U.S. patent application number 15/763673 was filed with the patent office on 2019-02-14 for antagonistic antibodies specifically binding human cd40 and methods of use.
The applicant listed for this patent is Janssen Biotech, Inc.. Invention is credited to Holger Babbe, Nathan Felix, Johan Fransson, Paul Kim, Michael Scully, Hong Zhou.
Application Number | 20190048089 15/763673 |
Document ID | / |
Family ID | 58409279 |
Filed Date | 2019-02-14 |
United States Patent
Application |
20190048089 |
Kind Code |
A1 |
Babbe; Holger ; et
al. |
February 14, 2019 |
Antagonistic Antibodies Specifically Binding Human CD40 and Methods
of Use
Abstract
The present invention relates to antagonistic antibodies
specifically binding human CD40, polynucleotides encoding the
antibodies or antigen-binding fragments thereof, and methods of
making and using the foregoing.
Inventors: |
Babbe; Holger; (Bryn Mawr,
PA) ; Felix; Nathan; (Yardley, PA) ; Fransson;
Johan; (Toronto, CA) ; Kim; Paul; (Del Mar,
CA) ; Scully; Michael; (Medford, NJ) ; Zhou;
Hong; (San Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Janssen Biotech, Inc. |
Horsham |
PA |
US |
|
|
Family ID: |
58409279 |
Appl. No.: |
15/763673 |
Filed: |
September 30, 2016 |
PCT Filed: |
September 30, 2016 |
PCT NO: |
PCT/US16/54671 |
371 Date: |
March 27, 2018 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
62234812 |
Sep 30, 2015 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2299/00 20130101;
A61K 47/6803 20170801; C07K 16/468 20130101; C07K 2317/92 20130101;
C07K 2317/565 20130101; C07K 2317/76 20130101; C07K 2317/94
20130101; A61K 39/39558 20130101; A61K 39/3955 20130101; A61P 35/00
20180101; C07K 2317/34 20130101; C07K 2317/21 20130101; A61K
47/6849 20170801; C07K 2317/52 20130101; A61K 45/06 20130101; A61K
2039/505 20130101; C07K 16/4208 20130101; C07K 16/2878 20130101;
C07K 2317/75 20130101; C07K 2317/55 20130101; C07K 2317/31
20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; C07K 16/46 20060101 C07K016/46; A61K 47/68 20060101
A61K047/68; A61K 45/06 20060101 A61K045/06; C07K 16/42 20060101
C07K016/42; A61K 39/395 20060101 A61K039/395 |
Claims
1) An isolated antagonistic antibody or an antigen-binding fragment
thereof specifically binding human CD40 of SEQ ID NO: 1, comprising
a heavy chain complementarity determining region (HCDR) 1 of SEQ ID
NO: 5, a HCDR2 of SEQ ID NO: 61, a HCDR3 of SEQ ID NO: 62, a light
chain complementarity determining region (LCDR) 1 of SEQ ID NO: 63,
a LCDR2 of SEQ ID NO: 9 and a LCDR3 of SEQ ID NO: 10.
2) The isolated antibody or the antigen-binding fragment thereof of
claim 1, wherein the antibody competes for binding to human CD40 of
SEQ ID NO: 1 with an antibody comprising a) a heavy chain variable
region (VH) of SEQ ID NO: 11 and a light chain variable region (VL)
of SEQ ID NO: 12; b) the VH of SEQ ID NO: 25 and the VL of SEQ ID
NO: 27; or c) the VH of SEQ ID NO: 26 and the VL of SEQ ID NO:
27.
3) The isolated antibody or the antigen-binding fragment thereof of
claim 1, wherein the antibody comprises the HCDR1, the HCDR2, the
HCDR3, the LCDR1, the LCDR2 and the LCDR3 of a) SEQ ID NOs: 5, 6,
7, 8, 9 and 10, respectively. b) SEQ ID NOs: 5, 15, 7, 20, 9 and
10, respectively; c) SEQ ID NOs: 5, 16, 7, 20, 9 and 10,
respectively; d) SEQ ID NOs: 5, 17, 7, 20, 9 and 10, respectively;
e) SEQ ID NOs: 5, 18, 7, 20, 9 and 10, respectively; f) SEQ ID NOs:
5, 18, 19, 20, 9 and 10, respectively; or g) SEQ ID NOs: 5, 17, 19,
20, 9 and 10, respectively.
4) The antibody or the antigen-binding fragment thereof of claim 1,
wherein the antibody binds human CD40 with a dissociation constant
(K.sub.D) of about 1.5.times.10.sup.-10 M or less, when the K.sub.D
is measured using ProteOn XPR36 system at 25.degree. C. in
Dulbecco's phosphate buffered saline containing 0.01% polysorbate
20 (PS-20) and 100 .mu.g/ml bovine serum albumin.
5) The isolated antibody or the antigen-binding fragment thereof of
claim 1, wherein the antibody inhibits soluble human CD40L-driven
human tonsillar B cell proliferation with an IC.sub.50 value of
less than about 1.times.10.sup.-9 M.
6) The isolated antibody or the antigen-binding fragment thereof of
claim 1, wherein the antibody inhibits soluble human CD40L-driven
production of IL-12p40 by human dendritic cells with an IC.sub.50
value of less than about 1.times.10.sup.-9 M.
7) The isolated antibody or the antigen-binding fragment thereof of
any of the claims 1-6, comprising the VH and the VL of a) SEQ ID
NOs: 11 and 12, respectively; b) SEQ ID NOs: 21 and 27,
respectively; c) SEQ ID NOs: 22 and 27, respectively; d) SEQ ID
NOs: 23 and 27, respectively; e) SEQ ID NOs: 24 and 27,
respectively; f) SEQ ID NOs: 25 and 27, respectively; or g) SEQ ID
NOs: 26 and 27, respectively.
8) The isolated antibody or the antigen-binding fragment thereof of
claim 1, which is an IgG1, IgG2, IgG3 or IgG4 isotype.
9) The isolated antibody or the antigen-binding fragment thereof of
claim 8, wherein the antibody comprises at least one mutation in an
Fc region that reduces binding of the antibody to a Fc.gamma.
receptor when compared to a wild-type Fc.
10) The isolated antibody or the antigen-binding fragment thereof
of claim 9, wherein the at least one mutation in the Fc region is a
S228P mutation, a F234A mutation, a L234A mutation, a L235A
mutation, a G237A mutation, a P238S mutation, a H268A mutation, a
A330S mutation or a P331S mutation, wherein residue numbering is
according to the EU Index.
11) The isolated antibody or the antigen-binding fragment thereof
of claim 9, wherein the antibody is an IgG1 isotype and comprises a
L234A/L235A/G237A/P238S/H268A/A330S/P331S mutation.
12) The isolated antibody or the antigen-binding fragment thereof
of claim 9, wherein the antibody is an IgG4 isotype and comprises a
S228P/F234A/L235A mutation.
13) The isolated antibody or the antigen-binding fragment thereof
of claim 1, comprising a heavy chain and a light chain of a) SEQ ID
NOs: 35 and 47, respectively; b) SEQ ID NOs: 36 and 48,
respectively; c) SEQ ID NOs: 37 and 48, respectively; d) SEQ ID
NOs: 38 and 48, respectively; e) SEQ ID NOs: 39 and 48,
respectively; f) SEQ ID NOs: 40 and 48, respectively; g) SEQ ID
NOs: 41 and 48, respectively; h) SEQ ID NOs: 42 and 48,
respectively; i) SEQ ID NOs: 43 and 48, respectively; j) SEQ ID
NOs: 44 and 48, respectively; k) SEQ ID NOs: 45 and 48,
respectively; or l) SEQ ID NOs: 46 and 48, respectively.
14) The antibody of any or the antigen-binding fragment thereof of
claim 1, wherein the antibody is a bispecific or a multispecific
antibody.
15) A pharmaceutical composition comprising the isolated antibody
or the antigen-binding fragment thereof of claim 1 and a
pharmaceutically acceptable carrier.
16) An immunoconjugate comprising the isolated antibody or the
antigen-binding fragment thereof of claim 1 linked to a cytotoxic
agent or an imaging agent.
17) An isolated polynucleotide encoding a) the VH of SEQ ID NOs:
11, 21, 22, 23, 24, 25 or 26; b) the VL of SEQ ID NOs: 12 or 27; or
c) the VH of SEQ ID NOs: 11, 21, 22, 23, 24, 25 or 26 and the VL of
SEQ ID NOs: 12 or 27.
18) An isolated polynucleotide comprising the polynucleotide
sequence of SEQ ID NOs: 13, 14, 28, 29, 30, 31, 32, 33, or 34.
19) A vector comprising the polynucleotide of claim 17.
20) A vector comprising the polynucleotide of claim 18.
21) A host cell comprising the vector of claim 19.
22) A host cell comprising the vector of claim 20.
23) A method of producing an antagonistic antibody or an
antigen-binding fragment thereof specifically binding human CD40 of
SEQ ID NO: 1, comprising culturing the host cell of claim 21 in
conditions wherein the antibody is expressed, and isolating the
antibody.
24) A method of treating a subject having an inflammatory disease,
comprising administering to the subject in need thereof the
isolated antibody or the antigen-binding fragment thereof of claim
1 for a time sufficient to treat the inflammatory disease.
25) The method of claim 24, wherein the inflammatory disease is an
autoimmune disease.
26) The method of claim 24, wherein the inflammatory disease is
Addison's disease, an ankylosing spondylitis, an atherosclerosis,
an autoimmune hepatitis, an autoimmune diabetes, Graves' disease,
Guillain-Barre syndrome, Hashimoto's disease, an idiopathic
thrombocytopenia, an inflammatory bowel disease (IBD), a systemic
lupus erythematosus, lupus nephritis, cutaneous lupus
erythematosus, a multiple sclerosis, a myasthenia gravis, a
psoriasis, an arthritis, a scleroderma, Sjogren's syndrome, a
systemic sclerosis, a transplantation, a kidney transplantation, a
skin transplantation, a bone marrow transplantation, a graft versus
host disease (GVHD) or a type I diabetes.
27) The method of claim 26, wherein the arthritis is a rheumatoid
arthritis, a juvenile arthritis, a psoriatic arthritis, Reiter's
syndrome, an ankylosing spondylitis, or a gouty arthritis.
28) The method of claim 26, wherein the IBD is Crohn's disease or
an ulcerative colitis.
29) The method of claim 24 further administering a second
therapeutic agent.
30) The method of claim 29, wherein the second therapeutic agent is
nonsteroidal anti-inflammatory drugs (NSAIDs), salicylates,
hydroxychloroquine, sulfasalazine, corticosteroids, cytotoxic
drugs, immunosuppressive drugs and/or antibodies.
31) The pharmaceutical composition of claim 15 for use in
therapy.
32) An anti-idiotypic antibody binding to the antibody or the
antigen-binding fragment thereof of claim 7.
33) A kit comprising the antibody or the antigen-binding fragment
thereof of claim 7.
34) The kit of claim 33, further comprising reagents for detecting
the antibody and instructions of use.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to antagonistic antibodies
specifically binding human CD40, polynucleotides encoding the
antibodies or antigen-binding fragments thereof, and methods of
making and using the foregoing.
BACKGROUND OF THE INVENTION
[0002] The cell surface CD40 molecule is a member of the tumor
necrosis factor receptor superfamily (TNFR) and a key regulator in
both innate and adaptive immune responses. CD40 is constitutively
expressed on antigen presenting cells, in particular B-cells,
dendritic cells and macrophages, but can also be found on
fibroblasts, synoviocytes, smooth muscle cells, endothelial cells
and epithelial cells.
[0003] The natural ligand of CD40, designated CD154 or CD40L, is
mainly expressed on activated T lymphocytes and platelets. The
interaction of CD40 with CD40L on T cells induces both humoral and
cell-mediated immune responses. CD40 regulates this ligand-receptor
pair to activate B cells and other antigen-presenting cells (APC)
including dendritic cells (DCs), driving T cell activation. For
example, activation of CD40 on B cells induces B cell
proliferation, somatic hypermutation, differentiation into antibody
secreting cells and isotype switching in germinal centers of
secondary lymphoid organs. In vitro studies have shown direct
effects of CD40 activation on cytokine production (e.g. IL-6,
IL-10, IL-12, TNF-.alpha.), expression of adhesion molecules and
costimulatory receptors (e.g. ICAM, CD23, CD80 and CD86), and
increased expression of MHC class I, MHC class II, and TAP
transporter by B lymphocytes.
[0004] Antibodies that modulate the CD40/CD40L interaction are of
interest in treating diseases such as inflammatory diseases,
including autoimmune diseases.
SUMMARY OF THE INVENTION
[0005] The invention provides for an isolated antagonistic antibody
or an antigen binding portion thereof specifically binding human
CD40 of SEQ ID NO: 1, comprising a heavy chain complementarity
determining region (HCDR) 1 of SEQ ID NO: 5, a HCDR2 of SEQ ID NO:
61, a HCDR3 of SEQ ID NO: 62, a light chain complementarity
determining region (LCDR) 1 of SEQ ID NO: 63, a LCDR2 of SEQ ID NO:
9 and a LCDR3 of SEQ ID NO: 10.
[0006] The invention also provides for an isolated antagonistic
antibody or an antigen binding portion thereof specifically binding
human CD40 of SEQ ID NO: 1, comprising certain HCDR1, HCDR2, HCDR3,
LCDR1, LCDR3, LCDR3, VH, VL, HC and/or LC sequences.
[0007] The invention also provides for a pharmaceutical composition
comprising the antibody of the invention and a pharmaceutically
acceptable carrier.
[0008] The invention also provides for an immunoconjugate
comprising the antibody of the invention linked to a therapeutic
agent or an imaging agent.
[0009] The invention also provides for an isolated polynucleotide
encoding [0010] the VH of SEQ ID NOs: 11, 21, 22, 23, 24, 25 or 26;
[0011] the VL of SEQ ID NOs: 12 or 27; or [0012] the VH of SEQ ID
NOs: 11, 21, 22, 23, 24, 25 or 26 and the VL of SEQ ID NOs: 12 or
27.
[0013] The invention also provides for an isolated polynucleotide
comprising the polynucleotide sequence of SEQ ID NOs: 13, 14, 28,
29, 30, 31, 32, 33, or 34.
[0014] The invention also provides for an isolated polynucleotide
encoding the heavy chain of SEQ ID NOs: 35, 36, 37, 38, 39, 40, 41,
42, 43, 44, 45 or 46.
[0015] The invention also provides for an isolated polynucleotide
encoding the light chain of SEQ ID NOs: 77 or 78.
[0016] The invention also provides for an isolated polynucleotide
encoding the heavy chain of SEQ ID NOs: 35, 36, 37, 38, 39, 40, 41,
42, 43, 44, 45 or 46 and a light chain of SEQ ID NOs: 77 or 78.
[0017] The invention also provides for an isolated polynucleotide
comprising the polynucleotide sequence of SEQ ID NOs: 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 77 or 78.
[0018] The invention also provides for a vector comprising the
polynucleotide of the invention.
[0019] The invention also provides for a host cell comprising the
vector of the invention.
[0020] The invention also provides for a method of producing an
antagonistic antibody or an antigen binding portion thereof
specifically binding human CD40 of SEQ ID NO: 1, comprising
culturing the host cell of the invention in conditions wherein the
antibody is expressed, and isolating the antibody.
[0021] The invention also provides for a method of treating a
subject having an inflammatory disease, comprising administering to
the subject in need thereof the isolated antibody of the invention
for a time sufficient to treat the inflammatory disease.
[0022] The invention also provides for the antibody of the
invention for use in therapy.
[0023] The invention also provides for an anti-idiotypic antibody
binding to the antibody of the invention.
[0024] The invention also provides for a kit comprising the
antibody of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] FIG. 1 shows that C40B16 as wild-type IgG1 demonstrated
comparable minimal agonism when compared to Fc effector silent
Antibody D. Fc effector silent ASKP-1240, CFZ533 and BMS-986090
mAbs demonstrated higher level of agonism when compared to C40B16.
Agonism was assessed in HEK-Blue.TM. CD40L NF-.kappa.B activation
assay.
[0026] FIG. 2 shows that C40B16 does not induce agonism in an assay
measuring antibody-mediated IL-12p40 production by human dendritic
cells (DC), whereas ASKP-1240, CFZ533 and BMS-986090 induce
IL-12p40 production. IL-12p40 production was evaluated at 6
different antibody concentrations, (350, 110, 35, 11, 3.5, and 1.1
nM) each represented by a separate column for each antibody in the
Figure. DC+CD40L: positive control. DC only: negative control.
[0027] FIG. 3A shows that 500 ng/ml concentrations of anti-CD40
antibodies C40B176, C40B179, C40B180 and C40B183 do not induce
activation of dendritic cells (DC), whereas 350 nM ASKP-1240
induces IL-12p40 production. DC activation was assessed by IL-12p40
production by DCs in the presence of antibody. PP1B40: IgG1sigma
isotype control; CNT09412: IgG4_PAA isotype control.
[0028] FIG. 3B shows that high concentrations of anti-CD40
antibodies C40B176, C40B179, C40B180 and C40B183 do not induce
proliferation of B cells, whereas 500 nM ASKP-1240 does. PP1B40:
IgG1sigma isotype control; CNT09412: IgG4_PAA isotype control. Two
separate dose response curves are shown for ASKP-1240.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0029] All publications, including but not limited to patents and
patent applications, cited in this specification are herein
incorporated by reference as though fully set forth.
[0030] It is to be understood that the terminology used herein is
for the purpose of describing particular embodiments only and is
not intended to be limiting. Unless defined otherwise, all
technical and scientific terms used herein have the same meaning as
commonly understood by one of ordinary skill in the art to which
the invention pertains.
[0031] As used herein, the singular forms "a," "and," and "the"
include plural reference unless the context clearly dictates
otherwise.
[0032] Although any methods and materials similar or equivalent to
those described herein may be used in the practice for testing of
the present invention, exemplary materials and methods are
described herein.
[0033] "Specific binding" or "specifically binds" or "binds" refers
to antibody binding to human CD40 with greater affinity than for
non-related antigens. Typically, the antibody binds to human CD40
with a dissociation constant (K.sub.D) of 1.times.10.sup.-8 M or
less, for example 1.times.10.sup.-9 M or less, 1.times.10.sup.-10 M
or less, 1.times.10.sup.-11 M or less, or 1.times.10.sup.-12 M or
less, typically with a K.sub.D that is at least one hundred fold
less than its K.sub.D for binding to a non-related antigen (for
example, BSA, casein). The dissociation constant may be measured
using standard procedures. Antibodies that specifically bind human
CD40 may, however, have cross-reactivity to other related antigens,
for example to the same antigen from other species (homologs), such
as human or monkey, for example Macaca fascicularis (cynomolgus,
cyno), Pan troglodytes (chimpanzee, chimp) or Callithrix jacchus
(common marmoset, marmoset). While a monospecific antibody
specifically binds one antigen or one epitope, a bispecific
antibody specifically binds two distinct antigens or two distinct
epitopes.
[0034] "Antibodies" is meant in a broad sense and includes
immunoglobulin molecules including monoclonal antibodies including
murine, human, humanized and chimeric monoclonal antibodies,
antigen-binding fragments, bispecific or multispecific antibodies,
dimeric, tetrameric or multimeric antibodies, single chain
antibodies, domain antibodies and any other modified configuration
of the immunoglobulin molecule that comprises an antigen binding
site of the required specificity. "Full length antibodies" are
comprised of two heavy (H) chains and two light (L) chains
inter-connected by disulfide bonds as well as multimers thereof
(for example IgM). Each heavy chain is comprised of a heavy chain
variable region (VH) and a heavy chain constant region (comprised
of domains CH1, hinge CH2 and CH3). Each light chain is comprised
of a light chain variable region (VL) and a light chain constant
region (CL). The VH and the VL regions may be further subdivided
into regions of hypervariability, termed complementarity
determining regions (CDR), interspersed with framework regions
(FR). Each VH and VL is composed of three CDRs and four FR
segments, arranged from amino-terminus to carboxy-terminus in the
following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
[0035] "Complementarity determining regions (CDR)" are "antigen
binding sites" in an antibody. CDRs may be defined using various
terms: (i) Complementarity Determining Regions (CDRs), three in the
VH (HCDR1, HCDR2, HCDR3) and three in the VL (LCDR1, LCDR2, LCDR3)
are based on sequence variability (Wu and Kabat, J Exp Med
132:211-50, 1970; Kabat et al., Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md., 1991). (ii) "Hypervariable
regions", "HVR", or "HV", three in the VH (H1, H2, H3) and three in
the VL (L1, L2, L3) refer to the regions of an antibody variable
domains which are hypervariable in structure as defined by Chothia
and Lesk (Chothia and Lesk, Mol Biol 196:901-17, 1987). The
International ImMunoGeneTics (IMGT) database (http://www_imgt_org)
provides a standardized numbering and definition of antigen-binding
sites. The correspondence between CDRs, HVs and IMGT delineations
is described in Lefranc et al., Dev Comparat Immunol 27:55-77,
2003. The term "CDR", "HCDR1", "HCDR2", "HCDR3", "LCDR1", "LCDR2"
and "LCDR3" as used herein includes CDRs defined by any of the
methods described supra, Kabat, Chothia or IMGT, unless otherwise
explicitly stated in the specification.
[0036] Immunoglobulins may be assigned to five major classes, IgA,
IgD, IgE, IgG and IgM, depending on the heavy chain constant domain
amino acid sequence. IgA and IgG are further sub-classified as the
isotypes IgA.sub.1, IgA.sub.2, IgG.sub.1, IgG.sub.2, IgG.sub.3 and
IgG.sub.4. Antibody light chains of any vertebrate species can be
assigned to one of two clearly distinct types, namely kappa
(.kappa.) and lambda (.lamda.), based on the amino acid sequences
of their constant domains.
[0037] "Antigen-binding fragment" refers to a portion of an
immunoglobulin molecule that retains the antigen binding properties
of the parental full length antibody. Exemplary antigen-binding
fragments are as heavy chain complementarity determining regions
(HCDR) 1, 2 and/or 3, light chain complementarity determining
regions (LCDR) 1, 2 and/or 3, a heavy chain variable region (VH),
or a light chain variable region (VL), Fab, F(ab')2, Fd and Fv
fragments as well as domain antibodies (dAb) consisting of either
one VH domain or one VL domain. VH and VL domains may be linked
together via a synthetic linker to form various types of single
chain antibody designs in which the VH/VL domains pair
intramolecularly, or intermolecularly in those cases when the VH
and VL domains are expressed by separate chains, to form a
monovalent antigen binding site, such as single chain Fv (scFv) or
diabody; described for example in Int. Pat. Publ. No. WO1998/44001,
Int. Pat. Publ. No. WO1988/01649; Int. Pat. Publ. No. WO1994/13804;
Int. Pat. Publ. No. WO1992/01047.
[0038] "Monoclonal antibody" refers to an antibody population with
single amino acid composition in each heavy and each light chain,
except for possible well known alterations such as removal of
C-terminal lysine from the antibody heavy chain. Monoclonal
antibodies typically bind one antigenic epitope, except that
multispecific monoclonal antibodies bind two or more distinct
antigens or epitopes. Bispecific monoclonal antibodies bind two
distinct antigenic epitopes. Monoclonal antibodies may have
heterogeneous glycosylation within the antibody population.
Monoclonal antibody may be monospecific or multispecific, or
monovalent, bivalent or multivalent. A multispecific antibody, such
as a bispecific antibody or a trispecific antibody is included in
the term monoclonal antibody.
[0039] "Isolated antibody" refers to an antibody or an
antigen-binding fragment thereof that is substantially free of
other antibodies having different antigenic specificities (e.g., an
isolated antibody specifically binding human CD40 is substantially
free of antibodies that specifically bind antigens other than human
CD40). In case of a bispecific antibody, the bispecific antibody
specifically binds two antigens of interest, and is substantially
free of antibodies that specifically bind antigens other that the
two antigens of interest. "Isolated antibody" encompasses
antibodies that are isolated to a higher purity, such as antibodies
that are 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% pure.
[0040] "Humanized antibodies" refers to antibodies in which the
antigen binding sites are derived from non-human species and the
variable region frameworks are derived from human immunoglobulin
sequences. Humanized antibodies may include intentionally
introduced mutations in the framework regions so that the framework
may not be an exact copy of expressed human immunoglobulin or
germline gene sequences.
[0041] "Human antibodies" refers to antibodies having heavy and
light chain variable regions in which both the framework and the
antigen binding site are derived from sequences of human origin. If
the antibody contains a constant region or a portion of the
constant region, the constant region also is derived from sequences
of human origin.
[0042] A human antibody comprises heavy or light chain variable
regions that are derived from sequences of human origin if the
variable regions of the antibody are obtained from a system that
uses human germline immunoglobulin or rearranged immunoglobulin
genes. Such exemplary systems are human immunoglobulin gene
libraries displayed on phage, and transgenic non-human animals such
as mice or rats carrying human immunoglobulin loci as described
herein. A human antibody typically contain amino acid differences
when compared to the human germline or rearranged immunoglobulin
sequences due to, for example naturally occurring somatic
mutations, intentional introduction of substitutions into the
framework or antigen binding site and amino acid changes introduced
during cloning and VDJ recombination in non-human animals.
Typically, a human antibody is at least about 80%, 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or 100% identical in amino acid sequence to an amino
acid sequence encoded by a human germline or rearranged
immunoglobulin gene. In some cases, a human antibody may contain
consensus framework sequences derived from human framework sequence
analyses, for example as described in Knappik et al., J Mol Biol
296:57-86, 2000, or synthetic HCDR3 incorporated into human
immunoglobulin gene libraries displayed on phage, for example as
described in Shi et al., J Mol Biol 397:385-96, 2010 and Int. Pat.
Publ. No. WO2009/085462.
[0043] Antibodies in which antigen binding sites are derived from a
non-human species are not included in the definition of human
antibody.
[0044] "Recombinant" includes antibodies and other proteins that
are prepared, expressed, created or isolated by recombinant
means.
[0045] "Epitope" refers to a portion of an antigen to which an
antibody specifically binds. Epitopes typically consist of
chemically active (such as polar, non-polar or hydrophobic) surface
groupings of moieties such as amino acids or polysaccharide side
chains and may have specific three-dimensional structural
characteristics, as well as specific charge characteristics. An
epitope may be composed of contiguous and/or discontiguous amino
acids that form a conformational spatial unit. For a discontiguous
epitope, amino acids from differing portions of the linear sequence
of the antigen come in close proximity in 3-dimensional space
through the folding of the protein molecule.
[0046] "Multispecific" refers to an antibody that specifically
binds at least two distinct antigens or two distinct epitopes
within the antigens, for example three, four or five distinct
antigens or epitopes.
[0047] "Bispecific" refers to an antibody that specifically binds
two distinct antigens or two distinct epitopes within the same
antigen. The bispecific antibody may have cross-reactivity to other
related antigens or can bind an epitope that is shared between two
or more distinct antigens.
[0048] "Variant" refers to a polypeptide or a polynucleotide that
differs from a reference polypeptide or a reference polynucleotide
by one or more modifications for example, substitutions, insertions
or deletions.
[0049] "Vector" refers to a polynucleotide capable of being
duplicated within a biological system or that can be moved between
such systems. Vector polynucleotides typically contain elements,
such as origins of replication, polyadenylation signal or selection
markers, that function to facilitate the duplication or maintenance
of these polynucleotides in a biological system. Examples of such
biological systems may include a cell, virus, animal, plant, and
reconstituted biological systems utilizing biological components
capable of duplicating a vector. The polynucleotide comprising a
vector may be DNA or RNA molecules or a hybrid of these.
[0050] "Expression vector" refers to a vector that can be utilized
in a biological system or in a reconstituted biological system to
direct the translation of a polypeptide encoded by a polynucleotide
sequence present in the expression vector.
[0051] "Polynucleotide" refers to a synthetic molecule comprising a
chain of nucleotides covalently linked by a sugar-phosphate
backbone or other equivalent covalent chemistry. cDNA is a typical
example of a polynucleotide.
[0052] "Polypeptide" or "protein" refers to a molecule that
comprises at least two amino acid residues linked by a peptide bond
to form a polypeptide. Small polypeptides of less than 50 amino
acids may be referred to as "peptides".
[0053] "CD40" or "huCD40" refers to the human CD40 protein. CD40 is
also known as Tumor necrosis factor receptor superfamily member 5
(TNFRSF5), CD40L receptor or CD154 receptor. The amino acid
sequence of the full length human CD40 is shown in SEQ ID NO: 1.
Human full length CD40 protein is a type I membrane protein with
277 amino acids. The signal sequence spans residues 1-20, the
extracellular domain spans residues 21-193, the transmembrane
domain spans residues 194-215, and the cytoplasmic domain spans
residues 216-277 of SEQ ID NO: 1. Throughout the specification, the
extracellular domain of CD40, "CD40-ECD", refers to the CD40
fragment of residues 21-193 of SEQ ID NO: 1.
[0054] "Antagonist" or "antagonistic" refers to an antibody that
specifically binds human CD40 and inhibits CD40 biological activity
in the presence of CD40L in cellular assays such as CD40L-driven
human B cell proliferation or CD40L-driven IL-12p40 production by
human dendritic cells. The antagonist may inhibit CD40 biological
activity in a statistically significant manner when compared to a
control sample without the antibody. Alternatively, the
antagonistic antibody specifically binding human CD40 may inhibit
CD40 biological activity with an IC.sub.50 value of about 1 nM or
less. CD40L in the assays may be provided as a soluble form or
membrane-bound (e.g. as cells expressing CD40L, such as Jurkat
cells).
[0055] "CD40 biological activity" refers to a measurable event in a
cell occurring as a result of binding of CD40L to CD40 on human
cells. CD40 biological activity may be for example proliferation of
human B cells or production of IL-12p40 by human dendritic cells,
or downstream activation of CD40 signaling pathways. CD40
biological activity may be measured using known methods and methods
described herein.
[0056] "About" means within an acceptable error range for the
particular value as determined by one of ordinary skill in the art,
which will depend in part on how the value is measured or
determined, i.e., the limitations of the measurement system. Unless
explicitly stated otherwise within the Examples or elsewhere in the
Specification in the context of a particular assay, result or
embodiment, "about" means within one standard deviation per
practice in the art, or a range of up to 5%, whichever is
larger.
[0057] "In combination with" means that two or more therapeutics
can be administered to a subject together in a mixture,
concurrently as single agents or sequentially as single agents in
any order.
[0058] "Cross-linking" refers higher order multimerization of CD40
on cells induced by an antibody specifically binding human CD40
binding to Fc.gamma.RIIb cis or trans, resulting in induction of
CD40 agonistic activity.
[0059] Conventional one and three-letter amino acid codes are used
herein as shown in Table 1.
TABLE-US-00001 TABLE 1 Amino acid Three-letter code One-letter code
Alanine Ala A Arginine Arg R Asparagine Asn N Aspartate Asp D
Cysteine Cys C Glutamate Gln E Glutamine Glu Q Glycine Gly G
Histidine His H Isoleucine Ile I Lysine Lys K Methionine Met M
Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T
Tryptophan Trp W Tyrosine Tyr Y Valine Val V
Antibodies of the Invention
[0060] The invention provides antagonistic antibodies specifically
binding human CD40, polynucleotides encoding the antibodies,
vectors, host cells and methods of using the antibodies.
[0061] The antibodies of the invention are potent inhibitors of
CD40 and have minimal agonistic activity. It is documented that
antagonistic anti-CD40 antibodies, albeit being antagonists, can
also have agonistic activity as a result of Fc-dependent
cross-linking (for example see U.S. Pat. No. 7,537,763) and
therefore pose a potential safety risk when administered to
subjects in which suppression of CD40 signaling is desired, such as
patients with autoimmune disease. Preferable anti-CD40 antibodies
for the treatment of conditions in which inhibition of CD40
biological function is desired are hence those that lack agonistic
activity, or have minimal agonistic activity. Suitable therapeutic
CD40 antibodies would thus be Fc-engineered to abolish Fc.gamma.R
binding, and as a result lack Fc-mediated cross-linking and
potential for Fc-mediated agonism. Such effector silent
Fc-engineered antibodies are for example ASKP-1240, CFZ533,
BMS-986090 and Antibody D ("benchmark antibodies"). ASKP-1240 and
CFZ533 are currently in clinical development for inflammatory or
autoimmune diseases.
[0062] The antibodies of the invention demonstrate improved
properties when compared to the benchmark antibodies. The antibody
C40B16 of the invention has minimal agonistic activity and
activates CD40 signaling to a lesser extend when compared to the
benchmark antibodies ASKP-1240, CFZ533 and BMS-986090. As
ASKP-1240, CFZ533 and BMS-986090 are Fc effector silent antibodies,
the observed agonism of these antibodies may be epitope-dependent.
C40B16 on the contrary is a wild-type IgG1 and therefore possesses
neither epitope-dependent nor Fc-dependent agonism. Further, Fc
engineered effector silent antibodies C40B176, C40B179, C40B180 and
C40B183 of the invention demonstrate only minimal agonism but also
up to 10-fold improved potency when compared to Antibody D.
[0063] The invention provides for an isolated antagonistic antibody
or an antigen-binding fragment thereof specifically binding human
CD40 of SEQ ID NO: 1, comprising a heavy chain complementarity
determining region (HCDR) 1 of SEQ ID NO: 5, a HCDR2 of SEQ ID NO:
61, a HCDR3 of SEQ ID NO: 62, a light chain complementarity
determining region (LCDR) 1 of SEQ ID NO: 63, a LCDR2 of SEQ ID NO:
9 and a LCDR3 of SEQ ID NO: 10.
[0064] SEQ ID NOs: 61, 62 and 63 represent the HCDR2, the HCDR3 and
the LCDR1, genus sequences of antagonistic antibodies specifically
binding CD40, the genera encompassing variants of a parental
antibody C40B16 in which putative sites for post-translational
modifications have been mutated. The antibodies within the genus
are expected to display no shift in epitope, e.g. the antibodies
comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2
and the LCDR3 of SEQ ID NOs: 5, 61, 62, 63, 9 and 10 are expected
to have similar characteristics when compared to the parental
C40B16 antibody. Exemplary such antibodies are antibodies
comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2
and the LCDR3 or the VH and the VL amino acid sequences of
antibodies C40M141, C40M152, C40M142, C40M153, C40M144, C40M155,
C40M148, C40M194, C40M198, C40M197, C40M201 or C40M126 as shown in
Table 2 and Table 7.
SEQ ID NO: 61 Genus HCDR2 Sequence:
[0065] TIX.sub.1X.sub.2X.sub.3GGGTYYADSVKG; wherein
X.sub.1 is N, D or Q;
X.sub.2 is N, Q or A; and
X.sub.3 is S or A.
[0066] SEQ ID NO: 62 genus HCDR3 sequence EGGKYYYYAX.sub.1DV;
wherein
X.sub.1 is M or L
[0067] SEQ ID NO: 63 genus LCDR1 sequence SGDKLGDKYAX.sub.1;
wherein
X.sub.1 is C or A.
TABLE-US-00002 [0068] TABLE 2 SEQ ID NO: mAb HCDR1 HCDR2 HCDR3
LCDR1 LCDR2 LCDR3 C40B16 5 6 7 8 9 10 C40B124 5 15 7 20 9 10
C40B135 5 15 7 20 9 10 C40B125 5 16 7 20 9 10 C40B136 5 16 7 20 9
10 C40B127 5 17 7 20 9 10 C40B138 5 17 7 20 9 10 C40B131 5 18 7 20
9 10 C40B176 5 18 19 20 9 10 C40B180 5 18 19 20 9 10 C40B179 5 17
19 20 9 10 C40B183 5 17 19 20 9 10
[0069] In some embodiments, the antibody competes for binding to
human CD40 of SEQ ID NO: 1 with an antibody comprising
[0070] a heavy chain variable region (VH) of SEQ ID NO: 11 and a
light chain variable region (VL) of SEQ ID NO: 12;
[0071] the VH of SEQ ID NO: 25 and the VL of SEQ ID NO: 27, or
[0072] the VH of SEQ ID NO: 26 and the VL of SEQ ID NO: 27.
[0073] In some embodiments, the antibody binds to the same epitope
on human CD40 of SEQ ID NO: 1 to which the antibody comprising
[0074] a heavy chain variable region (VH) of SEQ ID NO: 11 and a
light chain variable region (VL) of SEQ ID NO: 12;
[0075] the VH of SEQ ID NO: 25 and the VL of SEQ ID NO: 27, or
[0076] the VH of SEQ ID NO: 26 and the VL of SEQ ID NO: 27 binds
to.
[0077] In some embodiments, the antibody binds human CD40 with a
dissociation constant (K.sub.D) of about 1.5.times.10.sup.-10 M or
less, when the K.sub.D is measured using ProteOn XPR36 system at
25.degree. C. in Dulbecco's phosphate buffered saline containing
0.01% polysorbate 20 (PS-20) and 100 .mu.g/ml bovine serum
albumin.
[0078] In some embodiments, the antibody inhibits soluble human
CD40L-driven human tonsillar B cell proliferation with an IC.sub.50
value of less than about 1.times.10.sup.-9 M.
[0079] In some embodiments, the antibody inhibits soluble human
CD40L-driven production of IL-12p40 by human dendritic cells with
an IC.sub.50 value of less than about 1.times.10.sup.-9 M.
[0080] In some embodiments, the antibody binds human CD40 with a
dissociation constant (K.sub.D) of about 1.5.times.10.sup.-10 M or
less, when the K.sub.D is measured using ProteOn XPR36 system at
25.degree. C. in Dulbecco's phosphate buffered saline containing
0.01% PS-20 and 100 .mu.g/ml bovine serum albumin, inhibits soluble
human CD40L-driven human tonsillar B cell proliferation with an
IC.sub.50 value of less than about 1.times.10.sup.-9 M and inhibits
soluble human CD40L-driven production of IL-12p40 by human
dendritic cells with an IC.sub.50 value of less than about
1.times.10.sup.-9 M.
[0081] In some embodiments, the antibody is an IgG1, IgG2, IgG3 or
IgG4 isotype.
[0082] In some embodiments, the antibody is an IgG1 isotype.
[0083] In some embodiments, the antibody is an IgG2 isotype.
[0084] In some embodiments, the antibody is an IgG3 isotype.
[0085] In some embodiments, the antibody is an IgG4 isotype.
[0086] In some embodiments, the antibody is an IgG1 isotype,
optionally comprising a L234A/L235A/G237A/P238S/H268A/A330S/P331S
mutation when compared to the wild-type IgG1.
[0087] In some embodiments, the antibody is an IgG1/.lamda.
isotype, optionally comprising the
L234A/L235A/G237A/P238S/H268A/A330S/P331S mutation when compared to
the wild-type IgG1.
[0088] In some embodiments, the antibody is an IgG1/.lamda. isotype
comprising the L234A/L235A/G237A/P238S/H268A/A330S/P331S mutation
when compared to the wild-type IgG1.
[0089] In some embodiments, the antibody is an IgG4 isotype,
optionally comprising a S228P mutation when compared to the
wild-type IgG4.
[0090] In some embodiments, the antibody is an IgG4/.lamda.
isotype, optionally comprising the S228P mutation when compared to
the wild-type IgG4.
[0091] In some embodiments, the antibody is an IgG4/.lamda. isotype
comprising the S228P mutation when compared to the wild-type
IgG4.
[0092] In some embodiments, the antibody is an IgG4 isotype,
optionally comprising a S228P/F234A/L235A mutation when compared to
the wild-type IgG4.
[0093] In some embodiments, the antibody is an IgG4/.lamda.
isotype, optionally comprising the S228P/F234A/L235A mutation when
compared to the wild-type IgG4.
[0094] In some embodiments, the antibody is an IgG4/.lamda. isotype
comprising the S228P/F234A/L235A mutation when compared to the
wild-type IgG4.
[0095] In some embodiments, the antibody is a multispecific
antibody, such as a bispecific antibody.
[0096] The antibody is suitable for use in therapy, for example in
treating an inflammatory disease.
[0097] The antibody is suitable for use in therapy, for example in
treating an autoimmune disease.
[0098] The antibody is suitable for use in therapy, for example in
treating Addinson's disease.
[0099] The antibody is suitable for use in therapy, for example in
treating an ankylosing spondylitis.
[0100] The antibody is suitable for use in therapy, for example in
treating an atherosclerosis.
[0101] The antibody is suitable for use in therapy, for example in
treating an autoimmune hepatitis.
[0102] The antibody is suitable for use in therapy, for example in
treating an autoimmune diabetes.
[0103] The antibody is suitable for use in therapy, for example in
treating Graves' disease.
[0104] The antibody is suitable for use in therapy, for example in
treating Buillain-Barre syndrome.
[0105] The antibody is suitable for use in therapy, for example in
treating Hashimoto's disease.
[0106] The antibody is suitable for use in therapy, for example in
treating, an idiopathic thrombocytopenia.
[0107] The antibody is suitable for use in therapy, for example in
treating an inflammatory bowel disease (IBD).
[0108] The antibody is suitable for use in therapy, for example in
treating a systemic lupus erythematosus.
[0109] The antibody is suitable for use in therapy, for example in
treating a multiple sclerosis.
[0110] The antibody is suitable for use in therapy, for example in
treating a myasthenia gravis.
[0111] The antibody is suitable for use in therapy, for example in
treating a psoriasis.
[0112] The antibody is suitable for use in therapy, for example in
treating an arthritis.
[0113] The antibody is suitable for use in therapy, for example in
treating a scleroderma.
[0114] The antibody is suitable for use in therapy, for example in
treating Sjogren's syndrome.
[0115] The antibody is suitable for use in therapy, for example in
treating a systemic sclerosis.
[0116] The antibody is suitable for use in therapy, for example in
treating a transplantation.
[0117] The antibody is suitable for use in therapy, for example in
treating a kidney transplantation.
[0118] The antibody is suitable for use in therapy, for example in
treating a skin transplantation.
[0119] The antibody is suitable for use in therapy, for example in
treating a bone marrow transplantation.
[0120] The antibody is suitable for use in therapy, for example in
treating a graft versus host disease (GVHD).
[0121] The antibody is suitable for use in therapy, for example in
treating a type I diabetes.
[0122] The antibody is suitable for use in therapy, for example in
treating a rheumatoid arthritis.
[0123] The antibody is suitable for use in therapy, for example in
treating a juvenile arthritis.
[0124] The antibody is suitable for use in therapy, for example in
treating a psoriatic arthritis.
[0125] The antibody is suitable for use in therapy, for example in
treating Reiter's syndrome.
[0126] The antibody is suitable for use in therapy, for example in
treating a gouty arthritis.
[0127] The antibody is suitable for use in therapy, for example in
treating Crohn's disease.
[0128] The antibody is suitable for use in therapy, for example in
treating an ulcerative colitis.
[0129] The antibody is suitable for use in therapy, for example in
treating an inflammatory disease in combination with a second
therapeutic agent.
[0130] The antibody is suitable for use in therapy, for example in
treating an autoimmune disease in combination with a second
therapeutic agent.
[0131] The antibody is suitable for use in therapy, for example in
treating a rheumatoid arthritis in combination with a second
therapeutic agent.
[0132] The antibody is suitable for use in therapy, for example in
treating a systemic lupus erythematosus in combination with a
second therapeutic agent.
[0133] The invention also provides for an isolated antagonistic
antibody or an antigen-binding fragment thereof specifically
binding human CD40 of SEQ ID NO: 1, comprising the HCDR1, the
HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs:
5, 6, 7, 8, 9 and 10, respectively.
[0134] In some embodiments, the antibody comprises the VH and the
VL of SEQ ID NOs: 11 and 12, respectively.
[0135] In some embodiments, the VH is encoded by a polynucleotide
comprising a polynucleotide sequence of SEQ ID NO: 13 and the VL is
encoded by a polynucleotide comprising a polynucleotide sequence of
SEQ ID NO: 14.
[0136] In some embodiments, the antibody comprises a heavy chain of
SEQ ID NO: 35 and a light chain of SEQ ID NO: 47.
[0137] In some embodiments, the heavy chain is encoded by a
polynucleotide comprising a polynucleotide sequence of SEQ ID NO:
65 and the light chain is encoded by a polynucleotide comprising a
polynucleotide sequence of SEQ ID NO: 77.
[0138] The invention also provides for an isolated antagonistic
antibody or an antigen-binding fragment thereof specifically
binding human CD40 of SEQ ID NO: 1, comprising the HCDR1, the
HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs:
5, 15, 7, 20, 9 and 10, respectively.
[0139] In some embodiments, the antibody comprises the VH and the
VL of SEQ ID NOs: 21 and 27, respectively.
[0140] In some embodiments, the VH is encoded by a polynucleotide
comprising a polynucleotide sequence of SEQ ID NO: 28 and the VL is
encoded by a polynucleotide comprising a polynucleotide sequence of
SEQ ID NO: 34.
[0141] In some embodiments, the antibody comprises the heavy chain
of SEQ ID NO: 36 and the light chain of SEQ ID NO: 48.
[0142] In some embodiments, the heavy chain is encoded by a
polynucleotide comprising a polynucleotide sequence of SEQ ID NO:
66 and the light chain is encoded by a polynucleotide comprising a
polynucleotide sequence of SEQ ID NO: 78.
[0143] In some embodiments, the antibody comprises the heavy chain
of SEQ ID NO: 37 and the light chain of SEQ ID NO: 48.
[0144] In some embodiments, the heavy chain is encoded by a
polynucleotide comprising a polynucleotide sequence of SEQ ID NO:
67 and the light chain is encoded by a polynucleotide comprising a
polynucleotide sequence of SEQ ID NO: 78.
[0145] The invention also provides for an isolated antagonistic
antibody or an antigen-binding fragment thereof specifically
binding human CD40 of SEQ ID NO: 1, comprising the HCDR1, the
HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs:
5, 16, 7, 20, 9 and 10, respectively.
[0146] In some embodiments, the antibody comprises the VH and the
VL of SEQ ID NOs: 22 and 27, respectively.
[0147] In some embodiments, the VH is encoded by a polynucleotide
comprising a polynucleotide sequence of SEQ ID NO: 29 and the VL is
encoded by a polynucleotide comprising a polynucleotide sequence of
SEQ ID NO: 34.
[0148] In some embodiments, the antibody comprises the heavy chain
of SEQ ID NO: 38 and the light chain of SEQ ID NO: 48.
[0149] In some embodiments, the heavy chain is encoded by a
polynucleotide comprising a polynucleotide sequence of SEQ ID NO:
68 and the light chain is encoded by a polynucleotide comprising a
polynucleotide sequence of SEQ ID NO: 78.
[0150] In some embodiments, the antibody comprises the heavy chain
of SEQ ID NO: 39 and the light chain of SEQ ID NO: 48.
[0151] In some embodiments, the heavy chain is encoded by a
polynucleotide comprising a polynucleotide sequence of SEQ ID NO:
69 and the light chain is encoded by a polynucleotide comprising a
polynucleotide sequence of SEQ ID NO: 78.
[0152] The invention also provides for an isolated antagonistic
antibody or an antigen-binding fragment thereof specifically
binding human CD40 of SEQ ID NO: 1, comprising the HCDR1, the
HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs:
5, 17, 7, 20, 9 and 10, respectively.
[0153] In some embodiments, the antibody comprises the VH and the
VL of SEQ ID NOs: 23 and 27, respectively.
[0154] In some embodiments, the VH is encoded by a polynucleotide
comprising a polynucleotide sequence of SEQ ID NO: 30 and the VL is
encoded by a polynucleotide comprising a polynucleotide sequence of
SEQ ID NO: 34.
[0155] In some embodiments, the antibody comprises the heavy chain
of SEQ ID NO: 40 and the light chain of SEQ ID NO: 48.
[0156] In some embodiments, the heavy chain is encoded by a
polynucleotide comprising a polynucleotide sequence of SEQ ID NO:
70 and the light chain is encoded by a polynucleotide comprising a
polynucleotide sequence of SEQ ID NO: 78.
[0157] In some embodiments, the antibody comprises the heavy chain
of SEQ ID NO: 41 and the light chain of SEQ ID NO: 48.
[0158] In some embodiments, the heavy chain is encoded by a
polynucleotide comprising a polynucleotide sequence of SEQ ID NO:
71 and the light chain is encoded by a polynucleotide comprising a
polynucleotide sequence of SEQ ID NO: 78.
[0159] The invention also provides for an isolated antagonistic
antibody or an antigen-binding fragment thereof specifically
binding human CD40 of SEQ ID NO: 1, comprising the HCDR1, the
HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs:
5, 18, 7, 20, 9 and 10, respectively.
[0160] In some embodiments, the antibody comprises the VH and the
VL of SEQ ID NOs: 24 and 27, respectively.
[0161] In some embodiments, the VH is encoded by a polynucleotide
comprising a polynucleotide sequence of SEQ ID NO: 31 and the VL is
encoded by a polynucleotide comprising a polynucleotide sequence of
SEQ ID NO: 34.
[0162] In some embodiments, the antibody comprises the heavy chain
of SEQ ID NO: 42 and the light chain of SEQ ID NO: 48.
[0163] In some embodiments, the heavy chain is encoded by a
polynucleotide comprising a polynucleotide sequence of SEQ ID NO:
72 and the light chain is encoded by a polynucleotide comprising a
polynucleotide sequence of SEQ ID NO: 78.
[0164] The invention also provides for an isolated antagonistic or
an antigen-binding fragment thereof specifically binding human CD40
of SEQ ID NO: 1, comprising the HCDR1, the HCDR2, the HCDR3, the
LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 5, 18, 19, 20, 9 and
10, respectively.
[0165] In some embodiments, the antibody competes for binding to
human CD40 of SEQ ID NO: 1 with an antibody comprising
[0166] a heavy chain variable region (VH) of SEQ ID NO: 11 and a
light chain variable region (VL) of SEQ ID NO: 12;
[0167] the VH of SEQ ID NO: 25 and the VL of SEQ ID NO: 27, or
[0168] the VH of SEQ ID NO: 26 and the VL of SEQ ID NO: 27.
[0169] In some embodiments, the antibody binds to the same epitope
on human CD40 of SEQ ID NO: 1 to which the antibody comprising
[0170] a heavy chain variable region (VH) of SEQ ID NO: 11 and a
light chain variable region (VL) of SEQ ID NO: 12;
[0171] the VH of SEQ ID NO: 25 and the VL of SEQ ID NO: 27, or
[0172] the VH of SEQ ID NO: 26 and the VL of SEQ ID NO: 27 binds
to.
[0173] In some embodiments, the antibody binds human CD40 with a
dissociation constant (K.sub.D) of about 1.5.times.10.sup.-10 M or
less, when the K.sub.D is measured using ProteOn XPR36 system at
25.degree. C. in Dulbecco's phosphate buffered saline containing
0.01% PS-20 and 100 .mu.g/ml bovine serum albumin.
[0174] In some embodiments, the antibody inhibits soluble human
CD40L-driven human tonsillar B cell proliferation with an IC.sub.50
value of less than about 1.times.10.sup.-9 M.
[0175] In some embodiments, the antibody inhibits soluble human
CD40L-driven production of IL-12p40 by human dendritic cells with
an IC.sub.50 value of less than about 1.times.10.sup.-9 M.
[0176] In some embodiments, the antibody binds human CD40 with a
dissociation constant (K.sub.D) of about 1.5.times.10.sup.-10 M or
less, when the K.sub.D is measured using ProteOn XPR36 system at
25.degree. C. in Dulbecco's phosphate buffered saline containing
0.01% PS-20 and 100 .mu.g/ml bovine serum albumin, inhibits soluble
human CD40L-driven human tonsillar B cell proliferation with an
IC.sub.50 value of less than about 1.times.10.sup.-9 M and inhibits
soluble human CD40L-driven production of IL-12p40 by human
dendritic cells with an IC.sub.50 value of less than about
1.times.10.sup.-9 M.
[0177] In some embodiments, the antibody comprises the VH and the
VL of SEQ ID NOs: 25 and 27, respectively.
[0178] In some embodiments, the VH is encoded by a polynucleotide
comprising a polynucleotide sequence of SEQ ID NO: 32 and the VL is
encoded by a polynucleotide comprising a polynucleotide sequence of
SEQ ID NO: 34.
[0179] In some embodiments, the antibody is an IgG1, IgG2, IgG3 or
IgG4 isotype.
[0180] In some embodiments, the antibody is an IgG1 isotype.
[0181] In some embodiments, the antibody is an IgG2 isotype.
[0182] In some embodiments, the antibody is an IgG3 isotype.
[0183] In some embodiments, the antibody is an IgG4 isotype.
[0184] In some embodiments, the antibody is an IgG1 isotype,
optionally comprising a L234A/L235A/G237A/P238S/H268A/A330S/P331S
mutation when compared to the wild-type IgG1.
[0185] In some embodiments, the antibody is an IgG1/.lamda.
isotype, optionally comprising the
L234A/L235A/G237A/P238S/H268A/A330S/P331S mutation when compared to
the wild-type IgG1.
[0186] In some embodiments, the antibody comprises the VH of SEQ ID
NO: 25 and the VL of SEQ ID NO: 27 and is an IgG1/.lamda. isotype,
optionally comprising the L234A/L235A/G237A/P238S/H268A/A330S/P331S
mutation when compared to the wild-type IgG1.
[0187] In some embodiments, the antibody comprises the VH of SEQ ID
NO: 25 and the VL of SEQ ID NO: 27 and is an IgG1/.lamda. isotype
comprising the L234A/L235A/G237A/P238S/H268A/A330S/P331S mutation
when compared to the wild-type IgG1.
[0188] In some embodiments, the antibody is an IgG4 isotype,
optionally comprising a S228P mutation when compared to the
wild-type IgG4.
[0189] In some embodiments, the antibody is an IgG4/.lamda.
isotype, optionally comprising the S228P mutation when compared to
the wild-type IgG4.
[0190] In some embodiments, the antibody comprises the VH of SEQ ID
NO: 25 and the VL of SEQ ID NO: 27 and is an IgG4/.lamda. isotype,
optionally comprising the S228P mutation when compared to the
wild-type IgG4.
[0191] In some embodiments, the antibody comprises the VH of SEQ ID
NO: 25 and the VL of SEQ ID NO: 27 and is an IgG4/.lamda. isotype
comprising the S228P mutation when compared to the wild-type
IgG4.
[0192] In some embodiments, the antibody is an IgG4 isotype,
optionally comprising a S228P/F234A/L235A mutation when compared to
the wild-type IgG4.
[0193] In some embodiments, the antibody is an IgG4/.lamda.
isotype, optionally comprising the S228P/F234A/L235A mutation when
compared to the wild-type IgG4.
[0194] In some embodiments, the antibody comprises the VH of SEQ ID
NO: 25 and the VL of SEQ ID NO: 27 and is an IgG4/.lamda. isotype,
optionally comprising the S228P/F234A/L235A mutation when compared
to the wild-type IgG4.
[0195] In some embodiments, the antibody comprises the VH of SEQ ID
NO: 25 and the VL of SEQ ID NO: 27 and is an IgG4/.lamda. isotype
comprising the S228P/F234A/L235A mutation when compared to the
wild-type IgG4.
[0196] In some embodiments, the antibody comprises the heavy chain
of SEQ ID NO: 43 and the light chain of SEQ ID NO: 48.
[0197] In some embodiments, the heavy chain is encoded by a
polynucleotide comprising a polynucleotide sequence of SEQ ID NO:
73 and the light chain is encoded by a polynucleotide comprising a
polynucleotide sequence of SEQ ID NO: 78.
[0198] In some embodiments, the antibody comprises the heavy chain
of SEQ ID NO: 44 and the light chain of SEQ ID NO: 48.
[0199] In some embodiments, the heavy chain is encoded by a
polynucleotide comprising a polynucleotide sequence of SEQ ID NO:
74 and the light chain is encoded by a polynucleotide comprising a
polynucleotide sequence of SEQ ID NO: 78.
[0200] In some embodiments, the antibody is a multispecific
antibody, such as a bispecific antibody.
[0201] The antibody is suitable for use in therapy, for example in
treating an inflammatory disease.
[0202] The antibody is suitable for use in therapy, for example in
treating an autoimmune disease.
[0203] The antibody is suitable for use in therapy, for example in
treating Addinson's disease.
[0204] The antibody is suitable for use in therapy, for example in
treating an ankylosing spondylitis.
[0205] The antibody is suitable for use in therapy, for example in
treating an atherosclerosis.
[0206] The antibody is suitable for use in therapy, for example in
treating an autoimmune hepatitis.
[0207] The antibody is suitable for use in therapy, for example in
treating an autoimmune diabetes.
[0208] The antibody is suitable for use in therapy, for example in
treating Graves' disease.
[0209] The antibody is suitable for use in therapy, for example in
treating Buillain-Barre syndrome.
[0210] The antibody is suitable for use in therapy, for example in
treating Hashimoto's disease.
[0211] The antibody is suitable for use in therapy, for example in
treating, an idiopathic thrombocytopenia.
[0212] The antibody is suitable for use in therapy, for example in
treating an inflammatory bowel disease (IBD).
[0213] The antibody is suitable for use in therapy, for example in
treating a systemic lupus erythematosus.
[0214] The antibody is suitable for use in therapy, for example in
treating a multiple sclerosis.
[0215] The antibody is suitable for use in therapy, for example in
treating a myasthenia gravis.
[0216] The antibody is suitable for use in therapy, for example in
treating a psoriasis.
[0217] The antibody is suitable for use in therapy, for example in
treating an arthritis.
[0218] The antibody is suitable for use in therapy, for example in
treating a scleroderma.
[0219] The antibody is suitable for use in therapy, for example in
treating Sjogren's syndrome.
[0220] The antibody is suitable for use in therapy, for example in
treating a systemic sclerosis.
[0221] The antibody is suitable for use in therapy, for example in
treating a transplantation.
[0222] The antibody is suitable for use in therapy, for example in
treating a kidney transplantation.
[0223] The antibody is suitable for use in therapy, for example in
treating a skin transplantation.
[0224] The antibody is suitable for use in therapy, for example in
treating a bone marrow transplantation.
[0225] The antibody is suitable for use in therapy, for example in
treating a graft versus host disease (GVHD).
[0226] The antibody is suitable for use in therapy, for example in
treating a type I diabetes.
[0227] The antibody is suitable for use in therapy, for example in
treating a rheumatoid arthritis.
[0228] The antibody is suitable for use in therapy, for example in
treating a juvenile arthritis.
[0229] The antibody is suitable for use in therapy, for example in
treating a psoriatic arthritis.
[0230] The antibody is suitable for use in therapy, for example in
treating Reiter's syndrome.
[0231] The antibody is suitable for use in therapy, for example in
treating a gouty arthritis.
[0232] The antibody is suitable for use in therapy, for example in
treating Crohn's disease.
[0233] The antibody is suitable for use in therapy, for example in
treating an ulcerative colitis.
[0234] The antibody is suitable for use in therapy, for example in
treating an inflammatory disease, in combination with a second
therapeutic agent.
[0235] The antibody is suitable for use in therapy, for example in
treating an autoimmune disease, in combination with a second
therapeutic agent.
[0236] The antibody is suitable for use in therapy, for example in
treating a rheumatoid arthritis in combination with a second
therapeutic agent.
[0237] The antibody is suitable for use in therapy, for example in
treating a systemic lupus erythematosus in combination with a
second therapeutic agent.
[0238] The invention also provides for an isolated antagonistic
antibody or an antigen-binding fragment thereof specifically
binding human CD40 of SEQ ID NO: 1, comprising the HCDR1, the
HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs:
5, 17, 19, 20, 9 and 10, respectively.
[0239] In some embodiments, the antibody competes for binding to
human CD40 of SEQ ID NO: 1 with an antibody comprising
[0240] a heavy chain variable region (VH) of SEQ ID NO: 11 and a
light chain variable region (VL) of SEQ ID NO: 12;
[0241] the VH of SEQ ID NO: 25 and the VL of SEQ ID NO: 27, or
[0242] the VH of SEQ ID NO: 26 and the VL of SEQ ID NO: 27.
[0243] In some embodiments, the antibody binds to the same epitope
on human CD40 of SEQ ID NO: 1 to which the antibody comprising
[0244] a heavy chain variable region (VH) of SEQ ID NO: 11 and a
light chain variable region (VL) of SEQ ID NO: 12;
[0245] the VH of SEQ ID NO: 25 and the VL of SEQ ID NO: 27, or
[0246] the VH of SEQ ID NO: 26 and the VL of SEQ ID NO: 27 binds
to.
[0247] In some embodiments, the antibody binds human CD40 with a
dissociation constant (K.sub.D) of about 1.5.times.10.sup.-10 M or
less, when the K.sub.D is measured using ProteOn XPR36 system at
25.degree. C. in Dulbecco's phosphate buffered saline containing
0.01% PS-20 and 100 .mu.g/ml bovine serum albumin.
[0248] In some embodiments, the antibody inhibits soluble human
CD40L-driven human tonsillar B cell proliferation with an IC.sub.50
value of less than about 1.times.10.sup.-9 M.
[0249] In some embodiments, the antibody inhibits soluble human
CD40L-driven production of IL-12p40 by human dendritic cells with
an IC.sub.50 value of less than about 1.times.10.sup.-9 M.
[0250] In some embodiments, the antibody binds human CD40 with a
dissociation constant (K.sub.D) of about 1.5.times.10.sup.-10 M or
less, when the K.sub.D is measured using ProteOn XPR36 system at
25.degree. C. in Dulbecco's phosphate buffered saline containing
0.01% PS-20 and 100 .mu.g/ml bovine serum albumin, inhibits soluble
human CD40L-driven human tonsillar B cell proliferation with an
IC.sub.50 value of less than about 1.times.10.sup.-9 M and inhibits
soluble human CD40L-driven production of IL-12p40 by human
dendritic cells with an IC.sub.50 value of less than about
1.times.10.sup.-9 M.
[0251] In some embodiments, the antibody comprises the VH and the
VL of SEQ ID NOs: 26 and 27, respectively.
[0252] In some embodiments, the VH is encoded by a polynucleotide
comprising a polynucleotide sequence of SEQ ID NO: 33 and the VL is
encoded by a polynucleotide comprising a polynucleotide sequence of
SEQ ID NO: 34.
[0253] In some embodiments, the antibody is an IgG1, IgG2, IgG3 or
IgG4 isotype.
[0254] In some embodiments, the antibody is an IgG1 isotype.
[0255] In some embodiments, the antibody is an IgG2 isotype.
[0256] In some embodiments, the antibody is an IgG3 isotype.
[0257] In some embodiments, the antibody is an IgG4 isotype.
[0258] In some embodiments, the antibody is an IgG1 isotype,
optionally comprising a L234A/L235A/G237A/P238S/H268A/A330S/P331S
mutation when compared to the wild-type IgG1.
[0259] In some embodiments, the antibody is an IgG1/.lamda.
isotype, optionally comprising the
L234A/L235A/G237A/P238S/H268A/A330S/P331S mutation when compared to
the wild-type IgG1.
[0260] In some embodiments, the antibody comprises the VH of SEQ ID
NO: 26 and the VL of SEQ ID NO: 27 and is an IgG1/.lamda. isotype,
optionally comprising the L234A/L235A/G237A/P238S/H268A/A330S/P331S
mutation when compared to the wild-type IgG1.
[0261] In some embodiments, the antibody comprises the VH of SEQ ID
NO: 26 and the VL of SEQ ID NO: 27 and is an IgG1/.lamda. isotype
comprising the L234A/L235A/G237A/P238S/H268A/A330S/P331S mutation
when compared to the wild-type IgG1.
[0262] In some embodiments, the antibody is an IgG4 isotype,
optionally comprising a S228P mutation when compared to the
wild-type IgG4.
[0263] In some embodiments, the antibody is an IgG4/.lamda.
isotype, optionally comprising the S228P mutation when compared to
the wild-type IgG4.
[0264] In some embodiments, the antibody comprises the VH of SEQ ID
NO: 26 and the VL of SEQ ID NO: 27 and is an IgG4/.lamda. isotype,
optionally comprising the S228P mutation when compared to the
wild-type IgG4.
[0265] In some embodiments, the antibody comprises the VH of SEQ ID
NO: 26 and the VL of SEQ ID NO: 27 and is an IgG4/.lamda. isotype
comprising the S228P mutation when compared to the wild-type
IgG4.
[0266] In some embodiments, the antibody is an IgG4 isotype,
optionally comprising a S228P/F234A/L235A mutation when compared to
the wild-type IgG4.
[0267] In some embodiments, the antibody is an IgG4/.lamda.
isotype, optionally comprising the S228P/F234A/L235A mutation when
compared to the wild-type IgG4.
[0268] In some embodiments, the antibody comprises the VH of SEQ ID
NO: 26 and the VL of SEQ ID NO: 27 and is an IgG4/.lamda. isotype,
optionally comprising the S228P/F234A/L235A mutation when compared
to the wild-type IgG4.
[0269] In some embodiments, the antibody comprises the VH of SEQ ID
NO: 26 and the VL of SEQ ID NO: 27 and is an IgG4/.lamda. isotype
comprising the S228P/F234A/L235A mutation when compared to the
wild-type IgG4.
[0270] In some embodiments, the antibody comprises the heavy chain
of SEQ ID NO: 45 and the light chain of SEQ ID NO: 48.
[0271] In some embodiments, the heavy chain is encoded by a
polynucleotide comprising a polynucleotide sequence of SEQ ID NO:
75 and the light chain is encoded by a polynucleotide comprising a
polynucleotide sequence of SEQ ID NO: 78.
[0272] In some embodiments, the antibody comprises the heavy chain
of SEQ ID NO: 46 and the light chain of SEQ ID NO: 48.
[0273] In some embodiments, the heavy chain is encoded by a
polynucleotide comprising a polynucleotide sequence of SEQ ID NO:
76 and the light chain is encoded by a polynucleotide comprising a
polynucleotide sequence of SEQ ID NO: 78.
[0274] In some embodiments, the antibody is a multispecific
antibody, such as a bispecific antibody.
[0275] The antibody is suitable for use in therapy, for example in
treating an inflammatory disease.
[0276] The antibody is suitable for use in therapy, for example in
treating an autoimmune disease.
[0277] The antibody is suitable for use in therapy, for example in
treating Addinson's disease.
[0278] The antibody is suitable for use in therapy, for example in
treating an ankylosing spondylitis.
[0279] The antibody is suitable for use in therapy, for example in
treating an atherosclerosis.
[0280] The antibody is suitable for use in therapy, for example in
treating an autoimmune hepatitis.
[0281] The antibody is suitable for use in therapy, for example in
treating an autoimmune diabetes.
[0282] The antibody is suitable for use in therapy, for example in
treating Graves' disease.
[0283] The antibody is suitable for use in therapy, for example in
treating Buillain-Barre syndrome.
[0284] The antibody is suitable for use in therapy, for example in
treating Hashimoto's disease.
[0285] The antibody is suitable for use in therapy, for example in
treating, an idiopathic thrombocytopenia.
[0286] The antibody is suitable for use in therapy, for example in
treating an inflammatory bowel disease (IBD).
[0287] The antibody is suitable for use in therapy, for example in
treating a systemic lupus erythematosus.
[0288] The antibody is suitable for use in therapy, for example in
treating a multiple sclerosis.
[0289] The antibody is suitable for use in therapy, for example in
treating a myasthenia gravis.
[0290] The antibody is suitable for use in therapy, for example in
treating a psoriasis.
[0291] The antibody is suitable for use in therapy, for example in
treating an arthritis.
[0292] The antibody is suitable for use in therapy, for example in
treating a scleroderma.
[0293] The antibody is suitable for use in therapy, for example in
treating Sjogren's syndrome.
[0294] The antibody is suitable for use in therapy, for example in
treating a systemic sclerosis.
[0295] The antibody is suitable for use in therapy, for example in
treating a transplantation.
[0296] The antibody is suitable for use in therapy, for example in
treating a kidney transplantation.
[0297] The antibody is suitable for use in therapy, for example in
treating a skin transplantation.
[0298] The antibody is suitable for use in therapy, for example in
treating a bone marrow transplantation.
[0299] The antibody is suitable for use in therapy, for example in
treating a graft versus host disease (GVHD).
[0300] The antibody is suitable for use in therapy, for example in
treating a type I diabetes.
[0301] The antibody is suitable for use in therapy, for example in
treating a rheumatoid arthritis.
[0302] The antibody is suitable for use in therapy, for example in
treating a juvenile arthritis.
[0303] The antibody is suitable for use in therapy, for example in
treating a psoriatic arthritis.
[0304] The antibody is suitable for use in therapy, for example in
treating Reiter's syndrome.
[0305] The antibody is suitable for use in therapy, for example in
treating a gouty arthritis.
[0306] The antibody is suitable for use in therapy, for example in
treating Crohn's disease.
[0307] The antibody is suitable for use in therapy, for example in
treating an ulcerative colitis.
[0308] The antibody is suitable for use in therapy, for example in
treating an inflammatory disease in combination with a second
therapeutic agent.
[0309] The antibody is suitable for use in therapy, for example in
treating an autoimmune disease in combination with a second
therapeutic agent.
[0310] The antibody is suitable for use in therapy, for example in
treating a rheumatoid arthritis in combination with a second
therapeutic agent.
[0311] The antibody is suitable for use in therapy, for example in
treating a systemic lupus erythematosus in combination with a
second therapeutic agent.
[0312] Competition between binding to human CD40 with antibodies of
the invention comprising certain VH and VL sequences may be assayed
in vitro using following protocol: His-tagged recombinant soluble
human CD40 (CD40-ECD-his) is used in the assay. 5 .mu.L of anti-his
mAb (10 .mu.g/mL, R&D Systems, MAB050) is directly coated on
MSD HighBind plates for 2 hours at room temperature and then
blocked with 5% MSD Blocker A buffer for an additional 2 hours at
room temperature. 25 .mu.L of 10 .mu.g/mL CD40-ECD-his protein is
added to be captured by anti-his mAb. After incubation with gentle
shaking at room temperature 2 hours, plates are washed 3.times.
with 0.1 M HEPES buffer, pH 7.4, followed by the addition of the
mixture of 10 nM Ruthenium (Ru)-labeled reference anti-CD40 mAb or
a Fab portion thereof which is pre-incubated at room temperature
for 30 minutes with different concentrations, from 2 .mu.M to 1 nM,
of a test anti-CD40 antibody. After incubation with gentle shaking
at room temperature 1 hours, plates are washed 3.times. with 0.1M
HEPES buffer (pH 7.4). MSD Read Buffer T is diluted with distilled
water (4-fold) and dispensed into each well then analyzed with a
SECTOR Imager 6000 (Meso Scale Discovery, Gaithersburg, Md.). The
test antibody competes for binding to human CD40 with a reference
antibody (e.g. an antibody comprising the VH of SEQ ID NO: 11 and
the VL of SEQ ID NO: 12, the antibody comprising the VH of SEQ ID
NO: 25 and the VL of SEQ ID NO: 27 or the antibody comprising the
VH of SEQ ID NO: 26 and the VL of SEQ DI NO: 27) when the test
antibody reduces the MDS signal obtained in the above assay using
the Ru-labeled reference antibody or a Fab portion thereof by more
than 90%. Antibodies that compete for binding to CD40 with an
antibody comprising the VH of SEQ ID NO: 11 and the VL of SEQ ID
NO: 12, the VH of SEQ ID NO: 25 and the VL of SEQ ID NO: 27 or the
VH of SEQ ID NO: 26 and the VL of SEQ ID NO: 27 may be generated by
isolating antibodies specifically binding human CD40 using phage
display libraries, and screening the generated antibodies for their
ability to compete for binding to CD40 with the aforementioned
antibodies.
[0313] The CD40 epitope the antibody of the invention binds to may
be resolved for example using hydrogen/deuterium exchange (H/D
exchange) or by analyzing a crystal structure of the antibody in
complex with CD40. Two CD40 antibodies "bind the same epitope on
CD40" when 80% or more CD40 amino acid residues protected by the
antibody by at least 5% difference in deuteration levels through
H/D exchange are identical between the two antibodies, or when 80%
or more CD40 surface exposed amino acid residues determined to bind
the antibody in a crystal structure of a complex of the antibody
and CD40 are identical between the two antibodies. In the crystal
structure of a complex of the antibody and CD40, the epitope
residues are those CD40 residues that reside within 4 .ANG.
distance or less from any of the antibody CDR residues.
[0314] In an H/D exchange assay, CD40 protein is incubated in the
presence or absence of the antibody in deuterated water for
predetermined times resulting in deuterium incorporation at
exchangeable hydrogen atoms which are unprotected by the antibody,
followed by protease digestion of the protein and analyses of the
peptide fragments using LC-MS. In an exemplary assay, 5 .mu.L of
the test antibody (10 .mu.g) or 5 .mu.L of the complex of CD40 and
the test antibody (10 & 7.35 .mu.g, respectively) is incubated
with 120 .mu.L deuterium oxide labeling buffer (50 mM phosphate,
100 mM sodium chloride at pH 7.4) for 0 sec, 60 sec, 300 sec, 1800
sec, 7200 sec, and 14400 sec. Deuterium exchange is quenched by
adding 63 .mu.L of 5 M guanidine hydrochloride and final pH is 2.5.
The quenched sample is subjected to on-column pepsin/protease type
XIII digestion and LC-MS analysis. For pepsin/protease type XIII
digestion, 5 .mu.g of the samples in 125 .mu.L control buffer (50
mM phosphate, 100 mM sodium chloride at pH 7.4) are denatured by
adding 63 .mu.L of 5 M guanidine hydrochloride (final pH is 2.5)
and incubating the mixture for 3 min. Then, the mixture is
subjected to on-column pepsin/protease type XIII digestion and the
resultant peptides analyzed using an UPLC-MS system comprised of a
Waters Acquity UPLC coupled to a Q Exactive.TM. Hybrid
Quadrupole-Orbitrap Mass Spectrometer (Thermo). Raw MS data is
processed using HDX WorkBench, software for the analysis of H/D
exchange MS data. The deuterium levels are calculated using the
average mass difference between the deuteriated peptide and its
native form (t.sub.0). Peptide identification is done through
searching MS/MS data against the CD40 sequence with Mascot. The
mass tolerance for the precursor and product ions is 20 ppm and
0.05 Da, respectively.
[0315] For X-ray crystallography, CD40 and the test antibody are
expressed and purified using standard protocols. The CD40/test
antibody complex is incubated overnight at 4.degree. C.,
concentrated, and separated from the uncomplexed species using
size-exclusion chromatography. The complex is crystallized by the
vapor-diffusion method from various known test solutions for
example solutions containing PEG3350, ammonium citrate and
2-(N-morpholino)ethanesulfonic acid (MES).
[0316] Antibodies binding the same epitope on CD40 as a reference
antibody may be generated by isolating antibodies binding CD40
using phage display libraries, selecting those antibodies that
compete with the reference antibody for binding to CD40 by 100%,
and identifying the antibody epitope by H/D exchange or by X-ray
crystallography.
[0317] Alternatively, mice or rabbits may be immunized using
peptides encompassing the epitope residues, and the generated
antibodies may be evaluated for their binding within the recited
region.
[0318] The affinity of an antibody to human or cyno CD40 may be
determined experimentally using any suitable method. An exemplary
method utilizes ProteOn XPR36, Biacore 3000 or KinExA
instrumentation, ELISA or competitive binding assays known to those
skilled in the art. The measured affinity of a particular antibody
to CD40 may vary if measured under different conditions (e.g.,
osmolarity, pH). Thus, measurements of affinity and other binding
parameters (e.g., K.sub.D, K.sub.on, and Koff) are typically made
with standardized conditions and a standardized buffer, such as the
buffer described herein. Skilled in the art will appreciate that
the internal error for affinity measurements for example using
Biacore 3000 or ProteOn (measured as standard deviation, SD) can
typically be within 5-33% for measurements within the typical
limits of detection. Therefore the term "about" reflects the
typical standard deviation in the assay. For example, the typical
SD for a K.sub.D of 1.times.10.sup.-9 M is up to
.+-.0.33.times.10.sup.-9M.
[0319] In the B cell proliferation assay, 1.times.10.sup.5 human
tonsil B cells may be cultured in RPMI medium containing glutamax,
10% FBS and 1% Pen/Strep, titrations of each of the antibodies
specifically binding human CD40 may be added to the cells, followed
by addition of 0.5 .mu.g/ml soluble human CD154. Cells may be
cultured for 48 hours at 37.degree. C., pulsed with 3H-thymidine (1
.mu.Ci/well) in 50 .mu.l medium and cultured for 16-18 hours before
harvest and counting.
[0320] In the DC IL-12p40 production assay, human DCs may be
generated by culturing purified human monocytes
(2.5.times.10.sup.6/well 6-well plates) for 6 days in 3 ml RPMI
medium containing glutamax, 25 mM HEPES, 10% FBS, 1% Pen/Strep and
50 ng each GM-CSF and IL-4. On day 3, 1 ml of the medium may be
removed and replaced with 2 ml fresh medium containing 50 ng/ml
each of GM-CSF and IL-4. On day 6, DCs may be plated into 96-well
plates (100,000 cells/well) followed by titrations of each of the
antibodies specifically binding human CD40 and addition of 1
.mu.g/ml of soluble human CD154 to the cultures. Cells may be
cultured for 48 hours before collecting and analyzing supernatants
for IL-12p40 by MSD.
[0321] Immune effector properties of the antibodies of the
invention may be enhanced or silenced through Fc modifications. For
example, Fc effector functions such as C1q binding, complement
dependent cytotoxicity (CDC), antibody-dependent cell-mediated
cytotoxicity (ADCC), phagocytosis, down regulation of cell surface
receptors (e.g., B cell receptor; BCR), etc. may be provided and/or
controlled by modifying residues in the Fc responsible for these
activities.
[0322] In some embodiments, the antibody of the invention has
reduced binding to Fc.gamma.RI, Fc.gamma.RIIa, Fc.gamma.RIIb,
Fc.gamma.RIIIa or Fc.gamma.RIIIb.
[0323] "Reduced binding" refers to reduced binding of the antibody
of the invention having at least one mutation in the Fc region to
an Fc.gamma. receptor (Fc.gamma.R) when compared to the binding of
the parental antibody without the mutation to the same Fc.gamma.R.
"Reduced binding" may be at least about 100-fold, at least about
500-fold, at least about 1000-fold, at least about 5000-fold, at
least about 10,000-fold, or at least about 20,000-fold reduced
binding. In practice, antibodies exhibiting "reduced binding" to a
particular Fc.gamma.R refer to antibodies that have statistically
insignificant effector function mediated by the particular
Fc.gamma.R.
[0324] In some embodiments, the antibody of the invention comprises
at least one mutation in the Fc region that reduces binding of the
antibody to an Fc.gamma.R.
[0325] In some embodiments, the Fc.gamma.R is Fc.gamma.RI,
Fc.gamma.RIIa or Fc.gamma.RIIIa or Fc.gamma.RIIIb.
[0326] In some embodiments, the at least one mutation in the Fc
region is a L234A mutation, a L235A mutation, a G237A mutation, a
P238S mutation, a M252Y mutation, a S254T mutation, a T256E
mutation, a H268A mutation, a A330S mutation or a P331S mutation,
wherein residue numbering is according to the EU Index.
[0327] In some embodiments, the antibody of the invention comprises
a L234A/L235A/G237A/P238S/H268A/A330S/P331S mutation in the Fc
region, wherein residue numbering is according to the EU Index.
[0328] In some embodiments, the at least one mutation in the Fc
region is a V234A mutation, a G237A mutation, a P238S mutation, a
M252Y mutation, a S254T mutation, a T256E mutation, a H268A
mutation, a V309L mutation, an A330S mutation or a P331S mutation,
wherein residue numbering is according to the EU Index.
[0329] In some embodiments, the antibody of the invention comprises
a V234A/G237A/P238S/H268A/V309L/A330S/P331S mutation in the Fc
region, wherein residue numbering is according to the EU Index.
[0330] In some embodiments, the antibody of the invention comprises
a S228P mutation in the Fc region, wherein residue numbering is
according to the EU Index.
[0331] In some embodiments, the antibody of the invention comprises
a F234A mutation in the Fc region, wherein residue numbering is
according to the EU Index.
[0332] In some embodiments, the antibody of the invention comprises
a L235A mutation in the Fc region, wherein residue numbering is
according to the EU Index.
[0333] In some embodiments, the antibody of the invention comprises
a S228P/F234A/L235A mutation, wherein residue numbering is
according to the EU Index.
[0334] Binding of the antibodies of the invention to Fc.gamma.RI,
Fc.gamma.RIIa, Fc.gamma.RIIb, Fc.gamma.RIIIa and Fc.gamma.RIIIb may
be evaluated using recombinant soluble forms or cell-associated
forms of the Fc.gamma. receptors. For example, direct or indirect,
e.g., competitive binding, measurements may be applied for
assessing relative affinities and avidities of the antibodies of
the invention to various Fc.gamma.R. In an exemplary assay, a test
antibody binding to soluble Fc.gamma.R captured on a plate is
evaluated using competitive binding between 1 .mu.g/ml biotinylated
human IgG1 and serial dilutions of the test antibody pre-complexed
with antigen.
[0335] In some embodiments, the antibody of the invention
comprising at least one mutation in the Fc region has reduced
antibody dependent cellular cytotoxicity (ADCC), reduced
antibody-dependent cellular phagocytosis, ("ADCP") and/or reduced
complement dependent cytotoxicity (CDC).
[0336] "Antibody-dependent cellular cytotoxicity",
"antibody-dependent cell-mediated cytotoxicity" or "ADCC" is a
mechanism for inducing cell death that depends upon the interaction
of antibody-coated target cells with effector cells possessing
lytic activity, such as natural killer cells, monocytes,
macrophages and neutrophils via Fc gamma receptors (Fc.gamma.R)
expressed on effector cells. For example, NK cells express
Fc.gamma.RIIIa, whereas monocytes express Fc.gamma.RI, Fc.gamma.RII
and Fc.gamma.RIIIa. To assess ADCC activity of the antibodies of
the invention, the antibody may be added to target cells in
combination with immune effector cells, which may be activated by
the antigen antibody complexes resulting in cytolysis of the target
cell. Cytolysis is generally detected by the release of label (e.g.
radioactive substrates, fluorescent dyes or natural intracellular
proteins) from the lysed cells. Exemplary effector cells for such
assays include peripheral blood mononuclear cells (PBMC) and NK
cells. Exemplary target cells include cells expressing CD40.
[0337] "Antibody-dependent cellular phagocytosis" ("ADCP") refers
to a mechanism of elimination of antibody-coated target cells by
internalization by phagocytic cells, such as macrophages or
dendritic cells. ADCP may be evaluated using monocyte-derived
macrophages as effector cells and cells expressing CD40 engineered
to express GFP or other labeled molecule as target cells.
Effector:target cell ratio may be for example 4:1. Effector cells
may be incubated with target cells for 4 hours with or without the
test CD40 antibody. After incubation, cells may be detached using
accutase. Macrophages may be identified with anti-CD11b and
anti-CD14 antibodies coupled to a fluorescent label, and percent
phagocytosis may be determined based on % GFP fluorescent in the
CD11.sup.+CD14.sup.+ macrophages using standard methods.
[0338] "Complement-dependent cytotoxicity", or "CDC", refers to a
mechanism for inducing cell death in which an Fc effector domain of
a target-bound antibody binds and activates complement component
C1q which in turn activates the complement cascade leading to
target cell death. Activation of complement may also result in
deposition of complement components on the target cell surface that
facilitate ADCC by binding complement receptors (e.g., CR3) on
leukocytes. CDC of CD40-expressing cells may be measured for
example by plating cells expressing CD40 in an appropriate medium,
adding anti-CD40 antibodies into the mixture, followed by addition
of pooled human serum. After incubation period, percentage (%)
lysed cells may be detected as % propidium iodide stained cells in
FACS assay using standard methods.
[0339] "Reduced ADCC", "reduced CDC" and "reduced ADCP" refers to a
statistically significant reduction in ADCC, CDC and/or ADCP
mediated by the antibody of the invention comprising at least one
mutation in the Fc region when compared to the same antibody
without the mutation. ADCC, CDC and/or ADCP, such as assays
described herein and in assays described in U.S. Pat. No.
8,871,204.
[0340] Variants of the antibodies of the invention comprising the
VH or the VL amino acid sequences shown in Table 7 are within the
scope of the invention. For example, variants may comprise one,
two, three, four, five, six, seven, eight, nine, ten, eleven,
twelve, thirteen, fourteen or fifteen amino acid substitutions in
the VH and/or the VL that do not adversely affect the
characteristics of the antibodies. In some embodiments, the
sequence identity may be about 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98% or 99% to the antibody VH or the VL amino acid sequence of
the invention.
[0341] The percent identity between the two sequences is a function
of the number of identical positions shared by the sequences (i.e.,
% identity=# of identical positions/total # of positions
.times.100), taking into account the number of gaps, and the length
of each gap, which need to be introduced for optimal alignment of
the two sequences.
[0342] The percent identity between two amino acid sequences may be
determined using the algorithm of E. Meyers and W. Miller (Comput.
Appl. Biosci., 4:11-17 (1988)) which has been incorporated into the
ALIGN program (version 2.0), using a PAM120 weight residue table, a
gap length penalty of 12 and a gap penalty of 4. In addition, the
percent identity between two amino acid sequences may be determined
using the Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970))
algorithm which has been incorporated into the GAP program in the
GCG software package (available at http://_www_gcg_com), using
either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of
16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or
6.
[0343] In some embodiments, the antibody of the invention comprises
the VH that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99% or 100% identical to the VH of SEQ ID NOs: 11, 21, 22, 23,
24, 25 or 26 and the VL that is at least 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99% or 100% identical to the VL of SEQ ID NOs:
12 or 27, wherein the antibody exhibits one or more of the
following properties:
[0344] inhibits 0.5 .mu.g/ml human soluble CD40L-driven human
tonsillar B cell proliferation with an IC.sub.50 value of less than
about 1.times.10.sup.-9 M;
[0345] inhibits 1 .mu.g/ml human soluble CD40L-driven production of
IL-12p40 by human dendritic cells with an IC.sub.50 value of less
than about 1.times.10.sup.-9 M; or
[0346] binds to human CD40 with a dissociation constant (K.sub.D)
of about 5.times.10.sup.-9 M or less, [0347] when the K.sub.D is
measured using ProteOn XPR36 system using experimental design
described in Example 4.
[0348] In some embodiments, the antibody of the invention comprises
the VH and the VL which are at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or 100% identical to the VH of SEQ ID NO: 11 and
the VL of SEQ ID NO: 12.
[0349] In some embodiments, the antibody of the invention comprises
the VH and the VL which are at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or 100% identical to the VH of SEQ ID NO: 21 and
the VL of SEQ ID NO: 27.
[0350] In some embodiments, the antibody of the invention comprises
the VH and the VL which are at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or 100% identical to the VH of SEQ ID NO: 22 and
the VL of SEQ ID NO: 27.
[0351] In some embodiments, the antibody of the invention comprises
the VH and the VL which are at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or 100% identical to the VH of SEQ ID NO: 23 and
the VL of SEQ ID NO: 27.
[0352] In some embodiments, the antibody of the invention comprises
the VH and the VL which are at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or 100% identical to the VH of SEQ ID NO: 24 and
the VL of SEQ ID NO: 27.
[0353] In some embodiments, the antibody of the invention comprises
the VH and the VL which are at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or 100% identical to the VH of SEQ ID NO: 25 and
the VL of SEQ ID NO: 27.
[0354] In some embodiments, the antibody of the invention comprises
the VH and the VL which are at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or 100% identical to the VH of SEQ ID NO: 26 and
the VL of SEQ ID NO: 27.
[0355] In some embodiments, the antibody of the invention comprises
the VH of SEQ ID NO: 11 and the VL of SEQ ID NO: 12, wherein the
VH, the VL or both the VH and the VL comprise one, two, three,
four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen,
fourteen or fifteen conservative amino acid substitutions and the
antibody exhibits one or more of the following properties:
[0356] inhibits 0.5 .mu.g/ml human soluble CD40L-driven human
tonsillar B cell proliferation with an IC.sub.50 value of less than
about 1.times.10.sup.-9 M;
[0357] inhibits 1 .mu.g/ml human soluble CD40L-driven production of
IL-12p40 by human dendritic cells with an IC.sub.50 value of less
than about 1.times.10.sup.-9 M; or
[0358] binds to human CD40 with a dissociation constant (K.sub.D)
of about 5.times.10.sup.-9 M or less, [0359] when the K.sub.D is
measured using ProteOn XPR36 system using experimental design
described in Example 4.
[0360] In some embodiments, the antibody of the invention comprises
the VH of SEQ ID NO: 11 and the VL of SEQ ID NO: 12, wherein the
VH, the VL or both the VH and the VL comprise one, two, three,
four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen,
fourteen or fifteen conservative amino acid substitutions.
[0361] In some embodiments, the antibody of the invention comprises
the VH of SEQ ID NO: 21 and the VL of SEQ ID NO: 27, wherein the
VH, the VL or both the VH and the VL comprise one, two, three,
four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen,
fourteen or fifteen conservative amino acid substitutions.
[0362] In some embodiments, the antibody of the invention comprises
the VH of SEQ ID NO: 22 and the VL of SEQ ID NO: 27, wherein the
VH, the VL or both the VH and the VL comprise one, two, three,
four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen,
fourteen or fifteen conservative amino acid substitutions.
[0363] In some embodiments, the antibody of the invention comprises
the VH of SEQ ID NO: 23 and the VL of SEQ ID NO: 27, wherein the
VH, the VL or both the VH and the VL comprise one, two, three,
four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen,
fourteen or fifteen conservative amino acid substitutions.
[0364] In some embodiments, the antibody of the invention comprises
the VH of SEQ ID NO: 24 and the VL of SEQ ID NO: 27, wherein the
VH, the VL or both the VH and the VL comprise one, two, three,
four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen,
fourteen or fifteen conservative amino acid substitutions.
[0365] In some embodiments, the antibody of the invention comprises
the VH of SEQ ID NO: 25 and the VL of SEQ ID NO: 27, wherein the
VH, the VL or both the VH and the VL comprise one, two, three,
four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen,
fourteen or fifteen conservative amino acid substitutions.
[0366] In some embodiments, the antibody of the invention comprises
the VH of SEQ ID NO: 26 and the VL of SEQ ID NO: 27, wherein the
VH, the VL or both the VH and the VL comprise one, two, three,
four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen,
fourteen or fifteen conservative amino acid substitutions.
[0367] "Conservative modification" refers to amino acid
modifications that do not significantly affect or alter the binding
characteristics of the antibody containing the amino acid
sequences. Conservative modifications include amino acid
substitutions, additions and deletions. Conservative amino acid
substitutions are those in which the amino acid is replaced with an
amino acid residue having a similar side chain. The families of
amino acid residues having similar side chains are well defined and
include amino acids with acidic side chains (e.g., aspartic acid,
glutamic acid), basic side chains (e.g., lysine, arginine,
histidine), nonpolar side chains (e.g., alanine, valine, leucine,
isoleucine, proline, phenylalanine, methionine), uncharged polar
side chains (e.g., glycine, asparagine, glutamine, cysteine,
serine, threonine, tyrosine, tryptophan), aromatic side chains
(e.g., phenylalanine, tryptophan, histidine, tyrosine), aliphatic
side chains (e.g., glycine, alanine, valine, leucine, isoleucine,
serine, threonine), amide (e.g., asparagine, glutamine),
beta-branched side chains (e.g., threonine, valine, isoleucine) and
sulfur-containing side chains (cysteine, methionine). Furthermore,
any native residue in the polypeptide may be substituted with
alanine, as has been previously described for alanine scanning
mutagenesis (MacLennan et al., Acta Physiol. Scand. Suppl.
643:55-67, 1998; Sasaki et al., Adv. Biophys. 35:1-24, 1998). Amino
acid substitutions to the antibodies of the invention may be made
by well-known methods for example by PCR mutagenesis (U.S. Pat. No.
4,683,195). Alternatively, libraries of variants may be generated
using known methods, for example using random (NNK) or non-random
codons, for example DVK codons, which encode 11 amino acids (Ala,
Cys, Asp, Glu, Gly, Lys, Asn, Arg, Ser, Tyr, Trp). The resulting
antibody variants may be tested for their characteristics using
assays described herein.
[0368] Although the embodiments illustrated in the Examples
comprise pairs of variable regions, one from a heavy chain and one
from a light chain, a skilled artisan will recognize that
alternative embodiments may comprise single heavy or light chain
variable regions. The single variable region may be used to screen
for variable domains capable of forming a two-domain specific
antigen-binding fragment capable of for example specifically
binding to human CD40. The screening may be accomplished by phage
display screening methods using for example hierarchical dual
combinatorial approach disclosed in Int. Pat. Publ. No.
WO1992/01047.
[0369] Antibodies of the invention may be generated using various
technologies. For example, the hybridoma method of Kohler and
Milstein, Nature 256:495, 1975 may be used to generate monoclonal
antibodies. In the hybridoma method, a mouse or other host animal,
such as a hamster, rat, rabbit or monkey, is immunized with human,
marmoset or cyno CD40 or fragments of CD40, such as soluble form of
CD40, followed by fusion of spleen cells from immunized animals
with myeloma cells using standard methods to form hybridoma cells
(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103
(Academic Press, 1986)). Colonies arising from single immortalized
hybridoma cells are screened for production of antibodies with
desired properties, such as specificity of binding,
cross-reactivity or lack thereof, and affinity for the antigen.
[0370] Various host animals may be used to produce the antibodies
of the invention. For example, Balb/c mice may be used to generate
antibodies. The antibodies made in Balb/c mice and other non-human
animals may be humanized using various technologies to generate
more human-like sequences. Exemplary humanization techniques
including selection of human acceptor frameworks are known and
include CDR grafting (U.S. Pat. No. 5,225,539), SDR grafting (U.S.
Pat. No. 6,818,749), Resurfacing (Padlan, Mol Immunol 28:489-499,
1991), Specificity Determining Residues Resurfacing (U.S. Pat.
Publ. No. 20100261620), human-adaptation (or human framework
adaptation) (U.S. Pat. Publ. No. US2009/0118127), Superhumanization
(U.S. Pat. No. 7,709,226) and guided selection (Osbourn et al
(2005) Methods 36:61-68, 2005; U.S. Pat. No. 5,565,332). In these
methods, CDRs of parental antibodies are transferred onto human
frameworks that may be selected based on their overall homology to
the parental frameworks, based on framework CDR length, homology or
canonical structure information, or a combination thereof.
[0371] Humanized antibodies may be further optimized to improve
their selectivity or affinity to a desired antigen by incorporating
altered framework support residues to preserve binding affinity
(backmutations) by techniques such as those disclosed as described
in Int. Pat. Publ. No. WO90/007861 and in Int. Pat. Publ. No.
WO92/22653, or by introducing variation to any of the CDRs to
improve for example affinity of the antibody.
[0372] Transgenic mice carrying human immunoglobulin (Ig) loci in
their genome may be used to generate human antibodies against a
target protein, and are described in for example Int. Pat. Publ.
No. WO90/04036, U.S. Pat. No. 6,150,584, Int. Pat. Publ. No.
WO99/45962, Int. Pat. Publ. No. WO02/066630, Int. Pat. Publ. No.
WO02/43478, Lonberg et al (1994) Nature 368:856-9; Green et al
(1994) Nature Genet. 7:13-21; Green & Jakobovits (1998) Exp.
Med. 188:483-95; Lonberg and Huszar (1995) Int. Rev. Immunol.
13:65-93; Bruggemann et al (1991) Eur. J. Immunol. 21:1323-1326;
Fishwild et al (1996) Nat. Biotechnol. 14:845-851; Mendez et al
(1997) Nat. Genet. 15:146-156; Green (1999) J. Immunol. Methods
231:11-23; Yang et al (1999) Cancer Res. 59:1236-1243; Bruggemann
and Taussig (1997) Curr. Opin. Biotechnol. 8:455-458; Int. Pat.
Publ. No. WO02/043478). The endogenous immunoglobulin loci in such
mice may be disrupted or deleted, and at least one complete or
partial human immunoglobulin locus may be inserted into the mouse
genome using homologous or non-homologous recombination, using
transchromosomes, or using minigenes. Companies such as Regeneron
(http://_www_regeneron_com), Harbour Antibodies
(http://_www_harbourantibodies_com), Open Monoclonal Technology,
Inc. (OMT) (http://_www_omtinc_net), KyMab (http://_www_kymab_com),
Trianni (http://_www.trianni_com) and Ablexis
(http://_www_ablexis_com) may be engaged to provide human
antibodies directed against a selected antigen using technology as
described above.
[0373] Human antibodies may be selected from a phage display
library, where the phage is engineered to express human
immunoglobulins or portions thereof such as Fabs, single chain
antibodies (scFv), or unpaired or paired antibody variable regions
(Knappik et al., J. Mol Biol 296:57-86, 2000; Krebs et al., J
Immunol Meth 254:67-84, 2001; Vaughan et al., Nature Biotechnology
14:309-314, 1996; Sheets et al., PITAS (USA) 95:6157-6162, 1998;
Hoogenboom and Winter, J Mol Biol 227:381, 1991; Marks et al., J
Mol Biol 222:581, 1991). The antibodies of the invention may be
isolated for example from phage display libraries expressing
antibody heavy and light chain variable regions as fusion proteins
with bacteriophage pIX coat protein as described in Shi et al., J
Mol Biol 397:385-96, 2010 and Int. Pat. Publ. No. WO09/085462). The
libraries may be screened for phage binding to human and/or cyno
CD40 and the obtained positive clones may be further characterized,
the Fabs isolated from the clone lysates, and expressed as full
length IgGs. Such phage display methods for isolating human
antibodies are described in for example: U.S. Pat. Nos. 5,223,409;
5,403,484; and U.S. Pat. No. 5,571,698 to Ladner et al.; U.S. Pat.
Nos. 5,427,908 and 5, 580,717 to Dower et al.; U.S. Pat. Nos.
5,969,108 and 6,172,197 to McCafferty et al.; and U.S. Pat. Nos.
5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915 and 6,593,081
to Griffiths et al.
[0374] Preparation of immunogenic antigens and monoclonal antibody
production may be performed using any suitable technique, such as
recombinant protein production. The immunogenic antigens may be
administered to an animal in the form of purified protein, or
protein mixtures including whole cells or cell or tissue extracts,
or the antigen may be formed de novo in the animal's body from
nucleic acids encoding said antigen or a portion thereof.
[0375] The antibodies of the invention may be human or
humanized.
[0376] In some embodiments, the antibody of the invention comprises
a VH framework derived from human germline gene VH3_3-23 (SEQ ID
NO: 49).
[0377] In some embodiments, the antibody of the invention comprises
a VL framework derived from human germline gene VL3_3R (IGLV3-1)
(SEQ ID NO: 50).
[0378] The antibodies of the invention may be of IgA, IgD, IgE, IgG
or IgM type. The antibodies of the invention may be of IgG1, IgG2,
IgG3, IgG4 type.
[0379] The antibodies of the invention may further be engineered to
generate modified antibodies with similar or altered properties
when compared to the parental antibodies. The VH, the VL, the VH
and the VL, the constant regions, VH framework, VL framework, or
any or all of the six CDRs may be engineered in the antibodies of
the invention.
[0380] The antibodies of the invention may be engineered by CDR
grafting. One or more CDR sequences of the antibodies of the
invention may be grafted to a different framework sequence. CDR
grafting may be done using methods described herein. In some
embodiments, the antibodies of the invention comprise a VH that
comprises the HDCR1 of SEQ ID NO: 5, the HCDR2 of SEQ ID NO: 6, 15,
16, 17 or 18, the HCDR3 of SEQ ID NOs: 7 or 19, and the VL that
comprises the LCDR1 of SEQ ID NOs: 8 or 20, the LCDR2 of SEQ ID NO:
9 and/or the LCDR3 of SEQ ID NO: 10, wherein the VH framework is
not derived from VH3_3-23 (SEQ ID NO: 49) and the VL framework is
not derived from VL3_3R (IGLV3-1) (SEQ ID NO: 50). The framework
sequences to be used may be obtained from public DNA databases or
published references that include germline antibody gene sequences.
For example, germline DNA and the encoded protein sequences for
human heavy and light chain variable region genes can be found at
IMGT.RTM., the international ImMunoGeneTics information System.RTM.
http://_www-imgt_org. Framework sequences that may be used to
replace the existing framework sequences in the antibodies of the
invention are those that show the highest percent identity to
C40B16, C40B124, C40B135, C40B125, C40B136, C40B127, C40B138,
C40B131, C40B176, C40B180, C40B179 or C40B183 VH or VL at amino
acid level.
[0381] The framework sequences of the parental and engineered
antibodies may further be modified, for example by backmutations to
restore and/or improve binding of the resulting antibody to the
antigen as described for example in U.S. Pat. No. 6,180,370. The
framework sequences of the parental and engineered antibodies may
further be modified by mutating one or more residues within the
framework region, or even within one or more CDR regions, to remove
T-cell epitopes to thereby reduce the potential immunogenicity of
the antibody. This approach is also referred to as "deimmunization"
and described in further detail in U.S. Pat. Publ. No.
20030153043.
[0382] The CDR residues of the antibodies of the invention may be
mutated to improve one or more binding properties of the antibody
of interest. Site-directed mutagenesis or PCR-mediated mutagenesis
may be performed to introduce the mutation(s) and the effect on
antibody binding, or other functional property of interest, may be
evaluated in in vitro or in vivo assays as described herein and
provided in the Examples. Exemplary substitutions that may be
introduced are conservative modifications as discussed supra.
Moreover, typically no more than one, two, three, four or five
residues within a CDR region are altered.
[0383] Antibodies of the invention may be post-translationally
modified by processes such as glycosylation, isomerization,
deglycosylation or non-naturally occurring covalent modification
such as the addition of polyethylene glycol moieties (pegylation)
and lipidation. Such modifications may occur in vivo or in vitro.
For example, the antibodies of the invention may be conjugated to
polyethylene glycol (PEGylated) to improve their pharmacokinetic
profiles. Conjugation may be carried out by techniques known to
those skilled in the art. Conjugation of therapeutic antibodies
with PEG has been shown to enhance pharmacodynamics while not
interfering with function (Knigh et al., Platelets 15:409-18, 2004;
Leong et al., Cytokine 16:106-19, 2001; Yang et al., Protein Eng.
16:761-70, 2003).
[0384] Antibodies or antigen-binding fragments thereof of the
invention may be modified to improve stability, selectivity,
cross-reactivity, affinity, immunogenicity or other desirable
biological or biophysical property are within the scope of the
invention. Stability of an antibody is influenced by a number of
factors, including core packing of individual domains that affects
their intrinsic stability, protein/protein interface interactions
that have impact upon the HC and LC pairing, burial of polar and
charged residues, (4) H-bonding network for polar and charged
residues; and surface charge and polar residue distribution among
other intra- and inter-molecular forces (Worn et al., J Mol Biol
305:989-1010, 2001). Potential structure destabilizing residues may
be identified based upon the crystal structure of the antibody or
by molecular modeling in certain cases, and the effect of the
residues on antibody stability may be tested by generating and
evaluating variants harboring mutations in the identified residues.
One of the ways to increase antibody stability is to raise the
thermal transition midpoint (T.sub.m) as measured by differential
scanning calorimetry (DSC). In general, the protein T.sub.m is
correlated with its stability and inversely correlated with its
susceptibility to unfolding and denaturation in solution and the
degradation processes that depend on the tendency of the protein to
unfold (Remmele et al., Biopharm 13:36-46, 2000). A number of
studies have found correlation between the ranking of the physical
stability of formulations measured as thermal stability by DSC and
physical stability measured by other methods (Gupta et al., AAPS
PharmSci 5E8, 2003; Zhang et al., J Pharm Sci 93:3076-89, 2004; Maa
et al., Int J Pharm 140:155-68, 1996; Bedu-Addo et al., Pharm Res
21:1353-61, 2004; Remmele et al., Pharm Res 15:200-8, 1997).
Formulation studies suggest that a Fab T.sub.m has implication for
long-term physical stability of a corresponding mAb.
[0385] In some embodiments, the antibody of the invention is a
multispecific antibody.
[0386] In some embodiments, the antibody of the invention is a
bispecific antibody.
[0387] The monospecific antagonistic antibodies specifically
binding CD40 of the invention may be engineered into bispecific
antibodies which are also encompassed within the scope of the
invention.
[0388] Full length bispecific antibodies may be generated for
example using Fab arm exchange (e.g., half-molecule exchange,
exchanging one heavy chain-light chain pair) between two
monospecific bivalent antibodies by introducing substitutions at
the heavy chain CH3 interface in each half molecule to favor
heterodimer formation of two antibody half molecules having
distinct specificity either in vitro in cell-free environment or
using co-expression. The Fab arm exchange reaction is the result of
a disulfide-bond isomerization reaction and
dissociation-association of CH3 domains. The heavy chain disulfide
bonds in the hinge regions of the parental monospecific antibodies
are reduced. The resulting free cysteines of one of the parental
monospecific antibodies form an inter heavy-chain disulfide bond
with cysteine residues of a second parental monospecific antibody
molecule and simultaneously CH3 domains of the parental antibodies
release and reform by dissociation-association. The CH3 domains of
the Fab arms may be engineered to favor heterodimerization over
homodimerization. The resulting product is a bispecific antibody
having two Fab arms or half molecules which each bind a distinct
epitope.
[0389] Bispecific antibodies may also be generated using designs
such as the Triomab/Quadroma (Trion Pharma/Fresenius Biotech),
Knob-in-Hole (Genentech), CrossMAbs (Roche) and the
electrostatically-induced CH3 interaction (Chugai, Amgen,
NovoNordisk, Oncomed), the LUZ-Y (Genentech), the Strand Exchange
Engineered Domain body (SEEDbody)(EMD Serono), the Biclonic (Merus)
and as DuoBody.RTM. Products (Genmab A/S).
[0390] The Triomab quadroma technology may be used to generate full
length bispecific antibodies of the invention. Triomab technology
promotes Fab arm exchange between two parental chimeric antibodies,
one parental mAb having IgG2a and the second parental mAb having
rat IgG2b constant regions, yielding chimeric bispecific
antibodies.
[0391] The "knob-in-hole" strategy (see, e.g., Intl. Publ. No. WO
2006/028936) may be used to generate full length bispecific
antibodies. Briefly, selected amino acids forming the interface of
the CH3 domains in human IgG can be mutated at positions affecting
CH3 domain interactions to promote heterodimer formation. An amino
acid with a small side chain (hole) is introduced into a heavy
chain of an antibody specifically binding a first antigen and an
amino acid with a large side chain (knob) is introduced into a
heavy chain of an antibody specifically binding a second antigen.
After co-expression of the two antibodies, a heterodimer is formed
as a result of the preferential interaction of the heavy chain with
a "hole" with the heavy chain with a "knob". Exemplary CH3
substitution pairs forming a knob and a hole are (expressed as
modified position in the first CH3 domain of the first heavy
chain/modified position in the second CH3 domain of the second
heavy chain): T366Y/F405A, T366W/F405W, F405W/Y407A, T394W/Y407T,
T394S/Y407A, T366W/T394S, F405W/T394S and
T366W/T366S_L368A_Y407V.
[0392] The CrossMAb technology may be used to generate full length
bispecific antibodies of the invention. CrossMAbs, in addition to
utilizing the "knob-in-hole" strategy to promoter Fab arm exchange,
have in one of the half arms the CH1 and the CL domains exchanged
to ensure correct light chain pairing of the resulting bispecific
antibody (see e.g. U.S. Pat. No. 8,242,247).
[0393] Other cross-over strategies may be used to generate full
length bispecific antibodies by exchanging variable or constant, or
both domains between the heavy chain and the light chain or within
the heavy chain in the bispecific antibodies, either in one or both
arms. These exchanges include for example VH-CH1 with VL-CL, VH
with VL, CH3 with CL and CH3 with CH1 as described in Int. Patent
Publ. Nos. WO2009/080254, WO2009/080251, WO2009/018386 and
WO2009/080252.
[0394] Other strategies such as promoting heavy chain
heterodimerization using electrostatic interactions by substituting
positively charged residues at one CH3 surface and negatively
charged residues at a second CH3 surface may be used, as described
in US Patent Publ. No. US2010/0015133; US Patent Publ. No.
US2009/0182127; US Patent Publ. No. US2010/028637 or US Patent
Publ. No. US2011/0123532. In other strategies, heterodimerization
may be promoted by following substitutions (expressed as modified
position in the first CH3 domain of the first heavy chain/modified
position in the second CH3 domain of the second heavy chain):
L351Y_F405A_Y407V/T394W, T366I_K392M_T394W/F405A_Y407V,
T366L_K392M_T394W/F405A_Y407V, L351Y_Y407A/T366A_K409F,
L351Y_Y407A/T366V_K409F, Y407A/T366A_K409F, or
T350V_L351Y_F405A_Y407V/T350V_T366L_K392L_T394W as described in
U.S. Patent Publ. No. US2012/0149876 or U.S. Patent Publ. No.
US2013/0195849.
[0395] LUZ-Y technology may be utilized to generate bispecific
antibodies. In this technology, a leucine zipper is added into the
C terminus of the CH3 domains to drive the heterodimer assembly
from parental mAbs that is removed post-purification as described
in Wranik et al., (2012) J Biol Chem 287(52): 42221-9.
[0396] SEEDbody technology may be utilized to generate bispecific
antibodies. SEEDbodies have, in their constant domains, select IgG
residues substituted with IgA residues to promote
heterodimerization as described in U.S. Patent No.
US20070287170.
[0397] Bispecific antibodies may be generated in vitro in a
cell-free environment by introducing asymmetrical mutations in the
CH3 regions of two monospecific homodimeric antibodies and forming
the bispecific heterodimeric antibody from two parent monospecific
homodimeric antibodies in reducing conditions to allow disulfide
bond isomerization according to methods described in Int. Patent
Publ. No. WO2011/131746. In the methods, the first monospecific
bivalent antibody and the second monospecific bivalent antibody are
engineered to have certain substitutions at the CH3 domain that
promoter heterodimer stability; the antibodies are incubated
together under reducing conditions sufficient to allow the
cysteines in the hinge region to undergo disulfide bond
isomerization; thereby generating the bispecific antibody by Fab
arm exchange. Substitutions that may be used are F405L in one heavy
chain and K409R in the other heavy chain. The incubation conditions
may optimally be restored to non-reducing. Exemplary reducing
agents that may be used are 2-mercaptoethylamine (2-MEA),
dithiothreitol (DTT), dithioerythritol (DTE), glutathione,
tris(2-carboxyethyl)phosphine (TCEP), L-cysteine and
beta-mercaptoethanol. For example, incubation for at least 90 min
at a temperature of at least 20.degree. C. in the presence of at
least 25 mM 2-MEA or in the presence of at least 0.5 mM
dithiothreitol at a pH of from 5-8, for example at pH of 7.0 or at
pH of 7.4 may be used.
[0398] Substitutions are typically made at the DNA level to a
molecule such as the constant domain of the antibody using standard
methods.
[0399] The antibodies of the invention may be engineered into
various well known antibody forms.
[0400] In some embodiments, the bispecific antibodies include
recombinant IgG-like dual targeting molecules, wherein the two
sides of the molecule each contain the Fab fragment or part of the
Fab fragment of at least two different antibodies; IgG fusion
molecules, wherein full length IgG antibodies are fused to an extra
Fab fragment or parts of Fab fragment; Fc fusion molecules, wherein
single chain Fv molecules or stabilized diabodies are fused to
heavy-chain constant-domains, Fc-regions or parts thereof; Fab
fusion molecules, wherein different Fab-fragments are fused
together; ScFv- and diabody-based and heavy chain antibodies (e.g.,
domain antibodies, nanobodies) wherein different single chain Fv
molecules or different diabodies or different heavy-chain
antibodies (e.g. domain antibodies, nanobodies) are fused to each
other or to another protein or carrier molecule.
Polynucleotides, Vectors, Host Cells
[0401] The invention also provides for an isolated polynucleotide
encoding any of the antibody heavy chain variable regions, any of
the antibody light chain variable regions, or any of the antibody
heavy chains and/or the antibody light chains of the invention.
[0402] The invention also provides for an isolated polynucleotide
encoding the VH of SEQ ID NOs: 11, 21, 22, 23, 24, 25 or 26.
[0403] The invention also provides for an isolated polynucleotide
encoding the VL of SEQ ID NOs: 12 or 27.
[0404] The invention also provides for an isolated polynucleotide
encoding the VH of SEQ ID NOs: 11, 21, 22, 23, 24, 25 or 26 and the
VL of SEQ ID NOs: 12 or 27.
[0405] The invention also provides for an isolated polynucleotide
comprising the polynucleotide sequence of SEQ ID NOs: 13, 14, 28,
29, 30, 31, 32, 33, or 34.
[0406] The invention also provides for an isolated polynucleotide
encoding the heavy chain of SEQ ID NOs: 35, 36, 37, 38, 39, 40, 41,
42, 43, 44, 45 or 46.
[0407] The invention also provides for an isolated polynucleotide
encoding the light chain of SEQ ID NOs: 77 or 78.
[0408] The invention also provides for an isolated polynucleotide
encoding the heavy chain of SEQ ID NOs: 35, 36, 37, 38, 39, 40, 41,
42, 43, 44, 45 or 46 and a light chain of SEQ ID NOs: 77 or 78.
[0409] The invention also provides for an isolated polynucleotide
comprising the polynucleotide sequence of SEQ ID NOs: 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 77 or 78.
[0410] The polynucleotide sequences encoding the VH or the VL or a
fragment thereof of the antibodies of the invention or the heavy
chain and the light chain of the antibodies of the invention may be
operably linked to one or more regulatory elements, such as a
promoter or enhancer, that allow expression of the nucleotide
sequence in the intended host cell. The polynucleotide may be a
cDNA.
[0411] The invention also provides for a vector comprising the
polynucleotide of the invention. Such vectors may be plasmid
vectors, viral vectors, vectors for baculovirus expression,
transposon based vectors or any other vector suitable for
introduction of the synthetic polynucleotide of the invention into
a given organism or genetic background by any means. For example,
polynucleotides encoding light and/or heavy chain variable regions
of the antibodies of the invention, optionally linked to constant
regions, are inserted into expression vectors. The light and/or
heavy chains may be cloned in the same or different expression
vectors. The DNA segments encoding immunoglobulin chains may be
operably linked to control sequences in the expression vector(s)
that ensure the expression of immunoglobulin polypeptides. Such
control sequences include signal sequences, promoters (e.g.
naturally associated or heterologous promoters), enhancer elements,
and transcription termination sequences, and are chosen to be
compatible with the host cell chosen to express the antibody. Once
the vector has been incorporated into the appropriate host, the
host is maintained under conditions suitable for high level
expression of the proteins encoded by the incorporated
polynucleotides.
[0412] In some embodiments, the vector comprises the polynucleotide
of SEQ ID NO: 13 and the polynucleotide of SEQ ID NO: 14.
[0413] In some embodiments, the vector comprises the polynucleotide
of SEQ ID NO: 28 and the polynucleotide of SEQ ID NO: 34.
[0414] In some embodiments, the vector comprises the polynucleotide
of SEQ ID NO: 29 and the polynucleotide of SEQ ID NO: 34.
[0415] In some embodiments, the vector comprises the polynucleotide
of SEQ ID NO: 30 and the polynucleotide of SEQ ID NO: 34.
[0416] In some embodiments, the vector comprises the polynucleotide
of SEQ ID NO: 31 and the polynucleotide of SEQ ID NO: 34.
[0417] In some embodiments, the vector comprises the polynucleotide
of SEQ ID NO: 32 and the polynucleotide of SEQ ID NO: 34.
[0418] In some embodiments, the vector comprises the polynucleotide
of SEQ ID NO: 33 and the polynucleotide of SEQ ID NO: 34.
[0419] In some embodiments, the vector comprises the polynucleotide
of SEQ ID NO: 65 and the polynucleotide of SEQ ID NO: 77.
[0420] In some embodiments, the vector comprises the polynucleotide
of SEQ ID NO: 66 and the polynucleotide of SEQ ID NO: 78.
[0421] In some embodiments, the vector comprises the polynucleotide
of SEQ ID NO: 67 and the polynucleotide of SEQ ID NO: 78.
[0422] In some embodiments, the vector comprises the polynucleotide
of SEQ ID NO: 68 and the polynucleotide of SEQ ID NO: 78.
[0423] In some embodiments, the vector comprises the polynucleotide
of SEQ ID NO: 69 and the polynucleotide of SEQ ID NO: 78.
[0424] In some embodiments, the vector comprises the polynucleotide
of SEQ ID NO: 70 and the polynucleotide of SEQ ID NO: 78.
[0425] In some embodiments, the vector comprises the polynucleotide
of SEQ ID NO: 71 and the polynucleotide of SEQ ID NO: 78.
[0426] In some embodiments, the vector comprises the polynucleotide
of SEQ ID NO: 72 and the polynucleotide of SEQ ID NO: 78.
[0427] In some embodiments, the vector comprises the polynucleotide
of SEQ ID NO: 73 and the polynucleotide of SEQ ID NO: 78.
[0428] In some embodiments, the vector comprises the polynucleotide
of SEQ ID NO: 74 and the polynucleotide of SEQ ID NO: 78.
[0429] In some embodiments, the vector comprises the polynucleotide
of SEQ ID NO: 75 and the polynucleotide of SEQ ID NO: 78.
[0430] In some embodiments, the vector comprises the polynucleotide
of SEQ ID NO: 76 and the polynucleotide of SEQ ID NO: 78.
[0431] Suitable expression vectors are typically replicable in the
host organisms either as episomes or as an integral part of the
host chromosomal DNA. Commonly, expression vectors contain
selection markers such as ampicillin-resistance,
hygromycin-resistance, tetracycline resistance, kanamycin
resistance or neomycin resistance to permit detection of those
cells transformed with the desired DNA sequences.
[0432] Suitable promoter and enhancer elements are known in the
art. For expression in a eukaryotic cell, exemplary promoters
include light and/or heavy chain immunoglobulin gene promoter and
enhancer elements; cytomegalovirus immediate early promoter; herpes
simplex virus thymidine kinase promoter; early and late SV40
promoters; promoter present in long terminal repeats from a
retrovirus; mouse metallothionein-I promoter; and various art-known
tissue specific promoters. Selection of the appropriate vector and
promoter is well within the level of ordinary skill in the art.
[0433] Large numbers of suitable vectors and promoters are known to
those of skill in the art; many are commercially available for
generating a subject recombinant constructs. The following vectors
are provided by way of example. Bacterial: pBs, phagescript,
PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a
(Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3,
pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryotic: pWLneo,
pSV2cat, pOG44, PXR1, pSG (Stratagene) pSVK3, pBPV, pMSG and pSVL
(Pharmacia).
[0434] The invention also provides for a host cell comprising one
or more vectors of the invention. "Host cell" refers to a cell into
which a vector has been introduced. It is understood that the term
host cell is intended to refer not only to the particular subject
cell but to the progeny of such a cell, and also to a stable cell
line generated from the particular subject cell. Because certain
modifications may occur in succeeding generations due to either
mutation or environmental influences, such progeny may not be
identical to the parent cell, but are still included within the
scope of the term "host cell" as used herein. Such host cells may
be eukaryotic cells, prokaryotic cells, plant cells or archeal
cells.
[0435] Escherichia coli, bacilli, such as Bacillus subtilis, and
other enterobacteriaceae, such as Salmonella, Serratia, and various
Pseudomonas species are examples of prokaryotic host cells. Other
microbes, such as yeast, are also useful for expression.
Saccharomyces (e.g., S. cerevisiae) and Pichia are examples of
suitable yeast host cells. Exemplary eukaryotic cells may be of
mammalian, insect, avian or other animal origins. Mammalian
eukaryotic cells include immortalized cell lines such as hybridomas
or myeloma cell lines such as SP2/0 (American Type Culture
Collection (ATCC), Manassas, Va., CRL-1581), NS0 (European
Collection of Cell Cultures (ECACC), Salisbury, Wiltshire, UK,
ECACC No. 85110503), FO (ATCC CRL-1646) and Ag653 (ATCC CRL-1580)
murine cell lines. An exemplary human myeloma cell line is U266
(ATTC CRL-TIB-196). Other useful cell lines include those derived
from Chinese Hamster Ovary (CHO) cells such as CHO-K1SV (Lonza
Biologics, Walkersville, Md.), CHO-K1 (ATCC CRL-61) or DG44.
[0436] The invention also provides for a method of producing the
antibody of the invention comprising culturing the host cell of the
invention in conditions that the antibody is expressed, and
recovering the antibody produced by the host cell. Methods of
making antibodies and purifying them are known. Once synthesized
(either chemically or recombinantly), the whole antibodies, their
dimers, individual light and/or heavy chains, or other antibody
fragments such as VH and/or VL, may be purified according to
standard procedures, including ammonium sulfate precipitation,
affinity columns, column chromatography, high performance liquid
chromatography (HPLC) purification, gel electrophoresis, and the
like (see generally Scopes, Protein Purification (Springer-Verlag,
N.Y., (1982)). A subject antibody may be substantially pure, e.g.,
at least about 80% to 85% pure, at least about 85% to 90% pure, at
least about 90% to 95% pure, or at least about 98% to 99%, or more,
pure, e.g., free from contaminants such as cell debris,
macromolecules, etc. other than the subject antibody.
[0437] The invention also provides for a method for producing an
antagonistic antibody specifically binding CD40 of SEQ ID NO: 1,
comprising: [0438] incorporating the first polynucleotide encoding
the VH of the antibody and the second polynucleotide encoding the
VL of the antibody into an expression vector; [0439] transforming a
host cell with the expression vector; [0440] culturing the host
cell in culture medium under conditions wherein the VL and the VH
are expressed and form the antibody; and [0441] recovering the
antibody from the host cell or culture medium.
[0442] The polynucleotides encoding certain VH or VL sequences of
the invention may be incorporated into vectors using standard
molecular biology methods. Host cell transformation, culture,
antibody expression and purification are done using well known
methods.
Methods of Treatment
[0443] Antagonistic antibodies specifically binding CD40 of the
invention, for example antibodies C40B16, C40B124, C40B135,
C40B125, C40B136, C40B127, C40B138, C40B131, C40B176, C40B180,
C40B179 or C40B183, may be used for the treatment and/or prevention
of any condition or disease wherein antagonizing the effects of
CD40 may be therapeutically effective and may reduce the symptoms
of the disease. Examples thereof include the treatment of
inflammatory diseases such as autoimmune diseases wherein the
induction of tolerance and/or the suppression of humoral immunity
are therapeutically desirable. Diseases that may be treated with
the antibodies of the invention are autoimmune diseases, Addison's
disease, an ankylosing spondylitis, an atherosclerosis, an
autoimmune hepatitis, an autoimmune diabetes, Graves' disease,
Guillain-Barre syndrome, Hashimoto's disease, an idiopathic
thrombocytopenia, an inflammatory bowel disease (IBD), a systemic
lupus erythematosus, a multiple sclerosis, a myasthenia gravis, a
psoriasis, an arthritis, a scleroderma, Sjogren's syndrome, a
systemic sclerosis, a transplantation, a kidney transplantation, a
skin transplantation, a bone marrow transplantation, a graft versus
host disease (GVHD), a type I diabetes, a rheumatoid arthritis, a
juvenile arthritis, a psoriatic arthritis, Reiter's syndrome, an
ankylosing spondylitis, or a gouty arthritis, Crohn's disease or an
ulcerative colitis.
[0444] The invention also provides for a method of treating an
arthritis, comprising administering a therapeutically effective
amount of the antibody of the invention to a subject in need
thereof for a time sufficient to treat the arthritis.
[0445] In some embodiments, the arthritis is a juvenile arthritis,
a rheumatoid arthritis, a psoriatic arthritis, Reiter's syndrome,
an ankylosing spondylitis, or a gouty arthritis.
[0446] The invention also provides for a method of treating a
lupus, comprising administering a therapeutically effective amount
of the antibody of the invention to a subject in need thereof for a
time sufficient to treat the lupus.
[0447] In some embodiments, the lupus is a systemic lupus
erythematosus (SLE) or a cutaneous lupus erythematosus (CLE).
[0448] In some embodiments, the subject has lupus nephritis.
[0449] In some embodiments, the subject has a cutaneous lupus
erythematosus.
[0450] The invention also provides for a method of treating an
inflammatory bowel disease, comprising administering a
therapeutically effective amount of the antibody of the invention
to a subject in need thereof for a time sufficient to treat the
inflammatory bowel disease.
[0451] In some embodiments, the inflammatory bowel disease is
Crohn's disease.
[0452] In some embodiments, the inflammatory bowel disease is an
ulcerative colitis.
[0453] "Treatment" or "treat" refers to therapeutic treatment.
Subjects in need of treatment include those subjects diagnosed with
the disorder or experiencing at least one of the symptoms of the
disease. Subjects that may be treated also include those prone to
or susceptible to have the disorder, or those in which the disorder
is to be prevented. Beneficial or desired clinical results include
alleviation of symptoms, diminishment of extent of disease,
stabilized (i.e., not worsening) state of disease, delay or slowing
of disease progression, amelioration or palliation of the disease
state, and remission (whether partial or total), whether detectable
or undetectable. Beneficial treatment result include, in a subject
who has received treatment, reduction in the levels of inflammatory
cytokines, adhesion molecules, proteases, immunoglobulins,
combinations thereof, increased production of anti-inflammatory
proteins, a reduction in the number of autoreactive cells, an
increase in immune tolerance, inhibition of autoreactive cell
survival, and/or a decrease in one or more symptoms mediated by
stimulation of CD40-expressing cells by CD154.
[0454] Clinical response may be assessed using screening techniques
such as magnetic resonance imaging (MRI) scan, x-radiographic
imaging, computed tomographic (CT) scan, flow cytometry or
fluorescence-activated cell sorter (FACS) analysis, histology,
gross pathology, and blood chemistry, including but not limited to
changes detectable by ELISA, RIA, chromatography, and the like.
[0455] The methods of the invention may be used to treat a subject
belonging to any animal classification. Examples of subjects that
may be treated include mammals such as humans, rodents, dogs, cats
and farm animals.
[0456] The antibodies of the invention may be useful in the
preparation of a medicament for such treatment, wherein the
medicament is prepared for administration in dosages defined
herein.
[0457] The antibodies of the invention may be administered in
combination with a second therapeutic agent.
[0458] The second therapeutic agent may be any known therapy for
inflammatory diseases, such as autoimmune diseases, including any
agent or combination of agents that are known to be useful, or
which have been used or are currently in use for treatment. Such
therapies and therapeutic agents include surgery or surgical
procedures (e.g. splenectomy, lymphadenectomy, thyroidectomy,
plasmapheresis, leukophoresis, cell, tissue, or organ
transplantation, intestinal procedures, organ perfusion, and the
like), therapy such as steroid therapy and non-steroidal therapy,
hormone therapy, cytokine therapy, therapy with dermatological
agents (for example, topical agents used to treat skin conditions
such as allergies, contact dermatitis, and psoriasis),
immunosuppressive therapy, and other anti-inflammatory drugs
including monoclonal antibodies.
[0459] The second therapeutic agent may be a corticosteroid, an
immunosuppressant, a cytotoxic drug, or a B-cell modulator.
[0460] In some embodiments, the antibodies of the invention are
administered in combination with a second therapeutic agent.
Exemplary second therapeutic agents are corticosteroids,
nonsteroidal anti-inflammatory drugs (NSAIDs), salicylates,
hydroxychloroquine, sulfasalazine, cytotoxic drugs,
immunosuppressive drugs immunomodulatory antibodies, methotrexate,
cyclophosphamide, mizoribine, chlorambucil, cyclosporine,
tacrolimus (FK506; ProGrafrM), mycophenolate mofetil, and
azathioprine (6-mercaptopurine), sirolimus (rapamycin),
deoxyspergualin, leflunomide and its malononitriloamide analogs;
anti-CTLA4 antibodies and Ig fusions, anti-B lymphocyte stimulator
antibodies (e.g., LYMPHOSTAT-BTM) and CTLA4-Ig fusions, anti-CD80
antibodies, anti-T cell antibodies such as anti-CD3 (OKT3),
anti-CD4, corticosteroids such as, for example, clobetasol,
halobetasol, hydrocortisone, triamcinolone, betamethasone,
fluocinole, fluocinonide, prednisone, prednisolone,
methylprednisolone; non-steroidal anti-inflammatory drugs (NSAIDs)
such as, for example, sulfasalazine, medications containing
mesalamine (known as 5-ASA agents), celecoxib, diclofenac,
etodolac, fenprofen, flurbiprofen, ibuprofen, ketoprofen,
meclofamate, meloxicam, nabumetone, naproxen, oxaprozin, piroxicam,
rofecoxib, salicylates, sulindac, and tolmetin; phosphodiesterase-4
inhibitors, anti-TNF.alpha. antibodies REMICADE.RTM. (infliximab),
SIMPONI.RTM. (golimumab) and HUMIRA.RTM. (adalimumab), thalidomide
or its analogs such as lenalidomide.
[0461] The antibodies of the invention may be administered in
combination with a second therapeutic agent simultaneously,
sequentially or separately.
[0462] Treatment effectiveness or RA may be assessed using
effectiveness as measured by clinical responses defined by the
American College of Rheumatology criteria, the European League of
Rheumatism criteria, or any other criteria. See for example, Felson
et al. (1995) Arthritis Rheum. 38: 727-35 and van Gestel et al.
(1996) Arthritis Rheum. 39: 34-40.
Administration/Pharmaceutical Compositions
[0463] The invention also provides for pharmaceutical compositions
of the antagonistic antibodies specifically binding CD40 of the
invention and a pharmaceutically acceptable carrier. For
therapeutic use, the antibodies the invention may be prepared as
pharmaceutical compositions containing an effective amount of the
antibody as an active ingredient in a pharmaceutically acceptable
carrier. "Carrier" refers to a diluent, adjuvant, excipient, or
vehicle with which the active compound is administered. Such
vehicles may be liquids, such as water and oils, including those of
petroleum, animal, vegetable or synthetic origin, such as peanut
oil, soybean oil, mineral oil, sesame oil and the like. For
example, 0.4% saline and 0.3% glycine may be used. These solutions
are sterile and generally free of particulate matter. They may be
sterilized by conventional, well-known sterilization techniques
(e.g., filtration). The compositions may contain pharmaceutically
acceptable auxiliary substances as required to approximate
physiological conditions such as pH adjusting and buffering agents,
stabilizing, thickening, lubricating and coloring agents, etc. The
concentration of the antibodies of the invention in such
pharmaceutical compositions may vary widely, i.e., from less than
about 0.5%, usually to at least about 1% to as much as 5%, 10%,
15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% by weight and will be
selected primarily based on required dose, fluid volumes,
viscosities, etc., according to the particular mode of
administration selected. Suitable vehicles and formulations,
including other human proteins, e.g., human serum albumin, are
described, for example, in e.g. Remington: The Science and Practice
of Pharmacy, 21.sup.st Edition, Troy, D. B. ed., Lipincott Williams
and Wilkins, Philadelphia, Pa. 2006, Part 5, Pharmaceutical
Manufacturing pp 691-1092, See especially pp. 958-989.
[0464] The mode of administration of the antibodies of the
invention in the methods of the invention may be any suitable route
such as parenteral administration, e.g., intradermal,
intramuscular, intraperitoneal, intravenous or subcutaneous,
transmucosal (oral, intranasal, intravaginal, and rectal) or other
means appreciated by the skilled artisan, as well known in the
art.
[0465] The antibodies of the invention may be administered to the
subject by any suitable route, for example parentally by
intravenous (i.v.) infusion or bolus injection, intramuscularly or
subcutaneously or intraperitoneally. i.v. infusion may be given
over for, example, 15, 30, 60, 90, 120, 180, or 240 minutes, or
over 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hours.
[0466] The dose given to the subject is sufficient to alleviate or
at least partially arrest the disease being treated
("therapeutically effective amount") and may be from about 0.005
mg/kg to about 100 mg/kg, for example about 0.05 mg/kg to about 20
mg/kg, about 0.1 mg/kg to about 20 mg/kg, about 1 mg to about 20
mg/kg, about 4 mg/kg, about 8 mg/kg, about 16 mg/kg or about 24
mg/kg, or, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg/kg, but
may even higher, for example about 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 30, 40, 50, 60, 70, 80, 90 or 100 mg/kg.
[0467] A fixed unit dose may also be given, for example, 50, 100,
200, 500 or 1000 mg, or the dose may be based on the patient's
surface area, e.g., 500, 400, 300, 250, 200, or 100 mg/m.sup.2.
Usually between 1 and 8 doses, (e.g., 1, 2, 3, 4, 5, 6, 7 or 8) may
be administered to treat the inflammatory disease, such as an
autoimmune disease such or a rheumatoid arthritis, but 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20 or more doses may be given.
[0468] The administration of the antibodies of the invention may be
repeated after one day, two days, three days, four days, five days,
six days, one week, two weeks, three weeks, one month, five weeks,
six weeks, seven weeks, two months, three months, four months, five
months, six months or longer. Repeated courses of treatment are
also possible, as is chronic administration. The repeated
administration may be at the same dose or at a different dose. For
example, the antibodies of the invention may be administered at 0.1
mg/kg, at 1 mg/kg, at 5 mg/kg, at 8 mg/kg or at 16 mg/kg at weekly
interval for 8 weeks, followed by administration at 8 mg/kg or at
16 mg/kg every two weeks for an additional 16 weeks, followed by
administration at 8 mg/kg or at 16 mg/kg every four weeks by
intravenous infusion.
[0469] The antibodies of the invention may be provided by
maintenance therapy, such as, e.g., once a week, once a month, once
in two months, once in three months, once in four months, once in
five months, or once in six months over one or more years.
[0470] For example, the antibodies of the invention may be provided
as a daily dosage in an amount of about 0.1-100 mg/kg, such as 0.5,
0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45,
50, 60, 70, 80, 90 or 100 mg/kg, per day, on at least one of day 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, or 40, or alternatively, at least one of week 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 after
initiation of treatment, or any combination thereof, using single
or divided doses of every 24, 12, 8, 6, 4, or 2 hours, or any
combination thereof.
[0471] The antibodies of the invention may also be administered
prophylactically in order to reduce the risk of developing a
disease and/or delay the onset of the disease to be treated.
[0472] An exemplary pharmaceutical composition of the invention for
intramuscular injection may be prepared to contain 1 ml sterile
buffered water, and between about 1 ng to about 100 mg/kg, e.g.
about 50 ng to about 30 mg/kg or about 5 mg to about 25 mg/kg, of
the antibody of the invention.
[0473] An exemplary pharmaceutical composition of the invention for
intravenous infusion may be made up to contain about 200 ml of
sterile Ringer's solution, and about 8 mg to about 2400 mg, about
400 mg to about 1600 mg, or about 400 mg to about 800 mg of the
antibodies of the invention for administration to a 80 kg patient.
Methods for preparing parenterally administrable compositions are
well known and are described in more detail in, for example,
"Remington's Pharmaceutical Science", 15th ed., Mack Publishing
Company, Easton, Pa.
[0474] "Therapeutically effective amount" of the antibodies of the
invention effective in the treatment of a disease may be determined
by standard research techniques. For example, in vitro assays may
be employed to help identify optimal dosage ranges. Optionally, the
dosage of the antibodies of the invention that may be effective in
the treatment of a disease such as the inflammatory disease may be
determined by administering the antibodies to relevant animal
models well known in the art. Selection of a particular effective
dose may be determined (e.g., via clinical trials) by those skilled
in the art based upon the consideration of several factors. Such
factors include the disease to be treated or prevented, the
symptoms involved, the patient's body mass, the patient's immune
status and other factors known by the skilled artisan. The precise
dose will also depend on the route of administration, and the
severity of disease, and should be decided according to the
judgment of the practitioner and each patient's circumstances.
Effective doses can be extrapolated from dose-response curves
derived from in vitro or animal model test systems. The antibodies
of the invention may be tested for their efficacy and effective
dosage using any of the models described herein.
[0475] The antibodies of the invention may be lyophilized for
storage and reconstituted in a suitable carrier prior to use. This
technique has been shown to be effective with conventional protein
preparations and well known lyophilization and reconstitution
techniques can be employed.
Anti-Idiotypic Antibodies
[0476] The present invention provides for an anti-idiotypic
antibody binding to the antibody of the invention.
[0477] The invention also provides for an anti-idiotypic antibody
specifically binding the antibody comprising the VH of SEQ ID NO:
11 and the VL or SEQ ID NO: 12.
[0478] The invention also provides for an anti-idiotypic antibody
specifically binding the antibody comprising the VH of SEQ ID NO:
21 and the VL or SEQ ID NO: 27.
[0479] The invention also provides for an anti-idiotypic antibody
specifically binding the antibody comprising the VH of SEQ ID NO:
22 and the VL or SEQ ID NO: 27.
[0480] The invention also provides for an anti-idiotypic antibody
specifically binding the antibody comprising the VH of SEQ ID NO:
23 and the VL or SEQ ID NO: 27.
[0481] The invention also provides for an anti-idiotypic antibody
specifically binding the antibody comprising the VH of SEQ ID NO:
24 and the VL or SEQ ID NO: 27.
[0482] The invention also provides for an anti-idiotypic antibody
specifically binding the antibody comprising the VH of SEQ ID NO:
25 and the VL or SEQ ID NO: 27.
[0483] The invention also provides for an anti-idiotypic antibody
specifically binding the antibody comprising the VH of SEQ ID NO:
26 and the VL or SEQ ID NO: 27.
[0484] An anti-idiotypic (Id) antibody is an antibody which
recognizes the antigenic determinants (e.g. the paratope or CDRs)
of the antibody. The Id antibody may be antigen-blocking or
non-blocking. The antigen-blocking Id may be used to detect the
free antibody in a sample (e.g. CD40 antibody of the invention
described herein). The non-blocking Id may be used to detect the
total antibody (free, partially bond to antigen, or fully bound to
antigen) in a sample. An Id antibody may be prepared by immunizing
an animal with the antibody to which an anti-Id is being
prepared.
[0485] An anti-Id antibody may also be used as an immunogen to
induce an immune response in yet another animal, producing a
so-called anti-anti-Id antibody. An anti-anti-Id may be
epitopically identical to the original mAb, which induced the
anti-Id. Thus, by using antibodies to the idiotypic determinants of
a mAb, it is possible to identify other clones expressing
antibodies of identical specificity. Anti-Id antibodies may be
varied (thereby producing anti-Id antibody variants) and/or
derivatized by any suitable technique.
Immunoconjugates
[0486] An "immunoconjugate" refers to the antibody of the invention
conjugated to one or more heterologous molecule(s).
[0487] In some embodiments, the antibody of the invention is
conjugated to one or more cytotoxic agents or an imaging agent.
[0488] Exemplary cytotoxic agents include chemotherapeutic agents
or drugs, growth inhibitory agents, toxins (e.g., protein toxins,
enzymatically active toxins of bacterial, fungal, plant, or animal
origin, or fragments thereof), and radionuclides.
[0489] The cytotoxic agent may be one or more drugs, such as to a
mayatansinoid (see, e.g., U.S. Pat. Nos. 5,208,020, 5,416,06), an
auristatin such as monomethylauristatin drug moieties DE and DF
(MMAE and MMAF) (see, e.g., U.S. Pat. Nos. 5,635,483 and 5,780,588,
and 7,498,298), a dolastatin, a calicheamicin or derivative thereof
(see, e.g., U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739, 116,
5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman
et al., (1993) Cancer Res 53:3336-3342; and Lode et al., (1998)
Cancer Res 58:2925-2928); an anthracycline such as daunomycin or
doxorubicin (see, e.g., Kratz et al., (2006) Current Med. Chem
13:477-523; Jeffrey et al., (2006) Bioorganic & Med Chem
Letters 16:358-362; Torgov et al., (2005) Bioconj Chem 16:717-721;
Nagy et al., (2000) Proc Natl Acad Sci USA 97:829-834; Dubowchik et
al, Bioorg. & Med. Chem. Letters 12: 1529-1532 (2002); King et
al., (2002) J Med Chem 45:4336-4343; and U.S. Pat. No. 6,630,579),
methotrexate, vindesine, a taxane such as docetaxel, paclitaxel,
larotaxel, tesetaxel, and ortataxel.
[0490] The cytotoxic agent may also be an enzymatically active
toxin or fragment thereof, such as diphtheria A chain, nonbinding
active fragments of diphtheria toxin, exotoxin A chain (from
Pseudomonas aeruginosa), ricin A chain, modeccin A chain,
alpha-sarcin, Aleurites fordii proteins, dianthins, Phytolacca
americana proteins (PAPI, PAPII, and PAP-S), momordica charantia
inhibitor, curcin, crotin, sapaonaria officinalis inhibitor,
gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the
tricothecenes.
[0491] The cytotoxic agent or an imaging agent may also be a
radionuclide. Exemplary radionuclides include Ac-225, At-211,
1-131, I-125, Y-90, Re-186, Re-188, Sm-153, Bi-212, P-32, Pb-212
and radioactive isotopes of Lu. When the radioconjugate is used for
detection, it may comprise a radioactive atom for scintigraphic
studies, for example Tc-99m or I-123, or a spin label for nuclear
magnetic resonance (NMR) imaging (also known as magnetic resonance
imaging, mri), such as I-123, I-131, In-111, F-19, C-13, N-15 or
O-17.
[0492] Conjugates of the antibodies of the invention and the
heterologous molecule may be made using a variety of bifunctional
protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio)
propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)
cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional
derivatives of imidoesters (such as dimethyl adipimidate HQ),
active esters (such as disuccinimidyl suberate), aldehydes (such as
glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl)
hexanediamine), bis-diazonium derivatives (such as
bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as
toluene 2,6-diisocyanate), and bis-active fluorine compounds (such
as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin
immunotoxin may be prepared as described in Vitetta et al., (1987)
Science 238: 1098. Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody. See, e.g., WO94/11026. The linker
may be a "cleavable linker" facilitating release of a cytotoxic
drug in the cell. For example, an acid-labile linker,
peptidase-sensitive linker, photolabile linker, dimethyl linker or
disulfide-containing linker (Chari et al., (1992) Cancer Res 52:
127-131; U.S. Pat. No. 5,208,020) may be used.
[0493] Conjugates of the antibodies of the invention and the
heterologous molecule may be prepared with cross-linker reagents
such as BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA,
SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS,
sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB
(succinimidyl-(4-vinylsulfone)benzoate) which are commercially
available (e.g., from Pierce Biotechnology, Inc., Rockford, Ill.,
U.S.A).
[0494] The invention also provides for an immunoconjugate
comprising the antibody specifically binding CD40 of SEQ ID NO: 1
of the invention linked to a therapeutic agent or an imaging
agent.
Diagnostic Uses and Kits
Kits
[0495] The invention also provides for a kit comprising the
antagonistic antibody specifically binding human CD40 of the
invention.
[0496] The kit may be used for therapeutic uses and as diagnostic
kits.
[0497] The kit may be used to detect the presence of CD40 in a
biological sample.
[0498] In some embodiments, the kit comprises the antagonistic
antibody specifically binding human CD40 of the invention and
reagents for detecting the antibody. The kit can include one or
more other elements including: instructions for use; other
reagents, e.g., a label, a therapeutic agent, or an agent useful
for chelating, or otherwise coupling, an antibody to a label or
therapeutic agent, or a radioprotective composition; devices or
other materials for preparing the antibody for administration;
pharmaceutically acceptable carriers; and devices or other
materials for administration to a subject.
[0499] In some embodiments, the kit comprises the antibody of the
invention in a container and instructions for use of the kit.
[0500] In some embodiments, the antibody in the kit is labeled.
[0501] In some embodiments, the kit comprises the antibody C40B16,
C40B124, C40B135, C40B125, C40B136, C40B127, C40B138, C40B131,
C40B176, C40B180, C40B179 or C40B183.
Methods of Detecting CD40
[0502] The invention also provides for a method of detecting CD40
in a sample, comprising obtaining the sample, contacting the sample
with the antibody of the invention, and detecting the antibody
bound to CD40 in the sample.
[0503] In some embodiments, the sample may be derived from urine,
blood, serum, plasma, saliva, ascites, circulating cells,
circulating tumor cells, cells that are not tissue associated
(i.e., free cells), tissues (e.g., surgically resected tumor
tissue, biopsies, including fine needle aspiration), histological
preparations, and the like.
[0504] The antibodies of the invention may be detected using known
methods. Exemplary methods include direct labeling of the
antibodies using fluorescent or chemiluminescent labels, or
radiolabels, or attaching to the antibodies of the invention a
moiety which is readily detectable, such as biotin, enzymes or
epitope tags. Exemplary labels and moieties are ruthenium,
.sup.111In-DOTA, .sup.111In-diethylenetriaminepentaacetic acid
(DTPA), horseradish peroxidase, alkaline phosphatase and
beta-galactosidase, poly-histidine (HIS tag), acridine dyes,
cyanine dyes, fluorone dyes, oxazin dyes, phenanthridine dyes,
rhodamine dyes and Alexafluor.RTM. dyes.
[0505] The antibodies of the invention may be used in a variety of
assays to detect CD40 in the sample. Exemplary assays are western
blot analysis, radioimmunoassay, surface plasmon resonance,
immunoprecipitation, equilibrium dialysis, immunodiffusion,
electrochemiluminescence (ECL) immunoassay, and
immunohistochemistry, fluorescence-activated cell sorting (FACS) or
ELISA assay.
[0506] The present invention will now be described with reference
to the following specific, non-limiting examples.
General Materials and Methods
Generation of Antigens Used in the Studies
[0507] Cloning, expression and purification of the antigens was
done using standard methods. The amino acid sequences of the
proteins used are shown below.
TABLE-US-00003 Full length human CD40 (huCD40); SEQ ID NO: 1
MVRLPLQCVLWGCLLTAVHPEPPTACREKQYLINSQCCSLCQPGQKLVSD
CTEFTETECLPCGESEFLDTWNRETHCHQHKYCDPNLGLRVQQKGTSETD
TICTCEEGWHCTSEACESCVLHRSCSPGFGVKQIATGVSDTICEPCPVGF
FSNVSSAFEKCHPWTSCETKDLVVQQAGTNKTDVVCGPQDRLRALVVIPI
IFGILFAILLVLVFIKKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAP
VQETLHGCQPVTQEDGKESRISVQERQ Human CD40 extracellular domain
(huCD40-ECD); SEQ ID NO: 2
EPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTETECLPCGESEFLDT
WNRETHCHQHKYCDPNLGLRVQQKGTSETDTICTCEEGWHCTSEACESCV
LHRSCSPGFGVKQIATGVSDTICEPCPVGFFSNVSSAFEKCHPWTSCETK
DLVVQQAGTNKTDVVCGPQDRLR Macaca fascicularis (cynomolgous, herein
referred to as cyno) CD40 (cCD40); SEQ ID NO: 3
MVRLPLQCVLWGCLLTAVYPEPPTACREKQYLINSQCCSLCQPGQKLVSD
CTEFTETECLPCSESEFLDTWNRETRCHQHKYCDPNLGLRVQQKGTSETD
TICTCEEGLHCMSESCESCVPHRSCLPGFGVKQIATGVSDTICEPCPVGF
FSNVSSAFEKCRPWTSCETKDLVVQQAGTNKTDVVCGPQDRQRALVVIPI
CLGILFVILLLVLVFIKKVAKKPNDKAPHPKQEPQEINFLDDLPGSNPAA
PVQETLHGCQPVTQEDGKESRISVQERQ Cyno CD40 extracellular domain
(cCD40-ECD); SEQ ID NO: 4
EPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTETECLPCSESEFLDT
WNRETRCHQHKYCDPNLGLRVQQKGTSETDTICTCEEGLHCMSESCESCV
PHRSCLPGFGVKQIATGVSDTICEPCPVGFFSNVSSAFEKCRPWTSCETK
DLVVQQAGTNKTDVVCGPQDRQR Soluble human CD154; SEQ ID NO: 60
MQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLT
VKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTH
SSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL Full length human
CD154; SEQ ID NO: 64
MIETYNQTSPRSAATGLPISMKIFMYLLTVFLITQMIGSALFAVYLHRRL
DKIEDERNLHEDFVFMKTIQRCNTGERSLSLLNCEEIKSQFEGFVKDIML
NKEETKKENSFEMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSN
NLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGR
FERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHG TGFTSFGLL
Binding Assay in Primary Human and Cyno Dendritic Cells (DCs)
[0508] Human monocytes were isolated from either frozen/fresh PBMC
using CD14 negative isolation kit per manufacturer's protocol (MACS
Miltenyi). Cyno monocytes were isolated from fresh PBMC using CD14
positive isolation kit per manufacturer's protocol (MACS Miltenyi).
To generate DCs, monocytes were cultured for 5 days in complete
RPMI medium (Invitrogen) in the presence of 100 ng/ml human GM-CSF
and human IL-4 (Peprotech) and medium was replenished every 2 days.
On day 5, DCs were stimulated with 100 ng/ml LPS (Sigma) for 24
hours. Cells were then stained with each of the tested CD40
antibodies at different concentration in flow cytometry buffer
(PBS+1% FBS; BD Bioscience) in 100 .mu.l volume for 30 minutes on
ice followed with two washes with flow buffer. Cells were then
stained for an additional 30 minutes on ice with APC-conjugated
anti human IgG (Jackson ImmunoResearch) at the recommended dilution
(1:100) and washed twice with flow buffer. Cells were analyzed for
percent positive and Mean Fluorescence Intensity (MFI) to determine
the antibody binding using Fortessa (BD Bioscience).
Binding Assay in Raji (B Cells Lymphoma Cell Line) and HEK-Blue.TM.
CD40L NF-.kappa.B Cell Lines
[0509] Raji cells were obtained from ATCC and HEK-Blue.TM. CD40L
NF-.kappa.B cell line was obtained from Invivogen. Cells were
cultured in complete RPMI medium per company's recommendation.
Staining was done as described above for binding assay in primary
human and cyno DCs.
Human DC IL-12p40 Production Assay
[0510] Human DCs were generated by culturing purified human
monocytes (2.5.times.10.sup.6/well 6-well plates) for 6 days in 3
ml RPMI medium containing glutamax, 25 mM HEPES, 10% FBS, 1%
Pen/Strep and 50 ng each GM-CSF and IL-4. On day 3, 1 ml of the
medium was removed and replaced with 2 ml fresh medium containing
50 ng/ml each of GM-CSF and IL-4. For agonist assays, day 6 DCs
were plated into 96-well plates (100,000 cells/well) followed by
titrations of each of the CD40 antibodies. For antagonist assays,
day 6 DCs were plated into 96-well plates (100,000 cells/well)
followed by titrations of each of the CD40 antibodies and addition
of 1 .mu.g/ml of soluble human CD154 (R&D Systems) to the
cultures. For both assays, cells were cultured for 48 hours before
collecting and analyzing supernatants for IL-12p40 by MSD
(according to manufacturer's directions). For Jurkat D1.1 (ATCC)
antagonist assays, day 6 DCs were plated into 96-well plates
(10,000 cells/well) followed by titrations of each of the CD40
antibodies, after which irradiated Jurkat D1.1 cells (1,000 rads;
100,000/well) were added to the cultures. Cells were cultured for
24 hours before collecting and analyzing supernatants for IL-12p40
by MSD.
Human B Cell Proliferation Assay
[0511] Human tonsillar B cells were plated into 96-well plates
(100,000 cells/well) in RPMI medium containing glutamax, 10% FBS
and 1% Pen/Strep. For agonist assays, titrations of each of the
CD40 antibodies were added to the cells and cultured for 48 hours.
For antagonist assays, titrations of each of the CD40 antibodies
were added to the cells, followed by addition of 0.5 .mu.g/ml
soluble human CD154 (R&D Systems). Cells were cultured for 48
hours at 37 C. For Jurkat D1.1 antagonist assays, titrations of
each of the CD40 antibodies were added to the cells along with
IL-21 (100 ng/ml final, Thermo Fisher Scientific)), after which
irradiated Jurkat D1.1. cells (5,000 rads; 100,000/well) were added
to the cultures and incubated at 37 C for 48 hours. For all assays,
after 48 hours cells were pulsed with 3H-thymidine (1 uCi/well) in
50 .mu.l medium and cultured for 16-18 hours before harvest and
counting.
Cyno B Cell Proliferation Assay
[0512] Cyno spleen cells were plated into 96-well plates (100,000
cells/well) in RPMI medium containing glutamax, 10% FBS and 1%
Pen/Strep. Titrations of each of the CD40 antibodies were added to
the cells, followed by addition of 0.5 .mu.g/ml soluble human CD154
(R&D Systems). Cells were cultured for 48 hours at 37 C. After
48 hours, cells were pulsed with 3H-thymidine (1 uCi/well) in 50
.mu.l medium and cultured for 16-18 hours before harvest and
counting.
HEK-Blue.TM. CD40L NF-.kappa.B Activation Assay
[0513] HEK-Blue.TM. CD40L cell lines stably express human CD40 and
NF-.kappa.B-inducible secreted embryonic alkaline phosphatase
(SEAP). Activation of CD40 on HEK-Blue.TM. CD40L cells induce
downstream signaling events leading to activation of NF-.kappa.B
and secretion of SEAP, which can be measured by QUANTI-Blue.TM.
substrate conversion. These cells were used to assess the ability
of the antibodies to either block (antagonize) CD40-CD154
interaction or activate (agonize) CD40.
Inhibition of CD40 Dependent NF-kB Activation in HEK-Blue CD40L
Cell Line
[0514] HEK-Blue.TM. CD40L cells (Invivogen), which were maintained
according to the vendor's protocol, were seeded into 96 well tissue
culture plates in 100 .mu.l volume (2.5.times.10.sup.4 cells/well).
The assay plates were covered and incubated overnight (37.degree.
C., 5% CO.sub.2) to allow the cells to recover. On the following
day, a 4.times. solution of rhCD154-ECD-His (80 ng/ml final
concentration) and 4.times. solution of CD40 antibodies, or Fabs,
(1-25 .mu.g/ml final concentration) were pre-mixed and the
resulting 2.times. solution added to the 96 well assay plates
containing the cells (200 .mu.l/well final volumes). After 16-24 h
incubation (37.degree. C., 5% CO.sub.2), 40 .mu.l aliquots of the
supernatants were mixed with 160 .mu.l of pre-warmed
QUANTI-Blue.TM. (Invivogen) solution and incubated for 30-60
minutes prior to obtaining absorbance readings at 650 nm.
CD40 Dependent NF-kB Activation in HEK-Blue CD40L NFkB-SEAP Cell
Line
[0515] HEK-Blue.TM. CD40L cells were seeded as described above and
recovered overnight. On the following day, 2.times. CD40 antibody
solutions (1-25 .mu.g/ml final concentration) were added to the
plate at 100 .mu.l/well and incubated overnight (37.degree. C., 5%
CO.sub.2). After 16-24 h incubation (37.degree. C., 5% CO.sub.2),
40 .mu.l aliquots of the supernatants were analyzed as described
above.
Example 1. Isolation of Antibodies Specifically Binding Human CD40
Using Rats Expressing Human Immunoglobulin Loci
[0516] Antibodies were generated using transgenic rats expressing
human immunoglobulin loci, the OmniRat.RTM.; OMT, Inc. The
OmniRat.RTM. endogenous immunoglobulin loci are replaced by human
Ig.kappa. and Ig.lamda. loci and a chimeric human/rat IgH locus
with V, D and J segments of human origin linked to the rat C.sub.H
locus. The IgH locus contains 22 human V.sub.Hs, all human D and
J.sub.H segments in natural configuration linked to the rat C.sub.H
locus. Generation and characterization of the OmniRat.RTM. is
described in Osborn, et al. J Immunol 190: 1481-1490, 2013; and
Int. Pat. Publ. No. WO 14/093908.
[0517] Separate cohorts of five rats were immunized with
recombinant human and cyno CD40 ECD-His or human and cyno CD40
ECD-Fc proteins. Following a 31-34 day immunization regimen, lymph
nodes were harvested from two rats and used to generate hybridomas.
The generated hybridomas were screened for binding to both human
and cyno CD40-ECD. Hybridomas exhibiting statistically significant
binding to both human and cyno CD40-ECD following one-way ANOVA
with a Dunnett's mean comparison post-test were cloned and their V
regions sequenced using standard procedures
Example 2. Engineering of C40B16
[0518] C40B16 was generated by using the OmniRat.RTM. and
identified having antagonist activity in the HEK-Blue.TM. CD40L
NF-.kappa.B activation assay based on the criteria that an
antagonist had a signal that is lower than the 3.times. standard
deviation of the mean signal of HEK-Blue.TM. CD40L cells treated
with rhCD154-ECD-his alone.
[0519] C40B16 VH and VL were sequenced using standard methods and
the framework sequences compared to the closest germline gene
sequences in order to identify potential immunogenicity risks.
C40B16 VH amino acid sequence was most homologous to IGHV3-23 (SEQ
ID NO: 49) with 2 amino acid changes in the framework. C40B16 VL
framework was identical to that of IGLV3-1 (SEQ ID NO: 50).
[0520] The variable regions of C40B16 were engineered to reduce
possible immunogenicity and/or developability risk(s) by generating
mutations at positions R43, H82, N52, S54 and/or M108 in the VH
(residue numbering according to SEQ ID NO: 11) and at position C33
in the VL (residue numbering according to SEQ ID NO: 12) to
eliminate potential heterogeneity caused by unpaired cysteines. The
generated VH/VL domains were cloned as effector silent Fc isoforms
IgG1sigma or IgG4PAA. IgG1sigma contains mutations L234A, L235A,
G237A, P238S, H268A, A330S, and P331S when compared to the
wild-type IgG1. IgG4PAA contains mutations S228P, F234A and L235A
when compared to the wild-type IgG4. Residue numbering is according
to the EU Index. Table 3 shows the generated antibodies and
introduced mutations when compared to the parental C40B16 mAb. A
C33A mutation was engineered in the VL of all generated antibodies
when compared to the parental C40B16 VL.
[0521] Table 4 shows the HCDR1 and the HCDR2 amino acid sequences
of the antibodies.
[0522] Table 5 shows the HCDR3 and the LCDR1 amino acid sequences
of the antibodies.
[0523] Table 6 shows the LCDR2 and the LCDR3 amino acid sequences
of the antibodies.
[0524] Table 7 shows the SEQ ID NOs: of the amino acid and cDNA
sequences of the VH and the VL regions of the antibodies.
[0525] Table 8 shows the SEQ ID NOs: of the amino acid sequences of
the heavy chains and the light chains of the antibodies.
[0526] Table 9 shows the amino acid or polynucleotide sequences
corresponding to SEQ ID NOs: 11-14, 21-52 and 65-78.
TABLE-US-00004 TABLE 3 VH Mutation mAb compared to C40B16 Isotype
C40B16 parental IgG1 C40B124 R43K, H82Q, N52D, S54A IgG1Sigma
C40B135 R43K, H82Q, N52D, S54A IgG4PAA C40B125 R43K, H82Q, N52Q,
S54A IgG1Sigma C40B136 R43K, H82Q, N52Q, S54A IgG4PAA C40B127 R43K,
H82Q, N53Q, S54A IgG1Sigma C40B138 R43K, H82Q, N53Q, S54A IgG4PAA
C40B131 R43K, H82Q, N53A, S54A IgG4PAA C40B176 R43K, H82Q, N53A,
S54A, M108L IgG1Sigma C40B180 R43K, H82Q, N53A, S54A, M108L IgG4PAA
C40B179 R43K, H82Q, N53Q, S54A, M108L IgG1Sigma C40B183 R43K, H82Q,
N53Q, S54A, M108L IgG4PAA
TABLE-US-00005 TABLE 4 HCDR1 HCDR2 SEQ SEQ ID mAb Sequence ID NO:
Sequence NO: C40B16 SYAMS 5 TINNSGGGTYYADSVKG 6 C40B124 SYAMS 5
TIDNAGGGTYYADSVKG 15 C40B135 SYAMS 5 TIDNAGGGTYYADSVKG 15 C40B125
SYAMS 5 TIQNAGGGTYYADSVKG 16 C40B136 SYAMS 5 TIQNAGGGTYYADSVKG 16
C40B127 SYAMS 5 TINQAGGGTYYADSVKG 17 C40B138 SYAMS 5
TINQAGGGTYYADSVKG 17 C40B131 SYAMS 5 TINAAGGGTYYADSVKG 18 C40B176
SYAMS 5 TINAAGGGTYYADSVKG 18 C40B180 SYAMS 5 TINAAGGGTYYADSVKG 18
C40B179 SYAMS 5 TINQAGGGTYYADSVKG 17 C40B183 SYAMS 5
TINQAGGGTYYADSVKG 17
TABLE-US-00006 TABLE 5 HCDR3 LCDR1 SEQ SEQ ID mAb Sequence ID NO:
Sequence NO: C40B16 EGGKYYYYAMDV 7 SGDKLGDKYAC 8 C40B124
EGGKYYYYAMDV 7 SGDKLGDKYAA 20 C40B135 EGGKYYYYAMDV 7 SGDKLGDKYAA 20
C40B125 EGGKYYYYAMDV 7 SGDKLGDKYAA 20 C40B136 EGGKYYYYAMDV 7
SGDKLGDKYAA 20 C40B127 EGGKYYYYAMDV 7 SGDKLGDKYAA 20 C40B138
EGGKYYYYAMDV 7 SGDKLGDKYAA 20 C40B131 EGGKYYYYAMDV 7 SGDKLGDKYAA 20
C40B176 EGGKYYYYALDV 19 SGDKLGDKYAA 20 C40B180 EGGKYYYYALDV 19
SGDKLGDKYAA 20 C40B179 EGGKYYYYALDV 19 SGDKLGDKYAA 20 C40B183
EGGKYYYYALDV 19 SGDKLGDKYAA 20
TABLE-US-00007 TABLE 6 LCDR2 LCDR3 SEQ SEQ ID mAb Sequence ID NO:
Sequence NO: C40B16 QDSRRPS 9 QAWASSTVV 10 C40B124 QDSRRPS 9
QAWASSTVV 10 C40B135 QDSRRPS 9 QAWASSTVV 10 C40B125 QDSRRPS 9
QAWASSTVV 10 C40B136 QDSRRPS 9 QAWASSTVV 10 C40B127 QDSRRPS 9
QAWASSTVV 10 C40B138 QDSRRPS 9 QAWASSTVV 10 C40B131 QDSRRPS 9
QAWASSTVV 10 C40B176 QDSRRPS 9 QAWASSTVV 10 C40B180 QDSRRPS 9
QAWASSTVV 10 C40B179 QDSRRPS 9 QAWASSTVV 10 C40B183 QDSRRPS 9
QAWASSTVV 10
TABLE-US-00008 TABLE 7 VH amino VH VL acid DNA amino VL DNA VH SEQ
ID SEQ ID VL acid SEQ SEQ ID mAb name NO: NO: name ID NO: NO:
C40B16 11 13 12 14 C40B124 C40H60 21 28 C40L71 27 34 C40B135 C40H60
21 28 C40L71 27 34 C40B125 C40H61 22 29 C40L71 27 34 C40B136 C40H61
22 29 C40L71 27 34 C40B127 C40H63 23 30 C40L71 27 34 C40B138 C40H63
23 30 C40L71 27 34 C40B131 C40H56 24 31 C40L71 27 34 C40B176 C40H83
25 32 C40L71 27 34 C40B180 C40H83 25 32 C40L71 27 34 C40B179 C40H86
26 33 C40L71 27 34 C40B183 C40H86 26 33 C40L71 27 34
TABLE-US-00009 TABLE 8 HC HC LC LC protein DNA protein DNA SEQ SEQ
SEQ SEQ mAb ID NO: ID NO: ID NO: ID NO: C40B16 35 65 47 77 C40B124
36 66 48 78 C40B135 37 67 48 78 C40B125 38 68 48 78 C40B136 39 69
48 78 C40B127 40 70 48 78 C40B138 41 71 48 78 C40B131 42 72 48 78
C40B176 43 73 48 78 C40B180 44 74 48 78 C40B179 45 75 48 78 C40B183
46 76 48 78
TABLE-US-00010 TABLE 9 SEQ ID Protein Sequence NO: C40B16 VH
protein EVQLVESGGGLVQPGGSLRLSCAASGFTFSSY 11
AMSWVRQAPGRGLEWVSTINNSGGGTYYAD SVKGRFTISRDNSKNTLYLHMNSLRAEDTAV
YYCAKEGGKYYYYAMDVWGQGTTVTVSS C40B16 VL protein
SYELTQPPSVSVSPGQTASITCSGDKLGDKYA 12
CWYQQKPGQSPVLVIYQDSRRPSGIPERFSGS NSGNTATLTISGTQAMDEADYYCQAWASSTV
VFGGGTKLTVL C40B16 VH DNA GAGGTGCAGCTGGTGGAATCTGGCGGAGGA 13
CTGGTGCAGCCTGGCGGCAGCCTGAGACTG TCTTGTGCCGCCAGCGGCTTCACCTTCAGCA
GCTACGCTATGAGCTGGGTCCGACAGGCCC CTGGCAGAGGACTCGAGTGGGTGTCCACCA
TCAACAACAGCGGCGGAGGCACCTACTACG CCGACAGCGTGAAGGGCAGATTCACCATCA
GCCGGGACAACAGCAAGAACACCCTGTACC TGCACATGAACAGCCTGCGGGCCGAGGACA
CCGCCGTGTACTATTGTGCCAAAGAGGGCG GCAAGTACTACTACTATGCCATGGACGTGT
GGGGCCAGGGCACCACCGTGACAGTGTCAT CT C40B16 VL DNA
TCCTACGAGCTGACCCAGCCTCCCTCCGTGT 14 CTGTGTCTCCTGGCCAGACCGCCAGCATCAC
CTGTAGCGGCGACAAGCTGGGCGATAAGTA CGCCTGCTGGTATCAGCAGAAGCCCGGCCA
GAGCCCCGTGCTGGTCATCTACCAGGACAG CAGAAGGCCCAGCGGCATCCCCGAGAGATT
CAGCGGCAGCAACAGCGGCAATACCGCCAC CCTGACCATCAGCGGCACCCAGGCCATGGA
CGAGGCCGATTACTATTGTCAGGCCTGGGC CAGCAGCACCGTGGTGTTTGGCGGCGGAAC
AAAGCTGACCGTGCTG C40H60 protein EVQLVESGGGLVQPGGSLRLSCAASGFTFSSY 21
AMSWVRQAPGKGLEWVSTIDNAGGGTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAV
YYCAKEGGKYYYYAMDVWGQGTTVTVSS C40H61 protein
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSY 22 AMSWVRQAPGKGLEWVSTIQNAGGGTYYAD
SVKGRFTISRDNSKNTLYLQMNSLRAEDTAV YYCAKEGGKYYYYAMDVWGQGTTVTVSS C40H63
protein EVQLVESGGGLVQPGGSLRLSCAASGFTFSSY 23
AMSWVRQAPGKGLEWVSTINQAGGGTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAV
YYCAKEGGKYYYYAMDVWGQGTTVTVSS C40H56 protein
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSY 24 AMSWVRQAPGKGLEWVSTINAAGGGTYYAD
SVKGRFTISRDNSKNTLYLQMNSLRAEDTAV YYCAKEGGKYYYYAMDVWGQGTTVTVSS C40H83
protein EVQLVESGGGLVQPGGSLRLSCAASGFTFSSY 25
AMSWVRQAPGKGLEWVSTINAAGGGTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAV
YYCAKEGGKYYYYALDVWGQGTTVTVSS C40H86 protein
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSY 26 AMSWVRQAPGKGLEWVSTINQAGGGTYYAD
SVKGRFTISRDNSKNTLYLQMNSLRAEDTAV YYCAKEGGKYYYYALDVWGQGTTVTVSS C40L71
protein SYELTQPPSVSVSPGQTASITCSGDKLGDKYA 27
AWYQQKPGQSPVLVIYQDSRRPSGIPERFSGS NSGNTATLTISGTQAMDEADYYCQAWASSTV
VFGGGTKLTVL C40H60 DNA GAGGTGCAGCTGGTGGAGAGCGGCGGCGGC 28
CTGGTGCAGCCCGGCGGCAGCCTGCGGCTG AGCTGCGCCGCCAGCGGCTTCACCTTCAGC
AGCTACGCCATGAGCTGGGTGCGGCAGGCC CCCGGCAAGGGCCTGGAGTGGGTGAGCACC
ATCGACAACGCCGGCGGCGGCACCTACTAC GCCGACAGCGTGAAGGGCCGGTTCACCATC
AGCCGGGACAACAGCAAGAACACCCTGTAC CTGCAGATGAACAGCCTGCGGGCCGAGGAC
ACCGCCGTGTACTACTGCGCCAAGGAGGGC GGCAAGTACTACTACTACGCCATGGACGTG
TGGGGCCAGGGCACCACCGTGACCGTGAGC AGC C40H61 DNA
GAGGTGCAGCTGGTGGAGAGCGGCGGCGGC 29 CTGGTGCAGCCCGGCGGCAGCCTGCGGCTG
AGCTGCGCCGCCAGCGGCTTCACCTTCAGC AGCTACGCCATGAGCTGGGTGCGGCAGGCC
CCCGGCAAGGGCCTGGAGTGGGTGAGCACC ATCCAGAACGCCGGCGGCGGCACCTACTAC
GCCGACAGCGTGAAGGGCCGGTTCACCATC AGCCGGGACAACAGCAAGAACACCCTGTAC
CTGCAGATGAACAGCCTGCGGGCCGAGGAC ACCGCCGTGTACTACTGCGCCAAGGAGGGC
GGCAAGTACTACTACTACGCCATGGACGTG TGGGGCCAGGGCACCACCGTGACCGTGAGC AGC
C40H63 DNA GAGGTGCAGCTGGTGGAGAGCGGCGGCGGC 30
CTGGTGCAGCCCGGCGGCAGCCTGCGGCTG AGCTGCGCCGCCAGCGGCTTCACCTTCAGC
AGCTACGCCATGAGCTGGGTGCGGCAGGCC CCCGGCAAGGGCCTGGAGTGGGTGAGCACC
ATCAACCAGGCCGGCGGCGGCACCTACTAC GCCGACAGCGTGAAGGGCCGGTTCACCATC
AGCCGGGACAACAGCAAGAACACCCTGTAC CTGCAGATGAACAGCCTGCGGGCCGAGGAC
ACCGCCGTGTACTACTGCGCCAAGGAGGGC GGCAAGTACTACTACTACGCCATGGACGTG
TGGGGCCAGGGCACCACCGTGACCGTGAGC AGC C40H56 DNA
GAGGTGCAGCTGGTGGAGAGCGGCGGCGGC 31 CTGGTGCAGCCCGGCGGCAGCCTGCGGCTG
AGCTGCGCCGCCAGCGGCTTCACCTTCAGC AGCTACGCCATGAGCTGGGTGCGGCAGGCC
CCCGGCAAGGGCCTGGAGTGGGTGAGCACC ATCAACGCCGCCGGCGGCGGCACCTACTAC
GCCGACAGCGTGAAGGGCCGGTTCACCATC AGCCGGGACAACAGCAAGAACACCCTGTAC
CTGCAGATGAACAGCCTGCGGGCCGAGGAC ACCGCCGTGTACTACTGCGCCAAGGAGGGC
GGCAAGTACTACTACTACGCCATGGACGTG TGGGGCCAGGGCACCACCGTGACCGTGAGC AGC
C40H83 DNA GAGGTGCAGCTGGTGGAGAGCGGCGGCGGC 32
CTGGTGCAGCCCGGCGGCAGCCTGCGGCTG AGCTGCGCCGCCAGCGGCTTCACCTTCAGC
AGCTACGCCATGAGCTGGGTGCGGCAGGCC CCCGGCAAGGGCCTGGAGTGGGTGAGCACC
ATCAACGCCGCCGGCGGCGGCACCTACTAC GCCGACAGCGTGAAGGGCCGGTTCACCATC
AGCCGGGACAACAGCAAGAACACCCTGTAC CTGCAGATGAACAGCCTGCGGGCCGAGGAC
ACCGCCGTGTACTACTGCGCCAAGGAGGGC GGCAAGTACTACTACTACGCCCTGGACGTG
TGGGGCCAGGGCACCACCGTGACCGTGAGC AGC C40H86 DNA
GAGGTGCAGCTGGTGGAGAGCGGCGGCGGC 33 CTGGTGCAGCCCGGCGGCAGCCTGCGGCTG
AGCTGCGCCGCCAGCGGCTTCACCTTCAGC AGCTACGCCATGAGCTGGGTGCGGCAGGCC
CCCGGCAAGGGCCTGGAGTGGGTGAGCACC ATCAACCAGGCCGGCGGCGGCACCTACTAC
GCCGACAGCGTGAAGGGCCGGTTCACCATC AGCCGGGACAACAGCAAGAACACCCTGTAC
CTGCAGATGAACAGCCTGCGGGCCGAGGAC ACCGCCGTGTACTACTGCGCCAAGGAGGGC
GGCAAGTACTACTACTACGCCCTGGACGTG TGGGGCCAGGGCACCACCGTGACCGTGAGC AGC
C40L71 DNA AGCTACGAGCTGACCCAGCCCCCCAGCGTG 34
AGCGTGAGCCCCGGCCAGACCGCCAGCATC ACCTGCAGCGGCGACAAGCTGGGCGACAAG
TACGCCGCCTGGTACCAGCAGAAGCCCGGC CAGAGCCCCGTGCTGGTGATCTACCAGGAC
AGCCGGCGGCCCAGCGGCATCCCCGAGCGG TTCAGCGGCAGCAACAGCGGCAACACCGCC
ACCCTGACCATCAGCGGCACCCAGGCCATG GACGAGGCCGACTACTACTGCCAGGCCTGG
GCCAGCAGCACCGTGGTGTTCGGCGGCGGC ACCAAGCTGACCGTGCTG C40B16 heavy
chain EVQLVESGGGLVQPGGSLRLSCAASGFTFSSY 35 protein
AMSWVRQAPGRGLEWVSTINNSGGGTYYAD SVKGRFTISRDNSKNTLYLHMNSLRAEDTAV
YYCAKEGGKYYYYAMDVWGQGTTVTVSSAS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP
EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV
EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYV
DGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFF
LYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK C40B124 heavy chain
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSY 36 protein
AMSWVRQAPGKGLEWVSTIDNAGGGTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAV
YYCAKEGGKYYYYAMDVWGQGTTVTVSSAS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP
EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV
EPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPK DTLMISRTPEVTCVVVDVSAEDPEVKFNWYV
DGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPSSIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFF
LYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK C40B135 heavy chain
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSY 37 protein
AMSWVRQAPGKGLEWVSTIDNAGGGTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAV
YYCAKEGGKYYYYAMDVWGQGTTVTVSSAS TKGPSVFPLAPCSRSTSESTAALGCLVKDYFPE
PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSSLGTKTYTCNVDHKPSNTKVDKRV
ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSQEDPEVQFNWYVDGV
EVHNAKTKPREEQFNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKGLPSSIEKTISKAKGQPRE
PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSR
LTVDKSRWQEGNVFSCSVMHEALHNHYTQK SLSLSLGK C40B125 heavy chain
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSY 38 protein
AMSWVRQAPGKGLEWVSTIQNAGGGTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAV
YYCAKEGGKYYYYAMDVWGQGTTVTVSSAS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP
EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV
EPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPK DTLMISRTPEVTCVVVDVSAEDPEVKFNWYV
DGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPSSIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFF
LYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK C40B136 heavy chain
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSY 39 protein
AMSWVRQAPGKGLEWVSTIQNAGGGTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAV
YYCAKEGGKYYYYAMDVWGQGTTVTVSSAS TKGPSVFPLAPCSRSTSESTAALGCLVKDYFPE
PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSSLGTKTYTCNVDHKPSNTKVDKRV
ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSQEDPEVQFNWYVDGV
EVHNAKTKPREEQFNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKGLPSSIEKTISKAKGQPRE
PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDI
AVEWESNGQPENNYKTTPPVLDSDGSFFLYSR
LTVDKSRWQEGNVFSCSVMHEALHNHYTQK SLSLSLGK C40B127 heavy chain
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSY 40 protein
AMSWVRQAPGKGLEWVSTINQAGGGTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAV
YYCAKEGGKYYYYAMDVWGQGTTVTVSSAS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP
EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV
EPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPK DTLMISRTPEVTCVVVDVSAEDPEVKFNWYV
DGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPSSIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFF
LYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK C40B138 heavy chain
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSY 41 protein
AMSWVRQAPGKGLEWVSTINQAGGGTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAV
YYCAKEGGKYYYYAMDVWGQGTTVTVSSAS TKGPSVFPLAPCSRSTSESTAALGCLVKDYFPE
PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSSLGTKTYTCNVDHKPSNTKVDKRV
ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSQEDPEVQFNWYVDGV
EVHNAKTKPREEQFNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKGLPSSIEKTISKAKGQPRE
PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSR
LTVDKSRWQEGNVFSCSVMHEALHNHYTQK SLSLSLGK C40B131 heavy chain
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSY 42 protein
AMSWVRQAPGKGLEWVSTINAAGGGTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAV
YYCAKEGGKYYYYAMDVWGQGTTVTVSSAS TKGPSVFPLAPCSRSTSESTAALGCLVKDYFPE
PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSSLGTKTYTCNVDHKPSNTKVDKRV
ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSQEDPEVQFNWYVDGV
EVHNAKTKPREEQFNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKGLPSSIEKTISKAKGQPRE
PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSR
LTVDKSRWQEGNVFSCSVMHEALHNHYTQK SLSLSLGK C40B176 heavy chain
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSY 43 protein
AMSWVRQAPGKGLEWVSTINAAGGGTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAV
YYCAKEGGKYYYYALDVWGQGTTVTVSSAS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP
EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV
EPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPK DTLMISRTPEVTCVVVDVSAEDPEVKFNWYV
DGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPSSIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFF
LYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK C40B180 heavy chain
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSY 44 protein
AMSWVRQAPGKGLEWVSTINAAGGGTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAV
YYCAKEGGKYYYYALDVWGQGTTVTVSSAS TKGPSVFPLAPCSRSTSESTAALGCLVKDYFPE
PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSSLGTKTYTCNVDHKPSNTKVDKRV
ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSQEDPEVQFNWYVDGV
EVHNAKTKPREEQFNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKGLPSSIEKTISKAKGQPRE
PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSR
LTVDKSRWQEGNVFSCSVMHEALHNHYTQK SLSLSLGK C40B179 heavy chain
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSY 45 protein
AMSWVRQAPGKGLEWVSTINQAGGGTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAV
YYCAKEGGKYYYYALDVWGQGTTVTVSSAS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP
EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV
EPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPK DTLMISRTPEVTCVVVDVSAEDPEVKFNWYV
DGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPSSIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFF
LYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK C40B183 heavy chain
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSY 46 protein
AMSWVRQAPGKGLEWVSTINQAGGGTYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAV
YYCAKEGGKYYYYALDVWGQGTTVTVSSAS TKGPSVFPLAPCSRSTSESTAALGCLVKDYFPE
PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSSLGTKTYTCNVDHKPSNTKVDKRV
ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSQEDPEVQFNWYVDGV
EVHNAKTKPREEQFNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKGLPSSIEKTISKAKGQPRE
PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSR
LTVDKSRWQEGNVFSCSVMHEALHNHYTQK SLSLSLGK C40B16 light chain
SYELTQPPSVSVSPGQTASITCSGDKLGDKYA 47 protein
CWYQQKPGQSPVLVIYQDSRRPSGIPERFSGS NSGNTATLTISGTQAMDEADYYCQAWASSTV
VFGGGTKLTVLGQPKAAPSVTLFPPSSEELQA NKATLVCLISDFYPGAVTVAWKADSSPVKAG
VETTTPSKQSNNKYAASSYLSLTPEQWKSHRS YSCQVTHEGSTVEKTVAPTECS C40B124,
C40B135, SYELTQPPSVSVSPGQTASITCSGDKLGDKYA 48 C40B125, C40B136,
AWYQQKPGQSPVLVIYQDSRRPSGIPERFSGS C40B127, C40B138,
NSGNTATLTISGTQAMDEADYYCQAWASSTV C40B131, C40B176,
VFGGGTKLTVLGQPKAAPSVTLFPPSSEELQA C40B180. C40B179,
NKATLVCLISDFYPGAVTVAWKADSSPVKAG C40B183
VETTTPSKQSNNKYAASSYLSLTPEQWKSHRS Light chain protein
YSCQVTHEGSTVEKTVAPTECS IGHV3-23 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSY 49
AMSWVRQAPGKGLEWVSAISGSGGSTYYADS VKGRFTISRDNSKNTLYLQMNSLRAEDTAVY
YCAK IGLV3-1 SYELTQPPSVSVSPGQTASITCSGDKLGDKYA 50
CWYQQKPGQSPVLVIYQDSKRPSGIPERFSGS NSGNTATLTISGTQAMDEADYYCQAWDSSTA
IgG1sigma Fc ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY 51
FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK
KVEPKSCDKTHTCPPCPAPEAAGASSVFLFPP KPKDTLMISRTPEVTCVVVDVSAEDPEVKFN
WYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPSSIEKTISK
AKGQPREPQVYTLPPSREEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSD
GSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK IgG4PAA
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYF 52
PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKR
VESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSQEDPEVQFNWYVDG
VEVHNAKTKPREEQFNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKGLPSSIEKTISKAKGQPR
EPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYS
RLTVDKSRWQEGNVFSCSVMHEALHNHYTQ KSLSLSLGK C40B16 heavy chain
GAGGTGCAGCTGGTGGAATCTGGCGGAGGA 65 DNA
CTGGTGCAGCCTGGCGGCAGCCTGAGACTG TCTTGTGCCGCCAGCGGCTTCACCTTCAGCA
GCTACGCTATGAGCTGGGTCCGACAGGCCC CTGGCAGAGGACTCGAGTGGGTGTCCACCA
TCAACAACAGCGGCGGAGGCACCTACTACG CCGACAGCGTGAAGGGCAGATTCACCATCA
GCCGGGACAACAGCAAGAACACCCTGTACC TGCACATGAACAGCCTGCGGGCCGAGGACA
CCGCCGTGTACTATTGTGCCAAAGAGGGCG GCAAGTACTACTACTATGCCATGGACGTGT
GGGGCCAGGGCACCACCGTGACAGTGTCAT CTGCCTCCACCAAGGGCCCATCGGTCTTCCC
CCTGGCACCCTCCTCCAAGAGCACCTCTGGG GGCACAGCGGCCCTGGGCTGCCTGGTCAAG
GACTACTTCCCCGAACCGGTGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGC
ACACCTTCCCGGCTGTCCTACAGTCCTCAGG ACTCTACTCCCTCAGCAGCGTGGTGACCGTG
CCCTCCAGCAGCTTGGGCACCCAGACCTAC ATCTGCAACGTGAATCACAAGCCCAGCAAC
ACCAAGGTGGACAAGAAAGTTGAGCCCAAA TCTTGTGACAAAACTCACACATGCCCACCGT
GCCCAGCACCTGAACTCCTGGGGGGACCGT CAGTCTTCCTCTTCCCCCCAAAACCCAAGGA
CACCCTCATGATCTCCCGGACCCCTGAGGTC ACATGCGTGGTGGTGGACGTGAGCCACGAA
GACCCTGAGGTCAAGTTCAACTGGTACGTG GACGGCGTGGAGGTGCATAATGCCAAGACA
AAGCCGCGGGAGGAGCAGTACAACAGCAC GTACCGTGTGGTCAGCGTCCTCACCGTCCTG
CACCAGGACTGGCTGAATGGCAAGGAGTAC AAGTGCAAGGTCTCCAACAAAGCCCTCCCA
GCCCCCATCGAGAAAACCATCTCCAAAGCC AAAGGGCAGCCCCGAGAACCACAGGTGTAC
ACCCTGCCCCCATCCCGGGAGGAGATGACC AAGAACCAGGTCAGCCTGACCTGCCTGGTC
AAAGGCTTCTATCCCAGCGACATCGCCGTG GAGTGGGAGAGCAATGGGCAGCCGGAGAA
CAACTACAAGACCACGCCTCCCGTGCTGGA CTCCGACGGCTCCTTCTTCCTCTACAGCAAG
CTCACCGTGGACAAGAGCAGGTGGCAGCAG GGGAACGTCTTCTCATGCTCCGTGATGCATG
AGGCTCTGCACAACCACTACACGCAGAAGA GCCTCTCCCTGTCTCCGGGTAAA C40B124
heavy chain GAGGTGCAGCTGGTGGAGAGCGGCGGCGGC 66 DNA
CTGGTGCAGCCCGGCGGCAGCCTGCGGCTG AGCTGCGCCGCCAGCGGCTTCACCTTCAGC
AGCTACGCCATGAGCTGGGTGCGGCAGGCC CCCGGCAAGGGCCTGGAGTGGGTGAGCACC
ATCGACAACGCCGGCGGCGGCACCTACTAC GCCGACAGCGTGAAGGGCCGGTTCACCATC
AGCCGGGACAACAGCAAGAACACCCTGTAC CTGCAGATGAACAGCCTGCGGGCCGAGGAC
ACCGCCGTGTACTACTGCGCCAAGGAGGGC GGCAAGTACTACTACTACGCCATGGACGTG
TGGGGCCAGGGCACCACCGTGACCGTGAGC AGCGCCTCCACCAAGGGCCCATCGGTCTTCC
CCCTGGCACCCTCCTCCAAGAGCACCTCTGG GGGCACAGCGGCCCTGGGCTGCCTGGTCAA
GGACTACTTCCCCGAACCGGTGACGGTGTC GTGGAACTCAGGCGCCCTGACCAGCGGCGT
GCACACCTTCCCGGCTGTCCTACAGTCCTCA GGACTCTACTCCCTCAGCAGCGTGGTGACC
GTGCCCTCCAGCAGCTTGGGCACCCAGACC TACATCTGCAACGTGAATCACAAGCCCAGC
AACACCAAGGTGGACAAGAAAGTTGAGCCC AAATCTTGTGACAAAACTCACACATGCCCA
CCGTGCCCAGCACCTGAAGCAGCAGGGGCA TCTTCAGTCTTCCTCTTCCCCCCAAAACCCA
AGGACACCCTCATGATCTCCCGGACCCCTG AGGTCACATGCGTGGTGGTGGACGTGAGCG
CCGAAGACCCTGAGGTCAAGTTCAACTGGT ACGTGGACGGCGTGGAGGTGCATAATGCCA
AGACAAAGCCGCGGGAGGAGCAGTACAAC AGCACGTACCGTGTGGTCAGCGTCCTCACC
GTCCTGCACCAGGACTGGCTGAATGGCAAG GAGTACAAGTGCAAGGTCTCCAACAAAGCC
CTCCCATCCTCCATCGAGAAAACCATCTCCA AAGCCAAAGGGCAGCCCCGAGAACCACAG
GTGTACACCCTGCCCCCATCCCGGGAGGAG ATGACCAAGAACCAGGTCAGCCTGACCTGC
CTGGTCAAAGGCTTCTATCCCAGCGACATCG CCGTGGAGTGGGAGAGCAATGGGCAGCCGG
AGAACAACTACAAGACCACGCCTCCCGTGC TGGACTCCGACGGCTCCTTCTTCCTCTACAG
CAAGCTCACCGTGGACAAGAGCAGGTGGCA GCAGGGGAACGTCTTCTCATGCTCCGTGATG
CATGAGGCTCTGCACAACCACTACACGCAG AAGAGCCTCTCCCTGTCTCCGGGTAAA C40B135
heavy chain GAGGTGCAGCTGGTGGAGAGCGGCGGCGGC 67 DNA
CTGGTGCAGCCCGGCGGCAGCCTGCGGCTG AGCTGCGCCGCCAGCGGCTTCACCTTCAGC
AGCTACGCCATGAGCTGGGTGCGGCAGGCC CCCGGCAAGGGCCTGGAGTGGGTGAGCACC
ATCGACAACGCCGGCGGCGGCACCTACTAC GCCGACAGCGTGAAGGGCCGGTTCACCATC
AGCCGGGACAACAGCAAGAACACCCTGTAC CTGCAGATGAACAGCCTGCGGGCCGAGGAC
ACCGCCGTGTACTACTGCGCCAAGGAGGGC GGCAAGTACTACTACTACGCCATGGACGTG
TGGGGCCAGGGCACCACCGTGACCGTGAGC AGCGCCTCCACCAAGGGCCCATCCGTCTTCC
CCCTGGCGCCCTGCTCCAGGAGCACCTCCG AGAGCACAGCCGCCCTGGGCTGCCTGGTCA
AGGACTACTTCCCCGAACCGGTGACGGTGT CGTGGAACTCAGGCGCCCTGACCAGCGGCG
TGCACACCTTCCCGGCTGTCCTACAGTCCTC AGGACTCTACTCCCTCAGCAGCGTGGTGAC
CGTGCCCTCCAGCAGCTTGGGCACGAAAAC CTACACCTGCAACGTAGATCACAAGCCCAG
CAACACCAAGGTGGACAAGAGAGTTGAGTC CAAATATGGTCCCCCATGCCCACCATGCCCA
GCACCTGAGGCCGCCGGGGGACCATCAGTC TTCCTGTTCCCCCCAAAACCCAAGGACACTC
TCATGATCTCCCGGACCCCTGAGGTCACGTG CGTGGTGGTGGACGTGAGCCAGGAAGACCC
CGAGGTCCAGTTCAACTGGTACGTGGATGG CGTGGAGGTGCATAATGCCAAGACAAAGCC
GCGGGAGGAGCAGTTCAACAGCACGTACCG TGTGGTCAGCGTCCTCACCGTCCTGCACCAG
GACTGGCTGAACGGCAAGGAGTACAAGTGC AAGGTCTCCAACAAAGGCCTCCCGTCCTCC
ATCGAGAAAACCATCTCCAAAGCCAAAGGG CAGCCCCGAGAGCCACAGGTGTACACCCTG
CCCCCATCCCAGGAGGAGATGACCAAGAAC CAGGTCAGCCTGACCTGCCTGGTCAAAGGC
TTCTACCCCAGCGACATCGCCGTGGAGTGG GAGAGCAATGGGCAGCCGGAGAACAACTAC
AAGACCACGCCTCCCGTGCTGGACTCCGAC GGCTCCTTCTTCCTCTACAGCAGGCTAACCG
TGGACAAGAGCAGGTGGCAGGAGGGGAAT GTCTTCTCATGCTCCGTGATGCATGAGGCTC
TGCACAACCACTACACACAGAAGAGCCTCT CCCTGTCTCTGGGTAAA C40B125 heavy
chain GAGGTGCAGCTGGTGGAGAGCGGCGGCGGC 68 DNA
CTGGTGCAGCCCGGCGGCAGCCTGCGGCTG AGCTGCGCCGCCAGCGGCTTCACCTTCAGC
AGCTACGCCATGAGCTGGGTGCGGCAGGCC CCCGGCAAGGGCCTGGAGTGGGTGAGCACC
ATCCAGAACGCCGGCGGCGGCACCTACTAC GCCGACAGCGTGAAGGGCCGGTTCACCATC
AGCCGGGACAACAGCAAGAACACCCTGTAC CTGCAGATGAACAGCCTGCGGGCCGAGGAC
ACCGCCGTGTACTACTGCGCCAAGGAGGGC GGCAAGTACTACTACTACGCCATGGACGTG
TGGGGCCAGGGCACCACCGTGACCGTGAGC AGCGCCTCCACCAAGGGCCCATCGGTCTTCC
CCCTGGCACCCTCCTCCAAGAGCACCTCTGG GGGCACAGCGGCCCTGGGCTGCCTGGTCAA
GGACTACTTCCCCGAACCGGTGACGGTGTC GTGGAACTCAGGCGCCCTGACCAGCGGCGT
GCACACCTTCCCGGCTGTCCTACAGTCCTCA GGACTCTACTCCCTCAGCAGCGTGGTGACC
GTGCCCTCCAGCAGCTTGGGCACCCAGACC TACATCTGCAACGTGAATCACAAGCCCAGC
AACACCAAGGTGGACAAGAAAGTTGAGCCC AAATCTTGTGACAAAACTCACACATGCCCA
CCGTGCCCAGCACCTGAAGCAGCAGGGGCA TCTTCAGTCTTCCTCTTCCCCCCAAAACCCA
AGGACACCCTCATGATCTCCCGGACCCCTG AGGTCACATGCGTGGTGGTGGACGTGAGCG
CCGAAGACCCTGAGGTCAAGTTCAACTGGT ACGTGGACGGCGTGGAGGTGCATAATGCCA
AGACAAAGCCGCGGGAGGAGCAGTACAAC AGCACGTACCGTGTGGTCAGCGTCCTCACC
GTCCTGCACCAGGACTGGCTGAATGGCAAG GAGTACAAGTGCAAGGTCTCCAACAAAGCC
CTCCCATCCTCCATCGAGAAAACCATCTCCA AAGCCAAAGGGCAGCCCCGAGAACCACAG
GTGTACACCCTGCCCCCATCCCGGGAGGAG ATGACCAAGAACCAGGTCAGCCTGACCTGC
CTGGTCAAAGGCTTCTATCCCAGCGACATCG CCGTGGAGTGGGAGAGCAATGGGCAGCCGG
AGAACAACTACAAGACCACGCCTCCCGTGC TGGACTCCGACGGCTCCTTCTTCCTCTACAG
CAAGCTCACCGTGGACAAGAGCAGGTGGCA GCAGGGGAACGTCTTCTCATGCTCCGTGATG
CATGAGGCTCTGCACAACCACTACACGCAG AAGAGCCTCTCCCTGTCTCCGGGTAAA C40B136
heavy chain GAGGTGCAGCTGGTGGAGAGCGGCGGCGGC 69 DNA
CTGGTGCAGCCCGGCGGCAGCCTGCGGCTG AGCTGCGCCGCCAGCGGCTTCACCTTCAGC
AGCTACGCCATGAGCTGGGTGCGGCAGGCC CCCGGCAAGGGCCTGGAGTGGGTGAGCACC
ATCCAGAACGCCGGCGGCGGCACCTACTAC GCCGACAGCGTGAAGGGCCGGTTCACCATC
AGCCGGGACAACAGCAAGAACACCCTGTAC CTGCAGATGAACAGCCTGCGGGCCGAGGAC
ACCGCCGTGTACTACTGCGCCAAGGAGGGC GGCAAGTACTACTACTACGCCATGGACGTG
TGGGGCCAGGGCACCACCGTGACCGTGAGC AGCGCCTCCACCAAGGGCCCATCCGTCTTCC
CCCTGGCGCCCTGCTCCAGGAGCACCTCCG AGAGCACAGCCGCCCTGGGCTGCCTGGTCA
AGGACTACTTCCCCGAACCGGTGACGGTGT CGTGGAACTCAGGCGCCCTGACCAGCGGCG
TGCACACCTTCCCGGCTGTCCTACAGTCCTC AGGACTCTACTCCCTCAGCAGCGTGGTGAC
CGTGCCCTCCAGCAGCTTGGGCACGAAAAC CTACACCTGCAACGTAGATCACAAGCCCAG
CAACACCAAGGTGGACAAGAGAGTTGAGTC CAAATATGGTCCCCCATGCCCACCATGCCCA
GCACCTGAGGCCGCCGGGGGACCATCAGTC TTCCTGTTCCCCCCAAAACCCAAGGACACTC
TCATGATCTCCCGGACCCCTGAGGTCACGTG CGTGGTGGTGGACGTGAGCCAGGAAGACCC
CGAGGTCCAGTTCAACTGGTACGTGGATGG CGTGGAGGTGCATAATGCCAAGACAAAGCC
GCGGGAGGAGCAGTTCAACAGCACGTACCG TGTGGTCAGCGTCCTCACCGTCCTGCACCAG
GACTGGCTGAACGGCAAGGAGTACAAGTGC AAGGTCTCCAACAAAGGCCTCCCGTCCTCC
ATCGAGAAAACCATCTCCAAAGCCAAAGGG CAGCCCCGAGAGCCACAGGTGTACACCCTG
CCCCCATCCCAGGAGGAGATGACCAAGAAC CAGGTCAGCCTGACCTGCCTGGTCAAAGGC
TTCTACCCCAGCGACATCGCCGTGGAGTGG GAGAGCAATGGGCAGCCGGAGAACAACTAC
AAGACCACGCCTCCCGTGCTGGACTCCGAC GGCTCCTTCTTCCTCTACAGCAGGCTAACCG
TGGACAAGAGCAGGTGGCAGGAGGGGAAT GTCTTCTCATGCTCCGTGATGCATGAGGCTC
TGCACAACCACTACACACAGAAGAGCCTCT CCCTGTCTCTGGGTAAA C40B127 heavy
chain GAGGTGCAGCTGGTGGAGAGCGGCGGCGGC 70 DNA
CTGGTGCAGCCCGGCGGCAGCCTGCGGCTG AGCTGCGCCGCCAGCGGCTTCACCTTCAGC
AGCTACGCCATGAGCTGGGTGCGGCAGGCC CCCGGCAAGGGCCTGGAGTGGGTGAGCACC
ATCAACCAGGCCGGCGGCGGCACCTACTAC GCCGACAGCGTGAAGGGCCGGTTCACCATC
AGCCGGGACAACAGCAAGAACACCCTGTAC CTGCAGATGAACAGCCTGCGGGCCGAGGAC
ACCGCCGTGTACTACTGCGCCAAGGAGGGC GGCAAGTACTACTACTACGCCATGGACGTG
TGGGGCCAGGGCACCACCGTGACCGTGAGC AGCGCCTCCACCAAGGGCCCATCGGTCTTCC
CCCTGGCACCCTCCTCCAAGAGCACCTCTGG GGGCACAGCGGCCCTGGGCTGCCTGGTCAA
GGACTACTTCCCCGAACCGGTGACGGTGTC GTGGAACTCAGGCGCCCTGACCAGCGGCGT
GCACACCTTCCCGGCTGTCCTACAGTCCTCA GGACTCTACTCCCTCAGCAGCGTGGTGACC
GTGCCCTCCAGCAGCTTGGGCACCCAGACC TACATCTGCAACGTGAATCACAAGCCCAGC
AACACCAAGGTGGACAAGAAAGTTGAGCCC AAATCTTGTGACAAAACTCACACATGCCCA
CCGTGCCCAGCACCTGAAGCAGCAGGGGCA TCTTCAGTCTTCCTCTTCCCCCCAAAACCCA
AGGACACCCTCATGATCTCCCGGACCCCTG AGGTCACATGCGTGGTGGTGGACGTGAGCG
CCGAAGACCCTGAGGTCAAGTTCAACTGGT ACGTGGACGGCGTGGAGGTGCATAATGCCA
AGACAAAGCCGCGGGAGGAGCAGTACAAC AGCACGTACCGTGTGGTCAGCGTCCTCACC
GTCCTGCACCAGGACTGGCTGAATGGCAAG GAGTACAAGTGCAAGGTCTCCAACAAAGCC
CTCCCATCCTCCATCGAGAAAACCATCTCCA AAGCCAAAGGGCAGCCCCGAGAACCACAG
GTGTACACCCTGCCCCCATCCCGGGAGGAG ATGACCAAGAACCAGGTCAGCCTGACCTGC
CTGGTCAAAGGCTTCTATCCCAGCGACATCG CCGTGGAGTGGGAGAGCAATGGGCAGCCGG
AGAACAACTACAAGACCACGCCTCCCGTGC TGGACTCCGACGGCTCCTTCTTCCTCTACAG
CAAGCTCACCGTGGACAAGAGCAGGTGGCA GCAGGGGAACGTCTTCTCATGCTCCGTGATG
CATGAGGCTCTGCACAACCACTACACGCAG AAGAGCCTCTCCCTGTCTCCGGGTAAA C40B138
heavy chain GAGGTGCAGCTGGTGGAGAGCGGCGGCGGC 71 DNA
CTGGTGCAGCCCGGCGGCAGCCTGCGGCTG AGCTGCGCCGCCAGCGGCTTCACCTTCAGC
AGCTACGCCATGAGCTGGGTGCGGCAGGCC CCCGGCAAGGGCCTGGAGTGGGTGAGCACC
ATCAACCAGGCCGGCGGCGGCACCTACTAC GCCGACAGCGTGAAGGGCCGGTTCACCATC
AGCCGGGACAACAGCAAGAACACCCTGTAC CTGCAGATGAACAGCCTGCGGGCCGAGGAC
ACCGCCGTGTACTACTGCGCCAAGGAGGGC GGCAAGTACTACTACTACGCCATGGACGTG
TGGGGCCAGGGCACCACCGTGACCGTGAGC AGCGCCTCCACCAAGGGCCCATCCGTCTTCC
CCCTGGCGCCCTGCTCCAGGAGCACCTCCG AGAGCACAGCCGCCCTGGGCTGCCTGGTCA
AGGACTACTTCCCCGAACCGGTGACGGTGT CGTGGAACTCAGGCGCCCTGACCAGCGGCG
TGCACACCTTCCCGGCTGTCCTACAGTCCTC AGGACTCTACTCCCTCAGCAGCGTGGTGAC
CGTGCCCTCCAGCAGCTTGGGCACGAAAAC CTACACCTGCAACGTAGATCACAAGCCCAG
CAACACCAAGGTGGACAAGAGAGTTGAGTC CAAATATGGTCCCCCATGCCCACCATGCCCA
GCACCTGAGGCCGCCGGGGGACCATCAGTC TTCCTGTTCCCCCCAAAACCCAAGGACACTC
TCATGATCTCCCGGACCCCTGAGGTCACGTG CGTGGTGGTGGACGTGAGCCAGGAAGACCC
CGAGGTCCAGTTCAACTGGTACGTGGATGG CGTGGAGGTGCATAATGCCAAGACAAAGCC
GCGGGAGGAGCAGTTCAACAGCACGTACCG TGTGGTCAGCGTCCTCACCGTCCTGCACCAG
GACTGGCTGAACGGCAAGGAGTACAAGTGC AAGGTCTCCAACAAAGGCCTCCCGTCCTCC
ATCGAGAAAACCATCTCCAAAGCCAAAGGG CAGCCCCGAGAGCCACAGGTGTACACCCTG
CCCCCATCCCAGGAGGAGATGACCAAGAAC CAGGTCAGCCTGACCTGCCTGGTCAAAGGC
TTCTACCCCAGCGACATCGCCGTGGAGTGG GAGAGCAATGGGCAGCCGGAGAACAACTAC
AAGACCACGCCTCCCGTGCTGGACTCCGAC GGCTCCTTCTTCCTCTACAGCAGGCTAACCG
TGGACAAGAGCAGGTGGCAGGAGGGGAAT GTCTTCTCATGCTCCGTGATGCATGAGGCTC
TGCACAACCACTACACACAGAAGAGCCTCT CCCTGTCTCTGGGTAAA C40B131 heavy
chain GAGGTGCAGCTGGTGGAGAGCGGCGGCGGC 72 DNA
CTGGTGCAGCCCGGCGGCAGCCTGCGGCTG AGCTGCGCCGCCAGCGGCTTCACCTTCAGC
AGCTACGCCATGAGCTGGGTGCGGCAGGCC CCCGGCAAGGGCCTGGAGTGGGTGAGCACC
ATCAACGCCGCCGGCGGCGGCACCTACTAC GCCGACAGCGTGAAGGGCCGGTTCACCATC
AGCCGGGACAACAGCAAGAACACCCTGTAC CTGCAGATGAACAGCCTGCGGGCCGAGGAC
ACCGCCGTGTACTACTGCGCCAAGGAGGGC GGCAAGTACTACTACTACGCCATGGACGTG
TGGGGCCAGGGCACCACCGTGACCGTGAGC AGCGCCTCCACCAAGGGCCCATCCGTCTTCC
CCCTGGCGCCCTGCTCCAGGAGCACCTCCG AGAGCACAGCCGCCCTGGGCTGCCTGGTCA
AGGACTACTTCCCCGAACCGGTGACGGTGT
CGTGGAACTCAGGCGCCCTGACCAGCGGCG TGCACACCTTCCCGGCTGTCCTACAGTCCTC
AGGACTCTACTCCCTCAGCAGCGTGGTGAC CGTGCCCTCCAGCAGCTTGGGCACGAAAAC
CTACACCTGCAACGTAGATCACAAGCCCAG CAACACCAAGGTGGACAAGAGAGTTGAGTC
CAAATATGGTCCCCCATGCCCACCATGCCCA GCACCTGAGGCCGCCGGGGGACCATCAGTC
TTCCTGTTCCCCCCAAAACCCAAGGACACTC TCATGATCTCCCGGACCCCTGAGGTCACGTG
CGTGGTGGTGGACGTGAGCCAGGAAGACCC CGAGGTCCAGTTCAACTGGTACGTGGATGG
CGTGGAGGTGCATAATGCCAAGACAAAGCC GCGGGAGGAGCAGTTCAACAGCACGTACCG
TGTGGTCAGCGTCCTCACCGTCCTGCACCAG GACTGGCTGAACGGCAAGGAGTACAAGTGC
AAGGTCTCCAACAAAGGCCTCCCGTCCTCC ATCGAGAAAACCATCTCCAAAGCCAAAGGG
CAGCCCCGAGAGCCACAGGTGTACACCCTG CCCCCATCCCAGGAGGAGATGACCAAGAAC
CAGGTCAGCCTGACCTGCCTGGTCAAAGGC TTCTACCCCAGCGACATCGCCGTGGAGTGG
GAGAGCAATGGGCAGCCGGAGAACAACTAC AAGACCACGCCTCCCGTGCTGGACTCCGAC
GGCTCCTTCTTCCTCTACAGCAGGCTAACCG TGGACAAGAGCAGGTGGCAGGAGGGGAAT
GTCTTCTCATGCTCCGTGATGCATGAGGCTC TGCACAACCACTACACACAGAAGAGCCTCT
CCCTGTCTCTGGGTAAA C40B176 heavy chain
GAGGTGCAGCTGGTGGAGAGCGGCGGCGGC 73 DNA
CTGGTGCAGCCCGGCGGCAGCCTGCGGCTG AGCTGCGCCGCCAGCGGCTTCACCTTCAGC
AGCTACGCCATGAGCTGGGTGCGGCAGGCC CCCGGCAAGGGCCTGGAGTGGGTGAGCACC
ATCAACGCCGCCGGCGGCGGCACCTACTAC GCCGACAGCGTGAAGGGCCGGTTCACCATC
AGCCGGGACAACAGCAAGAACACCCTGTAC CTGCAGATGAACAGCCTGCGGGCCGAGGAC
ACCGCCGTGTACTACTGCGCCAAGGAGGGC GGCAAGTACTACTACTACGCCCTGGACGTG
TGGGGCCAGGGCACCACCGTGACCGTGAGC AGCGCCTCCACCAAGGGCCCATCGGTCTTCC
CCCTGGCACCCTCCTCCAAGAGCACCTCTGG GGGCACAGCGGCCCTGGGCTGCCTGGTCAA
GGACTACTTCCCCGAACCGGTGACGGTGTC GTGGAACTCAGGCGCCCTGACCAGCGGCGT
GCACACCTTCCCGGCTGTCCTACAGTCCTCA GGACTCTACTCCCTCAGCAGCGTGGTGACC
GTGCCCTCCAGCAGCTTGGGCACCCAGACC TACATCTGCAACGTGAATCACAAGCCCAGC
AACACCAAGGTGGACAAGAAAGTTGAGCCC AAATCTTGTGACAAAACTCACACATGCCCA
CCGTGCCCAGCACCTGAAGCAGCAGGGGCA TCTTCAGTCTTCCTCTTCCCCCCAAAACCCA
AGGACACCCTCATGATCTCCCGGACCCCTG AGGTCACATGCGTGGTGGTGGACGTGAGCG
CCGAAGACCCTGAGGTCAAGTTCAACTGGT ACGTGGACGGCGTGGAGGTGCATAATGCCA
AGACAAAGCCGCGGGAGGAGCAGTACAAC AGCACGTACCGTGTGGTCAGCGTCCTCACC
GTCCTGCACCAGGACTGGCTGAATGGCAAG GAGTACAAGTGCAAGGTCTCCAACAAAGCC
CTCCCATCCTCCATCGAGAAAACCATCTCCA AAGCCAAAGGGCAGCCCCGAGAACCACAG
GTGTACACCCTGCCCCCATCCCGGGAGGAG ATGACCAAGAACCAGGTCAGCCTGACCTGC
CTGGTCAAAGGCTTCTATCCCAGCGACATCG CCGTGGAGTGGGAGAGCAATGGGCAGCCGG
AGAACAACTACAAGACCACGCCTCCCGTGC TGGACTCCGACGGCTCCTTCTTCCTCTACAG
CAAGCTCACCGTGGACAAGAGCAGGTGGCA GCAGGGGAACGTCTTCTCATGCTCCGTGATG
CATGAGGCTCTGCACAACCACTACACGCAG AAGAGCCTCTCCCTGTCTCCGGGTAAA C40B180
heavy chain GAGGTGCAGCTGGTGGAGAGCGGCGGCGGC 74 DNA
CTGGTGCAGCCCGGCGGCAGCCTGCGGCTG AGCTGCGCCGCCAGCGGCTTCACCTTCAGC
AGCTACGCCATGAGCTGGGTGCGGCAGGCC CCCGGCAAGGGCCTGGAGTGGGTGAGCACC
ATCAACGCCGCCGGCGGCGGCACCTACTAC GCCGACAGCGTGAAGGGCCGGTTCACCATC
AGCCGGGACAACAGCAAGAACACCCTGTAC CTGCAGATGAACAGCCTGCGGGCCGAGGAC
ACCGCCGTGTACTACTGCGCCAAGGAGGGC GGCAAGTACTACTACTACGCCCTGGACGTG
TGGGGCCAGGGCACCACCGTGACCGTGAGC AGCGCCTCCACCAAGGGCCCATCCGTCTTCC
CCCTGGCGCCCTGCTCCAGGAGCACCTCCG AGAGCACAGCCGCCCTGGGCTGCCTGGTCA
AGGACTACTTCCCCGAACCGGTGACGGTGT CGTGGAACTCAGGCGCCCTGACCAGCGGCG
TGCACACCTTCCCGGCTGTCCTACAGTCCTC AGGACTCTACTCCCTCAGCAGCGTGGTGAC
CGTGCCCTCCAGCAGCTTGGGCACGAAAAC CTACACCTGCAACGTAGATCACAAGCCCAG
CAACACCAAGGTGGACAAGAGAGTTGAGTC CAAATATGGTCCCCCATGCCCACCATGCCCA
GCACCTGAGGCCGCCGGGGGACCATCAGTC TTCCTGTTCCCCCCAAAACCCAAGGACACTC
TCATGATCTCCCGGACCCCTGAGGTCACGTG CGTGGTGGTGGACGTGAGCCAGGAAGACCC
CGAGGTCCAGTTCAACTGGTACGTGGATGG CGTGGAGGTGCATAATGCCAAGACAAAGCC
GCGGGAGGAGCAGTTCAACAGCACGTACCG TGTGGTCAGCGTCCTCACCGTCCTGCACCAG
GACTGGCTGAACGGCAAGGAGTACAAGTGC AAGGTCTCCAACAAAGGCCTCCCGTCCTCC
ATCGAGAAAACCATCTCCAAAGCCAAAGGG CAGCCCCGAGAGCCACAGGTGTACACCCTG
CCCCCATCCCAGGAGGAGATGACCAAGAAC CAGGTCAGCCTGACCTGCCTGGTCAAAGGC
TTCTACCCCAGCGACATCGCCGTGGAGTGG GAGAGCAATGGGCAGCCGGAGAACAACTAC
AAGACCACGCCTCCCGTGCTGGACTCCGAC GGCTCCTTCTTCCTCTACAGCAGGCTAACCG
TGGACAAGAGCAGGTGGCAGGAGGGGAAT GTCTTCTCATGCTCCGTGATGCATGAGGCTC
TGCACAACCACTACACACAGAAGAGCCTCT CCCTGTCTCTGGGTAAA C40B179 heavy
chain GAGGTGCAGCTGGTGGAGAGCGGCGGCGGC 75 DNA
CTGGTGCAGCCCGGCGGCAGCCTGCGGCTG AGCTGCGCCGCCAGCGGCTTCACCTTCAGC
AGCTACGCCATGAGCTGGGTGCGGCAGGCC CCCGGCAAGGGCCTGGAGTGGGTGAGCACC
ATCAACCAGGCCGGCGGCGGCACCTACTAC GCCGACAGCGTGAAGGGCCGGTTCACCATC
AGCCGGGACAACAGCAAGAACACCCTGTAC CTGCAGATGAACAGCCTGCGGGCCGAGGAC
ACCGCCGTGTACTACTGCGCCAAGGAGGGC GGCAAGTACTACTACTACGCCCTGGACGTG
TGGGGCCAGGGCACCACCGTGACCGTGAGC AGCGCCTCCACCAAGGGCCCATCGGTCTTCC
CCCTGGCACCCTCCTCCAAGAGCACCTCTGG GGGCACAGCGGCCCTGGGCTGCCTGGTCAA
GGACTACTTCCCCGAACCGGTGACGGTGTC GTGGAACTCAGGCGCCCTGACCAGCGGCGT
GCACACCTTCCCGGCTGTCCTACAGTCCTCA GGACTCTACTCCCTCAGCAGCGTGGTGACC
GTGCCCTCCAGCAGCTTGGGCACCCAGACC TACATCTGCAACGTGAATCACAAGCCCAGC
AACACCAAGGTGGACAAGAAAGTTGAGCCC AAATCTTGTGACAAAACTCACACATGCCCA
CCGTGCCCAGCACCTGAAGCAGCAGGGGCA TCTTCAGTCTTCCTCTTCCCCCCAAAACCCA
AGGACACCCTCATGATCTCCCGGACCCCTG AGGTCACATGCGTGGTGGTGGACGTGAGCG
CCGAAGACCCTGAGGTCAAGTTCAACTGGT ACGTGGACGGCGTGGAGGTGCATAATGCCA
AGACAAAGCCGCGGGAGGAGCAGTACAAC AGCACGTACCGTGTGGTCAGCGTCCTCACC
GTCCTGCACCAGGACTGGCTGAATGGCAAG GAGTACAAGTGCAAGGTCTCCAACAAAGCC
CTCCCATCCTCCATCGAGAAAACCATCTCCA AAGCCAAAGGGCAGCCCCGAGAACCACAG
GTGTACACCCTGCCCCCATCCCGGGAGGAG ATGACCAAGAACCAGGTCAGCCTGACCTGC
CTGGTCAAAGGCTTCTATCCCAGCGACATCG CCGTGGAGTGGGAGAGCAATGGGCAGCCGG
AGAACAACTACAAGACCACGCCTCCCGTGC TGGACTCCGACGGCTCCTTCTTCCTCTACAG
CAAGCTCACCGTGGACAAGAGCAGGTGGCA GCAGGGGAACGTCTTCTCATGCTCCGTGATG
CATGAGGCTCTGCACAACCACTACACGCAG AAGAGCCTCTCCCTGTCTCCGGGTAAA C40B183
heavy chain GAGGTGCAGCTGGTGGAGAGCGGCGGCGGC 76 DNA
CTGGTGCAGCCCGGCGGCAGCCTGCGGCTG AGCTGCGCCGCCAGCGGCTTCACCTTCAGC
AGCTACGCCATGAGCTGGGTGCGGCAGGCC CCCGGCAAGGGCCTGGAGTGGGTGAGCACC
ATCAACCAGGCCGGCGGCGGCACCTACTAC GCCGACAGCGTGAAGGGCCGGTTCACCATC
AGCCGGGACAACAGCAAGAACACCCTGTAC CTGCAGATGAACAGCCTGCGGGCCGAGGAC
ACCGCCGTGTACTACTGCGCCAAGGAGGGC GGCAAGTACTACTACTACGCCCTGGACGTG
TGGGGCCAGGGCACCACCGTGACCGTGAGC AGCGCCTCCACCAAGGGCCCATCCGTCTTCC
CCCTGGCGCCCTGCTCCAGGAGCACCTCCG AGAGCACAGCCGCCCTGGGCTGCCTGGTCA
AGGACTACTTCCCCGAACCGGTGACGGTGT CGTGGAACTCAGGCGCCCTGACCAGCGGCG
TGCACACCTTCCCGGCTGTCCTACAGTCCTC AGGACTCTACTCCCTCAGCAGCGTGGTGAC
CGTGCCCTCCAGCAGCTTGGGCACGAAAAC CTACACCTGCAACGTAGATCACAAGCCCAG
CAACACCAAGGTGGACAAGAGAGTTGAGTC CAAATATGGTCCCCCATGCCCACCATGCCCA
GCACCTGAGGCCGCCGGGGGACCATCAGTC TTCCTGTTCCCCCCAAAACCCAAGGACACTC
TCATGATCTCCCGGACCCCTGAGGTCACGTG CGTGGTGGTGGACGTGAGCCAGGAAGACCC
CGAGGTCCAGTTCAACTGGTACGTGGATGG CGTGGAGGTGCATAATGCCAAGACAAAGCC
GCGGGAGGAGCAGTTCAACAGCACGTACCG TGTGGTCAGCGTCCTCACCGTCCTGCACCAG
GACTGGCTGAACGGCAAGGAGTACAAGTGC AAGGTCTCCAACAAAGGCCTCCCGTCCTCC
ATCGAGAAAACCATCTCCAAAGCCAAAGGG CAGCCCCGAGAGCCACAGGTGTACACCCTG
CCCCCATCCCAGGAGGAGATGACCAAGAAC CAGGTCAGCCTGACCTGCCTGGTCAAAGGC
TTCTACCCCAGCGACATCGCCGTGGAGTGG GAGAGCAATGGGCAGCCGGAGAACAACTAC
AAGACCACGCCTCCCGTGCTGGACTCCGAC GGCTCCTTCTTCCTCTACAGCAGGCTAACCG
TGGACAAGAGCAGGTGGCAGGAGGGGAAT GTCTTCTCATGCTCCGTGATGCATGAGGCTC
TGCACAACCACTACACACAGAAGAGCCTCT CCCTGTCTCTGGGTAAA C40B16 light chain
TCCTACGAGCTGACCCAGCCTCCCTCCGTGT 77 DNA
CTGTGTCTCCTGGCCAGACCGCCAGCATCAC CTGTAGCGGCGACAAGCTGGGCGATAAGTA
CGCCTGCTGGTATCAGCAGAAGCCCGGCCA GAGCCCCGTGCTGGTCATCTACCAGGACAG
CAGAAGGCCCAGCGGCATCCCCGAGAGATT CAGCGGCAGCAACAGCGGCAATACCGCCAC
CCTGACCATCAGCGGCACCCAGGCCATGGA CGAGGCCGATTACTATTGTCAGGCCTGGGC
CAGCAGCACCGTGGTGTTTGGCGGCGGAAC AAAGCTGACCGTGCTGGGTCAGCCCAAGGC
TGCACCCAGTGTCACTCTGTTCCCGCCCTCC TCTGAGGAGCTTCAAGCCAACAAGGCCACA
CTGGTGTGTCTCATAAGTGACTTCTACCCGG GAGCCGTGACAGTGGCCTGGAAGGCCGATA
GCAGCCCCGTCAAGGCGGGAGTGGAGACCA CCACACCCTCCAAACAAAGCAACAACAAGT
ACGCGGCCAGCAGCTATCTGAGCCTGACGC CTGAGCAGTGGAAGTCCCACAGAAGCTACA
GCTGCCAGGTCACGCATGAAGGGAGCACCG TGGAGAAGACAGTGGCCCCTACAGAATGTT CA
C40B124, C40B135, AGCTACGAGCTGACCCAGCCCCCCAGCGTG 78 C40B125,
C40B136, AGCGTGAGCCCCGGCCAGACCGCCAGCATC C40B127, C40B138,
ACCTGCAGCGGCGACAAGCTGGGCGACAAG C40B131, C40B176,
TACGCCGCCTGGTACCAGCAGAAGCCCGGC C40B180. C40B179,
CAGAGCCCCGTGCTGGTGATCTACCAGGAC C40B183
AGCCGGCGGCCCAGCGGCATCCCCGAGCGG Light chain DNA
TTCAGCGGCAGCAACAGCGGCAACACCGCC ACCCTGACCATCAGCGGCACCCAGGCCATG
GACGAGGCCGACTACTACTGCCAGGCCTGG GCCAGCAGCACCGTGGTGTTCGGCGGCGGC
ACCAAGCTGACCGTGCTGGGTCAGCCCAAG GCTGCACCCAGTGTCACTCTGTTCCCGCCCT
CCTCTGAGGAGCTTCAAGCCAACAAGGCCA CACTGGTGTGTCTCATAAGTGACTTCTACCC
GGGAGCCGTGACAGTGGCCTGGAAGGCCGA TAGCAGCCCCGTCAAGGCGGGAGTGGAGAC
CACCACACCCTCCAAACAAAGCAACAACAA GTACGCGGCCAGCAGCTATCTGAGCCTGAC
GCCTGAGCAGTGGAAGTCCCACAGAAGCTA CAGCTGCCAGGTCACGCATGAAGGGAGCAC
CGTGGAGAAGACAGTGGCCCCTACAGAATG TTCA
Example 3. Characterization of Antagonistic Anti-CD40
Antibodies
[0527] The parental C40B16 and the engineered variants were tested
for their antagonistic activity in a spectrum of assays including
ability of the antibodies to inhibit human soluble CD40L (sCD40L)
or membrane-bound CD40L (mCD40L)-mediated proliferation of human or
cyno B cells, and inhibition of IL-12p40 production by human DCs.
The experiments were conducted according to protocols described in
Example 1. Membrane-bound CD40L was provided on Jurkat cells in the
assays.
[0528] All antibodies demonstrated antagonism in the assays
performed.
[0529] Table 10 shows the IC.sub.50 values for inhibition of
soluble or membrane-bound CD40L-driven B cell proliferation. Table
11 shows the IC.sub.50 values for inhibition of soluble or
membrane-bound CD40L-driven IL-12p40 production by human dendritic
cells.
TABLE-US-00011 TABLE 10 B-cell proliferation IC.sub.50 nM +/- SD
sCD40L-driven mCD40L-driven sCD40L-driven human B-cell human B-cell
cyno B-cell mAb proliferation proliferation proliferation C40B16
C40B124 0.118 +/- 0.003 (4) C40B135 0.056 +/- 0.040 (3) 0.131 +/-
0.012 (4) 0.077 +/- 0.024 (2) C40B125 0.088 +/- 0.025 (6) 0.128 +/-
0.019 (6) 0.083 +/- 0.029 (2) C40B136 0.087 +/- 0.056 (6) 0.122 +/-
0.013 (4) 0.065 +/- 0.038 (2) C40B127 0.073 +/- 0.010 (2) 0.111 +/-
0.009 (4) 0.066 +/- 0.002 (2) C40B138 0.048 +/- 0.035 (2) 0.106 +/-
0.000 (2) 0.075 +/- 0.050 (2) C40B131 0.099 +/- 0.003 (4) C40B176
0.132 +/- 0.105 (4) 0.181 +/- 0.054 (4) 0.108 +/- 0.029 (4) C40B180
0.092 +/- 0.047 (4) 0.136 +/- 0.071 (4) 0.109 +/- 0.047 (4) C40B179
0.132 +/- 0.030 (4) 0.178 +/- 0.053 (4) 0.119 +/- 0.026 (4) C40B183
0.103 +/- 0.053 (4) 0.140 +/- 0.018 (4) 0.110 +/- 0.029 (4) sCD40L:
soluble CD40L mCD40L: membrane-bound CD40L The number in
parenthesis indicates the number of times the assay was
repeated
TABLE-US-00012 TABLE 11 DC IL-12p40 productionIC.sub.50 nM +/- SD
mAb sCD40L-driven mCD40L-driven C40B16 C40B124 0.387 +/- 0.09 (3)
0.507 +/- 0.07 (5) C40B135 0.329 +/- 0.02 (3) 0.430 +/- 0.10 (5)
C40B125 0.244 +/- 0.04 (5) 0.393 +/- 0.09 (5) C40B136 0.246 +/-
0.05 (5) 0.379 +/- 0.08 (5) C40B127 0.270 +/- 0.07 (3) 0.383 +/-
0.08 (5) C40B138 0.262 +/- 0.06 (3) 0.341 +/- 0.10 (5) C40B131
0.330 +/- 0.18 (3) 0.323 +/- 0.12 (5) C40B176 0.220 +/- 0.11 (3)
0.245 +/- 0.06 (2) C40B180 0.225 +/- 0.06 (3) 0.222 +/- 0.07 (2)
C40B179 0.184 +/- 0.07 (3) 0.297 +/- 0.06 (2) C40B183 0.208 +/-
0.09 (3) 0.291 +/- 0.07 (2) sCD40L: soluble CD40L mCD40L:
membrane-bound CD40L The number in parenthesis indicates the number
of times the assay was repeated
[0530] Affinity of the antibodies to human CD40 was assessed using
Surface Plasmon Resonance (SPR) using a ProteOn XPR36 system
(BioRad). A biosensor surface was prepared by coupling anti-Human
IgG Fc (Jackson cat#109-005-098) to the modified alginate polymer
layer surface of a GLC chip (BioRad, Cat#176-5011) using the
manufacturer instructions for amine-coupling chemistry.
Approximately 5000 RU (response units) of mAbs were immobilized.
The kinetic experiments were performed at 25.degree. C. in running
buffer (DPBS+0.01% P20+100 .mu.g/ml BSA). To perform kinetic
experiments, 200 RU of mAbs were captured followed by injections of
analytes (human or cyno CD40) at 5 concentrations (in a 4-fold
serial dilution). The association phase was monitored for 3 minutes
at 50 .mu.L/min, then followed by 15 minutes of buffer flow
(dissociation phase). The chip surface was regenerated with two 18
second pulses of 100 mM H.sub.3PO.sub.4 (Sigma, Cat#7961) at 100
.mu.L/min.
[0531] The collected data were processed using ProteOn Manager
software. First, the data was corrected for background using
inter-spots. Then, double reference subtraction of the data was
performed by using the buffer injection for analyte injections. The
kinetic analysis of the data was performed using a Langmuir 1:1
binding model. The results were reported in the format of Ka
(On-rate), Kd (Off-rate) and K.sub.D (equilibrium dissociation
constant).
[0532] Summary of kinetics affinity for the mAbs for binding to
human CD40 is shown in Table 12. The parameters reported in this
table were obtained from a 1:1 Langmuir binding model for all
samples. Affinity, K.sub.D=kd/ka.
TABLE-US-00013 TABLE 12 mAb ka (1/Ms) kd (1/s) K.sub.D (M) C40B16
4.27E+06 1.57E-04 3.68E-11 C40B124 2.30E+06 9.56E-05 4.16E-11
C40B135 2.93E+06 1.45E-04 4.96E-11 C40B125 3.89E+06 1.36E-04
3.49E-11 C40B136 3.30E+06 1.13E-04 3.43E-11 C40B127 3.79E+06
1.15E-04 3.03E-11 C40B138 3.58E+06 2.15E-04 5.99E-11 C40B131
3.52E+06 2.86E-04 8.12E-11 C40B176 2.86E+06 3.07E-04 1.07E-10
C40B180 2.67E+06 3.66E-04 1.37E-10 C40B179 2.86E+06 2.67E-04
9.33E-11 C40B183 2.91E+06 3.50E-04 1.21E-10
Example 4. Antagonistic Antibodies Specifically Binding Human CD40
are Potent Inhibitors of CD40/CD40L Pathway and Display Minimal
Agonistic Activity
[0533] Four antagonistic antibodies (C40B176, C40B179, C40B180 and
C40B183) were benchmarked against anti-CD40 antibodies currently in
clinical developments. The antibodies used in comparisons were:
Astellas/Kirin ASKP-1240 (HC SEQ ID NO: 53, LC SEQ ID NO: 54, Fc
silent IgG4 with S228P and L235E mutations), Antibody D (HC SEQ ID
NO: 55, LC SEQ ID NO: 56, Fc silent IgG1 with L234A and L235A
mutations; described in U.S. Pat. No. 8,591,900 as antibody B
IgG1KOb), Novartis CFZ533 (HC SEQ ID NO: 57, LC SEQ ID NO: 58, Fc
silent IgG1 with N297A mutation), and BMS-986090 (HC SEQ ID NO: 59,
IgG4 with S228P mutation).
TABLE-US-00014 (ASKP-1240 HC) SEQ ID NO: 53
QLQLQESGPGLLKPSETLSLTCTVSGGSISSPGYYGGWIRQPPGKGLEWI
GSIYKSGSTYHNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCTRP
VVRYFGWFDPWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLV
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTK
TYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKD
TLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNST
YRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVY
TLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (ASKP-1240 LC) SEQ
ID NO: 54 AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYD
ASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPTFGQG
TKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD
NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC
(Antibody D HC) SEQ ID NO: 55
EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYGMHWVRQAPGKGLEWVAY
ISSGNRIIYYADTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQD
GYRYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD
YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY
ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS
TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Antibody D LC)
SEQ ID NO: 56 DIVMTQSPDSLAVSLGEKVTINCKSSQSLLNSGNQKNYLTWHQQKPGQPP
KLLIYWTSTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYTY
PLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA
KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC
EVTHQGLSSPVTKSFNRGEC (CFZ533 HC) SEQ ID NO: 57
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAV
ISYEESNRYHADSVKGRFTISRDNSKITLYLQMNSLRTEDTAVYYCARDG
GIAAPGPDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK
DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT
YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP
KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYA
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ
VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (CFZ533 LC) SEQ
ID NO: 58 DIVMTQSPLSLTVTPGEPASISCRSSQSLLYSNGYNYLDWYLQKPGQSPQ
VLISLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQARQTP
FTFGPGTKVDIRRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK
VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE
VTHQGLSSPVTKSFNRGEC (BMS-986090 HC) SEQ ID NO: 59
EVQLLESGGGLVQPGGSLRLSCAASGFTFRDYEMWWVRQAPGKGLERVSA
INPQGTRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKLP
FRFSDRGQGTLVTVSSASTESKYGPPCPPCPAPEFLGGPSVFLFPPKPKD
TLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNST
YRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVY
TLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
[0534] Table 13 shows the IC.sub.50 values for inhibition of
soluble or membrane-bound CD40L-driven human or cyno B cell
proliferation for the tested antibodies. Table 14 shows the
IC.sub.50 values for inhibition of soluble or membrane-bound
CD40L-driven IL-12p40 production by human dendritic cells (DC) and
the affinity kinetics for each mAb. mAbs C40B176, C40B179, C40B180
and C40B183 exhibited comparable potency when compared to the best
performing benchmark mAb ASKP-1240.
TABLE-US-00015 TABLE 13 B cell proliferation; IC.sub.50 nM +/- SD
Soluble Membrane Soluble mAb CD40L human B-cells CD40L human
B-cells CD40L cyno B-cells C40B176 0.132 +/- 0.105 (4) 0.181 +/-
0.054 (4) 0.108 +/- 0.029 (4) C40B180 0.092 +/- 0.047 (4) 0.136 +/-
0.071 (4) 0.109 +/- 0.047 (4) C40B179 0.132 +/- 0.030 (4) 0.178 +/-
0.053 (4) 0.119 +/- 0.026 (4) C40B183 0.103 +/- 0.053 (4) 0.140 +/-
0.018 (4) 0.110 +/- 0.029 (4) ASKP-1240 0.079 +/- 0.017 (12) 0.132
+/- 0.042 (10) 0.146 +/- 0.063 (8) Antibody D 0.680 +/- 0.323 (4)
1.926 +/- 0.368 (2) CFZ533 0.223 +/- 0.117 (4) 0.279 +/- 0.062 (2)
BMS-986090 0.288 +/- 0.136 (4) 0.393 +/- 0.002 (2)
TABLE-US-00016 TABLE 14 Human DC IL-12p40 production; IC.sub.50 nM
+/- SD mAb Affinity K.sub.D (M) Soluble CD40L Membrane CD40L
C40B176 4.96E-11 0.220 +/- 0.11 (3) 0.245 +/- 0.06 (2) C40B180
5.99E-11 0.225 +/- 0.06 (3) 0.222 +/- 0.07 (2) C40B179 3.43E-11
0.184 +/- 0.07 (3) 0.297 +/- 0.06 (2) C40B183 1.07E-10 0.208 +/-
0.09 (3) 0.291 +/- 0.07 (2) ASKP-1240 1.81E-10 0.119 +/- 0.05 (6)
0.280 +/- 0.11 (7) Antibody D 5.8E-10 0.781 +/- 0.30 (3) 3.630 +/-
1.19 (5) CFZ533 9.36E-12 0.320 +/- 0.12 (3) 0.948 +/- 0.29 (5)
BMS-986090 8.7E-10 0.285 +/- 0.17 (2) 0.822 +/- 0.39 (5)
[0535] Possible undesired agonistic activity of the mAbs was
assessed using HEK-Blue.TM. CD40L NF-.kappa.B activation assay and
IL12p40 production by dendritic cells as readouts without
cross-linking using experimental protocols as described in Example
1. In addition to effector silent ASKP-1240, CFZ533, BMS-986090 and
Antibody D, the parental antibody C40B16 on wild-type IgG1 was
evaluated.
[0536] C40B16 as wild-type IgG1 and Antibody D on silent Fc
exhibited comparable non-existing or minimal agonism in the assay.
ASKP-1240, CFZ533 and BMS-986090 exhibited some agonism as
anti-CD40 antibody concentration increased. FIG. 1 shows the dose
response curves of the NF-.kappa.B reporter assay. FIG. 2 shows the
antibody-mediated IL12p40 production by DCs using antibody
concentrations between 350 nM and 1.1 nM.
[0537] The results suggest that ASKP-1240, CFZ533 and BMS-986090
may exhibit V-region driven agonism as the agonism was demonstrated
in effector silent Fc.
[0538] Agonism was also assessed at 500 ng/ml concentration of
antibodies in DC or B-cell agonistic assays, comparing ASKP-1240
and C40B176, C40B179, C40B180 and C40B183. At higher antibody
concentrations, ASKP-1240 induced IL-12p40 production by DCs (FIG.
3A) and proliferation of B cells (FIG. 3B), whereas C40B176,
C40B179, C40B180 and C40B183 did not.
[0539] Overall, the results suggest that C40B16 and its variants
C40B176, C40B179, C40B180 and C40B183, as well as Antibody D have
reduced agonist potential compared to ASKP-1240, CFZ533 and
BMS-986090. Notably, the results also suggest that the potency of
C40B176, C40B179, C40B180 and C40B183 in cell based assays is as
much as 10-fold better than the Antibody D molecule, and comparable
to the most potent benchmark molecule (ASKP-1240).
Sequence CWU 1
1
781277PRTHomo sapiens 1Met Val Arg Leu Pro Leu Gln Cys Val Leu Trp
Gly Cys Leu Leu Thr 1 5 10 15 Ala Val His Pro Glu Pro Pro Thr Ala
Cys Arg Glu Lys Gln Tyr Leu 20 25 30 Ile Asn Ser Gln Cys Cys Ser
Leu Cys Gln Pro Gly Gln Lys Leu Val 35 40 45 Ser Asp Cys Thr Glu
Phe Thr Glu Thr Glu Cys Leu Pro Cys Gly Glu 50 55 60 Ser Glu Phe
Leu Asp Thr Trp Asn Arg Glu Thr His Cys His Gln His 65 70 75 80 Lys
Tyr Cys Asp Pro Asn Leu Gly Leu Arg Val Gln Gln Lys Gly Thr 85 90
95 Ser Glu Thr Asp Thr Ile Cys Thr Cys Glu Glu Gly Trp His Cys Thr
100 105 110 Ser Glu Ala Cys Glu Ser Cys Val Leu His Arg Ser Cys Ser
Pro Gly 115 120 125 Phe Gly Val Lys Gln Ile Ala Thr Gly Val Ser Asp
Thr Ile Cys Glu 130 135 140 Pro Cys Pro Val Gly Phe Phe Ser Asn Val
Ser Ser Ala Phe Glu Lys 145 150 155 160 Cys His Pro Trp Thr Ser Cys
Glu Thr Lys Asp Leu Val Val Gln Gln 165 170 175 Ala Gly Thr Asn Lys
Thr Asp Val Val Cys Gly Pro Gln Asp Arg Leu 180 185 190 Arg Ala Leu
Val Val Ile Pro Ile Ile Phe Gly Ile Leu Phe Ala Ile 195 200 205 Leu
Leu Val Leu Val Phe Ile Lys Lys Val Ala Lys Lys Pro Thr Asn 210 215
220 Lys Ala Pro His Pro Lys Gln Glu Pro Gln Glu Ile Asn Phe Pro Asp
225 230 235 240 Asp Leu Pro Gly Ser Asn Thr Ala Ala Pro Val Gln Glu
Thr Leu His 245 250 255 Gly Cys Gln Pro Val Thr Gln Glu Asp Gly Lys
Glu Ser Arg Ile Ser 260 265 270 Val Gln Glu Arg Gln 275 2173PRTHomo
sapiens 2Glu Pro Pro Thr Ala Cys Arg Glu Lys Gln Tyr Leu Ile Asn
Ser Gln 1 5 10 15 Cys Cys Ser Leu Cys Gln Pro Gly Gln Lys Leu Val
Ser Asp Cys Thr 20 25 30 Glu Phe Thr Glu Thr Glu Cys Leu Pro Cys
Gly Glu Ser Glu Phe Leu 35 40 45 Asp Thr Trp Asn Arg Glu Thr His
Cys His Gln His Lys Tyr Cys Asp 50 55 60 Pro Asn Leu Gly Leu Arg
Val Gln Gln Lys Gly Thr Ser Glu Thr Asp 65 70 75 80 Thr Ile Cys Thr
Cys Glu Glu Gly Trp His Cys Thr Ser Glu Ala Cys 85 90 95 Glu Ser
Cys Val Leu His Arg Ser Cys Ser Pro Gly Phe Gly Val Lys 100 105 110
Gln Ile Ala Thr Gly Val Ser Asp Thr Ile Cys Glu Pro Cys Pro Val 115
120 125 Gly Phe Phe Ser Asn Val Ser Ser Ala Phe Glu Lys Cys His Pro
Trp 130 135 140 Thr Ser Cys Glu Thr Lys Asp Leu Val Val Gln Gln Ala
Gly Thr Asn 145 150 155 160 Lys Thr Asp Val Val Cys Gly Pro Gln Asp
Arg Leu Arg 165 170 3278PRTMacaca fascicularis 3Met Val Arg Leu Pro
Leu Gln Cys Val Leu Trp Gly Cys Leu Leu Thr 1 5 10 15 Ala Val Tyr
Pro Glu Pro Pro Thr Ala Cys Arg Glu Lys Gln Tyr Leu 20 25 30 Ile
Asn Ser Gln Cys Cys Ser Leu Cys Gln Pro Gly Gln Lys Leu Val 35 40
45 Ser Asp Cys Thr Glu Phe Thr Glu Thr Glu Cys Leu Pro Cys Ser Glu
50 55 60 Ser Glu Phe Leu Asp Thr Trp Asn Arg Glu Thr Arg Cys His
Gln His 65 70 75 80 Lys Tyr Cys Asp Pro Asn Leu Gly Leu Arg Val Gln
Gln Lys Gly Thr 85 90 95 Ser Glu Thr Asp Thr Ile Cys Thr Cys Glu
Glu Gly Leu His Cys Met 100 105 110 Ser Glu Ser Cys Glu Ser Cys Val
Pro His Arg Ser Cys Leu Pro Gly 115 120 125 Phe Gly Val Lys Gln Ile
Ala Thr Gly Val Ser Asp Thr Ile Cys Glu 130 135 140 Pro Cys Pro Val
Gly Phe Phe Ser Asn Val Ser Ser Ala Phe Glu Lys 145 150 155 160 Cys
Arg Pro Trp Thr Ser Cys Glu Thr Lys Asp Leu Val Val Gln Gln 165 170
175 Ala Gly Thr Asn Lys Thr Asp Val Val Cys Gly Pro Gln Asp Arg Gln
180 185 190 Arg Ala Leu Val Val Ile Pro Ile Cys Leu Gly Ile Leu Phe
Val Ile 195 200 205 Leu Leu Leu Val Leu Val Phe Ile Lys Lys Val Ala
Lys Lys Pro Asn 210 215 220 Asp Lys Ala Pro His Pro Lys Gln Glu Pro
Gln Glu Ile Asn Phe Leu 225 230 235 240 Asp Asp Leu Pro Gly Ser Asn
Pro Ala Ala Pro Val Gln Glu Thr Leu 245 250 255 His Gly Cys Gln Pro
Val Thr Gln Glu Asp Gly Lys Glu Ser Arg Ile 260 265 270 Ser Val Gln
Glu Arg Gln 275 4173PRTMacaca fascicularis 4Glu Pro Pro Thr Ala Cys
Arg Glu Lys Gln Tyr Leu Ile Asn Ser Gln 1 5 10 15 Cys Cys Ser Leu
Cys Gln Pro Gly Gln Lys Leu Val Ser Asp Cys Thr 20 25 30 Glu Phe
Thr Glu Thr Glu Cys Leu Pro Cys Ser Glu Ser Glu Phe Leu 35 40 45
Asp Thr Trp Asn Arg Glu Thr Arg Cys His Gln His Lys Tyr Cys Asp 50
55 60 Pro Asn Leu Gly Leu Arg Val Gln Gln Lys Gly Thr Ser Glu Thr
Asp 65 70 75 80 Thr Ile Cys Thr Cys Glu Glu Gly Leu His Cys Met Ser
Glu Ser Cys 85 90 95 Glu Ser Cys Val Pro His Arg Ser Cys Leu Pro
Gly Phe Gly Val Lys 100 105 110 Gln Ile Ala Thr Gly Val Ser Asp Thr
Ile Cys Glu Pro Cys Pro Val 115 120 125 Gly Phe Phe Ser Asn Val Ser
Ser Ala Phe Glu Lys Cys Arg Pro Trp 130 135 140 Thr Ser Cys Glu Thr
Lys Asp Leu Val Val Gln Gln Ala Gly Thr Asn 145 150 155 160 Lys Thr
Asp Val Val Cys Gly Pro Gln Asp Arg Gln Arg 165 170 55PRTArtificial
SequenceHCDR1 sequence 5Ser Tyr Ala Met Ser 1 5 617PRTArtificial
SequenceHCDR2 sequence 6Thr Ile Asn Asn Ser Gly Gly Gly Thr Tyr Tyr
Ala Asp Ser Val Lys 1 5 10 15 Gly 712PRTArtificial SequenceHCDR3
sequence 7Glu Gly Gly Lys Tyr Tyr Tyr Tyr Ala Met Asp Val 1 5 10
811PRTArtificial SequenceLCDR1 sequence 8Ser Gly Asp Lys Leu Gly
Asp Lys Tyr Ala Cys 1 5 10 97PRTArtificial SequenceLCDR2 sequence
9Gln Asp Ser Arg Arg Pro Ser 1 5 109PRTArtificial SequenceLCDR3
sequence 10Gln Ala Trp Ala Ser Ser Thr Val Val 1 5
11121PRTArtificial SequenceC40B16 VH 11Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser
Trp Val Arg Gln Ala Pro Gly Arg Gly Leu Glu Trp Val 35 40 45 Ser
Thr Ile Asn Asn Ser Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu His Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Lys Glu Gly Gly Lys Tyr Tyr Tyr Tyr Ala Met
Asp Val Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser 115
120 12106PRTArtificial SequenceC40B16 VL 12Ser Tyr Glu Leu Thr Gln
Pro Pro Ser Val Ser Val Ser Pro Gly Gln 1 5 10 15 Thr Ala Ser Ile
Thr Cys Ser Gly Asp Lys Leu Gly Asp Lys Tyr Ala 20 25 30 Cys Trp
Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr 35 40 45
Gln Asp Ser Arg Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50
55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala
Met 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Ala Trp Ala Ser Ser
Thr Val Val 85 90 95 Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100
105 13363DNAArtificial SequenceC40B16 VH DNA 13gaggtgcagc
tggtggaatc tggcggagga ctggtgcagc ctggcggcag cctgagactg 60tcttgtgccg
ccagcggctt caccttcagc agctacgcta tgagctgggt ccgacaggcc
120cctggcagag gactcgagtg ggtgtccacc atcaacaaca gcggcggagg
cacctactac 180gccgacagcg tgaagggcag attcaccatc agccgggaca
acagcaagaa caccctgtac 240ctgcacatga acagcctgcg ggccgaggac
accgccgtgt actattgtgc caaagagggc 300ggcaagtact actactatgc
catggacgtg tggggccagg gcaccaccgt gacagtgtca 360tct
36314318DNAArtificial SequenceC40B16 VL DNA 14tcctacgagc tgacccagcc
tccctccgtg tctgtgtctc ctggccagac cgccagcatc 60acctgtagcg gcgacaagct
gggcgataag tacgcctgct ggtatcagca gaagcccggc 120cagagccccg
tgctggtcat ctaccaggac agcagaaggc ccagcggcat ccccgagaga
180ttcagcggca gcaacagcgg caataccgcc accctgacca tcagcggcac
ccaggccatg 240gacgaggccg attactattg tcaggcctgg gccagcagca
ccgtggtgtt tggcggcgga 300acaaagctga ccgtgctg 3181517PRTArtificial
SequenceHCDR2 sequence 15Thr Ile Asp Asn Ala Gly Gly Gly Thr Tyr
Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly 1617PRTArtificial
SequenceHCDR2 sequence 16Thr Ile Gln Asn Ala Gly Gly Gly Thr Tyr
Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly 1717PRTArtificial
SequenceHCDR2 sequence 17Thr Ile Asn Gln Ala Gly Gly Gly Thr Tyr
Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly 1817PRTArtificial
SequenceHCDR2 sequence 18Thr Ile Asn Ala Ala Gly Gly Gly Thr Tyr
Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly 1912PRTArtificial
SequenceHCDR3 sequence 19Glu Gly Gly Lys Tyr Tyr Tyr Tyr Ala Leu
Asp Val 1 5 10 2011PRTArtificial SequenceLCDR1 sequence 20Ser Gly
Asp Lys Leu Gly Asp Lys Tyr Ala Ala 1 5 10 21121PRTArtificial
SequenceC40H60 protein 21Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Thr Ile Asp
Asn Ala Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Lys Glu Gly Gly Lys Tyr Tyr Tyr Tyr Ala Met Asp Val Trp
Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser 115 120
22121PRTArtificial SequenceC40H61 protein 22Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met
Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Thr Ile Gln Asn Ala Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Lys Glu Gly Gly Lys Tyr Tyr Tyr Tyr Ala
Met Asp Val Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser
115 120 23121PRTArtificial SequenceC40H63 protein 23Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ser Thr Ile Asn Gln Ala Gly Gly Gly Thr Tyr Tyr Ala Asp Ser
Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Glu Gly Gly Lys Tyr Tyr Tyr
Tyr Ala Met Asp Val Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val
Ser Ser 115 120 24121PRTArtificial SequenceC40H56 protein 24Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20
25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Ser Thr Ile Asn Ala Ala Gly Gly Gly Thr Tyr Tyr Ala
Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser
Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Glu Gly Gly Lys Tyr
Tyr Tyr Tyr Ala Met Asp Val Trp Gly 100 105 110 Gln Gly Thr Thr Val
Thr Val Ser Ser 115 120 25121PRTArtificial SequenceC40H83 protein
25Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ser Thr Ile Asn Ala Ala Gly Gly Gly Thr Tyr
Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Glu Gly Gly
Lys Tyr Tyr Tyr Tyr Ala Leu Asp Val Trp Gly 100 105 110 Gln Gly Thr
Thr Val Thr Val Ser Ser 115 120 26121PRTArtificial SequenceC40H86
protein 26Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ser Thr Ile Asn Gln Ala Gly Gly
Gly Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys
Glu Gly Gly Lys Tyr Tyr Tyr Tyr Ala Leu Asp Val Trp Gly 100 105 110
Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 27106PRTArtificial
SequenceC40L71 protein 27Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val
Ser Val Ser Pro Gly Gln 1 5 10 15 Thr Ala Ser Ile Thr Cys Ser Gly
Asp Lys Leu
Gly Asp Lys Tyr Ala 20 25 30 Ala Trp Tyr Gln Gln Lys Pro Gly Gln
Ser Pro Val Leu Val Ile Tyr 35 40 45 Gln Asp Ser Arg Arg Pro Ser
Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr
Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met 65 70 75 80 Asp Glu Ala
Asp Tyr Tyr Cys Gln Ala Trp Ala Ser Ser Thr Val Val 85 90 95 Phe
Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 28363DNAArtificial
SequenceC40H60 DNA 28gaggtgcagc tggtggagag cggcggcggc ctggtgcagc
ccggcggcag cctgcggctg 60agctgcgccg ccagcggctt caccttcagc agctacgcca
tgagctgggt gcggcaggcc 120cccggcaagg gcctggagtg ggtgagcacc
atcgacaacg ccggcggcgg cacctactac 180gccgacagcg tgaagggccg
gttcaccatc agccgggaca acagcaagaa caccctgtac 240ctgcagatga
acagcctgcg ggccgaggac accgccgtgt actactgcgc caaggagggc
300ggcaagtact actactacgc catggacgtg tggggccagg gcaccaccgt
gaccgtgagc 360agc 36329363DNAArtificial SequenceC40H61 DNA
29gaggtgcagc tggtggagag cggcggcggc ctggtgcagc ccggcggcag cctgcggctg
60agctgcgccg ccagcggctt caccttcagc agctacgcca tgagctgggt gcggcaggcc
120cccggcaagg gcctggagtg ggtgagcacc atccagaacg ccggcggcgg
cacctactac 180gccgacagcg tgaagggccg gttcaccatc agccgggaca
acagcaagaa caccctgtac 240ctgcagatga acagcctgcg ggccgaggac
accgccgtgt actactgcgc caaggagggc 300ggcaagtact actactacgc
catggacgtg tggggccagg gcaccaccgt gaccgtgagc 360agc
36330363DNAArtificial SequenceC40H63 DNA 30gaggtgcagc tggtggagag
cggcggcggc ctggtgcagc ccggcggcag cctgcggctg 60agctgcgccg ccagcggctt
caccttcagc agctacgcca tgagctgggt gcggcaggcc 120cccggcaagg
gcctggagtg ggtgagcacc atcaaccagg ccggcggcgg cacctactac
180gccgacagcg tgaagggccg gttcaccatc agccgggaca acagcaagaa
caccctgtac 240ctgcagatga acagcctgcg ggccgaggac accgccgtgt
actactgcgc caaggagggc 300ggcaagtact actactacgc catggacgtg
tggggccagg gcaccaccgt gaccgtgagc 360agc 36331363DNAArtificial
SequenceC40H56 DNA 31gaggtgcagc tggtggagag cggcggcggc ctggtgcagc
ccggcggcag cctgcggctg 60agctgcgccg ccagcggctt caccttcagc agctacgcca
tgagctgggt gcggcaggcc 120cccggcaagg gcctggagtg ggtgagcacc
atcaacgccg ccggcggcgg cacctactac 180gccgacagcg tgaagggccg
gttcaccatc agccgggaca acagcaagaa caccctgtac 240ctgcagatga
acagcctgcg ggccgaggac accgccgtgt actactgcgc caaggagggc
300ggcaagtact actactacgc catggacgtg tggggccagg gcaccaccgt
gaccgtgagc 360agc 36332363DNAArtificial SequenceC40H83 DNA
32gaggtgcagc tggtggagag cggcggcggc ctggtgcagc ccggcggcag cctgcggctg
60agctgcgccg ccagcggctt caccttcagc agctacgcca tgagctgggt gcggcaggcc
120cccggcaagg gcctggagtg ggtgagcacc atcaacgccg ccggcggcgg
cacctactac 180gccgacagcg tgaagggccg gttcaccatc agccgggaca
acagcaagaa caccctgtac 240ctgcagatga acagcctgcg ggccgaggac
accgccgtgt actactgcgc caaggagggc 300ggcaagtact actactacgc
cctggacgtg tggggccagg gcaccaccgt gaccgtgagc 360agc
36333363DNAArtificial SequenceC40H86 DNA 33gaggtgcagc tggtggagag
cggcggcggc ctggtgcagc ccggcggcag cctgcggctg 60agctgcgccg ccagcggctt
caccttcagc agctacgcca tgagctgggt gcggcaggcc 120cccggcaagg
gcctggagtg ggtgagcacc atcaaccagg ccggcggcgg cacctactac
180gccgacagcg tgaagggccg gttcaccatc agccgggaca acagcaagaa
caccctgtac 240ctgcagatga acagcctgcg ggccgaggac accgccgtgt
actactgcgc caaggagggc 300ggcaagtact actactacgc cctggacgtg
tggggccagg gcaccaccgt gaccgtgagc 360agc 36334318DNAArtificial
SequenceC40L71 DNA 34agctacgagc tgacccagcc ccccagcgtg agcgtgagcc
ccggccagac cgccagcatc 60acctgcagcg gcgacaagct gggcgacaag tacgccgcct
ggtaccagca gaagcccggc 120cagagccccg tgctggtgat ctaccaggac
agccggcggc ccagcggcat ccccgagcgg 180ttcagcggca gcaacagcgg
caacaccgcc accctgacca tcagcggcac ccaggccatg 240gacgaggccg
actactactg ccaggcctgg gccagcagca ccgtggtgtt cggcggcggc
300accaagctga ccgtgctg 31835451PRTArtificial SequenceC40B16 heavy
chain protein 35Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro
Gly Arg Gly Leu Glu Trp Val 35 40 45 Ser Thr Ile Asn Asn Ser Gly
Gly Gly Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu His Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala
Lys Glu Gly Gly Lys Tyr Tyr Tyr Tyr Ala Met Asp Val Trp Gly 100 105
110 Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230
235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350
Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 355
360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450
36451PRTArtificial SequenceC40B124 heavy chain protein 36Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20
25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Ser Thr Ile Asp Asn Ala Gly Gly Gly Thr Tyr Tyr Ala
Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser
Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Glu Gly Gly Lys Tyr
Tyr Tyr Tyr Ala Met Asp Val Trp Gly 100 105 110 Gln Gly Thr Thr Val
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150
155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys
Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Ala Ala Gly 225 230 235 240 Ala Ser Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Ala 260 265 270
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275
280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ser Ser Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser
Arg Glu Glu Met Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395
400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450 37448PRTArtificial
SequenceC40B135 heavy chain protein 37Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Thr Ile Asp Asn Ala Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Lys Glu Gly Gly Lys Tyr Tyr Tyr Tyr Ala Met
Asp Val Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Cys Ser
Arg Ser Thr Ser Glu Ser Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185
190 Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His
195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys
Tyr Gly 210 215 220 Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala
Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser Gln Glu Asp Pro 260 265 270 Glu Val Gln Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys
Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310
315 320 Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys
Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu 340 345 350 Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp
Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435
440 445 38451PRTArtificial SequenceC40B125 heavy chain protein
38Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ser Thr Ile Gln Asn Ala Gly Gly Gly Thr Tyr
Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Glu Gly Gly
Lys Tyr Tyr Tyr Tyr Ala Met Asp Val Trp Gly 100 105 110 Gln Gly Thr
Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135
140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr
Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val
Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr
Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 225 230 235 240 Ala Ser
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Ala 260
265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ser Ser Ile 325 330 335 Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 355 360 365
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370
375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450
39448PRTArtificial SequenceC40B136 heavy chain protein 39Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20
25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Ser Thr Ile Gln Asn Ala Gly Gly Gly Thr Tyr Tyr Ala
Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser
Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Glu Gly Gly Lys Tyr
Tyr Tyr Tyr Ala Met Asp Val Trp Gly 100 105 110 Gln Gly Thr Thr Val
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro
Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala 130 135 140 Ala
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150
155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr
Cys Asn Val Asp His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys
Arg Val Glu Ser Lys Tyr Gly 210 215 220 Pro Pro Cys Pro Pro Cys Pro
Ala Pro Glu Ala Ala Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro 260 265 270
Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275
280 285 Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val
Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
Ser Ser Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Gln Glu Glu
Met Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395
400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser
405 410 415 Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His
Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Leu Gly Lys 435 440 445 40451PRTArtificial SequenceC40B127
heavy chain protein 40Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Thr Ile Asn Gln
Ala Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Lys Glu Gly Gly Lys Tyr Tyr Tyr Tyr Ala Met Asp Val Trp Gly
100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly
Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215
220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly
225 230 235 240 Ala Ser Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Ser Ala 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ser Ser Ile 325 330 335
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340
345 350 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val
Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly
Lys 450 41448PRTArtificial SequenceC40B138 heavy chain protein
41Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ser Thr Ile Asn Gln Ala Gly Gly Gly Thr Tyr
Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Glu Gly Gly
Lys Tyr Tyr Tyr Tyr Ala Met Asp Val Trp Gly 100 105 110 Gln Gly Thr
Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val
Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala 130 135
140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Lys Thr
Tyr Thr Cys Asn Val Asp His 195 200 205 Lys Pro Ser Asn Thr Lys Val
Asp Lys Arg Val Glu Ser Lys Tyr Gly 210 215 220 Pro Pro Cys Pro Pro
Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser 225 230 235 240 Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro 260
265 270 Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr
Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Gly
Leu Pro Ser Ser Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Gln
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385
390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp
Lys Ser 405 410 415 Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val
Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Leu Gly Lys 435 440 445 42448PRTArtificial
SequenceC40B131 heavy chain protein 42Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Thr Ile Asn Ala Ala Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Lys Glu Gly Gly Lys Tyr Tyr Tyr Tyr Ala Met
Asp Val Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Cys Ser
Arg Ser Thr Ser Glu Ser Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185
190 Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His
195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys
Tyr Gly 210 215 220 Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala
Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser Gln Glu Asp Pro 260 265 270 Glu Val Gln Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys
Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310
315 320 Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys
Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu 340 345 350 Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp
Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435
440 445 43451PRTArtificial SequenceC40B176 heavy chain protein
43Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ser Thr Ile Asn Ala Ala Gly Gly Gly Thr Tyr
Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Glu Gly Gly
Lys Tyr Tyr Tyr Tyr Ala Leu Asp Val Trp Gly 100 105 110 Gln Gly Thr
Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135
140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr
Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val
Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr
Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 225 230 235 240 Ala Ser
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Ala 260
265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ser Ser Ile 325 330 335 Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr
Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420
425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser 435 440 445 Pro Gly Lys 450 44448PRTArtificial SequenceC40B180
heavy chain protein 44Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Thr Ile Asn Ala
Ala Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Lys Glu Gly Gly Lys Tyr Tyr Tyr Tyr Ala Leu Asp Val Trp Gly
100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly
Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser
Glu Ser Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser
Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His 195 200 205 Lys
Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly 210 215
220 Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser
225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser Gln Glu Asp Pro 260 265 270 Glu Val Gln Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu
Gln Phe Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys
Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr 325 330 335
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340
345 350 Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Arg Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Glu Gly Asn
Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 445
45451PRTArtificial SequenceC40B179 heavy chain protein 45Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20
25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Ser Thr Ile Asn Gln Ala Gly Gly Gly Thr Tyr Tyr Ala
Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser
Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Glu Gly Gly Lys Tyr
Tyr Tyr Tyr Ala Leu Asp Val Trp Gly 100 105 110 Gln Gly Thr Thr Val
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150
155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys
Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Ala Ala Gly 225 230 235 240 Ala Ser Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Ala 260 265 270
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275
280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ser Ser Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser
Arg Glu Glu Met Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395
400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450 46448PRTArtificial
SequenceC40B183 heavy chain protein 46Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Thr Ile Asn Gln Ala Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Lys Glu Gly Gly Lys Tyr Tyr Tyr Tyr Ala Leu
Asp Val Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Cys Ser
Arg Ser Thr Ser Glu Ser Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185
190 Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His
195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys
Tyr Gly 210 215 220 Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala
Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser Gln Glu Asp Pro 260 265 270 Glu Val Gln Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys
Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310
315 320 Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys
Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu 340 345 350 Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp
Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435
440 445 47212PRTArtificial SequenceC40B16 light chain protein 47Ser
Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln 1 5 10
15 Thr Ala Ser Ile Thr Cys Ser Gly Asp Lys Leu Gly Asp Lys Tyr Ala
20 25 30 Cys Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu Val
Ile Tyr 35 40 45 Gln Asp Ser Arg Arg Pro Ser Gly Ile Pro Glu Arg
Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile
Ser Gly Thr Gln Ala Met 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln
Ala Trp Ala Ser Ser Thr Val Val 85 90 95 Phe Gly Gly Gly Thr Lys
Leu Thr Val Leu Gly Gln Pro Lys Ala Ala 100 105 110 Pro Ser Val Thr
Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn 115 120 125 Lys Ala
Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala Val 130 135 140
Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly Val Glu 145
150 155 160 Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala
Ser Ser 165 170 175 Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His
Arg Ser Tyr Ser 180 185 190 Cys Gln Val Thr His Glu Gly Ser Thr Val
Glu Lys Thr Val Ala Pro 195 200 205 Thr Glu Cys Ser 210
48212PRTArtificial Sequencelight chain protein 48Ser Tyr Glu Leu
Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln 1 5 10 15 Thr Ala
Ser Ile Thr Cys Ser Gly Asp Lys Leu Gly Asp Lys Tyr Ala 20 25 30
Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr 35
40 45 Gln Asp Ser Arg Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly
Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr
Gln Ala Met 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Ala Trp Ala
Ser Ser Thr Val Val 85 90 95 Phe Gly Gly Gly Thr Lys Leu Thr Val
Leu Gly Gln Pro Lys Ala Ala 100 105 110 Pro Ser Val Thr Leu Phe Pro
Pro Ser Ser Glu Glu Leu Gln Ala Asn 115 120 125 Lys Ala Thr Leu Val
Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala Val 130 135 140 Thr Val Ala
Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly Val Glu 145 150 155 160
Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser 165
170 175 Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser Tyr
Ser 180 185 190 Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr
Val Ala Pro 195 200 205 Thr Glu Cys Ser 210 4998PRTHomo sapiens
49Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr
Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys 5095PRTHomo
sapiens 50Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro
Gly Gln 1 5 10 15 Thr Ala Ser Ile Thr Cys Ser Gly Asp Lys Leu Gly
Asp Lys Tyr Ala 20 25 30 Cys Trp Tyr Gln Gln Lys Pro Gly Gln Ser
Pro Val Leu Val Ile Tyr 35 40 45 Gln Asp Ser Lys Arg Pro Ser Gly
Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala
Thr Leu Thr Ile Ser Gly Thr Gln Ala Met 65 70 75 80 Asp Glu Ala Asp
Tyr Tyr Cys Gln Ala Trp Asp Ser Ser Thr Ala 85 90 95
51330PRTArtificial SequenceIgG1sigma Fc 51Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly
Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50
55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln
Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Ala Ala Gly Ala Ser
Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val
Ser Ala Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180
185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn 195 200 205 Lys Ala Leu Pro Ser Ser Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265
270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly
Lys 325 330 52327PRTArtificial SequenceIgG1PAA Fc 52Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr
Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35
40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn
Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro
Cys Pro Pro Cys Pro Ala Pro 100 105 110 Glu Ala Ala Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser
Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165
170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285
Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290
295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325
53448PRTArtificial SequenceASKP-1240 HC 53Gln Leu Gln Leu Gln Glu
Ser Gly Pro Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu
Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Pro 20 25 30 Gly Tyr
Tyr Gly Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu 35 40 45
Trp Ile Gly Ser Ile Tyr Lys Ser Gly Ser Thr Tyr His Asn Pro Ser 50
55 60 Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln
Phe 65 70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala
Val Tyr Tyr 85 90 95 Cys Thr Arg Pro Val Val Arg Tyr Phe Gly Trp
Phe Asp Pro Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Cys
Ser Arg Ser Thr Ser Glu Ser Thr Ala 130 135 140 Ala Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180
185 190 Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp
His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser
Lys Tyr Gly 210 215 220 Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe
Glu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser Gln Glu Asp Pro 260 265 270 Glu Val Gln Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr
Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val 290 295 300
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305
310 315 320 Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu
Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu 340 345 350 Pro Pro Ser Gln Glu Glu Met Thr Lys Asn
Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser 405 410 415 Arg
Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425
430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
435 440 445 54213PRTArtificial SequenceASKP-1240 LC 54Ala Ile Gln
Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ala 20 25
30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45 Tyr Asp Ala Ser Asn Leu Glu Ser Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Phe Asn Ser Tyr Pro Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg Thr Val Ala Ala Pro 100 105 110 Ser Val Phe Ile Phe Pro
Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120 125 Ala Ser Val Val
Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135 140 Val Gln
Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu 145 150 155
160 Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser
165 170 175 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
Tyr Ala 180 185 190 Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val
Thr Lys Ser Phe 195 200 205 Asn Arg Gly Glu Cys 210
55449PRTArtificial SequencemAb D HC 55Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Gly Met His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala
Tyr Ile Ser Ser Gly Asn Arg Ile Ile Tyr Tyr Ala Asp Thr Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Gln Asp Gly Tyr Arg Tyr Ala Met Asp Tyr
Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185
190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala
Ala Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310
315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435
440 445 Lys 56220PRTArtificial SequencemAb D LC 56Asp Ile Val Met
Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Lys
Val Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30
Gly Asn Gln Lys Asn Tyr Leu Thr Trp His Gln Gln Lys Pro Gly Gln 35
40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Thr Ser Thr Arg Glu Ser Gly
Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val
Tyr Tyr Cys Gln Asn 85 90 95 Asp Tyr Thr Tyr Pro Leu Thr Phe Gly
Gly Gly Thr Lys Val Glu Ile 100 105 110 Lys Arg Thr Val Ala Ala Pro
Ser Val Phe Ile Phe Pro Pro Ser Asp 115 120 125 Glu Gln Leu Lys Ser
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 130 135 140 Phe Tyr Pro
Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 145 150 155 160
Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 165
170 175 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr 180 185 190 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln
Gly Leu Ser 195 200 205 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu
Cys 210 215 220 57450PRTArtificial SequenceCFZ533HC 57Gln Val Gln
Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25
30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ala Val Ile Ser Tyr Glu Glu Ser Asn Arg Tyr His Ala Asp
Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Ile Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Thr Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Gly Gly Ile Ala Ala
Pro Gly Pro Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155
160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val
Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280
285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg
290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Glu
Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405
410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Pro 435 440 445 Gly Lys 450 58219PRTArtificial
SequenceCFZ533 LC 58Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Thr
Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser
Gln Ser Leu Leu Tyr Ser 20 25 30 Asn Gly Tyr Asn Tyr Leu Asp Trp
Tyr Leu Gln Lys Pro Gly Gln Ser 35 40
45 Pro Gln Val Leu Ile Ser Leu Gly Ser Asn Arg Ala Ser Gly Val Pro
50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr
Cys Met Gln Ala 85 90 95 Arg Gln Thr Pro Phe Thr Phe Gly Pro Gly
Thr Lys Val Asp Ile Arg 100 105 110 Arg Thr Val Ala Ala Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170
175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu
Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210
215 59348PRTArtificial SequenceBMS-986090 HC 59Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asp Tyr 20 25 30 Glu
Met Trp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Arg Val 35 40
45 Ser Ala Ile Asn Pro Gln Gly Thr Arg Thr Tyr Tyr Ala Asp Ser Val
50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Lys Leu Pro Phe Arg Phe Ser Asp Arg
Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Glu
Ser Lys Tyr Gly Pro Pro Cys Pro 115 120 125 Pro Cys Pro Ala Pro Glu
Phe Leu Gly Gly Pro Ser Val Phe Leu Phe 130 135 140 Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 145 150 155 160 Thr
Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe 165 170
175 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
180 185 190 Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val
Leu Thr 195 200 205 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val 210 215 220 Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu
Lys Thr Ile Ser Lys Ala 225 230 235 240 Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Gln 245 250 255 Glu Glu Met Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 260 265 270 Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 275 280 285 Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 290 295
300 Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu
305 310 315 320 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn His 325 330 335 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
Lys 340 345 60149PRTHomo sapiens 60Met Gln Lys Gly Asp Gln Asn Pro
Gln Ile Ala Ala His Val Ile Ser 1 5 10 15 Glu Ala Ser Ser Lys Thr
Thr Ser Val Leu Gln Trp Ala Glu Lys Gly 20 25 30 Tyr Tyr Thr Met
Ser Asn Asn Leu Val Thr Leu Glu Asn Gly Lys Gln 35 40 45 Leu Thr
Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr 50 55 60
Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser 65
70 75 80 Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu
Arg Ala 85 90 95 Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly Gln
Gln Ser Ile His 100 105 110 Leu Gly Gly Val Phe Glu Leu Gln Pro Gly
Ala Ser Val Phe Val Asn 115 120 125 Val Thr Asp Pro Ser Gln Val Ser
His Gly Thr Gly Phe Thr Ser Phe 130 135 140 Gly Leu Leu Lys Leu 145
6117PRTArtificial SequenceHCDR2 genus
sequenceMISC_FEATURE(3)..(3)Xaa may be Asn, Asp or
GlnMISC_FEATURE(4)..(4)Xaa may be Asn, Gln or
AlaMISC_FEATURE(5)..(5)Xaa may be Ser or Ala 61Thr Ile Xaa Xaa Xaa
Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly
6212PRTArtificial SequenceHCDR3 genusMISC_FEATURE(10)..(10)Xaa may
be Met or Leu 62Glu Gly Gly Lys Tyr Tyr Tyr Tyr Ala Xaa Asp Val 1 5
10 6311PRTArtificial SequenceLCDR1 genusMISC_FEATURE(11)..(11)Xaa
may be Cys ro Ala 63Ser Gly Asp Lys Leu Gly Asp Lys Tyr Ala Xaa 1 5
10 64259PRTHomo sapiens 64Met Ile Glu Thr Tyr Asn Gln Thr Ser Pro
Arg Ser Ala Ala Thr Gly 1 5 10 15 Leu Pro Ile Ser Met Lys Ile Phe
Met Tyr Leu Leu Thr Val Phe Leu 20 25 30 Ile Thr Gln Met Ile Gly
Ser Ala Leu Phe Ala Val Tyr Leu His Arg 35 40 45 Arg Leu Asp Lys
Ile Glu Asp Glu Arg Asn Leu His Glu Asp Phe Val 50 55 60 Phe Met
Lys Thr Ile Gln Arg Cys Asn Thr Gly Glu Arg Ser Leu Ser 65 70 75 80
Leu Leu Asn Cys Glu Glu Ile Lys Ser Gln Phe Glu Gly Phe Val Lys 85
90 95 Asp Ile Met Leu Asn Lys Glu Glu Thr Lys Lys Glu Asn Ser Phe
Glu 100 105 110 Met Gln Lys Gly Asp Gln Asn Pro Gln Ile Ala Ala His
Val Ile Ser 115 120 125 Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln
Trp Ala Glu Lys Gly 130 135 140 Tyr Tyr Thr Met Ser Asn Asn Leu Val
Thr Leu Glu Asn Gly Lys Gln 145 150 155 160 Leu Thr Val Lys Arg Gln
Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr 165 170 175 Phe Cys Ser Asn
Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser 180 185 190 Leu Cys
Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala 195 200 205
Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser Ile His 210
215 220 Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe Val
Asn 225 230 235 240 Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly
Phe Thr Ser Phe 245 250 255 Gly Leu Leu 651353DNAArtificial
SequenceC40B16 heavy chain DNA 65gaggtgcagc tggtggaatc tggcggagga
ctggtgcagc ctggcggcag cctgagactg 60tcttgtgccg ccagcggctt caccttcagc
agctacgcta tgagctgggt ccgacaggcc 120cctggcagag gactcgagtg
ggtgtccacc atcaacaaca gcggcggagg cacctactac 180gccgacagcg
tgaagggcag attcaccatc agccgggaca acagcaagaa caccctgtac
240ctgcacatga acagcctgcg ggccgaggac accgccgtgt actattgtgc
caaagagggc 300ggcaagtact actactatgc catggacgtg tggggccagg
gcaccaccgt gacagtgtca 360tctgcctcca ccaagggccc atcggtcttc
cccctggcac cctcctccaa gagcacctct 420gggggcacag cggccctggg
ctgcctggtc aaggactact tccccgaacc ggtgacggtg 480tcgtggaact
caggcgccct gaccagcggc gtgcacacct tcccggctgt cctacagtcc
540tcaggactct actccctcag cagcgtggtg accgtgccct ccagcagctt
gggcacccag 600acctacatct gcaacgtgaa tcacaagccc agcaacacca
aggtggacaa gaaagttgag 660cccaaatctt gtgacaaaac tcacacatgc
ccaccgtgcc cagcacctga actcctgggg 720ggaccgtcag tcttcctctt
ccccccaaaa cccaaggaca ccctcatgat ctcccggacc 780cctgaggtca
catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac
840tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga
ggagcagtac 900aacagcacgt accgtgtggt cagcgtcctc accgtcctgc
accaggactg gctgaatggc 960aaggagtaca agtgcaaggt ctccaacaaa
gccctcccag cccccatcga gaaaaccatc 1020tccaaagcca aagggcagcc
ccgagaacca caggtgtaca ccctgccccc atcccgggag 1080gagatgacca
agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac
1140atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac
cacgcctccc 1200gtgctggact ccgacggctc cttcttcctc tacagcaagc
tcaccgtgga caagagcagg 1260tggcagcagg ggaacgtctt ctcatgctcc
gtgatgcatg aggctctgca caaccactac 1320acgcagaaga gcctctccct
gtctccgggt aaa 1353661353DNAArtificial SequenceC40B124 heavy chain
DNA 66gaggtgcagc tggtggagag cggcggcggc ctggtgcagc ccggcggcag
cctgcggctg 60agctgcgccg ccagcggctt caccttcagc agctacgcca tgagctgggt
gcggcaggcc 120cccggcaagg gcctggagtg ggtgagcacc atcgacaacg
ccggcggcgg cacctactac 180gccgacagcg tgaagggccg gttcaccatc
agccgggaca acagcaagaa caccctgtac 240ctgcagatga acagcctgcg
ggccgaggac accgccgtgt actactgcgc caaggagggc 300ggcaagtact
actactacgc catggacgtg tggggccagg gcaccaccgt gaccgtgagc
360agcgcctcca ccaagggccc atcggtcttc cccctggcac cctcctccaa
gagcacctct 420gggggcacag cggccctggg ctgcctggtc aaggactact
tccccgaacc ggtgacggtg 480tcgtggaact caggcgccct gaccagcggc
gtgcacacct tcccggctgt cctacagtcc 540tcaggactct actccctcag
cagcgtggtg accgtgccct ccagcagctt gggcacccag 600acctacatct
gcaacgtgaa tcacaagccc agcaacacca aggtggacaa gaaagttgag
660cccaaatctt gtgacaaaac tcacacatgc ccaccgtgcc cagcacctga
agcagcaggg 720gcatcttcag tcttcctctt ccccccaaaa cccaaggaca
ccctcatgat ctcccggacc 780cctgaggtca catgcgtggt ggtggacgtg
agcgccgaag accctgaggt caagttcaac 840tggtacgtgg acggcgtgga
ggtgcataat gccaagacaa agccgcggga ggagcagtac 900aacagcacgt
accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc
960aaggagtaca agtgcaaggt ctccaacaaa gccctcccat cctccatcga
gaaaaccatc 1020tccaaagcca aagggcagcc ccgagaacca caggtgtaca
ccctgccccc atcccgggag 1080gagatgacca agaaccaggt cagcctgacc
tgcctggtca aaggcttcta tcccagcgac 1140atcgccgtgg agtgggagag
caatgggcag ccggagaaca actacaagac cacgcctccc 1200gtgctggact
ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg
1260tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca
caaccactac 1320acgcagaaga gcctctccct gtctccgggt aaa
1353671344DNAArtificial SequenceC40B135 heavy chain DNA
67gaggtgcagc tggtggagag cggcggcggc ctggtgcagc ccggcggcag cctgcggctg
60agctgcgccg ccagcggctt caccttcagc agctacgcca tgagctgggt gcggcaggcc
120cccggcaagg gcctggagtg ggtgagcacc atcgacaacg ccggcggcgg
cacctactac 180gccgacagcg tgaagggccg gttcaccatc agccgggaca
acagcaagaa caccctgtac 240ctgcagatga acagcctgcg ggccgaggac
accgccgtgt actactgcgc caaggagggc 300ggcaagtact actactacgc
catggacgtg tggggccagg gcaccaccgt gaccgtgagc 360agcgcctcca
ccaagggccc atccgtcttc cccctggcgc cctgctccag gagcacctcc
420gagagcacag ccgccctggg ctgcctggtc aaggactact tccccgaacc
ggtgacggtg 480tcgtggaact caggcgccct gaccagcggc gtgcacacct
tcccggctgt cctacagtcc 540tcaggactct actccctcag cagcgtggtg
accgtgccct ccagcagctt gggcacgaaa 600acctacacct gcaacgtaga
tcacaagccc agcaacacca aggtggacaa gagagttgag 660tccaaatatg
gtcccccatg cccaccatgc ccagcacctg aggccgccgg gggaccatca
720gtcttcctgt tccccccaaa acccaaggac actctcatga tctcccggac
ccctgaggtc 780acgtgcgtgg tggtggacgt gagccaggaa gaccccgagg
tccagttcaa ctggtacgtg 840gatggcgtgg aggtgcataa tgccaagaca
aagccgcggg aggagcagtt caacagcacg 900taccgtgtgg tcagcgtcct
caccgtcctg caccaggact ggctgaacgg caaggagtac 960aagtgcaagg
tctccaacaa aggcctcccg tcctccatcg agaaaaccat ctccaaagcc
1020aaagggcagc cccgagagcc acaggtgtac accctgcccc catcccagga
ggagatgacc 1080aagaaccagg tcagcctgac ctgcctggtc aaaggcttct
accccagcga catcgccgtg 1140gagtgggaga gcaatgggca gccggagaac
aactacaaga ccacgcctcc cgtgctggac 1200tccgacggct ccttcttcct
ctacagcagg ctaaccgtgg acaagagcag gtggcaggag 1260gggaatgtct
tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacacagaag
1320agcctctccc tgtctctggg taaa 1344681353DNAArtificial
SequenceC40B125 heavy chain DNA 68gaggtgcagc tggtggagag cggcggcggc
ctggtgcagc ccggcggcag cctgcggctg 60agctgcgccg ccagcggctt caccttcagc
agctacgcca tgagctgggt gcggcaggcc 120cccggcaagg gcctggagtg
ggtgagcacc atccagaacg ccggcggcgg cacctactac 180gccgacagcg
tgaagggccg gttcaccatc agccgggaca acagcaagaa caccctgtac
240ctgcagatga acagcctgcg ggccgaggac accgccgtgt actactgcgc
caaggagggc 300ggcaagtact actactacgc catggacgtg tggggccagg
gcaccaccgt gaccgtgagc 360agcgcctcca ccaagggccc atcggtcttc
cccctggcac cctcctccaa gagcacctct 420gggggcacag cggccctggg
ctgcctggtc aaggactact tccccgaacc ggtgacggtg 480tcgtggaact
caggcgccct gaccagcggc gtgcacacct tcccggctgt cctacagtcc
540tcaggactct actccctcag cagcgtggtg accgtgccct ccagcagctt
gggcacccag 600acctacatct gcaacgtgaa tcacaagccc agcaacacca
aggtggacaa gaaagttgag 660cccaaatctt gtgacaaaac tcacacatgc
ccaccgtgcc cagcacctga agcagcaggg 720gcatcttcag tcttcctctt
ccccccaaaa cccaaggaca ccctcatgat ctcccggacc 780cctgaggtca
catgcgtggt ggtggacgtg agcgccgaag accctgaggt caagttcaac
840tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga
ggagcagtac 900aacagcacgt accgtgtggt cagcgtcctc accgtcctgc
accaggactg gctgaatggc 960aaggagtaca agtgcaaggt ctccaacaaa
gccctcccat cctccatcga gaaaaccatc 1020tccaaagcca aagggcagcc
ccgagaacca caggtgtaca ccctgccccc atcccgggag 1080gagatgacca
agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac
1140atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac
cacgcctccc 1200gtgctggact ccgacggctc cttcttcctc tacagcaagc
tcaccgtgga caagagcagg 1260tggcagcagg ggaacgtctt ctcatgctcc
gtgatgcatg aggctctgca caaccactac 1320acgcagaaga gcctctccct
gtctccgggt aaa 1353691344DNAArtificial SequenceC40B136 heavy chain
DNA 69gaggtgcagc tggtggagag cggcggcggc ctggtgcagc ccggcggcag
cctgcggctg 60agctgcgccg ccagcggctt caccttcagc agctacgcca tgagctgggt
gcggcaggcc 120cccggcaagg gcctggagtg ggtgagcacc atccagaacg
ccggcggcgg cacctactac 180gccgacagcg tgaagggccg gttcaccatc
agccgggaca acagcaagaa caccctgtac 240ctgcagatga acagcctgcg
ggccgaggac accgccgtgt actactgcgc caaggagggc 300ggcaagtact
actactacgc catggacgtg tggggccagg gcaccaccgt gaccgtgagc
360agcgcctcca ccaagggccc atccgtcttc cccctggcgc cctgctccag
gagcacctcc 420gagagcacag ccgccctggg ctgcctggtc aaggactact
tccccgaacc ggtgacggtg 480tcgtggaact caggcgccct gaccagcggc
gtgcacacct tcccggctgt cctacagtcc 540tcaggactct actccctcag
cagcgtggtg accgtgccct ccagcagctt gggcacgaaa 600acctacacct
gcaacgtaga tcacaagccc agcaacacca aggtggacaa gagagttgag
660tccaaatatg gtcccccatg cccaccatgc ccagcacctg aggccgccgg
gggaccatca 720gtcttcctgt tccccccaaa acccaaggac actctcatga
tctcccggac ccctgaggtc 780acgtgcgtgg tggtggacgt gagccaggaa
gaccccgagg tccagttcaa ctggtacgtg 840gatggcgtgg aggtgcataa
tgccaagaca aagccgcggg aggagcagtt caacagcacg 900taccgtgtgg
tcagcgtcct caccgtcctg caccaggact ggctgaacgg caaggagtac
960aagtgcaagg tctccaacaa aggcctcccg tcctccatcg agaaaaccat
ctccaaagcc 1020aaagggcagc cccgagagcc acaggtgtac accctgcccc
catcccagga ggagatgacc 1080aagaaccagg tcagcctgac ctgcctggtc
aaaggcttct accccagcga catcgccgtg 1140gagtgggaga gcaatgggca
gccggagaac aactacaaga ccacgcctcc cgtgctggac 1200tccgacggct
ccttcttcct ctacagcagg ctaaccgtgg acaagagcag gtggcaggag
1260gggaatgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta
cacacagaag 1320agcctctccc tgtctctggg taaa 1344701353DNAArtificial
SequenceC40B127 heavy chain DNA 70gaggtgcagc tggtggagag cggcggcggc
ctggtgcagc ccggcggcag cctgcggctg 60agctgcgccg ccagcggctt caccttcagc
agctacgcca tgagctgggt gcggcaggcc 120cccggcaagg gcctggagtg
ggtgagcacc atcaaccagg ccggcggcgg cacctactac 180gccgacagcg
tgaagggccg gttcaccatc agccgggaca acagcaagaa caccctgtac
240ctgcagatga acagcctgcg ggccgaggac accgccgtgt actactgcgc
caaggagggc 300ggcaagtact actactacgc catggacgtg tggggccagg
gcaccaccgt gaccgtgagc 360agcgcctcca ccaagggccc atcggtcttc
cccctggcac cctcctccaa gagcacctct 420gggggcacag cggccctggg
ctgcctggtc aaggactact tccccgaacc ggtgacggtg 480tcgtggaact
caggcgccct gaccagcggc gtgcacacct tcccggctgt cctacagtcc
540tcaggactct actccctcag cagcgtggtg accgtgccct ccagcagctt
gggcacccag 600acctacatct gcaacgtgaa tcacaagccc agcaacacca
aggtggacaa gaaagttgag 660cccaaatctt gtgacaaaac tcacacatgc
ccaccgtgcc cagcacctga agcagcaggg 720gcatcttcag tcttcctctt
ccccccaaaa cccaaggaca ccctcatgat ctcccggacc 780cctgaggtca
catgcgtggt ggtggacgtg agcgccgaag accctgaggt caagttcaac
840tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga
ggagcagtac 900aacagcacgt accgtgtggt cagcgtcctc accgtcctgc
accaggactg gctgaatggc 960aaggagtaca agtgcaaggt ctccaacaaa
gccctcccat cctccatcga gaaaaccatc 1020tccaaagcca aagggcagcc
ccgagaacca caggtgtaca ccctgccccc atcccgggag 1080gagatgacca
agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac
1140atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac
cacgcctccc 1200gtgctggact ccgacggctc cttcttcctc tacagcaagc
tcaccgtgga caagagcagg 1260tggcagcagg ggaacgtctt ctcatgctcc
gtgatgcatg aggctctgca caaccactac 1320acgcagaaga gcctctccct
gtctccgggt aaa 1353711344DNAArtificial SequenceC40B138 heavy chain
DNA 71gaggtgcagc tggtggagag cggcggcggc ctggtgcagc ccggcggcag
cctgcggctg 60agctgcgccg ccagcggctt caccttcagc agctacgcca tgagctgggt
gcggcaggcc 120cccggcaagg gcctggagtg ggtgagcacc atcaaccagg
ccggcggcgg cacctactac 180gccgacagcg tgaagggccg gttcaccatc
agccgggaca acagcaagaa caccctgtac 240ctgcagatga acagcctgcg
ggccgaggac accgccgtgt actactgcgc caaggagggc 300ggcaagtact
actactacgc catggacgtg tggggccagg gcaccaccgt gaccgtgagc
360agcgcctcca ccaagggccc atccgtcttc cccctggcgc cctgctccag
gagcacctcc 420gagagcacag ccgccctggg ctgcctggtc aaggactact
tccccgaacc ggtgacggtg 480tcgtggaact caggcgccct gaccagcggc
gtgcacacct tcccggctgt cctacagtcc 540tcaggactct actccctcag
cagcgtggtg accgtgccct ccagcagctt gggcacgaaa 600acctacacct
gcaacgtaga tcacaagccc agcaacacca aggtggacaa gagagttgag
660tccaaatatg gtcccccatg cccaccatgc ccagcacctg aggccgccgg
gggaccatca 720gtcttcctgt tccccccaaa acccaaggac actctcatga
tctcccggac ccctgaggtc 780acgtgcgtgg tggtggacgt gagccaggaa
gaccccgagg tccagttcaa ctggtacgtg 840gatggcgtgg aggtgcataa
tgccaagaca aagccgcggg aggagcagtt caacagcacg 900taccgtgtgg
tcagcgtcct caccgtcctg caccaggact ggctgaacgg caaggagtac
960aagtgcaagg tctccaacaa aggcctcccg tcctccatcg agaaaaccat
ctccaaagcc 1020aaagggcagc cccgagagcc acaggtgtac accctgcccc
catcccagga ggagatgacc 1080aagaaccagg tcagcctgac ctgcctggtc
aaaggcttct accccagcga catcgccgtg 1140gagtgggaga gcaatgggca
gccggagaac aactacaaga ccacgcctcc cgtgctggac 1200tccgacggct
ccttcttcct ctacagcagg ctaaccgtgg acaagagcag gtggcaggag
1260gggaatgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta
cacacagaag 1320agcctctccc tgtctctggg taaa 1344721344DNAArtificial
SequenceC40B131 heavy chain DNA 72gaggtgcagc tggtggagag cggcggcggc
ctggtgcagc ccggcggcag cctgcggctg 60agctgcgccg ccagcggctt caccttcagc
agctacgcca tgagctgggt gcggcaggcc 120cccggcaagg gcctggagtg
ggtgagcacc atcaacgccg ccggcggcgg cacctactac 180gccgacagcg
tgaagggccg gttcaccatc agccgggaca acagcaagaa caccctgtac
240ctgcagatga acagcctgcg ggccgaggac accgccgtgt actactgcgc
caaggagggc 300ggcaagtact actactacgc catggacgtg tggggccagg
gcaccaccgt gaccgtgagc 360agcgcctcca ccaagggccc atccgtcttc
cccctggcgc cctgctccag gagcacctcc 420gagagcacag ccgccctggg
ctgcctggtc aaggactact tccccgaacc ggtgacggtg 480tcgtggaact
caggcgccct gaccagcggc gtgcacacct tcccggctgt cctacagtcc
540tcaggactct actccctcag cagcgtggtg accgtgccct ccagcagctt
gggcacgaaa 600acctacacct gcaacgtaga tcacaagccc agcaacacca
aggtggacaa gagagttgag 660tccaaatatg gtcccccatg cccaccatgc
ccagcacctg aggccgccgg gggaccatca 720gtcttcctgt tccccccaaa
acccaaggac actctcatga tctcccggac ccctgaggtc 780acgtgcgtgg
tggtggacgt gagccaggaa gaccccgagg tccagttcaa ctggtacgtg
840gatggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagtt
caacagcacg 900taccgtgtgg tcagcgtcct caccgtcctg caccaggact
ggctgaacgg caaggagtac 960aagtgcaagg tctccaacaa aggcctcccg
tcctccatcg agaaaaccat ctccaaagcc 1020aaagggcagc cccgagagcc
acaggtgtac accctgcccc catcccagga ggagatgacc 1080aagaaccagg
tcagcctgac ctgcctggtc aaaggcttct accccagcga catcgccgtg
1140gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc
cgtgctggac 1200tccgacggct ccttcttcct ctacagcagg ctaaccgtgg
acaagagcag gtggcaggag 1260gggaatgtct tctcatgctc cgtgatgcat
gaggctctgc acaaccacta cacacagaag 1320agcctctccc tgtctctggg taaa
1344731353DNAArtificial SequenceC40B176 heavy chain DNA
73gaggtgcagc tggtggagag cggcggcggc ctggtgcagc ccggcggcag cctgcggctg
60agctgcgccg ccagcggctt caccttcagc agctacgcca tgagctgggt gcggcaggcc
120cccggcaagg gcctggagtg ggtgagcacc atcaacgccg ccggcggcgg
cacctactac 180gccgacagcg tgaagggccg gttcaccatc agccgggaca
acagcaagaa caccctgtac 240ctgcagatga acagcctgcg ggccgaggac
accgccgtgt actactgcgc caaggagggc 300ggcaagtact actactacgc
cctggacgtg tggggccagg gcaccaccgt gaccgtgagc 360agcgcctcca
ccaagggccc atcggtcttc cccctggcac cctcctccaa gagcacctct
420gggggcacag cggccctggg ctgcctggtc aaggactact tccccgaacc
ggtgacggtg 480tcgtggaact caggcgccct gaccagcggc gtgcacacct
tcccggctgt cctacagtcc 540tcaggactct actccctcag cagcgtggtg
accgtgccct ccagcagctt gggcacccag 600acctacatct gcaacgtgaa
tcacaagccc agcaacacca aggtggacaa gaaagttgag 660cccaaatctt
gtgacaaaac tcacacatgc ccaccgtgcc cagcacctga agcagcaggg
720gcatcttcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat
ctcccggacc 780cctgaggtca catgcgtggt ggtggacgtg agcgccgaag
accctgaggt caagttcaac 840tggtacgtgg acggcgtgga ggtgcataat
gccaagacaa agccgcggga ggagcagtac 900aacagcacgt accgtgtggt
cagcgtcctc accgtcctgc accaggactg gctgaatggc 960aaggagtaca
agtgcaaggt ctccaacaaa gccctcccat cctccatcga gaaaaccatc
1020tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc
atcccgggag 1080gagatgacca agaaccaggt cagcctgacc tgcctggtca
aaggcttcta tcccagcgac 1140atcgccgtgg agtgggagag caatgggcag
ccggagaaca actacaagac cacgcctccc 1200gtgctggact ccgacggctc
cttcttcctc tacagcaagc tcaccgtgga caagagcagg 1260tggcagcagg
ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac
1320acgcagaaga gcctctccct gtctccgggt aaa 1353741344DNAArtificial
SequenceC40B180 heavy chain DNA 74gaggtgcagc tggtggagag cggcggcggc
ctggtgcagc ccggcggcag cctgcggctg 60agctgcgccg ccagcggctt caccttcagc
agctacgcca tgagctgggt gcggcaggcc 120cccggcaagg gcctggagtg
ggtgagcacc atcaacgccg ccggcggcgg cacctactac 180gccgacagcg
tgaagggccg gttcaccatc agccgggaca acagcaagaa caccctgtac
240ctgcagatga acagcctgcg ggccgaggac accgccgtgt actactgcgc
caaggagggc 300ggcaagtact actactacgc cctggacgtg tggggccagg
gcaccaccgt gaccgtgagc 360agcgcctcca ccaagggccc atccgtcttc
cccctggcgc cctgctccag gagcacctcc 420gagagcacag ccgccctggg
ctgcctggtc aaggactact tccccgaacc ggtgacggtg 480tcgtggaact
caggcgccct gaccagcggc gtgcacacct tcccggctgt cctacagtcc
540tcaggactct actccctcag cagcgtggtg accgtgccct ccagcagctt
gggcacgaaa 600acctacacct gcaacgtaga tcacaagccc agcaacacca
aggtggacaa gagagttgag 660tccaaatatg gtcccccatg cccaccatgc
ccagcacctg aggccgccgg gggaccatca 720gtcttcctgt tccccccaaa
acccaaggac actctcatga tctcccggac ccctgaggtc 780acgtgcgtgg
tggtggacgt gagccaggaa gaccccgagg tccagttcaa ctggtacgtg
840gatggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagtt
caacagcacg 900taccgtgtgg tcagcgtcct caccgtcctg caccaggact
ggctgaacgg caaggagtac 960aagtgcaagg tctccaacaa aggcctcccg
tcctccatcg agaaaaccat ctccaaagcc 1020aaagggcagc cccgagagcc
acaggtgtac accctgcccc catcccagga ggagatgacc 1080aagaaccagg
tcagcctgac ctgcctggtc aaaggcttct accccagcga catcgccgtg
1140gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc
cgtgctggac 1200tccgacggct ccttcttcct ctacagcagg ctaaccgtgg
acaagagcag gtggcaggag 1260gggaatgtct tctcatgctc cgtgatgcat
gaggctctgc acaaccacta cacacagaag 1320agcctctccc tgtctctggg taaa
1344751353DNAArtificial SequenceC40B179 heavy chain DNA
75gaggtgcagc tggtggagag cggcggcggc ctggtgcagc ccggcggcag cctgcggctg
60agctgcgccg ccagcggctt caccttcagc agctacgcca tgagctgggt gcggcaggcc
120cccggcaagg gcctggagtg ggtgagcacc atcaaccagg ccggcggcgg
cacctactac 180gccgacagcg tgaagggccg gttcaccatc agccgggaca
acagcaagaa caccctgtac 240ctgcagatga acagcctgcg ggccgaggac
accgccgtgt actactgcgc caaggagggc 300ggcaagtact actactacgc
cctggacgtg tggggccagg gcaccaccgt gaccgtgagc 360agcgcctcca
ccaagggccc atcggtcttc cccctggcac cctcctccaa gagcacctct
420gggggcacag cggccctggg ctgcctggtc aaggactact tccccgaacc
ggtgacggtg 480tcgtggaact caggcgccct gaccagcggc gtgcacacct
tcccggctgt cctacagtcc 540tcaggactct actccctcag cagcgtggtg
accgtgccct ccagcagctt gggcacccag 600acctacatct gcaacgtgaa
tcacaagccc agcaacacca aggtggacaa gaaagttgag 660cccaaatctt
gtgacaaaac tcacacatgc ccaccgtgcc cagcacctga agcagcaggg
720gcatcttcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat
ctcccggacc 780cctgaggtca catgcgtggt ggtggacgtg agcgccgaag
accctgaggt caagttcaac 840tggtacgtgg acggcgtgga ggtgcataat
gccaagacaa agccgcggga ggagcagtac 900aacagcacgt accgtgtggt
cagcgtcctc accgtcctgc accaggactg gctgaatggc 960aaggagtaca
agtgcaaggt ctccaacaaa gccctcccat cctccatcga gaaaaccatc
1020tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc
atcccgggag 1080gagatgacca agaaccaggt cagcctgacc tgcctggtca
aaggcttcta tcccagcgac 1140atcgccgtgg agtgggagag caatgggcag
ccggagaaca actacaagac cacgcctccc 1200gtgctggact ccgacggctc
cttcttcctc tacagcaagc tcaccgtgga caagagcagg 1260tggcagcagg
ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac
1320acgcagaaga gcctctccct gtctccgggt aaa 1353761344DNAArtificial
SequenceC40B183 heavy chain DNA 76gaggtgcagc tggtggagag cggcggcggc
ctggtgcagc ccggcggcag cctgcggctg 60agctgcgccg ccagcggctt caccttcagc
agctacgcca tgagctgggt gcggcaggcc 120cccggcaagg gcctggagtg
ggtgagcacc atcaaccagg ccggcggcgg cacctactac 180gccgacagcg
tgaagggccg gttcaccatc agccgggaca acagcaagaa caccctgtac
240ctgcagatga acagcctgcg ggccgaggac accgccgtgt actactgcgc
caaggagggc 300ggcaagtact actactacgc cctggacgtg tggggccagg
gcaccaccgt gaccgtgagc 360agcgcctcca ccaagggccc atccgtcttc
cccctggcgc cctgctccag gagcacctcc 420gagagcacag ccgccctggg
ctgcctggtc aaggactact tccccgaacc ggtgacggtg 480tcgtggaact
caggcgccct gaccagcggc gtgcacacct tcccggctgt cctacagtcc
540tcaggactct actccctcag cagcgtggtg accgtgccct ccagcagctt
gggcacgaaa 600acctacacct gcaacgtaga tcacaagccc agcaacacca
aggtggacaa gagagttgag 660tccaaatatg gtcccccatg cccaccatgc
ccagcacctg aggccgccgg gggaccatca 720gtcttcctgt tccccccaaa
acccaaggac actctcatga tctcccggac ccctgaggtc 780acgtgcgtgg
tggtggacgt gagccaggaa gaccccgagg tccagttcaa ctggtacgtg
840gatggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagtt
caacagcacg 900taccgtgtgg tcagcgtcct caccgtcctg caccaggact
ggctgaacgg caaggagtac 960aagtgcaagg tctccaacaa aggcctcccg
tcctccatcg agaaaaccat ctccaaagcc 1020aaagggcagc cccgagagcc
acaggtgtac accctgcccc catcccagga ggagatgacc 1080aagaaccagg
tcagcctgac ctgcctggtc aaaggcttct accccagcga catcgccgtg
1140gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc
cgtgctggac 1200tccgacggct ccttcttcct ctacagcagg ctaaccgtgg
acaagagcag gtggcaggag 1260gggaatgtct tctcatgctc cgtgatgcat
gaggctctgc acaaccacta cacacagaag 1320agcctctccc tgtctctggg taaa
134477636DNAArtificial SequenceC40B16 light chain DNA 77tcctacgagc
tgacccagcc tccctccgtg tctgtgtctc ctggccagac cgccagcatc 60acctgtagcg
gcgacaagct gggcgataag tacgcctgct ggtatcagca gaagcccggc
120cagagccccg tgctggtcat ctaccaggac agcagaaggc ccagcggcat
ccccgagaga 180ttcagcggca gcaacagcgg caataccgcc accctgacca
tcagcggcac ccaggccatg 240gacgaggccg attactattg tcaggcctgg
gccagcagca ccgtggtgtt tggcggcgga 300acaaagctga ccgtgctggg
tcagcccaag gctgcaccca gtgtcactct gttcccgccc 360tcctctgagg
agcttcaagc caacaaggcc acactggtgt gtctcataag tgacttctac
420ccgggagccg tgacagtggc ctggaaggcc gatagcagcc ccgtcaaggc
gggagtggag 480accaccacac cctccaaaca aagcaacaac aagtacgcgg
ccagcagcta tctgagcctg 540acgcctgagc agtggaagtc ccacagaagc
tacagctgcc aggtcacgca tgaagggagc 600accgtggaga agacagtggc
ccctacagaa tgttca 63678636DNAArtificial SequenceDNA of light chain
78agctacgagc tgacccagcc ccccagcgtg agcgtgagcc ccggccagac cgccagcatc
60acctgcagcg gcgacaagct gggcgacaag tacgccgcct ggtaccagca gaagcccggc
120cagagccccg tgctggtgat ctaccaggac agccggcggc ccagcggcat
ccccgagcgg 180ttcagcggca gcaacagcgg caacaccgcc accctgacca
tcagcggcac ccaggccatg 240gacgaggccg actactactg ccaggcctgg
gccagcagca ccgtggtgtt cggcggcggc 300accaagctga ccgtgctggg
tcagcccaag gctgcaccca gtgtcactct gttcccgccc 360tcctctgagg
agcttcaagc caacaaggcc acactggtgt gtctcataag tgacttctac
420ccgggagccg tgacagtggc ctggaaggcc gatagcagcc ccgtcaaggc
gggagtggag 480accaccacac cctccaaaca aagcaacaac aagtacgcgg
ccagcagcta tctgagcctg 540acgcctgagc agtggaagtc ccacagaagc
tacagctgcc aggtcacgca tgaagggagc 600accgtggaga agacagtggc
ccctacagaa tgttca 636
* * * * *
References