PSMA antibody and use thereof

Abstract

The present invention provides a PSMA antibody or an antigen binding fragment, a bispecific antibody comprising the antibody or the antigen binding fragment, a nucleic acid molecule encoding the antibody or the antigen binding fragment, an expression vector, a recombinant cell, a conjugate, a pharmaceutical composition, a kit and uses thereof.

Description

PSMA antibodies and their applications

[0001] Priority information

[0002] This application claims priority and benefits of patent application 202311699412.4 filed with the State Intellectual Property Office of China on December 11, 2023, and incorporates the entire text of it herein by reference. Technical Field

[0003] The present invention belongs to the field of biopharmaceutical technology, and specifically relates to PSMA antibodies and their applications. More specifically, the present invention relates to an antibody or antigen-binding fragment thereof, a bispecific antibody, a nucleic acid molecule, an expression vector, a recombinant cell, a conjugate, a pharmaceutical composition, a kit, and their uses. Background Art

[0004] Cancer is a major disease that affects human survival and development. According to the latest data, there are about 19 million new cancer cases and 10 million cancer deaths each year worldwide, and the incidence and mortality rates are on the rise.

[0005] Besides surgical resection, traditional cancer treatments such as chemotherapy and radiotherapy have significant side effects and are prone to recurrence. In recent years, immunotherapy, including tumor-targeted antibodies, immune checkpoint antibodies, and bispecific antibodies, has become a new hotspot and new hope in the fight against cancer.

[0006] In recent years, immunotherapies, exemplified by PD-1 / L1, have demonstrated tremendous potential. However, it's crucial to note that even the currently most widely approved PD-1 / L1 therapy has an overall response rate of only 30%, leaving many patients unable to benefit. Immune checkpoint molecules are inhibitory molecules expressed on the surface of immune cells, including T cells, natural killer cells, and monocytes and macrophages. Upon binding to their corresponding ligands, they transmit inhibitory signals to the immune cells, inhibiting their anti-cancer function. Due to the significant heterogeneity in the expression of immune checkpoint receptor ligands on tumors and tumor-infiltrating lymphocytes, a single type of immune checkpoint therapy cannot be suitable for all patients, and most patients will not benefit. Furthermore, some patients who have received immune checkpoint therapy will experience tumor recurrence and develop resistance to the therapy, rendering continued treatment ineffective. Furthermore, T cells recognize neoantigens (neoantigens)—antigens derived from tumor gene mutations—through their surface T cell receptors (TCRs). However, some tumors have a low frequency of genetic mutations and a limited number of neoantigens, often referred to as "cold tumors." Current immune checkpoint therapies, such as PD-1 / L1 therapy, achieve anti-cancer goals by restoring the function of T cells themselves. However, in "cold tumors", T cells cannot effectively recognize tumors, resulting in the ineffectiveness of immune checkpoint therapy for cold tumors.

[0007] Prostate cancer is one of the most common malignant tumors in Europe and the United States. With my country's economic growth and changing lifestyles, the incidence of prostate cancer is also on the rise. PSMA is a cell membrane protein that is barely expressed in most normal tissues but is specifically overexpressed in prostate cancer. Over 80% of prostate cancer patients have high PSMA expression, and this high expression is associated with a poor prognosis. Therefore, PSMA is a potential therapeutic target, and the development of PSMA-targeting antibodies, bispecific and multifunctional antibodies based on PSMA monoclonal antibodies, or CAR-T and CAR-NK therapies is highly valuable. Summary of the Invention

[0008] The present invention aims to at least partially address at least one of the technical problems existing in the prior art. To this end, the present invention provides an antibody or antigen-binding fragment targeting PSMA, which has high PSMA binding affinity and tumor killing activity.

[0009] In its first aspect, the present invention provides an antibody or antigen-binding fragment. According to an embodiment of the present invention, the antibody or antigen-binding fragment comprises at least one of the following CDRs: HCDR 1: GYSFTX1NW, wherein X1 is S or H; HCDR 2: IYPGDSDT; HCDR 3: ARQTGFLWSSDLWGRGT; LCDR 1: X2QDISX3A, wherein X2 is S or P, and X3 is S or Y; LCDR 2: DASX4, wherein X4 is S or W; LCDR 3: QQFNSYPLX5, wherein X5 is T or S; and X1 being S, X2 being S, X3 being S, X4 being S, and X5 being T cannot be present simultaneously. Compared to a wild-type antibody, the antibody or antigen-binding fragment exhibits high PSMA binding affinity and tumor cytotoxicity, and can detect PSMA.

[0010] In its second aspect, the present invention provides a multispecific antibody. According to an embodiment of the present invention, the multispecific antibody comprises: a first binding region comprising the antibody or antigen-binding fragment described in the first aspect; and a second binding region having binding activity for a first molecule. The multispecific antibody of the present invention has high PSMA binding affinity and tumor killing potency, and can effectively prevent and / or treat PSMA-mediated diseases, particularly prostate cancer.

[0011] In a third aspect, the present invention provides a nucleic acid molecule. According to embodiments of the present invention, the nucleic acid molecule encodes the antibody or antigen-binding fragment described in the first aspect, or the multispecific antibody described in the second aspect. The nucleic acid molecule of the present invention may encode the antibody or antigen-binding fragment described in the first aspect, or the multispecific antibody described in the second aspect.

[0012] In a fourth aspect, the present invention provides an expression vector. According to an embodiment of the present invention, the expression vector carries the nucleic acid molecule described in the third aspect. Thus, the expression vector of the present invention can effectively express the antibody or antigen-binding fragment of the first aspect or the multispecific antibody of the second aspect, thereby enabling the in vitro production of large quantities of the antibody or antigen-binding fragment or multispecific antibody.

[0013] In its fifth aspect, the present invention provides a recombinant cell. According to embodiments of the present invention, the recombinant cell comprises a nucleic acid molecule as described in the third aspect or an expression vector as described in the fourth aspect; or expresses the antibody or antigen-binding fragment as described in the first aspect, or the multispecific antibody as described in the second aspect. Under suitable conditions, the recombinant cell can effectively express the aforementioned antibody or antigen-binding fragment or multispecific antibody within the cell.

[0014] In its sixth aspect, the present invention provides a conjugate. According to an embodiment of the present invention, the conjugate comprises the antibody or antigen-binding fragment described in the first aspect, or the multispecific antibody described in the second aspect; and a coupling moiety, wherein the coupling moiety is linked to the antibody, antigen-binding fragment, or multispecific antibody. The conjugate of the present invention has high PSMA binding affinity and can be used to detect PSMA, or to prevent and / or treat PSMA-mediated diseases, particularly for detecting prostate cancer, or preventing and / or treating prostate cancer.

[0015] In its seventh aspect, the present invention provides a pharmaceutical composition. According to embodiments of the present invention, the pharmaceutical composition comprises the antibody or antigen-binding fragment described in the first aspect, the multispecific antibody described in the second aspect, the nucleic acid molecule described in the third aspect, the expression vector described in the fourth aspect, the recombinant cell described in the fifth aspect, or the conjugate described in the sixth aspect. The pharmaceutical composition of the present invention has high PSMA binding affinity and tumor killing potency, and can effectively prevent and / or treat PSMA-mediated diseases, particularly prostate cancer.

[0016] In an eighth aspect, the present invention provides a kit. According to an embodiment of the present invention, the kit comprises the antibody or antigen-binding fragment described in the first aspect, the multispecific antibody described in the second aspect, the nucleic acid molecule described in the third aspect, the expression vector described in the fourth aspect, the recombinant cell described in the fifth aspect, or the conjugate described in the sixth aspect. The kit of the present invention has high PSMA binding affinity and can effectively detect PSMA, detect PSMA-mediated related diseases, diagnose PSMA-mediated related diseases, stage PSMA-mediated related diseases, or assess the prognosis of PSMA-mediated related diseases.

[0017] In the ninth aspect of the present invention, the present invention provides the antibody or antigen-binding fragment of the first aspect, the multispecific antibody of the second aspect, the nucleic acid molecule of the third aspect, the expression vector of the fourth aspect, the recombinant cell of the fifth aspect, the conjugate of the sixth aspect, or the pharmaceutical composition of the seventh aspect, for preventing and / or treating PSMA-mediated related diseases, or having the following uses: preventing and / or treating PSMA-mediated related diseases (especially preventing and / or treating prostate cancer); and / or preparing a medicament for preventing and / or treating PSMA-mediated related diseases.

[0018] In the tenth aspect of the present invention, the present invention provides the antibody or antigen-binding fragment of the first aspect, the multispecific antibody of the second aspect, the nucleic acid molecule of the third aspect, the expression vector of the fourth aspect, the recombinant cell of the fifth aspect, the conjugate of the sixth aspect, or the kit of the eighth aspect, in the preparation of a kit for detecting PSMA, detecting a PSMA-mediated related disease, diagnosing a PSMA-mediated related disease, staging a PSMA-mediated related disease, or evaluating the prognosis of a PSMA-mediated related disease, or having the following uses: detecting PSMA, and / or detecting a PSMA-mediated related disease, and / or diagnosing a PSMA-mediated related disease, and / or staging a PSMA-mediated related disease, and / or evaluating the prognosis of a PSMA-mediated related disease, and / or preparing a kit for detecting PSMA, and / or preparing a kit for detecting a PSMA-mediated related disease, and / or preparing a kit for diagnosing a PSMA-mediated related disease, and / or preparing a kit for staging a PSMA-mediated related disease, and / or preparing a kit for evaluating the prognosis of a PSMA-mediated related disease.

[0019] In its eleventh aspect, the present invention provides a method for detecting PSMA. According to embodiments of the present invention, the method comprises contacting the antibody or antigen-binding fragment described in the first aspect, the multispecific antibody described in the second aspect, or the conjugate described in the sixth aspect with a sample to be tested to form an immune complex. The method of the present invention can detect PSMA and further detect prostate cancer, particularly for in vitro detection, and has advantages such as high detection accuracy.

[0020] In its twelfth aspect, the present invention provides a method for preventing and / or treating PSMA-mediated diseases. According to embodiments of the present invention, the method comprises administering to a subject a pharmaceutically acceptable amount of the antibody or antigen-binding fragment of the first aspect, the multispecific antibody of the second aspect, the conjugate of the sixth aspect, or the pharmaceutical composition of the seventh aspect. The method of the present invention is effective for treating or preventing tumors and / or cancers, particularly prostate cancer.

[0021] In its thirteenth aspect, the present invention provides a method for diagnosing PSMA-mediated diseases. According to an embodiment of the present invention, the method comprises: detecting PSMA in a test sample using the antibody or antigen-binding fragment described in the first aspect, the multispecific antibody described in the second aspect, or the conjugate described in the sixth aspect; and determining the PSMA content in the test sample based on the PSMA detection results. The antibody or antigen-binding fragment, or nucleic acid molecule, expression vector, or antibody or antigen-binding fragment expressed by a recombinant cell proposed in the present invention can effectively bind to human PSMA protein. Therefore, the method of the present invention can effectively detect the PSMA content in a test sample from a test individual and effectively diagnose PSMA-induced diseases.

[0022] In its fourteenth aspect, the present invention provides a method for staging PSMA-mediated diseases. According to an embodiment of the present invention, the method comprises: detecting PSMA in a test sample using the antibody or antigen-binding fragment described in the first aspect, the multispecific antibody described in the second aspect, or the conjugate described in the sixth aspect; and determining the PSMA content in the test sample based on the PSMA detection results. The antibodies or antigen-binding fragments, or nucleic acid molecules, expression vectors, or antibodies or antigen-binding fragments expressed by recombinant cells proposed in the present invention can effectively bind to human PSMA protein. Therefore, the method of the present invention can effectively detect the PSMA content in a test sample from a test individual and assess the stage of a PSMA-induced disease based on the PSMA content.

[0023] In its fifteenth aspect, the present invention provides a method for assessing the prognosis of a PSMA-mediated disease. According to an embodiment of the present invention, the method comprises: using the antibody or antigen-binding fragment described in the first aspect to detect PSMA in a test sample for staging a PSMA-mediated disease; and determining the PSMA content in the test sample based on the PSMA detection results. As previously mentioned, PSMA content has a significant impact on cancer. After treatment, individuals with related diseases can effectively assess the prognosis of such diseases by monitoring the PSMA content in their tissues or excretions, such as peripheral blood or urine. For example, the PSMA content in the subject can be compared before and after treatment, or the PSMA content in the treated subject can be compared with the PSMA level in a healthy individual or a diseased individual. The antibody or antigen-binding fragment of the first aspect of the present invention, or the nucleic acid molecule, expression vector, or antibody or antigen-binding fragment expressed by a recombinant cell, can effectively bind to human PSMA. Therefore, the method can effectively detect the PSMA content in a test sample from a test individual and assess the prognosis of a PSMA-induced disease based on the PSMA content. Beneficial effects:

[0024] 1) The affinity-matured PSMA antibody obtained in the present invention has a slower dissociation rate (Kd) than the parent antibody.

[0025] 2) The affinity-matured PSMA antibody obtained in the present invention binds to tumor cells more strongly than the parent antibody.

[0026] 3) The affinity-matured PSMA antibody obtained in the present invention has stronger ADCC activity than the parent antibody.

[0027] 4) The CD3×PSMA bispecific antibody obtained in the present invention has stronger binding activity and promoting killing activity than the parent antibody.

[0028] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0030] FIG1 is a comparison diagram of the CDR sequences of the affinity-matured PSMAv11 antibody and the parental mAb according to Example 2 of the present invention;

[0031] FIG2 is a graph showing the SPR results of affinity determination of the affinity-matured PSMAv11 antibody and parental mAb protein according to Example 3 of the present invention;

[0032] FIG3 is a flow cytometry result showing the binding of affinity-matured PSMAv11 antibody and parental mAb to 22Rv1 human prostate cancer cells according to Example 5 of the present invention;

[0033] FIG4 is a flow cytometry result showing the binding of affinity-matured PSMAv11 antibody and parental mAb to LNCaP human prostate cancer cells according to Example 5 of the present invention;

[0034] FIG5 is a graph showing the results of affinity-matured PSMAv11 antibody and parental mAb promoting PBMC to kill LNCaP human prostate cancer cells according to Example 6 of the present invention;

[0035] FIG6 is a graph showing the results of affinity-matured PSMAv11 antibody and parental mAb promoting PBMC to kill 22Rv1 human prostate cancer cells according to Example 6 of the present invention;

[0036] FIG7 is a flow cytometry result showing the binding of the CD3×PSMAv11 antibody and the CD3×parental PSMA bispecific antibody according to Example 7 of the present invention to 22Rv1 human prostate cancer cells;

[0037] FIG8 is a flow cytometry result showing the binding of the CD3×PSMAv11 antibody and the CD3×parental PSMA bispecific antibody according to Example 7 of the present invention to LNCaP human prostate cancer cells;

[0038] FIG9 is a graph showing the results of CD3×PSMAv11 antibody and CD3×parental PSMA bispecific antibody according to Example 8 of the present invention promoting PBMC to kill LNCaP human prostate cancer cells;

[0039] FIG10 is a graph showing the results of CD3×PSMAv11 antibody and CD3×parental PSMA bispecific antibody according to Example 8 of the present invention promoting PBMC to kill 22Rv1 human prostate cancer cells. DETAILED DESCRIPTION

[0040] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0041] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0042] Detailed description of the invention

[0043] Definitions and General Terms

[0044] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention, but not excluding other contents.

[0045] As used herein, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0046] As used herein, the term "fragment" refers to a target protein or polypeptide, as well as a target protein or polypeptide with N-terminal (N-terminus) or C-terminal (C-terminus) truncation, and / or internal deletion.

[0047] To facilitate understanding of the present invention, certain technical and scientific terms are defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. The abbreviations for amino acid residues are the standard three-letter and / or one-letter codes used in the art to designate one of the 20 commonly used L-amino acids.

[0048] The antibodies or antigen-binding fragments described herein are typically prepared by biosynthetic methods. Based on the nucleotide sequences described herein, those skilled in the art can readily prepare the encoding nucleic acids of the present invention using various known methods. These methods include, but are not limited to, PCR and artificial DNA synthesis. For specific methods, see J. Sambrook, "Molecular Cloning: A Laboratory Manual." As one embodiment of the present invention, the encoding nucleic acid sequence of the present invention can be constructed by synthesizing a nucleotide sequence in segments and then performing overlap extension PCR. The antibodies or antigen fragments are numbered and defined using the Kabat numbering system.

[0049] As used herein, the terms "identity," "homology," or "similarity" are used to describe an amino acid sequence or nucleic acid sequence relative to a reference sequence, and the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences is determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Foundation, Washington, DC). There are many algorithms for aligning sequences and determining sequence identity, including the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 10:106); the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 10:116; the similarity search method of Pearson et al. (1990) Proc. Natl. Acad. Sci. 10:117; the similarity search method of Pearson et al. (1990 ... .70:173-187 (1997); and BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410). Computer programs that utilize these algorithms are also available and include, but are not limited to, ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth. Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altschul et al., supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, Version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0050] As used herein, the term "at least 80% identity" refers to at least 80%, and may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity to the respective reference sequence.

[0051] As used herein, the term "at least 90% identity" refers to at least 90%, and may be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity to the respective reference sequence.

[0052] In this article, the term "expression vector" generally refers to a nucleic acid molecule that can be inserted into a suitable host and replicates itself, and the nucleic acid molecule contains a nucleotide sequence that can express a target protein, and the nucleic acid molecule can be transferred into a host cell and / or between host cells. The expression vector may include a vector mainly used to insert DNA or RNA into a cell, a vector mainly used to replicate DNA or RNA, and a vector mainly used for expression of transcription and / or translation of DNA or RNA. The expression vector also includes vectors with multiple of the above functions. The expression vector can be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Generally, the expression vector can produce a desired expression product by culturing a suitable host cell containing the expression vector.

[0053] In this article, the term "recombinant cell" generally refers to a cell in which the genetic material of a host cell is modified or reorganized using genetic engineering techniques or cell fusion techniques to obtain a cell with a unique trait of stable inheritance. Wherein, the term "host cell" refers to a prokaryotic cell or eukaryotic cell into which a recombinant expression vector can be introduced. The terms "transformed" or "transfected" as used herein refer to the introduction of a nucleic acid (e.g., an expression vector) into a cell by various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequence of the present invention and can be used for the expression and / or secretion of a target protein. Examples of suitable host cells that can be used in the present invention include immortalized hybridoma cells, NS / 0 myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, Cap cells (cells derived from human amniotic fluid), and CoS cells.

[0054] As used herein, the term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any of the methods well known in the pharmaceutical art. All methods include the step of bringing the active ingredient into association with a carrier which constitutes one or more accessory ingredients. Generally, the composition is prepared by uniformly and thoroughly combining the active antibody or antigen-binding fragment with a liquid carrier, a finely divided solid carrier, or both.

[0055] As used herein, the term "pharmaceutically acceptable excipient" may include any solvent, solid excipient, diluent, or other liquid excipient, etc., as appropriate for the particular intended dosage form. Except to the extent that any conventional excipient is incompatible with the antibodies or antigen-binding fragments of the present invention, such as by producing any adverse biological effects or by interacting in a deleterious manner with any other component of the pharmaceutically acceptable composition, their use is contemplated by the present invention.

[0056] As used herein, the term "administer" refers to the introduction of a predetermined amount of a substance into a patient via a suitable route. The antibodies, antigen-binding fragments, multispecific antibodies, or pharmaceutical compositions of the present invention can be administered via any common route, as long as it reaches the desired tissue. Various modes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, and the like, but the present invention is not limited to these exemplified modes of administration. Preferably, the compositions of the present invention are administered via intravenous or subcutaneous injection.

[0057] As used herein, the term "treatment" refers to any agent used to obtain a desired pharmacological and / or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. "Treatment" as used herein covers diseases in mammals, particularly humans, and includes: (a) preventing the occurrence of a disease or condition in an individual who is susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, such as arresting the progression of the disease; or (c) alleviating the disease, such as alleviating the symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a drug or antibody or antigen-binding fragment to an individual to treat, cure, alleviate, improve, reduce or inhibit the individual's disease, including but not limited to administering a drug containing the antibody or antigen-binding fragment described herein to an individual in need.

[0058] Detailed description of the anti-novel coronavirus antibodies and their applications

[0059] The present invention provides an antibody or antigen-binding fragment, a multispecific antibody, a nucleic acid molecule, an expression vector, a recombinant cell, a conjugate, a pharmaceutical composition, a kit, and uses thereof, which are described in detail below.

[0060] Antibodies or antigen-binding fragments

[0061] In one aspect, the present invention provides an antibody or antigen-binding fragment. According to an embodiment of the present invention, the antibody or antigen-binding fragment comprises at least one of the following CDRs: HCDR 1: GYSFTX1NW, wherein X1 is S or H; HCDR 2: IYPGDSDT; HCDR 3: ARQTGFLWSSDLWGRGT; LCDR 1: X2QDISX3A, wherein X2 is S or P, and X3 is S or Y; LCDR 2: DASX4, wherein X4 is S or W; LCDR 3: QQFNSYPLX5, wherein X5 is T or S; and X1 being S, X2 being S, X3 being S, X4 being S, and X5 being T cannot be present at the same time. Compared to a wild-type antibody, the antibody or antigen-binding fragment has high PSMA binding affinity and tumor killing activity, and can be used to detect PSMA or to prevent and / or treat PSMA-mediated diseases.

[0062] In this article, the term "antibody" is used in the broadest sense, which can include full-length monoclonal antibodies, multispecific antibodies, and chimeric antibodies. The specific structure is not limited as long as they exhibit the desired biological activity. It usually includes a light chain with a lighter molecular weight and a heavy chain with a heavier molecular weight. The heavy chain (H chain) and the light chain (L chain) are connected by a disulfide bond to form an antibody molecule. Among them, the amino acid sequence of the amino terminal (N-terminal) of the peptide chain varies greatly and is called the variable region (V region); the carboxyl terminal (C-terminal) is relatively stable and varies very little, and is called the constant region (C region). The V regions of the L chain and H chain are called VL and VH, respectively.

[0063] Herein, the heavy chain complementary determining regions (heavy chain variable region CDRs) are referred to as "HCDRs" or "HCDRs," which include HCDR1 (also known as CDR-H1), HCDR2 (also known as CDR-H2), and HCDR3 (also known as CDR-H3); the light chain complementary determining regions (light chain variable region CDRs) are referred to as "LCDRs" or "LCDRs," which include LCDR1 (also known as CDR-L1), LCDR2 (also known as CDR-L2), and LCDR3 (also known as CDR-L3). Commonly used CDR definition schemes in the art include: Kabat definition, Chothia definition, IMGT definition, Contact definition, and AbM definition.

[0064] As used herein, the "Kabat definition" refers to the definition system described in Kabat et al., U. S. Pat. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). For the "Chothia definition," see Chothia et al., J Mol Biol 196:901-917 (1987). Exemplary defined CDRs are listed in Table A below. The definitions in different documents vary slightly. Given the variable region amino acid sequence of an antibody, one skilled in the art can routinely determine which residues comprise a specific CDR. It should be noted that the CDRs in the present invention include but are not limited to CDRs defined by other methods in Table A. CDRs determined based on the heavy chain variable region and light chain variable region disclosed in this application using other rules disclosed in the art also fall within the scope of protection of the present disclosure.

[0065] Table A: CDR Definition 1

[0066] 1 The numbering of all CDR definitions in Table A is according to the Kabat numbering system (see below), with amino acid numbers on the heavy chain indicated by "H+numbers" and amino acid numbers on the light chain indicated by "L+numbers".

[0067] 2 "AbM" as used in Table A with a lowercase "b" refers to CDRs defined by Oxford Molecular's "AbM" antibody modeling software.

[0068] 3 If both H35A and H35B are absent, CDR-H1 ends at position 35; if only H35A is present, CDR-H1 ends at position 35A; if both H35A and H35B are present, CDR-H1 ends at position 35B.

[0069] 4 If both H35A and H35B are absent, CDR-H1 ends at position 32; if only H35A is present, CDR-H1 ends at position 33; if both H35A and H35B are present, CDR-H1 ends at position 34.

[0070] 5 If both H35A and H35B are absent, CDR-H1 ends at position 33; if only H35A is present, CDR-H1 ends at position 34; if both H35A and H35B are present, CDR-H1 ends at position 35.

[0071] Kabat et al. also defined a numbering system applicable to the variable region sequence of any antibody. Those skilled in the art can clearly map the Kabat numbering system to any variable region sequence without relying on any experimental data other than the sequence itself. As described herein, "Kabat numbering" refers to numbering using the numbering system described in Kabat et al., U.S. Patent No. 2002, "Sequence of Proteins of Immunological Interest" (1983). The HCDRs and LCDRs of the antibodies or antigen-binding fragments of the present invention are numbered using the above numbering system. For specific numbering results, see Table A. It should be noted that the polypeptide sequences in the sequence listing and Table B of the present invention are numbered according to the Kabat numbering system. However, those skilled in the art are fully capable of converting the sequence Kabat numbering in the sequence listing to "HCDRs" and / or "LCDRs" under other numbering systems, all of which are within the scope of protection of the present invention.

[0072] As used herein, the term "antigen-binding fragment" refers to a fragment comprising a portion or all of an antibody that lacks at least some of the amino acids present in the full-length chain but is still capable of specifically binding to an antigen. For example, the fragment may comprise a portion or all of an antibody CDR. Such fragments are biologically active because they bind to an antigen and can compete with other antigen-binding molecules (including intact antibodies) for binding to a given epitope. Such fragments are selected from Fab, Fv, scFv, or single-domain antibodies. Such fragments can be produced by recombinant nucleic acid technology or can be produced by enzymatic or chemical cleavage of antigen-binding molecules (including intact antibodies).

[0073] According to an embodiment of the present invention, the antibody or antigen-binding fragment may further include at least one of the following technical features:

[0074] According to an embodiment of the present invention, X1 is S.

[0075] According to an embodiment of the present invention, X1 is H.

[0076] According to an embodiment of the present invention, X2 is S.

[0077] According to an embodiment of the present invention, X2 is P.

[0078] According to an embodiment of the present invention, X3 is S.

[0079] According to an embodiment of the present invention, X3 is Y.

[0080] According to an embodiment of the present invention, X4 is S.

[0081] According to an embodiment of the present invention, X4 is W.

[0082] According to an embodiment of the present invention, X5 is T.

[0083] According to an embodiment of the present invention, X5 is S.

[0084] In one embodiment of the present invention, the antibody or antigen-binding fragment comprises one of the following sets of CDRs:

[0085] According to an embodiment of the present invention, the antibody or antigen-binding fragment comprises: HCDR1, HCDR2, and HCDR3 as represented by the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively; and LCDR1, LCDR2, and LCDR3 as represented by the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively. The CDRs are the same as those in group 18 in the table above.

[0086] According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof specifically recognizes PSMA.

[0087] According to an embodiment of the present invention, the antibody or antigen-binding fragment includes a heavy chain framework region and / or a light chain framework region.

[0088] According to an embodiment of the present invention, at least a portion of the heavy chain framework region and / or the light chain framework region is derived from at least one of a mouse antibody, a primate antibody, a bovine antibody, a horse antibody, a cow antibody, a porcine antibody, a sheep antibody, a goat antibody, a dog antibody, a cat antibody, a rabbit antibody, a camel antibody, a donkey antibody, a deer antibody, a mink antibody, a chicken antibody, a duck antibody, a goose antibody, a turkey antibody, a fighting cock antibody, or a mutant thereof.

[0089] According to an embodiment of the present invention, at least a portion of the heavy chain framework region and / or the light chain framework region is derived from at least one of a murine antibody and a human antibody.

[0090] According to an embodiment of the present invention, the antibody or antigen-binding fragment comprises: a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 7 or an amino acid sequence having at least 90% homology thereto; and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 8 or an amino acid sequence having at least 90% homology thereto.

[0091] According to an embodiment of the present invention, the antibody or antigen-binding fragment further comprises a heavy chain constant region and / or a light chain constant region.

[0092] According to an embodiment of the present invention, at least a portion of at least one of the heavy chain constant region and the light chain constant region is derived from at least one of a mouse antibody, a primate antibody, a bovine antibody, a horse antibody, a cow antibody, a porcine antibody, a sheep antibody, a goat antibody, a dog antibody, a cat antibody, a rabbit antibody, a camel antibody, a donkey antibody, a deer antibody, a mink antibody, a chicken antibody, a duck antibody, a goose antibody, a turkey antibody, a fighting cock antibody, or a mutant thereof.

[0093] According to an embodiment of the present invention, the heavy chain constant region includes a heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD.

[0094] According to an embodiment of the present invention, the light chain constant region comprises a light chain constant region selected from a κ type or a λ type.

[0095] According to an embodiment of the present invention, the light chain constant region and the heavy chain constant region are both derived from a murine antibody or a mutant thereof, and / or a human antibody or a mutant thereof.

[0096] According to an embodiment of the present invention, the N-terminus of the heavy chain constant region is connected to the C-terminus of the heavy chain variable region; and / or the N-terminus of the light chain constant region is connected to the C-terminus of the light chain variable region.

[0097] According to an embodiment of the present invention, the heavy chain constant region includes the heavy chain constant region shown in SEQ ID NO:9 or an amino acid sequence having at least 80% identity therewith; and / or the light chain constant region includes the light chain constant region shown in SEQ ID NO:10 or an amino acid sequence having at least 80% identity therewith.

[0098] According to an embodiment of the present invention, the antibody includes at least one of a polyclonal antibody, a full-length monoclonal antibody, a Fab antibody, a Fab' antibody, a F(ab')2 antibody, a Fv antibody, a single-chain antibody, a single-domain antibody and a minimum recognition unit; or the antigen-binding fragment includes at least one of a F(ab')2 fragment, a Fab' fragment, a Fab fragment, a F(ab)2 fragment, a Fv fragment, a scFv fragment, a scFv-Fc fusion protein, a scFv-Fv fusion protein and a minimum recognition unit.

[0099] As used herein, the terms "full-length antibody," "full-length monoclonal antibody," or "full-length monoclonal antibody" are composed of at least two identical light chains and at least two identical heavy chains connected by interchain disulfide bonds, such as immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD), or immunoglobulin E (IgE).

[0100] In this article, the terms "polyantibody" and "multispecific antibody" are synonymous and refer to antibodies that can recognize multiple antigenic epitopes, such as antibodies that can recognize two antigenic epitopes (bispecific antibodies, abbreviated as bispecific antibodies), antibodies that recognize three antigenic epitopes, or antibodies that recognize four antigenic epitopes. This is understood in a broad sense and the specific structure is not limited, as long as it can recognize multiple antigenic epitopes. In the present invention, at least one of the multiple antigenic epitopes is derived from PSMA.

[0101] Herein, the terms "single domain antibody", "nanoantibody" and "VHH antibody" are used interchangeably, which were originally described as the antigen-binding immunoglobulin (variable) domains of "heavy chain antibodies" (i.e., "antibodies lacking light chains") (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: "Naturally occurring antibodies devoid of light chains"; Nature 363, 446-448 (1993)), comprising a heavy chain variable region (VH) and conventional CH2 and CH3 regions, which specifically bind to an antigen protein (e.g., PSMA) through the heavy chain variable region.

[0102] As used herein, the term "Fab antibody" or "Fab fragment" generally refers to an antibody or fragment containing only the Fab molecule, which is composed of the VH and CH1 of the heavy chain and a complete light chain, with the light chain and heavy chain connected by a disulfide bond.

[0103] As used herein, the term "F(ab')2 antibody" or "F(ab')2 fragment" has two antigen-binding F(ab') portions linked together by a disulfide bond.

[0104] As used herein, the term "Fv antibody" or "Fv fragment" generally refers to an antibody or fragment consisting solely of a light chain variable region (VL) and a heavy chain variable region (VH) linked by non-covalent bonds. It is the smallest functional fragment of an antibody that retains a complete antigen-binding site.

[0105] As used herein, the terms "single-chain antibody" and "scFv fragment" refer to antibodies or fragments formed by connecting the heavy chain variable region and the light chain variable region of an antibody via a short peptide.

[0106] In this article, the terms "minimum recognition unit" and "MRU" both refer to antibodies or fragments consisting of only one CDR, which has a very small molecular weight of only about 1% of the complete antibody.

[0107] According to an embodiment of the present invention, the antibody or antigen-binding fragment comprises: a heavy chain having an amino acid sequence as shown in SEQ ID NO: 11 or an amino acid sequence having at least 80% homology thereto; and a light chain having an amino acid sequence as shown in SEQ ID NO: 12 or an amino acid sequence having at least 80% homology thereto.

[0108] Multispecific antibodies

[0109] In its second aspect, the present invention provides a multispecific antibody. According to an embodiment of the present invention, the multispecific antibody comprises: a first binding region comprising the antibody or antigen-binding fragment described in the first aspect; and a second binding region having binding activity for a first molecule. The multispecific antibody of the present invention has high PSMA binding affinity and tumor killing potency, and can effectively prevent and / or treat PSMA-mediated diseases, particularly prostate cancer.

[0110] According to an embodiment of the present invention, the first molecule is selected from at least one of immune cell surface antigens, tumor antigens, viruses, bacteria, endotoxins, cytokines and cytokine receptors.

[0111] In this article, "immune cell surface antigens" should be understood in a broad sense and may refer to immunogenic proteins on the surface of immune cells (such as T cells, NK cells, B cells, etc.), including but not limited to CD3, BCMA, CTLA-4, LAG-3, TIGIT, CD38, SLAMF7, B7-H3, CD19, CD20, CD30, CD33, CD47, CD52, CD133, RANKL, and CD16a.

[0112] In this article, "tumor antigen" should be understood broadly and can refer to immunogenic proteins on the surface of tumor cells, including but not limited to PD-L1, PD-1, TGF-β, CEA, GD2, and CD3.

[0113] In this article, "cytokine" should be understood broadly, referring to a class of proteins or small molecule polypeptides that can transmit information between cells and have immune regulatory and effector functions. This includes but is not limited to IL-10, VEGF (including at least one of VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, and PIGF), EpCAM, GM2, and RANKL.

[0114] In this article, "cytokine receptor" should be understood in a broad sense, and may refer to receptors on the cell surface that can bind to cytokines, including but not limited to Her2, EGFR, and IL-10R.

[0115] According to an embodiment of the present invention, the first molecule is selected from at least one of CD3, PD-L1, PD-1, IL-10, IL-10R, BCMA, VEGF, TGF-β, CTLA-4, LAG-3, TIGIT, CEA, CD38, SLAMF7, B7-H3, Her2, EpCAM, CD19, CD20, CD30, CD33, CD47, CD52, CD133, EGFR, GD2, CD3, GM2, RANKL and CD16a.

[0116] According to an embodiment of the present invention, the second binding region is the binding protein of the first molecule or a fragment thereof.

[0117] According to an embodiment of the present invention, the second binding region is at least one of the antibody or antigen-binding fragment of the first molecule, and the receptor fragment of the first molecule.

[0118] According to an embodiment of the present invention, the second binding region is a single-chain antibody of the first molecule.

[0119] According to an embodiment of the present invention, the second binding region further includes a first Fc fragment.

[0120] According to an embodiment of the present invention, the first binding region further includes a second Fc fragment.

[0121] As used herein, unless otherwise specified, an Fc fragment comprises a CH2, a CH3 region, and optionally a hinge region, such as a first Fc fragment and / or a second Fc fragment. In one embodiment of the present invention, the C-terminus of the CH2 region is connected to the N-terminus of the CH3 region. In another embodiment of the present invention, the C-terminus of the hinge region is connected to the N-terminus of the CH2 region, and the C-terminus of the CH2 region is connected to the N-terminus of the CH3 region.

[0122] According to an embodiment of the present invention, the first Fc fragment and the second Fc fragment are both human Fc peptide segments.

[0123] According to an embodiment of the present invention, the human Fc peptide segment is a human IgG1 Fc peptide segment.

[0124] According to an embodiment of the present invention, the first Fc fragment and the second Fc fragment are connected via a knob-into-hole structure.

[0125] In one embodiment of the present invention, the "knob into hole structure" is a knob (knob) and a hole (hole) mutation formed in the CH3 region of the antibody heavy chain constant region to facilitate the heavy chain bite to form a heterodimer, for example, by mutating the amino acids in the CH3 domain of the human IgG1 heavy chain constant region (T366S, L368A, Y407V, and Y349C mutations in one chain, i.e., the "hole"; T366W and S354C mutations in the other chain, i.e., the "knob").

[0126] According to an embodiment of the present invention, the C-terminus of the binding protein or fragment thereof of the first molecule is connected to the N-terminus of the first Fc fragment.

[0127] It should be noted that, when the first molecule of the binding protein or its fragment is an antibody containing two chains, it is preferred that the peptide chain containing the heavy chain is connected to the first Fc fragment.

[0128] According to an embodiment of the present invention, the second binding region further includes a connecting peptide.

[0129] According to an embodiment of the present invention, the C-terminus of the binding protein or fragment thereof of the first molecule is connected to the N-terminus of the connecting peptide, and the C-terminus of the connecting peptide is connected to the N-terminus of the first Fc fragment.

[0130] According to an embodiment of the present invention, the connecting peptide has an amino acid sequence as shown in (GGGGS)n, wherein n is an integer greater than or equal to 1, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0131] According to an embodiment of the present invention, the bispecific antibody includes a symmetric bispecific antibody or an asymmetric bispecific antibody, preferably an asymmetric bispecific antibody.

[0132] According to an embodiment of the present invention, the first molecule is CD3, and the second binding region includes an anti-CD3 antibody.

[0133] According to an embodiment of the present invention, the anti-CD3 antibody is selected from at least one of Fab antibody, Fab' antibody, F(ab')2 antibody, Fv antibody, single-chain antibody, single-domain antibody and minimum recognition unit.

[0134] According to an embodiment of the present invention, the anti-CD3 antibody is a CD3 single-chain antibody.

[0135] According to an embodiment of the present invention, the C-terminus of the CD3 single-chain antibody is connected to the N-terminus of the connecting peptide, and the C-terminus of the connecting peptide is connected to the N-terminus of the first Fc fragment.

[0136] According to an embodiment of the present invention, the CD3 single-chain antibody has the amino acid sequence shown in SEQ ID NO:13.

[0137] According to an embodiment of the present invention, the connecting peptide has an amino acid sequence as shown in SEQ ID NO:14.

[0138] According to an embodiment of the present invention, the first Fc fragment has the amino acid sequence shown in SEQ ID NO:15.

[0139] According to an embodiment of the present invention, the first binding region has an amino acid sequence as shown in SEQ ID NO:17.

[0140] According to an embodiment of the present invention, the antibody or antigen-binding fragment in the first binding region is selected from at least one of Fab antibody, Fab' antibody, F(ab')2 antibody, Fv antibody, single-chain antibody, single-domain antibody and minimum recognition unit, preferably Fab antibody or single-chain antibody.

[0141] According to an embodiment of the present invention, the antibody or antigen-binding fragment in the first binding region is a Fab antibody.

[0142] According to an embodiment of the present invention, the Fab antibody in the first binding region includes the CDRs in the antibody or antigen-binding fragment described in the first aspect.

[0143] As can be seen from the above definition, the Fab antibody comprises two chains, namely the heavy chain variable region + CH1, and the light chain variable region + CL, wherein the CDRs in the heavy chain variable region and the light chain variable region are consistent with the CDRs defined in the antibody or antigen-binding fragment described in the first aspect.

[0144] According to an embodiment of the present invention, the Fab antibody in the first binding region includes the heavy chain variable region and the light chain variable region in the antibody or antigen-binding fragment described in the first aspect.

[0145] As can be seen from the previous definition, the Fab antibody contains two chains, namely the heavy chain variable region + CH1, and the light chain variable region + CL (that is, the light chain constant region), wherein the heavy chain variable region and the light chain variable region are consistent with the heavy chain variable region and the light chain variable region defined in the antibody or antigen-binding fragment described in the first aspect.

[0146] According to an embodiment of the present invention, the CH1 in the first binding region has an amino acid sequence as shown in SEQ ID NO: 22.

[0147] According to an embodiment of the present invention, the C-terminus of CH1 in the Fab antibody in the first binding region is connected to the N-terminus of the second Fc fragment.

[0148] According to an embodiment of the present invention, the second Fc fragment has the amino acid sequence shown in SEQ ID NO:16.

[0149] According to an embodiment of the present invention, the first binding region includes: a first peptide chain having an amino acid sequence as shown in SEQ ID NO: 18; and a second peptide chain having an amino acid sequence as shown in SEQ ID NO: 12.

[0150] According to an embodiment of the present invention, the bispecific antibody comprises: a first peptide chain having an amino acid sequence as shown in SEQ ID NO: 18; a second peptide chain having an amino acid sequence as shown in SEQ ID NO: 12; and a third peptide chain having an amino acid sequence as shown in SEQ ID NO: 17.

[0151] Nucleic acid molecules, expression vectors and recombinant cells

[0152] In a third aspect, the present invention provides a nucleic acid molecule. According to embodiments of the present invention, the nucleic acid molecule encodes the antibody or antigen-binding fragment described in the first aspect, or the multispecific antibody described in the second aspect. The nucleic acid molecule of the present invention may encode the antibody or antigen-binding fragment described in the first aspect, or the multispecific antibody described in the second aspect.

[0153] According to an embodiment of the present invention, the nucleic acid molecule is DNA.

[0154] It should be noted that, for nucleic acid molecules mentioned herein, those skilled in the art will understand that they actually include either or both of the complementary double strands. For convenience, in this specification and claims, although only one strand is provided in most cases, the other complementary strand is also disclosed. In addition, the nucleic acid sequences in the present invention include DNA or RNA forms, and disclosure of one of them implies disclosure of the other.

[0155] In a fourth aspect, the present invention provides an expression vector. According to an embodiment of the present invention, the expression vector carries the nucleic acid molecule described in the third aspect. When the nucleic acid molecule is linked to the expression vector, the nucleic acid molecule can be directly or indirectly linked to control elements on the expression vector, as long as these control elements are capable of controlling translation and expression of the nucleic acid molecule. Of course, these control elements can be directly derived from the expression vector itself or exogenous, that is, not derived from the expression vector itself. Of course, the nucleic acid molecule and the control elements can be operably linked.

[0156] As used herein, "operably linked" means that a foreign gene is linked to an expression vector so that the control elements within the expression vector, such as transcriptional control sequences and translational control sequences, can function as intended to regulate the transcription and translation of the foreign gene. Commonly used expression vectors include plasmids, bacteriophages, and the like.

[0157] According to some specific embodiments of the present invention, after the expression vector is introduced into suitable recipient cells, it can effectively achieve the expression of the aforementioned antibody or antigen-binding fragment, the aforementioned recombinant protein or the aforementioned multispecific antibody under the mediation of the regulatory system, thereby achieving large-scale in vitro acquisition of the antibody or antigen-binding fragment, recombinant protein or multispecific antibody.

[0158] According to an embodiment of the present invention, the expression vector is selected from a eukaryotic expression vector or a prokaryotic expression vector.

[0159] In an optional embodiment of the present invention, the expression vector is a plasmid expression vector or a viral expression vector, such as a lentiviral expression vector.

[0160] In its fifth aspect, the present invention provides a recombinant cell. According to embodiments of the present invention, the recombinant cell comprises a nucleic acid molecule as described in the third aspect or an expression vector as described in the fourth aspect; or expresses the antibody or antigen-binding fragment as described in the first aspect, or the multispecific antibody as described in the second aspect. Under suitable conditions, the recombinant cell can effectively express the aforementioned antibody or antigen-binding fragment or multispecific antibody within the cell.

[0161] According to some specific embodiments of the present invention, the recombinant cells can efficiently and massively express antibodies or antigen-binding fragments thereof under appropriate conditions. The antibodies or antigen-binding fragments thereof have stronger specificity, longer half-life, and higher efficacy, and can deliver antibody drugs to target cells at a lower dosage, thereby achieving effective treatment or prevention of PSMA-mediated diseases, with low toxic side effects and higher safety.

[0162] It should be noted that "suitable conditions" refer to conditions suitable for the expression of the antibodies or antigen-binding fragments, recombinant proteins, or multispecific antibodies of the present invention. Those skilled in the art will readily appreciate that conditions suitable for the expression of the antibodies or antigen-binding fragments, recombinant proteins, or multispecific antibodies include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell status, suitable host cell density, suitable cell culture environment, and suitable cell culture time. "Suitable conditions" are not particularly limited, and those skilled in the art can optimize the most suitable conditions for the expression of the above-mentioned antibodies or antigen-binding fragments, recombinant proteins, or multispecific antibodies based on the specific laboratory environment.

[0163] According to an embodiment of the present invention, the recombinant cell is obtained by introducing the expression vector described in the fourth aspect into a host cell.

[0164] It should be noted that the recombinant cells of the present invention are not particularly limited and may be prokaryotic cells, eukaryotic cells, or bacteriophages. The prokaryotic cells may be Escherichia coli, Bacillus subtilis, Streptomyces, or Proteus mirabilis, among others. The eukaryotic cells include fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Trichoderma, insect cells such as fall armyworms, plant cells such as tobacco, and mammalian cells such as BHK cells, CHO cells, COS cells, and myeloma cells. In some embodiments, the recombinant cells of the present invention are preferably mammalian cells, including BHK cells, CHO cells, NSO cells, or COS cells, and do not include animal germ cells, fertilized eggs, or embryonic stem cells.

[0165] According to an embodiment of the present invention, the recombinant cell is a eukaryotic cell, preferably a mammalian cell.

[0166] Conjugates, pharmaceutical compositions and kits

[0167] In its sixth aspect, the present invention provides a conjugate. According to an embodiment of the present invention, the conjugate comprises the antibody or antigen-binding fragment described in the first aspect, or the multispecific antibody described in the second aspect; and a coupling moiety, wherein the coupling moiety is linked to the antibody, antigen-binding fragment, or multispecific antibody. The conjugate of the present invention has high PSMA binding affinity and can be used to detect PSMA, or to prevent and / or treat PSMA-mediated diseases, particularly for detecting prostate cancer, or preventing and / or treating prostate cancer.

[0168] According to an embodiment of the present invention, the coupling portion includes at least one selected from a carrier, a drug, a toxin, a cytokine, a protein tag and a modifier.

[0169] Herein, the carrier can be a substance that can be suspended or dispersed in a liquid phase (e.g., solid phase carriers such as particles and magnetic beads), or a solid phase that can contain or carry a liquid phase (e.g., supports such as plates, membranes, test tubes, and containers such as well plates, microfluidics, glass capillaries, nanocolumns, and monolithic columns); it can also be a labeling carrier for labeling antibodies or antigen-binding fragments, recombinant proteins, or multispecific antibodies, such as an enzyme (e.g., peroxidase, alkaline phosphatase, luciferin, β-galactosidase), an affinity substance (e.g., one of streptavidin and biotin, one of mutually complementary sense and antisense nucleic acids), a fluorescent substance (e.g., fluorescein, fluorescein isothiocyanate, rhodamine, green fluorescent protein, red fluorescent protein), a luminescent substance (e.g., luciferin, aequorin, acridinium ester, tris(2,2'-bipyridine)ruthenium, luminol), a radioactive isotope (e.g., 3 H. 14 C. 32 P. 35 S. 125 I) and gold colloid, etc.

[0170] According to an embodiment of the present invention, the drug is a small molecule drug that can bind to an antibody or antigen-binding fragment, a recombinant protein or a multispecific antibody.

[0171] According to an embodiment of the present invention, the protein tag includes but is not limited to a His tag, a Flag tag, a GST tag, an MBP tag, a SUMO tag, and a C-Myc tag.

[0172] According to an embodiment of the present invention, the modifier should be understood in a broad sense and may refer to a substance used to modify a protein, for example, polyethylene glycol or a derivative thereof.

[0173] It should be noted that methods known in the art can be used to bind the conjugated moiety to the antibody, antigen-binding fragment, recombinant protein, or multispecific antibody. Examples include physical adsorption, covalent binding, methods using affinity substances (e.g., biotin, streptavidin), and ion binding.

[0174] In its seventh aspect, the present invention provides a pharmaceutical composition. According to embodiments of the present invention, the pharmaceutical composition comprises the antibody or antigen-binding fragment described in the first aspect, the multispecific antibody described in the second aspect, the nucleic acid molecule described in the third aspect, the expression vector described in the fourth aspect, the recombinant cell described in the fifth aspect, or the conjugate described in the sixth aspect. The pharmaceutical composition of the present invention has high PSMA binding affinity and tumor killing potency, and can effectively prevent and / or treat PSMA-mediated diseases, particularly prostate cancer.

[0175] According to an embodiment of the present invention, the pharmaceutical composition further includes a pharmaceutically acceptable excipient.

[0176] Administration of the pharmaceutical compositions of the present invention can be carried out by any acceptable mode of administration. The pharmaceutical compositions of the present invention can be formulated into solid, semisolid, liquid, or gaseous dosage forms, such as injections or lyophilized powders. Current methods for preparing these dosage forms are known or will be apparent to those skilled in the art. Typical routes of administration of such pharmaceutical compositions include, but are not limited to, subcutaneous injection, intravenous, intramuscular, intradermal, intrasternal injection, or infusion techniques. The pharmaceutical compositions of the present invention are formulated so as to allow the bioactive ingredients contained therein to be bioavailable upon administration of the composition to a patient.

[0177] In its eighth aspect, the present invention provides a kit. According to embodiments of the present invention, the kit comprises the antibody or antigen-binding fragment described in the first aspect, the multispecific antibody described in the second aspect, the nucleic acid molecule described in the third aspect, the expression vector described in the fourth aspect, the recombinant cell described in the fifth aspect, or the conjugate described in the sixth aspect. The kit of the present invention has high PSMA binding affinity, can effectively detect PSMA, and can further diagnose tumors or cancers (particularly prostate cancer).

[0178] As previously described, the antibodies or antigen-binding fragments of certain embodiments of the present invention can effectively bind to human PSMA protein. Therefore, kits containing such antibodies or antigen-binding fragments can effectively detect human PSMA protein qualitatively or quantitatively. The kits provided herein can be used, for example, for immunoblotting, immunoprecipitation, and other assays that utilize the specific binding of human PSMA to antibodies. These kits may include any one or more of the following: antagonists, anti-PSMA antibodies, or reference drug materials; protein purification columns; immunoglobulin affinity purification buffers; cell assay diluents; instructions or literature, etc. Anti-PSMA antibodies can be used in various diagnostic tests, such as in vitro or in vivo detection of various diseases or the presence of drugs, toxins, or other proteins. For example, these antibodies can be tested in serum or blood of subjects for related diseases. The kits can also be used for scientific research, using the kits to detect human PSMA protein in test samples. Such related diseases can include PSMA-related diseases, such as cancer. Of course, the antibodies or antigen-binding fragments provided herein can also be used for radioimmunoassays and radioimmunotherapy for the aforementioned diseases. The binding molecules described above are also applicable to these applications and will not be further elaborated here.

[0179] According to some specific embodiments of the present invention, the kit may further include conventional materials for detecting PSMA, such as coating fluid.

[0180] use

[0181] In the ninth aspect of the present invention, the present invention provides the antibody or antigen-binding fragment of the first aspect, the multispecific antibody of the second aspect, the nucleic acid molecule of the third aspect, the expression vector of the fourth aspect, the recombinant cell of the fifth aspect, the conjugate of the sixth aspect, or the pharmaceutical composition of the seventh aspect, for use in preventing and / or treating PSMA-mediated related diseases, or having the following uses: for preventing and / or treating PSMA-mediated related diseases (especially preventing and / or treating prostate cancer); and / or for preparing a medicament for preventing and / or treating PSMA-mediated related diseases.

[0182] According to an embodiment of the present invention, the PSMA-mediated related diseases include tumors or cancers

[0183] According to an embodiment of the present invention, the cancer is at least one of prostate cancer, lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma and head and neck cancer.

[0184] According to an embodiment of the present invention, the cancer is prostate cancer.

[0185] In the tenth aspect of the present invention, the present invention provides the antibody or antigen-binding fragment of the first aspect, the multispecific antibody of the second aspect, the nucleic acid molecule of the third aspect, the expression vector of the fourth aspect, the recombinant cell of the fifth aspect, the conjugate of the sixth aspect, or the kit of the eighth aspect, for detecting PSMA, detecting a PSMA-mediated related disease, diagnosing a PSMA-mediated related disease, staging a PSMA-mediated related disease, or evaluating the prognosis of a PSMA-mediated related disease, or having the following uses: detecting PSMA, and / or detecting a PSMA-mediated related disease, and / or diagnosing a PSMA-mediated related disease, and / or staging a PSMA-mediated related disease, and / or evaluating the prognosis of a PSMA-mediated related disease, and / or preparing a kit for detecting PSMA, and / or preparing a kit for detecting a PSMA-mediated related disease, and / or preparing a kit for diagnosing a PSMA-mediated related disease, and / or preparing a kit for staging a PSMA-mediated related disease, and / or preparing a kit for evaluating the prognosis of a PSMA-mediated related disease.

[0186] According to an embodiment of the present invention, the PSMA-mediated related diseases include tumors and / or cancers.

[0187] According to an embodiment of the present invention, the cancer is at least one of prostate cancer, lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma and head and neck cancer.

[0188] According to an embodiment of the present invention, the cancer is prostate cancer.

[0189] method

[0190] In its eleventh aspect, the present invention provides a method for detecting PSMA. According to embodiments of the present invention, the method comprises contacting the antibody or antigen-binding fragment described in the first aspect, the multispecific antibody described in the second aspect, or the conjugate described in the sixth aspect with a sample to be tested to form an immune complex. The method of the present invention can detect PSMA and further detect prostate cancer, particularly for in vitro detection, and has advantages such as high detection accuracy.

[0191] According to an embodiment of the present invention, based on the signal of the immune complex, it is determined whether the sample to be tested contains the content of the new coronavirus.

[0192] According to an embodiment of the present invention, the immune complex further comprises a second antibody, which binds to the antibody or antigen-binding fragment.

[0193] According to an embodiment of the present invention, the immune complex further includes a second antibody, which binds to the new coronavirus.

[0194] In its twelfth aspect, the present invention provides a method for preventing and / or treating PSMA-mediated diseases. According to embodiments of the present invention, the method comprises administering to a subject a pharmaceutically acceptable amount of the antibody or antigen-binding fragment of the first aspect, the multispecific antibody of the second aspect, the conjugate of the sixth aspect, or the pharmaceutical composition of the seventh aspect. The method of the present invention is effective for treating or preventing tumors and / or cancers, particularly prostate cancer.

[0195] The effective amount of the antibody or antigen-binding fragment, multispecific antibody, pharmaceutical composition or conjugate of the present invention may vary depending on the mode of administration and the severity of the disease to be treated. The selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials). Such factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated, the patient's weight, the patient's immune status, the route of administration, etc. For example, depending on the urgency of the treatment condition, several divided doses may be administered daily, or the dose may be reduced proportionally.

[0196] The antibodies or antigen-binding fragments, multispecific antibodies, pharmaceutical compositions or conjugates of the present invention can be incorporated into drugs suitable for parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). These drugs can be prepared in various forms. For example, liquid, semisolid and solid dosage forms, including but not limited to liquid solutions (e.g., injection solutions and infusion solutions) or lyophilized powders. Typical drugs are in the form of injection solutions or infusion solutions. The aforementioned antibodies or antigen-binding fragments, multispecific antibodies, pharmaceutical compositions or conjugates can be administered by intravenous infusion or injection or intramuscular or subcutaneous injection.

[0197] As used herein, the term "subject" refers to a vertebrate, preferably a mammal, most preferably a human. Mammals include, but are not limited to, rodents, apes, humans, livestock, athletic animals, and pets. Tissues, cells, and progeny of biological entities obtained in vivo or cultured in vitro are also included.

[0198] According to an embodiment of the present invention, the administration route of the method is subcutaneous injection or intravenous injection.

[0199] According to an embodiment of the present invention, the cancer is at least one of prostate cancer, lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma and head and neck cancer.

[0200] In its thirteenth aspect, the present invention provides a method for diagnosing PSMA-mediated diseases. According to an embodiment of the present invention, the method comprises: detecting PSMA in a test sample using the antibody or antigen-binding fragment described in the first aspect, the multispecific antibody described in the second aspect, or the conjugate described in the sixth aspect; and determining the PSMA content in the test sample based on the PSMA detection results. The antibody or antigen-binding fragment, or nucleic acid molecule, expression vector, or antibody or antigen-binding fragment expressed by a recombinant cell proposed in the present invention can effectively bind to human PSMA protein. Therefore, the method of the present invention can effectively detect the PSMA content in a test sample from a test individual and effectively diagnose PSMA-induced diseases.

[0201] According to an embodiment of the present invention, the above-mentioned method for diagnosing a disease may further include at least one of the following additional technical features:

[0202] According to an embodiment of the present invention, the PSMA content in the test sample being no less than a minimum disease threshold indicates that the test sample is derived from a patient suffering from a PSMA-related disease. The minimum threshold value can be determined by performing a comparative analysis and verification of the PSMA content in test samples from a large number of individuals suffering from the PSMA-related disease and a large number of healthy individuals.

[0203] According to an embodiment of the present invention, the sample to be tested includes at least one of the following: blood, saliva, sweat, tissue, cells, blood, serum, plasma, feces and urine.

[0204] According to an embodiment of the present invention, the PSMA-mediated related diseases include cancer.

[0205] According to an embodiment of the present invention, the cancer is at least one of prostate cancer, lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma and head and neck cancer.

[0206] In its fourteenth aspect, the present invention provides a method for staging PSMA-mediated diseases. According to an embodiment of the present invention, the method comprises: detecting PSMA in a test sample using the antibody or antigen-binding fragment described in the first aspect, the multispecific antibody described in the second aspect, or the conjugate described in the sixth aspect; and determining the PSMA content in the test sample based on the PSMA detection results. The antibodies or antigen-binding fragments, or nucleic acid molecules, expression vectors, or antibodies or antigen-binding fragments expressed by recombinant cells proposed in the present invention can effectively bind to human PSMA protein. Therefore, the method of the present invention can effectively detect the PSMA content in a test sample from a test individual and assess the stage of a PSMA-induced disease based on the PSMA content.

[0207] According to an embodiment of the present invention, the above-mentioned method for staging a disease may further include at least one of the following additional technical features:

[0208] :

[0209] According to an embodiment of the present invention, a PSMA content in the test sample that is not less than the standard level for stage IV tumor disease indicates that the test sample is derived from a patient with stage IV tumor; a PSMA content in the test sample that is between the standard levels for stage IV and stage III tumor disease indicates that the test sample is derived from a patient with stage III tumor; a PSMA content in the test sample that is between the standard levels for stage III and stage II tumor disease indicates that the test sample is derived from a patient with stage II tumor; and a PSMA content in the test sample that is between the standard levels for stage I and stage II tumor disease indicates that the test sample is derived from a patient with stage I tumor. It will be understood by those skilled in the art that the PSMA levels at stage I, II, III, and IV tumor disease vary depending on the type of tumor, and the stage of the tumor can be determined by comparing the PSMA content in the test sample with the standard level of PSMA corresponding to the tumor stage, or by comparing the PSMA content in the test sample with the PSMA content in samples from individuals or groups with known disease stages. The values ​​of the standard levels of tumor stages I, II, III, and IV can be determined by comparative analysis and verification of the differences in PSMA content in test samples from a large number of individuals suffering from PSMA-related diseases and a large number of healthy individuals.

[0210] According to an embodiment of the present invention, the sample to be tested includes at least one of the following: blood, saliva, sweat, tissue, cells, blood, serum, plasma, feces and urine.

[0211] According to an embodiment of the present invention, the PSMA-mediated related diseases include cancer.

[0212] According to an embodiment of the present invention, the cancer is at least one of prostate cancer, lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma and head and neck cancer.

[0213] In its fifteenth aspect, the present invention provides a method for assessing the prognosis of a PSMA-mediated disease. According to an embodiment of the present invention, the method comprises: using the antibody or antigen-binding fragment described in the first aspect to detect PSMA in a test sample for staging a PSMA-mediated disease; and determining the PSMA content in the test sample based on the PSMA detection results. As previously mentioned, PSMA content has a significant impact on cancer. After treatment, individuals with related diseases can effectively assess the prognosis of such diseases by monitoring the PSMA content in their tissues or excretions, such as peripheral blood or urine. For example, the PSMA content in the subject can be compared before and after treatment, or the PSMA content in the treated subject can be compared with the PSMA level in a healthy individual or a diseased individual. The antibody or antigen-binding fragment of the first aspect of the present invention, or the nucleic acid molecule, expression vector, or antibody or antigen-binding fragment expressed by a recombinant cell, can effectively bind to human PSMA. Therefore, the method can effectively detect the PSMA content in a test sample from a test individual and assess the prognosis of a PSMA-induced disease based on the PSMA content.

[0214] According to an embodiment of the present invention, the above method for evaluating disease prognosis may further include at least one of the following additional technical features:

[0215] :

[0216] According to an embodiment of the present invention, the sample to be tested is derived from a patient suffering from a PSMA-mediated disease before or after treatment.

[0217] According to an embodiment of the present invention, the sample to be tested includes at least one of the following: blood, saliva, sweat, tissue, cells, blood, serum, plasma, feces and urine.

[0218] According to an embodiment of the present invention, the prognostic effect of a PSMA-mediated related disease is determined based on the PSMA content in a test sample of a patient suffering from a PSMA-mediated related disease before or after treatment.

[0219] According to an embodiment of the present invention, the PSMA-mediated related diseases include cancer.

[0220] According to an embodiment of the present invention, the cancer is at least one of prostate cancer, lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma and head and neck cancer.

[0221] The amino acid sequences involved in this article are shown in Table B:

[0222] Table B: Amino acid sequences

[0223] Below, the scheme of the present invention will be explained in conjunction with embodiment.It will be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.In the embodiment, if specific technology or conditions are not indicated, the technology or conditions described in the literature in this area or the product instructions are used.The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be obtained by commercial purchase.

[0224] Practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a skilled artisan. The technique is fully explained in the literature, for example, in Molecular Cloning: A Laboratory Manual, 2nd ed. (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); and PCR: The Polymerase Chain Reaction. Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (J.E. Colligan et al., eds., 2011), each of which is expressly incorporated herein by reference.

[0225] In the embodiments of the present invention, the nucleotide sequence used to prepare the expression vector can be obtained according to its amino acid sequence using conventional methods or conventional software (such as the online program Vectorbuilder (website: https: / / www.vectorbuilder.cn / tool / codon-optimization.html), GeneOptimizer online program, etc.).

[0226] Example 1: Preparation of antibodies

[0227] The steps for preparing the antibody are as follows:

[0228] (1) ExpiCHO cells (purchased from Thermo Fisher) were cultured using ExpiCHO Expression Medium (purchased from Thermo Fisher) and the cell concentration was adjusted to 6×10 6 / mL to obtain ExpiCHO cell solution.

[0229] (2) When the antibody is a monoclonal antibody, a pcDNA3.4 vector (commissioned by Nanjing GenScript for synthesis) containing nucleotide sequences encoding the antibody heavy chain and antibody light chain was added to 2 mL of OptiSFM medium (purchased from Thermo Fisher) at a ratio of 1:1 to obtain solution A;

[0230] When the antibody is a bispecific antibody, a pcDNA3.4 vector (commissioned to Nanjing GenScript for synthesis) containing nucleotide sequences encoding CD3 antibody, PSMA antibody heavy chain, and PSMA antibody light chain was added to 2 mL of OptiSFM medium (purchased from Thermo Fisher) at a ratio of 1:1:1 to obtain solution A.

[0231] (3) Add 160 μL of ExpiFectamine CHO transfection reagent (purchased from Thermo Fisher) to 2 mL of OptiSFM culture medium (purchased from Thermo Fisher) to obtain solution B.

[0232] (4) Solution A and Solution B were then mixed to obtain a transfection mixture, and the entire transfection mixture was added to 50 mL of the ExpiCHO cell solution within 5 minutes.

[0233] (5) After culturing at 37°C and 5% CO2 for 1 day, 8 mL of feed and 300 μL of Enhancer (purchased from Thermo Fisher) were added, and the cells were transferred to 32°C and 5% CO2 for 9 days. The culture supernatant was harvested, with 8 mL of feed added on the 5th day.

[0234] (6) The target antibody was obtained by affinity purification from the culture supernatant using a Protein A purification column (purchased from NanoMicro).

[0235] Example 2: Antibody affinity maturation

[0236] During affinity maturation of natural antibodies, somatic high-frequency mutations are primarily concentrated in the CDR region. By performing single-point saturation mutagenesis at each site in the CDR region in vitro, sufficient mutational diversity can be achieved without disrupting the protein structure. This approach can achieve in vitro reproduction that is most similar to the somatic high-frequency mutations of natural antibodies in vivo.

[0237] Single-site saturation mutagenesis was performed at every amino acid site in the CDR region to construct an unbiased single-site saturation mutagenesis plasmid library of the parent antibody. Mutation sites that enhanced antigen-specific binding were screened using ELISA, and these sites were then combined to generate candidate antibody mutation sequences.

[0238] This technology was used to optimize affinity for the parental PSMA mAb (prepared by the method described in Example 1, with a heavy chain variable region of SEQ ID NO:23 and a light chain variable region of SEQ ID NO:24, the heavy chain amino acid sequence of which is shown in SEQ ID NO:19, and the light chain amino acid sequence of which is shown in SEQ ID NO:20). This resulted in the slower-dissociating PSMA monoclonal antibody, PSMAv11 (prepared by the method described in Example 1, with a heavy chain amino acid sequence of which is shown in SEQ ID NO:11, and a light chain amino acid sequence of which is shown in SEQ ID NO:12). This antibody has the sequences of a heavy chain variable region (amino acid sequence of which is shown in SEQ ID NO:7) and a light chain variable region (amino acid sequence of which is shown in SEQ ID NO:8). A comparison of the CDR sequences of the parental mAb and the affinity-matured antibody, PSMAv11, is shown in Figure 1.

[0239] Furthermore, the parental mAb antibody and PSMAvll antibody prepared in this example were used to carry out the following examples 3 to 6:

[0240] Example 3: Antibody affinity detection

[0241] Biacore is a method for analyzing biomolecular interactions based on the principle of optical surface plasmon resonance (SPR). It can not only detect the specific binding between antigens and antibodies, but also obtain intermolecular association rate constant (Ka), dissociation rate constant (Kd), equilibrium dissociation constant (KD) and other very important data in drug development, thereby calculating the affinity of the antibody.

[0242] In a Biacore 1K (Cytiva) system, the antibody was diluted to 10 μg / mL in HBS-EP running buffer and coupled to a protein A (Cytiva, 29127556) chip at a flow rate of 10 μL / min. The kinetics and affinity data of antigen-antibody binding were measured at a flow rate of 30 μL / min, with an association time of 120 s and a dissociation time of 800 s.

[0243] The binding kinetics and affinity data of the parental mAb and the affinity-optimized antibody PSMAvll to PSMA were tested. The results are shown in Figure 2. Compared with the parental mAb, the affinity-optimized antibody PSMAvll had an approximately 1.8-fold increased affinity for PSMA and a 4.6-fold slower dissociation rate.

[0244] Note: Ka represents the association rate constant (the larger the value, the stronger the affinity); Kd represents the dissociation rate constant (the smaller the value, the stronger the affinity), which reflects the affinity of the compound for the target; KD represents Kd / Ka, which is the equilibrium dissociation constant (affinity constant). The smaller the KD, the less dissociation, which means the stronger the affinity.

[0245] Example 4: PSMA Antibody ELISA Binding Assay

[0246] The binding properties of the parental mAb and affinity-optimized PSMAvll antibody to PSMA were determined using ELISA. The inventors coated PSMA protein onto 96-well plates and used the signal strength after antibody addition to determine the binding properties of the antibody to PSMA.

[0247] PSMA protein (purchased from Acro) was diluted to 1 μg / ml in PBS buffer and plated at 100 μL / well in a 96-well plate. The plate was incubated at 4°C overnight. The PBS buffer was aspirated from the 96-well plate, and the plate was washed six times with PBST (PBS, pH 7.2, containing 0.1% Tween 20). The plate was then blocked with 200 μL / well of PBS containing 10% BSA and incubated at 37°C for 2 h. The blocking buffer was removed, and the plate was washed six times with PBST. Then, 100 μL / well of the parental mAb, affinity-optimized PSMAv11 antibody, and control IgG1 (purchased from Bio-Bio) serially diluted in PBST containing 0.05% BSA (maximum working concentration 20,000 ng / ml, 5-fold dilutions, 8 steps) were added and incubated at 37°C for 1 h. Aspirate the reaction mixture from the wells, wash the plate six times with PBST, and then add 100 μL / well of HRP (horseradish peroxidase)-labeled anti-human secondary antibody (Fab specific) (purchased from Sigma) diluted in PBST containing 0.05% BSA. Incubate at 37°C for 1 hour. Aspirate the secondary antibody, wash the plate six times with PBST, and add 80 μL / well of TMB (tetramethylbenzidine). Incubate at room temperature for 3 minutes. Terminate the reaction by adding 80 μL / well of 4 M sulfuric acid. Read the absorbance at 450 nm using a microplate reader.

[0248] The results showed that the affinity-optimized antibody PSMAv11 of the present invention can bind to the PSMA protein.

[0249] Example 5: PSMA Antibody Flow Cytometry Binding Experiment

[0250] Tumor cells (22RV1 or LNCaP human prostate cancer) were diluted to 2×10 6 / mL, 100μL / tube were added to 1.5ml EP tubes. 10μL / tube of goat serum was added and the cells were blocked at 4°C for 30 min. A serial dilution (maximum working concentration: 50μg / mL, 5-fold dilution, 10 steps) of the parental mAb, affinity-optimized PSMAv11 antibody, and control IgG1 (purchased from Bio-Bio) were added and incubated at 4°C for 30 min. 1mL of PBS was added to the EP tubes and the cells were centrifuged at 3500 rpm for 5 min at 4°C. The supernatant was discarded and the tubes were washed once with PBS. After centrifugation, the supernatant was discarded and the cells were resuspended in 100μL / tube of PBS. 1μL / tube of Alexa-647-labeled goat anti-human secondary antibody (purchased from Jackson Labs) was added and the cells were incubated at 4°C for 30 min in the dark. The cells were washed twice with PBS and centrifuged and the supernatant was discarded. The cells were resuspended in 200μL / tube of PBS and analyzed by flow cytometry.

[0251] The results are shown in Figures 3 and 4, which show that the affinity-optimized antibody PSMAv11 of the present invention binds to 22RV1 and LNCaP human prostate cancer cells with higher fluorescence intensity than the parent antibody, indicating that the affinity-optimized antibody PSMAv11 has higher binding activity.

[0252] Example 6: PSMA Antibody Promotes PBMC to Kill Tumor Cells

[0253] The ability of PSMA monoclonal antibody to promote PBMC to kill 22RV1 and LNCaP human prostate cancer cells was detected.

[0254] (1) Add complete RPMI-1640 medium to a 16-well RTCA plate at a volume of 50 μL / well and calibrate the plate.

[0255] (2) Dilute the tumor cells to 2×10 5 / mL, and added separately to the RTCA plate obtained in step (1) at a volume of 50 μL / well, and then the cell coefficient was detected using the xCELLigence RTCA MP device at 37°C and 5% CO2 for 24 h;

[0256] (3) Using complete RPMI-1640 medium, the parental mAb and affinity-optimized antibody PSMAvll were serially diluted and added to the RTCA plate obtained in step (2) at a volume of 20 μL / well;

[0257] (4) PBMC (purchased from Saili Bio) were diluted to 1.25×10 6 pcs / mL, added to the RTCA plate obtained in step (3), with an addition volume of 80 μL / well;

[0258] (5) The reaction system obtained in step (4) was incubated at 37° C. and 5% CO 2 for 24 h to detect the cell coefficient using an xCELLigence RTCA MP device.

[0259] As shown in Figures 5 and 6, the PSMAv11 antibody of the present invention promotes PBMC to kill tumors, and its killing function is stronger than that of the parental mAb.

[0260] Example 7: CD3×PSMA Antibody Flow Cytometry Binding Experiment

[0261] According to the method of Example 1, an experimental group of CD3×PSMAv11 bispecific antibodies (wherein the amino acid sequence of the CD3 antibody is shown in SEQ ID NO: 17, the amino acid sequence of the heavy chain is shown in SEQ ID NO: 18, and the amino acid sequence of the light chain is shown in SEQ ID NO: 12) and a control group of CD3×parental mAb bispecific antibodies (wherein the amino acid sequence of the CD3 antibody is shown in SEQ ID NO: 17, the amino acid sequence of the heavy chain is shown in SEQ ID NO: 21, and the amino acid sequence of the light chain is shown in SEQ ID NO: 20) were prepared. Flow cytometry binding experiments were then performed. The specific experimental steps were as follows:

[0262] Tumor cells were diluted to 2 × 10 6 / mL, 100μL / tube were added to 1.5mL EP tubes. 10μL / tube of goat serum was added and the cells were blocked at 4°C for 30 minutes. A serial dilution of CD3×parental PSMA bispecific antibody, CD3×PSMAvll bispecific antibody, and control IgG1LALA (purchased from Bio-Bio) were added and incubated at 4°C for 30 minutes. 1mL of PBS was added to the EP tubes and the cells were centrifuged at 3500 rpm for 5 minutes at 4°C. The supernatant was discarded and the tubes were washed once with PBS. After centrifugation, the supernatant was discarded and the cells were resuspended in 100μL / tube of PBS. 1μL / tube of Alexa-647-labeled goat anti-human secondary antibody (purchased from Jackson Labs) was added and the cells were incubated at 4°C for 30 minutes in the dark. The cells were washed twice with PBS and centrifuged and the supernatant was discarded. The cells were resuspended in 200μL / tube of PBS and analyzed by flow cytometry.

[0263] The results are shown in Figures 7 and 8, which indicate that the CD3×PSMAv11 of the present invention binds to 22RV1 and LNCaP human prostate cancer cells more strongly than the CD3×parental PSMA bispecific antibody.

[0264] Example 8: CD3×PSMA Antibody Promotes PBMC to Kill Tumor Cells

[0265] The ability of the CD3×PSMA bispecific antibody and the CD3×parental mAb bispecific antibody obtained in Example 7 to promote PBMC to kill 22RV1 and LNCaP human prostate cancer cells was tested. The specific steps are as follows:

[0266] (1) Add complete RPMI-1640 medium to a 16-well RTCA plate at a volume of 50 μL / well and calibrate the plate.

[0267] (2) Dilute the tumor cells to 2×10 5 / mL, and added to the RTCA plate obtained in step (1) at a volume of 50 μL / well, and then the cell coefficient was detected using the xCELLigence RTCA MP device at 37°C and 5% CO2 for 24 hours;

[0268] (3) CD3×parental PSMA bispecific antibody, CD3×PSMAvll bispecific antibody, and control IgG1LALA (purchased from Bio-Bio) were serially diluted in complete RPMI-1640 medium and added to the RTCA plate obtained in step (2) at a volume of 20 μL / well;

[0269] (4) PBMC (purchased from Saili Bio) were diluted to 1.25×10 6 pcs / mL, added to the RTCA plate obtained in step (3), with an addition volume of 80 μL / well;

[0270] (5) The reaction system obtained in step (4) was incubated at 37° C. and 5% CO 2 for 24 h to detect the cell coefficient using an xCELLigence RTCA MP device.

[0271] As shown in Figures 9 and 10, the CD3×PSMAv11 antibody of the present invention promotes PBMC to kill tumors much more effectively than the CD3×parental PSMA bispecific antibody.

[0272] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0273] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. An antibody or antigen-binding fragment, characterized in that comprising at least one of the following CDRs: HCDR 1: GYSFTX1NW, wherein X1 is S or H; HCDR 2: IYPGDSDT; HCDR 3:ARQTGFLWSSDLWGRGT; LCDR 1: X2QDISX3A, where X2 is S or P, and X3 is S or Y; LCDR 2: DASX4, where X4 is S or W; LCDR 3:QQFNSYPLX5, where X5 is T or S; X1 is S, X2 is S, X3 is S, X4 is S, and X5 is T cannot exist at the same time.

2. The antibody or antigen-binding fragment according to claim 1, characterized in that X1 is S; Optionally, X1 is H; Optionally, X2 is S; Optionally, X2 is P; Optionally, X3 is S; Optionally, X3 is Y; Optionally, X4 is S; Optionally, X4 is W; Optionally, X5 is T; Optionally, X5 is S; Optionally, the antibody or antigen-binding fragment comprises: HCDR1, HCDR2, HCDR3 as shown in the amino acid sequences of SEQ ID NOs: 1, 2 and 3, respectively; and LCDR1, LCDR2, LCDR3 as shown in the amino acid sequences of SEQ ID NOs: 4, 5 and 6, respectively; Optionally, the antibody or antigen-binding fragment thereof specifically recognizes PSMA.

3. The antibody or antigen-binding fragment according to claim 2, characterized in that: The antibody or antigen-binding fragment comprises a heavy chain framework region and / or a light chain framework region; Optionally, at least a portion of the heavy chain framework region and / or the light chain framework region is from at least one of a mouse antibody, a primate antibody, a bovine antibody, a horse antibody, a dairy cow antibody, a porcine antibody, a sheep antibody, a goat antibody, a dog antibody, a cat antibody, a rabbit antibody, a camel antibody, a donkey antibody, a deer antibody, a mink antibody, a chicken antibody, a duck antibody, a goose antibody, a turkey antibody, a fighting cock antibody, or a mutant thereof; Optionally, at least a portion of the heavy chain framework region and / or the light chain framework region is from at least one of a murine antibody and a human antibody; Optionally, the antibody or antigen-binding fragment comprises: A heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 7, or an amino acid sequence having at least 90% homology thereto; and The light chain variable region has an amino acid sequence as shown in SEQ ID NO: 8, or an amino acid sequence having at least 90% homology thereto.

4. The antibody or antigen-binding fragment according to any one of claims 1 to 3, characterized in that further comprising a heavy chain constant region and / or a light chain constant region; Optionally, at least a portion of at least one of the heavy chain constant region and the light chain constant region is from at least one of a mouse antibody, a primate antibody, a bovine antibody, a horse antibody, a dairy cow antibody, a porcine antibody, a sheep antibody, a goat antibody, a dog antibody, a cat antibody, a rabbit antibody, a camel antibody, a donkey antibody, a deer antibody, a mink antibody, a chicken antibody, a duck antibody, a goose antibody, a turkey antibody, a fighting cock antibody, or a mutant thereof; Optionally, the heavy chain constant region comprises a heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; or The light chain constant region comprises a light chain constant region selected from a κ type or a λ type; Optionally, the light chain constant region and the heavy chain constant region are both from a murine antibody or a mutant thereof, and / or a human antibody or a mutant thereof; Optionally, the N-terminus of the heavy chain constant region is connected to the C-terminus of the heavy chain variable region; and / or The N-terminus of the light chain constant region is connected to the C-terminus of the light chain variable region; Optionally, the heavy chain constant region comprises the heavy chain constant region shown in SEQ ID NO: 9, or an amino acid sequence having at least 80% identity thereto; and / or The light chain constant region comprises the light chain constant region shown in SEQ ID NO: 10 or an amino acid sequence having at least 80% identity thereto; Optionally, the antibody comprises at least one of a polyclonal antibody, a full-length monoclonal antibody, a Fab antibody, a Fab' antibody, a F(ab')2 antibody, a Fv antibody, a single-chain antibody, a single-domain antibody and a minimum recognition unit; or The antigen binding fragment comprises at least one of a F(ab')2 fragment, a Fab' fragment, a Fab fragment, a F(ab)2 fragment, a Fv fragment, a scFv fragment, a scFv-Fc fusion protein, a scFv-Fv fusion protein and a minimum recognition unit; Optionally, the antibody or antigen-binding fragment comprises: The heavy chain of the amino acid sequence shown in SEQ ID NO: 11 or an amino acid sequence having at least 80% homology thereto; and The light chain of the amino acid sequence shown in SEQ ID NO: 12 or an amino acid sequence having at least 80% homology thereto.

5. A multispecific antibody, characterized in that: include: A first binding region, the first binding region comprising the antibody or antigen-binding fragment of any one of claims 1 to 4; A second binding region, wherein the second binding region has a first molecule binding activity.

6. The multispecific antibody according to claim 5, characterized in that The first molecule is selected from at least one of immune cell surface antigens, tumor antigens, viruses, bacteria, endotoxins, cytokines, and cytokine receptors; Optionally, the first molecule is selected from at least one of CD3, PD-L1, PD-1, IL-10, IL-10R, BCMA, VEGF, TGF-β, CTLA-4, LAG-3, TIGIT, CEA, CD38, SLAMF7, B7-H3, Her2, EpCAM, CD19, CD20, CD30, CD33, CD47, CD52, CD133, EGFR, GD2, CD3, GM2, RANKL and CD16a; Optionally, the second binding region is a binding protein of the first molecule or a fragment thereof; Optionally, the second binding region is at least one of an antibody or antigen-binding fragment of the first molecule, and a receptor fragment of the first molecule; Optionally, the second binding region is a single chain antibody of the first molecule; Optionally, the second binding region further comprises a first Fc fragment; Optionally, the C-terminus of the binding protein or fragment thereof of the first molecule is connected to the N-terminus of the first Fc fragment; Optionally, the second binding region further comprises a connecting peptide; Optionally, the C-terminus of the binding protein or fragment thereof of the first molecule is connected to the N-terminus of the connecting peptide, and the C-terminus of the connecting peptide is connected to the N-terminus of the first Fc fragment; Optionally, the connecting peptide has an amino acid sequence as shown in (GGGGS)n, wherein n is an integer greater than or equal to 1, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; Optionally, the bispecific antibody comprises a symmetric bispecific antibody or an asymmetric bispecific antibody, preferably an asymmetric bispecific antibody; Optionally, the first molecule is CD3, and the second binding region comprises an anti-CD3 antibody; Optionally, the anti-CD3 antibody is selected from at least one of a Fab antibody, a Fab' antibody, a F(ab')2 antibody, a Fv antibody, a single-chain antibody, a single-domain antibody, and a minimum recognition unit; Optionally, the anti-CD3 antibody is a CD3 single-chain antibody; Optionally, the C-terminus of the CD3 single-chain antibody is connected to the N-terminus of the connecting peptide, and the C-terminus of the connecting peptide is connected to the N-terminus of the first Fc fragment; Optionally, the CD3 single-chain antibody has the amino acid sequence shown in SEQ ID NO: 13; Optionally, the connecting peptide has an amino acid sequence as shown in SEQ ID NO: 14; Optionally, the first Fc fragment has the amino acid sequence shown in SEQ ID NO: 15; Optionally, the first binding region has an amino acid sequence as shown in SEQ ID NO:

17.

7. The multispecific antibody according to any one of claims 5 to 6, characterized in that The antibody or antigen-binding fragment in the first binding region is selected from at least one of Fab antibody, Fab' antibody, F(ab')2 antibody, Fv antibody, single-chain antibody, single-domain antibody and minimum recognition unit, preferably Fab antibody or single-chain antibody; Optionally, the antibody or antigen-binding fragment in the first binding region is a Fab antibody; Optionally, the Fab antibody comprises CDRs in the antibody or antigen-binding fragment of any one of claims 1 to 4; Optionally, the Fab antibody comprises the heavy chain variable region and the light chain variable region of the antibody or antigen-binding fragment according to any one of claims 1 to 4; Optionally, the first binding region further comprises a second Fc fragment; Optionally, the C-terminus of CH1 in the Fab antibody is connected to the N-terminus of the second Fc fragment; Optionally, the second Fc fragment has the amino acid sequence shown in SEQ ID NO: 16; Optionally, the first binding region comprises: A first peptide chain having an amino acid sequence as shown in SEQ ID NO: 18; A second peptide chain having an amino acid sequence as shown in SEQ ID NO:

12.

8. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the antibody or antigen-binding fragment of any one of claims 1 to 4, or the multispecific antibody of any one of claims 5 to 7; Optionally, the nucleic acid molecule is DNA.

9. An expression vector, characterized in that Carrying the nucleic acid molecule according to claim 8; Optionally, the expression vector comprises a vector selected from a eukaryotic expression vector or a prokaryotic expression vector.

10. A recombinant cell, characterized in that include: Carrying the nucleic acid molecule according to claim 8 or the expression vector according to claim 9; or, Expressing the antibody or antigen-binding fragment of any one of claims 1 to 4, or the multispecific antibody of any one of claims 5 to 7; Optionally, the recombinant cell is obtained by introducing the expression vector of claim 9 into a host cell.

11. A conjugate, characterized in that: Include: The antibody or antigen-binding fragment of any one of claims 1 to 4 or the multispecific antibody of any one of claims 5 to 7; and a conjugate moiety, which is linked to the antibody or antigen-binding fragment or multispecific antibody; Optionally, the coupling moiety comprises at least one selected from a carrier, a drug, a toxin, a cytokine, a protein tag and a modifier.

12. A pharmaceutical composition, characterized in that include: The antibody or antigen-binding fragment of any one of claims 1 to 4, the multispecific antibody of any one of claims 5 to 7, the nucleic acid molecule of claim 8, the expression vector of claim 9, the recombinant cell of claim 10 or the conjugate of claim 11; Optionally, a pharmaceutically acceptable excipient is further included.

13. A kit, characterized in that: include: The antibody or antigen-binding fragment of any one of claims 1 to 4, the multispecific antibody of any one of claims 5 to 7, the nucleic acid molecule of claim 8, the expression vector of claim 9, the recombinant cell of claim 10 or the conjugate of claim 11.

14. The antibody or antigen-binding fragment of any one of claims 1 to 4, the multispecific antibody of any one of claims 5 to 7, the nucleic acid molecule of claim 8, the expression vector of claim 9, the recombinant cell of claim 10, the conjugate of claim 11 or the pharmaceutical composition of claim 12, for preventing and / or treating PSMA-mediated related diseases, or having the following uses: Prevention and / or treatment of PSMA-mediated diseases; and / or Preparation of drugs for preventing and / or treating PSMA-mediated related diseases; Optionally, the PSMA-mediated related diseases include tumors and / or cancers; Optionally, the cancer is prostate cancer.

15. The antibody or antigen-binding fragment of any one of claims 1 to 4, the multispecific antibody of any one of claims 5 to 7, the nucleic acid molecule of claim 8, the expression vector of claim 9, the recombinant cell of claim 10, the conjugate of claim 11 or the kit of claim 13, for detecting PSMA, detecting a PSMA-mediated related disease, diagnosing a PSMA-mediated related disease, staging a PSMA-mediated related disease, or evaluating the prognosis of a PSMA-mediated related disease, or having the following uses: PSMA testing, and / or Detection of PSMA-mediated diseases, and / or Diagnosis of PSMA-mediated diseases, and / or Staging of PSMA-mediated disease, and / or Assess PSMA-mediated disease prognosis, and / or Prepare a kit for detecting PSMA, and / or Preparation of a kit for detecting PSMA-mediated related diseases, and / or Preparation of a kit for diagnosing PSMA-mediated related diseases, and / or Preparation of a kit for staging PSMA-mediated related diseases, and / or Prepare a kit for evaluating the prognosis of PSMA-mediated related diseases; Optionally, the PSMA-mediated related diseases include tumors and / or cancers; Optionally, the cancer is prostate cancer.

16. A method for preventing and / or treating PSMA-mediated related diseases, characterized in that: include: Administering a pharmaceutically acceptable amount of the antibody or antigen-binding fragment of any one of claims 1 to 4, the multispecific antibody of any one of claims 5 to 7, the conjugate of claim 11, or the pharmaceutical composition of claim 12 to a subject; Optionally, the PSMA-mediated related diseases include tumors and / or cancers; Optionally, the cancer is prostate cancer.

17. A method for detecting PSMA, characterized in that: include: The antibody or antigen-binding fragment according to any one of claims 1 to 4, the multispecific antibody according to any one of claims 5 to 7, the conjugate according to claim 11 or the kit according to claim 13 is contacted with a sample to be detected to form an immune complex.

18. A method for diagnosing a PSMA-mediated disease, staging a PSMA-mediated disease, or evaluating the prognosis of a PSMA-mediated disease, characterized in that: include: Using the antibody or antigen-binding fragment of any one of claims 1 to 4, the multispecific antibody of any one of claims 5 to 7, or the conjugate of claim 11 to detect PSMA in the sample to be tested; Based on the detection result of PSMA, the content of PSMA in the sample to be tested is determined.

Images