Antigen binding protein targeting CD26 and drug application thereof

Abstract

Relates to antigen binding proteins combined with human CD26 and application of the antigen binding proteins in preparation of drugs for treating tumors with high CD26 expression. The invention for the first time discloses a CD26 antigen binding protein which recognizes the same CD26 fragment or competes with antigen binding proteins comprising HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, HCDR3 as shown in SEQ ID NO: 3, LCDR2 comprising LCDR1 as shown in SEQ ID NO: 4 and consisting of Arg Met Ser, and LCDR3 as shown in SEQ ID NO: 5. Compared with other mouse antibodies and corresponding chimeric antibodies or humanized antibodies, the CD26 antigen binding protein combined with amino acid residues in the amino acid sequence shown as SEQ ID NO: 59 or SEQ ID NO: 64 of CD26 shows better antigen affinity and good safety and effectiveness. And compared with the existing first-line treatment scheme (such as BAVENCIO for treating kidney cancer) of related tumors, the compound also shows more prominent effectiveness and safety.

Description

Antigen binding proteins targeting CD26 and their pharmaceutical applications Technical Field

[0001] The present invention relates to an antibody or antigen-binding protein binding to human CD26, and application thereof in preparing a medicine for treating tumors with high CD26 expression. Background Art

[0002] CD26 is a multifunctional type II transmembrane glycoprotein that can also be found in a soluble form in plasma. CD26 typically exists as a homodimer, with a monomer consisting of 766 amino acids and a relative molecular mass of approximately 110 kDa. CD26 amino acid residues are divided into five regions from the inside out: an intracellular region (1-6), a transmembrane region (7-28), a heavily glycosylated region (29-323), a cysteine-rich region (324-551), and a C-terminal catalytic domain (552-766). Its three-dimensional molecular structure is closely related to its function. CD26 (DPP4) inhibitors have been used clinically for decades to treat type 2 diabetes. The expression level of CD26 on the surface of various tumor cells is significantly increased, for example, malignant mesothelioma, renal cancer, prostate cancer, lung cancer, etc. For these types of tumors with high CD26 expression, CD26 is an important target (CD26 / DPP4-a potential biomarker and target for cancer therapy, Pharmacology & Therapeutics (2019), 198:135-159).

[0003] Currently, the most rapidly developing targeted anti-cancer drug targeting human CD26 is the monoclonal antibody YS110 from Y'S Therapeutics, which has completed Phase I / II clinical trials. However, according to analysis of existing literature, preclinical studies of YS110 have shown low in vitro and in vivo activity. For example, in the paper "A humanized anti-CD26 monoclonal antibody inhibits cell growth of malignant mesothelioma via retarded G2 / M cell cycle transition, Cancer Cell Int (2016) 16:35", at a concentration of 250ug / ml, after 48 hours of action, the inhibition rate on tumor cell growth was 18.3%, and the IC 50The value is far higher than 250ug / ml, indicating its low in vitro activity. For example, the patent "Anti-CD26 Antibodies and Methods of Use Thereof (Application Number: CN200680034937.4)" demonstrates YS110's ability to treat mouse tumor models harboring multiple CD26-overexpressing cell lines. YS110 only partially reduced tumor size and alleviated tumor growth, but failed to completely inhibit tumor growth and achieve a cure, demonstrating that its activity in animal models remains suboptimal. The results of a completed Phase II clinical study of YS110 showed good tolerability, but the disease control rate fell short of expectations, consistent with the low activity demonstrated in preclinical studies.

[0004] Although the widespread distribution of CD26 has been reported in the literature, the use of YS110 still has good safety, which shows that its safety in clinical use is well guaranteed. Among the 31 patients in the YS110 clinical trial, no patient died. The main side effects were 16.1% of patients had adverse reactions to the infusion, 9.7% of patients had hiccups, and 6.5% of patients had diarrhea. (Phase 2 Study of YS110, a Recombinant Humanized Anti-CD26 Monoclonal Antibody, in Japanese Patients With Advanced Malignant Pleural Mesothelioma, JTO Clinincal and Research Reports (2021), 2(6))

[0005] In summary, although the safety of the CD26 target has been verified in clinical trials, existing antibodies targeting CD26 are difficult to achieve good therapeutic effects on tumors.

[0006] Antigens play a crucial role in stimulating and initiating the immune response. Antigens are substances that stimulate a specific immune response and bind to the immune response products, antibodies or sensitized lymphocytes, to produce immune effects. These substances typically possess two inherent properties: immunogenicity and reactogenicity. In reality, antigens don't exert their effects as intact molecules; their function and specificity are typically achieved through their epitopes. Immune cells initiate immune responses by binding to and recognizing epitopes on antigen molecules.

[0007] An epitope, also known as an antigenic determinant, refers to a specific sequence of amino acid residues and their spatial structure within an antigen molecule that can induce an immune response. An antigen molecule contains multiple epitopes, including those that stimulate a protective immune response and those that are detrimental to protective immune responses, such as inhibitory and toxic epitopes. It is also recognized that the numerous epitopes within a molecule contribute differently to antigenicity, resulting in dominant and non-dominant epitopes. Furthermore, the relative position, conformational characteristics, and flanking sequence of epitopes are closely related to the expression of epitope function.

[0008] Summary of the Invention

[0009] To solve the above problems, the present invention provides an isolated polypeptide having an amino acid sequence identical to or at least 90% identical to the sequence shown in SEQ ID NO: 59 or SEQ ID NO: 64.

[0010] The present invention also provides a more effective and safer CD26 antibody or antigen-binding fragment. The antigen-binding protein of the present invention that binds to CD26 recognizes amino acid residues within the amino acid sequence of SEQ ID NO: 59 or SEQ ID NO: 64. The phrase "recognizing the region of the amino acid sequence of CD26 set forth in SEQ ID NO: 59 or SEQ ID NO: 64" refers to the ability to bind to some or all of the amino acid residues within the amino acid sequence of CD26 set forth in SEQ ID NO: 59 or SEQ ID NO: 64.

[0011] The antigen-binding protein of the present invention that binds to CD26 competes for CD26 binding with an antigen-binding protein comprising the HCDR1 of SEQ ID NO: 1, the HCDR2 of SEQ ID NO: 2, the HCDR3 of SEQ ID NO: 3, and the LCDR1 of SEQ ID NO: 4, the LCDR2 consisting of Arg Met Ser, and the LCDR3 of SEQ ID NO: 5, such as the mouse antibody No. 79 or 19G300 exemplified in the Examples.

[0012] The antigen-binding protein of the present invention is the same as the CD26 fragment recognized by the antigen-binding protein comprising HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, HCDR3 shown in SEQ ID NO: 3, LCDR1 shown in SEQ ID NO: 4, LCDR2 composed of Arg Met Ser, and LCDR3 shown in SEQ ID NO: 5.

[0013] The antigen-binding protein of the present invention binds to the amino acid residues in the amino acid sequence of SEQ ID NO: 64. The term "bind" means that it can bind to part or all of the amino acid residues in the amino acid sequence of SEQ ID NO: 64.

[0014] The antigen-binding protein of the present invention binds to the amino acid residues in the amino acid sequence of SEQ ID NO: 59. The term "bind" means that it can bind to part or all of the amino acid residues in the amino acid sequence of SEQ ID NO: 59.

[0015] Methods known in the art can be used to determine whether an antigen binding protein recognizes the same CD26 fragment as or competes with an antigen binding protein comprising HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, HCDR3 shown in SEQ ID NO: 3, and LCDR1 shown in SEQ ID NO: 4, LCDR2 consisting of Arg Met Ser, and LCDR3 shown in SEQ ID NO: 5. For example, an antigen-binding protein comprising an amino acid sequence comprising HCDR1 of SEQ ID NO:1, HCDR2 of SEQ ID NO:2, HCDR3 of SEQ ID NO:3, and LCDR1 of SEQ ID NO:4, LCDR2 consisting of Arg Met Ser, and LCDR3 of SEQ ID NO:5 is bound to CD26 protein until saturation is achieved. The binding ability of the antigen-binding protein to the CD26 protein is evaluated under this saturation state. If the antigen-binding protein is still able to bind to the CD26 protein, it can be concluded that the antigen-binding protein recognizes a different CD26 fragment than the antigen-binding protein comprising HCDR1 of SEQ ID NO:1, HCDR2 of SEQ ID NO:2, HCDR3 of SEQ ID NO:3, and LCDR1 of SEQ ID NO:4, LCDR2 consisting of Arg Met Ser, and LCDR3 of SEQ ID NO:5. Conversely, if the antigen-binding protein is unable to bind to the CD26 protein under the saturation state, then the antigen-binding protein recognizes a different CD26 fragment than the antigen-binding protein comprising HCDR1 of SEQ ID NO:1, HCDR2 of SEQ ID NO:2, HCDR3 of SEQ ID NO:3, and LCDR1 of SEQ ID NO:4, LCDR2 consisting of Arg Met Ser, and LCDR3 of SEQ ID NO:5. The HCDR3 shown in SEQ ID NO:3 and an antigen-binding protein comprising the LCDR1 shown in SEQ ID NO:4, the LCDR2 consisting of Arg Met Ser, and the LCDR3 shown in SEQ ID NO:5 can compete for binding to the same CD26 fragment. Other routine experiments (e.g., peptide mutagenesis and binding analysis) can then be performed to confirm whether the inability of an antigen-binding protein to bind to the CD26 protein under the above-mentioned saturation state is due to both recognizing the same CD26 fragment or due to steric hindrance. For example, ELISA, RIA, Biacore, flow cytometry, or any other quantitative or qualitative antibody binding assay known in the art can be used.

[0016] The identification of CD26 protein fragments recognized or bound by the antigen-binding protein can be carried out by methods such as epitope mapping, X-ray crystallography, enzymatic hydrolysis and chemical cleavage, short peptide synthesis, phage presentation of random peptide libraries, alanine scanning, HDX-MS, and bioinformatics epitope prediction. In specific Examples 26 and 27 of the present application, the binding of an antigen-binding protein comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, HCDR3 of SEQ ID NO: 3, LCDR1 of SEQ ID NO: 4, LCDR2 composed of Arg Met Ser, and LCDR3 of SEQ ID NO: 5, as well as another antigen-binding protein to CD26 fragments was verified by enzymatic cleavage of CD26 and synthesis of CD26 protein fragments.

[0017] To prepare antibodies that compete with an antigen-binding protein comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, HCDR3 of SEQ ID NO: 3, and LCDR1 of SEQ ID NO: 4, LCDR2 consisting of Arg Met Ser, and LCDR3 of SEQ ID NO: 5 for binding to CD26, or that recognize the same CD26 fragment, the animal immunization method described in Example 1 can be used. Specifically, a CD26 fragment recognized by an antigen-binding protein comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, HCDR3 of SEQ ID NO: 3, and LCDR1 of SEQ ID NO: 4, LCDR2 consisting of Arg Met Ser, and LCDR3 of SEQ ID NO: 5 is used as an immunogen to prepare CD26 monoclonal antibodies using existing methods, and then in vivo or in vitro experiments are performed to verify the anti-tumor efficacy and safety of the antibodies. In addition, other methods known in the art can also be used to prepare antibodies.

[0018] The antigen-binding protein of the present invention is a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, a chimeric antibody, a multispecific antibody or an antibody fragment thereof.

[0019] The antigen-binding protein of the present invention is a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment, a scFv, a diabody or a single-chain antibody molecule.

[0020] The antigen-binding protein of the present invention is an IgG1, IgG2, IgG3 or IgG4 antibody.

[0021] An isolated nucleic acid selected from any one of a), b) or c):

[0022] a) a DNA encoding an antigen-binding protein, which is the above-mentioned antigen-binding protein that binds to CD26;

[0023] b) a DNA encoding a polypeptide having an amino acid sequence at least 90% identical to SEQ ID NO: 59 or SEQ ID NO: 64;

[0024] c) a DNA completely complementary to the DNA described in a or b;

[0025] d) RNA of a, b, or c, wherein U replaces T.

[0026] A vector comprising the above nucleic acid.

[0027] A cell comprising the above-mentioned vector.

[0028] Nucleic acid molecules encoding target antigen-binding proteins can be administered as part of a viral or non-viral vector, using a vector to deliver the nucleic acid encoding the target antigen-binding protein to the subject. Various viral vectors that have been successfully used to introduce nucleic acids into hosts include retroviruses, adenoviruses, adeno-associated viruses (AAV), herpes viruses, and pox viruses, among others.

[0029] A composition comprising any one of the above antigen-binding proteins, nucleic acids or cells.

[0030] A method for treating a disease associated with elevated CD26 levels, comprising administering to a subject an effective amount of any one of the above-described antigen-binding proteins, nucleic acids, or cells. Diseases associated with elevated CD26 levels include, but are not limited to, tumors, and tumors with elevated CD26 levels include, but are not limited to, renal cancer, mesothelioma, lung cancer, liver cancer, and prostate cancer. The antibody or antigen-binding fragment is administered alone or in combination with other anticancer agents.

[0031] Compared with the existing technology, this application has the following advantages:

[0032] This patent discloses for the first time that antibodies that compete with an antigen-binding protein comprising HCDR1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, HCDR3 of SEQ ID NO: 3, LCDR1 of SEQ ID NO: 4, LCDR2 consisting of Arg Met Ser, and LCDR3 of SEQ ID NO: 5 for binding to CD26 or recognize the same CD26 fragment, and that CD26 antibodies that bind to amino acid residues within the amino acid sequence of CD26 as shown in SEQ ID NO: 59 or SEQ ID NO: 64 have better anti-tumor efficacy and safety. Specifically:

[0033] Firstly, the antibodies of the present application that bind to specific antigen fragments have obvious advantages in terms of effectiveness.

[0034] The data from Example 1 show that the lysis intensity of mouse antibody No. 62 on human tumor cells is much higher than that of other mouse antibodies, and its killing effect on human tumor cells is comparable to that of the humanized antibody YS110 that has completed phase II clinical trials.

[0035] In addition, the data of Examples 5, 8, and 10 show that the chimeric antibodies or humanized antibodies obtained by transformation based on the variable region of mouse antibody No. 62 have better dot hybridization activity and better affinity with the antigen than the corresponding antibodies derived from other mouse antibodies. In Example 11, when comparing the cytotoxic effects of PBMC mediated by different BiTE antibodies on different target cells in vitro, the BiTE derived from mouse antibody No. 62 was superior to the BiTE whose CD26 antibody sequence was derived from YS110 in killing a variety of tumor cells. The results of Examples 17-24 show that in a variety of animal tumor models, BiTE derived from mouse antibody No. 62 can significantly inhibit tumor growth, and some models can completely inhibit tumor growth, which is significantly better than the BiTE whose CD26 antibody sequence is derived from YS110. The data of Examples 23 and 24 show that in a variety of animal tumor models, BiTE derived from mouse antibody No. 62 showed that it was significantly better than the existing first-line treatment options for tumors (such as BAVENCIO for the treatment of renal cancer) in inhibiting tumor growth.

[0036] Secondly, the antibodies of the present application that bind to specific antigen fragments have obvious advantages in terms of safety.

[0037] Compared with other mouse antibody-derived BiTEs, the BiTE obtained by modifying the variable region of mouse antibody No. 62 of this application shows better safety (Example 13) and a lower risk of causing cytokine storm. The data in Example 12 show that the full-length anti-CD26 IgG antibody cannot effectively induce T cell activation. The CD26-CD3 bispecific antibody with the BiTE structure of this application does not cause obvious T cell activation before reaching the tumor site. Only after the antibody binds to the tumor cells will it cause T cell activation around the tumor tissue, thereby killing the tumor cells, thereby improving the safety of the effect. The data in Examples 23 and 24 show that the bispecific antibody with the BiTE structure of this application shows significantly better safety than the full-length IgG antibody YS110 in a variety of animal tumor models.

[0038] In addition, it comprises HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, HCDR3 shown in SEQ ID NO: 3, and LCDR1 shown in SEQ ID NO: 4, LCDR2 consisting of Arg Met Ser, and SEQ ID NO: 5. The antigen-binding protein of LCDR3 shown in NO:5, such as the different humanized bispecific antibodies (19G292, 19G293, 19G294) derived from mouse antibody No. 62 in Example 10 of this application, recognizes and binds to the CD26 protein without being affected by the binding of different CD3 antibody portions to the CD3 protein; the different humanized bispecific antibodies (19G294, 18G272) that bind to different CD3 proteins all have the advantage of inducing lower amounts of cytokine secretion by T cells (Example 13) and have the same level of in vitro efficacy (Example 16). In vivo experiments also demonstrated that the bispecific antibodies that bind to different CD3 proteins all significantly inhibited tumor growth and had better safety (Example 22), further indicating that the safety and effectiveness of the present application mainly come from the binding of the anti-CD26 antibody CDR region to the CD26 protein fragment. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1: Dot-blot hybridization assay for humanized bispecific antibodies

[0040] Figure 1a shows the dot-blot hybridization activity test of humanized bispecific antibodies 18G272 and 18G278, and Figure 1b shows the dot-blot hybridization activity test of humanized bispecific antibody 19G294.

[0041] Figure 2: Flow cytometry detection of antibody binding specificity to target cell antigens

[0042] Figure 3: 19G294 binds to CD26 and CD3 proteins simultaneously

[0043] Figure 4: Cytotoxic effect of humanized bispecific antibodies on PBMC cells

[0044] Figure 5: Tumor volume of 18G272 and 19G294 in the PC-3 cell NOD / SCID mouse subcutaneous transplantation model

[0045] Figure 6: Animal body weight in the PC-3 cell subcutaneous transplantation model of NOD / SCID mice treated with 18G272 and 19G294

[0046] Figure 7: Tumor volume of 18G272 in the NCI-H596 cell subcutaneous transplantation model in NOD / SCID mice

[0047] Figure 8: Tumor volume of 19G294 in the NCI-H226 cell NOD / SCID mouse subcutaneous transplantation model

[0048] Figure 9: Animal body weight in the NCI-H226 cell subcutaneous transplantation model of NOD / SCID mice

[0049] Figure 10: Tumor volume of 19G294 in A498 cell NOD / SCID mouse subcutaneous transplantation model

[0050] Figure 11: Tumor volume of 17G29 and 19G295 in the OS-RC-2 cell NOD / SCID mouse subcutaneous transplantation model

[0051] Figure 12: Tumor volume of animals in the 18G272 and 19G295 groups in the OS-RC-2 cell NOD / SCID mouse subcutaneous transplantation model

[0052] Figure 13: Survival rates of 18G272 and 19G295 in the OS-RC-2 cell NOD / SCID mouse subcutaneous transplantation model

[0053] Figure 14: Tumor volume of 18G272 in OS-RC-2 cell NOD / SCID mouse subcutaneous transplantation model

[0054] Figure 15: Tumor volume of 19G294 in OS-RC-2 cell NOD / SCID mouse subcutaneous transplantation model

[0055] Figure 16: Tumor volume of animals in the OS-RC-2 cell NOD / SCID mouse subcutaneous transplantation model with different doses of 19G294 and 21G587

[0056] Figure 17: Animal body weight in the OS-RC-2 cell NOD / SCID mouse subcutaneous transplantation model with different doses of 19G294 and 21G587

[0057] Figure 18: Tumor volume of 19G294, BAVENCIO, and axitinib in a subcutaneous transplant model of OS-RC-2 cells in NOD / SCID mice

[0058] Figure 19: Tumor volume of animals 26 days after inoculation with 19G294, BAVENCIO, and axitinib in the OS-RC-2 cell NOD / SCID mouse subcutaneous transplant model

[0059] Figure 20: Tumor volume of animals 26 days after inoculation with 19G294, BAVENCIO, and axitinib in the A498 cell NOD / SCID mouse subcutaneous transplant model

[0060] Figure 21: Tumor weight of animals 65 days after inoculation with 19G294, BAVENCIO, and axitinib in the A498 cell NOD / SCID mouse subcutaneous transplant model

[0061] Figure 22: TIC plot of trypsin digestion amino acid sequence coverage

[0062] Figure 23: Sample trypsin digestion sequence coverage

[0063] Figure 24: CD26 fragment display vector structure

[0064] Figure 25: Binding of 19G294 to CD26 complete extracellular domain and 21G572 display cells

[0065] Figure 26: Binding of 21G428-429 to cells displaying 21G572 and other different truncated CD26 fragments

[0066] Figure 27: Flow cytometry analysis of antibody binding differences in mixed cell systems

[0067] Figure 28: Competitive binding of 19G294, 19G300 and CD26

[0068] Figure 29: Competitive binding of 19G294, 17G29 and CD26 DETAILED DESCRIPTION

[0069] Unless otherwise specifically defined herein, the technical terms used herein have the meanings that are commonly understood by those of ordinary skill in the art.

[0070] Singular forms such as "a," "an," and "the" include their corresponding plural referents.

[0071] The term "or" means and is used interchangeably with "and / or".

[0072] The term "CD26" is also known as dipeptidyl peptidase 4 (DPP4). The amino acid sequence of human CD26 can be found in Genbank under the accession number NP_001926.2, and its cDNA sequence can be found in Genbank under the accession number NM_001935.3.

[0073] The term "conservative amino acid substitution" refers to the replacement of an original amino acid with a new amino acid that does not substantially alter the chemical, physical, and / or functional properties of the antibody or fragment, such as its binding affinity for CD26. Common conservative amino acid substitutions are well known in the art. For example, Ala can be conservatively substituted with Gly or Ser; Arg can be conservatively substituted with Lys or His; Asn can be conservatively substituted with Gln or His, etc. The term "affinity" refers to the strength of the interaction between an antibody and an antigen. Antibody variable regions interact with antigens through non-covalent forces; the greater the interaction, the stronger the affinity.

[0074] The term "antibody" refers to a family of immunoglobulins that can non-covalently, reversibly, and specifically bind to their respective antigens. For example, naturally occurring IgG antibodies are tetramers comprising at least two heavy chains and two light chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain: CL. VH and VL can be further subdivided into highly variable complementarity determining regions (CDRs, also known as hypervariable regions) and more conserved framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. HCDR1, HCDR2, and HCDR3 are the three heavy chain complementary determining regions, and LCDR1, LCDR2, and LCDR3 are the three light chain complementary determining regions. The positions of the CDRs and framework regions can be determined using various numbering systems well known in the art, such as Kabat, Chothia, IMGT, etc., and this application uses IMGT to determine. The heavy chain variable region (VH) and light chain variable region (VL) are responsible for antigen recognition, especially the complementary determining regions (CDRs) therein, which are generally specific for different epitopes of the antigen. The constant region is primarily responsible for effector functions.

[0075] The term "antigen-binding fragment" refers to an antigen-binding fragment of an antibody, that is, an antibody fragment that retains the ability to specifically bind to an antigen, such as a fragment that retains one or more CDR regions, including but not limited to Fab fragments, FV fragments, diabodies, linear antibodies, single-chain antibodies, nanobodies, multispecific antibodies, etc.

[0076] The term "monoclonal antibody" means a substantially homogeneous antibody population, i.e., except for a small amount of possible naturally occurring mutations, the amino acid sequences of the antibody molecules contained in the population are identical. In contrast, polyclonal antibodies typically include a variety of different antibodies with different amino acid sequences in their variable domains (particularly their complementary determining regions). Monoclonal antibodies can be obtained by methods well known to those skilled in the art. The application adopts hybridoma technology to obtain monoclonal antibodies, which is one of the many methods known to obtain monoclonal antibodies.

[0077] The term "murine antibody" refers to an antibody that contains only rat or mouse immunoglobulin sequences.

[0078] The term "chimeric antibody" refers to an antibody in which the variable regions are derived from non-human (eg, murine) antibody sequences, with the remainder derived from human antibody sequences.

[0079] The term "humanized antibody" refers to a antibody in which the CDR regions are derived from non-human (eg, murine) antibody sequences, while the remainder are derived from human antibody sequences.

[0080] The term "bispecific antibody" refers to an antibody that possesses two specific antigen-binding sites for the same or different antigens.

[0081] The term "BiTE" refers to a bispecific antibody, or bispecific T cell engager. It consists of two antigen-specific single-chain variable fragments (scFv) connected by a linker. One scFv targets a tumor-associated surface antigen, while the other targets CD3 on the surface of T cells. A single-chain variable fragment (scFv) is composed of an antibody heavy chain variable region and a light chain variable region connected by a linker. A "linker" is an amino acid sequence used to connect different protein fragments, and extensive research has been conducted on the design and selection of linkers. In a BiTE, the linker connects a heavy chain variable region and a light chain variable region to form an scFv. It also tandemly connects two scFvs and allows them to rotate freely. It typically consists of a repeating structure called GGGGS (S represents serine and G represents glycine). Glycine's small molecular weight and short side chain increase its flexibility, while serine's strong hydrophilicity increases the hydrophilicity of the peptide chain. Optimizing the number of repeat structures can not only help the light chain and heavy chain in scFv to associate in the correct conformation, but also promote the optimal interaction between T cells and target cells in the immune synapse by affecting the distance between the two scFvs (Zhou Chong et al. Research progress of bispecific single-chain antibody BiTE [J]. Journal of Biology, 2018). Common linkers connecting the heavy chain variable region and the light chain variable region in scFv are KESGSVSSEQLAQFRSLD, EGKSSGSGSESKST, GSTSGGGSGGGSGGGGSS (US20180326032), GSTSGSGKPGSGEGSTKG (Preclinical Development of Bivalent Chimeric Antigen Receptors Targeting Both CD19 and CD22), etc. In a specific embodiment of the present application, the flexible linker connecting a heavy chain and a light chain to form an scFv is composed of three GGGGS repeating structures, and the flexible linker connecting two scFvs in series is GGGGS. Those skilled in the art can select and determine from known linkers based on the teachings of existing technology through routine experimental methods and limited trials, and can optimize the number of GGGGS repeats in the two flexible linkers, or use flexible linkers other than GGGGS.

[0082] CD3 is an integral part of T cell signaling. When the BiTE molecule binds to T cells and tumor cells at the same time, the T cells are activated, promoting CD8+ T cells to directly secrete perforin and granzymes, and CD4+ T cells to secrete cytokines to further recruit activated killer T cells, thereby killing tumor cells. The CD3 antibody sequence can be selected from OKT-3, L2K, TR66, UCHT1, SP34, IORT3, Catumaxomab, Blinatumomab, Solitomab, WBP3311_2.306.4 (patent CN201880061333.1) and other CD3 antibody sequences known in the art. In the specific embodiments of the present application, the CD3 antibody sequences of Solitomab and WBP3311_2.306.4 are used. Those skilled in the art can select and determine from other known CD3 antibodies by conventional experimental methods and limited experiments, and are not limited to the specific CD3 antibody sequences in the specific embodiments of the present application.

[0083] The term "chimeric BiTE bispecific antibody" herein refers to a murine CD26 scFv linked to a humanized CD3 scFv via a linker.

[0084] The terms "administration," "administering," "treating," and "treatment" refer to the contacting of an exogenous pharmaceutical, therapeutic, or diagnostic agent with a tissue, organ, cell, or biological fluid of a subject. "Treatment" refers to slowing or arresting the development of clinical symptoms of a disease, or to alleviating or improving at least one physical parameter, or to preventing the progression of a disease.

[0085] Example 1. Preparation and screening of anti-human CD26 hybridoma cell lines

[0086] Step 1: Animal immunization

[0087] An rCD26 expression vector was designed based on the CD26 cDNA sequence published in GenBank (GenBank Accession No. NM_001935.3). A histidine tag was introduced at the 5' end of the codon, and the gene was synthesized. The gene was then ligated into the pCHO1.0 plasmid for expression and purification in CHO-S host cells. The recombinant CD26 target protein was immunized in female BALB / c mice according to standard immunization procedures. For detailed immunization procedures, please refer to the "Antibody Preparation and Use Laboratory Guide." Serum titers from immunized mice were monitored using an indirect ELISA assay. The mice with the highest serum titers were selected for fusion experiments using mouse spleen cells and mouse myeloma cells.

[0088] Step 2: Cell fusion

[0089] (1) Preparation of spleen cells

[0090] The immunized mice were sacrificed by cervical dislocation and soaked in 75% (v / v) alcohol for 10 minutes. The spleens were removed on a sterile operating table and placed on a cell sieve. The cells were thoroughly ground, passed through the sieve, and washed several times by centrifugation with sterile 1640 culture medium (purchased from Gibco). The cells were resuspended to make a single-cell suspension, counted, and set aside.

[0091] (2) Preparation of feeder cells

[0092] An 8- to 10-week-old female BALB / c mouse was eyeballed to obtain negative serum. The mouse was sacrificed by cervical dislocation and then immersed in 75% (v / v) alcohol for 10 minutes. The abdominal skin was aseptically opened to expose the peritoneum. Approximately 10 mL of 1640HT medium (purchased from SIGMA) was injected into the mouse's peritoneal cavity using a syringe. The abdomen was gently massaged and pipetted several times. The macrophage-containing medium was aspirated and injected into 20% 1640HAT medium for later use.

[0093] A 2- to 3-week-old female BALB / c mouse was sacrificed by cervical dislocation and then immersed in 75% (v / v) alcohol for 10 minutes. The thymus was aseptically removed and placed on a cell sieve, ground, and passed through the sieve to obtain thymocytes, which were placed in the above-mentioned 20% 1640HAT medium containing macrophages and set aside.

[0094] (3) Cell fusion

[0095] Select mouse myeloma cell line SP2 / 0 in the logarithmic growth phase, collect and count. 8 The above spleen cells were mixed with 2×10 7 Add the above SP2 / 0 cell lines to a fusion tube, mix, centrifuge at 1000 rpm for 10 minutes, discard the supernatant, and gently rub the tube back and forth on the palm of your hand to loosen the pellet. Add 1 mL of preheated PEG1450 (available from SIGMA) slowly, then rapidly, over 60 seconds. Stop the reaction by adding 30 mL of 1640HT medium. Centrifuge at 1000 rpm for 10 minutes, discard the supernatant, gently rub the pellet to loosen it, and add it to the 20% 1640HT medium obtained in step 2.

[0096] After thoroughly mixing the above HAT medium, aliquot 200 μL / well into a 96-well cell culture plate and culture in a cell culture incubator at 37°C and 5% CO2. After one week, replace the 20% 1640HAT medium with 10% 1640HT medium. After 3 days, collect the supernatant for analysis.

[0097] Step 3: Screening of anti-CD26 specific hybridoma cell lines

[0098] (1) Preparation of the test plate: Dilute the recombinant CD26 target protein to 1 μg / ml with CB coating solution, coat the 96-well ELISA plate at 100 μl / well, coat overnight at 2-8°C, wash once, and pat dry; block with PBST buffer containing 2% BSA (200 μl / well) at 37°C for 2 hours; pat dry and set aside.

[0099] (2) Screening of positive clones: 100 μl / well of the culture supernatant of the cells to be tested was added to the above-mentioned detection plate, incubated at 37°C for 30 minutes, washed and patted dry, 100 μl / well of diluted HRP-labeled goat anti-mouse IgG was added, incubated at 37°C for 30 minutes, washed and patted dry, 100 μl / well of TMB color development solution was added, incubated at 37°C in the dark for 15 minutes, 50 μl of 2M H2SO4 was added to each well to terminate the reaction, and the OD 450 Read the value at the positive well. 450 The positive value / negative control value is ≥ 2.1. Positive clones are selected for cell cloning screening. After three to four rounds of cloning screening, a single clone with a 100% positive rate is identified as a stable cell line and the cell line is then confirmed.

[0100] Step 4: Evaluation of CD26 Mouse Monoclonal Antibody Activity

[0101] In the 786-0 cell reaction system, 786-0 cells were labeled with green fluorescent signal using the fluorescent dye Calcein-AM. 786-0 cells were cultured at 6×10 5 cells / ml, 50 μl per well was inoculated into a U-shaped 96-well cell culture plate, and 3 replicates were set up for each group. 50 μl of CD26 antibody with a final concentration of 100 ng / ml was added to the reaction wells of the corresponding sample wells, 50 μl of culture medium was added to the blank control wells, and 50 μl of Triton X-100 with a final concentration of 1% was added to the positive control wells. The cells were incubated in a 37°C incubator for 30 min, and then 6×10 6 cells / ml of PBMC (peripheral blood mononuclear cells), the reaction system was placed at 37 ° C under carbon dioxide culture for 5 hours. After the reaction, the supernatant was centrifuged and placed in a new 96-well plate. After centrifugation again, 80 μl of the supernatant was placed in a black 96-well plate and detected using a microplate reader under the conditions of 470 nm excitation wavelength and 515 nm emission wavelength. The cell lysis rate formula = (V sample -V vehicle control ) / (V TritonX-100 -V vehicle control )×100%. (Where V sampleV is the average value of the fluorescence signal readings measured under the conditions of excitation light wavelength and emission light wavelength in the drug treatment group. vehicle control V is the average value of the fluorescence signal readings of the blank control group measured under the conditions of excitation light wavelength and emission light wavelength. TritonX-100 =The average value of the fluorescence signal readings of the positive control group measured under the conditions of excitation light wavelength and emission light wavelength. Table 1 Detection of antibody-mediated cytotoxic effect of PBMC on target cell 786-0

[0102] From the above results, it can be seen that under the conditions of an antibody concentration of 100 ng / ml and an action time of 5 hours, the cell lysis rate of PBMC mediated by mouse antibody No. 62 on target cell 786-0 was 53.1%, and the cell lysis rate of PBMC mediated by mouse antibody No. 79 on target cell 786-0 was 48.8%. The lysis intensity of target cell 786-0 was much higher than that of other mouse monoclonal antibodies 72, 160, 51, and 212, and was close to that of the humanized antibody YS110.

[0103] 786-0 is a human tumor cell. Theoretically, a successful humanized antibody would have a much greater affinity for human cells than the parental mouse antibody, resulting in a more robust lysis of these tumor cells. Mouse antibodies #62 and #79 demonstrated comparable cytotoxicity against human tumor cells to that of the humanized antibody YS110, which has completed Phase II clinical trials. This demonstrates the superiority of mouse antibodies #62 and #79. Those skilled in the art would reasonably expect that humanized antibodies using mouse antibodies #62 and #79 as parental antibodies would demonstrate superior cytotoxicity against human tumor cells to mouse antibodies #62 and #79 themselves, as well as to YS110. This is also demonstrated in Example 11.

[0104] Example 2. Sequencing and screening of variable region sequences of hybridoma cell lines

[0105] Step 1. Extraction of total RNA from CD26 hybridoma cells

[0106] The hybridoma cell lines were passaged into T75 culture flasks and cultured until the cells reached approximately 90% confluence. The cells were then digested and collected by centrifugation. Total RNA was extracted from the monoclonal hybridoma cell lines using an RNA extraction kit (purchased from Roche). The first-strand cDNA was then reverse transcribed and amplified using a cDNA reverse transcription kit (purchased from Thermo) using the total RNA as a template. The reaction products were stored at -20°C. For long-term storage, they should be stored at -70°C.

[0107] Step 2. PCR amplification of heavy and light chain variable region genes

[0108] Using the first-strand cDNA from hybridoma cells as a template, a 50 μl reaction system was added with 1 μl of cDNA, 5 μl of 10× PCR buffer, 1 μl of each upstream and downstream primer (25 pmol), 1 μl of dNTPs, 1 μl of 25 mmol / L MgCl₂, and 39 μl of H₂O. The reaction was pre-denatured at 95°C for 10 min, followed by the addition of 1 μl of Taq enzyme and PCR amplification. The reaction conditions were 94°C, denaturation for 1 min, 58°C, annealing for 1 min, and extension at 72°C for 1.5 min for 30 cycles, followed by a 10-min incubation at 72°C. Five μl of the PCR product was subjected to electrophoresis on a 1.2% agarose gel.

[0109] Step 3. Cloning and sequencing of heavy and light chain variable region genes

[0110] According to the instructions of the pGM-T Fast ligation kit (Beijing Tiangen Biochemical Technology Co., Ltd., VT207-02), the heavy and light chain variable region genes were ligated with the pGM-T vector, transformed into Escherichia coli Top10 competent cells, screened by blue-white spots, and cultured at 37°C for 12-16 h.

[0111] The resulting white colonies were inoculated into 1-5 ml of LB medium containing ampicillin at a final concentration of 100 μl. After shaking at 37°C for 3-4 hours, clones containing the correct insertion sequence were screened by PCR. Simultaneously, positive clones screened by PCR were sequenced, and the results were compared and analyzed using the IMGT computer network gene library to obtain the amino acid sequences of the antibody's heavy and light chain variable regions, as well as the CDR regions.

[0112] Mouse antibody No. 62 comprises the HCDR1 set forth in SEQ ID NO: 1, the HCDR2 set forth in SEQ ID NO: 2, the HCDR3 set forth in SEQ ID NO: 3; the LCDR1 set forth in SEQ ID NO: 4, the LCDR2 consisting of Arg Met Ser (wherein Arg represents arginine, Met represents methionine, and Ser represents serine); and the LCDR3 set forth in SEQ ID NO: 5. Its heavy chain variable region is set forth in SEQ ID NO: 31, and its light chain variable region is set forth in SEQ ID NO: 32.

[0113] Example 3. Design of chimeric BiTE bispecific antibodies

[0114] Based on the heavy and light chain sequences of the murine monoclonal antibodies 72, 160, 62, and 51 obtained in Example 1 and sequenced using the method in Example 2, a bispecific antibody targeting both CD26 and CD3 was designed:

[0115] The light chain variable region targeting the first antigen CD26 was connected to the heavy chain variable region targeting CD26 with a connecting short peptide (such as SEQ ID NO: 12) to form a CD26 single-chain antibody region, namely a single-chain antibody derived from mouse antibody No. 72 (such as SEQ ID NO: 16), a single-chain antibody derived from mouse antibody No. 160 (such as SEQ ID NO: 17), a single-chain antibody derived from mouse antibody No. 62 (such as SEQ ID NO: 18), and a single-chain antibody derived from mouse antibody No. 51 (such as SEQ ID NO: 19).

[0116] The single-chain antibody sequence targeting the second antigen CD3 protein is formed by connecting the heavy chain variable region (such as SEQ ID NO: 10) and the light chain variable region (such as SEQ ID NO: 11) through a connecting short peptide (such as SEQ ID NO: 12).

[0117] Four first recognition region sequences were fused with one second recognition region sequence via a connecting short peptide (e.g., SEQ ID NO: 13) to form a BiTE structure bispecific antibody, forming four BiTE sequences, namely 17G105 derived from mouse antibody No. 72 (e.g., SEQ ID NO: 20), 17G108 derived from mouse antibody No. 160 (e.g., SEQ ID NO: 21), 17G61 derived from mouse antibody No. 62 (e.g., SEQ ID NO: 22), and 17G131 derived from mouse antibody No. 51 (e.g., SEQ ID NO: 23).

[0118] Example 4. Construction of expression plasmid, expression and purification of anti-CD26-CD3 chimeric bispecific antibody

[0119] The mouse (Mus musculus) IgG kappa signal peptide sequence (such as SEQ ID NO: 24) was added upstream of the above-mentioned CD26 single-chain antibody amino acid sequence (SEQ ID NO: 16-19) and optimized according to the mammalian cell CHO codon preference to obtain the optimized CD26 single-chain antibody gene sequence (SEQ ID NO: 25-28). An AvrII restriction site and a Kozak sequence were introduced upstream. The Solitomab CD3 single-chain antibody amino acid sequence was codon-optimized to obtain an optimized gene sequence (SEQ ID NO: 29). A linker short peptide gene (such as SEQ ID NO: 30) was added upstream of the gene, and a stop codon and a BstZ17I restriction site were added downstream of the gene. That is, the optimized AvrII restriction site, kozak sequence, signal peptide, and CD26 single-chain antibody sequence were fused with the CD3 single-chain antibody, stop codon, and BstZ17I restriction site through a connecting peptide gene to form four chimeric BiTE genes. The above genes were synthesized and constructed into the pUC57 plasmid to form multiple long-term storage plasmids named pUC57-17G105, pUC57-17G108, pUC57-17G61, and pUC57-17G131.

[0120] The target gene was amplified, and the PCR product was recovered by 1% agarose electrophoresis. The final target gene was double-digested with AvrII and BstZ17I to recover the PCR product and the pZHK 2.0 vector. The backbone vector pCHO 1.0 was purchased from Invitrogen and was digested with SfiI to remove the "expression cassette 1" and then circularized with T4 ligase to form the pZHK2.0 vector.

[0121] The double-digested PCR product was ligated into the pZHK 2.0 vector using T4 ligase, transformed into Top10 competent cells, and plated on LB plates containing kanamycin resistance and cultured overnight at 37°C. Positive clones were screened the next day and sequenced, confirming they were completely consistent with the expected sequence.

[0122] A mammalian cell line stably expressing a high-expressing CD26-CD3 chimeric BiTE bispecific antibody was inoculated in Dynamis medium (A2617501, purchased from Thermo Fisher Scientific) and cultured in a fed-batch format at 37°C, 8% CO2, and 130 rpm. The supernatant was collected by cold centrifugation at 12,000 rpm for 15 minutes, filtered through a 0.45 μm filter, and purified by chromatography. The target product was approximately 55 kDa, indicating the correct size.

[0123] Example 5. Affinity Detection of Anti-CD26-CD3 Chimeric Bispecific Antibody and CD26 Protein

[0124] Fortebio Octet QK e The affinity of the chimeric antibody for the antigen protein CD26 was measured using a cytometry instrument. First, the amino-coupled biosensor AR2G was activated with EDC / s-NHS. The activated biosensor AR2G was then immobilized in a CD26 protein solution diluted with 10 mM sodium acetate (pH 5.0). The immobilized CD26 protein biosensor was quenched in a 1 M ethanolamine (pH 8.5) solution and then equilibrated in a 1× kinetic solution. After equilibration, the biosensor was bound to a humanized bispecific antibody solution and dissociated in a 1× kinetic solution. Data were analyzed using Fortebio Data Analysis 8.0 software to calculate the affinity constant (KD), association rate constant (kon), and dissociation rate constant (kdis). The response value indicates the number of antibody molecules that can bind to the antigen; kon indicates the binding rate of the antibody to the corresponding receptor; kdis indicates the dissociation rate of the antibody to the corresponding receptor; and NA indicates no valid data.

[0125] Table 2 Affinity test of chimeric antibodies and CD26 protein

[0126] The above data demonstrate that chimeric antibodies can bind and dissociate from the CD26 protein. 17G61 (an anti-CD26-CD3 chimeric bispecific antibody derived from mouse antibody No. 62) exhibited the highest response to CD26, indicating that at the same concentration, more 17G61 molecules bound to the CD26 protein. 17G105 (an anti-CD26-CD3 chimeric bispecific antibody derived from mouse antibody No. 72) and 17G131 (an anti-CD26-CD3 chimeric bispecific antibody derived from mouse antibody No. 51) exhibited significantly lower response values ​​than 17G61. The KD value for 17G61 is smaller, indicating a stronger affinity for CD26.

[0127] Example 6. Humanization of anti-CD26 mouse antibody

[0128] Humanized designs were performed based on the sequences of candidate murine antibodies No. 62 and No. 212, which exhibited high CD26 affinity and potent cytotoxicity against CD26-overexpressing tumor cells, as described in Example 1. Humanization analysis was performed on the variable regions of the heavy chain (shown in SEQ ID NO: 31) and light chain (shown in SEQ ID NO: 32) of murine antibody No. 62, and the variable regions of the heavy chain (shown in SEQ ID NO: 33) and light chain (shown in SEQ ID NO: 34) of murine antibody No. 212. Computational predictions revealed that each antibody produced multiple pairs of humanized heavy and light chain combinations. Humanization of mouse antibody No. 62 yielded four distinct heavy and light chain amino acid sequences: 18G272-VL (SEQ ID NO: 7), 18G272-VH (SEQ ID NO: 6), 19G292-VL (SEQ ID NO: 35), 19G292-VH (SEQ ID NO: 36), 19G293-VL (SEQ ID NO: 37), 19G293-VH (SEQ ID NO: 38), 19G294-VL (SEQ ID NO: 9), and 19G294-VH (SEQ ID NO: 8). The fully human antibody derived from humanization of mouse antibody No. 62 is designated 21G428-429, with its heavy chain amino acid sequence shown in SEQ ID NO: 70 and its light chain amino acid sequence shown in SEQ ID NO: 69. The fully human antibody obtained by humanizing mouse antibody No. 79 is denoted as 21G430-431, the heavy chain amino acid sequence of which is shown in SEQ ID NO: 72, and the light chain amino acid sequence is shown in SEQ ID NO: 71.

[0129] After humanization of 212, four groups of different heavy and light chain amino acid sequences were obtained: 18G278-VL (SEQ ID NO: 39), 18G278-VH (SEQ ID NO: 40), 19G295-VL (SEQ ID NO: 41), 19G295-VH (SEQ ID NO: 42), 19G296-VL (SEQ ID NO: 43), 19G296-VH (SEQ ID NO: 44), 19G297-VL (SEQ ID NO: 45), and 19G297-VH (SEQ ID NO: 46).

[0130] The humanized YS110 antibody was used as a positive control. The heavy and light chain codes and sequences are shown below: YS110-VL (SEQ ID NO: 47), YS110-VH (SEQ ID NO: 48).

[0131] Example 7. Construction of expression plasmid, stable expression and purification of humanized bispecific antibodies

[0132] The light chain and heavy chain of each group of humanized CD26 antibodies (18G272, 19G292, 19G293, 19G294, 18G278, 19G295, 19G296, and 19G297) were connected by a connecting peptide (SEQ ID NO: 12). A connecting peptide (SEQ ID NO: 13) was added downstream, and the optimized sequence was optimized according to the codon preference of mammalian cells (CHO). The optimized CD26 single-chain antibody gene sequence (SEQ ID NOs: 49-56) was obtained. An AvrII restriction site and a Kozak sequence were introduced upstream, and a CD3 single-chain antibody gene (SEQ ID NO: 29), a stop codon, and a BstZ17I restriction site were added downstream. The humanized CD26-CD3BiTE gene sequences were obtained, directly synthesized, and constructed into the pUC57 plasmids, which were named pUC57-18G272, pUC57-19G292, pUC57-19G293, pUC57-19G294, pUC57-18G278, pUC57-19G295, pUC57-19G296, and pUC57-19G297.

[0133] The light and heavy chains of the YS110 antibody were also linked via a linker peptide (SEQ ID NO: 12), with a linker peptide (SEQ ID NO: 13) added downstream. An AvrII restriction site and a Kozak sequence were introduced upstream, and a CD3 single-chain antibody gene (SEQ ID NO: 29), a stop codon, and a BstZ17I restriction site were added downstream. The resulting humanized CD26-CD3 BiTE gene sequence was directly synthesized and constructed into the pUC57 plasmid, designated pUC-17G29.

[0134] In addition, the 19G294 light chain and heavy chain were linked by a linker peptide (SEQ ID NO: 12), with a linker peptide (SEQ ID NO: 13) added downstream. This was then linked to a single-chain antibody (SEQ ID NO: 57) formed by linking the heavy and light chains of the CD3 antibody derived from WBP3311_2.306.4 via a linker peptide (SEQ ID NO: 12). The design was optimized based on CHO codon preference. An AvrII restriction site and a Kozak sequence were introduced upstream, and a stop codon and a BstZ17I restriction site were added downstream. The construct was directly synthesized into the pUC57 plasmid and named pUC57-21G587.

[0135] The target gene was amplified, and the PCR product was recovered by 1% agarose gel electrophoresis. The PCR product and the pZHK2.0 vector were double-digested with AvrII and BstZ17I. The double-digested product was ligated into the pZHK2.0 vector with T4 ligase and transformed into Top10 competent cells. The cells were plated on LB plates containing kanamycin resistance and cultured overnight at 37°C. The next day, positive clones were screened and sequenced for complete consistency with the expected sequence, thus obtaining the humanized CD26-CD3 BiTE bispecific antibody expression plasmid.

[0136] A mammalian cell line stably expressing a highly expressed CD26-CD3 humanized BiTE bispecific antibody was inoculated in Dynamis medium and cultured in a fed-batch format at 37°C, 8% CO2, and 130 rpm. The supernatant was harvested and centrifuged at 12,000 rpm for 15 minutes. The supernatant was filtered through a 0.45 μm filter to obtain the treated supernatant, which was then purified by chromatography. The target product was approximately 55 kDa.

[0137] A similar method was used to prepare the humanized BiTE bispecific antibody 19G300 derived from mouse antibody No. 79, the amino acid sequence of which is shown in SEQ ID NO: 58.

[0138] Example 8. Dot blot detection of purified humanized bispecific antibody samples

[0139] Purified samples were diluted to various concentrations, and 2 μl of each diluted sample was spotted onto NC membranes. 17G29 was used as a positive control sample. After complete absorption, the membranes were blocked with 5% skim milk powder blocking buffer on a horizontal shaker at room temperature for 1 hour, followed by three 5-min washes with TBST. CD3-HRP protein was added to 2.5% skim milk powder blocking buffer at a dilution of 1:1000 (50 μl CD3-HRP added to 50 mL of 2.5% skim milk powder blocking buffer), shaken at room temperature for 1 hour, followed by three 5-min washes with TBST. CD26-HRP protein was added to 2.5% skim milk powder blocking buffer at a dilution of 1:1000 (50 μl CD26-HRP added to 50 mL of 2.5% skim milk powder blocking buffer), shaken at room temperature for 1 hour, followed by three 5-min washes with TBST. The TBST solution on the membrane was blotted dry with paper towels, and a color development solution (SuperSignal West Pico Chemiluminescent Substrate) was added. The color was developed for 1 minute, and the color development solution was blotted dry with paper towels. The membrane was then exposed on a gel imaging system using a continuous exposure program set to 15 seconds per frame for 12 frames. Dot blot analysis of 18G272 (humanized mouse anti-antibody No. 62) and 18G278 (humanized mouse anti-antibody No. 212) revealed that the binding activity of 18G272 to CD26 and CD3 at various concentrations was close to that of the positive control 17G29 (a humanized anti-CD26-CD3 bispecific antibody derived from YS110), and significantly higher than that of 18G278 (Figure 1-a). Dot blot analysis was performed on a total of 6 samples, including 19G292-19G297 (humanized anti-CD26-CD3 bispecific antibodies derived from mouse antibodies No. 62 and No. 212). It was found that 19G292-19G297 all had binding activity to CD26 and CD3, and the binding activity of 19G292-19G296 to CD26 was close to that of the positive control 17G29, among which 19G294 was even better than the positive control (Figure 1-b).

[0140] Example 9 Study on the Specificity of Binding of Humanized Bispecific Antibodies to Target Cell Antigens

[0141] 1. Screening of CD26-positive and CD26-negative cells

[0142] The target cells were cultured in T75 cell culture flasks. When the cells were more than 80% confluent, the cells were digested with trypsin and collected. The cells were washed once with PBS and counted using a hemocytometer. The cells were divided into 5 × 10 5cells per aliquot; anti-CD26 monoclonal antibody was used as the primary antibody and incubated with the target cells at room temperature for approximately 40 minutes. After the incubation period, the supernatant was discarded by centrifugation, the cell pellet was resuspended with PBS, centrifuged again and the supernatant was discarded, and the cell pellet was collected; the corresponding antibody Alexa Fluor 488 mouse anti-human IgG1 was then used as the secondary antibody, the cell pellet was resuspended, and the cells were incubated in the dark at room temperature for approximately 30 minutes. After the incubation period, the cells were washed twice with PBS, centrifuged and the supernatant was discarded, and the cell pellet was collected; the cell pellet was resuspended in approximately 200 ul of PBS solution, and the CD26 positivity rate was detected and analyzed by flow cytometry within 1 hour.

[0143] Flow cytometry analysis revealed that CD26-positive cell lines included 786-0, OS-RC-2, A498, NCI-H226, NCI-H2052, NCI-H596, HCC 827, Huh-7, and PC-3, while CD26-negative cell lines included G401 and A-375. Their positive rates are shown in Table 4. The CD26-positive cell lines were consistent with those in the reference (Chinese Patent Application No. CN200680034937.4, CD26 / DPP4-a potential biomarker and target for cancer therapy, Pharmacology & Therapeutics (2019), 198:135-159).

[0144] Table 4 CD26 positive rate of target cells

[0145] 2. FITC-labeled antibodies:

[0146] Weigh fluorescein FITC and dissolve it in DMSO solution to a final concentration of 1 mg / ml. Take another 500 μl of 18G272 antibody, adjust it to a final concentration of 1 mg / ml, add 1 / 10 volume of 1M Na2CO3 solution and mix well. Add 15 μl of FITC solution and mix well. Incubate at room temperature in the dark for 3.5 hours. After incubation, centrifuge using an ultrafiltration concentrator with a cutoff diameter of 10 kDa to completely remove FITC that is not bound to 18G272, and collect the solution in the concentrator.

[0147] 3. Flow cytometry detection:

[0148] The cells cultured in T75 cell culture flasks until about 80% of the cells were digested with trypsin and collected; resuspended with an appropriate amount of PBS and counted using a hemocytometer, with 5×10 cells per aliquot. 5Cells were centrifuged and the cell pellet collected. Target cells were resuspended in FITC-labeled 18G272, placed on a homogenizer, and incubated at room temperature in the dark for 30 minutes. After incubation, the cells were resuspended in PBS, centrifuged, and the supernatant discarded. This process was repeated once. The cell pellet was resuspended in 500 μl of PBS. A negative control was set up: In a separate sample, the antibody sample was not added during the labeling process. All other steps were carried out in parallel. The resulting solution served as the FITC-labeled negative control. FITC-labeled positive rates were analyzed using a BD Accuri C6 flow cytometer.

[0149] The results showed that the humanized bispecific antibody 18G272 bound to CD26-positive 786-0 cells, NCI-H226 cells, PC-3 cells, and HCC 827 cells, but not to CD26-negative A375 cells, demonstrating its specific binding to the CD26 target.

[0150] The same method was used to test the specific binding of the humanized bispecific antibody 19G294 to target cell antigens. 19G294 bound to CD26-positive 786-0 cells, NCI-H226 cells, PC-3 cells, and OS-RC-2 cells, but not to CD26-negative G401 cells. Humanized bispecific antibody 19G294 bound to CD26-positive cells but not to CD26-negative cells, demonstrating specific binding to the CD26 antigen target.

[0151] Representative figures of the specificity of antibody binding to target cell antigens detected by flow cytometry are shown in Figure 2, and detailed data are shown in Tables 5 and 6.

[0152] Table 5 Specificity of binding of antibody 18G272 to target cell antigens detected by flow cytometry

[0153] Table 6 Flow cytometry detection of specificity of antibody 19G294 binding to target cell antigens

[0154] Other humanized bispecific antibodies of the present invention also bind to CD26-positive cells but not to CD26-negative cells, and have specific binding to CD26 antigen targets.

[0155] Example 10 Affinity Analysis of Humanized Bispecific Antibodies and CD26 Protein

[0156] 1. Affinity study with CD26 protein

[0157] First, the CD26 protein was biotinylated with EZ-link NHS-PEG12-Biotin; the biotinylated CD26 protein was immobilized using an SA biosensor and equilibrated in a 1× Kinetics solution; the protein was bound to a solution of the antibody molecule to be detected at a concentration gradient of 31.3 nM, 62.5 nM, 125 nM, 250 nM, and 500 nM; the protein was dissociated in a 1× Kinetics solution; and data were analyzed using Fortebio Data Analysis 8.0 software to calculate the affinity constant.

[0158] Table 7 Affinity of humanized bispecific antibodies to CD26 protein

[0159] The above data show that when the anti-CD3 moieties are identical, the affinity constants of the humanized antibodies derived from mouse antibody 62 (19G292, 19G293, and 19G294) are all lower than those derived from mouse antibody 212 or YS110, indicating that the former have a higher affinity for CD26. Furthermore, 19G294 and 21G587, which share the same anti-CD26 moiety but different anti-CD3 moieties, exhibit comparable affinity for the CD26 protein.

[0160] Combined with the affinity data for the above antibodies and CD26 protein (Table 7), when the CD3 antibody portion is the same, the humanized bispecific antibodies derived from 62 mouse antibodies (19G292, 19G293, and 19G294) show better CD26 protein binding ability than the humanized bispecific antibodies derived from 212 mouse antibodies (19G296 and 19G297) and the YS110 humanized bispecific (17G29). When the CD3 antibody portion is different, the humanized bispecific antibodies derived from 62 mouse antibodies (19G294 and 21G587) have the same level of binding ability to CD26 protein. The recognition and binding of the antibodies of the present application to the CD26 protein are not affected by the binding of different CD3 antibody portions to the CD3 protein.

[0161] 2. Observation of the simultaneous binding of bispecific antibodies to CD26 and CD3 proteins

[0162] First, the 19G294 protein was biotinylated with EZ-link NHS-PEG12-Biotin; the biotinylated 19G294 protein was immobilized using an SA biosensor and equilibrated in a 1× Kinetics solution. The protein was first bound to a 400 nM CD26 protein solution and then placed in a 400 nM CD26 solution containing 400 nM CD3 protein; or first bound to a 400 nM CD3 protein solution and then placed in a 400 nM CD3 solution containing 400 nM CD26 protein. The binding was observed using Fortebio Data Analysis 8.0 software.

[0163] As shown in Figure 3, 19G294 was immobilized on an SA sensor. After reaching saturation binding with CD26, it continued to bind to CD3 in a CD26 solution (curve shown in Figure 3, SensorG9). After reaching saturation binding with CD3, it continued to bind to CD26 in a CD3 solution (curve shown in Figure 3, SensorH9). Figure 3, SensorF9, shows a negative control, demonstrating the absence of nonspecific binding between the sensor and the solution. These results demonstrate that after 19G294 binds to either CD26 or CD3, it can continue to bind to either CD3 or CD26. There is no order of binding, and binding to CD3 does not affect the recognition and binding of the humanized bispecific antibody to CD26.

[0164] Example 11 Evaluation of Humanized Bispecific Antibody-Mediated Cytotoxicity of PBMCs Against Target Cells

[0165] 1. Human renal cancer cell 786-0 cell model

[0166] In the 786-0 cell reaction system, 786-0 cells were labeled with green fluorescent signal using the fluorescent dye Calcein-AM. 786-0 cells were cultured at 6×10 5 cells / ml, 50 μl per well was inoculated into a U-shaped 96-well cell culture plate, 50 μl of humanized bispecific antibodies with final concentrations of 100 ng / ml, 10 ng / ml, 1 ng / ml, 0.1 ng / ml, 0.01 ng / ml, and 0.001 ng / ml were added to the corresponding sample reaction wells, 50 μl of culture medium was added to the blank control wells, 50 μl of Triton X-100 with a final concentration of 1% was added to the positive control wells as a positive control, and 50 μl of 6×10 6cells / ml of PBMC cells, the reaction system was incubated at 37°C under carbon dioxide culture for 5 hours. After the reaction was completed, the supernatant was centrifuged and placed in a new 96-well plate. After centrifugation again, 80 μl of the supernatant was placed in a black 96-well plate and detected using a microplate reader under the conditions of 470 nm excitation wavelength and 515 nm emission wavelength.

[0167] 2. Human renal cancer cell OS-RC-2 cell model

[0168] In the OS-RC-2 cell reaction system, OS-RC-2 cells were labeled with green fluorescent signal using the fluorescent dye Calcein-AM. OS-RC-2 cells were cultured at 6×10 5 cells / ml, 50 μl per well was inoculated into a U-shaped 96-well cell culture plate, 50 μl of humanized bispecific antibodies with final concentrations of 100 ng / ml, 10 ng / ml, 1 ng / ml, 0.1 ng / ml, 0.01 ng / ml, and 0.001 ng / ml were added to the corresponding sample reaction wells, 50 μl of culture medium was added to the blank control wells, 50 μl of Triton X-100 with a final concentration of 1% was added to the positive control wells as a positive control, and 50 μl of 9×10 6 cells / ml of PBMC cells, the reaction system was incubated at 37°C under carbon dioxide culture for 5 hours. After the reaction was completed, the supernatant was centrifuged and placed in a new 96-well plate. After centrifugation again, 80 μl of the supernatant was placed in a black 96-well plate and detected using a microplate reader under the conditions of 470 nm excitation wavelength and 515 nm emission wavelength.

[0169] 3. Human mesothelioma NCI-H226 cell model

[0170] In the NCI-H226 cell reaction system, NCI-H226 cells were labeled with green fluorescent signal using the fluorescent dye Calcein-AM. NCI-H226 cells were cultured at 6×10 5 cells / ml, 50 μl per well was inoculated into a U-shaped 96-well cell culture plate, 50 μl of humanized bispecific antibody with a final concentration of 100 ng / ml, 10 ng / ml, 1 ng / ml, 0.1 ng / ml, 0.01 ng / ml, and 0.001 ng / ml were added to the corresponding sample reaction wells, 50 μl of culture medium was added to the blank control wells, 50 μl of Triton X-100 with a final concentration of 1% was added to the positive control wells as a positive control, and 50 μl of 9×10 6cells / ml of PBMC cells, the reaction system was incubated at 37°C under carbon dioxide culture for 5 hours. After the reaction was completed, the supernatant was centrifuged and placed in a new 96-well plate. After centrifugation again, 80 μl of the supernatant was placed in a black 96-well plate and detected using a microplate reader under the conditions of 470 nm excitation wavelength and 515 nm emission wavelength.

[0171] 4. Human mesothelioma NCI-H2052 cell model

[0172] In the NCI-H2052 cell reaction system, NCI-H2052 cells were labeled with green fluorescent signal using the fluorescent dye Calcein-AM. NCI-H2052 cells were cultured at 6×10 5 cells / ml, 50 μl per well was inoculated into a U-shaped 96-well cell culture plate, 50 μl of humanized bispecific antibodies with final concentrations of 100 ng / ml, 10 ng / ml, 1 ng / ml, 0.1 ng / ml, 0.01 ng / ml, and 0.001 ng / ml were added to the corresponding sample reaction wells, 50 μl of culture medium was added to the blank control wells, 50 μl of Triton X-100 with a final concentration of 1% was added to the positive control wells as a positive control, and 50 μl of 9×10 6 cells / ml of PBMC cells, incubate the reaction system at 37°C under carbon dioxide culture for 5 hours. After the reaction is completed, centrifuge the supernatant and place it in a new 96-well plate. After centrifugation again, take 80 μl of the supernatant and place it in a black 96-well enzyme plate, and use a microplate reader to detect under the conditions of 470 nm excitation wavelength and 515 nm emission wavelength.

[0173] 5. Human prostate cancer PC-3 cell model

[0174] In the PC-3 cell reaction system, PC-3 cells were labeled with green fluorescent signal using the fluorescent dye Calcein-AM. PC-3 cells were cultured at 6×10 5 cells / ml, 50 μl per well was inoculated into a U-shaped 96-well cell culture plate, 50 μl of humanized bispecific antibodies with final concentrations of 100 ng / ml, 10 ng / ml, 1 ng / ml, 0.1 ng / ml, 0.01 ng / ml, and 0.001 ng / ml were added to the corresponding sample reaction wells, 50 μl of culture medium was added to the blank control wells, 50 μl of Triton X-100 with a final concentration of 1% was added to the positive control wells as a positive control, and 50 μl of 9×10 6cells / ml of PBMC cells, the reaction system was incubated at 37°C under carbon dioxide culture for 5 hours. After the reaction was completed, the supernatant was centrifuged and placed in a new 96-well plate. After centrifugation again, 80 μl of the supernatant was placed in a black 96-well plate and detected using a microplate reader under the conditions of 470 nm excitation wavelength and 515 nm emission wavelength.

[0175] 6. Human non-small cell lung cancer HCC 827 cell model

[0176] In the HCC 827 cell reaction system, HCC 827 cells were labeled with green fluorescent signal using the fluorescent dye Calcein-AM. HCC 827 cells were cultured at 6×10 5 cells / ml, 50 μl per well was inoculated into a U-shaped 96-well cell culture plate, 50 μl of humanized bispecific antibodies with final concentrations of 100 ng / ml, 10 ng / ml, 1 ng / ml, 0.1 ng / ml, 0.01 ng / ml, and 0.001 ng / ml were added to the corresponding sample reaction wells, 50 μl of culture medium was added to the blank control wells, 50 μl of Triton X-100 with a final concentration of 1% was added to the positive control wells as a positive control, and 50 μl of 6×10 6 cells / ml of PBMC cells, incubate the reaction system at 37°C under carbon dioxide culture for 5 hours. After the reaction is completed, centrifuge the supernatant and place it in a new 96-well plate. After centrifugation again, take 80 μl of the supernatant and place it in a black 96-well enzyme plate, and use a microplate reader to detect under the conditions of 470 nm excitation wavelength and 515 nm emission wavelength.

[0177] 7. Experimental Results

[0178] Cell lysis rate = (V sample -V vehicle control ) / (V TritonX-100 -V vehicle control )×100%. (Where V sample is the average fluorescence signal reading of the drug-treated group, V vehicle control is the average value of the fluorescence signal reading of the solvent control group, V Triton-100 is the average value of the fluorescence signal readings of the positive control group.

[0179] The cell lysis rate and sample concentration were calculated using GraphPad Prism 7.00 software to calculate the IC of PBMC to target cells mediated by the sample. 50 value.

[0180] Table 9 IC values ​​of cytotoxic effects of PBMC on different target cells mediated by different humanized bispecific antibodies 50 Value (pg / ml) —: Indicates that the data has not been detected.

[0181] Humanized anti-CD26-CD3 bispecific antibodies derived from mouse antibody No. 62, such as 18G272, 19G292, 19G293, and 19G294, can mediate cytotoxic effects of PBMC cells against various CD26-positive tumor cells and exhibit killing activity in vitro. Humanized anti-CD26-CD3 bispecific antibodies derived from mouse antibody No. 212, such as 19G295, 19G296, and 19G297, cannot mediate cytotoxic effects of PBMC cells against CD26-positive tumor cells.

[0182] This indicates that, on the one hand, mouse antibody No. 62 exhibits superior cytotoxicity against CD26-positive tumor cells. The humanized anti-CD26-CD3 antibody derived from mouse antibody No. 62 demonstrates superior cytotoxicity against various CD26-positive tumors compared to 17G29 (an anti-CD26-CD3 humanized antibody derived from YS110), which shares the same CD3 antibody moiety, and the humanized anti-CD26-CD3 antibody derived from mouse antibody No. 212, which shares the same CD3 antibody moiety. Furthermore, the humanized mouse antibody No. 62 can be applied to bispecific antibodies with BiTE structures (18G272, 19G292, 19G293, 19G294, and 17G29), demonstrating superior cytotoxicity against various CD26-positive tumor cells compared to the full-length IgG antibody YS110.

[0183] Example 12 Activation of PBMC Cells by Humanized Bispecific Antibodies in Mediating Cytotoxic Effects of PBMC on Target Cells

[0184] PBMC cells + double antibody system: collect PBMC cells, count them, and resuspend them to 1×10 6 cells / ml, 200ul per well was added to a 12-well cell culture plate, the antibody was diluted to a final concentration of 1ng / ml, and incubated for 24h. In the PBMC cell + double antibody + OS-RC-2 cell system: PBMC cells were collected, counted, and resuspended to 6×10 5 cells / ml, 100ul / well was added to a 12-well cell culture plate, the antibody was diluted to a final concentration of 1ng / ml, and OS-RC-2 cells were diluted to 9×10 6 cells / ml, 100ul / well was added into the corresponding 12-well cell culture plate and incubated for 24h.

[0185] After incubation, the cell pellet was collected by centrifugation, resuspended in PBS, and anti-CD25-APC antibody (purchased from Miltenyl) and anti-CD69-PE antibody (purchased from Miltenyl) were added. The cells were incubated in a refrigerator at 4°C for 10 min, washed twice with PBS, resuspended in PBS, and detected by BD ACCURI C6 flow cytometer.

[0186] In the "PBMC cells + bispecific antibody" system, the bispecific antibody did not induce upregulation of CD25 and CD69 expression on PBMC cells. In the "PBMC cells + bispecific antibody + OS-RC-2 cells" system, the bispecific antibody induced upregulation of CD25 and CD69 expression on PBMC cells, promoting PBMC cell activation. In the "PBMC cells + YS110 + OS-RC-2 cells" system, YS110 did not induce upregulation of CD25 and CD69 expression on PBMC cells. See Table 10.

[0187] Table 10 Flow cytometry detection of the expression of CD25 and CD69 on PBMC cells during the process of antibody-mediated cytotoxic effect of PBMC on target cells

[0188] The same phenomenon was also observed in other CD26-positive tumor cells such as 786-0. That is, in the "PBMC cells + bispecific antibodies" system, the bispecific antibodies did not induce upregulation of CD25 and CD69 expression in PBMC cells; but in the "PBMC cells + bispecific antibodies + other CD26-positive tumor cells (786-0, etc.)" system, they induced upregulation of CD25 and CD69 expression in PBMC cells.

[0189] CD25 and CD69 are important markers of T cell activation. The above results demonstrate that the full-length IgG antibody YS110 cannot effectively induce T cell activation. The CD26-CD3 bispecific antibody does not cause significant T cell activation before reaching the tumor site. Only after the antibody binds to tumor cells will it activate T cells around the tumor tissue and subsequently kill the tumor cells, thereby improving the safety of the effect.

[0190] Example 13 Cytokine Detection in the Process of Humanized Bispecific Antibodies Mediating PBMC Cytotoxic Effects on Target Cells

[0191] Resuspend OS-RC-2 cells at 6 × 10 5 cells / ml, 100 μl was added to each well of a 12-well plate, and PBMC cells from individual donors were collected and resuspended at 9×10 6cells / ml, 100 μl was added to each well, followed by 100 μl of 19G294, 18G272, and 17G29 samples at 0 ng / ml and 3 ng / ml, respectively, for a final volume of 300 μl. The reaction system was incubated at 37°C for 2 h, 4 h, 6 h, and 21 h. The culture supernatant was collected, centrifuged, and the supernatant was analyzed by ELISA. ELISA kits were used to detect cytokines IFN-γ, TNF-α, L-4, and IL-10.

[0192] The cytokine content of each sample detected by ELISA kit is shown in the following table.

[0193] Table 11 ELISA detection of IFN-γ content (pg / ml)

[0194] Table 12 ELISA detection of TNF-α content (pg / ml)

[0195] Table 13 ELISA detection of IL-10 content (pg / ml)

[0196] Table 14 ELISA detection of IL-4 content (pg / ml) *Indicates that the reading is too high and exceeds the upper detection limit of the device.

[0197] In summary, during the humanized bispecific antibody-mediated PBMC killing of target cells, under the same dosage conditions, the anti-CD26-CD3 humanized bispecific antibodies 19G294 and 18G272 derived from mouse antibody 62 caused PBMC cells to secrete lower cytokines than 17G29 (an anti-CD26-CD3 humanized bispecific antibody derived from YS110) at all time points. This type of molecular structure that targets CD3 and activates T cells has higher safety when the cytokine storm caused by the secretion of cytokines by T cells is relatively low (A FAD oncology analysis of CD3 bispecific constructs and first-in-human dose selection (2017), Regulatory Toxicology and Pharmacology, 90:144-152.). Under the premise of having the same CD3 sequence recognition region, under the same conditions, compared with 17G29, the bispecific antibodies 19G294 and 18G272 of the present application have the advantage of inducing lower amounts of cytokine secretion by T cells. This may be related to the fact that the CD26 antigen fragments recognized by their CDR regions are different from those of 17G29. Compared with other CD26 antigen fragments, the antigen fragments recognized by the antibodies of the present application are related to reducing the risk of cytokine storm and improving safety.

[0198] Example 14 Humanized bispecific antibody mediates PBMC-to-PBMC toxicity

[0199] 1. 786-0 cell model

[0200] PBMC cells were labeled with green fluorescent signal using the fluorescent dye Calcein-AM, and 786-0 cells were cultured at 6×10 5 cells / ml, 50 μl per well was inoculated into a U-shaped 96-well cell culture plate, 50 μl of humanized bispecific antibody with a final concentration of 100 ng / ml, 10 ng / ml, 1 ng / ml, 0.1 ng / ml, 0.01 ng / ml, and 0.001 ng / ml were added to the corresponding sample reaction wells, 50 μl of culture medium was added to the blank control well, 50 μl of Triton X-100 with a final concentration of 1% was added to the positive control well, and then 50 μl of 9×10 6cells / ml of PBMC cells, incubate the reaction system at 37°C under carbon dioxide culture for 5 hours. After the reaction is completed, centrifuge the supernatant and place it in a new 96-well plate. After centrifugation again, take 80 μl of the supernatant and place it in a black 96-well enzyme plate, and use a microplate reader to detect under the conditions of 470 nm excitation wavelength and 515 nm emission wavelength.

[0201] 2. OS-RC-2 cell model

[0202] PBMC cells were labeled with green fluorescent signal using the fluorescent dye Calcein-AM, and OS-RC-2 cells were cultured at 6×10 5 cells / ml, 50 μl per well was inoculated into a U-shaped 96-well cell culture plate, and 50 μl of humanized bispecific antibody 18G272 with a final concentration of 100 ng / ml, 10 ng / ml, 1 ng / ml, 0.1 ng / ml, 0.01 ng / ml, and 0.001 ng / ml were added to the corresponding sample reaction wells, 50 μl of culture medium was added to the blank control wells, 50 μl of Triton X-100 with a final concentration of 1% was added to the positive control wells, and then 50 μl of 9×10 6 cells / ml of PBMC cells, incubate the reaction system at 37°C under carbon dioxide culture for 5 hours. After the reaction is completed, centrifuge the supernatant and place it in a new 96-well plate. After centrifugation again, take 80 μl of the supernatant and place it in a black 96-well enzyme plate, and use a microplate reader to detect under the conditions of 470 nm excitation wavelength and 515 nm emission wavelength.

[0203] 3. Results Analysis

[0204] Cell lysis rate = (V sample -V vehicle control ) / (V TritonX-100 -V vehicle control )×100%. (Where V sample is the average fluorescence signal reading of the drug-treated group, V vehicle control is the average value of the fluorescence signal reading of the solvent control group, V Triton-100 The IC value of PBMC cells mediated by each sample on PBMC cells was calculated using GraphPad Prism 7.00 software based on the cell lysis rate and sample concentration. 50 value.

[0205] In the process of PBMC killing target cells 786-0 mediated by antibody 18G272, the ability of PBMC to kill PBMC cells was extremely low. 50 The value was approximately 99,730,000 pg / ml; antibody 18G272 mediated PBMC (6#) had no cytotoxic effect on PBMC (6#) cells. The results are shown in Figure 4.

[0206] Antibody 18G272 mediated PBMC-to-PBMC cell killing of target OS-RC-2 cells, but did not mediate PBMC-to-PBMC cell killing. Antibody 18G272 mediated PBMC (1#) to PBMC (1#) cells had no cytotoxic effect; Antibody 18G272 mediated PBMC (3#) to PBMC (3#) cells had no cytotoxic effect.

[0207] In the process of PBMC killing target cells 786-0 mediated by antibody 19G294, the ability of PBMC to kill PBMC cells was extremely low. 50 The value is about 1915313pg / ml; the IC value of the cytotoxic effect of PBMC(3#) mediated by antibody 19G294 on PBMC(3#) cells is 50 The value is approximately 1947224pg / ml.

[0208] In the process of PBMC killing target cells OS-RC-2 mediated by antibody 19G294, the ability of PBMC to kill PBMC cells was extremely low. 50 The value is about 47920315pg / ml; the IC value of the cytotoxic effect of PBMC(3#) mediated by antibody 19G294 on PBMC(3#) cells is 50 The value is approximately 1406115138pg / ml.

[0209] In summary, the CD26-CD3 humanized bispecific antibody can accurately identify PBMCs. Although there are literature reports that T cells express CD26, the bispecific antibodies 18G272 and 19G294 of the present application do not mediate obvious cytotoxic effects of PBMC cells on PBMC cells, proving that they have good safety.

[0210] Example 15 Comparison of in vitro efficacy of 19G294 and drug B (BAVENCIO, PD-L1 antibody)

[0211] The target cells were cultured in T75 cell culture flasks. When the cells were more than 80% confluent, the cells were digested with trypsin and collected. The cells were washed once with PBS and counted using a hemocytometer. The cells were divided into 5 × 10 5 Target cells were incubated with PE-labeled anti-PD-L1 monoclonal antibody (purchased from Sino Biological) at room temperature in the dark for approximately 40 minutes. After incubation, the cells were centrifuged and the supernatant was discarded. The cell pellet was resuspended in PBS and centrifuged again to discard the supernatant. The cell pellet was collected and resuspended in approximately 200 μl of PBS solution. The PD-L1 positivity rate was detected and analyzed by flow cytometry within 1 hour.

[0212] Flow cytometry analysis showed that the PD-L1 positivity rate of 786-0 cell line was 66.2%, and that of A498 cell line was 29.6%.

[0213] Table 15 Detection of cell PD-L1 positivity rate

[0214] Human renal cancer 786-0-LUC cells (786-0 cells were transformed with the reporter gene Luciference) and human renal cancer A498-LUC cells (A498 cells were transformed with the reporter gene Luciference) were cultured at 3×10 5 cells / ml, 50 μl per well was inoculated into a white bottom opaque 96-well cell culture plate, 50 μl of antibodies with final concentrations of 100 ng / ml, 10 ng / ml, 1 ng / ml, 0.1 ng / ml, 0.01 ng / ml, and 0.001 ng / ml were added to the corresponding reaction wells, and then 50 μl of antibodies with a final concentration of 3×10 cells / ml was added at an E / T ratio of 10:1. 6 The reaction system was incubated at 37°C under carbon dioxide for 21 hours. After the reaction, the cells were centrifuged, the supernatant was discarded, and 100 μl of reporter gene detection reagent (purchased from Vazyme) was added. After shaking on a microplate shaker at 400 rpm for 5 minutes, the cells were immediately detected using a microplate reader under Luminescence conditions. The detection data were analyzed using GraphPad Prism 7.00 software. A four-parameter curve was drawn to plot the reporter gene reduction level against the antibody dose, and the EC was calculated. 50 The results are shown in Table 16.

[0215] Table 16 Antibody-mediated killing effect of PBMC on target cells (EC 50 pg / ml)

[0216] From the above data, we can see that 19G294-mediated PBMC cytotoxic effect on the two target cells EC50 The PD-L1 expression rates of 19G294 and 786-0 are much lower than those of BAVENCIO, 786-0 and A498, which are 66.2% and 29.6% respectively. It can be seen that regardless of the PD-L1 expression level of cells, the activity of 19G294 is significantly higher than that of BAVENCIO.

[0217] Example 16 Comparison of in vitro efficacy of 21G587 and 19G294

[0218] Human renal cancer 786-0-LUC cells (786-0 cells were transformed with the reporter gene Luciference) and human renal cancer A498-LUC cells (A498 cells were transformed with the reporter gene Luciference) were cultured at 3×10 5 cells / ml, 50 μl per well was inoculated into a white bottom opaque 96-well cell culture plate, 50 μl of antibodies with final concentrations of 100 ng / ml, 10 ng / ml, 1 ng / ml, 0.1 ng / ml, 0.01 ng / ml, and 0.001 ng / ml were added to the corresponding reaction wells, and then 50 μl of antibodies with a final concentration of 3×10 cells / ml was added at an E / T ratio of 10:1. 6 The reaction system was incubated at 37°C under carbon dioxide for 21 hours. After the reaction, the cells were centrifuged, the supernatant was discarded, and 100 μl of reporter gene detection reagent (purchased from Vazyme) was added. After shaking on a microplate shaker at 400 rpm for 5 minutes, the cells were immediately detected using a microplate reader under Luminescence conditions. The detection data were analyzed using GraphPad Prism 7.00 software. A four-parameter curve was drawn to plot the reporter gene reduction level against the antibody dose, and the EC was calculated. 50 The results are shown in Table 17.

[0219] Table 17 Antibody-mediated killing effect of PBMC on target cells (EC 50 pg / ml)

[0220] From the above data, it can be seen that the anti-CD26 antibody sequence of this patent is combined with different anti-CD3 sequences. The anti-CD26 part of 19G294 and 21G587 is the same, and the anti-CD3 part is different. Both have the same level of in vitro efficacy. The in vitro efficacy experiment of this experiment further proves that the recognition and binding of the antibody of the present application to the CD26 protein fragment is not affected by the binding of different CD3 antibody parts to the CD3 protein.

[0221] Example 17 Efficacy of humanized bispecific antibodies 18G272 and 19G294 in NOD / SCID mice bearing PC-3 (prostate cancer) xenografts

[0222] Methods: NOD / SCID mice weighing 18-22 g and aged 5-7 weeks were selected and 5×10 6 cells / 0.1ml PC-3 cell suspension with 1×10 7 cells / 0.1ml PBMC cell suspension was fully mixed in a ratio of 1:1 (volume ratio) and inoculated subcutaneously in NOD / SCID mice, with 0.2ml per mouse. The animals were randomly divided into three groups according to their body weight, namely Model group (PBS), 19G294 group (30μg / mouse / time), and 18G272 group (30μg / mouse / time), with 6 animals in each group. The Model group and each treatment group were intravenously administered, starting on the day of inoculation and continuing for five consecutive days. Two days after drug withdrawal, the second course of treatment was carried out. The first administration time was 1 hour after the cells were subcutaneously inoculated into the mice, that is, the first course of treatment was D1, D2, D3, D4, and D5; the second course of treatment was D8, D9, D10, D11, and D12. The frequency of administration was 1 day / time.

[0223] General clinical observation: Observation should be conducted at least once a day during the quarantine period and the trial period, including the effects of tumor growth and treatment on the normal behavior of the animals. Specific contents include death or dying of animals (tumor-bearing), mental state, behavioral activities and other abnormal conditions.

[0224] Body weight: Check 2 to 3 times a week.

[0225] Tumor volume: 2 to 3 times a week, using vernier calipers to measure the long diameter and short diameter of the tumor, and the tumor volume (mm 3 ) = major diameter * minor diameter 2 / 2. Relative tumor inhibition rate: TGI (%) = (1-T / C) × 100%. It is generally accepted that T represents the relative tumor volume of the drug group at a certain time point (the ratio of the tumor volume measured at that time to the tumor volume at the time of grouping), and C represents the relative tumor volume of the model group at a certain time point (the ratio of the tumor volume measured at that time to the tumor volume at the time of grouping). However, in the present application, the groups were grouped according to body weight on the day of inoculation. When calculating TGI, T and C represent the actual measured tumor volumes of the drug group and model group, respectively.

[0226] Tumor weight: After the last test, the animals were euthanized, the tumor was removed, rinsed with saline and dried with filter paper, the tumor was weighed and photographed. Relative tumor inhibition rate TGI (%) = (1-T TW / C TW )×100%, T TW represents the average tumor weight at the end of the treatment group experiment, C TW It represents the average tumor weight at the end of the experiment in the model group.

[0227] Conclusion: 18G272, at a dose of 30 μg / mouse, showed a trend toward inhibiting tumor growth in PC-3 human prostate cancer model mice. 19G294 almost completely inhibited tumor growth in PC-3 human prostate cancer model mice. The results are shown in Figure 5 and Table 18. At a dose of 30 μg / mouse, the body weights of the test articles 19G294 and 18G272 increased, comparable to those of the model group, and treatment was well tolerated. The results are shown in Figure 6 and Table 18.

[0228] Table 18 Tumor growth and body weight statistics in the PC-3 human prostate cancer cell subcutaneous transplantation model in mice Note: *: P < 0.05, vs Model group; **: P < 0.01, vs Model group.

[0229] Example 18 Efficacy of humanized bispecific antibody 18G272 in NOD / SCID mice bearing NCI-H596 (lung cancer) xenografts

[0230] Methods: NOD / SCID mice weighing 18-22 g and aged 5-7 weeks were selected and 8×10 6 cells / 0.1ml NCI-H596 cell suspension with 1.6×10 7 PBMC suspensions were thoroughly mixed at a 1:1 (volume ratio) and inoculated subcutaneously into NOD / SCID mice, with 0.2 ml per mouse. Animals were randomly divided into two groups based on body weight: the Model group (PBS, 6 mice / group) and the 18G272 group (30 μg / mouse / dose, 6 mice / group).

[0231] The route of administration and the administration cycle are the same as those in Example 17.

[0232] General clinical observations, body weight, and tumor volume measurements were the same as in Example 17.

[0233] Conclusion: Test article 18G272, at a dose of 30 μg / mouse, significantly inhibited tumor growth in a mouse model of human lung adenosquamous carcinoma. At a dose of 30 μg / mouse, the animals showed an increasing body weight trend and were well tolerated during treatment. The results are shown in Figure 7 and Table 19.

[0234] Table 19 Tumor growth and body weight statistics in the NCI-H596 human lung squamous cell carcinoma subcutaneous transplantation model in mice Note: *: P<0.05, vs Model group.

[0235] Example 19 Efficacy of humanized bispecific antibody 19G294 in NOD / SCID mice bearing NCI-H226 (mesothelioma) xenografts

[0236] Methods: NOD / SCID mice weighing 18-22 g and aged 5-7 weeks were selected and 5×10 6 cells / 0.1ml NCI-H226 cell suspension and 1.5×10 7 Cells / 0.1ml PBMC cell suspension was thoroughly mixed at a 1:1 (volume ratio) and inoculated subcutaneously into NOD / SCID mice, with 0.2ml per mouse. Animals were randomly divided into two groups based on body weight: a Model group (PBS, 5 mice / group) and a 19G294 group (60 μg / mouse / time, 5 mice / group). The route of administration and dosing cycle were the same as in Example 17.

[0237] General clinical observations, body weight, and tumor volume measurements were the same as in Example 17.

[0238] Conclusion: Test article 19G294, at a dose of 60 μg / mouse, significantly inhibited tumor growth in mice bearing a human lung adenosquamous carcinoma (mesothelioma) model, as shown in Figure 8. At a dose of 60 μg / mouse, test article 19G294 increased the body weight of the animals, and treatment was well tolerated. The results are shown in Figure 9 and Table 20.

[0239] Table 20 Tumor growth and body weight statistics in the NCI-H226 human lung squamous cell carcinoma (mesothelioma) cell subcutaneous transplantation model in mice Note: *: P < 0.05, vs Model group. a: TGI is calculated based on tumor weight on day 57 after cell inoculation.

[0240] Example 20 Efficacy of humanized bispecific antibody 19G294 in NOD / SCID mice bearing A498 (renal cancer) xenografts

[0241] Methods: NOD / SCID mice weighing 18-22 g and aged 5-7 weeks were selected and 1×10 7 cells / 0.1ml A498 cell suspension with 1×10 7 Cells / 0.1ml PBMC suspension was thoroughly mixed at a 1:1 (volume ratio) and inoculated subcutaneously into NOD / SCID mice, with 0.2ml per mouse. Animals were randomly divided into two groups based on body weight: the Model group (PBS) and the 19G294 group (30μg / mouse / dose), with 5 animals in each group.

[0242] The route of administration and the administration cycle are the same as those in Example 17.

[0243] General clinical observations, body weight, tumor volume, and tumor weight were the same as those in Example 17.

[0244] Conclusion: Test article 19G294 at a dose of 30 μg / mouse can completely inhibit tumor growth in human renal cancer model mice. The results are shown in Figure 10 and Table 21. At a dose of 30 μg / mouse, the animals did not experience weight loss and tolerated the treatment well.

[0245] Table 21 Tumor growth and body weight statistics in the A498 human renal cancer cell subcutaneous transplantation model in mice Note: *: P < 0.05, vs Model group; **: P < 0.01, vs Model group. a: TGI is calculated based on tumor weight on day 61 after cell inoculation.

[0246] Example 21 Efficacy of humanized bispecific antibodies 18G272, 19G294, etc. in NOD / SCID mice bearing OS-RC-2 (renal cancer) xenografts

[0247] Experiment 1:

[0248] Methods: NOD / SCID mice weighing 18-22 g and aged 5-7 weeks were selected and 5×10 6 cells / 0.1ml OS-RC-2 cell suspension with 1×10 7 Cells / 0.1ml PBMC suspension was thoroughly mixed at a 1:1 (volume ratio) and inoculated subcutaneously into NOD / SCID mice, with 0.2ml per mouse. Animals were randomly divided into three groups based on body weight: Model group (PBS), 17G29 group (15μg / mouse / dose), and 19G295 group (15μg / mouse / dose); each group consisted of 8 animals.

[0249] The route of administration and the administration cycle are the same as those in Example 17.

[0250] General clinical observations, body weight, and tumor volume measurements were the same as in Example 17.

[0251] Conclusion: 17G29 and 19G295, at a dose of 15 μg / mouse, both significantly inhibited tumor growth at D15 (see Figure 11). However, at D39, neither 17G29 nor 19G295 was able to inhibit tumor growth, with no significant difference between the two. Furthermore, in the OS-RC-2 human renal cancer model, which resulted in animal death during the experiment, both 17G29 and 19G295, at a dose of 15 μg / mouse, increased the median survival of mice, with no statistically significant difference between the two.

[0252] Table 22 Tumor growth and body weight statistics in the OS-RC-2 human renal cancer cell subcutaneous transplantation model in mice Note: *: P < 0.05, vs Model group; **: P < 0.01, vs Model group; After D15, animals in each group died or were euthanized due to tumor growth.

[0253] Experiment 2:

[0254] Methods: NOD / SCID mice weighing 18-22 g and aged 5-7 weeks were selected and 3×10 6 cells / 0.1ml OS-RC-2 cell suspension and 6×10 6 Cells / 0.1ml PBMC cell suspension was thoroughly mixed at a 1:1 (volume ratio) and inoculated subcutaneously into NOD / SCID mice, with 0.2ml per mouse. Animals were randomly divided into three groups based on body weight: the Model group (PBS, 6 mice), the 18G272 group (30μg / mouse / dose, 6 mice), and the 19G295 group (30μg / mouse / dose, 6 mice).

[0255] The route of administration and the administration cycle are the same as those in Example 17.

[0256] General clinical observations, body weight, and tumor volume measurements were the same as in Example 17.

[0257] Conclusion: Compared with the model group, 18G272 at a dose of 30 μg / mouse significantly inhibited tumor growth in mice on D22, while 19G295 showed a trend toward tumor suppression, but without significant differences. At D30, the 18G272 group still had a strong inhibitory effect on tumors, with a highly significant difference, while the 19G295 group showed a trend toward tumor suppression, but without significant differences compared with the model group (see Figure 12 and Table 23). The OS-RC-2 human renal cancer model caused animal mortality during the experiment, but no animal mortality occurred with the test article 18G272 at a dose of 30 μg / mouse, while some animals in the 19G295 group did die (see Figure 13 and Table 23). In summary, 18G272 has superior anti-tumor effects to 19G295.

[0258] Table 23 Tumor growth and survival statistics in the OS-RC-2 human renal cancer cell subcutaneous transplantation model in mice Note: *: P < 0.05, vs. Model group; **: P < 0.01, vs. Model group. Animals in each group died or were euthanized due to tumor growth on Day 22. a: TGI is calculated based on tumor volume on Day 22 after cell inoculation.

[0259] Experiment 3:

[0260] Methods: NOD / SCID mice weighing 18-22 g and aged 5-7 weeks were selected and 3×10 6cells / 0.1ml OS-RC-2 cell suspension and 6×10 6 A 1:1 (volume ratio) PBMC suspension was thoroughly mixed and inoculated subcutaneously into NOD / SCID mice, with 0.2 ml per mouse. Animals were randomly divided into two groups based on body weight: a Model group (PBS) and an 18G272 group (30 μg / mouse / dose); each group consisted of six animals.

[0261] The route of administration and the administration cycle are the same as those in Example 17.

[0262] General clinical observations, body weight, tumor volume, and tumor weight were the same as those in Example 17.

[0263] Conclusion: 18G272, at a dose of 30 μg / mouse, significantly inhibited tumor growth in OS-RC-2 human renal cancer model mice, as shown in Figure 14 and Table 24. The OS-RC-2 human renal cancer model can cause animal death during the experimental period. Under the conditions of this experiment, 18G272, at a dose of 30 μg / mouse, significantly prolonged the median survival time (MST) of OS-RC-2 human renal cancer model mice and reduced animal mortality. Treatment was well tolerated, as shown in Table 24.

[0264] Table 24 Statistics of tumor growth, body weight, and survival rate in the OS-RC-2 human renal cancer cell subcutaneous transplantation model in mice Note: *: P < 0.05, vs. Model group; **: P < 0.01, vs. Model group. Animals in each group died or were euthanized due to tumor growth after D15. a: TGI is calculated based on tumor weight on day 29 after cell inoculation.

[0265] Experiment 4:

[0266] Methods: NOD / SCID mice weighing 18-22 g and aged 5-7 weeks were selected and 3×10 6 cells / 0.1ml OS-RC-2 cell suspension and 6×10 6 A 1:1 (volume ratio) PBMC suspension was thoroughly mixed and inoculated subcutaneously into NOD / SCID mice, with 0.2 ml per mouse. Animals were randomly divided into two groups based on body weight: a Model group (PBS) and a 19G294 group (30 μg / mouse / dose); each group consisted of five animals.

[0267] The route of administration and the administration cycle are the same as those in Example 17.

[0268] General clinical observations, body weight, and tumor volume measurements were the same as in Example 17.

[0269] Conclusion: 19G294, at a dose of 30 μg / mouse, significantly inhibited tumor growth in the OS-RC-2 human renal cancer model mice. The results are shown in Figure 15 and Table 25. The OS-RC-2 human renal cancer model can cause animal death during the experimental period. Under these experimental conditions, 19G294 reduced animal mortality and prolonged animal survival. Treatment was well tolerated, as shown in Table 25.

[0270] Table 25 Statistics of tumor growth, body weight, and survival rate in the OS-RC-2 human renal cancer cell subcutaneous transplantation model in mice Note: *: P < 0.05, vs Model group; **: P < 0.01, vs Model group; animals began to die or be euthanized after D23

[0271] Combining the results of the four experiments described above, in a NOD / SCID mouse model bearing OS-RC-2 (renal cancer) xenografts, 17G29 demonstrated comparable efficacy to 19G295, while 19G295 was inferior to 18G272. 19G294 was comparable to 18G272, significantly and even completely inhibiting tumor growth. Furthermore, 17G29 demonstrated comparable safety to 19G295, while 19G295 was inferior to 18G272, and 19G294 was comparable to 18G272. This demonstrates that 18G272 and 19G294 exhibit superior in vivo efficacy and safety compared to 17G29.

[0272] Example 22 Efficacy of different doses of humanized bispecific antibodies 19G294 and 21G587 in NOD / SCID mice bearing OS-RC-2 (renal cancer) xenografts

[0273] Methods: NOD / SCID mice weighing 18-22 g and aged 5-7 weeks were selected and 3×10 6 cells / 0.1ml OS-RC-2 cell suspension and 6×10 6 PBMC cell suspensions were thoroughly mixed at a 1:1 (volume ratio) and inoculated subcutaneously into NOD / SCID mice, with 0.2 ml per mouse. Animals were randomly divided into five groups based on body weight: Model (PBS), 19G294 (5 μg / mouse / dose), 19G294 (15 μg / mouse / dose), 19G294 (30 μg / mouse / dose), and 21G587 (30 μg / mouse / dose); each group consisted of three animals.

[0274] The route of administration and the administration cycle are the same as those in Example 17.

[0275] General clinical observations, body weight, tumor volume, and tumor weight were the same as those in Example 17.

[0276] Conclusion: Test article 19G294 at doses of 15 and 30 μg / mouse, and 21G587 at a dose of 30 μg / mouse, significantly inhibited tumor growth in the OS-RC-2 human renal cancer model mice. The efficacy of 19G294 in the low, medium, and high dose groups was dose-dependent. The OS-RC-2 human renal cancer model can cause significant weight loss or death in animals during the experimental period. Under the conditions of this experiment, test articles 19G294 (5, 15, and 30 μg / mouse) and 21G587 (30 μg / mouse) improved the decline in animal condition caused by tumor growth and reduced animal mortality. Treatment was well tolerated. The results are shown in Figures 16 and 17, and Table 26.

[0277] Table 26 Statistics of tumor growth, body weight, and survival rate in the OS-RC-2 cell subcutaneous transplantation model in mice Note: *: P < 0.05, vs. Model group; **: P < 0.01, vs. Model group; animals began to die or be euthanized after D18. a: TGI is calculated based on tumor weight on day 33 after cell inoculation.

[0278] Example 23 Efficacy of humanized bispecific antibody 19G294, axitinib combined with BAVENCIO, and YS110 in NOD / SCID mice bearing OS-RC-2 (renal cancer) xenografts

[0279] Methods: NOD / SCID mice weighing 18-22 g and aged 5-7 weeks were selected and 3×10 6 cells / 0.1ml OS-RC-2 cell suspension and 6×10 6 PBMC suspensions were thoroughly mixed at a 1:1 (volume ratio) and inoculated subcutaneously into NOD / SCID mice, with 0.2 ml per mouse. Animals were randomly divided into five groups based on body weight: Model group (PBS), positive control group A (BAVENCIO, 1.8 mg / mouse / dose; axitinib, 30 μg / mouse / dose), positive control group B (BAVENCIO, 1.8 mg / mouse / dose; axitinib, 60 μg / mouse / dose), 19G294 group (30 μg / mouse / dose), and YS110 group (205 μg / mouse / dose); each group consisted of 3-4 animals. Axitinib was administered orally, while BAVENCIO, the test article, and PBS were administered intravenously. Axitinib, 19G294, and PBS were administered starting on the day of vaccination and continued for five consecutive days. After two days of discontinuation, the second course of treatment (five consecutive days) was carried out with a dosing frequency of 1 day / time (10 times in total). BAVENCIO was administered starting on the day of vaccination, once a week, for 2 consecutive weeks (2 times in total). YS110 was administered starting on the day of vaccination, twice a week, for 2 consecutive weeks (4 times in total).

[0280] General clinical observations, body weight, tumor volume, and tumor weight were the same as those in Example 17.

[0281] Conclusion: Test article 19G294 significantly inhibited tumor growth in OS-RC-2 human renal cell carcinoma model mice at a dose of 30 μg / mouse. BAVENCIO (1.8 mg / mouse) combined with axitinib (30 μg / mouse, 60 μg / mouse) and YS110 (205 μg / mouse) did not significantly inhibit tumor growth in OS-RC-2 human renal cell carcinoma model mice. The OS-RC-2 human renal cell carcinoma model causes animals to experience significant weight loss or death during the experimental period. Under the conditions of this experiment, test article 19G294 (30 μg / mouse) can improve the decline in animal condition caused by tumor growth and reduce animal mortality, and is well tolerated during treatment. BAVENCIO combined with axitinib (two dose groups) and YS110 did not have the above-mentioned improvement effects. In summary, under the conditions of this experiment, the pharmacological effect of 19G294 is superior to BAVENCIO combined with axitinib and YS110. The results are shown in Figures 18, 19, and Table 27.

[0282] Table 27 Tumor growth, body weight, and survival rate statistics in the OS-RC-2 human renal cancer cell subcutaneous transplantation model in mice Note: *: P < 0.05, vs. Model group; **: P < 0.01, vs. Model group; animals began to die or be euthanized after D26. a: TGI is calculated based on tumor weight on day 31 after cell inoculation.

[0283] Example 24 Efficacy of humanized bispecific antibody 19G294, axitinib combined with BAVENCIO, and YS110 in NOD / SCID mice bearing A498 (renal cancer) xenografts

[0284] Methods: NOD / SCID mice weighing 18-22 g and aged 5-7 weeks were selected and 7×10 6 cells / 0.1ml A498 cell suspension and 7×10 6PBMC suspensions were thoroughly mixed at a 1:1 (volume ratio) and inoculated subcutaneously into NOD / SCID mice at a 0.2 ml inoculation rate. Animals were randomly divided into five groups based on body weight: Model group (PBS), positive control group-B (BAVENCIO, 1.8 mg / mouse / dose; axitinib, 60 μg / mouse / dose), positive control group-C (BAVENCIO, 1.8 mg / mouse / dose; axitinib, 120 μg / mouse / dose), 19G294 group (30 μg / mouse / dose), and YS110 group (205 μg / mouse / dose); each group consisted of three animals. Axitinib was administered orally, while BAVENCIO, the test article, and PBS were administered intravenously. Axitinib, 19G294, and PBS were administered starting on the day of vaccination and continued for five consecutive days. After two days of discontinuation, the second course of treatment (five consecutive days) was carried out with a dosing frequency of 1 day / time (10 times in total). BAVENCIO was administered starting on the day of vaccination, once every 2 weeks (1 time in total). YS110 was administered starting on the day of vaccination, twice a week, for 2 consecutive weeks (4 times in total).

[0285] General clinical observations, body weight, tumor volume, and tumor weight were the same as those in Example 17.

[0286] Organ weight and organ coefficient: After the final test, animals were euthanized, and their organs were isolated, rinsed with saline, and dried with filter paper. Organs were weighed and photographed. Organ coefficient = organ weight / mouse body weight × 100% (organ weight and mouse body weight in g).

[0287] Conclusion: Test article 19G294 significantly inhibited tumor growth in the A498 human renal cell carcinoma model mice at a dose of 30 μg / mouse. Neither BAVENCIO (1.8 mg / mouse) nor axitinib (60 μg / mouse, 120 μg / mouse) nor YS110 (205 μg / mouse) significantly inhibited tumor growth in the A498 human renal cell carcinoma model mice. Test article 19G294 (30 μg / mouse), BAVENCIO combined with axitinib (two dose groups), and YS110 were well tolerated during treatment. Test article 19G294 (30 μg / mouse) and YS110 had no significant effects on the kidneys, spleen, liver, lungs, or corresponding organ counts in mice. BAVENCIO combined with axitinib (two dose groups) had significant effects on the liver and / or organ counts, but had no significant effects on the kidneys, spleen, lungs, or corresponding organ counts. In summary, under these experimental conditions, 19G294 demonstrated superior efficacy and safety compared to BAVENCIO combined with axitinib and YS110. The results are shown in Figures 20 and 21, and Tables 28 and 29.

[0288] Table 28 Tumor growth, body weight, and survival rate statistics in the A498 human renal cancer cell subcutaneous transplantation model in mice Note: *: P < 0.05, vs Model group; **: P < 0.01, vs Model group. a: TGI is calculated based on tumor weight on day 65 after cell inoculation.

[0289] Table 29-1 Statistics of organ weight and organ number in the A498 human renal cancer cell subcutaneous transplantation model in mice

[0290] Table 29-2 Statistics of organ weight and organ system number in the A498 human renal cancer cell subcutaneous transplantation model in mice Note: *: P < 0.05, vs Model group; **: P < 0.01, vs Model group

[0291] Example 25 Evaluation of Bispecific Antibody Activity

[0292] In the 786-0 cell reaction system, 786-0 cells were labeled with green fluorescent signal using the fluorescent dye Calcein-AM. 786-0 cells were cultured at 6×10 5 cells / ml, 50 μl per well was inoculated into a U-shaped 96-well cell culture plate, and 50 μl of humanized bispecific antibody with a final concentration of 100 ng / ml, 10 ng / ml, 1 ng / ml, 0.1 ng / ml, 0.01 ng / ml, and 0.001 ng / ml was added to the corresponding reaction wells, and then 50 μl of 9×10 6 cells / ml of PBMC cells, the reaction system was incubated at 37°C under carbon dioxide culture for 5 hours. After the reaction was completed, the supernatant was centrifuged and placed in a new 96-well plate. After centrifugation again, 80 μl of the supernatant was placed in a black 96-well plate and detected using a microplate reader under the conditions of 470 nm excitation wavelength and 515 nm emission wavelength.

[0293] The cell lysis rate is expressed as follows: (V sample -V vehicle control ) / (V Triton -100-V vehicle control )×100%. Where V sample is the fluorescence signal reading of the drug-treated group, V vehicle control is the average value of the fluorescence signal reading of the solvent control group, V Triton-100 It is the average value of the fluorescence signal reading of the positive control group.

[0294] The cell lysis rate and sample concentration were calculated using GraphPad Prism 7.00 software to calculate the IC of PBMC to target cells mediated by the sample. 50 value.

[0295] Table 30 IC values ​​of cytotoxic effects of PBMC mediated by mouse anti-79 bispecific antibody on different target cells 50 Value (pg / ml)

[0296] From the above results, it can be seen that 19G300 has certain in vitro activity against different types of tumor cells.

[0297] Example 26. Binding ability of full-length humanized antibodies in mixed cell systems

[0298] The binding ability of the full-length humanized antibodies 21G428-429 and 21G430-431 derived from mouse antibodies No. 62 and No. 79 in Example 6 was tested in a mixed cell system.

[0299] 786-0 cells were collected and counted, and the cell density was adjusted to 6 × 10 5 PBMC cells were collected and counted, and the cell density was adjusted to 6×10 5 786-0 cells / mL. Add 50 μL each of 786-0 and PBMC cells to an EP tube to form a mixed cell system. Add antibody diluted to a final concentration of 0.3 μg / mL, mix thoroughly with the mixed cell system, and incubate at 4°C for 1 hour.

[0300] After incubation, cells were collected by centrifugation, washed twice with PBS, and resuspended in PBS. Goat anti-human IgG fluorescent secondary antibody was added, and cells were incubated at 4°C for 1 hour. Cells were then washed twice with PBS, resuspended in PBS, and analyzed using a BD Accuri C6 flow cytometer.

[0301] The test results showed (Figure 27) that the full-length humanized antibodies 21G428-429 and 21G430-431 exhibited significantly higher binding levels in cancer cells 786-0 than in PBMCs, indicating that the two antibodies have greater selectivity for recognizing the CD26 target on tumor cells than on PBMCs, preferentially binding to the CD26 target on tumor cells and having extremely low binding to the CD26 target on PBMCs, thereby avoiding killing normal cells. Antibodies bind to different fragments of the antigen through their variable regions to exert specific functions. The CD26 target is distributed on both tumor cells and PBMCs. Therefore, 21G428-429 and 21G430-431 have a significant degree of differential binding to CD26 protein fragments on tumor cells and PBMCs, which is very meaningful for antibodies to more safely exert their anti-tumor effects.

[0302] Example 27 Competitive Binding of Antibodies to CD26 Protein

[0303] (I) Competitive binding of 19G294 (the parent antibody of the anti-CD26 part is mouse antibody No. 62) and 19G300 (the parent antibody of the anti-CD26 part is mouse antibody No. 79) to CD26 protein. CD26 was biotinylated with EZ-link NHS-PEG12-Biotin.

[0304] Two SA biosensors, SensorF4 and SensorG4, were used to immobilize biotinylated CD26 (A).

[0305] Equilibrate in 1× Kinetics solution (B);

[0306] Sensor F4 was first reacted with a 1× Kinetics solution containing 500 nM 19G294. After reaching equilibrium (C), it was reacted again with a 1× Kinetics solution containing 500 nM 19G300 (this solution also contained 500 nM 19G294) (D).

[0307] Sensor G4 was first reacted with a 1× Kinetics solution containing 500 nM 19G300. After reaching equilibrium (C), it was reacted again with a 1× Kinetics solution containing 500 nM 19G294 (this solution also contained 500 nM 19G300) (D).

[0308] The results are shown in Figure 28: CD26 binding to 19G294 before binding to 19G300 was significantly affected (no obvious binding signal in step D); CD26 binding to 19G300 before binding to 19G294 was also significantly affected (no obvious binding signal in step D). This indicates that 19G294 and 19G300 compete for binding and that their CD26 epitopes overlap to a certain extent.

[0309] (II) Competitive binding of 19G294 and 17G29 (the parent antibody of the anti-CD26 portion is YS110) to CD26 protein

[0310] CD26 was biotinylated using EZ-link NHS-PEG12-Biotin;

[0311] Two SA biosensors, SensorG12 and SensorH12, were used to immobilize biotinylated CD26 (A).

[0312] Equilibrate in 1× Kinetics solution (B);

[0313] SensorG12 was first reacted with a 1× Kinetics solution containing 500 nM 17G29. After reaching equilibrium (C), it was reacted again with a 1× Kinetics solution containing 500 nM 19G294 (D).

[0314] SensorH12 was first reacted with a 1× Kinetics solution containing 500 nM 19G294. After equilibrium was confirmed (C), it was reacted again with a 1× Kinetics solution containing 500 nM 17G29 (D).

[0315] The results are shown in Figure 29: CD26 binds to 19G294 before binding to 17G29 (clear binding signal in step D); CD26 binds to 17G29 before binding to 19G294 (clear binding signal in step D). There is no competitive binding between 19G294 and 17G29 (whose anti-CD26 end sequences are identical to the variable region sequences of YS110).

[0316] Example 28 Identification of Antibody-Antigen Binding Fragments (I)

[0317] Step 1:

[0318] The CD26 protein solution was replaced with a Tris-HCl (100 mM) solution system at a concentration of 25 μg / 50 μl, and 2.5 μl of Trypsin system was added. The enzyme digestion was carried out at 37°C for 17 h.

[0319] Step 2:

[0320] Establishing a database by mass spectrometry: Perform mass spectrometry analysis on the CD26 enzyme digestion product in step 1 to obtain enzyme-digested CD26 fragment data;

[0321] Step 3:

[0322] Antibody 19G294 was biotinylated with EZ-link NHS-PEG12-Biotin, coupled to the SA biosensor, and immersed in enzyme-cleaved CD26 to capture antibody fragments that could bind to it. The antibody was then eluted with 0.4 μl of 0.1% trifluoroacetic acid. This operation was repeated 10 times, and a total of 40 μl of elution product was collected.

[0323] Step 4

[0324] The eluted product obtained in step 3 is subjected to mass spectrometry analysis to obtain data, which is then compared with the database obtained in step 2 to determine the CD26 enzyme-cleaved fragment obtained.

[0325] The data are shown in Figures 22, 23, and Table 31. The results of the sample amino acid sequence coverage test indicate that only the GTWEVIGIEALTSDYLYYISNEYK peptide was identified. Therefore, it is inferred that the binding fragment of 19G294 to the CD26 protein is G400-K423 (GTWEVIGIEALTSDYLYYISNEYK, SEQ ID NO: 59). The antigen-binding fragment of 19G300 was identified using the same method described above, and binding to the GTWEVIGIEALTSDYLYYISNEYK peptide was also identified. Furthermore, Y'S Therapeutics' patent (application number: CN200680034937.4) discloses information on the antigen-binding fragment of the CD26 antibody, which differs from the binding position of 19G294 in the present application. 19G294 and 19G300 are derived from mouse antibody No. 62 and mouse antibody No. 79, respectively. Combined with the experimental results that the above mouse antibodies, humanized antibodies constructed based on the two mouse antibodies (21G428-429, 21G430-431), and bispecific antibodies (19G294, 19G300, etc.) have advantages over other CD26 antibodies such as YS110 in terms of in vitro and in vivo activity and safety, it shows that antibodies binding to the fragment G400~K423 have advantages.

[0326] Table 31 Sample antigen binding region peptide information

[0327] Example 29 Identification of Antibody-Antigen Binding Fragments (II)

[0328] 1. Vector Construction

[0329] Extracellular display vectors containing the CD26 extracellular domain sequence (29-766aa, SEQ ID NO: 60) and truncated regions of varying lengths were constructed. The CD26 signal peptide and CD26 transmembrane domain sequence (SEQ ID NO: 63) were fused to the full-length extracellular domain (SEQ ID NO: 60), and then fused to F2A (SEQ ID NO: 61) and GFP (SEQ ID NO: 62). The fused sequences were codon-optimized for mammalian cells and synthesized. PCR amplification was performed, and BamHI and GCCGCCACC (Kozak sequence) were added to the 5' end of the gene, while a stop codon and SalI site were added to the 3' end of the gene. The PCR product was ligated into the pWPT-GFP vector linearized by double enzyme digestion with BamHI and SalI through the BamHI and SalI restriction sites, replacing the GFP in the vector itself with CD26 signal peptide-CD26 transmembrane region-CD26 extracellular region-F2A peptide-GFP. The translated CD26 transmembrane region-CD26 extracellular region-F2A-GFP will be disconnected by the self-cleavage of the F2A peptide segment, and the GFP protein will be retained in the cell, while the CD26 extracellular region will be transported and displayed outside the membrane.

[0330] Similar to the construction scheme of CD26 complete extracellular region vector, the complete extracellular region of CD26 was replaced with different truncated fragments to construct 21G572 (29-497aa, SEQ ID NO: 64), 21G583 (29-300aa, SEQ ID NO: 66), 21G584 (29-237aa, SEQ ID NO: 67), 21G585 (29-182aa, SEQ ID NO: 68), and 23G645 (498-766aa, SEQ ID NO: 65) ( Figure 24 ).

[0331] The expression vector was confirmed to be correct after sequencing, and plasmid DNA was prepared using an endotoxin-free plasmid extraction kit.

[0332] 2. 293T Cell Transfection

[0333] The above plasmids were transformed into 293T cells in 6-well plates using the PEI method (PEI: plasmid = 10 μg: 2 μg). 293T cells treated with PEI alone served as a negative control. After 48–72 hours, cells were harvested and resuspended in 100 μl at a cell density of 1×10⁶ cells / ml. Cells were labeled with varying amounts of 19G294 or 21G428-429 antibodies, respectively. After labeling at room temperature for 1 hour, cells were washed three times with 1× PBS buffer, resuspended in 100 μl, and labeled with a goat anti-human fluorescent secondary antibody for 1 hour at room temperature. Cells were then washed three times with 1× PBS buffer, and analyzed using a BD C6 flow cytometer.

[0334] 3. Results Analysis

[0335] The results are shown in Figure 25. Untransfected 293T cells do not express CD26-F2AGFP, and no fluorescent signal is detected with or without 19G294 antibody labeling. Approximately 28% of 293T cells transfected with the full-length extracellular domain of CD26 (29-766aa) GFP are GFP-positive (positive in the FL1 channel) even without 19G294 labeling. Labeling with varying concentrations of 19G294 reveals that GFP-positive cells also appear positive in the FL4 channel, indicating that all cells expressing the full-length extracellular domain of CD26 (29-766aa) GFP are bound by 19G294. Approximately 40% of 293T cells transfected with 21G572 GFP are GFP-positive. After 19G294 labeling, this cell population exhibits a shift toward the FL4 channel consistent with the full-length CD26 extracellular domain, indicating that the 21G572 fragment (CD26 extracellular domain 29-497) contains a complete 19G294-binding fragment.

[0336] Further studies were conducted by transfecting 293T cells with 21G572, 21G583, 21G584, 21G585, and 23G645 vectors, respectively. After labeling with secondary antibodies, the fully human antibodies 21G430-431 from mouse anti-79 and 21G428-429 from mouse anti-62 were detected. It was found that 21G430-431, 21G428-429, and 21G572 had the same binding results as 19G294 (Figure 26), indicating that the binding fragments were consistent. However, 21G428-429 did not bind to 21G583 (29-300), 21G584 (29-237), 21G585 (29-182), and 23G645 (498-766) in the extracellular region of CD26.

Claims

1. An isolated polypeptide having an amino acid sequence that is identical or at least 90% identical to the sequence shown in SEQ ID NO: 59 or SEQ ID NO:

64.

2. An antigen binding protein that binds to CD26, which recognizes the amino acid sequence region of CD26 shown in SEQ ID NO: 59 or SEQ ID NO:

64.

3. An antigen binding protein that binds to CD26, and competes for binding to CD26 with an antigen binding protein comprising HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:2, HCDR3 shown in SEQ ID NO:3, LCDR1 shown in SEQ ID NO:4, LCDR2 composed of Arg Met Ser, and LCDR3 shown in SEQ ID NO:

5.

4. The antigen-binding protein of claim 3, which is the same as the CD26 fragment recognized by the antigen-binding protein comprising HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, HCDR3 shown in SEQ ID NO: 3, LCDR1 shown in SEQ ID NO: 4, LCDR2 composed of Arg Met Ser, and LCDR3 shown in SEQ ID NO:

5.

5. The antigen binding protein of claim 3 or claim 4, which binds to amino acid residues within the amino acid sequence shown in SEQ ID NO: 59 or SEQ ID NO:

64.

6. An isolated nucleic acid selected from any one of a), b) or c): a) a DNA encoding an antigen-binding protein, which is the CD26-binding antigen-binding protein according to any one of claims 2 to 5; b) a DNA encoding a polypeptide having an amino acid sequence at least 90% identical to SEQ ID NO:59 or SEQ ID NO:64; c) a DNA that is completely complementary to the DNA described in a or b; d) RNA of a, b or c, wherein U replaces T.

7. A vector comprising the nucleic acid according to claim 6. A cell comprising the vector according to claim 7.

9. A composition comprising the antigen binding protein of any one of claims 2 to 5, the nucleic acid of claim 6, the vector of claim 7 or the cell of claim 8.

10. A method for treating a disease associated with high CD26 levels, comprising administering to a subject an effective amount of the antigen binding protein of any one of claims 2 to 5, the nucleic acid of claim 6, the vector of claim 7 or the cell of claim 8; the disease associated with high CD26 levels includes but is not limited to tumors, and the tumors with high CD26 levels include but are not limited to renal cancer, mesothelioma, lung cancer, liver cancer, prostate cancer, etc.; the antibody or antigen binding fragment is administered alone or in combination with other anticancer agents.

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