Monoclonal antibodies targeting human cd70 and uses thereof

Abstract

The application provides a monoclonal antibody targeting human CD70 and application thereof. Specifically, the application obtains a group of monoclonal antibodies targeting human CD70 from B cells of mice immunized by the application, the monoclonal antibodies have high affinity with CD70 protein on the surface of cells, and can block the interaction between CD27 protein and CD70 protein. In addition, the application also provides a fusion protein, CAR-T / NK / macrophage, immunoconjugate and the like containing the antibody, and the CAR-T cell has specific killing function to cells expressing CD70.

Description

Technical Field

[0001] This invention belongs to the field of antibody technology, and more specifically, this invention relates to a monoclonal antibody targeting human CD70 and its application. Background Technology

[0002] CD70, a member of the tumor necrosis factor superfamily, exhibits abnormally high expression in various malignant tumors, including lung cancer, renal cell carcinoma, hematological malignancies (such as acute myeloid leukemia and diffuse large B-cell lymphoma), and central nervous system gliomas. Its expression level is significantly correlated with tumor cell proliferation, invasion and metastasis, and poor patient prognosis. Based on this characteristic, CD70 can serve as a novel biomarker for early cancer diagnosis, providing molecular evidence for precise clinical diagnosis. Simultaneously, its specific expression in tumor tissues makes it a key target for targeted therapy and disease prognosis monitoring. Currently, CD70-targeted therapies have entered the clinical stage in hematological malignancies and renal cell carcinoma, while related research on solid tumors such as breast cancer, ovarian cancer, and glioblastoma has also made progress in preclinical development, demonstrating broad clinical application potential.

[0003] Furthermore, in the field of allogeneic universal cell therapy (such as Universal CAR-T / CAR-NK), allogeneic cells are highly susceptible to recognition and rejection by the recipient host's immune system (such as allogeneic reactive T cells and NK cells), leading to rapid clearance of foreign cells in vivo and limiting their therapeutic durability and clinical efficacy. Studies have shown that activated immune cells in the host highly express CD70 when performing their rejection function. Therefore, developing CD70 antibodies with high affinity and specificity not only directly targets and eliminates CD70-positive tumor cells but also holds promise for overcoming immune rejection by targeting and eliminating activated CD70+ lymphocytes in the host, thus providing a novel strategy to improve the durability and efficacy of allogeneic universal cell therapy in vivo.

[0004] Currently, research on CD70 treatment still has many limitations, and there is an urgent need to develop new CD70 drugs and expand their clinical applications. Summary of the Invention

[0005] The purpose of this invention is to provide a treatment method for diseases that target CD70.

[0006] In a first aspect of the invention, an antibody or antigen-binding fragment thereof targeting CD70 is provided, characterized in that the antibody or antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region and the light chain variable region are selected from the group consisting of: (1) Heavy chain variable regions containing the following three CDRs: HCDR1, as shown in the amino acid sequence of SEQ ID NO. 91, HCDR2, as shown in the amino acid sequence of SEQ ID NO. 92, HCDR3, as shown in the amino acid sequence of SEQ ID NO. 93; and Light chain variable regions containing the following three CDRs: LCDR1, as shown in the amino acid sequence of SEQ ID NO. 94, LCDR2, as shown in the amino acid sequence of SEQ ID NO. 95, LCDR3, as shown in the amino acid sequence of SEQ ID NO. 96; or (2) Heavy chain variable regions containing the following three CDRs: HCDR1, as shown in the amino acid sequence of SEQ ID NO. 81, HCDR2, as shown in the amino acid sequence of SEQ ID NO. 82, HCDR3, as shown in the amino acid sequence of SEQ ID NO. 83; and Light chain variable regions containing the following three CDRs: LCDR1, as shown in the amino acid sequence of SEQ ID NO. 84, LCDR2, as shown in the amino acid sequence of SEQ ID NO. 85, LCDR3, as shown in the amino acid sequence of SEQ ID NO. 86; or (3) Heavy chain variable regions containing the following three CDRs: HCDR1, as shown in the amino acid sequence of SEQ ID NO. 71, HCDR2, as shown in the amino acid sequence of SEQ ID NO. 72, HCDR3, as shown in the amino acid sequence of SEQ ID NO. 73; and Light chain variable regions containing the following three CDRs: LCDR1, as shown in the amino acid sequence of SEQ ID NO. 74, LCDR2, as shown in the amino acid sequence of SEQ ID NO. 75, LCDR3, as shown in the amino acid sequence of SEQ ID NO. 76; or (4) Heavy chain variable regions containing the following three CDRs: HCDR1, as shown in the amino acid sequence of SEQ ID NO. 61, HCDR2, as shown in the amino acid sequence of SEQ ID NO. 62, HCDR3, as shown in the amino acid sequence of SEQ ID NO. 63; and Light chain variable regions containing the following three CDRs: LCDR1, as shown in the amino acid sequence of SEQ ID NO. 64, LCDR2, as shown in the amino acid sequence of SEQ ID NO. 65, LCDR3, as shown in the amino acid sequence of SEQ ID NO. 66; or (5) Heavy chain variable regions containing the following three CDRs: HCDR1, as shown in the amino acid sequence of SEQ ID NO. 51, HCDR2, as shown in the amino acid sequence of SEQ ID NO. 52, HCDR3, as shown in the amino acid sequence of SEQ ID NO. 53; and Light chain variable regions containing the following three CDRs: LCDR1, as shown in the amino acid sequence of SEQ ID NO. 54, LCDR2, as shown in the amino acid sequence of SEQ ID NO. 55, LCDR3, as shown in the amino acid sequence of SEQ ID NO. 56; or (6) Heavy chain variable regions containing the following three CDRs: HCDR1, as shown in the amino acid sequence of SEQ ID NO. 41, HCDR2, as shown in the amino acid sequence of SEQ ID NO. 42, HCDR3, as shown in the amino acid sequence of SEQ ID NO. 43; and Light chain variable regions containing the following three CDRs: LCDR1, as shown in the amino acid sequence of SEQ ID NO. 44, LCDR2, as shown in the amino acid sequence of SEQ ID NO. 45, LCDR3, as shown in the amino acid sequence of SEQ ID NO. 46; or (7) Heavy chain variable regions containing the following three CDRs: HCDR1, as shown in the amino acid sequence of SEQ ID NO. 31, HCDR2, as shown in the amino acid sequence of SEQ ID NO. 32, HCDR3, as shown in the amino acid sequence of SEQ ID NO. 33; and Light chain variable regions containing the following three CDRs: LCDR1, as shown in the amino acid sequence of SEQ ID NO. 34, LCDR2, as shown in the amino acid sequence of SEQ ID NO. 35, LCDR3, as shown in the amino acid sequence of SEQ ID NO. 36; or (8) Heavy chain variable regions containing the following three CDRs: HCDR1, as shown in the amino acid sequence of SEQ ID NO. 21, HCDR2, as shown in the amino acid sequence of SEQ ID NO. 22, HCDR3, as shown in the amino acid sequence of SEQ ID NO. 23; and Light chain variable regions containing the following three CDRs: LCDR1, as shown in the amino acid sequence of SEQ ID NO. 24, LCDR2, as shown in the amino acid sequence of SEQ ID NO. 25, LCDR3, as shown in the amino acid sequence of SEQ ID NO. 26; or (9) Heavy chain variable regions containing the following three CDRs: HCDR1, as shown in the amino acid sequence of SEQ ID NO. 11, HCDR2, as shown in the amino acid sequence of SEQ ID NO. 12, HCDR3, as shown in the amino acid sequence of SEQ ID NO. 13; and Light chain variable regions containing the following three CDRs: LCDR1, as shown in the amino acid sequence of SEQ ID NO. 14, LCDR2, as shown in the amino acid sequence of SEQ ID NO. 15, LCDR3, as shown in the amino acid sequence of SEQ ID NO. 16; or (10) Heavy chain variable regions containing the following three CDRs: HCDR1, as shown in the amino acid sequence of SEQ ID NO. 1, HCDR2, as shown in the amino acid sequence of SEQ ID NO. 2, HCDR3, as shown in the amino acid sequence of SEQ ID NO. 3; and Light chain variable regions containing the following three CDRs: LCDR1, as shown in the amino acid sequence of SEQ ID NO. 4, LCDR2, as shown in the amino acid sequence of SEQ ID NO. 5, LCDR3, as shown in the amino acid sequence of SEQ ID NO. 6.

[0007] In another preferred embodiment, the heavy chain variable region (VH) sequence of the antibody or its antigen-binding fragment is as shown in SEQ ID NO. 7, 17, 27, 37, 47, 57, 67, 77, 87, or 97, or has at least 90% sequence identity with the sequence shown in SEQ ID NO. 7, 17, 27, 37, 47, 57, 67, 77, 87, or 97.

[0008] In another preferred embodiment, the light chain variable region (VL) sequence of the antibody or its antigen-binding fragment is as shown in SEQ ID NO. 9, 19, 29, 39, 49, 59, 69, 79, 89, or 99, or has at least 90% sequence identity with the sequence shown in SEQ ID NO. 9, 19, 29, 39, 49, 59, 69, 79, 89, or 99.

[0009] In another preferred embodiment, the antibody further includes a constant region.

[0010] In another preferred embodiment, the constant region of the antibody is a humanized constant region.

[0011] In another preferred embodiment, the heavy chain variable region includes the three heavy chain CDRs and the human-derived heavy chain framework region for connecting the heavy chain CDRs.

[0012] In another preferred embodiment, the light chain variable region includes the three light chain CDRs and a human-sourced light chain framework region for connecting the light chain CDRs.

[0013] In another preferred embodiment, the antibody or its antigen-binding fragment is selected from the group consisting of: Fab fragment, Fab' fragment, F(ab)'2 fragment, F(ab)'3 fragment, Fv, single-chain Fv antibody (“scFv”), bisscFv, (scFv)2, microantibody, bifunctional antibody, trifunctional antibody, tetrafunctional antibody, disulfide-stabilized Fv protein (“dsFv”), or combinations thereof.

[0014] In another preferred embodiment, the antibody is a murine antibody, a murine-human chimeric antibody, or a humanized IgG1 antibody.

[0015] In another preferred embodiment, the antibody is a monovalent antibody, a bivalent antibody, a trivalent antibody, or a multivalent antibody.

[0016] In another preferred embodiment, the antibody includes monospecific, bispecific, trispecific, or multispecific antibodies.

[0017] In a second aspect, the present invention provides a fusion protein comprising: (i) an antibody or antigen-binding fragment thereof as described in the first aspect of the invention; and (ii) The portion fused with the antibody or its antigen-binding fragment, including tag sequences, signal peptides, and functional peptides that assist in expression and / or purification.

[0018] In another preferred embodiment, the tag sequence includes a 6His tag, a GGGS sequence, and a FLAG tag.

[0019] In another preferred embodiment, the fusion protein is a monospecific antibody, a bispecific antibody, or a multispecific antibody (such as a trispecific antibody).

[0020] In another preferred embodiment, the bispecific or multispecific antibody not only targets CD70 but also specifically binds to additional target antigens.

[0021] In another preferred embodiment, the fusion protein is a monomer, a dimer, or a polymer.

[0022] In a third aspect, the present invention provides a polynucleotide that encodes an antibody or an antigen-binding fragment thereof as described in the first aspect of the present invention, or a fusion protein as described in the second aspect of the present invention.

[0023] In a fourth aspect, the present invention provides a carrier containing the polynucleotide described in the third aspect of the present invention.

[0024] In another preferred embodiment, the vector is selected from the group consisting of plasmids, viruses (such as lentiviruses, adenoviruses, AAV viruses, retroviruses), granules, or combinations thereof.

[0025] In another preferred embodiment, the vector is a plasmid, preferably an expression vector.

[0026] In another preferred embodiment, the vector is a viral vector.

[0027] In a fifth aspect, the present invention provides a host cell containing the vector described in the fourth aspect of the present invention, or having an exogenous polynucleotide described in the third aspect of the present invention integrated into its genome.

[0028] In another preferred embodiment, the cells are isolated cells, and / or the cells are genetically engineered cells.

[0029] In another preferred embodiment, the cell is a somatic cell.

[0030] In another preferred embodiment, the cell is a mammalian cell.

[0031] In another preferred embodiment, the cell is an immune cell (such as an NK cell or a T cell).

[0032] In another preferred embodiment, the host cell is an engineered cell.

[0033] In a sixth aspect, the present invention provides a chimeric antigen receptor (CAR) whose antigen-binding domain contains a single-chain variable region sequence scFv targeting CD70, wherein the heavy chain variable region and light chain variable region contained in the scFv are as defined in the first aspect of the present invention, or the heavy chain sequence and light chain sequence contained in the scFv are as defined in the first aspect of the present invention.

[0034] In a seventh aspect, the present invention provides a CAR-NK cell, a CAR-T cell, or a CAR-macrophage, or a method for preparing the CAR-NK cell, CAR-T cell, or CAR-macrophage, wherein the CAR-NK cell, CAR-T cell, or CAR-macrophage expresses the chimeric antigen receptor described in the sixth aspect of the present invention.

[0035] In another preferred embodiment, the preparation method includes the following steps: The polynucleotide described in the third aspect of the present invention or the vector described in the fourth aspect of the present invention is introduced into NK cells, T cells, or macrophages to obtain the CAR-NK cells, CAR-T cells, or CAR-macrophages.

[0036] In an eighth aspect, the present invention provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, a recombinant protein as described in the second aspect of the present invention, a polynucleotide as described in the third aspect of the present invention, a carrier as described in the fourth aspect of the present invention, a host cell as described in the fifth aspect of the present invention, or a CAR-NK cell, CAR-T cell, or CAR-macrophage as described in the seventh aspect of the present invention, or a combination thereof, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0037] In another preferred embodiment, the pharmaceutical composition is a formulation, preferably a liquid formulation.

[0038] In another preferred embodiment, the dosage form of the pharmaceutical composition is an injection.

[0039] In a ninth aspect, the present invention provides an immunoconjugate comprising: (a) An antibody portion, said antibody portion being selected from the group consisting of: antibodies or antigen-binding fragments thereof as described in the first aspect of the invention, recombinant proteins as described in the second aspect of the invention, or combinations thereof; and (b) A conjugation portion conjugated to the antibody portion, the conjugation portion being selected from the group consisting of: detectable markers, drugs, toxins, cytokines, radionuclides, enzymes, or combinations thereof.

[0040] In another preferred embodiment, the conjugate is selected from: fluorescent or luminescent markers, radiolabels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes, radionuclides, biotoxins, cytokines (such as IL-2), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorobars, viral particles, liposomes, magnetic nanoparticles, prodrug-activating enzymes (e.g., DT-cardiacinase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (e.g., cisplatin), or any form of nanoparticles.

[0041] In a tenth aspect, the present invention provides the use of an antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, a recombinant protein as described in the second aspect of the present invention, a polynucleotide as described in the third aspect of the present invention, a vector as described in the fourth aspect of the present invention, a host cell as described in the fifth aspect of the present invention, a CAR-NK cell or CAR-T cell or CAR-macrophage as described in the seventh aspect of the present invention, a pharmaceutical composition as described in the eighth aspect of the present invention, or an immunoconjugate as described in the ninth aspect of the present invention, for the preparation of a drug or formulation, wherein the drug or formulation is used for: (a) Treatment of CD70-positive diseases or diseases with CD70 overexpression; and / or (b) In vitro detection of CD70 protein; and / or (c) Reduce immune rejection of allogeneic cell therapy products in subjects, thereby improving product durability and / or efficacy.

[0042] In another preferred embodiment, the allogeneic cell therapy product includes universal CAR-T and CAR-NK.

[0043] In another preferred embodiment, the disease is selected from: acute myeloid leukemia (AML), multiple myeloma, lymphoma, renal cell carcinoma (RCC), glioblastoma (GBM), systemic lupus erythematosus (SLE), systemic sclerosis (SSC), and primary Sjögren's syndrome (pSS).

[0044] In an eleventh aspect of the present invention, a method for preparing an anti-CD70 antibody is provided, the method comprising: (a) Culturing the host cells described in the fifth aspect of the present invention under suitable expression conditions; (b) Isolating an antibody from a culture, wherein the antibody is the antibody or antigen-binding fragment thereof described in the first aspect of the present invention.

[0045] In a twelfth aspect of the invention, a kit for detecting CD70 protein is provided, the kit comprising an antibody or an antigen-binding fragment thereof as described in the first aspect of the invention as a first detection reagent.

[0046] In another preferred embodiment, the kit further includes an instruction manual describing a method for detecting CD70 protein.

[0047] In another preferred embodiment, the kit comprises: A first container, and an antibody or antigen-binding fragment thereof as described in the first aspect of the present invention located in the first container.

[0048] In a thirteenth aspect of the invention, a treatment method for a disease caused by CD70 overexpression is provided, the method comprising administering to a subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof as described in the first aspect of the invention, a recombinant protein as described in the second aspect of the invention, a polynucleotide as described in the third aspect of the invention, a vector as described in the fourth aspect of the invention, a host cell as described in the fifth aspect of the invention, a CAR-NK cell or CAR-T cell or CAR-macrophage as described in the seventh aspect of the invention, a pharmaceutical composition as described in the eighth aspect of the invention, or an immunoconjugate as described in the ninth aspect of the invention.

[0049] 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

[0050] Figure 1 A flowchart of the mouse immunization process is shown.

[0051] Figure 2 The results show the detection of antibody titers in mouse serum after immunization using an enzyme-linked immunosorbent assay (ELISA).

[0052] Figure 3 The results show the detection of serum antibody titers in immunized mice using flow cytometry (FACS).

[0053] Figure 4 The agarose gel electrophoresis diagram of the PCR products of heavy and light chains is shown.

[0054] Figure 5 The SDS-PAGE results of ten monoclonal antibodies are shown.

[0055] Figure 6 The SEC-HPLC results for ten monoclonal antibodies are shown.

[0056] Figure 7 The EC50 results of flow cytometry assays were shown to detect the binding activity of ten monoclonal antibodies to CD70-expressing cells.

[0057] Figure 8 The results of affinity assays for CD70 and CD27 proteins are shown.

[0058] Figures 9a-9e The results show the affinity assays of antibodies numbered clone2, clone3, clone4, clone9, and clone10 for competing binding of CD27 protein with CD70 protein.

[0059] Figure 10 The IC50 results of antibody blocking the interaction between CD27 and CD70 proteins are shown for clones numbered 2, 3, 4, 9, and 10.

[0060] Figure 11 The expansion fold of CAR-T cells constructed with different CD70 antibody sequences is shown.

[0061] Figure 12 The viability (%) of CAR-T cells constructed with different CD70 antibody sequences is shown.

[0062] Figure 13 The diameter variations of CAR-T cells constructed with different CD70 antibody sequences are shown.

[0063] Figure 14The study demonstrated the cytotoxic function of CAR-T cells constructed with different CD70 antibody sequences against target cells (Molm-13).

[0064] Figure 15 The study demonstrated the cytotoxic function of CAR-T cells constructed with different CD70 antibody sequences against target cells (CD70-KO Molm-13).

[0065] Figure 16 The study demonstrated the specific killing function of CAR-T cells constructed with different CD70 antibody sequences against CD70-positive target cells (Molm-13) after removing non-specific background. Detailed Implementation

[0066] Through extensive and in-depth research, the inventors unexpectedly obtained a group of antibodies with high affinity and high specificity targeting CD70. Experimental results show that the antibodies of this invention have a strong affinity for the CD70 protein on the cell surface and can also block the interaction between CD27 protein and CD70 protein through a competitive binding mechanism. Furthermore, CAR-T cells constructed based on the single-chain variable region fragment derived from the antibodies of this invention exhibit excellent cytotoxic function and performance.

[0067] the term To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Before describing this invention, it should be understood that it is not limited to the specific methods and experimental conditions described, as such methods and conditions can be varied.

[0068] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0069] CD70 is a member of the tumor necrosis factor (TNF) superfamily, a type II transmembrane glycoprotein with a molecular weight of approximately 50 kDa. Its structure includes an extracellular binding domain, a transmembrane region, and a cytoplasmic region. Given its structural homology with TNF-α, CD70 also exists in a trimeric form. Early studies showed that CD70 expression is mainly limited to T cells and B cells strongly activated by antigens, and its expression level decreases with decreasing antigen stimulation. Furthermore, some mature dendritic cells can also express CD70 under the induction of CD40 and Toll-like receptor signaling pathways. CD70 is also abnormally expressed in various hematologic malignancies and a significant number of solid tumors (such as nasopharyngeal carcinoma, thymic carcinoma, and brain tumors).

[0070] The receptor for CD70 is CD27, a type I transmembrane glycoprotein with a molecular weight of approximately 55 kDa. CD27 exists as a dimer formed by two single strands bonded together by disulfide bonds and belongs to the TNF receptor superfamily. CD27 is widely expressed on T cells, NK cells, and B cells. In addition to its membrane-bound form, CD27 can also detach from the cell membrane to form soluble CD27 (sCD27) with a molecular weight of 28–32 kDa.

[0071] Single-cell antibody screening technology involves isolating a B cell library from an immune individual, screening at the single-cell level to obtain candidate antibody genes, and ultimately expressing fully functional antibodies. Traditional antibody development techniques (such as hybridoma and phage display) suffer from drawbacks such as long development cycles, low efficiency, and difficulty in preserving the natural antibody variable region pairing. To overcome these limitations, high-throughput antibody screening technology based on single B cells has emerged. Among them, the Beacon platform achieves precise and efficient antibody discovery through optofluidic chips and optical tweezers. This technology can directly isolate and analyze individual antigen-specific B cells, preserve the natural pairing of heavy chains (VH) and light chains (VL), and rapidly obtain their encoding genes, significantly shortening the antibody development cycle. Compared to traditional methods, Beacon technology has the advantages of high throughput, high specificity, and fully human antibody screening, showing great potential in the fields of infectious diseases, tumor immunotherapy, and autoimmune diseases.

[0072] In this invention, "EC50" (half-maximum effective concentration) refers to the antibody concentration required to induce 50% of the maximum biological effect. This parameter is used to measure the efficacy of the antibody.

[0073] In this invention, "IC50" (half-maximum inhibitory concentration) refers to the compound concentration at which 50% of the maximum inhibitory effect is achieved. This indicator is often used to evaluate the bioactivity of inhibitors.

[0074] As used herein, the term "antibody" (Ab) includes, but is not limited to, immunoglobulins that specifically bind to antigens and comprise at least two heavy (H) chains and two light (L) chains linked by disulfide bonds, or their antigen-binding portions. Each H chain contains a heavy chain variable region (abbreviated VH) and a heavy chain constant region. The heavy chain constant region contains three constant domains CH1, CH2, and CH3. Each light chain contains a light chain variable region (abbreviated VL) and a light chain constant region. The light chain constant region contains one constant domain CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each VH and VL contains three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen.

[0075] As used in this article, the terms “heavy chain variable region” and “VH” are used interchangeably.

[0076] As used in this article, the terms “light chain variable zone” and “VL” are used interchangeably.

[0077] As used herein, the term "antigen-binding domain" and the like encompasses any naturally occurring, enzymatically available, synthetic, or genetically modified polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Any suitable standard technique, such as proteolytic digestion, or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable domains and optionally antibody constant domains, can be used, for example, deriving antigen-binding fragments of antibodies from intact antibody molecules. Such DNA is known and / or readily available from, for example, commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated chemically or using molecular biology techniques, such as to arrange one or more variable domains and / or constant domains into a suitable layout, or to introduce codons, generate cysteine ​​residues, modify, add, or delete amino acids, etc.

[0078] As used herein, non-limiting examples of antigen-binding fragments or antigen-binding domains include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues mimicking the hypervariable region of an antibody (e.g., independent complementarity-determining regions (CDRs) such as CDR3 peptides) or constrained FR3-CDR3-FR4 peptides.

[0079] As used herein, antigen-binding fragments or antigen-binding domains will generally contain at least one variable domain. The variable domain can have any size or amino acid composition and will typically contain at least one CDR adjacent to or conforming to one or more frame sequences. In antigen-binding fragments having a VH domain associated with a VL domain, the VH and VL domains can be arranged opposite each other in any suitable configuration. For example, the variable region can be a dimer and contain VH-VH, VH-VL, or VL-VL dimers. Optionally, the antigen-binding domain can contain monomeric VH or VL domains.

[0080] In a given antibody's light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any of a number of known antibody CDR assignment systems or combinations thereof, including, for example: Chothia based on the antibody's three-dimensional structure and the topology of the CDR loop; Kabat (Kabat, E., et al., USDapartment of Health and Human Services, Sequences of Proteins of Immunological Interest, (1983)) based on antibody sequence variability; AbM (University of Bath); Contact (University College London); the International Immuno GeneTics database (IMGT); the EU numbering system; and Chothia definitions based on loop structural positions.

[0081] It should be understood that the precise amino acid sequence boundaries of the CDR in this invention can optionally be defined using the different assignment systems mentioned above. Preferably, unless otherwise stated, in this invention, when referring to the position of residues in the antibody variable region (including heavy chain variable region residues and light chain variable region residues), it refers to the numbering position according to the Kabat numbering system.

[0082] As used herein, the terms "antibody" or "immunoglobulin" refer to isotetraglycoproteins of approximately 150,000 Daltons with identical structural features, 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, although the number of disulfide bonds between heavy chains varies among different immunoglobulin isotypes. 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 multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other; the constant regions of the light chains are opposite the first constant region of the heavy chains, and the variable regions of the light chains are opposite the variable regions of the heavy chains. Specific amino acid residues form interfaces between the variable regions of the light and heavy chains.

[0083] As used herein, the term "variable" refers to the fact that certain portions of the variable region of an antibody differ sequentially, contributing to the binding and specificity of a particular antibody to its specific antigen. However, variability is not uniformly distributed throughout the entire variable region of an antibody. It is concentrated in three segments within the variable regions of the light and heavy chains, known as complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portions of the variable region are called framework regions (FRs). The variable regions of the native heavy and light chains each contain four FRs, which are generally β-sheet configurations linked by three CDRs forming a linking loop, and in some cases, partially β-sheet structures. The CDRs in each chain are tightly packed together by 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)). Constant regions do not directly participate in antibody-antigen binding, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity.

[0084] The present invention also provides other proteins or fusion expression products having the antibodies of the present invention. Specifically, the present invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a heavy chain containing a variable region, provided that the variable region is the same as or has at least 90% homology with the heavy chain variable region of the antibody of the present invention, preferably at least 95% homology.

[0085] This invention includes not only complete antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Therefore, this invention also includes fragments, derivatives, and analogs of said antibodies.

[0086] As used herein, the terms “fragment,” “derivative,” and “analyte” refer to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention. The polypeptide fragments, derivatives, or analogs of the present invention may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) polypeptides having substituent groups in one or more amino acid residues; or (iii) polypeptides formed by fusing a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (e.g., a leader sequence or secretion sequence, or a sequence used to purify this polypeptide, or a proteogenic sequence, or a fusion protein formed with a 6His tag). Based on the teachings herein, these fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0087] The term "antibody of the present invention" refers to a polypeptide having CD70 binding activity and including the aforementioned CDR region. This term also includes variants of polypeptides containing the aforementioned CDR region that have the same function as the antibodies of the present invention. These variants include (but are not limited to): deletions, insertions, and / or substitutions of one or more amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10), and the addition of one or more amino acids (typically less than 20, preferably less than 10, more preferably less than 5) to the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids of similar or comparable properties generally does not alter the function of the protein. Similarly, the addition of one or more amino acids to the C-terminus and / or N-terminus generally does not alter the function of the protein. This term also includes active fragments and active derivatives of the antibodies of the present invention.

[0088] The variant forms of this polypeptide include: homologous sequences, conserved variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibody of the present invention under high or low severity conditions, and polypeptides or proteins obtained using antiserum against the antibody of the present invention. These conserved variant polypeptides are preferably generated by amino acid substitutions according to Table A.

[0089] Table A The present invention also provides a polynucleotide molecule encoding the above-described antibody or a fragment thereof or a fusion 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.

[0090] 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.

[0091] 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.

[0092] 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 fusion protein.

[0093] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination 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.

[0094] Currently, the DNA sequence encoding the protein of this 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 this invention through chemical synthesis.

[0095] 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.

[0096] 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.

[0097] 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.

[0098] 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.

[0099] 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.

[0100] 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.

[0101] Detectable markers for diagnostic purposes include, but are not limited to: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing detectable products.

[0102] Therapeutic agents that can bind to or conjugate with the antibodies of this 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 flavinase (DTD) or biphenyl hydrolase-like protein (BPHL)), etc.

[0103] The present invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition containing the aforementioned antibody or its active fragment or fusion protein or its ADC or corresponding immune cell, and a pharmaceutically acceptable carrier. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5-8, preferably about 6-8, although the pH value may vary depending on the nature of the formulated substance and the condition to be treated.

[0104] The prepared pharmaceutical composition can be administered via conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or local administration. Typically, the pharmaceutical composition of the present invention is preferably administered by injection or oral administration. Injection administration preferably includes intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection, or subcutaneous injection. The pharmaceutical composition is in various dosage forms conventional in the art, preferably in solid, semi-solid, or liquid form, and can be an aqueous solution, non-aqueous solution, or suspension, more preferably tablets, capsules, granules, injections, or infusions.

[0105] The antibody described in this invention can also be expressed in cells by a nucleotide sequence for cell therapy, such as for chimeric antigen receptor T-cell immunotherapy (CAR-T).

[0106] The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-described monoclonal antibody (or conjugate thereof) of the present invention, and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other excipients. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 microgram / kg body weight to about 5 milligrams / kg body weight per day. Furthermore, the peptides of the present invention can also be used with other therapeutic agents.

[0107] In this invention, preferably, the pharmaceutical composition further includes one or more pharmaceutical carriers. The pharmaceutical carrier is a conventional pharmaceutical carrier in the art, and can be any suitable physiologically or pharmaceutically acceptable pharmaceutical excipient. The pharmaceutical excipient is a conventional pharmaceutical excipient in the art, preferably including pharmaceutically acceptable excipients, fillers, or diluents. More preferably, the pharmaceutical composition comprises 0.01–99.99% of the above-mentioned protein and 0.01–99.99% of the pharmaceutical carrier, where the percentage is a percentage by mass of the pharmaceutical composition.

[0108] In this invention, preferably, the dosage of the pharmaceutical composition is an effective amount, which is an amount capable of alleviating or delaying the progression of a disease, degenerative or damaging condition. The effective amount can be determined on an individual basis and will be partly based on considerations of the symptoms to be treated and the desired outcome. Those skilled in the art can determine the effective amount by using the aforementioned factors, such as individual baselines, and by using experiments not exceeding the conventional range.

[0109] When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to mammals. This safe and effective amount is typically at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 milligrams per kilogram of body weight. Preferably, the dose is about 10 micrograms per kilogram of body weight to about 20 milligrams per kilogram of body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of a skilled physician's expertise.

[0110] Compared with the prior art, the main advantages of the present invention include: 1. This invention, combined with the Beacon optical guidance platform, achieves efficient isolation, precise analysis, and specific screening of B cells at the single-cell level. Through screening positive single B cells, antibody nucleotide sequences capable of recognizing the CD70 protein were successfully obtained.

[0111] 2. This invention utilizes a mammalian cell expression system for the efficient expression and purification of antibodies. This expression system provides a post-translational modification environment highly similar to that of natural antibodies, resulting in monoclonal antibodies that are structurally and functionally closer to natural proteins, thus exhibiting superior biological activity and clinical application potential.

[0112] 3. The monoclonal antibody of this invention exhibits a strong binding affinity to the CD70 protein and effectively blocks the binding interaction between the CD27 and CD70 proteins. This provides new tools and ideas for targeted therapy and research in related diseases.

[0113] 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.

[0114] Reagents and Materials Freund's complete adjuvant (Sigma, CAT: F5881-6X10ML) Freund's incomplete adjuvant (Sigma, CAT: F5881-6X10ML) ACRO CD70 Protein (Bipsys, CAT: CDL-H52Da) Tris(hydroxymethyl)aminomethane (Sangon Biotech, CAT: 77-86-1) Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP (Global Genetics, CAT: US2108) LS Columns (Miltenyi, CAT: 130-042-401) ExpiFectamine™ 293 Transfection Kit (Thermo, CAT: A14524) Expi293™ expression medium (Gibco, CAT: A14351-01) MS Columns (Miltenyi, CAT: 130-042-201) Mini & MidiMACS™ Starting Kit (Miltenyi, CAT: 130-042-501) CD138+ Enrichment Kit, mouse (Miltenyi, CAT: 130-092-530) Wetting Additive (clear, 4°C) (x2) (BLI, CAT: 750-08091) Goat Anti Mouse IgG (Fcγ), AF647 (Jackson, CAT: 115-605-071) SA-Microbeads (Spherotech, CAT: SVP-60-5) Mouse Single Cell BCR IgG H / K Amplification Kit (Novizan, CAT: DD5101) DNA gel dye (Thermo, CAT: S33102) AO / PI (Countstar, CAT: RE010212) RPMIMedium 1640 basic (1×) (Gibco, CAT: C11875500BT) Fetal Bovine Serum (VivaCELL, CAT: C04001-500) AF647-conjugated Goat Anti-Mouse IgG, Fcγ Fragment Specific (Jackson, CAT: 115-605-071) D-PBS (Sangon Biotech, CAT: E607009-0500) APC anti-human CD70 Antibody (Biolegend, CAT: 355110) Buffer solution: HBS-EP+ buffer 10X (Cytiva, CAT: BR100826) pH 5.5 acetate buffer (Cytiva, CAT: BR100352) pH 1.5 glycine buffer (Cytiva, CAT: BR100354) pH 2.0 glycine buffer (Cytiva, CAT: BR100355) S-series CM5 chip (Cytiva, CAT: 29127557) TransAct, Human (miltenyi, CAT: 130-111-160) MACS LS Columns (miltenyi, CAT: 30-042-401) CytoSinct™ CD4 Nanobeads, human (Genscript, CAT: L00863-0.5) CytoSinct™ CD8 Nanobeads, human (Genscript, CAT: L00864-0.5) 0.5M EDTA solution (Invitrogen, CAT: AM9260G) 20% Human Serum Albumin Injection (Octapharma, CAT: S20180013) OptiVitro T cell SF medium (Excell, CAT: BA0032) OptiVitro T cell SFM supplement (Excell, CAT: BA0052) Recombinant Human IL-7 (novoprotein, CAT: GMP-CD47) Recombinant Human IL-15 (novoprotein, CAT: GMP-C016) Example 1. Immunization of mice and titer detection Immunogen preparation: The His-CD70 gene was synthesized and cloned into a vector. Mini-scale recombinant plasmid DNA was prepared for subsequent transfection. 293F cells were cultured in Gibco expression medium and Thermo's ExpiFectamine™ 293 transfection kit was used to express His-CD70 as a eukaryotic protein. The purified protein was then used as an immunogen.

[0115] Immunizing Balb / c mice: Immunization procedure as follows Figure 1 As shown, after the four immunizations, serum was collected and its potency was determined by enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FACS).

[0116] ELISA test for immunogenicity: (1) Coating: ACRO CD70 Protein was coated at 1 μg / mL, 100 μL / well, overnight at 4℃. The next day, the plate was washed (250 μL / well with washing buffer, washed 3 times) and spun dry (1000 rpm, 2 min).

[0117] (2) Sealing: Add 100 μL of sealing solution to each well, incubate at 37℃ for 1 hour, wash the plate and spin dry for later use.

[0118] (3) Primary antibody: The experimental serum was diluted 1 / 2000 and 2-fold in sequence. There were a total of 7 gradients. 100 μL was added to each well, and the plate was washed and dried at 37℃ for 1 h.

[0119] (4) Secondary antibody: The secondary antibody was diluted 1 / 5000, 100 μL / well was added, and the plate was washed and dried at 37℃ for 1 h.

[0120] (5) Color development termination: Add 100 μL of color development solution to each well and incubate at room temperature for 5 min; add 100 μL of termination solution to each well.

[0121] (6) Reading value: The value at 450nm detected by the microplate reader.

[0122] The results of ELISA for the antibody titers of mouse 4 immune serum are shown in Table 1 and 2. Figure 2 As shown: Table 1. Antibody titers in mouse serum The acceptable standard for mouse serum titer was that the OD450 value of diluted mouse serum was more than twice that of the control group. Table 1 and Figure 2 The results showed that the titers of the four immune immunizations were 32,000 for mice #1, #2, and #3, and 16,000 for serum #4.

[0123] FACS assay for immunogenicity: The experimental design scheme is shown in Table 2 below: Table 2: (1) Cell preparation: Count the cells from the cell suspension. Based on the counting results, collect the cells and centrifuge at 300g at 4℃ for 5 min, discarding the supernatant. Adjust the cell density to 3e6 / mL using buffer and transfer to a 96-well V plate, 3×10⁶ cells per well. 5 cells / 100uL.

[0124] (2) Serum incubation: Cell precipitation: Centrifuge the cells in the 96-well plate at 300g and discard the supernatant. Serum addition: Add the cells sequentially according to the experimental design and mix by pipetting. Incubation and washing: Incubate at room temperature for 30 min and wash twice with FACS Buffer.

[0125] (3) Fluorescent antibody incubation: Detection source-specific antibody: Add to the abundance verification group according to the antibody dilution ratio in the antibody instructions. Anti-IgG antibody: Add the prepared fluorescent secondary antibody to all serum incubation groups and part of the control group. Incubation and washing: Incubate at 4℃ for 60 min, and wash twice with FACS Buffer.

[0126] (4) BD Celesta on-machine testing.

[0127] FACS results of antibody titers in mouse 4-immune serum are as follows: Figure 3 As shown in the figure. Based on the MFI fluorescence value, there is a specific titer. Mice #4 and #1 have higher titers. Combined with the serum antibody titer results detected by ELISA, mice #1 can be selected for Beacon single B cell screening.

[0128] Example 2. Screening for single B-cell antibodies The experimental steps are as follows: Single B-cell isolation and Beacon screening: First, spleens from mice immunized with high titers as described in Example 1 were harvested, gently ground with a tissue grinder, and a single-cell suspension was prepared by filtering. Dead cells and debris were removed by Ficoll density gradient centrifugation. Subsequently, CD138 magnetic beads (Miltenyi kit) were used for positive selection enrichment of plasma cells to obtain a high-purity CD138+ population. The isolated plasma cells were loaded into a Beacon microfluidic chip (Nest1) according to the programmed procedure, and the reagent at position 2 was replaced with culture medium (CultureMedia) to infiltrate and calibrate the chip. Cell loading was completed in manual mode. After sealing the chip, the photoelectric positioning (OEP) program was run to introduce single cells into the nano-level chamber (NanoPen). The channels were flushed to remove excess cells, and cell distribution was confirmed by bright-field imaging. Next, microspheres coated with the target antigen and fluorescently labeled secondary antibody (detection channel: Cy5, exposure time 1800ms) were introduced into the Assay chip. After sealing, multiple rounds of dynamic imaging (3 min / round) were initiated to monitor the binding signal between the microspheres and the secreted antibody. Positive hits were screened using analysis software combined with manual verification. The detection was terminated when the number of hits and fluorescence intensity stabilized, and the channels were rinsed to prepare for the next round of experiments. Finally, target cells were precisely exported from the NanoPen to a 96-well plate containing lysis buffer (TCL + Oil) using OEP technology. The lysis products were collected by centrifugation (400g, 1 min, 20℃) for subsequent single-cell sequencing or antibody gene amplification.

[0129] Single-cell sequencing: After lysing single cells derived from Beacon, RNA was extracted from the cells. The RNA from positive B-cell cells was purified, and then subjected to reverse transcription and cDNA amplification. The antibody heavy chain variable region (VH) and light chain variable region (VL) were then amplified. The amplification products were sequenced and analyzed for VH and VL. The antibody heavy and light chain sequences were obtained.

[0130] like Figure 4 As shown, Beacon screening selected 24 single-cell samples, yielding 21 VH and 22 VL PCR products, with 21 pairs. All 21 VH / VL pairs have been sequenced. Sequencing of all 21 PCR products was successful.

[0131] Example 3. Monoclonal antibody expression and purification Ten pairs of monoclonal antibodies, selected through sequence alignment, had their heavy and light chain genes cloned into the vector pcDNA3.4. The heavy chain constant region of the antibodies was the mIgG1 subtype, and the light chain constant region was the mKappa subtype. The expression plasmid was transfected into CHO-S cells, and cell culture was performed using shake flasks or a bioreactor. The cell culture supernatant was collected and purified using a standard protein A affinity chromatography column. After purification, the antibody recombinant expression showed SDS-PAGE >95% and SEC-HPLC >95%. Results are as follows... Figure 5 , Figure 6 As shown. The obtained antibodies were stored at 80℃ for later use. Ten monoclonal antibodies were finally screened and numbered clone1, clone2, clone3, clone4, clone5, clone6, clone7, clone8, clone9, and clone10.

[0132] Example 4. Flow cytometry detection of the binding activity between monoclonal antibodies and target proteins (1) Two Jurkat cells and three CD70-Jurkat cells were resuscitated in liquid nitrogen and adjusted to the logarithmic growth phase using RPMI 1640 complete medium. CD70-Jurkat and Jurkat cells were placed in centrifuge tubes, centrifuged at 300×g for 5 min, and the supernatant was discarded. The cells were resuspended in FACS buffer (PBS + 2% FBS) at a density of 1.5×10⁻⁶ cells / mL. 6 / mL; Add 200μL of cell suspension to a 96-well plate, with a cell volume of 2×10⁶ cells / mL. 5 / well; 300×g, centrifuge for 5min, discard supernatant; (2) Primary antibody incubation: Ten monoclonal antibodies were serially diluted 3-fold, and 9 concentration gradients were prepared for each antibody: gradient (nM): 6666, 2222, 740.7, 246.9, 82.30, 8.230, 0.8230, 0.0823, 0.0082. According to the experimental group, the antibody dilution buffer of each concentration gradient was added, and the mixture was mixed by pipetting. The mixture was incubated at room temperature for 30 min, and washed twice with FACS Buffer (PBS + 2% FBS).

[0133] (3) Incubation of secondary antibody and direct label antibody: Add the 1:200 prepared secondary antibody to all primary antibody incubation groups; incubation and washing: incubate at 4℃ for 40 min, and wash twice with FACS Buffer.

[0134] (4) Resuspension: 180uL FACS Buffer per well for resuspension; flow cytometry for testing.

[0135] The results of flow cytometry analysis of the binding activity of ten monoclonal antibodies to their target proteins are as follows: Figure 7 As shown.

[0136] The EC50 (M) results of the binding activity between the monoclonal antibody and the target protein are shown in Table 3. Table 3: A smaller EC50 (M) value indicates higher binding activity between the monoclonal antibody and the target protein. For example... Figure 7 As shown in Table 3, all ten antibodies exhibited good binding activity. Among them, the monoclonal antibodies numbered clone2, clone3, clone5, clone8, and clone9 showed the highest binding activity to the target protein.

[0137] Example 5. Monoclonal antibody affinity detection Ligand coupling Ligand: CD70 protein Coupling buffer: 10mM Acetate 5.5 Affinity assay of ten antibodies to CD70 protein Preliminary experiment: Antibody concentration: 50 nM.

[0138] Flow rate: 30 μL / min Combination time: 120 s Dissociation time: 300 s Regeneration reagent: Glycine 2.0, 30S Antibody gradient dilution affinity assay experimental conditions: Antibody concentration: Based on the preliminary experiment, the initial concentration of clone 1 antibody was 50 nM, the initial concentration of clone 2, clone 4, and clone 6 antibodies was 100 nM, and the initial concentration of clone 3, clone 5, clone 7, clone 8, clone 9, and clone 10 antibodies was 200 nM. They were then continuously diluted in a 2-fold gradient, for a total of 7 concentration points.

[0139] Flow rate: 30 μL / min Combination time: 120s Dissociation time: 300s Regeneration reagent: Glycine 2.0, 30 seconds, twice The preliminary results of the affinity determination of ten plants are shown in Table 4: Table 4: The results of the affinity assay for ten strains using antibody gradient dilution are shown in Table 5. A KD(M) < 10⁻⁸ indicates good antibody affinity. Based on a comprehensive analysis of Tables 3, 4, and 5, the monoclonal antibodies numbered clone 2, clone 3, clone 4, clone 9, and clone 10 show good affinity values ​​and can be used for subsequent antibody-mediated blocking of CD27 protein binding to CD70 protein detection.

[0140] Example 6. Detection of recombination affinity between CD27 protein and CD70 Specific experimental procedures Ligand coupling Ligand: CD27 protein Coupling buffer: 10mM Acetate 5.5 Experimental conditions: Antibody concentration: The initial concentration of CD70 protein was 500 nM, and it was serially diluted in two-fold increments, with a total of 8 concentration points.

[0141] Flow rate: 30 μL / min Combination time: 120s Dissociation time: 300s Regeneration reagent: Glycine 2.0, 30 seconds, twice The affinity test results for CD70 and CD27 proteins were as follows: KD (M): 2.99 × 10⁻⁶ -9 ; The affinity assay results for CD70 and CD27 proteins are as follows: Figure 8As shown, CD70 and CD27 proteins have good affinity and can be used to detect the binding of CD27 protein to CD70 protein by monoclonal antibody as described in Example 7.

[0142] Example 7: Detection of CD27 protein binding to CD70 protein blocked by monoclonal antibody Specific experimental procedures Ligand coupling Ligand: CD27 protein Coupling buffer: 10mM Acetate 5.5 Experimental conditions: Antibody concentration: Based on the affinity results in Example 5, five monoclonal antibodies, clone2, clone3, clone4, clone9 and clone10, were screened. The antibody was initially 400 nM and serially diluted 2-fold, resulting in 10 concentration points. The antibody was then mixed with 400 nM CD70 protein at a 1:1 ratio and injected.

[0143] Flow rate: 30 μL / min Combination time: 120s Dissociation time: 300s Regeneration reagent: Glycine 2.0, 30 seconds, twice Affinity blocking assay results of clone2 Figure 9a As shown, the affinity blocking assay results for clone3 are as follows: Figure 9b As shown, the affinity blocking assay results for clone4 are as follows: Figure 9c As shown, the affinity blocking assay results for clone9 are as follows: Figure 9d As shown, the affinity blocking assay results for clone10 are as follows: Figure 9e As shown; specific values ​​are shown in Table 6: Table 6 The smaller the IC50 (M) value, the stronger the ability of the monoclonal antibody to block the binding of CD27 protein to the target protein. For example... Figure 9a -e、 Figure 10 As shown in Table 6, the monoclonal antibodies numbered clone2, clone3, clone4, clone9, and clone10 all have good ability to block CD27 protein from binding to CD70 protein. Among them, the antibody numbered clone9 has the lowest IC50 (M) value and the best blocking ability.

[0144] Since clone4 and clone9 have high sequence similarity, and clone2 and clone3 have high sequence similarity, clone3, clone9, clone10, and clone1 were selected as the preferred verification sources for the CAR gene structures of the single-chain variable region fragments derived from CD70 monoclonal antibodies in Example 8. The performance of CAR-T cells constructed from the CD70 antibody sequences in Example 8 was compared and their in vitro killing effect was evaluated.

[0145] Example 8. Performance comparison and in vitro killing evaluation of CAR-T cells constructed based on different CD70 antibody sequences The experimental procedure is as follows: PBMCs were resuscitated and counted, and one vial of human peripheral blood mononuclear cells (cell count 2 × 10⁻⁶) was extracted. 7 Cells (per branch) were rapidly revived in a 37°C water bath. The revived cells were then passed through a 70 μm cell sieve, and the sieve was washed with sorting buffer.

[0146] CD4 + / CD8 + T-cell immunomagnetic bead sorting: (1) Resuspend the centrifuged PBMCs in an appropriate amount of sorting buffer, with a resuspending volume of 200 μL. Add 20 μL each of CD4 and CD8 sorting beads, mix thoroughly, and incubate at 2-8℃ for 15 minutes. (2) After incubation, add 1-2 mL / 10 7 Add cells to sorting buffer, invert to mix 2-3 times, centrifuge at 300g for 10 minutes, discard the supernatant, and retain the cell pellet. Add cells according to the initial total cell count (2×10⁻⁶). 7 Add 0.5 mL / 10 (each) 8 Resuspend the cell pellet in sorting buffer. (3) Place the Miltenyi sorting column in the magnetic field of the Miltenyi separator and rinse the sorting column with 3 mL of sorting buffer. Add the above cell suspension to the sorting column and collect the effluent unlabeled cells (i.e., CD4). + / CD8 - and CD8 + / CD4 - (Mixed T cells). Wash the sorting column with 3 mL of sorting buffer, and repeat the washing twice after the liquid has drained. (4) Remove the sorting column from the separator and place it on a 15 mL centrifuge tube. Add 5 mL of sorting buffer, quickly push out the cells using the matching stopcock, and collect the cell suspension.

[0147] Cell activation (D0), 3 × 10 6 The sorted CD4 + / CD8 + T cells were cultured for activation. The cell density was adjusted to 1×10⁶. 6Cells / mL, resuspended in preheated OptiVitro T cell culture medium containing IL-7 / IL-15. Administer at 10 μL / 10 6 Add T cell activation reagent (T cell TransAct) to each cell, mix well, and incubate in an incubator at 37.0℃ and 5.0% CO2. This is recorded as day D0.

[0148] T cell transduction (D2): Based on the single-chain variable region fragments derived from four CD70 monoclonal antibodies screened in Example 7, CAR lentiviral expression plasmids were constructed and packaged. T cells were transduced at MOI=30, and the resulting CD70 CAR-T cells were named CD70-g1, CD70-g3, CD70-g9, and CD70-g10, respectively. Untransduced T cells (Mock-T group) and CD19 CAR-T cells were used as controls. After transduction, the cells were cultured in a 37.0℃, 5.0% CO2 incubator.

[0149] Cell medium change and expansion (D4): Cell samples were taken for counting, centrifuged to remove T cell TransAct and virus, and cultured with replenished medium. After approximately 48 hours of culture, cell growth was observed to be good. 20 μL of the cell suspension was gently mixed and counted to calculate the total cell count. 300g of cell suspension was centrifuged at room temperature for 5 min, the supernatant was discarded, and the cell density was adjusted to 0.5 × 10⁶ cells / mL. The cell pellet was resuspended in preheated culture medium, gently mixed, and cultured in a 37.0℃, 5.0% CO₂ incubator.

[0150] Samples were taken on day 0 (D0), day 2 (D2), day 4 (D4), day 6 (D6), day 8 (D8), day 11 (D11), day 14 (D14), and day 17 (D17) of culture for the following tests: cell count, cell viability, and changes in cell size.

[0151] The results are as follows Figure 11 , 12 As shown in Figure 13: (1) Cell expansion: Mock T and CD19 CAR-T cells expanded rapidly in the early stage, and cell expansion slowed down from D11 to D17. Among the CD70 CAR-T groups, CD70-g10 expanded the fastest; CD70-g1 expanded the slowest. By D17, CD70-g10 (11900 times) > CD70-g9 (5816 times) > CD70-g3 (5181 times) > CD19 (4077 times) > Mock T (3371 times) > CD70-g1 (859 times).

[0152] (2) Cell viability: Mock T, CD19 > 90%, and by D17, except for CD70-g1 < 70%, the other groups in the CD70 group were all between 70 and 90%, with CD70-g10 having the highest viability.

[0153] (3) Cell size: The cell diameter decreased faster in the group with faster expansion rate.

[0154] In vitro cytotoxic activity assay (Day 11) To evaluate the specific killing ability of the constructed CD70 CAR-T cells against CD70-positive target cells, the following experimental groups were set up, as shown in Table 7: Table 7: Co-culture of target cells: (1) Take cultured Molm 13 cells and Molm 13-CD70-KO cells, add CellTrace dye at a ratio of 1:20000, mix gently, and incubate at room temperature in the dark for 15 minutes to complete the target cell labeling. After labeling, plate the two types of target cells into 48-well plates, 1×10⁶ cells per well. 5 (1) Cells, each type of cell, are seeded in 18 wells. (2) Effector cells are prepared and co-cultured with effector and target cells. According to the different effector-target ratios in Table 7, the corresponding amount of effector cells is added to the target cells seeded in the plate. One sample is prepared for each group and each effector-target ratio. The target cell culture medium is added to 1 mL / well. The 48-well plate is placed in an incubator and cultured for 1 day. After co-culturing for 1 day, cell killing is detected by flow cytometry.

[0155] Results of in vitro lethality assessment are as follows Figure 14 , 15 As shown in Figure 16.

[0156] Figure 14 The results showed that when the target cells were Molm-13 (CD70 positive), the killing activity of the four CD70 CAR-T cells was significantly higher than that of the control group (CD19 CAR-T, Mock-T), indicating that the four CD70 CAR-T cells could efficiently kill CD70 positive Molm-13 cells. Among them, CD70-g3, CD70-g9, and CD70-g10 had much better killing ability than CD70-g1.

[0157] Figure 15 This indicates that when the target cells are CD70-KO Molm-13 (CD70 negative), the killing activity of all CD70 CAR-T cells drops sharply and no longer has high lysis capacity. This shows that the potent killing effect of CD70 CAR-T cells on target cells depends entirely on the CD70 antigen on the surface of the target cells. After the target cells lose CD70, the specific killing effect basically disappears, with only a small amount of non-specific lysis remaining.

[0158] Figure 16 This indicates that the non-specific killing effect of T cells themselves has been eliminated, and the difference in killing effect caused solely by CD70 antigen recognition is directly reflected. The results show that after removing non-specific background, the specific killing effect of the four CD70 CAR-T cells is ranked as follows: CD70-g10 > CD70-g9 > CD70-g3 > CD70-g1.

[0159] 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.

[0160] The sequence involved in this invention:

Claims

1. An antibody or antigen-binding fragment thereof targeting CD70, characterized in that, The antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region and the light chain variable region are selected from the group consisting of: (1) Heavy chain variable regions containing the following three CDRs: HCDR1, as shown in the amino acid sequence of SEQ ID NO. 91, HCDR2, as shown in the amino acid sequence of SEQ ID NO. 92, HCDR3, as shown in the amino acid sequence of SEQ ID NO. 93; and Light chain variable regions containing the following three CDRs: LCDR1, as shown in the amino acid sequence of SEQ ID NO. 94, LCDR2, as shown in the amino acid sequence of SEQ ID NO. 95, LCDR3, as shown in the amino acid sequence of SEQ ID NO. 96; or (2) Heavy chain variable regions containing the following three CDRs: HCDR1, as shown in the amino acid sequence of SEQ ID NO. 81, HCDR2, as shown in the amino acid sequence of SEQ ID NO. 82, HCDR3, as shown in the amino acid sequence of SEQ ID NO. 83; and Light chain variable regions containing the following three CDRs: LCDR1, as shown in the amino acid sequence of SEQ ID NO. 84, LCDR2, as shown in the amino acid sequence of SEQ ID NO. 85, LCDR3, as shown in the amino acid sequence of SEQ ID NO. 86; or (3) Heavy chain variable regions containing the following three CDRs: HCDR1, as shown in the amino acid sequence of SEQ ID NO. 71, HCDR2, as shown in the amino acid sequence of SEQ ID NO. 72, HCDR3, as shown in the amino acid sequence of SEQ ID NO. 73; and Light chain variable regions containing the following three CDRs: LCDR1, as shown in the amino acid sequence of SEQ ID NO. 74, LCDR2, as shown in the amino acid sequence of SEQ ID NO. 75, LCDR3, as shown in the amino acid sequence of SEQ ID NO. 76; or (4) Heavy chain variable regions containing the following three CDRs: HCDR1, as shown in the amino acid sequence of SEQ ID NO. 61, HCDR2, as shown in the amino acid sequence of SEQ ID NO. 62, HCDR3, as shown in the amino acid sequence of SEQ ID NO. 63; and Light chain variable regions containing the following three CDRs: LCDR1, as shown in the amino acid sequence of SEQ ID NO. 64, LCDR2, as shown in the amino acid sequence of SEQ ID NO. 65, LCDR3, as shown in the amino acid sequence of SEQ ID NO. 66; or (5) Heavy chain variable regions containing the following three CDRs: HCDR1, as shown in the amino acid sequence of SEQ ID NO. 51, HCDR2, as shown in the amino acid sequence of SEQ ID NO. 52, HCDR3, as shown in the amino acid sequence of SEQ ID NO. 53; and Light chain variable regions containing the following three CDRs: LCDR1, as shown in the amino acid sequence of SEQ ID NO. 54, LCDR2, as shown in the amino acid sequence of SEQ ID NO. 55, LCDR3, as shown in the amino acid sequence of SEQ ID NO. 56; or (6) Heavy chain variable regions containing the following three CDRs: HCDR1, as shown in the amino acid sequence of SEQ ID NO. 41, HCDR2, as shown in the amino acid sequence of SEQ ID NO. 42, HCDR3, as shown in the amino acid sequence of SEQ ID NO. 43; and Light chain variable regions containing the following three CDRs: LCDR1, as shown in the amino acid sequence of SEQ ID NO. 44, LCDR2, as shown in the amino acid sequence of SEQ ID NO. 45, LCDR3, as shown in the amino acid sequence of SEQ ID NO. 46; or (7) Heavy chain variable regions containing the following three CDRs: HCDR1, as shown in the amino acid sequence of SEQ ID NO. 31, HCDR2, as shown in the amino acid sequence of SEQ ID NO. 32, HCDR3, as shown in the amino acid sequence of SEQ ID NO. 33; and Light chain variable regions containing the following three CDRs: LCDR1, as shown in the amino acid sequence of SEQ ID NO. 34, LCDR2, as shown in the amino acid sequence of SEQ ID NO. 35, LCDR3, as shown in the amino acid sequence of SEQ ID NO. 36; or (8) Heavy chain variable regions containing the following three CDRs: HCDR1, as shown in the amino acid sequence of SEQ ID NO. 21, HCDR2, as shown in the amino acid sequence of SEQ ID NO. 22, HCDR3, as shown in the amino acid sequence of SEQ ID NO. 23; and Light chain variable regions containing the following three CDRs: LCDR1, as shown in the amino acid sequence of SEQ ID NO. 24, LCDR2, as shown in the amino acid sequence of SEQ ID NO. 25, LCDR3, as shown in the amino acid sequence of SEQ ID NO. 26; or (9) Heavy chain variable regions containing the following three CDRs: HCDR1, as shown in the amino acid sequence of SEQ ID NO. 11, HCDR2, as shown in the amino acid sequence of SEQ ID NO. 12, HCDR3, as shown in the amino acid sequence of SEQ ID NO. 13; and Light chain variable regions containing the following three CDRs: LCDR1, as shown in the amino acid sequence of SEQ ID NO. 14, LCDR2, as shown in the amino acid sequence of SEQ ID NO. 15, LCDR3, as shown in the amino acid sequence of SEQ ID NO. 16; or (10) Heavy chain variable regions containing the following three CDRs: HCDR1, as shown in the amino acid sequence of SEQ ID NO. 1, HCDR2, as shown in the amino acid sequence of SEQ ID NO. 2, HCDR3, as shown in the amino acid sequence of SEQ ID NO. 3; and Light chain variable regions containing the following three CDRs: LCDR1, as shown in the amino acid sequence of SEQ ID NO. 4, LCDR2, as shown in the amino acid sequence of SEQ ID NO. 5, LCDR3, as shown in the amino acid sequence of SEQ ID NO.

6.

2. A fusion protein, characterized in that, The fusion protein comprises: (i) the antibody or its antigen-binding fragment as described in claim 1; and (ii) A fusion portion fused to the antibody or its antigen-binding fragment, including a tag sequence, signal peptide, or functional peptide that assists in expression and / or purification.

3. A polynucleotide, characterized in that, The polynucleotide encodes the antibody or its antigen-binding fragment as described in claim 1, or the fusion protein as described in claim 2.

4. A carrier, characterized in that, The carrier contains the polynucleotide as described in claim 3.

5. A host cell, characterized in that, The host cell contains the vector of claim 4, or its genome is integrated with exogenous polynucleotides of claim 3.

6. A chimeric antigen receptor CAR, characterized in that, The antigen-binding domain of the chimeric antigen receptor contains a single-chain variable region sequence scFv targeting CD70, wherein the heavy chain variable region and light chain variable region contained in the scFv are as defined in claim 1, or the heavy chain sequence and light chain sequence contained in the scFv are as defined in claim 1.

7. A CAR-NK cell, CAR-T cell, or CAR-macrophage, characterized in that, The CAR-NK cells, CAR-T cells, or CAR-macrophages described herein express the chimeric antigen receptor as described in claim 6.

8. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the antibody or antigen-binding fragment thereof as claimed in claim 1, the recombinant protein as claimed in claim 2, the polynucleotide as claimed in claim 3, the carrier as claimed in claim 7, the host cell as claimed in claim 5, or the CAR-NK cell, CAR-T cell, or CAR-macrophage as claimed in claim 7, or a combination thereof, and a pharmaceutically acceptable carrier, diluent, or excipient.

9. An immunoconjugate, characterized in that, The immunoconjugate contains: (a) The antibody portion, wherein the antibody portion is selected from the group consisting of: The antibody or its antigen-binding fragment as described in claim 1, the recombinant protein as described in claim 2, or a combination thereof; and (b) A conjugation portion conjugated to the antibody portion, the conjugation portion being selected from the group consisting of: detectable markers, drugs, toxins, cytokines, radionuclides, enzymes, or combinations thereof.

10. Use of the antibody or antigen-binding fragment thereof as claimed in claim 1, the recombinant protein as claimed in claim 2, the polynucleotide as claimed in claim 3, the vector as claimed in claim 7, the host cell as claimed in claim 5, or the CAR-NK cell, CAR-T cell, or CAR-macrophage as claimed in claim 7, the pharmaceutical composition as claimed in claim 8, or the immunoconjugate as claimed in claim 9, characterized in that, Used in the preparation of a drug or formulation, said drug or formulation being used for: (a) Treatment of CD70-positive diseases or diseases with CD70 overexpression; and / or (b) In vitro detection of CD70 protein; and / or (c) Reduce immune rejection of allogeneic cell therapy products in subjects, thereby improving product durability and / or efficacy.

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