Combination of immune cell preparation and accelerant

Abstract

The present invention provides a combination comprising an immune cell preparation and a promoter comprising NAD + or a precursor thereof. In the immune cell treatment process, NAD < + > or a precursor thereof is used to promote the killing effect of immune cells.

Description

Technical Field

[0001] The present invention relates to the technical field of immune cell therapy, and more particularly to a combination of an immune cell preparation and a promoter. Background Art

[0002] Cancer is one of the major diseases that pose a serious threat to human health and life. Its high morbidity and mortality, to some extent, reflect the lack of effective clinical prevention and treatment options, posing a significant challenge to cancer prevention and treatment. With the deepening of our understanding of cancer, significant advances have been made in cancer treatment technologies, significantly improving overall cancer treatment outcomes. However, these technologies still present significant side effects and the development of drug resistance. Currently, cancer immunotherapy has emerged as a new therapeutic approach.

[0003] Immune cell therapy includes CAR-T immunotherapy, NK immunotherapy, DC immunotherapy, and TIL immunotherapy. During immune cell therapy, due to the immune escape mechanisms of tumor cells and the complex regulation of cellular immune responses, immune cell activity gradually decreases, proliferation and differentiation decreases, and long-term killing effects are affected. There are multiple approaches to improving the long-term killing effects of immune cells: first, during the immune cell preparation process, improving immune cell metabolism, increasing immune cell activity, and long-term expansion capacity; second, during immune cell therapy, improving the tumor microenvironment and combining it with immunomodulators; and third, for long-term immune cell killing, continuously stimulating immune cells to maintain their killing effects.

[0004] NAD+ (nicotinamide adenine dinucleotide, abbreviated as coenzyme I) is a coenzyme of many dehydrogenases in the body, connecting the tricarboxylic acid cycle and the respiratory chain. Its function is to transfer hydrogen removed during metabolism to flavoproteins. NAD+ can provide cellular energy supply and promote cell regeneration. Currently, there are documents that disclose the use of NAD+ or its precursors in the preparation of CAR-T cells. For example, CN115414375B discloses the use of NMN in the preparation of drugs for extending the lifespan of CD19-CAR-T cells, improving the proliferation ability of CD19-CAR-T cells, and increasing the ratio of Tscm and Tcm in CAR-T cells, and enhancing the efficacy of CD19-CAR-T cell immunotherapy.

[0005] There is currently no literature disclosing the application of NAD+ or its precursors in immune cell therapy. Summary of the Invention

[0006] The purpose of the present invention is to use NAD+ or its precursor in the process of immune cell therapy to promote the killing effect of immune cells.

[0007] The present invention provides a method for treating tumors, comprising:

[0008] (a) administering immune cells to a subject;

[0009] (b) administering to the subject an enhancer comprising NAD+ or a precursor thereof.

[0010] In some embodiments, the immune cell is an immune effector cell (killer cell) or an antigen presenting cell, for example selected from T cells, tumor infiltrating lymphocytes (TIL) cells, natural killer (NK) cells, natural killer T (NKT) cells, DC cells, macrophages and B cells.

[0011] In some embodiments, the immune cell further comprises a coding sequence for a chimeric antigen receptor and / or is capable of expressing a chimeric antigen receptor.

[0012] In some embodiments, the antigen-presenting cells are further loaded with tumor antigens or contain coding sequences for tumor antigens.

[0013] In some embodiments, the immune cells are CAR-T cells.

[0014] In some embodiments, the immune cells further comprise a coding sequence for an immunomodulatory agent and / or are capable of expressing an immunomodulatory agent. In some embodiments, the immunomodulatory agent is an immunosuppressant. In some embodiments, the immunomodulatory agent is an antibody. In some embodiments, the antibody is a single domain antibody. In some embodiments, the single domain antibody is a multispecific single domain antibody.

[0015] In some embodiments, the administration of immune cells is the administration of an immune cell preparation, which includes immune cells and pharmaceutically acceptable excipients, wherein the excipients include a diluent and a cryoprotectant.

[0016] In some embodiments, the diluent is selected from one or more of compound electrolyte injection, 0.9% NaCl injection, and sodium lactate Ringer's injection, preferably compound electrolyte injection.

[0017] In some embodiments, the cryoprotectant is selected from one or more of dextran glucose injection, human serum albumin solution, dimethyl sulfoxide, ethylene glycol, trehalose, sucrose, and glucose, preferably dextran glucose injection, human serum albumin solution, and dimethyl sulfoxide.

[0018] In some embodiments, the excipients include: 40-80% v / v compound electrolyte injection, 5-20% v / v dextran 40 glucose injection, 10-30% v / v human albumin solution, and 5-10% v / v dimethyl sulfoxide.

[0019] In some embodiments, the excipient further comprises vitamins. Preferably, the concentration of the vitamins is 1-20 mg / ml, more preferably 1-15 mg / ml, and more preferably 5-10 mg / ml.

[0020] In some embodiments, the content of the immune cells is a therapeutically effective amount. Preferably, the content of the immune cells in the immune cell preparation is 0.5×10 6 ~2×10 8 / mL, preferably 1.5×10 6 ~5×10 7 pieces / mL.

[0021] In some embodiments, the dose of immune cells administered includes 0.1×10 6 ~2×10 8 cells / kg patient body weight, preferably 1×10 6 ~2×10 7 cells / kg patient body weight, more preferably 5×10 6 ~2×10 7 cells / kg of patient body weight.

[0022] In some embodiments, the NAD+ precursor is selected from one or more of tryptophan, quinolinic acid, nicotinic acid (NA), nicotinamide (NAM), nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), reduced nicotinamide mononucleotide (NMNH), reduced nicotinamide riboside (NRH), reduced nicotinic acid riboside (NARH), reduced nicotinamide adenine dinucleotide (NADH), or a food- or pharmaceutically acceptable salt, derivative, or prodrug thereof; preferably nicotinamide mononucleotide (NMN) or reduced nicotinamide mononucleotide (NMNH).

[0023] In some embodiments, the amount of NAD+ or its precursor administered is 100-1200 mg / day.

[0024] In some embodiments, the enhancer further comprises flavonoids and / or coenzymes.

[0025] In some embodiments, the flavonoid is selected from one or more of quercetin, dihydroquercetin (DHQ), fisetin, catechin, gallin, resveratrol, and hesperetin, preferably quercetin and / or dihydroquercetin.

[0026] In some embodiments, the flavonoid is administered in an amount of 10-50 mg / day.

[0027] In some embodiments, the coenzyme is selected from at least one of glutathione or its derivatives, adenosine triphosphate and its derivatives, pyrroloquinoline quinone or its derivatives, adenosine methionine or its derivatives, coenzyme A or its derivatives, and coenzyme Q or its derivatives, preferably pyrroloquinoline quinone and / or coenzyme Q10.

[0028] In some embodiments, the coenzyme is administered in an amount of 10-100 mg / day.

[0029] In some embodiments, the administering of the immune cells and the administering of the enhancing agent are performed simultaneously.

[0030] In some embodiments, the booster is administered once daily for 3-10 days before and / or 3-30 days after administration of the immune cells.

[0031] In some embodiments, before administering immune cells, the NAD+ level in the subject's blood is assessed and a promoter is administered until the NAD+ level in the subject's blood is no less than 30%, 40%, 50%, 60%, 70% or 80% of the normal level for the age group, and then administration of immune cells is started.

[0032] In some embodiments, when administering immune cells and promoters, other drugs are also administered, such as immunosuppressants, PD-1 antibodies, CTLA4 antibodies, PD-1 / CTLA4 bispecific antibodies, etc.

[0033] In some embodiments, DC cells, a facilitating agent, and a PD-1 antibody, a CTLA4 antibody, or a PD-1 / CTLA4 bispecific antibody are administered.

[0034] The present invention also provides a combination comprising: an immune cell preparation and a promoter, wherein the promoter contains NAD+ or a precursor thereof.

[0035] In some embodiments, the immune cell preparation includes immune cells and pharmaceutically acceptable excipients, wherein the excipients include diluents and cryoprotectants.

[0036] In some embodiments, the diluent is selected from one or more of compound electrolyte injection, 0.9% NaCl injection, and sodium lactate Ringer's injection, preferably compound electrolyte injection.

[0037] In some embodiments, the cryoprotectant is selected from one or more of dextran glucose injection, human serum albumin solution, dimethyl sulfoxide, ethylene glycol, trehalose, sucrose, and glucose, preferably dextran glucose injection, human serum albumin solution, and dimethyl sulfoxide.

[0038] In some embodiments, the excipients include: 40-80% v / v compound electrolyte injection, 5-20% v / v dextran 40 glucose injection, 10-30% v / v human albumin solution, and 5-10% v / v dimethyl sulfoxide.

[0039] In some embodiments, the excipient further comprises vitamins. Preferably, the concentration of the vitamins is 1-20 mg / ml, more preferably 1-15 mg / ml, and more preferably 5-10 mg / ml.

[0040] In some embodiments, the immune cell preparation contains 0.5×10 6 ~2×10 8 / mL, preferably 1.5×10 6 ~5×10 7 pieces / mL.

[0041] In some embodiments, the NAD+ precursor is selected from one or more of tryptophan, quinolinic acid, nicotinic acid (NA), nicotinamide (NAM), nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), or alimentarily or pharmaceutically acceptable salts, derivatives or prodrugs thereof; preferably nicotinamide mononucleotide (NMN).

[0042] In some embodiments, the enhancer further comprises flavonoids and / or coenzymes.

[0043] In some embodiments, the flavonoid is selected from one or more of quercetin, dihydroquercetin (DHQ), fisetin, catechin, gallin, resveratrol, and hesperetin, preferably quercetin and / or dihydroquercetin.

[0044] In some embodiments, the coenzyme is selected from at least one of glutathione or its derivatives, adenosine triphosphate and its derivatives, pyrroloquinoline quinone or its derivatives, adenosine methionine or its derivatives, coenzyme A or its derivatives, and coenzyme Q or its derivatives, preferably pyrroloquinoline quinone and / or coenzyme Q10.

[0045] In some embodiments, in the promoter, the mass ratio of NAD+ or its precursor: flavonoid: coenzyme is 10-120: 1-5: 1-10. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is the result of single-shot killing of H226 tumor cells by CAR-T cells combined with NPQ.

[0047] Figure 2 The results show that CAR-T cells combined with NPQ can continuously kill H226 tumor cells.

[0048] Figure 3 The results show that CAR-T cells combined with NPQ continuously kill H226 tumor cells under serum starvation conditions. DETAILED DESCRIPTION

[0049] definition

[0050] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0051] The term "subject" refers to an animal, including but not limited to a mammal, such as a primate (e.g., a human, chimpanzee, or monkey), a rodent (e.g., a rat, mouse, guinea pig, gerbil, or hamster), a lagomorph (e.g., a rabbit), a bovine (e.g., a cow), a porcine (e.g., a pig), a caprine (e.g., a sheep), an equine (e.g., a horse), a canine (e.g., a dog), or a feline (e.g., a cat). The terms "subject" and "patient" are used interchangeably herein, e.g., to refer to a mammalian subject, e.g., a human subject.

[0052] The term "coding sequence" is defined herein as the portion of a nucleic acid sequence that directly determines the amino acid sequence of its protein product (e.g., CAR, single-chain antibody, hinge region, and transmembrane region). The boundaries of the coding sequence are typically determined by the ribosome binding site (for prokaryotes) immediately upstream of the 5' open reading frame of the mRNA and the transcription termination sequence immediately downstream of the 3' open reading frame of the mRNA. Coding sequences may include, but are not limited to, DNA, cDNA, and recombinant nucleic acid sequences.

[0053] The term "co-stimulatory molecule" refers to a molecule present on the surface of antigen-presenting cells that can bind to the co-stimulatory molecule receptors on Th cells to generate a co-stimulatory signal. The proliferation of lymphocytes requires not only the binding of antigens but also the reception of co-stimulatory molecule signals. Co-stimulatory signals are transmitted to T cells mainly through the binding of co-stimulatory molecules CD80 and CD86 expressed on the surface of antigen-presenting cells to CD28 molecules on the surface of T cells. B cells can receive co-stimulatory signals through common pathogen components such as LPS, or through complement components, or through CD40L on the surface of activated antigen-specific Th cells.

[0054] The term "specific binding" refers to the reaction between an antibody or antigen-binding fragment and the antigen against which it is directed. In certain embodiments, an antibody that specifically binds to an antigen (or is specific for an antigen) means that the antibody binds to the antigen with an affinity (KD) of less than about 10-5 M, such as less than about 10-6 M, 10-7 M, 10-8 M, or 10-9 M or less. "Specific recognition" has a similar meaning.

[0055] The term "chimeric antigen receptor" (CAR) is an artificially modified receptor that can anchor specific molecules (such as antibodies) that recognize cell surface antigens on immune cells, allowing immune cells to recognize cell surface antigens (such as tumor antigens or viral antigens) and kill tumor cells or virus-infected cells. CAR typically comprises an optional signal peptide, an extracellular antigen binding domain (extracellular recognition region, such as an antibody), a hinge region, a transmembrane region, and an intracellular signaling region in sequence. Typically, polypeptides that bind to tumor cell membrane antigens can bind to membrane antigens widely expressed by tumor cells with moderate affinity. The polypeptide that binds to tumor cell membrane antigens can be a natural polypeptide or an artificially synthesized polypeptide.

[0056] The "heavy chain antibodies" described herein are antibodies derived from camelids or cartilaginous fish. Compared to the above-mentioned four-chain antibodies, heavy chain antibodies lack light chains and heavy chain constant region 1 (CH1), and only contain two heavy chains consisting of variable regions (VHH) and other constant regions. The variable regions are connected to the constant regions by a hinge-like structure. Each heavy chain of the camelid heavy chain antibody contains one variable region (VHH) and two constant regions (CH2 and CH3), and each heavy chain of the cartilaginous fish heavy chain antibody contains one variable region and five constant regions (CH1-CH5). The antigen-binding fragment of the heavy chain antibody includes VHH or a single-chain heavy chain antibody. By fusing with the constant region of human IgG Fc, the heavy chain antibody can have the CH2 and CH3 of human IgG Fc.

[0057] "Antibody fragments" comprise a portion of an intact antibody, preferably the antigen-binding and / or variable region of an intact antibody. Antibody fragments are preferably antigen-binding fragments of an antibody. Examples of heavy chain antibody fragments include Fv fragments; diabodies; linear antibodies; single domain antibodies (VHH); single-chain antibody molecules; scFv-Fc fragments; and any fragments that can increase half-life by chemical modification or by incorporation into liposomes.

[0058] As used herein, the terms "nanobody," "single domain antibody," "heavy chain variable region domain of a heavy chain antibody," and "VHH" are used interchangeably to refer to a VHH that specifically recognizes and binds to an antigen. A VHH is the variable region of a heavy chain antibody. Typically, a VHH contains three CDRs and four FRs.

[0059] The term "NAD+ precursor" is any small molecule that results in an increase in NAD+, and the small molecule can exist in reduced or non-reduced form.

[0060] One of the purposes of the present invention is to provide a method for treating tumors, thereby enhancing the immune response of a subject and improving the killing effect of immune cells. The method comprises:

[0061] (a) administering immune cells to a subject;

[0062] (b) administering to the subject an enhancer comprising NAD+ or a precursor thereof.

[0063] immune cells

[0064] In some embodiments, the immune cell is an immune effector cell (killer cell) or an antigen presenting cell, for example selected from T cells, tumor infiltrating lymphocytes (TIL) cells, natural killer (NK) cells, natural killer T (NKT) cells, DC cells, macrophages and B cells.

[0065] In some embodiments, the immune cells are immune effector cells (killer cells), and the killer cells are T cells, TIL cells, or NK cells.

[0066] In some embodiments, the killer cell further contains the coding sequence of a chimeric antigen receptor and / or can express a chimeric antigen receptor. The CAR contains an optional signal peptide sequence, an extracellular target recognition region (antigen binding domain), a hinge region, a transmembrane region, an intracellular costimulatory domain, and an intracellular signal domain. The extracellular recognition region comprises an antibody targeting an antigen (e.g., a tumor antigen), such as a full-length antibody, an antigen binding fragment, a single domain antibody (nanoantibody), or a single-chain variable fragment (scFv).

[0067] In some embodiments, the immune cells are CAR-T cells.

[0068] The target antigens of the chimeric antigen receptor are not limited, including but not limited to: CD19, CD22, CD23, myeloproliferative leukemia protein (MPL), CD30, CD32, CD20, CD70, CD79b, CD99, CD123, CD138, CD179b, CD200R, CD276, CD324, Fc receptor-like 5 (FcRH5), CD171, CS-1 (signaling lymphocyte activation molecule family 7, SLAMF7), C-type lectin-like molecule-1 (CLL-1), CD33, cadherin 1, cadherin 6, cadherin 16, cadherin 17, cadherin 19, epidermal growth factor receptor variant III (EGFRviii), ganglioside GD2, ganglioside G D3, human leukocyte antigen A2 (HLA-A2), B cell maturation antigen (BCMA), Tn antigen, prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), FMS-like tyrosine kinase 3 (FLT3), fibroblast activation protein (FAP), tumor-associated glycoprotein (TAG)-72, CD38, CD44v6, carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), B7-H3 (CD276), KIT, interleukin-13 receptor subunit alpha-2 (IL-13Ra2), interleukin-11 receptor subunit alpha (IL11Ra), mesothelin (MSLN), prostate stem cell antigen (PSCA), vascular endothelial growth factor receptor 2 (VEGFR2), Lewis Y, CD24, platelet-derived growth factor receptor beta (PDGFR-β), protease serine 21 (PRSS21), sialoglycolipid stage-specific embryonic antigen 4 (SSEA-4), CD20, immunoglobulin Fc region, tissue factor, folate receptor alpha, epidermal growth factor receptor 2 (ERBB2), mucin 1 (MUC1), epidermal growth factor receptor (EGFR), neural small adhesion molecule (NCAM), protease, prostatic acid phosphatase (PAP), elongation factor 2 mutant (ELF2M), Ephrin B2, insulin-like growth factor I receptor (IGF-1 receptor), carbonic anhydrase IX (CAIX), latent membrane protein 2 (LMP2), melanocyte protein gpl00, bcr-abl, tyrosinase, erythropoietin-producing hepatocellular carcinoma A2 (EphA2), fucosylated monosialoganglioside (fucosylated GM1), sialyl Lewis a(sLea), ganglioside GM3, transglutaminase 5 (TGS5), high molecular weight melanoma-associated antigen (HMWMAA), o-acetyl GD2 ganglioside, folate receptor β, TEM1 / CD248, tumor endothelial marker-associated protein 7 (TEM7R), claudin6 (CLDN6), thyroid stimulating hormone receptor (TSHR), T cell receptor (TCR)-β1 constant chain, TCRβ2 constant chain, TCRγ-δ, G protein-coupled receptor class C group 5 member D (GPRC5D), CXORF61 protein, CD97, CD179a, anaplastic lymphoma kinase (ALK), polysialic acid, placenta-specific 1 (PLAC1), carbohydrate antigen GloboH, breast differentiation antigen NY-BR-1, uroplakin-2 (UPK2), hepatitis A virus cell receptor 1 (H AVCR1), adrenergic receptor beta 3 (ADRB3), pan-nexin 3 (PANX3), G protein-coupled receptor 20 (GPR20), lymphocyte antigen 6 family member K (LY6K), olfactory receptor family 51 subfamily E member 2 (OR51E2), T cell receptor gamma chain variable reading frame protein (TARP), Wilms tumor antigen 1 protein (WT1), tumor-testis antigen NY-ESO-1, tumor-testis antigen LAGE-1a, legumain, human papillomavirus (HPV) E6, HPV E7, human T-lymphotrophic virus (HTLV1)-Tax, Kaposi's sarcoma-associated herpesvirus glycoprotein (KSHV) K8.1 protein, Epstein-Barr virus (EBV)-encoded glycoprotein 350 (EBB gp350), HIV1 envelope glycoprotein gp120, Multiplexed Automated Genome Engineering (MAGE)-A1, Translocation-Ets-leukemia virus (ETV) protein 6-AML, sperm protein 17, X antigen family member (XAGE) 1, transmembrane tyrosine protein kinase receptor Tie 2. Melanoma tumor-testis antigen MAD-CT-1, melanoma tumor-testis antigen MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survival rate and telomerase, prostate cancer tumor antigen-1 (PCTA-1) / galectin 8, MelanA / MART1, Ras mutant, human telomerase reverse transcriptase (hTERT), delta-like 3 (DLL3), trophoblast cell surface antigen 2 (TROP2), protein tyrosine kinase 7 (PTK7), guanylate cyclase C (GCC), alpha-fetoprotein (AFP), sarcoma translocation breakpoint, melanoma inhibitor of apoptosis (ML-IAP), ERG (TMPRSS2ETS fusion gene), N-acetylglucosyltransferase V (NA17), paired box protein Pax-3 (PAX3), androgen receptor, cyclin B1, v-myc avian myelocytomas viral oncogene neuroblastoma-derived homolog (MYCN), Ras homology family member C (RhoC), tyrosinase-related protein 2 (TRP-2), cytochrome P4501B1 (CYP1B1), CCCTC-binding factor (zinc finger protein)-like (BORIS or Brother of the Regulator of Imprinted Sites), squamous cell carcinoma antigen recognized by T cells 3 (SART3), PAX5, preacrosomal protein-binding protein sp32 (OY-TES1), lymphocyte-specific protein tyrosine kinase (LCK), A kinase anchor protein 4 (AKAP-4), synovial sarcoma, X breakpoint 2 (SSX2), receptor for advanced glycation end products (RAGE-1), renal ubiquitous 1 (RU1), RU2, intestinal carboxylesterase, heat shock protein 70-2 mutant (mut hsp70-2), CD79a, CD79b, CD72, leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), IgA receptor Fc fragment (FCAR), leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2), CD300 molecule-like family member f (CD300LF), C-type lectin domain family 12 member A (CLEC12A), bone marrow stromal cell antigen 2 (BST2), EGF-like module-containing mucin-like hormone receptor-like 2 (EGF-like module-containing mucin-like hormone receptor-like 2, EMR2), lymphocyte antigen 75 (LY75), glypican-3 (GPC3), Fc receptor-like 5 (FCRL5), immunoglobulin lambda-like polypeptide 1 (IGLL1), FITC, luteinizing hormone receptor (LHR), follicle-stimulating hormone receptor (FSHR), chorionic gonadotropin receptor (CGHR), CC chemokine receptor 4 (CCR4), ganglioside GD3, signaling lymphocyte activation molecule (SLAM) family member 6 (SLAMF6), SLAMF4, luteinizing hormone receptor (LHR), follicle-stimulating hormone receptor (FSHR), chorionic gonadotropin receptor (CGHR), or any combination thereof.

[0069] In some embodiments, the extracellular recognition region is an anti-mesothelin single domain antibody, such as described in WO2022143550A1. In some embodiments, the extracellular recognition region is an anti-MUC1 single domain antibody. In some embodiments, the chimeric antigen receptor is as described in PCT / CN2022 / 143407. In some embodiments, the extracellular recognition region is an anti-BCMA single domain antibody. In some embodiments, the anti-BCMA single domain antibody is as described in CN202110301079.1. The entire text of the above-mentioned document or the above-mentioned patent application is incorporated herein by reference to each embodiment thereof specifically and individually.

[0070] The optional signal peptide on the CAR can be selected as needed. For example, a CD8 signal peptide, a CD28 signal peptide, a CD4 signal peptide or a light chain signal peptide, the sequence of which is within the knowledge of those skilled in the art. The CD8 signal peptide suitable for the present invention can be various human CD8 signal peptide sequences commonly used in CAR in the art.

[0071] The hinge region of CAR is selected from CD8α hinge region, IgD hinge region, IgG1 Fc CH2CH3 hinge region or IgG4 FcCH2CH3 hinge region, and its sequence is within the knowledge of those skilled in the art. The CD8 hinge region suitable for the present invention can be various human CD8 hinge region sequences commonly used in CAR in the art.

[0072] The transmembrane region of CAR is selected from one of the CD28 transmembrane region, CD8 transmembrane region, CD3ζ transmembrane region, CD134 transmembrane region, CD137 transmembrane region, ICOS transmembrane region and DAP10 transmembrane region; preferably the CD28 transmembrane region or CD8 transmembrane region, the sequence of which is within the knowledge of those skilled in the art. The human CD28 transmembrane region or CD8 transmembrane region suitable for the present invention can be various human CD28 transmembrane region or CD8 transmembrane region sequences commonly used in CAR in the art.

[0073] Suitable intracellular costimulatory domains can be selected as needed, including intracellular domains with costimulatory signal molecules, such as CD28, CD134 / OX40, CD137 / 4-1BB, lymphocyte-specific protein tyrosine kinase, inducible T cell costimulatory factor (ICOS) and DNAX activating protein 10, preferably CD28 costimulatory domain or 4-1BB costimulatory domain. Human CD28 costimulatory domains or 4-1BB costimulatory domains suitable for the present invention can be various human CD28 costimulatory domain sequences or 4-1BB costimulatory domain sequences commonly used in the art for CAR.

[0074] Similarly, the intracellular signaling domain of CAR can be selected as needed, including but not limited to CD3ζ intracellular signaling domain or FcεRIγ intracellular signaling domain. The CD3ζ intracellular signaling domain suitable for the present invention can be various CD3ζ intracellular signaling domains for CAR known in the art.

[0075] The above-mentioned parts that form the chimeric antigen receptor of the present invention, such as the CD8 signal peptide, antigen binding domain, CD8 hinge region, CD28 transmembrane region, CD28 costimulatory domain, CD3ζ intracellular signaling domain, etc., can be directly connected to each other, or can be connected through a linker sequence. The linker sequence can be a linker sequence suitable for antibodies well known in the art, such as a linker sequence containing G and S. Generally, the linker contains one or more repeated motifs. For example, the motif can be GGGS, GGGGS, SSSSG, GSGSA and GGSGG. Preferably, the motif is adjacent in the linker sequence, and there are no amino acid residues inserted between the repeats. The linker sequence can be composed of 1, 2, 3, 4 or 5 repeated motifs. The length of the linker can be 3 to 25 amino acid residues, for example, 3 to 15, 5 to 15, or 10 to 20 amino acid residues. In certain embodiments, the linker sequence is a polyglycine linker sequence. The number of glycine residues in the linker sequence is not particularly limited and is generally 2 to 20, for example, 2 to 15, 2 to 10, or 2 to 8. In addition to glycine and serine, the linker may also contain other known amino acid residues, such as alanine (A), leucine (L), threonine (T), glutamic acid (E), phenylalanine (F), arginine (R), and glutamine (Q). In certain embodiments, the linker sequence is (GGGGS)n, where n is an integer from 1 to 5.

[0076] In an exemplary embodiment, CAR contains, from N-terminus to C-terminus, a CD8 signal peptide, an anti-MSLN single domain antibody or an anti-MUC1 single domain antibody, a CD8 hinge region, a CD28 transmembrane region or a CD8 transmembrane region, a CD28 costimulatory domain or a 4-1BB costimulatory domain, and a CD3ζ intracellular signaling domain.

[0077] In some embodiments, the immune cell is a CAR-T cell targeting mesothelin. The structure of the CAR is as follows: from N-terminus to C-terminus, it contains a CD8α signal peptide, a mesothelin VHH 1444, a CD8α hinge region, a CD28 transmembrane region and an intracellular costimulatory signaling region, and a CD3ζ intracellular signaling domain; the amino acid sequence of the mesothelin VHH 1444 is shown in SEQ ID NO: 1, and the amino acid sequence of the CAR is shown in SEQ ID NO: 2.

[0078] In some embodiments, the immune cell is an antigen presenting cell.

[0079] In some embodiments, the antigen-presenting cells are further loaded with tumor antigens or contain coding sequences for tumor antigens.

[0080] In some embodiments, the immune cells further comprise coding sequences for a therapeutic agent; and / or are capable of expressing and / or secreting a therapeutic agent. In some embodiments, the therapeutic agent is an immunomodulatory agent, such as an antibody. In some embodiments, the therapeutic agent is an immune checkpoint inhibitor. In some embodiments, the antibody is a single domain antibody. In some embodiments, the single domain antibody is a multispecific single domain antibody.

[0081] The tumor antigen is selected from tumor-associated antigens and tumor neoantigens; preferably, the antigen-presenting cell expresses the tumor antigen; more preferably, the tumor antigen is selected from one or more of the following: hTERT, p53, Her2, Survivin, CEA, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-C1, MAGE-C2, MUC1, Wilms' tumor 1 (WT1), Her2-neu, P53, NY-ESO-1, hTERT, Mammaglobin-A, Folate Receptor α (FR-α), HPV16 / 18-E6, HPV16 / 18-E7, alpha-fetoprotein (AFP), Glypican3 (GPC3), prostate-specific antigen (PSA), prostatic acid phosphatase (PAP), prostate-specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), prostate transmembrane epithelial antigen 1 (STEAP1), B cell maturation antigen (BCMA), CMV pp65, gp100, PRAME; further preferably, the tumor antigens include any two or three of CEA, survivin and p53.

[0082] In some embodiments, the coding sequence of the tumor antigen is RNA, preferably mRNA.

[0083] In some embodiments, the antibody contains a functional region that targets a target.

[0084] In some embodiments, the multispecific single-domain antibody contains multiple functional regions that respectively target multiple targets, and each of the multiple functional regions is a single-domain antibody.

[0085] In some embodiments, the target is selected from the group consisting of immune checkpoint proteins, immune cell-associated antigens, tumor-associated antigens, immune co-stimulatory molecules, or their receptors. Immune checkpoint proteins include, but are not limited to, PD-1, CTLA4, PDL1, PDL2, PDL3, TIM3, LAG3, CD47, BTLA, TIGIT, CD160, LAIR1, B7-H1, B7-1, VSIR, and CD244. Immune cell-associated antigens include, but are not limited to, CD28, CD137, CD134, CD40, CD40L, ICOS, HVEM, CD2, CD27, CD30, GITR, LIGHT, DR3, SLAM, CD226, CD80, and CD86. Tumor-associated antigens include, but are not limited to, EIIIB fibronectin, Siglec15, VEGF(R), HER2, PSMA, AXL, MUC1, and MUC16.

[0086] In one or more embodiments, at least one of the plurality of functional domains is an immune checkpoint inhibitory single domain antibody.

[0087] In one or more embodiments, at least one of the plurality of functional domains is an activating antibody to an immune co-stimulatory molecule or its receptor.

[0088] In one or more embodiments, the multiple functional regions are different immune checkpoint inhibitory single domain antibodies.

[0089] In one or more embodiments, the multispecific single-domain antibody contains two or three functional regions that target two or three targets, respectively.

[0090] In some embodiments, the therapeutic agent is an antibody targeting PD-1, preferably a single-domain antibody targeting PD-1. The sequence of the single-domain antibody targeting PD-1 is the single-domain antibody targeting PD-1 described in any embodiment of patent CN202011582908.X, the entire contents of which are incorporated herein by reference.

[0091] In some embodiments, the sequence of the single-domain antibody targeting PD-1 is shown in any one of SEQ ID NOs: 3-6.

[0092] In some embodiments, the therapeutic agent is an antibody targeting CTLA-4, preferably a single-domain antibody targeting CTLA-4. The sequence of the single-domain antibody targeting CTLA-4 is the single-domain antibody targeting CTLA-4 described in any embodiment of patent CN202111152925.4, the entire contents of which are incorporated herein by reference.

[0093] In some embodiments, the sequence of the single-domain antibody targeting CTLA-4 is shown in SEQ ID NO:7.

[0094] In some embodiments, the therapeutic agent is a bispecific antibody comprising a first domain targeting PD-1 and a second domain targeting CTLA4. In some embodiments, the bispecific antibody is a bispecific antibody as described in any embodiment of patent CN202310338674.1, the entire contents of which are incorporated herein by reference.

[0095] In some embodiments, the coding sequence of the multispecific single-domain antibody is DNA or RNA, preferably mRNA or saRNA (self-replicating RNA).

[0096] In one or more embodiments, the multispecific Nanobody further contains an Fc region; preferably, the Fc region is the Fc region of IgG1, IgG2, IgG3 or IgG4; and the IgG1, IgG2, IgG3 or IgG4 is of human origin.

[0097] Since the Fc region can cause significant ADCC and CDC effects, it may cause immune cell damage and have negative pharmacological effects. Therefore, Fc is usually modified (also referred to as variant Fc in this article). By site mutation, the affinity constant of the multispecific nanobody to FcγRIIIa and / or C1q is reduced compared to before the mutation, thereby improving the efficacy of the antibody drug. The currently disclosed mutation sites, according to the EU numbering system, include: Fc region mutation sites in IgG1 include L234A, L235A, L235E, L235G, G236A, G237A, N297A, G318A, L320A, L322A; Fc region mutation sites in IgG3 include V234A, G237A, P238S, H28A, V309L, A330S, P331 S; IgG3 Fc region mutation sites include Leu281, Leu282, Gly283, Gly284, Asn344, Pro378; IgG4 Fc region mutation sites include S228P, E233P, F234V, L235A, F243L, D254A, R292P, Y300L, L309V, R409K. The variant Fc herein includes, but is not limited to, the Fc of each IgG having the above mutation sites.

[0098] In one or more embodiments, the Fc region is an IgG1 Fc region, and according to the EU numbering system, the Fc region has one or more of the following mutations: L234A, L235A, G237A. In one or more embodiments, the Fc region is an IgG4 Fc region, and according to the EU numbering system, the Fc region has one or more of the following mutations: S228P, E233P, F234V, L235A, D254A, L309V, R409K.

[0099] Immune cell preparations

[0100] The immune cells of the present invention can be administered alone or in combination with a diluent and / or with other components such as related cytokines or cell groups as a pharmaceutical composition. Briefly, the pharmaceutical composition of the present invention may include CAR-T cells as described herein, in combination with one or more pharmaceutically or physiologically acceptable adjuvants (e.g., carriers, diluents, or excipients). Such compositions may include buffers such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0101] In some embodiments, a pharmaceutical composition comprises the immune cells described in any embodiment herein and an adjuvant.

[0102] In some embodiments, excipients in the pharmaceutical composition include diluents and cryoprotectants.

[0103] The diluent preferably maintains the pH, osmotic pressure, and other balances of the pharmaceutical composition while performing a diluting effect. For example, the diluent may be selected from one or more of compound electrolyte injection, 0.9% NaCl injection, and sodium lactate Ringer's injection. The concentration of the diluent in the pharmaceutical composition may be 40-80% v / v, preferably 60-70% v / v.

[0104] The ingredients of compound electrolyte injection are well known in the art and may include sodium chloride, sodium gluconate, sodium acetate, potassium chloride, magnesium chloride, etc.

[0105] Cryoprotectants can prevent cell damage during cryopreservation of the pharmaceutical composition, and can be selected from, for example, one or more of dextran glucose injection, human albumin solution, dimethyl sulfoxide, ethylene glycol, trehalose, sucrose, glucose, and the like. The concentration of the cryoprotectant in the pharmaceutical composition can be 20-60% v / v, preferably 30-40% v / v.

[0106] Dextran is also known as dextran. Specific models of dextran include dextran-40 (also known as dextran 40, Dextran-40) and dextran-70 (also known as dextran 70, Dextran-70). Preferably, the dextran is dextran-40.

[0107] The human albumin solution is, for example, a human albumin injection, the ingredients of which are well known in the art and may include human albumin and optional other excipients (e.g., sodium octanoate, acetyltryptophan, sterile water for injection, etc.). In the human albumin solution, the concentration of human albumin is generally in the range of 5-25% (w / v). For example, human albumin injection can be used in different specifications, such as 2g (albumin concentration 5%), 40ml / bottle; 5g (20%), 25ml / bottle; 5g (10%), 50ml / bottle; 10g (20%), 50ml / bottle, etc.; 12.5g (25%), 50ml / bottle, etc. In some embodiments, the human albumin solution is 25% human albumin (i.e., the concentration of human albumin is 5-25% w / v).

[0108] In some embodiments, the cryoprotectant includes dextran glucose injection, human serum albumin solution, and dimethyl sulfoxide.

[0109] In some embodiments, the excipients include: 40-80% v / v compound electrolyte injection, 5-20% v / v dextran 40 glucose injection, 10-30% v / v human albumin solution (25% human albumin), and 5-10% v / v dimethyl sulfoxide. The human albumin solution may also be selected from other concentrations of human albumin, and its concentration in the excipients can be adjusted accordingly. In some embodiments, the excipients include: 60-70% v / v compound electrolyte injection, 5-15% v / v dextran 40 glucose injection, 15-25% v / v human albumin solution (25% human albumin), and 5-10% v / v dimethyl sulfoxide. In some embodiments, the excipients include: 65% v / v compound electrolyte injection, 10% v / v dextran 40 glucose injection, 20% v / v human albumin solution (25% human albumin) and 5% v / v dimethyl sulfoxide.

[0110] In some embodiments, the excipients may also include vitamins. Adding vitamins can further enhance the survival rate and proliferation of cells after recovery. Preferably, the vitamins include (but are not limited to): vitamin A, vitamin B, vitamin C, vitamin D, vitamin E, vitamin K, or a combination thereof. More preferably, the vitamin is vitamin C. Preferably, the concentration of the vitamins in the pharmaceutical composition is 1-20 mg / ml, preferably 1-15 mg / ml, and even more preferably 5-10 mg / ml.

[0111] In a preferred embodiment, the excipients include the following components in percentage by volume and concentration:

[0112] Human albumin 20-80mg / ml dimethyl sulfoxide 0.02-0.08ml / ml Dextran 3-12mg / ml glucose 2-10mg / ml Sodium chloride 2-5 mg / mL Sodium gluconate 2-5 mg / mL Sodium acetate 1-5 mg / mL potassium chloride 0.1-0.5 mg / mL magnesium chloride 0.1-0.5 mg / mL The balance is water.

[0113] Preferably, the excipient includes the following components in the following concentrations:

[0114]

[0115]

[0116] More preferably, the excipient comprises the following components in the following concentrations:

[0117] Human albumin 37.5-45 mg / mL dimethyl sulfoxide 0.05-0.055mL / mL Dextran 6-6.6 mg / mL glucose 5-5.5 mg / mL Sodium chloride 3.47-3.65 mg / mL Sodium gluconate 3.31-3.5 mg / mL Sodium acetate 2.43-2.56 mg / mL potassium chloride 0.24-0.26 mg / mL magnesium chloride 0.2-0.24 mg / mL and water.

[0118] In another preferred embodiment, the weight ratio of sodium chloride, sodium gluconate, sodium acetate, potassium chloride and magnesium chloride is (4.5-5.5): (4.5-5.5): (3.2-4.2): (0.32-0.42): (0.26-0.34).

[0119] In another preferred embodiment, the ratio of human serum albumin to dimethyl sulfoxide is (45-65 mg): (0.05-0.075 ml), preferably (37.5-45 mg): (0.05-0.055 ml).

[0120] In another preferred embodiment, the weight ratio of human serum albumin to dimethyl sulfoxide is 400-1000:1, preferably 680-900:1.

[0121] In another preferred embodiment, the weight ratio of human serum albumin to dextran is 3-12:1, preferably 4-10:1, and more preferably 5.5-7.5:1.

[0122] In another preferred embodiment, the weight ratio of dextran to glucose is 0.8-1.6, preferably 1.0-1.5.

[0123] In another preferred embodiment, the weight ratio of glucose to sodium chloride is 0.5-6:1, preferably 0.6-5:1.

[0124] In another preferred embodiment, the water is purified water or water for injection.

[0125] In some embodiments, the amount of CAR-T cells in the pharmaceutical composition can be adjusted according to the target dose (i.e., the number of CAR-T cells per kg of patient body weight). In some embodiments, the amount of CAR-T cells can be 0.5×10 6 ~2×108 / mL, for example 0.5×10 6 / mL, 1×10 6 / mL, 2×10 6 / mL, 3×10 6 / mL, 4×10 6 / mL, 5×10 6 / mL, 6×10 6 / mL, 7×10 6 / mL, 8×10 6 / mL, 9×10 6 / mL, 1×10 7 / mL, 2×10 7 / mL, 3×10 7 / mL, 4×10 7 / mL, 5×10 7 / mL, 6×10 7 / mL, 7×10 7 / mL, 8×10 7 / mL, 9×10 7 / mL, 1×10 8 / mL, 2×10 8 / mL, preferably 1×10 6 ~1×10 8 / mL, more preferably 5×10 6 ~2×10 7 pieces / mL.

[0126] In some embodiments, the pharmaceutical composition can be obtained by mixing the prepared immune cells with excipients. Taking CAR-T as an example, the cell stock solution preparation process is divided into four steps: sorting process, activation process, electroporation process, and culture process. The sorting process can be a single blood sample washed and then CD3 + T cells, CD3 antibody / 4-1bbLprotein as the first / second activation signal to activate CD3 +T cells are activated and then gene transduced by electroporation. After transduction, they are cultured and expanded, and the infusion dose is reached and centrifuged to obtain the prepared CAR-T cells (total viable cells). The density of the total viable cells in the pharmaceutical composition can be adjusted according to the target dose of CAR-T cells administered to the patient. In some embodiments, the density of viable cells in the pharmaceutical composition = (patient weight × target dose × loss multiple) ÷ CAR-positive cell ratio ÷ pharmaceutical composition volume. The loss multiple refers to the loss of cells during the preparation process and / or cryopreservation recovery of the pharmaceutical composition. Therefore, when preparing the composition, the target amount is multiplied by the loss multiple, for example, 1-1.5, for example, 1, 1.1, 1.2, 1.3, 1.4, 1.5. The proportion of CAR-positive cells can be detected by conventional methods in the art, such as flow cytometry.

[0127] Accelerator

[0128] In some embodiments, the NAD+ precursor is selected from one or more of tryptophan, quinolinic acid, nicotinic acid (NA), nicotinamide (NAM), nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), reduced nicotinamide mononucleotide (NMNH), reduced nicotinamide riboside (NRH), reduced nicotinic acid riboside (NARH), reduced nicotinamide adenine dinucleotide (NADH), or a food or pharmaceutically acceptable salt, derivative, or prodrug thereof; preferably nicotinamide mononucleotide (NMN) or reduced nicotinamide adenine dinucleotide (NADH). NMN, as a direct precursor of NAD+, can be easily converted directly into NAD+ by the NMNAT enzyme and is easily absorbed by cells.

[0129] In some embodiments, the enhancer further comprises flavonoids and / or coenzymes.

[0130] In some embodiments, flavonoids have anti-free radical and antioxidant effects, and can delay cell apoptosis. In some embodiments, the flavonoids are selected from one or more of quercetin, dihydroquercetin, fisetin, catechin, gallin, resveratrol, and hesperetin, preferably quercetin and / or dihydroquercetin. Among them, quercetin is a plant flavonol from the flavonoid group of polyphenols, which is present in many fruits, vegetables, leaves, seeds and grains. It is a naturally occurring polar auxin transport inhibitor. Dihydroquercetin is a dihydroflavonol compound that belongs to the vitamin P family. It is a widely used bioactive agent with multiple biological activities in the human body, including antioxidant, free radical scavenging and other effects.

[0131] In some embodiments, coenzyme is a general term for a large class of organic cofactors, which are essential factors for enzyme-catalyzed redox reactions, group transfer and isomerization reactions. In some embodiments, the coenzyme is selected from at least one of glutathione or its derivatives, adenosine triphosphate and its derivatives, pyrroloquinoline quinone or its derivatives, adenosine methionine or its derivatives, coenzyme A or its derivatives, coenzyme Q or its derivatives, preferably pyrroloquinoline quinone and / or coenzyme Q10. Pyrroloquinoline quinone (PQQ) is an oxidoreductase cofactor with the chemical formula C 14 H6N2O8 participates in a variety of redox reactions and mediates a series of cellular and molecular biochemical reactions by acting as a coenzyme, thereby affecting the physiological and metabolic processes of the human body.

[0132] Application method and dosage

[0133] In some embodiments, the pharmaceutical composition administered by the present invention can be prepared some time before administration to the patient (e.g., 15 to 30 days). After preparation, the pharmaceutical composition can be frozen (e.g., stored below -120°C) and resuscitated when needed so that the pharmaceutical composition can be infused when the patient is ready.

[0134] In some embodiments, a pharmaceutical composition according to any embodiment of the present invention is administered to a patient in need thereof, thereby delivering a dose of CAR-T cells to the patient. The dose may be 0.5×10 6 ~2×10 8 CAR-T cells / kg patient weight, for example 0.5×10 6 , 1×10 6 , 2×10 6 , 3×10 6 , 4×10 6 , 5×10 6 , 6×10 6 , 7×10 6 , 8×10 6 , 9×10 6 , 1×10 7 , 1.5×10 7 , 2×10 7 , 3×10 7 , 4×10 7 , 5×10 7 , 6×10 7 , 7×10 7 , 8×10 7 , 9×10 7 , 1×10 8 , 2×10 8 CAR-T cells / kg patient body weight. In a preferred embodiment, the dose is 1×10 6~2×10 7 CAR-T cells / kg patient weight, more preferably 5×10 6 ~2×10 7 CAR-T cells / kg patient body weight.

[0135] Administration of the pharmaceutical composition can be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation or transplantation. The compositions described herein can be administered to the patient subcutaneously, intradermally, intratumorally, intranodally, intraspinal, intramuscularly, by intravenous injection or intraperitoneally. In one embodiment, the pharmaceutical composition of the present invention is administered to the patient by intradermal or subcutaneous injection. In another embodiment, the pharmaceutical composition of the present invention is preferably administered by intravenous injection. The pharmaceutical composition can be injected directly into the tumor, lymph node or infection site.

[0136] In some embodiments, the treatment method comprises administering a single dose of the pharmaceutical composition to the patient, or administering the pharmaceutical composition multiple times. Optionally, the multiple administrations may be administered in an ascending dose or equal dose mode. The dosing interval may be selected based on actual conditions, preferably being at least 7 days, for example, 7 to 28 days. The number of administrations may be 1 to 3 times.

[0137] A period of time before administration of the pharmaceutical composition (e.g., 5-7 days before administration) may be preceded by a lymphocyte depletion regimen. In some embodiments, the lymphocyte depletion regimen comprises administering cyclophosphamide and / or fludarabine to the patient, e.g., administering 300 mg / m2 of cyclophosphamide to the patient. 2 / day and fludarabine 30 mg / m 2 / day for three days.

[0138] In some embodiments, when administering immune cells and promoters, other drugs are also administered, such as immunosuppressants, PD-1 antibodies, CTLA4 antibodies, PDL1 antibodies, PD-1 / CTLA4 bispecific antibodies, etc.

[0139] Administration of the enhancer can be performed in any convenient manner, including by spraying, injection, swallowing, infusion, implantation, or transplantation. Because the enhancer primarily comprises NAD+ or its precursors, flavonoids, and / or coenzymes, the enhancer components are relatively low in toxicity. Therefore, the enhancer components can be added to the immune cell preparation, and the immune cells and enhancer can be administered simultaneously.

[0140] In some embodiments, the facilitating agent is orally administered simultaneously with or on the same day as the immune cells are administered.

[0141] In some embodiments, the enhancing agent is administered 1-3 times daily within 3-10 days before and / or within 3-30 days after administration of the immune cells.

[0142] In some embodiments, before administering immune cells, the NAD+ level in the subject's blood is assessed and a promoter is administered until the NAD+ level in the subject's blood is no less than 30%, 40%, 50%, 60%, 70% or 80% of the normal level for the age group, and then administration of immune cells is started.

[0143] In some embodiments, the amount of NAD+ or its precursor administered is 100-1200 mg / day, such as 100 mg, 200 mg, 300 mg, 600 mg, 900 mg or 1200 mg.

[0144] In some embodiments, the flavonoid is administered in an amount of 10-50 mg / day, such as 10 mg, 20 mg, 30 mg, 40 mg, or 50 mg.

[0145] In some embodiments, the coenzyme is administered in an amount of 10-100 mg / day, such as 10 mg, 20 mg, 40 mg, 60 mg, 80 mg, or 100 mg.

[0146] combination

[0147] The present invention also provides a combination comprising: immune cells and a promoter, wherein the promoter comprises NAD+ or its precursor. In some embodiments, the combined preparation comprises: an immune cell preparation and a promoter, wherein the immune cell preparation is the immune cell preparation described in any of the above embodiments.

[0148] The immune cells and promoter can be packaged separately, or the promoter can be added to the composition during the preparation of the immune cell preparation.

[0149] In some embodiments, the promoter is added to the immune cell preparation during the preparation of the immune cell preparation, for example, before programmed cooling, or before administration of the immune cell preparation, to avoid prolonged contact of the promoter with the immune cells, resulting in consumption.

[0150] In some embodiments, the immune cell preparation includes immune cells and pharmaceutically acceptable excipients, wherein the excipients include diluents and cryoprotectants.

[0151] In some embodiments, the diluent is selected from one or more of compound electrolyte injection, 0.9% NaCl injection, and sodium lactate Ringer's injection, preferably compound electrolyte injection.

[0152] In some embodiments, the cryoprotectant is selected from one or more of dextran glucose injection, human serum albumin solution, dimethyl sulfoxide, ethylene glycol, trehalose, sucrose, and glucose, preferably dextran glucose injection, human serum albumin solution, and dimethyl sulfoxide.

[0153] In some embodiments, the excipients include: 40-80% v / v compound electrolyte injection, 5-20% v / v dextran 40 glucose injection, 10-30% v / v human albumin solution, and 5-10% v / v dimethyl sulfoxide.

[0154] In some embodiments, the excipient further comprises vitamins. Preferably, the concentration of the vitamins is 1-20 mg / ml, more preferably 1-15 mg / ml, and more preferably 5-10 mg / ml.

[0155] In some embodiments, the immune cell preparation contains 0.5×10 6 ~2×10 8 / mL, preferably 1.5×10 6 ~5×10 7 pieces / mL.

[0156] In some embodiments, the dosage form of the immune cell preparation is an immune cell injection.

[0157] In some embodiments, the NAD+ precursor is selected from one or more of tryptophan, quinolinic acid, nicotinic acid (NA), nicotinamide (NAM), nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), or alimentarily or pharmaceutically acceptable salts, derivatives or prodrugs thereof; preferably nicotinamide mononucleotide (NMN).

[0158] In some embodiments, the enhancer further comprises flavonoids and / or coenzymes.

[0159] In some embodiments, the flavonoid is selected from one or more of quercetin, dihydroquercetin (DHQ), fisetin, catechin, gallin, resveratrol, and hesperetin, preferably quercetin and / or dihydroquercetin.

[0160] In some embodiments, the coenzyme is selected from at least one of glutathione or its derivatives, adenosine triphosphate and its derivatives, pyrroloquinoline quinone or its derivatives, adenosine methionine or its derivatives, coenzyme A or its derivatives, and coenzyme Q or its derivatives, preferably pyrroloquinoline quinone and / or coenzyme Q10.

[0161] In some embodiments, the promoter is in an oral dosage form, and the mass ratio of NAD+ or its precursor: flavonoid: coenzyme is 10-120: 1-5: 1-10.

[0162] In some embodiments, the enhancer is a tablet comprising 300 mg NAD+ or its precursor, 10 mg flavonoids and 20 mg coenzyme.

[0163] In some embodiments, the enhancer is a tablet comprising 300 mg NMN, 10 mg PQQ, and 20 mg dihydroquercetin.

[0164] In some embodiments, the combined preparation of the present invention includes a packaged immune cell injection solution and a tablet enhancer.

[0165] The present invention also provides application of the combined preparation in treating tumors.

[0166] In the present invention, the tumor is a solid cancer, for example, selected from: mesothelioma, malignant pleural mesothelioma, non-small cell lung cancer, small cell lung cancer, squamous cell lung cancer, large cell lung cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, esophageal adenocarcinoma, breast cancer, glioblastoma, ovarian cancer, colorectal cancer, prostate cancer, cervical cancer, skin cancer, melanoma, kidney cancer, liver cancer, brain cancer, thymoma, sarcoma, carcinoma, uterine cancer, kidney cancer, gastrointestinal cancer, urothelial carcinoma, pharyngeal cancer, head and neck cancer, rectal cancer, esophageal cancer or bladder cancer, or one or more of their metastatic cancers.

[0167] In some embodiments, the cancer is a liquid cancer, for example, selected from the group consisting of chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), multiple myeloma, acute lymphocytic leukemia (ALL), Hodgkin lymphoma, B-cell acute lymphoblastic leukemia (BALL), T-cell acute lymphoblastic leukemia (TALL), small lymphocytic leukemia (SLL), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, diffuse large B-cell lymphoma (DLBCL), DLBCL associated with chronic inflammation, chronic myeloid leukemia, myeloproliferative neoplasms, follicular lymphoma, pediatric follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative disorders, MALT lymphoma (extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue), marginal Marginal zone lymphoma, myelodysplasia, myelodysplastic syndrome, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia, splenic marginal zone lymphoma, splenic lymphoma / leukemia, splenic diffuse red pulp small B-cell lymphoma, hairy cell leukemia variant, lymphoplasmacytic lymphoma, heavy chain disease, plasma cell myeloma, solitary plasmacytoma of bone, extraosseous plasmacytoma, marginal lymph node Primary mediastinal (thymic) large B-cell lymphoma, pediatric marginal zone lymphoma, primary cutaneous follicle center lymphoma, lymphomatoid granulomatosis, primary mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, ALK+ large B-cell lymphoma, large B-cell lymphoma arising in HHV8-associated multicentric Castleman disease, primary effusion lymphoma, B-cell lymphoma, acute myeloid leukemia (AML), or unclassifiable lymphoma.

[0168] The present invention will be described below by way of specific examples. It should be understood that these examples are merely illustrative and are not intended to limit the scope of the present invention. The methods and materials used in the examples are, unless otherwise stated, conventional materials and methods in the art.

[0169] Experimental methods:

[0170] Example 1: Preparation of Mesothelin-Targeted CAR-T Cells

[0171] Use CAR-T culture medium (AIM-V + 2% FBS + 100IU IL-2) and 5ug / mL anti-CD3 and anti-CD28 to coat 6-well plates or culture flasks at 4°C overnight. The revived PBMCs were inoculated into the coated 6-well plates or culture flasks and stimulated for 48 hours for activation. The cells were collected and electroporated using a Lonza 4D electroporator. The electroporation process was optimized according to the electroporator manual. Resuspend 5×10 6 to 7×10 6 For PBMC cells, 4ug P19V21 plasmid and 4ug P20S23 plasmid were added to the cell suspension. At the same time, 20ug PB mRNA (the amino acid sequence of the PB enzyme is shown in SEQ ID NO: 8) and 200IU Rnase inhibitor were added to the electroporation system, and electroporation was performed using the EO-115 program. After electroporation, the cells were allowed to stand at room temperature for 10 minutes. Afterwards, the PBMCs were transferred to a 6-well plate in the culture medium (which needed to be preheated to 37°C in advance), placed in an incubator (37°C; 5% CO2) and cultured for 24h-48h, and then the culture medium was supplemented to 4mL. The first passage was performed on the 3rd day after electroporation, the second passage was performed on the 6th day, and the CAR-T cell preparation was completed on the 10th day. The PBMCs during the passage process were cultured in CAR-T culture medium, and the cell density was 5×10 5 / ml.

[0172] The P19V21 plasmid contains the MSLN CAR plasmid. The MSLN CAR structure is as follows: from N-terminus to C-terminus, it contains the CD8α signal peptide, mesothelin VHH No. 1444, CD8α hinge region, CD28 transmembrane region and intracellular costimulatory signal region, and CD3ζ intracellular signal domain. The amino acid sequence of mesothelin VHH No. 1444 is shown in SEQ ID NO: 1, and the amino acid sequence of MSLN CAR is shown in SEQ ID NO: 2. The nucleotide sequence of the P19V21 plasmid is shown in SEQ ID NO: 9.

[0173] The above-mentioned P20S23 plasmid is a PD-1 nanobody plasmid, the nucleotide sequence of the plasmid is shown in SEQ ID NO: 10, and the PD-1 nanobody is human immunoglobulin κ light chain signal peptide and PD-1VHH in sequence; the amino acid sequence of PD-1VHH is shown in SEQ ID NO: 6.

[0174] Example 2, Preparation of Composition of CAR-T Preparation

[0175] After the culture is completed, a portion of the CAR-T cells is taken for quality control, and the remaining cells are prepared into a preparation (drug composition) and aliquoted into 25 mL Cryotore bags (Origen Biomedical, Cat# CS50), placed in a controlled rate CryoMed Freezers 7451 (Thermo Scientific), and then stored in liquid nitrogen. The preparation process is as follows:

[0176] ① Preparation of freezing solution

[0177] The maximum number of finished product bags was estimated based on the total cell volume, and the maximum volume of freezing solution was prepared. The freezing solution preparation ratio was: compound electrolyte: dextran glucose injection: 25% human serum albumin: dimethyl sulfoxide (DMSO) = 65:10:20:5.

[0178] ② Preparation: To a 500mL centrifuge cup, add 65mL of compound electrolytes, 10mL of dextran 40% glucose injection, 20mL of 25% human serum albumin, and 5mL of dimethyl sulfoxide per 100mL of cryopreservative solution. Mix thoroughly by pipetting 10-15 times with a 50mL pipette and store at 2-8°C until ready to use.

[0179] ③ Centrifuge and remove the supernatant

[0180] The cell stock solution was centrifuged. After centrifugation, the supernatant in the 500 mL centrifuge tube was discarded using a negative pressure pump.

[0181] ④Add freezing solution to adjust cell density

[0182] Filling viable cell density = (patient weight × target dose × 1.2 times) ÷ CD3 + MSLN CAR + The T cell ratio is divided into 25mL, and the cell volume of the preparation is actually canned at 1.2 times the target dose.

[0183] Use a 10mL pipette to add an appropriate amount of freezing solution to a 500mL centrifuge tube. After mixing thoroughly, transfer 500μL of the sample to a 1.5mL centrifuge tube and transfer it to the CAR-T cell preparation station through the rapid transfer port for counting. Use a pipette to accurately measure the volume of the cell suspension and calculate the total number of cells.

[0184] According to the cell counting results, fill the preparation according to the required live cell density and use a pipette to add freezing solution to adjust the live cell density. 6 ~1.1×10 8 cells / mL, according to 5×10 6 ~1.0×10 8 cells / mL for filling.

[0185] ⑤Filling

[0186] The mixed cell suspension was transferred into the freezing bag using a pipette through a 50 mL sterile syringe at a rate of 25 mL / bag. After filling, the suspension was immediately stored at 4°C.

[0187] ⑥Program-controlled cooling and freezing

[0188] The cells were placed in a programmed cooling apparatus for programmed cooling, and then stored in a liquid nitrogen tank at below -120°C.

[0189] Example 3: CAR-T cell combined with promoter to kill tumor cells in vitro

[0190] In this example, the in vitro pharmacodynamic properties of the CAR-T cell combination enhancer were evaluated by testing its effect on the specific killing ability of CAR-T cell target cells after co-culture with mesothelin-positive tumor cells. The target cells were H226, and the effector-target ratio (E:T) was 1:1.

[0191] The killing effect of CAR-T cells on tumor cells was detected using xCELLigence RTCA equipment (Roche Applied Science, Canada). The detection process was carried out according to the equipment operating instructions. 3 -2×10 4 Target cells (tumor cells) are resuspended in 50 μl of culture medium and inoculated into 16-well or 96-well microplates that come with the RTCA instrument. Tumor cells are cultured on the RTCA instrument for approximately 24 hours. When the cell index (CI, an indicator of cell growth) reaches approximately 1.5, CAR-T cells are resuspended in 50 μl of culture medium according to the effector-target ratio (effector cell: target cell). The corresponding promoter is added and mixed with the tumor cells. Culture is continued on the RTCA instrument for 3-5 days. Cell killing curves are recorded by the RTCA instrument, and the data are analyzed using RTCA Pro 2.3.0 software.

[0192] The combinations of CAR-T cells and promoters are shown in Table 1 below:

[0193] Table 1 Combinations of CAR-T cells and promoters

[0194]

[0195] Single kill results:

[0196] The cell lysis rate of the Ctrl group was set as 1, and the relative killing results of group 2 were as follows: Figure 1 As shown, the NPQ group can enhance the single-shot killing of tumor cells by CAR-T. The target cells are H226, and the effector-target ratio (E:T) is 1:1.

[0197] Continuous kill results:

[0198] After completing the single killing result, tumor cells were re-inoculated in the microplate, and then the CAR-T cells that completed the single killing were added to the microplate for culture to test the continuous killing effect of CAR-T cells. Figure 2 As shown in the figure, in the first round of killing (R1), the killing rates of the Ctrl and NPQ groups were 42.49% and 42.41%, respectively. In the second round of killing (R2), the killing rates of the Ctrl and NPQ groups were 15.88% and 25.30%, respectively. Therefore, the addition of NPQ can significantly enhance the continuous killing ability of CAR-T cells.

[0199] Example 4: Continuous killing of tumor cells under serum starvation conditions

[0200] Similar to Example 3, except that during the co-culture of CAR-T cells (or promoter combination) and tumor cells, no animal serum or SR (serum replacement) components were added to the culture medium to evaluate the continuous killing of tumor cells under serum starvation conditions.

[0201] The combinations of CAR-T cells and promoters are shown in Table 2 below:

[0202] Table 2 Combinations of CAR-T cells and promoters

[0203]

[0204]

[0205] Continuous kill results:

[0206] Comparison of results of groups 1-3 Figure 3 As shown, under serum-starved conditions (patient status), the tumor cell killing rate was high. Throughout the four rounds of killing (R1, R2, R3, and R4), the NPQ group consistently outperformed both the Ctrl and NMN groups. Therefore, the addition of NPQ can enhance the ability of CAR-T cells to continuously kill tumor cells under serum-starved conditions.

[0207] Partial sequence

[0208] SEQ ID NO:1_1444VHH

[0209] QVQVVESGGGFVQAGGSLRLSCAASTPIISIAYMGWYRQISEKERQLVATINSGGKTYYADSVKGRFTISRDNAKNTLYLQMNMLKPEDTG MYYCAASNKDYNDYDPDWGQGTQVTVSS

[0210] SEQ ID NO:2_MSLN CAR

[0211] MALPVTALLLPLALLLHAARPSQVQVVESGGGFVQAGGSLRLSCAASTPIISIAYMGWYRQISEKERQLVATINSGGKTYYADSVKGRFTISRDNAKNTLYLQMNMLKPEDTGMYYCAASNKDYNDYDPDWGQGTQVTVSSFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0212] SEQ ID NO:3_C43-z11

[0213] ELQLVESGGGLVQPGGSLRLSCAASGHSFSIYDMGWFRQAADKERESVAVINFGRGTTYYAESVKGRFTISRDNAKNTVYLQMNSLRAEDT AVYSCGIDRRQYGLGIPPLADHWGQGSQVTVSS

[0214] SEQ ID NO:4_C43-z14

[0215] QLQLVESGGGLVQPGGSLRLSCSASGHSFSIYDMGWFRQAPGKERESVAVINFGRGTTYYAESVKGRFTISRDNAKNTVYLQMNSLRAEDT AVYSCGIDRRQYGLGIPPLADHWGQGSQVTVSS

[0216] SEQ ID NO:5_C43-z15

[0217] QLQLVESGGGLVQPGGSLRLSCSASGHSFSIYDMGWFRQAPDKERESVAVINFGRGTTYYAESVKGRFTISRDNAKNTVYLQMNSLRAEDT AVYSCGIDRRQYGLGIPPLADHWGQGTQVTVSS

[0218] SEQ ID NO:6_1194nla

[0219] EVQLVESGGGLVQPGGSLRLSCAASGRPFSIYDMGWFRQAPDKERESVAVINLARGNTYYADSVKGRFTISRDNAKNTVYLQMNSLRAED TAVYSCGVDRRQYGLGIPPLADHWGQGTQVTVSS

[0220] SEQ ID NO:7_CTL-4

[0221] EVQLVESGGGLVQPGGSLRLSCAASGFSSDYYDIGWFRQAPGKEREMVSCIRSSGGSTKYADSVKGRFTISRDNSKNTVYLQMNSLRAEDT AVYYCGLAPISPVHAVCNQHYFGYWGQGTRVTVSS

[0222] SEQ ID NO:8_PB enzyme

[0223] MGSSLDDEHILSALLQSDDELVGEDSDSEVSDHVSEDDVQSDTEEAFIDEVHEVQPTSSGSEILDEQNVIEQPGSSLASNRILTLPQRTIRGKNKHCWSTSKPTRRSRVSALNIVRSQRGPTRMCRNIYDPLLCFKLFFTDEIISEIVKWTNAEISLKRRESMTSATFRDTNEDEIYAFFGILVMTAVRKDNHMSTDDLFDRSLSMVYVSVMSRDRFDFLIRCLRMDDKSIRPTLRENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFRVYIPNKPSKYGIKILMMCDSGTKYMINGMPYLGRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDNWFTSIPLAKNLLQEPYKLTIVGTVRSNKREIPEVLKNSRSRPVGTSMFCFDGPLTLVSYKPKPAKMVYLLSSCDEDASINESTGKPQMVMYYNQTKGGVDTLDQMCSVMTCSRKTNRWPMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMGLTSSFMRKRLEAPTLKRYLRDNISNILPKEVPGTSDDSTEEPVMKKRTYCTYCPSKIRRKASASCKKCKKVICREHNIDMCQSCF。

Claims

1. A combination, characterized in that, It includes an immune cell preparation and a promoter, wherein the promoter contains NAD+ or its precursor.

2. The combination according to claim 1, wherein The immune cells are immune effector cells and / or antigen presenting cells, wherein the immune effector cells are selected from tumor infiltrating lymphocytes (TIL) cells, natural killer (NK) cells or natural killer T (NKT) cells, and the antigen presenting cells are selected from DC cells, macrophages or B cells.

3. The combination according to claim 2, wherein: The immune effector cells further contain a coding sequence of a chimeric antigen receptor and / or are capable of expressing a chimeric antigen receptor; the antigen presenting cells further carry a tumor antigen or contain a coding sequence of a tumor antigen.

4. The combination according to claim 2 or 3, characterized in that The immune cell further contains an antibody coding sequence and / or is capable of expressing an antibody; preferably, the antibody is a single domain antibody, and more preferably, the single domain antibody is a multispecific single domain antibody.

5. The combination according to claim 4, wherein: The antibody contains a functional region targeting a target, and the target is selected from: an immune checkpoint protein, an immune cell-associated antigen, a tumor-associated antigen, an immune co-stimulatory molecule or its receptor; preferably, the immune checkpoint protein is selected from PD-1, CTLA4, PDL1, PDL2, PDL3, TIM3, LAG3, CD47, BTLA, TIGIT, CD160, LAIR1, B7-H1, B7-1, VSIR, CD244; more preferably, the immune checkpoint protein is selected from PD-1, CTLA4, PDL1.

6. The combination according to claim 1, wherein The immune cell preparation comprises immune cells and pharmaceutically acceptable excipients; Preferably, the excipients include a diluent and a cryoprotectant, wherein the diluent is selected from one or more of compound electrolyte injection, 0.9% NaCl injection, and sodium lactate Ringer's injection; and / or the cryoprotectant is selected from one or more of dextran glucose injection, human serum albumin solution, dimethyl sulfoxide, ethylene glycol, trehalose, sucrose, and glucose; More preferably, the excipients include: 40-80% v / v compound electrolyte injection, 5-20% v / v dextran 40 glucose injection, 10-30% v / v human albumin solution and 5-10% v / v dimethyl sulfoxide.

7. The combination according to any one of claims 1 to 6, characterized in that The NAD+ precursor is selected from tryptophan, quinolinic acid, nicotinic acid (NA), nicotinamide (NAM), nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), reduced nicotinamide mononucleotide (NMNH), reduced nicotinamide riboside (NRH), reduced nicotinic acid riboside (NARH), reduced nicotinamide adenine dinucleotide (NADH), or one or more of their food- or pharmaceutically acceptable salts, derivatives, or prodrugs; preferably nicotinamide mononucleotide (NMN) or reduced nicotinamide mononucleotide (NMNH).

8. The combination according to any one of claims 1 to 7, characterized in that The promoter further contains flavonoids and / or coenzymes; Preferably, the flavonoid is selected from one or more of quercetin, dihydroquercetin, fisetin, catechin, gallin, resveratrol, and hesperetin, preferably quercetin and / or dihydroquercetin; Preferably, the coenzyme is selected from at least one of glutathione or its derivatives, adenosine triphosphate and its derivatives, pyrroloquinoline quinone or its derivatives, adenosine methionine or its derivatives, coenzyme A or its derivatives, and coenzyme Q or its derivatives, preferably pyrroloquinoline quinone and / or coenzyme Q10.

9. The combination as claimed in claim 8, characterized in that In the promoter, the mass ratio of NAD+ or its precursor: flavonoid: coenzyme is 10-120: 1-5: 1-10.

10. Use of the combination according to any one of claims 1 to 9 in the preparation of a medicament for treating tumors.

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