Anti-human MPC antibody or antigen binding fragment thereof and application thereof

Abstract

The invention provides an anti-mitochondrial pyruvate vector (MPC) antibody or an antigen binding fragment thereof as well as a composition and application thereof. Specifically, the invention provides an anti-MPC antibody or an antigen binding fragment thereof, which can effectively bind to MPC. The invention also provides an antibody drug conjugate, a detection plate or a kit containing the antibody or the antigen binding fragment thereof, and the antibody drug conjugate, the detection plate or the kit can be applied to detection and marking of the MPC protein so as to be used for structure and function research or drug development of the MPC protein, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically the field of antibody technology, and relates to an antibody or its antigen-binding fragment capable of binding to human MPC, as well as combinations thereof and their applications. Background Technology

[0002] The mitochondrial pyruvate carrier (MPC), composed of two heterodimeric membrane proteins (mitochondrial pyruvate carrier 1 (MPC1, SLC54A1) and mitochondrial pyruvate carrier 2 (MPC2, SLC54A2)), is a specific protein complex located within the mitochondrial membrane. The MPC is the sole channel for the transfer of pyruvate from the mitochondrial interstitial space to the mitochondrial matrix. Within the mitochondria, pyruvate is converted to acetyl-CoA by the pyruvate dehydrogenase complex. Acetyl-CoA enters the tricarboxylic acid cycle (TCA cycle), participating in metabolism and energy production. Abnormal pyruvate metabolism due to MPC dysfunction is associated with cancer, heart failure, and type II diabetes. Furthermore, the nervous system requires a large amount of energy to maintain its function; therefore, mitochondrial energy supply is closely related to nerve function. Mitochondrial dysfunction can induce or exacerbate brain lesions. MPC is an important target for treating related diseases. In-depth research on its functional structure and regulatory mechanisms not only reveals the role of MPC in related diseases, but also provides a more effective approach for the diagnosis and treatment of these diseases.

[0003] However, to date, the existing libraries of detection / labeling antibodies for MPC carrier proteins are limited, and the limited number of detection antibodies or detection methods has hindered the progress of MPC research.

[0004] Therefore, there is an urgent need in this field to develop antibodies or antigen-binding fragments of MPC proteins with high specificity and high affinity, so that they can be used to further study MPC proteins in scientific research, diagnosis, detection and other technologies. Summary of the Invention

[0005] The purpose of this invention is to provide an antibody against human MPC or an antigen-binding fragment thereof, as well as combinations thereof and applications.

[0006] In a first aspect, the present invention provides an anti-human MPC antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment having three complementarity-determining regions (HCDRs) in the heavy chain variable region and three complementarity-determining regions (LCDRs) in the light chain variable region: (1) HCDR1 as shown in SEQ ID NO:59, HCDR2, as shown in SEQ ID NO:60, HCDR3, as shown in SEQ ID NO:61, LCDR1, as shown in SEQ ID NO:63, LCDR2, as shown in SEQ ID NO:64, and LCDR3, as shown in SEQ ID NO:65; (2) HCDR1 as shown in SEQ ID NO:69, HCDR2, as shown in SEQ ID NO:70, HCDR3, as shown in SEQ ID NO:71, LCDR1, as shown in SEQ ID NO:73, LCDR2, as shown in SEQ ID NO:74, and LCDR3 as shown in SEQ ID NO:75.

[0007] In another preferred embodiment, the antibody is a murine antibody, a chimeric antibody, or a humanized antibody.

[0008] In another preferred embodiment, the antigen-binding fragment includes: Fab fragment, Fab′-SH, Fv fragment, scFv fragment, and F(ab')2 fragment.

[0009] In another preferred embodiment, the heavy chain variable region and light chain variable region of the anti-human MPC antibody or its antigen-binding fragment are selected from the group consisting of: (a) The heavy chain variable region with the amino acid sequence shown in SEQ ID NO:58, and the light chain variable region with the amino acid sequence shown in SEQ ID NO:62; or (b) The heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:68, and the light chain variable region with an amino acid sequence as shown in SEQ ID NO:72.

[0010] In another preferred embodiment, the heavy chain of the antibody or its antigen-binding fragment further includes a heavy chain constant region; the light chain of the antibody or its antigen-binding fragment further includes a light chain constant region.

[0011] In another preferred embodiment, the antibody is a single-chain antibody, a double-chain antibody, or an antigen-binding fragment.

[0012] In another preferred embodiment, the heavy chain constant region is IgG1, IgG2a, IgG2b or IgM.

[0013] In another preferred embodiment, the constant regions of the heavy and light chains of the anti-human MPC antibody are the heavy chain constant region and light chain constant region sequences of human IgG1, respectively.

[0014] In another preferred embodiment, the constant regions of the heavy and light chains of the anti-human MPC antibody are the heavy chain constant region and light chain constant region sequences of mouse IgG1, respectively.

[0015] In another preferred embodiment, the heavy chain constant region is of human or mouse origin.

[0016] In another preferred embodiment, the light chain constant region is of human or mouse origin.

[0017] In another preferred embodiment, the antibody is a full-length antibody protein or an antigen-binding fragment.

[0018] In another preferred embodiment, the antibody is a monoclonal antibody.

[0019] In another preferred embodiment, the antibody is a partially or fully humanized monoclonal antibody.

[0020] In another preferred embodiment, the antibody further comprises a linker peptide located between the heavy chain variable region and the light chain variable region.

[0021] A second aspect of the present invention provides a recombinant protein having: (1) The anti-human MPC antibody or its antigen-binding fragment as described in the first aspect of the present invention; and (2) Optional tag sequence for expression and / or purification.

[0022] In another preferred embodiment, the tag includes: an Fc tag, a FLAG tag, a 6His tag, a Strep tag, a protein labeling or purification tag commonly used in the art, or a combination thereof.

[0023] In another preferred embodiment, the recombinant protein (or polypeptide) includes a fusion protein.

[0024] In another preferred embodiment, the recombinant protein is a monomer, a dimer, or a polymer.

[0025] In a third aspect, the present invention provides a polynucleotide expressing the anti-human MPC antibody or its antigen-binding fragment as described in the first aspect of the present invention.

[0026] In another preferred embodiment, the polynucleotide is DNA, RNA, or cDNA.

[0027] In another preferred embodiment, the polynucleotide comprises or consists of the following sequences: (a) The nucleotide sequences shown in SEQ ID NO:66 and SEQ ID NO:67; or (b) The nucleotide sequences shown in SEQ ID NO:76 and SEQ ID NO:77.

[0028] In a fourth aspect, the present invention provides a carrier containing the polynucleotide described in the third aspect of the present invention.

[0029] In another preferred embodiment, the vector includes: bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.

[0030] In another preferred embodiment, the vector is a eukaryotic expression vector.

[0031] In a fifth aspect, the present invention provides a host cell expressing the anti-human MPC antibody or its antigen-binding fragment as described in the first aspect of the present invention, having its genome integrated with the polynucleotides as described in the third aspect of the present invention, or containing the vector as described in the fourth aspect of the present invention.

[0032] In another preferred embodiment, the host cell is a eukaryotic cell or a prokaryotic cell.

[0033] In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, yeast cells, and mammalian cells.

[0034] In another preferred embodiment, the prokaryotic cell is Escherichia coli.

[0035] In another preferred embodiment, the mammalian cell is a human cell or a mouse cell.

[0036] A sixth aspect of the present invention provides an antibody-drug conjugate comprising: (I) An antibody portion comprising the anti-human MPC antibody or its antigen-binding fragment as described in the first aspect of the present invention; and (II) A coupling portion conjugated to the antibody or its antigen-binding fragment, the coupling portion being selected from the group consisting of: detectable markers, drugs, or combinations thereof.

[0037] In another preferred embodiment, the expression for the antibody-drug conjugate is: mAb-(XY)n, in, mAb is an anti-human MPC antibody or its antigen-binding fragment; X is a connector; Y represents the coupling portion, which is a detectable marker or drug; N is a positive integer ≤ 8; The conjugation portion is conjugated to the anti-human MPC antibody or its antigen-binding fragment via a linker.

[0038] In another preferred embodiment, the detectable markers include: radioactive isotopes, fluorescent markers, and biological substrate markers.

[0039] A seventh aspect of the present invention provides a pharmaceutical composition comprising: (a) The anti-human MPC antibody or its antigen-binding fragment as described in the first aspect of the present invention, the recombinant protein as described in the second aspect of the present invention, the polynucleotide as described in the third aspect of the present invention, the vector as described in the fourth aspect of the present invention, the host cell as described in the fifth aspect of the present invention, or the antibody-drug conjugate as described in the sixth aspect of the present invention; and (b) Pharmaceutically acceptable carriers, diluents or excipients.

[0040] In another preferred embodiment, the pharmaceutical composition is used to prepare a formulation or kit for treating diseases caused by MPC overexpression.

[0041] The eighth aspect of the present invention provides the use of the anti-human MPC antibody or its antigen-binding fragment as described in the first aspect of the present invention, the recombinant protein as described in the second aspect of the present invention, the polynucleotide as described in the third aspect of the present invention, the vector as described in the fourth aspect of the present invention, or the host cell as described in the fifth aspect of the present invention for the preparation of reagents, detection plates, and kits.

[0042] A ninth aspect of the present invention provides a method for preparing the anti-human MPC antibody or its antigen-binding fragment as described in the first aspect of the present invention, the method comprising the steps of: (s1) Under suitable expression conditions, host cells as described in the fifth aspect of the present invention are cultured to express the anti-human MPC antibody or its antigen-binding fragment.

[0043] In another preferred embodiment, the method further includes the step of: (s2) Separate and purify the anti-human MPC antibody or its antigen-binding fragment obtained in step (s1).

[0044] A tenth aspect of the present invention provides a method for detecting MPC protein in a sample, the method comprising the steps of: (y1) Contact the sample with the anti-human MPC antibody or its antigen-binding fragment as described in the first aspect of the present invention; (y2) Detect whether an antigen-antibody complex is formed, wherein if an antigen-antibody complex is formed, it indicates that the MPC protein is present in the sample.

[0045] In another preferred embodiment, the sample includes: animal tissue sample and exfoliated cell sample.

[0046] In another preferred embodiment, the sample comprises a tissue sample from a human.

[0047] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0048] In another preferred embodiment, the method is an in vitro method.

[0049] In another preferred embodiment, the method further includes step (y3) analyzing the affinity between the antibody and the antigen.

[0050] In another preferred embodiment, the MPC protein is a human MPC protein.

[0051] In an eleventh aspect of the present invention, a detection plate is provided, the detection plate comprising a substrate (support plate) and a test strip, the test strip containing the anti-human MPC antibody or its antigen-binding fragment as described in the first aspect of the present invention, the recombinant protein as described in the second aspect of the present invention, or the antibody-drug conjugate as described in the sixth aspect of the present invention.

[0052] In another preferred embodiment, the test strip also contains an antigen spotting area.

[0053] In another preferred embodiment, the test strip is composed of filter paper, chromatography material, nitrocellulose membrane and absorbent paper stacked in sequence.

[0054] A twelfth aspect of the present invention provides a kit comprising: (1) A first container containing the anti-human MPC antibody or its antigen-binding fragment as described in the first aspect of the present invention; and / or (2) A second container containing a second antibody against the anti-human MPC antibody or its antigen-binding fragment as described in the first aspect of the present invention; and / or (3) A third container containing a cell lysis reagent; or, The kit contains the detection plate described in the tenth aspect of the present invention.

[0055] In another preferred embodiment, the antibody or antigen-binding fragment in the first container is labeled with a detectable tag.

[0056] In another preferred embodiment, the second antibody in the second container is labeled with a detectable marker.

[0057] The thirteenth aspect of the present invention provides the use of the anti-human MPC antibody or its antigen-binding fragment as described in the first aspect of the present invention, the recombinant protein as described in the second aspect of the present invention, the polynucleotide as described in the third aspect of the present invention, the vector as described in the fourth aspect of the present invention, the host cell as described in the fifth aspect of the present invention, the detection plate as described in the eleventh aspect of the present invention, and the kit as described in the twelfth aspect of the present invention for detecting and labeling MPC proteins, and / or for structural and functional studies or drug development involving MPC proteins.

[0058] The fourteenth aspect of the present invention provides a method for treating a disease associated with MPC overexpression, comprising the steps of administering to a subject a therapeutically effective amount of the anti-human MPC antibody or its antigen-binding fragment as described in the first aspect of the present invention, the recombinant protein as described in the second aspect of the present invention, the polynucleotide as described in the third aspect of the present invention, the carrier as described in the fourth aspect of the present invention, the antibody-drug conjugate as described in the sixth aspect of the present invention, or the pharmaceutical composition as described in the seventh aspect of the present invention.

[0059] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0060] Figure 1 The prepared MPC1 and MPC2 protein SDS-PAGE images are shown.

[0061] Figure 2 The sorting flow cytometry of the control group (A) and the experimental group (B) is shown.

[0062] Figure 3 The heavy chain vector of human IgG used to construct the antibody is shown.

[0063] Figure 4 The light chain vector of human IgG used to construct the antibody is shown.

[0064] Figure 5 The graph shows the MFI values ​​of the ID2 antibody gradient and MPC protein binding by flow cytometry (ID2 and SJID2 refer to the same antibody).

[0065] Figure 6 The negative control group is shown as the MPC antigen and FACS flow cytometry binding profile (using beads medium); the negative control group was incubated with secondary antibody after adding MPC antigen but no antibody, with FACS solution as a control.

[0066] Figure 7 The flow cytometry plots of positive antibody ID2 (SJID2) binding to MPC antigen and the flow cytometry plots of MPC antigen and MPC-Fab antibody (ID2) (using bead media) are shown. In the positive control group, MPC antigen was added, followed by the addition of Fab antibody with a Strep tag, and then FITC Strep Tag II Antibody was added for detection.

[0067] Figure 8 The graph shows the MFI values ​​of the ID5 antibody gradient and MPC protein binding as detected by flow cytometry (ID5 and SJID5.5 refer to the same antibody).

[0068] Figure 9 The negative control group is shown as the MPC antigen and FACS flow cytometry binding profile (using beads medium); the negative control group was incubated with secondary antibody after adding MPC antigen but no antibody, with FACS solution as a control.

[0069] Figure 10 The flow cytometry plots of positive antibody ID5 (SJID5.5) binding to MPC antigen are shown. The flow cytometry plots of MPC antigen and MPC-Fab antibody (ID5) binding (using bead media) are also shown. In the positive control group, MPC antigen was added, followed by the addition of Fab antibody with a Strep tag, and then FITC Strep Tag II Antibody was added for detection. Detailed Implementation

[0070] It should be understood that the specific methods and experimental conditions of the present invention described below in varying degrees of detail are intended to provide a substantial understanding of the invention. Definitions of certain terms used in this specification are provided below. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0071] the term Through extensive and in-depth research and numerous screenings, the inventors unexpectedly obtained two anti-MPC antibodies. Experimental results demonstrate that the anti-MPC antibodies of this invention possess extremely high affinity, specificity, and good biological activity for MPC. These antibodies can be used for the specific binding and detection of human MPC. Based on this, the present invention was completed.

[0072] Where a numerical range is provided, unless the context clearly indicates otherwise, it should be understood that every intermediate integer of the value, every tenth of every intermediate integer of the value, any other intermediate value between the upper and lower limits of the range, and any other intermediate value within the specified range are included within the scope of this invention. The upper and lower limits of these smaller ranges may be independently included within the smaller range and also covered within the scope of this invention, but are subject to any express exclusions within the specified range. For example, "1 to 50" includes "2 to 25", "5 to 20", "25 to 50", "1 to 10", etc.

[0073] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed-ended. In other words, the terms also include “consistently made up of” or “composed of”.

[0074] As used herein, the term “and / or” refers to and covers any and all possible combinations of one or more of the related listed items.

[0075] As used herein, the terms "anti-human MPC antibody of the present invention or antigen-binding fragment thereof", "MPC antibody of the present invention" and "human MPC antibody of the present invention" are used interchangeably and all refer to antibodies against human MPC.

[0076] MPC The mitochondrial pyruvate carrier (MPC), composed of two heterodimeric membrane proteins (mitochondrial pyruvate carrier 1 (MPC1, SLC54A1) and mitochondrial pyruvate carrier 2 (MPC2, SLC54A2)), is a specific protein complex located within the mitochondrial membrane; when overexpressed, it can also be expressed on the cell membrane. The MPC is the only channel for the transfer of pyruvate from the mitochondrial interstitium to the mitochondrial matrix. Within the mitochondria, pyruvate is converted to acetyl-CoA by the pyruvate dehydrogenase complex. Acetyl-CoA enters the tricarboxylic acid cycle (TCA cycle), participating in metabolism and energy production. Abnormal pyruvate metabolism due to MPC dysfunction is associated with cancer, heart failure, and type II diabetes. Furthermore, the nervous system requires a large amount of energy to maintain its function; therefore, mitochondrial energy supply is closely related to nerve function. Mitochondrial dysfunction can induce or exacerbate brain lesions. MPC is an important target for treating related diseases. In-depth research on its functional structure and regulatory mechanisms not only reveals the role of MPC in related diseases, but also provides a more effective approach for the diagnosis and treatment of these diseases.

[0077] This invention, through the preparation of MPC monoclonal antibodies, not only provides a new tool for MPC scientific research and disease diagnosis, but also opens up new avenues for new drug development. With further research, anti-MPC monoclonal antibodies are expected to demonstrate their unique value and broad prospects in more fields.

[0078] Antibody This invention provides an MPC antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment has three complementary determinant HCDRs in the heavy chain variable region and three complementary determinant liquid crystal regions (LCDRs) in the light chain variable region: (1) HCDR1 as shown in SEQ ID NO:59, HCDR2, as shown in SEQ ID NO:60, HCDR3, as shown in SEQ ID NO:61, LCDR1, as shown in SEQ ID NO:63, LCDR2, as shown in SEQ ID NO:64, and LCDR3, as shown in SEQ ID NO:65; (2) HCDR1 as shown in SEQ ID NO:69, HCDR2, as shown in SEQ ID NO:70, HCDR3, as shown in SEQ ID NO:71, LCDR1, as shown in SEQ ID NO:73, LCDR2, as shown in SEQ ID NO:74, and LCDR3 as shown in SEQ ID NO:75.

[0079] In this invention, the terms "antibody (Ab)" and "immunoglobulin G (IgG)" refer to heterotetraglycoproteins with the same structural characteristics, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by a constant region, which consists of three domains: CH1, CH2, and CH3. Each light chain has a variable region (VL) at one end and a constant region at the other end, with the light chain constant region including a domain CL; the light chain constant region pairs with the CH1 domain of the heavy chain constant region, and the light chain variable region pairs with the heavy chain variable region. Constant regions do not directly participate in antibody-antigen binding, but they exhibit different effector functions, such as participating in antibody-dependent cell-mediated cytotoxicity (ADCC). Heavy chain constant regions include IgG1, IgG2, IgG3, and IgG4 isotypes; light chain constant regions include κ (Kappa) or λ (Lambda). The heavy and light chains of an antibody are covalently linked by disulfide bonds between the CH1 domain of the heavy chain and the CL domain of the light chain. The two heavy chains of an antibody are covalently linked by interpeptide disulfide bonds formed between their hinge regions.

[0080] In this invention, the terms "Fab" and "Fc" refer to the ability of papain to cleave an antibody into two identical Fab fragments and one Fc fragment. The Fab fragment consists of the VH and CH1 domains of the antibody's heavy chain and the VL and CL domains of its light chain. The Fc fragment, or crystallizable fragment, consists of the antibody's CH2 and CH3 domains. The Fc fragment lacks antigen-binding activity and is the site of interaction between the antibody and effector molecules or cells.

[0081] In this invention, the term "scFv" refers to a single-chain antibody fragment (scFv), which is composed of the variable regions of the antibody heavy chain and the variable regions of the light chain, typically linked by a short peptide (linker) of 15 to 25 amino acids.

[0082] In this invention, the term "variable" refers to the fact that certain portions of the variable region in an antibody differ in sequence, resulting in the binding and specificity of various specific antibodies to their specific antigens. However, variability is not uniformly distributed throughout the entire variable region of the antibody. It is concentrated in three segments within the variable regions of the heavy and light chains, known as complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portions of the variable regions are called frame regions (FRs). The variable regions of the natural heavy and light chains each contain four FR regions, which are generally β-sheet configurations, linked by three CDRs forming a linking loop, and in some cases may form a partial β-sheet structure. The CDRs in each chain are closely packed together through the FR regions and together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)).

[0083] As used herein, the term "frame region" (FR) refers to the amino acid sequence inserted between CDRs, that is, those portions of the variable regions of the light and heavy chains of immunoglobulins that are relatively conserved among different immunoglobulins within a single species. Each of the light and heavy chains of an immunoglobulin has four FRs, designated L-FR1, L-FR2, L-FR3, L-FR4 and H-FR1, H-FR2, H-FR3, H-FR4, respectively. Accordingly, the light chain variable domain can therefore be designated (L-FR1)-(LCDR1)-(L-FR2)-(LCDR2)-(L-FR3)-(LCDR3)-(L-FR4), and the heavy chain variable domain can therefore be represented as (H-FR1)-(HCDR1)-(H-FR2)-(HCDR2)-(H-FR3)-(HCDR3)-(H-FR4). Preferably, the FR of the present invention is a human antibody FR or a derivative thereof, wherein the derivative of the human antibody FR is substantially the same as the naturally occurring human antibody FR, that is, the sequence identity reaches 85%, 90%, 95%, 96%, 97%, 98% or 99%.

[0084] Knowing the amino acid sequence of the CDR, those skilled in the art can easily determine the framework regions L-FR1, L-FR2, L-FR3, L-FR4 and / or H-FR1, H-FR2, H-FR3, H-FR4.

[0085] As used herein, the term "human frame region" is a frame region that is substantially identical (approximately 85% or more, specifically 90%, 95%, 97%, 99%, or 100%) to the frame region of a naturally occurring human antibody.

[0086] As used herein, the term "linker" refers to an insertion into an immunoglobulin domain that provides sufficient mobility for the light and heavy chains to fold into one or more amino acid residues of an exchangeable dual variable region immunoglobulin. In this invention, preferred linkers are Linker1 and Linker2, wherein Linker1 links the VH and VL of a single-chain antibody (scFv), while Linker2 is used to link the scFv to the heavy chain of another antibody.

[0087] Suitable examples of linkers include monoglycine (Gly) or serine (Ser) residues, and the identification and sequence of amino acid residues in the linker can vary depending on the type of secondary structural element that needs to be achieved in the linker.

[0088] In this invention, the antibody also includes its conserved variants, which are polypeptides formed by replacing up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids with amino acids of similar or analogous properties compared to the amino acid sequence of the specific antibody of this invention. These conserved variant polypeptides are preferably generated by amino acid substitutions according to Table A.

[0089] Table A In this invention, the terms "antibody," "binding," and "specific binding" refer to a non-random binding reaction between two molecules, such as the reaction between an antibody and its targeted antigen. Typically, antibodies bind to the antigen with an equilibrium dissociation constant (KD) less than about 10⁻⁷ M, for example, less than about 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹ M, or even smaller. In this invention, the term "KD" refers to the equilibrium dissociation constant of a specific antibody-antigen interaction, which describes the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the stronger the antibody-antigen binding, and the higher the affinity between the antibody and the antigen. For example, the binding affinity between the antibody and the antigen can be determined using surface plasmon resonance (SPR) in a BIACORE instrument or using an ELISA to determine the relative affinity of antibody-antigen binding.

[0090] In this invention, the term "epitope" refers to a polypeptide determinant that specifically binds to an antibody. The epitopes of this invention are regions of an antigen that are bound to antibodies.

[0091] In some embodiments, the anti-pig CD19 antibody (or anti-pig B cell surface CD19 antibody) of this invention is immobilized on a solid support or substrate. In some embodiments, the anti-pig CD19 antibody of this invention is non-diffusionally immobilized on the solid support (e.g., the anti-pig CD19 antibody does not detach from the solid support). The solid support or substrate can be any physically separable solid on which the anti-pig CD19 antibody can be directly or indirectly attached, including but not limited to surfaces provided by microarrays and pores, and particles such as beads (e.g., paramagnetic beads, magnetic beads, microbeads, nanobeads), microparticles, and nanoparticles. Solid supports may also include, for example, chips, columns, optical fibers, wipes, filters (e.g., planar filters), one or more capillaries, glass and modified or functionalized glass (e.g., controlled-pore glass (CPG)), quartz, mica, diazotized membranes (paper or nylon), polyoxymethylene, cellulose, cellulose acetate, paper, ceramics, metals, metalloids, semiconductor materials, quantum dots, coated beads or particles, other chromatographic materials, magnetic particles; plastics (including acrylics, polystyrene, copolymers of styrene or other materials, polybutene, polyurethane, TEFLON™, polyethylene, polypropylene, polyamide, polyester, polyvinylidene fluoride (PVDF), etc.), polysaccharides, nylon or nitrocellulose, resins, silica or silica-based materials (including silicon, silica gel and modified silicon), carbon, metals (e.g., steel, gold, silver, aluminum, silicon and copper), inorganic glass, conductive polymers (including polymers such as polypyrrole and polyindole); micro or nanostructured surfaces such as nucleic acid tiling arrays. Nanotubes, nanowires, or nanoparticles decorate surfaces; or porous surfaces or gels such as methacrylates, acrylamide, sugar polymers, cellulose, silicates, or other fibrous or chain polymers. In some embodiments, the solid support or substrate may be coated with any number of materials, including polymers such as dextran, acrylamide, gelatin, or agarose, using a passive or chemically derived coating. Beads and / or particles may be free or connected to each other (e.g., sintered). In some embodiments, the solid support or substrate may be an aggregate of particles. In some embodiments, the particles may comprise silica, and the silica may comprise silicon dioxide. In some embodiments, the silica may be porous, and in some embodiments, the silica may be non-porous. In some embodiments, the particles also comprise a substance that imparts paramagnetism to the particles. In some embodiments, the substance comprises a metal, and in some embodiments, the substance is a metal oxide (e.g., iron or iron oxide, wherein the iron oxide contains a mixture of Fe2+ and Fe3+). Antibodies against porcine CD19 can be linked to solid supports via covalent bonds or non-covalent interactions, and can be linked to solid supports directly or indirectly (e.g., via intermediates such as spacer molecules or biotin).

[0092] The antibodies of the present invention can be used in any known assay method, such as flow cytometry, immunohistochemistry, immunofluorescence, mass cytometry (e.g., Cytof instruments), competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays.

[0093] Flow cytometry and mass flow cytometry assays involve using a single primary antibody to specifically recognize the presence of a target molecule expressed on the surface of a dispersion suspension of individual cells. The dispersed cells are typically obtained from biological fluid samples (e.g., blood) or can be obtained as single-cell dispersions prepared from free solid tissue samples (e.g., spleen, lymph node, or tumor biopsies). The primary antibody can be directly conjugated to a detectable moiety (e.g., a fluorophore such as phycoerythrin for flow cytometry or a heavy metal chelate for mass flow cytometry). Alternatively, the primary antibody can be unlabeled or labeled with an undetectable tag such as biotin, and then detected by a detectable secondary antibody that specifically recognizes the primary antibody itself or the tag on the primary antibody. The labeled cells are then analyzed in an instrument capable of single-cell detection (e.g., flow cytometer, mass flow cytometer, fluorescence microscope, or bright-field optical microscope) to identify target cells expressed in a dispersed population or tissue sample that are recognized by the primary antibody.

[0094] Sandwich assays involve the use of two antibodies, each capable of binding to a different immunogenic moiety or epitope of the protein being tested. In a sandwich assay, the test sample analyte binds to a first antibody immobilized on a solid support, followed by the binding of a second antibody to the analyte, forming an insoluble three-part complex. The second antibody itself can be labeled with the detectable moiety (direct sandwich assay) or can be measured using an anti-immunoglobulin antibody labeled with the detectable moiety (indirect sandwich assay). For example, one type of sandwich assay is an ELISA assay, in which the detectable moiety is an enzyme. In cell ELISA, a target cell population is attached to a solid support using antibodies that first attach to the support and recognize different cell surface proteins. These first antibodies capture the cells onto the support. CD19 on the cell surface can then be detected by adding an anti-porcine CD19 antibody to the captured cells and detecting the amount of CD19 antibody attached to the cells.

[0095] For immunohistochemistry, blood or tissue samples can be fresh or frozen, or embedded in paraffin and fixed with preservatives such as formalin. Antibodies can also be used for in vivo diagnostic assays. Typically, antibodies are labeled with radionuclides (such as 111In, 99Tc, 14C, 131I, 125I, 3H, 32P, or 35S) so that the bound target molecules can be located using immunoscintillation imaging.

[0096] Detection / diagnostic kits containing the anti-CD19 antibody of the present invention may, for convenience, be provided in a kit (e.g., a packaged combination of a predetermined amount of reagents and instructions for use in a diagnostic assay). When the antibody is labeled with a fluorophore, the kit will include an isotype-independent negative control antibody identical to the control that nonspecifically binds to the anti-CD19 antibody. When the antibody is labeled with an enzyme, the kit will include the substrate and cofactor required for the enzyme (e.g., a substrate precursor that can detect chromophores or fluorophores). Additionally, other additives may be included, such as stabilizers, buffers (e.g., blocking buffers or lysis buffers), etc. The relative amounts of various reagents can be widely varied to provide reagent solution concentrations that greatly optimize assay sensitivity. In particular, the reagents may be provided as dry powders (typically lyophilized), which include excipients that, upon dissolution, provide a reagent solution with an appropriate concentration.

[0097] Polynucleotides, vectors and host cells The present invention also provides a polynucleotide molecule encoding the aforementioned antibody or a fragment thereof or a recombinant protein thereof. The polynucleotide of the present invention may be in DNA or RNA form. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand.

[0098] The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence that encodes only the mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence of the mature polypeptide (and optional additional coding sequences) and a non-coding sequence.

[0099] The term "polynucleotide encoding a polypeptide" can refer to a polynucleotide that includes the polypeptide, or it can also include additional coding and / or non-coding sequences.

[0100] The present invention also relates to polynucleotides that hybridize with the above-described sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that hybridize with the polynucleotides described herein under stringent conditions. In the present invention, “stringent conditions” means: (1) hybridization and elution at lower ionic strength and higher temperatures, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with a denaturing agent, such as 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the identity between the two sequences is at least 90%, preferably at least 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0101] The full-length nucleotide sequence or fragments of the antibody of the present invention can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis. One feasible method is to synthesize the relevant sequence artificially, especially when the fragment length is short. Typically, long fragments can be obtained by first synthesizing multiple small fragments and then ligating them. Furthermore, the coding sequence of the heavy chain and an expression tag (such as 6His) can be fused together to form a recombinant protein.

[0102] Once the relevant sequence is obtained, it can be obtained in large quantities using recombinant methods. This typically involves cloning it into a vector, transforming it into cells, and then isolating the sequence from the proliferated host cells using conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in this invention include biomolecules existing in isolated forms.

[0103] Currently, the DNA sequence encoding the protein of the present invention (or a fragment thereof, or a derivative thereof) can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of the present invention through chemical synthesis.

[0104] The present invention also relates to vectors comprising the aforementioned suitable DNA sequences and suitable promoters or control sequences. These vectors can be used to transform suitable host cells to enable them to express proteins.

[0105] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; and animal cells of CHO, COS7, and 293 cells.

[0106] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as *E. coli*, competent cells capable of uptake DNA can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0107] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of this invention. Depending on the host cells used, the culture medium can be selected from various conventional media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature adjustment or chemical induction), and the cells are cultured for a further period.

[0108] The recombinant peptides used in the methods described above can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be separated and purified using various separation methods based on their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0109] The antibodies of the present invention can be used alone or in combination or conjugated with detectable markers (for diagnostic purposes), therapeutic agents, PK (protein kinase) modified parts, or any combination of the above substances.

[0110] Detectable markers for diagnostic purposes include, but are not limited to: fluorescent or luminescent markers, radioactive markers, biological substrate markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing detectable products. Radioactive markers are radioisotopes such as 35S, 14C, 125I, 3H, and 131I. Antibodies can be labeled with radioisotopes using techniques described, for example, those described in Current Protocols in Immunology, Volumes 1 and 2, edited by Coligen et al., Wiley-Interscience, New York, N.Y., Pubs. (1991), and the radioactivity can be measured using scintillation counting. Fluorescent markers include rare earth chelates (europium chelates) or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, erythroline, phycoerythrin, Texas red, and BrilliantViolet™. Fluorescent markers can be conjugated to antibodies using techniques disclosed, for example, those disclosed in Current Protocols in Immunology (ibid.). Fluorescence can be quantified using flow cytometry, imaging microscopy, or a fluorometer. Biological substrate markers, utilizing enzyme-substrate labeling systems such as the biotin-avidin system, serve as detection agents. Enzymes typically catalyze chemical changes in their substrates, which can be measured using various techniques. For example, enzymes can catalyze color changes in substrates, which can be measured spectrophotometrically. Alternatively, enzymes can alter the fluorescence or chemiluminescence of a substrate. Techniques for quantifying fluorescence changes are described above. Chemiluminescent substrates become electronically excited through a chemical reaction and then emit light that can be measured (e.g., using a chemiluminescence meter) or supply energy to a fluorescent acceptor. Examples of enzyme-labeled enzymes include luciferases (e.g., firefly luciferase and bacterial luciferase); luciferin, 2,3-dihydrophthalazinedione, malate dehydrogenase, urease, peroxidases (e.g., horseradish peroxidase (HRP)), alkaline phosphatase, β-galactosidase, glucosylamylase, lysozyme, sugar oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (e.g., uricase and xanthine oxidase), lactoperoxidase, microperoxidase, etc.

[0111] Therapeutic agents that can bind to or conjugate with the antibodies of the present invention include, but are not limited to: 1. radionuclides; 2. biotoxicants; 3. cytokines such as IL-2; 4. gold nanoparticles / nanorobars; 5. viral particles; 6. liposomes; 7. magnetic nanoparticles; 8. prodrug-activating enzymes (e.g., DT-cardiac flavin enzyme (DTD) or biphenyl hydrolase-like protein (BPHL)); 10. chemotherapeutic agents (e.g., cisplatin) or any form of nanoparticles. For example, by encapsulating the antibodies of the present invention into liposomes, cell delivery may be possible; furthermore, by conjugating the antibodies of the present invention with localization signals, intracellular mitochondrial delivery may be possible.

[0112] Antibody-drug conjugates (ADCs) The present invention also provides antibody-drug conjugates (ADCs) based on the antibodies of the present invention.

[0113] Typically, the antibody-drug conjugate comprises an antibody and an effector molecule, wherein the antibody is conjugated to the effector molecule, preferably chemically conjugated. The effector molecule is preferably a drug with therapeutic activity. Furthermore, the effector molecule may be one or more of a toxic protein, a chemotherapeutic agent, a small molecule drug, or a radionuclide.

[0114] The antibody and the effector molecule of the present invention can be coupled via a coupling agent. Examples of the coupling agent include any one or more of non-selective coupling agents, carboxyl-based coupling agents, peptide chains, and disulfide bonds. The non-selective coupling agent refers to a compound that covalently links the effector molecule and the antibody, such as glutaraldehyde. The carboxyl-based coupling agent can be any one or more of maleic aconitine-based coupling agents (e.g., maleic aconitine) and acylhydrazone-based coupling agents (with an acylhydrazone as the coupling site).

[0115] Certain residues on antibodies (such as Cys or Lys) are used to link to a variety of functional groups, including imaging reagents (e.g., chromophores and fluorophores), diagnostic reagents (e.g., MRI contrast agents and radioisotopes), stabilizers (e.g., ethylene glycol polymers), and therapeutic agents. Antibodies can be conjugated to functional agents to form antibody-functional agent conjugates. Functional agents (e.g., drugs, detection reagents, stabilizers) are conjugated (covalently linked) to antibodies. Functional agents can be directly attached to antibodies or indirectly through linkers.

[0116] Antibodies can be conjugated to drugs to form antibody-drug conjugates (ADCs). Typically, an ADC contains a linker between the drug and the antibody. The linker can be degradable or non-degradable. Degradable linkers are typically readily degraded in intracellular environments, such as at the target site, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzyme-degradable linkers, including peptide-containing linkers that can be degraded by intracellular proteases (such as lysosomal proteases or endosomal proteases), or sugar linkers, such as glucuronidase-containing linkers. Peptide linkers can include, for example, dipeptides, such as valine-citrulline, phenylalanine-lysine, or valine-alanine. Other suitable degradable linkers include, for example, pH-sensitive linkers (such as linkers that hydrolyze at pH less than 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (such as disulfide linkers). Non-degradable linkers typically release the drug under conditions where the antibody is hydrolyzed by proteases.

[0117] Prior to attachment to the antibody, the linker has a reactive group capable of reacting with certain amino acid residues, and the attachment is achieved through the reactive group. Thiol-specific reactive groups are preferred and include, for example, maleimide compounds, haloamides (e.g., iodinated, brominated, or chlorinated); haloesters (e.g., iodinated, brominated, or chlorinated); halomethyl ketones (e.g., iodinated, brominated, or chlorinated); benzyl halides (e.g., iodinated, brominated, or chlorinated); vinyl sulfones; pyridyl disulfides; mercury derivatives such as 3,6-di-(mercurymethyl)dioxane, with the counter ion being acetate, chloride, or nitrate; and polymethylene dimethyl sulfide thiosulfonate. The linker may include, for example, a maleimide attached to the antibody via a thiosuccinimide.

[0118] The drug can be any cytotoxic, cell growth-inhibiting, or immunosuppressive drug. Preferably, the linker connects the antibody and the drug, and the drug has a functional group that can bond with the linker. For example, the drug may have an amino, carboxyl, thiol, hydroxyl, or ketone group that can bond with the linker. In the case where the drug is directly linked to the linker, the drug has a reactive group before being linked to the antibody.

[0119] Useful drug classes include, for example, anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, etc. In this invention, the drug-linker can be used to form an ADC in a single, simple step. In other embodiments, bifunctional linker compounds can be used to form an ADC in two or more steps. For example, cysteine ​​residues react with the reactive portion of the linker in a first step, and in subsequent steps, functional groups on the linker react with the drug to form an ADC.

[0120] Typically, functional groups on the linker are selected to facilitate specific reaction with suitable reactive groups on the drug moiety. As a non-limiting example, azide-based moieties can be used to specifically react with reactive alkynyl groups on the drug moiety. The drug is covalently bound to the linker via a 1,3-dipolar cycloaddition between the azide and alkynyl groups. Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphines (suitable for reaction with azides); isocyanates and isothiocyanates (suitable for reaction with amines and alcohols); and activated esters, such as N-hydroxysuccinimide esters (suitable for reaction with amines and alcohols). These and other linking strategies, such as those described in Bioconjugation Techniques, Second Edition (Elsevier), are well known to those skilled in the art. Those skilled in the art will understand that for selective reaction between the drug moiety and the linker, when a complementary pair of reactive functional groups is selected, each member of that complementary pair can be used for either the linker or the drug.

[0121] The present invention also provides a method for preparing an ADC, which may further include: binding an antibody to a drug-adaptor compound under conditions sufficient to form an antibody-drug conjugate (ADC).

[0122] In some embodiments, the method of the present invention includes binding an antibody to a bifunctional adapter compound under conditions sufficient to form an antibody-adaptor conjugate. In these embodiments, the method of the present invention further includes binding the antibody-adaptor conjugate to a drug moiety under conditions sufficient to covalently link a drug moiety to the antibody via the adapter.

[0123] In some implementations, the antibody-drug conjugate (ADC) has the following molecular formula: in: Ab is an antibody. LU stands for connector; D is a drug; Furthermore, the subscript p is a value selected from 1 to 8.

[0124] Detection uses and kits The antibodies of this invention can be used in detection applications, such as for testing samples, to provide diagnostic information.

[0125] In this invention, the samples used include cells, tissue samples, and biopsy specimens. The term "biopsy" as used in this invention should include all types of biopsies known to those skilled in the art. Therefore, biopsies used in this invention can include, for example, tissue samples prepared by endoscopic methods or puncture or needle biopsy of organs.

[0126] The samples used in this invention include fixed or preserved cell or tissue samples.

[0127] The present invention also provides a kit containing the antibody (or fragment thereof) of the present invention. In a preferred embodiment of the present invention, the kit further includes a container, instructions for use, a buffer, etc. In a preferred embodiment, the antibody of the present invention can be immobilized on a detection plate.

[0128] Application of the antibody of this invention The anti-human MPC antibody of the present invention can be used for the study of MPC structure and function, as well as for the specific detection of human MPC protein in in vitro experiments, and can be used for drug development involving human MPC.

[0129] The main advantages of this invention include: (1) The anti-MPC antibody of the present invention can be used to label human MPC protein, and then can be used in various detection and analysis methods of human MPC protein in vitro, thereby specifically analyzing the specific binding of human MPC antigen.

[0130] (2) The anti-MPC antibody of the present invention has strong specificity for MPC protein and strong binding ability.

[0131] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0132] Example 1: Obtaining native conformation of human MPC protein Those skilled in the art, using conventional techniques, constructed the full-length human MPC1 gene sequence (Bgee: ENSG00000060762) and MPC2 gene sequence (Bgee: ENSG00000143158) into mammalian cell expression vectors, such as the pUC57 vector. Exogenous expression was performed using HEK293 cells to obtain large quantities of MPC1 and MPC2 protein samples. The expressed proteins were purified using conventional biochemical techniques, with optimized purification conditions to preserve the native conformation of the proteins as much as possible. Subsequently, the purified MPC1 and MPC2 proteins were packaged using Amphipol reagent to form an MPC complex, maximally mimicking the native conformation of MPC proteins on the mitochondrial membrane surface. This is intended for subsequent use as an antigen to immunize mice, etc. Alternatively, natural full-length human MPC protein can be purchased. Figure 1 This is an SDS-PAGE image of the prepared MPC1 and MPC2 proteins.

[0133] Example 2: Antigen preparation and mouse immunization, mouse B cell sorting The MPC protein obtained in Example 1 was used to immunize mice using conventional and known techniques. The immunization strategy was as follows: 10-12 week old C57BL / 6 mice were selected and divided into an immunization group and a control group. In the immunization group, each mouse was injected intraperitoneally and into the tarsal joint with 30 µg of the MPC protein (antigen) prepared in Example 1 and 20 µg of CpG OND 2395 (5'-tcgtcgttttcggcgcgcgccg-3') and an equal volume of AddaVax™ (InvivoGen). The control group was injected with 20 µg of CpG OND 2395 and an equal volume of AddaVax™. The mice were immunized once every 3-4 days for 4-6 times.

[0134] Sorting of mouse B cells: 3-7 days after the last immunization, mice in both the experimental and control groups were sacrificed, and spleen and lymph node tissues were obtained. Single-cell suspensions were prepared from the spleen and lymph nodes (popliteal fossa and groin), and then red blood cells were removed using erythrocyte lysis buffer (Sangon Biotech Cat.b541001-0100). After washing twice with 2% FBS in PBS (FACS buffer), the cells were stained. Subsequently, a Cytoflex SRT (Beckman Coulter) cell sorting system was used to sort the cells to obtain the target cells using the following staining gating strategy: DAPI-CD19+CD38+GL7-IgG1+.

[0135] The following reagents were used: DAPI (live / dead; Invitrogen Cat. D1306), anti-mouse CD19 (APC / Cyanine7; Biolegend Cat.115530), anti-mouse CD38 (PE / Cyanine7; Biolegend Cat.102718), anti-mouse / human GL7 Antigen (T and B cell Activation Marker), AlexaFluor® 488; Biolegend Cat.144612); anti-mouse IgG1 (APC; Biolegend Cat.406610).

[0136] Example results of flow sorting are as follows Figure 2 As shown in the figure, the control group yielded very few IgG1+ positive B cells (0.22%), while the experimental group, after immunization, obtained a large number of IgG1+ positive B cells (1.07%). The antigen-immunized group showed a significant immune response compared to the control group. Based on these results, this immunization protocol can effectively induce a sufficient number of antigen-specific memory B cells and germinal center B cells, and the sorting strategy can efficiently enrich antigen-specific memory B cells.

[0137] Example 3: Culture of mouse B cells The IgG1-positive B cells obtained from the above sorting were cultured. The culture method can be referred to Hanida and Kitamura, (2019). Induced Germinal Center B Cell Culture System, Bio-protocol 9 (4): e3163. DOI: 10.21769 / BioProtoc.3163. The feeding medium was: RPMI-1640, (Gibco, Cat.11875-093), 10% fetal bovine serum (FBS, VivaCell, Cat. C04002-500), 55 μM 2-mercaptoethanol (Thermo Fisher, Cat.21985), 100 units / mL penicillin (Biosharp), 100 μg / mL streptomycin (Biosharp), 10 mM HEPES (Thermo Fisher, Cat.15630-080), 1 mM sodium pyruvate (Thermo Fisher, (Cat.11360-070) and 0.1 mM MEM non-essential amino acids (Thermo 5 Fisher, Cat.11140-050). One day before sorting, feeder cells expressing CD40L and BAFF were seeded into 96-well plates at 800-1200 cells per well. Single IgG1-positive B cells were sorted into each well, and 2-10 ng / mL IL-4 was added to the feeder medium and cultured for two days. Then, 4-10 ng / mL IL-21 was added and cultured for another 6-8 days, changing the medium daily. On day 10, the supernatant was collected and stored at 4°C. The 96-well plates were stored at -80°C for subsequent lysis to obtain RNA from single B cells.

[0138] In the experiment, sorting was performed three times, with 100 plates sorted each time for culture. Flow cytometry data showed that the target memory B cell population accounted for approximately 95.6%, with an IgG1 positivity rate of approximately 1.07%, indicating that the sorting strategy can efficiently sort memory B cells. PBS with 20 μg of CpG OND 2395 and an equal volume of AddaVax™ served as the immunization control group. Subsequently, approximately 50 pairs of monoclonal antibody VDJ / VJ sequences were obtained using molecular cloning methods. However, after further flow cytometry verification using bead media to bind the antigen and antibody surfaces, only two highly specific monoclonal antibodies were obtained. MPCs are multiple transmembrane proteins, and technically, it is difficult to obtain high-affinity specific antibodies for multiple transmembrane proteins. In this experiment, through extensive work and technical expertise, two high-affinity MPC monoclonal antibodies were successfully obtained.

[0139] Example 4: ELISA Experiment, Results and Analysis MPC peptide protein was coated onto 96-well ELISA plates and incubated overnight at 4°C under humid conditions. After discarding the coating buffer, 100-120 μL of blocking buffer (1×PBS containing 4% BSA) was added to the plate and incubated at room temperature for 2 hours. After removing the blocking buffer, 20-60 μL of the antibody expression supernatant paired with the clones in Example 3 (antibody expression was prepared using conventional techniques in the art) was added to each well and incubated overnight at 4°C under humid conditions. After washing three times, 20-30 μL of secondary antibody (AKP goat anti-mouse IgG1, Southern Biotech, cat.1030-04) was added to each well and incubated at room temperature for 2 hours. After washing four times, 20-30 μL of chromogenic buffer containing disodium 4-nitrophenyl phosphate hexahydrate (CSNpharm, Cat.CSN66207) was added to each well. OD405 was measured using an MD SpectraMaxiD3. The results are shown in Tables 1-4.

[0140] Table 1. OD405 results of IgG1 positive controls on plate 1 Table 2. OD405 results of plate 1 antigen detection Table 3. OD405 results of IgG1 positive controls on plate 2 Table 4. OD405 results of plate 2 antigen detection The results show that the ELISA test results indicate that the supernatant of the cultured IgG1+ positive B cells contains antibodies that specifically bind to the MPC antigen. The color development varies due to differences in antibody binding strength or antibody concentration in the culture supernatant. Antigen-antibody specific binding will show a high value. After multiple batches of ELISA tests, nearly 10 highly specific antigen-antibody binding antibodies were obtained in all batches (this study used 300 96-well ELISA test plates; the table above does not provide all data, only some representative test values).

[0141] Example 5: Molecular cloning method and obtaining the monoclonal antibody VDJ / VJ sequence Based on the ELISA test results analysis of Example 4, cells from the positive wells (antigen-antibody binding) of a 96-well cell culture plate that were pre-frozen at -80℃ were selected for RNA extraction from the single B cells, and subsequent molecular cloning was performed to obtain the VDJ / VJ sequence.

[0142] Total RNA was extracted from B cells in 96-well cell culture plates that tested positive (antigen-antibody binding) using TRIzol Reagent (Thermo Fisher). Reverse transcription and PCR were performed according to existing techniques (Thomas Tilleret et al., *Journal of Immunological Methods*, 2009). In short, cDNA synthesis was performed using Maxima H Minus reverse transcriptase (Thermo Fisher) at 42°C, 5 min, 25°C, 10 min, 50°C, 60 min, and 94°C. Two rounds of semi-nested PCR were then performed using HotStar DNA polymerase (Qiagen) to enrich the heavy and light chains. The PCR products were purified and sequenced. Sequencing results were analyzed using IgBlast and the IMGT database. The VDJ / VJ fragment was amplified using gene-specific primers (Table 6), and the VDJ heavy chain and VJ light chain vectors were cloned using homologous recombination or T4 ligase ligation.

[0143] The primers used for the two-round semi-nested PCR are shown in Table 5. The PCR program was: 95℃ for 15 minutes, 95℃ for 30 seconds, 50-65℃ for 30 seconds, and 72℃ for 5 minutes.

[0144] Reverse transcription to cDNA: Add the following reagents sequentially to Mix 1: 1.4 µL RNase-free water; 1 µL 10 mM dNTP; 1 µL 100 µM Random Primers; 1 µL template RNA. Incubate at 65°C for 5 min. Add the following reagents sequentially to Mix 2: 4 µL 5×RT buffer; 0.1 µL RNase inhibitor; 0.25 µL reverse transcriptase; 11.25 µL RNase-free water. Combine Mix 1 and Mix 2 for reverse transcription PCR program: 42°C for 5 min; 25°C for 10 min; 50°C for 60 min; 85°C for 5 min; store at 4°C.

[0145] Table 5. Semi-nested PCR primers Table 6. Upstream and downstream primers for amplifying the VDJ / VJ fragment The PCR reaction systems involved in this embodiment are shown in Table 7-12. Each reaction system can be scaled up or down proportionally according to the required reaction system.

[0146] Table 7. Heavy chain first-round PCR reaction system Table 8. Heavy chain second-round PCR reaction system Table 9. Kappa light chain first-round PCR reaction system Table 10. Second-round PCR reaction system for Kappa light chain Table 11. Lambda light chain first-round PCR reaction system Table 12. Lambda light chain second-round PCR reaction system The primer mixes mentioned above for the heavy chain, Kappa chain, and lambda light chain refer to the mixture of the corresponding primers in List 5 above in the first and second rounds of semi-nested PCR for the corresponding heavy chain, Kappa chain, and lambda light chain. For example, in the IgK 1st PCR, 5′L-Vk mix Fw is a mixture of 5L-Vκ_3, 5L-Vκ_4, 5L-Vκ_5, 5L-Vκ_6, 5L-Vκ_6-8-9, 5L-Vκ_14, 5L-Vκ_19, and 5L-Vκ_20, with each primer at 10 μM. The mixes in other PCR systems are explained accordingly.

[0147] The VDJ / VJ-specific primers in this embodiment include homologous arms of the heavy / light chain vector, as well as specific portions of the VDJ / VJ fragment. Generally, the homologous arms of these specific primers will vary depending on the cloning vector used. This application is not limited to the specific primers used in the embodiments.

[0148] After reverse transcription and two rounds of semi-nested PCR, the nucleotide and amino acid sequences of the VDJ / VJ variable region of the antibody were sequenced to obtain antibodies numbered ID2 and ID5, whose sequences are shown in Table 13.

[0149] Table 13. MPC antibody sequences The vector map of the antibody for MPC prepared using the heavy chain constant region and light chain constant region vector of human IgG1 in this embodiment is shown in [reference needed]. Figure 3 and Figure 4 .

[0150] The sequences of the heavy and light chains of the humanized antibody prepared in this embodiment are shown in Table 14.

[0151] Table 14. Humanized antibody sequences Example 6: Detection of MPC antigen and antibody bead binding In this embodiment, the successfully paired antibody heavy and light chain plasmids were transfected into 293F cells for expression and purification using conventional techniques in the art, for subsequent experimental use. In the experiment, polystyrene Protein A (Spherotech) microspheres were resuspended in 1×PBS at room temperature, centrifuged, and then resuspended with 10 μg / mL human anti-flag protein antibody. The mixture was stirred and incubated for 15 minutes, washed twice with 1×PBS, and incubated for 1-2 hours with 2 μg / mL MPC-2% FACS solution. After washing twice with 2% FACS solution, 50 μL of Fab antibody diluted in 2% FACS solution was added, and incubation was performed for 1 hour. After washing twice with 1×PBS, 50 μL of secondary antibody (Monoclonal Mouse Strep Tag II Antibody, LSBio, Cat. LS-C203631) was added, and the mixture was incubated on ice for 15 minutes. All procedures were performed on ice. Flow cytometry analysis and MFI analysis were performed using a CytoFLEX LX (Beckman) system.

[0152] The result of ID2 is as follows Figure 5-7 As shown; the ID5 result is as follows Figure 8-10 As shown. Among them, for Figure 5 and Figure 8 The specific MFI numerical statistics of the eight gradient dilution points for flow cytometry analysis of antibody binding to MPC protein are shown in Table 15.

[0153] Table 15. MFI values ​​at eight serial dilution points for antibody-MPC protein binding as determined by flow cytometry. All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. An anti-human MPC antibody or its antigen-binding fragment, characterized in that, The antibody or its antigen-binding fragment has the following three complementarity-determining regions (HCDR) of the heavy chain variable region and three complementarity-determining regions (LCDR) of the light chain variable region: HCDR1, as shown in SEQ ID NO:69, HCDR2, as shown in SEQ ID NO:70, HCDR3, as shown in SEQ ID NO:71, LCDR1, as shown in SEQ ID NO:73, LCDR2, as shown in SEQ ID NO:74, and LCDR3 as shown in SEQ ID NO:

75.

2. The anti-human MPC antibody or its antigen-binding fragment as described in claim 1, characterized in that, The heavy chain variable region and light chain variable region of the anti-human MPC antibody or its antigen-binding fragment are the heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:68, and the light chain variable region with an amino acid sequence as shown in SEQ ID NO:

72.

3. A recombinant protein, characterized in that, The recombinant protein has the following characteristics: (1) The anti-human MPC antibody or its antigen-binding fragment as described in claim 1; and (2) Optional tag sequence for expression and / or purification.

4. A polynucleotide, characterized in that, The polynucleotide expression is the anti-human MPC antibody of claim 1 or its antigen-binding fragment.

5. A carrier, characterized in that, The carrier contains the polynucleotide as described in claim 4.

6. A host cell, characterized in that, The host cell expresses the anti-human MPC antibody of claim 1 or its antigen-binding fragment, and its genome is integrated with the polynucleotide of claim 4, or contains the vector of claim 5.

7. An antibody-drug conjugate, characterized in that, The antibody-drug conjugate contains: (I) Antibody portion, said antibody portion comprising the anti-human MPC antibody of claim 1 or its antigen-binding fragment; and (II) A coupling portion conjugated to the antibody or its antigen-binding fragment, the coupling portion being selected from the group consisting of: detectable markers, drugs, or combinations thereof.

8. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains: (a) The anti-human MPC antibody of claim 1 or its antigen-binding fragment, the recombinant protein of claim 3, the polynucleotide of claim 4, the vector of claim 5, the host cell of claim 6, or the antibody-drug conjugate of claim 7; and (b) Pharmaceutically acceptable carriers, diluents or excipients.

9. The use of the anti-human MPC antibody or its antigen-binding fragment according to claim 1, the recombinant protein according to claim 3, the polynucleotide according to claim 4, the vector according to claim 5, or the host cell according to claim 6, characterized in that, Used for preparing reagents, test plates, and kits.

10. A method for detecting MPC protein in a sample, characterized in that, The method includes the following steps: (y1) Contact the sample with the anti-human MPC antibody or its antigen-binding fragment as described in claim 1; (y2) Detect whether an antigen-antibody complex is formed, wherein if an antigen-antibody complex is formed, it indicates that the MPC protein is present in the sample.

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