CN113461821B - anti-CD 3 humanized antibodies - Google Patents

anti-CD 3 humanized antibodies Download PDF

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CN113461821B
CN113461821B CN202111028884.8A CN202111028884A CN113461821B CN 113461821 B CN113461821 B CN 113461821B CN 202111028884 A CN202111028884 A CN 202111028884A CN 113461821 B CN113461821 B CN 113461821B
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CN113461821A (en
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宋作伟
张清仪
王英明
李德彬
王米
秦照峰
房继旋
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Suzhou Inshore Protein Technology Co ltd
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Suzhou Inshore Protein Technology Co ltd
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Abstract

The invention provides a novel anti-CD 3 humanized antibody. The anti-CD 3 humanized antibody can specifically bind to human and monkey CD3 proteins and has good biological activity.

Description

anti-CD 3 humanized antibodies
Technical Field
The invention relates to the field of biomedicine, in particular to an anti-CD 3 humanized antibody.
Background
Activation of T cells is critical for stimulating an immune response. T cells exhibit immune specificity and direct most cellular immune responses. Although T cells do not secrete antibodies, they are necessary for B lymphocytes to secrete antibodies.
CD3 is a homodimeric or heterodimeric antigen expressed on T cells that is associated with the T cell receptor complex (TCR) and is essential for T cell activation. Functional CD3 is a dimer formed from two of four different chains (epsilon, zeta, delta, and gamma). The CD3 dimer arrangement includes γ/ε, δ/ε, and ζ/ζ. Antibodies to CD3 have been shown to aggregate CD3 on T cells, causing T cell activation in a manner similar to the involvement of TCR by peptide-loaded MHC molecules. Thus, anti-CD 3 antibodies have therapeutic purposes involving T cell activation. In addition, bispecific antibodies capable of binding to CD3 and a target antigen have been proposed as therapeutic uses involving targeting of T cell immune responses to tissues and cells expressing target antigen cells.
Existing bispecific antibodies that bind CD3 currently in clinical trials for cancer therapy are limited by their short half-life and/or variable efficacy. Although many attempts have been made to obtain anti-CD 3 antibodies with particularly advantageous properties, these attempts have been met with limited success to date.
Therefore, there remains an urgent need in the art to develop a novel anti-CD 3 humanized antibody.
Disclosure of Invention
The invention aims to provide a novel anti-CD 3 humanized antibody.
In a first aspect of the invention, there is provided an anti-CD 3 humanized antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region have 6 complementarity determining region CDRs selected from the group consisting of:
(A1) three complementarity determining regions, VH-CDRs, of the heavy chain variable region and three complementarity determining regions, VL-CDRs, of the light chain variable region:
huVH-CDR1： GFTFNKYA， SEQ ID NO. 1
huVH-CDR2： IRSKYNNYAT， SEQ ID NO. 2
huVH4-CDR3： HGNFGNSYISYWEY， SEQ ID NO. 3
huVL-CDR1： TGAVTSGNY， SEQ ID NO. 4
huVL-CDR2： GTK， SEQ ID NO. 5
huVL4-CDR3： VLWNSNRW， SEQ ID NO. 6；
(A2) three complementarity determining regions, VH-CDRs, of the heavy chain variable region and three complementarity determining regions, VL-CDRs, of the light chain variable region:
huVH-CDR1： GFTFNKYA， SEQ ID NO.1
huVH-CDR2： IRSKYNNYAT， SEQ ID NO.2
huVH3-CDR3： HGNFGNSYISYWRY， SEQ ID NO.10
huVL-CDR1： TGAVTSGNY， SEQ ID NO.4
huVL-CDR2： GTK， SEQ ID NO.5
huVL3-CDR3： VLWYSGRW， SEQ ID NO.11；
(A3) three complementarity determining regions, VH-CDRs, of the heavy chain variable region and three complementarity determining regions, VL-CDRs, of the light chain variable region:
huVH-CDR1： GFTFNKYA， SEQ ID NO.1
huVH-CDR2： IRSKYNNYAT， SEQ ID NO.2
huVH2-CDR3： HGNFGNSYISYWQY， SEQ ID NO.15
huVL-CDR1： TGAVTSGNY， SEQ ID NO.4
huVL-CDR2： GTK， SEQ ID NO.5
huVL2-CDR 3: VLWRSNRW, SEQ ID No. 16; or
(A4) Three complementarity determining regions, VH-CDRs, of the heavy chain variable region and three complementarity determining regions, VL-CDRs, of the light chain variable region:
huVH-CDR1： GFTFNKYA， SEQ ID NO.1
huVH-CDR2： IRSKYNNYAT， SEQ ID NO.2
huVH1-CDR3： HGNFGNTYISYWAY， SEQ ID NO.20
huVL-CDR1： TGAVTSGNY， SEQ ID NO.4
huVL-CDR2： GTK， SEQ ID NO.5
huVL1-CDR3： VLWYSKRW， SEQ ID NO.21。
in another preferred embodiment, any one of the above amino acid sequences further comprises a derivative sequence optionally having at least one amino acid added, deleted, modified and/or substituted, and capable of retaining CD3 binding affinity.
In another preferred embodiment, the antibody comprises a heavy chain and a light chain, wherein the heavy chain of the antibody comprises the three heavy chain CDRs and a heavy chain framework region for linking the heavy chain CDRs, and the light chain of the antibody comprises the three light chain CDRs and a light chain framework region for linking the light chain CDRs.
In another preferred embodiment, the heavy chain variable region has the sequence shown in SEQ ID number 7. In another preferred embodiment, the heavy chain of said antibody further comprises a heavy chain constant region.
In another preferred embodiment, the heavy chain constant region is of human, murine or rabbit origin, preferably of human origin.
In another preferred embodiment, the light chain variable region has the sequence shown in SEQ ID number 8. In another preferred embodiment, the light chain of the antibody further comprises a light chain constant region.
In another preferred embodiment, the light chain constant region is of human, murine or rabbit origin, preferably of human origin.
In another preferred embodiment, the antibody binds to a human CD3 epsilon polypeptide with a Kd of 250nM or less, preferably 100nM or less; preferably; less than or equal to 15 nM; preferably, less than or equal to 10 nM; more preferably less than or equal to 5 nM.
In another preferred embodiment, the antibody specifically binds to CD 3.
In another preferred embodiment, the CD3 is from human or cynomolgus monkey.
In another preferred embodiment, said CD3 is CD3 epsilon, CD3 zeta, CD3 delta, or CD3 gamma; preferably, the CD3 is CD3 epsilon or CD3 gamma.
In another preferred embodiment, the antibody specifically binds to human CD3 epsilon or cynomolgus monkey CD3 epsilon; or the antibody specifically binds to human CD3 gamma or cynomolgus monkey CD3 gamma.
In another preferred embodiment, the antibody is a double-chain antibody, or a single-chain antibody (scFv).
In another preferred embodiment, the antibody is a single chain antibody, wherein the single chain antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region have 6 complementarity determining regions CDRs selected from the group consisting of seq id nos:
(A1) three complementarity determining regions, VH-CDRs, of the heavy chain variable region and three complementarity determining regions, VL-CDRs, of the light chain variable region:
huVH-CDR1： GFTFNKYA， SEQ ID NO. 1
huVH-CDR2： IRSKYNNYAT， SEQ ID NO. 2
huVH4-CDR3： HGNFGNSYISYWEY， SEQ ID NO. 3
huVL-CDR1： TGAVTSGNY， SEQ ID NO. 4
huVL-CDR2： GTK， SEQ ID NO. 5
huVL4-CDR3： VLWNSNRW， SEQ ID NO. 6；
(A2) three complementarity determining regions, VH-CDRs, of the heavy chain variable region and three complementarity determining regions, VL-CDRs, of the light chain variable region:
huVH-CDR1： GFTFNKYA， SEQ ID NO.1
huVH-CDR2： IRSKYNNYAT， SEQ ID NO.2
huVH3-CDR3： HGNFGNSYISYWRY， SEQ ID NO.10
huVL-CDR1： TGAVTSGNY， SEQ ID NO.4
huVL-CDR2： GTK， SEQ ID NO.5
huVL3-CDR3： VLWYSGRW， SEQ ID NO.11；
(A3) three complementarity determining regions, VH-CDRs, of the heavy chain variable region and three complementarity determining regions, VL-CDRs, of the light chain variable region:
huVH-CDR1： GFTFNKYA， SEQ ID NO.1
huVH-CDR2： IRSKYNNYAT， SEQ ID NO.2
huVH2-CDR3： HGNFGNSYISYWQY， SEQ ID NO.15
huVL-CDR1： TGAVTSGNY， SEQ ID NO.4
huVL-CDR2： GTK， SEQ ID NO.5
huVL2-CDR 3: VLWRSNRW, SEQ ID No. 16; or
(A4) Three complementarity determining regions, VH-CDRs, of the heavy chain variable region and three complementarity determining regions, VL-CDRs, of the light chain variable region:
huVH-CDR1： GFTFNKYA， SEQ ID NO.1
huVH-CDR2： IRSKYNNYAT， SEQ ID NO.2
huVH1-CDR3： HGNFGNTYISYWAY， SEQ ID NO.20
huVL-CDR1： TGAVTSGNY， SEQ ID NO.4
huVL-CDR2： GTK， SEQ ID NO.5
huVL1-CDR3： VLWYSKRW， SEQ ID NO.21。
in another preferred embodiment, the single chain antibody comprises a light chain variable region, a linker and a heavy chain variable region in this order, or comprises a heavy chain variable region, a linker and a light chain variable region in this order.
In another preferred embodiment, the linker is (G4S) n, wherein n is an integer of 1-5, and the preferred linker is (G4S) 3.
In another preferred embodiment, the heavy chain variable region of the antibody comprises the amino acid sequence shown as SEQ ID number 7; and the light chain variable region of the antibody contains an amino acid sequence shown as SEQ ID number 8; or
The heavy chain variable region of the antibody contains an amino acid sequence shown by SEQ ID number 12; and the light chain variable region of the antibody contains an amino acid sequence shown by SEQ ID number 13; or
The heavy chain variable region of the antibody contains an amino acid sequence shown by SEQ ID number 17; and the light chain variable region of the antibody contains an amino acid sequence shown as SEQ ID number 18; or
The heavy chain variable region of the antibody contains an amino acid sequence shown by SEQ ID number 22; and the variable region of the light chain of the antibody contains an amino acid sequence shown as SEQ ID number 23.
In another preferred embodiment, the sequence of said single chain antibody is selected from the group consisting of:
SEQ ID number 9, SEQ ID number 22, SEQ ID number 19 or SEQ ID number 24.
In another preferred embodiment, the antibody is a monoclonal antibody.
In another preferred embodiment, the antibody comprises a monospecific, bispecific, or trispecific antibody.
In another preferred embodiment, the bispecific antibody comprises:
(1) an anti-CD 3 antibody as described in the first aspect of the invention;
(2) antibodies that bind other targets.
In another preferred embodiment, the other target points are selected from the group consisting of: BCMA, EGFR, CD38, CD123, CD19, CD20, CD22, B7-H3, GPC3, HER2, PMSA, CD28, 4-1BB, OX40, CD40, CD27, CD47, CTLA4, PD1, PDL 1.
In another preferred embodiment, the bispecific antibody comprises:
a first antigen binding domain (D1); and
a second antigen-binding domain (D2);
wherein, D1 specifically binds to the target molecule CD3 protein;
d2 specifically binds to the target molecule CD19 protein;
wherein the D1 is an antibody or antigen-binding fragment thereof that specifically binds to CD3 protein;
the D2 is an antibody or antigen-binding fragment thereof that specifically binds to CD19 protein;
wherein D1 comprises a heavy chain variable region and a light chain variable region having 6 complementarity determining regions CDRs selected from the group consisting of:
(A1) three complementarity determining regions, VH-CDRs, of the heavy chain variable region and three complementarity determining regions, VL-CDRs, of the light chain variable region:
huVH-CDR1： GFTFNKYA， SEQ ID NO. 1
huVH-CDR2： IRSKYNNYAT， SEQ ID NO. 2
huVH4-CDR3： HGNFGNSYISYWEY， SEQ ID NO. 3
huVL-CDR1： TGAVTSGNY， SEQ ID NO. 4
huVL-CDR2： GTK， SEQ ID NO. 5
huVL4-CDR3： VLWNSNRW， SEQ ID NO. 6；
(A2) three complementarity determining regions, VH-CDRs, of the heavy chain variable region and three complementarity determining regions, VL-CDRs, of the light chain variable region:
huVH-CDR1： GFTFNKYA， SEQ ID NO.1
huVH-CDR2： IRSKYNNYAT， SEQ ID NO.2
huVH3-CDR3： HGNFGNSYISYWRY， SEQ ID NO.10
huVL-CDR1： TGAVTSGNY， SEQ ID NO.4
huVL-CDR2： GTK， SEQ ID NO.5
huVL3-CDR3： VLWYSGRW， SEQ ID NO.11；
(A3) three complementarity determining regions, VH-CDRs, of the heavy chain variable region and three complementarity determining regions, VL-CDRs, of the light chain variable region:
huVH-CDR1： GFTFNKYA， SEQ ID NO.1
huVH-CDR2： IRSKYNNYAT， SEQ ID NO.2
huVH2-CDR3： HGNFGNSYISYWQY， SEQ ID NO.15
huVL-CDR1： TGAVTSGNY， SEQ ID NO.4
huVL-CDR2： GTK， SEQ ID NO.5
huVL2-CDR 3: VLWRSNRW, SEQ ID No. 16; or
(A4) Three complementarity determining regions, VH-CDRs, of the heavy chain variable region and three complementarity determining regions, VL-CDRs, of the light chain variable region:
huVH-CDR1： GFTFNKYA， SEQ ID NO.1
huVH-CDR2： IRSKYNNYAT， SEQ ID NO.2
huVH1-CDR3： HGNFGNTYISYWAY， SEQ ID NO.20
huVL-CDR1： TGAVTSGNY， SEQ ID NO.4
huVL-CDR2： GTK， SEQ ID NO.5
huVL1-CDR3： VLWYSKRW， SEQ ID NO.21；
wherein the structure of the antigen binding fragment is selected from the group consisting of: (i) a Fab fragment; (ii) f (ab')2A fragment; (iii) (ii) a fragment of Fd; (iv) fv fragment; (v) a scFv molecule; or (vi) a dAb fragment.
In another preferred embodiment, said D1 and/or said D2 is a single chain antibody (scFv).
In another preferred embodiment, the D1 and the D2 are connected by a linker, preferably the linker is a flexible linker.
In another preferred embodiment, the linker is (GGGGS) n, wherein n is an integer from 1 to 5, preferably the linker peptide is GGGGS.
In another preferred embodiment, the D1 is an anti-CD 3 single chain antibody and D2 is an anti-CD 19 single chain antibody.
In another preferred embodiment, the anti-CD 3 single chain antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID number 7; and the light chain variable region comprises an amino acid sequence shown as SEQ ID number 8; or
The heavy chain variable region contains an amino acid sequence shown by SEQ ID number 12; and the light chain variable region comprises an amino acid sequence shown as SEQ ID number 13; or
The heavy chain variable region contains an amino acid sequence shown by SEQ ID number 17; and the light chain variable region comprises an amino acid sequence shown as SEQ ID number 18; or
The heavy chain variable region contains an amino acid sequence shown by SEQ ID number 22; and the light chain variable region comprises an amino acid sequence shown as SEQ ID number 23.
In a second aspect of the present invention, there is provided a recombinant protein having:
(i) an antibody according to the first aspect of the invention; and
(ii) optionally a tag sequence to facilitate expression and/or purification.
In another preferred embodiment, the tag sequence comprises a 6His tag.
In another preferred embodiment, the recombinant protein (or polypeptide) comprises a fusion protein.
In another preferred embodiment, the recombinant protein is a monomer, dimer, or multimer.
In another preferred embodiment, the recombinant protein is a monospecific, bispecific or trispecific recombinant protein.
In another preferred embodiment, said recombinant protein further comprises an additional fusion element (or fusion polypeptide fragment) fused to said element (i).
In another preferred embodiment, the recombinant protein comprises:
(i) an antibody selected from the group consisting of,
the heavy chain variable region of the antibody contains an amino acid sequence shown by SEQ ID number 7; and the light chain variable region of the antibody contains an amino acid sequence shown as SEQ ID number 8; or
The heavy chain variable region of the antibody contains an amino acid sequence shown by SEQ ID number 12; and the light chain variable region of the antibody contains an amino acid sequence shown by SEQ ID number 13; or
The heavy chain variable region of the antibody contains an amino acid sequence shown by SEQ ID number 17; and the light chain variable region of the antibody contains an amino acid sequence shown as SEQ ID number 18; or
The heavy chain variable region of the antibody contains an amino acid sequence shown by SEQ ID number 22; and the light chain variable region of the antibody contains an amino acid sequence shown as SEQ ID number 23;
and
(ii) optionally a tag sequence to facilitate expression and/or purification.
In a third aspect of the invention, there is provided a CAR construct, the scFv segment of the antigen binding region of the CAR construct being a binding region that specifically binds to CD3 and having a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region have 6 complementarity determining region CDRs selected from the group consisting of:
(A1) three complementarity determining regions, VH-CDRs, of the heavy chain variable region and three complementarity determining regions, VL-CDRs, of the light chain variable region:
huVH-CDR1： GFTFNKYA， SEQ ID NO. 1
huVH-CDR2： IRSKYNNYAT， SEQ ID NO. 2
huVH4-CDR3： HGNFGNSYISYWEY， SEQ ID NO. 3
huVL-CDR1： TGAVTSGNY， SEQ ID NO. 4
huVL-CDR2： GTK， SEQ ID NO. 5
huVL4-CDR3： VLWNSNRW， SEQ ID NO. 6；
(A2) three complementarity determining regions, VH-CDRs, of the heavy chain variable region and three complementarity determining regions, VL-CDRs, of the light chain variable region:
huVH-CDR1： GFTFNKYA， SEQ ID NO.1
huVH-CDR2： IRSKYNNYAT， SEQ ID NO.2
huVH3-CDR3： HGNFGNSYISYWRY， SEQ ID NO.10
huVL-CDR1： TGAVTSGNY， SEQ ID NO.4
huVL-CDR2： GTK， SEQ ID NO.5
huVL3-CDR3： VLWYSGRW， SEQ ID NO.11；
(A3) three complementarity determining regions, VH-CDRs, of the heavy chain variable region and three complementarity determining regions, VL-CDRs, of the light chain variable region:
huVH-CDR1： GFTFNKYA， SEQ ID NO.1
huVH-CDR2： IRSKYNNYAT， SEQ ID NO.2
huVH2-CDR3： HGNFGNSYISYWQY， SEQ ID NO.15
huVL-CDR1： TGAVTSGNY， SEQ ID NO.4
huVL-CDR2： GTK， SEQ ID NO.5
huVL2-CDR 3: VLWRSNRW, SEQ ID No. 16; or
(A4) Three complementarity determining regions, VH-CDRs, of the heavy chain variable region and three complementarity determining regions, VL-CDRs, of the light chain variable region:
huVH-CDR1： GFTFNKYA， SEQ ID NO.1
huVH-CDR2： IRSKYNNYAT， SEQ ID NO.2
huVH1-CDR3： HGNFGNTYISYWAY， SEQ ID NO.20
huVL-CDR1： TGAVTSGNY， SEQ ID NO.4
huVL-CDR2： GTK， SEQ ID NO.5
huVL1-CDR3： VLWYSKRW， SEQ ID NO.21。
in a fourth aspect of the invention there is provided a recombinant immune cell expressing an exogenous CAR construct according to the third aspect of the invention.
In another preferred embodiment, the immune cell is selected from the group consisting of: NK cells, T cells.
In another preferred embodiment, the immune cell is from a human or non-human mammal (e.g., a mouse).
In a fifth aspect of the present invention, there is provided an antibody drug conjugate comprising:
(a) an antibody moiety selected from the group consisting of: an antibody according to the first aspect of the invention, or a recombinant protein according to the second aspect of the invention, or a combination thereof; and
(b) a coupling moiety coupled to the antibody moiety, the coupling moiety selected from the group consisting of: a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.
In another preferred embodiment, said antibody moiety is coupled to said coupling moiety by a chemical bond or a linker.
In a sixth aspect of the invention there is provided the use of an active ingredient selected from the group consisting of: an antibody according to the first aspect of the invention, or a recombinant protein according to the second aspect of the invention, a CAR construct according to the third aspect of the invention, an immune cell according to the fourth aspect of the invention, an antibody drug conjugate according to the fifth aspect of the invention, or a combination thereof, the active ingredient being for:
(a) preparing a detection reagent or a kit;
(b) preparing a medicament or preparation for preventing and/or treating CD3 related diseases; and/or
(c) Preparing a medicament or a preparation for preventing and/or treating CD3 related cancers or tumors.
In another preferred embodiment, the cancer or tumor is selected from the group consisting of: lung cancer, melanoma, colon cancer, pancreatic cancer, bladder cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, liver cancer, lymphoma, myeloma, leukemia.
In a seventh aspect of the present invention, there is provided a pharmaceutical composition comprising:
(i) an active ingredient selected from the group consisting of: an antibody according to the first aspect of the invention, or a recombinant protein according to the second aspect of the invention, a CAR construct according to the third aspect of the invention, an immune cell according to the fourth aspect of the invention, an antibody drug conjugate according to the fifth aspect of the invention, or a combination thereof; and
(ii) a pharmaceutically acceptable carrier.
In another preferred embodiment, the pharmaceutical composition is a liquid preparation.
In another preferred embodiment, the pharmaceutical composition is an injection.
In another preferred embodiment, the pharmaceutical composition is used for preparing a medicament for treating tumors selected from the group consisting of: lung cancer, melanoma, colon cancer, pancreatic cancer, bladder cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, liver cancer, lymphoma, myeloma, leukemia.
In an eighth aspect of the invention, there is provided a polynucleotide encoding a polypeptide selected from the group consisting of:
(1) an antibody according to the first aspect of the invention; or
(2) A recombinant protein according to the second aspect of the invention;
(3) a CAR construct according to the third aspect of the invention.
In a ninth aspect of the invention there is provided a vector comprising a polynucleotide according to the eighth aspect of the invention.
In another preferred embodiment, the carrier comprises: bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.
In a tenth aspect of the invention there is provided a genetically engineered host cell comprising a vector or genome according to the ninth aspect of the invention into which has been integrated a polynucleotide according to the eighth aspect of the invention.
In another preferred embodiment, the host cell is a mammalian cell, or a prokaryotic cell.
In another preferred embodiment, the mammalian cell is a Chinese Hamster Ovary (CHO) cell.
In another preferred embodiment, the prokaryotic cell is Escherichia coli.
In an eleventh aspect of the invention, there is provided an in vitro non-diagnostic method for detecting CD3 protein in a sample, the method comprising the steps of:
(1) contacting said sample in vitro with an antibody according to the first aspect of the invention;
(2) detecting the formation of an antigen-antibody complex, wherein the formation of the complex is indicative of the presence of CD3 protein in the sample.
In a twelfth aspect of the present invention, there is provided a detection panel comprising: a substrate (support plate) and a test strip comprising an antibody according to the first aspect of the invention or an antibody drug conjugate according to the fifth aspect of the invention.
In a thirteenth aspect of the present invention, there is provided a kit comprising:
(1) a first container comprising an antibody according to the first aspect of the invention; and/or
(2) A second container comprising a secondary antibody to the antibody of the first aspect of the invention;
alternatively, the kit comprises a detection plate according to the twelfth aspect of the invention.
In a fourteenth aspect of the present invention, there is provided a method for producing a recombinant polypeptide, the method comprising:
(a) culturing a host cell according to the tenth aspect of the invention under conditions suitable for expression;
(b) isolating a recombinant polypeptide from the culture, said recombinant polypeptide being an antibody according to the first aspect of the invention or a recombinant protein according to the second aspect of the invention.
In a fifteenth aspect of the invention, there is provided a method of treating a disorder associated with a CD3 molecule, comprising the steps of: administering to a subject in need of inhibition or in need of treatment an antibody according to the first aspect of the invention or a recombinant protein according to the second aspect of the invention or a pharmaceutical composition according to the seventh aspect of the invention.
In another preferred embodiment, the disease associated with the CD3 molecule comprises tumor or organ transplant immune rejection.
In another preferred embodiment, the subject is a mammal (including a human).
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
Figure 1 shows the affinity of CD3 antibody to recombinant human CD3 as determined by ELISA.
Figure 2 shows the affinity of CD3 antibody to recombinant monkey CD3 as determined by ELISA.
FIG. 3 shows the affinity of fortebio detection CD3 full-length antibody and human CD3E protein, wherein A, B, C, D is the affinity of CD3 full-length antibody CQ53, CQ52, CQ51, CQ50 and human CD3E protein respectively.
FIG. 4 shows the affinity of ELISA detection CD3 full-length antibody and monkey CD3E protein, wherein A, B, C, D is the affinity of CD3 full-length antibody CQ53, CQ52, CQ51, CQ50 and monkey CD3E protein respectively.
Fig. 5 shows the affinity of the full-length CD3 antibody to Jurkat cells, wherein A, B, C, D is the affinity of the full-length CD3 antibodies CQ53, CQ52, CQ51, CQ50 to Jurkat cells, respectively.
Fig. 6 shows competition ELISA detection profiles of the full-length CD3 antibody and OKT3, wherein A, B, C, D is a competition ELISA detection profile of the full-length CD3 antibodies CQ53, CQ52, CQ51, CQ50, and OKT3, respectively.
Detailed Description
The present inventors have conducted extensive and intensive studies and have unexpectedly obtained a CD3 antibody having high biological activity. Experiments have shown that the CD3 antibodies of the invention are capable of specifically binding to human and monkey CD 3. The CD3 antibody of the invention can be further combined with an antibody or a binding fragment thereof which binds to a target antigen CD19 to construct a bispecific antibody, and has higher tumor killing activity. The present invention has been completed based on this finding.
Term(s) for
As used herein, the terms "administration" and "treatment" refer to the application of an exogenous drug, therapeutic agent, diagnostic agent, or composition to an animal, human, subject, cell, tissue, organ, or biological fluid. "administration" and "treatment" may refer to therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. The treatment of the cells comprises contacting the reagent with the cells, and contacting the reagent with a fluid, and contacting the fluid with the cells. "administering" and "treating" also mean treating in vitro and ex vivo by a reagent, a diagnostic, a binding composition, or by another cell. "treatment" when applied to a human, animal or study subject refers to therapeutic treatment, prophylactic or preventative measures, research, and diagnosis; including contact of the CD3 antibody with a human or animal, subject, cell, tissue, physiological compartment, or physiological fluid.
As used herein, the term "treatment" refers to the administration of a therapeutic agent, either internally or externally, to a patient having one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect, comprising any one of the CD3 antibodies of the invention and compositions thereof. Typically, the therapeutic agent is administered to the patient in an amount effective to alleviate one or more symptoms of the disease (therapeutically effective amount).
As used herein, the term "optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable regions of a particular sequence may, but need not, be 1, 2 or 3.
Antibodies
As used herein, the term "antibody" refers to an immunoglobulin, a tetrapeptide chain structure made up of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The constant regions of immunoglobulin heavy chains differ in their amino acid composition and arrangement, and thus, their antigenicity. Accordingly, immunoglobulins can be classified into five classes, or different classes called immunoglobulins, i.e., IgM, IgD, IgG, IgA, and IgE, and the heavy chain constant regions corresponding to the different classes of immunoglobulins are referred to as α, δ, ε, γ, and μ, respectively. IgG represents the most important class of immunoglobulins, which can be divided into 4 subclasses due to differences in chemical structure and biological function: IgG1, IgG2, IgG3, and IgG 4. Light chains are classified as kappa or lambda chains by differences in the constant regions. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.
The sequences of the antibody heavy and light chains, near the N-terminus, are widely varied by about 110 amino acids, being variable regions (V-regions); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region (C-region). The variable regions include 3 hypervariable regions (HVRs) and 4 FR Regions (FRs) which are relatively conserved in sequence. The amino acid sequences of the 4 FRs are relatively conserved and do not directly participate in the binding reaction. The 3 hypervariable regions determine the specificity of the antibody, also known as Complementarity Determining Regions (CDRs). Each of the Light Chain Variable Region (LCVR) and Heavy Chain Variable Region (HCVR) is composed of 3 CDR regions and 4 FR regions, which are sequentially arranged from amino terminus to carboxy terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The 3 CDR regions of the light chain, the light chain hypervariable region (LCDR), designated LCDR1, LCDR2 and LCDR 3; the 3 CDR regions of the heavy chain, the hypervariable region of the Heavy Chain (HCDR), are referred to as HCDR1, HCDR2 and HCDR 3. The CDR amino acid residues in the LCVR and HCVR regions of the antibodies or antigen-binding fragments of the invention are in number and position in accordance with known Kabat numbering convention (LCDR1-3, HCDR2-3), or in accordance with Kabat and chothia numbering convention (HCDR 1). The four FR regions in the native heavy and light chain variable regions are in a substantially β -sheet configuration, connected by three CDRs that form a connecting loop, and in some cases may form part of a β -sheet structure. The CDRs in each chain are held together tightly by the FR regions and form the antigen binding site of the antibody with the CDRs of the other chain. It is possible to determine which amino acids constitute the FR or CDR regions by comparing the amino acid sequences of antibodies of the same type. The constant regions are not directly involved in the binding of antibodies to antigens, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of antibodies.
The term "antigen-binding fragment", as used herein, refers to a Fab fragment, a Fab 'fragment, F (ab')2A fragment, or a single Fv fragment. Fv antibodies contain the variable regions of the antibody heavy chain, the variable regions of the light chain, but no constant regions, and have the smallest antibody fragment of the entire antigen binding site. Generally, Fv antibodies also comprise a polypeptide linker between the VH and VL domains and are capable of forming the structures required for antigen binding. Non-limiting examples of antigen-binding fragments include: (i) a Fab fragment; (ii) f (ab')2A fragment; (iii) (ii) a fragment of Fd; (iv) (iv) an Fv fragment; (v) a scFv molecule; (vi) a dAb fragment; and (vii) a minimal recognition unit (e.g., an independent Complementarity Determining Region (CDR) such as a CDR3 peptide) consisting of amino acid residues that mimic a hypervariable region of an antibody or a constrained FR3-CDR3-FR4 peptide. As used herein, the expression "antigen-binding fragment" also internally encompasses other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), Small Modular Immunopharmaceuticals (SMIPs), and shark variable IgNAR domains.
As used herein, the term "antigenic determinant" refers to a three-dimensional spatial site on an antigen that is not contiguous and is recognized by an antibody or antigen-binding fragment of the invention.
The invention includes not only intact antibodies, but also fragments of antibodies with immunological activity or fusion proteins of antibodies with other sequences. Accordingly, the invention also includes fragments, derivatives and analogs of the antibodies.
In the present invention, antibodies include murine, chimeric, humanized or fully human antibodies prepared using techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, including human and non-human portions, can be prepared using recombinant DNA techniques well known in the art.
As used herein, the term "monoclonal antibody" refers to an antibody secreted by a clone obtained from a single cell source. Monoclonal antibodies are highly specific, being directed against a single epitope. The cell may be a eukaryotic, prokaryotic, or phage clonal cell line.
As used herein, the term "chimeric antibody" is an antibody molecule expressed by a host cell transfected with a vector by splicing a V region gene of a murine antibody to a C region gene of a human antibody into a chimeric gene. Not only retains the high specificity and affinity of the parent mouse antibody, but also ensures that the humanized Fc segment can effectively mediate the biological effect function.
As used herein, the term "humanized antibody", is a variable region engineered version of a murine antibody of the invention, having CDR regions derived from (or substantially derived from) a non-human antibody (preferably a mouse monoclonal antibody), and FR regions and constant regions substantially derived from human antibody sequences; that is, the CDR sequence of the mouse antibody is grafted to the framework sequences of different types of human germline antibodies. Because the CDR sequences are responsible for most of the antibody-antigen interactions, recombinant antibodies that mimic the properties of a particular naturally occurring antibody can be expressed by constructing an expression vector.
In the present invention, the antibody may be monospecific, bispecific, trispecific, or more multispecific.
In the present invention, the antibody of the present invention also includes conservative variants thereof, which means that at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids are replaced by amino acids having similar or similar properties as compared with the amino acid sequence of the antibody of the present invention to form a polypeptide. These conservative variants are preferably produced by amino acid substitutions according to Table A.
TABLE A
Figure 174160DEST_PATH_IMAGE001
anti-CD 3 antibodies
As used herein, the term "CD 3" generally refers to natural or recombinant human CD3, as well as non-human homologs of human CD 3. CD3 is a homodimeric or heterodimeric antigen expressed on T cells that is associated with the T cell receptor complex (TCR) and is essential for T cell activation. Functional CD3 is a dimer formed from two of four different chains (epsilon, zeta, delta, and gamma). The CD3 dimer arrangement includes γ/ε, δ/ε, and ζ/ζ. Thus, unless specifically indicated to be from a non-human species, e.g., "mouse CD3," "monkey CD3," etc., the term "CD 3" refers to human CD 3.
As used herein, "CD 3E" refers to the CD3 epsilon Extracellular domain (Extracellular domain).
The present invention provides a highly specific single chain antibody (scFv) against CD3 comprising a heavy chain variable region (VH), a light chain variable region (VL) and a linker attached.
The present invention also provides a highly specific antibody against CD3 comprising a heavy chain variable region (VH) amino acid sequence and a light chain comprising a light chain variable region (VL) amino acid sequence.
Preferably, the heavy chain variable region and the light chain variable region have 6 complementarity determining regions CDRs selected from the group consisting of:
(A1) three complementarity determining regions, VH-CDRs, of the heavy chain variable region and three complementarity determining regions, VL-CDRs, of the light chain variable region:
huVH-CDR1： GFTFNKYA， SEQ ID NO. 1
huVH-CDR2： IRSKYNNYAT， SEQ ID NO. 2
huVH4-CDR3： HGNFGNSYISYWEY， SEQ ID NO. 3
huVL-CDR1： TGAVTSGNY， SEQ ID NO. 4
huVL-CDR2： GTK， SEQ ID NO. 5
huVL4-CDR3： VLWNSNRW， SEQ ID NO. 6；
(A2) three complementarity determining regions, VH-CDRs, of the heavy chain variable region and three complementarity determining regions, VL-CDRs, of the light chain variable region:
huVH-CDR1： GFTFNKYA， SEQ ID NO.1
huVH-CDR2： IRSKYNNYAT， SEQ ID NO.2
huVH3-CDR3： HGNFGNSYISYWRY， SEQ ID NO.10
huVL-CDR1： TGAVTSGNY， SEQ ID NO.4
huVL-CDR2： GTK， SEQ ID NO.5
huVL3-CDR3： VLWYSGRW， SEQ ID NO.11；
(A3) three complementarity determining regions, VH-CDRs, of the heavy chain variable region and three complementarity determining regions, VL-CDRs, of the light chain variable region:
huVH-CDR1： GFTFNKYA， SEQ ID NO.1
huVH-CDR2： IRSKYNNYAT， SEQ ID NO.2
huVH2-CDR3： HGNFGNSYISYWQY， SEQ ID NO.15
huVL-CDR1： TGAVTSGNY， SEQ ID NO.4
huVL-CDR2： GTK， SEQ ID NO.5
huVL2-CDR 3: VLWRSNRW, SEQ ID No. 16; or
(A4) Three complementarity determining regions, VH-CDRs, of the heavy chain variable region and three complementarity determining regions, VL-CDRs, of the light chain variable region:
huVH-CDR1： GFTFNKYA， SEQ ID NO.1
huVH-CDR2： IRSKYNNYAT， SEQ ID NO.2
huVH1-CDR3： HGNFGNTYISYWAY， SEQ ID NO.20
huVL-CDR1： TGAVTSGNY， SEQ ID NO.4
huVL-CDR2： GTK， SEQ ID NO.5
huVL1-CDR3： VLWYSKRW， SEQ ID NO.21。
preferably, the sequence of the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID number 7; and the sequence of the light chain variable region comprises an amino acid sequence shown as SEQ ID number 8; or
The heavy chain variable region contains an amino acid sequence shown by SEQ ID number 12; and the light chain variable region comprises an amino acid sequence shown as SEQ ID number 13; or
The heavy chain variable region contains an amino acid sequence shown by SEQ ID number 17; and the light chain variable region comprises an amino acid sequence shown as SEQ ID number 18; or
The heavy chain variable region contains an amino acid sequence shown by SEQ ID number 22; and the light chain variable region comprises an amino acid sequence shown as SEQ ID number 23.
Wherein, any one of the above amino acid sequences further comprises a derivative sequence with CD3 binding affinity, which is added, deleted, modified and/or substituted with at least one (e.g., 1-5, 1-3, preferably 1-2, more preferably 1) amino acid.
In another preferred embodiment, the sequence formed by adding, deleting, modifying and/or substituting at least one amino acid sequence is preferably an amino acid sequence with homology of at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95%.
The antibody of the present invention may be a double-chain or single-chain antibody, and may be selected from an animal-derived antibody, a chimeric antibody, a humanized antibody, more preferably a humanized antibody, a human-animal chimeric antibody, and still more preferably a fully humanized antibody.
As used herein, the term "scFv" is a single chain antibody (scFv) consisting of an antibody heavy chain variable region and a light chain variable region linked, typically by a 15-25 amino acid linker.
As used herein, the term "linker" refers to an insertion into an immunoglobulin domain that provides sufficient mobility for the domains of the light and heavy chains to fold into one or more amino acid residues that exchange the dual variable region immunoglobulin. In the present invention, a preferred linker means a linker connecting VH and VL of a single chain antibody (scFv), or for linking a scFv to a heavy chain of another antibody.
Examples of suitable linkers include single glycine (Gly), or serine (Ser) residues, and the identity and sequence of the amino acid residues in the linker may vary depending on the type of secondary structural element that is desired to be implemented in the linker. For example, (G4S) n, where n is an integer from 1 to 5.
The antibody derivatives of the present invention may be single chain antibodies, and/or antibody fragments, such as: fab, Fab ', (Fab')2Or other known antibody derivatives in the art, and any one or more of IgA, IgD, IgE, IgG, and IgM antibodies or antibodies of other subtypes.
Among them, the animal is preferably a mammal such as a mouse.
The antibodies of the invention may be murine, chimeric, humanized, CDR grafted and/or modified antibodies targeting human CD 3.
In a preferred embodiment of the present invention, the above
SEQ ID number 1, SEQ ID number 2 and SEQ ID number 3;
SEQ ID number 1, SEQ ID number 2 and SEQ ID number 10;
SEQ ID number 1, SEQ ID number 2 and SEQ ID number 15; or
SEQ ID number 1, SEQ ID number 2 and SEQ ID number 20
Any one or more of the sequences or the sequence with CD3 binding affinity obtained by adding, deleting, modifying and/or substituting at least one amino acid is positioned in the CDR region of the heavy chain variable region (VH).
In a preferred embodiment of the present invention, the above
SEQ ID number 4, SEQ ID number 5 and SEQ ID number 6;
SEQ ID number 4, SEQ ID number 5 and SEQ ID number 11;
SEQ ID number 4, SEQ ID number 5 and SEQ ID number 16; or
SEQ ID number 4, SEQ ID number 5 and SEQ ID number 21;
any one or more of the sequences or the sequence with CD3 binding affinity obtained by adding, deleting, modifying and/or substituting at least one amino acid is positioned in a CDR region of a light chain variable region (VL).
In the above-mentioned aspect of the present invention, the number of amino acids to be added, deleted, modified and/or substituted is preferably not more than 40%, more preferably not more than 35%, more preferably 1 to 33%, more preferably 5 to 30%, more preferably 10 to 25%, and more preferably 15 to 20% of the total number of amino acids in the original amino acid sequence.
In the present invention, the number of the amino acids to be added, deleted, modified and/or substituted is usually 1, 2, 3, 4 or 5, preferably 1 to 3, more preferably 1 to 2, and most preferably 1.
As used herein, the term "bispecific antibody (or diabody)" refers to an antibody molecule that specifically binds two antigens (targets) or two epitopes simultaneously. Bispecific antibodies can be divided into structurally symmetric and asymmetric molecules according to symmetry. Bispecific antibodies can be classified into bivalent, trivalent, tetravalent, and multivalent molecules, depending on the number of binding sites.
The antibodies of the invention may be monospecific, bispecific, trispecific or multispecific. For example, the antibodies of the invention can be used to form bispecific antibodies with antibodies or active fragments that bind other targets. The antibody that binds to the other target may be an antibody or an active fragment thereof that targets CD19, CD47, CD73, CD47, CTLA4, PD-1, PD-L1, CD 28.
Preparation of antibodies
Any method suitable for producing monoclonal antibodies may be used to produce the anti-CD 3 antibodies of the invention. For example, an animal may be immunized with a linked or naturally occurring CD3 homodimer or fragment thereof. Suitable immunization methods, including adjuvants, immunostimulants, repeated booster immunizations, and one or more routes may be used.
Any suitable form of CD3 may be used as an immunogen (antigen) for the production of non-human antibodies specific for CD3, which antibodies are screened for biological activity. The challenge immunogen may be full-length mature human CD3, including natural homodimers, or a peptide containing a single/multiple epitope. The immunogen may be used alone or in combination with one or more immunogenicity enhancing agents known in the art. Immunogens can be purified from natural sources or produced in genetically modified cells. The DNA encoding the immunogen may be genomic or non-genomic in origin (e.g., cDNA). DNA encoding the immunogen may be expressed using suitable genetic vectors including, but not limited to, adenoviral vectors, adeno-associated viral vectors, baculovirus vectors, plasmids and non-viral vectors.
An exemplary method for producing an anti-human CD3 antibody of the invention is described in example 1.
Humanized antibodies may be selected from any class of immunoglobulins, including IgM, IgD, IgG, IgA, and IgE. In the present invention, the antibody is an IgG antibody, and an IgG1 subtype is used. Optimization of the sequence of the essential constant domains to produce the desired biological activity is readily achieved by screening antibodies using the biological assays described in the examples below.
Likewise, any type of light chain can be used in the compounds and methods herein. In particular, kappa, lambda chains or variants thereof are useful in the compounds and methods of the invention.
An exemplary method for humanizing an anti-human CD3 antibody of the invention is described in example 2.
The sequence of the DNA molecule of the antibody or fragment thereof of the present invention can be obtained by a conventional technique, for example, by PCR amplification or genomic library screening. Alternatively, the coding sequences for the light and heavy chains may be fused together to form a single chain antibody.
Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.
In addition, the sequence can be synthesized by artificial synthesis, especially when the fragment length is short. Generally, fragments with long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art.
The invention also relates to a vector comprising a suitable DNA sequence as described above and a suitable promoter or control sequence. These vectors may be used to transform an appropriate host cell so that it can express the protein.
The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Preferred animal cells include (but are not limited to): CHO-S, CHO-K1, HEK-293 cells.
The steps described in the present invention for transforming a host cell with a recombinant DNA can be performed using techniques well known in the art. The obtained transformant can be cultured by a conventional method, and the transformant expresses the polypeptide encoded by the gene of the present invention. Depending on the host cell used, it is cultured in a conventional medium under suitable conditions.
Typically, the transformed host cells are cultured under conditions suitable for expression of the antibodies of the invention. The antibody of the invention is then purified by conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography, using conventional separation and purification means well known to those skilled in the art.
The resulting monoclonal antibodies can be identified by conventional means. For example, the binding specificity of a monoclonal antibody can be determined by immunoprecipitation or by an in vitro binding assay, such as Radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).
Antibody-drug conjugates (ADC)
The invention also provides an antibody-conjugated drug (ADC) based on the antibody of the invention.
Typically, the antibody-conjugated drug comprises the antibody, and an effector molecule to which the antibody is conjugated, and preferably chemically conjugated. Wherein the effector molecule is preferably a therapeutically active drug. Furthermore, the effector molecule may be one or more of a toxic protein, a chemotherapeutic drug, a small molecule drug or a radionuclide.
The antibody of the invention may be conjugated to the effector molecule by a coupling agent. Examples of the coupling agent may be any one or more of a non-selective coupling agent, a coupling agent using a carboxyl group, a peptide chain, and a coupling agent using a disulfide bond. The non-selective coupling agent is a compound which enables effector molecules and antibodies to form covalent bonds, such as glutaraldehyde and the like. The coupling agent using carboxyl can be any one or more of a cis-aconitic anhydride coupling agent (such as cis-aconitic anhydride) and an acylhydrazone coupling agent (coupling site is acylhydrazone).
Certain residues on the antibody (e.g., Cys or Lys, etc.) are used to attach to a variety of functional groups, including imaging agents (e.g., chromophores and fluorophores), diagnostic agents (e.g., MRI contrast agents and radioisotopes), stabilizing agents (e.g., ethylene glycol polymers) and therapeutic agents. The antibody may be conjugated to a functional agent to form an antibody-functional agent conjugate. Functional agents (e.g., drugs, detection reagents, stabilizers) are coupled (covalently linked) to the antibody. The functional agent may be attached to the antibody directly, or indirectly through a linker.
Antibodies may be conjugated to drugs to form Antibody Drug Conjugates (ADCs). Typically, the ADC comprises a linker between the drug and the antibody. The linker may be degradable or non-degradable. Degradable linkers are typically susceptible to degradation in the intracellular environment, e.g., the linker degrades at the site of interest, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzymatically degradable linkers, including peptidyl-containing linkers that can be degraded by intracellular proteases (e.g., lysosomal proteases or endosomal proteases), or sugar linkers such as glucuronide-containing linkers that can be degraded by glucuronidase. The peptidyl linker may comprise, for example, a dipeptide such as valine-citrulline, phenylalanine-lysine or valine-alanine. Other suitable degradable linkers include, for example, pH sensitive linkers (e.g., linkers that hydrolyze at a pH of less than 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (e.g., disulfide linkers). Non-degradable linkers typically release the drug under conditions in which the antibody is hydrolyzed by a protease.
Prior to attachment to the antibody, the linker has a reactive group capable of reacting with certain amino acid residues, and attachment is achieved by the reactive group. Thiol-specific reactive groups are preferred and include: for example maleimide compounds, haloamides (for example iodine, bromine or chlorine); halogenated esters (e.g., iodo, bromo, or chloro); halomethyl ketones (e.g., iodo, bromo, or chloro), benzyl halides (e.g., iodo, bromo, or chloro); vinyl sulfone, pyridyl disulfide; mercury derivatives such as 3, 6-bis- (mercuric methyl) dioxane, and the counter ion is acetate, chloride or nitrate; and polymethylene dimethyl sulfide thiolsulfonate. The linker may comprise, for example, a maleimide linked to the antibody via a thiosuccinimide.
The drug may be any cytotoxic, cytostatic, or immunosuppressive drug. In embodiments, the linker links the antibody and the drug, and the drug has a functional group that can form a bond with the linker. For example, the drug may have an amino, carboxyl, thiol, hydroxyl, or keto group that may form a bond with the linker. In the case of a drug directly attached to a linker, the drug has a reactive group prior to attachment to the antibody.
Useful classes of drugs include, for example, anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, and the like. In the present invention, a drug-linker can be used to form an ADC in a single step. In other embodiments, bifunctional linker compounds may be used to form ADCs in a two-step or multi-step process. For example, a cysteine residue is reacted with a reactive moiety of a linker in a first step, and in a subsequent step, a functional group on the linker is reacted with a drug, thereby forming an ADC.
Generally, the functional group on the linker is selected to facilitate specific reaction with a suitable reactive group on the drug moiety. As a non-limiting example, azide-based moieties may be used to specifically react with reactive alkynyl groups on the drug moiety. The drug is covalently bound to the linker by 1, 3-dipolar cycloaddition between the azide and the alkynyl group. Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphines (suitable for reaction with azides); isocyanates and isothiocyanates (suitable for reaction with amines and alcohols); and activated esters, such as N-hydroxysuccinimide esters (suitable for reaction with amines and alcohols). These and other attachment strategies, such as those described in bioconjugation technology, second edition (Elsevier), are well known to those skilled in the art. It will be appreciated by those skilled in the art that for selective reaction of a drug moiety and a linker, each member of a complementary pair may be used for both the linker and the drug when the reactive functional group of the complementary pair is selected.
Applications of
The invention provides the use of an antibody of the invention, for example for the preparation of a diagnostic formulation, or for the preparation of a medicament for the prevention and/or treatment of a CD 3-related disease. The CD 3-related diseases include inflammatory diseases, autoimmune diseases, and the like, including but not limited to psoriasis, psoriatic arthritis, ankylosing spondylitis, multiple sclerosis, inflammatory bowel disease (e.g., crohn's disease, ulcerative colitis, and the like), osteoarthritis, Rheumatoid Arthritis (RA), rheumatoid arthritis or osteoporosis, inflammatory fibrosis (e.g., scleroderma, pulmonary fibrosis, and cirrhosis), asthma (including allergic asthma), allergies, and cancer.
Pharmaceutical composition
The invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition comprising an antibody or an active fragment thereof or a fusion protein thereof or an ADC thereof or a corresponding CAR-T cell as described above, and a pharmaceutically acceptable carrier. Generally, these materials will be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally from about 5 to about 8, preferably from about 6 to about 8, although the pH will vary depending on the nature of the material being formulated and the condition being treated. The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: intratumoral, intraperitoneal, intravenous, or topical administration.
The antibody of the present invention may also be used for cell therapy by intracellular expression of a nucleotide sequence, for example, for chimeric antigen receptor T cell immunotherapy (CAR-T) and the like.
The pharmaceutical composition of the invention can be directly used for binding CD3 protein molecules, and thus can be used for preventing and treating CD3 related diseases. In addition, other therapeutic agents may also be used simultaneously.
The pharmaceutical composition of the present invention comprises a safe and effective amount (e.g., 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the monoclonal antibody (or conjugate thereof) of the present invention as described above and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should be compatible with the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections, solutions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example from about 1 microgram per kilogram of body weight to about 5 milligrams per kilogram of body weight per day. In addition, the polypeptides of the invention may also be used with other therapeutic agents.
Where a pharmaceutical composition is used, a safe and effective amount of the pharmaceutical composition is administered to the mammal, wherein the safe and effective amount is generally at least about 10 micrograms/kg body weight, and in most cases does not exceed about 50 mg/kg body weight, preferably the dose is from about 10 micrograms/kg body weight to about 20 mg/kg body weight. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.
Detection use and kit
The antibodies of the invention are useful in detection applications, for example, for detecting a sample, thereby providing diagnostic information.
In the present invention, the specimen (sample) used includes cells, tissue samples and biopsy specimens. The term "biopsy" as used herein shall include all kinds of biopsies known to the person skilled in the art. Thus, a biopsy as used in the present invention may comprise a tissue sample prepared, for example, by endoscopic methods or by needle or needle biopsy of an organ.
Samples for use in the present invention include fixed or preserved cell or tissue samples.
The invention also provides a kit containing the antibody (or fragment thereof) of the invention, and in a preferred embodiment of the invention, the kit further comprises a container, instructions for use, a buffer, and the like. In a preferred embodiment, the antibody of the present invention may be immobilized on a detection plate.
The main advantages of the invention include
(1) The CD3 antibody of the invention specifically binds to CD3 and has high biological activity;
(2) the CD3 antibody of the invention can be used to construct bispecific antibodies with other antibodies.
The invention is further illustrated by the following examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specifying the detailed conditions in the following examples, generally followed by conventional conditions such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight.
EXAMPLE 1 Generation of humanized CD3 monoclonal antibody
A Balb/C mouse is immunized by recombinant human CD3E protein (C00E, Novoprotein), after immunization is completed, B cells are taken to construct a phage display library, candidate antibodies are obtained through screening, and a series of humanized antibodies are obtained by utilizing structural simulation and rational design. The obtained humanized antibody heavy chain variable regions were huVH1, huVH2, huVH3 and huVH4, and the obtained humanized antibody light chain variable regions were huVL1, huVL2, huVL3 and huVL 4. VH and VL of humanized sequences are connected through a (G4S) 3 linker, a 6HIS tag is added at the C terminal to construct a single-chain antibody form (scFv), and the single-chain antibody protein is obtained by expression of escherichia coli and nickel column purification. The sequences of the heavy chain variable region (VH) and the light chain variable region (VL) and the CDRs of the obtained humanized antibody are as follows:
wherein VH-CDR1 and VH-CDR2 are identical and have the following sequences:
VH-CDR1：GFTFNKYA (SEQ ID NO.1)
VH-CDR2：IRSKYNNYAT (SEQ ID NO. 2)；
the VH-CDR3 all differed in sequence as follows:
huVH4-CDR3：HGNFGNTYISYWAY (SEQ ID NO. 3)
huVH3-CDR3：HGNFGNSYISYWQY (SEQ ID NO. 10)
huVH2-CDR3：HGNFGNSYISYWRY (SEQ ID NO. 15)
huVH1-CDR3：HGNFGNSYISYWEY (SEQ ID NO. 20)。
wherein, VL-CDR1 and VL-CDR2 are identical and have the following sequences:
VL-CDR1：TGAVTSGNY (SEQ ID NO.4)
VL-CDR2：GTK (SEQ ID NO. 5)
the VL-CDR3 sequences all differ as follows:
huVL4-CDR3：VLWYSKRW (SEQ ID NO.6)
huVL3-CDR3：VLWRSNRW (SEQ ID NO.11)
huVL2-CDR3：VLWYSGRW (SEQ ID NO.16)
huVL1-CDR3：VLWNSNRW (SEQ ID NO.21)。
humanized CD3 antibody heavy chain variable region huVH4 (SEQ ID number 7)
EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWEYWGQGTLVTVSS
Humanized CD3 antibody heavy chain variable region huVH3 (SEQ ID NO.12)
EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWRYWGQGTLVTVSS
Humanized CD3 antibody heavy chain variable region huVH2 (SEQ ID NO.17)
EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWQYWGQGTLVTVSS
Humanized CD3 antibody heavy chain variable region huVH1 (SEQ ID NO.22)
EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNTYISYWAYWGQGTLVTVSS
Humanized CD3 antibody light chain variable region huVL4 (SEQ ID NO.8)
QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGDKAALTLSGVQPEDEAEYYCVLWNSNRWVFGGGTKLTVL
Humanized CD3 antibody light chain variable region huVL3 (SEQ ID NO.13)
QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSGRWVFGGGTKLTVL
Humanized CD3 antibody light chain variable region huVL2 (SEQ ID NO.18)
QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWRSNRWVFGGGTKLTVL
Humanized CD3 antibody light chain variable region huVL1 (SEQ ID NO.23)
QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSKRWVFGGGTKLTVL
Humanized CD3 Single chain antibody huVH4VL4 (SEQ ID number 9)
EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWEYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGDKAALTLSGVQPEDEAEYYCVLWNSNRWVFGGGTKLTVL
Humanized CD3 Single chain antibody huVH3VL3 (SEQ ID number 14)
EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWRYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSGRWVFGGGTKLTVL
Humanized CD3 Single chain antibody huVH2VL2 (SEQ ID number 19)
EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWQYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWRSNRWVFGGGTKLTVL
Humanized CD3 Single chain antibody huVH1VL1 (SEQ ID number 24)
EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNTYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSKRWVFGGGTKLTVL
Example 2 affinity detection of CD3 antibody
This example describes mainly the affinity detection of the single chain antibody form of CD3 antibody with human and monkey recombinant CD3E protein.
Affinity with human CD3 protein
The affinity of CD3 antibody to recombinant hCD3 protein (CP 19, Novoprotein) was determined by ELISA, recombinant hCD3 was plated and CD3 antibody was diluted in a 10-fold gradient (starting at 20 μ g/ml) as shown in figure 1.
The calculated EC50 results are shown in table 1.
TABLE 1 single chain antibody form of CD3 antibody human CD3 protein affinity EC50
Figure 294563DEST_PATH_IMAGE002
2.2 affinity to monkey CD3 protein
The affinity of CD3 antibody to recombinant monkey CD3 protein (CW 07, Novoprotein) was determined by ELISA, recombinant monkey CD3 was plated and CD3 antibody was diluted in a 10-fold gradient (starting at 20 μ g/ml) as shown in figure 2.
The calculated EC50 results are shown in table 2.
TABLE 2 EC50 of single-chain antibody form of CD3 antibody with monkey CD3 protein affinity
Figure 526830DEST_PATH_IMAGE003
Example 3 full Length antibody construction
Constructing a full-length antibody of CD3 by fusing the heavy chain variable region with the heavy chain constant region hIgG1 and the light chain variable region with the light chain constant region kappa chain; the full length antibodies of CD3 obtained were CQ50 (comprising huVH1 and huVL 1), CQ51 (comprising huVH2 and huVL 2), CQ52 (comprising huVH3 and huVL 3) and CQ53 (comprising huVH4 and huVL 4), respectively. The variable region of marketed drug OKT3 was selected as a control antibody fused to hIgG 1. The related sequences are as follows:
hIgG1 constant region (SEQ ID number 25)
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
kappa chain constant region (SEQ ID number 26)
LTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
Heavy chain of OKT3 antibody (SEQ ID NO.27)
QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSAKTTAPSVYPLAPVCGGTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Light chain of OKT3 antibody (SEQ ID NO.28)
QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINRADTAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC
Example 4 full-Length antibody affinity assay
This example relates to the binding of full-length antibodies to human monkey protein, and to the affinity of the human CD3+ cell line Jurkat.
Detection of full-length CD3 antibody affinity for human CD3E protein
Using the method of fortebio, protein A sensor was selected, CD3 antibody was immobilized, CD3E was diluted in a gradient, 7 gradients were diluted in half from 100nM, and the detection affinity was shown in FIG. 3.
The calculated affinities are as follows in table 3:
TABLE 3 affinity of full-length CD3 antibody to human CD3E protein
Figure 125302DEST_PATH_IMAGE004
Calculated affinity of CQ50 was 5.16X 10-8M; CQ51 has an affinity of 6.24X 10-8M；
CQ52 affinity of 3.11X 10-9M; CQ53 has an affinity of 2.02X 10-9M。
Detection of binding of full-length CD3 antibody to monkey CD3E by ELISA
The results are shown in figure 4 of the drawings,
CQ50 detected EC50=0.2004 μ g/ml,
CQ51 detected EC50= 0.3385. mu.g/ml,
CQ52 detected EC50=0.195 μ g/ml,
CQ53 detected EC50=0.0823 μ g/ml.
The results of the assays showed that CQ50, CQ51, CQ52 and CQ53 bound to monkey CD3E, whereas OKT3 did not bind.
Binding to human CD3 positive cells
Take 4X 105After 1h of incubation of individual cells Jurkat with a gradient of CD3 antibody, the cells were washed 3 times with PBS and Anti-hFC-APC (purchased from Jackson immunology) was added and detected on a flow machine. The results are plotted as an S-curve in FIG. 5.
The calculation of the EC50 is carried out,
the CQ50 was 0.01947. mu.g/ml, and OKT3 was 0.01. mu.g/ml. The affinity of the two to cells is not very different;
CQ51 was 0.0158. mu.g/ml, OKT3 was 0.01. mu.g/ml. The affinity of the two to cells is not very different;
the CQ52 was 0.02167. mu.g/ml, and OKT3 was 0.01. mu.g/ml. The affinity of the two to cells is not very different;
CQ53 was 0.013. mu.g/ml, OKT3 was 0.01. mu.g/ml. The affinity of the two to the cell is not very different.
EXAMPLE 5 epitope Primary assay for full-Length CD3 antibody
Given that the full-length CD3 antibody is functionally comparable to OKT3, this example was primarily to investigate whether the full-length CD3 antibody binds consistently with OKT3 antibody on the epitope of CD 3E. Specifically, the full-length CD3 antibody was coated, a certain amount of HIS-tagged CD3E (C578 Novoprotein) protein was added, and the results are shown in fig. 6, after adding gradient-diluted OKT3 antibody and Anti-HIS secondary antibody (from Biolegend) with reference to EC90 value of ELISA assay.
From the results, there was no competitive relationship between CQ50 and OKT3, and the epitopes of the two antibodies were not identical;
there was no competitive relationship between CQ51 and OKT3, and the epitopes of the two antibodies were not identical;
there was no competitive relationship between CQ52 and OKT3, and the epitopes of the two antibodies were not identical;
there was no competitive relationship between CQ53 and OKT3, and the epitopes of the two antibodies were not identical.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Sequence listing
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Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys Tyr
20 25 30
Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp
50 55 60
Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr
65 70 75 80
Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Ile Ser Tyr Trp
100 105 110
Arg Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly
115 120 125
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Thr Val Val
130 135 140
Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu
145 150 155 160
Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Ser Gly Asn Tyr Pro Asn
165 170 175
Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly
180 185 190
Thr Lys Phe Leu Ala Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu
195 200 205
Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val Gln Pro Glu Asp
210 215 220
Glu Ala Glu Tyr Tyr Cys Val Leu Trp Tyr Ser Gly Arg Trp Val Phe
225 230 235 240
Gly Gly Gly Thr Lys Leu Thr Val Leu
245
<210> 15
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 15
His Gly Asn Phe Gly Asn Ser Tyr Ile Ser Tyr Trp Gln Tyr
1 5 10
<210> 16
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 16
Val Leu Trp Arg Ser Asn Arg Trp
1 5
<210> 17
<211> 125
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 17
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys Tyr
20 25 30
Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp
50 55 60
Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr
65 70 75 80
Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Ile Ser Tyr Trp
100 105 110
Gln Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 18
<211> 109
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 18
Gln Thr Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly
1 5 10 15
Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Ser Gly
20 25 30
Asn Tyr Pro Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly
35 40 45
Leu Ile Gly Gly Thr Lys Phe Leu Ala Pro Gly Thr Pro Ala Arg Phe
50 55 60
Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val
65 70 75 80
Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Val Leu Trp Arg Ser Asn
85 90 95
Arg Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105
<210> 19
<211> 249
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 19
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys Tyr
20 25 30
Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp
50 55 60
Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr
65 70 75 80
Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Ile Ser Tyr Trp
100 105 110
Gln Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly
115 120 125
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Thr Val Val
130 135 140
Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu
145 150 155 160
Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Ser Gly Asn Tyr Pro Asn
165 170 175
Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly
180 185 190
Thr Lys Phe Leu Ala Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu
195 200 205
Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val Gln Pro Glu Asp
210 215 220
Glu Ala Glu Tyr Tyr Cys Val Leu Trp Arg Ser Asn Arg Trp Val Phe
225 230 235 240
Gly Gly Gly Thr Lys Leu Thr Val Leu
245
<210> 20
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 20
His Gly Asn Phe Gly Asn Thr Tyr Ile Ser Tyr Trp Ala Tyr
1 5 10
<210> 21
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 21
Val Leu Trp Tyr Ser Lys Arg Trp
1 5
<210> 22
<211> 125
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 22
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys Tyr
20 25 30
Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp
50 55 60
Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr
65 70 75 80
Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Val Arg His Gly Asn Phe Gly Asn Thr Tyr Ile Ser Tyr Trp
100 105 110
Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 23
<211> 109
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 23
Gln Thr Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly
1 5 10 15
Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Ser Gly
20 25 30
Asn Tyr Pro Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly
35 40 45
Leu Ile Gly Gly Thr Lys Phe Leu Ala Pro Gly Thr Pro Ala Arg Phe
50 55 60
Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val
65 70 75 80
Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Val Leu Trp Tyr Ser Lys
85 90 95
Arg Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105
<210> 24
<211> 249
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 24
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys Tyr
20 25 30
Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp
50 55 60
Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr
65 70 75 80
Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Val Arg His Gly Asn Phe Gly Asn Thr Tyr Ile Ser Tyr Trp
100 105 110
Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly
115 120 125
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Thr Val Val
130 135 140
Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu
145 150 155 160
Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Ser Gly Asn Tyr Pro Asn
165 170 175
Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly
180 185 190
Thr Lys Phe Leu Ala Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu
195 200 205
Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val Gln Pro Glu Asp
210 215 220
Glu Ala Glu Tyr Tyr Cys Val Leu Trp Tyr Ser Lys Arg Trp Val Phe
225 230 235 240
Gly Gly Gly Thr Lys Leu Thr Val Leu
245
<210> 25
<211> 330
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 25
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
225 230 235 240
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
325 330
<210> 26
<211> 107
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 26
Leu Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
1 5 10 15
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
20 25 30
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
35 40 45
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
50 55 60
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
65 70 75 80
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
85 90 95
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210> 27
<211> 450
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 27
Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr
20 25 30
Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe
50 55 60
Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Thr Leu Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val Tyr
115 120 125
Pro Leu Ala Pro Val Cys Gly Gly Thr Thr Gly Ser Ser Val Thr Leu
130 135 140
Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp
145 150 155 160
Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser Ser
180 185 190
Thr Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser
195 200 205
Ser Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly Lys
450
<210> 28
<211> 213
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 28
Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly
1 5 10 15
Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met
20 25 30
Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr
35 40 45
Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala His Phe Arg Gly Ser
50 55 60
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Gly Met Glu Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Phe Thr
85 90 95
Phe Gly Ser Gly Thr Lys Leu Glu Ile Asn Arg Ala Asp Thr Ala Pro
100 105 110
Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly
115 120 125
Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn
130 135 140
Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu Asn
145 150 155 160
Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser
165 170 175
Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr
180 185 190
Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser Phe
195 200 205
Asn Arg Asn Glu Cys
210

Claims (11)

1. An anti-CD 3 humanized antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region have 6 complementarity determining regions CDRs selected from the group consisting of:
(A3) three complementarity determining regions, VH-CDRs, of the heavy chain variable region and three complementarity determining regions, VL-CDRs, of the light chain variable region:
huVH-CDR1： GFTFNKYA， SEQ ID NO.1
huVH-CDR2： IRSKYNNYAT， SEQ ID NO.2
huVH2-CDR3： HGNFGNSYISYWQY， SEQ ID NO.15
huVL-CDR1： TGAVTSGNY， SEQ ID NO.4
huVL-CDR2： GTK， SEQ ID NO.5
huVL2-CDR 3: VLWRSNRW, SEQ ID No. 16; or
(A4) Three complementarity determining regions, VH-CDRs, of the heavy chain variable region and three complementarity determining regions, VL-CDRs, of the light chain variable region:
huVH-CDR1： GFTFNKYA， SEQ ID NO.1
huVH-CDR2： IRSKYNNYAT， SEQ ID NO.2
huVH1-CDR3： HGNFGNTYISYWAY， SEQ ID NO.20
huVL-CDR1： TGAVTSGNY， SEQ ID NO.4
huVL-CDR2： GTK， SEQ ID NO.5
huVL1-CDR3： VLWYSKRW， SEQ ID NO.21。
2. the antibody of claim 1, wherein said antibody comprises a heavy chain and a light chain, wherein said heavy chain of said antibody comprises said three complementarity determining regions VH-CDR and a heavy chain framework region for linking the VH-CDR, and said light chain of said antibody comprises said three complementarity determining regions VL-CDR and a light chain framework region for linking the VL-CDR.
3. The antibody of claim 1, wherein said antibody is a single chain antibody.
4. A bispecific antibody, wherein said bispecific antibody comprises:
a first antigen-binding domain D1; and
a second antigen-binding domain D2;
wherein, D1 specifically binds to the target molecule CD3 protein; d2 specifically binds to the target molecule CD19 protein;
wherein the D1 is an antibody or antigen-binding fragment thereof that specifically binds to CD3 protein;
the D2 is an antibody or antigen-binding fragment thereof that specifically binds to CD19 protein;
wherein D1 comprises a heavy chain variable region and a light chain variable region having 6 complementarity determining regions CDRs selected from the group consisting of:
(A3) three complementarity determining regions, VH-CDRs, of the heavy chain variable region and three complementarity determining regions, VL-CDRs, of the light chain variable region:
huVH-CDR1： GFTFNKYA， SEQ ID NO.1
huVH-CDR2： IRSKYNNYAT， SEQ ID NO.2
huVH2-CDR3： HGNFGNSYISYWQY， SEQ ID NO.15
huVL-CDR1： TGAVTSGNY， SEQ ID NO.4
huVL-CDR2： GTK， SEQ ID NO.5
huVL2-CDR 3: VLWRSNRW, SEQ ID No. 16; or
(A4) Three complementarity determining regions, VH-CDRs, of the heavy chain variable region and three complementarity determining regions, VL-CDRs, of the light chain variable region:
huVH-CDR1： GFTFNKYA， SEQ ID NO.1
huVH-CDR2： IRSKYNNYAT， SEQ ID NO.2
huVH1-CDR3： HGNFGNTYISYWAY， SEQ ID NO.20
huVL-CDR1： TGAVTSGNY， SEQ ID NO.4
huVL-CDR2： GTK， SEQ ID NO.5
huVL1-CDR3： VLWYSKRW， SEQ ID NO.21；
wherein the structure of the antigen binding fragment is selected from the group consisting of: (i) a Fab fragment; (ii) f (ab')2A fragment; (iii) (ii) a fragment of Fd; (iv) (iv) an Fv fragment; (v) a scFv molecule; or (vi) a dAb fragment.
5. A recombinant protein, said recombinant protein having:
(i) the antibody of claim 1; and
(ii) optionally a tag sequence to facilitate expression and/or purification.
6. An antibody conjugate, comprising:
(a) an antibody moiety selected from the group consisting of: the antibody of claim 1, or the recombinant protein of claim 5, or a combination thereof; and
(b) a coupling moiety coupled to the antibody moiety, the coupling moiety selected from the group consisting of: a detectable label, a radionuclide, or a combination thereof.
7. Use of an active ingredient selected from the group consisting of: the antibody of claim 1, or the recombinant protein of claim 5, the antibody conjugate of claim 6, or a combination thereof, the active ingredient for:
(a) preparing a detection reagent or a kit.
8. A polynucleotide encoding a polypeptide selected from the group consisting of:
(1) the antibody of claim 1; or
(2) The recombinant protein of claim 5.
9. A vector comprising the polynucleotide of claim 8.
10. A genetically engineered host cell comprising the vector or genome of claim 9 having the polynucleotide of claim 8 integrated therein.
11. An in vitro non-diagnostic method for detecting CD3 protein in a sample, said method comprising the steps of:
(1) contacting the sample with the antibody of claim 1 in vitro;
(2) detecting the formation of an antigen-antibody complex, wherein the formation of the complex is indicative of the presence of CD3 protein in the sample.
CN202111028884.8A 2021-09-03 2021-09-03 anti-CD 3 humanized antibodies Active CN113461821B (en)

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PCT/CN2021/118264 WO2023029089A1 (en) 2021-09-03 2021-09-14 Anti-cd3 humanized antibody
EP21955620.6A EP4397685A1 (en) 2021-09-03 2021-09-14 Anti-cd3 humanized antibody

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