Enhanced DLL3-protein-targeting chimeric antigen receptor and mutant, and use thereof

Abstract

Provided are an anti-DLL3 antibody or an antigen-binding fragment thereof, a chimeric antigen receptor and a mutant thereof, and an enhanced chimeric antigen receptor comprising a PDL1 antagonist and an mIL7 element. Further provided are a nucleic acid encoding same, an expression cassette, vector and cell comprising the nucleic acid, a preparation method, and a use for preventing, treating, detecting, or diagnosing diseases related to DLL3. In the enhanced DLL3 chimeric antigen receptor, the PDL1 antagonist and the cell membrane IL7 cytokine element play a critical role in a long-term anti-tumor process. Animal efficacy experiments have shown complete tumor regression in all mice, indicating broad application prospects in the pharmaceutical field.

Description

Enhanced chimeric antigen receptor targeting DLL3 protein and mutant and application TECHNICAL FIELD

[0001] The present application relates to the field of biomedical or biopharmaceutical technology, in particular to a chimeric antigen receptor targeting DLL3 protein and mutant and application. BACKGROUND

[0002] In recent years, due to factors such as diet, environment, and aging of the population, the global incidence of cancer is growing, and cancer is considered a major cause of death and an important obstacle to prolonging life expectancy in every country in the world. According to the National Cancer Center, the data released in the National Cancer Center Journal shows that the number of lung cancer cases in China accounts for 22% of all new cancer cases, which is much higher than the incidence of other cancers, and the number of lung cancer deaths accounts for 28.5% of all cancer deaths. The incidence and mortality of lung cancer increase with age. Small cell lung cancer (SCLC) is one of lung cancer, according to the NCCN guidelines, accounting for about 15% of all lung cancer, with the characteristics of rapid progression, high recurrence rate, early metastasis, and poor prognosis. However, 2 / 3 of patients are in the advanced stage when diagnosed and cannot be cured, and more than 75% of patients will relapse after radical treatment, with a 5-year survival rate of only about 5%. At present, the treatment for small cell lung cancer is limited, and traditional radiotherapy and chemotherapy can only provide short-term benefits, and there is no effective means to significantly prolong the survival time of patients, so innovative therapies such as immunotherapy are urgently needed to solve this problem.

[0003] The structure of chimeric antigen receptor (CAR) is composed of three parts: extracellular domain, transmembrane domain and intracellular domain. The extracellular domain is composed of a single-chain antibody (scFv) fragment of a monoclonal antibody (mAb) that recognizes tumor-associated antigens or tumor-specific antigens and a hinge, which is connected to the transmembrane domain. The intracellular domain contains the CD3 zeta domain immune receptor tyrosine activation motif in the T cell antigen receptor complex, which plays a role in intracellular signal transduction after recognizing tumor antigens. T cells are the main cells that can specifically kill tumors in the body, and the killing process mediated by them is an important mechanism for the body's immune system to participate in anti-tumor. CAR-T cell therapy is a method that uses genetic engineering to modify CAR to T cells, enabling them to specifically kill tumor cells.

[0004] Human DLL3 protein (delta-like ligand 3, DLL3) is a single transmembrane protein composed of 619 amino acids, belonging to the Notch ligand family. DLL3 affects tumor growth, is related to tumor blood vessels and tumor immunity, and clinical research data shows that DLL3 is highly expressed in small cell lung cancer (SCLC) and other neuroendocrine tumor patients, and the high expression of DLL3 in SCLC is negatively correlated with the survival of patients, and DLL3 is only expressed in a small number of normal cells, such as neuronal cells, pancreatic islet cells, pituitary, and only in the cytoplasm. Chimeric antigen receptor T cells targeting human DLL3 can stimulate T cells to kill tumor cells specifically by recognizing tumor surface DLL3 antigen. Therefore, chimeric receptor T cells targeting DLL3 protein are a potential treatment for SCLC and other DLL3-expressing cancers.

[0005] Transposon, also known as transposable element, is a kind of genetic factor that can "jump and move" in the genome. PiggyBac transposon (PB transposon) has wide transposition activity and can realize efficient gene transposition with less dependence on host factors. The gene transduction system based on piggybac (PB) transposon contains two parts: PB transposase (DNA or mRNA form) and plasmid carrying target gene (PB plasmid). Compared with the traditional CAR gene transduction method, the PB gene transduction system can deliver CAR-expressing genes into T cells without using viruses, and the delivered genetic material is more, and there is no risk related to lentiviral transduction system, and the safety is higher.

[0006] Since the 1990s, non-activated resting T cells (and mature B cells) have been shown to exhibit low levels of constitutive tonic signaling that modulate their function and survival in a homeostatic manner. It is known that tonic signaling in non-engineered endogenous T cells, mainly due to the T cell receptor (TCR), generates non-antigen-specific interactions that can enhance the responsiveness of T cells to foreign peptides (Garbi N, Hammerling GJ, Probst HC, van den Broek M. Tonic T cell signalling and T cell tolerance as opposite effects of self-recognition on dendritic cells. Curr Opin Immunol. 22:601-8. (2010). doi: 10.1016 / j.coi.2010.08.007.). Since CARs and TCRs have similar signaling and functions, researchers have found that non-antigen-dependent clustering of CAR-scFvs triggers a tonic CAR-CD3zeta phosphorylation that can induce early exhaustion of CAR-T cells, thus limiting the anti-tumor effect (Long, A. H. et al. 4-1BB costimulation ameliorates T cell exhaustion induced by tonic signaling of chimeric antigen receptors. Nat. Med. 21, 581-590. (2015). doi: 10.1038 / nm.3838.).Therefore, CAR-T cells also release weak and continuous signals in the absence of antigen, which stimulates T cells and may accelerate T cell exhaustion. In 2023, a research team led by Wang Hao-peng of Shanghai University of Science and Technology first revealed that the positively charged patch (PCP) on the surface of CAR receptor mediates CAR aggregation, and the electrostatic effect is an important mechanism for the generation of tonic signals of CAR-T cells. It is proposed to reduce PCP on CAR through mutation in vitro to reduce spontaneous CAR activation and alleviate CAR-T cell exhaustion (Chen, J., Qiu, S., Li, W., Wang, K., Zhang, Y., Yang, H., Liu, B., Li, G., Li, L., Chen, M., et al. Tuning charge density of chimeric antigen receptor optimizes tonic signaling and CAR-T cell fitness. Cell Res. 33, 341-354. (2023). doi: 10.1038 / s41422-023-00789-0). Therefore, adjusting the positively charged patch of CAR scFv to optimize tonic signaling becomes a strategy to improve CAR-T function.

[0007] T cell activation is mainly triggered by the binding of T cell receptor (TCR) to antigen peptide-MHC complex. When T cell receptor binds to antigen peptide-MHC complex, T cell will be activated and a series of signal transduction events will be triggered, and auxiliary signals will also have important influence on T cell activation, such as the binding between CD28 and B7-1 / B7-2, and the stimulation of cytokines (such as IL-2), etc. In addition, T cells may be continuously activated in a ligand-independent or non-dependent manner after transduction of CAR structure. Cell surface glycoprotein CD69 is a marker of T cell activation, which is one of the earliest markers up-regulated after T cell activation. CD25 is the alpha chain of IL-2 receptor, which remains elevated within a few days of stimulating TCR activation, and further produces IL-2 for T lymphocyte activation. These signal transduction events ultimately lead to T cell differentiation, proliferation and secretion of cytokines, etc., and play an important role in immune response.

[0008] T cell exhaustion refers to the decline in T cell function due to persistent stimulation of the TCR in the presence of persistent antigen exposure, in addition, chronic CAR signaling in the absence of antigen can also drive T cell exhaustion, leading to reduced T cell persistence and impaired anti-tumor activity. The process of T cell exhaustion encompasses a spectrum from highly proliferative T cells with stem cell-like properties to T cells that have completely lost effector function and replicative capacity, the characteristics of exhausted T cells include increased expression of inhibitory markers, and gradual loss of function, wherein the exhaustion markers include PD-1, TIM3, LAG3.

[0009] In view of the deficiencies of the prior art, the present application optimizes the structure of the CAR vector, especially the positive charge patch adjustment of the CAR scFv to optimize the tonic signaling, thereby improving the function of CAR-T, and at the same time endowing the immune cells with more functions. The present application further provides an enhanced CAR structure co-expressing an immunosuppressive molecule and a cell membrane type interleukin targeting human DLL3 antigen, which is expected to bring hope to the treatment of DLL3 antigen positive tumor patients.

[0010] SUMMARY

[0011] In order to provide more anti-tumor drugs, especially drugs for treating DLL3 antigen positive tumors, the purpose of the present application is to provide an anti-DLL3 antibody or antigen binding fragment thereof, a chimeric antigen receptor and its mutant, an enhanced chimeric antigen receptor comprising a PDL1 antagonist and an mIL7 element. The present application also provides a nucleic acid encoding the same, an expression cassette, a vector, a cell comprising the nucleic acid, a preparation method, and applications for preventing, treating, detecting or diagnosing diseases related to DLL3. The enhanced DLL3 chimeric antigen receptor of the present application, wherein the PDL1 antagonist and the mIL7 element play a key role in the long-term anti-tumor process, animal efficacy experiments show that all the mouse tumors completely regressed.

[0012] One aspect of the present application provides an anti-DLL3 antibody or antigen binding fragment thereof, said antibody or antigen binding fragment thereof comprising 3 light chain complementarity determining regions and 3 heavy chain complementarity determining regions, characterized in that:

[0013] The 3 light chain complementarity determining regions of the antibody or antigen binding fragment thereof comprise LCDR1 as shown in SEQ ID NO: 16, LCDR2 as shown in SEQ ID NO: 17 and LCDR3 as shown in SEQ ID NO: 18, and the 3 heavy chain complementarity determining regions of the antibody or antigen binding fragment thereof comprise HCDR1 as shown in SEQ ID NO: 14, HCDR2 as shown in SEQ ID NO: 3 and HCDR3 as shown in SEQ ID NO: 4; preferably, the antibody is numbered 616;

[0014] the three light chain complementarity determining regions of the antibody or antigen-binding fragment thereof comprise LCDR1 set forth in SEQ ID NO: 11, LCDR2 set forth in SEQ ID NO: 7, and LCDR3 set forth in SEQ ID NO: 12, and the three heavy chain complementarity determining regions of the antibody or antigen-binding fragment thereof comprise HCDR1 set forth in SEQ ID NO: 2, HCDR2 set forth in SEQ ID NO: 3, and HCDR3 set forth in SEQ ID NO: 4; preferably the antibody is numbered 603; or

[0015] the three light chain complementarity determining regions of the antibody or antigen-binding fragment thereof comprise LCDR1 set forth in SEQ ID NO: 6, LCDR2 set forth in SEQ ID NO: 7, and LCDR3 set forth in SEQ ID NO: 8, and the three heavy chain complementarity determining regions of the antibody or antigen-binding fragment thereof comprise HCDR1 set forth in SEQ ID NO: 2, HCDR2 set forth in SEQ ID NO: 3, and HCDR3 set forth in SEQ ID NO: 4; preferably the antibody is numbered 546.

[0016] Further, the antibody or antigen-binding fragment thereof comprises a light chain variable region that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 15, and / or a heavy chain variable region that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 13; preferably the antibody is numbered 616;

[0017] the antibody or antigen-binding fragment thereof comprises a light chain variable region that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 10, and / or a heavy chain variable region that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 9; preferably the antibody is numbered 603; or

[0018] the antibody or antigen-binding fragment thereof comprises a light chain variable region that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 5, and / or a heavy chain variable region that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 1; preferably the antibody is numbered 546.

[0019] Further, the antigen binding fragment includes a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody, a Fab, a Fab', a F(ab')2, a Fv, a scFv, or a dsFv fragment.

[0020] Further, the antibody comprises a heavy chain constant region as set forth in SEQ ID NO: 19, and / or a light chain constant region as set forth in SEQ ID NO: 20.

[0021] Another aspect of the present application provides a mutant of an anti-DLL3 antibody or an antigen binding fragment thereof, comprising a mutation based on a parent 616 DLL3 antibody, an amino acid sequence of a light chain variable region of the parent 616 DLL3 antibody being as set forth in SEQ ID NO: 15, and an amino acid sequence of a heavy chain variable region being as set forth in SEQ ID NO: 13; the mutation comprising:

[0022] (a) an up-regulation positive charge patch amino acid mutation, or

[0023] (b) a down-regulation positive charge patch amino acid mutation;

[0024] Further, the up-regulation positive charge patch amino acid mutation is a glutamine (Q) to lysine (K) mutation, and the down-regulation positive charge patch amino acid mutation is a lysine (K) to glutamine (Q) mutation.

[0025] Further, an amino acid sequence of a scFv of the parent 616 DLL3 antibody is as set forth in SEQ ID NO: 28, and the mutant is selected from any one of the following:

[0026] (1) a 616-mutl mutant: Q at positions 37, 38, 100 of a scFv of the parent 616 DLL3 antibody is mutated to K, and an amino acid sequence of a scFv of the 616-mutl mutant is as set forth in SEQ ID NO: 38;

[0027] (2) a 616-mut2 mutant: Q at positions 3, 37, 38, 100 of a scFv of the parent 616 DLL3 antibody is mutated to K, and an amino acid sequence of a scFv of the 616-mut2 mutant is as set forth in SEQ ID NO: 39;

[0028] (3) a 616-mut3 mutant: Q at positions 6, 37, 38, 100 of a scFv of the parent 616 DLL3 antibody is mutated to K, and an amino acid sequence of a scFv of the 616-mut3 mutant is as set forth in SEQ ID NO: 40;

[0029] (4) 616-mut4 mutant: Q at positions 79, 100 of the scFv of the parent 616 DLL3 antibody is mutated to K, the scFv amino acid sequence of the 616-mut4 mutant is shown as SEQ ID NO: 41;

[0030] (5) 616-mut5 mutant: Q at positions 3, 79, 100 of the scFv of the parent 616 DLL3 antibody is mutated to K, the scFv amino acid sequence of the 616-mut5 mutant is shown as SEQ ID NO: 42;

[0031] (6) 616-mut6 mutant: Q at positions 3, 38, 127, 161 of the scFv of the parent 616 DLL3 antibody is mutated to K, the scFv amino acid sequence of the 616-mut6 mutant is shown as SEQ ID NO: 43;

[0032] (7) 616-mut7 mutant: Q at positions 6, 38, 127, 161 of the scFv of the parent 616 DLL3 antibody is mutated to K, the scFv amino acid sequence of the 616-mut7 mutant is shown as SEQ ID NO: 44;

[0033] (8) 616-mut8 mutant: Q at positions 37, 38, 127, 161 of the scFv of the parent 616 DLL3 antibody is mutated to K, the scFv amino acid sequence of the 616-mut8 mutant is shown as SEQ ID NO: 45;

[0034] (9) 616-mut9 mutant: Q at positions 79, 127, 161 of the scFv of the parent 616 DLL3 antibody is mutated to K, the scFv amino acid sequence of the 616-mut9 mutant is shown as SEQ ID NO: 46;

[0035] (10) 616-mut10 mutant: Q at positions 3, 79, 127, 161 of the scFv of the parent 616 DLL3 antibody is mutated to K, the scFv amino acid sequence of the 616-mut10 mutant is shown as SEQ ID NO: 47;

[0036] (11) 616-mut11 mutant: Q at positions 100, 127, 128, 161 of the scFv of the parent 616 DLL3 antibody is mutated to K, the scFv amino acid sequence of the 616-mut11 mutant is shown as SEQ ID NO: 48;

[0037] (12) 616-mut12 mutant: Q at positions 125, 127, 128, 161 of the scFv of the parent 616 DLL3 antibody is mutated to K, the scFv amino acid sequence of the 616-mut12 mutant is shown as SEQ ID NO: 49;

[0038] (13) 616-mut13 mutant: Q at positions 37, 38, 79, 199 of the scFv of the parent 616 DLL3 antibody is mutated to K, the scFv amino acid sequence of the 616-mut13 mutant is shown as SEQ ID NO: 50;

[0039] (14) 616-mut14 mutant: Q at positions 100, 199 of the scFv of the parent 616 DLL3 antibody is mutated to K, the scFv amino acid sequence of the 616-mut14 mutant is shown as SEQ ID NO: 51;

[0040] (15) 616-mut15 mutant: Q at positions 3, 100, 199 of the scFv of the parent 616 DLL3 antibody is mutated to K, the scFv amino acid sequence of the 616-mut15 mutant is shown as SEQ ID NO: 52;

[0041] (16) 616-mut16 mutant: K at positions 45, 135, 165, 186 of the scFv of the parent 616 DLL3 antibody is mutated to Q, the scFv amino acid sequence of the 616-mut16 mutant is shown as SEQ ID NO: 53;

[0042] (17) 616-mut17 mutant: K at positions 103, 135, 165, 186 of the scFv of the parent 616 DLL3 antibody is mutated to Q, the scFv amino acid sequence of the 616-mut17 mutant is shown as SEQ ID NO: 54;

[0043] (18) 616-mut18 mutant: K at positions 107, 135, 165, 186 of the scFv of the parent 616 DLL3 antibody is mutated to Q, the scFv amino acid sequence of the 616-mut18 mutant is shown as SEQ ID NO: 55;

[0044] (19) 616-mut19 mutant: K at positions 42, 45, 197 of the scFv of the parent 616 DLL3 antibody is mutated to Q, the scFv amino acid sequence of the 616-mut19 mutant is shown as SEQ ID NO: 56;

[0045] (20) 616-mut20 mutant: K at positions 39, 42, 45, 197 of the scFv of the parent 616 DLL3 antibody is mutated to Q, the scFv amino acid sequence of the 616-mut20 mutant is shown as SEQ ID NO: 57;

[0046] (21) 616-mut21 mutant: K at positions 45, 165, 186, 197 of the scFv of the parent 616 DLL3 antibody is mutated to Q, the scFv amino acid sequence of the 616-mut21 mutant is shown as SEQ ID NO: 58;

[0047] (22) 616-mut22 mutant: K at positions 103, 165, 186, 197 of the scFv of the parent 616 DLL3 antibody is mutated to Q, the scFv amino acid sequence of the 616-mut22 mutant is shown as SEQ ID NO: 59;

[0048] (23) 616-mut23 mutant: K at positions 107, 165, 186, 197 of the scFv of the parent 616 DLL3 antibody is mutated to Q, the scFv amino acid sequence of the 616-mut23 mutant is shown as SEQ ID NO: 60;

[0049] (24) 616-mut24 mutant: K at positions 42, 103, 203 of the scFv of the parent 616 DLL3 antibody is mutated to Q, the scFv amino acid sequence of the 616-mut24 mutant is shown as SEQ ID NO: 61;

[0050] (25) 616-mut25 mutant: K at positions 39, 42, 103, 203 of the scFv of the parent 616 DLL3 antibody is mutated to Q, the scFv amino acid sequence of the 616-mut25 mutant is shown as SEQ ID NO: 62;

[0051] (26) 616-mut26 mutant: K at positions 45, 135, 197, 203 of the scFv of the parent 616 DLL3 antibody is mutated to Q, the scFv amino acid sequence of the 616-mut26 mutant is shown as SEQ ID NO: 63;

[0052] (27) 616-mut27 mutant: K at positions 103, 135, 197, 203 of the scFv of the parent 616 DLL3 antibody is mutated to Q, the scFv amino acid sequence of the 616-mut27 mutant is shown as SEQ ID NO: 64;

[0053] (28) 616-mut28 mutant: K at positions 135, 165, 197, 203 of the scFv of the parent 616 DLL3 antibody is mutated to Q, the scFv amino acid sequence of the 616-mut28 mutant is set forth in SEQ ID NO: 65;

[0054] (29) 616-mut29 mutant: K at positions 186, 197, 203 of the scFv of the parent 616 DLL3 antibody is mutated to Q, the scFv amino acid sequence of the 616-mut29 mutant is set forth in SEQ ID NO: 66; or

[0055] (30) 616-mut30 mutant: K at positions 39, 186, 197, 203 of the scFv of the parent 616 DLL3 antibody is mutated to Q, the scFv amino acid sequence of the 616-mut30 mutant is set forth in SEQ ID NO: 67.

[0056] Another aspect of the present application provides a chimeric antigen receptor (CAR) targeting DLL3 antigen, the CAR comprising a DLL3 antigen binding domain, a transmembrane domain and an intracellular signaling domain, wherein the DLL3 antigen binding domain is a scFv of any of the antibodies or antigen binding fragments thereof of any of the aspects of the present application or a scFv of any of the mutants of any of the aspects of the present application.

[0057] Further, the amino acid sequence of the light chain variable region of the scFv is set forth in SEQ ID NO: 15 and the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 13, or the amino acid sequence of the light chain variable region of the scFv is set forth in SEQ ID NO: 10 and the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 9, or the amino acid sequence of the light chain variable region of the scFv is set forth in SEQ ID NO: 5 and the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 1.

[0058] Further, the amino acid sequence of the scFv is set forth in any one of SEQ ID NOs: 26-28, 38-67.

[0059] Further, the CAR further comprises one or more of a hinge region, a signal peptide and a costimulatory signaling domain.

[0060] Further, the transmembrane domain is a CD8 transmembrane region, the hinge region is a CD8 hinge region, the intracellular signaling domain is a CD3ζ intracellular signaling domain, the signal peptide is a CD8α signal peptide, or the costimulatory signaling domain is a 4-1BB or a CD28 costimulatory signaling domain.

[0061] Further, the CAR comprises, in order from N-terminus to C-terminus, the following elements: a CD8a signal peptide, a DLL3 antibody scFv VL-linker-DLL3 antibody scFv VH, a CD8 hinge and transmembrane region, a 4-1BB costimulatory signal, and a CD3 zeta intracellular signaling domain; optionally, each element is connected by a linker.

[0062] Further, the CD8a signal peptide comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 22; the CD8 hinge and transmembrane region comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 23; the 4-1BB costimulatory signaling domain comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 24; or the CD3 zeta intracellular signaling domain comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 25.

[0063] Further, the CAR comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 29-31, 68-72.

[0064] Another aspect of the present application provides an enhanced chimeric antigen receptor (CAR) targeting DLL3 antigen, comprising, in order from N-terminus to C-terminus, the following elements:

[0065] (1) a CAR targeting DLL3 antigen as described in any one of the present application;

[0066] (2) an immunosuppressive molecule expression element; and

[0067] (3) a cell membrane type interleukin and / or a secreted type chemokine expression element;

[0068] Further, the element (2) is selected from one or more of: a PD1 antagonist and a PDL1 antagonist; the cell membrane type interleukin in the element (3) is selected from one or more of: a cell membrane type IL2 cytokine, a cell membrane type IL4 cytokine, a cell membrane type IL7 cytokine, a cell membrane type IL9 cytokine, a cell membrane type IL10 cytokine, a cell membrane type IL15 cytokine, a cell membrane type IL18 cytokine, a cell membrane type IL21 cytokine, a cell membrane type IL23 cytokine, a cell membrane type IL24 cytokine, and a cell membrane type IL36 cytokine; the secreted type chemokine in the element (3) is selected from one or more of: a secreted type CCL1 chemokine, a secreted type CCL2 chemokine, a secreted type CCL3 chemokine, a secreted type CCL5 chemokine, a secreted type CCL7 chemokine, a secreted type CCL15 chemokine, a secreted type CCL16 chemokine, a secreted type CCL19 chemokine, a secreted type CCL20 chemokine, a secreted type CCL21 chemokine, a secreted type CXCL4 chemokine, a secreted type CXCL9 chemokine, a secreted type CXCL10 chemokine, a secreted type CXCL11 chemokine, and a secreted type CXCL1 chemokine.

[0069] Further, the element (2) is selected from a PDL1 antagonist expression element, and / or the element (3) is selected from a cell membrane type IL7 cytokine expression element.

[0070] Further, the PDL1 antagonist expression element comprises, from N-terminus to C-terminus, a kappa leader signal peptide, an anti-PD-L1 antibody scfv, a connecting peptide 1, and a human IgG CH2CH3 fragment; and the cell membrane type IL7 cytokine expression element comprises, from N-terminus to C-terminus, a human IL-7 cytokine fragment, a connecting peptide 2, and a CD8 transmembrane region.

[0071] Further, the kappa leader signal peptide in the anti-PDL1 expression element comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO: 73; the anti-PDL1 antibody scfv comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO: 74; the amino acid sequence of the linker 1 is GGGGS; the human IgG CH2CH3 fragment comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO: 75; the human IL-7 cytokine fragment in the cell membrane type IL7 cytokine expression element comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO: 76; the amino acid sequence of the linker 2 is set forth in SEQ ID NO: 79; the CD8 transmembrane region amino acid sequence comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO: 77; optionally, the element (2) and the element (3) are connected by a 2A peptide 1, the amino acid sequence of the 2A peptide 1 is set forth in SEQ ID NO: 78; the element (1) and element (2) are connected by a 2A peptide 2, the amino acid sequence of the 2A peptide 2 is set forth in SEQ ID NO: 83;

[0072] Further, the enhanced DLL3 antigen targeting CAR is 616 DLL3 CAR.aPDL1.mIL7, wherein the amino acid sequence of the 616 DLL3 CAR is set forth in SEQ ID NO: 31, the amino acid sequence of the aPDL1 is set forth in SEQ ID NO: 80, the amino acid sequence of the mIL7 is set forth in SEQ ID NO: 81, the amino acid sequence of the aPDL1.mIL7 is set forth in SEQ ID NO: 82, and the amino acid sequence of the 616 DLL3 CAR.aPDL1.mIL7 is set forth in SEQ ID NO: 84.

[0073] Another aspect of the present application provides a recombinant nucleic acid encoding any of the antibodies or antigen binding fragments thereof of the present application, or any of the mutants of the present application, or any of the DLL3 antigen targeting CARs of the present application, or any of the enhanced DLL3 antigen targeting CARs of the present application.

[0074] Another aspect of the present application provides a recombinant expression cassette comprising the recombinant nucleic acid of the present application.

[0075] Another aspect of the present application provides a recombinant vector comprising the recombinant nucleic acid of the present application or the recombinant expression cassette of the present application.

[0076] Further, the recombinant vector is an expression vector or a cloning vector.

[0077] Further, the expression vector is an expression vector constructed by piggybac (PB) transposon.

[0078] Another aspect of the present application provides a recombinant cell comprising the antibody or antigen-binding fragment thereof of any one of the present application, or the mutant of any one of the present application, or the CAR targeting DLL3 antigen of any one of the present application, or the enhanced CAR targeting DLL3 antigen of any one of the present application, or the recombinant nucleic acid of the present application.

[0079] Further, the cell comprises a T cell, an NK cell, a macrophage, a B cell, a DC cell, or a non-immune cell; more preferably, the T cell is a primary-derived T cell or a T cell differentiated from an iPSC.

[0080] Further, the T cell differentiated from an iPSC is a γδ T cell, a DNT cell, or a NKT cell.

[0081] Another aspect of the present application provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of the present application, or the mutant of any one of the present application, or the CAR targeting DLL3 antigen of any one of the present application, or the enhanced CAR targeting DLL3 antigen of any one of the present application, or the recombinant nucleic acid of the present application, or the recombinant cell of the present application; optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0082] Another aspect of the present application provides use of the antibody or antigen-binding fragment thereof of any one of the present application, or the mutant of any one of the present application, or the CAR targeting DLL3 antigen of any one of the present application, or the enhanced CAR targeting DLL3 antigen of any one of the present application, or the recombinant nucleic acid of the present application, or the recombinant cell of the present application, or the pharmaceutical composition of the present application for preventing, treating, detecting, or diagnosing a disease related to DLL3.

[0083] Further, the disease related to DLL3 is a DLL3 high-expression disease.

[0084] Further, the disease is a DLL3 high-expression cancer or tumor.

[0085] Further, the cancer or tumor is selected from one or more of neuroendocrine tumors and other tumors, including small cell lung cancer, large cell neuroendocrine carcinoma, gastroenteropancreatic neuroendocrine tumor, pancreatic neuroendocrine tumor, small cell bladder cancer, glioblastoma multiforme, metastatic castration-resistant prostate cancer, melanoma, medullary thyroid cancer.

[0086] Another aspect of the present application provides a method of treating or preventing a disease, comprising administering to a subject in need thereof any of the antibodies or antigen-binding fragments thereof of the present application, or any of the mutants of the present application, or any of the CARs targeting DLL3 antigen of the present application, or any of the enhanced CARs targeting DLL3 antigen of the present application, or the recombinant nucleic acid of the present application, or the recombinant cell of the present application, or the pharmaceutical composition of the present application.

[0087] Further, the disease is a disease associated with DLL3.

[0088] Further, the disease associated with DLL3 is a DLL3 high expression disease.

[0089] Further, the disease is a DLL3 high expression cancer or tumor.

[0090] Further, the cancer or tumor is selected from one or more of neuroendocrine tumors and other tumors, including small cell lung cancer, large cell neuroendocrine carcinoma, gastroenteropancreatic neuroendocrine tumor, pancreatic neuroendocrine tumor, small cell bladder cancer, glioblastoma multiforme, metastatic castration-resistant prostate cancer, melanoma, medullary thyroid cancer.

[0091] Another aspect of the present application provides a method of diagnosis or detection, comprising administering to a subject or sample in need thereof any of the antibodies or antigen-binding fragments thereof of the present application, or any of the mutants of the present application, or any of the CARs targeting DLL3 antigen of the present application, or any of the enhanced CARs targeting DLL3 antigen of the present application, or the recombinant nucleic acid of the present application, or the recombinant cell of the present application, or the pharmaceutical composition of the present application.

[0092] Another aspect of the present application provides a method of preparing an engineered immune cell, characterized by comprising the following steps:

[0093] (1) providing an immune cell to be modified; and

[0094] (2) introducing the recombinant nucleic acid of the present application into the immune cell.

[0095] Further, the immune cell is a T cell.

[0096] Further, the T cells are primary-derived T cells or iPSC-differentiated T cells; most preferably, the iPSC-differentiated T cells are γδ T cells, DNT cells or NKT cells.

[0097] The anti-DLL3 antibody or antigen-binding fragment thereof, chimeric antigen receptor and mutants thereof, enhanced chimeric antigen receptor comprising a PDL1 antagonist and mIL7 element provided by the present application have the following advantages:

[0098] 1. The anti-DLL3 antibody or antigen-binding fragment thereof of the present application exhibits good binding activity with human DLL3-His protein. The KD of all antibodies of the present application is less than 1.0E-09 when detecting the affinity of the antibody with hDLL3-His protein, showing high affinity.

[0099] 2. The DLL3 CAR-T cells of the present application have a positive binding rate to DLL3 protein in the range of 27-46%. The SHP77 cells expressing DLL3 are specifically killed, and the killing is positively correlated with the effector-target ratio. When the effector-target ratio is 3:1, the killing efficiency after 24 hours is 50-60%, and the 293T cell strain with negative expression of human DLL3 is not specifically killed.

[0100] 3. The DLL3 CAR-T cells of the present application release at least 10 times more cytokine IFN-γ after killing the SHP77 cells expressing DLL3 than the negative control (T cells without CAR transduction), and no significant release of cytokine IFN-γ is observed after killing the 293T cells with negative expression of DLL3.

[0101] 4. Under the condition of 1:5 of effector cells: target cells, through 3 rounds of tumor cell stimulation, the 546 DLL3 CAR-T, 606 DLL3 CAR-T and 616 DLL3 CAR-T cells of the present application have obvious tumor cell clearance effect, and the T cells without CAR plasmid electroporation have no tumor cell clearance effect. The long-term clearance of tumor cells and self-expansion of the 616 DLL3 CAR-T cells are superior to those of the 546 DLL3 CAR-T and 606 DLL3 CAR-T cells.

[0102] 5. This invention involves amino acid mutations in the glutamine (Q) or lysine (K) residues in the frame region (FR) of antibody 616, specifically 616scFv. Different numbers of lysine residues result in different net charges on the antibody; the C-terminal lysine (K) carries a positive charge, while glutamine (Q) is a neutral amino acid. The positive charge patches in the scFv are modulated by replacing glutamine (Q) and lysine (K) residues in the 616scFv frame region (FR). The number of mutations includes, but is not limited to, single, two, three, and four point mutations. This allows for the design of a series of 616scFv variant structures with amino acid substitutions.

[0103] 6. The positive rate test of DLL3 protein binding of the mutant DLL3 CAR-T of the present invention showed that the positive rate of DLL3 CAR-T of different mutants ranged from 17% to 35%. The 616 DLL3 CAR-T with amino acid mutations showed good killing effect on DLL3-positive SHP77 cells. The amino acid mutation did not affect the short-term anti-tumor effect of 616 DLL3 CAR-T. Under the condition of effector-target ratio of 5:1, the killing efficiency after 24 hours was 47%-65%, while it had no specific killing effect on the human DLL3-negative 293T cell line.

[0104] 7. The activation signal CD69 on the surface of mutant DLL3 CAR-T cells (616-mut18 DLL3 CAR-T, 616-mut22 DLL3 CAR-T, 616-mut27 DLL3 CAR-T) with downregulated positively charged plaques through amino acid mutations was decreased to some extent, while the self-activation signal CD69 of mutant DLL3 CAR-T cells (616-mut8 DLL3 CAR-T, 616-mut11 DLL3 CAR-T) with upregulated positively charged plaques through amino acid mutations was significantly increased, indicating that the regulation of positively charged plaques is related to the self-activation of CAR-T cells.

[0105] 8. The depletion signals (TIM3, LAG3) on the surface of mutant DLL3 CAR-T cells (616-mut18 DLL3 CAR-T, 616-mut22 DLL3 CAR-T, 616-mut27 DLL3 CAR-T) with downregulated positively charged plaques through amino acid mutations decreased to some extent, while the self-depletion signals (TIM3, LAG3) of mutant DLL3 CAR-T cells (616-mut8 DLL3 CAR-T, 616-mut11 DLL3 CAR-T) with upregulated positively charged plaques through amino acid mutations increased significantly, indicating that the regulation of positively charged plaques is related to the self-depletion of CAR-T cells.

[0106] 9. The mutant DLL3 CAR-T cells with up-regulated positive charge patch by amino acid mutation (616-mut8 DLL3 CAR-T, 616-mut11 DLL3 CAR-T) have higher self-cytokine release than the mutant DLL3 CAR-T cells with down-regulated positive charge patch by amino acid mutation (616-mut18 DLL3 CAR-T, 616-mut22 DLL3 CAR-T, 616-mut27 DLL3 CAR-T) without tumor antigen stimulation.

[0107] 10. The mutant DLL3 CAR-T cells with down-regulated positive charge patch by amino acid mutation (616-mut18 DLL3 CAR-T, 616-mut22 DLL3 CAR-T, 616-mut27 DLL3 CAR-T) have better long-term anti-tumor effect than the 616 DLL3 CAR-T cells without amino acid mutation.

[0108] 11. The application constructs an enhanced CAR targeting DLL3 antigen, i.e. 616 DLL3 CAR.aPDL1.mIL7, and the experimental results show that the 616 DLL3 CAR.aPDL1.mIL7-T cells expressing cell membrane IL7 fusion protein can detect human IL7 protein on the surface of T cells, and the expression efficiency and CAR positive rate are comparable.

[0109] 12. The 616 DLL3 CAR.aPDL1.mIL7-T cells of the application can normally secrete aPDL1 fusion protein and release it into the cell supernatant, and the concentration is 79.64 ng / mL. In addition, in this embodiment, the condition of adding tumor cells for co-culture is set, and when the 616 DLL3 CAR.aPDL1.mIL7-T cells perform cell killing function by contacting tumor antigen, the secretion amount of aPDL1 fusion protein is significantly improved, which is about 3 times higher than that without adding tumor cells for culture.

[0110] 13. For the 616 DLL3 CAR.aPDL1.mIL7-T cells of the application, the mIL7 fusion protein structure can enhance the viability and persistence of the whole T cells, and has specific promoting effect on the expansion of CAR positive T cells, which can produce specific promoting effect on the expansion of the positive cell population co-expressing CAR, and can also produce a synergistic effect on the CAR negative cells in the cell population which do not express mIL7 structure, thereby maintaining the viability and cell state of the cells.

[0111] 14. With an effector cell:target cell ratio of 1:5, after four rounds of tumor cell stimulation, 616 DLL3 CAR.aPDL1.mIL7-T cells showed significantly better tumor cell clearance than 616 DLL3 CAR-T cells. 616 DLL3 CAR-T cells were unable to inhibit tumor growth after the third round of killing, while 616 DLL3 CAR.aPDL1.mIL7-T cells maintained a good tumor-suppressing effect even after the fourth round of killing. This indicates that aPDL1 fusion protein and mIL7 fusion protein play a key role in the long-term anti-tumor process.

[0112] 15. Animal efficacy experiments showed that all groups of DLL3 CAR-T cells had a certain inhibitory effect on SHP-77 tumors in mice. In the 616 DLL3 CAR-T cell group, one mouse (1 / 5) experienced complete tumor elimination. In the 616-mut18 DLL3 CAR-T cell group (amino acid mutation downregulating positively charged plaques), two mice (2 / 5) experienced complete tumor elimination. The 616-mut8 DLL3 CAR-T cell group (amino acid mutation upregulating positively charged plaques) failed to inhibit tumor growth in the later stages. Meanwhile, in the 616 DLL3 CAR.aPDL1.mIL7-T treatment group, based on the functions of the aPDL1 and mIL7 fusion proteins, all mice (5 / 5) experienced complete tumor regression. Furthermore, the percentage of human T cells in the blood of mice after administration showed that the 616 DLL3 CAR.aPDL1.mIL7-T cells expanded significantly more in vivo than other groups, showing a trend of first increasing and then decreasing as the tumor regressed. Attached Figure Description

[0113] Figure 1 shows the results of serum antibody titer detection in mice after the fourth DLL3 protein immunization.

[0114] Figure 2 shows the ELISA binding activity of antibodies 546, 603, 616, and the blank control Isotype to human DLL3-His protein.

[0115] Figure 3 shows the binding activity of antibodies 546, 603, 616, and the blank control Isotype antibody to CT26-hDLL3 cells.

[0116] Figure 4 shows a schematic diagram of the structure of the gene fragment targeting the DLL3 chimeric antigen receptor in Example 2.

[0117] Figure 5 shows a schematic diagram of the PB 616 DLL3 CAR plasmid structure.

[0118] Figures 6A-6D show the CAR positive flow cytometry results of detecting DLL3 CAR-T cells by DLL3 antigen, wherein Figure 6A shows the results of T cells without transduction of CAR, Figure 6B shows the results of 546 DLL3 CAR-T cells, Figure 6C shows the results of 603 DLL3 CAR-T cells, and Figure 6D shows the results of 616 DLL3 CAR-T cells.

[0119] Figure 7 shows the CAR positive rate results of detecting the DLL3 CAR-T cells of the present application and controls by human DLL3 antigen protein, wherein the DLL3 CAR-T cells of the present application include 546 DLL3 CAR-T cells, 603 DLL3 CAR-T cells, and 616 DLL3 CAR-T cells.

[0120] Figures 8A-8C show the results of the binding reactions of the DLL3 CAR-T cells of the present application to different domain recombinant proteins of DLL3 protein, wherein Figure 8A shows the results of the binding reactions of 546 DLL3 CAR-T cells to different domain recombinant proteins of DLL3 protein, Figure 8B shows the results of the binding reactions of 603 DLL3 CAR-T cells to different domain recombinant proteins of DLL3 protein, and Figure 8C shows the results of the binding reactions of 616 DLL3 CAR-T cells to different domain recombinant proteins of DLL3 protein.

[0121] Figures 9A-9B show the in vitro specific killing results of 546 DLL3 CAR-T cells, 603 DLL3 CAR-T cells, 616 DLL3 CAR-T cells, and control T cells to target cells, wherein Figure 9A shows the in vitro specific killing results of 546 DLL3 CAR-T cells, 603 DLL3 CAR-T cells, 616 DLL3 CAR-T cells, and control T cells to 293T cell strain which is negative for DLL3 expression, and Figure 9B shows the in vitro specific killing results of 546 DLL3 CAR-T cells, 603 DLL3 CAR-T cells, 616 DLL3 CAR-T cells, and control T cells to SHP77 cell strain which is positive for DLL3 expression.

[0122] FIGS. 10A-10B show the 546 DLL3 CAR-T, 603 DLL3 CAR-T, 616 DLL3 CAR-T cells and control system T kill tumor cells IFN-γ factor release levels, wherein FIG. 10A shows the IFN-γ release after co-culturing the 546 DLL3 CAR-T, 603 DLL3 CAR-T, 616 DLL3 CAR-T cells and control system T with the 293T cells which are negative for DLL3 expression, and FIG. 10B shows the IFN-γ release after co-culturing the 546 DLL3 CAR-T, 603 DLL3 CAR-T, 616 DLL3 CAR-T cells and control system T with the SHP77 cells which are positive for DLL3 expression.

[0123] FIGS. 11A-11B show the results of long-term anti-tumor effect of the 546 DLL3 CAR-T, 603 DLL3 CAR-T, 616 DLL3 CAR-T cells on the DLL3 expression positive cells SHP-77, wherein FIG. 11A shows the results of tumor cell number change of long-term anti-tumor effect of the 546 DLL3 CAR-T, 603 DLL3 CAR-T, 616 DLL3 CAR-T cells on the DLL3 expression positive cells SHP-77, and FIG. 11B shows the results of CAR positive T cell number change of long-term anti-tumor effect of the 546 DLL3 CAR-T, 603 DLL3 CAR-T, 616 DLL3 CAR-T cells on the DLL3 expression positive cells SHP-77.

[0124] FIGS. 12A-12G show the positive flow cytometry diagrams of the untransduced, non-mutant and different amino acid mutant DLL3 CAR-T recognizing the DLL3 protein, wherein FIG. 12A shows the results of T cells without transduced CAR, FIG. 12B shows the results of the 616 DLL3 CAR-T cells, FIG. 12C shows the results of the 616-mut18 DLL3 CAR-T cells, FIG. 12D shows the results of the 616-mut22 DLL3 CAR-T cells, FIG. 12E shows the results of the 616-mut27 DLL3 CAR-T cells, FIG. 12F shows the results of the 616-mut8 DLL3 CAR-T cells, and FIG. 12G shows the results of the 616-mut11 DLL3 CAR-T cells.

[0125] FIG. 13 shows the results of CAR positive rate of untransduced, non-mutant and different amino acid mutant DLL3 CAR-T cells detected by human DLL3 antigen protein.

[0126] FIGS. 14A-14B show the results of in vitro specific killing of target cells by DLL3 CAR-T cells of non-transduction, non-mutation and different amino acid mutants, FIG. 14A shows the results of in vitro specific killing of DLL3-negative 293T cell strains by DLL3 CAR-T cells of non-transduction, non-mutation and different amino acid mutants, and FIG. 14B shows the results of in vitro specific killing of DLL3-positive SHP77 cell strains by DLL3 CAR-T cells of non-transduction, non-mutation and different amino acid mutants.

[0127] FIG. 15 shows the changes in the expression of CD69 on the surface of CAR-positive cells of non-mutant and each amino acid mutant DLL3 CAR-T cells under non-antigen dependence.

[0128] FIGS. 16A-16C show the changes in the expression of surface exhaustion indicators of CAR-positive cells of non-mutant and each amino acid mutant DLL3 CAR-T cells under non-antigen dependence, FIG. 16A shows the changes in the expression of PD-1 on the surface of CAR-positive cells of non-mutant and each amino acid mutant DLL3 CAR-T cells under non-antigen dependence, FIG. 16B shows the changes in the expression of TIM3 on the surface of CAR-positive cells of non-mutant and each amino acid mutant DLL3 CAR-T cells under non-antigen dependence, and FIG. 16C shows the changes in the expression of LAG3 on the surface of CAR-positive cells of non-mutant and each amino acid mutant DLL3 CAR-T cells under non-antigen dependence.

[0129] FIG. 17 shows the secretion amount of self-IFN-γ cytokines of non-transduction, non-mutation and each amino acid mutant DLL3 CAR-T cells under non-antigen dependence.

[0130] FIGS. 18A-18B show the results of long-term anti-tumor effects of non-transduction, non-mutation and each amino acid mutant DLL3 CAR-T on DLL3-positive SHP-77 cells, wherein FIG. 18A shows the results of changes in the number of tumor cells of long-term anti-tumor effects of non-transduction, non-mutation and each amino acid mutant DLL3 CAR-T on DLL3-positive SHP-77 cells, and FIG. 18B shows the results of changes in the number of CAR-positive T cells of long-term anti-tumor effects of non-mutation and each amino acid mutant DLL3 CAR-T on DLL3-positive SHP-77 cells.

[0131] FIG. 19 shows a schematic diagram of the structure of a 616 DLL3 CAR.aPDL1.mIL7 plasmid vector.

[0132] FIGS. 20A-20C show flow cytometry diagrams of CAR positive expression of DLL3 CAR-T cells detected by DLL3 antigen, wherein FIG. 20A shows the results of T cells without transduction of CAR, FIG. 20B shows the results of 616 DLL3 CAR-T cells, and FIG. 20C shows the results of 616 DLL3 CAR.aPDLl.mIL7-T cells.

[0133] FIG. 21 shows the results of CAR positive rate of DLL3 CAR-T cells detected by human DLL3 antigen protein.

[0134] FIGS. 22A-22C show flow cytometry diagrams of cell membrane surface IL7 protein expression of DLL3 CAR-T cells detected by anti-human IL7 antibody, wherein FIG. 22A shows the results of T cells without transduction of CAR, FIG. 22B shows the results of DLL3 CAR-T cells, and FIG. 22C shows the results of 616 DLL3 CAR.aPDLl.mIL7-T cells.

[0135] FIG. 23 shows the results of cell membrane surface IL7 protein expression of DLL3 CAR-T cells detected by anti-human IL7 antibody.

[0136] FIG. 24 shows the results of concentration detection of aPDLl fusion protein in the supernatant of each group of DLL3 CAR-T cells.

[0137] FIGS. 25A-25C show the results of change of cell viability, the results of change of cell number, and the results of change of CAR positive rate of each group of DLL3 CAR-T cells under cytokine-free culture conditions, wherein FIG. 25A shows the results of change of cell viability of each group of DLL3 CAR-T cells under cytokine-free culture conditions, FIG. 25B shows the results of change of cell number of each group of DLL3 CAR-T cells under cytokine-free culture conditions, and FIG. 25C shows the results of change of CAR positive rate of each group of DLL3 CAR-T cells under cytokine-free culture conditions.

[0138] FIGS. 26A-26B show the change in expansion fold of CAR-positive T cells and CAR-negative cells in 616 DLL3 CAR-T and 616 DLL3 CAR.aPDL1.mIL7-T cell populations under cytokine-free culture conditions, wherein FIG. 26A shows the change in expansion fold of CAR-positive T cells in 616 DLL3 CAR-T and 616 DLL3 CAR.aPDL1.mIL7-T cell populations under cytokine-free culture conditions, and FIG. 26B shows the change in expansion fold of CAR-negative cells in 616 DLL3 CAR-T and 616 DLL3 CAR.aPDL1.mIL7-T cell populations under cytokine-free culture conditions.

[0139] FIGS. 27A-27B show the results of changes in the number of SHP77 tumor cells and the number of CAR-positive cells in repeated killing by 616 DLL3 CAR-T and 616 DLL3 CAR.aPDL1.mIL7-T cells, wherein FIG. 27A shows the results of changes in the number of SHP77 tumor cells in repeated killing by 616 DLL3 CAR-T and 616 DLL3 CAR.aPDL1.mIL7-T cells, and FIG. 27B shows the results of changes in the number of CAR-positive cells in repeated killing by 616 DLL3 CAR-T and 616 DLL3 CAR.aPDL1.mIL7-T cells.

[0140] FIGS. 28A-28E show the results of changes in tumor size in NCG mice with small cell lung cancer SHP-77 tumors after administration of each group of DLL3 CAR-T cells, wherein FIG. 28A shows the change in tumor size in a single mouse after administration of the T cell (T cells without CAR transduction) group, FIG. 28B shows the change in tumor size in a single mouse after administration of the 616 DLL3 CAR-T cell group, FIG. 28C shows the change in tumor size in a single mouse after administration of the 616-mut18 DLL3 CAR-T cell group, FIG. 28D shows the change in tumor size in a single mouse after administration of the 616-mut8 DLL3 CAR-T cell group, and FIG. 28E shows the change in tumor size in a single mouse after administration of the 616 DLL3 CAR.aPDL1.mIL7-T cell group.

[0141] FIG. 29 shows the change in the proportion of human T cells in the blood of mice after administration of each group of DLL3 CAR-T cells. DETAILED DESCRIPTION

[0142] The application will be further described in connection with specific embodiments. The embodiments described are part of the specific embodiments of the application, but not all the embodiments. It should be understood that the following embodiments are given to provide a more complete disclosure and a better understanding of the application to those skilled in the art, and are not intended to limit the scope of the application. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the application.

[0143] Example 1. Anti-DLL3 antibody production and molecular characterization of the antibodies

[0144] 1.1 Method materials

[0145] His-tagged DLL3 antigen protein was used to immunize mice by subcutaneous injection in the abdomen. The first immunization used Freund's complete adjuvant to emulsify the antigen, and the second to fourth immunizations used Freund's incomplete adjuvant to emulsify the antigen. After immunizing the mice with the proteins in Table 1, the results of the antibody titer detection of the mouse serum are shown in Figure 1, which shows the serum titer of the mouse (number Q11) immunized with DLL3 protein at 500x, 2500x, 12500x, and 62500x representing the serum dilution ratio.

[0146] Table 1-1 Immunization proteins used and mice

[0147] 1.2 Establishment of phage library

[0148] The mouse (number Q11) was sacrificed, the spleen was dissected out, and the spleen was ground with a syringe rubber plug and filtered with a filter screen. The filtered spleen cells were frozen and stored, and cDNA was obtained after RNA extraction. The establishment of the phage library was performed according to the conventional method. The library capacity data of the constructed library are shown in Table 1-2.

[0149] Table 1-2 Library capacity of phage library constructed from immunized mice

[0150] 1.3 Screening by both plate screening and magnetic bead screening

[0151] 1.3.1 Plate screening

[0152] The plate was coated with DLL3-His protein. The next day, the phage library was added and incubated for 2 hours, and then the specifically bound phage was eluted with elution buffer after 4-10 times of washing.

[0153] 1.3.2 Magnetic bead screening

[0154] DLL3-Fc protein was biotinylated according to the kit steps, then combined with Thermo magnetic beads, incubated with phage library for 2h after BSA blocking, washed 4-10 times, then eluted specific binding phage with Elution Buffer. The antibody clone number and source library obtained by screening are shown in Tables 1-3.

[0155] Table 1-3 Active antibody source

[0156] 1.4 Construction and production of complete anti-DLL3 antibody molecules

[0157] The antibody variable region gene was amplified by conventional molecular biology technology PCR (2xPhanta Max Master Mix manufacturer: Vazyme, product number: P515-P1-AA, batch number: 7E351H9), and the antibody heavy chain variable region gene was connected into the vector pCDNA3.4 (Life Technology) with the antibody heavy chain constant region sequence by homologous recombination, and the antibody light chain variable region gene was connected into the vector pCDNA3.4 with the antibody light chain constant region sequence. The variable region sequences of each antibody of the positive clone IgG1 constructed in the embodiment of the application are shown in Table 1-4, and the heavy chain and light chain constant region sequences are shown in Table 1-5.

[0158] After sequencing, the positive clone plasmid was extracted and co-transfected into HEK293 cells, which were cultured at 37°C, 8% CO2, and 125rpm in a shaker. After 7 days of transient expression, the supernatant was purified by Protein A affinity chromatography to obtain the antibody, and the antibody concentration was determined by UV280 combined with the theoretical extinction coefficient.

[0159] Table 1-4 Antibody variable region amino acid sequence Note: The underlined part in VL and VH is CDR, and the analysis system is IMGT system

[0160] Table 1-5 Heavy chain and light chain constant region sequences

[0161] 1.5 Characterization of anti-DLL3 monoclonal antibody molecules

[0162] 1.5.1 ELISA detection of antibody binding to human DLL3-His protein

[0163] Human DLL3-His protein was diluted to 0.1 μg / mL with pH 9.6 CBS to coat the enzyme-labeled plate, 100 μL / well, 4°C incubation overnight; after washing the plate, it was blocked with skimmed milk. After washing the plate, 100 μL of gradient-diluted antibody was added to each well, 37°C incubation for 1 h; after washing the plate, 100 μL of HRP-labeled goat anti-human IgG (H+L) antibody diluted with PBST was added, 37°C incubation for 1 h. After washing the plate, 100 μL of TMB was added to each well, and after 10 min, 50 μL of 2M H2SO4 was added to each well to stop the color development, and the OD was read with an enzyme-labeled instrument 450 Figure 2 is the ELISA binding activity of the antibodies 546, 603, 616 of the present application and the blank control Isotype to human DLL3-His protein. The results show that the three antibodies with the antibody IDs 546, 603 and 616 of the present application all exhibit good binding activity to human DLL3-His protein. The Isotype in Figure 2 is an irrelevant IgG1 antibody for other target points, which is used as a blank control. The Isotype does not show binding activity. Taking the antibody concentration of 0.1 μg / mL as an example, the OD450 of the Isotype is 0.127, and the OD450 of the antibody group is 450 respectively, antibody 546: 4.461; antibody 603: 4.259; antibody 616: 4.240, and in particular, the OD450 of antibody 616 is more than 33 times that of the Isotype group. 450

[0164] 1.5.2 Flow cytometry detection of the binding of the antibody to CT26-hDLL3 cells

[0165] ​Dilute CT26-hDLL3 cells (Kang Yuan Bochuang Company, KC-1129) in logarithmic growth phase to 2E6 / mL with PBS, mix the gradient-diluted antibody and cells: 100 μL system = 50 μL antibody + 50 μL cells, incubate at 4°C for 1 h. After centrifugation to discard the supernatant, wash the cells with PBS, add 100 μL / w secondary antibody FITC anti-human IgG Fc, mix well, and incubate at 4°C for 0.5 h in the dark. After washing the cells with PBS, resuspend the cells with 100 μL / well PBS, and detect with a flow cytometer. The binding activity curves of each antibody and cells are shown in FIG. 3, which shows the binding activity of antibodies 546, 603, and 616 of the application and the blank control Isotype antibody to CT26-hDLL3 cells. Isotype in FIG. 3 is an irrelevant IgG1 antibody of other target points self-made, as a blank control. Isotype does not show binding activity. Taking an antibody concentration of 10 μg / mL as an example, the MFI of Isotype is 10120, and the MFI of the antibody group is 74397 for antibody 546, 78408 for antibody 603, and 73976 for antibody 616. The MFI of the antibody group is 7.3-7.7 times that of Isotype, and the EC 50 values are shown in Tables 1-6. Antibody 616 specifically binds to CT26-hDLL3 cells, and has high binding activity, with a binding EC 50 of 0.628 μg / mL.

[0166] The EC 50

[0167] 1.5.3 Detection of the affinity of the antibody to hDLL3-His protein

[0168] Human DLL3-His protein was diluted with 1x HBS-EP+ Buffer 2-fold gradient dilution at 5 concentrations, 50 nM, 25 nM, 12.5 nM, 6.25 nM and 3.125 nM respectively. The antibody sample was diluted with 1x HBS-EP+ to 2 μg / mL, and the antibody sample was captured by ProA chip, and the instrument capture time was set to 70 s, and the flow rate was 10 μL / min; analyte: binding 60 s, flow rate 30 μL / min, dissociation 600 s; regeneration: 10 mM Glycine-HCl buffer at pH 1.5 for 30 s, flow rate 30 μL / min, Startup 3 times. The equilibrium dissociation constant (KD) was calculated using the 1:1 binding model (BIAcore Insight Evaluation Software version 2.0.15.12933). The affinity results of each antibody are shown in Tables 1-7, and the KD of all antibodies is less than 1.0E-09, showing high affinity.

[0169] Table 1-7 Affinity of each antibody to human DLL3-His protein

[0170] Example 2 Construction of Targeting DLL3 Chimeric Antigen Receptor Recombinant Plasmid

[0171] 2.1 Preparation of Targeting DLL3 Chimeric Antigen Receptor Gene Fragment (DLL3 CAR)

[0172] The CAR fusion gene fragment is designed according to the following order of coding genes: CD8 alpha signal peptide, DLL3 antibody scFv VL-linker-DLL3 antibody scFv VH, CD8 hinge region, CD8 transmembrane region, and 4-1BB costimulatory signal and CD3 zeta intracellular signaling domain. The targeting DLL3 chimeric antigen receptor gene fragment is synthesized by Nanjing Kingsriver Biotechnology Co., Ltd. in the order by gene synthesis technology to form the structure of scFv VL-linker-scFv VH-CD8 hinge-CD8 TM-4-IBB-CD3 zeta, and the structural diagram is shown in Figure 4. The linker amino acid sequence is (G4S)3, i.e. GGGGSGGGGSGGGGS (SEQ ID NO: 21), the CD8 alpha signal peptide amino acid sequence is SEQ ID NO: 22, the CD8 hinge region (CD8 hinge) and the transmembrane region (CD8 TM) amino acid sequence is SEQ ID NO: 23, the 4-1BB amino acid sequence is SEQ ID NO: 24, the CD3 zeta intracellular signaling domain amino acid sequence is SEQ ID NO: 25, the anti-DLL3 antibody scFv, i.e. 546 DLL3 antibody scFv, 603 DLL3 antibody scFv, and 616 DLL3 antibody scFv, has the amino acid sequences shown in SEQ ID NOs: 26-28, respectively, and the specific sequences are shown in Table 2-1. Through the above construction process, the antibodies numbered BA546 (i.e. antibody 546), BA603 (i.e. antibody 603), and BA616 (i.e. antibody 616) are constructed into the targeting DLL3 chimeric antigen receptor gene fragment, and the 546 DLL3 CAR, 603 DLL3 CAR, and 616 DLL3 CAR have the amino acid sequences shown in SEQ ID NOs: 29-31, respectively, and the specific sequences are shown in Table 2-2.

[0173] Table 2-1. Amino acid sequences of CAR partial elements

[0174] Table 2-2. Amino acid sequences of DLL3 CAR

[0175] 2.2 Construction of DLL3 CAR vector based on piggybac (PB) transposon system

[0176] The pBluescript II SK(+) synthesized by General Biosystems) was used as the starting backbone. In the MCS (multiple cloning site) region, the 3'ITR sequence, the kanamycin resistance gene sequence and the 5'ITR sequence were inserted. Between the 3'ITR and the 5'ITR, the 2xcHS4 insulator sequence was inserted. Between the 2xcHS4 sequence, the EF1a promoter and the bGH poly(A) signal sequence were inserted. Downstream of the EF1a promoter, the CD8a-DLL3 antibody ScFv HV-linker-ScFv VL-CD8 hinge-CD8TM-4-1BB-CD3zeta (DLL3 CAR) gene sequence was connected. The above sequences formed the PB DLL3 CAR plasmid vector. The specific sequences used in this part of the construction are shown in Tables 2-3. Through the above construction process, PB 546 DLL3 CAR plasmid vector, PB 603 DLL3 CAR plasmid vector, and PB 616 DLL3 CAR plasmid vector were constructed from 546 DLL3 CAR, 603 DLL3 CAR, and 616 DLL3 CAR, respectively. Figure 5 shows the schematic diagram of the PB 616 DLL3 CAR plasmid structure. The PB 546 DLL3 CAR, PB 603 DLL3 CAR, and PB 616 DLL3 CAR vector plasmids constructed were extracted (completed by Nanjing Kings River Company), and transfection-grade plasmids were obtained.

[0177] Table 2-3. DLL3 CAR vector sequence

[0178] Example 3. Preparation of DLL3 CAR-T cells

[0179] 3.1 Sorting of human T lymphocytes

[0180] Peripheral blood mononuclear cells (PBMCs) (Shanghai Auneng Biotech) were labeled with CD3 MicroBeads human-lyophilized Kit (Miltenyi Biotech) magnetic beads, and high-purity CD3-positive T lymphocytes were sorted positively. The proportion of CD3-positive T cells after sorting was more than 95%. The purified T cells were then activated and proliferated using Dynabeads Human T-Activator CD3 / CD28 (Thermo Fisher, 11132D).

[0181] 3.2 CAR gene transduction based on piggybac (PB) transposon system

[0182] In this example, 72-96 hours after T cell stimulation and activation, the 546 DLL3 CAR, 603 DLL3 CAR, 616 DLL3 CAR PB transposon vector plasmid DNA and transposase mRNA obtained above were used for electroporation. First, the cells for electroporation were resuspended, the cell clumps were dispersed using a pipette tip or pipette, the cell suspension was counted, and 5x10 6 Cells were used for single electroporation experiment. 5x10 6 Cells were diluted to 5ml, centrifuged at 300xg for 10 minutes at room temperature, and the supernatant was aspirated as much as possible without touching the cell pellet. The cells were resuspended and washed with 5ml DPBS, centrifuged at 300xg for 10 minutes at room temperature, and the supernatant was aspirated as much as possible without touching the cell pellet. 100ul of Entranster-E (Engreen, 98668-20) was added to resuspend the cells, and the cell suspension was transferred to a 1.5ml centrifuge tube. The components in the following table (Table 3-1) were added to the centrifuge tube and mixed well.

[0183] Table 3-1. Electroporation system

[0184] The 4D-Nucleofector cell nucleus transducer of Swiss Lonza was used for electroporation, and the cell / plasmid suspension was quickly transferred to the electroporation cup and gently knocked the electroporation cup to make the cell suspension fully balanced in the electroporation cup. The program EO115 was used for electroporation. After electroporation, the electroporation cup was carefully removed. 500ul of preheated T cell culture medium X-VIVO 15 (Lonza, 04-418Q) was added and balanced in a 37°C incubator for 5 minutes, and the cells were resuspended using a micropore loading pipette and gently blown 2-3 times. The cells were transferred to a 12-well plate with 2ml of preheated medium, and cultured at 37°C. The cells were changed 4-6 hours after electroporation to increase cell survival. The supernatant was carefully aspirated, and fresh preheated medium was added. Incubate at 37°C, 5% CO2 incubator for 48-72h until detection.

[0185] 3.3 DLL3 CAR gene transduction T cell binding positive rate test after T cell stimulation and activation

[0186] After the electric transformation of 48-72h, the T cells after the electric transformation of the PB 546 DLL3 CAR, PB 603 DLL3 CAR and PB 616 DLL3 CAR plasmid vectors (the above cells are referred to as: 546 DLL3 CAR-T, 603 DLL3 CAR-T and 616 DLL3 CAR-T) and the T cells without the electric transformation of the CAR plasmid (referred to as T) were subjected to the CAR positive rate flow analysis. The biotin-labeled human DLL3 antigen protein (ACRO, DL3-H82E4) was used as the CAR binding protein, and then the avidin-coupled PE fluorescent dye was used to detect the chimeric antigen receptor (CAR) expression by the flow cytometry, and the positive rate of the DLL3 protein binding was shown in Table 3-2, and the DLL3 CAR positive rate was in the range of 27-46%. The experimental results show that the DLL3 CAR-T cells prepared by the structures of the 546 DLL3 CAR, 603 DLL3 CAR and 616 DLL3 CAR in the application can specifically recognize the DLL3 target protein. FIGS. 6A-6D show the CAR positive flow diagrams for detecting the DLL3 CAR-T cells by the DLL3 antigen, wherein FIG. 6A shows the results of the T cells without the transduction of the CAR, FIG. 6B shows the results of the 546 DLL3 CAR-T cells, FIG. 6C shows the results of the 603 DLL3 CAR-T cells, and FIG. 6D shows the results of the 616 DLL3 CAR-T cells. FIG. 7 shows the CAR positive rate results of the human DLL3 antigen protein for detecting the DLL3 CAR-T cells.

[0187] Table 3-2. The target protein binding positive rate of the DLL3 CAR-T cells

[0188] Example 4. The recognition of the DLL3 CAR-T to the DLL3 protein domain

[0189] The human DLL3 protein consists of 619 amino acids (NCBI Accession No. NM_016941.4, the first amino acid from the N-terminal is marked as 1aa, and then the amino acids are sequentially marked), and the complete structure contains 1 DSL domain, 1 intracellular domain and 6 epidermal growth factor-like domains (EGF-like Repeats). To test the recognition domain of DLL3 CAR-T to DLL3 protein, the extracellular segment of DLL3 protein was expressed by recombinant protein expression technology, as follows: human DLL3 protein extracellular segment full length (27-492aa)-His tag, human DLL3 protein DSL+EGF1-6 (176-492aa)-His tag, human DLL3 protein EGF1-6 (216-492aa)-His tag, human DLL3 protein EGF2-6 (274-492aa)-His tag, human DLL3 protein EGF3-6 (312-492aa)-His tag, human DLL3 protein EGF4-6 (353-492aa)-His tag, human DLL3 protein EGF5-6 (391-465aa)-His tag, human DLL3 protein EGF6 (429-492aa)-His tag. The above recombinant proteins were incubated with 546 DLL3 CAR-T, 603 DLL3 CAR-T and 616 DLL3 CAR-T, and then incubated with fluorescently labeled anti-His fluorescent antibody. After flow cytometry, the binding reaction of 546 DLL3 CAR-T, 603 DLL3 CAR-T and 616 DLL3 CAR-T to the above proteins was detected, and the results are shown in Table 4-1. The recognition domain of 546 DLL3 CAR-T, 603 DLL3 CAR-T and 616 DLL3 CAR-T to human DLL3 protein antigen is EGF4 domain (353-389aa). FIGS. 8A-8C show the results of the binding reaction of DLL3 CAR-T to different domain recombinant proteins of DLL3 protein. FIG. 8A shows the results of the binding reaction of 546 DLL3 CAR-T to different domain recombinant proteins of DLL3 protein, FIG. 8B shows the results of the binding reaction of 603 DLL3 CAR-T to different domain recombinant proteins of DLL3 protein, and FIG. 8C shows the results of the binding reaction of 616 DLL3 CAR-T to different domain recombinant proteins of DLL3 protein.

[0190] Table 4-1. Positive rate of the binding reaction of DLL3 CAR-T to different domain recombinant proteins of DLL3 protein

[0191] Example 5 In vitro cytotoxicity test of DLL3 CAR-T cells

[0192] 5.1 DLL3 CAR-T cell in vitro short-term target cell killing test experiment

[0193] An in vitro pharmacodynamic test was established by simulating the mechanism of action (MOA) of the product. Human DLL3 protein expression negative 293T cells and human DLL3 protein expression positive SHP-77 cells were used as target cells for DLL3 CAR-T cell function verification, and the 546 DLL3 CAR-T, 603 DLL3 CAR-T, and 616 DLL3 CAR-T prepared above were used as effector cells. According to the setting of 3:1 effector-to-target ratio (effector cells: target cells), a co-culture system of CAR-T cells and target tumor cells was established, and the tumor cell killing rate was detected by luciferase activity method (kit purchased from Biyun Tian) to evaluate the biological efficacy of CAR-T, and a co-culture system of untransduced T cells and tumor cells was set up as a control (referred to as T). The test results are shown in Tables 5-1 and 5-2. FIGS. 9A-9B show the in vitro specific killing results of 546 DLL3 CAR-T, 603 DLL3 CAR-T, 616 DLL3 CAR-T cells and control system T on target cells. FIG. 9A shows the in vitro specific killing results of 546 DLL3 CAR-T, 603 DLL3 CAR-T, 616 DLL3 CAR-T cells and control system T on 293T cell strain with negative DLL3 expression, and FIG. 9B shows the in vitro specific killing results of 546 DLL3 CAR-T, 603 DLL3 CAR-T, 616 DLL3 CAR-T cells and control system T on SHP77 cell strain with positive DLL3 expression. The experimental results show that under the condition of a fixed number of tumor cells, DLL3 CAR-T cells produce specific killing on SHP77 with positive DLL3 expression, and are positively correlated with the effector-to-target ratio. Under the condition of an effector-to-target ratio of 3:1, the killing efficiency after 24 hours is 50-60%, and there is no specific killing effect on human DLL3 expression negative 293T cell strain.

[0194] The specific killing detection method is performed by using a firefly luciferase reporter gene detection kit (purchased from Biyun Tian). The firefly luciferase is a protein with a molecular weight of about 61 kD, which can catalyze the oxidation of luciferin to oxyluciferin in the presence of ATP, magnesium ions and oxygen. In the process of luciferin oxidation, bioluminescence is emitted. The bioluminescence can be determined by a luminometer or a liquid scintillation detector. The principle is to construct a luciferase gene into a reporter gene plasmid. Then the cells are transfected, and the CAR-T cells or appropriate drugs are used to treat the cells. The cell membrane structure damage caused by cell apoptosis or necrosis will lead to the release of the enzyme in the cell plasma into the culture medium. By detecting the activity of luciferase released into the culture medium from the cells with broken plasma membrane, the quantitative analysis of cytotoxicity can be realized.

[0195] Table 5-1 Killing effect of DLL3 CAR-T cells on 293T cell strains with negative DLL3 expression

[0196] Table 5-2 Killing effect of DLL3 CAR-T cells on SHP77 cell strains with positive DLL3 expression

[0197] 5.2 IFN-γ factor release of DLL3 CAR-T cell killing tumor cells

[0198] The co-culture supernatant of the short-term target cell killing test experiment of the DLL3 CAR-T cells of Example 5.1 was collected, and the content of the cytokine (INF-γ) secreted into the culture supernatant was detected by a human IFN-γ ELISA detection kit (purchased from Novusbio) to evaluate the biological efficacy of the CAR-T. The experimental results show that the cytokine IFN-γ release level after the DLL3 CAR-T killing of the DLL3 expression positive SHP77 cells is at least 10 times higher than that of the negative control (T cells without CAR transduction), and there is no obvious release of cytokine IFN-γ after the killing of the DLL3 expression negative 293T cells. Table 5-3 shows the IFN-γ release levels after the killing of human DLL3 expression negative 293T cells and human DLL3 expression positive SHP-77 cells by the DLL3 CAR-T cells of the application and the controls, respectively. FIGS. 10A-10B show the IFN-γ factor release levels after the killing of tumor cells by the 546 DLL3 CAR-T, 603 DLL3 CAR-T, 616 DLL3 CAR-T cells and the control T, wherein FIG. 10A shows the IFN-γ release after the co-culture of the 546 DLL3 CAR-T, 603 DLL3 CAR-T, 616 DLL3 CAR-T cells and the control T with the DLL3 expression negative 293T cells, and FIG. 10B shows the IFN-γ release after the co-culture of the 546 DLL3 CAR-T, 603 DLL3 CAR-T, 616 DLL3 CAR-T cells and the control T with the DLL3 expression positive SHP77 cells.

[0199] The cytokine detection method: a human IFN-γ ELISA detection kit (purchased from Novusbio) is used, which is based on the solidification of antigens or antibodies and the enzyme labeling of antigens or antibodies. The antigens or antibodies combined on the solid phase carrier still maintain their immunological activity, and the enzyme-labeled antigens or antibodies retain their immunological activity and enzyme activity. During detection, the detected substances (antigens or antibodies) in the sample are combined with the fixed antibodies or antigens. By washing the plate to remove the non-combined substances, and then adding the enzyme-labeled antigens or antibodies, at this time, the amount of enzyme that can be fixed is related to the amount of the detected substances in the sample. After adding the substrate that reacts with the enzyme, color development occurs, and the content of the substances in the sample can be judged according to the color depth for qualitative or quantitative analysis.

[0200] Table 5-3 DLL3 CAR-T cell cytokine IFN-γ release results after killing target cells

[0201] 5.3 DLL3 CAR-T cell in vitro repeated killing tumor ability test

[0202] To evaluate the repeated anti-tumor effect of 546 DLL3 CAR-T, 603 DLL3 CAR-T, 616 DLL3 CAR-T cells in vitro. The 546 DLL3 CAR-T, 603 DLL3 CAR-T, 616 DLL3 CAR-T prepared by the above method, and T cells without CAR plasmid electroporation were used as effector cells, and human small cell lung cancer SHP-77 cell strain was used as target cells, with an effector to target ratio (effector cells: target cells) of 1:5. The cell-free co-culture system was used, and 2 / 5 volume of mixed cells were taken out every 3 days, and tumor cells were added for stimulation. The number of CAR-T cells and tumor cells in the co-culture system was detected to evaluate the long-term tumor killing ability of DLL3 CAR-T cells. The test results are shown in Tables 5-4 and 5-5. FIGS. 11A-11B show the results of long-term anti-tumor effect of 546 DLL3 CAR-T, 603 DLL3 CAR-T, 616 DLL3 CAR-T cells on DLL3 positive cells SHP-77, wherein FIG. 11A shows the number of tumor cells in the long-term anti-tumor effect of 546 DLL3 CAR-T, 603 DLL3 CAR-T, 616 DLL3 CAR-T cells on DLL3 positive cells SHP-77, and FIG. 11B shows the number of CAR positive T cells in the long-term anti-tumor effect of 546 DLL3 CAR-T, 603 DLL3 CAR-T, 616 DLL3 CAR-T cells on DLL3 positive cells SHP-77. The experimental results show that under the condition of effector cells: target cells at 1:5, through 3 rounds of tumor cell stimulation, 546 DLL3 CAR-T, 606 DLL3 CAR-T, 616 DLL3 CAR-T cells have obvious tumor cell clearance effect, while T cells without CAR plasmid electroporation have no tumor cell clearance effect. Among them, 616 DLL3 CAR-T has better long-term tumor cell clearance and self expansion than 546 DLL3 CAR-T and 606 DLL3 CAR-T.

[0203] Table 5-4 Number of tumor cells after repeated killing of tumor cells by DLL3 CAR-T cells

[0204] Table 5-5 Number of CAR-T cells after repeated killing of tumor cells by DLL3 CAR-T cells

[0205] Example 6 DLL3 CAR scFv sequence amino acid mutation

[0206] In this embodiment, by making amino acid mutations to the glutamine (Q) or lysine (K) of the framework region (FR) of antibody 616, i.e., 616scFv, different numbers of lysine residues impart different net charges to the antibody, with the C-terminal lysine (K) imparting a positive charge, while glutamine (Q) is a neutral amino acid. The glutamine (Q) and lysine (K) of the framework region (FR) of the 616scFv sequence are replaced with each other to adjust the positive charge patch of the scFv, and the number of mutations includes but is not limited to single, two, three, four point mutations. A series of 616scFv variant structures with amino acid substitutions are thus designed. Table 6-1 lists part of the 616scFv sequence after amino acid mutation, wherein the mutated amino acid is underlined.

[0207] Table 6-1 616 scFv structure amino acid mutant sequence

[0208] Example 7 Construction of mutant DLL3 CAR recombinant plasmid

[0209] The CAR fusion gene fragment was designed in the following order of coding genes: CD8a signal peptide, DLL3 antibody scFv, CD8 hinge region, CD8 transmembrane region, and 4-1BB costimulatory signal and CD3 zeta intracellular signaling domain. The target DLL3 chimeric antigen receptor gene fragment was synthesized by gene synthesis technology in order. In this embodiment, 616-mut8, 616-mut11, 616-mut18, 616-mut22, and 616-mut27 mutants were used to construct target DLL3 chimeric antigen receptor gene fragments, and the amino acid sequences thereof are shown as SEQ ID NO: 68-72, and the specific sequences are shown in Table 7-1, wherein the underlined amino acids are mutated amino acids.

[0210] Meanwhile, the DLL3 CAR vector was constructed based on the piggybac (PB) transposon system according to Example 2, and PB 616-mut8 DLL3 CAR, PB 616-mut11 DLL3 CAR, PB 616-mut18 DLL3 CAR, PB 616-mut22 DLL3 CAR, and PB 616-mut27 DLL3 CAR plasmid vectors were obtained.

[0211] Table 7-1 Amino acid sequence of mutant DLL3 CAR

[0212] Example 8 Test of mutant DLL3 CAR-T cell binding positive rate to DLL3 protein

[0213] In this embodiment, 616-mut8 DLL3 CAR-T, 616-mut11 DLL3 CAR-T, 616-mut18 DLL3 CAR-T, 616-mut22 DLL3 CAR-T, and 616-mut27 DLL3 CAR-T cells were prepared by sorting human T lymphocytes, and transducing mutant DLL3 CAR genes. The 616 DLL3 CAR-T cell without amino acid mutation was used as a control to test the binding positive rate of DLL3 protein. The binding positive rate of DLL3 protein of the T cells is shown in Table 8-1, and T represents human T lymphocytes without transduction of CAR. The DLL3 CAR positive rate of different mutants ranges from 17 to 35%.

[0214] The experimental results show that the CAR-T cells prepared by the amino acid mutants based on the 616 sequence in the present application (616-mut8 DLL3 CAR-T, 616-mut11 DLL3 CAR-T, 616-mut18 DLL3 CAR-T, 616-mut22 DLL3 CAR-T, and 616-mut27 DLL3 CAR-T) can specifically recognize the DLL3 target protein. FIGS. 12A-12G show the positive flow chart of untransduced, non-mutated, and different amino acid mutant DLL3 CAR-T cells recognizing DLL3 protein. FIG. 12A shows the results of T cells without transduction of CAR, FIG. 12B shows the results of 616 DLL3 CAR-T cells, FIG. 12C shows the results of 616-mut18 DLL3 CAR-T cells, FIG. 12D shows the results of 616-mut22 DLL3 CAR-T cells, FIG. 12E shows the results of 616-mut27 DLL3 CAR-T cells, FIG. 12F shows the results of 616-mut8 DLL3 CAR-T cells, and FIG. 12G shows the results of 616-mut11 DLL3 CAR-T cells. FIG. 13 shows the CAR positive rate of untransduced, non-mutated, and different amino acid mutant DLL3 CAR-T cells for human DLL3 antigen protein detection.

[0215] Table 8-1 Binding positive rate of mutant DLL3 CAR to DLL3 protein

[0216] Example 9 In vitro cytotoxicity test of amino acid mutant DLL3 CAR-T cells

[0217] The present example takes human DLL3 protein expression negative 293T cells and human DLL3 protein expression positive SHP-77 cells as the target cells for DLL3 CAR-T cell function verification, and the above prepared 616-mut8 DLL3 CAR-T, 616-mut11 DLL3 CAR-T, 616-mut18 DLL3 CAR-T, 616-mut22 DLL3 CAR-T, 616-mut27 DLL3 CAR-T as effector cells, and 616 DLL3 CAR-T without amino acid mutation as a control. According to the setting of 5:1, 2.5:1, 1.25:1 effector to target ratio (effector cell: target cell), the co-culture system of CAR-T cells and target tumor cells was established, and the tumor cell killing rate was detected by luciferase activity method to evaluate the biological efficacy of CAR-T. The experimental setting was 3 replicate holes, and the test results were shown in Table 9-1 and Table 9-2, and T represented human T lymphocytes without transduction of CAR. The results showed that the 616 DLL3 CAR-T with amino acid mutation had good killing effect on DLL3 positive SHP77 cells, and the amino acid mutation did not affect the short-term anti-tumor effect of 616 DLL3 CAR-T. Under the condition of effector to target ratio of 5:1, the killing efficiency after 24 hours was 47-65%, and there was no specific killing effect on human DLL3 expression negative 293T cell strain. FIGS. 14A-14B show the in vitro specific killing results of untransduced, unmutated and different amino acid mutant DLL3 CAR-T cells on target cells. FIG. 14A shows the in vitro specific killing results of untransduced, unmutated and different amino acid mutant DLL3 CAR-T cells on DLL3 expression negative 293T cell strain. FIG. 14B shows the in vitro specific killing results of untransduced, unmutated and different amino acid mutant DLL3 CAR-T cells on DLL3 expression positive SHP77 cell strain.

[0218] Table 9-1 Killing effect of amino acid mutant DLL3 CAR-T cells on 293T cell strain with negative expression of DLL3

[0219] Table 9-2 Killing effect of amino acid mutant DLL3 CAR-T cells on SHP77 cell strain with positive expression of DLL3

[0220] Example 10 Test of self-activation and exhaustion state of DLL3 CAR-T cells under non-antigen dependence

[0221] 10.1 Test of self-activation state of DLL3 CAR-T cells under non-antigen dependence

[0222] The present example tests the self-activation of 616-mut8 DLL3 CAR-T, 616-mut11 DLL3 CAR-T, 616-mut18 DLL3 CAR-T, 616-mut22 DLL3 CAR-T, and 616-mut27 DLL3 CAR-T prepared above in a conventional cell culture (without antigen stimulation) environment.

[0223] At different culture days (5, 7, 9, 11, 13, 15, and 17) of the different amino acid mutant DLL3 CAR-T cells, the expression amount of human CD69 on the surface of CAR-positive T cells was detected by flow cytometry to evaluate the activation state of the T cells. The 616 DLL3 CAR-T without amino acid mutation was used as a control. The test results are shown in Table 10-1, and FIG. 15 shows the expression changes of CD69 on the surface of CAR-positive cells of the non-mutant and each amino acid mutant DLL3 CAR-T cells under non-antigen dependence. The test results show that the activation signal CD69 on the surface of the mutant DLL3 CAR-T cells (616-mut18 DLL3 CAR-T, 616-mut22 DLL3 CAR-T, and 616-mut27 DLL3 CAR-T) with down-regulated positive charge patches has a certain degree of decrease, while the self-activation signal CD69 of the mutant DLL3 CAR-T cells (616-mut8 DLL3 CAR-T and 616-mut11 DLL3 CAR-T) with up-regulated positive charge patches has a significant increase, indicating that the regulation of the positive charge patches is related to the self-activation of the CAR-T cells.

[0224] Table 10-1 Expression changes of CD69 on the surface of CAR-positive cells of amino acid mutant DLL3 CAR-T cells under non-antigen dependence

[0225] 10.2 Test of self-depletion state of DLL3 CAR-T cells under non-antigen dependence

[0226] The present example tests the self-activation of 616-mut8 DLL3 CAR-T, 616-mut11 DLL3 CAR-T, 616-mut18 DLL3 CAR-T, 616-mut22 DLL3 CAR-T, and 616-mut27 DLL3 CAR-T prepared above in a conventional cell culture (without antigen stimulation) environment.

[0227] The expression levels of human PD-1, TIM3, and LAG3 on the surface of the T cells were detected to evaluate the exhaustion state of the T cells at different culture days (5, 7, 9, 11, 13, 15, and 17) of the different amino acid mutant DLL3 CAR-T cells, and the 616 DLL3 CAR-T without amino acid mutation was used as a control. The test results are shown in Tables 10-2, 10-3, and 10-4. FIGS. 16A-16C show the expression changes of the surface exhaustion indicators of the CAR-positive cells of the non-mutant and each amino acid mutant DLL3 CAR-T cells under non-antigen dependence. FIG. 16A shows the expression changes of the surface PD-1 of the CAR-positive cells of the non-mutant and each amino acid mutant DLL3 CAR-T cells under non-antigen dependence. FIG. 16B shows the expression changes of the surface TIM3 of the CAR-positive cells of the non-mutant and each amino acid mutant DLL3 CAR-T cells under non-antigen dependence. FIG. 16C shows the expression changes of the surface LAG3 of the CAR-positive cells of the non-mutant and each amino acid mutant DLL3 CAR-T cells under non-antigen dependence. The test results show that the surface exhaustion signals (TIM3 and LAG3) of the mutant DLL3 CAR-T cells (616-mut18 DLL3 CAR-T, 616-mut22 DLL3 CAR-T, and 616-mut27 DLL3 CAR-T) with the down-regulated positive charge patch have a certain degree of decrease, and the self-exhaustion signals (TIM3 and LAG3) of the mutant DLL3 CAR-T cells (616-mut8 DLL3 CAR-T and 616-mut11 DLL3 CAR-T) with the up-regulated positive charge patch have a significant increase, indicating that the regulation of the positive charge patch is related to the self-exhaustion of the CAR-T cells.

[0228] Table 10-2 Expression changes of the surface PD-1 of the CAR-positive cells of the amino acid mutant DLL3 CAR-T cells under non-antigen dependence

[0229] Table 10-3 Expression changes of the surface TIM3 of the CAR-positive cells of the amino acid mutant DLL3 CAR-T cells under non-antigen dependence

[0230] Table 10-4 Expression changes of the surface LAG3 of the CAR-positive cells of the amino acid mutant DLL3 CAR-T cells under non-antigen dependence

[0231] 10.3 Self-cytokine release test of the DLL3 CAR-T cells under non-antigen dependence

[0232] The cell supernatant of the 616 DLL3 CAR-T, 616-mut8 DLL3 CAR-T, 616-mut11 DLL3 CAR-T, 616-mut18 DLL3 CAR-T, 616-mut22 DLL3 CAR-T, 616-mut27 DLL3 CAR-T prepared above was collected on day 9 in the conventional cell culture (without antigen stimulation) environment. The cytokine (INF-γ) content in the cell culture supernatant was detected by human IFN-γ ELISA detection kit to test the self-endogenous stimulation signal triggered by CAR activation, and the test results are shown in Table 10-5, and T represents human T lymphocytes without transduction of CAR. FIG. 17 shows the secretion amount of self-INF-γ cytokine of the untransduced, non-mutant and each amino acid mutant DLL3 CAR-T cell under non-antigen dependence. The experimental results show that under the condition of no tumor antigen stimulation, the self-cytokine release of the mutant DLL3 CAR-T cell with up-regulated positive charge patch by amino acid mutation (616-mut8 DLL3 CAR-T, 616-mut11 DLL3 CAR-T) is obviously higher than that of the amino acid mutant DLL3 CAR-T cell with down-regulated positive charge patch (616-mut18 DLL3 CAR-T, 616-mut22 DLL3 CAR-T, 616-mut27 DLL3 CAR-T).

[0233] Table 10-5: Secretion amount of self-INF-γ cytokine of each DLL3 CAR-T cell under non-antigen dependence

[0234] Example 11: Anti-tumor function test of the amino acid mutant DLL3 CAR-T cell

[0235] To evaluate the in vitro antitumor activity of DLL3 CAR-T cells with various amino acid mutations, 616-mut8 DLL3 CAR-T, 616-mut11 DLL3 CAR-T, 616-mut18 DLL3 CAR-T, 616-mut22 DLL3 CAR-T, 616-mut27 DLL3 CAR-T, and 616 DLL3 CAR-T cells without amino acid mutations were prepared as effector cells. Human small cell lung cancer SHP-77 cell line was used as the target cell line. A cytokine-free co-culture system was used with an effector-target ratio of 1:5. Every 3 days, 2 / 5 of the mixed cell volume was harvested, and tumor cells were added again for stimulation. The sustained tumor-killing ability of DLL3-expressing CAR-T cells was evaluated by detecting changes in the number of CAR-T cells and tumor cells in the co-culture system. The test results are shown in Tables 11-1 and 11-2, where T represents untransduced human T lymphocytes. Figures 18A and 18B show the long-term anti-tumor effects of untransduced, unmutated, and amino acid mutant DLL3 CAR-T cells on DLL3-expressing SHP-77 cells. Figure 18A shows the changes in the number of tumor cells in the long-term anti-tumor effects of untransduced, unmutated, and amino acid mutant DLL3 CAR-T cells on DLL3-expressing SHP-77 cells. Figure 18B shows the changes in the number of CAR-positive T cells in the long-term anti-tumor effects of unmutated and amino acid mutant DLL3 CAR-T cells on DLL3-expressing SHP-77 cells. The test results showed that mutant DLL3 CAR-T cells (616-mut18 DLL3 CAR-T, 616-mut22 DLL3 CAR-T, and 616-mut27 DLL3 CAR-T) with downregulated positively charged plaques through amino acid mutations had better long-term anti-tumor effects than non-mutated 616 DLL3 CAR-T cells, while mutant DLL3 CAR-T cells (616-mut8 DLL3 CAR-T and 616-mut11 DLL3 CAR-T) with upregulated positively charged plaques through amino acid mutations had worse long-term anti-tumor effects.

[0236] Table 11-1 Changes in tumor cell number after repeated tumor killing by CAR-T cells with amino acid mutant DLL3

[0237] Table 11-2 Changes in the number of CAR-positive cells after repeated tumor killing by CAR-T cells with the amino acid mutant DLL3.

[0238] Example 12: Construction of recombinant plasmid using the 616 DLL3 CAR.aPDL1.mIL7 structure.

[0239] 12.1 Preparation of aPDL1, mIL7, aPDL1-mIL7 fusion protein gene fragments

[0240] The aPDL1 fusion protein gene fragment comprises the following genes: kappa leader signal peptide, anti-PD-L1 antibody scfv, connecting peptide 1, human IgG CH2CH3 fragment. The amino acid sequence of the connecting peptide 1 is G4S, i.e. GGGGS (SEQ ID NO: 85), the amino acid sequence of the kappa leader signal peptide is SEQ ID NO: 72, the amino acid sequence of the anti-PD-L1 antibody scfv is SEQ ID NO: 74, and the amino acid sequence of the human IgG CH2CH3 fragment is SEQ ID NO: 75.

[0241] The mIL7 fusion protein gene fragment comprises the following genes: human IL-7 cytokine fragment, connecting peptide 2, CD8 transmembrane region. The amino acid sequence of the connecting peptide 2 is Ser-Gly Linker, i.e. SGGGSGGGGSGGGGSGGGGSGGGSLQ (SEQ ID NO: 79), the amino acid sequence of the human IL-7 cytokine fragment is SEQ ID NO: 76, and the amino acid sequence of the CD8 transmembrane region is SEQ ID NO: 77.

[0242] The aPDL1-mIL7 fusion protein gene fragment comprises the aPDL1 and mIL7 fusion protein gene fragments, which are connected by 2A peptide 1, and the 2A peptide 1 is (GSG)T2A, the amino acid sequence of which is GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 78). The specific sequences of SEQ ID NO: 1-5 are shown in Table 12-1.

[0243] The above-mentioned aPDL1, mIL7, and aPDL1-mIL7 fusion protein genes are synthesized by Nanjing Kingsriver Biotechnology Co., Ltd. through gene synthesis technology, and the amino acid sequences thereof are shown in SEQ ID NO: 80-82, and the specific sequences are shown in Table 12-2.

[0244] Table 12-1 Amino acid sequences of enhanced structural elements

[0245] Table 12-2 Amino acid sequences of enhanced function fusion proteins

[0246] 12.2 Construction of 616 DLL3 CAR-aPDL1-mIL7 vector based on piggybac (PB) transposon system

[0247] The aPDLl-mIL7 fusion protein sequence synthesized in Example 1.1 was inserted into the rear of the 616 DLL3 CAR sequence by homologous recombination on the 616 DLL3 CAR plasmid structure, connected by 2A peptide 2, which was (GSG)P2A, with an amino acid sequence of GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 83), to form the 616 DLL3 CAR.aPDLl.mIL7 target gene fragment structure, with an amino acid sequence of SEQ ID NO: 84, as shown in Table 12-3, and the plasmid vector structure is shown in FIG. 19. The constructed 616 DLL3 CAR.aPDLl.mIL7 plasmid vector was extracted (contracted to Nanjing Kings River Company to complete), to obtain a transfection level Piggybac transposon plasmid.

[0248] Table 12-3 Amino acid sequence of enhanced CAR structure fusion protein

[0249] Example 13 Preparation of 616 DLL3 CAR.aPDLl.mIL7-T cells and detection of DLL3 CAR positive rate

[0250] In this example, 616 DLL3 CAR.aPDLl.mIL7-T cells were prepared by sorting and activating human T lymphocytes, and transducing the 616 DLL3 CAR.aPDLl.mIL7 plasmid vector. Un- electroporated T cells and T cells electroporated with the 616 DLL3 CAR plasmid vector were used as controls for DLL3 protein binding positive rate testing. The DLL3 protein binding positive rate is shown in Table 13-1, and the DLL3 CAR positive rate of the 616 DLL3 CAR.aPDLl.mIL7-T cells was 25.5%.

[0251] FIGS. 20A-20C show CAR positive flow cytometry diagrams of DLL3 CAR-T cells expressing DLL3 CAR detected by DLL3 antigen, wherein FIG. 20A shows the results of T cells without transduction of CAR, FIG. 20B shows the results of 616 DLL3 CAR-T cells, and FIG. 20C shows the results of 616 DLL3 CAR.aPDLl.mIL7-T cells. FIG. 21 shows the CAR positive rate results of DLL3 CAR-T cells detected by human DLL3 antigen protein, wherein Mock T is the T cell group without transduction of CAR.

[0252] Table 13-1 CAR positive rate of DLL3 CAR-T cells

[0253] Example 14 Cell membrane IL7 (mIL7) detection of 616 DLL3 CAR.aPDL1.mIL7-T cells

[0254] Cell membrane surface IL7 fusion protein (mIL7) detection was performed on 616 DLL3 CAR-T, 616 DLL3 CAR.aPDL1.mIL7-T and T cells without CAR plasmid electroporation, biotin-labeled anti-human IL7 antibody was used as the binding antibody of human IL7 protein, and avidin-coupled PE fluorescent dye was used to detect the positive rate of T cell membrane surface IL7 protein by flow cytometry. The results are shown in Table 14-1. The experimental results show that 616 DLL3 CAR.aPDL1.mIL7-T cells expressing cell membrane IL7 fusion protein can detect human IL7 protein on the surface of T cells, and the expression efficiency and CAR positive rate are comparable, while 616 DLL3 CAR-T cells without co-expression of IL7 fusion protein and T cells without CAR plasmid electroporation cannot detect IL7 protein on the cell membrane surface.

[0255] FIGS. 22A-22C show flow cytometry diagrams of cell membrane surface IL7 protein expression of DLL3 CAR-T cells detected by anti-human IL7 antibody, wherein FIG. 22A shows the results of T cells without CAR transduction, FIG. 22B shows the results of DLL3 CAR-T cells, and FIG. 22C shows the results of 616 DLL3 CAR.aPDL1.mIL7-T cells. FIG. 23 shows the results of anti-human IL7 antibody detection of cell membrane surface IL7 protein expression of DLL3 CAR-T cells, wherein Mock T is the T cell group without CAR transduction.

[0256] Table 14-1 Positive rate of cell membrane IL7 protein detection of 616 DLL3 CAR.aPDL1.mIL7-T cells

[0257] Example 15 Culture of 616 DLL3 CAR.aPDL1.mIL7-T cells and detection of aPDL1 in supernatant after killing tumor cells

[0258] In this embodiment, in order to test the release amount of aPDL1 fusion protein of 616 DLL3 CAR.aPDL1.mIL7-T cells, culture conditions without adding DLL3 positive tumor cells and co-culture with adding DLL3 positive tumor cells were set up, respectively. In the culture condition without adding DLL3 positive tumor cells, 3x10 5CAR positive cells / 200uL volume were inoculated into 96-well culture plates. In the culture condition of adding DLL3 positive tumor cells (SHP77 cells), SHP77 cells were inoculated into 96-well culture plates at a volume of 1x10 5 cells / 100uL volume were inoculated into 96-well culture plates, and 616DLL3 CAR.aPDL1.mIL7-T cells were inoculated at a volume of 3x10 5 CAR positive cells / 100uL volume were added for co-culture, and 616 DLL3 CAR-T and T cells without CAR plasmid electroporation were set as controls. After 24 hours of culture, the cell supernatant was collected, and the concentration of aPDL1 fusion protein in the cell supernatant was detected by ELISA double antibody sandwich method. The detection results of the concentration of aPDL1 fusion protein in the cell supernatant of each group are shown in Table 15-1 and FIG. 24, wherein Mock T is the T cell group without CAR transduction. The experimental results show that 616 DLL3 CAR.aPDL1.mIL7-T cells can normally secrete aPDL1 fusion protein and release it into the cell supernatant, and the concentration is 79.64 ng / mL. In addition, in the co-culture condition of adding tumor cells in this embodiment, the secretion amount of aPDL1 fusion protein of 616 DLL3 CAR.aPDL1.mIL7-T cells significantly increases when they contact tumor antigens to perform cell killing function, and the secretion amount increases by about 3 times, which is 333.98 ng / mL, compared with the culture condition without adding tumor cells.

[0259] Table 15-1 Secretion of aPDL1 fusion protein by 616 DLL3 CAR.aPDL1.mIL7-T cells

[0260] Example 16 Culture of 616 DLL3 CAR.aPDL1.mIL7-T cells without addition of cytokines

[0261] In this embodiment, 616 DLL3 CAR-T, 616 DLL3 CAR.aPDL1.mIL7-T and T cells without CAR plasmid electroporation were cultured to day 9 (T cell sorting was day 0), the medium was removed by centrifugation to remove the cytokine human IL7 and IL15 components that promote T cell expansion, and the same cell density was inoculated into new medium without the addition of cytokine human IL7 and IL15, and the cell viability, cell expansion number and CAR positive rate were observed by cell counting every 3 days. The cell viability of each group of cells under cytokine-free culture conditions is shown in Table 16-1 and FIG. 25A, the cell number change of each group of cells under cytokine-free culture conditions is shown in Table 16-2 and FIG. 25B, and the CAR positive rate change of each group of cells under cytokine-free culture conditions is shown in Table 16-3 and FIG. 25C, wherein Mock T is a T cell group without CAR transduction. The experimental results show that: 616 DLL3 CAR-T without co-expression of mIL7 fusion protein and T cells without CAR plasmid electroporation gradually die under cytokine-free culture conditions, and the viability and number of viable cells decrease rapidly, and by day 9, the cell viability decreases by 70-80%, and the cell number decreases by 80-90%. While 616 DLL3 CAR.aPDL1.mIL7-T cells expressing mIL7 fusion protein still maintain for a long time under factor-free culture conditions, and the viability and number of viable cells decrease slowly, and by day 9, the cell viability only decreases by 20%, and the cell number increases by 1-fold. At the same time, by detecting the change of CAR positive rate, it is found that compared with 616 DLL3 CAR-T without co-expression of mIL7 fusion protein and T cells without CAR plasmid electroporation, the proportion of CAR positive cells of 616 DLL3 CAR.aPDL1.mIL7-T cells expressing mIL7 fusion protein increases greatly from 22.50% to 54.18%, and the above results show that the mIL7 fusion protein structure can enhance the viability and persistence of the whole T cells, and has specific expansion effect on CAR positive T cells.

[0262] Table 16-1 Cell viability change of each group of cells under cytokine-free culture conditions

[0263] Table 16-2 Cell number change of each group of cells under cytokine-free culture conditions

[0264] Table 16-3 CAR positive rate change of each group of cells under cytokine-free culture conditions

[0265] In addition, in this embodiment, the change in the expansion fold of CAR-positive T cells and CAR-negative cells in the 616 DLL3 CAR-T and 616 DLL3 CAR.aPDL1.mIL7-T cell populations under cytokine-free culture conditions was also counted, and the results are shown in Tables 16-4, 16-5 and FIGS. 26A, 26B. The experimental results show that the CAR-positive cell population and the CAR-negative cell population of 616 DLL3 CAR-T both showed a significant decrease in expansion under cytokine-free culture conditions, with a decrease of 70-90%. The CAR-positive cell population of 616 DLL3 CAR.aPDL1.mIL7-T cells showed a more significant change in expansion than the overall number of cells, with the number of cells expanded by 4.8 times. The CAR-negative cell population did not show a significant expansion under cytokine-free culture conditions, but could maintain the initial number of cells and did not show a significant decrease. The above results show that the mIL7 fusion protein structure can specifically promote the expansion of the CAR-positive cell population, and can also have a synergistic effect on the CAR-negative cells that do not express the mIL7 structure, maintaining the viability and state of the cells.

[0266] Table 16-4 Change in expansion fold of CAR-positive cells in each group of cells under cytokine-free culture conditions

[0267] Table 16-5 Change in expansion fold of CAR-negative cells in each group of cells under cytokine-free culture conditions

[0268] Example 17 Test of the in vitro sustained killing ability of 616 DLL3 CAR.aPDL1.mIL7-T cells on tumors

[0269] To evaluate the enhanced long-term anti-tumor effect of DLL3 CAR-T cells co-expressing aPDL1 fusion protein and mIL7 fusion protein in vitro, 616 DLL3 CAR-T and 616 DLL3 CAR.aPDL1.mIL7-T cells were used as effector cells, human small cell lung cancer SHP-77 cell line was used as target cells, and the effector-to-target ratio (effector cells (CAR-positive cells): target cells) was 1:5. The number of tumor cells and CAR-positive cells was detected every 3 days in a cytokine-free co-culture system. Two-fifths of the co-cultured cells were removed and replaced with new tumor cells for repeated stimulation. This process was repeated for 4 rounds of killing. The number of CAR-positive cells and tumor cells in the co-culture system was detected to evaluate the sustained tumor killing ability of 616 DLL3 CAR.aPDL1.mIL7-T cells. The results of the number of SHP77 tumor cells in repeated killing are shown in Table 17-1 and FIG. 27A, and the results of the number of CAR-positive cells in repeated killing are shown in Table 17-2 and FIG. 27B. The experimental results show that when the effector-to-target ratio is 1:5, the 616 DLL3 CAR.aPDL1.mIL7-T cells have a significantly better tumor cell clearance effect than the 616 DLL3 CAR-T cells after 4 rounds of tumor cell stimulation. The 616 DLL3 CAR-T cells cannot inhibit tumor growth after the third round of killing, while the 616 DLL3 CAR.aPDL1.mIL7-T cells still have good tumor inhibition effect after the fourth round of killing, indicating that the aPDL1 fusion protein and mIL7 fusion protein play a key role in the long-term anti-tumor process.

[0270] Table 17-1 Number of SHP77 tumor cells in repeated killing

[0271] Table 17-2 Number of CAR-positive cells in repeated killing

[0272] Example 18 Animal efficacy experiment

[0273] In this example, an immunodeficient mouse model of human small cell lung cancer tumor cell load was established to evaluate the anti-tumor effect of amino acid mutant DLL3 CAR-T and 616 DLL3 CAR.aPDL1.mIL7-T cells in mice. Based on the in vitro study, 5x10 6 The number of human small cell lung cancer cells SHP-77 was counted, and the SHP-77 cells were inoculated on the 7th day (tumor volume was 50-60 mm 3doses, a total of 4 groups were set, which were T cell (T cells without CAR transduction) group, 616 DLL3 CAR-T cell group, 616-mut18 DLL3 CAR-T cell group, 616-mut8 DLL3 CAR-T cell group, and 616 DLL3 CAR.aPDL1.mIL7-T cell group. Except for the T cell group, each group was administered with 2x10 6 CAR-T cell number / mouse dose, and the number of animals in all conditions was 5. After administration, the tumor was measured twice a week, the tumor growth curve was drawn, and the content of T cells in the blood of each group of mice was detected every 7 days. FIGS. 28A-28E show the results of changes in tumor size of NCG mice with small cell lung cancer SHP-77 tumor after administration of each group of DLL3 CAR-T cells. FIG. 28A shows the change in tumor size of a single mouse after administration of the T cell (T cells without CAR transduction) group, FIG. 28B shows the change in tumor size of a single mouse after administration of the 616 DLL3 CAR-T cell group, FIG. 28C shows the change in tumor size of a single mouse after administration of the 616-mut18 DLL3 CAR-T cell group, FIG. 28D shows the change in tumor size of a single mouse after administration of the 616-mut8 DLL3 CAR-T cell group, and FIG. 28E shows the change in tumor size of a single mouse after administration of the 616 DLL3 CAR.aPDL1.mIL7-T cell group. In addition, FIG. 29 shows the change in the proportion of human T cells in the blood of mice after administration of each group of DLL3 CAR-T cells, where T represents T cells (T cells without CAR transduction). The experimental results show that 56 days after administration, each group of DLL3 CAR-T cells has a certain inhibitory effect on the SHP-77 tumor in mice, among which one mouse (1 / 5) in the 616 DLL3 CAR-T cell group completely eliminated the tumor, two mice (2 / 5) in the 616-mut18 DLL3 CAR-T cell group with amino acid mutation down-regulating the positive charge patch completely eliminated the tumor, the 616-mut8 DLL3 CAR-T cell group with amino acid mutation up-regulating the positive charge patch failed to inhibit tumor growth in the later stage, and the 616 DLL3 CAR.aPDL1.mIL7-T administration group based on the functions of aPDL1 fusion protein and mIL7 fusion protein completely eliminated the tumor in all mice (5 / 5). In addition, the results of the proportion of human T cells in the blood of mice after administration shown in FIG. 29 also show that the expansion of 616 DLL3 CAR.aPDL1.mIL7-T cells in vivo is significantly higher than that of other groups, and with the elimination of the tumor, it shows a trend of first increasing and then decreasing.

Claims

An anti-DLL3 antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof comprising 3 light chain complementarity determining regions and 3 heavy chain complementarity determining regions, characterized in that, wherein: the three light chain complementarity determining regions of the antibody or antigen-binding fragment thereof comprise LCDR1 of SEQ ID NO: 16, LCDR2 of SEQ ID NO: 17, and LCDR3 of SEQ ID NO: 18, and the three heavy chain complementarity determining regions of the antibody or antigen-binding fragment thereof comprise HCDR1 of SEQ ID NO: 14, HCDR2 of SEQ ID NO: 3, and HCDR3 of SEQ ID NO: 4; the three light chain complementarity determining regions of the antibody or antigen-binding fragment thereof comprise LCDR1 of SEQ ID NO: 11, LCDR2 of SEQ ID NO: 7, and LCDR3 of SEQ ID NO: 12, and the three heavy chain complementarity determining regions of the antibody or antigen-binding fragment thereof comprise HCDR1 of SEQ ID NO: 2, HCDR2 of SEQ ID NO: 3, and HCDR3 of SEQ ID NO: 4; or the three light chain complementarity determining regions of the antibody or antigen-binding fragment thereof comprise LCDR1 of SEQ ID NO: 6, LCDR2 of SEQ ID NO: 7, and LCDR3 of SEQ ID NO: 8, and the three heavy chain complementarity determining regions of the antibody or antigen-binding fragment thereof comprise HCDR1 of SEQ ID NO: 2, HCDR2 of SEQ ID NO: 3, and HCDR3 of SEQ ID NO:

4. The antibody or antigen-binding fragment thereof of claim 1, wherein: the antibody or antigen-binding fragment thereof comprises a light chain variable region that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 15, and / or a heavy chain variable region that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 13; the antibody or antigen-binding fragment thereof comprises a light chain variable region that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 10, and / or a heavy chain variable region that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 9; or the antibody or antigen-binding fragment thereof comprises a light chain variable region that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 5, and / or a heavy chain variable region that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:

1. The antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that, The antigen-binding fragment includes a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody, a Fab, a Fab', a F(ab')2, a Fv, a scFv or a dsFv fragment. The antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that, The antibody comprises a heavy chain constant region as shown in SEQ ID NO: 19, and / or comprises a light chain constant region as shown in SEQ ID NO:

20. A mutant of an anti-DLL3 antibody or antigen-binding fragment thereof, characterized in that, The mutation comprises: (a) an up-regulating positive charge patch amino acid mutation, or (b) a down-regulating positive charge patch amino acid mutation; Preferably, the up-regulating positive charge patch amino acid mutation is glutamine (Q) to lysine (K), and the down-regulating positive charge patch amino acid mutation is lysine (K) to glutamine (Q). More preferably, the mutation is in the framework region of the antibody. The mutant according to claim 5, characterized in that The amino acid sequence of the scFv of the parent 616 DLL3 antibody is shown in SEQ ID NO: 28, and the mutant is selected from any one of the following: (1) 616-mut1 mutant: Q to K at positions 37, 38, 100 of the scFv of the parent 616 DLL3 antibody, The amino acid sequence of the scFv of the 616-mut1 mutant is shown in SEQ ID NO: 38; (2) 616-mut2 mutant: Q to K at positions 3, 37, 38, 100 of the scFv of the parent 616 DLL3 antibody, the amino acid sequence of the scFv of the 616-mut2 mutant is shown in SEQ ID NO: 39; (3) 616-mut3 mutant: Q to K at positions 6, 37, 38, 100 of the scFv of the parent 616 DLL3 antibody, the amino acid sequence of the scFv of the 616-mut3 mutant is shown in SEQ ID NO: 40; (4) 616-mut4 mutant: Q to K at positions 79, 100 of the scFv of the parent 616 DLL3 antibody, the amino acid sequence of the scFv of the 616-mut4 mutant is shown in SEQ ID NO: 41; (5) 616-mut5 mutant: Q to K at positions 3, 79, 100 of the scFv of the parent 616 DLL3 antibody, the amino acid sequence of the scFv of the 616-mut5 mutant is shown in SEQ ID NO: 42; (6) 616-mut6 mutant: Q to K at positions 3, 38, 127, 161 of the scFv of the parent 616 DLL3 antibody, the amino acid sequence of the scFv of the 616-mut6 mutant is shown in SEQ ID NO: 43; (7) 616-mut7 mutant: Q at positions 6, 38, 127, 161 of the scFv of the parent 616 DLL3 antibody is mutated to K, the scFv amino acid sequence of the 616-mut7 mutant is shown as SEQ ID NO: 44; (8) 616-mut8 mutant: Q at positions 37, 38, 127, 161 of the scFv of the parent 616 DLL3 antibody is mutated to K, the scFv amino acid sequence of the 616-mut8 mutant is shown as SEQ ID NO: 45; (9) 616-mut9 mutant: Q at positions 79, 127, 161 of the scFv of the parent 616 DLL3 antibody is mutated to K, the scFv amino acid sequence of the 616-mut9 mutant is shown as SEQ ID NO: 46; (10) 616-mut10 mutant: Q at positions 3, 79, 127, 161 of the scFv of the parent 616 DLL3 antibody is mutated to K, the scFv amino acid sequence of the 616-mut10 mutant is shown as SEQ ID NO: 47; (11) 616-mut11 mutant: Q at positions 100, 127, 128, 161 of the scFv of the parent 616 DLL3 antibody is mutated to K, the scFv amino acid sequence of the 616-mut11 mutant is shown as SEQ ID NO: 48; (12) 616-mut12 mutant: Q at positions 125, 127, 128, 161 of the scFv of the parent 616 DLL3 antibody is mutated to K, the scFv amino acid sequence of the 616-mut12 mutant is shown as SEQ ID NO: 49; (13) 616-mut13 mutant: Q at positions 37, 38, 79, 199 of the scFv of the parent 616 DLL3 antibody is mutated to K, the scFv amino acid sequence of the 616-mut13 mutant is shown as SEQ ID NO: 50; (14) 616-mut14 mutant: Q at positions 100, 199 of the scFv of the parent 616 DLL3 antibody is mutated to K, the scFv amino acid sequence of the 616-mut14 mutant is shown as SEQ ID NO: 51; (15) 616-mut15 mutant: Q at positions 3, 100, 199 of the scFv of the parent 616 DLL3 antibody is mutated to K, the scFv amino acid sequence of the 616-mut15 mutant is shown as SEQ ID NO: 52; (16) 616-mut16 mutant: K at positions 45, 135, 165, 186 of the scFv of the parent 616 DLL3 antibody is mutated to Q, the scFv amino acid sequence of the 616-mut16 mutant is shown as SEQ ID NO: 53; (17) 616-mut17 mutant: K at positions 103, 135, 165, 186 of the scFv of the parent 616 DLL3 antibody is mutated to Q, the scFv amino acid sequence of the 616-mut17 mutant is set forth in SEQ ID NO: 54; (18) 616-mut18 mutant: K at positions 107, 135, 165, 186 of the scFv of the parent 616 DLL3 antibody is mutated to Q, the scFv amino acid sequence of the 616-mut18 mutant is set forth in SEQ ID NO: 55; (19) 616-mut19 mutant: K at positions 42, 45, 197 of the scFv of the parent 616 DLL3 antibody is mutated to Q, the scFv amino acid sequence of the 616-mut19 mutant is set forth in SEQ ID NO: 56; (20) 616-mut20 mutant: K at positions 39, 42, 45, 197 of the scFv of the parent 616 DLL3 antibody is mutated to Q, the scFv amino acid sequence of the 616-mut20 mutant is set forth in SEQ ID NO: 57; (21) 616-mut21 mutant: K at positions 45, 165, 186, 197 of the scFv of the parent 616 DLL3 antibody is mutated to Q, the scFv amino acid sequence of the 616-mut21 mutant is set forth in SEQ ID NO: 58; (22) 616-mut22 mutant: K at positions 103, 165, 186, 197 of the scFv of the parent 616 DLL3 antibody is mutated to Q, the scFv amino acid sequence of the 616-mut22 mutant is set forth in SEQ ID NO: 59; (23) 616-mut23 mutant: K at positions 107, 165, 186, 197 of the scFv of the parent 616 DLL3 antibody is mutated to Q, the scFv amino acid sequence of the 616-mut23 mutant is set forth in SEQ ID NO: 60; (24) 616-mut24 mutant: K at positions 42, 103, 203 of the scFv of the parent 616 DLL3 antibody is mutated to Q, the scFv amino acid sequence of the 616-mut24 mutant is set forth in SEQ ID NO: 61; (25) 616-mut25 mutant: K at positions 39, 42, 103, 203 of the scFv of the parent 616 DLL3 antibody is mutated to Q, the scFv amino acid sequence of the 616-mut25 mutant is set forth in SEQ ID NO: 62; (26) 616-mut26 mutant: K at positions 45, 135, 197, 203 of the scFv of the parent 616 DLL3 antibody is mutated to Q, the scFv amino acid sequence of the 616-mut26 mutant is set forth in SEQ ID NO: 63; ​ (27) 616-mut27 mutant: K at positions 103, 135, 197, 203 of the scFv of the parent 616 DLL3 antibody is mutated to Q, the scFv amino acid sequence of the 616-mut27 mutant is set forth in SEQ ID NO: 64; (28) 616-mut28 mutant: K at positions 135, 165, 197, 203 of the scFv of the parent 616 DLL3 antibody is mutated to Q, the scFv amino acid sequence of the 616-mut28 mutant is set forth in SEQ ID NO: 65; (29) 616-mut29 mutant: K at positions 186, 197, 203 of the scFv of the parent 616 DLL3 antibody is mutated to Q, the scFv amino acid sequence of the 616-mut29 mutant is set forth in SEQ ID NO: 66; or (30) 616-mut30 mutant: K at positions 39, 186, 197, 203 of the scFv of the parent 616 DLL3 antibody is mutated to Q, the scFv amino acid sequence of the 616-mut30 mutant is set forth in SEQ ID NO:

67. A chimeric antigen receptor (CAR) targeting the DLL3 antigen, characterized in that, the CAR comprises a DLL3 antigen binding domain, a transmembrane domain and an intracellular signaling domain, wherein the DLL3 antigen binding domain is a scFv of the antibody or antigen binding fragment thereof of any one of claims 1-4 or a scFv of the mutant of any one of claims 5-6; Preferably, the amino acid sequence of the light chain variable region and the amino acid sequence of the heavy chain variable region of the scFv are set forth in SEQ ID NO: 15 and SEQ ID NO: 13, respectively, or the amino acid sequence of the light chain variable region and the amino acid sequence of the heavy chain variable region of the scFv are set forth in SEQ ID NO: 10 and SEQ ID NO: 9, respectively, or the amino acid sequence of the light chain variable region and the amino acid sequence of the heavy chain variable region of the scFv are set forth in SEQ ID NO: 5 and SEQ ID NO: 1, respectively; More preferably, the amino acid sequence of the scFv is set forth in any one of SEQ ID NOs: 26-28, 38-67. The chimeric antigen receptor according to claim 7, characterized in that the CAR further comprises one or more of a hinge region, a signal peptide and a costimulatory signaling domain; Preferably, the transmembrane domain is a CD8 transmembrane region, the hinge region is a CD8 hinge region, the intracellular signaling domain is a CD3ζ intracellular signaling domain, the signal peptide is a CD8α signal peptide, or the costimulatory signaling domain is a 4-1BB or a CD28 costimulatory signaling domain. The chimeric antigen receptor according to claim 8, characterized in that, the CAR comprises, in order from N-terminus to C-terminus, a CD8α signal peptide, a DLL3 antibody scFv VL-linker-DLL3 antibody scFv VH, a CD8 hinge region and transmembrane region, a 4-1BB costimulatory signaling domain and a CD3ζ intracellular signaling domain; optionally, each element is connected by a linker; Preferably, the CD8a signal peptide comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 22; the CD8 hinge and transmembrane region comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 23; the 4-1BB costimulatory signaling domain comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 24; or the CD3 zeta intracellular signaling domain comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 25; More preferably, the amino acid sequence of the CAR is set forth in any one of SEQ ID NOs: 29-31, 68-72. An enhanced chimeric antigen receptor (CAR) targeting DLL3 antigen, characterized in that, comprises, in order from N-terminus to C-terminus: (1) a CAR targeting DLL3 antigen according to any one of claims 7-9; (2) an immunosuppressive molecule expression element; and (3) a cell membrane type interleukin and / or a secreted chemokine expression element; Preferably, the element (2) is selected from one or more of a PD1 antagonist and a PDL1 antagonist; the cell membrane type interleukin in the element (3) is selected from one or more of a cell membrane type IL2 cytokine, a cell membrane type IL4 cytokine, a cell membrane type IL7 cytokine, a cell membrane type IL9 cytokine, a cell membrane type IL10 cytokine, a cell membrane type IL15 cytokine, a cell membrane type IL18 cytokine, a cell membrane type IL21 cytokine, a cell membrane type IL23 cytokine, a cell membrane type IL24 cytokine, and a cell membrane type IL36 cytokine; and the secreted chemokine in the element (3) is selected from one or more of a secreted CCL1 chemokine, a secreted CCL2 chemokine, a secreted CCL3 chemokine, a secreted CCL5 chemokine, a secreted CCL7 chemokine, a secreted CCL15 chemokine, a secreted CCL16 chemokine, a secreted CCL19 chemokine, a secreted CCL20 chemokine, a secreted CCL21 chemokine, a secreted CXCL4 chemokine, a secreted CXCL9 chemokine, a secreted CXCL10 chemokine, a secreted CXCL11 chemokine, and a secreted CXCL1 chemokine. The enhanced chimeric antigen receptor (CAR) targeting DLL3 antigen according to claim 10, characterized in that, The element (2) is selected from a PDL1 antagonist expression element, and / or the element (3) is selected from a cell membrane type IL7 cytokine expression element; Preferably, the PDL1 antagonist expression element comprises, in order from N-terminus to C-terminus, a kappa leader signal peptide, an anti-PD-L1 antibody scfv, a connecting peptide 1, and a human IgG CH2CH3 fragment; the cell membrane type IL7 cytokine expression element comprises, in order from N-terminus to C-terminus, a human IL-7 cytokine fragment, a connecting peptide 2, a CD8 transmembrane region; More preferably, the kappa leader signal peptide in the anti-PDL1 expression element comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 73; the anti-PDL1 antibody scfv comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 74; the amino acid sequence of connecting peptide 1 is set forth in SEQ ID NO: 85; the human IgG CH2CH3 fragment comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 75; the human IL-7 cytokine fragment in the cell membrane type IL7 cytokine expression element comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 76; the amino acid sequence of connecting peptide 2 is set forth in SEQ ID NO: 79; the CD8 transmembrane region amino acid sequence comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 77; optionally, the element (2) and the element (3) are connected by a 2A peptide 1, the amino acid sequence of the 2A peptide 1 is set forth in SEQ ID NO: 78; the element (1) and element (2) are connected by a 2A peptide 2, the amino acid sequence of the 2A peptide 2 is set forth in SEQ ID NO: 83; Most preferably, the enhanced targeting DLL3 antigen CAR is 616 DLL3 CAR.aPDL1.mIL7, wherein the amino acid sequence of the 616 DLL3 CAR is set forth in SEQ ID NO: 31, the amino acid sequence of the aPDL1 is set forth in SEQ ID NO: 80, the amino acid sequence of the mIL7 is set forth in SEQ ID NO: 81, the amino acid sequence of the aPDL1.mIL7 is set forth in SEQ ID NO: 82, the amino acid sequence of the 616 DLL3 CAR.aPDL1.mIL7 is set forth in SEQ ID NO:

84. Most preferably, the enhanced targeting DLL3 antigen CAR is 616 DLL3 CAR.aPDL1.mIL7, wherein the amino acid sequence of the 616 DLL3 CAR is set forth in SEQ ID NO: 31, the amino acid sequence of the aPDL1 is set forth in SEQ ID NO: 80, the amino acid sequence of the mIL7 is set forth in SEQ ID NO: 81, the amino acid sequence of the aPDL1.mIL7 is set forth in SEQ ID NO: 82, the amino acid sequence of the 616 DLL3 CAR.aPDL1.mIL7 is set forth in SEQ ID NO:

84. A recombinant nucleic acid encoding the antibody or antigen-binding fragment thereof of any one of claims 1-4, or the mutant of any one of claims 5-6, or the CAR targeting DLL3 antigen of any one of claims 7-9, or the enhanced CAR targeting DLL3 antigen of any one of claims 10-11. A recombinant cell comprising the antibody or antigen-binding fragment thereof of any one of claims 1-4, or the mutant of any one of claims 5-6, or the CAR targeting DLL3 antigen of any one of claims 7-9, or the enhanced CAR targeting DLL3 antigen of any one of claims 10-11, or the recombinant nucleic acid of claim 12; preferably, the cell comprises a T cell, a NK cell, a macrophage, a B cell, a DC cell, or a non-immune cell; more preferably, the T cell is a primary T cell or an iPSC differentiated T cell; most preferably, the iPSC differentiated T cell is a gd T cell, a DNT cell, or a NKT cell. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-4, or the mutant of any one of claims 5-6, or the CAR targeting DLL3 antigen of any one of claims 7-9, or the enhanced CAR targeting DLL3 antigen of any one of claims 10-11, or the recombinant nucleic acid of claim 12, or the recombinant cell of claim 13; optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-4, or the mutant of any one of claims 5-6, or the CAR targeting DLL3 antigen of any one of claims 7-9, or the enhanced CAR targeting DLL3 antigen of any one of claims 10-11, or the recombinant nucleic acid of claim 12, or the recombinant cell of claim 13, or the pharmaceutical composition of claim 14 for preventing, treating, detecting, or diagnosing a disease associated with DLL3; preferably, the disease associated with DLL3 is a DLL3 high expression disease; more preferably, the disease is a DLL3 high expression cancer or tumor; most preferably, the cancer or tumor is selected from one or more of neuroendocrine tumors and other tumors, including small cell lung cancer, large cell neuroendocrine carcinoma, gastroenteropancreatic neuroendocrine tumor, pancreatic neuroendocrine tumor, small cell bladder cancer, glioblastoma multiforme, metastatic castration-resistant prostate cancer, melanoma, and medullary thyroid cancer. A method of making an engineered immune cell, characterized in that, Comprising the steps of: (1) providing an immune cell to be engineered; and (2) introducing the recombinant nucleic acid of claim 12 into the immune cell; Preferably, the immune cell is a T cell; more preferably, the T cell is a primary T cell or an iPSC differentiated T cell; most preferably, the iPSC differentiated T cell is a gd T cell, a DNT cell, or a NKT cell.

Images