U.S. patent application number 16/409201 was filed with the patent office on 2020-01-16 for monoclonal antibodies activating cd40 and uses thereof.
The applicant listed for this patent is ABVISION, INC.. Invention is credited to Cheng-Chi Chao, Chang-Hsin CHEN, Guochen YAN, Gloria ZHANG.
Application Number | 20200017596 16/409201 |
Document ID | / |
Family ID | 68467585 |
Filed Date | 2020-01-16 |
United States Patent
Application |
20200017596 |
Kind Code |
A1 |
Chao; Cheng-Chi ; et
al. |
January 16, 2020 |
MONOCLONAL ANTIBODIES ACTIVATING CD40 AND USES THEREOF
Abstract
The present disclosure provides isolated monoclonal antibodies
or an antigen-binding portion thereof that specifically bind to
CD40 preferably human CD40 with high affinity, and that function as
CD40 agonists. The disclosed invention also relates to antibodies
that are chimeric, humanized, bispecific, derivatized, single chain
antibodies or portions of fusion proteins. Nucleic acid molecules
encoding the antibodies of the disclosed invention, hybridoma, and
methods for expressing the antibodies of the disclosed invention
are also provided. Pharmaceutical compositions comprising the
antibodies of the disclosed invention are also provided. This
disclosure also provides methods for regulating humoral and
cellular immune responses, as well as methods for treating cancer
using an anti-CD40 agonist antibody of the disclosed invention.
Inventors: |
Chao; Cheng-Chi; (Milpitas,
CA) ; CHEN; Chang-Hsin; (San Jose, CA) ;
ZHANG; Gloria; (Reno, NV) ; YAN; Guochen;
(Reno, NV) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ABVISION, INC. |
MILPITAS |
CA |
US |
|
|
Family ID: |
68467585 |
Appl. No.: |
16/409201 |
Filed: |
May 10, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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62669863 |
May 10, 2018 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/2878 20130101;
C07K 2317/75 20130101; C07K 2317/24 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28 |
Claims
1. A monoclonal antibody, or an antigen-binding portion thereof
comprising: (a) a heavy chain CDR1 amino acid sequence, CDR2 amino
acid sequence, and CDR3 amino acid sequence, respectively, of amino
acid residues 26-33, 51-58, and 97-111 of SEQ ID NO:1; and (b) a
light chain CDR1 amino acid sequence, CDR2 amino acid sequence, and
CDR3 amino acid sequence, respectively, of amino acid residues
27-38, 56-58, and 95-103 of SEQ ID NO:2, wherein said antibody or
portion specifically binds to and activates human CD40.
2. (canceled)
3. The antibody or antigen-binding portion according to claim 1
that is a Fab fragment, an F(ab').sub.2 fragment, an Fv fragment, a
single chain antibody, or a bispecific antibody.
4. A pharmaceutical composition comprising the antibody or
antigen-binding portion-according to claim 1 and a pharmaceutically
acceptable carrier.
5.-6. (canceled)
7. The monoclonal antibody, or an antigen-binding portion thereof
of claim 1, wherein the antibody stimulates an anti-tumor immune
response.
8. A monoclonal antibody or an antigen-binding portion thereof
comprising: a heavy chain variable domain comprising SEQ ID NO:1
and a light chain variable domain comprising SEQ ID NO:2, wherein
the antibody specifically binds to and activates human CD40.
9. (canceled)
10. The antibody or antigen-binding portion according to claim 8
that is a Fab fragment, an F(ab').sub.2 fragment, an Fv fragment, a
single chain antibody, or a bispecific antibody.
11.-13. (canceled)
14. The antibody of claim 8, wherein the antibody stimulates an
anti-tumor immune response.
15.-16. (canceled)
17. The antibody according to claim 1, that is an immunoglobulin G
(IgG), an IgM, an IgE, an IgA or an IgD molecule, or is derived
therefrom.
18. The monoclonal antibody according to claim 17, wherein said
antibody is a chimeric antibody or humanized antibody.
19. (canceled)
20. The antibody according to claim 8, that is an immunoglobulin G
(IgG), an IgM, an IgE, an IgA or an IgD molecule, or is derived
therefrom.
21. (canceled)
22. The monoclonal antibody according to claim 20, wherein said
antibody is a chimeric antibody or humanized antibody.
23.-24. (canceled)
Description
BACKGROUND OF THE INVENTION
[0001] The CD40 antigen is a 50 kDa cell surface glycoprotein which
belongs to the Tumor Necrosis Factor Receptor (TNF-R) family.
(Stamenkovic et al., EMBO J. 8: 1403-10 (1989).) CD40 is expressed
on B lymphocytes, dendritic cells, macrophages and other cell types
and is important for regulation of cell differentiation to effector
cells that promote immune responses to antigens. CD40 is expressed
in all B-lymphomas and in 70% of all solid tumors. Although
constitutively expressed, CD40 is up-regulated in antigen
presenting cells by maturation signals, such as LPS, IL-1.beta.p,
IFN-.gamma. and GM-CSF.
[0002] CD40 activation plays a critical role in regulating humoral
and cellular immune responses. Antigen presentation without CD40
activation can lead to tolerance, while CD40 signaling can reverse
such tolerance, enhance antigen presentation by all antigen
presenting cells (APCs), lead to secretion of helper cytokines and
chemokines, increase co-stimulatory molecule expression and
signaling, and stimulate the cytolytic activity of immune cells.
CD40 plays a critical role in B cell proliferation, maturation and
class switching. (Foy T. M. et al., Ann. Rev. of Immunol.
14:591-617 (1996).) CD40 engagement by CD40L leads to the
association of the CD40 cytoplasmic domain with TRAFs (TNF-R
associated factors). (Lee H. H. et al., Proc. Natl. Acad. Sci. USA
96:1421-6 (1999). In addition to enhancement of cellular and immune
function, the effects of CD40 activation include: Cell recruitment
and differentiation by chemokines and cytokines; activation of
monocytes; increased cytolytic activity of cytolytic T lymphocyte
(CTL) and natural killer (NK) cells; induction of apoptosis in CD40
positive tumors; enhancement of immunogenicity of CD40 positive
tumors; and tumor-specific antibody production.
[0003] CD40 activation relies on CD40 ligand (CD40L or CD154).
However, the majority of CD40L is expressed on CD4+ T cells or
platelets, which are not easy to manipulate under in vivo
physiological conditions. The disclosed antibodies targeting CD40
can bypass the need of CD40L for activation. The high stability of
antibodies possesses numerous advantages for use in potential
therapeutics.
[0004] There exists a need in the art for antibodies or an
antigen-binding portion thereof, including chimeric and humanized
antibodies, that bind CD40 and cause activation of CD40 downstream
signaling, and that can have applications in the treatment of
cancer and targeted vaccine adjuvants.
SUMMARY OF THE INVENTION
[0005] The present disclosure provides an isolated monoclonal
antibody, or an antigen-binding portion thereof, that specifically
binds to and activates human CD40. The newly developed antibodies
have a high affinity and can target different epitopes on human
CD40, compared to published antibodies.
[0006] In one aspect, the disclosed invention pertains to a
monoclonal antibody, or an antigen-binding portion thereof that
specifically binds to and activates human CD40, wherein said
antibody or portion thereof comprises heavy chains: CDR1 amino acid
sequence, CDR2 amino acid sequence, and CDR3 amino acid sequence,
respectively, of amino acid residues 26-33, 51-58, and 97-111 of
SEQ ID NO:1; and light chains: CDR1 amino acid sequence, CDR2 amino
acid sequence, and CDR3 amino acid sequence, respectively, of amino
acid residues 27-38, 56-58, and 95-103 of SEQ ID NO:2. In another
aspect, the disclosed invention pertains to a monoclonal antibody,
or an antigen-binding portion thereof that specifically binds to
and activates human CD40, wherein said antibody or portion thereof
comprises heavy chains: CDR1 amino acid sequence, CDR2 amino acid
sequence, and CDR3 amino acid sequence, respectively, of amino acid
residues 26-33, 51-58, and 97-104 of SEQ ID NO:10; and light
chains: CDR1 amino acid sequence, CDR2 amino acid sequence, and
CDR3 amino acid sequence, respectively, of amino acid residues
27-32, 50-52, and 89-97 of SEQ ID NO:11. In some embodiments, the
monoclonal antibody, or said antigen-binding portion thereof
stimulates an anti-tumor immune response. In some embodiments, the
monoclonal antibody can be a chimeric antibody or a humanized
antibody. In another aspect, anti-CD40 agonist antibodies can have
a significant higher half-life in serum than CD40L.
[0007] In another aspect, the disclosed invention pertains to an
isolated monoclonal antibody or an antigen-binding portion thereof
that specifically binds to and activates human CD40, wherein said
antibody comprises: a heavy chain variable domain comprising SEQ ID
NO:1 and a light chain variable domain comprising SEQ ID NO:2. In
another aspect, the disclosed invention pertains to an isolated
monoclonal antibody or an antigen-binding portion thereof that
specifically binds to and activates human CD40, wherein said
antibody comprises: a heavy chain variable domain comprising SEQ ID
NO:10 and a light chain variable domain comprising SEQ ID NO:11. In
some embodiments, the monoclonal antibody, or an antigen-binding
portion thereof stimulates an anti-tumor immune response. In some
embodiments, the monoclonal antibody can be a chimeric antibody or
a humanized antibody.
[0008] The antibodies of the disclosed invention can be further
engineered into formats suitable for human therapeutics by
modifications that minimize immunogenicity. Suitable antibodies
include, but are not limited to chimeric antibodies and humanized
antibodies. The affinity, stability and specificity of the
disclosed antibodies can also be further optimized by techniques
known to one of skill in the art such as light-chain shuffling.
Other formats can involve oligomerization, drug conjugation and
fusion of the disclosed antibodies with other functional
proteins.
[0009] In another aspect, the antibodies of the disclosed invention
can be, for example, full-length antibodies, for example of an
IgG1, IgG2, IgG3, or IgG4 isotype. Alternatively, the disclosed
antibodies can be antibody fragments, such as Fab, Fab' and
F(ab').sub.2 fragments, diabody, triabody, tetrabody, single-chain
variable region fragment (scFv), disulfide-stabilized variable
region fragment (dsFv), and half antibodies. Alternatively, the
disclosed antibodies can be bispecific antibodies.
[0010] In another aspect, this disclosure provides an isolated
monoclonal antibody, or antigen binding portion thereof comprising:
(a) a heavy chain variable region CDR1 comprising SEQ ID NO:3; (b)
a heavy chain variable region CDR2 comprising SEQ ID NO:4; (c) a
heavy chain variable region CDR3 comprising SEQ ID NO:5; (d) a
light chain variable region CDR1 comprising SEQ ID NO:6; (e) a
light chain variable region CDR2 comprising Trp-Ala-Ser; and (f) a
light chain variable region CDR3 comprising SEQ ID NO:7; wherein
said antibody specifically binds to and activates human CD40.
[0011] In another aspect, this disclosure provides an isolated
monoclonal antibody, or antigen binding portion thereof comprising:
(a) a heavy chain variable region CDR1 comprising SEQ ID NO:12; (b)
a heavy chain variable region CDR2 comprising SEQ ID NO:13; (c) a
heavy chain variable region CDR3 comprising SEQ ID NO:14; (d) a
light chain variable region CDR1 comprising SEQ ID NO:15; (e) a
light chain variable region CDR2 comprising Trp-Thr-Ser; and (f) a
light chain variable region CDR3 comprising SEQ ID NO:16; wherein
said antibody specifically binds to and activates human CD40.
[0012] In another aspect, a pharmaceutical composition comprising
isolated monoclonal antibody, or antigen-binding portion thereof
and a pharmaceutically acceptable carrier are also provided.
[0013] In another aspect, the disclosed invention pertains to
methods of stimulating immune responses using the anti-CD40 agonist
antibodies of the disclosed invention. For example, in one
embodiment, the disclosed invention provides a method for treating
a subject in need thereof, wherein said response is indicated by
activation of antigen presenting cells, comprising the step of
administering to the subject an effective amount of the antibody or
antigen-binding portion of the disclosed invention.
[0014] In another aspect, the disclosed invention provides a method
for treating cancer in a human comprising the step of administering
to the human the antibody or antigen-binding portion of the
disclosed invention in an amount effective to treat said
cancer.
[0015] In yet another aspect, the disclosed invention pertains to a
monoclonal antibody or an antigen-binding portion thereof that
specifically binds to and activates human CD40, wherein said
antibody comprises: a heavy chain variable domain amino acid
sequence encoded by a nucleic acid sequence comprising SEQ ID NO:8
or 17 and a light chain variable domain amino acid sequence encoded
by a nucleic acid sequence comprising SEQ ID NOs:9 or 18. In yet
another aspect, the invention also provides nucleic acid molecules
encoding the heavy and/or light chain, or antigen-binding portions
thereof, of an anti-CD40 antibody.
[0016] Other features and advantages of the instant disclosure will
be apparent from the following detailed description and examples,
which should not be construed as limiting. The contents of all
references, GenBank entries, patents and published patent
applications cited throughout this application are expressly
incorporated herein by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1 illustrates antibody heavy chain and light chain
variable domain protein sequences and corresponding DNA sequences
of the disclosed invention encoding the heavy chain and light chain
variable domains of anti-human CD40 agonistic mAb (clone #1E10-H2),
in one embodiment. Heavy chain V domain of 1E10-H2 is SEQ ID NO:1
and nucleic acid sequence is SEQ ID NO:8, Light chain V domain of
1E10-H2 is SEQ ID NO:2 and nucleic acid sequence is SEQ ID
NO:9.
[0018] FIG. 2 illustrates that anti-human CD40 antibodies of the
present disclosure induced activation marker CD83 expression from
human primary monocyte-derived dendritic cells in a co-cultured
reaction assay. Isotype antibody control: mouse anti-BrdU (isotype:
IgG1); mouse anti-CD40 antibodies (isotype: IgG1): clone
#2G9-B11.
[0019] FIG. 3 illustrates that anti-human CD40 antibodies of the
present disclosure induced human primary B-cells proliferation in a
co-cultured reaction assay. Isotype antibody control: mouse
anti-BrdU (isotype: IgG1); mouse anti-CD40 antibodies (isotype:
IgG1): clone #2G9-B11.
DETAILED DESCRIPTION OF THE INVENTION
[0020] The present disclosure relates to an isolated monoclonal
antibody which binds to CD40 and acts as a CD40 agonist. In certain
embodiments, the antibodies of the disclosed invention are derived
from identified heavy and light chain germline sequences and/or
comprise identified structural features such as CDR regions
comprising identified amino acid sequences. This disclosure
provides isolated antibodies, methods of making such antibodies and
antigen-binding portions thereof of the disclosed invention. This
disclosure also relates to methods of using the antibodies, such as
using the anti-CD40 agonist antibodies of the disclosed invention
to stimulate immune responses, alone or in combination with other
immunostimulatory antibodies. Accordingly, also provided are
methods of using the anti-CD40 agonist antibodies of the disclosed
invention for example, including but not limited to, treating
cancer in a human.
[0021] In order that the present disclosure may be more readily
understood, certain terms are first defined. Additional definitions
are set forth throughout the detailed description.
[0022] An "activating antibody" (also referred to herein as an
"agonist antibody") as used herein can refer to an antibody that
increases one or more CD40 activities by at least about 20% when
added to a cell, tissue or organism expressing CD40. In some
embodiments, the antibody activates CD40 activity by at least about
40%, about 50%, about 60%, about 70%, about 80%, and about 85%. In
some embodiments, the activating antibody is added in the presence
of CD40L. In some embodiments, the activity of the activating
antibody is measured using a whole blood surface molecule
upregulation assay. In another embodiment, the activity of the
activating antibody is measured using a dendritic cell assay to
measure IL-12 release. In another embodiment the activity of the
activating antibody is measured using an in vivo tumor model.
[0023] The term "epitope" as used herein can include any protein
determinant capable of specific binding to an immunoglobulin or
T-cell receptor. Epitopic determinants usually consist of
chemically active surface groupings of molecules such as amino
acids or sugar side chains and usually have specific
three-dimensional structural characteristics, as well as specific
charge characteristics. An antibody is said to specifically bind an
antigen when the equilibrium dissociation constant is .ltoreq.1
.mu.M, preferably .ltoreq.100 nM and most preferably .ltoreq.10
nM.
[0024] The term "immune response" as used herein can refer to the
action of, for example, lymphocytes, antigen presenting cells,
phagocytic cells, granulocytes, and soluble macromolecules produced
by the above cells or the liver (including antibodies, cytokines,
and complement) that results in selective damage to, destruction
of, or elimination from an organism of invading pathogens, cells or
tissues infected with pathogens, cancerous cells, or, in cases of
autoimmunity or pathological inflammation, normal organismal cells
or tissues.
[0025] An "antigen-specific T cell response" as used herein can
refer to responses by a T cell that result from stimulation of the
T cell with the antigen for which the T cell is specific.
Non-limiting examples of responses by a T cell upon
antigen-specific stimulation include, but are not limited to,
proliferation and cytokine production (e.g., IL-2 production).
[0026] The term "antibody" as used herein can include whole
antibodies, F(ab')2 fragment, diabody, triabody, tetrabody,
bispecific antibody, monomeric antibodies and any antigen binding
fragment (i.e., "antigen-binding portion") or single-chain variable
region fragment (scFv), or disulfide-stabilized variable region
fragment (dsFv) thereof. Whole antibodies are glycoproteins
comprising at least two heavy (H) chains and two light (L) chains
inter-connected by disulfide bonds. Each heavy chain is comprised
of a heavy chain variable region (abbreviated herein as VH) and a
heavy chain constant region. The heavy chain constant region is
comprised of three domains, CH1, CH2 and CH3. Each light chain is
comprised of a light chain variable region (abbreviated herein as
VL) and a light chain constant region (CL). The light chain
constant region is comprised of one domain, CL. The VH and VL
regions can be further subdivided into regions of hypervariability,
termed complementarity determining regions (CDR), interspersed with
regions that are more conserved, termed framework regions (FR).
Each VH and VL is composed of three CDRs and four FRs, arranged
from amino-terminus to carboxy-terminus in the following order:
FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the
heavy and light chains contain a binding domain that interacts with
an antigen. The constant regions of the antibodies can mediate the
binding of the immunoglobulin to host tissues or factors, including
various cells of the immune system (e.g., effector cells) and the
first component (Clq) of the classical complement system.
[0027] The term "antigen-binding portion" of an antibody (or simply
"antibody portion"), as used herein, can refer to one or more
fragments of an antibody that retain the ability to specifically
bind to an antigen (e.g., a CD40 protein). It has been shown that
the antigen-binding function of an antibody can be performed by
fragments of a full-length antibody. Examples of binding fragments
encompassed within the term "antigen binding portion" of an
antibody include (i) a Fab fragment, a monovalent fragment
consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab')2
fragment, a bivalent fragment comprising two Fab fragments linked
by a disulfide bridge at the hinge region; (iii) a Fab' fragment,
which is essentially a Fab with part of the hinge region (see,
FUNDAMENTAL IMMUNOLOGY (Paul ed., 3.sup.rd ed. 1993); (iv) a Fd
fragment consisting of the VH and CH1 domains; (v) a Fv fragment
consisting of the VL and VH domains of a single arm of an antibody,
(vi) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which
consists of a VH domain; (vii) an isolated complementarity
determining region (CDR); and (viii) a nanobody, a heavy chain
variable region containing a single variable domain and two
constant domains. Furthermore, although the two domains of the Fv
fragment, VL and VH are encoded by separate genes, they can be
joined, using recombinant methods, by a synthetic linker that
enables them to be made as a single protein chain in which the VL
and VH regions pair to form monovalent molecules (known as single
chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426;
and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883).
Such single chain antibodies are also intended to be encompassed
within the term "antigen-binding portion" of an antibody. These
antibody fragments are obtained using conventional techniques known
to those of skill in the art, and the fragments are screened for
utility in the same manner as intact antibodies.
[0028] An "isolated antibody", as used herein, can refer to an
antibody that is substantially free of other antibodies having
different antigenic specificities (e.g., an isolated antibody that
specifically binds a CD40 protein can be substantially free of
antibodies that specifically bind antigens other than CD40
proteins). An isolated antibody that specifically binds a human
CD40 protein can, however, have cross-reactivity to other antigens,
such as CD40 proteins from other species. Moreover, an isolated
antibody can be substantially free of other cellular material
and/or chemicals.
[0029] Anti-CD40 agonist antibody-producing cells, e.g.,
hybridomas, can be selected, cloned and further screened for
desirable characteristics, including robust growth, high antibody
production and desirable antibody characteristics, as further
discussed below. Hybridomas can be expanded in vivo in syngeneic
animals, in animals that lack an immune system, e.g., nude mice, or
in cell culture in vitro. Methods of selecting, cloning and
expanding hybridomas are well known to those of ordinary skill in
the art.
[0030] The terms "monoclonal antibody" or "monoclonal antibody
composition" as used herein can refer to a preparation of antibody
molecules of single molecular composition. A monoclonal antibody
composition displays a single binding specificity and affinity for
a particular epitope.
[0031] The term "recombinant human antibody", as used herein, can
refer to all human antibodies that are prepared, expressed, created
or isolated by recombinant means, such as (a) antibodies isolated
from an animal (e.g., a mouse) that is transgenic or
transchromosomal for human immunoglobulin genes or a hybridoma
prepared therefrom (described below), (b) antibodies isolated from
a host cell transformed to express the human antibody, e.g., from a
transfectoma, (c) antibodies isolated from a recombinant,
combinatorial human antibody library, and (d) antibodies prepared,
expressed, created or isolated by any other means that involve
splicing of human immunoglobulin gene sequences to other DNA
sequences. Such recombinant human antibodies have variable regions
in which the framework and CDR regions are derived from human
germline immunoglobulin sequences. In certain embodiments, however,
such recombinant human antibodies can be subjected to in vitro
mutagenesis (or, when an animal transgenic for human Ig sequences
is used, in vivo somatic mutagenesis) and thus the amino acid
sequences of the VH and VL regions of the recombinant antibodies
are sequences that, while derived from and related to human
germline VH and VL sequences, may not naturally exist within the
human antibody germline repertoire in vivo.
[0032] The term "isotype" can refer to the antibody class (e.g.,
IgM or IgG1) that is encoded by the heavy chain constant region
genes.
[0033] The phrases "an antibody recognizing an antigen" and "an
antibody specific for an antigen" are used interchangeably herein
with the term "an antibody which binds specifically to an
antigen."
[0034] The term "humanized antibody" can refer to antibodies in
which CDR sequences derived from the germline of another mammalian
species, such as a mouse, have been grafted onto human framework
sequences. Additional framework region modifications can be made
within the human framework sequences.
[0035] The term "chimeric antibody" can refer to antibodies in
which the variable region sequences can be derived from one species
and the constant region sequences can be derived from another
species, such as an antibody in which the variable region sequences
can be derived from a mouse antibody and the constant region
sequences can be derived from a human antibody.
[0036] The term "surface plasmon resonance", as used herein, can
refer to an optical phenomenon that allows for the analysis of
real-time biospecific interactions by detection of alterations in
protein concentrations within a biosensor matrix, for example using
the BIAcore.TM. system (Pharmacia Biosensor AB, Uppsala, Sweden and
Piscataway, N.J.). For further descriptions, see Jonsson U. et al.,
Ann. Biol. Clin. 51:19-26 (1993); Jonsson U. et al., Biotechniques
11:620-627 (1991); Jonsson B. et al., J. Mal. Recognit. 8:125-131
(1995); and Johnsson B. et al., Anal. Biochem. 198:268-277
(1991).
[0037] The term "K.sub.D" can refer to the equilibrium dissociation
constant of a particular antibody-antigen interaction.
[0038] As used herein, an antibody that "specifically binds human
CD40" can refer to an antibody that binds to a human CD40 protein
(and possibly a CD40 protein from one or more non-human species)
but does not substantially bind to non-CD40 proteins. Preferably,
the antibody binds to a human CD40 protein with "high affinity,"
namely with a EC.sub.50 of 1.times.10.sup.-7 M or less, more
preferably 5.times.10.sup.-8 M or less, more preferably
3.times.10.sup.-8 M or less, more preferably 1.times.10.sup.-8 M or
less, more preferably 5.times.10.sup.-9M or less or even more
preferably 1.times.10.sup.-9 M or less.
[0039] The term "does not substantially bind" to a protein or
cells, as used herein, can mean that it cannot bind or does not
bind with a high affinity to the protein or cells, i.e., binds to
the protein or cells with an EC.sub.50 of 2.times.10.sup.-6 M or
more, more preferably 1.times.10.sup.-5 M or more, more preferably
1.times.10.sup.-4 M or more, more preferably 1.times.10.sup.-3 M or
more, even more preferably 1.times.10.sup.-2 M or more.
[0040] The term "high affinity" for an IgG antibody can refer to an
antibody having an EC.sub.50 of 1.times.10.sup.-6 M or less, more
preferably 1.times.10.sup.-7 M or less, even more preferably
1.times.10.sup.-8 M or less, even more preferably 1.times.10.sup.-9
M or less, even more preferably 1.times.10.sup.-1.degree. M or less
for a target antigen. However, "high affinity" binding can vary for
other antibody isotypes.
[0041] The term "subject" can refer to any human or non-human
animal. The term "nonhuman animal" includes all vertebrates, e.g.,
mammals and non-mammals, such as nonhuman primates, sheep, dogs,
cats, cows, horses, chickens, rabbits, mice, rats, amphibians, and
reptiles, although mammals are preferred, such as non-human
primates, sheep, dogs, cats, cows and horses.
[0042] The binding of an antibody of the disclosed invention to
CD40 can be assessed using one or more techniques well established
in the art. For example, in a preferred embodiment, an antibody can
be tested by ELISA assays, for example using a recombinant CD40
protein. Still other suitable binding assays include but are not
limited to a flow cytometry assay in which the antibody is reacted
with a cell line that expresses human CD40, such as CHO cells that
have been transfected to express CD40 (e.g., human CD40) on their
cell surface. Additionally or alternatively, the binding of the
antibody, including the binding kinetics (e.g., K.sub.D value) can
be tested in BIAcore binding assays and the like.
[0043] Preferably, an antibody of the disclosed invention binds to
a CD40 protein with an EC.sub.50 of 5.times.10.sup.-8 M or less,
binds to a CD40 protein with a EC.sub.50 of 2.times.10.sup.-8 M or
less, binds to a CD40 protein with a EC.sub.50 of 5.times.10.sup.-9
M or less, binds to a CD40 protein with a EC.sub.50 of
4.times.10.sup.-9 M or less, binds to a CD40 protein with a
EC.sub.50 of 3.times.10.sup.-9 M or less, binds to a CD40 protein
with a EC.sub.50 of 2.times.10.sup.-9 M or less, binds to a CD40
protein with a EC.sub.50 of 1.times.10.sup.-9 M or less.
[0044] Antibodies can be isolated and structurally characterized as
described in Example 1. The VH amino acid sequence of clone
#1E10-H2 is shown in SEQ ID NO:1. The VL amino acid sequence of
clone #1E10-H2 is shown in SEQ ID NO:2. The VH amino acid sequence
of clone #2G9-B11 is shown in SEQ ID NO:10. The VL amino acid
sequence of clone #2G9-B11 is shown in SEQ ID NO:11.
[0045] Accordingly, in one aspect, this disclosure provides an
isolated monoclonal antibody, or antigen-binding portion thereof
comprising: (a) a heavy chain variable region comprising an amino
acid sequence comprising SEQ ID NO:1; and (b) a light chain
variable region comprising an amino acid sequence comprising SEQ ID
NO:2; wherein the antibody specifically binds and activates human
CD40.
[0046] In another aspect, this disclosure provides an isolated
monoclonal antibody, or antigen-binding portion thereof comprising:
(a) a heavy chain variable region comprising an amino acid sequence
comprising SEQ ID NO:10; and (b) a light chain variable region
comprising an amino acid sequence comprising SEQ ID NO:11; wherein
the antibody specifically binds and activates human CD40. In some
embodiments, the monoclonal antibody, or an antigen-binding portion
thereof stimulates an anti-tumor immune response. In some
embodiments, the monoclonal antibody can be a chimeric
antibody.
[0047] In another aspect, this disclosure provides antibodies that
comprise the heavy chain and light chain CDR1, CDR2 and CDR3 of
clone #1E10-H2. The amino acid sequence of the VH CDR1 of clone
#1E10-H2 is shown in SEQ ID NO:3. The amino acid sequence of the VH
CDR2 of clone #1E10-H2 is shown in SEQ ID NO:4. The amino acid
sequence of the VH CDR3 of clone #1E10-H2 is shown in SEQ ID NO:5.
The amino acid sequences of the VL CDR1 of clone #1E10-H2 is shown
in SEQ ID NO:6. The amino acid sequences of the VL CDR2s of clone
#1E10-H2 is Trp-Ala-Ser. The amino acid sequences of the VL CDR3s
of clone #1E10-H2 is shown in SEQ ID NO:7.
[0048] In another aspect, this disclosure provides antibodies that
comprise the heavy chain and light chain CDR1, CDR2 and CDR3 of
clone #2G9-B11. The amino acid sequence of the VH CDR1 of clone
#2G9-B11 is shown in SEQ ID NO:12. The amino acid sequence of the
VH CDR2 of clone #2G9-B11 is shown in SEQ ID NO:13. The amino acid
sequence of the VH CDR3 of clone #2G9-B11 is shown in SEQ ID NO:14.
The amino acid sequences of the VL CDR1 of clone #2G9-B11 is shown
in SEQ ID NO:15. The amino acid sequences of the VL CDR2s of clone
#2G9-B11 is Trp-Thr-Ser. The amino acid sequences of the VL CDR3s
of clone #2G9-B11 is shown in SEQ ID NO:16. The CDR regions can be
delineated using the Kabat system (Kabat et al. (1991) Sequences of
Proteins of Immunological Interest, Fifth Edition, U.S. Department
of Health and Human Services, NIH Publication No. 91-3242).
[0049] In another aspect, this disclosure provides polynucleotide
sequences encoding the heavy chain and light chain variable domains
of the monoclonal antibody clone #1E10-H2. In another aspect, this
disclosure provides polynucleotide sequences encoding the heavy
chain and light chain variable domains of the monoclonal antibody
clone #2G9-B11.
[0050] Antibodies can be affinity maturated by light-chain
shuffling combined with or without random mutagenesis of its heavy
chain variable domain and panning against CD40. The VL CDR1, CDR2
and CDR3 of the antibodies mentioned in this disclosed invention
can be optimized with light-chain shuffling to create other
anti-CD40 agonist binding molecules of the disclosed invention.
[0051] An antibody of the disclosed invention further can be
prepared using an antibody having one or more of the VH and/or VL
sequences disclosed herein as starting material to engineer a
modified antibody, which modified antibody can have altered
properties from the starting antibody. An antibody can be
engineered by modifying one or more residues within one or both
variable regions (i.e., VH and/or VL), for example within one or
more CDR regions and/or within one or more framework regions.
Additionally, or alternatively, an antibody can be engineered by
modifying residues within the constant region(s), for example to
alter the effector function(s) of the antibody.
[0052] In certain embodiments, CDR grafting can be used to engineer
variable regions of antibodies. Antibodies interact with target
antigens predominantly through amino acid residues that are located
in the six heavy and light chain complementarity determining
regions (CDRs). For this reason, the amino acid sequences within
CDRs can be more diverse between individual antibodies than
sequences outside of CDRs.
[0053] Because CDR sequences can be responsible for most
antibody-antigen interactions, it can be possible to express
recombinant antibodies that mimic the properties of specific
naturally occurring antibodies by constructing expression vectors
that include CDR sequences from the specific naturally occurring
antibody grafted onto framework sequences from a different antibody
with different properties (see, e.g., Riechmann et al. (1998)
Nature 332:323-327; Jones et al. (1986) Nature 321: 522-525; Queen
et al. (1989) Proc. Natl. Acad. See. U.S.A. 86: 10029-10033; U.S.
Pat. Nos. 5,225,539; 5,530,101; 5,585,089; 5,693,762 and
6,180,370.)
[0054] Accordingly, another embodiment of the disclosed invention
pertains to an isolated monoclonal antibody, or antigen-binding
portion thereof, comprising a heavy chain variable region
comprising CDR1, CDR2, and CDR3 sequences comprising an amino acid
sequence of SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5,
respectively, and a light chain variable region a comprising CDR1,
CDR2, and CDR3 sequences comprising an amino acid sequence of SEQ
ID NO:6, Trp-Ala-Ser, and SEQ ID NO:7, respectively. Another
embodiment of the disclosed invention pertains to an isolated
monoclonal antibody, or antigen-binding portion thereof, comprising
a heavy chain variable region comprising CDR1, CDR2, and CDR3
sequences comprising an amino acid sequence of SEQ ID NO:12, SEQ ID
NO:13, and SEQ ID NO:14, respectively, and a light chain variable
region a comprising CDR1, CDR2, and CDR3 sequences comprising an
amino acid sequence of SEQ ID NO:15, Trp-Thr-Ser, and SEQ ID NO:16,
respectively. Thus, such antibodies contain the VH and VL CDR
sequences of monoclonal antibody 1E10-H2 or 2G9-B11 and can contain
different framework sequences from these antibodies.
[0055] Such framework sequences can be obtained from public DNA
databases or published references that include germline antibody
gene sequences. For example, germline DNA sequences for human heavy
and light chain variable region genes can be found in the "VBase"
human germline sequence database (available on the Internet at
www.mrc-cpe.cam.ac.uk/vbase), as well as in Kabat et al. (1991),
cited supra; Tomlinson et al. (1992) "The Repertoire of Human
Germline VH Sequences Reveals about Fifty Groups of VH Segments
with Different Hypervariable Loops" J. Mol. Biol. 227:776-798; and
Cox et al. (1994) "A Directory of Human Germ-line VH Segments
Reveals a Strong Bias in their Usage" Eur. J. Immunol. 24:827-836;
the contents of each of which are expressly incorporated herein by
reference. As another example, the germline DNA sequences for human
heavy and light chain variable region genes can be found in the
GenBank database. For example, the following heavy chain germline
sequences found in the HCo7 HuMAb mouse are available in the
accompanying GenBankAccession NOS.:1-69 (NG_0010109, NT_024637
& BC070333), 3-33 (NG_0010109 & NT_024637) and 3-7
(NG_0010109 &NT_024637). As another example, the following
heavy chain germline sequences found in the HCo12 HuMAb mouse are
available in the accompanying GenBankAccession NOS.: 1-69
(NG_0010109, NT_024637 & BC070333), 5-51 (NG_0010109 &
NT_024637), 4-34 (NG_0010109 & NT_024637), 3-30.3 (CAJ556644)
& 3-23 (AJ406678).
[0056] Antibody protein sequences are compared against a compiled
protein sequence database using one of the sequence similarity
searching methods called the Gapped BLAST (Altschul et al. (1997),
supra), which is well known to those skilled in the art. The
compositions and methods of the presently disclosed invention are
not limited to variants of the exemplary sequences disclosed herein
but include those having at least 90%, at least 95% and at least
99% sequence identity to an exemplary sequence disclosed
herein.
[0057] A nucleic acid molecule encoding the heavy or entire light
chain of an anti-CD40 agonist antibody or portions thereof can be
isolated from any source that produces such an antibody. In various
embodiments, the nucleic acid molecules are isolated from a B cell
isolated from an animal immunized with CD40 or from an immortalized
cell derived from such a B cell that expresses an anti-CD40 agonist
antibody. Methods of isolating mRNA encoding an antibody are
well-known in the art. See, e.g., Sambrook et al. The mRNA may be
used to produce cDNA for use in the polymerase chain reaction (PCR)
or cDNA cloning of antibody genes. In a preferred embodiment, the
nucleic acid molecule is isolated from a hybridoma that has as one
of its fusion partners a human immunoglobulin producing cell from a
non-human transgenic animal. In another embodiment, the nucleic
acid can be isolated from a non-human, non-transgenic animal. The
nucleic acid molecules isolated from a non-human, nontransgenic
animal may be used, e.g., for humanized antibodies.
[0058] In another aspect, the present disclosure provides a
pharmaceutical composition comprising one or more antibodies of the
present invention formulated together with a pharmaceutically
acceptable carrier. The composition may optionally contain one or
more additional pharmaceutically active ingredients, such as
another antibody or a drug. The pharmaceutical compositions of the
disclosed invention also can be administered in combination therapy
with, for example, another immunostimulatory agent, an anti-cancer
agent, an antiviral agent, or a vaccine, such that the anti-CD40
agonist antibody enhances the immune response stimulated by the
vaccine.
[0059] The pharmaceutical composition can comprise any number of
excipients. Excipients that can be used include carriers, surface
active agents, thickening or emulsifying agents, solid binders,
dispersion or suspension aids, solubilizers, colorants, flavoring
agents, coatings, disintegrating agents, lubricants, sweeteners,
preservatives, isotonic agents, and combinations thereof. The
selection and use of suitable excipients are taught in Gennaro,
ed., Remington: The Science and Practice of Pharmacy, 20th Ed.
(Lippincott Williams & Wilkins 2003), the disclosure of which
is incorporated herein by reference.
[0060] Therapeutic compositions typically must be sterile and
stable under the conditions of manufacture and storage. The
composition can be formulated as a solution, microemulsion,
dispersion, liposome, or other ordered structure suitable to high
drug concentration. Sterile injectable solutions can be prepared by
incorporating the anti-CD40 agonist antibody in the required amount
in an appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the active
compound into a sterile vehicle that contains a basic dispersion
medium and the required other ingredients from those enumerated
above. In the case of sterile powders for the preparation of
sterile injectable solutions, the preferred methods of preparation
are vacuum drying and freeze-drying that yields a powder of the
active ingredient plus any additional desired ingredient from a
previously sterile-filtered solution thereof. The proper fluidity
of a solution can be maintained, for example, by the use of a
coating such as lecithin, by the maintenance of the required
particle size in the case of dispersion and by the use of
surfactants. Prolonged absorption of injectable compositions can be
brought about by including in the composition an agent that delays
absorption, for example, monostearate salts and gelatin.
[0061] The antibodies of the present invention can be administered
by a variety of methods known in the art, although for many
therapeutic applications, the preferred route/mode of
administration is subcutaneous, intramuscular, or intravenous
infusion. As will be appreciated by the skilled artisan, the route
and/or mode of administration will vary depending upon the desired
results.
[0062] In certain embodiments, the antibody compositions active
compound may be prepared with a carrier that will protect the
antibody against rapid release, such as a controlled release
formulation, including implants, transdermal patches, and
microencapsulated delivery systems. Biodegradable, biocompatible
polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Many methods for the preparation of such
formulations are patented or generally known to those skilled in
the art. See, e.g., Sustained and Controlled Release Drug Delivery
Systems (J. R. Robinson, ed., Marcel Dekker, Inc., New York,
1978).
[0063] In certain embodiments, an anti-CD40 agonist antibody of the
disclosed invention can be orally administered, for example, with
an inert diluent or an assimilable edible carrier. The compound
(and other ingredients, if desired) can also be enclosed in a hard
or soft shell gelatin capsule, compressed into tablets, or
incorporated directly into the subject's diet. For oral therapeutic
administration, the anti-CD40 agonist antibodies can be
incorporated with excipients and used in the form of ingestible
tablets, buccal tablets, troches, lozenge, capsules, elixirs,
suspensions, syrups, wafers, and the like. To administer a compound
of the disclosed invention by other than parenteral administration,
it may be necessary to coat the compound with, or co-administer the
compound with, a material to prevent its inactivation.
[0064] Additional active compounds also can be incorporated into
the disclosed compositions. In certain embodiments, an anti-CD40
agonist antibody of the disclosed invention is co-formulated with
and/or co-administered with one or more additional therapeutic
agents. These agents include, without limitation, antibodies that
bind other targets (e.g., antibodies that bind one or more growth
factors or cytokines or their cell surface receptors, such as
anti-CTL4-antibody), antineoplastic agents, antitumor agents,
chemotherapeutic agents, peptide analogues that activate CD40,
soluble CD40L, one or more chemical agents that activates CD40,
and/or other agents known in the art that can enhance an immune
response against tumor cells, e.g., IFN-1, IL-2, IL-8, IL-12,
IL-15, IL-18, IL-23, IFN-.gamma., and GM-CSF. Such combination
therapies may require lower dosages of the anti-CD40 agonist
antibody as well as the co-administered agents, thus avoiding
possible toxicities or complications associated with the various
monotherapies.
[0065] Agonist anti-CD40 antibodies of the disclosed invention and
compositions comprising them also may be administered in
combination with other therapeutic regimens, in particular in
combination with radiation treatment.
[0066] The pharmaceutical compositions of the disclosed invention
can include pharmaceutically acceptable salts. A "pharmaceutically
acceptable salt" can refer to a salt that retains the desired
biological activity of the parent compound and does not impart any
undesired toxicological effects. Examples of such salts include
acid addition salts and base addition salts. Acid addition salts
include those derived from nontoxic inorganic acids, such as
hydrochloric, nitric, phosphoric, sulfuric, hydrobromic,
hydroiodic, phosphorous and the like, as well as from nontoxic
organic acids such as aliphatic mono- and dicarboxylic acids,
phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic
acids, aliphatic and aromatic sulfonic acids and the like. Base
addition salts include those derived from alkaline earth metals,
such as sodium, potassium, magnesium, calcium and the like, as well
as from nontoxic organic amines, such as
N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,
choline, diethanolamine, ethylenediamine, procaine and the
like.
[0067] Dosage regimens are adjusted to provide the optimum desired
response (e.g., a therapeutic response). For example, a single
bolus can be administered, several divided doses can be
administered over time or the dose can be proportionally reduced or
increased as indicated by the exigencies of the therapeutic
situation. It is especially advantageous to formulate parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the
subjects to be treated; each unit contains a predetermined quantity
of active compound calculated to produce the desired therapeutic
effect in association with the required pharmaceutical carrier.
Alternatively, the antibody can be administered as a sustained
release formulation, in which case less frequent administration can
be required.
[0068] An exemplary, non-limiting range for a therapeutically or
prophylactically effective amount of an antibody or antibody
portion of the disclosed invention is 0.025 to 50 mg/kg, more
preferably 0.1 to 50 mg/kg, more preferably 0.1 to 25 mg/kg, 0.1 to
10 mg/kg and 0.1 to 3 mg/kg. It is to be noted that dosage values
may vary with the type and severity of the condition to be
alleviated. It is to be further understood that for any particular
subject, specific dosage regimens should be adjusted over time
according to the individual need and the professional judgment of
the person administering or supervising the administration of the
compositions, and that dosage ranges set forth herein are exemplary
only and are not intended to limit the scope or practice of the
claimed composition.
[0069] Bispecific antibodies or antigen-binding fragments can be
produced by a variety of methods including fusion of hybridomas or
linking of Fab' fragments. See, e.g., Songsivilai & Lachmarm,
Clin. Exp. Immunol. 79: 315-321 (1990), Kos-telny et al., J.
Immunol. 148:1547-1553 (1992). In addition, bispecific antibodies
can be formed as "diabodies" or "Janusins." In some embodiments,
the bispecific antibody binds to two different epitopes of CD40. In
some embodiments, the bispecific antibody has a first heavy chain
and a first light chain from monoclonal antibody 1E10-H2, and an
additional antibody heavy chain and light chain. In some
embodiments, the bispecific antibody has a first heavy chain and a
first light chain from monoclonal antibody 2G9-B11, and an
additional antibody heavy chain and light chain. In some
embodiments, the additional light chain and heavy chain also are
from one of the above-identified monoclonal antibodies, but can be
different from the first heavy and light chains.
Example 1. Immunization and Hybridoma Generation
[0070] Antibodies were created by electrofusion of human
CD40-immunized mouse splenocytes (Balb/c strain) with SP2/0-A14
cells (ATCC) (Immunization procedure; dose, adjuvants, time
course). Fused cells were seeded into 96-well plates and ELISA
screening was conducted to identify wells that contain antibodies
which interact with the human CD40 protein. Positive wells were
further expanded and followed a limited dilution to isolate
monoclonal hybridomas. Purified antibodies were used to test their
ability to trigger CD40 signaling by an in-house created reporter
system. Basically, an engineered 293T cell line was transduced to
stably express the human CD40 protein. When testing the agonistic
functions of CD40 antibodies, DNA plasmids of an NF-kB-driven
luciferase reporter and Fc-gamma receptors were transiently
transfected into established CD40-293T cells. Antibodies to be
tested were added to the culture and incubated for 24 hours.
Treated cells were harvested, lysed and analyzed on a plate reader
for the activity of luciferase. Anti-human CD40 mAb clone #1E10-H2
and clone #2G9-B11 were tested binding to and activating human
CD40. The sequences of the IgG heavy chain and light chain variable
domains of anti-human CD40 agonistic mAb clone #1E10-H2 are shown
in FIG. 1.
Example 2. Anti-Human CD40 Antibody In-Vitro Assay
[0071] Frozen human primary monocytes (CD14+; negative selection)
were differentiated into dendritic cells (DC) by culturing with
rIL-4 and rGM-CSF for 5 days. 5.times.10.sup.5 DCs were then
co-cultured with 1.times.10.sup.5 293 FT cells transient
transfected with FcgR in 24-well plate, with serial diluted dosages
of anti-CD40 2G9-B11 antibodies or fixed dosage of isotype control
antibody, for additional 2 days. Cells were blocked with TruStain
FcX antibody (BioLegend), followed by staining with PE/Cy5
anti-CD11c antibody (BioLegend, clone 3.9, mouse IgG1) and
PE/Dazzle 594 anti-CD83 antibody (BioLegend, clone HB15e, mouse
IgG1). Flow cytometry was performed using BD LSRII flow cytometer.
Results are shown in FIG. 2, which demonstrate that anti-CD40
antibodies induced activation marker CD83 expression from human
primary monocyte-derived dendritic cells in a co-cultured reaction
assay.
Example 3. Anti-Human CD40 Antibody In-Vitro Assay
[0072] Human primary B-cells isolated from human PBMC (Miltenyi
Biotec, negative selection) were labeled with CFSE (BioLegend) and
co-cultured with 1.times.10.sup.5 293 FT cells transient
transfected with FcgR in 24-well plate, with fixed dosage of
antibodies, for 7 days. Cells were blocked with TruStain FcX
antibody (BioLegend), followed by staining with APC goat anti-human
IgG (H+L) antibody (Jackson ImmunoResearch). Flow cytometry was
performed using BD LSRII flow cytometer. Results are shown in FIG.
3, which demonstrate that anti-CD40 antibodies induced human
primary B-cells proliferation in a co-cultured reaction assay.
[0073] Although the above invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it is apparent to those skilled in the art that
certain changes and modifications will be practiced. Therefore, the
description and examples should not be construed as limiting the
scope of the disclosed invention.
[0074] The disclosures of all publications, patents, patent
applications and published patent applications referred to herein
by an identifying citation are hereby incorporated herein by
reference in their entirety. Websites references using
"World-Wide-Web" at the beginning of the Uniform Resource Locator
(URL) can be accessed by replacing "World-Wide-Web" with www.
Sequence CWU 1
1
181121PRTArtificial Sequencemonoclonal antibody 1Gln Val Gln Leu
Gln Gln Ser Gly Ala Glu Leu Met Lys Pro Gly Ala1 5 10 15Ser Val Lys
Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe Ser Ser Tyr 20 25 30Trp Ile
Glu Trp Ile Lys Gln Arg Pro Gly His Val Leu Glu Trp Ile 35 40 45Gly
Glu Ile Leu Pro Gly Ser Gly Ser Ala Asn Tyr Asn Glu Arg Phe 50 55
60Lys Gly Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr65
70 75 80Met Gln Leu Thr Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr
Cys 85 90 95Ala Arg Leu Arg Tyr Tyr Gly Ser Ser Pro Trp Phe Ala Tyr
Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ala 115
1202113PRTArtificial Sequenceengineered mAb 2Asp Ile Val Met Ser
Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly1 5 10 15Glu Lys Val Thr
Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30Ser Asn Gln
Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45Ser Pro
Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60Pro
Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr65 70 75
80Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Leu Tyr Tyr Cys Gln Gln
85 90 95Tyr Tyr Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu
Ile 100 105 110Lys38PRTArtificial Sequenceengineered mAb 3Gly Tyr
Thr Phe Ser Ser Tyr Trp1 548PRTArtificial Sequenceengineered mAb
4Ile Leu Pro Gly Ser Gly Ser Ala1 5514PRTArtificial
Sequenceengineered mAb 5Ala Arg Leu Arg Tyr Tyr Gly Ser Ser Pro Trp
Phe Ala Tyr1 5 10612PRTArtificial Sequenceengineered mAb 6Gln Ser
Leu Leu Tyr Ser Ser Asn Gln Lys Asn Tyr1 5 1079PRTArtificial
Sequenceengineered mAb 7Gln Gln Tyr Tyr Ser Tyr Pro Tyr Thr1
58363DNAArtificial Sequencemonoclonal antibody plasmid 8caggttcagc
tgcagcagtc tggagctgaa ctgatgaagc ctggggcctc agtgaagata 60tcctgcaagg
ctactggcta cacattcagt agctactgga tagagtggat aaagcagagg
120cctggacatg tccttgagtg gattggagag attttacctg gaagtggtag
tgctaactac 180aatgaaaggt tcaagggcaa ggccacattc actgcagata
catcctccaa cacagcctac 240atgcaactca ccagcctgac atctgaggac
tctgccgtct attactgtgc aagattgagg 300tactacggta gtagcccctg
gtttgcttac tggggccaag ggactctggt cactgtctct 360gca
3639339DNAArtificial Sequencevector encoding the protein sequence
9gacattgtga tgtcacagtc tccatcctcc ctagctgtgt cagttggaga gaaggttact
60atgagctgca agtccagtca gagcctttta tatagtagca atcaaaagaa ctacttggcc
120tggtaccagc agaaaccagg gcagtctcct aaactgctga tttactgggc
atccactagg 180gaatctgggg tccctgatcg cttcacaggc agtggatctg
ggacagattt cactctcacc 240atcagcagtg tgaaggctga agacctggca
ctttattact gtcagcaata ttatagctat 300ccgtacacgt tcggaggggg
gaccaagctg gaaataaaa 33910115PRTArtificial SequencemAb 10Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser
Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser Ser Tyr 20 25
30Ala Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val
35 40 45Ala Thr Ile Ser Ser Gly Gly Asp Tyr Thr Tyr Tyr Pro Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr
Leu Tyr65 70 75 80Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala
Met Tyr Tyr Cys 85 90 95Ala Arg Arg Asn His Phe Thr Tyr Trp Gly Gln
Gly Thr Leu Val Thr 100 105 110Val Ser Ala 11511107PRTArtificial
SequencemAb 11Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr
Ser Val Gly1 5 10 15Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp
Val Val Thr Ser 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser
Pro Lys Leu Leu Ile 35 40 45Tyr Trp Thr Ser Thr Arg His Thr Gly Val
Pro Asp Arg Phe Thr Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Asn Val Gln Ser65 70 75 80Glu Asp Leu Ala Asp Tyr Phe
Cys Gln Gln Tyr Ser Ser Tyr Pro Leu 85 90 95Thr Phe Gly Ala Gly Thr
Lys Leu Glu Leu Lys 100 105128PRTArtificial SequencemAb 12Gly Phe
Ser Phe Ser Ser Tyr Ala1 5138PRTArtificial SequencemAb 13Ile Ser
Ser Gly Gly Asp Tyr Thr1 5148PRTArtificial SequencemAb 14Ala Arg
Arg Asn His Phe Thr Tyr1 5156PRTArtificial SequencemAb 15Gln Asp
Val Val Thr Ser1 5169PRTArtificial SequencemAb 16Gln Gln Tyr Ser
Ser Tyr Pro Leu Thr1 517345DNAArtificial SequencemAb DNA sequence
17gaagtgcagc tggtggagtc tgggggaggc ttagtgaagc ctggagggtc cctgaaactc
60tcctgtgcag cctctggatt ctctttcagt agctatgcca tgtcttgggt tcgccagact
120ccggagaaga ggctggagtg ggtcgcaacc attagtagtg gtggtgatta
cacctactat 180ccagacagtg tgaagggtcg attcaccatc tccagagaca
atgccaagaa caccctgtac 240ctgcaaatga ccagtctgag gtctgaggac
acggccatgt attactgtgc aagacgaaat 300cattttactt actggggcca
agggactctg gtcactgtct ctgca 34518321DNAArtificial SequencemAb DNA
sequence 18gacattgtga tgacccagtc tcacaaattc atgtccacat cagtaggaga
cagggtcagc 60atcacctgca aggccagtca ggatgtggtt acttctgttg cctggtatca
acagaaacca 120gggcaatctc ctaaactact gatttactgg acatccaccc
ggcacactgg agtccctgat 180cgcttcacag gcagtggatc tgggacagat
ttcactctca ccattagcaa tgtgcagtct 240gaagacttgg cagattattt
ctgtcagcaa tatagcagct atccgctcac gttcggtgct 300gggaccaagc
tggagctgaa a 321
* * * * *
References