Antibodies against human prostate-specific membrane antigens and uses thereof

Abstract

The invention belongs to the technical field of antibody preparation, and particularly relates to an anti-human prostate specific membrane antigen (PSMA) antibody and application thereof. The amino acid sequences of CDR1-3 on a light chain variable region of the antibody are respectively shown as SEQ ID NO.3-5, and the amino acid sequences of CDR1-3 on a heavy chain variable region of the antibody are respectively shown as SEQ ID NO.8-10. The antibody with the complementarity determining region sequence provided by the invention can specifically recognize PSMA expressed by cells and tissues, has good affinity in combination with the PSMA, has high recognition sensitivity, can effectively resist the interference of complex non-target protein components in the cells / tissues, has no specific combination with non-target antigens, is suitable for immunological detection of the PSMA, and has good application prospects. The kit is especially suitable for western blot detection and immunohistochemical methods, and can reduce the occurrence rate of false positive and false negative results and improve the accuracy of immunodiagnosis.

Description

Technical Field

[0001] The present invention relates to the technical field of antibody preparation, in particular to an antibody against human prostate-specific membrane antigen and its application. Background Art

[0002] Prostate-specific membrane antigen (PSMA) is a type II transmembrane glycoprotein located on the prostate cell membrane. Also known as folate hydrolase 1 (FOLH1), glutamate carboxypeptidase 2 (GCP2), and N-acetylated-α-linked acid dipeptidase I (NAALAD1), it belongs to the M28 peptidase family. The PSMA protein consists of 750 amino acids, including a 19-amino acid intracellular domain, a 24-amino acid transmembrane domain, and a 707-amino acid extracellular domain. The extracellular region possesses folate hydrolase and N-acetylated-α-linked acid dipeptidase activities, catalyzing the hydrolysis of N-acetylaspartate-glutamate (NAAG) to N-acetylaspartate (NAA) and glutamate. In the intestine, PSMA mediates folate uptake. In the brain, PSMA regulates excitatory neurotransmission. The extracellular region of PSMA can provide multiple antigenic epitopes and is the main binding site for antibodies, peptides, RNA aptamers and small molecules. Therefore, PSMA has become an important research target in tumor immunotherapy and molecular imaging.

[0003] Prostate-specific membrane antigen (PSMA) is expressed at low levels in normal prostate tissue and non-prostate cancer tissues (such as the lacrimal gland, nervous system, and duodenum). However, it is highly expressed in prostate cancer, reaching 100-1000 times the normal level. PSMA expression increases with prostate disease progression and metastasis, and is particularly high in hormone-refractory and castration-resistant prostate cancer. This high expression makes PSMA a specific marker for prostate cancer and an ideal therapeutic target. Its sensitivity and specificity for distinguishing prostate cancer from other types of malignant tumors are 65.9% and 94.5%, respectively. PSMA is also expressed in the neovascularization of various non-prostate solid tumors, such as gastric cancer, lung cancer, pancreatic cancer, breast cancer, bladder cancer, kidney cancer, and colorectal cancer. PSMA is highly specifically expressed on tumor vascular endothelial cells, suggesting that PSMA may serve as a target for anti-angiogenic therapy, providing new insights into the treatment of other tumors.

[0004] Immunohistochemistry (IHC) is widely used in tumor pathological diagnosis and differential diagnosis, and is one of the important means of prostate tissue biopsy. Antibodies specifically targeting PSMA bind to the PSMA protein in the prostate tissue, and then the site of the antigen-antibody binding is displayed using a labeled antibody. The PSMA expression level can be detected based on the staining intensity and staining range in the tissue. Studies have shown that increased PSMA expression was detected in dedifferentiated, metastatic or hormone-resistant prostate cancer by IHC, and the PSMA staining intensity is correlated with the Gleason score, which is of great significance for the early diagnosis, treatment and disease monitoring of prostate cancer. Therefore, the development of monoclonal antibodies targeting the extracellular domain of PSMA with high recognition sensitivity and / or specificity and good detection performance has broad application prospects. Summary of the Invention

[0005] To address the existing problems of a lack of highly sensitive and / or specific antibodies for detecting human prostate-specific membrane antigen (PSMA), the present invention provides antibodies against human prostate-specific membrane antigen, their encoding genes, and recombinant vectors. Furthermore, the present invention provides the use of such monoclonal antibodies or antibody conjugates in the preparation of human prostate-specific membrane antigen immunoassay kits, as well as related immunoassay kits. To achieve the aforementioned objectives, the present invention is specifically implemented through the following technical solutions:

[0006] In a first aspect, the present invention provides an anti-human prostate-specific membrane antigen (PSMA) antibody, comprising a light chain variable region and a heavy chain variable region, wherein the amino acid sequences of CDR1, CDR2, and CDR3 on the light chain variable region are shown in SEQ ID NOs. 3-5, respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 on the heavy chain variable region are shown in SEQ ID NOs. 8-10, respectively.

[0007] Furthermore, the amino acid sequence of the light chain variable region is shown as SEQ ID NO.2, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.7.

[0008] Furthermore, the amino acid sequence of the antibody light chain is shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.6.

[0009] Furthermore, the antibody is a full-length antibody or an antigen-binding region thereof; the antigen-binding region is selected from at least one of a Fab fragment, a F(ab)2 fragment, a Fv fragment, a (Fv)2 fragment, a scFv fragment and a sc(Fv)2 fragment.

[0010] The second aspect of the present invention provides a nucleic acid molecule or a recombinant vector comprising the nucleic acid molecule, wherein the nucleic acid molecule encodes the above-mentioned anti-human prostate-specific membrane antigen antibody.

[0011] The third aspect of the present invention provides the use of the above-mentioned anti-human prostate-specific membrane antigen antibody or its antibody conjugate in the preparation of a human prostate-specific membrane antigen immunoassay kit, wherein the antibody conjugate comprises the antibody and a detection label connected to the antibody.

[0012] In a fourth aspect, the present invention provides a human prostate-specific membrane antigen immunoassay kit, which comprises the anti-human prostate-specific membrane antigen antibody or its antibody conjugate as described above, wherein the antibody conjugate comprises the antibody and a detection label connected to the antibody.

[0013] Furthermore, the immunoassay kit is selected from an immunoblotting kit or an immunohistochemistry kit.

[0014] Furthermore, the immunoassay kit also includes an anti-rabbit IgG secondary antibody.

[0015] The advantages and positive effects of the present invention are:

[0016] The antibodies provided by the present invention having the above-mentioned complementary determining region sequences can specifically recognize PSMA expressed by cells and tissues, have good affinity for binding to PSMA and high recognition sensitivity, can effectively resist interference from complex non-target protein components in cells / tissues, have no specific binding to non-target antigens, are suitable for immunological detection of PSMA, especially for protein immunoblotting and immunohistochemistry, can reduce the incidence of false positive and false negative results, and improve the accuracy of immunodiagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0018] Figure 1 This is a map of the expression vectors used to construct an anti-human prostate-specific membrane antigen antibody in Example 1 of the present invention, including, from left to right, the pBR322 vectors pre-carrying the antibody light chain constant region and heavy chain constant region;

[0019] Figure 2 This is a diagram showing the results of immunoblotting using an anti-human prostate-specific membrane antigen antibody bound to different cells or tissues according to Example 2 of the present invention;

[0020] Figure 3 This is a diagram showing the immunohistochemical detection results of human colon and human prostate cancer tissue sections using an anti-human prostate-specific membrane antigen antibody in Example 2 of the present invention. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the embodiments. The embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0022] Given the information contained herein, it will be readily apparent to those skilled in the art that various modifications may be made to the precise description of the present invention without departing from the spirit and scope of the appended claims. It should be understood that the scope of the present invention is not limited to the processes, properties, or components defined herein, as these embodiments and other descriptions are intended only to illustrate specific aspects of the present invention. Indeed, various modifications to the embodiments of the present invention that are apparent to those skilled in the art or related fields are intended to be within the scope of the appended claims.

[0023] For a better understanding of the present invention and not to limit the scope of the present invention, all numerals and other numerical values ​​used in the present invention to express amounts, percentages, etc. should be understood as modified by the word "about" in all cases. Therefore, unless otherwise specified, the numerical parameters listed in the specification and the appended claims are approximate values, which may be changed according to the different ideal properties to be obtained. Each numerical parameter should at least be regarded as obtained based on the reported significant digits and by conventional rounding methods.

[0024] In addition, it should be noted that, unless otherwise defined, in the context of the present invention, the scientific and technical terms used should have the meanings commonly understood by those skilled in the art.

[0025] The terms "include", "comprising", "containing", "having" and the like are non-limiting in meaning, that is, other steps and other components that do not affect the results may be added.

[0026] The term "and / or" should be considered as a specific disclosure of each of the two specified features or components with or without the other. For example, "A and / or B" is considered to include the following situations: (i) A, (ii) B, and (iii) A and B.

[0027] The terms "rabbit monoclonal antibody," "monoclonal antibody," "rabbit-derived antibody," and similar expressions have synonymous meanings and, unless otherwise specified, refer to antibodies that specifically bind to human prostate-specific membrane antigen (PSMA). The terms "prostate-specific membrane antigen (PSMA)," "PSMA," "NAALAD1," and "FOLH1" have synonymous meanings. The modifier "rabbit" indicates that the complementarity determining regions (CDRs) of the antibody are derived from rabbit immunoglobulin sequences.

[0028] An antibody is an immunoglobulin molecule that is capable of specifically binding to a target antigen or epitope through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. In the present invention, the term "antibody" should be interpreted in the broadest sense and encompasses various antibody structures, including but not limited to so-called full-length antibodies, antibody fragments, and genetic or chemical modifications thereof, as long as they exhibit the desired antigen-binding activity. Antibody fragments can be one or more portions or fragments of a full-length antibody that retain the antibody's ability to specifically bind to the target antigen.

[0029] A typical antibody molecule (full-length antibody) consists of two identical light chains (L) and two identical heavy chains (H). Light chains can be divided into two types: kappa (κ) and lambda (λ); heavy chains can be classified into five types: μ, δ, γ, α, and ε, which define antibodies as IgM, IgD, IgG, IgA, and IgE, respectively. The amino acid sequences near the N-terminus of heavy and heavy chains vary greatly, while the remaining amino acid sequences are relatively constant. The regions of the light and heavy chains with the most variable amino acid sequences near the N-terminus are called the variable region (V), while the regions with relatively stable amino acid sequences near the C-terminus are called the constant region (C). The heavy chain variable region (VH) and light chain variable region (VL) are generally the most variable parts of antibodies and contain the antigen recognition site. The VH and VL regions can be further subdivided into hypervariable regions (HVRs) and framework regions (FRs). The HVRs, also known as complementarity-determining regions (CDRs), are circular structures. The heavy and light chain CDRs are closely aligned and interact with each other through the FRs, forming a surface that complements the three-dimensional structure of the target antigen or epitope. This determines the antibody's specificity and is the site of antigen recognition and binding. The FRs are the more conserved portions of the VH and VL sequences. They generally follow a β-pleated sheet configuration and are connected by three CDRs that form a connecting loop. Each VH and VL sequence typically consists of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0030] CDRs and FRs can be identified according to the Kabat definition, the Chothia definition, a cumulative of the Kabat and Chothia definitions, the AbM definition, the contact definition, the IMGT unique numbering definition and / or the conformational definition, or any CDR determination method known in the art. As used herein, the Kabat numbering system is used to define CDRs.

[0031] The light chain constant region (CL) and heavy chain constant region (CH) are not directly involved in antibody-antigen binding, but they exhibit different effector functions, such as antibody-dependent cellular cytotoxicity (ADCC). The CL length is generally consistent across different Ig types (κ or λ), but the CH length varies across different Ig classes. For example, IgG, IgA, and IgD comprise CH1, CH2, and CH3, while IgM and IgE comprise CH1, CH2, CH3, and CH4. The amino acid sequences of the heavy and light chain constant regions of antibodies are well known in the art and can be obtained by querying the IMGT database.

[0032] A full-length antibody is the most complete antibody molecular structure and has a typical Y-shaped molecular structure. Therefore, in the context of the present invention, "full-length antibody", "intact antibody" and "Y-shaped antibody" have the same meaning and can be used interchangeably.

[0033] Antibody fragments are one or more parts or fragments of a full-length antibody that essentially retain the same biological function or activity as the full-length form. Specifically, an antibody fragment includes at least the same CDR regions as the full-length antibody, and more preferably the same variable regions, thereby retaining complete antigen recognition and binding sites and being able to bind to the same antigen as the full-length antibody, particularly to the same epitope. Typical examples include: Fab, F(ab)2, Fab', F(ab')2, Fv, (Fv)2, scFv, and sc(Fv)2. These antibody fragments can be obtained using conventional techniques in the art.

[0034] (i) Fab: An antigen-binding fragment (Fab) is a monovalent fragment consisting of a complete light chain (variable and constant regions) and a portion of the heavy chain (variable and first constant regions). By proteolytic cleavage of the full-length antibody, fragments such as Fab, F(ab')2, and Fab' can be obtained. For example, IgG can be degraded into two Fab fragments and an Fc fragment by papain; and into an F(ab')2 fragment and a pFc' fragment by pepsin. The F(ab')2 fragment is further reduced to form two Fab' fragments. Because Fab contains both the antigen-binding region and a portion of the constant region, it not only possesses the same antibody-antigen affinity and excellent tissue penetration as scFv, but also has a more stable structure.

[0035] (ii) F(ab)2: A bivalent fragment consisting of two Fabs linked by a disulfide bridge at the hinge region.

[0036] (iii) Fv: The variable fragment (Fv) is located at the N-terminus of the antibody Fab fragment, contains only the variable region, and is composed of the variable regions of a light chain and a heavy chain. It is a dimer of VH and VL non-covalently bound (VH-VL dimer). The three CDRs of each variable region interact with each other to form an antigen binding site on the surface of the VH-VL dimer, which has the ability to recognize and bind to antigens, although the affinity is lower than that of the intact antibody.

[0037] (iv) (Fv)2: Consists of two covalently linked Fv fragments.

[0038] (v) scFv: A single-chain variable fragment (scFv) is an Fv fragment composed of a single polypeptide chain, consisting of a heavy chain variable region (VH) and a light chain variable region (VL) connected by a flexible linker (typically consisting of 10-25 amino acids). It retains the antigen-binding specificity of the original antibody. The linker in this invention is not particularly limited, as long as it does not hinder the expression of the antibody variable regions connected to it. Compared to full-length antibodies, scFv has a smaller molecular weight, resulting in higher penetration and lower immune side effects.

[0039] (vi) The sc(Fv)2 fragment is composed of two heavy chain variable regions and two light chain variable regions connected by a linker or the like.

[0040] In some embodiments, the full-length sequence of the antibody or antibody fragment of the present invention may include CDR regions and FR regions from rabbit immunoglobulin sequences. In other embodiments, the antibody may contain amino acid residues encoded by non-rabbit immunoglobulin sequences, for example, humanized antibodies, chimeric antibodies, etc., to reduce the body's rejection reaction while maintaining the required specificity and affinity. The term "chimeric antibody" refers to an antibody in which part of the antibody is derived from a specific source or species, while the rest is derived from a different source or species. The term "humanized antibody" is a chimeric antibody with a CDR region of a non-human antibody such as a rabbit antibody and a FR region from a human. In some cases, the variable region of a non-human antibody is combined with the constant region of a human antibody, such as a human-rabbit chimeric antibody; in other cases, the CDR region of a non-human antibody is combined with the FR region and constant region derived from a human antibody sequence, that is, the CDR region of a non-human antibody is grafted onto a human antibody framework (FR) sequence, and this framework sequence is derived from the FR sequence of a single or multiple other human antibody variable regions. In the present invention, the CDR regions in the chimeric or humanized antibodies are derived from rabbit-derived CDR regions.

[0041] The terms "monoclonal antibody" or "single antibody" and other similar terms are used interchangeably and refer to a homogeneous antibody population, i.e., the individual antibodies comprising the population are identical except for a small amount of mutations and / or post-translational modifications (e.g., isomerization, amidation) that may occur naturally. "Monoclonal antibodies" are highly specific and exhibit a single binding specificity and affinity for the same or substantially identical epitope on the antigen. The modifier "monoclonal" indicates that the antibody is obtained from a substantially homogeneous antibody population and should not be construed as limiting the source or preparation method of the antibody. The antibody can be prepared by a variety of methods, including but not limited to hybridoma methods, phage display methods, yeast display methods, recombinant DNA methods, single cell screening, or single cell sequencing methods.

[0042] The term "specific binding" is a well-known term in the art, and a molecule exhibits "specific binding" if it reacts with a specific target antigen or epitope more frequently, more rapidly, longer-lastingly, and / or with greater affinity than with other target antigens or epitopes. "Specific binding" or "preferential binding" does not necessarily require (although it can include) exclusive binding.

[0043] In order to make the objects and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0044] An embodiment of the present invention provides an anti-human prostate-specific membrane antigen (PSMA) antibody, comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region each include three complementarity determining regions (CDRs), respectively named CDR1, CDR2, and CDR3, wherein the amino acid sequences of CDR1, CDR2, and CDR3 on the light chain variable region are shown in SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5, respectively; the amino acid sequences of CDR1, CDR2, and CDR3 on the heavy chain variable region are shown in SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10, respectively.

[0045] The present invention uses the polypeptide sequence (118-137aa) of the extracellular domain of human prostate-specific membrane antigen (PSMA) as an immunogen for immunization, and then obtains rabbit monoclonal antibodies against human PSMA based on the monoclonal antibody development technology of single B lymphocyte screening and culture. The obtained antibody can specifically recognize PSMA expressed by cells and tissues, has good affinity for binding to PSMA, high recognition sensitivity, can effectively resist the interference of complex non-target protein components in cells / tissues, and has no specific binding to non-target antigens. Positive cells with high expression of PSMA are detected by protein immunoblotting, and the target band is detected in the positive cell lysate with a clear band signal, which is consistent with the expected size of PSMA protein; positive tissues with high expression of PSMA are detected by immunohistochemistry, which can accurately distinguish between positive and negative samples, with accurate coloring in positive tissues and a clean background, and no specific staining in negative tissues. The above results confirm that the antibody of the present invention has good detection performance and is suitable for immunological detection of PSMA. It has the advantages of high specificity, high accuracy, high sensitivity, and strong anti-interference ability. It is particularly suitable for protein immunoblotting and immunohistochemistry, which can reduce the incidence of false positive and false negative results and improve the accuracy of immunodiagnosis.

[0046] Optionally, the light chain variable region and the heavy chain variable region each include four framework regions (FRs), wherein the four FRs and three CDRs are arranged in a staggered order to form a variable region. The amino acid sequence of the light chain variable region (VL) of the antibody of the present invention is shown in SEQ ID NO. 2, and the amino acid sequence of the heavy chain variable region (VH) is shown in SEQ ID NO. 7.

[0047] Optionally, the antibodies of the present invention further comprise a light chain constant region (CL) and a heavy chain constant region (CH), wherein the CL and VL constitute a complete light chain (FL), and the CH and VH constitute a complete heavy chain (FH). Antibody constant regions are generally available through public searches, such as searching the IMGT online database (www.imgt.org) for rabbit IgG gamma C reign to obtain CH, and searching for rabbit IgG kappa C reign to obtain CL.

[0048] Specifically, the amino acid sequence of the antibody light chain is shown in SEQ ID NO. 1, and the amino acid sequence of the heavy chain is shown in SEQ ID NO. 6. Correspondingly, CL is of κ type, and CH is of IgG type.

[0049] It should be noted that the monoclonal antibody of the present invention can be a full-length antibody (having a typical Y-shaped molecular structure) or an antigen-binding region of the full-length antibody; the antigen-binding region refers to a polypeptide that substantially retains the same biological function or activity as the full-length form. Specifically, the antigen-binding region includes the CDR region as described above, and more preferably has the variable region as described above, thereby retaining a complete antigen recognition and binding site, and can bind to the same antigen as the full-length antibody, especially to the same epitope. Optionally, the antigen-binding region is selected from at least one of Fab, F(ab)2, Fab', F(ab')2, Fv, (Fv)2, scFv and sc(Fv)2. These antigen-binding regions can be obtained by conventional techniques in the art.

[0050] Yet another embodiment of the present invention provides a nucleic acid molecule, a recombinant vector comprising the nucleic acid molecule, or a host cell comprising the nucleic acid molecule, wherein the nucleic acid molecule encodes the above-mentioned anti-human prostate-specific membrane antigen antibody.

[0051] Nucleic acid molecules can be in the form of DNA (such as cDNA, genomic DNA or synthetic DNA) or RNA (such as mRNA or synthetic RNA). DNA can be single-stranded or double-stranded, and can be a coding strand or a non-coding strand.

[0052] The sequence of the nucleic acid molecule can be derived from the antibody AA sequence by conventional means such as codon coding rules. The full-length sequence of the nucleic acid molecule or its fragments can usually be obtained by PCR amplification, recombination or artificial synthesis.

[0053] The original vector for constructing the recombinant vector is a conventional various vectors in the art, as long as it can accommodate the nucleic acid molecule. Typical vectors include plasmids (such as pBR322, pUC series, pET series, pGEX series), viral vectors, phages (such as λgt4λB, λ-Charon, λΔz1 and M13), cosmids and minichromosomes. The vector can be a cloning vector (i.e., for transferring nucleic acid molecules into a host and multiplying them in large quantities in the host cell) or an expression vector (i.e., containing the necessary genetic elements to allow the nucleic acid molecules inserted into the vector to be expressed in the host cell). The nucleic acid molecule is inserted into a suitable vector to form a cloning vector or expression vector carrying the nucleic acid molecule, which is then introduced into the host cell and cultured under specific conditions to express the antibody. This is a well-known technology in the art and will not be described in detail here.

[0054] The nucleic acid molecules encoding the monoclonal antibodies FL and FH of the present invention can be inserted into two vectors, respectively, which can be introduced into the same or different host cells. When the heavy chain and light chain are expressed in different host cells, each chain can be isolated from the host cell in which it is expressed, and the isolated heavy and light chains are mixed and incubated under appropriate conditions to form antibodies. In other embodiments, the nucleic acid molecules of the monoclonal antibodies FL and FH can also be cloned into a single vector, with each nucleic acid sequence linked to a suitable promoter downstream; for example, each nucleic acid sequence encoding the heavy chain and light chain can be operably linked to a different promoter, or the nucleic acid sequence encoding the heavy chain and light chain can be operably linked to a single promoter so that both the heavy chain and the light chain can be expressed by the same promoter. The choice of expression vector / promoter depends on the type of host cell used to produce the antibody.

[0055] Conventional techniques are used to transfect or transform recombinant vectors into host cells. When the host is a prokaryotic organism such as Escherichia coli, competent cells capable of absorbing DNA are harvested after the exponential growth phase and treated with CaCl2 or MgCl2. Alternatively, transfection can be accomplished by microinjection, electroporation, or liposome packaging. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate coprecipitation, microinjection, electroporation, liposome packaging, or gene gun bombardment to achieve gene introduction.

[0056] The host cell can be a prokaryotic or eukaryotic cell. Examples of prokaryotic host cells that can be used in the present invention include, but are not limited to, Escherichia coli (e.g., DH5α, JM109, BL21, W3110), Bacillus (e.g., Bacillus subtilis, Bacillus thuringiensis), and Enterobacteriaceae strains (e.g., Salmonella typhimurium, Serratia marcescens), and Pseudomonas. Examples of eukaryotic host cells that can be used for transformation include, but are not limited to, yeast, insect cells, and animal cells, such as Drosophila S2 or Sf9 cells, mammalian CHO, CHO DG44, CHO-S, COS-7, 293 series cells, HepG2, Huh7, 3T3, RIN, MDCK, and HEK293 cell lines. After obtaining a host cell transfected or transformed with the recombinant vector described above, the antibody can be expressed by culturing under suitable conditions, and then separated to obtain purified antibodies.

[0057] In a preferred embodiment, the above-mentioned recombinant vector is a mammalian expression vector pBR322, and the host cell is a human kidney epithelial cell (293F cell).

[0058] In a typical embodiment, a method for preparing a monoclonal antibody comprises: concatenating the heavy and light chain genes of the antibody with a signal peptide, loading the genes separately into the expression vector pBR322, co-transfecting 293F cells, culturing and collecting the cell culture supernatant, and purifying the target antibody. The choice of signal peptide is designed based on the host cell and is not particularly limited in the present invention.

[0059] Yet another embodiment of the present invention provides the use of the above-mentioned anti-human prostate-specific membrane antigen antibody or its antibody conjugate in the preparation of a human prostate-specific membrane antigen immunoassay kit, wherein the antibody conjugate comprises the above-mentioned anti-human prostate-specific membrane antigen antibody and a detection label connected to the antibody.

[0060] The advantages of using the anti-human prostate-specific membrane antigen antibody or its antibody conjugate in preparing a human prostate-specific membrane antigen immunoassay kit are the same as the advantages of the anti-human prostate-specific membrane antigen antibody over the prior art as described above, and will not be repeated here.

[0061] It should be emphasized that the antibodies of the present invention can be used alone or linked (covalently or non-covalently) to a detection label to form an antibody conjugate. In some embodiments, the antibodies of the present invention are used as antigen-binding (or capture) antibodies that specifically recognize and bind to PSMA in the sample to be tested, and then the PSMA is qualitatively or quantitatively detected by analyzing the detection label signal attached thereto; in other embodiments, the anti-human PSMA antibody is not labeled (as a primary antibody or capture antibody), but a secondary antibody or other molecule that can bind to the primary antibody is labeled and conjugated (as a detection antibody). For example, if the anti-human PSMA antibody is a rabbit IgG antibody, the secondary antibody can be an anti-rabbit IgG antibody. Thus, by analyzing the changes in the detection label signal generated after the secondary antibody specifically binds to the antibody of the present invention, PSMA can be qualitatively or quantitatively detected, such as the multiple detection systems established in Example 2 below.

[0062] The detection label is used to generate a recognizable signal change, so as to identify the antibody of the present invention or its secondary antibody according to the signal change, and then identify the expression of PSMA in the sample to be tested through the specific reaction of the antigen and antibody. The detection label includes, but is not limited to: biotin, fluorescent dyes (such as umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride), fluorescent proteins (such as allophycocyanin, phycoerythrin, PerCP and phycocyanin), enzymes (such as alkaline phosphatase, acid phosphatase, β-galactosidase, glucose oxidase, horseradish peroxidase, acetylcholinesterase, avidin), colloidal gold, colored magnetic beads, latex particles, radionuclides, detection antibodies or combinations thereof.

[0063] The above-mentioned immunoassay methods include but are not limited to: enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunospot (ELISPOT), immunohistochemistry (IHC), immunofluorescence (IF), immunoblotting (WB), immunoprecipitation (IP) and flow cytometry (FC).

[0064] Based on the same inventive concept, an embodiment of the present invention further provides a human prostate-specific membrane antigen immunoassay kit, comprising the above-mentioned anti-human prostate-specific membrane antigen antibody or its antibody conjugate.

[0065] Optionally, the immunoassay kit is an enzyme-linked immunosorbent assay kit, an ELISpot kit, an immunohistochemistry kit, an immunofluorescence kit, an immunoblotting kit, an immunoprecipitation kit, or a flow cytometry kit. Preferably, the immunoassay kit is an immunoblotting kit or an immunohistochemistry kit.

[0066] Optionally, the immunoassay kit further comprises an anti-rabbit IgG secondary antibody.

[0067] The present invention will be further described below with reference to specific examples. Experimental methods in the following examples where specific conditions are not specified were generally performed under conventional conditions, such as those described in the Molecular Cloning Laboratory Manual (4th Edition) published by Cold Spring Harbor Laboratory, or under conditions recommended by the manufacturer.

[0068] Example 1 Preparation of rabbit monoclonal antibodies against human prostate-specific membrane antigen (PSMA)

[0069] 1.1. Antigen Preparation: In this example, the 118-137 amino acid (aa) fragment of the extracellular domain of PSMA protein conjugated to hemocyanin (KLH) (for the full amino acid sequence, see Uniprot Protein ID: Q04609, Gene ID: 2346, NCBI Protein Accession No.: NP_004467.1, NCBI Gene Accession No.: NM_004476.3) (sequence: SYPNKTHPNYISIINEDGNE (see SEQ ID NO. 15)) was used as the immunogen to immunize rabbits. Monoclonal antibodies were then prepared based on single B lymphocyte labeling and sorting techniques to obtain rabbit-derived antibodies against the PSMA protein.

[0070] The method for preparing the immunogen by coupling the PSMA protein 118-137aa fragment with KLH is as follows: 1) Preparation of SMCC solution: 50 mg of SMCC was dissolved in 10 mL of 1×PBS, and the solution was heated in a 55°C water bath for 5-6 minutes to dissolve, thereby preparing a 5 mg / mL SMCC solution; 2) Conjugation of KLH with thiol: KLH was weighed in proportion to the total mass of the polypeptide to be coupled, with a polypeptide:KLH ratio of 2:1, and dissolved in 1×PBS, maintaining the concentration at 3 mg / mL, and shaken well. Then, an appropriate volume of SMCC solution was taken according to the mass of KLH, with a KLH:SMCC ratio of 10:1.5, and added to the dissolved KLH. The solution was placed on a rotating incubator for mixing and shaking at room temperature for 1 hour, and the solution was activated by shaking and mixing with 1×PBS (pH 7.4) at a volume ratio of V PBS :V KLH =15:1, dialyze for 1 hour to remove free SMCC. 3) Activated KLH-coupled hapten: Add 100 μL of 1× PBS to the vial containing the hapten peptide to dissolve it. If complete dissolution is not achieved, dissolve the protein in 8 M urea to prepare a 10 mg / mL peptide solution. Slowly add this solution to the activated KLH (dialysis complete) in multiple portions. Place in a small shaker or rotary incubator at the lowest speed and observe every half hour. If precipitation occurs, continue adding urea and incubate at room temperature for 4 hours or at 4°C overnight.

[0071] 1.2 Animal Immunization: Three Japanese white rabbits were immunized with 300 μg of the immunogen per rabbit. Prior to the first immunization, the antigen was mixed with an equal volume of complete Freund's adjuvant (Sigma) to form an emulsion, which was then injected subcutaneously at multiple sites on the abdomen and back of the rabbits. Three weeks after the first immunization, 150 μg of the immunogen was mixed with an equal volume of incomplete Freund's adjuvant (Sigma) to form an emulsion, and the emulsion was injected subcutaneously at multiple sites on the abdomen and back of the rabbits for two booster immunizations. Serum samples were collected after each immunization and titers against human PSMA were determined by enzyme-linked immunosorbent assay (ELISA). Antibodies were purified from the final immunization serum and tested for binding specificity to endogenous samples (including tissue samples expressing PSMA protein and tissue samples negative for PSMA protein) by immunohistochemistry. Rabbits with high serum titers and the best endogenous detection were given a booster immunization with 150 μg of the immunogen at multiple sites. Three days later, the animals were sacrificed and their spleens were harvested.

[0072] 1.3. Isolation of B lymphocytes from the spleen and sorting of antigen-specific B lymphocytes: For related methods, please refer to the published patents "Method for Efficient Isolation of Single Antigen-Specific B Lymphocytes from Spleen Cells (Publication No.: CN110016462A, Publication Date: 2019-07-16)" and "A B Lymphocyte In Vitro Culture System and Application (Publication No.: CN111518765A, Publication Date: 2020-08-11)".

[0073] 1.4 Cloning of rabbit monoclonal antibody gene: The supernatant of cultured B lymphocytes was used to identify antigen-specific B lymphocytes by ELISA coated with PSMA protein. TM RNA was extracted using a MicroPrep kit (ZYMO, Cat. No. R1051) and reverse transcribed into cDNA. Using the aforementioned cDNA as a template, naturally paired rabbit monoclonal antibody light chain variable (VL) and heavy chain variable (VH) regions were amplified from the cDNA of the corresponding positive clones by PCR and sequenced. The PCR reaction system consisted of 4 μL of cDNA, 1 μL of a 10 mM forward primer, 1 μL of a 10 mM reverse primer, 12.5 μL of 2× Gloria HiFi (ABclonal, Cat. No. RK20717), and 6.5 μL of H2O. The PCR amplification protocol consisted of 98°C for 30 s, followed by 40 cycles of 98°C for 10 s, 64°C for 30 s, and 72°C for 30 s, and finally 72°C for 5 min. The reaction mixture was stored at 4°C. The primer sequences (5'-3') for amplifying the VL and VH genes are shown below, where F and R represent the forward primer and the reverse primer, respectively.

[0074] VL-Primer-F: 5'-tgaattcgagctcggtacccatggacacgagggccccac-3' (SEQ ID NO. 11);

[0075] VL-Primer-R: 5'-cacacacgatggtgactgttccagttgccacctgatcag-3' (SEQ IDNO.12);

[0076] VH-Primer-F: 5'-tgaattcgagctcggtacccatggagactgggctgcgctg-3' (SEQ IDNO.13);

[0077] VH-Primer-R: 5'-gtagcctttgaccaggcagcccagggtcaccgtggagctg-3' (SEQ ID NO. 14).

[0078] The amplified DNA products were sequenced to obtain the VL sequence shown in SEQ ID NO.2 and the VH sequence shown in SEQ ID NO.7; the IMGT online database (www.imgt.org) was then queried to obtain the sequence of the constant region, and an antibody with a complete light chain (FL) shown in SEQ ID NO.1 and a complete heavy chain (FH) shown in SEQ ID NO.6 was obtained.

[0079] Antibody sequencing was completed by Jinkairui Biotechnology Co., Ltd. The antibody amino acid (AA) sequence is shown in Table 1 , where LCDR1-3 represent the complementarity determining regions CDR1-3 of the light chain variable region, and HCDR1-3 represent the complementarity determining regions CDR1-3 of the heavy chain variable region, respectively. The variable region numbering system is the Kabat numbering system.

[0080] Table 1 Sequence information of rabbit monoclonal antibodies in this example

[0081]

[0082]

[0083] 1.5. Expression and large-scale production of antibodies: Through genetic engineering recombinant expression technology, the amplified VL and VH genes are inserted into the expression vector in series with the light chain constant region (CL) and the heavy chain constant region (CH), and monoclonal antibodies are produced in large quantities through recombinant expression of antibody genes. The specific operation is as follows: The mammalian expression vector pBR322 carrying the CL and CH genes is linearized with the restriction enzymes XbaI (955bp) and NheI (949bp), respectively, and the VL and VH genes with signal peptides upstream are constructed into the aforementioned expression vector by homologous recombination to obtain an expression vector containing complete light and heavy chain genes. The successful construction of the expression vector was verified by sequencing. The map of the mammalian expression vector pBR322 carrying the CL and CH genes of the rabbit monoclonal antibody is shown in Figure 1 In the figure, pBR322 origin and f1 origin are replication promoters, Ampcillin is a resistance gene, CMV promoter is a transcription promoter, SV40 PAterminator is a tailing signal, Light chain constant is the nucleic acid sequence of the light chain constant region (left figure), and Heavy chain constant is the nucleic acid sequence of the heavy chain constant region (right figure).

[0084] To facilitate antibody purification, secretory expression of the antibody is achieved by adding signal peptides upstream of the VL and VH genes. Signal peptides commonly used in the art can be used for antibody expression. For example, the patent "Anti-human interferon α2 rabbit monoclonal antibody and its application (Publication No.: CN116063487A, Publication Date: 2023-05-05)" and the patent "High-affinity human IL-5 rabbit monoclonal antibody and its application (Publication No.: CN115819578A, Publication Date: 2023-03-21)" have a signal peptide "MDTRAPTQLLGLLLLWLPGARC" upstream of the VL gene and a signal peptide "METGLRWLLLVAVLKGVQC" upstream of the VH gene. Of course, those skilled in the art can also replace other signal peptides for antibody expression after obtaining the antibody sequence of the present invention. Therefore, the signal peptide sequence is not shown in the antibody sequence of Table 1 of this example.

[0085] The constructed FL and FH expression vectors were co-transfected into 293F cells and cultured for 72-96 hours. The culture supernatant contained recombinant rabbit monoclonal antibodies that recognized human PSMA. The target antibodies were purified from the culture supernatant using Protein A affinity gel resin (purchased from Tiandi Renhe, Catalog No. SA023100). The antibody concentration was 1 mg / mL and the purity was ≥95%. The purified antibodies were aliquoted and stored at -20°C until needed.

[0086] Example 2 Establishment and Effect Evaluation of Immunoassay Method Based on Anti-Human PSMA Antibodies

[0087] In this example, an immunoblotting and immunohistochemistry analysis system was established for a rabbit-derived antibody against human PSMA to evaluate the performance of the antibody for immunoassay.

[0088] 2.1 Establishment of Western blot (WB) detection system

[0089] The positive samples in this example are human prostate cancer cells (LNCaP) that highly express PSMA, and the negative samples include human chronic myeloid leukemia cells (HAP1) that do not express PSMA, human monocytic leukemia cells (THP-1), mouse brain tissue, mouse kidney tissue, rat brain tissue, and rat spleen tissue. The WB experimental analysis operation is as follows: (1) Electrophoresis: cells or tissues are lysed to obtain protein lysate, and 6% polyacrylamide gel electrophoresis (PAGE) is performed; (2) Transfer: gel protein bands are transferred to NC membrane in an electrotransfer system according to conventional methods; (3) Blocking: the NC membrane is placed in TBST blocking solution containing 3% skim milk powder and blocked at room temperature for 1 hour; (4) Primary antibody incubation: the antibody prepared in Example 1 (primary antibody working concentration is 0.01 μg / mL) is added and incubated at 4°C overnight; (5) Secondary antibody incubation: the membrane is washed with TBST, HRP-conjugated Goat anti-Rabbit IgG (H+L) secondary antibody (purchased from ABclonal, product number AS014, secondary antibody dilution ratio 1:5000) is added and incubated at room temperature for 1 hour; (6) Color development: the membrane is washed with TBST, ECL supersensitive color development solution is added and developed.

[0090] WB results are shown in Figure 2 PSMA is a type II transmembrane glycoprotein composed of 750 amino acids. Its theoretical molecular weight is 84KDa. After glycosylation, its observed molecular weight is about 100KDa due to the increase in protein molecular weight. Figure 2 As can be seen, the negative sample lacked the target band, indicating no specific antigen-antibody binding reaction with non-target proteins, resulting in a negative signal. In the positive sample, a target band matching the expected molecular weight of PSMA was observed, indicating a positive signal. This demonstrates that the antibody can sensitively recognize PSMA protein in cell lysates, exhibiting high specificity and antigen-protein binding affinity, and effectively resisting interference from complex cellular components.

[0091] 2.2 Construction of Immunohistochemistry (IHC) Detection System

[0092] PSMA is a specific marker for prostate cancer and is mainly expressed in epithelial cells. In this example, the positive sample is human prostate cancer tissue, and the negative sample is human colon tissue. The IHC detection steps are as follows: (1) Baking: Immerse the paraffin tissue slices baked at a constant temperature of 56°C for 30 minutes in dewaxing solution 1. After 5 minutes, take out the slices and immerse the paraffin slices in the order of dewaxing solution 2, dewaxing solution 3, anhydrous ethanol 1, anhydrous ethanol 2, and anhydrous ethanol 3. Place them in the dewaxing solution for 5 minutes and in the anhydrous ethanol for 3 minutes; then wash the slices with running water for 3 minutes; dewaxing solutions 1-3 were purchased from Wuxi Jiangyuan Industrial Technology and Trade Co., Ltd.; (2) Antigen repair: 0.01M Tris-EDTA repair solution (pH 9.0) High-pressure heat repair; (3) Inactivation of endogenous peroxidase: Immerse the slides in PBS buffer for 3 times, 3 minutes each time, and remove the buffer on the slides; then immerse the slides in 3% hydrogen peroxide solution and incubate at room temperature for 10 minutes; (4) Blocking: Immerse the slides in PBS buffer for 3 times, 3 minutes each time, then remove the buffer, circle the area to be examined on the slide, add PBS blocking solution in the area to be examined, and incubate at room temperature for 30 minutes; (5) Primary antibody incubation: Remove the blocking solution, add the antibody dilution solution prepared in the example (primary antibody working concentration is 0.65 μg / mL, primary antibody dilution ratio is 1:900), and incubate at room temperature for 60 minutes; remove the antibody working solution, quickly rinse once with PBS buffer, and soak and wash three times, 3 minutes each time; (6) Secondary antibody incubation: Add ready-to-use secondary antibody working solution (purchased from DAKO, product number K5007), and incubate at room temperature for 25 minutes; remove the secondary antibody working solution. Liquid, PBS buffer rapid rinse once, soak and wash 3 times, each time for 3 minutes; (7) Color development: add DAB color development solution, observe the color change closely under the microscope, and after obtaining the appropriate staining intensity, immerse the slice in a large amount of distilled water to stop color development, and then rinse in running water for 10 minutes; (8) Restaining: immerse the slightly drained tissue slice in Mayer's hematoxylin for 1 minute, and rinse in running water for 3 minutes; (9) Rebluing: immerse the slightly drained slice in a saturated aqueous solution of lithium carbonate for 3 seconds, and rinse in running water for 3 minutes; (10) Dehydration: immerse the slice in anhydrous ethanol twice, lift it up and down several times during the immersion period, and take it out after 10 seconds; dry the slice at high temperature (54-58℃); (11) Sealing: add an appropriate amount of neutral gum to the center of the slice and cover it with a cover glass. The amount of glue added should be appropriate. After sealing the cover glass, the tissue should be completely covered and no glue should overflow. Finally, scan the slice.

[0093] Immunohistochemical staining results are classified as either positive or negative. A positive result is defined as the presence of brown staining at the antigen-expressing site in specific tissues and cells, with low or no background. A negative result is defined as the absence of brown staining in specific tissues and cells. Hematoxylin counterstaining of cell nuclei, which appears blue, is used to outline the background of the cells specifically immunostained. Figure 3From left to right, the IHC staining results for human colon tissue sections and human prostate cancer tissue are shown. The results show a clear positive signal in human prostate cancer tissue, localized to the cytoplasm and cell membrane, consistent with the transmembrane localization of PMSA. The staining is clear and the background is clean. Human colon tissue exhibits no specific staining, exhibiting a negative signal. This demonstrates that the antibodies of the present invention are highly specific and can effectively recognize naturally expressed PSMA proteins in tissue samples. Binding to the target protein is unaffected by the complexity of the tissue cells, resulting in high sensitivity, ensuring the accuracy and reliability of the test results and helping to reduce the incidence of false positive and negative results.

[0094] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements and improvements made to the present invention within the scope of implementation of the present invention should be included in the scope of protection of the present invention.

Claims

1. An anti-human prostate-specific membrane antigen antibody, characterized in that It includes a light chain variable region and a heavy chain variable region, the amino acid sequences of CDR1, CDR2 and CDR3 on the light chain variable region are shown as SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5, respectively, and the amino acid sequences of CDR1, CDR2 and CDR3 on the heavy chain variable region are shown as SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10, respectively.

2. The anti-human prostate-specific membrane antigen antibody according to claim 1, characterized in that The amino acid sequence of the light chain variable region is shown in SEQ ID NO.2, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.

7.

3. The anti-human prostate-specific membrane antigen antibody according to claim 2, characterized in that The amino acid sequence of the antibody light chain is shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.

6.

4. The anti-human prostate-specific membrane antigen antibody according to claim 1, characterized in that The antibody is a full-length antibody or an antigen-binding region thereof; the antigen-binding region is selected from at least one of a Fab fragment, a F(ab)2 fragment, a Fv fragment, a (Fv)2 fragment, a scFv fragment and a sc(Fv)2 fragment.

5. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the anti-human prostate-specific membrane antigen antibody according to any one of claims 1 to 4.

6. A recombinant vector, characterized in that The recombinant vector comprises the nucleic acid molecule according to claim 5.

7. Use of the anti-human prostate-specific membrane antigen antibody or its antibody conjugate according to any one of claims 1 to 4 in the preparation of a human prostate-specific membrane antigen immunoassay kit, characterized in that: The antibody conjugate comprises the antibody and a detection label linked to the antibody.

8. A human prostate-specific membrane antigen immunoassay kit, characterized in that: The invention comprises the anti-human prostate-specific membrane antigen antibody or its antibody conjugate according to any one of claims 1 to 4, wherein the antibody conjugate comprises the antibody and a detection label connected to the antibody.

9. The human prostate-specific membrane antigen immunoassay kit according to claim 8, characterized in that: The immunoassay kit is selected from an immunoblotting kit or an immunohistochemistry kit.

10. The human prostate-specific membrane antigen immunoassay kit according to claim 8, characterized in that: The immunoassay kit further comprises an anti-rabbit IgG secondary antibody.

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