CN118027209A

CN118027209A - Recombinant antibodies and uses thereof

Info

Publication number: CN118027209A
Application number: CN202311720119.1A
Authority: CN
Inventors: 曹国帅; 成赢; 李洋洋; 武玉伟
Original assignee: Hefei Tiangang Immune Drugs Co ltd
Current assignee: Hefei Tiangang Immune Drugs Co ltd
Priority date: 2023-12-13
Filing date: 2023-12-13
Publication date: 2024-05-14

Abstract

The CD3 and FAP bispecific antibody disclosed by the invention has weak binding with T cells, strong binding with FAP positive cells and high killing promoting activity, effectively promotes PBMC to kill FAP positive cells, and has good anticancer activity; the bispecific antibody has reduced performance of promoting the secretion of proinflammatory cytokines and T cell activation performance, higher safety and good clinical application and drug development value.

Description

Recombinant antibodies and uses thereof

Technical Field

The invention relates to the field of biological medicine, in particular to a recombinant antibody and application thereof.

Background

Cancer is a major disease affecting human survival and development, and according to the latest data, new cancer cases 1900 Mo Zuo are about annually worldwide, cancer cases 1000 ten thousand are about annually dead, and the occurrence rate and death rate are on an increasing trend. Besides surgical incision, the traditional cancer treatment means such as chemotherapy, radiotherapy and the like have large side effects and are easy to relapse. In recent years, immunotherapy, including tumor targeting antibodies, immune checkpoint antibodies, bispecific antibodies, and the like, has become a new hotspot and hope for anticancer. Immunotherapy represented by PD-1/L1 has demonstrated great potential, but even with the currently most widely approved PD-1/L1 therapies, the overall response rate is only 30%, and still more patients cannot benefit from it, one of the main reasons being that immune checkpoint therapies are ineffective against "cold tumors". T cells recognize neo-antigens (TCR) through their surface T cell receptors, i.e. antigens mutated in tumor genes, while partial tumors have low frequency of gene mutation and a small number of neo-antigen species, called "cold tumors". Current immune checkpoint therapies, such as PD-1/L1 therapies, achieve anti-cancer goals by restoring T cell self function, whereas in "cold tumors" T cells cannot effectively recognize the tumor, resulting in immune checkpoint therapies that are ineffective against "cold tumors.

Bispecific antibodies based on CD3 (hereinafter referred to as "CD3 bispecific antibodies") promote T cell activation and killing of tumors by recruiting T cells to tumor sites, bridging T cells and tumors, and these bispecific antibodies do not require neoantigens and can guide T cells to kill "cold tumors". CD3 bispecific antibodies trigger strong activation signals upon binding to T cells and tumor cells, thus also being able to "disregard" to some extent the inhibitory signals of immune checkpoint molecules; however, CD3 bispecific antibodies also promote the production of large amounts of pro-inflammatory cytokines, such as tnfα, IL-6, etc., eliciting strong cytokine storms and excessive immune responses, causing body damage, and severely potentially life threatening. Therefore, the bispecific antibody based on CD3 has good application prospect in clinic, but the safety is still to be further improved.

One way to solve the safety problem of CD3 bispecific antibodies is to increase the affinity of the CD3 bispecific antibody for binding to tumor targets, and simultaneously decrease its affinity for CD3, thereby allowing more distribution of CD3 bispecific antibody drug to tumor sites, increasing tumor local drug concentration, decreasing peripheral drug concentration, decreasing off-target toxicity, decreasing pro-inflammatory cytokine production levels, and decreasing on-target toxicity. Therefore, there is a need to develop CD3 bispecific antibodies with better safety and higher clinical application value in clinic.

Fibroblast Activation Protein (FAP) is a type II transmembrane protein, belongs to the serine protease family, has serine proteolytic enzyme activity, participates in extracellular matrix degradation, and is related to tumor growth, metastasis and the like. FAP is not expressed in normal tissues, is highly up-regulated in various cancers, is specifically expressed on the surfaces of tumor fibroblasts, can be hydrolyzed by other proteolytic enzymes, is changed into a soluble form to exist in tissue fluid and plasma, and promotes migration and infiltration of tumor cells through extracellular matrix hydrolysis. Because FAP has the characteristic of specific expression of tumor tissues, FAP-expressing tumor fibroblasts are directly targeted and killed to destroy tumor microenvironment, or FAP antibodies are used for targeting tumor tissues to deliver medicines, so that the FAP-expressing tumor cells become hot spots for development of immunotherapeutic medicines.

In view of the above, FAP is a potential therapeutic target, and it is very valuable to develop a highly safe and specific dual antibody against cd3xfap.

Disclosure of Invention

The present invention aims to solve at least one of the technical problems in the related art to some extent.

In a first aspect, the present invention provides a recombinant antibody comprising at least:

A first binding region comprising an antibody or antigen binding fragment that specifically recognizes CD 3; and

A second binding region comprising an antibody or antigen binding fragment that specifically recognizes FAP,

Wherein the method comprises the steps of

The antibody or antigen binding fragment of the first binding region specifically recognizing CD3 comprises a scFV region, a first Fc region,

The antibody or antigen binding fragment of the second binding region that specifically recognizes FAP includes a Fab region and a second Fc region.

The inventor improves the safety of the CD3 and FAP bispecific antibody, and obtains the CD3 and FAP bispecific antibody with a new configuration through a large number of screening and test verification. Further experimental results prove that: the CD3 and FAP bispecific antibody disclosed by the invention is weak in combination with T cells, strong in combination with FAP positive cells, high in killing promoting activity, and good in anticancer activity, and can effectively promote PBMC to kill FAP positive cells; the bispecific antibody has reduced performance of promoting the secretion of proinflammatory cytokines and T cell activation performance, higher safety and good clinical application and drug development value.

According to a specific embodiment of the present invention, the C-terminus of the scFV region of the first binding region is linked to the N-terminus of the first Fc region.

According to a specific embodiment of the present invention, the C-terminus of the heavy chain variable region of the scFV region is linked to the N-terminus of the light chain variable region via a linker peptide 1 and the C-terminus of the light chain variable region is linked to the N-terminus of the first Fc region via a linker peptide 2.

According to a specific embodiment of the invention, the antibody or antigen binding fragment that specifically recognizes FAP comprises:

The sequences of the light chain variable region CDR1, CDR2 and CDR3 shown in SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO. 14 are shown as the sequences of the heavy chain variable region CDR1, CDR2 and CDR3 shown in SEQ ID NO. 9, SEQ ID NO. 10 and SEQ ID NO. 11.

According to a specific embodiment of the invention, the antibody or antigen binding fragment specifically recognizing FAP has the light chain variable region shown in SEQ ID NO. 31 or an amino acid sequence having at least 80% identity to the sequence shown in SEQ ID NO. 31 and the heavy chain variable region shown in SEQ ID NO. 32 or an amino acid sequence having at least 80% identity to the sequence shown in SEQ ID NO. 32.

According to a specific embodiment of the invention, the antibody or antigen binding fragment specifically recognizing FAP has an amino acid sequence as shown in SEQ ID NO. 20 having at least 80% identity to the light chain as shown in SEQ ID NO. 20 and a heavy chain as shown in SEQ ID NO. 19 having at least 80% identity to the sequence as shown in SEQ ID NO. 19.

According to a specific embodiment of the invention, the antibody or antigen binding fragment specifically recognizing CD3 comprises:

Light chain variable region CDR1, CDR2 and CDR3 sequences shown as SEQ ID NO. 4, GTN and SEQ ID NO.5 respectively, heavy chain variable region CDR1, CDR2 and CDR3 sequences shown as SEQ ID NO. 1, SEQ ID NO.2 and SEQ ID NO.3 respectively,

Or alternatively

Light chain variable region CDR1, CDR2 and CDR3 sequences shown as SEQ ID NO. 4, GTN and SEQ ID NO.5 respectively, heavy chain variable region CDR1, CDR2 and CDR3 sequences shown as SEQ ID NO. 1, SEQ ID NO.2 and SEQ ID NO. 6 respectively,

Or alternatively

The sequences of the light chain variable region CDR1, CDR2 and CDR3 shown in SEQ ID NO. 4, GTN and SEQ ID NO. 8 are shown as the sequences of the heavy chain variable region CDR1, CDR2 and CDR3 shown in SEQ ID NO. 1, SEQ ID NO.2 and SEQ ID NO. 7 respectively.

According to a specific embodiment of the invention, the antibody or antigen binding fragment specifically recognizing CD3 comprises any one of the following:

(a) Has a light chain variable region shown in SEQ ID NO. 26 and a heavy chain variable region shown in SEQ ID NO. 27, or

(B) Has a light chain variable region shown in SEQ ID NO. 26 and a heavy chain variable region shown in SEQ ID NO. 28, or

(C) Has a light chain variable region shown in SEQ ID NO. 29 and a heavy chain variable region shown in SEQ ID NO. 30, or

An amino acid sequence having at least 80% sequence identity compared to any one of (a), (b) and (c).

According to a specific embodiment of the invention, the first Fc region and the second Fc region are linked by a knob-intoo-hole structure.

According to a specific embodiment of the invention, at least a portion of the first and second Fc regions are derived from at least one of a murine antibody, a primate-source antibody or a mutant thereof.

According to a specific embodiment of the invention, at least a portion of the first and second Fc regions are derived from human IgG1 or a mutant thereof.

According to a specific embodiment of the present invention, the first Fc region has an amino acid sequence as shown in SEQ ID NO. 33 and the second Fc region has an amino acid sequence as shown in SEQ ID NO. 34.

According to a specific embodiment of the invention, the connecting peptides 1,2 each independently have an amino acid sequence (GGGGS) n, wherein n is an integer greater than or equal to 1, preferably 1,2,3,4, 5, 6, 7, 8, 9 or 10.

According to a specific embodiment of the present invention, the connecting peptide 1 has an amino acid sequence as shown in SEQ ID NO. 35.

According to a specific embodiment of the present invention, the connecting peptide 2 has an amino acid sequence as shown in SEQ ID NO. 36.

According to a specific embodiment of the invention, the first binding region comprises an amino acid sequence as shown in any one of SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, or an amino acid sequence having at least 80% identity to SEQ ID NO. 15, SEQ ID NO. 16 or SEQ ID NO. 17.

According to a specific embodiment of the invention, the recombinant antibody comprises any one of the following:

(d) The amino acid sequences shown as SEQ ID NO. 15, SEQ ID NO. 19 and SEQ ID NO. 20, or

(E) The amino acid sequences shown as SEQ ID NO. 16, SEQ ID NO. 19 and SEQ ID NO. 20, or

(F) Amino acid sequences as shown in SEQ ID NO. 17, SEQ ID NO. 19 and SEQ ID NO. 20, or

An amino acid sequence having at least 80% sequence identity compared to any one of (d), (e) and (f).

According to a specific embodiment of the invention, the recombinant antibody further comprises a third binding region comprising an antibody or antigen-binding fragment that specifically recognizes FAP.

According to a specific embodiment of the present invention, the antibody or antigen binding fragment of the third binding region that specifically recognizes FAP comprises a Fab region, the heavy chain CH1 region of which is linked to the scFV region of the antibody or antigen binding fragment of the first binding region that specifically recognizes CD 3.

According to a specific embodiment of the present invention, the C-terminal end of the heavy chain CH1 region of the Fab region is linked to the heavy chain variable region of the scFV region of an antibody or antigen binding fragment of the first binding region that specifically recognizes CD 3.

According to a specific embodiment of the invention, the Fab region of the antibody or antigen binding fragment of the third binding region that specifically recognizes FAP is identical or different in amino acid sequence from the Fab region of the antibody or antigen binding fragment of the second binding region that specifically recognizes FAP.

In a second aspect the invention provides an isolated polynucleotide encoding the recombinant antibody of the first aspect.

According to an embodiment of the invention, the antibody or antigen binding fragment encoded by the polynucleotide is capable of specifically binding to FAP and/or CD 3.

According to an embodiment of the invention, the nucleic acid molecule of the polynucleotide is DNA and/or RNA.

It should be noted that, for the nucleic acids mentioned in the present specification and claims, one skilled in the art will understand that either one or both of the complementary double strands are actually included. For convenience, in the present description and claims, although only one strand is shown in most cases, the other strand complementary thereto is actually disclosed. In addition, the nucleic acid sequences of the present application include DNA forms or RNA forms, one of which is disclosed, meaning the other is also disclosed.

In a third aspect the invention provides an expression vector carrying a polynucleotide according to the second aspect.

According to embodiments of the invention, the expression vector may comprise an optional control sequence operably linked to the nucleic acid molecule. Wherein the control sequences are one or more control sequences that direct expression of the nucleic acid molecule in a host. The expression vectors presented in the embodiments of the present invention can efficiently express such antibodies or antigen-binding fragments in large amounts in suitable host cells.

In a fourth aspect, the invention provides a recombinant cell carrying a polynucleotide according to the second aspect, an expression vector according to the third aspect or capable of expressing a recombinant antibody according to the first aspect.

According to an embodiment of the invention, the recombinant cell is obtained by introducing the expression vector of the fourth aspect into a host cell.

According to an embodiment of the invention, the recombinant cell is a eukaryotic cell.

According to an embodiment of the invention, the recombinant cell is a mammalian cell.

It should be noted that the recombinant cells of the present invention are not particularly limited, and may be prokaryotic cells, eukaryotic cells, or phage. The prokaryotic cell can be escherichia coli, bacillus subtilis, streptomycete or proteus mirabilis and the like. The eukaryotic cells comprise fungi such as pichia pastoris, saccharomyces cerevisiae, schizosaccharomyces, trichoderma and the like, insect cells such as armyworm and the like, plant cells such as tobacco and the like, and mammalian cells such as BHK cells, CHO cells, COS cells, myeloma cells and the like. In some embodiments, the recombinant cells of the invention are preferably mammalian cells, including BHK cells, CHO cells, NSO cells, or COS cells, and do not include animal germ cells, fertilized eggs, or embryonic stem cells.

According to an embodiment of the invention, the expression efficiency of the recombinant antibody is higher when the cell is a eukaryotic cell, such as a mammalian cell.

In a fifth aspect, the invention provides a composition comprising at least one of the recombinant antibody according to the first aspect, the polynucleotide according to the second aspect, the expression vector according to the third aspect, and the recombinant cell according to the fourth aspect.

According to the embodiment of the invention, the recombinant antibody can effectively promote PBMC to kill FAP positive cells and has anticancer activity; can produce fewer pro-inflammatory cytokines, has higher safety, and has good clinical application value and drug development value. Thus, the obtained medicament can be further used for preventing and/or treating CD3 and/or FAP mediated related diseases.

According to an embodiment of the present invention, the recombinant antibody can effectively bind to FAP protein and CD3 and effectively inhibit proliferation of tumor cells, and thus, a composition comprising the same can also effectively bind to FAP and CD3 protein, has good effect of preventing and/or treating FAP-mediated diseases, and the kind of the composition is not particularly limited, and may be a food composition or a pharmaceutical composition.

The compositions of the invention may also be administered in combination with each other, or with one or more other therapeutic compounds, for example, with a chemotherapeutic agent. Thus, the composition may also contain a chemotherapeutic agent. The antibodies, or antigen-binding fragments thereof, or immunoconjugates of the invention may also be combined with a second therapeutic agent, exemplary agents of which include, but are not limited to, other agents that inhibit FAP activity (including other antibodies or antigen-binding fragments thereof, peptide inhibitors, small molecule antagonists, etc.) and/or agents that interfere with FAP upstream or downstream signal transduction.

It is noted that the compositions include combinations that are separated in time and/or space, so long as they are capable of co-acting to achieve the objects of the present invention. For example, the ingredients contained in the composition may be administered to the subject in whole or separately. When the components contained in the composition are separately administered to a subject, the individual components may be administered to the subject simultaneously or sequentially.

In a sixth aspect, the invention provides a method of producing a recombinant antibody according to the first aspect, comprising culturing a recombinant cell according to the fourth aspect, and isolating an antibody or antigen-binding fragment of the recombinant cell.

The seventh aspect of the present invention provides a medicament comprising at least one of the recombinant antibody of the first aspect, the polynucleotide of the second aspect, the expression vector of the third aspect, the recombinant cell of the fourth aspect, and the composition of the fifth aspect.

According to an eighth aspect of the present invention, there is provided a kit comprising at least one of the recombinant antibody according to the first aspect, the polynucleotide according to the second aspect, the expression vector according to the third aspect, and the recombinant cell according to the fourth aspect.

The ninth aspect of the present invention provides the use of a recombinant antibody according to the first aspect, a polynucleotide according to the second aspect, an expression vector according to the third aspect, a recombinant cell according to the fourth aspect or a composition according to the fifth aspect in the manufacture of a medicament for the prevention and/or treatment of FAP mediated related diseases.

According to an embodiment of the invention, the FAP-mediated related disease comprises a tumor, the FAP being positive in tumor-associated fibroblasts of the tumor.

According to an embodiment of the invention, the tumor-induced disease comprises lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, renal cancer, gastric cancer, melanoma, esophageal cancer, oral squamous cell carcinoma or head and neck cancer.

In a tenth aspect the invention provides a kit for detecting FAP and/or CD3 in a sample, the kit comprising at least one of a recombinant antibody according to the first aspect, a polynucleotide according to the second aspect, an expression vector according to the third aspect, a recombinant cell according to the fourth aspect.

According to embodiments of the present invention, kits comprising the antibodies or antigen binding fragments are effective for qualitative or quantitative detection of FAP protein and/or CD 3. The kit provided by the invention can be used for immunoblotting, immunoprecipitation and the like, and relates to a kit for detection by utilizing the specific binding performance of human FAP and antibodies. These kits may comprise any one or more of the following: an antagonist, an anti-FAP antibody, an anti-CD 3 antibody, or a drug reference material; a protein purification column; immunoglobulin affinity purification buffers; cell assay diluent; instructions, literature, etc. The anti-FAP antibodies and/or CD3 can be used in different types of diagnostic tests, for example, the presence of various diseases or drugs, toxins or other proteins can be detected in vitro or in vivo, for example, the presence of related diseases can be detected by detecting serum or blood of a subject, and scientific research can be performed, and the FAP protein in a sample to be detected can be detected by using the kit. Such related diseases may include FAP related diseases, such as cancer. Of course, the antibodies or antigen binding fragments provided herein may also be used in radioimmunoassays, radioimmunotherapy, and the like for the above-described diseases. For the above application scenario, the binding molecules are equally applicable and will not be described here.

According to an eleventh aspect of the invention there is provided the use of a recombinant antibody according to the first aspect, a polynucleotide according to the second aspect, an expression vector according to the third aspect, a recombinant cell according to the fourth aspect in the preparation of a kit for the detection of FAP and/or CD3.

In a twelfth aspect the invention provides an immunoconjugate comprising a therapeutic agent and the recombinant antibody of the first aspect coupled to the therapeutic agent.

According to an embodiment of the invention, the therapeutic agent comprises any one of a polypeptide, a radionuclide, a small molecule.

Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

Drawings

The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:

FIG. 1 is a schematic diagram of a CD3 FAP bispecific antibody of example 1 of the present invention;

FIG. 2 is a graph showing the results of flow cytometry for binding of antibodies to CD 8T cells according to example 2 of the present invention, wherein A in FIG. 2 is the binding ratio of antibodies to CD 8T cells, and B in FIG. 2 is the average fluorescence intensity;

FIG. 3 is a graph showing the results of flow cytometry for binding of antibodies to CD 8T cells of Cross3 XAHF 213391:1, cross313 XAHF 21339, cross316 XAHF 21339, and Cross325 XAHF 21339 of example 2 of the present invention, wherein A in FIG. 3 is the ratio of binding of antibodies to CD 8T cells and B in FIG. 3 is the average fluorescence intensity;

FIG. 4 is a graph showing the results of flow cytometry for binding of antibodies to A-375-FAP cells according to example 2 of the present invention, wherein A in FIG. 4 is the ratio of binding of antibodies to A-375-FAP cells, and B in FIG. 4 is the average fluorescence intensity;

FIG. 5 is a graph showing the results of flow cytometry for antibody binding to A-375-FAP cells of Cross3×AHF213391:1, cross313×AHF21339, cross316×AHF21339, cross325×AHF21339 of example 2 of the present invention, wherein A in FIG. 5 is the ratio of antibody binding to A-375-FAP cells and B in FIG. 5 is the average fluorescence intensity;

FIG. 6 is a graph showing the results of promoting spontaneous activation of secreted cytokines by PBMC in the absence of target protein (FAP) using antibodies Cross3×AHF213391:1 and UCHT1×AHF213391:1 in example 3 of the present invention, wherein A in FIG. 6 represents the secretion of IL-2, B in FIG. 6 represents the secretion of IFN- γ, C in FIG. 6 represents the secretion of IL-6, and D in FIG. 6 represents the secretion of TNFα;

FIG. 7 is a graph showing the results of promoting IL-6 secretion by PBMC from Cross3 XAHF 213391:1, cross313 XAHF 21339, cross316 XAHF 21339, and Cross325 XAHF 21339 antibodies in example 3 of the present invention;

FIG. 8 is a graph showing the results of promoting spontaneous activation of secreted cytokines by PBMC in the presence of target protein (FAP) in example 3 of the present invention, wherein A in FIG. 8 represents the secretion result of IL-2 and B in FIG. 8 represents the secretion result of IFN-gamma;

FIG. 9 is a graph showing the results of promoting IL-2 secretion by Cross3 XAHF 213391:1, cross313 XAHF 21339, cross316 XAHF 21339, and Cross325 XAHF 21339 antibodies in example 3 of the present invention in the co-incubation of PBMC with A375-FAP;

FIG. 10 is a graph showing the results of promoting IFN-. Gamma.secretion by Cross3 XAHF 213391:1, cross313 XAHF 21339, cross316 XAHF 21339, and Cross325 XAHF 21339 antibodies in example 3 of the present invention in the co-incubation of PBMC with A375-FAP;

FIG. 11 is a graph showing the results of promoting TNFα secretion by Cross3×AHF213391:1, cross313×AHF21339, cross316×AHF21339, and Cross325×AHF21339 antibodies in example 3 of the present invention in the co-incubation of PBMC with A375-FAP;

FIG. 12 is a graph showing the results of promoting IL-6 secretion by Cross3 XAHF 213391:1, cross313 XAHF 21339, cross316 XAHF 21339, and Cross325 XAHF 21339 antibodies in example 3 of the present invention in the co-incubation of PBMC with A375-FAP;

FIG. 13 is a graph showing the results of flow cytometry for promoting T cell proliferation by antibodies of examples 4 of the present invention, wherein A in FIG. 13 represents activation of CD4T cells by antibodies, and B in FIG. 13 represents activation of CD 8T cells by antibodies, wherein Cross 3X AHF213391:1, cross 3X AHF213392:1, UCHT 1X AHF213391:1, UCHT 1X AHF 213392:1;

FIG. 14 shows that antibodies of example 4 of the present invention, cross3×AHF213391:1, cross313×AHF21339, cross316×AHF21339, cross325×AHF21339, all promote CD 4T cell proliferation;

FIG. 15 shows that antibodies of example 4 of the present invention, cross3×AHF213391:1, cross313×AHF21339, cross316×AHF21339, cross325×AHF21339, all promote CD 8T cell proliferation;

FIG. 16 is a graph showing the results of promoting PBMC killing of A-375-FAP melanoma cells by antibodies to Cross3×AHF213391:1, cross3×AHF213392:1, UCHT1×AHF213391:1, UCHT1×AHF213392:1 in example 5 of the present invention;

FIG. 17 is a graph showing the results of the antibodies to Cross 3X AHF21339, cross 313X AHF21339, cross 316X AHF21339, and Cross 325X AHF21339 in example 5 of the present invention promoting the killing of A-375-FAP melanoma cells by PBMC;

FIG. 18 is a graph showing ELISA results for binding of antibodies to FAP protein by Cross 3X AHF21339, cross 313X AHF21339, cross 316X AHF21339, and Cross 325X AHF21339 in example 6 of the present invention;

FIG. 19 is a graph showing the results of ELISA for Cross3 XAHF 21339, cross313 XAHF 21339, cross316 XAHF 21339, and Cross325 XAHF 21339 antibodies binding to CD3E & D protein (CD 3E & CD 3D) in example 6 of the present invention;

FIG. 20 is a graph showing the results of promoting CD 4T cells to express CD25 by antibodies to Cross3×AHF213391:1, cross313×AHF21339, cross316×AHF21339, and Cross325×AHF21339 in example 7 of the present invention;

FIG. 21 is a graph showing the results of promoting CD 8T cells to express CD25 by antibodies to Cross3×AHF213391:1, cross313×AHF21339, cross316×AHF21339, and Cross325×AHF21339 in example 7 of the present invention;

FIG. 22 is a graph showing the results of promoting CD 4T cells to express CD69 by antibodies to Cross3×AHF213391:1, cross313×AHF21339, cross316×AHF21339, and Cross325×AHF21339 in example 7 of the present invention;

FIG. 23 is a graph showing the results of promoting CD69 expression by CD 8T cells using antibodies to Cross 3X AHF213391:1, cross 313X AHF21339, cross 316X AHF21339, and Cross 325X AHF21339 in example 7 of the present invention.

Detailed Description

Embodiments of the present invention are described in detail below. The following examples are illustrative only and are not to be construed as limiting the invention.

It should be noted that the terms "first," "second," and "second" are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implying a number of technical features being indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature. Further, in the description of the present invention, unless otherwise indicated, the meaning of "a plurality" is two or more.

The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.

In order that the invention may be more readily understood, certain technical and scientific terms are defined below. Unless clearly defined otherwise herein in this document, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The abbreviations for amino acid residues are standard 3-letter and/or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

In this document, the terms "comprise" or "include" are used in an open-ended fashion, i.e., to include what is indicated by the present invention, but not to exclude other aspects.

In this document, the terms "optionally," "optional," or "optionally" generally refer to the subsequently described event or condition may, but need not, occur, and the description includes instances in which the event or condition occurs, as well as instances in which the event or condition does not.

Antibodies or antigen binding fragments of the invention are typically prepared by biosynthetic methods. The coding nucleic acids according to the invention can be prepared by various known methods, conveniently by the person skilled in the art, based on the nucleotide sequences according to the invention. Such as, but not limited to: PCR, DNA synthesis, etc., and specific methods can be found in J.Sam Brookfield, guidelines for molecular cloning experiments. As one embodiment of the present invention, the coding nucleic acid sequence of the present invention can be constructed by a method of synthesizing nucleotide sequences in segments followed by overlap extension PCR. Wherein the antibody or antigen fragment is numbered and defined using the Kabat numbering system. Herein, the term "antibody" is an immunoglobulin molecule capable of binding to a specific antigen. Comprising two light chains of relatively light molecular weight and two heavy chains of relatively heavy molecular weight, the heavy (H) and light (L) chains being linked by disulfide bonds to form a tetrapeptide chain molecule. Among them, the amino-terminal (N-terminal) amino acid sequence of the peptide chain varies greatly, called variable region (V region), and the carboxyl-terminal (C-terminal) is relatively stable, and varies little, called constant region (C region). The V chains of the L chain and H chain are referred to as VL and VH, respectively. Certain regions in the variable region have a higher degree of variation in amino acid composition and arrangement sequence, referred to as hypervariable regions (Hypervariable region, HVR), which are the sites of antigen and antibody binding and are therefore also referred to as determinant-DETERMINING REGION (CDR). The heavy chain variable region and the light chain variable region each have three CDR regions.

Antibodies of the invention include murine antibodies, chimeric antibodies, humanized antibodies, preferably humanized antibodies.

As used herein, the term "antigen-binding fragment" or "antibody fragment" refers generally to an antigen-binding antibody fragment, which may include a portion of an intact antibody, typically an antigen-binding or variable region, examples of which include Fab, fab ', F (ab') 2, fv or scFv, diabodies, linear antibodies, single chain antibody molecules, and the like.

As used herein, the term "mutant" or "variant" may refer to a molecule comprising a mutation of one or more nucleotides or amino acids of any naturally occurring or engineered molecule.

As used herein, the term "chimeric antibody" refers to a recombinant antibody obtained by replacing the constant region amino acid sequence of a monoclonal antibody from one species (e.g., mouse) with the constant region of an antibody from another species (e.g., human) using recombinant DNA technology.

Herein, the term "humanized antibody" (humanized antibody) refers to a recombinant antibody obtained by replacing all of the non-CDR (Fv Framework Region (FR)) amino acid sequences of the constant and variable regions of a monoclonal antibody from one species (e.g., mouse) with the non-CDR amino acid sequences of the constant and variable regions of an antibody from another species (e.g., human) using recombinant DNA technology. That is, when the constant region of one antibody is humanized, it is called a chimeric antibody, and when the non-CDR amino acid sequences of the constant and variable regions are all humanized, it is called a humanized antibody.

Herein, the "full length antibody" is a tetrapeptide chain structure formed by connecting two identical light chains and two identical heavy chains through inter-chain disulfide bonds, such as immunoglobulin G (IgG), immunoglobulin a (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD), or immunoglobulin E (IgE). The same class of immunoglobulins can also be of different subclasses, e.g., igG1, igG2, igG3, igG4, depending on the amino acid composition. Immunoglobulin light chains are classified as either kappa chains or lambda chains depending on the constant region.

As used herein, the term "antibody affinity maturation" (antiboby affinity maturation) refers to a state of immune function that is normally present in the body. In humoral immunity, the average affinity of antibodies produced by the secondary response is higher than that of the primary immune response, a phenomenon known as antibody affinity maturation. During affinity maturation of natural antibodies, somatic high frequency mutations are mainly concentrated in the CDR regions. By performing single point saturation mutation at each site of the CDR region through in vitro experiments, enough mutation diversity is obtained, and meanwhile, the protein structure is not destroyed, and the in vitro reproduction of the high-frequency mutation of somatic cells in vivo, which is most similar to that of a natural antibody, can be realized by the method. Single-point saturation mutation is carried out on each amino acid site of the CDR region, and an unbiased single-point saturation mutation plasmid library of the parent antibody is constructed. ELISA is used for screening mutation hot spots with enhanced specific binding with antigen, and then the mutation hot spots are combined and screened to obtain candidate antibody mutation sequences.

In this context, the term "tumor-associated fibroblasts" (cancer-associated fibroblasts, CAFs) is one of the most important components in the tumor microenvironment, playing an essential role in the development and progression of tumors. Bone marrow and adipose localized tissue resident fibroblasts and mesenchymal stem cells are the primary precursor cells of CAFs origin. Numerous studies have shown that CAFs does not exist as a single cell around a tumor, but rather interacts with tumor cells, promoting tumor growth and survival and maintaining its malignant propensity.

As used herein, the term "operably linked" refers to the linkage of a foreign gene to a vector such that control elements within the vector, such as transcription and translation control sequences, and the like, are capable of performing their intended functions of regulating transcription and translation of the foreign gene. In the case of attaching the above-mentioned nucleic acid molecule to a vector, the nucleic acid molecule may be directly or indirectly attached to a control element on the vector, as long as the control element is capable of controlling translation, expression, etc. of the nucleic acid molecule. Of course, these control elements may be directly from the carrier itself or may be exogenous, i.e. not from the carrier itself. It will be appreciated by those skilled in the art that the nucleic acid molecules encoding the antibodies or antigen binding fragments may be inserted separately into different vectors, typically into the same vector. The usual vectors may be, for example, plasmids, phages and the like. Such as Plasmid-X.

In this context, the term "conservatively modified form of an amino acid sequence, such as" refers to an amino acid modification that does not significantly affect or alter the binding properties of an antibody comprising the amino acid sequence of that amino acid, including amino acid substitutions, additions and deletions. Modifications may be introduced into the antibodies of the invention by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are substitutions in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been identified in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

In this context, the term "identity" is used to describe the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences when compared to the amino acid sequence or nucleic acid sequence of a reference sequence, using conventional methods, e.g., see, ausubel et al, eds. (1995), current Protocols in Molecular Biology, chapter 19 (Greene Publishing and Wiley-Interscience, new York); and ALIGN program (Dayhoff(1978),Atlas ofProtein Sequence and Structure 5：Suppl.3(National Biomedical Research Foundation,Washington,D.C.). there are many algorithms for aligning sequences and determining sequence identity, including, needleman et al (1970) J.mol. Biol.48:443 homology comparison algorithm; smith et al (1981) adv.appl.Math.2:482, a local homology algorithm; pearson et al (1988) Proc.Natl. Acad.Sci.85:2444 similarity search method; computer programs utilizing the Smith-Waterman algorithm (Meth. Mol. Biol.70:173-187 (1997)), and BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al (1990) J. Mol. Biol. 215:403-410)) are also available and include, but are not limited to, ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al, meth. Enzyme, 266:460-480 (1996)); or GAP, BESTFIT, BLASTAltschul et al, supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, 8 th edition, madison, wisconsin, USA, and CLUSTAL in the PC/Gene program provided by Intelligenetics, mountain View, california.

The antibody "Cross 3X AHF213391:1" is herein identical to "Cross 3X AHF21339".

According to particular embodiments of the invention, one skilled in the art may replace, add and/or delete one or more (e.g., 1,2,3, 4, 5, 6, 7, 8, 9 or 10 or more) amino acids to the sequences of the invention to obtain variants of the sequences of the antibodies or functional fragments thereof without substantially affecting the activity of the antibodies (retaining at least 95% of the activity). They are all considered to be included within the scope of the present invention. Such as substitution of amino acids with similar properties in the variable region. The sequences of the variants of the invention may have at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity (or homology) to a reference sequence. Sequence identity as described herein can be measured using sequence analysis software. Such as computer programs BLAST, in particular BLASTP or TBLASTN, using default parameters. The amino acid sequences mentioned in the present invention are all shown in N-terminal to C-terminal fashion. It will be appreciated by those skilled in the art that the CDR sequences analyzed by different databases may vary, but that such variations are intended to be within the scope of the present invention.

According to particular embodiments of the invention, the antibodies of the invention may be full length (e.g., igG1 or IgG4 antibodies) or may comprise only antigen binding portions (e.g., fab, F (ab') 2, or scFv fragments), or may be modified to affect function. The invention includes anti-CD 3 antibodies having modified glycosylation patterns. In some applications, it may be useful to modify to remove undesired glycosylation sites, or antibodies in which no fucose moiety is present on the oligosaccharide chain, for example to enhance Antibody Dependent Cellular Cytotoxicity (ADCC) function. In other applications, galactosylation modifications may be made to alter Complement Dependent Cytotoxicity (CDC). After a series of modifications, the fragments of the invention still have CD3 binding activity. Preferably, the functional fragment will consist of or comprise a partial sequence of the heavy chain variable region or the light chain variable region of its source antibody, which partial sequence is sufficient to retain the same binding specificity and sufficient affinity as its source antibody, preferably at least equal to 1/100, more preferably at least equal to 1/10, for CD 3. Such functional fragments will comprise a minimum of 5 amino acids, preferably 10, 15, 25, 50 and 100 consecutive amino acids of the antibody sequence from which they are derived.

Recombinant antibodies

The present invention provides a recombinant antibody comprising at least:

first binding region: the first binding region comprises an antibody or antigen binding fragment that specifically recognizes CD 3; and

Second binding region: the second binding region comprises an antibody or antigen binding fragment that specifically recognizes FAP,

Wherein the method comprises the steps of

According to a specific embodiment of the present invention, the C-terminus of the heavy chain variable region of the scFV region is linked to the N-terminus of the light chain variable region by a linker peptide 1 and the C-terminus of the light chain variable region is linked to the N-terminus of the first Fc region by a linker peptide 2.

Or alternatively

According to one embodiment of the present invention, there is provided a recombinant protein comprising three polypeptide chains: the first polypeptide chain comprises a heavy chain variable region of a CD3 antibody, a light chain variable region of a CD3 antibody and a first Fc region from the N end to the C end; the second polypeptide chain comprises a heavy chain of a FAP antibody; the third polypeptide chain comprises the light chain of the FAP antibody.

According to one embodiment of the present invention, there is provided a recombinant protein comprising three polypeptide chains: the first polypeptide chain comprises, in order from the N-terminus to the C-terminus, a heavy chain variable region of a CD3 antibody (SEQ ID NO:27, SEQ ID NO:28 or SEQ ID NO: 30), a light chain variable region of a CD3 antibody (SEQ ID NO:26, SEQ ID NO: 29), and a first Fc region (SEQ ID NO: 33); the second polypeptide chain comprises the heavy chain of the FAP antibody (SEQ ID NO: 19); the third polypeptide chain comprises the light chain of the FAP antibody (SEQ ID NO: 20).

According to a specific embodiment of the invention, the amino acid sequence of the first polypeptide chain is shown as SEQ ID NO. 15, SEQ ID NO. 16 or SEQ ID NO. 17, the amino acid sequence of the second polypeptide chain is shown as SEQ ID NO. 19, and the amino acid sequence of the third polypeptide chain is shown as SEQ ID NO. 20.

According to one embodiment of the present invention, there is provided a recombinant protein comprising four polypeptide chains: the first polypeptide chain comprises a light chain of a FAP antibody; the second polypeptide chain comprises a heavy chain variable region of a CD3 antibody, a CH1 region of the CD3 antibody, a heavy chain variable region of the CD3 antibody, a light chain variable region of the CD3 antibody and a first Fc region from the N end to the C end in sequence; the second polypeptide chain comprises a heavy chain of a FAP antibody; the third polypeptide chain comprises the light chain of the FAP antibody.

According to one embodiment of the present invention, there is provided a recombinant protein comprising four polypeptide chains: the first polypeptide chain comprises the light chain of the FAP antibody (SEQ ID NO: 20); the second polypeptide chain comprises, in order from the N-terminus to the C-terminus, a heavy chain variable region of the FAP antibody (SEQ ID NO: 32), a CH1 region of the FAP antibody, a heavy chain variable region of the CD3 antibody (SEQ ID NO:27, SEQ ID NO:28 or SEQ ID NO: 30), a light chain variable region of the CD3 antibody (SEQ ID NO:26, SEQ ID NO: 29), and a first Fc region (SEQ ID NO: 33); the second polypeptide chain comprises the heavy chain of the FAP antibody (SEQ ID NO: 19); the third polypeptide chain comprises the light chain of the FAP antibody (SEQ ID NO: 20).

According to the embodiment of the disclosure, 2 kinds of CD3-FAP bispecific antibodies with different configurations are screened, even though antigen binding domains are the same, the antigen binding capacities among the antibodies with different configurations are different, and one purpose of the invention is to screen a set of CD3-FAP bispecific antibodies with better effect of promoting immune cells to kill tumors, which comprises three recombinant proteins with polypeptide chains: a recombinant protein comprising three polypeptide chains: the first polypeptide chain comprises a heavy chain variable region of a CD3 antibody, a light chain variable region of a CD3 antibody and a first Fc region from the N end to the C end; the second polypeptide chain comprises a heavy chain of a FAP antibody; the third polypeptide chain comprises the light chain of the FAP antibody. The antibody not only has better capability of promoting immune cells to kill HCC827 cells of non-small cell lung cancer, but also has better effect in killing other B7H7 positive cells.

According to the embodiment of the invention, FAP is specifically expressed on the surface of tumor fibroblast, belongs to a part of tumor microenvironment, and achieves the purpose of resisting cancer by destroying the tumor microenvironment. It should be noted that the anti-FAP antibody provided by the present invention may act on tumors expressing FAP in tumor fibroblasts in any tumor microenvironment, where the tumors are not limited, and may be any one or more of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, melanoma, glioma, renal cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

Nucleic acid molecules, recombinant vectors, immunoconjugates

In preparing or obtaining these antibodies, nucleic acid molecules expressing these antibodies may be used, linked to different vectors, and then expressed in different cells to obtain the corresponding antibodies.

Thus, the present invention also provides an isolated nucleic acid encoding the above antibody or antigen-binding fragment thereof, and recombinant vectors and transformants containing the nucleic acid. The nucleic acid molecule encodes the antibody or antigen binding fragment thereof described above, preferably the nucleic acid is an expression cassette obtained by genetic engineering means.

Recombinant vectors may be referred to as cloning vectors, or as expression vectors, and may be obtained by operably linking the nucleic acid to commercially available vectors (e.g., plasmid or viral vectors), commonly used plasmids include pSeTag2, PEE14, pMH3, and the like.

In some preferred embodiments, the nucleic acid molecule is species optimized for expression in mammalian cells.

The invention also provides an expression vector comprising the isolated nucleic acid molecule described above. In the case of ligating the above isolated polynucleotide to a vector, the polynucleotide may be directly or indirectly ligated to a control element on the vector, as long as the control element is capable of controlling translation, expression, etc. of the polynucleotide. Of course, these control elements may be directly from the carrier itself or may be exogenous, i.e. not from the carrier itself. Of course, the polynucleotide may be operably linked to a control element.

The immunoconjugates provided herein comprise a therapeutic agent coupled to an antibody or antigen-binding fragment thereof as described previously. The manner in which the antibody or antigen-binding fragment thereof is conjugated to the therapeutic agent may be in a conventional manner.

The present invention provides compositions comprising an antibody or antigen-binding fragment thereof as described above, and/or an immunoconjugate as described above, and a pharmaceutically acceptable carrier. In certain embodiments, the compositions comprise combinations that are separated in time and/or space, so long as they are capable of co-acting to achieve the objects of the invention. For example, the ingredients contained in the composition may be administered to the subject in whole or separately. When the components contained in the composition are separately administered to a subject, the individual components may be administered to the subject simultaneously or sequentially.

Cells

The present invention provides a cell. The cells carry the aforementioned nucleic acids or the aforementioned vectors or transformants, or express the aforementioned bispecific antibodies. According to an embodiment of the present invention, the cells are obtained by transfecting or transforming the vector or transformant, and the cells can efficiently express the aforementioned bispecific antibody under appropriate conditions.

According to an embodiment of the invention, the cell is a prokaryotic cell, a eukaryotic cell or a phage.

According to an embodiment of the invention, the prokaryotic cell is E.coli, bacillus subtilis, streptomyces or Proteus mirabilis.

According to an embodiment of the invention, the eukaryotic cell is a fungus, an insect cell, a plant cell or a mammalian cell.

According to an embodiment of the invention, the fungus is pichia pastoris, saccharomyces cerevisiae, schizosaccharomyces, or trichoderma.

According to an embodiment of the invention, the insect cell is a meadow myxoplasma cell; according to an embodiment of the invention, the plant cell is a tobacco plant cell; according to an embodiment of the invention, the mammalian cell is a BHK cell, CHO cell, COS cell, myeloma cell or human embryonic kidney 293 cell; and does not include animal germ cells, fertilized eggs, or embryonic stem cells.

According to an embodiment of the invention, the cell is a mammalian cell.

According to an embodiment of the invention, the cell is a BHK cell, CHO cell, COS cell or NSO cell.

The term "suitable conditions" as used herein refers to conditions suitable for expression of the antibodies or antigen-binding fragments thereof of the present application. Those skilled in the art will readily appreciate that conditions suitable for expression of an antibody or antigen-binding fragment thereof include, but are not limited to, suitable transformation or transfection means, suitable transformation or transfection conditions, healthy host cell status, suitable host cell density, suitable cell culture environment, suitable cell culture time. The "suitable conditions" are not particularly limited, and those skilled in the art can optimize the conditions for optimal expression of the recombinant antibody according to the specific environment of the laboratory.

Pharmaceutical composition

The invention provides a pharmaceutical composition. The pharmaceutical composition comprises: the bispecific antibody, the nucleic acid, the vector or transformant, or the cell. According to the embodiment of the invention, the bispecific antibody can effectively promote PBMC to kill tumor cells and has anticancer activity; can produce fewer pro-inflammatory cytokines, has higher safety, and has good clinical application value and drug development value. Thus, the obtained medicament can be further used for preventing and/or treating CD3 and/or FAP mediated related diseases.

According to an embodiment of the invention, further comprising pharmaceutically acceptable excipients.

According to an embodiment of the present invention, the auxiliary materials include: one or more pharmaceutically acceptable excipients, diluents, stabilizers or carriers.

According to an embodiment of the invention, the pharmaceutical composition is an injection.

It is noted that the pharmaceutical composition includes combinations that are separated in time and/or space, as long as they can co-act to achieve the object of the present invention. For example, the ingredients contained in the composition may be administered to the subject in whole or separately. When the components contained in the composition are separately administered to a subject, the individual components may be administered to the subject simultaneously or sequentially.

The medicine contains safe and effective amount of the active ingredients and pharmaceutically acceptable auxiliary materials. Such excipients include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. Generally, the pharmaceutical preparation is matched with the administration mode, and the dosage forms of the medicine are injection, oral preparation (tablet, capsule and oral liquid), transdermal agent and sustained release agent. For example, by using physiological saline or an aqueous solution containing glucose and other auxiliary agents by conventional methods. The medicament is preferably manufactured under aseptic conditions.

The effective amount of the active ingredient described herein may vary depending upon the mode of administration, the severity of the condition being treated, and the like. The selection of the preferred effective amount can be determined by one of ordinary skill in the art based on a variety of factors (e.g., by clinical trials). Such factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life etc.; the severity of the disease to be treated in the patient, the weight of the patient, the immune status of the patient, the route of administration, etc. For example, separate doses may be administered several times per day, or the dose may be proportionally reduced, as dictated by the urgent need for the treatment of the condition.

Pharmaceutically acceptable excipients described herein include (but are not limited to): water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptides, cellulose, nanogels, or combinations thereof. The choice of carrier should be compatible with the mode of administration and will be well known to those of ordinary skill in the art.

Kit for detecting a substance in a sample

The invention provides a kit. The kit comprises the bispecific antibody, the nucleic acid molecule, the vector or transformant or the cell. According to the embodiment of the invention, the kit can be combined with CD3 protein and/or FAP protein, and can effectively identify the CD3 protein and/or FAP protein.

As described above, the bispecific antibody of the present invention can specifically bind to CD3 and FAP, and the CD3 protein and/or FAP protein-related kit developed by using this property can be used for CD3 protein and/or FAP protein-related research, such as for detecting and/or enriching and/or separating and purifying CD3 protein and/or FAP protein of human or other mammals.

The kit can effectively detect, enrich or separate and purify the CD3 protein and/or the FAP protein in the biological sample, and is further used for scientific research, such as qualitative or quantitative detection of CD3 protein and/or FAP protein molecules in the biological sample. More specifically, it can be used for immunoblotting, immunoprecipitation, etc. involving a kit for detection using the specific binding properties of CD3 protein and/or FAP protein and antibody, etc. These kits may comprise any one or more of the following: antagonists, bispecific antibodies of the invention, or drug reference materials; a protein purification column; immunoglobulin affinity purification buffers; assay of cells diluent. Bispecific antibodies of the invention can be used in different types of diagnostic tests, for example, to detect the presence of a wide variety of diseases or drugs, toxins or other proteins, etc., in vitro or in vivo. For example, the test may be used to test for CD3 and/or FAP mediated related diseases by testing the serum or blood of a subject.

Use in the preparation of a kit

The present invention provides the use of the aforementioned bispecific antibody, the aforementioned nucleic acid molecule, the aforementioned vector or transformant or the aforementioned cell for the preparation of a kit for the detection of CD3 and/or FAP.

As previously described, the bispecific antibodies of embodiments of the present invention are capable of specifically binding to CD3 and FAP, and thus, the bispecific antibodies can be used to detect CD3 and/or FAP. Further, it can be used for preparing a CD3 and/or FAP related kit and for scientific research, such as qualitative or quantitative detection of CD3 and/or FAP protein molecules in biological samples. More specifically, it can be used for immunoblotting, immunoprecipitation, etc. involving a kit for detection using the specific binding properties of CD3 and/or FAP and an antibody, etc. These kits may comprise any one or more of the following: antagonists, bispecific antibodies of the invention, or drug reference materials; a protein purification column; immunoglobulin affinity purification buffers; assay of cells diluent. Bispecific antibodies of the invention can be used in different types of diagnostic tests, for example, to detect the presence of a wide variety of diseases or drugs, toxins or other proteins, etc., in vitro or in vivo. For example, the test may be used to test for CD3 and/or FAP mediated related diseases by testing the serum or blood of a subject.

Use in the preparation of a medicament

The present invention provides the use of the aforementioned bispecific antibody, the aforementioned nucleic acid molecule, the aforementioned vector or transformant, the aforementioned cell or the aforementioned pharmaceutical composition for the preparation of a medicament for the prevention and/or treatment of a CD3 and/or FAP mediated related disease. According to the embodiment of the invention, the bispecific antibody and the corresponding nucleic acid, vector or transformant or the pharmaceutical composition can be further prepared into medicines which can be clinically used for preventing or treating related diseases mediated by CD3 and/or FAP.

According to an embodiment of the invention, the CD3 mediated related disease comprises an autoimmune disease.

According to an embodiment of the invention, the autoimmune disease comprises at least one of the following: systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, autoimmune thyroid diseases, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, syndrome of lung hemorrhagic nephritis, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple cerebral spinal sclerosis, and acute idiopathic polyneuritis.

According to an embodiment of the invention, the FAP-mediated related disease is cancer, a disease caused by transplant rejection, an autoimmune disease, an infectious disease.

According to an embodiment of the invention, the cancer is at least one of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, renal cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma and head and neck cancer.

Method for treating disease

The present invention provides a method for preventing and/or treating CD3 and/or FAP mediated diseases. According to an embodiment of the invention, the method comprises: administering to a subject a pharmaceutically acceptable amount of the bispecific antibody of the foregoing, the nucleic acid molecule of the foregoing, the foregoing vector or transformant, the foregoing cell or the foregoing pharmaceutical composition.

It is noted that the terms "subject," "individual," and "patient" are used interchangeably herein to refer to a mammal being evaluated for treatment and/or being treated. In one embodiment, the mammal is a human. The terms "subject," "individual," and "patient" include, but are not limited to, individuals with cancer, individuals with autoimmune diseases, individuals with pathogen infection, and the like. The subject may be a human, but also includes other mammals, particularly mammals that may be used as laboratory models of human diseases, such as mice, rats, and the like.

The effective amount of the antibodies or antigen-binding fragments, conjugates, nucleic acids, vectors or transformants thereof or pharmaceutical compositions of the present invention may vary depending on the mode of administration and the severity of the disease to be treated, etc. The selection of the preferred effective amount can be determined by one of ordinary skill in the art based on a variety of factors (e.g., by clinical trials). Such factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life etc.; the severity of the disease to be treated in the patient, the weight of the patient, the immune status of the patient, the route of administration, etc. For example, separate doses may be administered several times per day, or the dose may be proportionally reduced, as dictated by the urgent need for the treatment of the condition.

In some embodiments, the CD 3-mediated related disease comprises an autoimmune disease.

In some embodiments, the autoimmune disease comprises at least one of the following: systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, autoimmune thyroid diseases, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, syndrome of lung hemorrhagic nephritis, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple cerebral spinal sclerosis, and acute idiopathic polyneuritis.

In some embodiments, the FAP-mediated disease is cancer, a disease caused by transplant rejection, an autoimmune disease, an infectious disease.

In some embodiments, the cancer is at least one of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, renal cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

Nucleic acids encoding the heavy and/or light chains of the antibodies of the invention are within the scope of the invention, and corresponding nucleic acid sequences can be readily obtained by the skilled artisan based on the amino acid sequences of the heavy and/or light chains, as shown in table 1.

TABLE 1

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The aspects of the present disclosure will be explained below with reference to examples. Those skilled in the art will appreciate that the following examples are illustrative of the present disclosure and should not be construed as limiting the scope of the present disclosure. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Example 1 production of antibodies

The specific experimental procedure for antibody production is as follows: (1) ExpiCHO cells (from Thermo Fisher) were cultured using ExpiCHO Expression Medium medium (from Thermo Fisher) and cell concentration was adjusted to 6X 10 ⁶/mL to obtain ExpiCHO cell solution. (2) When the diabody is in the a configuration (a in fig. 1), pcdna3.4 vector (delegated nanjing masui synthesis) containing CD3 antibody, FAP antibody heavy chain, FAP antibody light chain was used in a mass ratio of 1:1:1 in a ratio of 2mL OptiSFM medium (available from Thermo Fisher) to obtain solution a; or when the diabody is in the B configuration (B in fig. 1), pcdna3.4 vector containing FAP heavy chain-CD 3 antibody, FAP antibody heavy chain, FAP antibody light chain (delegated nanjing masui synthesis) was prepared according to 1:1:1 was added to 2mL OptiSFM medium (available from Thermo Fisher) to obtain solution a. (3) 160 μ L ExpiFectamineCHO transfection reagent (from Thermo Fisher) was added to 2mL OptiSFM medium (from Thermo Fisher) to obtain solution b. (4) Solution a and solution b were then mixed to obtain a transfection mixture, and the transfection mixture was added to the 50mL ExpiCHO cell solution in its entirety over 5 minutes. (5) After 1 day of incubation at 37℃under 5% CO ₂, 8mL of Feed, 300 μ L ENHANCER (available from Thermo Fisher) was added and the culture supernatant was harvested after 9 days of incubation at 32℃under 5% CO ₂, with 8mL of Feed added on day 5. (6) The target antibody was obtained by affinity purification from the culture supernatant using a Protein A purification column (from Nami).

In this example, 7 bispecific antibodies of CD3 and FAP in two configurations were prepared together to examine the properties of the bispecific antibodies of the present invention, the specific information of the 7 bispecific antibodies is shown in Table 2, wherein the A, B antibody configuration is shown in FIG. 1

TABLE 2

The 7 bispecific antibodies were respectively: cross3×AHF213391:1 (scFv+Fc amino acid sequence of CD3 is shown as SEQ ID NO: 18), FAP antibody heavy chain amino acid sequence is shown as SEQ ID NO:19, FAP antibody light chain amino acid sequence is shown as SEQ ID NO:22, FAP antibody light chain amino acid sequence is shown as SEQ ID NO: 19), cross3×AHF213392:1 (FAP antibody heavy chain variable region+CH 1 region and CD3 antibody scFv+Fc amino acid sequence is shown as SEQ ID NO:21, FAP antibody heavy chain amino acid sequence is shown as SEQ ID NO:19, and FAP antibody light chain amino acid sequence is shown as SEQ ID NO: 20), UCHT1×AHF213391:1 (scFv+Fc amino acid sequence of CD3 antibody is shown as SEQ ID NO:22, FAP antibody light chain amino acid sequence is shown as SEQ ID NO: 19), UCHT1×AHF213392:1 (FAP antibody heavy chain can be shown as SEQ ID NO:21, FAP chain amino acid sequence is shown as SEQ ID NO: 213P antibody heavy chain amino sequence is shown as SEQ ID NO: 16), FAP antibody heavy chain amino acid sequence is shown as SEQ ID NO: 213P antibody heavy chain amino sequence is shown as SEQ ID NO:16, FAP antibody heavy chain is shown as SEQ ID NO:20 Cross325×AHF21339 (scFv+Fc amino acid sequence of CD3 antibody shown in SEQ ID NO:17, FAP antibody heavy chain amino acid sequence shown in SEQ ID NO:19, and FAP antibody light chain amino acid sequence shown in SEQ ID NO: 20).

Example 2 antibody flow cytometry binding experiments

Flow cytometry experiments were used to detect the binding properties of bispecific antibodies, bispecific antibodies were added to cells, and the intensity of the signal after antibody addition was used to determine the binding properties of the antibodies and cells.

(1) PBMC were diluted to 2X 10 ⁶/ml with PBS and added to a 1.5ml EP tube at a volume of 100. Mu.l/tube, 10. Mu.l/tube goat serum was added thereto and blocked at 4℃for 30min. Gradient diluted bispecific antibody Cross3×AHF213391:1、Cross3×AHF213392:1、UCHT1×AHF213391:1、UCHT1×AHF213392:1、Cross313×AHF21339、Cross316×AHF21339、Cross325×AHF21339、 was added to control hIgG1LALA (purchased from Baiying organism) and incubated at 4℃for 30min. To the EP tube, 1ml of PBS was added, centrifuged at 3500rpm X5 min at 4℃and the supernatant was discarded and washed once with PBS. After centrifugation, the supernatant was discarded, and the cells were resuspended in 100. Mu.l/tube PBS, to which were added 1. Mu.l/tube Alexa-647 labeled goat anti-human IgG antibody secondary antibody (purchased from Jackson lab) and 0.5. Mu.l/tube PerCP-Cy5.5 labeled anti-human CD8 antibody, and incubated at 4℃for 30min in the absence of light. Washed twice with PBS and the supernatant was discarded after centrifugation. Cells were resuspended in 200 μl/tube PBS and examined by flow cytometry.

The binding strength and binding activity of 7 bispecific antibodies to CD 8T cells are shown in FIG. 2 and FIG. 3, wherein FIG. 2 shows that Cross3×AHF213391:1, cross3×AHF213392:1, UCHT1×AHF213391:1, UCHT1×AHF213392:1 antibodies are capable of binding to CD 8T cells, and B-configuration diabodies are less capable of binding to T cells than A-configuration diabodies, and FAP-based on Cross3 is less active than FAP-based on UCHT1 diabodies; FIG. 3 shows that Cross3 XAHF 213391:1, cross313 XAHF 21339, cross316 XAHF 21339, cross325 XAHF 21339 antibodies were able to bind CD 8T cells, ordered in Cross3 XAHF 213391:1, cross313 XAHF 21339, cross316 XAHF 21339, cross325 XAHF 21339 according to binding strength to T cells.

(2) Preparation of A-375-FAP cells: HEK293T cells were plated in six well plates at 5 x 10 ⁵ cells/well and incubated overnight with DMEM medium without diabodies. The medium was discarded before transfection and 1ml of fresh DMEM medium without diabody was added. pLVX-EF1a-FAP-IRES-puro (assigned to Nanjing gold-srey synthesis), pLVX-EF1a-IRES-puro vector (purchased from Ubbelo organism) was added to 200. Mu.l serum-free DMEM medium at a ratio of 2:1:1, then 12. Mu.g polyetherimide (PEI, purchased from Polysciences) was added, after mixing, the whole solution was added to six well plates with HEK293T cells, after 6h of culture, the medium was discarded, fresh complete DMEM medium was added, after 48h of transfection, cell culture supernatant was collected, and 0.45. Mu.m filter (purchased from Millipore) was used, i.e., virus supernatant was added to 1X 35A-containing 1:35, and finally, to a fresh medium (assigned to 3 ml) was added to the final well plates with fresh 37-35. Mu.5 ml of Polysciences), and then, after 6h of culture medium was removed.

A-375-FAP tumor cells were diluted to 2X 10 ⁶/ml with PBS and added to 1.5ml EP tube at a volume of 100. Mu.l/tube, 10. Mu.l/tube goat serum was added thereto and blocked at 4℃for 30min. Gradient diluted bispecific antibody Cross3×AHF213391:1、Cross3×AHF213392:1、UCHT1×AHF213391:1、UCHT1×AHF213392:1、Cross313×AHF21339、Cross316×AHF21339、Cross325×AHF21339、 was added to control hIgG1LALA (purchased from Baiying organism) and incubated at 4℃for 30min. To the EP tube, 1ml of PBS was added, centrifuged at 3500rpm X5 min at 4℃and the supernatant was discarded and washed once with PBS. After centrifugation, the supernatant was discarded, and the cells were resuspended in 100. Mu.l/tube PBS, to which 1. Mu.l/tube Alexa-647 labeled goat anti-human IgG antibody secondary antibody (purchased from Jackson lab) was added and incubated at 4℃for 30min in the absence of light. Washed twice with PBS and the supernatant was discarded after centrifugation. Cells were resuspended in 200 μl/tube PBS and examined by flow cytometry.

The binding strength and binding activity of 7 bispecific antibodies to A-375-FAP cells are shown in FIG. 4 and FIG. 5, wherein FIG. 4 shows that Cross3×AHF213391:1, cross3×AHF213392:1, UCHT1×AHF213391:1, UCHT1×AHF213392:1 antibodies are all capable of binding to A-375-FAP cells, and that the B-type diabody has lower activity (EC 50) to bind FAP than the A-type diabody; FIG. 5 shows that Cross3 XAHF 213391:1, cross313 XAHF 21339, cross316 XAHF 21339, cross325 XAHF 21339 antibodies were all capable of binding A-375-FAP cells and that the activity (EC 50) was comparable for all diabodies.

Example 3 experiments with bispecific antibodies to promote secretion of cytokines by PBMC

(1) Bispecific antibodies promote cytokine secretion by PBMC in the absence of target proteins (tumor cells free)

Adding a bispecific antibody into the PBMC culture system, culturing for 48 hours, collecting culture supernatant, and detecting the content of cytokines in the supernatant for judging the release characteristics of the cytokines triggered by the bispecific antibody under the condition of no target protein.

(A) Bispecific antibodies Cross3×AHF213391:1, UCHT1×AHF213391:1, cross313×AHF21339, cross316×AHF21339, cross325×AHF21339, control hIgG1LALA (from Baiying organism) were serially diluted in 96 well plates at 100 μl/well;

(b) PBMCs (purchased from siren) were grown using complete RPMI 1640 medium

Chimaphila) was diluted to 1X 10 ⁶/ml and added to a 96-well plate at 100. Mu.l/well;

(c) Culturing the 96-well plate in a 5% CO ₂ incubator at 37 ℃ for 48 hours;

(d) Centrifuging at room temperature of 300g×10min, and collecting cell culture supernatant;

(e) The supernatant was assayed for cytokine content using the CBA kit (purchased from BD).

The results are shown in FIGS. 6 and 7, where PBMC from each of the test groups in FIG. 6 were derived from the same donor, and PBMC from each of the test groups in FIG. 7 were derived from the same donor, and the PBMC from each of the test groups in FIG. 6 and FIG. 7 were derived from different donors.

When the donor of PBMC was identical, FIG. 6 shows that antibodies to Cross3×AHF213391:1 and UCHT1×AHF213391:1 do not cause spontaneous activation of PBMC in the absence of target protein (FAP), with some safety.

When the donor of PBMC is identical, FIG. 7 shows that the Cross313 XAHF 21339, cross316 XAHF 21339, cross325 XAHF 21339 antibodies do not cause spontaneous activation of PBMC in the absence of target protein (FAP), whereas Cross3 XAHF 213391:1 triggers release of pro-inflammatory cytokine IL-6 at high concentrations, indicating that the safety of Cross313 XAHF 21339, cross316 XAHF 21339, cross325 XAHF 21339 is higher than that of Cross3 XAHF 213391:1.

(2) Bispecific antibodies promote cytokine secretion by PBMC in the Presence of target proteins (tumor cells)

Adding the bispecific antibody into the co-incubation system of the PBMC and the A-375-FAP melanoma cells, culturing for 48 hours, collecting culture supernatant, and detecting the cytokine content in the supernatant to judge the characteristic of the bispecific antibody for triggering cytokine release.

(A) A-375-FAP cells were diluted to 1X 10 ⁵/ml with complete RPMI 1640 medium, added to 96-well plates and incubated in a 5% CO ₂ incubator at 37℃for 24h;

(b) Bispecific antibody Cross3×AHF213391:1、Cross3×AHF213392:1、UCHT1×AHF213391:1、UCHT1×AHF213392:1、Cross313×AHF21339、Cross316×AHF21339、Cross325×AHF21339、 was gradient diluted against hIgG1LALA (purchased from Baiying organism) in complete RPMI 1640 medium and added to a 96-well plate at 20 μl/well;

(c) PBMC (from Chimaphila) were diluted to 1.25X10 ⁶/ml with complete RPMI 1640 medium and added to 96-well plates at 80 μl/well;

(d) Culturing the 96-well plate in a 5% CO ₂ incubator at 37 ℃ for 48 hours;

(e) Centrifuging at room temperature of 300g×10min, and collecting cell culture supernatant;

(f) The supernatant was assayed for cytokine content using the CBA kit (purchased from BD).

The results are shown in FIGS. 8-12, wherein FIG. 8 shows that Cross3×AHF213391:1, cross3×AHF213392:1, UCHT1×AHF213391:1, UCHT1×AHF213392:1 antibodies are capable of activating PBMC in the presence of target protein (FAP), causing it to secrete cytokines IL-2, IFN-gamma, IL-6 and TNF-alpha, and that the activity of Cross3 diabodies is superior to UCHT1 diabodies, and that Cross3×AHF213391:1 activity is optimal; FIG. 9 shows that Cross3×AHF213391:1, cross313×AHF21339, cross316×AHF21339, and Cross325×AHF21339 antibodies are capable of activating PBMC and secreting IL-2 in the presence of target protein (FAP); FIG. 10 shows that Cross3×AHF213391:1, cross313×AHF21339, cross316×AHF21339, and Cross325×AHF21339 antibodies are capable of activating PBMC and secreting IFN-gamma in the presence of target protein (FAP); FIG. 11 shows that Cross3×AHF213391:1, cross313×AHF21339, cross316×AHF21339, and Cross325×AHF21339 antibodies are capable of activating PBMC and secreting TNFα in the presence of target protein (FAP); FIG. 12 shows that Cross3×AHF213391:1, cross313×AHF21339, cross316×AHF21339, and Cross325×AHF21339 antibodies are capable of activating PBMC and secreting the pro-inflammatory cytokine IL-6 in the presence of target protein (FAP).

In conclusion, the antibodies of Cross313×AHF21339, cross316×AHF21339 and Cross325×AHF21339 have higher safety and can also maintain higher tumor killing ability.

Example 4 experiments on bispecific antibodies to promote T cell proliferation

And (3) marking CFSE fluorescein by the PBMC cells, adding a bispecific antibody into a PBMC and A-375-FAP cell co-incubation system, culturing for 72 hours, and detecting CFSE fluorescence intensity in the CD 4T cells and the CD 8T cells by using a flow cytometry method for judging the characteristics of the bispecific antibody for inducing T cell proliferation.

(1) A-375-FAP cells were diluted to 1X 10 ⁵/ml using complete RPMI 1640 medium and added to 96-well plates;

(2) Bispecific antibody Cross3×AHF213391:1、Cross3×AHF213392:1、UCHT1×AHF213391:1、UCHT1×AHF213392:1、Cross313×AHF21339、Cross316×AHF21339、Cross325×AHF21339、 was gradient diluted against hIgG1LALA (purchased from Baiying organism) in complete RPMI 1640 medium and added to a 96-well plate at 20 μl/well;

(3) PBMCs were labeled with 5 μm CFSE, and after labeling, PBMCs (purchased from fitted organisms) were diluted to 1.25×10 ⁶/ml with complete RPMI 1640 medium and added to 96-well plates at 80 μl/well;

(4) Culturing the 96-well plate in a 5% CO ₂ incubator at 37 ℃ for 72 hours;

(5) PerCP-Cy5.5 labeled CD8 antibody and BV605 labeled CD4 antibody were added and incubated at 4℃for 30min in the absence of light.

(6) Washed twice with PBS and the supernatant was discarded after centrifugation.

(7) Cells were resuspended in 200 μl/tube PBS and examined by flow cytometry.

The results are shown in FIGS. 13-15, wherein FIG. 13 shows that Cross3 XAHF 213391:1, cross3 XAHF 213392:1, UCHT1 XAHF 213391:1, UCHT1 XAHF 213392:1 antibodies promote CD4 and CD 8T cell activation, and that Cross3 diabody activity is superior to UCHT1 diabody, and Cross3 XAHF 213391:1 activity is optimal; FIG. 14 shows that antibodies to Cross3×AHF213391:1, cross313×AHF21339, cross316×AHF21339, and Cross325×AHF21339 all promote CD 4T cell proliferation; FIG. 15 shows that antibodies to Cross3×AHF213391:1, cross313×AHF21339, cross316×AHF21339, and Cross325×AHF21339 all promote CD 8T cell proliferation.

Example 5: bispecific antibody-promoted PBMC killing FAP positive cell experiment

The ability of the bispecific antibody to promote killing of the A-375-FAP human melanoma cells by PBMC was tested.

(A) Adding complete RPMI-1640 medium into a 16-hole RTCA plate according to the volume of 50 mu L/hole, and calibrating on the machine;

(b) Diluting the A-375-FAP tumor cells to 2X 10 ⁵/mL with a complete RPMI-1640 medium, adding the diluted A-375-FAP tumor cells to the RTCA plate obtained in the step (1) according to the volume of 50 mu L/hole, and detecting the cell coefficients for 24 hours at 37 ℃ under the condition of 5% CO ₂ by using xCELLigence RTCAMP equipment;

(c) Gradient diluted bispecific antibody Cross3×AHF213391:1、Cross3×AHF213392:1、UCHT1×AHF213391:1、UCHT1×AHF213392:1、Cross313×AHF21339、Cross316×AHF21339、Cross325×AHF21339、 was added to the RTCA plate obtained in step (b) in a volume of 20 μl/well against hIgG1LALA (purchased from the english organism) using complete RPMI-1640 medium;

(d) Diluting PBMC (from Chimaphila) to 1.25X10 ⁶/ml with complete RPMI-1640 medium, adding to the RTCA plate obtained in step (c) in a volume of 80. Mu.l/well;

(e) The reaction system obtained in step (d) was subjected to cell factor detection at 37℃with 5% CO ₂ using xCELLigence RTCAMP apparatus for 24 hours.

As shown in FIG. 16, cross3×AHF213391:1, cross3×AHF213392:1, UCHT1×AHF213391:1, UCHT1×AHF213392:1 antibodies were able to promote PBMC killing A-375-FAP cells, the 1:1 configuration diabody was more potent than the 2:1 diabody, cross3 diabody was superior to UCHT1 diabody, and Cross3×AHF213391:1 had the highest killing promoting activity.

As shown in FIG. 17, the antibodies Cross3×AHF213391:1, cross313×AHF21339, cross316×AHF21339 and Cross325×AHF21339 all promoted PBMC killing of A-375-FAP cells, and the antibodies Cross313×AHF21339, cross316×AHF21339 and Cross325×AHF21339 all promoted complete killing with similar killing activity, but weaker than Cross3×AHF 213391:1.

Example 6: ELISA binding assay for antibodies of the invention

ELISA experiments were used to detect the binding properties of bispecific antibodies. The antigen protein is coated in a 96-well plate, and the intensity of the signal after the antibody is added is used for judging the binding property of the bispecific antibody and the antigen protein.

(1) FAP-His protein (available from Acro) was diluted to 2. Mu.g/ml with PBS buffer, added to 96-well plates at a volume of 100. Mu.l/well, and left overnight at 4 ℃. PBS buffer in 96-well plates was aspirated, and after 6 washes with PBST (pH 7.2 PBS containing 0.1% Tween 20), 200. Mu.l/well PBS/10% BSA was added and incubated for 2h at 37℃for blocking. After removing the blocking solution and washing the plate 6 times with PBST, 100. Mu.l/well of the bispecific antibody to be tested, cross3 XAHF 213391:1, cross313 XAHF 21339, cross316 XAHF 21339, cross325 XAHF 21339, control hIgG1LALA (from Baiying organism) diluted with a gradient of PBST/0.05% BSA, were added and incubated for 1h at 37 ℃. After the reaction system was removed and the plate was washed 6 times with PBST, HRP (horseradish peroxidase) -labeled anti-human IgG antibody secondary antibody (purchased from Jacksonlab) was diluted with 100. Mu.l/well of PBST/0.05% BSA and incubated at 37℃for 1 hour. After washing the plate 6 times with PBST, 80. Mu.l/well TMB (tetramethylbenzidine) was added, incubated at room temperature for 3min, and the reaction was stopped by adding 80. Mu.l/well 4M sulfuric acid. The absorbance was read with a microplate reader at 450 mm.

As a result, as shown in FIG. 18, the antibodies Cross3×AHF213391:1, cross313×AHF21339, cross316×AHF21339, and Cross325×AHF21339 were all capable of binding to FAP protein and were similar in binding ability.

(2) CD3E & D protein (from Acro) was diluted to 2. Mu.g/ml with PBS buffer, added to 96-well plates at a volume of 100. Mu.l/well, and left overnight at 4 ℃. PBS buffer in 96-well plates was aspirated, and after 6 washes with PBST (pH 7.2 PBS containing 0.1% Tween 20), 200. Mu.l/well PBS/10% BSA was added and incubated for 2h at 37℃for blocking. After removing the blocking solution and washing the plate 6 times with PBST, 100. Mu.l/well of the bispecific antibody to be tested, cross3 XAHF 213391:1, cross313 XAHF 21339, cross316 XAHF 21339, cross325 XAHF 21339, control hIgG1LALA (from Baiying organism) diluted with a gradient of PBST/0.05% BSA, were added and incubated for 1h at 37 ℃. After the reaction system was removed and the plate was washed 6 times with PBST, HRP (horseradish peroxidase) -labeled anti-human IgG antibody secondary antibody (available from Southern bioech) was diluted with 100. Mu.l/well of PBST/0.05% BSA and incubated at 37℃for 1h. After washing the plate 6 times with PBST, 80. Mu.l/well TMB (tetramethylbenzidine) was added, incubated at room temperature for 3min, and the reaction was stopped by adding 80. Mu.l/well 4M sulfuric acid. The absorbance was read with a microplate reader at 450 mm.

As a result, as shown in FIG. 19, the antibodies of the present invention, cross 3X AHF213391:1, cross 313X AHF21339, cross 316X AHF21339, and Cross 325X AHF21339, were all capable of binding CD3E & D protein, and the antibodies of Cross 313X AHF21339, cross 316X AHF21339, and Cross 325X AHF21339 were less active than Cross 3X AHF213391:1 in binding CD 3.

Example 7: bispecific antibody-promoted T cell activation assay

Adding a bispecific antibody into a PBMC and A-375-FAP cell co-incubation system, culturing for 48 hours, and detecting the expression of CD 4T cells and CD 8T cell surface activation markers CD25 and CD69 by using a flow cytometry method to judge the characteristic of the bispecific antibody for triggering T cell activation.

(2) Bispecific antibodies Cross3×AHF213391:1, cross313×AHF21339, cross316×AHF21339, cross325×AHF21339, control hIgG1LALA (purchased from Baiying organism) were gradient diluted in 96 well plates, 20 μl/well;

(3) PBMC (from Chimaphila) were diluted to 1.25X106/ml using complete RPMI 1640 medium and added to 96 well plates at 80. Mu.l/well;

(4) Culturing the 96-well plate in a 5% CO ₂ incubator at 37 ℃ for 48 hours;

(5) CD4, CD8, CD25, CD69 antibodies were added and incubated at 4 ℃ for 30min protected from light.

(7) Cells were resuspended in 200 μl/tube PBS and examined by flow cytometry.

The results are shown in FIGS. 20-23, wherein FIG. 20 shows that Cross3×AHF213391:1, cross313×AHF21339, cross316×AHF21339, and Cross325×AHF21339 antibodies all promote CD 4T cell activation and express CD25; FIG. 21 shows that Cross3 XAHF 213391:1, cross313 XAHF 21339, cross316 XAHF 21339, and Cross325 XAHF 21339 antibodies all promote CD 8T cell activation and express CD25; FIG. 22 shows that Cross3 XAHF 213391:1, cross313 XAHF 21339, cross316 XAHF 21339, and Cross325 XAHF 21339 antibodies all promote CD 4T cell activation and express CD69; FIG. 23 shows that antibodies to Cross3×AHF213391:1, cross313×AHF21339, cross316×AHF21339, and Cross325×AHF21339 all promote activation of CD 8T cells and express CD69.

The experimental result shows that the bispecific antibody can be combined with CD3 and FAP, so that T cell activation and cytokine secretion are promoted, PBMC (tumor cell killing) is effectively promoted, and the bispecific antibody has good anticancer activity; the bispecific antibody of the invention can realize high affinity binding with FAP, low affinity binding with CD3 and higher anticancer activity. In conclusion, the bispecific antibody disclosed by the invention can promote immune cells to resist cancer, has good anti-cancer activity, higher safety and good clinical application value and drug development value.

In the description of the present specification, the descriptions of the terms "one embodiment," "some embodiments," "examples," "particular examples," "some embodiments," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.

While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.

Claims

1. A recombinant antibody comprising at least:

Wherein the method comprises the steps of

2. The recombinant antibody according to claim 1, wherein the C-terminus of the scFV region in the first binding region is linked to the N-terminus of the first Fc region;

Optionally, the C-terminus of the heavy chain variable region of the scFV region is linked to the N-terminus of the light chain variable region by a linker peptide 1 and the C-terminus of the light chain variable region is linked to the N-terminus of the first Fc region by a linker peptide 2.

3. The recombinant antibody according to claim 1, wherein the antibody or antigen-binding fragment that specifically recognizes FAP comprises:

The sequences of the light chain variable regions CDR1, CDR2 and CDR3 shown in SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO. 14 are shown as the sequences of the heavy chain variable regions CDR1, CDR2 and CDR3 shown in SEQ ID NO. 9, SEQ ID NO. 10 and SEQ ID NO. 11;

Optionally, the antibody or antigen binding fragment that specifically recognizes FAP has a light chain variable region as set forth in SEQ ID NO. 31 or an amino acid sequence that is at least 80% identical to the sequence set forth in SEQ ID NO. 31 and a heavy chain variable region as set forth in SEQ ID NO. 32 or an amino acid sequence that is at least 80% identical to the sequence set forth in SEQ ID NO. 32;

Optionally, the antibody or antigen binding fragment that specifically recognizes FAP has an amino acid sequence that is at least 80% identical to the light chain shown in SEQ ID NO. 20 or to the sequence shown in SEQ ID NO. 20, and a heavy chain shown in SEQ ID NO. 19 or to the sequence shown in SEQ ID NO. 19.

4. The recombinant antibody according to claim 1, wherein the antibody or antigen-binding fragment that specifically recognizes CD3 comprises:

Or alternatively

5. The recombinant antibody according to any one of claims 1-4, wherein said antibody or antigen-binding fragment that specifically recognizes CD3 comprises any one of the following:

6. The recombinant antibody according to claim 1, wherein the first Fc region and the second Fc region are linked by a knob-intor-hole structure;

Optionally, at least a portion of the first and second Fc regions are derived from at least one of a murine antibody, a primates antibody, or a mutant thereof;

Optionally, at least a portion of the first and second Fc regions are from human IgG1 or a mutant thereof;

optionally, the first Fc region has an amino acid sequence as shown in SEQ ID NO. 33 and the second Fc region has an amino acid sequence as shown in SEQ ID NO. 34.

7. Recombinant antibody according to claim 2, characterized in that the connecting peptides 1,2 each independently have an amino acid sequence (GGGGS) n, wherein n is an integer greater than or equal to 1, preferably 1,2, 3, 4, 5, 6, 7, 8, 9 or 10;

optionally, the connecting peptide 1 has an amino acid sequence as shown in SEQ ID NO. 35;

Optionally, the linker peptide 2 has the amino acid sequence shown as SEQ ID NO. 36.

8. The recombinant antibody according to claim 1, wherein said first binding region comprises an amino acid sequence as set forth in any one of SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 17, or an amino acid sequence having at least 80% identity to SEQ ID No. 15, SEQ ID No. 16 or SEQ ID No. 17.

9. The recombinant antibody according to claim 1, wherein the recombinant antibody comprises any one of the following:

10. The recombinant antibody according to claim 1, further comprising a third binding region comprising an antibody or antigen-binding fragment that specifically recognizes FAP.

11. The recombinant antibody according to claim 10, wherein said antibody or antigen-binding fragment of said third binding region that specifically recognizes FAP comprises a Fab region, the heavy chain CH1 region of said Fab region being linked to the scFV region of said antibody or antigen-binding fragment of said first binding region that specifically recognizes CD 3.

12. The recombinant antibody according to claim 11, wherein the C-terminal end of the heavy chain CH1 region of said Fab region is linked to the heavy chain variable region of the scFV region of an antibody or antigen binding fragment of said first binding region that specifically recognizes CD 3;

Optionally, the Fab region of the antibody or antigen binding fragment of the third binding region that specifically recognizes FAP is identical to or different from the amino acid sequence of the Fab region of the antibody or antigen binding fragment of the second binding region that specifically recognizes FAP.

13. An isolated polynucleotide encoding the recombinant antibody of any one of claims 1-12.

14. An expression vector carrying the polynucleotide of claim 13.

15. A recombinant cell carrying the polynucleotide of claim 13 or the expression vector of claim 14; or (b)

A recombinant antibody according to any one of claims 1 to 12 capable of being expressed.

16. The recombinant cell according to claim 15, wherein the recombinant cell is obtained by introducing the expression vector of claim 14 into a host cell;

optionally, the recombinant cell is a eukaryotic cell;

optionally, the recombinant cell is a mammalian cell.

17. A composition comprising at least one of the recombinant antibody of any one of claims 1-12, the polynucleotide of claim 13, the expression vector of claim 14, and the recombinant cell of claim 15 or 16.

18. A method of producing the recombinant antibody of any one of claims 1-12, comprising culturing the recombinant cell of claim 15 or 16 and isolating the antibody or antigen-binding fragment of the recombinant cell.

19. A medicament comprising at least one of the recombinant antibody of any one of claims 1-12, the polynucleotide of claim 13, the expression vector of claim 14, the recombinant cell of claim 15 or 16, the composition of claim 17.

20. A kit comprising at least one of the recombinant antibody of any one of claims 1-12, the polynucleotide of claim 13, the expression vector of claim 14, the recombinant cell of claim 15 or 16.

21. Use of the recombinant antibody of any one of claims 1-12, the polynucleotide of claim 13, the expression vector of claim 14, the recombinant cell of claim 15 or 16, or the composition of claim 17 in the manufacture of a medicament for the prevention and/or treatment of FAP-mediated related diseases;

optionally, the FAP-mediated related disease comprises a tumor, the tumor having positive FAP in tumor-associated fibroblasts of the tumor;

Optionally, the tumor-derived disease comprises lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, renal cancer, gastric cancer, esophageal cancer, melanoma, oral squamous cell carcinoma, or head and neck cancer.

22. A kit for detecting FAP and/or CD3 in a sample, comprising at least one of the recombinant antibody of any one of claims 1-12, the polynucleotide of claim 13, the expression vector of claim 14, the recombinant cell of claim 15 or 16.

23. Use of the recombinant antibody of any one of claims 1-12, the polynucleotide of claim 13, the expression vector of claim 14, the recombinant cell of claim 15 or 16 in the preparation of a kit for detecting FAP and/or CD3.

24. An immunoconjugate comprising a therapeutic agent and the recombinant antibody of any one of claims 1-12 conjugated to the therapeutic agent.

25. The immunoconjugate of claim 24, wherein the therapeutic agent comprises any one of a polypeptide, a radionuclide, a small molecule.