Enhanced receptors, immune cells expressing enhanced receptors, and their use

Abstract

The present invention provides a TIGIT- and OX40-based potentiated receptor (TIGIT / OX40) that can increase the activation level and survival time of immune cells, and immune cells modified to express the potentiated receptor. The present invention also provides immune cells modified to express two different potentiated receptors, including the potentiated receptor.

Description

[Technical Field]

[0001] This invention belongs to the field of biopharmaceutical technology, and more particularly to the field of therapy using immune cells. [Background technology]

[0002] Switch receptors are fusion proteins that have recently attracted attention in the field of cell therapy. They are also called switch molecules, switch costimulatory receptors, or chimeric switch receptors. Switch receptors are transmembrane fusion proteins in which the extracellular segment binds to target cells and is derived from molecules that normally produce immunosuppressive signals (e.g., checkpoint molecules), while the intracellular segment is derived from molecules that produce immune-stimulating signals (e.g., costimulatory molecules). As a result, when immune cells are modified with such chimeric receptors, the suppressive signal that would normally be triggered when the extracellular segment of the immune receptor binds to target cells is converted into a stimulating signal by the chimeric receptor, thereby increasing the activity of immune cells.

[0003] WO2013 / 019615 describes various chimeric switch receptors having extracellular segments derived from CTLA4, PD-1, or BTLA, and intracellular segments derived from CD28 or ICOS. While CTLA4, PD-1, or BTLA are normally thought to generate an inhibitory signal when bound to their respective native ligands, the intracellular domain of CD28 or ICOS contained within the intracellular domain of the chimeric switch receptor converts this inhibitory signal into an activating signal. The application demonstrates that T cells modified by expressing this chimeric switch receptor exhibit an aggressive immune response, rather than the usual suppressive response, when exposed to tumor cells expressing BTLA or PD-1 ligands.

[0004] With the advancement of cell therapy and immunotherapy, there is a demand in this field for the development of chimeric switch receptors that exhibit superior effects. SUMMARY OF THE INVENTION

[0005] The present inventors have developed a novel enhanced receptor that exhibits significantly greater capability in activating immune cells compared to prior art chimeric switch receptors, thereby completing the present invention.

[0006] Accordingly, in a first aspect of the present invention, there is provided an enhanced receptor comprising an extracellular domain (ECD), a transmembrane domain (TM), and an intracellular domain (ICD). In this receptor, the extracellular domain is derived from the extracellular domain of TIGIT, and the intracellular domain is derived from the intracellular domain of OX40.

[0007] In a specific embodiment, the extracellular domain of TIGIT is the extracellular domain of human wild-type TIGIT, or a fragment or variant thereof, wherein the fragment or variant has ligand binding ability, and the ligand is CD155.

[0008] In a second aspect of the present invention, there is provided an immune cell modified to express the enhanced receptor of the first aspect.

[0009] In a third aspect of the present invention, there is provided an immune cell modified to express two different enhanced receptors, wherein the first enhanced receptor is the enhanced receptor according to the first aspect of the present invention. Preferably, the second enhanced receptor has an extracellular domain capable of specifically binding to PD-L1, and the second enhanced receptor has an intracellular domain derived from CD28. More preferably, the extracellular segment of the second enhanced receptor is PD1, or a variant thereof capable of specifically binding to PD-L1, or an anti-PD-L1 antibody.

[0010] In the fourth aspect of the present invention, the present invention provides methods for preparing the immune cells of the second and third aspects.

[0011] In the fifth aspect of the present invention, the present invention provides use of the immune cells of the second and third aspects in the treatment of diseases.

[0012] In the sixth aspect of the present invention, there is provided a pharmaceutical composition comprising the immune cells of the second aspect or the third aspect.

[0013] In the seventh aspect of the present invention, there is provided a nucleic acid molecule comprising a nucleotide sequence encoding the enhanced receptor of the first aspect. In a further embodiment, the nucleic acid molecule further comprises a nucleotide sequence encoding a second enhanced receptor, wherein the second enhanced receptor comprises 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 a T cell immune checkpoint molecule different from TIGIT, and the second intracellular domain is derived from a co-stimulatory molecule that mediates an immune cell activation signal.

[0014] In the eighth aspect of the present invention, there is provided an expression vector comprising the nucleic acid molecule of the seventh aspect.

[0015] In the ninth aspect of the present invention, there is provided a host cell comprising the nucleic acid molecule of the seventh aspect or the expression vector of the eighth aspect. [BRIEF DESCRIPTION OF THE DRAWINGS]

[0016] [Figure 1] A and B of Figure 1 show the results of the in vitro experiment of Example 3, which indicate the secretion levels of IL-2 (A) and INFγ (B) secreted by T cells after tumor-recognizing T cells are modified with different enhanced receptors. [Figure 2] These are the results of the in vivo experiment of Example 4, which show the change in tumor volume in different treatment groups and indicate the effect of the enhanced receptor on the tumor suppressive effect. [Figure 3]Figure 2 shows a magnified view of the data from the remaining four groups of animals after excluding the two controls (frozen medium and Mock-T). [Figure 4] This shows the results of an in vitro experiment in Example 5, which demonstrated the level of IL-2 secretion from T cells after modifying tumor-recognizing T cells using different enhanced receptors. [Figure 5] This shows the results of an in vitro experiment in Example 5, which demonstrated the level of INFγ secretion from T cells after modifying tumor-recognizing T cells using different enhanced receptors. [Figure 6] The results of the in vivo experiment in Example 5 show changes in tumor volume in different treatment groups and illustrate the effects of different reinforcing receptors on tumor suppression. [Figure 7] Figure 6 shows a magnified view of the data from the remaining four groups of animals after excluding the control (Mock-T) group. [Figure 8] This shows the in vivo experimental results of Example 6, illustrating the changes in tumor volume in different treatment groups. [Figure 9] This figure shows the changes in TCR copy number in peripheral blood of mice treated with different groups of therapeutic cells (TCR-T; PD1(108) / CD28-TCR-T; and PD1(108) / CD28-TIGIT / OX40-TCR-T) over time at different time points after injection. [Figure 10] The changes in TCR copy number in tumor tissue from mice treated with different groups of therapeutic cells (TCR-T;PD1(108) / CD28-TCR-T; and PD1(108) / CD28-TIGIT / OX40-TCR-T) are shown over time at different time points after injection. [Modes for carrying out the invention]

[0017] definition Unless otherwise noted, the methods disclosed herein are carried out using conventional techniques in immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, which are within the scope of the art. For example, refer to Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012), the series Current Protocols in Molecular Biology (FM Ausubel, et al. eds.), the series Methods In Enzymology (Academic Press, Inc.), PCR 2, A Practical Approach (MJ MacPherson, BD Hames and GR Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Culture of Animal Cells, A Manual of Basic Technique and Specialized Applications, 6th Edition (RI Freshney, ed. (2010)), etc.

[0018] The terms “about” or “approximately” mean within an acceptable margin of error for a particular value as determined by those skilled in the art, which will in part depend on how that value is measured or determined, i.e., the limits of the measuring system. Where specific values ​​are given in the specification and claims, unless otherwise stated, the term “about” should be presumed to mean within an acceptable margin of error for that particular value.

[0019] In this specification, “enhanced receptor” refers to a recombinant transmembrane protein comprising an extracellular domain (ECD), a transmembrane domain (TM), and an intracellular domain (ICD), wherein the extracellular domain is derived from an immune cell protein, which, in unmodified immune cells, induces an immune cell inactivation signal by binding to its ligand, and wherein the intracellular domain is derived from a co-stimulatory molecule that mediates an immune cell activation signal. The enhanced receptor of the present invention can be used to modify immune cells. Immune cells modified to have the enhanced receptor generate an immune cell activation signal through the binding of the extracellular domain of the enhanced receptor to the corresponding ligand, instead of the immune cell inactivation signal in the natural state, by the binding of the extracellular domain of the enhanced receptor to the corresponding ligand. In a specific embodiment, the extracellular domain of the enhanced receptor of the present invention is derived from TIGIT, and the intracellular domain is derived from OX40.

[0020] TIGIT stands for T cell immunoreceptor with Ig and immunoreceptor tyrosine-based inhibitory domains.

[0021] OX40, also known as CD134, is a co-stimulatory immune checkpoint molecule belonging to the TNF receptor superfamily.

[0022] PD-1 stands for programmed death receptor 1. PD-1 is an inhibitory immune checkpoint molecule expressed on the surface of B cells and T cells.

[0023] PD-L1 is one of the ligands for PD-1, and when it binds to PD-1, it emits an immunosuppressive signal.

[0024] CD28 is a receptor on the surface of T cells and provides a co-stimulatory signal for T cell activation.

[0025] The term "nucleotide" generally refers to a combination of a base, sugar, and phosphate. Nucleotides may include synthetic nucleotides. Nucleotides may also include synthetic nucleotide analogs. Nucleotides can be monomeric units of nucleic acid sequences (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide may include ribonucleoside triphosphates, adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), guanosine triphosphate (GTP), and deoxyribonucleoside triphosphates such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, and their derivatives. Such derivatives include, for example, "αS"dATP, 7-deaza-dGTP, and nucleotide derivatives that confer nuclease resistance to nucleic acid molecules containing them. As used herein, the term nucleotide may mean dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives. Exemplary examples of dideoxyribonucleoside triphosphates, but not limited to, include ddATP, ddCTP, ddGTP, ddITP, and ddTTP. Nucleotides may be unlabeled or detectably labeled by known methods. Labeling may be performed using quantum dots. Examples of detectable labels include radioisotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzyme labels.

[0026] The term “gene” means a nucleic acid (e.g., genomic DNA, and DNA such as cDNA) and the nucleotide sequence that encodes its corresponding RNA transcript. As used herein in relation to genomic DNA, the term may include intervening untranslated regions and regulatory regions, and may include the 5' and 3' ends. In some uses, the term includes the transcription region, which contains the 5' and 3' untranslated regions (5'-UTR and 3'-UTR), exons, and introns. In some genes, the transcription region will include the “open reading frame” that encodes the polypeptide. In some uses of the term, “gene” includes only the coding sequence (e.g., the “open reading frame” or “coding region”) necessary to encode the polypeptide. In some cases, a gene does not encode a polypeptide and is, for example, a ribosomal RNA gene (rRNA) and a transfer RNA (tRNA) gene. In some cases, the term “gene” includes not only the transcription sequence but also, additionally, the untranslated region, which includes upstream and downstream regulatory regions, enhancers, and promoters. The term "gene" can refer to an "endogenous gene" or native gene located in a natural position within the genome of an organism. It can also refer to an "exogenous gene" or non-native gene. A non-native gene can refer to a gene that is not normally found in a host organism but is introduced into the host organism through gene transfer. A non-native gene can also refer to a gene that is not in a natural position within the genome of an organism. Furthermore, a non-native gene may also include naturally occurring nucleic acid or polypeptide sequences containing mutations, insertions, and / or deletions (e.g., non-native sequences).

[0027] The terms “polynucleotide,” “oligonucleotide,” and “nucleic acid” are used to mean either a nucleotide, deoxyribonucleotide, or ribonucleotide, or an analog thereof, in a polymerized form of any length, interchangeably, whether in single-stranded, double-stranded, or multi-stranded form. Polynucleotides may be exogenous or endogenous to cells. Polynucleotides may exist in a cell-free environment. Polynucleotides may be genes or fragments thereof. Polynucleotides may be DNA. Polynucleotides may be RNA. Polynucleotides may have any three-dimensional structure and may perform any known or unknown function. Polynucleotides may contain one or more analogues (e.g., altered backbone, sugars, or nucleic acid bases).

[0028] The term "expression" refers to one or more processes by which polynucleotides are transcribed from a DNA template (e.g., mRNA or other RNA transcripts), and / or the process by which the transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides may collectively be referred to as "gene products." When polynucleotides are derived from genomic DNA, expression may include the splicing of mRNA within eukaryotic cells. In relation to expression, "up-regulated" generally means an increase in the expression level of polynucleotides (e.g., RNA such as mRNA) and / or polypeptides relative to their wild-type expression level, while "down-regulated" generally means a decrease in the expression level of polynucleotides (e.g., RNA such as mRNA) and / or polypeptides relative to their wild-type expression level.

[0029] As used herein, the term "regulating" in relation to expression or activity means altering the level of expression or activity. Regulation can occur at the transcriptional and / or translational levels.

[0030] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein to mean polymers of at least two amino acid residues linked by peptide bonds. These terms are not intended to imply a specific length of the polymer, nor to imply or distinguish whether a peptide is produced using recombinant techniques, chemical or enzymatic synthesis, or of natural origin. The terms apply to naturally occurring amino acid polymers as well as to amino acid polymers containing at least one modified amino acid. In some cases, polymers may also be interrupted by non-amino acids. The terms include amino acid chains of any length, including full-length proteins, or proteins with or without secondary and / or tertiary structures (e.g., domains). The terms also include amino acid polymers modified by any other operation, such as disulfide bond formation, glycosylation, lipid modification, acetylation, phosphorylation, oxidation, and conjugate with labeling components, for example.

[0031] The term "amino acid" generally refers to natural or non-natural amino acids, and includes, but is not limited to, modified amino acids and amino acid analogs. Modified amino acids may include natural amino acids and non-natural amino acids that have been chemically modified to contain groups or chemical sites that do not exist naturally. Amino acid analogs may refer to amino acid derivatives. The term "amino acid" includes both D-amino acids and L-amino acids.

[0032] Where used herein in relation to polypeptides, the terms “derivative,” “variant,” and “fragment” mean polypeptides that are related to the wild-type polypeptide by any of the following: amino acid sequence, structure (e.g., secondary and / or tertiary), activity (e.g., enzymatic activity), and / or function. Polypeptide derivatives, variants, and fragments may include one or more amino acid differences (e.g., mutations, insertions, and deletions), truncations, modifications, or combinations thereof compared to the wild-type polypeptide.

[0033] As used herein, “fusion” may mean a protein and / or nucleic acid containing one or more non-natural sequences (e.g., a portion). A fusion may contain one or more identical non-natural sequences. A fusion may contain one or more different non-natural sequences. A fusion may be a chimera. A fusion may include a nucleic acid affinity tag. A fusion may include a barcode. A fusion may include a peptide affinity tag. A fusion may provide site-specific intracellular localization of polypeptides (e.g., nuclear localization signals (NLS) targeting the nucleus, mitochondrial localization signals targeting mitochondria, chloroplast localization signals targeting chloroplasts, endoplasmic reticulum (ER) retention signals, etc.). A fusion may provide non-natural sequences (e.g., affinity tags) that can be used for tracking or purification. A fusion may include small molecules such as biotin or dyes, e.g., Alexa fluor® dyes, cyanine 3 dyes, cyanine 5 dyes.

[0034] The term "neoantigen" generally refers to tumor-specific antigens resulting from gene mutations. The resulting mutant proteins, or fragments thereof, can trigger an antitumor T cell response.

[0035] The term "genetic profile" refers to information about specific genes, including mutations and gene expression in an individual or certain type of tissue. Genetic profiles can be used for neoantigen selection. The term "somatic 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.

[0036] The term "antibody" refers to a protein-binding molecule with immunoglobulin-like function. The term "antibody" includes multiple antibodies (e.g., monoclonal and polyclonal antibodies), and their derivatives, variants, and fragments. Antibody derivatives, variants, or fragments may refer to functional derivatives or fragments that retain (e.g., completely and / or partially) the binding specificity of the corresponding antibody. Antigen-binding fragments include Fab, Fab', F(ab')2, variable fragments (Fv), single-chain variable post-fragment evant (scFv), minibodies, bispecific antibodies, and single-domain antibodies ("sdAb," "nanobodies," or "camelids"). The term "antibody" includes antibodies and antigen-binding fragments of optimized, engineered, or chemically conjugated antibodies. Examples of optimized antibodies include affinity-mature antibodies. Examples of engineered antibodies include Fc-optimized antibodies (e.g., antibodies optimized in crystalline fragment regions) and multispecific antibodies (e.g., bispecific antibodies).

[0037] As used herein, the term "artificial TCR" is understood to mean an exogenous T cell receptor (TCR) complex, which refers to a TCR complex in which one or more TCR chains are introduced into the genome of an immune cell that may or may not endogenously express a TCR. In some cases, an exogenous TCR complex may refer to a TCR complex in which one or more chains of an endogenous TCR complex have one or more mutant sequences, for example, at the nucleic acid level or the amino acid level. Expressing exogenous TCRs on immune cells can confer binding specificity to epitopes and antigens (e.g., epitopes and antigens that preferentially exist on the surface of cancer cells or other disease-causing cells or particles). An exogenous TCR complex may include the TCRα chain, TCRβ chain, CD3γ chain, CD3δ chain, CD3ζ chain, or any combination thereof, introduced into the genome. In some cases, the chains introduced into the genome may replace endogenously present chains.

[0038] Immune checkpoint molecules are molecules that regulate the immune response, and typically consist of receptor / ligand molecule pairs that can exert either an inhibitory or stimulating effect on the immune response.

[0039] In this specification, a co-stimulatory molecule means a cell surface molecule that amplifies or cancels the initial activation signal of a T cell. Unless otherwise specified, a co-stimulatory molecule in this specification is a positive co-stimulatory molecule, i.e., a co-stimulatory molecule that generates an activation signal.

[0040] The terms “subject,” “individual,” and “patient” are used interchangeably herein and are used to mean vertebrates, preferably mammals such as humans. Mammals include, but are not limited to, mice, monkeys, humans, livestock, sports animals, and pets. Tissues, cells, and their offspring of biological entities obtained in vivo or cultured in vitro are also included.

[0041] The terms “treatment” and “treating” mean, but are not limited to, an approach to obtain beneficial or desired results, including therapeutic and / or preventive benefits. For example, treatment may include administering cells, cell populations, or compositions disclosed herein. Therapeutic benefit means any improvement or effect, under treatment, relating to any treatment of one or more diseases, health conditions, or symptoms. For preventive benefit, the cells, cell populations, or compositions may be administered to subjects at risk of developing certain diseases, health conditions, or symptoms, or to subjects who have not yet developed a disease, health condition, or symptom but have reported one or more physiological symptoms of a disease.

[0042] The terms “effective amount” or “therapeutic effective amount” mean an amount of a composition, such as a composition containing immune cells of the present invention, such as lymphocytes (e.g., T lymphocytes and / or NK cells), that is sufficient to produce the desired activity when administered to a subject in need. The term “therapeutic effective” means an amount of a composition that is sufficient to delay the onset of symptoms, halt the progression of symptoms, or alleviate or reduce at least one symptom of a disease treated by the method of the present invention.

[0043] TIGIT / OX40 Enhanced Receptor TIGIT is a member of the poliovirus receptor (PVR) / nectin protein family and is an immune receptor primarily expressed on activated T cells, memory T cells, NK cells, and certain T regulatory cells (Tregs). TIGIT can bind to the high-affinity homologous ligand CD155 (also known as PVR) expressed on antigen-presenting cells (APCs), and this binding inhibits the immune response mediated by T cells, APCs, and NK cells. TIGIT has also been shown to compete with CD226 for binding to CD155, thereby balancing CD226-mediated co-stimulated T cell signaling. Due to these functions, TIGIT has become one of the popular targets in immunotherapy.

[0044] The TIGIT / OX40 enhanced receptor of the present invention comprises the extracellular domain of human TIGIT, or a fragment or variant thereof, wherein the fragment or variant has ligand-binding ability, and the ligand is CD155. Human TIGIT consists of 244 amino acids, with the extracellular domain comprising 141 amino acids and the transmembrane domain comprising 23 amino acids. In certain embodiments, the extracellular domain of the TIGIT / OX40 enhanced receptor of the present invention is the extracellular domain of human TIGIT, and has, for example, the amino acid sequence shown in SEQ ID NO: 9.

[0045] The TIGIT / OX40-enhanced receptor of the present invention comprises 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, including 37 amino acids in the cytoplasmic terminal, 217 amino acids in the extracellular domain, and 23 amino acids in the transmembrane domain. In certain embodiments, the intracellular domain of the TIGIT / OX40-enhanced receptor of the present invention is derived from the intracellular domain of wild-type human OX40 and has an amino acid sequence such as that shown in SEQ ID NO: 10.

[0046] In some embodiments, an enhanced receptor having TIGIT as its extracellular segment, where its intracellular segment is an intracellular domain derived from OX40, allows T cells to secrete more cytokines, such as IL-2 and IFNγ, and to survive longer in vivo. Therefore, this configuration is particularly preferred over the selection of intracellular domains derived from other co-stimulatory molecules, such as CD28 or ICOS.

[0047] The transmembrane region of the TIGIT / OX40-enhanced receptor of the present invention may optionally be derived from TIGIT or OX40, and for example, includes a transmembrane domain of TIGIT or OX40. In a preferred embodiment, the transmembrane domain is derived from OX40 and has the amino acid sequence shown in SEQ ID NO: 11.

[0048] Second reinforcement receptor Some embodiments of the present invention relate to immune cells modified to express two different enhanced receptors (also known as dual enhanced receptors), wherein the first enhanced receptor is the TIGIT / OX40 enhanced receptor of the present invention, and the second enhanced receptor comprises a second extracellular domain, a second transmembrane domain, and a second intracellular domain, wherein the second extracellular domain can specifically bind to a ligand of a T cell immune checkpoint molecule different from TIGIT, and the second intracellular domain is derived from a co-stimulatory molecule that mediates immune cell activation signals.

[0049] If the second extracellular domain is capable of specifically binding to a ligand of another T cell immune checkpoint molecule different from TIGIT, the other T cell immune checkpoint molecule is selected from the group consisting of transforming growth factor β receptor (TGF-β-R), programmed cell death 1 (PD-1), cytotoxic T lymphocyte-associated protein 4 (CTLA-4), B and T lymphocyte attenuation factor (BTLA), killer cell immunoglobulin-like receptor (KIR), indoleamine 2,3-dioxygenase (IDO), lymphocyte activation gene 3 (LAG3), and T cell immunoglobulin mucin 3 (TIM-3). For example, the second extracellular domain may be the extracellular domain of another T cell immune checkpoint molecule different from TIGIT, or a fragment thereof that retains ligand-binding ability. For example, the second extracellular domain may be an antibody or a fragment thereof, which specifically binds to a ligand of another T cell immune checkpoint molecule different from TIGIT, and for example, the antibody may be a single-chain antibody (scFv) or a single-domain antibody (VHH).

[0050] In a preferred embodiment, the other T cell immune checkpoint molecule is PD-1. Therefore, the second extracellular domain may be PD-1, or a variant or fragment thereof capable of binding to PD-L1; or it may be an anti-PD-L1 antibody or a fragment thereof capable of binding to PD-L1, such as a single-chain antibody against PD-L1 (scFv) or a single-domain antibody against PD-L1 (VHH). In a specific embodiment, the extracellular domain of the second enhanced receptor has the amino acid sequence shown in SEQ ID NO: 16. In a preferred embodiment, the extracellular domain of the second enhanced receptor is a variant of the extracellular domain of PD-1, the variant having improved affinity for PD-L1 compared to wild-type PD-1, and not binding to at least one known PD-1 antibody. For example, the extracellular domain of the second enhanced receptor has the amino acid sequence shown in SEQ ID NO: 12.

[0051] In some embodiments, the co-stimulatory molecule from which the second intracellular domain originates may 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. In some embodiments, the immune cell activation signal may be mediated by an activator. In some embodiments, the activator may be a soluble cytokine, a soluble chemokine, or a growth factor. In some embodiments, the activator is a soluble cytokine, where the soluble cytokine may be IL-1, IL-2, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, IL-21, TNF, TGF, IFN, or functional fragments or variants thereof. In some embodiments, the immune cell activation signal may include clonal increase of the modified immune cells, cytokine release by the modified immune cells, cytotoxicity of the modified immune cells, proliferation of the modified immune cells, differentiation, dedifferentiation, transdifferentiation of the modified immune cells, migration and / or transport of the modified immune cells, exhaustion and / or reactivation of the modified immune cells, and release of other intercellular molecules, metabolites, compounds, or combinations thereof by the modified immune cells.

[0052] In a preferred embodiment, the co-stimulatory molecule is CD28. In a specific embodiment, the intracellular domain of the second reinforcing receptor has the amino acid sequence shown in SEQ ID NO: 13.

[0053] In a specific embodiment, the second enhanced receptor is a PD1 / CD28 enhanced receptor comprising the following:

[0054] (a) A second extracellular domain derived from a variant or fragment thereof that is capable of binding to PD-1 or PD-L1, for example, an extracellular domain derived from a variant or fragment thereof that is capable of binding to PD-1 or PD-L1, preferably the second extracellular domain having an amino acid sequence such as that shown in SEQ ID NO:16 or SEQ ID NO:13;

[0055] (b) A second transmembrane domain derived from PD-1 or CD28, for example, a second transmembrane domain derived from a transmembrane domain of PD-1 or CD28, preferably a transmembrane domain derived from CD28, for example, a second transmembrane domain having the amino acid sequence shown in SEQ ID NO: 14; and

[0056] (c) A second intracellular domain derived from CD28, for example, a second intracellular domain having an amino acid sequence such as that shown in SEQ ID NO: 13, derived from the intracellular domain of CD28.

[0057] When cells possess two different enhanced receptor modifications, they can convert immunosuppressive signals mediated by different immune checkpoint molecules into activation signals. In this way, immune cells with two enhanced receptor modifications can exhibit greater activation and immune response compared to immune cells with one enhanced receptor modification. For example, the inventors have found that when immune cells possessing both TIGIT / OX40 enhanced receptor and PD-1(108) / CD28 enhanced receptor modifications are used, a synergistic effect can be achieved in tumor suppression, and this effect is significantly superior to that of immune cells with only one enhanced receptor modification used alone. Accordingly, the present invention also relates to a method for enhancing the effects (e.g., immune activation effect and immune response effect) of therapeutic immune cells using the TIGIT / OX40 enhanced receptor of the present invention, wherein the therapeutic immune cells may possess modifications other than the TIGIT / OX40 enhanced receptor, such as a second enhanced receptor and / or chimeric antigen receptor of the present invention.

[0058] In embodiments including a second enhanced receptor, the nucleotide sequence encoding the TIGIT / OX40 enhanced receptor and the nucleotide sequence encoding the second enhanced receptor can be introduced into immune cells for expression, either individually or in combination. For example, the nucleotide sequences encoding the two enhanced receptors can be linked by a self-cleaving peptide, such as the coding sequence for a 2A peptide. Examples of such 2A peptides include, but are not limited to, T2A, P2A, E2A, and F2A.

[0059] immune cells The enhanced receptor of the present invention is used to modify immune cells, particularly lymphocytes such as T cells, in order to enhance the immune-activating effect of said immune cells.

[0060] The immune cells prior to modification (in particular, before modification to express the enhanced receptor of the present invention) may originate from various sources and may be prepared by one or more of the following methods.

[0061] The immune cells that may be used in the present invention include, but are not limited to, peripheral blood mononuclear cells (PBMCs), peripheral blood lymphocytes (PBLs), and other blood cell subsets, including T cells, natural killer cells, monocytes, natural killer T cells, monocyte progenitor cells, hematopoietic stem cells, or non-pluripotent stem cells. In some cases, the cells can be any immune cell, such as T cells like CD3+ T cells, CD4+ T cells, CD8+ T cells, or any type of T cell, such as tumor-infiltrating lymphocytes (TILs). The T cells also include memory T cells, memory stem T cells, or other effector T cells.

[0062] Immune cells can be populations such as T cells, NK cells, and B cells obtained from a subject. T cells can be obtained from numerous sources including PBMCs, bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, and tissue from infection sites, ascites, pleural fluid, splenic tissue, and tumors. In some embodiments, T cells can be obtained from units of blood collected from a subject using any many techniques, such as Ficoll® isolation. In some embodiments, cells from an individual's circulating blood are obtained by apheresis. Products from apheresis typically include lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. Cells collected by apheresis may be washed to remove plasma fractions and to place cells suitable for subsequent processing steps into appropriate buffers or solvents.

[0063] T cells can also be selected from a mixed population, for example, by selecting T cells from whole blood. T cells can also be proliferated from a mixed population. In the process of selection and proliferation, T cells can also be skewed to a specific population and phenotype. For example, T cells can be phenotypically skewed to include CD45RO(-), CCR7(+), CD45RA(+), CD62L(+), CD27(+), CD28(+), and / or IL-7Rα(+). Appropriate cells containing one or more markers selected from the list including CD45RO(-), CCR7(+), CD45RA(+), CD62L(+), CD27(+), CD28(+), and / or IL-7Rα(+) can be selected.

[0064] Stem cells can also be used to obtain immune cells through differentiation. Stem cells include embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, neuronal stem cells, and mesenchymal stem cells. Immune cells may include any number of primary cells, such as human cells, non-human cells, and / or mouse cells.

[0065] Immune cells may originate from the subject being treated (e.g., the patient). Cells may originate from a healthy human donor. Immune cells may originate from a donor who is a direct relative of the subject, such as a donor who is HLA hemicompatible with the subject.

[0066] Preferably, immune cells have recognition specificity for target cells such as tumor cells. Such recognition specificity can be achieved by various methods, including, but is not limited to, genetic modification, co-culturing with antigen-presenting cells loaded with target cell antigens, and / or selection of specific cell populations.

[0067] In one embodiment, the immune cells of the present invention may be tumor-infiltrating lymphocytes (TILs). In some embodiments, the TILs may express at least one of PD-1, CD137, and TIM-3.

[0068] In some embodiments, the T cells may include a T cell receptor (TCR) complex that exhibits specific binding to a neoantigen. In some embodiments, the TCR complex may be an endogenous TCR complex. In some embodiments, the TCR complex may be an exogenous TCR complex.

[0069] In some embodiments, the neoantigen may be selected based on the genetic profile of a tumor sample from an individual. In some embodiments, the neoantigen may be selected based on the somatic mutation profile of a tumor sample from an individual.

[0070] In certain embodiments, the immune cells of the present invention are derived from peripheral blood mononuclear cells (PBMCs) and are sorted to enrich PBMCs with respect to PD1+ T cells. "Enrichment" means a process that results in an increase in the number or proportion of a particular cell or component in a population. In actual laboratory and clinical practice, cell preparation, sorting, or modification processes are often performed on samples (cell populations) containing multiple cells. Such enrichment operations are preferably performed so that the immune cells or immune cell populations used for further modification in the present invention have a higher proportion of PD1 than the unenriched PBMC population, before modifying the expression of the enhanced receptor. + This means that it contains T cells. For example, in patients with advanced solid tumors, peripheral blood PD1 + The proportion of T cells is approximately 28% on average. When immune cells are used for modification as immune cells of the present invention, it is preferable to further increase this proportion by enrichment, for example, sorting treatment. In a specific embodiment, the peripheral blood PBMCs are used, and PD1 + PD1 in sorted cell populations + The proportion of cells is 50% or more, preferably 60% or more, more preferably 70% or more, and especially preferably 80% or more.

[0071] After modification, immune cells obtained through the above method and source, particularly immune cells modified to express the enhanced receptor of the present invention, are referred to as modified immune cells.

[0072] Other modifications The modified immune cells of the present invention may have one or more other modifications in addition to enhanced receptor expression to confer further functions and / or properties.

[0073] The immune cells of the present invention may be modified to express a chimeric antigen receptor (CAR), the chimeric antigen receptor comprising (i) an antigen interaction domain capable of binding to B cell surface proteins; (ii) a transmembrane domain; and (iii) an intracellular signaling domain. Immune cells having a CAR can achieve immune cell expansion and / or activation after contact with B cell surface proteins.

[0074] In some embodiments, the B cell surface protein may be any protein that can be found on the surface of a B cell. Non-limiting examples include CD1d, CD5, CD10, CD11a, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD28, CD29, CD34, CD37, CD38, CD40, CD44, CD45, CD49b, CD69, CD72, CD74, CD80, CD83, CD84, CD86, CD93, CD95, CD117, CD127, CD138, CD147, CD148, CD185, CD270, CD284, and CD360. In some embodiments, the antigen-interacting domain of the CAR can bind to a surface protein on a non-B cell, provided that the binding to the surface protein does not significantly jeopardize the host's overall health or immune system. In some embodiments, the surface protein is a surface protein on an immune cell. In some embodiments, the surface protein is a surface protein on a cell other than an immune cell. In some embodiments, the surface protein is 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, CD6 2(E, L, P), CD63, CD64(A, B, C), CD66(a, b, c, d, e, f), CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD78, CD79(a, b), CD80, CD81, CD82, CD83, CD84, CD85(a, d, e, h, j, k), CD86, CD87, CD88, CD89, CD90, CD91, CD92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD100, CD1(ac), 1A, 1D, 1E, CD2, CD3(γ, δ, ε), CD4, CD5, CD6, CD7, 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, and CD186 may be selected. In a preferred embodiment, the B cell surface protein is selected from CD19, CD20, and CD22.

[0075] In some embodiments, the antigen-interacting domain of a CAR may be able to bind B cell surface proteins or fragments thereof on dead B cells. B cell apoptosis can occur before or after the onset of an immune response (e.g., an immune response against tumor cells). Therefore, dead B cells or their debris may still have B cell surface proteins or fragments thereof presented on their surface. The ability of CARs to target both living and dead B cells may increase the chances for immune cells containing CARs to (i) bind to B cell surface proteins and (i) initiate signal transduction of the intracellular signaling domain. In some cases, signal transduction of the intracellular signaling domain may promote the expansion (proliferation) of immune cells containing CARs.

[0076] In some embodiments, the antigen interaction domain of the CAR may be able to bind a B cell surface protein or a fragment thereof to the surface of a particle (e.g., nanoparticles) (e.g., via conjugated and / or non-conjugated bonds). The particle may be any particulate material, including organic and / or inorganic materials. The particle may have a variety of shapes and sizes. The particle may be about 1 nanometer (nm) to about 50 micrometers (μm) in at least one dimension. The particle may be at least about 1 nm, 5 nm, 10 nm, 50 nm, 100 nm, 500 nm, 1 μm, 5 μm, 10 μm, 50 μm, or more in at least one dimension. The particle may be at most 50 μm, 10 μm, 5 μm, 1 μm, 500 nm, 100 nm, 50 nm, 10 nm, 5 nm, 1 nm, or less in at least one dimension. The particles may be, for example, nanoparticles, microparticles, nanospheres, microspheres, nanorods, microrods, nanofibers, nanoribbons, etc. Examples of particles include, for example, metal nanoparticles (e.g., gold nanoparticles, silver nanoparticles, and iron nanoparticles), intermetallic compound nanosemiconductor nanoparticles, core-shell nanoparticles, particles having an inorganic core and a polymer shell, particles having an organic core and a polymer shell, or mixtures thereof. Alternatively, the particles may be, for example, crosslinked polymers, hydrogel polymers, biodegradable polymers, polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL), copolymers, polysaccharides, starch, cellulose, chitosan, polyhydroxyalkanoic acid (PHA), PHB, PHV, lipids, peptides, peptide amphiphiles, polypeptides (e.g., proteins), or combinations thereof. Particles presenting B cell surface proteins on their surface may be introduced in vitro into immune cells containing CARs that bind to B cell surface proteins. Alternatively, or additionally, particles displaying B cell surface proteins may be introduced in vivo (e.g., by localized or systemic injection) along with immune cells containing CARs.Such particles can be used in vitro or in vivo to increase populations of immune cells containing CARs.

[0077] The antigen-binding domain may include any protein or molecule capable of binding to an antigen, such as a B cell surface protein. Non-limiting examples of the antigen-binding domain include, but are not limited to, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, mouse antibodies, or functional derivatives, variants, or fragments thereof, which include, but are not limited to, Fab, Fab', F(ab')2, Fv, single-chain Fv(scFv), minibodies, bispecific antibodies, and single-domain antibodies, such as the heavy chain variable region (VH), light chain variable region (VL), and variable region (VHH) of a camel-derived nanobody. In some embodiments, the first antigen-binding domain includes at least one of Fab, Fab', F(ab')2, Fv, or scFv. In some embodiments, the antigen-binding domain includes an antibody mimetic. Antibody imitators are molecules capable of binding to target molecules with affinity equivalent to that of antibodies, and include, for example, single-chain binding molecules, cytochrome b562-based binding molecules, fibronectin or fibronectin-like protein backbones (e.g., adnectin), lipokine backbones, calixarene backbones, A-domains, and other backbones. In some embodiments, the antigen-binding domain includes a transmembrane receptor, or any derivative, variant, or fragment thereof. For example, the antigen-binding domain may include at least one ligand-binding domain of a transmembrane receptor.

[0078] In some embodiments, the antigen-binding domain may comprise an scFV. The scFV may be derived from an antibody whose variable region sequence is known. In some embodiments, the scFV may be derived from an antibody sequence obtained from an available mouse hybridoma. The scFV may be obtained from a whole exome sequence of tumor cells or primary cells. In some embodiments, the scFV can be modified. For example, the scFV can be modified in various ways. In some embodiments, the scFV can be mutated, thereby giving the scFV a higher affinity for its target. In some cases, the affinity of the scFV to its target can be optimized for targets that are expressed at low levels in normal tissues. This optimization may be done to minimize potential toxicity, such as hypercytokinemia. In other cases, cloning an scFV with higher affinity for a cell membrane-bound form of target may be preferred over its soluble form. This modification may be done when some targets can also be detected at varying levels in a soluble form, and their targeting may cause unintended toxicity, such as hypercytokinemia.

[0079] The antigen-binding domain of a CAR may bind to an intracellular signaling domain via a transmembrane domain. The transmembrane domain of the CAR may immobilize the CAR to the cell membrane of a cell, such as an immune cell. In some embodiments, the transmembrane segment comprises a polypeptide. The transmembrane polypeptide binding the antigen-binding domain and intracellular signaling domain of the CAR may have any suitable polypeptide sequence. In some cases, the transmembrane polypeptide comprises the polypeptide sequence of the transmembrane region of an endogenous or wild-type transmembrane protein. In some embodiments, the transmembrane polypeptide comprises a polypeptide sequence having at least one (e.g., at least two, three, four, five, six, seven, eight, nine, ten, or more) amino acid substitutions, deletions, and insertions compared to the transmembrane protein of an endogenous or wild-type transmembrane protein. In some embodiments, the transmembrane polypeptide comprises a non-native polypeptide sequence, such as the sequence of a polypeptide linker. The polypeptide linker may be mobile or rigid. The polypeptide linker may be structured or unstructured. In some embodiments, transmembrane polypeptides transmit signals from the extracellular to the intracellular region of a cell, for example, via an antigen-binding domain. The native transmembrane domain of CD28 may be used in CARs. In other cases, the native transmembrane domain of CD8 alpha may be used in CARs.

[0080] The CARs of the present invention may comprise signaling domains involved in immune cell signaling, or derivatives, variants, or fragments thereof. The intracellular signaling domains of the CARs may induce the activity of immune cells containing the CARs. The intracellular signaling domains may transmit effector function signals and instruct cells to perform specific functions. The signaling domains may comprise signaling domains of other molecules. In some cases, cleaved sites of signaling domains are used in the CARs.

[0081] In some embodiments, the intracellular signaling domain comprises multiple signaling domains involved in immune cell signaling, or derivatives, variants, or fragments thereof. For example, the intracellular signaling domain comprises at least two immune cell signaling domains, e.g., at least two, three, four, five, six, seven, eight, nine, or ten immune cell signaling domains. The immune cell signaling domain may be involved in the initial activation of the TCR complex, either in a stimulating or repressive manner. The intracellular signaling domain may also be the intracellular signaling domain of a T cell receptor (TCR) complex. The intracellular signaling domain of the CAR of the present invention may include the signaling domains of Fcγ receptor (FcγR), Fcε receptor (FcεR), FCα receptor (FcαR), neonatal Fc receptor (FcRn), CD3, CD3ζ, CD3γ, CD3δ, CD3ε, CD4, CD5, CD8, CD21, CD22, CD28, CD32, CD40L (CD154), CD45, CD66d, CD79a, CD79b, CD80, CD86, CD278 (also known as ICOS), CD247ζ, CD247η, DAP10, DAP12, FYN, LAT, Lck, MAPK, MHC complex, NFAT, NF-κB, PLC-γ, iC3b, C3dg, C3d, and Zap70. In some embodiments, the signaling domain may contain an immunoreceptor-activating tyrosine motif or ITAM. The ITAM-containing signaling domain may contain two repeats of the amino acid sequence YxxL / I, separated by 6-8 amino acids, where x is any amino acid independently, forming the conserved motif YxxL / Ix(6-8)YxxL / I. When the antigen-binding domain binds to an epitope, the ITAM-containing signaling domain may be modified, for example, by phosphorylation. Phosphorylated ITAM can function as a docking site for other proteins, such as proteins involved in various signaling pathways.In some embodiments, the primary signaling domain includes a modified ITAM domain, such as a mutated, cleaved, and / or optimized ITAM domain, the modified ITAM domain having altered (e.g., increased or decreased) activity compared to the natural ITAM domain.

[0082] In some embodiments, the intracellular signaling domain of the CAR of the present invention includes an FcγR signaling domain (e.g., ITAM). The FcγR signaling domain may be selected from FcγRI(CD64), FcγRIIA(CD32), FcγRIIB(CD32), FcγRIIIA(CD16a), and FcγRIIIB(CD16b). In some embodiments, the intracellular signaling domain includes an FcεR signaling domain (e.g., ITAM). The FcεR signaling domain may be selected from FcεRI and FcεRII(CD23). In some embodiments, the intracellular signaling domain includes an FcαR signaling domain (e.g., ITAM). The FcαR signaling domain may be selected from FcεRI(CD89) and Fcα / μR. In some embodiments, the intracellular signaling domain includes a CD3ζ signaling domain. In some embodiments, the primary signaling domain includes the ITAM of CD3ζ.

[0083] In some embodiments, the intracellular signaling domain of the CAR of the present invention may contain an immunoreceptor-activating tyrosine motif or ITIM. The signaling domain containing the ITIM may contain a conserved amino acid sequence (S / I / V / LxYxxI / V / L) found at the cytoplasmic end of several suppressor receptors of the immune system. Such a primary signaling domain containing the ITIM may be modified, for example, by an enzyme such as a member of the Src kinase family (e.g., Lck), such as by phosphorylation. Following phosphorylation, other proteins, including enzymes, may be added to the ITIM. These other proteins include, but are not limited to, the phosphorylated tyrosine phosphatases SHP-1 and SHP-2, an inositol monophosphate-degrading enzyme called SHIP, and proteins having one or more SH2 domains (e.g., ZAP70). The intracellular signaling domains include BTLA, CD5, CD31, CD66a, CD72, CMRF35H, DCIR, EPO-R, FcγRIIB (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), interleukin-4 receptor (IL4R), immunoglobulin superfamily receptor translocation-related 1 (IRTA1), immunoglobulin superfamily receptor translocation-related 2 (IRTA2), killer cell immunoglobulin-like receptor 2DL1 (KIR2DL1), and killer cell immunoglobulin-like receptor 2DL2 (KIR2DL2), killer cell immunoglobulin-like receptor; 2DL3 (KIR2DL3), killer cell immunoglobulin-like receptor; 2DL4 (KIR2DL4), killer cell immunoglobulin-like receptor; 2DL5 (KIR2DL5), killer cell immunoglobulin-like receptor; 3DL1 (KIR3DL1), killer cell immunoglobulin-like receptor; 3DL2 (KIR3DL2), leukocyte immunoglobulin-like receptor superfamily B member.1 (LIR1), Leukocyte Immunoglobulin-like Receptor Superfamily B Member 2 (LIR2), Leukocyte Immunoglobulin-like Receptor Superfamily B Member 3 (LIR3), Leukocyte Immunoglobulin-like Receptor Superfamily B Member 5 (LIR5), Leukocyte Immunoglobulin-like Receptor Superfamily B Member 8 (LIR8), Leukocyte-associated Immunoglobulin-like Receptor 1 (LAIR-1), Mast Cell Function-Associated Antigen (MAFA), NKG2A, Natural Cytotoxic Trigger Receptor 2 (NKp44), NTB-A, Programmed cell death protein 1 (PD-1), PILR, SIGLECL1, sialic acid-conjugated immunoglobulin-like lectin 2 (SIGLEC2 or CD22), sialic acid-conjugated immunoglobulin-like lectin 3 (SIGLEC3 or CD33), sialic acid-conjugated immunoglobulin-like lectin 5 (SIGLEC5 or CD170), sialic acid-conjugated immunoglobulin-like lectin 6 (SIGLEC6), sialic acid-conjugated immunoglobulin-like lectin 7 (SIGLEC7), sialic acid-conjugated immunoglobulin The signaling domains (e.g., ITAM) may include sialic acid-bound Ig-like lectin 10 (SIGLEC10), sialic acid-bound Ig-like lectin 11 (SIGLEC11), sialic acid-bound Ig-like lectin 4 (SIGLEC4), sialic acid-bound Ig-like lectin 8 (SIGLEC8), sialic acid-bound Ig-like lectin 9 (SIGLEC9), platelet and endothelial cell adhesion molecule 1 (PECAM-1), signal regulatory protein (SIRPα2), and signal threshold-regulated transmembrane adapter 1 (SIT). In some embodiments, the intracellular signaling domains include modified ITAM domains, such as mutated, cleaved, and / or optimized ITAM domains, which have altered (e.g., increased or decreased) activity compared to the native ITAM domain.

[0084] In some embodiments, the intracellular signaling domain includes at least two ITAM domains (e.g., at least three, four, five, six, seven, eight, nine, or ten ITAM domains). In some embodiments, the intracellular signaling domain includes at least two ITIM domains (e.g., at least three, four, five, six, seven, eight, nine, or ten ITIM domains) (e.g., at least two primary signaling domains). In some embodiments, the intracellular signaling domain includes both ITAM and ITIM domains.

[0085] In some cases, the intracellular signaling domain of the CAR of the present invention may include a co-stimulatory domain. In some embodiments, a co-stimulatory domain from, for example, a co-stimulatory molecule may provide a co-stimulatory signal for immune cell signaling, such as signaling from, for example, ITAM and / or ITIM domains, for example, for activation and / or inactivation of immune cell activity. In some embodiments, the co-stimulatory domain is operable to regulate proliferation and / or survival signals in immune cells. In some embodiments, the co-stimulatory signaling domain includes a signaling domain of an MHC class I protein, an MHC class II protein, a TNF receptor protein, an immunoglobulin-like protein, a cytokine receptor, an integrin, a signaling lymphocyte activator molecule (SLAM protein), an activated NK cell receptor, BTLA, or a Toll ligand receptor. In some embodiments, the co-stimulatory domains are 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 Ligands: TNFSF7, CD27 / TNFRSF7, CD28, CD29, CD2F-10 / SLAMF9, CD30; Ligands: TNFSF8, CD30 / TNFRSF8, CD300a / LMIR1, CD4, CD40; Ligands: TNFSF5, CD40 / TNFRSF5, CD48 / SLAMF2, CD49a, CD49D, CD49f, 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 HLA / TNFSF18, GITR / TNFRSF18, HLA-DR, HVEM / TNFRSF14, IA4, IC AM-1, ICOS / CD278, Ikaros, IL2Rβ, IL2Rγ, IL7Rα, α4 / CD49d α4β1 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), Lipoprotein Facilitator-1(LFA-1). NKG-α / TNF-β, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80(KLRF1), NTB-A / SLAMF6, OX40. 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, and TSLPThe intracellular signaling domain comprises a signaling domain of a molecule selected from the group consisting of R, VLA1, and VLA-6. In some embodiments, the intracellular signaling domain comprises multiple co-stimulatory domains, e.g., at least two, e.g., at least three, four, or five co-stimulatory domains. The co-stimulatory signaling region can provide a synergistic signal with the primary effector activation signal and can satisfy the requirements for T cell activation. In some embodiments, the addition of a co-stimulatory domain to the CAR can enhance the effects and persistence of the immune cells provided herein.

[0086] The binding of CARs to B cell surface proteins may enhance the proliferation of immune cells compared to immune cells without CARs. This proliferation may mean an increase in immune cells (expansion). This proliferation may also mean a change in the phenotype of the immune cells. The proliferation of immune cells containing CARs provided herein may be increased compared to that of equivalent immune cells without CARs exhibiting binding to B cell surface proteins. The proliferation of immune cells containing CARs is increased by approximately 5-10 times, 10-20 times, 20-30 times, 30-40 times, 40-50 times, 50-60 times, 60-70 times, 70-80 times, 80-90 times, 90-100 times, 100-200 times, 200-300 times, 300-400 times, 400-500 times, 500-600 times, and 600-700 times compared to the proliferation of equivalent immune cells without CARs. The proliferation of immune cells containing CARs can be increased by approximately 5-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-200, 200-300, 300-400, 400-500, 500-600, and 600-700 times compared to the proliferation of equivalent immune cells without CARs, and proliferation can be observed at least approximately 12, 24, 36, 48, 60, 72, 84, or 96 hours after contact of B cells with B cell surface proteins. Increased proliferation can be confirmed either in vitro or in vivo. In some embodiments, proliferation involves quantifying the number of immune cells. Quantifying the number of immune cells may include flow cytometry, trypan blue exclusion tests, and hematological counts. Proliferation may also be determined by phenotypic analysis of immune cells.

[0087] It is worth noting that the purpose of the CD19 CAR modification performed by the present invention is to assist in the increase and activation of immune cells (e.g., T cells) by binding to B cells. This is not entirely identical to the purpose and function of loading CD19 CARs onto T cells in general CD19 CAR-T technology, and the most important difference is that, in the present invention, the role of the CD19 CAR is not the recognition and binding of target cells, and therefore the modified immune cells of the present invention are not limited to the treatment of tumors associated with CD19-expressing B cells.

[0088] Preparation method Any suitable delivery method can be used to introduce the composition and molecules (e.g., polypeptides and / or nucleic acids encoding polypeptides of the present invention) into host cells, such as immune cells. Various components may be delivered simultaneously or separately and transiently. The choice of method may depend on the type of cells being transformed or the conditions under which the transformation occurs (e.g., in vitro, ex vivo, or in vivo).

[0089] The delivery method involves contacting a target polynucleotide with one or more nucleic acids containing nucleotide sequences encoding the enhanced receptor of the present invention or other modified molecules such as a chimeric antigen receptor, or introducing them into a cell (or cell population). The suitable nucleic acid containing the nucleotide sequences encoding the nucleotide sequences may include an expression vector, wherein the expression vector containing nucleotide sequences encoding one or more enhanced receptors or other modified molecules of the present invention is a recombinant expression vector.

[0090] Non-exclusive examples of delivery methods or transformations include, for example, viral or bacteriophage infection, gene transfer, binding, plasmofusion, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI) transfection, DEAE-dextran mediated transfection, liposome mediated transfection, particle-gun method, calcium phosphate precipitation, direct microinjection, and nanoparticle-mediated nucleic acid delivery.

[0091] In some embodiments, the present invention provides a method comprising delivering one or more polynucleotides, or one or more vectors described herein, or one or more transcripts thereof, and / or proteins translated therefrom, to a host cell. In some embodiments, the present invention further provides cells produced by such a method, or organisms (e.g., animals, plants, fungi, etc.) containing or produced from such cells.

[0092] Conventional viral and nonviral-based gene transfer methods may be used to introduce the nucleic acids into mammalian cells or target tissues. Such methods may be used to administer nucleic acids encoding the compositions of the present invention to cells or host organisms in culture. Nonviral vector delivery systems may include DNA plasmids, RNA (e.g., transcripts of vectors described herein), naked nucleic acids, and nucleic acids compounded with a delivery vehicle such as liposomes. Viral vector delivery systems may include DNA and RNA viruses, which may have genomes that are either episomal or integrated after delivery to cells.

[0093] Nonviral nucleic acid delivery methods may include lipofection, nucleofection, microinjection, particulate guns, viromosomes, liposomes, immunoliposomes, polycations, or lipid:nucleic acid complexes, naked DNA, artificial viral particles, and drug-enhancing DNA incorporation. Effective receptors for polynucleotides may be used for the recognition of cationic and neutral lipids suitable for lipofection. Delivery may be to cells (e.g., in vitro or ex vivo administration) or to target tissues (e.g., in vivo administration). Preparations of lipid:nucleic acid complexes, including target liposomes such as immunolipid complexes, may be used.

[0094] RNA or DNA virus-based systems can be used to target specific cells in the body and to deliver an effective viral payload to the cell nucleus. Viral vectors may be administered directly (in vivo), or they may be used to treat cells in vitro, and modified cells may optionally be administered (ex vivo). Virus-based systems may include retroviruses, lentiviruses, adenoviruses, and adeno-associated and herpes simplex virus vectors for gene transfer. Integration into the host genome can occur with retrovirus, lentivirus, and adeno-associated virus gene transfer methods, which can result in long-term expression of the inserted transgene. High transformation efficiencies can be observed in many different cell types and target tissues.

[0095] The affinity of retroviruses can be modified by incorporating exogenous envelope proteins, expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors that can transform or infect non-dividing cells and produce high viral titers. The choice of retroviral gene delivery may depend on the target tissue. Retroviral vectors may contain cis-acting terminal repeat sequences with the ability to package exogenous sequences up to 6–10 kb. The smallest cis-acting LTR is sufficient for vector replication and packaging, which can be used to deliver therapeutic genes to target cells for persistent transgene expression. Retroviral vectors may include those based on mouse leukemia virus (MuLV), gibbon leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof.

[0096] Adenovirus-based systems may be used. Adenovirus-based systems can induce transient expression of transgenes. Adenovirus-based vectors can have high transduction efficiency in cells and may not require cell division. High titers and levels of expression can be obtained with adenovirus-based vectors. Adenovirus-associated virus ("AAV") vectors can be used to transduce target nucleic acids into cells, for example, in the in vitro production of nucleic acids and peptides, and for in vivo or ex vivo gene therapy methods.

[0097] Packaging cells can be used to form viral particles capable of infecting host cells. Examples of such cells include 293 cells (e.g., for adenovirus packaging) and Psi2 or PA317 cells (e.g., for retrovirus packaging). Viral vectors can be created by producing cell lines that package nucleic acids into viral particles. The vector may contain the minimum viral sequence necessary for packaging and subsequent integration into a host. The vector may also contain other viral sequences that can be replaced by expression cassettes of polynucleotides (multiple polynucleotides) to be expressed. Missing viral functions can be supplied trans by the packaging cell line. For example, 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 within a cell line, which may include helper plasmids encoding other AAV genes, namely rep and cap, but lacking ITR sequences. Cell lines can also be infected with adenoviruses as helpers. Helper viruses can promote the replication of AAV vectors and the expression of AAV genes from helper plasmids. Adenovirus contamination can be reduced, for example, by heat treatment. Adenoviruses are more susceptible to heat treatment than AAV. Additional methods for delivering nucleic acids to cells, such as those described in U.S. Patent Application No. 20030087817, are also used and are incorporated herein by reference.

[0098] Host cells are transfected transiently or nontransiently with one or more vectors described herein. Cells may be transfected in a manner that occurs naturally in a subject. Cells may be taken from or derived from a subject and then transfected. Cells may be derived from cells taken from a subject, i.e., a cell line. In some embodiments, cells transfected with one or more vectors described herein are used to establish a new cell line containing sequences derived from one or more vectors. In some embodiments, cells transfected transiently with the compositions of the present invention (e.g., transient transfection with one or more vectors or transfection with RNA) are used to establish a new cell line containing cells that include modifications but do not contain other exogenous sequences.

[0099] Any suitable vector compatible with host cells can be used in the method of the present invention. Non-limiting examples of vectors for eukaryotic host cells include, for example, pXT1, pSG5 (Stratagene®), pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia®).

[0100] Contact with the cell composition may be made in any culture medium and under any culture conditions that promote cell viability. For example, cells may be suspended in a suitable nutrient solvent supplemented with Iscove's modified DMEM or RPMI 1640, fetal bovine serum or heat-inactivated goat serum (about 5-10%), L-glutamine, thiols, particularly 2-mercaptoethanol, and antibiotics such as penicillin and streptomycin. The culture medium may contain growth factors to which the cells respond. Growth factors as defined herein are molecules that can promote cell viability, growth, and / or differentiation in the culture medium or in untreated tissue through specific effects on transmembrane receptors. Growth factors may include polynucleotide factors and non-polynucleotide factors.

[0101] In many embodiments, the selected delivery system targets a specific tissue or cell type. In some cases, tissue or cell targeting of the delivery system is achieved by binding the delivery system to a tissue or cell-specific marker, such as a cell surface protein. Viral or nonviral delivery systems can be customized for the target tissue or cell type of interest.

[0102] When preparing a population of modified immune cells for therapeutic use, it is preferable that the proportion of cells possessing the desired modification, such as TIGIT / OX40, be at a level of 50% or higher, for example, about 60%, 70%, 80%, or 90% or higher. It should be understood that the modified immune cells and associated therapeutic methods of the present invention are individualized therapeutic strategies based on individual patients, and even when prepared in a similar manner, the proportion of effective cells in the final cell product may vary due to individual differences.

[0103] use Various target cells can be killed using immune cells modified with the enhanced receptor of the present invention. The target cells to which this method can be applied may include a wide range of cell types. Target cells may be in vitro. Target cells may be in vivo. Target cells may be ex vivo. Target cells may be isolated cells. Target cells may be cells inside organs. Target cells may be organs. Target cells may be cells in cell culture. Target cells may be a single cell in a collection of cells. Target cells may be mammalian cells or derived from mammalian cells. Target cells may be rodent cells or derived from rodents. Target cells may be human cells or derived from human cells. Target cells may be prokaryotic cells or derived from prokaryotic cells. Target cells may be bacterial cells or derived from bacterial cells. Target cells may be archaeal cells or derived from archaeal cells. Target cells may be eukaryotic cells or derived from eukaryotic cells. Target cells may 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 invertebrate cells or derived from invertebrate cells. The target cells may be vertebrate cells 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. The target cells may originate from a specific organ or tissue.

[0104] Target cells may be stem cells or derived from progenitor cells. Target cells may include stem cells (e.g., adult stem cells, embryonic stem cells, induced pluripotent stem (iPS) cells, etc.) and progenitor cells (e.g., cardiomyocyte progenitor cells, neural progenitor cells, etc.). Target cells may include mammalian stem cells and progenitor cells such as rodent stem cells, rodent progenitor cells, human stem cells, human progenitor cells, etc. Cloned cells may include progenitor cells. Target cells may contain target nucleic acids. Target cells may be within a living organism. Target cells may be genetically modified cells. Target cells may be host cells.

[0105] The target cells may be primary cells. For example, primary cells may be cultured 0, 1, 2, 4, 5, 10, 15 times or more. The cells may be unicellular organisms. The cells may grow in a culture medium.

[0106] Target cells may be disease cells. Disease cells may have altered metabolism, gene expression, and / or morphological characteristics. Disease cells may be cancer cells, diabetic cells, and apoptotic cells. Disease cells may be cells derived from a subject suffering from a disease. Exemplary diseases may include vascular disorders, cancer, metabolic disorders, eye diseases, organ disorders, musculoskeletal disorders, and heart disease.

[0107] Cancer cells are the specific object of interest. In some embodiments, the target cells are cancer cells. In some embodiments, the targeted cancer cells are solid tumor cells. In some embodiments, the cancer is of the hematopoietic spectrum, such as lymphoma. In certain embodiments, the lymphoma is a B-cell lymphoma. The B-cell lymphoma may be Hodgkin lymphoma or non-Hodgkin lymphoma, and this includes, but is not limited to, large B-cell lymphomas such as diffuse large B-cell lymphoma (DLBCL), non-specific (NOS) diffuse large B-cell lymphoma (DLBCL), mediastinal large B-cell lymphoma (PMBCL), follicular cell lymphoma, and mantle cell lymphoma. The tumor may be a refractory or recurrent tumor, for example, when the tumor patient has received prior therapy directed at the tumor.

[0108] In some embodiments, target cells form tumors. When treated with cells having the enhanced receptor of the present invention, this can result in stabilized tumor growth (e.g., one or more tumors do not increase in size by more than 1%, 5%, 10%, 15%, or 20%, and / or do not metastasize). In some embodiments, tumors are stabilized for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more weeks. In some embodiments, tumors are stabilized for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more months. In some embodiments, tumors are stabilized for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more years. In some embodiments, the size of the tumor or the number of tumor cells 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. In some embodiments, the tumor is completely eliminated or reduced to below detection levels. In some embodiments, the subject remains tumor-free (e.g., in remission) for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more weeks after treatment. In some embodiments, the subject remains tumor-free for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more months after treatment. In some embodiments, subjects maintain a tumor-free state for at least approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years or more after treatment.

[0109] Target cell death can be detected by any suitable method, for example, by measuring cells before and after treatment, or by measuring the levels of markers associated with live or dead cells (e.g., live or dead target cells), although these methods are not limited to these. The degree of cell death can be measured by any suitable method. In some embodiments, the degree of cell death is measured in relation to initial conditions. For example, an individual has a known initial amount of target cells, such as an initial cell mass of a known size or circulating target cells at a known concentration. In such cases, the degree of cell death can be expressed as the ratio of viable cells after treatment to the initial cell population. In some embodiments, the degree of cell death can be measured by a suitable cell death assay. A variety of cell death assays are available, and a variety of detection methodologies are available. Examples of detection methodologies include, but are not limited to, cell staining, microscopy, flow cytometry, cell sorting, and the use of combinations thereof.

[0110] If a tumor is subjected to surgical excision following the completion of a treatment period, the effectiveness of a procedure to measure tumor size can be measured by measuring the percentage of necrotic (i.e., dead) excised tissue. In some embodiments, the procedure is therapeutically effective when the percentage of necrotic excised tissue is greater than about 20% (e.g., at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%). In some embodiments, the percentage of necrotic excised tissue is 100%, meaning that there is no living tumor tissue or it is undetectable.

[0111] Exposure of target cells to immune cells or immune cell populations as invented herein can be carried out either in vitro or in vivo. Exposure of target cells to immune cells or immune cell populations in vitro can be achieved by co-culturing the target cells and immune cells. Target cells and immune cells can be co-culturned, for example, as adherent cells, or alternatively, in suspension. Target cells and immune cells can be co-culturned in various suitable types of cell culture solvents, for example, with adjuvants, growth factors, ions, etc. Exposure of target cells to immune cells or immune cell populations in vivo can, in some cases, be achieved by administering immune cells to a subject, for example, a human subject, so that the immune cells can localize to target cells via the circulatory system. In some cases, immune cells can be delivered immediately, for example, by direct injection, to the region where the target cells are localized.

[0112] Exposure may occur for any appropriate period of time, for example, 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 longer.

[0113] In therapeutic applications, the modified immune cells or immune cell populations invented herein may be administered to a subject already suffering from a disease or condition in a sufficient amount to cure or at least partially cessate the symptoms of the disease or condition, or to cure, heal, improve, or induce remission of the condition. The effective dose for this use may vary based on the severity and course of the disease or condition, previous treatments, the subject's health status, weight, and responsiveness to the drug, as well as the decision to administer the treatment.

[0114] When used for therapeutic purposes, the immune cells of the present invention having an enhanced receptor can be administered at about 1×10 7 to about 1×10 12 cells, preferably about 1×10 8 to about 1×10 11 cells, for example about 1×10 7 , about 1×10 8 , about 1×10 9 , about 1×10 10 , about 1×10 11 , about 1×10 12 to the subject at a dose. In some embodiments, the cell dosage is the total amount of cells administered.

[0115] The dose of therapeutic cells to be administered can also be determined based on the body weight of the subject. Specifically, the immune cells of the present invention having an enhanced receptor 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 cells / kg (subject body weight), about 5×10 6 cells / kg (subject body weight), about 6.7×10 6 cells / kg (subject body weight), about 1×10 7 cells / kg (subject body weight), about 3.3×10 7 cells / kg (subject body weight), about 5×10 7 cells / kg (subject body weight), about 6.7×10 7 cells / kg (subject body weight), about 1×10 8 cells / kg (subject body weight).

[0116] Immune cells of the present invention having a dual-enhancing receptor have a lower effective dose compared to immune cells having only a single-enhancing receptor. In some embodiments, the immune cells having a dual-enhancing receptor can achieve similar or superior therapeutic effects at a dose at least 50% lower, or even an order of magnitude lower, compared to immune cells having only a single-enhancing 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 PD1 / CD28-enhancing receptor or a PD1(108) / CD28-enhancing receptor. [Examples]

[0117] For a complete understanding and application of the present invention, the invention will be described in detail below with reference to embodiments and drawings, but embodiments are intended to illustrate the invention only and not to limit the scope of the invention. The scope of the invention is particularly limited by the appended claims.

[0118] Example 1 Preparation of a lentiviral vector expressing an enhanced receptor To determine the effects of the TIGIT / OX40 enhanced receptor of the present invention on other enhanced receptors, the following different lentiviral vectors were prepared by transfecting 293T cells with a fourth-generation lentiviral vector system (tetraplasmid system) using the PEI (Polysciences, 23966-1) reagent transfection reagent: 1) NY-ESO-1 TCR: An artificial transcatheter tumor receptor (TCR) that targets the tumor target NY-ESO-1; 2) CD19 CAR: A CAR that targets CD19; 3) TIGIT / OX40: These are reinforced receptors; ECD is derived from TIGIT, ICD from OX40, and TM from OX40; 4) TIGIT / OX40-TCR: The TIGIT / OX40 gene described in 3) above and the artificial TCR gene targeting the tumor target NY-ESO-1 described in 1) above are constructed in the same vector, the two gene sequences are linked with a T2A sequence, and the T2A sequence is sheared with a shear enzyme to individually express the TIGIT / OX40-enhanced receptor protein and the TCR protein on the cell membrane, thereby creating non-fusion expression of the two proteins; 5) TIGIT / OX40-CAR: Non-fusion expression of a CAR targeting TIGIT / OX40 as described in 3) above and CD19 as described in 2) above; 6) TIGIT / CD28-TCR: This is a non-fusion 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, and the ICD and TM are derived from CD28; 7) PD1 / CD28-TCR: This is a non-fusion expression of an artificial TCR targeting the enhanced receptor PD1 / CD28 and the tumor target NY-ESO-1. The enhanced receptor PD1 ECD was derived from wild-type PD1, while the ICD and TM were derived from CD28.

[0119] The preparation of the corresponding lentiviral vector was described using the example of TIGIT / OX40-TCR. The purpose of loading TCRs onto cells is to enable them to recognize tumor cells with the corresponding antigen. In animal (mouse) experiments, the mouse tumors were xenografts from humans, and modification of the TCR allowed therapeutic immune cells to recognize the xenografts. This was possible because it allowed for the mimicry of immune cells with modified tumor recognition properties in practical applications. In practice, tumor recognition properties are obtained not only by loading TCRs (e.g., preparing novel antigen-recognizing TCRs based on the individual's mutation profile), but also by isolating tumor-infiltrating lymphocytes (TILs) and extracting PD1 from peripheral blood to obtain cells with tumor recognition properties. + It should be understood that these can also be obtained by isolating T cells. In the latter two cases, loading of TCRs that recognize tumor antigens or novel antigens is not necessary, so this embodiment also describes a protocol for preparing only the TIGIT / OX40 enhanced receptor.

[0120] First, a major vector expressing the TIGIT / OX40-TCR construct was constructed. The TIGIT / OX40-TCR construct was constructed by spacing the TIGIT / OX40 enhanced receptor gene sequence and the TCR gene sequence within the same vector using a T2A sequence. TIGIT / OX40 consists of the TIGIT extracellular domain (TIGIT ECD), the OX40 transmembrane domain (OX40™), and the OX40 intracellular domain (OX40 ICD), and their nucleotide sequences are shown in Table 1 below.

[0121] Table 1 Sequence composition of the TIGIT / OX40-TCR construct The primary vector, TIGIT / OX40-TCR plasmid, the packaging vectors pMDL-gag plasmid and Rev plasmid, and the envelope vector pMD2.G plasmid were mixed in a 1:2:2:1 ratio and co-transfected into 293T (ATCC) cells using PEI reagent. After culturing the transfected cells at 37°C for 48 hours, the supernatant was collected and concentrated for lentivirus by ultracentrifugation. The lentivirus pellet obtained by centrifugation was resuspended in 1 mL of 1640 medium, dispensed into 10 x 1.5 mL EP tubes (100 μl each), and stored at -80°C.

[0122] The titer of the TIGIT / OX40-TCR enhanced receptor lentiviral vector was tested. 100 μl of the original virus solution was collected and serially diluted 3-fold, 9-fold, 27-fold, 81-fold, 243-fold, and 729-fold. 50 μl of virus sample was collected from each dilution and infected 293T cells for 48 to 72 hours. The culture supernatant was then removed, the cells were washed with PBS, and trypsin was added. The 293T cells were harvested, stained for TIGIT with anti-TIGIT antibody (Biolegend, 372714), and the percentage of TIGIT+ cells in cells infected with each dilution was analyzed by flow cytometry. The titer was calculated according to the following formula: Titer (TU / ml) = Initial number of 293T cells (approx. 40,000-45,000) × TIGIT + cell ratio × Dilution factor × 20 (The first viral dilution factor in which the percentage of TIGIT+ cells was less than 20% was used in the calculation.)

[0123] Using a similar approach, the seven constructs described above were constructed based on the amino acid and nucleotide sequences shown below. 293T cells were transfected using lentiviral vectors containing these constructs.

[0124] Table 2. Arrangement of other elements JPEG0007943704000002.jpg32170 Following the method described above, the percentage of antibody-labeled cells was detected by flow cytometry through different antibody labels, and the titer of each lentivirus was calculated. 7 If the above conditions are met, it will be ready to proceed to the next step.

[0125] Example 2 Preparation of tumor-recognizing immune cells equipped with enhanced receptors In this example, T cells were used as immune cells carrying enhanced receptors, and the T cells were modified to possess specific recognition properties for certain tumor cells, thereby achieving specific binding between immune cells and tumor cells in in vitro experiments.

[0126] To compare the effects of different enhanced receptors, we prepared a total of four different tumor-recognizing T cells, each possessing a NY-ESO-1-TCR that specifically recognizes NY-ESO-1. Here, ii) to iv) are modified with enhanced receptors. Details are as follows: i) NY-ESO-1-TCR-T: T cells modified to express the NY-ESO-1-TCR, which can target tumor cells expressing NY-ESO-1 via the artificial TCR, and are also called "TCR-T"; ii) TIGIT / OX40-TCR-T: T cells modified to express the enhanced receptor TIGIT / OX40 and the NY-ESO-1-TCR, which can target tumor cells expressing NY-ESO-1 via the artificial TCR; iii) TIGIT / CD28-TCR-T: T cells modified to express the enhanced receptor TIGIT / CD28 and the NY-ESO-1-TCR, which can target tumor cells expressing NY-ESO-1 via the artificial TCR; iv) PD1 / CD28-TCR-T: T cells modified to express the enhanced receptor PD1 / CD28 and the NY-ESO-1-TCR, which can target tumor cells expressing NY-ESO-1 via the artificial TCR.

[0127] As described above, TCRs confer tumor recognition characteristics to immune cells, and various enhanced receptors were used to enhance the efficacy of immune cells.

[0128] Peripheral blood mononuclear cells were collected from tumor patients by apheresis, and PD1 was obtained from the apheresis cells. + Cells were isolated and divided equally into five groups. CD3 / CD28 DynaBeads were added separately to each group and stimulated overnight. The following day, lentiviral vectors containing 1) NY-ESO-1-TCR, 4) TIGIT / OX40-TCR, 6) TIGIT / CD28-TCR, and 7) PD1 / CD28-TCR from Example 1 were added to four of the cell groups according to an MOI (number of lentiviruses / number of cells) of 2. The transfected cells (TCR-T cells, TIGIT / OX40-TCR-T cells, TIGIT / CD28-TCR-T cells, PD1 / CD28-TCR-T cells) were cultured and grown, and T cells without any added virus were used as a control (Mock-T).

[0129] Example 3: In vitro activation effect of TIGIT / OX40-enhanced receptor on T cells To confirm the function of the TIGIT / OX40-enhanced receptor, the tumor-associated antigen NY-ESO-1 was used as the target, and the human fibrosarcoma cell line HT1080 (donated by Beijing JOINN Laboratories (China) Co., Ltd.), which naturally expresses NY-ESO-1, was used as the target cell. Furthermore, the HLA-A*02:01 gene was expressed in the HT1080 cells. All four cells constructed in Example 2 expressed TCR-T targeting NY-ESO-1, and these cells were used as effector T cells. The tumor cells HT1080 and the T cells were co-cultured, and the activation levels of T cells modified with different modifications were measured by detecting the amounts of IFN-γ and IL-2 secreted from the T cells. The results are shown in Figures 1A-B.

[0130] As shown in Figures 1A-B, the amounts of IL-2 and IFN-γ secreted by TIGIT / OX40-TCR-T cells were significantly higher than those secreted by PD1 / CD28-TCR-T cells, TIGIT / CD28-TCR-T cells, and TCR-T cells. In vitro experiments showed that in the HT1080 tumor cell model, immune activation of TCR-T cells expressing enhanced receptors was stronger, and the effects of the two enhanced receptors with TIGIT as the extracellular domain were superior to those of the enhanced receptor with PD1 as the extracellular domain. Among these, the activation level by TIGIT / OX40 was shown to be the highest.

[0131] Example 4: Effects of TIGIT / OX40-enhanced receptor on T cell activation in vivo To further investigate the role of the TIGIT / OX40-type enhanced receptor, we first constructed a J82-NYESO1 (J82-NY) tumor cell line by loading and expressing the NY-ESO-1 tumor antigen in human bladder metastatic cancer cells J82 (purchased from the Chinese Academy of Medical Sciences, catalog number 1101HUM-PUMC000346). Specifically, a lentivirus containing NY-ESO-1 was added to J82 cells according to an MOI of 5, and after 72 hours, G418 was added to screen for positive cells. Approximately two weeks later, NY-ESO-1 expression was detected by flow cytometry. Flow cytometry results showed that more than 95% of the J82 cells expressed NY-ESO-1, indicating successful cell construction. Subsequently, a xenograft mouse model of J82-NY tumor cells was constructed.

[0132] 1 x 10 6 Individual tumor cells (J82-NY) were subcutaneously inoculated into NSG mice (purchased from Beijing IDMO Co., Ltd.). Tumor volume was 80-120 mm². 3 When the target was reached, 30 mice were selected and randomly divided into the following 6 groups (5 mice per group), and each group received one of the following 6 types of cell therapy: a. Frozen medium administration group (Frozen medium group); b. A group of T cells that have not undergone transduction (Mock-T group); c. A group of TCR cells transduced solely from NY-ESO-1 (TCR-T group); d. A group of TCR cells transduced with PD1 / CD28-enhanced receptors and NY-ESO-1 (PD1 / CD28-TCR-T group); e. TCR cell group transduced with TIGIT / CD28-enhanced receptor and NY-ESO-1 (PD1 / CD28-TCR-T group); f. A group of TCR cells transduced with TIGIT / OX40-enhanced receptor and NY-ESO-1 (TIGIT / OX40-TCR-T group).

[0133] Each group has a total of 8 x 10 6 Cells (effective cell count 6 × 10) 6200 μl of the solution was administered as a single injection via the tail vein, and the control group received the same amount of 0.2 ml of cell freezing medium. After administration, the size of the tumors and the condition of the mice were observed every 2-3 days. Tumor volume was calculated using the formula "tumor volume = 1 / 2 × major diameter × minor diameter × minor diameter," and the average tumor volume of 5 mice in each group was calculated. Observation continued for 70 days, and the results are shown in Figures 2 and 3.

[0134] As shown in Figures 2 and 3, the tumor volume of mice in the frozen medium group and the Mock-T group continued to increase. On day 44 of observation, mice in both groups were euthanized due to excessive tumor volume. In mice in groups c, d, e, and f, which were administered tumor-recognizing TCR-T cells, the tumor volume began to decrease after day 10 and showed the strongest efficacy around day 25. However, with the extension of the observation period, tumors began to recur in mice in the TCR-T group and gradually increased after day 35. By day 70, the tumor volume exceeded the tumor volume at the start of administration, and tumor progression was observed in 4 out of 5 mice in the TCR-T group, with the other mouse confirmed to have died on day 70. The average tumor volume of mice in the PD1 / CD28-TCR-T group also continued to increase after day 46. The efficacy was well maintained in the TIGIT / CD28-TCR-T group and the TIGIT / OX40-TCR-T group. As shown in the enlarged figure 3, in the TIGIT / OX40 group, the tumors in the mice remained completely absent until the end of the observation period and did not recur. However, tumor recurrence was observed in some mice in the TIGIT / CD28 group.

[0135] These results indicate that TIGIT / OX40 not only enhances the tumor-suppressing effect of TCR-T cells, but also plays a stronger role than the other two enhancing receptors in maintaining T cell function and efficacy.

[0136] Example 5 Experiment 1: Combination of enhanced receptors TIGIT / OX40 and PD1 / CD28 In this embodiment, T cells were introduced simultaneously with two enhanced receptors. The two enhanced receptors were (1) the enhanced receptor PD1(108) / CD28 constructed based on the PD1 mutant 108#, and (2) TIGIT / OX40 as described in Example 1.

[0137] Preparation of lentiviral vectors expressing the PD1(108) / CD28-TIGIT / OX40 dual-enhanced receptor. Compared to wild-type PD1, PD1 mutant 108# was able to bind to human PD-L1 with higher affinity, but did not bind to many anti-PD1 antibodies, including cintilimab, nivolumab, camrelizumab, pemlolizumab, and tripalimab.

[0138] To enable T cells to express both of the two enhanced receptors, the nucleotide sequence encoding PD1(108) / CD28 was ligated using the nucleotide sequence encoding TIGIT / OX40 and the nucleotide sequence encoding T2A (SEQ ID NO: 22), and this was introduced into a lentiviral vector as the dual enhanced receptor construct PD1(108) / CD28-TIGIT / OX40. In this way, after transfection in a lentiviral system, the two enhanced receptors are expressed in the transfected T cells with the help of the self-cleaving peptide T2A, and the two enhanced receptors function separately to co-regulate the T cells. The amino acid sequence and encoding nucleotide sequence of PD1(108) are shown in SEQ ID NO: 12 and SEQ ID NO: 4, respectively.

[0139] Lentiviral vectors were constructed in the same manner as in Example 1, transfected into 293T cells, and then measured for titer. Specifically, lentiviral vectors containing the following gene sequences were constructed: 1) PD1(108) / CD28-TIGIT / OX40-TCR: Non-fusion expression of the dual-enhancing receptor construct PD1(108) / CD28-TIGIT / OX40 and the tumor target NY-ESO-1 described in Example 1.

[0140] Each lentiviral titer was calculated. 3 × 10 7 A higher potency was considered more suitable for further use.

[0141] In vitro activation effect of PD1(108) / CD28-TIGIT / OX40 bi-enhancing receptor on T cells To confirm the function of the PD1(108) / CD28-TIGIT / OX40 dual-enhanced receptor, J82-NY cells overexpressing NY-ESO-1 were used as target cells and co-cultured with T cells possessing a TCR that specifically recognizes NY-ESO-1. By measuring the amounts of IFN-γ and IL-2 secreted from T cells with different modifications, the effect of modification by the enhanced receptor on T cells was detected on the degree of T cell activation.

[0142] To compare the effects of different enhanced receptors, four types of tumor-recognizing T cells, each possessing a NY-ESO-1-TCR that specifically recognizes NY-ESO-1, were prepared using the same method as in Example 2. Here, ii) to iv) had further different enhanced receptor modifications. Details are as follows: i) NY-ESO-1-TCR-T: T cells modified to express the NY-ESO-1-TCR, which can target tumor cells expressing NY-ESO-1 via an artificial TCR, and are also called TCR-T; ii) PD1 / CD28-TCR-T: T cells modified to express the enhanced receptor PD1 / CD28 and the NY-ESO-1-TCR, which can target tumor cells expressing NY-ESO-1 via the artificial TCR; iii) Anti-PDL1 / CD28-TCR-T: These are T cells modified to express the enhanced receptor anti-PDL1 / CD28 and NY-ESO-1-TCR, which can target tumor cells expressing NY-ESO-1 via the artificial TCR. Here, anti-PDL1 indicates that this enhanced receptor possesses an anti-PD-L1 single-chain antibody (PD-L1 scFv) as its ECD, and its amino acid and nucleotide sequences are shown in SEQ ID NO: 15 and SEQ ID NO: 7, respectively. iv) PD1(108) / CD28-TIGIT / OX40-TCR-T: These are T cells modified to express two enhanced receptors, PD1(108) / CD28 and TIGIT / OX40-TCR-T, and can target tumor cells expressing NY-ESO-1 via an artificial TCR.

[0143] The cells of species i) and ii) described above were prepared as described in Examples 1 and 2. The preparation of cells of species iii) was similar to that of cells of species ii) in that both enhanced receptors bind to PD-L1, but while ii) used the PD-L1 ligand receptor PD-1 as the ECD, iii) used the anti-PD-L1 antibody scFv as the ECD. The preparation of cells of species iv) was achieved using the lentiviral vector described in this example and through a transfection process similar to that in Example 2.

[0144] The density of J82-NY cells was adjusted to 1 × 10⁶ cells / mL, and 100 μl of this was placed in a U-shaped bottom 96-well plate. According to the effector cell:target cell ratio of 1:1 relative to J82-NY cells, 100 μl of each of the four types of T cells were collected and added to the U-shaped bottom 96-well plate containing the J82-NY cells. After co-culture at 37°C for 24 hours, the plate was placed in a centrifuge and centrifuged at 400 g for 5 minutes. When collecting 150 μl of the culture supernatant, care was taken to avoid aspirating cells from the bottom, and it was transferred to a new 96-well plate. After collecting the supernatant, the plate was sealed with a sealing film and stored at 4°C. ELISA was performed the following day.

[0145] ELISA was performed using the Human IL-2 Detection Kit (ELISA method) (Dakewe Biotech Co., Ltd., product code 1110203) following these steps: Microtiter plates were pre-coated with anti-human IL-2 capture antibody. Standards or samples and detection antibody (biotin-labeled anti-human IL-2 antibody) were added to the microtiter plates simultaneously, and horseradish peroxidase-labeled streptavidin (SA-HRP), which specifically binds to biotin in the immune complex, was added. The enzyme substrate tetramethylbenzidine (TMB) was added, resulting in a blue color. The intensity of the color was proportional to the IL-2 concentration in the standard or sample. After 5-10 minutes for color development, the reaction was terminated by adding the reaction termination solution, and the absorbance (OD) at 450 nm was measured using a multimode microplate reader. The concentration of human IL-2 was proportional to the OD value within a certain range, and a standard curve was obtained by ELISA using an IL-2 standard. The IL-2 concentration in each co-culture was calculated using this standard curve. The results of this experiment are shown in Figure 4.

[0146] ELISA was performed using the Human IFNγ Detection Kit (ELISA method) (Dakewe Biotech Co., Ltd., product code 1110003) following these steps: A standard or sample and detection antibody (biotin-labeled anti-human IFNγ antibody) were added simultaneously to a microtiter plate, and horseradish peroxidase-labeled streptavidin (SA-HRP), which specifically binds to biotin in the immune complex, was added. The enzyme substrate tetramethylbenzidine (TMB) was added, resulting in a blue color, the intensity of which correlated with the IFNγ concentration in the standard or sample. After 5-10 minutes for color development, the reaction was terminated by adding the reaction termination solution, and the absorbance (OD) at 450 nm was measured using a multimode microplate reader. The human IFNγ concentration was proportional to the OD value within a certain range, and a standard curve was obtained by ELISA using an IFNγ standard. The IFNγ concentration in each co-culture was calculated based on this standard curve. The results of this experiment are shown in Figure 5.

[0147] As shown in Figure 4, IL-2 secreted from PD1(108) / CD28-TIGIT / OX40-TCR-T cells was significantly higher than IL-2 secreted from PD1 / CD28-TCR-T cells, anti-PDL1 / CD28-TCR-T cells, and TCR-T cells. Furthermore, as shown in Figure 5, IFN-γ secreted from PD1(108) / CD28-TIGIT / OX40-TCR-T cells was significantly higher than IFN-γ secreted from anti-PDL1 / CD28-TCR-T cells and TCR-T cells, but there was no significant difference in the amount of IFN-γ secreted from PD1 / CD28-TCR-T cells. In vitro experiments revealed that TCR-T cells that underwent dual-enhancing receptor modification showed significantly higher levels of activation.

[0148] Animal experiments using NY-ESO-1 TCR-T cells expressing the PD1(108) / CD28-TIGIT / OX40 enhanced receptor. To investigate the role of the PD1(108) / CD28-TIGIT / OX40 bi-enhanced receptor, in vivo mouse experiments were performed using the same method as in Example 4.

[0149] 1 x 10 6 Individual tumor cells (J82-NY) were subcutaneously inoculated into NSG mice. The tumor volume was 80-120 mm². 3 Upon reaching this point, 25 mice were selected and randomly divided into the following 5 groups (5 mice per group), and each group received one of the following 5 types of cell therapy: a. A group of T cells that have not undergone transduction (Mock-T group); b. A group of TCR cells transduced solely from NY-ESO-1 (TCR-T group); c. A group of TCR cells transduced with PD1 / CD28-enhanced receptors and NY-ESO-1 (PD1 / CD28-TCR-T group); d. A group of TCR cells transduced with anti-PDL1 / CD28 enhanced receptor and NY-ESO-1 (anti-PDL1 / CD28-TCR-T group); e. A group of TCR cells transduced with PD1(108) / CD28-TIGIT / OX40 enhanced receptor and NY-ESO-1 (PD1(108) / CD28-TIGIT / OX40-TCR-T group).

[0150] Each group, except for the Mock-T group, received a single injection of 6 × 10⁶ TCR-positive active cells into the tail vein. The amount of cells injected into the tail vein of each mouse in the Mock-T group was the same as the total amount of cells administered to each mouse in the remaining groups. After administration, tumor size was measured and the condition of the mice was observed every 2-3 days. Tumor volume was calculated using the formula "tumor volume = 1 / 2 × major diameter × minor diameter × minor diameter". The observation period was 37 days, and the results are shown in Figures 6 and 7.

[0151] As shown in Figures 6 and 7, the tumor volume of mice in the Mock-T group continuously increased. On the other hand, the tumor volume of mice in groups b, c, d, and e initially increased but then decreased. Tumors in mice in the TCR-T group shrank the fastest in the early stages, but after day 28, they began to gradually increase again as the tumors progressed. The average tumor volume of mice in the PD1 / CD28-TCR-T group also showed an increasing trend after day 35. The PD1(108) / CD28-TIGIT / OX40-TCR-T group showed the highest efficacy. These results suggest that T cells possessing a dual-enhancing receptor and capable of specifically recognizing tumors exhibit superior tumor suppression effects in vivo compared to T cells possessing only an enhanced receptor that specifically binds to PD-L1.

[0152] Example 6 Experiment 2 using both enhanced receptors TIGIT / OX40 and PD1 / CD28 To verify the effects of using TIGIT / OX40 and PD1(108) / CD28 together, an example was created. 5 Animal experiments were conducted again using the same method.

[0153] Preparation of lentiviral vectors expressing the PD1(108) / CD28-TIGIT / OX40 dual-enhanced receptor. Examples 5 Similar to the construct, the PD1(108) / CD28-TIGIT / OX40 dual-enhanced receptor was constructed by concatenating the coding sequences of PD1(108) / CD28 and TIGIT / OX40 using the coding sequence of T2A. A safer fourth-generation lentiviral vector system was used for transduction. The main vector, PD1(108) / CD28-TIGIT / OX40, the packaging vector, pMDL-gag, Rev, and the envelope vector, pMD2.G, were co-introduced into HEK293T cells along with calcium phosphate or liposome-PEI. After 48 hours, the supernatant was collected and concentrated for lentivirus by centrifugation.

[0154] To determine the titer of the PD1(108) / CD28-TIGIT / OX40 enhanced receptor lentiviral vector, 50 μl of the vector was diluted threefold and used to infect 293T cells for 48 to 72 hours. The 293T cells were collected and stained for PD1(108). The percentage of PD1(108)-positive cells was analyzed by flow cytometry, and titer was calculated according to the formula: Titer (TU / ml) = Initial number of 293 T cells (approximately 40,000-45,000) × PD1(10⁸) + percentage of cells × dilution factor × 20 (the first viral dilution factor when the percentage of PD1(10⁸) + cells was less than 20% was used in the calculation).

[0155] Since the PD1(108) / CD28 enhanced receptor and the TIGIT / OX40 enhanced receptor are located on the same vector, the titer of the PD1(108) / CD28 enhanced receptor was used to represent the titer of the TIGIT / OX40 enhanced receptor.

[0156] A lentiviral vector containing the following gene sequence was constructed using a similar method: 1) Non-fusion expression of an artificial TCR targeting PD1(108) / CD28-TIGIT / OX40 and the tumor target NY-ESO-1, also known as PD1(108) / CD28-TIGIT / OX40-TCR; 2) An artificial TCR targeting PD1(108) / CD28 and the tumor target NY-ESO-1, also known as PD1(108) / CD28-TCR; 3) An artificial TCR targeting TIGIT / OX40 and the tumor target NY-ESO-1, also known as TIGIT / OX40-TCR; 4) Artificial TCR targeting the tumor target NY-ESO-1, also known as TCR.

[0157] Each lentiviral titer was calculated. 3 × 10 7 A higher potency was considered more suitable for further use.

[0158] Animal experiments using NY-ESO-1 TCR-T cells expressing the PD1(108) / CD28-TIGIT / OX40 bi-enhanced receptor. To investigate the role of the PD1(108) / CD28-TIGIT / OX40 dual-enhanced receptor, we used the tumor-associated antigen NY-ESO-1 as a target, and J82 tumor cells expressing NY-ESO-1 were used as target cells (J82-NY-ESO1, or J82-NY). TCR-T cells targeting NY-ESO-1 were used as effector T cells, and enhanced TCR-T cells were prepared by having the NY-ESO-1 targeting TCR-T cells simultaneously express a modified PD1(108) / CD28-TIGIT / OX40 receptor. 1 × 10⁶ tumor cells, J82-NY, were subcutaneously inoculated into NSG mice. When the tumor volume reached 80-120 mm³, 30 mice were selected and randomly divided into 6 groups, each receiving the following cell therapy: a. Frozen medium administration group (Frozen Medium Group); b. Non-transduced T cell group (Mock-T Group); c. TCR cell group transduced with NY-ESO-1 only (TCR-T Group); d. TCR cell group transduced with PD1(108) / CD28-enhanced receptor and NY-ESO-1 (PD1(108) / CD28-TCR-T Group); e. TCR cell group transduced with TIGIT / OX40-enhanced receptor and NY-ESO-1 (TIGIT / OX40-TCR-T Group); f. TCR cell groups transduced with PD1(108) / CD28-TIGIT / OX40 enhanced receptor and NY-ESO-1 (PD1(108) / CD28-TIGIT / OX40-TCR-T group). The element sequences contained in each group are the same as described above. All cells total 1.5 × 10⁻⁶ 7 pieces / fish(3×10 6 The drug was administered once via tail vein injection using individual TCR-positive cells per mouse. After administration, the tumor size was measured and the condition of the mice was observed every 2-3 days. Tumor volume was calculated using the formula "tumor volume = 1 / 2 × major diameter × minor diameter × minor diameter" and observed for 31 days.

[0159] The results are shown in Figure 8 and Table 3 (where tumor volume is the mean value for each group). Tumor volume continued to increase in the frozen medium group and the Mock-T group mice, while the tumor growth rate in the TCR-T group mice was slower than in the frozen medium group and the Mock-T group, indicating some degree of suppression of tumor growth. Tumor growth in all three groups of mice expressing both the enhanced receptor and TCR showed a decrease in tumor volume, suggesting that the expression of the enhanced receptor promotes the tumor suppressive function of TCR-T cells. Surprisingly, the PD1(108) / CD28-TIGIT / OX40-TCR-T group expressing the dual enhanced receptor showed a significant tumor suppressive effect and the longest duration of efficacy. The combination of the two enhanced receptors showed a synergistic effect in tumor suppression compared to cells containing only one of the enhanced receptor modifications.

[0160] Table 3 Tumor volume (mm³) at different time points in different treatment groups 3 ) JPEG0007943704000003.jpg58170

[0161] In vivo pharmacokinetic study of NY-ESO-1 TCR-T cells expressing the PD1(108) / CD28-TIGIT / OX40 enhanced receptor. To investigate the role of the PD1(108) / CD28-TIGIT / OX40 enhanced receptor, the in vivo metabolic status of TCR-T cells expressing the PD1(108) / CD28-TIGIT / OX40 enhanced receptor was evaluated using the J82-NY tumor model.

[0162] 1 x 10 6 Individual tumor cells (J82-NY) were subcutaneously inoculated into NSG mice. Tumor volume was 80-120 mm². 3At this point, 54 mice were selected and randomly divided into three groups, each receiving the following cell therapy: a. A group of TCR cells transduced with NY-ESO-1 alone (TCR-T group); b. A group of TCR cells transduced with PD1(108) / CD28-enhanced receptor and NY-ESO-1 (PD1(108) / CD28-TCR-T group); c. A group of TCR cells transduced with PD1(108) / CD28-TIGIT / OX40-enhanced receptor and NY-ESO-1 (PD1(108) / CD28-TIGIT / OX40-TCR-T group). In all cases, a single dose of 5 × 10⁶ TCR-positive cells per mouse was administered via tail vein injection. After reinjecting T cells, two mice were taken from each group on D0 (2 hours after injection), D1, D3, D7, D10, D14, D21, D28, and D35. The animals were anesthetized with isoflurane, euthanized, and whole blood (anticoagulated with EDTA) and tumor tissue were collected. The samples were stored at -80°C. Tumor size was measured and the condition of the mice was observed every 2-3 days. Tumor volume was calculated using the formula "tumor volume = 1 / 2 × major diameter × minor diameter × minor diameter" and observed for 35 days. The in vivo metabolic status of the administered therapeutic T cells in mice was evaluated by detecting the content of TCR genes in the peripheral blood and tumor tissue of different mice using qPCR.

[0163] As a result, as shown in Figure 9, TCR-T cells in the peripheral blood of mice did not show a significant proliferation phenomenon for 28 days after reinfusion, and an increase peak appeared at D35. On the other hand, PD1(108) / CD28-TCR-T cells and PD1(108) / CD28-TIGIT / OX40-TCR-T cells showed increase peaks at D10 and D14, respectively, and the degree of increase in peripheral blood was stronger for PD1(108) / CD28-TIGIT / OX40-TCR-T cells than for PD1(108) / CD28-TCR-T cells. This result suggests that the enhanced receptor PD1(108) / CD28-TIGIT / OX40 has a stronger promoting effect on the proliferation and survival of TCR-T cells in the peripheral blood of mice.

[0164] As a result, as shown in Figure 10, TCR genes could be detected in tumor tissue from D3 after reinjection of T cells, but their content was low. At D7, the content of TCR genes in tumor tissue increased in the TCR-T group and the PD1(108) / CD28-TCR-T group, but the content of TCR genes in tumor tissue of the PD1(108) / CD28-TIGIT / OX40-TCR-T group was lower than that of the other two groups. The peak increase in tumor tissue for the TCR-T group and the PD1(108) / CD28-TCR-T group appeared at D10, with the increase being stronger in the PD1(108) / CD28-TCR-T group than in the TCR-T group, and then decreasing thereafter. In tumor tissue from the PD1(108) / CD28-TIGIT / OX40-TCR-T group, the TCR gene content increased significantly from D10, peaking at D14. The increase was stronger than in the PD1(108) / CD28-TCR-T group, and the content remained high from D21 to D35, being the highest among the three groups. The enhanced receptor PD1(108) / CD28-TIGIT / OX40 showed a stronger effect in promoting the proliferation and survival of TCR-T cells in mouse tumor tissue.

[0165] Example 7: Effects of the enhanced receptor TIGIT / OX40 in human subjects. The effects of the enhanced receptor TIGIT / OX40 were further validated in clinical trials involving human subjects. Specifically, the effects of T cells (c610) modified with a dual-enhanced receptor using a combination of the TIGIT / OX40 enhanced receptor and PD1(108) / CD28 were compared with T cells (c410) modified with PD1(108) / CD28 alone. In this example, PD1(108) / CD28-TCR-T cells and PD1(108) / CD28-TIGIT / OX40-TCR-T cells were used in the same manner as described in Examples 4 to 7.

[0166] Preparation protocol for c410 (PD1(108) / CD28-CD19 CAR-T) cells and c610 (PD1(108) / CD28-TIGIT / OX40-CD19 CAR-T) cells

[0167] 1. Day 0: (1) PD1 + T cell sorting: a. Apheresis blood was collected from the patient, anti-PD1-Biotin antibody (BioLegend, 329934) was added, the mixture was thoroughly mixed, and incubated away from light; b. Washed with buffer and centrifuged; c. Resuspend in buffer, add 20 μl of anti-Biotin MicroBeads (Miltenyi Biotec, Germany, 170-076-709), mix well, and incubate away from light; d. Wash the cells with buffer, centrifuge, and resuspend them, while washing the column with buffer; e. After the cells have passed through the column, remove the adsorption column, place it in a new centrifuge tube, add buffer to the column, and quickly push the liquid out of the column with the piston, then PD1 + The cells were collected and counted; f. Depending on the needs of the experiment, an appropriate number of PD1 + The cells were centrifuged, the supernatant was discarded, and the cells were resuspended in complete culture medium.

[0168] (2) Anti-CD3 / CD28 magnetic beads were added to activate T cells: a. A suitable number of Dynabeads CD3 / CD28 (Thermo Fisher Scientific, 40203D) were taken according to a specific ratio and placed in a centrifuge tube; b. The magnetic beads were resuspended in X-VIVO medium (Lonza, BEBP02-054Q), washed, and allowed to stand on a magnetic rack until the beads adhered to the tube wall. After the beads had settled, the supernatant was removed by pipette. c. Cells and magnetic beads T SCMThe cells were resuspended in medium (X-VIVO + 2.5% CTS™ Immune Cell SR (Thermo Fisher Scientific, A2596102) + 2 U / ml Memory Stem Cell Expansion Factor (Shanghai Novoprotein Scientific Inc., GMP-1647)), transferred together to a culture flask, supplemented with TSCM medium, and the cell density was increased to 1.2 × 10⁶. 6 Adjusted to / ml; d. The cells were cultured in a 37°C, 5% CO2 incubator.

[0169] 2. Day 1: Addition of lentivirus a. Lentivirus transfection was performed within 24 hours of cell activation; b. Based on the type of cells to be prepared, the type of lentivirus to be used (410 or 610; where 410 is PD1(108) / CD28-CD19 CAR, and 610 is PD1(108) / CD28-TIGIT / OX40-CD19 CAR lentivirus) and MOI were determined, and the amount of lentivirus added was calculated from the number of cells as follows: lentivirus addition = number of cells × MOI / lentivirus titer. c. Remove the corresponding volume of lentivirus from a -80°C refrigerator, place it in ice, transfer it to the laboratory through a window, and leave it in the ice until it thaws; d. Cells to be treated with lentivirus were removed from the incubator, and the information of the cells to be treated with lentivirus, patient name, cell name, and preparation date was verified to confirm that there were no errors; e. The thawed lentivirus was added to the cells, mixed by blowing with a pipette, and then cultured in a 37°C, 5% incubator.

[0170] 3. Day 3: Replenish cells with fluid, and T according to the number of cells. SCM Culture medium was added.

[0171] 4. Day 5: Removal of magnetic beads a. In a biological safety cabinet, prepare magnetic racks and centrifuge tubes of the corresponding volume based on the volume of the magnetic bead culture medium (>20 ml: 50 ml centrifuge tube; ≤20 ml: 15 ml centrifuge tube; slightly less than 1 ml: EP tube). b. Cells containing magnetic beads were passed through a magnetic rack three times, the beads were removed, and the cell suspension was collected; c. The magnetic beads were washed 2-3 times, and the cell suspension was collected again; d. The cell suspensions obtained in b and c were combined, the total volume was recorded, and the cells were counted; e. The weights of the tubes were equalized, and the supernatant was discarded after centrifugation; f. Complete medium (X-VIVO + 2.5% CTS) TM Immune cells SR (Thermo Fisher Scientific, A2596102) + recombinant human interleukin 2 for injection (Beijing SL Pharmaceutical Co., Ltd., China National Drug Approval No. S19991010) were added, resuspended, transferred to culture flasks, and cell density was adjusted by supplementing with complete medium according to the counting results. The information was then labeled. g. Cultured in a 37°C, 5% CO2 incubator.

[0172] 5. Day 8: Cell count measured, fluid replenished. 6. Day 11: Cell count measured, fluid replenished. 7. Day 14:

[0173] (1) Recovery of finished cells: a. The cells to be harvested were removed from the incubator, the cell suspension was mixed with a pipette, samples were taken and used for counting cell number and viability, and some samples were stored for flow cytometry; b. Transfer the cell suspension to a 200 ml centrifuge tube / 50 ml centrifuge tube and centrifuge at 500 g for 8 minutes; c. After centrifugation, the supernatant was placed into two freezing tubes (one for mycoplasma, one for endotoxin, and one for the Gram test); d. Discard the supernatant, resuspend in physiological saline to a ratio of 200 ml / 50 ml, wash, and centrifuge at 600 g for 10 minutes; e. Discard the supernatant, resuspend the cells in quantitative saline, mix well, take a sample and use it to count the cell number and cell viability, centrifuge, and discard the supernatant; f. Cells were resuspended in a quantified amount of cell freezing medium product (Cryostor CS10, BioLife Solutions, 100-1061) according to the number of cells.

[0174] (2) Cryopreservation of cells: a. Cells with added freezing medium were quickly dispensed into freezing bags (each freezing bag needed to be labeled with patient name, number, cell details, freezing time, etc.), and the bags were placed in a programmed cooling box and frozen in a refrigerator at -80°C; b. After placing the bag in a -80°C refrigerator, it was transferred to liquid nitrogen for storage within 24 hours; Information such as the number of freezer bags was recorded.

[0175] Inclusion Criteria The study population enrolled in the c410 / c610 trial consisted of adult patients with relapsed or refractory CD19-positive large B-cell lymphoma following second-line or higher systemic therapy: c410 / c610 included nonspecific (NOS) diffuse large B-cell lymphoma (DLBCL), primary mediastinal large B-cell lymphoma (PMBCL), high-grade B-cell lymphoma and DLBCL transformed from follicular lymphoma, follicular cell lymphoma, and mantle cell lymphoma.

[0176] The dose increase group was 1 × 10 6 / kg, 3.3 × 10 6 / kg, 10 x 10 6 The subjects were divided into three groups based on their dose per kg, and based on the safety and initial efficacy of the dose escalation study, the dose escalation group was recommended as the cell dose for the expansion study.

[0177] The inclusion criteria were primarily: age 18 to under 70, regardless of sex, ECOG score of 0 or 1, peripheral blood PD1-positive T cells of 18% or higher of total T cells, basic cardiopulmonary reserve, and absence of serious hematological, hepatic, or renal abnormalities. The exclusion criteria were primarily as follows: active central nervous system lymphoma, coexistence of active central nervous system disease, coexistence of malignancy and immune system disease, severe cardiopulmonary impairment, presence of a clinical emergency, recent major surgery, and other unlisted investigational drugs or treatments.

[0178] Autologous peripheral blood mononuclear cells (PBMCs ≥ ​​3 × 10) were collected from subjects who passed the screening. 9 PD-1-positive T lymphocytes were obtained by sorting and used in the preparation of cell therapies.

[0179] Prior to cell reinfusion, subjects underwent detailed baseline examinations and lesion assessments. Subjects had received prior chemotherapy (FC regimen, cyclophosphamide 250-300 mg / m²). 2 and fludarabine 25-30 mg / m² 2 The patient received the treatment once a day for three days from D-5 to D-3, and the reinfusion of c410 / c610 cells was performed on D0.

[0180] Following cell reinfusion, subjects were followed up to evaluate the tolerability of c410 / c610 cells in the treatment of relapsed or refractory CD19-positive non-Hodgkin lymphoma, and dose-limiting toxicity (DLT), safety, and efficacy were observed.

[0181] result Table 4 shows the basic information and detailed data on treatment outcomes of the subjects who participated in the above clinical trial.

[0182] Following c410 / c610 cell therapy, all subjects experienced no dose-limiting toxicity and the treatment was well-tolerated, with CRS levels down to grade 2.

[0183] The objective response rate (ORR) was 0% (0 / 1, 0 / 3) in one case in the low-dose c410 cell group (1 × 10⁶ / kg) and 0% (0 / 1, 0 / 3) in three cases in the medium-dose group (3.3 × 10⁶ / kg). The high-dose group (10 × 10⁶ / kg) included a total of 6 cases, with a complete remission rate (CR) of 50% (3 / 6) at D28 and 33.3% (2 / 6) at W12. The ORR was 83.3% (5 / 6) at D28 and 33.3% (2 / 6) at W12.

[0184] Compared to c410, the complete response (CR) at D28 in the low-dose c610 cell group (1 × 10⁶ / kg) was 100% (2 / 2) in two subjects, which was significantly better than the treatment outcome with c410 cells.

[0185] As shown in the results in Table 4 below, the efficacy of immune cells containing only the PD1(108) / CD28 single receptor (c410) gradually increased with increasing dose (1E6 / kg in the low-dose group, 3.3E6 / kg in the medium-dose group, and 1E7 / kg in the high-dose group), but the risk of cytokine storm (CRS) also increased.

[0186] In contrast, immune cells containing the PD1(108) / CD28-TIGIT / OX40 dual-enhanced receptor (c610) showed significantly superior therapeutic efficacy and safety. Low-dose c610 (1E6 / kg) was far more effective than the comparable low-dose c410 group, significantly better than the high-dose c410 group, and had a lower CRS risk than each dose group of c410. The results of this clinical trial demonstrated that the TIGIT / OX40 enhanced receptor of the present invention, when used in combination with the PD1(108) / CD28 enhanced receptor, exhibits remarkable therapeutic effects against tumors. Table 4 JPEG0007943704000004.jpg240141

[0187] Sequence information JPEG0007943704000005.jpg240170JPEG0007943704000006.jpg240170JPEG0007943704000007.jpg238170JPEG0007943704000008.jpg239170JPEG0007943704000009.jpg228170

Claims

1. An enhanced receptor comprising an extracellular domain (ECD), a transmembrane domain (TM), and an intracellular domain (ICD), The aforementioned enhanced receptor is the TIGIT / OX40 enhanced receptor, The extracellular domain is the extracellular domain of human wild-type TIGIT. The intracellular domain is the intracellular domain of human OX40, An enhanced receptor in which the transmembrane domain is derived from the transmembrane domain of TIGIT or the transmembrane domain of OX40.

2. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the enhanced receptor described in claim 1, Isolated nucleic acid molecules containing one or more of the following nucleotide sequences: (1) The nucleotide sequence shown in SEQ ID NO: 1, which encodes the extracellular domain of TIGIT; (2) The nucleotide sequence shown in SEQ ID NO: 2, which codes for the intracellular domain of OX40; and (3) The nucleotide sequence shown in SEQ ID NO: 3, which encodes the transmembrane domain derived from OX40.

3. A recombinant expression vector comprising the isolated nucleic acid molecule described in claim 2.

4. A host cell comprising an isolated nucleic acid molecule according to claim 2, or a recombinant expression vector according to claim 3.

5. Immune cells, which, prior to the modification of the immune cells, were isolated from peripheral blood mononuclear cells (PBMCs) and subjected to PD-1 positive screening, and have been modified to express the enhanced receptor described in claim 1.

6. The extracellular domain of the TIGIT / OX40 enhanced receptor has the amino acid sequence shown in SEQ ID NO:

9. The immune cell according to claim 5, wherein the intracellular domain of the TIGIT / OX40 enhanced receptor has the amino acid sequence shown in SEQ ID NO:

10.

7. The immune cell according to claim 5, wherein the transmembrane domain of the TIGIT / OX40-enhanced receptor is the transmembrane domain of OX40 and has the amino acid sequence shown in SEQ ID NO:

11.

8. An immune cell, modified to further express a second enhanced receptor other than the enhanced receptor described in claim 1, wherein the second enhanced receptor comprises a second extracellular domain, a second transmembrane domain, and a second intracellular domain. Here, the second extracellular domain is capable of specifically binding to a ligand of a T cell immune checkpoint molecule different from TIGIT, and the second intracellular domain is derived from a co-stimulatory molecule that mediates immune cell activation signals. The immune cell according to claim 5, wherein the nucleotide sequence encoding the enhanced receptor according to claim 1 and the nucleotide sequence encoding the second enhanced receptor are introduced into the immune cell for expression, either alone or in combination.

9. The immune cell according to claim 8, wherein the extracellular domain of the second enhanced receptor can specifically bind to PD-L1.

10. The immune cell according to claim 9, wherein the intracellular domain of the second enhanced receptor is derived from CD28.

11. The immune cell according to claim 8, wherein the second extracellular domain is the extracellular domain of PD-1 and has the amino acid sequence shown in SEQ ID NO:

16.

12. The second extracellular domain is a variant of PD-1 that can bind to PD-L1, The immune cell according to claim 8, wherein the variant is a PD-1 variant (108) having the amino acid sequence shown in SEQ ID NO:

12.

13. The immune cell according to claim 8, wherein the second extracellular domain is a single-chain antibody (scFv) that can specifically bind to PD-L1.

14. Immune cells modified to express a chimeric antigen receptor (CAR), wherein the CAR is (a) Antigen interaction domain capable of binding to B cell surface proteins; (b) transmembrane domains; and (c) Intracellular signaling domain The immune cells according to claim 5, including the immune cells described in claim 5.

15. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the enhanced receptor described in claim 1 and a nucleotide sequence encoding a second enhanced receptor, The nucleotide sequence encoding the enhanced receptor described in claim 1 and the nucleotide sequence encoding the second enhanced receptor are linked by the coding sequence of the 2A peptide. The second enhanced receptor comprises a second extracellular domain, a second transmembrane domain, and a second intracellular domain. Here, the second extracellular domain is an isolated nucleic acid molecule that can specifically bind to a ligand of a T cell immune checkpoint molecule different from TIGIT, and the second intracellular domain is derived from a co-stimulatory molecule that mediates immune cell activation signals.

16. The isolated nucleic acid molecule according to claim 15, wherein the extracellular domain of the second enhanced receptor is the extracellular domain of PD-1 and has a nucleotide sequence shown in SEQ ID NO: 8 that encodes the amino acid sequence shown in SEQ ID NO:

16.

17. The isolated nucleic acid molecule according to claim 15, wherein the extracellular domain of the second enhanced receptor is a variant of PD-1 capable of binding to PD-L1, the variant being a PD-1 variant (108), and having a nucleotide sequence shown in SEQ ID NO: 4 that encodes the amino acid sequence shown in SEQ ID NO:

12.

18. The isolated nucleic acid molecule according to claim 15, wherein the second extracellular domain is a single-chain antibody (scFv) that can specifically bind to PD-L1.

19. The isolated nucleic acid molecule according to claim 15, wherein the intracellular domain of the second enhanced receptor is derived from CD28.

20. comprising an isolated nucleic acid molecule according to any one of claims 15 to 19, A recombinant expression vector that allows the nucleotide sequences encoding the TIGIT / OX40 enhanced receptor and the nucleotide sequence encoding the second enhanced receptor to be introduced into immune cells for expression.

21. An injectable pharmaceutical composition for the treatment of relapsed or refractory CD19-positive large B-cell lymphoma, comprising as an active ingredient immune cells as described in any one of claims 5 to 14, wherein the effective dose is 1 × 10⁻⁶ ６ / kg ~ 10 x 10 ６ A pharmaceutical composition characterized by being / kg body weight / day.

22. The pharmaceutical composition according to claim 21, wherein the relapsed or refractory CD19-positive large B-cell lymphoma includes nonspecific (NOS) diffuse large B-cell lymphoma (DLBCL), primary mediastinal large B-cell lymphoma (PMBCL), DLBCL derived from high-grade B-cell lymphoma and follicular lymphoma, follicular cell lymphoma, and mantle cell lymphoma.

23. The subjects received the FC regimen and cyclophosphamide 250-300 mg / m². ２ and fludarabine 25-30 mg / m² ２ The pharmaceutical composition according to claim 21, wherein the drug is administered once a day for three days from D-5 to D-3, and the reinjection of the immune cells is performed on D0.

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