Antibodies, chimeric antigen receptors targeting DLL3 and their applications

Abstract

The present invention relates to antibodies targeting DLL3, chimeric antigen receptors and their applications. Specifically, the present invention provides an antibody targeting DLL3 or an antigen-binding fragment thereof, a chimeric antigen receptor (CAR) fusion protein comprising an antigen-binding domain targeting DLL3, and an engineered immune cell (such as, CAR-T cell) expressing the CAR fusion protein. The CAR-T cells of the present invention have high affinity and biological activity, and good anti-tumor effects in vitro and in vivo.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to antibodies targeting DLL3, chimeric antigen receptors, and their applications. Background Art

[0002] Delta-like ligand 3 (DLL3) is a type I transmembrane Delta-like protein and functions as an inhibitory Notch ligand, mainly 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. SCLC accounts for approximately 15% of all lung cancer types. Although patients with this type of lung cancer have a relatively high response to first-line chemotherapy and radiotherapy, patients with extensive-stage disease often experience recurrence, and cases with a survival period of more than five years from the date of diagnosis are extremely rare. For recurrent or refractory small cell lung cancer, the choice of treatment options is extremely limited, and existing treatment methods are often accompanied by significant treatment-related toxicities. In addition to small cell lung cancer, DLL3 is also abnormally expressed in tumor cells such as large cell neuroendocrine carcinoma, gastrointestinal neuroendocrine tumor, glioblastoma multiforme, metastatic castration-resistant prostate cancer, small cell bladder cancer, and neuroendocrine lung tumor. Based on the high expression characteristics of DLL3 on the surface of homogeneous tumor cells and the low expression and cytoplasmic localization in normal cells, DLL3 is regarded as an ideal target for the treatment of SCLC and other neuroendocrine tumors.

[0003] However, existing anti-human DLL3 antibodies in the prior art have defects such as insufficient affinity and specificity for membrane surface DLL3, resulting in off-target effects and adverse reactions.

[0004] Therefore, it is of great significance in this field to develop antibodies and antigen chimeric receptors targeting DLL3 with better affinity and stronger specificity. Summary of the Invention

[0005] The present invention provides antibodies and antigen chimeric receptors targeting DLL3 with better affinity and stronger specificity.

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

[0007] (1) HCDR1 with the amino acid sequence shown in SEQ ID NO: 23,

[0008] HCDR2 with the amino acid sequence shown in SEQ ID NO: 24,

[0009] An HCDR3 having an amino acid sequence as shown in SEQ ID NO: 25,

[0010] An LCDR1 having an amino acid sequence as shown in SEQ ID NO: 27,

[0011] An LCDR2 having an amino acid sequence as shown in SEQ ID NO: 28, and

[0012] An LCDR3 having an amino acid sequence as shown in SEQ ID NO: 29;

[0013] (2) An HCDR1 having an amino acid sequence as shown in SEQ ID NO: 47,

[0014] An HCDR2 having an amino acid sequence as shown in SEQ ID NO: 48,

[0015] An HCDR3 having an amino acid sequence as shown in SEQ ID NO: 49,

[0016] An LCDR1 having an amino acid sequence as shown in SEQ ID NO: 51,

[0017] An LCDR2 having an amino acid sequence as shown in SEQ ID NO: 52, and

[0018] An LCDR3 having an amino acid sequence as shown in SEQ ID NO: 53;

[0019] (3) An HCDR1 having an amino acid sequence as shown in SEQ ID NO: 56,

[0020] An HCDR2 having an amino acid sequence as shown in SEQ ID NO: 57,

[0021] An HCDR3 having an amino acid sequence as shown in SEQ ID NO: 58,

[0022] An LCDR1 having an amino acid sequence as shown in SEQ ID NO: 60,

[0023] An LCDR2 having an amino acid sequence as shown in SEQ ID NO: 61, and

[0024] An LCDR3 having an amino acid sequence as shown in SEQ ID NO: 62;

[0025] (4) An HCDR1 having an amino acid sequence as shown in SEQ ID NO: 5,

[0026] An HCDR2 having an amino acid sequence as shown in SEQ ID NO: 6,

[0027] The HCDR3 with the amino acid sequence shown in SEQ ID NO: 7,

[0028] The LCDR1 with the amino acid sequence shown in SEQ ID NO: 9,

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

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

[0031] (5) The HCDR1 with the amino acid sequence shown in SEQ ID NO: 14,

[0032] The HCDR2 with the amino acid sequence shown in SEQ ID NO: 15,

[0033] The HCDR3 with the amino acid sequence shown in SEQ ID NO: 16,

[0034] The LCDR1 with the amino acid sequence shown in SEQ ID NO: 18,

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

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

[0037] (6) The HCDR1 with the amino acid sequence shown in SEQ ID NO: 32,

[0038] The HCDR2 with the amino acid sequence shown in SEQ ID NO: 33,

[0039] The HCDR3 with the amino acid sequence shown in SEQ ID NO: 34,

[0040] The LCDR1 with the amino acid sequence shown in SEQ ID NO: 36,

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

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

[0043] (7) The HCDR1 with the amino acid sequence shown in SEQ ID NO: 40,

[0044] The HCDR2 with the amino acid sequence shown in SEQ ID NO: 41,

[0045] The HCDR3 with the amino acid sequence shown in SEQ ID NO: 42,

[0046] the LCDR1 with the amino acid sequence shown in SEQ ID NO: 9,

[0047] the LCDR2 with the amino acid sequence shown in SEQ ID NO: 44, and

[0048] the LCDR3 with the amino acid sequence shown in SEQ ID NO: 11.

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

[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 the amino acid sequence shown in SEQ ID NO: 22, and the light chain variable region with the amino acid sequence shown in SEQ ID NO: 26;

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

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

[0054] (d) The heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 4, and the light chain variable region with the amino acid sequence 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 the amino acid sequence shown in SEQ ID NO: 31, and the light chain variable region with the amino acid sequence shown in SEQ ID NO: 35;

[0057] (g) The heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 39, and the light chain variable region with the amino acid sequence 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) A heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 64, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 65;

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

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

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

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

[0064] In another preferred example, the antibody or its antigen-binding fragment includes a whole antibody, Fab, single-chain antibody (scFv), or nanobody.

[0065] In another preferred example, 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 with any one of the above sequences.

[0066] In another preferred example, any one of the above amino acid sequences further includes a derivative sequence that is optionally added, deleted, modified and / or substituted with at least one amino acid and can retain the DLL3 binding affinity.

[0067] In another preferred example, the number of added, deleted, modified and / or substituted amino acids is 1 - 3, preferably 1 - 2, 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 example, the single-chain antibody sequentially includes a light chain variable region, a linker and a heavy chain variable region, or sequentially includes a heavy chain variable region, a linker and a light chain variable region.

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

[0071] In another preferred example, the antibody includes a monospecific, bispecific, or trispecific antibody.

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

[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) a transmembrane domain,

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

[0076] (iv) an activation domain.

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

[0078] L-scFv-H-TM-C-S (I)

[0079] In the formula,

[0080] each "-" is independently a linker peptide or a peptide bond;

[0081] L is an optional signal peptide sequence;

[0082] scFv is 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;

[0083] H is an optional hinge region;

[0084] TM is a transmembrane domain;

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

[0086] S is a cytoplasmic signaling sequence.

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

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

[0089] In another preferred embodiment, H is the 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 of a protein selected from the following group: 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, CRTAM, 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 a combination thereof.

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

[0092] In another preferred embodiment, the TM is a transmembrane region of a protein selected from the following group: 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 conduction sequence) is a cytoplasmic conduction sequence selected from the following group sources: CD3ζ, CD3γ, CD3δ, CD3ε, Fc receptor gamma chain, FcRβ, CD79a, CD79b, FcγRIIa, DAP10, DAP12, NKp44, NKp30, NKp46, NKG2D.

[0094] In the third aspect of the present invention, a recombinant protein is provided, and the recombinant protein has:

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

[0096] (ii) Optionally, a tag sequence for assisting expression and / or purification.

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

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

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

[0100] In the fourth aspect of the present invention, an antibody-drug conjugate is provided, and the antibody-drug conjugate contains:

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

[0102] (b) A conjugate moiety conjugated to the antibody moiety, and the conjugate moiety is selected from the group consisting of: a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

[0103] In another preferred example, the antibody moiety is conjugated to the conjugate moiety through a chemical bond or a linker.

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

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

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

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

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

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

[0110] In another preferred embodiment, the TGF-β DNR 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 comprises 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 comprises 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 comprises 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 switch 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, E2A peptide.

[0116] In another preferred embodiment, when the polynucleotide encodes the CAR fusion protein as described in the second aspect of the present 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 switch receptor (CSR) or dominant negative receptor (DNR); or a polynucleotide sequence encoding the chimeric switch 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 a CAR fusion protein as described in the second aspect of the present 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 a CAR fusion protein as described in the second aspect of the present 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 amino acid sequences.

[0119] In the sixth aspect of the present invention, there is provided a vector, which contains the polynucleotide as described in the fifth aspect of the present invention.

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

[0121] In the seventh aspect of the present invention, there is provided a genetically engineered host cell, which contains the vector as described in the sixth aspect of the present invention, or the polynucleotide as described in the fifth aspect of the present invention is integrated into the genome, or expresses the antibody or its antigen-binding fragment as described in the first aspect of the present invention or the chimeric antigen receptor fusion protein as described in the second aspect of the present invention.

[0122] In another preferred embodiment, the cell is a separated cell, and / or the cell is a genetically engineered cell.

[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 cell is a T cell, a macrophage or an NK cell.

[0126] In another preferred embodiment, the engineered immune cell includes a T cell, a macrophage or an NK cell, preferably (i) a chimeric antigen receptor T cell (CAR-T cell); or (ii) a chimeric antigen receptor NK cell (CAR-NK cell).

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

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

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

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

[0131] In another preferred embodiment, the PD-1 CSR comprises an amino acid sequence as shown in SEQ ID NO: 80, or comprises 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 comprises 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 comprises 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 present invention, the CAR fusion protein and the CSR or the DNR are co-expressed 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, E2A peptide.

[0136] In another preferred embodiment, the host cell expresses the CAR fusion protein and the PD-1 CSR, and its amino acid sequence 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, and its amino acid sequence 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 TGFbRII DNR, and its amino acid sequence comprises the amino acid sequence shown in SEQ ID NO: 86.

[0139] In the eighth aspect of the present invention, a method for preparing engineered immune cells is provided. The engineered immune cells express the CAR fusion protein as described in the second aspect of the present invention, and the method includes the following steps: transfecting the polynucleotide as described in the fifth aspect of the present invention or the vector as described in the sixth aspect of the present invention into T cells or NK cells to obtain the engineered immune cells.

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

[0141] In the ninth aspect of the present invention, there is provided the use of the antibody or its antigen-binding fragment as described in the first aspect of the present invention, the chimeric antigen receptor fusion protein as described in the second aspect of the present invention, the vector as described in the sixth aspect of the present invention, or the host cell as described in the seventh aspect of the present invention for preparing a drug or preparation for preventing and / or treating DLL3-positive cancer or tumor.

[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-resistant prostate cancer, small cell bladder cancer, neuroendocrine lung tumor, or a combination thereof.

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

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

[0145] (2) A pharmaceutically acceptable carrier.

[0146] In another preferred embodiment, the carrier is selected from the group consisting of: diluent, excipient, or a combination thereof.

[0147] In the eleventh aspect of the present invention, a kit for preparing the cell as described in the seventh aspect of the present invention is provided. The kit contains a container and the polynucleotide as described in the fifth aspect of the present invention or the vector as described in the sixth aspect of the present invention located in the container.

[0148] In a twelfth aspect of the present invention, there is provided a method for treating a disease, comprising administering to a subject in need an appropriate amount of the cells described in the seventh aspect of the present invention, or the preparation described in the tenth aspect of the present 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 expression of DLL3.

[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-resistant prostate cancer, small cell bladder cancer, neuroendocrine lung tumor, or a combination thereof.

[0152] It should be understood that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0153] Figure 1 Shows the DLL3 expression of 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] Figure 3 a- Figure 3 f shows the short-term killing comparison of CAR-T corresponding to D3001-D3034 against NCI-H82 and SHP-77 cells at effector-to-target ratios of 3:1, 1:1, and 0.3:1.

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

[0157] Figure 5 a- Figure 5 d shows the short-term killing comparison of CAR-T corresponding to D3005, D3012-D3016, D3018, D3022-D3025, D3027, D3028, D3031-D3034 against CHO-K1 cell lines overexpressing human DLL3, human DLL1, and human DLL4, as well as CHO-K1 cell lines, at effector-to-target ratios of 3:1, 1:1, and 0.3:1.

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

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

[0160] Figure 8 a- Figure 8 b shows the long-term killing test results of the preferred murine-derived CAR-T against NCI-H82-3D cell cultures.

[0161] Figure 9 Shows a. the growth trend of tumor masses in NPG mice in each group after CAR-T cell infusion; b. the body weight change curves of NPG mice in each group; c. the T cell metabolic kinetics curves in NPG mice; d. the CAR-T cell metabolic kinetics curves in NPG mice.

[0162] Figure 10 a- Figure 10 b shows the short-term and long-term killing effects of the preferred humanized CAR-T of D3018, D3031, and D3034 against NCI-H82 cells.

[0163] Figure 11 a- Figure 11 d shows the short-term killing effect of the preferred humanized CAR-T of D3018, D3031, and D3034 and the parental CAR-T against NCI-H82 cells.

[0164] Figure 12 a- Figure 12 d shows the long-term killing effect of the preferred humanized CAR-T of D3018, D3031, and D3034 and the parental CAR-T against NCI-H82 cells.

[0165] Figure 13 a- Figure 13d shows the long-term killing effects of the humanized CAR-Ts of D3018, D3031, and D3034 and the parental CAR-T on the NCI-H82-3D culture.

[0166] Figure 14 a- Figure 14 d shows the short-term killing comparison of the CAR-Ts corresponding to D3018, D3031, D3034, and their preferred humanized sequences against the CHO-K1 cell line overexpressing human DLL3, human DLL1, and human DLL4 and the CHO-K1 cell line at effector-to-target ratios of 3:1, 1:1, and 0.3:1.

[0167] Figure 15 a- Figure 15 b shows the short-term killing comparison of the CAR-Ts corresponding to D3018, D3031, D3034, and their preferred humanized sequences against the CHO-K1 cell line overexpressing human DLL3, human DLL1, and human DLL4 and the CHO-K1 cell line at effector-to-target ratios of 3:1, 1:1, and 0.3:1.

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

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

[0170] Figure 18 It shows the comparison of 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. a-d: Comparison of the 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. e-h: Comparison of the 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. i-l: Comparison of the 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 19Shows the in vivo safety verification of D3045 and D3058. a. Growth trend of tumor masses in NPG mice of each group after infusion of 10E6 CAR-T cells; b. Weight change curves of NPG mice in each group; c. T cell metabolic kinetics curve in NPG mice; d. CAR-T cell metabolic kinetics curve 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. Growth trend of tumor masses in NPG mice of each group after infusion of 1.5E6 CAR-T cells; b. Weight change curves of NPG mice in each group; c. T cell metabolic kinetics curve in NPG mice; d. CAR-T cell metabolic kinetics curve in NPG mice.

[0173] Figure 21 Shows the in vivo safety verification of a. D3059 and D3067. a. Growth trend of tumor masses in NPG mice of each group after infusion of 10E6 CAR-T cells; b. Weight change curves of NPG mice in each group; c. T cell metabolic kinetics curve in NPG mice; d. CAR-T cell metabolic kinetics curve in NPG mice.

[0174] Figure 22 Shows the structural pattern diagrams of DLL3 and its truncated forms.

[0175] Figure 23 Shows the short-term killing comparison of CAR-T corresponding to the preferred murine sequences D3018, D3031, and D3034 against the CHO-K1 cell line overexpressing the full-length human DLL3 and its truncated forms and the CHO-K1 cell line at an effector-to-target ratio of 3:1.

[0176] Figure 24 Shows the short-term killing comparison of CAR-T corresponding to the preferred humanized sequence D3045 against the CHO-K1 cell line overexpressing the full-length human DLL3 and its truncated forms and the CHO-K1 cell line at an effector-to-target ratio of 3:1.

[0177] Figure 25 a- Figure 25 d Shows the long-term killing effect of D3034, D3045 / PD-1 DNR, and D3045 / PD-1 CSR on NCI-H889 cells and the amplification curves of T cells / CAR-T cells.

[0178] Figure 26 a- Figure 26d shows the long-term killing effects of D3034 and D3045 / TGFbRII DNR on NCI-H889 cells and the amplification curves of T cells / CAR-T cells in the medium supplemented with 0 ng / mL TGF-β (-) and 5 ng / mL TGF-β (+). Detailed implementation manners

[0179] Through extensive and in-depth research and a large number of screenings, the present inventors have developed an antibody targeting DLL3 or its antigen-binding fragment (e.g., single-chain antibody), a chimeric antigen receptor comprising the single-chain antibody thereof, and a CAR-immune cell (such as a CAR-T cell) expressing the chimeric antigen receptor. The present invention also provides a humanized CAR-T cell with reduced immunogenicity.

[0180] Through the screening and careful design of the CAR element structure of the present invention (including but not limited to the screening of scFv suitable for CAR expression and tumor killing effects), the CAR-immune cells of the present invention can specifically kill tumor cells expressing DLL3 without killing normal cells, thereby improving safety. At the same time, the CAR-immune cells of the present invention have a high killing rate for tumor cells. The present invention provides a new approach for the treatment of DLL3-positive tumors. The present invention has been completed on this basis.

[0181] Specifically, the experiments of the present invention show that the murine antibodies corresponding to D3001 to D3034 of the present invention all have good affinity. Through in vitro killing experiments of short-term killing, long-term killing, and 3D cell cultures, it is found that the murine CAR-Ts corresponding to D3012, D3014, D3018, D3025, D3027, D3031, and D3034 of the present invention have good killing effects. Based on this, humanized CAR-Ts are constructed, and it is found that the humanized DLL3-targeting CAR-T cells corresponding to D3045, D3056, D3058, and D3059 of the present invention have excellent short-term and long-term killing effects on in vitro tumor cells and antitumor effects in vivo.

[0182] Terms

[0183] To more easily understand the present disclosure, certain terms are first defined. As used in this application, unless otherwise clearly 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" may refer to a value or a component within an acceptable error range of a specific value or component determined by a person of ordinary skill in the art, which will depend in part on how the value or component is measured or determined.

[0185] The term "administer" refers to the physical introduction of the product of the present invention into a subject using any of a variety of methods and delivery systems known to those of skill in the art, including intravenous, intramuscular, subcutaneous, intraperitoneal, intraspinal, or other parenteral routes of administration, such as by injection or infusion.

[0186] The term "isolated" refers to a substance that is substantially or essentially free of components that are normally associated with it in its natural state. The substance can be a cell or a macromolecule, such as a protein or a nucleic acid. For example, as used herein, an "isolated nucleic acid" refers to a polynucleotide that has been purified from the sequences that flank it in its natural state, such as a DNA fragment that has been removed from the sequences that are normally adjacent. Alternatively, as used herein, an "isolated antibody" or "isolated polypeptide" and the like refer to an antibody or polypeptide molecule that has been isolated and / or purified in vitro from its natural cellular environment and its association with other components of the cell.

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

[0188] It should be understood that the amino acid names are identified by a single English letter in the international common use, 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), Ile(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 cell

[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 DLL3-targeting CAR-immune cells described in the first aspect of the present invention.

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

[0192] The chimeric antigen receptor (CAR) of the present invention includes an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain includes a target-specific binding element (also referred to as an antigen-binding domain). The intracellular domain includes a co-stimulatory signaling region and a ζ-chain portion. The co-stimulatory signaling region refers to a part of the intracellular domain that includes a co-stimulatory molecule. Co-stimulatory molecules are cell surface molecules required for an effective response of lymphocytes to an antigen, rather than antigen receptors or their ligands.

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

[0194] As used herein, both "antigen-binding domain" and "single-chain antibody fragment" refer to a Fab fragment, Fab' fragment, F(ab')2 fragment, or a single Fv fragment having antigen-binding activity. An Fv antibody contains the variable region of the antibody heavy chain and the variable region of the light chain, but no constant region, and is the smallest antibody fragment having all antigen-binding sites. Generally, an Fv antibody also contains a polypeptide linker between the VH and VL domains and is capable of forming the structure required for antigen binding. The antigen-binding domain is usually a scFv (single-chain variable fragment). The size of an scFv is generally 1 / 6 of a complete antibody. The single-chain antibody is preferably an amino acid sequence of a single chain encoded by a single nucleotide chain. As a preferred embodiment of the present 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 embodiment, a transmembrane domain that is 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 interaction with other members of the receptor complex.

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

[0197] PD-L1 overexpressed on tumor cells can specifically recognize and bind to PD-1 on activated T cells, resulting in the loss of killing ability of T cells. Expressing a PD-1 dominant negative receptor (DNR) on the surface of CAR-T cells, PD-1 DNR can bind to PD-L1 on tumor cells and does not activate the PD-1 pathway inside T cells, which can overcome the inhibitory effect of PD-L1 on the surface of tumor cells on CAR-T cells. Furthermore, by replacing the natural inhibitory signal domain in the intracellular part of T cell PD-1 with an activating signal domain, such as the intracellular domain of CD28, the inhibitory signal of T cell PD-1 induced by PD-L1 on tumor cells can be converted into an activating signal for CAR-T, enhancing the function of CAR-T.

[0198] PD-1 CSR (PD-1 chimeric switch receptor) is a genetically engineered receptor that can specifically recognize and bind to the PD-L1 molecule. Different from the traditional PD-1 receptor, after binding to PD-L1, PD-1 CSR does not transmit inhibitory signals but instead 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 the anti-tumor ability of T cells, resulting in the loss of the ability of T cells to kill tumors. Expressing a TGFβ receptor II dominant negative receptor (DNR) on the surface of CAR-T cells, TGFβRIIDNR (or TFGbRII DNR) can bind to TGFβ on tumor cells but does not activate the TGFβ pathway inside T cells, which can overcome the inhibitory effect of TGFβ on CAR-T cells in the tumor immunosuppressive microenvironment.

[0200] vector

[0201] Nucleic acid sequences encoding the desired molecule can be obtained using recombinant methods known in the art, such as, for example, by screening libraries from cells expressing the gene, by obtaining the gene from a vector known to include the gene, or by directly isolating it from cells and tissues containing the gene using standard techniques. Optionally, the gene of interest can be produced synthetically.

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

[0203] Briefly, generally the expression cassette or nucleic acid sequence of the invention is operably linked to a promoter and incorporated into an expression vector. The vector is suitable for replication and integration in eukaryotic cells. Typical cloning vectors contain transcriptional and translational terminators, initiation sequences, and promoters that can be used to regulate the expression of the desired nucleic acid sequence.

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

[0205] The nucleic acid can be cloned into many types of vectors. For example, the nucleic acid can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Particular vectors of interest include expression vectors, replication vectors, probe production vectors, and sequencing vectors.

[0206] Furthermore, the expression vector can be provided to the cells in the form of a viral vector. Viral vector technology is well known in the art and is 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. Generally, suitable vectors contain an origin of replication that functions in at least one organism, a promoter sequence, convenient restriction enzyme sites, and one or more selectable markers (e.g., WO01 / 96584; WO01 / 29058; and U.S. Patent No. 6,326,193).

[0207] Numerous virus-based systems have been developed for gene transfer 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 ex vivo. 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 modulate the frequency of transcription initiation. Typically, these are located in the region between 30 - 110 bp upstream of the start site, although recently it has been shown that many promoters also contain functional elements downstream of the start site. The spacing between promoter elements is often flexible so that promoter function is maintained when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased by 50 bp before activity begins to decline. Depending on the promoter, it has been shown that individual elements can act 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 strong constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences can also be used, including but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein - Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters such as but not limited to actin promoter, myosin promoter, heme promoter, and creatine kinase promoter. Further, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention. The use of inducible promoters provides a molecular switch that can turn on the expression of a polynucleotide sequence operably linked to the inducible promoter when such expression is desired, or turn off the expression when the expression is not desired. Examples of inducible promoters include but are not limited to the metallothionein promoter, glucocorticoid promoter, progesterone promoter, and tetracycline promoter.

[0210] To evaluate the expression of a CAR polypeptide or a portion thereof, the expression vector introduced into the cells may also contain either or both of a selectable marker gene and a reporter gene to facilitate the identification and selection of expressing cells from the population of cells sought to be transfected or infected by the viral vector. In other aspects, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes such as neo and the like.

[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 not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifest by some readily detectable property such as enzymatic activity. After the DNA has been introduced into the recipient cells, the expression of the reporter gene is assayed at an appropriate time. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79-82). Suitable expression systems are well known and may be prepared using known techniques or obtained commercially. Generally, constructs having at least 5 flanking regions that show the highest level of reporter gene expression are identified as promoters. Such promoter regions may be ligated to a reporter gene and used to evaluate the ability of a reagent to modulate promoter-driven transcription.

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

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

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

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

[0216] In the case of using a non-viral delivery system, an exemplary delivery vehicle is a liposome. Lipid formulations are contemplated for introducing nucleic acids into host cells (in vitro, ex vivo, or in vivo). On the other hand, the nucleic acid can be associated with lipids. Nucleic acids associated with lipids can be encapsulated within the aqueous interior of liposomes, dispersed within the lipid bilayer of liposomes, attached to liposomes via a linking molecule associated with both the liposome and the oligonucleotide, entrapped within liposomes, complexed with liposomes, dispersed in a solution containing lipids, mixed with lipids, combined with lipids, included as a suspension within lipids, included within micelles 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 can exist in a bilayer structure, as micelles, or have a "collapsed" structure. They can also simply be dispersed in solution, possibly forming aggregates of non-uniform size or shape. Lipids are fatty substances, which can be naturally occurring or synthetic lipids. For example, lipids include lipid droplets, which occur naturally in the cytoplasm and in such 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 vector is a lentiviral vector.

[0218] Formulation

[0219] The present invention provides a formulation comprising the CAR-immune cells (e.g., CAR-T cells) described in the seventh aspect of the present invention, and a pharmaceutically acceptable carrier, diluent, or excipient. In one embodiment, the formulation is a liquid formulation. Preferably, the formulation is an injection. Preferably, the concentration of the CAR-T cells in the formulation is 1×10 3 -1×10 8cells / ml, more preferably 1×10 4 -1×10 7 cells / ml.

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

[0221] Therapeutic applications

[0222] The present invention includes therapeutic applications with cells (e.g., T cells) transduced with a lentiviral vector (LV) encoding the expression cassette of the present invention. The transduced T cells can target the tumor cell marker DLL3, can be used for autologous and allogeneic tumor treatment, can be prepared on a large scale, are of uniform and stable quality, and can be readily available for use in any patient.

[0223] Accordingly, the present invention also provides a method of stimulating a T cell-mediated immune response against a target cell population or tissue in a mammal, which comprises the step of administering the CAR-T cells of the present invention to the 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 the CAR-T cells are injected into a recipient in need thereof. The injected cells are capable of killing the tumor cells of the recipient. Unlike antibody therapies, CAR-T cells can replicate in vivo, resulting in long-term persistence 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 can persist for a sustained period of time. Additionally, the CAR-mediated immune response can be part of an adoptive immunotherapy procedure, wherein the CAR-modified T cells induce an immune response specific for 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 a lentiviral vector comprising an anti-DLL3 scFv, a CD8α hinge region and transmembrane region, and 4-1BB and CD3ζ signaling domains, the present invention is to be construed as including any number of variations to each of the components of the construct.

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

[0228] A solid tumor is an abnormal mass of tissue that generally does not contain cysts or areas of fluid. Solid tumors can be either benign or malignant. Different types of solid tumors are named for the cell types that form them (such as small cell lung cancer, large cell neuroendocrine carcinoma, gastrointestinal neuroendocrine tumor, glioblastoma multiforme, metastatic castration-resistant prostate cancer, small cell bladder cancer, neuroendocrine lung tumor, etc.).

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

[0230] For ex vivo immunization, at least one of the following occurs in vitro prior to administering the cells into a mammal: i) expanding the cells, ii) introducing a nucleic acid encoding the CAR into the cells, and / or iii) cryopreserving the cells.

[0231] Ex vivo procedures are well known in the art and are discussed more fully below. Briefly, cells are isolated from a mammal (preferably a human) and genetically modified (i.e., transduced or transfected in vitro) with a vector expressing the CAR disclosed herein. The CAR-modified cells can be administered to a mammalian recipient to provide a therapeutic benefit. The mammalian recipient can be a human, and the CAR-modified cells can be autologous with respect to the recipient. Optionally, the cells can be allogeneic, syngeneic, or xenogeneic with respect 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 an antigen in a patient.

[0233] The present invention provides a method of treating a tumor, which comprises administering to a subject in need thereof a therapeutically effective amount of the CAR-immune cells of the present invention.

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

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

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

[0237] Administration of the subject composition can be effected in any convenient manner, including by spraying, injection, swallowing, infusion, implantation or transplantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, by intravenous (i.v.) injection 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 i.v. injection. The composition of T cells can be directly injected into a tumor, lymph node or site of infection.

[0238] In certain embodiments of the present invention, cells activated and expanded using the methods described herein or other methods known in the art for expanding T cells to therapeutic levels are administered to a patient in combination (e.g., before, concurrently, or after) with any number of relevant treatment modalities, which include but are not limited to treatment with the following reagents: reagents such as bevacizumab, megestrol acetate dispersible tablets, paclitaxel injection, ifosfamide, and ifosfamide for injection in the treatment of ovarian cancer patients. In further embodiments, the CAR-immune cells of the present invention can be used in combination with: chemotherapy, radiation, immunosuppressive agents, such as cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunotherapeutic agents. In further embodiments, the cell compositions of the present invention are administered to a patient in combination (e.g., before, concurrently, or after) with bone marrow transplantation, using chemotherapeutic agents such as fludarabine, external beam radiotherapy (XRT), and cyclophosphamide. For example, in one embodiment, a 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 present invention. In an additional embodiment, the expanded cells are administered before or after surgery.

[0239] The doses of the above treatments administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. Dosage ratios for human administration can be practiced according to accepted practice in the art. Generally, for each treatment or course of treatment, 1×10 6 to 1×10 10 CAR-immune cells of the present invention can be administered to a patient, for example, by intravenous infusion.

[0240] The main advantages of the present invention include:

[0241] (a) The antibodies targeting DLL3 of the present invention all have optimized affinities (fine-tuned affinity, too high may lead to systemic toxicity, and too low may result in poor effects).

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

[0243] (c) The antibodies targeting DLL3 of the present invention all have species cross-reactivities that are conducive to safety evaluation.

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

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

[0246] (f) The humanized DLL3-targeted CAR-T of the present invention shows better killing effects and amplification trends than the sequence when fused with other elements (PD-1 DNR, PD-1 CSR, and TFGbRII DNR).

[0247] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight percentage and weight parts.

[0248] Example 1 Preparation and Screening of Anti-human DLL3 Antibody Hybridoma Cells

[0249] 1.1 Preparation of Hybridoma Cells

[0250] Immunization: Balb / c mice were immunized with the extracellular region recombinant protein of human DLL3 (sequence number: UniProtKB-Q9NYJ7, 27aa - 492aa), and the serum titer was detected by ELISA using a 96-well enzyme-linked immunosorbent assay plate coated with the recombinant protein of human DLL3-his (sequence number: UniProtKB-Q9NYJ7, 27aa - 492aa); the mice that met the fusion requirements were used for the next cell fusion.

[0251] Cell fusion and hybridoma preparation: Mice with the required titer were selected for booster immunization. Three days later, the spleens of the mice were aseptically taken to prepare B lymphocyte suspensions, which were fused with SP2 / 0 myeloma cells. The fused cells were resuspended in HAT medium and then aliquoted into 96-well cell culture plates. They were cultured in an incubator at 37°C and 5% CO2.

[0252] 1.2 Screening of Positive Hybridoma Binding

[0253] 10 - 14 days after fusion, coat an ELISA plate with the recombinant human DLL3-his extracellular domain protein (20 ng / ml) and incubate overnight at 4°C; after washing three times with PBS, block with 4% skim milk-PBS at room temperature for 1 hour; wash three times with PBS, add the hybridoma clone culture supernatant, and incubate at room temperature for 1 hour. Set the following controls: (1) Positive control (PC): Serum from immunized mice (diluted 1:1000 with PBS); (2) Negative control (NC): Serum from non-immunized mice (diluted 1:1000 with PBS). After washing three times with PBST (0.05% Tween-PBS) and twice with PBS, add HRP-conjugated goat anti-mouse IgG (Fcγ), and incubate at 37°C for 0.5 hour; then wash three times with PBST (0.05% Tween 20-PBS), add TMB chromogenic solution, develop color in the dark for 15 - 30 minutes, add ELISA stop solution to terminate the reaction; read the A450 value with an ELISA reader. Select the clones with higher readings according to the principle from high to low, and take the clone culture supernatant the next day for secondary ELISA confirmation.

[0254] Example 2: Sequencing of Murine Anti-Human DLL3 Antibody

[0255] After expanding the culture of hybridoma clone cells secreting anti-human DLL3 antibody, extract total cellular RNA according to the steps described in the TRIzol kit (Cat: 15596026, Invitrogen); reverse transcribe the total cellular RNA of hybridoma cells into cDNA using M-MuLV reverse transcriptase (Cat: M0253S, NEB); amplify the variable region of the antibody light chain IgVL(x) and the variable region of the heavy chain VH sequences using degenerate primers and the Phusion kit (Cat: EO553L, NEB); purify the PCR amplification products using a gel extraction kit (Cat: AP-GX-250, Axygen); ligate the amplified PCR products to the T vector according to the instructions of the T vector cloning kit (Cat: ZC205, Zhuangmeng Biotech) and transform Escherichia coli competent cells, and sequence the plasmid after strain amplification and plasmid extraction to obtain the variable region sequence and CDR sequence of the monoclonal antibody.

[0256] Example 3: Affinity Analysis of Murine Antibody

[0257] Using the extracellular region of human DLL3 (UniProtKB-Q9NYJ7) fused with mFc protein (DLL3-ECD-mFc) as an antigen, after fully emulsifying it with an equal volume of complete Freund's adjuvant (Sigma, F5581), 6- to 8-week-old Balb / c mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were immunized subcutaneously. The antigen immunization dose was 50 μg / mouse. Subsequently, every 2 weeks, the mice were immunized subcutaneously three times with the same dose of antigen fully emulsified with incomplete Freund's adjuvant (Sigma, F5506). After three immunizations, the serum titers of the mice were measured.

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

[0259] The DLL3 antigen protein was prepared into 7 concentrations with gradient dilutions of 100 nM, 50 nM, 25 nM, 12.5 nM, 6.5 nM, 3.13 nM, and 1.56 nM. Samples were loaded onto a 96-well plate. Human Protein G probe was selected, the stationary phase was the antigen, and the mobile phase was the DLL3 antibody. The binding time was set to 180 s, and the dissociation time was set to 300 s. The binding constant and dissociation constant of the human monoclonal antibody were detected, and the affinity was calculated.

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

[0261] Table 1

[0262]

[0263] Example 4 Humanization and Mutation Design of Anti-Human DLL3 Monoclonal Antibody

[0264] 4.1 Humanization of Murine Monoclonal Antibody

[0265] CDR Transplantation

[0266] First, comprehensively analyze the heavy chain sequence of the murine antibody to determine the Complementarity-determining regions (CDR) where the antibody binds to the antigen and the framework regions that support the conserved three-dimensional conformation of the antibody. Subsequently, based on the homology alignment results, select the most similar human antibody as the basic template, and combine with the results of the full-sequence blast to perform CDR transplantation. Retain the CDR regions of the murine antibody and replace the framework region sequence of the murine antibody with the framework region sequence of the human germline antibody.

[0267] Secondly, establish the structural model of the murine antibody, and compare each different amino acid site in the structural models of the human antibody and the corresponding murine antibody one by one. If using the human amino acid sequence at a certain site in the framework region does not cause the disruption or change of the spatial structure of the CDR region, then use the human amino acid sequence at this site; otherwise, use the corresponding murine sequence at this site (i.e., revert to the murine sequence).

[0268] According to the structural simulation, partially revert the amino acids in the framework region of the humanized antibody to the murine sequence. Finally, obtain the amino acid sequences of the variable regions of the heavy and light chains of the humanized antibody.

[0269] Example 5 Cultivation of Tumor Cell Lines

[0270] 5.1 Cell Line Cultivation

[0271] Six cell lines are used in the present invention, namely 293T, SUP-T1, NCI-H82, SHP-77, NCI-H889, and CHO-K1 cells. Among them, 293T cells are a human embryonic kidney epithelial cell line, used for studying the expression of foreign genes and virus preparation; SUP-T1 is a T lymphocyte cell line, used for detecting virus titer; NCI-H82, SHP-77, and NCI-H889 are human small cell lung cancer cell lines, which are the target cells of this study; CHO-K1 is a subcloned cell line of the Chinese hamster ovary cell line CHO, used for constructing an overexpression cell line of foreign genes. 293T cells are cultured in DMEM complete medium (DMEM + 10% FBS); SUP-T1, NCI-H82, SHP-77, and NCI-H889 cells are cultured in 1640 complete medium (1640 + 10% FBS); CHO-K1 is cultured in F12K complete medium (F12K + 10% FBS).

[0272] 5.2 Flow Cytometry Detection of the Expression of Tumor Cell DLL3 Target

[0273] Centrifuge the cells to be tested at 300g for 5 minutes to collect the cell pellet; wash the cells once with PBS. Take 2×10 5The cells were stained with 100 μL of the staining system. After resuspending the cells in the staining system, they were incubated in the dark at room temperature for 30 min. After the incubation, they were washed once with the flow cytometry buffer, resuspended in 200 μL of the flow cytometry buffer, and then subjected to analysis on the instrument.

[0274] The staining system was prepared as follows: 100 μL of the flow cytometry buffer + antibody.

[0275] The formula of the flow cytometry buffer was: PBS + 1% FBS + 2.5 mM EDTA.

[0276] The results of the flow cytometry detection were as Figure 1 shown. The cell surface of the small cell lung cancer cell lines NCI-H82 and SHP-77 highly expressed the DLL3 protein.

[0277] Example 6 Construction of a CAR based on a murine antibody-derived scFv

[0278] The CAR structure used in this example was a second-generation CAR structure. The specific structure was that at the 5' end was the CD8 signal peptide, the scFv sequence region, followed by the CD8 hinge region, the CD8 transmembrane region, the 4-1BB intracellular co-stimulatory domain, and the CD3ζ signal transduction domain. Among them, the scFv started from the 5' end with the variable light chain region (VL) of the antibody, G4S (SEQ ID NO: 79), a flexible linker polypeptide, and the variable heavy chain region (VH) of the antibody.

[0279] Example 7 Lentivirus production

[0280] 2×10 7 293T cells were seeded into a 15-cm cell culture dish with a medium volume of 20 mL. The next day, the expression plasmid, the helper plasmid pMDLg-pRRE, the helper plasmid pRSV-Rev, and the helper plasmid pMD2.G were mixed in a ratio of 2:1:1:1 in 1 mL of opti-MEM medium to prepare a plasmid-containing medium; additionally, 50 μg of PEI was added to 1 mL of opti-MEM medium, mixed well, and after standing at room temperature for 5 minutes, the PEI mixed solution was added dropwise to the plasmid mixed solution. After thorough mixing, it was left standing at room temperature for 15 minutes. This plasmid-PEI solution was evenly added to the 293T cell culture dish. After 16 h, the fresh 20 mL of complete DMEM medium was replaced. After 48 h, the culture medium supernatant was collected, filtered through a 0.45-μm filter membrane, and then ultracentrifuged at 30,000 g for 2 hours to obtain the virus precipitate. The virus precipitate was resuspended in PBS medium, and the virus solution was aliquoted. The virus titer of the virus solution was detected using SUP-T1 cells. The virus solution was stored long-term at -80 °C.

[0281] Example 8 Activation and virus transduction of T cells

[0282] 8.1 Peripheral blood mononuclear cell (PBMC) resuscitation

[0283] Turn on the water bath and preheat the temperature to 37°C. Take out a tube of PBMC from the liquid nitrogen tank, quickly place it in the water bath, and rapidly dissolve the cell cryopreservation solution. After dissolution, spray the cryotube with 75% alcohol and open it in the 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.

[0284] 8.2 Activation of PBMC cells

[0285] Resuspend the cells with AIM-V complete medium (5% FBS + 300 IU IL-2 / mL), and take a sample for counting. According to the counting result, dilute the cells to 2×10 6 / mL. Add 10 μL of TransAct T cell activator to every 2×10 6 PBMC cells. Place the cells in an incubator at 37°C and 5% carbon dioxide for activation culture for 48 hours.

[0286] 8.3 Collection of activated T cells and virus transduction

[0287] After 48 hours of activation culture of PBMC, collect the cells into a 15 mL centrifuge tube and centrifuge at 400 g for 5 minutes. After centrifugation, resuspend the cells with AIM-V complete medium (5% FBS + 300 IU / mL IL-2). Take a sample for counting.

[0288] Adjust the T cells to 1×10 6 / mL according to the counting result, and aliquot 0.5 mL per well into a 24-well plate; add the corresponding CAR-T lentivirus at MOI = 10, and add Polybrene at a concentration of 8 μg / mL. Mix well and centrifuge at 1000 g for 1 hour in a centrifuge.

[0289] After centrifugation, take out the 24-well plate from the centrifuge, pipette the precipitated cells to mix well and put them back into the incubator to culture overnight. The next day, replace with fresh AIM-V complete medium (5% FBS + 300 IU / mL IL-2). After culturing for five days, collect some cells and detect the CAR expression of the cells.

[0290] Example 9 Flow cytometry staining analysis

[0291] Centrifuge the cells to be tested at 300 g for 5 minutes to collect the cell pellet; wash the cells once with PBS. Take 2×10 5The cells were stained with 100 μL of the staining system. After resuspending the cells in the staining system, they were incubated in the dark at room temperature for 30 min. After the incubation, they were washed once with flow cytometry buffer, resuspended in 200 μL of flow cytometry buffer, and then subjected to on-machine analysis.

[0292] The staining system was prepared as follows: 100 μL of flow cytometry buffer + antibody.

[0293] The formula for the flow cytometry buffer was: PBS + 0.5% BSA + 2.5 mM EDTA.

[0294] The amount of antibody used was determined according to the antibody concentration and actual situation.

[0295] The positive rate of CAR-T cells was as Figure 2 shown. The proportion of CAR + T cells was 48.3%.

[0296] Example 10 Detection of the cytotoxic effect of DLL3-CAR-T cells on tumor cells (short-term killing)

[0297] Untransduced T cells (UNT) and the prepared DLL3-CAR T cells were co-incubated with NCI-H82 and SHP-77 cells at effector-to-target ratios of 0.3:1, 1:1, and 3:1 for 16 h, and then the death and lysis of tumor cells were detected using an LDH kit (Roche, 11644793001).

[0298] The results were as Figure 3 shown. The results showed that, compared with UNT, DLL3-CAR T cells had a good cytotoxic effect on both NCI-H82 and SHP-77 tumor cells. This indicates that DLL3-CAR T cells have a strong cytotoxic effect on solid tumor cells expressing DLL3.

[0299] Figure 3 a and Figure 3 d showed that the cytotoxic effects of the CAR-T corresponding to D3005, D3006, and D3007 on NCI-H82 and SHP-77 cells were relatively obvious and could proceed to the next comparison.

[0300] Figure 3 b and Figure 3 e showed that the cytotoxic effects of the CAR-T corresponding to D3012, D3014, D3015, D3016, D3017, D3018, and D3019 on NCI-H82 and SHP-77 cells were relatively obvious and could proceed to the next comparison.

[0301] Figure 3 c and Figure 3f shows that the cytotoxicity of CAR-T corresponding to D3028, D3031, and D3034 against NCI-H82 and SHP-77 cells is significant, and further comparison can be carried out.

[0302] Example 11 Long-Term Killing Experiment

[0303] Collect the prepared CAR-T cells, NCI-H82 cells, and SHP-77 cells respectively, centrifuge at 300g for 5 minutes, then resuspend with AIM-V medium (5% FBS), and take samples for counting. Adjust the CAR% of the prepared CAR-T cells to the same level with activated T cells from the same donor; seed 6×10 5 NCI-H82 cells and 6×10 5 SHP-77 cells into a 12-well plate, and then add the corresponding number of CAR-T cells according to the effector-to-target ratios E:T = 1:4 and 1:5 respectively.

[0304] Supplement the volume of each well to 1.5 mL with AIM-V medium (5% FBS). After co-culturing for 48 hours, detect the proportion of T cells. At the same time, add 6×10 5 target cells and continue co-culturing for the next round of detection. Stimulate with target cells continuously for 5 - 7 times, and analyze the continuous killing ability of CAR-T by detecting the proportion of T cells in each well.

[0305] In the last round, the higher the proportion of T cells means the stronger the continuous killing ability of CAR-T and the stronger the ability to resist T cell exhaustion. Similar to the short-term killing grouping, the present invention divides all binders into three batches for comparison of long-term killing, and finally collects the excellent binders in each group for a final comparison.

[0306] Figure 4 a and Figure 4 d show the trend graphs of the proportion of CD3+ cells in the long-term killing of CAR-T corresponding to D3001 - D3011 against NCI-H82 and SHP-77 cells. Combining microscopic observation and the results of short-term killing, the present invention selects D3005 for further comparison.

[0307] Figure 4 b and Figure 4 e show the trend graphs of the proportion of CD3+ cells in the long-term killing of CAR-T corresponding to D3012 - D3022 against NCI-H82 and SHP-77 cells. Select D3012, D3013, D3014, D3015, D3016, D3018, D3022 for further comparison.

[0308] Figure 4 c and Figure 4f shows the trend of the proportion of CD3+ cells in the long-term killing of NCI-H82 and SHP-77 cells by CAR-T corresponding to D3023~D3034. D3023, D3024, D3025, D3027, D3028, D3030, D3031, D3032, and D3034 were selected for the next step of comparison.

[0309] When rescreening 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 human-mouse crossover.

[0310] The results are as follows Figure 5 and Figure 6 As shown, the results showed that the above 17 sequences all had obvious toxic side effects on cell lines overexpressing human DLL3. In the human-mouse cross-validation, D3005, D3013 and D3015 did not have a killing effect on cell lines overexpressing mouse DLL3. In the specificity validation 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.

[0311] The above 17 CAR sequences were screened for long-term killing, and the results were as follows Figure 7 a- Figure 7 Combined with the results of microscopic observation and short-term killing, D3012, D3014, D3018, D3025, D3027, D3031, and D3034 were finally selected for in vitro killing of 3D culture and in vivo animal experiments as well as sequence humanization transformation.

[0312] Example 12 In vitro killing of 3D cell cultures

[0313] Collect NCI-H82 cells by centrifugation, take samples and count them for later use. Place the matrigel (ACRO, AC-M082704) in a 4°C refrigerator, fully dissolve it overnight, and then dilute it once with serum-free 1640 medium. Resuspend the NCI-H82 cells to a concentration of 50,000 cells / mL with matrigel medium; place the cell suspension on ice, and use a pre-cooled pipette tip to add 100 μL of NCI-H82 cell suspension to a 96-well plate, with three replicate wells per well and three counting wells. Place the 96-well plate in a 37°C carbon dioxide incubator and let it stand for 30 minutes to allow the matrigel suspension to fully solidify, then add 100 μL of NCI-H82 cell complete medium to each well and culture for 7-9 days. During this period, the medium can be replaced by half or full volume to maintain cell growth.

[0314] 3D cell culture counting: Discard the supernatant in the counting wells, wash twice with PBS, add 100 μL of Corning Dispase, and place it in a carbon dioxide incubator for 1 hour of digestion. Take out the plate and gently pipette until there is no sense of stickiness when pipetting. Add 100 μL of FACS buffer to the original system, centrifuge at 3000 rpm for 5 minutes, remove the supernatant, add 100 μL of FACS buffer, centrifuge at 3000 rpm for 5 minutes, and remove the supernatant completely. Add 200 μL of FACS buffer and take 20 μL for cell counting.

[0315] Addition of effector cells: Collect, count, and detect the CAR% of CAR-T cells cultured for 7 days after lentiviral infection. Correct the CAR% of CAR-T to the same level value. Culture NCI-H82 cells for about 8 days, carefully aspirate 100 μL of the supernatant from the culture wells of NCI-H82, and then add 100 μL of the CAR-T cell suspension to it at an effector-to-target ratio of 1:5. Change the medium once every 48 hours, and carefully aspirate when changing the medium to avoid sucking out the Matrigel.

[0316] After 6 days of co-culture with the addition of effector cells, discard the supernatant, add Corning Dispase to each well for digestion and counting. Resuspend the cells with 100 μL of staining buffer and add staining antibodies to detect the proportion of T cells. The more T cells, the less tumor cells, indicating better in vitro killing effect in the corresponding group and stronger infiltration ability of the corresponding T cells.

[0317] The detection results of the continuous killing of 3D cultures for 6 days are as Figure 8 shown. The CAR-T cells corresponding to the sequences in the figure all have good infiltration and killing effects on NCI-H82-3D cultures, and D3014 and D3027 have better effects.

[0318] Example 13 Experiment on anti-tumor ability in vivo - NCI-H82 / NPG animal model

[0319] NPG mice were purchased from Beijing Vitalstar Biotechnology Co., Ltd.; in this experiment, a subcutaneous injection tumorigenesis animal model was used. Each NPG mouse was subcutaneously inoculated with 1×10 7 of NCI-H82 cells. After tumor modeling, the long diameter and short diameter were measured with a vernier caliper three times a week, and the tumor was measured twice a week after grouping. Then calculate the tumor size according to the formula V = 1 / 2ab 2 ; where a is the long diameter and b is the short diameter. Weigh the body weight once every three days after starting tumor modeling and twice a week after grouping.

[0320] Seven days after inoculation with tumor cells, reinfuse 4×106 The CAR-T cells; On Day 1, Day 7, Day 14, Day 21, and Day 28 after reinfusion, blood was taken from the orbits of the mice to detect the dynamic content ratios of T cells and CAR-T cells in the peripheral blood.

[0321] Figure 9 Table a and Table 2 show the inhibitory effects on the growth of NCI-H82 tumors in NPG mice after reinfusion of CAR-T cells with different sequences. The CAR-T cells corresponding to D3018, D3031, and D3034 have good inhibitory effects on tumor growth.

[0322] Table 2

[0323]

[0324] Note: The numerical values in the table are the average volumes of tumors in each group of mice (rounded to the nearest whole number), and the unit is mm. 3 。

[0325] Figure 9 Figure b shows the change curves of the body weights of NPG mice in each group during the entire experiment. The body weights of the mice in each group increased steadily overall, without significant fluctuations.

[0326] Figure 9 Figure c and Figure 9 Figure d show the metabolic kinetic curves of T cells and CAR-T cells in NPG mice after reinfusion of CAR-T cells with different sequences. The CAR-T cells corresponding to D3018, D3031, and D3034 showed obvious in vivo amplification.

[0327] Example 14 Antibody Humanization and Sequence Screening

[0328] 14.1 Primary Screening for Short-Term and Long-Term Killing

[0329] After obtaining the plasmid of the antibody sequence after humanization modification, lentivirus was prepared and CAR-T cells were prepared; the preparation process was the same as above. The technical processes for detecting the short-term killing of each sequence by LDH and the long-term killing ability by flow cytometry were the same as above. After humanization, the present inventors mainly compared whether the humanized CAR sequence could maintain the same or better killing effect as the corresponding murine CAR sequence.

[0330] First, comparisons were made among the humanized sequences of each parent. The short-term killing and long-term killing results are as Figure 10As shown. Based on the results of short-term killing and long-term killing, among the humanized sequences corresponding to D3018, D3043 and D3045 were selected for subsequent comparison. Among the humanized sequences corresponding to D3031, D3056, D3057 and D3058 were selected for subsequent comparison. Among the humanized sequences corresponding to D3034, D3059 and D3060 were selected for subsequent comparison.

[0331] 14.2 Rescreening of short-term killing and long-term killing

[0332] Figure 11 a- Figure 11 d and Figure 12 a- Figure 12 d shows the comparison of short-term killing and long-term killing effects of D3018, D3031 and D3034 and their humanized sequences in two donors.

[0333] The results showed that the humanized sequences of D3018 and D3031 after screening both had good killing effects on NCI-H82. However, the humanized sequence of D3034 was not superior to D3034 itself. The killing effects after re-humanization design of D3034 are as Figure 11 d and Figure 12 d shows. Finally, D3059 and D3067 were selected for subsequent comparison.

[0334] 14.3 In vitro killing of 3D cultures

[0335] The in vitro killing process of 3D cultures is the same as above.

[0336] The results are as Figure 13 a- Figure 13 d shows. Combining the results of short-term killing and long-term killing and the transduction efficiency of the lentiviruses corresponding to each sequence, among the humanized sequences corresponding to D3018, D3045 was selected for subsequent in vivo comparison. Among the humanized sequences corresponding to D3031, D3056, D3057 and D3058 were selected for comparison in in vivo experiments. Among the humanized sequences corresponding to D3034, D3059 and D3067 were selected for comparison in in vivo experiments.

[0337] 14.4 In vivo killing

[0338] The constructed CHO-K1 cell lines overexpressing human DLL3, mouse DLL3, human DLL1 and human DLL4 and CHO-K1 cells were used to verify the human-mouse cross and target specificity of the candidate sequences for animal experiments.

[0339] The results are as Figure 14 a- Figure 14 d and Figure 15 a- Figure 15As shown in Figure b, the candidate sequence has a strong killing effect on human and mouse DLL3, but has no killing effect on human DLL1 and human DLL4. This indicates that the candidate sequence in the in vivo experiment has good human-mouse cross-reactivity and target specificity.

[0340] 14.5 Pharmacodynamic verification in NPG mice

[0341] 14.5.1 D3018 and D3031 humanized sequences

[0342] The NPG mice were modeled with the humanized sequences in the same way as above. The infusion doses were set as a low-dose group (1.5×10 6 ) and a high-dose group (3×10 6 ). Seven days after modeling, each mouse was infused with CAR-T cells via the tail vein.

[0343] Figure 16 a- Figure 16 d shows the inhibitory effects of CAR-T cells corresponding to the humanized sequences of D3045, D3031, and D3031 on tumors and the amplification curves of T cells and CAR-T cells in the low-dose group. In the low-dose group, the best suppression effect on tumor growth and T cell amplification was D3031, followed by the humanized sequence D3058 of D3031;

[0344] Figure 17 a- Figure 17 d shows the inhibitory effects of CAR-T cells corresponding to the humanized sequences of D3018 and D3031 and their humanized sequences on tumors and the amplification curves of T cells and CAR-T cells in the high-dose group. In the high-dose group, the best in vivo pharmacodynamic effect was the humanized sequence D3045 of D3018, followed by D3031 and its humanized sequence D3058; D3045 and D3058 were selected for further comparison in in vivo experiments. Three doses were set in the in vivo pharmacodynamics of D3045 and D3058, namely a low-dose group (0.5×10 6 ), a medium-dose group (1×10 6 ), and a high-dose group (2×10 6 ).

[0345] The results are as shown in Figure 18 a- Figure 18 l. D3045 showed pharmacodynamic effects in some mice in the low-dose group and showed good pharmacodynamic effects overall in the medium-dose group and the high-dose group. Compared with D3058, D3045 showed better effects in terms of in vivo pharmacodynamics and the metabolic kinetics of in vivo CAR-T cells, and the body weight was stable without significant fluctuations.

[0346] 14.5.2 Acute toxicity experiments of D3045 and D3058

[0347] The reinfusion dose for the acute toxicity experiment was 1×10 7 CAR-T cells to evaluate the in vivo safety of the optimized sequences. The results are as Figure 19 a- Figure 19 shown in d. Figure 19 a shows the inhibitory effect on the growth of NCI-H82 tumors in NPG mice after the reinfusion of D3045 and D3058 CAR-T cells. Figure 19 b shows the body weight growth trend of NPG mice after the reinfusion of D3045 and D3058 CAR-T cells. The body weight was stable without a significant downward trend. Figure 19 c-d show the metabolic kinetic curves of T cells and CAR-T cells in NPG mice after the reinfusion of D3045 and D3058 CAR-T cells, both of which have a good amplification trend, indicating that both D3045 and D3058 have good in vivo safety.

[0348] 4.5.3 D3018 and D3034 humanized sequences

[0349] Figure 20 a- Figure 20 d show the comparison of in vivo pharmacodynamic effects of the humanized sequence D3045 of D3018 and the optimized humanized sequences D3059 and D3067 of D3034 at a dose of 1.5×10 6 . D3059 showed more significant in vivo pharmacodynamic effects and metabolic kinetics of CAR-T cells than D3067, but the in vivo effect was still inferior to that of D3045.

[0350] Figure 21 a- Figure 21 d show the comparison of in vivo safety between D3059 and D3069. At a dose of 1×10 7 , both D3059 and D3069 showed tumor suppression effects and good in vivo amplification of T cells and CAR-T cells, and there was no sudden weight loss trend, indicating good in vivo safety.

[0351] Example 15 Exploration of CAR-T binding epitopes

[0352] Based on DLL3-negative CHO-K1 cells, a series of DLL3 overexpressing cell lines with full-length or domain-truncated DLL3 were constructed, as Figure 22 shown. The killing results of the optimized murine sequences D3018, D3031, D3034 and the optimized humanized sequence D3045 against the series of DLL3 overexpressing cell lines with full-length or domain-truncated DLL3 indicate that the main binding site of the CAR-T of the present invention to DLL3-positive cells is the EGF6 domain, and the results are as Figure 23 and Figure 24 shown.

[0353] Example 16 In vitro Pharmacodynamic Verification of PD-1 DNR and PD-1 CSR

[0354] D3045 was further optimized and modified to enable 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 verification, and the effector-to-target ratio was set at 1:3. The long-term killing method was the same as above.

[0355] The results are as Figure 25 shown. Figure 25 a shows the proportion of T cells in living cells during long-term killing. As the number of killing rounds increased, D3045 showed signs of fatigue, while D3045 / PD-1 DNR and D3045 / PD-1 CSR still showed good killing effects and showed better amplification effects than D3045, as Figure 25 shown in Figure 25 b. The CAR-T proportion and amplification curve are as

[0356] Example 17 In vitro Pharmacodynamic Verification of TGFbRII DNR

[0357] D3045 was optimized to enable T cells to express the CAR fusion protein and TGFbRII DNR (amino acid sequence as shown in SEQID NO: 82). SHP-77 was selected as the target cell, and the effector-to-target ratio was 1:4. 5 ng / mL TGF-β was added to the AIM-V medium (5% FBS), and a control with 0 ng / mL TGF-β added was also set. The long-term killing method was the same as above.

[0358] The results are as Figure 26 shown. Figure 26 a shows the proportion of T cells in living cells during long-term killing. Compared with D3045 in the group with 0 ng / mL TGF-β added, in the experimental group with 5 ng / mL TGF-β added, D3045 showed signs of exhaustion in the earlier killing rounds, verifying the inhibitory effect of TGFβ on T cells in the tumor immune microenvironment. The presence of TGFbRII DNR counteracted this inhibitory effect and showed a better T cell amplification trend, as Figure 26 shown in Figure 26 b. The CAR-T proportion and amplification curve are as Figure 26As shown in d, corresponding to the killing effect, in the experimental group with the addition of 5 ng / mL TGF-β, the amplification effect of D3045 / TGFbRII DNR was significantly better than that of D3045, and the CAR-T amplification effect was better without additional TGF-β in the medium.

[0359] The sequence information of the present invention is shown in Table 3 below.

[0360] Table 3

[0361]

[0362] All documents mentioned in the present invention are cited herein as references, as if each document was individually cited as a reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims

1. 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 are respectively: a heavy-chain variable region having an amino acid sequence as shown in SEQ ID NO: 64, and a light-chain variable region having an amino acid sequence as shown in SEQ ID NO: 65; (ii) a transmembrane domain, (iii) at least one co-stimulatory domain, and (iv) an activation domain.

2. The chimeric antigen receptor fusion protein according to claim 1, wherein The amino acid sequence of the chimeric antigen receptor fusion protein is as shown in SEQ ID NO:

63.

3. A polynucleotide, characterized in that, The polynucleotide encodes the chimeric antigen receptor fusion protein according to claim 1.

4. The polynucleotide according to claim 3, wherein The polynucleotide further comprises a polynucleotide sequence encoding a chimeric switch receptor (CSR) or a dominant negative receptor (DNR).

5. The polynucleotide according to claim 4, wherein The polynucleotide sequence encoding a chimeric switch receptor (CSR) or a dominant negative receptor (DNR) is selected from the group consisting of: a polynucleotide sequence encoding a PD-1 dominant negative receptor (PD-1 DNR), a polynucleotide sequence encoding a PD-1 chimeric switch receptor (PD-1 CSR), and a polynucleotide sequence encoding a TGF-β dominant negative receptor (TGF-β DNR).

6. A carrier, characterized in that, The vector contains the polynucleotide according to claim 3.

7. A genetically engineered host cell, characterized in that, The host cell contains the vector according to claim 6, or the polynucleotide according to claim 3 is integrated into the genome, or expresses the chimeric antigen receptor fusion protein according to claim 1.

8. Use of the chimeric antigen receptor fusion protein according to claim 1, the vector according to claim 6, or the host cell according to claim 7, characterized in that For the preparation of a drug or preparation for preventing and / or treating DLL3-positive cancer or tumor; wherein, the cancer or tumor is small cell lung cancer.

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