Anti-Trop2 / CD3 bispecific antibody and application thereof

Abstract

The invention relates to the technical field of engineering antibodies, in particular to an antibody combined with TROP2, a bispecific antibody targeting TROP2 and CD3 as well as a preparation method and application of the bispecific antibody. According to the present invention, the TROP2 antibody has good TROP2 binding ability, and has high affinity; the bispecific antibody combined with the TROP2 and the CD3 has excellent biological functions of anti-TROP2 and anti-CD3 antibodies, a bridge can be built between tumor cells and immune effector cells, the immune effector cells and oriented immune response are effectively activated, the tumor cell killing effect of the immune cells is remarkably enhanced, meanwhile, the ADCC effect is reduced to the maximum extent, and the tumor cell killing effect is improved. The compound has high safety and has a good application prospect in immunotherapy of tumors.

Description

[0001] The patent application with the application number 2024107888811, filed on June 18, 2024, is the priority application. TECHNICAL FIELD

[0002] The present application relates to an antibody, antigen binding fragment, anti-Trop2 / CD3 bispecific antibody specifically binding to Trop2, and a method of making and use thereof, in particular, its use in diagnosis, prevention and treatment of diseases associated with Trop2, including tumors, such as breast cancer, gastric cancer, pancreatic cancer, ovarian cancer, intestinal cancer, etc. BACKGROUND

[0003] Trop2 is a cell surface glycoprotein expressed by the TACSTD2 gene, full name human trophoblast cell surface antigens 2 (Trop2hoblast Cell Surface Antigens 2). Trop2 consists of a hydrophobic leader peptide, an extracellular domain, a transmembrane domain and a cytoplasmic tail, is a single transmembrane glycoprotein, with a size of 35.7KD, and is a class of calcium ion channel signal transducers. The N-terminal of Trop2 protein is the extracellular domain (Trop2 EC), which is connected with the intracellular short tail (Trop2 IC) through a one-way transmembrane helix (TM), thereby being fixed on the cell membrane. The cytoplasmic tail has a highly conserved phosphatidylinositol 4,5-bisphosphate (PIP2) binding sequence, indicating that PIP2 plays an important role in the signal transduction of Trop2. In addition to the PIP2 binding motif, it also contains conserved tyrosine and serine phosphorylation sites. Trop2 is not expressed or lowly expressed in normal tissues, and is overexpressed in many malignant tumors such as breast cancer, gastric cancer, colorectal cancer, pancreatic cancer, prostate cancer, cervical cancer, head and neck cancer and ovarian cancer, which can promote the occurrence, invasion, metastasis and diffusion of tumors, and plays a key role in the growth of tumors, so Trop2 is considered to be a target for tumor immunization. The current Trop2 antibody therapeutic agents entering the clinic are all anti-Trop2 antibody drug conjugates (anti-Trop2-ADC), and the first Trop2-ADC drug in the world is controversial, and its effect needs to be strengthened, and its safety is not perfect enough. For example, the Trop2-ADC product DS-1062 developed by AstraZeneca / First Sanjyo for advanced NSCLC patients in the phase III clinical trial achieved the main endpoint of PFS, but some grade 5 adverse events were observed, and DS-1062 has not yet shown the gold standard overall survival (OS) advantage. For example, the Trop2-ADC drug Trodelvy of Gilead in the ASCENT study, the incidence of adverse reactions above grade 3 reached 45%, and 55.9% of patients reported grade 3 and above treatment-related adverse events, and the two side effects of neutropenia and diarrhea were marked with black box warnings by the FDA. Because the wide expression of Trop2 in normal tissues determines that the development of drugs targeting Trop2 needs to target drug delivery to malignant tissues as much as possible to control the occurrence of off-target situations and to minimize the potential toxicity to normal tissues expressing high levels of Trop2, this requirement poses a challenge to anti-Trop2-ADC. Therefore, the safety and effectiveness of the current Trop2-ADC face not small challenges.

[0004] The current bispecific antibodies (bsAb) and

[0005] CAR-T (chimeric antigen receptor T cells) and the like are important directions for improving the efficacy of monoclonal antibodies. Among them, cell-bridging bispecific antibodies have a bridging effect of connecting immune T cells and cancer target cells, and have significantly improved tumor cell killing activity and specificity to Trop2, thereby having a breakthrough clinical value. Among them, cell-bridging bispecific antibodies based on bispecific antibody molecules are also known as engagers. If one of the targets of the bispecific antibody molecule is the signal receptor of the immune cell, such as CD3 expressed on the surface of human T cells, and the other target is the antigen on the surface of the tumor cell, the engager can simultaneously connect the immune cell and the tumor target cell to form a tumor cell-bispecific antibody molecule-T cell complex. The complex crosslinks and activates the immune cell receptor (such as CD3 of T cells). T cells thus enter an activated and proliferative state, in addition to an increase in number, they also release a variety of cytokines (such as IL2, TNF-α, IFN-γ, IL-6, etc.) and toxic molecules (granzyme B and perforin, etc.). The cytokine systemically mobilizes the immune system, and the toxic molecules become the weapons of T cells to specifically kill target cells. These engagers have shown a breakthrough clinical effect, and thus have received great attention in the treatment of diseases such as blood cancer. In summary, it is of great significance to develop a Trop2-targeted bispecific antibody, especially an engager immune bispecific antibody with potential good Trop2 / CD3 antigen binding.

[0006] The CD3 molecule on the surface of T cells is a 6-mer composed of 5 subunits, α, β, γ, δ, ε (two ε subunits in one mer), with molecular weights of 14.9 kDa, 15.3 kDa, 20.0 kDa, 18.7 kDa and 22.8 kDa, and lengths of 135, 139, 182, 171, 207 amino acid residues, respectively, which together form 6 peptide chains in the extracellular region, plus two ζ subunits in the intracellular region, to form a T cell receptor (TCR) complex. The TCR complex has the functions of signal transduction, T cell activation, and stabilization of the TCR structure. The CD3 cytoplasmic segment contains an immunoreceptor tyrosine-based activation motif (ITAM), the TCR recognizes and binds to an antigenic peptide presented by a major histo-compatibility complex (MHC) molecule, causing the TCR complex to oligomerize, resulting in the tyrosine residues in the conserved sequence of the ζ subunit and the ITAM of CD3 being phosphorylated by the tyrosine protein kinase p56lck in the T cell, and then recruiting other tyrosine protein kinases containing SH2 (Scr homology 2) domains (such as ZAP-70). Phosphorylation of the ITAM and binding to ZAP-70 are one of the important biochemical reactions in the early stage of the T cell activation signal transduction process. Similarly, antibodies such as OKT3 that bind to CD3 subunits (ε / γ / σ) can also cause oligomerization of the TCR complex and activate T cells.

[0007] Based on the function of CD3, it is of great significance to develop a bispecific antibody that can be used for immunotherapy, simultaneously binds to Trop2 and CD3, and has high-level tumor killing activity. However, there is currently a lack of satisfactory bispecific antibodies against Trop2 and CD3 in the art. Therefore, there is an urgent need in the art to develop new bispecific antibodies against Trop2 and CD3. Anti-Trop2 / CD3 bispecific antibodies with high-level tumor killing activity and improved safety are still an unmet clinical need. SUMMARY

[0008] A first object of the present application is to provide an anti-Trop2 antibody, an antigen-binding active fragment thereof, or a mutant thereof that specifically binds to trophoblast cell surface antigen 2 (Trop2).

[0009] A second object of the present application is to provide a bispecific antibody that specifically binds to Trop2 and CD3, and an active fragment thereof.

[0010] A third object of the present application is to provide a nucleic acid composition comprising nucleotide sequences encoding a light chain variable region and a heavy chain variable region of an anti-Trop2 antibody, or nucleic acid compositions encoding a light chain and a heavy chain; or a nucleic acid fusion comprising nucleotide sequences encoding a light chain variable region and a heavy chain variable region of an anti-Trop2 antibody; or a nucleic acid fusion comprising nucleotide sequences encoding a light chain and a heavy chain of an anti-Trop2 antibody; or a composition of a nucleic acid encoding a Trop2 binding arm of a bispecific antibody that specifically binds to Trop2 and CD3, and a nucleic acid encoding a CD3 binding arm.

[0011] A fourth object of the present application is to provide a pharmaceutical composition comprising an antibody that specifically binds to Trop2 and a Trop2 binding active fragment thereof or a mutant thereof, or a bispecific antibody that binds to Trop2 and CD3, or a nucleic acid encoding an anti-Trop2 antibody or a bispecific antibody that binds to Trop2 and CD3.

[0012] A fifth object of the present application is to provide a use of the above-mentioned anti-Trop2 antibody, an antigen-binding active fragment thereof, or a mutant thereof, a bispecific antibody that binds to Trop2 and CD3 or an active fragment thereof, or a pharmaceutical composition comprising the aforementioned anti-Trop2 antibody or a bispecific antibody that binds to Trop2 and CD3, in the manufacture of a medicament for the diagnosis, prevention, and treatment of a disease associated with Trop2 overexpression, and in the manufacture of an antibody-drug conjugate.

[0013] Specifically, the present application provides the following technical solutions:

[0014] In a first aspect, the present application provides an anti-Trop2 antibody that specifically binds to Trop2, an antigen-binding fragment thereof, or a mutant thereof, comprising a Trop2-specific heavy chain variable region (VHA) and a light chain variable region (VLA), wherein the VHA comprises a VHA CDR1 as shown in SEQ ID NO: 1, a VHA CDR2 as shown in SEQ ID NO: 2, and a VHA CDR3 as shown in SEQ ID NO: 3, and the VLA comprises a VLA CDR1 as shown in SEQ ID NO: 4, a VLA CDR2 as shown in SEQ ID NO: 5, and a VLA CDR3 as shown in SEQ ID NO: 6.

[0015] In some embodiments, the VHA further comprises a humanized heavy chain variable region framework region (VHAFR), and the VLA further comprises a humanized light chain variable region framework region (VLAFR).

[0016] In some embodiments, the VHA comprises an amino acid sequence selected from any one of SEQ ID NOs: 7-12.

[0017] In some embodiments, the VLA comprises an amino acid sequence selected from any one of SEQ ID NOs: 13-15.

[0018] Further, antibodies having variable region sequences as follows exhibit more excellent affinity for binding to Trop2:

[0019] In some embodiments, the VHA has an amino acid sequence of SEQ ID NO: 7, and the VLA has an amino acid sequence of SEQ ID NO: 13;

[0020] In some embodiments, the VHA has an amino acid sequence of SEQ ID NO: 8, and the VLA has an amino acid sequence of SEQ ID NO: 14;

[0021] In some embodiments, the VHA has an amino acid sequence of SEQ ID NO: 9, and the VLA has an amino acid sequence of SEQ ID NO: 14;

[0022] In some embodiments, the VHA has an amino acid sequence of SEQ ID NO: 10, and the VLA has an amino acid sequence of SEQ ID NO: 14;

[0023] In some embodiments, the VHA has an amino acid sequence of SEQ ID NO: 11, and the VLA has an amino acid sequence of SEQ ID NO: 14;

[0024] In some embodiments, the VHA has an amino acid sequence of SEQ ID NO: 12, and the VLA has an amino acid sequence of SEQ ID NO: 14;

[0025] In some embodiments, the VHA has an amino acid sequence of SEQ ID NO: 8, and the VLA has an amino acid sequence of SEQ ID NO: 15;

[0026] In some embodiments, the VHA has an amino acid sequence of SEQ ID NO: 9, and the VLA has an amino acid sequence of SEQ ID NO: 15;

[0027] In some embodiments, the VHA has an amino acid sequence of SEQ ID NO: 10, and the VLA has an amino acid sequence of SEQ ID NO: 15;

[0028] In some embodiments, the VHA has an amino acid sequence as set forth in SEQ ID NO: 11, and the VLA has an amino acid sequence as set forth in SEQ ID NO: 15.

[0029] In some embodiments, the VHA has an amino acid sequence as set forth in SEQ ID NO: 12, and the VLA has an amino acid sequence as set forth in SEQ ID NO: 15.

[0030] In some embodiments, the anti-Trop2 antibody is selected from the group consisting of a murine antibody, a chimeric antibody, a humanized antibody, a fully human antibody, or a combination thereof.

[0031] In some embodiments, the anti-Trop2 antibody is a single chain antibody, a Fab antibody, a minibody, or a full antibody immunoglobulin IgG antibody.

[0032] In some embodiments, the antibody is an antibody full-length protein, or an antigen binding fragment.

[0033] In some embodiments, the anti-Trop2 antibody is a fully human monoclonal antibody.

[0034] In some embodiments, the anti-Trop2 antibody further comprises a light chain constant region (CLA) and a heavy chain constant region (CHA).

[0035] Preferably, the heavy chain constant region of the anti-Trop2 antibody is of human IgG isotype.

[0036] Preferably, the light chain constant region of the anti-Trop2 antibody is of human k type or human λ type.

[0037] Most preferably, the heavy chain constant region of the anti-Trop2 antibody is of human IgG4 isotype.

[0038] Most preferably, the light chain constant region of the anti-Trop2 antibody is of human k type.

[0039] In some embodiments, the LA comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 22-24.

[0040] In some embodiments, the LA comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 22-24.

[0041] In some preferred embodiments, the present application provides an anti-Trop2 antibody having a heavy chain and a light chain full-length sequence as follows:

[0042] the heavy chain has the amino acid sequence set forth in SEQ ID NO: 16 and the light chain has the amino acid sequence set forth in SEQ ID NO: 22.

[0043] In some embodiments, the present application provides an anti-Trop2 antibody having the following heavy and light chain full length sequences:

[0044] the heavy chain has the amino acid sequence set forth in SEQ ID NO: 17 and the light chain has the amino acid sequence set forth in SEQ ID NO: 23; or, the heavy chain has the amino acid sequence set forth in SEQ ID NO: 18 and the light chain has the amino acid sequence set forth in SEQ ID NO: 23; or, the heavy chain has the amino acid sequence set forth in SEQ ID NO: 19 and the light chain has the amino acid sequence set forth in SEQ ID NO: 23; or, the heavy chain has the amino acid sequence set forth in SEQ ID NO: 20 and the light chain has the amino acid sequence set forth in SEQ ID NO: 23.

[0045] In some embodiments, the present application provides an anti-Trop2 antibody having the following heavy and light chain full length sequences:

[0046] the heavy chain has the amino acid sequence set forth in SEQ ID NO: 17 and the light chain has the amino acid sequence set forth in SEQ ID NO: 24; or, the heavy chain has the amino acid sequence set forth in SEQ ID NO: 18 and the light chain has the amino acid sequence set forth in SEQ ID NO: 24; or, the heavy chain has the amino acid sequence set forth in SEQ ID NO: 19 and the light chain has the amino acid sequence set forth in SEQ ID NO: 24; or, the heavy chain has the amino acid sequence set forth in SEQ ID NO: 20 and the light chain has the amino acid sequence set forth in SEQ ID NO: 24.

[0047] In some embodiments, the present application provides an antigen binding fragment that specifically binds to human Trop2, comprising one or both of the variable light region (VHA) and the variable heavy region (VLA) of the foregoing anti-Trop2 antibodies.

[0048] In some embodiments, the present application provides an antigen binding fragment that specifically binds to human Trop2, comprising one or both of the light chain (HA) and the heavy chain (LA) of the foregoing anti-Trop2 antibodies.

[0049] In some embodiments, the present application provides mutants of an antibody or antigen-binding fragment thereof that specifically binds human Trop2, the mutants comprising a conservatively altered amino acid variation of VHA and VLA that retains Trop2 binding specificity; the VHA and VLA have at least one of the following two: a) bind the same epitope; b) have an amino acid sequence that is greater than 90%, or greater than 95%, or greater than 97%, or greater than 98%, or greater than 99%, or greater than 99.5%, or greater than 99.8% sequence identity compared to the preceding VHA and VLA sequences.

[0050] In some embodiments, the VHA and VLA have an amino acid sequence as set forth in any one of SEQ ID NOs: 7-12 and any one of SEQ ID NOs: 13-15, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology thereto, respectively, wherein the heavy chain variable region variant and the light chain variable region variant bind the same Trop2 epitope as the VHA and VLA of the preceding anti-Trop2 antibody.

[0051] In some embodiments, the anti-Trop2 antibody, antigen-binding fragment or mutant thereof has one or more of the following properties selected from the group consisting of:

[0052] (1) specifically binds human or cynomolgus monkey Trop2;

[0053] (2) activity of not cross-binding with EpCAM of the same family;

[0054] (3) promotes endocytosis of Trop2 protein or tumor cells expressing Trop2 on the cell surface;

[0055] (4) binds recombinant human Trop2 protein with substantially the same or stronger KD as the reference antibody.

[0056] In some embodiments, the chimeric anti-human Trop2 antibody provided by the present application binds to recombinant human Trop2 protein and cynomolgus monkey Trop2 protein with an ELISA binding EC50 of no more than about 0.08 (nM) and binds to Trop2 on the cell membrane with a FACS binding EC50 of no more than 3.5 (nM).

[0057] In some embodiments, the humanized anti-human Trop2 antibody provided by the present application specifically binds to recombinant human Trop2 protein and cynomolgus monkey Trop2 protein in vitro with an ELISA binding EC50 of no more than 0.55 (nM).

[0058] The aforementioned anti-Trop2 antibody of the present invention binds to cells expressing Trop2, wherein the cells expressing Trop2 can be selected from any type of human epithelial malignant tumor cells (e.g., gastric cancer, cervical cancer, breast cancer, lung cancer, prostate cancer, colon cancer, esophageal cancer, pancreatic cancer, head and neck cancer, ovarian cancer, endometrial mucosal serous papillary carcinoma, etc.).

[0059] Thirdly, the present invention provides a bispecific antibody that specifically binds Trop2 and T cell antigens, comprising a first antigen-binding arm that specifically binds Trop2 and a second antigen-binding arm that specifically binds T cell surface antigens, wherein the first antigen-binding arm comprises VHA and VLA derived from the anti-Trop2 antibody described in the first or second aspect above and its antigen-binding fragment.

[0060] Fourthly, the present invention provides a bispecific antibody that specifically binds Trop2 and T cell surface antigen CD3, comprising: a first antigen-binding arm that specifically binds Trop2 and a second antigen-binding arm that specifically binds T cell surface antigen CD3, wherein the first antigen-binding arm comprises a heavy chain variable region (VHA) and a light chain variable region (VLA) of the antigen-binding fragment of the anti-Trop2 antibody described in the first or second aspect.

[0061] Fifthly, the present invention provides a bispecific antibody that specifically binds to Trop2 and CD3, comprising: a first antigen-binding arm that specifically binds to Trop2, and a second antigen-binding arm that specifically binds to the T cell surface antigen CD3, wherein:

[0062] a) The first antigen-binding arm comprises: i) an scFab(A) region and ii) an Fc(A) region, wherein the scFab(A) region is connected to the N-terminus of the Fc(A') region;

[0063] b) The second antigen-binding arm comprises: i) an scFab(B) region and ii) an Fc(B) region, wherein the scFab(B) region is connected to the N-terminus of the Fc(B) region; wherein,

[0064] c) wherein the ScFab(A) region contains a Trop2 binding site and the ScFab(B) region contains a CD3 binding site;

[0065] d) The Fc(A') and the Fc(B) interact to form a KIH structure, and the bispecific antibody forms a heterodimeric antibody through the KIH structure.

[0066] In some embodiments, the first antigen-binding arm and the second antigen-binding arm comprise either a) or b):

[0067] a) ScFab(A), which contains the amino acid sequence shown in SEQ ID NO:27, and

[0068] ScFab(B) contains the amino acid sequence shown in SEQ ID NO:36;

[0069] b) ScFab(A), which contains the amino acid sequence shown in SEQ ID NO:27, and

[0070] ScFab(B) contains an amino acid sequence as shown in SEQ ID NO:45.

[0071] In some embodiments, Fc(A) comprises an amino acid sequence as shown in SEQ ID NO:28; and Fc(B) comprises an amino acid sequence as shown in SEQ ID NO:37 or SEQ ID NO:46.

[0072] In a sixth aspect, the present invention provides a bispecific antibody that specifically binds to Trop2 and CD3, comprising: a first antigen-binding arm that specifically binds to Trop2 and a second antigen-binding arm that specifically binds to the T cell surface antigen CD3; wherein the first antigen-binding arm is a single-chain peptide chain containing a light chain A (LA) and a heavy chain A' (HA') that are specific to Trop2 binding, wherein the amino acid sequences of LA and HA' are as shown in SEQ ID NO:23 and SEQ ID NO:51, respectively; the second antigen-binding arm is a single-chain peptide chain containing a light chain B (LB) and a heavy chain B (HB) that are specific to CD3 binding, wherein LB contains the amino acid sequences as shown in SEQ ID NO:35 or SEQ ID NO:44, respectively, and HB contains the amino acid sequences as shown in SEQ ID NO:34 or SEQ ID NO:43, respectively.

[0073] In some embodiments, the first antigen-binding arm is a single-chain peptide chain with a LA-linker-HA' structure, in which LA and HA' are linked by a linker, and the second antigen-binding arm is a single-chain peptide chain with a LB-linker-HB structure, in which LB and HB are linked by a linker.

[0074] In some embodiments, the linker is a linker peptide with the amino acid sequence (GGGGX)n, where X is Gly or Ser, and n is a natural number from 1 to 8.

[0075] Preferably, the amino acid sequence of the linker peptide is shown in SEQ ID NO:52.

[0076] As an example of a bispecific antibody with the above structure, based on the above Trop2 antibody and CD3 binding arm, this invention constructs a bispecific antibody that simultaneously binds Trop2 and CD3, the structure and sequence of which are as follows:

[0077] Preferably, the heavy chain (HA') and light chain (LA) of the first antigen-binding arm are linked by a linker to form a single-chain peptide having the amino acid sequence shown in SEQ ID NO:29.

[0078] Preferably, the heavy chain (HB) and light chain (LB) of the second antigen-binding arm domain are linked by a linker peptide to form a single-chain peptide chain having the amino acid sequence shown in SEQ ID NO:38 or 47.

[0079] This invention reveals that, compared with bispecific antibodies with the above-mentioned amino acid sequence, the first antigen-binding arm and the second antigen-binding arm of the bispecific antibody can better retain the specific antigen-binding ability of the parent antibody in the first antigen-binding arm and the second antigen-binding arm, while having excellent biological functions of binding Trop2 and CD3, including significant advantages in terms of production process and anti-tumor pharmaceutical properties.

[0080] This invention develops a bispecific antibody that has the above-mentioned antibody molecular structure and specifically binds to Trop2 and CD3. This bispecific antibody has a specific dual-targeting effect and can efficiently stimulate a directed immune response to effectively kill tumor cells.

[0081] Therefore, antibodies containing the disclosed amino acid sequences, including those encoded by similar nucleotide sequences modified by conserved sequences or those containing similar amino acid sequences modified by conserved sequences, should all be considered within the scope of this invention.

[0082] In some embodiments, the bispecific antibody has one or more of the following activities:

[0083] (1) It can bind to human Trop2 in vitro.

[0084] (2) It can bind to the human CD3E&D complex in vitro;

[0085] (3) It can bind to monkey Trop2 protein;

[0086] (4) It has pM-level Trop2-dependent killing activity against Trop2-positive tumor cells;

[0087] (5) Mediates T cell activation;

[0088] (6) It can mediate the release of IL-2, IL-6, IL-10, IFN-γ and TNF-α from PBMCs;

[0089] (7) It can inhibit tumor growth and proliferation in animals.

[0090] In a seventh aspect, the present invention also provides a nucleic acid molecule encoding the Trop2 antibody or antigen-binding fragment, and a nucleic acid molecule encoding the bispecific antibody or antigen-binding arm thereof that binds Trop2 and CD3.

[0091] In some embodiments, those skilled in the art can modify the gene sequence encoding the aforementioned antibody based on the principle of codon degeneracy, without altering the amino acid sequence, to obtain a gene encoding the same antibody. Those skilled in the art can also artificially synthesize and modify genes based on the codon preference of the host expressing the antibody, thereby improving antibody expression efficiency.

[0092] Eighthly, the present invention also provides biological materials containing the nucleic acid molecules, said biological materials including recombinant DNA, expression cassettes, vectors, host cells, engineered bacteria or cell lines.

[0093] In some implementations, the nucleic acid molecule is operatively linked to an expression regulatory sequence.

[0094] In a ninth aspect, the present invention also provides a method for preparing the Trop2 antibody or the bispecific antibody binding Trop2 and CD3, comprising: introducing a nucleic acid encoding the antibody into a host cell to obtain a host cell stably expressing the bispecific antibody; culturing the host cell and obtaining the antibody by separation and purification.

[0095] When preparing the anti-Trop2 antibody or the bispecific antibody that specifically binds Trop2 and CD3, those skilled in the art may select, as needed, conventional host cells, expression vectors, methods for introducing the expression vector into the host cells, and antibody isolation and purification methods.

[0096] In a tenth aspect, the present invention also provides a pharmaceutical composition comprising:

[0097] (i) An active ingredient selected from the group consisting of: an anti-Trop2 antibody as described above, its antigen-binding fragment or a mutant thereof, or a bispecific antibody binding Trop2 and CD3 as described above, or a nucleic acid molecule as described above, or a biological material as described above, or an antibody-drug conjugate as described above, or a fusion protein comprising an anti-Trop2 antibody as described above or a bispecific antibody binding Trop2 and CD3; and

[0098] (ii) Pharmaceutically acceptable carriers.

[0099] The fusion proteins described above comprise a complex of the anti-Trop2 antibody or bispecific antibody provided by this invention and other proteins or polypeptide molecules having a certain function.

[0100] Specifically, the fusion protein can be constructed by linking an antibody gene with an immunotoxin or cytokine gene to form a recombinant expression vector, and the recombinant fusion protein molecule can be obtained through mammalian cells or other expression systems.

[0101] In some embodiments, the pharmaceutical composition or diagnostic reagent may also contain other active ingredients or excipients permitted in the pharmaceutical and diagnostic reagent fields (e.g., antibody components and pharmacologically acceptable delivery molecules or solutions).

[0102] In some embodiments, the anti-Trop2 antibody or antigen-binding fragment or its mutant, which is used as a therapeutic component, is sterile and can be lyophilized at low temperatures.

[0103] Eleventhly, the present invention provides any of the following applications of the anti-Trop2 antibody as described above, or the bispecific antibody as described above that binds Trop2 and CD3, or the nucleic acid molecule as described above, or the biological material as described above, or the antibody-drug conjugate as described above, or the pharmaceutical composition as described above:

[0104] (1) Use in the preparation of drugs for the diagnosis, prevention or treatment of diseases related to abnormal expression or function of Trop2;

[0105] (2) Use in the preparation of medicaments for the diagnosis, prevention or treatment of diseases targeting Trop2;

[0106] (3) Application in the preparation of drugs for killing cells with abnormal Trop2 expression;

[0107] (4) Application in the preparation of detection reagents for Trop2 and / or CD3;

[0108] (5) Application in the preparation of reagents suitable for CAR-T therapy;

[0109] (6) Application in the preparation of Trop2-ADC and Trop2 / CD3 bispecific ADC and immunotoxin or labeled anticonjugates.

[0110] Preferably, the drug is an antitumor drug.

[0111] In some embodiments, the Trop2-related disease is a malignant tumor that highly expresses Trop2.

[0112] In some embodiments, the malignant tumor is an epithelial malignant tumor.

[0113] In some embodiments, the malignant tumor is selected from male / female reproductive system tumors (e.g., endometrial cancer, uterine cancer, cervical cancer, breast cancer, ovarian cancer, prostate cancer), digestive system tumors (e.g., pancreatic cancer, colon cancer, gastric cancer, esophageal squamous cell carcinoma, esophageal cancer, bile duct cancer, intestinal cancer), head and neck tumors (e.g., oral squamous cell carcinoma, pharyngeal cancer), nervous system tumors (e.g., glioma), and respiratory system tumors (e.g., lung cancer, such as small cell lung cancer).

[0114] In some embodiments, the Trop2-related diseases described above are preferably tumors that highly or excessively express Trop2, particularly epithelial malignancies that express Trop2 (including but not limited to gastric cancer, cervical cancer, breast cancer, lung cancer, prostate cancer, colon cancer, esophageal cancer, pancreatic cancer, head and neck cancer, ovarian cancer, and endometrial serous papillary carcinoma).

[0115] In some embodiments, the method further includes administering other antitumor treatments to the patient, such as chemotherapy, antibodies targeting other tumor-specific antigens, or radiotherapy.

[0116] The anti-Trop2 antibody and its bispecific antibody provided by this invention can inhibit biological activities related to multiple cell signaling pathways that require Trop2 activation. All interference with Trop2 aberration expression should be considered equally as part of the purpose of this invention.

[0117] The beneficial effects of this invention are as follows:

[0118] (1) This invention provides a novel antibody that specifically recognizes Trop2, as well as an antigen-binding fragment and its conserved amino acid mutants, providing human Trop2-specific antibody candidate molecules for the development of anti-tumor antibody drugs targeting Trop2 and the prevention and treatment of tumors overexpressing Trop2.

[0119] (2) This invention utilizes genetic engineering and antibody engineering methods to construct a single-chain peptide chain containing a Trop-binding arm derived from the light and heavy chains of a complete anti-Trop2 monoclonal antibody, and a single-chain CD3-binding arm derived from the light and heavy chains of an anti-CD3 antibody. These are further assembled using a KIH structure to form a bispecific antibody binding to Trop2 and CD3. The KIH structure between the Fc regions of the two different antigen-binding arms effectively mimics heavy chain mismatch; it preserves the antigen-binding site of the complete monoclonal antibody structure; the linker structure in the light chain-linker-heavy chain binding arm effectively prevents light chain mismatch, resulting in a highly stable IgG-type symmetrical structure. The two different specific binding arms can be expressed using the same vector and the same host cell, and the assembly of the bispecific antibody is completed in the host cell. During host expression of the binding arms, the KIH structure significantly reduces the proportion of protein isoforms with other structures, thereby greatly reducing the difficulty of extraction and purification processes and simplifying the preparation method.

[0120] (3) The anti-Trop2 / CD3 bispecific antibody of the present invention can simultaneously possess the excellent biological functions of two monoclonal antibodies, anti-Trop2 and anti-CD3. It can build a bridge between tumor cells and immune effector cells, effectively activate immune effector cells and directed immune responses, thereby helping to direct T cell immune responses to be more accurately concentrated in tumor tissues. The high affinity of Trop2 significantly enhances the efficacy of immune cells in killing tumor cells, providing a treatment or improvement pathway for diseases related to abnormal high expression of Trop2, and has the application prospect of developing antibody drugs for various solid tumors.

[0121] (4) The bispecific antibody of the present invention simultaneously reduces Fc function to the greatest extent, prevents nonspecific activation of T cells induced by Fc function, and improves the safety of the bispecific antibody. Attached Figure Description

[0122] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0123] Figure 1 FACS detection diagram for identifying isolated monoclonal cell lines.

[0124] 1A showed that the proportion of Trop2-positive cells in the obtained Trop2-expressing mixed cell pool reached 78.6%;

[0125] Image 1B shows cell clone 1E11 with high expression of human Trop2, named CHO-hTrop2.

[0126] Figure 2ELISA was used to detect the binding activity of chimeric antibody ch3F8 and control antibody huRS7 to recombinant human Trop2 protein.

[0127] Figure 3 FACS detection results of .ch3F8 and reference antibody huRS7 binding to Trop2 on the cell membrane.

[0128] 3A. Results of FACS detection of the binding activity of chimeric antibody ch3F8 and reference antibody huRS7 to Trop2 protein on the surface of CHO-hTrop2 cells; 3B. Results of FACS detection of the binding activity of chimeric antibody ch3F8 and reference antibody huRS7 to Trop2 protein on the surface of MDA-MB-468 tumor cells.

[0129] Figure 4 Results of ELISA detection of the cross-binding activity of chimeric antibody ch3F8.

[0130] 4A. Results of ELISA detection of the binding activity of chimeric antibody ch3F8 and reference antibody huRS7 to Mouse-derived Trop2 recombinant protein.

[0131] 4B. Results of ELISA detection of the binding activity of chimeric antibody ch3F8 and reference antibody huRS7 to Rat-derived Trop2 recombinant protein.

[0132] The results of 4C..ELISA detection showed that the chimeric antibody ch3F8 and the reference antibody huRS7 could bind to the Rhesus-derived Trop2 recombinant protein.

[0133] Figure 5 Results of ELISA detection of the binding activity of chimeric antibody ch3F8 and reference antibody huRS7 to recombinant EpCAM proteins of the same family.

[0134] Figure 6 The results of ELISA detection of the binding activity of 3F8 chimeric antibody and humanized antibody to recombinant human Trop2 protein in vitro.

[0135] Figure 7 Results of FACS detection of the binding activity of 3F8 chimeric antibody and humanized antibody to human Trop2 on the cell surface.

[0136] 7A. Results of FACS detection of the binding activity of 3F8 chimeric antibody and humanized antibody to Trop2 protein on the surface of CHO-hTrop2 cells.

[0137] 7B. Results of FACS detection of the binding activity of 3F8 chimeric antibody and humanized antibody to Trop2 protein on the surface of MDA-MB-468 tumor cells.

[0138] Figure 8 Results of ELISA detection of binding levels of 3F8 chimeric antibody and humanized antibody to recombinant human Trop2 protein from other species.

[0139] 8A. ELISA was used to detect the binding activity of 3F8 chimeric and humanized antibodies to Rhesus-derived Trop2 protein.

[0140] 8B. ELISA was used to detect the binding activity of 3F8 chimeric antibody and humanized antibody to Mouse-derived Trop2 protein.

[0141] The binding activity of 3F8 chimeric antibody and humanized antibody to Rat-derived Trop2 protein was detected by 8C ELISA.

[0142] Figure 9 Results of ELISA detection of the binding activity of 3F8 chimeric antibody and humanized antibody to recombinant EpCAM protein of the same family.

[0143] Figure 10 The results of endocytic activity detection of the chimeric anti-Trop2 antibody and the humanized antibody obtained by modifying the murine antibody 3F8 in this invention.

[0144] Figure 11 Results of ELISA detection of the binding activity of the anti-Trop2 / CD3 bispecific antibody of this invention with recombinant human Trop2 protein and with human CD3E&D complex.

[0145] 11A. Results of ELISA detection of the binding activity between CD3 bispecific antibody and recombinant human Trop2 protein;

[0146] 11B. Results of ELISA detection of the binding activity of CD3 bispecific antibody to human CD3E&D complex.

[0147] Figure 12 Results of FACS detection of the binding activity of Trop2×CD3 bispecific antibody to Trop2 or CD3 on the cell surface

[0148] 12A. Results of FACS detection of the binding activity of Trop2×CD3 bispecific antibody to Trop2 on the surface of MDA-MB-468 cells.

[0149] Results of 12B.FACS detection of the binding activity of Trop2×CD3 bispecific antibody to the CD3 complex on the surface of Jurkat cells.

[0150] Figure 13Results of ELISA detection of the binding activity of Trop2×CD3 bispecific antibody and Trop2 protein and CD3E&D complex from other species.

[0151] 13A. ELISA detection of the binding activity between Trop2×CD3 bispecific antibody and recombinant monkey Trop2 protein.

[0152] 13B. ELISA was used to detect the binding activity of CD3 bispecific antibody to monkey CD3E&D complex.

[0153] Figure 14 Results of FACS detection of the in vitro tumor-killing activity of Trop2×CD3 bispecific antibody.

[0154] 14A. Results of detection of killing activity against Trop2-overexpressing BxPC-3 pancreatic cancer cells mediated by Trop2×CD3 bispecific antibody.

[0155] Results of 14B.Trop2×CD3 bispecific antibody-mediated killing activity against MDA-MB-468 breast cancer cells.

[0156] Results of 14C.Trop2×CD3 bispecific antibody-mediated killing activity against MDA-MB-231 breast cancer cells.

[0157] Results of 14D.Trop2×CD3 bispecific antibody-mediated killing activity against Trop2-negative A549 non-small cell lung cancer.

[0158] Figure 15 Results of FACS detection of the effect of Trop2×CD3 bispecific antibody-mediated T cell activation.

[0159] The activation effect of CD8+ T cells mediated by 15A.CD3 bispecific antibody.

[0160] The effect of 15B.CD3 bispecific antibody-mediated CD4+ T cell activation.

[0161] Figure 16 Flow cytometry was used to analyze the levels of bispecific antibody-mediated cytokine release into the supernatant.

[0162] 16A. Results of PBMC-mediated release of IL-2 cytokine levels.

[0163] Results of detection of IL-6 cytokine release levels mediated by 16B.CD3 bispecific antibody in PBMCs.

[0164] Results of detection of IL-10 cytokine release levels mediated by 16C.CD3 bispecific antibody in PBMCs.

[0165] Results of detection of IFN-γ cytokine release levels mediated by 16D.CD3 bispecific antibody from PBMCs.

[0166] Results of detection of TNF-α cytokine release levels mediated by 16E.CD3 bispecific antibody from PBMCs.

[0167] Figure 17 The curve showing the inhibitory effect of Trop2×CD3 Ab2 on tumor growth in a human triple-negative breast cancer MDA-MB-231 mouse model.

[0168] Figure 18 Schematic diagram of pHr-hκ plasmid structure.

[0169] Figure 19 Schematic diagram of the pHr-IgG4 plasmid structure. Detailed Implementation

[0170] Based on the information contained in this application, those skilled in the art can make various changes to the precise description of the invention without departing from the spirit and scope of the appended claims. It should be understood that the scope of the invention is not limited to the defined processes, properties, or components, as these embodiments and other descriptions are merely illustrative of specific aspects of the invention. In fact, various modifications to embodiments of the invention that will be apparent to those skilled in the art or related fields are covered within the scope of the appended claims.

[0171] To better understand the invention and not to limit its scope, all figures indicating amounts, percentages, and other numerical values ​​used in this application should, in all cases, be understood to be modified by the word "approximately." Therefore, unless specifically stated otherwise, the numerical parameters listed in the specification and appended claims are approximate values ​​and may vary depending on the desired properties being sought. Each numerical parameter should at least be considered as obtained based on reported significant figures and through conventional rounding methods.

[0172] In this document, "Trop2" refers to trophoblast cell surface antigen 2, and its variants, homologs, and analogs. For example, antibodies that specifically bind to human Trop2 can cross-react with the Trop2 protein of cynomolgus monkeys under certain conditions. Exemplarily, the Trop2 specifically bound by the anti-Trop2 antibody of the present invention comprises an amino acid sequence as shown in SEQ ID NO:48, encoded by a nucleic acid nucleotide sequence as shown in SEQ ID NO:49.

[0173] The hTrop2 polypeptide may contain an amino acid sequence (SEQ ID NO:86) encoding a nucleic acid as shown in SEQ ID NO:87:

[0174]

[0175]

[0176] The technical problem to be solved by the present invention is to overcome the shortcomings of the existing antibody drugs in terms of tumor killing efficacy and safety, and to provide an anti-Trop2 monoclonal antibody, an anti-Trop2 / CD3 bispecific antibody that can simultaneously and specifically target and bind to Trop2 and CD3, more competitively stimulate T cell immunity in solid tumor tissues with abnormal Trop2 overexpression, and its application in the preparation of drugs for the prevention and treatment of cancer or tumor diseases with abnormal Trop2 expression.

[0177] Through extensive and in-depth research, the inventors obtained a maternal clone 3F8 capable of binding to human Trop2 using the hybridoma method. Then, through nucleic acid-level manipulation, they constructed a coding nucleic acid encoding a full-length light chain containing the humanized light chain constant region and a coding nucleic acid encoding a full-length heavy chain containing the humanized heavy chain constant region. Subsequently, using genetic engineering techniques, they screened for mouse anti-Trop2 chimeric antibodies with EC50 values ​​comparable to or lower than huRS7 in in vitro binding to human and monkey Trop2, and with affinity for human Trop2 comparable to or higher than huRS7 (i.e., lower KD values ​​measured by biofilm thin-layer interferometry (BLI)). Furthermore, through CDR transplantation and reverse mutation humanization modifications, and extensive screening, they obtained Trop2 binding antibodies with affinity comparable to the chimeric antibodies. The invention further describes the development of a specific humanized antibody against Trop2 with reduced immunogenicity. Specifically, it involves designing nucleic acids encoding a light chain, a linker peptide, and a heavy chain containing a hole-modified binding arm containing an anti-Trop2 antibody, and a nucleic acid encoding a light chain, a linker peptide, and a heavy chain containing a knob-modified binding arm containing an anti-CD3 antibody. Through genetic engineering, the nucleic acid molecules encoding the two specific binding arms of Trop2 and CD3 are sequentially ligated into expression vectors to obtain recombinant expression vectors containing nucleic acids encoding the Trop2 binding arm and the CD3 binding arm of the bispecific antibody of this invention. These vectors are then combined and transfected to obtain recombinant mammalian expression cells expressing the bispecific antibody of this invention. Finally, the anti-Trop22 / CD3 bispecific antibody of this invention is cultured and purified.

[0178] The anti-Trop2 antibody of this invention specifically binds to human or monkey Trop2, but not to mouse or rat Trop2. Specifically, the anti-Trop2 antibody of this invention binds to human and monkey Trop2 in vitro with an EC50 value comparable to or lower than huRS7. Furthermore, this invention uses this humanized antibody as the parent antibody sequence source and further constructs and expresses a bispecific antibody with low cytokine storm levels and balanced efficacy with tumor-killing drugs, potentially providing a marketable Trop2 / CD3 bispecific antibody for clinical use.

[0179] Anti-Trop2 antibodies

[0180] This invention provides an anti-Trop2 antibody that can specifically bind to the extracellular domain of human Trop2 and has good targeting specificity and tumor killing activity, its antigen-binding fragment and mutant, which includes Trop2-specific heavy chain variable region A (VHA) and light chain variable region A (VLA).

[0181] Specifically, the anti-Trop2 antibodies of the present invention can bind to human or monkey Trop2 in vitro with an EC50 value comparable to or lower than that of huRS7. These anti-Trop2 antibodies can specifically bind to human Trop2 on the surface of CHO-hTrop2 cells, and the FACS binding EC50 of human Trop2 bound to the surface of CHO-hTrop2 cells is measured to be no higher than 2.7 nM. The resulting antibodies that can cross-bind to human and monkey Trop2 include chimeric antibodies and humanized antibodies.

[0182] In the following text, the variable domains of the heavy and light chains of the anti-Trop2 antibody of this invention may be referred to as "VHA" and "VLA," respectively. These domains are typically the most variable parts of the anti-Trop2 antibody (relative to other antibodies of the same type) and contain antigen-binding sites. Variable domains mediate antigen binding and define the specificity of a particular antibody for its specific antigen. The variability is concentrated in three segments called hypervariable regions (HVRs) (present in both the light and heavy chain variable domains), namely VHA CDR1, VHA CDR2, and VHA CDR3 in the heavy chain variable domain and VLA CDR1, VLA CDR2, and VLA CDR3 in the light chain variable domain. The highly conserved portions of the variable domains are called backbone regions (FRs). The variable domains of both the natural heavy and light chains each contain four FR regions (FR1, FR2, FR3, and FR4). The structure of VLA is FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4, and the structure of VHA is FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4. Various annotation schemes exist for the variable regions of antibodies, including Chothia, Kabat, IMGT, and Contact. This article uses the Kabat annotation scheme for the exemplary anti-Trop2 antibody.

[0183] In the anti-Trop2 antibody of the present invention, paired VHA and VLA together form a Trop2 antigen binding site.

[0184] In some embodiments, the anti-Trop2 antibody of the present invention is selected from at least one of murine antibodies, chimeric antibodies, human antibodies or humanized antibodies, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, single-chain antibodies, biantibodies, triantibodies, tetraantibodies or single-domain antibodies, or nanobodies or single-domain antibodies.

[0185] Preferably, the anti-Trop2 antibody of the present invention is a monoclonal antibody. Furthermore, the anti-Trop2 antibody of the present invention can be, for example, a murine, chimeric, or human monoclonal antibody, preferably a human antibody.

[0186] Trop2 monoclonal antibodies

[0187] In some preferred embodiments, the anti-Trop2 antibody of the present invention is a murine antibody. The murine anti-Trop2 monoclonal antibody can be prepared using methods well-known and commonly used by those skilled in the art, such as any method selected from hybridoma method, phage display method, yeast display method, recombinant DNA method, single-cell screening, or single-cell sequencing method.

[0188] In some embodiments, the present invention uses a hybridoma fusion method to prepare a parent clone of a mouse-derived anti-Trop2 monoclonal antibody, and uses ELISA and FACS methods to screen for high-titer, high-affinity, and high-specificity anti-Trop2 monoclonal antibodies, one of which is named 3F8.

[0189] The anti-Trop2 antibody or its antigen-binding portion of the present invention is a monoclonal antibody having the structural and chemical characteristics described below and in the following examples.

[0190] On one hand, the present invention provides an anti-Trop2 antibody or an antigen-binding fragment thereof, wherein the anti-Trop2 antibody or the antigen-binding fragment thereof comprises a heavy chain variable region A (VHA) and a light chain variable region A (VLA), wherein the VHA comprises heavy chain complementarity-determining regions (VHA CDRs) composed of VHA CDR1, VHA CDR2 and VHA CDR3, and the VLA comprises light chain complementarity-determining regions (VLA CDRs) composed of VLA CDR1, VLA CDR2 and VLA CDR3, wherein any one of VHA CDR1, VHA CDR2 and VHA CDR3, VLACDR1, VLA CDR2 and VLA CDR3 has the SEQ ID NO: specified in Table 1.

[0191] The anti-Trop2 antibody or its antigen-binding portion of the present invention is a monoclonal antibody with the structural and chemical characteristics described below and in the following examples.

[0192] In some embodiments, the amino acid sequences of the CDRs of the heavy / light chain variable regions of the anti-Trop2 antibodies of the present invention are summarized in Table 1 below, and some antibodies share the same VHA CDR or VLA CDR.

[0193] In some embodiments, the amino acid sequence of the CDR in the heavy / light chain variable region of the Trop2 antibody of the present invention is selected from one or more CDRs in Table 1(a)-(f) below:

[0194] Table 1 shows the VHA CDR and VLA CDR of the Trop2 antibody of this invention as defined by the Kabat numbering system.

[0195] SEQ ID NO: CDR SEQ ID NO: Sequence (a) VH ACDR1 SEQ ID NO: 1 DHYMH (b) VH ACDR2 SEQ ID NO: 2 YINCFNGAAGYNQKFKG (c) VH ACDR3 SEQ ID NO: 3 EGYDDGYAMDY (d) VL ACDR1 SEQ ID NO: 4 RSSQSLVHSYGNTYLH (e) VL ACDR2 SEQ ID NO: 5 KVSNRFS (f) VL ACDR3 SEQ ID NO: 6 SQSTHVYT

[0196] It is well known in the antibody field that the CDR3 domain, independent of the CDR1 and / or CDR2 domains, can independently determine the binding specificity of an antibody to a homologous antigen and can predictably generate a variety of antibodies with the same binding specificity based on a common CDR3 sequence.

[0197] The heavy chain variable region CDR2 (VHA CDR2) of an anti-Trop2 antibody can be CDRs that can bind to other antibodies against human Trop2, such as VHA CDR1 and / or VHA CDR3 from the heavy chain variable region A of different anti-Trop2 antibodies and / or VLA CDR1, VLA CDR2 and / or VLA CDR3 from the light chain variable region A.

[0198] In some embodiments, the anti-Trop2 antibody of the present invention may comprise the CDR2 of the VHA of the aforementioned anti-Trop2 antibody and at least the CDR3 of the VHA and / or VLA of the aforementioned anti-Trop2 antibody, or the CDR3 of the VHA and / or VLA of another anti-Trop2 antibody, wherein the antibody is capable of specifically binding to human Trop2. These antibodies preferably (a) retain the functional property of specifically binding to Trop2; (b) bind to the same epitopes; and / or (c) have a binding affinity similar to that of the anti-Trop2 antibody of the present invention.

[0199] In some embodiments, the anti-Trop2 antibody of the present invention may further comprise the CDR2 region of the aforementioned anti-Trop2 antibody in VHA / VLA or the CDR2 region of another anti-Trop2 antibody in VHA / VLA, wherein the antibody is capable of specifically binding to human Trop2.

[0200] In some embodiments, the anti-Trop2 antibody of the present invention may further comprise the CDR1 of the aforementioned anti-Trop2 antibody VHA and / or VLA, or the CDR1 of another anti-Trop2 antibody VHA and / or VLA, wherein the antibody is capable of specifically binding to human Trop2.

[0201] In some embodiments, the anti-Trop2 antibody or its antigen-binding portion of the present invention includes:

[0202] (a) VHA CDR1, VHA CDR2 and VHA CDR3 of the heavy chain variable regions listed in Table 1; and

[0203] (b) VLA CDR1, VLA CDR2 and VLA CDR3 of the light chain variable region listed in Table 1, or the CDR of the VLA of another anti-Trop2 antibody, wherein the antibody specifically binds to human Trop2.

[0204] In some embodiments, the murine anti-Trop2 antibody of the present invention (e.g., the anti-Trop2 antibody named 3F8) comprises VHA CDR1 with the amino acid sequence DHYMH (SEQ ID NO:1), VHA CDR2 with the amino acid sequence YINCFNGAAGYNQKFKG (SEQ ID NO:2), and VHA CDR3 with the amino acid sequence SQSTHVYT (SEQ ID NO:3), and VLA CDR1 with the amino acid sequence RSSQSLVHSYGNTYLH (SEQ ID NO:4), VLA CDR2 with the amino acid sequence KVSNRFS (SEQ ID NO:5), and VLA CDR3 with the amino acid sequence SQSTHVYT (SEQ ID NO:6).

[0205] In some embodiments, the Trop2 antibody or its antigen-binding portion of the present invention may include VHA and / or VLA sequences comprising the aforementioned CDR1, CDR2 and CDR3 sequences, which undergo one or more conserved modifications compared to the anti-Trop2 antibody of the present invention. The anti-Trop2 antibody of the present invention does not lose its specific binding ability to Trop2 after certain conserved sequence modifications.

[0206] The amino acid sequence of the antibody complementarity-determining region (CDR) determines the specificity and affinity of the antibody. The monoclonal antibody containing the above-mentioned CDR sequence described in this invention has the ability to specifically bind to Trop2 protein, including natural Trop2 protein and recombinant Trop2 protein. It can specifically recognize human Trop2 protein, cynomolgus monkey Trop2 protein, and cells and tissues expressing the corresponding Trop2 protein. It has strong versatility. ELISA and FACS detection can confirm that it has good specificity, high sensitivity, and strong affinity. It is suitable for targeted therapy of diseases with Trop2 overexpression and for immunological diagnosis and detection.

[0207] In some preferred embodiments, the anti-Trop2 antibody of the present invention is a chimeric antibody or a humanized antibody.

[0208] In some preferred embodiments, the anti-Trop2 antibody or its antigen-binding portion of the present invention may be, for example, a chimeric antibody.

[0209] In some specific embodiments, the applicant of this invention, based on the murine anti-Trop2 antibody 3F8, obtained new full-length heavy chain and light chain coding nucleic acids by linking the heavy chain variable region and light chain variable region coding nucleic acids of the 3F8 murine antibody to the human IgG4 heavy chain constant region coding nucleic acid and the human kappa light chain constant region coding nucleic acid, respectively. Furthermore, recombinant expression vectors for the chimeric antibody heavy chain and chimeric antibody light chain were constructed, and these vectors were transfected into host cells to obtain recombinant engineered cells, which expressed the chimeric anti-Trop2 antibody of this invention, named ch3F8.

[0210] In some preferred embodiments, the anti-Trop2 antibody or its antigen-binding portion of the present invention is humanized. The variable region of the chimeric anti-Trop2 antibody undergoes humanization mutation at the nucleic acid level, and the encoding nucleic acids of the heavy chain variable region and the light chain variable region are respectively linked to the encoding nucleic acids of the human IgG4 heavy chain constant region and the human kappa light chain constant region, resulting in new full-length heavy chain encoding nucleic acids and full-length light chain encoding nucleic acids. Further, recombinant expression vectors for the chimeric antibody heavy chain and the chimeric antibody light chain are constructed, transfected into host cells, and expressed in host cells. The resulting humanized anti-Trop2 antibody exhibits a conserved amino acid sequence relative to the chimeric antibody and a reduced immunogenicity, while simultaneously possessing affinity for human Trop2 protein and species cross-binding characteristics comparable to or close to those of the chimeric antibody.

[0211] Variable region modification is an antibody humanization technique that improves one or more binding properties (e.g., affinity) of a target antibody by mutating amino acid residues in the VHA and / or VLA regions.

[0212] The preparation of VHA and VLA of the humanized anti-Trop2 antibody of the present invention uses the sequence of the chimeric anti-Trop2 antibody VHA / VLA of the present invention as starting material, and generates variable region variants (i.e., VHA' and / or VLA') for generating modified antibodies by, for example, introducing one or two amino acid modifications into one or more frame regions.

[0213] The humanized anti-Trop2 antibody of the present invention can be prepared as an amino acid-modified antibody using a chimeric antibody comprising the VHA / VLA sequence of the anti-Trop2 antibody of the present invention as the starting material. Genetic modification can be performed to modify one or more amino acid residues within one or two variable regions (i.e., VHA and / or VLA) (e.g., in one or more CDR regions and / or one or more frame regions) to improve binding affinity and / or increase similarity to naturally occurring antibodies of certain species. For example, the frame region can be modified with conserved amino acids to form the frame region of the humanized antibody. Furthermore, the antibody can be genetically modified to produce residues in a modified constant region, ultimately altering, for example, the effector function of the antibody.

[0214] Some embodiments of the present invention relate to anti-Trop2 antibodies or their antigen-binding portions, comprising VHA and / or VLA, wherein the VHA comprises VHA CDR1, VHA CDR2, and VHA CDR3 having the sequences described above in the present invention, and the VLA comprises VLA CDR1, VLA CDR2, and VLA CDR3 having the sequences described above in the present invention. Although these antibodies comprise the VHA CDRs and VLA CDRs sequences of the monoclonal antibodies of the present invention, they may contain different frame (FR) sequences.

[0215] The preferred framework sequence for the conserved amino acid mutant used in the anti-Trop2 antibody of the present invention can also be a framework region that has a high degree of structural similarity to the framework sequence used in the antibody of the present invention.

[0216] Antibodies primarily interact with target antigens through amino acid residues located in the complementarity-determining regions (CDRs) of the six heavy and light chains. In some embodiments, CDR insertion can be used for gene modification of the nucleic acid encoding the variable region of the antibody. Specifically, this involves constructing an expression vector containing a framework sequence encoding a nucleic acid that can insert the CDR sequence of a specific natural antibody into antibodies with different properties, thereby expressing the variable region of a recombinant anti-Trop2 antibody that mimics the properties of a specific natural antibody.

[0217] Transplanting mouse antibody core receptors (CDRs) into the variable region light and heavy chain framework of human immunoglobulin molecules allows the humanized antibody to possess the antigen-binding specificity of the mouse monoclonal antibody while minimizing the heterology of the mouse monoclonal antibody. However, simple CDR transplantation is often insufficient to maintain the effective binding ability of the original mouse antibody to its antigens. When the human FR region is replaced, the ability of the variable region, where mouse CDRs and human FRs intercalate, to bind antigens decreases due to structural changes.

[0218] In some embodiments, the present invention introduces frame region mutations by performing CDR transplantation on chimeric antibodies followed by reversion mutation. The VHA CDR1, VHA CDR2, and VHA CDR3, VLA CDR1, VLA CDR2, and VLA CDR3 sequences can be inserted into a frame region having the same sequence as a germline immunoglobulin containing that germline frame region sequence, or the CDR sequence can be inserted into a highly homologous frame region containing one or more conserved mutations compared to the germline frame region sequence.

[0219] In some embodiments, beneficial framework sequences of mutations that maintain or enhance the antigen-binding specificity of antibodies can be obtained from publicly available DNA databases or publicly available references, including germline antibody gene sequences. Antibody protein sequences can be compared for homology with protein sequence databases using sequence similarity search methods well-known in the art.

[0220] More preferably, the present invention utilizes antibody structure simulation and optimization analysis software platforms well-known in the antibody field (e.g., Discovery Studio comprehensive antibody simulation computing platform, GeoBiologics, DeepAb), through CDR transplantation and reversion mutation, to obtain new heavy chain variable region and new light chain variable region encoded nucleic acids with affinity comparable to chimeric antibodies and simultaneously reduced immunogenicity. These are respectively linked to the 5' of the nucleic acid encoded by the human IgG4 heavy chain constant region and the nucleic acid encoded by the human kappa light chain constant region, to obtain new full-length heavy chain encoded nucleic acid and full-length light chain encoded nucleic acid, and further constructs and expresses the humanized anti-Trop2 antibody of the present invention.

[0221] In the context of this invention, "monoclonal antibody that specifically binds to Trop2 protein" and "monoclonal antibody against Trop2 protein" are used interchangeably and both refer to monoclonal antibodies that specifically bind to Trop2 protein (including human Trop2). "Specific binding" is a well-known term in the art. A molecule exhibits "specific binding" if it reacts more frequently, more rapidly, for a longer duration, and / or has a greater affinity for a particular target antigen or epitope than it reacts with other target antigens or epitopes. "Specific binding," or "preferential binding," does not necessarily require (although may include) exclusive binding.

[0222] Alternatively, antibodies can be modified by altering residues in the constant region, for example, to silence the Fc function of an antibody.

[0223] Antibody screening and optimization platforms are among the methods well-known to those skilled in the art for sequence similarity analysis and framework region screening. The applicant uses a screening and optimization platform to compare antibody protein sequences with compiled protein sequence databases. Preferably, framework region sequences with extremely high structural similarity to the framework sequences used in the antibodies of this invention are used as the framework sequences for the anti-Trop2 antibody of this invention.

[0224] The humanized anti-Trop2 antibody of the present invention comprises modifications to the framework residues within the VHA and / or VLA of the chimeric anti-Trop2 antibody of the present invention, for example, to improve the properties of the antibody. Typically, such framework modifications can increase antibody affinity and / or decrease antibody immunogenicity. For example, one method is to “reverse mutate” one or more framework residues of an anti-Trop antibody generated by CDR transplantation into the corresponding germline framework sequence of the CDR, such as a mouse germline. Such residues can be identified by comparing the mouse antibody framework sequence with the germline sequence of the derived antibody.

[0225] In some exemplary embodiments, the present invention uses the germline frames of IGKV2-30 and IGKV2-28 as templates for the light chain and selects four germline frames of IGHV1-46, IGHV1-3, IGHV1-2 and IGHV1-18 as templates for the heavy chain. The light chain variable region CDR and heavy chain variable region CDR sequences of the murine chimeric anti-Trop2 antibody ch3F8 are transplanted into the corresponding germline frames. After the CDR transplantation is completed, several key sites are selected for reversion mutation of the humanized sequence generated by each template, and finally the humanized heavy chain variable region sequence and the humanized light chain variable region sequence are obtained.

[0226] In some embodiments, the present invention obtains 5 humanized heavy chain variable region sequences and 2 humanized light chain variable region sequences by CDR transplantation and reverse mutation, and combines the light and heavy chains to obtain a total of 10 humanized variants.

[0227] Preferably, in the process of constructing the humanized anti-Trop2 antibody of the present invention, conserved modifications well known in the art are introduced through CDR transplantation and reversion mutation. The conserved modification mutation can be an amino acid substitution, addition, or deletion, but substitution is preferred. Furthermore, typically no more than one, two, three, four, or five residues within the CDR region are altered.

[0228] In some embodiments, the modifications introduced into the antibodies of the present invention can be achieved using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conserved amino acid substitution is the substitution of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains are known to those skilled in the art from textbooks.

[0229] Those skilled in the art can obtain information from well-known textbooks about the use of family amino acid residues with similar side chains for conserved modification of natural amino acids.

[0230] The anti-Trop2 antibody or its antigen-binding portion of the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region A (VHA) comprises an amino acid sequence of VHA from any row listed in Table 2;

[0231] In some embodiments, the light chain variable region A (VLA) comprises the amino acid sequence of the VLA listed in any row of Table 3, or the VLA region of another anti-Trop2 antibody that specifically binds to human Trop2.

[0232] Table 2. Heavy chain variable region A (VHA) of exemplary anti-Trop2 antibodies of the present invention.

[0233]

[0234] Table 3. Light chain variable region A (VLA) of exemplary anti-Trop2 antibodies of the present invention.

[0235]

[0236]

[0237] In some embodiments, the heavy chain variable region A comprises: (a) amino acid sequences as shown in any one of SEQ ID NO:7-12,

[0238] In some embodiments, the light chain variable region A comprises: (a) an amino acid sequence as shown in any one of SEQ ID NO:13-15,

[0239] In some embodiments, the heavy chain variable region A comprises the amino acid sequence shown in SEQ ID NO:7.

[0240] In some embodiments, the heavy chain variable region A comprises the amino acid sequence shown in SEQ ID NO:8.

[0241] In some embodiments, the heavy chain variable region A comprises the amino acid sequence shown in SEQ ID NO:9.

[0242] In some embodiments, the heavy chain variable region A comprises the amino acid sequence shown in SEQ ID NO:10.

[0243] In some embodiments, the heavy chain variable region A comprises the amino acid sequence shown in SEQ ID NO:11.

[0244] In some embodiments, the heavy chain variable region A comprises the amino acid sequence shown in SEQ ID NO:12.

[0245] In some embodiments, the light chain variable region A comprises the amino acid sequence shown in SEQ ID NO:13.

[0246] In some embodiments, the light chain variable region A comprises the amino acid sequence shown in SEQ ID NO:14.

[0247] In some embodiments, the light chain variable region A comprises the amino acid sequence shown in SEQ ID NO:15.

[0248] The VH and / or VL sequences (or CDR sequences) of other anti-Trop2 antibodies that bind to human Trop2 can be “mixed and paired” with the VHA and / or VLA sequences (or CDR sequences) of the anti-Trop2 antibodies of the present invention.

[0249] Preferably, when VHA and VLA (or their CDR) are mixed and paired, the VHA sequence in a particular VHA / VLA pair may be replaced by a structurally similar VH sequence. Similarly, it is preferred that the VLA sequence in a particular VHA / VLA pair be replaced by a structurally similar VL sequence.

[0250] In some embodiments, the present invention provides a mutant of the aforementioned anti-Trop2 antibody comprising a variant of the heavy chain variable region A (VHA') and a variant of the light chain variable region A (VLA'), wherein VHA' comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in any one of SEQ ID NO:7-12, or an amino acid sequence formed by altering, adding, or deleting one, two, three, four, or five amino acids in the amino acid sequence shown in any one of SEQ ID NO:7-12; wherein the VHA' has, compared to the aforementioned sequence VHA, at least one of the following: a) binding the same antigenic epitope; b) an amino acid sequence with sequence identity greater than 85%, 90%, 95%, 98%, or 99%.

[0251] In some embodiments, the present invention provides a mutant of the aforementioned anti-Trop2 antibody comprising a variant of the heavy chain variable region A (VHA') and a variant of the light chain variable region A (VLA'), wherein VLA' comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in any one of SEQ ID NO:13-15, or an amino acid sequence formed by altering, adding, or deleting one, two, three, four, or five amino acids in the amino acid sequence shown in any one of SEQ ID NO:13-15. The VLA' has, compared to the aforementioned sequence VLA, at least one of the following: a) binding to the same antigenic epitope; b) an amino acid sequence with sequence identity greater than 85%, 90%, 95%, 98%, or 99%.

[0252] In some embodiments, in the anti-Trop2 antibody of the present invention, the heavy chain variable region A (VHA) and the light chain variable region A (VLA) respectively comprise the amino acid sequences shown in any one of SEQ ID NO:7-12 and the amino acid sequences shown in any one of SEQ ID NO:13-15.

[0253] In some embodiments, the sequence combinations of VHA and VLA and the exemplary anti-Trop2 antibodies formed in the Trop2 antibody of the present invention are shown in any row of Table 4.

[0254] Table 4. Combinations of heavy chain variable region A (VHA) and light chain variable region A (VLA) of exemplary antibodies of the present invention

[0255] Anti-Trop2 antibody name SEQ ID NO: of VHA SEQ ID NO: of VLA ch3F8 SEQ ID NO: 7 SEQ ID NO: 13 hu3F8H1L1 SEQ ID NO: 8 SEQ ID NO: 14 hu3F8H2L1 SEQ ID NO: 9 SEQ ID NO: 14 hu3F8H3L1 SEQ ID NO: 10 SEQ ID NO: 14 hu3F8H4L1 SEQ ID NO: 11 SEQ ID NO: 14 hu3F8H5L1 SEQ ID NO: 12 SEQ ID NO: 14 hu3F8H1L2 SEQ ID NO: 8 SEQ ID NO: 15 hu3F8H2L2 SEQ ID NO: 9 SEQ ID NO: 15 hu3F8H3L2 SEQ ID NO: 10 SEQ ID NO: 15 hu3F8H4L2 SEQ ID NO: 11 SEQ ID NO: 15 hu3F8H5L2 SEQ ID NO: 12 SEQ ID NO: 15

[0256] In some embodiments, the anti-Trop2 antibody of the present invention comprises the VHA shown in SEQ ID NO:7 and the VLA shown in SEQ ID NO:13, exemplarily, such as a chimeric anti-Trop2 antibody named ch3F8.

[0257] In some embodiments, the anti-Trop2 antibody of the present invention comprises the VHA shown in SEQ ID NO:8 and the VLA shown in SEQ ID NO:14, exemplarily, such as a humanized anti-Trop2 antibody named hu3F8 H1L1.

[0258] In some embodiments, the anti-Trop2 antibody of the present invention comprises the VHA shown in SEQ ID NO:9 and the VLA shown in SEQ ID NO:14, exemplarily, such as a humanized anti-Trop2 antibody named hu3F8 H2L1.

[0259] In some embodiments, the anti-Trop2 antibody of the present invention comprises the VH shown in SEQ ID NO:10 and the VLA shown in SEQ ID NO:14, exemplarily, such as a humanized anti-Trop2 antibody named hu3F8 H3L1.

[0260] In some embodiments, the anti-Trop2 antibody of the present invention comprises the VH shown in SEQ ID NO:11 and the VLA shown in SEQ ID NO:14, exemplarily, such as a humanized anti-Trop2 antibody named hu3F8 H4L1.

[0261] In some embodiments, the anti-Trop2 antibody of the present invention comprises the VH shown in SEQ ID NO:12 and the VLA shown in SEQ ID NO:14, exemplarily, such as a humanized anti-Trop2 antibody named hu3F8 H5L1.

[0262] In some embodiments, the anti-Trop2 antibody of the present invention comprises the VH shown in SEQ ID NO:8 and the VLA shown in SEQ ID NO:15, exemplarily, such as a humanized anti-Trop2 antibody named hu3F8 H1L2.

[0263] In some embodiments, the anti-Trop2 antibody of the present invention comprises the VH shown in SEQ ID NO:9 and the VLA shown in SEQ ID NO:15, exemplarily, such as a humanized anti-Trop2 antibody named hu3F8 H2L2.

[0264] In some embodiments, the anti-Trop2 antibody of the present invention comprises the VH shown in SEQ ID NO:10 and the VLA shown in SEQ ID NO:15, exemplarily, such as a humanized anti-Trop2 antibody named hu3F8 H3L2.

[0265] In some embodiments, the anti-Trop2 antibody of the present invention comprises the VH shown in SEQ ID NO:11 and the VLA shown in SEQ ID NO:15, exemplarily, such as a humanized anti-Trop2 antibody named hu3F8 H4L2.

[0266] In some embodiments, the anti-Trop2 antibody of the present invention comprises the VH shown in SEQ ID NO:12 and the VLA shown in SEQ ID NO:15, exemplarily, such as a humanized anti-Trop2 antibody named hu3F8 H5L2.

[0267] In some embodiments, the anti-Trop2 antibody of the present invention, its antigen-binding fragment, or its mutant further comprises constant regions, such as a heavy chain constant region and a heavy chain variable region constituting a full-length heavy chain, and a light chain constant region and a light chain variable region constituting a full-length light chain. The constant region sequences of the antibody are generally available through public searches, such as searching the IMGT online database for heavy chain and light chain constant region sequences.

[0268] In some embodiments, the heavy chain constant region of the anti-Trop2 antibody of the present invention is the IgG heavy chain constant region, preferably an isotype of the human IgG heavy chain constant region.

[0269] In some embodiments, the human IgG heavy chain constant region is selected from one of the IgG1, IgG2, IgG3 or IgG4 antibody heavy chain constant regions.

[0270] In some preferred embodiments, the heavy chain constant region of the anti-Trop2 antibody of the present invention is the heavy chain constant region of human IgG4 antibody.

[0271] In some embodiments, the antibody light chain constant region is selected from either the human antibody κ or λ chain light chain constant regions.

[0272] In some embodiments, the antibody light chain constant region is the human antibody κ-type light chain constant region.

[0273] In one embodiment, the human IgG4 heavy chain constant region of the anti-Trop2 antibody of the present invention has, for example, the human IgG4 heavy chain constant region of the amino acid sequence shown in SEQ ID NO:25.

[0274] The constant region of the heavy chain contains the "Fc region" (crystallizable fragment region), also known as the "Fc domain" or simply "Fc". The Fc region refers to the C-terminal region of the antibody heavy chain, which mediates the binding of immunoglobulins to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells), or binding to the first component (C1q) of the classical complement system. In IgG antibody isotypes, the Fc region consists of two identical protein fragments from the CH2 and CH3 domains of both antibody heavy chains.

[0275] In some embodiments, the anti-Trop2 antibody of the present invention has, for example, the heavy chain constant region shown in SEQ ID NO:25 and the human κ constant region of the amino acid sequence shown in SEQ ID NO:26.

[0276] As an alternative to modifications within the framework or CDR region, the antibodies of the present invention can be genetically engineered to form a genetically modified Fc region to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or Fc receptor binding and / or antibody-dependent cytotoxicity (ADCC).

[0277] Preferably, the humanized anti-Trop2 antibody of the present invention can also be genetically engineered to introduce a combination of F234A and L235A site mutations, thereby weakening the function of the Fc terminus of the antibody.

[0278] Preferably, the humanized anti-Trop2 antibody of the present invention can also be genetically engineered to introduce S228P site mutation combinations, thereby preventing Fab exchange of IgG4 antibody.

[0279] In a second aspect, the present invention provides an anti-Trop2 antibody or an antigen-binding fragment thereof or a mutant thereof, comprising a heavy chain A (HA) and a light chain A (LA), wherein the HA comprises an amino acid sequence as shown in any one of SEQ ID NO:16-21 in Table 5, and the LA comprises an amino acid sequence as shown in any one of SEQ ID NO:22-24 in Table 6.

[0280] Table 5. Heavy chain A (HA) of the exemplary anti-Trop2 antibody of the present invention.

[0281]

[0282]

[0283] Table 6. Light chain A (LA) of exemplary anti-Trop2 antibodies of the present invention

[0284]

[0285] In some embodiments, heavy chain A comprises the amino acid sequence shown in SEQ ID NO:16. In some embodiments, heavy chain A comprises the amino acid sequence shown in SEQ ID NO:17. In some embodiments, heavy chain A comprises the amino acid sequence shown in SEQ ID NO:18.

[0286] In some embodiments, the heavy chain A comprises the amino acid sequence shown in SEQ ID NO:19.

[0287] In some embodiments, the heavy chain A comprises the amino acid sequence shown in SEQ ID NO:20.

[0288] In some embodiments, the heavy chain A comprises the amino acid sequence shown in SEQ ID NO:21.

[0289] In some embodiments, the light chain A comprises the amino acid sequence shown in SEQ ID NO:22.

[0290] In some embodiments, the light chain A comprises the amino acid sequence shown in SEQ ID NO:23.

[0291] In some embodiments, the light chain A comprises the amino acid sequence shown in SEQ ID NO:24.

[0292] In some embodiments, the anti-Trop2 antibody of the present invention comprises a heavy chain (HA) as shown in SEQ ID NO:16 and a light chain (LA) as shown in SEQ ID NO:22, exemplarily, such as a chimeric anti-Trop2 antibody named ch3F8.

[0293] In some embodiments, the anti-Trop2 antibody of the present invention comprises a heavy chain (HA) as shown in SEQ ID NO:17 and a light chain (LA) as shown in SEQ ID NO:23, exemplarily, such as a humanized anti-Trop2 antibody named hu3F8 H1L1.

[0294] In some embodiments, the anti-Trop2 antibody of the present invention comprises a heavy chain (HA) as shown in SEQ ID NO:18 and a light chain (LA) as shown in SEQ ID NO:23, exemplarily, such as a humanized anti-Trop2 antibody named hu3F8 H2L1.

[0295] In some embodiments, the anti-Trop2 antibody of the present invention comprises a heavy chain (HA) as shown in SEQ ID NO:19 and a light chain (LA) as shown in SEQ ID NO:23, exemplarily, such as a humanized anti-Trop2 antibody named hu3F8 H3L1.

[0296] In some embodiments, the anti-Trop2 antibody of the present invention comprises a heavy chain (HA) as shown in SEQ ID NO:20 and a light chain (LA) as shown in SEQ ID NO:23, exemplarily, such as a humanized anti-Trop2 antibody named hu3F8 H4L1.

[0297] In some embodiments, the anti-Trop2 antibody of the present invention comprises a heavy chain (HA) as shown in SEQ ID NO:21 and a light chain (LA) as shown in SEQ ID NO:23, exemplarily, such as a humanized anti-Trop2 antibody named hu3F8 H5L1.

[0298] In some embodiments, the anti-Trop2 antibody of the present invention comprises a heavy chain (HA) as shown in SEQ ID NO:17 and a light chain (LA) as shown in SEQ ID NO:24, exemplarily, such as a humanized anti-Trop2 antibody named hu3F8 H1L2.

[0299] In some embodiments, the anti-Trop2 antibody of the present invention comprises a heavy chain (HA) as shown in SEQ ID NO:18 and a light chain (LA) as shown in SEQ ID NO:24, exemplarily, such as a humanized anti-Trop2 antibody named hu3F8 H2L2.

[0300] In some embodiments, the anti-Trop2 antibody of the present invention comprises a heavy chain (HA) as shown in SEQ ID NO:19 and a light chain (LA) as shown in SEQ ID NO:24, exemplarily, such as a humanized anti-Trop2 antibody named hu3F8 H3L2.

[0301] In some embodiments, the anti-Trop2 antibody of the present invention comprises a heavy chain (HA) as shown in SEQ ID NO:20 and a light chain (LA) as shown in SEQ ID NO:24, exemplarily, such as a humanized anti-Trop2 antibody named hu3F8 H4L2.

[0302] In some embodiments, the anti-Trop2 antibody of the present invention comprises a heavy chain (HA) as shown in SEQ ID NO:21 and a light chain (LA) as shown in SEQ ID NO:24, exemplarily, such as a humanized anti-Trop2 antibody named hu3F8 H5L2.

[0303] In some embodiments, the amino acid sequence combination of HA and LA and the light chain in the Trop2 antibody of the present invention and the naming of the anti-Trop2 antibody are shown in any row of Table 7.

[0304] Table 7. Exemplary combinations of heavy chain A (HA) and light chain A (LA) of the anti-Trop2 antibody of the present invention

[0305]

[0306]

[0307] In some embodiments, the present invention provides a mutant of the aforementioned anti-Trop2 antibody, comprising a heavy chain A' (HA') and a light chain A' (LA'), wherein HA' comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in any one of SEQ ID NO:16-21, or an amino acid sequence formed by altering, adding, or deleting one, two, three, four, or five amino acids in the amino acid sequence shown in any one of SEQ ID NO:16-21; LA' comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in any one of SEQ ID NO:22-24, or an amino acid sequence formed by altering, adding, or deleting one, two, three, four, or five amino acids in the amino acid sequence shown in any one of SEQ ID NO:22-24. The HA' and LA' have, compared to the aforementioned sequences VHA and VLA, at least one of the following: a) binding to the same antigenic epitope; b) an amino acid sequence with sequence identity greater than 85%, 90%, 95%, 98%, or 99%.

[0308] Trop2 binding fragments

[0309] In some preferred embodiments, the present invention also provides antigen-binding fragments that specifically bind to Trop2, said antigen-binding fragments including at least one of Fab fragment, Fab' fragment, Fab 2 fragment, F(ab)'2 fragment, Fv fragment, scFv fragment and scFv-Fc fragment, dAb fragment, and disulfide-linked Fv (sdFv).

[0310] Antigen-binding fragments can be prepared using a variety of techniques, including but not limited to hydrolyzing intact antibody proteins and producing them through expression by host cells containing antigen-binding fragments.

[0311] In this invention, "fragments," "derivatives," and "analytes" are polypeptides that substantially retain Trop2 binding affinity function or activity. The Trop2 binding fragments, derivatives, or analogs of this invention can be: (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted; or (ii) polypeptides having substituent groups in one or more amino acid residues; or (iii) polypeptides formed by fusing an anti-Trop2 polypeptide with another compound (e.g., a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol); or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (e.g., a protein formed by fusing a leader sequence, secretion sequence, or tag sequence such as 6His). Based on the teachings herein, these fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0312] In some embodiments well known to those skilled in the art, the affinity of an antibody for an antigen can be increased by altering the level of glycosylation, and the biological (e.g., serum) half-life of the antibody can be increased by PEGylation. In some embodiments well known to those skilled in the art, a preferred class of active derivatives refers to polypeptides formed by replacing up to three, more preferably up to two, and more preferably up to one amino acid with amino acids of similar or analogous properties compared to the amino acid sequences in Tables 6 and 7. These conserved variant polypeptides are preferably produced by amino acid substitutions according to Table 2.

[0313] Activity of anti-Trop2 antibodies

[0314] The antibodies of this invention can be characterized by a variety of physical properties and detected by ELISA, FACS, antigen cross-binding assay, and biofilm thin-layer interferometry (BLI).

[0315] In some embodiments, the anti-Trop2 antibody of the present invention, its antigen-binding fragment, or a mutant thereof can specifically bind to human Trop2.

[0316] In some embodiments, the anti-Trop2 antibody is a chimeric antibody that binds to the human Trop2 protein via ELISA with EC50 at a concentration of no more than about 0.09 (nM).

[0317] In some embodiments, the anti-Trop2 antibody is a chimeric antibody that binds to EC50 and human Trop2 on the cell membrane at a FACS concentration of no more than 3.0 (nM).

[0318] In some embodiments, the anti-Trop2 antibody of the present invention, its antigen-binding fragment or its mutant or derivative thereof, can have cross-binding activity against Trop2 in humans and cynomolgus monkeys.

[0319] In some embodiments, the anti-Trop2 antibody is a chimeric antibody that binds to the cynomolgus monkey Trop2 protein with an ELISA concentration of no more than about 0.08 (nM) using EC50.

[0320] In some embodiments, the anti-Trop2 antibody is a chimeric antibody that cannot bind to the same family protein EpCAM.

[0321] In some embodiments, the anti-Trop2 antibody is a chimeric antibody that has an affinity for human Trop2 protein equivalent to or higher than that of the reference antibody huRS7.

[0322] In some embodiments, the KD(M) of the chimeric antibody against human Trop2 protein is determined to be no higher than 8.3E-10 using biofilm thin-layer interferometry (BLI).

[0323] In some embodiments, the anti-Trop2 antibody is a humanized antibody that specifically binds to the human Trop2 protein in vitro via ELISA with EC50 at a concentration of about 0.55 (nM), or about 0.45 (nM), or about 0.25 (nM), or about 0.18 (nM), or about 0.15 (nM).

[0324] In some embodiments, the anti-Trop2 antibody is a humanized antibody that specifically binds to the human Trop2 protein in vitro via ELISA with EC50 at a concentration of about 0.14 (nM), or about 0.13 (nM), or about 0.12 (nM), or about 0.11 (nM), or about 0.10 (nM).

[0325] In some embodiments, the anti-Trop2 antibody is a humanized antibody, which specifically binds to the recombinant human Trop2 protein in vitro by ELISA with EC50 at a concentration not higher than 0.09 (nM), or not higher than 0.08 (nM), or not higher than 0.075 (nM).

[0326] In some embodiments, the anti-Trop2 antibody is a humanized antibody that is capable of binding to Trop2 expressed on the surface of tumor cells in vitro.

[0327] In some embodiments, the humanized anti-Trop2 antibody of the present invention is able to bind to Trop2 expressed on the surface of CHO-K1 cells in vitro.

[0328] In some embodiments, the FACS binding activity of the humanized anti-Trop2 antibody of the present invention to Trop2 expressed on the surface of CHO-K1 cells in vitro is approximately no higher than 34 (nM), or approximately no higher than 30 (nM), or approximately no higher than 20 (nM), or approximately no higher than 10 (nM), or approximately no higher than 8 (nM), or approximately no higher than 7 (nM), or approximately no higher than 5 (nM), or approximately no higher than 4 (nM), or approximately no higher than 3 (nM), or approximately no higher than 2 (nM).

[0329] In some embodiments, the humanized anti-Trop2 antibody of the present invention is able to bind to Trop2 expressed on the surface of cancer cells in vitro.

[0330] In some embodiments, the humanized anti-Trop2 antibody of the present invention is able to bind to Trop2 expressed on the surface of breast cancer cells in vitro.

[0331] In some embodiments, the humanized anti-Trop2 antibody of the present invention can bind to TRROP2 expressed on the surface of MDA-MB-461 cells in vitro with FACS binding activity values ​​of approximately 37 (nM), approximately 30 (nM), approximately 20 (nM), approximately 10 (nM), approximately 6 (nM), approximately 5 (nM), approximately 4 (nM), or approximately 3 (nM).

[0332] In some embodiments, the humanized anti-Trop2 antibody has an affinity level for Trop2 comparable to that of the maternal chimeric antibody.

[0333] In some embodiments, the humanized antibody has a high affinity for human Trop2 protein comparable to that of the chimeric antibody ch3F8.

[0334] In some embodiments, the humanized anti-Trop2 antibody has a high affinity for cynomolgus monkey Trop2 protein comparable to that of the chimeric antibody ch3F8, and the ELISIA binding EC50 of cynomolgus monkey Trop2 protein is approximately no higher than 0.072 (nM), or approximately no higher than 0.06 (nM), or approximately no higher than 0.05 (nM), or approximately no higher than 0.04 (nM), or approximately no higher than 0.03 (nM), or approximately no higher than 0.02 (nM).

[0335] In some embodiments, the KD(M) of the humanized antibody against human Trop2 protein, as determined by BLI, is not higher than 1.25E-9, or not higher than 1.15E-9, or not higher than 1.05E-9, or not higher than 9.40E-10, or not higher than 8.80E-10, or not higher than 4.35E-10, or not higher than 3.90E-10, or not higher than 2.50E-10.

[0336] In some embodiments, the humanized anti-Trop2 antibody of the present invention, its antigen-binding fragment, or a mutant thereof does not cross-bind with other proteins of the same protein family.

[0337] In some embodiments, the humanized anti-Trop2 antibody of the present invention, its antigen-binding fragment, or a mutant thereof does not cross-bind with EpCAM of the same protein family.

[0338] In some embodiments, the anti-Trop2 antibody of the present invention, its antigen-binding fragment, or a mutant thereof has the activity of promoting tumor cell endocytosis.

[0339] Anti-Trop2 / T cell bispecific antibodies

[0340] Thirdly, the present invention provides a bispecific antibody against Trop2 / T cell antigen.

[0341] Bispecific antibodies (BsAbs) are artificial antibodies that can simultaneously and specifically bind to two different antigens or different antigenic epitopes, functioning as a bridge connecting two antigens (epitaxes). The mechanisms of action of bispecific antibodies include, for example: (1) simultaneously activating two different signaling pathways in the immune cell activation process to exert unique or overlapping functions, enhancing the growth, proliferation, and survival of immune cells; (2) simultaneously inhibiting two different signaling pathways in the immune cell exhaustion process to exert unique or overlapping functions, delaying immune cell functional exhaustion and survival; (3) simultaneously blocking two different growth signaling pathways in the carcinogenesis process to exert unique or overlapping functions, interfering with / inhibiting the growth, proliferation, and survival of tumor cells; (4) simultaneously targeting different antigens or epitopes on the cell surface, enhancing / expanding their specific binding to tumor cells and directly utilizing ADCC to kill tumor cells; (5) simultaneously targeting two proteins, bringing them closer through physical interaction, activating them, and allowing them to exert their biological functions.

[0342] One type of bispecific antibody is a bispecific antibody that cross-links immune T cells or NK cells with tumor cells, also known as a T cell / NK cell cross-linking agent / molecular glue (T / NK engager). It has great value in immunotherapy for tumors, autoimmune diseases and other diseases, and is also called a bispecific antibody.

[0343] The bispecific antibody provided by this invention is an immune bispecific antibody. The main mechanism of action of this immune bispecific antibody in antitumor therapy is as follows: the bispecific antibody binds to tumor-expressed antigens, forming molecular bundles, which then recruit (bind) T cells or NK cells, forming a target cell-bispecific antibody bundle-T or NK cell complex. At this point, the T cells or NK cells are activated, releasing cytokines and toxic molecules to kill nearby tumor cells.

[0344] Bispecific antibodies can be prepared using artificial construction techniques such as cell fusion or recombinant DNA technology. Due to their specificity and bifunctionality, they have become a research hotspot in antibody engineering, especially in the field of immunobispecific antibodies, which have broad application prospects in tumor treatment and autoimmune diseases. Whether a bispecific antibody product meets clinical efficacy and safety requirements, and whether its industrial production can be achieved, depends on the individual biological activities of the two antibody-binding units, the choice of affinity combination of the two protein-binding arms, and the bispecific antibody conformation formed by the selected two binding arms. Different bispecific antibody structures can affect antibody efficacy. Maintaining the ability of the bispecific antibody to simultaneously bind to its respective antigen is often fundamental to ensuring its biological function. Since the distance between the two protein-binding units in an immunobispecific antibody also significantly affects the overall efficacy of the molecule, including the intensity of stimulation of immune cells and the bispecific antibody-mediated killing ability, the immunobispecific antibody designed for a specific combination of anti-trop2 antibodies in this invention also requires designing the distance between the two antibody-binding units.

[0345] The bispecific antibody of the present invention has primary specificity against tumor antigens and secondary specificity against immune cells, and can induce immune cell-mediated cytotoxicity targeting tumor cells, thereby having an anti-tumor effect.

[0346] In some embodiments, the bispecific antibodies of the present invention include, but are not limited to, proteins on immune cells that can be targeted, such as CD3, CD2, CD28, CD134, CD137, CD152, and CD279.

[0347] In some preferred embodiments, the immune cells are T cells, and the immune cell surface protein is CD3.

[0348] Anti-Trop2 / CD3 bispecific antibodies

[0349] Another aspect of the present invention is to provide a novel bispecific antibody or its active fragment targeting Trop2 and CD3 using KIH technology, as well as its preparation method and application.

[0350] This invention also uses the aforementioned humanized anti-Trop2 antibody and another anti-CD3 antibody as parental antibodies. Through genetic engineering methods involving nucleic acid-level splicing and ligation, construction of recombinant vectors and transfection, and expression, a bispecific anti-Trop2 / CD3 antibody is obtained. This antibody contains a Trop2 binding arm with a hole-forming amino acid mutant combination and a CD3 binding arm with a knob-forming amino acid mutant combination. This antibody can form a bridge between tumor cells and T cells, recruit T cells to Trop2-overexpressing tumor sites, and achieves good anti-tumor efficacy by efficiently killing tumor cells while avoiding the side effects of excessive cytokine release. This invention has found that, compared with other bispecific antibodies, the bispecific antibody with the IgG (1+1) symmetrical structure of this invention retains the specific antigen-binding ability of both the first and second functional units, while exhibiting excellent drug-like properties and cell-bridging function that simultaneously binds to Trop2 and CD3-expressing cells. The bispecific antibody developed in this invention, which has a specific molecular structure and binds Trop2 and CD3, has a Trop2-specific tumor-targeting effect and can efficiently stimulate and guide T-cell immune responses at tumor sites with high Trop2 expression, thereby killing tumor cells with high Trop2 expression.

[0351] In some embodiments of the present invention, two exemplary anti-Trop2 / CD3 bispecific antibodies were screened and tested. Both of them adopted the IgG (1+1) symmetrical form.

[0352] Fourthly, this invention provides a bispecific antibody against Trop2 / CD3, with the following configuration:

[0353] ScFab(A)-Fc(A') / ScFab(B)-Fc(B) is a 1+1 symmetrical IgG bispecific antibody constructed using KIH technology. Its features include monovalent dual targets, ultra-stable structure, long half-life, and mammalian cell production. These advantages are due to the presence of ScFab and modified Fc moieties in its two binding arms.

[0354] In some embodiments, the ScFab(A)-Fc(A') / ScFab(B)-Fc(B) configuration of the anti-Trop2 / CD3 bispecific antibody of the present invention introduces a Knobs-into-Holes (KIH) mutant domain combination. The Knobs-into-Holes (KIH) method involves replacing a large amino acid (hole) with a small amino acid in one heavy chain of the bsAb, and vice versa in the other heavy chain (knob) of the bsAb, ultimately guiding the formation of a heterodimer according to electrostatic directing theory. This technology achieves a correct assembly rate of 90%-95% for the bispecific antibody heavy chains, meeting the requirements for large-scale production.

[0355] In some embodiments, the amino acid sequence fragments contained in the antigen-binding arm of the anti-Trop2 / CD3 bispecific antibody of the ScFab(A)-Fc(A') / ScFab(B)-Fc(B) configuration of the present invention are shown in Tables 8 and 9.

[0356] In some embodiments, the anti-Trop2 / CD3 bispecific antibody may include a single-chain peptide of ScFab(A)-Fc(A') formed by the fusion of a ScFab(A) fragment and an Fc(A') fragment (forming a VHA / VLA pair for the Trop2-specific binding site), while the CD3-binding arm includes a single-chain peptide of ScFab(B)-Fc(B) formed by the fusion of a ScFab(B) fragment and an Fc(B) fragment (containing a VHB / VLB pair for the CD3-specific binding site).

[0357] In some embodiments, the Trop2 binding arm may include a hole single-chain peptide with a ScFab(A)-Fc(A') structure consisting of a ScFab(A) single-chain peptide and an Fc(A') (containing a hole mutation), while the CD3 binding arm includes a knob single-chain peptide with a ScFab(B)-Fc(B) structure consisting of a ScFab(B) single-chain peptide and an Fc(B) (containing a knob mutation).

[0358] In some embodiments, the anti-Trop2 / CD3 dual antibody of the present invention is a hole single chain with a ScFab(A)-Fc(A') structure or a knob single chain with a ScFab(B)-Fc(B) structure.

[0359] In one exemplary embodiment, the Trop2-binding first antigen-binding arm of the anti-Trop2 / CD3 bispecific antibody of the present invention comprises ScFab(A) as shown in SEQ ID NO:27.

[0360] In one exemplary embodiment, the second antigen-binding arm of the anti-Trop2 / CD3 bispecific antibody of the present invention comprises ScFab(B) as shown in SEQ ID NO:36.

[0361] In one exemplary embodiment, the CD3-binding second antigen-binding arm of the anti-Trop2 / CD3 bispecific antibody of the present invention comprises ScFab(B) as shown in SEQ ID NO:45.

[0362] In some implementations, heavy chain mismatch can be prevented by introducing mutations into the Fc fragment at the site involved in Fc receptor binding.

[0363] In some implementations, the Fc(B) segment contains a knob mutation consisting of amino acid site mutations S354C and T366W (both according to EU numbers).

[0364] In some implementations, the Fc(A') segment contains a hole mutation consisting of amino acid site mutations Y349C, T366S, L368A, and Y407V (all according to EU numbers).

[0365] In some embodiments, the present invention introduces amino acid mutations into the heavy chain of the anti-Trop2 / CD3 bispecific antibody to give the antibody or antigen-binding fragment a weakened Fc function, such as a combination of amino acid mutations that weaken antibody-dependent cytotoxicity (ADCC) activity.

[0366] In some embodiments, the Fc(A') and Fc(B) fragments of the anti-Trop2 / CD3 bispecific antibody of the present invention have reduced effector function compared to wild-type IgG (e.g., IgG4) antibodies.

[0367] In some implementations, the effector function can be reduced by introducing amino acid mutations into the Fc fragment at the site involved in Fc receptor binding.

[0368] In some exemplary embodiments, the amino acid mutations in the IgG4-Fc(Knob) of the first antigen-binding arm and the IgG4-Fc(hole) of the second antigen-binding arm include the S228P mutation (Eu number) which reduces Fab exchange.

[0369] In a specific example, IgG4-Fc(B)(Knob) contains F234A and L235A mutations that weaken the function of the antibody Fc (both according to EU numbers).

[0370] In a specific example, IgG4-Fc(A')(hole) contains F234A and L235A mutations that weaken the function of the antibody Fc (both according to EU numbers).

[0371] In some embodiments, the Fc(B) segment may contain the amino acid sequence shown in SEQ ID NO:37.

[0372] In some embodiments, the Fc(B) segment may contain the amino acid sequence shown in SEQ ID NO:46.

[0373] In some embodiments, the Fc(A') segment may contain the amino acid sequence shown in SEQ ID NO:28.

[0374] In some embodiments, the Fc(B) segment may contain the amino acid sequence shown in SEQ ID NO:37, and the Fc(A') segment may contain the amino acid sequence shown in SEQ ID NO:28.

[0375] In some embodiments, the Fc(B) segment may contain the amino acid sequence shown in SEQ ID NO:46, and the Fc(A') segment may contain the amino acid sequence shown in SEQ ID NO:28.

[0376] In some exemplary embodiments, the first antigen-binding arm of the anti-Trop2 / CD3 bispecific antibody of the present invention specifically binds to Trop2, comprising ScFab(A) as shown in SEQ ID NO:27 and Fc(A') as shown in SEQ ID NO:28, with the exemplary parent antibody being hu3F8 H1L1; the second antigen-binding arm specifically binds to CD3, comprising ScFab(B) as shown in SEQ ID NO:36 and Fc(B) as shown in SEQ ID NO:37, with the exemplary parent antibody being hXR32; and the assembled exemplary bispecific antibody is anti-Trop2×CD3 Ab1.

[0377] In some exemplary embodiments, the first antigen-binding arm of the anti-Trop2 / CD3 bispecific antibody of the present invention specifically binds to Trop2, comprising ScFab(A) as shown in SEQ ID NO:27 and Fc(A') as shown in SEQ ID NO:28, with the exemplary parent antibody being hu3F8 H1L1; the second antigen-binding arm specifically binds to CD3, comprising ScFab(B) as shown in SEQ ID NO:45 and Fc(B) as shown in SEQ ID NO:46, with the exemplary parent antibody being diL2K; and the assembled exemplary bispecific antibody is anti-Trop2×CD3 Ab2.

[0378] The fragment numbers included in the exemplary anti-Trop2 binding arm and anti-CD3 binding arm are shown in Tables 8 and 9.

[0379] Table 8. Amino acid sequence number of the second antigen-binding arm and the anti-CD3 binding arm of the anti-Trop2 / CD3 bispecific antibody of the present invention.

[0380]

[0381]

[0382]

[0383] Table 9. Amino acid sequence of the first antigen-binding arm (Trop2-binding arm) of the anti-Trop2 / CD3 bispecific antibody of the present invention.

[0384]

[0385]

[0386]

[0387] Fifthly, the present invention provides a bispecific antibody against Trop2 / CD3, comprising a first binding arm specific to Trop2 binding and a second binding arm specific to CD3 binding. The first binding arm comprises a light chain variable region A (VLA), a light chain constant region A (CLA), and a heavy chain variable region A (VHA) from the anti-Trop2 antibody or its antigen-binding fragment from the first and second aspects described above, and further comprises a PAA+hole variant of the CH1 segment and (CH2-CH3) segment of the IgG4 heavy chain constant region. The second binding arm comprises a light chain variable region B (VLB), a light chain constant region B (CLB), and a heavy chain variable region B (VHB) from the anti-CD3 antibody, and further comprises a PAA+knob variant of the CH1 segment and (CH2-CH3) segment of the IgG4 heavy chain constant region.

[0388] In some embodiments, the VHA / VLA of the first antigen-binding arm is perfectly matched (VHA to VLA, VLA to VHA) with the VHA / VLA of the anti-Trop2 antibody. In some embodiments, the VHB / VLB of the second antigen-binding arm is perfectly matched (VHB to VLB, VLB to VHB) with the VHB / VLB of the anti-CD3 antibody.

[0389] In some exemplary embodiments, the first antigen-binding arm comprises a VLA having the amino acid sequence shown in SEQ ID NO:14, derived from the anti-Trop2 antibody of the present invention.

[0390] In some exemplary embodiments, the first antigen-binding arm comprises VHA derived from the anti-Trop2 antibody of the present invention, as shown in SEQ ID NO:8.

[0391] In some exemplary embodiments, the first antigen-binding arm comprises CH1 of the amino acid sequence shown in SEQ ID NO:53 of the anti-Trop2 antibody of the present invention.

[0392] In some exemplary embodiments, the first antigen-binding arm may comprise VLA as shown in SEQ ID NO:14, CLA as shown in SEQ ID NO:26, VHA as shown in SEQ ID NO:8, and CH1 as shown in SEQ ID NO:53, derived from the anti-Trop2 antibody of the present invention.

[0393] In some exemplary embodiments, the second antigen-binding arm comprises a VLB of the amino acid sequence of anti-CD3 Ab1 as shown in SEQ ID NO:30.

[0394] In some exemplary embodiments, the second antigen-binding arm comprises anti-CD3 Ab1 VHB having the amino acid sequence shown in SEQ ID NO:32.

[0395] In some exemplary embodiments, the second antigen-binding arm comprises CH1 of anti-CD3 Ab1 having the amino acid sequence shown in SEQ ID NO:53.

[0396] In some exemplary embodiments, the second antigen-binding arm comprises a VLB of the amino acid sequence of anti-CD3 Ab2 as shown in SEQ ID NO:39.

[0397] In some exemplary embodiments, the second antigen-binding arm comprises VHB of the amino acid sequence of anti-CD3 Ab2 as shown in SEQ ID NO:41.

[0398] In some exemplary embodiments, the second antigen-binding arm comprises CH1 of the amino acid sequence of anti-CD3 Ab2 as shown in SEQ ID NO:53.

[0399] In some embodiments, the first antigen-binding arm of the bispecific antibody further includes constant domains, such as a light chain constant region A (CLA) and a modified heavy chain constant region A (CHA'), or the second antigen-binding arm further includes constant domains, such as a light chain constant region B (CLB) and a heavy chain constant region B (CHB).

[0400] In some embodiments, CHA' and CHB are selected from one of the constant regions of human IgG1, IgG2, IgG3 or IgG4 isotype heavy chain.

[0401] In some embodiments, CHA' and CHB are constant regions of the human IgG4 isotype heavy chain.

[0402] In some embodiments, compared to wild-type IgG (e.g., IgG4) antibodies, the Fc fragment variants contained in the heavy chain portion of the first and second antigen-binding arms of the bispecific antibodies of the present invention have reduced effector function. This reduction in effector function can be achieved by introducing mutations into the Fc fragment at the site involved in Fc receptor binding.

[0403] In some implementations, the CHB may contain a knob mutation consisting of amino acid site mutations S354C and T366W.

[0404] In some implementations, the CHA may contain a hole mutation consisting of amino acid site mutations Y349C, T366S, L368A, and Y407V.

[0405] The knob mutation composed of S354C and T366W and the hole mutation composed of Y349C, T366S, L368A and Y407V can form a KIH structure to prevent heavy chain mismatch.

[0406] In some implementations, both CHA and CHB contain the amino acid site mutation S228P that reduces Fab exchange.

[0407] In some implementations, both CHA and CHB contain F234A and L235A (Eu number) that weaken the function of antibody Fc.

[0408] In some exemplary embodiments, the first antigen-binding arm comprises CHA', which is derived from the parental anti-Trop2 antibody hu3F8 H1L1 of the present invention, and has the amino acid sequence shown in SEQ ID NO:50.

[0409] In some exemplary embodiments, the second antigen-binding arm comprises the CHB of the parent antibody hXR32 with the amino acid sequence shown in SEQ ID NO:33.

[0410] In some exemplary embodiments, the second antigen-binding arm comprises the CHB of the parent antibody diL2K with the amino acid sequence shown in SEQ ID NO:42.

[0411] In some implementations, the CLA and CLB are human. The constant region of type or λ light chain.

[0412] In some implementations, the CLA and CLB are human. Type light chain constant region.

[0413] In some exemplary embodiments, the first antigen-binding arm comprises a CLA derived from the amino acid sequence shown in SEQ ID NO:26 of the anti-Trop2 parent antibody hu3F8 H1L1 of the present invention.

[0414] In some exemplary embodiments, the second antigen-binding arm comprises a CLB of the parent antibody hXR32 with the amino acid sequence shown in SEQ ID NO:31.

[0415] In some exemplary embodiments, the second antigen-binding arm comprises a CLB of the parent antibody diL2K with the amino acid sequence shown in SEQ ID NO:40.

[0416] In some exemplary embodiments, the parent antibody of the first antigen-binding arm is hu3F8 H1L1, which includes anti-Trop2 antibody as shown in SEQ ID NO:14 VLA, as shown in SEQ ID NO:26 CLA, as shown in SEQ ID NO:52 linker, as shown in SEQ ID NO:8 VHA and as shown in SEQ ID NO:50 CHA'.

[0417] In some exemplary embodiments, the parent antibody of the second antigen-binding arm is hXR32, which includes VLB as shown in SEQ ID NO:30, CLB as shown in SEQ ID NO:31, linker as shown in SEQ ID NO:52, VHB as shown in SEQ ID NO:32, and CHB as shown in SEQ ID NO:33.

[0418] In some exemplary embodiments, the parent antibody of the second antigen-binding arm is diL2K, comprising VLB as shown in SEQ ID NO:39, CLB as shown in SEQ ID NO:40, linker as shown in SEQ ID NO:52, VHB as shown in SEQ ID NO:41, and CHB as shown in SEQ ID NO:42.

[0419] In some exemplary embodiments, the first antigen-binding arm parent antibody is hu3F8 H1L1, comprising anti-Trop2 antibody as shown in SEQ ID NO:14 VLA, as shown in SEQ ID NO:26 CLA, as shown in SEQ ID NO:52 linker, as shown in SEQ ID NO:8 VHA and as shown in SEQ ID NO:50 CHA'; the second antigen-binding arm parent antibody is hXR32, comprising as shown in SEQ ID NO:30 VLB, as shown in SEQ ID NO:31 CLB, as shown in SEQ ID NO:52 linker, as shown in SEQ ID NO:32 VHB and as shown in SEQ ID NO:33 CHB; the assembled exemplary bispecific antibody is a bispecific antibody named Trop2×CD3 Ab1.

[0420] In some exemplary embodiments, the first antigen-binding arm parent antibody is hu3F8 H1L1, comprising anti-Trop2 antibody as shown in SEQ ID NO:14 VLA, as shown in SEQ ID NO:26 CLA, as shown in SEQ ID NO:52 linker, as shown in SEQ ID NO:8 VHA, and as shown in SEQ ID NO:50 CHA'; the second antigen-binding arm parent antibody is diL2K, comprising as shown in SEQ ID NO:39 VLB, as shown in SEQ ID NO:40 CL, as shown in SEQ ID NO:52 linker, as shown in SEQ ID NO:41 VHB, and as shown in SEQ ID NO:42 CHB; the assembled exemplary bispecific antibody is a bispecific antibody named Trop2×CD3 Ab2.

[0421] In some embodiments, those skilled in the art can further modify the antigen-binding arm, particularly the Trop2-binding arm, of the anti-Trop2 / CD3 bispecific antibody disclosed herein to distinguish it in its amino acid sequence from that of naturally occurring binding polypeptides derived therefrom. For example, the polypeptide or amino acid sequence derived from the specified protein may be similar, for example, having a certain percentage identity with the starting sequence, such as at least 85%, 90%, 95%, 98%, or 99% identity with the starting sequence.

[0422] In some embodiments, the modification is the deletion, addition, or substitution of individual amino acids.

[0423] In some embodiments, the modification is a conservative substitution of an amino acid.

[0424] In other embodiments, the modification may be the replacement of an amino acid string with a structurally similar string that differs in the order and / or composition of its side chain family members.

[0425] Methods for conserved modification of the antibody's specific binding functional sequence can be found in well-known publications in the field of antibody engineering.

[0426] In a sixth aspect, the present invention provides an anti-Trop2 / CD3 bispecific antibody comprising a first antigen-binding arm specific to Trop2 binding and a second antigen-binding arm specific to CD3 binding, wherein the anti-Trop2 portion comprises a single-chain peptide chain composed of a Trop2-binding specific light chain and heavy chain pair (LA-linker-HA), and the anti-CD3 portion may comprise a single-chain peptide chain composed of a CD3-binding specific light chain and heavy chain pair (LB-linker-HB), wherein the HA and HB interact to form a KIH structure, and the first antigen-binding arm and the second antigen-binding arm are assembled into a heterodimer through the KIH structure.

[0427] In some embodiments, the HA and the HB are selected from the same type of human IgG1, IgG2, IgG3 or IgG4.

[0428] In some implementations, the HA and HB are isotypes of human IgG4.

[0429] In some embodiments, compared to wild-type IgG (e.g., IgG4) antibodies, the heavy chain portion of the first and second antigen-binding arms of the bispecific antibodies of the present invention contains amino acid mutations that reduce effector function. For example, the reduction in effector function can be achieved by introducing mutations into the Fc fragment at sites involved in Fc receptor binding, such as amino acid mutations that weaken antibody-dependent cytotoxicity (ADCC) activity.

[0430] In some implementations, the Fc fragment of a bispecific antibody has reduced effector function compared to a wild-type IgG (e.g., IgG4) antibody.

[0431] In some exemplary embodiments, the amino acid mutation in the CH2 segment of the heavy chain constant region of the first antigen-binding arm and the second antigen-binding arm includes an S228P mutation (Eu number) that reduces antibody Fab exchange.

[0432] In a specific example, IgG4-Fc(B)(Knob) contains F234A and L235A (both according to EU numbers) that weaken the function of antibody Fc.

[0433] In a specific example, IgG4-Fc(A')(hole) contains F234A and L235A (both according to EU numbers) that weaken the function of antibody Fc.

[0434] In some implementations, HB may include a knob mutation consisting of amino acid site mutations S354C and T366W to prevent heavy chain mismatch.

[0435] In some embodiments, the HA may include a hole mutation consisting of amino acid site mutations Y349C, T366S, L368A, and Y407V to prevent heavy chain mismatch.

[0436] The Trop2-specific first antigen-binding arm and the CD3-specific second antigen-binding arm interact with the hole mutation region of HA' and the knob mutation region of HB to form a KIH structure, which is further assembled into a heterodimeric bispecific antibody through the KIH structure.

[0437] Those skilled in the art will also understand that the antibodies disclosed herein can be modified to differ in amino acid sequence from naturally occurring binding polypeptides derived therefrom. For example, a polypeptide or amino acid sequence derived from a specified protein may be similar, for example, having a certain percentage identity with the starting sequence, such as having at least 85%, 90%, 95%, 98%, or 99% identity with the starting sequence.

[0438] In some embodiments, the modification is the deletion, addition, or substitution of individual amino acids.

[0439] In some embodiments, the modification is a conserved substitution of the amino acid sequence.

[0440] In other embodiments, the conserved modification involves replacing an amino acid string with a structurally similar string that differs in the sequence and / or composition of its side chain family members, but maintains the same antigen-binding specificity as the unmodified starting sequence. Methods for conserving modifications to the antibody's specific binding functional sequence are available in publications in the field of antibody engineering.

[0441] In some implementations, the LA and LB are human κ-type light chains.

[0442] In some embodiments, the first antigen-binding arm of anti-Trop2 comprises a light chain (LA) of the amino acid sequence shown in SEQ ID NO:23.

[0443] In some embodiments, the first antigen-binding arm of anti-Trop2 comprises a modified heavy chain A' (HA') containing PAA and hole mutations of the amino acid sequence shown in SEQ ID NO:51.

[0444] In some embodiments, the second antigen-binding arm against CD3 comprises a light chain (LB) of the amino acid sequence shown in SEQ ID NO:35.

[0445] In some embodiments, the second antigen-binding arm against CD3 comprises a light chain (LB) of the amino acid sequence shown in SEQ ID NO:44.

[0446] In some embodiments, the second antigen-binding arm against CD3 comprises a heavy chain (HB) containing PAA and knob mutations of the amino acid sequence shown in SEQ ID NO:34.

[0447] In some embodiments, the second antigen-binding arm against CD3 comprises a heavy chain (HB) containing PAA and knob mutations of the amino acid sequence shown in SEQ ID NO:43.

[0448] In some embodiments, the first antigen-binding arm of anti-Trop2 comprises a light chain (LA) of the amino acid sequence shown in SEQ ID NO:23 and a modified heavy chain A' (HA') of the amino acid sequence shown in SEQ ID NO:51, with an exemplary parental monoclonal antibody being hu3F8H1L1; the second antigen-binding arm of anti-CD3 comprises a light chain (LB) of the amino acid sequence shown in SEQ ID NO:35 and a heavy chain (HB) of the amino acid sequence shown in SEQ ID NO:34, with an exemplary parental monoclonal antibody being hXR32 knob; an exemplary bispecific antibody such as Trop2×CD3 Ab1 is also included.

[0449] In some embodiments, the first antigen-binding arm of anti-Trop2 comprises a light chain (LA) of the amino acid sequence shown in SEQ ID NO:23 and a modified heavy chain A' (HA') of the amino acid sequence shown in SEQ ID NO:51, with an exemplary parental monoclonal antibody being hu3F8 H1L1; the second antigen-binding arm of anti-CD3 comprises a light chain (LB) of the amino acid sequence shown in SEQ ID NO:44 and a heavy chain (HB) of the amino acid sequence shown in SEQ ID NO:43, with an exemplary parental monoclonal antibody being diL2K knob; the exemplary bispecific antibody is named as Trop2×CD3 Ab2.

[0450] In some implementations, LA and HA' in the anti-CD3 section are connected by a linker, and LB and HB in the anti-Trop2 section are connected by a linker.

[0451] In some implementations, the linker is a linker peptide.

[0452] An exemplary connector includes multiple glycine (G) and serine (S).

[0453] In some embodiments, the linker peptide comprises at least 50%, 60%, 70%, or 80% glycine.

[0454] In some embodiments, the linker peptide comprises at least 80% glycine.

[0455] In some implementations, the linker is as shown in formula (GGGGS). nThe linker peptide with the structure shown.

[0456] In some embodiments, the linker peptide is no more than 16, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 amino acids in length. In some embodiments, the length is 3 to 90, 4 to 70, or 5 to 50 amino acid residues.

[0457] In some implementations, n can be, for example, 2 to 50.

[0458] In some embodiments, the length of the linker peptide may be at least 2 amino acids, or at least 3, 4, 5, 7, 8, 9, 10, 12, 15, 17, 20, 22 or 25 amino acids.

[0459] In some implementations, n can be, for example, 4 to 10.

[0460] In some implementations, a shorter linker results in more moderate T-cell binding activity.

[0461] In some embodiments, the linker has the structure shown in (GGGGS)4 (SEQ ID NO:52).

[0462] Encoding nucleic acids

[0463] In one aspect, the present invention also provides compositions or fusions comprising nucleic acid molecules encoding the anti-Trop2 antibody of the present invention, its antigen-binding fragment, its variants or derivatives.

[0464] In one aspect, the present invention also provides a composition comprising a nucleic acid molecule encoding a Trop-binding arm and a CD3-binding arm encoding the anti-Trop2 / CD3 bispecific antibody described above.

[0465] In some embodiments, the entire heavy and / or light chain of the anti-Trop2 antibody, Trop2 binding peptide, variant or derivative thereof of the present invention may be encoded on the same polynucleotide molecule or separate polynucleotide molecules.

[0466] In some embodiments, portions of the heavy chain variable region and / or light chain variable region of the Trop2 binding polypeptide of the present invention, its variants or derivatives may be encoded on the same polynucleotide molecule or separate polynucleotide molecules.

[0467] In some embodiments, the nucleic acid-encoding compositions of the present invention comprise a nucleic acid encoding a full-length Trop2-binding arm of a monoclonal antibody against Trop2 protein as described above or a bispecific antibody against Trop2 / CD3, and a nucleic acid encoding a CD3-binding arm, or a nucleic acid encoding HA' and / or LA, or VHA and / or VLA, of a Trop2 monoclonal antibody as described above. The nucleic acids of the present invention may be present in whole cells, in cell lysates, or in partially purified or substantially pure forms. The nucleic acids of the present invention may be, for example, DNA or RNA and may or may not contain intron sequences.

[0468] In a preferred embodiment, the nucleic acid is a cDNA molecule.

[0469] The nucleic acids of this invention can be obtained using standard molecular biology techniques.

[0470] In some embodiments, the murine anti-Trop2 monoclonal antibody of the present invention is an antibody expressed by a hybridoma (e.g., a hybridoma prepared from a transgenic mouse carrying a human immunoglobulin gene, as further described below), and the cDNA encoding the light and heavy chains of the antibody prepared by the hybridoma can be obtained by standard PCR amplification or cDNA cloning techniques.

[0471] In some implementations, once the DNA fragments encoding VHA and VLA are obtained, these fragments can be further manipulated using standard recombinant DNA techniques, such as converting variable region genes into full-length heavy chain genes, full-length light chain genes, Fab fragment genes, or scFv genes. In these operations, the DNA fragment encoding VHA or VLA is operatively ligated to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term "operatively ligated" means that the two DNA fragments are joined together such that the amino acid sequences encoded by both DNA fragments are within their reading frames.

[0472] Isolated DNA encoding the VHA region can be converted into a full-length heavy chain gene by operatively linking the VHA-encoding DNA to another DNA molecule encoding the heavy chain constant regions (CH1, CH2, and CH3). The sequences of human heavy chain constant region genes are known in the field, and DNA fragments including these regions can be obtained by standard PCR amplification.

[0473] In some preferred embodiments, the heavy chain constant region can be the human IgG1, IgG2, IgG3, or IgG4 constant region, preferably the human IgG4 heavy chain constant region. The DNA encoding the VH region of the heavy chain portion of the ScFab fragment can be operatively linked to another DNA molecule that encodes only the CH1 constant region of the heavy chain.

[0474] Isolated DNA encoding the VLA region can be converted into a full-length light chain gene by operatively linking the VLA-coding DNA to another DNA molecule encoding the light chain constant region (CLA). The sequences of human light chain constant region genes are known in the field, and DNA fragments including these regions can be obtained by standard PCR amplification.

[0475] In some preferred embodiments, the light chain constant region can be the human κ and human λ constant regions, preferably the human κ-type light chain constant region.

[0476] In some preferred embodiments, to create the gene encoding scFab(A), the DNA fragment encoding VLA and the DNA fragment encoding CLA can be operatively coupled with a flexible linker, such as a sequence encoding an amino acid (GGGGS). 4-6 DNA fragments encoding VHA and CH1 can be operatively linked into an expression frame of a promoter. Further, a DNA fragment encoding an insertion hinge region and Fc(A') is added downstream of the CH1-encoding DNA, allowing VLA, CLA, VLA, VHA, and CH1, the hinge region, and Fc(A') to be expressed as consecutive single-stranded proteins. The VHA and CLA regions are connected via this flexible linker to form a Trop2-specific first antigen-binding arm. In some preferred embodiments, to create the gene encoding scFab(B), DNA fragments encoding VLB and CLB can be operatively linked to a flexible linker, for example, encoding an amino acid sequence (Gly4-Ser). 4-6 The DNA fragments encoding VHB and CH1 can be operatively linked into an expression frame of a promoter. Furthermore, a DNA fragment encoding an insertion hinge region and Fc(B) is added downstream of the CH1-encoded DNA, so that VLB, CLB, VHB, CH1, the hinge region, and Fc(B) can be expressed as a continuous single-stranded protein, wherein the VHB and CLB regions are connected via this flexible linker to form a CD3-specific second antigen-binding arm.

[0477] In some preferred embodiments, to create the gene encoding LA-linker-HA', the C-terminus of the DNA fragment encoding LA can be operatively coupled to a flexible linker, such as an amino acid sequence (Gly4-Ser). 4-6Another segment is linked, and the other end of the flexible linker is then linked to the N-terminus of the nucleotide sequence encoding HA', so that HA' and LA sequences can be expressed as consecutive single-stranded proteins, wherein the LA and HA regions are linked through this flexible linker to form the first antigen-binding arm specific to Trop2. In some preferred embodiments, to create the gene encoding LB-linker-HB, the DNA segment encoding LB can be operatively linked to the encoding of the flexible linker, for example, the encoding of the amino acid sequence (Gly4-Ser). 4-6 Another segment is connected, and the other end of the flexible linker is then connected to the N-terminus of the nucleotide sequence encoding HB, so that the LB and HB regions are connected through the flexible linker, and the HB and LB sequences can be expressed as a continuous single-chain protein, wherein the LB and HB regions are connected through the flexible linker to form a CD3-specific second antigen binding arm.

[0478] Nucleic acid constructs

[0479] In one aspect, the present invention also relates to nucleic acid constructs containing the polynucleotide sequences described herein, and one or more regulatory sequences operatively linked to these sequences. The polynucleotide sequences described herein can be manipulated in various ways to ensure the expression of the antibody. The nucleic acid constructs can be manipulated according to the expression vector or requirements before insertion into the vector. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.

[0480] The regulatory sequence can be a suitable promoter sequence. The promoter sequence is typically operatively linked to the coding sequence of the protein to be expressed. The promoter can be any nucleotide sequence exhibiting transcriptional activity in the selected host cell, including mutant, truncated, and heterozygous promoters, and can be obtained from a gene encoding an extracellular or intracellular polypeptide homologous or heterologous to that of the host cell. The regulatory sequence can also be a suitable transcription terminator sequence, a sequence recognized by the host cell to terminate transcription. The terminator sequence is operatively linked to the 3' end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the selected host cell can be used in this invention. The regulatory sequence can also be a suitable leader sequence, the untranslated region of mRNA important for translation in the host cell. The leader sequence is operatively linked to the 5' end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the selected host cell can be used in this invention.

[0481] In some preferred embodiments, the nucleic acid construct is a vector.

[0482] In some implementations, the types of vectors commonly used in antibody construction genetic engineering can be, for example, plasmids, phage particles, phage derivatives, animal viruses, and granules, which can be specifically selected by those skilled in the art depending on the host cells to be introduced.

[0483] In one embodiment of the present invention, the nucleic acid construct is an expression vector, which is used to express the polynucleotides described above.

[0484] In some preferred embodiments, the nucleic acid construct used in the preparation of the Trop2 humanized antibody of the present invention is a pHr expression vector with a signal peptide at the N-terminus.

[0485] In some preferred embodiments, the pHr expression vector with an N-terminal signal peptide is pHr-hκ containing the hκ constant region gene, such as... Figure 18 As shown.

[0486] In some preferred embodiments, the pHr expression vector with an N-terminal signal peptide is pHr-hIgG4 containing the hIgG4 constant region gene, such as... Figure 19 As shown.

[0487] Cells

[0488] The host cells suitable for introducing the nucleic acid constructs described herein can be prokaryotic cells or higher eukaryotic cells, such as mammalian cells and immune cells, preferably immune effector cells.

[0489] In a preferred embodiment, the host cell used for introducing the nucleic acid constructs described herein is a eukaryotic cell.

[0490] Eukaryotic cells, especially mammalian cells, are more likely than prokaryotic cells to assemble and secrete properly folded and immunologically active antibodies.

[0491] In some preferred embodiments, preferred mammalian host cells for expressing the recombinant antibodies of the present invention include Chinese hamster ovary (CHO), NSO myeloma cells, COS cells, SP2 cells, and the GS gene expression system.

[0492] In some preferred embodiments, the host cell CHO cell is used to introduce the nucleic acid construct described herein.

[0493] In some exemplary embodiments, the host cell used for introducing the nucleic acid constructs described herein is an Expi CHO-S cell.

[0494] Method of making Trop2 monoclonal antibodies

[0495] In some embodiments, the anti-Trop2 mouse antibody of the present invention is prepared and screened using somatic cell hybridization (hybridoma) technology.

[0496] For example, human Trop2 protein with a 6×His tag fused to its C-terminus was emulsified with a rapid adjuvant and then immunized in BalB / C mice. Spleens were collected from mice whose serum titers met the fusion criteria, and single-cell suspensions were prepared. Hybridomas were generated by electrofusion with SP20 myeloma cells. The parent clone that could bind human Trop2 was screened, and single clones with Trop2 binding activity were isolated. The heavy chain variable region sequence and light chain variable region sequence of the mouse clone 3F8 of this invention were obtained by sequencing.

[0497] In some embodiments, the preparation of the anti-Trop2 antibody of the present invention utilizes techniques recognized in the art to modify the antigen-binding peptides, variants, or derivatives thereof disclosed herein to reduce their immunogenicity. For example, the antibody may be humanized or deimmunized. Alternatively, chimeric antibodies may be prepared; these types of antibodies are derived from non-human antibodies, typically murine antibodies, which retain or substantially retain the antigen-binding properties of the parent antibody but exhibit lower immunogenicity in humans. An exemplary anti-Trop2 antibody of the present invention is ch3F8.

[0498] In this art, reducing the immunogenicity of an antibody can be achieved by a variety of methods, including (a) transplanting the entire non-human variable domain into a human constant region to produce a chimeric antibody; (b) transplanting at least a portion of one or more non-human complementarity-determining regions (CDRs) into a human framework and constant region, with or without key framework residues; or (c) transplanting the entire non-human variable domain, but “masking” the non-human portion with a human-like portion by replacing surface residues.

[0499] In some embodiments, the humanized anti-Trop2 antibody of the present invention is prepared by constructing a recombinant encoding nucleic acid through genetic engineering, constructing an expression vector, transforming it into a host cell, expressing it in the host cell, and then extracting and purifying the expression product.

[0500] In one embodiment, the coding nucleic acid sequences for the heavy chain variable region (VHA) and light chain variable region (VLA) of the humanized anti-Trop2 antibody are obtained using a CDR transplantation followed by reversion mutation. Specifically, the light and heavy chain sequences of the 3F8 mouse antibody are first input into an antibody structure simulation and optimization analysis platform to search for homologous proteins in the variable regions. The results from several databases are compared, and the heavy chain template and light chain template with the highest homology are selected from the germLine framework. The CDR sequences of the mouse anti-3F8 light and heavy chain variable regions are then transplanted into the germLine framework sequence. After the CDR transplantation is completed, several key sites are selected from the humanized sequences generated from each template for reversion mutation, resulting in multiple humanized heavy chain variable region sequences and multiple humanized light chain variable region sequences. The selected light and heavy chains are then combined to obtain the final humanized antibody.

[0501] In some preferred embodiments, the light chain variable region coding nucleic acid of the humanized anti-Trop2 antibody of the present invention is designed using the germline frames IGKV2-30 and IGKV2-28 as templates for the light chain, and the heavy chain variable region coding nucleic acid is designed using four germline frames IGHV1-46, IGHV1-3, IGHV1-2, and IGHV1-18 as templates for the heavy chain. The heavy chain CDR and light chain CDR sequences of mouse anti-3F8 are respectively transplanted into the four germline frames GHV1-46, IGHV1-3, IGHV1-2, and IGHV1-18 of the heavy chain and the germline frames GKV2-30 and IGKV2-28 of the light chain. The generated humanized sequences are then subjected to reverse mutations, and the humanized antibody coding nucleic acid of the present invention is obtained through screening.

[0502] Regarding the preparation of Trop2 monoclonal antibodies or their Trop2-binding fragments, once the DNA fragments encoding VHA and / or VLA are obtained, these DNA fragments can be further manipulated using standard recombinant DNA techniques, such as converting variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these operations, the DNA fragment encoding VLA or VHA is operatively ligated to another DNA fragment encoding a different protein, such as the antibody constant region or a flexible linker. "Operationally ligated" means that the ligation of the two DNA fragments should ensure that the amino acid sequences encoded by both DNA fragments remain within the reading frame.

[0503] The isolated DNA region encoding VHA can be converted into a full-length heavy chain gene by operatively ligating the VHA-coding region to another DNA molecule (CH1, CH2, and CH3) encoding the human IgG heavy chain constant region. The sequences of human heavy chain constant region genes are known in the art, and DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be the human IgG1, IgG2, IgG3, or IgG4 constant region, preferably the IgG1 or IgG4 heavy chain constant region, and most preferably the human IgG4 heavy chain constant region. For Fab fragment heavy chain genes, the VHA-coding DNA can be efficiently ligated to another DNA molecule that encodes only the heavy chain CH1 constant region.

[0504] The isolated VLA DNA coding region can be converted into a full-length light chain gene by operably linking it to a DNA molecule encoding another light chain constant region (CLA). The sequences of human light chain constant region genes are known in the art, and DNA fragments containing these regions can be obtained by standard PCR amplification. The light chain constant region can be a human κ or human λ constant region. In some preferred embodiments, the light chain constant region can be a human κ constant region.

[0505] In some preferred embodiments, the full-length heavy chain encoding nucleic acid of the chimeric anti-Trop2 antibody of the present invention is generated by placing the nucleic acid encoding the VHA of the murine antibody 3F8 at the N-terminus of the constant region of the human IgG4 heavy chain; the full-length light chain encoding nucleic acid is generated by placing the nucleic acid encoding the VLA of the 3F8 murine antibody at the N-terminus of the constant region of the human light chain kappa.

[0506] In some preferred embodiments, the full-length heavy chain encoding nucleic acid of the humanized anti-Trop2 antibody of the present invention is generated by humanizing the nucleic acid encoding the variable region VHA of the heavy chain of a mouse antibody and placing it at the N-terminus of the constant region of the human IgG4 heavy chain; and the full-length light chain encoding nucleic acid is generated by humanizing the variable region VLA of the light chain of a mouse antibody and placing it at the N-terminus of the constant region of the human light chain kappa.

[0507] In some embodiments, the present invention encodes a nucleic acid that is operatively linked to construct an anti-Trop2 antigen-binding fragment.

[0508] For example, in order to construct an scFab gene, the DNA fragments encoding VL, CL, linker, VH, and CH1 are operatively linked into the scFab-encoding nucleic acid, which is then linked to another fragment encoding Fc, such that the scFab and Fc sequences can be formed from consecutive single-stranded protein regions connected by hinge regions.

[0509] For example, to construct the nucleic acid encoding the antigen-binding arm of a Trop2 / CD3 bispecific antibody, a DNA fragment encoding the amino acid sequences of VLA, CLA, linker, VHA, and CH1A is further linked upstream of another DNA fragment encoding a DNA fragment containing a hinge region and a (PAA+hole) mutant Fc(A'), thereby operably linking the nucleic acid encoding a single-stranded peptide chain of the (ScFab(A)-Fc(A')) structure, such that the scFab(A) and Fc(A') sequences can be... The DNA sequences encoding VLB, CLB, linker4, VHB, and CH1B, as well as the DNA sequences encoding the hinge region, are further linked upstream of another DNA sequence encoding the hinge region and the nucleic acid sequence encoding Fc(B), thereby operably linking the coding nucleic acids of the single-stranded peptide chain of the (ScFab(B)-Fc(B)) structure, so that the scFab(B) and Fc(B) sequences can be linked by the hinge region into a continuous CD3-specific single-arm binding region.

[0510] In the preparation of the anti-Trop2 antibody of the present invention, the nucleic acid encoding the light chain A and the nucleic acid encoding the heavy chain A' can be inserted into the same or different expression vectors, respectively. In a preferred embodiment, the nucleic acid encoding the variable region is used to generate a full-length antibody gene of any antibody isotype by inserting them into an expression vector that already encodes the heavy chain constant region and the light chain constant region of the desired isotype, such that the nucleic acid encoding the VHA segment is operatively linked to the CHA-terminal nucleic acid in the vector and the nucleic acid encoding the VLA segment is operatively linked to the CLA-terminal nucleic acid in the vector. Alternatively or additionally, the recombinant expression vector can encode a signal peptide that promotes the secretion of the antibody chain from the host cell. The genes encoding the antibody light chain and heavy chain can be cloned into a vector containing the signal peptide so that the signal peptide is linked within the N-terminal frame of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide derived from a non-immunoglobulin).

[0511] In some preferred embodiments, the heavy and light chain polynucleotides encoding a monoclonal antibody against the anti-Trop2 protein as described in this invention can be cloned into an expression vector, with each nucleotide sequence ligated downstream of a suitable promoter. For example, each nucleotide sequence encoding the heavy and light chains can be operatively ligated to different promoters, or the nucleotide sequences encoding the heavy and light chains can be operatively ligated to a single promoter such that both the heavy and light chains can be expressed by the same promoter.

[0512] In some embodiments, the nucleic acid encoding the Trop2-specific first antigen-binding arm of the anti-Trop2 / CD3 bispecific antibody protein as described in this invention and the nucleic acid encoding the CD3-specific second antigen-binding arm can be constructed onto two separate vectors or linked to a single expression vector, which can be introduced into the same host cell.

[0513] In a preferred embodiment, when the encoding nucleic acids of the first antigen-binding arm specific to Trop2 and the second antigen-binding arm specific to CD3 are ligated into the same expression vector, the resulting recombinant expression vector is expressed in a host cell. Each antigen-binding arm can be isolated from the host cell expressing it, and the isolated first antigen-binding arm specific to Trop2 and the second antigen-binding arm specific to CD3 can be assembled in the host cell to form the anti-Trop2 / CD3 bispecific antibody of the present invention, and purified to obtain the anti-Trop2 / CD3 bispecific antibody of the present invention.

[0514] In one embodiment, the recombinant vector used to express the anti-Trop2 / CD3 bispecific antibody of the present invention is a pHr expression vector, and the host cell is an Expi CHO-S cell.

[0515] The choice of the aforementioned expression vector / promoter depends on the type of host cell used to produce the antibody, which is well known in the art and will not be described in detail here.

[0516] Once the coding nucleic acid sequences for the relevant variable regions are obtained, the coding nucleic acid sequences for the light and heavy chains of the anti-Trop2 monoclonal antibody and the Trop2-binding and CD3-binding arms of the anti-Trop2 / CD3 bispecific antibody of the present invention can be obtained in large quantities using recombinant methods. This is typically done by cloning them into a vector, transforming them into cells, and then isolating the relevant sequences from the proliferated host cells using conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in the present invention include biomolecules existing in isolated forms. Currently, the DNA sequences encoding the proteins of the present invention (or fragments thereof, or derivatives thereof) can be obtained entirely through chemical synthesis. These DNA sequences can then be introduced into various existing DNA molecules (or vectors, etc.) known in the art. Furthermore, mutations can be introduced into the protein sequences of the present invention through chemical synthesis.

[0517] Methods for introducing nucleic acids or vectors into mammalian cells are known in the art. These vectors can be transferred into cells by physical, chemical, or biological methods. For example, for eukaryotic hosts, DNA transfection methods including, but not limited to, the following can be used: calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc. In an exemplary embodiment of the present invention, a nucleic acid encoding a heavy or light chain containing an anti-Trop2 antibody or its antigen-binding fragment, or an expression vector pHr containing VHA-encoded nucleic acid and VLA-encoded nucleic acid, is transiently transfected into host cells via a CHO-s transient transfection expression system.

[0518] The nucleic acid encoding the heavy or light chain of the anti-Trop2 antibody or its antigen-binding fragment in the above-described method, or a nucleic acid fusion containing VHA-encoding and VLA-encoding nucleic acids, or a nucleic acid encoding the anti-Trop2 binding arm, or a nucleic acid encoding the CD3 binding arm, or a nucleic acid fusion containing both Trop2-encoding and CD3-encoding nucleic acids, can be expressed intracellularly, on the cell membrane, or secreted extracellularly. In some preferred embodiments, the aforementioned nucleic acid fusion encoding the present invention's humanized anti-Trop2 antibody or bispecific antibody is secreted extracellularly into the host cell.

[0519] In some exemplary embodiments, the obtained recombinant cells can be cultured using conventional methods to express the antibodies encoded by the nucleic acid molecules of the present invention. Depending on the host cells used, the culture medium used in the culture can be selected from various conventional culture media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature change or chemical induction), and the cells are cultured for a further period. If desired, the target recombinant protein can be separated and purified from the culture medium using various separation methods based on its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, percolation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations thereof.

[0520] In one exemplary embodiment, the preparation of the chimeric or humanized anti-Trop2 antibody involves constructing a gene fragment encoding a fusion of heavy and light chain nucleic acids into a pHr expression vector with an N-terminal signal peptide, yielding anti-Trop2 and anti-CD3 single-arm antibody plasmids; then, the CHO-s transient transfection system is used to generate anti-Trop2 antibodies. The obtained recombinant pHr expression vector encoding the light and heavy chains, or the recombinant pHr expression vector encoding the first and second antigen-binding arms, is then co-transfected into Expi CHO-S cells using transfection reagents. Expi CHO-S cells are continuously cultured until cell viability is ≤70%, and the culture supernatant is collected. The target chimeric or humanized anti-Trop2 antibody, or anti-Trop2 / CD3 antibody, is purified from the culture supernatant using an affinity chromatography column.

[0521] In one embodiment, the preparation of the anti-Trop2 / CD3 bispecific antibody involves constructing a gene fragment encoding the first antigen-binding arm specific to Trop2 and the second antigen-binding arm specific to CD3 into a pHr expression vector with an N-terminal signal peptide, thereby obtaining anti-Trop2 and anti-CD3 single-arm antibody plasmids. The pHr expression vector is then transfected into Expi CHO-S cells, and the recombinant cells are cultured and purified to obtain the Trop2 / CD3 bispecific antibody of the present invention.

[0522] Functional properties of anti-Trop2 mAbs

[0523] The anti-Trop2 antibody of the present invention can bind to human Trop2 protein with high affinity, can bind to human Trop2 protein expressed in the in vitro environment and on the cell surface, cross-binds Trop2 protein of cynomolgus monkey in vitro, and has no cross-binding activity against EpCAM of the same protein family.

[0524] The anti-Trop2 antibodies of the present invention can be characterized by their various physical properties in order to detect and / or distinguish their different categories.

[0525] The term "antibody that specifically binds to human Trop2" as used in this article refers to antibodies that bind to human Trop2 protein (and Trop2 protein that may be derived from one or more non-human species) but do not substantially bind to non-human Trop2 protein.

[0526] Affinity (KD )

[0527] In some embodiments, the exemplary chimeric anti-Trop2 antibody ch3F8 of the present invention binds to human Trop2 protein with “high affinity”, i.e., with a KD(M) of 5.0E-9 or less, more preferably 1.0E-9 or less, and more preferably below 8.2E-10, exhibiting a higher affinity for human Trop2 protein than the control antibody huRS7.

[0528] In some embodiments, the humanized anti-Trop2 antibodies of the present invention include hu3F8 H1L1, hu3F8 H2L1, hu3F8H3L1, hu3F8 H4L1, hu3F8 H5L1, hu3F8 H1L2, hu3F8 H2L2, hu3F8 H3L2, and hu3F8. H4L2 and hu3F8H5L2 bind to human Trop2 protein with "high affinity," specifically with a KD(M) of 1.30E-9 or less, more preferably 1.25E-9 or less, more preferably 1.15E-9 or less, more preferably 9.5E-10 or less, more preferably 8.5E-10 or less, more preferably 5.5E-10 or less, more preferably 4.5E-10 or less, more preferably 4.0E-10 or less, more preferably 3.0E-10 or less, and most preferably below 2.5E-10. Their affinity for human Trop2 protein is comparable to that of the chimeric antibody ch3F8.

[0529] "Basically not binding to non-human Trop2" means that the KD(M) value of binding to non-human Trop2 is 1.0E-6 or greater, more preferably 1.0E-5 or greater, more preferably 1.0E-4 or greater, more preferably 1.0E-3 or greater, and even more preferably 1.0E-2 or greater.

[0530] As used in this article, “KD” is intended to refer to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e., Kd / Ka) and expressed as a molar concentration (M). Antibody values ​​can be determined using methods well-established in the art. The preferred detection method for determining antibody KD is through surface plasmon resonance, preferably using a biosensor system such as the Biacore™ system. The terms “Kassoc” or “Ka”, as used herein, refer to the binding rate of a specific antibody-antigen interaction, while the terms “Kdis” or “Kd”, as used herein, refer to the dissociation rate of a specific antibody-antigen interaction.

[0531] ELISA binding EC50 and FACS binding EC50 for Trop2 binding

[0532] The binding specificity of the anti-Trop2 antibody of the present invention can be determined by in vitro assays, such as enzyme-linked immunosorbent assay (ELISA) and flow cytometry.

[0533] In some embodiments, the Trop2-binding specific anti-Trop2 antibody or its antigen-binding fragment can bind to Trop2 in both humans and cynomolgus monkeys at an ELISA concentration of about 0.55 nM or lower via EC50.

[0534] In some embodiments, the anti-Trop2 antibody is a chimeric antibody, wherein the exemplary chimeric antibody ch3F8 binds to human Trop2 protein with EC50 at an ELISA concentration of no more than about 0.08 (nM), a binding level comparable to that of the control antibody huRS7.

[0535] In some embodiments, the anti-Trop2 antibody is a chimeric antibody, wherein an exemplary chimeric antibody ch3F8 binds to EC50 with a FACS concentration not exceeding 3.5 (nM) to human Trop2 expressed on the cell membrane.

[0536] In some embodiments, the anti-Trop2 antibody is a chimeric antibody, wherein an exemplary chimeric antibody ch3F8 cross-binds with cynomolgus monkey Trop2 in vitro by ELISA binding EC50 at a concentration not exceeding 0.082 (nM).

[0537] In some embodiments, the anti-Trop2 antibody is a humanized antibody. In some embodiments, the humanized anti-Trop2 antibodies of the present invention are hu3F8 H1L1, hu3F8 H2L1, hu3F8 H3L1, hu3F8 H4L1, hu3F8H5L1, hu3F8 H1L2, hu3F8 H2L2, hu3F8 H3L2, hu3F8 H4L2, and hu3F8... H5L2 binds specifically to human Trop2 protein in vitro using ELISA with EC50 at concentrations not exceeding 0.55 (nM), 0.45 (nM), 0.25 (nM), 0.18 (nM), 0.15 (nM), 0.14 (nM), 0.13 (nM), 0.12 (nM), 0.11 (nM), 0.10 (nM), 0.009 (nM), 0.008 (nM), or 0.0075 (nM).

[0538] In some embodiments, the anti-Trop2 antibody is a humanized antibody. In some embodiments, the humanized anti-Trop2 antibodies of the present invention are hu3F8 H1L1, hu3F8 H2L1, hu3F8 H3L1, hu3F8 H4L1, hu3F8 H5L1, hu3F8 H1L2, hu3F8 H2L2, hu3F8 H3L2, hu3F8 H4L2, and hu3F8... H5L2 binds specifically to human Trop2 protein on the surface of CHO-K1 cells in vitro with FACS at concentrations not exceeding 40 (nM), or not exceeding 35 (nM), or not exceeding 32 (nM), or not exceeding 25 (nM), or not exceeding 20 (nM), or not exceeding 10 (nM), or not exceeding 8.0 (nM), or not exceeding 7.0 (nM), or not exceeding 6.0 (nM), or not exceeding 5.0 (nM), or not exceeding 4.0 (nM), or not exceeding 3.0 (nM), or not exceeding 2.0 (nM).

[0539] In some embodiments, the humanized anti-Trop2 antibodies hu3F8 H1L1, hu3F8 H2L1, hu3F8 H3L1, hu3F8 H4L1, hu3F8 H5L1, hu3F8 H1L2, hu3F8 H2L2, hu3F8 H3L2, hu3F8 H4L2, and hu3F8 H5L2 of the present invention are used in ELISA to specifically cross-bind with monkey Trop2 at EC50 concentrations not exceeding 0.065 (nM), or not exceeding 0.055 (nM), or not exceeding 0.045 (nM), or not exceeding 0.035 (nM), or not exceeding 0.025 (nM), or not exceeding 0.015 (nM), or not exceeding 0.008 (nM).

[0540] Functional activity of anti-Trop2 / CD3 bispecific antibodies

[0541] The anti-Trop2 / CD3 bispecific antibody of the present invention has one or more of the following activities:

[0542] (1) In vitro binding to human Trop2 and human CD3E&D complex respectively;

[0543] (2) It can bind to monkey Trop2 protein;

[0544] (3) It has pM-level Trop2-dependent killing activity against Trop2-positive tumor cells;

[0545] (4) Mediates T cell activation;

[0546] (5) It can mediate the release of IL-2, IL-6, IL-10, IFN-γ and TNF-α from PBMCs;

[0547] (6) It can inhibit tumor growth and proliferation in animals.

[0548] Binding levels in vitro to human Trop2-His and human CD3E&D complex

[0549] In some embodiments, the anti-Trop2 / CD3 bispecific antibody of the present invention can bind to human Trop2-His and human CD3E&D complexes in vitro, respectively.

[0550] In some exemplary embodiments, the ELISA binding EC50 of the anti-Trop2 / CD3 bispecific antibody to human Trop2 in vitro is approximately no higher than 0.2 nM, or approximately no higher than 0.25 nM, or approximately no higher than 0.28 nM, or approximately no higher than 0.25 nM, or approximately no higher than 0.3 nM, or approximately no higher than 0.32 nM. In some exemplary embodiments, the ELISA binding EC50 of the anti-Trop2 / CD3 bispecific antibody to human CD3E&D in vitro is approximately no higher than 0.3 nM, or approximately no higher than 0.4 nM, or approximately no higher than 0.6 nM, or approximately no higher than 0.8 nM, or approximately no higher than 1 nM, or approximately no higher than 3 nM, or approximately no higher than 5 nM, or approximately no higher than 8 nM, or approximately no higher than 10 nM, or approximately no higher than 12 nM, or approximately no higher than 15 nM, or approximately no higher than 17 nM.

[0551] In some exemplary embodiments, the ELISA binding EC50 of the anti-Trop2 / CD3 bispecific antibody to human Trop2 in vitro is approximately no higher than 0.2 nM or no higher than 0.25 nM, and the ELISA binding EC50 of CD3E&D is approximately no higher than 10 nM, or no higher than 12 nM, or no higher than 15 nM or no higher than 17 nM.

[0552] Binding levels to Trop2-expressing cells and to human CD3E&D complex-expressing cells

[0553] In some embodiments, the anti-Trop2 / CD3 bispecific antibody can bind to cells expressing human Trop2 on their cell surface.

[0554] In some embodiments, the anti-Trop2 / CD3 bispecific antibody can bind to Trop2 on the surface of breast cancer cells.

[0555] In some exemplary embodiments, the FACS binding EC50 of the anti-Trop2 / CD3 bispecific antibody to Trop2 on the surface of MDA-MB-468 cells is no higher than 8 nM to no higher than 12 nM.

[0556] In some embodiments, the anti-Trop2 / CD3 bispecific antibody can bind to human CD3E&D expressed on the cell surface.

[0557] In some embodiments, the anti-Trop2 / CD3 bispecific antibody can bind to cells whose cell surface expresses the human CD3E&D complex.

[0558] In some embodiments, the FACS binding EC50 of the anti-Trop2 / CD3 bispecific antibody and the Jurkat cells (Clone E6-1 cells, human T-lymphocytic leukemia cells) expressing human CD3E&D is not higher than 2 nM.

[0559] In some embodiments, the FACS binding EC50 of the anti-Trop2 / CD3 bispecific antibody and the cells expressing human CD3E&DJurkat cells (Clone E6-1 cells, human T-lymphocytic leukemia cells) is not higher than 30 nM.

[0560] Cross-binding to monkey Trop2

[0561] In some embodiments, the anti-Trop2 / CD3 bispecific antibody binds to monkey Trop2 protein in vitro via EC50 binding, similar to an ELISA assay for human Trop2.

[0562] In some embodiments, the anti-Trop2 / CD3 bispecific antibody binds to the monkey Trop2 protein via EC50 using an ELISA at a concentration of no more than about 0.25 nM. In some embodiments, the anti-Trop2 / CD3 bispecific antibody binds to the monkey Trop2 protein via EC50 using an ELISA at a concentration of no more than about 0.22 nM.

[0563] In some embodiments, the anti-Trop2 / CD3 bispecific antibody can bind to monkey CD3E&D proteins in vitro.

[0564] In some embodiments, the anti-Trop2 / CD3 bispecific antibody binds to monkey CD3E&D protein via EC50 using an ELISA at a concentration not exceeding 0.3 nM. In some embodiments, the anti-Trop2 / CD3 bispecific antibody binds to monkey CD3E&D protein via EC50 using an ELISA at a concentration not exceeding 9 nM.

[0565] In vitro anti-tumor killing activity

[0566] In some embodiments, the anti-Trop2 / CD3 bispecific antibody exhibits cytotoxic activity against a variety of tumor cell lines in vitro.

[0567] In some embodiments, the anti-Trop2 / CD3 bispecific antibody exhibits strong killing activity against a variety of Trop2-positive tumor cell lines in vitro.

[0568] In some embodiments, the anti-Trop2 / CD3 bispecific antibody exhibits cytotoxic activity against pancreatic cancer adenocarcinoma cell lines in vitro.

[0569] In some embodiments, the anti-Trop2 / CD3 bispecific antibody exhibits cytotoxic activity against pancreatic adenocarcinoma cell lines in vitro, with an EC50 value of the cytotoxicity curve not exceeding 3.0 pM.

[0570] In some embodiments, the anti-Trop2 / CD3 bispecific antibody exhibits cytotoxic activity against pancreatic adenocarcinoma cell lines in vitro, with an EC50 value of the cytotoxicity curve not exceeding 4.0 pM.

[0571] In some embodiments, the anti-Trop2 / CD3 bispecific antibody exhibits cytotoxic activity against breast cancer cell lines in vitro.

[0572] In some embodiments, the anti-Trop2 / CD3 bispecific antibody exhibits cytotoxic activity against the breast cancer cell line MDA-MB-468 in vitro, with an EC50 value of the cytotoxicity curve not exceeding 0.14 pM.

[0573] In some embodiments, the anti-Trop2 / CD3 bispecific antibody exhibits cytotoxic activity against the breast cancer cell line MDA-MB-468 in vitro, with an EC50 value of the cytotoxicity curve not exceeding 0.32 pM.

[0574] In some embodiments, the anti-Trop2 / CD3 bispecific antibody exhibits cytotoxic activity against the breast cancer cell line MDA-MB-231 in vitro, with an EC50 value of the cytotoxicity curve not exceeding 1.8 pM.

[0575] In some embodiments, the anti-Trop2 / CD3 bispecific antibody exhibits cytotoxic activity against the breast cancer cell line MDA-MB-231 in vitro, with an EC50 value of the cytotoxicity curve not exceeding 6.5 pM.

[0576] Ability to specifically activate T cells in the context of cancer cells

[0577] The Trop2×CD3 bispecific antibody of the present invention can mediate T cell activation in a cancer cell environment.

[0578] In some embodiments, the mediated activation T cells include, but are not limited to, CD25+ T cells, CD69+ T cells, CD25+ / CD69+ double-positive T cells, CD4+ T cells, and CD8+ T cells.

[0579] In some embodiments, the Trop2×CD3 bispecific antibody of the present invention can mediate the activation of CD25+ / CD69+ double-positive cells.

[0580] In some embodiments, the FACS binding EC50 of the Trop2×CD3 bispecific antibody-mediated activation of CD25+ / CD69+ double-positive cells of the present invention is not higher than 1.4 pM.

[0581] In some embodiments, the FACS binding EC50 of the Trop2×CD3 bispecific antibody-mediated activation of CD25+ / CD69+ double-positive cells of the present invention is not higher than 2.1 pM.

[0582] In some embodiments, the FACS binding EC50 of the Trop2×CD3 bispecific antibody-mediated activation of CD4+ double-positive cells of the present invention is not higher than 7.2 pM.

[0583] In some embodiments, the FACS binding EC50 of the Trop2×CD3 bispecific antibody-mediated activation of CD4+ double-positive cells of the present invention is not higher than 7.8 pM.

[0584] Ability to mediate cytokine release in the context of cancer cells

[0585] The Trop2×CD3 bispecific antibody of the present invention can mediate the release of cytokines from PBMCs in a cancer cell environment.

[0586] In some embodiments, the cancer cells include breast cancer cells.

[0587] In some embodiments, the cytokines include, but are not limited to, IL-2, IL-6, IL-10, IFN-γ, and TNF-α.

[0588] In some embodiments, the FACS binding 50 of IL-2 released from PBMCs mediated by the Trop2×CD3 bispecific antibody is no higher than 2.7 pM, and the Emax is approximately 110-120 (pg / mL).

[0589] In some embodiments, the FACS detection of IL-2 release from PBMCs mediated by the Trop2×CD3 bispecific antibody has an F binding EC50 of no more than 8.0 pM and an Emax of approximately 600-650 (pg / mL).

[0590] In some embodiments, the binding EC50 of the Trop2×CD3 bispecific antibody-mediated PBMC release of IL-6 as detected by FACS is no higher than 0.62 pM, and the Emax is approximately 111,000-112,000 (pg / mL).

[0591] In some embodiments, the binding EC50 of the Trop2×CD3 bispecific antibody-mediated PBMC release of IL-6 as detected by FACS is no higher than 0.99 pM, and the Emax is approximately 118,000-120,000 (pg / mL).

[0592] In some embodiments, the binding EC50 of the Trop2×CD3 bispecific antibody-mediated PBMC release of IL-10 as detected by FACS is no higher than 3.1 pM, and the Emax is approximately 380-420 (pg / mL).

[0593] In some embodiments, the binding EC50 of the Trop2×CD3 bispecific antibody-mediated PBMC release of IL-10 as detected by FACS is no higher than 3.8 pM, and the Emax is approximately 480-520 (pg / mL).

[0594] In some embodiments, the Trop2×CD3 bispecific antibody Mediate The FACS detection of IFN-γ release from PBMCs showed an EC50 of no more than 1.1 pM and an Emax of approximately 103,000-105,000 (pg / mL).

[0595] In some embodiments, the Trop2×CD3 bispecific antibody Mediate The FACS detection of IFN-γ release from PBMCs showed an EC50 of no more than 1.4 pM and an Emax of approximately 16,000-164,000 (pg / mL).

[0596] In some embodiments, the Trop2×CD3 bispecific antibody Mediate The FACS detection of TNF-α release from PBMCs showed an EC50 of no more than 2.5 pM and an Emax of approximately 6000-6300 (pg / mL).

[0597] In some embodiments, the Trop2×CD3 bispecific antibody Mediate FACS detection of TNF-α release from PBMCs showed that the EC50 was not higher than 3.6 pM, and the Emax was approximately 8000-9000 (pg / mL).

[0598] In vivo tumor growth and proliferation inhibitory activity

[0599] In some embodiments, the anti-Trop2 antibody of the present invention can inhibit tumor growth and proliferation in Trop2-positive tumor animal models.

[0600] In some embodiments, the anti-Trop2 antibody of the present invention can inhibit tumor growth and proliferation in a mouse model of breast cancer.

[0601] In some embodiments, the anti-Trop2 antibody of the present invention can inhibit tumor growth and proliferation in a mouse model of MDA-MB-231 breast cancer.

[0602] In some embodiments, administration of the anti-Trop2 antibody of the present invention at a dose level of 0.5-2 mg / mL in animals was able to inhibit the growth and proliferation of MDA-MB-231 tumor breast cancer cells.

[0603] In some embodiments, administration of the anti-Trop2 antibody of the present invention at a dose level of 0.8-8 mg / mL in animals can inhibit the growth and proliferation of MDA-MB-231 tumor breast cancer cells.

[0604] In some embodiments, administration of the anti-Trop2 antibody of the present invention at a dose level of 1-5 mg / mL in animals can inhibit the growth and proliferation of MDA-MB-231 tumor breast cancer cells.

[0605] In some embodiments, administration of the anti-Trop2 antibody of the present invention at a dose level of 1 mg / mL to mice resulted in a TGI of 93.9% in inhibiting the growth and proliferation of Trop2-positive tumor cells.

[0606] In some embodiments, administration of the anti-Trop2 antibody of the present invention at a dose level of 1 mg / mL in mice can inhibit the growth and proliferation of Trop2-positive tumor cells.

[0607] In some embodiments, administration of the anti-Trop2 antibody of the present invention at a dose level of 5 mg / mL in mice resulted in a TGI of 137.8% that inhibited the growth and proliferation of Trop2-positive tumor cells.

[0608] Tumor growth inhibition value (TGI) is used to evaluate the inhibitory effect of a test drug on tumor growth in vivo (i.e., in animal experiments). The TGI value is calculated using formula (I).

[0609] TGI = (1 - tumor weight in the treatment group / tumor weight in the control group) * 100%, (I).

[0610] Compositions

[0611] In one aspect, the present invention also provides pharmaceutical compositions comprising a pharmaceutically acceptable effective dose of an anti-Trop2 antibody, an antigen-binding fragment, or a mutant or derivative thereof, or an anti-Trop2 / CD3 bispecific antibody of the present invention as an active ingredient, and a pharmaceutically acceptable carrier.

[0612] "Pharmaceuticalally acceptable effective dose" refers to the dose listed in a pharmacopoeia or other recognized pharmacopoeia for use in animals, especially for humans.

[0613] "Pharmaceutical acceptable carriers" are generally non-toxic solid, semi-solid, or liquid fillers, diluents, encapsulating materials, or any type of excipients that are well-known to those skilled in the art of pharmacy.

[0614] The anti-Trop2 antibody formulation of the present invention can be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0615] Pharmaceutical compositions can be in the form of sterile aqueous solutions or dispersions. They can also be formulated in microemulsions, liposomes, or other ordered structures suitable for high concentrations of drugs.

[0616] In some embodiments, the composition is formulated according to conventional procedures to be a pharmaceutical composition suitable for intravenous administration to humans. Typically, the composition for intravenous administration is a solution in a sterile isotonic buffer solution. These components are usually provided individually or mixed together in unit dosage forms, for example, as a dry lyophilized powder or anhydrous concentrate in a sealed container such as an ampoule or sachet, indicating the amount of active agent. When administered by infusion, the composition can be prepared with an infusion bottle containing sterile pharmaceutical-grade water or physiological saline. When administered by injection, a single ampoule of sterile water for injection or physiological saline can be provided, allowing for mixing prior to administration.

[0617] The dosage of the Trop2 antibody preparation of this invention can be specifically determined by clinicians based on the reduction of the severity of disease symptoms, the increase in the frequency and duration of the asymptomatic period, or the ability to prevent damage or incapacitation caused by the disease.

[0618] Preferably, the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or bolus). Depending on the route of administration, the active ingredient may be encapsulated in a material to protect it from acids and other natural conditions that may inactivate it; alternatively, the antibody of the present invention may be administered via a non-parenteral route, such as topical, epidermal, or mucosal administration, or local application.

[0619] In some embodiments, the pharmaceutical compositions of the present invention may also be further manufactured as sustained-release agents, including implants, transdermal patches, and microcapsule delivery systems.

[0620] In some embodiments, the pharmaceutical composition may be administered via medical devices, such as (1) needle-free subcutaneous injection devices; (3) transdermal drug delivery devices; and (5) permeation devices.

[0621] Uses and methods of use

[0622] Use in the manufacture of an antibody-drug conjugate that specifically binds to Trop2

[0623] In some embodiments, the anti-Trop2 antibody described in the foregoing aspects of the present invention, its antigen-binding fragment or variant, and / or the anti-CD3 / Trop2 bispecific antibody described in the foregoing aspects, its antigen-binding fragment, can be used to prepare antibody-drug conjugates.

[0624] In some embodiments, the antibody-drug conjugate may comprise an anti-Trop2 antibody or an anti-Trop2 / CD3 bispecific antibody as described above, or a therapeutic agent, prodrug, peptide, protein, enzyme, virus, lipid, biological response modifier, pharmaceutical agent, or PEG conjugated to the anti-Trop2 antibody or the anti-Trop2 / CD3 bispecific antibody.

[0625] In some embodiments, those skilled in the art can crosslink the anti-Trop2 antibody or anti-Trop2 / CD3 bispecific antibody of the present invention with a therapeutic agent to form an immunoconjugate, such as an antibody-drug conjugate (ADC).

[0626] In ADCs, antibodies and therapeutic agents are preferably cross-linked via a cleavable linker, such as a peptide linker, disulfide linker, or hydrazone linker. More preferably, the linker is a peptide linker, such as Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Pro-Val-Gly-Val-Val, Ala-Asn-Val, Val-Leu-Lys, Ala-Ala-Asn, Cit-Cit, Val-Lys, Lys, Cit, Ser, or Glu.

[0627] In some embodiments, the anti-Trop2 antibody may bind to or fuse with a therapeutic agent, wherein the therapeutic agent may include a detectable marker, such as a radioactive marker, an immunomodulator, a hormone, an enzyme, an oligonucleotide, a photoactive therapeutic or diagnostic agent, a cytotoxic agent (which may be a drug or a toxin), an ultrasound enhancer, or a non-radioactive marker.

[0628] Suitable therapeutic agents include cytotoxins, alkylating agents, DNA minor groove binding molecules, DNA intercalating agents, DNA cross-linking agents, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, antimitotic agents, topoisomerase I or II inhibitors, heat shock protein inhibitors, tyrosine kinase inhibitors, and antibiotics.

[0629] In some embodiments, the cytotoxic agent is preferably a DNA topoisomerase inhibitor, a microtubule disruptor, or an RNApol II inhibitor.

[0630] In some embodiments, the DNA topoisomerase I inhibitor includes sarsaparilla toxin and maytansin.

[0631] In some embodiments, the DNA topoisomerase I inhibitors are MMAE, MMAF, DM1, and DM4.

[0632] In some embodiments, the DNA topoisomerase I inhibitor is MMAE.

[0633] The technique of conjugating different therapeutic agents to antibodies is well known to those skilled in the art.

[0634] Using the aforementioned anti-Trop2 antibody or anti-CD3 / Trop2 bispecific antibody, those skilled in the art can detectably label it by conjugating it with a chemiluminescent compound, and then determine the presence of the chemiluminescently labeled antigen-binding peptide by detecting the luminescence that occurs during the chemical reaction.

[0635] Use for the manufacture of a medicament for the prevention and treatment of cancer

[0636] In one respect, as described herein, the aforementioned anti-Trop2 antibodies, variants or derivatives, anti-Trop2 / CD3 bispecific antibodies or their antigen-binding fragments, or compositions and antibody-drug conjugates of the present invention may be used for the prevention and treatment of certain tumors or cancers.

[0637] In some embodiments, the present invention provides the anti-Trop2 antibody described in the foregoing aspects and / or the anti-CD3 / Trop2 bispecific antibody described in the foregoing aspects for the preparation of medicaments for the prevention or treatment of diseases related to Trop2 overexpression or dysfunction, or diseases targeting Trop2.

[0638] In some embodiments, the present invention provides the anti-Trop2 antibody described in the foregoing aspects and / or the anti-CD3 / Trop2 bispecific antibody described in the foregoing aspects, which are used to prepare a drug for killing Trop2-overexpressing cells.

[0639] The coding sequences of anti-Trop2 monoclonal antibodies and anti-CD3 / Trop2 bispecific antibodies, nucleic acid constructs, and cells described herein, as well as compositions comprising the products described above, can be used to prepare drugs for the prevention or treatment of the various conditions and diseases described herein, wherein the conditions and diseases are diseases related to Trop2 expression or conditions that are directly or indirectly caused by abnormal Trop2 expression, usually referring to diseases caused by Trop2 overexpression, such as cancer or tumors.

[0640] In some embodiments, the Trop2-overexpressing or dysfunctional tumor is preferably a solid tumor.

[0641] In some embodiments, the solid tumors include, but are not limited to, breast cancer, colorectal cancer, gastric adenocarcinoma, esophageal cancer, hepatocellular carcinoma, non-small cell lung cancer, small cell lung cancer, ovarian epithelial cancer, prostate cancer, pancreatic ductal adenocarcinoma, head and neck cancer, squamous cell carcinoma, renal cell carcinoma, bladder tumor, cervical cancer, endometrial cancer, follicular thyroid cancer, and glioblastoma multiforme.

[0642] In some implementations, the Trop2 overexpressing cancers include any one or more of the following cancers: thyroid cancer, papillary carcinoma, breast cancer, squamous cell carcinoma of the lung, endometrial cancer, prostate cancer, colon cancer, lung adenocarcinoma, ovarian epithelial cancer (59%), gastric cancer, pancreatic cancer, and non-small cell lung cancer.

[0643] In some implementations, the Trop2 overexpressing cancers include any one or more of these cancers, such as breast cancer, gastric cancer, pancreatic cancer, and non-small cell lung cancer.

[0644] Therapeutic uses and methods of use

[0645] In some embodiments, the anti-Trop2 antibody, its antigen-binding fragment or mutant thereof, or the anti-Trop2 / CD3 bispecific antibody or its antigen-binding fragment thereof of the present invention can be used as a treatment for diseases with Trop2 overexpression or dysfunction.

[0646] In some implementations, the disease characterized by Trop2 overexpression or dysfunction is cancer, in which Trop2 is overexpressed in tumor cells.

[0647] In some implementations, the treatment method requires administering an effective amount of the invented antibody to the patient.

[0648] In some implementations, the Trop2 overexpressing cancers include any one or more of the following cancers: thyroid cancer, papillary carcinoma, breast cancer, squamous cell carcinoma of the lung, endometrial cancer, prostate cancer, colon cancer, lung adenocarcinoma, ovarian epithelial cancer (59%), gastric cancer, pancreatic cancer, and non-small cell lung cancer.

[0649] In some implementations, the Trop2 overexpressing cancers include any one or more of these cancers, such as breast cancer, gastric cancer, pancreatic cancer, and non-small cell lung cancer.

[0650] In some embodiments, the present invention provides a method for treating diseases associated with Trop2 overexpression or dysfunction, or Trop2-targeted cancers, in patients in need.

[0651] In some embodiments, the treatment methods further involve the administration of the antibodies of the present invention, comprising administering the antibodies of the present invention, or compositions comprising the antibodies of the present invention, to a patient (e.g., a human or animal model of tumor treatment) to treat one or more of the Trop2 expression abnormalities described herein. Therapeutic compounds of the present invention include, but are not limited to, the anti-Trop2 antibodies of the present disclosure (including variants and derivatives thereof as described herein) and nucleic acids or polynucleotides encoding antibodies of the present disclosure (including variants and derivatives thereof as described herein).

[0652] In one implementation, the treatment method requires administering an effective amount of the invented antibody to the patient.

[0653] In some implementations, at least one of the patient's cancer cells (e.g., stromal cells) expresses, overexpresses, or is induced to express the Trop2 tumor antigen.

[0654] In the treatment of tumors that overexpress or are induced to express Trop2, the specific dosage and dosing regimen for any particular patient will depend on a variety of factors. The dosage and treatment regimen of the Trop2 antibody, bispecific antibody or its derivatives or compositions of the present invention administered during treatment can be determined by those skilled in the art and clinicians based on factors including the specific antibody used, its variants or derivatives, the patient's age, weight, general health condition, sex and diet, as well as the timing of administration, excretion rate, drug combination and the severity of the specific disease being treated, combined with the individual patient to be treated, route of administration, type of formulation, characteristics of the compounds used, severity of the disease and expected effect, and in conjunction with pharmacological and pharmacokinetic principles well known in the art.

[0655] Methods of administration of formulations comprising the anti-Trop2 antibody, antigen-binding fragment, mutant, or derivative of the present invention include, but are not limited to, parenteral routes such as intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous. Formulations of Trop2 antigen-binding peptides or compositions may be administered via any convenient route, such as by infusion or rapid concentration, absorption through the epithelial or mucosal skin layer (e.g., oral mucosa, rectal and intestinal mucosa), and may be administered together with other bioactive agents. Therefore, pharmaceutical compositions containing the antigen-binding peptides of the present invention can be administered parenterally. As used herein, the term "parenteral" refers to administration methods including, but not limited to, intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, and intra-articular injections and infusions.

[0656] The anti-Trop2 antibody, encoding nucleic acid, or antibody-drug conjugate of the present invention can be administered alone or as a pharmaceutical composition in combination with a diluent and / or other components such as associated cytokines or cell populations. In this regard, the pharmaceutical composition can be prepared by mixing an active pharmaceutical agent of desired purity with an optional pharmaceutically acceptable carrier in the form of a lyophilized formulation or an aqueous solution. The pharmaceutically acceptable carrier is non-toxic to the recipient at the dose and concentration used and may include at least one of buffers (e.g., neutral buffered saline, sulfate buffered saline), antioxidants, preservatives, isotonic agents, stabilizers, chelating agents (e.g., EDTA or glutathione), adjuvants (e.g., aluminum hydroxide), and surfactants. Furthermore, for the pharmaceutical composition to be used for in vivo administration, it must be sterile. The pharmaceutical composition can be sterilized by filtration through a sterile filter membrane.

[0657] The amount of antibody active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the target population and the specific route of administration, and is generally the amount of the composition that produces a therapeutic effect. Typically, in one hundred percent, this amount will range from about 0.01% to about 99% of the active ingredient combined with a pharmaceutically acceptable carrier.

[0658] The composition can be applied in any convenient manner.

[0659] Skilled practitioners and clinicians in the art can adjust the dosing regimen to provide the optimal desired response (e.g., therapeutic response). For example, a single bolus injection can be administered, several separate doses can be administered over time, or the dose can be proportionally reduced or increased depending on the urgency of the treatment situation. Parenteral compositions can be formulated into dosing units to facilitate administration and uniformity of dosage.

[0660] Combination therapy

[0661] In another aspect, the present invention provides a combination therapy in which the pharmaceutical composition of the present invention is co-administered with one or more additional antibodies that effectively inhibit tumor growth in subjects.

[0662] In some embodiments, the present invention provides a method for inhibiting tumor growth in a subject, the method comprising administering to the subject a pharmaceutical composition of the present disclosure and one or more additional antibodies, including anti-LAG-3 antibodies, anti-PD-L1 antibodies, and anti-PD-1 antibodies. In some embodiments, the subject is a human. Trop2 pathway blockade may also be further combined with standard cancer treatment.

[0663] In some implementations, the subject is a human being.

[0664] In some embodiments, the pharmaceutical composition may contain at least one additive selected from: a cytotoxic agent, a chemotherapeutic agent, a cytokine, an immunosuppressant, a growth inhibitor, and an active pharmaceutical agent required for the specific indication to be treated. The specific amount of the additive may be adjusted as needed. Administration may include injection. The compositions described herein may be administered to patients subcutaneously, intradermally, intratumorally, intravenously, or intraperitoneally.

[0665] In one embodiment, the composition of the present invention is administered to a patient via intradermal or subcutaneous injection.

[0666] In other embodiments, the compositions of the present invention are preferably administered via intravenous injection. The T-cell compositions may be injected directly into the tumor site.

[0667] The dosage of the composition can range from about 0.0001 to 100 mg / kg. The "therapeuticly effective dose" of the anti-Trop2 antibody or its antigen-binding moiety, bispecific molecule, CAR-T cell, oncolytic virus, immunoconjugate, nucleic acid molecule, expression vector, or host cell administered in cancer treatment is preferably determined based on the need to reduce the severity of disease symptoms, increase the frequency and duration of asymptomatic periods, or prevent damage or disability caused by disease suffering. For example, for the treatment of tumor-bearing subjects, the "therapeuticly effective dose" preferably eliminates symptoms by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and even more preferably at least about 80% relative to untreated subjects.

[0668] The therapeutic composition comprising the anti-Trop2 antibody or anti-Trop2 / CD3 bispecific antibody of the present invention can be administered via a medical device.

[0669] In some embodiments, the monoclonal antibodies of the present invention can be formulated to ensure proper distribution in vivo. For example, to ensure that the therapeutic antibodies of the present disclosure or their antigen-binding portions cross the blood-brain barrier, they can be formulated in liposomes, which may additionally contain a targeting portion to enhance selective transport to specific cells or organs.

[0670] The combination of therapeutic agents discussed herein can be administered simultaneously as a single composition in a pharmaceutically acceptable carrier, or as a separate composition of each agent in a pharmaceutically acceptable carrier. In another embodiment, the combination of therapeutic agents can be administered sequentially.

[0671] Furthermore, if more than one dose of combination therapy is administered consecutively, the order of administration can be reversed or the same order can be maintained at each administration point. Sequential administration can be combined with simultaneous administration, or any combination thereof.

[0672] In this article, "anti-tumor effect" refers to a biological effect that can be represented by a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in the number of metastases, an increase in life expectancy, or an improvement in various cancer-related physiological symptoms.

[0673] The terms "patient," "subject," and "individual" are used interchangeably in this article to refer to a living organism, such as a mammal, that can elicit an immune response. Examples include, but are not limited to, humans.

[0674] Diagnosis, detection and kits

[0675] The binding molecules of this invention, due to their high affinity for Trop2, can be used for assays, such as binding assays, to detect and / or quantify Trop2 expressed in tissues or cells. Binding molecules, such as single-chain antibodies, can be used in studies further investigating the role of Trop2 in disease. The method for detecting Trop2 generally involves obtaining cell and / or tissue samples; detecting the level of Trop2 in the samples.

[0676] The Trop2 binding molecule of the present invention can be used for diagnostic purposes to detect, diagnose or monitor Trop2-related diseases and / or conditions.

[0677] This invention provides a method for detecting the presence of Trop2 in samples using classic immunohistochemical methods known to those skilled in the art, which can be performed in vivo or in vitro. Examples of methods suitable for detecting the presence of Trop2 include ELISA, FACS, and RIA.

[0678] For diagnostic applications, binders such as single-chain antibodies are typically labeled with detectable labeling groups. Suitable labeling groups include (but are not limited to) the following: radioisotopes or radionuclides, fluorescent groups (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic groups (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent groups, biotinylated groups, or predetermined polypeptide epitopes recognized by secondary reporter molecules (e.g., leucine zipper pairs, binding sites for secondary antibodies, metal-binding domains, epitope tags), MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents. Various methods for labeling proteins are known in the art and can be used to carry out this invention.

[0679] Another aspect of the invention provides a method for detecting the presence of a test molecule that competes with the antibody of the invention for binding to Trop2. An example of such determination would involve detecting the amount of free antibody in a solution containing a certain amount of Trop2, with or without the test molecule. An increase in the amount of free antibody (i.e., antibody not bound to Trop2) would indicate that the test molecule is able to compete with the antibody for binding to Trop2.

[0680] In one implementation, the antibody is labeled with a labeling group. Alternatively, the test molecule is labeled and the amount of free test molecule is monitored in the presence or absence of the antibody.

[0681] This invention also provides a detection kit for detecting Trop2 levels, comprising an antibody that recognizes the Trop2 protein, a lysis medium for dissolving samples, and universal reagents and buffers required for detection, such as various buffers, detection labels, and detection substrates. This detection kit can be used as an in vitro diagnostic device.

[0682] Although the invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations may be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

[0683] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified, the equipment and reagents used in the embodiments and experimental examples are commercially available. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0684] All figures and references, Genbank sequences, patents and published patent applications cited in this application are expressly incorporated herein by reference.

[0685] Experimental methods not specifically described in the following examples are generally performed under standard conditions, such as those described in Molecular Cloning: A Laboratory Manual (4th Edition) published by Cold Spring Harbor Laboratory, or generally under the conditions recommended by the manufacturer.

[0686] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified, the equipment and reagents used in the embodiments and experimental examples are commercially available. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0687] Example

[0688] Example 1: Construction of a human Trop2 overexpressing cell line

[0689] Human Trop2-overexpressing cell lines were constructed using liposome transfection. Lipofectamine was used... TMThe pCMV3 plasmid expressing human Trop2 (Invitrogen, L3000008) was transfected into CHO-K1 cells (hamster ovary cell subline, ATCC#CCL-61) using Invitrogen's 3000 transfection reagent (L3000008). Specifically, one day before transfection, CHO-K1 cells were seeded into 6-well plates at a density of 4 × 10⁵ cells / well and cultured overnight to allow cell adhesion. The next day, the transfection was performed according to the manufacturer's instructions, using 2.5 μg of DNA. After transfection, the cells were cultured in a CO₂ incubator. After 48 hours, the medium was replaced with one containing Hygromycin B (Invitrogen, 10687010). After 10 days of antibiotic-resistant selection under pressure, a mixed cell pool expressing human Trop2 was obtained. The mixed cell pool was then seeded using the limiting dilution method to obtain monoclonal cell lines. The isolated monoclonal cell lines were identified using flow cytometry (FACS). Specifically, after digesting the monoclonal cells to be identified, each cell was washed twice with FACS buffer (PBS + 1% BSA). Then, 50 μL of anti-HA-tagged mouse antibody (Essential Biotech, 100028-MM10) was added to each well, and the cells were incubated at 4°C for 30 min. After centrifugation at 500g for 3 min, the supernatant was discarded. 200 μL of FACS buffer was added to each well, and the cells were washed twice. 50 μL of PE-labeled goat anti-mouse Fc secondary antibody (Jackson, 115-115-164) was added to each well, and the cells were incubated at 4°C for 30 min. After centrifugation at 500g for 3 min, the supernatant was discarded. 200 μL of FACS buffer was added to each well, and the cells were washed twice. 50 μL of FACS buffer was added to each well to resuspend the cells, and the Trop2 expression level in the monoclonal cells was identified using flow cytometry (Beckman Coulter Biotechnology Co., Ltd., A00-1-1102).

[0690] Figure 1 A showed that the proportion of Trop2-positive cells in the obtained Trop2-expressing mixed cell pool reached 78.6%.

[0691] Figure 1 B showed that human Trop2-overexpressing cell clone 1E11 was successfully obtained and named CHO-hTrop2.

[0692] The Trop2 that the anti-Trop2 antibody of the present invention specifically binds to comprises an amino acid sequence as shown in SEQ ID NO:48 and is encoded by a nucleic acid nucleotide sequence as shown in SEQ ID NO:49.

[0693] Example 2: Generation and Identification of Anti-Trop2 Monoclonal Antibody

[0694] This invention employs hybridoma technology for screening anti-Trop2 antibodies. Specifically, human Trop2 protein (Bepsys, TR2-H5223) fused with a 6×His tag at its C-terminus was emulsified with a rapid adjuvant (Bio-Long, KX0210041) and then immunized with BalB / C mice (purchased from Spiefol Biotechnology Co., Ltd.). Serum titers were detected by ELISA. Mice that reached the fusion standard were euthanized, spleens were collected, and single-cell suspensions were prepared. Hybridomas were generated by electrofusion with SP20 myeloma cells (Nanjing Kebai, CBP60881). After the hybridomas grew for 10 days in 96-well cell culture plates, the parent clone 3F8 capable of binding human Trop2 was screened by ELISA and FACS. Single clones with Trop2 binding activity were isolated using limiting dilution and sequenced. The heavy chain variable region and light chain variable region sequences of the mouse clone 3F8 were obtained by sequencing, as shown in Tables 10, 11, and 12.

[0695] Table 10. VHA CDR and VLA CDR of the Trop2 antibody of the present invention as defined by the Kabat numbering system.

[0696] SEQ ID NO CDR SEQ ID NO Sequence (a) VH ACDR1 SEQ ID NO: 1 DHYMH (b) VH ACDR2 SEQ ID NO: 2 YINCFNGAAGYNQKFKG (c) VH ACDR3 SEQ ID NO: 3 EGYDDGYAMDY (d) VL ACDR1 SEQ ID NO: 4 RSSQSLVHSYGNTYLH (e) VL ACDR2 SEQ ID NO: 5 KVSNRFS (f) VL ACDR3 SEQ ID NO: 6 SQSTHVYT

[0697] Table 11. Heavy chain variable region A (VHA) of exemplary anti-Trop2 antibodies of the present invention

[0698]

[0699]

[0700] Table 12. Light chain variable region A (VLA) of exemplary anti-Trop2 antibodies of the present invention

[0701]

[0702] Example 3 Construction, Expression and Purification of Recombinant Trop2 Chimeric Antibody

[0703] First, the heavy chain and light chain variable regions of the 3F8 murine antibody were placed at the N-terminus of the human IgG4 heavy chain constant region (Uniprot ID: P01861) and light chain kappa constant region (Uniprot ID: P01834). The fused gene fragment was then constructed into a pHr expression vector with an N-terminal signal peptide. The obtained light and heavy chain expression vectors were then co-transfected into Expi CHO-S cells (Gibco, A29129) using a transfection reagent (Gibco, A29133). Specifically, Expi CHO-S cells were passaged according to the required transfection volume, and the cell density was adjusted to 4 × 10⁶ cells one day before transfection. 6Cells / mL. Continue culturing ExpiCHO-S cells to achieve a cell density of 6 × 10⁶ cells / mL in the culture on the day of transfection. 6 Cells / mL. Take 4% OptiPRO. TM SFM complexing medium (purchased from Gibco) was used as the transfection buffer. 0.8 μg of plasmid DNA was added to each milliliter of transfection buffer, mixed well, and then ExpiFectamine was added. TM Mix CHO reagent (purchased from Gibco) thoroughly. Gently pour the cationic transfection reagent / DNA mixture into the Expi CHO-S cell suspension. Gently mix and incubate overnight at 37°C with 5% CO2. After 18-22 hours of overnight incubation, add 24% of the transfected culture volume of ExpiCHO reagent to the culture flask. TM Excipients (purchased from Gibco) and 0.6% ExpiCHO by volume of the transfected culture. TM The cell supernatant (purchased from Gibco) was gently mixed and cultured continuously until day 10 or when cell viability was ≤70%. The supernatant was then collected. The target antibody was purified from the culture supernatant using a Protein A affinity chromatography column. Specifically, before purification, the collected cell supernatant was centrifuged at 10,000 rpm for 10 min, and filtered through a 0.22 μm filter. The Protein A column was equilibrated with 5 column volumes of equilibration buffer (10 mM PB, pH 7.0). The filtered supernatant was added to the purification column and equilibrated with 10 column volumes of equilibration buffer. 5 mL of elution buffer (100 mM Gly-NaCl, pH 3.8) was added, and the eluent was collected. The antibody concentration was detected using the A280 method, and the antibody purity was determined by SEC-HPLC. The recombinant chimeric antibody ch3F8 with a purity greater than 95% was then concentrated by ultrafiltration. The full-length amino acid sequence of the heavy chain of the chimeric antibody ch3F8 is shown in SEQ ID NO:16.

[0704] EVQLQQSGPELVKTGASVKISCKASGYSFTDHYMHWVKQSHGKSLEWIGYINCFNGAAGYNQKFKGKATLTVDTSTSTAYMQFNSLTSEDSAVYYCAREGYDDGYAMDYWGQGTSVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO:16).

[0705] The full-length amino acid sequence of the light chain of the chimeric antibody ch3F8 is shown in SEQ ID NO:22:

[0706] DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQ PEDFAVYYCQQHYITPLTFGAGTKVEIKRTRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:22).

[0707] The amino acid sequence of the variable region of the heavy chain of the chimeric antibody ch3F8 is shown in SEQ ID NO:16:

[0708] EVQLQQSGPELVKTGASVKISCKASGYSFTDHYMHWVKQSHGKSLEWIGYINCFNGAAGYNQKFKGKATLTVDTSTSTA YMQFNSLTSEDSAVYYCAREGYDDGYAMDYWGQGTSVTVSS (SEQ ID NO: 7).

[0709] The amino acid sequence of the light chain variable region of the chimeric antibody ch3F8 is shown in SEQ ID NO:22:

[0710] DIQLTQSPSSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQ PEDFAVYYCQQHYITPLTFGAGTKVEIKRT (SEQ ID NO: 13).

[0711] Example 4: Trop2 chimeric antibody ELISA binding activity analysis

[0712] Human Trop2-His (TR2-H5223) was diluted to 2 μg / mL with coating buffer (a mixture of 8 mL 0.2 mol / L Na2CO3 and 17 mL 0.2 mol / L NaHCO3, then 75 mL distilled water was added to adjust the pH to 9.6). 50 μL of this solution was added to each well of a Corning ELISA plate (3590) and incubated at 37°C for 2 hours. After discarding the liquid in the wells, 200 μL of washing buffer (PBS + 0.5‰ Tween-20) was added to each well, and the plate was washed three times. Then, 200 μL of blocking buffer (PBS + 0.5‰ Tween-20 + 5% skim milk powder) was added to each well, and the plate was incubated at 37°C for 2 hours. After discarding the liquid in the wells, 200 μL of washing buffer was added to each well, and the plate was washed three times. 50 μL of serially diluted ch3F8 antibody and reference antibody huRS7 (sequence derived from patent CN100360567C) were added to the wells of an ELISA plate. The initial antibody concentration was 400 nM, and the plate was serially diluted 5-fold, resulting in 11 concentration points. The plates were incubated at 37°C for 60 min. After discarding the liquid in the wells, 200 μL of washing buffer was added to each well, and the plates were washed 3 times. 50 μL of HRP-labeled goat anti-human Fc secondary antibody (Jackson, 109-035-170) was added to each well, and the plates were incubated at 37°C for 60 min. After discarding the liquid in the wells, 200 μL of washing buffer was added to each well, and the plates were washed 3 times. 50 μL of chromogenic reagent (Sigma, T4444) was added to each well, and the plates were incubated at room temperature for 2 min. Then, 50 μL of 2 mol / L H2SO4 was added to terminate the reaction. The OD450 values ​​were read using a ThermoScientific MμLtiskan FC, 8D-030A ELISA reader. Plot the graph based on OD450 values ​​and antibody concentrations. Figure 2 The results showed that ch3F8 could bind to recombinant human Trop2 protein, and its binding activity was close to that of the reference antibody huRS7.

[0713] The heavy chain variable region of the reference antibody huRS7 is shown in SEQ ID NO:54, and the light chain variable region is shown in SEQ ID NO:55.

[0714] QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTS VSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSS(SEQ ID NO:54);

[0715] DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTI SSLQPEDFAVYYCQQHYITPLTFGAGTKVEIKRT(SEQ ID NO:55).

[0716] The amino acid sequence of the full-length heavy chain of the chimeric antibody is shown in SEQ ID NO:16:

[0717] EVQLQQSGPELVKTGASVKISCKASGYSFTDHYMHWVKQSHGKSLEWIGYINCFNGAAGYNQKFKGKATLTVDTSTSTAYMQFNSLTSEDSAVYYCAREGYDDGYAMDYWGQGTSVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG(SEQ ID NO:16);

[0718] The amino acid sequence of the full-length light chain of the chimeric antibody is shown in SEQ ID NO:22:

[0719] DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSYGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTD FTLKISRVEAEDLGVYFCSQSTHVYTFGGGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:22).

[0720] Example 5: FACS Binding Activity Analysis of Trop2 Chimeric Antibody

[0721] The CHO-hTrop2 cell line and MDA-MB-468 tumor cell line (Wuhan Shangen, SNL-061) obtained in Example 1 were digested into single-cell suspensions using trypsin. The cells were washed twice with FACS buffer, and the density was adjusted to 2 × 10⁶ / mL. 100 μL was added to each well of a U-bottom 96-well plate (BIOFIL, 002096). After centrifugation at 500g for 3 min, the supernatant was discarded. 50 μL of serially diluted 3F8 chimeric antibody and reference antibody huRS7 were added to each well, starting at 400 nM and then 5-fold diluted sequentially for a total of 10 concentrations. The cells were incubated at 4°C for 30 min. After centrifugation at 500g for 3 min, the supernatant was discarded. 200 μL of FACS buffer was added to each well, and the cells were washed twice. 50 μL of PE-labeled goat anti-human Fc secondary antibody (Jackson, 109-115-098) was added to each well, and the cells were incubated at 4°C for 30 min. Centrifuge at 500g for 3 min and discard the supernatant. Add 200 μL of FACS buffer to each well and wash twice. Resuspend the cells in 50 μL of FACS buffer to each well and detect the fluorescence intensity of the cells using flow cytometry. Plot a graph with antibody concentration on the x-axis and PE fluorescence area on the y-axis.

[0722] Figure 3 The results showed that both ch3F8 and the reference antibody huRS7 could bind to Trop2 on the cell membrane, and the binding activity of the ch3F8 chimeric antibody was close to that of the reference antibody huRS7.

[0723] Example 6: Species Cross-Binding Activity Analysis of Trop2 Chimeric Antibodies

[0724] Rat Trop2-His (Essential Biotech, 86246-R08H), mouse Trop2-His (Bepsys, TR2-M52H6), and monkey Trop2-His (Bepsys, TR2-R52H3) were diluted to 2 μg / mL with coating buffer and added to each well of an ELISA plate at a rate of 50 μL / well. The plates were incubated at 37°C for 2 hours. The liquid in the wells was discarded, and 200 μL of washing buffer was added to each well for washing three times. 200 μL of blocking buffer was added to each well, and the plates were incubated at 37°C for 2 hours. The liquid in the wells was discarded again, and 200 μL of washing buffer was added to each well for washing three times. 50 μL of serially diluted ch3F8 antibody and reference antibody huRS7 were added to each well of the ELISA plate. The initial antibody concentration was 400 nM, and the plates were subsequently diluted 5-fold, resulting in 11 concentration points. The plates were incubated at 37°C for 60 min. After discarding the liquid in each well, add 200 μL of washing buffer and wash three times. Add 50 μL of HRP-labeled goat anti-human Fc secondary antibody to each well and incubate at 37°C for 60 min. After discarding the liquid in each well, add 200 μL of washing buffer to each well and wash three times. Add 50 μL of chromogenic buffer to each well and incubate at room temperature for 2 min. Then add 50 μL of 2 mol / L H₂SO₄ to stop the reaction and read the OD450 value on a microplate reader. Plot a graph with antibody concentration on the x-axis and OD450 value on the y-axis. Figure 4 As shown, both the ch3F8 antibody and the huRS7 reference antibody were able to bind to recombinant monkey Trop2 protein, but had no binding activity to mouse Trop2 and rat Trop2 proteins.

[0725] Example 7: Analysis of cross-binding activity between Trop2 chimeric antibody and EpCAM

[0726] Human EpCAM-His (EPM-H5223) was diluted to 2 μg / mL with coating buffer and added to each well at 50 μL. The plate was incubated at 37°C for 2 hours. The liquid in the wells was discarded, and 200 μL of washing buffer was added to each well for washing three times. 200 μL of blocking buffer was added to each well, and the plate was incubated at 37°C for 2 hours. The liquid in the wells was discarded again, and 200 μL of washing buffer was added to each well for washing three times. 50 μL of serially diluted ch3F8 antibody and reference antibody huRS7 were added to each well, starting at 400 nM and diluting 5-fold for a total of 11 concentrations. The plate was incubated at 37°C for 60 min. The liquid in the wells was discarded again, and 200 μL of washing buffer was added to each well for washing three times. 50 μL of HRP-labeled goat anti-human Fc secondary antibody was added to each well, and the plate was incubated at 37°C for 60 min. After discarding the liquid in each well, add 200 μL of washing buffer to each well and wash three times. Add 50 μL of chromogenic solution to each well and incubate at room temperature for 2 min. Then, add 50 μL of 2 mol / L H2SO4 to terminate the reaction and read the OD450 value on a microplate reader. Plot a graph with antibody concentration on the x-axis and OD450 value on the y-axis. Figure 5The results showed that neither the ch3F8 antibody nor the reference antibody huRS7 could bind to the recombinant EpCAM protein.

[0727] Example 8: Trop2 chimeric antibody affinity assay

[0728] The equilibrium dissociation constant (KD) of the Trop2 chimeric antibody binding to human Trop2 protein was determined using biofilm thin-layer interferometry (BLI). The specific procedures were as follows: One hour before the experiment, the Protein A sensor (Sartorius, 18-5010) was immersed in PBST solution (10 mM PBS + 0.1% BSA + 0.2‰ Tween-20, pH 7.4). Human Trop2-His (Bepsys, TR2-H5223) was serially diluted with PBST, starting at 100 nM and then sequentially diluted 2-fold, resulting in 7 concentration points. A zero-concentration control well was also included. The chimeric antibody ch3F8 and the reference antibody huRS7 were diluted to 5 μg / mL. The Fortebio molecular interaction analyzer (Sartorius, Octet R8) was set to the following operating conditions: temperature 30℃, Shake speed 1000 rpm. Antibodies were captured using a pre-coated Protein A probe for 180 s; binding to serially diluted antibody samples was performed for 120 s; dissociation was performed for 300 s; and regeneration was repeated three times with regeneration buffer (10 mM glycine, pH 1.7) for 30 s each time. Detection was performed using ForteBio's Octet System. After obtaining the sensor data, the binding constant (ka) and dissociation constant (kd) were analyzed using Octet BLIAnalysis software. An ideal binding-dissociation curve was fitted, and the equilibrium dissociation constant KD (kd / ka) between the antibody and antigen was calculated. As shown in Table 13, the affinity of ch3F8 was higher than that of the reference antibody huRS7.

[0729] Table 13. Equilibrium dissociation constants for Trop2 antibody detection by BLI method

[0730] Antibody Ka (1 / Ms) Kd (1 / s) KD (M) huRS7 2.94E+05 4.33E-04 1.47E-09 ch3F8 4.91E+05 4.14E-04 8.43E-10

[0731] Example 9: Trop2 mouse anti-humanization modification

[0732] Humanization design employed a CDR transplantation method. Specifically, the light and heavy chain sequences of the 3F8 mouse antibody were first input into an antibody structure simulation and optimization analysis platform to search for homologous proteins in the variable region. The results from several databases were compared, and finally, the germline frames of IGKV2-30 and IGKV2-28 were selected as templates for the light chain, and four germline frames of IGHV1-46, IGHV1-3, IGHV1-2, and IGHV1-18 were selected as templates for the heavy chain. The CDR sequences of the variable regions of the 3F8 mouse antibody light and heavy chains were transplanted into the germline frames. After CDR transplantation, several key sites were selected from the humanized sequences generated from each template for reversion mutation, resulting in 5 humanized heavy chain variable region sequences and 2 humanized light chain variable region sequences. The light and heavy chains were combined to obtain a total of 10 humanized antibodies, named hu3F8 H1L1, hu3F8 H2L1, hu3F8 H3L1, hu3F8 H4L1, hu3F8 H5L1, hu3F8H1L2, hu3F8 H2L2, hu3F8 H3L2, hu3F8 H4L2 and hu3F8 H5L2, respectively.

[0733] hu3F8 H1L1 and hu3F8 H1L2 contain the heavy chain shown in SEQ ID NO:17:

[0734] QVQLVQSGAEVKKPGASVKVSCKASGYTFTDHYMHWVRQAPGQGLEWMGYINCFNGAAGYNQKFKGRVTMTVDTSTSTA

[0735] YMELSSLRSEDTAVYYCAREGYDDGYAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVT

[0736] VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGG

[0737] PSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNG

[0738] KEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO:17);

[0739] hu3F8 H2L1 and hu3F8 H2L2 contain the heavy chains shown in SEQ ID NO:18:

[0740] QVQLVQSGAEVKKPGASVKVSCKASGYTFTDHYMHWVRQAPGQRLEWMGYINCFNGAAGYNQKFKGRVTITVDTSASTAYMELSSLRSEDTAVYYCAREGYDDGYAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID:18);

[0741] hu3F8 H3L1 and hu3F8 H3L2 contain the heavy chains shown in SEQ ID NO:19:

[0742] QVQLVQSGAEVKKPGASVKVSCKASGYTFTDHYMHWVRQAPGQGLEWMGYINCFNGAAGYNQKFKGRVTMTVDTSISTA

[0743] YMELSRLRSDDTAVYYCAREGYDDGYAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVT

[0744] VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGG

[0745] PSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNG

[0746] KEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG(SEQ ID: 19);

[0747] hu3F8 H4L1 and hu3F8 H4L2 contain the heavy chain shown in SEQ ID NO:20:

[0748] QVQLVQSGAEVKKPGASVKVSCKASGYTFTDHYMHWVRQAPGQGLEWMGYINCFNGAAGYNQKFKGRVTMTVDTSTSTAYMELRSLRSDDTAVYYCAREGYDDGYAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG(SEQ ID: 20);

[0749] hu3F8 H5L1 and hu3F8 H5L2 contain the heavy chain shown in SEQ ID NO:21:

[0750] QVQLVQSGAEVKKPGASVKVSCKASGYTFTDHYMHWVRQAPGQGLEWIGYINCFNGAAGYNQKFKGRATLTVDTSTSTAYMELSSLRSEDTAVYYCAREGYDDGYAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPE FLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID: 21);

[0751] hu3F8 H1L1, hu3F8 H2L1, hu3F8 H3L1, hu3F8 H4L1, hu3F8 H5L1 all contain the light chain LA1 shown in SEQ ID NO: 23:

[0752] DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSYGNTYLHWYQQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLK

[0753] ISRVEAEDLGVYFCSQSTHVYTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQID NO: 23);

[0754] hu3F8 H1L2, hu3F8 H2L2, hu3F8 H3L2, hu3F8 H4L2, hu3F8 H5L2 all contain the light chain LA2 shown in SEQ ID NO: 24:

[0755] DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSYGNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSG SGTDFTLKISRVEAEDLGVYFCSQSTHVYTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0756] Example 10: Determination of Affinity of Humanized Trop2 Antibody

[0757] The equilibrium dissociation constant (KD) of the humanized Trop2 antibody binding to human Trop2 protein was determined using surface plasmon resonance (SPR) technology. Specifically, a Biacore 8K (Cytiva) was used for detection. First, the humanized Trop2 antibody was conjugated to the surface of an activated Protein A chip (Cytiva, 29127556), and the Trop2 antibody concentration was adjusted to 2 μg / mL. The antibody was captured using the antibody-specific properties of the Protein A chip to obtain approximately 300-430 response units (RUs). Then, the analyte, human Trop2-His protein, was diluted to 200 nM using 1xHBS EP+ buffer (Cytiva, BR-1006-69) as the run buffer. The injection flow rate was 30 μL / min, the binding time was 120 s, and the dissociation time was 200 s. After reaching the preset time, the antigen-antibody complex on the chip surface was regenerated for 30 seconds using glycine (10mM, pH 1.5) regeneration buffer. The elution target was to restore the baseline response value to the initial response value level after coupling. After obtaining the sensor data, the binding constant (ka) and dissociation constant (kd) were analyzed using software. An ideal binding-dissociation curve was fitted, and the equilibrium dissociation constant KD (kd / ka) between the antibody and antigen was calculated. The equilibrium dissociation constant of the humanized antibody is shown in Table 14.

[0758] Table 14. Equilibrium dissociation constants for Trop2 antibody detection by SPR method

[0759]

[0760]

[0761] Example 11: ELISA Binding Activity Analysis of Humanized Trop2 Antibody

[0762] Human Trop2-His (TR2-H5223) was diluted to 2 μg / mL with coating buffer and added to each well of an ELISA plate at a rate of 50 μL / well. The plates were incubated at 37°C for 2 hours. The liquid in the wells was discarded, and 200 μL of washing buffer was added to each well for washing three times. 200 μL of blocking buffer was added to each well, and the plates were incubated at 37°C for 2 hours. The liquid in the wells was discarded again, and 200 μL of washing buffer was added to each well for washing three times. 50 μL of serially diluted ch3F8 antibody and humanized antibodies hu3F8 with different light and heavy chain combinations were added to each well of the ELISA plate. The initial antibody concentration was 400 nM, and the plates were diluted 5-fold sequentially, resulting in 11 concentration points. The plates were incubated at 37°C for 60 min. The liquid in the wells was discarded, and 200 μL of washing buffer was added to each well for washing three times. 50 μL of HRP-labeled goat anti-human Fc secondary antibody was added to each well, and the plates were incubated at 37°C for 60 min. After discarding the liquid in each well, add 200 μL of washing buffer to each well and wash three times. Add 50 μL of chromogenic solution to each well and incubate at room temperature for 2 min. Then, add 50 μL of 2 mol / L H2SO4 to terminate the reaction and read the OD450 value on a microplate reader. Plot a graph with antibody concentration on the x-axis and OD450 value on the y-axis.

[0763] according to Figure 6 The results showed that, except for hu3F8 H5L1, the binding activity of other humanized antibodies to recombinant human Trop2 protein was close to that of the maternal chimeric antibody.

[0764] Example 12: FACS Binding Activity Analysis of Humanized Trop2 Antibody

[0765] CHO-hTrop2 cells and MDA-MB-468 tumor cells were washed twice with FACS buffer, then the density was adjusted to 2×10⁶ / mL, and 100 μL was added to each well of a U-bottom 96-well plate. After centrifugation at 500g for 3 min, the supernatant was discarded, and 50 μL of serially diluted 3F8 chimeric antibody and humanized hu3F8 antibody with different light and heavy chain combinations were added to each well. The initial antibody concentration was 400 nM, and the antibodies were diluted 5-fold sequentially, for a total of 8 concentration spots. The cells were incubated at 4℃ for 30 min. After centrifugation at 500g for 3 min, the supernatant was discarded. 200 μL of FACS buffer was added to each well, and the cells were washed twice. 50 μL of PE-labeled goat anti-human Fc secondary antibody was added to each well, and the cells were incubated at 4℃ for 30 min. After centrifugation at 500g for 3 min, the supernatant was discarded. 200 μL of FACS buffer was added to each well, and the cells were washed twice. 50 μL of FACS buffer was added to each well, and the fluorescence intensity of the cells was detected by flow cytometry. Plot the antibody concentration on the x-axis and the PE fluorescence area on the y-axis.

[0766] according to Figure 7The results showed that, except for hu3F8 H5L1 and hu3F8 H5L2, the binding activity of the other humanized antibodies to human Trop2 at the cellular level was close to that of the maternal chimeric antibodies.

[0767] Example 13: Species Cross-Binding Activity Analysis of Humanized Trop2 Antibody

[0768] Rat Trop2-His (Essence, 86246-R08H), mouse Trop2-His (Bepsys, TR2-M52H6), and monkey Trop2-His (Bepsys, TR2-R52H3) were diluted to 2 μg / mL with coating buffer and added to 50 μL / well of an ELISA plate. The plates were incubated at 37°C for 2 hours. The liquid in the wells was discarded, and 200 μL of washing buffer was added to each well for washing three times. 200 μL of blocking buffer was added to each well, and the plates were incubated at 37°C for 2 hours. The liquid in the wells was discarded again, and 200 μL of washing buffer was added to each well for washing three times. 50 μL of serially diluted humanized 3F8 antibody and chimeric antibody ch3F8 with different light and heavy chain combinations were added to the wells of the ELISA plate. The initial antibody concentration was 400 nM, and the plates were serially diluted 5-fold, resulting in 11 concentration points. The plates were incubated at 37°C for 60 min. After discarding the liquid in each well, add 200 μL of washing buffer and wash three times. Add 50 μL of HRP-labeled goat anti-human Fc secondary antibody to each well and incubate at 37°C for 60 min. After discarding the liquid in each well, add 200 μL of washing buffer to each well and wash three times. Add 50 μL of chromogenic buffer to each well and incubate at room temperature for 2 min. Then add 50 μL of 2 mol / L H₂SO₄ to stop the reaction and read the OD450 value on a microplate reader. Plot a graph with antibody concentration on the x-axis and OD450 value on the y-axis.

[0769] The results are as follows Figure 8 As shown, the humanized antibody maintained its binding activity against monkey Trop2, but had no binding activity against either rat Trop2 or mouse Trop2.

[0770] Example 14: Cross-binding activity analysis of humanized Trop2 antibody with EpCAM

[0771] Human EpCAM-His (EPM-H5223) was diluted to 2 μg / mL with coating buffer and added to each well at 50 μL. The plate was incubated at 37°C for 2 hours. The liquid in the wells was discarded, and 200 μL of washing buffer was added to each well for washing three times. 200 μL of blocking buffer was added to each well, and the plate was incubated at 37°C for 2 hours. The liquid in the wells was discarded again, and 200 μL of washing buffer was added to each well for washing three times. 50 μL of serially diluted humanized 3F8 antibody and chimeric antibody ch3F8 with different light and heavy chain combinations were added to each well. The initial antibody concentration was 400 nM, and the plate was diluted 5-fold sequentially, resulting in 11 concentration points. The plate was incubated at 37°C for 60 min. The liquid in the wells was discarded, and 200 μL of washing buffer was added to each well for washing three times. 50 μL of HRP-labeled goat anti-human Fc secondary antibody was added to each well, and the plate was incubated at 37°C for 60 min. After discarding the liquid in each well, add 200 μL of washing buffer to each well and wash three times. Add 50 μL of chromogenic solution to each well and incubate at room temperature for 2 min. Then, add 50 μL of 2 mol / L H2SO4 to terminate the reaction and read the OD450 value on a microplate reader. Plot a graph with antibody concentration on the x-axis and OD450 value on the y-axis.

[0772] The results are as follows Figure 9 As shown, the humanized antibody did not bind to human EpCAM.

[0773] Example 15 Trop2 Antibody Internalization Activity Detection

[0774] This embodiment uses an endpoint method to detect the endocytic activity of the Trop2 antibody. Specifically, the density of digested MDA-MB-468 cells was adjusted to 1×10⁻⁶ cells one day before the experiment. 5 The antibody was seeded at a rate of 50 μL / well into 96-well cell culture plates and cultured overnight at 37°C with 5% CO2 to allow cell adhesion. The next day, the antibody was diluted to 24 μg / mL, and the goat anti-human Fc secondary antibody (SSA015) labeled with pHAbAmine and Thiol Reactive Dyes (Promega, G9841) was diluted to 72 μg / mL. 25 μL of the antibody and 25 μL of the labeled goat anti-human Fc secondary antibody were mixed and incubated at room temperature in the dark for 30 min before being added to the corresponding cell wells. After culturing the cells at 37°C with 5% CO2 for 24 hours, they were digested with trypsin and washed twice with FACS buffer. The mean fluorescence intensity of the PE signal in the cells was detected by flow cytometry and plotted.

[0775] Figure 10 The results showed that different combinations of light and heavy chains in humanization could affect the endocytic activity of antibodies to varying degrees.

[0776] Example 16 Construction of Trop2×CD3 bispecific antibody vector and protein expression and purification

[0777] 16.1 Construction of Trop2×CD3 bispecific antibody expression vector

[0778] In this embodiment, two anti-Trop2 / CD3 bispecific antibodies were constructed, named Trop2 / CD3 Ab1 and Trop2 / CD3 Ab2, respectively. The specific structures of the bispecific antibodies are as follows: The bispecific antibodies consist of two peptide chains. Peptide chain 1# is the amino acid sequence for binding Trop2. Specifically, the C-terminus of the anti-Trop2 VL is linked to the N-terminus derived from the human κ chain constant region. Following the C-terminus of the κ chain constant region is a (G4S)6 linker. Following the C-terminus of the linker is the N-terminus of the Trop2 antibody VH. The C-terminus of the anti-Trop2 VH is linked to the sequence of the human IgG4 constant region. Peptide chain 2# is the amino acid sequence for binding CD3. Specifically, the C-terminus of the anti-CD3 VL is linked to the N-terminus derived from the human κ chain constant region. Following the C-terminus of the κ chain constant region is a (G4S)6 linker. Following the C-terminus of the linker is the N-terminus of the anti-CD3 VH. The C-terminus of the anti-CD3 VH is linked to the sequence of the human IgG4 constant region.

[0779] A series of mutations were performed on the Fc of this bispecific antibody. Specifically, the knock-in-hole mutation, invented by Genentech in the 1990s, was used to prevent heavy chain mismatch. In one antibody heavy chain (knob chain), serine S at position 354 was mutated to cysteine ​​C, and serine T at position 366 was mutated to tryptophan W. In the other heavy chain (Hole chain), tyrosine Y at position 349 was mutated to cysteine ​​C, serine T at position 366 was mutated to serine S, leucine L at position 368 was mutated to alanine A, and amino acid at position 407 was mutated from tyrosine Y to valine V (Y349C, T366S, L368A, Y407V). To reduce antibody Fab exchange, serine S at position 228 was mutated to proline P. To weaken the function of the antibody Fc terminus, mutations were introduced at two sites: F234A and L235A. The anti-Trop2 and anti-CD3 gene fragments constructed above were respectively constructed into the pHr expression vector to obtain anti-Trop2 and anti-CD3 single-arm antibody plasmids.

[0780] 16.2 Expression and purification of Trop2×CD3 bispecific antibody

[0781] Bispecific antibodies were generated using the CHO-S transient transfection expression system. Specifically, Expi CHO-S cells were passaged according to the required transfection volume, and the cell density was adjusted to 4 × 10⁶ cells / mL the day before transfection. Expi CHO-S cells were cultured further until the cell density in the culture on the day of transfection was 6 × 10⁶ cells / mL. 4% OptiPRO was then used... TM SFM complexing medium was used as the transfection buffer. 0.8 μg of plasmid DNA was added to each milliliter of transfection buffer, mixed well, and then ExpiFectamine was added. TM Mix CHO reagent thoroughly, then gently pour the cationic transfection reagent / DNA mixture into the Expi CHO-S cell suspension. Gently mix and incubate overnight at 37°C with 5% CO2. After 18-22 hours of overnight incubation, add 24% of the transfected culture volume of ExpiCHO reagent to the culture flask. TM ExpiCHO was added as excipient and 0.6% of the volume of the transfected culture. TM Add the enhancer, mix gently, and culture continuously until day 10 or when cell viability is ≤70%, then collect the culture supernatant. Purify the target antibody from the culture supernatant using a Protein A affinity chromatography column. Specifically, before purification, centrifuge the collected cell supernatant at 10000 rpm for 10 min, and filter the supernatant through a 0.22 μm filter. Equilibrate the Protein A column with 5 column volumes of equilibration buffer. Add the filtered supernatant to the purification column and equilibrate with 10 column volumes of equilibration buffer. Add 5 mL of elution buffer and collect the eluent. The antibody concentration is detected using the A280 method, and the antibody purity is determined by SEC-HPLC. The obtained bispecific antibodies are then concentrated by ultrafiltration, ultimately yielding two bispecific antibodies: Trop2×CD3 Ab1 and Trop2×CD3 Ab2.

[0782] Example 17 Trop2×CD3 Bispecific Antibody ELISA Binding Activity Analysis

[0783] Human Trop2-His (TR2-H5223) and human CD3E&D complex (CDD-H52W1) were diluted to 2 μg / mL with coating buffer and added to 50 μL / well of an ELISA plate. The plates were incubated at 37°C for 2 hours. The liquid in the wells was discarded, and 200 μL of washing buffer was added to each well for washing three times. 200 μL of blocking buffer was added to each well, and the plates were incubated at 37°C for 2 hours. The liquid in the wells was discarded again, and 200 μL of washing buffer was added to each well for washing three times. 50 μL of serially diluted Trop2×CD3 Ab1 and Trop2×CD3 Ab2 antibodies were added to each well of the ELISA plate. The initial antibody concentration was 400 nM, and the plates were diluted 5-fold sequentially, resulting in 11 concentration points. The plates were incubated at 37°C for 60 min. The liquid in the wells was discarded, and 200 μL of washing buffer was added to each well for washing three times. Add 50 μL of HRP-labeled goat anti-human Fc secondary antibody to each well and incubate at 37°C for 60 min. Discard the liquid in the wells and add 200 μL of washing buffer to each well, washing three times. Add 50 μL of chromogenic solution to each well and incubate at room temperature for 2 min. Then, add 50 μL of 2 mol / L H2SO4 to stop the reaction and read the OD on a microplate reader. 450 Numerical values. OD is plotted on the x-axis as antibody concentration. 450 Plot the values ​​on the ordinate. The results are as follows: Figure 11 As shown, the two bispecific antibodies exhibited comparable binding activities to human Trop2, but differed in their binding activities to the human CD3E&D complex. The binding activity of Trop2×CD3 Ab2 to human CD3E&D was significantly lower than that of Trop2×CD3 Ab1, with the former's EC50 value for binding to human CD3E&D being approximately 50 times that of the latter.

[0784] Example 18: FACS Binding Activity Analysis of Trop2×CD3 Bispecific Antibody

[0785] Trypsin-digested MDA-MB-468 and Jurkat cells were collected by centrifugation and washed twice with FACS buffer. The densities of MDA-MB-468 and Jurkat cells were then adjusted to 2 × 10⁻⁶ cells per cell, respectively. 6 / mL and 5×10 5Add 100 μL of Trop2×CD3 Ab1 and Trop2×CD3 Ab2 antibodies to each well of a U-bottom 96-well plate. Centrifuge at 500g for 3 min, discard the supernatant, and add 50 μL of serially diluted Trop2×CD3 Ab2 antibodies to each well. For binding assays with MDA-MB-468 cells, the starting antibody concentration was 500 nM, with 5-fold dilutions for a total of 8 concentration points; for binding assays with Jurkat cells, the starting antibody concentration was 1000 nM, with 5-fold dilutions for a total of 8 concentration points. Incubate at 4°C for 30 min. Centrifuge at 500g for 3 min, discard the supernatant. Add 200 μL of FACS buffer to each well and wash twice. Add 50 μL of PE-labeled goat anti-human Fc secondary antibody to each well and incubate at 4°C for 30 min. Centrifuge at 500g for 3 min, discard the supernatant. Add 200 μL of FACS buffer to each well and wash twice. Cells were resuspended in 50 μL of FACS buffer in each well, and the fluorescence intensity of the cells was detected by flow cytometry. A graph was plotted with antibody concentration on the x-axis and PE fluorescence area on the y-axis.

[0786] The results are as follows Figure 12 As shown, the two bispecific antibodies exhibit comparable binding activity to human Trop2 at the cellular level. Due to differences in the binding epitopes of the CD3 binding arm to the CD3 complex, the two bispecific antibodies demonstrate different binding behaviors to the human CD3 complex at the cellular level. Compared to Trop2×CD3 Ab1, Trop2×CD3 Ab2 shows a lower EC50 value in its binding curve with Jurkat cells, and its maximum binding amount is also lower than that of Trop2×CD3 Ab1.

[0787] Example 19: Analysis of the cross-binding activity of Trop2×CD3 bispecific antibody species

[0788] Monkey Trop2-His (TR2-R52H3) and monkey CD3E&D complex (CDD-C52W4) were diluted to 2 μg / mL with coating buffer and added to 50 μL / well of an ELISA plate. The plates were incubated at 37°C for 2 hours. The liquid in the wells was discarded, and 200 μL of washing buffer was added to each well for washing three times. 200 μL of blocking buffer was added to each well, and the plates were incubated at 37°C for 2 hours. The liquid in the wells was discarded again, and 200 μL of washing buffer was added to each well for washing three times. 50 μL of serially diluted Trop2×CD3 Ab1 and Trop2×CD3 Ab2 antibodies were added to each well of the ELISA plate. The initial antibody concentration was 400 nM, and the plates were diluted 5-fold sequentially, resulting in 11 concentration points. The plates were incubated at 37°C for 60 min. The liquid in the wells was discarded, and 200 μL of washing buffer was added to each well for washing three times. Add 50 μL of HRP-labeled goat anti-human Fc secondary antibody to each well and incubate at 37°C for 60 min. Discard the liquid in the wells and add 200 μL of washing buffer to each well, washing three times. Add 50 μL of chromogenic solution to each well and incubate at room temperature for 2 min. Then, add 50 μL of 2 mol / L H2SO4 to stop the reaction and read the OD on a microplate reader. 450 Numerical values. OD is plotted on the x-axis as antibody concentration. 450 Plot the values ​​on the ordinate.

[0789] The results are as follows Figure 13 As shown, both bispecific antibodies can bind to the monkey Trop2 protein, and their binding activities are comparable. For the monkey CD3E&D protein, Trop2×CD3 Ab1 has binding activity, while Trop2×CD3 Ab2 cannot bind.

[0790] Example 20: Detection of the in vitro tumor-killing activity of Trop2×CD3 bispecific antibody

[0791] In this embodiment, the FACS method was used to detect the in vitro killing effect of Trop2×CD3 bispecific antibody-mediated T cells on different tumor cell lines. Specifically, human peripheral blood mononuclear cells (PBMCs, Shanghai Miaoshun Biotechnology Co., Ltd., PB010C) were resuscitated one day before the experiment and placed in complete culture medium RPIM 1640 (Gibco, 11875093) + 10% fetal bovine serum (Gibco, 10100147C). On the day of the experiment, the PBMC density was adjusted to 2×10⁶ cells / year. 6The bispecific antibody to be tested was serially diluted in RPMI 1640 complete medium, with a maximum concentration of 20 nM, in 5-fold serial dilutions, resulting in 9 concentration points. 50 μL of the serially diluted bispecific antibody was mixed with 50 μL of PBMC and incubated in a 37℃ CO2 incubator for 30 min. Trop2-overexpressing BxPC-3 pancreatic cancer cells (Wuhan Shangen Biotechnology, SNL-099), MDA-MB-468 breast cancer cells (Wuhan Shangen Biotechnology, SNL-061), MDA-MB-231 breast cancer cells (Nanjing Kebai Biotechnology, CBP60382), and Trop2-negative A549 non-small cell lung cancer cells (Nanjing Kebai Biotechnology, CBP60084) were digested into single cells using trypsin, stained with 5 μM CFSE (Invitrogen, 65-0850-84), and the cell density was adjusted to 2 × 10⁶ cells using RPMI 1640 medium. 5 Cells were seeded at a rate of 100 μL / well in 96-well low-adsorption U-bottom plates. The incubated PBMC and antibody mixture was then added to the corresponding wells. Cells were incubated at 37°C CO2 for 48 hours, centrifuged, and resuspended in FACS buffer containing Fixable Viability Dye (Invitrogen, 65-0865-18). Cells showing both CFSE and Fixable Viability Dye positivity were detected by flow cytometry, and the tumor target cell killing ratio was calculated.

[0792] Figure 14 The results showed that both bispecific antibodies exhibited very strong cytotoxic activity against different types of Trop2-positive tumor cells, with EC50 values ​​at the pM level. However, neither antibody showed cytotoxic activity against Trop2-negative A549 cells. These results indicate that the cytotoxic activity of the bispecific antibodies depends on Trop2 expression. Comparing the EC50 values ​​of the cytotoxic curves, the cytotoxic activity of Trop2×CD3 Ab2 was slightly lower than that of Trop2×CD3 Ab1, but both achieved the same maximum cytotoxic effect with increasing drug concentration.

[0793] Example 21: Analysis of the T cell activation function of Trop2×CD3 bispecific antibody

[0794] The effect of Trop2×CD3 bispecific antibody-mediated T cell activation was detected using the FACS method. Specifically, human peripheral blood mononuclear cells (PBMCs, Shanghai Miaoshun Biotechnology Co., Ltd., PB010C) were resuscitated into complete culture medium RPIM 1640 + 10% fetal bovine serum one day before the experiment. On the day of the experiment, the PBMC density was adjusted to 2×10⁶ cells / year. 6The bispecific antibody to be tested was serially diluted in RPMI 1640 complete medium, with a maximum concentration of 20 nM, in 5-fold serial dilutions, resulting in 8 concentration points. 50 μL of the serially diluted bispecific antibody was mixed with 50 μL of PBMC and incubated in a 37℃ CO2 incubator for 30 min. MDA-MB-468 and MDA-MB-231 breast cancer cells were digested into single-cell suspensions using trypsin, and the cell density was adjusted to 2 × 10⁶ cells / mL using RPMI 1640 medium. 5 PBMCs / mL were seeded at a rate of 100 μL / well into 96-well low-adsorption U-bottom plates. The incubated PBMC and antibody mixture was then added to the corresponding wells. The plates were incubated at 37°C in a CO2 incubator for 48 hours. After centrifugation and discarding the supernatant, PerCP anti-human CD45 (Biolegend, 304026), FITC anti-human CD2 Antibody (Biolegend, 309206), PE / Cyanine7 anti-human CD4 (Biolegend, 317414), APC anti-human CD8a (Biolegend, 301014), Brilliant Violet 421™ anti-human CD69 (Biolegend, 310929), and PE anti-human CD25 (Biolegend, 302606) were added, and the plates were incubated at 4°C for 30 minutes. The plates were washed twice with FACS buffer. The proportion of CD25 and CD69 dual-positive cells in CD4+ and CD8+ T cells was detected by flow cytometry, which is the activation ratio of CD4+ and CD8+ T cells.

[0795] Figure 15 The results showed that Trop2×CD3 Ab1 and Trop2×CD3 Ab2 could specifically activate T cells when co-cultured with triple-negative breast cancer cells, and the activation abilities of the two were comparable.

[0796] Figure 15 The study demonstrated the activation effect of CD4+ and CD8+ T cells mediated by CD3 bispecific antibody.

[0797] Example 22: Analysis of Trop2×CD3 Bispecific Antibody-Mediated Cytokine Release Levels

[0798] PBMC cell density was adjusted to 4 × 10⁶ cells / mL using RPMI 1640 complete medium. The bispecific antibody to be tested was serially diluted using RPMI 1640 complete medium, with a maximum concentration of 4 nM, in 5-fold serial dilutions, resulting in 8 concentration points. 50 μL of the serially diluted bispecific antibody was mixed with 50 μL of PBMC and incubated at 37°C CO₂ for 30 min. MDA-MB-468 breast cancer cells were digested into single cells using trypsin, and the cell density was adjusted to 2 × 10⁵ cells / mL using RPMI 1640 medium. Cells were seeded at 100 μL / well in low-adsorption U-bottom 96-well plates. The incubated PBMC and antibody mixture was added to the corresponding wells. The cells were incubated at 37°C CO₂ for 48 hours, and the cell supernatant was collected by centrifugation. The supernatant was then processed using LEGENDplex. TM The HU Th1Panel (5-plex) w / VbP V02 kit (Biolegend, 741036) was used to detect cytokine concentrations in the supernatant. The procedure was performed according to the manufacturer's instructions, and cytokine release from the supernatant was analyzed using flow cytometry.

[0799] from Figure 16 The results showed that both Trop2×CD3 Ab1 and Trop2×CD3 Ab2 could mediate the release of IL-2, IL-6, IL-10, IFN-γ and TNF-α from PBMCs when co-cultured with triple-negative breast cancer cells, and the level of cytokine release mediated by Trop2×CD3 Ab2 was lower than that mediated by Trop2×CD3 Ab1. Figure 16 The results of comparing the levels of cytokine release mediated by CD3 bispecific antibodies were presented.

[0800] Example 23: In vivo tumor-suppressive activity analysis of Trop2×CD3 bispecific antibody

[0801] This study investigated the inhibitory effect of the Trop2×CD3 Ab2 bispecific antibody on MDA-MB-231 tumors in NSG female mice (purchased from Spiford Biotechnology Co., Ltd.). Specifically, after the animals passed quarantine, MDA-MB-231 cells in the logarithmic growth phase were collected and the concentration was adjusted to 102. 8 The concentration of MDA-MB-231 cells was 1:1 (5 × 10⁶ cells / mL) and mixed with an equal volume of matrix gel before inoculation. 0.1 mL of the cell mixture was subcutaneously injected into the left anterior axilla of each mouse. Seven days after MDA-MB-231 cell inoculation, animals with significantly larger tumors were selected and intravenously injected with 0.1 mL of a 5 × 10⁶ cell mixture. 7 / mL PBMCs (cultured overnight in 10ng / ml IL-2 complete medium after resuscitation). When the animal tumor volume reaches approximately 100–150 mm². 3Animal models were randomly divided into three groups of five mice each, based on tumor volume. Drug administration was performed on days 0, 3, 7, 11, and 14 after grouping. Mice were intraperitoneally injected with either the negative control antibody hIgG or Trop2×CD3 Ab2, at doses of 1 mg / kg and 5 mg / kg, respectively. Tumor volume was measured twice weekly. After five administrations, the tumor inhibition rate (TGI%) was calculated.

[0802] according to Figure 17 As shown, Trop2×CD3 Ab2 inhibited tumor growth in a human triple-negative breast cancer MDA-MB-231 mouse model, exhibiting a dose-dependent effect. After 5 administrations, the TGI was 93.9% in the low-dose group and 137.8% in the high-dose group.

[0803] Figure 17 The in vivo tumor-suppressing effect of Trop2×CD3 Ab2 bispecific antibody.

[0804] Although the present invention has been described in detail above with general descriptions and specific embodiments, further modifications and improvements can be made based on the present invention. These modifications and improvements are obvious to those skilled in the art without requiring inventive effort. Therefore, all such modifications and improvements made without departing from the present invention fall within the scope of protection claimed by the present invention.

Claims

1. An antibody against trophoblast cell surface antigen 2 (Trop2), its antigen-binding fragment, or a mutant thereof, characterized in that, It includes a heavy chain variable region (VHA) and a light chain variable region (VLA), wherein the VHA includes VHA CDR1 as shown in SEQ ID NO:1, VHA CDR2 as shown in SEQ ID NO:2 and VHA CDR3 as shown in SEQ ID NO:3, and the VLA includes VLA CDR1 as shown in SEQ ID NO:4, VLA CDR2 as shown in SEQ ID NO:5 and VLA CDR3 as shown in SEQ ID NO:

6.

2. The anti-Trop2 antibody, its antigen-binding fragment, or a mutant thereof as described in claim 1, characterized in that, The VHA comprises an amino acid sequence as shown in any one of SEQ ID NO:7-12, or an amino acid sequence having a similarity of not less than 90% to an amino acid sequence shown in any one of SEQ ID NO:7-12; and / or the VLA comprises an amino acid sequence as shown in any one of SEQ ID NO:13-15, or an amino acid sequence having a similarity of not less than 90% to an amino acid sequence shown in any one of SEQ ID NO:13-15.

3. A bispecific antibody that specifically binds to Trop2 and T cells, comprising a first antigen-binding arm specifically binding to Trop2 and a second antigen-binding arm specifically binding to a T cell surface antigen; characterized in that, The first antigen-binding arm comprises VHA CDR1 shown in SEQ ID NO:1, VHA CDR2 shown in SEQ ID NO:2 and VHA CDR3 shown in SEQ ID NO:3, VLA CDR1 shown in SEQ ID NO:4, VLA CDR2 shown in SEQ ID NO:5 and VLA CDR3 shown in SEQ ID NO:6; The T cell surface antigens include or are selected from CD3, CD2, CD28, CD134, CD137, CD152, and CD279.

4. A bispecific antibody that specifically binds to Trop2 and CD3, comprising a first antigen-binding arm that specifically binds to Trop2 and a second antigen-binding arm that specifically binds to the T cell surface antigen CD3; characterized in that: The first antigen-binding arm is a single-chain peptide chain containing VLA, CLA, linker, VHA, CH1, and Fc(A') from the N-terminus to the C-terminus, and The second antigen-binding arm is a single-chain peptide chain containing VLB, CLB, linker, VHB, CH1 and Fc(B) from the N-terminus to the C-terminus; Wherein, VHA and VLA are respectively the VHA and VLA of the anti-Trop2 antibody of claim 1 or 2, or the antigen-binding fragment or its mutant, and Fc(A') is a variant generated by introducing a hole mutation in the Fc region of the heavy chain portion of the anti-Trop2 antibody of claim 1 or 2; VHB and VLB are respectively the CD3-specific heavy chain variable region and light chain variable region, and Fc(B) is a variant generated by introducing a knob mutation in the Fc region of the heavy chain portion of the anti-CD3 antibody; The VLA contains an amino acid sequence as shown in SEQ ID NO:14, the VHA contains an amino acid sequence as shown in SEQ ID NO:8, and the Fc(A') contains a variant of the amino acid sequence as shown in SEQ ID NO:28; Furthermore, the VLB contains an amino acid sequence as shown in SEQ ID NO:30 or SEQ ID NO:

39. The VHB contains an amino acid sequence as shown in SEQ ID NO:32 or SEQ ID NO:

41. The Fc(B) contains an amino acid sequence as shown in SEQ ID NO:37 or SEQ ID NO:

46.

5. A bispecific antibody that specifically binds to Trop2 and CD3, comprising a first antigen-binding arm that specifically binds to Trop2 and a second antigen-binding arm that specifically binds to the T cell surface antigen CD3, characterized in that: The first antigen-binding arm is a single-chain peptide chain containing a Trop2-specific light chain A (LA) and a heavy chain A' (HA'), wherein the amino acid sequences of the light chain A and the heavy chain A' are as shown in SEQ ID NO:23 and SEQ ID NO:51, respectively; the second antigen-binding arm is a single-chain peptide chain containing a CD3-specific light chain B (LB) and a heavy chain B (HB), wherein the light chain B contains the amino acid sequences as shown in SEQ ID NO:35 or SEQ ID NO:44, respectively, and the heavy chain B contains the amino acid sequences as shown in SEQ ID NO:34 or SEQ ID NO:43, respectively; wherein the first antigen-binding arm is a single-chain peptide chain with an LA-linker-HA' structure in which LA and HA' are linked by a linker, and the second antigen-binding arm is a single-chain peptide chain with an LB-linker-HB structure in which LB and HB are linked by a linker.

6. The bispecific antibody according to claim 4 or 5, characterized in that, The linker is a linker peptide having the structure shown in (GGGGS)n; wherein n is any integer selected from 1 to 10; or preferably, the linker peptide has an amino acid sequence as shown in SEQ ID NO:

52.

7. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: (i) An active ingredient selected from the group consisting of: an anti-Trop2 antibody as described in claim 1 or 2, an antigen-binding fragment thereof or a mutant thereof, or a bispecific antibody binding Trop2 and CD3 as described in claim 4, 5 or 6, and (ii) Pharmaceutically acceptable carriers.

8. Any one or more of the following applications of the anti-Trop2 antibody of claim 1 or 2, or the bispecific antibody binding Trop2 and CD3 of claim 5 or 6, or the pharmaceutical composition of claim 8: (1) Use in the preparation of drugs for the diagnosis, prevention or treatment of diseases related to Trop2 overexpression or dysfunction; (2) Use in the preparation of medicaments for the diagnosis, prevention or treatment of diseases targeting Trop2; (3) Application in the preparation of drugs for killing Trop2-overexpressing cells; (4) Application in the preparation of detection reagents for Trop2 and / or CD3; (5) Application in the preparation of antibody-drug conjugates that specifically bind to Trop2; (6) Application in the preparation of relevant reagents suitable for CAR-T therapy.

9. The application as described in claim 8, characterized in that, The diseases associated with Trop2 overexpression or dysfunction are cancers, including or selected from non-small cell lung cancer, colon cancer, rectal cancer, gastric cancer, invasive endometrial adenocarcinoma, ovarian cancer, squamous bladder cancer, choriocarcinoma, bronchial cancer, breast cancer, cervical cancer, pancreatic cancer, or seminal vesicle cancer. Or preferably, the diseases associated with Trop2 expression or dysfunction are selected from invasive breast cancer, invasive pancreatic cancer, or non-small cell lung cancer; Or more preferably, the disease associated with abnormal Trop2 expression or function is invasive breast cancer.

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