Modified inkt cell

Abstract

A modified iNKT cell, which, compared with an unmodified iNKT cell, lacks or inhibits the expression of a negative immunoregulatory gene, wherein the gene is selected from one or more of the group consisting of PD-1, Tim-3, CTLA-4, CD74 and TGF-βR, and the lack or inhibition of the expression of the negative immunoregulatory gene is realized by means of the technique of intracellular retention / degradation of a membrane protein. Further provided is the use of the iNKT cell and a composition thereof.

Description

Modified iNKT cells Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to a modified iNKT cell and its use. Background Art

[0002] Invariant natural killer T cells (iNKT cells) are innate-like CD1d-restricted T cells that express the invariant T cell receptor (iTCR). They undergo a unique differentiation program in the thymus, acquiring NK-like properties, including potent cytotoxicity, rapid cytokine secretion, and efficient tissue trafficking. iNKT cells are among the first cell types to respond to pathogenic stimuli, and their cytokine production helps determine the course of the immune response. Due to their anti-tumor effects, iNKT cells have recently emerged as a potential target for immunotherapy.

[0003] Membrane protein intracellular retention technology is a non-gene editing technique. Its principle is that the retention sequence has a corresponding receptor on the Golgi membrane. Once the sequence enters the Golgi, it binds to the KDEL receptor on the Golgi, forming a reflux vesicle that is transported back to the endoplasmic reticulum. Therefore, KDEL retention sequence-carrying antibodies can effectively retain the antibody in the endoplasmic reticulum, thereby downregulating the antibody complex on the cell surface, achieving a functional loss similar to gene editing, thereby preventing GvHD.

[0004] Currently, there are few studies on the development of universal CAR-iNKT cells using non-gene-edited membrane protein intracellular retention technology. There is an urgent need to combine the two technologies to develop related products for disease treatment. Summary of the Invention

[0005] The present application provides a modified iNKT cell, which lacks or inhibits the expression of immune negative regulatory genes compared to unmodified iNKT cells, and the genes are selected from one or more of the following groups: PD-1, Tim-3, CTLA-4, CD74 and TGF-βR, wherein the lack or inhibition of expression of immune negative regulatory genes is achieved by membrane protein intracellular retention technology. Compared with unmodified iNKT cells, the modified iNKT cells provided by the present application have significantly inhibited the expression of immune negative regulatory genes; have significant inhibitory and killing functions after secondary tumor cell stimulation; have a significantly increased survival rate in vitro; and after combined expression of cytokines, the cell's tumor killing ability is further enhanced. The modified iNKT cells provided by the present application can not only stably inhibit the expression of immune negative regulatory genes, but also have a strong tumor killing effect. At the same time, their survival rate has been significantly improved, with unexpected technical effects.

[0006] On the one hand, the present application provides a modified iNKT cell, which lacks or inhibits the expression of immune negative regulatory genes compared to unmodified iNKT cells, wherein the genes are selected from one or more of the following groups: PD-1, Tim-3, CTLA-4, CD74 and TGF-βR, wherein the lack or inhibition of expression of immune negative regulatory genes is achieved by membrane protein intracellular retention technology.

[0007] In certain embodiments, the membrane protein intracellular retention technology is to directly or indirectly connect a positioning sequence at the N-terminus or C-terminus of an immune negative regulatory gene. In certain embodiments, the membrane protein intracellular retention technology is to directly or indirectly connect a positioning sequence at the C-terminus of an immune negative regulatory gene. In certain embodiments, the membrane protein intracellular retention technology is to directly or indirectly connect a positioning sequence at the N-terminus of an immune negative regulatory gene. In certain embodiments, the indirect connection uses a (GGGGS)4 connection.

[0008] In certain embodiments, the immune negative regulatory gene is PD-1. In certain embodiments, the amino acid sequence of PD-1 is shown in SEQ ID NO: 12 or SEQ ID NO: 19.

[0009] In certain embodiments, the localization sequence is selected from the group consisting of an endoplasmic reticulum retention sequence, a Golgi apparatus retention sequence, an E3 ubiquitin ligase binding sequence, a proteasome localization sequence, and a lysosome localization sequence.

[0010] In certain embodiments, the localization sequence is an endoplasmic reticulum retention sequence. In certain embodiments, the localization sequence is a lysosomal localization sequence.

[0011] In certain embodiments, the amino acid sequence of the endoplasmic reticulum retention sequence comprises one or more amino acid sequences selected from the following group: KDEL (SEQ ID NO: 9), KKMP (SEQ ID NO: 10) and AEKDEL (SEQ ID NO: 11).

[0012] In certain embodiments, the amino acid sequence of the lysosomal localization sequence is shown in SEQ ID NO:8.

[0013] In certain embodiments, the iNKT cells express CAR.

[0014] In certain embodiments, the CAR comprises a targeting moiety that specifically binds to an antigen.

[0015] In certain embodiments, the antigen is one or more antigens selected from the group consisting of GPC3, CLDN18.2, ROR1, and AXL.

[0016] In certain embodiments, the antigen is GPC3. In certain embodiments, the antigen is ROR1.

[0017] In certain embodiments, the targeting moiety is a scFv. In certain embodiments, the targeting moiety is a VHH.

[0018] In certain embodiments, the CAR comprises a signal peptide.

[0019] In certain embodiments, the signal peptide is a signal peptide derived from CD8 protein.

[0020] In certain embodiments, the signal peptide is a signal peptide derived from IL-2.

[0021] In certain embodiments, the CAR comprises a hinge region comprising a hinge region derived from one or more proteins selected from the group consisting of CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT.

[0022] In certain embodiments, the hinge region is a hinge region derived from CD28. In certain embodiments, the hinge region is a hinge region derived from CD8.

[0023] In certain embodiments, the CAR comprises a transmembrane region comprising a transmembrane domain derived from one or more proteins selected from the group consisting of CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM.

[0024] In certain embodiments, the transmembrane region is a transmembrane region derived from CD28.

[0025] In certain embodiments, the CAR comprises a costimulatory domain, which comprises a costimulatory domain derived from one or more proteins selected from the group consisting of CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, a ligand of CD83, CD40, and MyD88.

[0026] In certain embodiments, the costimulatory domain is a costimulatory domain derived from 4-1BB.

[0027] In certain embodiments, the CAR comprises an intracellular signaling domain, which comprises an intracellular signaling domain derived from one or more proteins selected from the group consisting of CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, Kaposi's sarcoma herpes virus (HSKV), DAP10, DAP-12, and a domain comprising at least one ITAM.

[0028] In certain embodiments, the intracellular signaling domain is an intracellular signaling domain derived from CD3ζ.

[0029] In certain embodiments, the iNKT cells are CAR-iNKT cells, and the CAR is composed of a CD8 signal peptide, a GPC3 antibody, a CD8 hinge region, a CD28 transmembrane region, a 4-1BB costimulatory domain, a CD3ζ intracellular signaling domain, a GSG linker sequence, a P2A self-cleaving peptide, an IgGκ signal peptide, a PD-1 antibody, a V5 tag, (GGGGS) 4, and a positioning sequence connected in sequence. In certain embodiments, the amino acid sequence of the PD-1 antibody is shown in SEQ ID NO: 19, and the amino acid sequence of the positioning sequence is shown in SEQ ID NO: 8.

[0030] In certain embodiments, the iNKT cells are CAR-iNKT cells, and the CAR is composed of a CD8 signal peptide, a ROR1 antibody, a CD28 hinge region, a CD28 transmembrane region, a 4-1BB costimulatory domain, a CD3ζ intracellular signaling domain, a GSG linker sequence, a P2A self-cleaving peptide, an IgGκ signal peptide, a PD-1 antibody, a V5 tag, (GGGGS) 4, and a positioning sequence connected in sequence. In certain embodiments, the amino acid sequence of the PD-1 antibody is shown in SEQ ID NO: 19, and the amino acid sequence of the positioning sequence is shown in SEQ ID NO: 8.

[0031] In certain embodiments, the CAR comprises a cytokine domain.

[0032] In certain embodiments, the CAR comprises a cytokine domain.

[0033] In certain embodiments, the cytokine domain:

[0034] (1) Contains one or more cytokines and / or their functional fragments;

[0035] (2) comprising one or more cytokine receptors and / or functional fragments thereof; and

[0036] (3) Includes a combination of (1) and (2).

[0037] In certain embodiments, the cytokine domain is expressed on the membrane of an iNKT cell.

[0038] In certain embodiments, the cytokine domain is secreted and expressed from iNKT cells.

[0039] In certain embodiments, the cytokine is one or more factors selected from the group consisting of IL-15, IL-10, IL-18, and IL-21.

[0040] In certain embodiments, the cytokine is IL-15.

[0041] In certain embodiments, the cytokine receptor is IL-15R or its alpha subunit (IL-15Rα).

[0042] On the other hand, the present application provides a nucleic acid molecule encoding a nucleic acid sequence encoding an immune negative regulatory gene and a nucleic acid sequence encoding a localization sequence, wherein the immune negative regulatory gene is selected from one or more genes in the following group: PD-1, Tim-3, CTLA-4, CD74 and TGF-βR.

[0043] In certain embodiments, the immune negative regulatory gene is PD-1. In certain embodiments, the amino acid sequence of PD-1 is shown in SEQ ID NO: 12 or SEQ ID NO: 19.

[0044] In certain embodiments, the localization sequence is selected from the group consisting of an endoplasmic reticulum retention sequence, a Golgi apparatus retention sequence, an E3 ubiquitin ligase binding sequence, a proteasome localization sequence, and a lysosome localization sequence.

[0045] In certain embodiments, the localization sequence is an endoplasmic reticulum retention sequence.

[0046] In certain embodiments, the localization sequence is a lysosomal localization sequence.

[0047] In certain embodiments, the amino acid sequence of the endoplasmic reticulum retention sequence comprises one or more amino acid sequences selected from the group consisting of KDEL (SEQ ID NO: 9), KKMP (SEQ ID NO: 10), and AEKDEL (SEQ ID NO: 11).

[0048] In certain embodiments, the amino acid sequence of the lysosomal localization sequence is shown in SEQ ID NO:8.

[0049] In certain embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding a targeting antigen.

[0050] In certain embodiments, the antigen is one or more antigens selected from the group consisting of GPC3, CLDN18.2, ROR1, and AXL.

[0051] In certain embodiments, the antigen is GPC3. In certain embodiments, the antigen is ROR1.

[0052] In certain embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding a promoter.

[0053] In certain embodiments, the promoter is selected from the group consisting of EF1α, CMV, MSCV, SSFV, and UbC.

[0054] In certain embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding a signal peptide.

[0055] In certain embodiments, the signal peptide is a signal peptide derived from CD8 protein.

[0056] In certain embodiments, the signal peptide is a signal peptide derived from IL-2.

[0057] In certain embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding a hinge region, wherein the hinge region comprises a hinge region derived from one or more proteins selected from the group consisting of CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT.

[0058] In certain embodiments, the hinge region is a hinge region derived from CD28. In certain embodiments, the hinge region is a hinge region derived from CD8.

[0059] In certain embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding a transmembrane region, which comprises a transmembrane domain derived from one or more proteins selected from the group consisting of CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM.

[0060] In certain embodiments, the transmembrane region is a transmembrane region derived from CD28.

[0061] In certain embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding a costimulatory domain, which comprises a costimulatory domain derived from one or more proteins selected from the group consisting of CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, a ligand of CD83, CD40, and MyD88.

[0062] In certain embodiments, the costimulatory domain is an intracellular costimulatory domain derived from 4-1BB.

[0063] In certain embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an intracellular signaling domain, which comprises an intracellular signaling domain derived from one or more proteins selected from the group consisting of CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, Kaposi's sarcoma herpes virus (HSKV), DAP10, DAP-12, and a domain comprising at least one ITAM.

[0064] In certain embodiments, the intracellular signaling domain is an intracellular signaling domain derived from CD3ζ.

[0065] In certain embodiments, the localization sequence is located upstream of the sequence of the immune negative regulatory gene.

[0066] In certain embodiments, the localization sequence is located downstream of the sequence of the immune negative regulatory gene.

[0067] In certain embodiments, the nucleic acid molecule comprises a sequence encoding a cleavage peptide

[0068] In certain embodiments, the cleavage peptide is selected from one or more cleavage peptides of the following group: P2A, T2A, F2A, E2A, BmCPV2A, IRES and BmIFV2A.

[0069] In certain embodiments, the cleavage peptide is P2A.

[0070] In certain embodiments, the cleavage peptide P2A is linked to the linker sequence GSG.

[0071] In certain embodiments, the nucleic acid molecule can encode, in sequence, a CD8 signal peptide, a GPC3 antibody, a CD8 hinge region, a CD8 transmembrane region, a 4-1BB costimulatory domain, a CD3ζ intracellular signaling domain, a GSG linker sequence, a P2A self-cleavage peptide, an IgGκ signal peptide, a PD-1 antibody, a V5 tag, (GGGGS) 4, and a localization sequence. In certain embodiments, the amino acid sequence of the PD-1 antibody is shown in SEQ ID NO: 19, and the amino acid sequence of the localization sequence is shown in SEQ ID NO: 8.

[0072] In certain embodiments, the nucleic acid molecule can encode, in sequence, a CD8 signal peptide, a ROR1 antibody, a CD28 hinge region, a CD8 transmembrane region, a 4-1BB costimulatory domain, a CD3ζ intracellular signaling domain, a GSG linker sequence, a P2A self-cleavage peptide, an IgGκ signal peptide, a PD-1 antibody, a V5 tag, (GGGGS) 4, and a localization sequence. In certain embodiments, the amino acid sequence of the PD-1 antibody is shown in SEQ ID NO: 19, and the amino acid sequence of the localization sequence is shown in SEQ ID NO: 8.

[0073] In certain embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding a cytokine domain.

[0074] In certain embodiments, the nucleic acid sequence encoding the cytokine domain may be:

[0075] (1) Sequences capable of expressing one or more cytokines and / or their functional fragments;

[0076] (2) sequences capable of expressing one or more cytokine receptors and / or functional fragments thereof; and

[0077] (3) A sequence capable of expressing a combination of (1) and (2).

[0078] In certain embodiments, the cytokine is one or more factors selected from the group consisting of IL-15, IL-10, IL-18, and IL-21.

[0079] In certain embodiments, the cytokine is IL-15.

[0080] In certain embodiments, the cytokine receptor is IL-15R or its alpha subunit (IL-15Rα).

[0081] In another aspect, the present application provides a plasmid comprising the nucleic acid molecule described in the present application.

[0082] In certain embodiments, the plasmid includes viral plasmids and bacterial plasmids.

[0083] In certain embodiments, the plasmids are DNA plasmids and RNA plasmids.

[0084] In certain embodiments, the plasmid includes linear plasmids and circular plasmids.

[0085] On the other hand, the present application provides a pharmaceutical composition comprising the iNKT cells described herein, the nucleic acid molecules described herein, the plasmid described herein, and at least one pharmaceutically acceptable carrier.

[0086] On the other hand, the iNKT cells, the nucleic acid molecules, the plasmids, and the pharmaceutical compositions described herein are used in the preparation of drugs for preventing, alleviating, and / or treating diseases and / or conditions.

[0087] Those skilled in the art can easily discern other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of this application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application relates. Accordingly, the descriptions in the drawings and specification of this application are merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0088] The specific features of the inventions of this application are set forth in the appended claims. The features and advantages of the inventions of this application can be better understood by referring to the exemplary embodiments described in detail below and the accompanying drawings. A brief description of the drawings is as follows:

[0089] FIG1 shows the detection of PD1 expression levels after 3 days of co-culture of PD1 knockout and non-knockout ROR1CAR-iNKT cells with tumor cells described in the present application.

[0090] FIG2 shows the results of the ROR1CAR-iNKT cell knockout of TGFβRII as described in the present application.

[0091] Figures 3A, 3B, and 3C show the comparison of tumor cell killing after PD1 and TGFβRII knockout as described in the present application.

[0092] FIG4A shows the killing function test of the PD1 knockout and non-knockout ROR1CAR-iNKT cells described in the present application after secondary tumor cell stimulation.

[0093] FIG4B shows the survival rate detection of PD1 knockout and non-knockout ROR1CAR-iNKT cells during in vitro co-culture as described in the present application.

[0094] Figure 5A shows a schematic diagram of the construction of GPC3-CAR and GPC3-CAR PD1KO described in this application.

[0095] FIG5B shows the expression efficiency of the PD1 knockout and non-knockout constructs GPC3-CAR PD1KO on iNKT cells described in this application.

[0096] Figure 6 shows the tumor suppression effect detection of GPC3-CAR PD1 KO described in this application.

[0097] FIG7A shows a schematic diagram of the CAR framework of PD1 knockout and non-knockout GPC3CAR-iNKT cells expressing IL-15 described in the present application.

[0098] Figure 7B shows the expression detection results of the IL-15 enhanced CAR described in this application in PBMC.

[0099] FIG7C shows the results of detecting the secretion level of IL-15 in the supernatant 72 hours after transduction as described in the present application.

[0100] FIG7D shows the expression level detection of PD1 knockout and non-knockout GPC3CAR-iNKT cells described in the present application.

[0101] FIG7E shows the detection of IL-15 secretion levels in the supernatant 72 hours after transduction as described in the present application.

[0102] FIG8A shows the killing function test of secreted or membrane receptor-bound IL-15 on Hep3B and Huh7 cells expressing GPC3 as described in the present application.

[0103] Figure 8B shows that the secreted or membrane receptor-bound IL-15 described in this application enhances the secretion of IFN-γ

[0104] Figure 9 shows a schematic diagram of the three-in-one CAR architecture used in tumor killing described in this application to reduce PD1 expression in cells. DETAILED DESCRIPTION

[0105] The following describes the implementation of the present invention through specific embodiments. People familiar with this technology can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0106] Definition of terms

[0107] In this application, the term "iNKT cells" generally refers to a special T cell subset involved in innate immunity, which has a constant TCRα receptor. iNKT cells can recognize antigens in the presence of CD1d molecules, can specifically recognize α-GalCer, and iNKT cells can be labeled with CD1d / α-GalCer tetramers. iNKT cells can differentiate into NKT1, NKT2 and NKT17 subpopulations, which are similar to Th1, Th2 and Th17 subpopulations. iNKT cells contain a large amount of cytotoxic cytokines (such as IL-4, IL-10, IFN-γ, etc.), which are much higher than NK cells and T cells. When iNKT cells are stimulated, they can quickly release cytokines to kill tumor cells.

[0108] In this application, the term "membrane protein intracellular retention technology" generally refers to the technology of inhibiting gene expression by carrying specific proteins through positioning sequences.

[0109] In this application, the term "lack" generally refers to a level (eg, protein expression) that is substantially not expressed compared to the original level, or is unable to exert the original function.

[0110] In this application, the term "inhibit expression" generally refers to a decrease in the level (e.g., protein expression) compared to the original level. For example, the level of reduction can be judged by a P value, P < 0.05, P < 0.04, P < 0.03, P < 0.02, P < 0.01, P < 0.005, or P < 0.001. For example, the inhibited expression can be reduced by 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100% or more compared to the original level.

[0111] In the present application, the term "chimeric antigen receptor" is often referred to as "CAR", and generally refers to a fusion protein comprising an extracellular domain capable of binding to an antigen and at least one intracellular domain. In the present application, the CAR may include an intracellular domain, and the intracellular domain includes a signaling domain and / or a costimulatory domain. In the present application, a group of polypeptides of CAR may be located in the same polypeptide chain (e.g., comprising a chimeric fusion protein), or may be discontinuous with each other, for example, they may be located in different polypeptide chains. In the present application, the signal involved in induction can be transduced into the cytoplasm of T cells via CD3 and ζ chains. In the present application, the intracellular domain may include a primary signaling binding domain (e.g., the main signaling domain of CD3-zeta (ζ)). In one aspect, the cytoplasmic signaling domain may also include one or more costimulatory domains derived from at least one costimulatory molecule. For example, the costimulatory domain may be 4-1BB (i.e., CD137), CD27, ICOS and / or CD28. In the present application, CAR may include a chimeric fusion protein, for example, containing an optional leader sequence on the amino terminus (N-ter). Wherein, the leader sequence optionally cuts the antigen binding domain (for example, ScFv) during cell processing and positions CAR on the cell membrane.

[0112] In this application, the term "immune negative regulatory gene" generally refers to a gene that suppresses cellular immune responses through negative regulation. For example, immune checkpoint-related protein genes. In this application, immune negative regulatory genes include but are not limited to PD-1, Tim-3, CTLA-4, CD74, and TGF-βR.

[0113] As used herein, the term "localization sequence" generally refers to a localization sequence that directs a polypeptide to a subcellular structure. For example, an endoplasmic reticulum retention sequence, a Golgi apparatus retention sequence, an E3 ubiquitin ligase binding sequence, a proteasome localization sequence, or a lysosomal localization sequence. As used herein, a localization sequence can be directly linked to an adjacent polypeptide or can be linked via a linker sequence. As used herein, a localization sequence can be located at the N-terminus or the C-terminus of a polypeptide.

[0114] In this application, the term "endoplasmic reticulum retention sequence" generally refers to a short peptide signal sequence that has a corresponding receptor on the membrane of the Golgi apparatus and can be transported back to the endoplasmic reticulum through reflux vesicles. The endoplasmic reticulum retention sequence suitable for the present invention can be an endoplasmic reticulum retention sequence well known in the art. In this application, the endoplasmic reticulum retention sequence can be located at the C-terminus or the N-terminus of an adjacent polypeptide. In this application, the endoplasmic reticulum retention sequence includes but is not limited to KDEL (SEQ ID NO: 9), KKMP (SEQ ID NO: 10) and AEKDEL (SEQ ID NO: 11).

[0115] In this application, the term "Golgi retention sequence" generally refers to a short peptide signal sequence that can target the corresponding receptor on the membrane of the Golgi apparatus. In this application, Golgi retention sequences include but are not limited to KDEL (SEQ ID NO: 9), KKMP (SEQ ID NO: 10) and AEKDEL (SEQ ID NO: 11).

[0116] In this application, the term "E3 ubiquitin ligase binding sequence" generally refers to a short peptide signal sequence that can target human immunoglobulin G (IgG) constant region (Fc) genes (e.g., IgG1, IgG2 or IgG4).

[0117] As used herein, the term "proteasome localization sequence" generally refers to a short peptide signal sequence, which is achieved by linking an antibody sequence to a sequence containing an E3 ubiquitin ligase targeting domain and co-expressing a nucleic acid sequence for an E3 ubiquitin ligase protein. The E3 ubiquitin ligase protein binds to the Fc domain of an antibody with high affinity and can recruit the ubiquitin-proteosome complex to degrade molecules (e.g., proteins and peptides) bound to the antibody. As used herein, proteasome localization sequences include, but are not limited to, the PEST motif (SEQ ID NO: 22).

[0118] In this application, the term "lysosomal localization sequence" generally refers to a short peptide signal sequence. In this application, the lysosomal localization sequence includes but is not limited to the KFERQ motif (SEQ ID NO: 20). In this application, the lysosomal localization sequence includes but is not limited to the SFHDDSDEDLLHI motif (SEQ ID NO: 8).

[0119] In this application, the term "cytokine domain" generally refers to a domain that can be used to express a cytokine and / or its functional fragment, or can be used to express one or more cytokine receptors and / or their functional fragments, or can simultaneously express a cytokine and / or its functional fragment and one or more cytokine receptors and / or their functional fragments.

[0120] In this application, the term "targeting moiety" generally refers to a molecule or fragment that can specifically recognize and bind to a cell surface marker. Targeting moieties include, but are not limited to, antibodies or fragments thereof, peptides, hormones, growth factors, cytokines, and any other natural or non-natural ligands.

[0121] In this application, the term "signal peptide" generally refers to a propeptide present as an N-terminal peptide on a protein precursor. The function of the signal peptide may be to promote the translocation of the expressed polypeptide connected to the endoplasmic reticulum. The signal peptide is usually removed during this process. The signal peptide removed from the protein precursor may be heterologous or homologous to the organism used to produce the polypeptide.

[0122] In this application, the term "hinge region" generally refers to the connecting region between the antigen binding region and the immune cell Fc receptor (FcR) binding region. For example, the hinge region can be the region between the heavy chain CH1 and CH2 functional regions in an immunoglobulin.

[0123] In this application, the term "transmembrane region" generally refers to the region connecting the extracellular antigen binding domain and the intracellular signaling domain, which is generally composed of dimeric membrane proteins, mainly including CD3ζ, CD4, CD8, CD28, etc., and can anchor the CAR structure to the T cell membrane. Different designs of the transmembrane region can affect the expression of the introduced CAR gene.

[0124] In this application, the term "costimulatory domain" generally refers to a domain in CAR that passes through the cell membrane and is connected to an intracellular signal transduction domain to transmit signals.

[0125] In this application, the term "intracellular signaling domain" generally refers to a domain located inside a cell that can transduce a signal. In this application, the intracellular signaling domain can conduct a signal into the cell. For example, the intracellular signaling domain is the intracellular signaling domain of the chimeric antigen receptor.

[0126] In this application, the term "promoter" generally refers to a DNA sequence that can regulate the expression of a selected DNA sequence to which the promoter is operably linked, thereby affecting the expression of the selected DNA sequence in a cell.

[0127] In this application, the term "cleavage peptide" generally refers to a class of polypeptides that can achieve the function of cleaving proteins. For example, the cleavage peptide can achieve protein cleavage via ribosome skipping rather than proteolysis. For example, the cleavage peptide can be a cleavage 2A peptide, which can include T2A, F2A, P2A and / or E2A. In this application, the cleavage peptide can also be connected to a linker sequence, and the linker sequence can be GSG.

[0128] In the present application, the term "single-chain antibody" (ScFv) generally refers to an antibody composed of the heavy chain variable region and the light chain variable region connected by a linker.

[0129] In this application, the term "VHH" generally refers to an antibody comprising a variable antigen-binding domain of a heavy chain antibody. VHH may also be referred to as a nanobody (Nb) and / or a single domain antibody.

[0130] In this application, the term "plasmid" generally refers to any molecule that transfers coding information to a cell. For example, a plasmid can be a linear or circular autonomously replicating sequence, a genome-integrating sequence, a phage, or a nucleotide sequence derived from single-stranded or double-stranded DNA or RNA from any source.

[0131] As used herein, the term "pharmaceutical composition" generally refers to a preparation that is in a form that permits the biological activity of the active ingredient to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the preparation is to be administered.

[0132] In the present application, the term "pharmaceutically acceptable carrier" may include buffers, antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counterions, metal complexes and / or nonionic surfactants, etc.

[0133] In this application, the term "about" generally refers to a variation within a range of 0.5%-10% above or below the specified value, for example, a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value.

[0134] As used herein, the term "diagnosis" generally refers to the detection of a disease or condition, or the determination of the state or extent of a disease or condition, and also includes the detection of the cause of a disease or condition, the determination of the therapeutic effect of a drug therapy, or the prediction of a response pattern to a drug therapy or a xenobiotic. The diagnostic methods described herein can be used independently or in combination with other diagnostic and / or staging methods known in the medical field for a particular disease or condition.

[0135] As used herein, the term "prevent" generally refers to methods used to prevent, inhibit, or reduce the likelihood of the occurrence or recurrence of a disease or condition (e.g., cancer). It also refers to delaying the onset or recurrence of a disease or condition or delaying the onset or recurrence of symptoms of a disease or condition. "Prevent" and similar terms also include reducing the intensity, effects, symptoms, and / or burden of a disease or condition prior to its onset or recurrence.

[0136] As used herein, the term "treat" generally refers to any beneficial or desirable effect on the symptoms or pathology of a disease or pathological condition, and may even include a minimal reduction in one or more measurable markers of the disease or condition being treated (e.g., cancer). Treatment may optionally involve a reduction or improvement in the symptoms of a disease or condition, or a delay in the progression of a disease or condition. "Treatment" does not necessarily indicate complete eradication or cure of a disease or condition or its associated symptoms.

[0137] In this application, the term "proliferation" generally refers to the generation of multiple individual cells by division of a starting cell. The multiple individual cells can be of the same type or of different types. The starting cells used for proliferation need not be the same as the cells produced by proliferation. For example, the proliferated cells can be generated from the growth and differentiation of a starting cell population.

[0138] In this application, the term "differentiation" generally refers to the process by which a non-specific or less specific cell acquires specific cell characteristics. A differentiated or differentiation-induced cell is a cell that occupies a more specific position in a cell lineage.

[0139] In this application, the term "ex vivo" generally refers to operations performed on cells, tissues and / or organs that have been removed from an organism. In some embodiments, the cells, tissues and / or organs can be returned to the organism or introduced into another organism by certain methods.

[0140] In this application, the term "in vitro" generally refers to removing or releasing a part of an organism from the organism.

[0141] In this application, the term "include" generally means to include, encompass, contain or encompass. In some cases, it also means "to be", "to be composed of..."

[0142] In this application, the term "and / or" should be understood to mean any one, two, more than one or any combination of the alternatives.

[0143] In this application, the term "about" generally refers to a variation within a range of 0.5%-10% above or below the specified value, for example, a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value.

[0144] Detailed Description of the Invention

[0145] Modified iNKT cells

[0146] On the one hand, the present application provides a modified iNKT cell, which lacks or inhibits the expression of immune negative regulatory genes compared to unmodified iNKT cells, wherein the genes are selected from one or more of the following groups: PD-1, Tim-3, CTLA-4, CD74 and TGF-βR, wherein the lack or inhibition of expression of immune negative regulatory genes is achieved by membrane protein intracellular retention technology.

[0147] In the present application, the immune negative regulatory gene can be a gene related to a T cell immune checkpoint, a gene that inhibits T cell proliferation, a gene that inhibits tumor expression, or a gene that exerts a tumor suppressor effect by inducing cell cycle arrest and promoting cell apoptosis. For example, the immune negative regulatory gene can be PD-1. For example, the amino acid sequence of the immune negative regulatory gene can be as shown in SEQ ID NO: 12 or SEQ ID NO: 19.

[0148] In the present application, the modified iNKT cells may express chimeric antigen receptors (CARs).

[0149] In the present application, the CAR package may comprise a localization sequence, which can direct the polypeptide to a subcellular structure.

[0150] For example, the subcellular structure includes, but is not limited to, the Golgi apparatus or endoplasmic reticulum, the proteasome, the cell membrane, or the lysosome.

[0151] For example, the localization sequence can be selected from an endoplasmic reticulum retention sequence, a Golgi apparatus retention sequence, an E3 ubiquitin ligase binding sequence, a proteasome localization sequence, and a lysosome localization sequence.

[0152] For example, the localization sequence may be located at the N-terminus or the C-terminus of the polypeptide.

[0153] For example, the positioning sequence can be directly linked to its adjacent polypeptide, or can be linked via a linker sequence well known in the art, such as a linker sequence comprising G and S.

[0154] In the present application, the endoplasmic reticulum retention sequence is a short peptide signal sequence. The endoplasmic reticulum retention sequence has a corresponding receptor on the membrane of the Golgi apparatus and can be transported back to the endoplasmic reticulum through reflux vesicles.

[0155] For example, the endoplasmic reticulum retention sequence can be located in the C segment of the adjacent polypeptide, can be located in the N segment of the adjacent polypeptide, or can be located in the C segment and N terminus of the adjacent polypeptide.

[0156] For example, the endoplasmic reticulum retention sequence can be an endoplasmic reticulum retention sequence known in the art. For example, the "endoplasmic reticulum retention sequence" or "Golgi apparatus retention sequence" includes, but is not limited to, KDEL (SEQ ID NO: 9), KKMP (SEQ ID NO: 10), and AEKDEL (SEQ ID NO: 11). For example, the amino acid sequence of the endoplasmic reticulum retention sequence is shown in SEQ ID NO: 8. For example, the amino acid sequence of the lysosomal localization sequence is shown in SEQ ID NO: 8.

[0157] In the present application, the proteasome localization sequence can be achieved by linking the antibody sequence to a sequence containing an E3 ubiquitin ligase targeting domain and co-expressing a nucleic acid sequence for an E3 ubiquitin ligase protein. The E3 ubiquitin ligase protein binds to the Fc domain of the antibody with high affinity and can recruit the ubiquitin-proteosome complex to degrade molecules bound to the antibody. For example, the proteasome localization sequence includes, but is not limited to, the PEST (SEQ ID NO: 22) motif.

[0158] In the present application, the E3 ubiquitin ligase protein targeting domain sequence encodes an amino acid sequence selected from a human immunoglobulin G (IgG) constant region (Fc) gene (e.g., IgG1, IgG2, or IgG4), and is used to form a fusion protein comprising an antibody and an Fc domain. For example, the lysosomal localization sequence includes, but is not limited to, a KFERQ (SEQ ID NO: 20) motif.

[0159] In the present application, the CAR comprises a targeting moiety that specifically binds to an antigen. In the present application, the targeting moiety may comprise an antibody or an antigen binding fragment. The antigen binding fragment may be selected from the group consisting of Fab, Fab', F(ab)2, F(ab')2, Fv, VHH, and ScFv fragments. For example, the targeting moiety may be ScFv. For example, the targeting moiety may be VHH.

[0160] For example, the antigen is selected from one or more of the group consisting of GPC3, CLDN18.2, ROR1, and AXL. For example, the antigen may be GPC3. For example, the antigen may be CLDN18.2. For example, the antigen may be ROR1. For example, the antigen may be AXL. For example, the antigen may comprise the amino acid sequence set forth in SEQ ID NO:1, or an amino acid sequence having at least 80% homology thereto. For example, the antigen may comprise the amino acid sequence set forth in SEQ ID NO:15, or an amino acid sequence having at least 80% homology thereto.

[0161] In the present application, the CAR comprises a signal peptide. For example, the signal peptide may be derived from a CD8 protein. For example, the signal peptide may be derived from a CD8 protein. For example, the signal peptide may be derived from an IL-2 signal peptide. For example, the signal peptide may comprise the amino acid sequence shown in SEQ ID NO: 2.

[0162] In the present application, the CAR comprises a hinge region, which can be any oligopeptide or polypeptide that connects the transmembrane domain to the extracellular ligand binding domain to provide more flexibility and accessibility to the extracellular ligand binding domain, and can be a synthetic sequence corresponding to a naturally occurring hinge sequence, or the hinge region can be a completely synthetic hinge sequence.

[0163] For example, the hinge region comprises a hinge region derived from one or more proteins selected from the group consisting of CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT. For example, the hinge region may be a hinge region derived from CD28. For example, the hinge region may comprise the amino acid sequence shown in SEQ ID NO: 3.

[0164] In the present application, the CAR comprises a transmembrane region that can cross the cell membrane, anchor the CAR to the cell surface, and connect the extracellular domain to the intracellular signaling domain, thereby affecting the expression of CAR on the cell surface.

[0165] For example, the transmembrane domain comprises a transmembrane domain derived from one or more proteins selected from the group consisting of CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM. For example, the transmembrane region can be a transmembrane region derived from CD28. For example, the transmembrane domain can comprise the amino acid sequence shown in SEQ ID NO:4.

[0166] In the present application, the CAR comprises a costimulatory domain, which is derived from the intracellular signaling domain of a costimulatory protein, which can enhance cytokine production, proliferation, cytotoxicity and / or persistence in vivo.

[0167] For example, the costimulatory domain comprises a costimulatory domain derived from one or more proteins selected from the group consisting of CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, a ligand of CD83, CD40, and MyD88. For example, the costimulatory domain is a costimulatory domain derived from 4-1BB. For example, the costimulatory domain may comprise the amino acid sequence shown in SEQ ID NO:5.

[0168] In the present application, the CAR comprises an intracellular signaling domain that can transduce signals into cells.

[0169] For example, the intracellular signaling domain comprises an intracellular signaling domain derived from one or more proteins selected from the group consisting of CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, Kaposi's sarcoma herpesvirus (HSKV), DAP10, DAP-12, and a domain comprising at least one ITAM. For example, the intracellular signaling domain is an intracellular signaling domain derived from CD3ζ. For example, the intracellular signaling domain may comprise the amino acid sequence shown in SEQ ID NO: 6.

[0170] In the present application, the CAR comprises a cytokine domain, and the cytokine domain can:

[0171] (1) Contains one or more cytokines and / or their functional fragments;

[0172] (2) comprising one or more cytokine receptors and / or functional fragments thereof; and

[0173] (3) Includes a combination of (1) and (2).

[0174] In the present application, the cytokine domain is expressed on the iNKT cell membrane. In the present application, the cytokine domain is secreted and expressed in iNKT cells.

[0175] In the present application, the cytokines include interleukins, interferons and / or tumor necrosis factors.

[0176] For example, the cytokine is selected from one or more factors in the group consisting of IL-15, IL-10, IL-18, and IL-21. For example, the cytokine comprises IL-2, IL-4, IL-6, IL-7, IL-12, TNF-α, and / or IFNγ. For example, the cytokine is IL-15. For example, the cytokine receptor is IL-15R or its α subunit (IL-15Rα). For example, the cytokine domain may comprise the amino acid sequence shown in SEQ ID NO: 17 or SEQ ID NO: 18.

[0177] In the present application, the iNKT cells can be CAR-iNKT cells, and the CAR is composed of a CD8 signal peptide, a GPC3 antibody, a CD8 hinge region, a CD8 transmembrane region, a 4-1BB co-stimulatory domain, a CD3ζ intracellular signaling domain, a GSG linker sequence, a P2A self-cleaving peptide, an IgGκ signal peptide, a PD-1 antibody, a V5 tag, (GGGS)4 and a positioning sequence connected in sequence.

[0178] For example, the amino acid sequence of the CD8 signal peptide is shown in SEQ ID NO: 2, the amino acid sequence of the GPC3 antibody is shown in SEQ ID NO: 15, the amino acid sequence of the CD8 hinge region is shown in SEQ ID NO: 16, the amino acid sequence of the CD28 transmembrane region is shown in SEQ ID NO: 4, the amino acid sequence of the 4-1BB costimulatory domain is shown in SEQ ID NO: 5, the amino acid sequence of the CD3ζ intracellular signaling domain is shown in SEQ ID NO: 6, the amino acid sequence of the GSG linker sequence and the P2A self-cleaving peptide is shown in SEQ ID NO: 7, the amino acid sequence of the IgGκ signal peptide is shown in SEQ ID NO: 14, the amino acid sequence of the PD-1 antibody is shown in SEQ ID NO: 19, the amino acid sequence of the V5 tag is shown in SEQ ID NO: 13, the amino acid sequence of (GGGGS)4 is shown in SEQ ID NO: 21, and the amino acid sequence of the positioning sequence is shown in SEQ ID NO: 8.

[0179] For example, the amino acid sequence of the CD8 signal peptide is shown in SEQ ID NO: 2, the amino acid sequence of the ROR1 antibody is shown in SEQ ID NO: 1, the amino acid sequence of the CD28 hinge region is shown in SEQ ID NO: 3, the amino acid sequence of the CD28 transmembrane region is shown in SEQ ID NO: 4, the amino acid sequence of the 4-1BB costimulatory domain is shown in SEQ ID NO: 5, the amino acid sequence of the CD3ζ intracellular signaling domain is shown in SEQ ID NO: 6, the amino acid sequence of the GSG linker sequence and the P2A self-cleaving peptide is shown in SEQ ID NO: 7, the amino acid sequence of the IgGκ signal peptide is shown in SEQ ID NO: 14, the amino acid sequence of the PD-1 antibody is shown in SEQ ID NO: 19, the amino acid sequence of the V5 tag is shown in SEQ ID NO: 13, the amino acid sequence of (GGGS)4 is shown in SEQ ID NO: 21, and the amino acid sequence of the positioning sequence is shown in SEQ ID NO: 8.

[0180] For example, the amino acid sequence of the CD8 signal peptide is shown in SEQ ID NO: 2, the amino acid sequence of the GPC3 antibody is shown in SEQ ID NO: 15, the amino acid sequence of the CD8 hinge region is shown in SEQ ID NO: 16, the amino acid sequence of the CD28 transmembrane region is shown in SEQ ID NO: 4, the amino acid sequence of the 4-1BB costimulatory domain is shown in SEQ ID NO: 5, the amino acid sequence of the CD3ζ intracellular signaling domain is shown in SEQ ID NO: 6, the amino acid sequence of the GSG linker sequence and the P2A self-cleaving peptide is shown in SEQ ID NO: 7, the amino acid sequence of the IgGκ signal peptide is shown in SEQ ID NO: 14, the amino acid sequence of the PD-1 antibody is shown in SEQ ID NO: 12, the amino acid sequence of the V5 tag is shown in SEQ ID NO: 13, the amino acid sequence of (GGGGS)4 is shown in SEQ ID NO: 21, and the amino acid sequence of the positioning sequence is shown in SEQ ID NO: 11.

[0181] For example, the amino acid sequence of the CD8 signal peptide is shown in SEQ ID NO: 2, the amino acid sequence of the ROR1 antibody is shown in SEQ ID NO: 1, the amino acid sequence of the CD28 hinge region is shown in SEQ ID NO: 3, the amino acid sequence of the CD28 transmembrane region is shown in SEQ ID NO: 4, the amino acid sequence of the 4-1BB costimulatory domain is shown in SEQ ID NO: 5, the amino acid sequence of the CD3ζ intracellular signaling domain is shown in SEQ ID NO: 6, the amino acid sequence of the GSG linker sequence and the P2A self-cleaving peptide is shown in SEQ ID NO: 7, the amino acid sequence of the IgGκ signal peptide is shown in SEQ ID NO: 14, the amino acid sequence of the PD-1 antibody is shown in SEQ ID NO: 12, the amino acid sequence of the V5 tag is shown in SEQ ID NO: 13, the amino acid sequence of (GGGS)4 is shown in SEQ ID NO: 21, and the amino acid sequence of the positioning sequence is shown in SEQ ID NO: 11.

[0182] Nucleic acid molecules

[0183] On the other hand, the present application provides a nucleic acid molecule encoding the CAR described in the present application.

[0184] In the present application, the nucleic acid molecule comprises a nucleic acid sequence encoding a localization sequence, and the localization sequence is selected from the group consisting of an endoplasmic reticulum retention sequence, a Golgi apparatus retention sequence, an E3 ubiquitin ligase binding sequence, a proteasome localization sequence, and a lysosome localization sequence.

[0185] For example, the localization sequence is an endoplasmic reticulum retention sequence. For example, the amino acid sequence of the endoplasmic reticulum retention sequence comprises one or more amino acid sequences selected from the group consisting of KDEL (SEQ ID NO: 9), KKMP (SEQ ID NO: 10), and AEKDEL (SEQ ID NO: 11). For example, the localization sequence is a lysosomal localization sequence. For example, the amino acid sequence of the lysosomal localization sequence is as shown in SEQ ID NO: 8.

[0186] In the present application, the nucleic acid molecule comprises a nucleic acid sequence encoding an immune negative regulatory gene, and the gene is selected from one or more genes in the following group: PD-1, Tim-3, CTLA-4, CD74 and TGF-βR.

[0187] In the present application, the nucleic acid molecule comprises a nucleic acid sequence encoding a targeting antigen.

[0188] For example, the antigen is one or more antigens selected from the group consisting of GPC3, CLDN18.2, ROR1, and AXL. For example, the antigen is GPC3. For example, the antigen is CLDN18.2. For example, the antigen is ROR1. For example, the antigen is AXL.

[0189] In the present application, the nucleic acid molecule comprises a nucleic acid sequence encoding a promoter, which is generally operably linked to the coding sequence of the protein to be expressed. The promoter can be any nucleic acid sequence that shows transcriptional activity in the selected host cell, including mutant, truncated, and hybrid promoters, and can be obtained from a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to the host cell.

[0190] For example, the promoter is selected from the group consisting of EF1α, CMV, MSCV, SSFV and UbC.

[0191] In the present application, the nucleic acid molecule comprises a nucleic acid sequence encoding a signal peptide.

[0192] For example, the signal peptide is a signal peptide derived from CD8 protein.For example, the signal peptide is a signal peptide derived from IL-2 protein.

[0193] In the present application, the nucleic acid molecule comprises a nucleic acid sequence encoding a hinge region, and the hinge region comprises a hinge region derived from one or more proteins selected from the group consisting of CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT.

[0194] For example, the hinge region is a hinge region derived from CD28. For example, the hinge region is a hinge region derived from CD8.

[0195] In the present application, the nucleic acid molecule comprises a nucleic acid sequence encoding a transmembrane region, which comprises a transmembrane domain derived from one or more proteins selected from the group consisting of CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM.

[0196] For example, the transmembrane region is the transmembrane region derived from CD28.

[0197] In the present application, the nucleic acid molecule comprises a nucleic acid sequence encoding a costimulatory domain, and the costimulatory domain comprises a costimulatory domain derived from one or more proteins selected from the group consisting of CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, a ligand of CD83, CD40, and MyD88.

[0198] For example, the costimulatory domain is an intracellular costimulatory domain derived from 4-1BB.

[0199] In the present application, the nucleic acid molecule comprises a nucleic acid sequence encoding an intracellular signaling domain, and the intracellular signaling domain comprises an intracellular signaling domain derived from one or more proteins selected from the following groups: CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, Kaposi's sarcoma herpes virus (HSKV), DAP10, DAP-12 and a domain comprising at least one ITAM.

[0200] For example, the intracellular signaling domain is an intracellular signaling domain derived from CD3ζ.

[0201] In the present application, the nucleic acid molecule comprises a nucleic acid sequence encoding a cytokine domain, and the nucleic acid sequence encoding the cytokine domain can be:

[0202] (1) Sequences capable of expressing one or more cytokines and / or their functional fragments;

[0203] (2) sequences capable of expressing one or more cytokine receptors and / or functional fragments thereof; and

[0204] (3) A sequence capable of expressing a combination of (1) and (2).

[0205] For example, the cytokine is selected from one or more factors in the group consisting of IL-15, IL-10, IL-18, and IL-21. For example, the cytokine is IL-15. For example, the cytokine receptor is IL-15R or its α subunit (IL-15Rα).

[0206] In the present application, in the nucleic acid molecule, the localization sequence may be located upstream of the sequence of the targeting antigen.

[0207] In the present application, in the nucleic acid molecule, the localization sequence may be located downstream of the targeting antigen sequence.

[0208] In the present application, in the nucleic acid molecule, the localization sequence can be located both upstream and downstream of the sequence of the targeting antigen.

[0209] In the present application, the nucleic acid molecule comprises a sequence encoding a cleavage peptide, and the cleavage peptide is selected from one or more cleavage peptides in the following group: P2A, T2A, F2A, E2A, BmCPV2A, IRES and BmIFV2A.

[0210] For example, the cleavage peptide is P2A. For example, the cleavage peptide P2A is connected to the linker sequence GSG (SEQ ID NO: 7).

[0211] For example, the nucleic acid molecule can encode, in sequence, a CD8 signal peptide, a GPC3 antibody, a CD8 hinge region, a CD28 transmembrane region, a 4-1BB co-stimulatory domain, a CD3ζ intracellular signaling domain, a GSG linker sequence, a P2A self-cleaving peptide, an IgGκ signal peptide, a PD-1 antibody, a V5 tag, (GGGGS)4, and a localization sequence.

[0212] For example, the nucleic acid molecule can encode, in sequence, a CD8 signal peptide, a ROR1 antibody, a CD28 hinge region, a CD28 transmembrane region, a 4-1BB co-stimulatory domain, a CD3ζ intracellular signaling domain, a GSG linker sequence, a P2A self-cleaving peptide, an IgGκ signal peptide, a PD-1 antibody, a V5 tag, (GGGGS)4, and a localization sequence.

[0213] In the present application, the nucleic acid sequence of the nucleic acid molecule may be as shown in the examples of the present application and the drawings.

[0214] In the present application, the nucleic acid sequence of the nucleic acid molecule can be further modified according to the modified iNKT cells described in the detailed description of the invention in the specification of this application.

[0215] plasmids

[0216] In another aspect, the present application provides a plasmid comprising the nucleic acid molecule described in the present application.

[0217] In the present application, the plasmid includes viral plasmid and bacterial plasmid.

[0218] In the present application, the plasmid includes DNA plasmid and RNA plasmid.

[0219] In this application, the plasmids include linear plasmids and circular plasmids.

[0220] In the present application, the plasmid may be natural or artificially modified.

[0221] Pharmaceutical composition

[0222] On the other hand, the present application provides a pharmaceutical composition comprising the iNKT cells described herein, the nucleic acid molecules described herein, and / or the plasmid described herein, and at least one pharmaceutically acceptable carrier.

[0223] In the present application, the pharmaceutically acceptable carrier refers to a carrier, diluent and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient of the drug, including but not limited to: pH regulators, surfactants, carbohydrates, adjuvants, antioxidants, chelating agents, ionic strength enhancers and preservatives.

[0224] use

[0225] In another aspect, the present application provides a use of the composition provided herein in preparing a medicament for preventing, alleviating and / or treating a disease and / or condition.

[0226] On the other hand, the present application also provides a method for preventing and / or treating a disease, comprising administering the modified iNKT cells described in the present application to a subject in need thereof.

[0227] For example, the transformed iNKT cells of described administration can be prepared pharmaceutically according to any conventional method.For example, carrier, excipient or diluent can be used to mix or dilute active ingredient.The example of suitable carrier, excipient or diluent is lactose, dextrose, sucrose, sorbitol, mannitol, glycine, polyethylene glycol, starch, gum arabic, alginic acid, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.Described preparation can comprise filler, anti-agglomeration agent, lubricant, wetting agent, flavoring agent, emulsifier, preservative etc. additionally.Prepare the compositions of the present invention to provide quick, continuous or delayed release active ingredient after giving patient by using any method known in the art.

[0228] The cells herein can be administered by injection (e.g., intramuscular, intravenous, intraperitoneal, subcutaneous), or by other methods such as infusion to ensure that they enter the bloodstream in an effective form. The cells can also be administered via intratumoral, peritumoral, intralesional, or perilesional routes to exert local and systemic therapeutic effects. For example, they can be administered topically or intravenously.

[0229] In the present application, the administration dosage of the cells may be a single dose or multiple doses. For example, the actual administration amount of the cells may be determined based on a variety of relevant factors, such as the type of disease; the route of administration; the age, sex, and / or weight of the patient; and the severity of the patient's symptoms.

[0230] Without intending to be bound by any theory, the following examples are merely intended to illustrate the preparation method and application of the present application, and are not intended to limit the scope of the present invention.

[0231] Example

[0232] Example 1 Preparation of cells

[0233] Cell culture

[0234] HEK293T, MEC1, Hep3B, Huh7 and MDA-MB-231 cell lines were obtained from ATCC. MEC1-ROR1 cell line was generated in-house. HEK293T, Hep3B and Huh7 cell lines were maintained in full culture medium (DMEM containing 10% heat-inactivated FBS, 100 U / mL penicillin / streptomycin and 2 mM L-glutamine). MDA-MB-231, MEC1 and MEC1-ROR1 cell lines were maintained in full culture medium (IMDM containing 10% heat-inactivated FBS, 100 U / mL penicillin / streptomycin and 2 mM L-glutamine). PBMCs were isolated from whole blood of healthy donors using Ficoll-Paque and plated at 2x10 7 1 mL of sample was aliquoted at a concentration of 10 cells / mL and stored frozen in a liquid nitrogen tank. The culture medium was heat-inactivated FBS supplemented with 10% DMSO (vol / vol).

[0235] Clone construction

[0236] All relevant gene constructs were constructed and synthesized using GeneWiz. The gene construct encoding PD1A2VHH-PEBL-GSGP2A-ROR1CAR was inserted into the pALD expression plasmid via the SalI and Spel cloning sites. To ensure intracellular retention of the PD1 protein, an IgGk signal peptide (IgGKSP) was added to the N-terminus of PD1A2VHH (SEQ ID NO: 19), and a PEBL sequence (LSM: SEQ ID NO: 8) was added to the C-terminus.

[0237] Lentivirus preparation

[0238] All lentiviruses were produced using HEK293T cells. Freshly resuspended HEK293T cells were washed in ice-cold PBS and seeded into 10-cm culture dishes at a cell density of 90%-95% of the dish area. A mixture of lentiviral packaging plasmid and transfection plasmid was mixed with Lipofectamine 2000 / 3000 and transiently transfected into the seeded HEK293T cells. After 48 hours of culture, the supernatant was collected, filtered through a 0.45 μm filter, and aliquoted into 1 ml tubes for storage at -80°C.

[0239] T cell activation and transduction

[0240] To transduce PBMCs with lentivirus encoding the corresponding constructs, frozen PBMCs were thawed and placed in 24-well plates. CD3- and CD28-coupled magnetic beads and IL-2 (500 IU / ml) were added for 24 hours of activation. The activated PBMCs were then distributed across multiple wells of the 24-well plates. The corresponding lentivirus and polybrene (8 g / ml) were added and centrifuged at 2000 g for 2 hours in a benchtop centrifuge. The cells were then transferred to a 37°C incubator with 5% CO2. The transfected cells were then cultured in X-VIVO-15 cell culture medium supplemented with IL-2 (500 IU / ml) for 48 hours before subsequent experiments and analysis.

[0241] Flow cytometry

[0242] Flow cytometry was performed using a flow cytometer (Beckman Coulter) in plate format, and data were analyzed using FlowJo software. Cells were washed once with FACS buffer (PBS containing 0.5% BSA, 0.1% NaN3, 2 mM EDTA, pH 7.0) and resuspended to 5 × 10 7 Cells were washed at 400 μL of FACS buffer per well. The cells were centrifuged at 300 × g for 3 minutes and the supernatant was removed. After washing and resuspending in FACS buffer, the cells were analyzed by flow cytometry.

[0243] ELISA

[0244] ELISAMAX was used according to the manufacturer's instructions. TM Deluxe Set Human IL-15 (Biolegend; 435104) was used to measure the IL-15 concentration in the culture supernatant.

[0245] Experimental results:

[0246] The expression of related structures and the related structure results of PD1 knockout are shown in Figure 1. More than 90% of PD1 protein was successfully knocked out. The secretion of sIL15 and the membrane expression structure IL-15 are shown in Figure 3B. Both can be secreted and expressed normally.

[0247] Example 2: Membrane protein intracellular retention technology reduces PD1 expression in CAR-iNKT cells

[0248] This example studies the knockout effect of the membrane protein intracellular retention technology on the PD1 gene in CAR-iNKT cells.

[0249] 2.1 Tumor cell lines

[0250] MEC1 (B-cell chronic lymphocytic leukemia) tumor cells were engineered to stably express the ROR1 antigen. The MDA-MB-231 cell line already expresses the ROR1 antigen. These three tumor cell lines were transfected to express GFP and used in subsequent experiments.

[0251] 2.2 Co-culture of CAR-T cells and tumor cells

[0252] PBMC cells and tumor cells transfected with ROR1CAR v2 and ROR1CAR v2-PD1 KO were accurately counted respectively, with a ratio of 1:1 and 1=1x10 4 Cells were resuspended in 100 μl of cell culture medium at a density of 100 μl and seeded in a round-bottom 96-well plate. After 72 hours of culture, the cells were centrifuged and resuspended in FACS buffer. The cells were stained with PE-labeled ROR1 protein (Acro Biosystems; RO1-HP2E3) and BV421-labeled PD1 antibody (Biolegend; 367422) and analyzed by flow cytometry.

[0253] Experimental results:

[0254] As shown in Figure 1, PBMC cells transfected with the ROR1CARv2 structure induced PBMC to express PD1 (MEC1: 5.91%; MEC1-ROR1: 44.5%; MDA-MB-231: 29.1%) after co-culture with tumor cells.

[0255] After co-culture with tumor cells, PBMC cells transfected with ROR1CARv2-PD1KO structure showed significantly reduced PD1 expression compared with non-knockout CAR-iNKT cells (MEC1: 0.07%; MEC1-ROR1: 7.94%; MDA-MB-231: 0.99%).

[0256] The above experimental results indicate that the method provided in this application can construct stable iNKT cells that lack or suppress the expression of immune negative regulatory genes.

[0257] Example 3: Membrane protein intracellular retention technology reduces TGF-βR expression in CAR-iNKT cells

[0258] This example studies the knockout effect of TGF-βR gene in CAR-iNKT cells using membrane protein intracellular retention technology.

[0259] 3.1 Tumor cell lines

[0260] The MEC1 (B-cell chronic lymphocytic leukemia) tumor cell line was engineered to stably express the ROR1 antigen. The tumor cell line was transfected to express GFP and used in subsequent experiments.

[0261] 3.2 Verification of the knockout effect of TGFβRII in CAR-iNKT cells

[0262] After 72 hours of culture, the cells were centrifuged and resuspended in FACS buffer. The cells were stained with PE-labeled ROR1 protein (Acro Biosystems; RO1-HP2E3) and BV421-labeled TGFβRII antibody (Biolegend; 399710), and then detected by flow cytometry.

[0263] 3.3 Re-challenge experiment of CAR-iNKT cells and tumor cells (Incucyte instrument detection method)

[0264] PBMC cells and MEC1 and MEC1-ROR1 tumor cells transfected with ROR1CAR v2, ROR1CAR v2-PD1 KO and ROR1CAR v2-TGFβRII KO structures were accurately counted and counted at a ratio of 1:1 and 1 = 0.5x10 4 The cells were resuspended in 100 μl of cell culture medium and seeded in a flat-bottomed 96-well plate. The plate was then placed in an Incucyte instrument and the cell killing ability (the percentage of GFP tumor cell death) was tested according to the manufacturer's instructions. The TGFβ group was co-cultured with 100 mg / ml of TGFβ. After 48 hours of culture, 0.5x10 4 The tumor cells were added into the corresponding wells again, and the cell killing ability was continued to be tested.

[0265] Experimental results:

[0266] As shown in FIG2 , the knockout effect of TGFβRII was approximately 60%.

[0267] Compared to PBMC cells transfected with ROR1CARv2-PD1KO and ROR1CARv2-TGFβRIIKO structures, after co-culture with tumor cells MEC1-ROR1, the PD1 knockout cell group had a significantly better killing effect than the TGFβR knockout group (Figure 3B). In the group with TGFβ inhibition, the PD1 knockout cells also showed excellent antagonism to TGFβ inhibition, and their killing effect was also significantly better than the TGFβR knockout group.

[0268] The above experimental results show that the PD1 knockout-enhanced CAR T cells used in this application have better effects than other cells such as TGFβR knockout.

[0269] Example 4 PD1 knockout CAR-iNKT cells show improved function

[0270] This example studies the effect of membrane protein intracellular retention technology on the killing ability and survival ability of CAR-iNKT cells after PD1 gene knockout in CAR-iNKT cells.

[0271] 4.1 Tumor cell lines

[0272] MEC1 (B-cell chronic lymphocytic leukemia) tumor cells were engineered to stably express the ROR1 antigen. MDA-MB-231 (breast cancer) cell lines already express the ROR1 antigen. These three tumor cell lines were transfected to express GFP and used in subsequent experiments.

[0273] 4.2 Re-challenge experiment of CAR-iNKT cells and tumor cells (Incucyte instrument detection method)

[0274] PBMC cells transfected with ROR1CAR v2 and ROR1CAR v2-PD1 KO structures and MEC1 and MEC1-ROR1 tumor cells were accurately counted respectively, with a ratio of 1:3 and 1 = 0.25x10 4 The cells were resuspended in 100 μl of cell culture medium and seeded in a flat-bottom 96-well plate. The plate was then placed in the Incucyte instrument and the cell killing ability (the percentage of GFP tumor cell death) was tested according to the manufacturer's instructions. After 48 hours of culture, 0.75x10 4 The tumor cells were added into the corresponding wells again, and the cell killing ability was continued to be tested.

[0275] 4.3 Re-challenge experiment of CAR-iNKT cells and tumor cells (flow cytometry detection method)

[0276] PBMC cells transfected with ROR1CAR v2 and ROR1CAR v2-PD1 KO structures and MEC1, MEC1-ROR1 and MDA-MB-231 tumor cells were accurately counted respectively, with a ratio of 1:3 and 1 = 0.25x10 4 The cells were resuspended in 100 μl of cell culture medium and seeded into three flat-bottom 96-well plates. After three days of culture, 0.75×10 4Tumor cells were added to the remaining culture plates and cultured continuously. After 3, 7, and 11 days of culture, cells from one 96-well plate were centrifuged and resuspended in FACS buffer. The cells were stained with PE-labeled ROR1 protein (Acro Biosystems; RO1-HP2E3) and BV421-labeled PD1 antibody (Biolegend; 367422), respectively, and analyzed by flow cytometry.

[0277] Experimental results:

[0278] As shown in Figure 4A, compared with ROR1-CAR-iNKT cells without knockout, PD1-knockout CAR-iNKT cells still maintained strong inhibitory and killing functions after secondary tumor cell stimulation (D0, D3).

[0279] As shown in Figure 4B , compared with non-knockout CAR T cells, the survival rate of PD1-knockout CAR-iNKT cells was significantly improved during in vitro co-culture.

[0280] These experimental results demonstrate that the iNKT cells provided herein, which lack or suppress the expression of negative immune regulatory genes, exhibit stronger tumor suppressive and cytotoxic activity after secondary stimulation with tumor cells, compared to unmodified iNKT cells, and their survival rate is further enhanced. The unexpectedly enhanced cytotoxic activity and prolonged survival of the modified iNKT cells suggest that the iNKT cells provided herein have great potential for disease treatment.

[0281] Example 5 Construction and Effect Detection of GPC3-CAR PD-1KO

[0282] 5.1 Construction and Expression Detection of GPC3-CAR

[0283] All relevant gene constructs were constructed and synthesized by Jin Weizhi. The GPC3CAR-PD1KO gene construct encoding GPC3CAR--GSGP2A-PD1antibody-PEBL was inserted into the pALD expression plasmid via the SalI and SpeI cloning sites. To ensure that the PD1 protein is retained in the cell, an IgGk signal peptide (IgGKSP) was added to the N-terminus of the PD1antibody (SEQ ID NO: 19), and a PEBL sequence (LSM: SEQ ID NO: 8) signal sequence was added to the C-terminus.

[0284] Using the third-generation lentiviral packaging system, a mixture of packaging plasmid, target plasmid, and PEIpro was transiently transfected into 293T cells. After 48 hours of culture, the supernatant containing the lentivirus was collected and filtered using a 0.45 μm filter. The samples were aliquoted into 1 ml tubes and stored in a -80 ° C refrigerator. When used, the lentivirus of the above structure was melted and transduced into iNKT cells. After 72 hours of culture, PE-labeled GPC3 protein (Acro Biosystems; GP3-HP2E3) was used for staining, and the expression of the CAR structure was detected by flow cytometry.

[0285] Figure 5A is a schematic diagram of the construction of GPC3-CAR and GPC3-CAR PD1KO.

[0286] 5.2 In vivo efficacy evaluation of GPC3-CAR and GPC3-CAR PD-1KO

[0287] (1) Tumor cell culture and inoculation

[0288] Human hepatocellular carcinoma Hep3B cells were cultured in MEM medium supplemented with 10% FBS in a 37°C incubator containing 5% CO2. Before the tenth generation of cell culture, approximately 1×10 6 Hep3B cells were resuspended in 100 μL PBS, mixed with an equal volume of Matrigel (v / v 1:1), and inoculated subcutaneously into the right flank of NCG-hIL15 mice in a volume of 200 μL. Mice were anesthetized with 3-4% isoflurane before inoculation.

[0289] (2) Dosage regimen

[0290] The day of tumor cell inoculation was defined as day 0. Three days after inoculation, mice were divided into groups of six according to body weight and treated accordingly. Animals were dosed with a total cell count of 1.5E7 per mouse, adjusted to a volume of 200 μL per mouse, via the tail vein for three doses: on days 3, 10, and 17 after inoculation. Animals were observed daily, and tumor diameter and weight were measured twice weekly. Tumors from surviving animals were removed and weighed at the end of the experimental period.

[0291] Experimental results: As shown in Figure 5B, the PD1 knockout construct GPC3-CAR PD1KO had an expression efficiency of 50.1% on iNKTs, which is twice the expression efficiency of GPC3-CAR on iNKTs (26.1%). GPC3-CAR PD1KO can significantly improve the expression efficiency of GPC3-CAR on iNKTs.

[0292] In vivo experimental results:

[0293] As shown in Figure 6 , on day 26, the average tumor volumes of the PBS group, Mock-iNKT group, GPC3-CAR group, and GPC3-CAR PD1 KO group were 1141.9±124.51mm 3 ,814.7±164.3mm 3 , 635.6±79.8mm 3 and 645.8±146.3mm 3 The tumor volumes of the GPC3-CAR group and the GPC3-CAR PD1 KO group were smaller than those of the PBS group and the Mock-T group, but there was no significant difference between the two groups. As of day 42, the average tumor volumes of the PBS group, the Mock-iNKT group, the GPC3-CAR group, and the GPC3-CAR PD1 KO group were 2956.83±75.64mm, respectively. 3 ,2512±871.21mm 3 , 2763.49±329.59mm 3 and 1056.6±553.9mm 3 On day 42, the average tumor volume in the GPC3-CAR PD1 KO group was significantly different from that in the PBS group, the Mock-iNKT group, and the GPC3-CAR group (p < 0.05). The results indicate that GPC3-CAR PD1 KO has a clear advantage in long-term anti-tumor effects compared to GPC3-CAR.

[0294] The above results show that compared with GPC3-CAR, GPC3-CAR PD1 KO can not only increase the expression on iNKT, but also significantly enhance the tumor suppression effect.

[0295] Example 6 Preparation of three-in-one CAR architecture cells fused with PD1 knockout and IL-15 enhancement

[0296] The preparation process is based on the following steps:

[0297] 6.1 Construction of a three-in-one CAR structure integrating PD1 knockout and interleukin-15 enhancement

[0298] First, the DNA of different CAR structures is synthesized by a biotechnology company. In the present invention, taking GPC3CAR as an example, the design of using interleukin 15 to enhance the function of CAR can be to add secretory interleukin 15 after P2A, or to add membrane receptor-bound interleukin 15. The structural design is shown in Figure 7A. On the basis of the above structure, protein intracellular retention technology is also integrated to achieve PD1 knockout and IL-15 secretion at the same time, resulting in a three-in-one GPC3-CAR framework. In addition, we also designed a separate GPC3 second-generation CAR as an experimental reference group. After obtaining the synthetic plasmids of different CARs, the designed structural segments were connected to the pALD plasmid using double enzyme digestion for packaging lentivirus.

[0299] 6.2 Expression of a three-in-one CAR structure integrating PD1 knockout and IL-15 enhancement

[0300] Using the third-generation lentiviral packaging system, a liquid mixed with packaging plasmid, target plasmid and Lipofectamine3000 was transiently transfected into 293T cells. After 48 hours of culture, the supernatant containing the lentivirus was collected and filtered using a 0.45μm filter. The sample was aliquoted into 1ml tubes and stored in a -80°C refrigerator. When used, the lentivirus of the above structure was melted and transduced into Jurkat or activated PBMCs. After 72 hours of culture, PE or FITC-labeled GPC3 protein (Acro Biosystems; GP3-HP2E3 and GP3-HF2H1) was used for staining, and then the expression of the CAR structure was detected by flow cytometry. For the membrane receptor-linked IL-15 structure, an anti-FLAG tag antibody was used for staining to determine the expression of membrane receptor-linked IL-15 behind P2A. In addition, the supernatant of the cell culture was collected after 72 hours, concentrated 4-10 times using a protein concentrator as needed, and used for enzyme-linked immunosorbent assay to measure IL-15 secreted into the culture medium.

[0301] Experimental results:

[0302] As shown in Figure 7B, GPC3-CAR, GPC3CAR-sIL15, or GPC3CAR-TeIL15 were all expressed normally in PBMCs and bound to the antigen. In the supernatant 72 hours after transduction, the GPC3-CAR plus secreted IL-15 constructs in the above structures could detect a certain degree of IL-15 secretion (Figure 7C). Furthermore, the three-in-one construct fused with PD1 knockout and IL-15 enhancement also expressed the CAR construct in Jurkat cells (Figure 7D) and secreted IL-15 into the culture medium (Figure 7E).

[0303] The above experimental results show that the iNKT cells provided in this application, which lack or inhibit the expression of immune negative regulatory genes, can secrete cytokines normally after further modification, and can secrete more cytokines in the same period of time compared with unmodified iNKT cells (Figure 7E). This effect is unexpected and also shows that the iNKT cells provided in this application can be used to treat diseases by expressing various cytokines.

[0304] Example 7: Secreted or membrane receptor-bound IL-15 can enhance the tumor killing function of CAR-T cells

[0305] This example studies the effect of CAR-iNKT cells on tumor killing function after superposition of secretory IL-15 or membrane receptor-bound IL-15.

[0306] 7.1 Tumor cell lines

[0307] Hep3B and Huh7 hepatoma cells were tested for endogenous GPC3 expression. MDA-MB-231, a human breast cancer cell line, did not express GPC3. These three tumor cell lines were transfected to stably express GFP and used in subsequent experiments.

[0308] 7.2 In vitro killing of CAR-T cells (detected by Incucyte real-time imaging)

[0309] The above structures (GPC3-CAR; GPC3-CAR-sIL5 and GPC3-CAR-TeIL15) were transduced into PBMC cells respectively. After 72 hours of culture, the expression of each structural element was detected by flow cytometry. The effector cells and target cells were accurately counted, and the CAR expression rate was adjusted to achieve the same level for each structure. Then, the effector-target ratio was 1:3 and 1=7x10 3 Cells were resuspended at a density of 100 μl in 200 μl of cell culture medium and seeded into a flat-bottom 96-well plate. The 96-well plate was placed in the IncuCyte for real-time imaging, allowing the 48-hour growth curve of GFP-labeled target cells to be monitored. After the killing experiment, the 96-well plate was removed, centrifuged, and the supernatant was assayed for IFN-γ secretion using an enzyme-linked immunosorbent assay.

[0310] Experimental results:

[0311] As shown in Figure 8A, for the liver cancer target cells Hep3B and Huh7 expressing GPC3, both secretory and membrane receptor-bound IL-15 GPC3-CART have strong killing ability and tumor suppression effect. For MDA-MB231 that does not express GPC3, growth is not inhibited, reflecting good specificity. In the IFN-γ secretion assay (Figure 8B), secretory or membrane receptor-bound IL-15 can detect higher levels of IL-15 secretion, reflecting a strong tumor killing ability.

[0312] The above experimental results show that unmodified iNKT cells can express cytokines in different construction modes and have strong tumor-killing ability.

[0313] Example 8: A three-in-one structure combining PD1 knockout and IL-15 enhancement reduces PD1 expression in CAR-T cells

[0314] This example studies the effect of PD1 knockout in the process of killing tumors by a three-in-one structure that integrates PD1 knockout and IL-15 enhancement.

[0315] 8.1 Tumor cell lines

[0316] Hep3B and Huh7 hepatoma cells were tested for endogenous GPC3 expression. MDA-MB-231, a human breast cancer cell line, did not express GPC3. These three tumor cell lines were transfected to stably express GFP and used in subsequent experiments.

[0317] 8.2 Co-culture of CAR-iNKT Cells with Tumor Cells and Flow Cytometry Detection of PD1 Expression in Cells

[0318] The above structures (GPC3-CAR; GPC3-CAR-PD1 KO-sIL15) were transfected into Jurakt cells respectively. After 72 hours of culture, CAR expression was detected by flow cytometry and the cell density was adjusted to the same CAR expression rate. The effector cells and target cells were accurately counted and then the effector-target ratio was 1:1 and 1 = 5x10 4 The cells were resuspended in 200 μl of cell culture medium at a density of 100 μg / mL and seeded in a round-bottom 96-well plate. After 72 hours of co-culture, the cells were washed with PBS and stained with PE-labeled GPC3 protein (Acro Biosystems; GP3-HP2E3) and BV421-labeled PD1 antibody (Biolegend; 367422) before analysis by flow cytometry.

[0319] Experimental results:

[0320] As shown in Figure 9, after co-culture with tumor cells, the GPC3-CAR group can induce high levels of PD1 expression, especially in CART cells co-cultured with specific target cells, which showed approximately 50% PD1 expression. In contrast, our three-in-one CART architecture uses PD1 membrane protein retention technology to significantly reduce PD1 expression in cells, demonstrating a good knockout effect, making CAR-iNKT cells less susceptible to exhaustion and having a more sustained tumor killing effect.

[0321] The above experimental results indicate that the iNKT cells provided in the present application that lack or suppress the expression of immune negative regulatory genes have a more lasting tumor killing effect compared with unmodified iNKT cells.

Claims

1. An engineered iNKT cell that lacks or inhibits the expression of an immune negative regulatory gene compared to an unengineered iNKT cell, said gene being selected from one or more of the following groups: PD-1, Tim-3, CTLA-4, CD74, and TGF-βR, wherein said lack or inhibition of the expression of the immune negative regulatory gene is achieved by a membrane protein intracellular retention / degradation technique.

2. The iNKT cell according to claim 1, wherein said membrane protein intracellular retention technique is directly or indirectly linking a localization sequence to the N-terminus or C-terminus of the immune negative regulatory gene.

3. The iNKT cell according to any one of claims 1-2, wherein said membrane protein intracellular retention technique is directly or indirectly linking a localization sequence to the C-terminus of the immune negative regulatory gene.

4. The iNKT cell according to any one of claims 1-3, wherein said membrane protein intracellular retention technique is directly or indirectly linking a localization sequence to the N-terminus of the immune negative regulatory gene.

5. The iNKT cell according to any one of claims 2-4, wherein said indirect linking uses a (GGGGS)4 linker.

6. The iNKT cell according to any one of claims 1-5, wherein said immune negative regulatory gene is PD-1.

7. The iNKT cell according to claim 6, wherein the amino acid sequence of said PD-1 is as shown in SEQ ID NO:12 or SEQ ID NO:

19.

8. The iNKT cell according to any one of claims 2-7, wherein said localization sequence is selected from an endoplasmic reticulum retention sequence, a Golgi retention sequence, an E3 ubiquitin ligase binding sequence, a proteasome localization sequence, and a lysosome localization sequence.

9. The iNKT cell according to claim 8, wherein said localization sequence is an endoplasmic reticulum retention sequence or a lysosome localization sequence.

10. The iNKT cell according to any one of claims 8-9, wherein the amino acid sequence of said endoplasmic reticulum retention sequence comprises one or more amino acid sequences selected from the following groups: KDEL (SEQ ID NO:9), KKMP (SEQ ID NO:10), and AEKDEL (SEQ ID NO:11).

11. The iNKT cell according to any one of claims 8-9, wherein the amino acid sequence of said lysosome localization sequence is as shown in SFHDDSDEDLLHI (SEQ ID NO:8).

12. The iNKT cell according to any one of claims 1-11, wherein said iNKT cell expresses a CAR.

13. The iNKT cell according to claim 12, wherein said CAR comprises a targeting moiety that specifically binds an antigen.

14. The iNKT cell according to claim 13, said antigen being selected from one or more of the following antigens: GPC3, CLDN18.2, ROR1, and AXL.

15. The iNKT cell according to any one of claims 12-14, said antigen being GPC3 or ROR1.

16. The iNKT cell according to any one of claims 12 - 14, wherein the targeting moiety is a scFv.

17. The iNKT cell according to any one of claims 12 - 14, wherein the targeting moiety is a VHH.

18. The iNKT cell according to any one of claims 12 - 17, wherein the CAR comprises a signal peptide.

19. The iNKT cell according to claim 18, wherein the signal peptide is a signal peptide derived from the CD8 protein.

20. The iNKT cell according to claim 18, wherein the signal peptide is a signal peptide derived from IL - 2.

21. The iNKT cell according to any one of claims 12 - 20, wherein the CAR comprises a hinge region, and the hinge region comprises a hinge region derived from one or more proteins selected from the group consisting of: CD28, IgG1, IgG4, IgD, 4 - 1BB, CD4, CD27, CD7, CD8, PD - 1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT.

22. The iNKT cell according to claim 21, wherein the hinge region is a hinge region derived from CD28 or a hinge region derived from CD8.

23. The iNKT cell according to any one of claims 12 - 22, wherein the CAR comprises a transmembrane region, and the transmembrane region comprises a transmembrane domain derived from one or more proteins selected from the group consisting of: CD8, CD28, 4 - 1BB, CD4, CD27, CD7, PD - 1, TRAC, TRBC, CD3ε, CD3ζ, CTLA - 4, LAG - 3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM.

24. The iNKT cell according to claim 23, wherein the transmembrane region is a transmembrane region derived from CD28.

25. The iNKT cell according to any one of claims 12-24, wherein the CAR comprises a co-stimulatory domain, and the co-stimulatory domain comprises a co-stimulatory domain derived from one or more proteins selected from the group consisting of: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, the ligand of CD83, CD40, and MyD88.

26. The iNKT cell according to claim 23, wherein the co-stimulatory domain is a co-stimulatory domain derived from 4-1BB.

27. The iNKT cell according to any one of claims 12-25, wherein the CAR comprises an intracellular signaling domain, and the intracellular signaling domain comprises an intracellular signaling domain derived from one or more proteins selected from the group consisting of: CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma herpesvirus (HSKV), DAP10, DAP-12, and a domain comprising at least one ITAM.

28. The iNKT cell according to claim 27, wherein the intracellular signaling domain is an intracellular signaling domain derived from CD3ζ.

29. The iNKT cell according to any one of claims 1-28, wherein the iNKT cell is a CAR-iNKT cell, and the CAR consists of a CD8 signal peptide, a GPC3 antibody, a CD8 hinge region, a CD28 transmembrane region, a 4-1BB co-stimulatory domain, a CD3ζ intracellular signaling domain, a GSG linker sequence, a P2A self-cleaving peptide, an IgGκ signal peptide, a PD-1 antibody, a V5 tag, (GGGGS)4, and a localization sequence connected in sequence.

30. The iNKT cell according to any one of claims 1-28, wherein the iNKT cell is a CAR-iNKT cell, and the CAR consists of a CD8 signal peptide, a ROR1 antibody, a CD28 hinge region, a CD28 transmembrane region, a 4-1BB co-stimulatory domain, a CD3ζ intracellular signaling domain, a GSG linker sequence, a P2A self-cleaving peptide, an IgGκ signal peptide, a PD-1 antibody, a V5 tag, (GGGGS)4, and a localization sequence connected in sequence.

31. The CAR-iNKT cell according to any one of claims 29-30, wherein the amino acid sequence of the PD-1 antibody is as shown in SEQ ID NO:19, and the amino acid sequence of the localization sequence is as shown in SEQ ID NO:

8.

32. The iNKT cell according to any one of claims 12 - 31, wherein the CAR comprises a cytokine domain.

33. The iNKT cell according to claim 32, wherein the cytokine domain: (1) comprises one or more cytokines and / or their functional fragments; (2) comprises one or more cytokine receptors and / or their functional fragments; and (3) comprises a combination of (1) and (2).

34. The iNKT cell according to any one of claims 32 - 33, wherein the cytokine domain is expressed on the iNKT cell membrane.

35. The iNKT cell according to any one of claims 32 - 34, wherein the cytokine domain is secreted and expressed from the iNKT cell.

36. The iNKT cell according to any one of claims 32 - 35, wherein the cytokine is selected from one or more factors in the following group: IL - 15, IL - 10, IL - 18, and IL - 21.

37. The iNKT cell according to any one of claims 32 - 36, wherein the cytokine is IL - 15.

38. The iNKT cell according to any one of claims 32 - 36, wherein the cytokine receptor is IL - 15R or its α subunit (IL - 15Rα).

39. A nucleic acid molecule, which encodes a nucleic acid sequence comprising a nucleic acid sequence encoding an immune negative regulatory gene and a nucleic acid sequence encoding a localization sequence, and the immune negative regulatory gene is selected from one or more genes in the following group: PD - 1, Tim - 3, CTLA - 4, CD74, and TGF - βR.

40. The nucleic acid molecule according to claim 39, wherein the immune negative regulatory gene is PD - 1.

41. The nucleic acid molecule according to any one of claims 39 - 40, wherein the amino acid sequence of PD - 1 is as shown in SEQ ID NO:12 or SEQ ID NO:

19.

42. The nucleic acid molecule according to any one of claims 39 - 41, wherein the localization sequence is selected from an endoplasmic reticulum retention sequence, a Golgi retention sequence, an E3 ubiquitin ligase binding sequence, a proteasome localization sequence, and a lysosome localization sequence.

43. The nucleic acid molecule according to any one of claims 39 - 42, wherein the localization sequence is an endoplasmic reticulum retention sequence or a lysosome localization sequence.

44. The nucleic acid molecule according to any one of claims 42 - 43, wherein the amino acid sequence of the endoplasmic reticulum retention sequence comprises one or more amino acid sequences optionally selected from the following group: KDEL (SEQ ID NO:9), KKMP (SEQ ID NO:10), and AEKDEL (SEQ ID NO:11).

45. The nucleic acid molecule according to any one of claims 42 - 43, wherein the amino acid sequence of the lysosome localization sequence is as shown in SFHDDSDEDLLHI (SEQ ID NO:8).

46. The nucleic acid molecule according to any one of claims 39 - 45, which comprises a nucleic acid sequence encoding a target antigen.

47. The nucleic acid molecule according to claim 46, wherein the antigen is one or more antigens selected from the group consisting of GPC3, CLDN18.2, ROR1, and AXL.

48. The nucleic acid molecule according to any one of claims 46-47, wherein the antigen is GPC3 or ROR1.

49. The nucleic acid molecule according to any one of claims 39-48, which comprises a nucleic acid sequence encoding a promoter.

50. The nucleic acid molecule according to claim 49, wherein the promoter is selected from the group consisting of EF1α, CMV, MSCV, SSFV, and UbC.

51. The nucleic acid molecule according to any one of claims 39-50, which comprises a nucleic acid sequence encoding a signal peptide.

52. The nucleic acid molecule according to claim 51, wherein the signal peptide is a signal peptide derived from the CD8 protein.

53. The nucleic acid molecule according to claim 51, wherein the signal peptide is a signal peptide derived from IL-2.

54. The nucleic acid molecule according to any one of claims 39-53, which comprises a nucleic acid sequence encoding a hinge region, and the hinge region comprises a hinge region derived from one or more proteins selected from the group consisting of CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT.

55. The nucleic acid molecule according to claim 54, wherein the hinge region is a hinge region derived from CD28 or a hinge region derived from CD8.

56. The nucleic acid molecule according to any one of claims 39-55, which comprises a nucleic acid sequence encoding a transmembrane region, and the transmembrane region comprises a transmembrane domain derived from one or more proteins selected from the group consisting of CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM.

57. The nucleic acid molecule according to claim 56, wherein the transmembrane region is a transmembrane region derived from CD28.

58. A nucleic acid molecule according to any one of claims 39-57, which comprises a nucleic acid sequence encoding a co-stimulatory domain, said co-stimulatory domain comprising a co-stimulatory domain derived from one or more proteins selected from the group consisting of: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, the ligand of CD83, CD40 and MyD88.

59. The nucleic acid molecule according to claim 58, wherein the co-stimulatory domain is the intracellular co-stimulatory domain derived from 4-1BB.

60. A nucleic acid molecule according to any one of claims 39-59, which comprises a nucleic acid sequence encoding an intracellular signaling domain, said intracellular signaling domain comprising an intracellular signaling domain derived from one or more proteins selected from the group consisting of: CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma herpesvirus (HSKV), DAP10, DAP-12 and a domain comprising at least one ITAM.

61. The nucleic acid molecule according to claim 60, wherein the intracellular signaling domain is the intracellular signaling domain derived from CD3ζ.

62. The nucleic acid molecule according to any one of claims 39-61, wherein the localization sequence is upstream of the sequence of the immune negative regulatory gene.

63. The nucleic acid molecule according to any one of claims 39-61, wherein the localization sequence is downstream of the sequence of the immune negative regulatory gene.

64. The nucleic acid molecule according to any one of claims 39-63, which comprises a sequence encoding a cleavage peptide.

65. The nucleic acid molecule according to claim 64, wherein the cleavage peptide is selected from one or more cleavage peptides in the group consisting of: P2A, T2A, F2A, E2A, BmCPV2A, IRES and BmIFV2A.

66. The nucleic acid molecule according to any one of claims 64-65, wherein the cleavage peptide is P2A.

67. The nucleic acid molecule according to any one of claims 64-66, wherein the cleavage peptide P2A is linked to the linker sequence GSG.

68. The nucleic acid molecule according to any one of claims 39 - 67, which can successively encode a CD8 signal peptide, a GPC3 antibody, a CD8 hinge region, a CD28 transmembrane region, a 4 - 1BB co - stimulatory domain, a CD3ζ intracellular signaling domain, a GSG linker sequence, a P2A self - cleaving peptide, an IgGκ signal peptide, a PD - 1 antibody, a V5 tag, (GGGGS)4, and a localization sequence.

69. The nucleic acid molecule according to any one of claims 39 - 67, which can successively encode a CD8 signal peptide, a ROR1 antibody, a CD28 hinge region, a CD28 transmembrane region, a 4 - 1BB co - stimulatory domain, a CD3ζ intracellular signaling domain, a GSG linker sequence, a P2A self - cleaving peptide, an IgGκ signal peptide, a PD - 1 antibody, a V5 tag, (GGGGS)4, and a localization sequence.

70. The nucleic acid molecule according to any one of claims 68 - 69, wherein the amino acid sequence of the PD - 1 antibody is as shown in SEQ ID NO:19, and the amino acid sequence of the localization sequence is as shown in SEQ ID NO:

8.

71. The nucleic acid molecule according to any one of claims 39 - 70, which comprises a nucleic acid sequence encoding a cytokine domain.

72. The nucleic acid molecule according to claim 71, wherein the nucleic acid sequence encoding the cytokine domain can be: (1) a sequence capable of expressing one or more cytokines and / or their functional fragments; (2) a sequence capable of expressing one or more cytokine receptors and / or their functional fragments; and (3) a sequence capable of expressing a combination of (1) and (2).

73. The nucleic acid molecule according to any one of claims 71 - 72, wherein the cytokine is selected from one or more of the following factors: IL - 15, IL - 10, IL - 18, and IL - 21.

74. The nucleic acid molecule according to any one of claims 71 - 73, wherein the cytokine is IL - 15.

75. The nucleic acid molecule according to any one of claims 71 - 73, wherein the cytokine receptor is IL - 15R or its α subunit (IL - 15Rα).

76. A plasmid, which comprises the nucleic acid molecule according to any one of claims 39 - 75.

77. The plasmid according to claim 76, which includes a viral plasmid and a bacterial plasmid.

78. The plasmid according to claim 76, which includes a DNA plasmid and an RNA plasmid.

79. The plasmid according to claim 76, which includes a linear plasmid and a circular plasmid.

80. A pharmaceutical composition, which comprises the iNKT cells according to any one of claims 1 - 38, the nucleic acid molecule according to any one of claims 39 - 75, the plasmid according to any one of claims 76 - 79, and at least one pharmaceutically acceptable carrier. Use of the iNKT cells according to any one of claims 1-38, the nucleic acid molecule according to any one of claims 39-75, the plasmid according to any one of claims 76-79, and the pharmaceutical composition according to claim 80, in the preparation of a medicament for preventing, alleviating, and / or treating a disease and / or disorder.

Images