Antibodies targeting DLL3 and uses thereof

Abstract

The invention discloses a DLL3 targeting antibody and application thereof. The DLL3 antibody shows excellent specificity and binding capacity, and a chimeric antigen receptor derived from the DLL3 antibody shows excellent killing capacity on tumor cells, and is relatively low in cytotoxicity and low in side effect.

Description

Antibodies targeting DLL3 and their applications Technical Field

[0001] The present invention relates to the field of antibodies, and in particular to an antibody targeting DLL3 and its application. Background Art

[0002] DLL3, short for Delta-like protein 3, is a type I transmembrane protein encoded by the DLL3 gene. The DLL3 protein consists of 618 amino acid residues and is divided into an extracellular Delta / Serrate / Lag-2 (DSL) domain, six EGF-like domains, a transmembrane segment, and an intracellular segment.

[0003] During development, DLL3 protein is primarily expressed in the presomitic mesoderm during early embryonic development. Mutations in DLL3 lead to spondylocostal dysostosis in both humans and mice. In adult tissues, DLL3 mRNA can be detected in the brain, endocrine tissues, and testes, but protein is difficult to detect, suggesting that DLL3 protein has a good safety profile as a target for CAR-NK therapy. At the cellular level, DLL3 protein is primarily localized to the Golgi apparatus rather than the cell membrane. When DLL3 is abnormally overexpressed in cells, it can be detected on the cell membrane, and its overexpression is positively correlated with the expression of ASCL-1 (Achaete-scute homolog 1), a basic helix-loop-helix transcription factor involved in neuroendocrine cell fate determination.

[0004] DLL3 protein is highly expressed in a variety of solid tumors, including small cell lung cancer, large cell neuroendocrine carcinoma of the lung, gastroenteropancreatic neuroendocrine carcinoma, castration-resistant neuroendocrine prostate cancer, endometrial cancer, isocitrate dehydrogenase-mutant gliomas, and Merkel cell carcinoma. In small cell lung cancer and endometrial cancer, high DLL3 protein expression is associated with shorter overall survival. In terms of cancer pathogenesis, DLL3 acts as a ligand of the Notch signaling pathway, primarily through cis-inhibition, downregulating Notch receptor proteins in cells with high DLL3 expression and inhibiting the Notch signaling pathway. Knockdown of DLL3 in gastrointestinal neuroendocrine cancer cell lines inhibits cell proliferation.

[0005] In summary, DLL3 is a target that is widely expressed in neuroendocrine cancers and has a good safety profile. Targeting DLL3 protein using antibodies, antibody-drug conjugates, and CAR-NK has broad application prospects.

[0006] Summary of the Invention

[0007] The object of the present invention is to provide an antibody targeting DLL3 and its application.

[0008] In a first aspect of the present invention, an antibody or antigen-binding fragment thereof targeting DLL3 is provided, wherein 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:

[0009] (1) The heavy chain variable region includes the following complementarity determining regions (CDRs): (Ab.76)

[0010] HCDR1 shown in SEQ ID NO:37,

[0011] HCDR2 shown in SEQ ID NO: 38, and

[0012] HCDR3 shown in SEQ ID NO: 39;

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

[0014] LCDR1 shown in SEQ ID NO:40,

[0015] LCDR2 shown in SEQ ID NO:41, and

[0016] LCDR3 shown in SEQ ID NO:42; or

[0017] (2) The heavy chain variable region includes the following complementarity determining regions (CDRs): (Ab.55)

[0018] HCDR1 shown in SEQ ID NO:7,

[0019] HCDR2 shown in SEQ ID NO:8, and

[0020] HCDR3 shown in SEQ ID NO:9;

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

[0022] LCDR1 shown in SEQ ID NO: 10,

[0023] LCDR2 shown in SEQ ID NO: 11, and

[0024] LCDR3 shown in SEQ ID NO: 12; or

[0025] (3) The heavy chain variable region includes the following complementarity determining regions (CDRs): (Ab.67)

[0026] HCDR1 shown in SEQ ID NO: 19,

[0027] HCDR2 shown in SEQ ID NO: 20, and

[0028] HCDR3 shown in SEQ ID NO:21;

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

[0030] LCDR1 shown in SEQ ID NO:22,

[0031] LCDR2 shown in SEQ ID NO: 23, and

[0032] LCDR3 shown in SEQ ID NO: 24; or

[0033] (4) The heavy chain variable region includes the following complementarity determining regions (CDRs): (Ab.73)

[0034] HCDR1 shown in SEQ ID NO:31,

[0035] HCDR2 shown in SEQ ID NO: 32, and

[0036] HCDR3 shown in SEQ ID NO: 33;

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

[0038] LCDR1 shown in SEQ ID NO:34,

[0039] LCDR2 shown in SEQ ID NO: 35, and

[0040] LCDR3 shown in SEQ ID NO:36; or

[0041] (5) The heavy chain variable region includes the following complementarity determining regions (CDRs): (Ab.71)

[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: 29, and

[0048] LCDR3 shown in SEQ ID NO:30; or

[0049] (6) The heavy chain variable region includes the following complementarity determining regions (CDRs): (Ab.53)

[0050] HCDR1 shown in SEQ ID NO: 1,

[0051] HCDR2 shown in SEQ ID NO: 2, and

[0052] HCDR3 shown in SEQ ID NO: 3;

[0053] And the light chain variable region includes the following complementarity determining regions CDR:

[0054] LCDR1 shown in SEQ ID NO:4,

[0055] LCDR2 shown in SEQ ID NO:5, and

[0056] LCDR3 shown in SEQ ID NO: 6; or

[0057] (7) The heavy chain variable region includes the following complementarity determining regions (CDRs): (Ab.79)

[0058] HCDR1 shown in SEQ ID NO: 13,

[0059] HCDR2 shown in SEQ ID NO:43, and

[0060] HCDR3 shown in SEQ ID NO: 33;

[0061] And the light chain variable region includes the following complementarity determining regions CDR:

[0062] LCDR1 shown in SEQ ID NO:44,

[0063] LCDR2 shown in SEQ ID NO: 35, and

[0064] LCDR3 shown in SEQ ID NO:36; or

[0065] (8) The heavy chain variable region includes the following complementarity determining regions (CDRs): (Ab.63)

[0066] HCDR1 shown in SEQ ID NO: 13,

[0067] HCDR2 shown in SEQ ID NO: 14, and

[0068] HCDR3 shown in SEQ ID NO: 15;

[0069] And the light chain variable region includes the following complementarity determining regions CDR:

[0070] LCDR1 shown in SEQ ID NO: 16,

[0071] LCDR2 shown in SEQ ID NO: 17, and

[0072] LCDR3 shown in SEQ ID NO: 18;

[0073] The CDR sequences are based on the numbering scheme of Kabat.

[0074] 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 complementarity determining region (CDR) selected from the following group:

[0075] (1) The heavy chain variable region includes the following complementarity determining regions (CDRs): (Ab.76)

[0076] HCDR1 shown in SEQ ID NO:56,

[0077] HCDR2 shown in SEQ ID NO:57, and

[0078] HCDR3 shown in SEQ ID NO: 39;

[0079] And the light chain variable region includes the following complementarity determining regions CDR:

[0080] LCDR1 shown in SEQ ID NO:40,

[0081] LCDR2 shown in SEQ ID NO:41, and

[0082] LCDR3 shown in SEQ ID NO:42; or

[0083] (2) The heavy chain variable region includes the following complementarity determining regions (CDRs): (Ab.55)

[0084] HCDR1 shown in SEQ ID NO:47,

[0085] HCDR2 shown in SEQ ID NO:48, and

[0086] HCDR3 shown in SEQ ID NO:9;

[0087] And the light chain variable region includes the following complementarity determining regions CDR:

[0088] LCDR1 shown in SEQ ID NO: 10,

[0089] LCDR2 shown in SEQ ID NO: 11, and

[0090] LCDR3 shown in SEQ ID NO: 12; or

[0091] (3) The heavy chain variable region includes the following complementarity determining regions (CDRs): (Ab.67)

[0092] HCDR1 shown in SEQ ID NO:50,

[0093] HCDR2 shown in SEQ ID NO:51, and

[0094] HCDR3 shown in SEQ ID NO:21;

[0095] And the light chain variable region includes the following complementarity determining regions CDR:

[0096] LCDR1 shown in SEQ ID NO:22,

[0097] LCDR2 shown in SEQ ID NO: 23, and

[0098] LCDR3 shown in SEQ ID NO: 24; or

[0099] (4) The heavy chain variable region includes the following complementarity determining regions (CDRs): (Ab.73)

[0100] HCDR1 shown in SEQ ID NO:54,

[0101] HCDR2 shown in SEQ ID NO:55, and

[0102] HCDR3 shown in SEQ ID NO: 33;

[0103] And the light chain variable region includes the following complementarity determining regions CDR:

[0104] LCDR1 shown in SEQ ID NO:34,

[0105] LCDR2 shown in SEQ ID NO: 35, and

[0106] LCDR3 shown in SEQ ID NO:36; or

[0107] (5) The heavy chain variable region includes the following complementarity determining regions (CDRs): (Ab.71)

[0108] HCDR1 shown in SEQ ID NO:52,

[0109] HCDR2 shown in SEQ ID NO:53, and

[0110] HCDR3 shown in SEQ ID NO: 27;

[0111] And the light chain variable region includes the following complementarity determining regions CDR:

[0112] LCDR1 shown in SEQ ID NO: 28,

[0113] LCDR2 shown in SEQ ID NO: 29, and

[0114] LCDR3 shown in SEQ ID NO:30; or

[0115] (6) The heavy chain variable region includes the following complementarity determining regions (CDRs): (Ab.53)

[0116] HCDR1 shown in SEQ ID NO:45,

[0117] HCDR2 shown in SEQ ID NO:46, and

[0118] HCDR3 shown in SEQ ID NO: 3;

[0119] And the light chain variable region includes the following complementarity determining regions CDR:

[0120] LCDR1 shown in SEQ ID NO:4,

[0121] LCDR2 shown in SEQ ID NO:5, and

[0122] LCDR3 shown in SEQ ID NO: 6; or

[0123] (7) The heavy chain variable region includes the following complementarity determining regions (CDRs): (Ab.79)

[0124] HCDR1 shown in SEQ ID NO:58,

[0125] HCDR2 shown in SEQ ID NO:48, and

[0126] HCDR3 shown in SEQ ID NO: 33;

[0127] And the light chain variable region includes the following complementarity determining regions CDR:

[0128] LCDR1 shown in SEQ ID NO:44,

[0129] LCDR2 shown in SEQ ID NO: 35, and

[0130] LCDR3 shown in SEQ ID NO:36; or

[0131] (8) The heavy chain variable region includes the following complementarity determining regions (CDRs): (Ab.63)

[0132] HCDR1 shown in SEQ ID NO:49,

[0133] HCDR2 shown in SEQ ID NO:48, and

[0134] HCDR3 shown in SEQ ID NO: 15;

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

[0136] LCDR1 shown in SEQ ID NO: 16,

[0137] LCDR2 shown in SEQ ID NO: 17, and

[0138] LCDR3 shown in SEQ ID NO: 18;

[0139] Wherein, the CDR sequence is based on the Chothia numbering scheme.

[0140] In another preferred embodiment, the antibody includes a heavy chain and a light chain, the heavy chain includes the three heavy chain CDRs and a heavy chain framework region for connecting the heavy chain CDRs; the light chain includes the three light chain CDRs and a light chain framework region for connecting the light chain CDRs.

[0141] In another preferred embodiment, the antibody or antigen-binding fragment thereof targeting DLL3 is selected from the following group: camel Ig, Ig NAR, 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).

[0142] In another preferred embodiment, the light chain of the antibody further includes a light chain constant region.

[0143] In another preferred embodiment, the light chain constant region is of human, mouse or rabbit origin, preferably of human origin.

[0144] In another preferred embodiment, the heavy chain of the antibody further includes a heavy chain constant region.

[0145] In another preferred embodiment, the heavy chain constant region is of human, mouse or rabbit origin, preferably of human origin.

[0146] In another preferred embodiment, the antibody is a double-chain antibody or a single-chain antibody.

[0147] In another preferred embodiment, the antibody is a monoclonal antibody.

[0148] In another preferred embodiment, the antibody includes a monospecific, bispecific, trispecific antibody or a multispecific antibody.

[0149] In another preferred embodiment, the antibody specifically binds to DLL3.

[0150] In another preferred embodiment, the KD value (M) of the antibody's affinity for human DLL3 is 1.0E-12 to 1.0E-8.

[0151] In another preferred embodiment, the antibody is a single-chain antibody (scFv) having an amino acid sequence selected from the group consisting of SEQ ID NO. 59, 60, 61, 62, 63, 64, 65, and 66.

[0152] 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 with the amino acid sequence shown in SEQ ID NO. 59, 60, 61, 62, 63, 64, 65, and 66 (wherein the CDRs remain unchanged or substantially unchanged).

[0153] The second aspect of the present invention provides a recombinant protein, wherein the recombinant protein has:

[0154] (i) the antibody or antigen-binding fragment thereof according to the first aspect of the present invention;

[0155] and (ii) optionally a tag sequence to facilitate expression and / or purification.

[0156] In another preferred embodiment, the tag sequence includes a 6His tag.

[0157] In another preferred embodiment, the recombinant protein (or polypeptide) includes a fusion protein.

[0158] In another preferred embodiment, the recombinant protein is a monomer, a dimer, or a multimer.

[0159] In another preferred embodiment, the recombinant protein further comprises an additional fusion element (or fusion polypeptide fragment) fused with the element (i).

[0160] 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 comprises a single-chain variable region sequence scFv of an antibody targeting DLL3, and the heavy chain variable region and light chain variable region of the scFv comprise a complementarity determining region CDR selected from the following group:

[0161] (1) The heavy chain variable region includes the following complementarity determining regions (CDRs): (Ab.76)

[0162] HCDR1 shown in SEQ ID NO:37,

[0163] HCDR2 shown in SEQ ID NO: 38, and

[0164] HCDR3 shown in SEQ ID NO: 39;

[0165] And the light chain variable region includes the following complementarity determining regions CDR:

[0166] LCDR1 shown in SEQ ID NO:40,

[0167] LCDR2 shown in SEQ ID NO:41, and

[0168] LCDR3 shown in SEQ ID NO:42; or

[0169] (2) The heavy chain variable region includes the following complementarity determining regions (CDRs): (Ab.55)

[0170] HCDR1 shown in SEQ ID NO:7,

[0171] HCDR2 shown in SEQ ID NO:8, and

[0172] HCDR3 shown in SEQ ID NO:9;

[0173] And the light chain variable region includes the following complementarity determining regions CDR:

[0174] LCDR1 shown in SEQ ID NO: 10,

[0175] LCDR2 shown in SEQ ID NO: 11, and

[0176] LCDR3 shown in SEQ ID NO: 12; or

[0177] (3) The heavy chain variable region includes the following complementarity determining regions (CDRs): (Ab.67)

[0178] HCDR1 shown in SEQ ID NO: 19,

[0179] HCDR2 shown in SEQ ID NO: 20, and

[0180] HCDR3 shown in SEQ ID NO:21;

[0181] And the light chain variable region includes the following complementarity determining regions CDR:

[0182] LCDR1 shown in SEQ ID NO:22,

[0183] LCDR2 shown in SEQ ID NO: 23, and

[0184] LCDR3 shown in SEQ ID NO: 24; or

[0185] (4) The heavy chain variable region includes the following complementarity determining regions (CDRs): (Ab.73)

[0186] HCDR1 shown in SEQ ID NO:31,

[0187] HCDR2 shown in SEQ ID NO: 32, and

[0188] HCDR3 shown in SEQ ID NO: 33;

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

[0190] LCDR1 shown in SEQ ID NO:34,

[0191] LCDR2 shown in SEQ ID NO: 35, and

[0192] LCDR3 shown in SEQ ID NO:36; or

[0193] (5) The heavy chain variable region includes the following complementarity determining regions (CDRs): (Ab.71)

[0194] HCDR1 shown in SEQ ID NO: 25,

[0195] HCDR2 shown in SEQ ID NO: 26, and

[0196] HCDR3 shown in SEQ ID NO: 27;

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

[0198] LCDR1 shown in SEQ ID NO: 28,

[0199] LCDR2 shown in SEQ ID NO: 29, and

[0200] LCDR3 shown in SEQ ID NO:30; or

[0201] (6) The heavy chain variable region includes the following complementarity determining regions (CDRs): (Ab.53)

[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] (7) The heavy chain variable region includes the following complementarity determining regions (CDRs): (Ab.79)

[0210] HCDR1 shown in SEQ ID NO: 13,

[0211] HCDR2 shown in SEQ ID NO:43, and

[0212] HCDR3 shown in SEQ ID NO: 33;

[0213] And the light chain variable region includes the following complementarity determining regions CDR:

[0214] LCDR1 shown in SEQ ID NO:44,

[0215] LCDR2 shown in SEQ ID NO: 35, and

[0216] LCDR3 shown in SEQ ID NO:36; or

[0217] (8) The heavy chain variable region includes the following complementarity determining regions (CDRs): (Ab.63)

[0218] HCDR1 shown in SEQ ID NO: 13,

[0219] HCDR2 shown in SEQ ID NO: 14, and

[0220] HCDR3 shown in SEQ ID NO: 15;

[0221] And the light chain variable region includes the following complementarity determining regions CDR:

[0222] LCDR1 shown in SEQ ID NO: 16,

[0223] LCDR2 shown in SEQ ID NO: 17, and

[0224] LCDR3 shown in SEQ ID NO: 18;

[0225] The CDR sequences are based on the numbering scheme of Kabat.

[0226] In another preferred embodiment, the heavy chain variable region and the light chain variable region of the scFv include complementarity determining regions (CDRs) selected from the following groups:

[0227] (1) The heavy chain variable region includes the following complementarity determining regions (CDRs): (Ab.76)

[0228] HCDR1 shown in SEQ ID NO:56,

[0229] HCDR2 shown in SEQ ID NO:57, and

[0230] HCDR3 shown in SEQ ID NO: 39;

[0231] And the light chain variable region includes the following complementarity determining regions CDR:

[0232] LCDR1 shown in SEQ ID NO:40,

[0233] LCDR2 shown in SEQ ID NO:41, and

[0234] LCDR3 shown in SEQ ID NO:42; or

[0235] (2) The heavy chain variable region includes the following complementarity determining regions (CDRs): (Ab.55)

[0236] HCDR1 shown in SEQ ID NO:47,

[0237] HCDR2 shown in SEQ ID NO:48, and

[0238] HCDR3 shown in SEQ ID NO:9;

[0239] And the light chain variable region includes the following complementarity determining regions CDR:

[0240] LCDR1 shown in SEQ ID NO: 10,

[0241] LCDR2 shown in SEQ ID NO: 11, and

[0242] LCDR3 shown in SEQ ID NO: 12; or

[0243] (3) The heavy chain variable region includes the following complementarity determining regions (CDRs): (Ab.67)

[0244] HCDR1 shown in SEQ ID NO:50,

[0245] HCDR2 shown in SEQ ID NO:51, and

[0246] HCDR3 shown in SEQ ID NO:21;

[0247] And the light chain variable region includes the following complementarity determining regions CDR:

[0248] LCDR1 shown in SEQ ID NO:22,

[0249] LCDR2 shown in SEQ ID NO: 23, and

[0250] LCDR3 shown in SEQ ID NO: 24; or

[0251] (4) The heavy chain variable region includes the following complementarity determining regions (CDRs): (Ab.73)

[0252] HCDR1 shown in SEQ ID NO:54,

[0253] HCDR2 shown in SEQ ID NO:55, and

[0254] HCDR3 shown in SEQ ID NO: 33;

[0255] And the light chain variable region includes the following complementarity determining regions CDR:

[0256] LCDR1 shown in SEQ ID NO:34,

[0257] LCDR2 shown in SEQ ID NO: 35, and

[0258] LCDR3 shown in SEQ ID NO:36; or

[0259] (5) The heavy chain variable region includes the following complementarity determining regions (CDRs): (Ab.71)

[0260] HCDR1 shown in SEQ ID NO:52,

[0261] HCDR2 shown in SEQ ID NO:53, and

[0262] HCDR3 shown in SEQ ID NO: 27;

[0263] And the light chain variable region includes the following complementarity determining regions CDR:

[0264] LCDR1 shown in SEQ ID NO: 28,

[0265] LCDR2 shown in SEQ ID NO: 29, and

[0266] LCDR3 shown in SEQ ID NO:30; or

[0267] (6) The heavy chain variable region includes the following complementarity determining regions (CDRs): (Ab.53)

[0268] HCDR1 shown in SEQ ID NO:45,

[0269] HCDR2 shown in SEQ ID NO:46, and

[0270] HCDR3 shown in SEQ ID NO: 3;

[0271] And the light chain variable region includes the following complementarity determining regions CDR:

[0272] LCDR1 shown in SEQ ID NO:4,

[0273] LCDR2 shown in SEQ ID NO:5, and

[0274] LCDR3 shown in SEQ ID NO: 6; or

[0275] (7) The heavy chain variable region includes the following complementarity determining regions (CDRs): (Ab.79)

[0276] HCDR1 shown in SEQ ID NO:58,

[0277] HCDR2 shown in SEQ ID NO:48, and

[0278] HCDR3 shown in SEQ ID NO: 33;

[0279] And the light chain variable region includes the following complementarity determining regions CDR:

[0280] LCDR1 shown in SEQ ID NO:44,

[0281] LCDR2 shown in SEQ ID NO: 35, and

[0282] LCDR3 shown in SEQ ID NO:36; or

[0283] (8) The heavy chain variable region includes the following complementarity determining regions (CDRs): (Ab.63)

[0284] HCDR1 shown in SEQ ID NO:49,

[0285] HCDR2 shown in SEQ ID NO:48, and

[0286] HCDR3 shown in SEQ ID NO: 15;

[0287] And the light chain variable region includes the following complementarity determining regions CDR:

[0288] LCDR1 shown in SEQ ID NO: 16,

[0289] LCDR2 shown in SEQ ID NO: 17, and

[0290] LCDR3 shown in SEQ ID NO: 18;

[0291] Wherein, the CDR sequence is based on the Chothia numbering scheme.

[0292] In another preferred embodiment, the scFv further comprises a connecting peptide between the heavy chain variable region and the light chain variable region.

[0293] In another preferred embodiment, the connecting peptide is (G4S)3 or (G4S)4.

[0294] In another preferred embodiment, the scFv is represented by the following formula A or formula B:

[0295] VH-VL,(A); VL-VH,(B)

[0296] In the formula, VH is the antibody heavy chain variable region; VL is the antibody light chain variable region; and “-” is a connecting peptide or peptide bond.

[0297] In another preferred embodiment, the connecting peptide is (G4S)n, preferably n is 3-5; more preferably n is 3.

[0298] In another preferred embodiment, the scFv has an amino acid sequence selected from the group consisting of:

[0299] SEQ ID NO. 59, 60, 61, 62, 63, 64, 65, 66.

[0300] In another preferred embodiment, the antibody single-chain variable region includes human, mouse, or human-mouse chimeric antibody single-chain variable region.

[0301] In another preferred embodiment, the scFv is represented by formula A (VH-VL).

[0302] In another preferred embodiment, the antigen binding domain targets the extracellular region of DLL3.

[0303] In another preferred embodiment, the chimeric antigen receptor has the following structure: L-scFv-H-TM-C-CD3ζ (I)

[0304] Where,

[0305] Each "-" is independently a connecting peptide or a peptide bond;

[0306] L is none or a signal peptide sequence;

[0307] scFv is a scFv targeting DLL3;

[0308] H is an optional hinge region;

[0309] TM is the transmembrane domain;

[0310] C is a co-stimulatory signal molecule;

[0311] CD3ζ is the CD3ζ cytoplasmic signaling sequence.

[0312] In another preferred embodiment, the L is a signal peptide selected from the following histones: CD8, CD4, CD16, CD56, CD137, CSF2, DAP12, EF1, GM-CSF, IL-8, IL-21 or a combination thereof.

[0313] In another preferred embodiment, the L is derived from a signal peptide derived from CD8.

[0314] In another preferred embodiment, the scFv is represented by the following formula A or formula B: VH-VL, (A); VL-VH, (B)

[0315] In the formula, VH is the antibody heavy chain variable region; VL is the antibody light chain variable region; and “-” is a connecting peptide or peptide bond.

[0316] In another preferred embodiment, the scFv is represented by formula A (VH-VL).

[0317] In another preferred embodiment, the H is a hinge region selected from the group consisting of CD8, CD28, CD137, Fc, or a combination thereof.

[0318] In another preferred embodiment, the H is the hinge region derived from CD28.

[0319] 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.

[0320] In another preferred embodiment, the TM is a transmembrane region derived from CD28.

[0321] In another preferred embodiment, the 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.

[0322] In another preferred embodiment, C is a costimulatory signal molecule derived from CD28.

[0323] The fourth aspect of the present invention provides a polynucleotide encoding 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.

[0324] In another preferred embodiment, the polynucleotide is isolated.

[0325] The fifth aspect of the present invention provides a vector comprising the polynucleotide described in the fourth aspect of the present invention.

[0326] 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.

[0327] In another preferred embodiment, the vector is a retroviral vector.

[0328] The sixth aspect of the present invention provides a host cell, wherein the host cell contains the vector described in the fifth aspect of the present invention or the chromosome in which the exogenous polynucleotide described in the fourth aspect of the present invention is integrated.

[0329] In another preferred embodiment, the cell is an isolated cell, and / or the cell is a genetically engineered cell.

[0330] In another preferred embodiment, the cells are mammalian cells.

[0331] In another preferred embodiment, the cells are NK cells or T cells.

[0332] In another preferred embodiment, the host cell is an engineered immune cell.

[0333] 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.

[0334] In another preferred embodiment, the host cell is an immune cell, and the immune cell expresses or exposes outside the cell membrane the antibody according to the first aspect of the present invention or the chimeric antigen receptor according to the third aspect of the present invention.

[0335] In another preferred embodiment, the immune cells include NK cells and T cells.

[0336] In another preferred embodiment, the immune cells are from humans or non-human mammals (such as mice).

[0337] 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:

[0338] 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.

[0339] In the eighth aspect of the present invention, a pharmaceutical composition is provided, which contains 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 chimeric antigen receptor described in the third aspect of the present invention, the polynucleotide described in the fourth aspect of the present invention, the vector described in the fifth aspect of the present invention, or the host cell described in the sixth aspect of the present invention, and a pharmaceutically acceptable carrier, diluent or excipient.

[0340] In another preferred embodiment, the pharmaceutical composition is a preparation, preferably a liquid preparation.

[0341] In another preferred embodiment, the pharmaceutical composition is in the form of an injection.

[0342] In another preferred embodiment, the pharmaceutical composition is used to prepare a drug or preparation for preventing and / or treating cancer or tumor.

[0343] The ninth aspect of the present invention provides an immunoconjugate, wherein the immunoconjugate comprises:

[0344] (a) an antibody portion 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

[0345] (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.

[0346] 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.

[0347] 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.

[0348] (a) preparing a detection reagent or kit; and / or

[0349] (b) preparing a drug or preparation for preventing and / or treating a DLL3-related disease.

[0350] In another preferred embodiment, the DLL3-related disease is cancer or tumor.

[0351] In another preferred embodiment, the tumor is selected from the following group: hematological tumors, solid tumors, or a combination thereof.

[0352] 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.

[0353] In another preferred embodiment, the tumor is a DLL3-positive tumor, preferably selected from the following group: chronic lymphocytic leukemia, breast cancer, lung cancer, ovarian cancer, colorectal cancer, endometrial cancer, melanoma, small cell lung cancer (SCLC), large cell neuroendocrine lung carcinoma (LCNEC), gastroenteropancreatic neuroendocrine carcinoma, castrate resistant neuroendocrine prostate cancers (crNEPC), small-cell bladder cancer, neuroendocrine carcinoma of the uterine cervix, isocitrate dehydrogenase-mutant glioma, Merkel cell carcinoma and medullary thyroid carcinomas, or a combination thereof.

[0354] In another preferred embodiment, the detection is an immunoassay.

[0355] In another preferred embodiment, the immunoassay is ELISA immunoassay, immunochromatography assay, immunocytochemistry staining assay or immunohistochemistry staining assay.

[0356] In another preferred embodiment, the diagnostic reagent is a test piece or a test plate.

[0357] In another preferred embodiment, the reagents include chips and antibody-coated immune particles.

[0358] In an eleventh aspect of the present invention, a method for detecting DLL3 protein in a sample (including diagnostic or non-diagnostic) in vitro is provided, the method comprising the steps of:

[0359] (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;

[0360] (2) Detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of DLL3 protein in the sample.

[0361] In another preferred embodiment, the diagnostic reagent is a test piece or a test plate.

[0362] In another preferred embodiment, the method is a cell immunochemistry (ICC) detection method, an immunohistochemistry (IHC) detection method, a whole cell ELISA detection method, or a cell lysate ELISA detection method.

[0363] A twelfth aspect of the present invention provides a method for preparing a recombinant polypeptide, the method comprising:

[0364] (a) culturing the host cell according to the sixth aspect of the present invention under conditions suitable for expression;

[0365] (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.

[0366] 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 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 immunoconjugate described in the ninth aspect of the present invention, or a combination thereof.

[0367] A fourteenth aspect of the present invention provides a kit, comprising:

[0368] (1) a first container containing the antibody or antigen-binding fragment thereof according to the first aspect of the present invention; and / or

[0369] (2) a second container containing a secondary antibody against the antibody of the present invention;

[0370] Alternatively, the kit contains the detection plate described in the thirteenth aspect of the present invention.

[0371] 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.

[0372] In another preferred embodiment, the disease is cancer or tumor.

[0373] 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 described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0374] Figure 1 shows the results of SDS-PAGE of anti-DLL3 scFv-Fc antibody.

[0375] FIG2 shows the results of anti-DLL3 antibody BLI assay.

[0376] FIG3 shows the results of anti-DLL3 antibody EC50 assays.

[0377] Figure 4 shows the anti-DLL3 CAR molecular structure.

[0378] FIG5 shows the results of detecting DLL3 protein expression on the surface of tumor cell lines.

[0379] Figure 6 shows the results of CAR detection of anti-DLL3 CAR-NK92 cell lines after sorting.

[0380] Figure 7 shows the single-round killing results of anti-DLL3 CAR NK92 on SHP77 and NCI-H82 cells.

[0381] Figure 8A shows the results of multiple rounds of killing of SHP77 by anti-DLL3 CAR NK92, and Figure 8B shows the results of multiple rounds of killing of NCI-H82 cells. DETAILED DESCRIPTION

[0382] After extensive and in-depth research, the inventors unexpectedly discovered a series of antibodies targeting DLL3. Screening revealed excellent biological activity in these antibodies, leading to the construction of chimeric antigen receptors targeting DLL3. Natural killer (NK) cells expressing these chimeric antigen receptors demonstrated superior cytotoxicity against target cells. This led to the completion of the present invention.

[0383] the term

[0384] 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.

[0385] 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.

[0386] As used herein, the terms "comprising" or "including" may be open, semi-closed, or closed. In other words, the terms also include "consisting essentially of" or "consisting of."

[0387] Antibody

[0388] 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 complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL comprises three CDRs and four FRs, arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen.

[0389] As used herein, the terms "heavy chain variable region" and "VH" are used interchangeably.

[0390] As used herein, the terms "light chain variable region" and "VL" are used interchangeably.

[0391] As used herein, the term "antigen binding domain" or the like includes any naturally occurring, enzymatically obtainable, synthetic or genetically modified polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Any suitable standard technique such as proteolytic digestion or recombinant genetic engineering techniques involving manipulation and expression of DNA encoding antibody variable domains and optionally antibody constant domains can be used, for example, to derive an antigen-binding fragment of an antibody from a complete antibody molecule. Such DNA is known and / or readily available or 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 biology techniques, for example, to arrange one or more variable domains and / or constant domains into a suitable layout, or to introduce codons, produce cysteine ​​residues, modify, add or delete amino acids, etc.

[0392] 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., individual complementarity determining regions (CDRs) such as CDR3 peptides) or constrained FR3-CDR3-FR4 peptides.

[0393] As used herein, an antigen-binding fragment or antigen-binding domain will generally comprise at least one variable domain. The variable domain can have 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. In an antigen-binding fragment having a VH domain associated with a VL domain, the VH and VL domains can be arranged relative to each other in any suitable arrangement. For example, the variable region can be a dimer and contain a VH-VH, VH-VL, or VL-VL dimer. Alternatively, the antigen-binding domain can contain a monomeric VH or VL domain.

[0394] 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 ImmunoGeneTics database (IMGT), the EU numbering system, and the Chothia definition based on loop structural position.

[0395] It will be appreciated that the precise amino acid sequence boundaries of the CDRs of the present invention may optionally be defined using the various designation systems mentioned above. Preferably, unless otherwise indicated, in the present invention, when referring to residue positions in an antibody variable region (including heavy chain variable region residues and light chain variable region residues), the numbering positions are those according to the Kabat numbering system.

[0396] As used herein, the terms "antibody" or "immunoglobulin" are heterotetrameric glycoproteins of approximately 150,000 daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.

[0397] As used herein, the term "variable" refers to certain parts of the variable region in an antibody that are different in sequence, which form the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the entire antibody variable region. It is concentrated in three segments called complementary determining regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conservative parts of the variable region are called framework regions (FRs). The variable regions of natural heavy and light chains each contain four FR regions, which are generally in a -folded configuration, connected by three CDRs that form a connecting loop, and in some cases can form a partially folded structure. The CDRs in each chain are closely together through the FR region and form the antigen-binding site of the antibody together with the CDRs of the other chain (see Kabat et al., NIH Publ. No. 91-3242, Volume 1, pp. 647-669 (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.

[0398] 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.

[0399] 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. A polypeptide fragment, derivative, or analog of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, where such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide formed by fusion of a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) a polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence (e.g., 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). Based on the teachings herein, these fragments, derivatives, and analogs are well known to those skilled in the art.

[0400] The "light chains" of vertebrate antibodies (immunoglobulins) can be assigned to 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 are 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 structures and three-dimensional configurations of the different classes of immunoglobulins are well known in the art.

[0401] The present invention also provides other polypeptides, such as fusion proteins comprising human antibodies or fragments thereof. In addition to substantially full-length polypeptides, the present invention also encompasses fragments of the antibodies of the invention. Typically, the fragments comprise at least about 50 contiguous amino acids of the antibodies of the invention, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids.

[0402] In the present invention, the antibodies of the present invention also include conservative variants thereof, which refer to polypeptides in which up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids are replaced with amino acids having similar or similar properties compared to the amino acid sequence of the antibodies of the present invention. These conservative variant polypeptides are preferably generated by amino acid substitutions according to Table A.

[0403] Table A

[0404] Chimeric Antigen Receptor (CAR)

[0405] 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.

[0406] 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 costimulatory molecules. Costimulatory molecules are cell surface molecules required for the effective response of lymphocytes to antigens, rather than antigen receptors or their ligands.

[0407] Between the extracellular domain and the transmembrane domain of CAR, or between the cytoplasmic domain and the transmembrane domain of CAR, a linker can be incorporated. As used herein, the term "linker" 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 linker may include 0-300 amino acids, preferably 2 to 100 amino acids and most preferably 3 to 50 amino acids.

[0408] 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 DLL3. 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 them to not grow, be caused to die or be affected in other ways, and causing the patient's tumor burden to be reduced or eliminated. The antigen binding domain is preferably fused with an intracellular domain from one or more of the costimulatory molecules and the 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.

[0409] As used herein, "antigen-binding domain" and "single-chain antibody fragment" 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 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 scFv (single-chain variable fragment). The size of 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 DLL3, preferably a single-chain antibody.

[0410] 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 such a domain being bound to the transmembrane domain of the same or different surface membrane proteins, thereby minimizing the interaction with other members of the receptor complex.

[0411] 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 costimulatory factor signaling region and a CD3zeta signaling region (ζ chain portion), and the connection order is as follows: L-scFv-H-TM-C-CD3ζ (I)

[0412] NK cells

[0413] Natural killer (NK) cells are a major type of immune effector cell that protect the body from viral infection and tumor invasion through non-antigen-specific pathways. In recent years, NK cells have shown great promise in adoptive cellular immunotherapy. NK cells can be derived from a wide range of sources, including peripheral blood, umbilical cord blood, embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs).

[0414] NK-92 cells are an interleukin-2 (IL2)-dependent NK cell line derived from peripheral blood mononuclear cells of a 50-year-old Caucasian male patient with acute non-Hodgkin's lymphoma. Currently the only FDA-approved NK cell line for clinical trials, NK-92 cells are highly cytotoxic, economical, off-the-shelf, and easily scalable. They survive tumor cells for a short time and are easily expanded in vitro. The vast majority of treated patients do not reject NK-92 cells, and there is no risk of graft-versus-host reaction. NK-92 cells do not express KIRs and are constitutively activated, demonstrating a good clinical safety profile to date.

[0415] As used herein, the terms "CAR-NK cell", "CAR-NK", "CARNK", and "CAR-NK cell of the present invention" all refer to CAR-NK cells expressing the chimeric antigen receptor CAR of the first aspect of the present invention.

[0416] carrier

[0417] The nucleic acid sequence encoding the desired molecule can be obtained using recombinant methods known in the art, such as, for example, by screening libraries from cells expressing the gene, by obtaining the gene from a vector known to include the gene, or by directly isolating from cells and tissues containing the gene using standard techniques. Alternatively, the gene of interest can be produced synthetically.

[0418] The present invention also provides vectors into which the expression cassettes of the present invention are inserted. Vectors derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer because they allow for 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.

[0419] In brief summary, the expression cassette or nucleic acid sequence of the present invention is generally operably linked to a promoter and incorporated into an expression vector. Such vectors are suitable for replication and integration into eukaryotic cells. Typical cloning vectors contain transcriptional and translational terminators, initiation sequences, and promoters that can be used to regulate expression of the desired nucleic acid sequence.

[0420] 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, and 5,589,466, which are incorporated herein by reference in their entireties. In another embodiment, the present invention provides gene therapy vectors.

[0421] 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.

[0422] 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. The virus that can be used as a vector includes but is not limited to retrovirus, adenovirus, adeno-associated virus, herpes virus and slow virus. Generally, suitable vectors are included in at least one organism and work in the origin of replication, promoter sequence, convenient restriction enzyme site and one or more selectable markers (for example, WO01 / 96584; WO01 / 29058; and U.S. Patent number 6,326,193).

[0423] 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 the 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.

[0424] 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, promoter function is maintained. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased by 50 bp before activity begins to decline. Depending on the promoter, it has been shown that individual elements can work together or independently to initiate transcription.

[0425] 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 for high-level expression. 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 expression of a polynucleotide sequence operably linked to the 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, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.

[0426] In order to evaluate the expression of the CAR polypeptide or its portion, 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 that is sought to be transfected or infected by a viral vector. In other aspects, selectable markers can 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 to enable expression in host cells. Useful selectable markers include, for example, antibiotic resistance genes, such as neo and the like.

[0427] Reporter gene is used to identify the cells of potential transfection and to evaluate the functionality of regulatory sequences. Generally, reporter gene is following gene: it is not present in or is expressed by receptor organism or tissue, and its coded polypeptide, the expression of this polypeptide is clearly represented by some easily detectable properties such as enzymatic activity. After DNA has been introduced into receptor cells, the expression of reporter gene is measured under the appropriate time. Suitable reporter gene can comprise the gene (for example, Ui-Tei etc., 2000FEBS Letters479:79-82) of coding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase or green fluorescent protein. Suitable expression system is well known and can utilize known technology to prepare or obtain commercially. Generally, the construct with minimum 5 flanking regions showing the highest level of reporter gene expression is identified as promoter. Such promoter region can be connected to reporter gene and be used to evaluate the ability of reagent regulating promoter-driven transcription.

[0428] Methods for introducing genes into cells and expressing genes in cells are known in the art. In the context of expression vectors, the vectors can be readily 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.

[0429] 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.

[0430] 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 method for inserting genes into mammalian, for example, human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, among others. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0431] 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).

[0432] In the case of using non-viral delivery system, exemplary delivery means is liposome. Consider using lipid preparation, to introduce nucleic acid into host cell (in vitro, in vitro (ex vivo) or in vivo). On the other hand, this nucleic acid can be associated with lipid. The nucleic acid associated with lipid can be encapsulated in the aqueous interior of liposome, dispersed in the lipid bilayer of liposome, attached to liposome through the connecting molecule associated with liposome and oligonucleotide, trapped in liposome, compounded with liposome, dispersed in the solution comprising lipid, mixed with lipid, united with lipid, included in lipid as suspension, included in micelle or with micelle compound, or otherwise associated with lipid. The lipid associated with composition, lipid / DNA or lipid / expression vector are not limited to any specific structure in solution. For example, they can be present in bilayer structure, as micelle or have " collapsed (collapsed) " structure. They can also be simply dispersed in solution, may form 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.

[0433] In a preferred embodiment of the present invention, the vector is a retroviral vector.

[0434] Therapeutic applications

[0435] 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 induce CAR-mediated NK-cell or T cell responses.

[0436] 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 steps of administering to the mammal an NK cell expressing the CAR of the present invention.

[0437] In one embodiment, the present invention includes a type of cell therapy in which NK cells are genetically modified to express a CAR of the present invention, and the CAR-NK cells are infused into a recipient in need thereof. The infused cells are capable of killing the recipient's tumor cells. Unlike antibody therapies, CAR-NK cells are able to persist in the body, resulting in long-term persistence that can lead to sustained tumor control.

[0438] Treatable cancers include tumors that are not vascularized or substantially not yet vascularized, and vascularized tumors. Cancer may include non-solid tumors (such as hematological tumors, such as leukemia and lymphoma) or may include solid tumors. The types of cancer treated with the CAR of the present invention include but are not limited to cancer, blastoma and sarcoma, and certain leukemia or lymphoma, benign and malignant tumors and malignant tumors, such as sarcoma, cancer and melanoma. Also included are adult tumors / cancers and childhood tumors / cancers.

[0439] Solid tumors are abnormal masses of tissue that do not typically 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, and ovarian cancer.

[0440] 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.

[0441] Pharmaceutical composition

[0442] The antibody, fusion protein or CAR-modified NK cells of the present invention can be administered alone or as a pharmaceutical composition with a diluent and / or in combination with other components such as IL-2, IL-15, IL-18, IL-21 or other cytokines or cell groups. Briefly, the pharmaceutical composition of the present invention may include a target cell group as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may include buffers such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions of the present invention are preferably formulated for intravenous administration.

[0443] The pharmaceutical compositions of the present invention can be administered in a manner appropriate to 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 appropriate dosages can be determined by clinical trials.

[0444] When an "immunologically effective amount," "anti-tumor effective amount," "tumor-inhibitory effective amount," or "therapeutic amount" is indicated, the precise 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 compositions comprising the T cells described herein can be administered at a dose of 104 to 109 cells / kg body weight, preferably 105 to 106 cells / kg body weight (including all integer values ​​within those ranges). The T cell compositions can also be administered multiple times at these doses. The cells can be administered using infusion techniques well known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). The optimal dosage and treatment regimen for a particular patient can be readily determined by a person skilled in the medical field by monitoring the patient for signs of disease and adjusting treatment accordingly.

[0445] The main advantages of the present invention include

[0446] (1) The chimeric antigen receptor of the present invention has an extracellular antigen-binding domain that is a specific anti-DLL3 scFv. The specific anti-DLL3 scFv binds to a specific hinge region and an intracellular domain, and the resulting CAR exhibits a significant ability to kill tumor cells with minimal cytotoxicity and side effects.

[0447] (2) The CAR-NK cells of the present invention have a high degree of activation and have excellent cytotoxicity against DLL3-positive target cells.

[0448] (3) The antibodies of the present invention have excellent binding ability and affinity kinetics to target cells, which are significantly better than commercially available positive controls.

[0449] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which detailed conditions are not specified, were generally performed under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0450] Example 1 Screening of anti-DLL3 antibodies

[0451] DLL3-related antibodies were obtained by immunizing mice with antigen-specific humanized antibody-producing region gene fragments and then screening hybridomas. Candidate scFvs used the VL-(G4S)3-VH format, and their sequences are shown in Table 1. CDR sequences annotated using Kabat and Chothia are shown in Tables 2 and 3. The following experiments were performed using the scFv-human IgG Fc format.

[0452] The results of SDS-PAGE detection of anti-DLL3 scFv-Fc antibody expression and purification are shown in Figure 1. The results show that the antibody expression and purification results are good. The anti-DLL3 scFv from Amgen's patent was selected as Yangshen (AMG757).

[0453] The results of biolayer interferometry (BLI) assays of representative antibodies against human DLL3 protein are shown in Figure 2. All antibodies were analyzed by flow cytometry using two DLL3-positive cell lines, SHP77 and NCI-H82, and the results are shown in Figure 3. BLI data and EC50 data for all antibodies are summarized in Table 4.

[0454] Table 1 Anti-DLL3 antibody scFv sequences

[0455] Table 2 CDR sequences based on Kabat SEQ:SEQ ID NO.

[0456] Table 3 CDR sequences based on Chothia SEQ:SEQ ID NO.

[0457] Table 4 Summary of anti-DLL3 antibody kinetic data and EC50 results (SCFV-FC) *Ab.73 Since koff is lower than the detection limit of the instrument (1E-06 / s), it is expressed as less than the lower limit in the table.

[0458] Example 2 Anti-DLL3 CAR Structural Design

[0459] The inventors discovered that anti-DLL3 antibodies with high affinity for DLL3 may not necessarily produce the same CAR molecules as those with high cytotoxicity. Therefore, the inventors conducted extensive screening and, based on the antibodies obtained, designed and screened a large number of CAR molecules, ultimately identifying molecules with excellent cytotoxicity at the CAR level. Among them, the CARs corresponding to Ab.55, Ab.67, and Ab.76 demonstrated exceptional cytotoxicity across multiple rounds. The specific designs are as follows:

[0460] The scFv of the anti-DLL3 CAR structure is derived from some of the antibodies screened in Example 1 and is named according to the antibody name and the order of the light and heavy chains in the scFv.

[0461] The anti-DLL3 CAR structure is shown in Figure 4 and includes a signal peptide (SP), scFv, hinge region, transmembrane region (TM), costimulatory domain (CD), and CD3ζ domain. The signal peptide includes the following sequence (Table 5). The data of the present invention are based on CD8αSP, CD28hinge, CD28TM, and CD28CD structures. The scFv is composed of VL-Linker-VH or VH-Linker-VL, where the linker can be (G4S)3 or (G4S)4, etc.

[0462] Table 5 Signal peptide sequence

[0463] Example 3 Detection of DLL3 expression on tumor cell surface

[0464] Experimental methods: Flow cytometry was used to detect the expression of DLL3 on the surface of SHP77 and NCI-H82 tumor cells. The detection method is as follows.

[0465] Take 2E5-3E5 tumor cells to be tested, centrifuge at 300g, 4°C for 5 min, and discard the supernatant. 3.2 Add 200 μL FACS Buffer (1% FBS dissolved in PBS), centrifuge at 300g, 4°C for 5 min, and discard the supernatant.

[0466] Prepare primary antibody solution at a concentration of 1 μg / mL. Add 100 μL / well of primary antibody solution, mix thoroughly by pipetting, and incubate at 4°C for 30 min. After incubation, add 100 μL of FACS buffer, centrifuge at 300g, and 4°C for 5 min. Discard the supernatant.

[0467] Add 200 μL / well FACS Buffer, pipette to mix, centrifuge at 300g, 4°C for 5 min, and discard the supernatant. Repeat the above steps once. Add 100 μL / well secondary antibody solution, pipette to mix, and incubate at 4°C for 30 min. After incubation, add 100 μL FACS Buffer, centrifuge at 300g, 4°C for 5 min, and discard the supernatant. Add 200 μL / well FACS Buffer, pipette to mix, centrifuge at 300g, 4°C for 5 min, and discard the supernatant. Repeat the above steps once. Add 200 μL / well FACS Buffer, resuspend, and analyze using a flow cytometer.

[0468] The results showed that DLL3 protein was expressed on the surface of SHP77 and NCI-H82 cells ( FIG. 5 ).

[0469] Example 4 Virus packaging and NK92 cell infection

[0470] Experimental Methods: The retroviral packaging vectors were BaEV-TR and pCMV-gag-pol, and the vector carrying the CAR molecule was pMSCV. These vectors were designed by our laboratory and then delivered to GeneWeichi for synthesis and extraction. The viral packaging, cell infection, and sorting procedures are as follows:

[0471] HEK-293T cells in good condition were digested and resuspended in DMEM complete medium. The cells were seeded into 10 cm culture dishes at a concentration of 8E5 / mL (10 mL / dish). After culturing in an incubator for 16 hours, the cell density was observed. When the density reached approximately 90%, plasmid transfection was initiated. Take the centrifuge tube and add 500 μL of Opti-MEM TM I reduced serum medium (Gibco, 31985062), add 7.5 μg BaEV-TR, 10 μg pCMV-gag-pol and 20 μg corresponding CAR-expressing pMSCV vector to one centrifuge tube and mix evenly to obtain Opti-MEM-plasmid mixture; add 40 μL PEIpro solution (polyplus, 115-010) to another centrifuge tube and mix evenly to obtain Opti-MEM-PEI mixture.

[0472] Add the Opti-MEM®-plasmid mixture to the Opti-MEM®-PEI mixture and mix thoroughly by pipetting. Incubate at room temperature for 15 minutes to allow the transfection complex to form. After incubation, collect the cell culture supernatant. Concentrate the virus using a Lenti-X Concentrator (Takara, 631232).

[0473] Resuspend the virus in 100 μL of NK92 cell culture medium, remove 5 μL for titer determination, and store the remaining virus at 4°C. Titer determination was performed using K562 cells supplemented with 5 μg / mL polybrene (Sigma-Aldrich, TR-1003) during infection. The K562 positive rate was determined 48 hours after infection, and the viral titer was calculated.

[0474] 4E5 NK92 cells were plated in a 6-well plate and the virus concentrate was added at an MOI of 2. Polybrene (Sigma-Aldrich, TR-1003) was added to a final concentration of 5 μg / ml and mixed thoroughly. The cells were centrifuged at 800g for 1 hour at 32°C and cultured overnight. The NK92 cells were removed, centrifuged, resuspended in fresh NK92 culture medium, and cultured for another 4 days before flow cytometry analysis.

[0475] Mix the cultured DLL3-CAR NK92 cells thoroughly by pipetting and counting. Based on the count, transfer 1E7 cells to be sorted into a centrifuge tube and centrifuge at 300g for 5 minutes. Discard the supernatant. Resuspend the cells in 10mL of MACS buffer and centrifuge at 300g for 5 minutes. Discard the supernatant. Resuspend the cells in 2mL of 3ug / mL Biotinylated Huamn DLL3His, Avitag™ (Acro Biosystem, DL3-H82E4) diluted in PBS and incubate at 4°C for 30 minutes.

[0476] After incubation, wash and resuspend the cells, add 20 μL of Anti-Biotin MicroBeads (Miltenyi, 130-090-485), and mix thoroughly. Incubate at 4°C for 15 minutes. After incubation, wash and resuspend the cells. Wash the cells labeled with the magnetic beads by loading them onto an adsorption column LS (Miltenyi, 130-042-401) on a magnetic rack. Take 200 μL of the sorted cells and perform NC-200 counting.

[0477] Resuspend the sorted cells and incubate them at 37°C, 5% CO2 in a humidified incubator. Determine the CAR positivity rate 4 days after sorting. Centrifuge 1E5-2E5 cells at 700g for 2 minutes at 4°C and discard the supernatant. Wash, centrifuge, and discard the supernatant.

[0478] After mixing, add 100 μL of antigen protein suspension to each sample at a concentration of 2 μg / mL, mix thoroughly by pipetting, and incubate at 4°C for 30-60 minutes. After incubation, wash, centrifuge, and discard the supernatant.

[0479] Prepare the detection antibody streptavidin-PE (Biolegend, #405203) at a dilution ratio of 1:200. After mixing, add 100 μL of detection antibody to each sample, pipette to mix, and incubate at 4°C for 30 min-60 min.

[0480] After incubation, wash, centrifuge, and discard the supernatant. Add 200 μL / sample FACS Buffer to resuspend and analyze by flow cytometry.

[0481] Experimental results: The CAR detection results of the CAR-NK92 cell line after sorting are shown in Figure 6.

[0482] The results showed that after NK92 cells were infected with the virus and sorted by magnetic beads, the positive rates of anti-DLL3 CAR in all groups of NK92 cells reached more than 95%, except for the CAR55 positive rate of 94.7%.

[0483] Example 5 Detection of the killing ability of anti-DLL3 CAR NK92 on target cells

[0484] Experimental methods: The killing ability of anti-DLL3 CAR NK92 cells was tested using different methods.

[0485] 5.1 Single round damage

[0486] The luciferase reporter gene was used to detect the single-round killing ability of anti-DLL3 CAR NK92 against SHP77-Luciferase and NCI-H82-Luciferase target cells. The anti-CD19 CD19-CAR was used as a negative control. The specific operation steps are as follows:

[0487] IL-2 was withdrawn from the CAR NK92 effector cells used in the killing experiment 24 hours in advance.

[0488] Digest the target cells, neutralize them, pipette thoroughly to mix, count them, and then resuspend them in an appropriate volume of RPMI1640 complete medium. Transfer the target cells to a 96-well flat-bottom plate at 2E4 / well and 100μL / well. Count the effector cells by pipetting thoroughly to mix and count them. Take an appropriate number of effector cells, centrifuge at 300g for 5 minutes at room temperature, and resuspend them in an appropriate volume of RPMI1640 complete medium. Transfer the effector cells to a 96-well flat-bottom plate with target cells at 50μL / well and co-culture in a 37°C, 5% CO2 incubator for an appropriate time. Add 60μL ONE-Glo to each well and mix. After 3 minutes of reaction, mix thoroughly and take 130μL of the reaction solution to a 96-well white flat-bottom plate. Use a microplate reader to detect the luminescent signal intensity and then calculate the cytotoxicity.

[0489] The killing results recorded by the Luciferase reporter gene method are shown in Figure 7. It can be found that under this experimental condition, CAR55, CAR67, and CAR76 have a good killing effect on both target cells.

[0490] The killing results recorded by the Luciferase reporter gene method are shown in Figure 7. It can be found that under this experimental condition, CAR55, CAR67, and CAR76 have a good killing effect on both target cells.

[0491] 5.2 Multiple rounds of damage

[0492] The multi-round killing ability of anti-DLL3 CAR NK92 on SHP77-GFP and NCI-H82-GFP target cells was detected using a cell imaging multifunctional detection system (Cytation C7). The anti-CD19 CD19-CAR was used as a negative control. The specific operation steps are as follows:

[0493] IL-2 was withdrawn from the NK92 culture medium 24 h in advance.

[0494] Prepare a 96-well plate for imaging and coat it with poly-D-lysine. Digest and thoroughly disperse the target cells, then seed the plate at 2E4 / well (100 μL / well). Allow the cells to adhere at 37°C for 6 hours. Use a Cytation C7 to photograph the plate as a baseline. Slowly add 50 μL / well of effector cells according to the experimentally determined effector-target ratio (E:T). Place the plate in the Cytation C7 instrument and begin imaging after 30 minutes.

[0495] When the first round of killing is about to reach the target cell detection bottom line or the target cell reading is about to stop decreasing, start coating the 96-well plate used for the second round of killing and inoculate the target cells at a volume of 2E4 / well, 100 μL / well.

[0496] After the first round of killing reaches the target cell detection bottom line or the target cell reading no longer decreases, the solution in the wells of the first round of killing is thoroughly pipetted and mixed, and transferred to the coated 96-well plate used for the second round of killing. After 30 minutes, start taking pictures and recording.

[0497] Experimental results:

[0498] The single-round killing effect of anti-DLL3 CAR NK92 on SHP77 and NCI-H82 cells is shown in Figure 7, and the multiple-round killing effect is shown in Figures 8A and 8B. Under these experimental conditions, all groups of CAR NK92 had a good killing effect on target cells.

[0499] The results showed that at E:T=1:1, in the first round of killing (14 hours), CAR76 could exhibit a specific killing activity greater than 82% compared with Yangshen CAR757.

[0500] Based on the experiments of the present invention, CAR 76, CAR 55, and CAR 67 have excellent specific killing activity.

[0501] discuss:

[0502] In single-round and multi-round killing, CAR76 has better specific killing activity than Yangshen CAR757. However, surprisingly, the affinity (KD value) of the scFv antibody Ab76 corresponding to CAR76 is lower than that of Yangshen.

[0503] In addition, compared with Yangshen, Ab63 has a superior EC50 value against NCIH82, suggesting that it has excellent killing activity. In the single-round cell killing activity experiment, it has good CAR function short-term killing activity (Figure 7), but is inferior to Yangshen in terms of CAR function multi-round killing (Figures 8A, 8B).

[0504] Given the complex biological environment in which NK cells interact with target cells, the data on antibody target affinity (KD value) and EC50 for target cells are for reference only and do not clearly indicate that CAR NK cells constructed with high-affinity antibodies as elements will necessarily have potent tumor cell-killing properties.

[0505] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. An antibody or antigen-binding fragment thereof targeting DLL3, 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:37, HCDR2 shown in SEQ ID NO:38, and HCDR3 shown in SEQ ID NO:39; And the light chain variable region includes the following complementarity determining regions CDR: LCDR1 shown in SEQ ID NO:40, LCDR2 shown in SEQ ID NO:41, and LCDR3 shown in SEQ ID NO:42; or (2) The heavy chain variable region includes the following complementarity determining regions CDR: 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 (3) 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; or (4) The heavy chain variable region includes the following complementarity determining regions CDR: HCDR1 shown in SEQ ID NO:31, HCDR2 shown in SEQ ID NO:32, and HCDR3 shown in SEQ ID NO:33; And the light chain variable region includes the following complementarity determining regions CDR: LCDR1 shown in SEQ ID NO:34, LCDR2 shown in SEQ ID NO:35, and LCDR3 shown in SEQ ID NO:36; or (5) The heavy chain variable region includes the following complementarity determining regions (CDRs): HCDR1 shown in SEQ ID NO:25, HCDR2 shown in SEQ ID NO:26, and HCDR3 shown in SEQ ID NO:27; And the light chain variable region includes the following complementarity determining regions CDR: LCDR1 shown in SEQ ID NO:28, LCDR2 shown in SEQ ID NO:29, and LCDR3 shown in SEQ ID NO:30; or (6) The heavy chain variable region includes the following complementarity determining regions CDR: HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:2, and HCDR3 shown in SEQ ID NO:3; And the light chain variable region includes the following complementarity determining regions CDR: LCDR1 shown in SEQ ID NO:4, LCDR2 shown in SEQ ID NO:5, and LCDR3 shown in SEQ ID NO:6; or (7) The heavy chain variable region includes the following complementarity determining regions (CDRs): HCDR1 shown in SEQ ID NO:13, HCDR2 shown in SEQ ID NO:43, and HCDR3 shown in SEQ ID NO:33; And the light chain variable region includes the following complementarity determining regions CDR: LCDR1 shown in SEQ ID NO:44, LCDR2 shown in SEQ ID NO:35, and LCDR3 shown in SEQ ID NO:36; or (8) The heavy chain variable region includes the following complementarity determining regions (CDRs): HCDR1 shown in SEQ ID NO:13, HCDR2 shown in SEQ ID NO: 14, and HCDR3 shown in SEQ ID NO:15; And the light chain variable region includes the following complementarity determining regions CDR: LCDR1 shown in SEQ ID NO:16, LCDR2 shown in SEQ ID NO: 17, and LCDR3 shown in SEQ ID NO: 18; 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:56, HCDR2 shown in SEQ ID NO:57, and HCDR3 shown in SEQ ID NO:39; And the light chain variable region includes the following complementarity determining regions CDR: LCDR1 shown in SEQ ID NO:40, LCDR2 shown in SEQ ID NO:41, and LCDR3 shown in SEQ ID NO:42; or (2) The heavy chain variable region includes the following complementarity determining regions CDR: HCDR1 shown in SEQ ID NO:47, HCDR2 shown in SEQ ID NO:48, 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 (3) The heavy chain variable region includes the following complementarity determining regions CDR: HCDR1 shown in SEQ ID NO:50, HCDR2 shown in SEQ ID NO:51, 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; or (4) The heavy chain variable region includes the following complementarity determining regions CDR: HCDR1 shown in SEQ ID NO:54, HCDR2 shown in SEQ ID NO:55, and HCDR3 shown in SEQ ID NO:33; And the light chain variable region includes the following complementarity determining regions CDR: LCDR1 shown in SEQ ID NO:34, LCDR2 shown in SEQ ID NO:35, and LCDR3 shown in SEQ ID NO:36; or (5) The heavy chain variable region includes the following complementarity determining regions (CDRs): HCDR1 shown in SEQ ID NO:52, HCDR2 shown in SEQ ID NO:53, and HCDR3 shown in SEQ ID NO:27; And the light chain variable region includes the following complementarity determining regions CDR: LCDR1 shown in SEQ ID NO:28, LCDR2 shown in SEQ ID NO:29, and LCDR3 shown in SEQ ID NO:30; or (6) The heavy chain variable region includes the following complementarity determining regions CDR: HCDR1 shown in SEQ ID NO:45, HCDR2 shown in SEQ ID NO:46, and HCDR3 shown in SEQ ID NO:3; And the light chain variable region includes the following complementarity determining regions CDR: LCDR1 shown in SEQ ID NO:4, LCDR2 shown in SEQ ID NO:5, and LCDR3 shown in SEQ ID NO:6; or (7) The heavy chain variable region includes the following complementarity determining regions (CDRs): HCDR1 shown in SEQ ID NO:58, HCDR2 shown in SEQ ID NO:48, and HCDR3 shown in SEQ ID NO:33; And the light chain variable region includes the following complementarity determining regions CDR: LCDR1 shown in SEQ ID NO:44, LCDR2 shown in SEQ ID NO:35, and LCDR3 shown in SEQ ID NO:36; or (8) The heavy chain variable region includes the following complementarity determining regions (CDRs): HCDR1 shown in SEQ ID NO:49, HCDR2 shown in SEQ ID NO:48, and HCDR3 shown in SEQ ID NO:15; And the light chain variable region includes the following complementarity determining regions CDR: LCDR1 shown in SEQ ID NO:16, LCDR2 shown in SEQ ID NO: 17, and LCDR3 shown in SEQ ID NO: 18; 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) optionally a tag sequence to facilitate expression and / or purification.

3. [Corrected 23.07.2024 in accordance with Rule 26] 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 DLL3, 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:37, HCDR2 shown in SEQ ID NO:38, and HCDR3 shown in SEQ ID NO:39; And the light chain variable region includes the following complementarity determining regions CDR: LCDR1 shown in SEQ ID NO:40, LCDR2 shown in SEQ ID NO:41, and LCDR3 shown in SEQ ID NO:42; or (2) The heavy chain variable region includes the following complementarity determining regions CDR: 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 (3) 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; or (4) The heavy chain variable region includes the following complementarity determining regions CDR: HCDR1 shown in SEQ ID NO:31, HCDR2 shown in SEQ ID NO:32, and HCDR3 shown in SEQ ID NO:33; And the light chain variable region includes the following complementarity determining regions CDR: LCDR1 shown in SEQ ID NO:34, LCDR2 shown in SEQ ID NO:35, and LCDR3 shown in SEQ ID NO:36; or (5) The heavy chain variable region includes the following complementarity determining regions (CDRs): HCDR1 shown in SEQ ID NO:25, HCDR2 shown in SEQ ID NO:26, and HCDR3 shown in SEQ ID NO:27; And the light chain variable region includes the following complementarity determining regions CDR: LCDR1 shown in SEQ ID NO:28, LCDR2 shown in SEQ ID NO:29, and LCDR3 shown in SEQ ID NO:30; or (6) The heavy chain variable region includes the following complementarity determining regions CDR: HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:2, and HCDR3 shown in SEQ ID NO:3; And the light chain variable region includes the following complementarity determining regions CDR: LCDR1 shown in SEQ ID NO:4, LCDR2 shown in SEQ ID NO:5, and LCDR3 shown in SEQ ID NO:6; or (7) The heavy chain variable region includes the following complementarity determining regions (CDRs): HCDR1 shown in SEQ ID NO:13, HCDR2 shown in SEQ ID NO:43, and HCDR3 shown in SEQ ID NO:33; And the light chain variable region includes the following complementarity determining regions CDR: LCDR1 shown in SEQ ID NO:44, LCDR2 shown in SEQ ID NO:35, and LCDR3 shown in SEQ ID NO:36; or (8) The heavy chain variable region includes the following complementarity determining regions (CDRs): HCDR1 shown in SEQ ID NO:13, HCDR2 shown in SEQ ID NO: 14, and HCDR3 shown in SEQ ID NO:15; And the light chain variable region includes the following complementarity determining regions CDR: LCDR1 shown in SEQ ID NO:16, LCDR2 shown in SEQ ID NO: 17, and LCDR3 shown in SEQ ID NO: 18; 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:56, HCDR2 shown in SEQ ID NO:57, and HCDR3 shown in SEQ ID NO:39; And the light chain variable region includes the following complementarity determining regions CDR: LCDR1 shown in SEQ ID NO:40, LCDR2 shown in SEQ ID NO:41, and LCDR3 shown in SEQ ID NO:42; or (2) The heavy chain variable region includes the following complementarity determining regions CDR: HCDR1 shown in SEQ ID NO:47, HCDR2 shown in SEQ ID NO:48, 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 (3) The heavy chain variable region includes the following complementarity determining regions CDR: HCDR1 shown in SEQ ID NO:50, HCDR2 shown in SEQ ID NO:51, 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; or (4) The heavy chain variable region includes the following complementarity determining regions CDR: HCDR1 shown in SEQ ID NO:54, HCDR2 shown in SEQ ID NO:55, and HCDR3 shown in SEQ ID NO:33; And the light chain variable region includes the following complementarity determining regions CDR: LCDR1 shown in SEQ ID NO:34, LCDR2 shown in SEQ ID NO:35, and LCDR3 shown in SEQ ID NO:36; or (5) The heavy chain variable region includes the following complementarity determining regions (CDRs): HCDR1 shown in SEQ ID NO:52, HCDR2 shown in SEQ ID NO:53, and HCDR3 shown in SEQ ID NO:27; And the light chain variable region includes the following complementarity determining regions CDR: LCDR1 shown in SEQ ID NO:28, LCDR2 shown in SEQ ID NO:29, and LCDR3 shown in SEQ ID NO:30; or (6) The heavy chain variable region includes the following complementarity determining regions CDR: HCDR1 shown in SEQ ID NO:45, HCDR2 shown in SEQ ID NO:46, and HCDR3 shown in SEQ ID NO:3; And the light chain variable region includes the following complementarity determining regions CDR: LCDR1 shown in SEQ ID NO:4, LCDR2 shown in SEQ ID NO:5, and LCDR3 shown in SEQ ID NO:6; or (7) The heavy chain variable region includes the following complementarity determining regions (CDRs): HCDR1 shown in SEQ ID NO:58, HCDR2 shown in SEQ ID NO:48, and HCDR3 shown in SEQ ID NO:33; And the light chain variable region includes the following complementarity determining regions CDR: LCDR1 shown in SEQ ID NO:44, LCDR2 shown in SEQ ID NO:35, and LCDR3 shown in SEQ ID NO:36; or (8) The heavy chain variable region includes the following complementarity determining regions (CDRs): HCDR1 shown in SEQ ID NO:49, HCDR2 shown in SEQ ID NO:48, and HCDR3 shown in SEQ ID NO:15; And the light chain variable region includes the following complementarity determining regions CDR: LCDR1 shown in SEQ ID NO:16, LCDR2 shown in SEQ ID NO: 17, and LCDR3 shown in SEQ ID NO: 18; Wherein, the CDR sequence is based on the Chothia numbering scheme.

4. [Corrected 23.07.2024 in accordance with Rule 26] 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, comprising the vector according to claim 5 or a chromosome in 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 of the present invention or the vector according to claim 5 of the present invention 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, a radionuclide, an enzyme, or a combination thereof.

10. 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, (a) preparing a detection reagent or a kit; and / or (b) preparing a drug or preparation for preventing and / or treating a DLL3-related disease.

11. The use according to claim 10, characterized in that 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.

12. A method for detecting DLL3 protein in a sample in vitro (including diagnostic or non-diagnostic), 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 DLL3 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; (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 (support plate) 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 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 / or (2) a second container, wherein the second container contains a secondary antibody of the antibody of claim 1; Alternatively, the kit contains the detection plate described in claim 14.

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