WO2020219843A1 - Chimeric antigen receptor constructs and their use in car-t cells - Google Patents

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

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WO2020219843A1
WO2020219843A1 PCT/US2020/029768 US2020029768W WO2020219843A1 WO 2020219843 A1 WO2020219843 A1 WO 2020219843A1 US 2020029768 W US2020029768 W US 2020029768W WO 2020219843 A1 WO2020219843 A1 WO 2020219843A1
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car
cells
cell
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css
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French (fr)
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Lishan Su
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University of North Carolina at Chapel Hill
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University of North Carolina at Chapel Hill
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Priority to JP2021563033A priority Critical patent/JP2022529380A/ja
Priority to CN202080047342.2A priority patent/CN114269777A/zh
Priority to US17/605,890 priority patent/US12479890B2/en
Priority to EP20796240.8A priority patent/EP3959236A4/en
Publication of WO2020219843A1 publication Critical patent/WO2020219843A1/en
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Definitions

  • the present invention is directed to chimeric antigen receptor (CAR) compositions and methods of their use in cancer and anti-pathogen immunotherapy.
  • CAR chimeric antigen receptor
  • Chimeric antigen receptors also known as chimeric immunoreceptors, chimeric T cell receptors or artificial T cell receptors, are engineered receptors that combine a new specificity with an immune cell to target cancer cells. Typically, these receptors graft the specificity of a monoclonal antibody onto a T cell. The receptors are called chimeric because they are fusions of parts from different sources.
  • CAR-T cell therapy refers to a treatment that uses such transformed cells primarily for cancer therapy.
  • the basic principle of CAR-T cell design involves recombinant receptors that combine antigen-binding and T-cell activating functions. The general premise of CAR-T cells is to artificially generate T-cells targeted to markers found on diseased cells e.g., cancer cells.
  • T-cells from a person, genetically alter them, and put them back into the patient for them to attack the diseased cells. Once the T cell has been engineered to become a CAR-T cell, it acts as a“living drug”.
  • CAR-T cells create a link between an extracellular ligand recognition domain and an intracellular signaling molecule which in turn activates T cells.
  • the extracellular ligand recognition domain is usually a single-chain variable fragment (scFv).
  • scFv single-chain variable fragment
  • CAR-T cells can be derived from either a patient's own blood (autologous) or derived from another healthy donor (allogenic). 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 that are present on the surface of tumors. When they come in contact with the antigens on the tumors, the CAR-T cells are activated via the signal peptide, proliferate and become cytotoxic.
  • the CAR-T cells destroy the cancer cells through mechanisms such as extensive stimulated cell proliferation, increasing the degree to which the cell is toxic to other living cells, i.e., cytotoxicity, and by causing the increased production of factors that are secreted from cells in the immune system that have an effect on other cells in the organism. These factors are called cytokines and include interleukins, interferons and growth factors.
  • 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)).
  • cancer types e.g., solid tumors
  • diseases e.g., chronic viral infections (e.g., HIV)
  • the present invention provides compositions of a chimeric antigen receptor (CAR) and methods of using the same, wherein the co-stimulatory signal (CSS) comprises a herpes virus entry mediator (HVEM) protein, also called CD270.
  • CAR co-stimulatory signal
  • HVEM herpes virus entry mediator
  • the CSS in the CAR is important when modulating the immune activity of CAR-transduced T cells (CAR-T cells) which are employed in a wide variety of diseases such as cancer (e.g., solid tumors) and pathogen infection (e.g., chronic viral or bacterial infections).
  • a CAR comprising a HVEM CSS exhibited enhanced effector functions associated with significantly higher glycolysis and mitochondrial respiration, and induced equivalent proportions of central and effector memory subsets compared to a CAR comprising a CSS domain that does not comprise HVEM.
  • CAR-T cell function can be improved through HVEM co-stimulation by reprogramming CAR-T cell energy metabolism.
  • one aspect of the invention relates to a chimeric antigen receptor (CAR) comprising an antigen binding domain, a transmembrane domain, a T-cell receptor domain, and a costimulatory signal (CSS) domain comprising herpes virus entry mediator (HVEM) protein or a functional fragment or variant thereof having at least 90% identity thereto.
  • CAR chimeric antigen receptor
  • SCS costimulatory signal
  • HVEM herpes virus entry mediator
  • Another aspect of the invention relates to a nucleic acid molecule encoding the CAR of the invention.
  • An additional aspect of the invention relates to a vector comprising the nucleic acid molecule of the invention.
  • a further aspect of the invention relates to a cell comprising the CAR of the invention.
  • Another aspect of the invention relates to a cell comprising the nucleic acid molecule of the invention and/or the vector of the invention.
  • An additional aspect of the invention relates to a composition
  • a composition comprising 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.
  • a further aspect of the 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 invention, the nucleic acid molecule of the invention, the vector of the invention, and/or the cell of the invention, thereby providing an immune response against the target in the subject.
  • Another aspect of the 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 invention or the vector of the invention, culturing the transfected T-cells, and
  • FIG. 1A Schematic representation of CAR expressing lentiviral vector constructs. The CARs contain the extracellular domain of human CD4 (sCD4) that binds HIV Env protein as an antigen recognition domain and differ in the co-stimulatory signal sequence.
  • FIG. IB Gating strategy for the analysis of CAR expression in the transduced human T cell line. GFP + transduced cells were gated (lower panel) and used for the analysis of CAR expression (upper panel) with anti-c-myc tag antibody by flow cytometry. Typical dot plot and CAR histograms of each transduced cell are shown.
  • FIGS. 7A and 7B Similar transduction efficiency was achieved by lentiviral transduction (See also FIGS. 7A and 7B).
  • FIG. ID CAR (50kDa, upper panel), endogenous CD3z (18kDa, middle panel) and Actin (43kDa, lower panel) expression levels in the CAR transduced T cell line harboring different CSS were determined by western blot with hh ⁇ -E ⁇ 3z and anti-actin antibody, respectively.
  • the activated (CD69 + ) cell frequencies in transduced (CD3 + GFP + ) or un-transduced (CD3 + GFP ) T cells were determined by the co-culture assay with target cells expressing GFP (CHO-GFP) or HIV-Env/GFP (CHO-Env- GFP) (See also FIG. 7C).
  • target cells expressing GFP CHO-GFP
  • HIV-Env/GFP HIV-Env/GFP
  • FIG. IF IL-2 secretion in the co-culture assay was measured by ELISA.
  • FIG. 1G Linear regression analysis between frequencies of activated CAR-T cell (y-axis) and the levels of CAR expression (x-axis).
  • FIG. 1H Linear regression analysis between IL-2 secretion from antigen-stimulated CAR-T cell (y-axis) in the co-culture assay and the levels of CAR expression (x-axis).
  • FIG. 2B Comparison of CAR expression among CAR-T cells harboring different CSS. CAR expression in GFP + transduced primary human CD8 T cell was analyzed with anti-c-myc tag antibody by flow cytometry.
  • FIG. 2C Representative histograms of CAR expression (x-axis) in GFP + cells from two different donors are shown. The numbers in each histogram shows MFI of CAR.
  • FIG. 2C CAR (50kDa, upper panel), endogenous CD3z (18kDa, middle panel) and Actin (43kDa, lower panel) expression levels in human CAR-T cell harboring different CSS were determined by western blot with hh ⁇ 3z and anti-actin antibody, respectively.
  • FIG. 3A Human CAR-T cells harboring different CSS were co-cultured with CHO-GFP (open circle) or CHO-Env-GFP (filled circle) cells at effector: target ratio of 10: 1, 5: 1 and 1 : 1.
  • FIG. 3A Human CAR-T cells harboring different CSS were co-cultured with CHO-GFP (open circle) or CHO-Env-GFP (filled circle) cells at effector: target ratio of 10: 1, 5: 1 and 1 : 1.
  • the results shows the mean of two separate experiments with primary CD 8 T cells from two
  • FIG. 3C Comparison of cytotoxicity among human CAR-T cells harboring different CSS at effector: target ratio of 10: 1. ** P ⁇ 0.05 (one-way ANOVA; bonfferoni’s post-hoc).
  • FIG. 3D Linear regression analysis between TNF-a secretion (y-axis) in the co-culture assay and the levels of CAR expression (x-axis) among human CAR-T cells harboring different CSS. (See also FIGS. 8A-8C for linear regression analysis of IL-2, IFN-g or cytotoxicity).
  • FIGS 4A-4E 4- IBB and HVEM co-stimulation averts CAR-T cell exhaustion.
  • FIG. 4A Representative plots of cell-surface expression of PD-1 (x-axis) and LAG-3 (y-axis) on GFP + transduced cells harboring different CSS are shown. The numbers in each plot shows frequency of each population in the CAR-T cell.
  • FIGS. 9A-9C Linear regression analysis between TNF-a secretion (y-axis) in the co-culture assay and frequency of exhausted (PDl + /LAG-3 + ) CAR-T cells (x-axis) among human CAR-T cells harboring different CSS.
  • FIGS. 9A-9C Bar graph shows that HVEM CAR-T cells exhibited lower frequencies of the populations expressing either one or two inhibitory receptor.
  • FIG. 4E CAR-T cells with HVEM-derived CSSD averts CAR-T cell exhaustion. Representative plots shows cell-surface expression of PD-1 (x-axis) and LAG-3 (y-axis) on the CAR-T cells with different CSSD. The numbers in each plot show percentages of each population in the CAR-T cells with different CSSD.
  • FIG. 5A Representative plots of cell-surface expression of CD45RO (x-axis) and CCR7 (y- axis) on GFP + cells harboring different CSS are shown. The numbers in each plot shows frequency of each population in the CAR-T cell.
  • FIG. 5B Bar graph summarizes frequencies of naive (T N : CCR7 + CD45RO ), central memory (T CM : CCR7 + CD45RO + ), effector memory (TEM: CCR7 CD45RO + ) and terminally differentiated effector memory (T E MRA: CCR7 CD45RO) population in each CAR-T cell harboring different CSS.
  • FIG. 5C Bar graph shows T C M and T EM frequencies (y-axis) among CAR-T cells harboring different CSS.
  • FIG. 6A The oxygen consumption rates (OCRs) of human CAR-T cells harboring different CSS under basal metabolic conditions and in response to mitochondrial inhibitors. The data are the summary of two experiments performed with differentially sorted cells from two different healthy human donors and plotted as mean with standard deviations.
  • FIG.6B Basal OCR level measurements.
  • FIG. 6C Basal extracellular acidification rate (ECAR) level determination.
  • FIG. 6D ATP production defined as (last rate measurement before oligomycin addition) - (minimum rate measurement after oligomycin addition).
  • FIG. 6E Maximal OCR level measurements.
  • FIG. 6F Spare respiratory measurements.
  • FIG. 6G Non-mito respiration measurements.
  • FIG. 6H Proton leak measurements. Bar graph shows the mean with standard deviations of two separate experiments with cells from two different healthy human donors. ** P ⁇ 0.05 (one-way ANOVA;
  • FIG. 7A Typical dot plots of CAR-transduced Jurkat E 6.1 cells. Transduction efficiency and CAR expression were determined by GFP expression (y-axis) and anti-c-myc tag antibody staining (x-axis) followed by flow cytometry. The numbers in the plots show the frequency of each cell population.
  • FIG. 7C Gating strategy to determine CAR-T cell activation in the co-culture assay.
  • Target cells HAV gpl20 + GFP + CHO cells
  • CAR transduced Jurkat E6.1 cells CAR transduced Jurkat E6.1 cells.
  • the transduced cells were separated from target cells based on GFP/CD3 expression and used for the analysis of CD69 expression (middle and right panel).
  • the representative plots show the frequency of activated (CD69 + ) cells in un-transduced (CD3 + GFP , middle plot) or transduced (CD3 + GFP + , right plot) effector cells upon co-cultivation with target cells. The number in each plot shows the frequency of activated cell population.
  • FIG. 8A Linear regression analysis between effector functions and CAR expression level among human CAR-T cells harboring different CSS.
  • the graphs represents the results of linear regression analysis between IL-2 secretion (FIG. 8A), IFN-g secretion (FIG.
  • FIG. 9A Linear regression analysis between effector functions and human CAR-T cell exhaustion human CAR-T cells harboring different CSS.
  • the graphs represent the results of linear regression analysis between IL-2 secretion (FIG. 9A), IFN-g secretion (FIG.
  • FIG. 10A-10B Analysis of memory T cell subsets by surface expression of
  • FIG. 10A Increase in central memory T cells with CAR-T cells with different CSSDs.
  • FIG. 10B Increase in effector memory T cells and central memory T cells with CAR-T cells with CD28- and 4-lBB-derived CSSD, respectively.
  • FIG. 11 A Schematic representation of various CAR expressing lentiviral vector constructs.
  • the CARs contain an anti-CAIX-scFV that binds renal cell tumor-associated transmembrane protein carbonic anhydrase IX (CAIX) as an extracellular antigen recognition domain and differ in the co-stimulatory signal sequence.
  • FIG. 11B Typical dot plots of CAR-transduced human cells. Transduction efficiency and CAR expression were determined in CAR-T cells via anti-c-myc tag antibody staining (x-axis) followed by flow cytometry. The numbers in the plots show the frequency of each cell population.
  • FIG. 11C The mean fluorescence intensity (MFI) of c-myc in CAR in cells (y-axis) harboring different CSS as shown.
  • MFI mean fluorescence intensity
  • FIGS. 12A-12C HVEM-CAR exhibits the highest effector function in human CAR- T cells: Two human renal cancer cell lines expressing tumor-associated transmembrane carbonic anhydrase IX (CAIX) plus a control renal cancer cell line with no CAIX expression.
  • FIG. 12A Gating and analysis of CAR expression in the transduced human renal cancer cell lines ACHN, Ketr-3, and OSRC.
  • FIG. 12B Data showing kinetics of human CAR-T cells killing renal cancer cells.
  • FIG. 12C Summarized data showing human CAR-T cells killing renal cancer cells.
  • FIG. 12D IFN-g (left panel) and IL-2 (right panel) secretion measured by ELISA of human CAR-T cells harboring different CSS in an assay co-cultured with human renal cancer cell line Ketr-3.
  • FIG. 12E IFN-g (left panel) and IL-2 (right panel) secretion measured by ELISA of human CAR-T cells harboring different CSS in an assay co-cultured with human renal cancer cell line OSRC -2.
  • FIG. 13A-13C HVEM-CAR T-cells show higher metabolic activity.
  • FIG. 13A OCR of various CAR-T cells by Seahorse assays.
  • FIG. 13B Basal OCR.
  • FIG. 13C Maximal OCR.
  • Figures 14A-14C HVEM-CAR T-cells exhibiting efficient anti-kidney tumors in mice in vivo.
  • FIG. 14A Schematic diagram showing the human renal cancer (RC) treatment design in mice.
  • FIG. 14B Summarized data showing human CAR-T cells in peripheral blood in NSG mice on day 14.
  • FIG. 14C Overall survival of NSG mice with human renal cancer cells with lung metastasis following CAR-T therapy.
  • FIG. 15A-15C HVEM-CAR T-cells show better expansion in vivo.
  • FIG. 15A Percentage of CAR-T cells after in vitro transduction.
  • FIG. 14B Human CAR-T cells in peripheral blood in NSG mice on day 14.
  • FIG. 14C Summarized data of human T cells as percentage of total mouse blood cells (left panel) or human T cell count/100 m ⁇ of mouse blood (right panel).
  • Figure 16 Photographs of mouse lungs with reduced metastases at termination.
  • Figure 17 Histopathology of mouse lungs with reduced metastases at termination.
  • FIG. 18 Schematics of third generation CARs. All constructs contain the HVEM costimulatory domain along with the cytoplasmic domains from 4 IBB, CD28, ICOS, or 0X40 proteins. In the last three constructs, linkers were placed between the HVEM and 0X40 domains in the pRRL-VRC01-CD8-HVEM-OX40-CD3z constructs or the ITAM 3 motif in the CD3z domain was mutated to the PD-1 ITIM.
  • Nucleotide sequences are presented herein by single strand only, in the 5' to 3' direction, from left to right, unless specifically indicated otherwise. Nucleotides and amino acids are represented herein in the manner recommended by the IUPAC-IUB Biochemical Nomenclature Commission, or (for amino acids) by either the one-letter code, or the three letter code, both in accordance with 37 C.F.R. ⁇ 1.822 and established usage.
  • the term "about,” as used herein when referring to a measurable value such as an amount of an antibody, compound or agent of this invention, dose, time, temperature, and the like, is meant to encompass variations of ⁇ 10%, ⁇ 5%, ⁇ 1%, ⁇ 0.5%, or even ⁇ 0.1% of the specified amount.
  • the term "consisting essentially of (and grammatical variants), as applied to an amino and/or nucleotide sequence of this invention, means an amino and/or nucleotide sequence that consists of both the recited sequence (e.g, SEQ ID NO) and a total of ten or less (e.g, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional amino acids and/or nucleotides on the N-terminal end and/or C- terminal end and/or 5' and/or 3' ends of the recited sequence such that the ability of the an amino and/or nucleotide sequence to bind to its target is not materially altered.
  • the total of ten or less additional nucleotides includes the total number of additional nucleotides on both the 5' and 3' ends added together.
  • the term "materially altered,” as applied to the binding of the nucleotide sequence, refers to an increase or decrease in binding affinity of at least about 50% or more as compared to the binding affinity of a nucleotide sequence consisting of the recited sequence.
  • antibody“ refers to full-length immunoglobulins as well as to fragments thereof. Such full-length immunoglobulins may be monoclonal, polyclonal, chimeric, humanized, veneered or human antibodies.
  • antibody fragments comprises portions of a full-length immunoglobulin retaining the targeting specificity of said immunoglobulin. Many but not all antibody fragments lack at least partially the constant region (Fc region) of the full-length immunoglobulin. In some embodiments, antibody fragments are produced by digestion of the full-length immunoglobulin. An antibody fragment may also be a synthetic or recombinant construct comprising parts of the immunoglobulin or immunoglobulin chains (see e.g. Holliger, P. and Hudson, J. Engineered antibody fragments and the rise of single domains. Nature Biotechnology 2005, vol. 23, no. 9, p.
  • antibody fragments include, without being limited to, include scFv, Fab, Fv, Fab’, F(ab’) 2 fragments, dAb, VHH, nanobodies, V(NAR) or minimal recognition units.
  • Single chain variable fragments or“single chain antibodies” or“scFv” are one type of antibody fragment. scFv are fusion proteins comprising the VH and VL of immunoglobulins connected by a linker. They thus lack the constant Fc region present in full-length
  • immunoglobulins but retain the specificity of the original immunoglobulin.
  • the numbering system to identify amino acid residue positions in the VH and VL of the 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.
  • the publication further provides conversion tables between the AHo and the Kabat system (Kabat, E.A., et al. Sequences of Proteins of Immunological Interest. 5th edition. Edited by U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES. NIH Publications, 1991. p. 91- 3242).
  • nucleic acid As used herein,“nucleic acid,”“nucleotide sequence,” and“polynucleotide” are used interchangeably and encompass both RNA and DNA, including cDNA, genomic DNA, mRNA, synthetic (e.g, chemically synthesized) DNA or RNA and chimeras of RNA and DNA.
  • polynucleotide, nucleotide sequence, or nucleic acid refers to a chain of nucleotides without regard to length of the chain.
  • fragment will be understood to mean a nucleotide sequence of reduced length relative to a reference nucleic acid or nucleotide sequence and comprising, consisting essentially of and/or consisting of a nucleotide sequence of contiguous nucleotides identical to the reference nucleic acid or nucleotide sequence.
  • a nucleic acid fragment according to the invention may be, where appropriate, included in a larger polynucleotide of which it is a constituent.
  • such fragments can comprise, consist essentially or and/or consist of, oligonucleotides having a length of 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.
  • identity refers to the sequence match between two proteins or nucleic acids.
  • the protein or nucleic acid sequences to be compared are aligned to give maximum identity, for example using bioinformatics tools such as EMBOSS Needle (pair wise alignment; available at www.ebi.ac.uk).
  • EMBOSS Needle air wise alignment; available at www.ebi.ac.uk.
  • the percent identity is a function of the number of matching positions divided by the number of positions compared and multiplied by 100%. For instance, if 6 out of 10 sequence positions are identical, then the identity is 60%.
  • the percent identity between two protein sequences can, e.g., be determined using the
  • Needleman and Wunsch algorithm (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) which has been incorporated into EMBOSS Needle, using a BLOSUM62 matrix, 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 and an“end gap extend penalty” of 0.5.
  • Two molecules having the same primary amino acid or nucleic acid sequence are identical irrespective of any chemical and/or biological modification. For example, two antibodies having the same primary amino acid sequence but different glycosylation patterns are identical by this definition.
  • nucleic acids for example, two molecules having the same sequence but different linkage components such as thiophosphate instead of phosphate are identical by this definition.
  • the term“variant” refers to an amino acid or nucleic acid sequence which differs from the parental sequence by virtue of addition (including insertions), deletion and/or substitution of one or more amino acid residues or nucleobases while retaining at least one desired activity of the parent sequence disclosed herein. In the case of CARs such desired activity may include specific target binding. Similarly, a variant nucleic acid sequence may be modified when compared to the parent sequence by virtue of addition, deletion and/or substitution of one or more nucleobases, but the encoded CAR retains the desired activity as described above. Variants may be naturally occurring, such as allelic or splice variants, or may be artificially constructed.
  • “one or more” means one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.
  • a“subject” that may be treated by the present invention include both human subjects for medical and/or therapeutic purposes and animal subjects for veterinary and drug screening and development purposes.
  • Other suitable animal subjects are, in general, mammalian subjects such as primates, bovines, ovines, caprines, porcines, equines, felines, canines, lagomorphs, rodents ( e.g ., rats and mice), etc.
  • Human subjects are the most preferred. Human subjects include fetal, neonatal, infant, juvenile, adult and geriatric subjects.
  • anti -tumor effect refers to a biological effect which can be manifested by a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the proliferation rate, a decrease in the number of metastases, an increase in life expectancy, and/or amelioration of various physiological symptoms associated with the cancerous condition.
  • An "anti-tumor effect” can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies of the invention to delay the occurrence of tumor in the first place.
  • autologous is meant to refer to any material derived from the same individual to whom it is later to be re-introduced.
  • Allogeneic refers to a graft derived from a different animal of the same species.
  • Xenogeneic refers to a graft derived from an animal of a different species.
  • antigen-binding portion or "antigen-binding fragment” of an antibody (or simply “antibody portion” or “antibody fragment”), as used herein, refers to one or more fragments, portions or domains of an antibody that retain the ability to specifically bind to an antigen. It has been shown that fragments of a full-length antibody can perform the antigen binding function of an antibody.
  • binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) an Fab fragment, a monovalent fragment consisting of the VL, VH, CL1 and CHI domains; (ii) an F(ab') 2 fragment, a bivalent fragment comprising two F(ab)' fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CHI domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment (Ward et al.
  • antigen-binding portion of an antibody.
  • epitope refers to an antigenic determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule known as a paratope.
  • a single antigen may have more than one epitope.
  • Epitopes may be either conformational or linear.
  • a conformational epitope is produced by spatially juxtaposed amino acids from different segments of one (or more) linear polypeptide chain(s).
  • a linear epitope is an epitope produced by adjacent amino acid residues in a polypeptide chain.
  • an epitope may include other moieties, such as saccharides, phosphoryl groups, or sulfonyl groups on the antigen.
  • an "antibody heavy chain,” as used herein, refers to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations.
  • an "antibody light chain,” as used herein, refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations, K and l light chains refer to the two major antibody light chain isotypes.
  • antigen or "Ag” as used herein is defined as a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both.
  • antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises a nucleotide sequence or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an "antigen" as that term is used herein.
  • an antigen need not be encoded solely by a full length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a "gene” at all. It is readily apparent that an antigen can be synthesized or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid.
  • Amino acid refers to a compound having a free carboxyl group and a free unsubstituted amino group on the a carbon, which may be joined by peptide bonds to form a peptide active agent as described herein.
  • Amino acids may be standard or non-standard, natural or synthetic, with examples (and their abbreviations) including but not limited to:
  • Tic tetrahydroisoquinoline-3-carboxylic acid
  • Aib aminoisobutyric acid
  • Base amino acid refers to any amino acid that is positively charged at a pH of 6.0, including but not limited to R, K, and H.
  • Amatic amino acid refers to any amino acid that has an aromatic group in the side-chain coupled to the alpha carbon, including but not limited to F, Y, W, and H.
  • detectable moiety includes any suitable detectable group, such as radiolabels (e.g. S, I, I, etc.), enzyme labels (e.g, horseradish peroxidase, alkaline phosphatase, etc.), fluorescence labels (e.g., fluorescein, green fluorescent protein, etc.), etc., as are well known in the art and used in accordance with known techniques.
  • radiolabels e.g. S, I, I, etc.
  • enzyme labels e.g, horseradish peroxidase, alkaline phosphatase, etc.
  • fluorescence labels e.g., fluorescein, green fluorescent protein, etc.
  • an "immune response” refers to the reaction of a subject to the presence of an antigen, which may include at least one of the following: making antibodies, developing immunity, developing hypersensitivity to the antigen, and developing tolerance.
  • the term“enhance an immune response” as used herein implies that the reaction of a subject to the presence of an antigen is increased and/or amplified in the presence of a CAR of the invention compared to the reaction of a subject to the presence of an antigen in the absence of the CAR of the invention.
  • An“effective” amount as used herein is an amount that provides a desired effect.
  • A“therapeutically effective” amount as used herein is an amount that provides some improvement or benefit to the subject.
  • a“therapeutically effective” amount is an amount that will provide some alleviation, mitigation, or decrease in at least one clinical symptom in the subject.
  • the therapeutic effects need not be complete or curative, as long as some benefit is provided to the subject.
  • Chimeric antigen receptor is an artificial antigen receptor which consists of an antigen-binding domain and a signal transduction domain capable of mimicking the T cell receptor-mediated signaling pathway.
  • a natural ligand or the single-chain variable region of an antibody to the target molecule has been used as the antigen-binding domain of CAR.
  • MHC major histocompatibility complex
  • T cells expressing CAR might serve as a useful tool for adoptive immunotherapy of a wide range of patients (Dotti et al, Immunol. Rev. 257(1): 107 (2014)).
  • T cells expressing the first generation CAR that has CD3z as the signal transduction domain often become anergic and fail to elicit potent immune response (Kershaw et al, Clin. Cancer Res.
  • CARs that have one and two co-stimulatory 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 the modular structure have been shown to successfully mimic T cell receptor-mediated signal transduction upon antigen stimulation, leading to proliferation and activation of CAR-T cells (Maus et al, Blood 123(17):2625 (2014)).
  • SCS co-stimulatory signal
  • Co-stimulatory molecules are divided into two major families; the CD28 family which includes CD28 and ICOS, and the tumor necrosis factor receptor superfamily (TNFRSF) which includes 4- IBB (TNFRSF9) and herpes virus entry mediator (HVEM, TNFRSF 14).
  • CD28 tumor necrosis factor receptor superfamily
  • TNFRSF tumor necrosis factor receptor superfamily
  • HVEM herpes virus entry mediator
  • T cells expressing the second generation CAR with the 4-lBB-derived CSS domain persist for more than 6 months in the blood of most patients, whereas CAR-T cells with the CD28-derived CSS domain become mostly undetectable after 3 months (Zhang et al, Oncotarget 6(32):33961 (2015)).
  • 4-lBB-mediated co-stimulation selectively induced mitochondrial biogenesis and oxidative metabolism for energy production, resulting in enhanced differentiation and increased in vitro persistence of central memory T cells (Kawalekar et al, Immunity
  • HVEM another member of the TNFRSF
  • HVEM deficiency in CD8 + T cells is shown to profoundly impair effector CD8 + T cell survival and development of protective immune memory
  • B and T lymphocyte attenuator one of the ligands of HVEM
  • interaction with HVEM expressed on CD8 + T cells was also reported to promote survival and memory generation in response to a bacterial infection (Steinberg et al., PLoS One 8(10):e77992 (2013)).
  • the identification of effective CSS in the CAR module is one of the key requirements for applying CAR-T cell therapy against a wide variety of diseases such as solid tumors and pathogen infections (e.g., chronic viral or bacterial infections).
  • the present inventors developed second generation CARs which included the extracellular domain of CD4, known as soluble CD4 (sCD4), as the antigen-binding domain targeted to 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 (FIG. 1A).
  • 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 costimulatory signal (CSS) domain comprising herpes virus entry mediator (HVEM) protein or a functional fragment thereof.
  • CAR chimeric antigen receptor
  • SCS costimulatory signal
  • HVEM herpes virus entry mediator
  • the CSS domain is essential for promoting the intracellular signal of the T-cell receptor domain to initiate T cell activation and proliferation.
  • promotion of such a signal can depend upon the selected CSS domain and/or combinations thereof.
  • a CSS domain comprising a HVEM protein or a functional fragment or variant thereof promotes T cell activation and proliferation.
  • the HVEM CSS of the invention promotes enhanced effector function associated with increased glycolysis and mitochondrial respiration relative to a CAR that does not comprise the HVEM CSS domain of the invention.
  • CAR-T cells comprising the HVEM CSS domain of the invention have an increase in CAR-T glycolysis by 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%) compared to CAR-T cells that do not comprise the HVEM CSS domain of the invention.
  • CAR-T cells comprising the HVEM CSS domain of the invention have an increase in mitochondrial respiration by 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%) compared to CAR-Y cells that do not comprise the HVEM CSS domain of the invention.
  • the HVEM CSS domain comprises, consists essentially of, or consists of the amino acid sequence of: WVWWFLSGSL VIVIVCSTVG LIICVKRRKP RGDVVKVIVS VQRKRQEAEG EATVIEALQA PPDVTTVAVE ETIPSFTGRS PNH (SEQ ID NO:l) 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.
  • the HVEM CSS comprises, consists essentially of, or consists of a fragment of HVEM with the amino acid of: LVIVIVCSTV GLIICVKRRK PRGDVVKVIV SVQRKRQEAE GEATVIEALQ APPDVTTVAV EETIPSFT (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.
  • the CSS domain further comprises one or more additional CSS domains (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.), variants, and/or fragments thereof.
  • additional CSS domains e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.
  • Non-limiting examples include a CD28 CSS domain, a 4-1BB CSS domain, an OX-40 CSS domain, an ICOS domain, or any other CSS domain and/or functional fragment or variant thereof now known or later identified, having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity thereto, in any combination.
  • a linker may be present between two or more of the domains, e.g., a 3-12 residue linker, e.g., a 5-8 residue linker.
  • FIG. 18 Exemplary constructs are depicted in FIG. 18.
  • the T-cell receptor domain is a signaling domain that transduces the event of receptor ligand binding to an intracellular signal that partially activates the T cell. Absent appropriate co stimulatory signals, this event is insufficient for useful T cell activation and proliferation.
  • a non- limiting example of a T-cell receptor domain of this invention is the T cell receptor domain of the T cell receptor zeta chain (e.g., O ⁇ 3z).
  • the T-cell receptor domain of the CAR of the invention comprises, consists essentially of, or consists of a O ⁇ 3z signaling domain or a related T-cell receptor domain derived from a T cell receptor or functional fragment or variant thereof having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity thereto.
  • the IT AM 3 motif in the O ⁇ 3z domain may be mutated to the PD-1 ITIM.
  • T-cell receptors include, but are not limited to, ITAM containing primary cytoplasmic signaling sequences such as TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79B, and/or CD66d.
  • ITAM containing primary cytoplasmic signaling sequences such as TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79B, and/or CD66d.
  • the transmembrane domain is essential for the stability of the CAR as a whole.
  • the transmembrane domain may be a hydrophobic alpha helix that spans across the membrane of the cell (e.g., T-cell).
  • the transmembrane domain can be 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.
  • 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.
  • the antigen binding domain may be chosen to recognize a ligand (i.e., antigen) that acts as a cell surface marker on a target cell associated with a particular disease state.
  • the antigen binding domain comprises a monovalent antibody fragment.
  • the monovalent antibody fragment comprises a single chain variable fragment (scFv) or a Fab fragment.
  • 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).
  • the monovalent antibody fragment has a VH and VL domain connected in either orientation by a flexible linker (e.g., VL- linker-VH or VH-linker-VL).
  • a flexible linker e.g., VL- linker-VH or VH-linker-VL
  • the orientation is VL-linker-VH with the light chain variable region being at the N-terminal end and the heavy chain variable region being at the C-terminal end of the polypeptide.
  • the flexible linker typically comprises 10 to about 25 amino acids, e.g., glycine to confer flexibility and/or serines and/or threonines for improved solubility).
  • a (GGGGS)3 linker SEQ ID NO:3 or a variant thereof is used.
  • the disease state targeted by the antigen binding domain of the CAR of the invention may be cancer and/or pathogen infections (e.g., chronic viral or bacterial infections).
  • the antigen binding domain targets an antigen present on the surface of a cancer cell and/or viral particle. Exemplary cancer and/or tumor cell antigens are described in S. A. Rosenberg ⁇ Immunity 10:281 (1991)).
  • cancer and tumor antigens include, but are not limited to: BRCA1 gene product, BRCA2 gene product, gplOO, tyrosinase, GAGE- 1/2, BAGE, RAGE, LAGE, NY-ESO-1, CDK-4, b-catenin, MUM-1, Caspase-8, KIAA0205, HPVE, SART-1, PRAME, p 15, melanoma tumor antigens (Kawakami et al. (1994) Proc. Natl. Acad.
  • telomerases e.g., solid tumors
  • melanoma e.g., adenocarcinoma, thymoma, lymphoma (e.g, non-Hodgkin’s lymphoma, Hodgkin’s 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 cancer, neuroblastoma and any other cancer or malignant condition now known or later identified (see, e.g, Rosenberg (1996) Ann. Rev. Med. 47:481-91).
  • the antigen binding domain targets an antigen present on the surface of a cancer cell.
  • the antigen binding domain targets an antigen present on the surface of a kidney cancer cell.
  • Kidney cancer cells include, but are not limited to, cells from renal cell cancer, transitional cell cancer, Wilms tumor, renal sarcoma and/or metastatic kidney cancer.
  • the antigen binding domain targets an antigen present on the surface of a renal cell cancer (RCC) cell selected from, but not limited to, clear all RCC, papillary RCC, chromophobe RCC, collecting duct RCC, and/or unclassified RCC.
  • RCC renal cell cancer
  • the renal cell cancer cell is selected from, but is not limited to, a Ketr-3 and/or ORSC-2 or ACHN renal cancer cell line.
  • Exemplary kidney cancer antigens include, but are not limited to, any surface protein and/or polypeptide present on the surface of a kidney cancer cell known in the art or identified in the future.
  • the antigen binding domain of this invention targets surface protein carbonic anhydrase IX (CAIX).
  • the antigen binding domain of this invention targeting 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.
  • the antigen binding domain targets an antigen present on the surface of a neuroblastoma cell.
  • the neuroblastoma cell is from a PDX cell line although other neuroblastoma cell lines can also be employed.
  • neuroblastoma antigens include, but are not limited to, any surface protein and/or polypeptide present on the surface of a neuroblastoma cell known in the art or identified in the future.
  • the antigen binding domain of this invention targets surface protein disialoganglioside GD2.
  • the antigen binding domain of this invention targeting the surface protein disialoganglioside GD2 comprises a monovalent antibody fragment comprising the amino acid sequence of an aGD2 or a variant thereof having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity thereto.
  • the antigen binding domain targets an antigen present on the surface of a viral particle.
  • viral particles include, but are not limited to, influenza virus, equine infectious anemia virus, simian immunodeficiency virus (SIV), human
  • immunodeficiency virus lassa fever virus, herpes simplex virus, varicella zoster virus, cytomegalovirus, epstein-barr virus, variola virus, adeno virus, papilloma virus, parvo virus, measles virus, mumps virus, respiratory syncytial virus, para influenza virus, corona virus, rubella virus, rabies virus, human T-cell lymphotropic virus, picoma virus, hepa DNA virus, flavivirus, deltavirus, calicivirus, polio virus, zika virus, west nile virus, SARS, rubella, norovirus, human papillomavirus, malaria, human T-lymphotropic virus, and/or helicobacter pylori.
  • Exemplary viral antigens include, but are not limited to, any surface protein and/or polypeptide present on the surface of the above listed viral particles.
  • 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 El, Norovirus Group- 1 P-Domain, Norovirus Group-2 P-Domain, HPV 11, HPV 16, HPV 18, HPV 6, HPV16 E6, Malaria Pf. MSP1, Malaria Pv.
  • the viral particle is an HIV particle.
  • the antigen binding domain is an HIV binding domain.
  • the antigen binding domain of this invention targeting the surface envelope protein ENV of the 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.
  • the soluble CD4 protein is a human soluble CD4 protein comprising, consisting essentially of, or consisting of the amino acid sequence: MNRGVPFRHL LLVLQLALLP AATQGKKVVL
  • the antigen binding domain and the transmembrane domain are connected with a spacer.
  • the spacer is a short spacer which comprises less than 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 amino acids.
  • the spacer can include at least a portion of a Fc region, for example, a hinge portion of a human Fc region of a CH3 domain or variants thereof.
  • the spacer includes all or part of an immunoglobulin (e.g., IgGl, IgG2, IgG3, IgG4) hinge region i.e., the sequence that falls between the CHI and CH2 domains of an immunoglobulin, e.g., IgG4 Fc hinge or a CD8 hinge region.
  • an immunoglobulin e.g., IgGl, IgG2, IgG3, IgG4
  • IgG4 Fc hinge e.g., IgG4 Fc hinge or a CD8 hinge region.
  • Examples include, but are not limited to, CD8 hinge, CD28 hinge IgG4 (HL-CH3), or IgG4 (L235E, N297Q).
  • the spacer comprises, consists essentially of, or consists of a CD8 hinge region having an amino acid sequence of: AGEQKLISEE DLGALSNSIM YFSHFVPVFL
  • PAKPTTTPAP RPPTPAPTIA SQPLSLRPEA SRPAAGGAVH TRGLD (SEQ ID NO:5).
  • the CAR of the invention can further comprises a detectable moiety as would be known in the art and/or an effector molecule, nonlimiting examples of which include a drug, a toxin, a small molecule, an antibody, and/or an antibody fragment, singly or in any combination.
  • the CAR of the invention comprises an anti-c-myc tag.
  • the CAR of this invention comprises, consists essentially of, or consists of an amino acid sequence of: MNRGVPFRHL LLVLQLALLP AATQGKKVVL GKKGDTVELT CTASQKKSIQ FHWKNSNQIK ILGNQGSFLT KGP SKLNDRA DSRRSLWDQG NFPLIIKNLK IEDSDTYICE VEDQKEEVQL LVFGLTANSD THLLQGQSLTL TLESPPGSSP SVQCRSPRGK NIQGGKTLSV SQLELQDSGT WTCTVLQNQK KVEFKIDIVV LAAGEQKLIS EEDLGALSNS IMYFSHFVPV FLPAKPTTTP APRPPTPAPT IASQPLSLRP EASRPAAGGA VHTRGLDWVW WFLSGSLVIV IVCSTVGLII CVKRRKPRGD VVKVIVSVQR KRQEAEGEAT VIEALQAPPD VTTV
  • the present invention additionally provides a nucleic acid molecule encoding the CAR of this invention.
  • the nucleic acid molecule comprises, consists essentially of, or consists of a nucleotide sequence of:
  • Vectors include, but are not limited to plasmid vectors, phage vectors, virus vectors, or cosmid vectors.
  • T lymphocytes of this invention can be transduced, e.g., with a viral vector under conditions whereby the CAR is produced in the T lymphocyte.
  • the choice of vector will often depend on the host cell into which it is to be introduced.
  • the present invention provides a cell comprising the CAR of this invention and in some embodiments, the present invention provides a cell comprising the nucleic acid molecule and/or vector of this invention.
  • a cell of this invention include an abT cell, a natural killer (NK) cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, a natural killer T (NKT) cell, a Thl7 cell, a gdT cell, and any combination thereof.
  • the present invention provides a cytotoxic T lymphocyte comprising a CAR that recognizes and binds to an antigen present on the surface of a cancer cell and /or viral particle.
  • the cytotoxic T lymphocyte comprising a CAR that recognizes and binds to the surface envelope protein ENV of the human immunodeficiency virus HIV-1.
  • the cytotoxic T lymphocyte comprising a CAR that recognizes and binds to the surface protein of a cancer cell such as a surface protein of a renal cancer cell (i.e., CAIX) and/or a surface protein of a neuroblastoma cell (i.e., aGD2).
  • the cytotoxic T lymphocyte can then be transduced with a viral vector or transfected with a plasmid or nucleic acid construct comprising a nucleotide sequence encoding the CAR of this invention and in some embodiments the nucleotide sequence can be any lentiviral or retroviral vectors used in the clinics for other CAR-T cells.
  • the present invention includes T lymphocytes engineered to comprise a CAR comprising an antigen binding fragment specific for an antigen present on the surface of a cancer cell and/or viral particle (e.g., the surface envelope protein ENV of the human immunodeficiency virus HIV-1, CAIX and/or sGD2), a transmembrane domain (e.g., CD28), a T-cell receptor domain (e.g., CD3z), and a CSS domain comprising a HVEM protein of the invention or a functional fragment or variant thereof having at least 90% identity thereto.
  • a cancer cell and/or viral particle e.g., the surface envelope protein ENV of the human immunodeficiency virus HIV-1, CAIX and/or sGD2
  • a transmembrane domain e.g., CD28
  • T-cell receptor domain e.g., CD3z
  • CSS domain comprising a HVEM protein of the invention or a functional fragment or variant thereof having at least 90% identity thereto.
  • the monoclonal antibody fragment for the antigen e.g., the surface envelope protein ENV of the human immunodeficiency virus HIV-1, CAIX and/or sGD2
  • scFv single-chain variable fragment
  • the present invention provides cells specific for the antigen present on the surface of the cancer cell and /or viral particle (e.g., surface envelope protein ENV of the human immunodeficiency virus HIV-1, CAIX, aGD2), wherein said cells have a CAR on the cell surface that is produced by joining an extracellular antigen-binding domain derived from, for example, a CD4 protein, CAIX, aGD2 and/or fragment thereof to a T-cell receptor domain derived from the T-cell receptor zeta-chain, and a CSS domain comprising a HVEM protein or a functional fragment or variant thereof having at least 90% identity thereto.
  • a CD4 protein, CAIX, aGD2 and/or fragment thereof to a T-cell receptor domain derived from the T-cell receptor zeta-chain
  • a CSS domain comprising a HVEM protein or a functional fragment or variant thereof having at least 90% identity thereto.
  • a method for promoting responsiveness of a cell to an antigen comprising transfecting a cell with the nucleic acid molecule of the invention and/or the vector of the invention to produce a transfected cell that comprises an antigen binding domain on the cell surface, wherein the antigen binding domain specifically binds to the antigen, thereby promoting the responsiveness of the cell to the antigen.
  • “Responsiveness,” as used herein, refers to the ability of a cell to promote an immune response upon binding of an antigen.
  • Cells modified with CARs of the invention may promote an increased immune response (e.g., a stronger, faster, and/or more effective immune response) compared to cells with no CARs of the invention.
  • the antigen is present on the surface of a cancer cell and/or viral particle.
  • the antigen is present on a viral particle, e.g., an HIV particle.
  • the antigen is present on a cancer cell, e.g., a solid tumor (e.g., kidney cancer).
  • the cell is a cytotoxic T lymphocyte.
  • the present invention provides a composition (e.g., a pharmaceutical composition) comprising, consisting essentially of, or consisting of the CAR of this invention, the nucleic acid molecule of this invention, the vector of this invention and/or the cell of this invention, in a pharmaceutically acceptable carrier.
  • a composition e.g., a pharmaceutical composition
  • a pharmaceutical composition comprising, consisting essentially of, or consisting of the CAR of this invention, the nucleic acid molecule of this invention, the vector of this invention and/or the cell of this invention, in a pharmaceutically acceptable carrier.
  • the present invention provides methods of providing an immune response against a target e.g., cancer cell and/or infectious agent) in a subject in need thereof.
  • 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.
  • 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 the infection in the subject.
  • the target is a 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.
  • the cancer comprises a solid tumor.
  • the method comprises administering engineered T-cells (i.e., T lymphocytes) comprising the CAR of the invention.
  • engineered T-cells i.e., T lymphocytes
  • Methods for preparing engineered T lymphocytes comprising the CAR of the invention are well known to a skilled artisan.
  • cytotoxic lymphocytes i.e., T-cells
  • a subject having a compromised immunity against a target e.g., a cancer cell and/or infectious agent
  • a target e.g., a cancer cell and/or infectious agent
  • the subject has cancer. In some embodiments, the subject has an infection. In some embodiments, cytotoxic lymphocytes (i.e., T-cells) are isolated from peripheral blood using techniques well known in the art (e.g., Ficoll density gradient centrifugation followed by negative selection to remove undesired cells).
  • Cytotoxic lymphocytes can be engineered to express the CAR of the invention by transfecting a population of lymphocytes with an expression vector and/or nucleic acid molecule of the invention encoding the CAR of the invention.
  • Appropriate means for preparing a transfected population of lymphocytes expressing a CAR of the invention will be well known to the skilled artisan and include, but are not limited to, retrovirus, lentivirus (viral mediated CAR gene delivery system), sleeping beauty, and/or piggyback (transposon/transposase systems that include a non-viral mediated CAR gene delivery system).
  • the transfected lymphocytes are cultured in conditions that are suitable for a population of cells that will be introduced into a subject (e.g., a human). Specific considerations include the use of culture media that lacks any animal products, such as bovine serum. Other considerations include sterilized-conditions to avoid contamination of bacteria, fungi, and mycoplasma. In some embodiments, prior to being administered to a subject, the cultured transfected lymphocytes may be pelleted, washed, and re-suspended in a pharmaceutically acceptable carrier or diluent.
  • Administration of the transfected lymphocytes to the subject may provide or enhance an immune response against a target (e.g., cancer cell and/or infectious agent).
  • administration of the transfected lymphocytes to the subject may treat cancer and/or a pathogen infection in the subject.
  • the subject is a human.
  • the pathogen infection to be treated is a chronic viral infection, e.g., HIV.
  • the target to be treated is cancer.
  • the cancer to be treated comprises a solid tumor.
  • the cancer to be treated is kidney cancer.
  • Exemplary kidney cancers include, but are not limited to, renal cell cancer, transitional cell cancer, Wilms tumor, renal sarcoma and/or metastatic kidney cancer.
  • the kidney cancer is renal cell cancer (RCC).
  • renal cell cancer includes, but is not limited to, clear all RCC, papillary RCC, chromophobe RCC, collecting duct RCC, and/or unclassified RCC.
  • a method of the invention comprises administering to a subject in need thereof an effective amount of a 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., kidney cancer).
  • cancer i.e., kidney cancer
  • treatment results in a reduction in tumor size.
  • the tumor size/volume is reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% relative to untreated tumors.
  • treatment results in an increased survival rate of the subject.
  • the survival rate increases 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% relative to untreated subjects.
  • a method of the invention comprises administering to a subject in need thereof an effective amount of a 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.
  • 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 relative to untreated subjects.
  • CAR-modified T cells of the present invention may be administered either alone, or as a pharmaceutical composition in combination with diluents and/or with other components.
  • compositions of the present invention may comprise a cell population as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
  • Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline, sterile saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, 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, singly or in any combination.
  • buffers such as neutral buffered saline, phosphate buffered saline, sterile saline and the like
  • carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol
  • proteins polypeptides or amino acids
  • antioxidants antioxidants
  • chelating agents such as
  • compositions of the present invention can be administered in a manner appropriate to the disease to be treated and/or prevented.
  • the quantity and frequency of administration will be determined by such factors as the condition of the subject, as well as the type and severity of the subject's disease, although in some embodiments, appropriate dosages may be determined by clinical trials.
  • compositions of the present invention can be administered by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject).
  • a pharmaceutical composition comprising cells of this invention can be administered at a dosage of about 10 3 to about 10 10 cells/kg body weight, and in some embodiments, the dosage can be from about 10 5 to about 10 8 cells/kg body weight or from about 10 6 to about 10 8 including all integer values (e.g., 10 4 , 10 5 , 10 6 , 10 7 ,10 8 , 10 9 ) within those ranges.
  • the cell compositions of this invention can be administered multiple times (e.g., hourly, four times daily, three times daily, two times daily, daily, twice weekly, three times weekly, weekly, monthly, bi-monthly, semi-annually, annually, etc.) at these dosages.
  • the cells of this invention can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al. New Eng. ./. of Med. 319: 1676 (1988)).
  • the optimal dosage and treatment regimen for a particular subject can readily be determined by one skilled in the art of medicine by monitoring the subject for signs of disease and adjusting the treatment accordingly.
  • activated T cells may be desirable to administer activated T cells to a subject and then subsequently redraw blood (or have an apheresis performed), activate T cells therefrom as described herein, and reinfuse the subject with these activated and expanded T cells.
  • This process can be carried out multiple times, e.g., weekly or every few weeks.
  • T cells can be activated from blood draws of from about lOcc to about 400cc. In certain embodiments, T cells are activated from blood draws of 20cc, 30cc, 40cc, 50cc, 60cc, 70cc, 80cc, 90cc, or lOOcc. Not to be bound by theory, using this multiple blood draw/multiple reinfusion protocol may serve to select out certain populations of T cells.
  • compositions of this invention can be carried out in any manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation and/or
  • compositions of this invention can be administered to a patient
  • the T cell compositions of the present invention can be administered to a subject by intradermal or subcutaneous injection.
  • the T cell compositions of the present invention can be administered by i.v. injection.
  • the compositions of T cells can be injected directly into a tumor, lymph node and/or site of infection.
  • cells activated and expanded using the methods described herein, or other methods known in the art where T cells are expanded to therapeutic levels can be administered to a subject in conjunction with (e.g., before, concurrently and/or following) any number of relevant treatment modalities.
  • the CARs and/or nucleic acid molecules, and/or engineered T-cells of the invention may be used in combination with other treatment options (e.g., drugs and/or surgery) depending on the disease being treated.
  • EXAMPLE 1 Co-stimulatory signal determines CAR-T cell activity in human T cell line.
  • the human sCD4 molecule corresponding to the extracellular domain of CD4 was reported to selectively target HIV-infected cells through binding of HIV Env gpl20 (Chaudhary el ah, Nature 335(6188):369 (1988)).
  • HIV Env-targeting CAR lentiviral vector were made expressing sCD4-CAR in combination with different CSS (FIG.
  • target cells expressing HIV Env were co-cultured with target cells at 1 :2 ratio for 24 hours.
  • the cell containing supernatant was harvested and cleared by centrifugation for IL-2 ELISA.
  • the remaining cells were used for flow cytometric analysis to determine CD69 expression upon antigen-dependent stimulation.
  • Target cells CD3 GFP +
  • effector cells CD3 +
  • Both GFP and GFP + effector cell activation was determined by CD69 upregulation (FIG. 7C, middle and right panel).
  • EXAMPLE 2 HVEM co-stimulation exhibits the highest CAR expression in human CAR- T cells.
  • CAR-T cells were developed harboring different CSS using primary human CD8 + T cells.
  • the CAR-transduced CD8 + T cells were sorted based on GFP expression and used for the analysis of effector functions and characteristics.
  • the expression levels of CAR on cell surfaces and in whole cell lysate were different in CAR-T cells harboring different CSS (FIGS. 2A-2C), which was a similar trend to what was observed in CAR-transduced T cell lines (FIGS. IB and ID).
  • EXAMPLE 3 HVEM co-stimulation exhibits the highest effector functions in human CAR-T cells.
  • cytotoxic activity and cytokine secretion were measured upon co-cultivation with target cells.
  • T cells exhibited antigen-specific cytotoxic activity against HIV Env-expressing target cells.
  • cytotoxic activity was dependent on the CSS and the CAR-T cells harboring HVEM co-stimulation exhibited the highest cytotoxic activity among CAR-T cells tested in this study (FIG. 3C).
  • cytokine secretion such as IL-2, TNF-a and IFN-g (FIG. 3B).
  • EXAMPLE 4 TNFRSF co-stimulation averts CAR-T cell exhaustion.
  • T CM CD45RO + CCR7 +
  • T EM CD45RO + CCR7
  • FIG. 5C The most strikingly affected memory populations were central (T CM : CD45RO + CCR7 + ) and effector (T EM : CD45RO + CCR7 ) memory populations.
  • CAR-T cells harboring 4-1BB CSS included significantly enhanced T CM population whereas CD28 CSS induced less T CM population (FIG. 5D).
  • CAR-T cell harboring HVEM CSS included equivalent proportions of T CM and T EM population (FIG. 5D).
  • EXAMPLE 6 HVEM co-stimulation reprograms CAR-T cell energy metabolism to more energetically active state.
  • EXAMPLE 7 Discussion of results from EXAMPLES 1-6.
  • Anti -HIV- 1 CAR was developed using sCD4 as an antigen recognition domain with different CSS in the signaling domain. Using this system as a model, it was shown that the CSS in the CAR determines CAR-T cell activity in both a human T cell line and human primary CD8 + T cell-derived CAR-T cells. Also uncovered was the interplay among effector functions, development of memory subsets, T cell exhaustion and energy metabolism, suggesting that the CSS in the CAR significantly affects CAR-T cell functions and characteristics. Especially, HVEM co-stimulation enhanced effector functions of CAR-T cells through reprogramming energy metabolism. These studies shed light on the insight that the choice of CSS can impact the fate of CAR-T cell functions and characteristics and HVEM could be a promising candidate for generating effective CAR-T cells against not only solid tumors but also persistent infectious diseases.
  • CD28 co-stimulation induces the PI3K-Akt pathway whereas 4-1BB co-stimulation is primarily mediated by TNFR-associated factors (TRAFs) which activate c-Jun N-terminal kinase (INK) and p38 (Kim et al, Mol Cells 10(3):247 (2000); Cannons et al., J. Immunol. 165(11):6193 (2000)).
  • TNFRSF members including 4- 1BB and HVEM are each capable of activating the alternative NF-KB pathway (Hauer et al, Proc. Natl. Acad. Sci. USA 102(8):2874 (2005)). These changes in signaling pathways cause transcriptional and translational changes (Mehta et al., Nat. Rev. Immunol. 17(10):608 (2017)), leading to the difference in T cell activation status and CAR expression levels.
  • 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 while averted by 4- IBB co-stimulation
  • CD28 co-stimulation exhibited low energy metabolism with increased effector memory subset whereas 4-1BB co-stimulation induced higher mitochondrial respiration associated with increased central memory subset.
  • HVEM co-stimulation exhibit enhanced mitochondrial respiration and glycolysis associated with development of similar frequencies of central and effector memory subsets.
  • TNFRSF co-stimulations including 4-1BB and HVEM which have been tested in this study recruit different TRAFs and might activate the NF-KB pathway with different magnitude, leading to differential energy metabolism levels, memory subsets development and effector functions.
  • CAR expression level was also associated with energy status (FIGS. 2A-2C and 6A-6E), suggesting that different signaling pathways might impact the level of energy metabolism to meet metabolic demand.
  • HIV-1 infection can be controlled by anti -retroviral therapy (ART) but not eradicated because ART cannot eliminate latently-infected cells, indicating that HIV-infected patients should receive effective ART entire their life.
  • ART anti -retroviral therapy
  • Recent attempts of eradication focused on the killing of latently-infected cells which persist during ART by immunological mechanisms.
  • a HIV-specific immunotoxin or HIV-specific CAR-T cells have successfully been developed to kill HIV-infected cells (Denton et al, PLoS Pathog. 10(l):el003872 (2014); Sahu et al, Virology 446(l-2):268 (2013); Liu et al., J. Virol.
  • bNAbs Broadly neutralizing antibodies
  • CAR-T cells harboring 4-1BB co-stimulation have been shown to be more potent than CAR-T cells harboring CD28 co-stimulation in vitro and in vivo using an HIV-treatment model (Leibman et al, PLoS Pathog. 13(10):e 1006613 (2017)). It is shown here that CAR-T cells harboring HVEM co-stimulation are more potent than CAR-T cells harboring CD28 or 4- IBB co-stimulation, suggesting that CAR-T cells harboring HVEM co-stimulation might be more beneficial in controlling HIV replication. [0130] The present data showed that CSS in the CAR determines effector functions and characteristics of CAR-T cells.
  • HVEM co-stimulation induces enhanced effector functions associated with superior characteristics compared to the commonly used CSS in CAR design such as CD28 and 4-1BB. This has suggested that HVEM might be a promising candidate for development of CAR-T cells with superior function and characteristics. We might find more potent CSS by testing a panel of CSS in the context of CAR, extending the design of CAR for future CAR-T cell therapy.
  • PBMCs Peripheral blood mononuclear cells
  • M10, D10 and R10 were supplemented with 10% Fetal Bovine Serum (FBS) (Thermo Fisher Scientific), 2 mM Glutamine (Thermo Fisher Scientific), 10 U/mL penicillin and 10 pg/mL streptomycin (Thermo Fisher Scientific), named shortly M10, D10 and R10, respectively.
  • FBS Fetal Bovine Serum
  • 2 mM Glutamine Thermo Fisher Scientific
  • 10 U/mL penicillin and 10 pg/mL streptomycin Thermo Fisher Scientific
  • PBMCs were cultured in AIM-V (Thermo Fisher Scientific) supplemented with 5% FBS, 10 mM HEPES (complete AIM-V) overnight to remove plastic adherent monocytes.
  • AIM-V Thermo Fisher Scientific
  • Monocyte-depleted PBMCs were used in transduction experiments. Cells were grown at 37°C and 5% CO2.
  • MEM methicillin
  • DMEM fetal calf serum
  • RPMI fetal calf serum
  • CHO cells and their transfectants were maintained in M10 supplemented with non-essential amino acid (M10- NEAA).
  • 293FT cells were cultured in D10, and Jurkat E6.1 cells obtained from European Collection of Authenticated Cell Cultures through DS Pharma were maintained in R10.
  • PBMCs Human peripheral blood mononuclear cells from healthy donors were prepared by Ficoll- Paque (GE Healthcare Life Sciences, Pittsburgh, PA) density gradient and cultured in AIM-V (Thermo Fisher Scientific) supplemented with 5% FBS, 10 mM HEPES (shortly complete AIM- V) overnight to remove plastic adherent monocytes. Monocyte-depleted PBMCs were used to transduction experiments. All cells were grown at 37°C with 5% CO2.
  • the DNA linker was introduced into lentiviral vector plasmid pTK643-CMV-IRES-GFP/blastcidine(BSD).
  • the DNA linker containing XhcA-Xho I -As/ W I -Bs/B ⁇ -Bam H I restriction enzyme sites was made by incubating two oligo DNAs LI and L2 at molar ratio of 1 : 1 at 70°C for 10 mins and left at room temperature for 2 hours.
  • the linker DNA was inserted into XbaVBamHI digested pTK643-CMV-IRES- GFP/B SD (pTK643 -CMV-MC S-IRES-GFP/B SD).
  • sCD4 DNA fragment (1-148 amino acids of human CD4) DNA fragment
  • total RNA extracted from Jurkat E6.1 cells by ISOGEN Nippon Gene, Tokyo, Japan
  • ReverTra Ace TOYOBO, Osaka, Japan
  • sCD4 DNA fragment containing XbaVEcoKl site was amplified using KOD-FX (TOYOBO).
  • Purified DNA fragment was incubated with Ampli Taq (Thermo Fisher Scientific) at 72°C for 10 minutes to add A tails.
  • Ampli Taq Thermo Fisher Scientific
  • a tailed DNA fragment was ligated into pGEM-T easy vector (Promega, WI). The sequence was verified with BigDye Terminator v3.1 Cycle
  • Recombinant lentivirus was produced as previously described (Cockrell et al, Mol Ther. 14(2):276 (2006)) with some modifications. Briefly, 293FT cells were cultured on collagen coated 10 cm dish (IWAKI, Shizuoka, Japan) with 80 to 90% confluency. The culture medium was replaced with D10 containing 25 mM Chloroquine (SIGMA, Darmstadt, Germany) without antibiotics. Regarding vectors packed with ANRF, the following plasmid amounts were used: 15 pg lentiviral vector plasmid, 10 pg ANRF, and 5 pg 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 mM forskolin (Tokyo Chemical Industry, Tokyo, Japan).
  • the culture supernatants containing recombinant lentiviruses were harvested 48 hours after transfection, cleared by centrifugation and 0.45 pm filtration (Millipore, Darmstadt, Germany).
  • the recombinant lentiviruses were concentrated by high-speed centrifugation at 18000 rpm for 3 hours using Himac CR21N (Hitachi Koki, Tokyo, JAPAN).
  • the lentivirus containing supernatant was used to transduce CAR/GFP genes into Jurkat E6.1 cells and HIV Env gene into CHO cells, respectively. Briefly, 2 million Jurkat E6.1 cells or semi-confluent CHO cells in 6 well plates were exposed to 1 mL of un-concentrated lentivirus containing supernatant in the presence of polybrane at 8 pg/mL. The cells were centrifuged at 5500 rpm for 3 hrs at 22°C to enhance viral infection. After removal of the supernatant, the cells were cultured at 37°C in a CO2 incubator for 48 hours. The culture medium was replaced with media supplemented with 10 pg/mL BSD. Thereafter, transduced cells were maintained in media with 10 pg/mL BSD until following assays were performed.
  • Human primary CD8 T cells were isolated from monocyte-depleted PBMCs with anti human CD3-APC, CD4-PE and CD8PE/Cy7 antibody (Biolegend, San Diego, CA) by cell sorting on FACS Aria II (Becton Dickinson, Franklin Lakes, NJ), routinely achieving more than 95% purity.
  • the purified CD8 T cells were activated with anti-CD3/CD28 beads (Thermo Fisher Scientific) in a 3: 1 beadxell ratio with complete AIM-V supplemented with 40 U/mL recombinant human IL-2 (obtained through NIH AIDS Reagent Program, Division of AIDS, NIAID, from Dr.
  • Antibodies used in flow cytometry were obtained from Biolegend unless otherwise indicated.
  • CAR expression on transduced Jurkat E6.1 cells was analyzed with 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 the co-culture assay was analyzed with anti -human CD3-APC antibody, CD69-PE antibody and GFP (See FIGS. 7A and 7B for gating strategy).
  • CAR expression and T cell exhaustion of human CAR-T cells were analyzed with biotinylated anti-c-myc tag antibody (Biolegend) followed by streptavidin-PE (TONBO biosciences), anti-human PD-l-APC antibody, anti-human LAG-3-PE/Cy7 antibody (eBioscience) and GFP.
  • Memory phenotype of CAR-T cells was analyzed with CD45RO-PE, CD8-PE-Cy7, CCR7-APC and GFP.
  • the centrifuged cells were resuspended with antibody solution in FACS Buffer (PBS containing 2% FBS and 0.02% Sodium Azide) and incubated on ice for 30 min. After washing with ice-cold FACS Buffer, cells were fixed with 1%
  • Activation and IL-2 secretion of CAR-transduced Jurkat E6.1 cells were determined by co-culturing with target cells (CHO-GFP or CHO-Env-GFP). 0.1 million target cells were seeded in each well of 96 well flat-bottom plates. 0.2 million CAR-T cells were added and co cultured overnight. The next day, the culture supernatant including cells was harvested. The cells and cell-free supernatant were separated by centrifugation at 3,000 rpm for 5 min at 4°C. IL-2 secretion into the supernatant was measured using Human IL-2 ELISA MAX Deluxe (Biolegend) according to the manufacturer’s instruction. The remaining cells were used to determine CD69 expression by flow cytometry.
  • CAR-T cells 10 thousand target cells were seeded in each well of 96 well flat-bottom plates. CAR-T cells with different target: effector ratio were added and co-cultured in 0.2 mL per well of Phenol Red-free R10 with NEAA. After overnight incubation, the supernatant was collected and centrifuged at 3000 rpm for 10 min at 4°C to remove cellular debris. Lactate dehydrogenase release in the supernatant was assayed using CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI) according to the manufacturer’s instruction.
  • Mitochondrial function of the CAR-T cells was analyzed with an extracellular flux analyzer XFp (Agilent Technologies). Each well of the cell culture microplate was coated with CellTak (Coming) in accordance with the manufacturer’s instructions.
  • XFp Extracellular flux analyzer
  • sorted CAR-T cells harboring 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 three hundred thousand per well. The plate was centrifuged at 200 x g for 1 min and incubated at 37°C in a non-CCL incubator for 30-60 min.
  • Oxygen consumption rates were measured under basal conditions and following treatment with 1 mM oligomycin, 1 mM FCCP and 1 mM rotenone/antimycin A (XFp Cell Mito Stress Kit, Agilent technologies). Four measurements at each condition were performed. ATP production was defined as (last rate measurement before oligomycin addition) - (minimum rate measurement after oligomycin addition).
  • EXAMPLE 9 Chimeric antigen receptor T cell bearing herpes virus entry mediator costimulatory signal domain exhibits functional potency.
  • Chimeric antigen receptor comprises an extracellular antigen-binding domain combined with an intracellular signal transduction domain (Dotti et al, Immunol. Rev. 257: 107 (2014)).
  • the first generation CAR-transduced T (CAR-T) cells that have OI)3z as a signal transduction domain of the CAR often become anergic and fail to elicit a potent immune response (Kershaw et al, Clin. Cancer Res. 12:6106 (2006)).
  • the second and third generation CAR-T cells have been developed by adding one and two co-stimulatory signal domains (CSSDs) in the CAR, respectively (Dotti et al, Immunol. Rev. 257: 107 (2014)).
  • CSSDs co-stimulatory signal domains
  • Co-stimulatory molecules can be classified to two major families; the CD28 family including CD28 and ICOS, and the TNF receptor superfamily (TNFRSF) including 4- IBB and herpes virus entry mediator (HVEM). So far, the CSSD derived from CD28 or 4-1BB has commonly been used to construct CAR (Miller et al, Oncol. Res. Treat. 38:683 (2015)). A previous study has shown that the second generation CAR-T cells with 4-lBB-derived CSSD persist for more than 6 months in the blood of most patients, whereas the CAR-T cells with CD28-derived CSSD become mostly undetectable after 3 months (Zhang et al, Oncotarget 6:33961 (2015)).
  • 4-1BB co- stimulation induced enhanced differentiation to central memory subset with increased in vitro persistence (Kawalekar et al, Immunity 44:380 (2016)). 4-1BB co-stimulation has also been shown to increase mitochondrial biogenesis and oxidative metabolism for energy production and avert tonic signaling-induced T cell exhaustion (Long et al, Nat. Med. 21 :581 (2015)). Therefore, the CSSD derived from TNFRSF appears to function better than the one from CD28 family in the context of second generation CAR-T cells.
  • HVEM another member of TNFRSF
  • HVEM deficiency in CD8 + T cells has been shown to profoundly impair effector CD8 + T cell survival and development of protective immunological memory (Flynn et al, PLoS One 8:e77991 (2013)).
  • a interaction between HVEM expressed on CD8 + T cells and B- and T-lymphocyte attenuator has also been reported to promote survival and immunological memory generation in response to bacterial infection (Steinberg et al, PLoS One 8:e77992 (2013)).
  • HVEM serves as a potent co-stimulatory molecule in T cells, suggesting that the CSSD derived from HVEM may also be useful in the context of CAR-T cells.
  • EXAMPLE 10 CAR with HVEM-derived CSSD is efficiently expressed in a human T cell line.
  • a sCD4 corresponding 1-148 amino acids of human CD4 was reported to selectively target HIV-infected cells through binding to an HIV Env (Chaudhary et al, Nature 335:369 (1988)).
  • lentiviral vectors were constructed expressing the CAR in combination with CSSD derived from CD28, 4-1BB or HVEM (FIG.
  • EXAMPLE 11 Human T cell line expressing CAR with HVEM-derived CSSD is efficiently activated upon cognate antigen stimulation
  • CD3 target cells CHO-GFP or CHO-Env-GFP
  • CD3 + Jurkat E6.1 cells which had been transduced with CAR-expressing lentiviral vectors
  • FIG. 7C left panel
  • the GFP and successfully transduced GFP + Jurkat E6.1 cells were examined for CD69 upregulation, an indicator of T cell activation (FIG. 7C, right panel).
  • FIG. IE no significant activation was observed in the GFP or GFP + Jurkat E6.1 cells upon co-cultivation with control CHO-GFP cells.
  • the GFP + but not GFP Jurkat E6.1 cells were efficiently activated when CHO-Env-GFP cells were used as target cells (FIG. IE). Also, measurement of IL-2 in the culture supernatants showed that IL-2 secretion by the CAR-transduced Jurkat E6.1 cells was induced upon co-cultivation with CHO-Env-GFP cells but not with control CHO-GFP cells (FIG. IE). These data demonstrated that human T cells transduced with the CAR-expressing vectors constructed in this study were specifically activated upon cognate antigen stimulation. Linear regression analysis demonstrated that the percentage of activated T cell upon cognate antigen stimulation and IL-2 secretion were correlated with the levels of CAR expression on cell surface (FIGs.
  • EXAMPLE 12 Human primary T cell-derived CAR-T cells with HVEM-derived CSSD exhibit potent effector functions
  • EXAMPLE 13 CAR-T cells with HVEM-derived CSSD efficiently differentiate to both central and effector memory subsets
  • CD45RO + CCR7 CD45RO + CCR7 T cells
  • the CAR-T cells with HVEM-derived CSSD contained equivalent percentages of T CM and T EM subsets (FIG. 10B), suggesting that the HVEM-derived CSSD efficiently induced both T EM and T CM subsets.
  • EXAMPLE 14 CAR-T cell with HVEM-derived CSSD averts T cell exhaustion
  • Prolonged stimulation of T cells has been known to cause exhaustion characterized by decreased proliferation, lowered levels of cytokine production, high rates of apoptosis 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)).
  • PD-1 programmed cell death 1
  • LAG-3 lymphocyte activation gene 3
  • EXAMPLE 15 CAR-T cell with HVEM-derived CSSD exhibits reprogrammed energy metabolism
  • Mitochondrial respiration and glycolysis can be measured by oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), respectively.
  • OCR oxygen consumption rate
  • ECAR extracellular acidification rate
  • the CAR-T cells with HVEM-derived CSSD showed the highest level of basal OCR, followed by those with 4-lBB-derived CSSD and the control T cells, and those with CD28-derived CSSD showed the lowest level of basal OCR (FIGS. 6B-6E).
  • ATP-linked respiration was also measured by adding an ATP synthase inhibitor oligomycin and maximal OCR levels by adding carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP; uncoupling of oxygen consumption from ATP production) (FIG. 6A).
  • Lentiviral vectors were developed expressing the CAR consisting of sCD4 as an antigen recognition domain combined with CSSD derived from CD28, 4-1BB or HVEM. Transducing human T cell line and primary T cells with these vectors, it was demonstrated that the CSSD in CAR construct was a crucial determinant for the levels of CAR expression on the cell surface, which appeared to be correlated with the activities and effector functions of the CAR-T cells. It was also shown in this study that T cell exhaustion, energy metabolism and induction of the memory T cell subsets were also affected by the CSSD in CAR constructs.
  • the HVEM-derived CSSD led to the highest level of CAR expression, most potent effector functions of CAR-T cells, evasion of exhaustion and balanced induction of both central and effector memory T cell subsets, associated with elevated 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 a certain context.
  • CAR synthesis rather than trafficking might be affected by CSSD.
  • different CSSDs might activate different signaling pathways, resulting in distinct levels of gene expression.
  • CD28-mediated co-stimulation induces PI3K- Akt pathway
  • 4-lBB-mediated co-stimulation primarily activates JNK and p38 through TNFR-associated factors (Kim et al, Mol. Cells 10: 247 (2000); Cannons et al, ./. Immunol. 165: 6193 (2000)).
  • co-stimulation signals impacted differentiation of the CAR-T cells to central and effector memory T cell subsets, and that the HVEM-derived CSSD induced efficient and balanced differentiation.
  • Different co-stimulatory signals is likely to affect metabolic programs in distinct manners to differentiate specific memory T cell subsets.
  • CD4-based CAR-T cells with HVEM-derived CSSD which showed more potent effector functions than those with CD28- or 4-lBB-derived CSSD, may also be a useful tool for the“shock and kill” treatment of HIV infection.
  • the present results demonstrate that the CSSD in CAR is a crucial determinant for effector functions and characteristics of CAR-T cells, indicating that the CSSD in CAR is important for designing more potent CAR-T cells.
  • the HVEM-derived CAR-T cells may be a promising candidate for generating effective CAR-T cells.
  • FIG. 11 A FACS detection of c-myc+ human CAR-T cells transduced by the four constructs in shown in FIG. 11B.
  • Mean surface c-myc expression levels (MFI) is shown in FIG. 11C.
  • CAIX CAR-T cells Three target kidney/renal cancer cell lines were used to examine the effectiveness of the CAIX CAR-T cells.
  • ACHN is a negative control with no CAIX expression.
  • Ketr-3 expresses high and OSRC-2 expresses low CAIX as detected by FACS (FIG 12A).
  • CAIX-HVEV CAR-T cells killed CAIX+ target cells more effectively that other CAR-T cells (FIGS 12B and 12C).
  • CAIX-HVEV CAR-T cells also produced more IL-2 and IFN-g in response to co-culture with CAIX+ target cells than other CAR-T cells (Ketr-3 (FIG. 12D) and OSCR-2 (FIG. 12E)).
  • FIG. 13 A shows the oxygen consumption rate (OCR) of various CAR-T cells by Seahorse assay.
  • OCR oxygen consumption rate
  • FIG. 14A A renal cancer treatment study using the CAIX CAR-T cells of Example 16 was carried out in NPG mice.
  • the study design is shown in FIG. 14A.
  • Mice were injected intravenously with 1 x 10 6 OSCR-2 human renal cancer cells on day 1.
  • 1 x 10 7 CAR-T cells were injected intravenously on day 7.
  • FIG. 14B shows the level of human CAR-T cells in peripheral blood of the mice on day 14.
  • FIG. 14C The overall survival of the mice is shown in FIG. 14C.
  • HVEM-based CAIX CAR-T cells provided significantly better survival than the other CAR-T cells.
  • Lung metastatic tumors in 6 mice/group were examined at the indicated time points as shown in FIG. 16.
  • mice in the HVEM CAR-T group were terminated 90 days post tumor injection while mice in the other groups were terminated 50 days post tumor injection.
  • the white patches indicate metastatic tumors on the lung.
  • HVEM-based CAIX CAR-T cells provided significantly recued tumor metastases than the other CAR-T cells.
  • FIG. 17 shows representative hematoxylin/eosin lung histopathology of 1 mouse/group at termination. Only the HVEM CAR-T group showed functional lung structure while all other groups showed heavily infiltrated lungs with loss of lung structure and function.
  • FIG. 15A shows the percentage of CAR-T (myc+) cells after in vitro transduction.
  • FIG. 15B shows the level of human CAR-T cells in mouse blood on day 7 after CAR-T cell transfer.
  • FIG. 15C shows summarized data of human T cells as a percentage of total mouse blood cells (left) or human T cell count/100 m ⁇ of mouse blood. The HVEM CAR-T cells showed better expansion in vivo.

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