CN111303288B

CN111303288B - Separated protein combined with antigen PSMA and application thereof

Info

Publication number: CN111303288B
Application number: CN202010143681.2A
Authority: CN
Inventors: 黄冰; 何云; 戎一平
Original assignee: Heping Pharmaceutical Suzhou Co ltd
Current assignee: Nuona Biological Suzhou Co ltd
Priority date: 2020-03-04
Filing date: 2020-03-04
Publication date: 2020-12-25
Anticipated expiration: 2040-03-04
Also published as: CN111303288A

Abstract

The present application provides an isolated antigen binding protein comprising V having an amino acid sequence as set forth in SEQ ID NO. 15_HHCDR1, HCDR2 and HCDR 3; and it comprises V with an amino acid sequence shown as SEQ ID NO. 16_LLCDR1, LCDR2 and LCDR3 in (1). The present application also provides chimeric antigen receptors and immunoconjugates comprising the isolated antigen binding proteins, nucleic acids encoding the isolated antigen binding proteins, vectors comprising the isolated antigen binding proteins, cells comprising the nucleic acids or the vectors, methods of making the isolated antigen binding proteins, and uses of the isolated antigen binding proteins.

Description

Separated protein combined with antigen PSMA and application thereof

Technical Field

The application relates to the field of biomedicine, in particular to a separated protein combined with antigen PSMA and application thereof.

Background

Prostate cancer is the most commonly diagnosed cancer for men, and is the third most common cancer of death. Statistically, 16 million patients were diagnosed with this tumor in 2017, and caused 2 million 6 thousand deaths (Siegel RL)et al., （2017） CA Cancer J Clin. 67:7-30). Patients with localized cancer are often treated by surgery or radiation therapy (Walsh PC)et al., （2007） N Engl J Med. 357:2696–705). However, 20-40% of patients who receive radical prostatectomy, and 30-50% of patients who receive radiation therapy, experience relapse (Paller CJ)et al., （2013） Clin Adv Hematol Oncol. 11:14-23). The standard therapy for metastatic cancer is usually androgen blockade, by means of bilateral orchiectomy or chemocastration (e.g. administration of Luteinizing Hormone Receptor (LHR) agonists or antagonists) (Tannock IF)et al., （2004） N Engl J Med. 351:1502-12). Although androgen blockade is effective, with significant side effects, patients gradually develop castration-resistant prostate cancer (CRPC) (Petrylak DP)et al., （2004） N Engl J Med. 351: 1513-20). At present, no radical treatment method is available for metastatic CRPC (mCRPC), and the prognosis is poor.

In recent years, cancer vaccines, immune checkpoint blockades and tumor targeting antibodies have had a significant impact on the treatment of solid tumors. In 2010, the personalized cancer vaccine Sipuleucel-T targeting prostatic acid phosphatase was named the first FDA approved mCRPC immune preparation (Kantoff PWet al., （2010） N Engl J Med363: 411-22) that successfully whip clinical trials of vaccines targeting prostate-associated other antigens such as prostate specific membrane antigen (PMSA) and Prostate Specific Antigen (PSA).

Among the prostate cancer candidate markers that have been identified, the prostate specific membrane antigen (PMSA) appears to be the most prominent. PSMA is a homodimeric class II membrane glycoprotein expressed in different tissues, such as prostate, kidney, small intestine, central and peripheral nervous system, but mainly in prostate. PSMA is upregulated in prostate cancer (Schulke N et al, (2003)Proc Natl Acad Sci USA. 100:12590-5; Ross JS et al., （2003） Clin Cancer Res.6357-62) and increased with disease progression, with the highest expression level in metastatic, castration-resistant prostate cancer (Su SL)et al., （1995） Cancer Res. 55:1441-3). In addition, PSMA is also abundantly expressed in the neovasculature of most other solid tumors such as renal, breast, intestinal cancers, etc. (silver)r DA et al., （1997）, Clin Cancer Res. 3:81-5; Liu H et al., （1997）, Cancer Res. 57:3629-34; Chang SS et al., （1999） Cancer Res. 59:3192-8;Chang SS et al., （1999） Clin Cancer Res 5:2674-81; Chang SS et al., （2001）, Urology57:801-5). Most importantly, PSMA is rapidly and continuously internalized, delivering antibodies, antibody drug conjugates, etc. bound thereto to the interior of cells (Liu H)et al., （1998） Cancer Res. 58:4055-60; Henry MD et al., （2004） Cancer Res.64:7995-8001). These properties make PSMA an attractive target for immune antibody therapy of prostate and other cancers.

Among the antibodies used to treat prostate cancer, most studied is mab J591, which targets PSMA. Early experiments showed that J591 is well transported to prostate cancer transferase in bone and soft tissue (Nanus DM)et al., （2003） J Urol.170: S84-8). Phase 2 trials of J591 with low doses of interleukin-2 (IL-2) showed that this therapy was well tolerated, with 9 out of 16 patients having stable PSA (-50%<PSA variations<25%), but no patients showed a PSA reduction of more than 50%. While the phase 2 trial of J591 labeled with lutetium-177 showed more encouraging results, 59.6% of patients experienced a decrease in PSA after treatment, 1 of 12 patients had partial remission, and 8 patients were stable (Tagawa ST)et al., （2013） Clin Cancer Res.19:5182-91). In addition, BAY2010112, a double antibody specific for the CD3 receptor of T cells and PSMA, is also under investigation for prostate cancer treatment. Preclinical studies of BAY2010112 in a mouse model of prostate cancer found that administration of this antibody rapidly reduced tumor size and achieved complete remission (Friedrich M)et al., （2012） Mol Cancer Ther.11:2664-73). Clinical trials of BAY2010112 are currently in progress. In addition, conjugates of human IgG1 PSMA antibody with microtubule disrupter MMAE are being used in clinical trials for taxane-resistant mCRPC treatment. In phase 1 clinical trials, about 50% of treated patients show a decrease in PSAOr a decrease in hematological tumor cells (Petrylak DP)et al., （2013） J Clin Oncol.31:119). In the phase 2 clinical trial, 30% of patients show a 30% or more decrease in PSA, 14% of patients show a 50% or more decrease in PSA, 61% of patients have stable disease, 13% have partial remission, and 26% have disease progression (Petrylak DP)et al., （2015）, J Clin Oncol. 33:144）。

In addition, PSMA has also been shown to increase extracellular glutamate concentration either directly or indirectly, whereas pain caused by central or peripheral nervous system injury is associated with increased glutamate concentration. PSMA inhibition can reduce glutamate concentration and thereby reduce pain (Zhou J et al, (2005),Nature Reviews. Drug Discovery. 4（12）: 1015–26; Nagel J et al., （2006） Neuropharmacology. 51 （7–8）: 1163–71; Chen SRet al., （2002） The Journal of Pharmacology and Experimental Therapeutics. 300（2）: 662–7）。

despite advances in PSMA targeting, PSMA antibodies suffer from a number of considerable drawbacks, such as unknown epitope information for antibody binding, lack of cross-species reactivity data, and the like. Therefore, there is a need to develop more new antibodies to PMSA with desirable pharmaceutical properties.

Disclosure of Invention

In one aspect, the present application provides an isolated antigen binding protein comprising V as set forth in amino acid sequence SEQ ID NO. 15_HHCDR1, HCDR2 and HCDR 3; and which comprises V having the amino acid sequence SEQ ID NO 16_LLCDR1, LCDR2 and LCDR3 in (1).

In certain embodiments, the HCDR1 comprises the amino acid sequence set forth in SEQ ID No. 1, the HCDR2 comprises the amino acid sequence set forth in SEQ ID No. 2, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID No. 3.

In certain embodiments, the LCDR1 comprises the amino acid sequence set forth in SEQ ID No. 4, the LCDR2 comprises the amino acid sequence set forth in SEQ ID No. 5, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID No. 6.

In certain embodiments, the V is_HIncluding the framework regions H-FR1, H-FR2, H-FR3, and H-FR 4.

In certain embodiments, the C-terminus of H-FR1 is linked directly or indirectly to the N-terminus of HCDR1 and the H-FR1 comprises the amino acid sequence set forth in SEQ ID No. 7; the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 8; the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO 9; the N-terminal of the H-FR4 is linked to the C-terminal of the HCDR3, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO. 10.

In certain embodiments, the V is_LIncluding the framework regions L-FR1, L-FR2, L-FR3, and L-FR 4.

In certain embodiments, the C-terminus of L-FR1 is linked directly or indirectly to the N-terminus of LCDR1 and the L-FR1 comprises the amino acid sequence set forth in SEQ ID NO. 11; the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 comprises the amino acid sequence shown in SEQ ID NO. 12; the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 comprises the amino acid sequence shown in SEQ ID NO 13; the N-terminal of the L-FR4 is linked to the C-terminal of the LCDR3, and the L-FR4 comprises the amino acid sequence shown in SEQ ID NO. 14.

In certain embodiments, the isolated antigen binding protein comprises an antibody heavy chain constant region, and the antibody heavy chain constant region is derived from a human IgG heavy chain constant region.

In certain embodiments, the antibody heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO 19.

In certain embodiments, the isolated antigen binding protein comprises an antibody light chain constant region, and the antibody light chain constant region comprises a human Ig kappa constant region.

In certain embodiments, the antibody light chain constant region comprises the amino acid sequence set forth in SEQ ID NO 20.

In certain embodiments, the isolated antigen binding protein comprises an antibody heavy chain HC, and the HC comprises the amino acid sequence set forth in SEQ ID No. 21.

In certain embodiments, the isolated antigen binding protein comprises an antibody light chain LC, and the LC comprises the amino acid sequence set forth in SEQ ID NO. 22.

In certain embodiments, the isolated antigen binding protein comprises an antibody or antigen binding fragment thereof, wherein the antigen binding fragment comprises Fab, Fab', F (ab)₂Fv fragment, F (ab')₂scFv, di-scFv and/or dAb.

In certain embodiments, the isolated antigen binding protein has one or more of the following properties:

1) can be 1 × 10^-10M or less binds to PSMA protein, wherein the KD value is determined by Octet;

2) in FACS assays, capable of specifically binding to PSMA protein on the surface of HEK293T cells overexpressing human PSMA, CHO-K1 cells overexpressing monkey PSMA, or LNCAP cells;

3) (ii) capable of internalizing into HEK293T cells or LNCAP cells overexpressing human PSMA;

4) has ADCP activity on LNCAP cells.

In certain embodiments, the PSMA protein comprises a human PSMA protein or a monkey PSMA protein.

In another aspect, the present application provides a chimeric antigen receptor comprising the isolated antigen binding protein.

In another aspect, the present application provides an immunoconjugate comprising the isolated antigen binding protein.

In another aspect, the present application provides an isolated nucleic acid molecule or molecules encoding the isolated antigen binding protein or the chimeric antigen receptor.

In another aspect, the present application provides a vector comprising the nucleic acid molecule.

In another aspect, the present application provides a cell comprising said nucleic acid molecule or said vector.

In another aspect, the present application provides a pharmaceutical composition comprising the isolated antigen binding protein, the chimeric antigen receptor, the immunoconjugate, the nucleic acid molecule, the vector, and/or the cell, and optionally a pharmaceutically acceptable adjuvant.

In another aspect, the present application provides a method of making the isolated antigen binding protein, the method comprising culturing the cell under conditions such that the isolated antigen binding protein is expressed.

In another aspect, the present application provides the use of the isolated antigen binding protein, the chimeric antigen receptor, the immunoconjugate, the nucleic acid molecule, the vector, the cell and/or the pharmaceutical composition for the preparation of a medicament for the prevention, alleviation and/or treatment of tumors.

In certain embodiments, the tumor comprises prostate cancer.

In another aspect, the present application provides a method of detecting PSMA in a sample, comprising administering the isolated antigen binding protein.

Compared with the prior art, the isolated antigen binding protein has at least one of the following beneficial effects:

(1) the isolated antigen binding proteins described herein can be expressed at 1 × 10^-10K of M or less_DBinding to PSMA protein, wherein said K_DValues were determined by Octet.

(2) In FACS assays, the isolated antigen binding proteins described herein are capable of specifically binding to PSMA protein on the surface of HEK293T cells overexpressing human PSMA, CHO-K1 cells overexpressing monkey PSMA, or LNCAP cells.

(3) The isolated antigen binding proteins described herein are capable of internalizing into HEK293T cells or LNCAP cells that overexpress human PSMA.

(4) The isolated antigen binding proteins described herein have ADCP activity on LNCAP cells.

(5) The isolated antigen binding proteins described herein can be used to prepare a medicament that is effective in preventing, alleviating, and/or treating a tumor.

(6) The isolated antigen binding proteins described herein can be used to detect the presence or amount of PSMA in a sample.

Other aspects and advantages of the present application will be readily apparent to those skilled in the art from the following detailed description. Only exemplary embodiments of the present application have been shown and described in the following detailed description. As those skilled in the art will recognize, the disclosure of the present application enables those skilled in the art to make changes to the specific embodiments disclosed without departing from the spirit and scope of the invention as it is directed to the present application. Accordingly, the descriptions in the drawings and the specification of the present application are illustrative only and not limiting.

Drawings

The specific features of the invention to which this application relates are set forth in the appended claims. The features and advantages of the invention to which this application relates will be better understood by reference to the exemplary embodiments described in detail below and the accompanying drawings. The brief description of the drawings is as follows:

FIGS. 1A and 1B show binding of antibodies to human PSMA protein on the cell surface;

FIGS. 2A and 2B show binding of antibodies to monkey PSMA protein on the cell surface;

FIG. 3 shows the binding of antibodies to PSMA protein expressed on the surface of LNCAP cells;

FIG. 4 shows the survival of HEK293T/human PSMA cells under antibody treatment;

figure 5 shows the survival of LNCAP cells under antibody treatment;

FIG. 6 shows antibody-mediated phagocytosis of LNCAP in macrophages;

FIGS. 7A-7D show the effect of endocytosis of the antibody on the target cell.

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification.

The present application is further described below: in the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Also, protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology related terms, and laboratory procedures used herein are all terms and conventional procedures used extensively in the relevant art. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

In the present application, the term "isolated" generally refers to a product obtained from a natural state by artificial means. If an "isolated" substance or component occurs in nature, it may be altered from its natural environment, or it may be isolated from its natural environment, or both. For example, a polynucleotide or polypeptide that is not isolated naturally occurs in a living animal, and a polynucleotide or polypeptide that is the same in high purity and that is isolated from such a natural state is said to be isolated. The term "isolated" does not exclude the presence of other impurities which do not affect the activity of the substance, mixed with artificial or synthetic substances.

In the present application, the term "isolated antigen binding protein" generally refers to a protein having antigen binding ability obtained from a natural state by artificial means. The "isolated antigen binding protein" may comprise a portion that binds an antigen and, optionally, a scaffold or framework portion that allows the antigen binding portion to adopt a conformation that facilitates binding of the antigen binding portion to an antigen. The antigen binding protein may comprise, for example, an antibody-derived protein scaffold or an alternative protein scaffold or artificial scaffold with grafted CDRs or CDR derivatives. Such scaffolds include, but are not limited to, scaffolds comprising antibody sources introduced, for example, with mutations to stabilize the three-dimensional structure of the antigen binding protein and fully synthetic scaffolds comprising, for example, biocompatible polymers. See, e.g., Korndorfer et al, 2003, Proteins: Structure, Function, andBioinformation, 53(1): 121-. In addition, peptide antibody mimetics ("PAMs") as well as scaffolds based on antibody mimetics utilizing fibronectin components can be used as scaffolds.

In the present application, the terms "KD", "K_D”、“K _D"used interchangeably, and generally refers to the equilibrium dissociation constant," KD "is the ratio of the dissociation rate constant (kdis, also referred to as the" off-rate "(koff) or" KD ") to the association rate constant (kon, also referred to as the" association rate "(kon) or" ka "). The binding affinity of an antigen-binding protein (e.g., an antibody) for an antigen can be expressed using an association rate constant (kon), an dissociation rate constant (kdis), and an equilibrium dissociation constant (KD). Methods for determining binding and dissociation rate constants are well known in the art and include, but are not limited to, biofilm interference techniques (BLI), Radioimmunoassays (RIA), equilibrium dialysis, Surface Plasmon Resonance (SPR), Fluorescence Resonance Energy Transfer (FRET), Co-immunoprecipitation (Co-IP), and protein chip techniques. The measured affinity for a particular protein-protein interaction may be different if measured under different conditions (e.g., salt concentration, pH).

In the present application, the term "EC 50" or "EC₅₀"half maximal effect concentration, also known as half maximal effect concentration, generally refers to the concentration of antibody that causes 50% of the maximal effect.

In the present application, the term "PSMA" generally refers to the prostate specific membrane antigen, also known as type ii Glutamate Carboxypeptidase (GCPII) or NAAG peptidase. The term includes variants, homologues, analogues, orthologues and/or paralogues. For example, an antibody specific for human PSMA may cross-react with PSMA protein of another species, such as monkey, under certain circumstances. In other embodiments, an antibody specific for human PSMA protein may be completely specific for human PSMA protein without cross-reacting with other species or types of proteins, or may cross-react with PSMA proteins of some other species but not all other species.

In the present application, the term "human PSMA" generally refers to a PSMA protein having a human amino acid sequence, such as a PSMA protein having the amino acid sequence of Genbank accession No. NP _ 004467. The term "monkey PSMA" refers to a PSMA protein having the amino acid sequence of a monkey, which may be a cynomolgus monkey or a cynomolgus monkey. For example, the term "monkey PSMA" may refer to the cynomolgus PSMA protein having the amino acid sequence of Genbank accession number XP — 005579379.

In the present application, the term "specific binding" or "specific" generally refers to a measurable and reproducible interaction, such as binding between a target and an antibody, that can be determinative of the presence of the target in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antibody that specifically binds a target (which may be an epitope) is an antibody that binds that target with greater affinity, avidity, more readily, and/or for a greater duration than it binds other targets. In one embodiment, the extent of binding of the antibody to an unrelated target is less than about 10% of the binding of the antibody to the target, as measured, for example, by Radioimmunoassay (RIA). For example, in the present application, the isolated antigen binding protein can be<4x10^-10M or less dissociation constant (KD) binds to PSMA protein. In certain embodiments, the antibody specifically binds to an epitope on the protein that is conserved among proteins of different species. In another embodiment, specific binding may include, but is not required to be, exclusive binding.

In the present application, the term "tumor" generally refers to a neoplasm or solid lesion formed by abnormal cell growth. In the present application, the tumor may be a solid tumor or a hematological tumor. For example, in the present application, the tumor can be a PSMA-positive tumor, wherein the PSMA-positive tumor can include prostate cancer.

In the present application, the term "variable domain" generally refers to the amino-terminal domain of an antibody heavy or light chain. The variable domains of the heavy and light chains may be referred to as "V" respectively_H"and" V_L"(alternatively referred to as" VH "and" VL ", respectively). These domains are usually the most variable parts of an antibody (relative to other antibodies of the same type) and contain an antigen binding site.

In the present application, the term "variable" generally refers to the fact that certain segments of the variable domains differ greatly in sequence between antibodies. The V domain mediates antigen binding and determines the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domain. Instead, it is concentrated in three segments called hypervariable regions (CDRs or HVRs) in the light and heavy chain variable domains. The more highly conserved portions of the variable domains are called Framework Regions (FR). The variable domains of native heavy and light chains each comprise four FR regions, mostly in a β -sheet configuration, connected by three CDRs, which form a circular connection, and in some cases form part of a β -sheet structure. The CDRs in each chain are held together in close proximity by the FR regions, and the CDRs from the other chain together contribute to the formation of the antigen binding site of the antibody (see Kabat et al, Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not directly involved in binding of the antibody to the antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity.

In the present application, the term "antibody" generally refers to an immunoglobulin or a fragment or derivative thereof, and encompasses any polypeptide comprising an antigen binding site, whether produced in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, nonspecific, humanized, single chain, chimeric, synthetic, recombinant, hybrid, mutated, and grafted antibodies. Unless otherwise modified by the term "intact", as in "intact antibody", for the purposes of the present invention, the term "antibody" also includes antibody fragments, such as Fab, F (ab')₂Fv, scFv, Fd, dAb, and other antibody fragments that retain antigen binding function (e.g., specifically bind PSMA). Typically, such fragments should include an antigen binding domain. The basic 4 chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies consist of 5 elementary heterotetrameric units with another polypeptide called J chain and contain 10 antigen-binding sites, while IgA antibodies comprise 2-5 elementary 4-chain monomers that can polymerize in association with J chain to form multivalent combinationsAnd (5) Yuan. For IgG, the 4-chain unit is typically about 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has a variable domain (VH) at the N-terminus, followed by three constant domains (CH) for the alpha and gamma chains, respectively, and four CH domains for the mu and isotype. Each L chain has a variable domain (VL) at the N-terminus and a constant domain at its other end. VL corresponds to VH and CL to the first constant domain of the heavy chain (CH 1). Specific amino acid residues are believed to form an interface between the light and heavy chain variable domains. The VH and VL pair together to form a single antigen-binding site. For the structure and properties of antibodies of different classes see, e.g., Basic and Clinical Immunology, 8th Edition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds), Appleton&Lange, Norwalk, conn., 1994, page 71 and chapter 6. L chains from any vertebrate species can be classified into one of two distinctly different classes, termed κ and λ, based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of its heavy Chain (CH) constant domain, immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, with heavy chains designated α, γ and μ, respectively. Based on the relatively small differences in CH sequence and function, the γ and α classes are further divided into subclasses, e.g., humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgK 1.

In the present application, the term "CDR" generally refers to a region of an antibody variable domain whose sequence is highly variable and/or forms a structurally defined loop. Typically, an antibody comprises six CDRs; three in VH (HCDR1, HCDR2, HCDR3), and three in VL (LCDR1, LCDR2, LCDR 3). In natural antibodies, HCDR3 and LCDR3 show the majority of diversity of the six CDRs, and in particular HCDR3 is thought to play a unique role in conferring fine specificity to the antibody. See, e.g., Xu et al, Immunity 13:37-45 (2000); Johnson and Wu, in Methods in Molecular Biology 248:1-25 (Lo, ed., Human Press, Totowa, N.J., 2003). In fact, naturally occurring camel antibodies consisting of only heavy chains function normally and are stable in the absence of light chains. See, for example, Hamers-Casterman et al, Nature 363: 446-.

In this application, the term "FR" generally refers to a more highly conserved portion of an antibody variable domain, which is referred to as the framework region. Typically, the variable domains of native heavy and light chains each comprise four FR regions, namely four in the VH (H-FR1, H-FR2, H-FR3, and H-FR4), and four in the VL (L-FR1, L-FR2, L-FR3, and L-FR 4). For example, the VL of an isolated antigen binding protein described herein may comprise the framework regions L-FR1, L-FR2, L-FR3, and L-FR 4. The VH of an isolated antigen binding protein described herein may comprise the framework regions H-FR1, H-FR2, H-FR3, and H-FR 4.

In the present application, the term "antigen-binding fragment" generally refers to one or more fragments that have the ability to specifically bind an antigen (e.g., PSMA protein). In the present application, the antigen binding fragments may include Fab, Fab', F (ab)₂Fv fragment, F (ab')₂scFv, di-scFv and/or dAb.

In the present application, the term "monoclonal antibody" or "monoclonal antibody composition" generally refers to a preparation of antibody molecules of a single molecular composition. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope.

In this application, the term "human antibody" generally refers to antibodies in which the variable region framework and CDR regions are derived from human germline immunoglobulin sequences. In addition, if the antibody contains constant regions, it is also derived from human germline immunoglobulin sequences. The human antibodies of the present application may comprise amino acid residues not encoded by human germline immunoglobulin sequences, e.g., mutations introduced by random or point mutations in vitro or by somatic mutations in vivo. However, the term "human antibody" does not include antibodies in which CDR sequences from other mammalian species are inserted into the human framework sequences.

In this application, the term "murine antibody" generally refers to an antibody in which the variable region framework and CDR regions are derived from mouse germline immunoglobulin sequences. In addition, if the antibody contains constant regions, it is also derived from mouse germline immunoglobulin sequences. The murine antibodies of the present application may comprise amino acid residues not encoded by mouse germline immunoglobulin sequences, such as mutations introduced by random or point mutations in vitro or by somatic mutations in vivo. However, the term "murine antibody" does not include antibodies having CDR sequences from other mammalian species inserted into the mouse framework sequences.

In the present application, the term "chimeric antibody" generally refers to an antibody obtained by combining genetic material of non-human origin with genetic material of human origin. Or more generally, a chimeric antibody refers to an antibody that combines genetic material of one species with genetic material of another species.

In this application, the term "humanized antibody" generally refers to an antibody that is derived from a non-human species but whose protein sequence has been modified to increase its similarity to a naturally occurring human antibody.

In the present application, the terms "antibody recognizing an antigen" and "antibody specific to an antigen" are used herein interchangeably with the term "antibody specifically binding to an antigen".

In this application, the term "directly connected" is used in contrast to the term "indirectly connected," which generally refers to a direct connection. For example, the direct linkage may be a direct linkage without a spacer between the substances. The spacer may be a linker. For example, the linker may be a peptide linker. The term "indirectly linked" generally refers to a condition in which the substances are not directly linked to each other. For example, the indirect connection may be a connection via a spacer. For example, in the isolated antigen binding proteins described herein, the C-terminus of the L-FR1 and the N-terminus of the LCDR1 can be linked directly or indirectly.

In the present application, the terms "antibody-dependent cell-mediated phagocytosis" or "ADCP" are used interchangeably and generally refer to the binding of the Fc portion of an antibody to an Fc receptor on an effector cell (e.g., phagocytic cell) following binding of the antibody to a corresponding antigen on a target cell, thereby inducing phagocytosis of the target cell by the effector cell.

In the present application, the term "isolated nucleic acid molecule" generally refers to an isolated form of nucleotides, deoxyribonucleotides or ribonucleotides, of any length, or an analog isolated from its natural environment or synthesized synthetically.

In the present application, the term "vector" generally refers to a nucleic acid vehicle into which a polynucleotide encoding a protein can be inserted and the protein expressed. The vector may be transformed, transduced or transfected into a host cell so that the genetic material elements it carries are expressed in the host cell. By way of example, the carrier includes: a plasmid; phagemid; a cosmid; artificial chromosomes such as Yeast Artificial Chromosomes (YACs), Bacterial Artificial Chromosomes (BACs), or artificial chromosomes (PACs) derived from P1; bacteriophage such as lambda phage or M13 phage, animal virus, etc. Animal virus species used as vectors are retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e.g., herpes simplex virus), poxvirus, baculovirus, papilloma virus vacuolatum (e.g., SV 40). A vector may contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may contain a replication initiation site. The vector may also include components which assist its entry into the cell, such as viral particles, liposomes or protein coats, but not exclusively.

In this application, the term "cell" generally refers to a single cell, cell line or cell culture that may be or has been the recipient of a subject plasmid or vector, which includes a nucleic acid molecule of the invention or a vector of the invention. The cell may comprise progeny of a single cell. Progeny may not necessarily be identical (in morphology of the total DNA complement or in the genome) to the original parent cell due to natural, accidental, or deliberate mutation. The cells may comprise cells transfected in vitro with a vector described herein. The cell may be a bacterial cell (e.g., E.coli), yeast cell, or other eukaryotic cell, such as a COS cell, Chinese Hamster Ovary (CHO) cell, CHO-K1 cell, LNCAP cell, HeLa cell, HEK293 cell, COS-1 cell, NS0 cell. In certain embodiments, the cell is a mammalian cell. In certain embodiments, the mammalian cell is a HEK293 cell.

In the present application, the term "pharmaceutical composition" generally refers to a composition that is suitable for administration to a patient, preferably a human patient. For example, a pharmaceutical composition described herein, which can comprise an isolated antigen binding protein described herein, a nucleic acid molecule described herein, a vector described herein, and/or a cell described herein, and optionally a pharmaceutically acceptable adjuvant. In addition, the pharmaceutical composition may further comprise suitable formulations of one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and/or preservatives. The acceptable ingredients of the composition are preferably non-toxic to the recipient at the dosages and concentrations employed. The pharmaceutical compositions of the present invention include, but are not limited to, liquid, frozen and lyophilized compositions.

In the present application, the term "pharmaceutically acceptable adjuvant" generally refers to any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, which are compatible with pharmaceutical administration, are generally safe, non-toxic, and are neither biologically nor otherwise undesirable.

In this application, the term "subject" generally refers to a human or non-human animal, including but not limited to a cat, dog, horse, pig, cow, sheep, rabbit, mouse, rat, or monkey.

In the present application, the term "comprising" is generally intended to include the explicitly specified features, but not to exclude other elements.

In the present application, the term "about" generally means varying from 0.5% to 10% above or below the stated value, for example, varying from 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the stated value.

Isolated antigen binding proteins

In one aspect, the present application provides an isolated antigen binding protein comprising V having an amino acid sequence as set forth in SEQ ID NO. 15_HAt least one CDR of (a); and it comprises V with an amino acid sequence shown as SEQ ID NO. 16_LAt least one CDR of (a).

In the present application, the isolated antigen binding protein may comprise V having the amino acid sequence shown in SEQ ID NO. 15_HHCDR1 in (iii).

In the present application, the isolated antigen binding protein may comprise V having the amino acid sequence shown in SEQ ID NO. 15_HHCDR2 in (iii).

In the present application, the isolated antigen binding protein may comprise V having the amino acid sequence shown in SEQ ID NO. 15_HHCDR3 in (iii).

In the present application, the isolated antigen binding protein may comprise V having the amino acid sequence shown in SEQ ID NO 16_LLCDR1 in (1).

In the present application, the isolated antigen binding protein may comprise V having the amino acid sequence shown in SEQ ID NO 16_LLCDR2 in (1).

In the present application, the isolated antigen binding protein may comprise V having the amino acid sequence shown in SEQ ID NO 16_LLCDR3 in (1).

Properties of the isolated antigen binding protein

In the present application, the isolated antigen binding protein may have one or more of the following properties:

1) can be 1 × 10^-10K of M or less_DBinding to PSMA protein, wherein said K_DValues were determined by Octet;

4) has ADCP activity on LNCAP cells.

In bookIn the application, the isolated antigen binding protein can be expressed as 1 × 10^-10K of M or less_DBinding to PSMA protein, wherein said K_DValues were determined by Octet. For example, the isolated antigen binding proteins described herein bind to K derived from the human PSMA protein_DThe value may be ≦ 1 × 10^-10M、≤5×10^-11M、≤1×10^-11M、≤1×10^-12M、≤9×10^-13M、≤8×10^-13M、≤7×10^-13M、≤6×10^-13M、≤5×10^-13M、≤4×10^-13M、≤3×10^-13M、≤2×10^-13M or less than or equal to 1X 10^-13And M. For another example, an isolated antigen binding protein described herein binds to monkey-derived K of PSMA protein_DThe value may be ≦ 1 × 10^-10M、≤5×10^-11M、≤1×10^-11M、≤1×10^-12M、≤9×10^-13M、≤8×10^-13M、≤7×10^-13M、≤6×10^-13M、≤5×10^-13M、≤4×10^-13M、≤3×10^-13M、≤2×10^-13M or less than or equal to 1X 10^-13M。

In the present application, K is_DValues can also be determined by ELISA, competition ELISA or BIACORE or KINEXA.

In the present application, the isolated antigen binding protein is capable of specifically binding to PSMA protein on the surface of HEK293T cells, CHO-K1 cells, or LNCAP cells, as can be determined by FACS. For example, specific binding of an isolated antigen binding protein described herein to PSMA protein on the surface of HEK293T cells, CHO-K1 cells, or LNCAP cells can be reflected by EC50 in a FACS assay, e.g., a lower EC50 indicates better specific binding. For example, the isolated antigen binding protein can have an EC50 value of 0.01 μ g/ml to 0.10 μ g/ml, 0.01 μ g/ml to 0.30 μ g/ml, 0.01 μ g/ml to 0.50 μ g/ml, 0.01 μ g/ml to 0.70 μ g/ml, 0.01 μ g/ml to 0.90 μ g/ml, 0.01 μ g/ml to 1.00 μ g/ml, 0.01 μ g/ml to 1.30 μ g/ml, 0.01 μ g/ml to 1.35 μ g/ml, or 0.01 μ g/ml to 1.40 μ g/ml for binding to PSMA protein on the cell surface of HEK293T in FACS assay. For example, the isolated antigen binding protein may have an EC50 value of 0.01. mu.g/ml-0.10. mu.g/ml, 0.01. mu.g/ml-0.20. mu.g/ml, 0.01. mu.g/ml-0.30. mu.g/ml, 0.01. mu.g/ml-0.40. mu.g/ml, 0.01. mu.g/ml-0.50. mu.g/ml, 0.01. mu.g/ml-0.60. mu.g/ml, 0.01. mu.g/ml-0.70. mu.g/ml, 0.01. mu.g/ml-0.80. mu.g/ml, 0.01. mu.g/ml-0.90. mu.g/ml, 0.01. mu.g/ml-0.95. mu.g/ml, 0.01. mu.g/ml-1.00. mu.g/ml, or 0.01. mu.10. mu.g/ml, for binding to the PSMA protein on the cell surface of CHO-K1 in FACS assay. For example, the isolated antigen binding protein may have an EC50 value of 0.01. mu.g/ml-0.10. mu.g/ml, 0.01. mu.g/ml-0.20. mu.g/ml, 0.01. mu.g/ml-0.30. mu.g/ml, 0.01. mu.g/ml-0.40. mu.g/ml, 0.01. mu.g/ml-0.50. mu.g/ml, 0.01. mu.g/ml-0.60. mu.g/ml, 0.01. mu.g/ml-0.70. mu.g/ml, 0.01. mu.g/ml-0.80. mu.g/ml, 0.01. mu.g/ml-0.90. mu.g/ml, 0.01. mu.g/ml-0.95. mu.g/ml, 0.01. mu.g/ml-1.00. mu.g/ml, or 0.01. mu.g/ml-1. mu.10. mu.g/ml in a FACS assay for binding to the PSMA protein on the.

In the present application, the PSMA protein may comprise a human PSMA protein or a monkey PSMA protein. For example, the PSMA protein may comprise a PSMA protein having an amino acid sequence of Genbank accession No. NP _ 004467. As another example, the PSMA protein may comprise a PSMA protein having the amino acid sequence of Genbank accession number XP _ 014970879. As another example, the PSMA protein may comprise a PSMA protein having the amino acid sequence of Genbank accession number XP _ 005579379.

In the present application, the isolated antigen binding protein is capable of internalizing into HEK293T cells or LNCAP cells. For example, the isolated antigen binding proteins described herein can mediate internalization of cell surface expressed PSMA proteins by binding to the extracellular tail of PSMA. In certain embodiments, the rate of endocytosis of an isolated antigen binding protein described herein in LNCAP cells can be less than or equal to 2 hours, less than or equal to 1.5 hours, less than or equal to 1 hour, or less than or equal to 0.5 hours.

In the present application, the isolated antigen binding protein may exhibit ADCP activity on PSMA-positive cells (also denoted as "PSMA + cells"). The PSMA-positive cells can be LNCAP cells. For example, in certain embodiments, the isolated antigen binding protein is capable of inhibiting the growth of tumor cells by inducing antibody-dependent cell-mediated phagocytosis (ADCP). The tumor cell can be a PSMA-positive cell, e.g., a LNCAP cell.

The class of isolated antigen binding proteins

In the present application, the isolated antigen binding protein may comprise an antibody or antigen binding fragment thereof. For example, an isolated antigen binding protein described herein can include, but is not limited to, a recombinant antibody, a monoclonal antibody, a human antibody, a murine antibody, a humanized antibody, a chimeric antibody, a bispecific antibody, a single chain antibody, a diabody, a triabody, a tetrabody, an Fv fragment, an scFv fragment, an Fab 'fragment, an F (ab')₂Fragments and camelized single domain antibodies.

In the present application, the antibody may be a humanized antibody. In other words, an isolated antigen binding protein described herein, which can be an antibody or variant, derivative, analog, or fragment thereof that immunospecifically binds to an antigen of interest (e.g., human PSMA) and comprises a Framework (FR) region having substantially the amino acid sequence of a human antibody and a Complementarity Determining Region (CDR) having substantially the amino acid sequence of a non-human antibody. By "substantially" in the context of a CDR is meant that the amino acid sequence of the CDR is at least 80%, preferably at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of a CDR of a non-human antibody. The humanized antibody can comprise substantially all of at least one and typically two variable domains (Fab, Fab ', F (ab') 2, FabC, Fv), in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., antibody) and all or substantially all of the framework regions are framework regions having human immunoglobulin consensus sequences. Preferably, the humanized antibody further comprises at least a portion of an immunoglobulin constant region (e.g., Fc), typically that of a human immunoglobulin. In some embodiments, the humanized antibody contains a light chain and at least a variable domain of a heavy chain. The antibody may also include the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. In some embodiments, the humanized antibody contains only humanized light chains. In some embodiments, the humanized antibody contains only humanized heavy chains. In particular embodiments, the humanized antibody contains only the humanized variable domains of the light chain and/or the humanized heavy chain.

In the present application, the antigen binding fragments may include Fab, Fab', F (ab)₂Fv fragment, F (ab')₂scFv, di-scFv and/or dAb.

CDR

The CDRs of an antibody, also known as complementarity determining regions, are part of the variable region. The amino acid residues of this region contact the antigen or antigenic epitope. Antibody CDRs can be determined by a variety of coding systems, such as CCG, Kabat, Chothia, IMGT, combinations of Kabat/Chothia, and the like. These coding systems are known in the art and can be found in particular in http:// www.bioinf.org.uk/abs/index. One skilled in the art can determine the CDR regions using different coding systems depending on the sequence and structure of the antibody. The CDR regions may differ using different coding systems. The CDRs of the isolated antigen binding proteins described herein can be determined using Kabat.

In the present application, the HCDR1 can comprise the amino acid sequence shown in SEQ ID NO. 1.

In the present application, the HCDR2 can comprise the amino acid sequence shown in SEQ ID NO. 2.

In the present application, the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO. 3.

For example, the HCDR1 of the isolated antigen binding proteins described herein can comprise the amino acid sequence shown in SEQ ID NO. 1, the HCDR2 can comprise the amino acid sequence shown in SEQ ID NO. 2, and the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO. 3.

In the present application, the LCDR1 can comprise the amino acid sequence shown in SEQ ID NO. 4.

In the present application, the LCDR2 can comprise the amino acid sequence shown in SEQ ID NO. 5.

In the present application, the LCDR3 can comprise the amino acid sequence shown in SEQ ID NO. 6.

For example, the LCDR1 of the isolated antigen binding proteins described herein can comprise the amino acid sequence shown in SEQ ID NO. 4, LCDR2 can comprise the amino acid sequence shown in SEQ ID NO. 5, and LCDR3 can comprise the amino acid sequence shown in SEQ ID NO. 6.

As another example, the HCDR1 of an isolated antigen binding protein described herein can comprise the amino acid sequence set forth in SEQ ID NO. 1, HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO. 2, HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO. 3, and LCDR1 can comprise the amino acid sequence set forth in SEQ ID NO. 4, LCDR2 can comprise the amino acid sequence set forth in SEQ ID NO. 5, and LCDR3 can comprise the amino acid sequence set forth in SEQ ID NO. 6.

FR

In the present application, the VH of the isolated antigen binding protein may comprise the framework regions H-FR1, H-FR2, H-FR3, and H-FR 4.

In the present application, the C-terminus of the H-FR1 is linked directly or indirectly to the N-terminus of the HCDR1, and the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 7.

In the present application, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 8.

In the present application, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO. 9.

In the present application, the N-terminus of the H-FR4 is linked to the C-terminus of the HCDR3, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 10.

For example, H-FR1 of an isolated antigen binding protein described herein can comprise the amino acid sequence shown as SEQ ID NO. 7, H-FR2 can comprise the amino acid sequence shown as SEQ ID NO. 8, H-FR3 can comprise the amino acid sequence shown as SEQ ID NO. 9, and H-FR4 can comprise the amino acid sequence shown as SEQ ID NO. 10.

In the present application, the VL of the isolated antigen binding protein may comprise the framework regions L-FR1, L-FR2, L-FR3, and L-FR 4.

In the present application, the C-terminus of the L-FR1 may be linked directly or indirectly to the N-terminus of the LCDR1, and the L-FR1 may comprise the amino acid sequence shown in SEQ ID NO. 11.

In the present application, the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO. 12.

In the present application, the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO 13.

In the present application, the N-terminus of the L-FR4 is linked to the C-terminus of the LCDR3, and the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO. 14.

For example, L-FR1 of an isolated antigen binding protein described herein can comprise the amino acid sequence shown as SEQ ID NO. 11, L-FR2 can comprise the amino acid sequence shown as SEQ ID NO. 12, L-FR3 can comprise the amino acid sequence shown as SEQ ID NO. 13, and L-FR4 can comprise the amino acid sequence shown as SEQ ID NO. 14.

As another example, H-FR1 of an isolated antigen binding protein described herein can comprise the amino acid sequence set forth in SEQ ID NO. 7, H-FR2 can comprise the amino acid sequence set forth in SEQ ID NO. 8, H-FR3 can comprise the amino acid sequence set forth in SEQ ID NO. 9, H-FR4 can comprise the amino acid sequence set forth in SEQ ID NO. 10, and L-FR1 can comprise the amino acid sequence set forth in SEQ ID NO. 11, L-FR2 can comprise the amino acid sequence set forth in SEQ ID NO. 12, L-FR3 can comprise the amino acid sequence set forth in SEQ ID NO. 13, and L-FR4 can comprise the amino acid sequence set forth in SEQ ID NO. 14.

V_HAnd V_L

The isolated antigen binding proteins described herein may comprise an antibody light chain variable region V_HAnd antibody heavy chain variable region V_L. For example, the V_HCan comprise the amino acid sequence shown as SEQ ID NO. 15, and the V_LCan comprise the amino acid sequence shown as SEQ ID NO. 16.

Constant region, heavy chain and light chain

In the present application, the isolated antigen binding protein may comprise an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from a human IgG heavy chain constant region. In certain embodiments, the isolated antigen binding protein may comprise an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from a human IgG1 heavy chain constant region. For example, the antibody heavy chain constant region may comprise the amino acid sequence set forth in SEQ ID NO 19.

In the present application, the isolated antigen binding protein may comprise an antibody light chain constant region, and the antibody light chain constant region may comprise a human Ig kappa constant region. For example, the antibody light chain constant region may comprise the amino acid sequence set forth in SEQ ID NO 20.

In the present application, the isolated antigen binding protein may comprise an antibody heavy chain HC, and the HC may comprise the amino acid sequence shown in SEQ ID No. 21.

In the present application, the isolated antigen binding protein may comprise an antibody light chain LC, and the LC may comprise the amino acid sequence shown in SEQ ID NO. 22.

An isolated antigen binding protein described herein can comprise an antibody heavy chain and an antibody light chain.

For example, the heavy chain may comprise the amino acid sequence shown in SEQ ID NO. 21 and the light chain may comprise the amino acid sequence shown in SEQ ID NO. 22.

In the present application, the heavy chain of the isolated antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 21 and the light chain may comprise the amino acid sequence shown in SEQ ID NO. 22. Wherein the HCDR1 of the isolated antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO. 1, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 2, the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 3, and the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO. 4, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO. 5, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO. 6. Furthermore, H-FR1 of the isolated antigen binding protein may comprise the amino acid sequence shown as SEQ ID NO. 7, H-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 8, H-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 9, H-FR4 may comprise the amino acid sequence shown as SEQ ID NO. 10, and L-FR1 may comprise the amino acid sequence shown as SEQ ID NO. 11, L-FR2 may comprise the amino acid sequence shown as SEQ ID NO. 12, L-FR3 may comprise the amino acid sequence shown as SEQ ID NO. 13, L-FR4 may comprise SEQ ID NO. 11ID NO. 14. Further, the V_HCan comprise the amino acid sequence shown as SEQ ID NO. 15, and the V_LCan comprise the amino acid sequence shown as SEQ ID NO. 16. For example, the isolated antigen binding protein may be PR 001104.

Furthermore, it is contemplated that the isolated antigen binding proteins described herein may comprise heavy and/or light chain sequences that are modified with one or more conserved sequences from PR001104 antibodies. By "conservative sequence modification" is meant an amino acid modification that does not significantly affect or alter the binding properties of the antibody. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the isolated antigen binding proteins described herein by standard techniques known in the art, such as point mutations and PCR-mediated mutations. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Groups of amino acid residues having similar side chains are known in the art. These groups of amino acid residues include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In certain embodiments, one or more amino acid residues in a CDR region of an isolated antigen binding protein described herein can be replaced with other amino acid residues of the ipsilateral chain set. Those skilled in the art know that some conservative sequence modifications do not abolish antigen binding, as seen in particular, for example, Brummell et al, (1993) Biochem 32:1180-8; de Wildt et al, (1997) prot. Eng. 10:835-41; Komissarov et al, (1997) J. Biol. chem. 272:26864 26870; Hall et al, (1992) J. Immunol. 149:1605-12; Kelley and O' Connell (1993) Biochem.32:6862-35; Adib-Conquy et al, (1998) int. Immunol.10:341-6 and Beers et al, (2000) Clin. Can. Res. 6: 2835-43.

Chimeric antigen receptor, immune coupler, nucleic acid molecule, vector, cell and pharmaceutical composition

Chimeric antigen receptors

In another aspect, the present application provides chimeric antigen receptors, which may comprise the isolated antigen binding proteins described herein.

In certain embodiments, the isolated antigen binding proteins described herein can be included in a PSMA-specific CAR in the form of an scFv. The CAR containing the isolated antigen binding protein described herein can be contained in an immune cell, such as a T cell, NK cell.

Immunoconjugates

In another aspect, the present application also provides immunoconjugates that can comprise the isolated antigen binding proteins described herein.

In certain embodiments, the isolated antigen binding proteins described herein can be crosslinked to a therapeutic agent to form the immunoconjugate. Such as antibody-drug conjugates (ADCs). Suitable therapeutic agents include cytotoxins, alkylating agents, DNA minor groove binding molecules, DNA intercalators, DNA cross-linkers, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, inhibitors of topoisomerase I or II, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics, and antimitotic agents, such as SN-38. In an ADC, the antibody and therapeutic agent may be cross-linked by a linker that is cleavable, for example, a peptide linker, a disulfide linker, or a hydrazone linker. In certain embodiments, the linker may be a peptide linker, such as Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Pro-Val-Gly-Val-Val, Ala-Asn-Val, Val-Leu-Lys, Ala-Ala-Asn, Cit-Cit, Val-Lys, Cit, Ser, or Glu. ADCs can be prepared as described in U.S. Pat. Nos. 7,087,600, 6,989,452, and 7,129,261, PCT publication WO 02/096910, WO 07/038,658, WO 07/051,081, WO 07/059,404, WO 08/083,312, and WO 08/103,693, U.S. Pat. Nos. 20060024317, 20060004081, and 20060247295.

In addition, the isolated antigen binding proteins described herein can also be fused to other functional molecules (e.g., antibodies or receptor ligands) to form bispecific molecules. The bispecific molecule can specifically bind to at least two different binding sites or targeting molecules. The bispecific molecules can be prepared by genetic engineering, somatic hybridization, or chemical methods. See, for example, Kufer et al, cited supra, Cao and Suresh, Bioconjugate Chemistry, 9 (6), 635-644 (1998), and van Spriel et al, Immunology Today, 21 (8), 391-397 (2000).

Nucleic acid molecules

In another aspect, the present application also provides an isolated nucleic acid molecule or molecules that can encode an isolated antigen binding protein described herein or a chimeric antigen receptor described herein. The isolated nucleic acid molecule or molecules described herein can be an isolated form of nucleotides, deoxyribonucleotides or ribonucleotides, or an analog isolated from its natural environment or synthesized, of any length, but can encode an isolated antigen binding protein described herein or a chimeric antigen receptor described herein.

Carrier

In another aspect, the present application also provides a vector, which may comprise a nucleic acid molecule as described herein. The vector may be used to express the genetic material element carried by the vector in a host cell by transformation, transduction or transfection of the host cell. For example, the carrier may include: a plasmid; phagemid; a cosmid; artificial chromosomes such as Yeast Artificial Chromosomes (YACs), Bacterial Artificial Chromosomes (BACs), or artificial chromosomes (PACs) derived from P1; bacteriophage such as lambda phage or M13 phage, animal virus, etc. Animal virus species used as vectors are retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e.g., herpes simplex virus), poxvirus, baculovirus, papilloma virus vacuolatum (e.g., SV 40). As another example, the vector may contain various elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain a replication initiation site. In addition, the vector may include components that assist its entry into the cell, such as viral particles, liposomes or protein coats, but not exclusively.

Cells

In another aspect, the present application also provides a cell, which may comprise a nucleic acid molecule described herein or a vector described herein. The cell may comprise progeny of a single cell. Progeny may not necessarily be identical (in morphology of the total DNA complement or in the genome) to the original parent cell due to natural, accidental, or deliberate mutation. In certain embodiments, the cells may further comprise cells transfected in vitro with a vector of the invention. In certain embodiments, the cell can be a bacterial cell (e.g., E.coli), a yeast cell, or other eukaryotic cell, such as a COS cell, Chinese Hamster Ovary (CHO) cell, CHO-K1 cell, LNCAP cell, HeLa cell, HEK293 cell, COS-1 cell, NS0 cell, or myeloma cell. In certain embodiments, the cell may be a mammalian cell. In certain embodiments, the mammalian cell can be a HEK293 cell.

Pharmaceutical composition

In another aspect, the present application also provides a pharmaceutical composition that can comprise an isolated antigen binding protein described herein, a chimeric antigen receptor described herein, an immunoconjugate described herein, a nucleic acid molecule described herein, a vector described herein, and/or a cell described herein, and optionally a pharmaceutically acceptable adjuvant.

In certain embodiments, the pharmaceutical composition may further comprise suitable formulations of one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and/or preservatives. The acceptable ingredients of the composition are preferably non-toxic to the recipient at the dosages and concentrations employed. The pharmaceutical compositions of the present invention include, but are not limited to, liquid, frozen and lyophilized compositions.

In certain embodiments, the pharmaceutically acceptable adjuvants may include any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents that are compatible with pharmaceutical administration, are generally safe, non-toxic, and are neither biologically nor otherwise undesirable.

In certain embodiments, the pharmaceutical composition may comprise parenteral, transdermal, intracavity, intraarterial, intrathecal and/or intranasal administration or direct injection into tissue. For example, the pharmaceutical composition may be administered to a patient or subject by infusion or injection. In certain embodiments, the administration of the pharmaceutical composition may be performed by different means, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical, or intradermal administration. In certain embodiments, the pharmaceutical composition may be administered without interruption. The uninterrupted (or continuous) administration may be achieved by a small pump system worn by the patient to measure the therapeutic agent flow into the patient, as described in WO 2015/036583.

The dosage regimen for the pharmaceutical composition may be the administration of a bolus, multiple divided doses may be administered over time, or the dose may be reduced or increased in proportion to the criticality of the treatment condition. In certain embodiments, the treatment regimen may be administered once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months, or once every 3-6 months. In certain embodiments, the dosing regimen comprises intravenous administration, 1 mg/kg body weight or 3 mg/kg body weight, and the antibody is administered on one of the following dosing schedules: (i) administering six times every four weeks, and then once every three months; (ii) once every three weeks; (iii) once at 3 mg/kg body weight, then once every three weeks at 1 mg/kg body weight. In certain embodiments, the dose is adjusted to achieve a blood concentration of about 1-1000 μ g/ml, for example, may be about 25-300 μ g/ml.

Preparation method and application

Preparation method

In another aspect, the present application also provides methods of making an isolated antigen binding protein described herein, which methods can include culturing a cell described herein under conditions such that the isolated antigen binding protein described herein is expressed.

Use of

In another aspect, the present application also provides the use of the isolated antigen binding protein, the chimeric antigen receptor, the immunoconjugate, the nucleic acid molecule, the vector, the cell and/or the pharmaceutical composition for the preparation of a medicament for the prevention, alleviation and/or treatment of tumors.

In another aspect, the present application also provides a method of preventing, ameliorating, or treating a tumor, which may comprise administering to a subject in need thereof the isolated antigen binding protein, the chimeric antigen receptor, the immunoconjugate, the nucleic acid molecule, the vector, the cell, and/or the pharmaceutical composition described herein. In the present application, the administration can be carried out in different ways, for example intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.

In another aspect, the isolated antigen binding protein, the chimeric antigen receptor, the immunoconjugate, the nucleic acid molecule, the vector, the cell, and/or the pharmaceutical composition of the present application may be used for the prevention, amelioration, or treatment of a tumor.

In the present application, the tumor may be a solid tumor or a hematological tumor.

In the present application, the tumor may comprise a PSMA-positive tumor, which may comprise prostate cancer.

In the present application, the subject may include humans and non-human animals. For example, the subject may include, but is not limited to, a cat, dog, horse, pig, cow, sheep, rabbit, mouse, rat, or monkey.

In the present application, the isolated antigen binding protein may be administered with one or more additional antibodies in order to effectively inhibit tumor growth in a subject. In certain embodiments, the isolated antigen binding protein may be administered to a subject in combination with one or more other antibodies, such as LAG-3 antibodies, PD-1 antibodies, and/or CTLA-4 antibodies.

In the present application, the isolated antigen binding protein may be administered with a chemotherapeutic agent, which may be a cytotoxic agent, e.g., SN-38, epirubicin, oxaliplatin, and/or 5-FU.

In another aspect, the present application also provides a method of detecting PSMA in a sample comprising administering an isolated antigen binding protein described herein. In the present application, the administration can be carried out in different ways, for example intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.

Without wishing to be bound by any theory, the following examples are only intended to illustrate the protein molecules, preparation methods, uses, etc. of the present application, and are not intended to limit the scope of the invention of the present application. The examples do not include detailed descriptions of conventional methods, such as those used to construct vectors and plasmids, methods of inserting genes encoding proteins into such vectors and plasmids, or methods of introducing plasmids into host cells. Such methods are well known to those having ordinary skill in the art and are described in numerous publications, including Sambrook, j., Fritsch, e.f. and maniis, T. (1989) Molecular Cloning: a Laboratory Manual, 2nd edition, Cold spring Harbor Laboratory Press.

Examples

EXAMPLE 1 Generation and selection of monoclonal hybridoma cells

1.1 preparation of CHO-K1/cyno PSMA cell Stable transformant

The lentiviral particles (gecko gene, cat # LVCON 335) packed with a nucleic acid sequence encoding cynomolgus monkey PSMA (Genbank accession number XP _005579379 for the corresponding cynomolgus monkey PSMA amino acid sequence) infected CHO-K1 cells (ATCC, cat # CCL-61) at a ratio of m.o.i. =100 ((m.o.i. = (lentiviral particle titer volume)/number of infected cells), the cells after 48 hours of infection were transferred at a ratio of 1:10 instead of the F2K medium containing 8 μ g/ml and 10% (w/v) fetal bovine serum for about 1 week to the uninfected control group CHO-K1 were screened for killing, while the cells remained in the infected group CHO-K1. after digestion of the lentiviral infected group CHO-K1 cells were limitedly diluted to 0.5 cells per well in 96 well plates, and culture medium containing the screening for about 5 days was continued to see if the colonies survived, and (5) marking. The culture was continued in a 37 ℃ carbon dioxide incubator for about 1 week until 50% of the total volume was reached, and the colonies were expanded into 6 wells. Expression was detected by flow cytometry using an anti-PSMA Tab antibody (recombinant human IgG1 antibody prepared using VH and VL sequences of pasotuzumab). Clone CHO-K1/cyno PSMA B6 with high growth rate and FACS detection and high fluorescence intensity is continuously expanded and cultured, and is frozen and stored in liquid nitrogen.

1.2 mouse immunization, hybridoma cell fusion and antibody screening

The H2L2 transgenic mice (WO 2010/070263 Al) are capable of producing comparable immune responses and antibody titers to wild-type mice (e.g., BALB/C).

6-8 weeks old Harbour H2L2 transgenic mice were immunized with recombinant PSMA ECD-his (Sino-Biological, cat # 15877-H07H) as immunogen and bred under specific pathogen free conditions (SPF). In the first immunization, each mouse was co-injected with 50 μ g of immunogenic protein and 0.22 ml of complete Freund's adjuvant (CFA, Sigma, cat # F5881) in the peritoneal cavity and axillary and inguinal lymph nodes, respectively. To enhance the immune response, two weeks after the first immunization, 25 μ g of the immunogenic protein along with 200 μ l of Ribi (Sigma adjuvant system, Sigma, cat # S6322) were injected into the intraperitoneal and subcutaneous lymph nodes of each mouse, followed by 25 μ g of immunogen and 200 μ l of Ribi adjuvant every 2 weeks for a total of 6 injections per mouse plus the first immunization. One week after the fourth and sixth needles during the immunization period, respectively, blood was collected, diluted 10-fold to 6 concentrations (1: 100, 1:1000, 1:10000, 1:100000, 1: 1000000), and the titer of anti-human PMSA in the blood of mice was determined by enzyme-linked immunosorbent assay (ELISA) assay on ELISA plates coated with human PSMA ECD protein (Sino-Biological, cat # 15877-H07H), and the specific reactivity of 3 concentrations of blood of mice (1: 100, 1:1000, 1: 10000) to LNCAP cells (Cobioer, Nanjing, China) highly expressing human PSMA was examined by flow cytometry. Blank control (PB) was serum from pre-immunized mice.

After completion of the above steps, mice with a specific immune response to human PSMA were selected for the boost by intraperitoneal injection of 100 μ g of purified PSMA ECD-his before fusion. Three days later, the mice were sacrificed and spleen cells and lymph node cells were collected. Reacting NH₄OH was added to the spleen cells and lymph node samples at a final concentration of 1% (w/w) to lyse the red blood cells in the samples. The samples were centrifuged at 1000 rpm and washed three times with DMEM medium to determine the viability and number of cells. Mouse myeloma cells sp2/0 (ATCC, cat # CRL-1581) were washed twice with serum-free DMEM, and the survival rate and number of cells were determined. Viable splenocytes were then fused with mouse myeloma cell sp2/0 (ATCC, cat # CRL-1581) at a ratio of 5:1 using high efficiency electrofusion.

The fused cells were resuspended and adjusted to 10 concentrations in medium (hybrid-SFM, cat #12045084, Life Technologies) containing 20% ultra-low IgG FBS (ultra-low IgG, Total Bovine Serum, cat #16250086, Life Technologies) supplemented with 1 Xhypoxanthine, aminopterin, and thymidine (50X HAT supplement, cat #21060017, Life Technologies)⁵Individual cells/200 μ l. 200 μ l of fused cells were added to each well of a 96-well plate, and cultured at 37 ℃ under 5% carbon dioxide. Hybridoma supernatants were screened 14 days after cell fusion by ELISA to determine their ability to bind to PSMA ECD his protein.

The selected positive clone (OD 450> 2) is screened by a flow cytometer to be specifically combined with HEK293T/human PSMA cells (KYinno, Beijing, China) and CHO-K1/cyno PSMA B6 cells. After the fluorescence intensity is scored, 25 hybridoma mother clones with the strongest fluorescence intensity are selected, subcloning is carried out through a limiting dilution method, and the submonols which grow through screening are screened through ELISA and flow cytometry, so that the submonols which are most strongly combined with PSMA protein or PSMA on cells are found out. The sub-monoclonal, determined as an IgG subtype by mouse Ig typing Ready-SET-GO! ELISA (Life technologies, cat # 88-50640-88), will be subjected to sequencing analysis.

Example 2 sequencing, expression and purification of monoclonal PSMA antibodies

The monoclonal PSMA antibody selected in example 1 was sequenced and the amino acid sequence is shown in table 1.

TABLE 1 sequencing results of monoclonal PSMA antibodies

The heavy chain variable region sequence of the monoclonal PSMA antibody described above was subcloned into a pTT5 expression vector containing a signal peptide and the constant region of human heavy chain IgG1 (SEQ ID NO: 19). The light chain variable region sequence of the monoclonal PSMA antibody was subcloned into an expression vector containing a signal peptide and the human antibody light chain kappa constant region (SEQ ID NO: 20). After the sequencing verification, the recombinant plasmids were extracted with big sampling kit (Macherey-Nagel, NucleoBond Xtra Midi) to improve the purity and quality of the recombinant plasmids, and the plasmids were filtered through a 0.22 μm filter (millipore). The purified plasmid was used for transfection.

The plasmid coding the heavy chain of the antibody and the plasmid coding the light chain of the antibody are simultaneously transfected into a mammalian host cell (such as human embryonic kidney cell HEK 293), and the purified PSMA recombinant antibody with the correct pairing assembly of the light chain and the heavy chain can be obtained by utilizing the conventional recombinant protein expression and purification technology. Specifically, HEK293F cells (invitrogen, cat # A14527) were expanded in FreeStyle ™ F17 Expression Medium (Thermo # A1383504). Before transient transfection, cell concentration was adjusted to 6-8 x 10⁵Cells/ml, 8% CO at 37 ℃₂Cultured in a shaker for 24 hours at a cell concentration of 1.2X 10⁶Cells/ml. 30 ml of cultured cells were prepared. The above plasmid encoding the antibody heavy chain and plasmid encoding the antibody light chain were mixed at a ratio of 2:3 and 30. mu.g of the total plasmid was dissolved in 1.5 ml of Opti-MEM serum-reduced medium (Thermo, 31985088), and sterilized by filtration through a 0.22 μm filter. Further, 120. mu.l of 1 mg/ml PEI (Polysciences, Inc # 23966-2) was dissolved in 1.5 ml of Opti-MEM and allowed to stand for 5 minutes. Adding PEI slowly into the plasmid, incubating at room temperature for 10 min, shaking the culture flask while adding the mixed solution of PEI slowly, and heating at 37 °C 8% CO₂Cultured in a shaker for 5 days. Cell viability was observed after 5 days. Collecting the culture, centrifuging at 3300g for 10 min, and collecting the supernatant; the supernatant was then centrifuged at high speed to remove impurities. A gravity column (Bio-Rad # 7311550) containing the MabSelect chamber (GE Healthcare Life Science # 71-5020-91 AE) was equilibrated with PBS (pH 7.4), washed 2-5 column volumes. Passing the supernatant sample through a column; the column was washed with 5-10 column volumes of PBS, the target protein was eluted with 0.1M glycine pH3.5, then neutralized with Tris-HCl pH 8.0, and finally the exchange was concentrated to PBS buffer using an ultrafiltration tube (Millipore, UFC 901024) to obtain a purified recombinant protein solution. And finally, measuring the concentration by using a NanoDrop (Thermo Scientific polypeptide loop), measuring the purity of the antibody by using a high performance liquid chromatography-mass spectrometry method, measuring the endotoxin content by using an endotoxin detection kit (lonza), and subpackaging and storing for later use.

From the above experiments, a monoclonal PSMA antibody PR001104 (i.e., an isolated antigen binding protein as described herein) was obtained, which is an IgG1 subtype with the amino acid sequence of the variable region shown in table 1 above.

In addition, two comparative example antibodies were prepared for the subsequent examples, one being the comparative example 1 antibody PR001086 (homemade) and the other being the comparative example 2 antibody Tab (pasotuzumab, homemade).

Specifically, the constant region sequence of the antibody PR001086 of comparative example 1 was identical to PR001104, and the variable region sequence was different from PR001104, specifically, V thereof_HThe amino acid sequence of (A) is shown as SEQ ID NO. 17, V_LThe amino acid sequence of (A) is shown as SEQ ID NO. 18, the amino acid sequence of the heavy chain is shown as SEQ ID NO. 23, and the amino acid sequence of the light chain is shown as SEQ ID NO. 24.

Comparative example 2 antibody Tab is a comparative example antibody having human IgG1 subtype, which was self-made based on the sequence of AMgen's PSMA X CD3 Pasotuzumab, and the amino acid sequences of its heavy and light chains are shown in SEQ ID NOS: 25 and 26, respectively.

Example 3 binding Capacity of monoclonal PSMA antibodies to cell surface PSMA

HEK293T cells overexpressing human PSMA (HEK 293T/human PSMA, Kangyuan Bochuang, cat # K)C-1005) or CHO-K1 cells overexpressing cynomolgus monkey PSMA (CHO-K1/cyno PSMA) or tumor cells highly expressing human PSMA (LNCAP) were cultured and expanded in T-75 flasks, and after 90% fusion was achieved, the medium was aspirated and the cells were washed twice with PBS. The cells were treated with pancreatin (Invitrogen, cat # 15050065) for about 1 minute, and the pancreatin was neutralized with the medium. The cells were then washed twice with PBS, cell counts were determined, and cells were resuspended to 2X 10 with PBS⁶Cells/ml. 100 μ l of cell suspension was added to each well of a 96-well V-plate. Incubated with different concentrations of purified PSMA antibody or isotype control antibody for 1 hour on ice. Cells were washed twice with PBS and incubated with goat anti-human (H + L) -Alexa Fluor 647 (Life technology, cat # A21445) for 30-45 min at 4 ℃. After another PBS wash, the cells were analyzed for Median Fluorescence Intensity (MFI) on a FACS caton ii flow cytometer with the control group being human IgG 1.

As shown in fig. 1A, fig. 1B, and tables 2 and 3, higa 1k or higa 1 in the figure both represent the control group (i.e., human IgG 1), and it can be seen that the PSMA antibody PR001104 of the present application can specifically bind to human PSMA, and the detected binding capacity of the antibody is increased in positive correlation with the antibody concentration; in contrast, the comparative example 1 antibody PR001086 bound poorly to human PSMA. The PSMA antibody PR001104 of the present application had an EC50 value comparable to that of the Tab comparative example 2 antibody, but at the same concentration, the PSMA antibody PR001104 of the present application exhibited a higher Emax, indicating that the antibody is able to bind more human PSMA protein on HEK293T/human PSMA cells.

TABLE 2 EC50 values corresponding to FIG. 1A

TABLE 3 EC50 values corresponding to FIG. 1B

As shown in fig. 2A, fig. 2B, and tables 4 and 5, hIgG1, K, or hIgG1 all represent controls (i.e., human IgG 1), and it can be seen that PSMA antibody PR001104 of the present application can specifically bind to cynomolgus PSMA expressed on the surface of CHO-K1/cyno PSMA cells. And the binding EC50 value of this antibody was much lower than the control antibody Tab, indicating that the antibody binds monkey PSMA more sensitively at lower concentrations. And under the same concentration, the PSMA antibody PR001104 of the application shows higher Emax, which indicates that the antibody can bind more cyno PSMA protein (namely monkey PSMA protein) on CHO-K1/monkey PSMA cells. PR001086 bound poorly to monkey PSMA.

TABLE 4 EC50 values corresponding to FIG. 2A

TABLE 5 EC50 values corresponding to FIG. 2B

As shown in fig. 3 and table 6, hIgG1 represents the control group (i.e., human IgG 1), and it can be seen that PSMA antibody PR001104 of the present application specifically binds to LNCAP cell surface-expressed PSMA, and that the EC50 value of PSMA antibody PR001104 of the present application is comparable to that of Tab compared to the comparative example 2 antibody Tab, but at the same concentration, PSMA antibody PR001104 of the present application exhibits a higher Emax, indicating that the antibody can bind to more human PSMA protein on LNCAP cells.

TABLE 6 EC50 values corresponding to FIG. 3

Example 4 killing of target cells by antibody internalization

PSMA antibodies can mediate internalization of cell surface expressed PSMA proteins by binding to the extracellular tail of PSMA. In this example, the PSMA antibodies of the present application were tested for their degree of internalization and susceptibility of PSMA + cells to killing by PSMA antibodies.

HEK293T/human PSMA or LNCAP cells were cultured and expanded in T-75 flasks, and after 90% confluence was achieved, the medium was aspirated and the cells washed twice with PBS. The cells were treated with pancreatin (Invitrogen, cat # 15050065) for about 1 minute, and the pancreatin was neutralized with the medium. The cells were transferred to a 15 ml sterile centrifuge tube and centrifuged at 1000 rpm for 5 minutes at room temperature to pellet the cells. The medium was aspirated and the cells were resuspended in the respective medium. Gently blow the cells to obtain a single cell suspension. Counting with cell counting plate, then 2 x 10³LNCAP cells or HEK293T/human PSMA cells were added to black ViewPlate-96 TC (Perkin Elmer, cat # 6005225) plates. 37 ℃ and 5% CO₂Incubate overnight in incubator.

The next day, 10X concentration (100 nM) antibody solutions were prepared in FBS-free medium, 5-fold diluted, and 6 antibody concentrations were prepared. 10 μ l of each antibody sample was transferred to the cell plate, and the final volume of each well was 100 μ l. 2 mul of alpha HFc-CL-MMAF culture medium (alpha HFc-CL-MMAF kit, Cat #: AH-102AF, moradec) at 50 mug/ml was added to each well, so that the final concentration was 1 mug/ml. 37 ℃ and 5% CO₂Incubate for 4 days.

On the sixth day, 100. mu.l of CellTiter-Glo luminescent cell activity reagent (Promega, USA, cat # G7570) was added to each well, mixed for 2 minutes on a shaker, and cell lysis was induced. The 96-well plate was incubated at room temperature for 10 minutes to stabilize the light signal. The luminescence was recorded using a PE Enspire microplate reader (Perkin Elmer, Enspire) and the EC50 value was determined.

FIG. 4 shows the survival of HEK293T/human PSMA cells under antibody treatment, where hIgG1 represents the control group (i.e., human IgG 1). It can be seen that the cell survival rate was comparable to the comparative example 2 antibody Tab and its EC50 value was comparable to the comparative example 2 antibody under the treatment of PSMA antibody PR001104 of the present application (see table 7).

Fig. 5 shows the survival of LNCAP cells under antibody treatment, where hIgG1 represents the control group (i.e., human IgG 1). It can be seen that LNCAP cell survival was lower compared to the comparative example 2 antibody Tab and EC50 value was lower than the comparative example 2 antibody Tab (see table 8) under treatment with PSMA antibody PR001104 of the present application, indicating that it is able to achieve maximal antibody internalization effect at lower concentrations.

TABLE 7 EC50 values corresponding to FIG. 4

TABLE 8 EC50 values corresponding to FIG. 5

Example 5 determination of binding affinity and dissociation constant of PSMA antibody to recombinant PSMA protein

Affinity was determined using an Octet RED96 instrument (fortiebai) and an anti-human IgG Fc avidin sensor (AHC sensor, Pall ForteBio, cat # 18-5060) according to the detailed procedures and methods provided by the manufacturer. Specifically, human PSMA protein (Sino Biological, cat # 15877-H07H) was diluted to 200nM in PBS buffer (pH 7.4) containing 0.1% (w/w) BSA and 0.02% (v/v) Tween 20 and incubated with AHC sensor. The PSMA antibody at 40 nM was incubated with the AHC sensor loaded with human PSMA protein for 3 minutes at 30 ℃. The reaction mixture was incubated in PBS buffer (pH 7.4) containing 0.1% (v/w) BSA and 0.02% (v/v) Tween 20 at 30 ℃ for an additional 5 minutes. Octet Red96 records the binding and separation signals of PSMA antibodies to human PSMA protein in real time. Affinity, association and dissociation constants were determined by Octet using software and the results are shown in table 9.

As can be seen from table 9, the KD value for PR001104 antibody was lower than the Tab of the comparative example 2 antibody, indicating a stronger binding affinity for PSMA, which is about 100-fold lower than the comparative example 2 antibody.

TABLE 9 binding affinities of antibodies

Example 6 ADCP Activity of PSMA antibodies

CD14+ monocytes were isolated from PBMCs (Allcells, cat # PB005-C, Lot # LP 191125) using human CD14 sorting magnetic beads (Meltenyi, 130-⁶The suspension was resuspended in 10% FBS-containing RPMI1640 medium, and 100 ng/mL GM-CSF (PeproTech, cat # 300-03-A) was added. Take 2 x 10⁶The monocytes were cultured in 6-well plates per well for 9 days in a 37-degree carbon dioxide incubator to differentiate into macrophages. The solution (containing 100 ng/ml GM-CSF) was changed every 3-4 days. After 9 days the macrophages were digested with pancreatin and the pancreatin reaction was stopped with RPMI1640 containing 10% FBS. The cells were harvested, washed once with PBS and resuspended to a density of 1x 10 with PBS⁶And/ml. LNCAP cells were also harvested and resuspended at a density of 1x 10 with PBS⁶And/ml. The macrophages were stained with 0.1 μ M Far-red (life technologies, cat # C34572) and LNCAP cells were stained with 0.5 μ M CFSE (life technologies, cat # C34544) for 10 minutes at 4 degrees. Centrifuging the stained cells with>20 ml of RPMI1640+10% FBS medium were washed once. Resuspend the washed cells in 1% BSA-RPMI 1640 medium and adjust the cell density to 1.6 x 10⁶And/ml. 25 μ l LNCAP cells per well (4 × 10 cells per well) were added to a 96 well V-plate (Corning, cat # 3894)⁴) And 25 μ l macrophages (4 x 10 cells per well)⁴). The antibody was diluted with 1% BSA-RPMI 1640 to an intermediate concentration of 20 nM and further diluted 5-fold into 7 gradients. And adding 50 mu.l of diluted antibody into each well of the same 96-well V-shaped plate containing LNCAP and macrophages, and mixing completely. Incubate at 37 ℃ for 1 hour. The percentage of double positives for FITC + LNCAP cells and Alexa647+ macrophages was identified by flow cytometry using BD FACS Caton II (BD, Germany). Data were analyzed using FlowJo software (Tree Star, Ashland, OR) and the percentage of double-stained cells was used to determine ADCP-mediated cell killing.

FIG. 6 shows PSMA antibody PR001104 of the present application mediated phagocytosis of LNCAP in macrophages, where hIgG1 represents the control group (i.e., human IgG 1) and LNCAP + Macrophage represents the absence of antibody. As can be seen in combination with table 10, PR001104 antibody showed ADCP effect on LNCAP cells. Among them, in the case of PBMC of donor LP191125#, the highest killing% of antibody PR001104 was slightly higher than that of the comparative example 2 antibody Tab, as seen from the mean and standard error of specific killing rate, and its EC50 value was comparable to Tab, indicating that it was able to achieve the maximum killing power at lower concentration comparable to or higher than that of the comparative example 2 antibody.

TABLE 10 ADCP Effect of PSMA antibody PR001104 on LNCAP

Example 7 Epitope identification of antigen binding proteins (Epitope binding)

Epitope competition experiments were performed on antigen binding proteins Tab and PR001104 using an Octet Red96e instrument (Fortebio). Tab was diluted to 200nM with 1xkinetics buffer (Fortebio; cat # 18-1105) and the remaining antibodies to 100 nM. Firstly, capturing human PSMA protein (Sino Biological; Cat. No. 15877-H07H) with His label by using HIS1K sensor (Fortebio; Cat. No. 18-5120), wherein the capture height is 0.4 nm; immersing the sensor in the first antibody for 180 seconds, and recording the signal at the 180 th second as the 100% signal of the antibody; the sensor was then immersed in a mixture of primary and secondary antibodies for 180 seconds and the final signal was recorded as the signal of the secondary antibody. The inhibition rate was calculated by the formula, and the inhibition rate (%) = (A-B)/A100 (note: A: 100% signal of a certain antibody, and B: the antibody as the signal of the second antibody).

If the inhibition rate is greater than 80%, it means that the two antibodies have very similar epitopes; if the inhibition rate is between 40-80%, it means that the two antibodies have epitopes that are relatively close but do not completely overlap; if the inhibition rate is less than 40%, it means that the two antibodies have non-overlapping epitopes.

As a result, as shown in tables 11 and 12, it can be seen that antibodies PR001104 and Tab have different epitopes.

TABLE 11 epitope Competition assay signals

TABLE 12 competitive inhibition of epitopes

Example 8 Effect of the internalization of PSMA antibodies on target cells

PSMA antibodies can mediate internalization of cell surface expressed PSMA proteins by binding to the extracellular portion of PSMA. In this example, PSMA antibodies were tested for internalization at different time points over a short period of time (3 hours).

LNCAP cells were cultured and expanded in T-75 flasks in RPMI1640 medium (Life technologies, cat # 61870-. The cells were treated with pancreatin (Invitrogen, cat # 15050065) for about 1 minute, and the pancreatin was neutralized with the medium. The cells were transferred to a 15 ml sterile centrifuge tube and centrifuged at 1000 rpm for 5 minutes at room temperature to pellet the cells. The medium was aspirated and the cells were resuspended in the respective medium. Gently blow the cells to obtain a single cell suspension. Counting with cell counting plates, followed by resuspension of LNCAP cells to 2 x 10 with ice-cold FACS buffer (PBS +2% FBS)⁶cells/ml, 100 μ l per well of cell suspension was placed in a 96 well V-plate (Corning, 3894) and centrifuged at 400g for 5 minutes. After discarding the supernatant, 100. mu.l of FACS buffer containing PR001104 or the antibody Tab or hIgG1 of comparative example 2 (i.e., human IgG 1) at a final concentration of 100nM were added, respectively, and incubated at 4 ℃ for one hour. Unbound antibody was removed by washing twice with ice-cold FACS buffer. 100ul of ice cold FACS buffer was added to each well. The cells were left separately at 37 ℃ for 0/30/45/60/90/120/180 minutes, then transferred to 4 ℃ and pre-cooled PBS was added to prevent antibody endocytosis. Wash once more with ice cold FACS buffer. Cells were treated with 2 μ g/ml AF488 goat anti-human lgG (H + L) (Jackson ImmunoResearch) was placed in ice cold FACS buffer at 4 ℃ for half an hour. Unbound secondary antibody was removed by washing twice with ice-cold FACS buffer. Cells were resuspended in 200 μ l FACS buffer. The fluorescence intensity of the cells was analyzed by FACS canto II flow cytometer. Endocytosis ratio% = (1- (MFI)_37℃/ MFI_4℃））*100%。

The results are shown in fig. 7A-7D, where PR001104 had a better endocytosis effect than the reference antibody (i.e., Tab or hIgG 1) at saturating antibody binding concentrations within 2 hours in LNCAP, a PSMA positive tumor cell.

The foregoing detailed description is provided by way of illustration and example, and is not intended to limit the scope of the appended claims. Various modifications of the presently recited embodiments will be apparent to those of ordinary skill in the art and are intended to be within the scope of the appended claims and their equivalents.

Sequence listing

<110> and platinum medicine (Suzhou) Co., Ltd

<120> an isolated antigen PSMA-binding protein and uses thereof

<130> 0113-PA-009

<160> 26

<170> PatentIn version 3.5

<210> 1

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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<223> HCDR1 of PR001104

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Arg Asn Gly Met His

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Val Ile Trp His Asp Gly Ser Asn Lys Tyr Tyr Ser Asp Ser Val Lys

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Gly Glu Ser Thr Arg Ala Thr

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Gln Ala Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Ser

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Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala

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Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln

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Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg

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Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

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Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly

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Glu Arg Ala Thr Leu Ser Cys

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Trp Tyr Gln Leu Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr

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Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr

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Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys

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Gln Ala Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Ser Arg Asn

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Ala Val Ile Trp His Asp Gly Ser Asn Lys Tyr Tyr Ser Asp Ser Val

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Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

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Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly

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Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn

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Leu Ala Trp Tyr Gln Leu Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

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Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Ser Trp Pro Pro

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Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Arg Asn Tyr

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Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

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Ser Thr Ile Ser Asp Asn Ile Val Ser Thr Trp Tyr Ala Asp Ser Val

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Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr

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Glu Lys Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Gly Asn

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Arg Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Asn Trp Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 19

<211> 330

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> PR 001104/heavy chain constant region of antibody PR001086 of comparative example 1

<400> 19

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 20

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> light chain constant region of PR 001104/comparative example 1 antibody PR001086

<400> 20

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 21

<211> 452

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> heavy chain of PR001104

<400> 21

Gln Ala Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Ser Arg Asn

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp His Asp Gly Ser Asn Lys Tyr Tyr Ser Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Gln Tyr Ser Ser Gly Trp Val Asp Ala Phe Asp Ile Trp

100 105 110

Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215 220

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly Lys

450

<210> 22

<211> 215

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> light chain of PR001104

<400> 22

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn

20 25 30

Leu Ala Trp Tyr Gln Leu Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Gly Glu Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Ser Trp Pro Pro

85 90 95

Val Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala

100 105 110

Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser

115 120 125

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu

130 135 140

Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

145 150 155 160

Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu

165 170 175

Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val

180 185 190

Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys

195 200 205

Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 23

<211> 445

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> heavy chain of comparative example 1 antibody PR001086

<400> 23

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Arg Asn Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Thr Ile Ser Asp Asn Ile Val Ser Thr Trp Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr

65 70 75 80

Leu His Met Asn Ser Leu Arg Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Arg Ala Ala Val Asp Leu Trp Gly Gln Gly Thr Met Val Thr

100 105 110

Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

115 120 125

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val

130 135 140

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala

145 150 155 160

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

165 170 175

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

180 185 190

Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys

195 200 205

Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 24

<211> 214

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> light chain of comparative example 1 antibody PR001086

<400> 24

Glu Lys Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Gly Asn

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Arg Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Asn Trp Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 25

<211> 451

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> heavy chain of Tab antibody of comparative example 2

<400> 25

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Glu

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ile Ile Ser Asp Gly Gly Tyr Tyr Thr Tyr Tyr Ser Asp Ile Ile

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Phe Pro Leu Leu Arg His Gly Ala Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys

450

<210> 26

<211> 214

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> light chain of Tab of antibody of comparative example 2

<400> 26

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Asp Thr Asn

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Lys Ser Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr Ser Asp Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Ala Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ser

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Ser Tyr Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

Claims

1. An isolated PSMA antigen binding protein comprising HCDR1, HCDR2, and HCDR3, said HCDR1 is the amino acid sequence shown in SEQ ID NO. 1, said HCDR2 is the amino acid sequence shown in SEQ ID NO. 2, and said HCDR3 is the amino acid sequence shown in SEQ ID NO. 3; the polypeptide also comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 is an amino acid sequence shown in SEQ ID NO. 4, the LCDR2 is an amino acid sequence shown in SEQ ID NO. 5, and the LCDR3 is an amino acid sequence shown in SEQ ID NO. 6.

2. The isolated PSMA antigen binding protein of claim 1, comprising V_HSaid V is_HIncluding the framework regions H-FR1, H-FR2, H-FR3 and H-FR 4.

3. The isolated PSMA antigen-binding protein of claim 2, wherein the C-terminus of H-FR1 is linked directly or indirectly to the N-terminus of HCDR1, and the H-FR1 comprises the amino acid sequence set forth in SEQ ID No. 7; the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 8; the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO 9; the N-terminal of the H-FR4 is linked to the C-terminal of the HCDR3, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO. 10.

4. The isolated PSMA antigen binding protein of claim 1, comprising V_LSaid V is_LIncluding the framework regions L-FR1, L-FR2, L-FR3 and L-FR 4.

5. The isolated PSMA antigen binding protein of claim 4, wherein the C-terminus of L-FR1 is linked directly or indirectly to the N-terminus of the LCDR1 and the L-FR1 comprises the amino acid sequence set forth in SEQ ID NO. 11; the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 comprises the amino acid sequence shown in SEQ ID NO. 12; the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 comprises the amino acid sequence shown in SEQ ID NO 13; the N-terminal of the L-FR4 is linked to the C-terminal of the LCDR3, and the L-FR4 comprises the amino acid sequence shown in SEQ ID NO. 14.

6. The isolated PSMA antigen binding protein of claim 2, wherein the V_HComprises the amino acid sequence shown in SEQ ID NO. 15.

7. The isolated PSMA antigen binding protein of claim 4, wherein the V_LComprises the amino acid sequence shown as SEQ ID NO. 16.

8. The isolated PSMA antigen-binding protein of claim 1, comprising an antibody heavy chain constant region, and the antibody heavy chain constant region is derived from a human IgG heavy chain constant region.

9. The isolated PSMA antigen binding protein of claim 8, wherein the antibody heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO 19.

10. The isolated PSMA antigen-binding protein of claim 1, comprising an antibody light chain constant region, and the antibody light chain constant region comprises a human Ig kappa constant region.

11. The isolated PSMA antigen-binding protein of claim 10, wherein the antibody light chain constant region comprises the amino acid sequence set forth in SEQ ID No. 20.

12. The isolated PSMA antigen binding protein of claim 1, comprising an antibody heavy chain HC, and wherein the HC comprises the amino acid sequence set forth in SEQ ID NO: 21.

13. The isolated PSMA antigen binding protein of claim 1, comprising an antibody light chain LC, and the LC comprises the amino acid sequence set forth in SEQ ID NO. 22.

14. The isolated PSMA antigen binding protein of claim 1, comprising an antibody or antigen binding fragment thereof, wherein the antigen binding fragment comprises Fab, Fab', F (ab)₂Fv fragment, F (ab')₂scFv and/or di-scFv.

15. The isolated PSMA antigen-binding protein of claim 1, having one or more of the following properties:

4) has ADCP activity on LNCAP cells.

16. The isolated PSMA antigen-binding protein of claim 15, wherein the PSMA protein comprises a human PSMA protein or a monkey PSMA protein.

17. A chimeric antigen receptor comprising the isolated PSMA antigen-binding protein of any of claims 1-16.

18. An immunoconjugate comprising the isolated PSMA antigen binding protein of any of claims 1-16.

19. An isolated one or more nucleic acid molecules encoding the isolated PSMA antigen-binding protein of any of claims 1-16 or the chimeric antigen receptor of claim 17.

20. A vector comprising the nucleic acid molecule of claim 19.

21. A cell comprising the nucleic acid molecule of claim 19 or the vector of claim 20.

22. A pharmaceutical composition comprising the isolated PSMA antigen-binding protein of any of claims 1-16, the chimeric antigen receptor of claim 17, and/or the immunoconjugate of claim 18, and optionally a pharmaceutically acceptable adjuvant.

23. A method of making the isolated PSMA antigen-binding protein of any of claims 1-16, comprising culturing the cell of claim 21 under conditions such that the isolated PSMA antigen-binding protein of any of claims 1-16 is expressed.

24. Use of the isolated PSMA antigen-binding protein of any of claims 1-16, the chimeric antigen receptor of claim 17, the immunoconjugate of claim 18, and/or the pharmaceutical composition of claim 22, in the manufacture of a medicament for the prevention, amelioration, and/or treatment of a tumor that is prostate cancer.

25. Use of the isolated PSMA antigen-binding protein of any of claims 1-16 in the preparation of a reagent for detecting PSMA in a sample.