Recombinant antibody and use thereof

Abstract

The invention relates to the field of biomedicine, and specifically relates to a recombinant antibody and a use thereof. A CD3 and FAP bispecific antibody binds weakly to T cells and binds strongly to FAP-positive cells, and has high killing activity, effectively enhancing the killing of FAP-positive cells by PBMCs and demonstrating good anticancer activity. In addition, the bispecific antibody has reduced ability to promote secretion of pro-inflammatory cytokines and T-cell activation, thereby improving safety.

Description

Recombinant antibodies and their applications

[0001] Priority information

[0002] This application claims priority to the Chinese patent application with application number 202311720119.1 filed with the Chinese Patent Office on December 13, 2023, entitled “Recombinant Antibodies and Their Applications,” the entire contents of which are incorporated by reference into this application. Technical Field

[0003] The present invention relates to the field of biomedicine, and in particular to recombinant antibodies and applications thereof. Background Art

[0004] Cancer is a major disease that affects human survival and development. According to the latest data, approximately 19 million new cancer cases and 10 million cancer deaths occur worldwide each year, and both incidence and mortality rates are on the rise. Besides surgical resection, traditional cancer treatments such as chemotherapy and radiotherapy are associated with significant side effects and a high risk of recurrence. In recent years, immunotherapy, including tumor-targeted antibodies, immune checkpoint antibodies, and bispecific antibodies, has become a new hotspot and a new hope in the fight against cancer. Immunotherapy, exemplified by PD-1 / L1, has demonstrated tremendous potential. However, even the PD-1 / L1 therapy, currently the most widely approved, has an overall response rate of only 30%, leaving many patients unable to benefit. One major reason for this is that immune checkpoint therapy is ineffective against "cold tumors." T cells recognize neoantigens (neoantigens)—antigens expressed by tumor gene mutations—through the T cell receptors (TCRs) on their surface. However, some tumors have a low frequency of genetic mutations and a limited number of neoantigens, making them "cold tumors." Current immune checkpoint therapies, such as PD-1 / L1 therapy, achieve anti-cancer goals by restoring the function of T cells themselves. However, in "cold tumors", T cells cannot effectively recognize tumors, resulting in the ineffectiveness of immune checkpoint therapy for "cold tumors".

[0005] CD3-based bispecific antibodies (hereinafter referred to as "CD3 bispecific antibodies") recruit T cells to reach local tumors, bridge T cells and tumors, promote T cell activation, and kill tumors. This type of bispecific antibody does not require neoantigens and can guide T cells to kill "cold tumors." After binding to T cells and tumor cells, CD3 bispecific antibodies trigger strong activation signals, so they can also "ignore" the inhibitory signals of immune checkpoint molecules to a certain extent; however, CD3 bispecific antibodies also promote the production of a large number of pro-inflammatory cytokines, such as TNFα, IL-6, etc., triggering a strong cytokine storm and excessive immune response, causing damage to the body, and severe cases may be life-threatening. Therefore, CD3-based bispecific antibodies have good application prospects in clinical practice, but their safety needs to be further improved.

[0006] One approach to addressing the safety concerns of CD3 bispecific antibodies is to increase their affinity for tumor targets while simultaneously reducing their affinity for CD3. This allows for greater local distribution of CD3 bispecific antibodies to the tumor, increasing local tumor drug concentrations while reducing peripheral drug concentrations, reducing off-target toxicity, lowering the production of pro-inflammatory cytokines, and reducing on-target toxicity. Therefore, clinically, there is an urgent need to develop CD3 bispecific antibodies with improved safety and greater clinical application value.

[0007] Fibroblast activation protein (FAP) is a type II transmembrane protein belonging to the serine protease family. It possesses serine protease activity, participates in extracellular matrix degradation, and is associated with tumor growth and metastasis. FAP is not expressed in normal tissues but is highly upregulated in various cancers. It is specifically expressed on the surface of tumor fibroblasts and can be hydrolyzed by other proteases, becoming soluble in tissue fluid and plasma. It promotes tumor cell migration and infiltration through extracellular matrix hydrolysis. Due to FAP's tumor tissue-specific expression, directly targeting FAP-expressing tumor fibroblasts to disrupt the tumor microenvironment, or using FAP antibodies to deliver drugs to tumor tissue, has become a hot topic in immunotherapy drug development.

[0008] In summary, FAP is a potential therapeutic target, and it is very valuable to develop a CD3×FAP bispecific antibody with high safety and specificity. Summary of the Invention

[0009] The present invention aims to solve one of the technical problems in the related art at least to a certain extent.

[0010] 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 CD3; and a second binding region, comprising an antibody or antigen-binding fragment that specifically recognizes FAP; wherein the antibody or antigen-binding fragment that specifically recognizes CD3 in the first binding region comprises an scFV region and a first Fc region, and the antibody or antigen-binding fragment that specifically recognizes FAP in the second binding region comprises a Fab region and a second Fc region.

[0011] To improve the safety of recombinant CD3 and FAP antibodies, the present invention has developed a novel CD3 and FAP bispecific antibody through extensive screening and experimental verification. Further experimental results demonstrate that the CD3 and FAP bispecific antibody of the present invention weakly binds to T cells but strongly binds to FAP-positive cells, exhibits high cytotoxicity, and can effectively promote the killing of FAP-positive cells by PBMCs, demonstrating excellent anti-cancer activity. Furthermore, this bispecific antibody exhibits reduced pro-inflammatory cytokine secretion and T cell activation, resulting in enhanced safety and promising clinical application and drug development value.

[0012] According to an embodiment of the present invention, the above-mentioned recombinant antibody may further include at least one of the following additional technical features:

[0013] According to a specific embodiment of the present invention, the C-terminus of the scFV region in the first binding region is connected to the N-terminus of the first Fc region.

[0014] According to a specific embodiment of the present invention, the C-terminus of the heavy chain variable region of the scFV region is connected to the N-terminus of the light chain variable region through connecting peptide 1, and the C-terminus of the light chain variable region is connected to the N-terminus of the first Fc region through connecting peptide 2.

[0015] According to a specific embodiment of the present invention, the antibody or antigen-binding fragment that specifically recognizes FAP includes: light chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively, and / or heavy chain variable region CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively.

[0016] According to a specific embodiment of the present invention, the antibody or antigen-binding fragment that specifically recognizes FAP has a light chain variable region shown in SEQ ID NO: 31, or an amino acid sequence that is at least 80% identical to the sequence shown in SEQ ID NO: 31, and a heavy chain variable region shown in SEQ ID NO: 32, or an amino acid sequence that is at least 80% identical to the sequence shown in SEQ ID NO: 32.

[0017] According to a specific embodiment of the present invention, the antibody or antigen-binding fragment that specifically recognizes FAP has a light chain as shown in SEQ ID NO: 20, or an amino acid sequence that is at least 80% identical to the sequence shown in SEQ ID NO: 20, and a heavy chain as shown in SEQ ID NO: 19, or an amino acid sequence that is at least 80% identical to the sequence shown in SEQ ID NO: 19.

[0018] According to a specific embodiment of the present invention, the antibody or antigen-binding fragment that specifically recognizes CD3 comprises:

[0019] The light chain variable region CDR1, CDR2, and CDR3 sequences are as shown in SEQ ID NO: 4, GTN, and SEQ ID NO: 5, respectively; the heavy chain variable region CDR1, CDR2, and CDR3 sequences are as shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; or

[0020] The light chain variable region CDR1, CDR2, and CDR3 sequences are as shown in SEQ ID NO: 4, GTN, and SEQ ID NO: 5, respectively; the heavy chain variable region CDR1, CDR2, and CDR3 sequences are as shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 6, respectively; or

[0021] The light chain variable region CDR1, CDR2, and CDR3 sequences are shown in SEQ ID NO:4, GTN, and SEQ ID NO:8, respectively; and the heavy chain variable region CDR1, CDR2, and CDR3 sequences are shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:7, respectively.

[0022] According to a specific embodiment of the present invention, the antibody or antigen-binding fragment that specifically recognizes CD3 includes any one of the following:

[0023] (a) having a light chain variable region shown in SEQ ID NO: 26 and a heavy chain variable region shown in SEQ ID NO: 27, or,

[0024] (b) having a light chain variable region shown in SEQ ID NO: 26 and a heavy chain variable region shown in SEQ ID NO: 28, or,

[0025] (c) having a light chain variable region shown in SEQ ID NO: 29 and a heavy chain variable region shown in SEQ ID NO: 30, or,

[0026] An amino acid sequence having at least 80% sequence identity with any one of (a), (b), and (c).

[0027] According to a specific embodiment of the present invention, the first Fc region and the second Fc region are connected via a knob-into-hole structure.

[0028] According to a specific embodiment of the present invention, at least a portion of the first Fc region and / or the second Fc region is derived from at least one of a murine antibody, a primate antibody or a mutant thereof.

[0029] According to a specific embodiment of the present invention, at least a portion of the first Fc region and / or the second Fc region is derived from human IgG1 or a mutant thereof.

[0030] According to a specific embodiment of the present invention, the first Fc region has the amino acid sequence shown in SEQ ID NO: 33, and / or the second Fc region has the amino acid sequence shown in SEQ ID NO: 34.

[0031] According to a specific embodiment of the present invention, the connecting peptides 1 and 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.

[0032] 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.

[0033] 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.

[0034] According to a specific embodiment of the present 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 that has at least 80% identity with SEQ ID NO:15, SEQ ID NO:16 or SEQ ID NO:17.

[0035] According to a specific embodiment of the present invention, the recombinant antibody comprises any one of the following:

[0036] (d) the amino acid sequences shown in SEQ ID NO: 15, SEQ ID NO: 19 and SEQ ID NO: 20, or,

[0037] (e) the amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 19 and SEQ ID NO: 20, or,

[0038] (f) the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 19 and SEQ ID NO: 20, or,

[0039] An amino acid sequence having at least 80% sequence identity with any one of (d), (e), and (f).

[0040] According to a specific embodiment of the present invention, the recombinant antibody further comprises a third binding region, and the third binding region comprises an antibody or antigen-binding fragment that specifically recognizes FAP.

[0041] According to a specific embodiment of the present invention, the antibody or antigen-binding fragment that specifically recognizes FAP in the third binding region includes a Fab region, and the heavy chain CH1 region of the Fab region is connected to the scFV region of the antibody or antigen-binding fragment that specifically recognizes CD3 in the first binding region.

[0042] According to a specific embodiment of the present invention, the C-terminus of the heavy chain CH1 region of the Fab region is connected to the heavy chain variable region of the scFV region of the antibody or antigen-binding fragment that specifically recognizes CD3 of the first binding region.

[0043] According to a specific embodiment of the present invention, the amino acid sequence of the Fab region of the antibody or antigen-binding fragment that specifically recognizes FAP in the third binding region is the same as or different from the amino acid sequence of the Fab region of the antibody or antigen-binding fragment that specifically recognizes FAP in the second binding region.

[0044] The second aspect of the present invention provides an isolated polynucleotide encoding the recombinant antibody according to the first aspect.

[0045] According to an embodiment of the present invention, the isolated polynucleotide may further include at least one of the following additional technical features:

[0046] According to an embodiment of the present invention, the antibody or antigen-binding fragment encoded by the polynucleotide is capable of specifically binding to FAP and / or CD3.

[0047] According to an embodiment of the present invention, the nucleic acid molecule of the polynucleotide is DNA and / or RNA.

[0048] It should be noted that, for nucleic acids mentioned in the present specification and claims, those skilled in the art will understand that they actually include any one or both of the complementary double strands. For convenience, although only one strand is provided in most cases in this specification and claims, the other complementary strand is also disclosed. In addition, the nucleic acid sequences in this application include DNA or RNA forms, and disclosure of one of them means that the other is also disclosed.

[0049] The third aspect of the present invention provides an expression vector carrying the polynucleotide described in the second aspect.

[0050] According to an embodiment of the present invention, the expression vector may include optional control sequences that are operably linked to the nucleic acid molecule. The control sequences are one or more control sequences that direct the expression of the nucleic acid molecule in a host. The expression vectors proposed in the embodiments of the present invention can efficiently and abundantly express the antibody or antigen-binding fragment in suitable host cells.

[0051] The fourth aspect of the present invention provides a recombinant cell carrying the polynucleotide described in the second aspect, the expression vector described in the third aspect, or capable of expressing the recombinant antibody described in the first aspect.

[0052] According to an embodiment of the present invention, the above-mentioned recombinant cell may further include at least one of the following additional technical features:

[0053] According to an embodiment of the present invention, the recombinant cell is obtained by introducing the expression vector described in the fourth aspect into a host cell.

[0054] According to an embodiment of the present invention, the recombinant cell is a eukaryotic cell.

[0055] According to an embodiment of the present invention, the recombinant cell is a mammalian cell.

[0056] It should be noted that the recombinant cells of the present invention are not particularly limited and may be prokaryotic cells, eukaryotic cells, or bacteriophages. The prokaryotic cells may be Escherichia coli, Bacillus subtilis, Streptomyces, or Proteus mirabilis, among others. The eukaryotic cells include fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Trichoderma, insect cells such as fall armyworms, plant cells such as tobacco, and mammalian cells such as BHK cells, CHO cells, COS cells, and myeloma cells. In some embodiments, the recombinant cells of the present 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.

[0057] According to an embodiment of the present invention, when the cell is a eukaryotic cell, such as a mammalian cell, the expression efficiency of the recombinant antibody is higher.

[0058] The fifth aspect of the present invention provides a composition comprising at least one of the recombinant antibody described in the first aspect, the polynucleotide described in the second aspect, the expression vector described in the third aspect, or the recombinant cell described in the fourth aspect.

[0059] According to embodiments of the present invention, the recombinant antibody can effectively promote PBMCs to kill FAP-positive cells, exhibiting anti-cancer activity; it can also produce fewer pro-inflammatory cytokines, offering improved safety, and possessing promising clinical application and drug development value. The resulting drug can be further used to prevent and / or treat diseases mediated by CD3 and / or FAP.

[0060] According to an embodiment of the present invention, the recombinant antibody can effectively bind to FAP protein and CD3 and effectively inhibit the proliferation of tumor cells. Therefore, a composition comprising the above-mentioned substance can also effectively bind to FAP and CD3 proteins and has a good effect in preventing and / or treating FAP-mediated diseases. The type of the composition is not particularly limited and can be a food composition or a pharmaceutical composition.

[0061] The compositions of the present invention can also be administered in combination with each other or with one or more other therapeutic compounds, for example, in combination with a chemotherapeutic agent. Therefore, the composition may also contain a chemotherapeutic agent. The antibodies or antigen-binding fragments thereof, or immunoconjugates of the present invention may also be combined with a second therapeutic agent, exemplary 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.

[0062] It should be noted that the composition includes combinations separated in time and / or space, as long as they can work together to achieve the purpose of the present invention. For example, the components contained in the composition can be administered to the subject as a whole or separately. When the components contained in the composition are administered to the subject separately, each component can be administered to the subject simultaneously or sequentially.

[0063] The sixth aspect of the present invention provides a method for preparing the recombinant antibody of the first aspect, comprising culturing the recombinant cell of the fourth aspect, and isolating the antibody or antigen-binding fragment of the recombinant cell.

[0064] The seventh aspect of the present invention provides a drug comprising at least one of the recombinant antibody described in the first aspect, the polynucleotide described in the second aspect, the expression vector described in the third aspect, the recombinant cell described in the fourth aspect, and the composition described in the fifth aspect.

[0065] The eighth aspect of the present invention provides a kit, which contains at least one of the recombinant antibody described in the first aspect, the polynucleotide described in the second aspect, the expression vector described in the third aspect, and the recombinant cell described in the fourth aspect.

[0066] In a ninth aspect, the present invention provides use 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, or the composition of the fifth aspect in the preparation of a medicament for preventing and / or treating FAP-mediated related diseases.

[0067] According to an embodiment of the present invention, the above-mentioned use may further include at least one of the following additional technical features:

[0068] According to an embodiment of the present invention, the FAP-mediated related disease includes a tumor, and the FAP is positive in tumor-associated fibroblasts of the tumor.

[0069] According to an embodiment of the present invention, the disease caused by the tumor includes lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, melanoma, esophageal cancer, oral squamous cell carcinoma or head and neck cancer.

[0070] In a tenth aspect, the present invention provides a kit for detecting FAP and / or CD3 in a sample, the kit comprising at least one of the recombinant antibody described in the first aspect, the polynucleotide described in the second aspect, the expression vector described in the third aspect, and the recombinant cell described in the fourth aspect.

[0071] According to embodiments of the present invention, kits containing the antibodies or antigen-binding fragments can effectively detect FAP protein and / or CD3 qualitatively or quantitatively. The kits provided herein can be used, for example, for immunoblotting, immunoprecipitation, and other assays utilizing the specific binding properties of human FAP and antibodies. These kits may include any one or more of the following: antagonists, anti-FAP antibodies, anti-CD3 antibodies, or reference pharmaceutical materials; protein purification columns; immunoglobulin affinity purification buffers; cell assay diluents; instructions or literature, etc. Anti-FAP antibodies and / or CD3 can be used in various diagnostic tests, such as in vitro or in vivo detection of various diseases or the presence of drugs, toxins, or other proteins. For example, testing can be performed on serum or blood of a subject to detect related diseases. The kits can also be used for scientific research, using the kits to detect FAP protein in test samples. Such related diseases may include FAP-related diseases, such as cancer. Of course, the antibodies or antigen-binding fragments provided herein can also be used for radioimmunoassays and radioimmunotherapy for the aforementioned diseases. The binding molecules described above are also applicable to the aforementioned applications and are not further elaborated here.

[0072] In an eleventh aspect, the present invention provides use of the recombinant antibody of the first aspect, the polynucleotide of the second aspect, the expression vector of the third aspect, and the recombinant cell of the fourth aspect in preparing a kit for detecting FAP and / or CD3.

[0073] The twelfth aspect of the present invention provides an immunoconjugate, which contains a therapeutic agent and the recombinant antibody according to the first aspect coupled to the therapeutic agent.

[0074] According to an embodiment of the present invention, the therapeutic agent includes any one of a polypeptide, a radionuclide, and a small molecule.

[0075] The thirteenth aspect of the present invention provides the use of the recombinant antibody described in the first aspect, the polynucleotide described in the second aspect, the expression vector described in the third aspect, the recombinant cell described in the fourth aspect, the composition described in the fifth aspect or the drug described in the seventh aspect in preventing and / or treating CD3 and / or FAP-mediated related diseases.

[0076] In a fourteenth aspect, the present invention provides the recombinant antibody described in the first aspect, the polynucleotide described in the second aspect, the expression vector described in the third aspect, the recombinant cell described in the fourth aspect, the composition described in the fifth aspect, or the drug described in the seventh aspect, for use in preventing and / or treating CD3 and / or FAP-mediated related diseases.

[0077] The fifteenth aspect of the present invention provides use of the recombinant antibody of the first aspect, the polynucleotide of the second aspect, or the kit of the tenth aspect in diagnosing whether a subject suffers from a CD3 and / or FAP-mediated related disease.

[0078] In the sixteenth aspect, the present invention provides a method for preventing and / or treating CD3 and / or FAP-mediated related diseases, the method comprising administering to a subject a pharmaceutically acceptable amount of the recombinant antibody described in the first aspect, the polynucleotide described in the second aspect, the expression vector described in the third aspect, the recombinant cell described in the fourth aspect, the composition described in the fifth aspect, or the drug described in the seventh aspect.

[0079] A seventeenth aspect of the present invention provides a method for diagnosing whether a subject has a CD3 and / or FAP-mediated disease. According to an embodiment of the present invention, the method comprises detecting a test sample from the subject using the recombinant antibody of the first aspect, the polynucleotide of the second aspect, or the kit of the tenth aspect.

[0080] The eighteenth aspect of the present invention provides use of the recombinant antibody described in the first aspect, the polynucleotide described in the second aspect, or the kit described in the tenth aspect in detecting CD3 and / or FAP in a sample to be tested.

[0081] A nineteenth aspect of the present invention provides a method for detecting CD3 and / or FAP. According to an embodiment of the present invention, the method comprises detecting a sample using the recombinant antibody of the first aspect, the polynucleotide of the second aspect, or the kit of the tenth aspect.

[0082] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0084] FIG1 is a schematic diagram of the CD3×FAP bispecific antibody in Example 1 of the present invention;

[0085] Figure 2 is a flow cytometry result diagram of the binding of Cross3×AHF213391:1, Cross3×AHF213392:1, UCHT1×AHF213391:1, and UCHT1×AHF213392:1 antibodies to CD8 T cells in Example 2 of the present invention, wherein A in Figure 2 is the binding ratio of the antibody to CD8 T cells, and B in Figure 2 is the mean fluorescence intensity;

[0086] Figure 3 is a flow cytometry result diagram of the binding of Cross3×AHF21339 1:1, Cross313×AHF21339, Cross316×AHF21339, and Cross325×AHF21339 antibodies to CD8 T cells in Example 2 of the present invention, wherein A in Figure 3 is the binding ratio of the antibody to CD8 T cells, and B in Figure 3 is the mean fluorescence intensity;

[0087] Figure 4 is a graph showing flow cytometry results of the binding of Cross3×AHF213391:1, Cross3×AHF213392:1, UCHT1×AHF213391:1, and UCHT1×AHF213392:1 antibodies to A-375-FAP cells in Example 2 of the present invention, wherein A in Figure 4 is the binding ratio of the antibody to A-375-FAP cells, and B in Figure 4 is the mean fluorescence intensity;

[0088] Figure 5 is a flow cytometry result diagram of Cross3×AHF21339 1:1, Cross313×AHF21339, Cross316×AHF21339, and Cross325×AHF21339 antibodies binding to A-375-FAP cells in Example 2 of the present invention, wherein A in Figure 5 is the binding ratio of the antibody to A-375-FAP cells, and B in Figure 5 is the mean fluorescence intensity;

[0089] FIG6 is a graph showing the results of Cross3×AHF213391:1 and UCHT1×AHF213391:1 antibodies in Example 3 of the present invention promoting spontaneous activation of PBMCs to secrete cytokines in the absence of target protein (FAP), wherein FIG6A represents the secretion results of IL-2, FIG6B represents the secretion results of IFN-γ, FIG6C represents the secretion results of IL-6, and FIG6D represents the secretion results of TNFα;

[0090] FIG7 is a graph showing the results of Cross3×AHF213391:1, Cross313×AHF21339, Cross316×AHF21339, and Cross325×AHF21339 antibodies in promoting PBMC self-activation and IL-6 secretion in Example 3 of the present invention;

[0091] FIG8 is a graph showing the results of Cross3×AHF213391:1, Cross3×AHF213392:1, UCHT1×AHF213391:1, and UCHT1×AHF213392:1 in Example 3 of the present invention promoting spontaneous activation of PBMCs to secrete cytokines in the presence of target protein (FAP), wherein A in FIG8 represents the secretion result of IL-2, and B in FIG8 represents the secretion result of IFN-γ;

[0092] FIG9 is a graph showing the results of promoting IL-2 secretion by Cross3×AHF21339 1:1, Cross313×AHF21339, Cross316×AHF21339, and Cross325×AHF21339 antibodies in co-incubation of PBMC and A375-FAP in Example 3 of the present invention;

[0093] FIG10 is a graph showing the results of promoting IFN-γ secretion by Cross3×AHF21339 1:1, Cross313×AHF21339, Cross316×AHF21339, and Cross325×AHF21339 antibodies in co-incubation of PBMC and A375-FAP in Example 3 of the present invention;

[0094] FIG11 is a graph showing the results of promoting TNFα secretion by Cross3×AHF21339 1:1, Cross313×AHF21339, Cross316×AHF21339, and Cross325×AHF21339 antibodies in co-incubation of PBMC and A375-FAP in Example 3 of the present invention;

[0095] FIG12 is a graph showing the results of promoting IL-6 secretion by Cross3×AHF21339 1:1, Cross313×AHF21339, Cross316×AHF21339, and Cross325×AHF21339 antibodies in co-incubation of PBMC and A375-FAP in Example 3 of the present invention;

[0096] Figure 13 is a graph showing flow cytometry results of Cross3×AHF213391:1, Cross3×AHF213392:1, UCHT1×AHF213391:1, and UCHT1×AHF213392:1 antibodies promoting T cell proliferation in Example 4 of the present invention, wherein A in Figure 13 represents the activation of CD4 T cells by the antibodies, and B in Figure 13 represents the activation of CD8 T cells by the antibodies;

[0097] Figure 14 shows that Cross3×AHF213391:1, Cross313×AHF21339, Cross316×AHF21339, and Cross325×AHF21339 antibodies in Example 4 of the present invention can all promote CD4 T cell proliferation;

[0098] Figure 15 shows that the Cross3×AHF213391:1, Cross313×AHF21339, Cross316×AHF21339, and Cross325×AHF21339 antibodies in Example 4 of the present invention can all promote CD8 T cell proliferation;

[0099] Figure 16 is a graph showing the results of Cross3×AHF213391:1, Cross3×AHF213392:1, UCHT1×AHF213391:1, and UCHT1×AHF213392:1 antibodies promoting PBMC to kill A-375-FAP melanoma cells in Example 5 of the present invention;

[0100] FIG17 is a graph showing the results of Cross3×AHF21339, Cross313×AHF21339, Cross316×AHF21339, and Cross325×AHF21339 antibodies promoting PBMC to kill A-375-FAP melanoma cells in Example 5 of the present invention;

[0101] FIG18 is a graph showing the ELISA results of Cross3×AHF21339, Cross313×AHF21339, Cross316×AHF21339, and Cross325×AHF21339 antibodies binding to FAP protein in Example 6 of the present invention;

[0102] FIG19 is a graph showing the ELISA results of Cross3×AHF21339, Cross313×AHF21339, Cross316×AHF21339, and Cross325×AHF21339 antibodies binding to CD3E&D proteins (CD3E&CD3D) in Example 6 of the present invention;

[0103] Figure 20 is a graph showing the results of Cross3×AHF21339 1:1, Cross313×AHF21339, Cross316×AHF21339, and Cross325×AHF21339 antibodies promoting CD4 T cells to express CD25 in Example 7 of the present invention;

[0104] FIG21 is a graph showing the results of Cross3×AHF213391:1, Cross313×AHF21339, Cross316×AHF21339, and Cross325×AHF21339 antibodies promoting CD8 T cells to express CD25 in Example 7 of the present invention;

[0105] FIG22 is a graph showing the results of Cross3×AHF213391:1, Cross313×AHF21339, Cross316×AHF21339, and Cross325×AHF21339 antibodies promoting CD4 T cells to express CD69 in Example 7 of the present invention;

[0106] FIG23 is a graph showing the results of Cross3×AHF213391:1, Cross313×AHF21339, Cross316×AHF21339, and Cross325×AHF21339 antibodies promoting CD8 T cells to express CD69 in Example 7 of the present invention. DETAILED DESCRIPTION

[0107] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0108] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0109] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0110] To facilitate understanding of the present invention, certain technical and scientific terms are defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. The abbreviations for amino acid residues are the standard three-letter and / or one-letter codes used in the art to designate one of the 20 commonly used L-amino acids.

[0111] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention, but not excluding other contents.

[0112] As used herein, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0113] The antibodies or antigen-binding fragments described herein are typically prepared by biosynthetic methods. Based on the nucleotide sequences described herein, those skilled in the art can readily produce the encoding nucleic acids of the present invention using various known methods. These methods include, but are not limited to, PCR and artificial DNA synthesis. For specific methods, see J. Sambrook, "Molecular Cloning: A Laboratory Manual." As one embodiment of the present invention, the encoding nucleic acid sequences of the present invention can be constructed by synthesizing a nucleotide sequence in segments followed by overlap extension PCR. The antibodies or antigen fragments are numbered and defined using the Kabat numbering system. As used herein, the term "antibody" refers to an immunoglobulin molecule capable of binding to a specific antigen. It comprises two light chains with a relatively low molecular weight and two heavy chains with a relatively high molecular weight. The heavy chain (H chain) and light chain (L chain) are connected by disulfide bonds to form a tetrapeptide chain. The amino acid sequence at the amino terminus (N-terminus) of the peptide chain varies greatly and is referred to as the variable region (V region). The carboxyl terminus (C-terminus) is relatively stable and exhibits minimal variation and is referred to as the constant region (C region). The V regions of the L chain and H chain are referred to as VL and VH, respectively. Certain regions within the variable region have a higher degree of variation in amino acid composition and sequence, known as hypervariable regions (HVRs). These regions are where antigen and antibody bind, and are therefore also called complementarity-determining regions (CDRs). Both the heavy and light chain variable regions have three CDRs.

[0114] The antibodies of the present invention include murine antibodies, chimeric antibodies, and humanized antibodies, preferably humanized antibodies.

[0115] As used herein, the term "antigen-binding fragment" also refers to "antibody fragment". Antibody fragments generally refer to antigen-binding antibody fragments, which may include a portion of an intact antibody, generally the antigen-binding region or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, Fv or scFv, diabodies, linear antibodies, single-chain antibody molecules, etc.

[0116] As used herein, the term "mutant" or "variant" may refer to any naturally occurring or engineered molecule comprising one or more nucleotide or amino acid mutations.

[0117] As used herein, the term "chimeric antibody" refers to a recombinant antibody obtained by using recombinant DNA technology to replace the amino acid sequence of the constant region of a monoclonal antibody from one species (such as mouse) with the constant region of an antibody from another species (such as human).

[0118] As used herein, the term "humanized antibody" refers to a recombinant antibody obtained by using recombinant DNA technology to replace 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). In other words, when the constant region of an antibody is humanized, it is called a chimeric antibody, while when the non-CDR amino acid sequences of the constant and variable regions are completely humanized, it is called a humanized antibody.

[0119] As used herein, a "full-length antibody" is a tetrapeptide chain structure composed of two identical light chains and two identical heavy chains connected by interchain 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 divided into different subclasses based on their amino acid composition, such as IgG1, IgG2, IgG3, and IgG4. Immunoglobulin light chains are classified as either kappa or lambda chains based on their constant regions.

[0120] In this article, the term "antibody affinity maturation" refers to a normal immune function state of the body. In humoral immunity, the average affinity of the antibodies produced by the secondary response is higher than that of the primary immune response. This phenomenon is called antibody affinity maturation. During the affinity maturation process of natural antibodies, somatic high-frequency mutations are mainly concentrated in the CDR region. Through in vitro experiments, single-point saturation mutations are performed at each site in the CDR region to obtain sufficient mutation diversity without destroying the protein structure. This approach can achieve in vitro reproduction that is most similar to the somatic high-frequency mutations of natural antibodies in vivo. Single-point saturation mutations are performed on each amino acid site in the CDR region to construct an unbiased single-point saturation mutation plasmid library of the parent antibody. ELISA is used to screen out mutation hotspots that enhance specific binding to the antigen, and these mutation hotspots are then combined for screening to obtain candidate antibody mutation sequences.

[0121] In this article, the term "cancer-associated fibroblasts" (CAFs) refers to one of the most important components of the tumor microenvironment, playing an essential role in tumor development and progression. Local tissue-resident fibroblasts and mesenchymal stem cells in the bone marrow and adipose tissue are the primary precursor cells for CAFs. Numerous studies have demonstrated that CAFs do not exist as isolated cells in the tumor periphery but rather interact with tumor cells, promoting tumor growth and survival and maintaining their malignant potential.

[0122] As used herein, the term "operably linked" refers to connecting an exogenous gene to a vector so that the control elements within the vector, such as transcription control sequences and translation control sequences, can function as intended to regulate the transcription and translation of the exogenous gene. When connecting the above-mentioned nucleic acid molecule to a vector, the nucleic acid molecule can be directly or indirectly connected to the control elements on the vector, as long as these control elements are capable of controlling the translation and expression of the nucleic acid molecule. Of course, these control elements can be directly derived from the vector itself, or they can be exogenous, i.e., not derived from the vector itself. Those skilled in the art will appreciate that the nucleic acid molecules used to encode antibodies or antigen-binding fragments can be independently inserted into different vectors, but are commonly inserted into the same vector. Commonly used vectors can be, for example, plasmids, bacteriophages, etc. For example, the Plasmid-X plasmid.

[0123] As used herein, the term "conservatively modified amino acid sequence" refers to amino acid modifications that do not significantly affect or alter the binding properties of an antibody containing the amino acid sequence, including amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies of the present 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 (such as lysine, arginine, histidine), amino acids with acidic side chains (such as aspartic acid, glutamic acid), amino acids with uncharged polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with beta-branched side chains (such as threonine, valine, isoleucine), and amino acids with aromatic side chains (such as tyrosine, phenylalanine, tryptophan, histidine).

[0124] As used herein, the term "identity" is used to describe an amino acid sequence or nucleic acid sequence relative to a reference sequence, and the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences is determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Foundation, Washington, DC). There are many algorithms for aligning sequences and determining sequence identity, including the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:2444; 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). Computer programs that utilize these algorithms are also available and include, but are not limited to, ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth. Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altschul et al., supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, Version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0125] In this article, the antibody "Cross3×AHF21339 1:1" is the same as "Cross3×AHF21339".

[0126] According to a specific embodiment of the present invention, without substantially affecting the activity of the antibody (retaining at least 95% of the activity), those 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 in the sequence of the present invention to obtain variants of the sequence of the antibody or its functional fragment. They are all considered to be included in the scope of protection of the present invention. For example, amino acids with similar properties are replaced in the variable region. The sequence of the variant of the present invention may have at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity (or homology) with the reference sequence. The sequence identity of the present invention can be measured using sequence analysis software. For example, the computer program BLAST, especially BLASTP or TBLASTN, is used with default parameters. The amino acid sequences described in the present invention are all shown in an N-terminal to C-terminal manner. It should be understood by those skilled in the art that the CDR sequences analyzed from different databases may be different, but these changes should be included in the scope of protection of the present invention.

[0127] According to specific embodiments of the present invention, the antibodies of the present invention may be full-length (e.g., IgG1 or IgG4 antibodies) or may comprise only an antigen-binding portion (e.g., Fab, F(ab')2, or scFv fragments), or may be modified to affect function. The present invention includes anti-CD3 antibodies with modified glycosylation patterns. In some applications, modification to remove undesirable glycosylation sites may be useful, or antibodies lacking fucose moieties on oligosaccharide chains may be useful, for example, to enhance antibody-dependent cellular cytotoxicity (ADCC) function. In other applications, galactosylation modification may be performed to alter complement-dependent cytotoxicity (CDC). After a series of modifications, the fragments of the present invention still possess CD3 binding activity. Preferably, the functional fragment will consist of or comprise a partial sequence of the heavy chain variable region or light chain variable region of the antibody from which it is derived, the partial sequence being sufficient to retain the same binding specificity and sufficient affinity as the antibody from which it is derived, preferably at least 1 / 100, and more preferably at least 1 / 10, of the affinity of the antibody from which it is derived. Such functional fragments will comprise a minimum of 5 amino acids, and preferably 10, 15, 25, 50 and 100 contiguous amino acids of the antibody sequence from which they are derived.

[0128] recombinant antibodies

[0129] The present invention provides a recombinant antibody, comprising at least: a first binding region, wherein the first binding region comprises an antibody or antigen-binding fragment that specifically recognizes CD3; and a second binding region, wherein the second binding region comprises an antibody or antigen-binding fragment that specifically recognizes FAP; wherein the antibody or antigen-binding fragment that specifically recognizes CD3 in the first binding region comprises an scFV region and a first Fc region, and the antibody or antigen-binding fragment that specifically recognizes FAP in the second binding region comprises a Fab region and a second Fc region.

[0130] To improve the safety of a CD3 and FAP bispecific antibody, this application has obtained a novel recombinant CD3 and FAP antibody (bispecific antibody) through extensive screening and experimental verification. Further experimental results have shown that the CD3 and FAP bispecific antibody of the present invention has weak binding to T cells, strong binding to FAP-positive cells, and high cytotoxicity, effectively promoting PBMC to kill FAP-positive cells and having good anti-cancer activity. In addition, the bispecific antibody has reduced pro-inflammatory cytokine secretion and T cell activation, is safer, and has good clinical application and drug development value.

[0131] According to a specific embodiment of the present invention, the above-mentioned recombinant antibody may further include at least one of the following additional technical features:

[0132] According to a specific embodiment of the present invention, the C-terminus of the scFV region in the first binding region is connected to the N-terminus of the first Fc region.

[0133] According to a specific embodiment of the present invention, the C-terminus of the heavy chain variable region of the scFV region is connected to the N-terminus of the light chain variable region through connecting peptide 1, and the C-terminus of the light chain variable region is connected to the N-terminus of the first Fc region through connecting peptide 2.

[0134] According to a specific embodiment of the present invention, the antibody or antigen-binding fragment that specifically recognizes FAP comprises:

[0135] The light chain variable region CDR1, CDR2, and CDR3 sequences are shown in SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively, and / or the heavy chain variable region CDR1, CDR2, and CDR3 sequences are shown in SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively.

[0136] According to a specific embodiment of the present invention, the antibody or antigen-binding fragment that specifically recognizes CD3 comprises:

[0137] The light chain variable region CDR1, CDR2, and CDR3 sequences are as shown in SEQ ID NO: 4, GTN, and SEQ ID NO: 5, respectively; the heavy chain variable region CDR1, CDR2, and CDR3 sequences are as shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; or

[0138] The light chain variable region CDR1, CDR2, and CDR3 sequences are as shown in SEQ ID NO: 4, GTN, and SEQ ID NO: 5, respectively; the heavy chain variable region CDR1, CDR2, and CDR3 sequences are as shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 6, respectively; or

[0139] The light chain variable region CDR1, CDR2, and CDR3 sequences are shown in SEQ ID NO:4, GTN, and SEQ ID NO:8, respectively; and the heavy chain variable region CDR1, CDR2, and CDR3 sequences are shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:7, respectively.

[0140] According to a specific embodiment of the present invention, the present invention provides a recombinant protein comprising three polypeptide chains: the first polypeptide chain comprises, from N-terminus to C-terminus, the heavy chain variable region of the CD3 antibody, the light chain variable region of the CD3 antibody, and the first Fc region; the second polypeptide chain comprises the heavy chain of the FAP antibody; and the third polypeptide chain comprises the light chain of the FAP antibody.

[0141] According to a specific embodiment of the present invention, the present invention provides a recombinant protein comprising three polypeptide chains: the first polypeptide chain comprises, from N-terminus to C-terminus, the heavy chain variable region of the CD3 antibody (SEQ ID NO: 27, SEQ ID NO: 28 or SEQ ID NO: 30), the 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); and the third polypeptide chain comprises the light chain of the FAP antibody (SEQ ID NO: 20).

[0142] According to a specific embodiment of the present 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.

[0143] According to a specific embodiment of the present invention, the present invention provides a recombinant protein comprising four polypeptide chains: the first polypeptide chain comprises the light chain of the FAP antibody; the second polypeptide chain comprises, from N-terminus to C-terminus, the heavy chain variable region of the CD3 antibody, the CH1 region of the CD3 antibody, the heavy chain variable region of the CD3 antibody, the light chain variable region of the CD3 antibody, and the first Fc region; the second polypeptide chain comprises the heavy chain of the FAP antibody; and the third polypeptide chain comprises the light chain of the FAP antibody.

[0144] According to a specific embodiment of the present invention, the present invention provides 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, from N-terminus to C-terminus, the heavy chain variable region of the FAP antibody (SEQ ID NO: 32), the CH1 region of the FAP antibody, the heavy chain variable region of the CD3 antibody (SEQ ID NO: 27, SEQ ID NO: 28 or SEQ ID NO: 30), the light chain variable region of the CD3 antibody (SEQ ID NO: 26, SEQ ID NO: 29), and the first Fc region (SEQ ID NO: 33); the second polypeptide chain comprises the heavy chain of the FAP antibody (SEQ ID NO: 19); and the third polypeptide chain comprises the light chain of the FAP antibody (SEQ ID NO: 20).

[0145] According to the disclosed embodiments, the present invention has screened two CD3-FAP bispecific antibodies with different configurations. Even though the antigen-binding domains are identical, the antigen-binding abilities of the antibodies with different configurations vary. One objective of the present invention is to screen for a set of superior CD3-FAP bispecific antibodies that are more effective in promoting immune cell killing of tumors. These antibodies comprise a recombinant protein comprising three polypeptide chains: a first polypeptide chain comprising, from N-terminus to C-terminus, the heavy chain variable region of the CD3 antibody, the light chain variable region of the CD3 antibody, and a first Fc region; a second polypeptide chain comprising the heavy chain of the FAP antibody; and a third polypeptide chain comprising the light chain of the FAP antibody. These antibodies not only have a better ability to promote immune cell killing of non-small cell lung cancer HCC827 cells, but also exhibit improved efficacy in killing other B7H7-positive cells.

[0146] According to an embodiment of the present invention, FAP is specifically expressed on the surface of tumor fibroblasts and is part of the tumor microenvironment. Anti-cancer effects are achieved by disrupting the tumor microenvironment. It should be noted that the anti-FAP antibodies proposed in the present invention can act on any tumor fibroblasts expressing FAP in the tumor microenvironment. Tumors herein are not limited and can include any one or more of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, melanoma, glioma, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

[0147] Nucleic acid molecules, recombinant vectors, immunoconjugates

[0148] In the process of preparing or obtaining these antibodies, nucleic acid molecules expressing these antibodies can be connected to different vectors and then expressed in different cells to obtain the corresponding antibodies.

[0149] Therefore, the present invention also provides isolated nucleic acids encoding the above-mentioned recombinant antibodies, antibodies or antigen-binding fragments, as well as recombinant vectors and transformants containing the nucleic acids. The nucleic acid molecules encode the above-mentioned recombinant antibodies, antibodies or antigen-binding fragments, and the nucleic acids are preferably expression cassettes obtained by genetic engineering.

[0150] The recombinant vector may refer to a cloning vector or an expression vector, and can be obtained by operably linking the nucleic acid with a commercially available vector (such as a plasmid or viral vector). Commonly used plasmids include pSeTag2, PEE14, pMH3, and the like.

[0151] In some preferred embodiments, the nucleic acid molecule is species-optimized and is more easily expressed in mammalian cells.

[0152] The present invention also provides an expression vector comprising the isolated nucleic acid molecule described above. When the isolated polynucleotide is linked to the vector, the polynucleotide may be directly or indirectly linked to control elements on the vector, as long as these control elements are capable of controlling translation and expression of the polynucleotide. These control elements may be derived directly from the vector itself or exogenously, i.e., not derived from the vector itself. It suffices that the polynucleotide and the control elements are operably linked.

[0153] The immunoconjugate provided by the present invention comprises a therapeutic agent and the aforementioned recombinant antibody, antibody, or antigen-binding fragment coupled to the therapeutic agent. The recombinant antibody, antibody, or antigen-binding fragment can be coupled to the therapeutic agent in a conventional manner.

[0154] The composition provided by the present invention contains the recombinant antibody, antibody or antigen-binding fragment, and / or the above-mentioned immunoconjugate and a pharmaceutically acceptable carrier as described above. In certain embodiments, the composition includes a combination separated in time and / or space, as long as it can work together to achieve the purpose of the present invention. For example, the components contained in the composition can be applied to the subject as a whole, or separately. When the components contained in the composition are applied to the subject separately, each component can be applied to the subject simultaneously or sequentially.

[0155] recombinant cells

[0156] The present invention provides a recombinant cell. The recombinant cell carries the aforementioned nucleic acid, the aforementioned vector, or the aforementioned transformant, or expresses the aforementioned recombinant antibody. According to an embodiment of the present invention, the cell is obtained by transfecting or transforming the aforementioned vector or transformant, and the cell can efficiently express the aforementioned recombinant antibody under appropriate conditions.

[0157] According to a specific embodiment of the present invention, the above-mentioned recombinant cell may further include at least one of the following additional technical features:

[0158] According to an embodiment of the present invention, the cell is a prokaryotic cell, a eukaryotic cell or a bacteriophage.

[0159] According to an embodiment of the present invention, the prokaryotic cell is Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis.

[0160] According to an embodiment of the present invention, the eukaryotic cell is a fungus, an insect cell, a plant cell or a mammalian cell.

[0161] According to an embodiment of the present invention, the fungus is Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe or Trichoderma.

[0162] According to an embodiment of the present invention, the insect cell is a Spodoptera frugiperda cell; according to an embodiment of the present invention, the plant cell is a tobacco plant cell.

[0163] According to an embodiment of the present invention, the mammalian cells are BHK cells, CHO cells, COS cells, myeloma cells or human embryonic kidney 293 cells; and do not include animal germ cells, fertilized eggs or embryonic stem cells.

[0164] According to an embodiment of the present invention, the cell is a mammalian cell.

[0165] According to an embodiment of the present invention, the cell is a BHK cell, a CHO cell, a COS cell or a NSO cell.

[0166] It should be noted that the "suitable conditions" described in the specification of this application refer to conditions suitable for the expression of the recombinant antibodies, antibodies or antigen-binding fragments described in this application. It will be readily understood by those skilled in the art that conditions suitable for the expression of recombinant antibodies, antibodies or antigen-binding fragments include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell status, suitable host cell density, suitable cell culture environment, and suitable cell culture time. "Suitable conditions" are not particularly limited, and those skilled in the art can optimize the most suitable conditions for the expression of the recombinant antibodies, antibodies or antigen-binding fragments according to the specific environment of the laboratory.

[0167] Composition

[0168] The present invention provides a composition. The composition comprises: the aforementioned recombinant antibody, the aforementioned nucleic acid, the aforementioned vector or transformant, or the aforementioned cell. According to embodiments of the present invention, the recombinant antibody can effectively promote PBMCs to kill tumor cells, exhibiting anti-cancer activity; it can also produce less pro-inflammatory cytokines, exhibiting increased safety, and possessing excellent clinical application and drug development value. The resulting composition can be further used to prevent and / or treat diseases mediated by CD3 and / or FAP.

[0169] drug

[0170] The present invention provides a drug. The drug comprises: the aforementioned recombinant antibody, the aforementioned nucleic acid, the aforementioned vector or transformant, the aforementioned cell, or the aforementioned composition. According to embodiments of the present invention, the recombinant antibody can effectively promote PBMCs to kill tumor cells, exhibiting anti-cancer activity; it can produce less pro-inflammatory cytokines, has higher safety, and has excellent clinical application and drug development value. The resulting drug can be further used to prevent and / or treat diseases mediated by CD3 and / or FAP.

[0171] According to an embodiment of the present invention, the above-mentioned drug may further include at least one of the following additional technical features:

[0172] According to an embodiment of the present invention, the composition further comprises a pharmaceutically acceptable excipient.

[0173] According to an embodiment of the present invention, the excipients include: one or more pharmaceutically acceptable excipients, diluents, stabilizers or carriers.

[0174] According to an embodiment of the present invention, the medicine is an injection.

[0175] It should be noted that the medicament includes combinations separated in time and / or space, as long as they can work together to achieve the purpose of the present invention. For example, the components contained in the medicament can be administered to the subject as a whole or separately. When the components contained in the medicament are administered to the subject separately, the individual components can be administered to the subject simultaneously or sequentially.

[0176] The medicament of the present invention contains a safe and effective amount of the active ingredient of the present invention and pharmaceutically acceptable excipients. Such excipients include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. Generally, the pharmaceutical formulation should be compatible with the mode of administration. The dosage form of the medicament of the present invention is an injection, oral preparation (tablets, capsules, oral liquid), transdermal preparation, or sustained-release preparation. For example, it can be prepared using conventional methods using physiological saline or an aqueous solution containing glucose and other excipients. The medicament is preferably manufactured under sterile conditions.

[0177] The effective amount of the active ingredient of the present invention may vary depending on the mode of administration and the severity of the disease to be treated. The preferred effective amount can be determined by one of ordinary skill in the art based on various factors (e.g., through clinical trials). Such factors include, but are not limited to, the pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, the route of administration, and the like. For example, depending on the exigencies of the treatment, several divided doses may be administered daily, or the dose may be reduced proportionally.

[0178] The pharmaceutically acceptable excipients of the present invention 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, as is well known to those skilled in the art.

[0179] Reagent test kit

[0180] The present invention provides a kit. The kit comprises the aforementioned recombinant antibody, the aforementioned nucleic acid molecule, the aforementioned vector or transformant, or the aforementioned cell. According to an embodiment of the present invention, the kit can bind to CD3 protein and / or FAP protein and can effectively identify CD3 protein and / or FAP protein.

[0181] As described above, the recombinant antibodies of the embodiments of the present invention can specifically bind to CD3 and FAP. The CD3 protein and / or FAP protein-related kits developed using this property can be used for CD3 protein and / or FAP protein-related research, such as for detecting and / or enriching and / or isolating and purifying CD3 protein and / or FAP protein from humans or other mammals.

[0182] The kits can effectively detect, enrich, or isolate and purify CD3 and / or FAP proteins in biological samples for further use in scientific research, such as qualitative or quantitative detection of CD3 and / or FAP protein molecules in biological samples. More specifically, the kits can be used in immunoblotting, immunoprecipitation, and other assays that utilize the specific binding properties of CD3 and / or FAP proteins with antibodies. These kits may contain any one or more of the following: an antagonist, a recombinant antibody of the present invention, or a pharmaceutical reference material; a protein purification column; an immunoglobulin affinity purification buffer; and a cell assay diluent. The recombinant antibodies of the present invention can be used in various diagnostic tests, for example, to detect various diseases or the presence of drugs, toxins, or other proteins in vitro or in vivo. For example, they can be used to test for CD3 and / or FAP-mediated diseases by testing serum or blood in a subject.

[0183] Use in the preparation of kits

[0184] The present invention provides use of the aforementioned recombinant antibody, the aforementioned nucleic acid molecule, the aforementioned vector or transformant, or the aforementioned cell in preparing a kit for detecting CD3 and / or FAP.

[0185] As previously described, the recombinant antibodies of the present invention are capable of specifically binding to CD3 and FAP. Therefore, these recombinant antibodies can be used to detect CD3 and / or FAP. Furthermore, they can be used to prepare CD3 and / or FAP-related kits for scientific research, such as for the qualitative or quantitative detection of CD3 and / or FAP protein molecules in biological samples. More specifically, they can be used in kits for immunoblotting, immunoprecipitation, and other assays that utilize the specific binding properties of CD3 and / or FAP with antibodies. These kits may contain any one or more of the following: an antagonist, a recombinant antibody of the present invention, or a reference drug material; a protein purification column; an immunoglobulin affinity purification buffer; and a cell assay diluent. The recombinant antibodies of the present invention can be used in various diagnostic tests, for example, to detect various diseases or the presence of drugs, toxins, or other proteins in vitro or in vivo. For example, they can be used to test for CD3 and / or FAP-mediated diseases by testing serum or blood in a subject.

[0186] Use in preparing medicines

[0187] The present invention provides the use of the aforementioned recombinant antibodies, nucleic acid molecules, vectors or transformants, cells, or compositions in the preparation of medicaments for preventing and / or treating diseases mediated by CD3 and / or FAP. According to embodiments of the present invention, the recombinant antibodies and corresponding nucleic acids, vectors or transformants, or compositions of the present invention can be further prepared into medicaments that can be clinically used to prevent or treat diseases mediated by CD3 and / or FAP.

[0188] According to an embodiment of the present invention, the above-mentioned use may further include at least one of the following additional technical features:

[0189] According to an embodiment of the present invention, the CD3-mediated related diseases include autoimmune diseases.

[0190] According to an embodiment of the present invention, the autoimmune disease includes at least one of the following: systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis and acute idiopathic polyneuritis.

[0191] According to an embodiment of the present invention, the FAP-mediated related diseases are cancer, diseases caused by transplant rejection, autoimmune diseases, and infectious diseases.

[0192] According to an embodiment of the present 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, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma and head and neck cancer.

[0193] Uses and methods for preventing and / or treating diseases

[0194] The present invention provides the use of the aforementioned recombinant antibodies, nucleic acid molecules, vectors or transformants, cells, compositions, or drugs for preventing and / or treating diseases mediated by CD3 and / or FAP. As previously described, the recombinant antibodies can effectively promote PBMCs to kill FAP-positive cells and have good anti-cancer activity. Furthermore, the recombinant antibodies have reduced pro-inflammatory cytokine secretion and T cell activation, resulting in improved safety. Therefore, the recombinant antibodies, as well as substances capable of expressing the antibodies or antigen-binding fragments thereof under appropriate conditions, or substances containing the recombinant antibodies can be used to prevent and / or treat diseases mediated by CD3 and / or FAP.

[0195] According to an embodiment of the present invention, the above-mentioned use for treating diseases may further include at least one of the following additional technical features:

[0196] In some embodiments, the CD3-mediated related disease comprises an autoimmune disease.

[0197] In some embodiments, the autoimmune disease comprises at least one of systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis, and acute idiopathic polyneuritis.

[0198] In some embodiments, the FAP-mediated disease comprises a tumor wherein the tumor-associated fibroblasts are positive for FAP.

[0199] In some embodiments, the tumor-induced disease comprises lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, melanoma, oral squamous cell carcinoma, or head and neck cancer.

[0200] The present invention provides the aforementioned recombinant antibodies, nucleic acid molecules, vectors or transformants, cells, or pharmaceutical compositions for use in preventing and / or treating diseases mediated by CD3 and / or FAP. As previously described, the recombinant antibodies can effectively promote PBMCs to kill FAP-positive cells and exhibit good anti-cancer activity. Furthermore, the recombinant antibodies have reduced pro-inflammatory cytokine secretion and T cell activation, resulting in improved safety. Therefore, the recombinant antibodies, as well as substances capable of expressing the antibodies or antigen-binding fragments thereof under appropriate conditions, or substances containing the recombinant antibodies, can be used to prevent and / or treat diseases mediated by CD3 and / or FAP.

[0201] According to an embodiment of the present invention, the above-mentioned use for treating and / or preventing a disease may further include at least one of the following additional technical features:

[0202] In some embodiments, the CD3-mediated related disease comprises an autoimmune disease.

[0203] In some embodiments, the autoimmune disease comprises at least one of systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis, and acute idiopathic polyneuritis.

[0204] In some embodiments, the FAP-mediated disease comprises a tumor wherein the tumor-associated fibroblasts are positive for FAP.

[0205] In some embodiments, the tumor-induced disease comprises lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, melanoma, oral squamous cell carcinoma, or head and neck cancer.

[0206] The present invention provides a method for preventing and / or treating diseases mediated by CD3 and / or FAP. According to an embodiment of the present invention, the method comprises administering a pharmaceutically acceptable amount of the aforementioned recombinant antibody, nucleic acid molecule, vector or transformant, cell, composition, or drug to a subject. As described above, the recombinant antibody can effectively promote PBMCs to kill FAP-positive cells and exhibits excellent anti-cancer activity. Furthermore, the recombinant antibody has reduced pro-inflammatory cytokine secretion and T cell activation, resulting in improved safety. Therefore, the recombinant antibody, as well as substances capable of expressing the antibody or antigen-binding fragment thereof under appropriate conditions, or substances containing the recombinant antibody, can effectively prevent and / or treat diseases mediated by CD3 and / or FAP. The methods according to the embodiments of the present invention can effectively prevent and / or treat diseases mediated by CD3 and / or FAP.

[0207] It should be 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 can be a human, but also includes other mammals, particularly mammals that can be used as laboratory models of human diseases, such as mice, rats, and the like.

[0208] The effective amount of the recombinant antibody, antibody or antigen-binding fragment thereof, conjugate, nucleic acid, vector or transformant, composition or drug of the present invention may vary depending on the mode of administration and the severity of the disease to be treated. The selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials). The 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, the patient's weight, the patient's immune status, the route of administration, etc. For example, depending on the urgency of the treatment condition, several divided doses may be administered daily, or the dose may be reduced proportionally.

[0209] According to an embodiment of the present invention, the above-mentioned method for treating and / or preventing a disease may further include at least one of the following additional technical features:

[0210] In some embodiments, the CD3-mediated related disease comprises an autoimmune disease.

[0211] In some embodiments, the autoimmune disease comprises at least one of systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis, and acute idiopathic polyneuritis.

[0212] In some embodiments, the FAP-mediated disease comprises a tumor wherein the tumor-associated fibroblasts are positive for FAP.

[0213] In some embodiments, the tumor-induced disease comprises lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, melanoma, oral squamous cell carcinoma, or head and neck cancer.

[0214] Detection and diagnostic purposes

[0215] The present invention provides uses of the aforementioned recombinant antibodies, polynucleotides, or kits for detecting CD3 and / or FAP in a test sample. As previously described, the recombinant antibodies of the embodiments of the present invention are capable of specifically binding to CD3 and / or FAP. Therefore, the recombinant antibodies and related products can be used to detect CD3 and / or FAP, for example, in scientific research, for qualitative or quantitative detection of CD3 and / or FAP protein molecules in a test sample.

[0216] In some embodiments, the sample to be tested includes at least one of the following: tissue, cells, blood, serum, plasma, saliva, sweat, feces or urine.

[0217] The present invention provides the use of the aforementioned recombinant antibodies, the aforementioned polynucleotides, or the aforementioned kits in diagnosing whether a subject suffers from a CD3 and / or FAP-mediated disease. As previously described, the recombinant antibodies of the embodiments of the present invention can effectively bind to CD3 and / or FAP. Therefore, the recombinant antibodies can be used to detect the levels of CD3 and / or FAP. CD3 and / or FAP mediate a variety of diseases. Therefore, based on the levels of CD3 and / or FAP contained in a biological sample from a subject, it can be determined whether the subject suffers from a CD3 and / or FAP-mediated disease. In addition, the above substances can also be used to monitor the content of CD3 and / or FAP in a test sample from a subject, that is, the above substances can also be used to stage the disease in subjects suffering from CD3 and / or FAP-mediated diseases, and can also be used to evaluate the prognosis of CD3 and / or FAP-mediated diseases.

[0218] According to an embodiment of the present invention, the above-mentioned use for diagnosing a disease may further include at least one of the following additional technical features:

[0219] In some embodiments, the CD3-mediated related disease comprises an autoimmune disease.

[0220] In some embodiments, the autoimmune disease comprises at least one of systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis, and acute idiopathic polyneuritis.

[0221] In some embodiments, the FAP-mediated disease comprises a tumor wherein the tumor-associated fibroblasts are positive for FAP.

[0222] In some embodiments, the tumor-induced disease comprises lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, melanoma, oral squamous cell carcinoma, or head and neck cancer.

[0223] Detection and diagnostic methods

[0224] The present invention provides a method for detecting CD3 and / or FAP. According to an embodiment of the present invention, the method comprises using the aforementioned recombinant antibody, the aforementioned polynucleotide, or the aforementioned kit to detect a sample to be tested.

[0225] In some embodiments, the sample to be tested includes at least one of the following: tissue, cells, blood, serum, plasma, saliva, sweat, feces or urine.

[0226] The present invention provides a method for diagnosing whether a subject suffers from a disease mediated by CD3 and / or FAP. According to an embodiment of the present invention, the method comprises using the aforementioned recombinant antibody, the aforementioned polynucleotide, or the aforementioned kit to detect a test sample from the subject. As previously described, the recombinant antibodies of the embodiments of the present invention are capable of effectively binding to CD3 and / or FAP. Therefore, the recombinant antibodies can be used to detect the levels of CD3 and / or FAP. CD3 and / or FAP mediate a variety of diseases. Therefore, based on the levels of CD3 and / or FAP contained in a biological sample from a subject, it can be determined whether the subject suffers from a disease mediated by CD3 and / or FAP. In addition, the above-mentioned substances can also be used to monitor the content of CD3 and / or FAP in a test sample from a subject. That is, the above-mentioned substances can also be used to stage the disease in subjects suffering from a disease mediated by CD3 and / or FAP, and can also be used to assess the prognosis of a disease mediated by CD3 and / or FAP.

[0227] According to an embodiment of the present invention, the above-mentioned method for diagnosing a disease may further include at least one of the following additional technical features:

[0228] In some embodiments, the CD3-mediated related disease comprises an autoimmune disease.

[0229] In some embodiments, the autoimmune disease comprises at least one of systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis, and acute idiopathic polyneuritis.

[0230] In some embodiments, the FAP-mediated disease comprises a tumor wherein the tumor-associated fibroblasts are positive for FAP.

[0231] In some embodiments, the tumor-induced disease comprises lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, melanoma, oral squamous cell carcinoma, or head and neck cancer.

[0232] In some embodiments, the sample to be tested includes at least one of the following: tissue, cells, blood, serum, plasma, saliva, sweat, feces or urine.

[0233] Nucleic acids encoding the heavy and / or light chains of the antibodies of the present invention are within the scope of the present invention. Based on the amino acid sequences of the heavy and / or light chains, those skilled in the art can easily obtain the corresponding nucleic acid sequences, as shown in Table 1.

[0234] Table 1

[0235] The scheme of the present disclosure will be explained below in conjunction with the examples. Those skilled in the art will understand that the following examples are only used to illustrate the present disclosure and should not be considered to limit the scope of the present disclosure. Where specific techniques or conditions are not specified in the examples, they are carried out according to the techniques or conditions described in the literature in this area or according to the product instructions. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0236] Example 1 Production of Antibodies

[0237] The specific experimental procedures for antibody production are as follows: (1) ExpiCHO cells (purchased from Thermo Fisher) were cultured using ExpiCHO Expression Medium (purchased from Thermo Fisher) and the cell concentration was adjusted to 6×10 6 / mL to obtain ExpiCHO cell solution. (2) When the bispecific antibody is of A configuration (A in Figure 1), the pcDNA3.4 vector containing CD3 antibody, FAP antibody heavy chain, and FAP antibody light chain (commissioned by Nanjing GenScript for synthesis) is added to 2mL OptiSFM culture medium (purchased from Thermo Fisher) at a mass ratio of 1:1:1 to obtain solution a; or when the bispecific antibody is of B configuration (B in Figure 1), the pcDNA3.4 vector containing FAP heavy chain-CD3 antibody, FAP antibody heavy chain, and FAP antibody light chain (commissioned by Nanjing GenScript for synthesis) is added to 2mL OptiSFM culture medium (purchased from Thermo Fisher) at a mass ratio of 1:1:1 to obtain solution a. (3) 160μL ExpiFectamineCHO transfection reagent (purchased from Thermo Fisher) is added to 2mL OptiSFM culture medium (purchased from Thermo Fisher) to obtain solution b. (4) Solution a and solution b were then mixed to obtain a transfection mixture, and the entire transfection mixture was added to 50 mL of ExpiCHO cell solution within 5 minutes. (5) After culturing at 37°C, 5% CO2 for 1 day, 8 mL of feed and 300 μL of Enhancer (purchased from Thermo Fisher) were added, and the cells were transferred to 32°C, 5% CO2 for 9 days. The culture supernatant was harvested, with 8 mL of feed added on the 5th day. (6) The target antibody was obtained by affinity purification from the culture supernatant using a Protein A purification column (purchased from NanoMicro).

[0238] In this example, a total of 7 CD3 and FAP bispecific antibodies of two configurations were prepared to investigate the various properties of the recombinant antibodies of the present invention. The specific information of the 7 bispecific antibodies is shown in Table 2, and the configurations of the A and B class antibodies are shown in Figure 1.

[0239] Table 2

[0240] The seven bispecific antibodies are: Cross3×AHF213391:1 (the amino acid sequence of the scFv+Fc of CD3 is shown in SEQ ID NO: 18, the amino acid sequence of the heavy chain of the FAP antibody is shown in SEQ ID NO: 19, and the amino acid sequence of the light chain of the FAP antibody is shown in SEQ ID NO: 20), Cross3×AHF213392:1 (the amino acid sequence of the heavy chain variable region+CH1 region of the FAP antibody and the scFv+Fc of the CD3 antibody is shown in SEQ ID NO: 21, the amino acid sequence of the heavy chain of the FAP antibody is shown in SEQ ID NO: 19, and the amino acid sequence of the light chain of the FAP antibody is shown in SEQ ID NO: 20), UCHT1×AHF213391:1 (the amino acid sequence of the scFv+Fc of the CD3 antibody is shown in SEQ ID NO: 22, the amino acid sequence of the heavy chain of the FAP antibody is shown in SEQ ID NO: 19, and the amino acid sequence of the light chain of the FAP antibody is shown in SEQ ID NO: NO: 20), UCHT1×AHF213392:1 (the amino acid sequence of the heavy chain variable region + CH1 region of the FAP antibody and the scFv + Fc of the CD3 antibody is shown in SEQ ID NO: 23, the amino acid sequence of the FAP antibody heavy chain is shown in SEQ ID NO: 19, and the amino acid sequence of the FAP antibody light chain is shown in SEQ ID NO: 20), Cross313×AHF21339 (the amino acid sequence of the scFv + Fc of the CD3 antibody is shown in SEQ ID NO: 15, the amino acid sequence of the FAP antibody heavy chain is shown in SEQ ID NO: 19, and the amino acid sequence of the FAP antibody light chain is shown in SEQ ID NO: 20), Cross316×AHF21339 (the amino acid sequence of the scFv + Fc of the CD3 antibody is shown in SEQ ID NO: 16, the amino acid sequence of the FAP antibody heavy chain is shown in SEQ ID NO: 19, and the amino acid sequence of the FAP antibody light chain is shown in SEQ ID NO: NO: 20), Cross325×AHF21339 (the amino acid sequence of the scFv+Fc of the CD3 antibody is shown in SEQ ID NO: 17, the amino acid sequence of the FAP antibody heavy chain is shown in SEQ ID NO: 19, and the amino acid sequence of the FAP antibody light chain is shown in SEQ ID NO: 20).

[0241] Example 2 Antibody Flow Cytometry Binding Experiment

[0242] Flow cytometry experiments are used to detect the binding properties of bispecific antibodies. Bispecific antibodies are added to cells, and the strength of the signal after the addition of the antibody is used to determine the binding properties of the antibody and cells.

[0243] (1) Dilute PBMC to 2×10 6 / ml, added to a volume of 100μl / tube in a 1.5ml EP tube, and 10μl / tube of goat serum was added. Blocking was carried out at 4°C for 30 minutes. A serial dilution of bispecific antibodies (Cross3×AHF21339 1:1, Cross3×AHF21339 2:1, UCHT1×AHF21339 1:1, UCHT1×AHF21339 2:1, Cross313×AHF21339, Cross316×AHF21339, Cross325×AHF21339), and control hIgG1LALA (purchased from Bio-Bio) were added and incubated at 4°C for 30 minutes. 1ml of PBS was added to the EP tube, and the tube was centrifuged at 3500 rpm for 5 minutes at 4°C. The supernatant was discarded and the tube was washed once with PBS. After centrifugation, discard the supernatant and resuspend the cells in 100 μl / tube of PBS. Add 1 μl / tube of Alexa-647-labeled goat anti-human IgG secondary antibody (purchased from Jackson Lab) and 0.5 μl / tube of PerCP-Cy5.5-labeled anti-human CD8 antibody. Incubate at 4°C in the dark for 30 min. Wash twice with PBS, centrifuge, and discard the supernatant. Resuspend the cells in 200 μl / tube of PBS and analyze by flow cytometry.

[0244] The binding strength and binding activity of 7 bispecific antibodies to CD8 T cells are shown in Figures 2 and 3. Figure 2 shows that Cross3×AHF213391:1, Cross3×AHF213392:1, UCHT1×AHF213391:1, and UCHT1×AHF213392:1 antibodies can bind to CD8 T cells, and the B-configuration bispecific antibody has weaker T cell binding ability than the A-configuration bispecific antibody. The Cross3-based FAP bispecific antibody has weaker T cell binding activity than the UCHT1-based FAP bispecific antibody. Figure 3 shows that Cross3×AHF213391:1, Cross313×AHF21339, Cross316×AHF21339, and Cross325×AHF21339 antibodies can bind to CD8 T cells, ranked according to the strength of binding to T cells: Cross3×AHF213391:1, Cross313×AHF21339, Cross316×AHF21339, and Cross325×AHF21339.

[0245] (2) Preparation of A-375-FAP cells: HEK293T cells were prepared at 5×10 5Cells were plated / well in a six-well plate and cultured overnight in DMEM medium without the antibody. Before transfection, the medium was discarded and 1 ml of fresh DMEM medium without the antibody was added. The coding sequence (SEQ ID NO: 25) of human FAP protein (SEQ ID NO: 24) was inserted between the EcoRI and BamHI restriction sites of pLVX-EF1a-IRES-puro vector (purchased from Ubao Bio), pMD2G (purchased from Ubao Bio), and psPAX2 (purchased from Ubao Bio) vectors (3 μg in total) were added to 200 μl serum-free DMEM medium at a ratio of 2:1:1, and then 12 μg of polyetherimide (PEI, purchased from Polysciences) was added. After mixing, the mixture was allowed to stand for 16 minutes, and then all the liquid was added to a six-well plate containing HEK293T cells. After culturing for 6 hours, the medium was discarded and fresh complete DMEM medium was added for culturing. 48 hours after transfection, the cell culture supernatant was collected and filtered through a 0.45 μm filter (purchased from Millipore) to obtain the viral supernatant. All the viral supernatant was added to a plate containing 1×10 4 A-375 cells were cultured in a 6-well plate with polybrene (Sigma) at a final concentration of 4 μg / ml and incubated for 12 hours. The supernatant was then discarded and fresh complete DMEM medium was added. The resulting cells are A-375-FAP cells.

[0246] A-375-FAP tumor cells were diluted to 2 × 10 6 / ml, added to a volume of 100μl / tube in a 1.5ml EP tube, and 10μl / tube of goat serum was added. Blocking was carried out at 4°C for 30 minutes. A serial dilution of bispecific antibodies (Cross3×AHF21339 1:1, Cross3×AHF21339 2:1, UCHT1×AHF21339 1:1, UCHT1×AHF21339 2:1, Cross313×AHF21339, Cross316×AHF21339, Cross325×AHF21339), and control hIgG1LALA (purchased from Bio-Bio) were added and incubated at 4°C for 30 minutes. 1ml of PBS was added to the EP tube, and the tube was centrifuged at 3500 rpm for 5 minutes at 4°C. The supernatant was discarded and the tube was washed once with PBS. After centrifugation, discard the supernatant and resuspend the cells in 100 μl / tube of PBS. Add 1 μl / tube of Alexa-647-labeled goat anti-human IgG secondary antibody (purchased from Jackson Lab) and incubate at 4°C in the dark for 30 min. Wash twice with PBS and centrifuge. Discard the supernatant. Resuspend the cells in 200 μl / tube of PBS and analyze using flow cytometry.

[0247] The binding strength and binding activity of the seven bispecific antibodies to A-375-FAP cells are shown in Figures 4 and 5. Figure 4 shows that Cross3×AHF213391:1, Cross3×AHF213392:1, UCHT1×AHF213391:1, and UCHT1×AHF213392:1 antibodies can all bind to A-375-FAP cells, and the activity (EC50) of the B-type bispecific antibodies in binding to FAP is lower than that of the A-type bispecific antibodies; Figure 5 shows that Cross3×AHF213391:1, Cross313×AHF21339, Cross316×AHF21339, and Cross325×AHF21339 antibodies can all bind to A-375-FAP cells, and the activities (EC50) of all bispecific antibodies are comparable.

[0248] Example 3 Experiment on Bispecific Antibodies Promoting Cytokine Secretion by PBMC

[0249] (1) Experiment on the promotion of PBMC cytokine secretion by bispecific antibodies in the absence of target protein (no tumor cells)

[0250] The bispecific antibody was added to the PBMC culture system, and the culture supernatant was collected after 48 hours of culture. The cytokine content in the supernatant was detected to determine the characteristics of the bispecific antibody-induced cytokine release in the absence of target protein.

[0251] (a) The bispecific antibodies Cross3×AHF21339 (1:1), UCHT1×AHF21339 (1:1), Cross313×AHF21339, Cross316×AHF21339, Cross325×AHF21339, and control hIgG1LALA (purchased from Bio-Bio) were serially diluted in complete RPMI 1640 medium and added to a 96-well plate at 100 μl / well.

[0252] (b) PBMC (purchased from Saili Biotechnology) were diluted to 1×10 6 / ml, added to a 96-well plate, 100 μl / well, and the 96-well plate was cultured in a 37°C, 5% CO2 incubator for 48 h;

[0253] (c) Centrifugation at 300 g for 10 min at room temperature to collect the cell culture supernatant;

[0254] (d) The cytokine content in the supernatant was detected using a CBA kit (purchased from BD).

[0255] The results are shown in Figures 6 and 7, respectively. The PBMCs used in each experimental group in Figure 6 and Figure 7 were from the same donor, respectively. The PBMCs used in the experiments in Figures 6 and 7 were from different donors. Figure 6 demonstrates that the Cross3×AHF21339 1:1 and UCHT1×AHF21339 1:1 antibodies do not induce spontaneous PBMC activation in the absence of target protein (FAP), demonstrating a reasonable safety profile. Figure 7 shows that Cross313×AHF21339, Cross316×AHF21339, and Cross325×AHF21339 antibodies do not cause spontaneous activation of PBMCs in the absence of target protein (FAP), while Cross3×AHF213391:1 triggers the release of the proinflammatory cytokine IL-6 at high concentrations, indicating that Cross313×AHF21339, Cross316×AHF21339, and Cross325×AHF21339 are safer than Cross3×AHF213391:1.

[0256] (2) Experiment on the promotion of PBMC cytokine secretion by bispecific antibodies in the presence of target protein (tumor cells)

[0257] The bispecific antibody was added to the co-incubation system of PBMC and A-375-FAP melanoma cells. After 48 hours of culture, the culture supernatant was collected and the cytokine content in the supernatant was detected to determine the characteristics of cytokine release induced by the bispecific antibody.

[0258] (a) A-375-FAP cells were diluted to 1×10 5 / ml, added to 96-well plates, and cultured in a 37°C, 5% CO2 incubator for 24 h;

[0259] (b) The bispecific antibodies Cross3×AHF21339 1:1, Cross3×AHF21339 2:1, UCHT1×AHF21339 1:1, UCHT1×AHF21339 2:1, Cross313×AHF21339, Cross316×AHF21339, Cross325×AHF21339, and control hIgG1LALA (purchased from Bio-Bio) were serially diluted in complete RPMI 1640 medium and added to a 96-well plate at 20 μl / well.

[0260] (c) PBMC (purchased from Saili Biotechnology) were diluted to 1.25×10 6 / ml, added to 96-well plate, 80 μl / well;

[0261] (d) The 96-well plate was incubated in a 37°C, 5% CO2 incubator for 48 h;

[0262] (e) Centrifugation at 300 g for 10 min at room temperature to collect the cell culture supernatant;

[0263] (f) The cytokine content in the supernatant was detected using a CBA kit (purchased from BD).

[0264] The results are shown in Figures 8-12, wherein Figure 8 shows that Cross3×AHF213391:1, Cross3×AHF213392:1, UCHT1×AHF213391:1, and UCHT1×AHF213392:1 antibodies can activate PBMCs in the presence of target protein (FAP) and promote their secretion of cytokines IL-2, IFN-γ, IL-6, and TNF-α, and the activity of Cross3 bispecific antibody is better than that of UCHT1 bispecific antibody, and Cross3×AHF213391:1 has the best activity; Figure 9 shows that Cross3×AHF213391:1, Cross313×AHF21339, Cross316×AHF21339, and Cross325×AHF21339 antibodies can activate PBMCs in the presence of target protein (FAP) and promote their secretion of IL-2; Figure 10 shows that Cross3×AHF213 391:1, Cross313×AHF21339, Cross316×AHF21339, and Cross325×AHF21339 antibodies can activate PBMCs and secrete IFN-γ in the presence of target protein (FAP); Figure 11 shows the results of Cross3×AHF213391:1, Cross313×AHF21339, Cross316×AHF21339, and Cross325 ×AHF21339 antibody can activate PBMC and secrete TNFα in the presence of target protein (FAP); Figure 12 shows that Cross3×AHF213391:1, Cross313×AHF21339, Cross316×AHF21339, and Cross325×AHF21339 antibodies can activate PBMC and secrete the proinflammatory cytokine IL-6 in the presence of target protein (FAP).

[0265] In summary, Cross313×AHF21339, Cross316×AHF21339, and Cross325×AHF21339 antibodies have high safety while maintaining high tumor cell-killing ability.

[0266] Example 4 Bispecific Antibody Promotes T Cell Proliferation Experiment

[0267] PBMC cells were labeled with CFSE fluorescein, and the bispecific antibody was added to the PBMC and A-375-FAP cell co-incubation system. After 72 hours of culture, the CFSE fluorescence intensity in CD4 T cells and CD8 T cells was detected by flow cytometry to determine the characteristics of the bispecific antibody in inducing T cell proliferation.

[0268] (1) A-375-FAP cells were diluted to 1×10 using complete RPMI 1640 medium. 5 / ml, added to 96-well plate;

[0269] (2) The bispecific antibodies Cross3×AHF213391:1, Cross3×AHF213392:1, UCHT1×AHF213391:1, UCHT1×AHF213392:1, Cross313×AHF21339, Cross316×AHF21339, Cross325×AHF21339, and control hIgG1LALA (purchased from Bio-Bio) were serially diluted in complete RPMI 1640 medium and added to a 96-well plate at 20 μl / well.

[0270] (3) PBMCs were labeled with 5 μM CFSE. After labeling, PBMCs (purchased from Saili Biotechnology) were diluted to 1.25×10 6 / ml, added to 96-well plate, 80 μl / well;

[0271] (4) The 96-well plate was incubated in a 37°C, 5% CO2 incubator for 72 h;

[0272] (5) Add CD8 antibody labeled with PerCP-Cy5.5 and CD4 antibody labeled with BV605 and incubate at 4°C in the dark for 30 min.

[0273] (6) Wash twice with PBS and discard the supernatant after centrifugation.

[0274] (7) Resuspend the cells in 200 μl / tube of PBS and analyze them using a flow cytometer.

[0275] The results are shown in Figures 13-15, where Figure 13 shows that Cross3×AHF213391:1, Cross3×AHF213392:1, UCHT1×AHF213391:1, and UCHT1×AHF213392:1 antibodies can promote the activation of CD4 and CD8 T cells, and the activity of Cross3 bispecific antibody is better than that of UCHT1 bispecific antibody, and Cross3×AHF213391:1 has the best activity; Figure 14 shows that Cross3×AHF213391:1, Cross313×AHF21339, Cross316×AHF21339, and Cross325×AHF21339 antibodies can all promote the activation of CD4 T cell proliferation; Figure 15 shows that Cross3×AHF213391:1, Cross313×AHF21339, Cross316×AHF21339, and Cross325×AHF21339 antibodies can all promote CD8 T cell proliferation.

[0276] Example 5: Experiment on Bispecific Antibodies Promoting PBMC to Kill FAP-Positive Cells

[0277] The ability of bispecific antibodies to promote PBMC to kill A-375-FAP human melanoma cells was tested.

[0278] (a) Complete RPMI-1640 medium was added to a 16-well RTCA plate at a volume of 50 μL / well and calibrated on the instrument.

[0279] (b) A-375-FAP tumor cells were diluted to 2×10 5 / mL, and added to the RTCA plate obtained in step (1) at a volume of 50 μL / well, and then the cell coefficient was detected using the xCELLigence RTCAMP device at 37°C and 5% CO2 for 24 h;

[0280] (c) Add serially diluted bispecific antibodies Cross3×AHF21339 1:1, Cross3×AHF21339 2:1, UCHT1×AHF21339 1:1, UCHT1×AHF21339 2:1, Cross313×AHF21339, Cross316×AHF21339, Cross325×AHF21339, and control hIgG1LALA (purchased from Bio-Tech) to the RTCA plate obtained in step (b) using complete RPMI-1640 medium at a volume of 20 μl / well;

[0281] (d) PBMC (purchased from Saili Biotechnology) were diluted to 1.25×106 pcs / ml, and added to the RTCA plate obtained in step (c) at a volume of 80 μl / well;

[0282] (e) The reaction system obtained in step (d) was incubated at 37° C., 5% CO 2 and the cell coefficient was detected using an xCELLigence RTCAMP instrument for 24 h.

[0283] The results are shown in Figure 16. Cross3×AHF213391:1, Cross3×AHF213392:1, UCHT1×AHF213391:1, and UCHT1×AHF213392:1 antibodies can promote PBMC to kill A-375-FAP cells. The 1:1 bispecific antibody has a stronger killing ability than the 2:1 bispecific antibody. The Cross3 bispecific antibody is better than the UCHT1 bispecific antibody, and the Cross3×AHF213391:1 has the highest killing activity. The results are shown in Figure 17. Cross3×AHF21339 1:1, Cross313×AHF21339, Cross316×AHF21339, and Cross325×AHF21339 antibodies can all promote PBMC to kill A-375-FAP cells. The killing activities of Cross313×AHF21339, Cross316×AHF21339, and Cross325×AHF21339 antibodies are similar, weaker than Cross3×AHF21339 1:1, but all can promote complete killing.

[0284] Example 6: ELISA binding experiment of the antibody of the present invention

[0285] ELISA is used to test the binding properties of bispecific antibodies. The antigen protein is coated onto a 96-well plate, and the strength of the signal after the antibody is added is used to determine the binding properties of the bispecific antibody and the antigen protein.

[0286] (1) Dilute FAP-His protein (purchased from Acro) to 2 μg / ml with PBS buffer and add 100 μl / well to a 96-well plate. Place at 4°C overnight. Aspirate the PBS buffer in the 96-well plate, wash the plate 6 times with PBST (pH 7.2 PBS containing 0.1% Tween 20) buffer, add 200 μl / well PBS / 10% BSA, and incubate at 37°C for 2 hours for blocking. Remove the blocking solution, wash the plate 6 times with PBST, and add 100 μl / well of the bispecific antibodies to be tested, Cross3×AHF21339 1:1, Cross313×AHF21339, Cross316×AHF21339, Cross325×AHF21339, and control hIgG1LALA (purchased from Bio-Bio), which were serially diluted with PBST / 0.05% BSA, and incubate at 37°C for 1 hour. Remove the reaction mixture, wash the plate six times with PBST, and then add 100 μl / well of HRP (horseradish peroxidase)-labeled anti-human IgG secondary antibody (purchased from JacksonLab) diluted in PBST / 0.05% BSA. Incubate at 37°C for 1 hour. Wash the plate six times with PBST, then add 80 μl / well of TMB (tetramethylbenzidine), incubate at room temperature for 3 minutes, and terminate the reaction by adding 80 μl / well of 4M sulfuric acid. Read the absorbance at 450 nm using a microplate reader.

[0287] The results are shown in Figure 18. Cross3×AHF213391:1, Cross313×AHF21339, Cross316×AHF21339, and Cross325×AHF21339 antibodies can all bind to FAP protein with similar binding abilities.

[0288] (2) Dilute CD3E&D protein (purchased from Acro) to 2 μg / ml with PBS buffer and add it to a 96-well plate at a volume of 100 μl / well. Place it at 4°C overnight. Aspirate the PBS buffer in the 96-well plate, wash the plate 6 times with PBST (pH 7.2 PBS containing 0.1% Tween 20) buffer, add 200 μl / well PBS / 10% BSA, and incubate at 37°C for 2 hours for blocking. Remove the blocking solution, wash the plate 6 times with PBST, and add 100 μl / well of the bispecific antibodies to be tested Cross3×AHF21339 1:1, Cross313×AHF21339, Cross316×AHF21339, Cross325×AHF21339, and control hIgG1LALA (purchased from Bio-Bio) serially diluted with PBST / 0.05% BSA, and incubate at 37°C for 1 hour. Remove the reaction mixture, wash the plate six times with PBST, and then add 100 μl / well of HRP (horseradish peroxidase)-conjugated anti-human IgG secondary antibody (purchased from Southern Biotech) diluted in PBST / 0.05% BSA. Incubate at 37°C for 1 hour. Wash the plate six times with PBST, then add 80 μl / well of TMB (tetramethylbenzidine) and incubate at room temperature for 3 minutes. Terminate the reaction by adding 80 μl / well of 4 M sulfuric acid. Read the absorbance at 450 nm using a microplate reader.

[0289] As shown in Figure 19, the results of the present invention Cross3×AHF213391:1, Cross313×AHF21339, Cross316×AHF21339, and Cross325×AHF21339 antibodies can all bind to CD3E&D proteins, and the affinity of Cross313×AHF21339, Cross316×AHF21339, and Cross325×AHF21339 antibodies for binding to CD3 is weaker than that of Cross3×AHF213391:1.

[0290] Example 7: Bispecific Antibody Promotes T Cell Activation Experiment

[0291] The bispecific antibody was added to the PBMC and A-375-FAP cell co-incubation system. After 48 hours of culture, the expression of CD4 T cells and CD8 T cell surface activation markers CD25 and CD69 was detected by flow cytometry to determine the characteristics of bispecific antibody-induced T cell activation.

[0292] (1) A-375-FAP cells were diluted to 1×10 using complete RPMI 1640 medium. 5 / ml, added to 96-well plate;

[0293] (2) The bispecific antibodies Cross3×AHF21339 (1:1), Cross313×AHF21339, Cross316×AHF21339, Cross325×AHF21339, and control hIgG1LALA (purchased from Bio-Bio) were serially diluted in complete RPMI 1640 medium and added to a 96-well plate at 20 μl / well.

[0294] (3) PBMC (purchased from Saili Biotechnology) were diluted to 1.25 × 106 / ml using complete RPMI 1640 medium and added to a 96-well plate at 80 μl / well;

[0295] (4) Incubate the 96-well plate in a 37°C, 5% CO2 incubator for 48 h;

[0296] (5) Add CD4, CD8, CD25, and CD69 antibodies and incubate at 4°C in the dark for 30 min.

[0297] (6) Wash twice with PBS and discard the supernatant after centrifugation.

[0298] (7) Resuspend the cells in 200 μl / tube of PBS and analyze them using a flow cytometer.

[0299] The results are shown in Figures 20-23, wherein Figure 20 shows that Cross3×AHF213391:1, Cross313×AHF21339, Cross316×AHF21339, and Cross325×AHF21339 antibodies can all promote CD4 T cell activation and expression of CD25; Figure 21 shows that Cross3×AHF213391:1, Cross313×AHF21339, Cross316×AHF21339, and Cross325×AHF21339 antibodies can all promote CD8 T cell activation and expression of CD25; Figure 22 shows that Cross3×AHF213391:1, Cross313×AHF21339, Cross316×AHF21339, and Cross325×AHF21339 antibodies can all promote CD4 T cells are activated and express CD69; Figure 23 shows that Cross3×AHF213391:1, Cross313×AHF21339, Cross316×AHF21339, and Cross325×AHF21339 antibodies can all promote CD8 T cell activation and expression of CD69.

[0300] The above experimental results show that the bispecific antibody of the present invention can bind to CD3 and FAP, thereby promoting T cell activation and cytokine secretion, effectively promoting PBMC to kill tumor cells, and has excellent anti-cancer activity. It can achieve high-affinity binding to FAP and low-affinity binding to CD3, resulting in higher anti-cancer activity. In summary, the bispecific antibody of the present invention can promote the anti-cancer effect of immune cells, has good anti-cancer activity, and has a higher safety profile, with good clinical application value and drug development value.

[0301] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", "some implementation plans" or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0302] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A recombinant antibody, characterized in that: At least: First binding region: The first binding region includes an antibody or antigen binding fragment that specifically recognizes CD3; and Second binding region: The second binding region includes an antibody or antigen-binding fragment that specifically recognizes FAP, in The antibody or antigen-binding fragment that specifically recognizes CD3 in the first binding region includes a scFV region and a first Fc region. The antibody or antigen-binding fragment that specifically recognizes FAP in the second binding region includes a Fab region and a second Fc region.

2. The recombinant antibody according to claim 1, wherein the C-terminus of the scFV region in the first binding region is connected to the N-terminus of the first Fc region; Optionally, the C-terminus of the heavy chain variable region of the scFV region is connected to the N-terminus of the light chain variable region via a connecting peptide 1, and the C-terminus of the light chain variable region is connected to the N-terminus of the first Fc region via a connecting peptide 2.

3. The recombinant antibody according to claim 1, characterized in that The antibody or antigen-binding fragment that specifically recognizes FAP includes: The light chain variable region CDR1, CDR2, and CDR3 sequences are shown in SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively, and the heavy chain variable region CDR1, CDR2, and CDR3 sequences are shown in SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively; Optionally, the antibody or antigen-binding fragment that specifically recognizes FAP has a light chain variable region as shown in SEQ ID NO: 31, or an amino acid sequence that is at least 80% identical to the sequence shown in SEQ ID NO: 31, and a heavy chain variable region as shown in SEQ ID NO: 32, or an amino acid sequence that is at least 80% identical to the sequence shown in SEQ ID NO: 32; Optionally, the antibody or antigen-binding fragment that specifically recognizes FAP has a light chain as shown in SEQ ID NO:20 or an amino acid sequence that is at least 80% identical to the sequence shown in SEQ ID NO:20, and a heavy chain as shown in SEQ ID NO:19 or an amino acid sequence that is at least 80% identical to the sequence shown in SEQ ID NO:

19.

4. The recombinant antibody according to claim 1, characterized in that The antibody or antigen-binding fragment that specifically recognizes CD3 includes: The light chain variable region CDR1, CDR2, and CDR3 sequences are shown in SEQ ID NO:4, GTN, and SEQ ID NO:5, respectively; the heavy chain variable region CDR1, CDR2, and CDR3 sequences are shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; or, The light chain variable region CDR1, CDR2, and CDR3 sequences are shown in SEQ ID NO:4, GTN, and SEQ ID NO:5, respectively; the heavy chain variable region CDR1, CDR2, and CDR3 sequences are shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:6, respectively; or, The light chain variable region CDR1, CDR2, and CDR3 sequences are shown in SEQ ID NO:4, GTN, and SEQ ID NO:8, respectively; and the heavy chain variable region CDR1, CDR2, and CDR3 sequences are shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:7, respectively.

5. The recombinant antibody according to any one of claims 1 to 4, characterized in that The antibody or antigen-binding fragment that specifically recognizes CD3 includes any one of the following: (a) having 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) having 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) having 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 with any one of (a), (b) and (c).

6. The recombinant antibody according to claim 1, characterized in that The first Fc region and the second Fc region are connected via a knob-into-hole structure; Optionally, at least a portion of the first Fc region and the second Fc region are derived from at least one of a murine antibody, a primate antibody, or a mutant thereof; Optionally, at least a portion of the first Fc region and the second Fc region are derived from human IgG1 or a mutant thereof; Optionally, the first Fc region has the amino acid sequence shown in SEQ ID NO:33, and / or the second Fc region has the amino acid sequence shown in SEQ ID NO:

34.

7. The recombinant antibody according to claim 2, characterized in that The connecting peptides 1 and 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 connecting peptide 2 has an amino acid sequence as shown in SEQ ID NO:

36.

8. The recombinant antibody according to claim 1, characterized in that 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 that has at least 80% identity with SEQ ID NO:15, SEQ ID NO:16 or SEQ ID NO:

17.

9. The recombinant antibody according to claim 1, characterized in that The recombinant antibody comprises any one of the following: (d) the amino acid sequence shown in SEQ ID NO: 15, SEQ ID NO: 19 and SEQ ID NO: 20, or, (e) the amino acid sequence shown in SEQ ID NO: 16, SEQ ID NO: 19 and SEQ ID NO: 20, or, (f) the amino acid sequence 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 with any one of (d), (e) and (f).

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

11. The recombinant antibody according to claim 10, characterized in that The antibody or antigen-binding fragment that specifically recognizes FAP in the third binding region comprises a Fab region, and the heavy chain CH1 region of the Fab region is connected to the scFV region of the antibody or antigen-binding fragment that specifically recognizes CD3 in the first binding region.

12. The recombinant antibody according to claim 11, characterized in that The C-terminus of the heavy chain CH1 region of the Fab region of the antibody or antigen-binding fragment that specifically recognizes FAP in the third binding region is connected to the heavy chain variable region of the scFV region of the antibody or antigen-binding fragment that specifically recognizes CD3 in the first binding region; Optionally, the amino acid sequence of the Fab region of the antibody or antigen-binding fragment that specifically recognizes FAP of the third binding region is the same as or different from the amino acid sequence of the Fab region of the antibody or antigen-binding fragment that specifically recognizes FAP of the second binding region.

13. An isolated polynucleotide, characterized in that The polynucleotide encodes the recombinant antibody according to any one of claims 1 to 12.

14. An expression vector, characterized in that: Carrying the polynucleotide of claim 13.

15. A recombinant cell, characterized in that Carrying the polynucleotide of claim 13 or the expression vector of claim 14; or Capable of expressing the recombinant antibody according to any one of claims 1 to 12.

16. The recombinant cell according to claim 15, characterized in that The recombinant cell is obtained by introducing the expression vector according to 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, characterized in that The method comprises at least one of the recombinant antibody according to any one of claims 1 to 12, the polynucleotide according to claim 13, the expression vector according to claim 14, and the recombinant cell according to claim 15 or 16.

18. A method for preparing the recombinant antibody according to any one of claims 1 to 12, characterized in that: The method comprises culturing the recombinant cell of claim 15 or 16 and isolating the antibody or antigen-binding fragment of the recombinant cell.

19. A drug, characterized in that The method comprises at least one of the recombinant antibody according to any one of claims 1 to 12, the polynucleotide according to claim 13, the expression vector according to claim 14, the recombinant cell according to claim 15 or 16, or the composition according to claim 17.

20. A kit, characterized in that The kit comprises at least one of the recombinant antibody according to any one of claims 1 to 12, the polynucleotide according to claim 13, the expression vector according to claim 14, and the recombinant cell according to claim 15 or 16.

21. Use of the recombinant antibody according to any one of claims 1 to 12, the polynucleotide according to claim 13, the expression vector according to claim 14, the recombinant cell according to claim 15 or 16, or the composition according to claim 17 in the preparation of a medicament for preventing and / or treating CD3 and / or FAP-mediated related diseases; Optionally, the CD3-mediated related diseases include autoimmune diseases; Optionally, the autoimmune disease comprises at least one of the following: systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis and acute idiopathic polyneuritis; Optionally, the FAP-mediated related disease comprises a tumor, wherein the tumor-associated fibroblasts of the tumor are positive for FAP; Optionally, the tumor-induced disease comprises lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney 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, characterized in that: The kit comprises at least one of the recombinant antibody according to any one of claims 1 to 12, the polynucleotide according to claim 13, the expression vector according to claim 14, and the recombinant cell according to claim 15 or 16.

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

24. An immunoconjugate, characterized in that The immunoconjugate contains a therapeutic agent and the recombinant antibody according to any one of claims 1 to 12 coupled to the therapeutic agent.

25. The immunoconjugate according to claim 24, characterized in that The therapeutic agent includes any one of a polypeptide, a radionuclide, and a small molecule.

26. Use of the recombinant antibody according to any one of claims 1 to 12, the polynucleotide according to claim 13, the expression vector according to claim 14, the recombinant cell according to claim 15 or 16, or the composition according to claim 17 in preventing and / or treating CD3 and / or FAP-mediated related diseases.

27. The recombinant antibody according to any one of claims 1 to 12, the polynucleotide according to claim 13, the expression vector according to claim 14, the recombinant cell according to claim 15 or 16, or the composition according to claim 17, for use in preventing and / or treating CD3 and / or FAP-mediated related diseases.

28. Use of the recombinant antibody according to any one of claims 1 to 12, the polynucleotide according to claim 13, or the kit according to claim 22 in diagnosing whether a subject suffers from a CD3 and / or FAP-mediated related disease.

29. The use according to any one of claims 26 to 28, characterized in that The CD3-mediated related diseases include autoimmune diseases; Optionally, the autoimmune disease comprises at least one of the following: systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis and acute idiopathic polyneuritis; Optionally, the FAP-mediated related disease comprises a tumor, wherein the tumor-associated fibroblasts of the tumor are positive for FAP; Optionally, the tumor-induced disease comprises lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, melanoma, oral squamous cell carcinoma or head and neck cancer.

30. A method for preventing and / or treating CD3 and / or FAP-mediated related diseases, characterized in that: The method comprises: administering to a subject a pharmaceutically acceptable amount of the recombinant antibody of any one of claims 1 to 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, or the drug of claim 19.

31. A method for diagnosing whether a subject suffers from a CD3 and / or FAP-mediated disease, characterized in that: The method comprises using the recombinant antibody according to any one of claims 1 to 12, the polynucleotide according to claim 13 or the kit according to claim 22 to detect a test sample from the subject.

32. The method according to claim 30 or 31, characterized in that The CD3-mediated related diseases include autoimmune diseases; Optionally, the autoimmune disease comprises at least one of the following: systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis and acute idiopathic polyneuritis; Optionally, the FAP-mediated related disease comprises a tumor, wherein the tumor-associated fibroblasts of the tumor are positive for FAP; Optionally, the tumor-induced disease comprises lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, melanoma, oral squamous cell carcinoma or head and neck cancer.

33. Use of the recombinant antibody according to any one of claims 1 to 12, the polynucleotide according to claim 13, or the kit according to claim 22 in detecting CD3 and / or FAP in a sample to be tested.

34. A method for detecting CD3 and / or FAP, characterized in that: The method comprises using the recombinant antibody according to any one of claims 1 to 12, the polynucleotide according to claim 13 or the kit according to claim 22 to detect the sample to be tested.

35. The method according to claim 34, characterized in that The sample to be tested includes at least one of the following: tissue, cell, blood, serum, plasma, saliva, sweat, feces or urine.

Images