Chimeric antigen receptor constructs and their use in car-t cells

Abstract

To provide chimeric antigen receptor (CAR) compositions and methods of their use in cancer and anti-viral immunotherapy.SOLUTION: The CAR of the invention comprises a costimulatory signal (CSS) domain comprising herpes virus entry mediator protein (HVEM) or a functional fragment or variant thereof. CARs comprising such a HVEM CSS exhibit enhanced effector function.SELECTED DRAWING: Figure 1A

Description

Technical Field

[0001] [Statement of Priority] This application claims the benefit of U.S. Provisional Application No. 62 / 839,175, filed Apr. 26, 2019, under 35 U.S.C. § 119(e), the entire content of which is incorporated herein by reference.

[0002] [Statement Regarding Electronic Filing of Sequence Listing] A sequence listing in ASCII text format, submitted under 37 C.F.R. § 1.821, created on Apr. 22, 2020, and submitted via EFS-Web, having a size of 10,703 bytes and titled 5470-846WO_ST25.txt, is provided in lieu of a paper copy. This sequence listing is incorporated herein by reference for its disclosure.

[0003] [Field of the Invention] The present invention relates to chimeric antigen receptor (CAR) compositions and methods of using them in cancer and anti-pathogen immunotherapy.

[0004] [Government Support] This invention was made with government support under grant number AI077454 awarded by the National Institutes of Health. The government has certain rights in this invention.

Background Art

[0005] Chimeric antigen receptors (CARs), also known as chimeric immune receptors, chimeric T cell receptors or artificial T cell receptors, are engineered receptors that combine new specificities with immune cells to target cancer cells. Typically, these receptors fuse the specificity of monoclonal antibodies to T cells. The receptors are called chimeric because they are fusions of parts from different origins. CAR-T cell therapy refers to the treatment of using such transformed cells, mainly for cancer therapy.

[0006] The basic principle of CAR-T cell design involves a recombinant receptor that combines antigen binding and T cell activation functions. The general premise of CAR-T cells is to artificially produce T cells that are targeted against markers found on diseased cells such as cancer cells. Scientists can remove T cells from a human, genetically modify them, and then return them into the patient to attack diseased cells. When the T cells are modified to become CAR-T cells, they act as a "living drug".

[0007] CAR-T cells create a link between the extracellular ligand recognition domain and the intracellular signaling molecule, as a result activating the T cells. The extracellular ligand recognition domain is usually a single-chain variable fragment (scFv). An important aspect of the safety of CAR-T cell therapy is a way to ensure that only cancerous tumor cells are targeted and normal cells are not. The specificity of CAR-T cells is determined by the selection of the molecule to be targeted.

[0008] For example, in cancer treatment, CAR-T cells can be collected from the patient's own blood (autologous) or from another healthy donor (allogeneic). These T cells are genetically engineered to express an artificial T cell receptor through which they are targeted to cancer antigens. This process is MHC-independent and thus the targeting efficiency is greatly increased. CAR-T cells are programmed to target antigens present on the tumor surface. When in contact with the antigens on the tumor, CAR-T cells are activated via a signaling peptide, proliferate, and become cytotoxic. CAR-T cells destroy cancer cells by increasing the degree to which the cells are toxic to other living cells (i.e., cytotoxicity) through mechanisms such as extensive stimulated cell proliferation, and by increasing the production of factors that have effects on other cells within the organism and are secreted from cells in the immune system. These factors are called cytokines and include interleukins, interferons, and growth factors.

[0009] Recently, CAR-T cell therapy has been considered as a treatment option for other cancer types (e.g., solid tumors) and / or diseases (e.g., chronic viral infections (e.g., HIV)). Thus, due to the potential expansion of the applications related to CAR-T cell therapy, there is a continuing need in the art to improve current CAR-T cell therapies and to produce next-generation CAR-T cell therapy agents and their components.

Summary of the Invention

Problems to be Solved by the Invention

[0010] The present invention provides a chimeric antigen receptor (CAR) composition and methods of using the same, wherein the co-stimulatory signal (CSS) comprises a herpes virus entry mediator (HVEM) protein (also referred to as CD270). The CSS in the CAR is important when modulating the immune activity of CAR-transduced T cells (CAR-T cells) used in a variety of diseases such as cancer (e.g., solid tumors) and pathogen infections (e.g., chronic viral or bacterial infections). CARs containing the HVEM CSS exhibited significantly higher glycolysis and mitochondrial respiration associated with high effector function and induced comparable percentages of central and effector memory subsets compared to CARs containing a CSS domain without HVEM. Thus, the function of CAR-T cells can be improved through HVEM co-stimulation by reprogramming the energy metabolism of CAR-T cells.

Means for Solving the Problems

[0011] Accordingly, one aspect of the present invention relates to a chimeric antigen receptor (CAR) comprising an antigen-binding domain, a transmembrane domain, a T cell receptor domain, and a co-stimulatory signal (CSS) domain comprising a herpes virus entry mediator (HVEM) protein or a functional fragment or variant thereof having at least 90% identity thereto.

[0012] Another aspect of the present invention relates to a nucleic acid molecule encoding the CAR of the present invention.

[0013] A further aspect of the present invention relates to a vector comprising the nucleic acid molecule of the present invention.

[0014] A further aspect of the present invention relates to a cell comprising the CAR of the present invention.

[0015] Another aspect of the present invention relates to a cell comprising the nucleic acid molecule of the present invention and / or the vector of the present invention.

[0016] A further aspect of the present invention relates to a composition comprising the CAR of the present invention, the nucleic acid molecule of the present invention, the vector of the present invention, and / or the cell of the present invention in a pharmaceutically acceptable carrier.

[0017] A further aspect of the present invention relates to a method of providing an immune response against a target in a subject in need thereof, the method comprising administering to the subject an effective amount of the CAR of the present invention, the nucleic acid molecule of the present invention, the vector of the present invention, and / or the cell of the present invention, thereby providing an immune response against the target in the subject.

[0018] Another aspect of the present invention relates to a method of providing an immune response against a target in a subject in need thereof, the method comprising obtaining T cells from a subject having cancer and / or an infection, transfecting the T cells with the nucleic acid molecule of the present invention or the vector of the present invention, culturing the transfected T cells, and administering the cultured, transfected T cells to the subject, thereby providing an immune response against the target in the subject.

[0019] These and other aspects of the present invention are shown in more detail in the following description of the present invention.

Brief Description of the Drawings

[0020]

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Mode for Carrying Out the Invention

[0021] The present invention will now be described in more detail with reference to the accompanying drawings, in which preferred embodiments of the invention are shown. However, the invention can be implemented in different forms and should not be construed as limited to the embodiments shown herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In this specification, the technical terms used in the description of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention. All publications, patent applications, patents, patent documents, and other references cited herein are incorporated by reference in their entirety with respect to the teachings relevant to the passages and / or paragraphs in which the references are cited.

[0023] Unless otherwise clearly indicated, nucleotide sequences are shown herein only by single strands, in the 5' to 3' direction, from left to right. Nucleotides and amino acids are represented herein in the manner recommended by the IUPAC-IUB Biochemical Nomenclature Commission or, for amino acids, either by the one-letter code or the three-letter code, both in accordance with 37 C.F.R.§1.822 and established usage.

[0024] Unless otherwise indicated, standard methods known to those skilled in the art may be used for gene cloning, nucleic acid amplification and detection, etc. Such techniques are known to those skilled in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual 4th Ed. (Cold Spring Harbor, NY, 2012); Ausubel et al., Current Protocols in Molecular Biology (Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York).

[0025] As used in the description of the present invention and the appended claims, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise.

[0026] Also, as used herein, "and / or" refers to any and all possible combinations of one or more of the related listed items, and includes and, where optionally construed (as "or"), refers to and includes the omission of a combination.

[0027] Furthermore, as used herein, the term "about", when referring to measurable values such as amounts, dosages, times, temperatures, etc. of the antibodies, compounds or agents of the present invention, means encompassing a variation of ±10%, ±5%, ±1%, ±0.5%, or indeed ±0.1% of the specified amount.

[0028] The term "consisting essentially of" (and grammatical variations thereof) as applied to an amino acid and / or nucleotide sequence of the present invention means an amino acid and / or nucleotide sequence consisting of both the recited sequence (e.g., SEQ ID NO) and up to 10 additional amino acids and / or nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) at the N-terminus and / or C-terminus and / or 5' and / or 3' termini of the recited sequence, provided that the ability of the amino acid and / or nucleotide sequence to bind to its target is not substantially altered. For example, up to 10 additional nucleotides includes the total number of additional nucleotides added at both the 5' and 3' termini. The term "substantially altered" as applied to the binding of a nucleotide sequence refers to an increase or decrease in binding affinity of at least about 50% or higher compared to the binding affinity of the nucleotide sequence consisting of the recited sequence.

[0029] Within the scope of the present invention, the term "antibody" refers to full-length immunoglobulins and fragments thereof. Such full-length immunoglobulins may be monoclonal, polyclonal, chimeric, humanized, veneered or human antibodies.

[0030] The term "antibody fragment" includes portions of full-length immunoglobulins that maintain the targeting specificity of the immunoglobulin. Many, but not all, antibody fragments have at least a partial deletion of the constant region (Fc region) of the full-length immunoglobulin. In some embodiments, the antibody fragment is produced by digestion of a full-length immunoglobulin. The antibody fragment may also be a synthetic or recombinant construct that includes immunoglobulin portions or immunoglobulin chains (see, for example, Holliger, P. and Hudson, J. Engineered antibody fragments and the rise of single domains. Nature Biotechnology 2005, vol. 23, no. 9, p. 1126-1136). Examples of antibody fragments include, without limitation, scFv, Fab, Fv, Fab’, F(ab’)2 fragments, dAb, VHH, nanobodies, V(NAR), or minimal recognition units. "Single-chain variable fragment" or "single-chain antibody" or "scFv" is one type of antibody fragment. An scFv is a fusion protein that includes the VH and VL of an immunoglobulin linked by a linker. They thus lack the constant Fc region that is present within the full-length immunoglobulin, but maintain the specificity of the original immunoglobulin.

[0031] The numbering system used herein to identify amino acid residue positions in the VH and VL of an antibody corresponds to the "AHo" system described by Honegger, A. and Pluckthun, A. Yet another numbering scheme for immunoglobulin variable domains: An automatic modelling and analysis tool. Journal of Molecular Biology 2001, vol. 309, p. 657-670. That document further provides a conversion table between the AHo and Kabat systems (Kabat, E.A., et al. Sequences of Proteins of Immunological Interest. 5th edition., 1991. p. 91-3242, edited by U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES. NIH Publications).

[0032] As used herein, "nucleic acid", "nucleotide sequence" and "polynucleotide" are used interchangeably and include both RNA and DNA, including cDNA, genomic DNA, mRNA, synthetic (e.g., chemically synthesized) DNA or RNA, and chimeras of RNA and DNA. The terms polynucleotide, nucleotide sequence, or nucleic acid refer to a strand of nucleotides regardless of strand length.

[0033] The term "fragment" is understood to mean a nucleotide sequence of reduced length as compared to a reference nucleic acid or nucleotide sequence, and comprises, consists essentially of, and / or consists of a nucleotide sequence of contiguous nucleotides identical to the reference nucleic acid or nucleotide sequence. Such nucleic acid fragments according to the invention may, where appropriate, be contained within a larger polynucleotide and may be a constituent thereof. In some embodiments, such fragments can comprise, consist essentially of, and / or consist of oligonucleotides having at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive nucleotides of a nucleic acid or nucleotide sequence according to the invention.

[0034] As used herein, the term "identity" refers to sequence identity between two proteins or nucleic acids. The protein or nucleic acid sequences being compared are aligned to give maximum identity using bioinformatics tools such as EMBOSS Needle (pairwise alignment; available at www.ebi.ac.uk). If the same position within the sequences being compared is occupied by the same nucleobase or amino acid residue, then as a result, the respective molecules are identical at that position. Thus, "percent identity" is a function obtained by dividing the number of matching positions by the number of positions being compared and multiplying by 100%. For example, if 6 out of 10 sequence positions are identical, then as a result, the identity is 60%. The percent identity between two protein sequences can be determined using, for example, the Needleman and Wunsch algorithm incorporated in EMBOSS Needle (Needleman, S.B. and Wunsch, C.D. A general method applicable to the search for similarities in the amino acid sequence of two proteins. Journal of Molecular Biology 1970, vol. 48, p. 443-453), with a "gap open penalty" of 10, a "gap extend penalty" of 0.5, a false "end gap penalty", an "end gap open penalty" of 10, an "end gap extend penalty" of 0.5, and using the BLOSUM62 matrix. Two molecules having the same primary amino acid or nucleic acid sequence are identical regardless of any chemical and / or biological modifications. For example, two antibodies having the same primary amino acid sequence but different glycosylation patterns are identical according to this definition. In the case of nucleic acids, for example, two molecules having the same sequence but different binding components such as phosphorothioate instead of phosphate are identical according to this definition.

[0035] As used herein, the term "mutation" refers to an amino acid or nucleic acid sequence that differs from the parental sequence by the addition (including insertion), deletion, and / or substitution of one or more amino acid residues or nucleic acid bases while maintaining at least one desired activity of the parental sequence disclosed herein. In the case of a CAR, such desired activity may include specific target binding. Similarly, a mutant nucleic acid sequence may be modified compared to the parental sequence by the addition, deletion, and / or substitution of one or more nucleic acid bases, but the encoded CAR maintains the desired activity described above. Mutations may be of natural origin, such as alleles or splice variants, or may be artificially constructed.

[0036] Also, as used herein, "one or more" means 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.

[0037] As used herein, a "subject" that can be treated by the present invention includes both human subjects for medical and / or therapeutic purposes and animal subjects for veterinary and drug screening and development purposes. Other suitable animal subjects generally include mammalian subjects, such as primates, cows, sheep, goats, pigs, horses, cats, dogs, rabbits, rodents (e.g., rats and mice), etc. Human subjects are most preferred. Human subjects include fetal, neonatal, infant, juvenile, adult, and elderly subjects.

[0038] As used herein, the term "anti-tumor effect" refers to a biological effect that can be manifested by a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in the growth rate, a reduction in the number of metastases, an increase in the average lifespan, and / or remission of various physiological symptoms associated with the cancerous condition. The "anti-tumor effect" can also be manifested by the ability of the peptides, polynucleotides, cells, and antibodies of the present invention to delay the onset of tumors in the first place.

[0039] As used herein, the term "autologous" means referring to any material taken from the same individual that will be reintroduced later.

[0040] "Allogeneic" refers to transplanted tissue taken from different animals of the same species.

[0041] "Xenogeneic" refers to transplanted tissue taken from animals of different species.

[0042] As used herein, the term "antigen-binding portion" or "antigen-binding fragment" of an antibody (or simply "antibody portion" or "antibody fragment") refers to one or more fragments, portions or domains of an antibody that retain the ability to specifically bind to an antigen. Fragments of full-length antibodies have been shown to be capable of performing the antigen-binding function of the antibody. Examples of binding fragments that are included within the scope of the term "antigen-binding portion" of an antibody are: (i) Fab fragment (a monovalent fragment consisting of the VL, VH, CL1 and CH1 domains); (ii) F(ab')2 fragment (a divalent fragment containing two F(ab)' fragments linked by a disulfide bridge in the hinge region); (iii) Fd fragment consisting of the VH and CH1 domains; (iv) Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) dAb fragment consisting of the VH domain (Ward et al. (1989) Nature 241:544-546); and (vi) isolated complementarity determining regions (CDRs). Further, although the two domains of the Fv fragment (VL and VH) are encoded by separate genes, they can be linked by a synthetic linker that enables, using recombinant methods, the pair of VL and VH regions to be made as a single continuous chain forming a monovalent molecule (known as single-chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be included within the scope of the term "antigen-binding portion" of an antibody. Other forms of single-chain antibodies such as diabodies are also included (see, e.g., Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448).

[0043] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site within the variable region of an antibody molecule, known as a paratope. A single antigen can have more than one epitope. Epitopes can be either conformational or linear. Conformational epitopes are produced by spatially juxtaposed amino acids from different segments of one (or more) linear polypeptide chain(s). Linear epitopes are epitopes produced by adjacent amino acid residues within a polypeptide chain. In certain embodiments, an epitope may include on the antigen other moieties, such as saccharides, phosphoryl groups, or sulfonyl groups.

[0044] As used herein, "antibody heavy chain" refers to the larger of two types of polypeptide chains that exist in their native conformation within the whole antibody molecule.

[0045] As used herein, "antibody light chain" refers to the smaller of two types of polypeptide chains that exist in their native conformation within the whole antibody molecule, and κ and λ light chains refer to the two major antibody light chain isotypes.

[0046] As used herein, the term "antigen" or "Ag" is defined as a molecule that elicits an immune response. This immune response can involve either or both of antibody production or activation of specific immunocompetent cells. One of ordinary skill in the art understands that virtually any macromolecule, including substantially all proteins or peptides, can act as an antigen. Additionally, an antigen can be obtained from recombinant or genomic DNA. One of ordinary skill in the art understands that any DNA containing a nucleotide sequence or partial nucleotide sequence that encodes a protein that elicits an immune response thus encodes an "antigen" as the term is used herein. Further, one of ordinary skill in the art understands that an antigen need not be encoded only by the full-length nucleotide sequence of a gene. The present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and it is readily apparent that these nucleotide sequences can be arranged in various combinations to elicit a desired immune response. Further, one of ordinary skill in the art understands that an antigen need not be encoded by a "gene" at all. It is readily apparent that an antigen can be synthesized or obtained from a biological sample. Such biological samples can include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0047] As used herein, "amino acid" refers to a compound having a free carboxyl group and a free unsubstituted amino group on the α-carbon, which can be linked by peptide bonds to form the peptide activators described herein. Amino acids can be standard or non-standard, natural or synthetic, and examples (and their abbreviations) include, but are not limited to: Asp = D = Aspartic acid Ala = A = Alanine Arg = R = Arginine Asn = N = Asparagine Cys = C = Cysteine Gly = G = Glycine Glu = E = Glutamic acid Gln = Q = Glutamine His = H = Histidine Ile = I = Isoleucine Leu = L = Leucine Lys = K = Lysine Met = M = Methionine Phe = F = Phenylalanine Pro = P = Proline Ser = S = Serine Thr = T = Threonine Trp = W = Tryptophan Tyr = Y = Tyrosine Val = V = Valine Orn = Ornithine Nal = 2-Naphthylalanine Nva = Norvaline Nle = Norleucine Thi = 2-Thienylalanine Pcp = 4-Chlorophenylalanine Bth = 3-Benzothienylalanine (benzothienyalanine) Bip = 4,4’-Biphenylalanine Tic = Tetrahydroisoquinoline-3-carboxylic acid Aib = Aminoisobutyric acid Anb = α-Aminonormal butyric acid (aminonormal butyricacid) Dip = 2,2-Diphenylalanine Thz = 4-Thiazolylalanine

[0048] All peptide sequences referred to in this specification are written according to normal convention, and thus the N-terminal amino acid is on the left and the C-terminal amino acid is on the right. A short line (or no line) between two amino acid residues indicates a peptide bond.

[0049] "Basic amino acid" refers to any amino acid with a positive charge at pH 6.0, including but not limited to R, K, and H. "Aromatic amino acid" refers to any amino acid having an aromatic group within the side chain attached to the α-carbon, including but not limited to F, Y, W, and H.

[0050] As used herein, the term "detectable moiety" includes any suitable detectable group, such as radiolabels (e.g., 35 S, 125 I, 131 I, etc.), enzyme labels (e.g., horseradish peroxidase, alkaline phosphatase, etc.), fluorescent labels (e.g., fluorescein, green fluorescent protein, etc.), etc., which are well known in the art and used according to known techniques.

[0051] "Immune response" refers to the reaction of a subject to the presence of an antigen and may include at least one of the following: production of antibodies, development of immunity, development of hypersensitivity to the antigen, and development of tolerance.

[0052] As used herein, the term "enhancing an immune response" implies that the reaction of a subject to the presence of an antigen is increased and / or amplified in the presence of the CAR of the present invention as compared to the reaction of the subject to the presence of the antigen in the absence of the CAR of the present invention.

[0053] By the terms "treat", "treating" or "treatment of", it is intended that the severity of the condition of the subject is decreased or at least partially improved or modified, and that some alleviation, mitigation or reduction in at least one clinical symptom is achieved.

[0054] As used herein, an "effective" amount is an amount that provides the desired effect.

[0055] As used herein, a "therapeutically effective" amount is an amount that provides some improvement or benefit to the subject. Put another way, a "therapeutically effective" amount is an amount that provides some alleviation, mitigation, or reduction of at least one clinical symptom in the subject. One of ordinary skill in the art understands that the therapeutic effect need not be complete or curative as long as some benefit is provided to the subject.

[0056] A chimeric antigen receptor (CAR) is an artificial antigen receptor consisting of an antigen-binding domain and a signaling domain capable of mimicking the signaling pathway mediated by the T cell receptor. The single-chain variable region of an antibody against a target molecule or a natural ligand is used as the antigen-binding domain of the CAR. The advantage of CARs is that they can recognize a given target without the need for antigen processing or antigen presentation restricted by the major histocompatibility complex (MHC), which brings the potential for T cell-expressed CARs (CAR-T cells) to serve as a useful tool for adoptive immunotherapy in a wide range of patients (Dotti et al., Immunol. Rev. 257(1):107 (2014)). T cells expressing first-generation CARs with CD3ζ as the signaling domain often become anergic and are unable to induce a strong immune response (Kershaw et al., Clin. Cancer Res. 12(20Pt1):6106 (2006)). To address this problem, second- and third-generation CARs with one and two costimulatory signal (CSS) domains derived from CD28, 4-1BB, or ICOS have been developed (Dotti et al., Immunol. Rev. 257(1):107 (2014)). These CARs with a modular structure have been shown to successfully mimic the signaling mediated by the T cell receptor upon antigen stimulation, resulting in the proliferation and activation of CAR-T cells (Maus et al., Blood 123(17):2625 (2014)).

[0057] Clinical trials of adoptive immunotherapy for B cell malignancies using CD19-targeted CAR-T cells have shown promising results (Maude et al., Blood 125(26):4017(2015)), and the cells were approved by the US Food and Drug Administration in 2017, suggesting the possibility of further expansion for clinical application (Miller et al., Oncol.Res.Treat. 38(12):683(2015)). However, adoptive transfer of CAR-T cells has shown less obvious therapeutic effects on solid tumors than on lymphoid malignancies (Newick et al., Mol Ther.Oncolytics 3:16006(2016)). Since it has recently been reported that heparanase, which promotes tumor infiltration of CAR-T cells, increases the anti-tumor effect (Caruana et al., Nat.Med. 21(5):524(2015)), modifying the tumor microenvironment may help improve the therapeutic efficacy against solid tumors. To increase the usefulness of immunotherapy mediated by CAR-T cells, it may also be necessary to further improve the effector functions and characteristics of CAR-T cells. Co-stimulation is an important event for T cells to exhibit effective effector functions and is mediated by co-stimulatory molecules. Co-stimulatory molecules are divided into two main families; the CD28 family (including CD28 and ICOS), and the tumor necrosis factor receptor superfamily (TNFRSF) (including 4-1BB (TNFRSF9) and herpes virus entry mediator (HVEM, TNFRSF14)). So far, the CSS domain derived from CD28 or 4-1BB has generally been used to construct CARs. Previous studies have shown that T cells expressing second-generation CARs with the CSS domain derived from 4-1BB persist in the blood of most patients for more than 6 months, while CAR-T cells with the CSS domain derived from CD28 become almost undetectable after 3 months (Zhang et al., Oncotarget 6(32):33961(2015)).Furthermore, costimulation mediated by 4-1BB selectively induces oxidative metabolism for mitochondrial biogenesis and energy production, leading to high differentiation and increasing the in vitro persistence of central memory T cells (Kawalekar et al., Immunity 44(2):380(2016)). Additionally, costimulation mediated by 4-1BB avoids T cell exhaustion induced by tonic signaling (Long et al., Nat. Med. 21(6):581(2015)). Therefore, the CSS domain derived from TNFRSF is thought to function better than those derived from the CD28 family in the context of second-generation CARs.

[0058] Regarding the effector CD8 of HVEM, another member of TNFRSF + Reports have accumulated suggesting its role in the effector function of T cells and the development of memory T cells. CD8 + HVEM deficiency in CD8 + T cells has been shown to greatly impair the survival of effector CD8 + T cells and the development of protective immune memory (Flynn et al., PLoS One 8(10):e77991(2013)). Interaction of B and T lymphocyte attenuator (one of the ligands of HVEM) with HVEM expressed on CD8

[0059] The identification of effective CSS in CAR modules is one of the important requirements for applying CAR-T cell therapy to a variety of diseases, such as solid tumors and pathogen infections (e.g., chronic viral or bacterial infections). The inventors developed a second-generation CAR that includes the extracellular domain of CD4 (known as soluble CD4 (sCD4)) as an antigen-binding domain targeting the surface envelope protein (Env) of human immunodeficiency virus type 1 (HIV-1), in combination with a CSS domain derived from CD28, 4-1BB, or HVEM (Figure 1A). Using sCD4-CARs with different CSSs, the inventors observed the correlation between CAR-transduced T cell function and CAR expression on CAR-T cells generated from both human T cell lines and primary CD8 + T cells. This correlation was dependent on the CSS within the CAR, and HVEM CSS was shown to provide the most potent CSS. Phenotypic and metabolic analyses showed that HVEM CSS induced an equivalent proportion of central and effector memory phenotypes, along with significantly higher glycolysis and mitochondrial respiration. Furthermore, HVEM CSS avoided CAR-T cell exhaustion. These results were unexpected and surprisingly provide the first evidence that the CSS within the CAR affects the activity and properties of CAR-T cells through the regulation of energy metabolism, and that HVEM could be a useful CSS for developing effective CAR-T cells.

[0060] Accordingly, the present invention relates to a chimeric antigen receptor (CAR) comprising an antigen-binding domain, a transmembrane domain, a T cell receptor domain, and a co-stimulatory signal (CSS) domain comprising a herpes virus entry mediator (HVEM) protein or a functional fragment thereof.

[0061] The CSS domain is essential for promoting the intracellular signal of the T cell receptor domain to initiate T cell activation and proliferation. Thus, the promotion of such signals may depend on the selected CSS domain and / or its combination. For example, in the present invention, a CSS domain containing the HVEM protein or its functional fragment or variant promotes T cell activation and proliferation. In particular, the HVEM CSS of the present invention promotes high effector function associated with increased glycolysis and mitochondrial respiration compared to a CAR that does not contain the HVEM CSS domain of the present invention.

[0062] In some embodiments, CAR-T cells containing the HVEM CSS domain of the present invention have at least about 50% to about 100%, about 60% to about 90%, or about 70% to about 80% (or at least about 50%, about 60%, about 70%, about 80%, about 90%, or about 95%) increased CAR-T glycolysis compared to CAR-T cells that do not contain the HVEM CSS domain of the present invention. In some embodiments, CAR-T cells containing the HVEM CSS domain of the present invention have at least about 50% to about 100%, about 60% to about 90%, or about 70% to about 80% (or at least about 50%, about 60%, about 70%, about 80%, about 90%, or about 95%) increased mitochondrial respiration compared to CAR-Y cells that do not contain the HVEM CSS domain of the present invention.

[0063] In some embodiments, the HVEM CSS domain consists essentially of or consists of the amino acid sequence of WVWWFLSGSLVIVIVCSTVGLIICVKRRKPRGDVVKVIVSVQRKRQEAEGEATVIEALQAPPDVTTVAVEETIPSFTGRSPNH (SEQ ID NO: 1), or a functional fragment or variant thereof having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity thereto. In some embodiments, the HVEM CSS consists essentially of or consists of a fragment of HVEM having the amino acids of LVIVIVCSTVGLIICVKRRKPRGDVVKVIVSVQRKRQEAEGEATVIEALQAPPDVTTVAVEETIPSFT (SEQ ID NO: 2), or a variant thereof having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity thereto.

[0064] In some embodiments, the CSS domain further comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) additional CSS domains, variants, and / or fragments thereof. Non-limiting examples include the CD28 CSS domain, the 4-1BB CSS domain, the OX-40 CSS domain, the ICOS domain, or any other CSS domain known now or later identified and / or functional fragments or variants thereof having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity thereto, in any combination. In some embodiments, a linker may be present between two or more domains (e.g., a linker of 3 to 12 residues, e.g., a linker of 5 to 8 residues). An exemplary construct is shown in FIG. 18.

[0065] The T cell receptor domain is a signaling domain that transmits receptor ligand-binding events to intracellular signals that partially activate T cells. In the absence of an appropriate co-stimulatory signal, this event is insufficient for useful T cell activation and proliferation. Non-limiting examples of the T cell receptor domains of the present invention are the T cell receptor domains of the T cell receptor zeta chain (e.g., CD3ζ). In some embodiments, the T cell receptor domain of the CAR of the present invention consists essentially of or consists of the CD3ζ signaling domain or a related T cell receptor domain derived from the T cell receptor, or a functional fragment or variant thereof having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity thereto. In some embodiments, the ITAM3 motif within the CD3ζ domain can be mutated to a PD-1 ITIM. Examples of related T cell receptors include, but are not limited to, primary cytoplasmic signaling sequences containing an ITAM, such as TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79B, and / or CD66d.

[0066] The transmembrane domain is essential for the stability of the CAR as a whole. In some embodiments, the transmembrane domain may be a hydrophobic α helix that spans the membrane of a cell (e.g., a T cell). In some embodiments, the transmembrane domain may be derived from any type I transmembrane protein, such as CD4, CD28, or HVEM, or a functional fragment or variant thereof having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity thereto. In some embodiments, the transmembrane protein is CD28, or a functional fragment thereof having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity thereto.

[0067] The antigen-binding domain can be selected to recognize a ligand (i.e., an antigen) that acts as a cell surface marker on target cells associated with a particular medical condition. In some embodiments, the antigen-binding domain comprises a monovalent antibody fragment. In some embodiments, the monovalent antibody fragment comprises a single-chain variable fragment (scFv) or a Fab fragment. In some embodiments, the monovalent antibody fragment has a molecular weight of about 25 to about 30 kDa (or about 25 kDa, about 26 kDa, about 27 kDa, about 28 kDa, about 29 kDa, or 30 kDa). In some embodiments, the monovalent antibody fragment has VH and VL domains (e.g., VL-linker-VH or VH-linker-VL) linked in either direction by a flexible linker. In some embodiments, the orientation is VL-linker-VH, with the variable light chain at the N-terminus of the polypeptide and the variable heavy chain at the C-terminus of the polypeptide. The flexible linker typically comprises 10 to about 25 amino acids, for example, glycine confers flexibility and / or serine and / or threonine are for improved solubility). For example, in some embodiments, a (GGGGS)3 linker (SEQ ID NO: 3) or a variant thereof is used. Variations of the motif with 3 to 5 repeats may also be used. Further suitable linkers are described, for example, in Alfthan, K. Properties of a single-chain antibody containing different linker peptides. Protein Engineering 1995, vol. 8, no. 7, p. 725-731, which is incorporated herein by reference in its entirety.

[0068] The conditions targeted by the antigen-binding domain of the CAR of the present invention can be cancer and / or pathogen infection (e.g., chronic viral or bacterial infection). In some embodiments, the antigen-binding domain targets antigens present on the surface of cancer cells and / or viral particles. Exemplary cancer and / or tumor cell antigens are described in S. A. Rosenberg (Immunity 10:281 (1991)).Other exemplary cancers and tumor antigens include, but are not limited to: BRCA1 gene product, BRCA2 gene product, gp100, tyrosinase, GAGE-1 / 2, BAGE, RAGE, LAGE, NY-ESO-1, CDK-4, β-catenin, MUM-1, caspase-8, KIAA0205, HPVE, SART-1, PRAME, p15, melanoma tumor antigen (Kawakami et al. (1994) Proc. Natl. Acad. Sci. USA 91:3515; Kawakami et al., (1994) J. Exp. Med., 180:347; Kawakami et al. (1994) Cancer Res. 54:3124), MART-1, gp100 MAGE-1, MAGE-2, MAGE-3, CEA, TRP-1, TRP-2, P-15, tyrosinase (Brichard et al. (1993) J. Exp. Med. 178:489); HER-2 / neu gene product (U.S. Patent No. 4,968,603), CA125, LK26, FB5 (endosialin), TAG72, AFP, CA19-9, NSE, DU-PAN-2, CA50, SPan-1, CA72-4, HCG, STN (sialyl Tn antigen), c-erbB-2 protein, PSA, L-CanAg, estrogen receptor, milk fat globulin, p53 tumor suppressor protein (Levine (1993) Ann. Rev. Biochem. 62:623); mucin antigen (International Patent Publication No. WO90 / 05142); telomerase; nuclear matrix protein; prostate acid phosphatase; papillomavirus antigen; and / or antigens currently known or later discovered to be associated with the following cancers (e.g., solid tumors): melanoma, adenocarcinoma, thymoma, lymphoma (e.g., non-Hodgkin lymphoma, Hodgkin lymphoma), sarcoma, lung cancer, liver cancer, colon cancer, leukemia, uterine cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer, bladder cancer, kidney cancer, pancreatic cancer, brain tumor, neuroblastoma, and any other cancer or malignancy currently known or later identified (see, e.g., Rosenberg (1996) Ann. Rev. Med. 47:481-91).

[0069] In some embodiments, the antigen-binding domain targets an antigen present on the surface of cancer cells. In some embodiments, the antigen-binding domain targets an antigen present on the surface of renal cancer cells. Renal cancer cells include, but are not limited to, cells derived from renal cell carcinoma, transitional cell carcinoma, Wilms tumor, renal sarcoma, and / or metastatic renal cancer.

[0070] In some embodiments, the antigen-binding domain targets an antigen present on the surface of renal cell carcinoma (RCC) cells selected from, but not limited to, clear cell RCC, papillary RCC, chromophobe RCC, collecting duct RCC, and / or unclassified RCC. In some embodiments, the renal cell carcinoma cells are selected from, but not limited to, the Ketr-3 and / or ORSC-2 or ACHN renal cancer cell lines. Exemplary renal cancer antigens include, but are not limited to, any surface protein and / or polypeptide present on the surface of renal cancer cells known in the art or identified in the future.

[0071] In some embodiments, the antigen-binding domain of the present invention targets the surface protein carbonic anhydrase IX (CAIX). In some embodiments, the antigen-binding domain of the present invention that targets the surface protein carbonic anhydrase IX (CAIX) comprises a monovalent antibody fragment comprising the amino acid sequence of an anti-CAIX scFv or a variant thereof having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity thereto.

[0072] In some embodiments, the antigen-binding domain targets an antigen present on the surface of neuroblastoma cells. In some embodiments, the neuroblastoma cells are derived from a PDX cell line, although other neuroblastoma cell lines can also be used. Exemplary neuroblastoma antigens include, but are not limited to, any surface protein and / or polypeptide present on the surface of neuroblastoma cells known in the art or identified in the future. In some embodiments, the antigen-binding domain of the present invention targets the surface protein disialoganglioside GD2. In some embodiments, the antigen-binding domain of the present invention that targets the surface protein disialoganglioside GD2 (aGD2) comprises a monovalent antibody fragment containing the amino acid sequence of aGD2 or a variant thereof having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity thereto.

[0073] In some embodiments, the antigen-binding domain targets an antigen present on the surface of viral particles. Examples of viral particles include, but are not limited to, influenza virus, equine infectious anemia virus, simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), Lassa fever virus, herpes simplex virus, varicella-zoster virus, cytomegalovirus, Epstein-Barr virus, poxvirus, adenovirus, papillomavirus, parvovirus, measles virus, mumps virus, respiratory syncytial virus, parainfluenza virus, coronavirus, rubella virus, rabies virus, human T-cell tropic virus, picornavirus, hepadna (hepa DNA) virus, flavivirus, delta virus, calicivirus, poliovirus, dengue virus, West Nile virus, SARS, rubella, norovirus, human papillomavirus, malaria, human T-lymphotropic virus, and / or Helicobacter pylori.

[0074] Exemplary viral antigens include, but are not limited to, any surface protein and / or polypeptide present on the surface of the viral particles listed above. Examples of such surface proteins and / or polypeptides include, but are not limited to, Zika envelope domain-3, Zika envelope N, WNV envelope, WNV Pre-M, VZV ORF9, VZV ORF26, CoV-NL63, CoV-229E, Rubella E1, Norovirus group-1 P-domain, Norovirus group-2 P-domain, HPV11, HPV16, HPV18, HPV6, HPV16 E6, Malaria Pf.MSP1, Malaria Pv.MSP1, Lassa capsid, Lassa GP1, H1N1 Beijing, H1N1 California, H1N1 New Caledonia, HTLV-1 envelope, HTLV-1 gp21, HTLV-1 mosaic, HIV subtype O envelope, HIV subtype O gp41, HIV-1 envelope, HIV-1 gag p17, p24, HIV-1 gp120 CM, CagA pylori, Omp pylori, HP-NAP, HAV P2C, HAV P2C-P3A, HAV P2C-P3B, HAV P3C, HAV VP1, HAV VP1-P2A(669-782a.a), HAV VP1-P2A(722-830a.a.), HAV VP3, HAV VP4-VP2, HSV 2 gG, HSV-1 gD, and / or HSV-2 gB. In some embodiments, the viral particles are HIV particles. In some embodiments, the antigen-binding domain targets a surface receptor of the HIV particles such as, but not limited to, the envelope protein ENV of human immunodeficiency virus HIV-1.

[0075] In some embodiments, the antigen-binding domain of the invention that targets the surface envelope protein ENV of human immunodeficiency virus HIV-1 comprises a soluble CD4 protein or a functional fragment or variant thereof having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity thereto. In some embodiments, the soluble CD4 protein consists essentially of, or consists of, a human soluble CD4 protein comprising the amino acid sequence: MNRGVPFRHLLLVLQLALLPAATQGKKVVLGKKGDTVELTCTASQKKSIQFHWKNSNQIKILGNQGSFLTKGPSKLNDADSRRSLWDQGNFPLIIKNLKIEDSDTYICEVEDQKEEVQLLVFGLTANSDTHLLQGQSLTLTLESPPGSSPSVQCRSPRGKNIQGGKTLSVSQLELQDSGTWTCTVLQNQKKVEFKIDIVVLA (SEQ ID NO: 4), or a functional fragment or variant thereof having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity thereto.

[0076] In some embodiments, the antigen-binding domain and the transmembrane domain are linked using a spacer. A variety of different spacers can be used. For example, in some embodiments, the spacer is a short spacer comprising 100, 90, 80, 70, 60, 50, 40, 30, 20, or fewer than 10 amino acids. In some embodiments, the spacer can comprise at least a portion of the Fc region, for example, the hinge of the human Fc region of the CH3 domain or variants thereof. In some embodiments, the spacer comprises all or a portion of an immunoglobulin (e.g., IgG1, IgG2, IgG3, IgG4) hinge region, i.e., the sequence that enters between the CH1 and CH2 domains of the immunoglobulin, such as the IgG4 Fc hinge or the CD8 hinge region. Examples include, but are not limited to, CD8 hinge, CD28 hinge IgG4 (HL-CH3), or IgG4 (L235E, N297Q). In some embodiments, the spacer consists essentially of or consists of a CD8 hinge region having the amino acid sequence of AGEQKLISEEDLGALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLD (SEQ ID NO: 5).

[0077] In further embodiments, the CAR of the invention can further comprise a detectable moiety and / or effector molecule known in the art, non-limiting examples of which include drugs, toxins, small molecules, antibodies, and / or antibody fragments, alone or in any combination. In some embodiments, the CAR of the invention comprises an anti-c-myc tag.

[0078] In some embodiments, the CAR of the present invention comprises, consists essentially of, or consists of the amino acid sequence of MNRGVPFRHLLLVLQLALLPAATQGKKVVLGKKGDTVELTCTASQKKSIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRADSRRSLWDQGNFPLIIKNLKIEDSDTYICEVEDQKEEVQLLVFGLTANSDTHLLQGQSLTLTLESPPGSSPSVQCRSPRGKNIQGGKTLSVSQLELQDSGTWTCTVLQNQKKVEFKIDIVVLAAGEQKLISEEDLGALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDWVWWFLSGSLVIVIVCSTVGLIICVKRRKPRGDVVKVIVSVQRKRQEAEGEATVIEALQAPPDVTTVAVEETIPSFTGRSPNHRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 6) or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity thereto.

[0079]

[0080] Vectors containing the nucleic acid molecules of the present invention are further provided herein. The vectors include, but are not limited to, plasmid vectors, phage vectors, viral vectors, or cosmid vectors. The T lymphocytes of the present invention can be transduced, for example, using a viral vector under conditions where the CAR is produced in the T lymphocytes. The choice of vector often depends on the host cell to be introduced.

[0081] In some embodiments, the present invention provides cells comprising the CAR of the present invention, and in some embodiments, the present invention provides cells comprising the nucleic acid molecule and / or vector of the present invention. Non-limiting examples of the cells of the present invention include αβ T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, natural killer T (NKT) cells, Th17 cells, γδ T cells, and any combination thereof.

[0082] In some embodiments, the present invention provides cytotoxic T lymphocytes comprising a CAR that recognizes and binds to an antigen present on the surface of cancer cells and / or viral particles. For example, in some embodiments, cytotoxic T lymphocytes comprising a CAR that recognizes and binds to the surface envelope protein ENV of the human immunodeficiency virus HIV-1. In some embodiments, cytotoxic T lymphocytes comprising a CAR that recognizes and binds to a surface protein of cancer cells, such as a surface protein of renal cancer cells (i.e., CAIX) and / or a surface protein of neuroblastoma cells (i.e., aGD2). The cytotoxic T lymphocytes can then be transduced using a viral vector or transfected using a plasmid or nucleic acid construct comprising the nucleotide sequence encoding the CAR of the present invention. In some embodiments, the nucleotide sequence can be any lentiviral or retroviral vector used in the hospital for other CAR-T cells.

[0083] In certain embodiments, the invention comprises T lymphocytes modified to comprise a chimeric antigen receptor (CAR) comprising an antigen-binding fragment specific for an antigen present on the surface of cancer cells and / or viral particles (e.g., the surface envelope protein ENV of human immunodeficiency virus HIV-1, CAIX, and / or sGD2), a transmembrane domain (e.g., CD28), a T cell receptor domain (e.g., CD3ζ), and a CSS domain comprising the HVEM protein of the invention or a functional fragment or variant thereof having at least 90% identity thereto. In certain embodiments, the monoclonal antibody fragment for an antigen (e.g., the surface envelope protein ENV of human immunodeficiency virus HIV-1, CAIX, and / or sGD2) is a single-chain variable fragment (scFv).

[0084] In certain embodiments, the invention provides cells having on their cell surface a CAR produced by binding an extracellular antigen-binding domain derived from, e.g., the CD4 protein, CAIX, aGD2, and / or fragments thereof, to a T cell receptor domain derived from the T cell receptor ζ chain, and a CSS domain comprising the HVEM protein or a functional fragment or variant thereof having at least 90% identity thereto, to an antigen specific for cancer cells and / or viral particles (e.g., the surface envelope protein ENV of human immunodeficiency virus HIV-1, CAIX, aGD2).

[0085] In a further embodiment of the invention, a method for promoting the reactivity of a cell to an antigen is provided, the method comprising the step of transfecting a cell with a nucleic acid molecule of the invention and / or a vector of the invention to produce a transfected cell comprising an antigen-binding domain on the cell surface, wherein the antigen-binding domain specifically binds to the antigen, thereby promoting the reactivity of the cell to the antigen. As used herein, "reactivity" refers to the ability of a cell to promote an immune response upon binding of an antigen. Cells modified with the CAR of the invention can promote an increased (e.g., stronger, faster, and / or more effective) immune response as compared to cells not using the CAR of the invention. In some embodiments, the antigen is present on the surface of cancer cells and / or viral particles. In some embodiments, the antigen is present on a viral particle, e.g., an HIV particle. In some embodiments, the antigen is present on a cancer cell, e.g., a solid tumor (e.g., renal cancer). In some embodiments, the cell is a cytotoxic T lymphocyte.

[0086] In a further embodiment, the invention provides a composition (e.g., a pharmaceutical composition) consisting essentially of or consisting of the CAR of the invention, the nucleic acid molecule of the invention, the vector of the invention, and / or the cell of the invention in a pharmaceutically acceptable carrier.

[0087] In a further embodiment, the invention provides a method of providing an immune response against a target (e.g., cancer cells and / or infectious agents) in a subject in need thereof. In some embodiments, the method comprises administering to the subject an effective amount of the CAR of the invention and / or the nucleic acid molecule of the invention and / or the vector of the invention, and / or the cell of the invention, thereby providing an immune response against the target in the subject. In some embodiments, the target is an infectious agent, and administration of the CAR of the invention and / or the nucleic acid molecule of the invention and / or the vector of the invention, and / or the cell of the invention treats infection in the subject. In some embodiments, the target is cancer, and administration of the CAR of the invention and / or the nucleic acid molecule of the invention and / or the vector of the invention, and / or the cell of the invention treats cancer in the subject. In some embodiments, the cancer includes solid tumors.

[0088] In some embodiments, the method comprises administering modified T cells (i.e., T lymphocytes) comprising the CAR of the invention. Methods of preparing modified T lymphocytes comprising the CAR of the invention are well known to those skilled in the art. For example, in some embodiments, the cytotoxic lymphocytes (i.e., T cells) can be obtained from a subject who is susceptible to immune responses against a target (e.g., cancer cells and / or infectious agents) and / or at risk of becoming immunodeficient against the target. In some embodiments, the subject has cancer. In some embodiments, the subject has an infection. In some embodiments, the cytotoxic lymphocytes (i.e., T cells) are isolated from peripheral blood using techniques well known in the art (e.g., after Ficoll density gradient centrifugation, negative selection is performed to remove unwanted cells).

[0089] Cytotoxic lymphocytes can be modified to express the CARs of the present invention by transfecting a population of lymphocytes with the expression vectors and / or nucleic acid molecules of the present invention that encode the CARs of the present invention. Methods suitable for preparing a transfected population of lymphocytes that express the CARs of the present invention are well known to those skilled in the art and include, but are not limited to, retroviruses, lentiviruses (viral-mediated CAR gene delivery systems), sleeping beauty, and / or piggyback (transposon / transposase systems including non-viral-mediated CAR gene delivery systems).

[0090] The transfected lymphocytes are cultured under conditions appropriate for the population of cells to be introduced into a subject (e.g., a human). Specific considerations include the use of a culture medium that does not contain any animal products such as bovine serum. Other considerations include sterile conditions to avoid contamination with bacteria, fungi, and mycoplasma. In some embodiments, prior to administration to a subject, the cultured transfected lymphocytes may be pelleted, washed, and resuspended in a pharmaceutically acceptable carrier or diluent. Administration of the transfected lymphocytes to a subject can provide or enhance an immune response against a target (e.g., cancer cells and / or infectious agents). In some embodiments, administration of the transfected lymphocytes to a subject can treat cancer and / or pathogen infection in the subject. In some embodiments, the subject is a human. In some embodiments, the pathogen infection being treated is a chronic viral infection, e.g., HIV.

[0091] In some embodiments, the target to be treated is cancer. In some embodiments, the cancer to be treated includes solid tumors. In some embodiments, the cancer to be treated is renal cancer. Exemplary renal cancers include, but are not limited to, renal cell carcinoma, transitional cell carcinoma, Wilms tumor, renal sarcoma, and / or metastatic renal cancer. In some embodiments, the renal cancer is renal cell carcinoma (RCC). In some embodiments, renal cell carcinoma includes, but is not limited to, clear cell type RCC, papillary RCC, chromophobe RCC, collecting duct RCC, and / or unclassified RCC.

[0092] In some embodiments, the method of the invention comprises administering to a subject in need thereof an effective amount of the CAR of the invention and / or the nucleic acid molecule of the invention and / or the vector of the invention, and / or the cell of the invention, to treat cancer (i.e., renal cancer). In some embodiments, the treatment results in a decrease in tumor size. In some embodiments, the tumor size / volume is reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to the untreated tumor. In some embodiments, the treatment results in an increase in the survival rate of the subject. In some embodiments, the survival rate is increased by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% compared to the untreated subject.

[0093] In some embodiments, the method of the invention comprises administering to a subject in need thereof an effective amount of the CAR of the invention and / or the nucleic acid molecule of the invention and / or the vector of the invention, and / or the cell of the invention, to reduce the number of cancer cells present in the subject. In some embodiments, the number of cancer cells is reduced by at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold compared to the untreated subject.

[0094] The CAR-modified T cells of the present invention can be administered alone or as a pharmaceutical composition in combination with a diluent and / or other components. Briefly described, the pharmaceutical composition of the present invention may comprise the cell population described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may include buffers such as neutral buffered saline, phosphate buffered saline, sterile saline; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA and / or glutathione; adjuvants (e.g., aluminum hydroxide) and / or preservatives, alone or in any combination. The pharmaceutical composition of the present invention can be administered in a manner appropriate for the disease to be treated and / or prevented. The dosage and frequency of administration are determined by factors such as the condition of the subject and the type and severity of the disease of the subject, but in some embodiments, an appropriate dosage can be determined by clinical trials.

[0095] When an "immunologically effective amount", "anti-tumor effective amount", "tumor-inhibiting effective amount" or "therapeutic amount" is indicated, the exact amount of the composition of the present invention to be administered can be determined by a physician taking into account age, body weight, tumor size, degree of infection or metastasis, and individual differences in the condition of the patient (subject). In some embodiments, the pharmaceutical composition comprising the cells of the present invention can be administered at a dosage of about 10 3 ~ about 10 10 cells / kg body weight, and in some embodiments, the dosage can be about 10 5 ~ about 10 8 cells / kg body weight or about 10 6 ~ about 10 8 and may be, and all integer values within these ranges (e.g., 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 ) are included.

[0096] The cell composition of the present invention can be administered at these dosages multiple times (e.g., every hour, 4 times a day, 3 times a day, 2 times a day, daily, 2 times a week, 3 times a week, weekly, monthly, every other month, every six months, annually, etc.).

[0097] The cells of the present invention can be administered using infusion techniques commonly known in immunotherapy (see, e.g., Rosenberg et al. New Eng. J. of Med. 319:1676 (1988)). The optimal dosage and treatment regimen for a particular subject can be readily determined by those skilled in the medical arts by monitoring the subject for signs of the disease and adjusting the treatment accordingly.

[0098] In some embodiments, it may be desirable to administer activated T cells to a subject, then subsequently redraw blood (or perform apheresis), activate T cells therefrom as described herein, and reinfuse these activated and expanded T cells into the subject. This process can be performed multiple times, e.g., weekly or every few weeks. In certain embodiments, T cells can be activated from a blood draw of about 10 cc to about 400 cc. In certain embodiments, T cells are activated from a blood draw of 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, or 100 cc. Without being bound by theory, using this multiple blood draw / multiple reinfusion protocol can help select a particular population of T cells.

[0099] Administration of the compositions of the present invention can be performed in any manner including aerosol inhalation, injection, oral ingestion, transfusion, implantation and / or transplantation. The compositions of the present invention can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, and / or intraperitoneally. In some embodiments, the T cell compositions of the present invention can be administered to a subject by intradermal or subcutaneous injection. In another embodiment, the T cell compositions of the present invention can be administered by i.v. injection. In some embodiments, the composition of T cells can be directly injected into a tumor, lymph node and / or site of infection.

[0100] In some embodiments of the present invention, cells activated and expanded using the methods described herein or other methods known in the art in which the T cells are expanded to therapeutic levels can be administered to a subject in conjunction with any number of relevant therapeutic modalities (e.g., before, simultaneously and / or after). In some embodiments, the CARs and / or nucleic acid molecules, and / or modified T cells of the present invention can be used in combination with other therapeutic options (e.g., drugs and / or surgical procedures), depending on the disease being treated.

[0101] The present invention is described in more detail in the following non-limiting examples. The following examples are provided to more fully illustrate preferred embodiments of the present invention. However, they should in no way be construed as limiting the broad scope of the present invention.

Examples

[0102] [Example 1: Costimulatory signals determine CAR-T cell activity in human T cell lines.] The human sCD4 molecule corresponding to the extracellular domain of CD4 (amino acids 1-148) has been reported to selectively target HIV-infected cells through binding to HIV Env gp120 (Chaudhary et al., Nature 335(6188):369(1988)). To produce HIV Env-targeted CARs, lentiviral vectors expressing sCD4-CAR in combination with different CSSs were generated (Figure 1A). GFP + CAR expression in transduced Jurkat E6.1 cells was analyzed by flow cytometry using an anti-c-Myc tag antibody (Figures 1B-1C). The transduction efficiency did not clearly differ between transduced cells (Figures 7A-7B), but the expression levels of sCD4-CAR on the cell surface were found to differ between CAR-T cells with different CSSs (Figure 2C). Under similar expression levels of endogenous CD3ζ and actin between CAR-T cells with different CSSs, the expression levels of CAR in whole cell lysates also differed between CAR-T cells with different CSSs (Figure 2C). Therefore, these data suggested that the expression level of CAR is CSS-dependent.

[0103] To determine the functional activities between CAR-Ts with different CSSs, a co-culture assay was demonstrated using target cells expressing HIV Env. Target cells (CHO-GFP or CHO-Env-GFP) were co-cultured with CAR-T cells at a 1:2 ratio for 24 hours. The supernatant containing the cells was collected and clarified by centrifugation for IL-2 ELISA. The remaining cells were used for flow cytometry analysis to determine CD69 expression upon antigen-dependent stimulation. Target cells (CD3 - GFP + ) could be separated from effector cells (CD3 + ) (Figure 7C, left panel). GFP - and GFP + effector cell activation were both determined by CD69 upregulation (Figure 7C, middle and right panels). As shown in Figure 1E, minimal activation was observed upon co-culture with CHO-GFP cells for GFP- and GFP + was observed in both effector cells. Minimal activation was seen with GFP - in effector cells, but clear effector cell activation was seen in all CAR-T cells upon co-culture with CHO-Env-GFP cells. IL-2 secretion was also observed upon co-culture with target cells expressing HIV-Env, but not with target cells expressing only GFP (Figure 1F - 1H). These data suggested that the CAR-T cells generated in this study were activated in an antigen-dependent manner. Furthermore, both effector cell activation and IL-2 secretion were found to differ between CAR-T cells with different CSSs (Figure 3B). To determine the relationship between CAR expression level and CAR-T cell activity, linear regression analysis was performed. A clear correlation was seen for the frequency of activated CAR-T cells and IL-2 secretion with respect to CAR expression level (Figure 3D). Linear regression analysis clearly revealed that this relationship was dependent on the origin of the CSS. Collectively, these data suggested that CSS determines CAR-T cell activity in human T cell lines.

[0104] [Example 2: HVEM co-stimulation shows the highest CAR expression in human CAR-T cells.] To test whether the effect of CSS observed in T cell lines was also observed in human CAR-T cells, primary human CD8 + T cells were used to develop CAR-T cells with different CSSs. CAR-transduced CD8 + T cells were sorted based on GFP expression and used for analysis of effector function and characteristics. The expression levels of CAR on the cell surface and in whole cell lysates differed among CAR-T cells with different CSSs (Figure 2A - 2C), showing a similar trend to that observed in the CAR-transduced T cell line (Figure 1B and 1D). These data also suggested that HVEM co-stimulation resulted in the highest CAR expression in the human CAR-T cells tested in this study.

[0105] [Example 3: HVEM co-stimulation shows the highest effector function in human CAR-T cells.] To test whether differences in CAR expression levels correlate with the effector function of CAR-T cells in a primary cell setting, cytotoxic activity and cytokine secretion were measured during co-culture with target cells. As shown in Figure 3A, T cells showed antigen-specific cytotoxic activity against target cells expressing HIV Env. These data also suggested that cytotoxic activity was CSS-dependent and that CAR-T cells with HVEM co-stimulation showed the highest cytotoxic activity among the CAR-T cells tested in this study (Figure 3C). A similar trend was observed in cytokine secretion such as IL-2, TNF-α, and IFN-γ (Figure 3B). Interestingly, little cytokine secretion was observed in CAR-T cells with CD28 co-stimulation, which was similar to the previous reported results of GD2 CAR-T cells with CD28 CSS (Long et al., Nat. Med. 21(6):581(2015)). Linear regression analysis was performed to determine the relationship between CAR expression levels and CAR-T cell effector function. Consistent with the results from the human T cell line, a clear correlation was observed between CAR expression and CAR-T cell effector function (Figures 3D and 8A-8C). These correlations were dependent on the origin of the CSS. Therefore, these data suggested that the origin of CSS in the CAR could determine the functional activity of CAR-T cells and that HVEM co-stimulation showed the highest effector function against commonly used CSS in human CAR-T cells.

[0106] [Example 4: TNFRSF co-stimulation avoids CAR-T cell exhaustion.] As shown in FIGS. 3A-3D, the functional activity was dependent on the origin of the CSS in the CAR. We and other researchers have shown that CAR-T cells with CD28 co-stimulation have reduced effector function, leading us to the hypothesis that CSS in the CAR affects CAR-T cell exhaustion. Exhausted T cells have low proliferative and cytokine-producing capacity (associated with a high apoptosis rate) and express high levels of inhibitory receptors, such as PD-1 and LAG-3 (Virgin et al., Cell 2009;138(1):30(2009); Wherry, Nat. Immunol. 12(6):492(2011)). Therefore, the frequency of the exhausted population (PD-1 + / LAG-3 + ) in CAR-T cells with different CSSs was tested (FIG. 4A). CAR-T cells with CD28 CSS showed an increase in the exhausted population, while CAR-T cells with 4-1BB or HVEM CSS had significantly fewer exhausted populations (FIGS. 4B and 4D). Linear regression analysis showed a clear correlation between effector function and the frequency of the exhausted population among CAR-T cells with different CSSs, suggesting that TNFRSF co-stimulation avoids CAR-T cell exhaustion (FIGS. 4C and FIGS. 9A-9C).

[0107] [Example 5: CSS in the CAR affects the balance of memory T cell subsets.] The proportion of memory subsets in CAR-T cells has been suggested to affect the in vivo persistence of CAR-T cells (Sommermeyer et al., Leukemia 30(2):492(2016); Busch et al., Semin. Immunol. 28(1):28(2016)). To understand the relationship between CSS and the memory phenotype, the surface expression of CD45RO and CCR7 was analyzed as markers for memory T cell subsets (Boots et al., Nat. Rev. Rheumatol. 9(10):604(2013)). The results showed that CSS affects the generation of memory T cell subsets (Figures 5A and 5B). The most significantly affected memory populations were the central (T CM :CD45RO + CCR7 + ) and effector (T EM :CD45RO + CCR7 - ) memory populations (Figure 5C). CAR-T cells with 4-1BB CSS contained a significantly increased T CM population, while CD28 CSS induced a smaller T CM population (Figure 5D). Interestingly, CAR-T cells with HVEM CSS contained equivalent populations of T CM and T EM populations (Figure 5D). These data suggested that CSS in the CAR affects the balance of memory T cell populations.

[0108] [Example 6: HVEM co-stimulation reprograms CAR-T cell energy metabolism to a more energetically active stage.] Recent reports have suggested that the CAR signaling domain reprograms T cell metabolism (Kawalekar et al., Immunity 44(2):380 (2016)), but it is still unknown whether HVEM CSS can effect different regulation of cell metabolism. The oxygen consumption rate (OCR) of CAR-T cells and control T cells with different CSSs was measured under basal conditions, and then oligomycin (an inhibitor of ATP synthase), carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP; uncouples oxygen consumption from ATP production), and rotenone and antimycin A (inhibitors of the electron transport chain for complexes I and III, respectively) were added sequentially to analyze the relative contributions of mitochondrial and non-mitochondrial oxygen consumption mechanisms (Figure 6A). The results showed that the basal OCR levels, an indicator of mitochondrial respiration, were significantly different between CAR-T cells and control T cells with different CSSs. The OCR levels in CAR-T cells with CD28 CSS were lower than those in control T cells. In contrast, CAR-T cells with 4-1BB or HVEM CSS had higher OCRs compared to CAR-T cells with CD28 CSS and control T cells (Figure 6B). Surprisingly, the basal extracellular acidification rate (ECAR), an indicator of glycolysis, was significantly increased only in CAR-T cells with HVEM CSS. ATP production and the maximum OCR levels showed a similar trend to the results of the basal OCR levels (Figures 6C-6E). Further studies included measurements of spare respiratory capacity (Figure 6F), non-mito respiration (Figure 6G), and proton leak (Figure 6H). From these data, it was suggested that CAR-T cells with CD28 exhibited a low energy state, while 4-1BB CSS induced high mitochondrial respiration in CAR-T cells. Furthermore, CAR-T cells with HVEM CSS showed high glycolysis and mitochondrial respiration, suggesting that HVEM co-stimulation induced a more energetically active state in CAR-T cells.

[0109] [Example 7: Discussion of the results according to Examples 1-6.] Anti-HIV-1 CARs with different CSS within the signaling domain were developed using sCD4 as the antigen recognition domain. Using this system as one model, it was shown that the CSS within the CAR determines CAR-T cell activity in both human T cell lines and human primary CD8 + T cell-derived CAR-T cells. The interactions between effector function, generation of memory subsets, T cell exhaustion, and energy metabolism were also revealed, suggesting that the CSS within the CAR significantly affects the function and characteristics of CAR-T cells. In particular, HVEM co-stimulation enhanced the effector function of CAR-T cells by reprogramming energy metabolism. These tests revealed the insight that the choice of CSS can affect the outcome of the function and characteristics of CAR-T cells, and the insight that HVEM can be a promising candidate for generating effective CAR-T cells against not only solid tumors but also persistent infectious diseases.

[0110] These data suggested that CAR-T cell activity correlated with CAR expression in both CAR-transduced T cell lines and human CAR-T cells. This was not surprising, as higher CAR density on the cell surface within a certain range preferably induced higher activation of CAR-T cells upon antigen stimulation. Surprisingly, CAR expression levels and CAR-T cell activity also clearly differed among CAR-T cells with different CSSs. Western blot analysis using whole cell lysates also showed differences in CAR expression at different CSSs, suggesting that it was not stability but rather the CAR expression level itself that differed at different CSS settings. One explanation for this difference among CSSs could be due to the induction of different signaling pathways. CD28 co-stimulation induces the PI3K-Akt pathway, while 4-1BB co-stimulation is mainly mediated by TNF receptor-associated factor (TRAF) that activates c-Jun N-terminal kinase (JNK) and p38 (Kim et al., Mol. Cells 10(3):247(2000); Cannons et al., J. Immunol. 165(11):6193(2000)). Furthermore, TNFRSF members including 4-1BB and HVEM are each capable of activating alternative NF-κB pathways (Hauer et al., Proc. Natl. Acad. Sci. USA 102(8):2874(2005)). These differences in signaling pathways result in differences in transcription and translation (Mehta et al., Nat. Rev. Immunol. 17(10):608(2017)), leading to differences in T cell activation status and CAR expression levels.

[0111] Antigen-independent tonic signaling can be induced in some CAR-T cells during ex vivo expansion, leading to T cell differentiation and exhaustion. CAR-T cell exhaustion has been shown to be induced by CD28 co-stimulation but avoided by 4-1BB co-stimulation (Figures 4A and 4B) (Long et al., Nat. Med. 21(6):581(2015)). In these studies, HVEM co-stimulation also appears to avoid CAR-T cell exhaustion, suggesting that TNFRSF co-stimulation may be important for avoiding CAR-T cell exhaustion (Figures 4C and 9A-9C). A recent report showed that increased expression from the gamma-retroviral LTR promoter causes T cell apoptosis, but decreased CAR expression from the EF1α promoter within a self-inactivating lentiviral vector attenuates this toxicity (Gomes-Silva et al., Cell Rep. 21(1):17(2017)). Even when CAR expression was from the EF1α promoter within a lentiviral vector, CD28 co-stimulation induced CAR-T cell exhaustion. These data suggest that not only CAR expression levels but also the origin of CSS influence the induction of CAR-T cell exhaustion. To support this hypothesis, the PD-1 / Shp2 complex selectively binds to and dephosphorylates CD28, resulting in decreased T cell expansion and exhaustion (Hui et al., Science 355(6332):1428(2017)). Thus, CD28 co-stimulation during ex vivo expansion of CAR T cells can be engineered to induce T cell exhaustion.

[0112] Consistent with previous findings, CD28 co-stimulation has been shown to exhibit low energy metabolism along with an increase in the effector memory subset, while 4-1BB co-stimulation induces higher mitochondrial respiration associated with an increase in the central memory subset. These data also show that HVEM co-stimulation exhibits high mitochondrial respiration and glycolysis associated with the generation of central and effector memory subsets of similar frequencies. These data suggest that distinct co-stimulation regulates specific metabolic pathways and affects the generation of memory subsets (Kawalekar et al., Immunity 44(2):380(2016)). In particular, TNFRSF co-stimulation, including 4-1BB and HVEM tested in this study, can mobilize different TRAFs, activate the NF-κB pathway at different magnitudes, and result in different energy metabolism levels, generation of memory subsets, and effector functions. Furthermore, CAR expression levels are also associated with the energy state (Figs. 2A-2C and Figs. 6A-6E), suggesting that different signaling pathways can affect the energy metabolism levels to meet the metabolic requirements. For example, translation is the most energy-demanding process in cells (Lindqvist et al., Curr. Opin. Genet. Dev. 48:104(2018); Topisirovic et al., Cold Spring Harb. Symp. Quant. Biol. 76:355(2011); Buttgereit et al., Biochem. J. 312(Pt1):163(1995)).

[0113] HIV-1 infection can be controlled by antiretroviral therapy (ART), but ART cannot eradicate it because it cannot remove latently infected cells, indicating that HIV-infected patients must receive effective ART for life. Recent attempts at eradication have focused on killing latently infected cells that persist during ART by immunological mechanisms. To achieve this, HIV-specific immunotoxins or HIV-specific CAR-T cells have been successfully developed to kill HIV-infected cells (Denton et al., PLoS Pathog. 10(1):e1003872 (2014); Sahu et al., Virology 446(1-2):268 (2013); Liu et al., J. Virol. 89(13):6685 (2015); Ali et al., J. Virol. 90(15):6999 (2016); Liu et al., J Virol. 90(21):9712 (2016); Leibman et al., PLoS Pathog. 13(10):e1006613 (2017)). Additionally, broadly neutralizing antibodies (bNAbs) have been shown to remove HIV-infected cells by antibody-dependent cellular cytotoxicity (Bruel et al., Nat. Commun. 7:10844 (2016)). These studies indicate that HIV-infected cells can be removed by targeted cytotoxic therapies. Furthermore, the "shock and kill" approach, which is the killing of pharmacologically reactivated latently infected cells by HIV-specific CTLs or bNAbs, has been proposed (Halper-Stromberg et al., Cell 158(5):989 (2014)). However, it has been reported that a broad CTL response is required to clear latent HIV-1 due to escape mutations (Deng et al., Nature 517(7534):381 (2015)), suggesting that targeting conserved regions may be beneficial for clearing latent HIV-1.Consistent with this concept, recent reports have shown that CD4-based CAR-T cells can control HIV more efficiently than TCR-based or bNAb-based CAR-T cells. Additionally, CAR-T cells with 4-1BB costimulation have been shown to be more potent than CAR-T cells with CD28 costimulation in both in vitro and in vivo HIV treatment models (Leibman et al., PLoS Pathog. 13(10):e1006613 (2017)). In the present invention, it has been shown that CAR-T cells with HVEM costimulation are more potent than CAR-T cells with CD28 or 4-1BB costimulation, suggesting that CAR-T cells with HVEM costimulation may be more beneficial for controlling HIV replication.

[0114] This data demonstrated that the CSS within the CAR determines the effector function and characteristics of CAR-T cells. As shown in this study, HVEM costimulation induces high effector function associated with superior characteristics compared to the design of CSS commonly used in CARs, such as CD28 and 4-1BB. This suggests that HVEM could be a promising candidate for the development of CAR-T cells with excellent functions and characteristics. We may discover more potent CSS by testing a panel of CSS in the context of CARs and expanding the CAR design for future CAR-T cell therapies.

[0115] In summary, these results indicate that HVEM costimulation for generating CAR-T cells elucidates a CSS for designing more potent CAR-T cells compared to commonly used costimulations such as CD28 or 4-1BB. This study provides the first step towards modulating and generating CAR-T cells with desired functions and characteristics.

[0116] [Example 8: Details of Experimental Models and Subjects] A. Details of the Subject Collection of Human Samples Human peripheral blood samples were collected from healthy donors. Peripheral blood mononuclear cells (PBMCs) from healthy donors were prepared by Ficoll-Paque (GE Healthcare Life Sciences, Pittsburgh, PA) density gradient under a protocol approved by the Science Ethics Committee of Dokkyo Medical University. Informed consent was obtained from all subjects.

[0117] Cell Lines Culture media, MEM, DMEM, and RPMI (Thermo Fisher Scientific, Waltham, MA) were supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific), 2 mM glutamine (Thermo Fisher Scientific), 10 U / mL penicillin, and 10 μg / mL streptomycin (Thermo Fisher Scientific) and abbreviated as M10, D10, and R10, respectively. CHO cells and their transfectants were maintained in M10 supplemented with non-essential amino acids (M10-NEAA). 293FT cells were cultured in D10, and Jurkat E6.1 cells obtained from the European Collection of Authenticated Cell Culture through DS Pharma were maintained in R10. All cells were grown at 37 °C and 5% CO2.

[0118] Primary Cell Culture PBMCs were cultured overnight in AIM-V (Thermo Fisher Scientific) supplemented with 5% FBS and 10 mM HEPES (complete AIM-V) to remove plastic-adherent monocytes. Monocyte-derived PBMCs were used in the transduction experiments. Cells were grown at 37 °C and 5% CO2.

[0119] Another method is to use culture media, MEM, DMEM, and RPMI (Thermo Fisher Scientific, Waltham, MA), which were supplemented with 10% FBS (Thermo Fisher Scientific), 2 mM glutamine (Thermo Fisher Scientific), 10 U / mL penicillin, and 10 μg / mL streptomycin (Thermo Fisher Scientific), and were abbreviated as M10, D10, and R10, respectively. CHO cells and their transfectants were maintained in M10 (M10-NEAA) supplemented with non-essential amino acids. 293FT cells were cultured in D10, and Jurkat E6.1 cells obtained from the European Collection of Authenticated Cell Cultures through DS Pharma were maintained in R10. Human peripheral blood mononuclear cells (PBMCs) from healthy donors were prepared by Ficoll-Paque (GE Healthcare LifeSciences, Pittsburgh, PA) density gradient and cultured overnight in AIM-V (Thermo Fisher Scientific) supplemented with 5% FBS and 10 mM HEPES (abbreviated as complete AIM-V) to remove plastic-adherent monocytes. Monocyte-derived PBMCs were used for transduction experiments. All cells were grown at 37°C in 5% CO2.

[0120] B. Details of the Method Construction of Vectors To introduce multiple restriction enzyme sites, a DNA linker was introduced into the lentiviral vector plasmid pTK643-CMV-IRES-GFP / Blasticidin (BSD). A DNA linker containing XbaI-XhoI-BsiWI-BstBI-BamHI restriction enzyme sites was prepared by incubating two oligo DNAs L1 and L2 at a 1:1 molar ratio at 70 °C for 10 minutes and then leaving them at room temperature for 2 hours. The linker DNA was inserted into pTK643-CMV-IRES-GFP / BSD (pTK643-CMV-MCS-IRES-GFP / BSD) digested with XbaI / BamHI.

[0121] To generate the sCD4 (amino acids 1 - 148 of human CD4) DNA fragment, total RNA extracted from Jurkat E6.1 cells by ISOGEN (Nippon Gene, Tokyo, Japan) was used for cDNA synthesis according to the manufacturer's instructions by ReverTra Ace (Toyobo, Osaka, Japan). The sCD4 DNA fragment containing XbaI / EcoRI sites was amplified using KOD-FX (Toyobo). The purified DNA fragment was incubated with AmpliTaq (Thermo Fisher Scientific) at 72 °C for 10 minutes to add an A tail. The tailed DNA fragment was ligated into the pGEM-T Easy vector (Promega, WI). The sequence was confirmed using the BigDye Terminator v3.1 Cycle Sequencing kit (Thermo Fisher Scientific). To generate the sCD4-CAR-expressing lentiviral vector, the sCD4 DNA fragment digested with XbaI / EcoRI and the DNA fragment containing different CSSs (CD28, 4-1BB, or HVEM) artificially synthesized by Genscript (Piscataway, NJ) and digested with EcoRI / BamHI were ligated into pTK643-EF1a-IRES-GFP / BSD digested with XbaI / BamHI.

[0122] To generate an HIV Env (NL4-3 strain) expressing lentiviral vector plasmid, the pRE11-NL43 provided by Dr. Noriaki Hosoya digested with XbaI / XhoI was ligated into pTK643-CMV-MCS-IRES-GFP / BSD digested with XbaI / XhoI.

[0123] Production of Recombinant Lentiviruses Recombinant lentiviruses were produced with a slight modification of a previous description (Cockrell et al., Mol Ther. 14(2):276(2006)). Briefly, 293FT cells were cultured at a confluence of 80 - 90% on 10 cm dishes (Iwaki, Shizuoka, Japan) coated with collagen. The culture medium was replaced with D10 (antibiotic-free) containing 25 μM chloroquine (SIGMA, Darmstadt, Germany). For vectors packaged with ΔNRF, the following plasmid amounts were used: 15 μg of lentiviral vector plasmid, 10 μg of ΔNRF, and 5 μg of pMD.G. The plasmids were co-transfected into 293FT cells by polyethyleneimine "MAX" (Polysciences, Warrington, PA) at a DNA:PEI ratio of 2:1. The supernatant was replaced with D10 containing 5 mM sodium butylate (Wako, Osaka, Japan) and 10 μM forskolin (Tokyo Chemical Industry, Tokyo, Japan). The culture supernatant containing recombinant lentiviruses was collected 48 hours after transfection and cleared by centrifugation and 0.45 μm filtration (Millipore, Darmstadt, Germany). Recombinant lentiviruses were concentrated by high-speed centrifugation at 18,000 rpm for 3 hours using Himac CR21N (Hitachi Koki, Tokyo, Japan).

[0124] Transduction with Lentiviral Vectors The supernatant containing lentivirus was used to transduce the CAR / GFP gene into Jurkat E6.1 cells and the HIV Env gene into CHO cells, respectively. Briefly, 2 million Jurkat E6.1 cells or semi-confluent CHO cells in a 6-well plate were exposed to 1 mL of the supernatant containing unconcentrated lentivirus in the presence of 8 μg / mL of polybrene. The cells were centrifuged at 5500 rpm for 3 hours at 22 °C to enhance virus infection. After removing the supernatant, the cells were cultured in a CO2 incubator at 37 °C for 48 hours. The culture medium was replaced with medium supplemented with 10 μg / mL of BSD. Subsequently, the transduced cells were maintained in medium containing 10 μg / mL of BSD until the following assays were performed.

[0125] Human primary CD8 T cells were isolated from monocyte-derived PBMC using anti-human CD3-APC, CD4-PE, and CD8 PE / Cy7 antibodies (Biolegend, San Diego, CA) by cell sorting on a FACS Aria II (Becton Dickinson, Franklin Lakes, NJ) (routinely, purity higher than 95% is achieved). The purified CD8 T cells were activated for 3 days using anti-CD3 / CD28 beads (Thermo Fisher Scientific) at a bead:cell ratio of 3:1 with complete AIM-V supplemented with 40 U / mL of recombinant human IL-2 (obtained from Dr. Maurice Gately, Hoffmann-La Roche Inc. through the NIH AIDS Reagent Program, AIDS Branch, NIAID). After removing the anti-CD3 / CD28 beads, the activated CD8 T cells were transduced with a lentiviral vector on days 3 and 4 using plates coated with Retronectin (TAKARA, Shiga, Japan) according to the manufacturer's instructions. Subsequently, the medium containing 300 U / mL of IL-2 (complete AIM-V) was replaced every 2 - 3 days until a sufficient number of cells for cell sorting had grown.

[0126] Flow Cytometry Antibodies used in flow cytometry were obtained from Biolegend unless otherwise specified. CAR expression on transduced Jurkat E6.1 cells was analyzed using an anti-c-myc tag antibody (Santa Cruz Biotechnology, Dallas, TX) followed by anti-mouse Igs-PE (Agilent Technologies). Activation of CAR-transduced Jurkat E6.1 cells in co-culture assays was analyzed using anti-human CD3-APC antibody, CD69-PE antibody, and GFP (see Figures 7A and 7B for gating strategies). CAR expression and T cell exhaustion of human CAR-T cells were analyzed using a biotinylated anti-c-myc tag antibody (Biolegend) followed by streptavidin-PE (TONBO biosciences), anti-human PD-1-APC antibody, anti-human LAG-3-PE / Cy7 antibody (eBioscience), and GFP. The memory phenotype of CAR-T cells was analyzed using CD45RO-PE, CD8-PE-Cy7, CCR7-APC, and GFP. Centrifuged cells were resuspended in antibody solution in FACS buffer (PBS containing 2% FBS and 0.02% sodium azide) and incubated on ice for 30 minutes. After washing with ice-cold FACS buffer, cells were fixed using 1% paraformaldehyde / PBS. Stained cells were fixed and analyzed by FACS Calibur (Becton Dickinson).

[0127] Western Blot Five million cells were washed once with ice-cold PBS and resuspended in 150 μl of RIPA buffer (10 mM TrisHCl (pH 7.4), 1% NP-40, 0.1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 1 mM EDTA) supplemented with Complete Mini (Roche, Mannheim, Germany) and 1 mM PMSF, and incubated on ice for 30 minutes. The cleared cell lysate (20 μl) by centrifugation was mixed with NuPAGE LDS sample buffer (Thermo Fisher Scientific) and 0.1 M DTT, and boiled at 95°C for 5 minutes. The boiled sample was separated by electrophoresis in a NuPAGE 10% Bis-Tris gel (Thermo Fisher Scientific) and transferred to an Immobilon P membrane (Millipore). The membrane was blocked for 30 minutes using a blocking buffer (5% non-fat dry milk and 0.1% Tween-20 in TBS). The blocked membrane was incubated overnight at 4°C with an anti-actin antibody (I-19) or anti-CD3ζ antibody (F-3) (Santa Cruz Biotechnology) diluted in the blocking buffer. Then, the membrane was incubated with anti-goat IgG-HRP (Millipore) or anti-mouse IgG-HRP (GE Healthcare Life Sciences) diluted in the blocking buffer. After all antibody incubation steps, the membrane was washed with 0.1% Tween-20 / TBS. Immunoreactivity was visualized using Lumi-Light PLUS (Roche) as a substrate and detected using Light-Capture II (ATTO, Tokyo, Japan).

[0128] Co - culture Assay The activation of CAR-transduced Jurkat E6.1 cells and IL-2 secretion were determined by co-culturing with target cells (CHO-GFP or CHO-Env-GFP). 100,000 target cells were seeded into each well of a 96-well flat-bottom plate. 200,000 CAR-T cells were added and co-cultured overnight. The next day, the culture supernatant containing the cells was collected. The cells and cell-free supernatant were separated by centrifugation at 3,000 rpm for 5 minutes at 4°C. IL-2 secretion into the supernatant was measured using Human IL-2 ELISA MAX Deluxe (Biolegend) according to the manufacturer's instructions. CD69 expression was determined by flow cytometry using the remaining cells.

[0129] To determine the cytotoxicity of CAR-T cells, 10,000 target cells were seeded into each well of a 96-well flat-bottom plate. CAR-T cells were added at different target:effector ratios and co-cultured in phenol red-free NEAA-containing R10 at 0.2 mL / well. After overnight incubation, the supernatant was collected and centrifuged at 3,000 rpm for 10 minutes at 4°C to remove cell debris. Lactate dehydrogenase release in the supernatant was assayed using the CytoTox96 non-radioactive cytotoxicity assay (Promega, Madison, WI) according to the manufacturer's instructions. If the purity after cell sorting and culture is heterogeneous, untransduced T cells were added to ensure that the number of CAR + T cells and the total number of T cells were consistently maintained across the CAR-T cell population. IL-2, TNF-α, and IFN-γ secretion were also determined using the recovered supernatant with Human IL-2, TNF-α, IFN-γ ELISA MAX Deluxe (all from Biolegend) according to the manufacturer's instructions.

[0130] Analysis of Energy Metabolism The mitochondrial function of CAR-T cells was analyzed using an extracellular flux analyzer XFp (Agilent Technologies). Each well of the cell culture microplate was coated with CellTak (Corning) according to the manufacturer's instructions. To assay mitochondrial function, sorted CAR-T cells with different CSS were suspended in XF RPMI medium supplemented with 5.5 mM glucose, 2 mM L-glutamine, and 1 mM sodium pyruvate and seeded at 300,000 cells / well. The plate was centrifuged at 200 x g for 1 minute and incubated for 30 - 60 minutes in a non-CO2 incubator at 37°C. During incubation, the instrument XFp and its assay cartridge were calibrated according to the manufacturer's instructions. The oxygen consumption rate (OCR) was measured under basal conditions and after treatment with 1 μM oligomycin, 1 μM FCCP, and 1 μM rotenone / antimycin A (XFp Cell Mito Stress Kit, Agilent technologies). Four measurements were taken for each condition. ATP production was defined as (the last rate measurement value before oligomycin addition) - (the minimum rate measurement value after oligomycin addition).

[0131] Statistical Analysis Statistical analysis has been described previously (Nunoya et al., J. Infect. Dis. 209(7):1039(2014)). One-way or two-way analysis of variance (ANOVA) by Bonferroni multiple comparison test and linear regression analysis were performed, and R 2 values were calculated using GraphPad Prism (Graph Pad Software, San Diego, CA). A p-value of < 0.05 was considered statistically significant.

[0132] [Example 9: Chimeric antigen receptor T cells having a herpesvirus entry mediator - costimulatory signal domain exhibit functional efficacy.] Chimeric antigen receptors (CARs) contain an extracellular antigen-binding domain combined with an intracellular signaling domain (Dotti et al., Immunol. Rev. 257:107 (2014)). First-generation CAR-transduced T (CAR-T) cells having CD3ζ as the signaling domain of the CAR often become anergic and are unable to induce a strong immune response (Kershaw et al., Clin. Cancer Res. 12:6106 (2006)). Second-generation and third-generation CAR-T cells were developed by adding one and two costimulatory signaling domains (CSSDs), respectively, into the CAR (Dotti et al., Immunol. Rev. 257:107 (2014)). These CAR molecules having a modular structure have been shown to successfully mimic T cell receptor-mediated signaling upon stimulation with cognate antigen, leading to the proliferation and activation of CAR-T cells (Maus et al., Blood 123:2625 (2014)).

[0133] Immunotherapy using CD19-targeted CAR-T cells has shown significant efficacy against B cell malignancies (Maude et al., Blood 125:4017 (2015)). To expand the potential to various clinical applications (Maldini et al., Nat. Rev. Immunol. 18:605 (2018)), it may be necessary to further improve CAR-T cell efficacy. Signaling inferred by CSSD derived from co-stimulatory molecules is known to be an important event for showing potent CAR-T cell efficacy (Dotti et al., Immunol. Rev. 257:107 (2014)). However, much remains unclear about the effect of CSSD on the function and characteristics of CAR-T cells. Co-stimulatory molecules can be classified into two main families: the CD28 family including CD28 and ICOS, and the TNF receptor superfamily (TNFRSF) including 4-1BB and herpesvirus entry mediator (HVEM). So far, CSSD derived from CD28 or 4-1BB has been generally used to construct CARs (Miller et al., Oncol. Res. Treat. 38:683 (2015)). Previous studies have shown that second-generation CAR-T cells with 4-1BB-derived CSSD persist in the blood of most patients for more than 6 months, while CAR-T cells with CD28-derived CSSD become almost undetectable after 3 months (Zhang et al., Oncotarget 6:33961 (2015)). Furthermore, 4-1BB co-stimulation induces high differentiation into the central memory subset and increases persistence in vitro (Kawalekar et al., Immunity 44:380 (2016)). 4-1BB co-stimulation has also been shown to increase oxidative metabolism for mitochondrial biogenesis and energy production and avoid T cell exhaustion induced by tonic signaling (Long et al., Nat. Med. 21:581 (2015)). Therefore, CSSD derived from TNFRSF is thought to function better than that derived from the CD28 family in the context of second-generation CAR-T cells.

[0134] CD8 + Reports have accumulated suggesting a potential role of HVEM, a different member of TNFRSF, in the effector function and memory generation of T cells. For example, CD8 + Deficiency of HVEM in CD8 + T cells has been shown to greatly impair the survival of effector CD8 + T cells and the generation of protective immunity memory (Flynn et al., PLoS One 8: e77991 (2013)). Interactions between CD8

[0135] [Example 10: CARs with HVEM-derived CSSD are efficiently expressed in human T cell lines.] sCD4 corresponding to amino acids 1 - 148 of human CD4 has been reported to selectively target HIV-infected cells through binding to HIV Env (Chaudhary et al., Nature 335: 369 (1988)). To produce HIV Env-targeted CAR-T cells, lentiviral vectors expressing CARs in combination with CSSD derived from CD28, 4-1BB, or HVEM were constructed (Figure 1A). Flow cytometry analysis showed that the transduction rates of Jurkat E6.1 cells using different lentiviral vectors were similar to each other (Figures 7A - 7B). On the other hand, GFP +CAR expression levels on the cell surface of cells varied considerably (Figure 1B) and were highest on CAR-T cells with HVEM-derived CSSD (Figure 1C). Western blot analysis also revealed that the amount of CAR with HVEM-derived CSSD in whole cell lysates was greater than that of CAR with CD28 or 4-1BB-derived CSSD (Figure 1D).

[0136] [Example 11: Human T cell lines expressing CAR with HVEM-derived CSSD are efficiently activated upon autologous antigen stimulation.] To test the function of CAR-transduced Jurkat E6.1 cells with different CSSDs, CD3 - target cells (CHO-GFP or CHO-Env-GFP) were co-cultured with CD3 + Jurkat E6.1 cells transduced with a CAR-expressing lentiviral vector (Figure 7C, left panel). Then, GFP - and successfully transduced GFP + Jurkat E6.1 cells were tested for upregulation of CD69, an indicator of T cell activation (Figure 7C, right panel). As shown in Figure 1E, no significant activation was observed in GFP - or GFP + Jurkat E6.1 cells upon co-culture with control CHO-GFP cells. When CHO-Env-GFP cells were used as target cells, GFP - Jurkat E6.1 cells were not efficiently activated, while GFP +was efficiently activated (Figure 1E). Also, measurement of IL-2 in the culture supernatant showed that IL-2 secretion by CAR-transduced Jurkat E6.1 cells was induced upon co-culture with CHO-Env-GFP cells but not upon co-culture with control CHO-GFP cells (Figure 1E). These data demonstrated that human T cell transduction using the CAR expression vector constructed in this study was specifically activated upon autologous antigen stimulation. Linear regression analysis showed that the percentage of activated T cells and IL-2 secretion upon autologous antigen stimulation correlated with the CAR expression level on the cell surface (Figures 1G and 1H). Furthermore, the ability of CAR-T cells with HVEM-derived CSSD to induce antigen-dependent T cell activation exceeded that of CAR-T cells with CD28- or 4-1BB-derived CSSD in the context used in this study.

[0137] [Example 12: Human primary T cell-derived CAR-T cells with HVEM-derived CSSD exhibit potent effector functions.] To compare CAR-T cells with different CSSD in more detail, human primary CD8 +were transduced with a CAR-expressing lentiviral vector and analyzed for their effector functions. Similar to the observations in Jurkat E6.1 cells (Figure 1B-1D), the CAR expression levels on the cell surface and in whole cell lysates were highest for CAR-T cells with the HVEM-derived CSSD and lowest for those with the CD28-derived CSSD (Figure 2B-2C). The effector functions of CAR-T cells with different CSSDs were also compared. All three CAR-T cells elicited antigen-specific cytotoxic activity against HIV Env-expressing target cells, but the CAR-T cells with the HVEM-derived CSSD showed the highest activity (Figure 3A). Similarly, the highest levels of cytokines such as IL-2, TNF-α, and IFN-γ were secreted from CAR-T cells with the HVEM-derived CSSD compared to the other CAR-T cells tested in this study (Figure 3D). Similar to the observations in Jurkat E6.1 cells (Figure 1G and 1H), the effector functions of CAR-T cells measured by cytotoxic activity and cytokine secretion correlated with the CAR expression levels on the cell surface (Figure 3D and Figure 8A-8C). Thus, CAR-T cells with the HVEM-derived CSSD were considered to be potent.

[0138] [Example 13: CAR-T cells with the HVEM-derived CSSD efficiently differentiate into both central and effector memory subsets.] The relative proportions of memory subsets in CAR-T cells have been suggested to influence their functions and persistence in vivo (Sommermeyer et al., Leukemia 30:492 (2016); Busch et al., Semin. Immunol. 28:28 (2016)). Memory subsets that are particularly important for protective immunity are central memory (T CM , CD45RO + CCR7 + ) and effector memory (T EM , CD45RO + CCR7 -)Known as T cells (Boots et al., Nat. Rev. Rheumatol. 9:604 (2013)). Therefore, the proportion of memory T cell subsets was analyzed by surface expression of CD45RO and CCR7 (Figures 10A and 10B). Control T cells predominantly contained T EM subsets, while CAR-T cells with different CSSDs showed a larger percentage of T CM subsets (Figure 10A). In the comparison between CAR-T cells with different CSSDs, CAR-T cells with CD28- and 4-1BB-derived CSSDs predominantly contained T EM and T CM subsets, respectively (Figure 10B). Interestingly, CAR-T cells with HVEM-derived CSSD contained an equal percentage of T CM and T EM subsets (Figure 10B), suggesting that HVEM-derived CSSD efficiently induces both T EM and T CM subsets.

[0139] [Example 14: CAR-T cells with HVEM-derived CSSD avoid T cell exhaustion.] Long-term stimulation of T cells is known to cause exhaustion characterized by reduced proliferation, decreased cytokine production levels, high apoptosis rates, and expression of inhibitory receptors such as programmed cell death 1 (PD-1) and lymphocyte activation gene 3 (LAG-3) (Virgin et al., Cell 138:30 (2009); Wherry, Nat. Immunol. 12:492 (2011)). To determine the effect of CSSD on T cell exhaustion, PD-1 + , LAG-3 + and PD-1 + / LAG-3 +The percentage of exhausted T cell populations was tested (Figure 4E). The results showed that CAR-T cells with CD28-derived CSSD contained a relatively large percentage of exhausted T cells, while those with 4-1BB-derived CSSD contained a significantly lower percentage of exhausted T cells (Figures 9A - 9C). Furthermore, CAR-T cells with HVEM-derived CSSD contained the lowest percentage of exhausted T cells among the CAR-T cells tested in this study (Figures 9A - 9C). Interestingly, the percentage of + PD-1 + or LAG-3 cells was larger and smaller, respectively, for CAR-T cells with HVEM-derived CSSD than for those with 4-1BB-derived CSSD. Collectively, CAR-T cells with HVEM-derived CSSD avoided T cell exhaustion.

[0140] [Example 15: CAR-T cells with HVEM-derived CSSD exhibit reprogrammed energy metabolism.] T cell exhaustion has recently been shown to be associated with a defect in energy metabolism (Fisicaro et al., Nat. Med. 23:327 (2017); Bengsch et al., Immunity 45:358 (2016)). Therefore, the metabolic states of CAR-T cells with different CSSDs were compared after sequential addition of basal conditions and reagents to analyze the relative contributions of mitochondrial and non-mitochondrial oxygen consumption mechanisms (Figure 6A). Mitochondrial respiration and glycolysis can be measured by oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), respectively. CAR-T cells with HVEM-derived CSSD showed the highest level of basal OCR, followed by those with 4-1BB-derived CSSD, and control T cells and those with CD28-derived CSSD showed the lowest level of basal OCR (Figures 6B-6E). ATP-related respiration was also measured by adding the ATP synthase inhibitor oligomycin, and the maximum OCR level was measured by adding carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP; decouples oxygen consumption from ATP production) (Figure 6A). The results showed that ATP-related respiration and the maximum OCR level in different CAR-T cells increased as well as the basal OCR level (Figures 6C and 6D). Interestingly, basal ECAR significantly increased only for CAR-T cells with HVEM-derived CSSD (Figure 6E). These data indicated that CAR-T cells with CD28-derived CSSD showed a low energy state, while those with 4-1BB-derived CSSD induced higher mitochondrial respiration. Furthermore, CAR-T cells with HVEM-derived CSSD also induced a high glycolytic system. These results suggested that HVEM-derived CSSD can lead to reprogramming of energy metabolism.

[0141] [Example 16: Considerations of Examples 10-15] A lentiviral vector was developed that expresses a CAR consisting of sCD4 as an antigen recognition domain in combination with a CSSD derived from CD28, 4-1BB, or HVEM. Transduction of human T cell lines and primary T cells with these vectors showed that the CSSD in the CAR construct is a crucial determinant of the CAR expression level on the cell surface and is thought to correlate with the activity and effector function of CAR-T cells. Also, in this study, it was shown that T cell exhaustion, energy metabolism, and induction of memory T cell subsets are also affected by the CSSD in the CAR construct. Among the CSSDs tested in this study, the HVEM-derived CSSD resulted in the highest level of CAR expression, the most potent effector function of CAR-T cells, avoidance of exhaustion, and a balanced induction of both central and effector memory T cell subsets (associated with high glycolysis and mitochondrial respiration). The results of this study suggest that the HVEM-derived CSSD may be useful for generating effective CAR-T cells in certain situations.

[0142] How CSSD controls the CAR expression level on the cell surface remains unknown. Since the amount of CAR molecules in whole cell lysates correlated with the level of cell surface expression, CAR synthesis other than trafficking can be affected by CSSD. Among other possibilities, different CSSDs can activate different signaling pathways, leading to different levels of gene expression. For example, co-stimulation mediated by CD28 induces the PI3K-Akt pathway, while co-stimulation mediated by 4-1BB mainly activates JNK and p38 through TNFR-associated factors (Kim et al., Mol. Cells 10:247 (2000); Cannons et al., J. Immunol. 165:6193 (2000)). Also, it has been shown that 4-1BB and HVEM can activate an alternative NF-ΚB pathway (Hauer et al., Proc. Natl. Acad. Sci. USA 102:2874 (2005)). These differences in signaling pathways can affect CAR expression at the transcriptional or translational level (Mehta et al., Nat. Rev. Immunol. 17:608 (2017)).

[0143] Antigen-independent tonic signaling during ex vivo expansion has been shown to cause exhaustion of CAR-T cells with CD28-derived CSSD, while those with 4-1BB-derived CSSD are relatively refractory to exhaustion (Long et al., Nat. Med. 21:581 (2015)). This study not only confirmed these observations but also showed that HVEM-derived CSSD can prevent exhaustion of CAR-T cells. Furthermore, these data suggested that CSSD in the CAR construct can affect the degree of T cell exhaustion through different effects on inhibitory receptor expression (Figure 4D). Since 4-1BB and HVEM are classified within TNFRSF, CSSDs derived from TNFRSF may, unlike those derived from the CD28 family, enable CAR-T cells to prevent exhaustion.

[0144] Mitochondrial dynamics has been suggested to control the fate of T cells through metabolic programs (Buck et al., Cell 166:63 (2016)). Furthermore, costimulation mediated by 4-1BB has been reported to enhance mitochondrial respiration along with the expansion of the central memory subset (Kawalekar et al., Immunity 44:380 (2016)). The results of the present invention are consistent with these previous studies and also show that the HVEM-derived CSSD enhances both glycolysis and mitochondrial respiration and exhibits a relatively low level of T cell exhaustion. It has also been shown that various T cell subsets require distinct metabolic programs to support their functions (Araki et al., Nature 460:108 (2009); Pearce et al., Nature 460:103 (2009); Rao et al., Immunity 32:67 (2010)). The data of the present invention suggest that costimulatory signals affect the differentiation of CAR-T cells into central and effector memory T cell subsets and that the HVEM-derived CSSD induces efficient and balanced differentiation. Different costimulatory signals may influence the differentiation of metabolic programs into specific memory T cell subsets in distinct manners.

[0145] The development of novel anti-HIV drugs and progress in anti-retroviral therapy (ART) have dramatically improved the prognosis of HIV-infected patients. However, ART has not been successful in completely eliminating HIV from the patient's body due to the persistence of latently infected cells. To achieve this goal, a so-called "shock and kill" approach has been proposed that combines pharmacological reactivation of latently infected cells and subsequent immunotherapy with HIV-specific CTLs or broadly neutralizing antibodies (bNAbs) (Halper-Stromberg et al., Cell 158:989 (2014)). In this protocol, CD4-based CAR-T cells targeting conserved regions of HIV-1 Env may be more useful than TCR-based CAR-T cells or bNAb-based CAR-T cells that cannot recognize antigens with escape mutations. Indeed, recent studies have reported promising results with CAR-T cells having a 4-1BB-derived CSSD in in vitro and in vivo models of HIV treatment (Leibman et al., PLoS Pathog. 13:e1006613 (2017)). The results of the studies of the present invention strongly suggest that CD4-based CAR-T cells having an HVEM-derived CSSD, which showed more potent effector functions than those having a CD28- or 4-1BB-derived CSSD, may also be a useful tool for the "shock and kill" treatment of HIV infection.

[0146] In summary, the results of the present invention show that the CSSD in the CAR is a very important determinant regarding the effector functions and characteristics of CAR-T cells, and indicate that the CSSD in the CAR is important for designing more potent CAR-T cells. Furthermore, HVEM-derived CAR-T cells may be promising candidates for generating effective CAR-T cells.

[0147] [Example 17: CAIX CAR Expression Test] Various CAR constructs were prepared using anti-CAIX scFv and different CSS domains as shown in Figure 11A. FACS detection of c-myc+ human CAR-T cells transduced by four constructs is shown in Figure 11B. The average surface c-myc expression level (MFI) is shown in Figure 11C.

[0148] The efficacy of CAIX CAR-T cells was tested using three target kidney / renal cancer cell lines. ACHN is a negative control without CAIX expression. As detected by FACS, Ketr-3 has high CAIX expression and OSRC-2 has low CAIX expression (Figure 12A). CAIX-HVEV CAR-T cells killed CAIX+ target cells more efficiently than other CAR-T cells (Figures 12B and 12C). Also, CAIX-HVEV CAR-T cells produced more IL-2 and IFN-γ than other CAR-T cells in response to co-culture with CAIX+ target cells (Ketr-3 (Figure 12D) and OSCR-2 (Figure 12E)).

[0149] Purified c-myc+ CAR-T cells were analyzed by Seahorse assay to measure their metabolic activity. Figure 13A shows the oxygen consumption rate (OCR) of various CAR-T cells by Seahorse assay. CAIX CAR-T cells based on HVEM showed higher basal OCR (Figure 13B) and maximal OCR (Figure 13C).

[0150] [Example 18: Test of Renal Cancer In Vivo] A test of renal cancer treatment using the CAIX CAR-T cells of Example 16 was conducted in NPG mice. The test design is shown in Figure 14A. Mice were intravenously injected with 1×10 6 individual OSCR-2 human renal cancer cells on day 1. 1×10 7 individual CAR-T cells were intravenously injected on day 7. Figure 14B shows the level of human CAR-T cells in the peripheral blood of mice on day 14. The overall survival of the mice is shown in Figure 14C. CAIX CAR-T cells based on HVEM provided significantly better survival than other CAR-T cells.

[0151] Lung metastatic tumors in 6 mice / group were tested at the time points shown in Figure 16. Mice in the HVEM CAR-T group were terminated 90 days after tumor injection, while mice in the other groups were terminated 50 days after tumor injection. White patches indicate metastatic tumors in the lung. CAIX CAR-T cells based on HVEM provided significantly reduced tumor metastasis compared to other CAR-T cells. Figure 17 shows representative hematoxylin / eosin lung histopathology of 1 mouse / group at the end. Only the HVEM CAR-T group showed a functional lung structure, while all other groups showed severely infiltrated lungs with loss of lung structure and function.

[0152] In repeated experiments, similar results of HVEM-CAR T expansion were observed in vivo 14 days after tumor injection and 7 days after CAR-T cell transfer. Figure 15A shows the percentage of CAR-T (myc+) cells after in vitro transduction. Figure 15B shows the level of human CAR-T cells in mouse blood 7 days after CAR-T cell transfer. Figure 15C shows summary data of human T cells as a percentage of total mouse blood cells (left) or human T cell count / 100 μl mouse blood. HVEM CAR-T cells showed better expansion in vivo.

[0153] The foregoing are examples of the present invention and should not be construed as limiting it. The present invention is defined by the following claims, and equivalents of the claims are included herein.

Claims

1. The antibody comprises an antigen-binding domain, a transmembrane domain, a T-cell receptor domain, and a costimulatory signal (CSS) domain comprising a herpesvirus entry mediator (HVEM) protein, the amino acid sequence of which is set forth in SEQ ID NO:

2. Chimeric antigen receptor (CAR).

2. A method for the treatment of a herpesvirus infection comprising administering to a subject an antibody having an antigen-binding domain, a transmembrane domain, a T cell receptor domain, and a costimulatory signal (CSS) domain comprising a herpesvirus entry mediator (HVEM) protein, the antibody comprising the amino acid sequence of SEQ ID NO:

1. Chimeric antigen receptor (CAR).

3. The CAR of claim 1 or 2, The CSS domain further comprises a CD28 CSS domain, a 4-1BB CSS domain, an OX-40 CSS domain, an ICOS CSS domain, or any combination thereof. CAR.

4. The CAR of any one of claims 1 to 3, The T cell receptor domain comprises a CD3ζ signaling domain. CAR.

5. The CAR of any one of claims 1 to 4, the antigen-binding domain comprises a monovalent antibody fragment; CAR.

6. The CAR of claim 5, The monovalent antibody fragment comprises a single chain variable fragment (scFv) or a Fab fragment. CAR.

7. The CAR of any one of claims 1 to 6, the antigen-binding domain targets an antigen present on the surface of a viral particle and / or a cancer cell; CAR.

8. The CAR of any one of claims 1 to 7, the antigen-binding domain comprises a soluble CD4 protein; CAR.

9. The CAR of any one of claims 1 to 7, the antigen-binding domain comprises a monovalent antibody fragment targeting the surface protein CAIX; CAR.

10. The CAR of any one of claims 1 to 7, the antigen-binding domain comprises a monovalent antibody fragment targeting the surface protein disialoganglioside GD2; CAR.

11. Encoding the CAR of any one of claims 1 to 10. Nucleic acid molecule.

12. 12. The nucleic acid molecule of claim 11, Comprising the nucleotide sequence of SEQ ID NO: 7 or a sequence having at least 90% identity thereto; Nucleic acid molecule.

13. 13. The nucleic acid molecule of claim 11 or 12, vector.

14. The CAR of any one of claims 1 to 10. cell.

15. 14. A nucleic acid molecule according to claim 11 or 12 and / or a vector according to claim 13. cell.

16. 16. The cell of claim 14 or 15, The cells are selected from the group consisting of αβ T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, natural killer T (NKT) cells, Th17 cells, γδ T cells, and any combination thereof. cell.

17. 17. A method for producing a CAR according to any one of claims 1 to 10, a nucleic acid molecule according to claim 11 or 12, a vector according to claim 13, and / or a cell according to any one of claims 14 to 16, in a pharmaceutically acceptable carrier. composition.

18. 1. A composition for use in a method of providing an immune response against a target in a subject in need thereof, comprising: The composition comprises a CAR of any one of claims 1 to 10, a nucleic acid molecule of claim 11 or 12, a vector of claim 13, and / or a cell of any one of claims 14 to 16, The method includes administering to the subject an effective amount of the composition; thereby providing an immune response against said target in said subject. composition.

19. 1. A composition for use in a method of providing an immune response against a target in a subject in need thereof, comprising: The composition comprises the nucleic acid molecule of claim 11 or 12 or the vector of claim 13, The method comprises: Obtaining T cells from a subject with cancer and / or infection; transfecting said T cells with said composition; Culturing the transfected T cells; and administering the cultured, transfected T cells to the subject; Including, thereby providing an immune response against said target in said subject. composition.

20. 1. A composition for use in a method for enhancing cellular responsiveness to an antigen, comprising: The composition comprises the nucleic acid molecule of claim 11 or 12 or the vector of claim 13, The method comprises: transfecting a cell with the composition to produce a transfected cell comprising the antigen-binding domain on the cell surface; wherein the antigen-binding domain specifically binds to the antigen, thereby enhancing the responsiveness of the cells to the antigen. composition.

21. 1. A composition for use in a method of treating a chronic viral or bacterial infection in a subject in need thereof, comprising: The composition comprises a CAR of any one of claims 1 to 8, a nucleic acid molecule of claim 11 or 12, a vector of claim 13, and / or a cell of any one of claims 14 to 16, The method includes administering to the subject an effective amount of the composition; thereby treating said infection in said subject. composition.

22. 1. A composition for use in a method of treating a chronic viral or bacterial infection in a subject in need thereof, comprising: The composition comprises the nucleic acid molecule of claim 11 or 12 or the vector of claim 13, The method comprises: obtaining T cells from a subject with an infection; transfecting said T cells with said composition; Culturing the transfected T cells; and administering the cultured, transfected T cells to a subject with an infection; Including, thereby treating said infection in said subject. composition.

23. 1. A composition for use in a method of treating cancer in a subject in need thereof, comprising: The composition comprises a CAR of any one of claims 1 to 10, a nucleic acid molecule of claim 11 or 12, a vector of claim 13, and / or a cell of any one of claims 14 to 16, The method includes administering to the subject an effective amount of the composition; thereby treating cancer in said subject. composition.

24. 1. A composition for use in a method of treating cancer in a subject in need thereof, comprising: The composition comprises the nucleic acid molecule of claim 11 or 12 or the vector of claim 13, The method comprises: Obtaining T cells from a subject with cancer; transfecting said T cells with said composition; Culturing the transfected T cells; and administering the cultured, transfected T cells to a subject having a solid tumor; Including, thereby treating cancer in said subject. composition.

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