Fully humanized antibody polypeptide fragment of targeted folate receptor alpha and application of fully humanized antibody polypeptide fragment

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a fully humanized antibody polypeptide fragment of a targeted folate receptor alpha and application of the fully humanized antibody polypeptide fragment. The antibody polypeptide fragment comprises a heavy chain variable region and a light chain variable region, and complementarity determining region (CDR) sequences of the heavy chain variable region and the light chain variable region are respectively HCDR1 (SEQ ID NO: 11), HCDR2 (SEQ ID NO: 12), HCDR3 (SEQ ID NO: 13), LCDR1 (SEQ ID NO: 14), LCDR2 (SEQ ID NO: 15) and LCDR3 (SEQ ID NO: 16). And the framework region is selected from SEQ ID NO. 3 to 6 and SEQ ID NO. 7 to 10. The invention further discloses a chimeric antigen receptor constructed based on the antibody, a bispecific antibody and an antibody drug conjugate. The fully humanized antibody fragment provided by the invention has the characteristics of low immunogenicity and high affinity, and provides an innovative strategy for treatment of FRalpha positive malignant tumors (such as ovarian cancer, breast cancer and lung cancer).

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to a fully human antibody polypeptide fragment targeting folate receptor alpha and its applications. Background Art

[0002] Folate receptor alpha (FRα, also known as FOLR1) is a glycoprotein anchored to the cell membrane through glycosylphosphatidylinositol (GPI), and has the characteristics of binding folic acid with high affinity and completing folic acid transport through receptor-mediated endocytosis. Research shows that FRα is significantly overexpressed on the surfaces of various malignant tumor cells, including ovarian cancer (up to 90%), breast cancer, endometrial cancer, mesothelioma, and lung cancer, etc., while its expression level is extremely low in normal tissues, especially almost not expressed in non-malignant ovarian tissues. This differential expression pattern makes FRα a highly potential tumor treatment target, especially attracting much attention in the targeted treatment of ovarian cancer.

[0003] In recent years, targeted treatment strategies based on FRα have developed rapidly, covering antibody-drug conjugates (ADCs), bispecific antibodies (BiTEs), immune cells modified with chimeric antigen receptors (CARs) (such as T cells, NK cells), etc. For example, the ADC drug Mirvetuximab soravtansine (ELAHERE®) acceleratedly approved by the FDA in 2022 has been used for the treatment of FRα-positive platinum-resistant ovarian cancer, marking an important breakthrough in the clinical transformation of FRα-targeted drugs. However, existing therapies still have significant limitations: 1. Challenges of CAR-T cell therapy: Although CAR-T is highly effective in hematological malignancies, its application in solid tumors is limited by the long preparation cycle of autologous cells, the limitation of the patient's immune status, and the risk of graft-versus-host disease (GVHD). In addition, the murine or humanized single-chain antibodies (scFvs) relied on by traditional CAR-T may cause immunogenicity and affect long-term efficacy.

[0004] 2. Room for optimization of NK cell therapy: Although CAR-NK has become an emerging direction due to its high safety (no GVHD risk) and short-acting characteristics, its targeting efficiency and in vivo persistence still need to be improved, and the insufficient affinity or stability of the antibody fragments used in existing CAR constructs may limit the efficacy.

[0005] 3. Insufficient synergistic effect of bispecific antibodies: Existing bispecific antibody designs mostly rely on a single target or epitope, have limited ability to synergistically block immunosuppressive signals in the tumor microenvironment, and some antibodies may cause immune rejection due to species source problems.

[0006] 4. Improvement requirements for ADC drugs: Although the marketed FRα ADC drugs have verified the druggability of the target, the conjugation technology, toxin release efficiency, and the affinity of the antibody itself still need to be optimized to reduce off-target toxicity and expand the indication range.

[0007] In addition, most of the existing antibodies targeting FRα are humanized or murine antibodies, and their immunogenicity may limit repeated dosing. However, fully human antibody fragments have more advantages in constructing CAR, bispecific antibodies, or ADCs due to their low immunogenicity and high specificity. Currently, the development of fully human antibodies against FRα still faces technical bottlenecks such as low screening efficiency and insufficient affinity, which restricts their application in multi-platform therapies.

[0008] Therefore, developing fully human FRα antibody fragments with high affinity and low immunogenicity, and constructing novel CAR immune cells, bispecific antibodies, and ADC drugs based on them, are of great significance for improving the treatment effect of FRα-positive malignant tumors, reducing side effects, and expanding clinical application scenarios. Summary of the Invention

[0009] The object of the present invention is to provide a fully human antibody polypeptide fragment for targeting folate receptor α and its application. The fully human antibody fragment provided by the present invention has the characteristics of low immunogenicity and high affinity, providing an innovative strategy for the treatment of FRα-positive malignant tumors (such as ovarian cancer, breast cancer, and lung cancer).

[0010] In the first aspect of the present invention, a fully human antibody polypeptide fragment for targeting folate receptor α is provided, and the antibody polypeptide fragment comprises: (a) a heavy chain variable region, the amino acid sequence of which is as shown in SEQ ID NO.1; and (b) a light chain variable region, the amino acid sequence of which is as shown in SEQ ID NO.2; Wherein, the heavy chain variable region comprises complementary determining regions HCDR1, HCDR2, and HCDR3, and the amino acid sequences of HCDR1, HCDR2, and HCDR3 are respectively as shown in SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13; Wherein, the light chain variable region comprises complementary determining regions LCDR1, LCDR2, and LCDR3, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are respectively as shown in SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16.

[0011] In one embodiment, the framework region of the heavy chain variable region belongs to IGHV3-30 containing a sequence selected from SEQ ID NOs. 3-6, and the framework region of the light chain variable region belongs to IGKV3-20 containing a sequence selected from SEQ ID NOs. 7-10.

[0012] The second aspect of the present invention provides a chimeric antigen receptor targeting folate receptor α, and the composition of the chimeric antigen receptor sequentially includes: Signal peptide; The above-mentioned antibody polypeptide fragment; Hinge region; Transmembrane segment; Intracellular co-stimulatory domain; Intracellular signal transduction domain.

[0013] In one embodiment, the signal peptide is human CD8α signal peptide, and its amino acid sequence is as shown in SEQ ID NO. 17; The amino acid sequence of the hinge region is as shown in SEQ ID NO. 18; The transmembrane segment is selected from any one of the following: human CD28α transmembrane segment, human NKG2D transmembrane segment, human CD8α transmembrane segment; the amino acid sequence of the human CD28α transmembrane segment is as shown in SEQ ID NO. 19; the amino acid sequence of the human NKG2D transmembrane segment is as shown in SEQ ID NO. 20; the amino acid sequence of the human CD8α transmembrane segment is as shown in SEQ ID NO. 21; The intracellular co-stimulatory domain is selected from any one of the following: intracellular segment of human 2B4, intracellular segment of human CD28, intracellular segment of human 4-1BB; the amino acid sequence of the intracellular segment of human 2B4 is as shown in SEQ ID NO. 22; the amino acid sequence of the intracellular segment of human CD28 is as shown in SEQ ID NO. 23; the amino acid sequence of the intracellular segment of human 4-1BB is as shown in SEQ ID NO. 24; The intracellular signal transduction domain is the intracellular segment of CD3ζ, and its amino acid sequence is as shown in SEQ ID NO. 25.

[0014] The third aspect of the present invention provides a host cell, and the host cell contains an expression vector of the above chimeric antigen receptor.

[0015] In one embodiment, the host cell is selected from one of NK-92 cells, human primary T cells, human primary NK cells, mononuclear macrophages or cytokine-induced killer cells.

[0016] The fourth aspect of the present invention provides a bifunctional bispecific antibody, which comprises: (a) the above-mentioned fully human antibody polypeptide fragment; (b) a second antibody fragment.

[0017] In one embodiment, the second antibody targets an antigen selected from PD-1, CD3, CTLA4, EGFR, HER2, CD276 or Siglec9.

[0018] The fifth aspect of the present invention provides an antibody-drug conjugate comprising the above-mentioned fully human antibody polypeptide fragment.

[0019] The sixth aspect of the present invention provides the use of the above-mentioned fully human antibody polypeptide fragment, the above-mentioned chimeric antigen receptor targeting folate receptor α, the above-mentioned host cell, the above-mentioned bifunctional bispecific antibody or the above-mentioned antibody-drug conjugate in the preparation of a drug for preventing or treating folate receptor α-positive malignant tumors.

[0020] In one embodiment, the malignant tumor is ovarian cancer, endometrial cancer, breast cancer, lung cancer or colon cancer.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. Low immunogenicity: Compared with traditional murine / humanized antibodies, the fully human antibody polypeptide fragment (containing the heavy chain variable region of SEQ ID NO.1 and the light chain variable region of SEQ ID NO.2) of the present invention is completely derived from human genes, significantly reducing the risk of immune rejection and being suitable for repeated dosing scenarios.

[0022] 2. High affinity and specificity: Through phage library screening and NGS optimization, the antibody complementarity-determining regions (CDRs) are precisely designed (such as the specific sequences of HCDR1-3 and LCDR1-3), and the affinity for binding to FRα reaches the nanomolar level (such as KD = 3.82E-08 M in Example 1), enabling efficient targeting of tumor cells.

[0023] 3. In the present invention, by combining the antibody fragment with the CD8α signal peptide, co-stimulatory domains (such as 4-1BB, CD28) and the CD3ζ signal domain, the constructed CAR-NK / T cells significantly inhibit the growth of ovarian cancer in in vivo experiments (Example 2, Figure 6 ) and have high safety (no risk of GVHD).

[0024] 4. In the bifunctional bispecific antibody provided by the present invention, the fully human antibody polypeptide fragment (binding the anti-FRα fragment and the second antibody such as anti-PD-1 / CD3) can simultaneously block tumor targeting and immunosuppressive signals. For example, the αFR-siglec9 bispecific antibody relieves macrophage inhibition (Example 3, Figure 9), significantly prolonging the survival time of tumor-bearing mice.

[0025] 5. The antibody-drug conjugate provided by the present invention exhibits potent anti-tumor activity at a low dose (2 mg / kg) (Example 4), and the toxin release is controllable, reducing off-target toxicity.

[0026] 6. The fully human antibody polypeptide fragment, chimeric antigen receptor targeting folate receptor α, host cell, bispecific antibody, and the above-mentioned antibody-drug conjugate provided by the present invention target a variety of solid tumors with high expression of FRα (such as ovarian cancer, lung cancer, breast cancer, etc.). Through the universality verified by the SKOV3 model in the examples, it has broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is a bar graph of OD450 in the polyclonal phage ELISA detection in Example 1; Figure 2 It is an enrichment index graph of specific phage antibodies after screening in Example 1; Figure 3 It is a reduced SDS-PAGE electrophoresis diagram of the purified full-length antibody in Example 1; Figure 4 It is a bar graph of OD450 for detecting the binding specificity of the purified antibody in the ELISA detection in Example 1; Figure 5 It is a kinetic diagram of the combination of the antibody and the antigen in Example 1; Figure 6 It is a curve graph of the inhibition of mouse tumor growth by CAR-NK cells in Example 2; Figure 7 It is a flow chart of the synthesis of the bispecific antibody plasmid in Example 3; Figure 8 It is a reduced SDS-PAGE electrophoresis diagram of the purified full-length bispecific antibody in Example 3; Figure 9 It is a curve graph of the inhibition of mouse tumor growth by the bispecific antibody in Example 3; Figure 10 It is a reduced SDS-PAGE electrophoresis diagram of the purified full-length antibody in Example 4; Figure 11 It is a curve graph of the inhibition of tumor growth by the ADC drug in Example 4. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0028] In this text, a range expressed as "from one numerical value to another numerical value" is a summary representation that avoids listing all the numerical values within that range in the specification. Therefore, the recitation of a particular numerical range encompasses any numerical value within that range and any smaller numerical ranges defined by any numerical values within that range, as if such any numerical value and such smaller numerical ranges were expressly written in the specification.

[0029] In this application, unless otherwise specified, the use of "or" means "and / or". In the case of multiple dependent claims, "or" is used only in the alternative to refer to more than one of the foregoing independent or dependent claims.

[0030] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all employ conventional techniques in the fields of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields in the art.

[0031] To make the present invention easier to understand, certain terms are first defined. As used in this application, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below.

[0032] As used herein, the term "antibody" or "immunoglobulin" is a heterotetrameric glycoprotein of approximately 150 kDa having the same structural characteristics, which consists of two identical light chains (LC) and two identical heavy chains (HC). Each light chain is linked to a heavy chain by a covalent disulfide bond, and 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. There are two types of light chains, λ and κ. There are five main types of heavy chains, which determine the functional activity of the antibody molecule: IgM, IgD, IgG, IgA, and IgE. Each chain contains distinct sequence domains. The light chain includes two domains, a variable domain (VL) and a constant domain (CL). The heavy chain includes four domains, a heavy chain variable region (VH) and three constant regions (CH1, CH2, and CH3, collectively CH). The variable regions of both the light and heavy chains determine the binding recognition and specificity against antigens. The constant domain (CL) of the light chain and the constant regions (CH) of the heavy chain confer important biological properties such as antibody chain association, secretion, complement binding, and binding to Fc receptors (FcR). The Fv fragment is the N-terminal portion of the immunoglobulin Fab fragment and consists of the variable portions of one light chain and one heavy chain. The specificity of an antibody depends on the structural complementarity between the antibody binding site and the antigenic determinant. The antibody binding site is composed of residues mainly from the highly variable regions or complementarity-determining regions (CDR). Residues from the non-highly variable or framework regions (FR) sometimes affect the overall domain structure and thus the binding site. The complementarity-determining region or CDR is the amino acid sequence specific to the Fv region that defines the affinity and binding site for antigen binding. Each of the light and heavy chains of an immunoglobulin has three CDRs, designated CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3, respectively. The conventional antibody antigen binding site thus consists of six CDRs, comprising a set of CDRs from the variable regions (v regions) of each heavy and light chain.

[0033] As used herein, the term "variable" means that certain portions of the variable regions in an antibody are different in sequence, which forms the binding and specificity of various specific antibodies to their specific antigens. However, the variability is not evenly distributed throughout the antibody variable regions, and it is concentrated in three segments called complementarity-determining regions (CDR) or hypervariable regions in the variable regions of the light and heavy chains. The more conserved portions in the variable regions are called framework regions (FR). The variable regions of the native heavy and light chains each contain four FR regions, which are generally in a β-sheet conformation and are connected by three CDRs forming connecting loops, and in some cases can form a partially folded structure. The CDRs in each chain are brought closely together by the FR regions and together with the CDRs of the other chain form the antigen binding site of the antibody. The constant regions do not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions.

[0034] As used herein, the terms "heavy chain variable region" and "VH" are used interchangeably, the terms "light chain variable region" and "VL" are used interchangeably, and the terms "variable region" and "complementarity determining region (CDR)" are used interchangeably.

[0035] The present invention includes not only intact antibodies, but also antibody fragments with immunological activity or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives, and analogs of the said antibodies. As used herein, the terms "fragment", "derivative", and "analog" 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 conservative or non-conservative amino acid residues (preferably conservative 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 a substituent group in one or more amino acid residues, or (iii) polypeptides formed by fusing the mature polypeptide with another compound (such as a compound that prolongs the half-life of the polypeptide, for example, polyethylene glycol), or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (such as a leader sequence or a secretion sequence or a sequence used to purify this polypeptide or a proprotein sequence, or a fusion protein formed with a 6His tag). These fragments, derivatives, and analogs are within the scope well-known to those skilled in the art.

[0036] The present invention provides a fully human antibody polypeptide fragment targeting folate receptor α, and the antibody polypeptide fragment comprises: (a) a heavy chain variable region, the amino acid sequence of which is shown in SEQ ID NO.1; and (b) a light chain variable region, the amino acid sequence of which is shown in SEQ ID NO.2; wherein, the heavy chain variable region comprises complementarity determining regions HCDR1, HCDR2, and HCDR3, and the amino acid sequences of HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13 respectively; wherein, the light chain variable region comprises complementarity determining regions LCDR1, LCDR2, and LCDR3, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16 respectively.

[0037] In one embodiment, the framework region of the heavy chain variable region belongs to IGHV3-30 containing a sequence selected from SEQ ID NOs. 3-6, and the framework region of the light chain variable region belongs to IGKV3-20 containing a sequence selected from SEQ ID NOs. 7-10.

[0038] In the present invention, the above fragments are combined with other components of the chimeric antigen receptor (CAR) and introduced into immune cells such as T or NK or macrophages, or the above fragments are combined with other antibodies to obtain a bifunctional bispecific antibody, or the above fragments are conjugated with a drug to obtain an antibody-drug conjugate. These combinations can specifically recognize and kill human tumor cell lines and human ovarian cancer xenograft models that are positive for folate receptor a expression. The antibody fragments and antibody-cells, antibody-antibody, and antibody-drug combinations provided by the present invention have great application potential in the clinical treatment of folate receptor a-positive malignant tumors.

[0039] A chimeric antigen receptor targeting folate receptor α provided by the present invention, the composition of the chimeric antigen receptor sequentially includes: A signal peptide; The above antibody polypeptide fragment; A hinge region; A transmembrane segment; An intracellular co-stimulatory domain; An intracellular signal transduction domain.

[0040] In one embodiment, the signal peptide is a human CD8α signal peptide, and its amino acid sequence is as shown in SEQ ID NO. 17; the N-terminus of the antibody polypeptide fragment that binds to folate receptor α is connected to the C-terminus of the human CD8α signal peptide.

[0041] The amino acid sequence of the hinge region is as shown in SEQ ID NO. 18, and its N-terminus is connected to the C-terminus of the antibody polypeptide fragment that binds to folate receptor α.

[0042] The transmembrane segment is selected from any one of the following: human CD28α transmembrane segment, human NKG2D transmembrane segment, human CD8α transmembrane segment; the amino acid sequence of the human CD28α transmembrane segment is as shown in SEQ ID NO. 19; the amino acid sequence of the human NKG2D transmembrane segment is as shown in SEQ ID NO. 20; the amino acid sequence of the human CD8α transmembrane segment is as shown in SEQ ID NO. 21; the human CD28α transmembrane segment, human NKG2D transmembrane segment, and human CD8α transmembrane segment are all N-terminally connected to the C-terminus of the hinge region.

[0043] The intracellular co-stimulatory domain is selected from any of the following: the intracellular segment of human 2B4, the intracellular segment of human CD28, the intracellular segment of human 4-1BB; the amino acid sequence of the intracellular segment of human 2B4 is as shown in SEQ ID NO.22; the amino acid sequence of the intracellular segment of human CD28 is as shown in SEQ ID NO.23; the amino acid sequence of the intracellular segment of human 4-1BB is as shown in SEQ ID NO.24; for the intracellular segment of human 2B4, the intracellular segment of human CD28, and the intracellular segment of human 4-1BB, the N-terminus is connected to the C-terminus of the hinge region.

[0044] The intracellular signal transduction domain is the intracellular segment of CD3ζ, whose N-terminus is connected to the C-terminus of the intracellular co-stimulatory domain, and its amino acid sequence is as shown in SEQ ID NO.25.

[0045] The host cell provided by the present invention, the host cell contains the expression vector of the above chimeric antigen receptor. In one embodiment, the host cell is selected from one of NK-92 cells, human primary T cells, human primary NK cells, mononuclear macrophages or cytokine-induced killer cells.

[0046] The bispecific antibody provided by the present invention, the bispecific antibody comprises: (a)The above-mentioned fully human antibody polypeptide fragment; (b)A second antibody fragment.

[0047] In one embodiment, the second antibody targets an antigen selected from PD-1, CD3, CTLA4, EGFR, HER2, CD276 or Siglec9.

[0048] The antibody-drug conjugate provided by the present invention includes the above-mentioned fully human antibody polypeptide fragment. The antibody-drug conjugate provided by the present invention further includes a therapeutic agent bound or conjugated to the fully human antibody polypeptide fragment, and the therapeutic agent includes but is not limited to: 1. Radionuclide; 2. Biological toxin; 3. Cytokines such as IL-2, etc.; 4. Gold nanoparticles / nanorods; 5. Virus particles; 6. Liposomes; 7. Nanomagnetic particles; 8. Prodrug-activating enzymes, for example, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL); 9. Chemotherapeutic agents (for example, cisplatin) or any form of nanoparticles; 10. Nucleic acids such as siRNA or ASO, etc.

[0049] The present invention provides the use of the above-mentioned fully human antibody polypeptide fragment, the above-mentioned nucleic acid molecule, the above-mentioned expression vector, the above-mentioned host cell, the above-mentioned chimeric antigen receptor targeting folate receptor α, the above-mentioned host cell (the host cell contains the expression vector of the above chimeric antigen receptor), the above-mentioned bispecific antibody or the above-mentioned antibody-drug conjugate in the preparation of a drug for preventing or treating folate receptor α-positive malignant tumors.

[0050] The sequences involved in the present invention are as follows: Heavy chain variable region of the antibody polypeptide fragment, the amino acid sequence of which is shown in SEQ ID NO.1, SEQ ID NO.1: EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYAISWVRQAPGKGLEWVSSISSSGTYRYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRWFGEYWGSWFDPWGQGTLVTVSS Light chain variable region of the antibody polypeptide fragment, the amino acid sequence of which is shown in SEQ ID NO.2, SEQ ID NO.2: EIVLTQSPGTLSLSPGERATLSCRASQTIGRKLAWYQQKPGQAPRLLIYATSTRAAGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDDWPPITFGQGTKLEIK The framework region of the heavy chain variable region contains a sequence selected from SEQ ID NOs. 3-6; SEQ ID NO.3: EVQLLESGGGLVQPGGSLRLSCAASGFTFS SEQ ID NO.4: WVRQAPGKGLEWVS SEQ ID NO.5: RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR SEQ ID NO.6: WGQGTLVTVSS The framework region of the light chain variable region contains a sequence selected from SEQ ID NOs. 7-10: SEQ ID NO.7: EIVLTQSPGTLSLSPGERATLSC SEQ ID NO.8: WYQQKPGQAPRLLIY SEQ ID NO.9: GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC SEQ ID NO.10: FGQGTKLEIK The amino acid sequence of the heavy chain variable region containing complementarity-determining region HCDR1, SEQ ID NO. 11, is as follows: DYAIS.

[0051] The amino acid sequence of the heavy chain variable region containing complementarity-determining region HCDR2, SEQ ID NO. 12, is as follows: SISSSGTYRYYADSVKG.

[0052] The amino acid sequence of the heavy chain variable region containing complementarity-determining region HCDR3, SEQ ID NO. 13, is as follows: DRWFGEYWGSWFDP The amino acid sequence of the light chain variable region containing complementarity-determining region LCDR1, SEQ ID NO. 14: RASQTIGRKLA The amino acid sequence of the light chain variable region containing complementarity-determining region LCDR2, SEQ ID NO. 15: ATSTRAA The amino acid sequence of the light chain variable region containing complementarity-determining region LCDR3, SEQ ID NO. 16: QQYDDWPPIT The human CD8α signal peptide, the amino acid sequence of which is shown in SEQ ID NO. 17, SEQ ID NO. 17: MALPVTALLLPLALLLHAARP The amino acid sequence of the hinge region is shown in SEQ ID NO. 18, SEQ ID NO. 18: AKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD The amino acid sequence of the human CD28α transmembrane segment is shown in SEQ ID NO. 19, SEQ ID NO. 19: FWVLVVVGGVLACYSLLVTVAFIIFWV The amino acid sequence of the human NKG2D transmembrane segment is shown in SEQ ID NO. 20, SEQ ID NO. 20: PFFFCCFIAVAMGIRFIIMVT The amino acid sequence of the human CD8α transmembrane segment is shown in SEQ ID NO. 21, SEQ ID NO. 21: IYIWAPLAGTCGVLLLSLVITLYC The amino acid sequence of the human 2B4 intracellular segment is shown in SEQ ID NO. 22, SEQ ID NO. 22: WRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQSQSSAPTSQEPAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLSRKELENFDVYS The amino acid sequence of the intracellular segment of human CD28 is shown in SEQ ID NO.23, SEQ ID NO.23: RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS The amino acid sequence of the intracellular segment of human 4-1BB is shown in SEQ ID NO.24, SEQ ID NO.24: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL The amino acid sequence of the intracellular segment of CD3ζ is shown in SEQ ID NO.25, SEQ ID NO.25: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Example 1 Screening and Identification of a Fully Human Antibody Targeting Folate Receptor α 1.1 Screening of Folate Receptor α-Specific Phage Antibodies from a Fully Human Phage Antibody Library 1) Dilute recombinant human FRα to 10 μg / mL with coating buffer (50 mmol / L NaHCO 3 , pH 9.6). Take 3 mL and add it to an immunotube, and coat it overnight at 4°C.

[0053] 2) Wash the antigen-coated test tube 3 times with PBS.

[0054] 3) Fill the immunotube (5 mL) with 10% bovine serum blocking solution and block it at room temperature for 1 hour.

[0055] 4) Discard the blocking solution, wash it 3 times with PBS, pour PBS into the immunotube and then pour it out quickly. The following washing operations are the same.

[0056] 5) Mix the fully human phage Fab antibody library (titer about 10 13 pfu) preserved in the laboratory with the blocking solution. Add it to the immunotube, rotate it at room temperature for 60 minutes, and then let it stand at room temperature for 60 minutes.

[0057] 6) When performing the first round of screening, wash the tube 5 times with PBS containing 0.1% Tween-20, and then wash 5 times with PBS.

[0058] 7) First, add 1 mL of fresh XL1-Blue bacteria, incubate at 37 °C for 15 min, and transfer it into 9 mL of 2XYT medium (containing 20 μg / ml ampicillin and 20 μg / ml tetracycline).

[0059] 8) Wash twice with distilled water, add 1 mL of eluent, pipette intermittently in the middle, and add 40 μL of neutralizing solution to fully neutralize after 10 minutes.

[0060] 9) Add it to 10 mL of fresh XL1-Blue bacteria, incubate at 37 °C for 15 min, and then add it to 9 mL of 2XTY medium (containing 20 μg / mL ampicillin and 20 μg / mL tetracycline).

[0061] 10) Mix the bacterial solutions recovered twice, remove an appropriate amount and spread on 2XYT plates to determine the output. The remaining bacterial solution is cultured at 37 °C for 3 h. Then expand the volume to 50 mL, add helper phage and 50 μg / mL kanamycin, and culture overnight at 30 °C and 200 rpm in a shaker.

[0062] 1.2 Further affinity screening 1) Centrifuge the phage-infected bacterial solution in 1.1 to recover the supernatant, precipitate with PEG8000 to obtain the secondary phage antibody library. Take the above antibody library solution and perform logarithmic dilution according to 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 . Take 100 μL of the liquid at each dilution and mix it with the XL1-blue bacterial solution in the logarithmic growth phase, and co-incubate at 37 °C and 200 rpm in a shaker for 30 minutes. Coat it on a 2XYT culture plate with ampicillin resistance and culture overnight at 37 °C. Calculate the number of colonies the next day, and calculate the titer according to the number of clones contained in each ml of the antibody library.

[0063] 2) Take 1 mL of the above phage for the next round of affinity screening.

[0064] 3) Repeat the steps in 1.1. In step 10) of 1.1, for the second round of screening, wash 10 times with PBST and 10 times with PBS. For the third and fourth rounds of screening, wash 20 times with PBST and 20 times with PBS.

[0065] 1.3 Selection of random clones 1) Prepare 2XYT medium containing 100 μg / mL ampicillin and 10 μg / mL tetracycline, and add it to a 96-well deep-well culture plate, about 600 μL per well.

[0066] 2) Randomly pick single colonies on the culture plates output from the third and fourth rounds of screening, and inoculate them into a 96-well deep-well culture plate. Incubate overnight at 37 °C with shaking.

[0067] 3) The next day, transfer at a ratio of 1:10 into a new 96-well deep-well culture plate containing 600 μL of medium, and incubate at 37 °C with shaking for 3 hours. Add helper phage, incubate at 37 °C for 20 minutes, and then incubate at 30 °C with shaking for 8 hours.

[0068] 4) Centrifuge at 3000 rpm at room temperature for 10 minutes. The supernatant is used as the phage solution of the Fab antibody to be tested.

[0069] 1.4 Polyclonal phage ELISA 1) Dilute recombinant human FRα protein and bovine serum albumin (BSA) to 10 μg / mL with PBS, add 100 μL per well, and coat a 96-well ELISA plate overnight at 4 °C.

[0070] 2) Wash three times with PBS containing 0.1% Tween-20. Coat the plate with 200 μL of blocking solution (10% bovine serum in PBS) at 37 °C for 2 hours.

[0071] 3) Pour out the coating solution, add 200 μL of the phage solution of the Fab antibody in 1.3.4 and 50 μL of blocking solution to each well. Incubate at 37 °C for 1 hour. 4) Wash five times with PBS containing 0.1% Tween-20. Add 100 μL of anti-M13 monoclonal antibody diluted 1:4000 with blocking solution to each well, and incubate at room temperature for 1 hour.

[0072] 5) Wash six times with PBS containing 0.1% Tween-20. Prepare the substrate chromogenic solution and add 100 μL to each well, incubate at room temperature for 5 minutes. Add 50 μL of 0.1 M dilute sulfuric acid to each well to terminate the reaction.

[0073] 6) Measure OD650 and OD450, and use the value of OD450 minus the value of OD650 as the final test result.

[0074] Result: Draw a bar chart with the OD450 values of the sample wells, and screen out those with an absorbance value ≥ 10 times the ODBSA value, see Figure 1 .

[0075] 1.4 NGS (Next Generation Sequencing Technology) for sequencing phage antibodies 1) The bacterial liquid output from the first round of elution in step 10 of step 1.1 and the bacterial liquid obtained from 2 - 3 rounds of elution in 1.2 were subjected to high-throughput sequencing (NGS, with sequencing and analysis services provided by Suzhou GeneCreate Biotechnology Co., Ltd.). The high-affinity phages after elution were used to infect Escherichia coli. Plasmid DNA was extracted from the samples using a plasmid miniprep kit. The extracted nucleic acids were cut into small segments and sequencing adapters were added. Before sequencing, quality control of the library was carried out, including detection of parameters such as the concentration and size distribution of the library, to ensure that the quality of the library met the sequencing requirements. The prepared library was loaded onto a high-throughput sequencing instrument, such as the Illumina platform, for large-scale parallel sequencing to obtain a large amount of DNA sequence data. After sequencing, the instrument generates a large amount of raw sequencing data, which needs to be further processed to be converted into useful information. Quality control, alignment, variant detection and other analysis steps were performed on the raw sequencing data to obtain meaningful biological information.

[0076] 2) Recombinant analysis of the heavy and light chains of the antibody.

[0077] The results are shown in Figure 2 As shown, using human FRα as the antigen, four rounds of affinity screening were performed on the Fab phage antibody library. After each round of screening, the phage antibody harvest rate showed an increasing trend, indicating specific enrichment of anti-FRα phage antibodies.

[0078] 1.5 Expression and purification of the full-length antibody IgG1 1) According to the sequencing results in 1.4, the antibody gene fragments were fully synthesized and cloned into the pCDNA3.4 expression vector (Guangzhou Aiji Biotechnology Co., Ltd.).

[0079] 2) Expi293T cells were passaged into a 500 ml shake flask with a working volume of 100 ml and cultured in a shaker at 125 rpm, 37 °C, and 8% CO 2 under the condition of, and used for transient transfection when the cell density reached 3×10 6 cells / ml.

[0080] 3) The heavy chain plasmid and light chain plasmid of the antibody were mixed with Opti-MEM medium at a mass ratio of 1:2, and then transfection reagent was added and mixed well. After incubation at room temperature for 10 min, it was added to the Expi293T cells to be transfected.

[0081] 4) The transfected cells were cultured in a shake flask on a shaker, and feeding was carried out during the culture process to enable high expression of the cells. After 6 days of culture, the culture supernatant was collected and filtered through a 0.22 μm filter membrane.

[0082] 5) Pass the filtered supernatant through a gravity column of rProtein G Beads, wash the unbound fraction with PBS buffer, and elute the target antibody protein with a pH 11.0 alkaline buffer and immediately neutralize it.

[0083] 6) Dialyze the eluted antibody overnight in PBS and further purify it through a Superdex 200 16 / 60 molecular sieve, and collect the eluted fraction.

[0084] Results: After purification, the full-length antibody was detected by reducing SDS-PAGE electrophoresis. As Figure 3 can be seen, a high-purity full-length antibody protein was obtained.

[0085] 1.6 Detection of antibody binding specificity by ELISA 1) Dilute recombinant human FRα protein and bovine serum albumin (BSA) to 2 μg / mL with PBS, add 100 μL to each well, and coat a 96-well ELISA plate overnight at 4°C.

[0086] 2) Wash three times with PBS containing 0.1% Tween-20. Coat the plate with 350 μL of blocking solution (10% bovine serum in PBS) and incubate at 37°C for 2 h.

[0087] 3) Gradient dilute the antibody, and the final dilution concentrations are: 0 nM, 0.01 nM, 0.1 nM, 1 nM, 10 nM, 100 nM.

[0088] 3) Pour out the coating solution, add 100 μL of the gradient-diluted antibody to each well. Incubate at room temperature for 1 h. 4) Wash five times with PBS containing 0.1% Tween-20. Add 100 μL of HRP-labeled anti-human IgG monoclonal antibody diluted 1:10000 with the blocking solution to each well and incubate at room temperature for 1 h.

[0089] 5) Wash five times with PBS containing 0.1% Tween-20. After preparing the substrate chromogenic solution, add 100 μL to each well and incubate at room temperature for 5 min. Add 50 μL of 0.1 M dilute sulfuric acid to each well to terminate the reaction.

[0090] 6) Measure OD650 and OD450, and use the value of OD450 minus the value of OD650 as the final detection result.

[0091] Results: Draw a bar graph with the OD450 value of the sample wells, and screen out those with an absorbance value ≥ 10 times the ODBSA value, as shown in Figure 4 .

[0092] 1.7 Detection of affinity 1) According to the biolayer interferometry (BLI) technique, use the Fortebio Octet detection instrument to detect the antibody affinity. Equilibrate the streptavidinylated probe at room temperature for 20 minutes. The equilibration buffer is PBST.

[0093] 2) Gradient dilute the antibody, and the final dilution concentrations are: 6.25 nM, 12.5 nM, 25 nM, 50 nM, 100 nM. The binding time is 120 seconds and the dissociation time is 180 seconds. Add each reagent according to the instrument prompts and run the program.

[0094] 4) After the program ends, the kinetic graph of the combination of the antibody and the antigen can be seen. Calculate the equilibrium dissociation constant of the monoclonal antibody according to the model, such as Figure 5 .

[0095] Results: The detection data are shown in Table 1, and the affinities of the G6-IgG1 antibody are obtained respectively.

[0096] Example 2 Construction and anti-tumor activity of chimeric antigen receptor natural killer cells targeting folate receptor α 2.1 Lentivirus packaging 1) After digesting the cultured 293T cells with trypsin and washing them with PBS, resuspend the cells with DMEM complete medium (containing 10% FBS) to prepare a cell suspension, count the cells and calculate the cell suspension concentration.

[0097] 2) Inoculate 293T cells into a 10 cm culture dish at a density of 1×10 5 / cm 2 , place it in a carbon dioxide incubator, and culture it at 5% CO 2 at 37 °C overnight. Observe the cell density the next day and perform subsequent operations when the cell density reaches 70%-80%.

[0098] 3) Add 5 μg of the constructed plasmid DNA, 3.75 μg of pPAX2 plasmid, and 1.25 μg of pMd3G plasmid to 0.5 ml of Opti-MEM medium, and gently shake and mix well. According to the instructions of PEI (molecular weight 40,000), add 30 μg of PEI transfection reagent to the above plasmid mixture, and gently shake and mix well. Let it stand at room temperature for 15 minutes until the transfection complex is formed, and add the transfection complex drop by drop to the seeded 293T cells, and place it in the incubator, and culture it at 5% CO 2 at 37 °C.

[0099] 4) After 12 hours, remove the culture medium supernatant containing the transfection complex, and re-add fresh DMEM complete medium. After 48 and 72 hours, collect the culture medium supernatants of 293T cells respectively, filter them with a filter with a pore size of 0.45 μm, and reserve them for use.

[0100] 2.2 Lentivirus concentration: 1) Take 2 39 mL round polypropylene quick-seal centrifuge tubes (Type 70 Ti), and add approximately 32 mL of pre-treated virus supernatant to each centrifuge tube.

[0101] 2) Adjust the weight of each tube with PBS so that the weight difference between the corresponding centrifuge tubes does not exceed 0.01 g.

[0102] 3) Place the 2 centrifuge tubes into the Beckman Type 70 Ti ultracentrifuge rotor in sequence.

[0103] 4) Centrifuge at 25000 rpm (82700 g) for 2.5 hours. Carefully remove the tubes from the rotor. Pour out the supernatant and aspirate the remaining droplets. There should be visible precipitate at the bottom of the tube.

[0104] 5) Gently resuspend the precipitate by pipetting with a 200 μL pipette. Avoid generating foam. Collect the liquid in all tubes into an EP tube. Aliquot the concentrated virus suspension into 50 μL portions each and store in a finished product tube. Quick-freeze with dry ice chips and store at -80 °C.

[0105] Result: Through the above operations, the chimeric antigen receptor-expressing lentivirus targeting folate receptor α was prepared.

[0106] 2.3 Construction of chimeric antigen receptor-modified NK-92MI cells targeting folate receptor α 1) Seed NK-92MI cells in a 24-well plate at 5×10 6 cells per well. Incubate at 5% CO 2 , 37 °C for 24 hours.

[0107] 2) Add 50 μL of the concentrated chimeric antigen receptor-expressing lentivirus targeting folate receptor α prepared in 2.2.5 and 5 μg / mL polybrene to the NK-92MI cell culture wells, and mix well. Incubate at 5% CO 2 , 37 °C overnight.

[0108] 3) Remove the old culture supernatant, add fresh complete NK-92MI cell medium, and incubate at 5% CO 2 , 37 °C for 3 days.

[0109] 4) Add 3 μg / mL puromycin to the medium of NK-92MI cells infected with the chimeric antigen receptor-expressing lentivirus targeting folate receptor α, and incubate at 5% CO 2 , 37 °C for 14 days. Among them, each time when changing the medium or subculturing, add 3 μg / mL puromycin to the medium.

[0110] 5) Cells were collected and detected by flow cytometry to analyze the expression of chimeric antigen receptor targeting folate receptor α in NK-92MI cells. Genetically modified NK-92MI cells with GFP fluorescence were successfully obtained. Cells with GFP fluorescence in the drug-screened cells were detected by flow cytometry, and genetically modified NK-92MI cells were sorted by flow cytometry, that is, NK-92MI cells modified with chimeric antigen receptor targeting folate receptor α (αFR-CAR-NK).

[0111] 2.4 In vivo experiments to verify the anti-tumor effect of αFR-CAR-NK 1) A human ovarian cancer xenograft model was established in severe immunodeficient mice B-NDG using the human ovarian cancer cell line SKOV3, and the killing effect of αFR-CAR-NK on ovarian cancer cells was explored in vivo experiments.

[0112] 2) 16 B-NDG mice were intraperitoneally inoculated with 1×10 6 SK-OV-3 cells. Fourteen days after inoculation, the tumor-bearing mice were grouped to receive different treatments. Among them, the tumor-bearing mice in the phosphate buffered saline (PBS) group were intraperitoneally infused with phosphate buffered saline, the tumor-bearing mice in the NK-92-MI group were intraperitoneally infused with 1×10 6 NK-92-MI cells, and the tumor-bearing mice in the αFR-CAR-NK group were intraperitoneally infused with 1×10 6 αFR-CAR-NK cells. The above three treatment methods were carried out once every 3 days for a total of 3 times. The tumor size of the mice was measured weekly after injection. The length and width of the tumor were measured using a vernier caliper, and the tumor volume of the mice was obtained according to the formula tumor volume = long diameter (mm) * short diameter (mm) * short diameter (mm) * 1 / 2.

[0113] The results were as Figure 6 shown that αFR-CAR-NK could significantly prolong the survival time of tumor-bearing mice. αFR-CAR-NK could significantly inhibit tumor growth. The constructed αFR-CAR-NK could specifically and efficiently kill folate receptor α-positive tumor cells.

[0114] Example 3 Construction and anti-tumor activity of bispecific antibody targeting folate receptor α and Siglec-9 3.1 Construction of bispecific antibody plasmid 1) The structure of the bispecific antibody adopts the scFv-IgG structure. The anti-FRα full-length IgG1 antibody is derived from 1.5.1, and the anti-Siglec-9 antibody sequence is derived from the literature (Development of Siglec-9 Blocking Antibody to Enhance Anti-Tumor Immunity) and is fully synthesized by Guangzhou Aiji Biotechnology Co., Ltd. in the form of a single-chain antibody (scFv). The synthesis process is as Figure 7 shown.

[0115] 2) Cloning primers and PCR reaction conditions; 3) Prepare a 1.0% (w / v) agarose gel and electrophoretically detect the PCR products. Observe the DNA electrophoresis bands in a gel imager and recover the agarose gel of the target band.

[0116] 4) Overlap PCR is used to ligate the Anti-FRα-IgG1 heavy chain gene and the Anti-Siglec-scFv gene. The amplified products are subjected to agarose gel electrophoresis and gel recovery. The reaction system is shown in Table 2: The enzyme digestion system is shown in Table 3; The ligation system is shown in Table 4; Take competent DH5α cells, thaw them in an ice bath for 5 min, take 5 μl of the recombinant product, add it to 50 μl of competent cells, place it on ice for 30 min, heat shock at 42 °C for 45 s, add 450 μl of 2XYT medium without antibiotics, incubate at 37 °C for 60 min for full recovery, and then evenly spread the bacterial solution on a 2XYT plate containing ampicillin and culture it overnight at 37 °C.

[0117] Pick a single colony from the 2XYT plate containing colonies for colony PCR. The colony PCR reaction system is shown in Table 5.

[0118] Sequencing identification is performed on the single colony containing the specific fragment after PCR. After obtaining the sequencing results, compare them with the template nucleotide sequence, select the single colony that is consistent with the template sequence as the positive colony, and perform large-scale amplification and then extract the plasmid for subsequent experiments.

[0119] 3.2 Expression and purification of bispecific antibody 1) Passage Expi293T cells into a 500 ml shake flask with a working volume of 100 ml, and culture them in a shaker at 125 rpm, 37 °C, 8% CO 2Cultured under the conditions that when the cell density reaches 3×10 6 cells / ml, it is used for transient transfection.

[0120] 2) Mix the correctly sequenced heavy-chain plasmid and light-chain plasmid of the antibody extracted on March 9th in a mass ratio of 1:2 with Opti-MEM medium, then add the transfection reagent and mix well. Incubate at room temperature for 15 min, and then dropwise add it to the Expi293T cells to be transfected.

[0121] 3) The transfected cells are cultured in a shake flask on a shaker. During the culture process, nutrients are added to make the cells highly express. After 6 days of culture, collect the culture supernatant and filter it through a 0.22 μm filter membrane.

[0122] 4) Pass the filtered supernatant through a gravity column of rProtein G Beads. Wash the unbound components with PBS buffer, and elute the target antibody protein with a pH 11.0 alkaline buffer and immediately neutralize it.

[0123] 5) Dialyze the eluted antibody in PBS overnight, and further purify it through a Superdex 200 16 / 60 molecular sieve, and collect the eluted fractions.

[0124] Result: After purification, the full-length antibody was detected by reducing SDS-PAGE electrophoresis. As Figure 8 shown, a full-length antibody protein with high purity was obtained.

[0125] 3.3 In vivo experiment to verify the anti-tumor effect of the bispecific antibody 1) Establish a human ovarian cancer xenograft model by subcutaneously inoculating the human ovarian cancer cell line SKOV3 into 6-8-week-old female BALB / c SCID mice, and explore the killing effect on ovarian cancer cells in vivo experiments.

[0126] 2) Twenty female BALB / c SCID mice were subcutaneously inoculated with 1×10 6 SK-OV-3 cells. Fourteen days after inoculation, the tumor-bearing mice were intravenously injected with 3×10 6 THP-1 cells and then grouped to receive different treatments. Among them, the tumor-bearing mice in the phosphate buffered saline (PBS) group were intravenously infused with phosphate buffered saline, the tumor-bearing mice in the control antibody group were intravenously infused with the monoclonal antibody drugs αFR Ab or αsiglec9 Ab (5 mg / kg), and the tumor-bearing mice in the bispecific antibody group were intravenously infused with an equimolar amount of the bispecific antibody drug αFR-siglec9 Ab (6.8 mg / kg). The above three treatment methods were all carried out once every 3 days for a total of 3 times. The tumor size of the mice was measured weekly after injection.

[0127] The results are as Figure 9As shown, the αFR-siglec9 bispecific antibody can significantly prolong the survival time of tumor-bearing mice. The αFR-siglec9 bispecific antibody can significantly inhibit tumor growth.

[0128] Example 4 Construction and Antitumor Activity of Folate Receptor α-Targeted Drug Conjugates 4.1 Construction of Antibody Plasmids 1) The full-length IgG1 antibody against FRα is derived from 1.5.1, in which two heavy-chain cysteines (Cys220, Cys229; EU numbering) are mutated to serine.

[0129] 4.2 Expression and Purification of Antibodies 1) Expi293T cells are passaged in a 500 ml shake flask with a working volume of 100 ml and cultured in a shaker at 125 rpm, 37 °C, and 8% CO 2 under the condition of, and used for transient transfection when the cell density reaches 3×10 6 cells / ml.

[0130] 2) The antibody heavy-chain plasmid and light-chain plasmid with correct sequencing results in 4.1 are mixed with Opti-MEM medium at a mass ratio of 1:2, then mixed with transfection reagent, incubated at room temperature for 15 min, and then added dropwise to the Expi293T cells to be transfected.

[0131] 3) The transfected cells are cultured in a shake flask on a shaker, and nutrients are added during the culture process to enable high expression of the cells. After 6 days of culture, the culture supernatant is collected and filtered through a 0.22 μm filter membrane.

[0132] 4) The filtered supernatant is passed through an rProtein G Beads gravity column, the unbound fraction is washed with PBS buffer, and the target antibody protein is eluted with a pH 11.0 alkaline buffer and immediately neutralized.

[0133] 5) The eluted antibody is dialyzed overnight in PBS, the dialyzed antibody is collected, and further purified by a Superdex 200 16 / 60 molecular sieve using a protein purifier, and the eluted fractions are collected.

[0134] Result: After purification, the full-length antibody was detected by reducing SDS-PAGE electrophoresis. As Figure 10 shown, a high-purity full-length antibody protein was obtained.

[0135] 4.3 Antibody-Drug Conjugation 1) Add 80 µM tris(2-carboxyethyl)phosphine (TCEP) and 1 mM ethylenediaminetetraacetic acid (EDTA) to PBS, and reduce the antibody (10 µM) at 37 °C for 2 hours.

[0136] 2) The reducing antibody was cooled on ice and then 40 µM maleimide-valine-citrulline-p-aminobenzoylcarbonyl-monomethyl eosin E (MMAE) was added.

[0137] 3) After incubation on ice for 4 h, extensive dialysis was performed with PBS to remove unbound MMAE.

[0138] 4) The conjugated antibody was analyzed using size exclusion chromatography (Yarra 3000) and hydrophobic interaction chromatography (HIC; TOSOH TSKgel) to determine the drug-to-antibody ratio (DAR). In hydrophobic interaction chromatography, solvent A was 10 mM potassium dihydrogen phosphate with 1.5 M ammonium sulfate at pH 7.0, and solvent B was 10 mM potassium dihydrogen phosphate with 20% (v / v) isopropanol at pH 7.0. The gradient method used was 0 - 5 min (0% B), 5 - 15 min (0% - 100% B), and 15 - 20 min (100% B), with a flow rate of 0.5 mL / min.

[0139] 4.4 In vivo experiments to verify the antitumor effect of ADC 1) 1 x 10 6 SKOV3 cells were implanted subcutaneously into female BALB / c SCID mice, 6 - 8 weeks old.

[0140] 2) When the tumor volume reached 50 - 100 mm 3 , the mice were randomly grouped and injected intravenously with 2 mg / kg antibody, ADC, or vehicle twice, with a three-week interval between administrations.

[0141] 3) The tumors were measured every 3 - 4 days during the experiment. The length and width of the tumors were measured using vernier calipers, and the tumor volume of the mice was obtained according to the formula tumor volume = long diameter (mm) * short diameter (mm) * short diameter (mm) * 1 / 2.

[0142] 4) Mice with a tumor volume exceeding 1000 mm 3 were euthanized.

[0143] The results were as Figure 11 shown. The αFR-ADC drug could significantly prolong the survival time of tumor-bearing mice. The αFR-ADC drug could significantly inhibit tumor growth.

[0144] The above-disclosed are only the preferred embodiments of the present invention. The preferred embodiments do not describe in detail all the details, nor do they limit the invention to the specific embodiments described. Obviously, many modifications and variations can be made according to the content of this specification. These embodiments are selected and specifically described in this specification in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can make good use of the present invention. The present invention is only limited by the claims and their full scope and equivalents.

[0145] Under the teachings of the present invention and the above embodiments, those skilled in the art can easily foresee that the present invention can be implemented by each of the raw materials or their equivalent replacements, each processing method or their equivalent replacements listed or exemplified in the present invention, and the upper and lower limit values and interval values of the parameters of each raw material and processing method can also implement the present invention. Embodiments are not listed one by one here.

Claims

1. A fully human antibody polypeptide fragment targeting folate receptor α, characterized in that: Antibody polypeptide fragments include: (a) a heavy chain variable region, the amino acid sequence of which is shown in SEQ ID NO.1; and (b) a light chain variable region, the amino acid sequence of which is shown in SEQ ID NO.2; Wherein, the heavy chain variable region comprises complementary determining regions HCDR1, HCDR2 and HCDR3, and the amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13, respectively; Wherein, the light chain variable region comprises complementary determining regions LCDR1, LCDR2, and LCDR3, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively.

2. The fully human antibody polypeptide fragment targeting folate receptor α according to claim 1, characterized in that: The framework region of the heavy chain variable region belongs to HYPERLINK "https: / / www.imgt.org / IMGT_vquest / analysis" \l "sequence1_alv"IGHV3-30 and contains a sequence selected from SEQ ID NO.3-6, and the framework region of the light chain variable region belongs to IGKV3-20 and contains a sequence selected from SEQ ID NO.7-10.

3. A chimeric antigen receptor targeting folate receptor α, characterized in that: The composition of the chimeric antigen receptor includes: Signal peptide; The antibody polypeptide fragment according to any one of claims 1 or 2; Hinge area; Transmembrane segment; intracellular co-stimulatory domain; Intracellular signaling domain.

4. The chimeric antigen receptor targeting folate receptor α according to claim 3, characterized in that The signal peptide is a human CD8α signal peptide, and its amino acid sequence is shown in SEQ ID NO.17; The amino acid sequence of the hinge region is shown in SEQ ID NO.18; The transmembrane segment is selected from any one of the following: human CD28α transmembrane segment, human NKG2D transmembrane segment, human CD8α transmembrane segment; the amino acid sequence of the human CD28α transmembrane segment is shown in SEQ ID NO.19; the amino acid sequence of the human NKG2D transmembrane segment is shown in SEQ ID NO.20; The amino acid sequence of the transmembrane segment of human CD8α is shown in SEQ ID NO.21; The intracellular co-stimulatory domain is selected from any one of the following: human 2B4 intracellular segment, human CD28 intracellular segment, human 4-1BB intracellular segment; the amino acid sequence of the human 2B4 intracellular segment is shown in SEQ ID NO.22; the amino acid sequence of the human CD28 intracellular segment is shown in SEQ ID NO.23; the amino acid sequence of the human 4-1BB intracellular segment is shown in SEQ ID NO.24; The intracellular signal transduction domain is the CD3ζ intracellular segment, and its amino acid sequence is shown in SEQ ID NO.

25.

5. A host cell, characterized in that The host cell comprises the expression vector of the chimeric antigen receptor according to any one of claims 3 or 4.

6. The host cell according to claim 5, characterized in that The host cell is selected from one of NK-92 cells, human primary T cells, human primary NK cells, mononuclear macrophages or cytokine-induced killer cells.

7. A bifunctional bispecific antibody, characterized in that: The bispecific antibody comprises: (a) the fully human antibody polypeptide fragment of claim 1 or 2; (b) Secondary antibody fragment.

8. The bifunctional and bispecific antibody according to claim 7, characterized in that: The second antibody targets an antigen selected from PD-1, CD3, CTLA4, EGFR, HER2, CD276 or Siglec9.

9. An antibody-drug conjugate, characterized in that: Comprising the fully human antibody polypeptide fragment according to any one of claims 1 or 2.

10. Use of the fully human antibody polypeptide fragment of any one of claims 1-2, the chimeric antigen receptor targeting folate receptor α of any one of claims 3-4, the host cell of any one of claims 5-6, the bifunctional bispecific antibody of any one of claims 7-8, or the antibody-drug conjugate of claim 9 in the preparation of a drug for preventing or treating folate receptor α-positive malignant tumors.

11. The use according to claim 10, characterized in that: The malignant tumor is ovarian cancer, endometrial cancer, breast cancer, lung cancer or colon cancer.

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