Antibodies targeting ROR1 and their applications

Abstract

The present invention provides an antibody targeting ROR1 and its application. The ROR1 antibody of the present invention exhibits excellent specificity and binding ability, and the chimeric antigen receptor derived therefrom exhibits excellent killing ability against tumor cells, and has less cytotoxicity and low side effects.

Description

[0001] This application is a divisional application of a Chinese patent application filed on April 21, 2023, with application number 202310435343X, and invention name “Antibodies targeting ROR1 and their applications”. Technical Field

[0002] The present invention relates to the field of antibodies, and in particular to an antibody targeting ROR1 and an application thereof. Background Art

[0003] ROR1 protein is a receptor tyrosine kinase protein encoded by the ROR1 gene, and its full name is Inactive tyrosine-protein kinase transmembrane receptor ROR1 or Neurotrophic tyrosine kinase, receptor-related 1. ROR1 protein contains 937 amino acid residues, which can be divided into extracellular immunoglobulin-like domain, cysteine-rich domain and Kringle domain, transmembrane segment, intracellular tyrosine kinase domain, serine / threonine-rich domain, proline-rich domain and C-terminal serine / threonine-rich domain.

[0004] Under normal circumstances, ROR1 protein is mainly expressed during embryogenesis. In mouse embryos, ROR1 expression areas include the central nervous system, early limb buds, cartilage growth discs, heart, lungs, and mesonephros. In normal adult tissues, ROR1 protein is expressed in immature B lymphocytes, some pancreatic cells, and some gastric cells. ROR1 antibody Zilovertamab (also known as cirmtuzumab or UC-961) has also shown good safety in clinical trials.

[0005] ROR1 protein is highly expressed in a variety of tumor tissues of hematological tumors and solid tumors, including acute lymphoblastic leukemia, chronic lymphoblastic leukemia, diffuse large B-cell lymphoblastic leukemia, follicular cell lymphoblastic leukemia, and mantle cell lymphoblastic leukemia in hematological tumors, and breast cancer, gastric cancer, lung cancer, ovarian cancer, colorectal cancer, pancreatic cancer, endometrial cancer, melanoma, mesothelioma, etc. Among them, high expression of ROR1 is associated with reduced overall survival in patients with chronic lymphoblastic leukemia, breast cancer, lung cancer, ovarian cancer, colorectal cancer, endometrial cancer, and melanoma. In terms of the mechanism of cancer occurrence, ROR1 protein activates the non-classical WNT signaling pathway, MAPK-ERK signaling pathway, and PI3K signaling pathway after binding to WNT5A protein, and inhibits the phosphorylation of p38 protein, resulting in increased cell proliferation and epithelial-mesenchymal transition, and reduced cell apoptosis.

[0006] In summary, ROR1 is a target that is widely expressed in tumor tissues and has good safety. Targeting ROR1 protein using antibodies, antibody-drug conjugates, CAR-T and CAR-NK has broad application prospects. Summary of the invention

[0007] The purpose of the present invention is to provide an antibody targeting ROR1 and application thereof.

[0008] In a first aspect of the present invention, an antibody or an antigen-binding fragment thereof targeting ROR1 is provided, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprise a complementary determining region CDR selected from the following group:

[0009] (1) The heavy chain variable region includes the following complementarity determining region CDR: (Ab3)

[0010] HCDR1 shown in SEQ ID NO:7,

[0011] HCDR2 shown in SEQ ID NO:8, and

[0012] HCDR3 shown in SEQ ID NO:9;

[0013] And the light chain variable region includes the following complementarity determining regions CDR:

[0014] LCDR1 shown in SEQ ID NO:10,

[0015] LCDR2 shown in SEQ ID NO: 11, and

[0016] LCDR3 shown in SEQ ID NO:12; or

[0017] (2) The heavy chain variable region includes the following complementarity determining region CDR: (Ab4)

[0018] HCDR1 shown in SEQ ID NO:13,

[0019] HCDR2 shown in SEQ ID NO:14, and

[0020] HCDR3 shown in SEQ ID NO:15;

[0021] And the light chain variable region includes the following complementarity determining regions CDR:

[0022] LCDR1 shown in SEQ ID NO:16,

[0023] LCDR2 shown in SEQ ID NO: 17, and

[0024] LCDR3 shown in SEQ ID NO: 18; or

[0025] (3) The heavy chain variable region includes the following complementarity determining region CDR: (Ab1)

[0026] HCDR1 shown in SEQ ID NO:1,

[0027] HCDR2 shown in SEQ ID NO:2, and

[0028] HCDR3 shown in SEQ ID NO:3;

[0029] And the light chain variable region includes the following complementarity determining regions CDR:

[0030] LCDR1 shown in SEQ ID NO:4,

[0031] LCDR2 shown in SEQ ID NO:5, and

[0032] LCDR3 shown in SEQ ID NO:6; or

[0033] (4) The heavy chain variable region includes the following complementarity determining region CDR: (Ab6)

[0034] HCDR1 shown in SEQ ID NO:19,

[0035] HCDR2 shown in SEQ ID NO:20, and

[0036] HCDR3 shown in SEQ ID NO:21;

[0037] And the light chain variable region includes the following complementarity determining regions CDR:

[0038] LCDR1 shown in SEQ ID NO:22,

[0039] LCDR2 shown in SEQ ID NO:23, and

[0040] LCDR3 shown in SEQ ID NO:24; or

[0041] (5) The heavy chain variable region includes the following complementarity determining region CDR: (Ab7)

[0042] HCDR1 shown in SEQ ID NO:25,

[0043] HCDR2 shown in SEQ ID NO:26, and

[0044] HCDR3 shown in SEQ ID NO:27;

[0045] And the light chain variable region includes the following complementarity determining regions CDR:

[0046] LCDR1 shown in SEQ ID NO:28,

[0047] LCDR2 shown in SEQ ID NO:23, and

[0048] LCDR3 shown in SEQ ID NO:29; or

[0049] (6) The heavy chain variable region includes the following complementary determining region CDR: (Ab11) HCDR1 shown in SEQ ID NO: 25,

[0050] HCDR2 shown in SEQ ID NO:26, and

[0051] HCDR3 shown in SEQ ID NO:27;

[0052] And the light chain variable region includes the following complementarity determining regions CDR:

[0053] LCDR1 shown in SEQ ID NO:28,

[0054] LCDR2 shown in SEQ ID NO:23, and

[0055] LCDR3 shown in SEQ ID NO:29; or

[0056] (7) The heavy chain variable region includes the following complementary determining region CDR: (Ab12) HCDR1 shown in SEQ ID NO: 45,

[0057] HCDR2 shown in SEQ ID NO:46, and

[0058] HCDR3 shown in SEQ ID NO:47;

[0059] And the light chain variable region includes the following complementarity determining regions CDR:

[0060] LCDR1 shown in SEQ ID NO:48,

[0061] LCDR2 shown in SEQ ID NO:49, and

[0062] LCDR3 shown in SEQ ID NO:50; or

[0063] (8) The heavy chain variable region includes the following complementarity determining region CDR: (Ab8) HCDR1 shown in SEQ ID NO: 30,

[0064] HCDR2 shown in SEQ ID NO:31, and

[0065] HCDR3 shown in SEQ ID NO:32;

[0066] And the light chain variable region includes the following complementarity determining regions CDR:

[0067] LCDR1 shown in SEQ ID NO:33,

[0068] LCDR2 shown in SEQ ID NO:34, and

[0069] LCDR3 shown in SEQ ID NO:35; or

[0070] (9) The heavy chain variable region includes the following complementary determining regions (CDRs): (Ab9) HCDR1 shown in SEQ ID NO: 36,

[0071] HCDR2 shown in SEQ ID NO:37, and

[0072] HCDR3 shown in SEQ ID NO:38;

[0073] And the light chain variable region includes the following complementarity determining regions CDR:

[0074] LCDR1 shown in SEQ ID NO:39,

[0075] LCDR2 shown in SEQ ID NO:23, and

[0076] LCDR3 shown in SEQ ID NO:29; or

[0077] (10) The heavy chain variable region includes the following complementarity determining region CDR: (Ab13)

[0078] HCDR1 shown in SEQ ID NO:51,

[0079] HCDR2 shown in SEQ ID NO:52, and

[0080] HCDR3 shown in SEQ ID NO:53;

[0081] And the light chain variable region includes the following complementarity determining regions CDR:

[0082] LCDR1 shown in SEQ ID NO:54,

[0083] LCDR2 shown in SEQ ID NO:23, and

[0084] LCDR3 shown in SEQ ID NO:40;

[0085] Wherein, the CDR sequence is based on Kabat's numbering scheme.

[0086] In another preferred embodiment, the heavy chain variable region and the light chain variable region of the antibody or antigen-binding fragment thereof include a complementary determining region CDR selected from the following group:

[0087] (1) The heavy chain variable region includes the following complementarity determining region CDR: (Ab3)

[0088] HCDR1 shown in SEQ ID NO:57,

[0089] HCDR2 shown in SEQ ID NO:56, and

[0090] HCDR3 shown in SEQ ID NO:9;

[0091] And the light chain variable region includes the following complementarity determining regions CDR:

[0092] LCDR1 shown in SEQ ID NO:10,

[0093] LCDR2 shown in SEQ ID NO: 11, and

[0094] LCDR3 shown in SEQ ID NO:12; or

[0095] (2) The heavy chain variable region includes the following complementarity determining region CDR: (Ab4)

[0096] HCDR1 shown in SEQ ID NO:58,

[0097] HCDR2 shown in SEQ ID NO:59, and

[0098] HCDR3 shown in SEQ ID NO:15;

[0099] And the light chain variable region includes the following complementarity determining regions CDR:

[0100] LCDR1 shown in SEQ ID NO:16,

[0101] LCDR2 shown in SEQ ID NO: 17, and

[0102] LCDR3 shown in SEQ ID NO: 18; or

[0103] (3) The heavy chain variable region includes the following complementarity determining region CDR: (Ab1)

[0104] HCDR1 shown in SEQ ID NO:55,

[0105] HCDR2 shown in SEQ ID NO:56, and

[0106] HCDR3 shown in SEQ ID NO:3;

[0107] And the light chain variable region includes the following complementarity determining regions CDR:

[0108] LCDR1 shown in SEQ ID NO:4,

[0109] LCDR2 shown in SEQ ID NO:5, and

[0110] LCDR3 shown in SEQ ID NO:6; or

[0111] (4) the heavy chain variable region comprises the following complementarity determining region CDR: (Ab6) HCDR1 shown in SEQ ID NO: 57,

[0112] HCDR2 shown in SEQ ID NO:60, and

[0113] HCDR3 shown in SEQ ID NO:21;

[0114] And the light chain variable region includes the following complementarity determining regions CDR:

[0115] LCDR1 shown in SEQ ID NO:22,

[0116] LCDR2 shown in SEQ ID NO:23, and

[0117] LCDR3 shown in SEQ ID NO:24; or

[0118] (5) The heavy chain variable region includes the following complementarity determining region CDR: (Ab7) HCDR1 shown in SEQ ID NO: 61,

[0119] HCDR2 shown in SEQ ID NO:62, and

[0120] HCDR3 shown in SEQ ID NO:27;

[0121] And the light chain variable region includes the following complementarity determining regions CDR:

[0122] LCDR1 shown in SEQ ID NO:28,

[0123] LCDR2 shown in SEQ ID NO:23, and

[0124] LCDR3 shown in SEQ ID NO:29; or

[0125] (6) The heavy chain variable region includes the following complementarity determining region CDR: (Ab11) HCDR1 shown in SEQ ID NO: 57,

[0126] HCDR2 shown in SEQ ID NO:60, and

[0127] HCDR3 shown in SEQ ID NO:42;

[0128] And the light chain variable region includes the following complementarity determining regions CDR:

[0129] LCDR1 shown in SEQ ID NO:43,

[0130] LCDR2 shown in SEQ ID NO:23, and

[0131] LCDR3 shown in SEQ ID NO:44; or

[0132] (7) The heavy chain variable region includes the following complementary determining region CDR: (Ab12) HCDR1 shown in SEQ ID NO: 65,

[0133] HCDR2 shown in SEQ ID NO:66, and

[0134] HCDR3 shown in SEQ ID NO:47;

[0135] And the light chain variable region includes the following complementarity determining regions CDR:

[0136] LCDR1 shown in SEQ ID NO:48,

[0137] LCDR2 shown in SEQ ID NO:49, and

[0138] LCDR3 shown in SEQ ID NO:50; or

[0139] (8) The heavy chain variable region includes the following complementarity determining region CDR: (Ab8) HCDR1 shown in SEQ ID NO: 61,

[0140] HCDR2 shown in SEQ ID NO:64, and

[0141] HCDR3 shown in SEQ ID NO:32;

[0142] And the light chain variable region includes the following complementarity determining regions CDR:

[0143] LCDR1 shown in SEQ ID NO:33,

[0144] LCDR2 shown in SEQ ID NO:34, and

[0145] LCDR3 shown in SEQ ID NO:35; or

[0146] (9) The heavy chain variable region includes the following complementarity determining region CDR: (Ab9)

[0147] HCDR1 shown in SEQ ID NO:57,

[0148] HCDR2 shown in SEQ ID NO:60, and

[0149] HCDR3 shown in SEQ ID NO:38;

[0150] And the light chain variable region includes the following complementarity determining regions CDR:

[0151] LCDR1 shown in SEQ ID NO:39,

[0152] LCDR2 shown in SEQ ID NO:23, and

[0153] LCDR3 shown in SEQ ID NO:40; or

[0154] (10) The heavy chain variable region includes the following complementarity determining region CDR: (Ab13)

[0155] HCDR1 shown in SEQ ID NO:67,

[0156] HCDR2 shown in SEQ ID NO:60, and

[0157] HCDR3 shown in SEQ ID NO:53;

[0158] And the light chain variable region includes the following complementarity determining regions CDR:

[0159] LCDR1 shown in SEQ ID NO:54,

[0160] LCDR2 shown in SEQ ID NO:23, and

[0161] LCDR3 shown in SEQ ID NO:40;

[0162] Wherein, the CDR sequence is based on the Chothia numbering scheme.

[0163] In another preferred embodiment, the antibody comprises a heavy chain and a light chain, the heavy chain comprises the three heavy chain CDRs and a heavy chain framework region for connecting the heavy chain CDRs; the light chain comprises the three light chain CDRs and a light chain framework region for connecting the light chain CDRs.

[0164] In another preferred embodiment, the antibody or antigen-binding fragment thereof targeting ROR1 is selected from the following group: camel Ig, IgNAR, Fab fragment, Fab' fragment F(ab)'2 fragment, F(ab)'3 fragment, Fv, single-chain Fv antibody ("scFv"), double scFv, (scFv)2, miniantibody, bifunctional antibody, trifunctional antibody, tetrafunctional antibody, disulfide bond-stabilized Fv protein ("dsFv") and single-domain antibody (sdAb, nanoantibody).

[0165] In another preferred example, the light chain of the antibody further includes a light chain constant region.

[0166] In another preferred embodiment, the light chain constant region is of human, mouse or rabbit origin, preferably of human origin.

[0167] In another preferred embodiment, the heavy chain of the antibody further includes a heavy chain constant region.

[0168] In another preferred embodiment, the heavy chain constant region is of human, mouse or rabbit origin, preferably of human origin.

[0169] In another preferred embodiment, the antibody is a double-chain antibody or a single-chain antibody.

[0170] In another preferred embodiment, the antibody is a monoclonal antibody.

[0171] In another preferred embodiment, the antibody includes a monospecific, bispecific, trispecific antibody or a multispecific antibody.

[0172] In another preferred embodiment, the antibody specifically binds to ROR1.

[0173] In another preferred embodiment, the KD value (M) of the antibody's affinity for human ROR1 is 1.0E-10 to 1.0E-8.

[0174] In another preferred embodiment, the antibody is a single-chain antibody (scFv) having an amino acid sequence selected from the following group:

[0175] SEQ ID NO. 68, 69, 70, 71, 72, 73, 74, 75, 76, 77 or SEQ ID NO. 79, 80, 81, 82, 83, 84, 85, 86, 87, 88.

[0176] In another preferred embodiment, the single-chain antibody has an amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity (wherein the CDR remains unchanged or substantially unchanged) to the amino acid sequence shown in SEQ ID NO.68, 69, 70, 71, 72, 73, 74, 75, 76, 77 or SEQ ID NO.79, 80, 81, 82, 83, 84, 85, 86, 87, 88.

[0177] The second aspect of the present invention provides a recombinant protein, wherein the recombinant protein has:

[0178] (i) the antibody or antigen-binding fragment thereof according to the first aspect of the present invention;

[0179] and (ii) optionally a tag sequence to facilitate expression and / or purification.

[0180] In another preferred embodiment, the tag sequence includes a 6His tag.

[0181] In another preferred embodiment, the recombinant protein (or polypeptide) includes a fusion protein.

[0182] In another preferred embodiment, the recombinant protein is a monomer, a dimer, or a polymer.

[0183] In another preferred embodiment, the recombinant protein further comprises an additional fusion element (or fusion polypeptide fragment) fused with the element (i).

[0184] In a third aspect of the present invention, a chimeric antigen receptor CAR is provided, wherein the antigen binding domain of the chimeric antigen receptor contains an antibody single chain variable region sequence scFv targeting ROR1, and the heavy chain variable region and the light chain variable region of the scFv include a complementary determining region CDR selected from the following group:

[0185] (1) The heavy chain variable region includes the following complementarity determining region CDR: (Ab3)

[0186] HCDR1 shown in SEQ ID NO:7,

[0187] HCDR2 shown in SEQ ID NO:8, and

[0188] HCDR3 shown in SEQ ID NO:9;

[0189] And the light chain variable region includes the following complementarity determining regions CDR:

[0190] LCDR1 shown in SEQ ID NO:10,

[0191] LCDR2 shown in SEQ ID NO: 11, and

[0192] LCDR3 shown in SEQ ID NO:12; or

[0193] (2) The heavy chain variable region includes the following complementarity determining region CDR: (Ab4)

[0194] HCDR1 shown in SEQ ID NO:13,

[0195] HCDR2 shown in SEQ ID NO:14, and

[0196] HCDR3 shown in SEQ ID NO:15;

[0197] And the light chain variable region includes the following complementarity determining regions CDR:

[0198] LCDR1 shown in SEQ ID NO:16,

[0199] LCDR2 shown in SEQ ID NO: 17, and

[0200] LCDR3 shown in SEQ ID NO: 18; or

[0201] (3) The heavy chain variable region includes the following complementarity determining region CDR: (Ab1)

[0202] HCDR1 shown in SEQ ID NO:1,

[0203] HCDR2 shown in SEQ ID NO:2, and

[0204] HCDR3 shown in SEQ ID NO:3;

[0205] And the light chain variable region includes the following complementarity determining regions CDR:

[0206] LCDR1 shown in SEQ ID NO:4,

[0207] LCDR2 shown in SEQ ID NO:5, and

[0208] LCDR3 shown in SEQ ID NO:6; or

[0209] (4) the heavy chain variable region comprises the following complementarity determining region CDR: (Ab6) HCDR1 shown in SEQ ID NO: 19,

[0210] HCDR2 shown in SEQ ID NO:20, and

[0211] HCDR3 shown in SEQ ID NO:21;

[0212] And the light chain variable region includes the following complementarity determining regions CDR:

[0213] LCDR1 shown in SEQ ID NO:22,

[0214] LCDR2 shown in SEQ ID NO:23, and

[0215] LCDR3 shown in SEQ ID NO:24; or

[0216] (5) The heavy chain variable region includes the following complementarity determining region CDR: (Ab7) HCDR1 shown in SEQ ID NO: 25,

[0217] HCDR2 shown in SEQ ID NO:26, and

[0218] HCDR3 shown in SEQ ID NO:27;

[0219] And the light chain variable region includes the following complementarity determining regions CDR:

[0220] LCDR1 shown in SEQ ID NO:28,

[0221] LCDR2 shown in SEQ ID NO:23, and

[0222] LCDR3 shown in SEQ ID NO:29; or

[0223] (6) The heavy chain variable region includes the following complementary determining region CDR: (Ab11) HCDR1 shown in SEQ ID NO: 25,

[0224] HCDR2 shown in SEQ ID NO:26, and

[0225] HCDR3 shown in SEQ ID NO:27;

[0226] And the light chain variable region includes the following complementarity determining regions CDR:

[0227] LCDR1 shown in SEQ ID NO:28,

[0228] LCDR2 shown in SEQ ID NO:23, and

[0229] LCDR3 shown in SEQ ID NO:29; or

[0230] (7) The heavy chain variable region includes the following complementary determining region CDR: (Ab12) HCDR1 shown in SEQ ID NO: 45,

[0231] HCDR2 shown in SEQ ID NO:46, and

[0232] HCDR3 shown in SEQ ID NO:47;

[0233] And the light chain variable region includes the following complementarity determining regions CDR:

[0234] LCDR1 shown in SEQ ID NO:48,

[0235] LCDR2 shown in SEQ ID NO:49, and

[0236] LCDR3 shown in SEQ ID NO:50; or

[0237] (8) The heavy chain variable region includes the following complementarity determining region CDR: (Ab8)

[0238] HCDR1 shown in SEQ ID NO:30,

[0239] HCDR2 shown in SEQ ID NO:31, and

[0240] HCDR3 shown in SEQ ID NO:32;

[0241] And the light chain variable region includes the following complementarity determining regions CDR:

[0242] LCDR1 shown in SEQ ID NO:33,

[0243] LCDR2 shown in SEQ ID NO:34, and

[0244] LCDR3 shown in SEQ ID NO:35; or

[0245] (9) The heavy chain variable region includes the following complementarity determining region CDR: (Ab9)

[0246] HCDR1 shown in SEQ ID NO:36,

[0247] HCDR2 shown in SEQ ID NO:37, and

[0248] HCDR3 shown in SEQ ID NO:38;

[0249] And the light chain variable region includes the following complementarity determining regions CDR:

[0250] LCDR1 shown in SEQ ID NO:39,

[0251] LCDR2 shown in SEQ ID NO:23, and

[0252] LCDR3 shown in SEQ ID NO:29; or

[0253] (10) The heavy chain variable region includes the following complementarity determining region CDR: (Ab13)

[0254] HCDR1 shown in SEQ ID NO:51,

[0255] HCDR2 shown in SEQ ID NO:52, and

[0256] HCDR3 shown in SEQ ID NO:53;

[0257] And the light chain variable region includes the following complementarity determining regions CDR:

[0258] LCDR1 shown in SEQ ID NO:54,

[0259] LCDR2 shown in SEQ ID NO:23, and

[0260] LCDR3 shown in SEQ ID NO:40;

[0261] Wherein, the CDR sequence is based on Kabat's numbering scheme.

[0262] In another preferred embodiment, the heavy chain variable region and the light chain variable region of the scFv include a complementary determining region CDR selected from the following group:

[0263] (1) The heavy chain variable region includes the following complementarity determining region CDR: (Ab3)

[0264] HCDR1 shown in SEQ ID NO:57,

[0265] HCDR2 shown in SEQ ID NO:56, and

[0266] HCDR3 shown in SEQ ID NO:9;

[0267] And the light chain variable region includes the following complementarity determining regions CDR:

[0268] LCDR1 shown in SEQ ID NO:10,

[0269] LCDR2 shown in SEQ ID NO: 11, and

[0270] LCDR3 shown in SEQ ID NO:12; or

[0271] (2) The heavy chain variable region includes the following complementarity determining region CDR: (Ab4)

[0272] HCDR1 shown in SEQ ID NO:58,

[0273] HCDR2 shown in SEQ ID NO:59, and

[0274] HCDR3 shown in SEQ ID NO:15;

[0275] And the light chain variable region includes the following complementarity determining regions CDR:

[0276] LCDR1 shown in SEQ ID NO:16,

[0277] LCDR2 shown in SEQ ID NO: 17, and

[0278] LCDR3 shown in SEQ ID NO: 18; or

[0279] (3) The heavy chain variable region includes the following complementarity determining region CDR: (Ab1) HCDR1 shown in SEQ ID NO: 55,

[0280] HCDR2 shown in SEQ ID NO:56, and

[0281] HCDR3 shown in SEQ ID NO:3;

[0282] And the light chain variable region includes the following complementarity determining regions CDR:

[0283] LCDR1 shown in SEQ ID NO:4,

[0284] LCDR2 shown in SEQ ID NO:5, and

[0285] LCDR3 shown in SEQ ID NO:6; or

[0286] (4) the heavy chain variable region comprises the following complementarity determining region CDR: (Ab6) HCDR1 shown in SEQ ID NO: 57,

[0287] HCDR2 shown in SEQ ID NO:60, and

[0288] HCDR3 shown in SEQ ID NO:21;

[0289] And the light chain variable region includes the following complementarity determining regions CDR:

[0290] LCDR1 shown in SEQ ID NO:22,

[0291] LCDR2 shown in SEQ ID NO:23, and

[0292] LCDR3 shown in SEQ ID NO:24; or

[0293] (5) The heavy chain variable region includes the following complementarity determining region CDR: (Ab7) HCDR1 shown in SEQ ID NO: 61,

[0294] HCDR2 shown in SEQ ID NO:62, and

[0295] HCDR3 shown in SEQ ID NO:27;

[0296] And the light chain variable region includes the following complementarity determining regions CDR:

[0297] LCDR1 shown in SEQ ID NO:28,

[0298] LCDR2 shown in SEQ ID NO:23, and

[0299] LCDR3 shown in SEQ ID NO:29; or

[0300] (6) The heavy chain variable region includes the following complementarity determining region CDR: (Ab11) HCDR1 shown in SEQ ID NO: 57,

[0301] HCDR2 shown in SEQ ID NO:60, and

[0302] HCDR3 shown in SEQ ID NO:42;

[0303] And the light chain variable region includes the following complementarity determining regions CDR:

[0304] LCDR1 shown in SEQ ID NO:43,

[0305] LCDR2 shown in SEQ ID NO:23, and

[0306] LCDR3 shown in SEQ ID NO:44; or

[0307] (7) The heavy chain variable region includes the following complementarity determining region CDR: (Ab12)

[0308] HCDR1 shown in SEQ ID NO:65,

[0309] HCDR2 shown in SEQ ID NO:66, and

[0310] HCDR3 shown in SEQ ID NO:47;

[0311] And the light chain variable region includes the following complementarity determining regions CDR:

[0312] LCDR1 shown in SEQ ID NO:48,

[0313] LCDR2 shown in SEQ ID NO:49, and

[0314] LCDR3 shown in SEQ ID NO:50; or

[0315] (8) The heavy chain variable region includes the following complementarity determining region CDR: (Ab8)

[0316] HCDR1 shown in SEQ ID NO:61,

[0317] HCDR2 shown in SEQ ID NO:64, and

[0318] HCDR3 shown in SEQ ID NO:32;

[0319] And the light chain variable region includes the following complementarity determining regions CDR:

[0320] LCDR1 shown in SEQ ID NO:33,

[0321] LCDR2 shown in SEQ ID NO:34, and

[0322] LCDR3 shown in SEQ ID NO:35; or

[0323] (9) The heavy chain variable region includes the following complementarity determining region CDR: (Ab9)

[0324] HCDR1 shown in SEQ ID NO:57,

[0325] HCDR2 shown in SEQ ID NO:60, and

[0326] HCDR3 shown in SEQ ID NO:38;

[0327] And the light chain variable region includes the following complementarity determining regions CDR:

[0328] LCDR1 shown in SEQ ID NO:39,

[0329] LCDR2 shown in SEQ ID NO:23, and

[0330] LCDR3 shown in SEQ ID NO:40; or

[0331] (10) The heavy chain variable region includes the following complementarity determining region CDR: (Ab13)

[0332] HCDR1 shown in SEQ ID NO:67,

[0333] HCDR2 shown in SEQ ID NO:60, and

[0334] HCDR3 shown in SEQ ID NO:53;

[0335] And the light chain variable region includes the following complementarity determining regions CDR:

[0336] LCDR1 shown in SEQ ID NO:54,

[0337] LCDR2 shown in SEQ ID NO:23, and

[0338] LCDR3 shown in SEQ ID NO:40; wherein the CDR sequence is based on the Chothia numbering scheme. In another preferred embodiment, the scFv further comprises a connecting peptide between the heavy chain variable region and the light chain variable region.

[0339] In another preferred embodiment, the connecting peptide is (G4S)3 or (G4S)4.

[0340] In another preferred embodiment, the scFv is shown in the following formula A or formula B:

[0341] VH-VL, (A); VL-VH, (B)

[0342] In the formula, VH is the antibody heavy chain variable region; VL is the antibody light chain variable region; "-" is a connecting peptide or peptide bond.

[0343] In another preferred embodiment, the connecting peptide is (G4S) n ,, preferably n is 3-5; more preferably n is 3.

[0344] In another preferred embodiment, the scFv is as shown in formula B (VL-VH), and has an amino acid sequence selected from the following group:

[0345] SEQ ID NO. 68, 69, 70, 71, 72, 73, 74, 75, 76, 77; or

[0346] The scFv is shown in formula A (VH-VL) and has an amino acid sequence selected from the group consisting of:

[0347] SEQ ID NO. 79, 80, 81, 82, 83, 84, 85, 86, 87, 88.

[0348] In another preferred embodiment, the antibody single-chain variable region includes human, mouse, or human-mouse chimeric antibody single-chain variable regions.

[0349] In another preferred embodiment, the scFv is represented by formula A (VH-VL).

[0350] In another preferred embodiment, the antigen binding domain targets the extracellular region of ROR1.

[0351] In another preferred embodiment, the chimeric antigen receptor has the following structure:

[0352] L-scFv-H-TM-C-CD3ζ(I)

[0353] In the formula,

[0354] Each "-" is independently a connecting peptide or a peptide bond;

[0355] L is none or a signal peptide sequence;

[0356] scFv is a scFv targeting ROR1;

[0357] H is an optional hinge region;

[0358] TM is the transmembrane domain;

[0359] C is a co-stimulatory signal molecule;

[0360] CD3ζ is the CD3ζ cytoplasmic signaling sequence.

[0361] In another preferred embodiment, the L is a signal peptide selected from the following group of proteins: CD8, CD4, CD16, CD56, CD137, CSF2, DAP12, EF1, GM-CSF, IL-8, IL-21 or a combination thereof.

[0362] In another preferred embodiment, the L is derived from a signal peptide derived from CD8.

[0363] In another preferred embodiment, the scFv is shown in the following formula A or formula B:

[0364] VH-VL, (A); VL-VH, (B)

[0365] In the formula, VH is the antibody heavy chain variable region; VL is the antibody light chain variable region; "-" is a connecting peptide or peptide bond.

[0366] In another preferred embodiment, the scFv is represented by formula A (VH-VL).

[0367] In another preferred embodiment, the H is a hinge region selected from the following histones: CD8, CD28, CD137, Fc, or a combination thereof.

[0368] In another preferred embodiment, the H is the hinge region derived from CD28.

[0369] In another preferred embodiment, the TM is a transmembrane region of a protein selected from the group consisting of CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a combination thereof.

[0370] In another preferred embodiment, the TM is a transmembrane region derived from CD28.

[0371] In another preferred embodiment, C is a co-stimulatory signal molecule of a protein selected from the following group: OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1, Dap10, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), NKG2D, GITR, TLR2, or a combination thereof.

[0372] In another preferred embodiment, C is a co-stimulatory signal molecule derived from CD28.

[0373] The fourth aspect of the present invention provides a polynucleotide, which encodes the antibody or antigen-binding fragment thereof described in the first aspect of the present invention, the recombinant protein described in the second aspect of the present invention, or the chimeric antigen receptor CAR described in the third aspect of the present invention.

[0374] In another preferred embodiment, the polynucleotide is isolated.

[0375] The fifth aspect of the present invention provides a vector, wherein the vector contains the polynucleotide described in the fourth aspect of the present invention.

[0376] In another preferred embodiment, the vector is selected from the group consisting of DNA, RNA, plasmid, lentiviral vector, adenoviral vector, retroviral vector, transposon, or a combination thereof.

[0377] In another preferred embodiment, the vector is a retroviral vector.

[0378] The sixth aspect of the present invention provides a host cell, wherein the host cell contains the vector or chromosome of the fifth aspect of the present invention in which the exogenous polynucleotide of the fourth aspect of the present invention is integrated.

[0379] In another preferred embodiment, the cell is an isolated cell, and / or the cell is a genetically engineered cell.

[0380] In another preferred embodiment, the cell is a mammalian cell.

[0381] In another preferred embodiment, the cells are NK cells or T cells.

[0382] In another preferred embodiment, the host cell is an engineered immune cell.

[0383] In another preferred embodiment, the engineered immune cells include T cells or NK cells, preferably (i) chimeric antigen receptor T cells (CAR-T cells); or (ii) chimeric antigen receptor NK cells (CAR-NK cells), wherein the sources of NK cells include peripheral blood, umbilical cord blood, embryonic stem cells (ESC), induced pluripotent stem cells (iPSC), etc.

[0384] In another preferred embodiment, the host cell is an immune cell, and the immune cell expresses or exposes outside the cell membrane the antibody described in the first aspect of the present invention or the chimeric antigen receptor described in the third aspect of the present invention.

[0385] In another preferred embodiment, the immune cells include NK cells and T cells.

[0386] In another preferred embodiment, the immune cells are from humans or non-human mammals (such as mice).

[0387] A seventh aspect of the present invention provides a method for preparing a CAR-NK cell or a CAR-T cell, wherein the CAR-NK cell or the CAR-T cell expresses the chimeric antigen receptor according to the third aspect of the present invention, comprising the following steps:

[0388] The polynucleotide described in the fourth aspect of the present invention or the vector described in the fifth aspect of the present invention is transduced into NK cells or T cells to obtain the CAR-NK cells or CAR-T cells.

[0389] In the eighth aspect of the present invention, a pharmaceutical composition is provided, which contains the antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, the recombinant protein as described in the second aspect of the present invention, the chimeric antigen receptor as described in the third aspect of the present invention, the polynucleotide as described in the fourth aspect of the present invention, the vector as described in the fifth aspect of the present invention, or the host cell as described in the sixth aspect of the present invention, and a pharmaceutically acceptable carrier, diluent or excipient.

[0390] In another preferred embodiment, the pharmaceutical composition is a preparation, preferably a liquid preparation.

[0391] In another preferred embodiment, the pharmaceutical composition is in the form of an injection.

[0392] In another preferred embodiment, the pharmaceutical composition is used to prepare a drug or preparation for preventing and / or treating cancer or tumor.

[0393] The ninth aspect of the present invention provides an immunoconjugate, wherein the immunoconjugate comprises:

[0394] (a) an antibody portion, the antibody portion being selected from the group consisting of the antibody or antigen-binding fragment thereof according to the first aspect of the invention, the recombinant protein according to the second aspect of the invention, or a combination thereof; and

[0395] (b) a conjugated moiety conjugated to the antibody portion, wherein the conjugated moiety is selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

[0396] In another preferred embodiment, the conjugate is selected from: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computer tomography) contrast agents, or enzymes capable of producing detectable products, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorods, viral particles, liposomes, nanomagnetic particles, prodrug activating enzymes (for example, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (for example, cisplatin) or any form of nanoparticles, etc.

[0397] In a tenth aspect, the present invention provides a use of the antibody or antigen-binding fragment thereof according to the first aspect of the present invention, the recombinant protein according to the second aspect of the present invention, the chimeric antigen receptor according to the third aspect of the present invention, the polynucleotide according to the fourth aspect of the present invention, the vector according to the fifth aspect of the present invention, the host cell according to the sixth aspect of the present invention, the pharmaceutical composition according to the eighth aspect of the present invention, or the immunoconjugate according to the ninth aspect of the present invention,

[0398] (a) preparing a detection reagent or a kit; and / or

[0399] (b) preparing drugs or preparations for preventing and / or treating ROR1-related diseases.

[0400] In another preferred embodiment, the ROR1-related disease is cancer or tumor.

[0401] In another preferred embodiment, the tumor is selected from the following group: a blood tumor, a solid tumor, or a combination thereof.

[0402] In another preferred embodiment, the blood tumor is selected from the group consisting of acute lymphoblastic leukemia, chronic lymphoblastic leukemia, diffuse large B-cell lymphoma, follicular cell lymphoma, mantle cell lymphoma, or a combination thereof.

[0403] In another preferred embodiment, the solid tumor is selected from the group consisting of breast cancer, gastric cancer, lung cancer, ovarian cancer, colorectal cancer, pancreatic cancer, endometrial cancer, melanoma, mesothelioma, or a combination thereof.

[0404] In another preferred embodiment, the tumor is a ROR1-positive tumor; preferably selected from the following group: chronic lymphoma, breast cancer, lung cancer, ovarian cancer, colorectal cancer, endometrial cancer, melanoma, or a combination thereof.

[0405] In another preferred embodiment, the detection is an immunoassay.

[0406] In another preferred embodiment, the immunoassay is ELISA immunoassay, immunochromatography assay, immunocytochemical staining assay or immunohistochemical staining assay.

[0407] In another preferred embodiment, the diagnostic reagent is a test piece or a test plate.

[0408] In another preferred embodiment, the reagents include chips and immune particles coated with antibodies.

[0409] In an eleventh aspect of the present invention, a method for detecting ROR1 protein in a sample in vitro (including diagnostic or non-diagnostic) is provided, the method comprising the steps of:

[0410] (1) contacting a sample with the antibody or antigen-binding fragment thereof according to the first aspect of the present invention, or the recombinant protein according to the second aspect of the present invention;

[0411] (2) Detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of ROR1 protein in the sample.

[0412] In another preferred embodiment, the diagnostic reagent is a test piece or a test plate.

[0413] In another preferred embodiment, the method is a cell immunochemistry (Immunocytochemistry staning, ICC) detection method, or an immunohistochemistry (Immunohistochemistry IHC) detection method, or a whole cell ELISA detection method, or a cell lysate ELISA detection method.

[0414] A twelfth aspect of the present invention provides a method for preparing a recombinant polypeptide, the method comprising:

[0415] (a) culturing the host cell according to the sixth aspect of the present invention under conditions suitable for expression;

[0416] (b) isolating a recombinant polypeptide from the culture, wherein the recombinant polypeptide is the antibody or antigen-binding fragment thereof according to the first aspect of the present invention, or the recombinant protein according to the second aspect of the present invention.

[0417] The thirteenth aspect of the present invention provides a detection plate, which comprises: a substrate (support plate) and a test strip, wherein the test strip contains the antibody or its antigen-binding fragment described in the first aspect of the present invention, the recombinant protein described in the second aspect of the present invention, the immunoconjugate described in the ninth aspect of the present invention, or a combination thereof.

[0418] A fourteenth aspect of the present invention provides a kit, comprising:

[0419] (1) a first container, wherein the first container contains the antibody or antigen-binding fragment thereof according to the first aspect of the present invention; and / or

[0420] (2) a second container, wherein the second container contains a secondary antibody against the antibody of the present invention;

[0421] Alternatively, the kit contains the detection plate described in the thirteenth aspect of the present invention.

[0422] The fifteenth aspect of the present invention provides a method for treating a disease, comprising administering to a subject in need of treatment an appropriate amount of the antibody or antigen-binding fragment thereof described in the first aspect of the present invention, the recombinant protein described in the second aspect of the present invention, the host cell described in the twelfth aspect of the present invention, or the pharmaceutical composition described in the eighth aspect of the present invention.

[0423] In another preferred embodiment, the disease is cancer or tumor.

[0424] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0425] Figure 1 Anti-ROR1 scFv-Fc antibody SDS-PAGE results are shown.

[0426] Figure 2 Anti-ROR1 antibody BLI assay results are shown.

[0427] Figure 3 Anti-ROR1 antibody EC50 assay results are shown.

[0428] Figure 4 The anti-ROR1 CAR molecular structure is shown.

[0429] Figure 5 The results of ROR1 protein expression detection on the surface of tumor cell lines are shown.

[0430] Figure 6 The results of CAR molecule expression detection of CAR-NK92 cell line after sorting are shown.

[0431] Figure 7 The killing results of anti-ROR1 CAR NK92 on AGS, MDA-MB-231, and MDA-MB-468 cells are shown.

[0432] Figure 8 The results of multiple rounds of killing of JeKo-1-Luc cells by anti-ROR1 CAR NK92 are shown.

[0433] Fig. 9 The results of a single round of killing of JeKo-1-Luc cells by anti-ROR1 CAR NK92 are shown. DETAILED DESCRIPTION

[0434] After extensive and in-depth research and a large number of screenings, the inventors unexpectedly obtained a series of ROR1-targeted antibodies for the first time. The screened antibodies have excellent biological activity, and based on this, a chimeric antigen receptor structure targeting ROR1 was further constructed. NK cells expressing the chimeric antigen receptor showed excellent killing ability against target cells. On this basis, the present invention was completed.

[0435] the term

[0436] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0437] The term "about" can refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.

[0438] As used herein, the term "comprising" or "including (comprising)" may be open, semi-closed and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

[0439] The term "antibody" (Ab) shall include, but is not limited to, immunoglobulins that specifically bind to an antigen and comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or antigen-binding portions thereof. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three constant domains CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises one constant domain CL. The VH and VL regions can be further subdivided into hypervariable regions called complementary determining regions (CDRs), which are interspersed with more conservative regions called framework regions (FRs). Each VH and VL comprises three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens.

[0440] As used herein, the term "heavy chain variable region" is synonymous with "V H ” can be used interchangeably.

[0441] As used herein, the terms "light chain variable region" and "V L ” can be used interchangeably.

[0442] As used herein, the term "antigen binding domain" and the like include any naturally occurring, enzymatically available, synthetic or genetically modified polypeptides or glycoproteins that specifically bind to an antigen to form a complex. Any suitable standard technique such as proteolytic digestion or recombinant genetic engineering techniques involving manipulation and expression of DNA encoding antibody variable domains and optionally antibody constant domains can be used, for example, an antigen binding fragment of an antibody derived from a complete antibody molecule. Such DNA is known and / or easily available or can be synthesized from, for example, commercial sources, DNA libraries (including, for example, phage antibody libraries). The DNA can be sequenced and chemically or by using molecular biological techniques, for example, to arrange one or more variable domains and / or constant domains into a suitable layout, or to introduce codons, generate cysteine ​​residues, modify, add or delete amino acids, etc.

[0443] As used herein, non-limiting examples of antigen-binding fragments or antigen-binding domains include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues that mimic the hypervariable regions of antibodies (e.g., independent complementarity determining regions (CDRs) such as CDR3 peptides) or constrained FR3-CDR3-FR4 peptides.

[0444] As used herein, an antigen binding fragment or antigen binding domain will generally comprise at least one variable domain. A variable domain can be of any size or amino acid composition and will generally comprise at least one CDR adjacent to or in frame with one or more framework sequences. L Domain-associated V H In the antigen-binding fragment of the structural domain, V H and V L The domains may be arranged relative to each other in any suitable arrangement. For example, the variable region may be a dimer and contain V H -V H 、V H -V L or V L -V L Alternatively, the antigen binding domain may contain a monomeric V H or V L Structural domain.

[0445] In a given antibody light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of a number of well-known antibody CDR assignment systems, including, for example: Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loops, Kabat based on antibody sequence variability (Kabat, E., et al., US Department of Health and Human Services, Sequences of Proteins of Immunological Interest, (1983), AbM (University of Bath), Contact (University College London), the International Immuno GeneTics database (IMGT), the EU numbering system, and the Chothia definition based on loop structural position.

[0446] It should be understood that the precise amino acid sequence boundaries of the CDRs in the present invention may optionally be defined using the different assignment systems mentioned above. Preferably, unless otherwise indicated, in the present invention, when referring to residue positions in the antibody variable region (including heavy chain variable region residues and light chain variable region residues), it refers to the numbering positions according to the Kabat numbering system.

[0447] Antibody

[0448] As used herein, the term "antibody" or "immunoglobulin" is a heterotetrameric glycoprotein of about 150,000 daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.

[0449] As used herein, the term "variable" means that some parts of the variable region in an antibody are different in sequence, which forms the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three fragments called complementary determining regions (CDRs) or hypervariable regions in the variable regions of light and heavy chains. The more conservative part of the variable region is called the framework region (FR). The variable regions of natural heavy and light chains each contain four FR regions, which are roughly in a β-folded configuration, connected by three CDRs forming a connecting loop, and in some cases can form a partial β-folded structure. The CDRs in each chain are closely together through the FR region and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Volume I, 647-669 pages (1991)). The constant region does not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in the antibody's antibody-dependent cytotoxicity.

[0450] The present invention includes not only complete monoclonal antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies.

[0451] As used herein, the terms "fragment", "derivative" and "analog" refer to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention. The polypeptide fragments, derivatives or analogs of the present invention may be (i) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) polypeptides having a substitution group in one or more amino acid residues, or (iii) polypeptides formed by fusion of a mature polypeptide with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iv) polypeptides formed by fusion of an additional amino acid sequence to the polypeptide sequence (such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or a fusion protein formed with a 6His tag). According to the teachings herein, these fragments, derivatives and analogs are within the scope known to those skilled in the art.

[0452] The "light chains" of vertebrate antibodies (immunoglobulins) can be classified into one of two distinct classes (called kappa and lambda) based on the amino acid sequence of their constant regions. Immunoglobulins can be divided into different classes based on the amino acid sequence of their heavy chain constant regions. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant regions corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structure and three-dimensional configuration of the different classes of immunoglobulins are well known in the art.

[0453] The present invention also provides other polypeptides, such as fusion proteins comprising human antibodies or fragments thereof. In addition to almost full-length polypeptides, the present invention also includes fragments of antibodies of the present invention. Typically, the fragment has at least about 50 consecutive amino acids of the antibody of the present invention, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids.

[0454] In the present invention, the antibody of the present invention also includes its conservative variants, which refer to polypeptides formed by replacing at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids with amino acids of similar or similar properties compared with the amino acid sequence of the antibody of the present invention. These conservative variant polypeptides are preferably produced by amino acid substitution according to Table 1.

[0455] Table 1

[0456] Initial residue Representative replacement Preferred substitutions Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala Leu

[0457] Chimeric Antigen Receptor (CAR)

[0458] As used herein, the terms "chimeric antigen receptor of the present invention" and "CAR of the present invention" are used interchangeably to refer to the chimeric antigen receptor described in the third aspect of the present invention.

[0459] The chimeric antigen receptor (CAR) of the present invention includes an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain includes a target-specific binding element (also referred to as an antigen binding domain). The intracellular domain includes a costimulatory signaling region and a ζ chain portion. The costimulatory signaling region refers to a portion of the intracellular domain including a costimulatory molecule. Costimulatory molecules are cell surface molecules required for the effective response of lymphocytes to antigens, rather than antigen receptors or their ligands.

[0460] Between the extracellular domain and the transmembrane domain of CAR, or between the cytoplasmic domain and the transmembrane domain of CAR, a joint may be incorporated. As used herein, the term "joint" generally refers to any oligopeptide or polypeptide that acts to connect the transmembrane domain to the extracellular domain or cytoplasmic domain of a polypeptide chain. The joint may include 0-300 amino acids, preferably 2 to 100 amino acids and most preferably 3 to 50 amino acids.

[0461] In a preferred embodiment of the present invention, the extracellular domain of the CAR provided by the present invention includes an antigen binding domain targeting ROR1. When the CAR of the present invention is expressed in T cells, it can recognize antigens based on antigen binding specificity. When it binds to its associated antigen, it affects tumor cells, causing tumor cells to not grow, be caused to die or be affected in other ways, and causes the patient's tumor load to be reduced or eliminated. The antigen binding domain is preferably fused with one or more intracellular domains from a costimulatory molecule and a ζ chain. Preferably, the antigen binding domain is fused with an intracellular domain of a combination of a 4-1BB signaling domain and a CD3ζ signaling domain.

[0462] As used herein, "antigen binding domain" and "single-chain antibody fragment" all refer to Fab fragments, Fab' fragments, F(ab')2 fragments, or single Fv fragments with antigen binding activity. Fv antibodies contain the variable region of the antibody heavy chain and the variable region of the light chain, but no constant region, and are the smallest antibody fragments with all antigen binding sites. Generally, Fv antibodies also contain a polypeptide linker between the VH and VL domains, and are capable of forming the structure required for antigen binding. The antigen binding domain is usually a scFv (single-chain variable fragment). The size of a scFv is generally 1 / 6 of a complete antibody. A single-chain antibody is preferably an amino acid chain sequence encoded by a nucleotide chain. As a preferred embodiment of the present invention, the scFv comprises an antibody that specifically recognizes the extracellular region of ROR1, especially an antibody that specifically recognizes amino acid residues 24 to 41 of the ROR1 sequence, preferably a single-chain antibody.

[0463] For hinge region and transmembrane region (transmembrane domain), CAR can be designed to include a transmembrane domain fused to the extracellular domain of CAR. In one embodiment, a transmembrane domain naturally associated with one of the domains in CAR is used. In some examples, a transmembrane domain can be selected, or modified by amino acid replacement to avoid binding such a domain to the transmembrane domain of the same or different surface membrane proteins, thereby minimizing the interaction with other members of the receptor complex.

[0464] Preferably, the structure of the CAR of the present invention includes a signal peptide, an antigen recognition sequence (antigen binding domain), a connecting region, a transmembrane region, a co-stimulatory factor signaling region and a CD3zeta signaling region (ζ chain portion), and the connection order is as follows:

[0465] L-scFv-H-TM-C-CD3ζ(I)

[0466] NK cells

[0467] Natural killer (NK) cells are a major type of immune effector cells that protect the body from viral infection and tumor cell invasion through non-antigen specific pathways. In recent years, NK cells have shown great application prospects in adoptive cell immunotherapy. NK cells have a wide range of sources, including peripheral blood, umbilical cord blood, embryonic stem cells (ESC), induced pluripotent stem cells (iPSC), etc.

[0468] NK-92 cells are an interleukin-2 (IL2)-dependent NK cell line derived from peripheral blood mononuclear cells of a 50-year-old male patient with acute non-Hodgkin's lymphoma. NK-92 cells are currently the only NK cell line approved by the FDA for clinical trials. This cell line has strong cytotoxicity, is economical, off-the-shelf, and easy to prepare on a large scale. It has a short survival time after killing tumor cells and is easy to expand in vitro. The vast majority of patients who receive treatment do not reject NK-92 cells, and there is no risk of graft-versus-host reaction. It does not express KIRs and is in a constitutively activated state. So far, it has shown good clinical safety.

[0469] As used herein, the terms "CAR-NK cell", "CAR-NK", "CARNK", and "CAR-NK cell of the present invention" all refer to a CAR-NK cell expressing the chimeric antigen receptor CAR of the first aspect of the present invention.

[0470] Carrier

[0471] The nucleic acid sequence 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 known vector comprising the gene, or by directly isolating from cells and tissues comprising the gene using standard techniques. Alternatively, the gene of interest can be produced synthetically.

[0472] The present invention also provides vectors into which the expression cassette of the present invention is inserted. Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer because they allow long-term, stable integration of transgenes and their proliferation in daughter cells. Lentiviral vectors have advantages over vectors derived from oncogenic retroviruses such as murine leukemia viruses because they can transduce non-proliferating cells, such as hepatocytes. They also have the advantage of low immunogenicity.

[0473] In brief summary, the expression cassette or nucleic acid sequence of the present invention is usually operably connected to a promoter and incorporated into an expression vector. The vector is suitable for replication and integration into eukaryotic cells. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters that can be used to regulate the expression of the desired nucleic acid sequence.

[0474] The expression constructs of the present invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods of gene delivery are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, 5,589,466, which are incorporated herein by reference in their entirety. In another embodiment, the present invention provides a gene therapy vector.

[0475] The nucleic acid can be cloned into many types of vectors. For example, the nucleic acid can be cloned into such vectors, which include but are not limited to plasmids, phagemids, phage derivatives, animal viruses and cosmids. Specific vectors of interest include expression vectors, replication vectors, probe generation vectors and sequencing vectors.

[0476] Further, expression vector can be provided to cell in the form of viral vector. Viral vector technology is well known in the art and is described in, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses and slow viruses. Generally, suitable vectors are included in at least one organism that works on the origin of replication, promoter sequence, convenient restriction enzyme sites and one or more selectable markers (for example, WO01 / 96584; WO01 / 29058; and U.S. Patent number 6,326,193).

[0477] Many virus-based systems have been developed for transferring genes into mammalian cells. 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 a subject's cells in vivo or in vitro. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In one embodiment, a lentiviral vector is used.

[0478] Additional promoter elements, such as enhancers, can regulate the frequency of transcription initiation. Typically, these are located in the 30-110 bp 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. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it is shown that individual elements can work cooperatively or independently to start transcription.

[0479] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence that can drive any polynucleotide sequence operably connected thereto to express at a high level. Another example of a suitable promoter 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 tumor 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 application of constitutive promoters. Inducible promoters are also considered to be part of the present invention. The use of an inducible promoter provides a molecular switch that can turn on the expression of a polynucleotide sequence operably linked 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.

[0480] 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.

[0481] Reporter gene is used to identify the cells of potential transfection and to evaluate the functionality of regulatory sequences. Generally, reporter gene is the following gene: it is not present in or expressed by a receptor organism or tissue, and it encodes a polypeptide whose expression is clearly indicated by some easily detectable properties such as enzymatic activity. After DNA has been introduced into the receptor cell, the expression of the reporter gene is measured at the appropriate time. Suitable reporter gene can include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase or green fluorescent protein (for example, Ui-Tei et al., 2000FEBS Letters479:79-82). Suitable expression systems are well known and can be prepared using known techniques or commercially available. Generally, the construct with a minimum of 5 flanking regions showing the highest level of reporter gene expression is identified as a promoter. Such a promoter region can be connected to a reporter gene and used to evaluate the ability of a reagent to regulate promoter-driven transcription.

[0482] Methods for introducing genes into cells and expressing genes into cells are known in the art. In the context of expression vectors, vectors can be easily introduced into host cells, such as mammalian, bacterial, yeast or insect cells, by any method known in the art. For example, expression vectors can be transferred into host cells by physical, chemical or biological means.

[0483] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0484] Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used methods for inserting genes into mammalian cells such as human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, etc. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0485] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles and liposomes. An exemplary colloidal system used as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0486] In the case of using a non-viral delivery system, an exemplary delivery vehicle is a liposome. Consider using lipid preparations to introduce nucleic acid into a host cell (in vitro, ex vivo or in vivo). On the other hand, the nucleic acid can be associated with a lipid. Nucleic acids associated with lipids can be encapsulated in the aqueous interior of the liposome, dispersed in the lipid bilayer of the liposome, attached to the liposome through a connecting molecule associated with both the liposome and the oligonucleotide, trapped in the liposome, compounded with the liposome, dispersed in a solution comprising lipids, mixed with lipids, combined with lipids, included in lipids as a suspension, included in micelles or compounded with micelles, or otherwise associated with lipids. The lipids, lipid / DNA or lipid / expression vectors associated with the composition are not limited to any specific structure in the solution. For example, they can be present in a bilayer structure, as micelles or have a "collapsed (collapsed)" structure. They can also be simply dispersed in a solution, possibly forming aggregates of size or shape inhomogeneity. Lipid is a fatty substance, which can be a naturally occurring or synthetic lipid. For example, lipids include fat droplets that occur naturally in the cytoplasm as well as compounds that contain long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0487] In a preferred embodiment of the present invention, the vector is a retroviral vector.

[0488] Therapeutic applications

[0489] The present invention includes cells (eg, NK cells, T cells, etc.) transduced with a retroviral vector (RV) encoding an antibody or CAR of the present invention. Transduced NK cells can cause CAR-mediated NK-cell or T cell responses.

[0490] Therefore, the present invention also provides a method for stimulating an NK cell-mediated immune response to a target cell population or tissue in a mammal, comprising the following steps: administering to a mammal NK cells expressing the CAR of the present invention.

[0491] In one embodiment, the present invention includes a class of cell therapies in which NK cells are genetically modified to express the CAR of the present invention, and the CAR-NK cells are injected into a recipient in need thereof. The injected cells are capable of killing the recipient's tumor cells. Unlike antibody therapies, CAR-NK cells are able to persist in vivo, resulting in long-term persistence that can lead to sustained tumor control.

[0492] Treatable cancers include tumors that are not vascularized or substantially not vascularized, and vascularized tumors.Cancers may include non-solid tumors (such as hematological tumors, such as leukemia and lymphoma) or may include solid tumors.The types of cancers treated with the CAR of the present invention include, but are not limited to, cancer, blastoma and sarcoma, and certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignant tumors, such as sarcomas, cancers, and melanomas.Also include adult tumors / cancers and childhood tumors / cancers.

[0493] Hematological cancers are cancers of the blood or bone marrow. Examples of hematological (or hematogenous) cancers include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia, and myeloblastic, promyelocytic, myelo-monocytic, monocytic, and erythroleukemias), chronic leukemias (such as chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphomas, Hodgkin's disease, non-Hodgkin's lymphomas (indolent and high-grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.

[0494] Solid tumors are abnormal masses of tissue that usually do not contain cysts or fluid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named after the cell types that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors such as sarcomas and carcinomas include fibrosarcomas, myxosarcoma, liposarcoma, mesothelioma, lymphoid malignancies, pancreatic cancer, ovarian cancer.

[0495] The CAR-modified NK cells of the present invention can also be used as a vaccine type for ex vivo immunization and / or in vivo therapy of mammals. Preferably, the mammal is a human.

[0496] Pharmaceutical composition

[0497] The antibody, fusion protein or CAR-modified NK cells of the present invention may be administered alone or as a pharmaceutical composition in combination with a diluent and / or with other components such as IL-2, IL-15, IL-18, IL-21 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 a composition may include a buffer such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; protein; polypeptide or amino acid such as glycine; antioxidant; chelating agent such as EDTA or glutathione; adjuvant (e.g., aluminum hydroxide); and preservative. The composition of the present invention is preferably formulated for intravenous administration.

[0498] The pharmaceutical composition 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-although the appropriate dosage can be determined by clinical trials.

[0499] When an "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 a physician, who takes into account 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 10 4 Up to 10 9 The dosage is preferably 10 cells / kg body weight. 5 Up to 10 6 The T cell composition can also be administered at these doses multiple times. The cells can be administered by using well-known injection techniques in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). The optimal dose and treatment regimen for a specific patient can be easily determined by a medical technician by monitoring the patient's disease signs and adjusting the treatment accordingly.

[0500] The main advantages of the present invention include

[0501] (1) The chimeric antigen receptor of the present invention has an extracellular antigen binding domain that is a specific anti-ROR1 scFv. The specific anti-ROR1 scFv binds to a specific hinge region and an intracellular domain, and the formed CAR exhibits a great ability to kill tumor cells, with less cytotoxicity and low side effects.

[0502] (2) The CAR-NK cells of the present invention have a high degree of activation and have excellent cytotoxicity against ROR1-positive target cells.

[0503] (3) The antibodies of the present invention have excellent binding ability and affinity kinetics to target cells.

[0504] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify detailed conditions are usually based on conventional conditions such as Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989) or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0505] Example 1 ROR1 Antibody Characteristics

[0506] ROR1-related antibodies were obtained by humanizing mice with antigen-immunized antibody-producing region gene fragments and then screening hybridomas. The candidate scFv was in the form of VL-(G4S)3-VH, and the sequence is shown in Table 2. The CDR sequences annotated by Kabat and Chothia are shown in Tables 3 and 4. The following experiments were performed using antibodies in the form of scFv-human IgG Fc.

[0507] The results of SDS-PAGE detection of antibody expression and purification are as follows Figure 1 The results show that the antibody expression and purification results are good. The representative antibody biolayer interferometry (BLI) measurement results of human ROR1 protein are shown in Figure 2 shown.

[0508] All antibodies were analyzed by flow cytometry using human breast cancer cells MDA-MB-231. Figure 3 The BLI data and EC50 data of all antibodies are summarized in Table 5.

[0509] Table 2 Anti-ROR1 antibody scFv sequences

[0510]

[0511]

[0512] Table 3 CDR sequences based on Kabat

[0513]

[0514] Note: SEQ:SEQ ID NO.

[0515] Table 4 CDR sequences based on Chothia

[0516]

[0517] Note: SEQ:SEQ ID NO.

[0518] Table 5 Summary of anti-ROR1 antibody kinetic data and EC50 results

[0519]

[0520] The results showed that Ab.7, Ab.11, Ab.12, and Ab.1 all exhibited excellent ROR1 affinity.

[0521] Example 2 ROR1 CAR structure design

[0522] The scFv of the ROR1 CAR structure is derived from the antibodies partially screened in Example 1, and is named according to the antibody name and the order of the light chain and heavy chain in the scFv.

[0523] CAR1L represents that the scFv of the CAR molecule comes from antibody Ab.1 and adopts the structure of VL-Linker-VH, while CAR1H adopts the structure of VH-Linker-VL.

[0524] ROR1 CAR structure Figure 4 As shown, it contains a signal peptide (SP), scFv, hinge region, transmembrane region (TM), costimulatory domain (CD) and CD3ζ domain. The signal peptide includes the following sequence (Table 6), and the data of the present invention are based on CD8αSP, CD28hinge, CD28TM and CD28CD structures. scFv is composed of VL-Linker-VH or VH-Linker-VL, where linker can be (G4S)3 or (G4S)4, etc. Among them,

[0525] The scFv sequence of CAR1L is shown in SEQ ID NO.68;

[0526] The scFv sequence of CAR3L is shown in SEQ ID NO.69;

[0527] The scFv sequence of CAR4L is shown in SEQ ID NO.70;

[0528] The scFv sequence of CAR6L is shown in SEQ ID NO.71;

[0529] The scFv sequence of CAR7L is shown in SEQ ID NO.72;

[0530] The scFv sequence of CAR8L is shown in SEQ ID NO.73;

[0531] The scFv sequence of CAR9L is shown in SEQ ID NO.74;

[0532] The scFv sequence of CAR11L is shown in SEQ ID NO.75;

[0533] The scFv sequence of CAR12L is shown in SEQ ID NO.76;

[0534] The scFv sequence of CAR13L is shown in SEQ ID NO.77;

[0535] The scFv sequence of Velos-LH is shown in SEQ ID NO.78.

[0536] Table 6 Signal peptide sequence

[0537] Signal peptide name sequence SEQ ID No: IL-21SP MRSSPGNMERIVICLMVIFLGTLV 93 CD8αSP MALPVTALLLPLALLLHAARP 94 CSF2SP MWLQSLLLLGTVACSIS 95 DAP12SP MGGLEPCSRLLLLPLLLAVSG 96 CD16SP MWQLLLPTALLLLVSA 97 CD56SP MLQTKDLIWTLFFLGTAVS 98 IL-8SP MTSKLAVALLAAFLISAALC 99

[0538] Table 7 scFv sequences of some anti-ROR1 CAR molecules

[0539]

[0540]

[0541] Example 3 Detection of ROR1 expression on tumor cell surface

[0542] The expression of ROR1 on the surface of AGS, JeKo-1, MDA-MB-231 and MDA-MB-468 tumor cells was detected by flow cytometry. The detection method is as follows.

[0543] 3.1 Take 2E5-3E5 tumor cells to be tested, centrifuge at 300g, 4℃ for 5 min, and discard the supernatant.

[0544] 3.2 Add 200 μL FACS Buffer (1% FBS dissolved in PBS), centrifuge at 300g, 4°C for 5 min, and discard the supernatant.

[0545] 3.3 Prepare the primary antibody solution. ROR1 antibody uses PE anti-human ROR1 Antibody (Biolegend, 357805) at a dilution of 1:100.

[0546] 3.4 Add 100 μL / well primary antibody solution, pipette to mix, and incubate at 4°C for 30 min.

[0547] 3.5 After incubation, add 100 μL FACS Buffer, centrifuge at 300g, 4℃ for 5 min, and discard the supernatant.

[0548] 3.6 Add 200 μL / well FACS Buffer, pipette to mix, centrifuge at 300g, 4℃ for 5 min, and discard the supernatant.

[0549] 3.7 Repeat 3.6 once.

[0550] 3.8 Add 200 μL / well FACS Buffer, resuspend, and detect using flow cytometer.

[0551] The results showed that AGS, JeKo-1, MDA-MB-231 and MDA-MB-468 cells all expressed ROR1 protein ( Figure 5 ).

[0552] Example 4. Virus packaging and NK92 cell infection

[0553] The packaging vectors of retrovirus are BaEV-TR and pCMV-gag-pol, and the vector carrying CAR molecules is pMSCV. These vectors were designed by our laboratory and then handed over to GeneWeichi for synthesis and extraction. The process of virus packaging, cell infection and sorting is as follows:

[0554] 4.1 HEK-293T cells in good condition were digested and resuspended in DMEM complete medium, and seeded in a 10 cm culture dish at a concentration of 8E5 / mL (10 mL / dish).

[0555] 4.2 After culturing in the incubator for 16 hours, observe the cell density and start plasmid transfection when the density is about 90%.

[0556] 4.3 Take two 1.5ml centrifuge tubes and add 500μL Opti-MEM TM I reduced serum culture medium (Gibco, 31985062), add 7.5 μg BaEV-TR, 10 μg pCMV-gag-pol and 20 μg corresponding pMSCV vector expressing CAR into one centrifuge tube and mix well to obtain Opti-MEM-plasmid mixed solution; add 40 μL PEIpro solution (polyplus, 115-010) into another centrifuge tube and mix well to obtain Opti-MEM-PEI mixed solution.

[0557] 4.4 Add the Opti-MEM-plasmid mixture into the Opti-MEM-PEI mixture, pipette thoroughly to mix, and leave at room temperature for 15 minutes to form a transfection complex.

[0558] 4.5 Add the above transfection complex dropwise into the HEK-293T culture supernatant, shake to mix, and continue culturing for 4-6 hours.

[0559] 4.6 Aspirate the culture supernatant, add 15 mL of DMEM complete medium, and culture for 48 hours.

[0560] 4.7 Collect the culture supernatant, add 15 mL of DMEM complete medium, and continue culturing for 24 hours; the collected supernatant is stored at 4°C.

[0561] 4.8 Collect the cell culture supernatant and combine it with the corresponding 48h culture supernatant.

[0562] 4.9 Concentrate the virus using Lenti-X Concentrator (Takara, 631232).

[0563] 4.10 Resuspend the virus in 100 μL NK92 cell culture medium, take out 5 μL for titer determination, and store the remaining virus at 4°C.

[0564] 4.11 Titer determination was performed using K562 cells, supplemented with 5 μg / mL polybrene (Sigma-Aldrich, TR-1003) during infection, and the K562 positive rate was determined 48 hours after infection to calculate the virus titer.

[0565] 4.12 4E5 NK92 cells were placed in a 6-well plate, and the virus concentrate was added at an MOI of 2, and polybrene (Sigma-Aldrich, TR-1003) was added at a final concentration of 5 ug / ml, and mixed evenly;

[0566] 4.13 Centrifuge the cells at 32°C, 800g for 1 h and culture overnight;

[0567] 4.14 Remove the NK92 cells, centrifuge and resuspend them in fresh NK92 culture medium, continue culturing for 4 days and then perform flow cytometry detection.

[0568] 4.15 Pipette and mix the cultured ROR1-CAR NK92 cells thoroughly to disperse them into single cells. Take 1 mL into a 1.5 ml centrifuge tube for NC-200 cell counting.

[0569] 4.16 According to the counting results, take 1E7 cells to be sorted into a centrifuge tube, centrifuge at 300g for 5 minutes, and discard the supernatant.

[0570] 4.17 Resuspend the cells in 10 mL MACS Buffer, centrifuge at 300 g for 5 min, and discard the supernatant.

[0571] 4.18 Resuspend the cells with 2 mL of 3 ug / mL Biotinylated Huamn ROR1 His, AvitagTM (Acro Biosystem, RO1-H82E6) diluent (diluted in PBS) and incubate at 4°C for 30 min.

[0572] 4.19 After incubation, add 5 mL of MACS Buffer, centrifuge at 300 g for 5 min, and discard the supernatant.

[0573] 4.20 Add 5 mL MACS Buffer and wash again, then discard the supernatant.

[0574] 4.21 Add 80 μL MACS Buffer to resuspend the cells.

[0575] 4.22 Add 20 μL Anti-Biotin MicroBeads (Miltenyi, 130-090-485) and mix thoroughly.

[0576] 4.23 Incubate at 4°C for 15 min.

[0577] 4.24 After incubation, add 2 mL MACS Buffer to wash once.

[0578] 4.25 Resuspend in 500 μL MACS Buffer.

[0579] 4.26 Load the adsorption column LS (Miltenyi, 130-042-401) on the magnetic stand and rinse the column with 3 mL of MACS Buffer.

[0580] 4.27 Add cells from 4.25 to the column.

[0581] 4.28 Add 3 mL MACS Buffer to the column, and the cells that are not magnetically attracted will be eluted out.

[0582] 4.29 Repeat 4.28 twice.

[0583] 4.30 Take a 15 mL centrifuge tube, remove the column from the magnetic stand and place it on the centrifuge tube.

[0584] 4.31 Add 5 mL of MACS Buffer to the column and push the plunger into the column to flush out the cells labeled with magnetic beads.

[0585] 4.32 Mix the cells in the 15 mL tube thoroughly, and then take 200 μL of the sorted cells for NC-200 counting.

[0586] 4.33 The sorted cells were centrifuged at 300g for 5 min and the supernatant was discarded.

[0587] 4.34 Resuspend with an appropriate volume of NK92 culture medium and culture in a 37°C, 5% CO2 incubator.

[0588] 4.35 CAR positive rate was determined 4 days after sorting. 1E5-2E5 cells to be tested were centrifuged at 700g and 4°C for 2 min, and the supernatant was discarded.

[0589] 4.36 Add 200 μL FACS Buffer to wash once, centrifuge at 700g, 4℃ for 2 min, and discard the supernatant.

[0590] 4.37 After mixing, add 100 μL of antigen protein suspension to each sample, with an antigen protein concentration of 2 ug / mL, mix by pipetting, and incubate at 4°C for 30 min-60 min.

[0591] 4.38 After incubation, add 100 μL / sample FACS Buffer, centrifuge at 700g, 4℃ for 2 min, and discard the supernatant.

[0592] 4.39 Add 200 μL / sample FACS Buffer, centrifuge at 700g, 4℃ for 2 min, and discard the supernatant.

[0593] 4.40 Prepare the detection antibody streptavidin-PE (Biolegend, #405203) at a dilution ratio of 1:200. After mixing, add 100 μL of the detection antibody to each sample, pipette to mix, and incubate at 4°C for 30 min-60 min.

[0594] 4.41 After incubation, add 100 μL / sample FACS Buffer, centrifuge at 700g, 4℃ for 2 min, and discard the supernatant.

[0595] 4.42 Add 200 μL / sample FACS Buffer, centrifuge at 700g, 4℃ for 2 min, and discard the supernatant.

[0596] 4.43 Repeat step 4.42 once.

[0597] 4.44 Add 200 μL / sample FACS Buffer and resuspend, and detect by flow cytometry.

[0598] The flow cytometry results showed that after virus infection and magnetic bead sorting, except for the CAR3H positive rate of 91.1%, the positive rates of anti-ROR1 CAR molecules in NK92 cells in each group reached more than 95% ( Figure 6 ).

[0599] Example 5. Detection of the killing ability of ROR1-CAR NK92 on target cells

[0600] Real-time Cellular Analysis (RTCA) was used to detect the killing ability of anti-ROR1 CAR NK92 on AGS, MDA-MB-231 and MDA-MB-468 target cells. The specific steps are as follows:

[0601] 5.1.1 Remove IL-2 from NK92 culture medium 24 h in advance.

[0602] 5.1.2 Take out the RTCA E96 Plate, add 50 μL of the corresponding complete culture medium to each well, create a new experiment in the RTCA program, complete the relevant experimental settings, put the E-plate in and lock it, the instrument will automatically perform a baseline measurement, and the baseline measurement is usually completed within one minute.

[0603] 5.1.3 Digest the target cells and inoculate them into E-plates at a volume of 50 μL / well according to the cell amount of AGS cells-2E4 / well, MDA-MB-231-4E4 / well and MDA-MB-468-4E4 / well.

[0604] 5.1.4 After inoculation, place at room temperature for 30 minutes, put the E-plate into the recording tank, and scan at least once every 30 minutes to record cell growth.

[0605] 5.1.5 Observe the resistance of target cells and add anti-ROR1 CAR NK92 effector cells during the rapid growth phase.

[0606] 5.1.6 Add effector cells according to the effector-target ratio (E:T) set in the experiment, place the E-plate on the plate, and start recording the killing step after 10 minutes.

[0607] 5.1.7 After the first round of killing reaches the bottom line or the Cell Index no longer decreases, you can choose to prepare multiple rounds of target cell stimulation, treat the corresponding target cells, add target cells according to the set amount in 5.3, the added volume is 50μL / well, place the E-plate on the plate, and start recording the killing step after 10 minutes.

[0608] Kill results recorded by RTCA method are shown in Figure 7It can be found that under this experimental condition, each group of CAR-NK92 has a good killing effect on AGS, MDA-MB-231, and MDA-MB-468.

[0609] The Luciferase reporter gene was used to detect the killing ability of anti-ROR1 CAR NK92 on JeKo-1-Luciferase target cells. The specific operation steps are as follows:

[0610] 5.2.1 IL-2 was removed from CAR NK92 effector cells for killing experiments 24 h in advance.

[0611] 5.2.2 The target cells JeKo-1-Luc were thoroughly pipetted and mixed, counted, and then resuspended in an appropriate volume of RPMI1640 complete medium.

[0612] 5.2.3 Transfer the target cells to a 96-well flat-bottom plate, 2E4 / well, 100 μL / well.

[0613] 5.2.4 The effector cells were thoroughly pipetted and mixed and counted. An appropriate number of effector cells were taken, centrifuged at 300g for 5 min at room temperature, and resuspended in an appropriate volume of RPMI1640 complete medium.

[0614] 5.2.5 The effector cells were transferred to the target cells in a 96-well flat-bottom plate, 50 μL / well, and co-cultured in a 37°C, 5% CO2 incubator. For single-round killing results, the co-culture time was 6 hours. For multiple-round killing results, the co-culture time was 24 hours.

[0615] 5.2.6 Add 60 μL ONE-Glo to each well and mix well.

[0616] 5.2.7 After 3 minutes of reaction, take 100 μL of the reaction solution into a 96-well white flat-bottom plate and use an ELISA reader to detect the luminescent signal intensity.

[0617] 5.2.8 After the first round of effector cell and target cell co-culture for 24 hours, 2E4 / well, 50 μL / well JeKo-1 cells can be added for a second round of stimulation. The co-culture time for the second round of stimulation is 16 hours.

[0618] 5.2.9 Add 80 μL ONE-Glo to each well and mix well.

[0619] 5.2.10 After 3 minutes of reaction, take 200 μL of the reaction solution into a 96-well white flat-bottom plate, use an ELISA reader to detect the luminescent signal intensity, and then calculate the cytotoxicity.

[0620] The killing results recorded by the Luciferase reporter gene method are shown in Figure 8 and Fig. 9It can be found that under this experimental condition, each group of CAR-NK92 has a good killing effect on JeKo-1-Luciferase cells.

[0621] The results showed that the preferred CAR-NK92 is an NK cell expressing CAR3L, CAR4L, CAR1L, and CAR6H. Among them, CAR3L-NK, CAR4L-NK, CAR1L-NK, and CAR6H-NK can show a specific killing activity greater than 75% in the first round of killing (16 hours) at E:T=1:1; CAR3L-NK and CAR4L-NK can quickly show specific killing activity in a single round of killing of 6 hours at E:T=1:1.

[0622] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. An antibody or an antigen-binding fragment thereof targeting ROR1, characterized in that: The antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprise a complementarity determining region (CDR) selected from the group consisting of: (1) The heavy chain variable region includes the following complementarity determining regions (CDRs): HCDR1 shown in SEQ ID NO:7, HCDR2 shown in SEQ ID NO:8, and HCDR3 shown in SEQ ID NO:9; And the light chain variable region includes the following complementarity determining regions CDR: LCDR1 shown in SEQ ID NO:10, LCDR2 shown in SEQ ID NO: 11, and LCDR3 shown in SEQ ID NO:12; or (2) The heavy chain variable region includes the following complementarity determining regions CDR: HCDR1 shown in SEQ ID NO:19, HCDR2 shown in SEQ ID NO:20, and HCDR3 shown in SEQ ID NO:21; And the light chain variable region includes the following complementarity determining regions CDR: LCDR1 shown in SEQ ID NO:22, LCDR2 shown in SEQ ID NO:23, and LCDR3 shown in SEQ ID NO:24; wherein the CDR sequences are based on the numbering scheme of Kabat; or The heavy chain variable region and the light chain variable region of the antibody or antigen-binding fragment thereof include a complementarity determining region (CDR) selected from the group consisting of: (1) The heavy chain variable region includes the following complementarity determining regions (CDRs): HCDR1 shown in SEQ ID NO:57, HCDR2 shown in SEQ ID NO:56, and HCDR3 shown in SEQ ID NO:9; And the light chain variable region includes the following complementarity determining regions CDR: LCDR1 shown in SEQ ID NO:10, LCDR2 shown in SEQ ID NO: 11, and LCDR3 shown in SEQ ID NO:12; or (2) The heavy chain variable region includes the following complementarity determining regions CDR: HCDR1 shown in SEQ ID NO:57, HCDR2 shown in SEQ ID NO:60, and HCDR3 shown in SEQ ID NO:21; And the light chain variable region includes the following complementarity determining regions CDR: LCDR1 shown in SEQ ID NO:22, LCDR2 shown in SEQ ID NO:23, and LCDR3 shown in SEQ ID NO:24; Wherein, the CDR sequence is based on the Chothia numbering scheme.

2. A recombinant protein, characterized in that The recombinant protein has: (i) the antibody or antigen-binding fragment thereof according to claim 1; and (ii) a tag sequence to facilitate expression and / or purification.

3. A chimeric antigen receptor CAR, characterized in that: The antigen binding domain of the chimeric antigen receptor contains an antibody single-chain variable region sequence scFv targeting ROR1, and the heavy chain variable region and light chain variable region of the scFv include a complementary determining region CDR selected from the following group: (1) The heavy chain variable region includes the following complementarity determining regions (CDRs): HCDR1 shown in SEQ ID NO:7, HCDR2 shown in SEQ ID NO:8, and HCDR3 shown in SEQ ID NO:9; And the light chain variable region includes the following complementarity determining regions CDR: LCDR1 shown in SEQ ID NO:10, LCDR2 shown in SEQ ID NO: 11, and LCDR3 shown in SEQ ID NO:12; or (2) The heavy chain variable region includes the following complementarity determining regions CDR: HCDR1 shown in SEQ ID NO:19, HCDR2 shown in SEQ ID NO:20, and HCDR3 shown in SEQ ID NO:21; And the light chain variable region includes the following complementarity determining regions CDR: LCDR1 shown in SEQ ID NO:22, LCDR2 shown in SEQ ID NO:23, and LCDR3 shown in SEQ ID NO:24; wherein the CDR sequences are based on the numbering scheme of Kabat; or The heavy chain variable region and the light chain variable region of the antibody or antigen-binding fragment thereof include a complementarity determining region (CDR) selected from the group consisting of: (1) The heavy chain variable region includes the following complementarity determining regions (CDRs): HCDR1 shown in SEQ ID NO:57, HCDR2 shown in SEQ ID NO:56, and HCDR3 shown in SEQ ID NO:9; And the light chain variable region includes the following complementarity determining regions CDR: LCDR1 shown in SEQ ID NO:10, LCDR2 shown in SEQ ID NO: 11, and LCDR3 shown in SEQ ID NO:12; or (2) The heavy chain variable region includes the following complementarity determining regions CDR: HCDR1 shown in SEQ ID NO:57, HCDR2 shown in SEQ ID NO:60, and HCDR3 shown in SEQ ID NO:21; And the light chain variable region includes the following complementarity determining regions CDR: LCDR1 shown in SEQ ID NO:22, LCDR2 shown in SEQ ID NO:23, and LCDR3 shown in SEQ ID NO:24; Wherein, the CDR sequence is based on the Chothia numbering scheme.

4. A polynucleotide, characterized in that The polynucleotide encodes the antibody or antigen-binding fragment thereof according to claim 1, the recombinant protein according to claim 2, or the chimeric antigen receptor CAR according to claim 3.

5. A carrier, characterized in that The vector contains the polynucleotide according to claim 4.

6. A host cell, characterized in that The host cell contains the vector according to claim 5 or the chromosome of which the exogenous polynucleotide according to claim 4 is integrated.

7. A method for preparing CAR-NK cells or CAR-T cells, characterized in that: The CAR-NK cell or CAR-T cell expresses the chimeric antigen receptor according to claim 3, comprising the following steps: The polynucleotide according to claim 4 or the vector according to claim 5 is transduced into NK cells or T cells to obtain the CAR-NK cells or CAR-T cells.

8. A pharmaceutical composition, characterized in that The pharmaceutical composition contains the antibody or antigen-binding fragment thereof according to claim 1, the recombinant protein according to claim 2, the chimeric antigen receptor according to claim 3, the polynucleotide according to claim 4, the vector according to claim 5, or the host cell according to claim 6, and a pharmaceutically acceptable carrier, diluent or excipient.

9. An immunoconjugate, characterized in that: The immunoconjugate contains: (a) an antibody portion, the antibody portion being selected from the group consisting of: The antibody or antigen-binding fragment thereof according to claim 1, the recombinant protein according to claim 2, or a combination thereof; and (b) a conjugated moiety conjugated to the antibody portion, wherein the conjugated moiety is selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, an enzyme, or a combination thereof.

10. The immunoconjugate according to claim 9, characterized in that The coupling moiety is a radionuclide.

11. A use of the antibody or antigen-binding fragment thereof according to claim 1, the recombinant protein according to claim 2, the chimeric antigen receptor according to claim 3, the polynucleotide according to claim 4, the vector according to claim 5, the host cell according to claim 6, the pharmaceutical composition according to claim 8, or the immunoconjugate according to claim 9 or 10, characterized in that: (a) preparing a reagent or kit for detecting a ROR1-related disease; and / or (b) preparing drugs or preparations for preventing and / or treating ROR1-related diseases; The ROR1-related disease is a ROR1-positive tumor; The tumor is selected from the group consisting of a blood tumor, a solid tumor, or a combination thereof; wherein The blood tumor is selected from the group consisting of acute lymphoblastic leukemia, chronic lymphoblastic leukemia, diffuse large B-cell lymphoblastic leukemia, mantle cell lymphoblastic leukemia, or a combination thereof; The solid tumor is selected from the group consisting of breast cancer, gastric cancer, lung cancer, ovarian cancer, colorectal cancer, pancreatic cancer, endometrial cancer, melanoma, or a combination thereof.

12. A method for detecting ROR1 protein in a sample in vitro for non-disease treatment purposes or non-disease diagnosis purposes, characterized in that: The method comprises the steps of: (1) contacting a sample with the antibody or antigen-binding fragment thereof according to claim 1, or the recombinant protein according to claim 2; (2) Detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of ROR1 protein in the sample.

13. A method for preparing a recombinant polypeptide, characterized in that: The method includes: (a) culturing the host cell according to claim 6 under conditions suitable for expression; and (b) isolating a recombinant polypeptide from the culture, wherein the recombinant polypeptide is the antibody or antigen-binding fragment thereof according to claim 1, or the recombinant protein according to claim 2.

14. A detection board, characterized in that: The detection plate comprises: a substrate and a test strip, wherein the test strip contains the antibody or antigen-binding fragment thereof according to claim 1, the recombinant protein according to claim 2, the immunoconjugate according to claim 9 or 10, or a combination thereof.

15. A kit, characterized in that: The kit includes: (1) a first container, wherein the first container contains the antibody or antigen-binding fragment thereof according to claim 1; and (2) a second container, wherein the second container contains a secondary antibody against the antibody or antigen-binding fragment thereof of claim 1; Alternatively, the kit contains the detection plate described in claim 14.

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