WO2024046394A1 - 增强受体、表达增强受体的免疫细胞及其用途 - Google Patents

增强受体、表达增强受体的免疫细胞及其用途 Download PDF

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WO2024046394A1
WO2024046394A1 PCT/CN2023/115934 CN2023115934W WO2024046394A1 WO 2024046394 A1 WO2024046394 A1 WO 2024046394A1 CN 2023115934 W CN2023115934 W CN 2023115934W WO 2024046394 A1 WO2024046394 A1 WO 2024046394A1
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cells
receptor
domain
cell
tigit
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French (fr)
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赵洁
高斌
谷为岳
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Chineo Medical Technology Co Ltd
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Chineo Medical Technology Co Ltd
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Priority to EP23859423.8A priority Critical patent/EP4582456A4/en
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Definitions

  • the present invention belongs to the field of biomedicine technology, specifically to the field of immune cell therapy.
  • Switch receptor is a fusion protein that has attracted much attention in the field of cell therapy in recent years.
  • Switch receptor is sometimes called switch molecule, switch costimulatory receptor, or chimeric switch receptor. It is a transmembrane fusion protein in which the extracellular segment It is derived from molecules that bind target cells and, in their natural state, produce immunosuppressive signals, such as checkpoint molecules, while the intracellular segments are derived from molecules that produce immunostimulatory signals, such as costimulatory molecules.
  • immunosuppressive signals such as checkpoint molecules
  • the intracellular segments are derived from molecules that produce immunostimulatory signals, such as costimulatory molecules.
  • WO2013/019615 discloses a variety of chimeric switching receptors, the extracellular segments of which are derived from CTLA4, PD-1 or BTLA, and the intracellular segments are derived from CD28 or ICOS.
  • CTLA4, PD-1 or BTLA should produce an inhibitory signal after binding to its natural ligand, but CD28 or ICOS in the intracellular domain of the chimeric switching receptor converts this inhibitory signal into an activating signal.
  • the application demonstrates that by expressing this chimeric switch receptor on T cells, the modified T cells exhibit a positive immune response when exposed to tumor cells expressing BTLA or PD-1 ligands. rather than the normally seen inhibitory response.
  • the inventor of the present invention has developed a new enhanced receptor, which exhibits significantly stronger ability to activate immune cells than chimeric switching receptors in the prior art, thereby completing the present invention. invention.
  • the invention provides an enhanced receptor comprising an extracellular domain (ECD), a transmembrane domain (TM) and an intracellular domain (ICD), wherein the extracellular domain is derived from From the extracellular domain of TIGIT, the intracellular domain is derived from the intracellular domain of OX40.
  • ECD extracellular domain
  • TM transmembrane domain
  • ICD intracellular domain
  • the extracellular domain of TIGIT is the extracellular domain of human TIGIT. domain or a fragment or variant thereof having ligand binding ability, wherein the ligand is CD155.
  • the present invention provides an immune cell modified to express the enhancing receptor of the first aspect.
  • the present invention provides an immune cell that is modified to express two different enhancer receptors, wherein the first enhancer receptor is the enhancer receptor of the first aspect of the present invention.
  • the extracellular domain of the second enhancing receptor is capable of specifically binding PD-L1
  • the intracellular domain of the second enhancing receptor is derived from CD28.
  • the extracellular segment of the second enhancing receptor is PD1 or a variant thereof capable of specifically binding PD-L1, or an anti-PD-L1 antibody.
  • the present invention provides methods for preparing immune cells according to the second and third aspects.
  • the present invention provides uses of the immune cells of the second and third aspects in treating diseases.
  • the present invention provides a pharmaceutical composition comprising the immune cells of the second or third aspect.
  • the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the enhanced receptor of the first aspect.
  • the nucleic acid molecule further comprises a nucleotide sequence encoding a second enhancer receptor, the second enhancer receptor comprising a second extracellular domain, a second transmembrane domain and a second cellular domain.
  • Intracellular domain wherein the second extracellular domain is capable of specifically binding a ligand of a T cell immune checkpoint molecule different from TIGIT, wherein the second intracellular domain is derived from a common protein that mediates immune cell activation signals. Stimulating molecules.
  • the present invention provides an expression vector comprising the nucleic acid molecule of the seventh aspect.
  • the present invention provides a host cell comprising the nucleic acid molecule of the seventh aspect or the expression vector of the eighth aspect.
  • Figures 1A-B are the results of the in vitro experiments in Example 3, showing the secretion levels of IL-2 (A) and INF ⁇ (B) by T cells after using different enhancer receptors to modify tumor-recognizing T cells.
  • Figure 2 is the results of the in vivo experiment in Example 4, showing the changes in tumor volume in different treatment groups, illustrating the impact of enhanced receptors on the tumor inhibitory effect.
  • Figure 3 is an enlarged view of the remaining four groups of animal data after removing the two controls (Freezing medium and Mock-T) in Figure 2.
  • Figure 4 is the results of the in vitro experiment in Example 5, showing the use of different enhancing receptors to modify tumors. Levels of IL-2 secretion by T cells following recognition of T cells.
  • Figure 5 is the results of the in vitro experiment in Example 5, showing the secretion level of INF ⁇ by T cells after using different enhancer receptors to modify tumor-recognizing T cells.
  • Figure 6 is the results of the in vivo experiment in Example 5, showing the changes in tumor volume in different treatment groups and illustrating the impact of different enhancing receptors on the tumor inhibitory effect.
  • Figure 7 is an enlarged view of the remaining four groups of animal data after removing the control (Mock-T) in Figure 6.
  • Figure 8 is the results of the in vivo experiment in Example 6, showing the changes in tumor volume in different treatment groups.
  • Figure 9 shows treatment with different groups of cells (TCR-T; PD1(108)/CD28-TCR-T; and PD1(108)/CD28-TIGIT/OX40-TCR-T) at different time points after injection. Changes in TCR copy number over time in peripheral blood of treated mice.
  • Figure 10 shows treatment with different groups of cells (TCR-T; PD1(108)/CD28-TCR-T; and PD1(108)/CD28-TIGIT/OX40-TCR-T) at different time points after injection. Changes in TCR copy number over time in tumor tissues of treated mice.
  • enhanced receptor in this context refers to a recombinant transmembrane protein comprising an extracellular domain (ECD), a transmembrane domain (TM) and an intracellular domain (ICD), wherein said extracellular domain domain from immunity
  • ECD extracellular domain
  • TM transmembrane domain
  • ICD intracellular domain
  • a protein of a cell that in unmodified immune cells triggers an immune cell inactivation signal upon binding to its ligand, wherein the intracellular domain is derived from a costimulatory molecule that mediates immune cell activation signals.
  • the enhancing receptors of the present invention can be used to modify immune cells.
  • Immune cells modified with enhanced receptors generate immune cell activation signals by enhancing the binding of the extracellular domain of the receptor to the corresponding ligand, rather than the natural state of the extracellular domain after binding to the corresponding ligand. Immune cell deactivation signals.
  • the extracellular domain of the enhancer receptor of the invention is from TIGIT and the intracellular domain is from OX40.
  • TAGIT means T cell immunoreceptor with Ig and ITIM domains.
  • OX40 also known as “CD134” is a costimulatory immune checkpoint molecule belonging to the TNF receptor superfamily.
  • PD-1 refers to programmed death receptor 1.
  • PD-1 is an inhibitory immune checkpoint molecule expressed on the surface of B cells and T cells.
  • PD-L1 is one of the ligands of PD-1, which generates an immunosuppressive signal after binding to PD-1.
  • CD28 is a receptor on the surface of T cells that provides costimulatory signals for T cell activation.
  • nucleotide generally refers to a base-sugar-phosphate combination.
  • the nucleotides may include synthetic nucleotides.
  • Nucleotides may include synthetic nucleotide analogs.
  • Nucleotides can be monomeric units of nucleic acid sequences (eg, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)).
  • nucleotide may include ribonucleoside triphosphate adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), guanosine triphosphate (GTP) and deoxyribonucleoside triphosphates such as dATP, dCTP , dITP, dUTP, dGTP, dTTP or their derivatives.
  • These derivatives may include, for example, [ ⁇ S]dATP, 7-deaza-dGTP, and 7-deaza-dATP, as well as nucleotide derivatives that confer nuclease resistance to nucleic acid molecules containing them.
  • nucleotide may refer to dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives.
  • ddNTPs dideoxyribonucleoside triphosphates
  • 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. Labeling can also be done with quantum dots. Detectable labels may include, for example, radioisotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzymatic labels.
  • genomic DNA refers to a nucleic acid (eg, DNA, such as genomic DNA and cDNA) and its corresponding nucleotide sequence encoding an RNA transcript.
  • genomic DNA includes inserted non-coding regions as well as regulatory regions, and may include 5' and 3' ends.
  • the term includes transcribed sequences, including 5' and 3' untranslated regions (5'-UTR and 3'-UTR), exons and introns. in some bases Therefore, the transcribed region will contain the "open reading frame" encoding the polypeptide.
  • a “gene” includes only the coding sequence necessary to encode a polypeptide (eg, an "open reading frame” or “coding region”). In some cases, the gene does not encode a polypeptide, 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 may refer to an "endogenous gene” or a native gene in its natural location in the genome of an organism. Genes may refer to "foreign genes” or unnatural genes.
  • rRNA ribosomal RNA genes
  • tRNA transfer RNA
  • Non-native genes may refer to genes that are not normally found in the host organism but are introduced into the host organism through gene transfer. Non-native genes can also refer to genes that are not in their natural location in the genome of an organism. Non-natural genes may also refer to naturally occurring nucleic acid or polypeptide sequences that contain mutations, insertions and/or deletions (eg, non-natural sequences).
  • polynucleotide oligonucleotide
  • nucleic acid a polymeric form of nucleotides, deoxyribonucleotides, or ribonucleotides, or analogs thereof, of any length, which Can be single stranded, double stranded or multiple stranded.
  • Polynucleotides may be exogenous or endogenous to the cell. Polynucleotides can exist in a cell-free environment.
  • a polynucleotide may be a gene or fragment thereof.
  • the polynucleotide can be DNA.
  • the polynucleotide can be RNA.
  • Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. Polynucleotides may contain one or more analogs (eg, altered backbones, sugars, or nucleobases).
  • expression refers to one or more processes by which a polynucleotide is transcribed from a DNA template (eg, into mRNA or other RNA transcripts) and/or the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein.
  • Transcripts and encoded polypeptides may collectively be referred to as "gene products.” If the polynucleotide is derived from genomic DNA, expression may include splicing the mRNA in a eukaryotic cell.
  • Up-regulation of expression generally refers to an increase in the expression level of a polynucleotide (e.g., RNA, e.g., mRNA) and/or polypeptide sequence relative to its expression level in the wild-type state, while “down-regulation” generally refers to an increase in the expression level relative to its expression level in the wild-type state. Its expression in the wild-type state reduces the expression levels of polynucleotide (eg, RNA, eg, mRNA) and/or polypeptide sequences.
  • RNA e.g., mRNA
  • modulate with respect to expression or activity means changing the level of expression or activity. Regulation can occur at the transcriptional level and/or the translational level.
  • peptide refers to a polymer of at least two amino acid residues linked by peptide bonds.
  • the term does not imply a specific length of polymer, nor is it intended to imply or distinguish whether the peptide is produced using recombinant techniques, 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.
  • the polymer can be interrupted by non-amino acids.
  • the term includes amino acid chains of any length, including full-length proteins, and also includes proteins with or without secondary and/or tertiary structure (eg, 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 manipulation, such as conjugation to a labeling component.
  • amino acid generally refers 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 that have been chemically modified to include groups or chemical moieties that are not naturally present on the amino acid.
  • Amino acid analogs may refer to amino acid derivatives.
  • amino acid includes D-amino acids and L-amino acids.
  • the terms “derivative”, “variant” and “fragment” refer to a polypeptide that is similar to the wild-type polypeptide in terms of, for example, amino acid sequence, structure (e.g., secondary and/or tertiary), activity (e.g., secondary and/or tertiary), activity (e.g. , enzyme activity) and/or functionally related polypeptides.
  • Derivatives, variants and fragments of a polypeptide may contain one or more amino acid variations (eg, mutations, insertions and deletions), truncations, modifications, or combinations thereof as compared to the wild-type polypeptide.
  • fusion may refer to a protein and/or nucleic acid comprising one or more non-natural sequences (eg, portions). Fusions may contain one or more identical non-natural sequences. Fusions may contain one or more different non-natural sequences. Fusions may be chimeras. Fusions can contain nucleic acid affinity tags. Fusions may include barcodes. The fusion may contain a peptide affinity tag. Fusions can provide subcellular localization of the polypeptide (e.g., nuclear localization signal (NLS) for targeting to the nucleus, mitochondrial localization signal for targeting to mitochondria, chloroplast localization signal for targeting to chloroplasts, endoplasmic reticulum (ER) ) to retain signals, etc.). Fusions can provide non-natural sequences (eg, affinity tags) that can be used for tracking or purification. Fusions may contain small molecules such as biotin or dyes such as Alexa Fluor dye, Cyanine3 dye, Cyanine5 dye.
  • mutant proteins generally refers to tumor-specific antigens caused by genetic mutations.
  • the resulting mutant proteins or fragments thereof can trigger anti-tumor T cell responses.
  • genetic profile refers to information about a specific gene, including variation and gene expression in an individual or in a certain type of tissue. Genetic profiling can be used for neoantigen selection.
  • genetic profiling can be used for neoantigen selection.
  • genetic mutation profile refers to information about specific genes associated with somatic mutations, including but not limited to specific genes resulting from somatic mutations. Somatic mutation profiles can be used for neoantigen selection.
  • antibody refers to a protein-binding molecule with immunoglobulin-like functions.
  • the term antibody includes antibodies (eg, monoclonal and polyclonal antibodies), as well as derivatives, variants and fragments thereof.
  • the derivatives, variants or fragments may refer to functional derivatives or fragments that retain the binding specificity (eg, intact and/or partial) of the corresponding antibody, such as.
  • Antigen-binding fragments include Fab, Fab', F(ab')2, variable fragment (Fv), single-chain variable fragment (scFv), minibodies, diabodies, and single domain antibodies ("sdAb” or “nanobodies” ” or “camel”).
  • antibody includes antibodies and antigen-binding fragments of antibodies that have been optimized, engineered, or chemically conjugated.
  • antibodies that have been optimized include affinity matured antibodies.
  • antibodies that have been engineered include Fc-optimized antibodies (eg, antibodies optimized in fragment crystallizable regions) and multispecific antibodies (eg, bispecific antibodies).
  • an exogenous T cell receptor (TCR) complex refers to a TCR complex in which one or more chains of the TCR are introduced into the genome of an immune cell.
  • the TCR may or may not be expressed endogenously.
  • an exogenous TCR complex may refer to a TCR complex in which one or more chains of the endogenous TCR complex has one or more mutated sequences, such as at the nucleic acid or amino acid level.
  • Expression of exogenous TCRs on immune cells can confer binding specificity for epitopes or antigens (eg, epitopes or antigens that are preferentially present on the surface of cancer cells or other disease-causing cells or particles).
  • the exogenous TCR complex may comprise TCR- ⁇ , TCR- ⁇ chain, CD3- ⁇ chain, CD3- ⁇ chain, CD3- ⁇ chain or any combination thereof introduced into the genome.
  • strands introduced into the genome can replace endogenous strands.
  • Immuno checkpoint molecules are molecules that regulate the immune response, usually a pair of receptor/ligand molecules that can produce inhibitory or stimulatory effects on the immune response.
  • Co-stimulatory molecules refers to cell surface molecules that amplify or counteract the initial activation signal of a T cell. Unless otherwise specified, the “costimulatory molecule” in this article refers to a “positive costimulatory molecule”, that is, a costimulatory molecule that generates an activation signal.
  • subject refers to a vertebrate, preferably a mammal, such as a human.
  • Mammals include, but are not limited to, rats, apes, humans, farm animals, sporting animals, and pets. Also included are tissues, cells and their progeny of biological entities obtained in vivo or cultured in vitro.
  • treatment refers to a method used to obtain beneficial or desired results, including but not limited to therapeutic benefits and/or preventive benefits.
  • treatment may include administration of cells, cell populations, or compositions disclosed herein.
  • Therapeutic benefit refers to any treatment-related improvement or effect in one or more diseases, conditions, or symptoms being treated.
  • the cells, cell populations, or compositions may 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. subjects, even though the disease, condition or symptoms may not yet be manifest.
  • an effective amount refers to an amount of a composition sufficient to produce the desired activity when administered to a subject in need thereof, where the composition, for example, comprises an immune cell, such as a lymphocyte, of the present invention.
  • a composition of cells eg, T lymphocytes.
  • therapeutically effective refers to the disease to be treated For a disease, the amount of the composition is sufficient to delay its manifestations, prevent its progression, or reduce or relieve at least one symptom thereof.
  • TIGIT is a member of the poliovirus receptor (PVR)/zonulin family. It is an immune receptor that is mainly expressed on activated T cells, memory T cells, NK cells and certain T regulatory cells (Treg). TIGIT is able to bind to its high-affinity cognate ligand CD155 (also known as PVR) expressed on antigen-presenting cells (APCs). This binding inhibits immune responses through T cells and APCs, as well as through NK cells. TIGIT has also been shown to compete with CD226 for binding to CD155, thus balancing CD226-mediated costimulatory T cell signaling. Because of these functions, TIGIT has become one of the popular targets in immunotherapy.
  • PVR poliovirus receptor
  • the TIGIT/OX40 enhanced receptor of the present invention includes the extracellular domain of human TIGIT, or a fragment or variant thereof with ligand binding ability, wherein the ligand is CD155.
  • Human TIGIT consists of 244 amino acids, of which the extracellular domain contains 141 amino acids and the transmembrane domain contains 23 amino acids.
  • the extracellular domain of the TIGIT/OX40 enhancing receptor of the present invention is the extracellular domain of human TIGIT, for example, it has the amino acid sequence shown in SEQ ID NO: 9.
  • the TIGIT/OX40 enhanced receptor of the present invention contains the intracellular domain of human OX40, or a fragment or variant thereof that has the function of forming a signaling complex.
  • Human OX40 consists of 277 amino acids, of which the cytoplasmic end is 37 amino acids, the extracellular domain is 217 amino acids, and the transmembrane domain is 23 amino acids.
  • the intracellular domain of the TIGIT/OX40 enhancing receptor of the invention is derived from the intracellular domain of wild-type human OX40, for example, it has the amino acid sequence shown in SEQ ID NO: 10.
  • TIGIT is used as an enhanced receptor for the extracellular segment.
  • the intracellular segment is selected from the intracellular domain derived from OX40, T cells will be able to secrete more cytokines such as IL-2 and IFN ⁇ and Due to its longer persistence in the body, it is particularly advantageous to use intracellular domains derived from other co-stimulatory molecules such as CD28, ICOS, etc.
  • the transmembrane region of the TIGIT/OX40 enhancing receptor of the invention may optionally be derived from TIGIT or OX40, for example, comprise the transmembrane domain of TIGIT or OX40.
  • the transmembrane region is derived from OX40 and has the amino acid sequence shown in SEQ ID NO: 11.
  • Some embodiments of the invention involve modifications to express two different enhancer receptors (or dual enhancers). Strong receptor) immune cell, wherein the first enhanced receptor is the TIGIT/OX40 enhanced receptor of the present invention, and the second enhanced receptor includes a second extracellular domain, a second transmembrane domain and a second intracellular domain, wherein the second extracellular domain is capable of specifically binding to a ligand of an immune checkpoint molecule other than TIGIT for T cells, wherein the second intracellular domain is derived from a mediating immune cell Costimulatory molecules that activate signaling.
  • the other T cell immune checkpoint molecules may be selected from the group consisting of: transforming growth factor beta Receptor (TGF- ⁇ -R), programmed cell death 1 (PD-1), cytotoxic T lymphocyte-associated protein 4 (CTLA-4), B and T lymphocyte attenuating factor (BTLA), killer cell immune cells protein-like receptor (KIR), indoleamine 2,3-dioxygenase (IDO), lymphocyte activation gene 3 (LAG3) and T cell immunoglobulin mucin 3 (TIM-3).
  • TGF- ⁇ -R transforming growth factor beta Receptor
  • PD-1 programmed cell death 1
  • CTLA-4 cytotoxic T lymphocyte-associated protein 4
  • BTLA B and T lymphocyte attenuating factor
  • KIR killer cell immune cells protein-like receptor
  • IDO indoleamine 2,3-dioxygenase
  • LAG3 lymphocyte activation gene 3
  • TIM-3 T cell immunoglobulin mucin 3
  • the second extracellular domain may be an extracellular domain of another immune checkpoint molecule of a T cell that is different from TIGIT, or a fragment thereof that retains ligand binding ability.
  • the second extracellular domain may be an antibody or a fragment thereof that specifically binds to a ligand of other immune checkpoint molecules of T cells that are different from TIGIT; for example, the antibody is a single chain antibody ( scFv) or single domain antibody (VHH).
  • the T cell other immune checkpoint molecule is PD-1.
  • the second extracellular domain may be PD-1 or a variant or fragment thereof capable of binding PD-L1; or an anti-PD-L1 antibody or a fragment thereof capable of specifically binding PD-L1, such as an anti-PD-L1 antibody or a fragment thereof capable of specifically binding PD-L1.
  • the extracellular domain of the second enhanced receptor has an amino acid sequence such as SEQ ID NO: 16.
  • the extracellular domain of the second enhancing receptor is a variant of the extracellular domain of PD-1 and has improved affinity for PD-L1 relative to wild-type PD-1 And does not bind to at least one known PD-1 antibody, such as the second enhancing receptor having an amino acid sequence such as SEQ ID NO: 12.
  • the costimulatory molecule from which the second intracellular domain is derived can be interleukin 2 receptor (IL-2R), interleukin 12 receptor (IL-12R), CD2, CD3, CD4, CD7, CD8, CD27, CD28, CD30, CD40, 4-1BB/CD137, ICOS, lymphocyte function-associated antigen 1 (LFA-1), LIGHT, NKG2C or OX40.
  • the immune cell activation signal can be mediated by an activating factor.
  • the activating factor can be a soluble cytokine, a soluble chemokine, or a growth factor.
  • the activating factor can be a soluble cytokine, and wherein the soluble cytokine is IL-1, IL-2, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, IL-21, TNF, TGF, IFN or any functional fragment or variant thereof.
  • the immune cell activation signal can comprise clonal expansion of the modified immune cell; cytokine release by the modified immune cell; cytotoxicity of the modified immune cell; Proliferation of the modified immune cells; differentiation, dedifferentiation or transdifferentiation of the modified immune cells; movement and/or transport of the modified immune cells; depletion and/or depletion of the modified immune cells or reactivation; and release of other intercellular molecules, metabolites, chemical compounds, or combinations thereof from the modified immune cells.
  • the costimulatory molecule is CD28.
  • the intracellular domain of the second enhanced receptor has an amino acid sequence such as SEQ ID NO: 13.
  • the second enhancing receptor is a PD1/CD28 enhancing receptor, which includes:
  • a second extracellular domain derived from PD-1 or a variant or fragment thereof capable of binding PD-L1 for example, an extracellular domain derived from PD-1 or a variant thereof capable of binding PD-L1 Or a fragment, preferably having an amino acid sequence as shown in SEQ ID NO:16 or SEQ ID NO:13;
  • a second transmembrane domain derived from PD-1 or CD28 such as a transmembrane domain derived from PD-1 or CD28, preferably a transmembrane domain derived from CD28, for example having SEQ ID NO: The amino acid sequence shown in 14;
  • a second intracellular domain derived from CD28 such as an intracellular domain derived from CD28, such as having an amino acid sequence as shown in SEQ ID NO: 13.
  • immunosuppressive signals mediated by different immune checkpoint molecules can be converted into activating signals.
  • immune cells modified with dual enhancer receptors can exhibit further activation and immune responses compared to immune cells modified with only one enhancer receptor.
  • the inventors found that using immune cells modified with TIGIT/OX40-enhancing receptors and PD-1(108)/CD28-enhancing receptors can produce a synergistic effect in suppressing tumors, and the effect is significantly better than using cells with only TIGIT/OX40-enhancing receptors alone.
  • the present invention also relates to a method of using the TIGIT/OX40 enhancing receptor of the present invention to improve the effects of therapeutic immune cells, such as immune activation and immune response effects, wherein the therapeutic immune cells can have TIGIT/OX40 enhancing receptors Other modifications, such as the second enhanced receptor and/or chimeric antigen receptor of the present invention.
  • nucleotide sequence encoding the TIGIT/OX40 enhancing receptor and the nucleotide sequence encoding the second enhancing receptor may be introduced into the immune cells individually or in combination. cells for expression.
  • nucleotide sequences encoding two enhancer receptors can be linked with sequences encoding a self-cleaving peptide, such as the 2A peptide.
  • the 2A peptides include, but are not limited to, T2A, P2A, E2A, F2A, etc.
  • the enhanced receptor of the present invention is used to modify immune cells, especially lymphocytes, such as T cells, to enhance the immune activation effect of the immune cells.
  • the immune cells Before being modified, particularly before the immune cells are modified to express the enhanced receptors of the invention, the immune cells can be from a variety of sources and prepared by any one or more of a variety of methods. .
  • Immune cells that can be used in the present invention include peripheral blood mononuclear cells (PBMC), peripheral blood lymphocytes (PBL) and other blood cell subsets (such as but not limited to T cells, natural killer cells, monocytes, natural killer T cells, monocyte precursor cells, hematopoietic stem cells or non-pluripotent stem cells).
  • the cell can be any immune cell, including any T cell, such as tumor-infiltrating cells (TIL), CD3 + T cells, CD4 + T cells, or CD8 + T cells.
  • T cells can also include memory T cells, memory stem T cells, or effector T cells.
  • the immune cells may be a population of T cells, 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 sites of infection, ascites, pleural effusion, spleen tissue, and tumors.
  • T cells can be obtained from units of blood collected from a subject using a variety of techniques, such as Ficoll TM isolation.
  • cells from the individual's circulating blood are obtained by apheresis.
  • Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. Cells collected by apheresis can be washed to remove the plasma fraction and place the cells in an appropriate buffer or medium for subsequent processing steps.
  • T cells can be selected from a bulk population, such as from whole blood. T cells can be expanded from mixed populations. During selection and expansion, T cells can be skewed toward specific populations and phenotypes. For example, T cells can be skewed to phenotypically include CD45RO(-), CCR7(+), CD45RA(+), CD62L(+), CD27(+), CD28(+), and/or IL-7R ⁇ (+ ). Appropriate cells can be selected that contain one or more markers selected from a list including: CD45RO(-), CCR7(+), CD45RA(+), CD62L(+), CD27(+), CD28 (+) and/or IL-7R ⁇ (+).
  • Immune cells can also be obtained through differentiation using stem cells.
  • the stem cells such as embryonic stem cells, induced Guide pluripotent stem cells, hematopoietic stem cells, neuronal stem cells and mesenchymal stem cells.
  • Immune cells can include a variety of primary cells, such as human cells, non-human cells, and/or mouse cells.
  • Immune cells can be derived from the subject (eg, patient) to be treated. Immune cells can be derived from healthy human donors. The immune cells can be derived from a donor who is directly related to the subject, such as a donor who is HLA haploidentical to the subject.
  • the immune cells have recognition specificity for target cells, such as tumor cells.
  • This conferring of recognition specificity can be achieved by various methods, including but not limited to genetic modification, by incubation with antigen-presenting cells loaded with target cell antigens, and/or by sorting specific cell populations.
  • the immune cells of the invention may be tumor-infiltrating lymphocytes (TIL).
  • TIL tumor-infiltrating lymphocytes
  • the TIL can express at least one of PD-1, CD137, and TIM-3.
  • the T cell can comprise a T cell receptor (TCR) complex that exhibits specific binding to a neoantigen.
  • TCR T cell receptor
  • the TCR complex can be an endogenous TCR complex.
  • the TCR complex can be an exogenous TCR complex.
  • the neoantigen can be selected based on the genetic profile of a tumor sample from an individual. In some embodiments, the neoantigens can be selected based on the somatic mutation profile of tumor samples from the individual.
  • the immune cells of the invention are derived from peripheral blood mononuclear cells (PBMC) and sorted to enrich for PD1 + T cells in PBMC.
  • PBMC peripheral blood mononuclear cells
  • enrichment means processing to increase the number or proportion of a certain cell or component in a certain population. Since in actual laboratory and clinical operations, the process of cell preparation, sorting or modification is often performed on samples (cell populations) containing multiple cells, such enrichment operations mean that the expression of enhanced receptors is carried out. Before the modification, it is preferred that the immune cells or immune cell population used for further modification in the present invention contain a higher proportion of PD1 + T cells than the PBMC population that has not been enriched.
  • the proportion of peripheral blood PD1 + T cells averages around 28%.
  • the proportion of PD1 + cells is greater than 50%, preferably greater than 60%, more preferably greater than 70%, particularly preferably greater than 80% .
  • modified immune cell Any immune cell obtained through the above methods and sources, after being modified, especially modified to express the enhanced receptor of the present invention, is called a "modified immune cell”.
  • modified immune cells of the invention may also have one or more other modifications to confer further functions and/or properties.
  • the immune cells of the present invention can be modified to express a chimeric antigen receptor (CAR), which includes (i) an antigen-interacting domain capable of binding to B cell surface proteins; (ii) a transmembrane domain ; and (iii) intracellular signaling domain. After the immune cells carrying the CAR come into contact with B cell surface proteins, they can expand and/or activate the immune cells.
  • CAR chimeric antigen receptor
  • the B cell surface protein can be any protein 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.
  • the antigen-interacting domain of the CAR is capable of binding to a surface protein on a non-B cell, so long as binding to the surface protein does not significantly impair the overall health or immune system of the host.
  • the surface protein is a surface protein on an immune cell. In some embodiments, the surface protein is a surface protein on cells other than immune cells.
  • the surface protein may be selected from the group consisting of (provided that binding to the surface protein does not significantly impair the host's overall health or immune system) CD31, CD32A, CD32B, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42a, CD42b, CD42c, CD42d, 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,
  • the B cell surface protein is selected from the group consisting of CD19, CD20 and CD22.
  • the antigen-interacting domain of the CAR is capable of binding to B cell surface proteins or fragments thereof on dead B cells.
  • B cell apoptosis can occur before or after an immune response (eg, an immune response against tumor cells) is generated.
  • an immune response eg, an immune response against tumor cells
  • dead B cells or fragments thereof may still have B cell surface proteins or fragments thereof represented on the surface.
  • the ability of a CAR to target both live and dead B cells may increase the chance that a CAR-containing immune cell (i) binds to B cell surface proteins and (i) initiates signaling from intracellular signaling domains. In some cases, signaling by intracellular signaling domains may promote expansion (proliferation) of CAR-containing immune cells.
  • the antigen-interacting domain of the CAR is capable of binding a B cell surface protein or fragment thereof that is coupled to the particle (e.g., via covalent and/or non-covalent bonds) (e.g., nanoparticle) surface.
  • the particles may be any particulate material containing organic and/or inorganic materials. Particles can be of various shapes and sizes. The particles may range from about 1 nanometer (nm) to about 50 microns ( ⁇ m) 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 ⁇ m, 5 ⁇ m, 10 ⁇ m, 50 ⁇ m, or larger in at least one dimension.
  • the particles may be up to about 50 ⁇ m, 10 ⁇ m, 5 ⁇ m, 1 ⁇ m, 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, nanoribbons, etc.
  • particles examples include metallic nanoparticles (e.g., gold nanoparticles, silver nanoparticles, and iron nanoparticles), intermetallic nanosemiconductor nanoparticles, core-shell nanoparticles, particles with an inorganic core and a polymer shell, particles with an organic core and a polymer Particles of material shells, and their mixtures.
  • metallic nanoparticles e.g., gold nanoparticles, silver nanoparticles, and iron nanoparticles
  • intermetallic nanosemiconductor nanoparticles e.g., gold nanoparticles, silver nanoparticles, and iron nanoparticles
  • core-shell nanoparticles examples include particles with an inorganic core and a polymer shell, particles with an organic core and a polymer Particles of material shells, and their mixtures.
  • 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 (eg, protein), or combinations thereof.
  • Particles that display B cell surface proteins on their surface can be introduced in vitro into immune cells containing a CAR that binds B cell surface proteins.
  • particles presenting B cell surface proteins can be introduced in vivo (eg, locally or systemically injected) with CAR-containing immune cells. Such particles can be used to expand CAR-containing immune cell populations in vitro or in vivo.
  • An antigen-binding domain may comprise any protein or molecule capable of binding an antigen (eg, a B cell surface protein).
  • 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, which functional derivatives , variants or fragments including but not limited to Fab, Fab', F(ab')2, Fv, single chain Fv (scFv), small antibodies, diabodies and single domain antibodies (such as the heavy chain of camel-derived Nanobodies can variable domain (VH), light chain variable domain (VL) and variable domain (VHH)).
  • the first antigen binding domain comprises at least one of Fab, Fab', F(ab')2, Fv, and scFv.
  • the antigen binding domain comprises an antibody mimetic.
  • Antibody mimetics refer to molecules that bind target molecules with comparable affinity to antibodies and include single-chain binding molecules, cytochrome b562-based binding molecules, fibronectin or fibronectin-like protein scaffolds (e.g., adnectin), lipids Plasmopherin scaffolds, calixarene scaffolds, A domains and other scaffolds.
  • the antigen binding domain comprises a transmembrane receptor or any derivative, variant or fragment thereof.
  • the antigen-binding domain may comprise at least one ligand for a transmembrane receptor Binding domain.
  • the antigen binding domain may comprise scFV.
  • scFv can be derived from antibodies whose variable region sequences are known.
  • scFvs can be derived from antibody sequences obtained from available mouse hybridomas.
  • scFvs can be obtained from whole-exome sequencing of tumor cells or primary cells.
  • the scFv can be altered.
  • scFvs can be modified in a variety of ways.
  • the scFv can be mutated so that the scFv has higher affinity for its target.
  • the affinity of an scFv for its target can be optimized for targets that are expressed at low levels on normal tissues.
  • This optimization can be performed to minimize potential toxicities such as hypercytokineemia.
  • clones of scFvs with higher affinity for the membrane-bound form of the target may be superior to their soluble form counterparts.
  • This modification can be made if some targets are also detectable in soluble form at different levels and whose targeting may cause unwanted toxicity (such as hypercytokineemia).
  • the antigen-binding domain of the CAR can be linked to the intracellular signaling domain via a transmembrane domain.
  • the transmembrane domain of the CAR can anchor the CAR to the plasma membrane of cells, such as immune cells.
  • the transmembrane segment comprises a polypeptide.
  • the transmembrane polypeptide connecting the antigen-binding domain and the intracellular signaling domain of the CAR can have any suitable polypeptide sequence.
  • the transmembrane polypeptide comprises the polypeptide sequence of the transmembrane portion of an endogenous or wild-type transmembrane protein.
  • the transmembrane polypeptide comprises at least 1 (e.g., at least 2, 3, 4, 5, 6, 7) compared to the transmembrane portion of the endogenous or wild-type transmembrane protein. , 8, 9, 10 or more) polypeptide sequences with amino acid substitutions, deletions and insertions.
  • the transmembrane polypeptide comprises a non-natural polypeptide sequence, such as a sequence of a polypeptide linker.
  • Polypeptide linkers can be flexible or rigid. Polypeptide linkers can be structured or unstructured.
  • a transmembrane polypeptide transmits a signal from an extracellular region to an intracellular region of a cell, eg, via an antigen-binding domain.
  • the native transmembrane portion of CD28 can be used in CARs. In other cases, the native transmembrane portion of CD8 ⁇ can also be used in CARs.
  • a CAR of the present disclosure may comprise a signaling domain involved in immune cell signaling, or any derivative, variant or fragment thereof.
  • the intracellular signaling domain of CAR can induce the activity of CAR-containing immune cells.
  • Intracellular signaling domains can transduce effector functional signals and direct cells to perform specialized functions.
  • the signaling domain may contain signaling domains of other molecules. In some cases, truncated portions of the signaling domain are used in CARs.
  • the intracellular signaling domain comprises multiple signaling domains involved in immune cell signaling, or any derivatives, variants or fragments thereof.
  • intracellular signaling structures A domain may comprise at least 2 immune cell signaling domains, such as at least 2, 3, 4, 5, 7, 8, 9 or 10 immune cell signaling domains.
  • Immune cell signaling domains can participate in regulating the primary activation of TCR complexes in a stimulatory or inhibitory manner.
  • the intracellular signaling domain may be that of a T cell receptor (TCR) complex.
  • TCR T cell receptor
  • the intracellular signaling domain of the CAR of the present invention may include the following signaling domains: Fc ⁇ receptor (Fc ⁇ R), Fc ⁇ receptor (Fc ⁇ R), Fc ⁇ receptor (Fc ⁇ R), neonatal Fc receptor (FcRn), CD3, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD4, CD5, CD8, CD21, CD22, CD28, CD32, CD40L (CD154), CD45, CD66d, CD79a, CD79b, CD80, CD86, CD278 (also known as ICOS), CD247 ⁇ , CD247n, DAP10, DAP12, FYN, LAT, Lck, MAPK, MHC complex, NFAT, NF- ⁇ B, PLC- ⁇ , iC3b, C3dg, C3d and Zap70.
  • Fc ⁇ receptor Fc ⁇ receptor
  • Fc ⁇ R Fc ⁇ receptor
  • Fc ⁇ R Fc ⁇ receptor
  • Fc ⁇ R Fc ⁇ receptor
  • Fc ⁇ R neonatal
  • the signaling domain includes an immunoreceptor tyrosine-based activation motif or ITAM.
  • ITAM-containing signaling domain may comprise two repeats of the amino acid sequence YxxL/I 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.
  • the ITAM-containing signaling domain can be modified, for example, by phosphorylation. Phosphorylated ITAMs can serve as docking sites for other proteins, such as those involved in various signaling pathways.
  • the primary signaling domain comprises a modified ITAM domain (e.g., a mutated, truncated, and/or optimized ITAM domain) that is identical to a native ITAM domain. Have altered (e.g., increased or decreased) activity compared to.
  • a modified ITAM domain e.g., a mutated, truncated, and/or optimized ITAM domain
  • the intracellular signaling domain of a CAR of the invention comprises an Fc ⁇ R signaling domain (eg, ITAM).
  • the Fc ⁇ R signaling domain may be selected from Fc ⁇ RI (CD64), Fc ⁇ RIIA (CD32), Fc ⁇ RIIB (CD32), Fc ⁇ RIIIA (CD16a), and Fc ⁇ RIIIB (CD16b).
  • the intracellular signaling domain comprises an Fc ⁇ R signaling domain (eg, ITAM).
  • the Fc ⁇ R signaling domain may be selected from Fc ⁇ RI and Fc ⁇ RII (CD23).
  • the intracellular signaling domain comprises an FcaR signaling domain (eg, ITAM).
  • the Fc ⁇ R signaling domain may be selected from Fc ⁇ RI (CD89) and Fc ⁇ / ⁇ R.
  • the intracellular signaling domain comprises a CD3 ⁇ signaling domain.
  • the primary signaling domain comprises an ITAM of CD3 ⁇ .
  • the intracellular signaling domain of a CAR of the invention comprises an immunoreceptor tyrosine-based inhibitory motif or ITIM.
  • the ITIM-containing signaling domain may comprise a conserved sequence of amino acids found in the cytoplasmic tails of some inhibitory receptors of the immune system (S/I/V/LxYxxI/V/L). able to pass Enzymes such as members of the Src kinase family (eg, Lck) modify the ITIM-containing primary signaling domain, e.g., phosphorylate it. After phosphorylation, other proteins, including enzymes, can be recruited to ITIM.
  • proteins include, but are not limited to, enzymes such as the phosphotyrosine phosphatases SHP-1 and SHP-2, the inositol phosphatase known as SHIP, and proteins with one or more SH2 domains (eg, ZAP70).
  • enzymes such as the phosphotyrosine phosphatases SHP-1 and SHP-2, the inositol phosphatase known as SHIP, and proteins with one or more SH2 domains (eg, ZAP70).
  • Intracellular signaling domains may include the following signaling domains (e.g., ITIMs): BTLA, CD5, CD31, CD66a, CD72, CMRF35H, DCIR, EPO-R, Fc ⁇ RIIB (CD32), Fc receptor-like protein 2 ( FCRL2), Fc receptor-like protein 3 (FCRL3), Fc receptor-like protein 4 (FCRL4), Fc receptor-like protein 5 (FCRL5), Fc receptor-like protein 6 (FCRL6), protein G6b (G6B), leukocytes Interleukin 4 receptor (IL4R), immunoglobulin superfamily receptor transport-associated protein 1 (IRTA1), immunoglobulin superfamily receptor transport-associated protein 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
  • the intracellular signaling domain comprises a modified ITIM domain (e.g., a mutated, truncated, and/or optimized ITIM domain) that is consistent with the native ITIM structure. Domains have altered (eg, increased or decreased) activity compared to each other.
  • a modified ITIM domain e.g., a mutated, truncated, and/or optimized ITIM domain
  • the intracellular signaling domain comprises at least 2 ITAM domains (eg, at least 3, 4, 5, 6, 7, 8, 9, or 10 ITAM domains) .
  • the intracellular signaling domain includes at least 2 ITIM domains (e.g., at least 3, 4, 5, 6, 7, 8, 9 or 10 ITIM domains) (e.g., at least 2 primary signaling domains).
  • the intracellular signaling domain includes both an ITAM domain and an ITIM domain.
  • the intracellular signaling domain of the CAR of the invention may include a costimulatory domain.
  • a costimulatory domain e.g., from a costimulatory molecule
  • a costimulatory domain is operable to modulate proliferation and/or survival signaling in immune cells.
  • the costimulatory signaling domain includes the following signaling domains: MHC class I protein, MHC class II protein, TNF receptor protein, immunoglobulin-like protein, cytokine receptor, integrin, signal Transduction lymphocyte activation molecule (SLAM protein), activated NK cell receptor, BTLA or Toll ligand receptor.
  • the costimulatory domain comprises a signaling domain of a molecule selected from: 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
  • the intracellular signaling domain includes multiple costimulatory domains, such as at least two (eg, at least 3, 4, or 5) costimulatory domains.
  • Costimulatory signaling regions can provide signals that are synergistic with primary effector activation signals and can fulfill the requirements for T cell activation.
  • adding a costimulatory domain to a CAR can enhance the efficacy and persistence of immune cells provided herein.
  • the binding of CAR to B cell surface proteins can enhance the proliferation of immune cells compared with immune cells lacking CAR.
  • the proliferation of immune cells may refer to the expansion of immune cells.
  • the proliferation of immune cells may refer to the phenotypic changes of immune cells.
  • Immune cells containing CARs provided herein can proliferate more than comparable immune cells lacking a CAR that exhibits binding to B cell surface proteins.
  • Immune cells containing a CAR can proliferate from about 5-fold to about 10-fold, from about 10-fold to about 20-fold, from about 20-fold to about 30-fold, from about 30-fold to about 40-fold more than the proliferation of comparable immune cells lacking CAR.
  • Immune cells containing a CAR can proliferate from about 5-fold to about 10-fold, from about 10-fold to about 20-fold, from about 20-fold to about 30-fold, from about 30-fold to about 40-fold more than the proliferation of comparable immune cells lacking CAR.
  • proliferation can include quantifying the number of immune cells. Quantifying the number of immune cells can include flow cytometry, trypan blue exclusion, and/or blood cell counting. Proliferation can also be determined by phenotypic analysis of immune cells.
  • the purpose of the CD19 CAR modification carried out in the present invention is to assist the expansion and activation of immune cells such as T cells by binding to B cells, which is different from the common CD19 CAR-T technology for T cells.
  • the purpose and function of cells loaded with CD19 CAR are not exactly the same.
  • the modified immune cells of the present invention are not limited to the treatment of tumors related to CD19-expressing B cells, because the role of CD19 CAR in the present invention is not Target cell recognition and binding.
  • compositions and molecules may be introduced into host cells (eg, immune cells) using any suitable delivery method.
  • host cells eg, immune cells
  • the various components can be delivered simultaneously or separately.
  • the choice of method may depend on the type of cells to be transformed and the environment in which transformation occurs (eg, in vitro, ex vivo, or in vivo).
  • Delivery methods may include introducing into a cell or population of cells one or more nucleic acids comprising a nucleotide sequence encoding an enhancing receptor or other modified molecule of the invention, such as a chimeric antigen receptor.
  • Suitable nucleic acids comprising the nucleotide sequences encoding the nucleotide sequences may include expression vectors, wherein expression vectors comprising the nucleotide sequences encoding one or more enhancing receptors or other modifying molecules of the invention are recombinant expression vectors.
  • Non-limiting examples of delivery methods or transformations include, for example, viral or phage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethylenimine (PEI)-mediated transfection, DEAE dextran-mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, and nanoparticle-mediated nucleic acid delivery.
  • delivery methods or transformations include, for example, viral or phage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethylenimine (PEI)-mediated transfection, DEAE dextran-mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, and nanoparticle-mediated nucleic acid delivery.
  • PKI polyethylenimine
  • the invention provides a method comprising converting one or more polynucleotides or one or more vectors as described herein or one or more transcripts thereof and/or from The transcribed protein or proteins are delivered to the host cell.
  • the invention further provides cells produced by such methods, as well as organisms (eg, animals, plants, or fungi) comprising or produced from such cells.
  • Non-viral vector delivery systems can include DNA plasmids, RNA (eg, transcripts of the vectors described herein), naked nucleic acids, and nucleic acids complexed with delivery vehicles (eg, liposomes).
  • Viral vector delivery systems include DNA and RNA viruses, which can have episomal or integrated genomes after delivery to cells.
  • Non-viral delivery methods of nucleic acids may include lipofection, nucleofection, microinjection, gene gun, virions, liposomes, immunoliposomes, polycationic or lipid:nucleic acid conjugates, naked DNA, artificial Agents enhance uptake of virions and DNA.
  • Lipofection can be recognized using efficient receptors suitable for use with polynucleotides of cationic and neutral lipids. Delivery can be to cells (eg, in vitro or ex vivo administration) or target tissue (eg, in vivo administration).
  • Formulations of lipid:nucleic acid complexes may be used, including targeted liposomes, such as immunolipid complexes.
  • RNA or DNA viruses 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 optionally modified cells can be administered (ex vivo).
  • Virus-based systems can include retroviral, lentiviral, adenoviral, adeno-associated virus, and herpes simplex virus vectors for gene transfer. Integration into the host genome can occur using retroviral, lentiviral, and adeno-associated viral gene transfer methods, which may result in long-term expression of the inserted transgene. High transduction efficiencies can be observed in many different cell types and target tissues.
  • 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 may depend on the target tissue.
  • Retroviral vectors can contain cis-acting long terminal repeats, which have the ability to package foreign sequences up to 6-10 kb.
  • Minimal cis-acting LTR may be sufficient for replication and packaging of vectors that can be used to integrate therapeutic genes into target cells to provide durable transgene expression.
  • Retroviral vectors may include those based on murine leukemia virus (MuLV), gibbon leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof.
  • Adenovirus-based systems can be used. Adenovirus-based systems can lead to transient expression of transgenes. Adenovirus-based vectors can transduce cells with high efficiency and may not require cell division. Adenovirus-based vectors can be used to achieve high titers and expression levels. Adeno-associated virus (“AAV”) vectors can be used, for example, to transduce cells with target nucleic acids in the in vitro production of nucleic acids and peptides, as well as in in vivo and ex vivo gene therapy procedures.
  • AAV Adeno-associated virus
  • Packaging cells can be used to form viral particles capable of infecting host cells.
  • Such cells may include 293 cells (eg, for packaging adenovirus), and Psi2 cells or PA317 cells (eg, for packaging retroviruses).
  • Viral vectors can be produced by generating 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 to be replaced by an expression cassette for the polynucleotide or polynucleotides to be expressed.
  • Lost viral functions can be supplied in trans by packaging cell lines.
  • an AAV vector may contain ITR sequences from the AAV genome required for packaging and integration into the host genome.
  • Viral DNA can be packaged in cell lines,
  • the cell lines may contain helper plasmids encoding other AAV genes (ie, rep and cap) but lacking the ITR sequence.
  • Cell lines can also be infected with adenovirus as a helper.
  • Helper viruses can promote AAV vector replication and expression of AAV genes from the helper plasmid. Contamination by adenovirus can be reduced, for example, by heat treatment, to which adenovirus is more sensitive than AAV. Additional methods for delivering nucleic acids to cells may be used, for example as described in US 20030087817 (incorporated herein by reference).
  • Host cells can be transfected transiently or non-transiently with one or more vectors described herein.
  • the cells can be transfected due to their natural presence in the subject.
  • Cells can be taken from or derived from a subject and transfected.
  • the cells may be derived from cells obtained from a subject, such as a cell line.
  • cells transfected with one or more vectors described herein are used to establish new cell lines containing one or more vector-derived sequences.
  • cells transiently transfected with the compositions of the invention eg, by transient transfection of one or more vectors, or transfection with RNA
  • vectors for eukaryotic host cells include pXT1, pSG5 (StratageneTM), pSVK3, pBPV, pMSG and pSVLSV40 (PharmaciaTM).
  • Contacting the cells with the composition can occur in any culture medium and under any culture conditions that promote cell survival.
  • cells can be suspended in any suitable convenient nutrient medium, such as Iscove's modified DMEM or RPMI 1640 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 are molecules capable of promoting cell survival, growth and/or differentiation in culture or in intact tissue through specific actions on transmembrane receptors. Growth factors can include polypeptide and non-polypeptide factors.
  • the delivery system selected is targeted to a specific tissue or cell type.
  • tissue or cell targeting of the delivery system is achieved by conjugating the delivery system to a tissue or cell specific marker, such as a cell surface protein.
  • tissue or cell specific marker such as a cell surface protein.
  • Viral and non-viral delivery systems can be customized to target the tissue or cell type of interest.
  • the proportion of cells with the desired modification exceeds 50%, such as reaching about 60%, about 70%, or about 80%, Even levels around 90% or higher.
  • the modified immune cells and related treatment methods of the present invention are a personalized treatment strategy based on individual patients, even if they are prepared in a similar manner, The proportion of effective cells in the final cell product will also vary due to individual differences.
  • Immune cells modified with enhanced receptors of the present invention can kill a variety of target cells.
  • Target cells to which this method can be applied include various cell types.
  • Target cells can be in vitro.
  • the target cells can be in vivo.
  • Target cells can be ex vivo.
  • Target cells can be isolated cells.
  • the target cells may be cells in an organism.
  • the target cell can be an organism.
  • the target cells may be cells in cell culture.
  • the target cell can be a cell in a collection of cells.
  • the target cells may be mammalian cells or derived from mammalian cells.
  • the target cells may be rodent cells or derived from rodent cells.
  • the target cells may be human cells or derived from human cells.
  • the target cells may be prokaryotic cells or derived from prokaryotic cells.
  • the target cells may be bacterial cells or may be derived from bacterial cells.
  • the target cells may be or derived from archaeal cells.
  • the target cells may be eukaryotic cells or derived from eukaryotic cells.
  • the target cells can be pluripotent stem cells.
  • the target cells may be plant cells or derived from plant cells.
  • the target cells may be animal cells or derived from animal cells.
  • the target cells may be or derived from invertebrate cells.
  • the target cells may be or derived from vertebrate cells.
  • the target cells may be microbial cells or derived from microbial cells.
  • the target cells may be fungal cells or derived from fungal cells.
  • Target cells can be from a specific organ or tissue.
  • Target cells can be stem cells or progenitor cells.
  • Target cells can include stem cells (eg, adult stem cells, embryonic stem cells, induced pluripotent stem (iPS) cells) and progenitor cells (eg, cardiac progenitor cells, neural progenitor cells, etc.).
  • Target cells can include mammalian stem cells and progenitor cells, including rodent stem cells, rodent progenitor cells, human stem cells, human progenitor cells, and the like.
  • Clonal cells can contain the cells' descendants.
  • Target cells can contain target nucleic acids.
  • the target cells may be in living organisms.
  • the target cells can be genetically modified cells.
  • the target cell can be a host cell.
  • Target cells can be primary cells.
  • a culture of primary cells can be passaged 0, 1, 2, 4, 5, 10, 15, or more times.
  • Cells can be single-celled organisms.
  • Cells can be grown in culture.
  • Target cells can be diseased cells.
  • Diseased cells may have altered metabolic, gene expression, and/or morphological characteristics.
  • Diseased cells can be cancer cells, diabetic cells, and apoptotic cells.
  • Diseased cells may be cells from a diseased subject.
  • Example diseases may include blood disorders, cancer, metabolic disorders, eye disorders, organ disorders, musculoskeletal disorders, heart disease, etc.
  • the target cells are cancer cells.
  • the targeted cancer cells are solid tumor cells.
  • the cancer is of the hematopoietic lineage, such as lymphoma.
  • the lymphoma is B-cell lymphoma.
  • the B-cell lymphoma may be Hodgkin's lymphoma or non-Hodgkin's lymphoma, including but not limited to large B-cell lymphoma, such as diffuse large B-cell lymphoma (DLBCL), diffuse large B-cell lymphoma (DLBCL) not otherwise specified (NOS), mediastinal large B-cell lymphoma (PMBCL), follicular cell lymphoma, mantle cell lymphoma.
  • the tumor may be a refractory tumor or a relapsed tumor, for example, the tumor patient has received prior treatment for the tumor.
  • the target cells form tumors.
  • Treatment with enhanced receptors of the invention may stabilize tumor growth (eg, one or more tumors do not increase in size by more than 1%, 5%, 10%, 15%, or 20%, and/or do not metastasize).
  • the tumor is stabilized for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more weeks.
  • the tumor is stabilized for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more months.
  • the tumor is stabilized for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more years.
  • the tumor size or tumor cell number is reduced by at least about 5%, 10%, 15%, 20%, 25, 30%, 35%, 40%, 45%, 50%, 55%, 60% , 65%, 70%, 75%, 80%, 85%, 90%, 95% or more.
  • tumors are completely eliminated, or reduced below detection levels.
  • the subject remains tumor-free (eg, in remission) for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more weeks following treatment .
  • the subject remains tumor-free for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more months following treatment.
  • the subject remains tumor-free for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more years following treatment.
  • Target cell death can be determined by any suitable method, including, but not limited to, counting cells before and after treatment or measuring the level of a marker associated with live or dead cells (e.g., live target cells or dead target cells) .
  • the extent of cell death can be determined by any suitable method. In some embodiments, the extent of cell death is determined with respect to starting conditions. For example, an individual may have a known starting amount of target cells, such as a starting cell mass of known size or a known concentration of circulating target cells. In such cases, the extent of cell death can be expressed as the ratio of viable cells after treatment to the starting cell population. In some embodiments, the extent of cell death can be determined by a suitable cell death assay. A variety of cell death assays are available, and a variety of detection methods can be utilized. Examples of detection methods include, but are not limited to, the use of cell staining, microscopy, flow cytometry, cell sorting, and combinations of these.
  • necrosis i.e., death
  • the percentage of tissue removed determines the efficacy of treatment in reducing tumor size.
  • treatment is if the percent necrosis of the resected tissue is greater than about 20% (eg, at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) Treatment is effective.
  • the percentage of necrosis in the resected tissue is 100%, ie, no viable tumor tissue is present or detectable.
  • Exposure of target cells to the modified immune cells or immune cell populations of the invention can be performed in vitro or in vivo. Exposure of target cells to immune cells or immune cell populations in vitro can be accomplished by co-culture of target cells and immune cells. Target cells and immune cells can be co-cultured, for example as adherent cells or alternatively in suspension. Target cells and immune cells can be co-cultured in various suitable types of cell culture media with, for example, supplements, growth factors, ions, etc. In some cases, exposing target cells to immune cells or populations of immune cells in vivo can be accomplished by administering the immune cells to a subject (eg, a human subject) and allowing the immune cells to localize to the target cells via the circulation system. In some cases, immune cells can be delivered to nearby areas where target cells are located, such as by direct injection.
  • a subject eg, a human subject
  • immune cells can be delivered to nearby areas where target cells are located, such as by direct injection.
  • the exposure can be for any suitable length of time, such as at least 1 minute, at least 5 minutes, at least 10 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, At least 7 hours, at least 8 hours, at least 12 hours, at least 16 hours, at least 20 hours, at least 24 hours, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month or more.
  • cells or cell populations bearing enhanced receptors of the present invention may be administered to a person suffering from a disease or disorder in an amount sufficient to cure or at least partially arrest the symptoms of a disease or disorder or to cure, cure, ameliorate or ameliorate the disorder.
  • a subject of a disease or condition may vary depending on the severity and duration of the disease or condition, prior therapy, the subject's state of health, weight and response to the drug, and the judgment of the treating physician.
  • the immune cells with enhanced receptors of the present invention can be used at a dose of about 1 ⁇ 10 7 to about 1 ⁇ 10 12 cells, preferably about 1 ⁇ 10 8 to about 1 ⁇ 10 11 cells
  • a dose of about 1 ⁇ 10 7 , about 1 ⁇ 10 8 , about 1 ⁇ 10 9 , about 1 ⁇ 10 10 , about 1 ⁇ 10 11 , and about 1 ⁇ 10 12 are administered to the subject.
  • the cell dose is the total amount of cells administered.
  • the dose of therapeutic cells administered can also be determined based on the subject's body weight.
  • the immune cells with enhanced receptors of the present invention can be administered at a dose of about 1 ⁇ 10 5 to about 1 ⁇ 10 11 cells/kg subject body weight, preferably about 1 ⁇ 10 6 to about 1 ⁇ 10 8 cells/kg subject body weight, for example, about 1 ⁇ 10 6 cells/kg subject body weight, about 3.3 ⁇ 10 6 /kg subject body weight, about 5 ⁇ 10 6 /kg subject body weight , about 6.7 ⁇ 10 6 /kg tested Subject weight, approximately 1 ⁇ 10 7 cells/kg subject weight, approximately 3.3 ⁇ 10 7 /kg subject weight, approximately 5 ⁇ 10 7 /kg subject weight, approximately 6.7 ⁇ 10 7 /kg subject weight Subject's body weight, approximately 1 ⁇ 10 8 cells/kg subject's body weight.
  • the effective dose of immune cells with dual enhancer receptors of the present invention is lower than that of immune cells with only a single enhancer receptor.
  • the immune cells bearing dual boosting receptors are capable of achieving similar or better results at a dose that is at least 50% lower, or even an order of magnitude lower, than immune cells bearing only a single boosting receptor.
  • the dual enhancing receptor is preferably a combination of the TIGIT/OX40 enhancing receptor of the present invention and another different enhancing receptor, such as a combination of PD1/CD28 enhancing receptor or PD1(108)/CD28 enhancing receptor.
  • the fourth generation lentiviral vector system (four plasmid system) was used to transfer into 293T cells through PEI (Polysciences, 23966-1) reagent transfection reagent.
  • PEI Polysciences, 23966-1
  • NY-ESO-1 TCR artificial TCR targeting the tumor target NY-ESO-1;
  • CD19 CAR CAR targeting CD19
  • TIGIT/OX40 ECD is derived from TIGIT, ICD is OX40, and TM is the enhanced receptor of OX40;
  • TIGIT/OX40-TCR The TIGIT/OX40 gene in 3) above and the artificial TCR gene targeting the tumor target NY-ESO-1 in 1) above are constructed on the same vector, with T2A between the two gene sequences. Sequence connection, by cutting the T2A sequence with a shearing enzyme, the TIGIT/OX40 enhanced receptor protein and TCR protein can be expressed separately on the cell membrane, forming a non-fusion expression of the two proteins;
  • TIGIT/OX40-CAR Non-fusion expression of TIGIT/OX40 in 3) above and the CAR targeting CD19 in 2) above;
  • TIGIT/CD28-TCR Non-fused expression of the enhanced receptor TIGIT/CD28 and an artificial TCR targeting the tumor target NY-ESO-1, where the ECD of the enhanced receptor TIGIT/CD28 is derived from TIGIT, ICD and TM is derived from CD28;
  • PD1/CD28-TCR Non-fusion expression of the enhanced receptor PD1/CD28 and artificial TCR targeting the tumor target NY-ESO-1, in which the ECD of the enhanced receptor PD1 is derived from wild-type PD1, and the ICD and TM are derived from CD28.
  • TIGIT/OX40-TCR Take TIGIT/OX40-TCR as an example to illustrate the preparation of the corresponding lentiviral vector.
  • the purpose of loading TCRs into cells is to enable them to recognize tumor cells bearing corresponding antigens. This is because in animal (mouse) experiments, the tumors in the mice were xenografts from humans, and the TCR modification enabled the therapeutic immune cells to recognize the xenografts, thereby simulating the modification process in practical applications. Immune cells that previously had tumor recognition.
  • tumor cell recognition can not only be imparted by loading TCR (such as preparing neoantigen-recognizing TCR according to individual mutation profiles), but also by isolating tumor-infiltrating lymphocytes (TIL), from PD1 + T cells were isolated from peripheral blood to obtain cells with tumor recognition.
  • TIL tumor-infiltrating lymphocytes
  • TIGIT/OX40-TCR construct is constructed by spacing the TIGIT/OX40 enhanced receptor gene sequence and the TCR gene sequence on the same vector from the T2A sequence.
  • TIGIT/OX40 is composed of the TIGIT extracellular domain (TIGIT ECD) and the OX40 transmembrane structure. Domain (OX40TM) and OX40 intracellular domain (OX40ICD), whose sequences are shown in Table 1 below.
  • TIGIT/OX40-TCR plasmid Mix the main vector TIGIT/OX40-TCR plasmid, packaging vector pMDL-gag plasmid, Rev plasmid, and envelope vector pMD2.G plasmid at a ratio of 1:2:2:1 and use PEI reagent to co-transfect 293T (ATCC )cell. After the transfected cells were cultured at 37°C for 48 hours, the supernatant was collected and ultracentrifuged to concentrate the lentivirus. Resuspend the lentiviral particles obtained by centrifugation in 1 mL of 1640 culture medium, and distribute into 10 1.5 ml EP tubes, 100 ⁇ l per tube, and store at -80°C.
  • titer of TIGIT/OX40-TCR enhanced receptor lentiviral vector. Take 100 ⁇ l of the virus stock solution, dilute it 3 times, and dilute it 3, 9, 27, 81, 243, and 729 times respectively. 50 ⁇ l of each virus sample with different dilutions was taken to infect 293T cells for 48 hours to 72 hours. Remove the culture supernatant, wash the cells with PBS, add trypsin, collect 293T cells, use anti-TIGIT antibody (Biolegend, 372714) for TIGIT staining, and analyze TIGIT + cells in virus-infected cells with different dilutions by flow cytometry. ratio, and calculate the titer according to the following formula:
  • Titer (TU/ml) starting number of 293T cells (about 40,000 to 45,000) ⁇ TIGIT + cell percentage ⁇ dilution factor ⁇ 20 (take the dilution factor of the first group of viruses with TIGIT + cell percentage ⁇ 20% for calculation ).
  • T cells are used as immune cells loaded with enhanced receptors, and the T cells are modified to have specific recognition capabilities for specific tumor cells, so that specific binding of immune cells to tumor cells can be achieved in in vitro experiments. .
  • NY-ESO-1-TCR-T T cells modified to express NY-ESO-1-TCR, which can target NY-ESO-1 tumor cells through artificial TCR, and can also be referred to as TCR-T. ;
  • TIGIT/OX40-TCR-T T cells modified to express the enhanced receptor TIGIT/OX40 and NY-ESO-1-TCR, which can target tumor cells expressing NY-ESO-1 through artificial TCR;
  • TIGIT/CD28-TCR-T T cells modified to express the enhanced receptor TIGIT/CD28 and NY-ESO-1-TCR, which can target tumor cells expressing NY-ESO-1 through artificial TCR;
  • PD1/CD28-TCR-T modified to express enhanced receptors PD1/CD28 and NY-ESO-1-TCR T cells that can target tumor cells expressing NY-ESO-1 through artificial TCRs.
  • TCRs confer tumor recognition to immune cells, while various enhancer receptors are used to increase the effectiveness of immune cells.
  • TCR-T Culture and expand the above-mentioned transfected cells (TCR-T, TIGIT/OX40-TCR-T, TIGIT/CD28-TCR-T and PD1/CD28-TCR-T cells), and use T cells transfected without adding any virus.
  • a control Mock-T.
  • the tumor-associated antigen NY-ESO-1 was used as the target, and the human fibrosarcoma cell line HT1080 (Beijing Zhaoyan New Drug Research Center Co., Ltd.) that naturally expresses NY-ESO-1 was used. Gift) as target cells, and expressed the HLA-A*02:01 gene in HT1080 cells.
  • the four cells constructed in Example 2 all express TCR-T targeting NY-ESO-1, and these cells are used as effector T cells.
  • the activation levels of the differently modified T cells were determined, and the results are shown in the figure. 1A-B.
  • IL-2 and IFN- ⁇ secreted by TIGIT/OX40-TCR-T cells were significantly higher than those secreted by PD1/CD28-TCR-T, TIGIT/CD28-TCR-T and TCR-T cells.
  • IL-2 and IFN- ⁇ The results of in vitro experiments show that in the HT1080 tumor cell model, TCR-T cells expressing enhancer receptors have stronger immune activation, and the effect of two enhancer receptors with TIGIT as the extracellular domain is better than that with PD1 as the extracellular domain. Domain-enhancing receptors, among which TIGIT/OX40 has the highest activation level.
  • TIGIT/OX40 enhances the effect of receptors on activating T cells in vivo
  • mice 1 ⁇ 10 6 tumor cells J82-NY were subcutaneously inoculated into NSG mice (purchased from Beijing Aidemo Biotechnology Co., Ltd.). When the tumor volume reached 80-120mm 3 , 30 mice were selected and randomly divided into 6 groups as follows (5 animals in each group), and were given one of the following 6 types of cell treatments:
  • Freezing medium group Freezing medium group
  • TCR-T group Only the TCR cell group transduced with NY-ESO-1 (TCR-T group);
  • TCR cell group transduced with PD1/CD28 enhanced receptor and NY-ESO-1 (PD1/CD28-TCR-T group);
  • TCR cell group transduced with TIGIT/CD28 enhanced receptor and NY-ESO-1 TIGIT/CD28-TCR-T group
  • TCR cell group transduced with TIGIT/OX40 enhanced receptor and NY-ESO-1 TIGIT/OX40-TCR-T group.
  • the administration method for each group was a single tail vein infusion of 200 ⁇ l of a total of 8 ⁇ 10 6 cells (the effective number of cells was 6 ⁇ 10 6 ), and the control group was given 0.2 ml of cell cryopreservation solution accordingly.
  • tumor size was measured and the mouse status was observed every 2-3 days.
  • mice in the cryopreservation solution and Mock-T groups continued to grow.
  • mice in both groups were euthanized due to excessive tumor size.
  • the tumor volume of mice in groups c, d, e, and f that were administered tumor-recognizing TCR-T cells began to shrink after the 10th day, and the strongest drug effect was shown around the 25th day.
  • the tumors in the mice in the TCR-T group began to recur, and began to gradually become larger after the 35th day.
  • 4 of the 5 mice in the TCR-T group had tumors.
  • the tumor volume exceeded the tumor volume at the beginning of treatment, and tumor progression occurred, and another mouse was found dead on day 70.
  • the average tumor volume of mice in the PD1/CD28-TCR-T group also continued to increase after day 46.
  • the drug efficacy of the TIGIT/CD28-TCR-T group and TIGIT/OX40-TCR-T group was maintained well.
  • the results of the enlarged picture in Figure 3 show that the tumors of the mice in the TIGIT/OX40 group remained completely cleared without recurrence until the end of the observation, while some mice in the TIGIT/CD28 group had tumor recurrence.
  • TIGIT/OX40 can not only enhance the tumor suppressive effect of TCR-T, but also has a stronger function in maintaining T cell function and efficacy than the other two enhancing receptors.
  • Example 5 One of the combined experiments of enhancing receptors TIGIT/OX40 and PD1/CD28
  • the two enhanced receptors are (1) the enhanced receptor PD1(108)/CD28 constructed based on PD1 mutant 108#, and (2) TIGIT/OX40 as described in Example 1.
  • PD1 mutant 108# can bind to human PD-L1 with high affinity, but does not bind to drugs including sintilimab, nivolumab, camrelizumab ( A variety of anti-PD1 antibodies including Camrelizumab, Pemrolizumab and Toripalimab.
  • the nucleotide sequence encoding PD1(108)/CD28 and the nucleotide sequence encoding TIGIT/OX40 were replaced with the nucleotide sequence encoding T2A (SEQ ID NO:22) ligated and introduced into the lentiviral vector as the dual-enhanced receptor construct PD1(108)/CD28-TIGIT/OX40.
  • T2A SEQ ID NO:22
  • the lentiviral vector was constructed in the same manner as in Example 1 and used to transfect 293T cells, and then the titer was determined. Specifically, lentiviral vectors containing the following gene sequences were constructed:
  • PD1(108)/CD28-TIGIT/OX40-TCR Non-fusion expression of the above dual-enhanced receptor construct PD1(108)/CD28-TIGIT/OX40 and the targeted tumor target NY as described in Example 1 -Artificial TCR of ESO-1.
  • J82-NY cells overexpressing NY-ESO-1 were used as target cells, and cells with TCR that specifically recognized NY-ESO-1 were used.
  • T Cell co-culture by measuring the amount of IFN- ⁇ and IL-2 secreted by differently modified T cells, thereby detecting the impact of enhanced receptor modification on T cells on the degree of T cell activation.
  • NY-ESO-1-TCR-T T cells modified to express NY-ESO-1-TCR, which can target NY-ESO-1 tumor cells through artificial TCR, and can also be referred to as TCR-T. ;
  • PD1/CD28-TCR-T T cells modified to express enhanced receptors PD1/CD28 and NY-ESO-1-TCR, which can target tumor cells expressing NY-ESO-1 through artificial TCR;
  • antiPDL1/CD28-TCR-T T cells modified to express enhanced receptors antiPDL1/CD28 and NY-ESO-1-TCR, which can target NY-ESO-1 tumor cells expressing NY-ESO-1 through artificial TCR, in which antiPDL1 Represents the ECD using anti-PD-L1 single chain antibody (PD-L1 scFv) as the enhanced receptor, and its amino acid sequence and nucleotide sequence are shown in SEQ ID NO: 15 and SEQ ID NO: 7 respectively;
  • PD1(108)/CD28-TIGIT/OX40-TCR-T T cells modified to express two enhancer receptors, PD1(108)/CD28 and TIGIT/OX40-TCR-T, which can pass through artificial TCR targets to tumor cells expressing NY-ESO-1.
  • the above-mentioned cells of species i) and ii) are prepared as described in Examples 1 and 2.
  • the preparation of iii) cells is similar to ii), both enhancing receptors bind PD-L1, but ii) uses the receptor PD-1 of the PD-L1 ligand as the ECD, while iii) uses anti-PD- scFv of L1 antibody as ECD.
  • the preparation of iv) cells is completed using the lentiviral vector described in this example and following the same transfection process as in Example 2.
  • Adjust the J82-NY cell density to 1 ⁇ 10 6 cells/mL put 100 ⁇ l into a U-shaped bottom 96-well plate, and take out each of the above four T cells at a ratio of 1:1 to the J82-NY cell target ratio.
  • ELISA method Human IL-2 detection kit (ELISA method) (Daktronix, Cat. No. 1110203) and perform ELISA according to the following procedure.
  • Microplates were previously coated with anti-human IL-2 capture antibody.
  • SA-HRP horseradish peroxidase-labeled streptavidin
  • SA-HRP horseradish peroxidase-labeled streptavidin
  • ELISA method Use the human IFN ⁇ detection kit (ELISA method) (Daktronix, Cat. No. 1110003) and perform ELISA according to the following procedure. Add the standard or sample to be tested and the detection antibody (biotin-labeled anti-human IFN ⁇ antibody) to the microwell plate at the same time, and then add horseradish peroxidase-labeled streptavidin (SA-HRP), whose specificity Binds biotin in immune complexes. Add the enzyme substrate tetramethylbenzidine (TMB), and a blue color will appear. The depth of the color is related to the concentration of IFN ⁇ in the standard or sample. After 5-10 minutes of color development, add the stop solution to terminate the reaction, and read it with a multifunctional microplate reader.
  • SA-HRP horseradish peroxidase-labeled streptavidin
  • TMB enzyme substrate tetramethylbenzidine
  • IL-2 secreted by PD1(108)/CD28-TIGIT/OX40-TCR-T cells is significantly higher than that of PD1/CD28-TCR-T, antiPDL1/CD28-TCR-T and TCR-T cells.
  • Secreted IL-2 As shown in the results of Figure 5, IFN- ⁇ secreted by PD1(108)/CD28-TIGIT/OX40-TCR-T cells was significantly higher than that secreted by antiPDL1/CD28-TCR-T and TCR-T cells, while There was no difference in the amount of IFN- ⁇ secreted by PD1/CD28-TCR-T cells.
  • the results of in vitro experiments showed that TCR-T cells with double-enhancing receptor modification achieved significantly higher activation levels.
  • mice 1 ⁇ 10 6 tumor cells J82-NY were subcutaneously inoculated into NSG mice. When the tumor volume reached 80-120mm 3 , 25 mice were selected and randomly divided into the following 5 groups (5 mice in each group), and were given the following One of 5 types of cell therapy:
  • TCR-T group Only the TCR cell group transduced with NY-ESO-1 (TCR-T group);
  • TCR cell group transduced with PD1/CD28 enhanced receptor and NY-ESO-1 PD1/CD28-TCR- T group
  • TCR cell group transduced with antiPDL1/CD28 enhanced receptor and NY-ESO-1 (antiPDL1/CD28-TCR-T group);
  • TCR cell group transduced with PD1(108)/CD28-TIGIT/OX40 enhanced receptor and NY-ESO-1 PD1(108)/CD28-TIGIT/OX40-TCR-T group.
  • each group was a single tail vein infusion of 6 ⁇ 10 6 TCR-positive effective cells.
  • the amount of cells infused into the tail vein of each mouse in the Mock-T group was the same as that of each mouse in the other groups. Mice were given the same total amount of cells.
  • mice with dual enhancer receptors that specifically recognize tumors provide better tumor suppression in vivo than T cells with only enhancer receptors that specifically bind PD-L1. Effect.
  • the PD1(108)/CD28-TIGIT/OX40 dual- enhanced receptor is constructed by connecting the coding sequence of PD1(108)/CD28 and the coding sequence of TIGIT/OX40 with the coding sequence of T2A.
  • a safer fourth-generation lentiviral vector system was used for transduction.
  • PD1(108)/CD28-TIGIT/OX40 enhanced receptor lentiviral vector For titer detection of PD1(108)/CD28-TIGIT/OX40 enhanced receptor lentiviral vector, use 3-fold dilution and take 50 ⁇ l to infect 293T cells for 48 hours to 72 hours. Collect 293T cells into PD1(108) staining was performed. The proportion of PD1(108)-positive cells was analyzed by flow cytometry, and the titer was calculated according to the formula:
  • Titer (TU/ml) starting number of 293T cells (about 40,000 to 45,000) ⁇ PD1 (108) + cell percentage ⁇ dilution factor ⁇ 20 (take the first PD1 (108) + cell percentage ⁇ 20% for calculation ).
  • the titer of the PD1(108)/CD28 enhanced receptor represents the titer of the TIGIT/OX40 enhanced receptor.
  • Lentiviral vectors containing the following gene sequences were constructed in a similar manner:
  • PD1(108)/CD28 and artificial TCR targeting the tumor target NY-ESO-1, referred to as PD1(108)/CD28-TCR;
  • TIGIT/OX40-TCR "TIGIT/OX40" and artificial TCR targeting the tumor target NY-ESO-1, referred to as TIGIT/OX40-TCR;
  • TCR Artificial TCR targeting the tumor target NY-ESO-1
  • the tumor-associated antigen NY-ESO-1 was used as the target, and J82 tumor cells expressing NY-ESO-1 were used as the target cells (J82-NY -ESO1, or J82-NY), using TCR-T cells targeting NY-ESO-1 as effector T cells, allowing the TCR-T cells targeting NY-ESO-1 to simultaneously express PD1(108)/CD28- TIGIT/OX40 enhances receptor modification to prepare enhanced TCR-T cells.
  • 1 ⁇ 10 6 tumor cells J82-NY were subcutaneously inoculated into NSG mice.
  • mice When the tumor volume reached 80-120mm 3 , 30 mice were selected and randomly divided into 6 groups. They were given the following cell treatments: a. Cryopreserved solution group (Freezing medium); b. Untransduced T cell group (Mock-T group); c. TCR cell group transduced only with NY-ESO-1 (TCR-T group); d. PD1 transduced ( 108)/CD28 enhanced receptor and NY-ESO-1 TCR cell group (PD1(108)/CD28-TCR-T group); e. transduced TIGIT/OX40 enhanced receptor and NY-ESO-1 TCR Cell group (TIGIT/OX40-TCR-T group); f.
  • TCR cell group transduced with PD1(108)/CD28-TIGIT/OX40 enhanced receptor and NY-ESO-1 (PD1(108)/CD28-TIGIT /OX40-TCR-T group).
  • the sequences of elements included in each group are the same as above.
  • the tumor volume is the average of each group.
  • the tumor volume of the mice in the Freezing medium group and the Mock-T group continued to grow.
  • the tumor growth rate of the mice in the TCR-T group was higher than that of the Freezing medium group and the Mock-T group.
  • the Mock-T group was slower and tumor growth was inhibited to a certain extent.
  • the tumor growth of mice in the three groups that expressed enhanced receptors and TCR at the same time showed a decrease in tumor volume, indicating that the expression of enhanced receptors promotes the tumor suppressive function of TCR-T cells.
  • the PD1(108)/CD28-TIGIT/OX40-TCR-T group expressing dual-enhancement receptors showed significant tumor suppressive effects, and the drug effect was maintained for the longest time. Compared with the group containing only one of them, for cells modified by two enhancer receptors, the combination of two enhancer receptors demonstrates a synergistic effect in tumor suppression.
  • TCR-T group Only transduced TCR cell group of NY-ESO-1 (TCR-T group); b. TCR cell group transduced with PD1(108)/CD28 enhanced receptor and NY-ESO-1 (PD1(108)/CD28-TCR- T group); c. TCR cell group transduced with PD1(108)/CD28-TIGIT/OX40 enhanced receptor and NY-ESO-1 (PD1(108)/CD28-TIGIT/OX40- TCR-T group).
  • TCR-T cells did not proliferate significantly in the peripheral blood of mice within 28 days after reinfusion, and an expansion peak appeared on D35.
  • PD1(108)/CD28-TCR-T cells and PD1(108)/CD28-TIGIT/OX40-TCR-T cells showed expansion peaks at D10 and D14 respectively, and PD1(108)/CD28-TIGIT/OX40 -TCR-T cells expanded more strongly than PD1(108)/CD28-TCR-T cells in peripheral blood.
  • This result shows that the enhanced receptor PD1(108)/CD28-TIGIT/OX40 has a stronger promoting effect on the expansion and persistence of TCR-T cells in the peripheral blood of mice.
  • TCR genes can be detected in tumor tissues starting from D3 after T cell reinfusion, but the content is low.
  • the TCR gene content in the tumor tissue of the TCR-T group and the PD1(108)/CD28-TCR-T group increased, while the TCR gene content of the tumor tissue of the PD1(108)/CD28-TIGIT/OX40-TCR-T group increased. lower than the other two groups.
  • the amplification peak of TCR-T group and PD1(108)/CD28-TCR-T group in tumor tissue appeared at D10, and the amplification degree of PD1(108)/CD28-TCR-T group was stronger than that of TCR-T group , and then started to decline.
  • the TCR gene content in the tumor tissue of the PD1(108)/CD28-TIGIT/OX40-TCR-T group began to significantly amplify from D10, and the peak of amplification occurred on D14, and the degree of amplification was stronger than that of PD1(108)/CD28- In the TCR-T group, it still maintained at a high level from D21 to D35, with the content being the highest among the three groups.
  • Enhanced receptor PD1(108)/CD28-TIGIT/OX40 has a stronger promoting effect on the proliferation and persistence of TCR-T cells in mouse tumor tissues.
  • TIGIT/OX40 The receptor-enhancing effects of TIGIT/OX40 were further tested in clinical trials in human subjects. Specifically, T cells modified with dual enhancer receptors using the TIGIT/OX40 enhancer receptor in combination with PD1(108)/CD28 (c610) were compared to T cells modified with PD1(108)/CD28 alone ( c410) effect.
  • This example uses PD1(108)/CD28-TCR-T cells and PD1(108)/CD28-TIGIT/OX40-TCR-T cells similar to those described in Examples 4-7.
  • T SCM medium X-VIVO+2.5% CTS TM immune cell SR (Thermo Fisher Scientific, A2596102) + 2U/ml memory stem cell expansion factor (Shanghai Nearshore Biotechnology Co., Ltd., GMP-1647)
  • CTS TM immune cell SR Thermo Fisher Scientific, A2596102
  • 2U/ml memory stem cell expansion factor 2U/ml memory stem cell expansion factor (Shanghai Nearshore Biotechnology Co., Ltd., GMP-1647)
  • the lentiviral transfection time is within 24 hours of cell activation
  • cryopreservation bags Quickly pack the cells added with the cryopreservation solution into cryopreservation bags (the cryopreservation bags must be marked with the patient’s name, serial number, cell details, cryopreservation time and other information), and place the cryopreservation bags into the programmed cooling box. , -80°C refrigerator for freezing;
  • the study population of c410/c610 is adult patients with relapsed or refractory CD19-positive large B-cell lymphoma who have received second-line or above systemic therapy, including diffuse large B-cell lymphoma (DLBCL) not otherwise specified (NOS). , primary mediastinal large B-cell lymphoma (PMBCL), high-grade B-cell lymphoma and follicular lymphoma-transformed DLBCL, follicular cell lymphoma, mantle cell lymphoma, etc.
  • DLBCL diffuse large B-cell lymphoma
  • PMBCL primary mediastinal large B-cell lymphoma
  • high-grade B-cell lymphoma and follicular lymphoma-transformed DLBCL follicular cell lymphoma, mantle cell lymphoma, etc.
  • the dose escalation group is divided into three groups: 1 ⁇ 10 6 /kg, 3.3 ⁇ 10 6 /kg and 10 ⁇ 10 6 /kg.
  • the dose expansion group recommends the cell dose of the expansion trial based on the safety and preliminary efficacy of the dose escalation trial. .
  • the main inclusion criteria include: age between 18 and 70 years old, no gender limit, ECOG score of 0 or 1, peripheral blood PD1-positive T cells accounting for ⁇ 18% of total T cells, basic cardiopulmonary reserve, and no severe hematology , Abnormal liver and kidney function; the exclusion criteria mainly include: combined with active central nervous system lymphoma, active central nervous system disease; combined with malignant tumors and immune system diseases; severe cardiopulmonary function impairment; clinical emergencies; recent major surgery and Other unmarketed clinical research drugs or treatments, etc.
  • PBMCs peripheral blood mononuclear cells
  • the objective response rate (ORR) of 1 subject in the c410 cell low-dose group (1 ⁇ 10 6 /kg) and 3 subjects in the medium-dose group (3.3 ⁇ 10 6 /kg) was 0% (0/ 1, 0/3).
  • a total of 6 patients were enrolled in the high-dose group (10 ⁇ 10 6 /kg).
  • the D28 complete response rate (CR) was 50% (3/6) and the W12CR was 33.3% (2/6).
  • the ORR of D28 was 83.3% (5/6) and that of W12 was 33.3% (2/6).
  • the D28CR of 2 subjects in the c610 cell low-dose group (1 ⁇ 10 6 /kg) was 100% (2/2), which was significantly better than the results of c410 cell therapy.
  • immune cells (c610) containing PD1(108)/CD28-TIGIT/OX40 dual-enhancing receptors have significantly better therapeutic effects and safety, and c610 is far more effective at low doses (1E6/kg).
  • the efficacy is far better than that of the same low-dose group of c410, and even significantly better than that of the high-dose group of c410, and its CRS risk is lower than that of each dose group of c410.
  • the results of this clinical trial illustrate that the TIGIT/OX40 enhanced receptor of the present invention has a surprising therapeutic effect on tumors when combined with the PD1(108)/CD28 enhanced receptor.

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Abstract

提供能够提高免疫细胞的活化水平及生存时间的基于TIGIT和OX40的增强受体(TIGIT/OX40)，以及经修饰以表达所述增强受体的免疫细胞；还提供经修饰以表达包含所述增强受体在内的两种不同增强受体的免疫细胞。

Description

增强受体、表达增强受体的免疫细胞及其用途 技术领域
本发明属于生物医药技术领域，具体而言涉及免疫细胞治疗领域。
背景技术
转换受体(switch receptor)是近年来细胞治疗领域备受关注的一种融合蛋白。转换受体有时也被称为转换分子(switch molecule)、转换共刺激受体(switch costimulatory receptor)、嵌合转换受体(chimeric switch recptor)，其是一种跨膜融合蛋白，其中胞外段来自能够结合靶细胞并且在天然状态下会产生免疫抑制信号的分子，如检查点分子，而胞内段来自能够产生免疫刺激信号的分子，如共刺激分子。由此一来，通过用这样的嵌合受体修饰免疫细胞，在免疫受体的胞外段与靶细胞结合之后，原先会引发的抑制信号会被嵌合受体转换成刺激信号，由此提高免疫细胞的活性。
WO2013/019615公开了多种嵌合转换受体，其胞外段来自CTLA4、PD-1或BTLA，胞内段则来自CD28或ICOS。CTLA4、PD-1或BTLA与其天然配体结合之后本来应该产生抑制信号，但嵌合转换受体胞内结构域的CD28或ICOS会将这种抑制信号转换为激活信号。该申请证明了通过将这种嵌合转换受体表达于T细胞之上，经过修饰的T细胞在暴露于表达BTLA或PD-1的配体的肿瘤细胞时，T细胞展现出正免疫应答，而不是通常所见的抑制性应答。
随着细胞治疗和免疫治疗的发展，本领域仍然需要具有更好效果的嵌合转换受体。
发明内容
本发明的发明人开发了一种新的增强受体，其相较于现有技术中的嵌合转换受体，在对免疫细胞的激活上展现出显著更强的能力，由此完成了本发明。
因此，在第一方面，本发明提供一种增强受体，其包含胞外结构域(ECD)、跨膜结构域(TM)和胞内结构域(ICD)，其中所述胞外结构域源自TIGIT的胞外结构域，所述胞内结构域源自OX40的胞内结构域。
在一个具体的实施方案中，所述TIGIT的胞外结构域为人TIGIT的胞外结 构域或其具有配体结合能力的片段或变体，其中所述配体为CD155。
第二方面，本发明提供一种免疫细胞，其经修饰而表达第一方面的增强受体。
第三方面，本发明提供一种免疫细胞，其经修饰而表达两种不同的增强受体，其中第一增强受体为本发明第一方面的增强受体。优选地，第二增强受体的胞外结构域能够特异性结合PD-L1，且第二增强受体的胞内结构域源自CD28。优选地，所述第二增强受体的胞外段为PD1或其能够特异性结合PD-L1的变体，或为抗PD-L1抗体。
第四方面，本发明提供第二方面和第三方面的免疫细胞的制备方法。
第五方面，本发明提供第二方面和第三方面的免疫细胞在治疗疾病中的用途。
第六方面，本发明提供包含第二方面或第三方面的免疫细胞的药物组合物。
第七方面，本发明提供核酸分子，其包含编码第一方面的增强受体的核苷酸序列。在进一步的实施方案中，所述核酸分子进一步包含编码第二增强受体的核苷酸序列，所述第二增强受体包含第二胞外结构域、第二跨膜结构域和第二胞内结构域，其中所述第二胞外结构域能够特异性结合不同于TIGIT的T细胞免疫检查点分子的配体，其中所述第二细胞内结构域源自介导免疫细胞激活信号的共刺激分子。
第八方面，本发明提供表达载体，其包含第七方面的核酸分子。
第九方面，本发明提供宿主细胞，其包含第七方面的核酸分子或第八方面的表达载体。
附图说明
图1A-B为实施例3中体外实验的结果，显示了使用不同的增强受体修饰肿瘤识别性T细胞之后T细胞的IL-2(A)和INFγ(B)的分泌水平。
图2为实施例4中体内实验的结果，显示了不同处理组的肿瘤体积变化，说明了增强受体对肿瘤抑制效果的影响。
图3为图2中去除了两种对照(Freezing medium和Mock-T)之后，剩余四组动物数据的放大图。
图4为实施例5中体外实验的结果，显示了使用不同的增强受体修饰肿瘤 识别性T细胞之后T细胞的IL-2的分泌水平。
图5为实施例5中体外实验的结果，显示了使用不同的增强受体修饰肿瘤识别性T细胞之后T细胞的INFγ的分泌水平。
图6为实施例5中体内实验的结果，显示了不同的处理组的肿瘤体积变化，说明了不同的增强受体对肿瘤抑制效果的影响。
图7为图6中去除了对照(Mock-T)之后，剩余四组动物数据的放大图。
图8为实施例6中体内实验的结果，显示了不同处理组的肿瘤体积变化。
图9显示了在注射后的不同时间点，用不同组的治疗细胞(TCR-T；PD1(108)/CD28-TCR-T；和PD1(108)/CD28-TIGIT/OX40-TCR-T)处理的小鼠的外周血中TCR拷贝数随时间的变化。
图10显示了在注射后的不同时间点，用不同组的治疗细胞(TCR-T；PD1(108)/CD28-TCR-T；和PD1(108)/CD28-TIGIT/OX40-TCR-T)处理的小鼠的肿瘤组织中TCR拷贝数随时间的变化。
发明详述
定义
除非另有说明，否则本文公开的一些方法的实践采用免疫学，生物化学，化学，分子生物学，微生物学，细胞生物学，基因组学和重组DNA的常规技术，这些技术在本领域的技术范围内。参见例如Sambrook和Green，Molecular Cloning：A Laboratory Manual，4th Edition(2012)；系列分子生物学当前议定书(F.M.Ausubel等编)；系列方法在酶学(Academic Press,Inc.)，PCR 2：A Practical Approach(M.J.Machersrs,B.D.Hames and G.R.Taylor编(1995))，Harlow and Lane编(1988)Antibodies,A Laboratory Manual,and Culture of Animal Cells:A Manual of Basic Technique and Specialized Applications,6th Edition(R.I.Breshney,编(2010))。
术语“约”或“近似”意指在本领域普通技术人员确定的特定值的可接受误差范围内，这将部分取决于如何测量或确定该值，即测量系统的局限性。在申请和权利要求中描述特定值的情况下，除非另有说明，否则应当假定术语“约”意味着在特定值的可接受误差范围内。
术语“增强受体”在本文的上下文中指一种重组跨膜蛋白，其包含胞外结构域(ECD)、跨膜结构域(TM)和胞内结构域(ICD)，其中所述胞外结构域来自免疫 细胞的蛋白质，并且所述蛋白质在未经修饰的免疫细胞中在与其配体结合后引发免疫细胞失活信号，其中所述胞内结构域来自介导免疫细胞激活信号的共刺激分子。本发明的增强受体可用于修饰免疫细胞。经修饰而带有增强受体的免疫细胞通过增强受体胞外结构域在对应配体的结合，产生免疫细胞激活信号，而非天然状态下所述胞外结构域与对应配体结合后的免疫细胞失活信号。在具体的实施方案中，本发明的增强受体的胞外结构域来自TIGIT，且胞内结构域来自OX40。
“TIGIT”意指带有Ig和免疫受体酪氨酸抑制基序(ITIM)结构域的T细胞免疫受体(T cell immunoreceptor with Ig and ITIM domains)。
“OX40”也称为“CD134”，是一种属于TNF受体超家族的共刺激性免疫检查点分子。
“PD-1”是指程序性死亡受体1。PD-1是一种抑制性免疫检查点分子，表达于B细胞和T细胞的表面。
“PD-L1”是PD-1的配体之一，与PD-1结合之后产生免疫抑制信号。
“CD28”是T细胞表面的一种受体，为T细胞活化提供共刺激信号。
术语“核苷酸”通常是指碱-糖-磷酸盐组合。核苷酸可包含合成核苷酸。核苷酸可包含合成的核苷酸类似物。核苷酸可以是核酸序列的单体单元(例如脱氧核糖核酸(DNA)和核糖核酸(RNA))。术语核苷酸可包括核糖核苷三磷酸腺苷三磷酸(ATP)、尿苷三磷酸(UTP)、三磷酸胞嘧啶(CTP)、三磷酸鸟苷(GTP)和脱氧核糖核苷三磷酸如dATP、dCTP、dITP、dUTP、dGTP、dTTP或其衍生物。这些衍生物可包括，例如，[αS]dATP、7-脱氮-dGTP和7-脱氮-dATP，以及赋予含有它们的核酸分子核酸酶抗性的核苷酸衍生物。本文使用的术语核苷酸可以指双脱氧核糖核苷三磷酸(ddNTP)及其衍生物。双脱氧核糖核苷三磷酸的说明性实例可包括但不限于ddATP、ddCTP、ddGTP、ddITP和ddTTP。核苷酸可以通过众所周知的技术进行未标记或可检测标记。标记也可以用量子点进行。可检测标记可包括例如放射性同位素、荧光标记、化学发光标记、生物发光标记和酶标记。
术语“基因”是指核酸(例如DNA，例如基因组DNA和cDNA)及其相应的编码RNA转录物的核苷酸序列。如本文所用，关于基因组DNA的术语包括插入的非编码区以及调节区，并且可包括5'和3'末端。在一些用途中，该术语包括转录序列，包括5'和3'非翻译区(5'-UTR和3'-UTR)，外显子和内含子。在一些基 因中，转录区域将包含编码多肽的“开放阅读框”。在该术语的一些用途中，“基因”仅包含编码多肽所必需的编码序列(例如，“开放阅读框”或“编码区”)。在一些情况下，基因不编码多肽，例如核糖体RNA基因(rRNA)和转移RNA(tRNA)基因。在一些情况下，术语“基因”不仅包括转录序列，而且还包括非转录区域，包括上游和下游调节区，增强子和启动子。基因可以指生物基因组中其天然位置中的“内源基因”或天然基因。基因可以指“外源基因”或非天然基因。非天然基因可以指通常不在宿主生物体中发现但通过基因转移引入宿主生物体的基因。非天然基因也可以指不在生物体基因组中的天然位置的基因。非天然基因还可以指天然存在的核酸或多肽序列，其包含突变，插入和/或缺失(例如，非天然序列)。
术语“多核苷酸”，“寡核苷酸”和“核酸”可互换使用，指任何长度的聚合形式的核苷酸、脱氧核糖核苷酸或核糖核苷酸，或其类似物，其可以是单股、双股或多股形式。多核苷酸对细胞可以是外源的或内源的。多核苷酸可以存在于无细胞环境中。多核苷酸可以是其基因或片段。多核苷酸可以是DNA。多核苷酸可以是RNA。多核苷酸可以具有任何三维结构，并且可以执行已知或未知的任何功能。多核苷酸可包含一种或多种类似物(例如改变的主链，糖或核碱基)。
术语“表达”是指多核苷酸从DNA模板转录(例如转录成mRNA或其他RNA转录物)和/或转录的mRNA随后翻译成肽、多肽或蛋白质的一个或多个过程。转录物和编码的多肽可统称为“基因产物”。如果多核苷酸衍生自基因组DNA，则表达可包括在真核细胞中剪接mRNA。表达的“上调”通常是指相对于其在野生型状态下的表达水平，多核苷酸(例如，RNA，例如mRNA)和/或多肽序列的表达水平增加，而“下调”通常是指相对于其在野生型状态下的表达，多核苷酸(例如，RNA，例如mRNA)和/或多肽序列的表达水平降低。
如本文所用，关于表达或活性的术语“调节”是指改变表达或活性水平。调节可以在转录水平和/或翻译水平发生。
术语“肽”、“多肽”和“蛋白质”在本文中可互换使用，是指通过肽键连接的至少两个氨基酸残基的聚合物。该术语不意味着特定长度的聚合物，也不意在暗示或区分肽是使用重组技术、化学或酶促合成产生的，或是天然存在的。该术语适用于天然存在的氨基酸聚合物以及包含至少一个修饰的氨基酸的氨基酸聚合物。在某些情况下，聚合物可被非氨基酸中断。该术语包括任何长度的氨基酸链，包括全长蛋白质，还包括具有或不具有二级和/或三级结构的蛋白质(例如，结构域)。 该术语还包括已经被修饰的氨基酸聚合物，例如，通过二硫键形成、糖基化、脂化、乙酰化、磷酸化、氧化和任何其他操作，例如与标记组分的缀合。
术语“氨基酸”通常是指天然和非天然氨基酸，包括但不限于修饰的氨基酸和氨基酸类似物。经修饰的氨基酸可包括天然氨基酸和非天然氨基酸，其已经化学修饰而包括不天然存在于氨基酸上的基团或化学部分。氨基酸类似物可以指氨基酸衍生物。术语“氨基酸”包括D-氨基酸和L-氨基酸。
当在本文中关于多肽使用时，术语“衍生物”，“变体”和“片段”是指与野生型多肽在例如氨基酸序列、结构(例如，二级和/或三级)、活性(例如，酶活性)和/或功能方面相关联的多肽。与野生型多肽相比，多肽的衍生物、变体和片段可包含一个或多个氨基酸变异(例如，突变、插入和缺失)、截短、修饰或其组合。
术语“融合物”用于本文可以指包含一个或多个非天然序列(例如，部分)的蛋白质和/或核酸。融合物可包含一种或多种相同的非天然序列。融合物可包含一种或多种不同的非天然序列。融合物可以是嵌合体。融合物可以包含核酸亲和标签。融合物可以包括条形码。融合物可包含肽亲和标签。融合物可以提供多肽的亚细胞定位(例如，用于靶向细胞核的核定位信号(NLS)，用于靶向线粒体的线粒体定位信号，用于靶向叶绿体的叶绿体定位信号，内质网(ER)保留信号等)。融合物可以提供可以用于跟踪或纯化的非天然序列(例如，亲和标签)。融合物可以包含小分子，例如生物素或染料、例如Alexa氟染料、Cyanine3染料、Cyanine5染料。
术语“新抗原”通常是指由基因突变引起的肿瘤特异性抗原。得到的突变蛋白或其片段可以引发抗肿瘤T细胞应答。
术语“遗传谱”是指关于特定基因的信息，包括个体或某种类型组织中的变异和基因表达。遗传谱可用于新抗原选择。术语“体细胞突变谱”是指关于与体细胞突变相关的特定基因的信息，包括但不限于由体细胞突变产生的特定基因。体细胞突变谱可用于新抗原选择。
术语“抗体”是指具有免疫球蛋白样功能的蛋白质结合分子。术语抗体包括抗体(例如，单克隆和多克隆抗体)，以及其衍生物、变体和片段。所述衍生物、变体或片段可以指保留相应抗体的结合特异性(例如，完整和/或部分)的功能衍生物或片段，如。抗原结合片段包括Fab、Fab'、F(ab')2、可变片段(Fv)、单链可变片段(scFv)、微抗体、双抗体和单结构域抗体(“sdAb”或“纳米抗体”或“骆驼”)。 术语抗体包括已经优化、工程化或化学缀合的抗体和抗体的抗原结合片段。已经优化的抗体的实例包括亲和力成熟的抗体。已经改造的抗体的实例包括Fc优化的抗体(例如，在片段可结晶区域中优化的抗体)和多特异性抗体(例如，双特异性抗体)。
本文所用的短语“人工TCR”，可以理解为“外源性T细胞受体(TCR)复合物”，是指TCR复合物，其中TCR的一条或多条链被引入免疫细胞的基因组中。可能会也可能不会内源性地表达TCR。在一些情况下，外源TCR复合物可以指TCR复合物，其中内源TCR复合物的一条或多条链具有一个或多个突变序列，例如在核酸或氨基酸水平。外源性TCR在免疫细胞上的表达可赋予表位或抗原(例如，优先存在于癌细胞或其他致病细胞或颗粒表面上的表位或抗原)的结合特异性。外源TCR复合物可包含引入基因组的TCR-α，TCR-β链，CD3-γ链，CD3-δ链，CD3-ζ链或其任何组合。在一些情况下，引入基因组的链可以取代内源性链。
“免疫检查点分子”是调节免疫应答的分子，其通常为一对可以对免疫应答产生抑制性或刺激性效果的受体/配体分子。
“共刺激分子”在本文中指将T细胞的初始激活信号放大或抵销的细胞表面分子。在没有特别说明的情况下，本文的“共刺激分子”为“正共刺激分子”，即产生激活信号的共刺激分子。
术语“受试者”、“个体”和“患者”在本文中可互换使用，其指脊椎动物，优选哺乳动物，例如人。哺乳动物包括但不限于鼠类、猿猴、人类、农场动物、运动动物和宠物。还包括体内获得的或体外培养的生物实体的组织，细胞及其后代。
术语“治疗”是指用于获得有益或所需结果的方法，包括但不限于治疗益处和/或预防益处。例如，治疗可包括施用本文公开的细胞、细胞群体或组合物。治疗益处是指治疗中的一种或多种疾病、病症或症状的任何治疗相关的改善或作用。就预防益处而言，可以将所述细胞、细胞群体或组合物施用于有风险发展形成特定疾病、病症或症状的受试者，或施用于报告了疾病的一种或多种生理症状的受试者，即使疾病、病症或症状可能还没有表现出来。
术语“有效量”或“治疗有效量”是指组合物的某个量在给予有需要的受试者时足以产生所需的活性，其中所述组合物例如是包含本发明的免疫细胞如淋巴细胞(例如，T淋巴细胞)的组合物。术语“治疗上有效的”是指对于有待治疗的病 症而言，组合物的量足以延迟其表现，阻止其进展，或是减轻或缓解其至少一种症状。
TIGIT/OX40增强受体
TIGIT是脊髓灰质炎病毒受体(PVR)/连蛋白家族成员，其为一种免疫受体，主要在活化的T细胞、记忆T细胞、NK细胞和某些T调节细胞(Treg)上表达。TIGIT能够结合其在抗原呈递细胞(APC)上表达的高亲和力同源配体CD155(也称为PVR)，这种结合通过T细胞和APC，以及通过NK细胞抑制免疫反应。TIGIT还被证明与CD226竞争结合CD155，从而平衡CD226介导的共刺激T细胞信号传导。因为这些功能，TIGIT成为免疫治疗中的热门靶点之一。
本发明的TIGIT/OX40增强受体包含人TIGIT的胞外结构域，或其具有配体结合能力的片段或变体，其中所述配体为CD155。人TIGIT由244个氨基酸组成，其中胞外结构域包含141个氨基酸，跨膜结构域为23个氨基酸。在具体的实施方案中，本发明的TIGIT/OX40增强受体的胞外结构域为人TIGIT的胞外结构域，例如其具有如SEQ ID NO:9所示的氨基酸序列。
本发明的TIGIT/OX40增强受体包含人OX40的胞内结构域，或其具有形成信号传导复合物功能的片段或变体。人OX40由277个氨基酸组成，其中胞质端为37个氨基酸，胞外结构域为217个氨基酸，跨膜结构域为23个氨基酸。在具体的实施方案中，本发明的TIGIT/OX40增强受体的胞内结构域源自野生型人OX40的胞内结构域，例如其具有如SEQ ID NO:10所示的氨基酸序列。
在一些实施方案中，将TIGIT作为胞外段的增强受体，当其胞内段选用源自OX40的胞内结构域时，会使T细胞具有分泌更多细胞因子如IL-2和IFNγ以及在体内的存续时间更长等特性，因此特别地优于选用源自其他共刺激分子如CD28、ICOS等的胞内结构域。
本发明的TIGIT/OX40增强受体的跨膜区可以任选地源自TIGIT或OX40，例如包含TIGIT或OX40的跨膜结构域。在一个优选的实施方案中，所述跨膜区源自OX40并且具有如SEQ ID NO:11所示的氨基酸序列。
第二增强受体
本发明的一些实施方案涉及经修饰而表达两种不同的增强受体(或称双增 强受体)的免疫细胞，其中所述第一增强受体为本发明的TIGIT/OX40增强受体，且所述第二增强受体包含第二胞外结构域、第二跨膜结构域和第二胞内结构域，其中所述第二胞外结构域能够特异性结合不同于TIGIT的T细胞其他免疫检查点分子的配体，其中所述第二胞内结构域源自介导免疫细胞激活信号的共刺激分子。
对于所述能够特异性结合不同于TIGIT的T细胞其他免疫检查点分子的配体的第二胞外结构域而言，所述T细胞其他免疫检查点分子可以选自下组：转化生长因子β受体(TGF-β-R)、程序性细胞死亡1(PD-1)、细胞毒性T淋巴细胞相关蛋白4(CTLA-4)、B和T淋巴细胞衰减因子(BTLA)、杀伤细胞免疫球蛋白样受体(KIR)、吲哚胺2,3-双加氧酶(IDO)、淋巴细胞激活基因3(LAG3)和T细胞免疫球蛋白粘蛋白3(TIM-3)。例如，所述第二胞外结构域可以是不同于TIGIT的T细胞其他免疫检查点分子的胞外结构域，或其保留了配体结合能力的片段。例如，所述第二胞外结构域可以是抗体或其片段，所述抗体或其片段特异性结合不同于TIGIT的T细胞其他免疫检查点分子的配体；例如所述抗体为单链抗体(scFv)或单域抗体(VHH)。
在优选的实施方案中，所述T细胞其他免疫检查点分子是PD-1。相应地，所述第二胞外结构域可以是PD-1或其能够结合PD-L1的变体或片段；或是抗PD-L1抗体或其能够特异性结合PD-L1的片段，如抗PD-L1的单链抗体(scFv)或抗PD-L1的单域抗体(VHH)。在一个具体的实施方案中，所述第二增强受体的胞外结构域具有如SEQ ID NO:16的氨基酸序列。在一个优选的实施方案中，所述第二增强受体的胞外结构域是PD-1的胞外结构域的变体，并且相对于野生型PD-1具有改进的与PD-L1的亲和力且不与至少一种已知的PD-1抗体结合，例如所述第二增强受体的具有如SEQ ID NO:12的氨基酸序列。
在一些实施方案中，所述第二胞内结构域所来源的共刺激分子可以是白细胞介素2受体(IL-2R)、白细胞介素12受体(IL-12R)、CD2、CD3、CD4、CD7、CD8、CD27、CD28、CD30、CD40、4-1BB/CD137、ICOS、淋巴细胞功能相关抗原1(LFA-1)、LIGHT、NKG2C或OX40。在一些实施方案中，所述免疫细胞激活信号可以由激活因子介导。在一些实施方案中，所述激活因子可以是可溶性细胞因子、可溶性趋化因子或生长因子。在一些实施方案中，所述激活因子可以是可溶性细胞因子，并且其中所述可溶性细胞因子是IL-1、IL-2、IL-6、IL-7、 IL-8、IL-10、IL-12、IL-15、IL-21、TNF、TGF、IFN或其任何功能片段或变体。在一些实施方案中，所述免疫细胞激活信号可以包含所述经修饰的免疫细胞的克隆扩增；所述经修饰的免疫细胞的细胞因子释放；所述经修饰的免疫细胞的细胞毒性；所述经修饰的免疫细胞的增殖；所述经修饰的免疫细胞的分化、去分化或转分化；所述经修饰的免疫细胞的运动和/或运输；所述经修饰的免疫细胞的耗竭和/或再激活；以及所述经修饰的免疫细胞的其他细胞间分子、代谢产物、化学化合物或其组合的释放。
在优选的实施方案中，所述共刺激分子是CD28。在一个具体的实施方案中，所述第二增强受体的胞内结构域具有如SEQ ID NO:13的氨基酸序列。
在一个具体的实施方案中，所述第二增强受体为PD1/CD28增强受体，其包含：
(a)第二胞外结构域，其源自PD-1或其能够结合PD-L1的变体或片段，例如源自PD-1的胞外结构域或其能够结合PD-L1的变体或片段，优选具有如SEQ ID NO:16或SEQ ID NO:13所示的氨基酸序列；
(b)第二跨膜结构域，其源自PD-1或CD28，例如源自PD-1或CD28的跨膜结构域，优选源自CD28的跨膜结构域，例如具有如SEQ ID NO:14所示的氨基酸序列；和
(c)第二胞内结构域，其源自CD28，例如源自CD28的胞内结构域，例如具有如SEQ ID NO:13所示的氨基酸序列。
在带有两种不同的增强受体修饰的情况下，能够将不同的免疫检查点分子介导的免疫抑制信号转换为激活信号。如此一来，相对于仅带有一种增强受体的修饰的免疫细胞而言，双增强受体修饰的免疫细胞能够展现出更进一步的激活和免疫应答。例如，发明人发现，使用带有TIGIT/OX40增强受体和PD-1(108)/CD28增强受体修饰的免疫细胞能够在抑制肿瘤方面产生协同作用，其效果显著优于使用单独仅带有其中一种增强受体修饰的免疫细胞。因此，本发明还涉及使用本发明的TIGIT/OX40增强受体来提高治疗性免疫细胞的效果，如免疫激活和免疫应答效果的方法，其中所述治疗性免疫细胞可以具有TIGIT/OX40增强受体之外的修饰，如本发明的第二增强受体和/或嵌合抗原受体。
在包含第二增强受体的实施方案中，可以将编码TIGIT/OX40增强受体的核苷酸序列和编码第二增强受体的核苷酸序列单独或以组合的方式引入免疫细 胞中以进行表达。例如，可以将编码两种增强受体的核苷酸序列用自切割肽，如2A肽的编码序列连接。所述2A肽包括但不限于T2A、P2A、E2A、F2A等。
免疫细胞
本发明的增强受体用于修饰免疫细胞，特别是淋巴细胞，如T细胞，以增强所述免疫细胞的免疫活化效果。
在进行修饰之前，特别是在对所述免疫细胞进行修饰以表达本发明的增强受体之前，所述免疫细胞可以来自多种来源，且通过多种方法中的任意一种或多种来制备。
本发明可以使用的免疫细胞包括外周血单核细胞(PBMC)、外周血淋巴细胞(PBL)和其他血细胞亚组(例如但不限于T细胞、自然杀伤细胞、单核细胞、自然杀伤T细胞、单核细胞前体细胞、造血干细胞或非多能干细胞)。在一些情况下，细胞可以是任何免疫细胞，包括任何T细胞，如肿瘤浸润性细胞(TIL)、CD3+T细胞、CD4+T细胞或CD8+T细胞。T细胞还可以包括记忆T细胞、记忆干T细胞或效应T细胞。
免疫细胞可以是从受试者获得的T细胞、NK细胞、B细胞等的群体。T细胞可以从许多来源获得，所述来源包括PBMC、骨髓、淋巴结组织、脐带血、胸腺组织、以及来自感染部位的组织、腹水、胸腔积液、脾脏组织和肿瘤。在一些实施方案中，可以使用多种技术(如FicollTM分离)从自受试者收集的血液单位获得T细胞。在一个实施方案中，通过单采术获得来自个体循环血液的细胞。单采术产物通常含有淋巴细胞，包括T细胞、单核细胞、粒细胞、B细胞、其他有核白细胞、红细胞和血小板。可以将通过单采术收集的细胞洗涤，以去除血浆部分并将细胞置于适当的缓冲液或介质中进行后续处理步骤。
可以从混合群体(bulk population)中选择T细胞，例如从全血中选择T细胞。可以从混合群体中扩增T细胞。在选择和扩增过程中，可以使T细胞向特定群体和表型倾斜。例如，可以使T细胞倾斜以在表型上包含CD45RO(-)、CCR7(+)、CD45RA(+)、CD62L(+)、CD27(+)、CD28(+)和/或IL-7Rα(+)。可以选择合适的细胞，所述细胞包含选自包含以下的列表的一种或多种标记：CD45RO(-)、CCR7(+)、CD45RA(+)、CD62L(+)、CD27(+)、CD28(+)和/或IL-7Rα(+)。
也可以使用干细胞通过分化获得免疫细胞。所述干细胞如胚胎干细胞、诱 导多能干细胞、造血干细胞、神经元干细胞和间充质干细胞。免疫细胞可以包含多种原代细胞，如人细胞、非人细胞和/或小鼠细胞。
免疫细胞可以源自待治疗的受试者(例如，患者)。免疫细胞可以源自健康的人供体。免疫细胞可以源自于受试者有直系亲缘关系的供体，如与受试者HLA半相合的供体。
优选地，所述免疫细胞对于靶细胞，如肿瘤细胞，具有识别特异性。这种识别特异性的赋予可以通过各种方法来实现，包括但不限于遗传修饰、通过与加载了靶细胞抗原的抗原呈递细胞共同孵育和/或分选特定细胞群体来实现。
在一个实施方案中，本发明的免疫细胞可以是肿瘤浸润淋巴细胞(TIL)。在一些实施方案中，所述TIL可以表达PD-1、CD137和TIM-3中的至少一种。
在一些实施方案中，所述T细胞可以包含展现出与新抗原的特异性结合的T细胞受体(TCR)复合物。在一些实施方案中，所述TCR复合物可以是内源TCR复合物。在一些实施方案中，所述TCR复合物可以是外源TCR复合物。
在一些实施方案中，可以基于来自个体的肿瘤样品的遗传谱选择所述新抗原。在一些实施方案中，可以基于来自个体的肿瘤样品的体细胞突变谱选择所述新抗原。
在一种特别的实施方案中，本发明的免疫细胞源自外周血单个核细胞(PBMC)，并且经过分选以富集PBMC中的PD1+T细胞。“富集”意味着经过处理使某种细胞或成分在某个群体中的数量或占比得到提高。由于在实际的实验室和临床操作中，细胞制备、分选或修饰的过程往往都是针对包含多个细胞的样品(细胞群体)进行的，这样的富集操作意味着在进行表达增强受体的修饰之前，优选用于本发明中进行进一步修饰的所述免疫细胞或免疫细胞群体相较于未进行所述富集的PBMC群体而言，含有更高比例的PD1+T细胞。例如，对于晚期实体瘤患者而言，外周血PD1+T细胞的比例平均在28％左右。在作为本发明的免疫细胞以供修饰时，优选通过富集例如分选过程进一步提高这个比例。在具体的实施方案中，所述源自外周血PBMC且经过PD1+分选的细胞群体中，PD1+细胞的比例大于50％，优选大于60％，更优选大于70％，特别优选大于80％。
通过上述方法和来源获得的任何一种免疫细胞，在进行了修饰，特别是表达本发明的增强受体的修饰之后，被称作“经修饰的免疫细胞”。
其他修饰
本发明的经修饰的免疫细胞除了表达增强受体之外，还可以具有一种或多种其他修饰，以赋予进一步的功能和/或性质。
本发明的免疫细胞可以经修饰而表达嵌合抗原受体(CAR)，所述嵌合抗原受体包含(i)能够结合B细胞表面蛋白的抗原相互作用结构域；(ii)跨膜结构域；和(iii)细胞内信号传导结构域。带有所述CAR的免疫细胞在与B细胞表面蛋白接触之后，能够实现对所述免疫细胞的扩增和/或激活。
在一些实施方案中，B细胞表面蛋白可以是可在B细胞表面上发现的任何蛋白质。非限制性例子包括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和CD360。在一些实施方案中，CAR的抗原相互作用结构域能够结合在非B细胞上的表面蛋白，只要与该表面蛋白的结合不显著损害宿主的总体健康状态或免疫系统。在一些实施方案中，所述表面蛋白是免疫细胞上的表面蛋白。在一些实施方案中，所述表面蛋白是在除免疫细胞以外的细胞上的表面蛋白。在一些实施方案中，所述表面蛋白可以选自(条件是与该表面蛋白的结合不显著损害宿主的总体健康状态或免疫系统)CD31、CD32A、CD32B、CD33、CD34、CD35、CD36、CD37、CD38、CD39、CD40、CD41、CD42a、CD42b、CD42c、CD42d、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(a-c)、1A、1D、1E、CD2、CD3(γ、δ、ε)、CD4、CD5、CD6、CD7、CD8、a、CD9、CD10、CD11(a、b、c、d)、CD13、CD14、CD15、CD16A、CD16B、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、CD208、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和CD186。在优选的实施方案中，好所述B细胞表面蛋白选自CD19、CD20和CD22。
在一些实施方案中，CAR的抗原相互作用结构域能够结合在死的B细胞上的B细胞表面蛋白或其片段。B细胞凋亡可以发生在免疫应答(例如，针对肿瘤细胞的免疫应答)产生之前或之后。因此，死的B细胞或其碎片仍然可以具有在表面上呈现的B细胞表面蛋白或其片段。CAR靶向活的和死的B细胞的能力可能增加包含CAR的免疫细胞(i)结合B细胞表面蛋白和(i)启动细胞内信号传导结构域的信号传导的机会。在一些情况下，细胞内信号传导结构域的信号传导可能促进包含CAR的免疫细胞的扩增(增殖)。
在一些实施方案中，CAR的抗原相互作用结构域能够结合B细胞表面蛋白或其片段，所述B细胞表面蛋白或其片段(例如，经由共价和/或非共价键)偶联至颗粒(例如，纳米颗粒)表面。颗粒可以是包含有机和/或无机材料的任何颗粒材料。颗粒可以具有各种形状和尺寸。颗粒在至少一个维度上可以为约1纳米(nm)至约50微米(μm)。颗粒在至少一个维度上可以为至少约1nm、5nm、10nm、50nm、100nm、500nm、1μm、5μm、10μm、50μm或更大。颗粒在至少一个维度上可以为至多约50μm、10μm、5μm、1μm、500nm、100nm、50nm、10nm、5nm、1nm或更小。颗粒可以是纳米颗粒、微粒、纳米球、微球、纳米棒、微棒、纳米纤维、纳米带等。颗粒的例子包括金属纳米颗粒(例如，金纳米颗粒、银纳米颗粒和铁纳米颗粒)、金属间纳米半导体纳米颗粒、核壳纳米颗粒、具有无机核与聚合物壳的颗粒、具有有机核与聚合物壳的颗粒、及其混合物。可替代地，颗粒可以是有机纳米颗粒，如交联聚合物、水凝胶聚合物、可生物降解聚合物、聚丙交酯(PLA)、聚乙交酯(PGA)、聚己内酯(PCL)、共聚物、多糖、淀粉、纤维素、壳聚糖、聚羟基链烷酸酯(PHA)、PHB、PHV、脂质、肽、肽两亲物、多肽(例如，蛋白质)或其组合。可以将在表面上呈现B细胞表面蛋白的颗粒体外引入包含结合B细胞表面蛋白的CAR的免疫细胞中。可替代地或另外地，可以与包含CAR的免疫细胞一起体内引入(例如，局部或系统注射)呈现B细胞表面蛋白的颗粒。可以将此类颗粒用于在体外或在体内扩增包含CAR的免疫细胞群。
抗原结合结构域可以包含能够结合抗原(例如，B细胞表面蛋白)的任何蛋白质或分子。抗原结合结构域的非限制性例子包括但不限于单克隆抗体、多克隆抗体、重组抗体、人抗体、人源化抗体、鼠抗体或其功能衍生物、变体或片段，所述功能衍生物、变体或片段包括但不限于Fab、Fab'、F(ab')2、Fv、单链Fv(scFv)、小抗体、双抗体和单结构域抗体(如骆驼衍生纳米抗体的重链可变结构域(VH)、轻链可变结构域(VL)和可变结构域(VHH))。在一些实施方案中，第一抗原结合结构域包含Fab、Fab'、F(ab’)2、Fv和scFv中的至少一种。在一些实施方案中，抗原结合结构域包含抗体模拟物。抗体模拟物是指能以与抗体相当的亲和力结合靶分子的分子，并且包括单链结合分子、基于细胞色素b562的结合分子、纤连蛋白或纤连蛋白样蛋白支架(例如，adnectin)、脂质运载蛋白支架、杯芳烃支架、A结构域和其他支架。在一些实施方案中，抗原结合结构域包含跨膜受体或其任何衍生物、变体或片段。例如，抗原结合结构域可以包含跨膜受体的至少一个配体 结合结构域。
在一些实施方案中，抗原结合结构域可以包含scFV。scFv可以源自其可变区序列已知的抗体。在一些实施方案中，scFv可以源自从可用的小鼠杂交瘤获得的抗体序列。可以从肿瘤细胞或原代细胞的全外显子测序获得scFv。在一些实施方案中，scFv可以被改变。例如，能以多种方式修饰scFv。在一些情况下，可以将scFv突变，使得scFv可以对其靶标具有更高的亲和力。在一些情况下，可以针对在正常组织上以低水平表达的靶标来优化scFv对其靶标的亲和力。可以进行此优化以使潜在的毒性(如高细胞因子血症)最小化。在其他情况下，对靶标的膜结合形式具有更高亲和力的scFv的克隆可能优于其可溶性形式对应物。如果还可以在不同水平上以可溶形式检测到一些靶标，且其靶向可能引起不想要的毒性(如高细胞因子血症)，则可以进行此修饰。
可以将CAR的抗原结合结构域经由跨膜结构域连接至细胞内信号传导结构域。所述CAR的跨膜结构域可以将CAR锚定至细胞(例如免疫细胞)的质膜。在一些实施方案中，跨膜区段包含多肽。连接CAR的抗原结合结构域和细胞内信号传导结构域的跨膜多肽可以具有任何合适的多肽序列。在一些情况下，跨膜多肽包含内源或野生型跨膜蛋白的跨膜部分的多肽序列。在一些实施方案中，跨膜多肽包含与内源或野生型跨膜蛋白的跨膜部分相比具有至少1个(例如，至少2个、3个、4个、5个、6个、7个、8个、9个、10个或更多)氨基酸取代、缺失和插入的多肽序列。在一些实施方案中，跨膜多肽包含非天然多肽序列，如多肽接头的序列。多肽接头可以是柔性的或刚性的。多肽接头可以是结构化的或非结构化的。在一些实施方案中，跨膜多肽例如经由抗原结合结构域将信号从细胞的细胞外区域传递至细胞内区域。可以在CAR中使用CD28的天然跨膜部分。在其他情况下，还可以在CAR中使用CD8α的天然跨膜部分。
本公开文本的CAR可以包含参与免疫细胞信号传导的信号传导结构域或其任何衍生物、变体或片段。CAR的细胞内信号传导结构域可以诱导包含CAR的免疫细胞的活性。细胞内信号传导结构域可以转导效应子功能信号并指导细胞执行专门功能。信号传导结构域可以包含其他分子的信号传导结构域。在一些情况下，在CAR中使用信号传导结构域的截短部分。
在一些实施方案中，细胞内信号传导结构域包含参与免疫细胞信号传导的多个信号传导结构域或其任何衍生物、变体或片段。例如，细胞内信号传导结构 域可以包含至少2个免疫细胞信号传导结构域，例如至少2个、3个、4个、5个、7个、8个、9个或10个免疫细胞信号传导结构域。免疫细胞信号传导结构域能以刺激方式或抑制方式参与调节TCR复合物的初级激活。细胞内信号传导结构域可以是T细胞受体(TCR)复合物的细胞内信号传导结构域。本发明的CAR的细胞内信号传导结构域可以包含以下的信号传导结构域：Fcγ受体(FcγR)、Fcε受体(FcεR)、Fcα受体(FcαR)、新生儿Fc受体(FcRn)、CD3、CD3ζ、CD3γ、CD3δ、CD3ε、CD4、CD5、CD8、CD21、CD22、CD28、CD32、CD40L(CD154)、CD45、CD66d、CD79a、CD79b、CD80、CD86、CD278(也称为ICOS)、CD247ζ、CD247η、DAP10、DAP12、FYN、LAT、Lck、MAPK、MHC复合物、NFAT、NF-κB、PLC-γ、iC3b、C3dg、C3d和Zap70。在一些实施方案中，信号传导结构域包括免疫受体酪氨酸基激活基序或ITAM。包含ITAM的信号传导结构域可以包含被6-8个氨基酸分开的氨基酸序列YxxL/I的两个重复，其中每个x独立地是任何氨基酸，从而产生保守基序YxxL/Ix(6-8)YxxL/I。当抗原结合结构域与表位结合时，可以例如通过磷酸化来修饰包含ITAM的信号传导结构域。磷酸化的ITAM可以作为其他蛋白质(例如参与各种信号传导途径的蛋白质)的停泊位点。在一些实施方案中，初级信号传导结构域包含经修饰的ITAM结构域(例如，突变的、截短的和/或优化的ITAM结构域)，所述经修饰的ITAM结构域与天然ITAM结构域相比具有改变的(例如，增加的或减少的)活性。
在一些实施方案中，本发明的CAR的细胞内信号传导结构域包含FcγR信号传导结构域(例如，ITAM)。FcγR信号传导结构域可以选自FcγRI(CD64)、FcγRIIA(CD32)、FcγRIIB(CD32)、FcγRIIIA(CD16a)和FcγRIIIB(CD16b)。在一些实施方案中，细胞内信号传导结构域包含FcεR信号传导结构域(例如，ITAM)。FcεR信号传导结构域可以选自FcεRI和FcεRII(CD23)。在一些实施方案中，细胞内信号传导结构域包含FcαR信号传导结构域(例如，ITAM)。FcαR信号传导结构域可以选自FcαRI(CD89)和Fcα/μR。在一些实施方案中，细胞内信号传导结构域包含CD3ζ信号传导结构域。在一些实施方案中，初级信号传导结构域包含CD3ζ的ITAM。
在一些实施方案中，本发明的CAR的细胞内信号传导结构域包含免疫受体酪氨酸基抑制基序或ITIM。包含ITIM的信号传导结构域可以包含在免疫系统的一些抑制受体的胞质尾中发现的氨基酸保守序列(S/I/V/LxYxxI/V/L)。可以通过 诸如Src激酶家族成员(例如，Lck)等酶来修饰包含ITIM的初级信号传导结构域，例如使其磷酸化。磷酸化后，可以将其他蛋白质(包括酶)募集到ITIM。这些其他蛋白质包括但不限于酶，如磷酸酪氨酸磷酸酶SHP-1和SHP-2、称为SHIP的肌醇磷酸酶、以及具有一个或多个SH2结构域的蛋白质(例如，ZAP70)。细胞内信号传导结构域可以包含以下的信号传导结构域(例如，ITIM)：BTLA、CD5、CD31、CD66a、CD72、CMRF35H、DCIR、EPO-R、FcγRIIB(CD32)、Fc受体样蛋白2(FCRL2)、Fc受体样蛋白3(FCRL3)、Fc受体样蛋白4(FCRL4)、Fc受体样蛋白5(FCRL5)、Fc受体样蛋白6(FCRL6)、蛋白G6b(G6B)、白细胞介素4受体(IL4R)、免疫球蛋白超家族受体转运相关蛋白1(IRTA1)、免疫球蛋白超家族受体转运相关蛋白2(IRTA2)、杀伤细胞免疫球蛋白样受体2DL1(KIR2DL1)、杀伤细胞免疫球蛋白样受体2DL2(KIR2DL2)、杀伤细胞免疫球蛋白样受体2DL3(KIR2DL3)、杀伤细胞免疫球蛋白样受体2DL4(KIR2DL4)、杀伤细胞免疫球蛋白样受体2DL5(KIR2DL5)、杀伤细胞免疫球蛋白样受体3DL1(KIR3DL1)、杀伤细胞免疫球蛋白样受体3DL2(KIR3DL2)、白细胞免疫球蛋白样受体亚家族B成员1(LIR1)、白细胞免疫球蛋白样受体亚家族B成员2(LIR2)、白细胞免疫球蛋白样受体亚家族B成员3(LIR3)、白细胞免疫球蛋白样受体亚家族B成员5(LIR5)、白细胞免疫球蛋白样受体亚家族B成员8(LIR8)、白细胞相关免疫球蛋白样受体1(LAIR-1)、肥大细胞功能相关抗原(MAFA)、NKG2A、天然细胞毒性触发受体2(NKp44)、NTB-A、程序性细胞死亡蛋白1(PD-1)、PILR、SIGLECL1、唾液酸结合Ig样凝集素2(SIGLEC2或CD22)、唾液酸结合Ig样凝集素3(SIGLEC3或CD33)、唾液酸结合Ig样凝集素5(SIGLEC5或CD170)、唾液酸结合Ig样凝集素6(SIGLEC6)、唾液酸结合Ig样凝集素7(SIGLEC7)、唾液酸结合Ig样凝集素10(SIGLEC10)、唾液酸结合Ig样凝集素11(SIGLEC11)、唾液酸结合Ig样凝集素4(SIGLEC4)、唾液酸结合Ig样凝集素8(SIGLEC8)、唾液酸结合Ig样凝集素9(SIGLEC9)、血小板和内皮细胞粘附分子1(PECAM-1)、信号调节蛋白(SIRP 2)和信号传导阈值调节跨膜衔接因子1(SIT)。在一些实施方案中，细胞内信号传导结构域包含经修饰的ITIM结构域(例如，突变的、截短的和/或优化的ITIM结构域)，所述经修饰的ITIM结构域与天然ITIM结构域相比具有改变的(例如，增加的或减少的)活性。
在一些实施方案中，细胞内信号传导结构域包含至少2个ITAM结构域(例如，至少3个、4个、5个、6个、7个、8个、9个或10个ITAM结构域)。在一些实施方 案中，细胞内信号传导结构域包含至少2个ITIM结构域(例如，至少3个、4个、5个、6个、7个、8个、9个或10个ITIM结构域)(例如，至少2个初级信号传导结构域)。在一些实施方案中，细胞内信号传导结构域包含ITAM结构域和ITIM结构域两者。
在一些情况下，本发明的CAR的细胞内信号传导结构域可以包括共刺激结构域。在一些实施方案中，共刺激结构域(例如来自共刺激分子)可以提供共刺激信号用于免疫细胞信号传导，如来自ITAM和/或ITIM结构域的信号传导，例如用于免疫细胞活性的激活和/或失活。在一些实施方案中，共刺激结构域是可操作的以调节免疫细胞中的增殖和/或存活信号。在一些实施方案中，共刺激信号传导结构域包含以下的信号传导结构域：MHC I类蛋白、MHC II类蛋白、TNF受体蛋白、免疫球蛋白样蛋白、细胞因子受体、整合素、信号传导淋巴细胞激活分子(SLAM蛋白)、激活NK细胞受体、BTLA或Toll配体受体。在一些实施方案中，共刺激结构域包含选自以下的分子的信号传导结构域：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配体/TNFSF7、CD27/TNFRSF7、CD28、CD29、CD2F-10/SLAMF9、CD30配体/TNFSF8、CD30/TNFRSF8、CD300a/LMIR1、CD4、CD40配体/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配体/TNFSF18、GITR/TNFRSF18、HLA I类、HLA-DR、HVEM/TNFRSF14、IA4、ICAM-1、ICOS/CD278、Ikaros、IL2Rβ、IL2Rγ、IL7Rα、整合素α4/CD49d、整合素α4β1、整合素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)、淋巴细胞功能相关抗原1(LFA-1)、淋巴毒素-α/TNF-β、NKG2C、NKG2D、NKp30、NKp44、NKp46、NKp80(KLRF1)、NTB-A/SLAMF6、OX40 配体/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和VLA-6。在一些实施方案中，细胞内信号传导结构域包含多个共刺激结构域，例如至少两个(例如，至少3个、4个或5个)共刺激结构域。共刺激信号传导区域可以提供与初级效应子激活信号协同的信号，并且可以完成T细胞激活的要求。在一些实施方案中，向CAR添加共刺激结构域可以增强本文提供的免疫细胞的功效和持久性。
与缺少CAR的免疫细胞相比，CAR与B细胞表面蛋白的结合可以增强免疫细胞的增殖。免疫细胞的增殖可以是指免疫细胞的扩增。免疫细胞的增殖可以是指免疫细胞的表型变化。本文提供的包含CAR的免疫细胞的增殖可以比缺少展现出与B细胞表面蛋白的结合的CAR的可比较免疫细胞的增殖更多。包含CAR的免疫细胞的增殖可以比缺少CAR的可比较免疫细胞的增殖多了约5倍至约10倍、约10倍至约20倍、约20倍至约30倍、约30倍至约40倍、约40倍至约50倍、约50倍至约60倍、约60倍至约70倍、约70倍至约80倍、约80倍至约90倍、约90倍至约100倍、约100倍至约200倍、从约200倍至约300倍、从约300倍至约400倍、从约400倍至约500倍、从约500倍至约600倍、从约600倍至约700倍。包含CAR的免疫细胞的增殖可以比缺少CAR的可比较免疫细胞的增殖多了约5倍至约10倍、约10倍至约20倍、约20倍至约30倍、约30倍至约40倍、约40倍至约50倍、约50倍至约60倍、约60倍至约70倍、约70倍至约80倍、约80倍至约90倍、约90倍至约100倍、约100倍至约200倍、从约200倍至约300倍、从约300倍至约400倍、从约400倍至约500倍、从约500倍至约600倍、从约600倍至约700倍，并且其中在B细胞与B细胞表面蛋白接触之后至少约12、24、36、48、60、72、84或96小时确定增殖。可以在体外或在体内确定增强的增殖。在一些实施方案中，增殖可以包括定量免疫细胞的数量。定量免疫细胞的数量可以包括流式细胞术、台盼蓝排除和/或血细胞计数。也可以通过免疫细胞的表型分析来确定增殖。
值得注意的是，本发明进行的CD19 CAR修饰的目的在于通过结合B细胞来辅助免疫细胞如T细胞的扩增和激活，这与常见的CD19 CAR-T技术中为T 细胞加载CD19 CAR的目的和功能不完全相同，其中最重要的区别在于本发明的经修饰的免疫细胞不局限于治疗与表达CD19的B细胞相关的肿瘤，因为CD19 CAR在本发明中的作用不是靶细胞的识别和结合。
制备方法
可以使用任何合适的递送方法将组合物和分子(例如，本发明的增强受体多肽和/或编码该多肽的核酸)引入宿主细胞(如免疫细胞)中。可以将各种组分同时或分开递送。方法的选择可能取决于待转化的细胞的类型和发生转化的环境(例如，体外、离体或体内)。
递送方法可包括将包含编码本发明的增强受体或其他修饰分子如嵌合抗原受体的核苷酸序列的一种或多种核酸引入细胞或细胞群体中。包含编码所述核苷酸序列的合适核酸可以包括表达载体，其中包含编码本发明的一种或多种增强受体或其他修饰分子的核苷酸序列的表达载体是重组表达载体。
递送方法或转化的非限制性例子包括例如病毒或噬菌体感染、转染、缀合、原生质体融合、脂转染、电穿孔、磷酸钙沉淀、聚乙烯亚胺(PEI)介导的转染、DEAE葡聚糖介导的转染、脂质体介导的转染、粒子枪技术、磷酸钙沉淀、直接显微注射和纳米颗粒介导的核酸递送。
在一些方面，本发明提供一种方法，所述方法包括将一种或多种多核苷酸或如本文所述的一种或多种载体或其一种或多种转录物和/或从其转录的一种或多种蛋白质递送至宿主细胞。在一些方面，本发明进一步提供了通过此类方法产生的细胞，以及包含此类细胞或由此类细胞产生的生物体(如动物、植物或真菌)。
可以将常规的基于病毒和非病毒的基因转移方法用于将核酸引入哺乳动物细胞或靶组织中。可以将此类方法用于将编码本发明的组合物的核酸给予培养中或宿主生物体中的细胞。非病毒载体递送系统可以包括DNA质粒、RNA(例如本文所述的载体的转录物)、裸核酸和与递送媒介物(如脂质体)复合的核酸。病毒载体递送系统包括DNA和RNA病毒，其可以在递送至细胞之后具有游离型或整合的基因组。
核酸的非病毒递送方法可以包括脂转染、核转染、显微注射、基因枪、病毒体、脂质体、免疫脂质体、聚阳离子或脂质：核酸缀合物、裸DNA、人工病毒粒子和DNA的药剂增强摄取。可以使用适合用于多核苷酸的有效受体识别脂转染 的阳离子和中性脂质。递送可以是至细胞(例如体外或离体给予)或靶组织(例如体内给予)。可以使用脂质:核酸复合物的制剂，包括靶向脂质体，如免疫脂质复合物。
可以将基于RNA或DNA病毒的系统用于靶向体内的特定细胞，并且将病毒有效载荷运输到细胞核。可以直接(体内)给予病毒载体，或者可以将它们用于体外处理细胞，并且可以任选地(离体)给予经修饰的细胞。基于病毒的系统可以包括逆转录病毒、慢病毒、腺病毒、腺相关病毒和单纯疱疹病毒载体，用于基因转移。可以在使用逆转录病毒、慢病毒和腺相关病毒基因转移方法的情况下发生在宿主基因组中整合，这可能导致所插入转基因的长期表达。可以在许多不同的细胞类型和靶组织中观察到高转导效率。
逆转录病毒的向性可以通过掺入外来包膜蛋白来改变，从而扩大靶细胞的潜在靶群体。慢病毒载体是可以转导或感染非分裂细胞并产生高病毒滴度的逆转录病毒载体。逆转录病毒基因转移系统的选择可能取决于靶组织。逆转录病毒载体可以包含顺式作用长末端重复序列，其具有对多达6-10kb的外来序列的包装能力。最小顺式作用LTR对载体的复制和包装而言可能是足够的，可以将所述载体用于将治疗性基因整合至靶细胞中以提供持久的转基因表达。逆转录病毒载体可以包括基于以下的那些：鼠白血病病毒(MuLV)、长臂猿白血病病毒(GaLV)、猿猴免疫缺陷病毒(SIV)、人免疫缺陷病毒(HIV)及其组合。
可以使用基于腺病毒的系统。基于腺病毒的系统可以导致转基因的瞬时表达。基于腺病毒的载体可以在细胞中具有高转导效率，并且可能不需要细胞分裂。可以使用基于腺病毒的载体来获得高滴度和表达水平。可以将腺相关病毒(“AAV”)载体用于例如在核酸和肽的体外产生中用靶核酸转导细胞，以及用于体内和离体基因疗法程序。
可以将包装细胞用于形成能够感染宿主细胞的病毒颗粒。此类细胞可以包括293细胞(例如，用于包装腺病毒)、以及Psi2细胞或PA317细胞(例如，用于包装逆转录病毒)。病毒载体可以通过产生将核酸载体包装到病毒颗粒中的细胞系来产生。载体可以含有包装和随后整合到宿主中所需的最小病毒序列。载体可以含有待被待表达的一种或多种多核苷酸的表达盒替代的其他病毒序列。失去的病毒功能可以由包装细胞系反式供应。例如，AAV载体可以包含包装和整合到宿主基因组中所需的来自AAV基因组的ITR序列。可以将病毒DNA包装于细胞系中， 所述细胞系可以含有编码其他AAV基因(即，rep和cap)而缺少ITR序列的辅助质粒。细胞系还可以被作为辅助者的腺病毒感染。辅助病毒可以促进AAV载体的复制和来自辅助质粒的AAV基因的表达。腺病毒的污染可以通过例如热处理来减少，腺病毒对所述热处理比AAV更敏感。可以使用用于将核酸递送至细胞的另外方法，例如如US 20030087817(通过引用并入本文)中所述。
可以将宿主细胞用本文所述的一种或多种载体瞬时或非瞬时地转染。细胞由于其天然存在于受试者内而可以被转染。细胞可以取自或源自受试者并被转染。细胞可以源自从受试者取得的细胞，如细胞系。在一些实施方案中，将用本文所述的一种或多种载体转染的细胞用于建立包含一个或多个载体衍生序列的新细胞系。在一些实施方案中，将用本发明的组合物瞬时转染(例如通过一种或多种载体的瞬时转染，或用RNA的转染)的细胞用于建立新细胞系，所述新细胞系包含含有修饰但缺少任何其他外源序列的细胞。
可以将与宿主细胞相容的任何合适的载体与本发明的方法一起使用。用于真核宿主细胞的载体的非限制性例子包括pXT1、pSG5(StratageneTM)、pSVK3、pBPV、pMSG和pSVLSV40(PharmaciaTM)。
使细胞与的组合物接触可以在任何培养基中和在促进细胞存活的任何培养条件下发生。例如，可以将细胞悬浮于任何适当的方便的营养培养基中，如伊斯科夫(Iscove's)改良的DMEM或RPMI 1640，其补充有胎牛血清或热灭活的山羊血清(约5％-10％)、L-谷氨酰胺、硫醇(特别是2-巯基乙醇)和抗生素(例如青霉素和链霉素)。培养物可以含有细胞对其有反应的生长因子。如本文所定义，生长因子是能够通过对跨膜受体的特异性作用而在培养中或在完整组织中促进细胞存活、生长和/或分化的分子。生长因子可以包括多肽和非多肽因子。
在许多实施方案中，所选的递送系统被靶向特定的组织或细胞类型。在一些情况下，通过将递送系统与组织或细胞特异性标记(如细胞表面蛋白)结合来实现递送系统的组织或细胞靶向。可以定制病毒和非病毒递送系统，以靶向感兴趣的组织或细胞类型。
在制备用于治疗用途的修饰的免疫细胞群体时，优选其中带有期望的修饰，如带有TIGIT/OX40的细胞的比例超过50％，如达到约60％，约70％，约80％，甚至约90％或更高的水平。需要理解的是，本发明的修饰的免疫细胞以及相关的治疗方法是一种基于个体患者的个性化治疗策略，即使通过类似的方式进行制备， 也会因为个体化差异使得最终的细胞产品中有效细胞的比例有所不同。
用途
使用本发明的增强受体修饰的免疫细胞可以杀伤多种靶细胞。可以应用此方法的靶细胞包括各种细胞类型。靶细胞可以在体外。靶细胞可以在体内。靶细胞可以是离体的。靶细胞可以是分离的细胞。靶细胞可以是生物体内的细胞。靶细胞可以是生物体。靶细胞可以是细胞培养物中的细胞。靶细胞可以是细胞集合中的一个细胞。靶细胞可以是哺乳动物细胞或源自哺乳动物细胞。靶细胞可以是啮齿动物细胞或源自啮齿动物细胞。靶细胞可以是人细胞或源自人细胞。靶细胞可以是原核细胞或源自原核细胞。靶细胞可以是细菌细胞或可以源自细菌细胞。靶细胞可以是古细菌细胞或源自古细菌细胞。靶细胞可以是真核细胞或源自真核细胞。靶细胞可以是多能干细胞。靶细胞可以是植物细胞或源自植物细胞。靶细胞可以是动物细胞或源自动物细胞。靶细胞可以是无脊椎动物细胞或源自无脊椎动物细胞。靶细胞可以是脊椎动物细胞或源自脊椎动物细胞。靶细胞可以是微生物细胞或源自微生物细胞。靶细胞可以是真菌细胞或源自真菌细胞。靶细胞可以来自特定的器官或组织。
靶细胞可以是干细胞或祖细胞。靶细胞可以包括干细胞(例如，成年干细胞、胚胎干细胞、诱导多能干(iPS)细胞)和祖细胞(例如，心脏祖细胞、神经祖细胞等)。靶细胞可以包括哺乳动物干细胞和祖细胞，包括啮齿动物干细胞、啮齿动物祖细胞、人干细胞、人祖细胞等。克隆细胞可以包含细胞的后代。靶细胞可以包含靶核酸。靶细胞可以是在活的生物体中。靶细胞可以是基因修饰的细胞。靶细胞可以是宿主细胞。
靶细胞可以是原代细胞。例如，可以将原代细胞的培养物传代0次、1次、2次、4次、5次、10次、15次或更多次。细胞可以是单细胞生物体。细胞可以在培养中生长。
靶细胞可以是患病细胞。患病细胞可能具有改变的代谢、基因表达和/或形态特征。患病细胞可以是癌细胞、糖尿病细胞和凋亡细胞。患病细胞可以是来自患病受试者的细胞。示例性疾病可以包括血液障碍、癌症、代谢障碍、眼睛障碍、器官障碍、肌肉骨骼障碍、心脏病等。
特别感兴趣的是癌细胞。在一些实施方案中，靶细胞是癌细胞。在一些实施 方案中，靶向的癌细胞是实体瘤细胞。在一些实施方案中，癌症是造血谱系的，如淋巴瘤。在具体的实施方案中，淋巴瘤为B细胞淋巴瘤。所述B细胞淋巴瘤可以是霍奇金淋巴瘤或非霍奇金淋巴瘤，包括但不限于大B细胞淋巴瘤，如弥漫性大B细胞淋巴瘤(DLBCL)、弥漫性大B细胞淋巴瘤(DLBCL)非特指型(NOS)、纵隔大B细胞淋巴瘤(PMBCL)，滤泡细胞淋巴瘤，套细胞淋巴瘤。所述肿瘤可以是难治型肿瘤或复发型肿瘤，例如肿瘤患者接受过针对该肿瘤的在先治疗。
在一些实施方案中，靶细胞形成肿瘤。用带有本发明的增强受体治疗可以稳定肿瘤的生长(例如，一个或多个肿瘤的尺寸增加不超过1％、5％、10％、15％或20％，和/或不转移)。在一些实施方案中，使肿瘤稳定持续至少约1、2、3、4、5、6、7、8、9、10、11、12周或更多周。在一些实施方案中，使肿瘤稳定持续至少约1、2、3、4、5、6、7、8、9、10、11、12个月或更多个月。在一些实施方案中，使肿瘤稳定持续至少约1、2、3、4、5、6、7、8、9、10年或更多年。在一些实施方案中，使肿瘤尺寸或肿瘤细胞数减少至少约5％、10％、15％、20％、25、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或更多。在一些实施方案中，肿瘤被完全消除，或被减少到检测水平以下。在一些实施方案中，受试者在治疗后保持无肿瘤(例如缓解)持续至少约1、2、3、4、5、6、7、8、9、10、11、12周或更多周。在一些实施方案中，受试者在治疗后保持无肿瘤持续至少约1、2、3、4、5、6、7、8、9、10、11、12个月或更多个月。在一些实施方案中，受试者在治疗之后保持无肿瘤持续至少约1、2、3、4、5、6、7、8、9、10年或更多年。
可以通过任何合适的方法确定靶细胞的死亡，所述方法包括但不限于在治疗之前和之后对细胞计数或测量与活细胞或死细胞(例如活靶细胞或死靶细胞)相关的标记的水平。可以通过任何合适的方法确定细胞死亡的程度。在一些实施方案中，关于起始条件确定细胞死亡的程度。例如，个体可以具有靶细胞的已知起始量，如已知尺寸的起始细胞团或已知浓度的循环靶细胞。在此类情况下，细胞死亡的程度可以表示为治疗之后的存活细胞与起始细胞群的比率。在一些实施方案中，可以通过合适的细胞死亡测定来确定细胞死亡的程度。多种细胞死亡测定是可用的，并且可以利用多种检测方法。检测方法的例子包括但不限于使用细胞染色、显微镜检查、流式细胞术、细胞分选、以及这些的组合。
当在治疗期结束后对肿瘤进行手术切除时，可以通过测量坏死(即，死亡)的 切除组织的百分比来确定治疗在减小肿瘤尺寸方面的功效。在一些实施方案中，如果切除组织的坏死百分比为大于约20％(例如，至少约30％、40％、50％、60％、70％、80％、90％或100％)，则治疗是治疗有效的。在一些实施方案中，切除组织的坏死百分比为100％，即不存在或不可检测到活的肿瘤组织。
将靶细胞暴露于本发明的修饰的免疫细胞或免疫细胞群体可以在体外或在体内进行。通过将靶细胞和免疫细胞共培养可以完成在体外将靶细胞暴露于免疫细胞或免疫细胞群。可以将靶细胞和免疫细胞例如作为粘附细胞或可替代地在悬浮液中共培养。可以将靶细胞和免疫细胞与例如补充剂、生长因子、离子等在各种合适类型的细胞培养基中共培养。在一些情况下，可以通过将免疫细胞给予受试者(例如人类受试者)并允许免疫细胞经由循环系统定位于靶细胞来完成在体内将靶细胞暴露于免疫细胞或免疫细胞群。在一些情况下，可以例如通过直接注射将免疫细胞递送至靶细胞所定位的附近区域。
暴露可以进行任何合适的时间长度，例如至少1分钟、至少5分钟、至少10分钟、至少30分钟、至少1小时、至少2小时、至少3小时、至少4小时、至少5小时、至少6小时、至少7小时、至少8小时、至少12小时、至少16小时、至少20小时、至少24小时、至少2天、至少3天、至少4天、至少5天、至少6天、至少1周、至少2周、至少3周、至少1个月或更长时间。
在治疗应用中，可以将带有本发明的增强受体的细胞或细胞群体以足以治愈或至少部分阻止疾病或病症的症状或者足以治愈、痊愈、改进或改善所述病症的量给予已经患有疾病或病症的受试者。对此用途有效的量可以根据疾病或病症的严重程度和病程、既往疗法、受试者的健康状态、体重和对药物的反应、以及治疗医师的判断而变化。
在用于治疗用途时，本发明的带有增强受体的免疫细胞可以以约1×107至约1×1012个细胞，优选约1×108至约1×1011个细胞的剂量给予受试者，例如约1×107、约1×108、约1×109、约1×1010、约1×1011、约1×1012。在一些实施方案中，所述细胞剂量为施用的细胞总量。
也可以基于受试者体重来确定给予治疗性细胞的剂量。具体而言，本发明的带有增强受体的免疫细胞可以以约1×105至约1×1011个细胞/kg受试者体重的剂量施用，优选约1×106至约1×108个细胞/kg受试者体重，例如，约1×106个细胞/kg受试者体重、约3.3×106/kg受试者体重、约5×106/kg受试者体重、约6.7×106/kg受试 者体重、约1×107个细胞/kg受试者体重、约3.3×107/kg受试者体重、约5×107/kg受试者体重、约6.7×107/kg受试者体重、约1×108个细胞/kg受试者体重。
本发明的带有双增强受体的免疫细胞，相较于仅带有单个增强受体的免疫细胞的有效剂量更低。在一些实施方案中，所述带有双增强受体的免疫细胞与仅带有单个增强受体的免疫细胞相比，能够在低至少50％，甚至低一个数量级的剂量下，实现类似或更好的治疗效果。所述双增强受体优选为本发明的TIGIT/OX40增强受体和另一种不同的增强受体的组合，如PD1/CD28增强受体或PD1(108)/CD28增强受体的组合。
实施例
为了更全面地理解和应用本发明，下文将参考实施例和附图详细描述本发明，所述实施例仅是意图举例说明本发明，而不是意图限制本发明的范围。本发明的范围由后附的权利要求具体限定。
实施例1.表达增强受体的慢病毒载体的制备
为了测定本发明的TIGIT/OX40增强受体与其他增强受体的效果，采用第四代慢病毒载体系统(四质粒系统)通过PEI(Polysciences，23966-1)试剂转染试剂转入293T细胞中，制备了如下几种不同的慢病毒载体：
1)NY-ESO-1 TCR：靶向肿瘤靶点NY-ESO-1的人工TCR；
2)CD19 CAR：靶向CD19的CAR；
3)TIGIT/OX40：ECD源自TIGIT，ICD为OX40，TM为OX40的增强受体；
4)TIGIT/OX40-TCR：上述3)中的TIGIT/OX40基因与上述1)中靶向肿瘤靶点NY-ESO-1的人工TCR基因构建在同一个载体上，两个基因序列中间用T2A序列连接，通过剪切酶在T2A序列上进行剪切即可使TIGIT/OX40增强受体蛋白和TCR蛋白分别单独表达在细胞膜上，形成两个蛋白的非融合表达；
5)TIGIT/OX40-CAR：非融合表达上述3)中的TIGIT/OX40与上述2)中靶向CD19的CAR；
6)TIGIT/CD28-TCR：非融合表达增强受体TIGIT/CD28与靶向肿瘤靶点NY-ESO-1的人工TCR，其中增强受体TIGIT/CD28的ECD源自TIGIT，ICD和 TM源自CD28；
7)PD1/CD28-TCR：非融合表达增强受体PD1/CD28与靶向肿瘤靶点NY-ESO-1的人工TCR，其中增强受体PD1的ECD源自野生型PD1，ICD和TM源自CD28。
以TIGIT/OX40-TCR为例来说明对应慢病毒载体的制备。为细胞加载TCR的目的在于使其识别带有对应抗原的肿瘤细胞。这是因为在动物(小鼠)实验中，小鼠体内的肿瘤为来自人的异种移植物，通过TCR修饰使治疗用的免疫细胞能够识别该异种移植物，由此模拟在实际应用中在修饰之前具有肿瘤识别性的免疫细胞。应该理解的是，在实际应用中，不仅可以通过加载TCR(如根据个体的突变谱来制备新抗原识别性TCR)来赋予肿瘤细胞识别性，还可以通过分离肿瘤浸润淋巴细胞(TIL)、从外周血分离PD1+T细胞来获得具有肿瘤识别性的细胞。在后两种情况下，加载肿瘤抗原或新抗原识别性TCR不是必须，因此本实施例也介绍了单独制备TIGIT/OX40增强受体的方案。
首先，构建表达TIGIT/OX40-TCR构建体的主载体。所述TIGIT/OX40-TCR构建体由T2A序列将TIGIT/OX40增强受体基因序列和TCR基因序列间隔构建在同一载体上，TIGIT/OX40由TIGIT胞外结构域(TIGIT ECD)、OX40跨膜结构域(OX40TM)和OX40胞内结构域(OX40ICD)组成，其序列如下表1所示。
表1.TIGIT/OX40-TCR构建体序列组成
将主载体TIGIT/OX40-TCR质粒，及包装载体pMDL-gag质粒，Rev质粒，包膜载体pMD2.G质粒，以1:2:2:1的比例混合后采用PEI试剂共转染293T(ATCC)细胞。将经过转染的细胞在37℃培养48小时后，收集上清，并进行超速离心浓缩慢病毒。将离心得到的慢病毒颗粒用1mL 1640培养基重悬，并分装到10个1.5ml EP管中，每管100μl，-80℃保存。
对TIGIT/OX40-TCR增强受体慢病毒载体的滴度进行检测。取病毒原液100μl，采用3倍倍比稀释，分别稀释3、9、27、81、243、729倍之后，从每个 不同稀释倍数的病毒样本中各取50μl感染293T细胞，持续48小时至72小时。去除培养上清，用PBS清洗细胞后加入胰酶，收集293T细胞，用抗TIGIT抗体(Biolegend，372714)进行TIGIT染色，通过流式细胞术分析各个不同稀释倍数的病毒感染的细胞中TIGIT+细胞的比例，并按照如下公式进行滴度计算：
滴度(TU/ml)＝起始293T细胞数(约40000至45000个)×TIGIT+细胞百分比×稀释倍数×20(取第一个TIGIT+细胞百分比<20％的那组病毒稀释倍数进行计算)。
采用类似的方法，构建了上文所列的7种构建体，其氨基酸和核苷酸序列如下所示。使用带有这些构建体的慢病毒载体来转染293T细胞。
表2.其他元件的序列组成
按照如上所述方法，通过不同的抗体标记，流式细胞术检测抗体标记阳性细胞的比例，计算各慢病毒滴度，滴度>3×107可进行下一步使用。
实施例2.制备肿瘤识别性且负载增强受体的免疫细胞
实施例中使用T细胞作为负载增强受体的免疫细胞，并且对T细胞进行修饰以使其对特定肿瘤细胞具有特异性识别能力，以便在体外实验中能够实现免疫细胞与肿瘤细胞的特异性结合。
为了比较不同的增强受体的效果，总共制备了四种不同的肿瘤识别性T细胞，其均带有特异性识别NY-ESO-1的NY-ESO-1-TCR，其中ii)至iv)带有增强受体修饰，具体如下：
i)NY-ESO-1-TCR-T：经修饰而表达NY-ESO-1-TCR的T细胞，其可以通过人工TCR靶向表达NY-ESO-1肿瘤细胞，也可简称为TCR-T；
ii)TIGIT/OX40-TCR-T：经修饰而表达增强受体TIGIT/OX40和NY-ESO-1-TCR的T细胞，其可以通过人工TCR靶向表达NY-ESO-1肿瘤细胞；
iii)TIGIT/CD28-TCR-T：经修饰而表达增强受体TIGIT/CD28和NY-ESO-1-TCR的T细胞，其可以通过人工TCR靶向表达NY-ESO-1肿瘤细胞；
iv)PD1/CD28-TCR-T：经修饰而表达增强受体PD1/CD28和NY-ESO-1-TCR 的T细胞，其可以通过人工TCR靶向表达NY-ESO-1肿瘤细胞。
如上所述，TCR为免疫细胞赋予了肿瘤识别性，而各种增强受体用于提高免疫细胞的效力。
单采肿瘤患者外周血单个核细胞，将单采细胞中的PD1+细胞分离出来，平均分为5组。向每组分别加入CD3/CD28 dynabeads刺激过夜，第二天将其中4组细胞按照MOI(慢病毒数目/细胞数)＝2分别加入包含实施例1中1)NY-ESO-1-TCR、4)TIGIT/OX40-TCR、6)TIGIT/CD28-TCR和7)PD1/CD28-TCR的慢病毒载体。培养并扩增上述转染细胞(TCR-T、TIGIT/OX40-TCR-T、TIGIT/CD28-TCR-T和PD1/CD28-TCR-T细胞)，并以不添加任何病毒转染的T细胞作为对照(Mock-T)。
实施例3.TIGIT/OX40增强受体在体外活化T细胞的效果
为了验证TIGIT/OX40增强受体的功能，以肿瘤相关抗原NY-ESO-1作为靶点，用天然表达NY-ESO-1的人纤维肉瘤细胞细胞系HT1080(北京昭衍新药研究中心股份有限公司赠送)作为靶细胞，并在HT1080细胞中表达了HLA-A*02:01基因。实施例2中构建的四种细胞均表达靶向NY-ESO-1的TCR-T，将这些细胞作为效应T细胞。通过将所述肿瘤细胞HT1080与所述T细胞共培养，并检测T细胞分泌的IFN-γ和IL-2的量，由此测定经过不同修饰的T细胞的激活水平，并将结果示于图1A-B。
如图1A-B所示，TIGIT/OX40-TCR-T细胞分泌的IL-2和IFN-γ显著高于PD1/CD28-TCR-T、TIGIT/CD28-TCR-T和TCR-T细胞所分泌的IL-2和IFN-γ。体外实验的结果表明在HT1080肿瘤细胞模型中，表达增强受体的TCR-T细胞的免疫激活更强，并且以TIGIT作为胞外结构域的两种增强受体的效果优于以PD1作为胞外结构域的增强受体，其中TIGIT/OX40的激活水平最高。
实施例4.TIGIT/OX40增强受体在体内活化T细胞的效果
为了进一步验证TIGIT/OX40增强受体的作用，首先在人膀胱移行癌细胞J82(购自中国医学科学院，货号1101HUM-PUMC000346)中负载表达NY-ESO-1肿瘤抗原，构建了J82-NYESO1(J82-NY)肿瘤细胞系。具体而言，将携带NY-ESO-1的慢病毒，按照MOI＝5加入J82细胞中，72小时后加入G418筛选阳性细胞， 大约2个星期后，流式检测NY-ESO-1的表达情况。流式细胞仪检测结果显示，高达95％以上的J82细胞表达NY-ESO-1，说明细胞构建成功。随后，构建了J82-NY肿瘤细胞的异种移植物小鼠模型。
将1×106肿瘤细胞J82-NY皮下接种到NSG小鼠(购自北京艾德摩生物技术有限公司)，待肿瘤体积达到80～120mm3，选取30只小鼠，随机分为如下6组(每组5只)，并分别给予下述6种细胞治疗之一：
a.冻存液组(Freezing medium组)；
b.未转导的T细胞组(Mock-T组)；
c.仅转导了NY-ESO-1的TCR细胞组(TCR-T组)；
d.转导了PD1/CD28增强受体和NY-ESO-1的TCR细胞组(PD1/CD28-TCR-T组)；
e.转导了TIGIT/CD28增强受体和NY-ESO-1的TCR细胞组(TIGIT/CD28-TCR-T组)；
f.转导了TIGIT/OX40增强受体和NY-ESO-1的TCR细胞组(TIGIT/OX40-TCR-T组)。
每组的给药方式均为单次尾静脉输注200μl共8×106个细胞(有效细胞数量为6×106个)，对照组相应地给予0.2ml的细胞冻存液。给药后，每2-3天进行肿瘤大小的测量及小鼠状态的观察。按照“肿瘤体积＝1/2×长径×短径×短径”来计算肿瘤体积，并计算每组5只小鼠的平均肿瘤体积。持续观察70天并将结果示于图2和图3。
如图2、3所示，冻存液和Mock-T组小鼠肿瘤体积持续增长。观察至第44天时，由于小鼠肿瘤体积过大，对两组小鼠进行了安乐死。施用了肿瘤识别性TCR-T细胞的c、d、e、f组小鼠的肿瘤体积在第10天后开始缩小，在第25天左右显示出最强药效。然而，随着观察时间延长，TCR-T组小鼠的肿瘤开始复发，第35天后开始逐渐变大，观察至第70天时，TCR-T组的5只小鼠中有4只小鼠的肿瘤体积超过了治疗起始时的肿瘤体积，出现肿瘤进展的现象，另一只小鼠在第70天被发现死亡。PD1/CD28-TCR-T组小鼠平均肿瘤体积也在第46天后持续增大。TIGIT/CD28-TCR-T组和TIGIT/OX40-TCR-T组的药效维持较好。放大图图3的结果显示，TIGIT/OX40组至观察结束小鼠肿瘤都维持完全清除且未复发的状态，而TIGIT/CD28组部分小鼠出现肿瘤复发的现象。
以上结果说明TIGIT/OX40不仅能够增强TCR-T的肿瘤抑制效应，同时相较于其他两种增强受体对于T细胞功能维持和疗效维持具有更强的功能。
实施例5.增强受体TIGIT/OX40和PD1/CD28的联用实验之一
本实施例涉及同时用两种增强受体来修饰T细胞。所述两种增强受体为(1)基于PD1突变体108#构建的增强受体PD1(108)/CD28，和(2)如实施例1中所述的TIGIT/OX40。
表达PD1(108)/CD28-TIGIT/OX40双增强受体的慢病毒载体的制备
与野生型PD1相比，PD1突变体108#能够以高亲和力结合人PD-L1，但不结合包括信迪利单抗(Sintilimab)、纳武单抗(Nivolumab)、卡瑞利珠单抗(Camrelizumab)、帕博利珠单抗(Pemrolizumab)和特瑞普利单抗(Toripalimab)在内的多种抗PD1抗体。
为了使T细胞同时表达两种增强受体，将编码PD1(108)/CD28的核苷酸序列与编码TIGIT/OX40的核苷酸序列用编码T2A的核苷酸序列(SEQ ID NO:22)连接，作为双增强受体构建体PD1(108)/CD28-TIGIT/OX40引入慢病毒载体中。如此一来，在用慢病毒系统转染之后，借助于自切割肽T2A的作用，被转染的T细胞中将表达上述两个增强受体，两个增强受体将分别发挥功能，共同调控T。PD1(108)的氨基酸序列和编码核苷酸序列分别如SEQ ID NO:12和SEQ ID NO:4所示。
按照与实施例1相同的方式构建慢病毒载体并用于转染293T细胞，然后测定滴度。具体而言，构建了包含以下基因序列的慢病毒载体：
1)PD1(108)/CD28-TIGIT/OX40-TCR：非融合表达上述双增强受体构建体PD1(108)/CD28-TIGIT/OX40及如实施例1中所述的靶向肿瘤靶点NY-ESO-1的人工TCR。
计算各慢病毒滴度，滴度>3×107可进行下一步使用。
表达PD1(108)/CD28-TIGIT/OX40双增强受体在体外活化T细胞的效果
为了验证PD1(108)/CD28-TIGIT/OX40双增强受体的功能，用过表达NY-ESO-1的J82-NY细胞作为靶细胞，与带有特异性识别NY-ESO-1的TCR的T 细胞共培养，通过测量不同修饰的T细胞分泌IFN-γ和IL-2的量，由此检测T细胞上带有的增强受体修饰对T细胞活化程度的影响。
为了比较不同的增强受体的效果，按照与实施例2类似的方式，总共制备了四种不同的肿瘤识别性T细胞，其均带有特异性识别NY-ESO-1的NY-ESO-1-TCR，并且ii)至iv)还进一步带有不同的增强受体修饰，具体如下：
i)NY-ESO-1-TCR-T：经修饰而表达NY-ESO-1-TCR的T细胞，其可以通过人工TCR靶向表达NY-ESO-1肿瘤细胞，也可简称为TCR-T；
ii)PD1/CD28-TCR-T：经修饰而表达增强受体PD1/CD28和NY-ESO-1-TCR的T细胞，其可以通过人工TCR靶向表达NY-ESO-1肿瘤细胞；
iii)antiPDL1/CD28-TCR-T：经修饰而表达增强受体antiPDL1/CD28和NY-ESO-1-TCR的T细胞，其可以通过人工TCR靶向表达NY-ESO-1肿瘤细胞，其中antiPDL1代表以抗PD-L1单链抗体(PD-L1 scFv)作为该增强受体的ECD，其氨基酸序列和核苷酸序列分别如SEQ ID NO:15和SEQ ID NO:7所示；
iv)PD1(108)/CD28-TIGIT/OX40-TCR-T：经修饰而表达两种增强受体PD1(108)/CD28和TIGIT/OX40-TCR-T的T细胞，其可以通过人工TCR靶向表达NY-ESO-1肿瘤细胞。
上述第i)和第ii)种的细胞的制备如实施例1和2中所述。第iii)种细胞的制备与ii)类似，两种增强受体均结合PD-L1，但ii)使用了PD-L1配体的受体PD-1作为ECD，而iii)使用了抗PD-L1抗体的scFv作为ECD。第iv)种细胞的制备采用本实施例中描述的慢病毒载体按照与实施例2中相同的转染流程完成。
将J82-NY细胞密度调整为1×106个/mL，取100μl放入U形底96孔板中，按照与J82-NY细胞效靶比为1:1的比例取上述四种T细胞各100μl加入放有J82-NY细胞的U形底96孔板中。在37℃共培养24小时后将培养板放入离心机400g离心5分钟。取150μl培养上清移至一新的96孔板中，注意不要吸到底部细胞。吸完上清后，用封口膜封住四周并于4℃保存。第二天进行ELISA检测。
使用人IL-2检测试剂盒(ELISA法)(达科为，货号1110203)，按照如下流程进行ELISA。预先用抗人IL-2捕获抗体包被微孔板。将标准品或待检样品和检测抗体(生物素标记的抗人IL-2抗体)同时加入微孔板，然后加入辣根过氧化物酶标记的链霉亲和素(SA-HRP)，其特异性结合免疫复合物中的生物素。加入酶 底物四甲基联苯胺(TMB)，出现蓝色，颜色的深浅与标准品或样本中IL-2浓度相关，显色5-10分钟后加入终止液终止反应，通过多功能酶标仪读取450nm处光吸收值(OD)。在一定范围内人IL-2浓度与OD值成正比，使用IL-2标准品进行ELISA检测并得到了标准曲线。使用该标准曲线计算出了每种共培养物中的IL-2浓度，并将结果示于图4。
使用人IFNγ检测试剂盒(ELISA法)(达科为，货号1110003)，按照如下流程进行ELISA。将标准品或待检样品和检测抗体(生物素标记的抗人IFNγ抗体)同时加入微孔板，然后加入辣根过氧化物酶标记的链霉亲和素(SA-HRP)，其特异性结合免疫复合物中的生物素。加入酶底物四甲基联苯胺(TMB)，出现蓝色，颜色的深浅与标准品或样本中IFNγ浓度相关，显色5-10分钟后加入终止液终止反应，通过多功能酶标仪读取450nm处光吸收值(OD)。在一定范围内人IFNγ浓度与OD值成正比，使用IFNγ标准品进行ELISA检测并得到了标准曲线。使用该标准曲线计算出了每种共培养物中的IFNγ浓度,并将结果示于图5。
如图4所示，PD1(108)/CD28-TIGIT/OX40-TCR-T细胞分泌的IL-2要显著高于PD1/CD28-TCR-T、antiPDL1/CD28-TCR-T和TCR-T细胞所分泌的IL-2。如图5结果所示，PD1(108)/CD28-TIGIT/OX40-TCR-T细胞分泌的IFN-γ显著高于antiPDL1/CD28-TCR-T和TCR-T细胞所分泌的IFN-γ，而与PD1/CD28-TCR-T细胞所分泌的IFN-γ量没有差异。体外实验的结果表明带有双增强受体修饰的TCR-T细胞的实现了显著更高的活化水平。
表达PD1(108)/CD28-TIGIT/OX40增强受体的NY-ESO-1 TCR-T细胞的动 物实验
为了验证PD1(108)/CD28-TIGIT/OX40双增强受体的作用，采用与实施例4中类似的方法进行了小鼠体内实验。
将1×106肿瘤细胞J82-NY皮下接种到NSG小鼠，待肿瘤体积达到80-120mm3，选取25只小鼠，随机分为如下5组(每组5只)，并分别给予下述5种细胞治疗之一：
a.未转导的T细胞组(Mock-T组)；
b.仅转导了NY-ESO-1的TCR细胞组(TCR-T组)；
c.转导了PD1/CD28增强受体和NY-ESO-1的TCR细胞组(PD1/CD28-TCR- T组)；
d.转导了antiPDL1/CD28增强受体和NY-ESO-1的TCR细胞组(antiPDL1/CD28-TCR-T组)；
e.转导了PD1(108)/CD28-TIGIT/OX40增强受体和NY-ESO-1的TCR细胞组(PD1(108)/CD28-TIGIT/OX40-TCR-T组)。
除Mock-T组，每组的给药方式均为单次尾静脉输注6×106个TCR阳性有效细胞，Mock-T组每只小鼠尾静脉输注细胞量与其余几组每只小鼠给予的总细胞量相同。给药后，每2-3天进行肿瘤大小的测量及小鼠状态观察。按照“肿瘤体积＝1/2×长径×短径×短径”来计算肿瘤体积。持续观察37天，并将结果示于图6和图7。
结果如图6、7所示，Mock-T组小鼠肿瘤体积持续增长，b、c、d、e组小鼠肿瘤体积先增大后缩小。TCR-T组小鼠肿瘤前期缩小最快，但是到第28天后开始逐渐变大，出现肿瘤进展的现象。PD1/CD28-TCR-T组小鼠平均肿瘤体积也在第35天后持续增大。PD1(108)/CD28-TIGIT/OX40-TCR-T组的药效最好。这些结果说明，带有双增强受体且能够特异性识别肿瘤的T细胞相较于仅带有特异性结合PD-L1的增强受体的T细胞而言，在体内提供了更好的肿瘤抑制效果。
实施例6.增强受体TIGIT/OX40和PD1/CD28的联用实验之二
按照与实施例6类似的方法再次进行了动物实验，来验证TIGIT/OX40和PD1(108)/CD28联用的效果。
PD1(108)/CD28-TIGIT/OX40双增强受体的慢病毒载体的制备
与实施例6的构建体相同，PD1(108)/CD28-TIGIT/OX40双增强受体是通过将PD1(108)/CD28的编码序列与TIGIT/OX40的编码序列用T2A的编码序列连接来构建。采用了更安全的第四代慢病毒载体系统进行转导。使用主载体PD1(108)/CD28-TIGIT/OX40，以及包装载体pMDL-gag、Rev、包膜载体pMD2.G，并采用磷酸钙或是脂质体PEI共转染293T细胞，48小时收集上清，并进行超速离心以浓缩慢病毒。
对于PD1(108)/CD28-TIGIT/OX40增强受体慢病毒载体的滴度检测，采用3倍倍比稀释，并取50μl感染293T细胞48小时至72小时。收集293T细胞进 行PD1(108)染色。通过流式分析PD1(108)阳性细胞的比例，并按照公式进行滴度计算：
滴度(TU/ml)＝起始293T细胞数(约40000至45000个)×PD1(108)+细胞百分比×稀释倍数×20(取第一个PD1(108)+细胞百分比<20％进行计算)。
由于PD1(108)/CD28增强受体和TIGIT/OX40增强受体在同一个载体上，因此用PD1(108)/CD28增强受体的滴度即代表了TIGIT/OX40增强受体的滴度。
以类似的方法构建了包含以下基因序列的慢病毒载体：
1)非融合表达的“PD1(108)/CD28-TIGIT/OX40”以及靶向肿瘤靶点NY-ESO-1的人工TCR，简称PD1(108)/CD28-TIGIT/OX40-TCR；
2)“PD1(108)/CD28”以及靶向肿瘤靶点NY-ESO-1的人工TCR，简称PD1(108)/CD28-TCR；
3)“TIGIT/OX40”以及靶向肿瘤靶点NY-ESO-1的人工TCR，简称TIGIT/OX40-TCR；
4)靶向肿瘤靶点NY-ESO-1的人工TCR，简称TCR。
计算各慢病毒滴度，滴度>3×107可进行下一步使用。
表达PD1(108)/CD28-TIGIT/OX40双增强受体的NY-ESO-1 TCR-T细胞的 动物实验
为了验证PD1(108)/CD28-TIGIT/OX40双增强受体的作用，以肿瘤相关抗原NY-ESO-1作为靶点，用表达NY-ESO-1的J82肿瘤细胞作为靶细胞(J82-NY-ESO1，或称J82-NY)，用靶向NY-ESO-1的TCR-T细胞作为效应T细胞，让靶向NY-ESO-1的TCR-T细胞同时表达PD1(108)/CD28-TIGIT/OX40增强受体修饰以制备增强型TCR-T细胞。将1×106肿瘤细胞J82-NY皮下接种到NSG小鼠，待肿瘤体积达到80-120mm3，选取30只小鼠，随机分为6组，分别给予如下细胞治疗：a.冻存液组(Freezing medium)；b.未转导的T细胞组(Mock-T组)；c.仅转导了NY-ESO-1的TCR细胞组(TCR-T组)；d.转导了PD1(108)/CD28增强受体和NY-ESO-1的TCR细胞组(PD1(108)/CD28-TCR-T组)；e.转导了TIGIT/OX40增强受体和NY-ESO-1的TCR细胞组(TIGIT/OX40-TCR-T组)；f.转导了PD1(108)/CD28-TIGIT/OX40增强受体和NY-ESO-1的TCR细胞组(PD1(108)/CD28-TIGIT/OX40-TCR-T组)。各组中包含元件的序列与上文相同。 给药方式均为总细胞量1.5×107个细胞/只(3×106个TCR阳性细胞/只)，通过尾静脉输注给药一次。给药后，每2～3天进行肿瘤大小的测量及小鼠状态观察。按照“肿瘤体积＝1/2×长径×短径×短径”来计算肿瘤体积，并持续观察31天。
结果如图8和表3所示(其中肿瘤体积为各组的平均值)，Freezing medium组和Mock-T组小鼠肿瘤体积持续增长，TCR-T组小鼠肿瘤生长速率比Freezing medium组和Mock-T组慢，肿瘤生长受到了一定程度抑制。3组同时表达增强受体和TCR组小鼠肿瘤生长都出现了肿瘤体积下降的结果，说明表达增强受体促进了TCR-T细胞的肿瘤抑制功能。令人惊讶的是，表达双增强受体的PD1(108)/CD28-TIGIT/OX40-TCR-T组展现出显著的肿瘤抑制效应，且药效维持时间最长，相比于只包含其中一种增强受体修饰的细胞而言，两种增强受体的联用体现出了在肿瘤抑制方面的协同效果。
表3.不同处理组在注射后不同时间点的肿瘤体积(mm3)
表达PD1(108)/CD28-TIGIT/OX40增强受体的NY-ESO-1 TCR-T细胞的体 内药代动力学研究
为了验证PD1(108)/CD28-TIGIT/OX40增强受体的作用，以J82-NY肿瘤模型评价了表达PD1(108)/CD28-TIGIT/OX40增强受体的TCR-T细胞的体内代谢情况。
将1×106肿瘤细胞J82-NY皮下接种到NSG小鼠，待肿瘤体积达到80-120mm3，选取54只小鼠，随机分为3组给予细胞治疗，分别为：a.仅转导了NY-ESO-1的TCR细胞组(TCR-T组)；b.转导了PD1(108)/CD28增强受体和NY-ESO-1的TCR细胞组(PD1(108)/CD28-TCR-T组)；c.转导了PD1(108)/CD28-TIGIT/OX40增强受体和NY-ESO-1的TCR细胞组(PD1(108)/CD28-TIGIT/OX40- TCR-T组)。给药方式均为按照TCR阳性细胞5×106个/只的剂量，通过尾静脉输注给药一次。T细胞回输后每组在D0(回输后2h)、D1、D3、D7、D10、D14、D21、D28、D35分别取2只小鼠，用异氟烷麻醉动物后，实施安乐死，并采集全血(EDTA抗凝)和肿瘤组织，-80℃保存样本。每2-3天进行肿瘤大小的测量及小鼠状态观察。按照“肿瘤体积＝1/2×长径×短径×短径”来计算肿瘤体积，并持续观察35天。通过qPCR的方法检测不同小鼠的外周血和肿瘤组织中TCR基因的含量，从而评价施用的治疗性T细胞在小鼠体内的代谢情况。
结果如图9所示，TCR-T细胞回输后的28天内并未在小鼠外周血中出现大幅增殖的现象，至D35时出现扩增峰。而PD1(108)/CD28-TCR-T细胞和PD1(108)/CD28-TIGIT/OX40-TCR-T细胞在分别在D10和D14出现扩增峰，且PD1(108)/CD28-TIGIT/OX40-TCR-T细胞在外周血中的扩增程度要强于PD1(108)/CD28-TCR-T细胞。该结果表明增强受体PD1(108)/CD28-TIGIT/OX40对TCR-T细胞在小鼠外周血中的扩增和存续具有更强的促进作用。
结果如图10所示，T细胞回输后从D3开始在肿瘤组织中能检测到TCR基因，但含量较低。D7时TCR-T组和PD1(108)/CD28-TCR-T组在肿瘤组织中的TCR基因含量增加，而PD1(108)/CD28-TIGIT/OX40-TCR-T组肿瘤组织中TCR基因含量低于另外两组。TCR-T组和PD1(108)/CD28-TCR-T组在肿瘤组织中的扩增峰出现在D10，且PD1(108)/CD28-TCR-T组的扩增程度强于TCR-T组，随后开始下降。而PD1(108)/CD28-TIGIT/OX40-TCR-T组肿瘤组织中TCR基因含量从D10开始出现大幅扩增，扩增高峰出现在D14，且扩增程度强于PD1(108)/CD28-TCR-T组，在D21-D35依然维持在较高的水平，含量为3组中最高。增强受体PD1(108)/CD28-TIGIT/OX40对TCR-T细胞在小鼠肿瘤组织中的增殖和存续具有更强的促进作用。
实施例7.增强受体TIGIT/OX40在人受试者中的效果
进一步在人类受试者中通过临床试验测试了TIGIT/OX40增强受体的效果。具体而言，比较了使用TIGIT/OX40增强受体与PD1(108)/CD28的组合进行双增强受体修饰的T细胞(c610)相对于使用单独的PD1(108)/CD28修饰的T细胞(c410)的效果。本实施例使用类似于实施例4-7中所述的PD1(108)/CD28-TCR-T细胞和PD1(108)/CD28-TIGIT/OX40-TCR-T细胞。
c410(PD1(108)/CD28-CD19 CAR-T)细胞和c610(PD1(108)/CD28- TIGIT/OX40-CD19 CAR-T)细胞制备操作规程
1.第0天：
(1)PD1+T细胞分选：
a.取患者单采血，加入anti-PD1-Biotin抗体(BioLegend，329934)，混匀，避光孵育；
b.用缓冲液清洗，离心；
c.用缓冲液重悬，加20μl anti-Biotin MicroBeads(德国美天旎生物技术公司，170-076-709)，混匀，避光孵育；
d.用缓冲液清洗，离心，重悬细胞，同时用缓冲液润洗柱子；
e.细胞过柱子后将吸附柱取下，放入一新的离心管上，加入缓冲液于柱内，迅速用活塞将柱内液体推出，收集PD1+细胞，计数；
f.根据实验需要，取相应数量的PD1+细胞，离心，弃上清，完全培养基重悬细胞。
(2)加入抗-CD3/CD28磁珠激活T细胞：
a.按照一定比例，取相应数量的Dynabeads CD3/CD28(Thermo Fisher Scientific，40203D)放置于离心管中；
b.使用X-VIVO培养基(Lonza，BEBP02-054Q)重悬洗涤磁珠，于磁力架上吸附静置，待磁珠吸附到管壁后，移液管去除上清；
c.使用TSCM培养基(X-VIVO+2.5％CTSTM免疫细胞SR(Thermo Fisher Scientific，A2596102)+2U/ml记忆干细胞扩增因子(上海近岸生物有限公司，GMP-1647))重悬细胞和磁珠，一起转移至培养瓶中，补加TSCM培养基调整细胞密度至1.2×106/ml；
d.将细胞放置于37℃、5％CO2培养箱中培养。
2.第1天：加入慢病毒
a.慢病毒转染时间为细胞激活24小时内；
b.根据需要制备的细胞类型确定使用的慢病毒类型(410或610；其中410即PD1(108)/CD28-CD19 CAR，610即PD1(108)/CD28-TIGIT/OX40-CD19 CAR慢病毒)和MOI，根据细胞数量计算出所加慢病毒的体积＝细胞数量×MOI/慢病 毒滴度；
c.从-80℃冰箱取出对应体积的慢病毒，插入冰块中，从传递窗传进实验室，慢病毒静置于冰中融化；
d.从培养箱取出需要加慢病毒的细胞，核对需加慢病毒细胞信息，患者姓名、细胞名称、制备日期，确认无误；
e.将解冻后的慢病毒加入细胞中，用移液枪吹打混匀，放置37℃、5％培养箱培养。
3.第3天：细胞补液，根据细胞数量补加TSCM培养基
4.第5天：去磁珠
a.在生物安全柜中，根据去磁珠细胞的培养基体积，准备相应体积的磁力架，离心管(＞20ml：50ml离心管；≤20ml：15ml离心管；不到1ml：EP管)；
b.将带有磁珠的细胞过三次磁力架，去除磁珠，收集细胞悬液；
c.洗涤2-3次磁珠，再收集细胞悬液；
d.合并b和c获得的细胞悬液，记录总体积并计数；
e.配平离心，离心，弃上清；
f.加完全培养基(X-VIVO+2.5％CTSTM免疫细胞SR(Thermo Fisher Scientific，A2596102)+3000IU/ml注射用重组人白介素2(北京双鹭药业股份有限公司，国药准字S19991010))重悬后转移至培养瓶中，根据计数结果补加完全培养基调整细胞密度，标记信息；
g.放置于37℃，5％CO2培养箱中培养。
5.第8天：细胞计数、补液
6.第11天：细胞计数、补液
7.第14天：
(1)收集细胞成品：
a.从培养箱中取出需要收集的细胞，用移液枪混匀细胞悬液，取样计数及细胞活率，并留样进行流式检测；
b.将细胞悬液转移至200ml离心管/50ml离心管中，500g，8min离心；
c.离心后，取上清至冻存管，共2支(用于支原体、内毒素、革兰氏检测)；
d.弃上清，加生理盐水至200ml/50ml重悬洗涤，600g，10min离心；
e.弃上清，加定量生理盐水重悬混匀，取样计数及细胞活率，离心，弃上清；
f.根据细胞数量，用定量成品细胞冻存液(Cryostor CS10，BioLife Solutions，100-1061)重悬细胞。
(2)冻存细胞：
a.将加入冻存液的细胞，迅速分装至冻存袋中(冻存袋要标记患者姓名、编号、细胞明细，冻存时间等信息)，并将冻存袋放入程序降温盒中，-80℃冰箱进行冻存；
b.在-80℃冰箱放置24小时以内，将冻存袋转入至液氮中保存；
记录冻存管袋的数量等信息。
入组标准
c410/c610入组研究人群为既往接受二线或以上系统性治疗后复发或难治性CD19阳性的大B细胞淋巴瘤成人患者，包括弥漫性大B细胞淋巴瘤(DLBCL)非特指型(NOS)，原发纵隔大B细胞淋巴瘤(PMBCL)、高级别B细胞淋巴瘤和滤泡性淋巴瘤转化的DLBCL、滤泡细胞淋巴瘤、套细胞淋巴瘤等。
剂量递增组分为1×106/kg、3.3×106/kg和10×106/kg三个组别，剂量扩展组依据剂量递增试验的安全性和初步疗效，推荐扩展试验的细胞剂量。
入组标准主要包括：年龄在18至70岁之间，性别不限，ECOG评分为0或1，外周血PD1阳性T细胞占总T细胞比例≥18％，具有基本心肺储备，无严重血液学、肝肾功能异常；排除标准主要包括：合并活动期中枢神经系统淋巴瘤、活动性中枢神经系统疾病；合并恶性肿瘤及免疫系统疾病；具有严重心肺功能损害；存在临床急症；近期接受重大手术及其他未上市的临床研究药物或治疗等。
筛选合格后，受试者进行自体外周血单个核细胞(PBMCs≥3×109)采集，分选获取PD-1阳性T淋巴细胞，用于细胞制剂的制备。
细胞回输前进行全面的基线检查及病灶评估，D-5至D-3接受预处理化疗(FC方案，环磷酰胺250-300mg/m2和氟达拉滨25-30mg/m2，每日1次，共3天)，D0给予c410/c610细胞回输。
回输后进行随访，评价c410细胞/c610细胞在治疗CD19阳性的复发或难治性非霍奇金淋巴瘤受试者中的耐受性、观察剂量限制性毒性(DLT)，安全性和有效性。
结果
将上述临床试验的受试者基本信息和治疗结果的详细数据示于表4中。
c410/c610细胞治疗后所有受试者均耐受性良好，未发生剂量限制性毒性，CRS最高2级。
c410细胞低剂量组(1×106/kg)的1例受试者和中剂量组(3.3×106/kg)的3例受试者客观缓解率(ORR)均为0％(0/1、0/3)。高剂量组(10×106/kg)共入组6例，D28完全缓解率(CR)为50％(3/6)，W12CR为33.3％(2/6)。D28的ORR为83.3％(5/6)，W12ORR为33.3％(2/6)。
与c410相比，c610细胞低剂量组(1×106/kg)的2例受试者D28CR为100％(2/2)，显著优于c410细胞治疗的结果。
如下表4所示的结果可见，仅含PD1(108)/CD28单个增强受体的免疫细胞(c410)疗效随剂量加大(从低剂量组1E6/kg，到中剂量组3.3E6/kg，到高剂量组1E7/kg)而逐渐提升，但细胞因子风暴(CRS)风险也加大。
与之相比，包含PD1(108)/CD28-TIGIT/OX40双增强受体的免疫细胞(c610)有显著更好的治疗效果和安全性，c610在低剂量(1E6/kg)下的疗效远远优于c410的同等低剂量组疗效，甚至显著优于c410的高剂量组疗效，而且其CRS风险低于c410各剂量组。此项临床试验的结果说明了本发明的TIGIT/OX40增强受体与PD1(108)/CD28增强受体联用时对于肿瘤具有令人惊讶的治疗效果。
序列信息


Claims (31)

  1. 一种增强受体，其包含胞外结构域(ECD)、跨膜结构域(TM)和胞内结构域(ICD)，其中所述胞外结构域源自TIGIT的胞外结构域，所述胞内结构域源自OX40的胞内结构域。
  2. 权利要求1所述的增强受体，其中所述胞外结构域是人野生型TIGIT的胞外结构域，或其能够结合TIGIT配体的片段或变体。
  3. 权利要求2所述的增强受体，其中所述胞外结构域具有如SEQ ID NO:9所示的氨基酸序列。
  4. 权利要求1或3中任一项所述的增强受体，其中所受胞内结构域是人OX40的胞内结构域，或其片段或变体。
  5. 权利要求4所述的增强受体，其中所述胞内结构域具有如SEQ ID NO:10所示的氨基酸序列。
  6. 权利要求1至5中任一项所述的增强受体，其中所述跨膜结构域源自TIGIT或OX40的跨膜结构域。
  7. 权利要求6所述的增强受体，其中所述跨膜结构域源自OX40的跨膜结构域并且具有如SEQ ID NO:11所示的氨基酸序列。
  8. 一种分离的核酸分子，其包含编码权利要求1-7中任一项所述的增强受体的核苷酸序列。
  9. 权利要求8所述的分离的核酸分子，其包含如下核苷酸序列中的一种或多种，优选包含(1)和(2)，更优选包含(1)至(3)：
    (1)如SEQ ID NO:1所示的核苷酸序列，其编码源自TIGIT的胞外结构域；
    (2)如SEQ ID NO:2所示的核苷酸序列，其编码源自OX40的胞内结构域；和
    (3)如SEQ ID NO:3所示的核苷酸序列，其编码源自OX40的跨膜结构域。
  10. 一种重组表达载体，其包含权利要求8或9的分离的核酸分子。
  11. 一种宿主细胞，其包含权利要求8或9的分离的核酸分子或权利要求10的重组表达载体。
  12. 一种免疫细胞，其经过修饰而表达权利要求1-7中任一项的增强受体。
  13. 权利要求12的免疫细胞，所述免疫细胞的修饰通过向所述免疫细胞中引入权利要求8或9的分离的核酸分子或权利要求10的重组表达载体进行。
  14. 权利要求12或13的免疫细胞，其经修饰而进一步表达权利要求1-7中所述增强受体之外的第二增强受体，其中所述第二增强受体包含第二胞外结构域、第二跨膜结构域和第二胞内结构域，其中所述第二胞外结构域能够特异性结合不同于TIGIT的T细胞免疫检查点分子的配体，其中所述第二细胞内结构域源自介导免疫细胞激活信号的共刺激分子。
  15. 权利要求14的免疫细胞，其中所述第二增强受体的胞外结构域能够特异性结合PD-L1。
  16. 权利要求14或15的免疫细胞，其中所述第二增强受体的胞内结构域源自CD28。
  17. 权利要求15或16的免疫细胞，其中所述第二胞外结构域为PD-1的胞外结构域，或其能够结合PD-L1的变体或片段。
  18. 权利要求15或16的免疫细胞，所述第二胞外结构域是特异性结合PD-L1的抗体，如特异性结合PD-L1的单链抗体(scFv)。
  19. 权利要求17的免疫细胞，其中所述第二胞外结构域具有如SEQ ID NO:12或SEQ ID NO:16所示的氨基酸序列。
  20. 权利要求12至19中任一项的免疫细胞，其经修饰而表达嵌合抗原受体，其中所述CAR包含：
    (a)能够结合B细胞表面蛋白的抗原相互作用结构域；
    (b)跨膜结构域；和
    (c)细胞内信号传导结构域。
  21. 权利要求12至20中任一项的免疫细胞，其在经修饰之前分离自外周血单个核细胞(PBMC)。
  22. 权利要求21所述的免疫细胞，其在经修饰之前获得自PBMC并且经过PD-1阳性分选。
  23. 一种分离的核酸分子，其包含编码权利要求1-7中任一项所述的增强受体的核苷酸序列，和编码第二增强受体的核苷酸序列，其中所述第二增强受体包含第二胞外结构域、第二跨膜结构域和第二胞内结构域，其中所述第二胞外结构域能够特异性结合不同于TIGIT的T细胞免疫检查点分子的配体，其中所述第二细胞内结构域源自介导免疫细胞激活信号的共刺激分子。
  24. 权利要求23所述的分离的核酸分子，其中所述第二增强受体的胞外结构域能够特异性结合PD-L1。
  25. 权利要求23或24的分离的核酸分子，其中所述第二增强受体的胞内结构域源自CD28。
  26. 权利要求24或25的分离的核酸分子，其中所述第二胞外结构域为PD-1的胞外结构域，或其能够结合PD-L1的变体或片段。
  27. 权利要求24或25的分离的核酸分子，所述第二胞外结构域是特异性结合PD-L1的抗体，如特异性结合PD-L1的单链抗体(scFv)。
  28. 权利要求24或25的分离的核酸分子，其中所述第二胞外结构域具有如SEQ ID NO:12或SEQ ID NO:16所示的氨基酸序列。
  29. 权利要求28的分离的核酸分子，其中所述第二胞外结构域的编码核苷酸序列具有如SEQ ID NO:4或SEQ ID NO:8所示的核苷酸序列。
  30. 权利要求12至22中任一项的免疫细胞在制备用于治疗疾病的药物中的用途。
  31. 权利要求30的用途，所述疾病为肿瘤。
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WO2025191067A1 (en) * 2024-03-13 2025-09-18 T-Knife Gmbh A chimeric transmembrane receptor comprising at least one t-cell immunoreceptor with ig and itim domains (tigit) polypeptide region, t-cells expressing the chimeric human tigit switch receptor, vectors with nucleic acids encoding for the tigit receptor, kits for preparing the t-cells, as well as corresponding pharmaceutical compositions and methods for treating a patient having a disease and for increasing cytotoxicity of a t-cell in adoptive cell therapy
CN118725139A (zh) * 2024-06-07 2024-10-01 浙江大学 一种促巨噬细胞m2样极化的嵌合抗原受体、核酸分子及抗原依赖的m2样极化的巨噬细胞
CN118725139B (zh) * 2024-06-07 2025-11-04 浙江大学 一种促巨噬细胞m2样极化的嵌合抗原受体、核酸分子及抗原依赖的m2样极化的巨噬细胞

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