WO2021204204A1 - Antigen gene transfection cell vaccine and related immune cell - Google Patents

Abstract

Provided are a cell vaccine for an infection by a pathogenic micro-organism and an illness related to the infection, said vaccine can be prepared by means of in vitro transfection of autologous or allogeneic antigen-presenting cells with a pathogenic micro-organism antigen, and as such the antigen-presenting cells loaded with the pathogenic micro-organism antigen serve as a prophylactic or therapeutic vaccine. Further provided are an immune cell, comprising a T cell or an NK cell, which can be activated by the antigen-loaded vaccine of the present invention, or can be directly activated by a pathogenic micro-organism. Further provided is a composition simultaneously including the cell vaccine and immune cells, said composition being used for preventing or treating an infection caused by the pathogenic micro-organism and an illness related thereto.

Description

A cell vaccine transfected with antigen gene and related immune cells Technical field

The invention belongs to the field of biomedicine technology, in particular to the field of immune cell therapy.

Background technique

Humans can use nucleic acids such as DNA or RNA as nucleic acid vaccines to combat pathogenic infections (such as microbial infections) or tumors and other diseases. However, this type of vaccine technology has limitations. The main reason is that nucleic acid vaccines are unstable and easily degraded in the body, and the delivery system cannot ensure that they are delivered into antigen-presenting cells (APC), which are presented by APC to activate the immune system.

If APC loaded with antigen is used as a product, whether it is used for disease prevention or treatment, its effect will be more certain than that of nucleic acid vaccines.

Summary of the invention

In the first aspect, for diseases infected by pathogenic microorganisms such as viruses, this article proposes a cellular vaccine, which can be constructed by transfecting pathogenic microorganism antigen genes into the patient’s autologous or allogeneic antigen-presenting cells in vitro, and then load all the The pathogenic microbial antigens or antigen-presenting cells of tumor antigens are delivered to patients as preventive or therapeutic vaccines.

In the second aspect, this article provides an immune cell, such as T cell or NK cell, which can be activated by the antigen gene-loaded vaccine of the first aspect described above, or can be directly activated by pathogenic microorganisms.

Either the above-mentioned cell vaccines or immune cells can be used for the prevention and treatment of pathogenic microbial infections and infection-related diseases such as tumors.

Unless otherwise specified, the practice of some of the methods disclosed herein uses conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, which are within the technical scope of the art. Inside. See, for example, Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012); series of current protocols for molecular biology (FMAusubel et al.); series of methods in Enzymology (Academic Press, Inc.), PCR 2: A Practical Approach (MJMachersrs, BD Hames and GRTaylor (1995)), Harlow and Lane (1988) Antibodies, A Laboratory Manual, and Culture of Animal Cells: A Manual of Basic Technology and Specialized Applications, 6th Edition (RIBreshney, Edited (2010)).

The term "about" or "approximately" means within the acceptable error range of a particular value determined by a person of ordinary skill in the art, which will depend in part on how the value is measured or determined, that is, the limitations of the measurement system. For example, according to the practice in the art, "about" can mean within 1 or more than 1 standard deviation. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Or, especially for biological systems or processes, the term may represent an order of magnitude of the value, preferably within 5 times, more preferably within 2 times. Where specific values are described in the application and claims, unless otherwise stated, it should be assumed that the term "about" means within the acceptable error range of the specific value.

As used herein, "cell" can generally refer to a biological cell. A cell can be the basic structure, function, and/or biological unit of an organism. The cell can be derived from any organism that has one or more kinds of cells. Some non-limiting examples include: prokaryotic cells, eukaryotic cells, bacterial cells, archaeal cells, single-celled eukaryotic cells, protozoan cells, cells from plants, algae cells, seaweeds, fungal cells, animal cells, cells from Vertebrate cells (for example, Drosophila, cnidaria, echinoderms, nematodes, etc.), cells from vertebrates (for example, fish, amphibians, reptiles, birds, mammals) cells from mammals (for example , Pigs, cows, goats, sheep, rodents, rats, mice, non-human primary cells), humans, etc.), etc. Sometimes cells are not derived from natural organisms (for example, cells can be synthetic, sometimes called artificial cells).

The term "antigen" as used herein refers to a molecule or fragment thereof capable of being bound by a selective binding agent. For example, the antigen may be a ligand that can be bound by a selective binding agent such as a receptor. As another example, the antigen can be an antigen molecule, which can be bound by a selective binding agent such as an immune protein (eg, an antibody). Antigen can also refer to a molecule or fragment thereof that can be used in animals to generate antibodies capable of binding the antigen.

The term "gene" as used herein refers to nucleic acids (e.g., DNA, such as genomic DNA and cDNA, or RNA) and their corresponding nucleotide sequences encoding RNA transcripts. As used herein, the term referring to genomic DNA includes inserted non-coding regions and regulatory regions, and may include 5'and 3'ends. In some uses, the term includes transcribed sequences, including 5'and 3'untranslated regions (5'-UTR and 3'-UTR), exons and introns. In some genes, the transcribed region will contain an "open reading frame" encoding the polypeptide. In some uses of the term, a "gene" contains only the coding sequence necessary to encode a polypeptide (for example, an "open reading frame" or "coding region"). In some cases, genes do not encode polypeptides, such as ribosomal RNA genes (rRNA) and transfer RNA (tRNA) genes. In some cases, the term "gene" includes not only transcribed sequences, but also non-transcribed regions, including upstream and downstream regulatory regions, enhancers and promoters. A gene can refer to an "endogenous gene" or a natural gene in its natural location in the genome of an organism. Genes can refer to "foreign genes" or non-natural genes. Non-native genes may refer to genes that are not normally found in the host organism but are introduced into the host organism by gene transfer. Non-natural genes can also refer to genes that are not in their natural locations in the genome of an organism. Non-natural genes can also refer to naturally occurring nucleic acid or polypeptide sequences that contain mutations, insertions, and/or deletions (e.g., non-natural sequences). In a specific embodiment, the antigen gene of the present invention is a complete or partial fragment of a gene from a pathogenic microorganism.

The term "antibody" as used herein refers to protein binding molecules with immunoglobulin-like functions. The term antibody includes antibodies (eg, monoclonal and polyclonal antibodies), as well as derivatives, variants and fragments thereof. Antibodies include, but are not limited to, immunoglobulins (Ig) of different classes (ie, IgA, IgG, IgM, IgD, and IgE) and subclasses (eg, IgG1, IgG2, etc.). Derivatives, variants or fragments thereof may refer to functional derivatives or fragments that retain the binding specificity (e.g., complete and/or partial) of the corresponding antibody. Antigen-binding fragments include Fab, Fab', F(ab')2, variable fragments (Fv), single chain variable fragments (scFv), minibodies, diabodies, and single domain antibodies ("sdAb" or "nanobodies") "Or "camel"). The term antibody includes antibodies and antigen-binding fragments of antibodies that have been optimized, engineered, or chemically conjugated. Examples of antibodies that have been optimized include affinity matured antibodies. Examples of antibodies that have been engineered include Fc-optimized antibodies (e.g., antibodies optimized in fragment crystallizable regions) and multispecific antibodies (e.g., bispecific antibodies).

The term "nucleotide" as used herein generally refers to a base-sugar-phosphate combination. Nucleotides may include synthetic nucleotides. Nucleotides may include synthetic nucleotide analogs. Nucleotides may be monomeric units of nucleic acid sequences (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide may include ribonucleoside adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), guanosine triphosphate (GTP) and deoxyribonucleoside triphosphates such as dATP, dCTP , DITP, dUTP, dGTP, dTTP or derivatives thereof. These derivatives may include, for example, [αS]dATP, 7-deaza-dGTP and 7-deaza-dATP, and nucleotide derivatives that confer nuclease resistance to nucleic acid molecules containing them. The term nucleotide as used herein may refer to dideoxyribonucleoside triphosphate (ddNTP) and its derivatives. Illustrative examples of dideoxyribonucleoside triphosphates may include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. Nucleotides can be unlabeled or detectably labeled by well-known techniques. Marking can also be done with quantum dots. Detectable labels can include, for example, radioisotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzyme labels.

The terms "polynucleotide", "oligonucleotide" and "nucleic acid" are used interchangeably and refer to nucleotides, deoxyribonucleotides or ribonucleotides, or their analogues, of any length in polymerized form. It is single-, double-, or multi-strand form. The polynucleotide can be exogenous or endogenous to the cell. The polynucleotide may exist in a cell-free environment. The polynucleotide can be a gene or a fragment thereof. The polynucleotide may be DNA. The polynucleotide may be RNA. A polynucleotide can have any three-dimensional structure and can perform any function, known or unknown. A polynucleotide may contain one or more analogs (e.g., altered backbone, sugar or nucleobases).

The term "expression" refers to one or more processes in which polynucleotides are transcribed from a DNA template (for example, into mRNA or other RNA transcripts) and/or the transcribed mRNA is subsequently translated into peptides, polypeptides or proteins. The transcript and the encoded polypeptide can be collectively referred to as "gene product". If the polynucleotide is derived from genomic DNA, expression can include splicing mRNA in eukaryotic cells. With reference to expression, "up-regulation" generally refers to an increase in the expression level of polynucleotides (e.g., RNA, such as mRNA) and/or polypeptide sequences relative to its expression level in the wild-type state, and "down-regulation" generally refers to relative Due to its expression in the wild-type state, the expression level of polynucleotide (e.g., RNA, e.g. mRNA) and/or polypeptide sequence is reduced.

As used herein, the term "modulation" with respect to expression or activity refers to altering the level of expression or activity. Regulation can occur at the transcription level and/or translation level.

The terms "peptide", "polypeptide" and "protein" are used interchangeably herein and refer to a polymer of at least two amino acid residues connected by peptide bonds. The term does not mean a polymer of a specific length, nor does it mean to imply or distinguish whether the peptide is produced using recombinant technology, chemical or enzymatic synthesis, or is naturally occurring. The term applies to naturally occurring amino acid polymers as well as amino acid polymers containing at least one modified amino acid. In some cases, polymers can be interrupted by non-amino acids. The term includes amino acid chains of any length, including full-length proteins, and proteins with or without secondary and/or tertiary structure (e.g., domains). The term also includes amino acid polymers that have been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, oxidation, and any other operations, such as conjugation with labeling components. The terms "amino acid" and "amino acid" as used herein generally refer to natural and unnatural amino acids, including but not limited to modified amino acids and amino acid analogs. Modified amino acids may include natural amino acids and unnatural amino acids, which have been chemically modified to include groups or chemical moieties that do not naturally occur on amino acids. Amino acid analogs may refer to amino acid derivatives. The term "amino acid" includes D-amino acids and L-amino acids.

When used herein with respect to polypeptides, the terms "derivatives", "variants" and "fragments" refer to polypeptides related to wild-type polypeptides, for example by amino acid sequence, structure (e.g., secondary and/or tertiary) , Activity (for example, enzyme activity) and/or function. Compared with wild-type polypeptides, derivatives, variants and fragments of polypeptides may contain one or more amino acid variations (e.g., mutations, insertions and deletions), truncations, modifications, or combinations thereof.

As used herein, a "fusion" can refer to a protein and/or nucleic acid comprising one or more non-natural sequences (e.g., portions). The fusion may contain one or more of the same non-natural sequence. The fusion may contain one or more different non-natural sequences. The fusion may be a chimera. The fusion may contain a nucleic acid affinity tag. The fusion can include a barcode. The fusion may contain a peptide affinity tag. Fusions can provide polypeptides for subcellular localization, for example, nuclear localization signal (NLS) for targeting the nucleus, mitochondrial localization signal for targeting mitochondria, chloroplast localization signal for targeting chloroplast, endoplasmic reticulum (ER ) Reserved signal, etc. Fusion can provide non-native sequences (e.g., affinity tags) that can be used for tracking or purification. The fusion can be a small molecule, such as biotin or a dye, such as Alexa fluoro dye, Cyanine 3 dye, Cyanine 5 dye.

The phrase "artificial TCR" as used herein can be understood as "exogenous T cell receptor (TCR) complex" and refers to a TCR complex in which one or more chains of TCR are introduced into the genome of immune cells. It may or may not express TCR endogenously. In some cases, an exogenous TCR complex may refer to a TCR complex, where one or more strands of the endogenous TCR complex have one or more mutated sequences, for example at the nucleic acid or amino acid level. The expression of exogenous TCR on immune cells can confer epitopes or antigens (for example, epitopes or antigens preferentially present on the surface of cancer cells or other pathogenic cells or particles) binding specificity. The exogenous TCR complex may include TCR-α, TCR-β chain, CD3-γ chain, CD3-δ chain, CD3-ζ chain, or any combination thereof introduced into the genome. In some cases, the strand introduced into the genome can replace the endogenous strand.

The terms "subject", "individual" and "patient" are used interchangeably herein and refer to vertebrates, preferably mammals, such as humans. Mammals include, but are not limited to, rodents, apes, humans, farm animals, sports animals, and pets. It also includes tissues, cells and their progeny of biological entities obtained in vivo or cultured in vitro.

As used herein, the term "treatment" refers to methods used to obtain beneficial or desired results, including but not limited to therapeutic benefits and/or preventive benefits. For example, treatment can include administration of a system or cell population disclosed herein. Therapeutic benefit refers to any treatment-related improvement or effect of one or more diseases, disorders, or symptoms in treatment. In order to prevent benefits, the composition can be administered to subjects who are at risk of developing a particular disease, disorder, or symptom, or to subjects who report one or more physiological symptoms of the disease, even if the disease, disorder, or symptom may not yet exist. The same is true for performance.

The term "effective amount" or "therapeutically effective amount" refers to the amount of the composition, for example, a composition containing a cell vaccine of the present disclosure such as APC cells (for example, PBMC, T lymphocytes, and/or NK cells). Of the subject is sufficient to produce the desired activity. In the context of the present disclosure, the term "therapeutically effective" means that the amount of the composition is sufficient to delay manifestation, prevent progression, reduce or alleviate at least one symptom of the condition treated by the method of the present invention.

The antigen-producing cells (APC) described herein, also known as accessory cells, are immune cells that can ingest, process, process and present antigen information to lymphocytes during the immune response process. Antigen-presenting cells (APC) can be human natural APCs, including dendritic cells (DC), macrophages, B lymphocytes, T cells, peripheral blood mononuclear cells (PBMC), etc.; they can also be derived from non-human natural The exogenous cells of APC include K562 cells, B cells, NK cells, and DC cells that are engineered or immortalized or inactivated by irradiation. Antigen-presenting cells (APC) can be derived from the patient's own body or from allogeneic cells.

In some embodiments, a cell vaccine is provided, characterized in that the vaccine is an antigen-presenting cell (APC) transfected with pathogenic microorganisms or tumor antigen genes.

In some embodiments, the pathogenic microorganism may be one or more of all pathogenic microorganisms in humans, and may include HPV virus, Epstein-Barr virus, Helicobacter pylori (HP), hepatitis B virus (HBV), HIV ( HIV), any one or more of the new coronavirus (SARS-Cov-2). In a specific embodiment China, the pathogenic microorganism is any one of the multiple subtypes of the HPV virus, especially the highly pathogenic subtypes, such as hpv16, hpv18, hpv31, hpv33, hpv35, hpv45, hpv51, hpv52, hpv56, hpv58, hpv61, more particularly HPV16 or HPV18.

In certain embodiments, the pathogenic microorganism antigen gene is a complete or partial fragment of the E6 and/or E7 gene of the HPV virus, or the Spike gene of SARS-Cov-2 such as the S1 or S2 subunit, or the complete or partial fragment of other genes. Part of the fragment.

In some embodiments, the antigen may also include neoantigen, tumor-associated antigen (TAA), and antigens of tumors caused by the pathogenic microorganisms.

In some embodiments, the gene transfection process is electroporation, or adeno-associated virus transfection.

In some embodiments, the gene transfection process is a way to allow the gene to be expressed constantly, such as lentiviral transfection and gene editing.

In some embodiments, the vector design used to load the pathogenic microorganism antigens into the APC is to direct the expression of the pathogenic microorganism antigens to the lysosomes in the APC.

In some embodiments, the vector design used to load the pathogenic microorganism antigen into the APC is to direct the expression of the pathogenic microorganism antigen to the endoplasmic reticulum within the APC.

In some embodiments, the vector design used to load pathogenic microorganism antigens into APC is to simultaneously direct the expression of pathogenic microorganism antigens to the lysosome and endoplasmic reticulum in the APC.

In some embodiments, the vector design used to load pathogenic microorganism antigens into APC is to secrete and express the pathogenic microorganism antigens outside the APC cell, or express it across the membrane.

For example, the above-mentioned vector design may include specific vector elements such as signal sequences and promoters to achieve specific expression. In a specific embodiment, the coding sequence of the signal peptide of HLA-I is linked to the pathogenic microorganism antigen gene. In a more specific embodiment, the pathogenic microorganism antigen gene construct used for transfection is selected from the following group: (1) as SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO :7 or SEQ ID NO: 9; (2) encoding SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10 The nucleotide sequence of an amino acid sequence; or (3) has at least 90% homology, at least 95% homology, at least 96% homology, or at least 97% homology with the nucleotide sequence of (1) or (2) Homology, at least 98% homology, at least 99% homology of nucleotide sequences.

The vaccine provided by the present disclosure can be used for the prevention of infection by any of the pathogenic microorganisms among HPV, EB, HP, HBV, HIV, and the new coronavirus, or for the treatment of patients infected by the pathogenic microorganism, or It is used for the treatment of cancer patients or people with precancerous lesions related to pathogenic microorganism infection.

The present disclosure provides an immune cell, which is a T cell that responds to vaccines or pathogenic microorganisms or tumors.

In some embodiments, the immune cells are natural unmodified T cells obtained by co-cultivation with the vaccine described herein in vitro.

In some embodiments, the immune cells are modified T cells or NK cells, with artificially modified T cell receptors (TCR) and/or chimeric antigen receptors (CAR), and the TCR or CAR can be The vaccine or the pathogenic microorganism produces a specific response, or can specifically recognize any virus of HPV, EB, HP, HBV, HIV or protein fragments of the pathogenic microorganism presented by a specific MHC molecule.

In some embodiments, the immune cell is a CAR-T cell or a CAR-NK cell, and the CAR is composed of an extracellular binding domain fused with an immune cell signaling pathway protein, wherein the extracellular binding domain is a human cell surface receptor protein of a pathogenic microorganism All or part of it, including the human cell surface receptor of coronavirus-angiotensin-converting enzyme 2 (ACE2), the immune cell signaling pathway protein is any one of costimulatory signaling proteins such as CD28 or 41BBz or ICOS.

In some embodiments, the gene transfection process of the artificially modified CAR or TCR is electrotransfection or adeno-associated virus transfection. In some embodiments, the gene transfection process of the artificially modified CAR or TCR is a way to allow the gene to be expressed constantly, such as lentiviral transfection and gene editing.

In one aspect, the immune cells provided by the present disclosure comprise enhanced receptors. The booster receptor may comprise the extracellular domain (ECD) of the protein. ECD can be fused with the intracellular domain (ICD) of a costimulatory molecule that mediates immune cell activation signals. Enhancing the binding of receptors and ligands can generate immune cell activation signals in modified immune cells such as T cells, rather than immune cell inactivation signals.

The modified T cell may comprise a T cell receptor (TCR) complex, which exhibits specific binding to the neoantigen. In some embodiments, the TCR complex is an endogenous TCR complex. In some embodiments, the TCR is an exogenous TCR complex. The TCR complex (e.g., endogenous or exogenous) of the modified immune cell can confer antigen binding specificity (e.g., neoantigen binding) to the immune cell. In some embodiments, the present disclosure provides a modified T cell comprising an endogenous TCR complex that specifically binds a neoantigen, the modified T cell comprising a chimeric stimulatory molecule, wherein the chimeric stimulatory molecule Contains: The bound polypeptide extracellular domain (PED) includes but is not limited to the membrane protein on the cells of tumor cells, wherein the PED is fused with the intracellular domain (ICD) of a costimulatory molecule that mediates immune cell activation signals, Wherein the chimeric stimulating molecule is combined with the chimeric stimulating molecule. The membrane protein generates the immune cell activation signal in the modified T cell.

The binding of a modified immune cell (such as a modified T cell provided herein) to an antigen can activate the immune cell. Enhanced receptors for modified cells can be used to provide further control of immune cell activity, such as but not limited to immune cell activation and expansion. Enhancing the binding of the receptor to the ligand in the modified immune cell (for example, a modified T cell or a modified TIL) can trigger an immune cell activation signal in the modified immune cell instead of an immune cell inactivation signal. Triggering the immune cell activation signal in the modified immune cell instead of the immune cell inactivation signal can minimize the immunosuppressive effect in the immune cell. Minimizing immune suppression in immune cells can increase the effectiveness of immune cells in immune responses, for example, by increasing immune cell cytotoxicity against pathogenic microorganisms or target cells (eg, tumor cells or cells infected with pathogenic microorganisms).

The enhanced receptor may contain the extracellular domain (ECD) of the protein, which triggers an immune cell signal after binding to its ligand in an unmodified immune cell. The signal can be an inactivation signal, an activation signal, or Neither activation nor deactivation signal. The protein can be a signal transduction receptor or any functional fragment, derivative or variant thereof. In some cases, the signaling receptor may be a membrane-bound receptor. In response to ligand binding, signaling receptors can induce one or more signaling pathways in the cell. In some cases, the signaling receptor may be a non-membrane-bound receptor. The enhanced receptor may comprise a fragment of a receptor selected from G protein coupled receptors (GPCRs), such as extracellular domains; integrin receptors; cadherin receptors; catalytic receptors (such as kinases); death receptors ; Checkpoint receptors; cytokine receptors; chemokine receptors; growth factor receptors; hormone receptors; and immune receptors.

In some embodiments, the booster receptors comprise fragments of immune checkpoint receptors, which can be involved in the regulation of the immune system. Non-limiting examples of such receptors include, but are not limited to, programmed cell death 1 (PD-1, or PD1), cytotoxic T lymphocyte-associated protein 4 (CTLA-4), B and T lymphocyte attenuating agents (BTLA ), killer immunoglobulin-like receptor (KIR), indoleamine 2,3-dioxygenase (IDO), lymphocyte activation gene-3 (LAG3), T cell immunoglobulin mucin 3 (TIM- 3) T cell immune receptor (TIGIT) with Ig and ITIM domains, SIRPα, NKG2D.

In some embodiments, the enhanced receptor comprises at least an extracellular fragment of TCR, which can participate in recognizing neoantigens of target cells (eg, cancer cell antigens or tumor antigens). In some examples, the enhanced receptor may comprise the extracellular variable region of the TCR alpha and/or beta chain.

The enhanced receptor may comprise an immune checkpoint receptor or any derivative, variant or fragment thereof. The enhanced receptor may bind to an antigen comprising any suitable immune checkpoint receptor ligand or any derivative, variant or fragment thereof. Non-limiting examples of such ligands include, but are not limited to, B7-1, B7-H3, B7-H4, HVEM (herpes virus entry medium), AP2M1, CD80, CD86, SHP-2, PPP2R5A, MHC (e.g., I Class, Class II), CD47, CD70, PD-L1 (or PDL1) and PD-L2. The region that enhances the binding of receptors to such ligands may be the natural receptors of such ligands or monoclonal antibodies of such ligands.

In some embodiments, the enhanced receptor comprises a fragment of a cytokine receptor. Cytokine receptors can perform a variety of functions, non-limiting examples of which include immune cell regulation and mediation of inflammation. In some embodiments, the enhanced receptor comprises a cytokine receptor, such as a type I cytokine receptor or a type II cytokine receptor, or any derivative, variant or fragment thereof. In some embodiments, the enhanced receptor comprises an interleukin receptor (e.g., IL-2R, IL-3R, IL-4R, IL-5R, IL-6R, IL-7R, IL-9R, IL-11R, IL-12R, IL-13R, IL-15R, IL-21R, IL-23R, IL-27R and IL-31R), a colony stimulating factor receptor (for example, erythropoietin receptor, CSF-1R, CSF-2R, GM-CSFR and G-CSFR), hormone receptor/neuropeptide receptor (for example, growth hormone receptor, prolactin receptor and leptin receptor), or any derivative, variant or fragment thereof . In some embodiments, the enhanced receptor comprises a type II cytokine receptor, or any derivative, variant or fragment thereof. In some embodiments, enhanced receptors include interferon receptors (eg, IFNAR1, IFNAR2, and IFNGR), interleukin receptors (eg, IL-10R, IL-20R, IL-22R, and IL-28R), tissue Factor receptors. (Also known as platelet tissue factor), or any derivative, variant or fragment thereof.

In some embodiments, the extracellular binding domain of the enhanced receptor can be any antibody or fragment of an antibody, and the antigen to which the antibody binds can be a membrane protein widely expressed by human cells, or a membrane protein expressed by tumor cells. These membrane proteins include class I RTKs (for example, epidermal growth factor (EGF) receptor family including EGFR; ErbB family including ErbB-2, ErbB-3 and ErbB-4), class II RTKs (for example, insulin receptor Body family includes INSR, IGF-1R and IRR), type III RTK (for example, platelet-derived growth factor (PDGF) receptor family, including PDGFR-α, PDGFR-β, CSF-1R, KIT/SCFR and FLK2/FLT3) , Type IV RTK (for example, fibroblast growth factor (FGF) receptor family, including FGFR-1, FGFR-2, FGFR-3 and FGFR-4), Type V RTK (for example, vascular endothelial growth factor (VEGF) Receptor family, including VEGFR1, VEGFR2 and VEGFR3), type VI RTK (such as the hepatocyte growth factor (HGF) receptor family, including hepatocyte growth factor receptor (HGFR/MET) and RON), a VII RTK (such as , Tropomyosin receptor kinase (Trk) receptor family, including TRKA, TRKB and TRKC), class VIII RTK (for example, ephrin (Eph) receptor family, including EPHA1, EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHB1, EPHB2, EPHB3, EPHB4, EPHB5 and EPHB6), class IX RTK (for example, AXL receptor family such as AXL, MER and TRYO3), class X RTK (for example, LTK receptor family such as LTK and ALK) , Class XI RTK (for example, TIE receptor family such as TIE and TEK), class XII RTK (for example ROR receptor family ROR1 and ROR2), class XIII RTK (for example, discoid domain receptor (DDR) family such as DDR1 and DDR2), XIV RTK (for example, RET acceptor family such as RET), XV RTK (for example, KLG acceptor family, including PTK7), XVI RTK (for example, RYK acceptor family including Ryk), XVII RTK (for example, MuSK receptor family such as MuSK), CD47, CD70, NKG2D, or any derivative, variant or fragment thereof.

In some embodiments, the enhanced receptor may comprise at least one extracellular region of a catalytic receptor (e.g., receptor tyrosine kinase (RTK)) or any derivative, variant or fragment thereof (e.g., ligand binding domain ), or fragments containing the variable regions of antibodies with these fragments as antigens. In some embodiments, the enhanced receptor comprises a class I RTK (e.g., the epidermal growth factor (EGF) receptor family including EGFR; the ErbB family including ErbB-2, ErbB-3, and ErbB-4), a class II RTK ( For example, the insulin receptor family includes INSR, IGF-1R and IRR), class III RTK (for example, the platelet-derived growth factor (PDGF) receptor family, including PDGFR-α, PDGFR-β, CSF-1R, KIT/SCFR and FLK2/FLT3), class IV RTK (for example, fibroblast growth factor (FGF) receptor family, including FGFR-1, FGFR-2, FGFR-3 and FGFR-4), class V RTK (for example, vascular endothelial growth Factor (VEGF) receptor family, including VEGFR1, VEGFR2 and VEGFR3), VI type RTK (such as hepatocyte growth factor (HGF) receptor family, including hepatocyte growth factor receptor (HGFR/MET) and RON), a VII RTK-like (e.g., tropomyosin receptor kinase (Trk) receptor family, including TRKA, TRKB and TRKC), type VIII RTK (e.g., ephrin (Eph) receptor family, including EPHA1, EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHB1, EPHB2, EPHB3, EPHB4, EPHB5 and EPHB6), class IX RTK (for example, AXL receptor family such as AXL, MER and TRYO3), class X RTK (for example, LTK receptor family such as LTK and ALK), type XI RTK (e.g., TIE receptor family such as TIE and TEK), type XII RTK (e.g. ROR receptor family ROR1 and ROR2), type XIII RTK (e.g. discoid domain receptor (DDR) Family such as DDR1 and DDR2), XIV-type RTK (for example, RET acceptor family such as RET), XV-type RTK (for example, KLG acceptor family, including PTK7), XVI-type RTK (for example, RYK acceptor family including Ryk), XVII RTK (for example, MuSK receptor family such as MuSK), CD47, CD70, NKG2D, or any derivative, variant or fragment thereof.

The enhanced receptor includes RTK or any derivative, variant or fragment thereof. The enhanced receptor can bind to an antigen containing any suitable RTK ligand or any derivative, variant or fragment thereof, or a fragment containing the variable region of an antibody with these fragments as the antigen. Non-limiting examples of RTK ligands include growth factors, cytokines and hormones. Growth factors include, for example, members of the epidermal growth factor family (eg, epidermal growth factor or EGF, heparin-binding EGF-like growth factor or HB-EGF, transforming growth factor-α or TGF-α, amphiregulin or AR, epidermal growth factor Regulin or EPR, epigen, betacellulin or BTC, neuregulin-1 or NRG1, neuregulin-2 or NRG2, neuregulin-3 or NRG3, neuregulin-4 or NRG4), fibroblast growth factor family (Such as FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15/19, FGF16, FGF17, FGF18, FGF20, FGF21 and FGF23), vascular endothelial growth Factor families (e.g., VEGF-A, VEGF-B, VEGF-C, VEGF-D and PIGF) and platelet-derived growth factor families (e.g., PDGFA, PDGFB, PDGFC and PDGFD). Hormones include, for example, members of the insulin/IGF/relaxin family (e.g., insulin, insulin-like growth factors, relaxin family peptides, including relaxin 1, relaxin 2, relaxin 3, Leydig cell-specific insulin-like Peptide (gene INSL3), early placental insulin-like peptide (ELIP) (gene INSL4), insulin-like peptide 5 (gene INSL5) and insulin-like peptide 6).

In some embodiments, the enhanced receptor includes at least the extracellular region (eg, ligand binding domain) of the catalytic receptor, such as receptor threonine/serine kinase (RTSK), or any derivative, variant, or Fragments, or fragments comprising the variable regions of antibodies with these fragments as antigens. The enhanced receptor may comprise type I RTSK, type II RTSK or any derivative, variant or fragment thereof. The enhanced receptor may comprise a type I receptor, or any derivative, variant or fragment thereof, selected from: ALK1 (ACVRL1), ALK2 (ACVR1A), ALK3 (BMPR1A), ALK4 (ACVR1B), ALK5 (TGFβR1), ALK6 (BMPR1B) and ALK7 (ACVR1C). The enhanced receptor may comprise a type II receptor, or any derivative, variant or fragment thereof selected from the group consisting of TGFβR2, BMPR2, ACVR2A, ACVR2B and AMHR2 (AMHR). In some embodiments, the enhanced receptor comprises the TGF-β receptor or any derivative, variant or fragment thereof.

The enhanced receptor contains RTSK or any derivative, variant or fragment thereof. The enhanced receptor can bind to an antigen containing any suitable RTSK ligand or any derivative, variant or fragment thereof, or an antibody containing these fragments as the antigen. Fragments of variable regions.

The booster receptor may comprise an intracellular domain (ICD) of a costimulatory molecule that triggers an immune cell activation signal. Co-stimulatory molecules can bind ligands. In some cases, costimulatory molecules can be activated by ligand-responsive proteins. In some embodiments, costimulatory molecules can be used to modulate proliferation and/or survival signals in immune cells. In some embodiments, ICD is the intracellular domain of a costimulatory molecule, which is selected from MHC class I protein, MHC class II protein, TNF receptor protein, immunoglobulin-like protein, cytokine receptor, integrin, Signaling. Lymphocyte activation molecule (SLAM protein), activated NK cell receptor, BTLA or Toll ligand receptor. In some embodiments, the costimulatory molecule or costimulatory domain comprises a signaling domain of a molecule selected from the group consisting of: 2B4/CD244/SLAMF4, 4-1BB/TNFSF9/CD137, B7-1/CD80, B7-2 /CD86, B7-H1/PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BAFF R/TNFRSF13C, BAFF/BLyS/TNFSF13B, BLAME/SLAMF8, BTLA/CD272, CD100 (SEMA4D), CD103, CD11a, CD11b, CD11c, CD11d, CD150, CD160 (BY55), CD18, CD19, CD2, CD200, CD229/SLAMF3, CD27 ligand/TNFSF7, CD27/TNFRSF7, CD28, CD29, CD2F- 10/SLAMF9, CD3, CD30 ligand/TNFSF8, CD30/TNFRSF8, CD300a/LMIR1, CD4, CD40 ligand/TNFSF5, CD40/TNFRSF5, CD48/SLAMF2, CD49a, CD49D, CD49f, CD5, CD53, CD58/LFA- 3. CD69, CD7, CD8α, CD8β, CD82/Kai-1, CD84/SLAMF5, CD90/Thy1, CD96, CDS, CEACAM1, CRACC/SLAMF7, CRTAM, CTLA-4, DAP12, Dectin-1/CLEC7A, DNAM1( CD226), DPPIV/CD26, DR3/TNFRSF25, EphB6, GADS, Gi24/VISTA/B7-H5, GITR ligand/TNFSF18, GITR/TNFRSF18, HLA Class I, HLA-DR, HVEM/TNFRSF14, IA4, ICAM-1 , ICOS/CD278, Ikaros, IL2Rβ, IL2Rγ, IL7Rα, IL-12R, integrin α4/CD49d, integrin; 4β1, integrin α4β7/LPAM-1, IPO-3, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIRDS2, LAG-3, LAT, LIGHT/TNFSF14, LTBR, Ly108, Ly9 (CD229), lymphocyte function related antigen-1 (LFA-1), lymphotoxin-α/TNF -β, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80(KLRF1), NTB-A/S LAMF6, OX40 ligand/TNFSF4, OX40/TNFRSF4, PAG/Cbp, PD-1, PDCD6, PD-L2/B7-DC, PSGL1, RELT/TNFRSF19L, SELPLG (CD162), SLAM (SLAMF1), SLAM/CD150, SLAMF4(CD244), SLAMF6(NTB)-A), SLAMF7, SLP-76, TACI/TNFRSF13B, TCL1A, TCL1B, TIM-1/KIM-1/HAVCR, TIM-4, TL1A/TNFSF15, TNFRII/TNFRSF1B, TNF-α, TRANCE/RANKL, TSLP, TSLP R, VLA1 and VLA-6.

The ECD and ICD of the enhanced receptor can be connected by a transmembrane domain, for example by a transmembrane segment. In some embodiments, the transmembrane segment comprises a polypeptide. The transmembrane polypeptide can have any suitable polypeptide sequence. In some cases, the transmembrane polypeptide comprises the polypeptide sequence of the transmembrane portion of the endogenous or wild-type transmembrane protein. In some embodiments, compared with the transmembrane portion of the endogenous or wild-type transmembrane protein, the transmembrane polypeptide comprises at least 1, (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) amino acid substitutions, deletions and insertions of polypeptide sequences. In some embodiments, the transmembrane polypeptide comprises a non-natural polypeptide sequence, such as the sequence of a polypeptide linker. The polypeptide linker can be flexible or rigid. The polypeptide linker can be structured or unstructured. In some embodiments, the transmembrane polypeptide transmits a signal from the ECD to the ICD, such as a signal indicative of ligand binding. In some embodiments, the ECD comprises a transmembrane domain. In some embodiments, the ICD comprises a transmembrane domain.

In some embodiments, ICD can mediate the production of immune cell activation signals (or immune cell activation signals) in immune cells. In some embodiments, the immune cell activation signal is mediated by an activating factor. The activator can be an immunomodulatory molecule. Activating factors can bind, activate or stimulate T cells or other immune cells to regulate their activity. In some embodiments, the activating factor may be secreted from immune cells. The activating factor can be, for example, a soluble cytokine, a soluble chemokine or a growth factor molecule. Non-limiting examples of activating factors that can mediate the activation of immune cells include soluble cytokines, such as IL-1, IL-2, IL-6, IL-7, IL-8, IL-10, IL-12, IL- 15. IL-21, tumor necrosis factor (TNF), transforming growth factor (TGF), interferon (IFN) or any functional fragment or variant thereof.

Immune cell activation signals may include or result in clonal expansion of modified immune cells (eg, modified TIL or modified T cells); released by modified immune cells (eg, modified TIL or modified T cells) Cytokines; cytotoxicity of modified immune cells (e.g., modified TIL or modified T cells); proliferation of modified immune cells (e.g., modified TIL or modified T cells); modified immune cells (e.g., modified TIL or modified T cells) differentiation, dedifferentiation or transdifferentiation; movement and/or transportation of modified immune cells (for example, modified TIL or modified T cells); modified immune cells (for example, modified TIL Or modified T cells) depletion and/or reactivation; through the release of other intercellular molecules, metabolites, chemical compounds, or combinations thereof through modified immune cells (for example, modified TIL or modified T cells).

In some embodiments, the immune cell activation signal includes or results in the clonal expansion of immune cells. Clonal expansion can include the production of daughter cells produced by immune cells. Daughter cells resulting from clonal expansion may contain enhanced receptors. The clonal expansion of modified immune cells can be greater than the clonal expansion of comparable immune cells lacking enhanced receptors. The clonal expansion of modified immune cells is about 5 times to about 10 times, about 10 times to about 20 times, about 20 times to about 30 times, about 30 times to about comparable immune cells lacking enhanced receptors. About 40 times, about 40 times to about 50 times, about 50 times to about 60 times, about 60 times to about 70 times, about 70 times to about 80 times, about 80 times to about 90 times, about 90 times to about 100 Times, about 100 times to about 200 times, about 200 times to about 300 times, about 300 times to about 400 times, about 400 times to about 500 times, about 500 times to about 600 times, or about 600 times to about 700 times . In some embodiments, determining clonal expansion can include quantifying many immune cells, for example, with and without the enhanced receptor and after the ligand binds to the enhanced receptor. The quantification of many immune cells can be achieved by a variety of techniques, non-limiting examples of which include flow cytometry, trypan blue exclusion, and blood cell count.

In some embodiments, the immune cell activation signal comprises or causes the immune cell to release cytokines. In some embodiments, immune cell activity includes or results in the release of intercellular molecules, metabolites, chemical compounds, or combinations thereof. Cytokines released by modified immune cells may include the release of IL-1, IL-2, IL-4, IL-5, IL-6, IL-13, IL-17, IL-21, IL-22, IFNγ, TNFα , CSF, TGFβ, granzyme, etc. In some embodiments, cytokine release can be quantified using enzyme-linked immunosorbent assay (ELISA), flow cytometry, western blotting, and the like. The cytokine release of modified immune cells may be greater than that of comparable immune cells lacking enhanced receptors. The modified immune cells provided herein can produce about 1 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 150 times, 200 times, 250 times, or more than 300 times cytokine release, Comparable immune cells lacking enhanced receptors. When the binding of the receptor to the ligand is enhanced and the modified immune cell binds to the neoantigen present on the target cell, the modified immune cell can be compared with comparable immune cells lacking the enhanced receptor (e.g., unmodified) Shows increased secretion of cytokines. In some embodiments, the secreted cytokine is IFNγ or IL-2. In some embodiments, cytokine release can be quantified in vitro or in vivo.

In some embodiments, the immune cell activation signal comprises or causes the immune cell to produce cytotoxicity. In some cases, the cytotoxicity of the modified immune cells provided herein can be used to kill target cells. Immune cells or populations of immune cells expressing enhanced receptors can induce the death of target cells. Killing target cells can be used in a variety of applications, including but not limited to treating diseases or conditions in which cell populations need to be eliminated or whose proliferation is desired to be inhibited. Cytotoxicity can also refer to the release of cytotoxic cytokines by immune cells, such as IFNγ or granzyme. In some cases, the modified immune cells provided herein can alter (i) the release of cytotoxins, such as perforin, granzyme, and granulysin, and/or (ii) pass through Fas-between T cells and target cells. Fas ligand interaction induces apoptosis. In some embodiments, cytotoxicity can be quantified by cytotoxicity assays, including co-culture assays, ELISPOT, chromium release cytotoxicity assays, and the like. The cytotoxicity of the modified immune cells provided herein may be greater than the cytotoxicity of comparable immune cells lacking enhanced receptors. Compared with comparable immune cells lacking enhanced receptors (e.g., unmodified), when the enhanced receptor binds to the ligand and the modified immune cells bind to neoantigens present on the target, the modified immune cells can exhibit Increased cytotoxicity against target cells. Compared with comparable immune cells lacking enhanced receptors, the cytotoxicity of the modified immune cells of the present invention to target cells can be about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175% or 200% More. The modified immune cells of the present invention can induce target cell death, which is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% higher than comparable immune cells without enhanced receptors. 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175% or 200%. In some embodiments, the immune cells provided herein can induce apoptosis in target cells that display target epitopes (e.g., neoantigens) on their surface. In some embodiments, cytotoxicity can be determined in vitro or in vivo. In some embodiments, determining cytotoxicity may include determining the level of disease after administration of the modified immune cells provided herein compared to the level of disease before administration. In some embodiments, determining cytotoxicity may include determining the level of disease after administration of the modified immune cells provided herein and the level of disease after administration of comparable immune cells lacking enhanced receptors.

In some embodiments, the immune cell activation signal comprises or results in the proliferation of immune cells. The proliferation of immune cells may refer to the expansion of immune cells. The proliferation of immune cells can refer to the phenotypic changes of immune cells. The proliferation of the modified immune cells of the present disclosure may be greater than the proliferation of comparable immune cells lacking enhanced receptors. Compared with the proliferation of comparable immune cells lacking enhanced receptors, the proliferation of the modified immune cells provided herein can be about 5 to about 10 times, about 10 to about 20 times, about 20 to about 30 times , About 30 times to about 40 times, about 40 times to about 50 times, about 50 times to about 60 times, about 60 times to about 70 times, about 70 times to about 80 times, about 80 times to about 90 times, about 90 times to about 100 times, about 100 times to about 200 times, about 200 times to about 300 times, about 300 times to about 400 times, about 400 times to about 500 times, about 500 times to about 600 times, or about 600 times to about 700 times higher. In some embodiments, proliferation can be determined by quantifying many immune cells. Quantifying many immune cells can include flow cytometry, trypan blue exclusion, and/or blood count. Proliferation can also be determined by phenotypic analysis of immune cells.

In some embodiments, the immune cell activation signal may include or result in the differentiation, dedifferentiation, or transdifferentiation of immune cells. The differentiation, dedifferentiation or transdifferentiation of immune cells can be determined by flow cytometry to evaluate the phenotypic expression of markers of differentiation, dedifferentiation or transdifferentiation on the cell surface. In some embodiments, the modified immune cells provided herein have increased differentiation capacity compared to comparable immune cells lacking enhanced receptors. In some embodiments, the modified immune cells provided herein have increased dedifferentiation capacity compared to comparable immune cells lacking enhanced receptors. In some embodiments, the modified immune cells provided herein have increased dedifferentiation capacity compared to comparable immune cells lacking enhanced receptors. In some embodiments, the modified immune cells provided herein have greater transdifferentiation capacity than comparable immune cells lacking enhanced receptors.

In some embodiments, the immune cell activation signal may include or cause the movement and/or transportation of immune cells. In some embodiments, movement can be determined by quantifying the immune cells localized to the target site. For example, the modified immune cells provided herein can be quantified at the target site or at a site that is not the target site after administration. Quantification can be performed by isolating the lesion and quantifying a variety of immune cells containing enhanced receptors (eg, tumor infiltrating lymphocytes). The movement and/or transportation of immune cells containing enhanced receptors may be greater than the movement and/or transportation of comparable immune cells lacking enhanced receptors. In some embodiments, the number of immune cells containing enhanced receptors at the target site (e.g., tumor lesions) may be about 5 times, 10 times, 15 times the number of comparable immune cells lacking cells lacking enhanced receptors, 20 times, 25 times, 30 times, 35 times or 40 times. The transwell migration assay can also be used to determine transport in vitro. In some embodiments, the number of immune cells containing enhanced receptors at the target site, for example, in a transwell migration assay, can be about 5 times, 10 times, 15 times the number of comparable immune cells lacking enhanced receptors , 20 times, 25 times, 30 times, 35 times or 40 times.

In some embodiments, the immune cell activation signal may include or result in the depletion and/or activation of immune cells. The depletion and/or activation of immune cells can be determined by phenotypic analysis by flow cytometry or microscopic analysis. For example, the expression level of exhausted markers, such as programmed cell death protein 1 (PD1), lymphocyte activation gene 3 protein (LAG3), 2B4, CD160, Tim3, and T cell immune receptors with immunoglobulin and ITIM domains Body (TIGIT), quantitatively and/or qualitatively determined. In some cases, immune cells (such as T cells) lose their effector functions in a layered manner and become exhausted. Due to fatigue, functions such as IL-2 production and cytokine expression, as well as high proliferation capacity may be lost. Exhaustion may also be a defect in the production of IFNγ, TNF and chemokines and degranulation. The depletion or activation of the modified immune cells provided herein may be greater than the depletion or activation of comparable immune cells lacking enhanced receptors. In some embodiments, the immune cells provided herein can experience at least about 1 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, and 8 fold compared to comparable immune cells lacking enhanced receptors. , 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 150 Times, 200 times, 250 times or more than 300 times depletion or activation. In some embodiments, the immune cells provided herein can experience at least about 1 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, and 8 fold compared to comparable immune cells lacking enhanced receptors. , 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 150 Depletion or activation is reduced by 200 times, 200 times, 250 times or more than 300 times.

In some embodiments, the binding of the target cell to the enhanced receptor can generate an immune cell activation signal on immune cells modified with the enhanced receptor.

In some embodiments, after enhancing the binding of the receptor to the ligand, the immune cells modified with the enhanced receptor (eg, modified TIL or modified T cells) behave as compared to immune cells lacking the enhanced receptor Enhance the binding of new antigens.

In some embodiments, immune cells modified with enhanced receptors (such as TIL, neoantigen-reactive T cells, CAR-T, TCR-T, NK, etc.) and immune cells without enhanced modification (corresponding to Compared with the aforementioned TIL, neoantigen-reactive T cells, CAR-T, TCR-T, NK, etc.), it can better overcome the inhibitory signal from the tumor microenvironment on immune cells to a certain extent, and the inhibitory signal can come from The ligand or antigen that enhances the extracellular domain of the receptor may also come from the inhibitory signal of the immune cell from other tumor microenvironments other than the ligand or antigen.

In some embodiments, the receptor-enhancing ECD is PD1 or PDL1 monoclonal antibody, and ICD is any costimulatory molecule. The tumor suppressor signal that can be effectively overcome to a certain extent is not limited to PDL1, but can also include CD47, TIM -3 ligands such as Galectin-9, TIGIT ligands such as CD155 and CD122 or PVR, CTLA-4 ligands such as B7 inhibit immune cells.

In some embodiments, if the ECD of the enhanced receptor is SIRPα or CD47 monoclonal antibody, ICD is any costimulatory molecule. The tumor suppressor signal that can be effectively overcome to a certain extent is not only from CD47, but also from PDL1. , TIM-3 ligands such as Galectin-9, TIGIT ligands such as CD155 and CD122 or PVR, CTLA-4 ligands such as B77 inhibit immune cells.

In some embodiments, the receptor-enhancing ECD is a TIM-3 ligand monoclonal antibody such as Galectin-9 monoclonal antibody or TIM-3 monoclonal antibody, and ICD is any certain costimulatory molecule, which can be effectively overcome to a certain extent. Tumor suppression signals not only come from TIM-3 ligands such as Galectin-9, but can also include suppression signals from PDL1, TIGIT ligands such as CD155 and CD122 or PVR, and CTLA-4 ligands such as B77 on immune cells.

In some embodiments, the receptor-enhancing ECD is TIGIT or its ligand monoclonal antibody such as CD155 monoclonal antibody or CD122 monoclonal antibody or PVR monoclonal antibody, and ICD is any certain costimulatory molecule, which can be effectively overcome to a certain extent. The tumor suppressor signals of TIGIT not only come from TIGIT ligands such as CD155 and CD122 or PVR, but can also include inhibitory signals from PDL1, TIM-3 ligands such as Galectin-9, and CTLA-4 ligands such as B77 on immune cells.

In some embodiments, the ECD of the enhanced receptor is VISTA, and ICD is any costimulatory molecule, and the tumor suppressor signal that can be effectively overcome to a certain extent is not limited to the VISTA ligand; if the ECD of the enhanced receptor is CTLA -4, the tumor suppressor signals that can be effectively overcome to a certain extent are not only from CTLA-4 ligands such as B7, but also from PDL1, CD47, TIM-3 ligands such as Galectin-9, TIGIT ligands such as CD155 and CD122 or PVR, CTLA-4 ligand such as B77 inhibits the signal of immune cells.

In some embodiments, T cells carry T cell receptors (TCR) or chimeric antigen receptor CARs. When TCR or CAR can recognize target cells, T cells modified with enhanced receptors are better than those without enhanced receptors. T cells have a stronger immune cell activation function; when neither TCR nor CAR can recognize target cells, T cells modified with enhanced receptors do not have stronger immune cell activation functions than T cells that have not been modified with enhanced receptors .

In one aspect, the present disclosure provides modified immune cells comprising chimeric antigen receptor (CAR) and T cell receptor (TCR) complexes that exhibit specific binding to neoantigens. The CAR may contain an antigen interaction domain capable of binding B cell surface proteins, transmembrane domains, and intracellular signaling domains.

The T cell receptor (TCR) complex that exhibits specific binding to the neoantigen may be an endogenous TCR complex or an exogenous TCR complex. The TCR complex (e.g., endogenous or exogenous) of the modified immune cell can confer antigen binding specificity (e.g., neoantigen binding) to the immune cell.

In some embodiments, the immune cells have artificially modified chimeric antigen receptors (CARs). The CARs do not target pathogenic microbial antigens, but are used for the purpose of specifically amplifying immune cells in vitro or in vivo. It is called Amplification Factor (AF). The amplification factor CAR may include an antigen-interacting domain capable of binding to B cell surface proteins. The B cell surface protein can be any protein that can be found on the surface of B cells. Non-limiting examples include CD1d, CD5, CD10, CD11a, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD28, CD29, CD34, CD37, CD38, CD40, CD44, CD45, CD49b, CD69, CD72 , CD74, CD80, CD83, CD84, CD86, CD93, CD95, CD117, CD127, CD138, CD147, CD148, CD185, CD270, CD284 and CD360. In some embodiments, the antigen interaction domain of the CAR can bind to surface proteins on non-B cells, as long as the binding to the surface proteins does not significantly damage the general health of the host or the immune system. In some embodiments, the surface protein is a surf protein on immune cells. In some embodiments, the surface protein is a surface protein on cells other than immune cells. In some embodiments, the surface protein can be selected from, provided that the binding to the surface protein does not significantly damage the general health of the host or the immune system CD31, CD32, A, B, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42 (a, b, c, d), CD43, CD44, CD45, CD46, CD47, CD48, CD49 (a, b, c, d, e, f), CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CD61, CD62 (E, L, P), CD63, CD64 (A, B, C), CD66 (a, b, c, d, e, f), CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD78, CD79(a, b), CD80, CD81, CD82, CD83, CD84, CD85(a, d, e, h, j, k) , CD86, CD87, CD88, CD89, CD90, CD91, CD92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD100, CD1 (ac), 1A, 1D, 1E, CD2, CD3 (γ, δ, ε), CD4, CD5, CD6, CD7, CD8a, CD9, CD10, CD11 (a, b, c, d), CD13, CD14, CD15, CD16, A, B, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD101, CD102, CD103, CD104, CD105, CD106, CD107(a, b), CD108, CD109, CD110, CD111, CD112, CD113, CD114, CD115 , CD116, CD117, CD118, CD119, CD120(a,b), CD121(a,b), CD122, CD123, CD124, CD125, CD126, CD127, CD129, CD130, CD131, CD132, CD133, CD134, CD135, CD136 , CD137, CD138, CD140b, CD141, CD142, CD143, CD144, CD146, CD147, CD148, CD150, CD191, CD192, CD193, CD194, CD195, CD196, CD197, CDw198, CDw199, CD200, CD201, CD202b, CD204, CD205 , CD206, CD207, CD 208, CD209, CDw210(a, b), CD212, CD213a(1,2), CD217, CD218, (a, b), CD220, CD221, CD222, CD223, CD224, CD225, CD226, CD227, CD228, CD229, CD230, CD233, CD234, CD235(a, b), CD236, CD238, CD239, CD240CE, CD240D, CD241, CD243, CD244, CD246, CD247, CD248, CD249, CD252, CD253, CD254, CD256, CD257, CD258, CD261 , CD262, CD263, CD264, CD265, CD266, CD267, CD268, CD269, CD271, CD272, CD273, CD274, CD275, CD276, CD278, CD279, CD280, CD281, CD282, CD283, CD284, CD286, CD288, CD289, CD290 , CD292, CDw293, CD294, CD295, CD297, CD298, CD299, CD300A, CD301, CD302, CD303, CD304, CD305, CD306, CD307, CD309, CD312, CD314, CD315, CD316, CD317, CD318, CD320, CD321, CD322 , CD324, CD325, CD326, CD328, CD329, CD331, CD332, CD333, CD334, CD335, CD336, CD337, CD338, CD339, CD340, CD344, CD349, CD350, CD151, CD152, CD153, CD154, CD155, CD156 (a , B, c), CD157, CD158 (a, d, e, i, k), CD159(a, c), CD160, CD161, CD162, CD163, CD164, CD166, CD167(a, b), CD168, CD169 , CD170, CD171, CD172 (a, b, g), CD174, CD177, CD178, CD179 (a, b), CD180, CD181, CD182, CD183, CD184, CD185 and CD186.

In some embodiments, the antigen-interacting domain of the expansion factor CAR is capable of binding B cell surface proteins or fragments thereof on dead B cells. B cell apoptosis can occur before or after the development of an immune response (e.g., immune response to tumor cells). Therefore, dead B cells or fragments thereof can still present B cell surface proteins or fragments on the surface. The ability of the expansion factor CAR to target live and dead B cells can increase the chances of immune cells containing the expansion factor CAR to (i) bind to B cell surface proteins and (ii) initiate signals from intracellular signaling domains Conduction. In some cases, the signal transduction of the intracellular signaling domain can promote the expansion (proliferation) of immune cells containing the expansion factor CAR.

In some embodiments, the antigen-interacting domain of the amplification factor CAR is capable of binding B cell surface proteins or fragments thereof, which are coupled (e.g., via covalent and/or non-covalent bonds) to particles (e.g., nanoparticles )surface. The particles may be any particulate material including organic and/or inorganic materials. The particles can have various shapes and sizes. The particles may be about 1 nanometer (nm) to about 50 micrometers (μm) in at least one dimension. The particles may be at least about 1 nm, 5 nm, 10 nm, 50 nm, 100 nm, 500 nm, 1 μm, 5 μm, 10 μm, 50 μm or more in at least one dimension. The particles may be up to about 50 μm, 10 μm, 5 μm, 1 μm, 500 nm, 100 nm, 50 nm, 10 nm, 5 nm, 1 nm or less in at least one dimension. The particles can be nanoparticles, microparticles, nanospheres, microspheres, nanorods, microrods, nanofibers, nanobelts and the like. Examples of particles include metal nanoparticles (eg, gold nanoparticles, silver nanoparticles, and iron nanoparticles), intermetallic semiconductor nanoparticles, core-shell nanoparticles, particles with an inorganic core having a polymer shell, and having a polymer shell The organic core particles, and their mixtures. Alternatively, the particles may be organic nanoparticles, such as cross-linked polymers, hydrogel polymers, biodegradable polymers, polylactide (PLA), polyglycolide (PGA), polycaprolactone (PCL) , Copolymer, polysaccharide, starch, cellulose, chitosan, polyhydroxyalkanoate (PHA), PHB, PHV, lipid, peptide, peptide amphiphile, polypeptide (such as protein) or a combination thereof. The particles presenting the B cell surface protein on the surface can be introduced into immune cells containing the expansion factor CAR that binds to the B cell surface protein in vitro. Alternatively or in addition, the particles presenting the B cell surface protein can be introduced in vivo (for example, local or systemic injection) together with immune cells containing the expansion factor CAR. These particles can be used to expand immune cell populations containing the expansion factor CAR in vitro or in vivo.

The antigen binding domain may comprise any protein or molecule capable of binding antigen, such as B cell surface protein. Non-limiting examples of antigen binding domains include, but are not limited to, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, murine antibodies or functional derivatives, variants or fragments thereof. Including but not limited to Fab, Fab', F(ab')2, Fv, single chain Fv (scFv), minibody, diabody and single domain antibody such as heavy chain variable domain (VH), derived from camelid The light chain variable domain (VL) and variable domain (VHH) of Nanobodies. In some embodiments, the first antigen binding domain comprises at least one of Fab, Fab', F(ab')2, Fv and scFv. In some embodiments, the antigen binding domain comprises an antibody mimic. Antibody mimics are molecules that can bind to target molecules with an affinity equivalent to that of antibodies, including single-chain binding molecules, cytochrome b562-based binding molecules, fibronectin or fibronectin-like protein scaffolds (for example, adnectin), lipids Carrier protein scaffolds, calixarene scaffolds, A domains and other scaffolds. In some embodiments, the antigen binding domain comprises a transmembrane receptor or any derivative, variant or fragment thereof. For example, the antigen binding domain may comprise at least the ligand binding domain of a transmembrane receptor.

In some embodiments, the antigen-interacting domain of the expansion factor CAR is capable of binding B cell surface proteins or fragments thereof on dead B cells. B cell apoptosis can occur before or after the development of an immune response (e.g., immune response to tumor cells). Therefore, dead B cells or fragments thereof can still present B cell surface proteins or fragments on the surface. The ability of the expansion factor CAR to target live and dead B cells can increase the chances of immune cells containing the expansion factor CAR to (i) bind to B cell surface proteins and (ii) initiate signals from intracellular signaling domains Conduction. In some cases, the signal transduction of the intracellular signaling domain can promote the expansion (proliferation) of immune cells containing the expansion factor CAR.

In some embodiments, the antigen-interacting domain of the amplification factor CAR is capable of binding B cell surface proteins or fragments thereof, which are coupled (e.g., via covalent and/or non-covalent bonds) to particles (e.g., nanoparticles )surface. The particles may be any particulate material including organic and/or inorganic materials. The particles can have various shapes and sizes. The particles may be about 1 nanometer (nm) to about 50 micrometers (μm) in at least one dimension. The particles may be at least about 1 nm, 5 nm, 10 nm, 50 nm, 100 nm, 500 nm, 1 μm, 5 μm, 10 μm, 50 μm or more in at least one dimension. The particles may be up to about 50 μm, 10 μm, 5 μm, 1 μm, 500 nm, 100 nm, 50 nm, 10 nm, 5 nm, 1 nm or less in at least one dimension. The particles can be nanoparticles, microparticles, nanospheres, microspheres, nanorods, microrods, nanofibers, nanobelts and the like. Examples of particles include metal nanoparticles (eg, gold nanoparticles, silver nanoparticles, and iron nanoparticles), intermetallic semiconductor nanoparticles, core-shell nanoparticles, particles with an inorganic core having a polymer shell, and having a polymer shell The organic core particles, and their mixtures. Alternatively, the particles may be organic nanoparticles, such as cross-linked polymers, hydrogel polymers, biodegradable polymers, polylactide (PLA), polyglycolide (PGA), polycaprolactone (PCL) , Copolymer, polysaccharide, starch, cellulose, chitosan, polyhydroxyalkanoate (PHA), PHB, PHV, lipid, peptide, peptide amphiphile, polypeptide (such as protein) or a combination thereof. The particles presenting the B cell surface protein on the surface can be introduced into immune cells containing the expansion factor CAR that binds to the B cell surface protein in vitro. Alternatively or in addition, the particles presenting the B cell surface protein can be introduced in vivo (for example, local or systemic injection) together with immune cells containing the expansion factor CAR. These particles can be used to expand immune cell populations containing the expansion factor CAR in vitro or in vivo.

The antigen binding domain may comprise any protein or molecule capable of binding antigen, such as B cell surface protein. Non-limiting examples of antigen binding domains include, but are not limited to, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, murine antibodies or functional derivatives, variants or fragments thereof. Including but not limited to Fab, Fab', F(ab')2, Fv, single chain Fv (scFv), minibody, diabody and single domain antibody such as heavy chain variable domain (VH), derived from camelid The light chain variable domain (VL) and variable domain (VHH) of Nanobodies. In some embodiments, the first antigen binding domain comprises at least one of Fab, Fab', F(ab')2, Fv and scFv. In some embodiments, the antigen binding domain comprises an antibody mimic. Antibody mimics are molecules that can bind to target molecules with an affinity equivalent to that of antibodies, including single-chain binding molecules, cytochrome b562-based binding molecules, fibronectin or fibronectin-like protein scaffolds (for example, adnectin), lipids Carrier protein scaffolds, calixarene scaffolds, A domains and other scaffolds. In some embodiments, the antigen binding domain comprises a transmembrane receptor or any derivative, variant or fragment thereof. For example, the antigen binding domain may comprise at least the ligand binding domain of a transmembrane receptor.

In some embodiments, the antigen binding domain may comprise scFV. The scFv can be derived from antibodies with known variable region sequences. In some embodiments, scFv can be derived from antibody sequences obtained from available mouse hybridomas. The scFv can be obtained from the entire exon sequencing of tumor cells or primary cells. In some embodiments, the scFv can be changed. For example, the scFv can be modified in various ways. In some cases, the scFv can be mutated so that the scFv can have a higher affinity for its target. In some cases, the affinity of the scFv to its target can be optimized for targets that are expressed at low levels on normal tissues. This optimization can be performed to minimize potential toxicity, such as hypercytokineemia. In other cases, the cloned scFv with higher affinity for the target membrane-bound form may be better than its soluble form counterpart. If certain targets can also be detected in different levels of soluble forms, and their targeting can cause unintended toxicity, such as hypercytokineemia, this modification can be made.

The antigen binding domain of the CAR of this system can be connected to the intracellular signal transduction domain through the transmembrane domain. The transmembrane domain may be a transmembrane segment. The transmembrane domain of the subject's CAR can anchor the CAR to the plasma membrane of cells, such as immune cells. In some embodiments, the transmembrane segment comprises a polypeptide. The transmembrane polypeptide connecting the antigen binding domain of the CAR and the intracellular signal transduction domain can have any suitable polypeptide sequence. In some cases, the transmembrane polypeptide comprises the polypeptide sequence of the transmembrane portion of the endogenous or wild-type transmembrane protein. In some embodiments, compared with the transmembrane portion of the endogenous or wild-type transmembrane protein, the transmembrane polypeptide comprises at least 1, (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) amino acid substitutions, deletions or insertions in the polypeptide sequence. In some embodiments, the transmembrane polypeptide comprises a non-natural polypeptide sequence, such as the sequence of a polypeptide linker. The polypeptide linker can be flexible or rigid. The polypeptide linker can be structured or unstructured. In some embodiments, the transmembrane polypeptide transmits signals from the extracellular region of the cell to the intracellular region through the antigen binding domain. The natural transmembrane portion of CD28 can be used in CAR. In other cases, the natural transmembrane portion of CD8α can also be used in CAR.

The CAR of the present disclosure may comprise a signaling domain or any derivative, variant or fragment thereof involved in immune cell signaling. The intracellular signaling domain of CAR can induce the activity of immune cells containing CAR. Intracellular signaling domains can transduce effector function signals and direct cells to perform specialized functions. The signal domain may include the signal transduction domain of other molecules. In some cases, the truncated part of the signal domain is used for CAR.

In some embodiments, the intracellular signaling domain comprises multiple signaling domains involved in immune cell signaling, or any derivative, variant or fragment thereof. For example, the intracellular signaling domain may include at least 2 immune cell signaling domains, for example, at least 2, 3, 4, 5, 7, 8, 9 or 10 immune cell signaling domains. Immune cell signaling domains can participate in the regulation of the primary activation of the TCR complex in a stimulating or inhibitory manner. The intracellular signaling domain may be the signaling domain of the T cell receptor (TCR) complex. The intracellular signaling domains of the subject CAR may include Fcγ receptors (FcγR), Fcε receptors (FcεR), Fcα receptors (FcαR), neonatal Fc receptors (FcRn), CD3, CD3ζ, CD3γ signaling structures Domain, CD3δ, CD3ε, CD4, CD5, CD8, CD21, CD22, CD28, CD32, CD40L (CD154), CD45, CD66d, CD79a, CD79b, CD80, CD86, CD278 (also known as ICOS), CD247ζ, CD247η, DAP10 , DAP12, FYN, LAT, Lck, MAPK, MHC complex, NFAT, NF-κB, PLC-γ, iC3b, C3dg, C3d and Zap70. In some embodiments, the signaling domain includes an immunoreceptor tyrosine-based activation motif or ITAM. The ITAM-containing signal transduction domain may include two repeated amino acid sequences YxxL/I, which are separated by 6-8 amino acids, where each x is independently any amino acid, resulting in the conserved motif YxxL/Ix(6-8)YxxL /I. When the antigen binding domain binds to the epitope, the ITAM-containing signaling domain can be modified, for example, by phosphorylation. Phosphorylated ITAM can be used as a docking site for other proteins, such as proteins involved in various signal transduction pathways. In some embodiments, the primary signaling domain comprises a modified ITAM domain, such as a mutated, truncated, and/or optimized ITAM domain, which has altered (e.g., increased Or reduced) activity.

In some embodiments, the intracellular signaling domain of the subject's CAR comprises an FcyR signaling domain (e.g., ITAM). The FcyR signaling domain can be selected from FcyRI (CD64), FcyRIIA (CD32), FcyRIIB (CD32), FcyRIIIA (CD16a) and FcyRIIIB (CD16b). In some embodiments, the intracellular signaling domain comprises an FcεR signaling domain (e.g., ITAM). The FcεR signaling domain can be selected from FcεRI and FcεRII (CD23). In some embodiments, the intracellular signaling domain comprises an FcaR signaling domain (e.g., ITAM). The FcaR signaling domain can be selected from FcaRI (CD89) and Fca/μR. In some embodiments, the intracellular signaling domain comprises a CD3ζ signaling domain. In some embodiments, the primary signaling domain comprises the ITAM of CD3ζ.

In some embodiments, the intracellular signaling domain of the subject's CAR comprises an immunoreceptor tyrosine-based inhibitory motif or ITIM. The ITIM-containing signaling domain may include a conserved amino acid sequence (S/I/V/LxYxxI/V/L), which is present in the cytoplasmic tail of some inhibitory receptors of the immune system. The main signaling domain comprising ITIM can be modified by enzymes (e.g., members of the Src kinase family (e.g. Lck)), such as phosphorylation. After phosphorylation, other proteins, including enzymes, can be recruited to ITIM. These other proteins include, but are not limited to, enzymes such as phosphotyrosine phosphatase SHP-1 and SHP-2, inositol phosphatase called SHIP, and proteins with one or more SH2 domains (such as ZAP70). Intracellular signaling domains may include BTLA, CD5, CD31, CD66a, CD72, CMRF35H, DCIR, EPO-R, FcγRIIB (CD32), Fc receptor-like protein 2 (FCRL2), Fc receptor signaling domains ( For example, ITIM), similar protein 3 (FCRL3), Fc receptor-like protein 4 (FCRL4), Fc receptor-like protein 5 (FCRL5), Fc receptor-like protein 6 (FCRL6), protein G6b (G6B), interleukin IL-4 receptor (IL4R), immunoglobulin superfamily receptor translocation related 1 (IRTA1), immunoglobulin superfamily receptor translocation related 2 (IRTA2), killer cell immunoglobulin-like receptor 2DL1 (KIR2DL1) , Killer cell immunoglobulin-like receptor 2DL2 (KIR2DL2), Killer cell immunoglobulin-like receptor 2DL3 (KIR2DL3), Killer cell immunoglobulin-like receptor 2DL4 (KIR2DL4), Killer cell immunoglobulin-like receptor 2DL5 ( KIR2DL5), killer cell immunoglobulin-like receptor 3DL1 (KIR3DL1), killer cell immunoglobulin-like receptor 3DL2 (KIR3DL2), leukocyte immunoglobulin-like receptor subfamily B member 1 (LIR1), leukocyte immunoglobulin-like Receptor subfamily B member 2 (LIR2), leukocyte immunoglobulin-like receptor subfamily B member 3 (LIR3), leukocyte immunoglobulin-like receptor subfamily B member 5 (LIR5), leukocyte immunoglobulin-like receptor Subfamily B member 8 (LIR8), leukocyte-associated immunoglobulin-like receptor 1 (LAIR-1), mast cell function-associated antigen (MAFA), NKG2A, natural cytotoxicity trigger receptor 2 (NKp44), NTB-A, Programmed cell death protein 1 (PD-1), PILR, SIGLECL1, sialic acid binding Ig such as lectin 2 (SIGLEC2 or CD22), sialic acid binding Ig such as lectin 3 (SIGLEC3 or CD33), sialic acid binding Ig such as agglutination 5 (SIGLEC5 or CD170), sialic acid binding Ig such as lectin 6 (SIGLEC6), sialic acid binding Ig such as lectin 7 (SIGLEC7), sialic acid binding Ig such as lectin 10 (SIGLEC10), sialic acid binding Ig such as agglutination 11 (SIGLEC11), sialic acid binding Ig such as lectin 4 (SIGLEC4), sialic acid binding Ig such as lectin 8 (SIGLEC8), sialic acid binding Ig such as lectin 9 (SIGL EC9), platelet and endothelial cell adhesion Molecule 1 (PECAM-1), Signal Regulatory Protein (SIRP2) and Signal Threshold Regulation Transmembrane Adaptor 1 (SIT). In some embodiments, the intracellular signaling domain comprises a modified ITIM domain, such as a mutated, truncated and/or optimized ITIM domain, which has altered (eg, increased Or reduced) activity.

In some embodiments, the intracellular signaling domain comprises at least 2 ITAM domains (eg, at least 3, 4, 5, 6, 7, 8, 9 or 10 ITAM domains) . In some embodiments, the intracellular signaling domain comprises at least 2 ITIM domains (eg, at least 3, 4, 5, 6, 7, 8, 9 or 10 ITIM domains) (For example, at least 2 main signaling domains). In some embodiments, intracellular signaling domains include ITAM and ITIM domains.

In some cases, the intracellular signaling domain of the subject's CAR may include a costimulatory domain. In some embodiments, the costimulatory domain, for example from a costimulatory molecule, can provide a costimulatory signal for immune cell signal transduction, such as signal transduction from ITAM and/or ITIM domains, for example, for activation and/or deactivation. live. Immune cell activity. In some embodiments, the costimulatory domain can be used to modulate proliferation and/or survival signals in immune cells. In some embodiments, the costimulatory signaling domain comprises MHC class I protein, MHC class II protein, TNF receptor protein, immunoglobulin-like protein, cytokine receptor, integrin, signaling lymphocyte activation molecule ( SLAM protein), activated signaling domain, NK cell receptor, BTLA or Toll ligand receptor. In some embodiments, the costimulatory domain comprises a signaling domain of a molecule selected from the group consisting of: 2B4/CD244/SLAMF4, 4-1BB/TNFSF9/CD137, B7-1/CD80, B7-2/CD86, B7 -H1/PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BAFF R/TNFRSF13C, BAFF/BLyS/TNFSF13B, BLAME/SLAMF8, BTLA/CD272, CD100(SEMA4D) , CD103, CD11a, CD11b, CD11c, CD11d, CD150, CD160(BY55), CD18, CD19, CD2, CD200, CD229/SLAMF3, CD27 ligand/TNFSF7, CD27/TNFRSF7, CD28, CD29, CD2F-10/SLAMF9 , CD30 ligand/TNFSF8, CD30/TNFRSF8, CD300a/LMIR1, CD4, CD40 ligand/TNFSF5, CD40/TNFRSF5, CD48/SLAMF2, CD49A, CD49D, CD49f, CD5, CD53, CD58/LFA-3, CD69, CD7 , CD8α, CD8β, CD82/Kai-1, CD84/SLAMF5, CD90/Thy1, CD96, CDS, CEACAM1, CRACC/SLAMF7, CRTAM, CTLA-4, DAP12, Dectin-1/CLEC7A, DNAM1(CD226), DPPIV/ CD26, DR3/TNFRSF25, EPHB6, GADS, Gi24/VISTA/B7-H5, GITR ligand/TNFSF18, GITR/TNFRSF18, HLA class I, HLA-DR, HVEM/TNFRSF14, IA4, ICAM-1, ICOS/CD278, Ikaros gene, IL2Rβ, IL2Rγ, IL7Rα, integrin α4/CD49d, integrin α4β1, integrin in α4β7/LPAM-1, IPO-3, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIRDS2, LAG-3, LAT, LIGHT/TNFSF14, LTBR, Ly108, LY9 (CD229), lymphocyte function associated antigen-1 (LFA-1), lymphotoxin-α/TNF-β, NKG2C, NKG2D, NKp30 , NKp44, NKp46, NKp80 (KLRF1), NTB-A/SLAMF6, OX40 ligand/ TNFSF4, OX40/TNFRSF4, PAG/Cbp, PD-1, PDCD6, PD-L2/B7-DC, PSGL1, RELT/TNFRSF19L, SELPLG (CD162), SLAM (SLAMF1), SLAM/CD150, SLAMF4 (CD244), SLAMF6 (NTB-A), SLAMF7, SLP-76, TACI/TNFRSF13B, TCL1A, TCL1B, TIM-1/KIM-1/HAVCR, TIM-4, TL1A/TNFSF15, TNF RII/TNFRSF1B, TNF-α, TRANCE/RANKL , TSLP, TSLP R, VLA1 and VLA-6. In some embodiments, the intracellular signaling domain comprises multiple costimulatory domains, such as at least two, such as at least 3, 4, or 5 costimulatory domains. The costimulatory signal transduction area can provide a signal that is coordinated with the primary effector activation signal, and can fulfill the requirement of activating T cells. In some embodiments, the addition of a costimulatory domain to the CAR can enhance the efficacy and persistence of the immune cells provided herein.

Compared with immune cells lacking CAR, the combination of CAR and B cell surface proteins can enhance immune cell proliferation. The proliferation of immune cells may refer to the expansion of immune cells. The proliferation of immune cells can refer to the phenotypic changes of immune cells. The proliferation of immune cells comprising the CAR provided herein may be greater than the proliferation of immune cells lacking the CAR, which immune cells exhibit binding to B cell surface proteins. The proliferation of immune cells containing CAR may be about 5 times to about 10 times, about 10 times to about 20 times, about 20 times to about 30 times, about 30 times to about 30 times compared with the proliferation of equivalent immune cells lacking CAR. 40 times, about 40 times to about 50 times, about 50 times to about 60 times, about 60 times to about 70 times, about 70 times to about 80 times, about 80 times to about 90 times, about 90 times to about 100 times , About 100 times to about 200 times, about 200 times to about 300 times, about 300 times to about 400 times, about 400 times to about 500 times, about 500 times to about 600 times, about 600 times to about 700 times. The proliferation of immune cells containing CAR may be about 5 times to about 10 times, about 10 times to about 20 times, about 20 times to about 30 times, about 30 times to about 30 times compared with the proliferation of equivalent immune cells lacking CAR. 40 times, about 40 times to about 50 times, about 50 times to about 60 times, about 60 times to about 70 times, about 70 times to about 80 times, about 80 times to about 90 times, about 90 times to about 100 times , About 100 times to about 200 times, about 200 times to about 300 times, about 300 times to about 400 times, about 400 times to about 500 times, about 500 times to about 600 times, about 600 times to about 700 times. And wherein the proliferation is determined at least about 12, 24, 36, 48, 60, 72, 84 or 96 hours after the B cell is brought into contact with the B cell surface protein. The enhanced proliferation can be determined in vitro or in vivo. In some embodiments, proliferation can include quantifying the number of immune cells. Quantifying many immune cells can include flow cytometry, trypan blue exclusion, and/or blood count. Proliferation can also be determined by phenotypic analysis of immune cells.

In one aspect, the present disclosure provides modified immune cells that are responsive to pathogenic microorganisms, wherein the modified immune cells comprise: (a) a chimeric stimulatory molecule comprising a polypeptide extracellular structure that binds to a neoantigen Domain (PED), where PED is fused with the intracellular domain (ICD) of a costimulatory molecule that mediates immune cell activation signals, and where the combination of chimeric stimulatory molecules and neoantigens generates immune cell activation signals in modified immune cells , And (b) a chimeric antigen receptor, which comprises (i) an antigen interaction domain capable of binding to B cell surface proteins; (ii) a transmembrane domain; (iii) an intracellular signal transduction domain. In some embodiments, PED may be the extracellular domain of the surface protein of unmodified TIL. In some embodiments, examples of PED include antibodies, and derivatives, variants, and fragments thereof.

In one embodiment, the present disclosure provides modified immune cells that are responsive to pathogenic microorganisms, wherein the modified immune cells comprise: (a) natural TCR that can specifically bind to neoantigens or genetically engineered Exogenous TCR; (b) an enhanced receptor containing the extracellular domain (ECD) of a protein, wherein the ECD is fused to the intracellular domain (ICD) of a costimulatory molecule that mediates immune cell activation signals, and wherein the enhanced receptor Bodies and ligands generate immune cell activation signals instead of immune cell inactivation signals in modified immune cells, and (c) chimeric antigen receptors, which contain (i) antigen interaction domains capable of binding to B cell surface proteins (Ii) Transmembrane domain; (iii) Intracellular signal transduction domain; In the above three of a, b, and c, a is indispensable. In some embodiments, b or c may only exist One; in some embodiments, a, b, and c coexist.

In one embodiment, the present disclosure provides modified tumor infiltrating lymphocytes (TIL) that are responsive to pathogenic microorganisms, wherein the modified immune cells comprise: (a) natural TCR that can specifically bind to neoantigens; b) An enhanced receptor containing the extracellular domain (ECD) of the protein. ECD is fused with the intracellular domain (ICD) of a costimulatory molecule that mediates immune cell activation signals, and where the conversion of the molecule into a ligand generates an immune cell activation signal instead of an immune cell inactivation signal in the modified TIL, and ( c) A chimeric antigen receptor, which comprises (i) an antigen interaction domain capable of binding to B cell surface proteins; (ii) a transmembrane domain; (iii) an intracellular signal transduction domain. Among the above three of a, b, and c, a is indispensable. In some embodiments, only one of b or c may exist; in some embodiments, a, b, and c coexist.

In one aspect, the present disclosure provides modified immune cells that overexpress cytokines, such as chemokines, that are responsive to pathogenic microorganisms, wherein the immune cells are (i) tumor infiltrating lymphocytes (TIL); (ii) between Tumor-infiltrating lymphocytes (sTIL); or (iii) T cells that exhibit specific binding to the antigen. The modified immune cell overexpressing the chemokine can be any of the modified immune cells provided herein.

Cytokines refer to proteins released by cells (for example, chemokines, interferons, lymphokines, interleukins, and tumor necrosis factor), which can affect cell behavior. Cytokines are produced by a variety of cells, including immune cells such as macrophages, B lymphocytes, T lymphocytes and mast cells, as well as endothelial cells, fibroblasts and various stromal cells. A given cytokine can be produced by more than one type of cell. Cytokines can participate in the production of systemic or local immune regulation.

Certain cytokines can act as pro-inflammatory cytokines. Pro-inflammatory cytokines refer to cytokines involved in inducing or amplifying an inflammatory response. Pro-inflammatory cytokines can produce an immune response together with various cells of the immune system, such as neutrophils and leukocytes. Certain cytokines can act as anti-inflammatory cytokines. Anti-inflammatory cytokines refer to cytokines involved in reducing inflammation. In some cases, anti-inflammatory cytokines can modulate the pro-inflammatory cytokine response. Some cytokines can act as pro-inflammatory cytokines and anti-inflammatory cytokines. Certain cytokines, such as chemokines, can play a role in chemotaxis. Chemokines can induce targeted chemotaxis in nearby responding cells.

In some embodiments, the expression of cytokines with pro-inflammatory and/or chemotactic functions can be up-regulated in immune cells. Up-regulating the expression of cytokines with pro-inflammatory and/or chemotactic functions can be used, for example, in immunotherapy to stimulate an immune response against target cells.

Examples of cytokines that can be overexpressed by the immune cells provided herein include, but are not limited to, lymphokines, monocytes, and traditional polypeptide hormones. Cytokines include growth hormones, such as human growth hormone, N-methionyl human growth hormone and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; pro relaxin; glycoprotein hormones, such as stimulating hormone Follicle Hormone (FSH), Thyroid Stimulating Hormone (TSH) and Luteinizing Hormone (LH); Liver Growth Factor; Fibroblast Growth Factor; Prolactin; Placental Prolactin; Tumor Necrosis Factor-α; Mullerian Inhibitory Substance; Mice Gonadotropin-related peptide; Inhibin; Activin; Vascular endothelial growth factor; Integration; Thrombopoietin (TPO); Nerve growth factor such as NGF-α; Platelet growth factor; Transforming growth factor (TGFs), such as TGF-α, TGF-β, TGF-β1, TGF-β2 and TGF-β3; insulin-like growth factors-I and -II; erythropoietin (EPO); FLT-3L; stem cell factor (SCF); osteoinductive factors; interferon (IFN), such as IFN-α, IFN-β, IFN-γ; colony stimulating factors (CSFs), such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); Granulocyte-CSF (G-CSF); Macrophage Stimulating Factor (MSP); Interleukins (ILs), such as IL-1, IL-1a, IL-1b, IL-1RA, IL-18, IL-2 , IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL -15, IL-16, IL-17, IL-20; tumor necrosis factors such as CD154, LT-β, TNF-α, TNF-β, 4-1BBL, APRIL, CD70, CD153, CD178, GITRL, LIGHT, OX40L , TALL-1, TRAIL, TWEAK, TRANCE; and other polypeptide factors including LIF, Oncostatin M (OSM) and Kit ligand (KL). Cytokine receptor refers to a receptor protein that binds to cytokine. Cytokine receptors can be membrane-bound and soluble.

In some embodiments, the overexpressed cytokine is a member of the interleukin (IL-1) family (e.g., a ligand), a member of the IL-1 receptor family, a member of the interleukin-6 (IL-6) family (e.g., Ligand), IL-6 receptor, interleukin-10 (IL-10) family member (such as ligand), IL-10 receptor, interleukin-12 (IL-12) family member (such as ligand) ), IL-12 receptor, interleukin-17 (IL-17) family member (eg ligand) or IL-17 receptor.

In some embodiments, the overexpressed cytokine is a member of the interleukin-1 (IL-1) family or a related protein; a member of the tumor necrosis factor (TNF) family or a related protein; a member of the interferon (IFN) family or a related protein ; Interleukin-6 (IL-6) family members or related proteins; or chemokines or related proteins. In some embodiments, the cytokine is selected from IL18, IL18BP, IL1A, IL1B, IL1F10, IL1F3/IL1RA, IL1F5, IL1F6, IL1F7, IL1F8, IL1RL2, IL1F9, IL33, BAFF/BLyS/TNFSF138, 4-1BBL, CD153/ CD30L/TNFSF8, CD40LG, CD70, Fas ligand/FASLG/CD95L/CD178, EDA-A1, TNFSF14/LIGHT/CD258, TNFA, LTA/TNFB/TNFSF1, LTB/TNFC, CD70/CD27L/TNFSF7, TNFSF10/TRAIL/ APO-2L (CD253), RANKL/OPGL/TNFSF11 (CD254), TNFSF12, TNF-α/TNFA, TNFSF13, TL1A/TNFSF15, OX-40L/TNFSF4/CD252, CD40L/CD154/TNFSF5, IFNA1, IFNA10, IFNA13, IFNA14, IFNA2, IFNA4, IFNA7, IFNB1, IFNE, IFNG, IFNZ, IFNA8, IFNA5/IFNaG, IFNω/IFNW1, CLCF1, CNTF, IL11, IL31, IL6, Leptin, LIF, OSM, CCL1/TCA3, CCL11, CCL12/ MCP-5, CCL13/MCP-4, CCL14, CCL15, CCL16, CCL17/TARC, CCL18, CCL19, CCL2/MCP-1, CCL20, CCL21, CCL22/MDC, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL3L3, CCL4, CCL4L1/LAG-1, CCL5, CCL6, CCL7, CCL8, CCL9, CX3CL1, CXCL1, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL3, CXCL17, CXCL17, CXCL4, CXCL5, CXCL6, CXCL7/Ppbp, CXCL9, IL8/CX CL8, XCL1, XCL2, FAM19A1, FAM19A2, FAM19A3, FAM19A4 and FAM19A5.

A variety of methods can be used to assess cytokine expression. Cytokine expression can be determined by measuring the cell culture medium in which the modified immune cells are grown (e.g., in vitro production) or serum obtained from one or more sera obtained from subjects with modified immune cells (e.g., in vivo Production) to evaluate cytokines. Cytokine levels can be quantified in various suitable units using any suitable assay, including concentration. In some embodiments, cytokine proteins are detected. In some embodiments, the mRNA transcript of the cytokine is detected. Examples of cytokine assays include enzyme-linked immunosorbent assay (ELISA), immunoblotting, immunofluorescence assay, radioimmunoassay, antibody arrays that allow parallel detection of various cytokines in samples, bead-based arrays, quantitative PCR, microarrays Wait. Other suitable methods may include proteomics methods (2-D gel, MS analysis, etc.).

In some embodiments, the cytokine overexpressed by the modified immune cells provided herein is a chemokine. The chemokines can be, for example, CC chemokines, CXC chemokines, C chemokines, and CX3C chemokines. In some embodiments, the chemokine overexpressed by the modified immune cells is a CC chemokine selected from CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27 and CCL28. The chemokine is a CXC chemokine selected from CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16 and CXCL17. In some embodiments, the chemokine overexpressed by the modified immune cells is a C chemokine selected from XCL1 and XCL2. In some embodiments, the chemokine overexpressed by immune cells is a CX3C chemokine, and the CX3C chemokine is CX3CL1.

In one aspect, the present disclosure provides a method of expanding a T cell population, the method comprising: (a) providing a T cell population, which includes at least the modified immune cells of any one of the various embodiments of the aspect; (b) Expose the T cell population to B cell surface proteins to achieve expansion of the T cell population. In some embodiments, in (b), the T cell population is exposed to B cells containing B cell surface proteins.

In one aspect, the present disclosure provides a method of expanding a T cell population, which comprises: (a) introducing a nucleic acid encoding a chimeric antigen receptor (CAR) into the T cell population, thereby generating CAR-expressing cells or cell populations , Wherein the CAR contains (i) a domain that interacts with the antigen specifically recognized by the CAR; (ii) a transmembrane domain; (iii) an intracellular signaling domain; (b) contacting a cell population expressing CAR with the antigen , Resulting in an expanded and/or activated immune cell population. Wherein, the antigen is not specifically expressed by tumor target cells. The antigen may be a B cell surface protein such as CD19, BCMA, or other blood cell surface protein, or a mutant type of natural protein such as EGFRviii, or an artificially modified protein.

In one aspect, the present disclosure provides a composition comprising one or more polynucleotides encoding one or more of the following: (a) an enhanced receptor, wherein the enhanced receptor comprises an extracellular domain of a protein (ECD), in which the ECD is fused with the intracellular domain (ICD) of a costimulatory protein that mediates immune cell activation signals; (b) an antigen-specific T cell receptor complex, or one or more components thereof.

In one aspect, the present disclosure provides a composition comprising one or more polynucleotides encoding one or more of the following: (a) an antigen-specific T cell receptor complex, or one or more combinations thereof Points; (b) chimeric antigen receptor, which comprises (i) an antigen interaction domain capable of binding to B cell surface proteins; (ii) a transmembrane domain; (iii) an intracellular signal transduction domain.

In one aspect, the present disclosure provides a composition comprising one or more polynucleotides encoding one or more of the following: (a) an enhanced receptor, wherein the enhanced receptor comprises an extracellular domain of a protein (ECD), wherein the ECD is fused with an intracellular domain (ICD) of a costimulatory protein that mediates immune cell activation signals; (b) an antigen-specific T cell receptor complex, or one or more groups thereof Points; (c) Chimeric antigen receptors, which comprise (i) antigen interaction domains capable of binding to B cell surface proteins; (ii) transmembrane domains; (iii) intracellular signal transduction domains.

In various embodiments of the aspects herein, promoters that can be used include promoters that are active in eukaryotic, mammalian, non-human mammalian, or human cells. The promoter can be an inducible or constitutively active promoter. Alternatively or in addition, the promoter may be tissue or cell specific.

Non-limiting examples of suitable eukaryotic promoters (ie promoters that function in eukaryotic cells) may include those from early cytomegalovirus (CMV), herpes simplex virus (HSV) thymidine kinase, early and late SV40, Those with long terminal repeats (LTR). From the retrovirus, the human elongation factor-1 promoter (EF1), a hybrid construct containing the cytomegalovirus (CMV) enhancer fused to the chicken beta active promoter (CAG), and the murine stem cell virus promoter ( MSCV), phosphoglycerate kinase-1 locus promoter (PGK) and mouse metallothionein-I. The promoter may be a fungal promoter. The promoter may be a plant promoter. A database of plant promoters can be found (e.g., PlantProm). The expression vector may also contain a ribosome binding site for translation initiation and a transcription terminator. The expression vector may also include suitable sequences for amplifying expression.

In various embodiments of the aspects herein, the modified immune cell further comprises an inactivation switch (or called a suicide switch). In the case of severe toxicity, such as hypercytokineemia, the kill switch can be activated to eliminate immune cells. This can happen when the immune system reacts so strongly that many inflammatory cytokines are released, causing mild to severe symptoms, including fever, headache, rash, rapid heartbeat, low blood pressure, and difficulty breathing. The kill switch may be a drug-induced kill switch. The kill switch may comprise inducible caspase-9.

Various embodiments of the aspects herein include cells, such as modified immune cells. Cells, such as immune cells (eg, lymphocytes including T cells and NK cells) can be obtained from the subject. Non-limiting examples of subjects include humans, dogs, cats, mice, rats and their transgenic species. Examples of samples from subjects from which cells can be obtained include, but are not limited to, skin, heart, lung, kidney, bone marrow, breast, pancreas, liver, muscle, smooth muscle, bladder, gallbladder, colon, intestine, brain, prostate, Esophagus, thyroid, serum, saliva, urine, stomach and digestive juices, tears, feces, semen, vaginal fluid, interstitial fluid from tumor tissue, eye fluid, sweat, mucus, earwax, oil, glandular secretions, spinal cord Fluid, hair, nails, plasma, nasal swab or nasopharyngeal cleansing, spinal fluid, cerebrospinal fluid, tissue, throat swab, biopsy, placental fluid, amniotic fluid, umbilical cord blood, strong fluid, cavity fluid, sputum, pus , Microbiota, meconium, milk and/or other excreta or body tissues.

In some cases, the cells may be T cell populations, NK cells, B cells, etc. obtained from the subject. T cells can be obtained from many sources, including PBMC, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, and tissue from infection sites, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, any number of techniques, such as Ficoll ^™ isolation, can be used to obtain T cells from blood units collected from the subject. In one embodiment, cells from the circulating blood of the individual are obtained by apheresis. Apheresis blood component products usually contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells and platelets. The cells collected by apheresis can be washed to remove the plasma fraction and placed in a suitable buffer or medium for subsequent processing steps.

Any one of a variety of immune cells can be used in the invention. In some embodiments, immune cells include granulocytes, such as basophils, eosinophils, and neutrophils; mast cells; monocytes that can develop into macrophages; antigen-presenting cells, such as dendrites Shape cells; and lymphocytes, such as natural killer cells (NK cells), B cells and T cells. In some embodiments, the immune cell is an immune effector cell. Immune effector cells refer to immune cells that can perform specific functions in response to stimulation. In some embodiments, immune cells are immune effector cells that can induce cell death. In some embodiments, the immune cells are lymphocytes. In some embodiments, the lymphocytes are NK cells. In some embodiments, the lymphocytes are T cells. In some embodiments, the T cell is an activated T cell. T cells include naive and memory cells (such as central memory or TCM, effector memory or TEM and effector memory RA or TERA), effector cells (such as cytotoxic T cells or CTL or Tc cells), helper cells (such as Th1, Th2, Th3 , Th9, Th7, TFH), regulatory cells (such as Treg and Tr1 cells), natural killer T cells (NKT cells), tumor infiltrating lymphocytes (TIL), lymphocyte activated killer cells (LAK), αβT cells, γδT cells Similar to the unique category of the T cell lineage. T cells can be divided into two categories: CD8+ T cells and CD4+ T cells, based on the presence of proteins on the cell surface. T cells expressing the subject's system can perform a variety of functions, including killing infected cells and activating or recruiting other immune cells. CD8+ T cells are called cytotoxic T cells or cytotoxic T lymphocytes (CTL). The CTL expressing the subject's system can participate in the identification and removal of virus-infected cells and cancer cells. CTL has specialized compartments or particles that contain cytotoxins that cause apoptosis, such as programmed cell death. CD4+ T cells can be subdivided into four subgroups: Th1, Th2, Th17 and Treg. "Th" refers to "T helper cells", although other subgroups may exist. Th1 cells can coordinate the immune response against intracellular microorganisms, especially bacteria. They can produce and secrete molecules that alert and activate other immune cells, such as bacteria that ingest macrophages. Th2 cells are involved in coordinating the immune response against extracellular pathogens (such as worms (parasites)) by warning B cells, granulocytes and mast cells. Th17 cells can produce interleukin 17 (IL-17), a signaling molecule that activates immune and non-immune cells. Th17 cells are important for recruiting neutrophils.

In some embodiments, the immune cell populations provided herein may be heterogeneous. In some embodiments, the cells used may consist of a heterogeneous mixture of CD4 and CD8 T cells. CD4 and CD8 cells can have the phenotypic characteristics of circulating effector T cells. The CD4 and CD8 cells may also have the phenotypic characteristics of effector memory cells. In some embodiments, the cell may be a central memory cell.

In some embodiments, the cells include peripheral blood mononuclear cells (PBMC), peripheral blood lymphocytes (PBL) and other blood cell subpopulations, such as but not limited to T cells, natural killer cells, monocytes, natural cells, killer T cells, monocyte precursor cells, hematopoietic stem cells or non-pluripotent stem cells. In some cases, the cell may be any immune cell, including any T cell, such as tumor infiltrating cells (TIL), such as CD3+ T cells, CD4+ T cells, CD8+ T cells, or any other type of T-cells. T cells may also include memory T cells, memory stem T cells or effector T cells. T cells can also be selected from a large population, for example, T cells are selected from whole blood. T cells can also be expanded from large populations. T cells can also be biased towards specific populations and phenotypes. For example, T cells can be tilted to phenotypes, including CD45RO(-), CCR7(+), CD45RA(+), CD62L(+), CD27(+), CD28(+) and/or IL-7Rα(+). Appropriate cells can be selected, which contain one or more markers selected from the following list: CD45RO(-), CCR7(+), CD45RA(+), CD62L(+), CD27(+), CD28(+) ) And/or IL-7Rα(+). Cells also include stem cells, such as embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, neuronal stem cells, and mesenchymal stem cells. The cells may comprise any number of primary cells, such as human cells, non-human cells and/or mouse cells. The cell can be a progenitor cell. The cells can be derived from the subject to be treated (e.g., patient). The cells can be derived from human donors. The host cell can be a stem memory TSCM cell composed of CD45RO(-), CCR7(+), CD45RA(+), CD62L+ (L-selectin), CD27+, CD28+ and IL-7Rα+, and the stem memory cell can also be It expresses CD95, IL-2Rβ, CXCR3 and LFA-1, and shows many functional properties different from the stem storage cells. The host cell may be a central memory TCM cell containing L-selectin and CCR7, and the central memory cell may secrete, for example, IL-2, but not IFNγ or IL-4. The cells may also be effector memory TEM cells containing L-selectin or CCR7, and produce, for example, effector cytokines such as IFNγ and IL-4.

In various embodiments of the aspects herein, the immune cells comprise lymphocytes. In some embodiments, the lymphocytes are natural killer cells (NK cells). In some embodiments, the lymphocytes are T cells. T cells can be obtained from many sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, spleen tissue, umbilical cord and tumors. In some embodiments, any number of available T cell lines can be used. Immune cells such as lymphocytes (e.g., cytotoxic lymphocytes) may preferably be autologous cells, but heterologous cells may also be used. Any number of techniques, such as Ficoll separation, can be used to obtain T cells from blood units collected from the subject. Cells from the circulating blood of an individual can be obtained by apheresis or leukopenia. Apheresis blood component products usually contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells and platelets. The cells collected by apheresis can be washed to remove the plasma fraction, and the cells can be placed in a suitable buffer or medium, such as phosphate buffered saline (PBS), for subsequent processing steps. After washing, the cells can be resuspended in various biocompatible buffers, such as Ca-free Mg-free PBS. Alternatively, the undesired components in the apheresis blood component sample can be removed, and the cells can be directly resuspended in the culture medium. The sample may be provided directly by the subject, or indirectly through one or more intermediates, such as a sample collection service provider or a medical provider (such as a doctor or a nurse). In some embodiments, separating T cells from peripheral blood leukocytes may include lysing red blood cells and separating them from peripheral blood leukocytes from monocytes by, for example, PERCOL ^™ gradient centrifugation.

A specific subpopulation of T cells, such as CD4+ or CD8+ T cells, can be further separated by positive or negative selection techniques. For example, with a combination of antibodies against surface markers specific to negatively selected cells, negative selection of T cell populations can be achieved. One suitable technique includes cell sorting by negative magnetic immunoadhesion, which utilizes a mixture of monoclonal antibodies directed against cell surface markers present on negatively selected cells. For example, to isolate CD4+ cells, the monoclonal antibody mixture may include antibodies against CD14, CD20, CD11b, CD16, HLA-DR and CD8. The negative selection process can be used to generate a desired population of T cells that is mostly homogeneous. In some embodiments, the composition comprises a mixture of two or more (eg, 2, 3, 4, 5 or more) different types of T cells.

In some embodiments, immune cells are members of an enriched cell population. One or more desired cell types can be enriched by any suitable method, non-limiting examples of which include treatment of cell populations to trigger expansion and/or differentiation into desired cell types, and treatment to prevent the development of unwanted cell types. Treatment of growing, killing or lysing unwanted cell types, purifying desired cell types (e.g. purification on an affinity column to retain desired or unwanted cell types based on one or more cell surface markers). In some embodiments, the enriched cell population is a cell population rich in cytotoxic lymphocytes selected from cytotoxic T cells (also known as cytotoxic T lymphocytes, CTL, T killer cells, Cytolytic T cells, CD8+ T cells and killer T cells), natural killer (NK) cells and lymphokine activated killer (LAK) cells.

In order to isolate the desired cell population by positive or negative selection, the concentration of the cells and the surface (for example, particles such as beads) can be changed. In certain embodiments, it may be necessary to significantly reduce the volume at which the beads and cells are mixed together (ie, increase the cell concentration) to ensure maximum contact between the cells and the beads. For example, a concentration of 2 billion cells/mL can be used. In some embodiments, a concentration of 1 billion cells/mL is used. In some embodiments, more than 100 million cells/mL are used. Cell concentrations of 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500 or 50 million cells/mL can be used. In another embodiment, a cell concentration of 7500, 8000, 8500, 9000, 9500 or 100 million cells/mL can be used. In a further embodiment, a concentration of 125 or 150 million cells/mL can be used. Using high concentrations can lead to increased cell yield, cell activation and cell expansion.

The system and method of the present disclosure can be used to kill a variety of pathogenic microorganisms such as viruses, bacteria, and fungi. The death of pathogenic microorganisms can be determined by any suitable method, including but not limited to counting cells before and after treatment, or measuring the level of markers associated with live pathogenic microorganisms or dead pathogenic microorganisms. The degree of killing of pathogenic microorganisms can be determined by any suitable method. In some embodiments, the degree of killing is determined relative to the starting conditions.

The various domains of the enhanced receptor and CAR provided herein can be connected by chemical bonds, such as amide bonds or disulfide bonds; a small organic molecule (such as a hydrocarbon chain); -200 amino acid sequence), or a combination of small organic molecules and peptide linkers. The peptide linker can provide the required flexibility to allow the desired expression, activity and/or conformational positioning of the chimeric polypeptide. The peptide linker can have any suitable length to connect at least two domains of interest, and is preferably designed to be flexible enough to allow the correct folding and/or function and/or activity of the one or two domains to which it is connected. The peptide linker can have a length of at least 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 amino acids in length . In some embodiments, the length of the peptide linker is about 0 to 200 amino acids, about 10 to 190 amino acids, about 20 to 180 amino acids, about 30 to 170 amino acids, about 40 to 160 amino acids, about 50 to 150 Amino acids, about 60 to 140 amino acids, about 70 to 130 amino acids, about 80 to 120 amino acids, about 90 to 110 amino acids. In some embodiments, the linker sequence may comprise an endogenous protein sequence. In some embodiments, the linker sequence comprises glycine, alanine and/or serine amino acid residues. In some embodiments, the linker may contain a motif of GS, GGS, GGGGS, GGSG, or SGGG, such as multiple or repeated motifs. The linker sequence may include any naturally occurring amino acid, non-naturally occurring amino acid, or a combination thereof.

Any suitable delivery method may be used to introduce the compositions and molecules of the present disclosure (e.g., polypeptides and/or nucleic acids encoding polypeptides) into host cells, such as antigen-promoting cells or immune cells. The various components can be delivered simultaneously or separated in time. The choice of method may depend on the type of transformed cell and/or the environment in which the transformation occurs (e.g., in vitro, ex vivo, or in vivo).

The delivery method may include contacting a target polynucleotide or introducing one or more nucleic acids into a cell (or a population of cells such as immune cells), the nucleic acid comprising a nucleotide sequence encoding a composition of the invention. A suitable nucleic acid comprising a nucleotide sequence encoding a composition of the present disclosure may include an expression vector, wherein the expression vector comprising a nucleotide sequence encoding one or more compositions of the present disclosure is a recombinant expression vector.

Non-limiting examples of delivery methods or transformations include, for example, viral or phage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI) mediated Transfection, DEAE-dextran-mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection and nanoparticle-mediated nucleic acid delivery.

In some aspects, the present disclosure provides methods that include combining one or more polynucleotides, or one or more vectors as described herein, or one or more transcripts thereof, and/or one or more transcribed from them. Methods of delivering one or more proteins to host cells. In some aspects, the present disclosure also provides cells produced by these methods, as well as organisms (such as animals, plants, or fungi) that contain these cells or are produced by these cells.

Conventional viral and non-viral-based gene transfer methods can be used to introduce nucleic acids into mammalian cells or target tissues. Such methods can be used to administer a nucleic acid encoding a composition of the present disclosure to cells or host organisms in culture. Non-viral vector delivery systems may include DNA plasmids, RNA (e.g., transcripts of vectors described herein), naked nucleic acids, and nucleic acids complexed with delivery vehicles, such as liposomes. Viral vector delivery systems can include DNA and RNA viruses, which can have episomal or integrated genomes after delivery to cells.

Methods of non-viral delivery of nucleic acids may include lipofection, nucleofection, microinjection, bioprojectiles, virosomes, liposomes, immunoliposomes, polycations or lipids: nucleic acid conjugates, naked DNA , Artificial virus particles and DNA agents enhance uptake. Efficient receptors suitable for polynucleotides can be used to recognize lipofected cationic and neutral lipids. Delivery can be cellular (e.g. in vitro or ex vivo administration) or target tissue (e.g. in vivo administration). The preparation of lipids can be used: nucleic acid complexes, including targeted liposomes, such as immunolipid complexes.

RNA or DNA virus-based systems can be used to target specific cells in the body and transport the viral payload to the nucleus. Viral vectors can be administered directly (in vivo), or they can be used to treat cells in vitro, and can optionally be administered to modified cells (ex vivo). Virus-based systems can include retrovirus, lentivirus, adenovirus, adeno-associated and herpes simplex virus vectors for gene transfer. Using retrovirus, lentivirus and adeno-associated virus gene transfer methods can be integrated in the host genome, which can lead to long-term expression of the inserted transgene. High transduction efficiency can be observed in many different cell types and target tissues.

Lentivirus can integrate its genome into host cells (such as 293 cells, or T cells). Lentivirus can use a three-plasmid system or a four-plasmid system.

The tropism of retroviruses can be changed by incorporating foreign envelope proteins, thereby amplifying the potential target population of target cells. Lentiviral vectors are retroviral vectors that can transduce or infect non-dividing cells and produce high viral titers. The choice of retroviral gene transfer system can depend on the target tissue. The retroviral vector may contain a cis-acting long terminal repeat sequence, which has a packaging capacity of up to 6-10 kb of foreign sequence. Minimal cis-acting LTR can be sufficient for vector replication and packaging, which can be used to integrate therapeutic genes into target cells to provide permanent transgene expression. Retroviral vectors may include vectors based on murine leukemia virus (MuLV), gibbon leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof.

An adenovirus-based system can be used. Adenovirus-based systems can lead to transient expression of transgenes. Adenovirus-based vectors can have high transduction efficiency in cells and may not require cell division. High titers and expression levels can be obtained with adenovirus-based vectors. Adeno-associated virus ("AAV") vectors can be used to transduce cells with target nucleic acids, for example, to produce nucleic acids and peptides in vitro, and for in vivo and ex vivo gene therapy procedures.

Packaging cells can be used to form viral particles capable of infecting host cells. Such cells may include 293 cells (for example, for packaging lentivirus or adenovirus) and Psi2 cells or PA317 cells (for example, for packaging retrovirus). Viral vectors can be produced by producing cell lines that package nucleic acid vectors into viral particles. The vector may contain the minimal viral sequences required for packaging and subsequent integration into the host. The vector may contain other viral sequences, which are replaced by the expression cassette of the polynucleotide to be expressed. The missing viral functions can be provided in trans by the packaging cell line. For example, an AAV vector can contain an ITR sequence from the AAV genome, which is necessary for packaging and integration into the host genome. Viral DNA can be packaged in a cell line that contains helper plasmids encoding other AAV genes, namely rep and cap, but lacks the ITR sequence. Cell lines can also be infected with adenovirus as a helper cell. The helper virus can promote the replication of the AAV vector and the expression of the AAV gene from the helper plasmid. Contamination of adenovirus can be reduced by, for example, heat treatment in which adenovirus is more sensitive than AAV. Other methods for delivering nucleic acids to cells can be used, for example, as described in US20030087817, which is incorporated herein by reference.

The host cell can be transfected transiently or non-transiently with one or more of the vectors described herein. The cell can be transfected because it is naturally present in the subject. The cells can be taken or derived from the subject and transfected. The cell may be derived from a cell taken from a subject, such as a cell line. In some embodiments, cells transfected with one or more of the vectors described herein are used to establish new cell lines containing one or more vector-derived sequences. In some embodiments, cells transiently transfected with the composition of the present disclosure (e.g., by transient transfection of one or more vectors, or transfected with RNA) are used for establishment of cells containing modifications but lacking any other exogenous sequences New cell line of cells.

Any suitable vector that is compatible with the host cell can be used with the methods of the present disclosure. Non-limiting examples of vectors for eukaryotic host cells include pXT1, pSG5 (Stratagene™), pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia™).

Contacting the cells with the composition can occur in any medium and under any culture conditions that promote the survival of the cells. For example, the cells can be suspended in any convenient appropriate nutrient medium, such as Iscove's modified DMEM or RPMI1640, supplemented with fetal bovine serum or heat-inactivated goat serum (approximately 5-10%), L-glutamine, Thiols, especially 2-mercaptoethanol and antibiotics, such as penicillin and streptomycin. The culture may contain growth factors to which the cells respond. Growth factors as defined herein are molecules capable of promoting the survival, growth and/or differentiation of cells in culture or intact tissues through specific effects on transmembrane receptors. Growth factors can include polypeptide and non-polypeptide factors.

In many embodiments, the selected delivery system targets a specific tissue or cell type. In some cases, tissue or cell targeting of the delivery system is achieved by combining the delivery system with tissue or cell-specific markers (e.g., cell surface proteins). Viral and non-viral delivery systems can be customized to target tissues or cell types of interest.

Pharmaceutical compositions containing the molecules described herein (for example, polypeptides and/or nucleic acids or proteins encoding polypeptides) or immune cells can be administered for prophylactic and/or therapeutic treatments. In therapeutic applications, the composition can be administered to a subject already suffering from a disease or condition in an amount sufficient to cure or at least partially prevent the symptoms of the disease or condition, or cure, heal, improve or improve the condition. The amount effective for this purpose can vary depending on the severity and course of the disease or condition, previous treatment, the subject's health, weight and response to the drug, and the judgment of the treating physician.

The multiple therapeutic agents can be administered in any order or simultaneously. If at the same time, multiple therapeutic agents can be provided in a single, unified form or in multiple forms, for example, as multiple separate pills or cell solutions. The vaccine or cell solution can be packaged together or separately in a single package or in multiple packages. One or all therapeutic agents can be given in multiple doses. If not at the same time, the time between multiple doses can vary to about 1 to 24 months.

The vaccines or cells described herein can be administered before, during, or after the occurrence of the disease or condition, and the time of administration of the compound-containing composition can vary. For example, the pharmaceutical composition can be used as a preventive agent, and can be continuously administered to a subject who has a disease or disease tendency to prevent the occurrence of the disease or disease. The vaccine, cell and pharmaceutical composition of the present invention can be administered to the subject during the onset of symptoms or as soon as possible. The administration of the vaccine can be started within the first 48 hours of the onset of symptoms, within the first 24 hours of the onset of symptoms, within the first 6 hours of the onset of symptoms, or within 3 hours of the onset of symptoms. Onset of symptoms. The initial administration can be by any practical route, for example, by any of the routes described herein, using any of the formulations described herein. The vaccine can be administered as soon as feasible after the onset of the disease or condition is detected or suspected, and the length of time required to treat the disease, for example, about 1 month to about 3 months. The duration of treatment for each subject may be different.

In some cases, the dose of cells administered to the subject may be less than 1×10 ⁴ cells/kg, or about 1×10 ⁴ cells/kg, about 2×10 ⁴ cells/kg, about 3× 10 ⁴ cells/kg, about 4×10 ⁴ cells/kg, about 5×10 ⁴ cells/kg, about 6×10 ⁴ cells/kg, about 7×10 ⁴ cells/kg, about 8×10 ⁴ cells/kg, about 9×10 ⁴ cells, about 1×10 ⁵ cells/kg, about 2×10 ⁵ cells/kg, about 3×10 ⁵ cells/kg, about 4×10 ⁵ cells /kg, about 5×10 ⁵ cells/kg, about 6×10 ⁵ cells/kg, about 7×10 ⁵ cells/kg, about 8×10 ⁵ cells/kg, about 9×10 ⁵ cells , About 1×10 ⁶ cells/kg, about 2×10 ⁶ cells/kg, about 3×10 ⁶ cells/kg, about 4×10 ⁶ cells/kg, about 5×10 ⁶ cells/kg, About 6×10 ⁶ cells/kg, about 7×10 ⁶ cells/kg, about 8×10 ⁶ cells/kg, about 9×10 ⁶ cells, about 1×10 ⁷ cells/kg, about 5 ×10 ⁷ cells/kg, about 1×10 ⁸ cells/kg or more. Here, the cell dose can be calculated according to the number of effectively modified cells successfully transfected, or it can be calculated according to the total number of cells.

Virus or phage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran-mediated transfection Dyeing, liposome-mediated, can introduce biological compartments into cells. Transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery, etc.

The vaccines, immune cells, and combinations of vaccines and immune cells described herein can all be used to prevent infections of any pathogenic microorganisms among HPV, EB, HP, HBV, HIV, and new coronaviruses, or for For the treatment of patients infected by pathogenic microorganisms, or for the treatment of patients with cancer or precancerous lesions.

The molecules (eg, polypeptides and/or nucleic acids) described herein may be present in the composition in the range of about 1 mg to about 2000 mg; about 5 mg to about 1000 mg, about 10 mg to about 25 mg to 500 mg, about 50 mg to about 250 mg, about 100mg to about 200mg, about 1mg to about 50mg, about 1mg to about 50mg. 50 mg to about 100 mg, about 100 mg to about 150 mg, about 150 mg to about 200 mg, about 200 mg to about 250 mg, about 250 mg to about 300 mg, about 300 mg to about 350 mg. mg, about 350 mg to about 400 mg, about 400 mg to about 450 mg, about 450 mg to about 500 mg, about 500 mg to about 550 mg, about 550 mg to about 600 mg, about 600 mg to about 650 mg, about 650 mg to about 700 mg, about 700 mg to about 750 mg , About 750 mg to about 800 mg, about 800 mg to about 850 mg, about 850 mg to about 900 mg, about 900 mg to about 950 mg, or about 950 mg to about 1000 mg.

The molecules (e.g., polypeptides and/or nucleic acids) described herein can be used at about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, About 250mg, about 300mg, about 350mg, about 400mg, about 450mg, about 500mg, about 550mg, about 600mg, about 650mg, about 700mg, about 750mg, about 800mg, about 850mg, about 900mg, about 950mg, about 1000mg, about 1050mg , About 1100mg, about 1150mg, about 1200mg, about 1250mg, about 1300mg, about 1350mg, about 1400mg, about 1450mg, about 1500mg, about 1550mg, about 1600mg, about 1650mg, about 1700mg, about 1750mg, about 1800mg, About 1850 mg, about 1900 mg, about 195 mg, 0 mg, or about 2000 mg is present in the composition.

The molecules (e.g., polypeptides and/or nucleic acids) described herein can be present in providing at least 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 10 or more Multiple units of activity/mg molecule in the composition. The activity can be the regulation of gene expression. In some embodiments, the total number of active units of molecules delivered to the subject is at least 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 60,000, 70,000, 80,000, 90,000, 110,000, 120,000, 130,000, 140,000, 150,000, 160,000, 170,000 , 180,000, 190,000, 200,000, 210,000, 220,000, 230,000 or 250,000 or more units. In some embodiments, the total number of active units of molecules delivered to the subject is at most 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 60,000, 70,000, 80,000, 90,000, 110,000, 120,000, 130,000, 140,000, 150,000, 160,000 , 170,000, 180,000, 190,000, 200,000, 210,000, 220,000, 230,000 or 250,000 or more units.

Sequence description

SEQ ID NO: 1. The gene sequence of the fusion protein of human papillomavirus (HPV16) virus structural protein L1 and HLA-I signal peptide.

SEQ ID NO: 2. The amino acid sequence of the fusion protein of human papillomavirus (HPV16) virus structural protein L1 and HLA-I signal peptide.

SEQ ID NO: 3. The gene sequence of the fusion protein of human papillomavirus (HPV16) virus structural protein L2 and HLA-I signal peptide.

SEQ ID NO: 4. The amino acid sequence of the fusion protein of human papillomavirus (HPV16) virus structural protein L2 and HLA-I signal peptide.

SEQ ID NO: 5. The gene sequence of the human papillomavirus (HPV16) sequence rearranged E6, E7 protein and HLA-I signal peptide fusion protein.

SEQ ID NO: 6. The amino acid sequence of the human papillomavirus (HPV16) sequence rearranged E6, E7 protein and HLA-I signal peptide fusion protein.

SEQ ID NO: 7. The gene sequence of the new coronavirus S protein and HLA-I signal peptide fusion protein.

SEQ ID NO: 8. The amino acid sequence of the fusion protein of novel coronavirus S protein and HLA-I signal peptide.

SEQ ID NO: 9. The gene sequence of the three structural proteins (N, M, E) of the novel coronavirus in series and forming a fusion protein with the HLA-I signal peptide.

SEQ ID NO: 10. The amino acid sequence of the three structural proteins (N, M, E) of the novel coronavirus in series and forming a fusion protein with the HLA-I signal peptide.

SEQ ID NO: 11. The gene sequence of the fusion protein between CD8α and HLA-I signal peptide.

SEQ ID NO: 12. The amino acid sequence of the fusion protein formed by CD8α and HLA-I signal peptide.

Detailed ways

For a more comprehensive understanding and application of the present invention, the present invention will be described in detail below with reference to examples, which are only intended to illustrate the present invention and not to limit the scope of the present invention. The scope of the present invention is specifically defined by the appended claims.

Example 1. Preparation of human papillomavirus (HPV) vaccine

Based on the L1 and L2 gene sequences, rearranged E6 and E7 gene sequences in the HPV16 genome, synthetic DNA was designed as a template for mRNA preparation. Specifically, construct multiple plasmids for preparing RNA, which respectively contain the sequence of the fusion protein of the viral structural protein L1 and the HLA-I signal peptide (see SEQ ID NO: 1 for the nucleic acid sequence, and SEQ ID NO: 2 for the amino acid sequence) , The sequence of the fusion protein containing the viral structural protein L2 and the HLA-I signal peptide (see SEQ ID NO: 3 for the nucleic acid sequence, and SEQ ID NO: 4 for the amino acid sequence), including the E6 and E7 proteins rearranged and linked together The sequence of the fusion protein formed with the HLA-I signal peptide (see SEQ ID NO: 5 for nucleic acid sequence, and SEQ ID NO: 6 for amino acid sequence) is used to present viral antigen peptides to activate T cells.

The mRNA used as an experimental control is derived from the fusion protein formed by the extracellular segment of CD8α protein and the HLA-I signal peptide in series (see SEQ ID NO: 11 for the nucleic acid sequence, and SEQ ID NO: 12 for the amino acid sequence). The plasmid construction is the same as above.

Using the constructed plasmid as a template, ^{mRNA was prepared using mMESSAGE mMACHINE TM} T7 Transcription Kit (ThermoFisher Scientific).

(1) The single enzyme digested plasmid is used as a transcription template, and the reaction conditions are shown in the following table.

Plasmid	10-20μg
Cutsmart buffer (10x)	5-10μl
SpeI/HindIII	3μl
RNase-free H 2 O	Add to 50-100μl
total capacity	50-100μl
temperature	37°C
time	7h or overnight

(2) Recycling of digested products

(2.1). Add 500μl of Binding buffer, mix well, transfer to the column, room temperature for 1-2min, centrifuge at 13000xg for 1min

(2.2). Add 700μl SPW wash buffer and wash 3 times, centrifuge at 13000xg for 1min each time

(2.3). Idling for 2min

(2.4). 30μl RNase-free H ₂ O elution

(2.5). Nanodrop 2000 determines DNA concentration

(3). For transcription, the reaction conditions are shown in the table below.

DNA template	1μg
2x T7NTP/CAP	10μl
10x reaction buffer	2μl
T7 enzyme mix	2μl
RNase-free H 2 O	Add to 20μl
total capacity	20μl
temperature	37°C
time	Overnight or 4h

(4). Add 1℃ TURBO DNase, 37℃ 15-20min, digest the template

(5). Lithium chloride precipitation, the reaction conditions are shown in the table below.

20μl above reaction system	20μl
RNase-free H 2 O	30μl
Lithium Chloride	30μl
total capacity	80μl

temperature	-20℃
time	More than 30min

(6).14000xg, 4℃, centrifuge for 15min

(7). Wash once with 1ml of newly prepared 70% ethanol, 14000xg, 4℃, centrifuge for 5min

(8). Aspirate the supernatant and dry

(9). 33μl RNase-free H ₂ O to dissolve RNA, take 1μl and add 2.5μl RNase-free H ₂ O and 2.5μl loading buffer for DNA agarose gel electrophoresis analysis, take 1μl to measure the concentration

(10). Dispense and store at -80℃

Experimental results: Quantitative analysis results show that the total amount of RNA can be about 30-50mug per 20ml transcription system, and the prepared RNA appears as a single band in agarose gel electrophoresis, indicating the high purity of the product.

Using ELISpot method to detect whether human papillomavirus protein antigen peptide can activate T cells

method one:

(11). Collect 100ml of peripheral blood from healthy people by intravenously, and prepare PBMC by Ficoll separation

(12). Electrotransform viral protein mRNA or control mRNA on DC (BTX ECM 830, voltage: 310V, duration: 7ms), transfer cells to a 24-well plate, 1 X 10^6 cells/ml/well, culture for 2 hours

(13). Collect the cells and inoculate them on the pre-made ELISpot plate, 2 X 10^4PBMC/200ul RPMI-1640 complete medium/well, culture for 24 hours

(14). Detect and analyze T cell activation on S6 Entry ELISpot analyzer (CTL Analyzers, LLC)

Experimental results: In three repeated independent experiments, the viral protein mRNA transfected PBMC group showed more spots than the control CD8α mRNA transfected DC group, indicating that the tested human peripheral blood has T cells that naturally recognize the viral protein. This result It supports the design of the present invention, that is, the cell vaccine has the ability to successfully activate T cells and the cellular immunity mediated by them.

Method Two:

(11). Peripheral blood mononuclear cell (PBMC, peripheral blood mononuclear cell) count of healthy people is 1-2 X 10^9, divided into two equal parts, one part is frozen, and the other part is inoculated in culture at ^{1.5 X 10^6 cells/cm 2} The bottle, adhere to the wall for 2 hours, then remove the suspended cells, and gently add X-VIVO 15 (Lonza) medium containing 2U/ml DC culture factor (Nearshore Protein Technology Co., Ltd.)

(12). On the third day, add 20% of the volume of fresh X-VIVO 15 medium, which contains 2U/ml DC culture factor

(13). On the 5th day, add 33.3% of the culture medium volume of fresh X-VIVO 15 medium, calculate according to the total volume and add 2U/ml DC maturation factor (Nearshore Protein Technology Co., Ltd.)

(14). Resuscitate the cells on the 6th day and culture them overnight in RPMI-1640 (ThermoFisher Scientific) complete medium containing 10U/ml IL-2 at a cell concentration of 1 x 10^6/ml

(15). Collect DC on the 7th day, transfer viral protein mRNA or control mRNA to DC (BTX ECM 830, voltage: 310V, duration: 7ms), transfer cells to 24-well plate, 1 X 10^6 cells/ml/well , Incubate for 2 hours

(16). Collect the recovered PBMC on the 6th day, mix 1:2 with electroporation DC and inoculate it on the pre-prepared ELISpot plate, (2X 10^4 PBMC+4 X10^4 electroporation DC)/200ul RPMI-1640 complete medium /well, incubate for 24 hours

(17). Detect T cell activation on the 9th day (S6 Entry ELISpot analyzer, CTL Analyzers, LLC)

Experimental results: In three repeated independent experiments, the viral protein mRNA transfected DC group showed more spots than the control CD8α mRNA transfected DC group, indicating that the tested human peripheral blood has T cells that naturally recognize the viral protein. This The results support the design of the present invention, that is, the cell vaccine has the ability to successfully activate T cells and the cellular immunity mediated by them.

Example 2. Preparation of Novel Coronavirus Vaccine

According to the gene sequences of S protein (S1 subunit, S2 subunit), N protein, M protein and E protein in the COVID-19 genome, a synthetic DNA sequence was designed. The specific steps include: constructing multiple plasmids for preparing RNA, which are respectively the sequence of the fusion protein containing the viral protein S protein and the HLA-I signal peptide (see SEQ ID NO: 7 for the nucleic acid sequence, and SEQ ID NO: 8 for the amino acid sequence. ), the sequence of the fusion protein of the three structural proteins of N, M, E and the HLA-I signal peptide in series (see SEQ ID NO: 9 for nucleic acid sequence, and SEQ ID NO: 10 for amino acid sequence), used to present viral antigen peptide activation T cells.

The mRNA used as an experimental control is derived from the fusion protein formed by the extracellular segment of CD8α protein and the HLA-I signal peptide in series (see SEQ ID NO: 11 for the nucleic acid sequence, and SEQ ID NO: 12 for the amino acid sequence). The plasmid construction is the same as above.

Using the constructed plasmid as a template, use mMESSAGE mMACHINE ^TM T7 Transcription Kit (ThermoFisher Scientific) to prepare mRNA

(1). The single enzyme digested plasmid is used as a transcription template, and the reaction conditions are shown in the table below.

Plasmid	10-20μg
Cutsmart buffer (10x)	5-10μl
SpeI/HindIII	3μl
RNase-free H 2 O	To 50-100μl
total capacity	50-100μl
temperature	37°C
time	7h or overnight

(2) Recycling of digested products

(2.1). Add 500μl of binding buffer and mix well, transfer to the column, room temperature for 1-2min, centrifuge at 13000xg for 1min

(2.2). Add 700μl SPW washing buffer and wash 3 times, centrifuge at 13000xg for 1min each time

(2.3). Idling for 2min

(2.4). 30μl RNase-free H ₂ O elution

(2.5). Nanodrop 2000 determines DNA concentration

(3). For transcription, the reaction conditions are shown in the table below.

DNA template	1μg
2x T7 NTP/CAP	10μl
10x Reaction buffer	2μl
T7 Enzyme mix	2μl
RNase-free H 2 O	To 20μl
total capacity	20μl
temperature	37°C
time	Overnight or 4h

(4). Add 1℃ TURBO DNase, 37℃ 15-20min, digest the template

(5). Lithium chloride precipitation, the reaction conditions are shown in the table below.

20μl above reaction system	20μl
RNase-free H 2 O	30μl
Lithium Chloride	30μl
total capacity	80μl
temperature	-20℃
time	More than 30min

(6).14000xg, 4℃, centrifuge for 15min

(7). Wash once with 1ml of newly prepared 70% ethanol, 14000xg, 4℃, centrifuge for 5min

(8). Aspirate the supernatant and dry

(9). 33μl RNase-free H ₂ O to dissolve RNA, take 1μl and add 2.5μl RNase-free H ₂ O and 2.5μl loading buffer to DNA agarose gel electrophoresis analysis, take 1μl to measure the concentration

(10). Dispense and store at -80℃

Experimental results: Quantitative analysis results show that the total amount of RNA can be about 30-50mug per 20ml transcription system, and the prepared RNA appears as a single band in agarose gel electrophoresis, indicating the high purity of the product.

Using ELISpot method to detect whether the novel coronavirus protein antigen peptide can activate T cells

method one:

(11). Collect 100ml of peripheral blood from healthy people by intravenously, and prepare PBMC by Ficoll separation

(12). Electrotransform viral protein mRNA or control mRNA on DC (BTX ECM 830, voltage: 310V, duration: 7ms), transfer cells to a 24-well plate, 1 X 10^6 cells/ml/well, culture for 2 hours

(13). Collect the cells and inoculate them on the pre-prepared ELISpot plate, 2 X 10^4PBMC/200ul RPMI-1640 complete medium/well, culture for 24 hours

(14). Detect and analyze T cell activation on S6 Entry ELISpot analyzer (CTL Analyzers, LLC)

Experimental results: In three repeated independent experiments, the viral protein mRNA transfected PBMC group showed more spots than the control CD8α mRNA transfected DC group, indicating that the tested human peripheral blood has T cells that naturally recognize the viral protein. The results support the design of the present invention, that is, the cell vaccine has the ability to successfully activate T cells and the cellular immunity mediated by them.

Method Two:

(11). Peripheral blood mononuclear cell (PBMC, peripheral blood mononuclear cell) count of healthy people is 1-2 X 10^9, divided into two equal parts, one part is frozen, and the other part is inoculated in culture at ^{1.5 X 10^6 cells/cm 2} The bottle, adhere to the wall for 2 hours, then remove the suspended cells, and gently add X-VIVO 15 (Lonza) medium containing 2U/ml DC culture factor (Nearshore Protein Technology Co., Ltd.)

(12). On the third day, add 20% of the volume of fresh X-VIVO 15 medium, which contains 2U/ml DC culture factor

(13). On the 5th day, add 33.3% of the culture medium volume of fresh X-VIVO 15 medium, calculate according to the total volume and add 2U/ml DC maturation factor (Nearshore Protein Technology Co., Ltd.)

(14). Resuscitate the cells on the 6th day and culture them overnight in RPMI-1640 (ThermoFisher Scientific) complete medium containing 10U/ml IL-2 at a cell concentration of 1 x 10^6/ml

(15). Collect DC on the 7th day, transfer viral protein mRNA or control mRNA to DC (BTX ECM 830, voltage: 310V, duration: 7ms), transfer cells to 24-well plate, 1 X 10^6 cells/ml/well , Incubate for 2 hours

(16). Collect the recovered PBMC on the 6th day, mix 1:2 with electroporation DC and inoculate it on the pre-prepared ELISpot plate, (2 X 10^4PBMC+4 X10^4 electroporation DC)/200ul RPMI-1640 complete medium /well, incubate for 24 hours

(17). Detect T cell activation on the 9th day (S6 Entry ELISpot analyzer, CTL Analyzers, LLC)

Experimental results: In three repeated independent experiments, the viral protein mRNA transfected DC group showed more spots than the control CD8α mRNA transfected DC group, indicating that the tested human peripheral blood has T cells that naturally recognize the viral protein. This The results support the design of the present invention, that is, the cell vaccine has the ability to successfully activate T cells and the cellular immunity mediated by them.

Claims (26)

1. A cell vaccine, characterized in that the vaccine is an antigen presenting cell (APC) transfected with an antigen gene derived from a pathogenic microorganism.
2. The vaccine according to claim 1, wherein the pathogenic microorganism is selected from one or more of the following group: HPV virus, Epstein-Barr virus, Helicobacter pylori (HP), hepatitis B virus (HBV), HIV ( HIV), a novel coronavirus (SARS-Cov-2), and the antigen gene is a complete or partial fragment of the gene of the pathogenic microorganism.
3. The vaccine of claim 1 or 2, wherein the antigen gene of the pathogenic microorganism is a complete or partial fragment of the E6 and/or E7 gene of the HPV virus.
4. The vaccine according to claim 1 or 2, wherein the antigen gene of the pathogenic microorganism is a whole or a partial fragment of the Spike of SARS-Cov-2 or other genes.
5. The vaccine according to any one of claims 1 to 4, wherein the APC is derived from the patient's autologous or foreign body.
6. The vaccine according to any one of claims 1 to 5, wherein the APC is human natural APC, including DC cells, B cells, T cells, and PBMC.
7. The vaccine according to any one of claims 1 to 5, wherein the APC is an exogenous cell derived from a non-human natural APC, including engineered or immortalized or irradiated K562 cells, B Cells, NK cells, DC cells.
8. The vaccine according to any one of claims 1 to 7, wherein the gene transfection process is electrotransfection or adeno-associated virus transfection.
9. The vaccine according to any one of claims 1 to 7, wherein the gene transfection process is a way to allow constant gene expression, such as lentiviral transfection and gene editing.
10. The vaccine according to any one of claims 1-9, wherein the vector design used to load the pathogenic microorganism antigen gene into the APC is to direct the expression of the pathogenic microorganism antigen gene to the lysozyme in the APC Body or endoplasmic reticulum.
11. The vaccine according to any one of claims 1-9, wherein the vector design used to load the pathogenic microorganism antigen gene into the APC is designed to direct the expression of the pathogenic microorganism antigen gene into the APC at the same time. Lysosome and endoplasmic reticulum.
12. The vaccine according to any one of claims 1 to 9, wherein the vector design used to load the pathogenic microorganism antigen gene into the APC is to allow the pathogenic microorganism antigen gene to be secreted and expressed outside the APC cell , Or transmembrane expression.
13. The vaccine according to any one of claims 1 to 12, wherein the vaccine can be used for infection or infection of any pathogenic microorganism among HPV, EB, HP, HBV, HIV, and new coronavirus. The prevention of diseases related to the infection, such as cancer, or the treatment of patients infected by pathogenic microorganisms, or the treatment of diseases related to infection of pathogenic microorganisms, such as cancer patients or people with precancerous lesions.
14. An immune cell, characterized in that the immune cell is a T cell and NK cell that respond to the vaccine of claims 1 to 13 or the pathogenic microorganism of claims 1 to 3.
15. The immune cell according to claim 14, wherein the immune cell is a natural unmodified T cell obtained by co-cultivating with the vaccine according to claim 1 in vitro.
16. The immune cell according to claim 14, wherein the immune cell has an artificially modified T cell receptor (TCR) and/or chimeric antigen receptor (CAR), and the TCR or CAR can be 1. The vaccine or the pathogenic microorganism produces a specific reaction, or can specifically recognize the protein fragment of the pathogenic microorganism presented by a specific MHC molecule, and the pathogenic microorganism is selected from any of HPV, EB, HP, HBV, and HIV .
17. The immune cell according to claim 16, wherein the CAR is composed of a fusion of an extracellular binding domain and an immune cell signaling pathway protein, wherein the extracellular binding domain is the same as the human cell surface of the pathogenic microorganism according to claims 1 to 3. All or part of the receptor protein, including the human cell surface receptor of coronavirus-Angiotensin Converting Enzyme 2 (ACE2).
18. The immune cell according to claim 17, wherein the immune cell signal pathway protein is a costimulatory signal protein such as CD28 or 41BBz or any one of ICOS.
19. The immune cell according to claims 14-18, wherein the immune cell has an artificially modified enhanced receptor, and the enhanced receptor is composed of the fusion of the extracellular binding domain of the immune cell and the immune cell signal pathway protein.
20. The immune cell according to claim 19, wherein the receptor-enhancing immune cell signal pathway protein is a costimulatory signal protein such as CD28 or 41BBz or any one of ICOS.
21. The immune cell according to claim 19, wherein the extracellular binding domain of the enhanced receptor is a receptor, ligand, antibody of the membrane protein of the target cell of the immune cell, or a membrane protein capable of binding to the membrane protein. The complete sequence structure of any one of the other protein structures or the part containing its binding domain.
22. The immune cell according to claim 19, wherein the extracellular binding domain of the enhanced receptor is any one of PD1, CTLA4, NKG2D, SIRPa, LAG3 or other immune cell surface proteins.
23. The immune cell according to claim 19, wherein the extracellular binding domain of the enhanced receptor is PDL1 monoclonal antibody, B7 monoclonal antibody, HER2 monoclonal antibody, EGFR monoclonal antibody, CD47 monoclonal antibody, or antibodies to other target cell membrane proteins , Or a part that contains the binding domain of an antibody variable region.
24. The immune cell according to any one of claims 14 to 23, wherein the immune cell can be used to infect any of the pathogenic microorganisms among HPV, EB, HP, HBV, HIV, and new coronavirus. Prevention, or for the treatment of patients infected by pathogenic microorganisms, or for the treatment of diseases related to pathogenic microorganism infections, such as cancer patients or people with precancerous lesions.
25. A composition, characterized in that the composition includes the vaccine of any one of claims 1-13 and the immune cell of any one of claims 14-24.
26. The composition according to claim 25, wherein the composition can be used to infect or be related to any of the pathogenic microorganisms among HPV, EB, HP, HBV, HIV, and new coronavirus. For the prevention of cancer, or for the treatment of patients infected by pathogenic microorganisms, or for the treatment of diseases related to pathogenic microorganisms such as cancer patients or precancerous lesions.