Humanized Anti-alpha-2-delta-1 monoclonal antibody and application thereof

Abstract

Provided herein are a humanized anti-alpha-2-delta-1 monoclonal antibody and an application thereof. Specifically provided is a humanized antibody, or a biologically active fragment derived from the antibody, that binds to a voltage-gated calcium channel alpha-2-delta-1 subunit (subtype 5). The antibody or the biologically active fragment derived from the antibody, either alone or in combination with lenvatinib, is capable of significantly inhibiting the self-renewal capacity of liver cancer cells and the growth of transplanted tumors in animals. The antibody, either alone or in combination with lenvatinib, can be used for a combination of liver cancer drugs targeting liver cancer stem cells, and can also be used for the in vivo diagnosis of alpha-2-delta-1+ tumors.

Description

Humanized anti-alpha2delta1 monoclonal antibody and application thereof TECHNICAL FIELD

[0001] The present application relates to an anti-alpha2delta1 monoclonal antibody and application thereof, in particular to a humanized anti-alpha2delta1 monoclonal antibody and application thereof in preparing a medicament for treating and / or diagnosing a tumor or other diseases related to voltage-gated calcium ion channel alpha2delta1 subunit. BACKGROUND

[0002] Ion channels mediating ion into cells such as K + , Cl – , Ca 2+ channels play an important role in the functional activities of normal cells and the balance of the body. In recent years, more and more evidence shows that abnormal expression and / or function of genes mediating these ion channels are associated with the occurrence and development of diseases such as individual abnormal development, inflammation, hypertension and tumor, and targeting ion channels mediating ion into and out of cells has become an important strategy for intervention of these diseases. Current targeting strategies mainly use small molecule antagonists of these channels, but small molecule antagonists generally have poor targeting specificity and often target multiple molecular targets at the same time. Monoclonal antibodies have strong molecular targeting specificity and can induce antibody-dependent cell killing effect, but so far no monoclonal antibody targeting any ion channel has entered clinical application.

[0003] Voltage-gated calcium channel a2d1 subunit is an auxiliary subunit of voltage-gated calcium channel encoded by gene CACNA2D1. As a GPI (glycolphosphatidylinositol)-anchored protein, it can interact with a1 subunit of voltage-gated calcium channel and recruit and stabilize it on cell membrane, thus regulating the opening and closing of calcium channel to control the calcium ion into the cell. It is known that there are five subtypes of a2d1 in humans, which are formed by different exon splicing of the same gene. Among them, subtype 1 is expressed in skeletal muscle, subtype 2 is expressed in central nervous system, subtypes 2, 4 and 5 are expressed in neuroblastoma cells, and subtypes 4 and 5 are expressed in aorta (https: / / www.uniprot.org / UniProtKB / P54289 / entry*expression). a2d1-mediated calcium influx plays a key role in a variety of cellular activities such as gene expression regulation, cell metabolism, proliferation, movement, cell survival and death. Abnormal expression and / or function of a2d1 has been proved to be closely related to a variety of diseases such as neuropathic pain, epilepsy, etc. Small molecule drugs targeting this molecule such as Gabapentin have been clinically used for the treatment of epilepsy, neuropathic pain, etc.

[0004] The inventors' team in the prior research (CN103562403B) used Hep-11 and Hep-12 cell pairs derived from the primary and recurrent tissues of the same liver cancer patient, obtained a mouse monoclonal antibody 1B50-1 against the recurrent liver cancer-derived cell line by differential immunization and hybridoma technology, and the antibody specifically recognized voltage-gated calcium channel α2δ1 subunit subtype 5. It is further proved that the α2δ1 subunit is a surface marker and a therapeutic target of liver cancer stem cells, which plays a role in tumor stem cell self-renewal, tumorigenicity and other characteristics through regulating calcium influx to activate calcium signaling. The mouse 1B50-1 antibody can inhibit calcium influx and thus inhibit the expression of tumor stem cell stemness-related genes, self-renewal, drug resistance and tumorigenicity, so it has the potential to reduce the content of tumor stem cells and thus has application prospects in liver cancer treatment. Subsequent studies have shown that α2δ1 can also be used as a functional marker and therapeutic target of gastric cancer, breast cancer, non-small cell lung cancer, small cell lung cancer and pancreatic cancer tumor stem cells, and the mouse monoclonal antibody 1B50-1 targeting α2δ1 also has the effect of inhibiting tumor stem cells of these tumors and thus has a therapeutic effect on these tumors. These studies show that the 1B50-1 antibody is a candidate anti-tumor drug targeting voltage-gated calcium channels, but due to the immunogenicity of mouse antibodies applied to humans, it can activate the human immune system to cause human anti-mouse antibody (HAMA) reaction, reduce the effect of monoclonal antibody drugs and cause adverse reactions, thus limiting its feasibility as a clinical tumor treatment drug. Therefore, the development of humanized or fully human antibody drugs that specifically target α2δ1 is expected to provide new drugs for liver cancer, lung cancer, pancreatic cancer and other tumors. SUMMARY

[0005] One object of the present application is to provide a humanized anti-α2δ1 monoclonal antibody or a biologically active fragment derived from the monoclonal antibody that can specifically bind to human α2δ1, which has no or low immunogenicity in the human body.

[0006] Another object of the present application is to provide a gene encoding the humanized anti-α2δ1 monoclonal antibody or a biologically active fragment derived from the monoclonal antibody that can specifically bind to human α2δ1, and a vector or cell containing the gene.

[0007] Another object of the present application is to provide a method for preparing the humanized anti-α2δ1 monoclonal antibody or a biologically active fragment derived from the monoclonal antibody that can specifically bind to human α2δ1.

[0008] Another object of the present application is to provide the use of the humanized anti-α2δ1 monoclonal antibody or a biologically active fragment derived from the monoclonal antibody that can specifically bind to human α2δ1 in the preparation of a tumor treatment drug.

[0009] Another object of the present application is to provide a pharmaceutical composition containing the humanized anti-α2δ1 monoclonal antibody or a biologically active fragment derived from the monoclonal antibody capable of specifically binding to human α2δ1.

[0010] Another object of the present application is to provide a kit capable of detecting α2δ1.

[0011] In one aspect, the present application provides an anti-α2δ1 monoclonal antibody or a biologically active fragment derived from the antibody, and in particular, the present application provides an anti-α2δ1 monoclonal antibody or a biologically active fragment derived from the antibody which binds to any one or more of amino acid sites of K49, N55, Q56, D59, E62, K63, Y64, Q65, D66, R624, E695, Q698, N699, K703, Y782, Q784, K786, and N805 of human voltage-gated calcium channel α2δ1 subunit (subtype 5), wherein the protein sequence of the α2δ1 subunit (subtype 5) is shown in SEQ ID NO. 1.

[0012] According to a specific embodiment of the present application, the biologically active fragment in the present application is capable of specifically binding to human α2δ1.

[0013] According to a specific embodiment of the present application, the antibody in the present application can be a human antibody, a humanized antibody, a chimeric antibody, a single chain antibody, or a single domain antibody.

[0014] According to a specific embodiment of the present application, the antibody or a biologically active fragment derived from the antibody of the present application comprises:

[0015] (a) a heavy chain variable region having an amino acid sequence selected from the group consisting of: an amino acid sequence shown in SEQ ID No. 2 or SEQ ID No. 3, or an amino acid sequence having equivalent function which is formed by substitution, deletion, or addition of one or more amino acids based on the amino acid sequence shown in SEQ ID No. 2 or SEQ ID No. 3; and / or

[0016] (b) a light chain variable region having an amino acid sequence selected from the group consisting of: an amino acid sequence shown in SEQ ID No. 4 or SEQ ID No. 5, or an amino acid sequence having equivalent function which is formed by substitution, deletion, or addition of one or more amino acids based on the amino acid sequence shown in SEQ ID No. 4 or SEQ ID No. 5.

[0017] According to a specific embodiment of the present application, the antibody or the biologically active fragment derived from the antibody of the present application, the heavy chain variable region of the antibody comprises the complementarity determining region amino acid sequences as shown in SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8. Preferably, the heavy chain variable region amino acid sequence comprises the complementarity determining regions CDRH1 (SEQ ID No. 6), CDRH2 (SEQ ID No. 7), CDRH3 (SEQ ID No. 8).

[0018] According to a specific embodiment of the present application, the antibody or the biologically active fragment derived from the antibody of the present application, the light chain variable region of the antibody comprises the complementarity determining region amino acid sequences as shown in SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11. Preferably, the light chain variable region amino acid sequence of the antibody comprises the complementarity determining regions CDRL1 (SEQ ID No. 9), CDRL2 (SEQ ID No. 10), CDRL3 (SEQ ID No. 11).

[0019] According to a specific embodiment of the present application, the antibody or the biologically active fragment derived from the antibody of the present application, the amino acids at positions 35, 57, 58, 59, 66, 107, 110 of the heavy chain are S, Y, W, D, R, R, G respectively.

[0020] According to a specific embodiment of the present application, the antibody or the biologically active fragment derived from the antibody of the present application, the amino acids at positions 36, 38, 56, 67, 108, 114 of the light chain are G, S, Y, D, N, W respectively.

[0021] According to a specific embodiment of the present application, the antibody or the biologically active fragment derived from the antibody of the present application, the amino acid at position 37 of the light chain is T or R.

[0022] According to some specific embodiments of the present application, the heavy chain variable region amino acid sequence of the humanized anti-α2δ1 monoclonal antibody of the present application is as shown in SEQ ID No. 2, or an amino acid sequence with equivalent function formed by replacing, deleting or adding one or several amino acids of the sequence; the light chain variable region amino acid sequence of the antibody is as shown in SEQ ID No. 4, or an amino acid sequence with equivalent function formed by replacing, deleting or adding one or several amino acids of the sequence. The present application has been experimentally proven that the affinity of the humanized antibody to α2δ1 is not lower than the affinity of the corresponding murine antibody, and the humanized antibody can significantly inhibit the proliferation of hepatocellular carcinoma Hep-12 cells and α2δ1 +The in vitro self-renewal ability of liver cancer cells and the growth of liver cancer cell line animal transplanted tumors, especially the inhibitory effect is more remarkable when combined with Lenvatinib.

[0023] In another aspect, the present application also provides a polynucleotide encoding the anti-α2δ1 monoclonal antibody or the biologically active fragment derived from the antibody according to the present application. Preferably, the polynucleotide can be DNA or RNA.

[0024] According to a specific embodiment of the present application, the polynucleotide of the present application comprises: a nucleotide sequence encoding an amino acid sequence as shown in SEQ ID No. 2 or SEQ ID No. 3, and / or a nucleotide sequence encoding an amino acid sequence as shown in SEQ ID No. 4 or / and SEQ ID No. 5.

[0025] In another aspect, the present application also provides a vector containing the polynucleotide according to the present application.

[0026] In another aspect, the present application also provides a host cell containing the polynucleotide according to the present application or the vector according to the present application.

[0027] In another aspect, the present application also provides a fusion protein comprising the amino acid sequence of the anti-α2δ1 monoclonal antibody or the biologically active fragment derived from the antibody according to the present application. The fusion protein is formed by connecting the anti-α2δ1 monoclonal antibody or the biologically active fragment derived from the antibody according to the present application with other proteins, which can be directly connected or connected through a linker.

[0028] In another aspect, the present application also provides a chimeric antigen receptor T cell expressing the anti-α2δ1 monoclonal antibody or the biologically active fragment derived from the antibody according to the present application. According to a specific embodiment of the present application, the present application constructs a chimeric antigen receptor T cell of the anti-α2δ1 antibody, which has a specific killing effect on α2δ1+ liver cancer stem cells and a therapeutic effect on hepatocellular carcinoma in animal transplanted tumors.

[0029] In another aspect, the present application also provides a multispecific antibody comprising the anti-α2δ1 monoclonal antibody or the biologically active fragment derived from the antibody according to the present application. Preferably, the multispecific antibody is a bispecific antibody. According to a specific embodiment of the present application, the present application provides a bispecific antibody, which is a bispecific antibody formed by the anti-α2δ1 monoclonal antibody or the biologically active fragment derived from the antibody according to the present application and an antibody targeting CD3. The antibody targeting CD3 can use the CD3 antibody in the prior art.

[0030] In another aspect, the present application also provides a method for preparing the anti-α2δ1 monoclonal antibody or the biologically active fragment derived from the antibody according to the present application, which comprises the following steps:

[0031] (a) providing the vector according to the present application;

[0032] (b) introducing the vector according to step (a) into a cell;

[0033] (c) culturing the cell according to step (b) under conditions suitable for expression of the monoclonal antibody; and

[0034] (d) isolating and purifying the monoclonal antibody.

[0035] In another aspect, the present application provides use of the humanized anti-α2δ1 monoclonal antibody or the biologically active fragment derived from the monoclonal antibody capable of specifically binding to human α2δ1 according to the present application or the fusion protein according to the present application or the chimeric antigen receptor T cell according to the present application or the multispecific antibody according to the present application in the preparation of a medicament for preventing and treating tumors.

[0036] In another aspect, the present application also provides use of the anti-α2δ1 monoclonal antibody or the biologically active fragment derived from the antibody according to the present application or the polynucleotide according to the present application or the fusion protein according to the present application or the chimeric antigen receptor T cell according to the present application or the multispecific antibody according to the present application in the preparation of a medicament for treating and / or detecting tumors.

[0037] In another aspect, the present application also provides a method for treating tumors, which comprises administering to a subject in need an effective amount of the anti-α2δ1 monoclonal antibody or the biologically active fragment derived from the antibody according to the present application or the polynucleotide according to the present application or the fusion protein according to the present application or the chimeric antigen receptor T cell according to the present application or the multispecific antibody according to the present application.

[0038] According to some embodiments of the present application, the humanized anti-α2δ1 monoclonal antibody or the biologically active fragment derived from the monoclonal antibody capable of specifically binding to human α2δ1 according to the present application or the fusion protein according to the present application or the chimeric antigen receptor T cell according to the present application or the multispecific antibody according to the present application can inhibit self-renewal of tumor cells as a tumor treatment drug, thereby having a therapeutic effect, especially in combination with lenvatinib.

[0039] According to some embodiments of the present application, the tumor is liver cancer.

[0040] In another aspect, the present application also provides a pharmaceutical composition comprising: an effective amount of the anti-a2d 1 monoclonal antibody or a biologically active fragment derived from the antibody or a polynucleotide encoding the a2d 1 monoclonal antibody or a biologically active fragment capable of specifically binding to a2d 1 of the monoclonal antibody or the fusion protein or the chimeric antigen receptor T cell or the multispecific antibody according to the present application.

[0041] According to a specific embodiment of the present application, the pharmaceutical composition further comprises: other pharmaceutically active ingredients having synergistic effects in combination with the anti-a2d 1 monoclonal antibody or a biologically active fragment derived from the antibody or the polynucleotide or the fusion protein or the chimeric antigen receptor T cell or the multispecific antibody according to the present application; and a pharmaceutically acceptable solvent and / or carrier.

[0042] According to some specific embodiments of the present application, the other pharmaceutically active ingredients include lenvatinib.

[0043] In another aspect, the present application also provides a kit for detecting a2d 1 level, comprising: the anti-a2d 1 monoclonal antibody or a biologically active fragment derived from the antibody according to the present application.

[0044] According to a specific embodiment of the present application, the anti-a2d 1 monoclonal antibody or a biologically active fragment derived from the antibody in the kit of the present application is labeled with fluorescein or a radioactive marker.

[0045] According to a specific embodiment of the present application, the kit of the present application further comprises: a secondary antibody, an enzyme or fluorescent or radioactive marker for detection, and a buffer. Preferably, the secondary antibody can be an anti-antibody against the anti-a2d 1 monoclonal antibody.

[0046] In some embodiments of the present application, a particle complex of a humanized α2δ1 antibody and its antigen α2δ1 (subtype 5, the amino acid sequence of which is shown in SEQ ID N No. 1) is prepared, and the structure of the complex is analyzed by cryo-EM. The particle cryo-EM structure of the antigen-antibody complex shows that the binding of the humanized α2δ1 antibody to its antigen α2δ1 results in the flexibility of the region of α2δ1 that binds to the α1 subunit of the calcium ion channel, suggesting that the humanized anti-α2δ1 antibody can interfere with the binding of the α2δ1 subunit to the α1 subunit and thus inhibit the influx of calcium ions. To verify this hypothesis, an expression vector of α2δ1 (subtype 5) with an HA tag is introduced into Hep-11 cells, with or without the addition of a humanized anti-α2δ1 antibody, and then an anti-HA tag antibody is used for immunoprecipitation. In cells without the addition of the humanized anti-α2δ1 antibody, the anti-HA tag antibody can successfully co-precipitate the α1F (a subtype of the α1 subunit) subunit, while in cells with the addition of the humanized anti-α2δ1 antibody, the α1F precipitated by the anti-HA tag antibody is significantly reduced, proving that the humanized anti-α2δ1 antibody can indeed inhibit the binding of α2δ1 to the α1 subunit, which is the main molecular mechanism of its inhibition of calcium ion influx, in vitro self-renewal, and in vivo anticancer. The particle cryo-EM structure of the antigen-antibody complex also reveals the amino acid sites that mediate the interaction between the humanized anti-α2δ1 antibody and the α2δ1 subunit (subtype 5). The amino acid sites on the heavy chain of the humanized anti-α2δ1 antibody involved in the interaction are S35, Y57, W58, D59, D64, R66, R107, T109, G110, T113, and the amino acid sites on the light chain are Q27, S28, G36, S38, E55, Y56, D67, S70, T107, N108, S109, and W114; the amino acid sites of the α2δ1 subunit (subtype 5) that interact with the antibody are K49, N55, Q56, D59, E62, K63, Y64, Q65, D66, R624, E695, Q698, N699, K703, Y782, Q784, and K786, N805. Further determination of the affinity of the antigen-antibody interaction after mutation of these sites shows that the affinity of the α2δ1 subunit (subtype 5) N55A, D59K, E62K, K63D sites to the humanized anti-α2δ1 antibody is significantly reduced or disappears. The affinity of the humanized anti-α2δ1 antibody heavy chain S35D, Y57A, W58A, D59A, R66A, R107A, G110W and light chain G36W, S38W, Y56A, D67A, N108W, W114A sites to the α2δ1 subunit (subtype 5) is significantly reduced or disappears. The affinity of the light chain T37R site to the α2δ1 subunit (subtype 5) is significantly increased.Therefore, one feature of the humanized anti-a2d1 antibody of the present application is to recognize one or more of the amino acids in the voltage-gated calcium channel a2d1 subunit (subtype 5) K49, N55, Q56, D59, E62, K63, Y64, Q65, D66, R624, E695, Q698, N699, K703, Y782, Q784, K786, N805 amino acid sites, preferably at least N55, D59, E62, K63 amino acid sites. Another feature of the humanized anti-a2d1 antibody of the present application is that the amino acids at positions 35, 57, 58, 59, 66, 107, 110 of the heavy chain are S, Y, W, D, R, R, G, and the amino acids at positions 36, 38, 56, 67, 108, 114 of the light chain are G, S, Y, D, N, W. Preferably, the amino acid at position 37 of the light chain variable region sequence (SEQ ID No. 5) of the humanized anti-a2d1 antibody is R.

[0047] In general, the humanized anti-a2d1 monoclonal antibody provided by the present application can inhibit the binding of a2d1 to the voltage-gated calcium channel a1 subunit to inhibit the influx of calcium ions, thereby inhibiting the self-renewal of tumor cells, overcoming resistance to lenvatinib, having good application prospects in the treatment of liver cancer, especially in the treatment of targeting liver cancer stem cells, and in the in vivo tracing and in vitro tissue detection of a2d1.

[0048] Definitions

[0049] In the present application, some terms used have the following meanings:

[0050] The term "antibody" used in the present application refers to an antibody molecule capable of specifically binding to a target polypeptide or a target sequence, and encompasses an intact antibody molecule or a fragment thereof, including an antigen-binding fragment thereof.

[0051] The term "antigen-binding fragment" used in the present application refers to any antibody fragment that retains the ability to specifically bind to a target protein, polypeptide or sequence, which includes a single-chain antibody, a Fab fragment, a F(ab')2 fragment, a disulfide-linked single-chain antibody (sFv), and a fragment containing any one of a light chain variable region (VL) and / or a heavy chain variable region (VH) or a fragment containing a CDR that specifically binds to a target protein, polypeptide or sequence, as well as fragments obtained by various methods well known in the art for obtaining the above antibody fragments.

[0052] The term "complementarity determining region" (CDR) as used herein refers to the regions of an antibody that recognize and bind an antigen, directly determining the specificity of the antibody, or specific amino acid sequences that determine the antigen-binding activity of an antibody. The CDR sequences in the present application are obtained by the website www.abysis.org / abysis / index.html using the Kabat scheme, which can be different from the sequences predicted using other schemes such as Chothia, IMGT, etc.

[0053] The term "specifically recognizes" as used herein refers to the ability of an antibody or antigen-binding fragment thereof to specifically bind to a target protein, polypeptide or sequence. The antibody does not non-specifically bind to other polypeptides or proteins. Preferably, an antibody or antigen-binding fragment thereof that specifically recognizes α2δ1 (subtype 5) does not cross-react with other antigens.

[0054] The antibody heavy chain and light chain amino acid position numbering in the present application, unless otherwise specified, refers to the numbering of the antibody heavy chain and light chain variable region amino acids by the IMGT scheme (see Figure 1A-1D for IMGT scheme numbering of antibody sequence amino acid positions).

[0055] The term "effective amount" refers to a "therapeutically effective anti-proliferative amount" or a "prophylactically effective anti-proliferative amount". The term includes an amount effective, at dosages and for periods of time necessary, to achieve a desired result, such as sufficient to treat a cell proliferative disorder. The effective amount of a compound of the present application can vary according to factors such as the disease state, age, and weight of the subject, and the ability of the compound of the present application to elicit a desired response in the subject. Dosage regimens can be adjusted to provide the optimum therapeutic response. An effective amount is also one in which any toxic or detrimental effects (e.g., side effects) of the compound of the present application are outweighed by the therapeutically beneficial effects. BRIEF DESCRIPTION OF DRAWINGS

[0056] FIG. 1A-1D show the humanized a2d1 antibody heavy chain variable region and light chain variable region candidate sequences designed in the examples based on the murine anti-a2d1 antibody 1B50-1. FIG. 1A. Comparison of the designed candidate heavy chain variable region with the murine heavy chain variable region, germline antibody sequence, the background labeled amino acids show the amino acids that are different from the murine antibody; FIG. 1B. Comparison of the designed candidate heavy chain variable region with the murine heavy chain variable region, germline antibody sequence, the background labeled amino acids show the amino acids that are different from the germline antibody sequence; FIG. 1C. Comparison of the designed candidate light chain variable region with the murine light chain variable region, germline antibody sequence, the background labeled amino acids show the amino acids that are different from the murine antibody; FIG. 1D. Comparison of the designed candidate light chain variable region with the murine light chain variable region, germline antibody sequence, the background labeled amino acids show the amino acids that are different from the germline antibody sequence. 1B50-VH Parent: murine anti-a2d1 antibody 1B50-1 heavy chain variable region; 1B50-VH VH1: designed humanized anti-a2d1 antibody heavy chain variable region sequence 1; 1B50-VH VH2: designed humanized anti-a2d1 antibody heavy chain variable region sequence 2; 1B50-VH IGHV2-5*08 / IGHJ4*03: human germline heavy chain variable region gene locus sequence; 1B50-VL Parent: murine anti-a2d1 antibody 1B50-1 light chain variable region; 1B50-VL VL1: designed humanized anti-a2d1 antibody light chain variable region sequence 1; 1B50-VL VL2: designed humanized anti-a2d1 antibody light chain variable region sequence 2; 1B50-VL IGKV3D-15*01 / IGKJ2*01: human germline light chain variable region gene locus sequence.

[0057] FIG. 2A-2C show the gel electrophoresis and affinity assay results of the purified humanized anti-a2d1 antibody expressed in the examples. In FIG. 2A and FIG. 2B, the results of the denaturing and non-denaturing polyacrylamide gel electrophoresis analysis of the first (lane 1) and second (lane 2) purification products, respectively, it can be seen from the figures that there are no obvious impurity bands in the two purified antibodies, meeting the requirements of antibody affinity purification purity. FIG. 2C is a flow cytometry analysis to detect the affinity of the humanized antibody, the affinity constant pKa of each group of antibodies is less than 10 -9 M, meeting the requirements of therapeutic antibody affinity evaluation, wherein the affinity of the hIgG1 VH2 VL1 and hIgG3 VH1 VL1 type humanized antibodies to the murine monoclonal antibody m1B50-1 is the closest.

[0058] FIG. 3A-FIG. 3D show the mechanism of action revealed by the cryo-EM structure of the humanized anti-a2d1 antibody (Fab fragment) and antigen a2d1 complex particles and the results of co-immunoprecipitation verification. FIG. 3A is a structure diagram of the three-dimensional structure reconstruction of the antigen-antibody complex protein particles, FIG. 3B is a structure diagram of the antigen-antibody complex protein particles built by the software, FIG. 3C is the results of Western blot of the products immunoprecipitated by the HA tag antibody of the Hep-11 cells transfected with HA-a2d1 treated with control IgG and humanized anti-a2d1 antibody 1B50-1 respectively, and then subjected to Western blot with anti-a2d1 antibody and anti-CACNA1F antibody, which is an antibody against the a1 subunit of the voltage-gated calcium channel, indicating that the humanized anti-a2d1 antibody 1B50-1 can inhibit the binding of a2d1 and the a1 subunit CACNA1F subtype, and FIG. 3D is the results of Western blot of the products immunoprecipitated by the humanized anti-a2d1 antibody (h1B50-1) of the Hep-12 cells, and then subjected to Western blot with anti-a2d1 antibody and anti-CACNA1F antibody, indicating that the humanized anti-a2d1 antibody cannot immunoprecipitate the CACNA1F subunit. FIG. 3A-FIG. 3D show the mechanism of action revealed by the cryo-EM structure of the humanized anti-a2d1 antibody (Fab fragment) and antigen a2d1 complex particles and the results of co-immunoprecipitation verification. FIG. 3A is a structure diagram of the three-dimensional structure reconstruction of the antigen-antibody complex protein particles, FIG. 3B is a structure diagram of the antigen-antibody complex protein particles built by the software, FIG. 3C is the results of Western blot of the products immunoprecipitated by the HA tag antibody of the Hep-11 cells transfected with HA-a2d1 treated with control IgG and humanized anti-a2d1 antibody 1B50-1 respectively, and then subjected to Western blot with anti-a2d1 antibody and anti-CACNA1F antibody, which is an antibody against the a1 subunit of the voltage-gated calcium channel, indicating that the humanized anti-a2d1 antibody 1B50-1 can inhibit the binding of a2d1 and the a1 subunit CACNA1F subtype, and FIG. 3D is the results of Western blot of the products immunoprecipitated by the humanized anti-a2d1 antibody (h1B50-1) of the Hep-12 cells, and then subjected to Western blot with anti-a2d1 antibody and anti-CACNA1F antibody, indicating that the humanized anti-a2d1 antibody cannot immunoprecipitate the CACNA1F subunit.

[0059] FIG. 4A-FIG. 4D are the results of identification of the key amino acids of the humanized anti-a2d1 antibody and a2d1 in the embodiment. FIG. 4A is a density map of the amino acid interaction interface of the local region revealed by the cryo-EM particle complex structure of the antigen-antibody, and the amino acid numbers in the figure are the natural sequence numbers of the heavy chain (the signal peptide contains 19 amino acids) and the light chain (the signal peptide contains 20 amino acids) amino acids; FIG. 4B is the results of affinity determination of the key amino acid site mutant of a2d1 and the humanized anti-a2d1 antibody revealed by ITC; FIG. 4C is the affinity results of the key amino acid mutant of the heavy chain variable region of the humanized anti-a2d1 antibody and a2d1; and FIG. 4D is the affinity results of the key amino acid mutant of the light chain variable region of the humanized anti-a2d1 antibody and a2d1.

[0060] FIG. 5A-FIG. 5D are the results of the inhibitory effect of the humanized anti-a2d1 antibody 1B50-1, lenvatinib, and the combination of the two on the sphere-forming ability of hepatocellular carcinoma Hep-12 and a2d1 + cells sorted from PLC / PRF5, lenvatinib-resistant cell lines Huh7-LR and Hep3B-LR. FIG. 5A and FIG. 5C are representative phase contrast microscope photographs of the spheres, and FIG. 5B and FIG. 5D are histograms of the statistical results of the sphere-forming ability.

[0061] FIGs. 6A-6F show the inhibitory effects of humanized anti-a2d1 antibody h1B50-1, lenvatinib, and their combination on liver cancer cell line hepatocellular orthotopic tumor. Among them, FIGs. 6A-6C are the luciferase fluorescence imaging (FIG. 6A) and the quantitative time curve of the fluorescence intensity of the orthotopic tumor in mice (FIG. 6B) of PLC / PRF5 cell hepatocellular orthotopic tumor in mice before administration (Day 10), 10 days after administration (Day 20), and at the end of administration (Day 30), and FIG. 6C is the Kaplan-Meier survival curve showing the survival time of mice in different treatment groups; FIGs. 6D-6F are the luciferase fluorescence imaging (FIG. 6D) and the quantitative time curve of the fluorescence intensity of the orthotopic tumor in mice (FIG. 6E) of Huh7 cell hepatocellular orthotopic tumor in mice before administration (Day 7), 10 days after administration (Day 17), and at the end of administration (Day 27), and FIG. 6F is the Kaplan-Meier survival curve showing the survival time of mice in different treatment groups.

[0062] FIGs. 7A-7D show the inhibitory effects of chimeric antigen receptor T (CAR-T) cells constructed with humanized anti-a2d1 antibody variable region nucleic acid sequences and CD3-a2d1 bispecific antibodies on hepatocellular carcinoma Hep-12 cell tumor. Among them, FIG. 7A shows a schematic diagram of a third-generation CAT-T vector constructed based on the nucleic acid coding sequences of the heavy chain (VH) and light chain (VL) variable regions of the humanized anti-a2d1 antibody, FIG. 7B shows the tumor formation ability of humanized anti-a2d1 CAR-T effector cells mixed with target cells Hep-12 at different ratios and then inoculated subcutaneously in NOD / SCID mice, FIG. 7C shows the therapeutic effect of humanized anti-a2d1 CAR-T on Hep-12 cell subcutaneous tumor in NOD / SCID mice, and FIG. 7D shows the therapeutic effect of CD3-a2d1 bispecific antibodies on Hep-12 cell subcutaneous tumor in humanized mice, and FIG. 7D shows the growth curves of the control group and the bispecific antibody treatment group.

[0063] FIGs. 8A-8D show the experimental results of the application of humanized anti-a2d1 antibody in tumor diagnosis in the examples. Among them, FIGs. 8A-8C are 64 Cu-labeled humanized anti-a2d1 antibody Fab'2 fragment 64 Cu-1B50-1-Fab'2)(FIG. 8A), pre-injection with unlabeled humanized anti-a2d1 antibody and then injection of 64 Cu-labeled humanized anti-a2d1 antibody Fab'2 fragment 64 Cu-1B50-1-Fab'2block)(FIG. 8B), and 64The results of the radionuclide tracing of Cu-IgG-Fab'2 antibody (Figure 8C) in Hep-12 tumor-bearing mice; Figure 8D shows the detection effect of humanized anti-α2δ1 antibody on aneuploid endothelial cells (ApEC) and circulating tumor cells (ApCTC) in the peripheral blood of patients, wherein the humanized anti-α2δ1 antibody 1B50-1 is directly labeled with FITC and used for the detection of peripheral blood cells of tumor patients. DETAILED DESCRIPTION

[0064] The present application is further illustrated by way of examples, but the present application is not limited to the following examples only.

[0065] The experimental methods used in the following examples are conventional methods and conventional conditions, or the conditions recommended by the instrument manufacturer, unless otherwise specified.

[0066] The materials, reagents, etc. used in the following examples can be obtained commercially, unless otherwise specified.

[0067] The experimental materials in the examples are obtained as shown below:

[0068] HEK293FT, FreeStyle TM 293-F cells were purchased from Invitrogen company, hepatoma cell lines PLC / PRF / 5 and Hep-3B were from ATCC (Ameician Type Culture Collection), hepatocellular carcinoma cell line HuH7 was from Japan Science and Technology Agency, Hep-11 and Hep-12 hepatoma cell lines were established by the inventors (Xu XL, Xing BC, Han HB, Zhao W, Hu MH, Xu ZL, Li JY, Xie Y, Gu J, Wang Y, Zhang ZQ. The properties of tumor-initiating cells from a hepatocellular carcinoma patient's primary and recurrent tumor, Carcinogenesis, 2010; 31(2): 167-74). The sorafenib-resistant cell lines HuH7-LR and Hep3B-LR were established by the inventors by treating HuH7 and Hep3B with low concentration gradient increasing method, respectively. The above cell lines were preserved in the laboratory of the inventors.

[0069] RPMI-1640 medium and 100× double antibodies were purchased from Tianjin Baiyueke Company; FreeStyle TM293-F serum-free medium, fetal bovine serum (FBS) and 10x trypsin were purchased from Gibco company. The restriction endonuclease used in the examples was purchased from NEB company, T4-DNA ligase was purchased from Promaga company, trypsin was purchased from GIBCO company. E. coli. competent strain DH5a was purchased from Invitrogen company. The antibody used in the examples was provided by the laboratory of the inventor, HRP labeled goat anti-human IgG secondary antibody and FITC labeled goat anti-human IgG secondary antibody were purchased from Jackson company. NOD / SCID mice (female, 4-6 weeks old, 18-21 g) were purchased from Vivotan Experimental Animal Co., Ltd. and were raised by the animal center of Peking University Hospital of Clinical Oncology. All animal experiments were approved by the Peking University Experimental Animal Protection and Use Association.

[0070] Design and expression and purification of humanized anti-a2d1 antibody sequences

[0071] According to the amino acid sequences of the heavy chain and light chain variable regions of the murine anti-a2d1 antibody 1B50-1, two candidate humanized anti-a2d1 antibody heavy chain (VH1, VH2) and light chain (VL1, VL2) amino acid sequences were designed. The alignment of the candidate sequences with the sequences of the murine anti-a2d1 antibody parent heavy chain and light chain, and the human germline gene clone sequence is shown in Figures 1A-1D. According to these candidate humanized antibody heavy chain and light chain amino acid sequences, oligodeoxyribonucleic acid sequences (DNA) encoding these amino acid sequences after codon optimization were artificially synthesized, and these sequences were combined in different combinations (VH1VL1, VH1VL2, VH2VL1, VH2VL2) with the antibody constant region (Fc) amino acid encoding sequence of human IgG1 and IgG3, respectively, to form fusion genes and construct them into eukaryotic gene expression vectors.

[0072] VH1: SEQ ID No. 2; VH2: SEQ ID No. 3; VL1: SEQ ID No. 4; VL2: SEQ ID No. 5.

[0073] The above-mentioned antibody sequence-encoding plasmid vectors were transfected with DNA transfection reagent PEI at a PEI:DNA mass ratio of 3:1 into FreeStyle 293-F cells. TM293-F cells were cultured in serum-free medium at 37°C in a 8% CO2 incubator, and the culture supernatant was collected after about 5 days for subsequent antibody purification. The antibody was purified using an AKTA protein purification system (GE Company), in which the purification column was HiTrap Protein G HP (GE Company), the equilibrium liquid was 20 mM phosphate, pH 7.0, the eluent was 0.1 M glycine, pH 2.7, the neutralizing liquid was 1 M Tris-Cl, pH 8.0, and the flow rate was 1 ml / min. The eluted antibody was replaced with 50 ml ultrafiltration centrifuge tube and endotoxin-free 1xPBS. 0.5 μg of purified antibody was analyzed by non-denaturing and denaturing SDS-PAGE to analyze the quality of the purified antibody. The electrophoresis results of two representative purified products are shown in Figures 2A and 2B, and the purified products have no obvious impurity bands, reaching electrophoretic purity.

[0074] Affinity determination of candidate humanized anti-α2δ1 antibodies

[0075] The affinity of humanized anti-α2δ1 antibodies to the target antigen α2δ1 was determined by flow cytometry. The purified antibody was gradiently diluted with 2% FBS in 1xPBS to the following concentrations: 0, 0.01, 0.05, 0.1, 0.2, 0.5, 1, 2, 5 (unit: μg / ml), and then incubated with an equal amount of α2δ1 positive Hep12 cells, and then washed twice with 2% FBS in 1xPBS, resuspended the cells with PBS, added FITC-labeled goat anti-human secondary antibody, incubated at 4°C for 20 min, washed the cells twice with 2% FBS in 1xPBS, resuspended the cells with 300 μl PBS, and detected the fluorescence intensity of the cells in each tube by C6 flow cytometry (BD Biosciences), and calculated the affinity and affinity constant pKa of each group of antibodies to the α2δ1 antigen of Hep12 cells. TM C6 flow cytometry (BD Biosciences) was used to detect the fluorescence intensity of the cells in each tube, and the affinity and affinity constant pKa of each group of antibodies to the α2δ1 antigen of Hep12 cells were calculated.

[0076] Affinity constant pKa calculation formula:

[0077] Where C i is the concentration of the i-th group of antibodies, OD i is the fluorescence intensity of the i-th group, OD max is the saturated fluorescence intensity, and n is the number of samples.

[0078] Experimental results showed that all antibody groups exhibited high affinity, meeting the affinity requirements for humanization of murine monoclonal antibodies (Figure 2C). Among them, the affinity constant of the hIgG3VH1VL1 humanized antibody was closest to that of the murine m1B50-1 antibody. However, the affinity of the humanized antibody with the light chain variable region VL2 sequence was relatively low. Therefore, the combination of the two heavy chain variable regions VH1 and VH2 with the light chain variable region VL1, regardless of the IgG subclass, showed high affinity, thus all were successfully humanized anti-α2δ1 antibodies. For simplicity, subsequent examples will use the hIgG1VH1VL1 combination, collectively referred to as h1B50-1.

[0079] Cryo-electron microscopy structural analysis of the humanized anti-α2δ1 antibody-antigen α2δ1 binding complex particles

[0080] To investigate the epitopes of humanized anti-α2δ1 antibodies binding to antigen α2δ1, the key amino acid sites of antibody-antigen binding, and the possible molecular mechanisms of action of humanized anti-α2δ1 antibodies, the inventors of this invention prepared cryo-electron microscopy images of the particle-based complexes of humanized anti-α2δ1 antibodies and antigen α2δ1. The inventors first used FreeStyle... TM 293-F expressed a Flag-tagged truncated C-segment of α2δ1 (subtype 5) (32 amino acids removed, leaving 1052 amino acids to enable secretion of α2δ1 into the culture medium for easier expression and purification) and a humanized anti-α2δ1 antibody (hIgG1VH1VL1), which were then purified by affinity chromatography and molecular sieve. The human α2δ1 antibody was further digested with papain to prepare an antibody-antigen-binding fragment (Fab). The affinity between the antigen α2δ1 subunit (subtype 5) and the humanized antibody Fab fragment was detected by isothermal titration calorimetry, with a kd value of 20 nM, close to that of the intact antibody. After molecular sieve separation and purification, a high-purity and homogeneous humanized anti-α2δ1 antibody Fab fragment was obtained. The truncated α2δ1 fragment from the molecular sieve peak and the humanized anti-α2δ1 antibody Fab fragment were incubated at a ratio of 1:1.5. The complex was then subjected to further molecular sieve separation and purification. The protein at the peak of the complex was used for cryo-electron microscopy (cryo-EM) sample preparation. The high-resolution structure of the antigen-antibody complex was resolved using single-particle cryo-EM technology, and the binding site of the humanized monoclonal antibody 1B50-1 recognizing the voltage-dependent calcium channel α2δ1 subunit (subtype 5) was identified. Data from different angles and orientations of the obtained cryo-EM particle complex were collected. After three-dimensional reconstruction, the inventors resolved the resolution of the humanized anti-α2δ1 antibody-α2δ1 subunit complex. the overall structure of the complex (Fig. 3A, Fig. 3B). The optimized complex structure model contains 1115 amino acids in total; among which the antigen a2d1 subunit is 683 amino acids, and the humanized a2d1 antibody variable region is 432 amino acids (219 amino acids for the heavy chain variable region and 213 amino acids for the light chain variable region). From the structure of the complex, the Cache2, Cache3 and Cache4 domains of the antigen a2d1 subunit can be identified, but the Cache1 domain and the VWA structure in the antigen a2d1 subunit cannot be clearly recognized. The humanized anti-a2d1 antibody is mainly bound to the third Cache domain in the a2d1 subunit structure. Since the VWA domain and the Cache1 domain are located in the structure composed of the peptide loop on the extracellular side of the a1 subunit, which mainly mediates the interaction with the a1 subunit protein (Wu J, Yan Z, et al. Structure of the voltage-gated calcium channel Cav1.1 at 3.6 A resolution. Nature, 2016, (537): 191-196), therefore one possibility is that the humanized anti-a2d1 antibody binds to the a2d1 subunit complex and causes the flexibility of the domain and cannot bind to the a1 subunit. To verify this hypothesis, the inventors transfected the eukaryotic cell expression vector with HA tag a2d1 into Hep-11 cells, and used anti-HA tag antibody for immunoprecipitation and then Western blot analysis with an antibody against one subtype of a1 subunit CACNA1F in the presence and absence of anti-a2d1 antibody 1B50-1, and found that CACNA1F was present in the anti-HA tag antibody immunoprecipitated products of the group without any antibody or control IgG antibody, and the precipitated CACNA1F was significantly reduced when anti-a2d1 antibody 1B50-1 was added (Fig. 3C). At the same time, immunoprecipitation with anti-a2d1 antibody 1B50-1 in Hep-12 cell (a2d1 positive) lysate can only precipitate a2d1 itself, and there is no CACNA1F subunit in the precipitate (Fig. 3D). These results prove that the humanized anti-a2d1 antibody affects the binding of the a2d1 subunit to the a1 subunit and thus loses the regulatory effect on the voltage-gated calcium channel-mediated calcium ion influx.

[0081] The antigen-antibody complex structure at near-atomic resolution analyzed by the inventors successfully identified the binding sites of a2d1 and humanized anti-a2d1 antibody (Figure 4A). The amino acid sites on the heavy chain variable region of humanized anti-a2d1 antibody binding to a2d1 subunit are S35, Y57, W58, D59, D64, R66, R107, T109, G110, T113, and the amino acid sites on the light chain are Q27, S28, G36, S38, E55, Y56, D67, S70, T107, N108, S109, and W114. The amino acid sites on the antigen a2d1 subunit (subtype 5) recognizing humanized anti-a2d1 antibody are K49, N55, Q56, D59, E62, K63, Y64, Q65, D66, R624, E695, Q698, N699, K703, Y782, Q784, K786, and N805. Further, the inventors constructed mutants of these sites, respectively, and determined the affinity changes of the mutants and wild-type proteins (antibodies) after mutation of the antibody binding sites to wild-type a2d1 truncates, mutant a2d1 truncates, and humanized anti-a2d1 using isothermal titration calorimetry (MicroCal PEAQ-ITC). The results are shown in Figures 4B-4D. The affinity of a2d1 subunit (subtype 5) to humanized anti-a2d1 antibody significantly decreased or disappeared after mutation of N55A, D59K, E62K, and K63D sites. The affinity of humanized anti-a2d1 antibody heavy chain to a2d1 subunit (subtype 5) significantly decreased or disappeared after mutation of S35D, Y57A, W58A, D59A, R66A, R107A, G110W, and light chain G36W, S38W, Y56A, D67A, N108W, and W114A sites. The affinity of light chain T37R site to a2d1 subunit (subtype 5) significantly increased. It is indicated that these sites are the key sites mediating the mutual binding of the two. TM 293-F expression and purification of these mutants and wild-type proteins (antibodies), and determination of the affinity changes of the mutants and wild-type proteins (antibodies) after mutation of the antibody binding sites to wild-type a2d1 truncates, mutant a2d1 truncates, and humanized anti-a2d1 using isothermal titration calorimetry (MicroCal PEAQ-ITC). The results are shown in Figures 4B-4D. The affinity of a2d1 subunit (subtype 5) to humanized anti-a2d1 antibody significantly decreased or disappeared after mutation of N55A, D59K, E62K, and K63D sites. The affinity of humanized anti-a2d1 antibody heavy chain to a2d1 subunit (subtype 5) significantly decreased or disappeared after mutation of S35D, Y57A, W58A, D59A, R66A, R107A, G110W, and light chain G36W, S38W, Y56A, D67A, N108W, and W114A sites. The affinity of light chain T37R site to a2d1 subunit (subtype 5) significantly increased. It is indicated that these sites are the key sites mediating the mutual binding of the two.

[0082] EFFECT

[0083] Example 1: Humanized anti-a2d1 antibody inhibits the self-renewal ability of hepatocellular carcinoma

[0084] To investigate the application value of the humanized anti-a2d1 antibody invented by the inventor, the inventor used a serum-free spheroid formation experiment to detect the effect of the humanized anti-a2d1 antibody h1B50-1 on the spheroid formation ability of liver cancer a2d1 positive cells. The effect of a2d1 + Hep-12 liver cancer cells on the in vitro self-renewal ability. The spheroid formation experiment was carried out according to the method in the literature (literature: Zhao W, Wang L, Han HB, Lin N, Guo T, Chen YD, Jin K, Cheng HP, Lu FM, Fang WG, Yang Y, Xing BC, Zhang ZQ. 1B50-1, a mAb raised against recurrent tumor cells, targets liver tumor-initiating cells by binding to the calcium channel a2d1 subunit. Cancer Cell, 2013, 23:541-556): dilute the cells with serum-free culture medium, add them to the Corning low adsorption 96-well plate (Corning) according to 100 cells / well, 100 μl per well, add different drug treatments respectively, add fresh culture medium after about 7 days, cultivate in a 37℃, 5% CO2 cell incubator for 1-2 weeks, count the spheroids with a diameter of more than 100 μm, and calculate the spheroid formation rate. The results are shown in Figures 5A-5D: compared with the control IgG treatment group, whether it is lenvatinib or humanized anti-a2d1 antibody h1B50-1, it has a significant inhibitory effect on the spheroid formation ability of a2d1 positive liver cancer Hep-12 cells and a2d1 positive cells purified from PLC / PRF5 by flow cytometry, and the inhibitory effect of lenvatinib and h1B50-1 combined is more significant (Figures 5A, 5B); although lenvatinib has no significant inhibitory effect on the spheroid formation ability of lenvatinib-resistant Huh7-LR and Hep3B-LR cell lines, the spheroid formation efficiency of the h1B50-1 treatment group is significantly decreased, and the combination of lenvatinib and h1B50-1 can further reduce the spheroid formation efficiency of the drug-resistant cell lines (Figures 5C, 5D). The calculated synergistic q value of the combination of lenvatinib and h1B50-1 on Huh7-Resistant and Hep3B-Resistant is 1.45 and 1.65 respectively, showing a synergistic effect. These results show that the humanized anti-a2d1 antibody can inhibit the in vitro self-renewal ability of liver cancer stem cells and lenvatinib-resistant cells, and has a synergistic effect when combined with lenvatinib.

[0085] Example 2: Humanized anti-a2d1 antibody has a significant inhibitory effect on liver cancer transplanted tumors

[0086] To verify the therapeutic effect of humanized anti-a2d1 antibody on hepatocellular carcinoma, the inventors inoculated hepatocellular carcinoma cells PLC / PRF5 and Huh7 cells with luciferase gene into the liver of nude mice (Balb / c-nu, Beijing Vantoll Life Experimental Animal Technology Co., Ltd.) in situ, respectively. When the in situ liver tumor was visible by chemiluminescence imaging with luciferase substrate, the nude mice were randomly divided into control group, regorafenib treatment group, h1B50-1 treatment group and regorafenib combined with h1B50-1 treatment group. The control group was injected intraperitoneally with isotype control IgG (800 pg per mouse), the regorafenib treatment group was perfused with regorafenib (200 pg per mouse) through the stomach, the h1B50-1 treatment group was injected intraperitoneally with humanized anti-a2d1 antibody h1B50-1 (800 pg per mouse), and the combined treatment group was perfused with regorafenib (200 pg per mouse) through the stomach and injected intraperitoneally with humanized anti-a2d1 antibody h1B50-1 (800 pg per mouse). Drug administration was performed daily. The luciferin chemiluminescence intensity in the in situ liver tissue was measured before drug administration, 10 days after drug administration and 20 days after drug administration, and the body weight and survival of the mice in each group were recorded. The results are shown in Figures 6A-6F: compared with the control group, both regorafenib and humanized anti-a2d1 antibody h1B50-1 treatment can significantly inhibit the growth of PLC / PRF5 and Huh7 liver in situ transplanted tumors, and the combined treatment group has a more significant inhibitory effect. At the end of drug administration, in the h1B50-1 alone and combined treatment groups, 4 out of 5 mice had complete tumor disappearance (4 / 5) in the PLC / PRF5 liver in situ transplanted tumor, and 5 out of 8 mice had complete tumor disappearance (5 / 8) in the Huh7 in situ transplanted tumor. The Kaplan-Meier mouse survival curve results show that the h1B50-1 alone and combined treatment groups can significantly prolong the survival of mice. These results show that humanized anti-a2d1 antibody alone or in combination with regorafenib is an effective means of treating hepatocellular carcinoma and can be used as a drug for the treatment of hepatocellular carcinoma.

[0087] Example 3: Therapeutic effect of anti-a2d1 CAR-T on liver cancer Hep-12 animal transplanted tumor

[0088] Another application of humanized anti-α2δ1 antibodies is the construction of their encoded nucleotides into chimeric antigen receptor T cells (CAR-T cells) for tumor immunotherapy. The inventors constructed the encoded nucleotides of the variable region of the humanized anti-α2δ1 antibody into a third-generation CAR-T lentiviral expression plasmid vector using conventional DNA recombination technology, forming a fusion protein with the CD28 hinge region, transmembrane and intracellular regions, CD137 intracellular region, and CD3ζ (Figure 7A). The recombinant plasmid was transformed into 293FT packaging cells, and the lentiviral supernatant was collected and concentrated using PEG8000. The concentrated virus was used to infect activated T lymphocytes isolated from healthy individuals (MOI=5). Flow cytometry was used to sort gene-expressing positive T lymphocytes, and the cells were expanded to obtain chimeric antigen receptor T cells (CAR-T). The prepared CAR-T cells were mixed with Hep-12 hepatocellular carcinoma cells at 1:1 and 1:2 ratios, respectively, and then... 6 One Hep12 cell per mouse was subcutaneously inoculated into NOD / SCID mice, and T cells infected with the empty vector were used as a control to observe tumor formation. After mixing CAR-T cells targeting α2δ1 with Hep-12, only one mouse in the effector cell:target cell ratio of 1:2 developed tumors, while all mice in the control group developed tumors (Figure 7B). Further, Hep-12 cells were first inoculated at 2 × 10⁻⁶ cells per mouse. 6 One cell / mouse was subcutaneously inoculated into NOD / SCID mice, and the tumors were allowed to grow to 100 mm. 3 Mice were randomly divided into two groups. One group received an intratumoral injection of 100 μl of 1×10⁻⁶ mol / L. 7 One group of mice received one CAR-T cell and another group received an equal dose of control T cells (T cells infected with an empty vector). Seven days later, each mouse was injected with the same dose of either CAR-T or control T cells. Tumor size was measured with calipers every other day. Compared with the control group, injection of CAR-T cells targeting α2δ1 significantly inhibited the growth of Hep-12 suppressor tumors (Figure 7C). These results indicate that the nucleotides encoding the humanized anti-α2δ1 antibody can be used to construct CAR-T cells for the treatment of liver cancer.

[0089] Example of Implementation Results 4: Therapeutic Effect of Anti-α2δ1-CD3 Bispecific Antibody on Hep-12 Animal Xenografts of Liver Cancer

[0090] Bi-specific antibody is an antibody that can bind two antigens or antigen epitopes at the same time. In recent years, it has become one of the important directions of research and development of immunotherapy drugs for tumors. In particular, bi-specific antibodies formed with antibodies targeting CD3 can mediate T cell targeting and killing of tumor cells positive for another antigen. In another embodiment of the present application, a humanized antibody targeting a2d1 is combined with an antibody targeting another antigen, such as CD3, to form a bi-specific antibody for the treatment of a2d1-positive tumors such as liver cancer. The present inventors constructed a eukaryotic expression plasmid for a bi-specific antibody against a2d1 and CD3 by conventional DNA recombination technology, and transfected FreeStyle TM 293-F cells to express and purify the bi-specific antibody. In in vitro experiments, the anti-a2d1 and CD3 bi-specific antibody can effectively mediate cytotoxic killing. For human liver cancer cells Hep-12 that highly express a2d1, the EC 50 50 of the mediated killing effect is 1.9 pmol / L. For human liver cancer cells Hep-11 that lowly express a2d1, the bi-specific antibody cannot mediate cytotoxic killing. To further evaluate the inhibitory effect of the anti-a2d1 and CD3 bi-specific antibody on tumors, Hep-12 cells were inoculated subcutaneously into NSG mice (NOD.Cg-Prkd cscid IL2rgtm 1Wjl / SzJ) that have been reconstituted with the human immune system. When the tumor reached a size of 100 mm 3 3, the anti-a2d1 and CD3 bi-specific antibody was injected intraperitoneally every other day, and the tumor volume was measured with a vernier caliper. The results are shown in Figure 7D. In in vivo experiments, the anti-a2d1 and CD3 bi-specific antibody can significantly inhibit the growth of Hep-12 tumors. These results show that the anti-a2d1 humanized antibody can be combined with antibodies such as anti-CD3 to form bi-specific or multi-specific antibody drugs for the treatment of tumors.

[0091] Example 5: Application of anti-a2d1 humanized antibody in in vivo nuclear imaging and detection of circulating tumor cells

[0092] Voltage-gated calcium channel a2d1 (subtype 5) is a surface marker and therapeutic target for various tumors, including liver cancer, lung cancer, pancreatic cancer, and gastric cancer, and such cells are closely related to treatment resistance. Therefore, non-invasive, real-time monitoring of the presence of such cells has important application prospects for monitoring disease progression and treatment response in patients with related tumors. To explore the application feasibility of humanized antibodies targeting a2d1, the present inventors first transfected FreeStyle TM293-F cells were respectively expressed to purify humanized anti-α2δ1 antibody h1B50-1 and isotype control IgG1, and then the antibodies were enzymatically cut by Thermo Scientific Pierce F(ab')2 Preparation Kit to obtain F(ab')2 fragments of h1B50-1 and control antibody with antigen α2δ1 binding activity. After the purified F(ab')2 fragments were modified by NCS-Bz-NOTA, they were labeled by CuCl2 and purified by PD-10 desalting column for subsequent animal experiments. 64 CuCl2, and used for subsequent animal experiments after purification by PD-10 desalting column.

[0093] The digested human hepatoma Hep-12 cells were inoculated into female 5-6 week old Balb / C-nu / nu mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) at 2×10 6 cells per mouse subcutaneously. When the tumor volume reached about 100 mm 3 , 200 μl of 11.1 MBq of 64 Cu-NOTA-h1B50-1-F(ab')2 or an equal amount of 64 Cu-NOTA-IgG1-F(ab')2 was injected into the mice through the tail vein. For blocking experiments, an excess of unlabeled h1B50-1-F(ab')2 was injected through the tail vein 12 h in advance. At different time points after injection, the mice were scanned by PET / CT (Pine Medical Technology Co., Ltd.) and the standardized uptake values (SUV) of the tumor tissue were determined. As shown in FIGS. 8A-8C, after injection of the labeled antibody for 3 hours, 64 Cu-NOTA-h1B50-1-F(ab')2 was mainly distributed in the liver, kidney, heart and tumor, and pre-blocking with unlabeled h1B50-1-F(ab')2, 64 Cu-NOTA-h1B50-1-F(ab')2 was mainly distributed in the liver, kidney, heart, and the binding of tumor tissue was almost completely blocked; the control 64 Cu-NOTA-IgG1-F(ab')2 was mainly distributed in the liver and kidney tissue. With the extension of time, 64 Cu-NOTA-h1B50-1-F(ab')2 gradually disappeared in the heart, and the radioactive intensity in the tumor remained unchanged or even showed an increasing trend, and its distribution in the tumor tissue could be completely blocked by unlabeled h1B50-1-F(ab')2, but the change in liver and kidney tissue was not significant. Since the antibody is mainly metabolized by the liver and kidney, whether or not to block, 64 Cu-NOTA-h1B50-1-F(ab')2 showed no significant change in the distribution of these two tissues. These results indicate64 Cu-NOTA-h1B50-1-F(ab')2 can label tumor cells of α2δ1 in vivo, and can be used for nuclide imaging tracing of such cells.

[0094] Another example of the application of the humanized antibody in diagnosis is the labeling of the humanized anti-α2δ1 (sub-type 5) antibody with other markers such as fluorescein isothiocyanate (FITC) for the detection of circulating tumor cells (CTC) and circulating tumor endothelial cells (CTEC) in the peripheral blood of tumor patients. The inventors combined the FITC-labeled humanized anti-α2δ1 antibody with a commercial subtraction enrichment (SE) and immunofluorescent staining-fluorescence in situ hybridization (i·FISH) kit to detect CTC and CETC in the peripheral blood of tumor patients. The results showed that the aneuploid CETC (ApCEC) or CTC (ApCTC) detected in the blood of tumor patients were all found to be α2δ1 positive (Figure 8D). This indicates that the humanized antibody can be applied to the detection of α2δ1-positive aneuploid endothelial cells and tumor cells in the blood of patients.

Claims

1. A monoclonal antibody against α2δ1 or a bioactive fragment thereof that binds to one or more amino acid sites of the human voltage-gated calcium channel α2δ1 subunit (subtype 5) K49, N55, Q56, D59, E62, K63, Y64, Q65, D66, R624, E695, Q698, N699, K703, Y782, Q784, K786, N805, wherein the protein sequence of the α2δ1 subunit (subtype 5) is as shown in SEQ ID NO.1, and the bioactive fragment is capable of specifically binding to human α2δ1.

2. The antibody according to claim 1, or a bioactive fragment derived from the antibody, wherein, The antibody is a human antibody, a humanized antibody, a chimeric antibody, a single-chain antibody, or a single-domain antibody.

3. The antibody according to claim 1, or a biologically active fragment derived from the antibody, comprising: (a) The heavy chain variable region, the amino acid sequence of which is selected from: the amino acid sequence shown in SEQ ID No. 2 or SEQ ID No. 3, or an amino acid sequence with equivalent function formed by substitution, deletion or addition of one or more amino acids based on the amino acid sequence shown in SEQ ID No. 2 or SEQ ID No. 3; preferably, the amino acid sequence of the heavy chain variable region includes the complementarity-determining regions shown in SEQ ID No. 6, SEQ ID No. 7, and SEQ ID No. 8; and / or (b) Light chain variable region, the amino acid sequence of which is selected from: the amino acid sequence shown in SEQ ID No. 4, or an amino acid sequence with equivalent function formed by replacing, deleting or adding one or more amino acids to the amino acid sequence shown in SEQ ID No. 4; preferably, the amino acid sequence of the light chain variable region includes the complementarity-determining regions shown in SEQ ID No. 9, SEQ ID No. 10 and SEQ ID No. 11; More preferably, the amino acids at positions 35, 57, 58, 59, 66, 107, and 110 of the heavy chain are S, Y, W, D, R, R, and G, respectively. More preferably, the amino acid sites at positions 36, 38, 56, 67, 108, and 114 of the light chain are G, S, Y, D, N, and W, respectively; More preferably, the 37th amino acid site of the light chain is T or R.

4. A polynucleotide encoding the anti-α2δ1 monoclonal antibody as described in any one of claims 1 to 3 or a bioactive fragment derived from the antibody; Preferably, the polynucleotide is DNA or RNA; More preferably, the polynucleotide comprises: a nucleotide sequence encoding an amino acid shown in SEQ ID No. 2 or SEQ ID No. 3, and / or a nucleotide sequence encoding an amino acid shown in SEQ ID No. 4 or / and SEQ ID No.

5.

5. A carrier or host cell containing the polynucleotide of claim 4.

6. A fusion protein having an amino acid sequence comprising the amino acid sequence of the anti-α2δ1 monoclonal antibody as described in any one of claims 1 to 3 or a biologically active fragment derived from the antibody.

7. A chimeric antigen receptor T cell expressing the anti-α2δ1 monoclonal antibody as described in any one of claims 1 to 3 or a biologically active fragment derived from the antibody.

8. A multispecific antibody comprising the anti-α2δ1 monoclonal antibody as described in any one of claims 1 to 3 or a biologically active fragment derived from the antibody; Preferably, the multispecific antibody is a bispecific antibody; More preferably, the bispecific antibody is the anti-α2δ1 monoclonal antibody as described in any one of claims 1 to 3, or a bispecific antibody formed by a biologically active fragment of the antibody and an antibody targeting CD3.

9. A method for producing the anti-α2δ1 monoclonal antibody according to any one of claims 1 to 3, or a bioactive fragment derived from the antibody, the method comprising the following steps: (a) Providing the carrier as described in claim 5; (b) Introduce the vector described in step (a) into the cells; (c) Culture the cells obtained in step (b) under conditions suitable for the expression of the monoclonal antibody; and (d) The monoclonal antibody was obtained by separation and purification.

10. The use of the anti-α2δ1 monoclonal antibody according to any one of claims 1 to 3, or a biologically active fragment derived from the antibody, or the polynucleotide according to claim 4, or the fusion protein according to claim 6, or the chimeric antigen receptor T cell according to claim 7, or the multispecific antibody according to claim 8, in the preparation of a medicament for tumor treatment and / or detection; Preferably, the anti-α2δ1 monoclonal antibody of any one of claims 1 to 3, or a bioactive fragment derived from the antibody, or the polynucleotide of claim 4, or the fusion protein of claim 6, or the chimeric antigen receptor T cell of claim 7, or the multispecific antibody of claim 8, is used in combination with lenvatinib to prepare a drug for the treatment of malignant tumors; More preferably, the tumor is liver cancer.

11. A pharmaceutical composition comprising: The effective dose of the anti-α2δ1 monoclonal antibody as described in any one of claims 1 to 3, or a biologically active fragment derived from the antibody, or the polynucleotide as described in claim 4, or the fusion protein as described in claim 6, or the chimeric antigen receptor T cell as described in claim 7, or the multispecific antibody as described in claim 8; Preferably, the pharmaceutical composition further comprises: other active pharmaceutical ingredients that have a synergistic effect when used in combination with the anti-α2δ1 monoclonal antibody of any one of claims 1 to 3 or a bioactive fragment derived from the antibody, or the polynucleotide of claim 4, or the fusion protein of claim 6, or the chimeric antigen receptor T cell of claim 7, or the multispecific antibody of claim 8; and pharmaceutically acceptable solvents and / or carriers; Preferably, the other active pharmaceutical ingredients include lenvatinib.

12. A kit for detecting α2δ1 levels, comprising: an anti-α2δ1 monoclonal antibody as described in any one of claims 1 to 3 or a bioactive fragment derived from the antibody; Preferably, the anti-α2δ1 monoclonal antibody according to any one of claims 1 to 3, or a biologically active fragment derived from the antibody, is labeled with a fluorescein or a radiolabeler; Preferably, the kit further comprises: a second antibody, an enzyme or fluorescent or radiolabeled marker for detection, and a buffer solution; More preferably, the second antibody is an anti-antibody against the anti-α2δ1 monoclonal antibody.

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