPTFGGGTRLELK 71 17H12 HC-CDR1 NFDMA 72
17H12 HC-CDR2 SITYDGGSTSYRDSVKG 73 17H12 HC-CDR3 VESIATISTYFDY 74
17H12 LC-CDR1 RASQSVSMSRYDLIH 75 17H12 LC-CDR2 RASDLAS 76 17H12
LC-CDR3 QQTRKSPPT 77 Anti-ILT3 37C8
QVQLKESGPGLVQASETLSLTCTVSGFSLTSYCVNWVRQPSG parental HC
KGPEWLGRFWYDEGKVYNLTLESRLSISGDTSKNQVFLKMN variable domain
RLRTDDTGTYYCTRDRDTMGITGWFAYWGQGTLVTVSS 78 Anti-ILT3 37C8
ETVMTQSPTSLSASIGERVTLNCKASQSVGINVDWYQQTPGQ parental LC
SPKLLIYGSANRHTGVPDRFTGSGFGSGFTLTISNVEPEDLGV variable domain
YYCLQYGSVPYTFGPGTKLELK 79 37C8 HC-CDR1 SYCVN 80 37C8 HC-CDR2
RFWYDEGKVYNLTLES 81 37C8 HC-CDR3 DRDTMGITGWFAY 82 37C8 LC-CDR1
KASQSVGINVD 83 37C8 LC-CDR2 GSANRHT 84 37C8 LC-CDR3 LQYGSVPYT 85
Anti-ILT3 1G12 QVQMQQSGTELMKPGASMKISCKATGYTFSTYWIQWIKQRP parental
HC GHGLEWIGEILPGSGTTNYNENFKGKATFSADTSSNTAYIHLS variable domain
SLTSEDSAVFYCARRLGRGPFDYWGQGTTLTVSS 86 Anti-ILT3 1G12
DIQMTQSPSSLSASLGGKVTITCEASQDINKHIDWYQHQPGR parental LC
GPSLLIHYASILQPGIPSRFSGSGSGRDYSFSITSLEPEDIATYY variable domain
CLQYDNLLPTFGGGTKLEIK 87 1G12 HC-CDR1 TYWIQ 88 1G12 HC-CDR2
EILPGSGTTNYNENFKG 89 1G12 HC-CDR3 RLGRGPFDY 90 1G12 LC-CDR1
EASQDINKHID 91 1G12 LC-CDR2 YASILQP 92 1G12 LC-CDR3 LQYDNLLPT 93
Anti-ILT3 20E4 QVQLKESGPGLVQASETLSLTCTVSGFSLTSYSVNWVRQPSG parental
HC KGLEWMGRFWYDGGTAYNSTLESRLSISGDTSKNQVFLKM variable domain
NSLQTDDTGTYYCTRDRDTMGITGWFAYWGQGTLVTVSP 94 Anti-ILT3 20E4
ETVMTQSPTSLSASIGERVTLNCKASQSVGVNVDWYQQTPG parental LC
QSPKLLIYGSANRHTGVPDRFTGSGFGSDFTLTISNVEPEDLG variable domain
VYYCLQYGSVPYTFGAGTKLELK 95 20E4 HC-CDR1 SYSVN 96 20E4 HC-CDR2
RFWYDGGTAYNSTLES 97 20E4 HC-CDR3 DRDTMGITGWFAY 98 20E4 LC-CDR1
KASQSVGVNVD 99 20E4 LC-CDR2 GSANRHT 100 20E4 LC-CDR3 LQYGSVPYT 101
Anti-ILT3 24A4 QVQLKESGPGLVQASETLSLTCTVSGFSLTSYCVNWVRQPSG parental
HC KGPEWLGRFWYDEGKVYNLTLESRLSISGDTSKNQVFLKMN variable domain
RLRTDDTGTYYCTRDRDTLGITGWFAYWGQGTLVTVSS 102 Anti-ILT3 24A4
ETVMTQSPTSLSASIGERVTLNCKASQSVGINVDWYQQTPGQ parental LC
SPKLLIYGSANRHTGVPDRFTGSGFGSGFTLTISNVEPEDLGV variable domain
YYCLQYGSVPYTFGPGTKLELK 103 24A4 HC-CDR1 SYCVN 104 24A4 HC-CDR2
RFWYDEGKVYNLTLES 105 24A4 HC-CDR3 DRDTLGITGWFAY 106 24A4 LC-CDR1
KASQSVGINVD 107 24A4 LC-CDR2 GSANRHT 108 24A4 LC-CDR3 LQYGSVPYT 109
Leader sequence A MEWSWVFLFFLSVTTGVHS 110 Leader sequence B
MSVPTQVLGLLLLWLTDARC 111 Mouse Anti-ILT3
EVQLVESGGDLVKPGGSLKLSCAASGFTFSNYGMSWVRQTP p52B8 parental HC:
DRRLEWVATISGGGDYTNYPDSMRGRFTISRDNAKNTLYLQ Murine IgG2a
MSSLKSEDTAMYYCGRRLWFRSLYYAMDYWGQGTSVTVSS heavy chain
AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTW
NSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNV
AHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPP
KIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTA
QTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKD
LPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMV
TDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKL
RVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK 112 Mouse Anti-ILT3
NIVLTQSPASLAVSLGQRATISCRASEKVDSFGNSFMHWYQQ p52B8 parental LC:
KPGQPPKLLIYLTSNLDSGVPARFSGSGSRTDFALTIDPVEAD murine Kappa light
DAATYYCQQNNEDPYTFGGGTKLEIKRADAAPTVSIFPPSSE chain
QLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWT
DQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIV KSFNRNEC 113 Chimeric
Anti- EVQLVESGGDLVKPGGSLKLSCAASGFTFSNYGMSWVRQTP ILT3 mouse 52B8
DRRLEWVATISGGGDYTNYPDSMRGRFTISRDNAKNTLYLQ VH parental/human
MSSLKSEDTAMYYCGRRLWFRSLYYAMDYWGQGTSVTVSS IgG4 (S228P)
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA
LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSN
TKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISR
TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNS
TYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQP
REPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP
ENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEA LHNHYTQKSLSLSLGK 114
Chimeric Anti- EVQLVESGGDLVKPGGSLKLSCAASGFTFSNYGMSWVRQTP ILT3 mouse
52B8 DRRLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNTLYLQ VH M64V/human
MSSLKSEDTAMYYCGRRLWFRSLYYAMDYWGQGTSVTVSS IgG4 (S228P)
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA
LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSN
TKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISR
TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNS
TYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQP
REPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP
ENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEA LHNHYTQKSLSLSLGK 115
Mouse Anti-ILT3 EVQLVESGGDLVKPGGSLKLSCAASGFTFSNYGMSWVRQTP 52B8 VH
DRRLEWVATISGGGDYTNYPDSLRGRFTISRDNAKNTLYLQ M64L/human IgG4
MSSLKSEDTAMYYCGRRLWFRSLYYAMDYWGQGTSVTVSS (S228P)
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA
LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSN
TKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISR
TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNS
TYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQP
REPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP
ENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEA LHNHYTQKSLSLSLGK 116
Chimeric Anti- NIVLTQSPASLAVSLGQRATISCRASEKVDSFGNSFMHWYQQ ILT3
mouse 52B8 KPGQPPKLLIYLTSNLDSGVPARFSGSGSRTDFALTIDPVEAD parental VL/
DAATYYCQQNNEDPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQ human Kappa
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS
KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE C 117 Humanized 52B8
EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variable
GKGLEWVATISGGGDYTNYPDSMRGRFTISRDNAKNSLYLQ domain VH1
MNSLRAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVSS 118 Humanized 52B8
EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variable
GKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1
MNSLRAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVSS (M64V) 119 Humanized 52B8
EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variable
GKGLEWVATISGGGDYTNYPDSLRGRFTISRDNAKNSLYLQ domain VH1
MNSLRAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVSS (M64L) 120 Humanized 52B8
EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variable
GKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1
MNSLRAEDTAVYYCGRRLFFRSLYYAMDYWGQGTLVTVSS (M64V, W101F) 121
Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC
variable GKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1
MNSLRAEDTAVYYCGRRLYFRSLYYAMDYWGQGTLVTVSS (M64V, W101Y) 122
Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC
variable GKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1
MNSLRAEDTAVYYCGRRLQFRSLYYAMDYWGQGTLVTVSS (M64V, W101Q) 123
Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC
variable GKGLEWVATISGGGDYTNYPDSMRGRFTISRDNAKNSLYLQ domain VH2
MNSLKAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVSS 124 Humanized 52B8
EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variable
GKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH2
MNSLKAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVSS (M64V) 125 Humanized 52B8
EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variable
GKGLEWVATISGGGDYTNYPDSLRGRFTISRDNAKNSLYLQ domain VH2
MNSLKAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVSS (M64L) 126 Humanized 52B8
DIVLTQSPDSLAVSLGERATINCRASEKVDSFGNSFMHWYQQ LC variable
KPGQPPKLLIYLTSNLDSGVPDRFSGSGSRTDFTLTISSLQAED domain VL1
VAVYYCQQNNEDPYTFGQGTKLEIK 127 Humanized 52B8
DIVLTQSPDSLAVSLGERATINCRASEKVDSFGNSFMHWYQQ LC variable
KPGQPPKLLIYLTSNLDSGVPDRFSGSGSGTDFTLTISSLQAED domain VL2
VAVYYCQQNNEDPYTFGQGTKLEIK 128 Humanized 52B8
EIVLTQSPATLSLSPGERATLSCRASEKVDSFGNSFMHWYQQ LC variable
KPGQAPRLLIYLTSNLDSGVPARFSGSGSRTDFTLTISSLEPED domain VL3
FAVYYCQQNNEDPYTFGQGTKLEIK 129 Humanized 52B8
EIVLTQSPATLSLSPGERATLSCRASEKVDSFGNSFMHWYQQ LC variable
KPGQAPRLLIYLTSNLDSGIPARFSGSGSGTDFTLTISSLEPED domain VL4
FAVYYCQQNNEDPYTFGQGTKLEIK 130 Humanized 52B8
DIQLTQSPSSLSASVGDRVTITCRASEKVDSFGNSFMHWYQQ LC variable
KPGKAPKLLIYLTSNLDSGVPSRFSGSGSGTDFTLTISSLQPED domain VL5
FATYYCQQNNEDPYTFGQGTKLEIK 131 Humanized 52B8
DIQMTQSPSSLSASVGDRVTITCRASEKVDSFGNSFMHWYQQ LC variable
KPGKAPKLLIYLTSNLDSGVPSRFSGSGSRTDFTLTISSLQPED domain VL6
FATYYCQQNNEDPYTFGQGTKLEIK 132 Humanized 52B8
DIQLTQSPSSLSASVGDRVTITCRASEKVDSFGNSFMHWYQQ LC variable
KPGKAPKLLIYLTSNLDSGVPSRFSGSGSRTDFTLTISSLQPED domain VL7
FATYYCQQNNEDPYTFGQGTKLEIK 133 Humanized 52B8
DIQLTQSPSSLSASVGDRVTITCRASEKVDSFGNSFMHWYQQ LC variable
KPGKAPKLLIYLTSNLDSGVPARFSGSGSRTDFTLTISSLQPED domain VL8
FATYYCQQNNEDPYTFGQGTKLEIK 134 Humanized 52B8
DIVLTQSPDSLAVSLGERATINCRASEKVDSFGNAFMHWYQQ LC variable
KPGQPPKLLIYLTSNLDSGVPDRFSGSGSGTDFTLTISSLQAED domain VL2,
VAVYYCQQNNEDPYTFGQGTKLEIK (S35A) 135 Humanized 52B8
DIVLTQSPDSLAVSLGERATINCRASEKVDSFGNNFMHWYQQ LC variable
KPGQPPKLLIYLTSNLDSGVPDRFSGSGSGTDFTLTISSLQAED domain VL2,
VAVYYCQQNNEDPYTFGQGTKLEIK (S35N) 136 Humanized 52B8
DIVLTQSPDSLAVSLGERATINCRASEKVDSFGQSFMHWYQQ LC variable
KPGQPPKLLIYLTSNLDSGVPDRFSGSGSGTDFTLTISSLQAED domain VL2,
VAVYYCQQNNEDPYTFGQGTKLEIK (N34Q) 137 Humanized 52B8
DIVLTQSPDSLAVSLGERATINCRASEKVDSFGDSFMHWYQQ
LC variable KPGQPPKLLIYLTSNLDSGVPDRFSGSGSGTDFTLTISSLQAED domain
VL2, VAVYYCQQNNEDPYTFGQGTKLEIK (N34D) 138 Humanized 52B8
DIQLTQSPSSLSASVGDRVTITCRASEKVDSFGNAFMHWYQQ LC variable
KPGKAPKLLIYLTSNLDSGVPSRFSGSGSGTDFTLTISSLQPED domain VL5,
FATYYCQQNNEDPYTFGQGTKLEIK (S35A) 139 Humanized 52B8
DIQLTQSPSSLSASVGDRVTITCRASEKVDSFGNNFMHWYQQ LC variable
KPGKAPKLLIYLTSNLDSGVPSRFSGSGSGTDFTLTISSLQPED domain VL5,
FATYYCQQNNEDPYTFGQGTKLEIK (S35N) 140 Humanized 52B8
DIQLTQSPSSLSASVGDRVTITCRASEKVDSFGQSFMHWYQQ LC variable
KPGKAPKLLIYLTSNLDSGVPSRFSGSGSGTDFTLTISSLQPED domain VL5
FATYYCQQNNEDPYTFGQGTKLEIK (N34Q) 141 Humanized 52B8
DIQLTQSPSSLSASVGDRVTITCRASEKVDSFGDSFMHWYQQ LC variable
KPGKAPKLLIYLTSNLDSGVPSRFSGSGSGTDFTLTISSLQPED domain VL5,
FATYYCQQNNEDPYTFGQGTKLEIK (N34D) 142 Humanized 52B8
EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variable
GKGLEWVATISGGGDYTNYPDSMRGRFTISRDNAKNSLYLQ domain
MNSLRAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVSS VH1/Human IgG4
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSG (S228P) constant
ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS domain
NTKVDKRVESKYGPPCP CPAPEFLGGPSVFLFPPKPKDTLMIS
RTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQ
PREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ
PENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLGK 143
Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC
variable GKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1
MNSLRAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVSS (M64V)/Human
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSG IgG4 (S228P)
ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS constant domain
NTKVDKRVESKYGPPCP CPAPEFLGGPSVFLFPPKPKDTLMIS
RTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQ
PREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ
PENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLGK 144
Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC
variable GKGLEWVATISGGGDYTNYPDSLRGRFTISRDNAKNSLYLQ domain VH1
MNSLRAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVSS (M64L)/Human
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSG IgG4 (S228P)
ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS constant domain
NTKVDKRVESKYGPPCP CPAPEFLGGPSVFLFPPKPKDTLMIS
RTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQ
PREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ
PENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLGK 145
Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC
variable GKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1
MNSLRAEDTAVYYCGRRLFFRSLYYAMDYWGQGTLVTVSS (M64V,
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA W101F)/Human
LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSN IgG4 (S228P)
TKVDKRVESKYGPPCP CPAPEFLGGPSVFLFPPKPKDTLMISR constant domain
TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNS
TYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQP
REPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP
ENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEA LHNHYTQKSLSLSLGK 146
Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC
variable GKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1
MNSLRAEDTAVYYCGRRLYFRSLYYAMDYWGQGTLVTVSS (M64V,
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA W101Y)/Human
LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSN IgG4 (S228P)
TKVDKRVESKYGPPCP CPAPEFLGGPSVFLFPPKPKDTLMISR constant domain
TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNS
TYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQP
REPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP
ENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEA LHNHYTQKSLSLSLGK 147
Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC
variable GKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1
MNSLRAEDTAVYYCGRRLQFRSLYYAMDYWGQGTLVTVSS (M64V,
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA W101Q)/Human
LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTC1VVDHKPSN IgG4 (S228P)
TKVDKRVESKYGPPCP CPAPEFLGGPSVFLFPPKPKDTLMISR constant domain
TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNS
TYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQP
REPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP
ENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEA LHNHYTQKSLSLSLGK 148
Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC
variable GKGLEWVATISGGGDYTNYPDSMRGRFTISRDNAKNSLYLQ domain
MNSLKAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVSS VH2/Human IgG4
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSG (S228P) constant
ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS domain
NTKVDKRVESKYGPPCP CPAPEFLGGPSVFLFPPKPKDTLMIS
RTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQ
PREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ
PENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLGK 149
Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC
variable GKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH2
MNSLKAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVSS (M64V)/Human
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSG IgG4 (S228P)
ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS constant domain
NTKVDKRVESKYGPPCP CPAPEFLGGPSVFLFPPKPKDTLMIS
RTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQ
PREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ
PENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLGK 150
Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC
variable GKGLEWVATISGGGDYTNYPDSLRGRFTISRDNAKNSLYLQ domain VH2
MNSLKAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVSS (M64L)/Human
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSG IgG4 (S228P)
ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS constant domain
NTKVDKRVESKYGPPCP CPAPEFLGGPSVFLFPPKPKDTLMIS
RTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQ
PREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ
PENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLGK 151
Humanized 52B8 DIVLTQSPDSLAVSLGERATINCRASEKVDSFGNSFMHWYQQ LC
variable KPGQPPKLLIYLTSNLDSGVPDRFSGSGSRTDFTLTISSLQAED domain
VL1/kappa VAVYYCQQNNEDPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQL constant
domain KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK
DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 152 Humanized 52B8
DIVLTQSPDSLAVSLGERATINCRASEKVDSFGNSFMHWYQQ LC variable
KPGQPPKLLIYLTSNLDSGVPDRFSGSGSGTDFTLTISSLQAED domain VL2/kappa
VAVYYCQQNNEDPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQL constant domain
KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK
DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 153 Humanized 52B8
EIVLTQSPATLSLSPGERATLSCRASEKVDSFGNSFMHWYQQ LC variable
KPGQAPRLLIYLTSNLDSGVPARFSGSGSRTDFTLTISSLEPED domain VL3/kappa
FAVYYCQQNNEDPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQL constant domain
KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK
DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 154 Humanized 52B8
EIVLTQSPATLSLSPGERATLSCRASEKVDSFGNSFMHWYQQ LC variable
KPGQAPRLLIYLTSNLDSGIPARFSGSGSGTDFTLTISSLEPEDF domain VL4/kappa
AVYYCQQNNEDPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLK constant domain
SGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD
STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 155 Humanized 52B8
DIQLTQSPSSLSASVGDRVTITCRASEKVDSFGNSFMHWYQQ LC variable
KPGKAPKLLIYLTSNLDSGVPSRFSGSGSGTDFTLTISSLQPED domain VL5/kappa
FATYYCQQNNEDPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQL constant domain
KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK
DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 156 Humanized 52B8
DIQMTQSPSSLSASVGDRVTITCRASEKVDSFGNSFMHWYQQ LC variable
KPGKAPKLLIYLTSNLDSGVPSRFSGSGSRTDFTLTISSLQPED domain VL6/kappa
FATYYCQQNNEDPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQL constant domain
KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK
DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 157 Humanized 52B8
DIQLTQSPSSLSASVGDRVTITCRASEKVDSFGNSFMHWYQQ LC variable
KPGKAPKLLIYLTSNLDSGVPSRFSGSGSRTDFTLTISSLQPED domain VL7/kappa
FATYYCQQNNEDPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQL constant domain
KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK
DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 158 Humanized 52B8
DIQLTQSPSSLSASVGDRVTITCRASEKVDSFGNSFMHWYQQ LC variable
KPGKAPKLLIYLTSNLDSGVPARFSGSGSRTDFTLTISSLQPED domain VL8/kappa
FATYYCQQNNEDPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQL constant domain
KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK
DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 159 Humanized 52B8
DIVLTQSPDSLAVSLGERATINCRASEKVDSFGNAFMHWYQ LC variable
QKPGQPPKLLIYLTSNLDSGVPDRFSGSGSGTDFTLTISSLQAE domain VL2
DVAVYYCQQNNEDPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQ (S35A)/kappa
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS constant domain
KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 160 Humanized 52B8
DIVLTQSPDSLAVSLGERATINCRASEKVDSFGNNFMHWYQ LC variable
QKPGQPPKLLIYLTSNLDSGVPDRFSGSGSGTDFTLTISSLQAE domain VL2
DVAVYYCQQNNEDPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQ (S35N)/kappa
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS constant domain
KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 161 Humanized 52B8
DIVLTQSPDSLAVSLGERATINCRASEKVDSFGQSFMHWYQQ LC variable
KPGQPPKLLIYLTSNLDSGVPDRFSGSGSGTDFTLTISSLQAED domain VL2
VAVYYCQQNNEDPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQL (N34Q)/kappa
KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK constant domain
DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 162 Humanized 52B8
DIVLTQSPDSLAVSLGERATINCRASEKVDSFGDSFMHWYQQ LC variable
KPGQPPKLLIYLTSNLDSGVPDRFSGSGSGTDFTLTISSLQAED domain VL2
VAVYYCQQNNEDPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQL (N34D)/kappa
KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK constant domain
DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 163 Humanized 52B8
DIQLTQSPSSLSASVGDRVTITCRASEKVDSFGNAFMHWYQQ LC variable
KPGKAPKLLIYLTSNLDSGVPSRFSGSGSGTDFTLTISSLQPED domain VL5
FATYYCQQNNEDPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQL (S35A)/kappa
KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK constant domain
DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 164 Humanized 52B8
DIQLTQSPSSLSASVGDRVTITCRASEKVDSFGNNFMHWYQQ LC variable
KPGKAPKLLIYLTSNLDSGVPSRFSGSGSGTDFTLTISSLQPED domain VL5
FATYYCQQNNEDPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQL (S35N)/kappa
KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK constant domain
DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 165 Humanized 52B8
DIQLTQSPSSLSASVGDRVTITCRASEKVDSFGQSFMHWYQQ LC variable
KPGKAPKLLIYLTSNLDSGVPSRFSGSGSGTDFTLTISSLQPED domain VL5
FATYYCQQNNEDPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQL (N34Q)/kappa
KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK constant domain
DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 166 Humanized 52B8
DIQLTQSPSSLSASVGDRVTITCRASEKVDSFGDSFMHWYQQ LC variable
KPGKAPKLLIYLTSNLDSGVPSRFSGSGSGTDFTLTISSLQPED domain VL5
FATYYCQQNNEDPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQL (N34D)/kappa
KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK constant domain
DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 167 Humanized 52B8
EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variable
GKGLEWVATISGGGDYTNYPDSMRGRFTISRDNAKNSLYLQ domain VH1/
MNSLRAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVSS Human IgG1 HC
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG (L234A L235A
ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS D265S) constant
NTKVDKKVEPKSCDKTHTCPPCPAPE GGPSVFLFPPKPKDT domain LMISRTPEVTCVVV
VSHEDPEVKFNWYVDGVEVHNAKTKPREE
QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA
KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK
168 Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC
variable GKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1
MNSLRAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVSS (M64V)/Human
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG IgG1 HC
ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS (L234A, L235A,
NTKVDKKVEPKSCDKTHTCPPCPAPE GGPSVFLFPPKPKDT D265S) constant
LMISRTPEVTCVVV VSHEDPEVKFNWYVDGVEVHNAKTKPREE domain
QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA
KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV
MHEALHNHYTQKSLSLSPGK
169 Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC
variable GKGLEWVATISGGGDYTNYPDSLRGRFTISRDNAKNSLYLQ domain VH1
MNSLRAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVSS (M64L)/Human
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG IgG1 HC
ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS (L234A, L235A,
NTKVDKKVEPKSCDKTHTCPPCPAPE GGPSVFLFPPKPKDT D265S) constant
LMISRTPEVTCVVV VSHEDPEVKFNWYVDGVEVHNAKTKPREE domain
QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA
KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK
170 Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC
variable GKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1
MNSLRAEDTAVYYCGRRLFFRSLYYAMDYWGQGTLVTVSS (M64V, W101F)/
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA Human IgG1 HC
LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN (L234A, L235A,
TKVDKKVEPKSCDKTHTCPPCPAPE GGPSVFLFPPKPKDTL D265S) constant
MISRTPEVTCVVV VSHEDPEVKFNWYVDGVEVHNAKTKPREEQ domain
YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESN
GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK
171 Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC
variable GKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1
MNSLRAEDTAVYYCGRRLYFRSLYYAMDYWGQGTLVTVSS (M64V, W101Y)/
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA Human IgG1 HC
LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN (L234A, L235A,
TKVDKKVEPKSCDKTHTCPPCPAPE GGPSVFLFPPKPKDTL D265S) constant
MISRTPEVTCVVV VSHEDPEVKFNWYVDGVEVHNAKTKPREEQ domain
YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESN
GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK
172 Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC
variable GKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1
MNSLRAEDTAVYYCGRRLQFRSLYYAMDYWGQGTLVTVSS (M64V, W101Q)/
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA Human IgG1 HC
LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN (L234A, L235A,
TKVDKKVEPKSCDKTHTCPPCPAPE GGPSVFLFPPKPKDTL D265S) constant
MISRTPEVTCVVV VSHEDPEVKFNWYVDGVEVHNAKTKPREEQ domain
YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESN
GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK
173 Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC
variable GKGLEWVATISGGGDYTNYPDSMRGRFTISRDNAKNSLYLQ domain VH2/
MNSLKAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVSS Human IgG1 HC
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG (L234A, L235A,
ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS D265S) constant
NTKVDKKVEPKSCDKTHTCPPCPAPE GGPSVFLFPPKPKDT domain LMISRTPEVTCVVV
VSHEDPEVKFNWYVDGVEVHNAKTKPREE
QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA
KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK
174 Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC
variable GKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH2
MNSLKAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVSS (M64V)/Human
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG IgG1 HC
ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS (L234A, L235A,
NTKVDKKVEPKSCDKTHTCPPCPAPE GGPSVFLFPPKPKDT D265S) constant
LMISRTPEVTCVVV VSHEDPEVKFNWYVDGVEVHNAKTKPREE domain
QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA
KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK
175 Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC
variable GKGLEWVATISGGGDYTNYPDSLRGRFTISRDNAKNSLYLQ domain VH2
MNSLKAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVSS (M64L)/Human
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG IgG1 HC
ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS (L234A, L235A,
NTKVDKKVEPKSCDKTHTCPPCPAPE GGPSVFLFPPKPKDT D265S) constant
LMISRTPEVTCVVV VSHEDPEVKFNWYVDGVEVHNAKTKPREE domain
QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA
KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK
176 Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC
variable GKGLEWVATISGGGDYTNYPDSMRGRFTISRDNAKNSLYLQ domain
MNSLRAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVSS VH1/Human IgG4
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSG (S228P) (K-)
ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS constant domain
NTKVDKRVESKYGPPCP CPAPEFLGGPSVFLFPPKPKDTLMIS
RTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQ
PREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ
PENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLG 177
Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC
variable GKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1
MNSLRAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVSS (M64V)/Human
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSG IgG4 (S228P) (K-)
ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS constant domain
NTKVDKRVESKYGPPCP CPAPEFLGGPSVFLFPPKPKDTLMIS
RTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQ
PREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ
PENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLG 178
Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC
variable GKGLEWVATISGGGDYTNYPDSLRGRFTISRDNAKNSLYLQ domain VH1
MNSLRAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVSS (M64L)/Human
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSG IgG4 (S228P) (K-)
ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS constant domain
NTKVDKRVESKYGPPCP CPAPEFLGGPSVFLFPPKPKDTLMIS
RTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQ
PREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ
PENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLG 179
Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC
variable GKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1
MNSLRAEDTAVYYCGRRLFFRSLYYAMDYWGQGTLVTVSS (M64V),
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA W101F/Human
LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSN IgG4 (S228P) (K-)
TKVDKRVESKYGPPCP CPAPEFLGGPSVFLFPPKPKDTLMISR constant domain
TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNS
TYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQP
REPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP
ENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEA LHNHYTQKSLSLSLG 180
Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC
variable GKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1
MNSLRAEDTAVYYCGRRLYFRSLYYAMDYWGQGTLVTVSS (M64V,
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA W101Y)/Human
LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSN IgG4 (S228P) (K-)
TKVDKRVESKYGPPCP CPAPEFLGGPSVFLFPPKPKDTLMISR constant domain
TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNS
TYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQP
REPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP
ENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEA LHNHYTQKSLSLSLG 181
Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC
variable GKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1
MNSLRAEDTAVYYCGRRLQFRSLYYAMDYWGQGTLVTVSS (M64V,
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA 101Q)/Human IgG4
LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSN (S228P) (K-)
TKVDKRVESKYGPPCP CPAPEFLGGPSVFLFPPKPKDTLMISR constant domain
TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNS
TYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQP
REPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP
ENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEA LHNHYTQKSLSLSLG 182
Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC
variable GKGLEWVATISGGGDYTNYPDSMRGRFTISRDNAKNSLYLQ domain
MNSLKAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVSS VH2/Human IgG4
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSG (S228P) (K-)
ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS constant domain
NTKVDKRVESKYGPPCP CPAPEFLGGPSVFLFPPKPKDTLMIS
RTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQ
PREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ
PENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLG 183
Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC
variable GKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH2
MNSLKAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVSS (M64V)/Human
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSG IgG4 (S228P) (K-)
ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS constant domain
NTKVDKRVESKYGPPCP CPAPEFLGGPSVFLFPPKPKDTLMIS
RTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQ
PREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ
PENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLG 184
Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC
variable GKGLEWVATISGGGDYTNYPDSLRGRFTISRDNAKNSLYLQ domain VH2
MNSLKAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVSS (M64L)/Human
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSG IgG4 (S228P) (K-)
ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS constant domain
NTKVDKRVESKYGPPCP CPAPEFLGGPSVFLFPPKPKDTLMIS
RTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQ
PREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ
PENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLG 185
Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC
variable GKGLEWVATISGGGDYTNYPDSMRGRFTISRDNAKNSLYLQ domain VH1/
MNSLRAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVSS Human IgG1 HC
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG (L234A, L235A,
ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS D265S) (K-)
NTKVDKKVEPKSCDKTHTCPPCPAPE GGPSVFLFPPKPKDT constant domain
LMISRTPEVTCVVV VSHEDPEVKFNWYVDGVEVHNAKTKPREE
QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA
KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPG
186 Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC
variable GKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1
MNSLRAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVSS (M64V)/Human
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG IgG1 HC
ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS (L234A, L235A,
NTKVDKKVEPKSCDKTHTCPPCPAPE GGPSVFLFPPKPKDT D265S) (K-)
LMISRTPEVTCVVV VSHEDPEVKFNWYVDGVEVHNAKTKPREE constant domain
QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA
KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPG
187 Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC
variable GKGLEWVATISGGGDYTNYPDSLRGRFTISRDNAKNSLYLQ domain VH1
MNSLRAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVSS (M64L)/Human
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG IgG1 HC
ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS (L234A, L235A,
NTKVDKKVEPKSCDKTHTCPPCPAPE GGPSVFLFPPKPKDT D265S) (K-)
LMISRTPEVTCVVV VSHEDPEVKFNWYVDGVEVHNAKTKPREE constant domain
QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA
KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPG
188 Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC
variable GKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1
MNSLRAEDTAVYYCGRRLFFRSLYYAMDYWGQGTLVTVSS (M64V, W101F)/
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA Human IgG1 HC
LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN (L234A, L235A,
TKVDKKVEPKSCDKTHTCPPCPAPE GGPSVFLFPPKPKDTL D265S) (K-)
MISRTPEVTCVVV VSHEDPEVKFNWYVDGVEVHNAKTKPREEQ constant domain
YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESN
GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPG 189
Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC
variable GKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1
MNSLRAEDTAVYYCGRRLYFRSLYYAMDYWGQGTLVTVSS (M64V, W101Y)/
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA Human IgG1 HC
LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN (L234A, L235A,
TKVDKKVEPKSCDKTHTCPPCPAPE GGPSVFLFPPKPKDTL D265S) (K-)
MISRTPEVTCVVV VSHEDPEVKFNWYVDGVEVHNAKTKPREEQ constant domain
YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESN
GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV
MHEALHNHYTQKSLSLSPG 190 Humanized 52B8
EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variable
GKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1
MNSLRAEDTAVYYCGRRLQFRSLYYAMDYWGQGTLVTVSS (M64V, W101Q)/
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA Human IgG1 HC
LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN (L234A, L235A,
TKVDKKVEPKSCDKTHTCPPCPAPE GGPSVFLFPPKPKDTL D265S) (K-)
MISRTPEVTCVVV VSHEDPEVKFNWYVDGVEVHNAKTKPREEQ constant domain
YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESN
GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPG 191
Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC
variable GKGLEWVATISGGGDYTNYPDSMRGRFTISRDNAKNSLYLQ domain VH2/
MNSLKAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVSS Human IgG1 HC
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG (L234A, L235A,
ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS D265S) (K-)
NTKVDKKVEPKSCDKTHTCPPCPAPE GGPSVFLFPPKPKDT constant domain
LMISRTPEVTCVVV VSHEDPEVKFNWYVDGVEVHNAKTKPREE
QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA
KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPG
192 Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC
variable GKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH2
MNSLKAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVSS M64V/Human
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG IgG1 HC
ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS (L234A, L235A,
NTKVDKKVEPKSCDKTHTCPPCPAPE GGPSVFLFPPKPKDT D265S) (K-)
LMISRTPEVTCVVV VSHEDPEVKFNWYVDGVEVHNAKTKPREE constant domain
QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA
KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPG
193 Humanized 52B8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC
variable GKGLEWVATISGGGDYTNYPDSLRGRFTISRDNAKNSLYLQ domain VH2
MNSLKAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVSS M64L/Human
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG IgG1 HC
ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS (L234A, L235A,
NTKVDKKVEPKSCDKTHTCPPCPAPE GGPSVFLFPPKPKDT D265S) (K-)
LMISRTPEVTCVVV VSHEDPEVKFNWYVDGVEVHNAKTKPREE constant domain
QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA
KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPG
194 Chimeric Anti- QVQLKESGPGLVQASETLSLTCTVSGFSLTSYSINWVRQSSG ILT3
rat 40A6 KGPEWMGRFWYDEGIAYNLTLESRLSISGDTSKNQVFLKMN parental HC
SLRTGDTGTYYCTRDRDTVGITGWFAYWGQGTLVTVSSAST variable
KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS domain/human
GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV IgG4 (S228P)
DKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISR constant domain
TPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST
YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP
REPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPE
NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK 195
Chimeric Anti- ETVMTQSPTSLSASIGERVTLNCKASQSVGVNVDWYQQTPG ILT3 rat
40A6 QSPKLLIYGSANRHTGVPDRFTGSGFGSDFTLTISDVEPEDLG parental LC
VYYCLQYGSVPYTFGAGTKLELKRTVAAPSVFIFPPSDEQLKS variable
GTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS domain/human
TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC kappa 196 Chimeric
Anti- QVQLKESGPGLVQASETLSLTCTVSGFSLTNYCVNWVRQPS ILT3 rat 16B1
GKGPEWLGRFWFDEGKAYNLTLESRLSISGDTSKNQVFLRM parental HC
NSLRADDTGTYYCTRDRDTVGITGWFAYWGQGTLVTVSSAS variable
TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT domain/human
SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK IgG4 (S228P)
VDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMIS constant domain
RTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS
TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP
REPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPE
NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK 197
Chimeric Anti- ETVMTQSPTSLSASIGERVTLNCKASQSVGINVDWYQQTPGQ ILT3 rat
16B1 SPKLLIYGSANRHTGVPDRFTGSGFGSDFTLTISNVEPEDLGV parental LC
YYCLQYGSVPYTFGPGTKLELKRTVAAPSVFIFPPSDEQLKSGT variable
ASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY domain/human
SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC kappa 198 Chimeric Anti-
QVQLQQSGAELMKPGASVKISCKATGYTFRTYWIEWVKQRP ILT3 mouse 11D1
GHGLEWIGEILPGNGNTHFNENFKDKATFTADTSSNAAYMQ parental HC
LSSLTSEDSAVYYCVRRLGRGPFDFWGQGTTLTVSSASTKGP variable
SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH domain/human
TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK IgG4 (S228P)
VEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPE constant domain
VTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV
VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP
QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK 199
Chimeric Anti- DIQMTQSPSSLSVSLGGKVTITCKASQDINEYIGWYQRKPGK ILT3
mouse 11D1 GPRLLIHYTSTLQSGIPSRFSGSGSGRDYSLSISNLEPEDIATYY parental
LC CLQYANPLPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTAS variable
VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL domain/human
SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC kappa 200 Chimeric Anti-
EVQLVESGGGLVQPGRSMKLSCAASGFTFSNFDMAWVRQA ILT3 rat 17H12
PTRGLEWVSSITYDGGSTSYRDSVKGRFTISRDNAKGTLYLQ parental HC
MDSLRSEDTATYYCTTVESIATISTYFDYWGQGVMVTVSSAS variable
TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT domain/human
SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK IgG4 (S228P)
VDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMIS constant domain
RTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS
TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP
REPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPE
NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK 201
Chimeric Anti- DIVLTQSPALAVSLGQRATISCRASQSVSMSRYDLIHWYQQK ILT3 rat
17H12 PGQQPKLLIFRASDLASGIPARFSGSGSGTDFTLTINPVQADDI parental LC
ATYYCQQTRKSPPTFGGGTRLELKRTVAAPSVFIFPPSDEQLKS variable
GTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS domain/human
TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC kappa 202 Chimeric
Anti- QVQLKESGPGLVQASETLSLTCTVSGFSLTSYCVNWVRQPSG ILT3 rat 37C8
KGPEWLGRFWYDEGKVYNLTLESRLSISGDTSKNQVFLKMN parental HC
RLRTDDTGTYYCTRDRDTMGITGWFAYWGQGTLVTVSSAST variable
KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS domain/human
GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV IgG4 (S228P)
DKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISR constant domain
TPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST
YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP
REPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPE
NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK 203
Chimeric Anti- ETVMTQSPTSLSASIGERVTLNCKASQSVGINVDWYQQTPGQ ILT3 rat
37C8 SPKLLIYGSANRHTGVPDRFTGSGFGSGFTLTISNVEPEDLGV parental LC
YYCLQYGSVPYTFGPGTKLELKRTVAAPSVFIFPPSDEQLKSGT variable
ASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY domain/human
SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC kappa 204 Chimeric Anti-
QVQMQQSGTELMKPGASMKISCKATGYTFSTYWIQWIKQRP ILT3 mouse 1G12
GHGLEWIGEILPGSGTTNYNENFKGKATFSADTSSNTAYIHLS parental HC
SLTSEDSAVFYCARRLGRGPFDYWGQGTTLTVSSASTKGPSV variable
FPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF domain/human
PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE IgG4 (S228P)
PKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVT constant domain
CVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT
TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK 205
Chimeric Anti- DIQMTQSPSSLSASLGGKVTITCEASQDINKHIDWYQHQPGR ILT3
mouse 1G12 GPSLLIHYASILQPGIPSRFSGSGSGRDYSFSITSLEPEDIATYY parental
LC CLQYDNLLPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTAS variable
VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL domain/human
SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC kappa 206 Chimeric Anti-
QVQLKESGPGLVQASETLSLTCTVSGFSLTSYSVNWVRQPSG ILT3 rat 20E4
KGLEWMGRFWYDGGTAYNSTLESRLSISGDTSKNQVFLKM parental HC
NSLQTDDTGTYYCTRDRDTMGITGWFAYWGQGTLVTVSPAS variable
TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT domain/human
SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK IgG4 (S228P)
VDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMIS constant domain
RTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS
TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP
REPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPE
NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK 207
Chimeric Anti- ETVMTQSPTSLSASIGERVTLNCKASQSVGVNVDWYQQTPG ILT3 rat
20E4 QSPKLLIYGSANRHTGVPDRFTGSGFGSDFTLTISNVEPEDLG parental LC
VYYCLQYGSVPYTFGAGTKLELKRTVAAPSVFIFPPSDEQLKS variable
GTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS domain/human
TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC kappa 208 Chimeric
Anti- QVQLKESGPGLVQASETLSLTCTVSGFSLTSYCVNWVRQPSG ILT3 rat 24A4
KGPEWLGRFWYDEGKVYNLTLESRLSISGDTSKNQVFLKMN parental HC
RLRTDDTGTYYCTRDRDTLGITGWFAYWGQGTLVTVSSAST variable
KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS domain/human
GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV IgG4 (S228P)
DKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISR constant domain
TPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST
YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP
REPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPE
NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK 209
Chimeric Anti- ETVMTQSPTSLSASIGERVTLNCKASQSVGINVDWYQQTPGQ ILT3 rat
24A4 SPKLLIYGSANRHTGVPDRFTGSGFGSGFTLTISNVEPEDLGV parental LC
YYCLQYGSVPYTFGPGTKLELKRTVAAPSVHFPPSDEQLKSGT variable
ASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY domain/human
SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC kappa 210 Humanized 52B8
EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAP HC variable
GKGLEWVATISGGGDYTNYPDSVRGRFTISRDNAKNSLYLQ domain VH1
MNSLRAEDTAVYYCGRRLWFRSLYYAMDYWGQGTLVTVSS (M64V)/Human
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG IgG1 HC
ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS (N297A) constant
NTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL domain
MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
QYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA
KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK
211 Human IgG1 HC ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA
constant domain LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN
(N297A; shown in TKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL
bold-face type) MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QY
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA
KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK
212 Chimeric anti-ILT3 QVQLKESGPGLVQASETLSLTCTVSGFSLTSYSINWVRQSSG
40A6 rat VH/ KGPEWMGRFWYDEGIAYNLTLESRLSISGDTSKNQVFLKMN human IgG1
SLRTGDTGTYYCTRDRDTVGITGWFAYWGQGTLVTVSSAST (N297A)
KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS
GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV
DKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR
TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYAS
TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP
REPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPE
NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK 213
Chimeric anti-ILT3 QVQLKESGPGLVQASETLSLTCTVSGFSLTNYCVNWVRQPS 16B1
rat VH/ GKGPEWLGRFWFDEGKAYNLTLESRLSISGDTSKNQVFLRM human IgG1
NSLRADDTGTYYCTRDRDTVGITGWFAYWGQGTLVTVSSAS (N297A)
TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT
SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS
RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYA
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ
PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQP
ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK 214
Chimeric anti-ILT3 QVQLQQSGAELMKPGASVKISCKATGYTFRTYWIEWVKQRP 11D1
mouse VH/ GHGLEWIGEILPGNGNTHFNENFKDKATFTADTSSNAAYMQ human IgG1
LSSLTSEDSAVYYCVRRLGRGPFDFWGQGTTLTVSSASTKGP (N297A)
SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH
TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK
VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE
VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYR
VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE
PQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN
NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK 215
Chimeric anti-ILT3 EVQLVESGGGLVQPGRSMKLSCAASGFTFSNFDMAWVRQA 17H12
rat VH/ PTRGLEWVSSITYDGGSTSYRDSVKGRFTISRDNAKGTLYLQ human IgG1
MDSLRSEDTATYYCTTVESIATISTYFDYWGQGVMVTVSSAS (N297A)
TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT
SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS
RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYA
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ
PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQP
ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK 216
Chimeric anti-ILT3 QVQLKESGPGLVQASETLSLTCTVSGFSLTSYCVNWVRQPSG 37C8
rat VH/ KGPEWLGRFWYDEGKVYNLTLESRLSISGDTSKNQVFLKMN human IgG1
RLRTDDTGTYYCTRDRDTMGITGWFAYWGQGTLVTVSSAST (N297A)
KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS
GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV
DKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR
TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYAS
TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP
REPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPE
NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK 217
Chimeric anti-ILT3 QVQMQQSGTELMKPGASMKISCKATGYTFSTYWIQWIKQRP 1G12
mouse VH/ GHGLEWIGEILPGSGTTNYNENFKGKATFSADTSSNTAYIHLS human IgG1
SLTSEDSAVFYCARRLGRGPFDYWGQGTTLTVSSASTKGPSV (N297A)
FPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE
PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT
CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVV
SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ
VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK
TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK 218
Chimeric anti-ILT3 QVQLKESGPGLVQASETLSLTCTVSGFSLTSYSVNWVRQPSG 20E4
rat VH/ KGLEWMGRFWYDGGTAYNSTLESRLSISGDTSKNQVFLKM human IgG1
NSLQTDDTGTYYCTRDRDTMGITGWFAYWGQGTLVTVSPAS (N297A)
TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT
SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS
RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYA
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ
PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQP
ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK 219
Chimeric anti-ILT3 QVQLKESGPGLVQASETLSLTCTVSGFSLTSYCVNWVRQPSG 24A4
rat VH/ KGPEWLGRFWYDEGKVYNLTLESRLSISGDTSKNQVFLKMN human IgG1
RLRTDDTGTYYCTRDRDTLGITGWFAYWGQGTLVTVSSAST (N297A)
KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS
GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV
DKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR
TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYAS
TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP
REPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPE
NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK 220
Chimeric anti-ILT3 QVQLKESGPGLVQASETLSLTCTVSGFSLTSYSINWVRQSSG 40A6
rat VH/ KGPEWMGRFWYDEGIAYNLTLESRLSISGDTSKNQVFLKMN human IgG1
SLRTGDTGTYYCTRDRDTVGITGWFAYWGQGTLVTVSSAST (N297A)
KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS
GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV
DKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR
TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYAS
TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP
REPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPE
NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK 221
Residues after LC- FGXG CDR3 Xaa is any amino acid 222 Residues
before CXXX HC-CDR1 Xaa is any amino acid 223 Residues before LEWIG
HC-CDR1 224 Residues after HC- WGXG CDR3 Xaa is any residue 225
Pembrolizumab QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQ Heavy Chain
APGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAY
MELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSS
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCN
VDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKP
KDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNA
KTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKG
LPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK
GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTV
DKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 226 Pembrolizumab
EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQ Light Chain
KPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPED
FAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQL
KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE
QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC 227 Human IgG1
HC ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA constant domain
LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN (N297A, D265A;
TKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL shown in bold-face
MISRTPEVTCVVV VSHEDPEVKFNWYVDGVEVHNAKTKPREE type) QY
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA
KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK
Constant regions are shown in italics. Amino acid sequences
underlined are CDRs.
[0299] While the present invention is described herein with
reference to illustrated embodiments, it should be understood that
the invention is not limited hereto. Those having ordinary skill in
the art and access to the teachings herein will recognize
additional modifications and embodiments within the scope thereof.
Therefore, the present invention is limited only by the claims
attached herein.
Sequence CWU 1
1
2271246PRTArtificial SequenceHuman ILT3 (LILRB4) extracellular
domain with C-terminal His Tag 1Gln Ala Gly Pro Leu Pro Lys Pro Thr
Leu Trp Ala Glu Pro Gly Ser1 5 10 15Val Ile Ser Trp Gly Asn Ser Val
Thr Ile Trp Cys Gln Gly Thr Leu 20 25 30Glu Ala Arg Glu Tyr Arg Leu
Asp Lys Glu Glu Ser Pro Ala Pro Trp 35 40 45Asp Arg Gln Asn Pro Leu
Glu Pro Lys Asn Lys Ala Arg Phe Ser Ile 50 55 60Pro Ser Met Thr Glu
Asp Tyr Ala Gly Arg Tyr Arg Cys Tyr Tyr Arg65 70 75 80Ser Pro Val
Gly Trp Ser Gln Pro Ser Asp Pro Leu Glu Leu Val Met 85 90 95Thr Gly
Ala Tyr Ser Lys Pro Thr Leu Ser Ala Leu Pro Ser Pro Leu 100 105
110Val Thr Ser Gly Lys Ser Val Thr Leu Leu Cys Gln Ser Arg Ser Pro
115 120 125Met Asp Thr Phe Leu Leu Ile Lys Glu Arg Ala Ala His Pro
Leu Leu 130 135 140His Leu Arg Ser Glu His Gly Ala Gln Gln His Gln
Ala Glu Phe Pro145 150 155 160Met Ser Pro Val Thr Ser Val His Gly
Gly Thr Tyr Arg Cys Phe Ser 165 170 175Ser His Gly Phe Ser His Tyr
Leu Leu Ser His Pro Ser Asp Pro Leu 180 185 190Glu Leu Ile Val Ser
Gly Ser Leu Glu Asp Pro Arg Pro Ser Pro Thr 195 200 205Arg Ser Val
Ser Thr Ala Ala Gly Pro Glu Asp Gln Pro Leu Met Pro 210 215 220Thr
Gly Ser Val Pro His Ser Gly Leu Arg Arg His Trp Glu His His225 230
235 240His His His His His His 2452237PRTArtificial SequenceMacaca
mulatta (Rhesus) ILT3 (LILRB4) extracellular domain (sequence
obtained from GenBank NP_001035766) 2Gln Ala Gly Pro Leu Pro Lys
Pro Thr Ile Trp Ala Glu Pro Gly Ser1 5 10 15Val Ile Ser Trp Gly Ser
Pro Val Thr Ile Trp Cys Gln Gly Thr Leu 20 25 30Asp Ala Gln Glu Tyr
Tyr Leu Asp Lys Glu Gly Ser Pro Ala Pro Trp 35 40 45Asp Thr Gln Asn
Pro Leu Glu Pro Arg Asn Lys Ala Lys Phe Ser Ile 50 55 60Pro Ser Met
Thr Gln His Tyr Ala Gly Arg Tyr Arg Cys Tyr Tyr His65 70 75 80Ser
His Pro Asp Trp Ser Glu Asp Ser Asp Pro Leu Asp Leu Val Met 85 90
95Thr Gly Ala Tyr Ser Lys Pro Ile Leu Ser Val Leu Pro Ser Pro Leu
100 105 110Val Thr Ser Gly Glu Ser Val Thr Leu Leu Cys Gln Ser Gln
Ser Pro 115 120 125Met Asp Thr Phe Leu Leu Phe Lys Glu Gly Ala Ala
His Pro Leu Pro 130 135 140Arg Leu Arg Ser Gln His Gly Ala Gln Leu
His Trp Ala Glu Phe Pro145 150 155 160Met Gly Pro Val Thr Ser Val
His Gly Gly Thr Tyr Arg Cys Ile Ser 165 170 175Ser Arg Ser Phe Ser
His Tyr Leu Leu Ser Arg Pro Ser Asp Pro Val 180 185 190Glu Leu Thr
Val Leu Gly Ser Leu Glu Ser Pro Ser Pro Ser Pro Thr 195 200 205Arg
Ser Ile Ser Ala Ala Gly Pro Glu Asp Gln Ser Leu Met Pro Thr 210 215
220Gly Ser Asp Pro Gln Ser Gly Leu Arg Arg His Trp Glu225 230
23536PRTArtificial SequenceHuman ILT3 peptide A 3Ile Ser Trp Gly
Asn Ser1 546PRTArtificial SequenceHuman ILT3 peptide B 4Ile Pro Ser
Met Thr Glu1 556PRTArtificial SequenceHuman ILT3 peptide C 5Met Thr
Gly Ala Tyr Ser1 568PRTArtificial SequenceHuman ILT3 peptide D 6Gln
Ser Arg Ser Pro Met Asp Thr1 578PRTArtificial SequenceHuman ILT3
peptide E 7Ala Gln Gln His Gln Ala Glu Phe1 584PRTArtificial
SequenceHuman ILT3 peptide F 8Leu Leu Ser His19327PRTArtificial
SequenceHuman IgG4 HC Constant domain (S228P) 9Ala Ser Thr Lys Gly
Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg1 5 10 15Ser Thr Ser Glu
Ser 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 Lys Thr65 70 75
80Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala
Pro 100 105 110Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys 115 120 125Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val 130 135 140Asp Val Ser Gln Glu Asp Pro Glu Val
Gln Phe Asn Trp Tyr Val Asp145 150 155 160Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200
205Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
210 215 220Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met
Thr Lys225 230 235 240Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp 245 250 255Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys 260 265 270Thr Thr Pro Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285Arg Leu Thr Val Asp
Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300Cys Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser305 310 315
320Leu Ser Leu Ser Leu Gly Lys 32510326PRTArtificial SequenceHuman
IgG4 HC Constant domain (S228P)(K-) 10Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro Cys Ser Arg1 5 10 15Ser Thr Ser Glu Ser 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 Lys Thr65 70 75 80Tyr
Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90
95Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro
100 105 110Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys 115 120 125Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val 130 135 140Asp Val Ser Gln Glu Asp Pro Glu Val Gln
Phe Asn Trp Tyr Val Asp145 150 155 160Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175Asn Ser Thr Tyr Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205Pro
Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215
220Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr
Lys225 230 235 240Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp 245 250 255Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys 260 265 270Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285Arg Leu Thr Val Asp Lys
Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300Cys Ser Val Met
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser305 310 315 320Leu
Ser Leu Ser Leu Gly 32511330PRTArtificial SequenceHuman IgG1 HC
constant domain 11Ala 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 95Lys 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 Asp Glu225 230 235 240Leu
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 33012330PRTArtificial SequenceHuman IgG1 HC
Constant domain (L234A L235A D265S) 12Ala 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
95Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110Pro Ala Pro Glu Ala Ala 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 Ser 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 Asp
Glu225 230 235 240Leu 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 33013329PRTArtificial
SequenceHuman IgG1 HC Constant domain (K-) (L234A L235A D265S)
13Ala 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 95Lys Val Glu Pro Lys Ser Cys Asp Lys
Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Ala Ala 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
Ser 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 Asp Glu225 230 235 240Leu 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
32514107PRTArtificial SequenceHuman LC Kappa Constant domain 14Arg
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 10515122PRTArtificial SequenceAnti-ILT3 52B8 parental HC
variable domain 15Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val
Lys Pro Gly Gly1 5 10 15Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Ser Asn Tyr 20 25 30Gly Met Ser Trp Val Arg Gln Thr Pro Asp
Arg Arg Leu Glu Trp Val 35 40 45Ala Thr Ile Ser Gly Gly Gly Asp Tyr
Thr Asn Tyr Pro Asp Ser Met 50 55 60Arg Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ala Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95Gly Arg Arg Leu Trp Phe Arg Ser Leu Tyr Tyr Ala Met Asp Tyr
Trp 100 105 110Gly Gln Gly Thr Ser Val Thr Val Ser Ser 115
12016111PRTArtificial SequenceAnti-ILT3 52B8 parental LC variable
domain 16Asn Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser
Leu Gly1 5 10 15Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Lys Val
Asp Ser Phe 20 25 30Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro
Gly Gln Pro Pro 35 40 45Lys Leu Leu Ile Tyr Leu Thr Ser Asn Leu Asp
Ser Gly Val Pro Ala 50 55 60Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp
Phe Ala Leu Thr Ile Asp65 70 75 80Pro Val Glu Ala Asp Asp Ala Ala
Thr Tyr Tyr Cys Gln Gln Asn Asn 85 90 95Glu Asp Pro Tyr Thr Phe Gly
Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110175PRTArtificial
Sequence52B8 HC-CDR1 17Asn Tyr Gly Met Ser1 51817PRTArtificial
Sequence52B8 HC-CDR2VARIANT(15)..(15)Xaa is M, V, or L 18Thr Ile
Ser Gly Gly Gly Asp Tyr Thr Asn Tyr Pro Asp Ser Xaa Arg1 5 10
15Gly1917PRTArtificial Sequence52B8 HC-CDR2 M 19Thr Ile Ser Gly Gly
Gly Asp Tyr Thr Asn Tyr Pro Asp Ser Met Arg1 5 10
15Gly2017PRTArtificial Sequence52B8 HC-CDR2 V 20Thr Ile Ser Gly Gly
Gly Asp Tyr Thr Asn Tyr Pro Asp Ser Val Arg1 5 10
15Gly2117PRTArtificial Sequence52B8 HC-CDR2 L 21Thr Ile Ser Gly Gly
Gly Asp Tyr Thr Asn Tyr Pro Asp Ser Leu Arg1 5 10
15Gly2213PRTArtificial Sequence52B8 HC-CDR3VARIANT(3)..(3)Xaa3 is
W, Y, Q, or F 22Arg Leu Xaa Phe Arg Ser Leu Tyr Tyr Ala Met Asp
Tyr1 5 102313PRTArtificial Sequence52B8 HC-CDR3 23Arg Leu Trp Phe
Arg Ser Leu Tyr Tyr Ala Met Asp Tyr1 5 102413PRTArtificial
Sequence52B8 HC-CDR3 24Arg Leu Tyr Phe Arg Ser Leu Tyr Tyr Ala Met
Asp Tyr1 5 102513PRTArtificial Sequence52B8 HC-CDR3 25Arg Leu Gln
Phe Arg Ser Leu Tyr Tyr Ala Met Asp Tyr1 5 102613PRTArtificial
Sequence52B8 HC-CDR3 26Arg Leu Phe Phe Arg Ser Leu Tyr Tyr Ala Met
Asp Tyr1 5 102715PRTArtificial Sequence52B8
LC-CDR1VARIANT(11)..(11)Xaa is N, D, or QVARIANT(12)..(12)Xaa is S,
N, or A 27Arg Ala Ser Glu Lys Val Asp Ser Phe Gly Xaa Xaa Phe Met
His1 5 10 152815PRTArtificial Sequence52B8 LC-CDR1
NVARIANT(12)..(12)Xaa is S, N, or A 28Arg Ala Ser Glu Lys Val Asp
Ser Phe Gly Asn Xaa Phe Met His1 5 10 152915PRTArtificial
Sequence52B8 LC-CDR1 DVARIANT(12)..(12)Xaa is S, N, or A 29Arg Ala
Ser Glu Lys Val Asp Ser Phe Gly Asp Xaa Phe Met His1 5 10
153015PRTArtificial Sequence52B8 LC-CDR1 QVARIANT(12)..(12)Xaa is
S, N, or A 30Arg Ala Ser Glu Lys Val Asp Ser Phe Gly Gln Xaa Phe
Met His1 5 10 153115PRTArtificial Sequence52B8 LC-CDR1
SVARIANT(11)..(11)Xaa is N, D, or Q 31Arg Ala Ser Glu Lys Val Asp
Ser Phe Gly Xaa Ser Phe Met His1 5 10 153215PRTArtificial
Sequence52B8 LC-CDR1 NVARIANT(11)..(11)Xaa is N, D, or Q 32Arg Ala
Ser Glu Lys Val Asp Ser Phe Gly Xaa Asn Phe Met His1 5 10
153315PRTArtificial Sequence52B8 LC-CDR1 AVARIANT(11)..(11)Xaa is
N, D, or Q 33Arg Ala Ser Glu Lys Val Asp Ser Phe Gly Xaa Ala Phe
Met His1 5 10 153415PRTArtificial Sequence52B8 LC-CDR1 NN 34Arg Ala
Ser Glu Lys Val Asp Ser Phe Gly Asn Asn Phe Met His1 5 10
153515PRTArtificial Sequence52B8 LC-CDR1 DN 35Arg Ala Ser Glu Lys
Val Asp Ser Phe Gly Asp Asn Phe Met His1 5 10 153615PRTArtificial
Sequence52B8 LC-CDR1 QN 36Arg Ala Ser Glu Lys Val Asp Ser Phe Gly
Gln Asn Phe Met His1 5 10 153715PRTArtificial Sequence52B8 LC-CDR1
NS 37Arg Ala Ser Glu Lys Val Asp Ser Phe Gly Asn Ser Phe Met His1 5
10 153815PRTArtificial Sequence52B8 LC-CDR1 DS 38Arg Ala Ser Glu
Lys Val Asp Ser Phe Gly Asp Ser Phe Met His1 5 10
153915PRTArtificial Sequence52B8 LC-CDR1 NA 39Arg Ala Ser Glu Lys
Val Asp Ser Phe Gly Asn Ala Phe Met His1 5 10 154015PRTArtificial
Sequence52B8 LC-CDR1 DA 40Arg Ala Ser Glu Lys Val Asp Ser Phe Gly
Asp Ala Phe Met His1 5 10 154115PRTArtificial Sequence52B8 LC-CDR1
QS 41Arg Ala Ser Glu Lys Val Asp Ser Phe Gly Gln Ser Phe Met His1 5
10 154215PRTArtificial Sequence52B8 LC-CDR1 AF 42Arg Ala Ser Glu
Lys Val Asp Ser Phe Gly Gln Ala Phe Met His1 5 10
15437PRTArtificial Sequence52B8 LC-CDR2 43Leu Thr Ser Asn Leu Asp
Ser1 5449PRTArtificial Sequence52B8 LC-CDR3 44Gln Gln Asn Asn Glu
Asp Pro Tyr Thr1 545121PRTArtificial SequenceAnti-ILT3 40A6
parental HC variable domain 45Gln Val Gln Leu Lys Glu Ser Gly Pro
Gly Leu Val Gln Ala Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val
Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30Ser Ile Asn Trp Val Arg Gln
Ser Ser Gly Lys Gly Pro Glu Trp Met 35 40 45Gly Arg Phe Trp Tyr Asp
Glu Gly Ile Ala Tyr Asn Leu Thr Leu Glu 50 55 60Ser Arg Leu Ser Ile
Ser Gly Asp Thr Ser Lys Asn Gln Val Phe Leu65 70 75 80Lys Met Asn
Ser Leu Arg Thr Gly Asp Thr Gly Thr Tyr Tyr Cys Thr 85 90 95Arg Asp
Arg Asp Thr Val Gly Ile Thr Gly Trp Phe Ala Tyr Trp Gly 100 105
110Gln Gly Thr Leu Val Thr Val Ser Ser 115 12046107PRTArtificial
SequenceAnti-ILT3 40A6 parental LC variable domain 46Glu Thr Val
Met Thr Gln Ser Pro Thr Ser Leu Ser Ala Ser Ile Gly1 5 10 15Glu Arg
Val Thr Leu Asn Cys Lys Ala Ser Gln Ser Val Gly Val Asn 20 25 30Val
Asp Trp Tyr Gln Gln Thr Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40
45Tyr Gly Ser Ala Asn Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly
50 55 60Ser Gly Phe Gly Ser Asp Phe Thr Leu Thr Ile Ser Asp Val Glu
Pro65 70 75 80Glu Asp Leu Gly Val Tyr Tyr Cys Leu Gln Tyr Gly Ser
Val Pro Tyr 85 90 95Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100
105475PRTArtificial Sequence40A6 HC-CDR1 47Ser Tyr Ser Ile Asn1
54816PRTArtificial Sequence40A6 HC-CDR2 48Arg Phe Trp Tyr Asp Glu
Gly Ile Ala Tyr Asn Leu Thr Leu Glu Ser1 5 10 154913PRTArtificial
Sequence40A6 HC-CDR3 49Asp Arg Asp Thr Val Gly Ile Thr Gly Trp Phe
Ala Tyr1 5 105011PRTArtificial Sequence40A6 LC-CDR1 50Lys Ala Ser
Gln Ser Val Gly Val Asn Val Asp1 5 10517PRTArtificial Sequence40A6
LC-CDR2 51Gly Ser Ala Asn Arg His Thr1 5529PRTArtificial
Sequence40A6 LC-CDR3 52Leu Gln Tyr Gly Ser Val Pro Tyr Thr1
553121PRTArtificial SequenceAnti-ILT3 16B1 parental HC variable
domain 53Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Gln Ala
Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu
Thr Asn Tyr 20 25 30Cys Val Asn Trp Val Arg Gln Pro Ser Gly Lys Gly
Pro Glu Trp Leu 35 40 45Gly Arg Phe Trp Phe Asp Glu Gly Lys Ala Tyr
Asn Leu Thr Leu Glu 50 55 60Ser Arg Leu Ser Ile Ser Gly Asp Thr Ser
Lys Asn Gln Val Phe Leu65 70 75 80Arg Met Asn Ser Leu Arg Ala Asp
Asp Thr Gly Thr Tyr Tyr Cys Thr 85 90 95Arg Asp Arg Asp Thr Val Gly
Ile Thr Gly Trp Phe Ala Tyr Trp Gly 100 105 110Gln Gly Thr Leu Val
Thr Val Ser Ser 115 12054107PRTArtificial SequenceAnti-ILT3 16B1
parental LC variable domain 54Glu Thr Val Met Thr Gln Ser Pro Thr
Ser Leu Ser Ala Ser Ile Gly1 5 10 15Glu Arg Val Thr Leu Asn Cys Lys
Ala Ser Gln Ser Val Gly Ile Asn 20 25 30Val Asp Trp Tyr Gln Gln Thr
Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45Tyr Gly Ser Ala Asn Arg
His Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60Ser Gly Phe Gly Ser
Asp Phe Thr Leu Thr Ile Ser Asn Val Glu Pro65 70 75 80Glu Asp Leu
Gly Val Tyr Tyr Cys Leu Gln Tyr Gly Ser Val Pro Tyr 85 90 95Thr Phe
Gly Pro Gly Thr Lys Leu Glu Leu Lys 100 105555PRTArtificial
Sequence16B1 HC-CDR1 55Asn Tyr Cys Val Asn1 55616PRTArtificial
Sequence16B1 HC-CDR2 56Arg Phe Trp Phe Asp Glu Gly Lys Ala Tyr Asn
Leu Thr Leu Glu Ser1 5 10 155713PRTArtificial Sequence16B1 HC-CDR3
57Asp Arg Asp Thr Val Gly Ile Thr Gly Trp Phe Ala Tyr1 5
105811PRTArtificial Sequence16B1 LC-CDR1 58Lys Ala Ser Gln Ser Val
Gly Ile Asn Val Asp1 5 10597PRTArtificial Sequence16B1 LC-CDR2
59Gly Ser Ala Asn Arg His Thr1 5609PRTArtificial Sequence16B1
LC-CDR3 60Leu Gln Tyr Gly Ser Val Pro Tyr Thr1 561118PRTArtificial
SequenceAnti-ILT3 11D1 parental HC variable domain 61Gln Val Gln
Leu Gln Gln Ser Gly Ala Glu Leu Met Lys Pro Gly Ala1 5 10 15Ser Val
Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe Arg Thr Tyr 20 25 30Trp
Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile 35 40
45Gly Glu Ile Leu Pro Gly Asn Gly Asn Thr His Phe Asn Glu Asn Phe
50 55 60Lys Asp Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser Asn Ala Ala
Tyr65 70 75 80Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val
Tyr Tyr Cys 85 90 95Val Arg Arg Leu Gly Arg Gly Pro Phe Asp Phe Trp
Gly Gln Gly Thr 100 105 110Thr Leu Thr Val Ser Ser
11562107PRTArtificial SequenceAnti-ILT3 11D1 parental LC variable
domain 62Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Val Ser
Leu Gly1 5 10 15Gly Lys Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile
Asn Glu Tyr 20 25 30Ile Gly Trp Tyr Gln Arg Lys Pro Gly Lys Gly Pro
Arg Leu Leu Ile 35 40 45His Tyr Thr Ser Thr Leu Gln Ser Gly Ile Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Arg Asp Tyr Ser Leu Ser
Ile Ser Asn Leu Glu Pro65 70 75 80Glu Asp Ile Ala Thr Tyr Tyr Cys
Leu Gln Tyr Ala Asn Pro Leu Pro 85 90 95Thr Phe Gly Gly Gly Thr Lys
Leu Glu Ile Lys 100 105635PRTArtificial Sequence11D1 HC-CDR1 63Thr
Tyr Trp Ile Glu1 56417PRTArtificial Sequence11D1 HC-CDR2 64Glu Ile
Leu Pro Gly Asn Gly Asn Thr His Phe Asn Glu Asn Phe Lys1 5 10
15Asp6510PRTArtificial Sequence11D1 HC-CDR3 65Arg Arg Leu Gly Arg
Gly Pro Phe Asp Phe1 5 106611PRTArtificial Sequence11D1 LC-CDR1
66Lys Ala Ser Gln Asp Ile Asn Glu Tyr Ile Gly1 5 10677PRTArtificial
Sequence11D1 LC-CDR2 67Tyr Thr Ser Thr Leu Gln Ser1
5689PRTArtificial Sequence11D1 LC-CDR3 68Leu Gln Tyr Ala Asn Pro
Leu Pro Thr1 569122PRTArtificial SequenceAnti-ILT3 17H12 parental
HC variable domain 69Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Arg1 5 10 15Ser Met Lys Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Asn Phe 20 25 30Asp Met Ala Trp Val Arg Gln Ala Pro
Thr Arg Gly Leu Glu Trp Val 35 40 45Ser Ser Ile Thr Tyr Asp Gly Gly
Ser Thr Ser Tyr Arg Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Gly Thr Leu Tyr65 70 75 80Leu Gln Met Asp Ser
Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95Thr Thr Val Glu
Ser Ile Ala Thr Ile Ser Thr Tyr Phe Asp Tyr Trp 100 105 110Gly Gln
Gly Val Met Val Thr Val Ser Ser 115 12070110PRTArtificial
SequenceAnti-ILT3 17H12 parental LC variable domain 70Asp Ile Val
Leu Thr Gln Ser Pro Ala Leu Ala Val Ser Leu Gly Gln1 5 10 15Arg Ala
Thr Ile Ser Cys Arg Ala Ser Gln Ser Val Ser Met Ser Arg 20 25 30Tyr
Asp Leu Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Gln Pro Lys 35 40
45Leu Leu Ile Phe Arg Ala Ser Asp Leu Ala Ser Gly Ile Pro Ala Arg
50 55 60Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn
Pro65 70 75 80Val Gln Ala Asp Asp Ile Ala Thr Tyr Tyr Cys Gln Gln
Thr Arg Lys 85 90 95Ser Pro Pro Thr Phe Gly Gly Gly Thr Arg Leu Glu
Leu Lys 100 105 110715PRTArtificial Sequence17H12 HC-CDR1 71Asn Phe
Asp Met Ala1 57217PRTArtificial Sequence17H12 HC-CDR2 72Ser Ile Thr
Tyr Asp Gly Gly Ser Thr Ser Tyr Arg Asp Ser Val Lys1 5 10
15Gly7313PRTArtificial Sequence17H12 HC-CDR3 73Val Glu Ser Ile Ala
Thr Ile Ser Thr Tyr Phe Asp Tyr1 5 107415PRTArtificial
Sequence17H12 LC-CDR1 74Arg Ala Ser Gln Ser Val Ser Met Ser Arg Tyr
Asp Leu Ile His1 5 10 15757PRTArtificial Sequence17H12 LC-CDR2
75Arg Ala Ser Asp Leu Ala Ser1 5769PRTArtificial Sequence17H12
LC-CDR3 76Gln Gln Thr Arg Lys Ser Pro Pro Thr1 577121PRTArtificial
SequenceAnti-ILT3 37C8 parental HC variable domain 77Gln Val Gln
Leu Lys Glu Ser Gly Pro Gly Leu Val Gln Ala Ser Glu1 5 10 15Thr Leu
Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30Cys
Val Asn Trp Val Arg Gln Pro Ser Gly Lys Gly Pro Glu Trp Leu 35 40
45Gly Arg Phe Trp Tyr Asp Glu Gly Lys Val Tyr Asn Leu Thr Leu Glu
50 55 60Ser Arg Leu Ser Ile Ser Gly Asp Thr Ser Lys Asn Gln Val Phe
Leu65 70 75 80Lys Met Asn Arg Leu Arg Thr Asp Asp Thr Gly Thr Tyr
Tyr Cys Thr 85 90 95Arg Asp Arg Asp Thr Met Gly Ile Thr Gly Trp Phe
Ala Tyr Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser 115
12078107PRTArtificial SequenceAnti-ILT3 37C8 parental LC variable
domain 78Glu Thr Val Met Thr Gln Ser Pro Thr Ser Leu Ser Ala Ser
Ile Gly1 5 10 15Glu Arg Val Thr Leu Asn Cys Lys Ala Ser Gln Ser Val
Gly Ile Asn 20 25 30Val Asp Trp Tyr Gln Gln Thr Pro Gly Gln Ser Pro
Lys Leu Leu Ile 35 40 45Tyr Gly Ser Ala Asn Arg His Thr Gly Val Pro
Asp Arg Phe Thr Gly 50 55 60Ser Gly Phe Gly Ser Gly Phe Thr Leu Thr
Ile Ser Asn Val Glu Pro65 70 75 80Glu Asp Leu Gly Val Tyr Tyr Cys
Leu Gln Tyr Gly Ser Val Pro Tyr 85 90 95Thr Phe Gly Pro Gly Thr Lys
Leu Glu Leu Lys 100 105795PRTArtificial Sequence37C8 HC-CDR1 79Ser
Tyr Cys Val Asn1 58016PRTArtificial Sequence37C8 HC-CDR2 80Arg Phe
Trp Tyr Asp Glu Gly Lys Val Tyr Asn Leu Thr Leu Glu Ser1 5 10
158113PRTArtificial Sequence37C8 HC-CDR3 81Asp Arg Asp Thr Met Gly
Ile Thr Gly Trp Phe Ala Tyr1 5 108211PRTArtificial Sequence37C8
LC-CDR1 82Lys Ala Ser Gln Ser Val Gly Ile Asn Val Asp1 5
10837PRTArtificial Sequence37C8 LC-CDR2 83Gly Ser Ala Asn Arg His
Thr1
5849PRTArtificial Sequence37C8 LC-CDR3 84Leu Gln Tyr Gly Ser Val
Pro Tyr Thr1 585118PRTArtificial SequenceAnti-ILT3 1G12 parental HC
variable domain 85Gln Val Gln Met Gln Gln Ser Gly Thr Glu Leu Met
Lys Pro Gly Ala1 5 10 15Ser Met Lys Ile Ser Cys Lys Ala Thr Gly Tyr
Thr Phe Ser Thr Tyr 20 25 30Trp Ile Gln Trp Ile Lys Gln Arg Pro Gly
His Gly Leu Glu Trp Ile 35 40 45Gly Glu Ile Leu Pro Gly Ser Gly Thr
Thr Asn Tyr Asn Glu Asn Phe 50 55 60Lys Gly Lys Ala Thr Phe Ser Ala
Asp Thr Ser Ser Asn Thr Ala Tyr65 70 75 80Ile His Leu Ser Ser Leu
Thr Ser Glu Asp Ser Ala Val Phe Tyr Cys 85 90 95Ala Arg Arg Leu Gly
Arg Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110Thr Leu Thr
Val Ser Ser 11586107PRTArtificial SequenceAnti-ILT3 1G12 parental
LC variable domain 86Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Leu Gly1 5 10 15Gly Lys Val Thr Ile Thr Cys Glu Ala Ser
Gln Asp Ile Asn Lys His 20 25 30Ile Asp Trp Tyr Gln His Gln Pro Gly
Arg Gly Pro Ser Leu Leu Ile 35 40 45His Tyr Ala Ser Ile Leu Gln Pro
Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Arg Asp Tyr
Ser Phe Ser Ile Thr Ser Leu Glu Pro65 70 75 80Glu Asp Ile Ala Thr
Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Leu Pro 85 90 95Thr Phe Gly Gly
Gly Thr Lys Leu Glu Ile Lys 100 105875PRTArtificial Sequence1G12
HC-CDR1 87Thr Tyr Trp Ile Gln1 58817PRTArtificial Sequence1G12
HC-CDR2 88Glu Ile Leu Pro Gly Ser Gly Thr Thr Asn Tyr Asn Glu Asn
Phe Lys1 5 10 15Gly899PRTArtificial Sequence1G12 HC-CDR3 89Arg Leu
Gly Arg Gly Pro Phe Asp Tyr1 59011PRTArtificial Sequence1G12
LC-CDR1 90Glu Ala Ser Gln Asp Ile Asn Lys His Ile Asp1 5
10917PRTArtificial Sequence1G12 LC-CDR2 91Tyr Ala Ser Ile Leu Gln
Pro1 5929PRTArtificial Sequence1G12 LC-CDR3 92Leu Gln Tyr Asp Asn
Leu Leu Pro Thr1 593121PRTArtificial SequenceAnti-ILT3 20E4
parental HC variable domain 93Gln Val Gln Leu Lys Glu Ser Gly Pro
Gly Leu Val Gln Ala Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val
Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30Ser Val Asn Trp Val Arg Gln
Pro Ser Gly Lys Gly Leu Glu Trp Met 35 40 45Gly Arg Phe Trp Tyr Asp
Gly Gly Thr Ala Tyr Asn Ser Thr Leu Glu 50 55 60Ser Arg Leu Ser Ile
Ser Gly Asp Thr Ser Lys Asn Gln Val Phe Leu65 70 75 80Lys Met Asn
Ser Leu Gln Thr Asp Asp Thr Gly Thr Tyr Tyr Cys Thr 85 90 95Arg Asp
Arg Asp Thr Met Gly Ile Thr Gly Trp Phe Ala Tyr Trp Gly 100 105
110Gln Gly Thr Leu Val Thr Val Ser Pro 115 12094107PRTArtificial
SequenceAnti-ILT3 20E4 parental LC variable domain 94Glu Thr Val
Met Thr Gln Ser Pro Thr Ser Leu Ser Ala Ser Ile Gly1 5 10 15Glu Arg
Val Thr Leu Asn Cys Lys Ala Ser Gln Ser Val Gly Val Asn 20 25 30Val
Asp Trp Tyr Gln Gln Thr Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40
45Tyr Gly Ser Ala Asn Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly
50 55 60Ser Gly Phe Gly Ser Asp Phe Thr Leu Thr Ile Ser Asn Val Glu
Pro65 70 75 80Glu Asp Leu Gly Val Tyr Tyr Cys Leu Gln Tyr Gly Ser
Val Pro Tyr 85 90 95Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100
105955PRTArtificial Sequence20E4 95Ser Tyr Ser Val Asn1
59616PRTArtificial Sequence20E4 96Arg Phe Trp Tyr Asp Gly Gly Thr
Ala Tyr Asn Ser Thr Leu Glu Ser1 5 10 159713PRTArtificial
Sequence20E4 97Asp Arg Asp Thr Met Gly Ile Thr Gly Trp Phe Ala Tyr1
5 109811PRTArtificial Sequence20E4 98Lys Ala Ser Gln Ser Val Gly
Val Asn Val Asp1 5 10997PRTArtificial Sequence20E4 99Gly Ser Ala
Asn Arg His Thr1 51009PRTArtificial Sequence20E4 100Leu Gln Tyr Gly
Ser Val Pro Tyr Thr1 5101121PRTArtificial SequenceAnti-ILT3 24A4
parental HC variable domain 101Gln Val Gln Leu Lys Glu Ser Gly Pro
Gly Leu Val Gln Ala Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val
Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30Cys Val Asn Trp Val Arg Gln
Pro Ser Gly Lys Gly Pro Glu Trp Leu 35 40 45Gly Arg Phe Trp Tyr Asp
Glu Gly Lys Val Tyr Asn Leu Thr Leu Glu 50 55 60Ser Arg Leu Ser Ile
Ser Gly Asp Thr Ser Lys Asn Gln Val Phe Leu65 70 75 80Lys Met Asn
Arg Leu Arg Thr Asp Asp Thr Gly Thr Tyr Tyr Cys Thr 85 90 95Arg Asp
Arg Asp Thr Leu Gly Ile Thr Gly Trp Phe Ala Tyr Trp Gly 100 105
110Gln Gly Thr Leu Val Thr Val Ser Ser 115 120102107PRTArtificial
SequenceAnti-ILT3 24A4 parental LC variable domain 102Glu Thr Val
Met Thr Gln Ser Pro Thr Ser Leu Ser Ala Ser Ile Gly1 5 10 15Glu Arg
Val Thr Leu Asn Cys Lys Ala Ser Gln Ser Val Gly Ile Asn 20 25 30Val
Asp Trp Tyr Gln Gln Thr Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40
45Tyr Gly Ser Ala Asn Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly
50 55 60Ser Gly Phe Gly Ser Gly Phe Thr Leu Thr Ile Ser Asn Val Glu
Pro65 70 75 80Glu Asp Leu Gly Val Tyr Tyr Cys Leu Gln Tyr Gly Ser
Val Pro Tyr 85 90 95Thr Phe Gly Pro Gly Thr Lys Leu Glu Leu Lys 100
1051035PRTArtificial Sequence24A4 HC-CDR1 103Ser Tyr Cys Val Asn1
510416PRTArtificial Sequence24A4 HC-CDR2 104Arg Phe Trp Tyr Asp Glu
Gly Lys Val Tyr Asn Leu Thr Leu Glu Ser1 5 10 1510513PRTArtificial
Sequence24A4 HC-CDR3 105Asp Arg Asp Thr Leu Gly Ile Thr Gly Trp Phe
Ala Tyr1 5 1010611PRTArtificial Sequence24A4 LC-CDR1 106Lys Ala Ser
Gln Ser Val Gly Ile Asn Val Asp1 5 101077PRTArtificial Sequence24A4
LC-CDR2 107Gly Ser Ala Asn Arg His Thr1 51089PRTArtificial
Sequence24A4 LC-CDR3 108Leu Gln Tyr Gly Ser Val Pro Tyr Thr1
510919PRTArtificial SequenceLeader sequence A 109Met Glu Trp Ser
Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly1 5 10 15Val His
Ser11020PRTArtificial SequenceLeader sequence B 110Met Ser Val Pro
Thr Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr1 5 10 15Asp Ala Arg
Cys 20111452PRTArtificial SequenceMouse Anti-ILT3 p52B8 parental HC
Murine IgG2a heavy chain 111Glu Val Gln Leu Val Glu Ser Gly Gly Asp
Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Lys Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Asn Tyr 20 25 30Gly Met Ser Trp Val Arg Gln Thr
Pro Asp Arg Arg Leu Glu Trp Val 35 40 45Ala Thr Ile Ser Gly Gly Gly
Asp Tyr Thr Asn Tyr Pro Asp Ser Met 50 55 60Arg Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Ser
Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95Gly Arg Arg
Leu Trp Phe Arg Ser Leu Tyr Tyr Ala Met Asp Tyr Trp 100 105 110Gly
Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr Ala Pro 115 120
125Ser Val Tyr Pro Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser
130 135 140Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro
Val Thr145 150 155 160Leu Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly
Val His Thr Phe Pro 165 170 175Ala Val Leu Gln Ser Asp Leu Tyr Thr
Leu Ser Ser Ser Val Thr Val 180 185 190Thr Ser Ser Thr Trp Pro Ser
Gln Ser Ile Thr Cys Asn Val Ala His 195 200 205Pro Ala Ser Ser Thr
Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro 210 215 220Thr Ile Lys
Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu225 230 235
240Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu
245 250 255Met Ile Ser Leu Ser Pro Ile Val Thr Cys Val Val Val Asp
Val Ser 260 265 270Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val
Asn Asn Val Glu 275 280 285Val His Thr Ala Gln Thr Gln Thr His Arg
Glu Asp Tyr Asn Ser Thr 290 295 300Leu Arg Val Val Ser Ala Leu Pro
Ile Gln His Gln Asp Trp Met Ser305 310 315 320Gly Lys Glu Phe Lys
Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro 325 330 335Ile Glu Arg
Thr Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln 340 345 350Val
Tyr Val Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val 355 360
365Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val
370 375 380Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn
Thr Glu385 390 395 400Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Met
Tyr Ser Lys Leu Arg 405 410 415Val Glu Lys Lys Asn Trp Val Glu Arg
Asn Ser Tyr Ser Cys Ser Val 420 425 430Val His Glu Gly Leu His Asn
His His Thr Thr Lys Ser Phe Ser Arg 435 440 445Thr Pro Gly Lys
450112218PRTArtificial SequenceMouse Anti-ILT3 p52B8 parental LC
murine Kappa light chain 112Asn Ile Val Leu Thr Gln Ser Pro Ala Ser
Leu Ala Val Ser Leu Gly1 5 10 15Gln Arg Ala Thr Ile Ser Cys Arg Ala
Ser Glu Lys Val Asp Ser Phe 20 25 30Gly Asn Ser Phe Met His Trp Tyr
Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45Lys Leu Leu Ile Tyr Leu Thr
Ser Asn Leu Asp Ser Gly Val Pro Ala 50 55 60Arg Phe Ser Gly Ser Gly
Ser Arg Thr Asp Phe Ala Leu Thr Ile Asp65 70 75 80Pro Val Glu Ala
Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asn Asn 85 90 95Glu Asp Pro
Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110Ala
Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln 115 120
125Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr
130 135 140Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu
Arg Gln145 150 155 160Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp
Ser Lys Asp Ser Thr 165 170 175Tyr Ser Met Ser Ser Thr Leu Thr Leu
Thr Lys Asp Glu Tyr Glu Arg 180 185 190His Asn Ser Tyr Thr Cys Glu
Ala Thr His Lys Thr Ser Thr Ser Pro 195 200 205Ile Val Lys Ser Phe
Asn Arg Asn Glu Cys 210 215113449PRTArtificial SequenceChimeric
Anti-ILT3 mouse 52B8 VH parental/human IgG4 (S228P) 113Glu Val Gln
Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu
Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30Gly
Met Ser Trp Val Arg Gln Thr Pro Asp Arg Arg Leu Glu Trp Val 35 40
45Ala Thr Ile Ser Gly Gly Gly Asp Tyr Thr Asn Tyr Pro Asp Ser Met
50 55 60Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu
Tyr65 70 75 80Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met
Tyr Tyr Cys 85 90 95Gly Arg Arg Leu Trp Phe Arg Ser Leu Tyr Tyr Ala
Met Asp Tyr Trp 100 105 110Gly Gln Gly Thr Ser Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro 115 120 125Ser Val Phe Pro Leu Ala Pro Cys
Ser Arg Ser Thr Ser Glu Ser Thr 130 135 140Ala Ala Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr145 150 155 160Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185
190Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp
195 200 205His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser
Lys Tyr 210 215 220Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe
Leu Gly Gly Pro225 230 235 240Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser 245 250 255Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser Gln Glu Asp 260 265 270Pro Glu Val Gln Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285Ala Lys Thr
Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val 290 295 300Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu305 310
315 320Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu
Lys 325 330 335Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr 340 345 350Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn
Gln Val Ser Leu Thr 355 360 365Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu 370 375 380Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu385 390 395 400Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys 405 410 415Ser Arg
Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425
430Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
435 440 445Lys114449PRTArtificial SequenceChimeric Anti-ILT3 mouse
52B8 VH M64V/human IgG4 (S228P) 114Glu Val Gln Leu Val Glu Ser Gly
Gly Asp Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Lys Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30Gly Met Ser Trp Val Arg
Gln Thr Pro Asp Arg Arg Leu Glu Trp Val 35 40 45Ala Thr Ile Ser Gly
Gly Gly Asp Tyr Thr Asn Tyr Pro Asp Ser Val 50 55 60Arg Gly Arg Phe
Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr65 70 75 80Leu Gln
Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95Gly
Arg Arg Leu Trp Phe Arg Ser Leu Tyr Tyr Ala Met Asp Tyr Trp 100 105
110Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
115 120 125Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu
Ser Thr 130 135 140Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr145 150 155 160Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro 165 170 175Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190Val Pro Ser Ser Ser
Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp 195 200 205His Lys Pro
Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr 210 215 220Gly
Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro225
230 235 240Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser 245 250 255Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser Gln Glu Asp 260 265 270Pro Glu Val Gln Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn 275 280 285Ala Lys Thr Lys Pro Arg Glu Glu
Gln Phe Asn Ser Thr Tyr Arg Val 290 295 300Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys Glu305 310 315 320Tyr Lys Cys
Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys 325 330 335Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345
350Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
355 360 365Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu 370 375 380Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu385 390 395 400Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Arg Leu Thr Val Asp Lys 405 410 415Ser Arg Trp Gln Glu Gly Asn
Val Phe Ser Cys Ser Val Met His Glu 420 425 430Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 435 440
445Lys115449PRTArtificial SequenceMouse Anti-ILT3 52B8 VH
M64L/human IgG4 (S228P) 115Glu Val Gln Leu Val Glu Ser Gly Gly Asp
Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Lys Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Asn Tyr 20 25 30Gly Met Ser Trp Val Arg Gln Thr
Pro Asp Arg Arg Leu Glu Trp Val 35 40 45Ala Thr Ile Ser Gly Gly Gly
Asp Tyr Thr Asn Tyr Pro Asp Ser Leu 50 55 60Arg Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Ser
Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95Gly Arg Arg
Leu Trp Phe Arg Ser Leu Tyr Tyr Ala Met Asp Tyr Trp 100 105 110Gly
Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120
125Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr
130 135 140Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr145 150 155 160Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly
Val His Thr Phe Pro 165 170 175Ala Val Leu Gln Ser Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val Thr 180 185 190Val Pro Ser Ser Ser Leu Gly
Thr Lys Thr Tyr Thr Cys Asn Val Asp 195 200 205His Lys Pro Ser Asn
Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr 210 215 220Gly Pro Pro
Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro225 230 235
240Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln
Glu Asp 260 265 270Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn 275 280 285Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
Asn Ser Thr Tyr Arg Val 290 295 300Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu305 310 315 320Tyr Lys Cys Lys Val
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys 325 330 335Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350Leu
Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360
365Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu385 390 395 400Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg
Leu Thr Val Asp Lys 405 410 415Ser Arg Trp Gln Glu Gly Asn Val Phe
Ser Cys Ser Val Met His Glu 420 425 430Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Leu Gly 435 440
445Lys116218PRTArtificial SequenceChimeric Anti-ILT3 mouse 52B8
parental VL / human Kappa 116Asn Ile Val Leu Thr Gln Ser Pro Ala
Ser Leu Ala Val Ser Leu Gly1 5 10 15Gln Arg Ala Thr Ile Ser Cys Arg
Ala Ser Glu Lys Val Asp Ser Phe 20 25 30Gly Asn Ser Phe Met His Trp
Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45Lys Leu Leu Ile Tyr Leu
Thr Ser Asn Leu Asp Ser Gly Val Pro Ala 50 55 60Arg Phe Ser Gly Ser
Gly Ser Arg Thr Asp Phe Ala Leu Thr Ile Asp65 70 75 80Pro Val Glu
Ala Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asn Asn 85 90 95Glu Asp
Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105
110Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
115 120 125Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
Phe Tyr 130 135 140Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn
Ala Leu Gln Ser145 150 155 160Gly Asn Ser Gln Glu Ser Val Thr Glu
Gln Asp Ser Lys Asp Ser Thr 165 170 175Tyr Ser Leu Ser Ser Thr Leu
Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190His Lys Val Tyr Ala
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205Val Thr Lys
Ser Phe Asn Arg Gly Glu Cys 210 215117122PRTArtificial
SequenceHumanized 52B8 HC variable domain VH1 117Glu Val Gln 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 Ser Asn Tyr 20 25 30Gly Met
Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala
Thr Ile Ser Gly Gly Gly Asp Tyr Thr Asn Tyr Pro Asp Ser Met 50 55
60Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65
70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Gly Arg Arg Leu Trp Phe Arg Ser Leu Tyr Tyr Ala Met Asp
Tyr Trp 100 105 110Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
120118122PRTArtificial SequenceHumanized 52B8 HC variable domain
VH1 M64V 118Glu Val Gln 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
Ser Asn Tyr 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45Ala Thr Ile Ser Gly Gly Gly Asp Tyr Thr Asn
Tyr Pro Asp Ser Val 50 55 60Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Gly Arg Arg Leu Trp Phe Arg
Ser Leu Tyr Tyr Ala Met Asp Tyr Trp 100 105 110Gly Gln Gly Thr Leu
Val Thr Val Ser Ser 115 120119122PRTArtificial SequenceHumanized
52B8 HC variable domain VH1 M64L 119Glu Val Gln 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 Ser Asn Tyr 20 25 30Gly Met Ser Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Thr Ile Ser Gly
Gly Gly Asp Tyr Thr Asn Tyr Pro Asp Ser Leu 50 55 60Arg Gly Arg Phe
Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Gly
Arg Arg Leu Trp Phe Arg Ser Leu Tyr Tyr Ala Met Asp Tyr Trp 100 105
110Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
120120122PRTArtificial SequenceHumanized 52B8 HC variable domain
VH1 M64V W101F 120Glu Val Gln 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 Ser Asn Tyr 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45Ala Thr Ile Ser Gly Gly Gly Asp Tyr
Thr Asn Tyr Pro Asp Ser Val 50 55 60Arg Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Gly Arg Arg Leu Phe
Phe Arg Ser Leu Tyr Tyr Ala Met Asp Tyr Trp 100 105 110Gly Gln Gly
Thr Leu Val Thr Val Ser Ser 115 120121122PRTArtificial
SequenceHumanized 52B8 HC variable domain VH1 M64V W101Y 121Glu Val
Gln 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 Ser Asn Tyr 20 25
30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ala Thr Ile Ser Gly Gly Gly Asp Tyr Thr Asn Tyr Pro Asp Ser
Val 50 55 60Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Gly Arg Arg Leu Tyr Phe Arg Ser Leu Tyr Tyr
Ala Met Asp Tyr Trp 100 105 110Gly Gln Gly Thr Leu Val Thr Val Ser
Ser 115 120122122PRTArtificial SequenceHumanized 52B8 HC variable
domain VH1 M64V W101Q 122Glu Val Gln 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 Ser Asn Tyr 20 25 30Gly Met Ser Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Thr Ile Ser Gly Gly Gly
Asp Tyr Thr Asn Tyr Pro Asp Ser Val 50 55 60Arg Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Gly Arg Arg
Leu Gln Phe Arg Ser Leu Tyr Tyr Ala Met Asp Tyr Trp 100 105 110Gly
Gln Gly Thr Leu Val Thr Val Ser Ser 115 120123122PRTArtificial
SequenceHumanized 52B8 HC variable domain VH2 123Glu Val Gln 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 Ser Asn Tyr 20 25 30Gly Met
Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala
Thr Ile Ser Gly Gly Gly Asp Tyr Thr Asn Tyr Pro Asp Ser Met 50 55
60Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65
70 75 80Leu Gln Met Asn Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Gly Arg Arg Leu Trp Phe Arg Ser Leu Tyr Tyr Ala Met Asp
Tyr Trp 100 105 110Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
120124122PRTArtificial SequenceHumanized 52B8 HC variable domain
VH2 M64V 124Glu Val Gln 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
Ser Asn Tyr 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45Ala Thr Ile Ser Gly Gly Gly Asp Tyr Thr Asn
Tyr Pro Asp Ser Val 50 55 60Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Lys Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Gly Arg Arg Leu Trp Phe Arg
Ser Leu Tyr Tyr Ala Met Asp Tyr Trp 100 105 110Gly Gln Gly Thr Leu
Val Thr Val Ser Ser 115 120125122PRTArtificial SequenceHumanized
52B8 HC variable domain VH2 M64L 125Glu Val Gln 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 Ser Asn Tyr 20 25 30Gly Met Ser Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Thr Ile Ser Gly
Gly Gly Asp Tyr Thr Asn Tyr Pro Asp Ser Leu 50 55 60Arg Gly Arg Phe
Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln
Met Asn Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Gly
Arg Arg Leu Trp Phe Arg Ser Leu Tyr Tyr Ala Met Asp Tyr Trp 100 105
110Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
120126111PRTArtificial SequenceHumanized 52B8 LC variable domain
VL1 126Asp Ile Val Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu
Gly1 5 10 15Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Glu Lys Val Asp
Ser Phe 20 25 30Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly
Gln Pro Pro 35 40 45Lys Leu Leu Ile Tyr Leu Thr Ser Asn Leu Asp Ser
Gly Val Pro Asp 50 55 60Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe
Thr Leu Thr Ile Ser65 70 75 80Ser Leu Gln Ala Glu Asp Val Ala Val
Tyr Tyr Cys Gln Gln Asn Asn 85 90 95Glu Asp Pro Tyr Thr Phe Gly Gln
Gly Thr Lys Leu Glu Ile Lys 100 105 110127111PRTArtificial
SequenceHumanized 52B8 LC variable domain VL2 127Asp Ile Val Leu
Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly1 5 10 15Glu Arg Ala
Thr Ile Asn Cys Arg Ala Ser Glu Lys Val Asp Ser Phe 20 25 30Gly Asn
Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45Lys
Leu Leu Ile Tyr Leu Thr Ser Asn Leu Asp Ser Gly Val Pro Asp 50 55
60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser65
70 75 80Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Asn
Asn 85 90 95Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
Lys 100 105 110128111PRTArtificial SequenceHumanized 52B8 LC
variable domain VL3 128Glu 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
Glu Lys Val Asp Ser Phe 20 25 30Gly Asn Ser Phe Met His Trp Tyr Gln
Gln Lys Pro Gly Gln Ala Pro 35 40 45Arg Leu Leu Ile Tyr Leu Thr Ser
Asn Leu Asp Ser Gly Val Pro Ala 50 55 60Arg Phe Ser Gly Ser Gly Ser
Arg Thr Asp Phe Thr Leu Thr Ile Ser65 70 75 80Ser Leu Glu Pro Glu
Asp Phe Ala Val Tyr Tyr Cys Gln Gln Asn Asn 85 90 95Glu Asp Pro Tyr
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105
110129111PRTArtificial SequenceHumanized 52B8 LC variable domain
VL4 129Glu 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 Glu Lys Val Asp Ser Phe 20 25 30Gly Asn Ser Phe Met His Trp Tyr
Gln Gln Lys Pro Gly Gln Ala Pro 35 40 45Arg Leu Leu Ile Tyr Leu Thr
Ser Asn Leu Asp Ser Gly Ile Pro Ala 50 55 60Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser65 70 75 80Ser Leu Glu Pro
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Asn Asn 85 90 95Glu Asp Pro
Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105
110130111PRTArtificial SequenceHumanized 52B8 LC variable domain
VL5 130Asp 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 Glu Lys Val Asp
Ser Phe 20 25 30Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro 35 40 45Lys Leu Leu Ile Tyr Leu Thr Ser Asn Leu Asp Ser
Gly Val Pro Ser 50 55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser65 70 75 80Ser Leu Gln Pro Glu Asp Phe Ala Thr
Tyr Tyr Cys Gln Gln Asn Asn 85 90 95Glu Asp Pro Tyr Thr Phe Gly Gln
Gly Thr Lys Leu Glu Ile Lys 100 105 110131111PRTArtificial
SequenceHumanized 52B8 LC variable domain VL6 131Asp 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 Glu Lys Val Asp Ser Phe 20 25 30Gly Asn
Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45Lys
Leu Leu Ile Tyr Leu Thr Ser Asn Leu Asp Ser Gly Val Pro Ser 50 55
60Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Ser65
70 75 80Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asn
Asn 85 90 95Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
Lys 100 105 110132111PRTArtificial SequenceHumanized 52B8 LC
variable domain VL7 132Asp 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
Glu Lys Val Asp Ser Phe 20 25 30Gly Asn Ser Phe Met His Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro 35 40 45Lys Leu Leu Ile Tyr Leu Thr Ser
Asn Leu Asp Ser Gly Val Pro Ser 50 55 60Arg Phe Ser Gly Ser Gly Ser
Arg Thr Asp Phe Thr Leu Thr Ile Ser65 70 75 80Ser Leu Gln Pro Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asn Asn 85 90 95Glu Asp Pro Tyr
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105
110133111PRTArtificial SequenceHumanized 52B8 LC variable domain
VL8 133Asp 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 Glu Lys Val Asp
Ser Phe 20 25 30Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro 35 40 45Lys Leu Leu Ile Tyr Leu Thr Ser Asn Leu Asp Ser
Gly Val Pro Ala 50 55 60Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe
Thr Leu Thr Ile Ser65 70 75 80Ser Leu Gln Pro Glu Asp Phe Ala Thr
Tyr Tyr Cys Gln Gln Asn Asn 85 90 95Glu Asp Pro Tyr Thr Phe Gly Gln
Gly Thr Lys Leu Glu Ile Lys 100 105 110134111PRTArtificial
SequenceHumanized 52B8 LC variable domain VL2 S35A 134Asp Ile Val
Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly1 5 10 15Glu Arg
Ala Thr Ile Asn Cys Arg Ala Ser Glu Lys Val Asp Ser Phe 20 25 30Gly
Asn Ala Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40
45Lys Leu Leu Ile Tyr Leu Thr Ser Asn Leu Asp Ser Gly Val Pro Asp
50 55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser65 70 75 80Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln
Gln Asn Asn 85 90 95Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu
Glu Ile Lys 100 105 110135111PRTArtificial SequenceHumanized 52B8
LC variable domain VL2 S35N 135Asp Ile Val Leu Thr Gln Ser Pro Asp
Ser Leu Ala Val Ser Leu Gly1 5 10 15Glu Arg Ala Thr Ile Asn Cys Arg
Ala Ser Glu Lys Val Asp Ser Phe 20 25 30Gly Asn Asn Phe Met His Trp
Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45Lys Leu Leu Ile Tyr Leu
Thr Ser Asn Leu Asp Ser Gly Val Pro Asp 50 55 60Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser65 70 75 80Ser Leu Gln
Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Asn Asn 85 90 95Glu Asp
Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105
110136111PRTArtificial SequenceHumanized 52B8 LC variable domain
VL2 N34Q 136Asp Ile Val Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser
Leu Gly1 5 10 15Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Glu Lys Val
Asp Ser Phe 20 25 30Gly Gln Ser Phe Met His Trp Tyr Gln Gln Lys Pro
Gly Gln Pro Pro 35 40 45Lys Leu Leu Ile Tyr Leu Thr Ser Asn Leu Asp
Ser Gly Val Pro Asp 50 55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser65 70 75 80Ser Leu Gln Ala Glu Asp Val Ala
Val Tyr Tyr Cys Gln Gln Asn Asn 85 90 95Glu Asp Pro Tyr Thr Phe Gly
Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110137111PRTArtificial
SequenceHumanized 52B8 LC variable domain VL2 N34D 137Asp Ile Val
Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly1 5 10 15Glu Arg
Ala Thr Ile Asn Cys Arg Ala Ser Glu Lys Val Asp Ser Phe 20 25 30Gly
Asp Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40
45Lys Leu Leu Ile Tyr Leu Thr Ser Asn Leu Asp Ser Gly Val Pro Asp
50 55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser65 70 75 80Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln
Gln Asn Asn 85 90 95Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu
Glu Ile Lys 100 105 110138111PRTArtificial SequenceHumanized 52B8
LC variable domain VL5 S35A 138Asp 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 Glu Lys Val Asp Ser Phe 20 25 30Gly Asn Ala Phe Met His Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45Lys Leu Leu Ile Tyr Leu
Thr Ser Asn Leu Asp Ser Gly Val Pro Ser 50 55 60Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser65 70 75 80Ser Leu Gln
Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asn Asn 85 90 95Glu Asp
Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105
110139111PRTArtificial SequenceHumanized 52B8 LC variable domain
VL5 S35N 139Asp 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 Glu Lys Val
Asp Ser Phe 20 25 30Gly Asn Asn Phe Met His Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro 35 40 45Lys Leu Leu Ile Tyr Leu Thr Ser Asn Leu Asp
Ser Gly Val Pro Ser 50 55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser65 70 75 80Ser Leu Gln Pro Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Asn Asn 85 90 95Glu Asp Pro Tyr Thr Phe Gly
Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110140111PRTArtificial
SequenceHumanized 52B8 LC variable domain VL5 N34Q 140Asp 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 Glu Lys Val Asp Ser Phe 20 25 30Gly
Gln Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40
45Lys Leu Leu Ile Tyr Leu Thr Ser Asn Leu Asp Ser Gly Val Pro Ser
50 55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser65 70 75 80Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln
Gln Asn Asn 85 90 95Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu
Glu Ile Lys 100 105 110141111PRTArtificial SequenceHumanized 52B8
LC variable domain VL5 N34D 141Asp 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 Glu Lys Val Asp Ser Phe 20 25 30Gly Asp Ser Phe Met His Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45Lys Leu Leu Ile Tyr Leu
Thr Ser Asn Leu Asp Ser Gly Val Pro Ser 50 55 60Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser65 70 75 80Ser Leu Gln
Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asn Asn 85 90 95Glu Asp
Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105
110142449PRTArtificial SequenceHumanized 52B8 HC variable domain
VH1/Human IgG4 (S228P) constant domain 142Glu Val Gln 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 Ser Asn Tyr 20 25 30Gly Met Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Thr Ile
Ser Gly Gly Gly Asp Tyr Thr Asn Tyr Pro Asp Ser Met 50 55 60Arg Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Gly Arg Arg Leu Trp Phe Arg Ser Leu Tyr Tyr Ala Met Asp Tyr
Trp 100 105 110Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro 115 120 125Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser
Thr Ser Glu Ser Thr 130 135 140Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr145 150 155 160Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190Val Pro
Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp 195 200
205His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr
210 215 220Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly
Gly Pro225 230 235 240Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser 245 250 255Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Ser Gln Glu Asp 260 265 270Pro Glu Val Gln Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His Asn 275 280 285Ala Lys Thr Lys Pro
Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val 290 295 300Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu305 310 315
320Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys
325 330 335Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr 340 345 350Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
Val Ser Leu Thr 355 360 365Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu 370 375 380Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu385 390 395 400Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys 405 410 415Ser Arg Trp
Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 435 440
445Lys143449PRTArtificial SequenceHumanized 52B8 HC variable domain
VH1 M64V/Human IgG4 (S228P) constant domain 143Glu Val Gln 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 Ser Asn Tyr 20 25 30Gly Met Ser
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Thr
Ile Ser Gly Gly Gly Asp Tyr Thr Asn Tyr Pro Asp Ser Val 50 55 60Arg
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Gly Arg Arg Leu Trp Phe Arg Ser Leu Tyr Tyr Ala Met Asp Tyr
Trp 100 105 110Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro 115 120 125Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser
Thr Ser Glu Ser Thr 130 135 140Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr145 150 155 160Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190Val Pro
Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp 195 200
205His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr
210 215 220Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly
Gly Pro225 230 235 240Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser 245 250 255Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Ser Gln Glu Asp 260 265 270Pro Glu Val Gln Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His Asn 275 280 285Ala Lys Thr Lys Pro
Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val 290 295 300Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu305 310 315
320Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys
325 330 335Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr 340 345 350Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
Val Ser Leu Thr 355 360 365Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu 370 375 380Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu385 390 395 400Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys 405 410 415Ser Arg Trp
Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430Ala
Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Leu Gly 435 440 445Lys144449PRTArtificial
SequenceHumanized 52B8 HC variable domain VH1 M64L/Human IgG4
(S228P) constant domain 144Glu Val Gln 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 Ser Asn Tyr 20 25 30Gly Met Ser Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Thr Ile Ser Gly Gly Gly
Asp Tyr Thr Asn Tyr Pro Asp Ser Leu 50 55 60Arg Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Gly Arg Arg
Leu Trp Phe Arg Ser Leu Tyr Tyr Ala Met Asp Tyr Trp 100 105 110Gly
Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120
125Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr
130 135 140Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr145 150 155 160Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly
Val His Thr Phe Pro 165 170 175Ala Val Leu Gln Ser Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val Thr 180 185 190Val Pro Ser Ser Ser Leu Gly
Thr Lys Thr Tyr Thr Cys Asn Val Asp 195 200 205His Lys Pro Ser Asn
Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr 210 215 220Gly Pro Pro
Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro225 230 235
240Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln
Glu Asp 260 265 270Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn 275 280 285Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
Asn Ser Thr Tyr Arg Val 290 295 300Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu305 310 315 320Tyr Lys Cys Lys Val
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys 325 330 335Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350Leu
Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360
365Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu385 390 395 400Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg
Leu Thr Val Asp Lys 405 410 415Ser Arg Trp Gln Glu Gly Asn Val Phe
Ser Cys Ser Val Met His Glu 420 425 430Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Leu Gly 435 440
445Lys145449PRTArtificial SequenceHumanized 52B8 HC variable domain
VH1 M64V W101F/Human IgG4 (S228P) constant domain 145Glu Val Gln
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 Ser Asn Tyr 20 25 30Gly
Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ala Thr Ile Ser Gly Gly Gly Asp Tyr Thr Asn Tyr Pro Asp Ser Val
50 55 60Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu
Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Gly Arg Arg Leu Phe Phe Arg Ser Leu Tyr Tyr Ala
Met Asp Tyr Trp 100 105 110Gly Gln Gly Thr Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro 115 120 125Ser Val Phe Pro Leu Ala Pro Cys
Ser Arg Ser Thr Ser Glu Ser Thr 130 135 140Ala Ala Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr145 150 155 160Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185
190Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp
195 200 205His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser
Lys Tyr 210 215 220Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe
Leu Gly Gly Pro225 230 235 240Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser 245 250 255Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser Gln Glu Asp 260 265 270Pro Glu Val Gln Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285Ala Lys Thr
Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val 290 295 300Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu305 310
315 320Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu
Lys 325 330 335Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr 340 345 350Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn
Gln Val Ser Leu Thr 355 360 365Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu 370 375 380Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu385 390 395 400Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys 405 410 415Ser Arg
Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425
430Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
435 440 445Lys146449PRTArtificial SequenceHumanized 52B8 HC
variable domain VH1 M64V W101Y/Human IgG4 (S228P) constant domain
146Glu Val Gln 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 Ser Asn
Tyr 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ala Thr Ile Ser Gly Gly Gly Asp Tyr Thr Asn Tyr Pro
Asp Ser Val 50 55 60Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Gly Arg Arg Leu Tyr Phe Arg Ser Leu
Tyr Tyr Ala Met Asp Tyr Trp 100 105 110Gly Gln Gly Thr Leu Val Thr
Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125Ser Val Phe Pro Leu
Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr 130 135 140Ala Ala Leu
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr145 150 155
160Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
165 170 175Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr 180 185 190Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr
Cys Asn Val Asp 195 200 205His Lys Pro Ser Asn Thr Lys Val Asp Lys
Arg Val Glu Ser Lys Tyr 210 215 220Gly Pro Pro Cys Pro Pro Cys Pro
Ala Pro Glu Phe Leu Gly Gly Pro225 230 235 240Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp 260 265 270Pro
Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280
285Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val
290 295 300Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys Glu305 310 315 320Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
Ser Ser Ile Glu Lys 325 330 335Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr 340 345 350Leu Pro Pro Ser Gln Glu Glu
Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu385 390 395
400Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys
405 410 415Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met
His Glu 420 425 430Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Leu Gly 435 440 445Lys147449PRTArtificial SequenceHumanized
52B8 HC variable domain VH1 M64V W101Q/Human IgG4 (S228P) constant
domain 147Glu Val Gln 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
Ser Asn Tyr 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45Ala Thr Ile Ser Gly Gly Gly Asp Tyr Thr Asn
Tyr Pro Asp Ser Val 50 55 60Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Gly Arg Arg Leu Gln Phe Arg
Ser Leu Tyr Tyr Ala Met Asp Tyr Trp 100 105 110Gly Gln Gly Thr Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125Ser Val Phe
Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr 130 135 140Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr145 150
155 160Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
Pro 165 170 175Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val Thr 180 185 190Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr
Thr Cys Asn Val Asp 195 200 205His Lys Pro Ser Asn Thr Lys Val Asp
Lys Arg Val Glu Ser Lys Tyr 210 215 220Gly Pro Pro Cys Pro Pro Cys
Pro Ala Pro Glu Phe Leu Gly Gly Pro225 230 235 240Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp 260 265
270Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr
Arg Val 290 295 300Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu305 310 315 320Tyr Lys Cys Lys Val Ser Asn Lys Gly
Leu Pro Ser Ser Ile Glu Lys 325 330 335Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350Leu Pro Pro Ser Gln
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu385 390
395 400Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp
Lys 405 410 415Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val
Met His Glu 420 425 430Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Leu Gly 435 440 445Lys148449PRTArtificial
SequenceHumanized 52B8 HC variable domain VH2/Human IgG4 (S228P)
constant domain 148Glu Val Gln 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 Ser Asn Tyr 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45Ala Thr Ile Ser Gly Gly Gly Asp Tyr
Thr Asn Tyr Pro Asp Ser Met 50 55 60Arg Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu
Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Gly Arg Arg Leu Trp
Phe Arg Ser Leu Tyr Tyr Ala Met Asp Tyr Trp 100 105 110Gly Gln Gly
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125Ser
Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr 130 135
140Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr145 150 155 160Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro 165 170 175Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr 180 185 190Val Pro Ser Ser Ser Leu Gly Thr
Lys Thr Tyr Thr Cys Asn Val Asp 195 200 205His Lys Pro Ser Asn Thr
Lys Val Asp Lys Arg Val Glu Ser Lys Tyr 210 215 220Gly Pro Pro Cys
Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro225 230 235 240Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250
255Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp
260 265 270Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val
His Asn 275 280 285Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
Thr Tyr Arg Val 290 295 300Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly Lys Glu305 310 315 320Tyr Lys Cys Lys Val Ser Asn
Lys Gly Leu Pro Ser Ser Ile Glu Lys 325 330 335Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350Leu Pro Pro
Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375
380Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu385 390 395 400Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
Thr Val Asp Lys 405 410 415Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
Cys Ser Val Met His Glu 420 425 430Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Leu Gly 435 440 445Lys149449PRTArtificial
SequenceHumanized 52B8 HC variable domain VH2 M64V/Human IgG4
(S228P) constant domain 149Glu Val Gln 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 Ser Asn Tyr 20 25 30Gly Met Ser Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Thr Ile Ser Gly Gly Gly
Asp Tyr Thr Asn Tyr Pro Asp Ser Val 50 55 60Arg Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Gly Arg Arg
Leu Trp Phe Arg Ser Leu Tyr Tyr Ala Met Asp Tyr Trp 100 105 110Gly
Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120
125Ser Val Phe Pro Leu Ala Pro
Cys Ser Arg Ser Thr Ser Glu Ser Thr 130 135 140Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr145 150 155 160Val Ser
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170
175Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
180 185 190Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn
Val Asp 195 200 205His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val
Glu Ser Lys Tyr 210 215 220Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro
Glu Phe Leu Gly Gly Pro225 230 235 240Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp 260 265 270Pro Glu Val
Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285Ala
Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val 290 295
300Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu305 310 315 320Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
Ser Ile Glu Lys 325 330 335Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr Thr 340 345 350Leu Pro Pro Ser Gln Glu Glu Met
Thr Lys Asn Gln Val Ser Leu Thr 355 360 365Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu385 390 395 400Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys 405 410
415Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Leu Gly 435 440 445Lys150449PRTArtificial SequenceHumanized 52B8 HC
variable domain VH2 M64L/Human IgG4 (S228P) constant domain 150Glu
Val Gln 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 Ser Asn Tyr
20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ala Thr Ile Ser Gly Gly Gly Asp Tyr Thr Asn Tyr Pro Asp
Ser Leu 50 55 60Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Lys Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Gly Arg Arg Leu Trp Phe Arg Ser Leu Tyr
Tyr Ala Met Asp Tyr Trp 100 105 110Gly Gln Gly Thr Leu Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125Ser Val Phe Pro Leu Ala
Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr 130 135 140Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr145 150 155 160Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170
175Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
180 185 190Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn
Val Asp 195 200 205His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val
Glu Ser Lys Tyr 210 215 220Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro
Glu Phe Leu Gly Gly Pro225 230 235 240Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp 260 265 270Pro Glu Val
Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285Ala
Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val 290 295
300Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu305 310 315 320Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
Ser Ile Glu Lys 325 330 335Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr Thr 340 345 350Leu Pro Pro Ser Gln Glu Glu Met
Thr Lys Asn Gln Val Ser Leu Thr 355 360 365Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu385 390 395 400Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys 405 410
415Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Leu Gly 435 440 445Lys151218PRTArtificial SequenceHumanized 52B8 LC
variable domain VL1/kappa constant domain 151Asp Ile Val Leu Thr
Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly1 5 10 15Glu Arg Ala Thr
Ile Asn Cys Arg Ala Ser Glu Lys Val Asp Ser Phe 20 25 30Gly Asn Ser
Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45Lys Leu
Leu Ile Tyr Leu Thr Ser Asn Leu Asp Ser Gly Val Pro Asp 50 55 60Arg
Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Ser65 70 75
80Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Asn Asn
85 90 95Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
Arg 100 105 110Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp Glu Gln 115 120 125Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu
Leu Asn Asn Phe Tyr 130 135 140Pro Arg Glu Ala Lys Val Gln Trp Lys
Val Asp Asn Ala Leu Gln Ser145 150 155 160Gly Asn Ser Gln Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175Tyr Ser Leu Ser
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190His Lys
Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200
205Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210
215152218PRTArtificial SequenceHumanized 52B8 LC variable domain
VL2/kappa constant domain 152Asp Ile Val Leu Thr Gln Ser Pro Asp
Ser Leu Ala Val Ser Leu Gly1 5 10 15Glu Arg Ala Thr Ile Asn Cys Arg
Ala Ser Glu Lys Val Asp Ser Phe 20 25 30Gly Asn Ser Phe Met His Trp
Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45Lys Leu Leu Ile Tyr Leu
Thr Ser Asn Leu Asp Ser Gly Val Pro Asp 50 55 60Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser65 70 75 80Ser Leu Gln
Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Asn Asn 85 90 95Glu Asp
Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105
110Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
115 120 125Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
Phe Tyr 130 135 140Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn
Ala Leu Gln Ser145 150 155 160Gly Asn Ser Gln Glu Ser Val Thr Glu
Gln Asp Ser Lys Asp Ser Thr 165 170 175Tyr Ser Leu Ser Ser Thr Leu
Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190His Lys Val Tyr Ala
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205Val Thr Lys
Ser Phe Asn Arg Gly Glu Cys 210 215153218PRTArtificial
SequenceHumanized 52B8 LC variable domain VL3/kappa constant domain
153Glu 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 Glu Lys Val Asp Ser
Phe 20 25 30Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln
Ala Pro 35 40 45Arg Leu Leu Ile Tyr Leu Thr Ser Asn Leu Asp Ser Gly
Val Pro Ala 50 55 60Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr
Leu Thr Ile Ser65 70 75 80Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr
Tyr Cys Gln Gln Asn Asn 85 90 95Glu Asp Pro Tyr Thr Phe Gly Gln Gly
Thr Lys Leu Glu Ile Lys Arg 100 105 110Thr Val Ala Ala Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125Leu Lys Ser Gly Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140Pro Arg Glu
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser145 150 155
160Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
165 170 175Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
Glu Lys 180 185 190His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly
Leu Ser Ser Pro 195 200 205Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215154218PRTArtificial SequenceHumanized 52B8 LC variable
domain VL4/kappa constant domain 154Glu 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 Glu Lys Val Asp Ser Phe 20 25 30Gly Asn Ser Phe Met His
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 35 40 45Arg Leu Leu Ile Tyr
Leu Thr Ser Asn Leu Asp Ser Gly Ile Pro Ala 50 55 60Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser65 70 75 80Ser Leu
Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Asn Asn 85 90 95Glu
Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105
110Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
115 120 125Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
Phe Tyr 130 135 140Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn
Ala Leu Gln Ser145 150 155 160Gly Asn Ser Gln Glu Ser Val Thr Glu
Gln Asp Ser Lys Asp Ser Thr 165 170 175Tyr Ser Leu Ser Ser Thr Leu
Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190His Lys Val Tyr Ala
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205Val Thr Lys
Ser Phe Asn Arg Gly Glu Cys 210 215155218PRTArtificial
SequenceHumanized 52B8 LC variable domain VL5/kappa constant domain
155Asp 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 Glu Lys Val Asp Ser
Phe 20 25 30Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro 35 40 45Lys Leu Leu Ile Tyr Leu Thr Ser Asn Leu Asp Ser Gly
Val Pro Ser 50 55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser65 70 75 80Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln Asn Asn 85 90 95Glu Asp Pro Tyr Thr Phe Gly Gln Gly
Thr Lys Leu Glu Ile Lys Arg 100 105 110Thr Val Ala Ala Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125Leu Lys Ser Gly Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140Pro Arg Glu
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser145 150 155
160Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
165 170 175Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
Glu Lys 180 185 190His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly
Leu Ser Ser Pro 195 200 205Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215156218PRTArtificial SequenceHumanized 52B8 LC variable
domain VL6/kappa constant domain 156Asp 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 Glu Lys Val Asp Ser Phe 20 25 30Gly Asn Ser Phe Met His
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45Lys Leu Leu Ile Tyr
Leu Thr Ser Asn Leu Asp Ser Gly Val Pro Ser 50 55 60Arg Phe Ser Gly
Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Ser65 70 75 80Ser Leu
Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asn Asn 85 90 95Glu
Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105
110Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
115 120 125Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
Phe Tyr 130 135 140Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn
Ala Leu Gln Ser145 150 155 160Gly Asn Ser Gln Glu Ser Val Thr Glu
Gln Asp Ser Lys Asp Ser Thr 165 170 175Tyr Ser Leu Ser Ser Thr Leu
Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190His Lys Val Tyr Ala
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205Val Thr Lys
Ser Phe Asn Arg Gly Glu Cys 210 215157218PRTArtificial
SequenceHumanized 52B8 LC variable domain VL7/kappa constant domain
157Asp 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 Glu Lys Val Asp Ser
Phe 20 25 30Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro 35 40 45Lys Leu Leu Ile Tyr Leu Thr Ser Asn Leu Asp Ser Gly
Val Pro Ser 50 55 60Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr
Leu Thr Ile Ser65 70 75 80Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln Asn Asn 85 90 95Glu Asp Pro Tyr Thr Phe Gly Gln Gly
Thr Lys Leu Glu Ile Lys Arg 100 105 110Thr Val Ala Ala Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125Leu Lys Ser Gly Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140Pro Arg Glu
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser145 150 155
160Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
165 170 175Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
Glu Lys 180 185 190His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly
Leu Ser Ser Pro 195 200 205Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215158218PRTArtificial SequenceHumanized 52B8 LC variable
domain VL8/kappa constant domain 158Asp 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 Glu Lys Val Asp Ser Phe 20 25 30Gly Asn Ser Phe Met His
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45Lys Leu Leu Ile Tyr
Leu Thr Ser Asn Leu Asp Ser Gly Val Pro Ala 50 55 60Arg Phe Ser Gly
Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Ser65
70 75 80Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asn
Asn 85 90 95Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
Lys Arg 100 105 110Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro
Ser Asp Glu Gln 115 120 125Leu Lys Ser Gly Thr Ala Ser Val Val Cys
Leu Leu Asn Asn Phe Tyr 130 135 140Pro Arg Glu Ala Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln Ser145 150 155 160Gly Asn Ser Gln Glu
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175Tyr Ser Leu
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190His
Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200
205Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210
215159218PRTArtificial SequenceHumanized 52B8 LC variable domain
VL2 S35A/kappa constant domain 159Asp Ile Val Leu Thr Gln Ser Pro
Asp Ser Leu Ala Val Ser Leu Gly1 5 10 15Glu Arg Ala Thr Ile Asn Cys
Arg Ala Ser Glu Lys Val Asp Ser Phe 20 25 30Gly Asn Ala Phe Met His
Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45Lys Leu Leu Ile Tyr
Leu Thr Ser Asn Leu Asp Ser Gly Val Pro Asp 50 55 60Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser65 70 75 80Ser Leu
Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Asn Asn 85 90 95Glu
Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105
110Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
115 120 125Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
Phe Tyr 130 135 140Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn
Ala Leu Gln Ser145 150 155 160Gly Asn Ser Gln Glu Ser Val Thr Glu
Gln Asp Ser Lys Asp Ser Thr 165 170 175Tyr Ser Leu Ser Ser Thr Leu
Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190His Lys Val Tyr Ala
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205Val Thr Lys
Ser Phe Asn Arg Gly Glu Cys 210 215160218PRTArtificial
SequenceHumanized 52B8 LC variable domain VL2 S35N/kappa constant
domain 160Asp Ile Val Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser
Leu Gly1 5 10 15Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Glu Lys Val
Asp Ser Phe 20 25 30Gly Asn Asn Phe Met His Trp Tyr Gln Gln Lys Pro
Gly Gln Pro Pro 35 40 45Lys Leu Leu Ile Tyr Leu Thr Ser Asn Leu Asp
Ser Gly Val Pro Asp 50 55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser65 70 75 80Ser Leu Gln Ala Glu Asp Val Ala
Val Tyr Tyr Cys Gln Gln Asn Asn 85 90 95Glu Asp Pro Tyr Thr Phe Gly
Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110Thr Val Ala Ala Pro
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125Leu Lys Ser
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140Pro
Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser145 150
155 160Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr 165 170 175Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys 180 185 190His Lys Val Tyr Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser Pro 195 200 205Val Thr Lys Ser Phe Asn Arg Gly Glu
Cys 210 215161218PRTArtificial SequenceHumanized 52B8 LC variable
domain VL2 N34Q/kappa constant domain 161Asp Ile Val Leu Thr Gln
Ser Pro Asp Ser Leu Ala Val Ser Leu Gly1 5 10 15Glu Arg Ala Thr Ile
Asn Cys Arg Ala Ser Glu Lys Val Asp Ser Phe 20 25 30Gly Gln Ser Phe
Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45Lys Leu Leu
Ile Tyr Leu Thr Ser Asn Leu Asp Ser Gly Val Pro Asp 50 55 60Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser65 70 75
80Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Asn Asn
85 90 95Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
Arg 100 105 110Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp Glu Gln 115 120 125Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu
Leu Asn Asn Phe Tyr 130 135 140Pro Arg Glu Ala Lys Val Gln Trp Lys
Val Asp Asn Ala Leu Gln Ser145 150 155 160Gly Asn Ser Gln Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175Tyr Ser Leu Ser
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190His Lys
Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200
205Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210
215162218PRTArtificial SequenceHumanized 52B8 LC variable domain
VL2 N34D/kappa constant domain 162Asp Ile Val Leu Thr Gln Ser Pro
Asp Ser Leu Ala Val Ser Leu Gly1 5 10 15Glu Arg Ala Thr Ile Asn Cys
Arg Ala Ser Glu Lys Val Asp Ser Phe 20 25 30Gly Asp Ser Phe Met His
Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45Lys Leu Leu Ile Tyr
Leu Thr Ser Asn Leu Asp Ser Gly Val Pro Asp 50 55 60Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser65 70 75 80Ser Leu
Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Asn Asn 85 90 95Glu
Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105
110Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
115 120 125Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
Phe Tyr 130 135 140Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn
Ala Leu Gln Ser145 150 155 160Gly Asn Ser Gln Glu Ser Val Thr Glu
Gln Asp Ser Lys Asp Ser Thr 165 170 175Tyr Ser Leu Ser Ser Thr Leu
Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190His Lys Val Tyr Ala
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205Val Thr Lys
Ser Phe Asn Arg Gly Glu Cys 210 215163218PRTArtificial
SequenceHumanized 52B8 LC variable domain VL5 S35A/kappa constant
domain 163Asp 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 Glu Lys Val
Asp Ser Phe 20 25 30Gly Asn Ala Phe Met His Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro 35 40 45Lys Leu Leu Ile Tyr Leu Thr Ser Asn Leu Asp
Ser Gly Val Pro Ser 50 55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser65 70 75 80Ser Leu Gln Pro Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Asn Asn 85 90 95Glu Asp Pro Tyr Thr Phe Gly
Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110Thr Val Ala Ala Pro
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125Leu Lys Ser
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140Pro
Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser145 150
155 160Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr 165 170 175Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys 180 185 190His Lys Val Tyr Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser Pro 195 200 205Val Thr Lys Ser Phe Asn Arg Gly Glu
Cys 210 215164218PRTArtificial SequenceHumanized 52B8 LC variable
domain VL5 S35N/kappa constant domain 164Asp 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 Glu Lys Val Asp Ser Phe 20 25 30Gly Asn Asn Phe
Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45Lys Leu Leu
Ile Tyr Leu Thr Ser Asn Leu Asp Ser Gly Val Pro Ser 50 55 60Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser65 70 75
80Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asn Asn
85 90 95Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
Arg 100 105 110Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp Glu Gln 115 120 125Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu
Leu Asn Asn Phe Tyr 130 135 140Pro Arg Glu Ala Lys Val Gln Trp Lys
Val Asp Asn Ala Leu Gln Ser145 150 155 160Gly Asn Ser Gln Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175Tyr Ser Leu Ser
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190His Lys
Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200
205Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210
215165218PRTArtificial SequenceHumanized 52B8 LC variable domain
VL5 N34Q/kappa constant domain 165Asp 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 Glu Lys Val Asp Ser Phe 20 25 30Gly Gln Ser Phe Met His
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45Lys Leu Leu Ile Tyr
Leu Thr Ser Asn Leu Asp Ser Gly Val Pro Ser 50 55 60Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser65 70 75 80Ser Leu
Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asn Asn 85 90 95Glu
Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105
110Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
115 120 125Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
Phe Tyr 130 135 140Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn
Ala Leu Gln Ser145 150 155 160Gly Asn Ser Gln Glu Ser Val Thr Glu
Gln Asp Ser Lys Asp Ser Thr 165 170 175Tyr Ser Leu Ser Ser Thr Leu
Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190His Lys Val Tyr Ala
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205Val Thr Lys
Ser Phe Asn Arg Gly Glu Cys 210 215166218PRTArtificial
SequenceHumanized 52B8 LC variable domain VL5 N34D/kappa constant
domain 166Asp 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 Glu Lys Val
Asp Ser Phe 20 25 30Gly Asp Ser Phe Met His Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro 35 40 45Lys Leu Leu Ile Tyr Leu Thr Ser Asn Leu Asp
Ser Gly Val Pro Ser 50 55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser65 70 75 80Ser Leu Gln Pro Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Asn Asn 85 90 95Glu Asp Pro Tyr Thr Phe Gly
Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110Thr Val Ala Ala Pro
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125Leu Lys Ser
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140Pro
Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser145 150
155 160Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr 165 170 175Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys 180 185 190His Lys Val Tyr Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser Pro 195 200 205Val Thr Lys Ser Phe Asn Arg Gly Glu
Cys 210 215167452PRTArtificial SequenceHumanized 52B8 HC variable
domain VH1/ Human IgG1 HC (L234A L235A D265S) constant domain
167Glu Val Gln 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 Ser Asn
Tyr 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ala Thr Ile Ser Gly Gly Gly Asp Tyr Thr Asn Tyr Pro
Asp Ser Met 50 55 60Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Gly Arg Arg Leu Trp Phe Arg Ser Leu
Tyr Tyr Ala Met Asp Tyr Trp 100 105 110Gly Gln Gly Thr Leu Val Thr
Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125Ser Val Phe Pro Leu
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140Ala Ala Leu
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr145 150 155
160Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
165 170 175Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr 180 185 190Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val Asn 195 200 205His Lys Pro Ser Asn Thr Lys Val Asp Lys
Lys Val Glu Pro Lys Ser 210 215 220Cys Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Ala Ala225 230 235 240Gly Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Ser Val Ser 260 265 270His
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280
285Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
290 295 300Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn305 310 315 320Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro 325 330 335Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln 340 345 350Val Tyr Thr Leu Pro Pro Ser
Arg Asp Glu Leu Thr Lys Asn Gln Val 355 360 365Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro385 390 395
400Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
405 410 415Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val 420 425 430Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu 435 440 445Ser Pro Gly Lys 450168452PRTArtificial
SequenceHumanized 52B8 HC variable domain VH1 M64V/ Human IgG1 HC
(L234A L235A D265S) constant domain 168Glu Val
Gln 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 Ser Asn Tyr 20 25
30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ala Thr Ile Ser Gly Gly Gly Asp Tyr Thr Asn Tyr Pro Asp Ser
Val 50 55 60Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Gly Arg Arg Leu Trp Phe Arg Ser Leu Tyr Tyr
Ala Met Asp Tyr Trp 100 105 110Gly Gln Gly Thr Leu Val Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro 115 120 125Ser Val Phe Pro Leu Ala Pro
Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr145 150 155 160Val Ser
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170
175Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
180 185 190Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
Val Asn 195 200 205His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val
Glu Pro Lys Ser 210 215 220Cys Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Ala Ala225 230 235 240Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Ser Val Ser 260 265 270His Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295
300Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn305 310 315 320Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro 325 330 335Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln 340 345 350Val Tyr Thr Leu Pro Pro Ser Arg
Asp Glu Leu Thr Lys Asn Gln Val 355 360 365Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro385 390 395 400Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410
415Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
420 425 430Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu 435 440 445Ser Pro Gly Lys 450169452PRTArtificial
SequenceHumanized 52B8 HC variable domain VH1 M64L/ Human IgG1 HC
(L234A L235A D265S) constant domain 169Glu Val Gln 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 Ser Asn Tyr 20 25 30Gly Met Ser Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Thr Ile Ser
Gly Gly Gly Asp Tyr Thr Asn Tyr Pro Asp Ser Leu 50 55 60Arg Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95Gly Arg Arg Leu Trp Phe Arg Ser Leu Tyr Tyr Ala Met Asp Tyr Trp
100 105 110Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro 115 120 125Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr 130 135 140Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr145 150 155 160Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205His
Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215
220Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala
Ala225 230 235 240Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu 245 250 255Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Ser Val Ser 260 265 270His Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu 275 280 285Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300Tyr Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn305 310 315 320Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330
335Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
340 345 350Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
Gln Val 355 360 365Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val 370 375 380Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro385 390 395 400Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430Met His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445Ser
Pro Gly Lys 450170452PRTArtificial SequenceHumanized 52B8 HC
variable domain VH1 M64V W101F/ Human IgG1 HC (L234A L235A D265S)
constant domain 170Glu Val Gln 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 Ser Asn Tyr 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45Ala Thr Ile Ser Gly Gly Gly Asp Tyr
Thr Asn Tyr Pro Asp Ser Val 50 55 60Arg Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Gly Arg Arg Leu Phe
Phe Arg Ser Leu Tyr Tyr Ala Met Asp Tyr Trp 100 105 110Gly Gln Gly
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125Ser
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135
140Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr145 150 155 160Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro 165 170 175Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr 180 185 190Val Pro Ser Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205His Lys Pro Ser Asn Thr
Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220Cys Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala225 230 235 240Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250
255Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Ser Val Ser
260 265 270His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu 275 280 285Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr 290 295 300Tyr Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn305 310 315 320Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350Val Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 355 360 365Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375
380Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro385 390 395 400Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr 405 410 415Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val 420 425 430Met His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445Ser Pro Gly Lys
450171452PRTArtificial SequenceHumanized 52B8 HC variable domain
VH1 M64V W101Y/ Human IgG1 HC (L234A L235A D265S) constant domain
171Glu Val Gln 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 Ser Asn
Tyr 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ala Thr Ile Ser Gly Gly Gly Asp Tyr Thr Asn Tyr Pro
Asp Ser Val 50 55 60Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Gly Arg Arg Leu Tyr Phe Arg Ser Leu
Tyr Tyr Ala Met Asp Tyr Trp 100 105 110Gly Gln Gly Thr Leu Val Thr
Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125Ser Val Phe Pro Leu
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140Ala Ala Leu
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr145 150 155
160Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
165 170 175Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr 180 185 190Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val Asn 195 200 205His Lys Pro Ser Asn Thr Lys Val Asp Lys
Lys Val Glu Pro Lys Ser 210 215 220Cys Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Ala Ala225 230 235 240Gly Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Ser Val Ser 260 265 270His
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280
285Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
290 295 300Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn305 310 315 320Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro 325 330 335Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln 340 345 350Val Tyr Thr Leu Pro Pro Ser
Arg Asp Glu Leu Thr Lys Asn Gln Val 355 360 365Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro385 390 395
400Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
405 410 415Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val 420 425 430Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu 435 440 445Ser Pro Gly Lys 450172452PRTArtificial
SequenceHumanized 52B8 HC variable domain VH1 M64V W101Q/ Human
IgG1 HC (L234A L235A D265S) constant domain 172Glu Val Gln 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 Ser Asn Tyr 20 25 30Gly Met Ser
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Thr
Ile Ser Gly Gly Gly Asp Tyr Thr Asn Tyr Pro Asp Ser Val 50 55 60Arg
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Gly Arg Arg Leu Gln Phe Arg Ser Leu Tyr Tyr Ala Met Asp Tyr
Trp 100 105 110Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro 115 120 125Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr 130 135 140Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr145 150 155 160Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190Val Pro
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200
205His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
210 215 220Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Ala Ala225 230 235 240Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu 245 250 255Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Ser Val Ser 260 265 270His Glu Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn305 310 315
320Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
325 330 335Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln 340 345 350Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val 355 360 365Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val 370 375 380Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro385 390 395 400Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430Met
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440
445Ser Pro Gly Lys 450173452PRTArtificial SequenceHumanized 52B8 HC
variable domain VH2/ Human IgG1 HC (L234A L235A D265S) constant
domain 173Glu Val Gln 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
Ser Asn Tyr 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45Ala Thr Ile Ser Gly Gly Gly Asp Tyr Thr Asn
Tyr Pro Asp Ser Met 50 55 60Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Lys Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Gly Arg Arg Leu Trp Phe Arg
Ser Leu Tyr Tyr Ala Met Asp Tyr Trp 100 105 110Gly Gln Gly Thr Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120
125Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
130 135 140Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr145 150 155 160Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly
Val His Thr Phe Pro 165 170 175Ala Val Leu Gln Ser Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val Thr 180 185 190Val Pro Ser Ser Ser Leu Gly
Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205His Lys Pro Ser Asn
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220Cys Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala225 230 235
240Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
245 250 255Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Ser
Val Ser 260 265 270His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu 275 280 285Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr 290 295 300Tyr Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn305 310 315 320Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350Val
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 355 360
365Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
370 375 380Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro385 390 395 400Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr 405 410 415Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val 420 425 430Met His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445Ser Pro Gly Lys
450174452PRTArtificial SequenceHumanized 52B8 HC variable domain
VH2 M64V/ Human IgG1 HC (L234A L235A D265S) constant domain 174Glu
Val Gln 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 Ser Asn Tyr
20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ala Thr Ile Ser Gly Gly Gly Asp Tyr Thr Asn Tyr Pro Asp
Ser Val 50 55 60Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Lys Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Gly Arg Arg Leu Trp Phe Arg Ser Leu Tyr
Tyr Ala Met Asp Tyr Trp 100 105 110Gly Gln Gly Thr Leu Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125Ser Val Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr145 150 155 160Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170
175Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
180 185 190Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
Val Asn 195 200 205His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val
Glu Pro Lys Ser 210 215 220Cys Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Ala Ala225 230 235 240Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Ser Val Ser 260 265 270His Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295
300Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn305 310 315 320Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro 325 330 335Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln 340 345 350Val Tyr Thr Leu Pro Pro Ser Arg
Asp Glu Leu Thr Lys Asn Gln Val 355 360 365Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro385 390 395 400Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410
415Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
420 425 430Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu 435 440 445Ser Pro Gly Lys 450175452PRTArtificial
SequenceHumanized 52B8 HC variable domain VH2 M64L/ Human IgG1 HC
(L234A L235A D265S) constant domain 175Glu Val Gln 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 Ser Asn Tyr 20 25 30Gly Met Ser Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Thr Ile Ser
Gly Gly Gly Asp Tyr Thr Asn Tyr Pro Asp Ser Leu 50 55 60Arg Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu
Gln Met Asn Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95Gly Arg Arg Leu Trp Phe Arg Ser Leu Tyr Tyr Ala Met Asp Tyr Trp
100 105 110Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro 115 120 125Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr 130 135 140Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr145 150 155 160Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205His
Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215
220Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala
Ala225 230 235 240Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu 245 250 255Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Ser Val Ser 260 265 270His Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu 275 280 285Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300Tyr Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn305 310 315 320Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330
335Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
340 345 350Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
Gln Val 355 360 365Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val 370 375 380Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro385 390 395 400Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430Met His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445Ser
Pro Gly Lys 450176448PRTArtificial SequenceHumanized 52B8 HC
variable domain VH1/Human IgG4 (S228P) (K-) constant domain 176Glu
Val Gln 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 Ser Asn Tyr
20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ala Thr Ile Ser Gly Gly Gly Asp Tyr Thr Asn Tyr Pro Asp
Ser Met 50 55 60Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Gly Arg Arg Leu Trp Phe Arg Ser Leu Tyr
Tyr Ala Met Asp Tyr Trp 100 105 110Gly Gln Gly Thr Leu Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125Ser Val Phe Pro Leu Ala
Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr 130 135 140Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr145 150 155 160Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170
175Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
180 185 190Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn
Val Asp 195 200 205His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val
Glu Ser Lys Tyr 210 215 220Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro
Glu Phe Leu Gly Gly Pro225 230 235 240Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp 260 265 270Pro Glu Val
Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285Ala
Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val 290 295
300Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu305 310 315 320Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
Ser Ile Glu Lys 325 330 335Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr Thr 340 345 350Leu Pro Pro Ser Gln Glu Glu Met
Thr Lys Asn Gln Val Ser Leu Thr 355 360 365Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu385 390 395 400Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys 405 410
415Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Leu Gly 435 440 445177448PRTArtificial SequenceHumanized 52B8 HC
variable domain VH1 M64V/Human IgG4 (S228P) (K-) constant domain
177Glu Val Gln 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 Ser Asn
Tyr 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ala Thr Ile Ser Gly Gly Gly Asp Tyr Thr Asn Tyr Pro
Asp Ser Val 50 55 60Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Gly Arg Arg Leu Trp Phe Arg Ser Leu
Tyr Tyr Ala Met Asp Tyr Trp 100 105 110Gly Gln Gly Thr Leu Val Thr
Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125Ser Val Phe Pro Leu
Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr 130 135 140Ala Ala Leu
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr145 150 155
160Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
165 170 175Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr 180 185 190Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr
Cys Asn Val Asp 195 200 205His Lys Pro Ser Asn Thr Lys Val Asp Lys
Arg Val Glu Ser Lys Tyr 210 215 220Gly Pro Pro Cys Pro Pro Cys Pro
Ala Pro Glu Phe Leu Gly Gly Pro225 230 235 240Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp 260 265 270Pro
Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280
285Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val
290 295 300Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys Glu305 310 315 320Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
Ser Ser Ile Glu Lys 325 330 335Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr 340 345 350Leu Pro Pro Ser Gln Glu Glu
Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu385 390 395
400Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys
405 410 415Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met
His Glu 420 425 430Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Leu Gly 435 440 445178448PRTArtificial SequenceHumanized
52B8 HC variable domain VH1 M64L/Human IgG4 (S228P) (K-) constant
domain 178Glu Val Gln 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
Ser Asn Tyr 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45Ala Thr Ile Ser Gly Gly Gly Asp Tyr Thr Asn
Tyr Pro Asp Ser Leu 50 55 60Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Gly Arg Arg Leu Trp Phe Arg
Ser Leu Tyr Tyr Ala Met Asp Tyr Trp 100 105 110Gly Gln Gly Thr Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125Ser Val Phe
Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr 130 135 140Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr145 150
155 160Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
Pro 165 170 175Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val Thr 180 185 190Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr
Thr Cys Asn Val Asp 195 200 205His Lys Pro Ser Asn Thr Lys Val Asp
Lys Arg Val Glu Ser Lys Tyr 210 215 220Gly Pro Pro Cys Pro Pro Cys
Pro Ala Pro Glu Phe Leu Gly Gly Pro225 230 235 240Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp 260
265 270Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn 275 280 285Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr
Tyr Arg Val 290 295 300Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys Glu305 310 315 320Tyr Lys Cys Lys Val Ser Asn Lys
Gly Leu Pro Ser Ser Ile Glu Lys 325 330 335Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350Leu Pro Pro Ser
Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375
380Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu385 390 395 400Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
Thr Val Asp Lys 405 410 415Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
Cys Ser Val Met His Glu 420 425 430Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Leu Gly 435 440 445179448PRTArtificial
SequenceHumanized 52B8 HC variable domain VH1 M64V W101F/Human IgG4
(S228P) (K-) constant domain 179Glu Val Gln 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 Ser Asn Tyr 20 25 30Gly Met Ser Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Thr Ile Ser Gly Gly
Gly Asp Tyr Thr Asn Tyr Pro Asp Ser Val 50 55 60Arg Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Gly Arg
Arg Leu Phe Phe Arg Ser Leu Tyr Tyr Ala Met Asp Tyr Trp 100 105
110Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
115 120 125Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu
Ser Thr 130 135 140Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr145 150 155 160Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro 165 170 175Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190Val Pro Ser Ser Ser
Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp 195 200 205His Lys Pro
Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr 210 215 220Gly
Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro225 230
235 240Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser 245 250 255Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
Gln Glu Asp 260 265 270Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His Asn 275 280 285Ala Lys Thr Lys Pro Arg Glu Glu Gln
Phe Asn Ser Thr Tyr Arg Val 290 295 300Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys Glu305 310 315 320Tyr Lys Cys Lys
Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys 325 330 335Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345
350Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
355 360 365Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu 370 375 380Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu385 390 395 400Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Arg Leu Thr Val Asp Lys 405 410 415Ser Arg Trp Gln Glu Gly Asn
Val Phe Ser Cys Ser Val Met His Glu 420 425 430Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 435 440
445180448PRTArtificial SequenceHumanized 52B8 HC variable domain
VH1 M64V W101Y/Human IgG4 (S228P) (K-) constant domain 180Glu Val
Gln 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 Ser Asn Tyr 20 25
30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ala Thr Ile Ser Gly Gly Gly Asp Tyr Thr Asn Tyr Pro Asp Ser
Val 50 55 60Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Gly Arg Arg Leu Tyr Phe Arg Ser Leu Tyr Tyr
Ala Met Asp Tyr Trp 100 105 110Gly Gln Gly Thr Leu Val Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro 115 120 125Ser Val Phe Pro Leu Ala Pro
Cys Ser Arg Ser Thr Ser Glu Ser Thr 130 135 140Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr145 150 155 160Val Ser
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170
175Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
180 185 190Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn
Val Asp 195 200 205His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val
Glu Ser Lys Tyr 210 215 220Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro
Glu Phe Leu Gly Gly Pro225 230 235 240Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp 260 265 270Pro Glu Val
Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285Ala
Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val 290 295
300Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu305 310 315 320Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
Ser Ile Glu Lys 325 330 335Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr Thr 340 345 350Leu Pro Pro Ser Gln Glu Glu Met
Thr Lys Asn Gln Val Ser Leu Thr 355 360 365Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu385 390 395 400Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys 405 410
415Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Leu Gly 435 440 445181448PRTArtificial SequenceHumanized 52B8 HC
variable domain VH1 M64V W101Q/Human IgG4 (S228P) (K-) constant
domain 181Glu Val Gln 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
Ser Asn Tyr 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45Ala Thr Ile Ser Gly Gly Gly Asp Tyr Thr Asn
Tyr Pro Asp Ser Val 50 55 60Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Gly Arg Arg Leu Gln Phe Arg
Ser Leu Tyr Tyr Ala Met Asp Tyr Trp 100 105 110Gly Gln Gly Thr Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125Ser Val Phe
Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr 130 135 140Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr145 150
155 160Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
Pro 165 170 175Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val Thr 180 185 190Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr
Thr Cys Asn Val Asp 195 200 205His Lys Pro Ser Asn Thr Lys Val Asp
Lys Arg Val Glu Ser Lys Tyr 210 215 220Gly Pro Pro Cys Pro Pro Cys
Pro Ala Pro Glu Phe Leu Gly Gly Pro225 230 235 240Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp 260 265
270Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr
Arg Val 290 295 300Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu305 310 315 320Tyr Lys Cys Lys Val Ser Asn Lys Gly
Leu Pro Ser Ser Ile Glu Lys 325 330 335Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350Leu Pro Pro Ser Gln
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu385 390
395 400Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp
Lys 405 410 415Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val
Met His Glu 420 425 430Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Leu Gly 435 440 445182448PRTArtificial
SequenceHumanized 52B8 HC variable domain VH2/Human IgG4 (S228P)
(K-) constant domain 182Glu Val Gln 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 Ser Asn Tyr 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Thr Ile Ser Gly Gly Gly Asp
Tyr Thr Asn Tyr Pro Asp Ser Met 50 55 60Arg Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Gly Arg Arg Leu
Trp Phe Arg Ser Leu Tyr Tyr Ala Met Asp Tyr Trp 100 105 110Gly Gln
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120
125Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr
130 135 140Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr145 150 155 160Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly
Val His Thr Phe Pro 165 170 175Ala Val Leu Gln Ser Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val Thr 180 185 190Val Pro Ser Ser Ser Leu Gly
Thr Lys Thr Tyr Thr Cys Asn Val Asp 195 200 205His Lys Pro Ser Asn
Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr 210 215 220Gly Pro Pro
Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro225 230 235
240Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln
Glu Asp 260 265 270Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn 275 280 285Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
Asn Ser Thr Tyr Arg Val 290 295 300Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu305 310 315 320Tyr Lys Cys Lys Val
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys 325 330 335Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350Leu
Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360
365Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu385 390 395 400Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg
Leu Thr Val Asp Lys 405 410 415Ser Arg Trp Gln Glu Gly Asn Val Phe
Ser Cys Ser Val Met His Glu 420 425 430Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Leu Gly 435 440 445183448PRTArtificial
SequenceHumanized 52B8 HC variable domain VH2 M64V/Human IgG4
(S228P) (K-) constant domain 183Glu Val Gln 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 Ser Asn Tyr 20 25 30Gly Met Ser Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Thr Ile Ser Gly Gly
Gly Asp Tyr Thr Asn Tyr Pro Asp Ser Val 50 55 60Arg Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met
Asn Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Gly Arg
Arg Leu Trp Phe Arg Ser Leu Tyr Tyr Ala Met Asp Tyr Trp 100 105
110Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
115 120 125Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu
Ser Thr 130 135 140Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr145 150 155 160Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro 165 170 175Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190Val Pro Ser Ser Ser
Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp 195 200 205His Lys Pro
Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr 210 215 220Gly
Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro225 230
235 240Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser 245 250 255Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
Gln Glu Asp 260 265 270Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His Asn 275 280 285Ala Lys Thr Lys Pro Arg Glu Glu Gln
Phe Asn Ser Thr Tyr Arg Val 290 295 300Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys Glu305 310 315 320Tyr Lys Cys Lys
Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys 325 330 335Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345
350Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
355 360 365Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu 370 375 380Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu385 390 395 400Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Arg Leu Thr Val Asp Lys 405 410 415Ser Arg Trp Gln Glu Gly Asn
Val Phe Ser Cys Ser Val Met His Glu 420
425 430Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu
Gly 435 440 445184448PRTArtificial SequenceHumanized 52B8 HC
variable domain VH2 M64L/Human IgG4 (S228P) (K-) constant domain
184Glu Val Gln 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 Ser Asn
Tyr 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ala Thr Ile Ser Gly Gly Gly Asp Tyr Thr Asn Tyr Pro
Asp Ser Leu 50 55 60Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Lys Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Gly Arg Arg Leu Trp Phe Arg Ser Leu
Tyr Tyr Ala Met Asp Tyr Trp 100 105 110Gly Gln Gly Thr Leu Val Thr
Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125Ser Val Phe Pro Leu
Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr 130 135 140Ala Ala Leu
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr145 150 155
160Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
165 170 175Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr 180 185 190Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr
Cys Asn Val Asp 195 200 205His Lys Pro Ser Asn Thr Lys Val Asp Lys
Arg Val Glu Ser Lys Tyr 210 215 220Gly Pro Pro Cys Pro Pro Cys Pro
Ala Pro Glu Phe Leu Gly Gly Pro225 230 235 240Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp 260 265 270Pro
Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280
285Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val
290 295 300Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys Glu305 310 315 320Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
Ser Ser Ile Glu Lys 325 330 335Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr 340 345 350Leu Pro Pro Ser Gln Glu Glu
Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu385 390 395
400Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys
405 410 415Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met
His Glu 420 425 430Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Leu Gly 435 440 445185451PRTArtificial SequenceHumanized
52B8 HC variable domain VH1/ Human IgG1 HC (L234A L235A D265S) (K-)
constant domain 185Glu Val Gln 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 Ser Asn Tyr 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45Ala Thr Ile Ser Gly Gly Gly Asp Tyr
Thr Asn Tyr Pro Asp Ser Met 50 55 60Arg Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Gly Arg Arg Leu Trp
Phe Arg Ser Leu Tyr Tyr Ala Met Asp Tyr Trp 100 105 110Gly Gln Gly
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125Ser
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135
140Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr145 150 155 160Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro 165 170 175Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr 180 185 190Val Pro Ser Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205His Lys Pro Ser Asn Thr
Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220Cys Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala225 230 235 240Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250
255Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Ser Val Ser
260 265 270His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu 275 280 285Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr 290 295 300Tyr Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn305 310 315 320Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350Val Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 355 360 365Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375
380Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro385 390 395 400Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr 405 410 415Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val 420 425 430Met His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445Ser Pro Gly
450186451PRTArtificial SequenceHumanized 52B8 HC variable domain
VH1 M64V/ Human IgG1 HC (L234A L235A D265S) (K-) constant domain
186Glu Val Gln 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 Ser Asn
Tyr 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ala Thr Ile Ser Gly Gly Gly Asp Tyr Thr Asn Tyr Pro
Asp Ser Val 50 55 60Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Gly Arg Arg Leu Trp Phe Arg Ser Leu
Tyr Tyr Ala Met Asp Tyr Trp 100 105 110Gly Gln Gly Thr Leu Val Thr
Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125Ser Val Phe Pro Leu
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140Ala Ala Leu
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr145 150 155
160Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
165 170 175Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr 180 185 190Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val Asn 195 200 205His Lys Pro Ser Asn Thr Lys Val Asp Lys
Lys Val Glu Pro Lys Ser 210 215 220Cys Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Ala Ala225 230 235 240Gly Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Ser Val Ser 260 265 270His
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280
285Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
290 295 300Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn305 310 315 320Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro 325 330 335Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln 340 345 350Val Tyr Thr Leu Pro Pro Ser
Arg Asp Glu Leu Thr Lys Asn Gln Val 355 360 365Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro385 390 395
400Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
405 410 415Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val 420 425 430Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu 435 440 445Ser Pro Gly 450187451PRTArtificial
SequenceHumanized 52B8 HC variable domain VH1 M64L/ Human IgG1 HC
(L234A L235A D265S) (K-) constant domain 187Glu Val Gln 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 Ser Asn Tyr 20 25 30Gly Met Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Thr Ile
Ser Gly Gly Gly Asp Tyr Thr Asn Tyr Pro Asp Ser Leu 50 55 60Arg Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Gly Arg Arg Leu Trp Phe Arg Ser Leu Tyr Tyr Ala Met Asp Tyr
Trp 100 105 110Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro 115 120 125Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr 130 135 140Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr145 150 155 160Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190Val Pro
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200
205His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
210 215 220Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Ala Ala225 230 235 240Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu 245 250 255Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Ser Val Ser 260 265 270His Glu Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn305 310 315
320Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
325 330 335Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln 340 345 350Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val 355 360 365Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val 370 375 380Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro385 390 395 400Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430Met
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440
445Ser Pro Gly 450188451PRTArtificial SequenceHumanized 52B8 HC
variable domain VH1 M64V W101F/ Human IgG1 HC (L234A L235A D265S)
(K-) constant domain 188Glu Val Gln 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 Ser Asn Tyr 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Thr Ile Ser Gly Gly Gly Asp
Tyr Thr Asn Tyr Pro Asp Ser Val 50 55 60Arg Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Gly Arg Arg Leu
Phe Phe Arg Ser Leu Tyr Tyr Ala Met Asp Tyr Trp 100 105 110Gly Gln
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120
125Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
130 135 140Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr145 150 155 160Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly
Val His Thr Phe Pro 165 170 175Ala Val Leu Gln Ser Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val Thr 180 185 190Val Pro Ser Ser Ser Leu Gly
Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205His Lys Pro Ser Asn
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220Cys Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala225 230 235
240Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
245 250 255Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Ser
Val Ser 260 265 270His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu 275 280 285Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr 290 295 300Tyr Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn305 310 315 320Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350Val
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 355 360
365Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
370 375 380Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro385 390 395 400Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr 405 410 415Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val 420 425 430Met His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445Ser Pro Gly
450189451PRTArtificial SequenceHumanized 52B8 HC variable domain
VH1 M64V W101Y/ Human IgG1 HC (L234A L235A D265S) (K-) constant
domain 189Glu Val Gln 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
Ser Asn Tyr 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45Ala Thr Ile Ser Gly Gly Gly Asp Tyr Thr Asn
Tyr Pro Asp Ser Val 50 55 60Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95Gly Arg Arg Leu Tyr Phe Arg Ser Leu Tyr Tyr Ala Met Asp Tyr Trp
100 105 110Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro 115 120 125Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr 130 135 140Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr145 150 155 160Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205His
Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215
220Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala
Ala225 230 235 240Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu 245 250 255Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Ser Val Ser 260 265 270His Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu 275 280 285Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300Tyr Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn305 310 315 320Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330
335Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
340 345 350Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
Gln Val 355 360 365Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val 370 375 380Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro385 390 395 400Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430Met His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445Ser
Pro Gly 450190451PRTArtificial SequenceHumanized 52B8 HC variable
domain VH1 M64V W101Q/ Human IgG1 HC (L234A L235A D265S) (K-)
constant domain 190Glu Val Gln 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 Ser Asn Tyr 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45Ala Thr Ile Ser Gly Gly Gly Asp Tyr
Thr Asn Tyr Pro Asp Ser Val 50 55 60Arg Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Gly Arg Arg Leu Gln
Phe Arg Ser Leu Tyr Tyr Ala Met Asp Tyr Trp 100 105 110Gly Gln Gly
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125Ser
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135
140Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr145 150 155 160Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro 165 170 175Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr 180 185 190Val Pro Ser Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205His Lys Pro Ser Asn Thr
Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220Cys Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala225 230 235 240Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250
255Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Ser Val Ser
260 265 270His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu 275 280 285Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr 290 295 300Tyr Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn305 310 315 320Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350Val Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 355 360 365Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375
380Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro385 390 395 400Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr 405 410 415Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val 420 425 430Met His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445Ser Pro Gly
450191451PRTArtificial SequenceHumanized 52B8 HC variable domain
VH2/ Human IgG1 HC (L234A L235A D265S) (K-) constant domain 191Glu
Val Gln 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 Ser Asn Tyr
20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ala Thr Ile Ser Gly Gly Gly Asp Tyr Thr Asn Tyr Pro Asp
Ser Met 50 55 60Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Lys Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Gly Arg Arg Leu Trp Phe Arg Ser Leu Tyr
Tyr Ala Met Asp Tyr Trp 100 105 110Gly Gln Gly Thr Leu Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125Ser Val Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr145 150 155 160Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170
175Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
180 185 190Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
Val Asn 195 200 205His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val
Glu Pro Lys Ser 210 215 220Cys Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Ala Ala225 230 235 240Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Ser Val Ser 260 265 270His Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295
300Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn305 310 315 320Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro 325 330 335Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln 340 345 350Val Tyr Thr Leu Pro Pro Ser Arg
Asp Glu Leu Thr Lys Asn Gln Val 355 360 365Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro385 390 395 400Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410
415Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
420 425 430Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu 435 440 445Ser Pro Gly 450192451PRTArtificial
SequenceHumanized 52B8 HC variable domain VH2 M64V/ Human IgG1 HC
(L234A L235A D265S) (K-) constant domain 192Glu Val Gln 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 Ser Asn Tyr 20 25 30Gly Met Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Thr Ile
Ser Gly Gly Gly Asp Tyr Thr Asn Tyr Pro Asp Ser Val 50 55 60Arg Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Gly Arg Arg Leu Trp Phe Arg Ser Leu Tyr Tyr Ala Met Asp Tyr
Trp 100 105 110Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro 115 120 125Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr 130 135 140Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr145 150 155 160Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190Val Pro
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200
205His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
210 215 220Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Ala Ala225 230 235 240Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu 245 250 255Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Ser Val Ser 260 265 270His Glu Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn305 310 315
320Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
325 330 335Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln 340 345 350Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val 355 360 365Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val 370 375 380Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro385 390 395 400Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430Met
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440
445Ser Pro Gly 450193451PRTArtificial SequenceHumanized 52B8 HC
variable domain VH2 M64L/ Human IgG1 HC (L234A L235A D265S) (K-)
constant domain 193Glu Val Gln 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 Ser Asn Tyr 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45Ala Thr Ile Ser Gly Gly Gly Asp Tyr
Thr Asn Tyr Pro Asp Ser Leu 50 55 60Arg Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu
Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Gly Arg Arg Leu Trp
Phe Arg Ser Leu Tyr Tyr Ala Met Asp Tyr Trp 100 105 110Gly Gln Gly
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125Ser
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135
140Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr145 150 155 160Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro 165 170 175Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr 180 185 190Val Pro Ser Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205His Lys Pro Ser Asn Thr
Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220Cys Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala225 230 235 240Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250
255Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Ser Val Ser
260 265 270His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu 275 280 285Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr 290 295 300Tyr Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn305 310 315 320Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350Val Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 355 360 365Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375
380Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro385 390 395 400Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr 405 410 415Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val 420 425 430Met His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445Ser Pro Gly
450194451PRTArtificial SequenceChimeric Anti-ILT3 rat 40A6 parental
HC variable domain/human IgG4 (S228P) constant domain 194Gln Val
Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Gln Ala Ser Glu1 5 10 15Thr
Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25
30Ser Ile Asn Trp Val Arg Gln Ser Ser Gly Lys Gly Pro Glu Trp Met
35 40 45Gly Arg Phe Trp Tyr Asp Glu Gly Ile Ala Tyr Asn Leu Thr Leu
Glu 50 55 60Ser Arg Leu Ser Ile Ser Gly Asp Thr Ser Lys Asn Gln Val
Phe Leu65 70 75 80Lys Met Asn Ser Leu Arg Thr Gly Asp Thr Gly Thr
Tyr Tyr Cys Thr 85 90 95Arg Asp Arg Asp Thr Val Gly Ile Thr Gly Trp
Phe Ala Tyr Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155 160Ser Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170
175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
180 185 190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His 195 200 205Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
Pro Lys Ser Cys 210 215 220Asp Lys Thr His Thr Cys Pro Pro Cys
Pro
Ala Pro Glu Ala Ala Gly225 230 235 240Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Ser Val Ser His 260 265 270Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295
300Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly305 310 315 320Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile 325 330 335Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 340 345 350Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 355 360 365Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro385 390 395 400Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410
415Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
420 425 430His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 435 440 445Pro Gly Lys 450195214PRTArtificial
SequenceChimeric Anti-ILT3 rat 40A6 parental LC variable
domain/human kappa 195Glu Thr Val Met Thr Gln Ser Pro Thr Ser Leu
Ser Ala Ser Ile Gly1 5 10 15Glu Arg Val Thr Leu Asn Cys Lys Ala Ser
Gln Ser Val Gly Val Asn 20 25 30Val Asp Trp Tyr Gln Gln Thr Pro Gly
Gln Ser Pro Lys Leu Leu Ile 35 40 45Tyr Gly Ser Ala Asn Arg His Thr
Gly Val Pro Asp Arg Phe Thr Gly 50 55 60Ser Gly Phe Gly Ser Asp Phe
Thr Leu Thr Ile Ser Asp Val Glu Pro65 70 75 80Glu Asp Leu Gly Val
Tyr Tyr Cys Leu Gln Tyr Gly Ser Val Pro Tyr 85 90 95Thr Phe Gly Ala
Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala 100 105 110Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120
125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu
Cys 210196451PRTArtificial SequenceChimeric Anti-ILT3 rat 16B1
parental HC variable domain/human IgG4 (S228P) constant domain
196Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Gln Ala Ser Glu1
5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn
Tyr 20 25 30Cys Val Asn Trp Val Arg Gln Pro Ser Gly Lys Gly Pro Glu
Trp Leu 35 40 45Gly Arg Phe Trp Phe Asp Glu Gly Lys Ala Tyr Asn Leu
Thr Leu Glu 50 55 60Ser Arg Leu Ser Ile Ser Gly Asp Thr Ser Lys Asn
Gln Val Phe Leu65 70 75 80Arg Met Asn Ser Leu Arg Ala Asp Asp Thr
Gly Thr Tyr Tyr Cys Thr 85 90 95Arg Asp Arg Asp Thr Val Gly Ile Thr
Gly Trp Phe Ala Tyr Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155
160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr Lys Val Asp Lys Lys
Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Ala Ala Gly225 230 235 240Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Ser Val Ser His 260 265 270Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280
285His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
290 295 300Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly305 310 315 320Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile 325 330 335Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val 340 345 350Tyr Thr Leu Pro Pro Ser Arg
Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro385 390 395
400Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
405 410 415Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met 420 425 430His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser 435 440 445Pro Gly Lys 450197214PRTArtificial
SequenceChimeric Anti-ILT3 rat 16B1 parental LC variable
domain/human kappa 197Glu Thr Val Met Thr Gln Ser Pro Thr Ser Leu
Ser Ala Ser Ile Gly1 5 10 15Glu Arg Val Thr Leu Asn Cys Lys Ala Ser
Gln Ser Val Gly Ile Asn 20 25 30Val Asp Trp Tyr Gln Gln Thr Pro Gly
Gln Ser Pro Lys Leu Leu Ile 35 40 45Tyr Gly Ser Ala Asn Arg His Thr
Gly Val Pro Asp Arg Phe Thr Gly 50 55 60Ser Gly Phe Gly Ser Asp Phe
Thr Leu Thr Ile Ser Asn Val Glu Pro65 70 75 80Glu Asp Leu Gly Val
Tyr Tyr Cys Leu Gln Tyr Gly Ser Val Pro Tyr 85 90 95Thr Phe Gly Pro
Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala 100 105 110Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120
125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu
Cys 210198448PRTArtificial SequenceChimeric Anti-ILT3 mouse 11D1
parental HC variable domain/human IgG4 (S228P) constant domain
198Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Met Lys Pro Gly Ala1
5 10 15Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe Arg Thr
Tyr 20 25 30Trp Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu
Trp Ile 35 40 45Gly Glu Ile Leu Pro Gly Asn Gly Asn Thr His Phe Asn
Glu Asn Phe 50 55 60Lys Asp Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser
Asn Ala Ala Tyr65 70 75 80Met Gln Leu Ser Ser Leu Thr Ser Glu Asp
Ser Ala Val Tyr Tyr Cys 85 90 95Val Arg Arg Leu Gly Arg Gly Pro Phe
Asp Phe Trp Gly Gln Gly Thr 100 105 110Thr Leu Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn145 150 155
160Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
Ser Ser 180 185 190Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
His Lys Pro Ser 195 200 205Asn Thr Lys Val Asp Lys Lys Val Glu Pro
Lys Ser Cys Asp Lys Thr 210 215 220His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Ala Ala Gly Gly Pro Ser225 230 235 240Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255Thr Pro Glu
Val Thr Cys Val Val Val Ser Val Ser His Glu Asp Pro 260 265 270Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280
285Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
290 295 300Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu Tyr305 310 315 320Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr 325 330 335Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu 340 345 350Pro Pro Ser Arg Asp Glu Leu
Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp385 390 395
400Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
405 410 415Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
Glu Ala 420 425 430Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro Gly Lys 435 440 445199214PRTArtificial SequenceChimeric
Anti-ILT3 mouse 11D1 parental LC variable domain/human kappa 199Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Val Ser Leu Gly1 5 10
15Gly Lys Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Glu Tyr
20 25 30Ile Gly Trp Tyr Gln Arg Lys Pro Gly Lys Gly Pro Arg Leu Leu
Ile 35 40 45His Tyr Thr Ser Thr Leu Gln Ser Gly Ile Pro Ser Arg Phe
Ser Gly 50 55 60Ser Gly Ser Gly Arg Asp Tyr Ser Leu Ser Ile Ser Asn
Leu Glu Pro65 70 75 80Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr
Ala Asn Pro Leu Pro 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro
Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys
Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170
175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys 210200452PRTArtificial
SequenceChimeric Anti-ILT3 rat 17H12 parental HC variable
domain/human IgG4 (S228P) constant domain 200Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg1 5 10 15Ser Met Lys Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Phe 20 25 30Asp Met Ala
Trp Val Arg Gln Ala Pro Thr Arg Gly Leu Glu Trp Val 35 40 45Ser Ser
Ile Thr Tyr Asp Gly Gly Ser Thr Ser Tyr Arg Asp Ser Val 50 55 60Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Gly Thr Leu Tyr65 70 75
80Leu Gln Met Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys
85 90 95Thr Thr Val Glu Ser Ile Ala Thr Ile Ser Thr Tyr Phe Asp Tyr
Trp 100 105 110Gly Gln Gly Val Met Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro 115 120 125Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr 130 135 140Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr145 150 155 160Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190Val Pro
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200
205His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
210 215 220Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Ala Ala225 230 235 240Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu 245 250 255Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Ser Val Ser 260 265 270His Glu Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn305 310 315
320Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
325 330 335Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln 340 345 350Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val 355 360 365Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val 370 375 380Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro385 390 395 400Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430Met
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440
445Ser Pro Gly Lys 450201217PRTArtificial SequenceChimeric
Anti-ILT3 rat 17H12 parental LC variable domain/human kappa 201Asp
Ile Val Leu Thr Gln Ser Pro Ala Leu Ala Val Ser Leu Gly Gln1 5 10
15Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val Ser Met Ser Arg
20 25 30Tyr Asp Leu Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Gln Pro
Lys 35 40 45Leu Leu Ile Phe Arg Ala Ser Asp Leu Ala Ser Gly Ile Pro
Ala Arg 50 55 60Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Asn Pro65 70 75 80Val Gln Ala Asp Asp Ile Ala Thr Tyr Tyr Cys
Gln Gln Thr Arg Lys 85 90 95Ser Pro Pro Thr Phe Gly Gly Gly Thr Arg
Leu Glu Leu Lys Arg Thr 100 105 110Val Ala Ala Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125Lys Ser Gly Thr Ala Ser
Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140Arg Glu Ala Lys
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly145 150
155 160Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
Tyr 165 170 175Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
Glu Lys His 180 185 190Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly
Leu Ser Ser Pro Val 195 200 205Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215202451PRTArtificial SequenceChimeric Anti-ILT3 rat 37C8
parental HC variable domain/human IgG4 (S228P) constant domain
202Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Gln Ala Ser Glu1
5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser
Tyr 20 25 30Cys Val Asn Trp Val Arg Gln Pro Ser Gly Lys Gly Pro Glu
Trp Leu 35 40 45Gly Arg Phe Trp Tyr Asp Glu Gly Lys Val Tyr Asn Leu
Thr Leu Glu 50 55 60Ser Arg Leu Ser Ile Ser Gly Asp Thr Ser Lys Asn
Gln Val Phe Leu65 70 75 80Lys Met Asn Arg Leu Arg Thr Asp Asp Thr
Gly Thr Tyr Tyr Cys Thr 85 90 95Arg Asp Arg Asp Thr Met Gly Ile Thr
Gly Trp Phe Ala Tyr Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155
160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr Lys Val Asp Lys Lys
Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Ala Ala Gly225 230 235 240Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Ser Val Ser His 260 265 270Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280
285His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
290 295 300Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly305 310 315 320Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile 325 330 335Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val 340 345 350Tyr Thr Leu Pro Pro Ser Arg
Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro385 390 395
400Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
405 410 415Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met 420 425 430His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser 435 440 445Pro Gly Lys 450203214PRTArtificial
SequenceChimeric Anti-ILT3 rat 37C8 parental LC variable
domain/human kappa 203Glu Thr Val Met Thr Gln Ser Pro Thr Ser Leu
Ser Ala Ser Ile Gly1 5 10 15Glu Arg Val Thr Leu Asn Cys Lys Ala Ser
Gln Ser Val Gly Ile Asn 20 25 30Val Asp Trp Tyr Gln Gln Thr Pro Gly
Gln Ser Pro Lys Leu Leu Ile 35 40 45Tyr Gly Ser Ala Asn Arg His Thr
Gly Val Pro Asp Arg Phe Thr Gly 50 55 60Ser Gly Phe Gly Ser Gly Phe
Thr Leu Thr Ile Ser Asn Val Glu Pro65 70 75 80Glu Asp Leu Gly Val
Tyr Tyr Cys Leu Gln Tyr Gly Ser Val Pro Tyr 85 90 95Thr Phe Gly Pro
Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala 100 105 110Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120
125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu
Cys 210204448PRTArtificial SequenceChimeric Anti-ILT3 mouse 1G12
parental HC variable domain/human IgG4 (S228P) constant domain
204Gln Val Gln Met Gln Gln Ser Gly Thr Glu Leu Met Lys Pro Gly Ala1
5 10 15Ser Met Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe Ser Thr
Tyr 20 25 30Trp Ile Gln Trp Ile Lys Gln Arg Pro Gly His Gly Leu Glu
Trp Ile 35 40 45Gly Glu Ile Leu Pro Gly Ser Gly Thr Thr Asn Tyr Asn
Glu Asn Phe 50 55 60Lys Gly Lys Ala Thr Phe Ser Ala Asp Thr Ser Ser
Asn Thr Ala Tyr65 70 75 80Ile His Leu Ser Ser Leu Thr Ser Glu Asp
Ser Ala Val Phe Tyr Cys 85 90 95Ala Arg Arg Leu Gly Arg Gly Pro Phe
Asp Tyr Trp Gly Gln Gly Thr 100 105 110Thr Leu Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn145 150 155
160Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
Ser Ser 180 185 190Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
His Lys Pro Ser 195 200 205Asn Thr Lys Val Asp Lys Lys Val Glu Pro
Lys Ser Cys Asp Lys Thr 210 215 220His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Ala Ala Gly Gly Pro Ser225 230 235 240Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255Thr Pro Glu
Val Thr Cys Val Val Val Ser Val Ser His Glu Asp Pro 260 265 270Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280
285Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
290 295 300Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu Tyr305 310 315 320Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr 325 330 335Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu 340 345 350Pro Pro Ser Arg Asp Glu Leu
Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp385 390 395
400Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
405 410 415Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
Glu Ala 420 425 430Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro Gly Lys 435 440 445205214PRTArtificial SequenceChimeric
Anti-ILT3 mouse 1G12 parental LC variable domain/human kappa 205Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly1 5 10
15Gly Lys Val Thr Ile Thr Cys Glu Ala Ser Gln Asp Ile Asn Lys His
20 25 30Ile Asp Trp Tyr Gln His Gln Pro Gly Arg Gly Pro Ser Leu Leu
Ile 35 40 45His Tyr Ala Ser Ile Leu Gln Pro Gly Ile Pro Ser Arg Phe
Ser Gly 50 55 60Ser Gly Ser Gly Arg Asp Tyr Ser Phe Ser Ile Thr Ser
Leu Glu Pro65 70 75 80Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr
Asp Asn Leu Leu Pro 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro
Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys
Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170
175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys 210206451PRTArtificial
SequenceChimeric Anti-ILT3 rat 20E4 parental HC variable
domain/human IgG4 (S228P) constant domain 206Gln Val Gln Leu Lys
Glu Ser Gly Pro Gly Leu Val Gln Ala Ser Glu1 5 10 15Thr Leu Ser Leu
Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30Ser Val Asn
Trp Val Arg Gln Pro Ser Gly Lys Gly Leu Glu Trp Met 35 40 45Gly Arg
Phe Trp Tyr Asp Gly Gly Thr Ala Tyr Asn Ser Thr Leu Glu 50 55 60Ser
Arg Leu Ser Ile Ser Gly Asp Thr Ser Lys Asn Gln Val Phe Leu65 70 75
80Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Gly Thr Tyr Tyr Cys Thr
85 90 95Arg Asp Arg Asp Thr Met Gly Ile Thr Gly Trp Phe Ala Tyr Trp
Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Pro Ala Ser Thr Lys
Gly Pro Ser 115 120 125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200
205Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
210 215 220Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala
Ala Gly225 230 235 240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met 245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Ser Val Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly305 310 315
320Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
325 330 335Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val 340 345 350Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
Asn Gln Val Ser 355 360 365Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu 370 375 380Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro385 390 395 400Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440
445Pro Gly Lys 450207214PRTArtificial SequenceChimeric Anti-ILT3
rat 20E4 parental LC variable domain/human kappa 207Glu Thr Val Met
Thr Gln Ser Pro Thr Ser Leu Ser Ala Ser Ile Gly1 5 10 15Glu Arg Val
Thr Leu Asn Cys Lys Ala Ser Gln Ser Val Gly Val Asn 20 25 30Val Asp
Trp Tyr Gln Gln Thr Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45Tyr
Gly Ser Ala Asn Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55
60Ser Gly Phe Gly Ser Asp Phe Thr Leu Thr Ile Ser Asn Val Glu Pro65
70 75 80Glu Asp Leu Gly Val Tyr Tyr Cys Leu Gln Tyr Gly Ser Val Pro
Tyr 85 90 95Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val
Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn
Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn
Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu
Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu
Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200
205Phe Asn Arg Gly Glu Cys 210208451PRTArtificial SequenceChimeric
Anti-ILT3 rat 24A4 parental HC variable domain/human IgG4 (S228P)
constant domain 208Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val
Gln Ala Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe
Ser Leu Thr Ser Tyr 20 25 30Cys Val Asn Trp Val Arg Gln Pro Ser Gly
Lys Gly Pro Glu Trp Leu 35 40 45Gly Arg Phe Trp Tyr Asp Glu Gly Lys
Val Tyr Asn Leu Thr Leu Glu 50 55 60Ser Arg Leu Ser Ile Ser Gly Asp
Thr Ser Lys Asn Gln Val Phe Leu65 70 75 80Lys Met Asn Arg Leu Arg
Thr Asp Asp Thr Gly Thr Tyr Tyr Cys Thr 85 90 95Arg Asp Arg Asp Thr
Leu Gly Ile Thr Gly Trp Phe Ala Tyr Trp Gly 100 105 110Gln Gly Thr
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125Val
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135
140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr Lys
Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly225 230 235 240Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250
255Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Ser Val Ser His
260 265 270Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val 275 280 285His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr 290 295 300Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly305 310 315
320Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
325 330 335Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val 340 345 350Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
Asn Gln Val Ser 355 360 365Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu 370 375 380Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro385 390 395 400Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440
445Pro Gly Lys 450209214PRTArtificial SequenceChimeric Anti-ILT3
rat 24A4 parental LC variable domain/human kappa 209Glu Thr Val Met
Thr Gln Ser Pro Thr Ser Leu Ser Ala Ser Ile Gly1 5 10 15Glu Arg Val
Thr Leu Asn Cys Lys Ala Ser Gln Ser Val Gly Ile Asn 20 25 30Val Asp
Trp Tyr Gln Gln Thr Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45Tyr
Gly Ser Ala Asn Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55
60Ser Gly Phe Gly Ser Gly Phe Thr Leu Thr Ile Ser Asn Val Glu Pro65
70 75 80Glu Asp Leu Gly Val Tyr Tyr Cys Leu Gln Tyr Gly Ser Val Pro
Tyr 85 90 95Thr Phe Gly Pro Gly Thr Lys Leu Glu Leu Lys Arg Thr Val
Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn
Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn
Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu
Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu
Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200
205Phe Asn Arg Gly Glu Cys 210210452PRTArtificial SequenceHumanized
52B8 HC variable domain VH1 M64V/ Human IgG1 HC (N297A) constant
domain 210Glu Val Gln 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
Ser Asn Tyr 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45Ala Thr Ile Ser Gly Gly Gly Asp Tyr Thr Asn
Tyr Pro Asp Ser Val 50 55 60Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Gly Arg Arg Leu Trp Phe Arg
Ser Leu Tyr Tyr Ala Met Asp Tyr Trp 100 105 110Gly Gln Gly Thr Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125Ser Val Phe
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr145 150
155 160Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
Pro 165 170 175Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val Thr 180 185 190Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr
Ile Cys Asn Val Asn 195 200 205His Lys Pro Ser Asn Thr Lys Val Asp
Lys Lys Val Glu Pro Lys Ser 210 215 220Cys Asp Lys Thr His Thr Cys
Pro Pro Cys Pro Ala Pro Glu Leu Leu225 230 235 240Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265
270His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
275 280 285Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala
Ser Thr 290 295 300Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn305 310 315 320Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro 325 330 335Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350Val Tyr Thr Leu Pro
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 355 360 365Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro385 390
395 400Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr 405 410 415Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val 420 425 430Met His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu 435 440 445Ser Pro Gly Lys
450211330PRTArtificial SequenceHuman IgG1 HC constant domain
(N297A) 211Ala 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 95Lys 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 Ala 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 Asp Glu225 230 235 240Leu 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 330212451PRTArtificial SequenceChimeric anti-ILT3 40A6 rat
VH /human IgG1 (N297A) 212Gln Val Gln Leu Lys Glu Ser Gly Pro Gly
Leu Val Gln Ala Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser
Gly Phe Ser Leu Thr Ser Tyr 20 25 30Ser Ile Asn Trp Val Arg Gln Ser
Ser Gly Lys Gly Pro Glu Trp Met 35 40 45Gly Arg Phe Trp Tyr Asp Glu
Gly Ile Ala Tyr Asn Leu Thr Leu Glu 50 55 60Ser Arg Leu Ser Ile Ser
Gly Asp Thr Ser Lys Asn Gln Val Phe Leu65 70 75 80Lys Met Asn Ser
Leu Arg Thr Gly Asp Thr Gly Thr Tyr Tyr Cys Thr 85 90 95Arg Asp Arg
Asp Thr Val Gly Ile Thr Gly Trp Phe Ala Tyr Trp Gly 100 105 110Gln
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120
125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly225 230 235
240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 275 280 285His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Ala Ser Thr Tyr 290 295 300Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly305 310 315 320Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350Tyr
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360
365Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
370 375 380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro385 390 395 400Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val 405 410 415Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met 420 425 430His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445Pro Gly Lys
450213451PRTArtificial SequenceChimeric anti-ILT3 16B1 rat VH
/human IgG1 (N297A) 213Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu
Val Gln Ala Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly
Phe Ser Leu Thr Asn Tyr 20 25 30Cys Val Asn Trp Val Arg Gln Pro Ser
Gly Lys Gly Pro Glu Trp Leu 35 40 45Gly Arg Phe Trp Phe Asp Glu Gly
Lys Ala Tyr Asn Leu Thr Leu Glu 50 55 60Ser Arg Leu Ser Ile Ser Gly
Asp Thr Ser Lys Asn Gln Val Phe Leu65 70 75 80Arg Met Asn Ser Leu
Arg Ala Asp Asp Thr Gly Thr Tyr Tyr Cys Thr 85 90 95Arg Asp Arg Asp
Thr Val Gly Ile Thr Gly Trp Phe Ala Tyr Trp Gly 100 105 110Gln Gly
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120
125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly225 230 235
240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 275 280 285His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Ala Ser Thr Tyr 290 295 300Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly305 310 315 320Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350Tyr
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360
365Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
370 375 380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro385 390 395 400Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val 405 410 415Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met 420 425 430His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445Pro Gly Lys
450214448PRTArtificial SequenceChimeric anti-ILT3 11D1 mouse VH
/human IgG1 (N297A) 214Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu
Met Lys Pro Gly Ala1 5 10 15Ser Val Lys Ile Ser Cys Lys Ala Thr Gly
Tyr Thr Phe Arg Thr Tyr 20 25 30Trp Ile Glu Trp Val Lys Gln Arg Pro
Gly His Gly Leu Glu Trp Ile 35 40 45Gly Glu Ile Leu Pro Gly Asn Gly
Asn Thr His Phe Asn Glu Asn Phe 50 55 60Lys Asp Lys Ala Thr Phe Thr
Ala Asp Thr Ser Ser Asn Ala Ala Tyr65 70 75 80Met Gln Leu Ser Ser
Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Val Arg Arg Leu
Gly Arg Gly Pro Phe Asp Phe Trp Gly Gln Gly Thr 100 105 110Thr Leu
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120
125Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
Trp Asn145 150 155 160Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
Pro Ala Val Leu Gln 165 170 175Ser Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val Thr Val Pro Ser Ser 180 185 190Ser Leu Gly Thr Gln Thr Tyr
Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205Asn Thr Lys Val Asp
Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220His Thr Cys
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser225 230 235
240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
Asp Pro 260 265 270Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala 275 280 285Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala
Ser Thr Tyr Arg Val Val 290 295 300Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr305 310 315 320Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350Pro
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360
365Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser 370 375 380Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp385 390 395 400Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425
430Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445215452PRTArtificial SequenceChimeric anti-ILT3 17H12 rat
VH /human IgG1 (N297A) 215Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Arg1 5 10 15Ser Met Lys Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Asn Phe 20 25 30Asp Met Ala Trp Val Arg Gln Ala
Pro Thr Arg Gly Leu Glu Trp Val 35 40 45Ser Ser Ile Thr Tyr Asp Gly
Gly Ser Thr Ser Tyr Arg Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Gly Thr Leu Tyr65 70 75 80Leu Gln Met Asp
Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95Thr Thr Val
Glu Ser Ile Ala Thr Ile Ser Thr Tyr Phe Asp Tyr Trp 100 105 110Gly
Gln Gly Val Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120
125Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
130 135 140Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr145 150 155 160Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly
Val His Thr Phe Pro 165 170 175Ala Val Leu Gln Ser Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val Thr 180 185 190Val Pro Ser Ser Ser Leu Gly
Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205His Lys Pro Ser Asn
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220Cys Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu225 230 235
240Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
245 250 255Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser 260 265 270His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu 275 280 285Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Ala Ser Thr 290 295 300Tyr Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn305 310 315 320Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350Val
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 355 360
365Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
370 375 380Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro385 390 395 400Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr 405 410 415Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val 420 425 430Met His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445Ser Pro Gly Lys
450216451PRTArtificial SequenceChimeric anti-ILT3 37C8 rat VH
/human IgG1 (N297A) 216Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu
Val Gln Ala Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly
Phe Ser Leu Thr Ser Tyr 20 25 30Cys Val Asn Trp Val Arg Gln Pro Ser
Gly Lys Gly Pro Glu Trp Leu 35 40 45Gly Arg Phe Trp Tyr Asp Glu Gly
Lys Val Tyr Asn Leu Thr Leu Glu 50 55 60Ser Arg Leu Ser Ile Ser Gly
Asp Thr Ser Lys Asn Gln Val Phe Leu65 70 75 80Lys Met Asn Arg Leu
Arg Thr Asp Asp Thr Gly Thr Tyr Tyr Cys Thr 85 90 95Arg Asp Arg Asp
Thr Met Gly Ile Thr Gly Trp Phe Ala Tyr Trp Gly 100 105 110Gln Gly
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120
125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly225 230 235
240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 275 280 285His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Ala Ser Thr Tyr 290 295 300Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly305 310 315 320Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350Tyr
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360
365Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
370 375 380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro385 390 395 400Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val 405 410 415Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met 420 425 430His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445Pro Gly Lys
450217448PRTArtificial SequenceChimeric anti-ILT3 1G12 mouse VH
/human IgG1 (N297A) 217Gln Val Gln Met Gln Gln Ser Gly Thr Glu Leu
Met Lys Pro Gly Ala1 5 10 15Ser Met Lys Ile Ser Cys Lys Ala Thr Gly
Tyr Thr Phe Ser Thr Tyr 20 25 30Trp Ile Gln Trp Ile Lys Gln Arg Pro
Gly His Gly Leu Glu Trp Ile 35 40 45Gly Glu Ile Leu Pro Gly Ser Gly
Thr Thr Asn Tyr Asn Glu Asn Phe 50 55 60Lys Gly Lys Ala Thr Phe Ser
Ala Asp Thr Ser Ser Asn Thr Ala Tyr65 70 75 80Ile His Leu Ser Ser
Leu Thr Ser Glu Asp Ser Ala Val Phe Tyr Cys 85 90 95Ala Arg Arg Leu
Gly Arg Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110Thr Leu
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120
125Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
Trp Asn145 150 155 160Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
Pro Ala Val Leu Gln 165 170 175Ser Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val Thr Val Pro Ser Ser 180 185 190Ser Leu Gly Thr Gln Thr Tyr
Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205Asn Thr Lys Val Asp
Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220His Thr Cys
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser225 230 235
240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
Asp Pro 260 265 270Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala 275 280 285Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala
Ser Thr Tyr Arg Val Val 290 295 300Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr305 310 315 320Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350Pro
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360
365Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp385 390 395 400Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser 405 410 415Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met His Glu Ala 420 425 430Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445218451PRTArtificial
SequenceChimeric anti-ILT3 20E4 rat VH /human IgG1 (N297A) 218Gln
Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Gln Ala Ser Glu1 5 10
15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr
20 25 30Ser Val Asn Trp Val Arg Gln Pro Ser Gly Lys Gly Leu Glu Trp
Met 35 40 45Gly Arg Phe Trp Tyr Asp Gly Gly Thr Ala Tyr Asn Ser Thr
Leu Glu 50 55 60Ser Arg Leu Ser Ile Ser Gly Asp Thr Ser Lys Asn Gln
Val Phe Leu65 70 75 80Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Gly
Thr Tyr Tyr Cys Thr 85 90 95Arg Asp Arg Asp Thr Met Gly Ile Thr Gly
Trp Phe Ala Tyr Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser
Pro Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe Pro Leu Ala Pro
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly Cys
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155 160Ser
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170
175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
180 185 190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His 195 200 205Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
Pro Lys Ser Cys 210 215 220Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly225 230 235 240Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr 290 295
300Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly305 310 315 320Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile 325 330 335Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 340 345 350Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 355 360 365Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro385 390 395 400Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410
415Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
420 425 430His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 435 440 445Pro Gly Lys 450219451PRTArtificial
SequenceChimeric anti-ILT3 24A4 rat VH /human IgG1 (N297A) 219Gln
Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Gln Ala Ser Glu1 5 10
15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr
20 25 30Cys Val Asn Trp Val Arg Gln Pro Ser Gly Lys Gly Pro Glu Trp
Leu 35 40 45Gly Arg Phe Trp Tyr Asp Glu Gly Lys Val Tyr Asn Leu Thr
Leu Glu 50 55 60Ser Arg Leu Ser Ile Ser Gly Asp Thr Ser Lys Asn Gln
Val Phe Leu65 70 75 80Lys Met Asn Arg Leu Arg Thr Asp Asp Thr Gly
Thr Tyr Tyr Cys Thr 85 90 95Arg Asp Arg Asp Thr Leu Gly Ile Thr Gly
Trp Phe Ala Tyr Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe Pro Leu Ala Pro
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly Cys
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155 160Ser
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170
175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
180 185 190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His 195 200 205Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
Pro Lys Ser Cys 210 215 220Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly225 230 235 240Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr 290 295
300Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly305 310 315 320Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile 325 330 335Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val 340 345 350Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser 355 360 365Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro385 390 395 400Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410
415Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
420 425 430His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 435 440 445Pro Gly Lys 450220451PRTArtificial
SequenceChimeric anti-ILT3 40A6 rat VH /human IgG1 (N297A) 220Gln
Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Gln Ala Ser Glu1 5 10
15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr
20 25 30Ser Ile Asn Trp Val Arg Gln Ser Ser Gly Lys Gly Pro Glu Trp
Met 35 40 45Gly Arg Phe Trp Tyr Asp Glu Gly Ile Ala Tyr Asn Leu Thr
Leu Glu 50 55 60Ser Arg Leu Ser Ile Ser Gly Asp Thr Ser Lys Asn Gln
Val Phe Leu65 70 75
80Lys Met Asn Ser Leu Arg Thr Gly Asp Thr Gly Thr Tyr Tyr Cys Thr
85 90 95Arg Asp Arg Asp Thr Val Gly Ile Thr Gly Trp Phe Ala Tyr Trp
Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser 115 120 125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200
205Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
210 215 220Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu Gly225 230 235 240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met 245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr 290 295 300Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly305 310 315
320Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
325 330 335Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val 340 345 350Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
Asn Gln Val Ser 355 360 365Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu 370 375 380Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro385 390 395 400Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440
445Pro Gly Lys 4502214PRTArtificial SequenceResidues after
LC-CDR3VARIANT(3)..(3)X is any amino acid 221Phe Gly Xaa
Gly12224PRTArtificial SequenceResidues before
HC-CDR1VARIANT(2)..(4)X is any amino acid 222Cys Xaa Xaa
Xaa12235PRTArtificial SequenceResidues before HC-CDR2 223Leu Glu
Trp Ile Gly1 52244PRTArtificial SequenceResidues after
HC-CDR2VARIANT(3)..(3)X is any amino acid 224Trp Gly Xaa
Gly1225447PRTArtificial SequencePembrolizumab Heavy Chain 225Gln
Val Gln Leu Val Gln Ser Gly Val Glu Val Lys Lys Pro Gly Ala1 5 10
15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Met 35 40 45Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu
Lys Phe 50 55 60Lys Asn Arg Val Thr Leu Thr Thr Asp Ser Ser Thr Thr
Thr Ala Tyr65 70 75 80Met Glu Leu Lys Ser Leu Gln Phe Asp Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Arg Arg Asp Tyr Arg Phe Asp Met Gly
Phe Asp Tyr Trp Gly Gln 100 105 110Gly Thr Thr Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val 115 120 125Phe Pro Leu Ala Pro Cys
Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala 130 135 140Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser145 150 155 160Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170
175Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp
His Lys 195 200 205Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser
Lys Tyr Gly Pro 210 215 220Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe
Leu Gly Gly Pro Ser Val225 230 235 240Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255Pro Glu Val Thr Cys
Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 260 265 270Val Gln Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285Thr
Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 290 295
300Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys305 310 315 320Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile
Glu Lys Thr Ile 325 330 335Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro 340 345 350Pro Ser Gln Glu Glu Met Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu 355 360 365Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser385 390 395 400Asp
Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 405 410
415Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
420 425 430His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
Lys 435 440 445226218PRTArtificial SequencePembrolizumab Light
Chain 226Glu 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 Lys Gly Val
Ser Thr Ser 20 25 30Gly Tyr Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro
Gly Gln Ala Pro 35 40 45Arg Leu Leu Ile Tyr Leu Ala Ser Tyr Leu Glu
Ser Gly Val Pro Ala 50 55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser65 70 75 80Ser Leu Glu Pro Glu Asp Phe Ala
Val Tyr Tyr Cys Gln His Ser Arg 85 90 95Asp Leu Pro Leu Thr Phe Gly
Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 110Thr Val Ala Ala Pro
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125Leu Lys Ser
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140Pro
Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser145 150
155 160Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr 165 170 175Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys 180 185 190His Lys Val Tyr Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser Pro 195 200 205Val Thr Lys Ser Phe Asn Arg Gly Glu
Cys 210 215227330PRTArtificial sequenceHuman IgG1 HC constant
domain (N297A; D265A) 227Ala 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 95Lys 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 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 Ala 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 Asp Glu225 230 235
240Leu 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 325 330
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