Antibodies targeting dll3 and uses thereof

Abstract

An antibody targeting DLL3 and its application. The DLL3 antibody shows excellent specificity and binding capacity, and the chimeric antigen receptor derived therefrom shows excellent killing capacity to tumor cells, and has less cytotoxicity and low side effects.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of antibodies. Specifically, it relates to an antibody targeting DLL3 and its use. BACKGROUND

[0002] DLL3 protein, also known as Delta-like protein 3, is a type I transmembrane protein encoded by DLL3 gene. DLL3 protein contains 618 amino acid residues, which can be divided into extracellular Delta / Serrate / Lag-2 (DSL) domain, 6 EGF-like domains, transmembrane segment and intracellular segment.

[0003] During development, DLL3 protein is mainly expressed in presomitic mesoderm in early embryonic development. Its mutation can cause Spondylocostal dysostosis in both humans and mice. In adult tissues, the mRNA of DLL3 can be detected in brain, endocrine tissue and testis, but the protein is difficult to detect, suggesting that DLL3 protein has good safety as a CAR-NK treatment target. At the cellular level, DLL3 protein is mainly located in the Golgi apparatus, not on the cell membrane surface. When the DLL3 of the cell is abnormally overexpressed, DLL3 protein can be detected on the cell membrane surface, 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 the determination of neural endocrine cell fate.

[0004] DLL3 protein is highly expressed in various 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 glioma and Merkel cell carcinoma, etc. In small cell lung cancer and endometrial cancer, high expression of DLL3 protein is associated with shorter overall survival of patients. In terms of cancer mechanism, DLL3, as a ligand of Notch signaling pathway, mainly down-regulates the Notch receptor protein of DLL3 highly expressed cells through cis-inhibition, and inhibits the Notch signaling pathway. Knocking down DLL3 in gastrointestinal neuroendocrine cancer cell lines can inhibit cell proliferation.

[0005] In summary, DLL3 is a target that is widely expressed in neuroendocrine type cancer and has good safety. Targeting DLL3 protein using antibodies, antibody conjugated drugs and CAR-NK has broad application prospects. SUMMARY

[0006] An object of the present application is to provide an antibody targeting DLL3 and use thereof.

[0007] In a first aspect of the present application, there is provided an antibody or antigen-binding fragment thereof targeting DLL3, comprising a heavy chain variable region and a light chain variable region, which comprise complementarity determining regions (CDRs) selected from the group consisting of:

[0008] (1) the heavy chain variable region comprises the following complementarity determining regions (CDRs): (Ab.76)

[0009] HCDR1 as set forth in SEQ ID NO: 37,

[0010] HCDR2 as set forth in SEQ ID NO: 38, and

[0011] HCDR3 as set forth in SEQ ID NO: 39;

[0012] and the light chain variable region comprises the following complementarity determining regions (CDRs):

[0013] LCDR1 as set forth in SEQ ID NO: 40,

[0014] LCDR2 as set forth in SEQ ID NO: 41, and

[0015] LCDR3 as set forth in SEQ ID NO: 42; or

[0016] (2) the heavy chain variable region comprises the following complementarity determining regions (CDRs): (Ab.55)

[0017] HCDR1 as set forth in SEQ ID NO: 7,

[0018] HCDR2 as set forth in SEQ ID NO: 8, and

[0019] HCDR3 as set forth in SEQ ID NO: 9;

[0020] and the light chain variable region comprises the following complementarity determining regions (CDRs):

[0021] LCDR1 as set forth in SEQ ID NO: 10,

[0022] LCDR2 as set forth in SEQ ID NO: 11, and

[0023] LCDR3 as set forth in SEQ ID NO: 12; or

[0024] (3) the heavy chain variable region comprises the following complementarity determining regions (CDRs): (Ab.67)

[0025] HCDR1 set out in SEQ ID NO: 19,

[0026] HCDR2 set out in SEQ ID NO: 20, and

[0027] HCDR3 set out in SEQ ID NO: 21 ;

[0028] and the light chain variable region comprises the complementarity determining regions CDRs:

[0029] LCDR1 set out in SEQ ID NO: 22,

[0030] LCDR2 set out in SEQ ID NO: 23, and

[0031] LCDR3 set out in SEQ ID NO: 24; or

[0032] (4) the heavy chain variable region comprises the complementarity determining regions CDRs: (Ab.73)

[0033] HCDR1 set out in SEQ ID NO: 31,

[0034] HCDR2 set out in SEQ ID NO: 32, and

[0035] HCDR3 set out in SEQ ID NO: 33;

[0036] and the light chain variable region comprises the complementarity determining regions CDRs:

[0037] LCDR1 set out in SEQ ID NO: 34,

[0038] LCDR2 set out in SEQ ID NO: 35, and

[0039] LCDR3 set out in SEQ ID NO: 36; or

[0040] (5) the heavy chain variable region comprises the complementarity determining regions CDRs: (Ab.71)

[0041] HCDR1 set out in SEQ ID NO: 25,

[0042] HCDR2 set out in SEQ ID NO: 26, and

[0043] HCDR3 set out in SEQ ID NO: 27;

[0044] and the light chain variable region comprises the complementarity determining regions CDRs:

[0045] LCDR1 set forth in SEQ ID NO: 28,

[0046] LCDR2 set forth in SEQ ID NO: 29, and

[0047] LCDR3 set forth in SEQ ID NO: 30; or

[0048] (6) the heavy chain variable region comprises the complementarity determining regions CDRs: (Ab.53)

[0049] HCDR1 set forth in SEQ ID NO: 1,

[0050] HCDR2 set forth in SEQ ID NO: 2, and

[0051] HCDR3 set forth in SEQ ID NO: 3;

[0052] and the light chain variable region comprises the complementarity determining regions CDRs:

[0053] LCDR1 set forth in SEQ ID NO: 4,

[0054] LCDR2 set forth in SEQ ID NO: 5, and

[0055] LCDR3 set forth in SEQ ID NO: 6; or

[0056] (7) the heavy chain variable region comprises the complementarity determining regions CDRs: (Ab.79)

[0057] HCDR1 set forth in SEQ ID NO: 13,

[0058] HCDR2 set forth in SEQ ID NO: 43, and

[0059] HCDR3 set forth in SEQ ID NO: 33;

[0060] and the light chain variable region comprises the complementarity determining regions CDRs:

[0061] LCDR1 set forth in SEQ ID NO: 44,

[0062] LCDR2 set forth in SEQ ID NO: 35, and

[0063] LCDR3 set forth in SEQ ID NO: 36; or

[0064] (8) the heavy chain variable region comprises the complementarity determining regions CDRs: (Ab.63)

[0065] HCDR1 set forth in SEQ ID NO: 13,

[0066] HCDR2 of SEQ ID NO: 14, and

[0067] HCDR3 of SEQ ID NO: 15;

[0068] and the light chain variable region comprises the complementarity determining regions (CDRs) of:

[0069] LCDR1 of SEQ ID NO: 16,

[0070] LCDR2 of SEQ ID NO: 17, and

[0071] LCDR3 of SEQ ID NO: 18;

[0072] wherein the CDR sequences are numbered according to the Kabat numbering scheme.

[0073] In another preferred embodiment, the heavy chain variable region and the light chain variable region of the antibody or antigen-binding fragment thereof comprise the complementarity determining regions (CDRs) selected from the group consisting of:

[0074] (1) the heavy chain variable region comprises the complementarity determining regions (CDRs) of: (Ab.76)

[0075] HCDR1 of SEQ ID NO: 56,

[0076] HCDR2 of SEQ ID NO: 57, and

[0077] HCDR3 of SEQ ID NO: 39;

[0078] and the light chain variable region comprises the complementarity determining regions (CDRs) of:

[0079] LCDR1 of SEQ ID NO: 40,

[0080] LCDR2 of SEQ ID NO: 41, and

[0081] LCDR3 of SEQ ID NO: 42; or

[0082] (2) the heavy chain variable region comprises the complementarity determining regions (CDRs) of: (Ab.55)

[0083] HCDR1 of SEQ ID NO: 47,

[0084] HCDR2 of SEQ ID NO: 48, and

[0085] HCDR3 of SEQ ID NO: 9;

[0086] and the light chain variable region comprises the complementarity determining regions CDRs:

[0087] LCDR1 set forth in SEQ ID NO: 10,

[0088] LCDR2 set forth in SEQ ID NO: 11, and

[0089] LCDR3 set forth in SEQ ID NO: 12; or

[0090] (3) the heavy chain variable region comprises the complementarity determining regions CDRs: (Ab.67)

[0091] HCDR1 set forth in SEQ ID NO: 50,

[0092] HCDR2 set forth in SEQ ID NO: 51, and

[0093] HCDR3 set forth in SEQ ID NO: 21;

[0094] and the light chain variable region comprises the complementarity determining regions CDRs:

[0095] LCDR1 set forth in SEQ ID NO: 22,

[0096] LCDR2 set forth in SEQ ID NO: 23, and

[0097] LCDR3 set forth in SEQ ID NO: 24; or

[0098] (4) the heavy chain variable region comprises the complementarity determining regions CDRs: (Ab.73)

[0099] HCDR1 set forth in SEQ ID NO: 54,

[0100] HCDR2 set forth in SEQ ID NO: 55, and

[0101] HCDR3 set forth in SEQ ID NO: 33;

[0102] and the light chain variable region comprises the complementarity determining regions CDRs:

[0103] LCDR1 set forth in SEQ ID NO: 34,

[0104] LCDR2 set forth in SEQ ID NO: 35, and

[0105] LCDR3 set forth in SEQ ID NO: 36; or

[0106] (5) the heavy chain variable region comprises the following complementarity determining regions CDRs: (Ab.71)

[0107] HCDR1 set forth in SEQ ID NO: 52,

[0108] HCDR2 set forth in SEQ ID NO: 53, and

[0109] HCDR3 set forth in SEQ ID NO: 27;

[0110] and the light chain variable region comprises the following complementarity determining regions CDRs:

[0111] LCDR1 set forth in SEQ ID NO: 28,

[0112] LCDR2 set forth in SEQ ID NO: 29, and

[0113] LCDR3 set forth in SEQ ID NO: 30; or

[0114] (6) the heavy chain variable region comprises the following complementarity determining regions CDRs: (Ab.53)

[0115] HCDR1 set forth in SEQ ID NO: 45,

[0116] HCDR2 set forth in SEQ ID NO: 46, and

[0117] HCDR3 set forth in SEQ ID NO: 3;

[0118] and the light chain variable region comprises the following complementarity determining regions CDRs:

[0119] LCDR1 set forth in SEQ ID NO: 4,

[0120] LCDR2 set forth in SEQ ID NO: 5, and

[0121] LCDR3 set forth in SEQ ID NO: 6; or

[0122] (7) the heavy chain variable region comprises the following complementarity determining regions CDRs: (Ab.79)

[0123] HCDR1 set forth in SEQ ID NO: 58,

[0124] HCDR2 set forth in SEQ ID NO: 48, and

[0125] HCDR3 set forth in SEQ ID NO: 33;

[0126] and the light chain variable region comprises the following complementarity determining regions CDRs:

[0127] LCDR1 as set forth in SEQ ID NO:44,

[0128] LCDR2 as set forth in SEQ ID NO:35, and

[0129] LCDR3 as set forth in SEQ ID NO:36; or

[0130] (8) the heavy chain variable region comprises the following complementarity determining regions CDRs: (Ab.63)

[0131] HCDR1 as set forth in SEQ ID NO:49,

[0132] HCDR2 as set forth in SEQ ID NO:48, and

[0133] HCDR3 as set forth in SEQ ID NO: 15;

[0134] and the light chain variable region comprises the following complementarity determining regions CDRs:

[0135] LCDR1 as set forth in SEQ ID NO: 16,

[0136] LCDR2 as set forth in SEQ ID NO: 17, and

[0137] LCDR3 as set forth in SEQ ID NO: 18;

[0138] wherein the CDR sequences are based on the numbering scheme of Chothia.

[0139] In another preferred embodiment, the antibody comprises a heavy chain comprising the three heavy chain CDRs and a heavy chain framework region for connecting the heavy chain CDRs; and a light chain comprising the three light chain CDRs and a light chain framework region for connecting the light chain CDRs.

[0140] In another preferred embodiment, the antibody or antigen-binding fragment thereof that targets DLL3 is selected from the group consisting of a camel Ig, IgNAR, Fab fragment, Fab' fragment, F(ab)'2 fragment, F(ab)'3 fragment, Fv, single chain Fv antibody ("scFv"), bis-scFv, (scFv)2, minibody, diabody, triabody, tetrabody, disulfide stabilized Fv protein ("dsFv"), and single domain antibody (sdAb, nanobody).

[0141] In another preferred embodiment, the light chain of the antibody further comprises a light chain constant region.

[0142] In another preferred embodiment, the light chain constant region is of human, murine, or rabbit origin, preferably human origin.

[0143] In another preferred embodiment, the heavy chain of the antibody further comprises a heavy chain constant region.

[0144] In another preferred embodiment, the heavy chain constant region is of human, murine or rabbit origin, preferably human origin.

[0145] In another preferred embodiment, the antibody is a diabody, or a single chain antibody.

[0146] In another preferred embodiment, the antibody is a monoclonal antibody.

[0147] In another preferred embodiment, the antibody is a monospecific, bispecific, trispecific or multispecific antibody.

[0148] In another preferred embodiment, the antibody specifically binds to DLL3.

[0149] In another preferred embodiment, the antibody has a KD value (M) of 1.0E-12 to 1.0E-8 for the affinity to human DLL3.

[0150] 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, 66.

[0151] In another preferred embodiment, the single chain antibody has an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity (wherein the CDRs are unchanged or substantially unchanged) to the amino acid sequence set forth in SEQ ID NO. 59, 60, 61, 62, 63, 64, 65, 66.

[0152] In a second aspect of the present application, there is provided a recombinant protein, said recombinant protein having:

[0153] (i) an antibody or antigen binding fragment thereof as described in the first aspect of the present application; and

[0154] (ii) optionally, a tag sequence to assist expression and / or purification.

[0155] In another preferred embodiment, the tag sequence comprises a 6His tag.

[0156] In another preferred embodiment, the recombinant protein (or polypeptide) comprises a fusion protein.

[0157] In another preferred embodiment, the recombinant protein is a monomer, dimer, or multimer.

[0158] In another preferred embodiment, the recombinant protein further comprises an additional fusion element (or fusion polypeptide fragment) fused to the element (i).

[0159] In a third aspect, the present application provides a chimeric antigen receptor (CAR) comprising an antigen binding domain comprising a single chain variable region sequence (scFv) of an antibody targeting DLL3, wherein the heavy chain variable region and the light chain variable region of the scFv comprise complementarity determining regions (CDRs) selected from the group consisting of:

[0160] (1) the heavy chain variable region comprises the following complementarity determining regions (CDRs): (Ab.76)

[0161] HCDR1 set forth in SEQ ID NO: 37,

[0162] HCDR2 set forth in SEQ ID NO: 38, and

[0163] HCDR3 set forth in SEQ ID NO: 39;

[0164] and the light chain variable region comprises the following complementarity determining regions (CDRs):

[0165] LCDR1 set forth in SEQ ID NO: 40,

[0166] LCDR2 set forth in SEQ ID NO: 41, and

[0167] LCDR3 set forth in SEQ ID NO: 42; or

[0168] (2) the heavy chain variable region comprises the following complementarity determining regions (CDRs): (Ab.55)

[0169] HCDR1 set forth in SEQ ID NO: 7,

[0170] HCDR2 set forth in SEQ ID NO: 8, and

[0171] HCDR3 set forth in SEQ ID NO: 9;

[0172] and the light chain variable region comprises the following complementarity determining regions (CDRs):

[0173] LCDR1 set forth in SEQ ID NO: 10,

[0174] LCDR2 set forth in SEQ ID NO: 11, and

[0175] LCDR3 set forth in SEQ ID NO: 12; or

[0176] (3) the heavy chain variable region comprises the following complementarity determining regions (CDRs): (Ab.67)

[0177] HCDR1 set forth in SEQ ID NO: 19,

[0178] HCDR2 set forth in SEQ ID NO: 20, and

[0179] HCDR3 set forth in SEQ ID NO: 21 ;

[0180] and the light chain variable region comprises the complementarity determining regions CDRs:

[0181] LCDR1 set forth in SEQ ID NO: 22,

[0182] LCDR2 set forth in SEQ ID NO: 23, and

[0183] LCDR3 set forth in SEQ ID NO: 24; or

[0184] (4) the heavy chain variable region comprises the complementarity determining regions CDRs: (Ab.73)

[0185] HCDR1 set forth in SEQ ID NO: 31,

[0186] HCDR2 set forth in SEQ ID NO: 32, and

[0187] HCDR3 set forth in SEQ ID NO: 33;

[0188] and the light chain variable region comprises the complementarity determining regions CDRs:

[0189] LCDR1 set forth in SEQ ID NO: 34,

[0190] LCDR2 set forth in SEQ ID NO: 35, and

[0191] LCDR3 set forth in SEQ ID NO: 36; or

[0192] (5) the heavy chain variable region comprises the complementarity determining regions CDRs: (Ab.71)

[0193] HCDR1 set forth in SEQ ID NO: 25,

[0194] HCDR2 set forth in SEQ ID NO: 26, and

[0195] HCDR3 set forth in SEQ ID NO: 27;

[0196] and the light chain variable region comprises the complementarity determining regions CDRs:

[0197] LCDR1 shown in SEQ ID NO: 28,

[0198] LCDR2 shown in SEQ ID NO: 29, and

[0199] LCDR3 shown in SEQ ID NO:30; or

[0200] (6) The heavy chain variable region includes the following complementarity determining regions (CDRs): (Ab.53)

[0201] HCDR1 shown in SEQ ID NO: 1,

[0202] HCDR2 shown in SEQ ID NO: 2, and

[0203] HCDR3 shown in SEQ ID NO: 3;

[0204] And the light chain variable region includes the following complementarity determining regions CDR:

[0205] LCDR1 shown in SEQ ID NO:4,

[0206] LCDR2 shown in SEQ ID NO:5, and

[0207] LCDR3 shown in SEQ ID NO: 6; or

[0208] (7) The heavy chain variable region includes the following complementarity determining regions (CDRs): (Ab.79)

[0209] HCDR1 shown in SEQ ID NO: 13,

[0210] HCDR2 shown in SEQ ID NO:43, and

[0211] HCDR3 shown in SEQ ID NO: 33;

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

[0213] LCDR1 shown in SEQ ID NO:44,

[0214] LCDR2 shown in SEQ ID NO: 35, and

[0215] LCDR3 shown in SEQ ID NO:36; or

[0216] (8) The heavy chain variable region includes the following complementarity determining regions (CDRs): (Ab.63)

[0217] HCDR1 shown in SEQ ID NO: 13,

[0218] HCDR2 of SEQ ID NO: 14, and

[0219] HCDR3 of SEQ ID NO: 15;

[0220] and the light chain variable region comprises the complementarity determining regions (CDRs) of:

[0221] LCDR1 of SEQ ID NO: 16,

[0222] LCDR2 of SEQ ID NO: 17, and

[0223] LCDR3 of SEQ ID NO: 18.

[0224] wherein the CDR sequences are numbered according to the Kabat numbering scheme.

[0225] In another preferred embodiment, the heavy chain variable region and the light chain variable region of the scFv comprise the complementarity determining regions (CDRs) selected from the group consisting of:

[0226] (1) the heavy chain variable region comprises the complementarity determining regions (CDRs) of: (Ab. 76)

[0227] HCDR1 of SEQ ID NO: 56,

[0228] HCDR2 of SEQ ID NO: 57, and

[0229] HCDR3 of SEQ ID NO: 39;

[0230] and the light chain variable region comprises the complementarity determining regions (CDRs) of:

[0231] LCDR1 of SEQ ID NO: 40,

[0232] LCDR2 of SEQ ID NO: 41, and

[0233] LCDR3 of SEQ ID NO: 42; or

[0234] (2) the heavy chain variable region comprises the complementarity determining regions (CDRs) of: (Ab. 55)

[0235] HCDR1 of SEQ ID NO: 47,

[0236] HCDR2 of SEQ ID NO: 48, and

[0237] HCDR3 of SEQ ID NO: 9;

[0238] and the light chain variable region comprises the complementarity determining regions CDRs:

[0239] LCDR1 set forth in SEQ ID NO: 10,

[0240] LCDR2 set forth in SEQ ID NO: 11, and

[0241] LCDR3 set forth in SEQ ID NO: 12; or

[0242] (3) the heavy chain variable region comprises the complementarity determining regions CDRs: (Ab.67)

[0243] HCDR1 set forth in SEQ ID NO: 50,

[0244] HCDR2 set forth in SEQ ID NO: 51, and

[0245] HCDR3 set forth in SEQ ID NO: 21 ;

[0246] and the light chain variable region comprises the complementarity determining regions CDRs:

[0247] LCDR1 set forth in SEQ ID NO: 22,

[0248] LCDR2 set forth in SEQ ID NO: 23, and

[0249] LCDR3 set forth in SEQ ID NO: 24; or

[0250] (4) the heavy chain variable region comprises the complementarity determining regions CDRs: (Ab.73)

[0251] HCDR1 set forth in SEQ ID NO: 54,

[0252] HCDR2 set forth in SEQ ID NO: 55, and

[0253] HCDR3 set forth in SEQ ID NO: 33;

[0254] and the light chain variable region comprises the complementarity determining regions CDRs:

[0255] LCDR1 set forth in SEQ ID NO: 34,

[0256] LCDR2 set forth in SEQ ID NO: 35, and

[0257] LCDR3 set forth in SEQ ID NO: 36; or

[0258] (5) the heavy chain variable region comprises the complementarity determining regions CDRs: (Ab.71)

[0259] HCDR1 set forth in SEQ ID NO: 52,

[0260] HCDR2 set forth in SEQ ID NO: 53, and

[0261] HCDR3 set forth in SEQ ID NO: 27;

[0262] and the light chain variable region comprises the complementarity determining regions CDRs:

[0263] LCDR1 set forth in SEQ ID NO: 28,

[0264] LCDR2 set forth in SEQ ID NO: 29, and

[0265] LCDR3 set forth in SEQ ID NO: 30; or

[0266] (6) the heavy chain variable region comprises the complementarity determining regions CDRs: (Ab.53)

[0267] HCDR1 set forth in SEQ ID NO: 45,

[0268] HCDR2 set forth in SEQ ID NO: 46, and

[0269] HCDR3 set forth in SEQ ID NO: 3;

[0270] and the light chain variable region comprises the complementarity determining regions CDRs:

[0271] LCDR1 set forth in SEQ ID NO: 4,

[0272] LCDR2 set forth in SEQ ID NO: 5, and

[0273] LCDR3 set forth in SEQ ID NO: 6; or

[0274] (7) the heavy chain variable region comprises the complementarity determining regions CDRs: (Ab.79)

[0275] HCDR1 set forth in SEQ ID NO: 58,

[0276] HCDR2 set forth in SEQ ID NO: 48, and

[0277] HCDR3 set forth in SEQ ID NO: 33;

[0278] and the light chain variable region comprises the complementarity determining regions CDRs:

[0279] LCDR1 as set forth in SEQ ID NO: 44,

[0280] LCDR2 as set forth in SEQ ID NO: 35, and

[0281] LCDR3 as set forth in SEQ ID NO: 36; or

[0282] (8) the heavy chain variable region comprises the following complementarity determining regions CDRs: (Ab.63)

[0283] HCDR1 as set forth in SEQ ID NO: 49,

[0284] HCDR2 as set forth in SEQ ID NO: 48, and

[0285] HCDR3 as set forth in SEQ ID NO: 15;

[0286] and the light chain variable region comprises the following complementarity determining regions CDRs:

[0287] LCDR1 as set forth in SEQ ID NO: 16,

[0288] LCDR2 as set forth in SEQ ID NO: 17, and

[0289] LCDR3 as set forth in SEQ ID NO: 18;

[0290] wherein the CDR sequences are based on the numbering scheme of Chothia.

[0291] In another preferred embodiment, the scFv further comprises a linker peptide between the heavy chain variable region and the light chain variable region.

[0292] In another preferred embodiment, the linker peptide is (G4S)3 or (G4S)4.

[0293] In another preferred embodiment, the scFv is represented by Formula A or Formula B:

[0294] VH-VL, (A); VL-VH, (B)

[0295] wherein VH is the antibody heavy chain variable region; VL is the antibody light chain variable region; and “-” is a linker peptide or a peptide bond.

[0296] In another preferred embodiment, the linker peptide is (G4S)n, preferably n is 3-5; more preferably n is 3.

[0297] In another preferred embodiment, the scFv has an amino acid sequence selected from the group consisting of:

[0298] SEQ ID NO. 59, 60, 61, 62, 63, 64, 65, 66.

[0299] In another preferred embodiment, the antibody single chain variable region comprises a human-derived, murine-derived, human-murine chimeric antibody single chain variable region.

[0300] In another preferred embodiment, the scFv is as shown in Formula A (VH-VL).

[0301] In another preferred embodiment, the antigen binding domain targets an extracellular region of DLL3.

[0302] In another preferred embodiment, the chimeric antigen receptor has the following Formula I structure:

[0303] L-scFv-H-TM-C-CD3ζ (I)

[0304] wherein,

[0305] each “-” is independently a linking peptide or a peptide bond;

[0306] L is nothing or a signal peptide sequence;

[0307] scFv is a scFv targeting DLL3;

[0308] H is an optional hinge region;

[0309] TM is a transmembrane domain;

[0310] C is a costimulatory signaling molecule;

[0311] CD3ζ is a CD3ζ cytoplasmic signaling sequence.

[0312] In another preferred embodiment, the L is a signal peptide selected from the group of proteins consisting of 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 as shown in Formula A or Formula B:

[0315] VH-VL, (A); VL-VH, (B)

[0316] wherein VH is the antibody heavy chain variable region; VL is the antibody light chain variable region; and “-” is a linking peptide or a peptide bond.

[0317] In another preferred embodiment, the scFv is as shown in Formula A (VH-VL).

[0318] In another preferred embodiment, H is a hinge region of a protein selected from the group consisting of CD8, CD28, CD137, Fc, or a combination thereof.

[0319] In another preferred embodiment, H is a CD28-derived hinge region.

[0320] In another preferred embodiment, 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.

[0321] In another preferred embodiment, TM is a CD28-derived transmembrane region.

[0322] In another preferred embodiment, C is a costimulatory signaling molecule of a protein selected from the group consisting of 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.

[0323] In another preferred embodiment, C is a CD28-derived costimulatory signaling molecule.

[0324] In a fourth aspect, the present application provides a polynucleotide encoding the antibody or antigen binding fragment thereof of the first aspect, the recombinant protein of the second aspect, or the chimeric antigen receptor CAR of the third aspect of the present application.

[0325] In another preferred embodiment, the polynucleotide is isolated.

[0326] In a fifth aspect, the present application provides a vector comprising the polynucleotide of the fourth aspect of the present application.

[0327] In another preferred embodiment, the vector is selected from the group consisting of DNA, RNA, plasmid, lentivirus vector, adenovirus vector, retrovirus vector, transposon, or a combination thereof.

[0328] In another preferred embodiment, the vector is a retrovirus vector.

[0329] In a sixth aspect, the present application provides a host cell comprising the vector of the fifth aspect or the polynucleotide of the fourth aspect of the present application integrated into the chromosome of the host cell.

[0330] In another preferred embodiment, the cell is an isolated cell, and / or the cell is a genetically engineered cell.

[0331] In another preferred embodiment, the cell is a mammalian cell.

[0332] In another preferred embodiment, the cell is an NK cell or a T cell.

[0333] In another preferred embodiment, the host cell is an engineered immune cell.

[0334] In another preferred embodiment, the engineered immune cell comprises a T cell or an NK cell, preferably (i) a chimeric antigen receptor T cell (CAR-T cell); or (ii) a chimeric antigen receptor NK cell (CAR-NK cell), wherein the NK cell is derived from peripheral blood, umbilical cord blood, embryonic stem cells (ESC), induced pluripotent stem cells (iPSC), etc.

[0335] In another preferred embodiment, the host cell is an immune cell expressing or externally exposed on the cell membrane with the antibody of the first aspect of the present application or the chimeric antigen receptor of the third aspect of the present application.

[0336] In another preferred embodiment, the immune cell comprises an NK cell, a T cell.

[0337] In another preferred embodiment, the immune cell is derived from a human or a non-human mammal (e.g., a mouse).

[0338] In a seventh aspect of the present application, a method for preparing a CAR-NK cell or a CAR-T cell expressing the chimeric antigen receptor of the third aspect of the present application is provided, comprising the following steps:

[0339] Transducing the polynucleotide of the fourth aspect of the present application or the vector of the fifth aspect of the present application into an NK cell or a T cell, thereby obtaining the CAR-NK cell or the CAR-T cell.

[0340] In an eighth aspect of the present application, a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of the first aspect of the present application, the recombinant protein of the second aspect of the present application, the chimeric antigen receptor of the third aspect of the present application, the polynucleotide of the fourth aspect of the present application, the vector of the fifth aspect of the present application, or the host cell of the sixth aspect of the present application, and a pharmaceutically acceptable carrier, diluent, or excipient is provided.

[0341] In another preferred embodiment, the pharmaceutical composition is a preparation, preferably a liquid preparation.

[0342] In another preferred embodiment, the pharmaceutical composition is in the form of an injection.

[0343] In another preferred embodiment, the pharmaceutical composition is used for preparing a medicament or a preparation for preventing and / or treating cancer or tumor.

[0344] In a ninth aspect of the present application, there is provided an immunoconjugate comprising:

[0345] (a) an antibody moiety selected from the group consisting of an antibody or an antigen binding fragment thereof according to the first aspect of the present application, a recombinant protein according to the second aspect of the present application, or a combination thereof; and

[0346] (b) a conjugated moiety conjugated to the antibody moiety, the conjugated moiety being selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

[0347] In another preferred embodiment, the conjugate is selected from the group consisting of a fluorescent or luminescent label, a radioactive label, an MRI (magnetic resonance imaging) or CT (computed tomography) contrast agent, or an enzyme capable of generating a detectable product, a radionuclide, a biological toxin, a cytokine (such as IL-2, etc.), an antibody, an antibody Fc fragment, an antibody scFv fragment, a gold nanoparticle / nanorod, a viral particle, a liposome, a nanomagnetic particle, a prodrug-activating enzyme (e.g., DT-diaphorase (DTD) or benzylphenyl hydroxylase-like protein (BPHL)), a chemotherapeutic agent (e.g., cisplatin), or any form of nanoparticle, etc.

[0348] In a tenth aspect of the present application, there is provided a use of an antibody or an antigen binding fragment thereof according to the first aspect of the present application, a recombinant protein according to the second aspect of the present application, a chimeric antigen receptor according to the third aspect of the present application, a polynucleotide according to the fourth aspect of the present application, a vector according to the fifth aspect of the present application, a host cell according to the sixth aspect of the present application, a pharmaceutical composition according to the eighth aspect of the present application, or an immunoconjugate according to the ninth aspect of the present application,

[0349] (a) preparing a detection reagent or a kit; and / or

[0350] (b) preparing a medicament or a preparation for preventing and / or treating a DLL3-related disease.

[0351] In another preferred embodiment, the DLL3-related disease is cancer or tumor.

[0352] In another preferred embodiment, the tumor is selected from the group consisting of a hematological tumor, a solid tumor, or a combination thereof.

[0353] 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.

[0354] In another preferred embodiment, the tumor is a DLL3-positive tumor; preferably selected from the group consisting of chronic lymphocytic cancer, 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.

[0355] In another preferred embodiment, the detecting is an immunoassay.

[0356] In another preferred embodiment, the immunoassay is an ELISA immunoassay, an immunochromatographic assay, an immunocytochemical staining assay, or an immunohistochemical staining assay.

[0357] In another preferred embodiment, the diagnostic reagent is a test strip or a test plate.

[0358] In another preferred embodiment, the reagent comprises a chip, an antibody-coated immunoparticle.

[0359] In an eleventh aspect of the present application, there is provided a method for detecting (including diagnosing or non-diagnosing) a DLL3 protein in a sample in vitro, the method comprising the steps of:

[0360] (1) contacting the sample with the antibody or antigen-binding fragment thereof of the first aspect of the present application, or the recombinant protein of the second aspect of the present application;

[0361] (2) detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of DLL3 protein in the sample.

[0362] In another preferred embodiment, the diagnostic reagent is a test strip or a test plate.

[0363] In another preferred embodiment, the method is an immunocytochemistry (ICC) detection method, an immunohistochemistry (IHC) detection method, a whole cell ELISA detection method, or a cell lysate ELISA detection method.

[0364] In a twelfth aspect of the present application, a method for preparing a recombinant polypeptide is provided, the method comprising:

[0365] (a) culturing the host cell of the sixth aspect of the present application under conditions suitable for expression;

[0366] (b) isolating the recombinant polypeptide from the culture, wherein the recombinant polypeptide is the antibody or antigen-binding fragment thereof of the first aspect of the present application, or the recombinant protein of the second aspect of the present application.

[0367] In a thirteenth aspect of the present application, a test plate is provided, wherein the test plate comprises: a substrate (support plate) and a test strip, and the test strip comprises the antibody or antigen-binding fragment thereof of the first aspect of the present application, the recombinant protein of the second aspect of the present application, the immunoconjugate of the ninth aspect of the present application, or a combination thereof.

[0368] In a fourteenth aspect of the present application, a kit is provided, wherein the kit comprises:

[0369] (1) a first container, wherein the first container comprises the antibody or antigen-binding fragment thereof of the first aspect of the present application; and / or

[0370] (2) a second container, wherein the second container comprises a secondary antibody against the antibody of the present application.

[0371] Alternatively, the kit comprises the test plate of the thirteenth aspect of the present application.

[0372] In a fifteenth aspect of the present application, a method for treating a disease is provided, comprising administering to a subject in need thereof an appropriate amount of the antibody or antigen-binding fragment thereof of the first aspect of the present application, the recombinant protein of the second aspect of the present application, the host cell of the twelfth aspect of the present application, or the pharmaceutical composition of the eighth aspect of the present application.

[0373] In another preferred embodiment, the disease is cancer or tumor.

[0374] It should be understood that, within the scope of the present application, all the technical features described above and the technical features described in detail hereinafter (such as the examples) can be combined with each other to form new or preferred technical solutions. Due to the limited space, they will not be listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0375] Figure 1 SDS-PAGE results of anti-DLL3 scFv-Fc antibodies are shown.

[0376] Figure 2 BLI assay results of anti-DLL3 antibodies are shown.

[0377] Figure 3 EC50 assay results of anti-DLL3 antibodies are shown.

[0378] Figure 4 Molecular structure of anti-DLL3 CAR is shown.

[0379] Figure 5 Results of detection of DLL3 protein expression on the surface of tumor cell lines are shown.

[0380] Figure 6 Results of CAR detection of anti-DLL3 CAR-NK92 cell lines after sorting are shown.

[0381] Figure 7 Results of single round killing of SHP77, NCI-H82 cells by anti-DLL3 CAR NK92 are shown.

[0382] Figure 8 A is the result of multiple rounds of killing of SHP77, Figure 8 B is the result of multiple rounds of killing of NCI-H82 cells. DETAILED DESCRIPTION

[0383] The present inventors have made extensive and in-depth studies and for the first time unexpectedly obtained a series of anti-DLL3 antibodies. The screened antibodies have excellent biological activity, and further based on this, a chimeric antigen receptor structure targeting DLL3 is constructed, and the NK cells expressing the chimeric antigen receptor show superior killing ability to target cells. On this basis, the present application is completed.

[0384] TERMS

[0385] Unless otherwise defined, 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 application belongs.

[0386] The term "about" can mean a value or composition that is within an acceptable error range for the particular value or composition determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.

[0387] As used herein, the term "comprising" or "including" can be open-ended, semi- closed, and / or closed. In other words, the term can include "consisting essentially of" or "consisting of."

[0388] Antibody

[0389] The term "antibody" (Ab) shall include, but is not limited to, an immunoglobulin which specifically binds an antigen and comprises at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen-binding portion 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 L 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 regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL comprises three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.

[0390] As used herein, the term "heavy chain variable region" is used interchangeably with "VH".

[0391] As used herein, the term "light chain variable region" is used interchangeably with "VL".

[0392] As used herein, the term "antigen binding domain" and the like includes any naturally occurring, enzymatically obtainable, synthetic, or genetically modified polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antigen binding fragments of an antibody can be derived from an intact antibody molecule using any suitable standard techniques, such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains. Such DNA is known and / or readily available from, for example, commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and / or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.

[0393] Non-limiting examples of antigen binding fragments or antigen binding domains, as used herein, 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 the amino acid residues that mimic the hypervariable region of an antibody (e.g., an individual complementarity determining regions (CDRs) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide.

[0394] As used herein, an antigen binding fragment or antigen binding domain will generally comprise at least one variable domain. The variable domain can be of any size or amino acid composition and will generally comprise at least one CDR adjacent to or in-frame with one or more framework sequences. In antigen binding fragments having a VH domain associated with a VL domain, the VHand VL domains can be situated relative to one another in any suitable arrangement. For example, the variable region can be dimeric and contain VH-VH, VH-VL, or VL-VL dimers. Alternatively, the antigen binding domain can contain a monomeric VHor VL domain.

[0395] The precise amino acid sequence boundaries of each CDR in a given antibody light chain variable region or heavy chain variable region amino acid sequence can be determined using any of a number of well-known antibody CDR assignment systems, or combinations thereof, including, for example: Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loops, Kabat based on the variability of antibody sequences, KABAT, E., et al., U.S. Department of Health and Human Services, Sequences of Proteins of Immunological Interest, (1983), AbM (University of Bath), Contact (University College London), the international Immuno GeneTics database (IMGT), the EU numbering system, and Chothia definition based on loop structure position.

[0396] It is understood that the precise amino acid sequence boundaries of the CDRs in the application can optionally be defined using the different assignment systems mentioned above. Preferably, unless otherwise indicated, in the application when referring to residue positions in an antibody variable region, including heavy chain variable region residues and light chain variable region residues, the numbering of the positions is according to the Kabat numbering system.

[0397] As used herein, the term "antibody" or "immunoglobulin" is a heterotetrameric glycoprotein of about 150,000 daltons having the same structural characteristics, consisting of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds between the heavy chains varies among the different immunoglobulin isotypes. There are also interchain disulfide bonds at regular intervals along the polypeptide chains. Each of the heavy chains and light chains has regularly spaced intrachain disulfide bonds. At one end of each heavy chain is a variable region (VH) followed by a number of constant regions. At one end of each light chain is a variable region (VL) followed by a constant region; the constant region of the light chain is aligned with the first constant region of the heavy chain, and the variable region of the light chain is aligned with the variable region of the heavy chain. Particular amino acid residues at the interface between the variable regions of the light and heavy chains form the interface.

[0398] As used herein, the term "variable" indicates certain portions of the variable regions of antibodies differ in sequence, which contributes to the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable regions of an antibody. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the variable regions of the light and heavy chains. The more conserved portions of the variable regions are called the framework regions (FRs). The variable regions of the heavy and light chains each comprise four FR regions, joined by three CDRs, which are generally in the order of -folded conformation, connected by the linking loops, which in some cases can form partial folded structures. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs of the other chain, contribute to the formation of a binding site of the antibody for an antigen (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pages 647-669 (1991)). The constant regions are not directly involved in the binding of an antibody to an antigen, but exhibit different effector functions, such as participation in antibody-dependent cellular cytotoxicity.

[0399] The present application includes not only intact monoclonal antibodies, but also fragments of antibodies that are immunologically active or fusions of antibodies with other sequences. Thus, the present application also includes fragments, derivatives, and analogs of the antibodies.

[0400] As used herein, the terms "fragment," "derivative," and "analog" refer to polypeptides that substantially retain the same biological function or activity of the antibodies of the present application. Polypeptide fragments, derivatives, or analogs of the present application can be (i) polypeptides having one or more conservative or non-conservative amino acid substitutions, preferably conservative amino acid substitutions, wherein such substituted amino acid residues can or can not be encoded by the genetic code, or (ii) polypeptides having a substituent group at one or more amino acid residues, or (iii) polypeptides formed by fusing the mature polypeptide to another compound, such as a compound that increases the half-life of the polypeptide, for example, a polyethylene glycol, or (iv) polypeptides formed by adding additional amino acid sequences to the polypeptide, such as leader sequences or secretion sequences or sequences or proteins for purification of the polypeptide or prosequences, or fusion proteins with a 6His tag. These fragments, derivatives, and analogs are within the scope of one skilled in the art in light of the teachings herein.

[0401] The "light chains" of a vertebrate antibody (immunoglobulin) can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant regions. Depending on the amino acid sequences of their heavy chain constant regions, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes) e.g., IgGl, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant regions that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.

[0402] The present application also provides other polypeptides, such as fusion proteins comprising a human antibody or fragment thereof. In addition to substantially full-length polypeptides, the present application also includes fragments of the antibodies of the present application. Typically, the fragment has at least about 50 contiguous amino acids of an antibody of the present application, 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.

[0403] In the present application, the antibodies of the present application also include conservative variants thereof, which are polypeptides having up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids replaced with similar or nearly similar amino acids as compared to the amino acid sequence of the antibodies of the present application. These conservative variant polypeptides are preferably generated by making amino acid replacements according to Table A.

[0404] Table A

[0405]

[0406] Chimeric antigen receptor (CAR)

[0407] As used herein, the terms "chimeric antigen receptor of the invention", "CAR of the invention" can be used interchangeably to refer to the chimeric antigen receptor of the third aspect of the invention.

[0408] The chimeric antigen receptor (CAR) of the invention comprises an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain comprises a target-specific binding element (also referred to as an antigen binding domain). The intracellular domain comprises a costimulatory signaling region and a zeta chain portion. The costimulatory signaling region refers to a portion of the intracellular domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules required for the efficient response of lymphocytes to antigen, other than antigen receptors or their ligands.

[0409] A linker can be incorporated between the extracellular domain and the transmembrane domain of the CAR, or between the cytoplasmic domain and the transmembrane domain of the CAR. As used herein, the term "linker" generally refers to any oligo- or polypeptide that serves to link the transmembrane domain to the extracellular domain or cytoplasmic domain of the polypeptide chain. The linker can comprise 0-300 amino acids, preferably 2 to 100 amino acids and most preferably 3 to 50 amino acids.

[0410] In a preferred embodiment of the invention, the extracellular domain of the CAR provided by the invention comprises an antigen binding domain targeting DLL3. The CAR of the invention, when expressed in a T cell, is capable of antigen recognition based on antigen binding specificity. When it binds to its cognate antigen, it affects tumor cells, causing them to not grow, to be caused to die, or to otherwise be affected, and leading to a reduction or elimination of the tumor burden of the patient. The antigen binding domain is preferably fused to an intracellular domain from one or more of a costimulatory molecule and a zeta chain. Preferably, the antigen binding domain is fused to an intracellular domain in combination with a 4-1 BB signaling domain, and a CD3 zeta signaling domain.

[0411] As used herein, "antigen binding domain" "single chain antibody fragment" each refers to a Fab fragment, a Fab' fragment, a F(ab')2 fragment, or a single Fv fragment having antigen binding activity. Fv antibody contains the variable region of antibody heavy chain, light chain variable region, but no constant region, and has the smallest antibody fragment with all antigen binding sites. Generally, Fv antibody also contains a polypeptide linker between the VHand VLdomains, and is capable of forming a structure required for antigen binding. The antigen binding domain is usually a scFv (single-chain variable fragment). The size of scFv is generally 1 / 6 of a complete antibody. The single chain antibody is preferably an amino acid chain sequence encoded by a single nucleotide chain. As a preferred mode of the invention, the scFv comprises an antibody, preferably a single chain antibody, which specifically recognizes the extracellular region of DLL3.

[0412] For the hinge region and transmembrane region (transmembrane domain), the CAR can be designed to include a transmembrane domain fused to the extracellular domain of the CAR. In one embodiment, a transmembrane domain naturally associated with one of the domains in the CAR is used. In some examples, the transmembrane domain can be selected, or modified by amino acid substitution, to avoid binding such domain to a transmembrane domain of the same or a different surface membrane protein, thereby minimizing interactions with other members of the receptor complex.

[0413] Preferably, the structure of the CAR of the present application comprises a signal peptide, an antigen recognition sequence (antigen binding domain), a linker region, a transmembrane region, a costimulatory factor signal region, and a CD3 zeta signaling region (zeta chain portion), in the following order:

[0414] L-scFv-H-TM-C-CD3ζ (I)

[0415] NK cells

[0416] Natural killer (NK) cells are a major class of immune effector cells that protect the body from viral infection and tumor cell invasion through a non-antigen-specific pathway. In recent years, NK cells have shown great application prospects in adoptive cellular immunotherapy. The sources of NK cells are wide, including peripheral blood, umbilical cord blood, embryonic stem cells (ESC), induced pluripotent stem cells (iPSC), etc.

[0417] NK-92 cells are a strain of interleukin-2 (IL2)-dependent NK cells derived from peripheral blood mononuclear cells of a 50-year-old white male patient with acute non-Hodgkin's lymphoma. NK-92 cells are the only NK cell line approved by the FDA for clinical trials so far. This strain of cells has strong cytotoxicity, is economical, off-the-shelf, easy to scale up, has a short survival time after killing tumor cells, is easy to expand in vitro, and most patients receiving treatment do not reject NK-92 cells, have no risk of graft-versus-host reaction, do not express KIRs, are in a constitutively activated state, and have shown good clinical safety so far.

[0418] As used herein, the terms "CAR-NK cell", "CAR-NK", "CAR NK", "CAR-NK cell of the present application" all refer to a CAR-NK cell expressing a chimeric antigen receptor CAR of the first aspect of the present application

[0419] Vector

[0420] Nucleic acid sequences encoding a 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 the gene from cells and tissues that contain the gene using standard techniques. Alternatively, a gene of interest can be produced synthetically.

[0421] The present application also provides vectors into which a cassette of the present application is inserted. Vectors derived from retroviruses, such as lentivirus, are suitable tools for achieving long-term gene transfer, as they allow long-term, stable integration of the transgene and its propagation in daughter cells. Lentiviral vectors have advantages over vectors derived from onco- retroviruses, such as murine leukemia viruses, as they can transduce non-proliferating cells, such as hepatocytes. They also have the advantage of being less immunogenic.

[0422] Briefly, an expression cassette or nucleic acid sequence of the present application is typically operably linked to a promoter and incorporated into an expression vector. The vector is suitable for replication and integration into eukaryotic cells. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of expression of the desired nucleic acid sequence.

[0423] The expression constructs of the present application 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, e.g., U.S. Patent Nos. 5,399,346, 5,580,859, 5,589,466, which are hereby incorporated by reference in their entireties. In another embodiment, the present application provides a gene therapy vector.

[0424] The nucleic acid can be cloned into a number of types of vectors. For example, the nucleic acid can be cloned into a vector including, but not limited to, a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Particular vectors of interest include expression vectors, replication vectors, probe production vectors, and sequencing vectors.

[0425] Further, expression vectors can be provided to cells in the form of viral vectors. Viral vector technology is well known in the art and described in, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. Typically, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction enzyme sites, and one or more selectable markers (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193).

[0426] Many viral-based systems have been developed for the transfer of genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A 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 cells in vivo or ex vivo. Many retroviral systems are known in the art. In some embodiments, an adenoviral vector is used. Many adenoviral vectors are known in the art. In one embodiment, a lentiviral vector is used.

[0427] Additional promoter elements, such as enhancers, can modulate the frequency of transcription initiation. Typically, these are located in the region 30-110 bp upstream of the initiation site, although it has recently been shown that many promoters also contain functional elements downstream of the initiation site. The spacing between promoter elements is often flexible, such that a promoter functions when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased by as much as 50 bp before activity begins to decline. Depending on the promoter, individual elements can appear to act cooperatively or independently to initiate transcription.

[0428] One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high level expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is elongation factor- 1 alpha (EF-1 alpha). However, other constitutive promoter sequences can also be used, including but not limited to the simian virus 40 (SV40) early promoter, the mouse mammary tumor virus (MMTV), the human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia virus promoter, the Epstein-Barr virus immediate early promoter, the Rous Sarcoma Virus promoter, and human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. Further, the present application should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present application. The use of inducible promoters provides a molecular switch that can turn on expression of a polynucleotide sequence operably linked to an inducible promoter when such expression is desired, or turn off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.

[0429] To assess expression of a CAR polypeptide or portion thereof, the expression vector introduced into a cell can also comprise either or both of a selectable marker gene or a reporter gene to facilitate identification and selection of expressing cells from a population of cells sought to be transfected or infected by the viral vector. In other aspects, a selectable marker can be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene can be flanked by appropriate regulatory sequences to enable expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes, such as neo and the like.

[0430] Reporter genes are used to identify potentially transfected cells and to assess functionality of regulatory sequences. Generally, a reporter gene is one that is not present or expressed by the recipient organism or tissue and which encodes a polypeptide whose expression is clearly indicated by some readily detectable property, such as enzyme activity. After the DNA has been introduced into the recipient cells, expression of the reporter gene is determined at an appropriate time. Suitable reporter genes can include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79-82). Suitable expression systems are well known and can be prepared using known techniques or obtained commercially. Generally, constructs having a minimum of 5 flanking regions that show the highest levels of reporter gene expression are identified as promoters. Such promoter regions can be linked to a reporter gene and used to assess the ability of an agent to modulate promoter-driven transcription.

[0431] Methods of introducing genes into cells and expressing genes into cells are known in the art. In the context of expression vectors, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell, by any of a number of methods in the art. For example, the expression vector can be transferred into a host cell by physical, chemical, or biological means.

[0432] Physical methods of introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods of producing cells that include vectors and / or exogenous nucleic acids are well known in the art. See, e.g., Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). The preferred method of introducing a polynucleotide into a host cell is calcium phosphate transfection.

[0433] Biological methods of introducing a polynucleotide of interest into a host cell 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, e.g., human, cells. Other viral vectors can be derived from lentivirus, poxvirus, herpes simplex virus I, adenovirus, and adeno-associated virus, among others. See, e.g., U.S. Patent Nos. 5,350,674 and 5,585,362.

[0434] Chemical means of introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, and beads; and lipid- based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as an in vitro and in vivo delivery vehicle is a liposome (e.g., a artificial membrane vesicle).

[0435] In the case of using a non-viral delivery system, an exemplary delivery vehicle is a liposome. It is contemplated that a lipid formulation is used to introduce a nucleic acid into a host cell (in vitro, ex vivo, or in vivo). In another aspect, the nucleic acid can be associated with a lipid. The nucleic acid associated with a lipid can be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution comprising lipids, mixed with lipids, associated with lipids, contained in lipids as a suspension, contained in or complexed with micelles, or otherwise associated with lipids. The lipids, lipid / DNA, or lipid / expression vector associated with the composition are not limited to any particular structure in solution. For example, they can exist in a bilayer structure, as micelles, or have a "collapsed" structure. They can also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances, which can be naturally occurring or synthetic. For example, lipids include fat droplets, which naturally occur in the cytoplasm and comprise long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes, among such compounds.

[0436] In a preferred embodiment of the application, the vector is a retroviral vector.

[0437] Therapeutic applications

[0438] The present application includes cells (e.g., NK cells, T cells, etc.) transduced with a retroviral vector (RV) encoding an antibody, CAR of the present application. The transduced NK cells can elicit a CAR-mediated NK-cell or T-cell response.

[0439] Accordingly, the present application also provides a method of stimulating an NK cell-mediated immune response to a target cell population or tissue in a mammal, comprising the step of administering to the mammal an NK cell expressing a CAR of the present application.

[0440] In one embodiment, the present application includes a class of cell therapy in which NK cells are genetically modified to express a CAR of the present application, and the CAR-NK cells are infused into a recipient in need thereof. The infused cells are capable of killing tumor cells of the recipient. Unlike antibody therapy, CAR-NK cells are capable of persisting in vivo, resulting in long-term persistence that can lead to sustained tumor control.

[0441] Cancers that can be treated include tumors that are not vascularized or are substantially not vascularized, as well as vascularized tumors. The cancer can include non-solid tumors (such as hematological tumors, e.g., leukemias and lymphomas) or can include solid tumors. Types of cancer treated with the CARs of the present application include, but are not limited to, carcinomas, blastomas, and sarcomas, and certain leukemias or lymphoid malignancies, benign and malignant tumors, and malignomas, e.g., sarcomas, carcinomas, and melanomas. Adult and pediatric tumors / cancers are also included.

[0442] A solid tumor is an abnormal mass of tissue that usually does not contain cysts or fluid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named for the type of cells that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors such as sarcomas and carcinomas include fibrosarcoma, myxosarcoma, liposarcoma, mesothelioma, lymphoid malignancy, pancreatic cancer, ovarian cancer.

[0443] CAR-modified NK cells of the present application can also be used as a vaccine type for ex vivo immunization and / or in vivo therapy of a mammal. Preferably, the mammal is a human.

[0444] Pharmaceutical Compositions

[0445] The antibodies, fusion proteins, or CAR-modified NK cells of the present application can be administered alone or in combination with a diluent and / or with other components such as IL-2, IL-15, IL-18, IL-21, or other cytokines or cell populations as a pharmaceutical composition. Briefly, a pharmaceutical composition of the present application can include a target cell population as described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions can include buffers such as neutral buffered saline, sulfate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose, or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions of the present application are preferably formulated for intravenous administration.

[0446] The pharmaceutical composition of the present application can be administered in a manner appropriate for the disease to be treated (or prevented). The amount and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease - although appropriate dosages can be determined by clinical trials.

[0447] When referring to an "immunologically effective amount", an "anti-tumor effective amount", a "tumor-inhibiting effective amount", or a "therapeutic amount", the precise amount of the composition of the present application to be administered will be determined by a physician, taking into account the age, weight, tumor size, extent of infection or metastasis, and the individuality of the patient's condition. It can be generally stated that the pharmaceutical composition comprising the T cells described herein can be administered at a dose of 104to 109cells / kg body weight, preferably 105to 106cells / kg body weight, including all integer values within those ranges. The T cell composition can also be administered multiple times at these doses. The cells can be administered by using infusion techniques well known in the art of immunotherapy (see, e.g., 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 monitoring the patient's disease signs and adjusting the treatment accordingly by a person skilled in the medical art.

[0448] The main advantages of the present application include

[0449] (1) The chimeric antigen receptor of the present application, whose extracellular antigen binding domain is a specific anti-DLL3 scFv, which binds to a specific hinge region and intracellular domain, the CAR formed shows great killing ability to tumor cells, and has less cytotoxicity and lower side effects.

[0450] (2) The CAR-NK cells of the present application have a high degree of activation, and have excellent cytotoxicity to DLL3-positive target cells.

[0451] (3) The binding ability and affinity kinetics of the antibody of the present application to target cells are excellent, significantly better than the commercially available positive control.

[0452] The present application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and not used to limit the scope of the present application. The experimental methods in the following examples, for which the detailed conditions are not specified, are generally carried out according to the conventional conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions suggested by the manufacturer. Unless otherwise specified, percentages and parts are by weight.

[0453] Example 1 Screening of Anti-DLL3 Antibodies

[0454] DLL3 related antibodies were generated by immunizing antigen-immune antibody production region gene fragment humanized mice, and then hybridoma screening was performed. The candidate scFv was in the form of VL-(G4S)3-VH, and the sequence is shown in Table 1. The CDR sequences annotated using Kabat and Chothia are shown in Table 2 and Table 3. The following experiments were performed using antibodies in the form of scFv-human IgG Fc.

[0455] SDS-PAGE was used to detect the expression and purification of anti-DLL3 scFv-Fc antibodies, and the results are shown in Figure 1 . The results show that the antibody expression and purification are good. The anti-DLL3 scFv derived from the patent of Amgen was selected as the positive control (AMG757).

[0456] The results of Biolayer Interferometry (BLI) assay of representative antibodies against human DLL3 protein are shown in Figure 2 . Flow cytometry binding analysis was performed on all antibodies using two DLL3-positive cell lines, SHP77 and NCI-H82, and the results are shown in Figure 3 . The BLI data and EC50 data of all antibodies are summarized in Table 4.

[0457] Table 1 scFv sequence of anti-DLL3 antibody

[0458]

[0459]

[0460] Table 2 CDR sequence based on Kabat

[0461]

[0462] SEQ: SEQ ID NO.

[0463] Table 3 CDR sequence based on Chothia

[0464]

[0465] SEQ: SEQ ID NO.

[0466] Table 4 Summary of kinetic data and EC50 results of anti-DLL3 antibodies (SCFV-FC)

[0467]

[0468] *Ab.73 Since the koff is lower than the lower limit of instrument detection (1E-06 / s), it is represented as less than the lower limit in the table.

[0469] Design of anti-DLL3 CAR structure

[0470] The inventors found that anti-DLL3 antibodies with high affinity to DLL3 do not necessarily result in high killing performance of the corresponding CAR molecules. Therefore, the inventors performed a large number of screenings, and on the basis of the obtained antibodies, a large number of CAR molecules were designed and screened, and finally obtained molecules with excellent killing performance at the CAR level. Among them, the corresponding CARs of Ab.55, Ab.67 and Ab.76 have exceptionally excellent performance in multiple rounds of killing. The specific designs are as follows:

[0471] The scFv of the anti-DLL3 CAR structure is derived from some of the antibodies obtained in Example 1, and the corresponding antibody names and the order of light chains and heavy chains in the scFv are named. Among them,

[0472] The anti-DLL3 CAR structure is shown in Figure 4 , which comprises a signal peptide (SP), a scFv, a hinge region, a transmembrane region (TM), a costimulatory domain (CD), and a CD3ζ domain. The signal peptide includes the following sequence (Table 5), and the data of the present application is based on CD8αSP, CD28hinge, CD28TM, and CD28CD structure. The scFv is composed of VL-Linker-VH or VH-Linker-VL, wherein the linker can be (G4S)3 or (G4S)4, etc.

[0473] Table 5 Signal peptide sequence

[0474]

[0475] Example 3 Detection of tumor cell surface DLL3 expression

[0476] Experimental method: The expression of DLL3 on the surface of SHP77 and NCI-H82 tumor cells was detected by flow cytometry, and the detection method is as follows.

[0477] Take 2E5-3E5 tumor cells to be tested, 300g, 4℃ centrifugation for 5min, discard the supernatant. 3.2 Add 200μL FACS Buffer (1% FBS dissolved in PBS), 300g, 4℃ centrifugation for 5min, discard the supernatant.

[0478] Prepare a primary antibody solution with a concentration of 1μg / mL. Add 100μL / well of primary antibody solution, mix well by blowing, and incubate at 4℃ for 30min. After incubation, add 100μL FACS Buffer, 300g, 4℃ centrifugation for 5min, discard the supernatant.

[0479] Add 200 μL / well FACS Buffer, blow and suck to mix, 300g, 4°C centrifuge for 5 min, discard the supernatant. Repeat the above steps once. Add 100 μL / well secondary antibody solution, blow and mix, 4°C incubate for 30 min. After incubation, add 100 μL FACS Buffer, 300g, 4°C centrifuge for 5 min, discard the supernatant. Add 200 μL / well FACS Buffer, blow and suck to mix, 300g, 4°C centrifuge for 5 min, discard the supernatant. Repeat the above steps once. Add 200 μL / well FACS Buffer, resuspend, and use a flow cytometer for detection.

[0480] The results show that SHP77 and NCI-H82 cells express DLL3 protein on the cell surface. Figure 5

[0481] Example 4 Virus packaging and NK92 cell infection

[0482] Experimental method: The packaging vectors of the retrovirus are BaEV-TR and pCMV-gag-pol, and the vector carrying the CAR molecule is pMSCV. These vectors are designed by the laboratory and synthesized and extracted by Jinweizhi. The virus packaging, cell infection and sorting process are as follows:

[0483] Resuspend the well-conditioned HEK-293T cells after digestion in DMEM complete culture medium, and inoculate 10 mL / dish of 8E5 / mL in a 10 cm culture dish. After incubation in the incubator for 16 h, observe the cell density, and when the density is about 90%, start the plasmid transfection. Take a centrifuge tube, add 500 μL Opti-MEM TM I serum-free medium (Gibco, 31985062), and in one of the centrifuge tubes, add 7.5 μg BaEV-TR, 10 μg pCMV-gag-pol and 20 μg of the corresponding pMSCV vector expressing CAR, mix well to form an Opti-MEM-plasmid mixture; in another centrifuge tube, add 40 μL PEIpro solution (polyplus, 115-010), mix well to form an Opti-MEM-PEI mixture.

[0484] Add the Opti-MEM-plasmid mixture to the Opti-MEM-PEI mixture, and mix well by blowing and sucking, and let stand at room temperature for 15 min to form a transfection complex. After incubation, collect the cell culture supernatant. Use Lenti-X Concentrator (Takara, 631232) to concentrate the virus.

[0485] ​The virus was resuspended with 100 μL NK92 cell culture medium, 5 μL was taken for titer determination, and the remaining virus was temporarily stored at 4°C. The titer determination was performed using K562 cells, and 5 μg / mL polybrene (Sigma-Aldrich, TR-1003) was supplemented during infection. The K562 positive rate was determined 48 h after infection, and the virus titer was calculated.

[0486] 4E5 NK92 cells were taken in a 6-well plate, and the virus concentrate was added at an MOI of 2, and polybrene (Sigma-Aldrich, TR-1003) was added at a final concentration of 5 ug / ml, and mixed evenly. The cells were centrifuged at 32°C, 800g for 1h, and cultured overnight. The NK92 was taken out, centrifuged and resuspended in fresh NK92 culture medium, and flow detection was performed after 4 days of continuous culture.

[0487] The DLL3-CAR NK92 cells in culture were thoroughly blown and mixed, and counted. According to the counting results, 1E7 cells to be sorted were taken in a centrifuge tube, centrifuged at 300g for 5min, and the supernatant was discarded. The cells were resuspended with 10mL MACS Buffer, centrifuged at 300g for 5min, and the supernatant was discarded. The cells were resuspended with 2mL 3ug / mL Biotinylated Huamn DLL3His, AvitagTM (AcroBiosystem, DL3-H82E4) diluent (diluted in PBS), and incubated at 4°C for 30min.

[0488] After incubation, the cells were washed and resuspended, and 20 μL Anti-Biotin MicroBeads (Miltenyi, 130-090-485) was added and mixed thoroughly. Incubate at 4°C for 15min. After incubation, wash and resuspend. The cells labeled with magnetic beads were washed out by loading the adsorption column LS (Miltenyi, 130-042-401) on the magnetic stand. Take 200 μL of the sorted cells for NC-200 counting.

[0489] The sorted cells were resuspended and cultured in a 37°C, 5% CO2 incubator. CAR positive rate determination was performed 4 days after sorting. Take 1E5-2E5 cells to be tested, centrifuge at 700g, 4°C for 2min, and discard the supernatant. Wash, centrifuge, and discard the supernatant.

[0490] After mixing, 100 μL of antigen protein suspension was added to each sample, the antigen protein concentration was 2ug / mL, and the mixture was blown and mixed, and incubated at 4°C for 30min-60min. After incubation, wash, centrifuge, and discard the supernatant.

[0491] The detection antibody streptavidin-PE (Biolegend, #405203) was prepared at a dilution ratio of 1:200, 100 μL of the detection antibody was added to each sample after mixing, and the mixture was mixed by blowing and then incubated at 4°C for 30-60 min.

[0492] After incubation, the samples were washed, centrifuged, and the supernatant was discarded. 200 μL / sample of FACS Buffer was added for resuspension, and the samples were detected by flow cytometry.

[0493] Experimental results: The CAR detection results of the sorted CAR-NK92 cell lines are shown in Table 1. Figure 6

[0494] The results show that after the NK92 cells are infected with the virus and sorted by magnetic beads, the positive rate of each group of NK92 cells with anti-DLL3 CAR reaches more than 95%, except for the CAR55 positive rate of 94.7%.

[0495] Example 5: Detection of the killing ability of anti-DLL3 CAR NK92 on target cells

[0496] Experimental method: The killing ability of anti-DLL3 CAR NK92 cells was detected in different ways.

[0497] 5.1 Single round of killing

[0498] The single round of killing ability of anti-DLL3 CAR NK92 on SHP77-Luciferase and NCI-H82-Luciferase target cells was detected using the Luciferase reporter gene, and CD19-CAR with anti-CD19 was used as a negative control. The specific operation steps are as follows,

[0499] The CAR NK92 effector cells used in the killing experiment were removed from IL-2 24 h in advance.

[0500] The target cells were digested, neutralized, mixed by blowing and counting, and then resuspended in an appropriate volume of RPMI1640 complete medium. The target cells were transferred to a 96-well flat-bottom plate at 2E4 / well and 100 μL / well. The effector cells were mixed by blowing and counted, and the appropriate number of effector cells were taken, centrifuged at 300g for 5 min at room temperature, and then resuspended in an appropriate volume of RPMI1640 complete medium. The effector cells were transferred to the target cell 96-well flat-bottom plate at 50 μL / well, and incubated at 37°C in a 5% CO2 incubator for an appropriate time. 60 μL of ONE-Glo was added to each well and mixed. After 3 min of reaction, mix well, take 130 μL of the reaction solution to a 96-well white flat-bottom plate, and use a microplate reader to detect the luminescence signal intensity, and then calculate the cytotoxicity.

[0501] ​The killing results recorded by Luciferase reporter gene method are shown in Figure 7 It can be found that CAR55, CAR67, CAR76 have good killing effect on two kinds of target cells under this experimental condition.

[0502] The killing results recorded by Luciferase reporter gene method are shown in Figure 7 It can be found that CAR55, CAR67, CAR76 have good killing effect on two kinds of target cells under this experimental condition.

[0503] 5.2 Multi-round killing

[0504] The multi-round killing ability of anti-DLL3 CAR NK92 on SHP77-GFP and NCI-H82-GFP target cells was detected by using a cell imaging multifunctional detection system (Cytation C7), and CD19-CAR for anti-CD19 was used as a negative control. The specific operation steps are as follows,

[0505] IL-2 in NK92 culture medium was removed 24h in advance.

[0506] A 96-well plate for shooting was taken and coated with poly-D-lysine for use. The target cells were digested and dispersed thoroughly, and then inoculated in the well plate at 2E4 / well, 100μL / well. After inoculation, the plate was attached at 37℃ for 6 hours. The well plate was photographed by using Cytation C7 as the baseline. According to the experimental setting of effector target ratio (E:T), 50μL / well of effector cells were slowly added, and the well plate was placed in the Cytation C7 instrument, and the photographing record was started after 30 minutes.

[0507] After the first round of killing reached the detection bottom line of the target cells or the reading value of the target cells was no longer reduced, the 96-well plate for the second round of killing was coated and the target cells were inoculated at 2E4 / well, 100μL / well.

[0508] After the first round of killing reached the detection bottom line of the target cells or the reading value of the target cells was no longer reduced, the solution in the first round of killing was thoroughly blown and mixed, and transferred to the coated 96-well plate for the second round of killing, and the photographing record was started after 30 minutes.

[0509] Experimental results:

[0510] The single round of killing of anti-DLL3 CAR NK92 on SHP77 and NCI-H82 cells is shown in Figure 7 The multi-round killing is shown in Figure 8 A, 8B. Under this experimental condition, the CAR NK92 of each group has good killing effect on the target cells.

[0511] The results show that: in the first round of killing (14 hours) at E:T = 1:1, compared with the Xiangshen CAR757, the CAR76 can exhibit more than 82% specific killing activity.

[0512] Based on the experiments of the present application, the CAR 76, CAR 55, and CAR 67 have excellent specific killing activity.

[0513] Discussion:

[0514] In single round, multiple rounds of killing, CAR76 has superior specific killing activity to Xiangshen CAR757. However, surprisingly, the affinity (KD value) of the corresponding scFv antibody Ab76 of CAR76 is lower than that of the Xiangshen.

[0515] In addition, compared with the Xiangshen, Ab63 has excellent EC50 value against NCIH82, suggesting that it has excellent killing activity. In the single round of cell killing activity experiment, it has good CAR function short-time killing activity ( Figure 7 ), but it is not as good as the Xiangshen in the CAR function multiple rounds of killing ( Figure 8 A, 8B).

[0516] Based on the complex biological environment of the interaction between NK cells and target cells, the target affinity (KD value) of the antibody and the EC50 data of the target cells are only for reference and cannot obviously indicate that the CAR NK cells constructed with high-affinity antibodies as elements will also have strong tumor cell killing performance.

[0517] All the documents mentioned in the present application are cited as references in the present application, as if each document is cited as a reference individually. In addition, it should be understood that after reading the above teaching of the present application, those skilled in the art can make various modifications or modifications to the present application, and these equivalent forms also fall within the scope defined by the claims attached to the present application.

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 comprising complementarity determining regions (CDRs) selected from the group consisting of: (1) the heavy chain variable region comprises the following complementarity determining regions (CDRs): HCDR1 set forth in SEQ ID NO: 19, HCDR2 set forth in SEQ ID NO: 20, and HCDR3 set forth in SEQ ID NO: 21 ; and the light chain variable region comprises the following complementarity determining regions (CDRs): LCDR1 set forth in SEQ ID NO: 22, LCDR2 set forth in SEQ ID NO: 23, and LCDR3 set forth in SEQ ID NO: 24; or (2) the heavy chain variable region comprises the following complementarity determining regions (CDRs): HCDR1 set forth in SEQ ID NO: 7, HCDR2 set forth in SEQ ID NO: 8, and HCDR3 set forth in SEQ ID NO: 9; and the light chain variable region comprises the following complementarity determining regions (CDRs): LCDR1 set forth in SEQ ID NO: 10, LCDR2 set forth in SEQ ID NO: 11, and LCDR3 set forth in SEQ ID NO: 12; or (3) the heavy chain variable region comprises the following complementarity determining regions (CDRs): HCDR1 set forth in SEQ ID NO: 31, HCDR2 set forth in SEQ ID NO: 32, and HCDR3 set forth in SEQ ID NO: 33; and the light chain variable region comprises the following complementarity determining regions (CDRs): LCDR1 set forth in SEQ ID NO: 34, LCDR2 set forth in SEQ ID NO: 35, and LCDR3 set forth in SEQ ID NO: 36; or (4) the heavy chain variable region comprises the following complementarity determining regions (CDRs): HCDR1 set forth in SEQ ID NO: 13, HCDR2 set forth in SEQ ID NO: 43, and HCDR3 set forth in SEQ ID NO: 33; and the light chain variable region comprises the following complementarity determining regions (CDRs): LCDR1 set forth in SEQ ID NO: 44, LCDR2 set forth in SEQ ID NO: 35, and LCDR3 set forth in SEQ ID NO: 36; wherein the CDR sequences are numbered according to 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 comprise complementarity determining regions (CDRs) selected from the group consisting of: (1) the heavy chain variable region comprises the following complementarity determining regions (CDRs): HCDR1 set forth in SEQ ID NO: 50, HCDR2 set forth in SEQ ID NO: 51, and HCDR3 set forth in SEQ ID NO: 21 ; and the light chain variable region comprises the following complementarity determining regions (CDRs): LCDR1 set forth in SEQ ID NO: 22, LCDR2 set forth in SEQ ID NO: 23, and LCDR3 set forth in SEQ ID NO: 24; or (2) the heavy chain variable region comprises the following complementarity determining regions (CDRs): HCDR1 set forth in SEQ ID NO: 7, HCDR2 set forth in SEQ ID NO: 8, and HCDR3 set forth in SEQ ID NO: 9; and the light chain variable region comprises the following complementarity determining regions (CDRs): LCDR1 set forth in SEQ ID NO: 10, LCDR2 set forth in SEQ ID NO: 11, and LCDR3 set forth in SEQ ID NO: 12; or (3) the heavy chain variable region comprises the following complementarity determining regions (CDRs): HCDR1 set forth in SEQ ID NO: 31, HCDR2 set forth in SEQ ID NO: 32, and HCDR3 set forth in SEQ ID NO: 33; and the light chain variable region comprises the following complementarity determining regions (CDRs): LCDR1 set forth in SEQ ID NO: 34, LCDR2 set forth in SEQ ID NO: 35, and LCDR3 set forth in SEQ ID NO: 36; or (4) the heavy chain variable region comprises the following complementarity determining regions (CDRs): HCDR1 set forth in SEQ ID NO: 13, HCDR2 set forth in SEQ ID NO: 43, and HCDR3 set forth in SEQ ID NO: 33; and the light chain variable region comprises the following complementarity determining regions (CDRs): LCDR1 set forth in SEQ ID NO: 44, LCDR2 set forth in SEQ ID NO: 35, and LCDR3 set forth in SEQ ID NO: 36; wherein the CDR sequences are numbered according to the numbering scheme of Kabat; or (2) the heavy chain variable region comprises the following complementarity determining regions CDRs: HCDR1 set forth in SEQ ID NO: 47, HCDR2 set forth in SEQ ID NO: 48, and HCDR3 set forth in SEQ ID NO: 9; and the light chain variable region comprises the following complementarity determining regions CDRs: LCDR1 set forth in SEQ ID NO: 10, LCDR2 set forth in SEQ ID NO: 11, and LCDR3 set forth in SEQ ID NO: 12; or (3) the heavy chain variable region comprises the following complementarity determining regions CDRs: HCDR1 set forth in SEQ ID NO: 54, HCDR2 set forth in SEQ ID NO: 55, and HCDR3 set forth in SEQ ID NO: 33; and the light chain variable region comprises the following complementarity determining regions CDRs: LCDR1 set forth in SEQ ID NO: 34, LCDR2 set forth in SEQ ID NO: 35, and LCDR3 set forth in SEQ ID NO: 36; or (4) the heavy chain variable region comprises the following complementarity determining regions CDRs: HCDR1 set forth in SEQ ID NO: 58, HCDR2 set forth in SEQ ID NO: 48, and HCDR3 set forth in SEQ ID NO: 33; and the light chain variable region comprises the following complementarity determining regions CDRs: LCDR1 set forth in SEQ ID NO: 44, LCDR2 set forth in SEQ ID NO: 35, and LCDR3 set forth in SEQ ID NO: 36; wherein the CDR sequences are based on the numbering scheme of Chothia.

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

3. A chimeric antigen receptor (CAR) comprising, the antigen binding domain of the chimeric antigen receptor contains a single chain variable region sequence scFv of an antibody targeting DLL3, the heavy chain variable region and the light chain variable region of the scFv comprising the following complementarity determining regions CDRs: (1) the heavy chain variable region comprises the following complementarity determining regions CDRs: HCDR1 set forth in SEQ ID NO: 19, HCDR2 set forth in SEQ ID NO: 20, and HCDR3 set forth in SEQ ID NO: 21 ; and the light chain variable region comprises the following complementarity determining regions CDRs: LCDR1 set forth in SEQ ID NO: 22, LCDR2 set forth in SEQ ID NO: 23, and LCDR3 set forth in SEQ ID NO: 24; or (2) the heavy chain variable region comprises the following complementarity determining regions CDRs: HCDR1 set forth in SEQ ID NO: 7, HCDR2 set forth in SEQ ID NO: 8, and HCDR3 set forth in SEQ ID NO: 9; and the light chain variable region comprises the following complementarity determining regions CDRs: LCDR1 set forth in SEQ ID NO: 10, LCDR2 set forth in SEQ ID NO: 11, and LCDR3 as set forth in SEQ ID NO: 12; or (3) the heavy chain variable region comprises the complementarity determining regions (CDRs) of: HCDR1 as set forth in SEQ ID NO: 13, HCDR2 as set forth in SEQ ID NO: 43, and HCDR3 as set forth in SEQ ID NO: 33; and the light chain variable region comprises the complementarity determining regions (CDRs) of: LCDR1 as set forth in SEQ ID NO: 44, LCDR2 as set forth in SEQ ID NO: 35, and LCDR3 as set forth in SEQ ID NO: 36; or (4) the heavy chain variable region comprises the complementarity determining regions (CDRs) of: HCDR1 as set forth in SEQ ID NO: 13, HCDR2 as set forth in SEQ ID NO: 43, and HCDR3 as set forth in SEQ ID NO: 33; and the light chain variable region comprises the complementarity determining regions (CDRs) of: LCDR1 as set forth in SEQ ID NO: 44, LCDR2 as set forth in SEQ ID NO: 35, and LCDR3 as set forth in SEQ ID NO: 36; wherein the CDR sequences are numbered according to the Kabat numbering scheme; or the heavy chain variable region and the light chain variable region of the antibody or antigen-binding fragment thereof comprise the complementarity determining regions (CDRs) selected from the group consisting of: (1) the heavy chain variable region comprises the complementarity determining regions (CDRs) of: HCDR1 as set forth in SEQ ID NO: 50, HCDR2 as set forth in SEQ ID NO: 51, and HCDR3 as set forth in SEQ ID NO: 21; and the light chain variable region comprises the complementarity determining regions (CDRs) of: LCDR1 as set forth in SEQ ID NO: 22, LCDR2 as set forth in SEQ ID NO: 23, and LCDR3 as set forth in SEQ ID NO: 24; or (2) the heavy chain variable region comprises the complementarity determining regions (CDRs) of: HCDR1 as set forth in SEQ ID NO: 47, HCDR2 as set forth in SEQ ID NO: 48, and HCDR3 as set forth in SEQ ID NO: 9; and the light chain variable region comprises the complementarity determining regions (CDRs) of: LCDR1 as set forth in SEQ ID NO: 10, LCDR2 as set forth in SEQ ID NO: 11, and LCDR3 as set forth in SEQ ID NO: 12; or (3) the heavy chain variable region comprises the complementarity determining regions (CDRs) of: HCDR1 as set forth in SEQ ID NO: 54, HCDR2 as set forth in SEQ ID NO: 55, and HCDR3 as set forth in SEQ ID NO: 33; and the light chain variable region comprises the complementarity determining regions (CDRs) of: LCDR1 as set forth in SEQ ID NO: 34, LCDR2 as set forth in SEQ ID NO: 35, and LCDR3 as set forth in SEQ ID NO: 36; or (4) the heavy chain variable region comprises the complementarity determining regions (CDRs) of: HCDR1 as set forth in SEQ ID NO: 58, HCDR2 as set forth in SEQ ID NO: 59, and HCDR3 as set forth in SEQ ID NO: 33; and the light chain variable region comprises the complementarity determining regions (CDRs) of: LCDR1 as set forth in SEQ ID NO: 44, LCDR2 as set forth in SEQ ID NO: 35, and LCDR3 as set forth in SEQ ID NO:

36. HCDR2 of SEQ ID NO: 48, and HCDR3 of SEQ ID NO: 33; and the light chain variable region comprises the following complementarity determining regions (CDRs): LCDR1 of SEQ ID NO: 44, LCDR2 of SEQ ID NO: 35, and LCDR3 of SEQ ID NO: 36; wherein the CDR sequences are based on the numbering scheme of Chothia.

4. A polynucleotide comprising a nucleic acid sequence encoding a polypeptide of claim 1. The polynucleotide encodes the antibody or antigen-binding fragment thereof of claim 1, the recombinant protein of claim 2, or the chimeric antigen receptor (CAR) of claim 3.

5. A vector, characterized in that, The vector comprises the polynucleotide of claim 4.

6. A host cell comprising the vector of claim 5 or having integrated into its chromosome the exogenous polynucleotide of claim 4.

7. A method of making a CAR-NK cell or a CAR-T cell, characterized in that, The CAR-NK cell or CAR-T cell expresses the chimeric antigen receptor of claim 3, comprising the steps of: transducing the polynucleotide of claim 4 or the vector of claim 5 into a NK cell or a T cell, thereby obtaining the CAR-NK cell or the CAR-T cell.

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

9. An immunoconjugate, comprising, The immunoconjugate comprises: (a) an antibody moiety selected from the group consisting of: the antibody or antigen-binding fragment thereof of claim 1, the recombinant protein of claim 2, or a combination thereof; and (b) a conjugated moiety conjugated to the antibody moiety, the conjugated moiety selected from the group consisting of: a detectable label, a drug, or a combination thereof.

10. An immunoconjugate, comprising, The immunoconjugate comprises: (a) an antibody moiety selected from the group consisting of: the antibody or antigen-binding fragment thereof of claim 1, the recombinant protein of claim 2, or a combination thereof; and (b) a conjugated moiety conjugated to the antibody moiety, the conjugated moiety selected from the group consisting of: a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

11. Use of the antibody or antigen-binding fragment thereof of claim 1, the recombinant protein of claim 2, the chimeric antigen receptor of claim 3, the polynucleotide of claim 4, the vector of claim 5, the host cell of claim 6, the pharmaceutical composition of claim 8, or the immunoconjugate of claim 9 or 10, (a) preparing a detection reagent or kit for detecting a DLL3-associated disease; and / or (b) preparing a medicament or preparation for treating a DLL3-associated disease; the DLL3-associated disease is a tumor, and the tumor is a DLL3-positive tumor. wherein, The DLL3-positive tumor is a solid tumor 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 of non-diagnostic in vitro detection of DLL3 protein in a sample, comprising, The method comprises the steps of: (1) contacting a sample with the antibody or antigen-binding fragment thereof of claim 1, or the recombinant protein of 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 of producing a recombinant polypeptide, comprising, The method comprises: (a) culturing the host cell of claim 6 under conditions suitable for expression; (b) isolating from the culture the recombinant polypeptide, which is the antibody or antigen-binding fragment thereof of claim 1, or the recombinant protein of claim 2.

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

15. A kit comprising, The kit comprises: (1) a first container containing the antibody or antigen-binding fragment thereof of claim 1; and / or (2) a second container containing a secondary antibody to the antibody of claim 1; Alternatively, the kit contains the detection plate of claim 14.

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