U.S. patent application number 17/290754 was filed with the patent office on 2022-01-06 for bispecific antibody binding to cd20 and cd3 and uses thereof.
The applicant listed for this patent is AMPSOURCE BIOPHARMA SHANGHAI INC.. Invention is credited to Si Chen, Xueyuan Cui, Shixiang Jia, Qiang Li, Xinlu MA, Yuhua Zhang.
Application Number | 20220002431 17/290754 |
Document ID | / |
Family ID | |
Filed Date | 2022-01-06 |
United States Patent
Application |
20220002431 |
Kind Code |
A1 |
Li; Qiang ; et al. |
January 6, 2022 |
BISPECIFIC ANTIBODY BINDING TO CD20 AND CD3 AND USES THEREOF
Abstract
Disclosed is a bispecific antibody that specifically binds to
surface antigens CD3 of immune cells and CD20 antigens on the
surfaces of tumor cells, and that can bind to human CD3 with high
affinity, inducing T cell proliferation, and mediating tumor cell
killing. The bispecific antibody in an in vitro test was able to
mediate the specific killing of target cells by T cells. The
construction method thereof is simple, avoiding the possibility of
mismatch between two sets of light chains and heavy chains of
heterobispecific antibodies, thereby reducing the difficulty of
antibody purification. The affinity of the obtained antibody is
high, the side effects caused by cytokines are small, and safety is
high.
Inventors: |
Li; Qiang; (Shanghai,
CN) ; Jia; Shixiang; (Shanghai, CN) ; MA;
Xinlu; (Shanghai, CN) ; Cui; Xueyuan;
(Shanghai, CN) ; Zhang; Yuhua; (Shanghai, CN)
; Chen; Si; (Shanghai, CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AMPSOURCE BIOPHARMA SHANGHAI INC. |
Shanghai |
|
CN |
|
|
Appl. No.: |
17/290754 |
Filed: |
October 29, 2019 |
PCT Filed: |
October 29, 2019 |
PCT NO: |
PCT/CN2019/113930 |
371 Date: |
April 30, 2021 |
International
Class: |
C07K 16/28 20060101
C07K016/28; C07K 16/32 20060101 C07K016/32; C07K 16/46 20060101
C07K016/46; C12N 15/85 20060101 C12N015/85; A61P 35/00 20060101
A61P035/00 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 1, 2018 |
CN |
201811294887.4 |
Claims
1. A bispecific antibody, which is a tetravalent homodimer formed
by two identical polypeptide chains that bind to each other by a
covalent bond, wherein each of the polypeptide chains comprises a
first single-chain Fv that specifically binds to tumor antigen
CD20, a second single-chain Fv that specifically binds to effector
cell antigen CD3 and an Fc fragment in sequence from N-terminus to
C-terminus; wherein the first single-chain Fv is linked to the
second single-chain Fv by a linker peptide, the second single-chain
Fv is linked to the Fc fragment directly or by a linker peptide,
and the Fc fragment has no effector functions comprising CDC, ADCC
and ADCP.
2. The bispecific antibody according to claim 1, wherein the first
single-chain Fv comprises a VH domain and a VL domain that are
linked by a linker peptide which has an amino acid sequence of
(GGGGX)n, wherein X comprises Ser or Ala, and n is a natural number
from 1 to 5.
3. The bispecific antibody according to claim 1, wherein the first
single-chain Fv is selected from the group consisting of: (1) a VH
domain comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 1,
2 and 3, respectively or having sequences that are least 80%, 85%,
90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more
amino acid substitutions than any of SEQ ID NOs: 1, 2 and 3; and a
VL domain comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs:
4, 5 and 6, respectively or having sequences that are least 80%,
85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or
more amino acid substitutions any of SEQ ID NOs: 4, 5 and 6; (2) a
VH domain comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs:
7, 8 and 9, respectively or having sequences that are at least 80%,
85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or
more amino acid substitutions than any of SEQ ID NOs: 7, 8 and 9;
and a VL domain comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ
ID NOs: 10, 11 and 12, respectively or having sequences that are at
least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or
have one or more amino acid substitutions than any of SEQ ID NOs:
10, 11 and 12; (3) a VH domain comprising HCDR1, HCDR2 and HCDR3 as
shown in SEQ ID NOs: 13, 14 and 15, respectively or having
sequences that are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99%
or more similar to or have one or more amino acid substitutions
than any of SEQ ID NOs: 13, 14 and 15; and a VL domain comprising
LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 16, 17 and 18,
respectively or having sequences that are at least 80%, 85%, 90%,
92%, 95%, 97%, 98%, 99% or more similar to or have one or more
amino acid substitutions than any of SEQ ID NOs: 16, 17 and 18; and
(4) a VH domain comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ
ID NOs: 19, 20 and 21, respectively or having sequences that are at
least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or
have one or more amino acid substitutions any of SEQ ID NOs: 19, 20
and 21; and a VL domain comprising LCDR1, LCDR2 and LCDR3 as shown
in SEQ ID NOs: 22, 23 and 24, respectively or having sequences that
are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar
to or have one or more amino acid substitutions than any of SEQ ID
NOs: 22, 23 and 24.
4. The bispecific antibody according to claim 1, wherein the first
single-chain Fv is selected from the group consisting of: (1) a VH
domain comprising an amino acid sequence as shown in SEQ ID NO: 25
or having a sequence that is at least 80%, 85%, 90%, 92%, 95%, 97%,
98%, 99% or more similar to or has one or more amino acid
substitutions than SEQ ID NO: 25; and a VL domain comprising an
amino acid sequence as shown in SEQ ID NO: 26 or having a sequence
that is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more
similar to or has one or more amino acid substitutions SEQ ID NO:
26; (2) a VH domain comprising an amino acid sequence as shown in
SEQ ID NO: 27 or having a sequence that is at least 80%, 85%, 90%,
92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino
acid substitutions than SEQ ID NO: 27; and a VL domain comprising
an amino acid sequence as shown in SEQ ID NO: 28 or having a
sequence that is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or
more similar to or has one or more amino acid substitutions SEQ ID
NO: 28; (3) a VH domain comprising an amino acid sequence as shown
in SEQ ID NO: 29 or having a sequence that is at least 80%, 85%,
90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more
amino acid substitutions than SEQ ID NO: 29; and a VL domain
comprising an amino acid sequence as shown in SEQ ID NO: 30 or
having a sequence that is at least 80%, 85%, 90%, 92%, 95%, 97%,
98%, 99% or more similar to or has one or more amino acid
substitutions than SEQ ID NO: 30; and (4) a VH domain comprising an
amino acid sequence as shown in SEQ ID NO: 31 or having a sequence
that is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more
similar to or has one or more amino acid substitutions than SEQ ID
NO: 31; and a VL domain comprising an amino acid sequence as shown
in SEQ ID NO: 32 or having a sequence that is at least 80%, 85%,
90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or more
amino acid substitutions than SEQ ID NO: 32.
5. The bispecific antibody according to claim 1, wherein the second
single-chain Fv comprises a VH domain and a VL domain that are
linked by a linker peptide which has an amino acid sequence of
(GGGGX).sub.n, wherein X comprises Ser or Ala, and n is a natural
number from 1 to 5.
6. The bispecific antibody according to claim 1, wherein the second
single-chain Fv binds to an effector cell at an EC.sub.50 value
greater than about 50 nM, or greater than 100 nM, or greater than
300 nM, or greater than 500 nM in an in vitro binding affinity
assay; more preferably, the second single-chain Fv of the
bispecific antibody is capable of binding to human CD3 and
specifically binding to CD3 of a cynomolgus monkey or a rhesus
monkey.
7. The bispecific antibody according to claim 6, wherein the second
single-chain Fv is selected from the group consisting of: (1) a VH
domain comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs:
34, 35 and 36, respectively or having sequences that are at least
80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have
one or more amino acid substitutions than any of SEQ ID NOs: 34, 35
and 36; and a VL domain comprising LCDR1, LCDR2 and LCDR3 as shown
in SEQ ID NOs: 37, 38 and 39, respectively or having sequences that
are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar
to or have one or more amino acid substitutions than any of SEQ ID
NOs: 37, 38 and 39; and (2) a VH domain comprising HCDR1, HCDR2 and
HCDR3 as shown in SEQ ID NOs: 40, 41 and 42, respectively or having
sequences that are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99%
or more similar to or have one or more amino acid substitutions
than any of SEQ ID NOs: 40, 41 and 42; and a VL domain comprising
LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 43, 44 and 45,
respectively or having sequences that are at least 80%, 85%, 90%,
92%, 95%, 97%, 98%, 99% or more similar to or have one or more
amino acid substitutions than any of SEQ ID NOs: 43, 44 and 45.
8. The bispecific antibody according to claim 7, wherein the second
single-chain Fv is selected from the group consisting of: (1) a VH
domain comprising an amino acid sequence as shown in SEQ ID NO: 46
or having a sequence that is at least 80%, 85%, 90%, 92%, 95%, 97%,
98%, 99% or more similar to or has one or more amino acid
substitutions than SEQ ID NO: 43; and a VL domain comprising an
amino acid sequence as shown in SEQ ID NO: 47 or having a sequence
that is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more
similar to or has one or more amino acid substitutions than SEQ ID
NO: 47; and (2) a second single-chain Fv that specifically binds to
CD3; wherein the VH domain thereof contains an amino acid sequence
as shown in SEQ ID NO: 48 or has a sequence that is at least 80%,
85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or
more amino acid substitutions than SEQ ID NO: 48; and the VL domain
thereof contains an amino acid sequence as shown in SEQ ID NO: 49
or has a sequence that is at least 80%, 85%, 90%, 92%, 95%, 97%,
98%, 99% or more similar to or has one or more amino acid
substitutions than SEQ ID NO: 49.
9. The bispecific antibody according to claim 1, wherein the linker
peptide that links the first single-chain Fv to the second
single-chain Fv consists of a flexible peptide and a rigid peptide;
wherein the flexible peptide comprises two or more amino acids, and
preferably selected from the following amino acids: Gly(G), Ser(S),
Ala(A) and Thr(T); more preferably, the flexible peptide comprises
G and S residues; most preferably, an amino acid composition
structure of the flexible peptide has a general formula of
G.sub.xS.sub.y(GGGGS).sub.z, wherein x, y and z are integers
greater than or equal to 0 and x+y+z.gtoreq.1; the rigid peptide is
derived from a full-length sequence consisting of amino acids 118
to 145 at carboxyl terminus of natural human chorionic gonadotropin
.beta.-subunit or a truncated fragment thereof; preferably, the
rigid peptide comprises SSSSKAPPPS.
10. The bispecific antibody according to claim 9, wherein the
linker peptide contains an amino acid sequence as shown in SEQ ID
NO: 52.
11. The bispecific antibody according to claim 1, wherein the
linker peptide that links the Fc fragment to the second
single-chain Fv comprises 1-20 amino acids, and preferably selected
from the following amino acids: Gly(G), Ser(S), Ala(A) and Thr(T);
more preferably selected from Gly (G) and Ser (S); further
preferably, the linker peptide consists of (GGGGS)n, wherein n=1,
2, 3 or 4.
12. The bispecific antibody according to claim 1, wherein the Fc
fragment comprises a hinge region, a CH2 domain and a CH3 domain
derived from a human immunoglobulin heavy chain constant region;
preferably, the Fc fragment is selected from heavy chain constant
regions of human IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD and
IgE; more preferably, the Fc fragment is selected from heavy chain
constant regions of human IgG1, IgG2, IgG3 and IgG4; further
preferably, the Fc fragment is selected from a heavy chain constant
region of human IgG1 or IgG4; and compared to a natural sequence
from which the Fc fragment is derived, the Fc fragment has one or
more amino acid substitutions, deletions or additions selected form
the group consisting of: (i) amino acid substitutions
L234A/L235A/P331S that are determined according to an EU numbering
system; (ii) amino acid substitutions M428L, T250Q/M428L/N434S or
M252Y/S254T/T256E determined according to the EU numbering system;
(iii) an amino acid substitution N297A determined according to the
EU numbering system; and (iv) an amino acid deletion K447
determined according to the EU numbering system.
13-16. (canceled)
17. The bispecific antibody according to claim 12, wherein the Fc
fragment has an amino acid sequence as shown in SEQ ID NO: 57 that
has six amino acid substitutions or replacements
L234A/L235A/N297A/P331S/T250Q/M428L determined according to the EU
numbering system and a deleted or removed K447 determined according
to the EU numbering system compared to the natural sequence from
which the Fc fragment is derived.
18. The bispecific antibody according to claim 1, wherein the
bispecific antibody binds to human CD20 and CD3 and has an amino
acid sequence as follows: (1) a sequence as shown in SEQ ID NO: 50;
(2) a sequence having one or more substitutions, deletions or
additions relative to the sequence as shown in SEQ ID NO: 50; or
(3) a sequence having at least 80%, at least 85%, at least 90%, at
least 91%, at least 92%, at least 93%, at least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99% or 100%
sequence identity relative to the sequence as shown in SEQ ID NO:
50.
19. A DNA molecule encoding the bispecific antibody according to
claim 1.
20. The DNA molecule according to claim 19, which has a nucleotide
sequence as shown in SEQ ID NO: 51.
21-22. (canceled)
23. A pharmaceutical composition, comprising the bispecific
antibody according to claim 1 and a pharmaceutically acceptable
excipient, carrier or diluent.
24. A method for preparing the bispecific antibody according to
claim 1, comprising: (a) obtaining a fusion gene of the bispecific
antibody, and constructing an expression vector of the bispecific
antibody; (b) transfecting the expression vector into a host cell
by a genetic engineering method; (c) culturing the host cell under
conditions that allow the bispecific antibody to be generated; (d)
separating and purifying the bispecific antibody; wherein the
expression vector in step (a) is one or more selected from a
plasmid, a bacterium and a virus; preferably, the expression vector
is a pCDNA3.4 vector; wherein the host cell into which the
constructed vector is transfected by a genetic engineering method
in step (b) comprises a prokaryotic cell, a yeast or a mammalian
cell, such as a CHO cell, an NS0 cell or another mammalian cell,
preferably a CHO cell; and wherein the bispecific antibody is
separated and purified in step (d) by a conventional immunoglobulin
purification method comprising protein A affinity chromatography
and ion exchange, hydrophobic chromatography or molecular sieve
chromatography.
25-27. (canceled)
28. A method for preventing/treating, delaying development of, or
reducing/inhibiting recurrence of a disease including diseases or
disorders comprising an immune-related disease, a tumor, an
autoimmune disease, an inflammatory disease or a transplant
rejection-related disease or disorder, comprising administering an
effective amount of the bispecific antibody of claim 1 to an
individual suffering from the diseases or disorders, wherein the
tumor comprises acute myeloid leukemia (AML), chronic myeloid
leukemia (CML), B acute lymphocytic leukemia (B-ALL), B chronic
lymphocytic leukemia (B-CLL), B-cell lymphoma (BCL), T-cell
lymphoma (TCL) (such as skin), myelodysplastic syndrome (MDS),
small lymphocytic lymphoma (SLL), hairy cell leukemia (HCL),
marginal zone lymphoma (MZL) (such as extranodal or splenic),
follicular lymphoma (FL) (such as pediatric or gastrointestinal),
B-cell prolymphocytic leukemia (B-PLL), mantle cell lymphoma (MCL),
lymphoplasmacytic lymphoma (LPL)/Waldenstrom's macroglobulinemia
(WM), lymphoblastic leukemia (ALL) (such as B cell), lymphoblastic
lymphoma (LBL) (such as B cell), plasmablastic lymphoma (PBL) (such
as B cell), Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse
large B-cell lymphoma (DLBCL) (for example, primary or
inflammation-related), Burkitt's lymphoma (BL), multiple myeloma,
anaplastic large-cell lymphoma, HIV-related lymphoma and
Waldenstrom's macroglobulinemia, the autoimmune or inflammatory
disease is selected from rheumatoid arthritis (RA), osteoarthritis,
reactive arthritis, systemic lupus erythematosus (SLE), Crohn's
disease, multiple sclerosis, scleroderma, psoriasis, psoriatic
arthritis, ulcerative colitis (such as chronic), insulin-dependent
diabetes (such as juvenile), thyroiditis (such as chronic),
hyperthyroidism, asthma, allergic diseases, sarcoidosis, autoimmune
hemolytic anemia, pernicious anemia, graft-versus-host disease,
dermatomyositis, chronic hepatitis, microscopic renal vasculitis,
chronic active hepatitis, uveitis, intestinal synovitis, autoimmune
intestinal disease, idiopathic leukopenia, autoimmune
glomerulonephritis, autoimmune hemolytic anemia, autoimmune
hepatitis, interstitial pneumonia, chronic pemphigus, pemphigus
vulgaris, arteritis, polyarteritis nodosa and ankylosing
spondylitis.
Description
CROSS-REFERENCE TO RELATED APPLICATION(S)
[0001] This application claims priority to Chinese Patent
Application No. 201811294887.4 filed Nov. 1, 2018, the disclosure
of which is incorporated herein by reference in its entirety.
TECHNICAL FIELD
[0002] The present disclosure relates to the field of immunology
and, in particular, to a bispecific antibody against CD20 and CD3
and use thereof, especially use thereof for treating tumors.
BACKGROUND
[0003] T lymphocytes play an important role in the process of
cellular immunity. T cell-mediated cellular immunity is mainly that
a T cell receptor (TCR) specifically recognizes an antigen peptide
presented by a major histocompatibility complex (MHC) on the
surface of cells so that intracellular signals of T cells are
activated and target cells are specifically killed. This is very
important for the timely clearance of abnormal cells in a body and
the prevention of tumorigenesis. Tumors are developed as a result
of immune escape due to the down-regulation or absence of MHC on
the surface of most tumor cells.
[0004] T cell-engaging bispecific antibodies (TCBs) represent a
very effective manner of re-directing activated cytotoxic T cells
to tumors. CD3, which is expressed in mature T cells as a part of
the T cell receptor, can transduce activation signals generated
during antigen recognition by TCR. TCBs can simultaneously bind to
tumor surface antigens and CD3c subunits of the T cell receptor to
establish physical links between T cells and tumor cells, so that T
cells in a resting state are effectively activated to kill tumor
cells, thereby achieving the effect of treating tumors (Smits N C,
Sentman C L, Journal of Clinical Oncology, 2016: JCO 649970). TCBs
activate T cells without relying on the traditional antigen
presentation by MHC and double activation signals of costimulatory
molecules. Therefore, TCBs eliminate the need for tumor-specific
immune and overcome the dysfunction of T cells in a tumor
microenvironment.
[0005] In recent years, to achieve correct assembly of two
different semi-antibodies, scientists have designed and developed
bispecific antibodies with various structures. These bispecific
antibodies are generally divided into two categories. One category
of bispecific antibodies contains no Fc region, such as BiTE, DART,
TrandAbs and bi-Nanobody. Such bispecific antibodies have the
advantages of a small molecular weight and an ability to be
expressed in prokaryotic cells without concerning the problem of
correct assembly. However, due to the absence of an Fc fragment and
a relatively small molecular weight, these bispecific antibodies
have relatively short half-lives. Moreover, bispecific antibodies
in this form are easy to polymerize, have poor stability, and are
expressed at a low level so that their clinical applications are
limited. The other category of bispecific antibodies reserves an Fc
domain, such as Triomabs, kih IgG, Cross-mab, orthoFab IgG, DVD
IgG, IgG scFv and scFv.sub.2-Fc. These bispecific antibodies form
IgG-like structures with larger molecular structures and have
longer half-lives due to cell endocytosis and recycling processes
mediated by FcRn. Some or all of effector functions mediated by Fc
are reserved, such as antibody-dependent cellular cytotoxicity
(ADCC), complement-dependent cytotoxicity (CDC) and
antibody-dependent cellular phagocytosis (ADCP). However, this
category of bispecific antibodies cannot completely eliminate the
production of mismatched products, either. Residual fractions of
any mismatched molecules are difficult to separate from the
products. Moreover, this method requires a lot of genetic
engineering modifications such as mutations for two antibody
sequences and is not simple and universal.
[0006] CD20 is a non-glycosylated type III transmembrane protein
and mainly involved in mediating the proliferation and
differentiation of B lymphocytes. As typical antigens on the
surface of B cells in a human immune system, CD20 is only present
on the surface of pre-B cells and mature B cells. It is expressed
in more than 95% of B cell lymphomas, and not expressed in
hematopoietic stem cells, plasma cells and other normal tissues.
Therefore, CD20 can serve as a target for the study on B cell
tumors and autoimmune diseases. At present, multiple CD20
monoclonal antibody drugs have been marketed for the treatment of
non-Hodgkin's lymphoma (NHL), chronic lymphocytic leukemia (CLL)
and rheumatoid arthritis (RA) with good effects. The main
mechanisms of the CD20 monoclonal antibody drugs are mainly
complement-dependent cytotoxicity reactions, antibody-dependent
cellular cytotoxicity, and increased sensitivity to cytotoxicity so
that tumor cells expressing CD20 are killed. Although anti-CD20
tumor targeting strategies have shown great potential in clinical
applications, not all patients respond to anti-CD20 treatment, and
some patients show resistance to anti-CD20 treatment (e.g.,
resistance to rituximab). Therefore, a bispecific antibody
targeting CD20 and CD3 can enhance the killing effect of immune
cells on tumor cells and be used for treating CD20-positive B cell
malignancies, such as non-Hodgkin's lymphoma and chronic
lymphocytic leukemia. The bispecific antibody targeting CD20 and
CD3 has a broad application prospect in the field of tumor
immunology.
[0007] Therefore, the present disclosure aims to develop a CD20
bispecific molecule with improved performance in terms of
half-life, stability, safety and productibility.
SUMMARY
[0008] An object of the present disclosure is to provide a
tetravalent, homodimer-type bispecific antibody molecule targeting
immune effector cell antigen CD3 and tumor antigen CD20. The
bispecific antibody can significantly inhibit or kill tumor cells
in vivo and has significantly reduced non-specific killing effect
on normal cells with the low expression of CD20 and controlled
toxic side effects due to the over-activation of effector cells and
significantly improved physicochemical and in vivo stability.
[0009] Specifically, in a first aspect of the present disclosure,
there is disclosed a bispecific antibody, which is a tetravalent
homodimer formed by two identical polypeptide chains that bind each
other by a covalent bond, wherein each of the polypeptide chains
includes a first single-chain Fv that specifically binds to tumor
antigen CD20, a second single-chain Fv that specifically binds to
effector cell antigen CD3 and an Fc fragment in sequence from
N-terminus to C-terminus; wherein the first single-chain Fv is
linked to the second single-chain Fv by a linker peptide, the
second single-chain Fv is linked to the Fc fragment directly or by
a linker peptide, and the Fc fragment has no effector functions
comprising CDC, ADCC and ADCP.
[0010] The first single-chain Fv has specificity to
tumor-associated antigen CD20 and includes a VH domain and a VL
domain linked by a linker peptide (L1), wherein VH, L1 and VL are
arranged in order of VH-L1-VL or VL-L1-VH and the linker peptide L1
has an amino acid sequence of (GGGGX).sub.n, wherein X includes Ser
or Ala, preferably Ser; and n is a natural number from 1 to 5,
preferably 3;
[0011] For example, some preferred amino acid sequences of the VH
domain and its complementarity determining regions (HCDR1, HCDR2
and HCDR3) and amino acid sequences of the VL domain and its
complementarity determining regions (LCDR1, LCDR2 and LCDR3) of the
first single-chain Fv against the tumor-associated antigen are
exemplarily listed in Table 6-1 of the present disclosure.
[0012] In a preferred embodiment of the present disclosure, the
amino acid sequence of the linker peptide L1 is (GGGGS)3. In other
preferred embodiments, the amino acid sequence of the linker
peptide L1 also includes (GGGGS)1, (GGGGS)2, (GGGGS)4, (GGGGS)5,
(GGGGA)1, (GGGGA)2, (GGGGA)3, (GGGGA)4 or (GGGGA)5.
[0013] Preferably, the first single-chain Fv is selected from the
group consisting of:
[0014] (i) a VH domain comprising HCDR1, HCDR2 and HCDR3 as shown
in SEQ ID NOs: 1, 2 and 3, respectively or having sequences that
are substantially identical to (for example, are at least 80%, 85%,
90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more
amino acid substitutions (such as conservative substitutions) than)
any of SEQ ID NOs: 1, 2 and 3; and a VL domain comprising LCDR1,
LCDR2 and LCDR3 as shown in SEQ ID NOs: 4, 5 and 6, respectively or
having sequences that are substantially identical to (for example,
are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar
to or have one or more amino acid substitutions (such as
conservative substitutions) than) any of SEQ ID NOs: 4, 5 and
6;
[0015] (ii) a VH domain comprising HCDR1, HCDR2 and HCDR3 as shown
in SEQ ID NOs: 7, 8 and 9, respectively or having sequences that
are substantially identical to (for example, are at least 80%, 85%,
90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more
amino acid substitutions (such as conservative substitutions) than)
any of SEQ ID NOs: 7, 8 and 9; and a VL domain comprising LCDR1,
LCDR2 and LCDR3 as shown in SEQ ID NOs: 10, 11 and 12, respectively
or having sequences that are substantially identical to (for
example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or
more similar to or have one or more amino acid substitutions (such
as conservative substitutions) than) any of SEQ ID NOs: 10, 11 and
12;
[0016] (iii) a VH domain comprising HCDR1, HCDR2 and HCDR3 as shown
in SEQ ID NOs: 13, 14 and 15, respectively or having sequences that
are substantially identical to (for example, are at least 80%, 85%,
90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more
amino acid substitutions (such as conservative substitutions) than)
any of SEQ ID NOs: 13, 14 and 15; and having VL domain comprising
LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 16, 17 and 18,
respectively or having sequences that are substantially identical
to (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%,
99% or more similar to or have one or more amino acid substitutions
(such as conservative substitutions) than) any of SEQ ID NOs: 16,
17 and 18; and
[0017] (iv) a VH domain comprising HCDR1, HCDR2 and HCDR3 as shown
in SEQ ID NOs: 19, 20 and 21, respectively or having sequences that
are substantially identical to (for example, are at least 80%, 85%,
90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or more
amino acid substitutions (such as conservative substitutions) than)
any of SEQ ID NOs: 19, 20 and 21; and a VL domain comprising LCDR1,
LCDR2 and LCDR3 as shown in SEQ ID NOs: 22, 23 and 24, respectively
or having sequences that are substantially identical to (for
example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or
more similar to or have one or more amino acid substitutions (such
as conservative substitutions) than) any of SEQ ID NOs: 22, 23 and
24.
[0018] More preferably, the first single-chain Fv is selected from
the group consisting of:
[0019] (i) a VH domain comprising an amino acid sequence as shown
in SEQ ID NO: 25 or having a sequence that is substantially
identical to (for example, is at least 80%, 85%, 90%, 92%, 95%,
97%, 98%, 99% or more similar to or has one or more amino acid
substitutions (such as conservative substitutions) than) SEQ ID NO:
25; and a VL domain comprising an amino acid sequence as shown in
SEQ ID NO: 26 or having a sequence that is substantially identical
to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99%
or more similar to or has one or more amino acid substitutions
(such as conservative substitutions) than) SEQ ID NO: 26;
[0020] (ii) a VH domain comprising an amino acid sequence as shown
in SEQ ID NO: 27 or having a sequence that is substantially
identical to (for example, is at least 80%, 85%, 90%, 92%, 95%,
97%, 98%, 99% or more similar to or has one or more amino acid
substitutions (such as conservative substitutions) than) SEQ ID NO:
27; and a VL domain comprising an amino acid sequence as shown in
SEQ ID NO: 28 or having a sequence that is substantially identical
to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99%
or more similar to or has one or more amino acid substitutions
(such as conservative substitutions) than) SEQ ID NO: 28;
[0021] (iii) a VH domain comprising an amino acid sequence as shown
in SEQ ID NO: 29 or having a sequence that is substantially
identical to (for example, is at least 80%, 85%, 90%, 92%, 95%,
97%, 98%, 99% or more similar to or has one or more amino acid
substitutions (such as conservative substitutions) than) SEQ ID NO:
29; and a VL domain comprising an amino acid sequence as shown in
SEQ ID NO: 30 or having a sequence that is substantially identical
to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99%
or more similar to or has one or more amino acid substitutions
(such as conservative substitutions) than) SEQ ID NO: 30; and
[0022] (iv) a VH domain comprising an amino acid sequence as shown
in SEQ ID NO: 31 or having a sequence that is substantially
identical to (for example, is at least 80%, 85%, 90%, 92%, 95%,
97%, 98%, 99% or more similar to or has one or more amino acid
substitutions (such as conservative substitutions) than) SEQ ID NO:
31; and a VL domain comprising an amino acid sequence as shown in
SEQ ID NO: 32 or having a sequence that is substantially identical
to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99%
or more similar to or has one or more amino acid substitutions
(such as conservative substitutions) than) SEQ ID NO: 32.
[0023] The linker peptide (L2) that links the first single-chain Fv
and the second single-chain Fv of the present disclosure consists
of a flexible peptide and a rigid peptide.
[0024] Further, the flexible peptide includes two or more amino
acids, and preferably selected from the following amino acids:
Gly(G), Ser(S), Ala(A) and Thr(T). More preferably, the flexible
peptide includes G and S residues. Most preferably, an amino acid
composition structure of the flexible peptide has a general formula
of G.sub.xS.sub.y(GGGGS).sub.z, wherein x, y and z are integers
greater than or equal to 0 and x+y+z.gtoreq.1. For example, in a
preferred embodiment, the amino acid sequence of the flexible
peptide is G.sub.2(GGGGS).sub.3.
[0025] Further, the rigid peptide is derived from a full-length
sequence (as shown in SEQ ID NO: 33) consisting of amino acids 118
to 145 at the carboxyl terminus of natural human chorionic
gonadotropin .beta.-subunit or a truncated fragment thereof
(hereinafter collectively referred to as a CTP). Preferably, the
CTP rigid peptide includes 10 amino acids at the N-terminus of SEQ
ID NO: 33, that is, SSSSKAPPPS (CTP.sup.1); or the CTP rigid
peptide includes 14 amino acids at the C-terminus of SEQ ID NO: 33,
that is, SRLPGPSDTPILPQ (CTP.sup.2); as another example, in another
embodiment, the CTP rigid peptide includes 16 amino acids at the
N-terminus of SEQ ID NO: 33, that is, SSSSKAPPPSLPSPSR (CTP.sup.3);
as another example, in some other embodiments, the CTP rigid
peptide includes 28 amino acids which begin at position 118 and end
at position 145 of the human chorionic gonadotropin .beta.-subunit,
that is, SSSSKAPPPSLPSPSRLPGPSDTPILPQ (CTP.sup.4).
[0026] For example, some preferred amino acid sequences of the
linker peptide L2 that links the first single-chain Fv and the
second single-chain Fv are exemplarily listed in Table 3 of the
present disclosure.
[0027] In a preferred embodiment of the present disclosure, the
linker peptide L2 has an amino acid sequence as shown in SEQ ID NO:
52, wherein the amino acid composition of the flexible peptide
thereof is G.sub.2(GGGGS)3, and the amino acid composition of the
rigid peptide thereof is SSSSKAPPPS (i.e. CTP.sup.1).
[0028] The second single-chain Fv has specificity to immune
effector cell antigen CD3 and includes a VH domain and a VL domain
linked by a linker peptide (L3), wherein VH, L3 and VL are arranged
in order of VH-L3-VL or VL-L3-VH and the linker peptide L3 has an
amino acid sequence of (GGGGX).sub.n, wherein X includes Ser or
Ala, preferably Ser, and n is a natural number from 1 to 5,
preferably 3.
[0029] Preferably, the second single-chain Fv of the bispecific
antibody binds to effector cells at an EC.sub.50 value greater than
50 nM, or greater than 100 nM, or greater than 300 nM, or greater
than 500 nM in an in vitro FACS binding assay; more preferably, the
second single-chain Fv of the bispecific antibody is capable of
binding to human CD3 and specifically binding to CD3 of a
cynomolgus monkey or a rhesus monkey. In a preferred embodiment of
the present disclosure, the bispecific antibody specifically binds
to effector cells at an EC.sub.50 value of 132.3 nM.
[0030] For example, some preferred amino acid sequences of the VH
domain and its complementarity determining regions (HCDR1, HCDR2
and HCDR3) and amino acid sequences of the VL domain and its
complementarity determining regions (LCDR1, LCDR2 and LCDR3) of the
anti-CD3 scFv are exemplarily listed in Table 2 of the present
disclosure.
[0031] Preferably, the second single-chain Fv specifically binds to
CD3; the VH domain of the second single-chain Fv contains HCDR1,
HCDR2 and HCDR3 as shown in SEQ ID NOs: 34, 35 and 36, respectively
or has sequences that are substantially identical to (for example,
are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar
to or have one or more amino acid substitutions (such as
conservative substitutions) than) any of SEQ ID NOs: 34, 35 and 36;
and the VL domain of the second single-chain Fv contains LCDR1,
LCDR2 and LCDR3 as shown in SEQ ID NOs: 37, 38 and 39, respectively
or has sequences that are substantially identical to (for example,
are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar
to or have one or more amino acid substitutions (such as
conservative substitutions) than) any of SEQ ID NOs: 37, 38 and
39.
[0032] Preferably, the second single-chain Fv specifically binds to
CD3; the VH domain of the second single-chain Fv contains HCDR1,
HCDR2 and HCDR3 as shown in SEQ ID NOs: 40, 41 and 42, respectively
or has sequences that are substantially identical to (for example,
are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar
to or have one or more amino acid substitutions (such as
conservative substitutions) than) any of SEQ ID NOs: 40, 41 and 42;
and the VL domain of the second single-chain Fv contains LCDR1,
LCDR2 and LCDR3 as shown in SEQ ID NOs: 43, 44 and 45, respectively
or has sequences that are substantially identical to (for example,
are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar
to or have one or more amino acid substitutions (such as
conservative substitutions) than) any of SEQ ID NOs: 43, 44 and
45.
[0033] More preferably, the second single-chain Fv specifically
binds to CD3; the VH domain of the second single-chain Fv contains
an amino acid sequence as shown in SEQ ID NO: 46 or has a sequence
that is substantially identical to (for example, is at least 80%,
85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or
more amino acid substitutions (such as conservative substitutions)
than) SEQ ID NO: 46; and the VL domain of the second single-chain
Fv contains an amino acid sequence as shown in SEQ ID NO: 47 or has
a sequence that is substantially identical to (for example, is at
least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or
has one or more amino acid substitutions (such as conservative
substitutions) than) SEQ ID NO: 47.
[0034] More preferably, the second single-chain Fv specifically
binds to CD3; the VH domain of the second single-chain Fv contains
an amino acid sequence as shown in SEQ ID NO: 48 or has a sequence
that is substantially identical to (for example, is at least 80%,
85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or
more amino acid substitutions (such as conservative substitutions)
than) SEQ ID NO: 48; and the VL domain of the second single-chain
Fv contains an amino acid sequence as shown in SEQ ID NO: 49 or has
a sequence that is substantially identical to (for example, is at
least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or
has one or more amino acid substitutions (such as conservative
substitutions) than) SEQ ID NO: 49.
[0035] In a preferred embodiment of the present disclosure, the
amino acid sequence of the linker peptide L3 is (GGGGS)3. In other
preferred embodiments, the amino acid sequence of the linker
peptide L3 also includes (GGGGS)1, (GGGGS)2, (GGGGS)4, (GGGGS)5,
(GGGGA)1, (GGGGA)2, (GGGGA)3, (GGGGA)4 or (GGGGA)5.
[0036] The Fc fragment of the present disclosure is linked to the
second single-chain Fv directly or by the linker peptide L4, and
the linker peptide L4 includes 1-20 amino acids, and preferably
selected from the following amino acids: Gly(G), Ser(S), Ala(A) and
Thr(T); more preferably, the linker peptide L4 is selected from Gly
(G) and Ser (S); further preferably, the linker peptide L4 consists
of (GGGGS)n, wherein n=1, 2, 3 or 4. In a preferred embodiment of
the present disclosure, the Fc fragment is directly linked to the
second single-chain Fv.
[0037] In another aspect, the Fc fragment of the present disclosure
includes a hinge region, a CH2 domain and a CH3 domain from a human
immunoglobulin heavy chain constant region. For example, in some
embodiments, the Fc fragment of the present disclosure is selected
from, for example, heavy chain constant regions of human IgG1,
IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE; particularly
selected from heavy chain constant regions of human IgG1, IgG2,
IgG3 and IgG4, and more particularly selected from a heavy chain
constant region of human IgG1 or IgG4; and the Fc fragment has one
or more amino acid substitutions, deletions or additions (for
example, at most 20, at most 15, at most 10, or at most 5
substitutions, deletions, or additions) than a natural sequence
from which the Fc fragment is derived.
[0038] In some preferred embodiments, the Fc fragment is changed,
for example, mutated to modify the properties of the bispecific
antibody molecule of the present disclosure (for example, to change
one or more of the following properties: Fc receptor binding,
antibody glycosylation, an effector cell function or a complement
function).
[0039] For example, the bispecific antibody provided by the present
disclosure includes an Fc variant containing amino acid
substitutions, deletions or additions that change (for example,
reduce or eliminate) effector functions. Fc region of the antibody
mediates several important effector functions such as ADCC, ADCP
and CDC. Methods for changing the affinity of the antibody to an
effector ligand (such as Fc.gamma.R or a complement C1q) by
substituting amino acid residues in the Fc region of the antibody
to change the effector functions are known in the art (see, for
example, EP388151A1; U.S. Pat. Nos. 5,648,260; 5,624,821; Natsume A
et al., Cancer Res., 68: 3863-3872, 2008; Idusogie E E et al., J.
Immunol., 166: 2571-2575, 2001; Lazar G A et al., PNAS, 103:
4005-4010, 2006; Shields R L et al., JBC, 276: 6591-6604, 2001;
Stavenhagen J B et al., Cancer Res., 67: 8882-8890, 2007;
Stavenhagen J B et al., Advan. Enzyme. Regul., 48: 152-164, 2008;
Alegre M L et al., J. Immunol., 148: 3461-3468, 1992; Kaneko E et
al., Biodrugs, 25: 1-11, 2011). In some preferred embodiments of
the present disclosure, amino acid L235 (EU numbering) in the
constant region of the antibody is modified to change an
interaction with an Fc receptor, such as L235E or L235A. In some
other preferred embodiments, amino acids 234 and 235 in the
constant region of the antibody are modified simultaneously, such
as L234A and L235A (L234A/L235A) (EU numbering).
[0040] For example, the bispecific antibody provided by the present
disclosure may include an Fc variant containing amino acid
substitutions, deletions or additions that extend a circulating
half-life. Studies show that M252Y/S254T/T256E, M428L/N434S or
T250Q/M428L can extend the half-life of the antibody in primates.
For more mutation sites included in the Fc variant with enhanced
binding affinity to a neonatal receptor (FcRn), see Chinese
invention patent CN 201280066663.2, US 2005/0014934A1, WO 97/43316,
U.S. Pat. Nos. 5,869,046, 5,747,03 and WO 96/32478. In some
preferred embodiments of the present disclosure, amino acid M428
(EU numbering) in the constant region of the antibody is modified
to enhance the binding affinity to the FcRn receptor, such as
M428L. In some other preferred embodiments, amino acids 250 and 428
(EU numbering) in the constant region of the antibody are modified
simultaneously, such as T250Q and M428L (T250Q/M428L).
[0041] For example, the bispecific antibody provided by the present
disclosure may also include an Fc variant containing amino acid
substitutions, deletions or additions that can reduce or eliminate
Fc glycosylation. For example, the Fc variant contains reduced
glycosylation of the N-linked glycan normally present at amino acid
site 297 (EU numbering). The glycosylation at position N297 has a
great effect on the activity of IgG. If the glycosylation at this
position is eliminated, the conformation of the upper half of CH2
of an IgG molecule is affected, thus losing the ability of binding
to Fc.gamma.Rs and affecting the biological activity related to the
antibody. In some preferred embodiments of the present disclosure,
amino acid N297 (EU numbering) in the constant region of human IgG
is modified to avoid the glycosylation of the antibody, such as
N297A.
[0042] For example, the bispecific antibody provided by the present
disclosure may also include an Fc variant containing amino acid
substitutions, deletions or additions that eliminate charge
heterogeneity. Various post-translational modifications during
expression in engineered cells will cause the charge heterogeneity
of monoclonal antibodies. The heterogeneity of lysine at C-terminus
of an IgG antibody is one of the main reasons for charge
heterogeneity. Lysine at C-terminus of a heavy chain may be deleted
at a certain proportion during the production of the antibody,
resulting in charge heterogeneity and affecting the stability,
effectiveness, immunogenicity or pharmacokinetic of the antibody.
In some preferred embodiments of the present disclosure, K447 (EU
numbering) at the C-terminus of the IgG antibody is removed or
deleted to eliminate the charge heterogeneity of the antibody and
improve the homogeneity of the expressed product.
[0043] Some preferred amino acid sequences of the Fc fragment are
exemplarily listed in Table 4 of the present disclosure. Compared
with a bispecific antibody containing the Fc region of wild-type
human IgG, the bispecific antibody provided by the present
disclosure contains an Fc fragment that exhibits reduced affinity
to at least one of human Fc.gamma.Rs (Fc.gamma.RI, Fc.gamma.RIIa,
or Fc.gamma.RIIIa) and C1q and has reduced effector cell functions
or complement functions. For example, in a preferred embodiment of
the present disclosure, the bispecific antibody includes an Fc
fragment that is derived from human IgG1, has L234A and L235A
substitutions (L234A/L235A), and exhibits reduced binding ability
to Fc.gamma.RI. In addition, the Fc fragment included in the
bispecific antibody provided by the present disclosure may also
contain amino acid substitutions that change one or more other
characteristics (such as an ability of binding to the FcRn
receptor, the glycosylation of the antibody or the charge
heterogeneity of the antibody). For example, in a preferred
embodiment of the present disclosure, the Fc fragment has an amino
acid sequence as shown in SEQ ID NO: 57, which has amino acid
substitutions L234A/L235A/T250Q/N297A/P331S/M428L and a deleted or
removed K447 compared with the natural sequence from which it is
derived.
[0044] The bispecific antibody molecule of the present disclosure
is a tetravalent homodimer formed by two identical polypeptide
chains that bind to each other by an interchain disulfide bond in
hinge regions of Fc fragments, and each polypeptide chain consists
of an anti-CD20 scFv, a linker peptide, an anti-CD3 scFv and a Fc
fragment in sequence from the N-terminus to the C-terminus.
[0045] In a preferred embodiment of the present disclosure, the
bispecific antibody binds to human CD20 and CD3 and has an amino
acid sequence as follows:
[0046] (1) a sequence as shown in SEQ ID NO: 50;
[0047] (2) a sequence having one or more substitutions, deletions
or additions (such as 1, 2, 3, 4 or 5 substitutions, deletions or
additions) relative to the sequence as shown in SEQ ID NO: 50;
or
[0048] (3) a sequence having at least 80%, at least 85%, at least
90%, at least 91%, at least 92%, at least 93%, at least 94%, at
least 95%, at least 96%, at least 97%, at least 98%, at least 99%
or 100% sequence identity relative to the sequence as shown in SEQ
ID NO: 50;
[0049] In some preferred embodiments, the substitutions in (2) are
conservative substitutions.
[0050] In a second aspect of the present disclosure, there is
provided a DNA molecule encoding the bispecific antibody as
described above.
[0051] In a preferred embodiment of the present disclosure, the DNA
molecule encoding the bispecific antibody as described above has a
nucleotide sequence as shown in SEQ ID NO: 51.
[0052] In a third aspect of the present disclosure, there is
provided a vector including the DNA molecule as described
above.
[0053] In a fourth aspect of the present disclosure, there is
provided a host cell including the vector as described above. The
host cell includes a prokaryotic cell, a yeast or a mammalian cell.
Preferably, the host cell is a mammalian cell such as a CHO cell,
an NS0 cell or other mammalian cells. Further preferably, the host
cell is a CHO cell.
[0054] In a fifth aspect of the present disclosure, there is
provided a pharmaceutical composition including the bispecific
antibody as described above and a pharmaceutically acceptable
excipient, carrier or diluent.
[0055] In a sixth aspect of the present disclosure, there is
provided a method for preparing the bispecific antibody of the
present disclosure, including:
[0056] (a) obtaining a fusion gene of the bispecific antibody, and
constructing an expression vector of the bispecific antibody;
[0057] (b) transfecting the expression vector into a host cell by a
genetic engineering method;
[0058] (c) culturing the host cell under conditions that allow the
bispecific antibody to be generated; and
[0059] (d) separating and purifying the bispecific antibody.
[0060] The expression vector in step (a) is one or more selected
from a plasmid, a bacterium and a virus; preferably, the expression
vector is a plasmid; more preferably, the expression vector is
pCDNA3.1;
[0061] The host cell into which the constructed vector is
transfected by a genetic engineering method in step (b) includes a
prokaryotic cell, a yeast or a mammalian cell; preferably, the host
cell is a mammalian cell such as a CHO cell, an NS0 cell or other
mammalian cells; further preferably, the host cell is a CHO
cell.
[0062] The bispecific antibody is separated and purified in step
(d) by a conventional immunoglobulin purification method including
protein A affinity chromatography and ion exchange, hydrophobic
chromatography or molecular sieve.
[0063] In a seventh aspect of the present disclosure, there is
provided use of the bispecific antibody for preparing a drug for
treating, preventing or alleviating a tumor; Examples of the tumor
include, but are not limited to, acute myeloid leukemia (AML),
chronic myeloid leukemia (CML), B acute lymphocytic leukemia
(B-ALL), B chronic lymphocytic leukemia (B-CLL), B-cell lymphoma
(BCL), T-cell lymphoma (TCL) (such as skin), myelodysplastic
syndrome (MDS), small lymphocytic lymphoma (SLL), hairy cell
leukemia (HCL), marginal zone lymphoma (MZL) (such as extranodal or
splenic), follicular lymphoma (FL) (such as pediatric or
gastrointestinal), B-cell prolymphocytic leukemia (B-PLL), mantle
cell lymphoma (MCL), lymphoplasmacytic lymphoma (LPL)/Waldenstrom's
macroglobulinemia (WM), lymphoblastic leukemia (ALL) (such as B
cell), lymphoblastic lymphoma (LBL) (such as B cell), plasmablastic
lymphoma (PBL) (such as B cell), Hodgkin's lymphoma, non-Hodgkin's
lymphoma, diffuse large B-cell lymphoma (DLBCL) (for example,
primary or inflammation-related), Burkitt's lymphoma (BL), multiple
myeloma, anaplastic large-cell lymphoma, HIV-related lymphoma and
Waldenstrom's macroglobulinemia.
[0064] In an eighth aspect of the present disclosure, there is
provided use of the bispecific antibody for preparing a drug for
treating, preventing or alleviating an autoimmune or inflammatory
disease, wherein the autoimmune or inflammatory disease is selected
from rheumatoid arthritis (RA), osteoarthritis, reactive arthritis,
systemic lupus erythematosus (SLE), Crohn's disease, multiple
sclerosis, scleroderma, psoriasis, psoriatic arthritis, ulcerative
colitis (such as chronic), insulin-dependent diabetes (such as
juvenile), thyroiditis (such as chronic), hyperthyroidism, asthma,
allergic diseases, sarcoidosis, autoimmune hemolytic anemia,
pernicious anemia, graft-versus-host disease, dermatomyositis,
chronic hepatitis, microscopic renal vasculitis, chronic active
hepatitis, uveitis, intestinal synovitis, autoimmune intestinal
disease, idiopathic leukopenia, autoimmune glomerulonephritis,
autoimmune hemolytic anemia, autoimmune hepatitis, interstitial
pneumonia, chronic pemphigus, pemphigus vulgaris, arteritis,
polyarteritis nodosa and ankylosing spondylitis.
[0065] In a ninth aspect of the present disclosure, there is
provided a method for enhancing or stimulating an immune response
or function, comprising administering a therapeutically effective
amount of the bispecific antibody or the pharmaceutical composition
to an individual.
[0066] In a tenth aspect of the present disclosure, there is
provided a method for preventing/treating, delaying development of,
or reducing/inhibiting recurrence of a disease including diseases
or disorders comprising an immune-related disease, a tumor, an
autoimmune disease, an inflammatory disease or a transplant
rejection-related disease or disorder, comprising administering an
effective amount of the bispecific antibody or the pharmaceutical
composition to an individual suffering from the diseases or
disorders.
[0067] Preferably, the method may be used in combination with one
or more other additional therapies, wherein the additional
therapies are selected from the group consisting of a surgery,
chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA
therapy, RNA therapy, nano therapy, viral therapy, adjuvant therapy
and a combination thereof.
[0068] The technical solutions provided by the present disclosure
have beneficial effects summarized as follows:
[0069] 1. The bispecific antibody provided by the present
disclosure includes anti-CD20 scFv that is located at the N
terminus of the bispecific antibody and has changed spatial
conformation, so that the bispecific antibody has reduced binding
ability to CD20 under some conditions, especially that the
bispecific antibody is difficult to bind to normal cells with weak
expression or low expression of CD20, thereby exhibiting reduced
non-specific killing effect. However, the binding specificity to
cells with over-expression or high expression of CD20 is not
significantly reduced, and the bispecific antibody exhibits a good
killing effect in vivo. It can be known that when a target antigen
is merely expressed on tumor cells or the bispecific antibody of
the present disclosure specifically binds to only tumor cells
over-expressing the target antigen, immune effector cells are
activated restrictively and merely in target cell tissues, which
can minimize the non-specific killing of the bispecific antibody on
normal cells and the accompanying release of cytokines and reduce
the toxic side effects of the bispecific antibody in clinical
treatment.
[0070] 2. The anti-CD3 scFv selected by the bispecific antibody
provided by the present disclosure specifically binds to effector
cells with weak binding affinity (EC.sub.50 value greater than 50
nM, or greater than 100 nM, or greater than 300 nM, or greater than
500 nM). In addition, the anti-CD3 scFv is embedded between the
anti-CD20 scFv and Fc, and the CTP rigid peptide contained in the
linker peptide L3 at its N terminus and the Fc fragment located at
its C terminus partially "cover" or "shield" the antigen binding
domain of the anti-CD3 scFv. Such steric hindrance effect makes the
anti-CD3 scFv bind to CD3 with weaker binding affinity (for
example, greater than 1 .mu.M), which reduces its ability to
activate and stimulate T cells, limits the excessive release of
cytokines, and provides higher safety. In addition, the anti-CD3
scFv used in the present disclosure can bind to CD3 natural
antigens of human and a cynomolgus monkey and/or a rhesus monkey at
the same time, so that no alternative molecule needs to be
constructed for preclinical toxicology evaluation and the effective
dose, toxic dose and toxic side effects obtained are more objective
and accurate and can be directly converted into a clinical dose to
reduce the risk of clinical studies.
[0071] 3. The bispecific antibody provided by the present
disclosure creatively adopts a divalent anti-CD3 scFv, which avoids
the asymmetric structure of a heterodimer-type (including a
monovalent anti-CD3 scFv) commonly used in the existing art in
terms of the configuration design of the bispecific antibody and
solves the problem of heavy chain mismatches, thereby simplifying
downstream purification steps. Moreover, unexpectedly, non-specific
binding of the anti-CD3 scFv to T cells is not observed in an in
vitro cell binding assay, and the degree of cell activation (the
release of cytokines such as IL-2) is controlled within a safe and
effective range. That is, the bivalent anti-CD3 scFv structure used
in the present disclosure has not induced over-activation of T
cells in a non-antigen-dependent manner, whereas for other
bispecific antibodies including bivalent anti-CD3 domains, the
uncontrollable over-activation of T cells is common and thus
anti-CD3 bispecific antibodies are generally designed to avoid the
introduction of a bivalent anti-CD3 structure.
[0072] 4. The modified Fc fragment included in the bispecific
antibody provided by the present disclosure has no ability of
binding to Fc.gamma.R, avoiding the systemic activation of T cells
mediated by Fc.gamma.R and allowing immune effector cells to be
activated restrictively and merely in target cell tissues.
[0073] 5. The bispecific antibody provided by the present
disclosure is homodimeric without mismatches of heavy chains and
light chains. The bispecific antibody is produced by a stable
downstream process and purified by simple and efficient steps, with
a homogeneous expression product and significantly improved
physicochemical and in vivo stability.
DETAILED DESCRIPTION
Abbreviations and Definitions
[0074] BiAb Bispecific antibody [0075] CDR Complementarity
determining region in a variable region of an immunoglobulin,
defined by a Kabat numbering system [0076] EC.sub.50 A
concentration at which 50% efficacy or binding is generated [0077]
ELISA Enzyme-linked immunosorbent assay [0078] FR Framework region
of an antibody: a variable region of an immunoglobulin excluding
CDRs [0079] HRP Horseradish peroxidase [0080] IL-2 Interleukin 2
[0081] IFN Interferon [0082] IC.sub.50 A concentration at which 50%
inhibition is generated [0083] IgG Immunoglobulin G [0084] Kabat
Immunoglobulin comparison and numbering system advocated by Elvin A
Kabat [0085] mAb Monoclonal antibody [0086] PCR Polymerase chain
reaction [0087] V region IgG chain segment whose sequence is
variable among different antibodies. It extends to Kabat residue
109 of the light chain and residue 113 of the heavy chain. [0088]
VH Heavy chain variable region of an immunoglobulin [0089] VK
.kappa. light chain variable region of an immunoglobulin [0090]
K.sub.D Equilibrium dissociation constant [0091] k.sub.a
Association rate constant [0092] k.sub.d Dissociation rate
constant
[0093] In the present disclosure, unless otherwise specified, the
scientific and technical terms used herein have meanings generally
understood by those skilled in the art. The antibody or fragments
thereof used in the present disclosure may be further modified
using conventional techniques known in the art alone or in
combination, such as amino acid deletion, insertion, substitution,
addition, and/or recombination and/or other modification methods. A
method for introducing such modifications into a DNA sequence of an
antibody according to the amino acid sequence of the antibody is
well known to those skilled in the art. See, for example, Sambrook,
Molecular cloning: a laboratory manual, Cold Spring Harbor
Laboratory (1989) N.Y. Such modifications are preferably performed
at a nucleic acid level. Meanwhile, for a better understanding of
the present disclosure, the definitions and explanations of related
terms are provided below.
[0094] "CD20", a specific marker molecule on the surface of B
lymphocytes, is expressed in more than 90% of B lymphoma cells and
normal B lymphocytes and not expressed in hematopoietic stem cells,
primary B lymphocytes, normal blood cells and other tissues. CD20
is not significantly internalized and shed off and no antigen
modulation is occurred upon binding to an antibody. CD20 may be
used as an ideal target for the treatment of B-cell lymphoma. CD20
mainly exerts an anti-tumor effect through ADCC and CDC. In recent
years, indications for the target CD20 have been increasingly
expanded, which include, for example, autoimmune diseases
(including multiple sclerosis, Crohn's disease) and inflammatory
diseases (e.g., ulcerative colitis), etc. The indications for the
target CD20 further include other related diseases or disorders
that are found in the existing art and will be found in the future.
The term also includes any variants, isoforms, derivatives and
species homologues of CD20 that are naturally expressed by cells
including tumor cells or expressed by cells transfected with CD20
genes or cDNA.
[0095] CD3 molecule is an important differentiation antigen on the
T cell membrane and a characteristic marker of mature T cells. It
is composed of six peptide chains, and these chains are associated
with the T cell antigen receptor (TCR) with a non-covalent bond to
constitute a TCR-CD3 complex. CD3 molecule not only participates in
the intracytoplasmic assembly of the TCR-CD3 complex but also
transmits antigen stimulation signals through the immunoreceptor
tyrosine-based activation motif (ITAM) of the cytoplasmic regions
of polypeptide chains. The main functions of CD3 molecule are to
stabilize TCR structure and transmit T cell activation signal. When
TCR specifically recognizes and binds to the antigen, CD3 is
involved in signal transduction into T cell cytoplasm as the first
signal to induce T cell activation and plays a very important role
in T cell antigen recognition and immune response generation.
[0096] "CD3" refers to a part of a T-cell receptor complex and
consists of three different chains CD3.epsilon., CD3.delta. and
CD3.gamma.. CD3 is clustered on T cells by, for example, being
immobilized by an anti-CD3 antibody, leading to the activation of T
cells, which is similar to T cell receptor-mediated activation but
independent of the specificity of TCR clones. Most anti-CD3
antibodies recognize the chain CD3.epsilon.. The second functional
domain that specifically recognizes the T cell surface receptor CD3
in the present disclosure is not specifically limited as long as it
can specifically recognize CD3, for example, but not limited to,
CD3 antigens mentioned in the following patents: U.S. Pat. Nos.
7,994,289; 6,750,325; 6,706,265; 5,968,509; 8,076,459; 7,728,114;
and U.S. 20100183615. Preferably, the antibody against human CD3
used in the present disclosure is cross-reactive with cynomolgus
monkeys and/or rhesus monkeys, for example, but not limited to, CD3
antigens mentioned in the following patents: WO 2016130726, U.S.
20050176028, WO 2007042261, or WO 2008119565. The term also
includes any variants, isotypes, derivatives, and species
homologues of CD3 that are naturally expressed by cells or
expressed by cells transfected with a gene or cDNA encoding the
preceding chains.
[0097] The term "antibody" specifically includes a monoclonal
antibody, a polyclonal antibody and an antibody-like polypeptide,
such as a chimeric antibody and a humanized antibody. An "antigen
binding fragment" includes fragments provided by any known
technique such as enzymatic cleavage, peptide synthesis and
recombination techniques. Some antigen binding fragments consist of
intact antibody moieties that retain the antigen binding
specificity of a parent antibody molecule. For example, the antigen
binding fragment may include at least one variable region (heavy or
light chain variable region) or one or more CDRs of an antibody
that is known to bind to a particular antigen. Suitable examples of
antigen binding fragments include, but are not limited to, a
bispecific antibody, a single-chain molecule, an Fab molecule, an
F(ab')2 molecule, an Fc molecule, an Fabc molecule, an Fv molecule,
a single-chain (Sc) antibody, a individual antibody light chain, a
individual antibody heavy chain, a chimeric fusion between an
antibody chain or CDR and other proteins, a protein scaffold, a
heavy chain monomer or dimer, a light chain monomer or dimer, a
dimer consisting of one heavy chain and one light chain, a
monovalent fragment consisting of VL, VH, CL and CH1 domains, or a
monovalent antibody as described in WO 2007059782, a divalent
fragment comprising two Fab fragments linked by a disulfide bond in
the hinge region, and an Fd fragment consisting essentially of VH
and CH1 domains; an Fv fragment consisting essentially of VL and VH
domains of a single antibody arm, a dAb fragment (Ward et al.,
Nature, 1989, 341: 544-54) consisting essentially of a VH domain
and also known as a domain antibody (Holt et al., Trends
Biotechnol. 2003, 21 (11): 484-90); a nanobody (Revets et al.,
Expert Opin Biol Ther. 2005 January; 5 (1): 111-24); or an isolated
complementarity determining region (CDR), etc. All antibody
isotypes may be used for producing antigen binding fragments.
Additionally, the antigen binding fragment may include a
non-antibody protein framework that can successfully incorporate a
polypeptide fragment in an orientation that imparts affinity to a
given antigen of interest (such as a protein scaffold). The antigen
binding fragment may be recombinantly produced or produced through
enzymatic or chemical cleavage of an intact antibody. The term
"antibody or an antigen binding fragment thereof" may be used for
representing that a given antigen binding fragment incorporates one
or more amino acid fragments of the antibody referred to in the
phrase.
[0098] The term "hypervariable region", "CDR" or "complementarity
determining region" refers to amino acid residues of an antibody,
which are responsible for antigen binding, and is a discontinuous
amino acid sequence. CDR sequences are amino acid residues in the
variable region that may be defined by the IMGT, Kabat, Chothia or
AbM method or identified by any CDR sequence determination method
well known in the art. For example, the hypervariable region
includes the following amino acid residues: amino acid residues
from a "complementarity determining region" or "CDR" defined by
sequence comparison, for example, residues at positions 24-34 (L1),
50-56 (L2) and 89-97 (L3) in a light chain variable domain and
residues at positions 31-35 (H1), 50-65 (H2) and 95-102 (H3) in a
heavy chain variable domain (see Kabat et al., 1991, Sequences of
Proteins of Immunological Interest (5th edition), Public Health
Service, National Institutes of Health, Bethesda, Md.), and/or
amino acid residues from a "hypervariable loop" (HVL) defined
according to the structure, for example, residues at positions
26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable
domain and residues at positions 26-32 (H1), 53-55 (H2) and 96-101
(H3) in the heavy chain variable domain (see Chothia and Leskl, J.
Mol Biol, 196: 901-917, 1987). "Framework" residues or "FR"
residues refer to variable domain residues other than the
hypervariable region residues as defined in the present disclosure.
In some embodiments, the antibody or the antigen-binding fragment
thereof in the present disclosure is preferably determined through
the Kabat, Chothia or IMGT numbering system. Those skilled in the
art may explicitly assign each system to any variable domain
sequence without relying on any experimental data beyond the
sequence itself. For example, the Kabat residue numbering method of
a given antibody may be determined by comparing the sequence of the
given antibody to each "standard" numbered sequence. Based on the
numbers of the sequences provided herein, the numbering scheme of
determining any variable region sequence in the sequence table is
entirely within the conventional technical scope of those skilled
in the art.
[0099] The term "single-chain Fv antibody" (or "scFv antibody")
refers to an antibody fragment comprising VH and VL domains of an
antibody. It is a fusion protein of the variable regions of the
heavy (VH) and light chains (VL) connected with a linker. The
linker enables these two domains to be cross-linked to form an
antigen-binding site, and the sequence of the linker generally
consists of a flexible peptide, for example, but not limited to,
G.sub.2(GGGGS).sub.3. The size of scFv is generally 1/6 of an
intact antibody. The single-chain antibody is preferably an amino
acid chain sequence encoded by a nucleotide chain. For the review
of scFv, reference may be made to Pluckthun (1994), The
Pharmacology of Monoclonal Antibodies, Vol. 113, edited by
Rosenburg and Moore, Springer-Verlag, New York, pages 269-315.
Reference may also be made to International Patent Application
Publication No. WO 88/01649 and U.S. Pat. Nos. 4,946,778 and
5,260,203.
[0100] The term "Fab fragment" consists of a light chain and a CH1
and a variable domain of each of a heavy chain. The heavy chain of
the Fab molecule cannot form a disulfide bond with another heavy
chain molecule. The size of "Fab antibody" is 1/3 of an intact
antibody, and "Fab antibody" includes only one antigen-binding
site.
[0101] The term "Fab' fragment" contains a light chain, and a VH
domain and a CH1 domain of a heavy chain, and a constant region
between CH1 and CH2 domains.
[0102] The term "F(ab')2 fragment" contains two light chains, VH
domains and CH1 domains of two heavy chains, and constant regions
between CH1 and CH2 domains, so that an interchain disulfide bond
is formed between the two heavy chains. Therefore, the F(ab').sub.2
fragment is composed of two Fab' fragments held together by the
disulfide bond between the two heavy chains.
[0103] The term "Fc" region refers to the antibody heavy chain
constant region fragment, including at least a hinge region and CH2
and CH3 domains.
[0104] The term "Fv region" includes variable regions from the
heavy chain and the light chain but lacks the constant regions, and
is the minimum fragment containing a complete antigen recognition
and binding site.
[0105] The term "Fd fragment" consists of CH1 and variable regions
of a heavy chain and is the heavy chain part of a Fab fragment
excluding the light chain.
[0106] The term "linker peptide" refers to a peptide linking two
polypeptides, wherein the linker peptide may be two immunoglobulin
variable regions or one variable region. The length of the linker
peptide may be 0 to 30 amino acids or 0 to 40 amino acids. In some
embodiments, the linker peptide may be in the length of 0 to 25, 0
to 20, or 0 to 18 amino acids. In some embodiments, the linker
peptide may be a peptide having no more than 14, 13, 12, 11, 10, 9,
8, 7, 6, or 5 amino acids. In other embodiments, the linker peptide
may include 0 to 25, 5 to 15, 10 to 20, 15 to 20, 20 to 30, or 30
to 40 amino acids. In other embodiments, the linker peptide may
have about 0, 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 or 30 amino
acids. The linker peptide is known to those skilled in the art. The
linker peptide may be prepared by any method in the art. For
example, the linker peptide may be originated from synthesis.
[0107] The term "heavy chain constant region" includes an amino
acid sequence from the immunoglobulin heavy chain. The polypeptide
comprising the heavy chain constant region includes at least one
of: a CH1 domain, a hinge domain (e.g., an upper hinge region, an
intermediate hinge region, and/or a lower hinge region), a CH2
domain, a CH3 domain, or a variant or fragment thereof. For
example, the antigen-binding polypeptide used herein may include a
polypeptide chain having a CH1 domain; a polypeptide having a CH1
domain, at least part of a hinge domain, and a CH2 domain; a
polypeptide chain having a CH1 domain and a CH3 domain; a
polypeptide chain having a CH1 domain, at least part of a hinge
domain, and a CH3 domain; or a polypeptide chain having a CH1
domain, at least part of a hinge domain, a CH2 domain, and a CH3
domain. In another embodiment, the polypeptide of the present
application includes a polypeptide chain having a CH3 domain. In
addition, the antibody used in the present application may lack at
least part of a CH2 domain (e.g., all or part of a CH2 domain). As
described above, it is appreciated by those of ordinary skill in
the art that heavy chain constant regions may be modified such that
they differ in amino acid sequence from naturally immunoglobulin
molecules.
[0108] The term "light chain constant region" includes an amino
acid sequence from the antibody light chain. Preferably, the light
chain constant region includes at least one of a constant kappa
domain and a constant lambda domain.
[0109] The term "VH domain" includes an amino-terminal variable
domain of the immunoglobulin heavy chain, while the term "CH1
domain" includes a first (mostly amino-terminal) constant region of
the immunoglobulin heavy chain. The CH1 domain is adjacent to the
VH domain and is the amino-terminal of the hinge region of the
immunoglobulin heavy chain molecule.
[0110] The term "hinge region" includes the portion of a heavy
chain molecule that links the CH1 domain to the CH2 domain. The
hinge region contains about 25 residues and is flexible so that two
N-terminal antigen binding regions move independently. The hinge
region may be divided into three different domains: upper, middle
and lower hinge domains (Roux K H et al., J. Immunol., 161: 4083,
1998).
[0111] The term "disulfide bond" includes a covalent bond formed
between two sulfur atoms. The amino acid cysteine contains a
sulfhydryl group which can form a disulfide bond or a bridge with a
second sulfhydryl group. In most naturally occurring IgG molecules,
CH1 and CK regions are linked by a disulfide bond and two heavy
chains are linked by two disulfide bonds at positions 239 and 242
according to the Kabat numbering system (position 226 or 229, EU
numbering system).
[0112] "Binding" is defined as an affinity interaction between a
particular epitope on an antigen and its corresponding antibody and
generally understood as "specific recognition". "Specific
recognition" means that the bispecific antibody of the present
disclosure does not cross-react with or substantially does not
cross-react with any polypeptide other than the target antigen. The
degree of specificity may be determined by immunological
techniques, including, but not limited to, immunoblotting,
immunoaffinity chromatography and flow cytometry, etc. In the
present disclosure, the specific recognition is preferably
determined through flow cytometry. In specific cases, the criteria
for the specific recognition may be determined by those having
ordinary skill in the art according to the common knowledge in the
art.
[0113] The term "bispecific antibody" refers to the bispecific
antibody of the present disclosure, for example, an anti-Her2
antibody or an antigen-binding fragment thereof, which may be
derivatized or linked to another functional molecule, for example,
another peptide or protein (e.g., TAA, a cytokine and a cell
surface receptor), to generate a bispecific antibody that binds to
at least two different binding sites or target molecules. To
produce the bispecific molecule of the present disclosure, the
antibody of the present disclosure may be functionally linked
(e.g., by chemical coupling, gene fusion, non-covalent binding or
other means) to one or more other binding molecules, for example,
another antibody, antibody fragment, peptide or binding mimetic, to
produce the bispecific molecule. For example, the "bispecific
antibody" refers to one including two variable domains or ScFv
units such that the antibody obtained recognizes two different
antigens. Various different forms and uses of the bispecific
antibody are known in the art (Chames P et al., Curr. Opin. Drug
Disc. Dev., 12:276, 2009; Spiess C et al., Mol. Immunol., 67:
95-106, 2015).
[0114] The term "hCG-.beta. carboxy terminal peptide (CTP)" is a
short peptide from the carboxy terminus of a human chorionic
gonadotropin (hCG) .beta.-subunit. Four reproduction-related
polypeptide hormones, follicle-stimulating hormone (FSH),
luteinizing hormone (LH), thyroid-stimulating hormone (TSH), and
human chorionic gonadotropin (hCG), each contain the same
.alpha.-subunit and their respective specific .beta.-subunits. The
in vivo half-life of hCG is significantly longer than those of the
other three hormones, mainly due to the specific carboxy terminal
peptide (CTP) on the .beta.-subunit of hCG. The CTP includes 37
amino acid residues and four O-glycosylation sites, in which sugar
side chain terminals are sialic acid residues. The electronegative
highly-sialyl CTP can resist renal clearance and thus extend the
half-life in vivo (Fares F A et al., Proc Natl Acad. Sci. USA,
1992, 89: 4304-4308, 1992).
[0115] The term "glycosylation" means that an oligosaccharide (a
carbohydrate containing two or more monosaccharides that are linked
together, e.g., a carbohydrate containing 2 to about 12
monosaccharides that are linked together) is attached to form a
glycoprotein. The oligosaccharide side chains are generally linked
to the backbone of the glycoprotein via N- or O-linkages. The
oligosaccharides of the antibodies disclosed herein are generally
CH2 domains linked to the Fc region as N-linked oligosaccharides.
"N-linked glycosylation" refers to carbohydrate moiety attachment
to an asparagine residue of a glycoprotein chain. For example, the
skilled artisan can recognize a single site useful for N-linked
glycosylation at residue 297 of each of CH2 domains of murine IgG1,
IgG2a, IgG2b and IgG3 and human IgG1, IgG2, IgG3, IgG4, IgA and
IgD.
[0116] The term "conservative modification" is intended to mean
that an amino acid modification does not significantly affect or
change the binding characteristics of the antibody containing an
amino acid sequence. Such conservative modifications include amino
acid substitutions, additions and deletions. A modification may be
introduced into the antibody of the present disclosure by using a
standard technology known in the art, such as a site-directed
mutagenesis and a PCR-mediated mutagenesis. A conservative amino
acid substitution refers to the substitution of an amino acid
residue with an amino acid residue with a similar side chain.
Families of amino acid residues with similar side chains have been
described in detail in the art. These families include amino acids
with basic side chains (such as lysine, arginine and histidine),
amino acids with acidic side chains (such as aspartic acid and
glutamic acid), amino acids with uncharged polar side chains (such
as glycine, asparagine, glutamine, serine, threonine, tyrosine,
cysteine and tryptophan), amino acids with non-polar side chains
(such as alanine, valine, leucine, isoleucine, proline,
phenylalanine and methionine), amino acids with .beta.-branched
side chains (such as threonine, valine and isoleucine) and amino
acids with aromatic side chains (such as tyrosine, phenylalanine,
tryptophan and histidine). Therefore, one or more amino acid
residues in the CDR of the antibody of the present disclosure may
be substituted with other amino acid residues from the same side
chain family.
Fc Variant with Altered Binding Affinity for the Neonatal Receptor
(FcRn)
[0117] "FcRn" used herein refers to a protein that binds to at
least part of the Fc region of the IgG antibody and that is encoded
by the FcRn gene. FcRn may be derived from any organism including,
but not limited to, humans, mice, rats, rabbits or monkeys. The
functional FcRn protein includes two polypeptides that often
referred to as heavy and light chains, in which the light chain is
.beta.-2-microglobulin and the heavy chain is encoded by the FcRn
gene.
[0118] The present disclosure relates to an antibody whose binding
to FcRn is regulated (the regulation includes to increase or
decrease the binding). For example, in some cases, increased
binding may result in cell recirculating antibodies, and thus
extends, for example, the half-life of the therapeutic antibody.
Sometimes, it is desirable to decrease the FcRn binding, for
example, when the antibody is used as a diagnostic or therapeutic
antibody including a radiolabel. In addition, antibodies exhibiting
increased binding to FcRn and altered binding to other Fc receptors
such as Fc.gamma. Rs may be used in the present disclosure.
[0119] The present application relates to an antibody including an
amino acid modification that regulates the binding to FcRn. Of
particular interest is that at lower pH, the binding affinity for
FcRn exhibits an increase, while at higher pH, the binding
basically does not exhibit an altered antibody that minimally
includes the Fc region or functional variants thereof.
[0120] Fc variant with enhanced binding affinity for the neonatal
receptor (FcRn)
[0121] The plasma half-life of IgG depends on its binding to FcRn,
where IgG generally binds to FcRn at a pH of 6.0 and dissociates
from FcRn at a pH of 7.4 (the pH of plasma). Through studies on the
binding site, a binding site of IgG to FcRn is modified to increase
the binding capacity at the pH of 6.0. It has been proved that
mutations of some residues of a human Fc.gamma. domain, which are
essential to the binding to FcRn, can increase the serum half-life.
It has been reported that mutations at T250, M252, 5254, T256,
V308, E380, M428, and N434 (EU numbering) can increase or decrease
the binding affinity for FcRn (Roopenian et al., Nat. Rev.
Immunol., 7: 715-725, 2007). Korean Patent No. KR 10-1027427
discloses trastuzumab (Herceptin, Genentech) variants with enhanced
binding affinity for FcRn, where these variants include one or more
amino acid modifications selected from 257C, 257M, 257L, 257N,
257Y, 279Q, 279Y, 308F, and 308Y. Korean Patent Publication No. KR
2010-0099179 provides bevacizumab (Avastin, Genentech) variants,
where these variants exhibit an increased in vivo half-life through
amino acid modifications included in N434S, M252Y/M428L,
M252Y/N434S, and M428L/N434S. In addition, Hinton et al. have found
that T250Q and M428L mutants increase the binding to FcRn threefold
and sevenfold, respectively. At the same time, the mutation of two
sites increases the binding 28-fold. In rhesus monkeys, the M428L
or T250QM/428 L mutant exhibits the plasma half-life increased
twofold (Hinton P. R. et al., J. Immunol., 176: 346-356, 2006). For
more mutational sites included in the Fc variant with the enhanced
binding affinity for the neonatal receptor (FcRn), see Chinese
invention patent CN 201280066663.2. In addition, studies have shown
that through the T250Q/M428L mutation on Fc fragments of five
humanized antibodies, the interaction between Fc and FcRn is
improved, and in subsequent in vivo pharmacokinetic assays, the
pharmacokinetic parameters of the Fc-mutation antibody are improved
compared with the wild-type antibody through subcutaneous
administration, for example, the in vivo exposure is increased, the
clearance rate is reduced, and the subcutaneous bioavailability is
increased (Datta-Mannan A et al., MAbs. Taylor & Francis, 4:
267-273, 2012).
[0122] Other mutational sites capable of enhancing the affinity of
the antibody of the present disclosure for FcRn include, but are
not limited to, the following amino acid modifications: 226, 227,
230, 233, 239, 241, 243, 246, 259, 264, 265, 267, 269, 270, 276,
284, 285, 288, 289, 290, 291, 292, 294, 298, 299, 301, 302, 303,
305, 307, 309, 311, 315, 317, 320, 322, 325, 327, 330, 332, 334,
335, 338, 340, 342, 343, 345, 347, 350, 352, 354, 355, 356, 359,
360, 361, 362, 369, 370, 371, 375, 378, 382, 383, 384, 385, 386,
387, 389, 390, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401,
403, 404, 408, 411, 412, 414, 415, 416, 418, 419, 420, 421, 422,
424, 426, 433, 438, 439, 440, 443, 444, 445, and 446, where the
numbers of the amino acids in the Fc region is numbers of the EU
indexes in Kabat.
[0123] Fc variants with enhanced binding affinity for FcRn also
include all other known amino acid modification sites as well as
amino acid modification sites that have not yet been found.
[0124] In an optional embodiment, the IgG variant can be optimized
to gain increased or decreased affinity for FcRn and increased or
decreased affinity for human Fc.gamma.R including, but not limited
to, Fc.gamma.RI, Fc.gamma.RIIa, Fc.gamma.RIIb, Fc.gamma.RIIc,
Fc.gamma.RIIIa and Fc.gamma.RIIIb, including allelic variants
thereof.
[0125] Preferentially, the Fc ligand specificity of an IgG variant
determines its therapeutic application. The given IgG variant for
therapeutic purposes depends on the epitope or form of the target
antigen as well as the to-be-treated disease or indication.
Enhanced FcRn binding may be more preferred for most targets and
indications because enhanced FcRn binding may result in extended
serum half-life. A relatively long serum half-life allows
administration at relatively low frequencies and doses during
treatment. This property may be particularly preferred when the
therapeutic agent is administered in order to respond to
indications requiring repeated administration. For some targets and
indications, the reduced affinity for FcRn may be particularly
preferred when the variant Fc is required to have an increased
clearance or reduced serum half-life, for example, when the Fc
polypeptide is used as an imaging agent or a radiotherapy
agent.
[0126] Fc Alterations to Extend the Half-Life
[0127] As used herein, "Fc alterations to extend the half-life"
refers to an alteration to extend the in vivo half-life of a
protein that includes an altered Fc polypeptide in the Fc
polypeptide chain as compared with the half-life of a protein that
includes the same Fc polypeptide but does not include any altered
but similar Fc. These alterations may be included in the Fc
polypeptide chain as part of the bispecific antibody. Alterations
T250Q, M252Y, S254T and T256E (alteration of threonine at position
250 to glutamine; alteration of methionine at position 252 to
tyrosine; alteration of serine at position 254 to threonine; and
alteration of threonine at position 256 to glutamic acid; where
numbers are based on EU numbering) is an Fc alteration to extend
half-life and may be used jointly, alone or in any combination.
These and other alterations are described in detail in U.S. Pat.
No. 7,083,784. The section about this alteration described in U.S.
Pat. No. 7,083,784 is incorporated herein by reference.
[0128] Similarly, M428L and N434S are Fc alterations that extend
the half-life and can be used jointly, alone or in any combination.
These alterations and other alterations are described in detail in
U.S. Patent Application Publication No. 2010/0234575 and U.S. Pat.
No. 7,670,600. Sections of such alterations described in U.S.
Patent Application Publication No. 2010/0234575 and U.S. Pat. No.
7,670,600 are incorporated herein by reference.
[0129] In addition, according to the meaning herein, any
substitution at one of the following positions can be considered to
be an Fc alternation that extends the half-life: 250, 251, 252,
259, 307, 308, 332, 378, 380, 428, 430, 434, and 436. Each of these
alterations or a combination of these alterations may be used to
extend the half-life of the bispecific antibody described herein.
Other alternations that can be used to extend the half-life are
described in detail in International Application No.
PCT/US2012/070146 (Publication No. WO 2013/096221) filed Dec. 17,
2012. The section about the above alterations of this application
is incorporated herein by reference.
[0130] Fc alternations that extend the half-life also include sites
and modifications thereof that are well known in the art or may be
discovered in the future.
[0131] Fc may be derived from any organism including, but not
limited to, humans, mice, rats, rabbits or monkeys.
[0132] Nucleic Acid Encoding Bispecific Antibody
[0133] Using the therapeutic agent, the antibody or the antibody
fragment described herein, those skilled in the art may easily
construct multiple clones containing functionally equivalent
nucleic acids (e.g., nucleic acids with different sequences but
encoding the same effector moiety or antibody sequence). Therefore,
the present disclosure provides bispecific antibodies, nucleic
acids encoding the antibodies, antibody fragments and conjugates
and fusion protein thereof, and variants, derivatives and species
homologues of the nucleic acids.
[0134] Many nucleic acid sequences encoding immunoglobulin regions
including VH, VL, hinge, CH1, CH2, CH3 and CH4 regions are known in
the art. See, for example, Kabat et al., Sequences of Proteins of
Immunological Interest, Public Health Service N.I.H., Bethesda,
Md., 1991. According to the teachings provided herein, those
skilled in the art may combine the nucleic acid sequences and/or
other nucleic acid sequences known in the art to construct a
nucleic acid sequence encoding the bispecific antibody of the
present disclosure. An exemplary nucleotide encoding the bispecific
antibody of the present disclosure includes SEQ ID NO: 51.
[0135] In addition, based on the amino acid sequences provided
herein and elsewhere and the common knowledge in the art, those
skilled in the art may determine the nucleic acid sequence encoding
the bispecific antibody of the present disclosure. In addition to
more conventional methods for producing cloned DNA fragments
encoding particular amino acid sequences, companies such as DNA 2.0
(Menlo Park, Calif., USA) and Blue Heron (Bothell, Wash., USA)
generally employ chemical synthesis to produce DNA that has any
size of genes arranged in a desired order, thus simplifying the
process of producing the DNA.
[0136] Method for Preparing a Bispecific Antibody
[0137] The bispecific antibody of the present disclosure may be
prepared by any method known in the art. Early methods for
constructing the bispecific antibody include chemical cross-linking
or hybridoma heterozygosis or quadroma method (e.g., Staerz U D et
al., Nature, 314: 628-31, 1985; Milstein C et al., Nature, 305:
537-540, 1983; Karpovsky B et al., J. Exp. Med., 160: 1686-1701,
1984). The chemical coupling method is to connect two different
monoclonal antibodies by chemical coupling to prepare a bispecific
monoclonal antibody. For example, two different monoclonal
antibodies chemically bind to each other, or two antibody
fragments, for example, two Fab fragments chemically bind to each
other. The heterozygosis-hybridoma method is to prepare a
bispecific monoclonal antibody by a cell hybridization method or a
ternary hybridoma method, where the cell hybridoma or the ternary
hybridoma is obtained by the fusion of constructed hybridomas or
the fusion of a constructed hybridoma and lymphocytes derived from
mice. Although these techniques are used to manufacture BiAb,
various generation problems make such complexes difficult to use,
such as the generation of mixed populations containing different
combinations of antigen-binding sites, difficulties in protein
expression, the need for purifying the target BiAb, low yields, and
high production costs.
[0138] Recent methods utilize genetically engineered constructs
that can produce a single homogeneous product of BiAb so that there
is no need for thorough purification to remove unwanted
by-products. Such constructs include tandem scFv, diabodies, tandem
diabodies, double variable domain antibodies, and heterdimeric
antibodies using the Ch1/Ck domain or DNLTM motifs (Chames &
Baty, Curr. Opin. Drug. Discov. Devel., 12: 276-83, 2009; Chames
& Baty, mAbs, 1: 539-47). Related purification techniques are
well known.
[0139] The antibody may also be produced by cloning and expressing
immunoglobulin variable region cDNAs produced from a single
lymphocyte selected to produce the specific antibody using a single
lymphocyte antibody method, for example, by a method described in
Babcook J et al., Proc. Natl. Acad. Sci. USA. 93: 7843-7848, 1996;
WO92/02551; WO2004/051268 and WO2004/106377.
[0140] Antigen polypeptides for producing, for example, antibodies
for immunizing hosts or for screening, such as antibodies for phage
display (or the expression on the surface of yeast cells or
bacterial cells) may be prepared from genetically engineered host
cells including expression systems by methods well known in the
art, or they may be recycled from natural biological sources. For
example, nucleic acids encoding one or two polypeptide chains of
the bispecific antibody may be introduced into cultured host cells
by a variety of known methods (such as transformation,
transfection, electroporation and bombardment with nucleic
acid-coated microparticles, etc.). In some embodiments, the nucleic
acids encoding the bispecific antibody may be inserted into a
vector suitable to be expressed in the host cell before introduced
into the host cell. A typical vector may include sequence elements
that enable the inserted nucleic acids to be expressed at RNA and
protein levels.
[0141] The vector is well known in the art and many vectors are
commercially available. The host cell containing the nucleic acids
may be cultured under conditions that enable the cell to express
the nucleic acids, and the resulting BiAb may be collected from a
cell population or a culture medium. Optionally, the BiAb may be
produced in vivo, for example, in a plant leaf (see, for example,
Scheller J et al., Nature Biotechnol., 19: 573-577, 2001 and
references cited therein), in a bird egg (see, for example, Zhu L
et al., Nature Biotechnol., 23: 1159-1169, 2005 and references
cited therein) or in mammalian milk (see, for example, Laible G et
al., Reprod. Fertil. Dev., 25: 315, 2012).
[0142] Various cultured host cells that may be used include, for
example, prokaryotic cells, eukaryotic cells, bacterial cells (such
as Escherichia coli or Bacillus stearothermophilus), fungal cells
(such as Saccharomyces cerevisiae or Pichia pastoris), insect cells
(such as lepidopteran insect cells including Spodoptera frugiperda
cells), or mammalian cells (such as Chinese hamster ovary (CHO)
cells, NS0 cells, baby hamster kidney (BHK) cells, monkey kidney
cells, Hela cells, human hepatocellular carcinoma cells or 293
cells).
[0143] The bispecific antibody may be prepared by immunizing an
appropriate subject (such as a rabbit, a goat, a mouse, or other
mammals including transgenic and knocked-out mammals) with an
immunogenic preparation of a bispecific antigen. An appropriate
immunogenic preparation may be, for example, a chemically
synthesized or recombinantly expressed bispecific antigen. The
preparation may further include adjuvants such as Freund's complete
or incomplete adjuvants or similar immunostimulatory compounds.
Furthermore, the bispecific antigen of the present disclosure may
be used alone or preferably used as a conjugate with a vector
protein when used for preparing antibodies, in particular by in
vivo immunization. Such methods of enhancing an antibody response
are well known in the art. Depending on different antibodies
required, different animal hosts may be used for in vivo
immunization. A host that expresses an endogenous antigen of
interest itself may be used, or a host that has been caused
deficient in an endogenous antigen of interest may be used.
[0144] The bispecific antibody may be prepared by a combination of
the methods as described above.
[0145] The bispecific antibody molecule of the present disclosure
may act as a monoclonal antibody (MAb) for each target. In some
embodiments, the antibody is chimeric, humanized or fully
human.
[0146] The monoclonal antibody may be prepared by any method known
in the art, such as a hybridoma technique (Kohler & Milstein,
Nature, 256: 495-497, 1975), a trioma technique, a human B-cell
hybridoma technique (Kozbor D et al., Immunology Today, 4: 72,
1983), and an EBV-hybridoma technique (Cole S P C et al.,
Monoclonal Antibodies and Cancer Therapy, pp 77-96, Alan R Liss,
Inc., 1985).
[0147] The bispecific antibody or a portion thereof in the present
disclosure may be used for detecting any or all of these antigens
(for example, in biological samples such as serum or plasma) by
conventional immunological analysis methods such as an
enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA)
or tissue immunohistochemistry. The present disclosure provides a
method for detecting an antigen in a biological sample, comprising
contacting a biological sample with the bispecific antibody or the
antibody portion of the present disclosure that specifically
recognizes the antigen, and detecting an antigen-bound antibody (or
antibody portion) or a non-bound antibody (or antibody portion), so
as to detect the antigen in the biological sample. The antibody is
directly or indirectly labeled with a detectable substance to
facilitate the detection of the bound or non-bound antibody.
Suitable detectable substances include various enzymes, repair
groups, fluorescent substances, luminescent substances and
radioactive substances. Examples of suitable enzymes include
horseradish peroxidase, alkaline phosphatase, .beta.-galactosidase
and acetylcholinesterase; examples of suitable repair group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent substances include 7-hydroxycoumarin,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of the luminescent substance includes
3-aminophthalhydrazide; examples of suitable radioactive substances
include I.sup.125, I.sup.131, .sup.35S or .sup.3H.
[0148] Pharmaceutical Composition
[0149] The bispecific antibody of the present disclosure or a
nucleic acid or polynucleotide encoding the antibody of the present
application may be applied to the preparation of the pharmaceutical
composition or a sterile composition. For example, the bispecific
antibody is mixed with a pharmaceutically acceptable carrier,
excipient or stabilizer. The pharmaceutical composition may include
one bispecific antibody or a combination of (for example, two or
more different) bispecific antibodies of the present disclosure.
For example, the pharmaceutical composition of the present
disclosure may include a combination of antibodies or antibody
fragments (or immunoconjugates) that have complementary activity
and bind to different epitopes on a target antigen. Formulations of
therapeutic and diagnostic agents may be prepared by mixing the
antibody with the pharmaceutically acceptable carrier, excipient or
stabilizer in the form of, for example, lyophilized powder, slurry,
an aqueous solution or a suspension.
[0150] The term "pharmaceutically acceptable" means that a
molecule, molecule fragment or composition does not produce
unfavorable, allergic or other adverse effects when properly
administered to animals or humans. Specific examples of some
substances that may act as the pharmaceutically acceptable carriers
or components thereof include sugars (such as lactose), starch,
cellulose and its derivatives, vegetable oils, gelatin, polyols
(such as propylene glycol), alginic acid and the like.
[0151] In some preferred embodiments, the pharmaceutical
composition of the present disclosure is used for treating,
preventing or alleviating diseases including, but not limited to,
cancer, lymphatic system diseases, autoimmune diseases,
inflammatory diseases, infectious diseases, immunodeficiency
syndromes and other related diseases or symptoms.
[0152] The bispecific antibody or the nucleic acid or
polynucleotide encoding the antibody of the present application may
be linked to the preceding pharmaceutically acceptable carriers or
components thereof (as immune complexes) or administered separately
from the preceding pharmaceutically acceptable carriers or the
components thereof. In the latter case, the bispecific antibody or
the nucleic acid or polynucleotide encoding the antibody of the
present application may be administered before, after or together
with the preceding pharmaceutically acceptable carriers or the
components thereof or may be administered with other known
therapies (such as anticancer therapies or radiation).
[0153] The compositions of the present disclosure may be in various
forms. The forms include, for example, liquid, semi-solid and solid
dosage forms such as liquid solutions (e.g. injectable and
non-molten solutions), dispersants or suspension tablets, pills,
powders, liposomes and suppositories. A preferred manner depends on
the mode of administration and the therapeutic use. Preferred
typical compositions are injectable or non-molten solutions, such
as those compositions that passively immunize humans like other
antibodies. The compositions of the present disclosure may be
administered through various routes including oral, rectal,
transmucosal, trans-intestinal, parenteral, intramuscular,
subcutaneous, intradermal, intramedullary, intrathecal, direct
intraventricular, intravenous, intraperitoneal, intranasal,
intraocular, inhalation, insufflation, topical, skin, transdermal,
or intraarterial. A preferred mode of administration is parenteral
(e.g., intravenous, subcutaneous, intraperitoneal, intramuscular).
In a preferred embodiment, the antibody is administered by
intravenous infusion or injection. In another preferred embodiment,
the antibody is injected intramuscularly or subcutaneously.
[0154] The above combination methods, treatment methods and
administration methods are well known. Combination, treatment and
administration methods that may be developed in the future are also
included.
[0155] The pharmaceutical composition of the present disclosure may
be a combination of two drugs or used in combination with an
already marketed product having a similar function or a product
that increases a therapeutic effect.
[0156] Combination Therapy
[0157] The present disclosure relates to uses of a combination of
the bispecific antibody or nucleic acids or polynucleotides
encoding the antibody of the present disclosure or immunoconjugates
or pharmaceutical compositions and one or more active therapeutic
agents (e.g., chemotherapeutic agents) or other prophylactic or
therapeutic modes (e.g., radiation). In such combination therapies,
the various active agents often have different complementary
mechanisms of action, and the combination therapy may lead to
synergistic effects. The combination therapy includes therapeutic
agents that affect immune responses (e.g., an enhanced or activated
response) and therapeutic agents that affect (e.g., inhibit or
kill) tumor/cancer cells. The combination therapy may reduce the
likelihood of drug-resistant cancer cells. The combination therapy
may allow the dose reduction of one or more reagents to reduce or
eliminate adverse effects associated with the one or more reagents.
Such combination therapies may have synergistic therapeutic or
prophylactic effects on underlying diseases, disorders or
symptoms.
[0158] The "combination" includes therapies that can be
administered separately, for example, separate formulations for
individual administration (e.g., which may be provided in a kit),
and therapies that can be administered together in a single
formulation (i.e., "co-formulation"). In some embodiments, the
bispecific antibody of the present disclosure or nucleic acids or
polynucleotides encoding the antibody of the present disclosure or
immunoconjugates or pharmaceutical compositions may be administered
sequentially. In some embodiments, the bispecific antibody of the
present disclosure or nucleic acids or polynucleotides encoding the
antibody of the present disclosure or immunoconjugates or
pharmaceutical compositions may be administered simultaneously. The
bispecific antibody or nucleic acids or polynucleotides encoding
the antibody of the present disclosure or immunoconjugates or
pharmaceutical compositions may be used in any manner in
combination with at least one other (active) agent.
[0159] Treatment with the bispecific antibody of the present
disclosure may be combined with other treatments that are effective
against the to-be-treated disease. Non-limiting examples of
antibody combination therapies of the present disclosure include
surgery, chemotherapy, radiation therapy, immunotherapy, gene
therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, and
adjuvant therapy.
[0160] Combination therapies also include all other combination
therapies known in the art or developed in the future.
BRIEF DESCRIPTION OF DRAWINGS
[0161] FIG. 1 illustrates SEC-HPLC test results of a purified
sample of bispecific antibody AP062.
[0162] FIG. 2 illustrates SDS-PAGE electrophoresis results of a
purified sample of bispecific antibody AP062.
[0163] FIG. 3 illustrates SDS-PAGE results of bispecific antibody
AP062 in an acceleration experiment at 5.degree. C.
[0164] FIG. 4 illustrates SDS-PAGE results of bispecific antibody
AP062 in an acceleration experiment at 25.degree. C.
[0165] FIG. 5 illustrates an ability, detected by flow cytometry,
of bispecific antibody AP062 to bind to CD20-positive tumor
cells.
[0166] FIG. 6 illustrates an ability of bispecific antibody AP062
to mediate effector cells to kill Raji-luc cells.
[0167] FIG. 7 illustrates abilities, detected by a reporter gene
assay, of bispecific antibodies AP062 and AB7K7 to activate Jurkat
NFATRE Luc cells.
[0168] FIG. 8 illustrates an in vivo anti-tumor effect of
bispecific antibody AP062 in an NPG mouse model of transplanted
tumor constructed by subcutaneously co-inoculating human CIK cells
and human Burkkit's lymphoma Raji cells.
[0169] FIG. 9 illustrates an in vivo anti-tumor effect of
bispecific antibody AP062 in an NPG mouse model of transplanted
tumor constructed by subcutaneously co-inoculating human CIK cells
and human Burkkit's lymphoma Daudi cells.
[0170] FIG. 10 illustrates changes of leukocytes and lymphocytes in
normal cynomolgus monkeys administered with bispecific antibody
AP062 multiple times.
DETAILED DESCRIPTION
[0171] The present disclosure is further described through examples
which should not be construed as further limitations. All drawings,
all reference documents, and the contents of patents and published
patent applications cited in the entire application are expressly
incorporated herein by reference.
[0172] In the following examples, materials used in experiments may
be purchased or prepared with reference to techniques disclosed in
the existing art; materials whose sources and specifications are
unidentified are commercially available; various processes and
methods not described in detail are conventional methods well known
in the art.
Example 1 Construction of an Expression Vector of a Bispecific
Antibody Molecule
[0173] The bispecific antibody molecule constructed in the present
disclosure is a tetravalent homodimer formed by two identical
polypeptide chains binding to each other by an interchain disulfide
bond in hinge regions of Fc fragments, wherein each polypeptide
chain consists of an anti-CD20 scFv, a linker peptide L2, an
anti-CD3 scFv and an Fc fragment in sequence from N-terminus to
C-terminus, wherein VH and VL in the anti-CD20 scFv are linked by a
linker peptide L1, and VH and VL in the anti-CD3 scFv are linked by
a linker peptide L3. Some preferred amino acid sequences of the VH
domain and its complementarity determining regions (HCDR1, HCDR2
and HCDR3) and amino acid sequences of the VL domain and its
complementarity determining regions (LCDR1, LCDR2 and LCDR3) of a
first single-chain Fv against CD20 are listed in Table 1, wherein
amino acid residues contained in the CDRs are defined according to
a Kabat rule. The amino acid composition of the linker peptide L1
between VH and VL of the anti-CD20 scFv is (GGGGS)n, wherein n=1,
2, 3, 4 or 5.
TABLE-US-00001 TABLE 1 Amino acid sequences of the anti-CD20 scFv
contained in the bispecific antibody and amino acid sequences of
its CDRs CD20 SEQ ID NO: 1 HCDR1 SYNMH SEQ ID NO: 2 HCDR2
AIYPGNGDTSYNQKFKG SEQ ID NO: 3 HCDR3 STYYGGDWYFNV SEQ ID NO: 4
LCDR1 RASSSVSYIH SEQ ID NO: 5 LCDR2 ATSNLAS SEQ ID NO: 6 LCDR3
QQWTSNPPT SEQ ID NO: 25 VH QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMH
WVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLT
ADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWY FNVWGAGTTVTVSA SEQ ID NO: 26
VL QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQK
PGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRV
EAEDAATYYCQQWTSNPPTFGGGTKLEIK SEQ ID NO: 7 HCDR1 NYYIH SEQ ID NO: 8
HCDR2 WIYPGDGNTKYNEKFKG SEQ ID NO: 9 HCDR3 DSYSNYYFDY SEQ ID NO: 10
LCDR1 RASSSVSYMH SEQ ID NO: 11 LCDR2 APSNLAS SEQ ID NO: 12 LCDR3
QQWSFNPPT SEQ ID NO: 27 VH EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIHW
VRQAPGQGLEWIGWIYPGDGNTKYNEKFKGRATLTA
DTSTSTAYLELSSLRSEDTAVYYCARDSYSNYYFDYW GQGTLVTVSS SEQ ID NO: 28 VL
DIQMTQSPSSLSASVGDRVTITCRASSSVSYMHWYQQ
KPGKAPKPLIYAPSNLASGVPSRFSGSGSGTDFTLTISS
LQPEDFATYYCQQWSFNPPTFGQGTKVEIK SEQ ID NO: 13 HCDR1 YSWIN SEQ ID NO:
14 HCDR2 RIFPGDGDTDYNGKFKG SEQ ID NO: 15 HCDR3 NVFDGYWLVY SEQ ID
NO: 16 LCDR1 RSSKSLLHSNGITYLY SEQ ID NO: 17 LCDR2 QMSNLVS SEQ ID
NO: 18 LCDR3 AQNLELPYT SEQ ID NO: 29 VH
QVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWINW
VRQAPGQGLEWMGRIFPGDGDTDYNGKFKGRVTITA
DKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVY WGQGTLVTVSS SEQ ID NO: 30 VL
DIVMTQTPLSLPVTPGEPASISCRSSKSLLHSNGITYLY
WYLQKPGQSPQLLIYQMSNLVSGVPDRFSGSGSGTDF
TLKISRVEAEDVGVYYCAQNLELPYTFGGGTKVEIK SEQ ID NO: 19 HCDR1 DYAMH SEQ
ID NO: 20 HCDR2 TISWNSGSIGYADSVKG SEQ ID NO: 21 HCDR3 DIQYGNYYYGMDV
SEQ ID NO: 22 LCDR1 RASQSVSSYLA SEQ ID NO: 23 LCDR2 DASNRAT SEQ ID
NO: 24 LCDR3 QQRSNWPIT SEQ ID NO: 31 VH
EVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHW
VRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRD
NAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYYYG MDVWGQGTTVTVSS SEQ ID NO: 32 VL
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQ
KPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISS
LEPEDFAVYYCQQRSNWPITFGQGTRLEIK
[0174] The anti-CD3 scFv binds to an effector cell at an EC.sub.50
value greater than about 50 nM, or greater than 100 nM, or greater
than 300 nM, or greater than 500 nM in an in vitro FACS binding
assay; more preferably, the second single-chain Fv of the
bispecific antibody is capable of binding to human CD3 and
specifically binding to CD3 of a cynomolgus monkey or a rhesus
monkey.
[0175] Some preferred amino acid sequences of the VH domain and its
complementarity determining regions (HCDR1, HCDR2 and HCDR3) and
amino acid sequences of the VL domain and its complementarity
determining regions (LCDR1, LCDR2 and LCDR3) of the anti-CD3 scFv
are listed in Table 2, wherein amino acid residues contained in the
CDRs are defined according to the Kabat rule. The amino acid
composition of the linker peptide L3 between VH and VL of the
anti-CD3 scFv is (GGGGS)n, wherein n=1, 2, 3, 4 or 5.
TABLE-US-00002 TABLE 2 Amino acid sequences of the anti-CD3 scFv
contained in the bispecific antibody and amino acid sequences of
its CDRs CD3-3 SEQ ID NO: 34 HCDR1 TYAMN SEQ ID NO: 35 HCDR2
RIRSKYNNYATYYADSVKD SEQ ID NO: 36 HCDR3 HGNFGNSYVSWFAY SEQ ID NO:
37 LCDR1 RSSTGAVTTSNYAN SEQ ID NO: 38 LCDR2 GTNKRAP SEQ ID NO: 39
LCDR3 ALWYSNLWV SEQ ID NO: 46 VH EVQLLESGGGLVQPGGSLKLSCAASGFTF
NTYAMNWVRQAPGKGLEWVARIRSKYNNY ATYYADSVKDRFTISRDDSKNTAYLQMNN
LKTEDTAVYYCVRHGNFGNSYVSWFAYWG QGTLVTVSS SEQ ID NO: 47 VL
ELVVTQEPSLTVSPGGTVTLTCRSSTGAV TTSNYANWVQQKPGQAPRGLIGGTNKRA
PGTPARFSGSLLGGKAALTLSGVQPEDE AEYYCALWYSNLWVFGGGTKLTVL CD3-4 SEQ ID
NO: 40 HCDR1 KYAMN SEQ ID NO: 41 HCDR2 RIRSKYNNYATYYADSVKD SEQ ID
NO: 42 HCDR3 HGNFGNSYISYWAY SEQ ID NO: 43 LCDR1 GSSTGAVTSGYYPN SEQ
ID NO: 44 LCDR2 GTKFLAP SEQ ID NO: 45 LCDR3 ALWYSNRWV SEQ ID NO: 48
VH EVQLLESGGGLVQPGGSLKLSCAASGFTF NKYAMNWVRQAPGKGLEWVARIRSKYNNY
ATYYADSVKDRFTISRDDSKNTAYLQMNN LKTEDTAVYYCVRHGNFGNSYISYWAYWG
QGTLVTVSS SEQ ID NO: 49 VL ELVVTQEPSLTVSPGGTVTLTCGSSTGAV
TSGYYPNWVQQKPGQAPRGLIGGTKFLAP GTPARFSGSLLGGKAALTLSGVQPEDEAE
YYCALWYSNRWVFGGGTKLTVL
[0176] The linker peptide that links the anti-CD20 scFv to the
anti-CD3 scFv consists of a flexible peptide and a rigid peptide;
preferably, an amino acid composition structure of the flexible
peptide has a general formula of G.sub.xS.sub.y(GGGGS).sub.z,
wherein x, y and z are integers greater than or equal to 0 and
x+y+z.gtoreq.1. The rigid peptide is derived from a full-length
sequence (as shown in SEQ ID NO: 33) consisting of amino acids 118
to 145 at the carboxyl terminus of natural human chorionic
gonadotropin .beta.-subunit or a truncated fragment thereof;
preferably, the composition of the CTP rigid peptide is SSSSKAPPPS
(CTP.sup.1). Some preferred amino acid sequences of the linker
peptide L2 that links the anti-CD20 scFv and the anti-CD3 scFv are
listed in Table 3.
TABLE-US-00003 TABLE 3 Amino acid sequences of the linker peptide
that links the anti-CD20 scFv to the anti-CD3 scFv SEQ ID NO: 52
G2(GGGGS)3CTP1 GGGGGGSGGGGSGGGGSSSS SKAPPPS SEQ ID NO: 53
(GGGGS)3CTP1 GGGGSGGGGSGGGGSSSSSK APPPS SEQ ID NO: 54
GS(GGGGS)2CTP1 GSGGGGSGGGGSSSSSKAPP PS SEQ ID NO: 55 (GGGGS)1CTP4
GGGGSSSSSKAPPPSLPSPS RLPGPSDTPILPQ
[0177] The Fc fragment is linked to the anti-CD3 scFv directly or
by a linker peptide, wherein the linker peptide includes 1-20 amino
acids, and preferably selected from the following amino acids:
Gly(G), Ser(S), Ala(A) and Thr(T), more preferably selected from
Gly (G) and Ser (S), and most preferably, the linker peptide
consists of (GGGGS)n, wherein n=1, 2, 3 or 4.
[0178] The Fc fragment is preferably selected from heavy chain
constant regions of human IgG1, IgG2, IgG3 and IgG4 and more
particularly selected from heavy chain constant regions of human
IgG1 or IgG4; and Fc is mutated to modify the properties of the
bispecific antibody molecule, e.g., reduced affinity to at least
one of human Fc.gamma.Rs (Fc.gamma.RI, Fc.gamma.RIIa or
Fc.gamma.RIIIa) and C1q, a reduced effector cell function, or a
reduced complement function. In addition, the Fc fragment may also
contain amino acid substitutions that change one or more other
characteristics (such as an ability of binding to an FcRn receptor,
the glycosylation of the antibody or the charge heterogeneity of
the antibody).
[0179] Some amino acid sequences of the Fc fragment with one or
more amino acid mutations are listed in Table 4.
TABLE-US-00004 TABLE 4 Amino acid sequences of Fc from human IgG
Amino acid sequence of constant region of IgG1 Fc (L234A/L235A)
mutant (EU numbering) SEQ ID NO: 56 DKTHTCPPCP APEAAGGPSV
FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY
RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK
NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG
NVFSCSVMHE ALHNHYTQKS LSLSPGK Amino acid sequence of constant
region of IgG1 (L234A/L235A/T250Q/N297A/P331S/M428L/K447-) mutant
(EU numbering) SEQ ID NO: 57 DKTHTCPPCP APEAAGGPSV FLFPPKPKDQ
LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYASTY RVVSVLTVLH
QDWLNGKEYK CKVSNKALPA SIEKTISKAK GQPREPQVYT LPPSRDELTK NQVSLTCLVK
GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVLHE
ALHNHYTQKS LSLSP
[0180] Exemplarily, the amino acid sequence of the preferred
bispecific antibody AP062 is shown by SEQ ID NO: 50, and the
corresponding nucleotide sequence is shown by SEQ ID NO: 51.
[0181] A gene encoding the bispecific antibody was synthesized by a
conventional molecular biology method, and cDNA encoding the
obtained fusion gene was separately inserted into corresponding
restriction sites of a eukaryotic expression plasmid pCMAB2M
modified by PCDNA3.1. The plasmid pCMAB2M also contains a selective
marker to have kanamycin resistance in bacteria and G418 resistance
in mammalian cells. In addition, when host cells are deficient in
the expression of DHFR genes, the expression vector pCMAB2M
contains mouse dihydrofolate reductase (DHFR) genes so that target
genes and the DHFR genes can be co-amplified in the presence of
methotrexate (MTX) (see U.S. Pat. No. 4,399,216).
Example 2 Expression of the Bispecific Antibody Molecule
[0182] The constructed expression plasmid was transfected into a
mammalian host cell line to express the bispecific antibody. To
achieve stable and high-level expression, a preferred host cell
line is DHFR deficient CHO-cells (see U.S. Pat. No. 4,818,679). In
this example, a CHO-derived cell strain DXB11 was selected as the
host cell. A preferred method of transfection is electroporation,
and other methods, including calcium phosphate co-precipitation and
lipofection, may also be used. During electroporation, 50 .mu.g of
DNA of the expression vector plasmid were added to 5.times.10.sup.7
cells in a cuvette with a Gene Pulser electroporator (Bio-Rad
Laboratories, Hercules, Calif.) set at an electric field of 300 V
and a capacitance of 1500 .mu.Fd. Two days after transfection, the
medium was replaced with a growth medium containing 0.6 mg/mL G418.
Transfectants were subcloned by limiting dilution and the secretion
rate of each cell line was measured through an ELISA. The cell
strain that expressed the bispecific antibody at a high level was
screened.
[0183] To achieve the higher-level expression of fusion proteins,
DHFR genes inhibited by MTX should be used for co-amplification.
The transfected fusion protein genes were co-amplified with the
DHFR genes in growth media containing MTX with increased
concentrations. Subclones with the positive expression of DHFR were
subjected to limiting dilution with gradually increased pressure to
screen transfectants capable of growing in media with MTX of up to
6 .mu.M. The secretion rates of the transfectants were determined
and a cell line with high foreign protein expression was screened.
A cell line with a secretion rate greater than 5 (preferably about
15).mu.g/10.sup.6 (millions) cells/24 h was adaptively suspended
using a serum-free medium. Cell supernatants were collected and the
bispecific antibody was separated and purified.
Example 3 Purification Process and Stability Test of the Bispecific
Antibody
[0184] 3.1 Purification of the Bispecific Antibody
[0185] The bispecific antibody was purified by three-step
chromatography. The three-step chromatography was respectively
affinity chromatography, hydroxyapatite chromatography and anion
exchange chromatography. (The protein purifier used in this example
was AKTA pure 25 M from GE in the U.S. Reagents used in this
example were purchased from Sinopharm Chemical Reagent Co., Ltd and
had purity at an analytical grade).
[0186] In a first step, affinity chromatography was performed.
Sample capture and concentration and the removal of partial
pollutants were performed using AT Protein A Diamond from Bestchrom
or other commercially available affinity media (such as MabSelect
Sure from GE). First, chromatography columns were equilibrated with
3-5 column volumes (CVs) of an equilibration buffer (20 mM PB, 150
mM NaCl, pH 7.4) at a linear flow rate of 100-200 cm/h. The
clarified fermentation broth was loaded at a linear flow rate of
100-200 cm/h with a load not higher than 20 mg/mL. After loading,
the chromatography columns were equilibrated with 3-5 column
volumes (CVs) of an equilibration buffer (20 mM PB, 150 mM NaCl, pH
7.4) at a linear flow rate of 100-200 cm/h to remove unbound
components. The chromatography columns were rinsed with 3-5 column
volumes decontamination buffer 1 (50 mM NaAc-HAc, 1 M NaCl, pH 5.0)
at a linear flow rate of 100-200 cm/h to remove partial pollutants.
The chromatography columns were equilibrated with 3-5 column
volumes (CVs) of decontamination buffer 2 (50 mM NaAc-HAc, pH 5.0)
at a linear flow rate of 100-200 cm/h. The target product was
eluted using an elution buffer (50 mM NaAc-HAc, pH 3.5) at a linear
flow rate not higher than 100 cm/h and target peaks were
collected.
[0187] In a second step, hydroxyapatite chromatography was
performed. Intermediate purification was performed using CHT TypeII
from BIO-RAD or other commercially available hydroxyapatite media
to reduce the content of polymers. After the target proteins were
polymerized, the polymers and monomers differed in property such as
charge characteristics and calcium ion chelation. The polymers and
the monomers were separated with differences between charge
characteristics. First, chromatography columns were equilibrated
with 3-5 column volumes (CVs) of an equilibration buffer (20 mM PB,
pH 7.4) at a linear flow rate of 100-200 cm/h. The target proteins
separated through the affinity chromatography in the first step
were loaded after its pH was adjusted to 7.0, with a load
controlled to be less than 5 mg/mL. After loading, the
chromatography columns were rinsed with 3-5 column volumes (CVs) of
an equilibration buffer (20 mM PB, pH 7.0) at a linear flow rate of
100-200 cm/h. Finally, the target proteins were eluted using 10
column volumes (CVs) of an elution buffer (20 mM PB, 1M NaCl, pH
7.0) at a linear flow rate not higher than 100 cm/h with a gradient
of 0-25%. Eluted fractions were collected and sent for SEC-HPLC,
respectively. Target components with the percentage of monomers
being greater than 95% were combined for chromatography in the next
step.
[0188] In a third step, anion exchange chromatography was
performed. Fine purification was performed by using DEAE Sepharose
Fast Flow from GE or other commercially available anion exchange
chromatography media (such as Q HP of Bestchrom, Toyopearl GigaCap
Q-650 of TOSOH, DEAE Beads 6FF of Smart-Lifesciences, Generik MC-Q
of Sepax Technologies, Inc, Fractogel EMD TMAE of Merck, and Q
Ceramic HyperD F of Pall) to further remove pollutants such as HCP
and DNA. First, chromatography columns were rinsed with 3-5 column
volumes (CVs) of an equilibration buffer (20 mM PB, 0.15 M NaCl, pH
7.0) at a linear flow rate of 100-200 cm/h. The target proteins
separated through hydroxyapatite chromatography in the second stop
were loaded and through-flow was collected. After loading, the
chromatography columns were rinsed with 3-5 column volumes (CVs) of
an equilibration buffer (20 mM PB, 0.15 M NaCl, pH 7.0) at a linear
flow rate of 100-200 cm/h. The through-flow components were
collected and sent for the detection of protein content, SEC-HPLC
and electrophoresis.
[0189] The SEC-HPLC purity results and SDS-PAGE electrophoresis
results of the samples are shown in FIG. 1 and FIG. 2. The SEC-HPLC
results showed that the purity of the main peak of the bispecific
antibody was more than 99.0% after three-step chromatography. The
band pattern in the SDS-PAGE electrophoresis was as expected, where
a band was shown at 160 KDa in the non-reducing electrophoresis and
a clear single-chain band (80 KDa) was obtained after
reduction.
[0190] 3.2 Stability Test of the Bispecific Antibody
[0191] The stability of a bispecific antibody AP062 in histidine
salts (20 mM histidine, 8% sucrose, 0.02% Tween-80) at different pH
(pH 5.5, 6.0 and 6.5) was investigated and the effect of shaking on
the stability of the sample was explored. The bispecific antibody
AP062 was stored for two weeks under accelerated conditions at
5.degree. C. and 25.degree. C. for the evaluation of protein
stability.
[0192] AP062 was transferred to histidine buffers of pH 5.5, 6.0
and 6.5, separately. Samples were taken at different time points
for detection and analysis. The detection items included SEC-HPLC
and SDS-PAGE.
[0193] SEC-HPLC and SDS-PAGE test results of three preparations
stored for 0-2 weeks at 5.degree. C. and 25.degree. C. are shown in
Tables 5 and 6 and FIGS. 3 and 4. After the samples were placed
still for two weeks, the proportions of the polymers, main peaks,
shoulder peaks and fragments in the SEC-HPLC test results have no
significant changes and differ little at different pH. There is no
significant difference in SEC-HPLC results over two weeks in the
histidine buffers of pH 5.5, 6.0 and 6.5.
TABLE-US-00005 TABLE 5 SEC-HPLC results of acceleration experiments
at 5.degree. C. SEC-polymer % SEC-main peak % SEC-fragment % T 0 1
W 2 W T 0 1 W 2 W T 0 1 W 2 W pH 5.5 2.5 1.1 2.7 95.6 97.4 96.6 1.8
1.6 0.7 pH 6.0 1.9 0.7 2.8 96.3 97.9 96.6 1.8 1.3 0.7 pH 6.5 2.3
0.8 2.9 95.6 97.4 96.4 2.1 1.8 0.7
TABLE-US-00006 TABLE 6 SEC-HPLC results of acceleration experiments
at 25.degree. C. SEC-polymer % SEC-main peak % SEC-fragment % T 0 1
W 2 W T 0 1 W 2 W T 0 1 W 2 W pH 5.5 2.5 0.8 2.1 95.6 97.5 97.2 1.8
1.7 0.8 pH 6.0 1.9 0.6 2.4 96.3 97.7 96.9 1.8 1.7 0.7 pH 6.5 2.3
0.6 2.2 95.6 97.6 97.0 2.1 1.8 0.9
Example 4 In Vitro Pharmacodynamic Study of the Bispecific
Antibody
[0194] 4.1 Detection of the Binding Activity of AP062 to
CD20-Positive Tumor Cells by Flow Cytometry
[0195] Raji cells (purchased from the cell bank of Chinese Academy
of Sciences) were cultured and collected by centrifugation. The
collected cells were resuspended with 1% PBSB and placed in 96-well
plates, 100 .mu.l (i.e., 2.times.10.sup.5 cells) per well, after
the cell density was adjusted to (2.times.10.sup.6) cells/ml.
Diluted bispecific antibodies with a series of concentrations were
added and incubated for 1 hour at 4.degree. C. The cells were
centrifuged to discard the supernatant and then washed three times
using a PBS solution with 1% BSA (PBSB). Diluted AF488-labeled goat
anti-human IgG antibodies (Jackson Immuno Research Inc., Cat. No.
109-545-088) or mouse anti-6.times.His IgG antibodies (R&D
Systems, Cat. No. IC050P) were added to the cells, and the cells
were incubated for 1 hour at 4.degree. C. in the dark. The obtained
cells were centrifuged to discard the supernatant and then washed
twice with 1% PBSB, and cells in each well were resuspended with
100 .mu.l of 1% paraformaldehyde. The signal intensity was detected
by flow cytometry. The analysis was performed with the average
fluorescence intensity as the Y-axis and the antibody concentration
as the X-axis through software GraphPad to calculate the EC.sub.50
value for the binding of AP062 to Raji cells.
[0196] As shown in FIG. 5, AP062 bound well to CD20-positive cells,
the signal intensity was proportional to the antibody
concentration, and the EC.sub.50 value for AP062 binding to Raji
cells was calculated, which was about 69.97 nM.
[0197] 4.2 AP062 Mediating Effector Cells to Target and Kill
CD20-Positive Tumor Cells
[0198] Normally cultured Raji-luc cells (purchased from Beijing
Biocytogen Biotechnology Co., Ltd.) were added to 96-well white
plates after the cell density was adjusted to 1.times.10.sup.5
cells/ml, 40 .mu.l per well. AP062 antibodies were diluted into a
series of gradients and added to the 96-well white plates. After
the CIK cell density was adjusted to 5.times.10.sup.5 cells/ml, the
CIK cells were added to the 96-well white plates, 40 .mu.l per
well, to make the effector:target ratio (E:T) equal to 5:1, and
cultured for 24 hours at 37.degree. C. After 24 hours, the white
plates were taken out, 100 .mu.l of One-Glo (Promega, Cat. No.
E6120) solution was added to each well, and then the white plates
were placed for at least three minutes at room temperature. The
luminescence value was measured by a microplate reader. The
analysis was performed with the fluorescence intensity as the
Y-axis and the antibody concentration as the X-axis through
software GraphPad to calculate the EC.sub.50 value of AP062 killing
Raji-luc cells.
[0199] As shown in FIG. 6, the EC.sub.50 for AP062 mediating
effector cells to kill Raji-luc cells was only 42.8 ng/ml and AP062
had target specificity, while the EC.sub.50 of isotype antibody as
a negative control was 229.5 ng/ml and isotype antibody had little
killing effect on Raji-luc cells.
[0200] 4.3 Evaluation of Abilities of Bispecific Antibodies to
Activate T Cells Through Reporter Gene Cell Strains
[0201] Jurkat T cells containing NFAT RE reporter genes (BPS
Bioscience, Cat. No. 60621) can overexpress luciferase in the
presence of bispecific antibodies and CD20-positive Raji cells, and
the degree of activation of the Jurkat T cells can be quantified by
detecting the activity of the luciferase. A four-parameter curve
was fitted using the concentration of bispecific antibody as the
X-axis and the fluorescein signal as the Y-axis.
[0202] As shown in FIG. 7, AP062 can specifically activate Jurkat
NFATRE Luc cells, wherein the EC.sub.50 value was 0.2006 .mu.g/ml
and its concentration was proportional to signal intensity, while
AB7K7 as a negative control had little ability to activate T
cells.
Example 5 In Vivo Pharmacodynamic Study of the Bispecific
Antibody
[0203] 5.1 NPG Mouse Model of Transplanted Tumor Established by
Co-Inoculating Subcutaneously Human CIK Cells and Human Burkkit's
Lymphoma Raji Cells
[0204] Human Burkkit's lymphoma Raji cells with positive CD20
expression were selected to observe the in vivo anti-tumor effect
of the bispecific antibody in a NPG mouse model of transplanted
tumor that was established by co-inoculating subcutaneously human
CIK cells and human Burkkit's lymphoma Raji cells.
[0205] The peripheral blood of a normal human was taken. Human
PBMCs were separated through density gradient centrifugation
(Lymphoprep.TM., human lymphocyte isolation solution, STEMCELL),
then re-suspended in a RPMI-1640 culture medium added with 10%
inactivated FBS, and added with OKT3 at a final concentration of 1
.mu.g/mL and human IL-2 at 250 IU/mL. After three days of culture,
the mixture was centrifuged at 300 g for 5 min, and the medium was
replaced with RPMI-1640 added with 10% inactivated FBS for cell
culture and human IL-2 at 250 IU/mL. A fresh medium was then added
every 2 days, and CIK cells were collected on the tenth day of
culture. Female NPG mice at the age of seven to eight weeks
(purchased from Beijing Vitalstar Biotechnology Co., Ltd.) were
selected and Raji cells in the logarithmic growth stage were
collected. 4.times.10.sup.6 Raji cells and 8.times.10.sup.5 CIK
cells were mixed and inoculated subcutaneously on the right back of
each NPG mouse. One hour later, the mice were randomly divided into
5 groups with 6 mice in each group according to their weight. They
were intraperitoneally administered with corresponding drugs. All
administration groups were administered twice per week. The
positive control monoclonal antibody Rituxan (Merlot, Roche
Pharmaceuticals) and the bispecific antibody AP062 were separately
administered at a dose of 1 mg/kg and a dose of 0.1 mg/kg. The day
of administration was recorded as Day 0. The maximum diameter (D)
and the minimum diameter (d) of the tumor were measured twice per
week. The volume of the tumor was calculated using the following
formula: volume (mm.sup.3)=[Dxd.sup.2]/2. The tumor growth
inhibition rate was calculated for each administration group using
the following formula: TGI (%)=(1-the volume of the administration
group/the volume of the control group).times.100%.
[0206] As shown in FIG. 8, on Day 24 of administration, the average
tumor volume of the PBS control group was 1766.84.+-.155.62
mm.sup.3; the average tumor volume of the treated group
administrated with Rituxan at a dose of 1 mg/kg was
647.92.+-.277.11 mm.sup.3, and TGI was 63.33%, which was
significantly different from that of the control group (P<0.01);
the average tumor volume of the treated group administrated with
Rituxan at a dose of 0.1 mg/kg was 1893.81.+-.186.99 mm.sup.3, and
Rituxan herein exhibited no efficacy; the average tumor volume of
the treated group administrated with AP062 at a dose of 1 mg/kg was
116.18.+-.39.50 mm.sup.3, and TGI was 93.42%, which was
significantly different from that of the control group (P<0.01);
the average tumor volume of the treated group administrated with
AP062 at a dose of 0.1 mg/kg was 1226.03.+-.340.05 mm.sup.3, and
TGI was 30.61%, which was not significantly different from that of
the control group. The results show that the bispecific antibody
AP062 could inhibit the growth of tumor cells by activating human
immune cells in animals; and at the same dose, the efficacy of the
bispecific antibody was better than the efficacy of the monoclonal
antibody Rituxan, and the bispecific antibody exhibited great
anti-tumor effects.
[0207] 5.2 NPG Mouse Model of Transplanted Tumor Constructed by
Subcutaneously Co-Inoculating Human CIK Cells and Human Burkkit's
Lymphoma Daudi Cells
[0208] CD20-positive human Burkkit's lymphoma Daudi cells were
selected to study the inhibiting effect of bispecific antibodies on
tumor growth in vivo in an NPG mouse model of transplanted tumor
constructed by subcutaneously co-inoculating human CIK cells and
human Burkkit's lymphoma Daudi cells.
[0209] CIK cells were prepared in the method as described in
Example 5.1. Female NPG mice at the age of seven to eight weeks
were selected, and Daudi cells in the logarithmic growth stage were
collected. 4.times.10.sup.6 Daudi cells and 8.times.10.sup.5 CIK
cells were mixed and inoculated subcutaneously on the right back of
each NPG mouse. One hour later, the mice were randomly divided into
five groups with six mice in each group according to their weights
and intraperitoneally administered with corresponding drugs. All
treated groups were administrated twice a week. Rituxan and
bispecific antibody AP062 were both administered at doses of 1
mg/kg and 0.1 mg/kg, respectively. The day of administration was
recorded as Day 0. The maximum diameter (D) and the minimum
diameter (d) of the tumor were measured weekly. The volume
(mm.sup.3) of the tumor of each group and the tumor growth
inhibition rate (TGI) (%) of each treated group were calculated
using the formulas as shown in Example 5.1.
[0210] As shown in FIG. 9, on Day 30 of administration, the average
tumor volume of the PBS control group was 889.68.+-.192.13
mm.sup.3; the average tumor volume of the treated group
administrated with Rituxan at a dose of 1 mg/kg was 241.51.+-.44.91
mm.sup.3, and TGI was 72.85%, which was significantly different
from that of the control group (P<0.01); the average tumor
volume of the treated group administrated with Rituxan at a dose of
0.1 mg/kg was 746.11.+-.299.71 mm.sup.3, which was not
significantly different from that of the control group; the average
tumor volume of the treated group administrated with AP062 at a
dose of 1 mg/kg was 72.05.+-.11.89 mm.sup.3, and TGI was 91.9%,
which was significantly different from that of the control group
(P<0.01); the average tumor volume of the treated group
administrated with AP062 at a dose of 0.1 mg/kg was 75.36.+-.11.81
mm.sup.3, and TGI was 91.53%, which was significantly different
from that of the control group (P<0.01). The results show that
the bispecific antibody AP062 could inhibit the growth of tumor
cells by activating human immune cells in animals; and at the same
dose, the efficacy of the bispecific antibody was better than the
efficacy of the monoclonal antibody Rituxan, and AP062 exhibited
good anti-tumor effects even at a low dose.
Example 6 Evaluation of the Safety of Bispecific Antibodies
[0211] The toxicity of AP062 was evaluated to determine appropriate
dose ranges and observation indicators for subsequent toxicity
tests. Adult Female cynomolgus monkeys (purchased from Guangzhou
Xiangguan Biotechnology Co., Ltd.) at the age of 3-4 years and with
the weight of 3-4 kg were divided into two groups with one mouse in
each group, wherein the two groups were a vehicle control group and
an AP062 treated group. The groups were administrated via
intravenous drip by a peristaltic pump for 1 hour. The dose amount
and volume administered are shown in Table 7. The groups were
administrated on Day 0 (D0), Day 7 (D7), Day 21 (D21), and Day 28
(D28), respectively, for a total of four doses, and the drug dose
was gradually escalated each time. The monkeys were weighed
weekly.
TABLE-US-00007 TABLE 7 Dosing schedule for cynomolgus monkey acute
toxicity evaluation To-be-tested Group drugs name Dose volume Dose
amount G1 Vehicle D0: 5 mL/kg N/A control group D7: 5 mL/kg D21: 10
mL/kg D28: 10 mL/kg G2 AP062 D0: 5 mL/kg D0: 0.06 mg/kg D7: 5 mL/kg
D7: 0.3 mg/kg D21: 10 mL/kg D21: 1.5 mg/kg D28: 10 mL/kg D28: 3
mg/kg
[0212] During the test, animals were periodically monitored for
clinical symptoms, body weight, food consumption, body temperature,
electrocardiogram, blood pressure, clinicopathological indexes
(blood cell count, coagulation function measure, and blood
biochemistry), lymphocyte subsets, cytokines, drug plasma
concentration measure, and toxicokinetics analyses. After
administration of AP062, the physical signs of cynomolgus monkeys
exhibited no abnormal reaction, the body weight was relatively
stable, the body temperature fluctuation was similar to the body
temperature fluctuation of the vehicle control group, and no death
or impending death was observed among animals during the
administration period. As shown in FIG. 10, after administration,
the white blood cell changes of cynomolgus monkeys in the AP062
group were similar to the white blood cell changes in the control
group; the first administration of AP062 at a dose of 0.06 mg/kg
had little effect on lymphocytes; 1 hour to 6 hours after the
second administration, the number of lymphocytes in the animals of
the treated group decreased sharply and recovered to normal after
24 hours; as the number of administrations increased, the effect of
AP062 on the decrease in the number of lymphocytes was weaker and
weaker despite increasing doses. In addition, after the first
administration of AP062, the release of IL-2, IL-6 and TNF-.alpha.
factors was promoted and the release of IL-5 was slightly
stimulated, but the release of IFN-.gamma. was not stimulated; as
the number of administrations increased, the release-promoting
effect of AP062 on cytokines became less and less significant,
indicating that the body had already been adapted to the
stimulation by bispecific antibodies.
Example 7 Pharmacokinetics Evaluation of Anti-CD20.times.CD3
Bispecific Antibodies
[0213] Female cynomolgus monkeys with the weight of 3-4 kg were
divided into two groups with one in one monkey in each group. The
first group was a blank control group, and the second group was an
AP062 treated group administrated at a dose of 0.3 mg/kg. The blood
sampling time points were Minute 15, Hour 1, Hour 3, Hour 6, Hour
10, Hour 24, Hour 30, Hour 48, Hour 54, Hour 72, Hour 96, and Hour
144, respectively, a total of 13 time points. Serum was collected
from blood and frozen at -80.degree. C.
[0214] The drug concentration of AP062 in serum was determined by
ELISA. The pharmacokinetics parameters were calculated using
software PKSolver. Specific parameters are shown in Table 8. The
results show that Ti/2 of AP062 in normal cynomolgus monkeys was
about 8.5 hours.
TABLE-US-00008 TABLE 8 Pharmacokinetics parameters of bispecific
antibody AP062 in cynomolgus monkeys AUC Vz_obs Cl_obs t.sub.1/2
0-inf_obs (.mu.g/kg)/ (.mu.g/kg)/ AP062 (h) (.mu.g/mL*h) (.mu.g/mL)
(.mu.g/mL)/h Pharmacokinetics 8.45 168.63 21.68 1.78 parameter
[0215] Though the preferred examples of the present disclosure are
illustrated and described, it should be understood that those
skilled in the art may make various changes in accordance with the
teachings herein within the scope of the present disclosure.
[0216] All the publications mentioned in the present disclosure are
incorporated herein by reference as if each publication is
separately incorporated herein by reference. In addition, it should
be understood that those skilled in the art, who have read the
preceding content of the present disclosure, may make various
changes or modifications on the present disclosure, and these
equivalent forms fall within the scope of the appended claims.
Sequence CWU 1
1
5715PRTArtificial Sequenceanti-CD20 antibody 1 HCDR1 amino acid
sequence 1Ser Tyr Asn Met His1 5217PRTArtificial Sequenceanti-CD20
antibody 1 HCDR2 amino acid sequence 2Ala Ile Tyr Pro Gly Asn Gly
Asp Thr Ser Tyr Asn Gln Lys Phe Lys1 5 10 15Gly312PRTArtificial
Sequenceanti-CD20 antibody 1 HCDR3 amino acid sequence 3Ser Thr Tyr
Tyr Gly Gly Asp Trp Tyr Phe Asn Val1 5 10410PRTArtificial
Sequenceanti-CD20 antibody 1 LCDR1 amino acid sequence 4Arg Ala Ser
Ser Ser Val Ser Tyr Ile His1 5 1057PRTArtificial Sequenceanti-CD20
antibody 1 LCDR2 amino acid sequence 5Ala Thr Ser Asn Leu Ala Ser1
569PRTArtificial Sequenceanti-CD20 antibody 1 LCDR3 amino acid
sequence 6Gln Gln Trp Thr Ser Asn Pro Pro Thr1 575PRTArtificial
Sequenceanti-CD20 antibody 2 HCDR1 amino acid sequence 7Asn Tyr Tyr
Ile His1 5817PRTArtificial Sequenceanti-CD20 antibody 2 HCDR2 amino
acid sequence 8Trp Ile Tyr Pro Gly Asp Gly Asn Thr Lys Tyr Asn Glu
Lys Phe Lys1 5 10 15Gly910PRTArtificial Sequenceanti-CD20 antibody
2 HCDR3 amino acid sequence 9Asp Ser Tyr Ser Asn Tyr Tyr Phe Asp
Tyr1 5 101010PRTArtificial Sequenceanti-CD20 antibody 2 LCDR1 amino
acid sequence 10Arg Ala Ser Ser Ser Val Ser Tyr Met His1 5
10117PRTArtificial Sequenceanti-CD20 antibody 2 LCDR2 amino acid
sequence 11Ala Pro Ser Asn Leu Ala Ser1 5129PRTArtificial
Sequenceanti-CD20 antibody 2 LCDR3 amino acid sequence 12Gln Gln
Trp Ser Phe Asn Pro Pro Thr1 5135PRTArtificial Sequenceanti-CD20
antibody 3 HCDR1 amino acid sequence 13Tyr Ser Trp Ile Asn1
51417PRTArtificial Sequenceanti-CD20 antibody 3 HCDR2 amino acid
sequence 14Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys
Phe Lys1 5 10 15Gly1510PRTArtificial Sequenceanti-CD20 antibody 3
HCDR3 amino acid sequence 15Asn Val Phe Asp Gly Tyr Trp Leu Val
Tyr1 5 101616PRTArtificial Sequenceanti-CD20 antibody 3 LCDR1 amino
acid sequence 16Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Ile Thr
Tyr Leu Tyr1 5 10 15177PRTArtificial Sequenceanti-CD20 antibody 3
LCDR2 amino acid sequence 17Gln Met Ser Asn Leu Val Ser1
5189PRTArtificial Sequenceanti-CD20 antibody 3 LCDR3 amino acid
sequence 18Ala Gln Asn Leu Glu Leu Pro Tyr Thr1 5195PRTArtificial
Sequenceanti-CD20 antibody 4 HCDR1 amino acid sequence 19Asp Tyr
Ala Met His1 52017PRTArtificial Sequenceanti-CD20 antibody 4 HCDR2
amino acid sequence 20Thr Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr
Ala Asp Ser Val Lys1 5 10 15Gly2113PRTArtificial Sequenceanti-CD20
antibody 4 HCDR3 amino acid sequence 21Asp Ile Gln Tyr Gly Asn Tyr
Tyr Tyr Gly Met Asp Val1 5 102211PRTArtificial Sequenceanti-CD20
antibody 4 LCDR1 amino acid sequence 22Arg Ala Ser Gln Ser Val Ser
Ser Tyr Leu Ala1 5 10237PRTArtificial Sequenceanti-CD20 antibody 4
LCDR2 amino acid sequence 23Asp Ala Ser Asn Arg Ala Thr1
5249PRTArtificial Sequenceanti-CD20 antibody 4 LCDR3 amino acid
sequence 24Gln Gln Arg Ser Asn Trp Pro Ile Thr1 525121PRTArtificial
Sequenceanti-CD20 antibody 1 VH amino acid sequence 25Gln Val Gln
Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala1 5 10 15Ser Val
Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Asn
Met His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu Glu Trp Ile 35 40
45Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe
50 55 60Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala
Tyr65 70 75 80Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val
Tyr Tyr Cys 85 90 95Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe
Asn Val Trp Gly 100 105 110Ala Gly Thr Thr Val Thr Val Ser Ala 115
12026106PRTArtificial Sequenceanti-CD20 antibody 1 VL amino acid
sequence 26Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser
Pro Gly1 5 10 15Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val
Ser Tyr Ile 20 25 30His Trp Phe Gln Gln Lys Pro Gly Ser Ser Pro Lys
Pro Trp Ile Tyr 35 40 45Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Val
Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile
Ser Arg Val Glu Ala Glu65 70 75 80Asp Ala Ala Thr Tyr Tyr Cys Gln
Gln Trp Thr Ser Asn Pro Pro Thr 85 90 95Phe Gly Gly Gly Thr Lys Leu
Glu Ile Lys 100 10527119PRTArtificial Sequenceanti-CD20 antibody 2
VH amino acid sequence 27Glu Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Asn Tyr 20 25 30Tyr Ile His Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Trp Ile Tyr Pro Gly Asp
Gly Asn Thr Lys Tyr Asn Glu Lys Phe 50 55 60Lys Gly Arg Ala Thr Leu
Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Leu Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp
Ser Tyr Ser Asn Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110Thr
Leu Val Thr Val Ser Ser 11528106PRTArtificial Sequenceanti-CD20
antibody 2 VL amino acid sequence 28Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Ser Ser Val Ser Tyr Met 20 25 30His Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Pro Leu Ile Tyr 35 40 45Ala Pro Ser Asn Leu
Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu65 70 75 80Asp Phe
Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Phe Asn Pro Pro Thr 85 90 95Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys 100 10529119PRTArtificial
Sequenceanti-CD20 antibody 3 VH amino acid sequence 29Gln Val Gln
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Tyr Ser 20 25 30Trp
Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40
45Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe
50 55 60Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala
Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr
Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
11530112PRTArtificial Sequenceanti-CD20 antibody 3 VL amino acid
sequence 30Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr
Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu
Leu His Ser 20 25 30Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys
Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu
Val Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val
Gly Val Tyr Tyr Cys Ala Gln Asn 85 90 95Leu Glu Leu Pro Tyr Thr Phe
Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 11031122PRTArtificial
Sequenceanti-CD20 antibody 4 VH amino acid sequence 31Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asp Tyr 20 25 30Ala
Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ser Thr Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val
50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Ser Leu
Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu
Tyr Tyr Cys 85 90 95Ala Lys Asp Ile Gln Tyr Gly Asn Tyr Tyr Tyr Gly
Met Asp Val Trp 100 105 110Gly Gln Gly Thr Thr Val Thr Val Ser Ser
115 12032107PRTArtificial Sequenceanti-CD20 antibody 4 VL amino
acid sequence 32Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu
Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser
Val Ser Ser Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala
Pro Arg Leu Leu Ile 35 40 45Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile
Pro Ala Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Glu Pro65 70 75 80Glu Asp Phe Ala Val Tyr Tyr
Cys Gln Gln Arg Ser Asn Trp Pro Ile 85 90 95Thr Phe Gly Gln Gly Thr
Arg Leu Glu Ile Lys 100 1053328PRTHomo sapiens 33Ser Ser Ser Ser
Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg1 5 10 15Leu Pro Gly
Pro Ser Asp Thr Pro Ile Leu Pro Gln 20 25345PRTArtificial
Sequenceanti-CD3 antibody 3 HCDR1 amino acid sequence 34Thr Tyr Ala
Met Asn1 53519PRTArtificial Sequenceanti-CD3 antibody 3 HCDR2 amino
acid sequence 35Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr
Ala Asp Ser1 5 10 15Val Lys Asp3614PRTArtificial Sequenceanti-CD3
antibody 3 HCDR3 amino acid sequence 36His Gly Asn Phe Gly Asn Ser
Tyr Val Ser Trp Phe Ala Tyr1 5 103714PRTArtificial Sequenceanti-CD3
antibody 3 LCDR1 amino acid sequence 37Arg Ser Ser Thr Gly Ala Val
Thr Thr Ser Asn Tyr Ala Asn1 5 10387PRTArtificial Sequenceanti-CD3
antibody 3 LCDR2 amino acid sequence 38Gly Thr Asn Lys Arg Ala Pro1
5399PRTArtificial Sequenceanti-CD3 antibody 3 LCDR3 amino acid
sequence 39Ala Leu Trp Tyr Ser Asn Leu Trp Val1 5405PRTArtificial
Sequenceanti-CD3 antibody 4 HCDR1 amino acid sequence 40Lys Tyr Ala
Met Asn1 54119PRTArtificial Sequenceanti-CD3 antibody 4 HCDR2 amino
acid sequence 41Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr
Ala Asp Ser1 5 10 15Val Lys Asp4214PRTArtificial Sequenceanti-CD3
antibody 4 HCDR3 amino acid sequence 42His Gly Asn Phe Gly Asn Ser
Tyr Ile Ser Tyr Trp Ala Tyr1 5 104314PRTArtificial Sequenceanti-CD3
antibody 4 LCDR1 amino acid sequence 43Gly Ser Ser Thr Gly Ala Val
Thr Ser Gly Tyr Tyr Pro Asn1 5 10447PRTArtificial Sequenceanti-CD3
antibody 4 LCDR2 amino acid sequence 44Gly Thr Lys Phe Leu Ala Pro1
5459PRTArtificial Sequenceanti-CD3 antibody 4 LCDR3 amino acid
sequence 45Ala Leu Trp Tyr Ser Asn Arg Trp Val1 546125PRTArtificial
Sequenceanti-CD3 antibody 3 VH amino acid sequence 46Glu Val Gln
Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30Ala
Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp
50 55 60Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn
Thr65 70 75 80Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr
Ala Val Tyr 85 90 95Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr
Val Ser Trp Phe 100 105 110Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ser 115 120 12547109PRTArtificial Sequenceanti-CD3 antibody
3 VL amino acid sequence 47Glu Leu Val Val Thr Gln Glu Pro Ser Leu
Thr Val Ser Pro Gly Gly1 5 10 15Thr Val Thr Leu Thr Cys Arg Ser Ser
Thr Gly Ala Val Thr Thr Ser 20 25 30Asn Tyr Ala Asn Trp Val Gln Gln
Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45Leu Ile Gly Gly Thr Asn Lys
Arg Ala Pro Gly Thr Pro Ala Arg Phe 50 55 60Ser Gly Ser Leu Leu Gly
Gly Lys Ala Ala Leu Thr Leu Ser Gly Val65 70 75 80Gln Pro Glu Asp
Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90 95Leu Trp Val
Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 10548125PRTArtificial
Sequenceanti-CD3 antibody 4 VH amino acid sequence 48Glu Val Gln
Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys Tyr 20 25 30Ala
Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp
50 55 60Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn
Thr65 70 75 80Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr
Ala Val Tyr 85 90 95Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr
Ile Ser Tyr Trp 100 105 110Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ser 115 120 12549109PRTArtificial Sequenceanti-CD3 antibody
4 VL amino acid sequence 49Glu Leu Val Val Thr Gln Glu Pro Ser Leu
Thr Val Ser Pro Gly Gly1 5 10 15Thr Val Thr Leu Thr Cys Gly Ser Ser
Thr Gly Ala Val Thr Ser Gly 20 25 30Tyr Tyr Pro Asn Trp Val Gln Gln
Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45Leu Ile Gly Gly Thr Lys Phe
Leu Ala Pro Gly Thr Pro Ala Arg Phe 50 55 60Ser Gly Ser Leu Leu Gly
Gly Lys Ala Ala Leu Thr Leu Ser Gly Val65 70 75 80Gln Pro Glu Asp
Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90 95Arg Trp Val
Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 10550744PRTArtificial
SequenceAP062 amino acid sequence 50Gln Val Gln Leu Gln Gln Pro Gly
Ala Glu Leu Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Met Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Asn Met His Trp Val Lys
Gln Thr Pro Gly Arg Gly Leu Glu Trp Ile 35 40 45Gly Ala Ile Tyr Pro
Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe 50 55 60Lys Gly Lys Ala
Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75 80Met Gln
Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly 100 105
110Ala Gly Thr Thr Val Thr Val Ser Ala Gly Gly Gly Gly Ser Gly Gly
115 120 125Gly Gly Ser Gly Gly Gly Gly Ser Gln Ile Val Leu Ser Gln
Ser Pro 130 135 140Ala Ile Leu Ser Ala Ser Pro Gly Glu Lys Val Thr
Met Thr Cys Arg145 150 155 160Ala Ser Ser Ser Val Ser Tyr Ile His
Trp Phe Gln Gln Lys Pro Gly 165 170 175Ser Ser Pro Lys Pro Trp Ile
Tyr Ala Thr Ser Asn Leu Ala Ser Gly 180 185 190Val Pro Val Arg Phe
Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu 195 200 205Thr Ile Ser
Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln 210
215 220Gln Trp Thr Ser Asn Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu
Glu225 230 235 240Ile Lys Gly Gly Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly Gly 245 250 255Gly Gly Ser Ser Ser Ser Ser Lys Ala Pro
Pro Pro Ser Glu Val Gln 260 265 270Leu Leu Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly Ser Leu Lys 275 280 285Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Asn Thr Tyr Ala Met Asn 290 295 300Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile305 310 315 320Arg
Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys 325 330
335Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Ala Tyr Leu
340 345 350Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr
Cys Val 355 360 365Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp
Phe Ala Tyr Trp 370 375 380Gly Gln Gly Thr Leu Val Thr Val Ser Ser
Gly Gly Gly Gly Ser Gly385 390 395 400Gly Gly Gly Ser Gly Gly Gly
Gly Ser Glu Leu Val Val Thr Gln Glu 405 410 415Pro Ser Leu Thr Val
Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Arg 420 425 430Ser Ser Thr
Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp Val Gln 435 440 445Gln
Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr Asn Lys 450 455
460Arg Ala Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly
Gly465 470 475 480Lys Ala Ala Leu Thr Leu Ser Gly Val Gln Pro Glu
Asp Glu Ala Glu 485 490 495Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu
Trp Val Phe Gly Gly Gly 500 505 510Thr Lys Leu Thr Val Leu Asp Lys
Thr His Thr Cys Pro Pro Cys Pro 515 520 525Ala Pro Glu Ala Ala Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 530 535 540Pro Lys Asp Gln
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val545 550 555 560Val
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 565 570
575Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
580 585 590Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
Leu His 595 600 605Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys 610 615 620Ala Leu Pro Ala Ser Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln625 630 635 640Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Asp Glu Leu 645 650 655Thr Lys Asn Gln Val
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 660 665 670Ser Asp Ile
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 675 680 685Tyr
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 690 695
700Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val705 710 715 720Phe Ser Cys Ser Val Leu His Glu Ala Leu His Asn
His Tyr Thr Gln 725 730 735Lys Ser Leu Ser Leu Ser Pro Gly
740512232DNAArtificial SequenceAP062 nucleotide sequence
51caagtgcagc tgcagcagcc tggagctgag ctggtgaaac ccggcgcctc cgtcaagatg
60tcctgtaagg cctccggata caccttcacc agctacaaca tgcactgggt gaaacagacc
120cctggaaggg gcctggagtg gatcggcgcc atttaccctg gcaacggcga
tacctcctat 180aatcagaagt ttaagggcaa ggccaccctg accgctgata
agagcagcag cacagcctac 240atgcagctgt ccagcctgac ctccgaggac
agcgccgtgt actactgtgc tcggagcacc 300tactacggcg gcgactggta
ctttaacgtg tggggagctg gcaccacagt gaccgtgagc 360gccggaggag
gaggatctgg aggaggaggc tccggaggag gaggaagcca gatcgtgctg
420agccagagcc ccgccatcct gagcgccagc cccggcgaga aggtgaccat
gacctgccgc 480gccagcagca gcgtgagcta catccactgg ttccagcaga
agcccggcag cagccccaag 540ccctggatct acgccaccag caacctggcc
agcggcgtgc ccgtgcgctt cagcggcagc 600ggcagcggca ccagctacag
cctgaccatc agccgcgtgg aggccgagga cgccgccacc 660tactactgcc
agcagtggac cagcaacccc cccaccttcg gcggcggcac caagctggag
720atcaagggtg gcggcggtgg aggatccggc ggtggaggta gcggcggagg
cggtagctcc 780agctctagta aagctccccc tccttccgag gtgcagctgc
tggagtccgg aggaggactg 840gtgcagccag gaggctccct gaagctgagc
tgtgctgcct ctggctttac cttcaacaca 900tatgccatga attgggtgcg
gcaggctcca ggcaagggac tggagtgggt ggctaggatc 960aggtctaagt
acaacaatta tgccacctac tatgctgatt ccgtgaagga caggttcacc
1020atctcccgcg acgatagcaa gaacacagcc tacctgcaga tgaacaatct
gaagaccgag 1080gataccgccg tgtactactg cgtgagacat ggcaactttg
gcaatagcta cgtgtcctgg 1140ttcgcttact ggggacaggg caccctggtc
acagtgagct ctggaggagg aggatctgga 1200ggaggaggct ccggaggagg
aggaagcgag ctggtggtga cccaggagcc atctctgaca 1260gtgtcccccg
gcggcacagt gaccctgaca tgtagatcca gcaccggcgc cgtgaccaca
1320tccaactacg ctaattgggt gcagcagaag ccaggacagg ctccaagggg
actgatcgga 1380ggaaccaaca agagggctcc tggaacacca gctcggttta
gcggatctct gctgggaggc 1440aaggctgccc tgaccctgtc cggagtgcag
ccagaggatg aggccgagta ttattgcgct 1500ctgtggtata gcaatctgtg
ggtgttcgga ggaggaacca agctgacagt gctggacaag 1560acccatacat
gcccaccatg ccctgcccct gaagccgccg gaggaccttc cgtgttcctg
1620ttccctccca agccaaaaga tcagctgatg atctctagaa cccccgaagt
cacctgcgtg 1680gtcgtcgacg tgtcccatga ggaccctgaa gtcaagttca
actggtacgt ggacggtgtc 1740gaagtccaca acgccaagac caagcctagg
gaggagcagt atgccagcac ataccgggtg 1800gtgtctgtgc tgaccgtgct
gcatcaggat tggctgaatg gcaaggaata taaatgtaag 1860gtgagcaata
aggctctgcc ggctagcatt gaaaaaacca tttccaaggc taagggccag
1920cccagggagc ctcaggtcta caccctgcct ccatctagag atgaactgac
caaaaaccag 1980gtgagcctga cttgcctggt caaaggcttc taccccagcg
acattgccgt ggagtgggag 2040tctaatggcc agcctgaaaa taactacaaa
actacccctc ctgtgctgga ctctgatggc 2100tccttctttc tgtactctaa
actgaccgtg gacaagtctc gctggcagca gggtaacgtg 2160ttttcttgct
ccgtgctgca cgaggctctg cataaccatt acacccagaa gagcctgtct
2220ctgtccccag ga 22325227PRTArtificial SequenceG2(GGGGS)3CTP1
linker peptide 52Gly Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly1 5 10 15Ser Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser
20 255325PRTArtificial Sequence(GGGGS)3CTP1 linker peptide 53Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser1 5 10
15Ser Ser Ser Lys Ala Pro Pro Pro Ser 20 255422PRTArtificial
SequenceGS(GGGGS)2CTP1 linker peptide 54Gly Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Ser Ser Ser Ser1 5 10 15Lys Ala Pro Pro Pro Ser
205533PRTArtificial Sequence(GGGGS)1CTP4 linker peptide 55Gly Gly
Gly Gly Ser Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu1 5 10 15Pro
Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro 20 25
30Gln56227PRTArtificial SequenceIgG1 Fc(L234A,L235A) mutant
constant region amino acid sequence 56Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Ala Ala Gly1 5 10 15Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr65 70 75 80Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90
95Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
100 105 110Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val 115 120 125Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
Asn Gln Val Ser 130 135 140Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu145 150 155 160Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215
220Pro Gly Lys22557226PRTArtificial SequenceIgG1(L234A,L235A,
T250Q,N297A,P331S,M428L, K447-) mutant constant region amino acid
sequence 57Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala
Ala Gly1 5 10 15Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Gln Leu Met 20 25 30Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His 35 40 45Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val 50 55 60His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Ala Ser Thr Tyr65 70 75 80Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly 85 90 95Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala Ser Ile 100 105 110Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125Tyr Thr Leu
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu145 150
155 160Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro 165 170 175Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val 180 185 190Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Leu 195 200 205His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser 210 215 220Pro Gly225
* * * * *