PD1-CD28 chimeric signal conversion receptor and application thereof in immune cell therapy

Abstract

The invention relates to a PD1-CD28 chimeric signal conversion receptor and application of the PD1-CD28 chimeric signal conversion receptor in immune cell therapy, and particularly discloses a fusion protein containing a PD-L1 binding region derived from PD1 and a hinge region, a transmembrane region and an intracellular region derived from CD28, homodimers formed by the fusion protein, nucleic acid encoding the fusion protein and a carrier containing the nucleic acid. Also disclosed are immune cells, vector systems, compositions and kits that co-express the fusion proteins and chimeric antigen receptors (CARs), as well as methods of using the immune cells and vector systems to treat diseases in a subject.

Description

[0001] This application claims priority to Chinese Patent Application No. 2025101254018, filed on January 26, 2025, the entire contents of which are incorporated herein by reference for all purposes. Technical Field

[0002] This invention relates to chimeric signal transduction receptors and their uses. Background Technology

[0003] Cell-based immunotherapy is a promising treatment for cancer. T cells and other immune cells are modified to target tumor antigens by introducing genetic material encoding artificial or synthetic receptors that target the antigen. These receptors are called chimeric antigen receptors (CARs) and are specific to the selected antigens. In recent years, cell-based immunotherapy (especially CAR-T therapy) has achieved great success in cancer treatment. In particular, CAR-T cell therapy targeting the CD19 antigen has shown significant and durable tumor suppression effects in the treatment of hematologic malignancies (such as leukemia and lymphoma), becoming an effective clinical treatment for hematologic malignancies.

[0004] Traditional CAR-T therapy involves adding a single CAR structure targeting a tumor antigen (e.g., CD19) to T cells. However, patients may experience relapse and drug resistance due to immunosuppression in the tumor microenvironment and the short lifespan of T cells, leading to immune escape by tumor cells. Research indicates that blocking PD-L1 / PD1-mediated immunosuppression is key to overcoming the immunosuppression in the tumor microenvironment and successfully treating malignant tumors with CAR-T cell therapy.

[0005] Therefore, in order to further improve the efficacy of CAR-T cell therapy, it is necessary to develop a strategy that can block PD-L1 / PD1-mediated immunosuppression. Summary of the Invention

[0006] The inventors have discovered that inserting a cysteine-containing motif (e.g., PCS, CPSCPAP) between the CD28 hinge region and transmembrane region of the PD1-CD28 chimeric signal transducer receptor can more effectively convert PD-L1 / PD1-mediated immunosuppressive signals into activating signals compared to chimeric receptors without this cysteine ​​motif, thereby enabling T cells to exert a stronger tumor cell killing ability. It is known that CD28 signal activation requires the formation of a homodimer. The insertion of the cysteine ​​motif introduces new intermolecular disulfide bonds. The introduction of these additional intermolecular disulfide bonds makes the dimer structure formed by the PD1-CD28 chimeric signal transducer receptor more stable, resulting in a stronger downstream CD28 activation signal and a more powerful CAR-T cell killing effect. In vitro experiments show that the insertion of the cysteine ​​motif significantly enhances the anti-tumor effect of CAR-T cells, indicating that the PD1-CD28 chimeric signal transducer receptor with the cysteine ​​motif of this invention can be used to enhance the therapeutic efficacy of CAR-T and other immunocellular therapies.

[0007] Accordingly, in one aspect, the present invention provides a fusion protein comprising a PD-L1 binding region derived from PD1 and a hinge region, a transmembrane domain, and an intracellular domain derived from CD28, wherein the hinge region and the transmembrane domain derived from CD28 contain a cysteine ​​motif capable of stabilizing the homodimer formed by the fusion protein.

[0008] In some embodiments, the cysteine-containing motif comprises PCS or CPSCPAP.

[0009] In some embodiments, the PD-L1 binding region derived from PD1 comprises the human PD1 extracellular domain or its N-terminal fragment, preferably the N-terminal fragment comprising at least 120 amino acids.

[0010] In some implementations, the PD-L1 binding region derived from PD1 includes:

[0011] (i) The amino acid sequence of SEQ ID NO: 18 or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 18; or

[0012] (ii) The amino acid sequence of SEQ ID NO: 19 or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 19.

[0013] In some embodiments, the hinge region derived from CD28 is a human CD28 hinge region, preferably, the CD28 hinge region comprises the amino acid sequence of SEQ ID NO: 20 or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 20.

[0014] In some embodiments, the transmembrane domain derived from CD28 is a human CD28 transmembrane domain. Preferably, the CD28 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 16.

[0015] In some embodiments, the intracellular domain derived from CD28 is a human CD28 intracellular domain. Preferably, the CD28 intracellular domain comprises the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 17.

[0016] In some embodiments, the hinge region, transmembrane domain, and intracellular domain derived from CD28 comprise the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 21.

[0017] In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 1. In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 26 or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 26.

[0018] In some implementations, the fusion protein forms a homodimer when expressed on the cell surface.

[0019] In some embodiments, the fusion protein, when expressed on the cell surface, is able to bind to PD-L1 and convert inhibitory signals into activation signals.

[0020] In another aspect, the present invention provides a homodimer formed from the fusion protein described herein.

[0021] In another aspect, the present invention provides a nucleic acid comprising a nucleotide sequence encoding the fusion protein described herein.

[0022] In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 2 or a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 2. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 27 or a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 27.

[0023] In another aspect, the present invention provides a carrier comprising the nucleic acid described herein.

[0024] In another aspect, the present invention provides an immune cell comprising the fusion protein described herein.

[0025] In some implementations, the immune cells are tumor-infiltrating lymphocytes (TILs) or T cells.

[0026] In some implementations, the immune cells also contain chimeric antigen receptors (CARs).

[0027] In another aspect, the present invention provides an immune cell that co-expresses the fusion protein and chimeric antigen receptor (CAR) described herein.

[0028] In some embodiments, the immune cells are selected from tumor-infiltrating lymphocytes (TILs), T cells, NK cells, and macrophages.

[0029] In some implementations, the CAR includes an antigen-binding domain that binds to CD19, a hinge region derived from CD28, a transmembrane domain derived from CD28, an intracellular co-stimulatory domain derived from 4-1BB, and an intracellular signal transduction domain derived from CD3ζ.

[0030] In some embodiments, the antigen-binding domain is anti-CD19 scFv and contains the amino acid sequence of SEQ ID NO: 22.

[0031] In some embodiments, the hinge region comprises the amino acid sequence of SEQ ID NO: 15 or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 15.

[0032] In some embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 16.

[0033] In some embodiments, the intracellular co-stimulatory domain comprises the amino acid sequence of SEQ ID NO: 23 or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 23.

[0034] In some embodiments, the intracellular signal transduction domain comprises the amino acid sequence of SEQ ID NO: 24 or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 24.

[0035] In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 5.

[0036] In another aspect, the present invention provides a vector system comprising, on one or more vectors, a nucleotide sequence encoding the fusion protein described herein and a nucleotide sequence encoding the CAR described herein.

[0037] In some embodiments, the nucleotide sequence encoding the fusion protein comprises a nucleotide sequence of SEQ ID NO: 2 or higher that has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 2. In some embodiments, the nucleotide sequence encoding the fusion protein comprises a nucleotide sequence of SEQ ID NO: 27 or higher that has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 27.

[0038] In some embodiments, the nucleotide sequence encoding the CAR comprises a nucleotide sequence of SEQ ID NO: 6 that has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 6.

[0039] In some embodiments, the vector system comprises a nucleotide sequence encoding the fusion protein and a nucleotide sequence encoding the CAR on a vector, and the vector comprises an EF1α promoter upstream of the nucleotide sequence encoding the CAR and an MSCV promoter upstream of the nucleotide sequence encoding the fusion protein.

[0040] In some implementations, the vector is selected from lentiviral vectors, retroviral vectors, adenovirus vectors, adeno-associated virus (AAV) vectors, plasmids, DNA vectors, mRNA vectors, transposon-based vectors, and artificial chromosomes, with lentiviral vectors being preferred.

[0041] In another aspect, the present invention provides a composition comprising the fusion protein, homodimer, nucleic acid, carrier, immune cell, or carrier system described herein.

[0042] In another aspect, the present invention provides a kit comprising the fusion protein, homodimer, nucleic acid, vector, immune cell, or vector system described herein.

[0043] In another aspect, the present invention provides a method for treating a disease in a subject, comprising administering to the subject an effective amount of the immune cell or carrier system described herein, wherein the disease is selected from cancer, autoimmune diseases, inflammatory diseases, and infectious diseases.

[0044] In some embodiments, the cancer is a blood cancer (e.g., lymphoma, leukemia, or multiple myeloma) or a solid tumor (e.g., lung cancer). In a preferred embodiment, the cancer is Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, or chronic myeloid leukemia. In other preferred embodiments, the cancer is lung cancer.

[0045] In some implementations, the cells are autologous or allogeneic to the subject.

[0046] In some implementations, the method includes the following steps:

[0047] (i) Isolate a cell-containing sample from the subject;

[0048] (ii) Transducing or transfecting the cells using the vector system according to any one of claims 20-24; and

[0049] (iii) The cells obtained in step (ii) are administered to the subject.

[0050] In some embodiments, the method further includes administering a second therapeutic agent, preferably selected from antibodies, chemotherapeutic agents, and small molecule drugs. Attached Figure Description

[0051] Figure 1 The spectrum of the pCDH-MSCV-PD1 / CD28-EF1-CD19 CAR vector (5#, without PCS motif) is shown.

[0052] Figure 2The spectrum of the pCDH-MSCV-PD1 / CD28(PCS)-EF1-CD19 CAR vector (5V5#, containing the PCS motif) is shown.

[0053] Figure 3 The spectrum of the pCDH-MSCV-PD1 / CD28(CPSCPAP)-EF1-CD19 CAR vector (5V6#, containing the CPSCPAP motif) is shown.

[0054] Figure 4 The gel electrophoresis results of plasmids extracted from DH5α competent cell culture medium transformed from vectors 5#, 5V5#, and 5V6# are shown.

[0055] Figure 5 The percentage of CAR-positive and PD1-positive cells in T cells infected with lentiviral vectors 5# and 5V5# is shown.

[0056] Figure 6A The killing rate of CD19 CAR-T cells co-expressing 5V5# PD1-CD28 chimera (test-CART) and CD19 CAR-T cells co-expressing 5# PD1-CD28 chimera (con-CART) on target cells was shown after 24 hours and 48 hours of co-incubation with JVM-3 target cells.

[0057] Figure 6B The study showed the killing rate of CD19 CAR-T cells co-expressing 5#, 5V5#, or 5V6# PD1-CD28 chimeras on target cells after 24 and 48 hours of co-incubation with JVM-3 target cells.

[0058] Figure 7 The results of Western blot analysis of T cell lysates expressing chimeric receptors 5#, 5V5#, or 5V6# after co-incubation with Raji-PDL1 cells are shown.

[0059] Figure 8 The study showed the killing rate of target cells by T cells expressing chimeric receptors 5#, 5V5#, or 5V6# after co-incubation with Raji-PD-L1-Luc cells for 24 and 48 hours.

[0060] Figure 9 The study showed the IFN-γ release levels in the culture supernatant of TIL cells expressing chimeric receptors 5#, 5V5#, or 5V6# after 24 and 48 hours of co-incubation with TIL-homogeneous tumor cells. Detailed Implementation

[0061] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0062] It should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention.

[0063] In this application specification and claims, unless otherwise expressly stated herein, the singular forms “an,” “a,” and “the” include the plural forms.

[0064] Unless otherwise stated or defined, the terms “comprising,” “including,” and “containing” should be understood to mean that the said element or step or group of elements or steps are included, but do not exclude any other element or step or group of elements or steps.

[0065] The term "and / or" as used herein includes any one of two or more related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected using at least two conjunctions selected from "and / or," it should be understood that, in this application, the technical solution undoubtedly includes solutions connected by "logical AND," and also undoubtedly includes solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, AND / OR, B, AND / OR, C, AND / OR, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0066] When a numerical range is given in the implementation scheme, it should be understood that, unless otherwise stated in the present invention, the two endpoints of each numerical range and any value between the two endpoints may be selected.

[0067] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. Apart from the specific methods, apparatus, and materials used in the embodiments, based on the prior art mastery of one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, used, or made by those of ordinary skill in the art in implementing this invention may be used to implement this invention, and all such methods, apparatus, and materials shall fall within the protection scope of this invention. The embodiments of this invention are described in more detail below.

[0068] I. Definition

[0069] PD-1 (programmed death receptor-1), also known as CD279, is a type I transmembrane glycoprotein belonging to the B7-CD28 family. Full-length human PD-1 consists of 288 amino acids, comprising a signal peptide (SP, 1-23 AA), an extracellular domain (ECD, 24-170 AA), a transmembrane domain (TMD, 171-191 AA), and an intracellular domain (ICD, 192-288 AA). The extracellular domain contains an N-terminal IgV-like domain and a C-terminal IgC-like domain, used for binding to ligands PD-L1 / PD-L2. The transmembrane domain separates the extracellular and intracellular domains. The intracellular domain contains the immunoreceptor tyrosine inhibitory motif (ITIM) and the immunoreceptor tyrosine switching motif (ITSM), each containing one tyrosine residue. PD-1 is primarily found on the surface of immune cells, such as T cells, B cells, and NK cells. It inhibits T cell activity, reduces T cell response to antigens, promotes self-tolerance, and avoids excessive inflammatory response by binding to the ligand PD-L1 to form a PD-1 / PD-L1 complex.

[0070] The amino acid sequence of the wild-type full-length human PD1 molecule and the amino acid sequences of each domain are shown below:

[0071] Full-length sequence:

[0072] MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRV TERRAEVPTAHSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL (SEQ IDNO: 7)

[0073] Signal peptide:

[0074] MQIPQAPWPVVWAVLQLGWRPGW (SEQ ID NO: 8)

[0075] Extracellular domains:

[0076] FLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLV (SEQ ID NO: 9)

[0077] Transmembrane domains:

[0078] VGVVGGLLGSLVLLVWVLAVI (SEQ ID NO: 10)

[0079] Intracellular domains:

[0080] CSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL (SEQ ID NO: 11)

[0081] As used herein, the term "PD-L1 binding region derived from PD1" includes the portion of PD1 that binds to its ligand PD-L1, such as the extracellular domain of PD1 or a fragment thereof.

[0082] CD28 is a co-stimulatory molecule expressed on the surface of T lymphocytes, playing a crucial role in T cell activation. It binds to the B7 molecule on antigen-presenting cells, mediating T cell co-stimulation and promoting their survival, proliferation, and cytokine production. CD28 is a type I transmembrane protein, a homodimer composed of two 44kDa polypeptides linked by disulfide bonds, with a molecular weight of 90kDa. The full-length human CD28 molecule has a single polypeptide chain of 220 amino acids, consisting of a signal peptide (SP, 1-18AA), an extracellular domain (ECD, 19-152AA), a transmembrane domain (TMD, 153-179AA), and an intracellular domain (ICD, 180-220AA). The extracellular domain contains two immunoglobulin-like domains responsible for binding to ligands CD80 and CD86, and also includes a hinge region (114-152AA). The transmembrane domain enables CD28 to anchor to the cell membrane and undergo homodimerization via specific transmembrane motifs, which is crucial for its signal transduction function. The intracellular domain of CD28 contains multiple signal transduction motifs, which are phosphorylated by tyrosine kinases after CD28 activation, initiating downstream signal transduction.

[0083] The amino acid sequence of the wild-type full-length human CD28 molecule and the amino acid sequences of each domain are shown below:

[0084] Full-length sequence:

[0085] MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFC KIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS(SEQ ID NO: 12)

[0086] Signal peptide:

[0087] MLRLLLALNLFPSIQVTG (SEQ ID NO: 13)

[0088] Extracellular domains:

[0089] NKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ IDNO: 14)

[0090] Hinge area:

[0091] IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 15)

[0092] Transmembrane domains:

[0093] FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 16)

[0094] Intracellular domains:

[0095] RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 17)

[0096] As used herein, the term "cysteine-containing motif" refers to a polypeptide segment containing cysteine ​​residues, which is capable of forming a disulfide bond between two polypeptide chains containing the motif, and is 3-10 amino acids in length.

[0097] As used herein, the term "sequence identity" refers to the degree to which two (nucleotide or amino acid) sequences have identical residues at the same positions in an alignment, and is typically expressed as a percentage. Preferably, identity is determined over the overall length of the sequences being compared. Thus, two copies of identical sequences have 100% identity, but sequences with low conservation and deletions, additions, or substitutions may have a lower degree of identity. Those skilled in the art will recognize that several algorithms can be used to determine sequence identity using standard parameters, such as Blast (Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402), Blast2 (Altschul et al. (1990) J. Mol. Biol. 215: 403-410), Smith-Waterman (Smith et al. (1981) J. Mol. Biol. 147: 195-197), and Clustal W.

[0098] As used herein, the terms “nucleic acid,” “nucleic acid sequence,” and “polynucleotide” are intended to be synonymous with each other. Those skilled in the art will understand that, due to the degeneracy of the genetic code, many different polynucleotides and nucleic acids can encode the same polypeptide. Furthermore, it should be understood that those skilled in the art can use conventional techniques to perform nucleotide substitutions that do not affect the sequence of the polypeptide encoded by the polynucleotide described herein, reflecting the codon usage of any specific host organism in which the polypeptide is to be expressed. Nucleic acids according to the invention can comprise DNA or RNA. They can be single-stranded or double-stranded. They can also be polynucleotides comprising synthetic or modified nucleotides. Many different types of modifications to oligonucleotides are known in the art. These include methylphosphonate and thiophosphate backbones with the addition of an acridine or polylysine chain at the 3' and / or 5' ends of the molecule. For the purposes described herein, it should be understood that polynucleotides can be modified by any method available in the art. Such modifications can be made to enhance the in vivo activity or lifetime of the polynucleotide of interest.

[0099] As used herein, the term "vector" refers to a nucleic acid molecule that serves as a medium for transferring (exogenous) genetic material into a host cell, where the nucleic acid molecule, acting as a vector, can, for example, be replicated and / or expressed.

[0100] As used herein, the term "chimeric antigen receptor" or "CAR" refers to a molecule engineered to contain an antigen-binding domain that targets a specific antigen, and upon binding to that antigen, activates immune cells (such as T cells or NK cells, including naive T cells, central memory T cells, effector memory T cells, or combinations thereof) to attack and destroy cells carrying that antigen. When these antigens are present on tumor cells, CAR-expressing immune cells can target and kill the tumor cells.

[0101] A classic chimeric antigen receptor (CAR) is a chimeric type I transmembrane protein that links an extracellular antigen-binding domain to an intracellular signaling domain. The antigen-binding domain is typically a single-chain variable fragment (scFv) derived from a monoclonal antibody (mAb), but it can be based on other forms containing antibody-like antigen-binding sites or on a native ligand derived from the antigen. A hinge domain is usually required to separate the antigen-binding domain from the membrane and allow for its proper orientation. A common hinge domain used is the Fc of IgG1. Depending on the antigen, more compact spacer regions may be suitable, such as the stem from CD8α, and even just the IgG1 hinge alone. The transmembrane domain anchors the protein within the cell membrane and connects the hinge domain to the intracellular domain (intramocyte domain).

[0102] According to at least one non-limiting viewpoint, at least three “generations” of CAR molecules have existed. In first-generation CARs, they are designed with an intracellular domain having an intracellular portion of the γ chain derived from FcεR1 or the intracellular portion of CD3ζ. Therefore, these first-generation CARs deliver an immune signal¹ sufficient to trigger T cell killing of homologous target cells, but cannot fully activate T cell proliferation and survival. To overcome this limitation, second-generation CARs have been constructed with a complex intracellular domain resulting from the fusion of the intracellular portion of a T cell costimulatory molecule with the intracellular portion of CD3ζ, thus enabling the simultaneous delivery of activation and costimulatory signals after antigen recognition. The most commonly used costimulatory domain is the CD28 costimulatory domain. This provides the most potent costimulatory signal—i.e., an immune signal²—that triggers T cell proliferation. Several CARs have also been described that include intracellular domains of the TNF receptor family, such as the closely associated OX40 and 41BB, which deliver survival signals. Even more potent third-generation CARs have now been described, possessing intracellular domains capable of delivering activation, proliferation, and survival signals.

[0103] Therefore, a CAR typically comprises: (i) an antigen-binding domain; (ii) a hinge domain; (iii) a transmembrane domain; and (iv) an intracellular domain that includes a signal transduction domain and one or more co-stimulatory domains.

[0104] As used herein, the term "antigen-binding domain" refers to the portion of a chimeric antigen receptor that recognizes an antigen. In a classic CAR, the antigen-binding domain comprises a single-chain variable fragment (scFv) derived from a monoclonal antibody. CARs have also been generated using domain-dependent antibodies (dAbs), VHH antigen-binding domains, or antigen-binding domains derived from native ligands of the antigen.

[0105] The term "antigen" refers to any molecule that elicits an immune response or can be bound by an antibody or antigen-binding molecule. An immune response may involve antibody production or activation of specific immune-active cells, or both. Those skilled in the art will readily understand that any macromolecule (including virtually all proteins or peptides) can act as an antigen. Antigens can be endogenously expressed, i.e., expressed from genomic DNA, or they can be recombinantly expressed. Antigens may be specific to certain tissues, such as cancer cells, or they may be widely expressed. For example, an antigen may be a tumor-associated antigen, such as all or fragments of CD19 or CD20.

[0106] In the context of CARs, as used herein, the term "hinge region" refers to the extracellular domain of a CAR molecule that lies between the antigen-binding domain and the transmembrane domain, spatially separating the antigen-binding domain from the intracellular domain. The hinge region may also be referred to as a "hinge domain" or "spacer region." Hinges can contribute to receptor expression, activity, and / or stability. Hinges can also provide flexibility in binding target antigens, allowing the antigen-binding domain to be oriented differently to facilitate binding. Hinge domains can be derived from natural, synthetic, semi-synthetic, or recombinant sources. Hinge domains can contain the amino acid sequence of naturally occurring immunoglobulin hinge regions or modified immunoglobulin hinge regions. Exemplary hinge domains suitable for CARs described herein include hinge regions derived from the extracellular regions of type 1 membrane proteins such as CD8 (e.g., CD8α), CD4, CD28, 4-1BB, and CD7, which may be wild-type hinge regions from these molecules or may be modified.

[0107] In the context of CARs, a "transmembrane domain" refers to a domain in a molecule that, when present on the cell surface or at the cell membrane (e.g., spanning part or all of the cell membrane), possesses properties of being present in the membrane. A transmembrane domain can be any protein structure that is thermodynamically stable in the membrane. This is typically an alpha helix containing several hydrophobic residues. A transmembrane domain of any transmembrane protein can be used to provide the transmembrane portion of a chimeric receptor. The presence and span of a protein's transmembrane domain can be determined by a domain technician using the TMHMM algorithm (http: / / www.cbs.dtu.dk / services / TMHMM-2.0 / ). Alternatively, artificially designed TM domains can be used. Commonly used transmembrane domains can be transmembrane regions or fragments of transmembrane proteins (e.g., type I transmembrane proteins such as CD28, CD8α) or other transmembrane proteins), artificial hydrophobic sequences, or combinations thereof.

[0108] In the context of CARs, the intracellular domain (intrinsic domain) is the signaling portion of a chimeric antigen receptor. It contains a signal transduction domain and one or more co-stimulatory domains. Upon antigen recognition, the receptor clusters native CD45 and CD148 are expelled from the synapse, and signals are transduced into the cell, thereby activating one or more immune cell effector functions (e.g., innate immune cell effector functions). The most commonly used intracellular domain component is the CD3ζ intracellular domain component containing three ITAMs. Upon antigen binding, it transmits an activation signal to T cells. CD3ζ may not provide a completely sufficient activation signal, and additional co-stimulatory signal transduction may be required. Co-stimulatory signals promote T cell proliferation and survival. There are two main types of co-stimulatory signals: those belonging to the Ig family (CD28, ICOS) and the TNF family (OX40, 41BB, CD27, GITR, etc.).

[0109] "Intracellular signal transduction domain" refers to a portion of a CAR polypeptide that participates in transducing information about the binding of an effective CAR to a target antigen into immune effector cells to trigger effector cell functions, such as activation, cytokine production, proliferation, and cytotoxic activity, including the release of cytotoxic factors into CAR-bound target cells, or other cellular responses triggered upon antigen binding to the extracellular CAR domain. Non-limiting examples of intracellular signal transduction domains containing immune receptor tyrosine-based activation motifs (ITAMs) include those derived from TCRζ, FcRγ, FcRβ, CD3γ, CD3θ, CD3δ, CD3η, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

[0110] "Intracellular costimulatory domain" refers to the intracellular signal transduction domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that provide a second signal required for effective activation and function of T lymphocytes when binding to antigens. Costimulatory domains can be, for example, 4-1BB, CD27, CD28, or OX40.

[0111] As used herein, the term “treatment” includes therapeutic or preventative treatment in subjects in need. “Therapeutic or preventative treatment” includes preventative treatment aimed at the complete prevention of clinical and / or pathological manifestations, or therapeutic treatment aimed at improving or alleviating clinical and / or pathological manifestations. Therefore, the term “treatment” also includes improving or preventing disease. For example, treatment may include: (i) preventing a disease, disorder, and / or condition in patients who may be susceptible to such disease, disorder, and / or condition but have not yet been diagnosed with it; (ii) suppressing the disease, disorder, and / or condition, i.e., preventing its development; or (iii) alleviating the disease, disorder, and / or condition, i.e., causing its remission.

[0112] As used herein, the term "effective amount" refers to the amount of a therapeutic agent sufficient to achieve such treatment or prevention when administered to a subject for the treatment or prevention of a disease. "Effective amount" can vary depending on the active ingredient, the disease and its severity, and the age and weight of the subject to be treated. "Therapeutic effective amount" refers to an effective amount for therapeutic treatment. "Prophylactic effective amount" refers to an effective amount for prophylactic treatment.

[0113] As used herein, the term "administration" means the physical introduction of a pharmaceutical agent into a subject using any of the various methods and delivery systems known to those skilled in the art. Exemplary routes of administration of the active agents disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral administration routes, such as by injection or infusion.

[0114] As used herein, the terms “subject,” “individual,” and “patient” are well-known in the art and are used interchangeably herein to refer to any subject requiring treatment, particularly a mammalian subject. Examples include, but are not limited to, humans and other primates, including non-human primates such as chimpanzees and other ape and monkey species. The terms individual, subject, and patient do not, in themselves, indicate a specific age, sex, race, etc.

[0115] The term "autologous" refers to any substance derived from the same individual into which it is later reintroduced. For example, the treatment methods described herein involve collecting lymphocytes from a patient, then engineering them to express, for example, the CAR and fusion protein of the present invention, before administering them back to the same patient.

[0116] The term "allogeneic" refers to any substance that originates from one individual and is then introduced into another individual of the same species, such as allogeneic T-cell transplantation.

[0117] As used herein, the term "transfection" refers to the process of introducing nucleic acid molecules or polynucleotides (including vectors) into target cells. The term "transduction" is generally used to describe the transfer of virus-mediated nucleic acid molecules or polynucleotides. Transfection of animal cells typically involves opening transient pores or "holes" in the cell membrane to allow material uptake. Transfection can be performed using calcium phosphate, via electroporation, via cell extrusion, or by mixing cationic lipids with the material to create liposomes that fuse with the cell membrane and deposit their carriers inside. Exemplary techniques for transfecting eukaryotic host cells include lipid vesicle-mediated uptake, heat shock-mediated uptake, calcium phosphate-mediated transfection (calcium phosphate / DNA coprecipitation), microinjection, and electroporation.

[0118] II. Fusion proteins and their homodimers

[0119] A chimeric protein formed by fusing the extracellular domain of PD1 with the transmembrane and intramembrane domains of CD28 can bind to PD-L1 but promotes T cell activation, including increased cytokine release, increased cell proliferation, and enhanced cytotoxicity. Reinfusion of T cells expressing the PD1-CD28 chimeric receptor into tumor-bearing mice completely eliminated the tumor. This demonstrates that the different functional regions of these two proteins can be well-compatible, combining to form a novel artificial receptor capable of regulating T cell immune responses. Further research revealed that the PD1-CD28 chimeric receptor can convert the PD-L1 / PD1 signal, which normally inhibits T cell activity, into a signal that stimulates T cell activation. Moreover, tumor tissue expresses a large amount of PD-L1, and the PD1-CD28 chimeric receptor, with the help of the signal provided by PD-L1, converts the inhibitory signal into a stimulatory signal, promoting the anti-tumor activity of T cells.

[0120] The inventors introduced a new intermolecular disulfide bond by inserting a cysteine ​​motif (e.g., PCS, CPSCPAP) between the CD28 hinge region and transmembrane region of the PD1-CD28 chimeric signal transducer, making the dimer structure formed by the PD1-CD28 chimeric signal transducer more stable, the downstream activation signal of CD28 stronger, and the killing power of CAR-T cells stronger. Accordingly, the inventors constructed an exemplary PD1-CD28 chimeric signal transduction receptor (5V5# and 5V6# PD1-CD28 chimeras). Both the CD28 and PD1 portions of the PD1-CD28 chimera are derived from human wild-type sequences. The PD1 portion contains 155 amino acids (including a signal peptide) at the N-terminus of the PD1 sequence, which is 15 amino acids shorter than the full length of the extracellular domain of wild-type PD1. The CD28 portion contains a portion of the CD28 hinge region (12 amino acids at the C-terminus), the PCS motif / CPSCPAP motif, the transmembrane domain (27 amino acids), and the intracellular domain (41 amino acids).

[0121] In one aspect, the present invention provides a fusion protein comprising a PD-L1 binding region derived from PD1 and a hinge region, a transmembrane domain, and an intracellular domain derived from CD28, wherein the hinge region and transmembrane domain derived from CD28 contain a cysteine ​​motif capable of stabilizing a homodimer formed from the fusion protein.

[0122] In some embodiments, the cysteine-containing motif has a length of 3-10 amino acids, preferably 3-8 amino acids, for example, 3, 4, 5, 6, 7 or 8 amino acids.

[0123] In some embodiments, the cysteine-containing motif comprises PCS or CPSCPAP. In some embodiments, the cysteine-containing motif consists of PCS or CPSCPAP.

[0124] In some embodiments, the PD-L1 binding region derived from PD1 is derived from mammals, such as humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, or cattle. In some embodiments, the PD-L1 binding region derived from PD1 is derived from humans.

[0125] In some embodiments, the PD-L1 binding region derived from PD1 comprises the extracellular domain of PD1 or its N-terminal fragment. In some embodiments, the PD-L1 binding region derived from PD1 comprises the complete extracellular domain of PD1. In some embodiments, the PD-L1 binding region derived from PD1 comprises at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 131, or at least 132 amino acids at the N-terminus of the extracellular domain of PD1. In a preferred embodiment, the PD-L1 binding region derived from PD1 comprises the amino acid sequence of SEQ ID NO: 18 (FLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAH) or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 18.

[0126] In some embodiments, the PD-L1 binding region derived from PD1 further comprises a signal peptide. In some embodiments, the PD-L1 binding region derived from PD1 comprises a PD1 signal peptide and an intact extracellular domain. In some embodiments, the PD-L1 binding region derived from PD1 comprises at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 131, or at least 132 amino acids at the N-terminus of the PD1 signal peptide and the extracellular domain. In a preferred embodiment, the PD-L1 binding region derived from PD1 comprises the amino acid sequence of SEQ ID NO: 19 (MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAH) or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 19.

[0127] In some embodiments, the CD28 hinge region is derived from mammals, such as humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, or cattle. In some embodiments, the CD28 hinge region is derived from humans.

[0128] In some embodiments, the CD28 hinge region comprises the complete CD28 hinge region or its C-terminal segment. In some embodiments, the CD28 hinge region comprises at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, or at least 35 amino acids at the C-terminus of the CD28 hinge region. In a preferred embodiment, the CD28 hinge region comprises the amino acid sequence of SEQ ID NO: 20 (CPSPLFPGPSKP) or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 20.

[0129] In some embodiments, the CD28 transmembrane domain is derived from mammals, such as humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, or cattle. In some embodiments, the CD28 transmembrane domain is derived from humans.

[0130] In some embodiments, the CD28 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 16.

[0131] In some embodiments, the CD28 intracellular domain is derived from mammals, such as humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, or cattle. In some embodiments, the CD28 intracellular domain is derived from humans.

[0132] In some embodiments, the CD28 intracellular domain comprises the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 17.

[0133] In some embodiments, the hinge region, transmembrane domain, and intracellular domain derived from CD28 are derived from mammals, such as humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, or cattle, preferably from humans. In some embodiments, the hinge region, transmembrane domain, and intracellular domain derived from CD28 comprise the amino acid sequence of SEQ ID NO: 21 (CPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS) or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 21.

[0134] In some embodiments, the fusion protein comprises the following structure from the N-terminus to the C-terminus:

[0135] The structure consists of a PD-L1 binding region derived from PD1, a CD28 hinge region, a cysteine ​​motif, a CD28 transmembrane domain, and a CD28 intracellular domain; the domains are connected by optional linkers.

[0136] In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 1. In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 26 or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 26.

[0137] In some embodiments, the fusion protein forms a homodimer when expressed on the cell surface. In some embodiments, the fusion protein, when expressed on the cell surface, is capable of binding PD-L1 and converting an inhibitory signal into an activating signal.

[0138] On the other hand, the present invention provides a homodimer formed from the fusion protein described herein. The fusion protein disclosed herein forms a homodimer when expressed on the cell surface. The insertion of a cysteine ​​motif introduces new intermolecular disulfide bonds, making the homodimer structure formed by the fusion protein more stable. When it binds to PD-L1 on the surface of target cells, it can effectively convert PD-L1 / PD1-mediated immunosuppressive signals into activating signals, thereby exerting a stronger antitumor effect.

[0139] III. Nucleic Acids and Vectors

[0140] The present invention provides a nucleic acid comprising a nucleotide sequence encoding a fusion protein disclosed herein.

[0141] The term "nucleic acid" includes single-stranded and double-stranded nucleotide polymers. Nucleic acids can be ribonucleotides or deoxyribonucleotides or modified forms of any type of nucleotide. These modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2',3'-dideoxyribose, and nucleotide bond modifications such as thiophosphates, dithiophosphates, selenophosphates, diselenophosphates, phenylthiophosphates, aniline phosphates, and aminophosphates.

[0142] In some embodiments, the nucleic acid is ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). In some embodiments, the present invention provides ribonucleic acid (RNA) comprising a nucleotide sequence encoding a fusion protein disclosed herein. In some embodiments, the present invention provides deoxyribonucleic acid (DNA) comprising a deoxynucleotide sequence encoding a fusion protein disclosed herein. In some embodiments, the nucleic acid of the present invention comprises the nucleotide sequence of SEQ ID NO: 2 or a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 2. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 27 or a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 27.

[0143] In some embodiments, deoxyribonucleic acid (DNA) can be introduced into human cells in vivo. In some embodiments, the deoxyribonucleic acid (DNA) of the present invention is contained in a vector or delivery agent. In some embodiments, the deoxyribonucleic acid (DNA) of the present invention is integrated into the genome of a cell. In some embodiments, ribonucleic acid (RNA) can be introduced into human cells in vivo. In some embodiments, the ribonucleic acid (RNA) of the present invention is contained in a vector or delivery agent.

[0144] The present invention also provides vectors comprising the nucleic acids disclosed herein. In some embodiments, the vector is an expression vector capable of expressing the fusion proteins disclosed herein. For example, the present invention provides an expression vector comprising any of the nucleic acid molecules described above.

[0145] Any vector may be used in this disclosure. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a retroviral vector, a DNA vector, a murine leukemia virus vector, an SFG vector, a plasmid, an RNA vector, an adenovirus vector, a baculovirus vector, an Epstein-Barr virus vector, a papillomavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-associated vector (AAV), a lentiviral vector, or any combination thereof. Suitable exemplary vectors include, for example, pGAR, pBABE-puro, pBABE-neo largeTcDNA, pBABE-hygro-hTERT, pMKO.1 GFP, MSCV-IRES-GFP, pMSCV PIG (Puro IRES GFP empty plasmid), pMSCV-loxp-dsRed-loxp-eGFP-Puro-WPRE, MSCV IRES luciferase, pMIG, MDH1-PGK-GFP_2.0, TtRMPVIR, pMSCV-IRES-mCherry FP, pRetroX GFP T2A Cre, pRXTN, pLncEXP, and pLXIN-Luc.

[0146] The expression vector can be any suitable recombinant expression vector. Suitable vectors include those designed for propagation and amplification or for expression, or both, such as plasmids and viruses. For example, vectors can be selected from the pUC series (Fermentas LifeSciences, Glen Burnie, Md.), pBluescript series (Stratagene, LaJolla, Calif.), pET series (Novagen, Madison, Wis.), pGEX series (Pharmacia Biotech, Uppsala, Sweden), and pEX series (Clontech, Palo Alto, Calif.). Phage vectors such as λGT10, λGT11, λZapII (Stratagene), λEMBL4, and λNM1149 can also be used. Examples of plant expression vectors that can be used in this disclosure include pBI01, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors that can be used in this disclosure include pcDNA, pEUK-Cl, pMAM, and pMAMneo (Clontech).

[0147] Recombinant expression vectors can be prepared using standard recombinant DNA techniques described, for example, in Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY 2001; and Ausubel et al., *Current Protocols in Molecular Biology*, Greene Publishing Associates and John Wiley & Sons, NY, 1994. Circular or linear expression vector constructs can be prepared to contain the functionality of a replication system in prokaryotic or eukaryotic host cells. The replication system can be derived from, for example, COLEL, 2μ plasmid, λ, SV40, bovine papillomavirus, etc.

[0148] For example, the vector may be a lentiviral vector containing a nucleotide sequence encoding the fusion protein disclosed herein. The vector may be administered to a subject and then enter the subject's cells, thereby integrating the nucleotide sequence encoding the fusion protein disclosed herein into the cell's genome, whereby the cells subsequently express the fusion protein disclosed herein.

[0149] IV. Immune cells

[0150] This invention provides an immune cell comprising the fusion protein disclosed herein. In some embodiments, the immune cell further comprises a chimeric antigen receptor (CAR).

[0151] The present invention also provides an immune cell that co-expresses the fusion protein and chimeric antigen receptor (CAR) disclosed herein.

[0152] In some embodiments, the immune cells are selected from tumor-infiltrating lymphocytes (TILs), T cells, NK cells, and macrophages. In some embodiments, the immune cells are T cells. The T cells can be any type of T cell, such as cultured T cells, for example, primary T cells or T cells derived from cultured T cell lines, such as Jurkat, SupTl, etc., or T cells obtained from mammals. If obtained from mammals, the T cells can be obtained from many sources, including but not limited to blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. The T cells may also be enriched or purified. Preferably, the T cells are human T cells. More preferably, the T cells are T cells isolated from humans. T cells can be any type of T cell and can be T cells at any developmental stage, including but not limited to CD4+ / CD8+ double-positive T cells, CD4+ helper T cells, such as Th1 and Th2 cells, CD4+ T cells, CD8+ T cells (e.g., cytotoxic T cells), tumor-infiltrating lymphocytes (TILs), memory T cells (e.g., central memory T cells and effector memory T cells), naive T cells, etc. In a preferred embodiment, the immune cell is a TIL.

[0153] A "chimeric antigen receptor" or "CAR" is a molecule engineered to contain an antigen-binding domain that targets a specific antigen. Upon binding to that antigen, it activates immune cells (such as T cells or NK cells, including naive T cells, central memory T cells, effector memory T cells, or combinations thereof) to attack and destroy cells carrying that antigen. When these antigens are present on tumor cells, CAR-expressing immune cells can target and kill the tumor cells.

[0154] CARs typically include: (i) an antigen-binding domain; (ii) a hinge domain; (iii) a transmembrane domain; and (iv) an intracellular domain that includes a signal transduction domain and one or more co-stimulatory domains.

[0155] In some embodiments, the CAR of the present invention includes an antigen-binding domain, a transmembrane domain, an intracellular co-stimulatory domain, and an intracellular signal transduction domain.

[0156] In some embodiments, the antigen-binding domain binds to one or more antigens selected from tumor-associated antigens (TAAs) and viral antigens. In some embodiments, the TAA is selected from 5T4, alpha-fetoprotein, BCMA, CA-125, carcinoembryonic antigen, CD19, CD20, CD22, CD23, CD30, CD33, CD40, CD56, CD79, CD78, CD123, CD138, c-Met, CSPG4, ROR1, GPC3, Tyrp-1, TACI, ALK, CLL-1, EGFR, EGFRvIII, ERBB2, FLT3, melanoma-associated antigen, mesothelin, MUC-1, and VEGFR2, preferably CD19. In some embodiments, the viral antigen is selected from HPV E6 or E7 antigen, CMV antigen, HBV antigen, EBV antigen, herpesvirus antigen, human immunodeficiency virus (HIV) antigen, influenza virus antigen, and coronavirus antigen.

[0157] In some embodiments, the antigen-binding domain is selected from antigen-binding domains derived from antibodies against the antigen and antigen-binding domains derived from natural ligands of the antigen. In some embodiments, the antibody-derived antigen-binding domain is in the form of scFv, Fab, or domain antibody (dAb).

[0158] In some embodiments, the antigen-binding domain is anti-CD19 scFv and contains the amino acid sequence of SEQ ID NO: 22 (DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS).

[0159] In some embodiments, the transmembrane domain comprises a transmembrane domain selected from the α chain of the T cell receptor (TCR), the β chain of the TCR, the ζ chain of the TCR, CD3ε, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD19, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137(41BB), CD152, CD154, and PD1, or any combination thereof, preferably comprising a transmembrane domain of CD28. In some embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 16.

[0160] In some embodiments, the intracellular co-stimulatory domain comprises an intracellular signaling region selected from 4-1BB (CD137), OX40 (CD134), ICOS (CD278), 2B4, HVEM, LAG3, DAP10, DAP12, CD27, CD28, CD30, CD40, glucocorticoid-induced tumor necrosis factor receptor (GITR), lymphocyte function-associated antigen-1 (LFA-1), MyD88, CD2, CD4, CD7, LIGHT, NKG2C, and B7-H3, or any combination thereof, preferably comprising an intracellular signaling region of 4-1BB. In some embodiments, the intracellular co-stimulatory domain comprises the amino acid sequence of SEQ ID NO: 23 (KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL) or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 23.

[0161] In some embodiments, the intracellular signal transduction domain comprises a signal transduction region selected from TCRξ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD79a, CD79b, ICOS (CD278), and CD66d, or any combination thereof, preferably comprising a CD3ζ signal transduction region. In some embodiments, the intracellular signal transduction domain comprises the amino acid sequence of SEQ ID NO: 24 (RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR) or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 24.

[0162] In some embodiments, the CAR further includes a hinge region. In some embodiments, the hinge region includes a hinge region or fragment thereof selected from CD8, CD28, IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, and IgM, preferably including a hinge region of CD28. In some embodiments, the hinge region includes the amino acid sequence of SEQ ID NO: 15 or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 15.

[0163] In some embodiments, the CAR further comprises a signal peptide. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 25 (MLLLVTSLLLCELPHPAFLLIP) or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 25.

[0164] In some embodiments, the CAR includes a signal peptide, an antigen-binding domain that binds to CD19, a hinge region derived from CD28, a transmembrane domain derived from CD28, an intracellular co-stimulatory domain derived from 4-1BB, and an intracellular signal transduction domain derived from CD3ζ.

[0165] In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 25, the antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 22, the hinge region comprises the amino acid sequence of SEQ ID NO: 15 or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 15, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 16, the intracellular co-stimulatory domain comprises the amino acid sequence of SEQ ID NO: 23 or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 23, and the intracellular signal transduction domain comprises the amino acid sequence of SEQ ID NO: 24 or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 24.

[0166] In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 5.

[0167] V. Carrier System

[0168] The present invention provides a vector system comprising, on one or more vectors, a nucleotide sequence encoding a fusion protein disclosed herein and a nucleotide sequence encoding a CAR disclosed herein.

[0169] In some embodiments, the nucleotide sequence encoding the fusion protein comprises a nucleotide sequence of SEQ ID NO: 2 or higher that has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 2. In some embodiments, the nucleotide sequence encoding the fusion protein comprises a nucleotide sequence of SEQ ID NO: 27 or higher that has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 27.

[0170] In some embodiments, the nucleotide sequence encoding the CAR comprises a nucleotide sequence of SEQ ID NO: 6 that has at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 6.

[0171] In some embodiments, the vector system comprises a nucleotide sequence encoding the fusion protein and a nucleotide sequence encoding the CAR on a single vector. In some embodiments, the vector is a dual-promoter vector, with one promoter located upstream of the nucleotide sequence encoding the CAR and the other upstream of the nucleotide sequence encoding the fusion protein. In some embodiments, the promoters are each independently selected from the EF1α promoter, the MSCV promoter, and the PGK promoter. In some embodiments, the EF1α promoter is upstream of the nucleotide sequence encoding the CAR, and the MSCV promoter is upstream of the nucleotide sequence encoding the fusion protein. In some embodiments, the MSCV promoter is upstream of the nucleotide sequence encoding the CAR, and the EF1α promoter is upstream of the nucleotide sequence encoding the fusion protein. In some embodiments, the vector is selected from lentiviral vectors, retroviral vectors, adenoviral vectors, adeno-associated virus (AAV) vectors, plasmids, DNA vectors, mRNA vectors, transposon-based vectors, and artificial chromosomes, preferably lentiviral vectors.

[0172] In other embodiments, the vector system comprises a first vector and a second vector, wherein the first vector contains a nucleotide sequence encoding the fusion protein, and the second vector contains a nucleotide sequence encoding the CAR. In some embodiments, the first vector and the second vector are each independently selected from lentiviral vectors, retroviral vectors, adenoviral vectors, adeno-associated virus (AAV) vectors, plasmids, DNA vectors, mRNA vectors, transposon-based vectors, and artificial chromosomes.

[0173] VI. Composition

[0174] The present invention provides a composition comprising the fusion protein, homodimer, nucleic acid, vector, immune cell or vector system disclosed herein.

[0175] The fusion proteins, homodimers, nucleic acids, carriers, immune cells, or carrier systems of the present invention (also referred to herein as “active ingredients”) can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically contain the active ingredient as well as a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delay agents compatible with drug administration. Preferred examples of such carriers or excipients include, but are not limited to, water, saline, Ringer's solution, dextran solution, and 5% human serum albumin. Liposomes and non-aqueous media such as non-volatile oils may also be used. The use of such media and agents for pharmaceutical active substances is well known in the art. Unless any conventional media or agent is incompatible with the active compound, its use in the composition is considered. Additional active compounds may also be incorporated into the composition.

[0176] In some embodiments, the pharmaceutical composition further comprises a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from antibodies, chemotherapeutic agents, and small molecule drugs.

[0177] Preferred examples of second-line therapeutic agents include known anticancer drugs such as cisplatin, maytansine derivatives, rachelmycin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodium photofrin II, temozolomide, topotecan, trimetreate glucuronate, and orlistatine E. E) vincristine and doxorubicin; and peptide cytotoxins, such as ricin, diphtheria toxin, Pseudomonas aeruginosa exotoxin A, DNases and RNases; radionuclides, such as iodine-131, rhenium-186, indium-111, iridium-90, bismuth-210 and 213, actinium-225 and astatine-213; prodrugs, such as antibody-directed enzyme prodrugs; immunostimulants, such as IL-2, chemokines such as IL-8, platelet-4; antibodies or fragments thereof, such as anti-CD3 antibodies or fragments thereof; complement activators; viral / bacterial protein domains and viral / bacterial peptides.

[0178] The pharmaceutical compositions of the present invention can be formulated to be compatible with their intended routes of administration. Examples of routes of administration include parenteral administration, such as intravenous, intradermal, subcutaneous, transdermal, mucosal, and rectal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous application may include the following components: sterile diluents, such as water for injection, saline solution, non-volatile oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as tetraethylamine oxalate (EDTA); buffers, such as acetate, citrate, or phosphate; and agents for adjusting tension, such as sodium chloride or dextran. The pH value can be adjusted with an acid or base, such as hydrochloric acid or sodium hydroxide. Parenteral preparations may be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0179] Suitable pharmaceutical compositions for injectable applications include sterile aqueous solutions (if water-soluble) or dispersions and sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, antibacterial water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and should be a fluid present in a manner easily injectable. It must be stable under preparation and storage conditions and must be protected against contamination by microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Appropriate flowability can be maintained, for example, by using coatings such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using surfactants. Microbial action can be prevented by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is preferable to include isotonic agents in the composition, such as sugars, polyols like mannitol, sorbitol, and sodium chloride. The absorption of injectable compositions can be prolonged by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0180] Sterile injectable solutions can be prepared by incorporating the desired amount of the active compound with one or a combination of the ingredients listed above (as needed) into a suitable solvent, followed by filtration and sterilization. Typically, dispersions are prepared by introducing the active compound into a sterile medium containing a basic dispersion medium and other desired ingredients from those listed above. In the case of sterile powders used to prepare sterile injectable solutions, the preparation method is vacuum drying and freeze-drying to produce a powder of the active ingredient plus any other desired ingredients from its previously sterile filtered solution.

[0181] Systemic application can also be performed via transmucosal or transdermal routes. For transmucosal or transdermal application, a penetrant suitable for the target penetration barrier is used in the formulation. Such penetrants are generally known in the art and include, for example, detergents, bile salts, and fusidic acid derivatives used for transmucosal application. Transmucosal application can be accomplished using nasal sprays or suppositories. For transdermal application, the active compound is formulated as an ointment, cream, gel, or lotion known in the art.

[0182] In one embodiment, the active ingredient is prepared together with a carrier (such as a controlled-release formulation, including implants and microencapsulated delivery systems) that protects the active ingredient from rapid elimination from the body. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be apparent to those skilled in the art.

[0183] VII. Reagent Kit

[0184] This invention provides a kit comprising the fusion protein, homodimer, nucleic acid, vector, immune cell, or vector system disclosed herein.

[0185] In some cases, the kit may include instructions for use, which include information about the fusion protein, homodimer, nucleic acid, vector, immune cell or vector system in the kit, as well as the dosage form and instructions on how to use them. Typically, this information helps patients and physicians use the encapsulated fusion protein, homodimer, nucleic acid, vector, immune cell or vector system effectively and safely. Containers used in such kits may typically contain at least one vial, test tube, flask, bottle, syringe, or other suitable container.

[0186] In some embodiments, the present invention provides a kit that may comprise the fusion protein, homodimer, nucleic acid, carrier, immune cell, or carrier system disclosed herein, instructions for use for administering the substance, and an administration device for administering the substance. This administration device can introduce a substance into a patient via a parenteral route (e.g., intramuscular, subcutaneous, or intravenous). For example, the administration device may be a syringe (e.g., a pre-filled syringe containing the fusion protein or immune cell of the present invention, such as an auto-injector), which may include a syringe and needle (which can be used to pierce the skin and / or blood vessels) for containing the fluid to be injected (e.g., the fusion protein or immune cell of the present invention). The administration method can be varied. Administration routes may include intravenous injection, intramuscular injection, subcutaneous injection, rectal administration, etc.

[0187] VIII. Methods and Applications

[0188] The present invention provides a method for treating a disease in a subject, comprising administering to the subject an effective amount of a fusion protein, homodimer, nucleic acid, carrier, immune cell, carrier system or composition disclosed herein, wherein the disease is selected from cancer, autoimmune diseases, inflammatory diseases and infectious diseases.

[0189] The present invention also provides the use of the fusion proteins, homodimers, nucleic acids, carriers, immune cells, carrier systems or compositions disclosed herein in the preparation of medicaments for treating a disease in a subject, said disease being selected from cancer, autoimmune diseases, inflammatory diseases and infectious diseases.

[0190] The present invention also provides fusion proteins, homodimers, nucleic acids, vectors, immune cells, vector systems or compositions disclosed herein for the treatment of a subject’s disease selected from cancer, autoimmune diseases, inflammatory diseases and infectious diseases.

[0191] In some embodiments, the cancer is a blood cancer (e.g., lymphoma, leukemia, or multiple myeloma) or a solid tumor (e.g., lung cancer). In some embodiments, the cancer is a CD19-positive cancer. In some embodiments, the cancer is a B-cell malignancy. In some embodiments, the cancer is lymphoma, leukemia, or multiple myeloma. In some embodiments, the cancer is selected from Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, and chronic myeloid leukemia. In a preferred embodiment, the cancer is diffuse large B-cell lymphoma, follicular lymphoma, or chronic myeloid leukemia. In other preferred embodiments, the cancer is Burkitt lymphoma. In other preferred embodiments, the cancer is lung cancer.

[0192] In an embodiment of administering immune cells co-expressing the fusion protein and CAR described herein, the CAR comprises an antigen-binding domain that binds to CD19 and the cancer is a CD19-positive cancer (e.g., B-cell malignancy, preferably lymphoma, leukemia).

[0193] In some implementation methods, the dose administered to the subject may vary depending on the implementation method, the drug used, the method of administration, and the site of treatment and the subject. However, the dose should be sufficient to provide a therapeutic response. Clinicians can determine the effective amount to administer to humans or other subjects to treat a medical condition. The precise amount required for effective treatment may depend on many factors, such as the activity of the active ingredient and the route of administration.

[0194] The dosage of the fusion protein, homodimer, nucleic acid, vector, immune cell, vector system, or composition described herein can be administered to mammals in a single dose or in a series of sub-dose over an appropriate period of time, such as daily, bi-weekly, weekly, bi-weekly, bi-weekly, bi-monthly, semi-annually, or annually as needed. Dosage units containing an effective amount of the fusion protein, homodimer, nucleic acid, vector, immune cell, vector system, or composition can be administered as a single daily dose, or the total daily dose can be administered as needed in two, three, four, or more daily sub-dose administrations.

[0195] The appropriate route of administration can be chosen by the physician. Administration routes may include parenteral administration, such as by injection, nasal administration, or percutaneous administration. Systemic or local administration can be performed via intravenous injection, intramuscular injection, intraperitoneal injection, or subcutaneous injection. In some embodiments, fusion proteins, homodimers, nucleic acids, carriers, immune cells, carrier systems, or combinations are selected for parenteral delivery, inhalation, or delivery via the digestive tract, such as oral administration. The dosage and method of administration can vary depending on the subject's weight, age, condition, etc., and can be appropriately selected.

[0196] In some embodiments, the method includes administering an effective amount of the immune cells disclosed herein to a subject, wherein the cells are autologous or allogeneic to the subject.

[0197] In the methods described herein, samples containing, for example, T cells can be isolated from the subject or from other sources, such as from the patient's own peripheral blood (first party), or from a hematopoietic stem cell graft from a donor's peripheral blood (second party), or from peripheral blood from an unrelated donor (third party).

[0198] In some embodiments, the method includes the steps of: (i) isolating a cell-containing sample from the subject; (ii) transducing or transfecting the cells using a vector system disclosed herein; and (iii) administering the cells obtained in step (ii) to the subject.

[0199] In some embodiments, the method further includes administering a second therapeutic agent. In some embodiments, the fusion protein, homodimer, nucleic acid, carrier, immune cell, carrier system, or composition disclosed herein is used in combination with the second therapeutic agent. In some embodiments, the fusion protein, homodimer, nucleic acid, carrier, immune cell, carrier system, or composition disclosed herein is administered before, substantially simultaneously with, or after the administration of the second therapeutic agent. In some embodiments, the second therapeutic agent is selected from antibodies, chemotherapeutic agents, and small molecule drugs.

[0200] Preferred examples of second-line therapeutic agents include known anticancer drugs such as cisplatin, maytansine derivatives, rachelmycin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodium photofrin II, temozolomide, topotecan, trimetreate glucuronate, and orlistatine E. E) vincristine and doxorubicin; and peptide cytotoxins, such as ricin, diphtheria toxin, Pseudomonas aeruginosa exotoxin A, DNases and RNases; radionuclides, such as iodine-131, rhenium-186, indium-111, iridium-90, bismuth-210 and 213, actinium-225 and astatine-213; prodrugs, such as antibody-directed enzyme prodrugs; immunostimulants, such as IL-2, chemokines such as IL-8, platelet-4; antibodies or fragments thereof, such as anti-CD3 antibodies or fragments thereof; complement activators; viral / bacterial protein domains and viral / bacterial peptides.

[0201] IX. Examples

[0202] The following embodiments are provided for the purpose of illustrating various embodiments of the invention, but are not intended to limit the invention in any way. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Variations and other uses will be apparent to those skilled in the art within the spirit of the invention as defined by the claims. Experimental methods not specifically described in the following embodiments were performed under conventional conditions in the art or as recommended by the manufacturer. Unless otherwise stated, the experimental materials and reagents used in the following embodiments are commercially available.

[0203] Example 1. Vector construction and plasmid extraction

[0204] The coding sequences of the PD1-CD28 chimeric signal transduction receptor (CTRT) containing (but not containing) the PCS motif, and containing the CPSCPAP motif, were cloned together with the CD19 CAR coding sequence into a dual-promoter lentiviral expression vector. CD19 CAR expression was driven by the EF1a promoter, and PD1-CD28 chimeric expression was driven by the MSCV promoter. The constructed plasmid vector was then transformed into competent bacterial cells to obtain recombinant engineered strains. These strains were fermented, and the plasmids were isolated, purified, and analyzed by gel electrophoresis. The specific experimental steps are as follows.

[0205] 1.1. Carrier Construction

[0206] Based on the amino acid sequences of the PD1-CD28 chimeric signal transduction receptor (CTR) without the PCS motif, with the PCS motif, and with the CPSCPAP motif, codon-optimized nucleotide coding sequences were designed, and the full-length nucleotide sequence of the PD1 / CD28-EF1a promoter-CD19 CAR was synthesized. This sequence was cloned into the vector pCDH-MSCV-MCS-EF1-copGFP-T2A-Puro (System Bioscience, CD713B-1), successfully constructing the pCDH-MSCV-PD1 / CD28-EF1-CD19CAR vector (named 5#, without the PCS motif), the pCDH-MSCV-PD1 / CD28(PCS)-EF1-CD19 CAR vector (named 5V5#, with the PCS motif), and the pCDH-MSCV-PD1 / CD28(CPSCPAP)-EF1-CD19 CAR vector (named 5V6#, with the CPSCPAP motif). The vector spectra of 5#, 5V5#, and 5V6# are as follows: Figure 1 , Figure 2 and Figure 3 As shown below, the amino acid and nucleotide sequences of the PD1-CD28 chimera and CD19 CAR in vectors 5#, 5V5#, and 5V6# are as follows:

[0207] 5V5# PD1-CD28 chimeric signal transducer amino acid sequence

[0208] Signal peptide-PD1-CD28 (PCS)

[0209]

[0210] The signal peptide sequence is shown underlined, the extracellular region of PD1 is shown in bold, the CD28 hinge region is shown in italics, and the transmembrane and intracellular domains of CD28 are shown in bold and underlined.

[0211] 5V5# PD1-CD28 chimeric signal transduction receptor nucleotide sequence

[0212] ATGCAGATTCCCCAGGCCCCTTGGCCTGTGGTGTGGGCCGTGCTGCAGCTGGGATGGAGGCCTGGCTGGTTCCTGGACAGCCCCGACAGACCCTGGAATCCCCCCACCTTTTCCCCTGCCCTGCTCGTGGTGACAGAGGGCGACAACGCCACCTTCACCTGCAGCTTCTCCAACACCAG CGAGAGCTTCGTCCTGAACTGGTACAGGATGAGCCCCAGCAACCAGACCGACAAGCTGGCCGCCTTCCCTGAGGACAGAAGCCAGCCCGGCCAGGACTGCAGGTTTAGAGTGACCCAGCTGCCCAATGGCAGAGACTTCCACATGAGCGTGGTGAGGGCCAGAAGGAATGACAGCGGCA CCTATCTGTGCGGCGCCATCAGCCTGGCTCCTAAAGCCCAGATCAAGGAGTCCCTGAGGGCCGAGCTGAGGGTGACCGAGAGGAGAGCTGAAGTGCCCACCGCTCACTGCCCCTCCCCTCTGTTTCCCGGCCCTAGCAAACCCCCTTGCAGCTTCTGGGTGCTGGTGGTGGTGGGCGGA GTGCTGGCCTGCTACAGCCTCCTGGTGACCGTCGCCTTCATCATCTTCTGGGTGAGAAGCAAGAGGAGCAGACTCCTGCACAGCGACTACATGAACATGACCCCTAGGAGGCCTGGCCCCACCAGAAAGCATTACCAGCCATATGCCCCCCCCAGAGACTTTGCCGCCTACAGGTCCTGA (SEQ ID NO: 2)

[0213] 5# PD1-CD28 chimeric signal transduction receptor amino acid sequence

[0214] Signal peptide-PD1-CD28

[0215]

[0216] The signal peptide sequence is shown underlined, the extracellular region of PD1 is shown in bold, the CD28 hinge region is shown in italics, and the transmembrane and intracellular domains of CD28 are shown in bold and underlined.

[0217] 5# PD1-CD28 chimeric signal transduction receptor nucleotide sequence

[0218] ATGCAGATTCCCCAGGCCCCTTGGCCTGTGGTGTGGGCCGTGCTGCAGCTGGGATGGAGGCCTGGCTGGTTCCTGGACAGCCCCGACAGACCCTGGAATCCCCCCACCTTTTCCCCTGCCCTGCTCGTGGTGACAGAGGGCGACAACGCCACCTTCACCTGCAGCTTCTCCAACACC AGCGAGAGCTTCGTCCTGAACTGGTACAGGATGAGCCCCAGCAACCAGACCGACAAGCTGGCCGCCTTCCCTGAGGACAGAAGCCAGCCCGGCCAGGACTGCAGGTTTAGAGTGACCCAGCTGCCCAATGGCAGAGACTTCCACATGAGCGTGGTGAGGGCCAGAAGGAATGACAGC GGCACCTATCTGTGCGGCGCCATCAGCCTGGCTCCTAAAGCCCAGATCAAGGAGTCCCTGAGGGCCGAGCTGAGGGTGACCGAGAGGAGAGCTGAAGTGCCCACCGCTCACTGCCCCTCCCCTCTGTTTCCCGGCCCTAGCAAACCCTTCTGGGTGCTGGTGGTGGTGGGCGGAGTG CTGGCCTGCTACAGCCTCCTGGTGACCGTCGCCTTCATCTTCTGGGTGAGAAGCAAGAGGAGCAGACTCCTGCACAGCGACTACATGAACATGACCCCTAGGAGGCCTGGCCCCACCAGAAAGCATTACCAGCCATATGCCCCCCCCAGAGACTTTGCCGCCTACAGGTCCTGA (SEQ ID NO: 4)

[0219] 5V6# PD1-CD28 chimeric signal transducer amino acid sequence

[0220] Signal peptide-PD1-CD28 (CPSCPAP)

[0221]

[0222] The signal peptide sequence is shown underlined, the extracellular region of PD1 is shown in bold, the CD28 hinge region is shown in italics, and the transmembrane and intracellular domains of CD28 are shown in bold and underlined.

[0223] 5V6# PD1-CD28 Chimeric Signal Transduction Receptor Nucleotide Sequence

[0224] ATGCAGATTCCCCAGGCCCCTTGGCCTGTGGTGTGGGCCGTGCTGCAGCTGGGATGGAGGCCTGGCTGGTTCCTGGACAGCCCCGACAGACCCTGGAATCCCCCCACCTTTTCCCCTGCCCTGCTCGTGGTGACAGAGGGCGACAACGCCACCTTCACCTGCAGCTTCTCCAACACCAGCGAGAGCTTCGTCCTGAACTGGTACAGGATGAGCCCCAGCAACCAGACCGACAAGCTGGCCGCCTTCCCTGAGGACAGAAGCCAGCCCGGCCAGGACTGCAGGTTTAGAGTGACCCAGCTGCCCAATGGCAGAGACTTCCACATGAGCGTGGTGAGGGCCAGAAGGAATGACAGCGGCACCTATCTGTGCGGCGCCATCAGCCTGGCTCCTAAAGCCCAGATCAAGGAGTCCCTGAGGGCCGAGCTGAGGGTGACCGAGAGGAGAGCTGAAGTGCCCACCGCTCACTGCCCCTCCCCTCTGTTTCCCGGCCCTAGCAAACCCTGTCCTAGCTGTCCTGCCCCATTCTGGGTGCTGGTGGTGGTGGGCGGAGTGCTGGCCTGCTACAGCCTCCTGGTGACCGTCGCCTTCATCATCTTCTGGGTGAGAAGCAAGAGGAGCAGACTCCTGCACAGCGACTACATGAACATGACCCCTAGGAGGCCTGGCCCCACCAGAAAGCATTACCAGCCATATGCCCCCCCCAGAGACTTTGCCGCCTACAGGTCCTGA (SEQ ID NO: 27)

[0225] CD19 CAR Amino Acid Sequences in 5#, 5V5#, and 5V6#

[0226] Signal Peptide - anti-CD19 scFv - CD28 Hinge - CD28 TM - 4 - 1BB - CD3ζ

[0227]

[0228] The signal peptide sequence is shown underlined, anti-CD19 scFv is shown in bold, the CD28 hinge region is shown in italics, the CD28 transmembrane region is shown in bold and underlined, the 4-1BB intracellular domain is shown in bold and italics, and the CD3ζ intracellular domain is shown in italics and underlined.

[0229] CD19 CAR nucleotide sequences in 5#, 5V5# and 5V6#

[0230]

[0231] 1.2. Competent cell transformation and plasmid extraction

[0232] DH5α competent cells were transformed with the constructed vectors 5#, 5V5#, and 5V6#, plated on Amp+LB plates, and incubated overnight at 37°C. Single colonies growing on the LB plates were picked and inoculated into 4 ml of Amp+LB liquid medium, and cultured at 37°C and 200 rpm for 24 hours. 250 μl of the bacterial culture was added to 200 ml of Amp+LB liquid medium and cultured at 37°C and 200 rpm for 24 hours. Large-scale plasmid extraction was performed using an Endo-Free Plasmid Kit, followed by gel electrophoresis. The plasmid large-scale extraction electrophoresis results are shown below. Figure 4 As shown.

[0233] Example 2. Preparation of Lentiviral Vectors

[0234] In this embodiment, a four-plasmid system was used to package lentivirus in HEK293FT cells. The specific experimental steps are as follows.

[0235] HEK293FT cells were revived and passaged until approximately 90% confluence was achieved, at which point they were transfected with plasmids to produce virus. Virus packaging helper plasmids pLP1, pLP2, and pLP-VSVG (all purchased from Invitrogen) were added to 500 μL of Opti-MEM medium at a ratio of pLP1:pLP2:pLP-VSVG = 2:2:1. Simultaneously, the target vector plasmid constructed in Example 1 was added (the mass ratio of packaging helper plasmid mixture to target plasmid was 9:5), and the mixture was thoroughly mixed to obtain a plasmid mixture. PEI was added to 500 μL of Ppti-MEM medium at a mass ratio of plasmid to PEI of 1:3, and the mixture was thoroughly mixed to obtain a PEI mixture. The plasmid mixture and PEI mixture were incubated at room temperature for 10 min. Finally, the PEI mixture and plasmid mixture were combined and added to HEK293FT cells. Forty-eight hours after plasmid transfection, the culture supernatant containing virus particles was collected, centrifuged, concentrated, and stored at -80°C.

[0236] Example 3. Determination of lentiviral vector T-cell infection efficiency

[0237] Peripheral blood was collected from healthy donors, and PBMCs were obtained through ficollography. T cells were then obtained through Untouch magnetic bead sorting. The T cells were cultured for 3 days at 37°C and 5% CO2 in a medium containing CD3+ / CD28+ (X-vivo + 10 μg / L IL-2 + 5 μg / L IL-7 + 5 μg / L IL-15 + 2.5% AB plasma) before lentiviral infection. The infection system consisted of 1x102 cells. 6Add 100 μl of the lentiviral vectors 5# and 5V5# prepared in Example 2 to each T cell, along with 8 μg / ml Polybrene and 3 mg / ml Polybrene Boost. After mixing, continue culturing at 37°C and 5% CO2 for 3 days. Take 2 x 10 5 Post-infection T cells were stained with APC-labeled human CD19 (20-291) protein (Beijing Baipusai Biotechnology Co., Ltd.) and BB515 mouse anti-human CD279 (PD-1) (BD Medical Devices (Shanghai) Co., Ltd.), and CAR+% and PD-1+ were analyzed by flow cytometry.

[0238] Results of lentiviral vector T-cell infection efficiency assay as follows: Figure 5 As shown in the figure. The results indicate that the vector constructed in this invention can successfully infect T cells, thereby obtaining CAR-T cells that efficiently express CD19 CAR and PD1-CD28 chimeric signal transduction receptor.

[0239] Example 4. Assay of T cell killing ability co-expressing PD1-CD28 chimera and CD19 CAR

[0240] 4.1. 5V5# chimera

[0241] To determine the cytotoxic effect of CD19 CAR-T cells expressing two different PD1-CD28 chimeras on tumor cells, JVM-3 cells (Yikang (Beijing) Pharmaceutical Technology Co., Ltd.), a human chronic myeloid leukemia cell line endogenously expressing CD19 and PD-L1, were used as target cells. CD19 CAR-T cells co-expressing the PD1-CD28 chimera were used as effector cells. Here, con-CART represents CD19 CAR-T cells co-expressing the 5# PD1-CD28 chimera without the PCS motif, and test-CART represents CD19 CAR-T cells co-expressing the 5V5# PD1-CD28 chimera containing the PCS motif.

[0242] JVM-3 cells were resuscitated and passaged. Target cells were collected, the original culture medium was discarded, and the cells were resuspended in PBS. Effector cells and target cells were labeled with CFSE and Did, respectively. Target cells were adjusted to a density of 1x10⁻⁶ cells / cells using RPMI 1640 complete culture medium. 6 / mL, add 50 μl to each well of a 96-well plate (50,000 cells), and adjust the density of effector cells to 1x10⁶ cells / well using RPMI 1640 complete culture medium. 6 / mL, 2.5x10 5 / mL, 5x10 4 / mL, 100 μl was added to each well of a 96-well plate (effect cell to target cell ratios of 2:1, 1:2, and 1:10); simultaneously, control groups containing only effector cells and control groups containing only target cells were set up, with culture medium added to bring the volume to 150 μl / well, 5 replicates for each group, and cultured in a cell incubator for 24 h and 48 h, respectively. Absolute counting microspheres were added, cells were collected, Live / Dead cell dye was added, and viable cell counts were performed using FACS, and the killing rate was calculated at 24 h and 48 h, respectively. The killing results are shown below. Figure 6A As shown.

[0243] Figure 6A The results showed that CD19 CAR-T cells expressing the 5V5# chimera of the present invention exhibited significantly stronger killing effects than CD19 CAR-T cells expressing the 5# chimera at all three effector-target ratios after co-culturing with JVM-3 for 24 or 48 hours. This indicates that the introduction of the PCS motif into the PD1-CD28 chimera significantly improved the antitumor activity of CAR-T cells.

[0244] 4.2. 5V5# and 5V6# chimeras

[0245] To determine the cytotoxic effect of CD19 CAR-T cells expressing three different PD1-CD28 chimeras on tumor cells, JVM-3 cells (Yikang (Beijing) Pharmaceutical Technology Co., Ltd.), a human chronic myeloid leukemia cell line endogenously expressing CD19 and PD-L1, were used as target cells. CD19 CAR-T cells co-expressing the PD1-CD28 chimera were used as effector cells. Among them, 5# is CD19 CAR-T cells co-expressing the PD1-CD28 chimera without the PCS and CPSCPAP motifs, 5V5# is CD19 CAR-T cells co-expressing the PD1-CD28 chimera containing the PCS motif, and 5V6# is CD19 CAR-T cells co-expressing the PD1-CD28 chimera containing the CPSCPAP motif.

[0246] JVM-3 cells were resuscitated and passaged. Target cells were collected, the original culture medium was discarded, and the cells were resuspended in PBS. Effector cells and target cells were labeled with CFSE and Did, respectively. Target cells were adjusted to a density of 1x10⁻⁶ cells / cells using RPMI 1640 complete culture medium. 6 / mL, add 50 μl to each well of a 96-well plate (50,000 cells), and adjust the density of effector cells to 1x10⁶ cells / well using RPMI 1640 complete culture medium. 6 / mL, 2.5x10 5 / mL, 5x10 4 / mL, 100 μl was added to each well of a 96-well plate (effect cell to target cell ratios of 2:1, 1:2, and 1:10); simultaneously, control groups containing only effector cells and control groups containing only target cells were set up, with culture medium added to bring the volume to 150 μl / well, 5 replicates for each group, and cultured in a cell incubator for 24 h and 48 h, respectively. Absolute counting microspheres were added, cells were collected, Live / Dead cell dye was added, and viable cell counts were performed using FACS, and the killing rate was calculated at 24 h and 48 h, respectively. The killing results are shown below. Figure 6B As shown.

[0247] Figure 6B The results showed that CD19 CAR-T cells expressing the 5V5# or 5V6# chimera of the present invention, after being co-cultured with JVM-3 for 24 or 48 hours, exhibited stronger killing effects than CD19 CAR-T cells expressing the 5# chimera at all three effector-to-target ratios; and CD19 CAR-T cells expressing the 5V5# chimera had stronger killing effects than CD19 CAR-T cells expressing the 5V6# chimera.

[0248] Example 5. Activation of CD28 downstream signaling molecules by the PD1-CD28 chimeric receptor

[0249] Plasmid vectors were constructed and lentiviral vectors were prepared using a method similar to that in Examples 1-2, except that the plasmid vectors contained only 5#, 5V5#, and 5V6# chimeras and no CD19 CAR, to avoid Raji expressing CD19 activating T cells by stimulating CD19 CAR. The plasmid vectors containing only 5#, 5V5#, and 5V6# chimeras were transformed into competent cells and plasmids were extracted for lentiviral vector preparation. The lentiviral vectors were then used to infect T cells, and the infected T cells were used as effector cells for subsequent experiments.

[0250] 1x10 6 Raji-PDL1 cells (Yikang (Beijing) Pharmaceutical Technology Co., Ltd.) were seeded in 48-well plates pre-treated with 100 μg / ml poly-L-lysine at a density of 2.5 x 10⁻⁶ cells per well. 5 10 cells. 4 hours later, 2.5 x 10⁻⁶ cells were cultured in X-VIVO15 medium without serum and factors. 5T cells and target cells were co-incubated in each well (E:T=1:1) for 20 min. T cells were then collected, and total protein was extracted using 30 μL of RIPA lysis buffer (Beijing Pulilai Gene Technology Co., Ltd.) before SDS-PAGE gel electrophoresis. Electrophoresis was performed using a constant voltage method, with the voltage set at 80 V. After the sample reached the separating gel, the voltage was increased to 120 V. The electrophoretic position of the sample was observed, and electrophoresis was stopped at the appropriate time. The gel was then removed from the gel casting plate for transfer. After transfer, the PVDF membrane was placed in 5% skim milk powder and incubated on a shaker at room temperature for 1 h. The PVDF membrane was then placed in primary antibody diluted 1:1000 with blocking buffer and incubated overnight at 4°C. The membrane was then washed with 0.2% PBST, with rapid shaking on a shaker for 10 minutes each time, three times at room temperature. Next, the PVDF membrane was placed in secondary antibody diluted 1:5000 with blocking buffer and incubated at room temperature for 1 hour. The membrane was then washed with 0.2% PBST, with rapid shaking on a shaker for 10 minutes each time, three times at room temperature. Color development was performed using ECL chemiluminescence buffer (Shanghai Yisheng Biotechnology Co., Ltd.) (solutions A and B were mixed evenly in a 1:1 ratio). Imaging was performed using a chemiluminescence imager. The imaging results are shown below. Figure 7 As shown.

[0251] The experimental results showed that, compared with the control group, the levels of phosphorylated AKT and ERK in T cells expressing chimeric receptors 5#, 5V5#, or 5V6# were significantly increased. The levels in the 5V5# and 5V6# chimeric receptor groups were higher than those in the 5# chimeric receptor group, and the levels in the 5V5# chimeric receptor group were higher than those in the 5V6# chimeric receptor group. These results indicate that, compared with the control group, the three PD1-CD28 chimeric receptors expressed on the surface of T cells can activate the downstream CD28 signaling molecules AKT and ERK after binding to PDL1-expressing tumor cells, with 5V5# and 5V6# showing higher activation than 5#, and 5V5# showing higher activation than 5V6#.

[0252] Example 6. In vitro killing assay of T cells expressing PD1-CD28 chimera

[0253] Plasmid vectors were constructed and lentiviral vectors were prepared using a method similar to that in Examples 1-2, except that the plasmid vectors contained only 5#, 5V5#, and 5V6# chimeras and no CD19 CAR, to prevent Raji expressing CD19 from activating T cells by stimulating CD19 CAR. T cells were infected with the lentiviral vectors containing only 5#, 5V5#, and 5V6# chimeras, and the infected T cells were used as effector cells for subsequent experiments.

[0254] Raji-PD-L1-Luc cells (human Burkitt's lymphoma tumor cells transfected with luciferase and PD-L1) (Yikang (Beijing) Pharmaceutical Technology Co., Ltd.) were revived and passaged. Target cells were collected when the cell count reached the experimental target. Target cells were seeded at 5000 cells per well in 96-well plates. 10000 and 2500 effector cells (effector-to-target ratios of 2:1 and 1:2, respectively) were added to each well (5#, 5V5#, and 5V6#). Control groups containing only effector cells and control groups containing only target cells were also set up, with 5 replicates in each well. Cells were cultured for 24 h and 48 h, respectively. After culture, 100 mL of BRITELITE PLUS KIT reagent (PerkinElmer, 6066761) was added, and the luminescence value was measured using a microplate reader. The killing rate was calculated at 24 h and 48 h. The killing results are shown below. Figure 8 As shown.

[0255] Figure 8 The results showed that T cells expressing the 5V5# or 5V6# chimera of the present invention, after being co-cultured with Raji-PD-L1-Luc for 24h or 48h, exhibited stronger killing effects than T cells expressing the 5# chimera at both effector-to-target ratios; and T cells expressing the 5V5# chimera were more effective at killing target cells than T cells expressing the 5V6# chimera.

[0256] Example 7. INF-γ release from TIL cells expressing PD1-CD28 chimera

[0257] Plasmid vectors were constructed and lentiviral vectors were prepared using a method similar to that used in Examples 1-2, except that the plasmid vectors contained only 5#, 5V5# and 5V6# chimeras and did not contain CD19 CAR.

[0258] Tumor tissue obtained surgically from lung cancer patients was partially cut into 1–3 mm pieces inside a biosafety cabinet. 3 One tissue fragment was seeded per well in a 24-well plate, and 1 mL of TIL culture medium (X-vivo + 3000 IU / ml IL-2) was added to each well. The plates were then incubated for 14 days. The remaining tumor tissue was processed into a single-cell suspension using the Tumor Dissociation Kit human (Medini, 130-095-929) for use as target cells and then cryopreserved in liquid nitrogen.

[0259] After 14 days of culture of the tissue blocks, TILs cells were collected as effector cells and transduced using the prepared lentiviral vectors 5#, 5V5#, and 5V6#, respectively. After transduction, the cells were cultured in an incubator for another 72 hours. The transduced TILs cells expressing the 5#, 5V5#, and 5V6# chimeras were collected and seeded at 10,000 cells per well in 96-well plates. Tumor cell suspensions of 20,000 and 40,000 cells per well (effect-to-target ratio of 1:2 and 1:4, respectively) were added for co-culture. A control group containing only TILs cells was also included. Each group had 5 replicates and was cultured in a cell culture incubator for 24 hours.

[0260] After co-culturing, the 96-well plate was placed in a centrifuge and centrifuged at 600g for 10 min. 120–150 μl of the supernatant was collected and the IFN-γ release level in the supernatant was detected using the LEGEND MAX™ Human IFN-γ ELISA Kit (Biolegend 430107). The IFN-γ release results are as follows: Figure 9 As shown.

[0261] Figure 9 The results showed that the IFN-γ release of TILs cells expressing the three chimeras at 24h and 48h was significantly higher than that of the TILs control group without chimeras; the IFN-γ release level of TILs cells expressing 5V5# or 5V6# chimeras was higher than that of TILs cells expressing 5# chimeras; and the IFN-γ release level of TILs cells expressing 5V5# chimeras was higher than that of TILs cells expressing 5V6# chimeras.

Claims

1. A fusion protein comprising a PD-L1 binding region derived from PD1 and a hinge region, a transmembrane domain, and an intracellular domain derived from CD28, wherein the hinge region and transmembrane domain derived from CD28 contain a cysteine ​​motif capable of stabilizing a homodimer formed from the fusion protein.

2. The fusion protein according to claim 1, wherein the cysteine ​​motif comprises PCS or CPSCPAP.

3. The fusion protein according to claim 1 or 2, wherein the PD-L1 binding region derived from PD1 comprises the human PD1 extracellular domain or its N-terminal fragment, preferably the N-terminal fragment comprising at least 120 amino acids.

4. The fusion protein of claim 3, wherein the PD-L1 binding region derived from PD1 comprises: (i) The amino acid sequence of SEQ ID NO: 18 or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 18; or (ii) The amino acid sequence of SEQ ID NO: 19 or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO:

19.

5. The fusion protein according to any one of claims 1-4, wherein The hinge region derived from CD28 is a human CD28 hinge region. Preferably, the CD28 hinge region contains the amino acid sequence of SEQ ID NO:20 or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO:

20. The transmembrane domain derived from CD28 is a human CD28 transmembrane domain. Preferably, the CD28 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 16; and / or The intracellular domain derived from CD28 is a human CD28 intracellular domain. Preferably, the CD28 intracellular domain comprises the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO:

17.

6. The fusion protein according to claim 5, wherein the hinge region, transmembrane domain and intracellular domain derived from CD28 comprise the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence having at least 80%, at least 85%, at least 90% or at least 95% sequence identity with SEQ ID NO:

21.

7. The fusion protein according to any one of claims 1-6, wherein the fusion protein comprises: (i) The amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 1; or (ii) The amino acid sequence of SEQ ID NO: 26 or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO:

26.

8. The fusion protein according to any one of claims 1-7, wherein the fusion protein forms a homodimer when expressed on the cell surface.

9. The fusion protein according to any one of claims 1-8, wherein the fusion protein, when expressed on the cell surface, is capable of binding PD-L1 and converting an inhibitory signal into an activation signal.

10. A homodimer formed from a fusion protein according to any one of claims 1-9.

11. A nucleic acid comprising a nucleotide sequence encoding a fusion protein according to any one of claims 1-9, preferably, the nucleic acid comprising (i) the nucleotide sequence of SEQ ID NO: 2 or a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 2 or (ii) the nucleotide sequence of SEQ ID NO: 27 or a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO:

27.

12. A vector comprising the nucleic acid according to claim 11.

13. An immune cell comprising the fusion protein according to any one of claims 1-9.

14. The immune cell according to claim 13, wherein the immune cell is a tumor-infiltrating lymphocyte (TIL) or a T cell.

15. The immune cell of claim 13, further comprising a chimeric antigen receptor (CAR).

16. An immune cell that co-expresses the fusion protein and chimeric antigen receptor (CAR) according to any one of claims 1-9.

17. The immune cell according to claim 15 or 16, wherein the immune cell is selected from tumor-infiltrating lymphocytes (TILs), T cells, NK cells, and macrophages.

18. The immune cell according to any one of claims 15-17, wherein the CAR comprises an antigen-binding domain that binds to CD19, a hinge region derived from CD28, a transmembrane domain derived from CD28, an intracellular co-stimulatory domain derived from 4-1BB, and an intracellular signal transduction domain derived from CD3ζ.

19. The immune cell according to claim 18, wherein The antigen-binding domain is anti-CD19 scFv and contains the amino acid sequence of SEQ ID NO:

22. The hinge region contains the amino acid sequence of SEQ ID NO: 15 or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO:

15. The transmembrane domain comprises the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO:

16. The intracellular co-stimulatory domain comprises the amino acid sequence of SEQ ID NO: 23 or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 23, and / or The intracellular signal transduction domain comprises the amino acid sequence of SEQ ID NO: 24 or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO:

24.

20. The immune cell of claim 19, wherein the CAR comprises the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO:

5.

21. A vector system comprising, on one or more vectors, a nucleotide sequence encoding a fusion protein according to any one of claims 1-9 and a nucleotide sequence encoding a CAR as defined in any one of claims 15-20.

22. The vector system of claim 21, wherein the nucleotide sequence encoding the fusion protein comprises: (i) A nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 2; or (ii) The nucleotide sequence of SEQ ID NO: 27 or a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO:

27.

23. The vector system according to claim 21 or 22, wherein the nucleotide sequence encoding the CAR comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with the nucleotide sequence of SEQ ID NO:

6.

24. The vector system according to any one of claims 21-23, wherein the vector system comprises a nucleotide sequence encoding the fusion protein and a nucleotide sequence encoding the CAR on a vector, and the vector comprises an EF1α promoter upstream of the nucleotide sequence encoding the CAR and an MSCV promoter upstream of the nucleotide sequence encoding the fusion protein.

25. The vector system according to any one of claims 21-24, wherein the vector is selected from lentiviral vectors, retroviral vectors, adenovirus vectors, adeno-associated virus (AAV) vectors, plasmids, DNA vectors, mRNA vectors, transposon-based vectors, and artificial chromosomes, preferably lentiviral vectors.

26. A composition comprising the fusion protein of any one of claims 1-9, the homodimer of claim 10, the nucleic acid of claim 11, the vector of claim 12, the immune cell of any one of claims 13-20, or the vector system of any one of claims 21-25.

27. A kit comprising the fusion protein of any one of claims 1-9, the homodimer of claim 10, the nucleic acid of claim 11, the vector of claim 12, the immune cells of any one of claims 13-20, or the vector system of any one of claims 21-25.

28. A method for treating a disease in a subject, comprising administering to the subject an effective amount of an immune cell according to any one of claims 13-20 or a carrier system according to any one of claims 21-25, wherein the disease is selected from cancer, autoimmune diseases, inflammatory diseases, and infectious diseases.

29. The method of claim 28, wherein the cancer is a hematologic malignancy (e.g., lymphoma, leukemia, or multiple myeloma) or a solid tumor (e.g., lung cancer), preferably Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, and chronic myeloid leukemia.

30. The method of claim 28 or 29, wherein the cells are autologous or allogeneic to the subject.

31. The method of claim 30, further comprising the following steps: (i) Isolate a cell-containing sample from the subject; (ii) Transducing or transfecting the cells using the vector system of any one of claims 21-25; and (iii) The cells obtained in step (ii) are administered to the subject.

32. The method according to any one of claims 28-31, further comprising administering a second therapeutic agent, preferably selected from antibodies, chemotherapeutic agents, and small molecule drugs.

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