WO2024036167A2 - Methods for enhancing the anti-tumor activity of car t cells by co-expression of ch25h - Google Patents
Methods for enhancing the anti-tumor activity of car t cells by co-expression of ch25h Download PDFInfo
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- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
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- C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
Definitions
- Trogocytosis is an evolutionarily conserved process of extraction and subsequent transfer of fragments of cell membrane along with associated proteins and other biomolecules between cells that are in close contact with each other. As a result of this transfer, a gain or loss of function for either acceptor or donor cells (or both) may occur.
- the functional consequences of trogocytosis in T cells depend on the context and nature of partner cells.
- priming T-cell receptor- dependent trogocytosis results in T cells acquiring membrane fragments along with the MHC class I and class II proteins and co-stimulatory and other molecules from the antigen-presenting cells.
- the effector trogocytosis between activated CD8+ T cells (cytotoxic T lymphocytes, CTLs) and target cells expressing specific antigen leads to a transfer of MHC-antigen complexes onto CTLs.
- effector trogocytosis between target malignant cells and therapeutic CD8+ T cells expressing a chimeric antigen receptor (CAR) is implicated in decreased clinical benefits of 41744090.2 1 Attorney Docket No.046483-7386WO1(03225) CTLs.
- Trogocytosis is associated with decreased viability and activity of chimeric antigen- expressing recipient CTLs due to their exhaustion as well as killing by other CAR CTLs in a process called fratricide. Additionally, the loss of antigen on surviving donor malignant cells masks them from subsequent attack by CAR T cells. These intricate and robust mechanisms help malignant cells to withstand CAR T cell adoptive therapies. Tumors are adept at utilizing evolutionarily conserved processes for evading immune surveillance. However, it is not clear whether the described above mechanisms are also effective in naturally occurring anti-tumor CTLs. Furthermore, how effector trogocytosis is regulated and how this regulation can be altered during tumor growth is not well understood. There is a need in the art for improved CAR T cell therapies.
- the invention includes a modified immune cell or precursor cell thereof, comprising a nucleic acid encoding a chimeric antigen receptor (CAR) and cholesterol 25- hydroxylase (CH25H) gene, wherein the CAR and CH25H are expressed in the cell.
- the CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular domain.
- the CAR comprises an antigen binding domain selected from the group consisting of an antibody, an scFv, and a Fab.
- the CAR comprises an antigen binding domain comprising specificity for a tumor associated antigen (TAA).
- TAA tumor associated antigen
- the TTA is selected from the group consisting of CD19, CD20, CD22, ROR1, Mesothelin, CD33/IL3Ra, c-Met, PSMA, PSCA, Glycolipid F77, and EGFRvIII.
- the CAR further comprises a hinge domain.
- the CAR comprises a hinge domain selected from the group consisting of an Fc fragment of an antibody, a hinge region of an antibody, a CH2 region of an antibody, a CH3 region of an antibody, an artificial hinge domain, a hinge comprising an amino acid sequence of CD8, or any combination thereof.
- the CAR comprises a transmembrane domain selected from the group consisting of an artificial hydrophobic sequence and transmembrane domain of a type I transmembrane protein, an alpha, beta, or zeta chain of a T cell receptor, CD28, CD3 epsilon, 41744090.2 2 Attorney Docket No.046483-7386WO1(03225) CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.
- the CAR comprises an intracellular domain comprising at least one co-stimulatory domain selected from the group consisting of co-stimulatory domains of proteins in the TNFR superfamily, CD28, 4-1BB (CD137), OX40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, ICOS, NKG2C, and B7-H3.
- co-stimulatory domains of proteins in the TNFR superfamily CD28, 4-1BB (CD137), OX40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, ICOS, NKG2C, and B7-H3.
- the CAR comprises an intracellular domain selected from the group consisting of cytoplasmic signaling domains of a human CD3 zeta chain, FcyRIII, FcsRI, a cytoplasmic tail of an Fc receptor, an immunoreceptor tyrosine-based activation motif (ITAM) bearing cytoplasmic receptors, TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d.
- ITAM immunoreceptor tyrosine-based activation motif
- the nucleic acid comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 10. In certain embodiments, the nucleic acid comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 8. In certain embodiments, the nucleic acid comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 15. In certain embodiments, the modified cell is an autologous cell.
- the modified cell is a cell isolated from a human subject. In certain embodiments, the modified cell is a modified T cell. Another aspect of the invention includes a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject any of the modified immune cells or precursor cells thereof contemplated herein. In certain embodiments, the disease or disorder is cancer. Another aspect of the invention includes a method of treating cancer in a subject in need thereof, the method comprising administering to the subject i) a modified T cell comprising a CAR, and ii) an agent that upregulates CH25H.
- nucleic acid comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR) and a nucleotide sequence encoding a cholesterol 25-hydroxylase (Ch25h) gene.
- the nucleic acid comprises a nucleotide 41744090.2 3 Attorney Docket No.046483-7386WO1(03225) sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 8, 10, or 15.
- FIGs.1A-1I Tumor-derived factors (TDFs) downregulate 25-hydroxycholesterol and stimulate trogocytosis between effector CTL and malignant cells.
- TDFs Tumor-derived factors
- FIG.1C Volcano plot of differentially expressed genes in CTLs treated as in FIG.1B.
- FIGs.2A-2M CH25H is a pivotal regulator of CTL trogocytosis, survival and activity.
- FIG.2D A schematic of experiment for assessing the role of Ch25h in viability of CD8 + T cells in vivo (upper panel). Lower panel depicts the flow cytometry analysis of the percentage of CFSE + CD8 + or CFSE- CD8 + T cells isolated from the MC38 or MC38-OVA tumors 24 hr after inoculation.
- FIGs.3A-3J Downregulation of CH25H in CTLs attenuates the immune responses and promote tumor growth.
- FIG.3A Correlation between CH25H expression and CD8 + T cell infiltration in human melanoma tumor.
- FIG.3B Association between CH25H expression and progress free survival in human melanoma patients.
- FIG.3C Association between CH25H expression and overall survival in human melanoma patients.
- FIG.3E Representative images of B16F10 tumor size (left) and weight (right) at day 15 from experiment described in FIG.3D.
- FIG.3H Flow cytometry analysis of percentage of CD69-expressing CTLs from experiment described in FIG.3G.
- FIG. 3I Flow cytometry analysis of percentage of PD-1-expressing CTLs from experiment described in FIG.3G.
- FIG.3J Flow cytometry analysis of percentage of Annexin V-expressing CTLs from experiment described in FIG.3G. Data are presented as mean ⁇ SEM.
- FIGs.4A-4J ATF3 regulates effector trogocytosis, activity and viability of CTLs, and tumor growth in a CH25H-dependent manner.
- FIG.5A-5I ATF3 and CH25H control trogocytosis and activity of CAR T cells.
- FIGs.6A-6H TAK981 sumoylation inhibitor upregulates CH25H, inhibits trogocytosis and augments CAR T viability and anti-tumor activities.
- FIG.6A Immunoblot analysis of ATF3 sumoylation in ATF3 immunoprecipitates from lysates from activated OT-I CD8+ T cells treated with TCM in the presence or absence of TAK981 (0.1 ⁇ M for 8 hr) using the indicated antibodies. Loading controls in starting lysates used for immunoprecipitation were verified by immunoblot using anti- ⁇ -tubulin antibody.
- PBS Vehicle
- anti-PD1 antibody i.p, 5mg/kg every 4 days
- TAK981 i.v, 15mg/kg once a week
- FIGs.7A-7L CARs designed to re-express CH25H inhibit trogocytosis and increase therapeutic efficacy.
- FIG.7A Design of anti-Meso-CAR, anti-Meso-Ch25h CAR, anti-CD19 CAR and anti-CD19-Ch25h CAR constructs.
- FIG.7E A schematic of experiment for comparing the efficacy of anti-Meso-CAR and anti-MESO-Ch25h T cells in vivo.
- FIG.7H Volume of B16F10-hCD19 tumors (inoculated s.c.
- FIG.7I A schematic of experiment for comparing the efficacy of human anti-CD19 and anti-CD19-Ch25h CAR T cells in the model of NALM6 acute lymphoblastic leukemia in NSG mice.
- FIGs.8A-8F Tumor-derived factors (TDFs) downregulate 25-hydroxycholesterol and stimulate trogocytosis between effector CTL and malignant cells.
- FIG.8A A schematic of experiment for assessing DiD transfer between CD8 + T cells and cancer cells.
- VEGF 50ng/ml
- PGE2 10nM
- FCM fibroblast cell medium
- IECM intestinal epithelial cell medium
- TCM Tumor conditioned medium
- FIGs.9A-9J CH25H is a pivotal regulator of CTL trogocytosis, survival and activity.
- FIG.9E Gating strategy of experiments for assessment of the role of Ch25h on trogocytosis in vivo.
- FIG.9H Flow cytometry analysis of transfer of OVA-MHC-I complexes from MC38OVA or control MC38 cells onto co-cultured (for 4 hr) WT or Ch25h -/- CTLs.
- FIGs.10A-10I Downregulation of CH25H in CTLs attenuates the immune responses and promote tumor growth.
- FIG.10A Correlation between expression of CH25H and CD8A expression in tumor tissues from human melanoma, colon and pancreatic cancer.
- FIG.10E Growth of MC38 tumors (inoculated s.c.
- FIG.10G Growth of Hepa1-6 tumors (inoculated s.c.
- FIGs.11A-11J ATF3 regulates effector trogocytosis, activity and viability of CTLs, and tumor growth in a CH25H-dependent manner.
- FIG.11G Representative images of B16F10 tumors that grew in WT, Ch25h ⁇ CD8 , Atf3 ⁇ CD8 or Ch25h;Atf3 ⁇ CD8 mice.
- FIG.11H Flow cytometry analysis of percentage of CD3 + CD8 + T cells in B16F10 tumors from WT, Ch25h ⁇ CD8 , Atf3 ⁇ CD8 or Ch25h;Atf3 ⁇ CD8 mice.
- FIGs.12A-12C ATF3 and CH25H control trogocytosis and activity of CAR T cells.
- FIG.12A Detection of the anti-CD19 CAR expression levels in indicated CAR T cells 48 hr after transduction.
- FIG.13A-13F TAK981 sumoylation inhibitor upregulates CH25H, inhibits trogocytosis and augments CAR T viability and anti-tumor activities.
- FIG.14A-14O CARs designed to re-express CH25H inhibit trogocytosis and increase therapeutic efficacy.
- FIG.14A Flow cytometry analysis of percentage of mCherry positive CD8 + T cells 48 hr after transduced with anti-MESO CAR that also expresses mCherry.
- FIG.14E Flow cytometric analysis of CD3 + CD8 + CAR T cells in tumor or blood from NSG mice treated as in FIG.14D.
- FIG.14F Flow cytometric analysis of anti-CD19 CAR expression in WT or Ch25h -/- T cells 48 hr after transduced with anti-CD19 CAR or anti-CD19-Ch25h CAR retrovirus.
- FIG.14M A schematic of experiment aimed at comparing the efficacy of anti-CD19 and anti-CD19-Ch25h CAR T cells against B16F10- hCD19 tumors.
- compositions and methods for modified immune cells or precursors thereof comprising a nucleic acid encoding a chimeric antigen 41744090.2 Attorney Docket No.046483-7386WO1(03225) receptor (CAR) and a cholesterol 25-hydroxylase (CH25H) gene.
- Methods for treating diseases or disorders are also provided.
- CAR T cells targeting tumor-specific antigens e.g. CD19 have displayed remarkable efficacy for the treatment of some patients with hematologic malignancies. However, despite high response rates, many patients relapse and, unfortunately, CAR-T immunotherapy has exhibited little success for the treatment of solid tumors.
- TME tumor microenvironment
- TTLs cytotoxic T lymphocytes
- trogocytosis involves the transfer of fragments of lipid membranes containing cholesterol and its metabolites, this study focused on identifying factors in the cholesterol metabolism pathway which control trogocytosis.
- cholesterol 41744090.2 Attorney Docket No.046483-7386WO1(03225) 25-hydroxylase (CH25H) was identified as a pivotal negative regulator of trogocytosis.
- CH25H is a gene whose enzymatic product 25-hydroxycholesterol (25HC) alters membrane fluidity and inhibits membrane fusion, which is essential for incorporating malignant cell membrane fragments into CTL during trogocytosis.
- 25HC 25-hydroxycholesterol
- CAR constructs were devised herein that co-express CH25H in a T cell.
- Previously described anti-mesothelin and anti- CD19 CARs (Milone et al., (2009) Mol Ther 17, 1453-1464) were redesigned to enable co- expression of CH25H.
- Mesolthlin is a cell-surface antigen implicated in tumor invasion and is highly expressed in mesothelioma, lung, pancreas, breast, ovarian, and other solid tumor cancers.
- CTLs harboring conventional or CH25H-expressing anit-MESO CAR revealed that expression of CH25H significantly increased the efficacy of this adoptive cell therapy as seen from assessment of tumor volume and weight and animal survival (FIG.7F).
- T cells harboring CH25H-expressing CAR displayed increased numbers in tumors and blood compared to control CAR T cells (FIG. 7G).
- “Activation,” as used herein, refers to the state of a T cell that has been sufficiently stimulated to induce detectable cellular proliferation. Activation can also be associated with induced cytokine production, and detectable effector functions.
- the term “activated T cells” refers to, among other things, T cells that are undergoing cell division.
- a disease means reducing the severity of one or more symptoms of the disease.
- 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 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. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA.
- any DNA which comprises a nucleotide sequences 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.
- an antigen need not be encoded by a “gene” at all.
- an antigen can be generated synthesized or can be derived from a biological sample.
- a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid. 41744090.2 Attorney Docket No.046483-7386WO1(03225)
- autologous is meant to refer to any material derived from the same individual to which it is later to be re-introduced into the individual.
- a “co-stimulatory molecule” refers to the cognate binding partner on a T cell that specifically binds with a co-stimulatory ligand, thereby mediating a co-stimulatory response by the T cell, such as, but not limited to, proliferation.
- Co-stimulatory molecules include but are not limited to an MHC class I molecule, BTLA and a Toll ligand receptor.
- a “disease” is a state of health of an animal (e.g., a mammal, e.g., a human) wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate.
- a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health.
- the term “downregulation” as used herein refers to the decrease or elimination of gene expression of one or more genes or of their RNA or/and protein products. “Effective amount” or “therapeutically effective amount” are used interchangeably herein, and refer to an amount of a compound, formulation, material, or composition, as described herein effective to achieve a particular biological result or provides a therapeutic or prophylactic benefit.
- Such results may include but are not limited to an amount that when administered to an animal (e.g., a mammal, e.g., a human), causes a detectable level of immune suppression or tolerance compared to the immune response detected in the absence of the composition of the invention.
- the immune response can be readily assessed by a plethora of art- recognized methods.
- the skilled artisan would understand that the amount of the composition administered herein varies and can be readily determined based on a number of factors such as the disease or condition being treated, the age and health and physical condition of the animal (e.g., mammal, e.g., human) being treated, the severity of the disease, the particular compound being administered, and the like.
- Encoding refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of 41744090.2 Attorney Docket No.046483-7386WO1(03225) other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
- a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system.
- Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
- endogenous refers to any material from or produced inside an organism, cell, tissue or system.
- epitope as used herein is defined as a small chemical molecule on an antigen that can elicit an immune response, inducing B and/or T cell responses.
- An antigen can have one or more epitopes. Most antigens have many epitopes; i.e., they are multivalent.
- an epitope is roughly about 10 amino acids and/or sugars in size.
- the epitope is about 4- 18 amino acids, more preferably about 5-16 amino acids, and even more most preferably 6-14 amino acids, more preferably about 7-12, and most preferably about 8-10 amino acids.
- the overall three-dimensional structure, rather than the specific linear sequence of the molecule is the main criterion of antigenic specificity and therefore distinguishes one epitope from another.
- a peptide used in the present invention can be an epitope.
- the term “exogenous” refers to any material introduced from or produced outside an organism, cell, tissue or system.
- expand refers to increasing in number, as in an increase in the number of T cells.
- the T cells that are expanded ex vivo increase in number relative to the number originally present in the culture.
- the T cells that are expanded ex vivo increase in number relative to other cell types in the culture.
- ex vivo refers to cells that have been removed from a living organism, (e.g., a human) and propagated outside the organism (e.g., in a culture dish, test tube, or bioreactor).
- expression as used herein is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter.
- Expression vector refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed.
- An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system.
- Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., Sendai viruses, lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.
- “Identity” as used herein refers to the subunit sequence identity between two polymeric molecules particularly between two amino acid molecules, such as, between two polypeptide molecules. When two amino acid sequences have the same residues at the same positions; e.g., if a position in each of two polypeptide molecules is occupied by an arginine, then they are identical at that position.
- the identity or extent to which two amino acid sequences have the same residues at the same positions in an alignment is often expressed as a percentage.
- the identity between two amino acid sequences is a direct function of the number of matching or identical positions; e.g., if half (e.g., five positions in a polymer ten amino acids in length) of the positions in two sequences are identical, the two sequences are 50% identical; if 90% of the positions (e.g., 9 of 10), are matched or identical, the two amino acids sequences are 90% identical.
- the term “immune response” as used herein is defined as a cellular response to an antigen that occurs when lymphocytes identify antigenic molecules as foreign and induce the formation of antibodies and/or activate lymphocytes to remove the antigen.
- immunosuppressive is used herein to refer to reducing overall immune response.
- isolated means altered or removed from the natural state.
- a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.”
- An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
- a “lentivirus” as used herein refers to a genus of the Retroviridae family.
- Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the 41744090.2 Attorney Docket No.046483-7386WO1(03225) most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses offer the means to achieve significant levels of gene transfer in vivo. By the term “modified” as used herein, is meant a changed state or structure of a molecule or cell of the invention. Molecules may be modified in many ways, including chemically, structurally, and functionally.
- Cells may be modified through the introduction of nucleic acids.
- modulating is meant mediating a detectable increase or decrease in the level of a response in a subject compared with the level of a response in the subject in the absence of a treatment or compound, and/or compared with the level of a response in an otherwise identical but untreated subject.
- the term encompasses perturbing and/or affecting a native signal or response thereby mediating a beneficial therapeutic response in a subject, preferably, a human.
- the following abbreviations for the commonly occurring nucleic acid bases are used.
- oligonucleotide typically refers to short polynucleotides.
- nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence.
- nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).
- parenteral administration of an immunogenic composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrasternal injection, or infusion techniques.
- polynucleotide as used herein is defined as a chain of nucleotides.
- nucleic acids are polymers of nucleotides.
- nucleic acids and polynucleotides as used herein are interchangeable.
- nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric “nucleotides.” The monomeric nucleotides can be hydrolyzed into nucleosides.
- polynucleotides 41744090.2 Attorney Docket No.046483-7386WO1(03225) include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR, and the like, and by synthetic means.
- recombinant means i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR, and the like, and by synthetic means.
- the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds.
- a protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence.
- Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds.
- the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types.
- Polypeptides include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others.
- the polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.
- specifically binds as used herein with respect to an antibody, is meant an antibody which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species.
- an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen.
- cross reactivity does not itself alter the classification of an antibody as specific.
- the terms “specific binding” or “specifically binding,” can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally.
- an antibody is specific for epitope “A”
- the presence of a molecule containing epitope A or free, 41744090.2 Attorney Docket No.046483-7386WO1(03225) unlabeled A
- stimulation is meant a primary response induced by binding of a stimulatory molecule (e.g., a TCR/CD3 complex) with its cognate ligand thereby mediating a signal transduction event, such as, but not limited to, signal transduction via the TCR/CD3 complex.
- Stimulation can mediate altered expression of certain molecules, such as downregulation of TGF-beta, and/or reorganization of cytoskeletal structures, and the like.
- a “stimulatory molecule,” as the term is used herein, means a molecule on a T cell that specifically binds with a cognate stimulatory ligand present on an antigen presenting cell.
- a “stimulatory ligand,” as used herein, means a ligand that when present on an antigen presenting cell (e.g., an aAPC, a dendritic cell, a B-cell, and the like) can specifically bind with a cognate binding partner (referred to herein as a “stimulatory molecule”) on a T cell, thereby mediating a primary response by the T cell, including, but not limited to, activation, initiation of an immune response, proliferation, and the like.
- an antigen presenting cell e.g., an aAPC, a dendritic cell, a B-cell, and the like
- a cognate binding partner referred to herein as a “stimulatory molecule”
- Stimulatory ligands are well-known in the art and encompass, inter alia, an MHC Class I molecule loaded with a peptide, an anti-CD3 antibody, a superagonist anti-CD28 antibody, and a superagonist anti-CD2 antibody.
- the term “subject” is intended to include living organisms in which an immune response can be elicited (e.g., animals, e.g., mammals, e.g., humans).
- a “subject” or “patient,” as used therein, may be an animal, a human, or non-human mammal.
- Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline and murine mammals.
- the subject is human.
- target site refers to a nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule may specifically bind under conditions sufficient for binding to occur.
- a target sequence refers to a genomic nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule may specifically bind under conditions sufficient for binding to occur.
- T cell receptor or “TCR” refers to a complex of membrane proteins that participate in the activation of T cells in response to the presentation of antigen. The TCR is responsible for recognizing antigens bound to major histocompatibility complex molecules.
- TCR is composed of a heterodimer of an alpha ( ⁇ ) and beta ( ⁇ ) chain, or gamma and delta ( ⁇ / ⁇ ) chains.
- TCRs may exist in alpha/beta and gamma/delta forms, which are structurally 41744090.2 Attorney Docket No.046483-7386WO1(03225) similar but have distinct anatomical locations and functions.
- Each chain is composed of two extracellular domains, a variable and constant domain.
- the TCR may be modified on any cell comprising a TCR, including, for example, a helper T cell, a cytotoxic T cell, a memory T cell, regulatory T cell, natural killer T cell, and gamma delta T cell.
- transplant refers to a biocompatible lattice or a donor tissue, organ or cell, to be transplanted.
- An example of a transplant may include but is not limited to skin cells or tissue, bone marrow, and solid organs such as heart, pancreas, kidney, lung and liver.
- a transplant can also refer to any material that is to be administered to a host.
- a transplant can refer to a nucleic acid or a protein.
- transfected or “transformed” or “transduced” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell.
- a “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid.
- the cell includes the primary subject cell and its progeny.
- To “treat” a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.
- a “vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell.
- vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses.
- the term “vector” includes an autonomously replicating plasmid or a virus.
- the term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like.
- viral vectors include, but are not limited to, Sendai viral vectors, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.
- the present invention provides modified immune cells or precursors thereof (e.g., T cells) for use in immunotherapy (e.g. CAR T cells).
- modified immune cells or precursors thereof e.g., T cells
- immunotherapy e.g. CAR T cells
- the invention provides a modified immune cell or precursor cell thereof (e.g., T cell) comprising a nucleic acid encoding a chimeric antigen receptor (CAR) and a cholesterol 25-hydroxylase (CH25H) gene.
- TDFs tumor-derived factors
- ATF3 is an early stress-responsive gene that can be induced by many factors in the tumor microenvironment including hypoxia and nutrient deprivation.
- ATF3 has been shown to suppress CH25h expression in macrophages.
- TDF-induced ATF3 in CTLs downregulates expression of CH25H.
- the latter catalyzes monooxygenation of cholesterol into 25-hydroxycholesterol (25HC), which interferes with trogocytosis stimulated by TDFs.
- Dysregulation of the ATF3-CH25H axis in the intratumoral natural or CAR-expressing CTLs stimulates effector trogocytosis and undermines viability and anti-tumor activity of these CTLs.
- pharmacological, or genetic restoration of CH25H expression in CTLs inhibits trogocytosis, suppresses tumor growth and increases the efficacy of immunotherapies.
- the invention provides a modified immune cell or precursor cell thereof (e.g., T cell) comprising a nucleic acid encoding a chimeric antigen receptor (CAR) and a cholesterol 25-hydroxylase (CH25H), wherein the cell is capable of expressing the CAR and CH25H.
- the modified immune cell or precursor cell thereof e.g., T cell
- the cell comprises a CAR and CH25H.
- CH25H is encoded by a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 10.
- the cell comprises a nucleic acid that encodes CH25H and an anti-mesothelin CAR. In certain embodiments, the cell comprises a nucleic acid that encodes CH25H and an anti-CD19 CAR. In certain embodiments, the cell comprises a nucleic acid comprising a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 10.
- the cell comprises a nucleic acid comprising a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 8 or SEQ ID NO: 15.
- the cell comprises a first nucleic acid encoding CH25H and a second nucleic acid encoding a CAR.
- the immune cell or precursor cell thereof is a T cell.
- the T cell is a human T cell.
- the cell is an autologous cell (e.g. an autologous T cell).
- the provided immune cells inhibit trogocytosis when administered in vivo to a subject. In some embodiments, this inhibition in the subject upon administration is greater as compared to that which would be achieved by alternative methods, such as those involving administration of cells genetically engineered by methods in which T cells do not encode a CAR and CH5H.
- the immune cells in the composition retain a phenotype of the immune cell or cells compared to the phenotype of cells in a corresponding or reference composition when assessed under the same conditions.
- cells in the composition include naive cells, effector memory cells, central memory cells, stem central memory cells, effector memory cells, and long-lived effector memory cells.
- the percentage of T cells, or T cells expressing the CAR and CH25H exhibit a non-activated, long-lived memory or central memory phenotype that is the same or substantially the same as a corresponding or reference population or composition of cells.
- such property, activity or phenotype can be measured in an in vitro assay, such as by incubation of the cells in the presence of an antigen targeted by the CAR, a cell expressing the antigen and/or an antigen-receptor activating substance.
- any of the assessed activities, properties or phenotypes can be assessed at various days following 41744090.2 Attorney Docket No.046483-7386WO1(03225) electroporation or other introduction of the agent, such as after or up to 3, 4, 5, 6, 7 days.
- activity, property or phenotype is retained by at least 80%, 85%, 90%, 95% or 100% of the cells in the composition compared to the activity of a corresponding composition.
- a "corresponding composition” or a “corresponding population of immune cells” refers to immune cells (e.g., T cells) obtained, isolated, generated, produced and/or incubated under the same or substantially the same conditions, except that the immune cells or population of immune cells were not introduced with the agent.
- immune cells e.g., T cells
- such immune cells are treated identically or substantially identically as immune cells that have been introduced with the agent, such that any one or more conditions that can influence the activity or properties of the cell, including the upregulation or expression of the inhibitory molecule, is not varied or not substantially varied between the cells other than the introduction of the agent.
- T cell markers Methods and techniques for assessing the expression and/or levels of T cell markers are known in the art. Antibodies and reagents for detection of such markers are well known in the art, and readily available. Assays and methods for detecting such markers include, but are not limited to, flow cytometry, including intracellular flow cytometry, ELISA, ELISPOT, cytometric bead array or other multiplex methods, Western Blot and other immunoaffinity-based methods. In some embodiments, CAR-expressing cells can be detected by flow cytometry or other immunoaffinity based method for expression of a marker unique to such cells, and then such cells can be co-stained for another T cell surface marker or markers.
- the population of cells containing T cells can be cells that have been obtained from a subject, such as obtained from a peripheral blood mononuclear cells (PBMC) sample, an unfractionated T cell sample, a lymphocyte sample, a white blood cell sample, an apheresis product, or a leukapheresis product.
- T cells can be separated or selected to enrich T cells in the population using positive or negative selection and enrichment methods.
- the population contains CD4+, CD8+ or CD4+ and CD8+ T cells.
- the administered cells can be detected or quantified after administration to a subject.
- qPCR quantitative PCR
- persistence is quantified as copies of DNA or 41744090.2 Attorney Docket No.046483-7386WO1(03225) plasmid encoding the exogenous receptor per microgram of DNA, or as the number of receptor- expressing cells per microliter of the sample, e.g., of blood or serum, or per total number of peripheral blood mononuclear cells (PBMCs) or white blood cells or T cells per microliter of the sample.
- PBMCs peripheral blood mononuclear cells
- flow cytometric assays detecting cells expressing the receptor generally using antibodies specific for the receptors also can be performed.
- Cell-based assays may also be used to detect the number or percentage of functional cells, such as cells capable of binding to and/or neutralizing and/or inducing responses, e.g., cytotoxic responses, against cells of the disease or condition or expressing the antigen recognized by the receptor.
- the extent or level of expression of another marker associated with the modified cell can be used to distinguish the administered cells from endogenous cells in a subject.
- C. Chimeric Antigen Receptors The present invention provides compositions and methods for cell-based immunotherapies.
- the cell-based immunotherapy comprises a modified immune cell or precursor thereof, e.g., modified T cell, comprising a chimeric antigen receptor (CAR) and CH25H.
- the immune cell has been genetically modified to express the CAR and CH25H.
- CARs of the present invention comprise an antigen binding domain, a transmembrane domain, and an intracellular domain.
- the antigen binding domain may be operably linked to another domain of the CAR, such as the transmembrane domain or the intracellular domain, both described elsewhere herein, for expression in the cell.
- a first nucleic acid sequence encoding the antigen binding domain is operably linked to a second nucleic acid encoding a transmembrane domain, and further operably linked to a third a nucleic acid sequence encoding an intracellular domain.
- the antigen binding domains described herein can be combined with any of the transmembrane domains described herein, any of the intracellular domains or cytoplasmic domains described herein, or any of the other domains described herein that may be included in a CAR of the present invention.
- a subject CAR of the present invention may also include a hinge domain as described herein.
- a subject CAR of the present invention may also include a spacer domain as described herein.
- each of the antigen binding domain, transmembrane domain, and intracellular domain is separated by a linker. 41744090.2 Attorney Docket No.046483-7386WO1(03225) Antigen Binding Domain
- the antigen binding domain of a CAR is an extracellular region of the CAR for binding to a specific target antigen including proteins, carbohydrates, and glycolipids.
- the CAR comprises affinity to a target antigen on a target cell.
- the target antigen may include any type of protein, or epitope thereof, associated with the target cell.
- the CAR may comprise affinity to a target antigen on a target cell that indicates a particular disease state of the target cell.
- the target cell antigen is a tumor associated antigen (TAA).
- TAAs tumor associated antigens
- TAAs include but are not limited to, differentiation antigens such as MART-1/MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2 and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor-suppressor genes such as p53, Ras, HER-2/neu; unique tumor antigens resulting from chromosomal translocations; such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as the Epstein Barr virus antigens EBVA and the human papillomavirus (HPV) antigens E6 and E
- the antigen binding domain of the CAR targets an antigen that includes but is not limited to CD19, CD20, CD22, ROR1, Mesothelin, CD33/IL3Ra, c-Met, PSMA, PSCA, Glycolipid F77, EGFRvIII, GD-2, NY-ESO-1 TCR, MAGE A3 TCR, and the like.
- the CAR of the invention can be engineered to include the appropriate antigen binding domain that is specific to the desired antigen target. For example, if CD19 is the desired antigen that is to be targeted, an antibody for CD19 can be used as the antigen bind moiety for incorporation into the CAR of the invention.
- a CAR having affinity for any target antigen is suitable for use in a composition or method of the present invention.
- a CAR of the present disclosure having affinity for a specific target antigen on a target cell may comprise a target-specific binding domain.
- the target-specific binding domain is a murine target-specific binding domain, e.g., the target- specific binding domain is of murine origin.
- the target-specific binding domain is a human target-specific binding domain, e.g., the target-specific binding domain is of human origin.
- a CAR of the present disclosure having affinity for CD19 on a target cell may comprise a CD19 binding domain.
- a CAR of the present disclosure may have affinity for one or more target antigens on one or more target cells.
- a CAR may have affinity for one or more target antigens on a target cell.
- the CAR is a bispecific CAR, or a multispecific CAR.
- the CAR comprises one or more target-specific binding domains that confer affinity for one or more target antigens.
- the CAR comprises one or more target-specific binding domains that confer affinity for the same target antigen.
- a CAR comprising one or more target-specific binding domains having affinity for the same target antigen could bind distinct epitopes of the target antigen.
- the binding domains may be arranged in tandem and may be separated by linker peptides.
- the binding domains are connected to each other covalently on a single polypeptide chain, through an oligo- or polypeptide linker, an Fc hinge region, or a membrane hinge region.
- the antigen binding domain is selected from the group consisting of an antibody, an antigen binding fragment (Fab), and a single-chain variable fragment (scFv).
- a CD19 binding domain of the present invention can be selected from the group consisting of a CD19-specific antibody, a CD19-specific Fab, and a CD19-specific scFv.
- a CD19 binding domain is a CD19-specific antibody.
- a CD19 binding domain is a CD19-specific Fab.
- a CD19 binding domain is a CD19-specific scFv (e.g. SEQ ID NO: 12).
- a mesothelin binding domain is a mesothelin-specific antibody.
- a mesothelin binding domain is a mesothelin- 41744090.2 Attorney Docket No.046483-7386WO1(03225) specific Fab.
- a mesothelin binding domain is a mesothelin-specific scFv (e.g. SEQ ID NO: 2).
- the antigen binding domain can include any domain that binds to the antigen and may include, but is not limited to, a monoclonal antibody, a polyclonal antibody, a synthetic antibody, a human antibody, a humanized antibody, a non-human antibody, and any fragment thereof.
- the antigen binding domain portion comprises a mammalian antibody or a fragment thereof.
- single-chain variable fragment or “scFv” is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of an immunoglobulin (e.g., mouse or human) covalently linked to form a VH::VL heterodimer.
- the heavy (VH) and light chains (VL) are either joined directly or joined by a peptide-encoding linker, which connects the N- terminus of the VH with the C-terminus of the VL, or the C-terminus of the VH with the N- terminus of the VL.
- the antigen binding domain (e.g., CD19 binding domain) comprises an scFv having the configuration from N-terminus to C-terminus, VH – linker – VL. In some embodiments, the antigen binding domain comprises an scFv having the configuration from N-terminus to C-terminus, VL – linker – VH.
- the linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility. The linker can link the heavy chain variable region and the light chain variable region of the extracellular antigen-binding domain.
- Non-limiting examples of linkers are disclosed in Shen et al., Anal. Chem.80(6):1910-1917 (2008) and WO 2014/087010, the contents of which are hereby incorporated by reference in their entireties.
- Various linker sequences are known in the art, including, without limitation, glycine serine (GS) linkers such as (GS)n, (GSGGS)n (SEQ ID NO:21), (GGGS)n (SEQ ID NO:22), and (GGGGS)n (SEQ ID NO:23), where n represents an integer of at least 1.
- Exemplary linker sequences can comprise amino acid sequences including, without limitation, GGSG (SEQ ID NO:24), GGSGG (SEQ ID NO:25), GSGSG (SEQ ID NO:26), GSGGG (SEQ ID NO:27), GGGSG (SEQ ID NO:28), GSSSG (SEQ ID NO:29), GGGGS (SEQ ID NO:30), GGGGSGGGGSGGGGS (SEQ ID NO:31) and the like.
- GGSG SEQ ID NO:24
- GGSGG SEQ ID NO:25
- GSGSG SEQ ID NO:26
- GSGGG SEQ ID NO:27
- GSSSG SEQ ID NO:29
- GGGGSGGGGSGGGGS SEQ ID NO:31
- an antigen binding domain of the present invention comprises a 41744090.2 Attorney Docket No.046483-7386WO1(03225) heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL is separated by the linker sequence having the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO:31), which may be encoded by the nucleic acid sequence GGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCT (SEQ ID NO:32).
- scFv proteins retain the specificity of the original immunoglobulin.
- Single chain Fv polypeptide antibodies can be expressed from a nucleic acid comprising VH- and VL-encoding sequences as described by Huston, et al. (Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988). See, also, U.S. Patent Nos. 5,091,513, 5,132,405 and 4,956,778; and U.S. Patent Publication Nos.20050196754 and 20050196754.
- Antagonistic scFvs having inhibitory activity have been described (see, e.g., Zhao et al., Hyrbidoma (Larchmt) 200827(6):455-51; Peter et al., J Cachexia Sarcopenia Muscle 2012 August 12; Shieh et al., J Imunol 2009183(4):2277-85; Giomarelli et al., Thromb Haemost 2007 97(6):955-63; Fife eta., J Clin Invst 2006116(8):2252-61; Brocks et al., Immunotechnology 19973(3):173-84; Moosmayer et al., Ther Immunol 19952(10:31-40).
- Fab refers to a fragment of an antibody structure that binds to an antigen but is monovalent and does not have a Fc portion, for example, an antibody digested by the enzyme papain yields two Fab fragments and an Fc fragment (e.g., a heavy (H) chain constant region; Fc region that does not bind to an antigen).
- an antibody digested by the enzyme papain yields two Fab fragments and an Fc fragment (e.g., a heavy (H) chain constant region; Fc region that does not bind to an antigen).
- F(ab′)2 refers to an antibody fragment generated by pepsin digestion of whole IgG antibodies, wherein this fragment has two antigen binding (ab′) (bivalent) regions, wherein each (ab′) region comprises two separate amino acid chains, a part of a H chain and a light (L) chain linked by an S—S bond for binding an antigen and where the remaining H chain portions are linked together.
- a “F(ab′)2” fragment can be split into two individual Fab′ fragments.
- the antigen binding domain may be derived from the same species in which the CAR will ultimately be used.
- the antigen binding domain of the CAR may comprise a human antibody or a fragment thereof.
- the antigen binding domain may be derived from a different species in which the 41744090.2 Attorney Docket No.046483-7386WO1(03225) CAR will ultimately be used.
- the antigen binding domain of the CAR may comprise a murine antibody or a fragment thereof.
- Transmembrane Domain CARs of the present invention may comprise a transmembrane domain that connects the antigen binding domain of the CAR to the intracellular domain of the CAR.
- the transmembrane domain of a subject CAR is a region that is capable of spanning the plasma membrane of a cell (e.g., an immune cell or precursor thereof).
- the transmembrane domain is for insertion into a cell membrane, e.g., a eukaryotic cell membrane.
- the transmembrane domain is interposed between the antigen binding domain and the intracellular domain of a CAR.
- the transmembrane domain is naturally associated with one or more of the domains in the CAR.
- the transmembrane domain can be selected or modified by one or more amino acid substitutions to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins, to minimize interactions with other members of the receptor complex.
- the transmembrane domain may be derived either from a natural or a synthetic source.
- the domain may be derived from any membrane-bound or transmembrane protein, e.g., a Type I transmembrane protein.
- the transmembrane domain may be any artificial sequence that facilitates insertion of the CAR into a cell membrane, e.g., an artificial hydrophobic sequence. Examples of the transmembrane domain of particular use in this invention include, without limitation, transmembrane domains derived from (i.e.
- the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine.
- transmembrane domains described herein can be combined with any of the antigen binding domains described herein, any of the intracellular domains described herein, or any of the other domains described herein that may be included in a subject CAR.
- the transmembrane domain further comprises a hinge region.
- a subject CAR of the present invention may also include a hinge region.
- the hinge region of the CAR is a hydrophilic region which is located between the antigen binding domain and the transmembrane domain.
- this domain facilitates proper protein folding for the CAR.
- the hinge region is an optional component for the CAR.
- the hinge region may include a domain selected from Fc fragments of antibodies, hinge regions of antibodies, CH2 regions of antibodies, CH3 regions of antibodies, artificial hinge sequences or combinations thereof. Examples of hinge regions include, without limitation, a CD8a hinge, artificial hinges made of polypeptides which may be as small as, three glycines (Gly), as well as CH1 and CH3 domains of IgGs (such as human IgG4).
- a subject CAR of the present disclosure includes a hinge region that connects the antigen binding domain with the transmembrane domain, which, in turn, connects to the intracellular domain.
- the hinge region is preferably capable of supporting the antigen binding domain to recognize and bind to the target antigen on the target cells (see, e.g., Hudecek et al., Cancer Immunol. Res. (2015) 3(2): 125-135).
- the hinge region is a flexible domain, thus allowing the antigen binding domain to have a structure to optimally recognize the specific structure and density of the target antigens on a cell such as tumor cell (Hudecek et al., supra).
- the flexibility of the hinge region permits the hinge region to adopt many different conformations.
- the hinge region is an immunoglobulin heavy chain hinge region.
- the hinge region is a hinge region polypeptide derived from a receptor (e.g., a CD8-derived hinge region).
- the hinge region can have a length of from about 4 amino acids to about 50 amino acids, e.g., from about 4 aa to about 10 aa, from about 10 aa to about 15 aa, from about 15 aa to about 20 aa, from about 20 aa to about 25 aa, from about 25 aa to about 30 aa, from about 30 aa to about 40 aa, or from about 40 aa to about 50 aa.
- the hinge region can have a length of greater than 5 aa, greater than 10 aa, greater than 15 aa, greater than 20 aa, greater 41744090.2 Attorney Docket No.046483-7386WO1(03225) than 25 aa, greater than 30 aa, greater than 35 aa, greater than 40 aa, greater than 45 aa, greater than 50 aa, greater than 55 aa, or more.
- Suitable hinge regions can be readily selected and can be of any of a number of suitable lengths, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids.
- Suitable hinge regions can have a length of greater than 20 amino acids (e.g., 30, 40, 50, 60 or more amino acids).
- hinge regions include glycine polymers (G) n , glycine-serine polymers (including, for example, (GS) n , (GSGGS) n (SEQ ID NO:21) and (GGGS) n (SEQ ID NO:22), where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art.
- Glycine and glycine-serine polymers can be used; both Gly and Ser are relatively unstructured, and therefore can serve as a neutral tether between components.
- Glycine polymers can be used; glycine accesses significantly more phi-psi space than even alanine, and is much less restricted than residues with longer side chains (see, e.g., Scheraga, Rev. Computational. Chem. (1992) 2: 73-142).
- Exemplary hinge regions can comprise amino acid sequences including, but not limited to, GGSG (SEQ ID NO:24), GGSGG (SEQ ID NO:25), GSGSG (SEQ ID NO:26), GSGGG (SEQ ID NO:27), GGGSG (SEQ ID NO:28), GSSSG (SEQ ID NO:29), and the like.
- the hinge region is an immunoglobulin heavy chain hinge region.
- the hinge region can comprise an amino acid sequence of a human IgG1, IgG2, IgG3, or IgG4, hinge region.
- the hinge region can include one or more amino acid substitutions and/or insertions and/or deletions compared to a wild-type (naturally- occurring) hinge region.
- Intracellular Signaling Domain A subject CAR of the present invention also includes an intracellular signaling domain.
- the terms “intracellular signaling domain” and “intracellular domain” are used interchangeably herein.
- the intracellular signaling domain of the CAR is responsible for activation of at least one of the effector functions of the cell in which the CAR is expressed (e.g., immune cell).
- the intracellular signaling domain transduces the effector function signal and directs the cell (e.g., immune cell) to perform its specialized function, e.g., harming and/or destroying a target cell.
- the cell e.g., immune cell
- Examples of an intracellular domain for use in the invention include, but are not limited to, the cytoplasmic portion of a surface receptor, co-stimulatory molecule, and any molecule that acts in concert to initiate signal transduction in the T cell, as well as any derivative or variant of these elements and any synthetic sequence that has the same functional capability.
- intracellular signaling domain examples include, without limitation, the ⁇ chain of the T cell receptor complex or any of its homologs, e.g., ⁇ chain, FcsRI ⁇ and ⁇ chains, MB 1 (Iga) chain, B29 (Ig) chain, etc., human CD3 zeta chain, CD3 polypeptides ( ⁇ , ⁇ and ⁇ ), syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.), and other molecules involved in T cell transduction, such as CD2, CD5 and CD28.
- the ⁇ chain of the T cell receptor complex or any of its homologs e.g., ⁇ chain, FcsRI ⁇ and ⁇ chains, MB 1 (Iga) chain, B29 (Ig) chain, etc.
- human CD3 zeta chain CD3 polypeptides ( ⁇ , ⁇ and ⁇ )
- the intracellular signaling domain may be human CD3 zeta chain (e.g. SEQ ID NO: 5), FcyRIII, FcsRI, cytoplasmic tails of Fc receptors, an immunoreceptor tyrosine-based activation motif (ITAM) bearing cytoplasmic receptors, and combinations thereof.
- the intracellular signaling domain of the CAR includes any portion of one or more co-stimulatory molecules, such as at least one signaling domain from CD2, CD3 (e.g. SEQ ID NO:5), CD8, CD27, CD28, ICOS, 4-1BB (e.g. SEQ ID NO:4), PD-1, any derivative or variant thereof, any synthetic sequence thereof that has the same functional capability, and any combination thereof.
- intracellular domain examples include a fragment or domain from one or more molecules or receptors including, but not limited to, TCR, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD86, common FcR gamma, FcR beta (Fc Epsilon RIb), CD79a, CD79b, Fcgamma RIIa, DAP10, DAP12, T cell receptor (TCR), CD8, CD27, CD28, 4-1BB (CD137), OX9, OX40, CD30, CD40, PD-1, ICOS, a KIR family protein, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, 41744090.2 Attorney Dock
- intracellular domains include, without limitation, intracellular signaling domains of several types of various other immune signaling receptors, including, but not limited to, first, second, and third generation T cell signaling proteins including CD3, B7 family costimulatory, and Tumor Necrosis Factor Receptor (TNFR) superfamily receptors (see, e.g., Park and Brentjens, J. Clin. Oncol. (2015) 33(6): 651-653). Additionally, intracellular signaling domains may include signaling domains used by NK and NKT cells (see, e.g., Hermanson and Kaufman, Front. Immunol.
- NKp30 B7-H6
- DAP 12 see, e.g., Topfer et al., J. Immunol. (2015) 194(7): 3201-3212
- NKG2D NKp44
- NKp46 NKp46
- DAP10 CD3z
- Intracellular signaling domains suitable for use in a subject CAR of the present invention include any desired signaling domain that provides a distinct and detectable signal (e.g., increased production of one or more cytokines by the cell; change in transcription of a target gene; change in activity of a protein; change in cell behavior, e.g., cell death; cellular proliferation; cellular differentiation; cell survival; modulation of cellular signaling responses; etc.) in response to activation of the CAR (i.e., activated by antigen and dimerizing agent).
- the intracellular signaling domain includes at least one (e.g., one, two, three, four, five, six, etc.) ITAM motifs as described below.
- the intracellular signaling domain includes DAP10/CD28 type signaling chains.
- the 41744090.2 Attorney Docket No.046483-7386WO1(03225) intracellular signaling domain is not covalently attached to the membrane bound CAR, but is instead diffused in the cytoplasm.
- Intracellular signaling domains suitable for use in a subject CAR of the present invention include immunoreceptor tyrosine-based activation motif (ITAM)-containing intracellular signaling polypeptides.
- ITAM immunoreceptor tyrosine-based activation motif
- an ITAM motif is repeated twice in an intracellular signaling domain, where the first and second instances of the ITAM motif are separated from one another by 6 to 8 amino acids.
- the intracellular signaling domain of a subject CAR comprises 3 ITAM motifs.
- intracellular signaling domains includes the signaling domains of human immunoglobulin receptors that contain immunoreceptor tyrosine based activation motifs (ITAMs) such as, but not limited to, FcgammaRI, FcgammaRIIA, FcgammaRIIC, FcgammaRIIIA, FcRL5 (see, e.g., Gillis et al., Front. Immunol. (2014) 5:254).
- a suitable intracellular signaling domain can be an ITAM motif-containing portion that is derived from a polypeptide that contains an ITAM motif.
- a suitable intracellular signaling domain can be an ITAM motif-containing domain from any ITAM motif-containing protein.
- a suitable intracellular signaling domain need not contain the entire sequence of the entire protein from which it is derived.
- suitable ITAM motif-containing polypeptides include, but are not limited to: DAP12, FCER1G (Fc epsilon receptor I gamma chain), CD3D (CD3 delta), CD3E (CD3 epsilon), CD3G (CD3 gamma), CD3Z (CD3 zeta), and CD79A (antigen receptor complex-associated protein alpha chain).
- the intracellular signaling domain is derived from DAP12 (also known as TYROBP; TYRO protein tyrosine kinase binding protein; KARAP; PLOSL; DNAX- activation protein 12; KAR-associated protein; TYRO protein tyrosine kinase-binding protein; killer activating receptor associated protein; killer-activating receptor-associated protein; etc.).
- DAP12 also known as TYROBP; TYRO protein tyrosine kinase binding protein; KARAP; PLOSL; DNAX- activation protein 12; KAR-associated protein; TYRO protein tyrosine kinase-binding protein; killer activating receptor associated protein; killer-activating receptor-associated protein; etc.
- the intracellular signaling domain is derived from FCER1G (also known as FCRG; Fc epsilon receptor I gamma chain; Fc receptor gamma-chain; fc-epsilon RI-gamma; fcRgamma; fceRl gamma; high affinity immunoglobulin epsilon receptor subunit gamma; immunoglobulin E receptor, high affinity, gamma chain; etc.).
- FCER1G also known as FCRG
- Fc epsilon receptor I gamma chain Fc receptor gamma-chain
- fcRgamma fceRl gamma
- high affinity immunoglobulin epsilon receptor subunit gamma immunoglobulin E receptor, high affinity, gamma chain; etc.
- the intracellular signaling domain is derived from T-cell surface glycoprotein CD3 delta chain (also known as CD3D; CD3-DELTA; T3D; CD3 antigen, delta subunit; CD3 delta; CD3d antigen, delta polypeptide (TiT3 complex); OKT3, delta chain; T-cell receptor T3 delta chain; T-cell 41744090.2 Attorney Docket No.046483-7386WO1(03225) surface glycoprotein CD3 delta chain; etc.).
- T-cell surface glycoprotein CD3 delta chain also known as CD3D; CD3-DELTA; T3D; CD3 antigen, delta subunit; CD3 delta; CD3d antigen, delta polypeptide (TiT3 complex); OKT3, delta chain; T-cell receptor T3 delta chain; T-cell 41744090.2 Attorney Docket No.046483-7386WO1(03225) surface glycoprotein CD3 delta chain; etc.
- the intracellular signaling domain is derived from T-cell surface glycoprotein CD3 epsilon chain (also known as CD3e, T- cell surface antigen T3/Leu-4 epsilon chain, T-cell surface glycoprotein CD3 epsilon chain, AI504783, CD3, CD3epsilon, T3e, etc.).
- the intracellular signaling domain is derived from T-cell surface glycoprotein CD3 gamma chain (also known as CD3G, T-cell receptor T3 gamma chain, CD3-GAMMA, T3G, gamma polypeptide (TiT3 complex), etc.).
- the intracellular signaling domain is derived from T-cell surface glycoprotein CD3 zeta chain (also known as CD3Z, T-cell receptor T3 zeta chain, CD247, CD3-ZETA, CD3H, CD3Q, T3Z, TCRZ, etc.).
- the intracellular signaling domain is derived from CD79A (also known as B-cell antigen receptor complex-associated protein alpha chain; CD79a antigen (immunoglobulin-associated alpha); MB-1 membrane glycoprotein; ig- alpha; membrane-bound immunoglobulin-associated protein; surface IgM-associated protein; etc.).
- an intracellular signaling domain suitable for use in an FN3 CAR of the present disclosure includes a DAP10/CD28 type signaling chain. In one embodiment, an intracellular signaling domain suitable for use in an FN3 CAR of the present disclosure includes a ZAP70 polypeptide. In some embodiments, the intracellular signaling domain includes a cytoplasmic signaling domain of TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, or CD66d. In one embodiment, the intracellular signaling domain in the CAR includes a cytoplasmic signaling domain of human CD3 zeta.
- intracellular signaling domain While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal.
- the intracellular signaling domain includes any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.
- the intracellular signaling domains described herein can be combined with any of the antigen binding domains described herein, any of the transmembrane domains described herein, or any of the other domains described herein that may be included in the CAR. The invention should be construed to include any CAR known in the art and/or disclosed herein.
- Exemplary CARs include, but are not limited to, those disclosed herein, those disclosed in US10357514B2, US10221245B2, US10603378B2, US8916381B1, US9394368B2, 41744090.2 Attorney Docket No.046483-7386WO1(03225) US20140050708A1, US9598489B2, US9365641B2, US20210079059A1, US9783591B2, WO2016028896A1, US9446105B2, WO2016014576A1, US20210284752A1, WO2016014565A2, WO2016014535A1, and US9272002B2, and any other CAR generally disclosed in the art.
- the modified immune cells described herein may be included in a composition for immunotherapy.
- the composition may include a pharmaceutical composition and further include a pharmaceutically acceptable carrier.
- a therapeutically effective amount of the pharmaceutical composition comprising the modified cells may be administered to a subject in need thereof.
- the invention includes a method for adoptive cell transfer therapy comprising administering to a subject in need thereof a modified cell (e.g., CAR T cell comprising CH25H) of the present invention.
- the invention includes a method of treating a disease or condition in a subject comprising administering to a subject in need thereof a population of modified cells cell (e.g., CAR T cell comprising CH25H).
- the invention includes a method of treating a disease or condition in a subject comprising administering to a subject in need thereof a CAR T cell and an agent that up- regulates CH25H.
- the agent is reserpine as described in e.g., Ortiz A. et al. (2019) Cancer Cell, 35: 33-45; Lu Z., et al (2021) J Clin Invest, 131: 144225.
- Methods for administration of immune cells for adoptive cell therapy are known and may be used in connection with the provided methods and compositions.
- the cell therapy e.g., adoptive T cell therapy is carried out by autologous transfer, in which the cells are isolated and/or otherwise prepared from the subject who is to receive the cell therapy, or from a sample derived from such a subject.
- the cells are derived from a subject, e.g., patient, in need of a treatment and the cells, following isolation and processing are administered to the same subject.
- the cell therapy e.g., adoptive T cell therapy
- the cells then are administered to a different subject, e.g., a second subject, of the same species.
- the first and second subjects are genetically identical. 41744090.2 Attorney Docket No.046483-7386WO1(03225)
- the first and second subjects are genetically similar.
- the second subject expresses the same HLA class or supertype as the first subject.
- the subject has been treated with a therapeutic agent targeting the disease or condition, e.g. the tumor, prior to administration of the cells or composition containing the cells.
- the subject is refractory or non-responsive to the other therapeutic agent.
- the subject has persistent or relapsed disease, e.g., following treatment with another therapeutic intervention, including chemotherapy, radiation, and/or hematopoietic stem cell transplantation (HSCT), e.g., allogenic HSCT.
- the administration effectively treats the subject despite the subject having become resistant to another therapy.
- the subject is responsive to the other therapeutic agent, and treatment with the therapeutic agent reduces disease burden.
- the subject is initially responsive to the therapeutic agent, but exhibits a relapse of the disease or condition over time. In some embodiments, the subject has not relapsed.
- the subject is determined to be at risk for relapse, such as at a high risk of relapse, and thus the cells are administered prophylactically, e.g., to reduce the likelihood of or prevent relapse.
- the subject has not received prior treatment with another therapeutic agent.
- the subject has persistent or relapsed disease, e.g., following treatment with another therapeutic intervention, including chemotherapy, radiation, and/or hematopoietic stem cell transplantation (HSCT), e.g., allogenic HSCT.
- the administration effectively treats the subject despite the subject having become resistant to another therapy.
- the modified immune cells of the present invention can be administered to an animal, preferably a mammal, even more preferably a human, to treat a cancer.
- the cells of the present invention can be used for the treatment of any condition related to a cancer, especially a cell-mediated immune response against a tumor cell(s), where it is desirable to treat or alleviate the disease.
- the types of cancers to be treated with the modified cells or pharmaceutical compositions of the invention include, carcinoma, blastoma, and sarcoma, and certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignancies e.g., sarcomas, carcinomas, and melanomas.
- cancers include but are not limited breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin 41744090.2 Attorney Docket No.046483-7386WO1(03225) cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, thyroid cancer, and the like.
- the cancers may be non-solid tumors (such as hematological tumors) or solid tumors.
- the cancer is a solid tumor or a hematological tumor.
- the cancer is a carcinoma.
- the cancer is a sarcoma.
- the cancer is a leukemia.
- the cancer is a solid tumor.
- Solid tumors are abnormal masses of tissue that usually do not contain cysts or liquid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named for the type of cells that form them (such as sarcomas, carcinomas, and lymphomas).
- solid tumors such as sarcomas and carcinomas
- solid tumors include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms
- Carcinomas that can be amenable to therapy by a method disclosed herein include, but are not limited to, esophageal carcinoma, hepatocellular carcinoma, basal cell carcinoma (a form of skin cancer), squamous cell carcinoma (various tissues), bladder carcinoma, including transitional cell carcinoma (a malignant neoplasm of the bladder), bronchogenic carcinoma, colon carcinoma, colorectal carcinoma, gastric carcinoma, lung carcinoma, including small cell carcinoma and non-small cell carcinoma of the lung, adrenocortical carcinoma, thyroid carcinoma, pancreatic carcinoma, breast carcinoma, ovarian carcinoma, prostate carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, 41744090.2 Attorney Docket No.046483-7386WO1(03225) papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, renal cell carcinoma, ductal carcinoma in situ or bile duct carcinoma, choriocarcinoma, seminoma
- Sarcomas that can be amenable to therapy by a method disclosed herein include, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, chordoma, osteogenic sarcoma, osteosarcoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, and other soft tissue sarcomas.
- the modified immune cells of the invention are used to treat a myeloma, or a condition related to myeloma.
- myeloma or conditions related thereto include, without limitation, light chain myeloma, non-secretory myeloma, monoclonal gamopathy of undertermined significance (MGUS), plasmacytoma (e.g., solitary, multiple solitary, extramedullary plasmacytoma), amyloidosis, and multiple myeloma.
- MGUS monoclonal gamopathy of undertermined significance
- plasmacytoma e.g., solitary, multiple solitary, extramedullary plasmacytoma
- amyloidosis e.g., solitary, multiple solitary, extramedullary plasmacytoma
- multiple myeloma e.g., solitary, multiple solitary, extramedullary plasmacytoma
- a method of the present disclosure is used to treat relapsed myeloma.
- the modified immune cells of the invention are used to treat a melanoma, or a condition related to melanoma.
- melanoma or conditions related thereto include, without limitation, superficial spreading melanoma, nodular melanoma, lentigo maligna melanoma, acral lentiginous melanoma, amelanotic melanoma, or melanoma of the skin (e.g., cutaneous, eye, vulva, vagina, rectum melanoma).
- a method of the present disclosure is used to treat cutaneous melanoma. In one embodiment, a method of the present disclosure is used to treat refractory melanoma. In one embodiment, a method of the present disclosure is used to treat relapsed melanoma. In yet other exemplary embodiments, the modified immune cells of the invention are used to treat a sarcoma, or a condition related to sarcoma.
- sarcoma or conditions related thereto include, without limitation, angiosarcoma, chondrosarcoma, Ewing’s sarcoma, fibrosarcoma, gastrointestinal stromal tumor, leiomyosarcoma, liposarcoma, malignant peripheral nerve sheath tumor, osteosarcoma, pleomorphic sarcoma, rhabdomyosarcoma, and synovial sarcoma.
- a method of the present disclosure is used to treat 41744090.2 Attorney Docket No.046483-7386WO1(03225) synovial sarcoma.
- a method of the present disclosure is used to treat liposarcoma such as myxoid/round cell liposarcoma, differentiated/dedifferentiated liposarcoma, and pleomorphic liposarcoma.
- a method of the present disclosure is used to treat myxoid/round cell liposarcoma.
- a method of the present disclosure is used to treat a refractory sarcoma.
- a method of the present disclosure is used to treat a relapsed sarcoma.
- the cells of the invention to be administered may be autologous, with respect to the subject undergoing therapy.
- the administration of the cells of the invention may be carried out in any convenient manner known to those of skill in the art.
- the cells of the present invention may be administered to a subject by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation.
- the compositions described herein may be administered to a patient transarterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally.
- the cells of the invention are injected directly into a site of inflammation in the subject, a local disease site in the subject, alymph node, an organ, a tumor, and the like.
- the cells, or individual populations of sub-types of cells are administered to the subject at a range of about one million to about 100 billion cells, such as, e.g., 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), such as about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values), and in some cases about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million
- the dose of total cells and/or dose of individual sub-populations of cells is within a range of between at or about 1x10 5 cells/kg to about 1x10 11 cells/kg 10 4 and at or 41744090.2 Attorney Docket No.046483-7386WO1(03225) about 10 11 cells/kilograms (kg) body weight, such as between 10 5 and 10 6 cells / kg body weight, for example, at or about 1 x 10 5 cells/kg, 1.5 x 10 5 cells/kg, 2 x 10 5 cells/kg, or 1 x 10 6 cells/kg body weight.
- the cells are administered at, or within a certain range of error of, between at or about 10 4 and at or about 10 9 T cells/kilograms (kg) body weight, such as between 10 5 and 10 6 T cells / kg body weight, for example, at or about 1 x 10 5 T cells/kg, 1.5 x 10 5 T cells/kg, 2 x 10 5 T cells/kg, or 1 x 10 6 T cells/kg body weight.
- a suitable dosage range of modified cells for use in a method of the present disclosure includes, without limitation, from about 1x10 5 cells/kg to about 1x10 6 cells/kg, from about 1x10 6 cells/kg to about 1x10 7 cells/kg, from about 1x10 7 cells/kg about 1x10 8 cells/kg, from about 1x10 8 cells/kg about 1x10 9 cells/kg, from about 1x10 9 cells/kg about 1x10 10 cells/kg, from about 1x10 10 cells/kg about 1x10 11 cells/kg.
- a suitable dosage for use in a method of the present disclosure is about 1x10 8 cells/kg.
- a suitable dosage for use in a method of the present disclosure is about 1x10 7 cells/kg. In other embodiments, a suitable dosage is from about 1x10 7 total cells to about 5x10 7 total cells. In some embodiments, a suitable dosage is from about 1x10 8 total cells to about 5x10 8 total cells. In some embodiments, a suitable dosage is from about 1.4x10 7 total cells to about 1.1x10 9 total cells. In an exemplary embodiment, a suitable dosage for use in a method of the present disclosure is about 7x10 9 total cells. In some embodiments, a dose of modified cells is administered to a subject in need thereof, in a single dose or multiple doses.
- a dose of modified cells is administered in multiple doses, e.g., once a week or every 7 days, once every 2 weeks or every 14 days, once every 3 weeks or every 21 days, once every 4 weeks or every 28 days.
- a single dose of modified cells is administered to a subject in need thereof.
- a single dose of modified cells is administered to a subject in need thereof by rapid intravenous infusion.
- the appropriate dosage may depend on the type of disease to be treated, the type of cells or recombinant receptors, the severity and course of the disease, whether the cells are administered for preventive or therapeutic purposes, previous therapy, the subject's clinical history and response to the cells, and the discretion of the attending physician.
- compositions and cells are in some embodiments suitably administered to the subject at one time or over a series of treatments. 41744090.2 Attorney Docket No.046483-7386WO1(03225)
- the cells are administered as part of a combination treatment, such as simultaneously with or sequentially with, in any order, another therapeutic intervention, such as an antibody or engineered cell or receptor or agent, such as a cytotoxic or therapeutic agent.
- another therapeutic intervention such as an antibody or engineered cell or receptor or agent, such as a cytotoxic or therapeutic agent.
- the cells in some embodiments are co-administered with one or more additional therapeutic agents or in connection with another therapeutic intervention, either simultaneously or sequentially in any order.
- the cells are co-administered with another therapy sufficiently close in time such that the cell populations enhance the effect of one or more additional therapeutic agents, or vice versa.
- the cells are administered prior to the one or more additional therapeutic agents. In some embodiments, the cells are administered after the one or more additional therapeutic agents. In some embodiments, the one or more additional agents includes a cytokine, such as IL-2, for example, to enhance persistence. In some embodiments, the methods comprise administration of a chemotherapeutic agent. Following administration of the cells, the biological activity of the engineered cell populations in some embodiments is measured, e.g., by any of a number of known methods. Parameters to assess include specific binding of an engineered or natural T cell or other immune cell to antigen, in vivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flow cytometry.
- the ability of the engineered cells to destroy target cells can be measured using any suitable method known in the art, such as cytotoxicity assays described in, for example, Kochenderfer et al., J. Immunotherapy, 32(7): 689-702 (2009), and Herman et al. J. Immunological Methods, 285(1): 25-40 (2004).
- the biological activity of the cells is measured by assaying expression and/or secretion of one or more cytokines, such as CD 107a, IFN ⁇ , IL-2, and TNF.
- the biological activity is measured by assessing clinical outcome, such as reduction in tumor burden or load.
- the subject is provided a secondary or alternative treatment.
- Secondary/alternative treatments include but are not limited to chemotherapy, radiation, surgery, and medications.
- the subject can be administered a conditioning therapy , such as a lymphodepletion step, prior to adoptive cell therapy.
- the conditioning therapy comprises administering an effective amount of cyclophosphamide to the subject.
- the conditioning therapy comprises administering an effective amount of fludarabine to the subject.
- the conditioning therapy comprises 41744090.2 Attorney Docket No.046483-7386WO1(03225) administering an effective amount of a combination of cyclophosphamide and fludarabine to the subject.
- Administration of a conditioning therapy prior to CAR T cell therapy may increase the efficacy of the CAR T cell therapy.
- the present disclosure provides methods for producing or generating a modified immune cell or precursor thereof (e.g., CAR T cells comprising CH25H) of the invention for tumor immunotherapy, e.g., adoptive immunotherapy.
- the cells generally are engineered by introducing one or more genetically engineered nucleic acids encoding a CAR and CH25H.
- the invention provides a method for generating a modified immune cell or precursor cell thereof, comprising introducing into an immune or precursor cell a a nucleic acid encoding a CAR and a CH25H gene.
- the CAR and CH25H are introduced into a cell by an expression vector.
- Expression vectors comprising a nucleic acid sequence encoding a CAR of the present invention are provided herein.
- Suitable expression vectors include lentivirus vectors, gamma retrovirus vectors, foamy virus vectors, adeno associated virus (AAV) vectors, adenovirus vectors, engineered hybrid viruses, naked DNA, including but not limited to transposon mediated vectors, such as Sleeping Beauty, Piggybak, and Integrases such as Phi31.
- Some other suitable expression vectors include Herpes simplex virus (HSV) and retrovirus expression vectors.
- the nucleic acid encoding a CAR and a CH25H gene is introduced into the cell via viral transduction.
- the viral transduction comprises contacting the immune or precursor cell with a viral vector comprising the nucleic acid encoding a CAR and a CH25H gene.
- the viral vector is an adeno- associated viral (AAV) vector.
- the AAV vector comprises a Woodchuck Hepatitis Virus post-transcriptional regulatory element (WPRE).
- WPRE Woodchuck Hepatitis Virus post-transcriptional regulatory element
- the AAV vector comprises a polyadenylation (polyA) sequence.
- the polyA sequence is a bovine growth hormone (BGH) polyA sequence.
- Adenovirus expression vectors are based on adenoviruses, which have a low capacity for integration into genomic DNA but a high efficiency for transfecting host cells.
- Adenovirus expression vectors contain adenovirus sequences sufficient to: (a) support packaging of the 41744090.2 Attorney Docket No.046483-7386WO1(03225) expression vector and (b) to ultimately express the CAR in the host cell.
- the adenovirus genome is a 36 kb, linear, double stranded DNA, where a foreign DNA sequence (e.g., a nucleic acid encoding a CAR) may be inserted to substitute large pieces of adenoviral DNA in order to make the expression vector of the present invention (see, e.g., Danthinne and Imperiale, Gene Therapy (2000) 7(20): 1707-1714).
- AAV adeno associated virus
- retrovirus expression vectors are capable of integrating into the host genome, delivering a large amount of foreign genetic material, infecting a broad spectrum of species and cell types and being packaged in special cell lines.
- the retroviral vector is constructed by inserting a nucleic acid (e.g., a nucleic acid encoding a CAR and a CH25H gene) into the viral genome at certain locations to produce a virus that is replication defective.
- a nucleic acid e.g., a nucleic acid encoding a CAR and a CH25H gene
- Lentiviral vectors are derived from lentiviruses, which are complex retroviruses that, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural function (see, e.g., U.S. Patent Nos.6,013,516 and 5,994, 136).
- Lentiviral vectors have been generated by multiply attenuating the HIV virulence genes, for example, the genes env, vif, vpr, vpu and nef are deleted making the vector biologically safe. Lentiviral vectors are capable of infecting non-dividing cells and can be used for both in vivo and ex vivo gene transfer and expression, e.g., of a nucleic acid encoding a CAR (see, e.g., U.S. Patent No.5,994,136).
- Expression vectors including a nucleic acid of the present disclosure can be introduced into a host cell by any means known to persons skilled in the art.
- the expression vectors may include viral sequences for transfection, if desired.
- the expression vectors may be introduced by fusion, electroporation, biolistics, transfection, lipofection, or the like.
- the host 41744090.2 Attorney Docket No.046483-7386WO1(03225) cell may be grown and expanded in culture before introduction of the expression vectors, followed by the appropriate treatment for introduction and integration of the vectors. The host cells are then expanded and may be screened by virtue of a marker present in the vectors.
- the host cell an immune cell or precursor thereof, e.g., a T cell, an NK cell, or an NKT cell.
- the present invention also provides genetically engineered cells which include and stably express a CAR and CH25H.
- the genetically engineered cells are genetically engineered T-lymphocytes (T cells), naive T cells (TN), memory T cells (for example, central memory T cells (TCM), effector memory cells (TEM)), natural killer cells (NK cells), and macrophages capable of giving rise to therapeutically relevant progeny.
- T cells genetically engineered T-lymphocytes
- TN naive T cells
- TCM central memory T cells
- TEM effector memory cells
- NK cells natural killer cells
- macrophages capable of giving rise to therapeutically relevant progeny.
- the genetically engineered cells are autologous cells.
- the modified cell is resistant to T cell exhaustion.
- Modified cells e.g., comprising a CAR and CH25H
- Additional methods for generating a modified cell of the present disclosure include, without limitation, chemical transformation methods (e.g., using calcium phosphate, dendrimers, liposomes and/or cationic polymers), non-chemical transformation methods (e.g., electroporation, optical transformation, gene electrotransfer and/or hydrodynamic delivery) and/or particle-based methods (e.g., impalefection, using a gene gun and/or magnetofection).
- Transfected cells expressing a CAR and CH25H of the present disclosure may be expanded ex vivo.
- Physical methods for introducing an expression vector into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like.
- dimyristyl phosphatidylcholine can be obtained from Sigma, St. Louis, MO; dicetyl phosphate (“DCP”) can be obtained from K & K Laboratories (Plainview, NY); cholesterol (“Choi”) can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL).
- Stock solutions of lipids in chloroform or chloroform/methanol can be stored at about -200C. Chloroform may be used as the only solvent since it is more readily evaporated than methanol.
- Liposome is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10).
- compositions that have different structures in solution than the normal vesicular structure are also encompassed.
- the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules.
- lipofectamine-nucleic acid complexes are also contemplated. Regardless of the method used to introduce exogenous nucleic acids into a host cell or otherwise expose a cell to the inhibitor of the present invention, in order to confirm the presence of the nucleic acids in the host cell, a variety of assays may be performed.
- the nucleic acids introduced into the host cell are RNA.
- the RNA is mRNA that comprises in vitro transcribed RNA or synthetic RNA. The RNA may be produced by in vitro transcription using a polymerase chain reaction (PCR)- generated template.
- PCR polymerase chain reaction
- DNA of interest from any source can be directly converted by PCR into a template for in vitro mRNA synthesis using appropriate primers and RNA polymerase.
- the 41744090.2 Attorney Docket No.046483-7386WO1(03225) source of the DNA may be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence or any other appropriate source of DNA.
- PCR may be used to generate a template for in vitro transcription of mRNA which is then introduced into cells. Methods for performing PCR are well known in the art. Primers for use in PCR are designed to have regions that are substantially complementary to regions of the DNA to be used as a template for the PCR.
- “Substantially complementary,” as used herein, refers to sequences of nucleotides where a majority or all of the bases in the primer sequence are complementary. Substantially complementary sequences are able to anneal or hybridize with the intended DNA target under annealing conditions used for PCR.
- the primers can be designed to be substantially complementary to any portion of the DNA template. For example, the primers can be designed to amplify the portion of a gene that is normally transcribed in cells (the open reading frame), including 5' and 3' UTRs. The primers may also be designed to amplify a portion of a gene that encodes a particular domain of interest.
- the primers are designed to amplify the coding region of a human cDNA, including all or portions of the 5' and 3' UTRs.
- Primers useful for PCR are generated by synthetic methods that are well known in the art.
- “Forward primers” are primers that contain a region of nucleotides that are substantially complementary to nucleotides on the DNA template that are upstream of the DNA sequence that is to be amplified.
- Upstream is used herein to refer to a location 5, to the DNA sequence to be amplified relative to the coding strand.
- “Reverse primers” are primers that contain a region of nucleotides that are substantially complementary to a double-stranded DNA template that are downstream of the DNA sequence that is to be amplified.
- Downstream is used herein to refer to a location 3' to the DNA sequence to be amplified relative to the coding strand. Chemical structures that have the ability to promote stability and/or translation efficiency of the RNA may also be used.
- the RNA preferably has 5' and 3' UTRs. In one embodiment, the 5' UTR is between zero and 3000 nucleotides in length.
- the length of 5' and 3' UTR sequences to be added to the coding region can be altered by different methods, including, but not limited to, designing primers for PCR that anneal to different regions of the UTRs. Using this approach, one of ordinary skill in the art can modify the 5' and 3' UTR lengths required to achieve optimal translation efficiency following transfection of the transcribed RNA.
- the 5' and 3' UTRs can be the naturally occurring, endogenous 5' and 3' UTRs for the gene of interest.
- UTR sequences that are not endogenous to the gene of interest 41744090.2 Attorney Docket No.046483-7386WO1(03225) can be added by incorporating the UTR sequences into the forward and reverse primers or by any other modifications of the template.
- the use of UTR sequences that are not endogenous to the gene of interest can be useful for modifying the stability and/or translation efficiency of the RNA. For example, it is known that AU-rich elements in 3' UTR sequences can decrease the stability of mRNA.
- 3' UTRs can be selected or designed to increase the stability of the transcribed RNA based on properties of UTRs that are well known in the art.
- the 5' UTR can contain the Kozak sequence of the endogenous gene.
- a consensus Kozak sequence can be redesigned by adding the 5' UTR sequence.
- Kozak sequences can increase the efficiency of translation of some RNA transcripts, but does not appear to be required for all RNAs to enable efficient translation. The requirement for Kozak sequences for many mRNAs is known in the art.
- the 5' UTR can be derived from an RNA virus whose RNA genome is stable in cells.
- various nucleotide analogues can be used in the 3' or 5' UTR to impede exonuclease degradation of the mRNA.
- a promoter of transcription should be attached to the DNA template upstream of the sequence to be transcribed. When a sequence that functions as a promoter for an RNA polymerase is added to the 5' end of the forward primer, the RNA polymerase promoter becomes incorporated into the PCR product upstream of the open reading frame that is to be transcribed.
- the promoter is a T7 polymerase promoter, as described elsewhere herein.
- Other useful promoters include, but are not limited to, T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for T7, T3 and SP6 promoters are known in the art.
- the mRNA has both a cap on the 5' end and a 3' poly(A) tail which determine ribosome binding, initiation of translation and stability mRNA in the cell.
- RNA polymerase produces a long concatameric product which is not suitable for expression in eukaryotic cells.
- phage T7 RNA polymerase can extend the 3' end of the transcript beyond the last base of the template (Schenborn and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003).
- the polyA/T segment of the transcriptional DNA template can be produced during PCR by using a reverse primer containing a polyT tail, such as 100T tail (size can be 50-5000 T), or after PCR by any other method, including, but not limited to, DNA ligation or in vitro recombination.
- Poly(A) tails also provide stability to RNAs and reduce their degradation. Generally, the length of a poly(A) tail positively correlates with the stability of the transcribed RNA. In one embodiment, the poly(A) tail is between 100 and 5000 adenosines.
- Poly(A) tails of RNAs can be further extended following in vitro transcription with the use of a poly(A) polymerase, such as E.
- RNAs produced by the methods disclosed herein include a 5' cap.
- the 5' cap is provided using techniques known in the art and described herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444 (2001); Stepinski, et al., RNA, 7:1468-95 (2001); Elango, et al., Biochim. Biophys. Res. Commun., 330:958-966 (2005)).
- the RNA is electroporated into the cells, such as in vitro transcribed RNA. Any solutes suitable for cell electroporation, which can contain factors facilitating cellular permeability and viability such as sugars, peptides, lipids, proteins, antioxidants, and surfactants can be included.
- a nucleic acid encoding a CAR of the present disclosure will be RNA, e.g., in vitro synthesized RNA.
- Methods for in vitro synthesis of RNA are known in the art; any known method can be used to synthesize RNA comprising a sequence encoding a CAR.
- Methods for introducing RNA into a host cell are known in the art. See, e.g., Zhao et al. Cancer Res. (2010) 15: 9053.
- Introducing RNA comprising a nucleotide sequence encoding a CAR into 41744090.2 Attorney Docket No.046483-7386WO1(03225) a host cell can be carried out in vitro, ex vivo or in vivo.
- a host cell e.g., an NK cell, a cytotoxic T lymphocyte, etc.
- RNA comprising a nucleotide sequence encoding a CAR and CH25H.
- the disclosed methods can be applied to the modulation of T cell activity in basic research and therapy, in the fields of cancer, stem cells, acute and chronic infections, and autoimmune diseases, including the assessment of the ability of the genetically modified T cell to kill a target cancer cell.
- the methods also provide the ability to control the level of expression over a wide range by changing, for example, the promoter or the amount of input RNA, making it possible to individually regulate the expression level.
- RNA transfection is essentially transient and a vector-free.
- An RNA transgene can be delivered to a lymphocyte and expressed therein following a brief in vitro cell activation, as a minimal expressing cassette without the need for any additional viral sequences. Under these conditions, integration of the transgene into the host cell genome is unlikely. Cloning of cells is not necessary because of the efficiency of transfection of the RNA and its ability to uniformly modify the entire lymphocyte population.
- IVT-RNA in vitro-transcribed RNA
- RNA polymerase promoter enabling RNA transcription, followed by a gene of interest which is flanked either 3' and/or 5' by untranslated regions (UTR), and a 3' polyadenyl cassette containing 50-70 A nucleotides.
- UTR untranslated regions
- 3' polyadenyl cassette containing 50-70 A nucleotides.
- the circular plasmid Prior to in vitro transcription, the circular plasmid is linearized downstream of the polyadenyl cassette by type II 41744090.2 Attorney Docket No.046483-7386WO1(03225) restriction enzymes (recognition sequence corresponds to cleavage site).
- the polyadenyl cassette thus corresponds to the later poly(A) sequence in the transcript.
- the RNA construct is delivered into the cells by electroporation. See, e.g., the formulations and methodology of electroporation of nucleic acid constructs into mammalian cells as taught in US 2004/0014645, US 2005/0052630A1, US 2005/0070841A1, US 2004/0059285A1, US 2004/0092907A1.
- electroporation may also be used for transfection of cells in vitro as described e.g. in US20070128708A1. Electroporation may also be utilized to deliver nucleic acids into cells in vitro. Accordingly, electroporation-mediated administration into cells of nucleic acids including expression constructs utilizing any of the many available devices and electroporation systems known to those of skill in the art presents an exciting new means for delivering an RNA of interest to a target cell.
- the immune cells e.g.
- T cells can be incubated or cultivated prior to, during and/or subsequent to introducing the nucleic acid molecule encoding the CAR and CH25H gene.
- the cells e.g. T cells
- the cells can be incubated or cultivated prior to, during or subsequent to the introduction of the nucleic acid molecule, such as prior to, during or subsequent to the transduction of the cells with a viral vector (e.g. lentiviral vector) encoding the CAR and CH25H gene.
- a viral vector e.g. lentiviral vector
- a nucleic acid of the present disclosure is provided for the production of a CAR and CH25H as described herein, e.g., in a mammalian cell.
- a nucleic acid of the present disclosure provides for amplification of the CAR and CH25H-encoding nucleic acid.
- the nucleic acid comprises a linker sequence.
- the linker comprises a nucleic acid sequence that encodes for a self-cleaving peptide.
- a “self-cleaving peptide” or “2A peptide” refers to an oligopeptide that allow multiple proteins to be encoded as polyproteins, which dissociate into component proteins upon translation. Use of the term “self-cleaving” is not intended to imply a proteolytic cleavage reaction.
- 2A peptides are known to those of skill in the art, including, without limitation, those found in members of the Picornaviridae virus family, e.g., foot-and- mouth disease virus (FMDV), equine rhinitis A virus (ERAV0, Thosea asigna virus (TaV), and porcine tescho virus-1 (PTV-1); and carioviruses such as Theilovirus and encephalomyocarditis viruses.
- FMDV foot-and- mouth disease virus
- EAV0 equine rhinitis A virus
- TaV Thosea asigna virus
- PTV-1 porcine tescho virus-1
- carioviruses such as Theilovirus and encephalomyocarditis viruses.
- 2A peptides derived from FMDV, ERAV, PTV-1, and TaV are referred to herein as “F2A,” “E2A,” “P2A,” and “T2A,” respectively.
- the linker comprises a nucleic acid sequence encoding a combination of a Furin cleavage site and a 2A peptide.
- examples include, without limitation, a linker comprising a nucleic acid sequence encoding Furin and F2A, a linker comprising a nucleic acid sequence encoding Furin and E2A, a linker comprising a nucleic acid sequence encoding Furin and P2A, a linker comprising a nucleic acid sequence encoding Furin and T2A.
- Those of skill in the art would be able to select the appropriate combination for use in the present invention.
- the linker may further comprise a spacer sequence between the Furin and 2A peptide.
- spacer sequences are known in the art, including, without limitation, glycine serine (GS) spacers such as (GS)n, (GSGGS)n (SEQ ID NO:21) and (GGGS)n (SEQ ID NO:22), where n represents an integer of at least 1.
- Exemplary spacer sequences can comprise amino acid sequences including, without limitation, GGSG (SEQ ID NO:24), GGSGG (SEQ ID NO:25), GSGSG (SEQ ID NO:26), GSGGG (SEQ ID NO:27), GGGSG (SEQ ID NO:28), GSSSG (SEQ ID NO:29), and the like.
- a nucleic acid of the present disclosure may be operably linked to a transcriptional control element, e.g., a promoter, and enhancer, etc. Suitable promoter and enhancer elements are known to those of skill in the art.
- the nucleic acid encoding a CAR is in operable linkage with a promoter.
- the promoter is a phosphoglycerate kinase-1 (PGK) promoter.
- suitable promoters include, but are not limited to, lacI, lacZ, T3, T7, gpt, lambda P and trc.
- suitable promoters include, but are not limited to, light and/or heavy chain immunoglobulin gene promoter and enhancer elements; cytomegalovirus immediate early promoter; herpes simplex virus thymidine kinase promoter; early and late SV40 promoters; promoter present in long terminal repeats from a retrovirus; mouse metallothionein-I promoter; and various art-known tissue specific promoters.
- Suitable reversible promoters including reversible inducible promoters are known in the art. Such reversible promoters may be isolated and derived from many organisms, e.g., eukaryotes and prokaryotes. Modification of reversible promoters derived from a first organism for use in a second organism, e.g., a first prokaryote and a second a eukaryote, a first eukaryote and a second a prokaryote, etc., is well known in the art.
- Such reversible promoters, and systems based on such reversible promoters but also comprising additional control proteins include, but are not limited to, alcohol regulated promoters (e.g., alcohol dehydrogenase I (alcA) gene promoter, promoters responsive to alcohol transactivator proteins (A1cR), etc.), tetracycline regulated promoters, (e.g., promoter systems including TetActivators, TetON, TetOFF, etc.), steroid regulated promoters (e.g., rat glucocorticoid receptor promoter systems, human estrogen receptor promoter systems, retinoid promoter systems, thyroid promoter systems, ecdysone promoter systems, mifepristone promoter systems, etc.), metal regulated promoters (e.g., metallothionein promoter systems, etc.), pathogenesis-related regulated promoters (e.g., salicylic acid regulated promoters, ethylene regulated promoters
- the promoter is a CD8 cell-specific promoter, a CD4 cell-specific promoter, a neutrophil-specific promoter, or an NK-specific promoter.
- a CD4 gene promoter can be used; see, e.g., Salmon et al. Proc. Natl. Acad. Sci. USA (1993) 90:7739; and 41744090.2 Attorney Docket No.046483-7386WO1(03225) Marodon et al. (2003) Blood 101:3416.
- a CD8 gene promoter can be used.
- NK cell-specific expression can be achieved by use of an NcrI (p46) promoter; see, e.g., Eckelhart et al.
- a suitable promoter is a constitutive promoter such as an ADH1 promoter, a PGK1 promoter, an ENO promoter, a PYK1 promoter and the like; or a regulatable promoter such as a GAL1 promoter, a GAL10 promoter, an ADH2 promoter, a PHOS promoter, a CUP1 promoter, a GALT promoter, a MET25 promoter, a MET3 promoter, a CYC1 promoter, a HIS3 promoter, an ADH1 promoter, a PGK promoter, a GAPDH promoter, an ADC1 promoter, a TRP1 promoter, a URA3 promoter, a LEU2 promoter, an ENO promoter, a TP1 promoter, and AOX1 (e.g., for use in Pichia).
- a constitutive promoter such as an ADH1 promoter, a PGK1 promoter, an ENO promoter,
- Suitable promoters for use in prokaryotic host cells include, but are not limited to, a bacteriophage T7 RNA polymerase promoter; a trp promoter; a lac operon promoter; a hybrid promoter, e.g., a lac/tac hybrid promoter, a tac/trc hybrid promoter, a trp/lac promoter, a T7/lac promoter; a trc promoter; a tac promoter, and the like; an araBAD promoter; in vivo regulated promoters, such as an ssaG promoter or a related promoter (see, e.g., U.S.
- Patent Publication No.20040131637 discloses a pagC promoter (Pulkkinen and Miller, J. Bacteriol. (1991) 173(1): 86-93; Alpuche-Aranda et al., Proc. Natl. Acad. Sci. USA (1992) 89(21): 10079-83), a nirB promoter (Harborne et al. Mol. Micro. (1992) 6:2805-2813), and the like (see, e.g., Dunstan et al., Infect. Immun. (1999) 67:5133-5141; McKelvie et al., Vaccine (2004) 22:3243-3255; and Chatfield et al., Biotechnol.
- sigma70 promoter e.g., a consensus sigma70 promoter (see, e.g., GenBank Accession Nos. AX798980, AX798961, and AX798183); a stationary phase promoter, e.g., a dps promoter, an spv promoter, and the like; a promoter derived from the pathogenicity island SPI-2 (see, e.g., WO96/17951); an actA promoter (see, e.g., Shetron-Rama et al., Infect. Immun.
- rpsM promoter see, e.g., Valdivia and Falkow Mol. Microbiol. (1996).22:367)
- a tet promoter see, e.g., Hillen, W. and Wissmann, A. (1989) In Saenger, W. and Heinemann, U. (eds), Topics in Molecular and Structural Biology, Protein--Nucleic Acid Interaction. Macmillan, London, UK, Vol.10, pp.143-162
- SP6 promoter see, e.g., Melton et al., Nucl. Acids Res. (1984) 12:7035); and the like.
- Suitable strong promoters for use in prokaryotes such as Escherichia coli include, but are not limited to Trc, Tac, T5, T7, and PLambda.
- operators for use in bacterial host cells include a lactose promoter operator (LacI 41744090.2 Attorney Docket No.046483-7386WO1(03225) repressor protein changes conformation when contacted with lactose, thereby preventing the Lad repressor protein from binding to the operator), a tryptophan promoter operator (when complexed with tryptophan, TrpR repressor protein has a conformation that binds the operator; in the absence of tryptophan, the TrpR repressor protein has a conformation that does not bind to the operator), and a tac promoter operator (see, e.g., deBoer et al., Proc.
- CMV immediate early cytomegalovirus
- constitutive promoter sequences may also be used, including, but not limited to a simian virus 40 (SV40) early promoter, a mouse mammary tumor virus (MMTV) or human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, a MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, the EF-1 alpha promoter, as well as human gene promoters such as, but not limited to, an actin promoter, a myosin promoter, a hemoglobin promoter, and a creatine kinase promoter. Further, the invention should not be limited to the use of constitutive promoters.
- SV40 simian virus 40
- MMTV mouse mammary tumor virus
- HV human immunodeficiency virus
- LTR long terminal repeat
- MoMuLV promoter an avian leukemia virus promoter
- inducible promoters are also contemplated as part of the invention.
- the use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired.
- inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.
- the locus or construct or transgene containing the suitable promoter is irreversibly switched through the induction of an inducible system.
- Suitable systems for induction of an irreversible switch are well known in the art, e.g., induction of an irreversible switch may make use of a Cre-lox-mediated recombination (see, e.g., Fuhrmann-Benzakein, et al., Proc. Natl. Acad. Sci. USA (2000) 28:e99, the disclosure of which is incorporated herein by reference). Any suitable combination of recombinase, endonuclease, ligase, recombination sites, etc. known to the art may be used in generating an irreversibly switchable promoter.
- a nucleic acid of the present disclosure further comprises a nucleic acid sequence encoding a CAR inducible expression cassette.
- the inducible expression cassette is for the production of a transgenic polypeptide product that is released upon CAR signaling. See, e.g., Chmielewski and Abken, Expert Opin. Biol. Ther. (2015) 15(8): 1145- 1154; and Abken, Immunotherapy (2015) 7(5): 535-544.
- a nucleic acid of the present disclosure further comprises a nucleic acid sequence encoding a cytokine operably linked to a T-cell activation responsive promoter.
- the cytokine operably linked to a T-cell activation responsive promoter is present on a separate nucleic acid sequence.
- the cytokine is IL-12.
- a nucleic acid of the present disclosure may be present within an expression vector and/or a cloning vector.
- An expression vector can include a selectable marker, an origin of replication, and other features that provide for replication and/or maintenance of the vector.
- Suitable expression vectors include, e.g., plasmids, viral vectors, and the like. Large numbers of suitable vectors and promoters are known to those of skill in the art; many are commercially available for generating a subject recombinant construct.
- Bacterial Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden).
- Expression vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences encoding heterologous proteins.
- a selectable marker operative in the expression host may be present.
- Suitable expression vectors include, but are not limited to, viral vectors (e.g. viral vectors based on vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., Invest. Opthalmol. Vis. Sci.
- a retroviral vector e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus; and the like.
- Additional expression vectors suitable for use are, e.g., without limitation, a lentivirus vector, a gamma retrovirus vector, a foamy virus vector, an adeno-associated virus vector, an adenovirus vector, a pox virus vector, a herpes virus vector, an engineered hybrid virus vector, a transposon mediated vector, and the like.
- Viral vector technology is well known in the art and is described, for example, in Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY), and in other virology and molecular biology manuals.
- Viruses which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno- associated viruses, herpes viruses, and lentiviruses.
- a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No.6,326,193).
- an expression vector e.g., a lentiviral vector
- an immune cell or precursor thereof e.g., a T cell.
- an expression vector (e.g., a lentiviral vector) of the present invention may comprise a nucleic acid encoding for a CAR.
- the expression vector (e.g., lentiviral vector) will comprise additional elements that will aid in the functional expression of the CAR encoded therein.
- an expression vector comprising a nucleic acid encoding for a CAR further comprises a mammalian promoter.
- the vector further comprises an elongation-factor-1-alpha promoter (EF-1 ⁇ promoter).
- an EF-1 ⁇ promoter may increase the efficiency in expression of downstream transgenes (e.g., a CAR encoding 41744090.2 Attorney Docket No.046483-7386WO1(03225) nucleic acid sequence).
- Physiologic promoters e.g., an EF-1 ⁇ promoter
- Other physiological promoters suitable for use in a vector e.g., lentiviral vector are known to those of skill in the art and may be incorporated into a vector of the present invention.
- the vector (e.g., lentiviral vector) further comprises a non-requisite cis acting sequence that may improve titers and gene expression.
- a non-requisite cis acting sequence is the central polypurine tract and central termination sequence (cPPT/CTS) which is important for efficient reverse transcription and nuclear import.
- CPS central polypurine tract and central termination sequence
- Other non-requisite cis acting sequences are known to those of skill in the art and may be incorporated into a vector (e.g., lentiviral vector) of the present invention.
- the vector further comprises a posttranscriptional regulatory element. Posttranscriptional regulatory elements may improve RNA translation, improve transgene expression and stabilize RNA transcripts.
- a posttranscriptional regulatory element is the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE).
- WPRE woodchuck hepatitis virus posttranscriptional regulatory element
- a vector for the present invention further comprises a WPRE sequence.
- Various posttranscriptional regulator elements are known to those of skill in the art and may be incorporated into a vector (e.g., lentiviral vector) of the present invention.
- a vector of the present invention may further comprise additional elements such as a rev response element (RRE) for RNA transport, packaging sequences, and 5’ and 3’ long terminal repeats (LTRs).
- RRE rev response element
- LTRs long terminal repeats
- LTRs generally provide functions required for the expression of retroviral genes (e.g., promotion, initiation and polyadenylation of gene transcripts) and to viral replication.
- a vector (e.g., lentiviral vector) of the present invention includes a 3’ U3 deleted LTR.
- a vector (e.g., lentiviral vector) of the present invention may comprise any combination of the elements described herein to enhance the efficiency of functional expression of transgenes.
- a vector (e.g., lentiviral vector) of the present invention may comprise a WPRE sequence, cPPT sequence, RRE sequence, 5’LTR, 3’ U3 deleted LTR’ in addition to a nucleic acid encoding a CAR.
- Vectors of the present invention may be self-inactivating vectors.
- self-inactivating vector refers to vectors in which the 3’ LTR enhancer promoter region (U3 region) has been modified (e.g., by deletion or substitution).
- a self-inactivating vector may 41744090.2 Attorney Docket No.046483-7386WO1(03225) prevent viral transcription beyond the first round of viral replication. Consequently, a self- inactivating vector may be capable of infecting and then integrating into a host genome (e.g., a mammalian genome) only once, and cannot be passed further. Accordingly, self-inactivating vectors may greatly reduce the risk of creating a replication-competent virus.
- a nucleic acid of the present invention may be RNA, e.g., in vitro synthesized RNA.
- Methods for in vitro synthesis of RNA are known to those of skill in the art; any known method can be used to synthesize RNA comprising a sequence encoding a CAR of the present disclosure.
- Methods for introducing RNA into a host cell are known in the art. See, e.g., Zhao et al. Cancer Res. (2010) 15: 9053.
- Introducing RNA comprising a nucleotide sequence encoding a CAR of the present disclosure into a host cell can be carried out in vitro, ex vivo or in vivo.
- a host cell e.g., an NK cell, a cytotoxic T lymphocyte, etc.
- RNA comprising a nucleotide sequence encoding a CAR of the present disclosure.
- the expression vector to be introduced into a cell may also contain either a selectable marker gene or a reporter gene, or both, to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors.
- the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure.
- Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells.
- Useful selectable markers include, without limitation, antibiotic-resistance genes.
- Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences.
- a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assessed at a suitable time after the DNA has been introduced into the recipient cells.
- Suitable reporter genes may include, without limitation, genes encoding luciferase, beta- galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82).
- G. Sources of Immune Cells 41744090.2 Attorney Docket No.046483-7386WO1(03225) Prior to expansion, a source of immune cells can be obtained from a subject for ex vivo manipulation. Sources of target cells for ex vivo manipulation may also include, e.g., autologous or heterologous donor blood, cord blood, or bone marrow.
- the source of immune cells may be from the subject to be treated with the modified immune cells of the invention, e.g., the subject's blood, the subject's cord blood, or the subject's bone marrow.
- subjects include humans, dogs, cats, mice, rats, and transgenic species thereof.
- the subject is a human.
- Immune cells can be obtained from a number of sources, including blood, peripheral blood mononuclear cells, bone marrow, lymph node tissue, spleen tissue, umbilical cord, lymph, or lymphoid organs.
- Immune cells are cells of the immune system, such as cells of the innate or adaptive immunity, e.g., myeloid or lymphoid cells, including lymphocytes, typically T cells and/or NK cells.
- Other exemplary cells include stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs).
- the cells are human cells. With reference to the subject to be treated, the cells may be allogeneic and/or autologous.
- the cells typically are primary cells, such as those isolated directly from a subject and/or isolated from a subject and frozen.
- the immune cell is a T cell, e.g., a CD8+ T cell (e.g., a CD8+ naive T cell, central memory T cell, or effector memory T cell), a CD4+ T cell, a natural killer T cell (NKT cells), a regulatory T cell (Treg), a stem cell memory T cell, a lymphoid progenitor cell a hematopoietic stem cell, a natural killer cell (NK cell) or a dendritic cell.
- a CD8+ T cell e.g., a CD8+ naive T cell, central memory T cell, or effector memory T cell
- a CD4+ T cell e.g., a CD4+ T cell, a natural killer T cell (NKT cells), a regulatory T cell (Treg), a stem cell memory T cell, a lymphoid progenitor cell a hematopoietic stem cell, a natural killer cell (NK cell) or
- the cells are monocytes or granulocytes, e.g., myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and/or basophils.
- the target cell is an induced pluripotent stem (iPS) cell or a cell derived from an iPS cell, e.g., an iPS cell generated from a subject, manipulated to alter (e.g., induce a mutation in) or manipulate the expression of one or more target genes, and differentiated into, e.g., a T cell, e.g., a CD8+ T cell (e.g., a CD8+ naive T cell, central memory T cell, or effector memory T cell), a CD4+ T cell, a stem cell memory T cell, a lymphoid progenitor cell or a hematopoietic stem cell.
- iPS induced pluripotent stem
- the cells include one or more subsets of T cells or other cell types, such as whole T cell populations, CD4+ cells, CD8+ cells, and subpopulations thereof, such as those defined by function, activation state, maturity, potential for differentiation, expansion, recirculation, localization, and/or persistence capacities, antigen- specificity, type of antigen 41744090.2 Attorney Docket No.046483-7386WO1(03225) receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and/or degree of differentiation.
- T cells or other cell types such as whole T cell populations, CD4+ cells, CD8+ cells, and subpopulations thereof, such as those defined by function, activation state, maturity, potential for differentiation, expansion, recirculation, localization, and/or persistence capacities, antigen- specificity, type of antigen 41744090.2 Attorney Docket No.046483-7386WO1(03225) receptor, presence in a particular organ or compartment, marker or cytokine secretion
- T cells and/or of CD4+ and/or of CD8+ T cells are naive T (TN) cells, effector T cells (TEFF), memory T cells and sub-types thereof, such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM), or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa- associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha/beta T cells, and delta/gamma T cells.
- TN naive T
- TSCM stem cell memory T
- TCM central memory T
- TEM effector memory T
- TIL tumor-infiltrating lymphocyte
- any number of T cell lines available in the art may be used.
- the methods include isolating immune cells from the subject, preparing, processing, culturing, and/or engineering them.
- preparation of the engineered cells includes one or more culture and/or preparation steps.
- the cells for engineering as described may be isolated from a sample, such as a biological sample, e.g., one obtained from or derived from a subject.
- the subject from which the cell is isolated is one having the disease or condition or in need of a cell therapy or to which cell therapy will be administered.
- the subject in some embodiments is a human in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and/or engineered.
- the cells in some embodiments are primary cells, e.g., primary human cells.
- the samples include tissue, fluid, and other samples taken directly from the subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic engineering (e.g. transduction with viral vector), washing, and/or incubation.
- the biological sample can be a sample obtained directly from a biological source or a sample that is processed.
- Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.
- the sample from which the cells are derived or isolated is blood or a blood-derived sample, or is or is derived from an apheresis or leukapheresis product.
- exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, 41744090.2
- PBMCs peripheral blood mononuclear cells
- Samples include, in the context of cell therapy, e.g., adoptive cell therapy, samples from autologous and allogeneic sources.
- the cells are derived from cell lines, e.g., T cell lines.
- the cells in some embodiments are obtained from a xenogeneic source, for example, from mouse, rat, non- human primate, and pig.
- isolation of the cells includes one or more preparation and/or non-affinity based cell separation steps.
- cells are washed, centrifuged, and/or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich for desired components, lyse or remove cells sensitive to particular reagents.
- cells are separated based on one or more property, such as density, adherent properties, size, sensitivity and/or resistance to particular components.
- cells from the circulating blood of a subject are obtained, e.g., by apheresis or leukapheresis.
- the samples contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and/or platelets, and in some aspects contains cells other than red blood cells and platelets.
- the blood cells collected from the subject are washed, e.g., to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps.
- the cells are washed with phosphate buffered saline (PBS).
- a washing step is accomplished by tangential flow filtration (TFF) according to the manufacturer's instructions.
- the cells are resuspended in a variety of biocompatible buffers after washing.
- components of a blood cell sample are removed and the cells directly resuspended in culture media.
- the methods include density-based cell separation methods, such as the preparation of white blood cells from peripheral blood by lysing the red blood cells and centrifugation through a Percoll or Ficoll gradient.
- immune are obtained cells from the circulating blood of an individual are obtained by apheresis or leukapheresis.
- the apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets.
- the cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media, such as phosphate buffered saline (PBS) or wash solution lacks calcium and may lack magnesium or may lack 41744090.2 Attorney Docket No.046483-7386WO1(03225) many if not all divalent cations, for subsequent processing steps.
- PBS phosphate buffered saline
- wash solution lacks calcium and may lack magnesium or may lack 41744090.2
- Attorney Docket No.046483-7386WO1(03225) many if not all divalent cations, for subsequent processing steps.
- the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca-free, Mg-free PBS.
- the undesirable components of the apheresis sample may be removed and the cells directly resuspended in culture media.
- the isolation methods include the separation of different cell types based on the expression or presence in the cell of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acid. In some embodiments, any known method for separation based on such markers may be used.
- the separation is affinity- or immunoaffinity-based separation.
- the isolation in some aspects includes separation of cells and cell populations based on the cells' expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner.
- Such separation steps can be based on positive selection, in which the cells having bound the reagents are retained for further use, and/or negative selection, in which the cells having not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for further use.
- negative selection can be particularly useful where no antibody is available that specifically identifies a cell type in a heterogeneous population, such that separation is best carried out based on markers expressed by cells other than the desired population.
- the separation need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker.
- positive selection of or enrichment for cells of a particular type such as those expressing a marker, refers to increasing the number or percentage of such cells, but need not result in a complete absence of cells not expressing the marker.
- negative selection, removal, or depletion of cells of a particular type, such as those expressing a marker refers to decreasing the number or percentage of such cells, but need not result in a complete removal of all such cells.
- multiple rounds of separation steps are carried out, where the positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection.
- a single separation step can deplete cells expressing multiple markers simultaneously, such as by incubating cells with a 41744090.2 Attorney Docket No.046483-7386WO1(03225) plurality of antibodies or binding partners, each specific for a marker targeted for negative selection.
- multiple cell types can simultaneously be positively selected by incubating cells with a plurality of antibodies or binding partners expressed on the various cell types.
- one or more of the T cell populations is enriched for or depleted of cells that are positive for (marker+) or express high levels (marker hlgh ) of one or more particular markers, such as surface markers, or that are negative for (marker -) or express relatively low levels (marker low ) of one or more markers.
- specific subpopulations of T cells such as cells positive or expressing high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and/or CD45RO+ T cells, are isolated by positive or negative selection techniques.
- such markers are those that are absent or expressed at relatively low levels on certain populations of T cells (such as non-memory cells) but are present or expressed at relatively higher levels on certain other populations of T cells (such as memory cells).
- the cells such as the CD8+ cells or the T cells, e.g., CD3+ cells
- the cells are enriched for (i.e., positively selected for) cells that are positive or expressing high surface levels of CD45RO, CCR7, CD28, CD27, CD44, CD 127, and/or CD62L and/or depleted of (e.g., negatively selected for) cells that are positive for or express high surface levels of CD45RA.
- cells are enriched for or depleted of cells positive or expressing high surface levels of CD 122, CD95, CD25, CD27, and/or IL7-Ra (CD 127).
- CD8+ T cells are enriched for cells positive for CD45RO (or negative for CD45RA) and for CD62L.
- CD3+, CD28+ T cells can be positively selected using CD3/CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3/CD28 T Cell Expander).
- T cells are separated from a PBMC sample by negative selection of markers expressed on non-T cells, such as B cells, monocytes, or other white blood cells, such as CD 14.
- a CD4+ or CD8+ selection step is used to separate CD4+ helper and CD8+ cytotoxic T cells.
- Such CD4+ and CD8+ populations can be further sorted into sub- populations by positive or negative selection for markers expressed or expressed to a relatively higher degree on one or more naive, memory, and/or effector T cell subpopulations.
- CD8+ cells are further enriched for or depleted of naive, central memory, effector memory, and/or central memory stem cells, such as by positive or negative selection based on surface antigens associated with the respective subpopulation.
- enrichment 41744090.2 Attorney Docket No.046483-7386WO1(03225) for central memory T (TCM) cells is carried out to increase efficacy, such as to improve long- term survival, expansion, and/or engraftment following administration, which in some aspects is particularly robust in such sub-populations.
- combining TCM-enriched CD8+ T cells and CD4+ T cells further enhances efficacy.
- memory T cells are present in both CD62L+ and CD62L- subsets of CD8+ peripheral blood lymphocytes.
- PBMC can be enriched for or depleted of CD62L-CD8+ and/or CD62L+CD8+ fractions, such as using anti-CD8 and anti-CD62L antibodies.
- a CD4+ T cell population and a CD8+ T cell sub-population e.g., a sub- population enriched for central memory (TCM) cells.
- the enrichment for central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and/or CD 127; in some aspects, it is based on negative selection for cells expressing or highly expressing CD45RA and/or granzyme B.
- isolation of a CD8+ population enriched for TCM cells is carried out by depletion of cells expressing CD4, CD 14, CD45RA, and positive selection or enrichment for cells expressing CD62L.
- enrichment for central memory T (TCM) cells is carried out starting with a negative fraction of cells selected based on CD4 expression, which is subjected to a negative selection based on expression of CD 14 and CD45RA, and a positive selection based on CD62L.
- TCM central memory T
- the same CD4 expression-based selection step used in preparing the CD8+ cell population or subpopulation also is used to generate the CD4+ cell population or sub-population, such that both the positive and negative fractions from the CD4- based separation are retained and used in subsequent steps of the methods, optionally following one or more further positive or negative selection steps.
- CD4+ T helper cells are sorted into naive, central memory, and effector cells by identifying cell populations that have cell surface antigens.
- CD4+ lymphocytes can be obtained by standard methods.
- naive CD4+ T lymphocytes are CD45RO-, CD45RA+, CD62L+, CD4+ T cells.
- central memory CD4+ cells are CD62L+ and CD45RO+.
- effector CD4+ cells are CD62L- and CD45RO.
- a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CDl lb, CD16, HLA-DR, and CD8.
- the antibody or binding partner is bound to a solid support or matrix, such as a 41744090.2 Attorney Docket No.046483-7386WO1(03225) magnetic bead or paramagnetic bead, to allow for separation of cells for positive and/or negative selection.
- the cells are incubated and/or cultured prior to or in connection with genetic engineering.
- the incubation steps can include culture, cultivation, stimulation, activation, and/or propagation.
- the compositions or cells are incubated in the presence of stimulating conditions or a stimulatory agent. Such conditions include those designed to induce proliferation, expansion, activation, and/or survival of cells in the population, to mimic antigen exposure, and/or to prime the cells for genetic engineering, such as for the introduction of a recombinant antigen receptor.
- the conditions can include one or more of particular media, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and/or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells.
- the stimulating conditions or agents include one or more agent, e.g., ligand, which is capable of activating an intracellular signaling domain of a TCR complex.
- the agent turns on or initiates TCR/CD3 intracellular signaling cascade in a T cell.
- Such agents can include antibodies, such as those specific for a TCR component and/or costimulatory receptor, e.g., anti-CD3, anti-CD28, for example, bound to solid support such as a bead, and/or one or more cytokines.
- the expansion method may further comprise the step of adding anti-CD3 and/or anti CD28 antibody to the culture medium (e.g., at a concentration of at least about 0.5 ng/ml).
- the stimulating agents include IL-2 and/or IL-15, for example, an IL-2 concentration of at least about 10 units/mL.
- T cells are isolated from peripheral blood by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLLTM gradient.
- T cells can be isolated from an umbilical cord.
- a specific subpopulation of T cells can be further isolated by positive or negative selection techniques.
- the cord blood mononuclear cells so isolated can be depleted of cells expressing certain antigens, including, but not limited to, CD34, CD8, CD14, CD19, and CD56. Depletion of these cells can be accomplished using an isolated antibody, a biological sample comprising an antibody, such as ascites, an antibody bound to a physical support, and a cell bound antibody.
- Enrichment of a T cell population by negative selection can be accomplished using a combination of antibodies directed to surface markers unique to the negatively selected cells.
- a preferred method is cell sorting and/or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected.
- a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8.
- concentration of cells and surface e.g., particles such as beads
- a concentration of 2 billion cells/ml is used.
- a concentration of 1 billion cells/ml is used.
- greater than 100 million cells/ml is used.
- a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/ml is used.
- a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells/ml is used.
- concentrations of 125 or 150 million cells/ml can be used.
- T cells can also be frozen after the washing step, which does not require the monocyte- removal step. While not wishing to be bound by theory, the freeze and subsequent thaw step provides a more uniform product by removing granulocytes and to some extent monocytes in the cell population. After the washing step that removes plasma and platelets, the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and will be useful in this context, in a non-limiting example, one method involves using PBS containing 20% DMSO and 8% human serum albumin, or other suitable cell freezing media.
- the cells are then frozen to -800C at a rate of 10C per minute and stored in the vapor phase of a liquid nitrogen storage tank.
- Other methods of controlled freezing may be used as well as uncontrolled freezing immediately at -200C or in liquid nitrogen.
- the population of T cells is comprised within cells such as peripheral blood mononuclear cells, cord blood cells, a purified population of T cells, and a T cell line.
- peripheral blood mononuclear cells comprise the population of T cells.
- purified T cells comprise the population of T cells.
- T regulatory cells (Tregs) can be isolated from a sample.
- the sample can include, but is not limited to, umbilical cord blood or peripheral blood.
- the Tregs are isolated by flow-cytometry sorting.
- the sample can be enriched for Tregs prior to isolation by any means known in the art.
- the isolated Tregs can be cryopreserved, and/or expanded prior to use. Methods for isolating Tregs are described in U.S. Patent Numbers: 7,754,482, 8,722,400, and 9,555,105, and U.S. Patent Application No.13/639,927, contents of which are incorporated herein in their entirety. H.
- the cells can be activated and expanded in number using methods as described, for example, in U.S. Patent Nos.6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681 ; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Publication No.20060121005.
- the T cells of the invention may be expanded by contact with a surface having attached thereto an agent that stimulates a CD3/TCR complex associated signal and a ligand that stimulates a co-stimulatory molecule on the surface of the T cells.
- T cell populations may be stimulated by contact with an anti-CD3 antibody, or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore.
- a ligand that binds the accessory molecule is used for co-stimulation of an accessory molecule on the surface of the T cells.
- T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions appropriate for stimulating proliferation of the T cells.
- an anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France) and these can be used in the invention, as can other methods and reagents known in the art (see, e.g., ten Berge et al., Transplant Proc. (1998) 30(8): 3975-3977; Haanen et al., J. Exp. Med. (1999) 190(9): 1319-1328; and Garland et al., J. Immunol. Methods (1999) 227(1-2): 53-63).
- Expanding T cells by the methods disclosed herein can be multiplied by about 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold, 200 fold, 300 fold, 400 41744090.2
- Attorney Docket No.046483-7386WO1(03225) fold 500 fold, 600 fold, 700 fold, 800 fold, 900 fold, 1000 fold, 2000 fold, 3000 fold, 4000 fold, 5000 fold, 6000 fold, 7000 fold, 8000 fold, 9000 fold, 10,000 fold, 100,000 fold, 1,000,000 fold, 10,000,000 fold, or greater, and any and all whole or partial integers therebetween.
- the T cells expand in the range of about 20 fold to about 50 fold.
- the T cells can be incubated in cell medium in a culture apparatus for a period of time or until the cells reach confluency or high cell density for optimal passage before passing the cells to another culture apparatus.
- the culturing apparatus can be of any culture apparatus commonly used for culturing cells in vitro.
- the level of confluence is 70% or greater before passing the cells to another culture apparatus. More preferably, the level of confluence is 90% or greater.
- a period of time can be any time suitable for the culture of cells in vitro.
- the T cell medium may be replaced during the culture of the T cells at any time. Preferably, the T cell medium is replaced about every 2 to 3 days.
- the invention includes cryopreserving the expanded T cells.
- the cryopreserved T cells are thawed prior to introducing nucleic acids into the T cell.
- the method comprises isolating T cells and expanding the T cells.
- the invention further comprises cryopreserving the T cells prior to expansion.
- the cryopreserved T cells are thawed for electroporation with the RNA encoding the chimeric membrane protein. Another procedure for ex vivo expansion cells is described in U.S. Pat. No.5,199,942 (incorporated herein by reference).
- Expansion such as described in U.S. Pat. No.5,199,942 can be an alternative or in addition to other methods of expansion described herein.
- ex vivo culture and expansion of T cells comprises the addition to the cellular growth factors, such as those described in U.S. Pat. No.5,199,942, or other factors, such as flt3-L, IL-1, IL-3 and c-kit ligand.
- expanding the T cells comprises culturing the T cells with a factor selected from the group consisting of flt3-L, IL-1, IL-3 and c-kit ligand.
- the culturing step as described herein can be very short, for example less than 24 hours such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours.
- the culturing step as described 41744090.2 Attorney Docket No.046483-7386WO1(03225) further herein (contact with agents as described herein) can be longer, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more days.
- Various terms are used to describe cells in culture.
- Cell culture refers generally to cells taken from a living organism and grown under controlled condition.
- a primary cell culture is a culture of cells, tissues or organs taken directly from an organism and before the first subculture.
- Cells are expanded in culture when they are placed in a growth medium under conditions that facilitate cell growth and/or division, resulting in a larger population of the cells.
- the rate of cell proliferation is typically measured by the amount of time required for the cells to double in number, otherwise known as the doubling time.
- Each round of subculturing is referred to as a passage.
- cells are subcultured, they are referred to as having been passaged.
- a specific population of cells, or a cell line is sometimes referred to or characterized by the number of times it has been passaged. For example, a cultured cell population that has been passaged ten times may be referred to as a P10 culture.
- the primary culture i.e., the first culture following the isolation of cells from tissue
- P0 The primary culture
- the cells are described as a secondary culture (P1 or passage 1).
- P2 or passage 2 After the second subculture, the cells become a tertiary culture (P2 or passage 2), and so on.
- P1 or passage 1 the primary culture following the isolation of cells from tissue
- P2 or passage 2 the cells are described as a secondary culture (P1 or passage 1).
- P2 or passage 2 tertiary culture
- the expansion of cells (i.e., the number of population doublings) during the period between passaging depends on many factors, including but is not limited to the seeding density, substrate, medium, and time between passaging.
- the cells may be cultured for several hours (about 3 hours) to about 14 days or any hourly integer value in between.
- Conditions appropriate for T cell culture include an appropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 15, (Lonza)) that may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-gamma, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGF-beta, and TNF- ⁇ or any other additives for the growth of cells known to the skilled artisan.
- serum e.g., fetal bovine or human serum
- IL-2 interleukin-2
- insulin IFN-gamma
- IL-4 interleukin-7
- GM-CSF GM-CSF
- IL-10 interleukin-12
- IL-15 TGF-beta
- additives for the growth of cells include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol.
- Media can include RPMI 1640, AIM-V, DMEM, MEM, ⁇ -MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, and vitamins, either serum-free or 41744090.2 Attorney Docket No.046483-7386WO1(03225) supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and/or an amount of cytokine(s) sufficient for the growth and expansion of T cells.
- Antibiotics e.g., penicillin and streptomycin
- the target cells are maintained under conditions necessary to support growth, for example, an appropriate temperature (e.g., 37°C) and atmosphere (e.g., air plus 5% CO 2 ).
- the medium used to culture the T cells may include an agent that can co-stimulate the T cells.
- an agent that can stimulate CD3 is an antibody to CD3
- an agent that can stimulate CD28 is an antibody to CD28.
- a cell isolated by the methods disclosed herein can be expanded approximately 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold, 200 fold, 300 fold, 400 fold, 500 fold, 600 fold, 700 fold, 800 fold, 900 fold, 1000 fold, 2000 fold, 3000 fold, 4000 fold, 5000 fold, 6000 fold, 7000 fold, 8000 fold, 9000 fold, 10,000 fold, 100,000 fold, 1,000,000 fold, 10,000,000 fold, or greater.
- the T cells expand in the range of about 20 fold to about 50 fold, or more.
- human T regulatory cells are expanded via anti-CD3 antibody coated KT64.86 artificial antigen presenting cells (aAPCs).
- the method of expanding the T cells can further comprise isolating the expanded T cells for further applications.
- the method of expanding can further comprise a subsequent electroporation of the expanded T cells followed by culturing.
- the subsequent electroporation may include introducing a nucleic acid encoding an agent, such as a transducing the expanded T cells, transfecting the expanded T cells, or electroporating the expanded T cells with a nucleic acid, into the expanded population of T cells, wherein the agent further stimulates the T cell.
- compositions comprising populations modified cells (e.g., CAR T cells comprising CH25H) for use in immunotherapy.
- the population of cells can be generated by any of the methods contemplated herein.
- therapeutic methods for administering the cells and compositions to subjects e.g., patients.
- compositions including the cells for administration including pharmaceutical compositions and formulations, such as unit dose form compositions including the number of cells for administration in a given dose or fraction thereof.
- compositions and formulations generally include one or more optional pharmaceutically acceptable carrier or excipient.
- the composition includes at least one additional therapeutic agent.
- pharmaceutical formulation refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.
- pharmaceutically acceptable carrier refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject.
- a pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative. In some aspects, the choice of carrier is determined in part by the particular cell and/or by the method of administration.
- the pharmaceutical composition can contain preservatives.
- Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride.
- a mixture of two or more preservatives is used.
- the preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition.
- Carriers are described, e.g., by Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).
- Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arg
- Buffering agents in some aspects are included in the compositions. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffering agents is used. The buffering agent or mixtures thereof are typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail in, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).
- the formulations can include aqueous solutions.
- the formulation or composition may also contain more than one active ingredient useful for the particular indication, disease, or condition being treated with the cells, preferably those with activities complementary to the cells, where the respective activities do not adversely affect one another.
- active ingredients are suitably present in combination in amounts that are effective for the purpose intended.
- the pharmaceutical composition further includes other pharmaceutically active agents or drugs, such as chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, and/or vincristine.
- chemotherapeutic agents e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, and/or vincristine.
- the pharmaceutical composition in some embodiments contains the cells in amounts effective to treat or prevent the disease or condition, such as a therapeutically effective or prophylactically effective amount.
- the desired dosage can be delivered by a single bolus administration of the cells, by multiple bolus administrations of the cells, or by continuous infusion administration of the cells.
- Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration.
- the cell populations are administered parenterally.
- parenteral includes intravenous, intramuscular, subcutaneous, rectal, 41744090.2 Attorney Docket No.046483-7386WO1(03225) vaginal, and intraperitoneal administration.
- the cells are administered to the subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.
- Compositions in some embodiments are provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may in some aspects be buffered to a selected pH.
- Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues.
- Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyoi (for example, glycerol, propylene glycol, liquid polyethylene glycol) and suitable mixtures thereof.
- Sterile injectable solutions can be prepared by incorporating the cells in a solvent, such as in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like.
- compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, and/or colors, depending upon the route of administration and the preparation desired. Standard texts may in some aspects be consulted to prepare suitable preparations.
- auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, and/or colors, depending upon the route of administration and the preparation desired. Standard texts may in some aspects be consulted to prepare suitable preparations.
- Various additives which enhance the stability and sterility of the compositions including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlor
- Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.
- the formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.
- TDFs tumor-derived factors
- TDFs induced ATF3 transcription factor that suppressed the expression of 25HC-regulating gene - cholesterol 25-hydroxylase (CH25H).
- Stimulation of trogocytosis in the intratumoral CTL by the ATF3-CH25H axis attenuated anti-tumor immunity, stimulated tumor growth, and impeded the efficacy of chimeric antigen receptor (CAR) T cell adoptive therapy.
- CAR chimeric antigen receptor
- Timer 2.0 (Li et al., (2020) Nucleic Acids Res 48, W509-W514) was used to analyze the correlation between immune cell infiltration and Ch25h expression in human melanoma.
- the expression of Ch25h in T cells was analyzed while controlling for age, gender, and disease stage. Those tumors that showed a survival benefit associated with increased Ch25h expression in T cells were then further analyzed for tumor purity and Ch25h expression.
- Ch25h allele was created by flanking the single exon of the Ch25h gene with the loxP sites inserted into the non-conservative regions (Lu et al., (2021) Nature 588, 693-698).
- OT-I WT and OT-I Ch25h -/- mice were generated by crossing OT- I mice with WT or Ch25h -/- mice. Atf3 f/f mice were previously described (Wolford et al., (2013) J Clin Invest 123, 2893-2906) and kindly provided by Dr. Tsonwin Hai (Ohio State University).
- Ch25h ⁇ CD8 or Atf3 ⁇ CD8 or Ch25h;Atf3 ⁇ CD8 mice were generated by intercrossing Cd8-Cre mice with conditional Ch25h f/f or Atf3 f/f mice. All animal experiments were performed using both male and female littermates of 6-8 weeks of age. All mice except NSG were in the C57BL/6J background.
- NALM6 acute lymphoblastic leukemia from Michael C.
- B16F10-hCD19-luc melanoma cells were generated by stable expression of luciferase in B16F10-hCD19 cells.293T cell and all cancer cells were cultured at 37°C with 5% CO2 in DMEM including 10% heat-inactivated Fetal Bovine Serum (FBS), 100U/ml penicillin-streptomycin and L-glutamine.
- FBS heat-inactivated Fetal Bovine Serum
- penicillin-streptomycin L-glutamine
- Phoenix packaging cells (from ATCC) were maintained at 37°C with 5% CO2 in RPMI-1640 including 10% heat- inactivated Fetal Bovine Serum (FBS), 50U/ml penicillin-streptomycin and L-glutamine.
- TCM Tumor- condition medium
- FCM NIH3T3 fibroblast-conditioned media
- CAR T cells vector generation, T cell isolation, expansion, and CAR T cell generation: The second generation anti-MESO CAR lentiviral construct, which included mCherry reporter, CD8 hinge, meso scFv, 4-1BB and CD3zeta signaling domains (described in Milone et al., (2009) Mol Ther 17, 1453-1464) was a gift from Michael C. Milone (University of Pennsylvania).
- a cassette encoding a 54 nucleotide self-cleaving peptide (T2A) positioned 5’ of Ch25h was commercially manufactured (Genscript, NJ) with 5’ AvrII and 3’ SalI restriction sites flanking the sequence and inserted into this vector as shown in FIG.7A.
- a similar strategy was employed to modify the anti-CD19 CAR retrovirus backbone consisting of a CD8 hinge, CD19 scFv, 4-1BB and CD3zeta signaling domains (obtained from Andy J. Minn, University of Pennsylvania).
- T cells were stimulated with Dynabeads Mouse T-Activator CD3/CD28 beads (Gibco, cat#11453D) or Dynabeads Human T-Activator CD3/CD28 beads (Gibco, cat#11132D) for 48 hr and were separated from beads prior to transduction.
- Transduction efficient, expression of anti-MESO and anti-CD19 CARs and expression of Ch25h were assessed using flow cytometry and quantitative RT-PCR (qPCR) analysis, respectively.
- OT-I-WT or -Ch25h -/- splenocytes were in vitro stimulated with OVA peptide (0.5 ⁇ g/ml for 48 hr) followed by treatment with vehicle or FCM or TCM or tumor derived factors such as VEGF165 or Prostaglandin E2 in the presence or absence of 25HC (4 ⁇ M, Sigma, cat#H1015) for 8 hr. Then, these differentially treated-CTLs were co-cultured with target MC38OVA cells expressing luciferase at indicated E/T ratio for 4 hr.
- CAR T cells setting anti-MESO or anti-CD19 CAR T cells were generated and treated with vehicle or 25HC at 4 ⁇ M for 8 hr. After that, CAR T cells were co-cultured with target EM- Meso-GFP-Luc or B16F10-hCD19-luc cells at indicated E/T ratios for 4 hr followed by the luciferase-based cytotoxicity assay.
- target cells alone were set as negative control with spontaneous death RLU and target cells with water were set as positive control with maximal killing RLU.
- luciferase substrate Promega Bright-Glo, cat# E6120
- OT-I or CAR T CTLs were co-cultured with respective target cells for 2, 4, 8 or 16 hr, washed once with PBS once and analyzed by flow cytometry using appropriate antibodies such as: anti-CD8-APC/Cy7(BioLegend, cat#100714), anti-CD69-PE (BioLegend, cat# 104507), anti-PD-1-BV605 (BioLegend, cat#135219), anti- LAG3-PE/Cy7 (BioLegend, cat#125226), anti-CD336-BV421 (BioLegend, cat#134019) and 41744090.2 Attorney Docket No.046483-7386WO1(03225) anti-Annexin V-FITC (BioLegend, cat#640906).
- tumor tissue was dissected and digested with 1 mg/ml Collagenase D (Roche, cat#11088882001) plus with 100 ⁇ g/ml DNase I (Roche, cat#10104159001) in RPMI medium with 2% FBS for 1 hr with continuous agitation at 37 °C.
- Digestion mixture was passed through 100 ⁇ m cell strainer to prepare single cell suspensions and washed with PBS supplemented with 2mM EDTA and 1% FBS.
- Single cells were stained with cell surface antibodies: anti-CD45-APC/Cy7 (BioLegend, cat#157204), anti- CD3-PE (BioLegend, cat#100206), anti-CD8-AF700 (BioLegend, cat#100730), anti- CD69- BV421 (BioLgend, cat#104545, anti-PD-1-PE/Cy7 (BioLegend, cat#109110), anti-LAG3- BV650 (BioLegend, cat#125227), anti-CD336-BV421 (BioLegend, cat#134019) and Annexin V-APC (BioLegend, cat#640941).
- Red cells were removed using RBC buffer. After that, single cells were incubated with anti-CD8-AF700 (Biolegnd, cat#100714), anti-CD4 (BioLegend, cat#100414), anti-CD19 (BioLegend, cat#115507), anti-NK1.1 (Biolegend, cat#108709), anti-CD69, anti-PD-1 and anti- Annexin V antibodies.
- the samples were prepared as previous described (Lu et al., (2021) J Clin Invest 131) and the Biotin anti-mouse CD3 antibody (Biolegend, cat#100243; 1:100), Biotin anti-mouse CD8 antibody (Biolegend, cat#100704), Alexa Fluor-594 streptavidin antibody (Biolegend,cat# 405240; 1:500), and Alexa Fluor-488 anti-mouse (Biolegend, 100723; 1:100) were used.
- OT-I WT T cells were isolated and stimulated with OVA peptide (0.5 ⁇ g/ml for 48 hr) followed by treatment with Vehicle or TCM in the presence or absence of TAK981 (0.1 ⁇ M) for 12 hr.
- OVA peptide 0.5 ⁇ g/ml for 48 hr
- Vehicle or TCM in the presence or absence of TAK981 (0.1 ⁇ M) for 12 hr.
- Cells were lysed, and 1 mg of pre-cleared protein lysates from each sample were taken into immunoprecipitation using rabbit anti-mouse ATF3 antibody (Cell Signaling Technology, cat#18665S). Immunoprecipitation was carried out as previously described (Huangfu et al., (2012) Oncogene 31, 161-172).
- the resulting immune-precipitates were eluted with glycine-containing buffer (pH 2.6) and analyzed by immunoblot using primary antibodies against SUMO1 (Invitrogen, cat#33-2400, 3 ⁇ g/ml), ATF3 (CST cat#18665S) and secondary antibodies (VeriBlot for IP Detection Reagent HRP, abcam, cat#ab131366, 1:200). Loading in initial samples was controlled by direct immunoblot using antibody against ⁇ -tubulin (Cell Signaling Technology, cat# 2146, 1:1000) and goat anti-mouse HRP-conjugated antibody (Cell Signaling Technology, cat#7076S, 1:5000).
- OT-I and CAR T settings were used to assess the extent of trogocytosis.
- OVA peptide 0.5 ⁇ g/ml for 48 hr
- Vehicle IECM
- PGE2 Sigma, cat#P0409, 10nM
- VEGF165 Sigma, cat#V5765, 50ng/ml
- TCM TCM in the presence or absence of 25HC (Sigma, cat#H1015, 4 ⁇ M) or GW3965 (Sigma, cat# G6295, 2 ⁇ M) for 8 hr as indicated.
- a total of 1x10 5 treated-CTLs were co-cultured with DiD-labeled target cells in 96-well plates at a 5:1 of ratio for indicated times.
- DiD label MC38 or MC38OVA cells were stained with 0.3 ⁇ M DiD (1,1′-dioctadecyl- 3,3,3′,3′- tetramethylindodicarbocyanine, Biotium, cat#60014) at 37 o C for 10 min followed by PBS washes for 3 times.
- CTLs or CAR T cells were pre-treated with vehicle of latrunculin A (1 ⁇ M, Sigma-Aldrich, cat#L5163) at 37°C for 20 min before co-incubation with target cells.
- latrunculin A 1 ⁇ M, Sigma-Aldrich, cat#L5163
- parental or DiD-labeled MC38 were seeded into the upper portion of Transwell chambers (Millipore, cat# CLS3422-48EA) and T cells were seeded on the bottom of these plates.
- Quantitative Real-time PCR Total RNA of CD8 + T cells which was treated with Vehicle or TCM or isolated from tumor tissue was extracted using RNA isolation kit (Qiagen, cat#74004).
- RNA Concentrations of RNA were measured by nanodrop2000 and the mRNA expression of Ch25h and Atf3 were tested using real-time PCR.
- Primers are follows: Ch25h forward: TGCTACAACGGTTCGGAGC (SEQ ID N: 34) and Reverse: AGAAGCCCACGTAAGTGATGAT (SEQ ID NO: 35); Atf3 forward: TTACCGTCAACAACAGACCC (SEQ ID NO: 36) and Reverse: TCAGCTCAGCATTCACACTC (SEQ ID NO: 37); AbcA1 forward: AGTGATAATCAAAGTCAAAGGCACAC (SEQ ID NO:38) and Reverse: AGCAACTTGGCACTAGTAACTCTG (SEQ ID NO:39); AbcG1 Forward: TTCATCGTCCTGGGCATCTT (SEQ ID NO:40) and Reverse: CGGATTTTGTATCTGAGGACGAA (SEQ ID NO:41); ApoE Forward: ACAGATCAGCTCGAGTGGCAAA (SEQ ID
- Chromatin immunoprecipitation (ChIP) analysis OT-I WT T cells were isolated from spleen of OT-I WT mice and stimulated with OVA (0.5 ⁇ g/ml) for 48 hr followed by treatment with FCM or TCM for 12hr. T cells were harvested and the Immunoprecipitation (IP) was conducted following instruction from SimpleChIP Plus Enzymatic Chromatin IP Kit (Cell signaling technology, cat# 9005S). ATF3 antibody used for the IP was Rabbit anti-mouse ATF3 antibody (Cell signaling technology, cat#18665S, 1:50).
- the ChIP-qPCR for the Ch25h promoter used the following primers: forward 5-TAGCAGCCCATGCTGAGACTATGT-3 (SEQ ID NO:46) and reverse primer, 5 TTCTTTAGCAGGGAAAGGGAGGTG-3 (SEQ ID NO:47). 41744090.2 Attorney Docket No.046483-7386WO1(03225) Tumorigenesis studies: For the syngeneic subcutaneous tumor model, B16F10 (1x10 6 ), MC38 (1x10 6 ), MH6499c4 (1x10 6 ) and Hepa1-6 (2x10 6 ) were s.c inoculated into right flanks of mice and tumor sizes were measured every other day using calipers.
- TAK981 tumor volume was calculated as width x width x length x 0.5 and mice survival was tracked until tumor volume reached ⁇ 1000 mm 3 .
- MC38 (1x10 6 ) cells were suspended into 100 ⁇ l PBS and inoculated into the right flank of Ch25h f/f and Ch25h ⁇ CD8 mice at day 0.
- TAK981 was dissolved in 20% hydroxypropyl beta-cyclodextrin and formulated as previously described (Lightcap et al., (2021) Sci Transl Med 13, eaba7791).
- Vehicle or anti-PD-1 i.p, 5mg/kg every 4 days
- TAK981 i.v, 15mg/kg, twice a week
- Tumor tissues were collected at day 25 for immune profiling analysis.
- mice were euthanized when tumor volume reached ⁇ 1000 mm 3 .
- human EM-Meso-GFP-Luc cells s.c., 1x10 6 ) were inoculated into the right flank of NSG mice at day 0.
- mice Human anti-MESO-CAR or anti-MESO-Ch25h CAR T cells (generated as described herein) were i.v. injected into tumor bearing mice at a dose of 1x10 6 /mouse at days 16, 23, 30 and 37; PBS was used as control. Tumor tissue was collected at day 45 for immune profiling. For the survival analysis, mice were euthanized when tumor volume reached ⁇ 1000 mm 3 .
- B16F10-hCD19 cells s.c, 0.3x10 6
- Mouse anti-CD19 CAR or anti- CD19-Ch25h CAR T cells were generated and i.v. injected into tumor bearing mice at dose of 2x10 6 /mouse at days 7 and 13. Tumor tissues were digested at day 19 for immune profiling analysis. For the survival analysis, mice were euthanized when tumor volume reached ⁇ 1000 mm 3 .
- GFP + NALM6 cancer cells were inoculated into NSG mice (i.v, 1x10 6 ) at day 0.
- Human anti-CD19 CAR or anti-CD19- Ch25h CAR T cells were injected (1x10 6 ) into tumor bearing mice at day 10, and PBS injection was used as the control group.
- mice were euthanized when they became moribund. 41744090.2 Attorney Docket No.046483-7386WO1(03225) Trogocytosis and viability assay in vivo: MC38 or MC38OVA cells were inoculated into the right flanks of Rag1 -/- mice (1x10 6 , s.c). WT OT-I and Ch25h -/- OT-I splenocytes were stimulated in vitro with OVA peptide (0.5 ⁇ g/ml for 48 hr).
- Ch25h -/- CTLs were stained with CFSE (1nM, Biolegend, cat#79898) at 37°C for 10 min followed by 3 washes with PBS. Following the washes, WT CTLs (1x10 6 , CFSE negative) were mixed with CFSE- labeled Ch25h -/- CTLs at a ratio of 1:1 and re-suspended in 50 ⁇ l PBS. This cell mixture was injected into MC38 or MC38OVA tumors.
- tumor tissues were digested into single cell suspensions; and percentages of CD8 + CFSE + , CD8 + CFSE-, OVA- MHCI + CFSE + and OVA-MHC + CFSE- cells were analyzed using flow cytometry.
- TAK981 For analysis of the effects of TAK981 on trogocytosis, 1x10 6 MC38-OVA cells were injected (s.c) into right flanks of Rag1 -/- mice.13 days after tumor inoculation, tumor bearing mice received OT1 CD8 + T cells injection (2x10 7 /mouse, intratumorally) and TAK981 injection (15mg/kg, i.v) simultaneously.24 hr after injection, tumor tissues were harvested and trogocytosis and T cells exhaustion were analyzed with flow cytometry.
- RNA sequencing analysis WT OT-I or Ch25h -/- OT-I splenocytes were stimulated with OVA peptide (0.5 ⁇ g/ml for 48 hr) followed by treatment with FCM or TCM for 8 hr in vitro. Total RNA was extracted with RNeasy Plus Mini Kit (QIAGEN). These samples were then used for RNA sequencing carried out as previously described (Amorim et al., (2019) Sci Transl Med 11). The adaptor-trimmed reads were aligned to the mouse genome mm10 by STAR v.2.7.2d with default parameters. Expression levels for each gene were counted by VERSE v.0.1.4.
- Samples were extracted using a biphasic extraction (Tambellini et al., (2013) Metabolites 3, 592-605) and then subjected to lipidomics analysis by spotting on a 24 well PTFE slide and analyzed by desorption electrospray ionization (Takats et al., (2004) Science 306, 471-473), on a Waters Xevo G2-XS qtof instrument. Pixels from technical replicates were averaged and 8564 features were detected. A targeted analysis was performed for 25HC m/z 385.3465, representing the [M+H-H 2 0] + ion of hydroxysterols (DeBarber et al., (2008) Anal Biochem 381, 151-153).
- Example 1 Tumor-derived factors (TDFs) downregulate 25-hydroxycholesterol and stimulate trogocytosis between effector CTLs and malignant cells Effector trogocytosis that transfers antigenic complexes to specific CTLs from malignant cells, undermines their killing due to antigen loss and CTL fratricide. Mechanisms regulating the extent of effector trogocytosis are not well understood. Factors present in the tumor 41744090.2 Attorney Docket No.046483-7386WO1(03225) microenvironment and how they affect the extent of trogocytosis were analyzed herein ( Figure 8A).
- TDFs tumor-derived factors
- PGE2 prostaglandin E2
- VEGF vascular endothelial growth factor
- FCM fibroblasts
- IECM primary mouse intestinal epithelial cells
- cholesterol 25-hydroxylase (Ch25h) was the top downregulated gene ( Figure 8E).
- Expression of Ch25h was notably decreased among all genes affected by TCM ( Figures 1C-1D).
- CH25H was focused on because of its enzymatic role in production of 25HC.25HC is known to alter the fluidity of lipid membranes and to inhibit their fusion, which is essential for incorporating malignant cell membrane fragments into CTL during trogocytosis.
- Example 3 Downregulation of CH25H in CTLs attenuates the immune response and promotes tumor growth Because of downregulation of CH25H in the CTLs isolated from mouse tumors ( Figures 1H, 8F) and decreased viability of antigen-experienced CH25H-deficient CTLs ( Figures 2F, 2G, 2K, 2L), it is plausible that partial loss of CH25H in the microenvironment of human cancers may be linked with a decrease in tumor-infiltrating lymphocytes. Thus, a putative association between the intratumoral expression of CH25H and CD8A, a gene primarily expressed by tumor infiltrating CTLs, was searched for.
- Example 4 ATF3 regulates effector trogocytosis, activity and viability of CTLs, and tumor growth in a CH25H-dependent manner
- ATF3 Activating transcription factor-3 (ATF3) is a key negative regulator of CH25H expression in macrophages.
- Chromatin immunoprecipitation assay demonstrated that ATF3 was present on the promoter of the Ch25h gene in mouse OT-I CD8+ T cells and treatment of these cells with TDFs increased this binding ( Figure 4A).
- ATF3 is well known as an early stress-responsive gene that could be induced by many factors of tumor microenvironment.
- ATF3 was induced in CTLs treated with media conditioned by MC38 colon adenocarcinoma cells but not by normal intestinal cells or fibroblasts ( Figure 1D, 11A).
- specific 41744090.2 Attorney Docket No.046483-7386WO1(03225) TDFs such as PGE2, VEGF and tumor-derived extracellular vesicles were all capable of increasing Atf3 expression in CD8+ T cells in vitro (Figure 11A).
- Atf3 ⁇ CD8 mice were generated, which lacked ATF3 in CD8+ cells but did not differ from Atf3 f/f controls in the composition of immune cells in spleen or blood (Figure 11D). Nevertheless, CTL from Atf3 ⁇ CD8 mice did not downregulate CH25H in response to in vitro treatment with TDFs (Figure 4E). Importantly, downregulation of CH25H in the CTLs isolated from MC38 tumors was less pronounced when these tumors grew in Atf3 ⁇ CD8 mice ( Figure 4F). Similar results were obtained in other tumor types (Figure 11E) suggesting that ATF3 contributes to decreased expression of CH25H in the intratumoral CTLs.
- Example 5 ATF3 and CH25H control trogocytosis and activity of CAR T cells To directly examine the role of the ATF3-Ch25h regulatory axis in effector trogocytosis as well as viability and function of CTLs, WT, Ch25h ⁇ CD8 , Atf3 ⁇ CD8 , and Ch25h; Atf3 ⁇ CD8 murine T cells were generated and stably transduced with retrovirus for expression of a CAR that 41744090.2 Attorney Docket No.046483-7386WO1(03225) targets human CD19 (hCD19, Figure 12A).
- CAR T cells from Atf3 ⁇ CD8 mice exhibited a greater anti-tumor activity as manifested by changes in tumor volume and weight (Figure 5H) and animal survival (Figure 5I).
- knockout of CH25H attenuated the efficacy of CAR T cell therapy in any genetic context tested here ( Figures 5H-5I).
- TAK981 sumoylation inhibitor upregulates CH25H, inhibits trogocytosis and augments CAR T cell viability and anti-tumor activities
- a pharmacologic approach to suppressing the effector trogocytosis was sought in order to increase viability and activity of the intratumoral CTLs.
- 25HC worked well in vitro Figures 2A-2M
- its suboptimal bioavailability prompted the search for a medically relevant small molecule agent that could interfere with ATF3-driven downregulation of CH25H and restore CH25H levels in the intratumoral CTLs.
- ATF3 directly suppresses transcription of CH25H and transcriptional repression is often associated with protein sumoylation;
- de-sumoylation of some transcriptional regulators is required for induction of ATF3;
- sumoylation of ATF3 itself has been implicated in its transcriptional suppressive activities;
- activation of innate and adaptive immunity was observed upon genetic or pharmacologic disruption of the sumoylation pathway.
- treatment of activated OT-I CD8+ T cells with TDFs increased the levels of both unmodified and sumoylated ATF3 protein ( Figure 6A).
- TAK981 novel, selective and potent sumoylation inhibitor TAK981 were then tested on TDFs-exposed CTLs.
- Pre-treatment of CD8+ T cells with TAK981 in vitro partially attenuated the TDFs-induced increase in ATF3 mRNA expression (Figure 13A) and in levels of sumoylated and unmodified ATF3 protein ( Figure 6A).
- TAK981 robustly prevented downregulation of CH25H expression ( Figure 6B). While TDFs stimulated trogocytosis (assessed by a transfer of MHC-I-OVA complex from MC38OVA target tumor cells onto OT-I CTLs), adding TAK981 prevented this stimulation.
- TAK981 restored the numbers of WT OT-I cells in the culture.
- 41744090.2 Attorney Docket No.046483-7386WO1(03225) compared to WT cells, the numbers of co-cultured CH25H-deficient OT-I CTLs were notably lower, and they did not increase in response to TAK981 treatment ( Figure 13B).
- TAK981 restored the tumoricidal activity of WT (but not Ch25h-null) OT-I CTLs against target MC38OVA tumor cells otherwise suppressed by TCM (Figure 13C).
- TAK981 prevents trogocytosis and associated decrease in viability and activity of CTLs in a CH25H-dependent manner.
- monotherapy of MC38 tumors in WT mice with TAK981 elicited a modest therapeutic effect
- the combination of this agent with anti-PD1 checkpoint inhibitor notably suppressed tumor growth.
- treatment with TAK981 alone or combined with anti-PD1 increased expression of CD69 and decreased levels of PD-1 and annexin-V on the intratumoral CTLs ( Figures 6D-6E).
- TAK981+anti-PD1 combination significantly decreased tumor volume and weight and prolonged animal survival (Figures 6F- 6H).
- Example 7 Armored CARs designed to re-express CH25H inhibit trogocytosis and increase therapeutic efficacy
- anti-MESO and anti-CD19 CARs were re-designed to enable the co-expression of CH25H ( Figure 7A).
- mice CTLs 41744090.2 Attorney Docket No.046483-7386WO1(03225) expressing conventional anti-MESO CARs to kill target EM-Meso-GFP-Luc cells (Figure 14A) was notably attenuated by knockout of CH25H but restored with treatment with 25HC in vitro ( Figure 14B).
- Figure 14C Experiments comparing CTLs harboring either conventional or CH25H- expressing CAR ( Figure 14C) revealed that re-expression of CH25H notably inhibited the extent of trogocytosis ( Figure 7B), and increased viability (Figure 7C) and tumoricidal activity (Figure 7D) of CAR T cells.
- CAR T cells that co-express CH25H exhibited a greater killing activity (Figure 14H), and decreased trogocytosis manifested by transfer of antigen onto CTLs (Figure 14I) or by disappearance of antigen from target tumor cells (Figure 14J).
- CH25H-expressing CAR T cells also displayed lesser exhaustion ( Figure 14K), and improved viability ( Figure 14L).
- CAR T cells expressing CH25H demonstrated a greater therapeutic efficacy against B16F10-hCD19 tumors (Figure 7H, 14M), better infiltrated these tumors ( Figure 14N), and expressed lower levels of markers of exhaustion and apoptosis (Figure 14O).
- Example 8 Discussion 41744090.2 Attorney Docket No.046483-7386WO1(03225) Data presented herein reveal that activation of ATF3 and ensuing suppression of CH25H in the tumor microenvironment stimulate the extent of effector trogocytosis between tumor- specific CTLs and malignant cells. These events also limit viability and activity of the intratumoral CTLs thereby stimulating tumor growth and eliciting resistance against adoptive cell transfer therapies including CAR T cell-based regimens. Maintaining expression of CH25H by administration of TAK981 sumoylation inhibitor or co-expressing CH25H along with a CAR leads to restricted trogocytosis, increased survival and activity of CTLs, and improved efficacy of CAR T cell treatment.
- TDFs elicited diverse and numerous changes in CTLs’ gene expression profile as well as distinct alterations in levels of lipid species ( Figures 1A-1I).
- ATF3 is an early stress-responsive gene that could be induced by many factors in the tumor microenvironment, it is plausible that many redundant factors besides PGE2 and VEGF are capable of downregulating CH25H and promoting trogocytosis.
- Results of studies utilizing TAK981 suggest that protein sumoylation may contribute to both induction of ATF3 and to ATF3-dependent suppression of CH25H.
- 25HC also implicated in suppression of uptake of the tumor-derived extracellular vesicles, 41744090.2 Attorney Docket No.046483-7386WO1(03225) which were proposed to contribute to the process of transfer of T cell receptor-containing complexes.
- 25-hydroxylation along with other modifications generates ligands for the LXR pathway.
- Inhibition of trogocytosis in T cells treated with LXR agonist is suggestive of a role for the LXR pathway – potentially downstream of the ATF3-CH25H axis.
- 25HC also acts as a negative regulator of the cell membrane cholesterol; and reduction of cholesterol levels in CD8+ T cells prevents their exhaustion in the tumor microenvironment.
- suppressing CH25H expression and stimulating effector trogocytosis might represent an evolutionarily important mechanism that attenuates the function of the immune system and may play a protective role to minimize tissue damage during inflammation and T cell-driven autoimmune reactions. If that is true, it would not be the first time, when such protective mechanism is appropriated by a growing tumor to evade the T cell immunity and perhaps even develop a resistance to therapies that involve adoptive transfer of CTLs.
- the trogocytosis-driven loss of antigen on target malignant cells that persist following an encounter with CTL is likely temporary and reversible.
- This agent acts to re-activate the anti-tumor immunity and is currently investigated in several oncology clinical trials (including NCT03648372, NCT04074330, NCT04776018 and NCT04381650).
- Data herein domonstrate that the efficacy of TAK981 relates to its ability to prevent downregulation of CH25H expression in the CD8 + T cells (FIGs.6A-6H). While clinical testing of this agent and its combinations with different types of immune therapies is underway, it is important to stratify these efforts and focus on scenarios where trogocytosis in CTLs acts to promote tumor growth.
- trogocytosis-mediated cross-priming and of trogocytosis-dependent anti-tumor effects elicited by neutrophils or macrophages.
- protein sumoylation affect numerous important cellular processes and continuous use of sumoylation inhibitors may complicate the efforts to manage oncological disease due to toxicity. Given these considerations, it would be more attractive to restrict the inhibition of trogocytosis to CTLs.
- Engineering novel human CAR T cells that either lack ATF3 (for example, via CRISPR/Cas9-driven knockout) or re-express CH25H represent such strategy.
- Embodiment 1 provides a modified immune cell or precursor cell thereof, comprising a nucleic acid encoding a chimeric antigen receptor (CAR) and cholesterol 25-hydroxylase (CH25H) gene, wherein the CAR and CH25H are expressed in the cell.
- Embodiment 2 provides the modified immune cell or precursor cell thereof of embodiment 1, wherein the CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular domain.
- Embodiment 3 provides the modified immune cell or precursor cell of any preceding embodiment, wherein the CAR comprises an antigen binding domain selected from the group consisting of an antibody, an scFv, and a Fab.
- Embodiment 4 provides the modified immune cell or precursor cell of any preceding embodiment, wherein the CAR comprises an antigen binding domain comprising specificity for a tumor associated antigen (TAA).
- TTA tumor associated antigen
- Embodiment 5 provides the modified immune cell or precursor cell of embodiment 4, wherein the TTA is selected from the group consisting of CD19, CD20, CD22, ROR1, Mesothelin, CD33/IL3Ra, c-Met, PSMA, PSCA, Glycolipid F77, and EGFRvIII.
- Embodiment 6 provides the modified immune cell or precursor cell of any preceding embodiment, wherein the CAR further comprises a hinge domain.
- Embodiment 7 provides the modified immune cell or precursor cell of any preceding embodiment, wherein the CAR comprises a hinge domain selected from the group consisting of an Fc fragment of an antibody, a hinge region of an antibody, a CH2 region of an antibody, a CH3 region of an antibody, an artificial hinge domain, a hinge comprising an amino acid sequence of CD8, or any combination thereof.
- the CAR comprises a hinge domain selected from the group consisting of an Fc fragment of an antibody, a hinge region of an antibody, a CH2 region of an antibody, a CH3 region of an antibody, an artificial hinge domain, a hinge comprising an amino acid sequence of CD8, or any combination thereof.
- Embodiment 8 provides the modified immune cell or precursor cell of any preceding embodiment, wherein the exogenous CAR comprises a transmembrane domain selected from the group consisting of an artificial hydrophobic sequence and transmembrane domain of a type I transmembrane protein, an alpha, beta, or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.
- the exogenous CAR comprises a transmembrane domain selected from the group consisting of an artificial hydrophobic sequence and transmembrane domain of a type I transmembrane protein, an alpha, beta, or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86
- Embodiment 9 provides the modified immune cell or precursor cell of any preceding embodiment, wherein the CAR comprises at least one co-stimulatory domain selected from the group consisting of co-stimulatory domains of proteins in the TNFR superfamily, CD28, 4-1BB 41744090.2 Attorney Docket No.046483-7386WO1(03225) (CD137), OX40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, ICOS, NKG2C, and B7-H3.
- the CAR comprises at least one co-stimulatory domain selected from the group consisting of co-stimulatory domains of proteins in the TNFR superfamily, CD28, 4-1BB 41744090.2
- Attorney Docket No.046483-7386WO1(03225) CD137
- OX40 CD134
- Embodiment 10 provides the modified immune cell or precursor cell of any preceding embodiment, wherein the CAR comprises an intracellular domain comprising an intracellular domain selected from the group consisting of cytoplasmic signaling domains of a human CD3 zeta chain, FcyRIII, FcsRI, a cytoplasmic tail of an Fc receptor, an immunoreceptor tyrosine- based activation motif (ITAM) bearing cytoplasmic receptors, TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d.
- ITAM immunoreceptor tyrosine- based activation motif
- Embodiment 11 provides the modified immune cell or precursor cell of any preceding embodiment, wherein the nucleic acid comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 10.
- Embodiment 12 provides the modified immune cell or precursor cell of any preceding embodiment, wherein the nucleic acid comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 8.
- Embodiment 13 provides the modified immune cell or precursor cell of any preceding embodiment, wherein the nucleic acid comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 15.
- Embodiment 14 provides the modified immune cell or precursor cell of any preceding embodiment, wherein the modified cell is an autologous cell.
- Embodiment 15 provides the modified immune cell or precursor cell of any preceding embodiment, wherein the modified cell is a cell isolated from a human subject.
- Embodiment 16 provides the modified immune cell or precursor cell of any preceding embodiment, wherein the modified cell is a modified T cell.
- Embodiment 17 provides a method of treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject the modified immune cell or precursor cell thereof of any of the preceding claims.
- Embodiment 18 provides the method of embodiment 17, wherein the disease or disorder is cancer.
- Embodiment 19 provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject i) a modified T cell comprising a CAR, and ii) an agent that upregulates CH25H.
- Embodiment 20 provides a nucleic acid comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR) and a nucleotide sequence encoding a cholesterol 25- hydroxylase (Ch25h) gene.
- Embodiment 21 provides the nucleic acid of embodiment 20, wherein the nucleic acid comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 8, 10, or 15.
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Abstract
The present disclosure provides modified immune cells or precursors thereof (e.g., T cells) comprising a CAR and CH25H. Compositions and methods of treatment are also provided.
Description
Attorney Docket No.046483-7386WO1(03225) METHODS FOR ENHANCING THE ANTI-TUMOR ACTIVITY OF CAR T CELLS BY CO-EXPRESSION OF CH25H CROSS-REFERENCE TO RELATED APPLICATION The present application is entitled to priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63/370,877, filed August 9, 2022, which is hereby incorporated by reference in its entirety herein. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under CA240814, CA247803 and CA165997 awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND OF THE INVENTION Intercellular transfer of biomolecules contributes to organization of collective behavior of cells in a multi-cellular organism. This transfer can be mediated by a number of mechanisms including trogocytosis, which plays an important role in the regulation of immunity and other vital biological processes. Trogocytosis is an evolutionarily conserved process of extraction and subsequent transfer of fragments of cell membrane along with associated proteins and other biomolecules between cells that are in close contact with each other. As a result of this transfer, a gain or loss of function for either acceptor or donor cells (or both) may occur. The functional consequences of trogocytosis in T cells depend on the context and nature of partner cells. For example, in the course of antigen presentation, priming T-cell receptor- dependent trogocytosis results in T cells acquiring membrane fragments along with the MHC class I and class II proteins and co-stimulatory and other molecules from the antigen-presenting cells. The effector trogocytosis between activated CD8+ T cells (cytotoxic T lymphocytes, CTLs) and target cells expressing specific antigen leads to a transfer of MHC-antigen complexes onto CTLs. Intriguingly, effector trogocytosis between target malignant cells and therapeutic CD8+ T cells expressing a chimeric antigen receptor (CAR) is implicated in decreased clinical benefits of 41744090.2 1
Attorney Docket No.046483-7386WO1(03225) CTLs. Trogocytosis is associated with decreased viability and activity of chimeric antigen- expressing recipient CTLs due to their exhaustion as well as killing by other CAR CTLs in a process called fratricide. Additionally, the loss of antigen on surviving donor malignant cells masks them from subsequent attack by CAR T cells. These intricate and robust mechanisms help malignant cells to withstand CAR T cell adoptive therapies. Tumors are adept at utilizing evolutionarily conserved processes for evading immune surveillance. However, it is not clear whether the described above mechanisms are also effective in naturally occurring anti-tumor CTLs. Furthermore, how effector trogocytosis is regulated and how this regulation can be altered during tumor growth is not well understood. There is a need in the art for improved CAR T cell therapies. The present invention addresses this need. SUMMARY OF THE INVENTION In one aspect, the invention includes a modified immune cell or precursor cell thereof, comprising a nucleic acid encoding a chimeric antigen receptor (CAR) and cholesterol 25- hydroxylase (CH25H) gene, wherein the CAR and CH25H are expressed in the cell. In certain embodiments, the CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular domain. In certain embodiments, the CAR comprises an antigen binding domain selected from the group consisting of an antibody, an scFv, and a Fab. In certain embodiments, the CAR comprises an antigen binding domain comprising specificity for a tumor associated antigen (TAA). In certain embodiments, the TTA is selected from the group consisting of CD19, CD20, CD22, ROR1, Mesothelin, CD33/IL3Ra, c-Met, PSMA, PSCA, Glycolipid F77, and EGFRvIII. In certain embodiments, the CAR further comprises a hinge domain. In certain embodiments, the CAR comprises a hinge domain selected from the group consisting of an Fc fragment of an antibody, a hinge region of an antibody, a CH2 region of an antibody, a CH3 region of an antibody, an artificial hinge domain, a hinge comprising an amino acid sequence of CD8, or any combination thereof. In certain embodiments, the CAR comprises a transmembrane domain selected from the group consisting of an artificial hydrophobic sequence and transmembrane domain of a type I transmembrane protein, an alpha, beta, or zeta chain of a T cell receptor, CD28, CD3 epsilon, 41744090.2 2
Attorney Docket No.046483-7386WO1(03225) CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. In certain embodiments, the CAR comprises an intracellular domain comprising at least one co-stimulatory domain selected from the group consisting of co-stimulatory domains of proteins in the TNFR superfamily, CD28, 4-1BB (CD137), OX40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, ICOS, NKG2C, and B7-H3. In certain embodiments, the CAR comprises an intracellular domain selected from the group consisting of cytoplasmic signaling domains of a human CD3 zeta chain, FcyRIII, FcsRI, a cytoplasmic tail of an Fc receptor, an immunoreceptor tyrosine-based activation motif (ITAM) bearing cytoplasmic receptors, TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. In certain embodiments, the nucleic acid comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 10. In certain embodiments, the nucleic acid comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 8. In certain embodiments, the nucleic acid comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 15. In certain embodiments, the modified cell is an autologous cell. In certain embodiments, the modified cell is a cell isolated from a human subject. In certain embodiments, the modified cell is a modified T cell. Another aspect of the invention includes a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject any of the modified immune cells or precursor cells thereof contemplated herein. In certain embodiments, the disease or disorder is cancer. Another aspect of the invention includes a method of treating cancer in a subject in need thereof, the method comprising administering to the subject i) a modified T cell comprising a CAR, and ii) an agent that upregulates CH25H. Another aspect of the invention includes a nucleic acid comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR) and a nucleotide sequence encoding a cholesterol 25-hydroxylase (Ch25h) gene. In certain embodiments, the nucleic acid comprises a nucleotide 41744090.2 3
Attorney Docket No.046483-7386WO1(03225) sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 8, 10, or 15. BRIEF DESCRIPTION OF THE DRAWINGS The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings. FIGs.1A-1I: Tumor-derived factors (TDFs) downregulate 25-hydroxycholesterol and stimulate trogocytosis between effector CTL and malignant cells. FIG.1A: Analysis of transfer of DiD dye from DID-labeled OVA-expressing MC38 cells onto co-cultured (for 4 hr) OT-I CD8+ T cells pre-treated with VEGF (50ng/ml), PGE2 (10nM), FCM (fibroblast cell medium), IECM (intestinal epithelial cell medium) or TCM (Tumor conditioned medium) for 8 hr as indicated(n=5). FIG.1B: KEGG enrichment analysis of altered pathways in CTLs treated with TCM (compared to FCM) for 8 hr (n=3). FIG.1C: Volcano plot of differentially expressed genes in CTLs treated as in FIG.1B. FIG.1D: Heatmap of differentially expressed genes in CTLs treated as in FIG.1B (n=3). FIG.1E: Heatmap of changes in lipid species that occurred in CTLs treated as in FIG.1B (n=4). FIG.1F: Levels of 25HC in CTLs treated as in FIG.1B (n=4). FIG. 1G: qPCR analysis of Ch25h mRNA expression in CTLs treated with Vehicle, media conditioned by primary mouse intestinal epithelial cells (IECM), or fibroblasts (FCM), or MC38 tumor cells (TCM), or PGE2 (10nM), VEGF (50ng/ml) or MC38 tumor-derived extracellular vesicles (TEVs, 20 µg/mL) for 8 hr (n=4-5). FIG.1H: qPCR analysis of Ch25h mRNA expression in CD8+ T cells isolated from MC38 tumors or spleens from naïve or MC38 tumor bearing mice (n=5). Na-sp, spleen from naïve mice; TB-sp, spleen from tumor bearing mice. FIG.1I: Pairwise comparison of Ch25h mRNA expression in CD8+ T cells isolated from tumor and spleen of individual mice (n=5). Data are presented as mean±SEM. Statistical analysis was performed using 2-tailed Students’ t test. n.s, not significant. FIGs.2A-2M: CH25H is a pivotal regulator of CTL trogocytosis, survival and activity. FIG.2A: Analysis of transfer of OVA-MHC-I complexes from OVA-expressing MC38 cells onto co-cultured (for 4 hr) OT-I CD8+ T cells pre-treated with FCM or TCM (with or without 25HC, 4μM or GW3965, 2μM) for 8 hr (n=4). FIG.2B: Number of live CD8+ CTLs after co- culture experiment described in FIG.2A (n=4). FIG.2C: Luciferase activity-based analysis of 41744090.2 4
Attorney Docket No.046483-7386WO1(03225) lysis of MC38OVA-luc cells incubated with OT-I CD8+ T cells pre-treated with FCM or TCM (with or without 25HC, 4μM) for 8hr (n=5). FIG.2D: A schematic of experiment for assessing the role of Ch25h in viability of CD8+ T cells in vivo (upper panel). Lower panel depicts the flow cytometry analysis of the percentage of CFSE+ CD8+or CFSE- CD8+ T cells isolated from the MC38 or MC38-OVA tumors 24 hr after inoculation. FIG.2E: Quantification of viable CFSE+ or CFSE- CD8+ T cells (left) and of transfer of OVA-MHC-I complexes on indicated CTLs (right) from experiment described in FIG.2D (n=5). FIG.2F: Analysis of number of live WT or Ch25h-/- OT-I CD8+ T cells cultured alone or with MC38OVA target cells for indicated time (n=3-5). FIG.2G: Analysis of percentage of apoptotic (Annexin V+) T cells treated as in FIG.2F (n=3-5). FIG.2H: Luciferase-based analysis of lysis of MC38OVA-luc cells incubated with WT or CH25H-null OT-I CD8+ T cells at indicated E:T ratios for 4 hr (n=8-10). FIG.2I: Flow cytometry analysis of expression of indicated markers on the surface of WT or CH25H-null CD8+ OT-I T cells after co-culture with MC38OVA target cells or MC38 cells (as an antigen- lacking control) for 8 hr (n=5). FIG.2J: Quantification of transfer of OVA-MHC-I complexes from MC38OVA target cells on WT or CH25H-null CD8+ OT-I T cells pre-treated or not with 25HC (4μM for 8 hr) (n=5). FIG.2K: Numbers of live CTLs from experiment described in FIG. 2J (n=5). FIG.2L: Percentage of apoptotic CTLs from experiment described in FIG.2J (n=5). FIG.2M: Luciferase-based analysis of lysis of MC38OVA-luc cells incubated with WT or CH25H-null OT-I CD8+ T cells pre-treated or not with 25HC (4μM for 8hr) (n=5). Data are presented as mean±SEM. Statistical analysis was performed using 2-tailed Students’ t test (FIG. 2A and FIG.2C) or 1-way ANOVA with Tukey’s multiple-comparison test(FIG.2B, FIG.2E, FIG.2G, FIG.2H, FIG.2I, FIG.2J, FIG.2K, FIG.2L and FIG.2M). n.s, not significant. FIGs.3A-3J: Downregulation of CH25H in CTLs attenuates the immune responses and promote tumor growth. FIG.3A: Correlation between CH25H expression and CD8+ T cell infiltration in human melanoma tumor. FIG.3B: Association between CH25H expression and progress free survival in human melanoma patients. FIG.3C: Association between CH25H expression and overall survival in human melanoma patients. FIG.3D: Growth of B16F10 melanoma tumors (inoculated s.c. at 1x106) in Ch25hf/f and Ch25hΔCD8Cre mice (left; n=9-10) and survival analysis for tumor-bearing animals (right; n=7-8). FIG.3E: Representative images of B16F10 tumor size (left) and weight (right) at day 15 from experiment described in FIG.3D. FIG.3F: Immunofluorescence analysis and its quantification for numbers of CD3+CD8+ T cells 41744090.2 5
Attorney Docket No.046483-7386WO1(03225) in MC38 tumors from Ch25hf/f and Ch25hΔCD8Cre mice (n=7-9). Scale bar: 100 μm. FIG.3G: Flow cytometry analysis of numbers and percentage of CD3+CD8+ T cells in MC38 tumors and spleens from tumor bearing Ch25hf/f and Ch25hΔCD8Cre mice (n=5). FIG.3H: Flow cytometry analysis of percentage of CD69-expressing CTLs from experiment described in FIG.3G. FIG. 3I: Flow cytometry analysis of percentage of PD-1-expressing CTLs from experiment described in FIG.3G. FIG.3J: Flow cytometry analysis of percentage of Annexin V-expressing CTLs from experiment described in FIG.3G. Data are presented as mean±SEM. Statistical analysis was performed using 2-tailed Students’ t test (FIG.3E and FIG.3F) or 1-way ANOVA with Tukey’s multiple-comparison test (FIGs.3G-3J) or 2-way ANOVA with Sidak’s multiple-comparison test (FIG.3D) or log-rank (Mantel-Cox) test (FIGs.3B-3D). n.s, not significant. FIGs.4A-4J: ATF3 regulates effector trogocytosis, activity and viability of CTLs, and tumor growth in a CH25H-dependent manner. FIG.4A: ChIP-qPCR analysis of ATF3 binding to Ch25h promoter in OT-I CD8+ T cells treated or not with MC38 TCM for 12 hr (n=3). FIG.4B: Overall (upper) and pairwise (below) qPCR analysis of Atf3 mRNA levels in CD8+ T cells from MC38 tumor tissue and spleen of naïve or tumor bearing mice (n=5). FIG.4C: Flow cytometry analysis of ATF3 protein level in CD8+ T cells from MC38 tumor tissue and spleen of naïve or tumor bearing mice (n=5). FIG.4D: Linear regression analysis of Atf3 and Ch25h mRNA expression in CD8+ T cells isolated from MC38 tumors (n=10). FIG.4E: Analysis of Ch25h expression in Atf3f/f or Atf3ΔCD8 CD8+ T cells treated in vitro with TCM or FCM for 8 hr (n=5). FIG.4F: Analysis of Ch25h expression in CD8+T cells isolated from MC38 tumor that grew in Atf3f/f or Atf3ΔCD8 mice (n=5). FIG.4G: Volume and weight of MC38 s.c. tumors that grew in Atf3f/f or Atf3ΔCD8 mice (n=5). FIG.4H: Flow cytometry assay of percentage of CD3+CD8+ T MC38 tumors from Atf3f/f or Atf3ΔCD8 mice (n=5). FIG.4I: Volume and weight of B16F10 tumors that grew in WT, Ch25hΔCD8, Atf3ΔCD8 or Ch25h;Atf3ΔCD8 mice (n=5). FIG.4J: Percentage of CD3+CD8+
T cells V-positive CD3+CD8+ T cells from experiment described in FIG.4I (n=5). Data are presented as mean±SEM. Statistical analysis was performed using 2-tailed Students’ t test (FIG.4B, FIG.4C and FIG.4G), linear regression analysis (FIG. 4D), 1-way ANOVA with Tukey’s multiple-comparison test (FIG.4A, FIG.4E-4J) or 2-way ANOVA with Sidak’s multiple-comparison test (FIG.4G and FIG.4I). n.s, not significant. FIGs.5A-5I: ATF3 and CH25H control trogocytosis and activity of CAR T cells. FIG. 5A: Flow cytometry analysis of transfer of CD19 from B16F10-hCD19 target cells onto anti- 41744090.2 6
Attorney Docket No.046483-7386WO1(03225) CD19 CAR T cells (co-incubated for 4 hr) produced from WT, Ch25hΔCD8, Atf3ΔCD8 or Atf3;Ch25h ΔCD8 splenic T cells (n=5). FIG.5B: Flow cytometry analysis of the percentage of CD8+Tim3+ anti-CD19-CAR T cells processed as in Panel A (n=5). FIG.5C: Flow cytometry analysis of the percentage of CD8+Annexin V+ anti-CD19-CAR T cells processed as in Panel A (n=5). FIG.5D: Luciferase-based analysis of lysis of B16F10-hCD19-luc cells incubated with indicated anti-CD19 CAR T cells in vitro (n=5). FIG.5E: Percentage of CD19+ CD8+ T cells isolated from B16F10-hCD19 tumors that grew in Rag1-/- mice treated with WT, Ch25hΔCD8, Atf3ΔCD8 or Ch25h;Atf3ΔCD8 anti-CD19 CAR T cells (n=5). FIG.5F: Percentage of CD8+ PD-1+ T cells and CD8+Tim3+ T cells isolated from B16F10-hCD19 tumors treated as in FIG.5E (n=5). FIG.5G: Percentage and absolute number of CD8+ anti-CD19 CAR+ T cells infiltrated into B16F10-hCD19 tumors treated as in FIG.5E (n=5). FIG.5H: Volume and weight of B16F10- hCD19 tumor that grew in Rag1-/- mice treated with indicated anti-CD19 CAR T cells (n=5). FIG.5I: Survival analysis for mice described in FIG.5H (n=5). Data are presented as mean±SEM. Statistical analysis was performed using 1-way ANOVA with Tukey’s multiple- comparison test (FIGs.5A-5H) or 2-way ANOVA with Sidak’s multiple-comparison test (FIG. 5H) or Kaplan-Meier test (FIG.5I). n.s, not significant. FIGs.6A-6H: TAK981 sumoylation inhibitor upregulates CH25H, inhibits trogocytosis and augments CAR T viability and anti-tumor activities. FIG.6A: Immunoblot analysis of ATF3 sumoylation in ATF3 immunoprecipitates from lysates from activated OT-I CD8+ T cells treated with TCM in the presence or absence of TAK981 (0.1μM for 8 hr) using the indicated antibodies. Loading controls in starting lysates used for immunoprecipitation were verified by immunoblot using anti-β-tubulin antibody. FIG.6B: qPCR analysis of Ch25h expression in CTLs treated with FCM or TCM in the presence or absence of TAK981 (0.1μM for 8 hr) (n=4). FIG.6C: Transfer of OVA-MHC-I complexes from MC38OVA cells onto WT or Ch25h-/- OT-I CTLs pre-treated or not with FCM or TCM (in presence or absence of 0.1μM TAK981) (n=4). FIG.6D: Expression of CD69 by the intratumoral CTLs isolated from MC38 tumors that grew in Ch25hf/f or Ch25hΔCD8 mice treated with Vehicle (PBS), anti-PD1 antibody (i.p, 5mg/kg every 4 days) and/or TAK981 (i.v, 15mg/kg once a week) as indicated (n=5). FIG.6E: Expression of PD-1 and Annexin V by the intratumoral CTLs from experiments described in FIG.6D (n=5). FIG.6F: Volume of MC38 tumors from experiments described in FIG.6D (n=5). FIG.6G: Weight of MC38 tumors from experiments described in FIG.6D (n=5). FIG.6H: Survival 41744090.2 7
Attorney Docket No.046483-7386WO1(03225) analysis of animals from experiments described in FIG.6D. Mice were euthanized when tumor volume reached ~2000 mm3 (n=5). Data are presented as mean±SEM. Statistical analysis was performed using 1-way ANOVA with Tukey’s multiple-comparison test (FIGs.6B-6E and 6G) or 2-way ANOVA with Sidak’s multiple-comparison test (FIG.6F) or Kaplan-Meier test (FIG. 6H). n.s, not significant. FIGs.7A-7L: CARs designed to re-express CH25H inhibit trogocytosis and increase therapeutic efficacy. FIG.7A: Design of anti-Meso-CAR, anti-Meso-Ch25h CAR, anti-CD19 CAR and anti-CD19-Ch25h CAR constructs. FIG.7B: Transfer of MESO from EM-Meso-GFP- Luc cells onto WT or CH25H-null anti-MESO or anti-MESO-Ch25h CAR T cells co-cultured for 4 hr (n=5). FIG.7C: Numbers of live WT or CH25H-null anti-MESO or anti-MESO-Ch25h CAR T cells after co-culture with EM-Meso-GFP-Luc target cells for 8 hr (n=5). FIG.7D: Luciferase-based analysis of killing of EM-Meso-GFP-Luc target cells by WT or CH25H-null anti-MESO or anti-MESO-Ch25h CAR T cells (n=5). FIG.7E: A schematic of experiment for comparing the efficacy of anti-Meso-CAR and anti-MESO-Ch25h T cells in vivo. FIG.7F: Volume of EM-Meso-GFP-Luc tumors (inoculated s.c. at 1x106) in NSG mice treated with vehicle, Meso-CAR T cells or Meso-Ch25h CAR T cells as indicated in FIG.7E (n=6). Kaplan- Meier survival analysis of survival of tumor–bearing mice (euthanized when tumors reached 1000 mm3) is shown on the right (n=7). FIG.7G: Quantification of percentage (left) and absolute numbers (right) of CD3+CD8+ T cells (i.e. CAR T cells) in EM-Meso-GFP-Luc tumors (upper panels) and blood (bottom panels) from NSG mice treated as in FIG.7E (n=5). FIG.7H: Volume of B16F10-hCD19 tumors (inoculated s.c. at 0.3x106) growing in Rag1-/- mice treated with vehicle or indicated CAR T cells generated from WT or Ch25h-/- splenocytes using anti-CD19 CAR or anti-CD19-Ch25h CAR constructs as indicated (upper panel). Kaplan-Meier survival analysis of survival of tumor–bearing mice (euthanized when tumors reached 1000 mm3) is shown on the bottom (n=5). FIG.7I: A schematic of experiment for comparing the efficacy of human anti-CD19 and anti-CD19-Ch25h CAR T cells in the model of NALM6 acute lymphoblastic leukemia in NSG mice. FIG.7J: Numbers of indicated CAR T cells in blood of NSG mice at day 17, 24 and 31 after injection of NALM6 leukemic cells (n=6). FIG.7K: Numbers of NALM6 cell in blood of NSG mice at day 17, 24 and 31 after injection of NALM6 leukemic cells (n=5-6). FIG.7L: Survival analysis of NALM6 leukemia cells-bearing mice treated with PBS or indicated CAR T cells (n=5). Data are presented as mean±SEM. Statistical 41744090.2 8
Attorney Docket No.046483-7386WO1(03225) analysis was performed using 1-way ANOVA with Tukey’s multiple-comparison test (FIGs.7B- 7D and 7G) or 2-way ANOVA with Sidak’s multiple-comparison test (FIGs.7F, 7H and 7L) or Kaplan-Meier test (FIGs.7F, 7H and 7L) or 2-tailed Students’ t test (FIGs.7J-7K). n.s, not significant. FIGs.8A-8F: Tumor-derived factors (TDFs) downregulate 25-hydroxycholesterol and stimulate trogocytosis between effector CTL and malignant cells. FIG.8A: A schematic of experiment for assessing DiD transfer between CD8+T cells and cancer cells. FIG.8B: Flow cytometry analysis of transfer of DiD dye from DID-labeled OVA-expressing MC38 cells onto co-cultured (for 4 hr) OT-I CD8+ T cells pre-treated with VEGF (50ng/ml), PGE2 (10nM), FCM (fibroblast cell medium), IECM (intestinal epithelial cell medium) or TCM (Tumor conditioned medium) for 8 hr as indicated (n=5). FIG.8C: Flow cytometry analysis of transfer of DiD dye from DID-labeled OVA-expressing MC38 cells onto OT-I CD8+ T cells co-cultured for 4 hr to allow contact (with or without Latrunculin A, 1μM) or separated in Transwell settings (n=5). FIG.8D: Flow cytometry analysis of transfer of DiD dye from DID-labeled MC38 or MC38OVA cells onto co-cultured (for 4 hr) OT-I CD8+ T cells pre-treated with FCM or TCM for 8 hr as indicated (n=5). FIG.8E: Heatmap of fold changes and P values in expression of cholesterol metabolism-associated genes in CTLs treated with TCM versus FCM for 8 hr (n=3). FIG.8F: qPCR analysis of Ch25h mRNA expression in CD8+ T cells isolated from B16F10 tumor, MH6499c4 tumor and Hepa1-6 tumor and spleen of naïve or tumor bearing mice (n=5). Data are presented as mean±SEM. Statistical analysis was performed using 2-tailed Students’ t test (FIG. 8F) or 1-way ANOVA with Tukey’s multiple-comparison test (FIGs.8C-8D). n.s, not significant. FIGs.9A-9J: CH25H is a pivotal regulator of CTL trogocytosis, survival and activity. FIG.9A: Flow cytometry analysis of transfer of OVA-MHC-I complexes from OVA-expressing MC38 cells onto co-cultured (for 4 hr) OT-I CD8+ T cells pre-treated with FCM or TCM (with or without 25HC, 4μM or GW3965, 2μM) for 8 hr (n=5). FIG.9B: qPCR analysis of mRNA expression of indicated LXR-stimulated genes in CD8+ T cells treated with FCM or TCM (with or without 25HC, 4μM or GW3965, 2μM) for 8 hr (n=4). FIG.9C: Flow cytometry analysis (and its quantification) of effector markers CD69, IFN-γ, Perforin and Granzyme B on the surface of WT or CH25H-null OT-I CTLs stimulated with OVA peptide (0.5μg/ml for 48 hr) (n=4-5). FIG. 9D: Flow cytometry analysis (and its quantification) of exhaustion (PD-1, LAG3, and TIM3) and apoptosis (Annexin V) markers on the surface of WT or CH25H-null OT-I CTLs stimulated with 41744090.2 9
Attorney Docket No.046483-7386WO1(03225) OVA peptide (0.5μg/ml for 48 hr) (n=5). FIG.9E: Gating strategy of experiments for assessment of the role of Ch25h on trogocytosis in vivo. FIG.9F: Flow cytometry of percentage of CD8+DiD+ T cells after WT or Ch25h-/- CTLs were co-cultured with MC38-OVA cells for 4 hr (n=5). FIG.9G: Flow cytometry analysis of transfer of DiD from DiD-labelled MC38 or MC38- OVA cell onto co-cultured (for 4 hr) WT or Ch25h-/-CTLs. Quantification is shown on the right (n=5). FIG.9H: Flow cytometry analysis of transfer of OVA-MHC-I complexes from MC38OVA or control MC38 cells onto co-cultured (for 4 hr) WT or Ch25h-/-CTLs. Quantification is shown on the right (n=5). FIG.9I: Effects of Latrunculin A treatment (1μM for 20 min) on DiD transfer under conditions described in FIG.9G (n=5). FIG.9J: Effects of Latrunculin A treatment (1μM for 20 min) on OVA-MHC-I complexes transfer under conditions described in FIG.9H (n=5). Data are presented as mean±SEM. Statistical analysis was performed using 2-tailed Students’ t test (FIGs.9B-9D and 9F) or 1-way ANOVA with Tukey’s multiple-comparison test (FIGs.9G- 9J). n.s, not significant. FIGs.10A-10I: Downregulation of CH25H in CTLs attenuates the immune responses and promote tumor growth. FIG.10A: Correlation between expression of CH25H and CD8A expression in tumor tissues from human melanoma, colon and pancreatic cancer. FIG.10B: A schematic of experiment for generating Ch25hΔCD8 mice (top). Genotyping analysis of Ch25hΔCD8 or Ch25h f/f mice (left bottom). Expression of Ch25h in splenic CD8+ and CD4+ T cells (right bottom; n=5). FIG.10C: Comparison of percentage of CD3+CD8+ T cells, CD3+CD4+ T cells, B cells and NK cells in spleens from Ch25hf/f and Ch25hΔCD8 mice (n=4). FIG.10D: Comparison of percentage of CD3+CD8+ T cells, CD3+CD4+ T cells, B cells and NK cells in blood from Ch25hf/f and Ch25hΔCD8 mice (n=4). FIG.10E: Growth of MC38 tumors (inoculated s.c. at 1x106) in Ch25hf/f and Ch25hΔCD8Cre (left) and Kaplan-Meier survival analysis of tumor– bearing mice euthanized when tumors reached ~1000 mm3 (right; n=5). FIG.10F: Growth of MH6499c4 tumors (inoculated s.c. at 1x106) in Ch25hf/f and Ch25hΔCD8Cre (left) and Kaplan- Meier survival analysis of tumor–bearing mice euthanized when tumors reached ~1000 mm3 (right; n=5-6). FIG.10G: Growth of Hepa1-6 tumors (inoculated s.c. at 2x106) in Ch25hf/f and Ch25hΔCD8Cre (left) and Kaplan-Meier survival analysis of tumor–bearing mice euthanized when tumors reached ~1000 mm3 (right; n=6). FIG.10H: Flow cytometry analysis of percentage of CD3+CD8+T cells (left), CD8+PD-1+ T cells (middle) and CD8+Annexin V + T cells (right) in MH6499c4 tumors from Ch25hf/f and Ch25hΔCD8 (n=5). FIG.10I: Flow cytometry analysis of 41744090.2
Attorney Docket No.046483-7386WO1(03225) percentage of CD3+CD8+T cells (left), CD8+PD-1+ T cells (middle) and CD8+Annexin V + T cells (right) in Hepa1-6 tumors from Ch25hf/f and Ch25hΔCD8 (n=5). Data are presented as mean±SEM. Statistical analysis was performed using 1-way ANOVA with Tukey’s multiple- comparison test (FIG.10B) or 2-tailed
(FIGs.10C, 10D, 10H and 10I) or 2-way ANOVA with Sidak’s multiple-comparison test (FIGs.10E-10G) or log-rank (Mantel-Cox) test (FIGs.10E-10G). n.s, not significant. FIGs.11A-11J: ATF3 regulates effector trogocytosis, activity and viability of CTLs, and tumor growth in a CH25H-dependent manner. FIG.11A: q-PCR analysis of Atf3 mRNA levels in CTLs treated with Vehicle, IECM, FCM, TCM, PGE2 (10nM), VEGF (50ng/ml) or MC38 tumor-derived extracellular vesicles (TEVs, 20 µg/mL) for 8 hr (n=4-5). FIG.11B: Overall (top) and pairwise comparison of Atf3 mRNA levels in CD8+ T cells from B16F10 (left), MH6499c4 (middle) and Hepa1-6 (right) tumors and spleens of naïve or corresponding tumor bearing mice (n=5). FIG.11C: Linear regression analysis of Atf3 and Ch25h mRNA expression in CD8+ T cells isolated from B16F10 (upper), MH6499c4 (middle) and Hepa1-6 (below) tumors (n=10). FIG.11D: A schematic of experiment for generating Atf3ΔCD8 mice, their genotyping and qPCR analysis of Atf3 mRNA levels in splenic CD4+ and CD8+ T cells. The lower panel depicts analysis of percentage of CD3+CD8+ T cells, CD3+CD4+ T cells, B cells and NKs cell in spleens and blood from Atf3f/f or Atf3ΔCD8 mice (n=4-5). FIG.11E: Analysis of Ch25h expression in CD8+ T cells isolated from B16F10, MH6499c4 or Hepa1-6 tumors or matched spleens from Atf3 or Atf3ΔCD8 mice (n=4). FIG.11F: Analysis of percentage of CD8+ Tim3+ T cells and CD8+ Annexin V+ T cells in MC38 tumors from Atf3f/f or Atf3ΔCD8 mice (n=5). FIG.11G: Representative images of B16F10 tumors that grew in WT, Ch25hΔCD8, Atf3ΔCD8 or Ch25h;Atf3ΔCD8 mice. FIG.11H: Flow cytometry analysis of percentage of CD3+CD8+ T cells in B16F10 tumors from WT, Ch25hΔCD8, Atf3ΔCD8 or Ch25h;Atf3ΔCD8 mice. FIG.11I: Flow cytometry analysis of the numbers of CD3+CD8+ T cells and percentage of CD8+Tim3+ and CD8+Annexin V+ T cells in B16F10 tumors grown in indicated mice (n=5). FIG.11J: Analysis of percentage or numbers of CD3+CD8+ T cells and the percentage of CD8+PD-1+, CD8+Tim3+ and CD8+Annexin V+ T cells in the spleens from B16F10 tumor-bearing or Ch25h;Atf3ΔCD8 mice
(n=5). Data are presented as mean±SEM. Statistical analysis was performed using linear regression analysis (FIG.11C), 1-way ANOVA with Tukey’s multiple-comparison test (FIGs. 41744090.2
Attorney Docket No.046483-7386WO1(03225) 11D, 11E, 11F, 11I and 11J) or 2-tailed Students’ t test (FIGs.11A, 11B and 11D).n.s, not significant. FIGs.12A-12C: ATF3 and CH25H control trogocytosis and activity of CAR T cells. FIG.12A: Detection of the anti-CD19 CAR expression levels in indicated CAR T cells 48 hr after transduction. FIG.12B: Flow cytometry analysis of the percentage of CD19+ CD8+ T cells isolated from B16F10-CD19 tumors that grew in Rag1-/- mice treated with WT, Ch25hΔCD8, Atf3ΔCD8 or Ch25h;Atf3ΔCD8 anti-CD19 CAR T cells (n=5). FIG.12C: Flow cytometry analysis of the percentage of CD8+ PD-1+ T cells and CD8+Tim3+ T cells isolated from B16F10-CD19 tumors that grew in Rag1-/- mice treated with WT, Ch25hΔCD8, Atf3ΔCD8 or Ch25h;Atf3ΔCD8 anti- CD19 CAR T cells (n=5). Data were analyzed using flowJo7.6. FIG.13A-13F: TAK981 sumoylation inhibitor upregulates CH25H, inhibits trogocytosis and augments CAR T viability and anti-tumor activities. FIG.13A: qPCR analysis of Atf3 expression in CTLs treated with FCM or TCM in the presence or absence of TAK981 (0.1μM for 8 hr) (n=5). FIG.13B: Analysis of live WT or Ch25h-/- OT-I CTLs pre-treated or not with FCM or TCM (in presence or absence of 0.1μM TAK981) and then cultured alone or with target MC38OVA cells for 4 hr (n=4). FIG.13C: Luciferase activity-based analysis of lysis of MC38OVA-luc cells incubated with WT or Ch25h-/- OT-I CD8+ T cells pre-treated with FCM or TCM (with or without 0.1μM TAK981) (n=4-5). FIG.13D: qPCR analysis of Atf3 and Ch25h expression in the intratumoral CTLs in WT host mice bearing MC38 tumors and treated with Vehicle, anti-PD-1, TAK981 or anti-PD-1+TAK981 as indicated (n=6). FIG.13E: Flow cytometry analysis of CD3+CD8+ T cells infiltrating MC38 tumors that grew in Ch25hf/f or Ch25hΔCD8 mice treated with Vehicle, anti-PD-1, TAK981 or anti-PD-1+TAK981. Quantification of percentage or absolute number of these cells is shown on the right (n=5). FIG.13F: Analysis of percentage of CD3+CD8+ T cells, CD8+CD69+ T cells, CD8+PD-1+ T cells and absolute number of CD3+CD8+ T cells in the spleens from MC38 tumor-bearing mice treated with Vehicle, anti-PD-1, TAK981 or anti-PD-1+TAK981 (n=5). Data are presented as mean±SEM. Statistical analysis was performed using 2-tailed Students’ t test (FIG.13A) or 1-way ANOVA with Tukey’s multiple-comparison test (FIGs.13B-13D). n.s, not significant. FIG.14A-14O: CARs designed to re-express CH25H inhibit trogocytosis and increase therapeutic efficacy. FIG.14A: Flow cytometry analysis of percentage of mCherry positive CD8+ T cells 48 hr after transduced with anti-MESO CAR that also expresses mCherry. FIG. 41744090.2
Attorney Docket No.046483-7386WO1(03225) 14B: Luciferase-based analysis of killing of EM-Meso-GFP-Luc target cells by WT or Ch25h-/- anti-Meso-CAR T cells pre-treated or not with 25HC (4μM for 8hr) (n=5). FIG.14C: qPCR analysis of Ch25h expression in WT or Ch25h-/- T cells transduced with anti-Meso CAR or anti- Meso-Ch25h CARs (n=4). FIG.14D: Representative images and weight quantification of EM- Meso-GFP-Luc tumors from NSG mice treated with vehicle, anti-Meso-CAR T cells or anti- Meso-Ch25h CAR T cells (n=5). FIG.14E: Flow cytometric analysis of CD3+CD8+ CAR T cells in tumor or blood from NSG mice treated as in FIG.14D. FIG.14F: Flow cytometric analysis of anti-CD19 CAR expression in WT or Ch25h-/- T cells 48 hr after transduced with anti-CD19 CAR or anti-CD19-Ch25h CAR retrovirus. FIG.14G: qPCR analysis of Ch25h expression in WT or Ch25h-/- T cells transduced with anti-CD19 CAR or anti-CD19-Ch25h CAR (n=4). FIG. 14H: Luciferase-based analysis of killing of B16F10-hCD19-luc cells by WT or Ch25h-/- anti- CD19 CAR or anti-CD19-Ch25h CAR T cells co-cultured with target cell at 1:2 ratio (n=5). FIG. 14I: Flow cytometry analysis of CD19transfer from B16F10-hCD19 target cells onto WT or Ch25h-/- anti-CD19 CAR or anti-CD19-Ch25h CAR T cells after 4 hr of co-culture (n=5). FIG. 14J: Flow cytometric analysis of remaining levels of CD19 antigen on B16F10-hCD19 cells 4 hr after co-culture with vehicle (PBS) or indicated anti-CD19 or anti-CD19-Ch25h CAR T cells (n=5). FIG.14K: Detection of percentage of CD19+CD8+Tim3+ T cells 4 hr after co-culture of indicated CAR T cells with B16F10-hCD19 target cells (n=5). FIG.14L: Percentage of CD19 antigen-positive live (left) and apoptotic (Annexin V+, right) indicated CAR T cells after 8 hr of co-culture with B16F10-hCD19 target cells (n=4). FIG.14M: A schematic of experiment aimed at comparing the efficacy of anti-CD19 and anti-CD19-Ch25h CAR T cells against B16F10- hCD19 tumors. FIG.14N: Analysis of intratumoral CD3+CD8+T cells isolated from B16F10- hCD19 tumors that grew in mice treated with indicated CAR T cells or vehicle (n=5). FIG.14O: Percentage of CD8+ Tim3+, CD8+LAG3+, CD8+PD-1+ and CD8+Annexin V+ T cells in B16F10- hCD19 tumors from mice treated with Vehicle or indicated CAR T cells (n=5). Data are presented as mean±SEM. Statistical analysis was performed using 1-way ANOVA with Tukey’s multiple-comparison test (FIGs.14B, 14C, 14G-14L, 14N and 14O). N.s, not significant. DETAILED DESCRIPTION The present disclosure provides compositions and methods for modified immune cells or precursors thereof (e.g., modified T cells) comprising a nucleic acid encoding a chimeric antigen 41744090.2
Attorney Docket No.046483-7386WO1(03225) receptor (CAR) and a cholesterol 25-hydroxylase (CH25H) gene. Methods for treating diseases or disorders (e.g. cancer) are also provided. CAR T cells targeting tumor-specific antigens e.g. CD19 have displayed remarkable efficacy for the treatment of some patients with hematologic malignancies. However, despite high response rates, many patients relapse and, unfortunately, CAR-T immunotherapy has exhibited little success for the treatment of solid tumors. In addition to the immune suppressive effects of the tumor microenvironment (TME) mediated by tumors, other mechanisms by which tumor cells decrease antigen density to evade targeted immunotherapy are beginning to be uncovered. One such mechanism is trogocytosis (named from the ancient Greek trogo-, meaning gnaw or nibble), which is a fast process of intercellular transfer of cell surface molecules and membrane fragments. It can affect immune responses by promoting tumor antigen escape and subverting CAR T killing ability. This process of therapeutic evasion involves the transfer of a fragment of the lipid plasma membrane along with target antigens from donor malignant cells to recipient cytotoxic T lymphocytes (CTLs). The transfer reduces antigen density on cancer cells while tagging the T cell with the target antigen. This results in T cell exhaustion, T cell fratricide, and tumor escape. The frequency of trogocytosis in immunotherapy treatments is currently unknown, however, data regarding the frequence of antigen loss in patients relapsing after immunotherapy treatments is starting to come to light. For example, 10%-20% of pediatic B-cell acute lymphoblastic leukemia (B-ALL) patients who hav relapsed after treatment with CART-19 present with antigen loss and 30% - 70% of relapsed and/or refractory ALL patients who have recurrent disease after treatment with CART present with a downregulation or loss of CD19 antigen. Similar trends have been observed in multiple myeloma, glioblastoma, and breast cancer patients. In order to reduce the relapse rate in CAR-T cell treatment of both hematological malignancies and solid rumors, many strategies are now relying on targeting multipe antigens. While this may be effective for tumors that present antigens in sufficient density for being targeted, strategies are needed to combat antigen loss and transfer from tumor cells. Since trogocytosis involves the transfer of fragments of lipid membranes containing cholesterol and its metabolites, this study focused on identifying factors in the cholesterol metabolism pathway which control trogocytosis. Through a series of in vitro studies, cholesterol 41744090.2
Attorney Docket No.046483-7386WO1(03225) 25-hydroxylase (CH25H) was identified as a pivotal negative regulator of trogocytosis. CH25H is a gene whose enzymatic product 25-hydroxycholesterol (25HC) alters membrane fluidity and inhibits membrane fusion, which is essential for incorporating malignant cell membrane fragments into CTL during trogocytosis. To address the issues of trogocytosis in an immunotherapy setting, CAR constructs were devised herein that co-express CH25H in a T cell. Previously described anti-mesothelin and anti- CD19 CARs (Milone et al., (2009) Mol Ther 17, 1453-1464) were redesigned to enable co- expression of CH25H. The effects of co-expression of CH25H with CARs on the efficacy of adoptive transfer of human CAR T cells were tested in the NALM6 B cell lymphoblastic leukemial (B-ALL) model. Under these conditions, CAR T cells expressing CH25H persisted in the host (FIG.7J) and exhibited greater anti-tumor effect as manifested by decreased number of leukemic cells in the blood (FIG.7K) and prolonged animal survival (FIG.7L). In addition to the B-ALL mouse model, a CH25H-expressing anti-mesothelin CAR was designed and interrogated against a solid tumor model. Mesolthlin (MESO) is a cell-surface antigen implicated in tumor invasion and is highly expressed in mesothelioma, lung, pancreas, breast, ovarian, and other solid tumor cancers. In vivo treatment of NSG mice harboring EM- Meso-GFP-Luc tumors, replicating mesothelioma, with CTLs harboring conventional or CH25H-expressing anit-MESO CAR revealed that expression of CH25H significantly increased the efficacy of this adoptive cell therapy as seen from assessment of tumor volume and weight and animal survival (FIG.7F). Under these conditions, T cells harboring CH25H-expressing CAR displayed increased numbers in tumors and blood compared to control CAR T cells (FIG. 7G). It is to be understood that the methods described in this disclosure are not limited to particular methods and experimental conditions disclosed herein as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. Furthermore, the experiments described herein, unless otherwise indicated, use conventional molecular and cellular biological and immunological techniques within the skill of the art. Such techniques are well known to the skilled worker, and are explained fully in the literature. See, e.g., Ausubel, et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, N.Y. (1987-2008), including all supplements, Molecular Cloning: A Laboratory 41744090.2
Attorney Docket No.046483-7386WO1(03225) Manual (Fourth Edition) by MR Green and J. Sambrook and Harlow et al., Antibodies: A Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor (2013, 2nd edition). Methods and techniques using T Cells with chimeric antigen receptors (CAR T cells) are described in e.g., Milone et al., (2009) Mol Ther 17, 1453-1464; Ruella, et al., (2016) J. Clin. Invest., 126(10):3814-3826; and Kalos, et al., (2011) 3 (95), 95ra73:1-11, the contents of which are hereby incorporated by reference in their entireties. A. Definitions Unless otherwise defined, scientific and technical terms used herein have the meanings that are commonly understood by those of ordinary skill in the art. In the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The use of “or” means “and/or” unless stated otherwise. The use of the term “including,” as well as other forms, such as “includes” and “included,” is not limiting. Generally, nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein is well-known and commonly used in the art. The methods and techniques provided herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. Enzymatic reactions and purification techniques are performed according to manufacturer’s specifications, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well- known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients. That the disclosure may be more readily understood, select terms are defined below. 41744090.2
Attorney Docket No.046483-7386WO1(03225) The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods. “Activation,” as used herein, refers to the state of a T cell that has been sufficiently stimulated to induce detectable cellular proliferation. Activation can also be associated with induced cytokine production, and detectable effector functions. The term “activated T cells” refers to, among other things, T cells that are undergoing cell division. As used herein, to “alleviate” a disease means reducing the severity of one or more symptoms of the disease. “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. The term “antigen” 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. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises a nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an “antigen” as that term is used herein. Furthermore, one skilled in the art will understand that 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 generated 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. 41744090.2
Attorney Docket No.046483-7386WO1(03225) As used herein, the term “autologous” is meant to refer to any material derived from the same individual to which it is later to be re-introduced into the individual. A “co-stimulatory molecule” refers to the cognate binding partner on a T cell that specifically binds with a co-stimulatory ligand, thereby mediating a co-stimulatory response by the T cell, such as, but not limited to, proliferation. Co-stimulatory molecules include but are not limited to an MHC class I molecule, BTLA and a Toll ligand receptor. A “co-stimulatory signal”, as used herein, refers to a signal, which in combination with a primary signal, such as TCR/CD3 ligation, leads to T cell proliferation and/or upregulation or downregulation of key molecules. A “disease” is a state of health of an animal (e.g., a mammal, e.g., a human) wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate. In contrast, a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health. The term “downregulation” as used herein refers to the decrease or elimination of gene expression of one or more genes or of their RNA or/and protein products. “Effective amount” or “therapeutically effective amount” are used interchangeably herein, and refer to an amount of a compound, formulation, material, or composition, as described herein effective to achieve a particular biological result or provides a therapeutic or prophylactic benefit. Such results may include but are not limited to an amount that when administered to an animal (e.g., a mammal, e.g., a human), causes a detectable level of immune suppression or tolerance compared to the immune response detected in the absence of the composition of the invention. The immune response can be readily assessed by a plethora of art- recognized methods. The skilled artisan would understand that the amount of the composition administered herein varies and can be readily determined based on a number of factors such as the disease or condition being treated, the age and health and physical condition of the animal (e.g., mammal, e.g., human) being treated, the severity of the disease, the particular compound being administered, and the like. “Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of 41744090.2
Attorney Docket No.046483-7386WO1(03225) other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA. As used herein “endogenous” refers to any material from or produced inside an organism, cell, tissue or system. The term “epitope” as used herein is defined as a small chemical molecule on an antigen that can elicit an immune response, inducing B and/or T cell responses. An antigen can have one or more epitopes. Most antigens have many epitopes; i.e., they are multivalent. In general, an epitope is roughly about 10 amino acids and/or sugars in size. Preferably, the epitope is about 4- 18 amino acids, more preferably about 5-16 amino acids, and even more most preferably 6-14 amino acids, more preferably about 7-12, and most preferably about 8-10 amino acids. One skilled in the art understands that generally the overall three-dimensional structure, rather than the specific linear sequence of the molecule, is the main criterion of antigenic specificity and therefore distinguishes one epitope from another. Based on the present disclosure, a peptide used in the present invention can be an epitope. As used herein, the term “exogenous” refers to any material introduced from or produced outside an organism, cell, tissue or system. The term “expand” as used herein refers to increasing in number, as in an increase in the number of T cells. In one embodiment, the T cells that are expanded ex vivo increase in number relative to the number originally present in the culture. In another embodiment, the T cells that are expanded ex vivo increase in number relative to other cell types in the culture. The term "ex vivo," as used herein, refers to cells that have been removed from a living organism, (e.g., a human) and propagated outside the organism (e.g., in a culture dish, test tube, or bioreactor). The term “expression” as used herein is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter. 41744090.2
Attorney Docket No.046483-7386WO1(03225) “Expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., Sendai viruses, lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide. “Identity” as used herein refers to the subunit sequence identity between two polymeric molecules particularly between two amino acid molecules, such as, between two polypeptide molecules. When two amino acid sequences have the same residues at the same positions; e.g., if a position in each of two polypeptide molecules is occupied by an arginine, then they are identical at that position. The identity or extent to which two amino acid sequences have the same residues at the same positions in an alignment is often expressed as a percentage. The identity between two amino acid sequences is a direct function of the number of matching or identical positions; e.g., if half (e.g., five positions in a polymer ten amino acids in length) of the positions in two sequences are identical, the two sequences are 50% identical; if 90% of the positions (e.g., 9 of 10), are matched or identical, the two amino acids sequences are 90% identical. The term “immune response” as used herein is defined as a cellular response to an antigen that occurs when lymphocytes identify antigenic molecules as foreign and induce the formation of antibodies and/or activate lymphocytes to remove the antigen. The term “immunosuppressive” is used herein to refer to reducing overall immune response. “Isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell. A “lentivirus” as used herein refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the 41744090.2
Attorney Docket No.046483-7386WO1(03225) most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses offer the means to achieve significant levels of gene transfer in vivo. By the term “modified” as used herein, is meant a changed state or structure of a molecule or cell of the invention. Molecules may be modified in many ways, including chemically, structurally, and functionally. Cells may be modified through the introduction of nucleic acids. By the term “modulating,” as used herein, is meant mediating a detectable increase or decrease in the level of a response in a subject compared with the level of a response in the subject in the absence of a treatment or compound, and/or compared with the level of a response in an otherwise identical but untreated subject. The term encompasses perturbing and/or affecting a native signal or response thereby mediating a beneficial therapeutic response in a subject, preferably, a human. In the context of the present invention, the following abbreviations for the commonly occurring nucleic acid bases are used. “A” refers to adenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine. The term “oligonucleotide” typically refers to short polynucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, C, G), this also includes an RNA sequence (i.e., A, U, C, G) in which “U” replaces “T.” Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s). “Parenteral” administration of an immunogenic composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrasternal injection, or infusion techniques. The term “polynucleotide” as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric “nucleotides.” The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides 41744090.2
Attorney Docket No.046483-7386WO1(03225) include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR, and the like, and by synthetic means. As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof. By the term “specifically binds,” as used herein with respect to an antibody, is meant an antibody which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. But, such cross-species reactivity does not itself alter the classification of an antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific. In some instances, the terms “specific binding” or “specifically binding,” can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope “A”, the presence of a molecule containing epitope A (or free, 41744090.2
Attorney Docket No.046483-7386WO1(03225) unlabeled A), in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled A bound to the antibody. By the term “stimulation,” is meant a primary response induced by binding of a stimulatory molecule (e.g., a TCR/CD3 complex) with its cognate ligand thereby mediating a signal transduction event, such as, but not limited to, signal transduction via the TCR/CD3 complex. Stimulation can mediate altered expression of certain molecules, such as downregulation of TGF-beta, and/or reorganization of cytoskeletal structures, and the like. A “stimulatory molecule,” as the term is used herein, means a molecule on a T cell that specifically binds with a cognate stimulatory ligand present on an antigen presenting cell. A “stimulatory ligand,” as used herein, means a ligand that when present on an antigen presenting cell (e.g., an aAPC, a dendritic cell, a B-cell, and the like) can specifically bind with a cognate binding partner (referred to herein as a “stimulatory molecule”) on a T cell, thereby mediating a primary response by the T cell, including, but not limited to, activation, initiation of an immune response, proliferation, and the like. Stimulatory ligands are well-known in the art and encompass, inter alia, an MHC Class I molecule loaded with a peptide, an anti-CD3 antibody, a superagonist anti-CD28 antibody, and a superagonist anti-CD2 antibody. The term “subject” is intended to include living organisms in which an immune response can be elicited (e.g., animals, e.g., mammals, e.g., humans). A “subject” or “patient,” as used therein, may be an animal, a human, or non-human mammal. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline and murine mammals. Preferably, the subject is human. A “target site” or “target sequence” refers to a nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule may specifically bind under conditions sufficient for binding to occur. In some embodiments, a target sequence refers to a genomic nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule may specifically bind under conditions sufficient for binding to occur. As used herein, the term “T cell receptor” or “TCR” refers to a complex of membrane proteins that participate in the activation of T cells in response to the presentation of antigen. The TCR is responsible for recognizing antigens bound to major histocompatibility complex molecules. TCR is composed of a heterodimer of an alpha ( ^) and beta (β) chain, or gamma and delta (γ/δ) chains. TCRs may exist in alpha/beta and gamma/delta forms, which are structurally 41744090.2
Attorney Docket No.046483-7386WO1(03225) similar but have distinct anatomical locations and functions. Each chain is composed of two extracellular domains, a variable and constant domain. In some embodiments, the TCR may be modified on any cell comprising a TCR, including, for example, a helper T cell, a cytotoxic T cell, a memory T cell, regulatory T cell, natural killer T cell, and gamma delta T cell. The term “therapeutic” as used herein means a treatment and/or prophylaxis. A therapeutic effect is obtained by suppression, remission, or eradication of a disease state. “Transplant” refers to a biocompatible lattice or a donor tissue, organ or cell, to be transplanted. An example of a transplant may include but is not limited to skin cells or tissue, bone marrow, and solid organs such as heart, pancreas, kidney, lung and liver. A transplant can also refer to any material that is to be administered to a host. For example, a transplant can refer to a nucleic acid or a protein. The term “transfected” or “transformed” or “transduced” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny. To “treat” a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject. A “vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, Sendai viral vectors, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like. Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically 41744090.2
Attorney Docket No.046483-7386WO1(03225) disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range. B. Modified Immune Cells The present invention provides modified immune cells or precursors thereof (e.g., T cells) for use in immunotherapy (e.g. CAR T cells). In one aspect, the invention provides a modified immune cell or precursor cell thereof (e.g., T cell) comprising a nucleic acid encoding a chimeric antigen receptor (CAR) and a cholesterol 25-hydroxylase (CH25H) gene. It was demonstrated herein that tumor-derived factors (TDFs) stimulate trogocytosis and decrease viability and activity of anti-tumor CTLs by reprogramming the regulatory axis that involves activating transcription factor-3 (ATF3) and cholesterol 25-hydroxylase (CH25H). ATF3 is an early stress-responsive gene that can be induced by many factors in the tumor microenvironment including hypoxia and nutrient deprivation. ATF3 has been shown to suppress CH25h expression in macrophages. Data herein demonstrate that TDF-induced ATF3 in CTLs downregulates expression of CH25H. The latter catalyzes monooxygenation of cholesterol into 25-hydroxycholesterol (25HC), which interferes with trogocytosis stimulated by TDFs. Dysregulation of the ATF3-CH25H axis in the intratumoral natural or CAR-expressing CTLs stimulates effector trogocytosis and undermines viability and anti-tumor activity of these CTLs. Conversely, pharmacological, or genetic restoration of CH25H expression in CTLs inhibits trogocytosis, suppresses tumor growth and increases the efficacy of immunotherapies. In one aspect, the invention provides a modified immune cell or precursor cell thereof (e.g., T cell) comprising a nucleic acid encoding a chimeric antigen receptor (CAR) and a cholesterol 25-hydroxylase (CH25H), wherein the cell is capable of expressing the CAR and CH25H. The modified immune cell or precursor cell thereof (e.g., T cell) can comprise any of the CARs disclosed herein, or any CAR known in the art. In certain embodiments, the cell comprises a CAR and CH25H. In certain embodiments CH25H is encoded by a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 10. In certain 41744090.2
Attorney Docket No.046483-7386WO1(03225) embodiments, the cell comprises a nucleic acid that encodes CH25H and an anti-mesothelin CAR. In certain embodiments, the cell comprises a nucleic acid that encodes CH25H and an anti-CD19 CAR. In certain embodiments, the cell comprises a nucleic acid comprising a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 10. In certain embodiments, the cell comprises a nucleic acid comprising a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 8 or SEQ ID NO: 15. In certain embodiments, the cell comprises a first nucleic acid encoding CH25H and a second nucleic acid encoding a CAR. In certain embodiments, the immune cell or precursor cell thereof is a T cell. In certain embodiments, the T cell is a human T cell. In certain embodiments, the cell is an autologous cell (e.g. an autologous T cell). Thus, provided are cells, compositions and methods that enhance immune cell, such as T cell, function in adoptive cell therapy, including those offering improved efficacy, such as inhibiting trogocytosis, increasing viability, increasing anti-tumor activity. In some embodiments, the provided immune cells inhibit trogocytosis when administered in vivo to a subject. In some embodiments, this inhibition in the subject upon administration is greater as compared to that which would be achieved by alternative methods, such as those involving administration of cells genetically engineered by methods in which T cells do not encode a CAR and CH5H. In some embodiments, the immune cells in the composition retain a phenotype of the immune cell or cells compared to the phenotype of cells in a corresponding or reference composition when assessed under the same conditions. In some embodiments, cells in the composition include naive cells, effector memory cells, central memory cells, stem central memory cells, effector memory cells, and long-lived effector memory cells. In some embodiments, the percentage of T cells, or T cells expressing the CAR and CH25H exhibit a non-activated, long-lived memory or central memory phenotype that is the same or substantially the same as a corresponding or reference population or composition of cells. In some embodiments, such property, activity or phenotype can be measured in an in vitro assay, such as by incubation of the cells in the presence of an antigen targeted by the CAR, a cell expressing the antigen and/or an antigen-receptor activating substance. In some embodiments, any of the assessed activities, properties or phenotypes can be assessed at various days following 41744090.2
Attorney Docket No.046483-7386WO1(03225) electroporation or other introduction of the agent, such as after or up to 3, 4, 5, 6, 7 days. In some embodiments, such activity, property or phenotype is retained by at least 80%, 85%, 90%, 95% or 100% of the cells in the composition compared to the activity of a corresponding composition. As used herein, reference to a "corresponding composition" or a "corresponding population of immune cells" (also called a "reference composition" or a "reference population of cells") refers to immune cells (e.g., T cells) obtained, isolated, generated, produced and/or incubated under the same or substantially the same conditions, except that the immune cells or population of immune cells were not introduced with the agent. In some aspects, except for not containing introduction of the agent, such immune cells are treated identically or substantially identically as immune cells that have been introduced with the agent, such that any one or more conditions that can influence the activity or properties of the cell, including the upregulation or expression of the inhibitory molecule, is not varied or not substantially varied between the cells other than the introduction of the agent. Methods and techniques for assessing the expression and/or levels of T cell markers are known in the art. Antibodies and reagents for detection of such markers are well known in the art, and readily available. Assays and methods for detecting such markers include, but are not limited to, flow cytometry, including intracellular flow cytometry, ELISA, ELISPOT, cytometric bead array or other multiplex methods, Western Blot and other immunoaffinity-based methods. In some embodiments, CAR-expressing cells can be detected by flow cytometry or other immunoaffinity based method for expression of a marker unique to such cells, and then such cells can be co-stained for another T cell surface marker or markers. The population of cells containing T cells can be cells that have been obtained from a subject, such as obtained from a peripheral blood mononuclear cells (PBMC) sample, an unfractionated T cell sample, a lymphocyte sample, a white blood cell sample, an apheresis product, or a leukapheresis product. In some embodiments, T cells can be separated or selected to enrich T cells in the population using positive or negative selection and enrichment methods. In some embodiments, the population contains CD4+, CD8+ or CD4+ and CD8+ T cells. In some embodiments, the administered cells can be detected or quantified after administration to a subject. For example, in some aspects, quantitative PCR (qPCR) is used to assess the quantity of cells expressing the CAR and/or CH25H in the blood or organ or tissue (e.g., disease site) of the subject. In some aspects, persistence is quantified as copies of DNA or 41744090.2
Attorney Docket No.046483-7386WO1(03225) plasmid encoding the exogenous receptor per microgram of DNA, or as the number of receptor- expressing cells per microliter of the sample, e.g., of blood or serum, or per total number of peripheral blood mononuclear cells (PBMCs) or white blood cells or T cells per microliter of the sample. In some embodiments, flow cytometric assays detecting cells expressing the receptor generally using antibodies specific for the receptors also can be performed. Cell-based assays may also be used to detect the number or percentage of functional cells, such as cells capable of binding to and/or neutralizing and/or inducing responses, e.g., cytotoxic responses, against cells of the disease or condition or expressing the antigen recognized by the receptor. In any of such embodiments, the extent or level of expression of another marker associated with the modified cell can be used to distinguish the administered cells from endogenous cells in a subject. C. Chimeric Antigen Receptors The present invention provides compositions and methods for cell-based immunotherapies. In certain embodiments, the cell-based immunotherapy comprises a modified immune cell or precursor thereof, e.g., modified T cell, comprising a chimeric antigen receptor (CAR) and CH25H. Thus, in some embodiments, the immune cell has been genetically modified to express the CAR and CH25H. CARs of the present invention comprise an antigen binding domain, a transmembrane domain, and an intracellular domain. The antigen binding domain may be operably linked to another domain of the CAR, such as the transmembrane domain or the intracellular domain, both described elsewhere herein, for expression in the cell. In one embodiment, a first nucleic acid sequence encoding the antigen binding domain is operably linked to a second nucleic acid encoding a transmembrane domain, and further operably linked to a third a nucleic acid sequence encoding an intracellular domain. The antigen binding domains described herein can be combined with any of the transmembrane domains described herein, any of the intracellular domains or cytoplasmic domains described herein, or any of the other domains described herein that may be included in a CAR of the present invention. A subject CAR of the present invention may also include a hinge domain as described herein. A subject CAR of the present invention may also include a spacer domain as described herein. In some embodiments, each of the antigen binding domain, transmembrane domain, and intracellular domain is separated by a linker. 41744090.2
Attorney Docket No.046483-7386WO1(03225) Antigen Binding Domain The antigen binding domain of a CAR is an extracellular region of the CAR for binding to a specific target antigen including proteins, carbohydrates, and glycolipids. In some embodiments, the CAR comprises affinity to a target antigen on a target cell. The target antigen may include any type of protein, or epitope thereof, associated with the target cell. For example, the CAR may comprise affinity to a target antigen on a target cell that indicates a particular disease state of the target cell. In one embodiment, the target cell antigen is a tumor associated antigen (TAA). Examples of tumor associated antigens (TAAs), include but are not limited to, differentiation antigens such as MART-1/MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2 and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor-suppressor genes such as p53, Ras, HER-2/neu; unique tumor antigens resulting from chromosomal translocations; such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as the Epstein Barr virus antigens EBVA and the human papillomavirus (HPV) antigens E6 and E7. Other large, protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG- 72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-Catenin, CDK4, Mum-1, p 15, p 16, 43- 9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-associated protein, TAAL6, TAG72, TLP, and TPS. In a preferred embodiment, the antigen binding domain of the CAR targets an antigen that includes but is not limited to CD19, CD20, CD22, ROR1, Mesothelin, CD33/IL3Ra, c-Met, PSMA, PSCA, Glycolipid F77, EGFRvIII, GD-2, NY-ESO-1 TCR, MAGE A3 TCR, and the like. Depending on the desired antigen to be targeted, the CAR of the invention can be engineered to include the appropriate antigen binding domain that is specific to the desired antigen target. For example, if CD19 is the desired antigen that is to be targeted, an antibody for CD19 can be used as the antigen bind moiety for incorporation into the CAR of the invention. 41744090.2
Attorney Docket No.046483-7386WO1(03225) This should not be construed as limiting in any way, as a CAR having affinity for any target antigen is suitable for use in a composition or method of the present invention. As described herein, a CAR of the present disclosure having affinity for a specific target antigen on a target cell may comprise a target-specific binding domain. In some embodiments, the target-specific binding domain is a murine target-specific binding domain, e.g., the target- specific binding domain is of murine origin. In some embodiments, the target-specific binding domain is a human target-specific binding domain, e.g., the target-specific binding domain is of human origin. For example, a CAR of the present disclosure having affinity for CD19 on a target cell may comprise a CD19 binding domain. In some embodiments, a CAR of the present disclosure may have affinity for one or more target antigens on one or more target cells. In some embodiments, a CAR may have affinity for one or more target antigens on a target cell. In such embodiments, the CAR is a bispecific CAR, or a multispecific CAR. In some embodiments, the CAR comprises one or more target-specific binding domains that confer affinity for one or more target antigens. In some embodiments, the CAR comprises one or more target-specific binding domains that confer affinity for the same target antigen. For example, a CAR comprising one or more target-specific binding domains having affinity for the same target antigen could bind distinct epitopes of the target antigen. When a plurality of target-specific binding domains is present in a CAR, the binding domains may be arranged in tandem and may be separated by linker peptides. For example, in a CAR comprising two target-specific binding domains, the binding domains are connected to each other covalently on a single polypeptide chain, through an oligo- or polypeptide linker, an Fc hinge region, or a membrane hinge region. In some embodiments, the antigen binding domain is selected from the group consisting of an antibody, an antigen binding fragment (Fab), and a single-chain variable fragment (scFv). For example, a CD19 binding domain of the present invention can be selected from the group consisting of a CD19-specific antibody, a CD19-specific Fab, and a CD19-specific scFv. In one embodiment, a CD19 binding domain is a CD19-specific antibody. In one embodiment, a CD19 binding domain is a CD19-specific Fab. In one embodiment, a CD19 binding domain is a CD19- specific scFv (e.g. SEQ ID NO: 12). In one embodiment, a mesothelin binding domain is a mesothelin-specific antibody. In one embodiment, a mesothelin binding domain is a mesothelin- 41744090.2
Attorney Docket No.046483-7386WO1(03225) specific Fab. In one embodiment, a mesothelin binding domain is a mesothelin-specific scFv (e.g. SEQ ID NO: 2). The antigen binding domain can include any domain that binds to the antigen and may include, but is not limited to, a monoclonal antibody, a polyclonal antibody, a synthetic antibody, a human antibody, a humanized antibody, a non-human antibody, and any fragment thereof. In some embodiments, the antigen binding domain portion comprises a mammalian antibody or a fragment thereof. The choice of antigen binding domain may depend upon the type and number of antigens that are present on the surface of a target cell. As used herein, the term “single-chain variable fragment” or “scFv” is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of an immunoglobulin (e.g., mouse or human) covalently linked to form a VH::VL heterodimer. The heavy (VH) and light chains (VL) are either joined directly or joined by a peptide-encoding linker, which connects the N- terminus of the VH with the C-terminus of the VL, or the C-terminus of the VH with the N- terminus of the VL. In some embodiments, the antigen binding domain (e.g., CD19 binding domain) comprises an scFv having the configuration from N-terminus to C-terminus, VH – linker – VL. In some embodiments, the antigen binding domain comprises an scFv having the configuration from N-terminus to C-terminus, VL – linker – VH. Those of skill in the art would be able to select the appropriate configuration for use in the present invention. The linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility. The linker can link the heavy chain variable region and the light chain variable region of the extracellular antigen-binding domain. Non-limiting examples of linkers are disclosed in Shen et al., Anal. Chem.80(6):1910-1917 (2008) and WO 2014/087010, the contents of which are hereby incorporated by reference in their entireties. Various linker sequences are known in the art, including, without limitation, glycine serine (GS) linkers such as (GS)n, (GSGGS)n (SEQ ID NO:21), (GGGS)n (SEQ ID NO:22), and (GGGGS)n (SEQ ID NO:23), where n represents an integer of at least 1. Exemplary linker sequences can comprise amino acid sequences including, without limitation, GGSG (SEQ ID NO:24), GGSGG (SEQ ID NO:25), GSGSG (SEQ ID NO:26), GSGGG (SEQ ID NO:27), GGGSG (SEQ ID NO:28), GSSSG (SEQ ID NO:29), GGGGS (SEQ ID NO:30), GGGGSGGGGSGGGGS (SEQ ID NO:31) and the like. Those of skill in the art would be able to select the appropriate linker sequence for use in the present invention. In one embodiment, an antigen binding domain of the present invention comprises a 41744090.2
Attorney Docket No.046483-7386WO1(03225) heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL is separated by the linker sequence having the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO:31), which may be encoded by the nucleic acid sequence GGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCT (SEQ ID NO:32). Despite removal of the constant regions and the introduction of a linker, scFv proteins retain the specificity of the original immunoglobulin. Single chain Fv polypeptide antibodies can be expressed from a nucleic acid comprising VH- and VL-encoding sequences as described by Huston, et al. (Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988). See, also, U.S. Patent Nos. 5,091,513, 5,132,405 and 4,956,778; and U.S. Patent Publication Nos.20050196754 and 20050196754. Antagonistic scFvs having inhibitory activity have been described (see, e.g., Zhao et al., Hyrbidoma (Larchmt) 200827(6):455-51; Peter et al., J Cachexia Sarcopenia Muscle 2012 August 12; Shieh et al., J Imunol 2009183(4):2277-85; Giomarelli et al., Thromb Haemost 2007 97(6):955-63; Fife eta., J Clin Invst 2006116(8):2252-61; Brocks et al., Immunotechnology 19973(3):173-84; Moosmayer et al., Ther Immunol 19952(10:31-40). Agonistic scFvs having stimulatory activity have been described (see, e.g., Peter et al., J Bioi Chem 2003 25278(38):36740-7; Xie et al., Nat Biotech 199715(8):768-71; Ledbetter et al., Crit Rev Immunol 199717(5-6):427-55; Ho et al., BioChim Biophys Acta 20031638(3):257-66). As used herein, “Fab” refers to a fragment of an antibody structure that binds to an antigen but is monovalent and does not have a Fc portion, for example, an antibody digested by the enzyme papain yields two Fab fragments and an Fc fragment (e.g., a heavy (H) chain constant region; Fc region that does not bind to an antigen). As used herein, “F(ab′)2” refers to an antibody fragment generated by pepsin digestion of whole IgG antibodies, wherein this fragment has two antigen binding (ab′) (bivalent) regions, wherein each (ab′) region comprises two separate amino acid chains, a part of a H chain and a light (L) chain linked by an S—S bond for binding an antigen and where the remaining H chain portions are linked together. A “F(ab′)2” fragment can be split into two individual Fab′ fragments. In some embodiments, the antigen binding domain may be derived from the same species in which the CAR will ultimately be used. For example, for use in humans, the antigen binding domain of the CAR may comprise a human antibody or a fragment thereof. In some embodiments, the antigen binding domain may be derived from a different species in which the 41744090.2
Attorney Docket No.046483-7386WO1(03225) CAR will ultimately be used. For example, for use in humans, the antigen binding domain of the CAR may comprise a murine antibody or a fragment thereof. Transmembrane Domain CARs of the present invention may comprise a transmembrane domain that connects the antigen binding domain of the CAR to the intracellular domain of the CAR. The transmembrane domain of a subject CAR is a region that is capable of spanning the plasma membrane of a cell (e.g., an immune cell or precursor thereof). The transmembrane domain is for insertion into a cell membrane, e.g., a eukaryotic cell membrane. In some embodiments, the transmembrane domain is interposed between the antigen binding domain and the intracellular domain of a CAR. In some embodiments, the transmembrane domain is naturally associated with one or more of the domains in the CAR. In some embodiments, the transmembrane domain can be selected or modified by one or more amino acid substitutions to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins, to minimize interactions with other members of the receptor complex. The transmembrane domain may be derived either from a natural or a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein, e.g., a Type I transmembrane protein. Where the source is synthetic, the transmembrane domain may be any artificial sequence that facilitates insertion of the CAR into a cell membrane, e.g., an artificial hydrophobic sequence. Examples of the transmembrane domain of particular use in this invention include, without limitation, transmembrane domains derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134 (OX-40), CD137 (4-1BB), CD154 (CD40L), Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9. In some embodiments, the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. Preferably a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain. 41744090.2
Attorney Docket No.046483-7386WO1(03225) The transmembrane domains described herein can be combined with any of the antigen binding domains described herein, any of the intracellular domains described herein, or any of the other domains described herein that may be included in a subject CAR. In some embodiments, the transmembrane domain further comprises a hinge region. A subject CAR of the present invention may also include a hinge region. The hinge region of the CAR is a hydrophilic region which is located between the antigen binding domain and the transmembrane domain. In some embodiments, this domain facilitates proper protein folding for the CAR. The hinge region is an optional component for the CAR. The hinge region may include a domain selected from Fc fragments of antibodies, hinge regions of antibodies, CH2 regions of antibodies, CH3 regions of antibodies, artificial hinge sequences or combinations thereof. Examples of hinge regions include, without limitation, a CD8a hinge, artificial hinges made of polypeptides which may be as small as, three glycines (Gly), as well as CH1 and CH3 domains of IgGs (such as human IgG4). In some embodiments, a subject CAR of the present disclosure includes a hinge region that connects the antigen binding domain with the transmembrane domain, which, in turn, connects to the intracellular domain. The hinge region is preferably capable of supporting the antigen binding domain to recognize and bind to the target antigen on the target cells (see, e.g., Hudecek et al., Cancer Immunol. Res. (2015) 3(2): 125-135). In some embodiments, the hinge region is a flexible domain, thus allowing the antigen binding domain to have a structure to optimally recognize the specific structure and density of the target antigens on a cell such as tumor cell (Hudecek et al., supra). The flexibility of the hinge region permits the hinge region to adopt many different conformations. In some embodiments, the hinge region is an immunoglobulin heavy chain hinge region. In some embodiments, the hinge region is a hinge region polypeptide derived from a receptor (e.g., a CD8-derived hinge region). The hinge region can have a length of from about 4 amino acids to about 50 amino acids, e.g., from about 4 aa to about 10 aa, from about 10 aa to about 15 aa, from about 15 aa to about 20 aa, from about 20 aa to about 25 aa, from about 25 aa to about 30 aa, from about 30 aa to about 40 aa, or from about 40 aa to about 50 aa. In some embodiments, the hinge region can have a length of greater than 5 aa, greater than 10 aa, greater than 15 aa, greater than 20 aa, greater 41744090.2
Attorney Docket No.046483-7386WO1(03225) than 25 aa, greater than 30 aa, greater than 35 aa, greater than 40 aa, greater than 45 aa, greater than 50 aa, greater than 55 aa, or more. Suitable hinge regions can be readily selected and can be of any of a number of suitable lengths, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids. Suitable hinge regions can have a length of greater than 20 amino acids (e.g., 30, 40, 50, 60 or more amino acids). For example, hinge regions include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, (GSGGS)n (SEQ ID NO:21) and (GGGS)n (SEQ ID NO:22), where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers can be used; both Gly and Ser are relatively unstructured, and therefore can serve as a neutral tether between components. Glycine polymers can be used; glycine accesses significantly more phi-psi space than even alanine, and is much less restricted than residues with longer side chains (see, e.g., Scheraga, Rev. Computational. Chem. (1992) 2: 73-142). Exemplary hinge regions can comprise amino acid sequences including, but not limited to, GGSG (SEQ ID NO:24), GGSGG (SEQ ID NO:25), GSGSG (SEQ ID NO:26), GSGGG (SEQ ID NO:27), GGGSG (SEQ ID NO:28), GSSSG (SEQ ID NO:29), and the like. In some embodiments, the hinge region is an immunoglobulin heavy chain hinge region. Immunoglobulin hinge region amino acid sequences are known in the art; see, e.g., Tan et al., Proc. Natl. Acad. Sci. USA (1990) 87(1):162-166; and Huck et al., Nucleic Acids Res. (1986) 14(4): 1779-1789. The hinge region can comprise an amino acid sequence of a human IgG1, IgG2, IgG3, or IgG4, hinge region. In one embodiment, the hinge region can include one or more amino acid substitutions and/or insertions and/or deletions compared to a wild-type (naturally- occurring) hinge region. For example, His229 of human IgG1 hinge can be substituted with Tyr; see, e.g., Yan et al., J. Biol. Chem. (2012) 287: 5891-5897. In one embodiment, the hinge region can comprise an amino acid sequence derived from human CD8, or a variant thereof. 41744090.2
Attorney Docket No.046483-7386WO1(03225) Intracellular Signaling Domain A subject CAR of the present invention also includes an intracellular signaling domain. The terms “intracellular signaling domain” and “intracellular domain” are used interchangeably herein. The intracellular signaling domain of the CAR is responsible for activation of at least one of the effector functions of the cell in which the CAR is expressed (e.g., immune cell). The intracellular signaling domain transduces the effector function signal and directs the cell (e.g., immune cell) to perform its specialized function, e.g., harming and/or destroying a target cell. Examples of an intracellular domain for use in the invention include, but are not limited to, the cytoplasmic portion of a surface receptor, co-stimulatory molecule, and any molecule that acts in concert to initiate signal transduction in the T cell, as well as any derivative or variant of these elements and any synthetic sequence that has the same functional capability. Examples of the intracellular signaling domain include, without limitation, the ζ chain of the T cell receptor complex or any of its homologs, e.g., η chain, FcsRIγ and β chains, MB 1 (Iga) chain, B29 (Ig) chain, etc., human CD3 zeta chain, CD3 polypeptides (Δ, δ and ε), syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.), and other molecules involved in T cell transduction, such as CD2, CD5 and CD28. In one embodiment, the intracellular signaling domain may be human CD3 zeta chain (e.g. SEQ ID NO: 5), FcyRIII, FcsRI, cytoplasmic tails of Fc receptors, an immunoreceptor tyrosine-based activation motif (ITAM) bearing cytoplasmic receptors, and combinations thereof. In one embodiment, the intracellular signaling domain of the CAR includes any portion of one or more co-stimulatory molecules, such as at least one signaling domain from CD2, CD3 (e.g. SEQ ID NO:5), CD8, CD27, CD28, ICOS, 4-1BB (e.g. SEQ ID NO:4), PD-1, any derivative or variant thereof, any synthetic sequence thereof that has the same functional capability, and any combination thereof. Other examples of the intracellular domain include a fragment or domain from one or more molecules or receptors including, but not limited to, TCR, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD86, common FcR gamma, FcR beta (Fc Epsilon RIb), CD79a, CD79b, Fcgamma RIIa, DAP10, DAP12, T cell receptor (TCR), CD8, CD27, CD28, 4-1BB (CD137), OX9, OX40, CD30, CD40, PD-1, ICOS, a KIR family protein, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, 41744090.2
Attorney Docket No.046483-7386WO1(03225) CD160, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CDlib, ITGAX, CD11c, ITGBl, CD29, ITGB2, CD18, LFA- 1, ITGB7, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, NKp44, NKp30, NKp46, NKG2D, Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, other co-stimulatory molecules described herein, any derivative, variant, or fragment thereof, any synthetic sequence of a co-stimulatory molecule that has the same functional capability, and any combination thereof. Additional examples of intracellular domains include, without limitation, intracellular signaling domains of several types of various other immune signaling receptors, including, but not limited to, first, second, and third generation T cell signaling proteins including CD3, B7 family costimulatory, and Tumor Necrosis Factor Receptor (TNFR) superfamily receptors (see, e.g., Park and Brentjens, J. Clin. Oncol. (2015) 33(6): 651-653). Additionally, intracellular signaling domains may include signaling domains used by NK and NKT cells (see, e.g., Hermanson and Kaufman, Front. Immunol. (2015) 6: 195) such as signaling domains of NKp30 (B7-H6) (see, e.g., Zhang et al., J. Immunol. (2012) 189(5): 2290-2299), and DAP 12 (see, e.g., Topfer et al., J. Immunol. (2015) 194(7): 3201-3212), NKG2D, NKp44, NKp46, DAP10, and CD3z. Intracellular signaling domains suitable for use in a subject CAR of the present invention include any desired signaling domain that provides a distinct and detectable signal (e.g., increased production of one or more cytokines by the cell; change in transcription of a target gene; change in activity of a protein; change in cell behavior, e.g., cell death; cellular proliferation; cellular differentiation; cell survival; modulation of cellular signaling responses; etc.) in response to activation of the CAR (i.e., activated by antigen and dimerizing agent). In some embodiments, the intracellular signaling domain includes at least one (e.g., one, two, three, four, five, six, etc.) ITAM motifs as described below. In some embodiments, the intracellular signaling domain includes DAP10/CD28 type signaling chains. In some embodiments, the 41744090.2
Attorney Docket No.046483-7386WO1(03225) intracellular signaling domain is not covalently attached to the membrane bound CAR, but is instead diffused in the cytoplasm. Intracellular signaling domains suitable for use in a subject CAR of the present invention include immunoreceptor tyrosine-based activation motif (ITAM)-containing intracellular signaling polypeptides. In some embodiments, an ITAM motif is repeated twice in an intracellular signaling domain, where the first and second instances of the ITAM motif are separated from one another by 6 to 8 amino acids. In one embodiment, the intracellular signaling domain of a subject CAR comprises 3 ITAM motifs. In some embodiments, intracellular signaling domains includes the signaling domains of human immunoglobulin receptors that contain immunoreceptor tyrosine based activation motifs (ITAMs) such as, but not limited to, FcgammaRI, FcgammaRIIA, FcgammaRIIC, FcgammaRIIIA, FcRL5 (see, e.g., Gillis et al., Front. Immunol. (2014) 5:254). A suitable intracellular signaling domain can be an ITAM motif-containing portion that is derived from a polypeptide that contains an ITAM motif. For example, a suitable intracellular signaling domain can be an ITAM motif-containing domain from any ITAM motif-containing protein. Thus, a suitable intracellular signaling domain need not contain the entire sequence of the entire protein from which it is derived. Examples of suitable ITAM motif-containing polypeptides include, but are not limited to: DAP12, FCER1G (Fc epsilon receptor I gamma chain), CD3D (CD3 delta), CD3E (CD3 epsilon), CD3G (CD3 gamma), CD3Z (CD3 zeta), and CD79A (antigen receptor complex-associated protein alpha chain). In one embodiment, the intracellular signaling domain is derived from DAP12 (also known as TYROBP; TYRO protein tyrosine kinase binding protein; KARAP; PLOSL; DNAX- activation protein 12; KAR-associated protein; TYRO protein tyrosine kinase-binding protein; killer activating receptor associated protein; killer-activating receptor-associated protein; etc.). In one embodiment, the intracellular signaling domain is derived from FCER1G (also known as FCRG; Fc epsilon receptor I gamma chain; Fc receptor gamma-chain; fc-epsilon RI-gamma; fcRgamma; fceRl gamma; high affinity immunoglobulin epsilon receptor subunit gamma; immunoglobulin E receptor, high affinity, gamma chain; etc.). In one embodiment, the intracellular signaling domain is derived from T-cell surface glycoprotein CD3 delta chain (also known as CD3D; CD3-DELTA; T3D; CD3 antigen, delta subunit; CD3 delta; CD3d antigen, delta polypeptide (TiT3 complex); OKT3, delta chain; T-cell receptor T3 delta chain; T-cell 41744090.2
Attorney Docket No.046483-7386WO1(03225) surface glycoprotein CD3 delta chain; etc.). In one embodiment, the intracellular signaling domain is derived from T-cell surface glycoprotein CD3 epsilon chain (also known as CD3e, T- cell surface antigen T3/Leu-4 epsilon chain, T-cell surface glycoprotein CD3 epsilon chain, AI504783, CD3, CD3epsilon, T3e, etc.). In one embodiment, the intracellular signaling domain is derived from T-cell surface glycoprotein CD3 gamma chain (also known as CD3G, T-cell receptor T3 gamma chain, CD3-GAMMA, T3G, gamma polypeptide (TiT3 complex), etc.). In one embodiment, the intracellular signaling domain is derived from T-cell surface glycoprotein CD3 zeta chain (also known as CD3Z, T-cell receptor T3 zeta chain, CD247, CD3-ZETA, CD3H, CD3Q, T3Z, TCRZ, etc.). In one embodiment, the intracellular signaling domain is derived from CD79A (also known as B-cell antigen receptor complex-associated protein alpha chain; CD79a antigen (immunoglobulin-associated alpha); MB-1 membrane glycoprotein; ig- alpha; membrane-bound immunoglobulin-associated protein; surface IgM-associated protein; etc.). In one embodiment, an intracellular signaling domain suitable for use in an FN3 CAR of the present disclosure includes a DAP10/CD28 type signaling chain. In one embodiment, an intracellular signaling domain suitable for use in an FN3 CAR of the present disclosure includes a ZAP70 polypeptide. In some embodiments, the intracellular signaling domain includes a cytoplasmic signaling domain of TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, or CD66d. In one embodiment, the intracellular signaling domain in the CAR includes a cytoplasmic signaling domain of human CD3 zeta. While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal. The intracellular signaling domain includes any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal. The intracellular signaling domains described herein can be combined with any of the antigen binding domains described herein, any of the transmembrane domains described herein, or any of the other domains described herein that may be included in the CAR. The invention should be construed to include any CAR known in the art and/or disclosed herein. Exemplary CARs include, but are not limited to, those disclosed herein, those disclosed in US10357514B2, US10221245B2, US10603378B2, US8916381B1, US9394368B2, 41744090.2
Attorney Docket No.046483-7386WO1(03225) US20140050708A1, US9598489B2, US9365641B2, US20210079059A1, US9783591B2, WO2016028896A1, US9446105B2, WO2016014576A1, US20210284752A1, WO2016014565A2, WO2016014535A1, and US9272002B2, and any other CAR generally disclosed in the art. Exemplary Nucleotide Sequences: Mesothelin CAR (Meso ScFv-CD8 Hinge-CD8 TM-4-1BB ICD-Zeta ICD-mCherry-WPRE (SEQ ID NO: 1)) meso scfv (SEQ ID NO: 2) CD8 HINGE (SEQ ID NO: 3) CD8 TM (SEQ ID NO: 16) 4-1BB ICD (SEQ ID NO: 4) Zeta ICD (SEQ ID NO: 5) mCherry (SEQ ID NO: 6) WPRE (SEQ ID NO: 7) ggatcccaggtacaactgcagcagtctgggcctgagctggagaagcctggcgcttcagtgaagatatcctgcaaggcttctggttactcattcactgg ctacaccatgaactgggtgaagcagagccatggaaagagccttgagtggattggacttattactccttacaatggtgcttctagctacaaccagaagt tcaggggcaaggccacattaactgtagacaagtcatccagcacagcctacatggacctcctcagtctgacatctgaagactctgcagtctatttctgt gcaagggggggttacgacgggaggggttttgactactggggccaagggaccacggtcaccgtctcctcaggtggaggcggttcaggcggcggtg gctctagcggtggtggatcggacatcgagctcactcagtctccagcaatcatgtctgcatctccaggggagaaggtcaccatgacctgcagtgccag ctcaagtgtaagttacatgcactggtaccagcagaagtcaggcacctcccccaaaagatggatttatgacacatccaaactggcttctggagtccca ggtcgcttcagtggcagtgggtctggaaactcttactctctcacaatcagcagcgtggaggctgaagatgatgcaacttattactgccagcagtggag taagcaccctctcacgtacggtgctgggacaaagttggaaatcaaaGCTAGCGATTATAAAGATGATGATGATAAA TCTAGCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGC AGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACA CGAGGGGGCTGGACTTCGCCTGTGATTccggaATCTACATCTGGGCGCCCTTGGCCGGGACT TGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAAC TCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGAT GGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTT CAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCA ATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATG GGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAA GATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCAC GATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAG GCCCTGCCCCCTCGCCCTAGGATGGTGAGCAAGGGCGAGGAGGATAACATGGCCATCATCA AGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGAT CGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGAC CAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCA AGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGC TTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACT CCTCCCTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCC GACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACC CCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCC ACTACGACGCTGAGGTCAAGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGC CTACAACGTCAACATCAAGTTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAAC AGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAGTAGGtc 41744090.2
Attorney Docket No.046483-7386WO1(03225) gacaaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatca tgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgt gcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcgg aactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaagctgacgtcctttccttgg ctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccg
Mesothelin CAR with CH25H expression (Meso ScFv-CD8 Hinge-CD8 TM-4-1BB ICD-Zeta ICD-2A- CH25H-WPRE (SEQ ID NO: 8)) meso scfv (SEQ ID NO: 2) CD8 HINGE (SEQ ID NO: 3) CD8 TM (SEQ ID NO: 16) 4-1BB ICD (SEQ ID NO: 4) Zeta ICD (SEQ ID NO: 5) T2A (SEQ ID NO: 9) CH25H (SEQ ID NO: 10) WPRE (SEQ ID NO: 7) ggatcccaggtacaactgcagcagtctgggcctgagctggagaagcctggcgcttcagtgaagatatcctgcaaggcttctggttactcattcactgg ctacaccatgaactgggtgaagcagagccatggaaagagccttgagtggattggacttattactccttacaatggtgcttctagctacaaccagaagt tcaggggcaaggccacattaactgtagacaagtcatccagcacagcctacatggacctcctcagtctgacatctgaagactctgcagtctatttctgt gcaagggggggttacgacgggaggggttttgactactggggccaagggaccacggtcaccgtctcctcaggtggaggcggttcaggcggcggtg gctctagcggtggtggatcggacatcgagctcactcagtctccagcaatcatgtctgcatctccaggggagaaggtcaccatgacctgcagtgccag ctcaagtgtaagttacatgcactggtaccagcagaagtcaggcacctcccccaaaagatggatttatgacacatccaaactggcttctggagtccca ggtcgcttcagtggcagtgggtctggaaactcttactctctcacaatcagcagcgtggaggctgaagatgatgcaacttattactgccagcagtggag taagcaccctctcacgtacggtgctgggacaaagttggaaatcaaaGCTAGCGATTATAAAGATGATGATGATAAA TCTAGCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGC AGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACA CGAGGGGGCTGGACTTCGCCTGTGATTccggaATCTACATCTGGGCGCCCTTGGCCGGGACT TGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAAC TCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGAT GGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTT CAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCA ATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATG GGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAA GATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCAC GATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAG GCCCTGCCCCCTCGCCCTAGGAGAGCCGAGGGCAGAGGCAGCCTGCTGACCTGCGGCGACG TGGAGGAGAACCCAGGCCCCATGGGCTGCTACAACGGTTCGGAGCTCCAAGACCTGGGC TGTTCCAGCCAGCTGCTCCTGCAGCCCCTCTGGGACACCATAAGGACAAGGGAGGCGT TCACGCGCTCACCCATCTTCCCAGTCACCTTTTCTATCATCACTTACGTGGGCTTCTGC CTACCGTTCGTGGTGCTGGACGTCCTGTATCCCTGGGTCCCCATCCTGCGACGCTACA AGATCCACCCGGACTTCTCGCCTTCCGTAAAGCAGCTTCTGCCTTGCCTGGGGCTGAC ACTCTACCAGCACCTGGTGTTCGTGTTCCCGGTGACGCTGCTGCACTGGGTGCGCAGC CCGGCGCTCCTCCCCCAGGAGGCCCCTGAGCTCGTCCAGCTCCTAAGTCACGTCCTGA TCTGCCTGCTGCTCTTCGACACCGAGATCTTCGCGTGGCACCTGCTGCACCATAAGGT GCCCTGGCTGTACCGCACCTTCCACAAGGTGCATCACCAGAACTCGTCCTCCTTCGCG CTGGCGACCCAATACATGAGCTTCTGGGAGCTGCTTTCGCTGACCTTCTTCGACGTGCT GAACGTCGCGGTGCTTCGGTGTCACCCACTCACCATCTTTACCTTTCACGTGATTAACA TCTGGCTGTCGGTGGAGGACCACTCGGGCTATGACTTCCCGTGGTCCACTCACAGACT 41744090.2
Attorney Docket No.046483-7386WO1(03225) TGTGCCCTTTGGCTGGTACGGGGGCGTGGCTCACCACGACATGCATCACTCTCAGTTT AACTGCAATTTTGCTCCTTACTTCACACACTGGGACAAAATGCTGGGCACTCTGCGGTC CGCGCCCCTGCCAGAGAGCCTTTGCGCCTGCGGTGAGCGCTGCGTGAACTCCAGAGAG CGATGCGCTGTACACTTGATCCAGAAGAAGAAACAGACTACGCGTACGCGGCCGCTCT AAGAGCAGAAACTCATCTCAGAAGAGGATCTGGCAGCAAATGATATCCTGGATTACAA GGATGACGACGATAAGGtcgacaaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacg ctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttg tggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggac tttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggt gttgtcggggaagctgacgtcctttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatcca gcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctcccc gcctg
ID CD8 Hinge (SEQ ID NO: 20) CD8 TM (SEQ ID NO: 13) 4-1BB ICD (SEQ ID NO: 4) Zeta ICD (SEQ ID NO: 14) Restriction Sites (SEQ ID NO: 17) LTR (SEQ ID NO: 18) Atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccggacatccagatgacacagactacatcctccctgtc tgcctctctgggagacagagtcaccatcagttgcagggcaagtcaggacattagtaaatatttaaattggtatcagcagaaaccagatggaactgtt aaactcctgatctaccatacatcaagattacactcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccattagcaac ctggagcaagaagatattgccacttacttttgccaacagggtaatacgcttccgtacacgttcggaggggggaccaagctggagatcacaggtggc ggtggctcgggcggtggtgggtcgggtggcggcggatctgaggtgaaactgcaggagtcaggacctggcctggtggcgccctcacagagcctgtc cgtcacatgcactgtctcaggggtctcattacccgactatggtgtaagctggattcgccagcctccacgaaagggtctggagtggctgggagtaatat ggggtagtgaaaccacatactataattcagctctcaaatccagactgaccatcatcaaggacaactccaagagccaagttttcttaaaaatgaacag tctgcaaactgatgacacagccatttactactgtgccaaacattattactacggtggtagctatgctatggactactggggccaaggaacctcagtcac cgtctcctcaAccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccg gccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttct cctgtcactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaa gaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaaCtgagagtgaagttcagcaggagcgcagacgcccccg cgtacaagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggacc ctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtga gattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgccct tcacatgcaggccctgccccctcgctaaGcggccgcggatccccgggtaccgagctcgaattctgcagtcgacggtaccgcgggcccgggatccga taaaataaaagattttatttagtctccagaaaaaggggggaatgaaagaccccacctgtaggtttggcaagctagcttaagtaacgccattttgcaaggcat ggaaaatacataactgagaatagagaagttcagatcaaggttaggaacagagagacagcagaatatgggccaaacaggatatctgtggtaagcagttcc tgccccggctcagggccaagaacagatggtccccagatgcggtcccgccctcagcagtttctagagaaccatcagatgtttccagggtgccccaagga cctgaaaatgaccctgtgccttatttgaactaaccaatcagttcgcttctcgcttctgttcgcgcgcttctgctccccgagctcaataaaagagcccacaacc cctcactcggcgcgccagtcctccgatagactgcgtcgcccgggtacccgtgtatccaataaaccctcttgcagttgcatccgacttgtggtctcgctgttc cttgggagggtctcctctgagtgattgactacccgtcagcgggggtctttca CD19 with CH25H expression (CD19- CD8 TM-4-1BB IC-Zeta-Restriction Sites-2A_CH25H- Restriction sites-3' LTR (SEQ ID NO: 15)) CD19 scfv (SEQ ID NO: 12) CD8 Hinge (SEQ ID NO: 20) CD8 TM (SEQ ID NO: 13) 41744090.2
Attorney Docket No.046483-7386WO1(03225) 4-1BB ICD (SEQ ID NO: 4) Zeta ICD (SEQ ID NO: 14) Restriction Sites (SEQ ID NO: 48 & 49) T2A (SEQ ID NO: 19)
ctggagcaagaagatattgccacttacttttgccaacagggtaatacgcttccgtacacgttcggaggggggaccaagctggagatcacaggtggc ggtggctcgggcggtggtgggtcgggtggcggcggatctgaggtgaaactgcaggagtcaggacctggcctggtggcgccctcacagagcctgtc cgtcacatgcactgtctcaggggtctcattacccgactatggtgtaagctggattcgccagcctccacgaaagggtctggagtggctgggagtaatat ggggtagtgaaaccacatactataattcagctctcaaatccagactgaccatcatcaaggacaactccaagagccaagttttcttaaaaatgaacag tctgcaaactgatgacacagccatttactactgtgccaaacattattactacggtggtagctatgctatggactactggggccaaggaacctcagtcac cgtctcctcaAccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccg gccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttct cctgtcactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaa gaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaaCtgagagtgaagttcagcaggagcgcagacgcccccg cgtacaagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggacc ctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtga gattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgccct tcacatgcaggccctgccccctcgccggccgcggatccccgggtaccgagctcgaattctgcagtcgaCCTAGGAGAGCCGAGGG CAGAGGCAGCCTGCTGACCTGCGGCGACGTGGAGGAGAACCCAGGCCCCATGGGCTGCTA CAACGGTTCGGAGCTCCAAGACCTGGGCTGTTCCAGCCAGCTGCTCCTGCAGCCCCTC TGGGACACCATAAGGACAAGGGAGGCGTTCACGCGCTCACCCATCTTCCCAGTCACCT TTTCTATCATCACTTACGTGGGCTTCTGCCTACCGTTCGTGGTGCTGGACGTCCTGTAT CCCTGGGTCCCCATCCTGCGACGCTACAAGATCCACCCGGACTTCTCGCCTTCCGTAA AGCAGCTTCTGCCTTGCCTGGGGCTGACACTCTACCAGCACCTGGTGTTCGTGTTCCC GGTGACGCTGCTGCACTGGGTGCGCAGCCCGGCGCTCCTCCCCCAGGAGGCCCCTGA GCTCGTCCAGCTCCTAAGTCACGTCCTGATCTGCCTGCTGCTCTTCGACACCGAGATCT TCGCGTGGCACCTGCTGCACCATAAGGTGCCCTGGCTGTACCGCACCTTCCACAAGGT GCATCACCAGAACTCGTCCTCCTTCGCGCTGGCGACCCAATACATGAGCTTCTGGGAG CTGCTTTCGCTGACCTTCTTCGACGTGCTGAACGTCGCGGTGCTTCGGTGTCACCCACT CACCATCTTTACCTTTCACGTGATTAACATCTGGCTGTCGGTGGAGGACCACTCGGGCT ATGACTTCCCGTGGTCCACTCACAGACTTGTGCCCTTTGGCTGGTACGGGGGCGTGGC TCACCACGACATGCATCACTCTCAGTTTAACTGCAATTTTGCTCCTTACTTCACACACT GGGACAAAATGCTGGGCACTCTGCGGTCCGCGCCCCTGCCAGAGAGCCTTTGCGCCTG CGGTGAGCGCTGCGTGAACTCCAGAGAGCGATGCGCTGTACACTTGATCCAGAAGAAG AAACAGACTACGCGTACGCGGCCGCTCTAAGAGCAGAAACTCATCTCAGAAGAGGATC TGGCAGCAAATGATATCCTGGATTACAAGGATGACGACGATAAGGtcgacacggtaccgcgggccc gggatccgataaaataaaagattttatttagtctccagaaaaaggggggaatgaaagaccccacctgtaggtttggcaagctagcttaagtaacgcca
gagcccacaacccctcactcggcgcgccagtcctccgatagactgcgtcgcccgggtacccgtgtatccaataaaccctcttgcagttgcatccgacttg tggtctcgctgttccttgggagggtctcctctgagtgattgactacccgtcagcgggggtctttca 41744090.2
Attorney Docket No.046483-7386WO1(03225) D. Methods of Treatment The modified immune cells described herein (e.g., CAR T cells comprising CH25H) may be included in a composition for immunotherapy. The composition may include a pharmaceutical composition and further include a pharmaceutically acceptable carrier. A therapeutically effective amount of the pharmaceutical composition comprising the modified cells may be administered to a subject in need thereof. In one aspect, the invention includes a method for adoptive cell transfer therapy comprising administering to a subject in need thereof a modified cell (e.g., CAR T cell comprising CH25H) of the present invention. In another aspect, the invention includes a method of treating a disease or condition in a subject comprising administering to a subject in need thereof a population of modified cells cell (e.g., CAR T cell comprising CH25H). In another aspect, the invention includes a method of treating a disease or condition in a subject comprising administering to a subject in need thereof a CAR T cell and an agent that up- regulates CH25H. In certain embodiments, the agent is reserpine as described in e.g., Ortiz A. et al. (2019) Cancer Cell, 35: 33-45; Lu Z., et al (2021) J Clin Invest, 131: 144225. Methods for administration of immune cells for adoptive cell therapy are known and may be used in connection with the provided methods and compositions. For example, adoptive T cell therapy methods are described, e.g., in US Patent Application Publication No.2003/0170238 to Gruenberg et al; US Patent No.4,690,915 to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85). See, e.g., Themeli et al. (2013) Nat Biotechnol.31(10): 928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1): 84-9; Davila et al. (2013) PLoS ONE 8(4): e61338. In some embodiments, the cell therapy, e.g., adoptive T cell therapy is carried out by autologous transfer, in which the cells are isolated and/or otherwise prepared from the subject who is to receive the cell therapy, or from a sample derived from such a subject. Thus, in some aspects, the cells are derived from a subject, e.g., patient, in need of a treatment and the cells, following isolation and processing are administered to the same subject. In some embodiments, the cell therapy, e.g., adoptive T cell therapy, is carried out by allogeneic transfer, in which the cells are isolated and/or otherwise prepared from a subject other than a subject who is to receive or who ultimately receives the cell therapy, e.g., a first subject. In such embodiments, the cells then are administered to a different subject, e.g., a second subject, of the same species. In some embodiments, the first and second subjects are genetically identical. 41744090.2
Attorney Docket No.046483-7386WO1(03225) In some embodiments, the first and second subjects are genetically similar. In some embodiments, the second subject expresses the same HLA class or supertype as the first subject. In some embodiments, the subject has been treated with a therapeutic agent targeting the disease or condition, e.g. the tumor, prior to administration of the cells or composition containing the cells. In some aspects, the subject is refractory or non-responsive to the other therapeutic agent. In some embodiments, the subject has persistent or relapsed disease, e.g., following treatment with another therapeutic intervention, including chemotherapy, radiation, and/or hematopoietic stem cell transplantation (HSCT), e.g., allogenic HSCT. In some embodiments, the administration effectively treats the subject despite the subject having become resistant to another therapy. In some embodiments, the subject is responsive to the other therapeutic agent, and treatment with the therapeutic agent reduces disease burden. In some aspects, the subject is initially responsive to the therapeutic agent, but exhibits a relapse of the disease or condition over time. In some embodiments, the subject has not relapsed. In some such embodiments, the subject is determined to be at risk for relapse, such as at a high risk of relapse, and thus the cells are administered prophylactically, e.g., to reduce the likelihood of or prevent relapse. In some aspects, the subject has not received prior treatment with another therapeutic agent. In some embodiments, the subject has persistent or relapsed disease, e.g., following treatment with another therapeutic intervention, including chemotherapy, radiation, and/or hematopoietic stem cell transplantation (HSCT), e.g., allogenic HSCT. In some embodiments, the administration effectively treats the subject despite the subject having become resistant to another therapy. The modified immune cells of the present invention (e.g., CAR T cell comprising CH25H) can be administered to an animal, preferably a mammal, even more preferably a human, to treat a cancer. In addition, the cells of the present invention can be used for the treatment of any condition related to a cancer, especially a cell-mediated immune response against a tumor cell(s), where it is desirable to treat or alleviate the disease. The types of cancers to be treated with the modified cells or pharmaceutical compositions of the invention include, carcinoma, blastoma, and sarcoma, and certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignancies e.g., sarcomas, carcinomas, and melanomas. Other exemplary cancers include but are not limited breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin 41744090.2
Attorney Docket No.046483-7386WO1(03225) cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, thyroid cancer, and the like. The cancers may be non-solid tumors (such as hematological tumors) or solid tumors. Adult tumors/cancers and pediatric tumors/cancers are also included. In one embodiment, the cancer is a solid tumor or a hematological tumor. In one embodiment, the cancer is a carcinoma. In one embodiment, the cancer is a sarcoma. In one embodiment, the cancer is a leukemia. In one embodiment the cancer is a solid tumor. Solid tumors are abnormal masses of tissue that usually do not contain cysts or liquid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named for the type of cells that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, melanoma, and CNS tumors (such as a glioma (such as brainstem glioma and mixed gliomas), glioblastoma (also known as glioblastoma multiforme) astrocytoma, CNS lymphoma, germinoma, medulloblastoma, Schwannoma craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, neuroblastoma, retinoblastoma and brain metastases). Carcinomas that can be amenable to therapy by a method disclosed herein include, but are not limited to, esophageal carcinoma, hepatocellular carcinoma, basal cell carcinoma (a form of skin cancer), squamous cell carcinoma (various tissues), bladder carcinoma, including transitional cell carcinoma (a malignant neoplasm of the bladder), bronchogenic carcinoma, colon carcinoma, colorectal carcinoma, gastric carcinoma, lung carcinoma, including small cell carcinoma and non-small cell carcinoma of the lung, adrenocortical carcinoma, thyroid carcinoma, pancreatic carcinoma, breast carcinoma, ovarian carcinoma, prostate carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, 41744090.2
Attorney Docket No.046483-7386WO1(03225) papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, renal cell carcinoma, ductal carcinoma in situ or bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical carcinoma, uterine carcinoma, testicular carcinoma, osteogenic carcinoma, epithelial carcinoma, and nasopharyngeal carcinoma. Sarcomas that can be amenable to therapy by a method disclosed herein include, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, chordoma, osteogenic sarcoma, osteosarcoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, and other soft tissue sarcomas. In certain exemplary embodiments, the modified immune cells of the invention are used to treat a myeloma, or a condition related to myeloma. Examples of myeloma or conditions related thereto include, without limitation, light chain myeloma, non-secretory myeloma, monoclonal gamopathy of undertermined significance (MGUS), plasmacytoma (e.g., solitary, multiple solitary, extramedullary plasmacytoma), amyloidosis, and multiple myeloma. In one embodiment, a method of the present disclosure is used to treat multiple myeloma. In one embodiment, a method of the present disclosure is used to treat refractory myeloma. In one embodiment, a method of the present disclosure is used to treat relapsed myeloma. In certain exemplary embodiments, the modified immune cells of the invention are used to treat a melanoma, or a condition related to melanoma. Examples of melanoma or conditions related thereto include, without limitation, superficial spreading melanoma, nodular melanoma, lentigo maligna melanoma, acral lentiginous melanoma, amelanotic melanoma, or melanoma of the skin (e.g., cutaneous, eye, vulva, vagina, rectum melanoma). In one embodiment, a method of the present disclosure is used to treat cutaneous melanoma. In one embodiment, a method of the present disclosure is used to treat refractory melanoma. In one embodiment, a method of the present disclosure is used to treat relapsed melanoma. In yet other exemplary embodiments, the modified immune cells of the invention are used to treat a sarcoma, or a condition related to sarcoma. Examples of sarcoma or conditions related thereto include, without limitation, angiosarcoma, chondrosarcoma, Ewing’s sarcoma, fibrosarcoma, gastrointestinal stromal tumor, leiomyosarcoma, liposarcoma, malignant peripheral nerve sheath tumor, osteosarcoma, pleomorphic sarcoma, rhabdomyosarcoma, and synovial sarcoma. In one embodiment, a method of the present disclosure is used to treat 41744090.2
Attorney Docket No.046483-7386WO1(03225) synovial sarcoma. In one embodiment, a method of the present disclosure is used to treat liposarcoma such as myxoid/round cell liposarcoma, differentiated/dedifferentiated liposarcoma, and pleomorphic liposarcoma. In one embodiment, a method of the present disclosure is used to treat myxoid/round cell liposarcoma. In one embodiment, a method of the present disclosure is used to treat a refractory sarcoma. In one embodiment, a method of the present disclosure is used to treat a relapsed sarcoma. The cells of the invention to be administered may be autologous, with respect to the subject undergoing therapy. The administration of the cells of the invention may be carried out in any convenient manner known to those of skill in the art. The cells of the present invention may be administered to a subject by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions described herein may be administered to a patient transarterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In other instances, the cells of the invention are injected directly into a site of inflammation in the subject, a local disease site in the subject, alymph node, an organ, a tumor, and the like. In certain embodiments, the cells, or individual populations of sub-types of cells, are administered to the subject at a range of about one million to about 100 billion cells, such as, e.g., 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), such as about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values), and in some cases about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells) or any value in between these ranges. In some embodiments, the dose of total cells and/or dose of individual sub-populations of cells is within a range of between at or about 1x105 cells/kg to about 1x1011 cells/kg 104 and at or 41744090.2
Attorney Docket No.046483-7386WO1(03225) about 1011 cells/kilograms (kg) body weight, such as between 105 and 106 cells / kg body weight, for example, at or about 1 x 105 cells/kg, 1.5 x 105 cells/kg, 2 x 105 cells/kg, or 1 x 106 cells/kg body weight. For example, in some embodiments, the cells are administered at, or within a certain range of error of, between at or about 104 and at or about 109 T cells/kilograms (kg) body weight, such as between 105 and 106 T cells / kg body weight, for example, at or about 1 x 105 T cells/kg, 1.5 x 105 T cells/kg, 2 x 105 T cells/kg, or 1 x 106 T cells/kg body weight. In other exemplary embodiments, a suitable dosage range of modified cells for use in a method of the present disclosure includes, without limitation, from about 1x105 cells/kg to about 1x106 cells/kg, from about 1x106 cells/kg to about 1x107 cells/kg, from about 1x107 cells/kg about 1x108 cells/kg, from about 1x108 cells/kg about 1x109 cells/kg, from about 1x109 cells/kg about 1x1010 cells/kg, from about 1x1010 cells/kg about 1x1011 cells/kg. In an exemplary embodiment, a suitable dosage for use in a method of the present disclosure is about 1x108 cells/kg. In an exemplary embodiment, a suitable dosage for use in a method of the present disclosure is about 1x107 cells/kg. In other embodiments, a suitable dosage is from about 1x107 total cells to about 5x107 total cells. In some embodiments, a suitable dosage is from about 1x108 total cells to about 5x108 total cells. In some embodiments, a suitable dosage is from about 1.4x107 total cells to about 1.1x109 total cells. In an exemplary embodiment, a suitable dosage for use in a method of the present disclosure is about 7x109 total cells. In some embodiments, a dose of modified cells is administered to a subject in need thereof, in a single dose or multiple doses. In some embodiments, a dose of modified cells is administered in multiple doses, e.g., once a week or every 7 days, once every 2 weeks or every 14 days, once every 3 weeks or every 21 days, once every 4 weeks or every 28 days. In an exemplary embodiment, a single dose of modified cells is administered to a subject in need thereof. In an exemplary embodiment, a single dose of modified cells is administered to a subject in need thereof by rapid intravenous infusion. For the prevention or treatment of disease, the appropriate dosage may depend on the type of disease to be treated, the type of cells or recombinant receptors, the severity and course of the disease, whether the cells are administered for preventive or therapeutic purposes, previous therapy, the subject's clinical history and response to the cells, and the discretion of the attending physician. The compositions and cells are in some embodiments suitably administered to the subject at one time or over a series of treatments. 41744090.2
Attorney Docket No.046483-7386WO1(03225) In some embodiments, the cells are administered as part of a combination treatment, such as simultaneously with or sequentially with, in any order, another therapeutic intervention, such as an antibody or engineered cell or receptor or agent, such as a cytotoxic or therapeutic agent. The cells in some embodiments are co-administered with one or more additional therapeutic agents or in connection with another therapeutic intervention, either simultaneously or sequentially in any order. In some contexts, the cells are co-administered with another therapy sufficiently close in time such that the cell populations enhance the effect of one or more additional therapeutic agents, or vice versa. In some embodiments, the cells are administered prior to the one or more additional therapeutic agents. In some embodiments, the cells are administered after the one or more additional therapeutic agents. In some embodiments, the one or more additional agents includes a cytokine, such as IL-2, for example, to enhance persistence. In some embodiments, the methods comprise administration of a chemotherapeutic agent. Following administration of the cells, the biological activity of the engineered cell populations in some embodiments is measured, e.g., by any of a number of known methods. Parameters to assess include specific binding of an engineered or natural T cell or other immune cell to antigen, in vivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flow cytometry. In certain embodiments, the ability of the engineered cells to destroy target cells can be measured using any suitable method known in the art, such as cytotoxicity assays described in, for example, Kochenderfer et al., J. Immunotherapy, 32(7): 689-702 (2009), and Herman et al. J. Immunological Methods, 285(1): 25-40 (2004). In certain embodiments, the biological activity of the cells is measured by assaying expression and/or secretion of one or more cytokines, such as CD 107a, IFNγ, IL-2, and TNF. In some aspects the biological activity is measured by assessing clinical outcome, such as reduction in tumor burden or load. In certain embodiments, the subject is provided a secondary or alternative treatment. Secondary/alternative treatments include but are not limited to chemotherapy, radiation, surgery, and medications. In some embodiments, the subject can be administered a conditioning therapy , such as a lymphodepletion step, prior to adoptive cell therapy. In some embodiments, the conditioning therapy comprises administering an effective amount of cyclophosphamide to the subject. In some embodiments, the conditioning therapy comprises administering an effective amount of fludarabine to the subject. In preferred embodiments, the conditioning therapy comprises 41744090.2
Attorney Docket No.046483-7386WO1(03225) administering an effective amount of a combination of cyclophosphamide and fludarabine to the subject. Administration of a conditioning therapy prior to CAR T cell therapy may increase the efficacy of the CAR T cell therapy. Methods of conditioning patients for T cell therapy are described in U.S. Patent No.9,855,298, which is incorporated herein by reference in its entirety. E. Methods of Producing Genetically Modified Immune Cells The present disclosure provides methods for producing or generating a modified immune cell or precursor thereof (e.g., CAR T cells comprising CH25H) of the invention for tumor immunotherapy, e.g., adoptive immunotherapy. The cells generally are engineered by introducing one or more genetically engineered nucleic acids encoding a CAR and CH25H. In certain embodiments, the invention provides a method for generating a modified immune cell or precursor cell thereof, comprising introducing into an immune or precursor cell a a nucleic acid encoding a CAR and a CH25H gene. In some embodiments, the CAR and CH25H are introduced into a cell by an expression vector. Expression vectors comprising a nucleic acid sequence encoding a CAR of the present invention are provided herein. Suitable expression vectors include lentivirus vectors, gamma retrovirus vectors, foamy virus vectors, adeno associated virus (AAV) vectors, adenovirus vectors, engineered hybrid viruses, naked DNA, including but not limited to transposon mediated vectors, such as Sleeping Beauty, Piggybak, and Integrases such as Phi31. Some other suitable expression vectors include Herpes simplex virus (HSV) and retrovirus expression vectors. In certain embodiments, the nucleic acid encoding a CAR and a CH25H gene is introduced into the cell via viral transduction. In certain embodiments, the viral transduction comprises contacting the immune or precursor cell with a viral vector comprising the nucleic acid encoding a CAR and a CH25H gene. In certain embodiments, the viral vector is an adeno- associated viral (AAV) vector. In certain embodiments, the AAV vector comprises a Woodchuck Hepatitis Virus post-transcriptional regulatory element (WPRE). In certain embodiments, the AAV vector comprises a polyadenylation (polyA) sequence. In certain embodiments, the polyA sequence is a bovine growth hormone (BGH) polyA sequence. Adenovirus expression vectors are based on adenoviruses, which have a low capacity for integration into genomic DNA but a high efficiency for transfecting host cells. Adenovirus expression vectors contain adenovirus sequences sufficient to: (a) support packaging of the 41744090.2
Attorney Docket No.046483-7386WO1(03225) expression vector and (b) to ultimately express the CAR in the host cell. In some embodiments, the adenovirus genome is a 36 kb, linear, double stranded DNA, where a foreign DNA sequence (e.g., a nucleic acid encoding a CAR) may be inserted to substitute large pieces of adenoviral DNA in order to make the expression vector of the present invention (see, e.g., Danthinne and Imperiale, Gene Therapy (2000) 7(20): 1707-1714). Another expression vector is based on an adeno associated virus (AAV), which takes advantage of the adenovirus coupled systems. This AAV expression vector has a high frequency of integration into the host genome. It can infect nondividing cells, thus making it useful for delivery of genes into mammalian cells, for example, in tissue cultures or in vivo. The AAV vector has a broad host range for infectivity. Details concerning the generation and use of AAV vectors are described in U.S. Patent Nos.5,139,941 and 4,797,368. Retrovirus expression vectors are capable of integrating into the host genome, delivering a large amount of foreign genetic material, infecting a broad spectrum of species and cell types and being packaged in special cell lines. The retroviral vector is constructed by inserting a nucleic acid (e.g., a nucleic acid encoding a CAR and a CH25H gene) into the viral genome at certain locations to produce a virus that is replication defective. Though the retroviral vectors are able to infect a broad variety of cell types, integration and stable expression of the CAR and CH25H requires the division of host cells. Lentiviral vectors are derived from lentiviruses, which are complex retroviruses that, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural function (see, e.g., U.S. Patent Nos.6,013,516 and 5,994, 136). Some examples of lentiviruses include the Human Immunodeficiency Viruses (HIV-1, HIV-2) and the Simian Immunodeficiency Virus (SIV). Lentiviral vectors have been generated by multiply attenuating the HIV virulence genes, for example, the genes env, vif, vpr, vpu and nef are deleted making the vector biologically safe. Lentiviral vectors are capable of infecting non-dividing cells and can be used for both in vivo and ex vivo gene transfer and expression, e.g., of a nucleic acid encoding a CAR (see, e.g., U.S. Patent No.5,994,136). Expression vectors including a nucleic acid of the present disclosure can be introduced into a host cell by any means known to persons skilled in the art. The expression vectors may include viral sequences for transfection, if desired. Alternatively, the expression vectors may be introduced by fusion, electroporation, biolistics, transfection, lipofection, or the like. The host 41744090.2
Attorney Docket No.046483-7386WO1(03225) cell may be grown and expanded in culture before introduction of the expression vectors, followed by the appropriate treatment for introduction and integration of the vectors. The host cells are then expanded and may be screened by virtue of a marker present in the vectors. Various markers that may be used are known in the art, and may include hprt, neomycin resistance, thymidine kinase, hygromycin resistance, etc. As used herein, the terms "cell," "cell line," and "cell culture" may be used interchangeably. In some embodiments, the host cell an immune cell or precursor thereof, e.g., a T cell, an NK cell, or an NKT cell. The present invention also provides genetically engineered cells which include and stably express a CAR and CH25H. In some embodiments, the genetically engineered cells are genetically engineered T-lymphocytes (T cells), naive T cells (TN), memory T cells (for example, central memory T cells (TCM), effector memory cells (TEM)), natural killer cells (NK cells), and macrophages capable of giving rise to therapeutically relevant progeny. In certain embodiments, the genetically engineered cells are autologous cells. In certain embodiments, the modified cell is resistant to T cell exhaustion. Modified cells (e.g., comprising a CAR and CH25H) may be produced by stably transfecting host cells with an expression vector including a nucleic acid of the present disclosure. Additional methods for generating a modified cell of the present disclosure include, without limitation, chemical transformation methods (e.g., using calcium phosphate, dendrimers, liposomes and/or cationic polymers), non-chemical transformation methods (e.g., electroporation, optical transformation, gene electrotransfer and/or hydrodynamic delivery) and/or particle-based methods (e.g., impalefection, using a gene gun and/or magnetofection). Transfected cells expressing a CAR and CH25H of the present disclosure may be expanded ex vivo. Physical methods for introducing an expression vector into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells including vectors and/or exogenous nucleic acids are well- known in the art. See, e.g., Sambrook et al. (2001), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York. Chemical methods for introducing an expression vector into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. 41744090.2
Attorney Docket No.046483-7386WO1(03225) Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine (“DMPC”) can be obtained from Sigma, St. Louis, MO; dicetyl phosphate (“DCP”) can be obtained from K & K Laboratories (Plainview, NY); cholesterol (“Choi”) can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform/methanol can be stored at about -20⁰C. Chloroform may be used as the only solvent since it is more readily evaporated than methanol. “Liposome” is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). Compositions that have different structures in solution than the normal vesicular structure are also encompassed. For example, the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules. Also contemplated are lipofectamine-nucleic acid complexes. Regardless of the method used to introduce exogenous nucleic acids into a host cell or otherwise expose a cell to the inhibitor of the present invention, in order to confirm the presence of the nucleic acids in the host cell, a variety of assays may be performed. Such assays include, for example, molecular biology assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; biochemistry assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the invention. In one embodiment, the nucleic acids introduced into the host cell are RNA. In another embodiment, the RNA is mRNA that comprises in vitro transcribed RNA or synthetic RNA. The RNA may be produced by in vitro transcription using a polymerase chain reaction (PCR)- generated template. DNA of interest from any source can be directly converted by PCR into a template for in vitro mRNA synthesis using appropriate primers and RNA polymerase. The 41744090.2
Attorney Docket No.046483-7386WO1(03225) source of the DNA may be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence or any other appropriate source of DNA. PCR may be used to generate a template for in vitro transcription of mRNA which is then introduced into cells. Methods for performing PCR are well known in the art. Primers for use in PCR are designed to have regions that are substantially complementary to regions of the DNA to be used as a template for the PCR. “Substantially complementary,” as used herein, refers to sequences of nucleotides where a majority or all of the bases in the primer sequence are complementary. Substantially complementary sequences are able to anneal or hybridize with the intended DNA target under annealing conditions used for PCR. The primers can be designed to be substantially complementary to any portion of the DNA template. For example, the primers can be designed to amplify the portion of a gene that is normally transcribed in cells (the open reading frame), including 5' and 3' UTRs. The primers may also be designed to amplify a portion of a gene that encodes a particular domain of interest. In one embodiment, the primers are designed to amplify the coding region of a human cDNA, including all or portions of the 5' and 3' UTRs. Primers useful for PCR are generated by synthetic methods that are well known in the art. “Forward primers” are primers that contain a region of nucleotides that are substantially complementary to nucleotides on the DNA template that are upstream of the DNA sequence that is to be amplified. “Upstream” is used herein to refer to a location 5, to the DNA sequence to be amplified relative to the coding strand. “Reverse primers” are primers that contain a region of nucleotides that are substantially complementary to a double-stranded DNA template that are downstream of the DNA sequence that is to be amplified. “Downstream” is used herein to refer to a location 3' to the DNA sequence to be amplified relative to the coding strand. Chemical structures that have the ability to promote stability and/or translation efficiency of the RNA may also be used. The RNA preferably has 5' and 3' UTRs. In one embodiment, the 5' UTR is between zero and 3000 nucleotides in length. The length of 5' and 3' UTR sequences to be added to the coding region can be altered by different methods, including, but not limited to, designing primers for PCR that anneal to different regions of the UTRs. Using this approach, one of ordinary skill in the art can modify the 5' and 3' UTR lengths required to achieve optimal translation efficiency following transfection of the transcribed RNA. The 5' and 3' UTRs can be the naturally occurring, endogenous 5' and 3' UTRs for the gene of interest. Alternatively, UTR sequences that are not endogenous to the gene of interest 41744090.2
Attorney Docket No.046483-7386WO1(03225) can be added by incorporating the UTR sequences into the forward and reverse primers or by any other modifications of the template. The use of UTR sequences that are not endogenous to the gene of interest can be useful for modifying the stability and/or translation efficiency of the RNA. For example, it is known that AU-rich elements in 3' UTR sequences can decrease the stability of mRNA. Therefore, 3' UTRs can be selected or designed to increase the stability of the transcribed RNA based on properties of UTRs that are well known in the art. In one embodiment, the 5' UTR can contain the Kozak sequence of the endogenous gene. Alternatively, when a 5' UTR that is not endogenous to the gene of interest is being added by PCR as described above, a consensus Kozak sequence can be redesigned by adding the 5' UTR sequence. Kozak sequences can increase the efficiency of translation of some RNA transcripts, but does not appear to be required for all RNAs to enable efficient translation. The requirement for Kozak sequences for many mRNAs is known in the art. In other embodiments the 5' UTR can be derived from an RNA virus whose RNA genome is stable in cells. In other embodiments various nucleotide analogues can be used in the 3' or 5' UTR to impede exonuclease degradation of the mRNA. To enable synthesis of RNA from a DNA template without the need for gene cloning, a promoter of transcription should be attached to the DNA template upstream of the sequence to be transcribed. When a sequence that functions as a promoter for an RNA polymerase is added to the 5' end of the forward primer, the RNA polymerase promoter becomes incorporated into the PCR product upstream of the open reading frame that is to be transcribed. In one embodiment, the promoter is a T7 polymerase promoter, as described elsewhere herein. Other useful promoters include, but are not limited to, T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for T7, T3 and SP6 promoters are known in the art. In one embodiment, the mRNA has both a cap on the 5' end and a 3' poly(A) tail which determine ribosome binding, initiation of translation and stability mRNA in the cell. On a circular DNA template, for instance, plasmid DNA, RNA polymerase produces a long concatameric product which is not suitable for expression in eukaryotic cells. The transcription of plasmid DNA linearized at the end of the 3' UTR results in normal sized mRNA which is not effective in eukaryotic transfection even if it is polyadenylated after transcription. 41744090.2
Attorney Docket No.046483-7386WO1(03225) On a linear DNA template, phage T7 RNA polymerase can extend the 3' end of the transcript beyond the last base of the template (Schenborn and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003). The polyA/T segment of the transcriptional DNA template can be produced during PCR by using a reverse primer containing a polyT tail, such as 100T tail (size can be 50-5000 T), or after PCR by any other method, including, but not limited to, DNA ligation or in vitro recombination. Poly(A) tails also provide stability to RNAs and reduce their degradation. Generally, the length of a poly(A) tail positively correlates with the stability of the transcribed RNA. In one embodiment, the poly(A) tail is between 100 and 5000 adenosines. Poly(A) tails of RNAs can be further extended following in vitro transcription with the use of a poly(A) polymerase, such as E. coli polyA polymerase (E-PAP). In one embodiment, increasing the length of a poly(A) tail from 100 nucleotides to between 300 and 400 nucleotides results in about a two-fold increase in the translation efficiency of the RNA. Additionally, the attachment of different chemical groups to the 3' end can increase mRNA stability. Such attachment can contain modified/artificial nucleotides, aptamers and other compounds. For example, ATP analogs can be incorporated into the poly(A) tail using poly(A) polymerase. ATP analogs can further increase the stability of the RNA. 5' caps also provide stability to RNA molecules. In a preferred embodiment, RNAs produced by the methods disclosed herein include a 5' cap. The 5' cap is provided using techniques known in the art and described herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444 (2001); Stepinski, et al., RNA, 7:1468-95 (2001); Elango, et al., Biochim. Biophys. Res. Commun., 330:958-966 (2005)). In some embodiments, the RNA is electroporated into the cells, such as in vitro transcribed RNA. Any solutes suitable for cell electroporation, which can contain factors facilitating cellular permeability and viability such as sugars, peptides, lipids, proteins, antioxidants, and surfactants can be included. In some embodiments, a nucleic acid encoding a CAR of the present disclosure will be RNA, e.g., in vitro synthesized RNA. Methods for in vitro synthesis of RNA are known in the art; any known method can be used to synthesize RNA comprising a sequence encoding a CAR. Methods for introducing RNA into a host cell are known in the art. See, e.g., Zhao et al. Cancer Res. (2010) 15: 9053. Introducing RNA comprising a nucleotide sequence encoding a CAR into 41744090.2
Attorney Docket No.046483-7386WO1(03225) a host cell can be carried out in vitro, ex vivo or in vivo. For example, a host cell (e.g., an NK cell, a cytotoxic T lymphocyte, etc.) can be electroporated in vitro or ex vivo with RNA comprising a nucleotide sequence encoding a CAR and CH25H. The disclosed methods can be applied to the modulation of T cell activity in basic research and therapy, in the fields of cancer, stem cells, acute and chronic infections, and autoimmune diseases, including the assessment of the ability of the genetically modified T cell to kill a target cancer cell. The methods also provide the ability to control the level of expression over a wide range by changing, for example, the promoter or the amount of input RNA, making it possible to individually regulate the expression level. Furthermore, the PCR-based technique of mRNA production greatly facilitates the design of the mRNAs with different structures and combination of their domains. One advantage of RNA transfection methods of the invention is that RNA transfection is essentially transient and a vector-free. An RNA transgene can be delivered to a lymphocyte and expressed therein following a brief in vitro cell activation, as a minimal expressing cassette without the need for any additional viral sequences. Under these conditions, integration of the transgene into the host cell genome is unlikely. Cloning of cells is not necessary because of the efficiency of transfection of the RNA and its ability to uniformly modify the entire lymphocyte population. Genetic modification of T cells with in vitro-transcribed RNA (IVT-RNA) makes use of two different strategies both of which have been successively tested in various animal models. Cells are transfected with in vitro-transcribed RNA by means of lipofection or electroporation. It is desirable to stabilize IVT-RNA using various modifications in order to achieve prolonged expression of transferred IVT-RNA. Some IVT vectors are known in the literature which are utilized in a standardized manner as template for in vitro transcription and which have been genetically modified in such a way that stabilized RNA transcripts are produced. Currently protocols used in the art are based on a plasmid vector with the following structure: a 5' RNA polymerase promoter enabling RNA transcription, followed by a gene of interest which is flanked either 3' and/or 5' by untranslated regions (UTR), and a 3' polyadenyl cassette containing 50-70 A nucleotides. Prior to in vitro transcription, the circular plasmid is linearized downstream of the polyadenyl cassette by type II 41744090.2
Attorney Docket No.046483-7386WO1(03225) restriction enzymes (recognition sequence corresponds to cleavage site). The polyadenyl cassette thus corresponds to the later poly(A) sequence in the transcript. As a result of this procedure, some nucleotides remain as part of the enzyme cleavage site after linearization and extend or mask the poly(A) sequence at the 3' end. It is not clear, whether this nonphysiological overhang affects the amount of protein produced intracellularly from such a construct. In another aspect, the RNA construct is delivered into the cells by electroporation. See, e.g., the formulations and methodology of electroporation of nucleic acid constructs into mammalian cells as taught in US 2004/0014645, US 2005/0052630A1, US 2005/0070841A1, US 2004/0059285A1, US 2004/0092907A1. The various parameters including electric field strength required for electroporation of any known cell type are generally known in the relevant research literature as well as numerous patents and applications in the field. See e.g., U.S. Pat. No.6,678,556, U.S. Pat. No.7,171,264, and U.S. Pat. No.7,173,116. Apparatus for therapeutic application of electroporation are available commercially, e.g., the MedPulser™ DNA Electroporation Therapy System (Inovio/Genetronics, San Diego, Calif.), and are described in patents such as U.S. Pat. No.6,567,694; U.S. Pat. No.6,516,223, U.S. Pat. No.5,993,434, U.S. Pat. No.6,181,964, U.S. Pat. No.6,241,701, and U.S. Pat. No.6,233,482; electroporation may also be used for transfection of cells in vitro as described e.g. in US20070128708A1. Electroporation may also be utilized to deliver nucleic acids into cells in vitro. Accordingly, electroporation-mediated administration into cells of nucleic acids including expression constructs utilizing any of the many available devices and electroporation systems known to those of skill in the art presents an exciting new means for delivering an RNA of interest to a target cell. In some embodiments, the immune cells (e.g. T cells) can be incubated or cultivated prior to, during and/or subsequent to introducing the nucleic acid molecule encoding the CAR and CH25H gene. In some embodiments, the cells (e.g. T cells) can be incubated or cultivated prior to, during or subsequent to the introduction of the nucleic acid molecule, such as prior to, during or subsequent to the transduction of the cells with a viral vector (e.g. lentiviral vector) encoding the CAR and CH25H gene. F. Nucleic Acids and Expression Vectors The present disclosure provides a nucleic acid encoding a CAR and a CH25H gene. 41744090.2
Attorney Docket No.046483-7386WO1(03225) In some embodiments, a nucleic acid of the present disclosure is provided for the production of a CAR and CH25H as described herein, e.g., in a mammalian cell. In some embodiments, a nucleic acid of the present disclosure provides for amplification of the CAR and CH25H-encoding nucleic acid. In some embodiments, the nucleic acid comprises a linker sequence. In some embodiments, the linker comprises a nucleic acid sequence that encodes for a self-cleaving peptide. As used herein, a “self-cleaving peptide” or “2A peptide” refers to an oligopeptide that allow multiple proteins to be encoded as polyproteins, which dissociate into component proteins upon translation. Use of the term “self-cleaving” is not intended to imply a proteolytic cleavage reaction. Various self-cleaving or 2A peptides are known to those of skill in the art, including, without limitation, those found in members of the Picornaviridae virus family, e.g., foot-and- mouth disease virus (FMDV), equine rhinitis A virus (ERAV0, Thosea asigna virus (TaV), and porcine tescho virus-1 (PTV-1); and carioviruses such as Theilovirus and encephalomyocarditis viruses. 2A peptides derived from FMDV, ERAV, PTV-1, and TaV are referred to herein as “F2A,” “E2A,” “P2A,” and “T2A,” respectively. Those of skill in the art would be able to select the appropriate self-cleaving peptide for use in the present invention. In some embodiments, the linker comprises a nucleic acid sequence encoding a combination of a Furin cleavage site and a 2A peptide. Examples include, without limitation, a linker comprising a nucleic acid sequence encoding Furin and F2A, a linker comprising a nucleic acid sequence encoding Furin and E2A, a linker comprising a nucleic acid sequence encoding Furin and P2A, a linker comprising a nucleic acid sequence encoding Furin and T2A. Those of skill in the art would be able to select the appropriate combination for use in the present invention. In such embodiments, the linker may further comprise a spacer sequence between the Furin and 2A peptide. Various spacer sequences are known in the art, including, without limitation, glycine serine (GS) spacers such as (GS)n, (GSGGS)n (SEQ ID NO:21) and (GGGS)n (SEQ ID NO:22), where n represents an integer of at least 1. Exemplary spacer sequences can comprise amino acid sequences including, without limitation, GGSG (SEQ ID NO:24), GGSGG (SEQ ID NO:25), GSGSG (SEQ ID NO:26), GSGGG (SEQ ID NO:27), GGGSG (SEQ ID NO:28), GSSSG (SEQ ID NO:29), and the like. Those of skill in the art would be able to select the appropriate spacer sequence for use in the present invention. 41744090.2
Attorney Docket No.046483-7386WO1(03225) In some embodiments, a nucleic acid of the present disclosure may be operably linked to a transcriptional control element, e.g., a promoter, and enhancer, etc. Suitable promoter and enhancer elements are known to those of skill in the art. In certain embodiments, the nucleic acid encoding a CAR is in operable linkage with a promoter. In certain embodiments, the promoter is a phosphoglycerate kinase-1 (PGK) promoter. For expression in a bacterial cell, suitable promoters include, but are not limited to, lacI, lacZ, T3, T7, gpt, lambda P and trc. For expression in a eukaryotic cell, suitable promoters include, but are not limited to, light and/or heavy chain immunoglobulin gene promoter and enhancer elements; cytomegalovirus immediate early promoter; herpes simplex virus thymidine kinase promoter; early and late SV40 promoters; promoter present in long terminal repeats from a retrovirus; mouse metallothionein-I promoter; and various art-known tissue specific promoters. Suitable reversible promoters, including reversible inducible promoters are known in the art. Such reversible promoters may be isolated and derived from many organisms, e.g., eukaryotes and prokaryotes. Modification of reversible promoters derived from a first organism for use in a second organism, e.g., a first prokaryote and a second a eukaryote, a first eukaryote and a second a prokaryote, etc., is well known in the art. Such reversible promoters, and systems based on such reversible promoters but also comprising additional control proteins, include, but are not limited to, alcohol regulated promoters (e.g., alcohol dehydrogenase I (alcA) gene promoter, promoters responsive to alcohol transactivator proteins (A1cR), etc.), tetracycline regulated promoters, (e.g., promoter systems including TetActivators, TetON, TetOFF, etc.), steroid regulated promoters (e.g., rat glucocorticoid receptor promoter systems, human estrogen receptor promoter systems, retinoid promoter systems, thyroid promoter systems, ecdysone promoter systems, mifepristone promoter systems, etc.), metal regulated promoters (e.g., metallothionein promoter systems, etc.), pathogenesis-related regulated promoters (e.g., salicylic acid regulated promoters, ethylene regulated promoters, benzothiadiazole regulated promoters, etc.), temperature regulated promoters (e.g., heat shock inducible promoters (e.g., HSP-70, HSP-90, soybean heat shock promoter, etc.), light regulated promoters, synthetic inducible promoters, and the like. In some embodiments, the promoter is a CD8 cell-specific promoter, a CD4 cell-specific promoter, a neutrophil-specific promoter, or an NK-specific promoter. For example, a CD4 gene promoter can be used; see, e.g., Salmon et al. Proc. Natl. Acad. Sci. USA (1993) 90:7739; and 41744090.2
Attorney Docket No.046483-7386WO1(03225) Marodon et al. (2003) Blood 101:3416. As another example, a CD8 gene promoter can be used. NK cell-specific expression can be achieved by use of an NcrI (p46) promoter; see, e.g., Eckelhart et al. Blood (2011) 117:1565. For expression in a yeast cell, a suitable promoter is a constitutive promoter such as an ADH1 promoter, a PGK1 promoter, an ENO promoter, a PYK1 promoter and the like; or a regulatable promoter such as a GAL1 promoter, a GAL10 promoter, an ADH2 promoter, a PHOS promoter, a CUP1 promoter, a GALT promoter, a MET25 promoter, a MET3 promoter, a CYC1 promoter, a HIS3 promoter, an ADH1 promoter, a PGK promoter, a GAPDH promoter, an ADC1 promoter, a TRP1 promoter, a URA3 promoter, a LEU2 promoter, an ENO promoter, a TP1 promoter, and AOX1 (e.g., for use in Pichia). Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art. Suitable promoters for use in prokaryotic host cells include, but are not limited to, a bacteriophage T7 RNA polymerase promoter; a trp promoter; a lac operon promoter; a hybrid promoter, e.g., a lac/tac hybrid promoter, a tac/trc hybrid promoter, a trp/lac promoter, a T7/lac promoter; a trc promoter; a tac promoter, and the like; an araBAD promoter; in vivo regulated promoters, such as an ssaG promoter or a related promoter (see, e.g., U.S. Patent Publication No.20040131637), a pagC promoter (Pulkkinen and Miller, J. Bacteriol. (1991) 173(1): 86-93; Alpuche-Aranda et al., Proc. Natl. Acad. Sci. USA (1992) 89(21): 10079-83), a nirB promoter (Harborne et al. Mol. Micro. (1992) 6:2805-2813), and the like (see, e.g., Dunstan et al., Infect. Immun. (1999) 67:5133-5141; McKelvie et al., Vaccine (2004) 22:3243-3255; and Chatfield et al., Biotechnol. (1992) 10:888- 892); a sigma70 promoter, e.g., a consensus sigma70 promoter (see, e.g., GenBank Accession Nos. AX798980, AX798961, and AX798183); a stationary phase promoter, e.g., a dps promoter, an spv promoter, and the like; a promoter derived from the pathogenicity island SPI-2 (see, e.g., WO96/17951); an actA promoter (see, e.g., Shetron-Rama et al., Infect. Immun. (2002) 70:1087- 1096); an rpsM promoter (see, e.g., Valdivia and Falkow Mol. Microbiol. (1996).22:367); a tet promoter (see, e.g., Hillen, W. and Wissmann, A. (1989) In Saenger, W. and Heinemann, U. (eds), Topics in Molecular and Structural Biology, Protein--Nucleic Acid Interaction. Macmillan, London, UK, Vol.10, pp.143-162); an SP6 promoter (see, e.g., Melton et al., Nucl. Acids Res. (1984) 12:7035); and the like. Suitable strong promoters for use in prokaryotes such as Escherichia coli include, but are not limited to Trc, Tac, T5, T7, and PLambda. Non-limiting examples of operators for use in bacterial host cells include a lactose promoter operator (LacI 41744090.2
Attorney Docket No.046483-7386WO1(03225) repressor protein changes conformation when contacted with lactose, thereby preventing the Lad repressor protein from binding to the operator), a tryptophan promoter operator (when complexed with tryptophan, TrpR repressor protein has a conformation that binds the operator; in the absence of tryptophan, the TrpR repressor protein has a conformation that does not bind to the operator), and a tac promoter operator (see, e.g., deBoer et al., Proc. Natl. Acad. Sci. U.S.A. (1983) 80:21-25). Other examples of suitable promoters include the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto. Other constitutive promoter sequences may also be used, including, but not limited to a simian virus 40 (SV40) early promoter, a mouse mammary tumor virus (MMTV) or human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, a MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, the EF-1 alpha promoter, as well as human gene promoters such as, but not limited to, an actin promoter, a myosin promoter, a hemoglobin promoter, and a creatine kinase promoter. Further, the invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention. The use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter. In some embodiments, the locus or construct or transgene containing the suitable promoter is irreversibly switched through the induction of an inducible system. Suitable systems for induction of an irreversible switch are well known in the art, e.g., induction of an irreversible switch may make use of a Cre-lox-mediated recombination (see, e.g., Fuhrmann-Benzakein, et al., Proc. Natl. Acad. Sci. USA (2000) 28:e99, the disclosure of which is incorporated herein by reference). Any suitable combination of recombinase, endonuclease, ligase, recombination sites, etc. known to the art may be used in generating an irreversibly switchable promoter. Methods, mechanisms, and requirements for performing site-specific recombination, described elsewhere herein, find use in generating irreversibly switched promoters and are well known in the art, see, 41744090.2
Attorney Docket No.046483-7386WO1(03225) e.g., Grindley et al. Annual Review of Biochemistry (2006) 567-605; and Tropp, Molecular Biology (2012) (Jones & Bartlett Publishers, Sudbury, Mass.), the disclosures of which are incorporated herein by reference. In some embodiments, a nucleic acid of the present disclosure further comprises a nucleic acid sequence encoding a CAR inducible expression cassette. In one embodiment, the inducible expression cassette is for the production of a transgenic polypeptide product that is released upon CAR signaling. See, e.g., Chmielewski and Abken, Expert Opin. Biol. Ther. (2015) 15(8): 1145- 1154; and Abken, Immunotherapy (2015) 7(5): 535-544. In some embodiments, a nucleic acid of the present disclosure further comprises a nucleic acid sequence encoding a cytokine operably linked to a T-cell activation responsive promoter. In some embodiments, the cytokine operably linked to a T-cell activation responsive promoter is present on a separate nucleic acid sequence. In one embodiment, the cytokine is IL-12. A nucleic acid of the present disclosure may be present within an expression vector and/or a cloning vector. An expression vector can include a selectable marker, an origin of replication, and other features that provide for replication and/or maintenance of the vector. Suitable expression vectors include, e.g., plasmids, viral vectors, and the like. Large numbers of suitable vectors and promoters are known to those of skill in the art; many are commercially available for generating a subject recombinant construct. The following vectors are provided by way of example, and should not be construed in anyway as limiting: Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene) pSVK3, pBPV, pMSG and pSVL (Pharmacia). Expression vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences encoding heterologous proteins. A selectable marker operative in the expression host may be present. Suitable expression vectors include, but are not limited to, viral vectors (e.g. viral vectors based on vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., Invest. Opthalmol. Vis. Sci. (1994) 35: 2543-2549; Borras et al., Gene Ther. (1999) 6: 515-524; Li and Davidson, Proc. Natl. Acad. Sci. USA (1995) 92: 7700-7704; Sakamoto et al., H. Gene Ther. (1999) 5: 1088-1097; WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655); adeno-associated 41744090.2
Attorney Docket No.046483-7386WO1(03225) virus (see, e.g., Ali et al., Hum. Gene Ther. (1998) 9: 81-86, Flannery et al., Proc. Natl. Acad. Sci. USA (1997) 94: 6916-6921; Bennett et al., Invest. Opthalmol. Vis. Sci. (1997) 38: 2857- 2863; Jomary et al., Gene Ther. (1997) 4:683690, Rolling et al., Hum. Gene Ther. (1999) 10: 641-648; Ali et al., Hum. Mol. Genet. (1996) 5: 591-594; Srivastava in WO 93/09239, Samulski et al., J. Vir. (1989) 63: 3822-3828; Mendelson et al., Virol. (1988) 166: 154-165; and Flotte et al., Proc. Natl. Acad. Sci. USA (1993) 90: 10613-10617); SV40; herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshi et al., Proc. Natl. Acad. Sci. USA (1997) 94: 10319- 23; Takahashi et al., J. Virol. (1999) 73: 7812-7816); a retroviral vector (e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and the like. Additional expression vectors suitable for use are, e.g., without limitation, a lentivirus vector, a gamma retrovirus vector, a foamy virus vector, an adeno-associated virus vector, an adenovirus vector, a pox virus vector, a herpes virus vector, an engineered hybrid virus vector, a transposon mediated vector, and the like. Viral vector technology is well known in the art and is described, for example, in Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno- associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No.6,326,193). In some embodiments, an expression vector (e.g., a lentiviral vector) may be used to introduce the CAR into an immune cell or precursor thereof (e.g., a T cell). Accordingly, an expression vector (e.g., a lentiviral vector) of the present invention may comprise a nucleic acid encoding for a CAR. In some embodiments, the expression vector (e.g., lentiviral vector) will comprise additional elements that will aid in the functional expression of the CAR encoded therein. In some embodiments, an expression vector comprising a nucleic acid encoding for a CAR further comprises a mammalian promoter. In one embodiment, the vector further comprises an elongation-factor-1-alpha promoter (EF-1α promoter). Use of an EF-1α promoter may increase the efficiency in expression of downstream transgenes (e.g., a CAR encoding 41744090.2
Attorney Docket No.046483-7386WO1(03225) nucleic acid sequence). Physiologic promoters (e.g., an EF-1α promoter) may be less likely to induce integration mediated genotoxicity, and may abrogate the ability of the retroviral vector to transform stem cells. Other physiological promoters suitable for use in a vector (e.g., lentiviral vector) are known to those of skill in the art and may be incorporated into a vector of the present invention. In some embodiments, the vector (e.g., lentiviral vector) further comprises a non- requisite cis acting sequence that may improve titers and gene expression. One non-limiting example of a non-requisite cis acting sequence is the central polypurine tract and central termination sequence (cPPT/CTS) which is important for efficient reverse transcription and nuclear import. Other non-requisite cis acting sequences are known to those of skill in the art and may be incorporated into a vector (e.g., lentiviral vector) of the present invention. In some embodiments, the vector further comprises a posttranscriptional regulatory element. Posttranscriptional regulatory elements may improve RNA translation, improve transgene expression and stabilize RNA transcripts. One example of a posttranscriptional regulatory element is the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). Accordingly, in some embodiments a vector for the present invention further comprises a WPRE sequence. Various posttranscriptional regulator elements are known to those of skill in the art and may be incorporated into a vector (e.g., lentiviral vector) of the present invention. A vector of the present invention may further comprise additional elements such as a rev response element (RRE) for RNA transport, packaging sequences, and 5’ and 3’ long terminal repeats (LTRs). The term “long terminal repeat” or “LTR” refers to domains of base pairs located at the ends of retroviral DNAs which comprise U3, R and U5 regions. LTRs generally provide functions required for the expression of retroviral genes (e.g., promotion, initiation and polyadenylation of gene transcripts) and to viral replication. In one embodiment, a vector (e.g., lentiviral vector) of the present invention includes a 3’ U3 deleted LTR. Accordingly, a vector (e.g., lentiviral vector) of the present invention may comprise any combination of the elements described herein to enhance the efficiency of functional expression of transgenes. For example, a vector (e.g., lentiviral vector) of the present invention may comprise a WPRE sequence, cPPT sequence, RRE sequence, 5’LTR, 3’ U3 deleted LTR’ in addition to a nucleic acid encoding a CAR. Vectors of the present invention may be self-inactivating vectors. As used herein, the term “self-inactivating vector” refers to vectors in which the 3’ LTR enhancer promoter region (U3 region) has been modified (e.g., by deletion or substitution). A self-inactivating vector may 41744090.2
Attorney Docket No.046483-7386WO1(03225) prevent viral transcription beyond the first round of viral replication. Consequently, a self- inactivating vector may be capable of infecting and then integrating into a host genome (e.g., a mammalian genome) only once, and cannot be passed further. Accordingly, self-inactivating vectors may greatly reduce the risk of creating a replication-competent virus. In some embodiments, a nucleic acid of the present invention may be RNA, e.g., in vitro synthesized RNA. Methods for in vitro synthesis of RNA are known to those of skill in the art; any known method can be used to synthesize RNA comprising a sequence encoding a CAR of the present disclosure. Methods for introducing RNA into a host cell are known in the art. See, e.g., Zhao et al. Cancer Res. (2010) 15: 9053. Introducing RNA comprising a nucleotide sequence encoding a CAR of the present disclosure into a host cell can be carried out in vitro, ex vivo or in vivo. For example, a host cell (e.g., an NK cell, a cytotoxic T lymphocyte, etc.) can be electroporated in vitro or ex vivo with RNA comprising a nucleotide sequence encoding a CAR of the present disclosure. In order to assess the expression of a polypeptide or portions thereof, the expression vector to be introduced into a cell may also contain either a selectable marker gene or a reporter gene, or both, to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In some embodiments, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, without limitation, antibiotic-resistance genes. Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assessed at a suitable time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include, without limitation, genes encoding luciferase, beta- galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82). G. Sources of Immune Cells 41744090.2
Attorney Docket No.046483-7386WO1(03225) Prior to expansion, a source of immune cells can be obtained from a subject for ex vivo manipulation. Sources of target cells for ex vivo manipulation may also include, e.g., autologous or heterologous donor blood, cord blood, or bone marrow. For example the source of immune cells may be from the subject to be treated with the modified immune cells of the invention, e.g., the subject's blood, the subject's cord blood, or the subject's bone marrow. Non-limiting examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. Preferably, the subject is a human. Immune cells can be obtained from a number of sources, including blood, peripheral blood mononuclear cells, bone marrow, lymph node tissue, spleen tissue, umbilical cord, lymph, or lymphoid organs. Immune cells are cells of the immune system, such as cells of the innate or adaptive immunity, e.g., myeloid or lymphoid cells, including lymphocytes, typically T cells and/or NK cells. Other exemplary cells include stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs). In some aspects, the cells are human cells. With reference to the subject to be treated, the cells may be allogeneic and/or autologous. The cells typically are primary cells, such as those isolated directly from a subject and/or isolated from a subject and frozen. In certain embodiments, the immune cell is a T cell, e.g., a CD8+ T cell (e.g., a CD8+ naive T cell, central memory T cell, or effector memory T cell), a CD4+ T cell, a natural killer T cell (NKT cells), a regulatory T cell (Treg), a stem cell memory T cell, a lymphoid progenitor cell a hematopoietic stem cell, a natural killer cell (NK cell) or a dendritic cell. In some embodiments, the cells are monocytes or granulocytes, e.g., myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and/or basophils. In an embodiment, the target cell is an induced pluripotent stem (iPS) cell or a cell derived from an iPS cell, e.g., an iPS cell generated from a subject, manipulated to alter (e.g., induce a mutation in) or manipulate the expression of one or more target genes, and differentiated into, e.g., a T cell, e.g., a CD8+ T cell (e.g., a CD8+ naive T cell, central memory T cell, or effector memory T cell), a CD4+ T cell, a stem cell memory T cell, a lymphoid progenitor cell or a hematopoietic stem cell. In some embodiments, the cells include one or more subsets of T cells or other cell types, such as whole T cell populations, CD4+ cells, CD8+ cells, and subpopulations thereof, such as those defined by function, activation state, maturity, potential for differentiation, expansion, recirculation, localization, and/or persistence capacities, antigen- specificity, type of antigen 41744090.2
Attorney Docket No.046483-7386WO1(03225) receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and/or degree of differentiation. Among the sub-types and subpopulations of T cells and/or of CD4+ and/or of CD8+ T cells are naive T (TN) cells, effector T cells (TEFF), memory T cells and sub-types thereof, such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM), or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa- associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha/beta T cells, and delta/gamma T cells. In certain embodiments, any number of T cell lines available in the art, may be used. In some embodiments, the methods include isolating immune cells from the subject, preparing, processing, culturing, and/or engineering them. In some embodiments, preparation of the engineered cells includes one or more culture and/or preparation steps. The cells for engineering as described may be isolated from a sample, such as a biological sample, e.g., one obtained from or derived from a subject. In some embodiments, the subject from which the cell is isolated is one having the disease or condition or in need of a cell therapy or to which cell therapy will be administered. The subject in some embodiments is a human in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and/or engineered. Accordingly, the cells in some embodiments are primary cells, e.g., primary human cells. The samples include tissue, fluid, and other samples taken directly from the subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic engineering (e.g. transduction with viral vector), washing, and/or incubation. The biological sample can be a sample obtained directly from a biological source or a sample that is processed. Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom. In some aspects, the sample from which the cells are derived or isolated is blood or a blood-derived sample, or is or is derived from an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, 41744090.2
Attorney Docket No.046483-7386WO1(03225) stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, or other organ, and/or cells derived therefrom. Samples include, in the context of cell therapy, e.g., adoptive cell therapy, samples from autologous and allogeneic sources. In some embodiments, the cells are derived from cell lines, e.g., T cell lines. The cells in some embodiments are obtained from a xenogeneic source, for example, from mouse, rat, non- human primate, and pig. In some embodiments, isolation of the cells includes one or more preparation and/or non-affinity based cell separation steps. In some examples, cells are washed, centrifuged, and/or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich for desired components, lyse or remove cells sensitive to particular reagents. In some examples, cells are separated based on one or more property, such as density, adherent properties, size, sensitivity and/or resistance to particular components. In some examples, cells from the circulating blood of a subject are obtained, e.g., by apheresis or leukapheresis. The samples, in some aspects, contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and/or platelets, and in some aspects contains cells other than red blood cells and platelets. In some embodiments, the blood cells collected from the subject are washed, e.g., to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some aspects, a washing step is accomplished by tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, the cells are resuspended in a variety of biocompatible buffers after washing. In certain embodiments, components of a blood cell sample are removed and the cells directly resuspended in culture media. In some embodiments, the methods include density-based cell separation methods, such as the preparation of white blood cells from peripheral blood by lysing the red blood cells and centrifugation through a Percoll or Ficoll gradient. In one embodiment, immune are obtained cells from the circulating blood of an individual are obtained by apheresis or leukapheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. The cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media, such as phosphate buffered saline (PBS) or wash solution lacks calcium and may lack magnesium or may lack 41744090.2
Attorney Docket No.046483-7386WO1(03225) many if not all divalent cations, for subsequent processing steps. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca-free, Mg-free PBS. Alternatively, the undesirable components of the apheresis sample may be removed and the cells directly resuspended in culture media. In some embodiments, the isolation methods include the separation of different cell types based on the expression or presence in the cell of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acid. In some embodiments, any known method for separation based on such markers may be used. In some embodiments, the separation is affinity- or immunoaffinity-based separation. For example, the isolation in some aspects includes separation of cells and cell populations based on the cells' expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner. Such separation steps can be based on positive selection, in which the cells having bound the reagents are retained for further use, and/or negative selection, in which the cells having not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for further use. In some aspects, negative selection can be particularly useful where no antibody is available that specifically identifies a cell type in a heterogeneous population, such that separation is best carried out based on markers expressed by cells other than the desired population. The separation need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection of or enrichment for cells of a particular type, such as those expressing a marker, refers to increasing the number or percentage of such cells, but need not result in a complete absence of cells not expressing the marker. Likewise, negative selection, removal, or depletion of cells of a particular type, such as those expressing a marker, refers to decreasing the number or percentage of such cells, but need not result in a complete removal of all such cells. In some examples, multiple rounds of separation steps are carried out, where the positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection. In some examples, a single separation step can deplete cells expressing multiple markers simultaneously, such as by incubating cells with a 41744090.2
Attorney Docket No.046483-7386WO1(03225) plurality of antibodies or binding partners, each specific for a marker targeted for negative selection. Likewise, multiple cell types can simultaneously be positively selected by incubating cells with a plurality of antibodies or binding partners expressed on the various cell types. In some embodiments, one or more of the T cell populations is enriched for or depleted of cells that are positive for (marker+) or express high levels (markerhlgh) of one or more particular markers, such as surface markers, or that are negative for (marker -) or express relatively low levels (markerlow) of one or more markers. For example, in some aspects, specific subpopulations of T cells, such as cells positive or expressing high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and/or CD45RO+ T cells, are isolated by positive or negative selection techniques. In some cases, such markers are those that are absent or expressed at relatively low levels on certain populations of T cells (such as non-memory cells) but are present or expressed at relatively higher levels on certain other populations of T cells (such as memory cells). In one embodiment, the cells (such as the CD8+ cells or the T cells, e.g., CD3+ cells) are enriched for (i.e., positively selected for) cells that are positive or expressing high surface levels of CD45RO, CCR7, CD28, CD27, CD44, CD 127, and/or CD62L and/or depleted of (e.g., negatively selected for) cells that are positive for or express high surface levels of CD45RA. In some embodiments, cells are enriched for or depleted of cells positive or expressing high surface levels of CD 122, CD95, CD25, CD27, and/or IL7-Ra (CD 127). In some examples, CD8+ T cells are enriched for cells positive for CD45RO (or negative for CD45RA) and for CD62L. For example, CD3+, CD28+ T cells can be positively selected using CD3/CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3/CD28 T Cell Expander). In some embodiments, T cells are separated from a PBMC sample by negative selection of markers expressed on non-T cells, such as B cells, monocytes, or other white blood cells, such as CD 14. In some aspects, a CD4+ or CD8+ selection step is used to separate CD4+ helper and CD8+ cytotoxic T cells. Such CD4+ and CD8+ populations can be further sorted into sub- populations by positive or negative selection for markers expressed or expressed to a relatively higher degree on one or more naive, memory, and/or effector T cell subpopulations. In some embodiments, CD8+ cells are further enriched for or depleted of naive, central memory, effector memory, and/or central memory stem cells, such as by positive or negative selection based on surface antigens associated with the respective subpopulation. In some embodiments, enrichment 41744090.2
Attorney Docket No.046483-7386WO1(03225) for central memory T (TCM) cells is carried out to increase efficacy, such as to improve long- term survival, expansion, and/or engraftment following administration, which in some aspects is particularly robust in such sub-populations. In some embodiments, combining TCM-enriched CD8+ T cells and CD4+ T cells further enhances efficacy. In some embodiments, memory T cells are present in both CD62L+ and CD62L- subsets of CD8+ peripheral blood lymphocytes. PBMC can be enriched for or depleted of CD62L-CD8+ and/or CD62L+CD8+ fractions, such as using anti-CD8 and anti-CD62L antibodies. In some embodiments, a CD4+ T cell population and a CD8+ T cell sub-population, e.g., a sub- population enriched for central memory (TCM) cells. In some embodiments, the enrichment for central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and/or CD 127; in some aspects, it is based on negative selection for cells expressing or highly expressing CD45RA and/or granzyme B. In some aspects, isolation of a CD8+ population enriched for TCM cells is carried out by depletion of cells expressing CD4, CD 14, CD45RA, and positive selection or enrichment for cells expressing CD62L. In one aspect, enrichment for central memory T (TCM) cells is carried out starting with a negative fraction of cells selected based on CD4 expression, which is subjected to a negative selection based on expression of CD 14 and CD45RA, and a positive selection based on CD62L. Such selections in some aspects are carried out simultaneously and in other aspects are carried out sequentially, in either order. In some aspects, the same CD4 expression-based selection step used in preparing the CD8+ cell population or subpopulation, also is used to generate the CD4+ cell population or sub-population, such that both the positive and negative fractions from the CD4- based separation are retained and used in subsequent steps of the methods, optionally following one or more further positive or negative selection steps. CD4+ T helper cells are sorted into naive, central memory, and effector cells by identifying cell populations that have cell surface antigens. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, naive CD4+ T lymphocytes are CD45RO-, CD45RA+, CD62L+, CD4+ T cells. In some embodiments, central memory CD4+ cells are CD62L+ and CD45RO+. In some embodiments, effector CD4+ cells are CD62L- and CD45RO. In one example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CDl lb, CD16, HLA-DR, and CD8. In some embodiments, the antibody or binding partner is bound to a solid support or matrix, such as a 41744090.2
Attorney Docket No.046483-7386WO1(03225) magnetic bead or paramagnetic bead, to allow for separation of cells for positive and/or negative selection. In some embodiments, the cells are incubated and/or cultured prior to or in connection with genetic engineering. The incubation steps can include culture, cultivation, stimulation, activation, and/or propagation. In some embodiments, the compositions or cells are incubated in the presence of stimulating conditions or a stimulatory agent. Such conditions include those designed to induce proliferation, expansion, activation, and/or survival of cells in the population, to mimic antigen exposure, and/or to prime the cells for genetic engineering, such as for the introduction of a recombinant antigen receptor. The conditions can include one or more of particular media, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and/or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells. In some embodiments, the stimulating conditions or agents include one or more agent, e.g., ligand, which is capable of activating an intracellular signaling domain of a TCR complex. In some aspects, the agent turns on or initiates TCR/CD3 intracellular signaling cascade in a T cell. Such agents can include antibodies, such as those specific for a TCR component and/or costimulatory receptor, e.g., anti-CD3, anti-CD28, for example, bound to solid support such as a bead, and/or one or more cytokines. Optionally, the expansion method may further comprise the step of adding anti-CD3 and/or anti CD28 antibody to the culture medium (e.g., at a concentration of at least about 0.5 ng/ml). In some embodiments, the stimulating agents include IL-2 and/or IL-15, for example, an IL-2 concentration of at least about 10 units/mL. In another embodiment, T cells are isolated from peripheral blood by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL™ gradient. Alternatively, T cells can be isolated from an umbilical cord. In any event, a specific subpopulation of T cells can be further isolated by positive or negative selection techniques. The cord blood mononuclear cells so isolated can be depleted of cells expressing certain antigens, including, but not limited to, CD34, CD8, CD14, CD19, and CD56. Depletion of these cells can be accomplished using an isolated antibody, a biological sample comprising an antibody, such as ascites, an antibody bound to a physical support, and a cell bound antibody. 41744090.2
Attorney Docket No.046483-7386WO1(03225) Enrichment of a T cell population by negative selection can be accomplished using a combination of antibodies directed to surface markers unique to the negatively selected cells. A preferred method is cell sorting and/or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected. For example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8. For isolation of a desired population of cells by positive or negative selection, the concentration of cells and surface (e.g., particles such as beads) can be varied. In certain embodiments, it may be desirable to significantly decrease the volume in which beads and cells are mixed together (i.e., increase the concentration of cells), to ensure maximum contact of cells and beads. For example, in one embodiment, a concentration of 2 billion cells/ml is used. In one embodiment, a concentration of 1 billion cells/ml is used. In a further embodiment, greater than 100 million cells/ml is used. In a further embodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/ml is used. In yet another embodiment, a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells/ml is used. In further embodiments, concentrations of 125 or 150 million cells/ml can be used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion. T cells can also be frozen after the washing step, which does not require the monocyte- removal step. While not wishing to be bound by theory, the freeze and subsequent thaw step provides a more uniform product by removing granulocytes and to some extent monocytes in the cell population. After the washing step that removes plasma and platelets, the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and will be useful in this context, in a non-limiting example, one method involves using PBS containing 20% DMSO and 8% human serum albumin, or other suitable cell freezing media. The cells are then frozen to -80⁰C at a rate of 1⁰C per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing may be used as well as uncontrolled freezing immediately at -20⁰C or in liquid nitrogen. In one embodiment, the population of T cells is comprised within cells such as peripheral blood mononuclear cells, cord blood cells, a purified population of T cells, and a T cell line. In 41744090.2
Attorney Docket No.046483-7386WO1(03225) another embodiment, peripheral blood mononuclear cells comprise the population of T cells. In yet another embodiment, purified T cells comprise the population of T cells. In certain embodiments, T regulatory cells (Tregs) can be isolated from a sample. The sample can include, but is not limited to, umbilical cord blood or peripheral blood. In certain embodiments, the Tregs are isolated by flow-cytometry sorting. The sample can be enriched for Tregs prior to isolation by any means known in the art. The isolated Tregs can be cryopreserved, and/or expanded prior to use. Methods for isolating Tregs are described in U.S. Patent Numbers: 7,754,482, 8,722,400, and 9,555,105, and U.S. Patent Application No.13/639,927, contents of which are incorporated herein in their entirety. H. Expansion of Immune Cells Whether prior to or after modification of cells to express a CAR and CH25H, the cells can be activated and expanded in number using methods as described, for example, in U.S. Patent Nos.6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681 ; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Publication No.20060121005. For example, the T cells of the invention may be expanded by contact with a surface having attached thereto an agent that stimulates a CD3/TCR complex associated signal and a ligand that stimulates a co-stimulatory molecule on the surface of the T cells. In particular, T cell populations may be stimulated by contact with an anti-CD3 antibody, or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore. For co-stimulation of an accessory molecule on the surface of the T cells, a ligand that binds the accessory molecule is used. For example, T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions appropriate for stimulating proliferation of the T cells. Examples of an anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France) and these can be used in the invention, as can other methods and reagents known in the art (see, e.g., ten Berge et al., Transplant Proc. (1998) 30(8): 3975-3977; Haanen et al., J. Exp. Med. (1999) 190(9): 1319-1328; and Garland et al., J. Immunol. Methods (1999) 227(1-2): 53-63). Expanding T cells by the methods disclosed herein can be multiplied by about 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold, 200 fold, 300 fold, 400 41744090.2
Attorney Docket No.046483-7386WO1(03225) fold, 500 fold, 600 fold, 700 fold, 800 fold, 900 fold, 1000 fold, 2000 fold, 3000 fold, 4000 fold, 5000 fold, 6000 fold, 7000 fold, 8000 fold, 9000 fold, 10,000 fold, 100,000 fold, 1,000,000 fold, 10,000,000 fold, or greater, and any and all whole or partial integers therebetween. In one embodiment, the T cells expand in the range of about 20 fold to about 50 fold. Following culturing, the T cells can be incubated in cell medium in a culture apparatus for a period of time or until the cells reach confluency or high cell density for optimal passage before passing the cells to another culture apparatus. The culturing apparatus can be of any culture apparatus commonly used for culturing cells in vitro. Preferably, the level of confluence is 70% or greater before passing the cells to another culture apparatus. More preferably, the level of confluence is 90% or greater. A period of time can be any time suitable for the culture of cells in vitro. The T cell medium may be replaced during the culture of the T cells at any time. Preferably, the T cell medium is replaced about every 2 to 3 days. The T cells are then harvested from the culture apparatus whereupon the T cells can be used immediately or cryopreserved to be stored for use at a later time. In one embodiment, the invention includes cryopreserving the expanded T cells. The cryopreserved T cells are thawed prior to introducing nucleic acids into the T cell. In another embodiment, the method comprises isolating T cells and expanding the T cells. In another embodiment, the invention further comprises cryopreserving the T cells prior to expansion. In yet another embodiment, the cryopreserved T cells are thawed for electroporation with the RNA encoding the chimeric membrane protein. Another procedure for ex vivo expansion cells is described in U.S. Pat. No.5,199,942 (incorporated herein by reference). Expansion, such as described in U.S. Pat. No.5,199,942 can be an alternative or in addition to other methods of expansion described herein. Briefly, ex vivo culture and expansion of T cells comprises the addition to the cellular growth factors, such as those described in U.S. Pat. No.5,199,942, or other factors, such as flt3-L, IL-1, IL-3 and c-kit ligand. In one embodiment, expanding the T cells comprises culturing the T cells with a factor selected from the group consisting of flt3-L, IL-1, IL-3 and c-kit ligand. The culturing step as described herein (contact with agents as described herein or after electroporation) can be very short, for example less than 24 hours such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours. The culturing step as described 41744090.2
Attorney Docket No.046483-7386WO1(03225) further herein (contact with agents as described herein) can be longer, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more days. Various terms are used to describe cells in culture. Cell culture refers generally to cells taken from a living organism and grown under controlled condition. A primary cell culture is a culture of cells, tissues or organs taken directly from an organism and before the first subculture. Cells are expanded in culture when they are placed in a growth medium under conditions that facilitate cell growth and/or division, resulting in a larger population of the cells. When cells are expanded in culture, the rate of cell proliferation is typically measured by the amount of time required for the cells to double in number, otherwise known as the doubling time. Each round of subculturing is referred to as a passage. When cells are subcultured, they are referred to as having been passaged. A specific population of cells, or a cell line, is sometimes referred to or characterized by the number of times it has been passaged. For example, a cultured cell population that has been passaged ten times may be referred to as a P10 culture. The primary culture, i.e., the first culture following the isolation of cells from tissue, is designated P0. Following the first subculture, the cells are described as a secondary culture (P1 or passage 1). After the second subculture, the cells become a tertiary culture (P2 or passage 2), and so on. It will be understood by those of skill in the art that there may be many population doublings during the period of passaging; therefore the number of population doublings of a culture is greater than the passage number. The expansion of cells (i.e., the number of population doublings) during the period between passaging depends on many factors, including but is not limited to the seeding density, substrate, medium, and time between passaging. In one embodiment, the cells may be cultured for several hours (about 3 hours) to about 14 days or any hourly integer value in between. Conditions appropriate for T cell culture include an appropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 15, (Lonza)) that may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-gamma, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGF-beta, and TNF-α or any other additives for the growth of cells known to the skilled artisan. Other additives for the growth of cells include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol. Media can include RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, and vitamins, either serum-free or 41744090.2
Attorney Docket No.046483-7386WO1(03225) supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and/or an amount of cytokine(s) sufficient for the growth and expansion of T cells. Antibiotics, e.g., penicillin and streptomycin, are included only in experimental cultures, not in cultures of cells that are to be infused into a subject. The target cells are maintained under conditions necessary to support growth, for example, an appropriate temperature (e.g., 37°C) and atmosphere (e.g., air plus 5% CO2). The medium used to culture the T cells may include an agent that can co-stimulate the T cells. For example, an agent that can stimulate CD3 is an antibody to CD3, and an agent that can stimulate CD28 is an antibody to CD28. A cell isolated by the methods disclosed herein can be expanded approximately 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold, 200 fold, 300 fold, 400 fold, 500 fold, 600 fold, 700 fold, 800 fold, 900 fold, 1000 fold, 2000 fold, 3000 fold, 4000 fold, 5000 fold, 6000 fold, 7000 fold, 8000 fold, 9000 fold, 10,000 fold, 100,000 fold, 1,000,000 fold, 10,000,000 fold, or greater. In one embodiment, the T cells expand in the range of about 20 fold to about 50 fold, or more. In one embodiment, human T regulatory cells are expanded via anti-CD3 antibody coated KT64.86 artificial antigen presenting cells (aAPCs). Methods for expanding and activating T cells can be found in U.S. Patent Numbers: 7,754,482, 8,722,400, and 9,555, 105, contents of which are incorporated herein in their entirety. In one embodiment, the method of expanding the T cells can further comprise isolating the expanded T cells for further applications. In another embodiment, the method of expanding can further comprise a subsequent electroporation of the expanded T cells followed by culturing. The subsequent electroporation may include introducing a nucleic acid encoding an agent, such as a transducing the expanded T cells, transfecting the expanded T cells, or electroporating the expanded T cells with a nucleic acid, into the expanded population of T cells, wherein the agent further stimulates the T cell. The agent may stimulate the T cells, such as by stimulating further expansion, effector function, or another T cell function. I. Pharmaceutical Compositions Also provided are composition comprising populations modified cells (e.g., CAR T cells comprising CH25H) for use in immunotherapy. The population of cells can be generated by any of the methods contemplated herein. Among the compositions are pharmaceutical compositions 41744090.2
Attorney Docket No.046483-7386WO1(03225) and formulations for administration, such as for adoptive cell therapy. Also provided are therapeutic methods for administering the cells and compositions to subjects, e.g., patients. Also provided are compositions including the cells for administration, including pharmaceutical compositions and formulations, such as unit dose form compositions including the number of cells for administration in a given dose or fraction thereof. The pharmaceutical compositions and formulations generally include one or more optional pharmaceutically acceptable carrier or excipient. In some embodiments, the composition includes at least one additional therapeutic agent. The term "pharmaceutical formulation" refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative. In some aspects, the choice of carrier is determined in part by the particular cell and/or by the method of administration. Accordingly, there are a variety of suitable formulations. For example, the pharmaceutical composition can contain preservatives. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Carriers are described, e.g., by Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other 41744090.2
Attorney Docket No.046483-7386WO1(03225) carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG). Buffering agents in some aspects are included in the compositions. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffering agents is used. The buffering agent or mixtures thereof are typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail in, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005). The formulations can include aqueous solutions. The formulation or composition may also contain more than one active ingredient useful for the particular indication, disease, or condition being treated with the cells, preferably those with activities complementary to the cells, where the respective activities do not adversely affect one another. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended. Thus, in some embodiments, the pharmaceutical composition further includes other pharmaceutically active agents or drugs, such as chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, and/or vincristine. The pharmaceutical composition in some embodiments contains the cells in amounts effective to treat or prevent the disease or condition, such as a therapeutically effective or prophylactically effective amount. Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of treated subjects. The desired dosage can be delivered by a single bolus administration of the cells, by multiple bolus administrations of the cells, or by continuous infusion administration of the cells. Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, the cell populations are administered parenterally. The term "parenteral," as used herein, includes intravenous, intramuscular, subcutaneous, rectal, 41744090.2
Attorney Docket No.046483-7386WO1(03225) vaginal, and intraperitoneal administration. In some embodiments, the cells are administered to the subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection. Compositions in some embodiments are provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may in some aspects be buffered to a selected pH. Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyoi (for example, glycerol, propylene glycol, liquid polyethylene glycol) and suitable mixtures thereof. Sterile injectable solutions can be prepared by incorporating the cells in a solvent, such as in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, and/or colors, depending upon the route of administration and the preparation desired. Standard texts may in some aspects be consulted to prepare suitable preparations. Various additives which enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, and sorbic acid. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin. The formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes. The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited herein, are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to 41744090.2
Attorney Docket No.046483-7386WO1(03225) physically incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other physical and electronic documents. While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods described herein may be made using suitable equivalents without departing from the scope of the embodiments disclosed herein. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto. Having now described certain embodiments in detail, the same will be more clearly understood by reference to the following examples, which are included for purposes of illustration only and are not intended to be limiting. EXPERIMENTAL EXAMPLES The invention is now described with reference to the following Examples. These Examples are provided for the purpose of illustration only, and the invention is not limited to these Examples, but rather encompasses all variations that are evident as a result of the teachings provided herein. Effector trogocytosis between malignant cells and tumor-specific cytotoxic T lymphocytes (CTLs) contributes to immune evasion through antigen loss on target cells and killing of antigen-experienced CTLs by other CTLs (i.e., fratricide). The mechanisms regulating these events in tumors remain poorly understood. Herein, it was demonstrated that tumor-derived factors (TDFs) act to stimulate effector trogocytosis and restrict CTLs’ tumoricidal activity and viability. TDFs robustly altered the CTL’s lipid profile including depletion of 25- hydroxycholesterol (25HC).25HC inhibited trogocytosis and prevented CTL’s inactivation and fratricide. Mechanistically, TDFs induced ATF3 transcription factor that suppressed the expression of 25HC-regulating gene - cholesterol 25-hydroxylase (CH25H). Stimulation of trogocytosis in the intratumoral CTL by the ATF3-CH25H axis attenuated anti-tumor immunity, stimulated tumor growth, and impeded the efficacy of chimeric antigen receptor (CAR) T cell adoptive therapy. Through use of armored CAR constructs or pharmacologic agents restoring 41744090.2
Attorney Docket No.046483-7386WO1(03225) CH25H expression these phenotypes were reversed and the efficacy of immunotherapies improved. Materials and Methods: Human databases analysis: Kendall Correlation analysis between CD8A expression, which is used for measuring CD8+ T cell presence in the tumor microenvironment, and CH25H expression in human cancer tissues from GENT2, was performed. Weak positive association between CD8A and CH25H was generated using the ggpubr (ggpubr: 'ggplot2' Based Publication Ready Plots. R package version 0.4.0.) and ggplot2 (ggplot2: Elegant Graphics for Data Analysis. Springer-Verlag New York, 2016) packages in the graphical and statistical software R (R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria). Timer 2.0 (Li et al., (2020) Nucleic Acids Res 48, W509-W514) was used to analyze the correlation between immune cell infiltration and Ch25h expression in human melanoma. The expression of Ch25h in T cells was analyzed while controlling for age, gender, and disease stage. Those tumors that showed a survival benefit associated with increased Ch25h expression in T cells were then further analyzed for tumor purity and Ch25h expression. Animal studies: NSG, WT, Ch25h-/-, Rag1-/-, Cd8 Cre and OT-I mice were purchased from Jackson Laboratory. The conditional Ch25h allele was created by flanking the single exon of the Ch25h gene with the loxP sites inserted into the non-conservative regions (Lu et al., (2021) Nature 588, 693-698). OT-I WT and OT-I Ch25h-/- mice were generated by crossing OT- I mice with WT or Ch25h-/- mice. Atf3f/f mice were previously described (Wolford et al., (2013) J Clin Invest 123, 2893-2906) and kindly provided by Dr. Tsonwin Hai (Ohio State University). Ch25h∆CD8 or Atf3∆CD8 or Ch25h;Atf3∆CD8 mice were generated by intercrossing Cd8-Cre mice with conditional Ch25hf/f or Atf3f/f mice. All animal experiments were performed using both male and female littermates of 6-8 weeks of age. All mice except NSG were in the C57BL/6J background. Cell culture, intestinal epithelial cell medium (IECM) and Tumor-conditioned medium (TCM) preparation: Human 293T cell and B16F10 cell were purchased from ATCC. NALM6 acute lymphoblastic leukemia (from Michael C. Milone, University of Pennsylvania), MC38/MC38OVA colon adenocarcinoma (from Susan Ostrand-Rosenberg, University of 41744090.2
Attorney Docket No.046483-7386WO1(03225) Maryland), B16F10-hCD19 melanoma (B16F10 melanoma stably expressing human CD19 - from Andy Minn, University of Pennsylvania), human EM-Meso-GFP-Luc cell line (from Edmund Moon, University of Pennsylvania), MH6499c4 pancreatic ductal adenocarcinoma (from Ben Stanger, University of Pennsylvania) and Hepa1-6 hepatocellular carcinoma (from Malay Haldar, University of Pennsylvania) were kindly gifted. B16F10-hCD19-luc melanoma cells were generated by stable expression of luciferase in B16F10-hCD19 cells.293T cell and all cancer cells were cultured at 37°C with 5% CO2 in DMEM including 10% heat-inactivated Fetal Bovine Serum (FBS), 100U/ml penicillin-streptomycin and L-glutamine. Phoenix packaging cells (from ATCC) were maintained at 37°C with 5% CO2 in RPMI-1640 including 10% heat- inactivated Fetal Bovine Serum (FBS), 50U/ml penicillin-streptomycin and L-glutamine. Tumor- condition medium (TCM) and NIH3T3 fibroblast-conditioned media (FCM) was prepared as previously reported (Gui et al., (2020) Nat Cancer 1, 603-619). Mouse intestinal epithelial cells were obtained and cultured as previously described (Katlinskaya et al., (2016) Mol Cell Biol 36, 1124-1135). Briefly, they were scraped gently using rubber blade and cultured in Advanced DMEM/F12 medium containing 10mM HEPES, 1XPenicillin-Streptomycin, 2 mM GlutaMAX, 1XN2 supplement, 50ng/ml mEGF, and 1mg/ml Rspondin for 12 hr. After that, complementary medium was replaced with serum-free DMEM/F12 medium for 24 hr and the medium was collected for following study. CAR T cells: vector generation, T cell isolation, expansion, and CAR T cell generation: The second generation anti-MESO CAR lentiviral construct, which included mCherry reporter, CD8 hinge, meso scFv, 4-1BB and CD3zeta signaling domains (described in Milone et al., (2009) Mol Ther 17, 1453-1464) was a gift from Michael C. Milone (University of Pennsylvania). For generation of the anti-Meso-Ch25h CAR plasmid, a cassette encoding a 54 nucleotide self-cleaving peptide (T2A) positioned 5’ of Ch25h was commercially manufactured (Genscript, NJ) with 5’ AvrII and 3’ SalI restriction sites flanking the sequence and inserted into this vector as shown in FIG.7A. A similar strategy was employed to modify the anti-CD19 CAR retrovirus backbone consisting of a CD8 hinge, CD19 scFv, 4-1BB and CD3zeta signaling domains (obtained from Andy J. Minn, University of Pennsylvania). The final constructs were verified using AvrII and SalI restriction digests and Sanger sequencing of the T2A-Ch25h insert using the final anti-MESO-Ch25h or anti-CD19-Ch25h constructs as the templates. 41744090.2
Attorney Docket No.046483-7386WO1(03225) Mouse T cells or CD8+ T cells were isolated from spleens of different genetic mice with T cells (Stem cell, cat#19851) or CD8+ T cells (Stem cell, cat#19853). Human T cells were isolated from heathy donors by the Human Immunology Core at the University of Pennsylvania. T cells were stimulated with Dynabeads Mouse T-Activator CD3/CD28 beads (Gibco, cat#11453D) or Dynabeads Human T-Activator CD3/CD28 beads (Gibco, cat#11132D) for 48 hr and were separated from beads prior to transduction. Activated T cells were transduced with viruses at a titer of MOI=3000 for 48 hr. Transduction efficient, expression of anti-MESO and anti-CD19 CARs and expression of Ch25h were assessed using flow cytometry and quantitative RT-PCR (qPCR) analysis, respectively. Cytotoxicity, Viability and Exhaustion assays: OT-I-WT or -Ch25h-/- splenocytes were in vitro stimulated with OVA peptide (0.5μg/ml for 48 hr) followed by treatment with vehicle or FCM or TCM or tumor derived factors such as VEGF165 or Prostaglandin E2 in the presence or absence of 25HC (4μM, Sigma, cat#H1015) for 8 hr. Then, these differentially treated-CTLs were co-cultured with target MC38OVA cells expressing luciferase at indicated E/T ratio for 4 hr. For CAR T cells setting, anti-MESO or anti-CD19 CAR T cells were generated and treated with vehicle or 25HC at 4μM for 8 hr. After that, CAR T cells were co-cultured with target EM- Meso-GFP-Luc or B16F10-hCD19-luc cells at indicated E/T ratios for 4 hr followed by the luciferase-based cytotoxicity assay. For the luciferase-based cytotoxicity assay, target cells alone were set as negative control with spontaneous death RLU and target cells with water were set as positive control with maximal killing RLU. After 4 hr co-culture, 100 μl of luciferase substrate (Promega Bright-Glo, cat# E6120) was added to each well containing CTLs and target cells. After 10 min incubation, luminescence was measured using the EnVision (PerkinElmer) plate reader. The percentage of killing was calculated using the formula: % lysis=100 X (spontaneous death RLU- tested RLU)/(spontaneous death RLU- maximal killing RLU). For the viability and exhaustion assay, OT-I or CAR T CTLs were co-cultured with respective target cells for 2, 4, 8 or 16 hr, washed once with PBS once and analyzed by flow cytometry using appropriate antibodies such as: anti-CD8-APC/Cy7(BioLegend, cat#100714), anti-CD69-PE (BioLegend, cat# 104507), anti-PD-1-BV605 (BioLegend, cat#135219), anti- LAG3-PE/Cy7 (BioLegend, cat#125226), anti-CD336-BV421 (BioLegend, cat#134019) and 41744090.2
Attorney Docket No.046483-7386WO1(03225) anti-Annexin V-FITC (BioLegend, cat#640906). Following staining, cells were washed with FACS buffer and mixed with counting beads (Invitrogen, cat#C36950) followed by flow cytometry analysis. Flow cytometry of tumors or spleens immune profile, immunofluorescence, immunoprecipitation and immunoblotting: For solid tumors, tumor tissue was dissected and digested with 1 mg/ml Collagenase D (Roche, cat#11088882001) plus with 100 μg/ml DNase I (Roche, cat#10104159001) in RPMI medium with 2% FBS for 1 hr with continuous agitation at 37 °C. Digestion mixture was passed through 100 μm cell strainer to prepare single cell suspensions and washed with PBS supplemented with 2mM EDTA and 1% FBS. Single cells were stained with cell surface antibodies: anti-CD45-APC/Cy7 (BioLegend, cat#157204), anti- CD3-PE (BioLegend, cat#100206), anti-CD8-AF700 (BioLegend, cat#100730), anti- CD69- BV421 (BioLgend, cat#104545, anti-PD-1-PE/Cy7 (BioLegend, cat#109110), anti-LAG3- BV650 (BioLegend, cat#125227), anti-CD336-BV421 (BioLegend, cat#134019) and Annexin V-APC (BioLegend, cat#640941). Following staining, cells were washed with PBS and subjected to flow cytometry analysis. For leukemia model, mouse blood was collected through retro-orbital bleeding and red cells were removed using RBC lysis buffer (BioLegend, cat#420302). After washed with PBS, cells were stained with anti-hCD19 (Biolegend, cat#392504), anti-human CD19 CAR (Acrobiosystems, cat#FM3-HPY53), anti-human CD8 (BioLegend, cat#344750) on the ice for 30 min. Following staining, cells were washed with PBS and subjected to flow cytometry analysis. For spleen immune profile assay, spleens were ground up and passed through 40μm cell strainer. Red cells were removed using RBC buffer. After that, single cells were incubated with anti-CD8-AF700 (Biolegnd, cat#100714), anti-CD4 (BioLegend, cat#100414), anti-CD19 (BioLegend, cat#115507), anti-NK1.1 (Biolegend, cat#108709), anti-CD69, anti-PD-1 and anti- Annexin V antibodies. For immunofluorescence analyses, the samples were prepared as previous described (Lu et al., (2021) J Clin Invest 131) and the Biotin anti-mouse CD3 antibody (Biolegend, cat#100243; 1:100), Biotin anti-mouse CD8 antibody (Biolegend, cat#100704), Alexa Fluor-594 streptavidin antibody (Biolegend,cat# 405240; 1:500), and Alexa Fluor-488 anti-mouse (Biolegend, 100723; 1:100) were used. 41744090.2
Attorney Docket No.046483-7386WO1(03225) For analysis of ATF3 sumoylation, OT-I WT T cells were isolated and stimulated with OVA peptide (0.5μg/ml for 48 hr) followed by treatment with Vehicle or TCM in the presence or absence of TAK981 (0.1μM) for 12 hr. Cells were lysed, and 1 mg of pre-cleared protein lysates from each sample were taken into immunoprecipitation using rabbit anti-mouse ATF3 antibody (Cell Signaling Technology, cat#18665S). Immunoprecipitation was carried out as previously described (Huangfu et al., (2012) Oncogene 31, 161-172). The resulting immune-precipitates were eluted with glycine-containing buffer (pH 2.6) and analyzed by immunoblot using primary antibodies against SUMO1 (Invitrogen, cat#33-2400, 3μg/ml), ATF3 (CST cat#18665S) and secondary antibodies (VeriBlot for IP Detection Reagent HRP, abcam, cat#ab131366, 1:200). Loading in initial samples was controlled by direct immunoblot using antibody against β-tubulin (Cell Signaling Technology, cat# 2146, 1:1000) and goat anti-mouse HRP-conjugated antibody (Cell Signaling Technology, cat#7076S, 1:5000). Flow cytometry analysis of trogocytosis: OT-I and CAR T settings were used to assess the extent of trogocytosis. In OT-I settings, splenocytes isolated from OT-I WT (Ch25h+/+) or OT-I Ch25h-/- mice were stimulated with OVA peptide (0.5μg/ml for 48 hr) followed by treatment with Vehicle, IECM, PGE2 (Sigma, cat#P0409, 10nM), VEGF165 (Sigma, cat#V5765, 50ng/ml) or TCM in the presence or absence of 25HC (Sigma, cat#H1015, 4μM) or GW3965 (Sigma, cat# G6295, 2μM) for 8 hr as indicated. A total of 1x105 treated-CTLs were co-cultured with DiD-labeled target cells in 96-well plates at a 5:1 of ratio for indicated times. For DiD label, MC38 or MC38OVA cells were stained with 0.3μM DiD (1,1′-dioctadecyl- 3,3,3′,3′- tetramethylindodicarbocyanine, Biotium, cat#60014) at 37oC for 10 min followed by PBS washes for 3 times. After co-culture, cells were washed with PBS and stained with anti- CD8-APC/Cy7 (BioLegend, cat#100714), PE-anti-mouse H-2Kb bounds to SIINFEKL (SEQ ID NO:33) antibody (BioLegend, cat#141603) on ice for 30 min. Following staining, cells were washed with FACS buffer and subjected to flow cytometry. Trogocytosis was tested by the acquisition of either DiD or OVA-MHC-I complexes by the CD8+ CTLs. In the CAR T setting, a total of 1x105 CAR T cells were co-cultured with target cells at a 1:2 ratio for 4 hr. After co-culture, cells were washed with PBS and stained with anti-CD8- APC/Cy7, anti-CD19 CAR and anti-hCD19 on the ice for 30 min. Following staining, cells were washed with FACS buffer and subjected to flow cytometry. Trogocytosis was tested by the 41744090.2
Attorney Docket No.046483-7386WO1(03225) acquisition of either MESO or hCD19 antigens by the CAR T cells and the loss of the CD19 antigen on target tumor cells. For the inhibition of trogocytosis, CTLs or CAR T cells were pre-treated with vehicle of latrunculin A (1μM, Sigma-Aldrich, cat#L5163) at 37°C for 20 min before co-incubation with target cells. In the Transwell setting, parental or DiD-labeled MC38 were seeded into the upper portion of Transwell chambers (Millipore, cat# CLS3422-48EA) and T cells were seeded on the bottom of these plates. Quantitative Real-time PCR: Total RNA of CD8+ T cells which was treated with Vehicle or TCM or isolated from tumor tissue was extracted using RNA isolation kit (Qiagen, cat#74004). Concentrations of RNA were measured by nanodrop2000 and the mRNA expression of Ch25h and Atf3 were tested using real-time PCR. Primers are follows: Ch25h forward: TGCTACAACGGTTCGGAGC (SEQ ID N: 34) and Reverse: AGAAGCCCACGTAAGTGATGAT (SEQ ID NO: 35); Atf3 forward: TTACCGTCAACAACAGACCC (SEQ ID NO: 36) and Reverse: TCAGCTCAGCATTCACACTC (SEQ ID NO: 37); AbcA1 forward: AGTGATAATCAAAGTCAAAGGCACAC (SEQ ID NO:38) and Reverse: AGCAACTTGGCACTAGTAACTCTG (SEQ ID NO:39); AbcG1 Forward: TTCATCGTCCTGGGCATCTT (SEQ ID NO:40) and Reverse: CGGATTTTGTATCTGAGGACGAA (SEQ ID NO:41); ApoE Forward: ACAGATCAGCTCGAGTGGCAAA (SEQ ID NO:42) and Reverse: ATCTTGCGCAGGTGTGTGGAGA (SEQ ID NO:43); Lbp Forward: GATCACCGACAAGGGCCTG (SEQ ID NO:44) and Reverse: GGCTATGAAACTCGTACTGCC (SEQ ID NO:45). Chromatin immunoprecipitation (ChIP) analysis: OT-I WT T cells were isolated from spleen of OT-I WT mice and stimulated with OVA (0.5μg/ml) for 48 hr followed by treatment with FCM or TCM for 12hr. T cells were harvested and the Immunoprecipitation (IP) was conducted following instruction from SimpleChIP Plus Enzymatic Chromatin IP Kit (Cell signaling technology, cat# 9005S). ATF3 antibody used for the IP was Rabbit anti-mouse ATF3 antibody (Cell signaling technology, cat#18665S, 1:50). The ChIP-qPCR for the Ch25h promoter used the following primers: forward 5-TAGCAGCCCATGCTGAGACTATGT-3 (SEQ ID NO:46) and reverse primer, 5 TTCTTTAGCAGGGAAAGGGAGGTG-3 (SEQ ID NO:47). 41744090.2
Attorney Docket No.046483-7386WO1(03225) Tumorigenesis studies: For the syngeneic subcutaneous tumor model, B16F10 (1x106), MC38 (1x106), MH6499c4 (1x106) and Hepa1-6 (2x106) were s.c inoculated into right flanks of mice and tumor sizes were measured every other day using calipers. Tumor volume was calculated as width x width x length x 0.5 and mice survival was tracked until tumor volume reached ~1000 mm3. Combination therapy and CAR T therapies: For the therapeutic combination of TAK981 with PD-1, MC38 (1x106) cells were suspended into 100 μl PBS and inoculated into the right flank of Ch25hf/f and Ch25h∆CD8 mice at day 0. TAK981 was dissolved in 20% hydroxypropyl beta-cyclodextrin and formulated as previously described (Lightcap et al., (2021) Sci Transl Med 13, eaba7791). Vehicle or anti-PD-1 (i.p, 5mg/kg every 4 days) or TAK981 (i.v, 15mg/kg, twice a week) or combination were injected into tumor-bearing mice when the tumor volume reached ~50-80 mm3. Tumor tissues were collected at day 25 for immune profiling analysis. For the survival analysis, mice were euthanized when tumor volume reached ~1000 mm3. For anti-MESO/anti-MESO-Ch25h CAR T therapy, human EM-Meso-GFP-Luc cells (s.c., 1x106) were inoculated into the right flank of NSG mice at day 0. Human anti-MESO-CAR or anti-MESO-Ch25h CAR T cells (generated as described herein) were i.v. injected into tumor bearing mice at a dose of 1x106/mouse at days 16, 23, 30 and 37; PBS was used as control. Tumor tissue was collected at day 45 for immune profiling. For the survival analysis, mice were euthanized when tumor volume reached ~1000 mm3. For anti-CD19/anti-CD19-Ch25h CAR T therapy, B16F10-hCD19 cells (s.c, 0.3x106) were inoculated into the right flank of Rag1-/- mice at day 0. Mouse anti-CD19 CAR or anti- CD19-Ch25h CAR T cells were generated and i.v. injected into tumor bearing mice at dose of 2x106/mouse at days 7 and 13. Tumor tissues were digested at day 19 for immune profiling analysis. For the survival analysis, mice were euthanized when tumor volume reached ~1000 mm3. For the CAR T therapy of acute lymphoblastic leukemia model, GFP+NALM6 cancer cells were inoculated into NSG mice (i.v, 1x106) at day 0. Human anti-CD19 CAR or anti-CD19- Ch25h CAR T cells were injected (1x106) into tumor bearing mice at day 10, and PBS injection was used as the control group. Peripheral blood was collected via retro-orbital bleeding at days 17, 24 and 31 and lymphocytes were used for flow cytometric analysis. For the survival analysis, mice were euthanized when they became moribund. 41744090.2
Attorney Docket No.046483-7386WO1(03225) Trogocytosis and viability assay in vivo: MC38 or MC38OVA cells were inoculated into the right flanks of Rag1-/- mice (1x106, s.c). WT OT-I and Ch25h-/- OT-I splenocytes were stimulated in vitro with OVA peptide (0.5μg/ml for 48 hr). After stimulation, Ch25h-/- CTLs were stained with CFSE (1nM, Biolegend, cat#79898) at 37°C for 10 min followed by 3 washes with PBS. Following the washes, WT CTLs (1x106, CFSE negative) were mixed with CFSE- labeled Ch25h-/- CTLs at a ratio of 1:1 and re-suspended in 50μl PBS. This cell mixture was injected into MC38 or MC38OVA tumors. Twenty-four hr after injection, tumor tissues were digested into single cell suspensions; and percentages of CD8+CFSE+, CD8+CFSE-, OVA- MHCI+CFSE+ and OVA-MHC+CFSE- cells were analyzed using flow cytometry. For analysis of the effects of TAK981 on trogocytosis, 1x106 MC38-OVA cells were injected (s.c) into right flanks of Rag1-/- mice.13 days after tumor inoculation, tumor bearing mice received OT1 CD8+ T cells injection (2x107/mouse, intratumorally) and TAK981 injection (15mg/kg, i.v) simultaneously.24 hr after injection, tumor tissues were harvested and trogocytosis and T cells exhaustion were analyzed with flow cytometry. RNA sequencing analysis: WT OT-I or Ch25h-/- OT-I splenocytes were stimulated with OVA peptide (0.5 μg/ml for 48 hr) followed by treatment with FCM or TCM for 8 hr in vitro. Total RNA was extracted with RNeasy Plus Mini Kit (QIAGEN). These samples were then used for RNA sequencing carried out as previously described (Amorim et al., (2019) Sci Transl Med 11). The adaptor-trimmed reads were aligned to the mouse genome mm10 by STAR v.2.7.2d with default parameters. Expression levels for each gene were counted by VERSE v.0.1.4. Normalization and differential expression (DE) analysis was conducted by DESeq2 v.1.28.1 using Vehicle-treated group as the control. Significantly DE genes were defined as those with Bonferroni adjusted P-values < 0.01 and log2 (fold change) > 0.58 or < -0.58. Pathway enrichment analysis involved the statistical identification of particular biological function/process categories that were overrepresented in the specified gene collection. Total DE or down-/ up-regulated genes were submitted to the Kyoto Encyclopedia of Genes and Genomes, respectively. Significantly enriched pathways (P-value < 0.05) were determined through gene set enrichment analysis integrated in the R/Bioconductor package cluster Profiler v.3.14.3. Seven KEGG pathways (‘00100’, ‘00120’, ‘00900’, ‘04975’, ‘04976’, ‘04979’, ‘05417’) were defined as the cholesterol related collection, of which P-values and fold changes were used for 41744090.2
Attorney Docket No.046483-7386WO1(03225) downstream heatmap plotting. Data have been submitted to Gene Expression Omnibus (GSE190702). Metabolomics studies: WT OT-I+ or Ch25h-/- splenocytes were stimulated with OVA peptide(0.5μg/ml) for 48 hr followed by treatment with Vehicle or TCM for 8 hr in vitro. These cells then were spun down at 250g for 5 min followed by washing with 1ml PBS twice. After this, cell pellets were suspended in 1 mL of ice-cold (-48 °C) 80% (v/v) methanol: water followed by centrifuging the solution at -9°C, 11,500 g for 10 min. Finally, cell pellets were frozen in liquid nitrogen for 30s and stored until analyzed. Samples were extracted using a biphasic extraction (Tambellini et al., (2013) Metabolites 3, 592-605) and then subjected to lipidomics analysis by spotting on a 24 well PTFE slide and analyzed by desorption electrospray ionization (Takats et al., (2004) Science 306, 471-473), on a Waters Xevo G2-XS qtof instrument. Pixels from technical replicates were averaged and 8564 features were detected. A targeted analysis was performed for 25HC m/z 385.3465, representing the [M+H-H20]+ ion of hydroxysterols (DeBarber et al., (2008) Anal Biochem 381, 151-153). Quantification and statistical analysis: All experiments described herein are the representative of at least three independent experiments (n≥5 mice for each group unless specifically indicated). For in vitro experiments, cells or tissues from each of these animals were processed (at least) in biological triplicates. All data were shown as average ± S.E.M. Statistical analysis between two groups was conducted with 2-tailed Student t test and multiple comparisons were performed using one-way ANOVA or two-way ANOVA analysis with Tukey’s multiple-comparison. Tumor growth curve analysis was conducted with repeated- measure two-way ANOVA (mixed-model) with Tukey’s multiple-comparison. The Kaplan- Meier curves were used to analyze the survival data, and Cox regression was used to compute hazard ratio. P values < 0.05 were considered significant. The results of the experiments are now described: Example 1: Tumor-derived factors (TDFs) downregulate 25-hydroxycholesterol and stimulate trogocytosis between effector CTLs and malignant cells Effector trogocytosis that transfers antigenic complexes to specific CTLs from malignant cells, undermines their killing due to antigen loss and CTL fratricide. Mechanisms regulating the extent of effector trogocytosis are not well understood. Factors present in the tumor 41744090.2
Attorney Docket No.046483-7386WO1(03225) microenvironment and how they affect the extent of trogocytosis were analyzed herein (Figure 8A). CTLs were exposed to tumor-derived factors (TDFs) produced by MC38 colon adenocarcinoma cells and present within the tumor cell-conditioned media (TCM), as well as purified factors such as prostaglandin E2 (PGE2) and vascular endothelial growth factor (VEGF), which are known to contribute to the immune suppressive properties of the tumor microenvironment. As controls, analogous treatment with vehicle, or media conditioned by fibroblasts (FCM) or by primary mouse intestinal epithelial cells (IECM), were used. OT-I CTLs were pretreated with these agents prior to incubating these CTLs with OVA- expressing MC38 colon adenocarcinoma cells labeled with DiD lipophilic dye. An increased transfer of DiD from MC38OVA onto co-incubated OT-I CTLs was detected after their exposure to TCM, PGE2 or VEGF (Figures 1A, 8B). This transfer was likely a result of trogocytosis because it was notably inhibited by the loss of cell-cell contact due to separation of CTLs and MC38OVA cells in a Transwell setting (Figure 8C). Furthermore, treatment of CTLs with inhibitor of actin polymerization Latrunculin A (previously shown to inhibit trogocytosis) or use of parental MC38 cells lacking OVA antigen, notably inhibited DiD transfer onto CTLs (Figure 8C-8D) indicating that this antigen-dependent transfer stimulated by the tumor-derived factors (TDFs) was a result of trogocytosis. To delineate the mechanisms underlying TDF-dependent increase in effector trogocytosis, gene expression was profiled in CTLs treated with TCM. This treatment altered expression of genes involved in several key biological processes including cholesterol metabolism pathway, which was notably suppressed (Figure 1B). Cholesterol controls many crucially important properties of the lipid bilayers of biological membranes such as their compressibility, fluidity, intrinsic curvature and propensity for transfer and fusion. Given that these characteristics are instrumental for trogocytosis, this pathway was focused on. Among the genes involved in cholesterol metabolism, cholesterol 25-hydroxylase (Ch25h) was the top downregulated gene (Figure 8E). Furthermore, expression of Ch25h was notably decreased among all genes affected by TCM (Figures 1C-1D). CH25H was focused on because of its enzymatic role in production of 25HC.25HC is known to alter the fluidity of lipid membranes and to inhibit their fusion, which is essential for incorporating malignant cell membrane fragments into CTL during trogocytosis. Intriguingly, the lipidomics profiling of CTLs treated with TCM revealed numerous alterations in the content of specific lipid species 41744090.2
Attorney Docket No.046483-7386WO1(03225) (Figure 1E) such as a notable decrease in the levels of hydroxysterols including 25HC (Figures 1E-1F). Downregulation of Ch25h levels seen in CTLs exposed to TDFs in vitro (Figure 1G) was also observed in the intratumoral CTLs compared to splenic CTLs (Figures 1H-1I). Similar results were obtained in CTLs isolated from tumors/spleens in other syngeneic mouse tumor models including B16F10 melanoma, MH6499c4 pancreatic ductal adenocarcinoma and Hepa1- 6 hepatocellular carcinoma (Figure 8F). These results indicate that downregulation of CH25H in CTLs is induced by TDFs and occurs in the tumor microenvironment. Example 2: CH25H is a pivotal regulator of CTL trogocytosis, survival and activity Next, the effects of 25HC on CTLs exposed to TDFs in vitro were determined. Stimulation of trogocytosis (assessed by the transfer of OVA antigen complexed with MHC-I from MC38OVA cells onto OT-I CTLs) by TCM was inhibited by 25HC exposure (Figures 9A, 2A). Importantly, 25HC treatment abolished a decrease in viability elicited by TDFs in the antigen-experienced but not antigen-naive CTLs (Figure 2B). Furthermore, this treatment also reversed the suppressive effects of TDFs on the ability of CTLs to kill MC38OVA target cells (Figure 2C) highlighting the importance of 25HC in the regulation of CTL’s viability and function. Given that 25HC can directly inhibit fusion of lipid membranes, these effects may explain the ability of 25HC to disrupt trogocytosis. However, additional mechanisms such as contribution of 25HC to the activation of the liver X receptor (LXR) cannot be ruled out. Indeed, treatment of TCM-exposed CD8+ T cells with 25HC upregulated known LXR-stimulated genes such as Abca1, Abcg1, Lbp and Apoe. Similar results were obtained using a bona fide LXR agonist GW3965 (Figure 9B). Importantly, treatment with LXR agonist GW3965 inhibited the extent of trogocytosis (Figures 2A, 9A). These results demonstrate that 25HC in CTLs can contribute to activation of the LXR-dependent pathway and indirectly implicate LXR in the regulation of trogocytosis. Given that CH25H is downregulated in the CTLs infiltrating tumor microenvironment (Figure 1H-1I, 8F), the importance of this regulation was determined using cells from OT-I mice lacking Ch25h alleles. Knockout of this gene did not significantly affect activation (Figure 9C), proliferation, exhaustion or apoptosis (Figure 9D) of CD8+ T cells cultured alone. Thus, the 41744090.2
Attorney Docket No.046483-7386WO1(03225) extent of trogocytosis and CTL viability was assessed in vivo. To this end, a mixture of selectively labeled WT and CH25H-deficient OT-I CTLs was injected into either control MC38 or antigen-bearing MC38OVA tumors grown in Rag1-/- mice (Figures 2D, 9E). Whereas no difference was detected a day later in MC38 tumors, a notable decrease in numbers and increase in transfer of MHC-I-OVA was seen in Ch25h-/- CTLs that were exposed to OVA antigen in MC38OVA tumors (Figures 2D-2E). These results suggest that CH25H protects CTLs from trogocytosis and decrease in viability upon antigen exposure. Next, these phenotypes were recapitulated in vitro. Indeed, knockout of Ch25h notably increased the ability of CTLs to undergo trogocytosis (Figure 9F), and these effects were antigen-dependent and sensitive to Latrunculin A (Figures 9G-9J). Importantly, when co- cultured with MC38OVA, the increased trogocytosis in Ch25h-deficient CTL was concomitant with decreased viability (Figure 2F), increased apoptosis (Figure 2G) and attenuated tumoricidal activity (Figure 2H). Furthermore, these cells displayed a reduced CD69 activation marker, increased LAG3 and PD-1 exhaustion markers and increased apoptosis (Figure 2I). These results were further supported by experiments, in which adding 25HC to the antigen- experienced CTLs reverted the effects of Ch25h ablation on the extent of trogocytosis (Figure 2J), viability (Figure 2K), apoptosis (Figure 2L) and tumoricidal activity (Figure 2M). Collectively, these results demonstrate that inactivation of CH25H in the tumor microenvironment stimulates trogocytosis and impedes viability and activity of the antigen- experienced CTLs. Example 3: Downregulation of CH25H in CTLs attenuates the immune response and promotes tumor growth Because of downregulation of CH25H in the CTLs isolated from mouse tumors (Figures 1H, 8F) and decreased viability of antigen-experienced CH25H-deficient CTLs (Figures 2F, 2G, 2K, 2L), it is plausible that partial loss of CH25H in the microenvironment of human cancers may be linked with a decrease in tumor-infiltrating lymphocytes. Thus, a putative association between the intratumoral expression of CH25H and CD8A, a gene primarily expressed by tumor infiltrating CTLs, was searched for. Meta analysis of published data (Liu et al., (2020) Nature 588, 693-698; Park et al., (2019) BMC Med Genomics 12, 101) revealed a weak yet positive correlation between expression of CH25H and CD8A in tumors from human 41744090.2
Attorney Docket No.046483-7386WO1(03225) patients with melanoma, colon and pancreatic cancers (Figure 10A). Accordingly, additional available data revealed a similar correlation between CH25H levels and tumor infiltration by CD8+ T cells (Figure 3A). Importantly, low levels of CH25H expression were correlated with poor prognosis manifested by lower progression-free and overall survival in patients with melanoma (Figure 3B-3C). In all, these results indirectly associate CH25H expression with infiltration of human tumors with CTLs and lesser progression of human cancers. Driven by these data from human cancer patients, the importance of expression of CH25H in CTLs for tumor growth was assessed. To this end, Cd8a-Cre and Ch25hf/f mice were crossed to generate Ch25hΔCD8 animals (Figure 10B). Whereas no major differences with WT mice was found in spleen or blood of these cells (Figure 10C-10D), Ch25hΔCD8 mice supported a notably accelerated growth of B16F10 melanoma tumors (Figure 3D-3E). Similar results were obtained in other tumor types including, MC38 (Figure 10E), MH6499c4 (Figure 10F) and Hepa1-6 (Figure 10G). Critically, tumors obtained from Ch25hΔCD8 mice displayed reduced number and a lower frequency of tumor infiltrating CD8+ T cells (Figure 3F-3G and 10H-10I). Of note, these CH25H-deficient intratumoral (but not splenic) CTLs expressed less CD69 (Figure 3H and 10H-10I), more PD-1 (Figure 3I and 10H-10I), and exhibited a greater rate of apoptosis (Figure 3J and 10H-10I). These results indicate that the expression of CH25H in intratumoral CTLs plays an important role in their activity and viability. Conversely, inactivation of CH25H in CTLs attenuates the anti-tumor immune responses and promotes tumor growth. Example 4: ATF3 regulates effector trogocytosis, activity and viability of CTLs, and tumor growth in a CH25H-dependent manner Next, the mechanisms underlying downregulation of CH25H in the intratumoral CTLs were delineated. Activating transcription factor-3 (ATF3) is a key negative regulator of CH25H expression in macrophages. Chromatin immunoprecipitation assay demonstrated that ATF3 was present on the promoter of the Ch25h gene in mouse OT-I CD8+ T cells and treatment of these cells with TDFs increased this binding (Figure 4A). Importantly, ATF3 is well known as an early stress-responsive gene that could be induced by many factors of tumor microenvironment. ATF3 was induced in CTLs treated with media conditioned by MC38 colon adenocarcinoma cells but not by normal intestinal cells or fibroblasts (Figure 1D, 11A). Furthermore, specific 41744090.2
Attorney Docket No.046483-7386WO1(03225) TDFs such as PGE2, VEGF and tumor-derived extracellular vesicles were all capable of increasing Atf3 expression in CD8+ T cells in vitro (Figure 11A). Likewise, analysis of CD8+ T cells isolated from MC38 tumors revealed an increase in ATF3 mRNA and protein levels compared to such cells from spleens of either tumor-bearing or naïve mice (Figure 4B-4C). ATF3 induction was also noted in the intratumoral CTLs from B16F10, MH6499c4 and Hepa1-6 tumors (Figure 11B). Importantly, in these models, the expression of Atf3 in the intratumoral CTLs often negatively correlated with Ch25h levels (Figure 4D, 11C). These results are consistent with a hypothesis wherein ATF3 induced in the intratumoral CTLs by TDFs plays an important role in downregulation of CH25H. To definitively test this possibility, Atf3ΔCD8 mice were generated, which lacked ATF3 in CD8+ cells but did not differ from Atf3f/f controls in the composition of immune cells in spleen or blood (Figure 11D). Nevertheless, CTL from Atf3ΔCD8 mice did not downregulate CH25H in response to in vitro treatment with TDFs (Figure 4E). Importantly, downregulation of CH25H in the CTLs isolated from MC38 tumors was less pronounced when these tumors grew in Atf3ΔCD8 mice (Figure 4F). Similar results were obtained in other tumor types (Figure 11E) suggesting that ATF3 contributes to decreased expression of CH25H in the intratumoral CTLs. Examination of tumor volumes from these experiments revealed a significantly slower growth of MC38 tumors in Atf3ΔCD8 mice compared to Atf3f/f controls (Figure 4G). Furthermore, genetic ablation of ATF3 in CTLs increased frequency of these cells within tumors (Figure 4H) and decreased expression of markers of exhaustion and apoptosis on these cells in the tumor microenvironment (Figure 11F). Similar results were found in CTL from B16F10 tumors (Figures 4I-J, 11G-11I) but not from spleens of B16F10 bearing mice (Figure 11J). Importantly, all phenotypes associated with ablation of ATF3 in CD8+ T cells were reversed upon concurrent ablation of CH25H (Figures 4I-J, 11G-11I). These results indicate that ATF3- driven downregulation of CH25H in the intratumoral CTLs decreases their viability, suppresses the anti-tumor immune responses, and stimulates tumor growth. Example 5: ATF3 and CH25H control trogocytosis and activity of CAR T cells To directly examine the role of the ATF3-Ch25h regulatory axis in effector trogocytosis as well as viability and function of CTLs, WT, Ch25hΔCD8, Atf3ΔCD8, and Ch25h; Atf3ΔCD8 murine T cells were generated and stably transduced with retrovirus for expression of a CAR that 41744090.2
Attorney Docket No.046483-7386WO1(03225) targets human CD19 (hCD19, Figure 12A). Upon incubation with target B16F10-hCD19 cells, trogocytosis was observed by a transfer of hCD19 onto CAR T cells. Similar to results obtained in the OVA-OT-I model (Figures 2A-2M), knockout of CH25H stimulated trogocytosis (Figure 5A). Furthermore, under these conditions, CAR T cells lacking CH25H exhibited greater signs of exhaustion (Figure 5B) and apoptosis (Figure 5C) and were less effective in lysis of target B16F10-hCD19 cells (Figure 5D). Importantly, effector trogocytosis was inhibited in ATF3- deficient CAR T cells, whereas these cells were less exhausted/apoptotic (Figures 5B-5C) and more effective in killing antigen-expressing malignant cells (Figure 5D). Importantly, all these phenotypes associated with ATF3 deletion were largely reverted by genetic ablation of CH25H (Figures 5A-5D). These results suggest a critical role for the ATF3-CH25H axis in the regulation of the extent of trogocytosis, viability and activity of CAR T cells in vitro. These results were corroborated by adoptive transfer of CAR WT, Ch25hΔCD8, Atf3ΔCD8, or Ch25h; Atf3ΔCD8 T cells into Rag1-/- mice bearing B16F10-hCD19 tumors. CAR T cells were then isolated from tumors and their status analyzed. A greater extent of trogocytosis (manifested by frequency of hCD19+ CD8+ T cells) and increase in exhaustion markers were noted in CH25H-null CAR T cells (Figures 5E-5F and 12B-12C). Whereas the opposite trends were seen in the intratumoral Atf3ΔCD8 CAR T cells, these effects of ATF3 deletion were abolished by additional knockout of CH25H (Figures 5E-5F and 12B-12C) suggesting an important role for ATF3-CH25H in regulating CAR T viability/activity in vivo. Indeed, evaluation of TILs in these B16F10-hCD19 tumors revealed a greater infiltration of tumors by ATF3-deficient CAR T cells whereas knockout of CH25H decreased the numbers and frequencies of intratumoral CTLs regardless of the status of ATF3 (Figure 5G). Accordingly, CAR T cells from Atf3ΔCD8 mice exhibited a greater anti-tumor activity as manifested by changes in tumor volume and weight (Figure 5H) and animal survival (Figure 5I). Importantly, knockout of CH25H attenuated the efficacy of CAR T cell therapy in any genetic context tested here (Figures 5H-5I). These results characterize the ATF3-CH25H axis as a pivotal regulator of effector trogocytosis and associated changes in CAR T cell viability and efficacy of their anti-tumor effects. 41744090.2
Attorney Docket No.046483-7386WO1(03225) Example 6: TAK981 sumoylation inhibitor upregulates CH25H, inhibits trogocytosis and augments CAR T cell viability and anti-tumor activities A pharmacologic approach to suppressing the effector trogocytosis was sought in order to increase viability and activity of the intratumoral CTLs. Although 25HC worked well in vitro (Figures 2A-2M), its suboptimal bioavailability prompted the search for a medically relevant small molecule agent that could interfere with ATF3-driven downregulation of CH25H and restore CH25H levels in the intratumoral CTLs. After extensive analysis of literature, an inhibitor of the sumoylation pathway was chosen because of the following factors: (i) ATF3 directly suppresses transcription of CH25H and transcriptional repression is often associated with protein sumoylation; (ii) Furthermore, de-sumoylation of some transcriptional regulators is required for induction of ATF3; (iii) Importantly, sumoylation of ATF3 itself has been implicated in its transcriptional suppressive activities; and (iv) in addition, activation of innate and adaptive immunity was observed upon genetic or pharmacologic disruption of the sumoylation pathway. Intriguingly, treatment of activated OT-I CD8+ T cells with TDFs increased the levels of both unmodified and sumoylated ATF3 protein (Figure 6A). The effects of novel, selective and potent sumoylation inhibitor TAK981 were then tested on TDFs-exposed CTLs. Pre-treatment of CD8+ T cells with TAK981 in vitro partially attenuated the TDFs-induced increase in ATF3 mRNA expression (Figure 13A) and in levels of sumoylated and unmodified ATF3 protein (Figure 6A). Furthermore, TAK981 robustly prevented downregulation of CH25H expression (Figure 6B). While TDFs stimulated trogocytosis (assessed by a transfer of MHC-I-OVA complex from MC38OVA target tumor cells onto OT-I CTLs), adding TAK981 prevented this stimulation. Importantly, this effect of TAK981 was seen in WT OT-I but not in Ch25h-/- OT-I cells (Figure 6C) indicating that this sumoylation inhibitor can suppress trogocytosis by maintaining the levels of CH25H. Given that effector trogocytosis of CTLs has been associated with decreased viability and functionality, the effects of TAK981 (along with tumor-derived factors) on these parameters was examined. These treatments did not affect numbers of OT-I CTLs which were not exposed to OVA antigen. However, the numbers of WT OT-I CTLs decreased upon their incubation with TCM in the presence of target MC38OVA tumor cells (Figure 13B). Under these conditions, treatment with TAK981 restored the numbers of WT OT-I cells in the culture. Importantly, 41744090.2
Attorney Docket No.046483-7386WO1(03225) compared to WT cells, the numbers of co-cultured CH25H-deficient OT-I CTLs were notably lower, and they did not increase in response to TAK981 treatment (Figure 13B). Likewise, TAK981 restored the tumoricidal activity of WT (but not Ch25h-null) OT-I CTLs against target MC38OVA tumor cells otherwise suppressed by TCM (Figure 13C). These results demonstrate that TAK981 prevents trogocytosis and associated decrease in viability and activity of CTLs in a CH25H-dependent manner. Whereas monotherapy of MC38 tumors in WT mice with TAK981 elicited a modest therapeutic effect, the combination of this agent with anti-PD1 checkpoint inhibitor notably suppressed tumor growth. In Ch25hf/f mice under the same conditions, treatment with TAK981 alone or combined with anti-PD1 increased expression of CD69 and decreased levels of PD-1 and annexin-V on the intratumoral CTLs (Figures 6D-6E). TAK981+anti-PD1 combination significantly decreased tumor volume and weight and prolonged animal survival (Figures 6F- 6H). Importantly, analysis of intratumoral CD8+ T cells revealed that TAK981 treatment downregulated ATF3 and upregulated CH25H in vivo (Figure 13D). Furthermore, consistent with the ability of TAK981 to inhibit trogocytosis (Figure 6B) and apoptosis (Figure 6E), inclusion of this agent into therapeutic regimens also increased infiltration of MC38 tumors with CTLs (Figure 13E). Importantly, these effects were limited to the intratumoral CTLs and were not observed in the spleens from the same animals (Figure 13F). Given that protein sumoylation affects numerous biologically important processes, these phenotypes cannot be attributed entirely to the effects of TAK981 on the levels of CH25H and on the extent of effector trogocytosis. However, ablation of CH25H in CD8+ T cells rendered these cells and their host mice much less sensitive to effects of TAK981, anti-PD1 and their combination (Figures 6D-6H). These results are consistent with the hypothesis that, albeit not essential, the prevention of CH25H downregulation in the intratumoral CTLs represents an important mechanism of action for anti-cancer therapies that involve TAK981. Example 7: Armored CARs designed to re-express CH25H inhibit trogocytosis and increase therapeutic efficacy To counteract downregulation of CH25H in the intratumoral CTLs, previously described anti-MESO and anti-CD19 CARs (Milone et al., (2009) Mol Ther 17, 1453-1464) were re- designed to enable the co-expression of CH25H (Figure 7A). The ability of mouse CTLs 41744090.2
Attorney Docket No.046483-7386WO1(03225) expressing conventional anti-MESO CARs to kill target EM-Meso-GFP-Luc cells (Figure 14A) was notably attenuated by knockout of CH25H but restored with treatment with 25HC in vitro (Figure 14B). Experiments comparing CTLs harboring either conventional or CH25H- expressing CAR (Figure 14C) revealed that re-expression of CH25H notably inhibited the extent of trogocytosis (Figure 7B), and increased viability (Figure 7C) and tumoricidal activity (Figure 7D) of CAR T cells. Accordingly, treatment of NSG mice harboring EM-Meso-GFP- Luc tumors (Figure 7E) with CTLs harboring conventional or CH25H-expressing anti-MESO CAR revealed that expression of CH25H significantly increased the efficacy of this adoptive cell therapy as seen from assessment of tumor volume and weight and animal survival (Figure 7F and 14D). Under these conditions, T cells harboring CH25H-expressing CARs displayed increased numbers of CD8+ T cells in tumors and blood compared to control CAR T cells (Figure 7G and 14E). To corroborate these data, another set of CARs, designed against human CD19, were used (Figure 7A, 14F-14G). When tested in vitro, CAR T cells that co-express CH25H exhibited a greater killing activity (Figure 14H), and decreased trogocytosis manifested by transfer of antigen onto CTLs (Figure 14I) or by disappearance of antigen from target tumor cells (Figure 14J). CH25H-expressing CAR T cells also displayed lesser exhaustion (Figure 14K), and improved viability (Figure 14L). Furthermore, when tested in vivo, CAR T cells expressing CH25H demonstrated a greater therapeutic efficacy against B16F10-hCD19 tumors (Figure 7H, 14M), better infiltrated these tumors (Figure 14N), and expressed lower levels of markers of exhaustion and apoptosis (Figure 14O). The effects of co-expressing CH25H on the efficacy of adoptive transfer of human CAR T cells harboring anti-CD19 CAR were tested in the model of NALM6 acute B cell leukemia (Figure 7I). Under these conditions, CAR T cells expressing CH25H better persisted in the host (Figure 7J) and exhibited a greater anti-tumor efficacy as manifested by decreased number of leukemic cells in the blood (Figure 7K) and prolonged animal survival (Figure 7L). Collectively, these data demonstrate that re-expression of CH25H in adoptively transferred CAR T cells protects them from trogocytosis and associated exhaustion and death and increases their tumoricidal activity and therapeutic potential. Example 8: Discussion 41744090.2
Attorney Docket No.046483-7386WO1(03225) Data presented herein reveal that activation of ATF3 and ensuing suppression of CH25H in the tumor microenvironment stimulate the extent of effector trogocytosis between tumor- specific CTLs and malignant cells. These events also limit viability and activity of the intratumoral CTLs thereby stimulating tumor growth and eliciting resistance against adoptive cell transfer therapies including CAR T cell-based regimens. Maintaining expression of CH25H by administration of TAK981 sumoylation inhibitor or co-expressing CH25H along with a CAR leads to restricted trogocytosis, increased survival and activity of CTLs, and improved efficacy of CAR T cell treatment. It is, however, important to note that TDFs elicited diverse and numerous changes in CTLs’ gene expression profile as well as distinct alterations in levels of lipid species (Figures 1A-1I). Given that ATF3 is an early stress-responsive gene that could be induced by many factors in the tumor microenvironment, it is plausible that many redundant factors besides PGE2 and VEGF are capable of downregulating CH25H and promoting trogocytosis. Results of studies utilizing TAK981 suggest that protein sumoylation may contribute to both induction of ATF3 and to ATF3-dependent suppression of CH25H. It is plausible that sumoylation can occur on ATF3 itself (as was shown for its ability to suppress expression of Tp53 tumor suppressor gene), and on other regulators that may contribute to the regulation of ATF3 and CH25H levels. Whereas results herein demonstrate that TAK981 decreases the levels of total and sumoylated ATF3, additional studies could unravel interesting and important mechanisms leading to identification of additional targets and small molecule agents that could counteract trogocytosis associated with downregulation of CH25H in CTLs. Data herein further suggest a mechanism by which CH25H inhibits trogocytosis. Although inactivation of CH25H can channel cholesterol monooxygenation toward formation of other biologically and immunologically active oxysterols (such as 27-hydroxycholesterol), it’s hypothesized herein that negative regulation of trogocytosis by CH25H is conferred by production of 25HC. Indeed, a decrease in 25HC was observed in CTLs exposed to TDFs (Figures 1A-1I). Furthermore, the data demonstrate a direct inhibitory effect of 25HC on trogocytosis and associated defects in viability and activity of CTLs (Figures 2A-2M). Furthermore, 25HC is known to inhibit lipid membrane fusion, which must happen for incorporating malignant cell membrane fragments into CTL during trogocytosis. These activities of 25HC are also implicated in suppression of uptake of the tumor-derived extracellular vesicles, 41744090.2
Attorney Docket No.046483-7386WO1(03225) which were proposed to contribute to the process of transfer of T cell receptor-containing complexes. Importantly, 25-hydroxylation along with other modifications generates ligands for the LXR pathway. Inhibition of trogocytosis in T cells treated with LXR agonist is suggestive of a role for the LXR pathway – potentially downstream of the ATF3-CH25H axis. Finally, 25HC also acts as a negative regulator of the cell membrane cholesterol; and reduction of cholesterol levels in CD8+ T cells prevents their exhaustion in the tumor microenvironment. It is plausible that suppressing CH25H expression and stimulating effector trogocytosis might represent an evolutionarily important mechanism that attenuates the function of the immune system and may play a protective role to minimize tissue damage during inflammation and T cell-driven autoimmune reactions. If that is true, it would not be the first time, when such protective mechanism is appropriated by a growing tumor to evade the T cell immunity and perhaps even develop a resistance to therapies that involve adoptive transfer of CTLs. The trogocytosis-driven loss of antigen on target malignant cells that persist following an encounter with CTL is likely temporary and reversible. Nevertheless, these events provide cancer cells with a window of opportunity for evading future killing through numerous mechanisms including downregulation of MHC-I, immunoediting and loss of antigen expression, production of additional immune-suppressive ligands and substances (e.g. PD-L1, adenosine, tumor-derived extracellular vesicles, and other mechanisms. Restricting trogocytosis by re-expression of CH25H in CTLs and production of 25HC closes this temporal window and prevent resistance to adoptive cell transfer therapies. Besides masking malignant cells through decreasing antigen density, trogocytosis also affects killing cells themselves. A number of consequences ensue for an effector CTL that did not manage to kill target malignant cell and, instead, engaged in trogocytosis. Among these consequences are exposure to other CTLs and subsequent fratricide as well as trogocytosis- associated exhaustion described for both CAR T and natural effector CD8+ T cells is likely that both consequences richly contribute to reduced killing capacity of CTLs in vitro and decreased efficacy of these cells in the therapeutic settings. Regardless of the mechanism, current data suggest that restricting trogocytosis via inducing CH25H expression using small molecule agents or CAR constructs engineered to re- express CH25H in CTLs lessens the time and chance for malignant cells to undergo immunoediting and generate the immune-suppressive tumor microenvironment. This strategy 41744090.2
Attorney Docket No.046483-7386WO1(03225) increases the efficacy of anti-cancer therapies involving adoptive transfer of CTLs such as CAR T cells (as demonstrated in FIGs.7A-7L). 25HC robustly inhibits trogocytosis in vitro, however, this oxysterol has limited stability and suboptimal bioavailability complicating its use in vivo. Data presented herein reveal that sumoylation inhibitor TAK981 can decrease the extent of trogocytosis (FIGs.6A-6H). This agent acts to re-activate the anti-tumor immunity and is currently investigated in several oncology clinical trials (including NCT03648372, NCT04074330, NCT04776018 and NCT04381650). Data herein domonstrate that the efficacy of TAK981 relates to its ability to prevent downregulation of CH25H expression in the CD8+ T cells (FIGs.6A-6H). While clinical testing of this agent and its combinations with different types of immune therapies is underway, it is important to stratify these efforts and focus on scenarios where trogocytosis in CTLs acts to promote tumor growth. Otherwise, indiscriminate suppression of trogocytosis may negate the benefits of trogocytosis-mediated cross-priming and of trogocytosis-dependent anti-tumor effects elicited by neutrophils or macrophages. Besides these considerations, protein sumoylation affect numerous important cellular processes and continuous use of sumoylation inhibitors may complicate the efforts to manage oncological disease due to toxicity. Given these considerations, it would be more attractive to restrict the inhibition of trogocytosis to CTLs. Engineering novel human CAR T cells that either lack ATF3 (for example, via CRISPR/Cas9-driven knockout) or re-express CH25H represent such strategy. However, ATF3 is a key transcription factors, which regulates many important genes, and whose effects on tumor growth and progression are complex. Thus, a novel third generation CAR that co- expresses CH25H was designed herein to guard against its downregulation in the tumor microenvironment. The present study demonstrates a that such an approach works. Expression of CH25H within CAR indeed protects CAR T cells from trogocytosis and associated decrease in survival and activity and increases therapeutic efficacy of mouse and human CAR T cells (FIGs. 7A-7L). Enumerated Embodiments The following enumerated embodiments are provided, the numbering of which is not to be construed as designating levels of importance. 41744090.2
Attorney Docket No.046483-7386WO1(03225) Embodiment 1 provides a modified immune cell or precursor cell thereof, comprising a nucleic acid encoding a chimeric antigen receptor (CAR) and cholesterol 25-hydroxylase (CH25H) gene, wherein the CAR and CH25H are expressed in the cell. Embodiment 2 provides the modified immune cell or precursor cell thereof of embodiment 1, wherein the CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular domain. Embodiment 3 provides the modified immune cell or precursor cell of any preceding embodiment, wherein the CAR comprises an antigen binding domain selected from the group consisting of an antibody, an scFv, and a Fab. Embodiment 4 provides the modified immune cell or precursor cell of any preceding embodiment, wherein the CAR comprises an antigen binding domain comprising specificity for a tumor associated antigen (TAA). Embodiment 5 provides the modified immune cell or precursor cell of embodiment 4, wherein the TTA is selected from the group consisting of CD19, CD20, CD22, ROR1, Mesothelin, CD33/IL3Ra, c-Met, PSMA, PSCA, Glycolipid F77, and EGFRvIII. Embodiment 6 provides the modified immune cell or precursor cell of any preceding embodiment, wherein the CAR further comprises a hinge domain. Embodiment 7 provides the modified immune cell or precursor cell of any preceding embodiment, wherein the CAR comprises a hinge domain selected from the group consisting of an Fc fragment of an antibody, a hinge region of an antibody, a CH2 region of an antibody, a CH3 region of an antibody, an artificial hinge domain, a hinge comprising an amino acid sequence of CD8, or any combination thereof. Embodiment 8 provides the modified immune cell or precursor cell of any preceding embodiment, wherein the exogenous CAR comprises a transmembrane domain selected from the group consisting of an artificial hydrophobic sequence and transmembrane domain of a type I transmembrane protein, an alpha, beta, or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. Embodiment 9 provides the modified immune cell or precursor cell of any preceding embodiment, wherein the CAR comprises at least one co-stimulatory domain selected from the group consisting of co-stimulatory domains of proteins in the TNFR superfamily, CD28, 4-1BB 41744090.2
Attorney Docket No.046483-7386WO1(03225) (CD137), OX40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, ICOS, NKG2C, and B7-H3. Embodiment 10 provides the modified immune cell or precursor cell of any preceding embodiment, wherein the CAR comprises an intracellular domain comprising an intracellular domain selected from the group consisting of cytoplasmic signaling domains of a human CD3 zeta chain, FcyRIII, FcsRI, a cytoplasmic tail of an Fc receptor, an immunoreceptor tyrosine- based activation motif (ITAM) bearing cytoplasmic receptors, TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. Embodiment 11 provides the modified immune cell or precursor cell of any preceding embodiment, wherein the nucleic acid comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 10. Embodiment 12 provides the modified immune cell or precursor cell of any preceding embodiment, wherein the nucleic acid comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 8. Embodiment 13 provides the modified immune cell or precursor cell of any preceding embodiment, wherein the nucleic acid comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 15. Embodiment 14 provides the modified immune cell or precursor cell of any preceding embodiment, wherein the modified cell is an autologous cell. Embodiment 15 provides the modified immune cell or precursor cell of any preceding embodiment, wherein the modified cell is a cell isolated from a human subject. Embodiment 16 provides the modified immune cell or precursor cell of any preceding embodiment, wherein the modified cell is a modified T cell. Embodiment 17 provides a method of treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject the modified immune cell or precursor cell thereof of any of the preceding claims. Embodiment 18 provides the method of embodiment 17, wherein the disease or disorder is cancer. Embodiment 19 provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject i) a modified T cell comprising a CAR, and ii) an agent that upregulates CH25H. 41744090.2
Attorney Docket No.046483-7386WO1(03225) Embodiment 20 provides a nucleic acid comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR) and a nucleotide sequence encoding a cholesterol 25- hydroxylase (Ch25h) gene. Embodiment 21 provides the nucleic acid of embodiment 20, wherein the nucleic acid comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 8, 10, or 15. The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited herein, are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other physical and electronic documents. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations. 41744090.2
Claims
Attorney Docket No.046483-7386WO1(03225) CLAIMS What is claimed: 1. A modified immune cell or precursor cell thereof, comprising a nucleic acid encoding a chimeric antigen receptor (CAR) and cholesterol 25-hydroxylase (CH25H) gene, wherein the CAR and CH25H are expressed in the cell. 2. The modified immune cell or precursor cell thereof of claim 1, wherein the CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular domain. 3. The modified immune cell or precursor cell of any preceding claim, wherein the CAR comprises an antigen binding domain selected from the group consisting of an antibody, an scFv, and a Fab. 4. The modified immune cell or precursor cell of any preceding claim, wherein the CAR comprises an antigen binding domain comprising specificity for a tumor associated antigen (TAA). 5. The modified immune cell or precursor cell of claim 4, wherein the TTA is selected from the group consisting of CD19, CD20, CD22, ROR1, Mesothelin, CD33/IL3Ra, c-Met, PSMA, PSCA, Glycolipid F77, and EGFRvIII. 6. The modified immune cell or precursor cell of any preceding claim, wherein the CAR further comprises a hinge domain. 7. The modified immune cell or precursor cell of any preceding claim, wherein the CAR comprises a hinge domain selected from the group consisting of an Fc fragment of an antibody, a hinge region of an antibody, a CH2 region of an antibody, a CH3 region of an 41744090.2
Attorney Docket No.046483-7386WO1(03225) antibody, an artificial hinge domain, a hinge comprising an amino acid sequence of CD8, or any combination thereof. 8. The modified immune cell or precursor cell of any preceding claim, wherein the CAR comprises a transmembrane domain selected from the group consisting of an artificial hydrophobic sequence and transmembrane domain of a type I transmembrane protein, an alpha, beta, or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. 9. The modified immune cell or precursor cell of any preceding claim, wherein the CAR comprises at least one co-stimulatory domain selected from the group consisting of co- stimulatory domains of proteins in the TNFR superfamily, CD28, 4-1BB (CD137), OX40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, ICOS, NKG2C, and B7-H3. 10. The modified immune cell or precursor cell of any preceding claim, wherein the CAR comprises an intracellular domain comprising an intracellular domain selected from the group consisting of cytoplasmic signaling domains of a human CD3 zeta chain, FcyRIII, FcsRI, a cytoplasmic tail of an Fc receptor, an immunoreceptor tyrosine-based activation motif (ITAM) bearing cytoplasmic receptors, TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. 11. The modified immune cell or precursor cell of any preceding claim, wherein the nucleic acid comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 10. 12. The modified immune cell or precursor cell of any preceding claim, wherein the nucleic acid comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 8. 41744090.2
Attorney Docket No.046483-7386WO1(03225) 13. The modified immune cell or precursor cell of any preceding claim, wherein the nucleic acid comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 15. 14. The modified immune cell or precursor cell of any preceding claim, wherein the modified cell is an autologous cell. 15. The modified immune cell or precursor cell of any preceding claim, wherein the modified cell is a cell isolated from a human subject. 16. The modified immune cell or precursor cell of any preceding claim, wherein the modified cell is a modified T cell. 17. A method of treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject the modified immune cell or precursor cell thereof of any of the preceding claims. 18. The method of claim 17, wherein the disease or disorder is cancer. 19. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject i) a modified T cell comprising a CAR, and ii) an agent that upregulates CH25H. 20. A nucleic acid comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR) and a nucleotide sequence encoding a cholesterol 25-hydroxylase (Ch25h) gene. 21. The nucleic acid of claim 20, wherein the nucleic acid comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 8, 10, or 15. 41744090.2
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