PSCA-targeting nanobody and use thereof

Abstract

Provided are a PSCA-targeting nanobody and the use thereof. The PSCA-targeting nanobody comprises a heavy chain variable region, the heavy chain variable region comprising CDR1, CDR2 and CDR3, the CDR1 comprising an amino acid sequence shown as SEQ ID NO: 27, the CDR2 comprising an amino acid sequence shown as SEQ ID NO: 28, and the CDR3 comprising an amino acid sequence shown as SEQ ID NO: 29. The provided nanobody can specifically bind to PSCA, and has high specificity, effectiveness and safety, and thus has good application prospects in the treatment of tumors.

Description

Nanobodies targeting PSCA and uses thereof

[0001] This application claims priority to Chinese patent application 2024112774539 with a filing date of 2024 / 9 / 12. This application incorporates the entirety of the aforementioned Chinese patent application. TECHNICAL FIELD

[0002] The present application relates to the technical field of antibody drugs, and particularly relates to nanobodies targeting PSCA and uses thereof. BACKGROUND

[0003] Prostate stem cell antigen (PSCA) is a very attractive target for the treatment and detection of prostate cancer, bladder cancer and pancreatic cancer. It is composed of 123 amino acids, is a glycosylphosphatidylinositol (GPI) anchored protein, belongs to the Thy-1 / Ly-6 family, and its expression is related to cancer clinical stage, invasion and metastasis [1] .

[0004] At present, a number of research teams have developed specific double antibodies, CAR-T, and PET probes targeting PSCA, and most of the proteins that bind to the extracellular segment of PSCA are monoclonal antibodies or antibody scfv fragments [2,3] . However, these antibodies have some limitations, for example, the long half-life of antibodies leads to long retention of drugs in the blood, which may cause toxic side effects. In addition, the large size of antibodies limits their ability to penetrate tumor core tissues, reducing drug delivery efficiency. An effective method is to combine antibodies and therapeutic drugs to achieve precise targeted drug delivery and reduce non-specific toxicity.

[0005] Nanobodies, as the smallest antigen-binding fragments in nature, have a stable structure and a relatively short half-life because their molecular weight is below the threshold for glomerular filtration. Nanobodies have been used in a number of studies to develop nanobody-drug conjugates (NDCs), however, there is currently no relevant data on NDCs for treating cancer targeting PSCA.

[0006] REFERENCES

[0007] [1] REITER R E, GU Z, WATABE T, et al. Prostate stem cell antigen: a cell surface marker overexpressed in prostate cancer [J]. Proc Natl Acad Sci U S A, 1998, 95(4): 1735-40.

[0008] [2]DORFF T B, BLANCHARD M S, ADKINS L N, et al. PSCA-CAR T cell therapy in metastatic castration-resistant prostate cancer: a phase 1 trial [J]. Nat Med, 2024.

[0009] [3]STRIESE F, NEUBER C, GRASSEL S, et al. Preclinical Characterization of the (177) Lu-Labeled Prostate Stem Cell Antigen (PSCA)-Specific Monoclonal Antibody 7F5 [J]. Int J Mol Sci, 2023, 24(11). SUMMARY

[0010] In view of the lack of antibodies targeting PSCA with high specificity, effectiveness and safety in the prior art, the present application provides a nanobody targeting PSCA, which has high specificity, effectiveness and safety. The present application uses phage display technology to screen nanobodies targeting PSCA, which has a molecular weight of 15 kDa. In addition, the present application couples the nanobody with the drug vc-MMAE, and the nanobody-drug conjugate (NDCs) shows significant tumor inhibition effect in vitro, has obvious killing effect on tumor cells such as prostate cancer, bladder cancer and pancreatic cancer, and has concentration dependence.

[0011] The present application solves the above technical problems through the following technical solutions.

[0012] The present application provides a nanobody targeting PSCA in the first aspect, which comprises a heavy chain variable region, the heavy chain variable region comprising CDR1, CDR2 and CDR3, the CDR1 comprising an amino acid sequence as shown in SEQ ID NO: 27, the CDR2 comprising an amino acid sequence as shown in SEQ ID NO: 28, and the CDR3 comprising an amino acid sequence as shown in SEQ ID NO: 29.

[0013] In some embodiments of the application, the CDR1 has an amino acid sequence as set forth in SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, or SEQ ID NO: 26; the CDR2 has an amino acid sequence as set forth in SEQ ID NO: 17 or SEQ ID NO: 20; and, the CDR3 has an amino acid sequence as set forth in SEQ ID NO: 18, SEQ ID NO: 22, or SEQ ID NO: 24.

[0014] In some preferred embodiments of the application, the CDR1 has an amino acid sequence as set forth in SEQ ID NO: 16, the CDR2 has an amino acid sequence as set forth in SEQ ID NO: 17, and the CDR3 has an amino acid sequence as set forth in SEQ ID NO: 18; or,

[0015] the CDR1 has an amino acid sequence as set forth in SEQ ID NO: 19, the CDR2 has an amino acid sequence as set forth in SEQ ID NO: 20, and the CDR3 has an amino acid sequence as set forth in SEQ ID NO: 18; or,

[0016] the CDR1 has an amino acid sequence as set forth in SEQ ID NO: 21, the CDR2 has an amino acid sequence as set forth in SEQ ID NO: 20, and the CDR3 has an amino acid sequence as set forth in SEQ ID NO: 22; or,

[0017] the CDR1 has an amino acid sequence as set forth in SEQ ID NO: 23, the CDR2 has an amino acid sequence as set forth in SEQ ID NO: 20, and the CDR3 has an amino acid sequence as set forth in SEQ ID NO: 24; or,

[0018] the CDR1 has an amino acid sequence as set forth in SEQ ID NO: 25, the CDR2 has an amino acid sequence as set forth in SEQ ID NO: 20, and the CDR3 has an amino acid sequence as set forth in SEQ ID NO: 24; or,

[0019] the CDR1 has an amino acid sequence as set forth in SEQ ID NO: 26, the CDR2 has an amino acid sequence as set forth in SEQ ID NO: 20, and the CDR3 has an amino acid sequence as set forth in SEQ ID NO: 18; or,

[0020] the CDR1 has an amino acid sequence as set forth in SEQ ID NO: 21, the CDR2 has an amino acid sequence as set forth in SEQ ID NO: 20, and the CDR3 has an amino acid sequence as set forth in SEQ ID NO: 24.

[0021] In some embodiments of the present application, the nanobody comprises (1) or (2) below:

[0022] (1) the heavy chain variable region comprises an amino acid sequence as set forth in any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7;

[0023] (2) the heavy chain variable region comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1, preferably an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1 in the FR region, or,

[0024] the heavy chain variable region comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 2, preferably an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 2 in the FR region; or,

[0025] the heavy chain variable region comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 3, preferably an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 3 in the FR region; or,

[0026] the heavy chain variable region comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 4, preferably an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 4 in the FR region; or,

[0027] the heavy chain variable region comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 5, preferably an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 5 in the FR region; or,

[0028] the heavy chain variable region comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 6, preferably an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 6 in the FR region; or,

[0029] The heavy chain variable region comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 7, preferably an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity in FR regions compared to SEQ ID NO: 3.

[0030] In some embodiments of the present application, the nanobody is a monoclonal antibody or a polyclonal antibody; and / or, the nanobody is a humanized antibody.

[0031] The second aspect of the present application provides a PSCA binding molecule, which comprises one, two or more nanobodies as described in the first aspect.

[0032] In some embodiments of the present application, the PSCA binding molecule further satisfies at least one of the following characteristics:

[0033] (1) the PSCA binding molecule is a heavy chain antibody; the heavy chain antibody comprises a nanobody as described in any one of the first aspect and a heavy chain constant region, the heavy chain constant region comprising CH2 and / or CH3; the heavy chain constant region is preferably derived from a heavy chain constant region of a human antibody;

[0034] (2) the PSCA binding molecule is a recombinant fusion protein, which comprises a first functional domain of a nanobody as described in the first aspect, and further comprises a second functional domain for prolonging the half-life in vivo or targeting another target, the structure of the second functional domain being, for example, Fab, scFv, VHH, Fc region of IgG or albumin;

[0035] (3) the PSCA binding molecule is a mono-specific antibody, a bi-specific antibody or a multi-specific antibody.

[0036] In some optional embodiments of the present application, the Fc region of IgG is a Fc region of IgG1.

[0037] In some optional embodiments of the present application, the Fc region of IgG1 has the amino acid sequence shown in SEQ ID NO: 30.

[0038] In some specific optional embodiments of the present application, the Fc region of IgG1 has the amino acid sequence shown in SEQ ID NO: 30.

[0039] The third aspect of the present application provides an isolated nucleic acid, which encodes a nanobody as described in the first aspect or a PSCA binding molecule as described in the second aspect.

[0040] In some embodiments of the present application, the nucleic acid comprises a nucleotide sequence as set forth in any one of SEQ ID NOs: 8-14.

[0041] In some embodiments of the present application, the nucleic acid comprises a nucleotide sequence as set forth in any one of SEQ ID NOs: 8-14.

[0042] In some embodiments of the present application, the nucleic acid further comprises a coding sequence of the Fc region of the IgG when the nucleic acid encodes the PSCA binding molecule.

[0043] In some embodiments of the present application, the coding sequence of the Fc region of the IgG is as set forth in SEQ ID NO: 31.

[0044] The fourth aspect of the present application provides a recombinant expression vector comprising the nucleic acid of the third aspect.

[0045] In some embodiments of the present application, the backbone of the recombinant expression vector is a plasmid, a cosmid, a phage or a viral vector.

[0046] In some preferred embodiments of the present application, the backbone of the plasmid is pComb.

[0047] In some specific embodiments of the present application, the backbone of the plasmid is pComb3X.

[0048] The fifth aspect of the present application provides a transformant comprising the nucleic acid of the third aspect or the recombinant expression vector of the fourth aspect.

[0049] In some embodiments of the present application, the host cell of the transformant is a prokaryotic cell, a yeast cell, an insect cell or an animal cell.

[0050] In some preferred embodiments of the present application, the host cell is E. coli.

[0051] In some specific embodiments of the present application, the host cell is HB2151 E. coli.

[0052] The sixth aspect of the present application provides an antibody drug conjugate comprising the nanobody of the first aspect or the PSCA binding molecule of the second aspect, and a cytotoxic agent or a label.

[0053] In some embodiments of the present application, the cytotoxic agent is MMAE or MMAF.

[0054] The seventh aspect of the present application provides a chimeric antigen receptor comprising the nanobody of the first aspect or the PSCA binding molecule of the second aspect.

[0055] In a eighth aspect, the present application provides a genetically modified cell, comprising the chimeric antigen receptor of the seventh aspect.

[0056] In some embodiments of the present application, the genetically modified cell is derived from a eukaryotic cell.

[0057] In some preferred embodiments of the present application, the genetically modified cell is derived from an isolated human cell.

[0058] In some embodiments of the present application, the genetically modified cell is derived from an immune cell.

[0059] In some preferred embodiments of the present application, the genetically modified cell is derived from a T cell or a NK cell.

[0060] In a ninth aspect, the present application provides a method of producing the nanobody of the first aspect or the PSCA binding molecule of the second aspect, comprising the steps of culturing the transformant of the fifth aspect, and obtaining the nanobody or the PSCA binding molecule from the culture.

[0061] In a tenth aspect, the present application provides a pharmaceutical composition comprising the nanobody of the first aspect, the PSCA binding molecule of the second aspect, the drug conjugate of the antibody of the sixth aspect, the chimeric antigen receptor of the seventh aspect, and / or the cell of the eighth aspect, and a pharmaceutically acceptable carrier or excipient.

[0062] In an eleventh aspect, the present application provides a drug delivery device comprising the nanobody of the first aspect, the PSCA binding molecule of the second aspect, the drug conjugate of the antibody of the sixth aspect, the chimeric antigen receptor of the seventh aspect, the cell of the eighth aspect, and / or the pharmaceutical composition of the tenth aspect.

[0063] In some embodiments of the present application, the drug delivery device further comprises a means for administering the nanobody, the PSCA binding molecule, the drug conjugate of the antibody, the chimeric antigen receptor, and / or the pharmaceutical composition to a subject, such as a syringe or an infusion device.

[0064] The twelfth aspect of the present application provides use of the nanobodies according to the first aspect, the PSCA binding molecules according to the second aspect, the nucleic acids according to the third aspect, the recombinant expression vectors according to the fourth aspect, the transformants according to the fifth aspect, the antibody drug conjugates according to the sixth aspect, the chimeric antigen receptors according to the seventh aspect, the cells according to the eighth aspect or the pharmaceutical compositions according to the tenth aspect in the preparation of a medicament for preventing, treating, diagnosing and / or prognosticating a PSCA high expression related disease.

[0065] In some embodiments of the present application, the PSCA high expression related disease is a tumor.

[0066] In some specific embodiments of the present application, the PSCA high expression related disease is prostate cancer, bladder cancer or pancreatic cancer.

[0067] The thirteenth aspect of the present application provides a method for detecting PSCA, which comprises the following steps:

[0068] (1) contacting a sample to be tested with the nanobodies according to the first aspect or the PSCA binding molecules according to the second aspect;

[0069] (2) qualitatively or quantitatively judging according to the binding of the sample to be tested with the nanobodies or the PSCA binding molecules.

[0070] In some embodiments of the present application, the method is not for diagnostic purposes.

[0071] In some embodiments of the present application, the method is in vitro.

[0072] The fourteenth aspect of the present application provides a method for preventing, treating, diagnosing and / or prognosticating a PSCA high expression related disease, which comprises administering to a patient the nanobodies according to the first aspect, the PSCA binding molecules according to the second aspect, the antibody drug conjugates according to the sixth aspect, the chimeric antigen receptors according to the seventh aspect, the pharmaceutical compositions according to the tenth aspect or the drug delivery devices according to the eleventh aspect.

[0073] In some embodiments of the present application, the PSCA high expression related disease is a tumor.

[0074] In some specific embodiments of the present application, the PSCA high expression related disease is prostate cancer, bladder cancer or pancreatic cancer.

[0075] The fifteenth aspect of the present application provides the nanobody of the first aspect, the PSCA binding molecule of the second aspect, the nucleic acid of the third aspect, the recombinant expression vector of the fourth aspect, the transformant of the fifth aspect, the drug conjugate of the antibody of the sixth aspect, the chimeric antigen receptor of the seventh aspect, the cell of the eighth aspect, the pharmaceutical composition of the tenth aspect or the administration device of the eleventh aspect for use in preventing, treating, diagnosing and / or prognosticating a PSCA high expression related disease.

[0076] In some embodiments of the present application, the PSCA high expression related disease is a tumor.

[0077] In some specific embodiments of the present application, the PSCA high expression related disease is prostate cancer, bladder cancer or pancreatic cancer.

[0078] In the present application, the amino acid sequences of the listed complementarity determining regions (CDRs) are shown according to the definition of the Kabat numbering convention (Table 1). However, it is well known in the art that CDRs of an antibody can be defined in various ways in the art, for example, Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loops (Chothia et al. (1989) Nature 342: 877-883, Al-Lazikani et al., “Standard conformations for the canonical structures of immunoglobulins”, Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on the variability of the antibody sequence (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Ed., U.S. Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the international ImMunoGeneTics database (IMGT, http: / / imgt.cines.fr / ), and North CDR definition based on affinity propagation clustering using a large number of crystal structures. It will be understood by a person skilled in the art that, unless otherwise specified, the terms “CDR” and “complementarity determining region” of a given antibody or region thereof (e.g. variable region) are to be understood as encompassing complementarity determining regions defined as by any of the above known approaches described in the present application.

[0079] Table 1. Antibody CDR definition methods of the present application (see http: / / bioinf.org.uk / abs / )

[0080] wherein Haa-Hbb can refer to the amino acid sequence from the N-terminus of the heavy chain of the antibody, aa position (Kabat numbering convention) to bb position (Chothia numbering convention). For example, H31-H35 can refer to the amino acid sequence from the N-terminus of the heavy chain of the antibody, from position 31 to position 35 according to the Kabat numbering convention.

[0081] Thus, where an antibody is defined by reference to specific CDR sequences as defined herein, the scope of the antibody also encompasses antibodies whose variable region sequences comprise the specified CDR sequences but whose CDR boundaries differ from those defined herein as a result of a different numbering system being employed (e.g. a different assignment system or combination). Although the scope of antibodies claimed herein is based on the sequences shown defined according to the Kabat numbering convention, amino acid sequences corresponding to the CDRs according to other numbering conventions should also fall within the scope of the present application.

[0082] In the present application, "PSCA binding molecules" are proteins having the function of recognizing and binding to PSCA, including but not limited to antibodies, antigen-binding fragments of antibodies, heavy chain antibodies, nanobodies, minibodies, affibodies, target binding regions of receptors, cell adhesion molecules, ligands, enzymes, cytokines and chemokines.

[0083] In the present application, "single domain antibodies", "VHH" and "nanobodies" are used interchangeably, all referring to nanobodies that specifically recognize and bind to PSCA. Nanobodies are variable regions of heavy chain antibodies. Typically, nanobodies contain three CDRs and four FRs. Nanobodies are the smallest functional antigen-binding fragments. Typically, after obtaining antibodies naturally lacking light chains and heavy chain constant region 1 (CH1), the variable region of the antibody heavy chain is cloned to construct a nanobody consisting of only one heavy chain variable region.

[0084] In the present application, binding molecules containing two or more nanobodies are multivalent nanobodies; binding molecules containing two or more nanobodies of different specificity are multispecific nanobodies. Multivalent nanobodies or multispecific nanobodies connect multiple nanobodies through linkers. The linkers are usually composed of 1-15 amino acids selected from G and S, for example (G4S)3.

[0085] In the present application, "multispecific antibodies" refer to a class of antibodies that can recognize and bind to at least two different antigen epitopes at the same time, for example bispecific antibodies.

[0086] In the present application, "fusion proteins" refer to proteins formed by genetically linking two or more different protein or polypeptide sequences together. Among them, the structure of Fc fusion protein is composed of two parts, namely the Fc fragment of immunoglobulin and the nanobody described in the present application. The two have relatively independent domains and functions, and can affect the physicochemical properties and biological activities of themselves from different aspects. Fc fusion protein dimerization can form heavy chain antibodies.

[0087] In the present invention, "heavy chain antibody" is an antibody derived from a Camelidae organism or a Chondrichthyes organism. In comparison with the above-mentioned full-length antibody, the heavy chain antibody lacks a light chain and a heavy chain constant region 1 (CH1), and only contains 2 heavy chains composed of a variable region (VHH) and other constant regions, with the variable region being connected to the constant region by a hinge-like region structure. Each heavy chain of the Camelidae heavy chain antibody contains 1 variable region (VHH) and 2 constant regions (CH2 and CH3), and each heavy chain of the Chondrichthyes heavy chain antibody contains 1 variable region and 5 constant regions (CH1-CH5). The antigen-binding fragment of the heavy chain antibody includes VHH and single-chain heavy chain antibody. By being fused with the constant region of human IgG Fc, the heavy chain antibody can have CH2 and CH3 of human IgG Fc.

[0088] In the present invention, heavy chain antibody and antibody are intended to distinguish different combination manners of antibodies.

[0089] Without materially affecting the activity of the antibody, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acids can be changed by those skilled in the art to obtain a variant of the sequence of the antibody or a functional fragment thereof. These variants include, but are not limited to, deletion, insertion, and / or substitution of one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acids, and addition of one or a few (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminal and / or N-terminal. In the art, when a conservative substitution is made with an amino acid having similar or similar properties, the function of the protein is usually not changed. For example, substitution of amino acids with similar properties in the FR and / or CDR regions of the variable region. Amino acid residues that can be subjected to conservative substitution are well known in the art. Such substituted amino acid residues can or can not be encoded by the genetic code. For another example, addition of one or a few amino acids at the C-terminal and / or N-terminal usually does not change the function of the protein. They are all considered to be included in the scope of protection of the present invention.

[0090] The variant forms of the various antibodies of the present invention include homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize to the coding DNA of the various antibodies of the present invention under high or low stringency conditions, and polypeptides or proteins obtained using antiserum against the various antibodies of the present invention.

[0091] In some embodiments, the sequences of the variants described herein can have at least 95%, 96%, 97%, 98%, or 99% identity to the sequence from which they are derived. Sequence identity as described herein can be measured using sequence analysis software, for example, the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. The present application also includes molecules having an antibody heavy chain variable region with CDRs, provided that the CDRs have more than 90% (more preferably more than 95%, most preferably more than 98%) sequence identity to the CDRs identified herein.

[0092] Antibodies of the present application can be prepared using methods conventional in the art, such as hybridoma technology, which is well known in the art. Nanobodies and heavy chain antibodies of the present application can be prepared using methods conventional in the art, such as phage display technology, which is well known in the art. Alternatively, various antibodies of the present application can be expressed in other cell lines. Suitable mammalian host cells can be transformed with sequences encoding the various antibodies of the present application. Transformation can be performed using any known method, including, for example, packaging the polynucleotide in a virus (or viral vector) and transducing the host cell with the virus (or vector). The transformation procedure used depends on the host to be transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art, including dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide in liposomes, and direct microinjection of the DNA into nuclei, among others. Mammalian cell lines useful as hosts for expression are well known in the art, including, but not limited to, a variety of immortalized cell lines available from the American Type Culture Collection (ATCC), including, but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and the like. Particularly preferred cell lines are selected by determining which cell lines have high expression levels and produce antibodies with the essential GPC3 binding properties.

[0093] In the present application, "nucleic acid" refers to a chain of nucleotides of any length and includes DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a chain by the action of a DNA or RNA polymerase.

[0094] In the present invention, the term "recombinant expression vector" means a genetically modified oligonucleotide or polynucleotide construct that permits expression of an mRNA, protein, polypeptide, or peptide by a host cell when the construct comprises a nucleotide sequence encoding the mRNA, protein, polypeptide, or peptide, and the vector is contacted with a cell under conditions sufficient for the mRNA, protein, polypeptide, or peptide to be expressed in the cell. The vectors of the present invention are not naturally occurring in general. However, portions of the vectors can be naturally occurring. The recombinant expression vectors of the present invention can comprise any type of nucleotide, including but not limited to DNA and RNA, which can be single-stranded or double-stranded, synthetic or obtained in part from natural sources, and which can contain natural, non-natural or altered nucleotides. The recombinant expression vectors can comprise naturally occurring or non-naturally occurring internucleotide linkages, or both types of linkages. In exemplary aspects, the altered nucleotides or non-naturally occurring internucleotide linkages do not hinder transcription or replication of the vector.

[0095] The recombinant expression vectors of the present invention can be any suitable recombinant expression vector that can be used to transform or transfect to deliver one or more genes or sequences of interest into any suitable host cell and preferably express the genes or sequences in the host cell. Suitable vectors include those designed for expansion and amplification or for expression or both, examples of vectors include but are not limited to viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or bacteriophage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.

[0096] In the present invention, the term "host cell" refers to any type of cell that can contain the nucleic acids or vectors described herein. In exemplary aspects, the host cell is a eukaryotic cell, such as a plant, animal, fungus, or alga; or can be a prokaryotic cell, such as a bacterium or protozoan.

[0097] In the present invention, the pharmaceutical composition can comprise a suitable pharmaceutically acceptable carrier, such as pharmaceutical adjuvants, as known in the art, pharmaceutical excipients, including buffers. "Pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. A pharmaceutical composition comprising the pharmaceutical composition described in the present invention, preferably in lyophilized form or in aqueous solution, can be prepared by mixing the antibody of the present invention having the desired degree of purity with one or more optional pharmaceutical adjuvants (Remington's Pharmaceutical Sciences, 16thEdition, Osol, A. Ed. (1980)), preferably in the form of a lyophilized formulation or an aqueous solution.

[0098] The pharmaceutical composition of the present application can also comprise more than one active ingredient, which is required for the particular indication being treated, preferably those having complementary activities that do not adversely affect each other. For example, it is desirable to further provide other active ingredients, such as other antibodies, antiviral agents, small molecule drugs or immunomodulators, etc. The active ingredients are present in amounts that are effective for the purpose intended. Sustained-release preparations can be prepared. Suitable examples include semi-permeable matrices of solid hydrophobic polymers containing the antibody of the present application, which matrices are in the form of shaped articles, e.g., films or microcapsules.

[0099] In the present application, a chimeric antigen receptor (CAR) is an engineered transmembrane protein that combines the specificity of an antigen-specific antibody with the function of a T cell receptor. Generally, a CAR comprises an extracellular domain, a transmembrane domain, and an intracellular domain. In exemplary aspects, the extracellular domain of a CAR comprises an antigen recognition region, which can be a scFV of an antigen-specific antibody.

[0100] In the present application, the term "Antibody-Drug Conjugate" generally refers to an antibody linked to a cytotoxic drug, a radionuclide or a label having biological activity through a stable linking unit. The "cytotoxic drug" can include a toxin, such as a small molecule toxin or an enzymatically active toxin of bacterial, fungal, plant or animal origin, a toxic drug, a chemotherapeutic drug, an antibiotic or a nucleolytic enzyme, or a derivative thereof.

[0101] In the present application, the application scenario of "non-diagnostic purposes" includes but is not limited to, for example, in vitro detection of the presence or absence of an antigen in a laboratory; or as a positive antibody to screen other antibodies targeting the same; or competing with other antibodies targeting the same to detect whether there is competition between antibodies, i.e., whether the antigen epitopes are the same or similar, etc.

[0102] On the basis of common general knowledge in the art, the above-mentioned preferred conditions can be combined in any manner, i.e., to obtain each preferred example of the present application.

[0103] The reagents and raw materials used in the present application are commercially available.

[0104] The positive progress effect of the present application is that a nanobody targeting PSCA with high specificity, effectiveness and safety is provided, and the antibody drug conjugate obtained on the basis of the nanobody has a significant killing effect on tumor (e.g., prostate cancer, bladder cancer and pancreatic cancer) cells, and has a good application prospect in the treatment of tumors. BRIEF DESCRIPTION OF DRAWINGS

[0105] Figure 1 is a SDS-PAGE gel electrophoresis diagram of PSCA nanobody.

[0106] Figure 2 is the binding of PSCA Nanobodies to CHO-K1, CHO-K1 PSCA cells.

[0107] Figure 3 is the binding ELISA of PSCA Nanobodies to human PSCA antigen.

[0108] Figure 4 is the binding ELISA of PSCA Nanobodies to mouse PSCA antigen.

[0109] Figure 5 is the binding of PSCA Nanobodies SIPI20028, SIPI20029, SIPI20031, SIPI20032 and SIPI20033 to CHO-K1 PSCA cells.

[0110] Figure 6 is the binding of PSCA Nanobodies SIPI20030 and SIPI20034 to CHO-K1 PSCA cells.

[0111] Figure 7 is the BLI detection results of PSCA Nanobody SIPI20028.

[0112] Figure 8 is the BLI detection results of PSCA Nanobody SIPI20029.

[0113] Figure 9 is the BLI detection results of PSCA Nanobody SIPI20030.

[0114] Figure 10 is the BLI detection results of PSCA Nanobody SIPI20031.

[0115] Figure 11 is the BLI detection results of PSCA Nanobody SIPI20032.

[0116] Figure 12 is the BLI detection results of PSCA Nanobody SIPI20033.

[0117] Figure 13 is the BLI detection results of PSCA Nanobody SIPI20034.

[0118] Figure 14 is the internalization of PSCA Nanobody SIPI20028 in CHO-K1 PSCA cells.

[0119] Figure 15 is the internalization of PSCA Nanobody SIPI20029 in CHO-K1 PSCA cells.

[0120] Figure 16 is the internalization of PSCA Nanobody SIPI20030 in CHO-K1 PSCA cells.

[0121] Figure 17 is the internalization of PSCA Nanobody SIPI20032 in CHO-K1 PSCA cells.

[0122] Figure 18 is the internalization of PSCA Nanobody SIPI20033 in CHO-K1 PSCA cells.

[0123] Figure 19 is the internalization of PSCA Nanobody SIPI20034 in CHO-K1 PSCA cells.

[0124] Figure 20 is the mass spectrometry result of SIPI20028 Nanobody conjugated with vc-MMAE.

[0125] Figure 21 is the killing effect of PSCA Nanobody SIPI20028 and antibody conjugated with vc-MMAE on PSCA positive RT4 cells.

[0126] Figure 22 is the killing effect of PSCA Nanobody SIPI20029 and antibody conjugated with vc-MMAE on PSCA positive RT4 cells.

[0127] Figure 23 is the killing effect of PSCA Nanobody SIPI20030 and antibody conjugated with vc-MMAE on PSCA positive RT4 cells.

[0128] Figure 24 is the killing effect of PSCA Nanobody SIPI20034 and antibody conjugated with vc-MMAE on PSCA positive RT4 cells.

[0129] Figure 25 is the killing effect of PSCA Nanobody SIPI20028 and antibody conjugated with vc-MMAE on PSCA negative CHO-K1 cells.

[0130] Figure 26 is the killing effect of PSCA Nanobody SIPI20029 and antibody conjugated with vc-MMAE on PSCA negative CHO-K1 cells.

[0131] Figure 27 is the killing effect of PSCA Nanobody SIPI20030 and antibody conjugated with vc-MMAE on PSCA negative CHO-K1 cells.

[0132] Figure 28 is the killing effect of PSCA Nanobody SIPI20034 and antibody conjugated with vc-MMAE on PSCA negative CHO-K1 cells.

[0133] Figure 29 is the killing effect of PSCA Nanobody SIPI20028 and antibody conjugated with vc-MMAE on PSCA overexpressing PC-3 cells.

[0134] Figure 30 is the killing effect of PSCA Nanobody SIPI20028 and antibody conjugated with vc-MMAE on PSCA overexpressing DU145 cells.

[0135] Figure 31 is the anti-tumor effect of SIPI20028 nanobody conjugated vc-MMAE in human prostate cancer xenograft model. DETAILED DESCRIPTION

[0136] The application will be further described by way of examples without limiting the application thereto. The experimental methods in the following examples, unless otherwise specified, are carried out according to the conventional methods and conditions, or according to the instructions of the commercial product.

[0137] Example 1 Screening, identification, sequencing and purification of antibodies

[0138] Screening of phage library

[0139] Day 1

[0140] (I) Preparation of electrotransformation competent cells:

[0141] 1. 10 mL of TG1 bacteria solution of overnight culture was inoculated into 1 L of 2xYT medium, and cultured at 37°C, 250 rpm for about 1 h.

[0142] 2. After 1 h of culture, 600 μL was taken and its OD was measured by NanoDrop 600 (Use cuvette, select Cell culture, check Use cuvette), and the medium was used as blank control. When the OD 600 was between 0.4 and 0.5, the culture was stopped.

[0143] 3. The bacteria solution with OD 600 of 0.4 was immediately placed in prepared ice water, and ice bathed for 30 min.

[0144] 4. The centrifuge was pre-cooled, 500 mL centrifuge bottles were pre-cooled in the 4°C refrigerator, and ultrapure water and 10% glycerol ice were pre-cooled.

[0145] 5. The bacteria solution in ice bath was taken out and poured into the pre-cooled centrifuge bottle. After balancing, it was centrifuged at 5500 x g, 4°C for 10 min.

[0146] 6. After centrifugation, the supernatant was discarded, about 50 mL of pre-cooled ultrapure water was added, and the centrifuge bottle was immersed in ice water after tightly closing the lid and shaking in the same direction. The intensity should not be too large, so that the bacteria were resuspended.

[0147] 7. Add pre-cooled ultrapure water to 500 mL, balance, and centrifuge at 5500 x g, 4°C for 10 min.

[0148] 8. Carefully discard the supernatant, and wash the bacteria with 500 mL of ultrapure water in the same way.

[0149] 9. After washing the bacterial cells twice, carefully pour off the supernatant, add about 50 mL of pre-cooled 10% glycerol, tightly cap the centrifuge bottle, and immerse it in ice water while oscillating in the same direction. Be careful not to oscillate too hard, so as to resuspend the bacterial cells.

[0150] 10. Transfer the bacterial solution to a pre-cooled 50 mL centrifuge tube, tightly cap the tube, and centrifuge at 5000 x g for 5 min at 4°C.

[0151] 11. Pour off the supernatant, and use a pipette to remove the residual solution.

[0152] 12. Add 0.8 mL of pre-cooled 10% glycerol, and use a gun head to stir and suspend the bacterial cells. The preparation of electro-competent cells is complete.

[0153] (II) Purification and concentration of the ligation product (QIAquick PCR Purification Kit, item number: 28104)

[0154] 1. Mix 1 volume of the ligation product of the antibody library and pcomb3x vector with 5 volumes of Buffer PB, and then load the mixture onto the column and centrifuge at 17900 x g for 1 min;

[0155] 2. Discard the liquid in the collection tube. Add 750 μL of Buffer PE to the column, and centrifuge at 17900 x g for 1 min;

[0156] 3. Repeat step 2, and then centrifuge the empty column at 17900 x g for 1 min;

[0157] 4. Replace the 1.5 mL EP tube with 30-50 μL of H2O, and let it stand for 4 min, and then centrifuge at 17900 x g for 1 min.

[0158] 5. Purify the ligation product using the above kit, and finally elute it with 100 μL of H2O. The detection concentration is 56.4 ng / μL, 260 / 280 is 1.92, and 260 / 230 is 1.86. Perform electrophoresis identification before and after purification of the ligation product.

[0159] (III) Transformation of the ligation product

[0160] 1. Prepare 100 mL of non-resistant 2 x YT medium + 10 x SOC glucose salt solution (Sigma, item number: 1S797), and incubate at 37°C on a shaker.

[0161] 2. Mix the purified and concentrated ligation product with the electro-competent cells at a volume ratio of 1:10, and then ice-bath for 5 min. Take 200 μL to a pre-cooled electro-transformation cup, and then ice-bath for another 10 min.

[0162] 3. Set the electroporator to Eco 2, and after the shock, quickly add 1 mL of 37°C 2YT medium, and then add to 75 mL of 37°C 2YT medium after blowing and sucking.

[0163] 4. After 20 min of 37°C, 250 rpm culture of the electroporated competent cells, make 10 -3 , 10 -4 , 10 -5 , and 10 -6 dilutions (add 10 μL per point), and take 100 μL of 10 -4 bacterial solution to spread on Amp plates, and calculate the capacity and sequencing after overnight culture at 37°C.

[0164] (IV) Rescue phage

[0165] 1. After 20 min of 37°C, 250 rpm culture of the electroporated competent cells, add 2xYT medium to 600 mL, add 600 μL of Amp (100 mg / mL), and measure the OD value after 2 h of 37°C, 250 rpm culture.

[0166] 2. When the OD 600 is 0.8, take 300 mL and add 20 times the amount of helper phage (M13K07), and let stand at 37°C for 45 min, during which time mix well every 15 min. The remaining 300 mL of bacterial solution is directly cultured overnight at 30°C, 220 rpm.

[0167] 3. Let stand for 45 min, and after the end, centrifuge at 4000 rpm for 10 min. Discard the supernatant and replace with 2xYT medium 500 mL, add Amp and Kana, and culture overnight at 30°C, 220 rpm.

[0168] The next day

[0169] 1. Centrifuge 300 mL of the bacterial solution directly cultured overnight at 8000 rpm for 5 min, discard the supernatant, and add 9.38 mL of 40% glycerol to resuspend the bacterial cells, and finally store 15 mL of bacterial cells with 25% glycerol at -80°C.

[0170] 2. Centrifuge 500 mL of the bacterial solution in 2xYT medium at 8000 rpm for 15 min, and take the supernatant.

[0171] 3. Add 1 / 4 volume of PEG / NaCl solution (2.5 M NaCl solution containing 20% (mass percent) PEG8000) to the supernatant, mix well, and let stand at 4°C for 1 h.

[0172] 4. Centrifuge at 8000 x g at 4°C for 15 min, discard the supernatant, and let stand for 5 min to allow the precipitate to gather together.

[0173] 5. Dissolve the pellet in 30 mL of PBST containing 10% glycerol, and vortex with 1 mL of a pipette tip.

[0174] 6. Centrifuge at 10000 x g, 4°C for 10 min, remove the residual cell debris, and aliquot the supernatant into 1.5 mL EP tubes. Measure the absorbance at 268 nm using the UV-Vis option of a Nanodrop, and obtain the OD value of the phage. Indicate the library capacity and OD value, and store at -80°C.

[0175] 7. Take the phage library phage lib and inoculate into 50 mL of 2xYT medium (containing 2% glucose and 100 μg / mL of ampicillin amp) with an initial OD600 of 0.05-0.1, and incubate at 30°C, 220 rpm for 3 h until the OD600 is about 0.5. 600 about 0.5.

[0176] 8. Add helper phage in an amount calculated according to [(OD 600 x 8 x 10 8 mL -1 x 50 mL (volume of bacterial solution) x 20) ÷ titer of helper phage] mL, and incubate at 37°C for 1 h, and centrifuge at 3800 rpm for 10 min. Resuspend the bacteria in 250 mL of 2xYT medium (containing 100 μg / mL of ampicillin amp and 50 μg / mL of kanamycin Kana), and incubate at 30°C, 220 rpm overnight (more than 16 h).

[0177] 9. Centrifuge the phage library after overnight incubation at 12000 rpm for 30 min, mix the supernatant with the precipitant in a volume ratio of 4:1, and the precipitant is a 2.5 M NaCl solution containing 20% (mass percentage) PEG8000. Incubate at 4°C for 2-3 h, centrifuge at 8000 rpm for 30 min, discard the supernatant, and dry the tube by tapping on sterile paper. Resuspend the phage precipitate in 1 mL of PBS, and centrifuge several times at 12000 rpm until no cell debris precipitates.

[0178] 10. Titer detection: dilute the phage in PBS by 10-fold gradient, mix 10 μL of the phage at a dilution gradient of 1:10000 with TG1 competent cells (OD600 about 0.5), incubate at 37°C for 30 min, and then plate (2xYT agar medium containing 100 μg / mL of ampicillin amp) and incubate at 37°C overnight. Observe the phage plaques on the plate.

[0179] II. Screening of the phage library:

[0180] (I) First round of screening

[0181] 1. One day before screening, TG1 competent cells were seeded into 20 mL of 2×YT medium and cultured at 37°C and 250 rpm until the OD600 was approximately 0.6. The cells were then stored at 4°C.

[0182] 2. Add 500 μL of phage library to a 1.5 mL EP tube, add 500 μL of blocking buffer (1×PBS buffer containing 6% skim milk) to the phage solution, add 5 μg of biotinylated PSCA-Fc protein (laboratory expression) to the phage-blocking buffer, and incubate at room temperature by rotation for 2 h.

[0183] 3. Take 200 μL of magnetic beads (nanomicroorganisms, catalog number: MPHCAS-300) into a 1.5 mL EP tube, place it on a magnetic rack, and wait for the magnetic beads to be completely adsorbed onto the tube wall. Then, remove the supernatant, wash twice with PBS, resuspend the magnetic beads in 500 μL of 1×PBS buffer, add blocking solution at a 1:1 ratio, and incubate at room temperature for 1 h by rotation.

[0184] 4. Place the EP tube containing the magnetic beads on a magnetic rack, discard the solution, add the blocked phage-protein solution, resuspend and mix well, and incubate at room temperature for 30 minutes by rotation.

[0185] 5. Place the EP tube on a magnetic rack, aspirate the solution, wash 5 times with 0.05% PBST solution, and finally wash 2 times with 1×PBS buffer.

[0186] 6. After resuspending the cleaned magnetic beads in 1 mL of TG1 competent cells (OD600 approximately 0.5), add them to 20 mL of TG1 competent cells for inoculation. Incubate at 37°C for 45 min, inverting and mixing once every 15 min.

[0187] 7. Titer Detection: Dilute 1 μL of phage solution infecting TG1 competent cells 1:100 and plate it on 2×YT agar medium (containing 100 μg / mL ampicillin amprolium). Incubate overnight at 37°C. Observe the phage plaques on the plate. Add ampicillin amprolium and glucose to the remaining bacterial solution to make the final solution contain 2% glucose and 100 μg / mL ampicillin amprolium by weight. Incubate at 37°C and 250 rpm for 1 h.

[0188] 8. Add the amount according to [(OD 600 ×8×10 8 mL -1×50mL (bacterial liquid volume) ×20) ÷ helper phage titer] mL of calculated helper phage, 37°C for 45 min, and inverted mixing every 15 min. Centrifugation at 3800 rpm for 10 min, and resuspension of the bacterial body in 20 mL of 2×YT medium (containing 100 μg / mL of ampicillin amp and 50 μg / mL of kanamycin Kana), 30°C, 220 rpm overnight culture (more than 16 h).

[0189] (II) Second round of screening

[0190] 1. The next day, centrifuge the overnight bacterial liquid at 8000 rpm for 15 min, and take the supernatant.

[0191] 2. Add 1 / 4 volume of PEG / NaCl solution to the supernatant, mix well, and then place at 4°C for 20 min.

[0192] 3. Centrifuge at 8000×g at 4°C for 15 min, discard the supernatant, and then empty for 5 min to make the precipitate gather together.

[0193] 4. Dissolve the precipitate with 1 mL of PBS, and blow with 1 mL of a gun head.

[0194] 5. Take 300 μL of the resuspension liquid, add 1:1 blocking solution (6% skim milk dissolved in PBS), and continue the operation steps of the first round (i.e., the aforementioned step (II) phage library screening).

[0195] Among them, the initial addition of PSCA-Fc antigen in the second, third and fourth rounds is 2 μg, 1 μg and 0.5 μg, respectively, and the elution times of the magnetic beads adsorbed phage in the volume percentage of 0.05% PBST are gradually increased from 5 times, 10 times and 15 times.

[0196] III. ELISA detection of phage

[0197] I. Phage amplification

[0198] 1. Dilute the phage library obtained in the last round and coat it in a large flat plate containing amp, and incubate at 37°C overnight.

[0199] 2. Add 100 μL of 2×YT medium to each well of the round-bottom plate, and pick a single colony from the last round of coating plate into the round-bottom plate, and incubate at 37°C, 250 rpm for 4 h to the logarithmic growth phase.

[0200] 3. Add the corresponding amount of helper phage to each well, and incubate at 37°C for 45 min, and then incubate at 250 rpm for 1 h.

[0201] 4. Add 100 μL 2xYT medium (containing 200 μg / mL ampicillin amp and 100 μg / mL kanamycin Kana) to each well, and incubate at 30°C, 220 rpm overnight.

[0202] II. Antigen coating and ELISA detection

[0203] 1. Coat 50 ng / 100 μL antigen PSCA-Fc protein (100 mM NaHCO3, pH 9.6) and BSA (100 mM NaHCO3, pH 9.6 or PBS) at 4°C overnight.

[0204] 2. Remove the coating solution, wash 3 times with 0.05% PBST solution, add 200 μL blocking solution (3% skim milk) to each well, and incubate at 37°C for 1 h.

[0205] 3. Centrifuge the bacterial solution incubated overnight in step I, 4, at 3800 rpm for 10 min, and retain the supernatant as the phage solution.

[0206] 4. Remove the blocking solution, wash 3 times with 0.05% PBST solution, add 100 μL phage solution to each well, and incubate at 37°C for 2 h.

[0207] 5. Remove the phage solution, wash 5 times with 0.05% PBST solution, add 50 μL M13 phage mouse monoclonal anti-M13 (Yiqiao Shenzhou, item number: 11973-MM05T-H) solution (1:15000 diluted in blocking solution) to each well, and incubate at 37°C for 1 h.

[0208] 6. Remove the M13 phage mouse monoclonal anti-M13 solution, wash 5 times with 0.05% PBST solution, add 50 μL TMB color developing solution (avoid light), and after waiting for about 5 min (until the color changes moderately), add 50 μL color developing termination solution.

[0209] 7. Measure the OD450nm absorbance value of the plate by an enzyme labeler. The obtained positive clones are sent to a company for sequencing to obtain the sequence encoding the nanobody. The sequence is analyzed and processed to obtain the following nanobodies, wherein the variable region sequence is defined according to the Kabat numbering system, see Table 2.

[0210] Table 2 CDR sequences of nanobodies

[0211] CDR1: X1X2X3MN (SEQ ID NO: 27), wherein X1 is N, K or R, X2 is F or Y, and X3 is A, P or G;

[0212] CDR2: VISHDGTX4KYYADSVKG (SEQ ID NO: 28), wherein X4 is V or T;

[0213] CDR3: WGTLX5X6SGX7X8GPSEYFEH (SEQ ID NO: 29), wherein X5 is Y or H, X6 is A or G, X7 is R or S, and X8 is S or Y.

[0214] The sequences of the Nanobodies of the application are shown below, with the CDR regions defined according to the Kabat numbering scheme shown in "bold + underlined" in the amino acid sequences:

[0215] >SIPI20028

[0216] Amino acid sequence (SEQ ID NO: 1):

[0217] Nucleotide sequence (SEQ ID NO: 8):

[0218] >SIPI20029

[0219] Amino acid sequence (SEQ ID NO: 2):

[0220] Nucleotide sequence (SEQ ID NO: 9):

[0221] >SIPI20030

[0222] Amino acid sequence (SEQ ID NO: 3):

[0223] Nucleotide sequence (SEQ ID NO: 10):

[0224] >SIPI20031

[0225] Amino acid sequence (SEQ ID NO: 4):

[0226] Nucleotide sequence (SEQ ID NO: 11):

[0227] >SIPI20032

[0228] Amino acid sequence (SEQ ID NO: 5):

[0229] Nucleotide sequence (SEQ ID NO: 12):

[0230] >SIPI20033

[0231] Amino acid sequence (SEQ ID NO: 6):

[0232] Nucleotide sequence (SEQ ID NO: 13):

[0233] >SIPI20034

[0234] Amino acid sequence (SEQ ID NO: 7):

[0235] Nucleotide sequence (SEQ ID NO: 14):

[0236] >PSCA-Fc (antigen)

[0237] Amino acid sequence (SEQ ID NO: 15):

[0238] Example 2 Activity identification of Nanobody

[0239] (I) Expression of PSCA Nanobody

[0240] The plasmid of the positive clone sequenced above was transformed into HB2151 for expression. The HB2151 strain was taken out from the -80°C refrigerator and placed on ice to melt. 1 μL of the plasmid was removed using a gun head and gently mixed in the bacterial solution. After being placed on ice for 15 min, it was heat shocked at 42°C for 90 s, and then placed on ice again for 5 min. The bacterial solution was streaked on a plate and incubated at 37°C overnight. The next day, one colony was picked from the plate and cultured in a shaker, and then sent for testing. The remaining bacterial solution was shaken to an OD value of 0.6-0.8, at which time IPTG was added to the bacterial solution at a ratio of 1:1000, and then the temperature condition was 30°C, 220 rpm overnight shaking.

[0241] On the third day, the culture medium was collected and centrifuged (8000 rpm, 5 min) and the supernatant was discarded. The bacteria were resuspended with DPBS (+ 250 mM NaCl), and then polimyxin B sulfate buffer (100x) was added, and the cells were lysed by shaking at 37°C for 0.5 h. The supernatant was collected by centrifugation (8000 rpm, 20 min). First, 0.8 mL of Ni-NTA agarose (brand: Thermo Scientific TM, lot: XL354095), after the solution flowed naturally, 10 mL of water was added to wash twice, then the antibody supernatant was added, and the effluent was discarded. DPBS (+ 250 mM NaCl + 20 mM imidazole, 10 CV) was added to remove non-specific binding proteins. DPBS (+ 250 mM NaCl + 40 mM imidazole, 10 CV) was added to remove non-specific binding proteins. DPBS (+ 250 mM NaCl + 200 mM imidazole, 20 CV) was added to exchange the antibodies bound to the agarose, and the target antibodies were collected.A 280 The antibody concentration was measured, SDS-PAGE gel electrophoresis and protein staining: 5 μg of protein sample (5x Protein Loading was added) was added to each well of the protein precast gel, and electrophoresis was performed at 140 V for 40 min. After the gel was removed, the protein staining solution was shaken for 30 min, and water was shaken overnight for washing. The results are shown in Figure 1.

[0242] (ii) FACS analysis of the binding activity of PSCA nanobodies to cell surface PSCA

[0243] CHO-K1 PSCA cells (a PSCA overexpressing cell line constructed in the laboratory) were divided into 5x10 5 cells / sample / 100 μL, and the test antibody was added at a final concentration of 1 μM. Incubate on ice for 30 min, and wash the cells with cold PBS buffer twice; add Alexa Fluor-488 labeled anti-VHH secondary antibody (Jackson, Cat: 128-545-232), incubate on ice for 30 min, wash the cells with ice-cold 1x PBS buffer twice, resuspend in 100 μL of 1x PBS buffer, and detect the mean fluorescence intensity (MFI) of the cells by flow cytometry. Aurora TM ) The mean fluorescence intensity (MFI) of the cells was detected. In the above detection, CHO-K1 PSCA cells labeled only with fluorescent secondary antibody were used as blank controls. At the same time, CHO-K1 cells were used as PSCA negative cells, and the test antibody was added at a final concentration of 1 μM, and the experimental operation was consistent with the above experiment. The mean fluorescence intensity (MFI) of the cells was detected by flow cytometry. Aurora TM ) The mean fluorescence intensity (MFI) of the cells was detected. In the above detection, CHO-K1 PSCA cells labeled only with fluorescent secondary antibody were used as blank controls. At the same time, CHO-K1 cells were used as PSCA negative cells, and the test antibody was added at a final concentration of 1 μM, and the experimental operation was consistent with the above experiment. The mean fluorescence intensity (MFI) of the cells was detected by flow cytometry.

[0244] The detection results are shown in Figure 2, which show that the SIPI20028, SIPI20029, SIPI20030, SIPI20031, SIPI20032, SIPI20033 and SIPI20034 nanobodies have good binding ability to CHO-K1 PSCA cells, and the above nanobodies do not bind to PSCA negative CHO-K1 cells.

[0245] (III) ELISA detection of the EC of the antibody to PSCA protein 50

[0246] The protein human PSCA-Fc (SEQ ID NO: 15) and Recombinant Mouse PSCA Protein (Yiqiao, item number: 5A1731-M08H) were diluted to 500 ng / mL with PBS, 100 μL / well coated the enzyme-linked plate, and coated overnight at 4°C; 3% skim milk blocking solution was blocked in a 37°C incubator for 60 min, and the plate was washed with PBST for 3 times; SIPI20028, SIPI20029, SIPI20030, SIPI20031, SIPI20032, SIPI20033, and SIPI20034 were gradiently diluted, 1 μM starting with 5-fold dilution for 8 gradients, 37°C reaction for 2 h, PBST washing the plate for 3 times; diluted HRP anti-VHH secondary antibody (Chengdu Critical Point, item number 077-201-005) was added for reaction for 1 h, PBST washing the plate for 3 times; finally, 50 μL TMB substrate was added for color development, color development for 10 min at room temperature in the dark, 50 μL stop solution was added to stop the reaction, and the absorbance of the well plate at 450 nm was read and recorded by the microplate reader.

[0247] The results are shown in FIGS. 3 and 4, and the EC of the different antibodies to human PSCA was calculated 50 (as shown in Table 3).

[0248] Table 3 EC of the antibody to human PSCA 50

[0249] (IV) FACS detection of the EC of the antibody to CHO-K1 PSCA 50

[0250] The CHO-K1 PSCA cells were digested, and after counting, the cells were diluted to 5 mL with PBS, and then 50 μL of the diluted cells were added to each well of a 96-well plate using a syringe. The PSCA VH nanobody was diluted, with a concentration starting from 2 μM and 5-fold dilution. 50 μL of the cell suspension was mixed with 50 μl of the diluted antibody, and incubated at 4°C for 30 min. PBS was used to wash twice. Then 50 μL of 1:200 diluted AF488 anti-VHH (brand: Jackson, item number: 128-545-230) was added, and incubated at 4°C for 30 min. PBS was used to wash twice. Then flow cytometry analysis was performed, and the results are shown in FIGS. 5-6 and Table 4.

[0251] According to the data processing, the EC of SIPI20028 was 13.82 nM, the EC of SIPI20029 was 50 13.82 nM, the EC of SIPI20029 was 50EC50 for 35.50 nM, EC50 of SIPI20030 50 EC50 for 85.16 nM, EC50 of SIPI20031 50 EC50 for 82.42 nM, EC50 of SIPI20032 50 EC50 for 23.30 nM, EC50 of SIPI20033 50 EC50 for 34.89 nM, EC50 of SIPI20034 50 EC50 for 69.39 nM.

[0252] Table 4 EC50 of antibodies to CHO-K1 PSCA

[0253] Example 3 BLI (Bio-Layer Interferometry) verification of antibody affinity constant

[0254] BLI (Bio-Layer Interferometry) is a label-free, real-time monitoring optical detection technology, mainly used for comprehensive quantitative analysis of interactions between biomolecules and protein concentration determination. BLI can monitor the entire molecular binding process in real time and calculate important data such as affinity (KD), binding rate (ka), dissociation rate (kd) between molecules. BLI uses a probe-type biosensor to directly detect samples without any fluorescent labeling of the sample being detected. By emitting white light to the sensor surface and collecting reflected light, different frequency reflected light spectra are affected by the thickness of the bio-sensor film layer and form interference. Using a new generation of biofilm interferometry analyzer (BLI, Gator Bio), the specific operation is as follows: the antibody is diluted to 5 μg / mL with PBST buffer, and flows through the surface of the His probe (Gator probe) for 120 s. The antigen is used as the mobile phase, with a binding time of 240 s and a dissociation time of 360 s. The initial concentration is 500 nM, 2-fold dilution, and 4 gradients.

[0255] Add the antibody to be tested in the black hole plate adapted by the gator instrument, and set the antigen at four concentration groups of 500 nM, 250 nM, 125 nM and 62.5 nM, respectively, and use the His probe for detection. According to the result analysis, the BLI detection KD of SIPI20028 is 1.08 x 10 -9 M, the BLI detection KD of SIPI20029 is 5.67 x 10 -9 M, the BLI detection KD of SIPI20030 is 3.07 x 10 -9 M, the BLI detection KD of SIPI20031 is 2.79 x 10 -9 M, the BLI detection KD of SIPI20032 is 1.53 x 10 -9M, BLI detected KD of SIPI20033: 6.02 x 10 -9 M, BLI detected KD of SIPI20034: 3.96 x 10 - 9 M (Figures 7-13).

[0256] Table 5 Affinity of antibodies

[0257] Example 4 Internalization of PSCA Nanobody in PSCA Positive Cells

[0258] Invitrogen TM Zenon TM pHrodo TM iFL IgG Labeling Reagents is a pH sensitive dye that does not emit fluorescence outside of the cell. When the fluorescent reagent binds to the antibody, the antibody is engulfed into the cell interior, and in the acidic environment (including lysosomes), it emits bright red fluorescence. Because the pH sensitive fluorescent dye binds to the Fc segment, it cannot directly label the VH antibody. This example uses the fluorescent dye to label the PSCA VH-Fc antibody, and uses flow cytometry to verify the internalization of the PSCA VH-Fc antibody in PSCA positive cells. The receptor cell surface does not have Fc receptors, and the detection is still the endocytosis mediated by the binding of the PSCA nanobody to its antigen.

[0259] (1) Digest CHO-K1 PSCA cells, and after counting, dilute 100w cells to 1 mL.

[0260] (2) Take PSCA VH-Fc antibody, and dilute to a working concentration of 240 nM.

[0261] (3) Prepare 500 μΐ of Zenon working solution, incubate with the antibody at room temperature for 5 min, and then add to the cells.

[0262] (4) Incubate the cells in each group at 37°C for 0 h, 1 h, 2 h, and 4 h, respectively. Then perform flow cytometry analysis.

[0263] The results are as follows: As shown in Figures 14-19, it can be seen that the SIPI20028, SIPI20029, SIPI20030, SIPI20032, SIPI20033, and SIPI20034 PSCA VH-Fc can be better internalized into CHO-K1 PSCA cells.

[0264] IgG1 Fc Base Sequence

[0265] IgG1 Fc Amino Acid Sequence

[0266] Example 5 CCK8 method verifies the cell killing ability of nanobody-chemical drug conjugate

[0267] (I) Construction of pcomb3x-VHH-Cys expression plasmid

[0268] Based on the obtained nanobody plasmid (the plasmid backbone is pcomb3x vector), a G4S-Cys sequence is added at the C-terminal of the antibody sequence.

[0269] (1) According to the sequence of the recombinant vector, the corresponding upstream and downstream primers are designed (synthesized by Anshengda Company, diluted to a final concentration of 10 μM), 1 μL of upstream and downstream primers, 5 μg of plasmid template, 25 μL of 2x PrimeSTAR (Takara, Catalog No: R045) are added, and ddH2O is added to make up the volume of the PCR reaction system to 50 μL.

[0270] (2) Agarose gel electrophoresis and product purification: prepare 1% agarose gel in advance. After mixing the PCR reaction product with 5 μL of 10x DNA loading buffer (brand: Takara, Catalog No: 9157, Batch No: AJ71984A), it is loaded and electrophoresed at 110 V for 25 min. After the gel runs, the gel block is placed under the nucleic acid ultraviolet imager to observe the band position, and the target band is cut off according to the correct single target band position and placed in a sterile 1.5 mL EP tube. Use the agarose gel DNA recovery kit (Zhuangmeng Biological, Catalog No: ZPN202-3) to recover the target band according to the instructions, use 20 μL of elution buffer to dissolve the recovered target band, and determine the concentration.

[0271] (3) Homologous recombination: the above obtained product can be catalyzed by homologous recombination cloning kit (Yixing Biological, Catalog No: 10922ES50) to carry out DNA seamless cloning. According to the molar ratio of vector to insert fragment 1:2, that is, the amount of vector used (ng) = 0.02 x the number of base pairs of the vector; the amount of insert used (ng) = 0.04 x the number of base pairs of the vector, 5 μL of homologous recombination enzyme is added, ddH2O is added to make up the volume of the reaction system to 10 μL, and 55°C is connected for 20 min.

[0272] (4) Transformation: Thaw DH5α competent strains (Sangon Biotech, catalog number: B528413-0100) stored at -80℃ on ice. Add 10 μL of the enzyme ligation product to 50 μL of competent strains, gently stir with your fingers 10 times to mix thoroughly, and incubate on ice for 10 min. After incubation, heat shock at 42℃ for 90 s, and immediately incubate on ice for another 5 min. Plate the mixture onto 2×YT solid medium containing 0.1% ampicillin and incubate upside down at 37℃ for 12-15 h. The next day, randomly select morphologically healthy single clones and send them to the company for sequencing.

[0273] (II) Protein Purification

[0274] (1) Transformation: Transform the correctly sequenced plasmid into E. coli HB2151 competent cells, following the same steps as above.

[0275] (2) IPTG-induced protein expression in Escherichia coli: Morphologically sound monoclonal strains were selected, picked up with a pipette tip, and injected into 300 mL of 2×YT medium containing 0.1% ampicillin. The culture was carried out at 37°C and 250 rpm until OD500. 600 The concentration was approximately 0.6. 1 mM IPTG was added to induce expression, and the mixture was induced overnight at 30°C and 220 rpm.

[0276] (3) Nickel column purification: The next day, collect bacterial cells and centrifuge at 8000 rpm for 10 min. After removing the supernatant, resuspend the bacterial cells in 80 mL of PBS, then add 800 μL of polymyxin. Lyse the bacterial cells at 37°C and 250 rpm for 1 h. Centrifuge the lysed bacterial solution at 10000 rpm and 4°C for 20 min. Filter the supernatant through a 0.45 μm filter membrane for column purification. Because the pcomb3x plasmid carries a His tag, the imidazole group on His can react with Ni... 2+ The His-tagged protein binds to metal ions by forming coordinate bonds. The His-tagged protein bound to the medium can be competitively eluted by increasing the imidazole concentration in the buffer, resulting in a higher purity His-tagged protein. First, the nickel column is washed with ten column volumes of pure water at a flow rate of 50–150 cm / h, then equilibrated with ten column volumes of binding buffer PBS at a flow rate of 150–600 cm / h. After centrifugation and filtration, the sample is loaded at a low flow rate. It is then washed with 20 mL of washing buffer (containing 20 mM and 40 mM imidazole, respectively) at a flow rate of 150 cm / h. Elution is then performed with 7 mL of elution buffer (500 mM imidazole) at a low flow rate. Finally, the column is washed with pure water and stored in 20% ethanol at 4°C.

[0277] (4) Buffer replacement: The eluate was transferred into an ultrafiltration tube with a molecular weight cut-off of 3000 D, centrifuged at 4000 rpm at 4°C for 20 min, and the liquid in the collection tube was discarded. PBS was added to the ultrafiltration tube and centrifuged again. This was repeated three times.

[0278] (5) SDS-PAGE gel electrophoresis and protein staining: 5 μg of protein sample (5x Protein Loading has been added) was added to each well of the protein precast gel, and electrophoresis was performed at 140 V for 40 min. After the gel was removed, the protein staining solution was oscillated for 30 min, and then washed with water overnight.

[0279] (Three) VcMMAE conjugation

[0280] 1. Take 1.5 mg of protein, add 40 μL of 10 mM TCEP reducing agent at a ratio of 1:4 of molecular weight, mix well, and stand at room temperature for 2 h. Replace the solvent with PBS for 2-3 times to remove the reducing agent. Add 500 μL of 1 mM VcMMAE (MCE, item number: HY-15575, 1 mg dissolved in 759.5 μL of DMSO) at a ratio of 1:5 of molecular weight, and mix well at room temperature for 1 h.

[0281] 2. Replace the solvent with PBS for 3 times to remove DMSO.

[0282] (Four) Mass spectrometry to identify DAR value

[0283] The DAR value of the antibody-drug conjugate is determined based on the difference in molecular weight of ADC with different drug loadings. The intact ADC or the subunit after enzyme / chemical cleavage is introduced into high-resolution mass spectrometry (such as QTOF, Orbitrap) by soft ionization (such as ESI), and the accurate molecular weight of each DAR component is detected. Knowing the mass of the antibody itself and the mass difference Δm of a single drug molecule, the mass spectrometry signal can be deconvoluted to distinguish the DAR0 and DAR1 peaks, and the average DAR value can be calculated according to the weighted average formula.

[0284] We took the reduced antibody (as a control group) and the antibody after reduction and conjugation for mass spectrometry, and calculated the average DAR value. In order to eliminate the influence of protein glycosidase on the molecular weight, Fast PNGase F, N-glycosidase F (fast version) (brand: Yixing, item number: 20406ES20) was used for treatment:

[0285] (1) Mix 100 μg of antibody with H2O to a total volume of 16 μL;

[0286] (2) Add 4 μL of Fast PNGase F Buffer (5x) to make the total reaction volume 20 μL;

[0287] (3) Add 1 μL of Fast PNGase F;

[0288] (4) Incubate at 50°C for 10 minutes.

[0289] The mass spectrometry results are shown in Figure 20. The average DAR value of SIPI20028-Cys conjugated with vc-MMAE was calculated to be 0.9.

[0290] (V) Cell killing experiment

[0291] RT4 (PSCA positive bladder cancer cells), PC-3 PSCA (prostate cancer cells overexpressing PSCA), DU145 PSCA (prostate cancer cells overexpressing PSCA) and CHO-K1 (PSCA negative cells) cells were plated into 96-well plates one day in advance, and the drug concentration was diluted in a three-fold concentration gradient with eight gradients, and the initial concentration was 1 μM. The diluted antibody was added to the cell plated well plate. At the same time, a blank control group (with medium, no cells, no drugs) and a negative control group (with cells, with medium, no drugs) were set up. Incubate at 37°C, 5% CO2 for 48h. Then aspirate the culture medium, add the CCK8 reagent detection (APExBIO, item number: K1018) prepared in advance, and incubate at 37°C for 1h. Then use the microplate reader to read at 450nm. Calculate the cell survival rate (%) = (sample group average - blank group average) / (negative control group average - blank group average) x 100% according to the formula to calculate the cell survival rate, and use Graphpad to calculate the IC 50 (Table 6).

[0292] According to the results, SIPI20028, SIPI20029, SIPI20030 and SIPI20034 conjugated with vc-MMAE have obvious killing effect on PSCA positive RT4 cells (Figures 21-28), and SIPI20028 conjugated with vc-MMAE has obvious killing effect on PC-3 and DU145 cells overexpressing PSCA (Figures 29-30).

[0293] Table 6 IC of antibodies on RT4 cells 50

[0294] (Six) Anti-tumor effect of PSCA nanobody conjugated drugs in human prostate cancer nude mouse xenograft model

[0295] PC-3 PSCA cells were cultured to the logarithmic growth phase, then trypsinized and resuspended in culture medium for use. 200 μL of cell suspension (1:1 PBS / matrix glue, containing 5 x 10 6 cells) was injected subcutaneously into the right back of the nude mice. After inoculation, the tumor growth of the nude mice was continuously observed, and when the tumor grew to 100-150 mm3 After modeling is successful and the state is uniform, the nude mice are randomly grouped (usually 2-3 weeks), each group of 2, and then administered by tail vein injection, the administration dose is 6 mg / kg, once every two days for 4 weeks. The tumor size is measured every week, the tumor volume is calculated (formula: V = (L x W 2 ) / 2, L is the long diameter, W is the short diameter), the body weight and survival status of the nude mice are recorded every week, so as to evaluate the drug toxicity and draw the survival curve. The results are shown in Table 7 and FIG. 31.

[0296] Table 7 Change of tumor volume of human prostate cancer nude mouse xenograft model

[0297] The actual embodiments in the present application are only for the explanation and illustration of the present application, and are not limited to the application, and the protection scope is still subject to the scope of the claims of the present application patent.

Claims

1. A Nanobody targeting PSCA comprising a heavy chain variable region comprising a CDR1, a CDR2 and a CDR3, characterized in that, the CDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 27, the CDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 28, and the CDR3 comprises an amino acid sequence as set forth in SEQ ID NO:

29.

2. The Nanobody of claim 1, wherein the amino acid sequence of the CDR1 is as set forth in SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, or SEQ ID NO: 26; the amino acid sequence of the CDR2 is as set forth in SEQ ID NO: 17 or SEQ ID NO: 20; and, the amino acid sequence of the CDR3 is as set forth in SEQ ID NO: 18, SEQ ID NO: 22, or SEQ ID NO: 24; and / or, the nanobody is a monoclonal antibody or a polyclonal antibody; and / or, the nanobody is a humanized antibody; preferably, the amino acid sequence of the CDR1 is as set forth in SEQ ID NO: 16, the amino acid sequence of the CDR2 is as set forth in SEQ ID NO: 17, and the amino acid sequence of the CDR3 is as set forth in SEQ ID NO: 18; or, the amino acid sequence of the CDR1 is as set forth in SEQ ID NO: 19, the amino acid sequence of the CDR2 is as set forth in SEQ ID NO: 20, and the amino acid sequence of the CDR2 is as set forth in SEQ ID NO: 18; or, the amino acid sequence of the CDR1 is as set forth in SEQ ID NO: 21, the amino acid sequence of the CDR2 is as set forth in SEQ ID NO: 20, and the amino acid sequence of the CDR3 is as set forth in SEQ ID NO: 22; or, the amino acid sequence of the CDR1 is as set forth in SEQ ID NO: 23, the amino acid sequence of the CDR2 is as set forth in SEQ ID NO: 20, and the amino acid sequence of the CDR3 is as set forth in SEQ ID NO: 24; or, the amino acid sequence of the CDR1 is as set forth in SEQ ID NO: 25, the amino acid sequence of the CDR2 is as set forth in SEQ ID NO: 20, and the amino acid sequence of the CDR3 is as set forth in SEQ ID NO: 24; or, the amino acid sequence of the CDR1 is as set forth in SEQ ID NO: 26, the amino acid sequence of the CDR2 is as set forth in SEQ ID NO: 20, and the amino acid sequence of the CDR3 is as set forth in SEQ ID NO: 18; or, the amino acid sequence of the CDR1 is as set forth in SEQ ID NO: 21, the amino acid sequence of the CDR2 is as set forth in SEQ ID NO: 20, and the amino acid sequence of the CDR3 is as set forth in SEQ ID NO:

24.

3. The Nanobody according to claim 1 or 2, characterized in that, the nanobody comprises (1) or (2) below: the CDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 27, the CDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 28, and the CDR3 comprises an amino acid sequence as set forth in SEQ ID NO:

29. (1) the heavy chain variable region comprises an amino acid sequence as set forth in any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7; (2) the heavy chain variable region comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1, preferably an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1 in FR regions, or, the heavy chain variable region comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 2, preferably an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 2 in FR regions; or, the heavy chain variable region comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 3, preferably an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 3 in FR regions; or, the heavy chain variable region comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 4, preferably an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 4 in FR regions; or, the heavy chain variable region comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 5, preferably an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 5 in FR regions; or, the heavy chain variable region comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 6, preferably an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 6 in FR regions; or, the heavy chain variable region comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 7, preferably an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 3 in FR regions.

4. A PSCA binding molecule, characterized in that, the PSCA binding molecule comprises one, two or more nanobodies as claimed in any one of claims 1-3; Preferably, the PSCA binding molecule further satisfies at least any one of the following characteristics: (1) the PSCA binding molecule is a heavy chain antibody; the heavy chain antibody comprises a nanobody as claimed in any one of claims 1-3, and a heavy chain constant region, the heavy chain constant region comprising CH2 and / or CH3; the heavy chain constant region is preferably derived from a heavy chain constant region of a human antibody; (2) the PSCA binding molecule is a recombinant fusion protein, the recombinant fusion protein comprises a first functional domain of a nanobody as claimed in any one of claims 1-3, and further comprises a second functional domain for prolonging in vivo half-life or targeting another target, the structure of the second functional domain being, for example, Fab, scFv, VHH, Fc region of IgG or albumin; (3) the PSCA binding molecule is a mono-specific antibody, a bi-specific antibody or a multi-specific antibody; Optionally, the Fc region of the IgG is an Fc region of IgG1, for example having the amino acid sequence set forth in SEQ ID NO:

30.

5. An isolated nucleic acid, comprising, The nucleic acid encodes the Nanobody of any one of claims 1-3 or the PSCA binding molecule of claim 4; Preferably, the nucleic acid comprises a nucleotide sequence as set forth in any one of SEQ ID NOs: 8-14; and / or, when the nucleic acid encodes the PSCA binding molecule, the nucleic acid further comprises a coding sequence of the Fc region of the IgG, preferably as set forth in SEQ ID NO:

31.

6. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid of claim 5; Preferably, the backbone of the recombinant expression vector is a plasmid, a cosmid, a bacteriophage or a viral vector; More preferably, the backbone of the plasmid is pComb, for example pComb3X.

7. A transformant characterized in that, The transformant comprises the nucleic acid of claim 5 or the recombinant expression vector of claim 6; Preferably, the host cell of the transformant is a prokaryotic cell, a yeast cell, an insect cell or an animal cell; More preferably, the host cell is E. coli, for example HB2151 E. coli.

8. An antibody drug conjugate, characterized in that, The antibody drug conjugate comprises the Nanobody of any one of claims 1-3 or the PSCA binding molecule of claim 4, and a cytotoxic agent or a label; Preferably, the cytotoxic agent is MMAE or MMAF.

9. A chimeric antigen receptor, characterized in that, The chimeric antigen receptor comprises the Nanobody of any one of claims 1-3 or the PSCA binding molecule of claim 4.

10. A genetically modified cell, characterized in that, The cell comprises the chimeric antigen receptor of claim 9; Preferably, the genetically modified cell is derived from a eukaryotic cell, preferably an isolated human cell; and / or, The genetically modified cell is derived from an immune cell, such as a T cell or an NK cell.

11. A method of preparing a Nanobody according to any one of claims 1-3 or a PSCA binding molecule according to claim 4, characterized in that, The method comprises the step of culturing the transformant of claim 7 to obtain the Nanobody or the PSCA binding molecule from the culture.

12. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises the Nanobody of any one of claims 1-3, the PSCA binding molecule of claim 4, the antibody drug conjugate of claim 8, the chimeric antigen receptor of claim 9 and / or the cell of claim 10, and a pharmaceutically acceptable carrier or excipient.

13. A drug delivery device characterized in that, The administration device comprises the Nanobody of any one of claims 1-3, the PSCA binding molecule of claim 4, the antibody drug conjugate of claim 8, the chimeric antigen receptor of claim 9 and / or the pharmaceutical composition of claim 12; Preferably, the administration device further comprises a means for administering the Nanobody, the PSCA binding molecule, the antibody drug conjugate, the chimeric antigen receptor and / or the pharmaceutical composition to a subject, for example a syringe or an infusion device.

14. Use of the Nanobody according to any one of claims 1-3, the PSCA binding molecule according to claim 4, the nucleic acid according to claim 5, the recombinant expression vector according to claim 6, the transformant according to claim 7, the drug conjugate of the antibody according to claim 8, the chimeric antigen receptor according to claim 9, the cell according to claim 10, or the pharmaceutical composition according to claim 12 in the preparation of a medicament for preventing, treating, diagnosing and / or prognosticating a PSCA high expression related disease. Preferably, the PSCA high expression related disease is a tumor, such as prostate cancer, bladder cancer or pancreatic cancer.

15. A method of detecting PSCA, comprising contacting a sample with an antibody of claim 1 and detecting the formation of an antibody-antigen complex. 15 The method comprises the following steps: (1) contacting a sample to be tested with the Nanobody according to any one of claims 1-3 or the PSCA binding molecule according to claim 4; (2) qualitatively or quantitatively judging according to the binding of the sample to be tested with the Nanobody or the PSCA binding molecule; Preferably, the method is for non-diagnostic purposes; and / or, the method is in vitro.

16. A method for preventing, treating, diagnosing and / or prognosing a disease associated with overexpression of PSCA, comprising administering a PSCA-binding agent to a subject in need thereof. The method comprises administering to a patient the Nanobody according to any one of claims 1-3, the PSCA binding molecule according to claim 4, the drug conjugate of the antibody according to claim 8, the chimeric antigen receptor according to claim 9, the pharmaceutical composition according to claim 12, or the administration device according to claim 13; Preferably, the PSCA high expression related disease is a tumor, such as prostate cancer, bladder cancer or pancreatic cancer.

17. The Nanobody according to any one of claims 1-3, the PSCA binding molecule according to claim 4, the nucleic acid according to claim 5, the recombinant expression vector according to claim 6, the transformant according to claim 7, the drug conjugate of the antibody according to claim 8, the chimeric antigen receptor according to claim 9, the cell according to claim 10, the pharmaceutical composition according to claim 12, or the administration device according to claim 13 for use in preventing, treating, diagnosing and / or prognosticating a PSCA high expression related disease. Preferably, the PSCA high expression related disease is a tumor, such as prostate cancer, bladder cancer or pancreatic cancer.

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