CN118027203A

CN118027203A - PSMA antibodies and their applications

Info

Publication number: CN118027203A
Application number: CN202311699412.4A
Authority: CN
Inventors: 曹国帅; 成赢; 李洋洋; 武玉伟
Original assignee: Hefei Tiangang Immune Drugs Co ltd
Current assignee: Hefei Tiangang Immune Drugs Co ltd
Priority date: 2023-12-11
Filing date: 2023-12-11
Publication date: 2024-05-14
Also published as: WO2025124255A1

Abstract

The invention provides an antibody or antigen binding fragment, which comprises HCDR1, HCDR2 and HCDR3 sequences respectively shown as amino acid sequences of SEQ ID NO.1, 2 and 3; and LCDR1, LCDR2, LCDR3 sequences shown as the amino acid sequences of SEQ ID NO.4, 5 and 6, respectively. Compared with wild type antibodies, the antibodies of the invention have higher PSMA affinity, can effectively kill tumors or cancers, and especially can prevent and/or treat prostate cancer.

Description

PSMA antibodies and uses thereof

Technical Field

The invention belongs to the technical field of biological pharmacy, and particularly relates to a PSMA antibody and application thereof. More specifically, the invention relates to an antibody or antigen binding fragment thereof, bispecific antibodies, nucleic acid molecules, expression vectors, recombinant cells, conjugates, pharmaceutical compositions, kits and uses thereof.

Background

Cancer is a major disease affecting human survival and development, and according to the latest data, new cancer cases 1900 Mo Zuo are about annually worldwide, cancer cases 1000 ten thousand are about annually dead, and the occurrence rate and death rate are on an increasing trend.

Besides surgical incision, the traditional cancer treatment means such as chemotherapy, radiotherapy and the like have large side effects and are easy to relapse. In recent years, immunotherapy, including tumor targeting antibodies, immune checkpoint antibodies, bispecific antibodies, and the like, has become a new hotspot and hope for anticancer.

In recent years, immunotherapy represented by PD-1/L1 has demonstrated great potential, but it cannot be ignored that even though the overall response rate of PD-1/L1 therapy, which is currently the most widely approved therapy, is still only 30%, more patients cannot benefit from it. The immune checkpoint molecule is an inhibitory molecule expressed on the surface of immune cells including T, NK, mononuclear macrophages and the like, and after being combined with corresponding ligands, the immune checkpoint molecule transmits inhibitory signals into the immune cells to inhibit the anticancer function of the immune cells. Since immune checkpoint receptor ligand expression of tumor, tumor infiltrating lymphocytes has a very large heterogeneity, a single class of immune checkpoint therapy cannot be applied to all patients, from which most patients cannot benefit; on the other hand, patients who have partially received immune checkpoint therapy may relapse into tumors and develop tolerance to the immune checkpoint therapy, and continued administration may not produce efficacy; in addition, T cells recognize neo-antigen (TCR) through surface T cell receptors, i.e., antigens in which tumor genes are mutated, while partial tumors have low frequency of gene mutation and a small number of neoantigens, which are called "cold tumors". Current immune checkpoint therapies, such as PD-1/L1 therapies, achieve anti-cancer goals by restoring the function of the T cells themselves, whereas in "cold tumors" T cells are unable to effectively recognize the tumor, resulting in immune checkpoint therapies that are ineffective against cold tumors.

. PSMA is a seed cell membrane protein that is hardly expressed in most human normal tissues, exhibits a high expression of specificity in prostate cancer, and is highly expressed in more than 80% of prostate cancer patients, and its high expression is associated with poor prognosis. Thus, PSMA is a potential therapeutic target, and it would be of great value to develop antibodies targeting PSMA or bi-and multifunctional antibodies based on PSMA mab or CAR-T, CAR-NK therapies.

Disclosure of Invention

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

In a first aspect of the invention, the invention provides an antibody or antigen binding fragment. According to an embodiment of the invention, the antibody or antigen binding fragment comprises CDRs selected from at least one of the following: HCDR 1 GYSFTX ₁ NW, wherein X ₁ is S or H; HCDR 2:IYPGDSDT; HCDR 3 ARQTGFLWSSDLWGRGT; LCDR 1:X ₂QDISX₃ a, wherein X ₂ is S or P, X ₃ is S or Y; LCDR 2 DASX ₄, wherein X ₄ is S or W; LCDR 3 is QQFNSPYLX ₅, wherein X ₅ is T or S; x ₁ is S, X ₂, S, X ₃ is S, X ₄, S, X ₅ is T are not present at the same time. The antibodies or antigen binding fragments have high PSMA binding affinity and tumor killing compared to wild-type antibodies, and PSMA can be detected.

In a second aspect of the invention, the invention provides a multispecific antibody. According to an embodiment of the invention, the multispecific antibody comprises: a first binding region comprising an antibody or antigen binding fragment of the first aspect; a second binding region, said second binding region having a first molecular binding activity. The multispecific antibody has high PSMA binding affinity and tumor killing power, and can effectively prevent and/or treat PSMA-mediated related diseases, especially prostate cancer.

In a third aspect of the invention, the invention provides a nucleic acid molecule. According to an embodiment of the invention, the nucleic acid molecule encodes an antibody or antigen-binding fragment according to the first aspect, a multispecific antibody according to the second aspect. The nucleic acid molecule of the invention may encode an antibody or antigen-binding fragment of the first aspect, a multispecific antibody of the second aspect.

In a fourth aspect of the invention, the invention provides an expression vector. According to an embodiment of the invention, the expression vector carries a nucleic acid molecule according to the third aspect. Therefore, the expression vector can effectively realize the expression of the antibody or antigen binding fragment of the first aspect or the multispecific antibody of the second aspect, and further realize the in-vitro mass acquisition of the antibody or antigen binding fragment or the multispecific antibody.

In a fifth aspect of the invention, the invention provides a recombinant cell. According to an embodiment of the invention, the recombinant cell comprises an expression vector carrying the nucleic acid molecule of the third aspect or the fourth aspect; or expressing the antibody or antigen-binding fragment of the first aspect, or the multispecific antibody of the second aspect. The recombinant cell can be used to efficiently express the aforementioned antibody or antigen-binding fragment or multispecific antibody in a cell under appropriate conditions.

In a sixth aspect of the invention, the invention provides a conjugate. According to an embodiment of the invention, the conjugate comprises an antibody or antigen-binding fragment according to the first aspect, or a multispecific antibody according to the second aspect; and a coupling moiety attached to the antibody or antigen binding fragment or multispecific antibody. The conjugates of the invention have high PSMA binding affinity and are useful for detecting PSMA, or for preventing and/or treating PSMA-mediated related diseases, in particular for detecting prostate cancer, or for preventing and/or treating prostate cancer.

In a seventh aspect of the invention, the invention provides a pharmaceutical composition. According to an embodiment of the invention, the pharmaceutical composition comprises an antibody or antigen binding fragment according to the first aspect, a multispecific antibody according to the second aspect, a nucleic acid molecule according to the third aspect, an expression vector according to the fourth aspect, a recombinant cell according to the fifth aspect or a conjugate according to the sixth aspect. The pharmaceutical composition of the present invention has high PSMA binding affinity and tumor killing power, and can be used for effectively preventing and/or treating PSMA mediated related diseases, particularly for preventing and/or treating prostate cancer.

In an eighth aspect of the invention, the invention provides a kit. According to an embodiment of the invention, the kit comprises an antibody or antigen binding fragment according to the first aspect, a multispecific antibody according to the second aspect, a nucleic acid molecule according to the third aspect, an expression vector according to the fourth aspect, a recombinant cell according to the fifth aspect or a conjugate according to the sixth aspect. The kit provided by the invention has high PSMA binding affinity, and can be used for effectively detecting PSMA, detecting PSMA-mediated related diseases, diagnosing PSMA-mediated related diseases, staging PSMA-mediated related diseases or evaluating PSMA-mediated related disease prognosis.

In a ninth aspect, the invention provides the use of an antibody or antigen binding fragment according to the first aspect, a multispecific antibody according to the second aspect, a nucleic acid molecule according to the third aspect, an expression vector according to the fourth aspect, a recombinant cell according to the fifth aspect, a conjugate according to the sixth aspect or a pharmaceutical composition according to the seventh aspect for the preparation of a medicament for the prevention and/or treatment of a PSMA-mediated related disease, in particular for the prevention and/or treatment of prostate cancer.

In a tenth aspect of the invention, the invention provides the use of an antibody or antigen binding fragment according to the first aspect, a multispecific antibody according to the second aspect, a nucleic acid molecule according to the third aspect, an expression vector according to the fourth aspect, a recombinant cell according to the fifth aspect or a conjugate according to the sixth aspect for 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 assessing a PSMA-mediated related disease prognosis.

The beneficial effects are that:

1) The affinity matured PSMA antibodies obtained in the present invention have a slower off-rate (Kd) than the parent antibody.

2) The affinity matured PSMA antibodies obtained in the present invention have higher binding strength to tumor cells than the parent antibodies.

3) The affinity matured antibody PSMA antibody obtained by the invention has stronger ADCC activity compared with a parent antibody.

4) The obtained CD3 xPSMA diabody has stronger binding activity and killing promoting activity compared with a parent antibody.

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

Drawings

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

FIG. 1 is a CDR sequence comparison of affinity matured PSMAv antibody, parent antibody parental mAb according to example 2 of the present invention;

FIG. 2 is a graph showing SPR results of affinity assays for affinity matured PSMAv antibody, parent antibody parental mAb protein according to example 3 of the present invention;

FIG. 3 is a graph showing the results of flow cytometry for binding of affinity matured PSMAv antibody, maternal antibody parental mAb, to 22Rv1 human prostate cancer cells according to example 5 of the present invention;

FIG. 4 is a graph of flow cytometry results of binding of affinity matured PSMAv antibody, maternal antibody parental mAb, to LNCaP human prostate cancer cells according to example 5 of the present invention;

FIG. 5 is a graph showing the results of affinity maturation PSMAv antibody, maternal antibody parental mAb to promote PBMC killing of LNCaP human prostate cancer cells according to example 6 of the invention;

FIG. 6 is a graph showing the results of affinity maturation PSMAv antibody, maternal antibody parental mAb to promote PBMC killing of 22Rv1 human prostate cancer cells according to example 6 of the present invention;

FIG. 7 is a graph showing the results of flow cytometry for binding of CD 3X PSMAv11 antibody, CD 3X PARENTAL PSMA bispecific antibody to 22Rv1 human prostate cancer cells according to example 7 of the present invention;

FIG. 8 is a graph showing the results of flow cytometry for binding of CD 3X PSMAv11 antibodies, CD 3X PARENTAL PSMA bispecific antibodies to LNCaP human prostate cancer cells according to example 7 of the present invention;

FIG. 9 is a graph showing the results of the CD 3X PSMAv11 antibody, CD 3X PARENTAL PSMA bispecific antibody and promoting the killing of LNCaP human prostate cancer cells by PBMC according to example 8 of the present invention;

FIG. 10 is a graph showing the results of the CD 3X PSMAv11 antibody, CD 3X PARENTAL PSMA bispecific antibody promoting PBMC killing of 22Rv1 human prostate cancer cells according to example 8 of the present invention.

Detailed Description

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

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

Description

Definitions and general terms

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

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

As used herein, the term "fragment" refers to a protein or polypeptide of interest, as well as a protein or polypeptide of interest having an N-terminal (N-terminal) or C-terminal (C-terminal) truncation, and/or internal deletion.

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

Antibodies or antigen binding fragments of the invention are typically prepared by biosynthetic methods. The coding nucleic acids according to the invention can be prepared by various known methods, conveniently by the person skilled in the art, based on the nucleotide sequences according to the invention. Such as, but not limited to: PCR, DNA synthesis, etc., and specific methods can be found in J.Sam Brookfield, guidelines for molecular cloning experiments. As one embodiment of the present invention, the coding nucleic acid sequence of the present invention can be constructed by a method of synthesizing nucleotide sequences in segments followed by overlap extension PCR. Wherein the antibody or antigen fragment is numbered and defined using the Kabat numbering system.

In this context, the terms "identity", "homology" or "similarity" are used to describe the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences when compared to the amino acid sequence or nucleic acid sequence of a reference sequence, using conventional methods, e.g., see Ausubel et al, editions (1995), current Protocols in Molecular Biology, chapter 19 (Greene Publishing and Wiley-Interscience, new York); and ALIGN program (Dayhoff(1978),Atlas of Protein Sequence and Structure5：Suppl.3(National Biomedical Research Foundation,Washington,D.C.). there are many algorithms for aligning sequences and determining sequence identity, including, needleman et al (1970) J.mol. Biol.48:443 homology comparison algorithm; smith et al (1981) adv.appl.Math.2:482, a local homology algorithm; pearson et al (1988) Proc.Natl. Acad.Sci.85:2444 similarity search method; computer programs utilizing the Smith-Waterman algorithm (Meth. Mol. Biol.70:173-187 (1997)), and BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al (1990) J. Mol. Biol. 215:403-410)), are also available and include, but are not limited to, ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al, meth. Enzyme, 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altschul et al, supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, 8 th edition, madison, wisconsin, USA, and CLUSTAL in the PC/Gene programs provided by Intelligenetics, mountain View, california.

The term "at least 80% identity" as used herein refers to at least 80% identity to each reference sequence, which may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity.

As used herein, the term "at least 90% identity" refers to at least 90% identity to each reference sequence, which may be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity.

In this context, the term "expression vector" generally refers to a nucleic acid molecule capable of insertion into a suitable host for self-replication, which nucleic acid molecule comprises a nucleotide sequence capable of expressing a protein of interest, and which nucleic acid molecule is capable of being transferred into and/or between host cells. The expression vector may include a vector mainly used for inserting DNA or RNA into cells, a vector mainly used for replicating 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 having a plurality of the above functions. The expression vector may be a polynucleotide capable of transcription and translation into a polypeptide when introduced into a suitable host cell. Typically, the expression vector will produce the desired expression product by culturing a suitable host cell containing the expression vector.

As used herein, the term "recombinant cell" generally refers to a cell that has been modified or recombined with genetic material of a host cell using genetic engineering techniques or cell fusion techniques to obtain a unique trait that is stably inherited. Wherein the term "host cell" refers to a prokaryotic or eukaryotic cell into which a recombinant expression vector may be introduced. The term "transformed" or "transfected" as used herein refers 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 sequences of the invention and can be used for 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 (human amniotic fluid derived cells) and CoS cells.

The term "pharmaceutical composition" as used herein generally refers to unit dosage forms and may be prepared by any of the methods well known in the pharmaceutical arts. All methods include the step of bringing the active ingredient into association with the carrier which constitutes one or more accessory ingredients. Generally, the compositions are prepared by uniformly and sufficiently combining the active antibody or antigen-binding fragment with a liquid carrier, a finely divided solid carrier, or both.

As used herein, the term "pharmaceutically acceptable excipients" may include any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for the particular dosage form of interest. In addition to the extent to which any conventional adjuvant is incompatible with the antibodies or antigen binding fragments of the invention, such as any adverse biological effects produced or interactions with any other component of the pharmaceutically acceptable composition in a deleterious manner, their use is also contemplated by the present invention.

As used herein, the term "administering" refers to introducing a predetermined amount of a substance into a patient by some suitable means. The antibody or antigen-binding fragment, multispecific antibody or pharmaceutical composition of the invention may be administered by any common route so long as it can reach the desired tissue. Various modes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, etc., but the invention is not limited to these illustrated modes of administration. Preferably, the compositions of the present invention are administered by intravenous or subcutaneous injection.

In this context, the term "treatment" refers to the use to obtain a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing the disease or symptoms thereof, and/or may be therapeutic in terms of partially or completely curing the disease and/or adverse effects caused by the disease. As used herein, "treating" encompasses diseases in mammals, particularly humans, including: (a) Preventing the occurrence of a disease or disorder in an individual susceptible to the disease but not yet diagnosed with the disease; (b) inhibiting disease, e.g., arresting disease progression; or (c) alleviating a disease, e.g., alleviating symptoms associated with a disease. As used herein, "treating" encompasses any administration of a drug or antibody or antigen-binding fragment to an individual to treat, cure, alleviate, ameliorate, reduce or inhibit a disease in the individual, including, but not limited to, administration of a drug comprising an antibody or antigen-binding fragment as described herein to an individual in need thereof.

Detailed description of the anti-novel coronavirus antibodies of the invention and their use

The 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 each described in detail below.

Antibodies or antigen binding fragments

In one aspect of the invention, the invention provides an antibody or antigen binding fragment. According to an embodiment of the invention, the antibody or antigen binding fragment comprises CDRs selected from at least one of the following: HCDR 1 GYSFTX ₁ NW, wherein X ₁ is S or H; HCDR 2:IYPGDSDT; HCDR 3 ARQTGFLWSSDLWGRGT; LCDR 1:X ₂QDISX₃ a, wherein X ₂ is S or P, X ₃ is S or Y; LCDR 2 DASX ₄, wherein X ₄ is S or W; LCDR 3 is QQFNSPYLX ₅, wherein X ₅ is T or S; x ₁ is S, X ₂, S, X ₃ is S, X ₄, S, X ₅ is T are not present at the same time. The antibodies or antigen binding fragments have high PSMA binding affinity and tumor killing compared to wild-type antibodies, and are useful for detecting PSMA, or for preventing and/or treating PSMA-mediated related diseases.

As used herein, the term "antibody" is used in its broadest sense and may include full length monoclonal antibodies, multispecific antibodies, and chimeric antibodies, and the specific structure is not limited so long as they exhibit the desired biological activity. It generally comprises a light chain of relatively light molecular weight and a heavy chain of relatively heavy molecular weight, the heavy chain (H chain) and the light chain (L chain) being linked by disulfide bonds to form an antibody molecule. Wherein the amino-terminal (N-terminal) amino acid sequence of the peptide chain varies greatly and is called variable region (V region); the carboxy terminus (C-terminus) is relatively stable with little variation and is referred to as the constant region (C-region). The V chains of the L chain and H chain are referred to as VL and VH, respectively.

Herein, the heavy chain complementarity determining region (heavy chain variable region CDR) is denoted by "HCDRs" or "HCDR", which includes HCDR1 (also known as CDR-H1), HCDR2 (also known as CDR-H2) and HCDR3 (also known as CDR-H3); light chain complementarity determining regions (light chain variable region CDRs) are denoted by "LCDRs" or "LCDRs," and include LCDR1 (also known as CDR-L1), LCDR2 (also known as CDR-L2) and LCDR3 (also known as CDR-L3). CDR definition schemes commonly used in the art include: kabat definition, chothia definition, IMGT definition, contact definition, and AbM definition.

As used herein, "Kabat definition" refers to the definition system described by Kabat et al, U.S. Dept. Of HEALTH AND Human Services, "Sequence of Proteinsof Immunological Interest" (1983). "Chothia definition" see Chothia et al, J Mol Biol 196:901-917 (1987). Exemplary defined CDRs are listed in table a below, with the definitions in the different documents being slightly different, and one of skill in the art can routinely determine which residues comprise a particular CDR given the variable region amino acid sequence of an antibody. It is noted that CDRs of the present application include CDRs not limited to those defined by other methods in table a, and CDRs determined using other rules disclosed in the art based on the heavy chain variable region and the light chain variable region of the present disclosure are also within the scope of the present disclosure.

Table a: CDR definition ¹

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

² The "AbM" as used in table a has a lower case "b" referring to CDRs defined by the "AbM" antibody modeling software of Oxford Molecular.

³ If neither H35A nor H35B is present, then CDR-H1 ends at position 35; if only H35A is present, then CDR-H1 ends at position 35A; if H35A and H35B are present at the same time, then CDR-H1 ends at position 35B.

⁴ If neither H35A nor H35B is present, then CDR-H1 ends at position 32; if only H35A is present, then CDR-H1 ends at position 33; if H35A and H35B are present at the same time, then CDR-H1 ends at position 34.

⁵ If neither H35A nor H35B is present, then CDR-H1 ends at position 33; if only H35A is present, then CDR-H1 ends at position 34; if H35A and H35B are present at the same time, then CDR-H1 ends at position 35.

Kabat et al also define a numbering system for variable region sequences suitable for use with any antibody. The Kabat numbering system can be specifically mapped to any variable region sequence by one of ordinary skill in the art without relying on any experimental data outside of the sequence itself. As used herein, "Kabat numbering" refers to numbering of HCDRs and LCDRs of an antibody or antigen-binding fragment of the invention using the numbering system described in Kabat et al, U.S. Dept. Of HEALTH AND HumanServices, "Sequence of Proteins of ImmunologicalInterest" (1983), the specific numbering results being set forth in Table A. It should be noted that the polypeptide sequences in the sequence table and the table B of the present invention are numbered according to the Kabat numbering system. However, it is fully within the scope of the present invention for a person of ordinary skill in the art to be able to convert the sequence Kabat numbering of the sequence listing to "HCDRs" and/or "LCDRs" under other numbering systems.

As used herein, the term "antigen binding fragment" is 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, e.g., the fragment may comprise a portion or all of the CDRs of an antibody. Such fragments are biologically active in that 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 may be produced by recombinant nucleic acid techniques, or may be produced by enzymatic or chemical cleavage of antigen binding molecules, including intact antibodies.

According to an embodiment of the present invention, the antibody or antigen binding fragment may further comprise at least one of the following technical features:

According to an embodiment of the invention, X ₁ is S.

According to an embodiment of the invention, X ₁ is H.

According to an embodiment of the invention, X ₂ is S.

According to an embodiment of the invention, X ₂ is P.

According to an embodiment of the invention, X ₃ is S.

According to an embodiment of the invention, X ₃ is Y.

According to an embodiment of the invention, X ₄ is S.

According to an embodiment of the invention, X ₄ is W.

According to an embodiment of the invention, X ₅ is T.

According to an embodiment of the invention, X ₅ is S.

In one embodiment of the invention, the antibody or antigen binding fragment comprises CDRs of one of the following sets:

According to an embodiment of the invention, the antibody or antigen binding fragment comprises: HCDR1, HCDR2, HCDR3 shown as 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 NO 4, 5 and 6, respectively. Wherein the CDRs are the same as set 18 in the above table.

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

According to embodiments of the invention, the antibody or antigen binding fragment comprises a heavy chain framework region and/or a light chain framework region.

According to an embodiment of the 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, a primates antibody, a bovine antibody, a equine antibody, a dairy bovine antibody, a porcine antibody, a ovine antibody, a caprine antibody, a canine antibody, a feline 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 bucket chicken antibody, or a mutant thereof.

According to an embodiment of the 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.

According to an embodiment of the invention, the antibody or antigen binding fragment comprises: a heavy chain variable region of an amino acid sequence as set forth 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 set forth in SEQ ID NO. 8 or an amino acid sequence having at least 90% homology thereto.

According to embodiments of the invention, the antibody or antigen binding fragment further comprises a heavy chain constant region and/or a light chain constant region.

According to an embodiment of the 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 murine antibody, a primates antibody, a bovine antibody, a equine antibody, a dairy bovine antibody, a porcine antibody, a ovine antibody, a caprine antibody, a canine antibody, a feline 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 bullfight antibody, or a mutant thereof.

According to an embodiment of the invention, the heavy chain constant region comprises a heavy chain constant region selected from the group consisting of IgG1, igG2, igG3, igG4, igA, igM, igE, or IgD.

According to embodiments of the invention, the light chain constant region comprises a light chain constant region selected from the group consisting of kappa-type and lambda-type.

According to an embodiment of the 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.

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

According to an embodiment of the invention, the heavy chain constant region comprises a heavy chain constant region as 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 a light chain constant region as set forth in SEQ ID NO. 10 or an amino acid sequence having at least 80% identity thereto.

According to an embodiment of the invention, the antibody comprises at least one of a polyclonal antibody, a full length monoclonal antibody, a Fab 'antibody, a F (ab') ₂ antibody, a Fv antibody, a single chain antibody, a single domain antibody, and a minimal 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, an Fv fragment, an scFv-Fc fusion protein, an scFv-Fv fusion protein, and a minimal recognition unit.

As used herein, the term "full length antibody", "full length monoclonal antibody" or "full length monoclonal antibody" is made up of at least two identical light chains and at least two identical heavy chains joined by interchain disulfide bonds, such as immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD) or immunoglobulin E (IgE).

The terms "polyclonal antibody" and "multispecific antibody" are synonymous herein, and refer to an antibody that recognizes multiple epitopes, for example, an antibody that recognizes two epitopes (bispecific antibody, abbreviated as "diabody"), an antibody that recognizes three epitopes, or an antibody that recognizes four epitopes, which is understood in a broad sense, and the specific structure is not limited, and may recognize multiple epitopes. In the present invention, at least one of the plurality of epitopes is derived from PSMA.

The terms "single domain antibody," "nanobody," and "VHH antibody" are used interchangeably herein, and are initially described as an antigen-binding immunoglobulin (variable) domain (Hamers-Casterman C,AtarhouchT,Muyldermans S,Robinson G,Hamers C,Songa EB,Bendahman N,Hamers R.:"Naturallyoccurring antibodies devoid of light chains";Nature 363,446-448(1993)), of a "heavy chain antibody" (i.e., an "antibody lacking a light chain") comprising a heavy chain variable region (VH) and conventional CH2 and CH3 regions, which specifically bind to an antigen protein (e.g., PSMA) via the heavy chain variable region.

In this context, the term "Fab antibody" or "Fab fragment" generally refers to an antibody or fragment comprising only Fab molecules, consisting of VH and CH1 of the heavy chain and the complete light chain, linked by a disulfide bond between the light and heavy chains.

As used herein, the term "F (ab ') ₂ antibody" or "F (ab ') ₂ fragment" has two antigen-binding F (ab ') moieties linked together by disulfide bonds.

As used herein, the term "Fv antibody" or "Fv fragment" generally refers to an antibody or fragment consisting of only the light chain variable region (VL) and the heavy chain variable region (VH) joined by a non-covalent bond, which is the smallest functional fragment of an antibody that retains the intact antigen-binding site.

As used herein, the terms "single chain antibody", "scFv fragment" are antibodies or fragments that are made up of antibody heavy and light chain variable regions linked by short peptides.

As used herein, the terms "minimal recognition unit" and "MRU" refer to antibodies or fragments consisting of only one CDR, which have a molecular weight that is sufficiently small to account for only about 1% of the total antibody.

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

Multispecific antibodies

According to an embodiment of the invention, the first molecule is selected from at least one of an immune cell surface antigen, a tumor antigen, a virus, a bacterium, an endotoxin, a cytokine and a cytokine receptor.

In this context, an "immune cell surface antigen" is to be understood in a broad sense and may refer to a protein that is immunogenic on the surface of an immune cell (e.g. T cell, NK cell, B cell, 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, CD16a, and the like.

In this context, "tumor antigen" is to be understood in a broad sense and may refer to a protein that is immunogenic on the surface of tumor cells. Including but not limited to PD-L1, PD-1, TGF-beta, CEA, GD2, CD3, and the like.

In this context, "cytokine" is understood in a broad sense and may refer to a class of proteins or small molecule polypeptides that are capable of transferring information between cells, and have immunomodulatory and effector functions. Including but 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, etc.

In this context, a "cytokine receptor" is to be understood in a broad sense and may refer to a receptor on the cell surface that can bind to a cytokine. Including but not limited to Her2, EGFR, IL-10R, and the like.

According to an embodiment of the 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 CD16 a.

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

According to an embodiment of the invention, 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.

According to an embodiment of the invention, the second binding region is a single chain antibody of the first molecule.

According to an embodiment of the invention, the second binding region further comprises a first Fc fragment.

According to an embodiment of the invention, the first binding region further comprises a second Fc fragment.

In this context, the Fc fragment includes a CH2, CH3 region and optionally a hinge region, such as a first Fc fragment and/or a second Fc fragment, unless specifically stated. In one embodiment of the invention, the C-terminal of the CH2 region is linked to the N-terminal of the CH3 region. In another embodiment of the present invention, the C-terminal of the hinge region is connected to the N-terminal of the CH2 region, and the C-terminal of the CH2 region is connected to the N-terminal of the CH3 region.

According to an embodiment of the invention, the first and second Fc fragment are both human Fc peptide fragments.

According to an embodiment of the invention, the human Fc peptide fragment is a human IgG1 Fc peptide fragment.

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

In one embodiment of the invention, the "knob intohole structure" is a button (Knob) snap (hole) mutation in the CH3 region of the heavy chain constant region of an antibody, facilitating heavy chain engagement, and heterodimer formation, for example, by mutating amino acids in the CH3 domain of the heavy chain constant region of a human IgG1 (T366S, L368A, Y407V, Y C mutation in one chain, i.e., "hole", and T366W, S354C mutation in the other chain, i.e., "knob").

According to an embodiment of the invention, the C-terminus of the binding protein of the first molecule or fragment thereof is linked to the N-terminus of the first Fc fragment.

When the binding protein or fragment thereof of the first molecule is an antibody having two chains, it is preferable to link a peptide chain having a heavy chain to the first Fc fragment.

According to an embodiment of the invention, the second binding region further comprises a connecting peptide.

According to an embodiment of the invention, the C-terminus of the binding protein of the first molecule or fragment thereof is linked to the N-terminus of the linker peptide, which C-terminus is linked to the N-terminus of the first Fc fragment.

According to an embodiment of the 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.

According to an embodiment of the invention, the bispecific antibody comprises a symmetric bispecific antibody or an asymmetric bispecific antibody, preferably an asymmetric bispecific antibody.

According to an embodiment of the invention, the first molecule is CD3 and the second binding region comprises an anti-CD 3 antibody.

According to an embodiment of the invention, the anti-CD 3 antibody is selected from at least one of Fab antibodies, fab 'antibodies, F (ab') ₂ antibodies, fv antibodies, single chain antibodies, single domain antibodies, and minimal recognition units.

According to an embodiment of the invention, the anti-CD 3 antibody is a CD3 single chain antibody.

According to an embodiment of the invention, the C-terminus of the CD3 single chain antibody is linked to the N-terminus of the linker peptide, which is linked to the N-terminus of the first Fc fragment.

According to an embodiment of the invention, the CD3 single chain antibody has an amino acid sequence as shown in SEQ ID NO. 13.

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

According to an embodiment of the invention, the first Fc fragment has the amino acid sequence shown as SEQ ID NO. 15.

According to an embodiment of the invention, the first binding region has the amino acid sequence shown as SEQ ID NO. 17.

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

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

According to an embodiment of the invention, the Fab antibody in the first binding region comprises CDRs in the antibody or antigen binding fragment of the first aspect.

As can be seen from the preceding definition, fab antibodies comprise two chains, namely a heavy chain variable region +ch1, and a light chain variable region +cl, wherein the CDRs in the heavy chain variable region and the light chain variable region correspond to the CDRs defined in the antibody or antigen binding fragment of the first aspect.

According to an embodiment of the invention, the Fab antibody in the first binding region comprises the heavy chain variable region and the light chain variable region of the antibody or antigen binding fragment of the first aspect.

As can be seen from the preceding definition, the Fab antibody comprises two chains, namely a heavy chain variable region +ch1, and a light chain variable region +cl (i.e. being a light chain constant region), wherein the heavy chain variable region and the light chain variable region correspond to the heavy chain variable region and the light chain variable region defined in the antibody or antigen binding fragment of the first aspect.

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

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

According to an embodiment of the invention, the second Fc fragment has the amino acid sequence shown as SEQ ID NO. 16.

According to an embodiment of the invention, 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 the amino acid sequence shown in SEQ ID NO. 12.

According to an embodiment of the 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.

Nucleic acid molecules, expression vectors and recombinant cells

According to an embodiment of the invention, the nucleic acid molecule is DNA.

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

In a fourth aspect of the invention, the invention provides an expression vector. According to an embodiment of the invention, the expression vector carries a nucleic acid molecule according to the third aspect. In the case of ligating the above-mentioned nucleic acid molecule to an expression vector, the nucleic acid molecule may be directly or indirectly linked to control elements on the expression vector, as long as these control elements are capable of controlling translation, expression, etc. of the nucleic acid molecule. These control elements may of course be derived directly from the expression vector itself or may be exogenous, i.e. not derived from the expression vector itself. Of course, the nucleic acid molecule may be operably linked to a control element.

"Operably linked" herein refers to linking the exogenous gene to the expression vector such that control elements within the expression vector, such as transcription control sequences and translation control sequences, and the like, are capable of performing their intended functions of regulating transcription and translation of the exogenous gene. The expression vector used in common can be, for example, a plasmid, phage, or the like.

According to some embodiments of the present invention, after the expression vector is introduced into a suitable recipient cell, the expression of the aforementioned antibody or antigen-binding fragment, the aforementioned recombinant protein, or the aforementioned multispecific antibody can be effectively achieved under the mediation of a regulatory system, thereby achieving in vitro mass acquisition of the antibody or antigen-binding fragment, the recombinant protein, or the multispecific antibody.

According to an embodiment of the invention, the expression vector comprises a vector selected from eukaryotic expression vectors or prokaryotic expression vectors.

In an alternative embodiment of the invention, the expression vector is a plasmid expression vector, a viral expression vector, such as a lentiviral expression vector.

According to some embodiments of the invention, the recombinant cells can efficiently and abundantly express antibodies or antigen-binding fragments thereof under suitable conditions, which have stronger specificity, longer half-life and higher efficacy, can deliver antibody drugs to target cells with smaller doses, achieve effective treatment or prevention of PSMA-mediated diseases, have lower toxic side effects, and have higher safety.

The term "suitable conditions" refers to conditions suitable for the antibody or antigen-binding fragment, recombinant protein or multispecific antibody of the present invention. Those skilled in the art will readily appreciate that conditions suitable for expression of the antibody or antigen-binding fragment, recombinant protein, or multispecific antibody include, but are not limited to, suitable transformation or transfection modes, suitable transformation or transfection conditions, healthy host cell states, suitable host cell densities, suitable cell culture environments, suitable cell culture times. The "suitable conditions" are not particularly limited, and those skilled in the art can optimize the conditions for expression of the above-described antibodies or antigen-binding fragments, recombinant proteins, or multispecific antibodies optimally according to the specific circumstances of the laboratory.

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

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

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

Conjugate, pharmaceutical composition and kit

According to an embodiment of the invention, the coupling moiety comprises at least one selected from the group consisting of a carrier, a drug, a toxin, a cytokine, a protein tag, and a modification.

Herein, the carrier may be a substance capable of being suspended or dispersed in a liquid phase (for example, a solid-phase carrier such as particles or magnetic beads), or a solid phase capable of accommodating or carrying a liquid phase (for example, a support such as a plate, a membrane, a test tube, or a container such as an orifice plate, a microchannel, a glass capillary, a nanopillar, a monolith, or the like); also useful are labeling vectors for labeling antibodies or antigen binding fragments, recombinant proteins, or multispecific antibodies, such as enzymes (e.g., peroxidase, alkaline phosphatase, luciferase (luciferin), β -galactosidase), affinity substances (e.g., one of streptavidin and biotin, one of the nucleic acids of the sense and antisense strands that are complementary to each other), fluorescent substances (e.g., fluorescein isothiocyanate, rhodamine, green fluorescent protein, red fluorescent protein), luminescent substances (e.g., luciferin, aequorin (Aequorin), acridine, tris (2, 2' -bipyridine) ruthenium, luminol), radioisotopes (e.g., ³H、¹⁴C、³²P、³⁵S、¹²⁵ I), gold colloids, and the like.

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

According to an embodiment of the present invention, the protein tag includes, but is not limited to, his tag, flag tag, GST tag, MBP tag, SUMO tag, C-Myc tag, and the like.

According to embodiments of the present invention, the modification is to be understood in a broad sense and may refer to a substance for modifying a protein. Illustratively, polyethylene glycol or derivatives thereof may be used.

The method of binding the conjugate moiety to the antibody or antigen-binding fragment, recombinant protein or multispecific antibody may be carried out using methods known in the art. Examples thereof include physical adsorption, covalent bonding, and methods using affinity substances (e.g., biotin and streptavidin), and ion bonding.

According to an embodiment of the invention, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

Administration of the pharmaceutical compositions of the present invention may be carried out by any acceptable mode of administration. The pharmaceutical compositions of the present invention may be formulated as solid, semi-solid, liquid or gaseous forms of formulation, such as injections, freeze-dried powders, the current methods of preparing such dosage forms being known or obvious to those skilled in the art. Typical routes of administration of such pharmaceutical compositions include, but are not limited to, subcutaneous injections, intravenous, intramuscular, intradermal, intrasternal injection or infusion techniques. The pharmaceutical composition of the present invention is formulated so as to allow the biologically active ingredient contained therein to be bioavailable upon administration of the composition to a patient.

In an eighth aspect of the invention, the invention provides a kit. According to an embodiment of the invention, the kit comprises an antibody or antigen binding fragment according to the first aspect, a multispecific antibody according to the second aspect, a nucleic acid molecule according to the third aspect, an expression vector according to the fourth aspect, a recombinant cell according to the fifth aspect or a conjugate according to the sixth aspect. The kit provided by the invention has high PSMA binding affinity, can effectively detect PSMA, and can further diagnose tumors or cancers (especially prostate cancer).

As previously mentioned, the antibodies or antigen-binding fragments of some embodiments of the invention are capable of efficiently binding to human PSMA protein, and thus, kits comprising the antibodies or antigen-binding fragments are capable of efficiently performing qualitative or quantitative detection of human PSMA protein. The kit provided by the invention can be used for immunoblotting, immunoprecipitation and the like, and relates to a kit for detection by utilizing the specific binding property of human PSMA and antibodies. These kits may comprise any one or more of the following: an antagonist, an anti-PSMA antibody, or a drug reference material; a protein purification column; immunoglobulin affinity purification buffers; cell assay diluent; instructions, literature, etc. anti-PSMA antibodies can be used in various types of diagnostic tests, for example, to detect a variety of diseases or drugs, toxins or other proteins in vitro or in vivo, for example, by detecting serum or blood from a subject to test for a disease of interest, and for research studies using the kit to detect human PSMA protein in a sample to be tested. Such related diseases may include PSMA-related diseases, such as cancer. Of course, the antibodies or antigen binding fragments provided herein may also be used in radioimmunoassays, radioimmunotherapy, and the like for the above-described diseases. For the above application scenario, the binding molecules are equally applicable and will not be described here.

According to some embodiments of the invention, the kit may further comprise a reagent conventionally used for detecting PSMA, such as a coating solution, etc.

Use of the same

According to an embodiment of the invention, the PSMA-mediated related disease comprises a tumor or a cancer

According to an embodiment of the 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, stomach cancer, esophageal cancer, oral squamous cell carcinoma and head and neck cancer.

According to an embodiment of the invention, the cancer is prostate cancer.

According to embodiments of the invention, the PSMA-mediated related diseases include tumors and/or cancers.

According to an embodiment of the invention, the cancer is prostate cancer.

Method of

In a twelfth aspect of the invention, the invention features a method of detecting PSMA. According to an embodiment of the invention, the method comprises: contacting the sample to be tested with the antibody or antigen binding fragment of the first aspect, the multispecific antibody of the second aspect, or the conjugate of the sixth aspect to form an immune complex. The method provided by the invention has the advantages of PSMA detection, further detection of prostate cancer, in particular in-vitro detection, high detection accuracy and the like.

According to an embodiment of the present invention, it is determined whether the sample to be tested contains a novel coronavirus content based on the signal of the immunocomplex.

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

According to an embodiment of the invention, the immune complex further comprises a second antibody, which binds to the novel coronavirus.

In a thirteenth aspect of the invention, the invention provides a method of preventing and/or treating cancer. According to an embodiment of the invention, the method comprises: administering to a subject a pharmaceutically acceptable amount of an antibody or antigen-binding fragment of the first aspect, a multispecific antibody of the second aspect, or a conjugate of the sixth aspect. The methods of the invention are effective in the treatment or prevention of tumors and/or cancers, particularly in the prevention and/or treatment of prostate cancer.

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

The antibodies or antigen-binding fragments, multispecific antibodies, pharmaceutical compositions, or conjugates of the invention may be incorporated into a medicament suitable for parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). These drugs can be prepared in various forms. Such as liquid, semi-solid, and solid dosage forms, and the like, including but not limited to liquid solutions (e.g., injection solutions and infusion solutions) or lyophilized powders. The drug is typically in the form of an injection solution or infusion solution. The aforementioned antibodies or antigen-binding fragments, multispecific antibodies, pharmaceutical compositions or conjugates may be administered by intravenous infusion or injection or intramuscular or subcutaneous injection.

Herein, the term "subject" refers to a vertebrate, preferably a mammal, most preferably a human. Mammals include, but are not limited to, mice, apes, humans, domestic animals, athletic animals, and pets. Tissues, cells and their progeny of the biological entity obtained in vivo or cultured in vitro are also included.

According to embodiments of the invention, the route of administration of the method employs subcutaneous or intravenous injection.

In a fourteenth aspect of the invention, the invention features a method of diagnosing a PSMA-mediated disease. According to an embodiment of the invention, the method comprises: detecting PSMA in a sample to be tested using the antibody or antigen-binding fragment of the first aspect, the nucleic acid molecule of the third aspect, the expression vector of the fourth aspect, or the recombinant cell of the fifth aspect; and determining the content of PSMA in the sample to be detected based on the detection result of the PSMA. The antibody or antigen binding fragment, or the nucleic acid molecule, the expression vector and the antibody or antigen binding fragment expressed by the recombinant cells can be effectively combined with the human PSMA protein, so that the method can be used for effectively detecting the content of PSMA in a sample to be detected from a tested individual and effectively diagnosing related diseases caused by the PSMA.

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

According to an embodiment of the present invention, the level of PSMA in the test sample is not less than the minimum level of illness is an indication that the test sample is derived from a patient suffering from a PSMA-induced related disease. The value of the minimum standard can be determined by comparing and analyzing the difference between the amounts of PSMA in a test sample of a large number of individuals suffering from the PSMA-induced related disorder and a large number of healthy individuals, and verifying the difference.

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

According to embodiments of the invention, the PSMA-mediated related disease includes cancer.

In a fifteenth aspect of the invention, the invention features a method of staging PSMA-mediated diseases. According to an embodiment of the invention, the method comprises: detecting PSMA in a sample to be tested using the antibody or antigen-binding fragment of the first aspect, the nucleic acid molecule of the third aspect, the expression vector of the fourth aspect, or the recombinant cell of the fifth aspect; and determining the content of PSMA in the sample to be detected based on the detection result of the PSMA. The antibody or antigen binding fragment, or the nucleic acid molecule, the expression vector and the antibody or antigen binding fragment expressed by the recombinant cells can be effectively combined with human PSMA protein, so that the method can be used for effectively detecting the content of PSMA in a sample to be detected from a tested individual and evaluating the period of related diseases caused by the PSMA based on the content of the PSMA.

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

According to the embodiment of the invention, the content of PSMA in the sample to be tested is not lower than the standard level of tumor stage IV disease, which is an indication that the sample to be tested is derived from a patient suffering from tumor stage IV, and the content of PSMA in the sample to be tested is between the standard levels of tumor stage IV and stage III disease, which is an indication that the sample to be tested is derived from a patient suffering from tumor stage III; the content of PSMA in the sample to be tested is between the standard levels of stage III and stage II tumor diseases, which is an indication that the sample to be tested is derived from a patient suffering from stage II tumor; the level of PSMA in the test sample between the standard levels of stage I and stage II disease is an indication that the test sample was derived from a patient with stage I tumor. It will be appreciated by those skilled in the art that the level of PSMA in the case of stage I, II, III, IV disease of a tumor will vary depending on the type of tumor, and that the stage of the tumor may be determined by comparing the amount of PSMA in the test sample with the standard level of PSMA corresponding to the stage of the tumor, or by comparing the amount of PSMA in the test sample with the amount of PSMA in samples derived from individuals or groups of known disease stages. The values of the standard levels of tumor stage I, stage II, stage III and stage IV can be determined by comparing, analyzing and verifying the difference in the amounts of PSMA in the samples to be tested of a number of individuals suffering from the PSMA-induced related disease and a number of healthy individuals.

In a fifteenth aspect of the invention, the invention features a method of assessing the prognosis of a PSMA-mediated related disease. According to an embodiment of the invention, the method comprises: detecting PSMA in a sample to be tested using the antibody or antigen-binding fragment of the first aspect, the nucleic acid molecule of the third aspect, the expression vector of the fourth aspect, or the recombinant cell of the fifth aspect; and determining the content of PSMA in the sample to be detected based on the detection result of the PSMA. As previously mentioned, the amount of PSMA has an important effect on cancer, and individuals suffering from the disease can be effectively combined with human PSMA by monitoring the amount of PSMA in tissues or excretions thereof, such as peripheral blood, urine, etc., and thus, the method of the invention can effectively detect the amount of PSMA in a test sample derived from a subject, and evaluate the prognosis of the disease caused by PSMA based on the amount of PSMA, for example, by comparing the amount of PSMA in a subject before and after treatment with the amount of PSMA in a normal individual or a diseased individual.

According to an embodiment of the present invention, the above method of assessing disease prognosis may further comprise at least one of the following additional technical features:

According to an embodiment of the invention, the test sample is derived from a patient suffering from PSMA-mediated related disease before or after treatment.

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

The amino acid sequences referred to herein are shown in table B:

Table B: amino acid sequence

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

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 ability of a person skilled in the art. This technique is well explained in the literature, as is the case for molecular cloning: laboratory Manual (Molecular Cloning: A Laboratory Manual), second edition (Sambrook et al, 1989); oligonucleotide Synthesis (Oligonucleotide Synthesis) (M.J.Gait, eds., 1984); animal cell Culture (ANIMAL CELL Culture) (r.i. freshney, 1987); the methods include methods of enzymology (Methods in Enzymology) (academic Press, inc. (ACADEMIC PRESS, inc.), manual of experimental immunology (Handbook of Experimental Immunology) (D.M.Weir and C.C. Blackwell, inc.), gene transfer Vectors for mammalian cells (GENE TRANSFER vector for MAMMALIAN CELLS) (J.M.Miller and M.P.Calos, inc., 1987), methods of contemporary molecular biology (Current Protocols in Molecular Biology) (F.M.Ausubel, et al, 1987), polymerase chain reaction (PCR: the Polymerase Chain Reaction) (Mullis, et al, 1994), and methods of contemporary immunology (Current Protocols in Immunology) (J.E.Coligan, et al, 2011), each of which are expressly incorporated herein by reference.

In the present embodiment, the nucleotide sequence used for preparing the expression vector may be obtained by a conventional method or a conventional software (e.g., on-line program Vectorbuilder (website: https:// www.vectorbuilder.cn/tool/code-optimization. Html), geneOptimizer on-line program, etc.) according to the amino acid sequence thereof.

Example 1: preparation of antibodies

The preparation steps of the antibody are as follows:

(1) ExpiCHO cells (from Thermo Fisher) were cultured using ExpiCHO Expression Medium medium (from Thermo Fisher) and cell concentration was adjusted to 6X 10 ⁶/mL to obtain ExpiCHO cell solution.

(2) When the antibody is a monoclonal antibody, the pcDNA3.4 vector (attorney docket Nanjin Style synthesis) containing nucleotide sequences encoding the heavy and light chains of the antibody was prepared according to 1:1 in a ratio of 2mL OptiSFM medium (available from Thermo Fisher) to obtain solution A;

When the antibody is a bispecific antibody, the pcdna3.4 vector (delegated nanjing gold strep synthesis) containing nucleotide sequences encoding the CD3 antibody, PSMA antibody heavy chain, and PSMA antibody light chain was prepared according to 1:1:1 was added to 2mL OptiSFM medium (available from Thermo Fisher) to obtain solution A.

(3) 160 Μ L ExpiFectamineCHO transfection reagent (from Thermo Fisher) was added to 2mL OptiSFM medium (from Thermo Fisher) to obtain solution B.

(4) Solution a and solution B were then mixed to obtain a transfection mixture, and the transfection mixture was added to the 50mL ExpiCHO cell solution in its entirety over 5 minutes.

(5) After 1 day of incubation at 37℃under 5% CO ₂, 8mL of Feed, 300 μ L ENHANCER (available from Thermo Fisher) was added and the culture supernatant was harvested after 9 days of incubation at 32℃under 5% CO ₂, with 8mL of Feed added on day 5.

(6) The target antibody was obtained by affinity purification from the culture supernatant using a Protein A purification column (from Nami).

Example 2 affinity maturation of antibodies

During affinity maturation of natural antibodies, somatic high frequency mutations are mainly concentrated in the CDR regions. By performing single point saturation mutation at each site of the CDR region through in vitro experiments, enough mutation diversity is obtained, and meanwhile, the protein structure is not destroyed, and the in vitro reproduction of the high-frequency mutation of somatic cells in vivo, which is most similar to that of a natural antibody, can be realized by the method.

Single-point saturation mutation is carried out on each amino acid site of the CDR region, and an unbiased single-point saturation mutation plasmid library of the parent antibody is constructed. ELISA is used for screening mutation sites with enhanced specific binding with antigen, and then the sites are combined and screened to obtain candidate antibody mutation sequences.

By this route, parent PSMA antibody parental mAb (prepared by the method described in example 1, heavy chain variable region SEQ ID NO:23 and light chain variable region SEQ ID NO:24, heavy chain amino acid sequence shown as SEQ ID NO:19, light chain amino acid sequence shown as SEQ ID NO: 20) was affinity-optimized to obtain PSMA monoclonal antibody PSMAvll (prepared by the method described in example 1, heavy chain amino acid sequence shown as SEQ ID NO:11, light chain amino acid sequence shown as SEQ ID NO: 12) having heavy chain variable region (amino acid sequence shown as SEQ ID NO: 7) and light chain variable region (amino acid sequence shown as SEQ ID NO: 8) sequences. A comparison of CDR sequences of parent antibody parental mAb and affinity matured antibody PSMAvll is shown in fig. 1.

Further, the following examples 3 to 6 were carried out using the parental mAb antibody and the PSMAvll antibody prepared in this example:

Example 3 antibody affinity detection

Biacore is a method for analyzing biomolecular interactions based on the principle of optical Surface Plasmon Resonance (SPR), and not only can detect specific binding between antigen and antibody, but also can obtain important data such as intermolecular binding rate constant (Ka), dissociation rate constant (Kd), equilibrium dissociation constant (KD) and the like in drug development, so as to calculate and obtain the affinity of the antibody.

In the Biacore 1K ((Cytiva) system, the antibodies were diluted to 10. Mu.g/mL with running buffer (HBS-EP) and conjugated to protein A ((Cytiva, 29127556) chips at a flow rate of 10. Mu.L/min. Kinetics of antigen binding to antibodies and affinity data were measured at a flow rate of 30. Mu.L/min, the binding time set at 120s and dissociation time at 800s.

Kinetics and affinity data for binding of parent antibody parental mAb and affinity-optimized antibody PSMAvll to PSMA were examined and the results are shown in fig. 2, which shows that affinity-optimized antibody PSMAvll has an approximately 1.8-fold increase in affinity for PSMA and a 4.6-fold decrease in dissociation rate compared to parent antibody parental mAb.

Note that: ka represents the binding rate constant (larger values represent stronger affinities); kd represents the dissociation rate constant (smaller value, stronger affinity), reflecting the size of the affinity of the compound for the target; KD stands for KD/Ka, for equilibrium dissociation constant (affinity constant), smaller KD indicates less dissociation and represents stronger affinity.

Example 4: PSMA antibody ELISA binding assay

The binding properties of antibody PSMAvll and PSMA were optimized using ELISA to detect maternal antibody parental mAb and affinity. The inventors coated PSMA protein into 96-well plates, and the intensity of the signal after antibody addition was used to determine the binding properties of the antibody and PSMA.

PSMA protein (purchased from Acro) was diluted to 1 μg/ml with PBS buffer, added to 96-well plates at a volume of 100 μl/well, and left overnight at 4 ℃. The PBS buffer in the 96-well plate was aspirated, and after washing the plate 6 times with PBST (i.e., 0.1 vol% Tween 20 in PBS at pH 7.2), 200. Mu.L/well of PBS containing 10% BSA was added and incubated at 37℃for 2 hours for blocking. After removing the blocking solution and washing the plate 6 times with PBST, 100. Mu.L/well of maternal antibody parental mAb diluted with a gradient of PBST containing 0.05% BSA (maximum working concentration 20000ng/ml, 5-fold dilution, 8 gradients), affinity optimized antibody PSMAvll, control IgG1 (purchased from Baiying organism) were added and incubated for 1h at 37 ℃. The reaction system in the wells was blotted, and after washing the plate 6 times with PBST, HRP (horseradish peroxidase) -labeled anti-human antibody secondary antibody (Fab-specific) (purchased from Sigma) was diluted with 100. Mu.L/well of PBST containing 0.05% BSA and incubated at 37℃for 1h. After the secondary antibody in the wells was blotted off and the plates were washed 6 times with PBST, 80. Mu.L/well TMB (tetramethylbenzidine) was added, incubated at room temperature for 3min, and the reaction was stopped by adding 80. Mu.L/well 4M sulfuric acid. The absorbance was read with a microplate reader at 450 mm.

The results indicate that affinity optimized antibody PSMAv of the present invention is capable of binding PSMA protein.

Example 5: PSMA antibody flow cytometry binding experiments

Tumor cells (22 RV1 or LNCaP human prostate cancer) were diluted to 2X 10 ⁶/mL with PBS, added to 1.5mL EP tube at a volume of 100. Mu.L/tube, 10. Mu.L/tube goat serum was added thereto, and blocked at 4℃for 30min. A gradient dilution (maximum working concentration of 50 μg/ml, 5-fold dilution, 10 gradients) of maternal antibody parental mAb, affinity optimized antibody PSMAvll, control IgG1 (purchased from the hundred english organism) was added and incubated at 4 ℃ for 30min. To the EP tube, 1mL of PBS was added, centrifuged at 3500rpm at 4℃for 5min, the supernatant was discarded, and the tube was washed once with PBS. After centrifugation, the supernatant was discarded, and the cells were resuspended in 100. Mu.l/tube PBS, to which 1. Mu.l/tube Alexa-647 labeled goat anti-human antibody secondary antibody (purchased from Jackson lab) was added and incubated at 4℃for 30min in the absence of light. Washed twice with PBS and the supernatant was discarded after centrifugation. Cells were resuspended in 200 μl/tube PBS and examined by flow cytometry.

The results are shown in fig. 3 and 4, and show that the affinity optimized antibody PSMAv11 of the invention binds to 22RV1, and that LNCaP human prostate cancer cells have higher fluorescence intensity than the parent antibody, indicating that the affinity optimized antibody PSMAv11 has higher binding activity.

Example 6 PSMA antibody promotes PBMC killing of tumor cells

PSMA mab was tested for its ability to promote PBMC killing of 22RV1, LNCaP human prostate cancer cells.

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

(2) Tumor cells were diluted to 2X 10 ⁵/mL with complete RPMI-1640 medium, added individually to the RTCA plate obtained in addition step (1) in a volume of 50. Mu.L/well, and then tested for cell factor 24h using xCELLigence RTCA MP apparatus at 37℃under 5% CO ₂;

(3) Gradient diluting a parent antibody parental mAb and an affinity optimization antibody PSMAvll by using a complete RPMI-1640 culture medium, and adding the mixture into the RTCA plate obtained in the step (2), wherein the addition volume is 20 mu L/hole;

(4) Diluting PBMC (from Chimaphila) to 1.25X10 ⁶/mL with complete RPMI-1640 medium, adding to the RTCA plate obtained in step (3) in a volume of 80. Mu.L/well;

(5) The reaction system obtained in the step (4) was subjected to cell factor detection at 37℃with 5% CO ₂ using xCELLigence RTCA MP apparatus for 24 hours.

As shown in fig. 5 and 6, the PSMAv antibody of the invention promotes PBMC to kill tumor, and the killing promoting function is stronger than that of the parent antibody parental mAb.

Example 7: CD3 XPSMA antibody flow cytometry binding experiments

Experimental group CD 3X PSMAv11 bispecific antibodies (wherein the amino acid sequence of the CD3 antibody is shown as SEQ ID NO:17, the amino acid sequence of the heavy chain is shown as SEQ ID NO:18, the amino acid sequence of the light chain is shown as SEQ ID NO: 12) and control group CD 3X parental mAb bispecific antibodies (wherein the amino acid sequence of the CD3 antibody is shown as SEQ ID NO:17, the amino acid sequence of the heavy chain is shown as SEQ ID NO:21, the amino acid sequence of the light chain is shown as SEQ ID NO: 20) were prepared respectively according to the method of example 1. Then, a flow cytometry combination experiment is carried out, and the specific experimental steps are as follows:

Tumor cells were diluted to 2X 10 ⁶/mL with PBS, added to 1.5mL EP tube at a volume of 100. Mu.L/tube, 10. Mu.L/tube goat serum was added thereto, and blocked at 4℃for 30min. Gradient dilutions of CD 3X PARENTAL PSMA bispecific antibody, CD 3X PSMAvll bispecific antibody, control IgG1LALA (purchased from Baiying organism) were added and incubated for 30min at 4 ℃. 1mL of PBS was added to the EP tube, centrifuged at 3500 rpm.times.5 min at 4℃and the supernatant was discarded and washed once with PBS. After centrifugation, the supernatant was discarded, and the cells were resuspended in 100. Mu.l/tube PBS, to which 1. Mu.l/tube Alexa-647 labeled goat anti-human antibody secondary antibody (purchased from Jackson lab) was added and incubated at 4℃for 30min in the absence of light. Washed twice with PBS and the supernatant was discarded after centrifugation. Cells were resuspended in 200 μl/tube PBS and examined by flow cytometry.

The results are shown in fig. 7 and 8, and demonstrate that the CD 3x PSMAv11 binding 22RV1, LNCaP human prostate cancer cells of the present invention are stronger than the CD 3x PARENTAL PSMA bispecific antibody.

Example 8 CD3×PSMA antibodies promote PBMC killing of tumor cells

The CD3 x PSMA bispecific antibody and CD3 x parental mAb bispecific antibody obtained in example 7 were tested for their ability to promote PBMC killing 22RV1, LNCaP human prostate cancer cells. The method comprises the following specific steps:

(3) Gradient dilution of CD 3X PARENTAL PSMA bispecific antibody, CD 3X PSMAvll bispecific antibody, control IgG1LALA (purchased from Baiying organism) with complete RPMI-1640 medium, adding into RTCA plate obtained in step (2) with addition volume of 20 μl/well;

As shown in fig. 9, 10, the CD3 x PSMAv11 antibody of the invention promoted PBMC killing of tumors much more than the CD3 x PARENTAL PSMA bispecific antibody.

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

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

Claims

1. An antibody or antigen-binding fragment comprising CDRs selected from at least one of the following:

HCDR 1 GYSFTX ₁ NW, wherein X ₁ is S or H;

HCDR 2:IYPGDSDT；

HCDR 3:ARQTGFLWSSDLWGRGT；

LCDR 1:X ₂QDISX₃ a, wherein X ₂ is S or P, X ₃ is S or Y;

LCDR 2 DASX ₄, wherein X ₄ is S or W;

LCDR 3 is QQFNSPYLX ₅, wherein X ₅ is T or S;

X ₁ is S, X ₂, S, X ₃ is S, X ₄, S, X ₅ is T are not present at the same time.

2. The antibody or antigen-binding fragment of claim 1, wherein X ₁ is S;

Optionally, X ₁ is H;

optionally, X ₂ is S;

Optionally, X ₂ is P;

optionally, X ₃ is S;

optionally, X ₃ is Y;

optionally, X ₄ is S;

Optionally, X ₄ is W;

optionally, X ₅ is T;

Optionally, X ₅ is S;

optionally, the antibody or antigen binding fragment comprises:

HCDR1, HCDR2, HCDR3 shown as amino acid sequences of SEQ ID NOs 1,2 and 3, respectively; and

LCDR1, LCDR2, LCDR3 shown as 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 of claim 2, wherein 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 light chain framework region is derived from at least one of a murine antibody, a primatized antibody, a bovine antibody, a equine antibody, a dairy-bovine antibody, a porcine antibody, a ovine antibody, a caprine antibody, a canine antibody, a feline 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 bucket chicken antibody, or a mutant thereof;

optionally, 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;

optionally, the antibody or antigen binding fragment comprises:

A heavy chain variable region of an amino acid sequence as set forth in SEQ ID NO. 7 or an amino acid sequence having at least 90% homology thereto; and

The amino acid sequence shown in SEQ ID NO. 8 or an amino acid sequence having at least 90% homology thereto.

4. The antibody or antigen-binding fragment of any one of claims 1-3, 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 and light chain constant regions is derived from at least one of a murine, primates, bovine, equine, dairy, porcine, ovine, caprine, canine, feline, rabbit, camel, donkey, deer, mink, chicken, duck, goose, turkey, bucket, or mutant thereof;

optionally, the heavy chain constant region comprises a heavy chain constant region selected from the group consisting of IgG1, igG2, igG3, igG4, igA, igM, igE, or IgD; or alternatively

The light chain constant region comprises a light chain constant region selected from the group consisting of kappa-type or lambda-type;

optionally, the light chain constant region and the heavy chain constant region are both derived from a murine antibody or mutant thereof, and/or a human antibody or mutant thereof;

Optionally, the N-terminus of the heavy chain constant region is linked to the C-terminus of the heavy chain variable region; and/or

The N-terminal of the light chain constant region is connected with the C-terminal of the light chain variable region;

optionally, the heavy chain constant region comprises a heavy chain constant region as set forth in SEQ ID No. 9 or an amino acid sequence having at least 80% identity thereto; and/or

The light chain constant region comprises a light chain constant region as 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 F (ab') ₂ antibody, an Fv antibody, a single chain antibody, a single domain antibody, and a minimal recognition unit; or alternatively

The antigen binding fragments include at least one of a F (ab ') ₂ fragment, a Fab' fragment, a Fab fragment, a F (ab) ₂ fragment, an Fv fragment, an scFv-Fc fusion protein, an scFv-Fv fusion protein, and a minimal recognition unit;

optionally, the antibody or antigen binding fragment comprises:

a heavy chain of the amino acid sequence shown as SEQ ID NO. 11 or an amino acid sequence having at least 80% homology thereto; and

An amino acid sequence as set forth in SEQ ID NO. 12 or an amino acid sequence having at least 80% homology thereto.

5. A multispecific antibody comprising:

A first binding region comprising the antibody or antigen binding fragment of any one of claims 1-4;

a second binding region, said second binding region having a first molecular binding activity.

6. The multispecific antibody of claim 5, wherein the first molecule is selected from at least one of an immune cell surface antigen, a tumor antigen, a virus, a bacterium, an endotoxin, a cytokine, and a cytokine receptor;

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 CD16 a;

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 of the first molecule or fragment thereof is linked to the N-terminus of the first Fc fragment;

Optionally, the second binding region further comprises a linking peptide;

optionally, the C-terminus of the binding protein of the first molecule or fragment thereof is linked to the N-terminus of the linker peptide, which is linked 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-CD 3 antibody;

optionally, the anti-CD 3 antibody is selected from at least one of a Fab antibody, a Fab 'antibody, a F (ab') ₂ antibody, an Fv antibody, a single chain antibody, a single domain antibody, and a minimal recognition unit;

Optionally, the anti-CD 3 antibody is a CD3 single chain antibody;

optionally, the C-terminus of the CD3 single chain antibody is linked to the N-terminus of the linker peptide, which is linked to the N-terminus of the first Fc fragment;

Optionally, the CD3 single chain antibody has an amino acid sequence as shown in SEQ ID NO. 13;

Optionally, the linker peptide has an amino acid sequence as shown in SEQ ID NO. 14;

optionally, the first Fc fragment has an amino acid sequence as shown in SEQ ID NO. 15;

optionally, the first binding region has the amino acid sequence shown as SEQ ID NO. 17.

7. The multispecific antibody according to any one of claims 5 to 6, wherein the antibody or antigen-binding fragment in the first binding region 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 minimal recognition unit, preferably is a Fab antibody or a 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 a heavy chain variable region and a light chain variable region in the antibody or antigen binding fragment of 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 linked to the N-terminus of the second Fc fragment;

Optionally, the second Fc fragment has an amino acid sequence as 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 the amino acid sequence shown in SEQ ID NO. 12.

8. A nucleic acid molecule encoding 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 carrying the nucleic acid molecule of claim 8;

Optionally, the expression vector comprises a vector selected from eukaryotic expression vectors or prokaryotic expression vectors.

10. A recombinant cell comprising:

Carrying the nucleic acid molecule of claim 8 or the expression vector of claim 9; or alternatively, the first and second heat exchangers may be,

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, comprising:

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 coupling moiety linked to the antibody or antigen binding fragment or multispecific antibody;

optionally, the conjugate moiety comprises at least one member selected from the group consisting of a carrier, a drug, a toxin, a cytokine, a protein tag, and a modification.

12. A pharmaceutical composition comprising:

The antibody or antigen-binding fragment of any one of claims 1-4, the multispecific antibody of any one of claims 5-7, 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, further comprising pharmaceutically acceptable excipients.

13. A kit, comprising:

the antibody or antigen binding fragment of any one of claims 1-4, the multispecific antibody of any one of claims 5-7, 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. Use 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 nucleic acid molecule of claim 8, the expression vector of claim 9, the recombinant cell of claim 10 or the conjugate of claim 11 in the preparation of a medicament for the prevention and/or treatment of a PSMA-mediated related disease;

optionally, the PSMA-mediated related disease comprises a tumor and/or cancer;

optionally, the cancer is prostate cancer.

15. Use of the antibody or antigen-binding fragment of any one of claims 1-4, the multispecific antibody of any one of claims 5-7, 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 composition of claim 12 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 assessing a PSMA-mediated related disease prognosis;

optionally, the PSMA-mediated related disease comprises a tumor and/or cancer;

optionally, the cancer is prostate cancer.