DLL3-targeting antibody, chimeric antigen receptor, and use thereof

Abstract

Provided are a DLL3-targeting antibody or an antigen-binding fragment thereof, a chimeric antigen receptor (CAR) fusion protein comprising a DLL3-targeting antigen-binding domain, and an engineered immune cell (e.g., CAR-T cell) expressing the CAR fusion protein. The CAR-T cell has high affinity and biological activity and possesses good anti-tumor effects in vivo and in vitro.

Description

Antibodies targeting DLL3, chimeric antigen receptors and their applications Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to antibodies targeting DLL3, chimeric antigen receptors, and their applications. Background Technology

[0002] Delta-like ligand 3 (DLL3) is a type I transmembrane Delta-like protein that acts as an inhibitory Notch ligand and is primarily expressed on the cell membrane. Only a few normal cell types express DLL3 (e.g., neurons, pancreatic islet cells, and pituitary cells). However, DLL3 is highly expressed on the surface of small cell lung cancer (SCLC) cells, which account for approximately 15% of all lung cancer types. Although patients with this type of lung cancer respond well to first-line chemotherapy and radiotherapy, patients with extensive-stage disease often experience relapse, and survival beyond five years from diagnosis is extremely rare. Treatment options for relapsed or refractory small cell lung cancer are extremely limited, and existing treatments are often accompanied by significant treatment-related toxicities. Besides small cell lung cancer, DLL3 is also abnormally expressed in tumor cells such as large cell neuroendocrine carcinoma, gastrointestinal neuroendocrine tumors, glioblastoma multiforme, metastatic castration prostate cancer, small cell bladder cancer, and neuroendocrine lung tumors. Based on the high expression of DLL3 on the surface of homogeneous tumor cells and its low expression in normal cells and its limitation to the cytoplasm, DLL3 is considered an ideal target for the treatment of SCLC and other neuroendocrine tumors.

[0003] However, existing anti-human DLL3 antibodies have drawbacks such as insufficient affinity and specificity for DLL3 on the membrane surface, leading to off-target effects and adverse reactions.

[0004] Therefore, developing antibodies and chimeric antigen receptors that target DLL3 with better affinity and higher specificity is of great significance to this field. Summary of the Invention

[0005] This invention provides antibodies and antigen-chimeric receptors that target DLL3 with better affinity and stronger specificity.

[0006] In a first aspect of the invention, an antibody or antigen-binding fragment thereof targeting DLL3 is provided, said antibody or antigen-binding fragment having a heavy chain variable region and a light chain variable region, wherein the complementarity-determining region HCDR of the heavy chain variable region and the complementarity-determining region LCDR of the light chain variable region are selected from the group consisting of:

[0007] (1) The amino acid sequence of HCDR1 is shown in SEQ ID NO:23.

[0008] The amino acid sequence HCDR2 is shown in SEQ ID NO:24.

[0009] The amino acid sequence is HCDR3 as shown in SEQ ID NO:25.

[0010] The amino acid sequence of LCDR1 is shown in SEQ ID NO:27.

[0011] The amino acid sequence is LCDR2 as shown in SEQ ID NO:28, and

[0012] The amino acid sequence is LCDR3 as shown in SEQ ID NO:29;

[0013] (2) The amino acid sequence of HCDR1 is shown in SEQ ID NO:47.

[0014] The amino acid sequence HCDR2 is shown in SEQ ID NO:48.

[0015] The amino acid sequence HCDR3 is shown in SEQ ID NO:49.

[0016] The amino acid sequence of LCDR1 is shown in SEQ ID NO:51.

[0017] The amino acid sequence is LCDR2 as shown in SEQ ID NO:52, and

[0018] The amino acid sequence is LCDR3 as shown in SEQ ID NO:53;

[0019] (3) The amino acid sequence of HCDR1 is as shown in SEQ ID NO:56.

[0020] The amino acid sequence HCDR2 is shown in SEQ ID NO:57.

[0021] The amino acid sequence HCDR3 is shown in SEQ ID NO:58.

[0022] The amino acid sequence of LCDR1 is shown in SEQ ID NO:60.

[0023] The amino acid sequence is LCDR2 as shown in SEQ ID NO:61, and

[0024] The amino acid sequence is LCDR3 as shown in SEQ ID NO:62;

[0025] (4) The amino acid sequence of HCDR1 is as shown in SEQ ID NO:5.

[0026] The amino acid sequence HCDR2 is shown in SEQ ID NO:6.

[0027] The amino acid sequence is HCDR3 as shown in SEQ ID NO:7.

[0028] The amino acid sequence of LCDR1 is shown in SEQ ID NO:9.

[0029] The amino acid sequence is LCDR2 as shown in SEQ ID NO:10, and

[0030] The amino acid sequence is LCDR3 as shown in SEQ ID NO:11;

[0031] (5) The amino acid sequence of HCDR1 is shown in SEQ ID NO:14.

[0032] The amino acid sequence HCDR2 is shown in SEQ ID NO:15.

[0033] The amino acid sequence HCDR3 is shown in SEQ ID NO:16.

[0034] The amino acid sequence of LCDR1 is shown in SEQ ID NO:18.

[0035] The amino acid sequence is LCDR2 as shown in SEQ ID NO:19, and

[0036] The amino acid sequence is LCDR3 as shown in SEQ ID NO:20;

[0037] (6) The amino acid sequence of HCDR1 is as shown in SEQ ID NO:32.

[0038] The amino acid sequence is HCDR2 as shown in SEQ ID NO:33.

[0039] The amino acid sequence HCDR3 is shown in SEQ ID NO:34.

[0040] The amino acid sequence of LCDR1 is shown in SEQ ID NO:36.

[0041] The amino acid sequence is LCDR2 as shown in SEQ ID NO:19, and

[0042] The amino acid sequence is LCDR3 as shown in SEQ ID NO:37;

[0043] (7) The amino acid sequence of HCDR1 is as shown in SEQ ID NO:40.

[0044] The amino acid sequence HCDR2 is shown in SEQ ID NO:41.

[0045] The amino acid sequence HCDR3 is shown in SEQ ID NO:42.

[0046] The amino acid sequence of LCDR1 is shown in SEQ ID NO:9.

[0047] The amino acid sequence is LCDR2 as shown in SEQ ID NO:44, and

[0048] The amino acid sequence is LCDR3 as shown in SEQ ID NO:11.

[0049] In another preferred embodiment, the antibody or its antigen-binding fragment is of murine or human origin.

[0050] In another preferred embodiment, when the antibody or its antigen-binding fragment is murine, the heavy chain variable region and the light chain variable region are selected from the group consisting of:

[0051] (a) The heavy chain variable region with amino acid sequence as shown in SEQ ID NO:22, and the light chain variable region with amino acid sequence as shown in SEQ ID NO:26;

[0052] (b) The heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:46, and the light chain variable region with an amino acid sequence as shown in SEQ ID NO:50;

[0053] (c) The heavy chain variable region with amino acid sequence as shown in SEQ ID NO:55, and the light chain variable region with amino acid sequence as shown in SEQ ID NO:59;

[0054] (d) The heavy chain variable region with amino acid sequence as shown in SEQ ID NO:4, and the light chain variable region with amino acid sequence as shown in SEQ ID NO:8;

[0055] (e) The heavy chain variable region with the amino acid sequence shown in SEQ ID NO:13, and the light chain variable region with the amino acid sequence shown in SEQ ID NO:17;

[0056] (f) The heavy chain variable region with amino acid sequence as shown in SEQ ID NO:31, and the light chain variable region with amino acid sequence as shown in SEQ ID NO:35;

[0057] (g) The heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:39, and the light chain variable region with an amino acid sequence as shown in SEQ ID NO:43.

[0058] In another preferred embodiment, when the antibody or its antigen-binding fragment is humanized, the heavy chain variable region and the light chain variable region are selected from the group consisting of:

[0059] (a) The heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:64, and the light chain variable region with an amino acid sequence as shown in SEQ ID NO:65;

[0060] (b) The heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:67, and the light chain variable region with an amino acid sequence as shown in SEQ ID NO:68;

[0061] (c) The heavy chain variable region with amino acid sequence as shown in SEQ ID NO:67, and the light chain variable region with amino acid sequence as shown in SEQ ID NO:70;

[0062] (d) The heavy chain variable region with amino acid sequence as shown in SEQ ID NO:72, and the light chain variable region with amino acid sequence as shown in SEQ ID NO:73.

[0063] In another preferred embodiment, the antibody is a murine antibody, a chimeric antibody, or a humanized antibody.

[0064] In another preferred embodiment, the antibody or its antigen-binding fragment comprises an intact antibody, Fab, single-chain antibody (scFv), or nanobody.

[0065] In another preferred embodiment, the CDR region of the antibody or its antigen-binding fragment contains an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95% sequence similarity to any of the above sequences.

[0066] In another preferred embodiment, any of the above-mentioned amino acid sequences further includes a derived sequence that has optionally been added, deleted, modified and / or substituted at least one amino acid and is capable of retaining DLL3 binding affinity.

[0067] In another preferred embodiment, the number of added, deleted, modified and / or substituted amino acids is 1-3, more preferably 1-2, and even more preferably 1.

[0068] In a preferred embodiment, the antibody or its antigen-binding fragment is a single-chain antibody (scFv).

[0069] In another preferred embodiment, the single-chain antibody sequentially comprises a light chain variable region, a linker, and a heavy chain variable region, or sequentially comprises a heavy chain variable region, a linker, and a light chain variable region.

[0070] In another preferred embodiment, the antibody is a monoclonal antibody.

[0071] In another preferred embodiment, the antibody includes monospecific, bispecific, or trispecific antibodies.

[0072] In a second aspect of the invention, a chimeric antigen receptor (CAR) fusion protein is provided, the chimeric antigen receptor fusion protein comprising, from the N-terminus to the C-terminus:

[0073] (i) A single-chain antibody, wherein the heavy chain variable region and the light chain variable region of the single-chain antibody respectively comprise the HCDR and LCDR described in the first aspect of the present invention;

[0074] (ii) Transmembrane structural domains

[0075] (iii) at least one co-stimulatory domain, and

[0076] (iv) Activate the structural domain.

[0077] In another preferred embodiment, the chimeric antigen receptor has the structure shown in Formula I:

[0078] L-scFv-H-TM-CS(I)

[0079] In the formula,

[0080] Each "-" independently represents a linking peptide or peptide bond;

[0081] L is an optional signal peptide sequence;

[0082] scFv is a single-chain antibody, and the heavy chain variable region and light chain variable region of the single-chain antibody respectively include HCDR and LCDR as described in the first aspect of the present invention;

[0083] H is an optional hinge area;

[0084] TM represents a transmembrane domain;

[0085] C is a co-stimulatory signaling molecule;

[0086] S represents the cytoplasmic signal transduction sequence.

[0087] In another preferred embodiment, the signal peptide of L is CD8.

[0088] In another preferred embodiment, the L comprises an amino acid sequence as shown in SEQ ID NO:74.

[0089] In another preferred embodiment, H is a hinge region of a protein selected from the group consisting of CD8, CD28, or a combination thereof.

[0090] In another preferred embodiment, C is a co-stimulatory signaling molecule selected from the group consisting of: 4-1BB, CD27, CD28, OX40, CD30, CD40, CD40L, CD70, CD2, LFA-1, LIGHT, NKG2C, B7-H3, PD-1, ICOS, CDS, ICAM-1, GITR, BAFFR, HVEM, SLAMF7, CD7, NKp80, CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1 (CD11a / CD18), ITGAM, CD11b, ITGAX, CD11c, ITGB1, ITGB2, KLRC2, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TNFRSF18, TNFRSF14, TRANCE / RANKL, DNAM1, SLAMF4, CD84, CD96, CEACAM1, CRT AM, Ly9, CD160, PSGL1, CD100, CD69, SLAMF6, SLAM, BLAME, SELPLG, LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, HAVCR1, LGALS9, Dap10, DAP12, CDS, ICAM-1, NKG2D, GITR, TLR2, TMIGD2, or combinations thereof.

[0091] In another preferred embodiment, the amino acid sequence of C is shown in SEQ ID NO:77.

[0092] In another preferred embodiment, the TM is a transmembrane region of a protein selected from the group consisting of: CD8, CD28, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a combination thereof.

[0093] In another preferred embodiment, the S (cytoplasmic transduction sequence) is a cytoplasmic transduction sequence selected from the following groups: CD3ζ, CD3γ, CD3δ, CD3ε, Fc receptor γ chain, FcRβ, CD79a, CD79b, FcγRIIa, DAP10, DAP12, NKp44, NKp30, NKp46, NKG2D.

[0094] In a third aspect of the invention, a recombinant protein is provided, said recombinant protein having:

[0095] (i) an antibody or antigen-binding fragment thereof as described in the first aspect of the invention; and

[0096] (ii) Optional tag sequences to assist in expression and / or purification.

[0097] In another preferred embodiment, the tag sequence includes a 6His tag.

[0098] In another preferred embodiment, the recombinant protein (or polypeptide) includes a fusion protein.

[0099] In another preferred embodiment, the recombinant protein is a monomer, a dimer, or a polymer.

[0100] In a fourth aspect of the invention, an antibody-drug conjugate is provided, the antibody-drug conjugate comprising:

[0101] (a) an antibody or antigen-binding fragment thereof as described in the first aspect of the invention, or a chimeric antigen receptor fusion protein as described in the second aspect of the invention; and

[0102] (b) A conjugation portion conjugated to the antibody portion, the conjugation portion being selected from the group consisting of: detectable markers, drugs, toxins, cytokines, radionuclides, enzymes, or combinations thereof.

[0103] In another preferred embodiment, the antibody portion is coupled to the coupling portion via a chemical bond or a linker.

[0104] In a fifth aspect of the invention, a polynucleotide is provided, said polynucleotide encoding a polypeptide selected from the group consisting of:

[0105] (1) The antibody or antigen-binding fragment thereof as described in the first aspect of the present invention;

[0106] (2) The chimeric antigen receptor fusion protein as described in the second aspect of the present invention; or

[0107] (3) The recombinant protein as described in the third aspect of the present invention.

[0108] In another preferred embodiment, when the polynucleotide encodes a chimeric antigen receptor fusion protein as described in the second aspect of the invention, the polynucleotide further comprises a polynucleotide sequence encoding a chimeric switching receptor (CSR) or a dominant-negative receptor (DNR).

[0109] In another preferred embodiment, the polynucleotide sequence encoding the chimeric switching receptor (CSR) or dominant-negative receptor (DNR) is selected from the group consisting of: a polynucleotide sequence encoding the PD-1 dominant-negative receptor (PD-1 DNR), a polynucleotide sequence encoding the PD-1 chimeric switching receptor (PD-1 CSR), and a polynucleotide sequence encoding the TGF-β dominant-negative receptor (TGF-βDNR).

[0110] In another preferred embodiment, the TGF-βDNR is TGFbRII DNR.

[0111] In another preferred embodiment, the PD-1 DNR comprises an amino acid sequence as shown in SEQ ID NO:80, or an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:80.

[0112] In another preferred embodiment, the PD-1 CSR comprises an amino acid sequence as shown in SEQ ID NO:81, or an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:81.

[0113] In another preferred embodiment, the TGFbRII DNR comprises an amino acid sequence as shown in SEQ ID NO:82, or an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:82.

[0114] In another preferred embodiment, the polynucleotide sequence encoding the chimeric switching receptor (CSR) or dominant-negative receptor (DNR) is linked to the polynucleotide sequence encoding the chimeric antigen receptor fusion protein via a polynucleotide sequence encoding a 2A self-cleaving peptide.

[0115] In another preferred embodiment, the 2A self-cleaving peptide is selected from the group consisting of: T2A peptide, P2A peptide, F2A peptide, and E2A peptide.

[0116] In another preferred embodiment, when the polynucleotide encodes the CAR fusion protein as described in the second aspect of the invention, the polynucleotide comprises, in the 5' to 3' direction: a polynucleotide sequence encoding the CAR fusion protein, a polynucleotide sequence encoding the 2A self-cleaving peptide, and a polynucleotide sequence encoding the chimeric switching receptor (CSR) or dominant-negative receptor (DNR); or a polynucleotide sequence encoding the chimeric switching receptor (CSR) or dominant-negative receptor (DNR), a polynucleotide sequence encoding the 2A self-cleaving peptide, and a polynucleotide sequence encoding the CAR fusion protein.

[0117] In another preferred embodiment, when the polynucleotide encodes the CAR fusion protein as described in the second aspect of the invention, the polynucleotide comprises, in the 5' to 3' direction, a polynucleotide sequence encoding the chimeric antigen receptor fusion protein, a polynucleotide sequence encoding the 2A self-cleaving peptide, and a polynucleotide sequence encoding PD-1 DNR, a polynucleotide sequence encoding PD-1 CSR, or a polynucleotide sequence encoding TGFbRII DNR.

[0118] In another preferred embodiment, when the polynucleotide encodes the CAR fusion protein as described in the second aspect of the invention, the amino acid sequence encoded by the polynucleotide is selected from the group consisting of: SEQ ID NO:84 (CAR-T2A-PD-1 DNR), SEQ ID NO:85 (CAR-T2A-PD-1 CSR), SEQ ID NO:86 (CAR-T2A-TGRbRII DNR); or an amino acid sequence having at least 95% sequence identity with the above-described amino acid sequences.

[0119] In a sixth aspect of the invention, a carrier is provided, said carrier containing the polynucleotide as described in the fifth aspect of the invention.

[0120] In another preferred embodiment, the vector includes: bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, lentiviruses, exosomes, or other vectors.

[0121] In a seventh aspect of the invention, a genetically engineered host cell is provided, the host cell containing the vector described in the sixth aspect of the invention, or having a genome integrated with a polynucleotide as described in the fifth aspect of the invention, or expressing an antibody or its antigen-binding fragment as described in the first aspect of the invention, or a chimeric antigen receptor fusion protein as described in the second aspect of the invention.

[0122] In another preferred embodiment, the cells are isolated cells, and / or the cells are genetically engineered cells.

[0123] In another preferred embodiment, the cell is a mammalian cell.

[0124] In another preferred embodiment, the host cell is an engineered immune cell.

[0125] In another preferred embodiment, the engineered immune cells are T cells, macrophages, or NK cells.

[0126] In another preferred embodiment, the engineered immune cells include T cells, macrophages or NK cells, preferably (i) chimeric antigen receptor T cells (CAR-T cells); or (ii) chimeric antigen receptor NK cells (CAR-NK cells).

[0127] In another preferred embodiment, the host cell also expresses CSR or DNR.

[0128] In another preferred embodiment, the CSR is a PD-1 CSR.

[0129] In another preferred embodiment, the DNR is PD-1 DNR or TGF-βDNR.

[0130] In another preferred embodiment, the DNR is a PD-1 DNR or a TGFbRII DNR.

[0131] In another preferred embodiment, the PD-1 CSR comprises an amino acid sequence as shown in SEQ ID NO:80, or an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:80.

[0132] In another preferred embodiment, the PD-1 DNR comprises an amino acid sequence as shown in SEQ ID NO:81, or an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:81.

[0133] In another preferred embodiment, the TGFbRII DNR comprises an amino acid sequence as shown in SEQ ID NO:82, or an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:82.

[0134] In another preferred embodiment, when the host cell expresses the CAR fusion protein as described in the second aspect of the invention, the CAR fusion protein is co-expressed with the CSR or the DNR via a 2A self-cleaving peptide.

[0135] In another preferred embodiment, the 2A self-cleaving peptide is selected from the group consisting of: T2A peptide, P2A peptide, F2A peptide, and E2A peptide.

[0136] In another preferred embodiment, the host cell expresses the CAR fusion protein and the PD-1 CSR, the amino acid sequence of which comprises the amino acid sequence shown in SEQ ID NO:84.

[0137] In another preferred embodiment, the host cell expresses the CAR fusion protein and the PD-1 DNR, the amino acid sequence of which comprises the amino acid sequence shown in SEQ ID NO:85.

[0138] In another preferred embodiment, the host cell expresses the CAR fusion protein and the TGFbRIIDNR, the amino acid sequence of which comprises the amino acid sequence shown in SEQ ID NO:86.

[0139] In an eighth aspect of the present invention, a method for preparing engineered immune cells expressing a CAR fusion protein as described in the second aspect of the present invention is provided, comprising the steps of: transducing a polynucleotide as described in the fifth aspect of the present invention or a vector as described in the sixth aspect of the present invention into T cells or NK cells, thereby obtaining the engineered immune cells.

[0140] In another preferred embodiment, the method further includes a step of testing the function and effectiveness of the obtained engineered immune cells.

[0141] In a ninth aspect of the invention, the use of an antibody or antigen-binding fragment thereof as described in the first aspect of the invention, a chimeric antigen receptor fusion protein as described in the second aspect of the invention, a vector as described in the sixth aspect of the invention, or a host cell as described in the seventh aspect of the invention, for the preparation of a medicament or preparation for the prevention and / or treatment of DLL3-positive cancers or tumors, is provided.

[0142] In another preferred embodiment, the cancer or tumor is selected from the group consisting of: small cell lung cancer, large cell neuroendocrine carcinoma, gastrointestinal neuroendocrine tumor, glioblastoma multiforme, metastatic castration prostate cancer, small cell bladder cancer, neuroendocrine lung tumor, or a combination thereof.

[0143] In a tenth aspect of the present invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising:

[0144] (1) An active ingredient selected from the group consisting of: the antibody or its antigen-binding fragment described in the first aspect of the present invention, the chimeric antigen receptor fusion protein described in the second aspect of the present invention, the vector described in the sixth aspect of the present invention, or the host cell described in the seventh aspect of the present invention; and

[0145] (2) Pharmaceutically acceptable carrier.

[0146] In another preferred embodiment, the carrier is selected from the group consisting of diluents, excipients, or combinations thereof.

[0147] In an eleventh aspect of the invention, a kit is provided for preparing cells as described in the seventh aspect of the invention, the kit comprising a container and a polynucleotide as described in the fifth aspect of the invention or a carrier as described in the sixth aspect of the invention located within the container.

[0148] In a twelfth aspect of the invention, a method for treating a disease is provided, comprising administering to a subject in need an appropriate amount of the cells described in the seventh aspect of the invention, or the preparation described in the tenth aspect of the invention.

[0149] In another preferred embodiment, the disease is a DLL3-positive cancer or tumor.

[0150] In another preferred embodiment, the disease is a cancer or tumor with high DLL3 expression.

[0151] In another preferred embodiment, the cancer or tumor is selected from the group consisting of: small cell lung cancer, large cell neuroendocrine carcinoma, gastrointestinal neuroendocrine tumor, glioblastoma multiforme, metastatic castration prostate cancer, small cell bladder cancer, neuroendocrine lung tumor, or a combination thereof.

[0152] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0153] Figure 1 shows the expression of DLL3 in NCI-H82 and SHP-77 cells; the left peak is the control peak, and the right peak is the DLL3 positive peak.

[0154] Figure 2 shows the positive rate of CAR-T cells.

[0155] Figures 3a-3f show the short-term killing effect of CAR-T cells corresponding to D3001-D3034 on NCI-H82 and SHP-77 cells at effective target ratios of 3:1, 1:1 and 0.3:1.

[0156] Figures 4a-4f show the trend of CD3+ cell proportion in the long-term killing of NCI-H82 and SHP-77 cells by CAR-T cells corresponding to D3001 to D3034.

[0157] Figures 5a-5d show the comparison of short-term killing effects of CAR-T cells overexpressing human DLL3, human DLL1, and human DLL4 on CHO-K1 cell lines and CHO-K1 cell lines at effective target ratios of 3:1, 1:1, and 0.3:1, corresponding to D3005, D3012-D3016, D3018, D3022-D3025, D3027, D3028, and D3031-D3034.

[0158] Figures 6a-6b show the comparison of short-term killing effects of CAR-T cells overexpressing human DLL3 and mouse DLL3 in CHO-K1 cell lines at effective target ratios of 3:1, 1:1 and 0.3:1, corresponding to D3005, D3012-D3016, D3018, D3022-D3025, D3027, D3028 and D3031-D3034.

[0159] Figures 7a-7d show the trend of CD3+ cell proportion in the long-term killing of NCI-H82 and SHP-77 cells by CAR-T cells corresponding to D3005, D3012-D3016, D3018, D3022-D3025, D3027, D3028, and D3031-D3034.

[0160] Figures 8a and 8b show the long-term killing effect of the preferred mouse CAR-T on NCI-H82-3D cell cultures.

[0161] Figure 9 shows: a. growth trend of tumors in NPG mice after CAR-T cell infusion; b. weight change curves of NPG mice in each group; c. metabolic kinetics curves of T cells in NPG mice; d. metabolic kinetics curves of CAR-T cells in NPG mice.

[0162] Figures 10a-10b show the short-term and long-term killing effects of humanized CAR-T cells containing the preferred D3018, D3031, and D3034 on NCI-H82 cells.

[0163] Figures 11a-11d show the short-term killing effects of humanized CAR-T cells of preferred D3018, D3031 and D3034 and parental CAR-T cells on NCI-H82 cells.

[0164] Figures 12a-12d show the long-term killing effects of humanized CAR-T cells of preferred D3018, D3031 and D3034 and maternal CAR-T cells on NCI-H82 cells.

[0165] Figures 13a-13d show the long-term killing effects of humanized CAR-T cells of preferred D3018, D3031 and D3034, and parental CAR-T cells, on NCI-H82-3D cultures.

[0166] Figures 14a-14d show the comparison of short-term killing effects of CAR-T cells corresponding to D3018, D3031, D3034 and their preferred humanized sequences on CHO-K1 cell lines overexpressing human DLL3, human DLL1 and human DLL4 at effective target ratios of 3:1, 1:1 and 0.3:1.

[0167] Figures 15a-15b show the comparison of short-term killing effects of CAR-T cells corresponding to D3018, D3031, D3034 and their preferred humanized sequences on CHO-K1 cell lines overexpressing human DLL3, human DLL1 and human DLL4 at effective target ratios of 3:1, 1:1 and 0.3:1.

[0168] Figure 16 shows a. the growth trend of tumors in NPG mice after 1.5E6 CAR-T cell infusion; b. the body weight change curves of NPG mice in each group; c. the metabolic kinetics curves of T cells in NPG mice; and d. the metabolic kinetics curves of CAR-T cells in NPG mice.

[0169] Figure 17 shows a. the growth trend of tumors in NPG mice after 3E6 CAR-T cell infusion; b. the body weight change curves of NPG mice in each group; c. the metabolic kinetics curves of T cells in NPG mice; and d. the metabolic kinetics curves of CAR-T cells in NPG mice.

[0170] Figure 18 shows the in vivo efficacy of D3045 and D3058 and the metabolic kinetics of T cells / CAR-T cells in NPG mice at doses of 0.5E6, 1E6, and 2E6. ad: In vivo efficacy of D3045 and D3058 and the metabolic kinetics of T cells / CAR-T cells in NPG mice at a dose of 0.5E6. eh: In vivo efficacy of D3045 and D3058 and the metabolic kinetics of T cells / CAR-T cells in NPG mice at a dose of 1E6. il: In vivo efficacy of D3045 and D3058 and the metabolic kinetics of T cells / CAR-T cells in NPG mice at a dose of 2E6.

[0171] Figure 19 shows the in vivo safety validation of D3045 and D3058. a. Tumor growth trend in NPG mice after 10E6 CAR-T cell infusion; b. Body weight change curves of NPG mice in each group; c. T cell metabolic kinetics curves in NPG mice; d. CAR-T cell metabolic kinetics curves in NPG mice.

[0172] Figure 20 shows the in vivo efficacy comparison of the preferred humanized sequences D3059 and D3067 of D3045 and D3034. a. Tumor growth trend in NPG mice after 1.5E6 CAR-T cell infusion; b. Body weight change curves of NPG mice in each group; c. T cell metabolic kinetics curves in NPG mice; d. CAR-T cell metabolic kinetics curves in NPG mice.

[0173] Figure 21 shows a. in vivo safety validation of D3059 and D3067. a. growth trend of tumors in NPG mice after 10E6 CAR-T cell infusion; b. body weight change curves of NPG mice in each group; c. T cell metabolic kinetics curves in NPG mice; d. CAR-T cell metabolic kinetics curves in NPG mice.

[0174] Figure 22 shows a schematic diagram of DLL3 and its truncated body structure.

[0175] Figure 23 shows a comparison of the short-term killing effect of CAR-T cells corresponding to the preferred mouse sequences D3018, D3031, and D3034 on CHO-K1 cell lines overexpressing full-length human DLL3 and its truncated variants, as well as the CHO-K1 cell line itself, at an effector-to-target ratio of 3:1.

[0176] Figure 24 shows a comparison of the short-term killing effect of CAR-T cells corresponding to the preferred humanized sequence D3045 at an effective target ratio of 3:1 on CHO-K1 cell lines overexpressing full-length human DLL3 and its truncated form, as well as the CHO-K1 cell line.

[0177] Figures 25a-25d show the long-term killing effects of D3034, D3045 / PD-1 DNR, and D3045 / PD-1 CSR on NCI-H889 cells and the expansion curves of T cells / CAR-T cells.

[0178] Figures 26a-26d show the long-term killing effect of D3034 and D3045 / TGFbRII DNR on NCI-H889 cells and the expansion curves of T cells / CAR-T cells in media supplemented with 0 ng / mL TGF-β(-) and 5 ng / mL TGF-β(+). Detailed Implementation

[0179] Through extensive and in-depth research and screening, the inventors have developed an antibody or antigen-binding fragment (e.g., a single-chain antibody) targeting DLL3, a chimeric antigen receptor comprising the single-chain antibody, and CAR-immune cells (e.g., CAR-T cells) expressing the chimeric antigen receptor. This invention also provides humanized CAR-T cells with reduced immunogenicity.

[0180] This invention, through the screening and careful design of CAR element structures (including but not limited to the screening of scFvs suitable for CAR expression and tumor-killing effects), enables the CAR-immune cells of this invention to specifically kill DLL3-expressing tumor cells without killing normal cells, thus improving safety. Simultaneously, the CAR-immune cells of this invention exhibit a high killing rate against tumor cells. This invention provides a new approach for the treatment of DLL3-positive tumors. This invention was completed based on these findings.

[0181] Specifically, experiments of this invention show that the murine antibodies corresponding to D3001–D3034 of this invention all have good affinity. Short-term killing, long-term killing, and in vitro killing experiments with 3D cell cultures revealed that the murine CAR-T cells corresponding to D3012, D3014, D3018, D3025, D3027, D3031, and D3034 of this invention have good killing effects. Based on this, humanized CAR-T cells were constructed, and the results showed that the humanized DLL3-targeting CAR-T cells corresponding to D3045, D3056, D3058, and D3059 of this invention have excellent short-term and long-term killing effects on tumor cells in vitro, as well as in vivo anti-tumor activity.

[0182] the term

[0183] To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.

[0184] The term “about” can refer to a value or composition within an acceptable range of error for a particular value or composition as determined by a person skilled in the art, which will depend in part on how the value or composition is measured or determined.

[0185] The term “administration” means the physical introduction of the product of the present invention into a subject using any of the various methods and delivery systems known to those skilled in the art, including intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, such as by injection or infusion.

[0186] The term "isolation" refers to the fact that a substance is substantially or substantially free of the components that normally accompany it in its natural state. The substance can be a cell or a macromolecule, such as a protein or nucleic acid. For example, as used herein, "isolated nucleic acid" refers to a polynucleotide purified from a sequence located laterally in its natural state, such as a DNA fragment removed from its normally adjacent sequence. Alternatively, as used herein, "isolated antibody" or "isolated polypeptide" and similar terms refer to antibody or polypeptide molecules that have been isolated and / or purified in vitro from their natural cellular environment and association with other components of the cell.

[0187] The term "antibody" (Ab) should include, but is not limited to, immunoglobulins that specifically bind to antigens and comprise at least two heavy (H) chains and two light (L) chains linked by disulfide bonds, or their antigen-binding portions. Each H chain contains a heavy chain variable region (abbreviated VH) and a heavy chain constant region. The heavy chain constant region contains three constant domains CH1, CH2, and CH3. Each light chain contains a light chain variable region (abbreviated VL) and a light chain constant region. The light chain constant region contains one constant domain CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each VH and VL contains three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen.

[0188] It should be understood that the amino acid names in this article adopt the internationally accepted single-letter identifiers, and the corresponding three-letter abbreviations of the amino acid names are: Ala (A), Arg (R), Asn (N), Asp (D), Cys (C), Gln (Q), Glu (E), Gly (G), His (H), I1e (I), Leu (L), Lys (K), Met (M), Phe (F), Pro (P), Ser (S), Thr (T), Trp (W), Tyr (Y), Val (V).

[0189] Chimeric antigen receptor (CAR)-immune cells

[0190] As used herein, the terms “chimeric antigen receptor (CAR)-immune cell”, “CAR-immune cell”, and “immune cell of the present invention” are used interchangeably and all refer to the specific CAR-immune cell targeting DLL3 described in the first aspect of the present invention.

[0191] The CAR-immune cells of the present invention, except for a specific extracellular binding domain, all have the structure of chimeric antigen receptors conventional in the art.

[0192] The chimeric antigen receptor (CAR) of this invention comprises an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain includes a target-specific binding element (also referred to as an antigen-binding domain). The intracellular domain includes a co-stimulatory signaling region and a ζ-chain portion. The co-stimulatory signaling region refers to a portion of the intracellular domain containing a co-stimulatory molecule. The co-stimulatory molecule is a cell surface molecule required for an effective lymphocyte response to an antigen, rather than an antigen receptor or its ligands.

[0193] A linker may be incorporated between the extracellular and transmembrane domains of the CAR, or between the cytoplasmic and transmembrane domains of the CAR. As used herein, the term "linker" generally refers to any oligopeptide or polypeptide that functions to link the transmembrane domain to the extracellular or cytoplasmic domain of the polypeptide chain. Linkers may comprise 0-300 amino acids, preferably 2 to 100 amino acids, and most preferably 3 to 50 amino acids.

[0194] As used herein, "antigen-binding domain" and "single-chain antibody fragment" refer to Fab fragments, Fab' fragments, F(ab')2 fragments, or single Fv fragments with antigen-binding activity. Fv antibodies contain variable regions of the antibody heavy chain and light chain, but no constant region, and are the smallest antibody fragments possessing all antigen-binding sites. Generally, Fv antibodies also contain a polypeptide linker between the VH and VL domains and are capable of forming the structure required for antigen binding. The antigen-binding domain is typically scFv (single-chain variable fragment). The size of an scFv is generally 1 / 6 that of a complete antibody. Single-chain antibodies are preferably a single amino acid chain sequence encoded by a single nucleotide chain. As a preferred embodiment of the invention, the scFv contains an antibody that specifically recognizes DLL3, preferably a single-chain antibody.

[0195] 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 implementation, 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 a domain to the transmembrane domain of the same or different surface membrane proteins, thereby minimizing interactions with other members of the receptor complex.

[0196] PD-1 DNR, PD-1 CSR and TFGbRII DNR

[0197] Overexpressed PD-L1 on tumor cells can specifically recognize and bind to PD-1 on activated T cells, leading to the loss of T cell killing ability. Expressing a dominant-negative PD-1 receptor (DNR) on the surface of CAR-T cells allows PD-1 DNR to bind to PD-L1 on tumor cells without activating the internal PD-1 pathway on T cells, thus overcoming the inhibitory effect of tumor cell surface PD-L1 on CAR-T cells. Furthermore, by replacing the native inhibitory signaling domain of T cell PD-1 with an activating signaling domain, such as the CD28 intracellular domain, the PD-L1-induced T cell PD-1 inhibitory signal can be converted into an activating signal for CAR-T cells, enhancing CAR-T function.

[0198] PD-1 CSR (PD-1 chimeric conversion receptor) is a genetically engineered receptor that specifically recognizes and binds to PD-L1 molecules. Unlike traditional PD-1 receptors, PD-1 CSR does not transmit inhibitory signals after binding to PD-L1; instead, it triggers the activation and proliferation of CAR-T cells.

[0199] TGFβ is a key molecule in the tumor immunosuppressive microenvironment. By binding to the TGFβ receptor on the surface of T cells, it transmits signals that inhibit T cells' anti-tumor activity, causing T cells to lose their ability to kill tumor cells. CAR-T cells express a dominant-negative TGFβ receptor II (DNR), TGFβRIIDNR (or TFGbRII DNR), which can bind to TGFβ on tumor cells but does not activate the TGFβ pathway within T cells. This can overcome the inhibitory effect of TGFβ on CAR-T cells in the tumor immunosuppressive microenvironment.

[0200] carrier

[0201] The nucleic acid sequence encoding the desired molecule can be obtained using recombination methods known in the art, such as, for example, by screening a library from a cell expressing the gene, by obtaining the gene from a vector known to contain the gene, or by directly isolating the gene from cells and tissues containing the gene using standard techniques. Optionally, the gene of interest can be synthesized.

[0202] This invention also provides vectors in which the expression cassette of this invention is inserted. Vectors derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer because they allow for long-term, stable integration of transgenes and their proliferation in daughter cells. Lentiviral vectors have advantages over vectors derived from oncogenic retroviruses, such as murine leukemia viruses, because they can transduce non-proliferating cells, such as hepatocytes. They also have the advantage of low immunogenicity.

[0203] In short, the expression cassette or nucleic acid sequence of this invention is typically operatively linked to a promoter and incorporated into an expression vector. This vector is suitable for replication and integration into eukaryotic cells. A typical cloning vector contains transcription and translation terminators, an initial sequence, and a promoter that can be used to regulate the expression of the desired nucleic acid sequence.

[0204] The expression constructs of the present invention can also be used with standard gene delivery protocols for nucleic acid immunotherapy and gene therapy. Methods of gene delivery are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, and 5,589,466, which are incorporated herein by reference in their entirety. In another embodiment, the present invention provides a gene therapy vector.

[0205] This nucleic acid can be cloned into many types of vectors. For example, this nucleic acid can be cloned into vectors including, but not limited to, plasmids, phage particles, phage derivatives, animal viruses, and granules. Specific vectors of interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0206] Furthermore, the expression vector can be provided to cells in the form of a viral vector. Viral vector technology is well known in the art and has been described, for example, in 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, herpesviruses, and lentiviruses. Typically, a suitable vector contains at least one origin of replication functioning in an organism, a promoter sequence, a convenient restriction enzyme site, and one or more optional markers (e.g., WO01 / 96584; WO01 / 29058; and U.S. Patent No. 6,326,193).

[0207] Many virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Selected genes can be inserted into vectors and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to target cells in vivo or in vitro. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In one embodiment, lentiviral vectors are used.

[0208] Additional promoter elements, such as enhancers, can regulate the frequency of transcription initiation. These are typically located in a 30–110 bp region upstream of the start site, although recent studies have shown that many promoters also contain functional elements downstream of the start site. The spacing between promoter elements is often flexible to maintain promoter function when an element is inverted or moved relative to another. In the thymidine kinase (TK) promoter, the spacing between promoter elements can be increased to 50 bp before activity begins to decline. Depending on the promoter, individual elements can function cooperatively or independently to initiate transcription.

[0209] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strongly constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operatively linked thereto. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to the early promoter of simian virus 40 (SV40), mouse mammary cancer virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Russ's sarcoma virus promoter, and human gene promoters, such as, but not limited to, actin promoter, myosin promoter, heme promoter, and creatine kinase promoter. Furthermore, the invention should not be limited to the application of constitutive promoters. Inducible promoters are also considered as part of the invention. The use of inducible promoters provides a molecular switch that can turn on the expression of a polynucleotide sequence operatively linked to the inducible promoter when such expression is desired, or turn off expression when expression is undesirable. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0210] To assess the expression of CAR peptides or portions thereof, the expression vector introduced into cells may also contain either or both of an optional marker gene or a reporter gene to facilitate the identification and selection of expressing cells from a population of cells seeking transfection or infection via a viral vector. Alternatively, the optional marker may be carried on a separate DNA segment and used in co-transfection procedures. Both the optional marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in host cells. Useful optional markers include, for example, antibiotic resistance genes such as neo.

[0211] Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. Typically, a reporter gene is a gene that is either absent from or expressed by the recipient organism or tissue, and that encodes a polypeptide whose expression is clearly indicated by readily detectable properties such as enzyme activity. After DNA has been introduced into the recipient cells, reporter gene expression is measured at an appropriate time. Suitable reporter genes may include those encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secretory 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 are commercially available. Typically, a construct with at least five flanking regions exhibiting the highest level of reporter gene expression is identified as a promoter. Such promoter regions can be ligated into reporter genes and used to evaluate the ability of reagents to regulate promoter-driven transcription.

[0212] Methods for introducing genes into cells and expressing genes into cells are known in the art. Within the scope of expression vectors, the vector can be readily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method in the art. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0213] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipid transfection, particle bombardment, microinjection, electroporation, and so on. Methods for producing cells comprising vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). Calcium phosphate transfection is a preferred method for introducing polynucleotides into host cells.

[0214] Biological approaches to introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian cells, such as human cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, among others. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0215] Chemical means of introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, and beads; and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as a delivery vehicle in both in vitro and in vivo is the liposome (e.g., an artificial membrane capsule).

[0216] In the case of using a non-viral delivery system, an exemplary delivery tool is a liposome. Consider using a lipid formulation to introduce nucleic acid into host cells (in vitro, ex vivo, or in vivo). Alternatively, the nucleic acid may be associated with a lipid. Lipid-associated nucleic acid can be encapsulated within the aqueous interior of a liposome, dispersed within the lipid bilayer of the liposome, attached to the liposome via a linker molecule associated with both the liposome and the oligonucleotide, trapped within the liposome, complexed with the liposome, dispersed in a solution containing lipids, mixed with lipids, conjugated 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 specific structure in solution. For example, they may be present in a bilayer structure, as micelles, or have a “collapsed” structure. They may also be simply dispersed in solution, possibly forming aggregates of varying sizes or shapes. Lipids are fatty substances and can be naturally occurring or synthetic lipids. For example, lipids include fat droplets, which occur naturally in the cytoplasm and in compounds containing long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols and aldehydes.

[0217] In a preferred embodiment of the present invention, the carrier is a lentivirus carrier.

[0218] preparation

[0219] This invention provides a formulation containing CAR-immune cells (e.g., CAR-T cells) as described in the seventh aspect of this invention, and a pharmaceutically acceptable carrier, diluent, or excipient. In one embodiment, the formulation is a liquid formulation. Preferably, the formulation is an injectable formulation. Preferably, the concentration of the CAR-T cells in the formulation is 1 × 10⁻⁶. 3 -1×10 8 Cells / ml, more optimal 1×10 4 -1×10 7 Cells / ml

[0220] In one embodiment, the formulation may include buffer solutions such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol; proteins; peptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The formulations of the present invention are preferably formulated for intravenous administration.

[0221] Therapeutic applications

[0222] This invention includes therapeutic applications using cells (e.g., T cells) transduced with a lentiviral vector (LV) encoding the expression cassette of this invention. The transduced T cells can target the tumor cell marker DLL3, can be used for autologous and allogeneic tumor therapy, can be mass-produced, are of uniform and stable quality, and are readily available for use by any patient.

[0223] Therefore, the present invention also provides a method for stimulating a T-cell-mediated immune response against a target cell population or tissue of a mammal, comprising the step of administering the CAR-T cells of the present invention to a mammal.

[0224] In one embodiment, the present invention includes a class of cell therapies in which T cells are genetically modified to express the CAR of the present invention, and CAR-T cells are injected into a recipient in need of them. The injected cells are able to kill the recipient's tumor cells. Unlike antibody therapies, CAR-T cells are able to replicate in vivo, producing long-lasting efficacy that can lead to sustained tumor control.

[0225] In one embodiment, the CAR-T cells of the present invention can undergo robust in vivo T cell expansion and sustain for an extended period of time. Additionally, the CAR-mediated immune response can be part of an adoptive immunotherapy step, wherein CAR-modified T cells induce an immune response specific to the antigen-binding domain in the CAR. For example, anti-DLL3 CAR-T cells elicit a specific immune response against cells expressing DLL3.

[0226] Although the data disclosed herein specifically disclose lentiviral vectors including anti-DLL3 scFv, CD8α hinge region and transmembrane region, and 4-1BB and CD3ζ signaling domains, the present invention should be interpreted as including any number of variations in each of the construct components.

[0227] Treatable cancers include tumors that are not vascularized or are substantially not vascularized, as well as vascularized tumors. Cancers can include solid tumors, as well as adult tumors / cancers and childhood tumors / cancers.

[0228] Solid tumors are abnormal masses of tissue that do not typically contain cysts or fluid-filled areas. Solid tumors can be benign or malignant. Different types of solid tumors are named after the cell types that form them (such as small cell lung cancer, large cell neuroendocrine carcinoma, gastrointestinal neuroendocrine tumor, glioblastoma multiforme, metastatic castration prostate cancer, small cell bladder cancer, neuroendocrine lung tumors, etc.).

[0229] The CAR-immune cells of the present invention can also be used as a type of vaccine for in vitro immunization and / or in vivo therapy in mammals. Preferably, the mammal is human.

[0230] For in vitro immunization, at least one of the following occurs in vitro before the cells are administered into a mammal: i) cell expansion, ii) introduction of nucleic acid encoding CAR into the cells, and / or iii) cryopreservation of the cells.

[0231] In vitro procedures are well known in the art and are discussed more fully below. Simply put, cells are isolated from a mammal (preferably human) and genetically modified (i.e., transduced or transfected in vitro) using a vector expressing a CAR disclosed herein. The CAR-modified cells can be administered to a mammalian recipient to provide therapeutic benefit. The mammalian recipient can be human, and the CAR-modified cells can be autologous relative to the recipient. Alternatively, the cells can be allogeneic, syngeneic, or xenogeneic relative to the recipient.

[0232] In addition to using cell-based vaccines for ex vivo immunization, the present invention also provides compositions and methods for in vivo immunization to elicit an immune response against antigens in a patient.

[0233] The present invention provides a method for treating tumors, comprising administering a therapeutically effective amount of the CAR-immune cells of the present invention to a subject in need of the treatment.

[0234] The CAR-immune cells of the present invention can be administered alone or as a pharmaceutical composition in combination with a diluent and / or other components such as IL-2, IL-17, or other cytokines or cell populations. In short, the pharmaceutical compositions of the present invention may include target cell populations as described herein, combined with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may include buffers such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol; proteins; peptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions of the present invention are preferably formulated for intravenous administration.

[0235] The pharmaceutical compositions of the present invention can be administered in a manner suitable for the disease to be treated (or prevented). The amount and frequency of administration will be determined by factors such as the patient's condition, and the type and severity of the patient's disease—although the appropriate dosage can be determined by clinical trials.

[0236] When referring to "immunologically effective amount," "antitumor effective amount," "tumor-suppressive effective amount," or "therapeutic amount," the precise amount of the composition of the invention to be administered can be determined by a physician, taking into account individual differences in the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and disease condition. It can generally be indicated that a pharmaceutical composition including T cells described herein can be administered in doses of 10... 4 Up to 10 9 A dose of cells / kg body weight, preferably 10. 5 Up to 10 6 The T-cell composition can be administered at a dose of cells per kg of body weight (including all integer values ​​within those ranges). These doses can also be administered multiple times. The cells can be administered using infusion techniques known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dose and treatment regimen for a specific patient can be readily determined by a physician skilled in the medical field by monitoring the patient's disease signs and thus adjusting the treatment accordingly.

[0237] The application of the target composition can be performed in any convenient manner, including by spraying, injection, swallowing, infusion, implantation, or transplantation. The compositions described herein can be administered to patients subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, intravenously (iv), or intraperitoneally. In one embodiment, the T-cell composition of the present invention is administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T-cell composition of the present invention is preferably administered by intravenous injection. The T-cell composition can be injected directly into the tumor, lymph node, or site of infection.

[0238] In some embodiments of the invention, activated and expanded cells, using the methods described herein or other methods known in the art for expanding T cells to a therapeutic level, are administered to a patient in combination with any number of relevant therapeutic modalities (e.g., before, simultaneously with, or after), including but not limited to treatment with agents such as bevacizumab, megestrol acetate dispersible tablets, paclitaxel injection, ifosfamide, and ifosfamide for injection for the treatment of ovarian cancer patients. In further embodiments, the CAR-immune cells of the invention may be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, and FK506, antibodies, or other immunotherapeutic agents. In further embodiments, the cell composition of the invention is administered to a patient in combination with bone marrow transplantation, chemotherapy agents such as fludarabine, external beam radiotherapy (XRT), or cyclophosphamide (e.g., before, simultaneously with, or after). For example, in one embodiment, the subject may undergo standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, after transplantation, the subject receives an infusion of the expanded immune cells of the invention. In an additional implementation, the expanded cells are administered before or after surgery.

[0239] The dosage of the above treatments administered to patients will vary depending on the precise nature of the condition being treated and the recipient of the treatment. The dosage ratios administered to individuals can be implemented according to accepted practices in the field. Typically, 1 × 10⁻⁶ ppm can be administered per treatment or per course of treatment. 6 One to 1×10 10 The CAR-immune cells of the present invention are administered to a patient, for example, via intravenous infusion.

[0240] The main advantages of this invention include:

[0241] (a) The antibodies targeting DLL3 of the present invention all have optimized affinity (too high affinity may lead to systemic toxicity, while too low affinity results in poor efficacy).

[0242] (b) The antibodies targeting DLL3 of the present invention all have good targeting specificity.

[0243] (c) The antibodies targeting DLL3 of the present invention all have species crossover that is beneficial for safety evaluation.

[0244] (d) The mouse CAR-T targeting DLL3 of the present invention has good tumor cell killing effect, including short-term killing and long-term killing (D3012, D3014, D3018, D3025, D3027, D3031 and D3034).

[0245] (e) The humanized DLL3-targeting CAR-T of the present invention has good tumor killing effect and in vivo safety both in vivo and in vitro, and can significantly inhibit tumor growth (D3045, D3056, D3058, D3059).

[0246] (f) The humanized CAR-T targeting DLL3 of the present invention exhibits better killing effect and amplification trend than the sequence when other elements are fused together (PD-1 DNR, PD-1 CSR and TFGbRIIDNR).

[0247] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0248] Example 1: Preparation and Screening of Hybridoma Cells with Anti-Human DLL3 Antibody

[0249] 1.1 Preparation of hybridoma cells

[0250] Immunization: Balb / c mice were immunized with recombinant human DLL3 extracellular region protein (serial number: UniProtKB-Q9NYJ7, 27aa-492aa). Serum titers were detected by ELISA using 96-well microplates coated with recombinant human DLL3-his protein (serial number: UniProtKB-Q9NYJ7, 27aa-492aa). Mice that met the fusion requirements were used for the next step of cell fusion.

[0251] Cell fusion and hybridoma preparation: Mice with the required titer were selected and subjected to a shock immunization. Three days later, the spleens of the mice were aseptically harvested, and a suspension of B lymphocytes was prepared and fused with SP2 / 0 myeloma cells. The fused cells were resuspended in HAT medium and aliquoted into 96-well cell culture plates. The plates were then incubated at 37°C in a 5% CO2 incubator.

[0252] 1.2 Positive hybridoma combination screening

[0253] 10-14 days after fusion, the ELISA plate was coated with recombinant human DLL3-his extracellular region protein (20 ng / ml) and incubated overnight at 4°C. After washing three times with PBS, the plate was blocked with 4% skim milk powder-PBS at room temperature for 1 hour. After washing three times with PBS, the supernatant of the hybridoma clone culture was added and the plate was incubated at room temperature for 1 hour. The following controls were set up: (1) Positive control (PC): serum of mice after immunization (diluted with PBS 1:1000); (2) Negative control (NC): serum of mice before immunization (diluted with PBS 1:1000). The plate was washed three times with PBST (0.05% Tween-PBS), washed twice with PBS, and HRP goat anti-mouse IgG (FcY) was added. The plate was incubated at 37°C for 0.5 hours. After washing three times with PBST (0.05% Tween 20-PBS), TMB chromogenic solution was added and the plate was incubated in the dark for 15-30 minutes. The ELISA stop solution was added to terminate the reaction. The A450 value was read by the ELISA reader. Following the principle of selecting clones with high read values ​​from highest to lowest, the culture supernatant of the clones was collected the following day for a second ELISA confirmation.

[0254] Example 2: Sequencing of mouse-derived anti-human DLL3 antibody

[0255] After expanding the culture of hybridoma clones secreting anti-human DLL3 antibodies, total RNA was extracted from the cells according to the instructions of the TRIzol kit (Cat:15596026, Invitrogen). The total RNA from the hybridoma cells was reverse transcribed into cDNA using M-MuLV reverse transcriptase (Cat:M0253S, NEB). The antibody light chain variable region IgVL(x) and heavy chain variable region VH sequences were amplified using degenerate primers and the Phusion kit (Cat:EO553L, NEB). The PCR amplification products were purified using a gel extraction kit (Cat:AP-GX-250, Axygen). The amplified PCR products were ligated into a T vector according to the instructions of the T vector cloning kit (Cat:ZC205 Zhuangmeng Biotechnology) and transformed into competent E. coli cells. After amplification and plasmid extraction, DNA sequencing was performed to obtain the monoclonal antibody variable region sequence and CDR sequence.

[0256] Example 3: Affinity Analysis of Mouse Antibodies

[0257] A fusion protein (DLL3-ECD-mFc) of the extracellular region of human DLL3 (UniProtKB-Q9NYJ7) and mFc was used as the antigen. After thorough emulsification with an equal volume of complete Freund's adjuvant (Sigma, F5581), the antigen was subcutaneously immunized into 6-8 week old Balb / c mice (purchased from Joinn Laboratories (Suzhou) New Drug Research Center Co., Ltd.). The antigen dose was 50 μg per mouse. Subsequently, every 2 weeks, the same dose of antigen was emulsified with incomplete Freund's adjuvant (Sigma, F5506) and subcutaneously immunized three times. Serum titers were measured after each immunization.

[0258] Using PEG Hybri Max (Sigma, 7181) as a fusion agent, mouse spleen cells and SP2 / 0 cells were mixed at a ratio of 4:1, and the fused cells were added to 96-well plates (1×10⁻⁶). 5 Each well contained 0.1 mL of 1× HAT (Invitrogen, 21060-017) medium. On day 3, 0.1 mL of HAT (Invitrogen, 11067-030) medium was added. On day 7, the medium in the 96-well plate was aspirated, and 0.2 mL of fresh HAT medium was added. On day 9, the supernatant was collected for various screening and testing.

[0259] DLL3 antigen protein was prepared into seven serially diluted concentrations of 100 nM, 50 nM, 25 nM, 12.5 nM, 6.5 nM, 3.13 nM, and 1.56 nM, and sampled into 96-well plates. Human Protein G probe was selected, with the antigen as the stationary phase and DLL3 antibody as the mobile phase. The binding time was set to 180 s and the dissociation time to 300 s. The binding and dissociation constants of the human monoclonal antibody were measured, and the affinity was calculated.

[0260] The kinetic constants are shown in Table 1 below. The results in Table 1 show that all clones were correctly cloned and could bind to human DLL3 recombinant protein.

[0261] Table 1

[0262] Example 4: Humanization and Mutation Design of Anti-human DLL3 Monoclonal Antibody

[0263] 4.1 Humanization of murine monoclonal antibodies

[0264] CDR Porting

[0265] First, a comprehensive analysis of the murine antibody heavy chain sequence was performed to identify the complementarity-determining regions (CDRs) for antibody-antigen binding and the framework regions supporting the conserved three-dimensional conformation of the antibody. Then, based on homology alignment results, the most similar human antibody was selected as the template, and CDR transplantation was performed using the full-sequence BLAST results. The CDR regions of the murine antibody were preserved, and the framework region sequence of the murine antibody was replaced with the framework region sequence of the human germline antibody.

[0266] Secondly, a structural model of the murine antibody was established, and each different amino acid site in the structural models of the human antibody and the corresponding murine antibody was compared one by one. If using a human amino acid sequence at a certain site in the frame region did not cause damage or change to the spatial structure of the CDR region, then the human amino acid sequence was used at that site; otherwise, the corresponding murine sequence was used at that site (i.e., the murine sequence was reversed).

[0267] Based on structural simulations, some amino acids in the framework region of the humanized antibody were reverted to mouse sequences. Finally, the variable region amino acid sequences of the heavy and light chains of the humanized antibody were obtained.

[0268] Example 5: Tumor Cell Line Culture

[0269] 5.1 Cell line culture

[0270] This invention utilizes six cell lines: 293T, SUP-T1, NCI-H82, SHP-77, NCI-H889, and CHO-K1. 293T cells are human embryonic kidney epithelial cell lines used to study exogenous gene expression and virus preparation; SUP-T1 is a T lymphocyte line used to detect viral titers; NCI-H82, SHP-77, and NCI-H889 are human small cell lung cancer cell lines, serving as the target cells in this study; and CHO-K1 is a subclonal cell line of the Chinese hamster ovary cell line CHO, used to construct an overexpression cell line for exogenous genes. 293T cells were cultured in DMEM complete medium (DMEM + 10% FBS); SUP-T1, NCI-H82, SHP-77 and NCI-H889 cells were cultured in 1640 complete medium (1640 + 10% FBS); CHO-K1 cells were cultured in F12K complete medium (F12K + 10% FBS).

[0271] 5.2 Flow cytometry detection of DLL3 target expression in tumor cells

[0272] Centrifuge the cells to be tested at 300g for 5 minutes and collect the cell pellet; wash the cells once with PBS. Take 2×10⁶ cells. 5 Cells were stained using a 100 μL staining system. After resuspending the cells in the staining system, they were incubated at room temperature in the dark for 30 min. After incubation, the cells were washed once with flow cytometry buffer, resuspended in 200 μL of flow cytometry buffer, and then subjected to flow cytometry analysis.

[0273] The staining system was prepared as follows: 100 μL flow cytometry buffer + antibody.

[0274] The flow cytometry buffer formulation is: PBS + 1% FBS + 2.5mM EDTA.

[0275] The flow cytometry results are shown in Figure 1. The small cell lung cancer cell lines NCI-H82 and SHP-77 highly express DLL3 protein on their cell surface.

[0276] Example 6: Construction of CAR based on murine antibody-derived scFv

[0277] The CAR structure used in this embodiment is a second-generation CAR structure, specifically consisting of a CD8 signal peptide at the 5' end, an scFv sequence region, followed by a CD8 hinge region, a CD8 transmembrane region, a 4-1BB intracellular co-stimulatory domain, and a CD3ζ signal transduction domain. The scFv structure, starting from the 5' end, comprises the antibody's light chain variable region (VL), G4S (SEQ ID NO:79), and a flexible linker peptide and antibody heavy chain variable region (VH).

[0278] Example 7 Lentiviral Production

[0279] 2×10 7 293T cells were seeded into 15cm cell culture dishes with 20mL of culture medium. The next day, the expression plasmid, helper plasmid pMDLg-pRRE, helper plasmid pRSV-Rev, and helper plasmid pMD2.G were mixed in a 2:1:1:1 ratio in 1mL of opti-MEM medium to prepare a plasmid-containing medium. Separately, 50μg of PEI was added to 1mL of opti-MEM medium, mixed, and incubated at room temperature for 5 minutes. The PEI mixture was then added dropwise to the plasmid mixture, mixed thoroughly, and incubated at room temperature for 15 minutes. This plasmid-PEI solution was then uniformly added to the 293T cell culture dishes. After 16 hours, the medium was replaced with 20mL of fresh DMEM complete medium. After 48 hours, the supernatant was collected, filtered through a 0.45μm filter, and centrifuged at 30000g for 2 hours to obtain the viral pellet. The viral pellet was resuspended in PBS medium and aliquoted. Viral titers were detected in the viral solution using SUP-T1 cells. The viral solution was stored at -80°C for an extended period.

[0280] Example 8: T cell activation and viral transduction

[0281] 8.1 Peripheral blood mononuclear cell (PBMC) resuscitation

[0282] Preheat the water bath to 37°C. Remove one PBMC vial from the liquid nitrogen tank and quickly place it in the water bath to rapidly thaw the cell cryopreservation solution. Once thawed, spray the cryopreservation tube with 75% alcohol and open it in a biosafety cabinet. Use a pipette to add the PBMC suspension to 14 mL of PBS, mix well, and centrifuge at 400 g for 5 minutes.

[0283] 8.2 Activation of PBMC cells

[0284] Cells were resuspended in AIM-V complete medium (5% FBS + 300 IU IL-2 / mL) and counted. Based on the counting results, the cells were diluted to 2 × 10⁻⁶. 6 / mL. Every 2×10 6 PBMC cells were supplemented with 10 μL of TransAct T cell activator. The cells were then placed in a 37°C, 5% CO2 incubator for 48 hours for activation.

[0285] 8.3 Collection of activated T cells and viral transduction

[0286] After PBMC activation and culture for 48 hours, cells were collected into 15 mL centrifuge tubes and centrifuged at 400 g for 5 minutes. After centrifugation, cells were resuspended in AIM-V complete medium (5% FBS + 300 IU / mL IL-2). Samples were taken for cell counting.

[0287] Based on the count results, the T cell count was adjusted to 1 × 10⁶. 6 / mL, distribute 0.5mL to each well of a 24-well plate; add the corresponding CAR-T lentivirus at MOI=10, add Polybrene at a concentration of 8μg / mL, mix well, and centrifuge at 1000g for 1 hour.

[0288] After centrifugation, remove the 24-well plate from the centrifuge, mix the cell pellet by pipetting, and return it to the incubator for overnight culture. The next day, replace the medium with fresh AIM-V complete medium (5% FBS + 300 IU / mL IL-2). After five days of culture, collect a portion of the cells and detect CAR expression.

[0289] Example 9 Flow Cytometry Staining Analysis

[0290] Centrifuge the cells to be tested at 300g for 5 minutes and collect the cell pellet; wash the cells once with PBS. Take 2×10⁶ cells. 5 Cells were stained using a 100 μL staining system. After resuspending the cells in the staining system, they were incubated at room temperature in the dark for 30 min. After incubation, the cells were washed once with flow cytometry buffer, resuspended in 200 μL of flow cytometry buffer, and then subjected to flow cytometry analysis.

[0291] The staining system was prepared as follows: 100 μL flow cytometry buffer + antibody.

[0292] The flow cytometry buffer formulation is: PBS + 0.5% BSA + 2.5mM EDTA.

[0293] The dosage of antibodies used depends on the antibody concentration and the actual situation.

[0294] The positivity rate of CAR-T cells is shown in Figure 2. +T cells accounted for 48.3%.

[0295] Example 10: Detection of the cytotoxic effect of DLL3-CAR-T cells on tumor cells (short-term killing)

[0296] Untransduced T cells (UNT) and prepared DLL3-CAR T cells were co-incubated with NCI-H82 and SHP-77 cells at effector-target ratios of 0.3:1, 1:1, and 3:1 for 16 h. The death and lysis of tumor cells were then detected using an LDH kit (Roche, 11644793001).

[0297] The results, shown in Figure 3, indicate that DLL3-CAR T cells exhibited significantly better cytotoxicity against both NCI-H82 and SHP-77 tumor cells compared to UNT. This demonstrates that DLL3-CAR T cells possess strong cytotoxicity against solid tumor cells expressing DLL3.

[0298] Figures 3a and 3d show a clear comparison of the cytotoxic effects of CAR-T cells corresponding to D3005, D3006, and D3007 on NCI-H82 and SHP-77 cells, which can be used for further comparison.

[0299] Figures 3b and 3e show that the CAR-T cells corresponding to D3012, D3014, D3015, D3016, D3017, D3018, and D3019 have significantly different cytotoxic effects on NCI-H82 and SHP-77 cells, which can be used for further comparison.

[0300] Figures 3c and 3f show that the CAR-T cells corresponding to D3028, D3031, and D3034 have significantly different cytotoxic effects on NCI-H82 and SHP-77 cells, which can be further compared in the next step.

[0301] Example 11 Long-term lethality experiment

[0302] Prepared CAR-T cells, NCI-H82 cells, and SHP-77 cells were collected separately, centrifuged at 300g for 5 minutes, and then resuspended in AIM-V medium (5% FBS). Samples were then counted. The CAR% percentage of the prepared CAR-T cells was corrected to the same level using activated T cells from the same donor; 6 × 10⁶ cells were then... 5 NCI-H82 cells and 6×10 5 SHP-77 cells were seeded into 12-well plates, and then the corresponding number of CAR-T cells were added at effector-target ratios of E:T = 1:4 and 1:5, respectively.

[0303] The volume of each well was increased to 1.5 mL using AIM-V medium (5% FBS), and after co-culturing for 48 hours, the percentage of T cells was measured. Simultaneously, 6 × 10⁶ cells were added to each well. 5 The target cells were co-cultured for the next round of testing. Stimulation with target cells was repeated 5-7 times, and the sustained killing ability of CAR-T cells was analyzed by detecting the percentage of T cells in each well.

[0304] In the final round, a higher proportion of T cells indicates a stronger sustained killing ability of CAR-T cells and a stronger ability to resist T cell exhaustion. Similar to the short-term killing grouping, this invention divides all binders into three batches for long-term killing comparison, and finally collects the best binders from each group for a final comparison.

[0305] Figures 4a and 4d show the trend of CD3+ cell proportion during the long-term killing of NCI-H82 and SHP-77 cells by CAR-T cells corresponding to D3001 to D3011. Based on microscopic observation and short-term killing effect, this invention selected D3005 for the next step of comparison.

[0306] Figures 4b and 4e show the trend of CD3+ cell proportion during the long-term killing of NCI-H82 and SHP-77 cells by CAR-T cells corresponding to D3012 to D3022. D3012, D3013, D3014, D3015, D3016, D3018, and D3022 were selected for the next step of comparison.

[0307] Figures 4c and 4f show the trend of CD3+ cell proportion during the long-term killing of NCI-H82 and SHP-77 cells by CAR-T cells corresponding to D3023 to D3034. D3023, D3024, D3025, D3027, D3028, D3030, D3031, D3032, and D3034 were selected for the next step of comparison.

[0308] When re-screening the remaining sequences, CHO-K1 cell lines overexpressing human DLL3, mouse DLL3, human DLL1, and human DLL4 were constructed to verify the target specificity of the CAR sequence for DLL3 and the human-mouse crossover.

[0309] The results are shown in Figures 5 and 6. The results show that all 17 sequences have significant toxic side effects on cell lines overexpressing human DLL3. In the human-mouse crossover validation, D3005, D3013, and D3015 did not have a killing effect on cell lines overexpressing mouse DLL3. In the validation of the specificity of the DLL3 target, D3005 had non-specific killing effect on human DLL1, D3023 had non-specific killing effect on human DLL4, and D3005 and D3015 had non-specific killing effect on CHO-K1.

[0310] The 17 CAR sequences were re-screened for long-term killing effects, and the results are shown in Figures 7a-7d. Based on microscopic observation and short-term killing results, D3012, D3014, D3018, D3025, D3027, D3031, and D3034 were finally selected for in vitro killing effects in 3D cultures, in vivo animal experiments, and sequence humanization modifications.

[0311] Example 12 In vitro killing effect of 3D cell cultures

[0312] Collect NCI-H82 cells by centrifugation, sample and count, and set aside for later use. Dissolve the matrix gel (ACRO, AC-M082704) overnight at 4°C, then dilute it 1 / 2 with serum-free 1640 medium. Resuspend the NCI-H82 cells in matrix gel medium to a concentration of 50,000 cells / mL. Place the cell suspension on ice and add 100 μL of NCI-H82 cell suspension to each well using a pre-cooled pipette tip, with three replicates per well and three counting wells. Incubate the 96-well plate in a 37°C CO2 incubator for 30 minutes to allow the matrix gel suspension to solidify completely. Then add 100 μL of complete NCI-H82 cell culture medium to each well and incubate for 7-9 days, changing half or all of the medium during this period to maintain cell growth.

[0313] 3D cultured cell counting: Discard the supernatant from the counting wells, wash twice with PBS, add 100 μL Corning Dispase, and incubate in a CO2 incubator for 1 hour. Remove the plate and gently pipette until there is no sticky residue. Add 100 μL FACS buffer to the original system, centrifuge at 3000 rpm for 5 minutes, remove the supernatant, add another 100 μL FACS buffer, centrifuge at 3000 rpm for 5 minutes, and remove the supernatant completely. Add 200 μL FACS buffer, and take 20 μL for cell counting.

[0314] Effector cell addition: CAR-T cells cultured for 7 days after lentiviral infection were collected, counted, and their CAR% was measured. The CAR% of CAR-T cells was corrected to the same level. After approximately 8 days of NCI-H82 cell culture, 100 μL of supernatant was carefully aspirated from each well, and then 100 μL of CAR-T cell suspension was added at an effector-to-target ratio of 1:5. The medium was changed every 48 hours, carefully aspirating to avoid removing the matrix gel.

[0315] After co-culturing effector cells for 6 days, the supernatant was discarded, and Corning Dispase was added to each well for digestion and counting. The cells were resuspended in 100 μL of staining buffer, and the percentage of T cells was detected by adding staining antibody. A higher T cell count indicates a lower tumor cell count, suggesting a better in vitro killing effect and stronger T cell infiltration ability in the corresponding group.

[0316] Figure 8 shows the results of the CAR-T cell assay for sustained killing of NCI-H82-3D culture over 6 days. The sequences in the figure correspond to all CAR-T cells that showed good infiltration and killing effects on the NCI-H82-3D culture, with D3014 and D3027 showing particularly good results.

[0317] Example 13: Experiment on in vivo antitumor activity - NCI-H82 / NPG animal model

[0318] NPG mice were purchased from Beijing Vitonda Biotechnology Co., Ltd.; this experiment used a subcutaneous tumor-forming animal model, with each NPG mouse subcutaneously injected with 1×10⁻⁶ NPG. 7 NCI-H82 cells were used for modeling. After modeling, the major and minor diameters were measured three times a week using calipers. After grouping, tumor measurements were performed twice a week. Then, the formula V = 1 / 2ab was applied. 2 The size of the tumor was calculated; where a is the major axis and b is the minor axis. After modeling began, the patient was weighed every three days, and after grouping, weighed twice a week.

[0319] Seven days after tumor cell inoculation, 4×10⁴ cells were reinfused via the tail vein. 6 CAR-T cells were infused; after infusion, blood was collected from the orbital cavity of mice on Day 1, Day 7, Day 14, Day 21, and Day 28 to detect the dynamic content and proportion of T cells and CAR-T cells in peripheral blood.

[0320] Figure 9a and Table 2 show the inhibitory effects of different sequence CAR-T cells on the growth of NCI-H82 tumors in NPG mice. CAR-T cells corresponding to D3018, D3031 and D3034 showed good inhibitory effects on tumor growth.

[0321] Table 2

[0322] Note: The values ​​in the table represent the average tumor volume of each group of mice (accurate to the nearest whole number), in mm. 3 .

[0323] Figure 9b shows the weight change curves of each group of NPG mice throughout the experiment. The weight of each group of mice increased steadily overall without significant fluctuations.

[0324] Figures 9c and 9d show the metabolic kinetics curves of T cells and CAR-T cells in NPG mice after infusion of different CAR-T sequences. The CAR-T cells corresponding to D3018, D3031 and D3034 showed the most significant in vivo expansion.

[0325] Example 14 Antibody Humanization and Sequence Screening

[0326] 14.1 Initial screening for short-term and long-term lethality

[0327] After obtaining the plasmid containing the humanized antibody sequence, lentiviruses and CAR-T cells were prepared; the preparation process was the same as above. The techniques for LDH detection of short-term killing ability and flow cytometry detection of long-term killing ability for each sequence were also the same as above. After humanization, the inventors mainly compared whether the humanized CAR sequences maintained the same or superior killing effect as the corresponding mouse CAR sequences.

[0328] First, comparisons were made between the humanized sequences of each parent genome. The short-term and long-term kill effects are shown in Figure 10. Based on the short-term and long-term kill effects, D3043 and D3045 were selected from the humanized sequences corresponding to D3018 for further comparison; D3056, D3057, and D3058 were selected from the humanized sequences corresponding to D3031 for further comparison; and D3059 and D3060 were selected from the humanized sequences corresponding to D3034 for further comparison.

[0329] 14.2 Rescreening for short-term and long-term lethality

[0330] Figures 11a-11d and 12a-12d show a comparison of the short-term and long-term killing effects of D3018, D3031, and D3034 with their humanized sequences in two donors.

[0331] The results showed that the humanized sequences of D3018 and D3031, after screening, had good killing effects against NCI-H82. However, the humanized sequence of D3034 was not superior to D3034 itself. The killing effect of D3034 after redesigning it for humanization is shown in Figures 11d and 12d. Finally, D3059 and D3067 were selected for further comparison.

[0332] 14.3 In vitro killing effect of 3D cultures

[0333] The in vitro killing process of 3D cultures is the same as above.

[0334] The results are shown in Figures 13a-13d. Based on the results of short-term and long-term killing effects and the transduction efficiency of the lentiviruses corresponding to each sequence, D3045 was selected from the humanized sequences corresponding to D3018 for subsequent in vivo comparison; D3056, D3057, and D3058 were selected from the humanized sequences corresponding to D3031 for in vivo experimental comparison; and D3059 and D3067 were selected from the humanized sequences corresponding to D3034 for in vivo experimental comparison.

[0335] 14.4 Internal lethality

[0336] The human-mouse crossover and target specificity were verified using the constructed CHO-K1 cell lines overexpressing human DLL3, mouse DLL3, human DLL1, and human DLL4, as well as the candidate sequences for animal experiments on CHO-K1 cells.

[0337] The results, shown in Figures 14a-14d and 15a-15b, indicate that the candidate sequences exhibit strong killing activity against DLL3 in humans and mice, but no killing activity against human DLL1 and DLL4. This demonstrates that the candidate sequences in the in vivo experiments possess excellent human-mouse cross-reactivity and target specificity.

[0338] 14.5 In vivo efficacy validation of NPG in mice

[0339] 14.5.1 Humanized sequences D3018 and D3031

[0340] The NPG mouse model was established using the same method as above. The infusion dose was set at a low dose (1.5 × 10⁻⁶). 6 ) and high-dose group (3×10 6 Seven days after modeling, CAR-T cells were reinfused into each mouse via the tail vein.

[0341] Figures 16a-16d show the tumor-suppressing effects of CAR-T cells corresponding to the humanized sequences of D3045, D3031, and D3031 in the low-dose group, as well as the expansion curves of T cells and CAR-T cells. In the low-dose group, D3031 showed the best tumor growth suppression effect and T cell expansion, followed by the humanized sequence of D3031, D3058.

[0342] Figures 17a-17d illustrate the tumor-suppressive effects of CAR-T cells corresponding to D3018 and D3031 and their humanized sequences in the high-dose group, as well as the expansion curves of T cells and CAR-T cells. In the high-dose group, the humanized sequence D3045 of D3018 showed the best in vivo efficacy, followed by D3031 and its humanized sequence D3058. D3045 and D3058 were selected for further in vivo comparison. Three dosages were set up for the in vivo efficacy of D3045 and D3058, namely the low-dose group (0.5 × 10⁻⁶).6 ), medium dose group (1×10) 6 ) and high-dose group (2×10 6 ).

[0343] The results are shown in Figures 18a-18l. D3045 showed efficacy in some mice in the low-dose group, and good overall efficacy in the medium- and high-dose groups. Compared with D3058, D3045 showed better efficacy and CAR-T cell metabolic kinetics in vivo, and the mice maintained stable body weight without significant fluctuations.

[0344] 14.5.2 Acute poisoning test of D3045 and D3058

[0345] The acute toxicity test infusion dose was 1×10 7 CAR-T cells were used to evaluate the in vivo safety of the preferred sequences. The results are shown in Figures 19a-19d. Figure 19a shows the inhibitory effect of D3045 and D3058 CAR-T infusion on NPG mouse NCI-H82 tumor growth. Figure 19b shows the weight gain trend of NPG mice after D3045 and D3058 CAR-T infusion; the weight remained stable without a significant decrease. Figures 19c-d show the metabolic kinetics of T cells and CAR-T cells in NPG mice after D3045 and D3058 CAR-T infusion, both showing a good expansion trend, indicating that D3045 and D3058 have good in vivo safety.

[0346] 4.5.3 Humanized sequences D3018 and D3034

[0347] Figures 20a-20d show the results at 1.5 × 10⁻⁶. 6 Comparison of in vivo efficacy of preferred humanized sequences D3059 and D3067 of D3018 at the indicated doses. D3059 showed more significant in vivo efficacy and CAR-T cell metabolic kinetics than D3067, but its in vivo effect was still inferior to that of D3045.

[0348] Figures 21a-21d show a comparison of the in vivo safety of D3059 and D3069 at 1×10⁻⁶. 7 At the indicated doses, both D3059 and D3069 demonstrated tumor suppression and good in vivo expansion of T cells and CAR-T cells, without a rapid weight loss trend, indicating good in vivo safety.

[0349] Example 15: CAR-T binding epitope exploration

[0350] Based on DLL3-negative CHO-K1 cells, a series of DLL3-overexpressing cell lines with full-length or truncated DLL3 domains were constructed, as shown in Figure 22. The killing effect of preferred mouse sequences D3018, D3031, and D3034, and the preferred humanized sequence D3045 on the DLL3-overexpressing cell lines with full-length or truncated DLL3 domains indicates that the main binding site between the CAR-T cells described in this invention and DLL3-positive cells is the EGF6 domain, as shown in Figures 23 and 24.

[0351] Example 16: In vitro efficacy validation of PD-1 DNR and PD-1 CSR

[0352] D3045 was further optimized to allow T cells to express the CAR fusion protein and PD-1 DNR (amino acid sequence as shown in SEQ ID NO:80) or PD-1 CSR (amino acid sequence as shown in SEQ ID NO:81). NCI-H889 was selected as the target cell for this long-term killing in vitro validation, with an effector-target ratio of 1:3, and the long-term killing method was the same as above.

[0353] The results are shown in Figure 25. Figure 25a shows the proportion of T cells in living cells during long-term killing. With the increase of killing rounds, D3045 showed signs of fatigue, while D3045 / PD-1 DNR and D3045 / PD-1 CSR still showed better killing effects and better amplification effects than D3045, as shown in Figure 25b. The CAR-T cell proportion and amplification curves are shown in Figures 25c-d. The CAR-T cell amplification effects of D3045 / PD-1 DNR and D3045 / PD-1 CSR were significantly better than those of D3045.

[0354] Example 17: In vitro efficacy validation of TGFbRII DNR

[0355] D3045 cells were optimized to express the CAR fusion protein and TGFbRII DNR (amino acid sequence shown in SEQ ID NO:82). SHP-77 cells were selected as target cells with an effector-target ratio of 1:4. 5 ng / mL TGF-β was added to AIM-V medium (5% FBS), while a control with 0 ng / mL TGF-β was set up. The long-term killing method was the same as above.

[0356] The results are shown in Figure 26. Figure 26a shows the proportion of T cells in living cells during long-term killing. Compared with D3045 in the group supplemented with 0 ng / mL TGF-β, D3045 showed exhaustion in the experimental group supplemented with 5 ng / mL TGF-β in an earlier killing cycle, verifying the inhibitory effect of TGF-β on T cells in the tumor immune microenvironment. The presence of TGFbRII DNR offset this inhibitory effect and showed a better T cell expansion trend, as shown in Figure 26b. The CAR-T proportion and expansion curves are shown in Figures 26c-26d. Corresponding to the killing effect, in the experimental group supplemented with 5 ng / mL TGF-β, the expansion effect of D3045 / TGFbRII DNR was significantly better than that of D3045. The CAR-T expansion effect was better in the culture medium without additional TGF-β.

[0357] The sequence information of the present invention is shown in Table 3 below.

[0358] Table 3

[0359] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

An antibody or antigen-binding fragment thereof targeting DLL3, said antibody or antigen-binding fragment having a heavy chain variable region and a light chain variable region, characterized in that... The complementary determinant region HCDR of the heavy chain variable region and the complementary determinant region LCDR of the light chain variable region are selected from the following group: (1) The amino acid sequence of HCDR1 is shown in SEQ ID NO:

23. The amino acid sequence HCDR2 is shown in SEQ ID NO:

24. The amino acid sequence is HCDR3 as shown in SEQ ID NO:

25. The amino acid sequence of LCDR1 is shown in SEQ ID NO:

27. The amino acid sequence is LCDR2 as shown in SEQ ID NO:28, and The amino acid sequence is LCDR3 as shown in SEQ ID NO:29; (2) The amino acid sequence of HCDR1 is shown in SEQ ID NO:

47. The amino acid sequence HCDR2 is shown in SEQ ID NO:

48. The amino acid sequence HCDR3 is shown in SEQ ID NO:

49. The amino acid sequence of LCDR1 is shown in SEQ ID NO:

51. The amino acid sequence is LCDR2 as shown in SEQ ID NO:52, and The amino acid sequence is LCDR3 as shown in SEQ ID NO:53; (3) The amino acid sequence of HCDR1 is as shown in SEQ ID NO:

56. The amino acid sequence HCDR2 is shown in SEQ ID NO:

57. The amino acid sequence HCDR3 is shown in SEQ ID NO:

58. The amino acid sequence of LCDR1 is shown in SEQ ID NO:

60. The amino acid sequence is LCDR2 as shown in SEQ ID NO:61, and The amino acid sequence is LCDR3 as shown in SEQ ID NO:62; (4) The amino acid sequence of HCDR1 is as shown in SEQ ID NO:

5. The amino acid sequence HCDR2 is shown in SEQ ID NO:

6. The amino acid sequence HCDR3 is shown in SEQ ID NO:

7. The amino acid sequence of LCDR1 is shown in SEQ ID NO:

9. The amino acid sequence is LCDR2 as shown in SEQ ID NO:10, and The amino acid sequence is LCDR3 as shown in SEQ ID NO:11; (5) The amino acid sequence of HCDR1 is shown in SEQ ID NO:

14. The amino acid sequence HCDR2 is shown in SEQ ID NO:

15. The amino acid sequence HCDR3 is shown in SEQ ID NO:

16. The amino acid sequence of LCDR1 is shown in SEQ ID NO:

18. The amino acid sequence is LCDR2 as shown in SEQ ID NO:19, and The amino acid sequence is LCDR3 as shown in SEQ ID NO:20; (6) The amino acid sequence of HCDR1 is as shown in SEQ ID NO:

32. The amino acid sequence is HCDR2 as shown in SEQ ID NO:

33. The amino acid sequence HCDR3 is shown in SEQ ID NO:

34. The amino acid sequence of LCDR1 is shown in SEQ ID NO:

36. The amino acid sequence is LCDR2 as shown in SEQ ID NO:19, and The amino acid sequence is LCDR3 as shown in SEQ ID NO:37; (7) The amino acid sequence of HCDR1 is as shown in SEQ ID NO:

40. The amino acid sequence HCDR2 is shown in SEQ ID NO:

41. The amino acid sequence HCDR3 is shown in SEQ ID NO:

42. The amino acid sequence of LCDR1 is shown in SEQ ID NO:

9. The amino acid sequence is LCDR2 as shown in SEQ ID NO:44, and The amino acid sequence is LCDR3 as shown in SEQ ID NO:

11. A chimeric antigen receptor (CAR) fusion protein, characterized in that, The chimeric antigen receptor fusion protein comprises, from the N-terminus to the C-terminus: (i) A single-chain antibody, wherein the heavy chain variable region and the light chain variable region of the single-chain antibody respectively comprise the HCDR and LCDR as described in claim 1; (ii) Transmembrane structural domains (iii) at least one co-stimulatory domain, and (iv) Activate the structural domain. A recombinant protein, characterized in that, The recombinant protein has the following characteristics: (i) the antibody or antigen-binding fragment thereof as described in claim 1; and (ii) Tag sequences that assist in expression and / or purification. An antibody-drug conjugate, characterized in that, The antibody-drug conjugate contains: (a) The antibody or its antigen-binding fragment as claimed in claim 1, or the chimeric antigen receptor fusion protein as claimed in claim 2; as well as (b) A conjugation portion conjugated to the antibody portion, the conjugation portion being selected from the group consisting of: detectable markers, drugs, toxins, cytokines, radionuclides, enzymes, or combinations thereof. A polynucleotide, characterized in that, The polynucleotides encode polypeptides selected from the following group: (1) The antibody or its antigen-binding fragment as described in claim 1; (2) The chimeric antigen receptor fusion protein as described in claim 2; or (3) The recombinant protein as described in claim 3. The polynucleotide as described in claim 5, characterized in that, When the polynucleotide encodes the chimeric antigen receptor fusion protein as described in claim 2, the polynucleotide further comprises a polynucleotide sequence encoding a chimeric switching receptor (CSR) or a dominant-negative receptor (DNR). The polynucleotide as described in claim 6, characterized in that, The polynucleotide sequence encoding the chimeric switching receptor (CSR) or dominant-negative receptor (DNR) is selected from the group consisting of: polynucleotide sequences encoding the PD-1 dominant-negative receptor (PD-1DNR), polynucleotide sequences encoding the PD-1 chimeric switching receptor (PD-1CSR), and polynucleotide sequences encoding the TGF-β dominant-negative receptor (TGF-βDNR). A carrier, characterized in that, The carrier contains the polynucleotide as described in claim 5. A genetically engineered host cell, characterized in that, The host cell contains the vector of claim 6, or has integrated the polynucleotide of claim 5 into its genome, or expresses the antibody of claim 1 or its antigen-binding fragment, or the chimeric antigen receptor fusion protein of claim 2. The use of the antibody or its antigen-binding fragment as described in claim 1, the chimeric antigen receptor fusion protein as described in claim 2, the vector as described in claim 8, or the host cell as described in claim 9, is characterized in that, Drugs or preparations used to prevent and / or treat DLL3-positive cancers or tumors; The cancer or tumor is selected from the group consisting of: small cell lung cancer, large cell neuroendocrine carcinoma, gastrointestinal neuroendocrine tumor, glioblastoma multiforme, metastatic castration prostate cancer, small cell bladder cancer, neuroendocrine lung tumor, or a combination thereof.

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