Compositions of recombinant fusion proteins and methods of using same to generate CAR immune cells

Abstract

Provided herein are recombinant chimeric antigen receptor (CAR) fusion proteins, methods of modifying immune cells into CAR immune cells by treating the immune cells with the recombinant CAR fusion proteins, and methods of treating cancer by administering CAR immune cells to a subject in need of treatment.

Description

[Technical field]

[0001] [CROSS REFERENCE TO RELATED APPLICATIONS] This application claims priority to U.S. Patent Application No. 63 / 320,885, filed March 17, 2022, the entire contents of which are incorporated herein by reference for all purposes.

[0002] [Reference to sequence listing] The sequence listing submitted as an XML file named "F285929_Sequence listing as filed" having a size of 98.5KB created on March 16, 2023 is incorporated herein by reference.

[0003] Provided herein are recombinant chimeric antigen receptor (CAR) fusion proteins, methods of modifying immune cells into CAR immune cells by treating the immune cells with the recombinant CAR fusion proteins, and methods of treating cancer by administering CAR immune cells to a subject in need of treatment. [Background technology]

[0004] Chimeric antigen receptor (CAR) technology has been developed to engineer immune cells into cancer-specific stimulated immune cells. CAR-T cells or CAR-NK cells have been widely evaluated in various clinical trials, but few have been prescribed in the clinic. CAR consists of an extracellular scFv domain that targets cancer, an intracellular costimulatory domain, and a transmembrane domain. CAR immune cells can recognize cancer cells by binding the extracellular scFv antibody to the target antigen on the cancer cells, which further triggers the stimulation of immune cells to exert stronger anti-cancer activity. Conventional CAR technology is limited in its use because it requires the use of viral vectors to introduce the CAR gene, it is not a suitable option for patients who need immediate treatment due to its long production time, and more importantly, it needs to be isolated, engineered, expanded ex vivo, and then reinfused into the patient. Summary of the Invention

[0005] To overcome the above limitations, the present disclosure provides novel recombinant CAR fusion proteins that can modify immune cells into CAR immune cells without the need for genetic modification.

[0006] In particular, a recombinant chimeric antigen receptor (CAR) fusion protein is disclosed that includes, in order, an immune cell targeting domain, a cleavable peptide, a membrane targeting domain, an extracellular cancer targeting domain, a transmembrane domain, and an intracellular signaling domain. In one embodiment, the immune cell targeting domain is a NK cell targeting domain or a T cell targeting domain. In another embodiment, the NK cell targeting domain is a NK cell targeting scFv antibody, a NK cell targeting full antibody, a NK cell targeting minibody, a NK cell targeting Fab, or a peptide comprising the VH, IgG hinge, IgG CH2, and IgG CH3 of anti-NKp46. In one embodiment, the cleavable peptide is a peptide configured to be enzymatically cleaved. In one embodiment, the membrane targeting domain is a peptide from interleukin-1 receptor type 1, 4F2 cell surface antigen heavy chain, linker for activation of T-cells family member 1, junctophilin-1, anti-listerial bacteriocin subtilosin biosynthesis protein AlbG, calcitonin receptor, gamma-secretase subunit APH-1A, or adiponectin receptor protein 2. In one embodiment, the extracellular cancer targeting domain is an anti-PD-L1 Vh, anti-HER2, anti-HER3, anti-TROP2, or any other cancer-specific antigen targeting antibody. In another embodiment, the recombinant CAR fusion protein further comprises a hinge region between the extracellular cancer targeting domain and the transmembrane domain. In one embodiment, the transmembrane domain is NKG2D. In another embodiment, the intracellular signaling domain comprises a CD3 zeta (CD3ζ) signaling domain and a costimulatory signaling region selected from the group consisting of 2B4, DAP12, GITR, CD137, and OX40. In one embodiment, the recombinant CAR fusion protein further comprises a tag sequence. In another embodiment, the tag sequence is selected from the group consisting of a histidine tag, a glutathione-S-transferase tag, or a hemagglutinin tag.

[0007] The present disclosure also provides a method of modifying immune cells into chimeric antigen receptor (CAR) immune cells, comprising treating the immune cells with a recombinant CAR fusion protein as discussed above. In one embodiment, the CAR fusion protein is at a concentration of about 1000 nM to about 2000 nM.

[0008] The present disclosure also provides a method of treating cancer, comprising administering to a subject in need of treatment CAR immune cells prepared by treating immune cells with a recombinant CAR fusion protein.

[0009] The present disclosure further provides a method of treating cancer comprising administering to a subject in need of treatment a recombinant CAR fusion protein as discussed above. In one embodiment, the CAR fusion protein is at a concentration of about 1000 nM to about 2000 nM.

[0010] The present disclosure also provides a method of treating cancer, comprising administering to a subject in need of treatment (i) a recombinant chimeric antigen receptor (CAR) fusion protein comprising, in order, an immune cell targeting domain, a cleavable peptide, a membrane targeting domain, an extracellular cancer targeting domain, a transmembrane domain, and an intracellular signaling domain, and (ii) a CAR immune cell. In one embodiment, the CAR immune cell is prepared by treating an immune cell with the recombinant CAR fusion protein.

[0011] Additional embodiments are described herein. [Brief description of the drawings]

[0012] [Figure 1] Figure 2 shows the amino acid sequence of the recombinant CAR fusion protein against anti-PD-L1. [Diagram 2] FIG. 1 shows the structure of the recombinant CAR fusion protein of the present disclosure and illustrates how it engineered immune cells into chimeric antigen receptor (CAR) immune cells. [Diagram 3]FIG. 1 shows the results of SDS-PAGE and Western blotting. [Figure 4] Figure 1 shows FACS data confirming NK cell uptake of recombinant CAR fusion protein against anti-PD-L1. [Diagram 5] Figures 5A and 5B show FACS data comparing the uptake efficiency of recombinant CAR fusion proteins for anti-PD-L1 by NK cells at various concentrations. [Figure 6] FIG. 1 shows FACS data demonstrating that CAR-NK cells had higher PE intensity, indicating that more PD-L1 protein was bound to CAR-NK cells by the CAR protein. [Figure 7] FIG. 1 shows data comparing cytotoxic / cytolytic cytokine secretion upon PD-L1 binding when CAR-NK cells were treated and when NK cells were treated. [Figure 8] FIG. 1 shows data comparing the anti-cancer activity of NK cells with that of CAR-NK cells. [Figure 9] FIG. 1 shows data comparing cytokine secretion when CAR-NK cells were treated and when NK cells were treated. [Figure 10] Figure 10A, Figure 10B, and Figure 10C show data regarding in vivo CAR-NK retention in vitro. [Figure 11] FIG. 1 shows in vivo tumor suppression by in vivo CAR protein. [Figure 12] FIG. 1 shows data on human cytokines in tumor tissue and plasma. [Figure 13] FIG. 1 shows data on in vivo CAR engineering on blood human NK cells and blood human T cells. [Figure 14] FIG. 1 shows data confirming that injection of in vivo CAR results in higher NK cell cytolysis. [Figure 15] FIG. 1 shows data on early liver toxicity studies. [Figure 16] Figure 16A, Figure 16B, and Figure 16C show data confirming that in vivo CAR proteins generate comprehensive immune responses within the TME. [Figure 17] FIG. 1 shows data on early immunogenicity studies. [Figure 18] FIG. 1 shows in vivo CAR-NK biodistribution data. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] definition Although any method and material similar or equivalent to those described herein can be used to carry out or test the embodiments described herein, some preferred methods, compositions, devices, and materials are described herein. However, before describing the materials and methods of the present invention, it should be understood that the present invention is not limited to the specific molecules, compositions, methodologies, or protocols described herein, as these may vary according to routine experimentation and optimization. It should also be understood that the terms used in the detailed description are only intended to describe specific forms or embodiments, and are not intended to limit the scope of the embodiments described herein.

[0014] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. However, in case of conflict, the present specification, including definitions, shall control. Therefore, in the context of the embodiments described herein, the following definitions apply.

[0015] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a peptide" is a reference to one or more peptides and equivalents thereof known to those skilled in the art, and so forth.

[0016] As used herein, the term "comprise" and linguistic variants thereof indicate the presence of the recited feature(s), element(s), method step(s), etc., but do not exclude the presence of additional feature(s), element(s), method step(s), etc. In contrast, the term "consisting of" and linguistic variants thereof indicate the presence of the recited feature(s), element(s), method step(s), etc., and excludes any unrecited feature(s), element(s), method step(s), etc., except for impurities normally associated therewith. The phrase "consisting essentially of" refers to the recited feature(s), element(s), method step(s), etc., as well as any additional feature(s), element(s), method step(s), etc. that do not materially affect the basic nature of the composition, system, or method. Many embodiments herein are described using the open-ended term "comprising." Such embodiments encompass a number of the open-ended "consisting of" and / or "consisting essentially of" embodiments, which may alternatively be claimed or described in the specification using such terms.

[0017] As used herein, the term "treatment" refers to partially or completely alleviating, ameliorating, alleviating, delaying the onset of, inhibiting the progression of, reducing the severity of, and / or reducing the incidence of one or more symptoms or characteristics of a particular disease, disorder, and / or condition. For example, "treating" cancer can refer to inhibiting the growth and / or spread of cancer cells, killing cancer cells, or shrinking cancer cells. Treatment may be administered to subjects who do not show signs of a disease, disorder, and / or condition and / or to subjects who show only early signs of a disease, disorder, and / or condition, with the aim of reducing the risk of developing a condition associated with the disease, disorder, and / or condition.

[0018] As used herein, the terms "about," "approximate," "at or about," and "substantially" mean that the relevant amount or value may be the exact value set forth in the claims or taught herein, or a value that will produce an equivalent result or effect. That is, it is understood that the amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and / or larger or smaller, if desired, to produce an equivalent result or effect, reflecting tolerances, conversion factors, rounding, measurement errors, and the like, and other factors known to those skilled in the art. In some circumstances, it may not be possible to reasonably determine a value that will produce an equivalent result or effect. In such cases, generally, as used herein, "about" and "at or about" are understood to mean a variation of ±10% of the nominal value given, unless otherwise indicated or implied. In general, amounts, sizes, formulations, parameters, or other quantities or characteristics are "about," "approximate," or "at or about," whether or not expressly stated as such. When "about," "approximate," or "at or about" is used before or after a quantitative value, it is understood that the parameter also includes the particular quantitative value itself, unless specifically stated otherwise.

[0019] The term "antibody" as used herein refers to an immunoglobulin molecule that specifically binds to an antigen. Antibodies can be intact immunoglobulins derived from natural or recombinant sources or can be immunoreactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. Antibodies in the present invention may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab, and F(ab)2, as well as single chain antibodies and humanized antibodies (Harlow et al., 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, NY; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0020] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide contains at least two amino acids, and there is no limit to the maximum number of amino acids that may comprise a protein or peptide sequence. A polypeptide includes any peptide or protein that contains two or more amino acids linked together by peptide bonds. As used herein, the term refers to both short chains, commonly referred to in the art as peptides, oligopeptides and oligomers, and longer chains, commonly referred to in the art as proteins, of which there are many varieties. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, mutants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. A polypeptide includes natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0021] The term "specifically binds" as used herein with respect to an antibody means an antibody that recognizes a particular antigen but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to antigens from one or more species. However, such cross-species reactivity does not in itself change the specific classification of the antibody. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of that antigen. However, such cross-reactivity does not in itself change the specific classification of the antibody. In some cases, the terms "specific binding" or "specifically binds" may be used in reference to the interaction of an antibody, protein, or peptide with a second chemical species to mean that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species, e.g., the antibody recognizes and binds to a particular protein structure rather than proteins in general. If an antibody is specific for epitope "A", then the presence of a molecule containing epitope A (i.e., free, unlabeled A) in a reaction involving labeled "A" and an antibody will result in a decrease in the amount of labeled A bound to the antibody.

[0022] The term "stimulation" refers to a primary response induced by the binding of a stimulatory molecule (e.g., the TCR / CD3 complex) with its cognate ligand, thereby mediating a signal transduction event, such as, but not limited to, signal transduction through the TCR / CD3 complex. Stimulation may mediate changes in the expression of certain molecules, such as downregulation of TGF-β and / or rearrangement of cytoskeletal structures.

[0023] "Stimulatory molecule," as that term is used herein, means a molecule on a T cell or NK cell that specifically binds to a cognate stimulatory ligand present on an antigen-presenting cell.

[0024] As used herein, a "stimulatory ligand" refers to a ligand that, when present on an antigen-presenting cell (e.g., APC, dendritic cell, B cell, etc.), can specifically bind to a cognate binding partner (herein referred to as a "stimulatory molecule") on a T cell or NK cell, thereby mediating a primary response by the T cell, including, but not limited to, activation, initiation of an immune response, proliferation, etc. Stimulatory ligands are known in the art and include, inter alia, peptide-loaded MHC class I molecules, anti-CD3 antibodies, superagonist anti-CD28 antibodies, superagonist anti-CD2 antibodies, etc.

[0025] The term "isolated" means altered or removed from the natural state. For example, a nucleic acid or peptide that is naturally present in a living animal is not "isolated," but a nucleic acid or peptide that is partially or completely separated from the materials that coexist in the natural state of the same nucleic acid or peptide is "isolated." An isolated nucleic acid or protein may exist in a substantially purified form, or it may exist in a non-native environment, such as, for example, a host cell.

[0026] The term "cancer" as used herein is defined as a disease characterized by rapid and uncontrollable growth of abnormal cells. Cancer cells may spread locally or to other parts of the body through the bloodstream and lymphatic system. Examples of various cancers include, but are not limited to, kidney cancer, spleen cancer, lung cancer, liver cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, etc.

[0027] The term "therapeutically effective amount" means an amount of a therapeutic, prophylactic, and / or diagnostic agent (e.g., a CAR) that is sufficient when administered to a subject suffering from or susceptible to a disease, disorder, and / or condition to treat, alleviate, ameliorate, relieve the symptoms of, prevent, delay the onset of, inhibit the progression of, reduce the severity of, and / or reduce the incidence of, the disease, disorder, and / or condition.

[0028] The terms "subject," "patient," "individual," and the like are used interchangeably herein and refer to any animal or cells thereof amenable to the methods described herein, whether in vitro or in situ. In certain non-limiting embodiments, a patient, subject, or individual is a human.

[0029] Detailed Description 1. Recombinant Chimeric Antigen Receptor (CAR) Fusion Proteins The present disclosure provides a recombinant chimeric antigen receptor (CAR) fusion protein, which in turn comprises an immune cell targeting domain, a cleavable peptide, a membrane targeting domain, an extracellular cancer targeting domain, a transmembrane domain, and an intracellular signaling domain. The structure of an exemplary embodiment of the recombinant CAR fusion protein of the present disclosure is described in FIG. 2. In this embodiment, the recombinant CAR fusion protein was designed to modify NK cells into CAR NK cells through in vivo application. As described in FIG. 2, the recombinant CAR fusion protein can target NK cells by the NK cell targeting domain (i.e., immune cell targeting domain) and enter the cytoplasm of the NK cell by antigen-antibody binding. Once the recombinant CAR fusion protein enters the cytoplasm, the recombinant CAR fusion protein is separated into the NK cell targeting domain and the remaining recombinant CAR fusion protein domain by the cleavable peptide (enzymatically cleaved). The membrane targeting domain then guides the remaining recombinant CAR fusion protein domain (i.e., the membrane targeting domain, the extracellular cancer targeting domain, the transmembrane domain, and the intracellular signaling domain, also referred to as the CAR domain) to be located in the cell membrane. Thus, the CAR domain can enhance the anti-cancer activity of NK cells upon binding to cancer cells.

[0030] The recombinant CAR fusion protein of the present disclosure has advantages over conventional CAR technology because CAR immune cells can be generated either ex vivo or in vivo within one day without the use of viral vectors.More importantly, this technology enables a safer and cheaper cell-free immunotherapy than conventional CAR immune cells.

[0031] Immune Cell Targeting Domain In one embodiment, the immune cell targeting domain recognizes the target immune cell so that the recombinant CAR fusion protein enters the target immune cell through antigen-antibody binding. The immune cell targeting domain is selected from the group consisting of a NK cell targeting domain, a T cell targeting domain, a dendritic cell targeting domain, and a macrophage targeting domain.

[0032] For example, the recombinant CAR fusion protein may include an NK cell targeting domain so that the recombinant CAR fusion protein recognizes and enters NK cells. The NK cell targeting domain may be an antibody, antibody fragment, or antigen-binding fragment that targets NK cells. For example, the NK cell targeting domain may be an NK cell targeting antibody, an NK cell targeting scFv antibody, or a peptide that includes the VH, IgG hinge, IgG CH2, and IgG CH3 of an NK cell targeting antibody. The NK cell targeting antibody may be anti-NKp46, anti-NKp30, anti-NKp44, anti-NKp80, anti-NKG2A, anti-NKG2C, anti-NKG2D, anti-CD16, anti-CD56, anti-KIR-s, or anti-CD122.

[0033] Furthermore, the recombinant CAR fusion protein may include a T cell targeting domain so that the recombinant CAR fusion protein can recognize and enter T cells. For example, the T cell targeting domain may be a T cell targeting antibody, a T cell targeting scFv antibody, or a peptide comprising the VH, IgG hinge, IgG CH2, and IgG CH3 of a T cell targeting antibody. The T cell targeting antibody may be anti-CD3, anti-CD8, anti-CD45R, anti-NKp46, anti-NKp30, anti-TIM3, anti-TIGIT, anti-LAG3, or anti-CTLA4.

[0034] In another embodiment, when recombinant CAR fusion proteins are used to modify immune cells into chimeric antigen receptor (CAR) immune cells in ex vivo applications, specific immune cells can be directly treated with recombinant CAR fusion proteins, so the immune cell targeting domain can be selected from any protein transduction domain that is not specific to the target immune cells but allows the recombinant CAR fusion protein to be non-specifically introduced into any cell. In one embodiment, immune cell targeting domains (not specific to a certain immune cell) applied ex vivo can include Tat or Tat peptide, polyarginine, Antennapedia (Antp) or Antp peptide, penetratin, SAP, PTD-5, K-FGF (SN50 peptide), HIV-1 Rev, FHV, HTLV-II, NLS, transportan, and pVEC. Examples of these domains include, but are not limited to, Tat (YGRKKRRQRRR, SEQ ID NO:2) or Tat peptide (RKKRRQRRR, SEQ ID NO:3), polyarginine (RRRRRR, SEQ ID NO:4, RRRRRRRR, SEQ ID NO:5, RRRRRRRRR, SEQ ID NO:6), antennapedia (Antp) or Antp peptide (RQIKIWFQNRRMKW, SEQ ID NO:7), penetratin (RQIKIWFQNRRMKWKK, SEQ ID NO:8), SAP (VRLPPPVRLPPPVRLPPP, SEQ ID NO:9), PTD-5 (RRQRRTSKLMKR, SEQ ID NO:10), K-FGF (SN50 peptide) (AAVALLPAVLLALLAP, SEQ ID NO:11), Rev of HIV-1 (TRQARRNRRRRWRERQR, SEQ ID NO:12), FHV (RRRRNRTRRNRRRVR, SEQ ID NO:13), HTLV-II (TRRQRTRRATTNR, SEQ ID NO:14), NLS (KRPAAIKKAGQAKKKK, SEQ ID NO:15), Transportan (GWTLNSAGYLLGKINLKALAALAKKIL, SEQ ID NO:16), pVEC (LLIILRRRIRKQAHAHSK, SEQ ID NO:17). See also Table 1 below.

[0035] [Table 1]

[0036] Cleavable peptides In one embodiment, once the recombinant CAR fusion protein is inside an immune cell, the cleavable peptide is enzymatically cleaved to separate the immune cell targeting domain from the remaining recombinant CAR fusion protein domain.

[0037] The cleavable peptide may be a protease cleavage site: Gly-Gly-Phe-Gly (GGFG, SEQ ID NO: 33), a furin cleavage site (RRAR, SEQ ID NO: 34), a capthesin cleavage site (Phe-Lys (FK), Ala-Ala-Asn (AAN), Gly-Phe-Leu-Gly (GFLG, SEQ ID NO: 35)), or a legumain cleavage site (Ala-Leu-Ala-Leu (ALAL, SEQ ID NO: 36)).

[0038] Membrane targeting domain In one embodiment, when the immune cell targeting domain is separated from the remaining recombinant CAR fusion protein domain by enzymatic cleavage of the cleavable peptide, the membrane targeting domain directs the remaining recombinant CAR fusion protein domain to be located within the cell membrane (triggering exocytosis of the remaining recombinant CAR fusion protein domain towards the outer cell membrane).

[0039] The membrane targeting domain may be a peptide from T cell surface glycoprotein CD8 alpha chain (MALPVTALLLPLALLLHAARP, SEQ ID NO:37), interleukin-1 receptor type 1, 4F2 cell surface antigen heavy chain, linker for activation of T cell family members 1, junctophilin-1, antilisterial bacteriocin subtilosin biosynthesis protein AlbG, calcitonin receptor, gamma-secretase subunit APH-1A, adiponectin receptor protein 2. Examples of these peptides include, but are not limited to, interleukin-1 receptor type 1 (HMIGICVTLTVIIVCSVFIY, SEQ ID NO: 38), 4F2 cell surface antigen heavy chain (LLLLFWLGWLGMLAGAVVIIV, SEQ ID NO: 39), linker for activation of T cell family members 1 (ALSPVELGLLLLPFVVMLLAALCV, SEQ ID NO: 40), junctophilin-1 (IMIVLVMLLNIGLAILFVHFL, SEQ ID NO: 41), the antilisterial bacteriocin subtilosin biosynthesis protein AlbG (STVFTVLLLLLGMAAYSFGWV (SEQ ID NO: 42), GLLACIAAVLMLPAFLYLHYV (SEQ ID NO: 43), TYVMAAVLCQVIIFGCMFEIV, TPPIVSTGMALLLILYLLFYM (SEQ ID NO: 44), IGWMLSFTISELLFLIILAAI (SEQ ID NO: 45)), calcitonin B (STVFTVLLLLLGMAAYSFGWV (SEQ ID NO: 46), GLLACIAAVLMLPAFLYLHYV (SEQ ID NO: 47), TYVMAAVLCQVIIFGCMFEIV, TPPIVSTGMALLLILYLLFYM (SEQ ID NO: 48), IGWMLSFTISELLFLIILAAI (SEQ ID NO: 49), calcitonin B (STVFTVLLLLLGMAAYSFGWV (SEQ ID NO: 50), GLLACIAAVLMLPAFLYLHYV (SEQ ID NO: 51), TYVMAAVLCQVIIFGCMFEIV, TPPIVSTGMALLLILYLLFYM (SEQ ID NO: 52), IGWMLSFTISELLFLIILAAI (SEQ ID NO: 53), calcitonin B (STVFTVLLLLLGMAAYSFGWV (SEQ ID NO: 5 Nin receptor (VGHSLSIFTLVISLGIFVFF (SEQ ID NO: 46), VTLHKNMFLTYILNSMIIII (SEQ ID NO: 47), ILHFFHQYMMACNYFWMLCEGIY (SEQ ID NO: 48), WYYLLGWGFPLVPTTIHAIT (SEQ ID NO: 49), LLYIIHGPVMAALVVNFFFLLNIV (SEQ ID NO: 50), ATMILVPLLGIQFVVFPW (SEQ ID NO: 51), YVMHSLIHFQGFFVATIYCFCN (SEQ ID NO: 52)), gamma-secretase subunit APH-1A (AAVFFGCTFVAFGPAFALFLI (SEQ ID NO: 53), VIILVAGAFFWLVSLLLASVV (SEQ ID NO: 54), YGLLIFGAAVSVLLQEVFRFA (SEQ ID NO: 55), YVSGLSFGIISGVFSVINILA (SEQ ID NO: 56), TSAFLTAAIILLHTFWGVVFF (SEQ ID NO: 57),ATMILVPLLGIQFVVFPW (SEQ ID NO: 58), LLPIYAVTVSMGLWAFITAGG (SEQ ID NO: 59)), or adiponectin receptor protein 2 (NIWTHLLGCVFFLCLGIFYMF (SEQ ID NO: 60), VVFGLFFLGAILCLSFSWLFH (SEQ ID NO: 61), LFSKLDYSGIALLIMGSFVPW (SEQ ID NO: 62), CFIYLIVICVLGIAAIIVSQW (SEQ ID NO: 63), YRGVRAGVFLGLGLSGIIPTL (SEQ ID NO: 64), QIGWLMLMASLYITGAALYAA (SEQ ID NO: 65), QLFHIFVVAGAFVHFHGVSNL (SEQ ID NO: 66)).

[0040] Extracellular Cancer Targeting Domains In one embodiment, when immune cells are engineered into chimeric antigen receptor (CAR) immune cells with a recombinant CAR fusion protein, the extracellular cancer targeting domain recognizes and binds to antigens on cancer cells.

[0041] The extracellular cancer targeting domain can be an antibody, an antibody fragment, or an antigen-binding fragment. In one embodiment, the extracellular cancer targeting domain is a PD-L1 antigen-binding domain, an anti-PD-L1 Vh(EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTPSSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK, SEQ ID NO: 67), HER2, CD19, CD33, CD20, CD30, CD138, CD123, CD79b, CD37, FOLR1, TROP2, DLL3, ENPP3, CA6, B-cell maturation antigen (BCMA), carbonic anhydrase IX (CAIX), CD171, carcinoembryonic antigen (CEA), ERBB2, EGFR, EGFRvIII, GD2, alphaFR, GP100, Lewis Y, melanoma antigen 1 recognized by T cells (MART1), melanoma antigen A3 (MAGEA3), NYSEO1, P53, prostate specific membrane antigen (PSMA), mucin 16 (MUC16), glypican 3 (GPC3), mesothelin antigen binding domain.

[0042] Hinge Area In one embodiment, the recombinant CAR fusion protein further comprises a hinge region. In some aspects, the hinge region may be located between the extracellular cancer targeting domain and the transmembrane domain. The hinge region can increase the distance between the extracellular cancer targeting domain and the transmembrane domain and provide flexibility. In one embodiment, the hinge region may comprise 12 to 45 amino acids. For example, the hinge region may be CD8 hinge (FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD, SEQ ID NO: 68), CD28, IgG1, and IgG4.

[0043] Transmembrane domain In one embodiment, during the process of exocytosis of the remaining recombinant CAR fusion protein domain towards the extracellular membrane, the transmembrane domain is positioned at the plasma membrane, such that the intracellular signaling domain is positioned on the inside of the intracellular membrane.

[0044] In some embodiments, the transmembrane domain can be NKG2D, an immunoglobulin Fc domain, a CD8α domain, CD3ζ, FcεR1γ, CD4, CD7, CD28, OX40, H2-Kb, ICOS, NKG2D, NKp44, and CD16.

[0045] Intracellular signaling domains In one embodiment, the intracellular signaling domain comprises a CD3 zeta (CD3ζ) signaling domain and a costimulatory signaling region selected from the group consisting of 2B4, DAP12, GITR, CD137, OX40, CD27, ICOS, CD40, 41BB, and DAP10.

[0046] Tag sequence In one embodiment, the recombinant CAR fusion protein further comprises a tag sequence. In some aspects, the tag sequence can be placed at the C-terminus or N-terminus of the recombinant CAR fusion protein, optionally via a linker. The linker can be a serine-glycine linker, such as GGGGS (SEQ ID NO: 69), GGGSS (SEQ ID NO: 70), GGGSG (SEQ ID NO: 71), or multiple variants thereof, such as GGGGSGGGGS (SEQ ID NO: 72) or (GGGGS (SEQ ID NO: 69)) m, (GGGSS (SEQ ID NO: 70)) m, (GGGSG (SEQ ID NO: 71)) m, where m is an integer between 1 and 5, 1 and 4, or 1 and 3. In a preferred embodiment, m is 2.

[0047] In some embodiments, the tag sequence can be, but is not limited to, a histidine tag, a glutathione-S-transferase tag, a maltose binding protein tag, a Strep tag, or a hemagglutinin tag. Examples of these tags include, but are not limited to, histidine tags (HHHHHH (SEQ ID NO: 73), HHHHHHHH (SEQ ID NO: 74), HHHHHHHHHH (SEQ ID NO: 75)), glutathione-S-transferase tags (MSPILGYWKI KGLVQPTRLLLEYLEEKYEEHLYERDEGDK WRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALD VVLYMDPMCL DAFPKLVCFKKRIEAIPQID KYLKSSKYIAWPLQGWQATF GGGDHPPK, SEQ ID NO: 76), maltose binding protein tag (MKIKTGARILALSALTTMMFSASALAKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKY DIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTRITK, SEQ ID NO: 77), or a Strep tag (WSHPQFEK, SEQ ID NO: 78).

[0048] The tag sequence can be used to isolate the recombinant CAR protein from E. coli or other cells during protein production and to verify the presence of the recombinant CAR protein in NK cells.

[0049] Linker In one embodiment, a linker may be included between the immune cell targeting domain and the cleavable peptide. For example, the linker may be a serine-glycine linker such as GGGGSGGGSGGGGS (SEQ ID NO: 79), GGGGS (SEQ ID NO: 69), GGGSS (SEQ ID NO: 70), GGGSG (SEQ ID NO: 71), or multiple variants thereof such as GGGGSGGGGS (SEQ ID NO: 72) or (GGGGS (SEQ ID NO: 69)) m, (GGGSS (SEQ ID NO: 70)) m, (GGGSG (SEQ ID NO: 71)) m, where m is an integer from 1 to 5, 1 to 4, or 1 to 3. In a preferred embodiment, m is 2.

[0050] 2. Methods for modifying immune cells into chimeric antigen receptor (CAR) immune cells The present disclosure also provides a method of modifying immune cells into chimeric antigen receptor (CAR) immune cells, comprising treating the immune cells with a recombinant CAR fusion protein as discussed above.

[0051] In one embodiment, the above method can be applied both in vivo and ex vivo.

[0052] In in vivo applications, the immune cell targeting domain can be an antibody, antibody fragment, or antigen-binding fragment that targets the specific immune cell to be modified into a CAR immune cell. For example, to modify NK cells into CAR NK cells in in vivo applications, the immune cell targeting domain can be an NK cell targeting domain. Furthermore, to modify T cells into CAR T cells in in vivo applications, the immune cell targeting domain can be a T cell targeting domain.

[0053] The NK cell targeting domain may be an antibody, antibody fragment, or antigen-binding fragment that targets NK cells. For example, the NK cell targeting domain may be an NK cell targeting antibody, an NK cell targeting scFv antibody, or a peptide comprising the VH, IgG hinge, IgG CH2, and IgG CH3 of an NK cell targeting antibody. The NK cell targeting antibody may be anti-NKp46, anti-NKp30, anti-NKp44, anti-NKp80, anti-NKG2A, anti-NKG2C, anti-NKG2D, anti-CD16, anti-CD56, anti-KIR-s, and / or anti-CD122.

[0054] Additionally, the T cell targeting domain may be a T cell targeting antibody, a T cell targeting scFv antibody, a peptide comprising the VH, IgG hinge, IgG CH2, and IgG CH3 of a T cell targeting antibody. The T cell targeting antibody may be anti-CD3, anti-CD8, anti-CD45R, anti-NKp46, anti-NKp30, anti-TIM3, anti-TIGIT, anti-LAG3, or anti-CTLA4.

[0055] In ex vivo applications, specific immune cells (modified into CAR immune cells) can be directly treated with recombinant CAR fusion protein, so the immune cell targeting domain can be selected from any protein transduction domain that is not specific to the target immune cells but allows the recombinant CAR fusion protein to be non-specifically introduced into any cell. In one embodiment, the immune cell targeting domain (not specific to a certain immune cell) applied ex vivo can include Tat or Tat peptide, polyarginine, Antennapedia (Antp) or Antp peptide, penetratin, SAP, PTD-5, K-FGF (SN50 peptide), HIV-1 Rev, FHV, HTLV-II, NLS, transportan, and pVEC. Examples of these domains include, but are not limited to, Tat (YGRKKRRQRRR, SEQ ID NO:2) or Tat peptide (RKKRRQRRR, SEQ ID NO:3), polyarginine (RRRRRR, SEQ ID NO:4, RRRRRRRR, SEQ ID NO:5, RRRRRRRRR, SEQ ID NO:6), antennapedia (Antp) or Antp peptide (RQIKIWFQNRRMKW, SEQ ID NO:7), penetratin (RQIKIWFQNRRMKWKK, SEQ ID NO:8), SAP (VRLPPPVRLPPPVRLPPP, SEQ ID NO:9), PTD-5 (RRQRRTSKLMKR, SEQ ID NO:10), K-FGF (SN50 peptide) (AAVALLPAVLLALLAP, SEQ ID NO:11), Rev of HIV-1 (TRQARRNRRRRWRERQR, SEQ ID NO:12), FHV (RRRRNRTRRNRRRVR, SEQ ID NO:13), HTLV-II (TRRQRTRRATTNR, SEQ ID NO:14), NLS (KRPAAIKKAGQAKKKK, SEQ ID NO:15), Transportan (GWTLNSAGYLLGKINLKALAALAKKIL, SEQ ID NO:16), pVEC (LLIILRRRIRKQAHAHSK, SEQ ID NO:17). See also Table 1 above.

[0056] In one embodiment, the recombinant CAR fusion protein can be treated to immune cells at a concentration effective for modifying immune cells into CAR immune cells, taking into account the efficiency of uptake of immune cells. For example, the lower limit of such a concentration may be a concentration sufficient for the modified CAR immune cells to exhibit a therapeutic effect against cancer. Furthermore, the upper limit of such a concentration may be a concentration at which the uptake of the recombinant CAR fusion protein by immune cells is saturated. For example, the recombinant CAR fusion protein may be at a concentration of 1000 nM to 2000 nM. In another embodiment, the recombinant CAR fusion protein may be at a concentration of about 1000 nM to about 2000 nM. In another embodiment, the lower limit may be about 1100 nM, about 1200 nM, about 1300 nM, about 1400 nM, or about 1500 nM. In another embodiment, the upper limit may be about 1900 nM, about 1800 nM, about 1700 nM, or about 1600 nM. In another embodiment, the recombinant CAR fusion protein may be at a concentration of about 1000 nM.

[0057] 3. Methods of treating cancer by administering CAR immune cells to a subject in need of treatment The present disclosure also provides a method of treating cancer, comprising administering to a subject in need of treatment CAR immune cells prepared by treating immune cells with a recombinant CAR fusion protein of the present disclosure.

[0058] In one embodiment, CAR immune cells are prepared by ex vivo applications as discussed in this disclosure and administered to a subject to treat cancer.

[0059] In one embodiment, the cancer being treated may be kidney cancer, spleen cancer, lung cancer, liver cancer, breast cancer, lung cancer, B-cell cancer, prostate cancer, lymphoma, chronic lymphocytic leukemia (CLL), B-cell acute lymphocytic leukemia (B-ALL), non-Hodgkin's lymphoma (NHL), acute lymphoblastic leukemia (ALL), myeloid malignancies, multiple myeloma, renal cell carcinoma (RCC), EGFR-positive solid tumors, glioblastoma, neuroblastoma, Ewing's sarcoma, osteosarcoma, acute myeloid leukemia (AML), melanoma, esophageal cancer, synovial sarcoma, sarcoma, colorectal cancer, hepatocellular carcinoma, non-small cell lung cancer, pancreatic cancer, triple-negative invasive breast cancer, ovarian cancer, or mesothelioma.

[0060] In another embodiment, the CAR immune cells are administered to a subject in a therapeutically effective amount to treat cancer.

[0061] In another embodiment, the method may further comprise administering a therapeutic agent, for example, the therapeutic agent may be a chemotherapeutic drug, a proteasome inhibitor, an immunomodulatory agent, a histone deacetylase inhibitor, a monoclonal antibody, a bispecific antibody, a recombinant antibody, or an immune checkpoint inhibitor.

[0062] The CAR immune cells can be administered to the subject according to any method. Such methods are known to those skilled in the art and include, but are not limited to, oral, transdermal, inhalation, intranasal, topical, intravaginal, intraocular, intraaural, intracerebral, rectal, sublingual, buccal, and parenteral administration, including injectables such as intravenous, intraarterial, intramuscular, and subcutaneous administration. Administration can be continuous or intermittent. The preparation can be administered for therapeutic purposes, i.e., to treat an existing disease or condition. The preparation can also be administered for prophylactic purposes, i.e., to prevent cancer, such as blood cancer or solid tumors.

[0063] An effective amount can be administered in one or more administrations, applications, or dosages, and is not intended to be limited to a particular formulation or route of administration. In one embodiment, administration occurs over a course of treatment that includes multiple treatment cycles and multiple rest periods.

[0064] 4. Methods of treating cancer by administering a recombinant CAR fusion protein to a subject in need of treatment The present disclosure also provides a method of treating cancer, comprising administering a recombinant CAR fusion protein to a subject in need of treatment.

[0065] In one embodiment, the cancer being treated may be kidney cancer, spleen cancer, lung cancer, liver cancer, breast cancer, lung cancer, B-cell cancer, prostate cancer, lymphoma, chronic lymphocytic leukemia (CLL), B-cell acute lymphocytic leukemia (B-ALL), non-Hodgkin's lymphoma (NHL), acute lymphoblastic leukemia (ALL), myeloid malignancies, multiple myeloma, renal cell carcinoma (RCC), EGFR-positive solid tumors, glioblastoma, neuroblastoma, Ewing's sarcoma, osteosarcoma, acute myeloid leukemia (AML), melanoma, esophageal cancer, synovial sarcoma, sarcoma, colorectal cancer, hepatocellular carcinoma, non-small cell lung cancer, pancreatic cancer, triple-negative invasive breast cancer, ovarian cancer, or mesothelioma.

[0066] In another embodiment, the recombinant CAR fusion protein can be administered to a subject at a concentration effective for modifying immune cells into CAR immune cells, taking into account the efficiency of immune cell uptake. For example, the lower limit of such a concentration may be a concentration sufficient for the modified CAR immune cells to exhibit a therapeutic effect against cancer. Furthermore, the upper limit of such a concentration may be a concentration at which the uptake of the recombinant CAR fusion protein by immune cells is saturated. For example, the recombinant CAR fusion protein may be at a concentration of 1000 nM to 2000 nM. In another embodiment, the recombinant CAR fusion protein may be at a concentration of about 1000 nM to about 2000 nM. In another embodiment, the lower limit may be about 1100 nM, about 1200 nM, about 1300 nM, about 1400 nM, or about 1500 nM. In another embodiment, the upper limit may be about 1900 nM, about 1800 nM, about 1700 nM, or about 1600 nM. In another embodiment, the recombinant CAR fusion protein may be at a concentration of about 1000 nM.

[0067] The recombinant CAR fusion protein can be administered to a subject according to any method. Such methods are known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, intranasal administration, topical administration, intravaginal administration, intraocular administration, intraaural administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injections such as intravenous administration, intraarterial administration, intramuscular administration, and subcutaneous administration. Administration can be continuous or intermittent. The preparation can be administered for therapeutic purposes, i.e., it can be administered to treat an existing disease or condition. The preparation can also be administered for prophylactic purposes, i.e., it can be administered to prevent cancer, such as blood cancer or solid tumors.

[0068] An effective amount can be administered in one or more administrations, applications, or dosages, and is not intended to be limited to a particular formulation or route of administration. In one embodiment, administration occurs over a course of treatment that includes multiple treatment cycles and multiple rest periods.

[0069] 5. Methods of treating cancer by administering recombinant CAR fusion proteins and CAR immune cells to a subject in need of treatment The present disclosure also provides a method of treating cancer by administering both the recombinant CAR fusion protein and CAR immune cells discussed above. In particular, in one embodiment, the present disclosure provides a method of treating cancer, comprising administering to a subject in need of treatment (i) a recombinant chimeric antigen receptor (CAR) fusion protein, the recombinant chimeric antigen receptor (CAR) fusion protein comprising, in order, an immune cell targeting domain, a cleavable peptide, a membrane targeting domain, an extracellular cancer targeting domain, a transmembrane domain, and an intracellular signaling domain, and (ii) a CAR immune cell.

[0070] In one embodiment, the CAR immune cells are prepared by treating immune cells with a recombinant CAR fusion protein. In another embodiment, the CAR immune cells may be CAR immune cells prepared by any other method. EXAMPLES

[0071] Example 1 (A) Preparation of recombinant CAR fusion protein against anti-PD-L1 A recombinant CAR fusion protein against anti-PD-L1 was prepared with the following structure: Anti-NKp46 Vh-IgG hinge-IgG CH2-IgG CH3-linker-Furin-membrane-targeted-Anti-PD-L1 Vh-CD8 hinge-NKG2D TM-2B4-CD3 zeta-His tag

[0072] In particular, the amino acid sequence of the recombinant CAR fusion protein for anti-PD-L1 was as shown in Figure 1 (SEQ ID NO:1). The bolded letters next to M represent the Vh-IgG hinge-IgG CH2-IgG CH3 of anti-NKp46 (i.e., immune cell targeting domain). The italicized letters represent the GGGGSGGGSGGGGGS (SEQ ID NO:79) linker and the GGGGS (SEQ ID NO:69) linker. The bolded underlined letters represent the Furin cleavage site (RRAR (SEQ ID NO:34), i.e., a cleavable peptide). The italicized underlined letters represent the membrane targeting domain. The regular capitalized letters represent the Vh of anti-PD-L1 (i.e., the extracellular cancer targeting domain). The bolded italicized letters represent the CD8 hinge (i.e., the hinge region). The underlined letters represent the NKG2D (i.e., the transmembrane domain). The smaller letters represent the 2B4-CD3 zeta (i.e., the intracellular signaling domain). The C-terminal HHHHHH (SEQ ID NO: 73) is a histidine tag (ie, a tag sequence).

[0073] (B) SDS-PAGE and Western blot analysis The in vivo CAR protein was tested by SDS-PAGE and Western blot to visualize its molecular weight. The molecular weight of the in vivo CAR is 127 kDa, and the SDS-PAGE and Western blot data show that the band of the in vivo CAR protein is near the 120 kDa molecular weight marker, indicating that the in vivo CAR protein was produced properly. See Figure 3.

[0074] (C) Confirmation of NK cell uptake of recombinant anti-PD-L1 CAR fusion protein The prepared recombinant CAR fusion protein against anti-PD-L1 was injected into NK-92 cells (1 μM of CAR fusion protein against anti-PD-L1, 1 × 10 per 0.5 mL) 6100 cells) for 24 hours. The cells were harvested, fixed, and permeabilized. Then, anti-his tag antibody conjugated to PE was treated to label the his tag on the recombinant CAR fusion protein against anti-PD-L1. As shown in Figure 4, it was found that NK cells treated with recombinant CAR fusion protein against anti-PD-L1 exhibited higher PE intensity. Since 6x histidine does not naturally occur in cells, the histidine labeled with PE was from the recombinant CAR fusion protein against anti-PD-L1.

[0075] (D) Optimization of delivery of recombinant CAR fusion protein To optimize the concentration for transfection of the recombinant CAR fusion protein, recombinant CAR fusion protein with various concentrations was injected into NK-92 cells (1 × 10 per 0.5 mL). 6 The cells were treated with 1000 nM of 1500 nM of 10 ...

[0076] (E) PD-L1 binding of recombinant CAR fusion protein After generating CAR-NK cells, PD-L1 protein (20ng / mL) was treated to both CAR-NK cells and NK cells and incubated for 2 hours. Because PD-L1 protein has 6x histidine protein, anti-his tag antibody conjugated to PE was used to identify PD-L1 protein-bound cells. In this experiment, cells were not permeabilized to stain the external his tag conjugated to PD-L1 protein. As shown in Figure 6, CAR-NK cells were found to have higher PE intensity, indicating that more PD-L1 protein was bound to CAR-NK cells by CAR protein.

[0077] (F) Cytokine release after PD-L1 binding with PD-L1 treatment alone without co-incubation of cancer cells. To verify the increased anti-cancer activity of CAR fusion protein, cytotoxic / cytolytic cytokine secretion upon PD-L1 binding was analyzed. After PD-L1 binding to NK cells or CAR-NK cells (treatment with 20ng / mL PD-L1 protein for 2 hours), cell supernatants were collected and ELISA was performed. As shown in Figure 7, it was confirmed that the cytokine secretion of CAR-NK cells was significantly increased compared with NK cells. This result suggested that CAR-NK cells may have stronger anti-cancer activity than NK cells.

[0078] (G) Anti-cancer activity of NK cells: cancer cell death The anti-cancer activity of CAR-NK cells and NK cells was compared through the induction of cancer cell death. Specifically, cancer cells treated with NK cells, cancer cells treated with CAR-NK cells, and cancer cells not treated with either NK or CAR-NK cells were compared. The experimental conditions were as follows: MDA-MB-231 cancer cells labeled with CellTracker Blue™ Effector:Target (E:T) = 5:1 Co-incubation of CellTracker Blue-labeled MDA-MB-231 with NK / in vivo CAR-NK for 4 hours

[0079] After co-incubation, cells were harvested and stained with Alexa Fluor-488 Annexin V to label dead cells. The cells were subjected to flow cytometry, and the number of cells (cancer cells) labeled with CellTracker Blue was analyzed by the intensity of Alexa Fluor 488 (Figure 8A). Cells stained with Alexa Fluor 488-Annexin V indicated dead cancer cells. The higher the number of boxes with number 2, the more dead cancer cells there were. The number of boxes with number 2 in Figure 8A is summarized in Figure 8B. These results showed that CAR-NK cells have stronger anti-cancer activity compared to NK cells.

[0080] (H) Anti-cancer activity of NK cells: NK cell cytokines After 4 hours of co-incubation of cancer cells with NK cells and cancer cells with CAR-NK cells, cytokine secretion from NK cells and cytokine secretion from CAR-NK cells were measured from cell supernatant. Here, the control group refers to cancer cells that were not treated with NK cells or CAR-NK cells. The results are shown in Figure 9. Upon transplantation of cancer cells, more cytotoxic / cytolytic cytokines were secreted from CAR-NK cells than from NK cells. It was confirmed that the increase in cancer cell death of CAR-NK cells was induced by the increase in cytokine secretion. Therefore, it was confirmed that the recombinant CAR fusion protein of the present disclosure can modify NK cells to have a CAR system without viral vector-mediated gene transfer and gene expression time, resulting in stronger anti-cancer activity.

[0081] (I) In vitro and in vivo retention of CAR-NK The retention of CAR-NK was confirmed by the presence of his-tag in cells at various time points. In vivo CAR-NK cells were generated under the same conditions as other in vitro experiments. Unbound in vivo CAR protein was removed on day +1 using a centrifugation process. After removal of in vivo CAR protein, in vivo CAR-NK cells were incubated in basic culture conditions for NK cells. In vivo CAR-NK cells were harvested at various time points after in vivo CAR protein treatment. Harvested cells were fixed, permeabilized, and his-tag was labeled with anti-his-tag antibody conjugated to PE. In vivo CAR-NK cells were detected up to day +9. See Figure 10A, Figure 10B, and Figure 10C.

[0082] The in vivo CAR delivery conditions were 1 μM in vivo CAR protein, 1 × 10 per 0.5 mL. 6 cells, NK-92 culture medium.

[0083] (J) In vivo tumor suppression by in vivo CAR protein Five million MDA-MB-231 cells were inoculated subcutaneously in humanized NSG mice. The control group received DPBS, and the test groups were injected intravenously with 1.7 mg / Kg or 11 mg / Kg of in vivo CAR protein once a week for 4 weeks. Tumor volume and body weight were measured twice a week. Tumor volume was measured by caliper measurement (v=0.5ab 2 , a=long dimension, b=short dimension). (A) Relative tumor volume, (B) relative body weight, and (C) survival rate. Circles: control group, squares: 1.7 mg / Kg in vivo CAR protein, and triangles: 11 mg / Kg in vivo CAR protein. (D) Individual relative tumor volumes for each group.

[0084] The 11 mg / Kg in vivo CAR group showed significant tumor suppression and improved survival rate. The in vivo CAR protein did not affect body weight, indicating safety and non-toxicity. These in vivo experiments demonstrate the therapeutic efficacy and dose-dependence of the in vivo CAR protein. See Figure 11.

[0085] (K) Human cytokines in tumor tissue and plasma After tumor volume and body weight were measured on day +28, animals were sacrificed to collect blood and organs (tumor, liver, kidney, spleen, and lungs). Plasma was then collected by centrifugation with gradient medium. To obtain tumor lysate, tumor tissue was homogenized and lysed in RIPA buffer. Supernatant was collected by centrifugation to remove cell debris. Tumor lysate and plasma were tested for human cytokines with ELISA kits. In vivo CAR treatment group showed increased cytokine secretion. It was hypothesized that increased concentration of cytokines suppresses tumor progression. This result suggests that in vivo CAR protein induces immune system activation and is responsible for its therapeutic efficacy. See Figure 12.

[0086] (L) In vivo CAR modification of blood human NK cells and blood human T cells Blood was collected on day +28, 7 days after the last injection of in vivo CAR protein. Immune cells in the blood were collected through centrifugation with gradient medium and separation of human NK cells or human T cells. Separated human NK cells and human T cells were fixed, permeabilized, and his-tag labeled with PE anti-his tag antibody to detect in vivo CAR-NK cells or in vivo CAR-T cells. In vivo CAR-NK cells were significantly detected in the in vivo CAR-injected group, and a dose-dependent pattern was observed. However, the difference in in vivo CAR-T numbers was negligible between groups. This result indicates that the current in vivo CAR protein specifically modifies NK cells due to its NK cell targeting domain. Since the in vivo CAR protein has a modular design, the NK cell targeting domain can be replaced with the T cell targeting domain for in vivo CAR-T applications. See Figure 13.

[0087] (M) In vivo CAR injection results in higher cytolytic activity of NK cells Human NK cells isolated from blood were analyzed for their cytolytic properties. + NK cells and CD107a + NK cells have the cytotoxic properties of antibody-dependent cellular cytotoxicity (ADCC) and degranulation, respectively. The isolated human NK cells were treated with anti-human CD16 antibody or anti-human CD107a antibody. In the 11 mg / Kg in vivo CAR protein injection group, human CD16 + The number of NK cells was increased. Human CD107a + NK cell numbers increased. These results suggest that in vivo CAR proteins stimulate NK cells to have cytolytic properties. See Figure 14.

[0088] (N) Early liver toxicity study The levels of alanine transaminase (ALT) and creatine kinase (CK) in blood were analyzed to determine whether in vivo CAR protein causes liver toxicity. Plasma was tested by ELISA to measure the concentrations of ALK and CK. In vivo CAR protein injection did not result in an increase in the levels of ALT or CK. This result indicates that in vivo CAR protein injection does not cause liver toxicity. See Figure 15.

[0089] (O) In vivo CAR proteins generate comprehensive immune responses within the TME Comprehensive profiling of immune cells in tumor tissues was performed to characterize the immune response after administration of in vivo CAR proteins. Tumor tissues were homogenized and dissociated by collagenase to obtain single cells. Single cells were labeled with various antibodies: anti-human CD45 (human hematopoietic marker), anti-human CD56 (human NK cell marker), anti-human CD3 (human T cell marker), anti-human CD4 (helper / regulatory human T cell marker), anti-human CD8 (cytotoxic human T cell marker), anti-human CD68 (human macrophage marker), anti-CD80 (human M1 macrophage marker), and anti-CD206 (human M2 macrophage marker). Administration of in vivo CAR proteins increased the number of tumor-infiltrating human immune cells in the tumor microenvironment (TME) compared to the control group. In vivo CAR proteins specifically modified human NK cells, and human CAR-NK cells secreted cytokines that reacted with other human immune cells. Furthermore, in both in vivo CAR protein-injected groups, human CD8 + / CD4 + The ratio of T cells and human M1 / M2 macrophages was significantly increased, thus confirming that the in vivo CAR protein induces a comprehensive immune response within the TME. + The number of NK cells was also increased. These results were consistent with the results of human CD107a +This is consistent with the data confirming an increase in NK cell numbers. Collectively, these results indicate that in vivo CAR proteins generate a comprehensive immune response within the TME, which accounts for their promising therapeutic efficacy. See Figures 16A, 16B, and 16C.

[0090] (P) Early immunogenicity studies To investigate the potential immunogenicity of CAR proteins in vivo, blood CD8 + / CD4 + The proportion of human T cells was analyzed. + This leads to proliferation of T cells, which in turn leads to CD8 + / CD4 + The ratio of human T cells will change. The in vivo CAR-injected group has higher CD8 T cell counts than the control group. + / CD4 + They were found to have only minor differences in the proportion of human T cells, indicating that they did not cause immunogenicity, see Figure 17.

[0091] (Q) In vivo biodistribution of CAR-NK To analyze the biodistribution of in vivo CAR-NK cells, organs including kidney, spleen, lung, liver, and tumor were isolated on day +28, 7 days after the final in vivo CAR injection. Tumor tissue was homogenized and dissociated with collagenase to obtain single cells. Cells were fixed and permeabilized to label intracellular his tag. Anti-his tag antibody conjugated to PE was treated to cells, and cells were analyzed by flow cytometry. In the 11 mg / Kg in vivo CAR injection group, the number of in vivo CAR-NK cells was increased in tumor tissue compared to other tissues. This data indicates that in vivo CAR protein modifies NK cells into CAR-NK cells and reliably directs them to tumors. See Figure 18.

[0092] As discussed above, recombinant CAR fusion proteins against anti-PD-L1 expressed on various cancer cells have been made for the first time and shown in this disclosure. It was verified that this recombinant CAR-PDL1 protein spontaneously modifies NK cells into CAR-NK PDL1. The resulting CAR-NK PDL1 captures PD-L1 protein (antigen) more efficiently than naive NK cells, resulting in increased secretion of cytotoxic / cytolytic cytokines upon PD-L1 binding. This result indicates that CAR-NK PDL1 was successfully made and can recognize its complementary antigen. It was also verified that co-culture of CAR-NK PDL1 with triple-negative breast cancer cells expressing PD-L1 resulted in more than two-fold stronger anti-cancer activity and significantly increased cytokine secretion. Thus, it was confirmed that the recombinant CAR fusion protein can modify immune cells into CAR immune cells and be developed for both in vivo and ex vivo applications.

[0093] Example 2 (A) Preparation of recombinant CAR fusion protein against anti-PD-L1 Recombinant CAR fusion proteins for T cells against anti-PD-L1 are designed by mixing T cell targeting antibodies and intracellular stimulatory domains from the table below.

[0094] [Table 2]

[0095] A CAR fusion protein for T cells is prepared with the following structure: Vh of anti-T cell targeting antibody-IgG hinge-IgG CH2-IgG CH3-linker-Furin-membrane targeting-Vh of anti-PD-L1-CD8 hinge-transmembrane domain-intracellular domain 1-CD3 zeta-His tag

[0096] (B) Confirmation of T cell uptake of recombinant anti-PD-L1 CAR fusion protein Recombinant CAR fusion protein against anti-PD-L1 was administered to T cells (1 μM CAR fusion protein against anti-PD-L1, 1 × 10 per 0.5 mL) 6 The T cells are incubated with 6× histidine (1000× 1000 cells) for 24 hours. The cells are harvested, fixed, and permeabilized. The his-tag antibody conjugated to PE is then treated to label the his-tag on the recombinant CAR fusion protein against anti-PD-L1. T cells treated with recombinant CAR fusion protein against anti-PD-L1 are expected to show higher PE intensity. The PE-labeled histidine is from the recombinant CAR fusion protein against anti-PD-L1 because 6× histidine does not naturally occur in cells.

[0097] (C) Optimization of delivery of recombinant CAR fusion protein To optimize the concentration for transfection of the recombinant CAR fusion protein, recombinant CAR fusion protein with various concentrations was administered to T cells (1 × 10 per 0.5 mL). 6 The cells are treated with 1000 nM of CAR fusion protein for 24 hours. The protein introduction is confirmed by the same method as discussed in (B) of Example 2 above. As a result, similar to the introduction of CAR fusion protein into NK cells, the protein uptake is expected to be saturated at more than 1500 nM, and 1000 nM is selected as the optimal introduction concentration.

[0098] (D) PD-L1 binding of recombinant CAR fusion protein After generating CAR-T cells, PD-L1 protein is treated to both CAR-T cells and T cells and incubated for 2 hours. Since PD-L1 protein has 6x histidine protein, anti-his tag antibody conjugated to PE is used to identify PD-L1 protein-bound cells. In this experiment, cells are not permeabilized to stain the external his tag conjugated to PD-L1 protein. CAR-T cells are expected to have higher PE intensity, indicating that more PD-L1 protein is bound to CAR-T cells by CAR protein than normal T cells.

[0099] (E) Cytokine release after PD-L1 binding by PD-L1 protein treatment without co-incubation of cancer cells. To verify the increased anti-cancer activity of CAR fusion proteins, we analyze the cytotoxic / cytolytic cytokine secretion upon PD-L1 binding to CAR-T cells and T cells. After PD-L1 binding to T cells or CAR-T cells (20ng / mL PD-L1 protein treatment for 2 hours), cell supernatants were collected and cytokine ELISA was performed similarly to the CAR-NK cell analysis. It is expected that the cytokine secretion of CAR-T cells is significantly increased compared to T cells. This result suggests that CAR-T cells have stronger anti-cancer activity than T cells.

[0100] (F) Anticancer activity of T cells: cancer cell death The anti-cancer activity of CAR-T cells and T cells is compared through the induction of cancer cell death. The experimental conditions are as follows: MDA-MB-231 cancer cells labeled with CellTracker Blue™ Effector:Target (E:T) = 5:1 Co-incubation of CellTracker Blue™-labeled MDA-MB-231 with T cells / in vivo CAR-T cells for 4 hours

[0101] After co-incubation, cells are harvested and stained with Alexa Fluor-488 Annexin V to label dead cells. Cells are subjected to flow cytometry to analyze the number of cells (cancer cells) labeled with CellTracker Blue™ by the intensity of Alexa Fluor 488. Cells stained with Alexa Fluor 488-Annexin V indicate dead cancer cells. The higher the number of cells stained with Alexa Fluor 488, the more dead cancer cells there are. It is predicted that CAR-T cells have stronger anti-cancer activity compared to T cells.

[0102] (G) Anti-cancer activity of T cells: T cell cytokines After 4 hours of co-incubation of cancer cells with T cells or CAR-T cells, cytokine secretion from T cells and cytokine secretion from CAR-T cells are measured from cell supernatant. The results are expected to show that when cancer cells are transplanted, more cytotoxic / cytolytic cytokines are secreted from CAR-T cells than from T cells. The cytokine results are expected to correlate with cancer cell death, demonstrating that increased cancer cell death of CAR-T cells is induced by increased cytokine secretion. Thus, recombinant CAR fusion proteins can modify T cells to have a CAR system without viral vector-mediated gene transfer and gene expression time, which is expected to result in stronger anti-cancer activity.

Claims

1. In order, an immune cell targeting domain; A cleavable peptide; a membrane targeting domain; an extracellular cancer targeting domain; A transmembrane domain; an intracellular signaling domain; 1. A recombinant chimeric antigen receptor (CAR) fusion protein comprising:

2. 2. The recombinant CAR fusion protein of claim 1, wherein the immune cell targeting domain is a NK cell targeting domain or a T cell targeting domain.

3. 3. The recombinant CAR fusion protein of claim 2, wherein the NK cell targeting domain is an NK cell targeting scFv antibody or a peptide comprising the VH, IgG hinge, IgG CH2, and IgG CH3 of anti-NKp46.

4. 2. The recombinant CAR fusion protein of claim 1, wherein the cleavable peptide is a peptide configured to be enzymatically cleaved.

5. 2. The recombinant CAR fusion protein of claim 1, wherein the membrane targeting domain is a peptide from interleukin-1 receptor type 1, 4F2 cell surface antigen heavy chain, linker for activation of T-cell family members 1, junctophilin-1, antilisterial bacteriocin subtilosin biosynthesis protein AlbG, calcitonin receptor, gamma-secretase subunit APH-1A, or adiponectin receptor protein 2.

6. 2. The recombinant CAR fusion protein of claim 1, wherein the extracellular cancer targeting domain is an anti-PD-L1 Vh.

7. 2. The recombinant CAR fusion protein of claim 1, further comprising a hinge region between said extracellular cancer targeting domain and said transmembrane domain.

8. 2. The recombinant CAR fusion protein of claim 1, wherein the transmembrane domain is NKG2D.

9. 2. The recombinant CAR fusion protein of claim 1, wherein the intracellular signaling domain comprises a CD3 zeta (CD3ζ) signaling domain and a costimulatory signaling region selected from the group consisting of 2B4, DAP12, GITR, CD137, and OX40.

10. The recombinant CAR fusion protein of claim 1, further comprising a tag sequence.

11. The recombinant CAR fusion protein of claim 10, wherein the tag sequence is selected from the group consisting of a histidine tag, a glutathione-S-transferase tag, or a hemagglutinin tag.

12. 13. A method of modifying an immune cell into a chimeric antigen receptor (CAR) immune cell, comprising treating the immune cell with a recombinant CAR fusion protein of claim 1.

13. 13. The method of claim 12, wherein the recombinant CAR fusion protein is at a concentration of about 1000 nM to about 2000 nM.

14. 13. A method of treating cancer, comprising administering to a subject in need of treatment a CAR immune cell of claim 12.

15. 13. A method of treating cancer, comprising administering to a subject in need of treatment the recombinant CAR fusion protein of claim 1.

16. 16. The method of claim 15, wherein the recombinant CAR fusion protein is at a concentration of about 1000 nM to about 2000 nM.

17. 1. A method of treating cancer, comprising: To a subject in need of treatment, (i) in order, an immune cell targeting domain; A cleavable peptide; a membrane targeting domain; an extracellular cancer targeting domain; A transmembrane domain; an intracellular signaling domain; A recombinant chimeric antigen receptor (CAR) fusion protein comprising: (ii) CAR immune cells, The method of claim 1, further comprising administering

18. 18. The method of claim 17, wherein the CAR immune cell is prepared by treating an immune cell with the recombinant CAR fusion protein.

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