Antibody or fragment of targeting human Nectin4 protein and application of antibody or fragment

Abstract

The invention discloses an antibody targeting a human Nectin4 protein and an application of the antibody. The antibody comprises five groups of heavy chain variable regions and five groups of light chain variable regions, and amino acid sequences of the five groups of heavy chain variable regions can be respectively combined with amino acid sequences of the five groups of light chain variable regions for double-chain use. The antibody can specifically recognize a human Nectin4 antigen, has good specificity in human normal tissues, is only combined with Nectin4 positive tissues, does not recognize other tissues and organs, and has no off-target risk; meanwhile, the antibody has good targeting performance on a natural configuration state and a recombinant configuration state, and has good application prospects of antibody drugs, multi-resistance drugs and recombinant antibody proteins.

Description

[0001] This application is a divisional application with application number 2023105880083, application date May 24, 2023, and application name “Antibodies or fragments thereof targeting human Nectin4 protein and applications”. Technical Field

[0002] The present invention relates to the field of biological cell technology, and in particular to an antibody or a fragment thereof, a biological material, a biological preparation and an application in preparing a drug component targeting a human nectin4 protein. Background Art

[0003] Nectin1, Nectin2 and Nectin3 are widely expressed in normal adult tissues, but Nectin4, also known as PVRL4 (poliovirus receptor-like 4), is only present during embryogenesis and its expression level decreases in normal tissues after adulthood. However, Nectin4 is overexpressed in a variety of tumor cells, such as urothelial carcinoma, breast cancer, pancreatic cancer, triple-negative breast cancer and bladder cancer. Nectin4 promotes tumor cell proliferation, differentiation, migration and invasion by activating the PI3K / Akt pathway. Using Nectin4 as a therapeutic target may be an effective strategy for treating cancers with high Nectin4 expression, but in actual treatment, this target cannot be specifically identified. Summary of the invention

[0004] Based on this, the problem to be solved by the present invention is to provide an antibody or a fragment thereof targeting human Nectin4-CAR that can specifically recognize Nectin4.

[0005] The technical solution of the present invention is as follows:

[0006] An antibody or a fragment thereof targeting human Nectin4-CAR, comprising a heavy chain and a light chain; the heavy chain and the light chain respectively comprise three heavy chain complementary determining regions 1 to 3 (e.g., HCDR1, HCDR2 and HCDR3) and three light chain complementary determining regions 1 to 3 (e.g., LCDR1, LCDR2 and LCDR3); wherein the amino acid sequences of the heavy chain complementary determining regions and the light chain complementary determining regions are respectively selected from one of the following combinations:

[0007] (i) the amino acid sequence of the heavy chain variable region HCDR1 is SEQ ID NO: 11, the amino acid sequence of the heavy chain variable region HCDR2 is SEQ ID NO: 12, and the amino acid sequence of the heavy chain variable region HCDR3 is SEQ ID NO: 13; the amino acid sequence of the light chain variable region LCDR1 is SEQ ID NO: 14, the amino acid sequence of the light chain variable region LCDR2 is SEQ ID NO: 15, and the amino acid sequence of the light chain variable region LCDR3 is SEQ ID NO: 16; or

[0008] (ii) the amino acid sequence of the heavy chain variable region HCDR1 is as shown in SEQ ID NO:21, the amino acid sequence of the heavy chain variable region HCDR2 is as shown in SEQ ID NO:22, and the amino acid sequence of the heavy chain variable region HCDR3 is as shown in SEQ ID NO:23; the amino acid sequence of the light chain variable region LCDR1 is as shown in SEQ ID NO:24, the amino acid sequence of the light chain variable region LCDR2 is as shown in SEQ ID NO:25, and the amino acid sequence of the light chain variable region LCDR3 is as shown in SEQ ID NO:26; or

[0009] (iii) the amino acid sequence of the heavy chain variable region HCDR1 is as shown in SEQ ID NO:31, the amino acid sequence of the heavy chain variable region HCDR2 is as shown in SEQ ID NO:32, and the amino acid sequence of the heavy chain variable region HCDR3 is as shown in SEQ ID NO:33; the amino acid sequence of the light chain variable region LCDR1 is as shown in SEQ ID NO:34, the amino acid sequence of the light chain variable region LCDR2 is as shown in SEQ ID NO:35, and the amino acid sequence of the light chain variable region LCDR3 is as shown in SEQ ID NO:36; or

[0010] (iv) the amino acid sequence of the heavy chain variable region HCDR1 is as shown in SEQ ID NO:41, the amino acid sequence of the heavy chain variable region HCDR2 is as shown in SEQ ID NO:42, and the amino acid sequence of the heavy chain variable region HCDR3 is as shown in SEQ ID NO:43; the amino acid sequence of the light chain variable region LCDR1 is as shown in SEQ ID NO:44, the amino acid sequence of the light chain variable region LCDR2 is as shown in SEQ ID NO:45, and the amino acid sequence of the light chain variable region LCDR3 is as shown in SEQ ID NO:46; or

[0011] (v) the amino acid sequence of the heavy chain variable region HCDR1 is shown in SEQ ID NO:51, the amino acid sequence of the heavy chain variable region HCDR2 is shown in SEQ ID NO:52, and the amino acid sequence of the heavy chain variable region HCDR3 is shown in SEQ ID NO:53; the amino acid sequence of the light chain variable region LCDR1 is shown in SEQ ID NO:54, the amino acid sequence of the light chain variable region LCDR2 is shown in SEQ ID NO:55, and the amino acid sequence of the light chain variable region LCDR3 is shown in SEQ ID NO:56.

[0012] In the above antibody or fragment thereof, the heavy chain and light chain respectively comprise a heavy chain variable region (VH) and a light chain variable region (VL); the heavy chain complementary determining regions 1 to 3 (HCDR1, HCDR2 and HCDR3) are from the heavy chain variable region, and the light chain complementary determining regions 1 to 3 (LCDR1, LCDR2 and LCDR3) are from the light chain variable region; wherein:

[0013] In group (i), the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 10; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 17; or

[0014] In group (ii), the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 20; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 27; or

[0015] In group (iii), the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 30; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 37; or

[0016] In group (iv), the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:40; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:47; or

[0017] In group (v), the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:50; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:57.

[0018] The above-mentioned antibody or fragment thereof is a monoclonal antibody, a single-chain antibody or a double-chain antibody against Nectin4 protein; preferably, the antibody or fragment thereof is a monoclonal antibody against Nectin4 protein.

[0019] Preferably, when the antibody or its fragment is humanized or otherwise derived from an animal, the change in the amino acid sequence in the three heavy chain or light chain complementary determining regions does not exceed 20%; or, when the monoclonal antibody is humanized or otherwise derived from an animal, the amino acid sequence change in the three heavy chain complementary determining regions or the three light chain complementary determining regions is 0.

[0020] The present invention also provides nucleic acid molecules containing the above antibodies or fragments thereof, and expression boxes, recombinant vectors, host cells, recombinant microorganisms and / or recombinant cell lines containing the nucleic acid molecules.

[0021] The present invention also provides biological preparations containing antibodies or fragments thereof, nucleic acid molecules, expression cassettes, recombinant vectors, host cells, recombinant microorganisms and / or recombinant cell lines, etc.

[0022] The present invention also provides a biomaterial, which comprises an expression cassette, a recombinant vector, a recombinant protein, a recombinant microorganism or a recombinant cell line constructed by a nucleic acid sequence or an amino acid sequence, and the nucleic acid sequence or the amino acid sequence is derived from the above-mentioned monoclonal antibody; in the biomaterial, the recombinant vector comprises a gene recombination expression vector and a chimeric antigen receptor.

[0023] Preferably, the biological agent, antibody or fragment thereof can be widely used in preparing drug components for preventing and / or treating tumors and cancers.

[0024] Preferably, the biological preparation includes reagents for detecting the concentration of human Nectin4 protein, reagents for detecting the expression level of human Nectin4 protein on the surface of tumor cells, antibodies coupled to toxins to kill human Nectin4 positive cells, antibodies coupled to other antibodies to produce polyclonal antibodies targeting human Nectin4 and other antigens, and antibodies coupled to other proteins to produce functional recombinant proteins targeting human Nectin4.

[0025] The present invention provides an antibody or a fragment thereof targeting human Nectin4 protein, which has the following advantages:

[0026] 1. It has the ability to specifically recognize human Nectin4 protein. Some combinations have dual species specificity between humans and mice. The efficacy and toxicity of dual species specificity in mice are more similar to those in humans, which makes it easier to map the reactions in animals to humans and thus obtain some guiding information for clinical trials.

[0027] 2. The antibody has good tissue specificity and only recognizes tissues expressing Nectin4, but not other tissues and organs, and has good prospects for detection;

[0028] 3. The CAR-T prepared by this antibody has a specific killing function on Nectin4-positive target cells and has good application prospects in cell immunotherapy;

[0029] 4. The antibody has good targeting properties in both its natural and recombinant configurations, and has good application prospects in the preparation of antibody drugs (ADCs), multi-antibody drugs and recombinant antibody proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The positive rate flow cytometric graph of 293T cell expression in Example 2;

[0031] Figure 2 This is a flow cytometric graph of the positive rate of 293T-hNectin4 cell expression in Example 2;

[0032] Figure 3 The positive rate flow cytometric graph of 293T-mNectin4 cell expression in Example 2;

[0033] Figure 4 Affinity test results of humanized antibodies targeting P177, Nectin4-E1, Nectin4-E8, Nectin4-F10, Nectin4-H1, and Nectin4-H12 cells;

[0034] Figure 5 This is a specificity curve diagram of the binding activity of humanized antibodies to 293T cells;

[0035] Figure 6 It is a specificity curve diagram of the binding activity of humanized antibody to 293T-hNectin4 cells;

[0036] Figure 7 It is a specificity curve diagram of the binding activity of humanized antibody to 293T-mNectin4 cells;

[0037] Figure 8 Design drawings for 6 CAR-Nectin4 structures;

[0038] Fig. 9 They are the amplification curves of CAR-T-NT, CAR-T-E1, CAR-T-H1, CAR-T-E8, CAR-T-F10, CAR-T-H12, and CAR-T-177 immune cell cultures;

[0039] Fig.10 This is the flow cytometric graph of the positive rate of CAR expression in NT cells (control blank sample);

[0040] Fig.11 This is the flow cytometry graph of the positive rate of CAR expression in CAR-T-E1 cells;

[0041] Fig.12 This is the flow cytometric graph of the positive rate of CAR expression in CAR-T-H1 cells;

[0042] Fig.13 This is the flow cytometry graph of the positive rate of CAR expression in CAR-T-E8 cells;

[0043] Fig.14 This is the flow cytometry graph of the positive rate of CAR expression in CAR-T-F10 cells;

[0044] Fig.15 This is the flow cytometry graph of the positive rate of CAR expression in CAR-T-H12 cells;

[0045] Fig.16 This is the flow cytometry graph of the positive rate of CAR expression in CAR-T-177 cells;

[0046] Fig.17 Killing curves of NT, AR-T-NT, CAR-T-E1, CAR-T-H1, CAR-T-E8, CAR-T-F10, CAR-T-H12, and CAR-T-177 cells against 293T cells, respectively;

[0047] Fig.18 These are the killing curves of NT, AR-T-NT, CAR-T-E1, CAR-T-H1, CAR-T-E8, CAR-T-F10, CAR-T-H12, and CAR-T-177 cells against 293T-hNectin4 cells, respectively. DETAILED DESCRIPTION

[0048] The antibody of the present invention is a monoclonal antibody (also referred to as MAb) or a fragment thereof, which is derived from bacteriophage, and the technical scheme is described in detail based on this.

[0049] The present invention provides a monoclonal antibody or a fragment thereof targeting human Nectin4 protein, comprising a heavy chain and a light chain; the heavy chain comprises three heavy chain complementary determining regions 1 to 3 (HCDR1, HCDR2 and HCDR3), the light chain comprises three light chain complementary determining regions 1 to 3 (HCDR1, HCDR2 and HCDR3), and the heavy chain further comprises a heavy chain variable region VH, and the light chain further comprises a light chain variable region VL; wherein:

[0050] Among the three heavy chain complementary determining regions (HCDR1, HCDR2 and HCDR3), the amino acid sequence of HCDR1 is shown in SEQ ID NO:11, SEQ ID NO:21, SEQ ID NO:31, SEQ ID NO:41 or SEQ ID NO:51; the amino acid sequence of HCDR2 is shown in SEQ ID NO:12, SEQ ID NO:22, SEQ ID NO:32, SEQ ID NO:42 or SEQ ID NO:52; the amino acid sequence of HCDR3 is shown in SEQ ID NO:13, SEQ ID NO:23, SEQ ID NO:33, SEQ ID NO:43 or SEQ ID NO:53;

[0051] Among the three light chain complementary determining regions (LCDR1, LCDR2 and LCDR3), the amino acid sequence of LCDR1 is shown in SEQ ID NO:14, SEQ ID NO:24, SEQ ID NO:34, SEQ ID NO:44 or SEQ ID NO:54; the amino acid sequence of LCDR2 is shown in SEQ ID NO:15, SEQ ID NO:25, SEQ ID NO:35, SEQ ID NO:45 or SEQ ID NO:55; the amino acid sequence of LCDR3 is shown in SEQ ID NO:16, SEQ ID NO:26, SEQ ID NO:36, SEQ ID NO:46 or SEQID NO:56.

[0052] Any amino acid sequence in the above three heavy chain complementary determining regions (HCDR1, HCDR2 and HCDR3) can be used in a double-chain combination with any amino acid sequence in the three light chain complementary determining regions (LCDR1, LCDR2 and LCDR3), or any amino acid sequence in the three heavy chain complementary determining regions (HCDR1, HCDR2 and HCDR3) can be used in a single-chain, or any amino acid sequence in the three light chain complementary determining regions (LCDR1, LCDR2 and LCDR3) can be used in a single-chain. Preferably, the amino acid sequences of the heavy chain complementary determining regions and the light chain complementary determining regions are selected from one of the following combinations.

[0053] Group i

[0054] The amino acid sequence of HV is shown in SEQ ID NO: 10:

[0055] QVQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGG IIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC ARESEYSGYGPFDY WGQGTLVTVSS; wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO: 11: GGTFSSYA ; The amino acid sequence of HCDR2 is shown in SEQ ID NO: 12: IIPIFGTA The amino acid sequence of HCDR3 is shown in SEQ ID NO: 13: ARESEYSGYGPFDY ;

[0056] The amino acid sequence of LV is shown in SEQ ID NO: 17:

[0057] QSALTQPPSASGTPGQRVTISCSGS SSNIGSQT VNWYQQFPGTAPKLLIY SNN QRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYC AAWDDSLNGWV FGGGTKLTVL; wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO: 14: SSNIGSQT; and the amino acid sequence of LCDR2 is shown in SEQ ID NO: 15: SNN ; The amino acid sequence of LCDR3 is shown in SEQ ID NO:16: AAWDDSLNGWV.

[0058] Group ii

[0059] The amino acid sequence of HV is shown in SEQ ID NO: 20:

[0060] QMQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGW INPNGDGT NLAQNFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYC ARDGLLIRYDSSGYLYQYGMDV WGQGTLVTVSS; wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO: 21: GGTFSSYA ; The amino acid sequence of HCDR2 is shown in SEQ ID NO: 22: INPNGDGT The amino acid sequence of HCDR3 is shown in SEQ ID NO: 23: ARDGLLIRYDSSGYLYQYGMDV .

[0061] The amino acid sequence of LV is shown in SEQ ID NO: 27:

[0062] DIVMTQSPSSVSASVGDRVTITCRAS QGISSWLAWYQQKPGKAPKLLIY AAS SLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC HQSYRTPET FGQGTKVEIK; wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO: 24 QGISSW ; The amino acid sequence of LCDR2 is shown in SEQ ID NO: 25 AAS ; The amino acid sequence of LCDR3 is shown in SEQ ID NO: 26 HQSYRTPET .

[0063] Group iii

[0064] The amino acid sequence of HV is shown in SEQ ID NO:30:

[0065] EVQLVESGGGLVKPGGSLRLSCAAS GFTFSDYY MSWIRQAPGKGLEWVSY ISSSSSYT NYADSV KGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC ARDIDILTGYYDY WGQGTLVTVSS; wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO:31: GFTFSDYY The amino acid sequence of LCDR2 is shown in SEQ ID NO:32: ISSSSSYT The amino acid sequence of LCDR3 is shown in SEQ ID NO:30: ARDIDILTGYYDY ;

[0066] The amino acid sequence of LV is shown in SEQ ID NO:37:

[0067] DIVMTQSPSSVSASVGDRVTIACRAS QSISTW LAWYQQKPGKGPKLLIY GAS SLQSGVPARFSASGSGTDFTLTITSLQPEDFATYYC QQAHSLPLT FGQGTRLEIK; wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO: 34: QSISTW The amino acid sequence of LCDR2 is shown in SEQ ID NO:35: GAS The amino acid sequence of LCDR3 is shown in SEQ ID NO: 36: QQAHSLPLT .

[0068] Group iv

[0069] The amino acid sequence of HV is shown in SEQ ID NO:40:

[0070] EVQLVQSGAEVKKPGESLKISCEGF GYSFPNYW IGWVRQMPGKGLEWMGI IYPGDSDT RYSPSFQGQVTISADKSISTAYLQWSSLKASDTAIYYC ARLPVYCSSTGCDNYYYYGMDV WGQGTLVTVSS; wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO:41: GYSFPNYW The amino acid sequence of HCDR2 is shown in SEQ ID NO:42: IYPGDSDT The amino acid sequence of HCDR3 is shown in SEQ ID NO:43: ARLPVYCSSTGCDNYYYYGMDV ;

[0071] The amino acid sequence of LV is shown in SEQ ID NO:47:

[0072] DIVMTQPPLSLPVTPGEPASISC RSSRSLLYRNGDNL LSWYLQKPGQSPQLLIY LGS SRASGVPDRISGSGSGTDFTLKISRVEAEDVGVYY CMQGLETPYT FGQGTKLEIK; wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO: 44: RSLLYRNGDNL The amino acid sequence of LCDR2 is shown in SEQ ID NO:45: LGS The amino acid sequence of LCDR3 is shown in SEQ ID NO:46: MQGLETPYT .

[0073] Group V

[0074] The amino acid sequence of HV is shown in SEQ ID NO:50:

[0075] QVQLVQSGAEVKKPGASVKVSCKAS GYTFTSYG ISWVRQAPGQGLEWMGW ISAYNGNT NYAQKLQGRVTMTTDTSSTAYMELRSLRSDDTAVYYC ARDLYNWNDAEGWSGGMDV WGQGTTVTVSS; wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO:51: GYTFTSYG The amino acid sequence of HCDR2 is shown in SEQ ID NO:52: ISAYNGNT ; The amino acid sequence of HCDR3 is shown in SEQ ID NO:53: ARDLYNWNDAEGWSGGMDV ;

[0076] The amino acid sequence of LV is shown in SEQ ID NO:57:

[0077] DIVMTQSPATLSLSPGERATLSCRAS QSVISNY LSWYQQRLGQAPRLLIY GTS TRATGVPARFSGSGSGTTDFTLTISSLQPEDFAVYYC QQDYNLPYN FGPGTKLEIK; wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO:54: QSVISNY The amino acid sequence of LCDR2 is shown in SEQ ID NO:55: GTS The amino acid sequence of LCDR3 is shown in SEQ ID NO:56: QQDYNLPYN .

[0078] In the present invention, when the amino acid sequences of the corresponding complementary determining regions in the heavy and light chain variable regions of the monoclonal antibody are used in pairs and expressed as humanized antibodies, the preferred expression pairing group is as follows:

[0079] 1). The amino acid sequences were paired according to the above group i, and the expressed and purified humanized antibody was named E1;

[0080] 2). The amino acid sequences were paired according to the above group ii, and the expressed and purified humanized antibody was named H1;

[0081] 3). The amino acid sequences were paired according to the above group iii, and the expressed and purified humanized antibody was named E8;

[0082] 4). The amino acid sequences were paired according to the above group iv, and the expressed and purified humanized antibody was named F10;

[0083] 5). The amino acid sequences were paired according to the above group v, and the expressed and purified humanized antibody was named H12.

[0084] After constructing the plasmid expression frame of the humanized antibody gene, the expression frame is transferred into 293T cells through a delivery system for antibody expression; wherein the delivery system can be one of lentivirus, retrovirus, common plasmid vector, episomal vector, nano-delivery system, electrotransduction and transposon.

[0085] In one embodiment, the fermented purified humanized antibody can be co-incubated with Nectin4-positive cells to verify the affinity and specificity of the humanized antibody; or, the fermented purified humanized antibody can be subjected to immunohistochemical detection with tissue sections and normal tissue chips to verify the tissue cross-reaction specificity of the humanized antibody.

[0086] In one embodiment, the amino acid sequences of the complementary determining regions corresponding to the heavy and light chain variable regions of the humanized antibody can be concatenated into a scFv fragment sequence to construct a chimeric antigen receptor targeting human nectin4; the chimeric antigen receptor includes an extracellular domain, a transmembrane domain and an intracellular domain; wherein:

[0087] 1) The extracellular domain of the chimeric antigen receptor includes an antigen binding domain; the antigen binding domain includes scFv, and the scFv is the heavy and light chain variable regions corresponding to E1, H1, E8, F10, and H12 respectively;

[0088] 2) The transmembrane domain of the chimeric antigen receptor CAR includes the hinge region of CD28 and the transmembrane domain of CD28, the hinge region of CD8 and the transmembrane domain of 4-1BB, the hinge region of CD8 and the transmembrane domain of ICOS, etc.; wherein, the transmembrane domain of the chimeric antigen receptor is the hinge region of CD28 and the transmembrane domain of CD28, the hinge region of CD8 and the transmembrane domain of 4-1BB or the co-stimulatory domain of 4-1BB or ICOS;

[0089] 3) The intracellular domain of the chimeric antigen receptor includes a co-stimulatory signal transduction region, a cytokine receptor intracellular region and a CD3ζ chain portion; wherein the co-stimulatory signal transduction region refers to a portion of the intracellular domain that includes a co-stimulatory molecule; co-stimulatory molecules are cell surface molecules required for the effective response of lymphocytes to antigens; the intracellular domain can be a combination of any one of the co-stimulatory domains of CD28, 4-1BB, ICOS and the intracellular activation signal CD3zeta.

[0090] In the present invention, the recombinant cell line is an immune cell, which may be any of T cells, NK cells, NKT cells, macrophages, gamma-delta T cells, TIL cells, and TCR-T cells. Specifically, when the immune cells express chimeric antigen receptors CAR, NK cells, NKT cells, TILs, and gamma-delta T cells are equivalent to T cells (or T cells can replace NK cells).

[0091] Any of the above monoclonal antibodies targeting human Nectin4 protein of the present invention can be prepared into pharmaceutically acceptable carriers, diluents or excipients, all of which have good shaping effects while maintaining good drug efficacy; for example, they can be applied to biomaterials and / or biological preparations.

[0092] When the monoclonal antibody of the present invention is used in a biological preparation, the biological preparation comprises an expression cassette (e.g., a gene expression cassette), a recombinant vector (e.g., a plasmid), a recombinant protein (e.g., a fusion protein, an antibody protein, etc.), a recombinant microorganism (e.g., Escherichia coli, a bacteriophage, etc.) or a recombinant cell line (e.g., an immune cell, a CHO cell, etc.) constructed by the above-mentioned nucleic acid sequence or amino acid sequence, and these nucleic acid sequences or amino acid sequences are derived from the above-mentioned monoclonal antibody; wherein the recombinant vector includes a gene recombinant expression vector and a chimeric antigen receptor.

[0093] When monoclonal antibodies are used in biological preparations, they exist as components of biological preparation configuration, and the biological preparation components also include reagents for detecting the concentration of human Nectin4 protein, reagents for detecting the expression level of human Nectin4 protein on the surface of tumor cells, antibodies coupled to toxins to kill human Nectin4 positive cells, antibodies coupled to other antibodies to produce polyclonal antibodies targeting human Nectin4 and other antigens, and antibodies coupled to other proteins to produce functional recombinant proteins targeting human Nectin4.

[0094] In summary, compared with the prior art, the monoclonal antibody provided by the present invention has the following advantages:

[0095] 1. It has the ability to specifically recognize and target human Nectin4 protein, and some combinations have dual species specificity for humans and mice;

[0096] 2. It has good tissue specificity and only recognizes tissues expressing Nectin4, but not other tissues and organs, indicating that the antibody of the present invention has good prospects for detection applications;

[0097] 3. CAR-T prepared by antibodies has a specific killing function on Nectin4-positive target cells and has good application prospects in cell immunotherapy;

[0098] 4. This antibody has good targeting properties in both its natural and recombinant configurations, and has good application prospects in antibody drugs (ADC), multi-antibody drugs and recombinant antibody proteins.

[0099] Taking CAR-T cells as an example, the expanded application of the monoclonal antibody targeting human Nectin4 protein of the present invention is representatively described in detail below.

[0100] The chimeric antigen receptor (CAR) of the present invention includes an extracellular domain, a transmembrane domain and an intracellular domain. Among them, the extracellular domain includes an antigen binding domain, the intracellular domain includes a costimulatory signal transduction region, a cytokine receptor intracellular region and a CD3ζ chain portion, and the costimulatory signal transduction region refers to a part of the intracellular domain including a costimulatory molecule, and the costimulatory molecule is a cell surface molecule required for the effective response of lymphocytes to antigens.

[0101] In a preferred embodiment, the extracellular domain of the CAR provided by the present invention includes a humanized antibody heavy and light chain variable region antigen binding domain targeting Nectin4. When the CAR of the present invention is expressed in T cells, antigen recognition can be performed based on antigen binding specificity or protein receptor binding. The antigen binding domain is preferably fused with the intracellular domain from the co-stimulatory molecule and the CD3zeta chain. Preferably, the antigen binding domain is fused with the intracellular domain of the combination of CD28, 4-1BB, ICOS signaling domain and CD3ζ signaling domain, respectively.

[0102] In the CAR of the present invention, the intracellular domain includes the signaling domains of CD28, 4-1BB, ICOS and the signaling domain of CD3zeta.

[0103] The vector of the expression cassette is selected from one or more of DNA, RNA, plasmid, lentiviral vector, adenoviral vector, retroviral vector, transposon, and other gene transfer systems. Preferably, the vector is a viral vector.

[0104] The nucleic acid sequence of the vector encoding the desired molecule can be obtained using recombinant methods known in the art, such as, for example, by screening a library from a cell expressing the gene, by obtaining the gene from a vector known to include the gene, or by directly isolating from cells and tissues containing the gene using standard techniques. Alternatively, the gene of interest can be produced synthetically.

[0105] The present invention also provides a vector for inserting an expression cassette, which is derived from a retrovirus, such as a lentivirus vector, and is characterized by long-term and stable integration of the target gene into cells; can transduce non-proliferating cells, such as hepatocytes; has low immunogenicity; and is highly safe. A typical cloning vector contains transcription and translation terminators, initiation sequences, and promoters that can be used to regulate the expression of the desired nucleic acid sequence.

[0106] The expression vector can be provided to the cell in the form of a viral vector, and viruses that can be used as vectors include but are not limited to retroviruses, adenoviruses, adeno-associated viruses, herpes viruses and slow viruses. Typically, suitable vectors contain a replication origin that works in at least one organism, a promoter sequence, a convenient restriction enzyme site and one or more selectable markers. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the subject cell in vivo or in vitro. In one embodiment, a slow virus vector is used.

[0107] Additional promoter elements, such as enhancers, can regulate the frequency of transcription initiation. Typically, these are located in the 30-110bp region upstream of the start site, although recently it has been shown that many promoters also contain functional elements downstream of the start site. The spacing between promoter elements is often flexible, so that when an element is inverted or moved relative to another, the promoter function is maintained. An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence, and another example is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to simian virus 40 (SV40) early promoter, mouse mammary cancer virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr (Epstein-Barr) virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, such as but not limited to actin promoter, myosin promoter, heme promoter, and creatine kinase promoter. Further, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also considered to be a part of the present invention. The use of inducible promoters provides a molecular switch that can turn on the expression of a polynucleotide sequence operably connected to an inducible promoter when such expression is desired, or turn off expression when expression is undesirable. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0108] In order to evaluate the expression of CAR polypeptides or parts thereof, the expression vector introduced into the cell may also include any one or both of a selectable marker gene or a reporter gene to facilitate identification and selection of expressing cells from a cell population seeking to be transfected or infected by a viral vector. In other aspects, selectable markers may be carried on a single DNA segment and used for co-transfection procedures. Both selectable markers and reporter genes may be flanked by appropriate regulatory sequences so that they can be expressed in host cells. Useful selectable markers include, for example, antibiotic resistance genes, such as neo and the like.

[0109] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and / or exogenous nucleic acids are well known in the art. A preferred method for introducing polynucleotides into host cells is lipofection. Nucleic acids can be associated with lipids, and nucleic acids associated with lipids can be encapsulated into the aqueous interior of liposomes, dispersed within the lipid bilayer of liposomes, attached to liposomes via linker molecules associated with both liposomes and oligonucleotides, entrapped in liposomes, complexed with liposomes, dispersed in solutions containing lipids, and lipids are fatty substances that can be naturally occurring or synthetic lipids. For example, lipids include fat droplets that occur naturally in the cytoplasm and such compounds that contain long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0110] The present invention provides CAR-T cells containing the above-described human Nectin4 protein targeting protein and a pharmaceutically acceptable carrier, diluent or excipient.

[0111] In one embodiment, the preparation is a liquid preparation. Preferably, the preparation is an injection. Preferably, the concentration of the CAR-T cells in the preparation is 1×10 3 ~1×10 8 / ml, preferably, the concentration of CAR-T cells is 1×10 4 ~1×10 7 In one embodiment, the formulation may include a buffer such as neutral buffered saline, sulfate buffered saline, etc.; a carbohydrate such as glucose, mannose, sucrose or dextran, mannitol; a protein; a polypeptide or an amino acid such as glycine; an antioxidant; a chelating agent such as EDTA or glutathione; an adjuvant (e.g., aluminum hydroxide); and a preservative. The formulation of the present invention is preferably formulated for intravenous administration.

[0112] The present invention includes therapeutic applications of cells (e.g., T cells) transduced with lentiviral vectors encoding nucleic acid constructs of the antibodies of the present invention. Transduced T cells can cause CAR-mediated T-cell responses. The injected cells are capable of killing the recipient's tumor cells, and the CAR-T cells are capable of replicating in vivo, producing long-term persistence that can lead to sustained tumor control. The CAR-T cell membrane of the present invention can express the humanized antibody scFv structure antigen chimeric receptor targeting Nectin4 of the present invention, specifically killing Nectin4-positive target cells without off-target risk.

[0113] In one embodiment, the immune cells of the present invention can undergo robust in vivo T cell expansion and can sustain an extended amount of time. In addition, the CAR-mediated immune response can be part of an adoptive immunotherapy step, in which CAR-modified T cells induce an immune response specific to the antigen binding domain in the CAR. For example, Nectin4 CAR-T cells cause a specific immune response against cells expressing Nectin4.

[0114] The symptoms that can be treated include tumor cells that positively express Nectin 4. The symptoms that may be caused by tumor cells include, but are not limited to, carcinoma, blastoma, and sarcoma.

[0115] Immune cell CAR modified T cells using antibodies of the present invention may be administered alone or in combination with other drugs, pharmaceutical compositions, diluents and / or with other components such as IL-2, IL-17 or other cytokines or cell groups. Simply put, the pharmaceutical composition of the present invention may include a target cell group as described herein, combined with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may include buffers such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The composition of the present invention is preferably formulated for intravenous administration.

[0116] Pharmaceutical compositions made with immune cells using antibodies of the present invention can be administered in a manner suitable for the disease to be treated (or prevented). The amount and frequency of administration will be determined by factors such as the patient's condition, and the type and severity of the patient's disease, and by the clinical protocol. When "immunologically effective amount", "anti-tumor effective amount", "tumor-suppressive effective amount" or "therapeutic amount" is indicated, the exact amount of the composition of the present invention to be administered can be determined by the physician, who takes into account the individual differences in the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and condition. It can be generally stated that the pharmaceutical composition comprising the T cells described herein can be administered in an amount of 1×10 4 ~1×10 9 The dosage is 1×10 cells / kg body weight, preferably 1×10 5 ~1×10 6 The T cell composition can also be administered at these doses multiple times. The optimal dosage and treatment regimen for a specific patient can be determined by a medical technician by monitoring the patient's disease signs and adjusting the treatment accordingly.

[0117] Administration of the formulations of the present invention can be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation or transplantation. The compositions described herein can be administered to the patient subcutaneously, intradermally, intratumorally, intranodally, intraspinal, intramuscularly, by intravenous (iv) injection or intraperitoneally. In one embodiment, the T cell composition of the present invention is administered to the patient by intradermal or subcutaneous injection. In another embodiment, the T cell composition of the present invention is preferably administered by intravenous (iv) injection. The composition of the T cell can be directly injected into a tumor, a lymph node or an infection site.

[0118] The following is a detailed description through specific embodiments.

[0119] Example 1 Preparation of monoclonal antibodies targeting human nectin4

[0120] 1.1. Acquisition of humanized antibody sequences

[0121] Using PCR technology, mRNA is extracted from normal human peripheral blood cells and reverse transcribed into cDNA. A complete set of antibody heavy chain variable region (VH) and light chain variable region (VL) genes are amplified from cDNA, cloned into a phage vector and expressed in the form of fusion proteins on the surface of its shell to prepare a phage library.

[0122] There are antibody genes in phage DNA, and antibody molecules are expressed on its surface. Multiple rounds of antigen affinity-adsorption-elution-amplification process steps are used to finally screen out the specific human Nectin4 targeted clone antibody, and the final monoclonal antibody is screened out by ELISA, which is then fermented and evaluated for its function. The gene of the humanized antibody finally screened is extracted and the expression plasmid of the antibody heavy and light chain variable regions is constructed and fermented, and then the antibody is purified and the CAR plasmid is constructed.

[0123] 1.2. Fermentation and purification of humanized antibodies

[0124] The constructed expression plasmids of the heavy and light chain variable regions of the antibody were paired, and PEI reagent was used as a transfection reagent. At the same time, a pair of expression plasmids of the heavy and light chain variable regions were transiently transfected into 293T cells for antibody protein fermentation. The heavy and light chain variable regions of the antibody were naturally paired during the fermentation process, and the culture fluid was collected. The culture fluid was filtered using a 0.22μm sterile filter membrane, and the culture supernatant was purified by affinity chromatography.

[0125] The purification method is as follows: (1) Start AKTA pure25 and use affinity column binding buffer (20mM PB, pH7.0) to equilibrate HiTrap rProtein A FF 1ml column at a rate of 1.0ml / min; (2) 50ml of the filtered culture supernatant is added at 1ml / min and re-equilibrated with affinity column binding buffer; (3) Elution buffer (0.1M glycine-HCl, pH2.7) is used to elute the sample at a rate of 1ml / min, and 700ul of the 280nm absorption peak is collected; (4) Superdex200 Increase 10 / 300GL column is equilibrated with molecular sieve chromatography buffer (20-50mM PB, 150mM NaCl, pH6.7) at a rate of 1ml / min, sample is loaded, and 2ml of the 280nm absorption peak is collected.

[0126] Example 2 Cell line culture

[0127] Cell line not expressing nectin4: 293T (human embryonic kidney cells), purchased from ATCC.

[0128] The cell line expressing human Nectin4 (i.e., hNectin4): 293T-hNectin4, constructed by self-produced lentivirus infection.

[0129] Cell line expressing mouse Nectin4 (i.e., mNectin4): 293T-mNectin4, constructed by self-produced lentivirus infection.

[0130] Packaging cells: 293T (human embryonic kidney cell line), purchased from ATCC.

[0131] Establishment of cell lines overexpressing human nectin4 and mouse nectin4: The base sequences expressing hNectin4 and mNectin4 were cloned into the PHBLV lentiviral vector backbone, respectively, and placed under the promoter of EF1α (EF-1α), forming PHBLV-EF1 α-hNectin4 viral vector and PHBLV-EF1 α-mNectin4 viral vector, respectively; PHBLV-EF1 α-hNectin4 and PHBLV-EF1 α-mNectin4 were mixed with the lentiviral envelope plasmid pMD2.G (Addgene, Plasmid#12259) and the lentiviral packaging plasmid psPAX2 (Addgene Plasmid#12260), respectively, and transferred into 293T using the transfection agent Lipofectamine3000 to prepare two kinds of lentiviral complete expression vectors; the viral supernatants were collected at 48h and 72h, respectively, and concentrated by ultracentrifugation (Merck Millipore); the concentrated virus was then used to infect 293T cells, and finally a 293T monoclonal cell line overexpressing hNectin4 and a 293T monoclonal cell line overexpressing mNectin4 were obtained, which were named 293T-hNectin4 cells and 293T-mNectin4 cells, respectively.

[0132] All the above cell culture media were cultured: adherent 293T, 293T-hNectin4 cells and 293T-mNectin4 cells were cultured in DMEM medium supplemented with 10% (v / v) fetal bovine serum (FBS).

[0133] like Figures 1 to 3 In the figure, there are flow cytometry images of 293T cells (blank control cells), 293T-hNectin4 cells and 293T-mNectin4 cells expressing human or mouse Nectin4, respectively; Figure 1 The middle is the result of 293T cells tested with anti-human hNectin4 antibody. The peak is on the left side of the vertical line, indicating that the test result is negative for hNectin4. The right side shows a positive rate of 0.58%, indicating that the construction of hNectin4 by NT blank cells was unsuccessful. Figure 2 The middle is the detection result of 293T-hNectin4 cells using anti-human hNectin4 antibody. The peak position in the figure is on the right side of the vertical line, indicating that the detection result is hNectin4 positive; Figure 3 The middle shows the detection result of 293T-mNectin4 cells using anti-mouse mNectin4 antibody. The peak position in the figure is on the right side of the vertical line, indicating that the detection result is mNectin4 positive.

[0134] Depend on Figures 1 to 3It can be seen that 293T-hNectin4 cells and 293T-mNectin4 cells have successfully constructed 293T overexpression cell lines.

[0135] Example 3 Evaluation of affinity and species specificity of humanized antibodies

[0136] 3.1. The affinity and species specificity of the monoclonal antibody in Example 1 were evaluated using the 293T, 293T-hNectin4 and 293T-mNectin4 cells in Example 2.

[0137] Using the 293T-hNectin4 cells in Example 2 as positive cells and the P177 antibody (ie) as a positive control, the EC50 (concentration for 50% of maximal effect) affinity of the Nectin4 humanized antibodies E1, H1, E8, F10, and H12 were detected. The results are as follows: Figure 4 shown. Figure 4 The results showed that the EC50 affinities of E1, H1, E8, F10, and H12 were close to or higher than the control antibody P177, indicating that the effects of humanized antibodies E1, H1, E8, F10, and H12 were close to or better than the positive control antibody, and their safety was close to or better than the existing positive antibodies.

[0138] 3.2. Using the 293T, 293T-hNectin4, and 293T-mNectin4 cells in Example 2 as target cells, the species specificity of the humanized Nectin4 antibodies E1, H1, E8, F10, and H12 was detected. The test results are as follows: Figures 5 to 7 shown.

[0139] in, Figure 5 In the figure, the peaks of binding of humanized antibodies E1, H1, E8, F10, and H12 to 293T cells are all on the left side of the vertical line, indicating that their binding is negative; Figure 6 The binding peaks of E1, H1, E8, F10, and H12 to 293T-hNectin4 cells are all on the right side of the vertical line, indicating that their binding is positive; Figure 7 The binding peaks of E1, H1, E8, F10, and H12 with 293T-mNectin4 cells are on the right side of the vertical line, indicating positive binding.

[0140] The above results show that humanized antibodies E1, H1, E8, F10, and H12 can specifically bind to human Nectin4, and all antibodies can bind to mouse Nectin4; therefore, the monoclonal antibody targeting Nectin4 of the present invention has both human and mouse species specificity.

[0141] Example 4 Isolation of peripheral blood PBMC and cultivation of T cells

[0142] Monocytes were isolated from the peripheral blood of donors and subjected to density gradient centrifugation using ficol. T cells were enriched using a T cell sorting kit (CD3 MicroBeads, human-lyophilized, 130-097-043). T cells were activated, cultured and expanded using magnetic beads coupled with anti-CD3 / anti-CD28. The culture medium used was TexMACS GMP Medium (MiltenyiBiotec, 170-076-309), which contained 10% (v / v) FBS, 2mM L-glutamine and 100IU / ml rh-IL2. The cells were cultured in a constant temperature incubator at 37°C and 5% CO2, and T cells were obtained after the culture.

[0143] Example 5 CAR structure design and lentiviral packaging

[0144] 5.1. Nectin4-CAR structure, i.e., the design of CAR structure targeting Nectin4:

[0145] In this example, five groups of amino acid sequences of purified humanized antibodies E1, H1, E8, F10, and H12 were targeted to the heavy and light chain variable region scFv sequences of Nectin4 humanized antibodies, and the scFv sequences were respectively constructed into an expression frame of the second-generation CAR, and were named CAR-E1, CAR-H1, CAR-E8, CAR-F10, and CAR-H12, respectively. The expression frame of CAR constructed by the heavy and light chain variable region scFv of the control targeting Nectin4 antibody was used as a control, and was named CAR-177. Figure 8 shown.

[0146] Figure 8 In the figure, 1# to 6# express the CAR expression frame structures of different humanized antibodies, representing chimeric antigen receptors expressed on the cell membranes of 6 types of immune cells, collectively referred to as Nectin4CAR; the details are as follows:

[0147] 1#, CD8 SP / E1scFv / CD8Hing / CD8TM / 4-1BB / CD3eta;

[0148] 2#, CD8 SP / H1scFv / CD8Hing / CD8TM / 4-1BB / CD3eta;

[0149] 3#, CD8 SP / E8scFv / CD8Hing / CD8TM / 4-1BB / CD3eta;

[0150] 4#, CD8 SP / F10scFv / CD8Hing / CD8TM / 4-1BB / CD3eta;

[0151] 5#, CD8 SP / H12scFv / CD8Hing / CD8TM / 4-1BB / CD3eta;

[0152] 6#, CD8 SP / 17scFv / CD8Hing / CD8TM / 4-1BB / CD3eta.

[0153] 5.2 Lentivirus packaging

[0154] The expression cassette was cloned into the PHBLV lentiviral vector backbone and placed under the promoter of EF1α (EF-1α) to form PHBLV-EF1α-CAR-E1, PHBLV-EF1α-CAR-H1, PHBLV-EF1α-CAR-E8, PHBLV-EF1α-CAR-F10 and PHBLV-EF1α-CAR-H12; PHBLV-EF1α-CAR-E1, PHBLV-EF1α-CAR-H1, PHBLV-EF1α-CAR-E8, PHBLV-EF1α-CAR-F10 and PHBLV-EF1α-CAR-H12 were respectively fused with the lentiviral envelope plasmid pMD2 .G (Addgene, Plasmid #12259) and lentiviral packaging plasmid psPAX2 (Addgene Plasmid #12260), and then transferred into 293T using the transfection agent Lipofectamine 3000 to prepare 6 complete lentiviral expression vectors, namely: Lv-CAR-E1, Lv-CAR-H1, Lv-CAR-E8, Lv-CAR-F10, Lv-CAR-H12, and Lv-CAR-177; after the cells were cultured for 24h and 48h, they were transferred to centrifuge tubes for centrifugation, and the viral supernatant was collected after the centrifugation stopped, and the supernatant was concentrated by ultracentrifugation (Merck Millipore); the concentrated virus can be used to infect T cells.

[0155] Example 6 CAR-T cell preparation

[0156] 6.1 Lentivirus infection

[0157] After the primary T cells isolated and purified in Example 4 were reactivated for 1 day, the 6 lentiviruses packaged in Example 5, such as Lv-CAR-E1, Lv-CAR-H1, Lv-CAR-E8, Lv-CAR-F10, Lv-CAR-H12, Lv-CAR-177), were infected with lentiviral vectors at an MOI (1-10), and the 6 virus-infected T cells were transferred to cell culture flasks and cultured in a constant temperature incubator at 37° C. and 5% CO2.

[0158] 6.2. Cell proliferation and CAR positive rate detection

[0159] Samples were taken on the 6th, 8th, 10th and 13th days after infection of the six types of T cells to detect the cell number; the CAR positivity of T cells was detected on the 6th day, and then culture medium was added every 1-2 days for cell subculture.

[0160] Using the six lentiviral vectors in Example 5, six CAR-T cells were successfully constructed, named CAR-E1, CAR-H1, CAR-E8, CAR-F10, CAR-H12, and CAR-177, respectively, and T cells not infected by the lentivirus were used as blank controls (i.e., NT cells).

[0161] Fig. 9 The figure shows the proliferation rate curves of seven types of T cells, such as NT, CAR-E1, CAR-H1, CAR-E8, CAR-F10, CAR-H12, and CAR-177. Fig. 9 It can be seen that under the same culture conditions, there is no significant difference in the proliferation rates of the seven T cells. The proliferation rates of CAR-E1, CAR-H1, CAR-E8, CAR-F10, and CAR-H12 cells prepared by the present invention are consistent with those of positive CAR-177 cells, indicating that antibodies or fragments thereof targeting human Nectin4 protein have no effect on the proliferation of constructed chimeric antigen receptor immune T cells.

[0162] like Figures 10 to 16 The figure shows the CAR positive rate flow cytometry of NT cells, CAR-E1, CAR-H1, CAR-E8, CAR-F10, CAR-H12, and CAR-177 sampled and tested on the 6th day. The right side of the vertical line in each figure is the CAR positive rate, which is Figures 10 to 16 It can be seen that in the CAR structure, different scFv fragments have no significant effect on the CAR positivity rate, indicating that replacing scFv does not affect the successful preparation of CAR-T cells. In other words, antibodies or their fragments targeting human Nectin4 protein have no effect on the proliferation of CAR-T cells after constructing CAR-T cells.

[0163] Example 7 CAR-T cell killing test on 293T-hNectin4 cells

[0164] The six CAR-T cells and NT cells obtained in Example 6 were subjected to an in vitro killing experiment. The killing effect of CAR-T cells was detected by RTCA equipment. The target cells and effector cells were co-incubated for 24 hours. The killing efficiency was as follows: Fig.17 and 18 shown.

[0165] Fig.17 It is shown in the figure that NT, CAR-E1, CAR-H1, CAR-E8, CAR-F10, CAR-H12, and CAR-177 cells have no killing effect on 293T cells, indicating that they have no killing activity on non-target cells; Fig.18 The results showed that CAR-E1, CAR-H1, CAR-E8, CAR-F10, CAR-H12, and CAR-177 cells constructed with antibodies targeting human Nectin4 protein or its fragments all had good targeted killing rates against 293T-hNectin4 cells, especially the killing efficiency of 50:1 was as high as over 90%, which was much higher than the killing rate of NT cells against 293T-hNectin4 cells (21.6%).

[0166] It should be understood that the above description of the preferred embodiments of the present invention is relatively detailed and cannot be regarded as limiting the scope of patent protection of the present invention. The scope of patent protection of the present invention shall be based on the attached claims.

Claims

1. An antibody or a fragment thereof targeting human Nectin4 protein, characterized in that: The antibody or fragment thereof comprises a heavy chain and a light chain, wherein the amino acid sequences of heavy chain complementary determining regions 1 to 3 (HCDR1, HCDR2 and HCDR3) comprised in the heavy chain are shown in SEQ ID NO:41, SEQ ID NO:42 and SEQ ID NO:43, respectively; the amino acid sequences of light chain complementary determining regions 1 to 3 (LCDR1, LCDR2 and LCDR3) comprised in the light chain are shown in SEQ ID NO:44, SEQ ID NO:45 and SEQ ID NO:46, respectively.

2. The antibody or fragment thereof according to claim 1, characterized in that The heavy chain and light chain respectively comprise a heavy chain variable region (VH) and a light chain variable region (VL); the heavy chain complementary determining regions 1 to 3 (HCDR1, HCDR2 and HCDR3) are from the heavy chain variable region, and the light chain complementary determining regions 1 to 3 (LCDR1, LCDR2 and LCDR3) are from the light chain variable region; wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:40; the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:

47.

3. The antibody or fragment thereof according to claim 1, characterized in that The antibody or its fragment is a monoclonal antibody, a single-chain antibody or a double-chain antibody directed against the Nectin4 protein.

4. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the antibody or fragment thereof according to any one of claims 1 to 3.

5. An expression cassette, characterized in that The expression cassette contains the nucleic acid molecule according to claim 4.

6. A recombinant vector, characterized in that: The recombinant vector contains the nucleic acid molecule described in claim 4, or contains the expression cassette described in claim 5.

7. A host cell, characterized in that The host cell contains the nucleic acid molecule according to claim 4, or contains the expression cassette according to claim 5.

8. A recombinant microorganism, characterized in that The recombinant microorganism contains the nucleic acid molecule described in claim 4, or contains the expression cassette described in claim 5, or contains the recombinant vector described in claim 6.

9. A recombinant cell line, characterized in that The recombinant cell line contains the nucleic acid molecule according to claim 4, or the expression cassette according to claim 5, or the recombinant vector according to claim 6.

10. A biological agent, characterized in that: The biological preparation contains the nucleic acid molecule described in claim 4, or the expression cassette described in claim 5, or the recombinant vector described in claim 6, or the host cell described in claim 7, or the recombinant microorganism described in claim 8, or the recombinant cell line described in claim 9.

11. The biological agent according to claim 10, characterized in that The biological preparation is a reagent for detecting the concentration of human Nectin4 protein, or a reagent for detecting the expression level of human Nectin4 protein on the surface of tumor cells, or a reagent for antibody-mediated complement-dependent or cell-dependent cytotoxic reaction on human Nectin4 positive cells.

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