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  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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  • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
  • A61K40/31—Chimeric antigen receptors [CAR]
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  • A61K40/41—Vertebrate antigens
  • A61K40/42—Cancer antigens
  • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
  • A61K40/421—Immunoglobulin superfamily
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  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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  • C07K14/70521—CD28, CD152
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  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
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  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
  • A61K2039/585—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation wherein the target is cancer
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  • A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the structure of the chimeric antigen receptor [CAR]
  • A61K2239/22—Intracellular domain
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• • A—HUMAN NECESSITIES
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  • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
  • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the dose, timing or administration schedule
• • A—HUMAN NECESSITIES
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
  • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
  • A61K2239/48—Blood cells, e.g. leukemia or lymphoma
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  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
  • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
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  • C07K2319/00—Fusion polypeptide
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  • C12N2510/00—Genetically modified cells

Definitions

• the disclosure generally relates to chimeric antigen receptors that target PD l ligands, and genetically engineered T cells expressing the same, for the treatment of cancer.
• the chimeric antigen receptor may include a co-stimulatory domain, such as Dap 10, to enhance efficacy of the treatment.
• CARs chimeric antigen receptors
• CAR-modified T cells for cancer therapy include the ability to recognize a broad range of tumor types, overcome the mechanisms that tumors use to escape immune detection, and enhance T-cell function. However, the up-regulation of inhibitory receptor expression on T cells and expression of inhibitory ligands in the tumor
• PD l programmed death receptor 1
• CD279 programmed death receptor 1
• the PD l receptor binds to two different ligands, programmed death ligand 1 (PDL1 , B7-H1 , CD274) and programmed death ligand 2 (PDL2, B7-DC, CD273), both of which are overexpressed on many types of solid tumors and haematological malignancies, including lymphoma. Effective therapies targeting the PDl ligands are needed.
• the present disclosure provides CARs that target PD l ligands and are suitable for adoptive T cell therapy.
• One aspect of the disclosure provides a CAR polypeptide comprising an extracellular binding domain specific for at least one of programmed death ligand 1 (PDL 1 ) and programmed death ligand 2 (PDL2); a transmembrane domain; and a cytoplasmic signaling domain.
• the extracellular domain is a programmed death receptor 1 (PD l ) extracellular domain.
• the cytoplasmic signaling domain is a ⁇ 3 ⁇ cytoplasmic domain.
• the CAR polypeptide further comprises a DNAX-activating protein 10 (Dap 10) co-stimulatory domain.
• the polypeptide comprises a sequence at least 90% identical to SEQ ID NO: 3.
• T lymphocytes genetically modified to express a CAR according to the disclosure.
• compositions for adoptive cell transfer comprising T lymphocytes of the disclosure and a pharmaceutically acceptable carrier.
• the composition further comprises one or more chemotherapeutic or radiotherapeutic agents.
• Another aspect of the disclosure provides a method of treating cancer in a subject in need thereof, wherein cells of said cancer express at least one of PDL l and PDL2, comprising administering to the subject a therapeutically effective amount of a composition for adoptive cell transfer according to the disclosure.
• the cancer is selected from the group consisting of lymphoma, melanoma, myeloma, pancreatic cancer, breast cancer, and ovarian cancer.
• FIG. 1A-F Chimeric programmed death 1 (chPD l ) T cells lyse programmed death ligand (PDL) -expressing RMA cells in a PD 1 -dependent manner, (a) Representative vector map of the chPD l -Dap l O, chPD l -CD28, and wild-type (wt) PD l receptors, (b) Effector murine non-transduced, wtPD l , or chPD l (black) T cells were stained with anti-PD l or isotype control antibodies or (c) murine chPD l T cells were stained with anti-PDL l or anti-PDL2 or isotype control antibodies and were analysed using flow cytometry, (d) RMA cells were stained with anti-PDL l or -PDL2 or isotype control antibodies and were analysed using flow cytometry, (e) Non-transduced (squares), wtPD l (triangles) or chPD
• chPD l T cells had significantly higher specific lysis at all E : T ratios compared with non-transduced or wtPD l T cells (*P ⁇ 0 0001 ).
• wtPD l or chPD l T cells were incubated with anti-PD l antibodies (open symbols), or with control IgG antibodies (closed symbols) before incubation with tumor cells. Blocking the PD l receptor significantly reduced the cytotoxicity of chPD l T cells against tumor cells at all ratios compared with control (*P ⁇ 0 001 ). Data are presented as mean + SD and are representative of at least three experiments.
• FIG. 2A-B Culture of RMA cells with chimeric programmed death 1 (chPD l ) T cells results in secretion of pro-inflammatory cytokines.
• Non-transduced, wild-type (wt) PD 1 -expressing (black), or chPD l -expressing (open) T cells were cultured with RMA cells or media.
• cytokine IL- 10 secretion of (a) pro-inflammatory cytokines interferon-y (IFN-y), tumor necrosis factor-cc (TNF-a), granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-2 (IL-2) and (b) anti-inflammatory cytokine IL- 10 was measured in cell-free supernatants by ELISA.
• the chPD l T cells produced higher levels of pro-inflammatory cytokines and decreased levels of anti-inflammatory cytokines compared with wtPD l T cells when cultured with RMA cells (*P ⁇ 0 001 ). Data are presented as mean + SD and are representative of at least three experiments.
• FIG. 3A-D Treatment with chimeric programmed death 1 (chPD l ) T cells leads to a reduction in tumor burden and an increase in survival of RMA-GFP-bearing mice.
• chPD l chimeric programmed death 1
• RMA-GFP cells (2 x 10 6 ) were injected intravenously (i. v.) into B6 mice on day 0.
• Mice were treated i.v. with a single treatment of PBS or wild-type (wt) PD 1 (black) or chPD l (open) T cells (5 x 10 6 ) after (a) 2 days, (b) 5 days or (c) two doses of wtPD l or chPD l T cells after 5 and 8 days.
• Mice were treated i.v.
• chPD l T cells significantly reduced RMA tumor burden and increased survival compared with wtPD l T cells or PBS (*P ⁇ 0 01 ). Data are presented as mean + SD and are representative of two independent experiments.
• FIG. 4A-C Chimeric programmed death 1 (chPD l ) -Dap 10 T cells secrete increased levels of pro-inflammatory cytokines and decreased levels of anti-inflammatory cytokines compared with chPD 1 -CD28 T cells.
• T cells expressing wild-type (wt) PD 1 - (black), chPD l -Dap l O (open), or chPD l -CD28 were cultured with RMA cells or media. After 24 hr, secretion of (a) cytokines and (b) chemokines was measured in cell-free supernatants by ELISA or LEGENDplex analysis.
• the chPD l -Dap l O T cells produced higher levels of pro-inflammatory cytokines and decreased levels of anti-inflammatory cytokines compared with wtPD l - or chPD l -CD28 T cells when cultured with RMA cells ⁇ *P ⁇ 0- 01 ).
• T cells were used as effector cells with RMA cells at the indicated effetor tp target (E : T) ratios ( 1 : 1 , 5 : 1 , 25 : 1 ) and cell lysis was measured using a lactate dehydrogenase assay. Data are presented as mean + SD and are representative of at least three experiments.
• FIG. 5A-B Inclusion of Dap 10 co-stimulatory domain induces a central memory phenotype in chimeric programmed death 1 (chPD l ) T cells.
• T cells expressing wild-type (wt) PD l - (black), chPD l -Dap l O (open), or chPD l -CD28 were cultured with RMA cells or media.
• RMA cells or media After 24 hr, (a) expression of genes that control effector and central memory differentiation was measured by RT-PCR or (b) cell surface marker expression was measured by flow cytometry. Stimulation with RMA cells altered gene or cell surface marker expression compared with culturing in media (*P ⁇ 0 01 ). Data are presented as mean + SD and are representative of at least two experiments.
• FIG. 6A-C Treatment with chimeric programmed death 1 (chPD l ) -Dap 10 T cells leads to a greater reduction in tumor burden and increased survival of RMA-GFP-bearing mice compared with treatment with chPD l -CD28 T cells.
• RMA-GFP cells (2 x 10 6 ) were injected intravenously (i.v.) into B6 mice on day 0. Mice were treated i.v.
• the chPD l -CD28 T cells significantly reduced RMA tumor burden and increased survival compared with wtPD l T cells (*P ⁇ 0 01 ).
• chPD l -Dap l O T cells significantly reduced RMA tumor burden and increased survival better than chPD l -CD28 T cells ( # P ⁇ 0 01 ).
• RMA-bearing mice were treated with 5 x 10 6 Ly5. 1 + chPD l -Dap l 0 (open) or chPD l -CD28 T cells i.v. 5 days after tumor cell injection. Spleen and lymph node cells were isolated at various time-points after T-cell injection and the percentage of Ly5.1 + CD3 + cells was calculated (n - 4).
• chPD l -Dap l O T cells had increased persistence in vivo compared with chPD l -CD28 T cells (*P ⁇ 0 01 ). Data are presented as mean + SD and are representative of two independent experiments.
• T cells protect individuals from disease by targeting and eliminating diseased cells.
• Tumor-specific T cells can be isolated, followed by activation and expansion outside the body, and then re-infused back into the patient to mediate cancer regression, a process termed adoptive T cell therapy.
• CARs that target PD l ligands, i.e. that target at least one of PDL1 or PDL2.
• the chimeric PD l receptor (chPD l ) polypeptide may comprise an extracellular binding domain specific for at least one of PDL1 and PDL2; a transmembrane domain; and a cytoplasmic signaling domain.
• 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 include a protein's or peptide's sequence.
• Polypeptides include any peptide or protein having 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.
• the extracellular binding domain specific for (i.e. that targets, recognizes, or binds) at least one of PDL1 and PDL2 may be the extracellular domain of the PD l (CD279) receptor.
• the PD 1 receptor is a protein found on the surface of cells, such as T lymphocytes, that has a role in regulating the immune system's response to an individual's own cells. In humans, the PD l protein is encoded by the PDCDI gene.
• a representative amino acid sequence for PD l is provided in SEQ ID NO: 1. In some embodiments, the domain comprises or consists of amino acids 1 - 155 of PD l .
• the transmembrane domain includes a hydrophobic polypeptide that spans the cellular membrane.
• the transmembrane domain spans from one side of a cell membrane (extracellular) through to the other side of the cell membrane (intracellular or cytoplasmic).
• the transmembrane domain may be in the form of an alpha helix or a beta barrel, or combinations thereof.
• the transmemebrane domain may include a polytopic protein, which has many transmembrane segments, each alpha-helical, beta sheets, or combinations thereof.
• the transmembrane domain that naturally is associated with one of the domains in the CAR is used.
• the transmembrane domain can be selected or modified by amino acid substitution 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.
• a transmembrane domain includes a transmembrane domain of a T-cell receptor a or ⁇ chain, a CD3 zeta chain, CD28, CD3s, CD45, CD4, CD5, CD8, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD 137, ICOS, CD 154, functional derivatives thereof, and combinations thereof.
• An artificially designed transmembrane domain is a polypeptide mainly comprising hydrophobic residues such as leucine and valine.
• phenylalanine, tryptophan and valine is found at each end of the synthetic transmembrane domain.
• a cytoplasmic signaling domain of a CAR may be responsible for intracellular signaling following the binding of extracellular ligand binding domain to the target resulting in the activation or inhibition of the immune cell and immune response.
• the signal transducing domain may be responsible for the activation or inactivation of at least one of the normal effector functions of the immune cell in which the CAR is expressed.
• the term "cytoplasmic signaling domain” refers to the portion of a protein which transduces the effector signal function signal and directs the cell to perform a specialized function.
• Examples of signal transducing domain for use in CARs of the present disclosure can be the cytoplasmic sequences of the T cell receptor and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any synthetic sequence that has the same functional capability.
• Cytoplasmic signaling sequences can comprise signaling motifs which are known as immunoreceptor tyrosine-based activation motifs of ITAMs.
• ITAMs are well defined signaling motifs found in the intracytoplasmic tail of a variety of receptors that serve as binding sites for syk/zap70 class tyrosine kinases.
• Examples of IT AM can include as non limiting examples those derived from TCRzeta, FcRgamma, FcRbeta, FcRepsilon,
• the signaling transducing domain of the CAR comprises a CD3zeta signaling domain. In some embodiments, the signaling transducing domain comprises or consists of amino acids 52- 164 of CD3zeta.
• the inhibitory domains of PD1 are replaced with the activating domains of the cytoplasmic signaling domain, thus switching the negative PD 1 signal to become an activating signal for the T cells. This should reduce the immune suppressive effects of PD 1 and instead induce anti-tumor immunity upon interaction with PD 1 ligands.
• Inclusion of co-stimulatory domains in CAR T cells can enhance T-cell functions, including cytokine secretion, differentiation, cytotoxicity, proliferation and
• Co-stimulatory receptors that are compatible with CARs of the present disclosure include, but are not limited to, functional domains from Dap 10, CD28, OX40, ICOS, 4- I BB (CD 137), NKG2C, and NKG2D, and active fragments, functional derivatives, and combinations thereof.
• CD28 and Dap 10 activate many similar pathways including phosphatidylinositol-3 kinase, AKT/Protein Kinase B and
• CD28 and Dap 10 stimulation seem to have unique effects on effector T cells, including differential activation of signal transduction pathways including ?-catenin, nuclear factor- ⁇ B and mammalian target of rapamycin
• Dap 10 co-stimulation through Dap 10 induces CD8 T-cell memory differentiation and secretion of pro-inflammatory but not anti-inflammatory cytokines, both of which seem to be preferable characteristics for successful CAR T-cell therapy.
• inclusion of the Dap 10 co-stimulatory domain in CARs may be preferential to CD28.
• Functional-conservative derivatives or variants of a polypeptide as disclosed herein may result from modifications and changes that may be made in the structure of the polypeptide (and in the DNA sequence encoding it), and still obtain a functional molecule with desirable characteristics (e.g. tumoricidal and/or immunostimulatory effects).
• Functional-conservative derivatives may also consist of a fragment of a polypeptide that retains its functionality.
• functional-conservative derivatives or variants are those in which a given amino acid residue in a protein has been changed without altering the overall conformation and function of the polypeptide, including, but not limited to, replacement of an amino acid with one having similar properties (such as, for example, polarity, hydrogen bonding potential, acidic, basic, hydrophobic, aromatic, and the like).
• Amino acids other than those indicated as conserved may differ in a protein so that the percent protein or amino acid sequence similarity between any two proteins of similar function may vary and may be, for example, from 70% to 99% as determined according to an alignment scheme such as by the Cluster Method, wherein similarity is based on the MEGALIGN algorithm.
• a functional- conservative derivative also includes a polypeptide which has at least 70%, 75%, 80%, 857c, 90%, or 95 or more amino acid identity as determined by BLAST or FASTA algorithms and which has the same or substantially similar properties or functions as the native or parent protein to which it is compared.
• certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of tumoricidal effects. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid substitutions can be made in a protein sequence, and, of course, in its DNA encoding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated that various changes may be made in the polypeptide sequences of the disclosure, or corresponding DNA sequences which encode said polypeptides, without appreciable loss of their biological activity. Said tumoricidal activity and
• immunostimuolatory activity can be assessed by various techniques well-known in the art, such as for instance the assays referred to in the Examples.
• conservative amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
• Exemplary substitutions which take several of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine;
• a polynucleotide of the disclosure can be cloned into a vector.
• a "vector” is a composition of matter which includes an isolated polynucleotide and which can be used to deliver the isolated polynucleotide 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, phagemid, cosmid, and viruses.
• Viruses include phages, phage derivatives.
• the term "vector” includes an autonomously replicating plasmid or a virus.
• viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.
• vectors include cloning vectors, expression vectors, replication vectors, probe generation vectors, integration vectors, and sequencing vectors.
• the vector is a viral vector.
• the viral vector is a retroviral vector or a lentiviral vector.
• an engineered cell is virally transduced to express the polynucleotide sequence.
• 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 endomiclease sites, and one or more selectable markers.
• chimeric antigen receptor polynucleotide may be achieved using, for example, expression vectors including, but not limited to, at least one of a SFFV or human elongation factor 1 l a (EF) promoter, CAG (chicken beta-actin promoter with CMV enhancer) promoter human elongation factor la (EF) promoter.
• EF human elongation factor 1 l a
• CAG chicken beta-actin promoter with CMV enhancer
• EF elongation factor la
• Examples of less-strong/ lower- expressing promoters utilized may include, but is not limited to, the simian virus 40 (SV40) early promoter, cytomegalovirus (CMV) immediate-early promoter, Ubiquitin C (UBC) promoter, and the phosphoglycerate kinase 1 (PGK) promoter, or a part thereof.
• SV40 simian virus 40
• CMV cytomegalovirus
• Inducible expression of chimeric antigen receptor may be achieved using, for example, a tetracycline responsive promoter, including, but not limited to, TRE3GV (Tet-response element, including all generations and preferably, the 3rd generation), inducible promoter (Clontech
• the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art.
• the expression vector can be transferred into a host cell by physical, chemical, or biological means.
• Adoptive T cell therapy involves isolating T cells from an individual's blood and genetically engineering those T cells to express a CAR of the disclosure.
• the engineered T cells are then grown ex vivo and infused back into the individual.
• the CAR T cells can then bind the targeted antigen on cancer cells and kill them.
• An embodiment of the disclosure provides an engineered cell, e.g. a T
• an “engineered cell” means any cell of any organism that is modified, transformed, or manipulated by addition or modification of a gene, a DNA or RNA sequence, or protein or polypeptide.
• Isolated cells, host cells, and genetically engineered cells of the present disclosure include isolated immune cells, such as NK cells and T cells that contain the DNA or RNA sequences encoding a CAR or CAR complex and express the chimeric receptor on the cell surface.
• Isolated host cells and engineered cells may be used, for example, for enhancing an NK cell activity or a T lymphocyte activity, treatment of cancer, and treatment of infectious diseases. Any cell capable of expressing and/or capable of integrating the chimeric antigen receptor polypeptide, as disclosed herein, into its membrane may be used.
• the engineered cell includes immunoregulatory cells.
• Immunoregulatory cells include T-cells (or T lymphocytes), such as CD4 T-cells (Helper T-cells), CD8 T-cells (Cytotoxic T-cells, CTLs), and memory T cells or memory stem cell T cells.
• T-cells include Natural Killer T-cells (NK T-cells).
• An embodiment of the disclosure provides a method of treating cancer in a subject in need thereof, wherein cells of said cancer express at least one of PDL l and PDL2, comprising administering to the subject a therapeutically effective amount of a composition comprising T cells genetically engineered to express a CAR according to the disclosure.
• the cells may be introduced into a host organism, e.g. a mammal, in a wide variety of ways.
• the cells may be introduced at the site of the tumor, in specific embodiments, although in alternative embodiments the cells hone to the cancer or are modified to hone to the cancer.
• the number of cells that are employed will depend upon a number of circumstances, the purpose for the introduction, the lifetime of the cells, the protocol to be used, for example, the number of administrations, the ability of the cells to multiply, the stability of the recombinant construct, and the like.
• the cells may be applied as a dispersion, generally being injected at or near the site of interest.
• the cells may be in a physiologically-acceptable medium.
• the route of administration may be intravenous, intraarterial, intraperitoneal, or subcutaneous, for example. Multiple administrations may be by the same route or by different routes. In some embodiments, multiple doses, e.g. 2, 3, 4, 5, or more doses are given over a period of time, e.g. over 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days.
• CRS cytokine release syndrome
• a co- stimulatory domain that does not induce elevation of one or more cytokines selected from IFN-y, GM-CSF, IL- 10 and IL-6 is used.
• a CRS therapy such as tocilizumab (an IL-6 antagonist) is administered concomitantly or sequentially with the CAR of the disclosure.
• radiotherapy and/or chemotherapy is administered concomitantly or sequentially with the CAR of the disclosure.
• cancer refers to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth.
• hyperproliferative and neoplastic disease states may be categorized as pathologic, i.e., characterizing or constituting a disease state, or may be categorized as non-pathologic, i.e., a deviation from normal but not associated with a disease state.
• pathologic i.e., characterizing or constituting a disease state
• non-pathologic i.e., a deviation from normal but not associated with a disease state.
• the term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness.
• cancer metastasis has its general meaning in the art and refers to the spread of a tumor from one organ or part to another non-adjacent organ or part.
• Any cancer or metastatic cancer expressing at least one of PDL 1 and PDL2 may be targeted using the inventive therapy including, but not limited to, lymphoma, melanoma, myeloma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, brain cancer, liver cancer, kidney cancer, lung cancer, spleen cancer, gall bladder cancer, anal cancer, testicular cancer, ovarian cancer, cervical cancer, skin cancer, bone cancer, and colon cancer.
• subject and “patient” are used interchangeably herein, and refer to an animal such as a mammal, which is afflicted with or suspected of having, at risk of, or being pre-disposed to cancer.
• the terms may refer to a human.
• the terms also include domestic animals bred for food, sport, or as pets, including horses, cows, sheep, poultry, fish, pigs, cats, dogs, and zoo animals, goats, apes (e.g. gorilla or chimpanzee), and rodents such as rats and mice.
• Typical subjects include persons susceptible to, suffering from or that have suffered from cancer.
• treating means reversing, alleviating, inhibiting the progress of, or ameliorating the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition.
• the treatment of the disclosure may slow the growth of said cancer, reduce the number of tumor cells in said cancer, reduce tumor load, or eliminate said cancer.
• a “therapeutically effective amount” is meant a sufficient amount of the molecule to treat a cancer, (for example, to limit tumor growth or to slow or block tumor metastasis) at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of the molecules and compositions of the present disclosure will be decided by the attending physician within the scope of sound medical judgment.
• the specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific polypeptide employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific polypeptide employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.
• T cell therapy described herein may be combined with standard-of-care treatments
• the T lymphocytes of the disclosure may be administered sequentially or concomitantly with one or more of
• said chemotherapeutic or radiotherapeutic agents are a therapeutic active agent used as anticancer agent.
• said anticancer agents include but are not limited to fludarabine, gemcitabine, capecitabine, methotrexate, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosoureas, platinum complexes such as cisplatin, carboplatin and oxaliplatin, mitomycin, dacarbazine, procarbazine, epipodophyllotoxins such as etoposide and teniposide, camptothecins such as irinotecan and topotecan, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, L-asparaginase, doxorubicin,
• alkylating agents alkylating agents, plant alkaloids, DNA topoisomerase inhibitors, anti-folates, pyrimidine analogs, purine analogs, DNA
• antimetabolites include taxanes, podophyllotoxins, hormonal therapies, retinoids, photosensitizers or photodynamic therapies, angiogenesis inhibitors, antimitotic agents, isoprenylation inhibitors, cell cycle inhibitors, actinomycin, bleomycin, anthracyclines, MDR inhibitors and Ca 2+ ATPase inhibitors.
• Additional anticancer agents may be selected from, but are not limited to, cytokines, chemokines, growth factors, growth inhibitory factors, hormones, soluble receptors, decoy receptors, monoclonal or polyclonal antibodies, mono-specific, bi-specific or multi-specific antibodies, monobodies, polybodies.
• antiemetic agents include, but are not limited to, metoclopramide, domperidone, prochlorperazine, promethazine, chlorpromazine, trimethobenzamide, ondansetron, granisetron, hydroxyzine, acetylleucine, alizapride, azasetron, benzquinamide, bietanautine, bromopride, buclizine, clebopride, cyclizine, dimenhydrinate, diphenidol, dolasetron, meclizine, methallatal, metopimazine, nabilone, pipamazine, scopolamine, sulpiride, tetrahydrocannabinols, thiethylperazine, thioproperazine and tropisetron.
• the antiemetic agent is granisetron or ondansetron.
• the other therapeutic active agent can be an opioid or non-opioid analgesic agent.
• opioid analgesic agents include, but are not limited to, morphine, heroin, hydromorphone, hydrocodone, oxymorphone, oxycodone, metopon, apomorphine, buprenorphine, meperidine, loperamide, ethoheptazine, betaprodine, diphenoxylate, fentanyl, sufentanil, alfentanil, remifentanil, levorphanol, dextromethorphan, phenazone, pentazocine, cyclazocine, methadone, isomethadone and propoxyphene.
• Suitable non-opioid analgesic agents include, but are not limited to, aspirin, celecoxib, rofecoxib, diclofenac, diflunisal, etodolac, fenoprofen, flurbiprofen, ibuprofen, ketoprofen,
• the further therapeutic active agent can be an anxiolytic agent.
• Suitable anxiolytic agents include, but are not limited to, buspirone, and
• benzodiazepines such as diazepam, lorazepam, oxazapam, clorazepate, clonazepam, chlordiazepoxide and alprazolam.
• radiotherapeutic agent as used herein, is intended to refer to any radiotherapeutic agent known to one of skill in the art to be effective to treat or ameliorate cancer, without limitation.
• the radiotherapeutic agent can be an agent such as those administered in brachytherapy or radionuclide therapy.
• Such methods can optionally further comprise the administration of one or more additional cancer therapies, such as, but not limited to, chemotherapies, and/or another radiotherapy.
• Another aspect of the disclosure relates to a pharmaceutical composition
• a pharmaceutical composition comprising a T lymphocyte according to the disclosure and a pharmaceutically acceptable carrier.
• compositions that do not produce an adverse, allergic or other untoward reaction when administered to a subject, such as a human, as appropriate.
• a pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
• the T lymphocytes of the disclosure may be contained in physiological saline, phosphate buffered saline (PBS), culture medium, or the like in order to maintain stability.
• PBS phosphate buffered saline
• the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
• vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
• These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
• the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
• the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
• Solutions comprising compounds of the disclosure as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
• Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
• inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like.
• Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine,
• the carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils.
• the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
• the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
• isotonic agents for example, sugars or sodium chloride.
• Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.
• Sterile injectable solutions are prepared by incorporating the T lymphocytes in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization.
• dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
• the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
• solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
• the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.
• aqueous solutions For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
• aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
• sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
• compositions of the disclosure formulated for parenteral administration such as intravenous or intramuscular injection
• other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; liposomal formulations; time release capsules; and any other form currently used.
• kits Any of the compositions described herein may be comprised in a kit.
• one or more cells for use in cell therapy that harbors recombinantly expressed CARs according to the disclosure and/or the reagents to generate one or more cells for use in cell therapy may be comprised in a kit.
• the kit components are provided in suitable container means.
• the kits comprises recombinant engineering reagents, such as vectors, primers, enzymes (restriction enzymes, ligase, polymerases, etc.), buffers, nucleotides, etc.
• kits may be packaged either in aqueous media or in lyophilized form.
• the container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there are more than one component in the kit, the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial.
• the kits of the present disclosure also will typically include a means for containing the components in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.
• EXAMPLE 1 Adoptive transfer of murine T cells expressing a chimeric-PD 1 -Dap 10 receptor as an immunotherapy for lymphoma.
• chPD l murine chimeric PD 1 receptor
• CD3C cytoplasmic domain of CD3C
• chimeric antigen receptor therapies use various co-stimulatory domains to enhance efficacy.
• the inclusion of a Dap 10 or CD28 co-stimulatory domain in the chPD l receptor was compared to determine which domain induced optimal anti-tumor immunity in a mouse model of lymphoma.
• the chPDl T cells secreted pro-inflammatory cytokines and lysed RMA lymphoma cells.
• chPD l T cells Adoptive transfer of chPD l T cells significantly reduced established tumors and led to tumor-free survival in lymphoma-bearing mice.
• both receptors induced secretion of pro-inflammatory cytokines; however, chPD l -CD28 T cells also secreted anti-inflammatory cytokines whereas chPD l-Dap lO T cells did not.
• chPD l -Dap l O induced a central memory T-cell phenotype compared with chPD l -CD28, which induced an effector memory phenotype.
• chPD l -Dap l O T cells also had enhanced in vivo persistence and anti-tumor efficacy compared with chPD l -CD28 T cells. Therefore, adoptive transfer of chPD l T cells represents a novel therapy for lymphoma and inclusion of the DaplO co-stimulatory domain in chimeric antigen receptors may induce a preferential cytokine profile and T-cell differentiation phenotype for anti-tumor therapies.
• Murine cDNA clones of CD3 ⁇ , PD 1 , CD28 and Dap l O were purchased from OriGene (Rockville, MD).
• the chPD l -Dap l O and chPDl -CD28 receptors were created by overlapping PCR using Phusion® high fidelity DNA polymerase (New England BioLabs, Ipswich, MA).
• the extracellular domain of the murine PD 1 receptor [amino acids (aa) 1-155] was fused in frame to the transmembrane region of CD28 (aa 141- 177) and the cytoplasmic domains of Dap l O (aa 57-79) and CD3 ⁇ (aa 52-164).
• the extracellular domain of the murine PD 1 receptor (aa 1-155) was fused in frame to the transmembrane (aa 141 -177) and cytoplasmic (aa 178-218) domains of CD28 and ⁇ 03 ⁇ (aa 52-164).
• the extracellular and transmembrane domain of the PD 1 receptor (aa 1-190) was used. All constructs were cloned into the pQCXIN retroviral expression vector using Notl and EcoRl digestion of the plasmid and constructs and were subsequently ligated into the vector. Ecotropic retroviral supernatants were expressed using the EcoPack 2-293 cell line according to the
• RetroX Concentrator was used to concentrate the ecotropic retroviral supernatants before transduction of primary murine T cells.
• mice Male C57BL/6 (B6) and 6.SJL-Ptprc M (Ly5.1 congenic) mice were purchased from Taconic Biosciences (Hudson, NY). Mice were between 8 and 12 weeks of age at the start of each experiment. All animal work was performed in accordance and with approval from Longwood University's Institutional Animal Use and Care Committee. Splenocytes from B6 or Ly5.1 congenic mice were activated with concanavalin A (1 ng/ml) for 18 hr.
• T cells (0-5 x 10 6 cells/ml) were transduced by centrifugation at 1000 g for 1 hr in the presence of 8 ⁇ ig/ml polybrene and 25 U/ml recombinant human interleukin-2 (IL-2) and were subsequently cultured for 6 hr before retroviral supernatants were removed and replaced with fresh complete RPMI- 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, 100 ⁇ ig/ml streptomycin, 1 mM pyruvate, 10 mM HEPES, 0 ⁇ 1 mM non-essential amino acids and 50 ⁇ 2-mercaptoethanol. Two days after infection, T cells were selected in complete RPMI-1640 medium containing G418 (0- 5 mg/ml) plus 25 U/ml recombinant human IL-2 for an additional 3 days. Viable cells were isolated using
• Histopaque-1083 (Sigma, St Louis, MO) and expanded for an additional 2 days without G418 before functional analysis.
• 100 ng of cDNA was used to measure gene expression of PDL1 , PDL2 and ⁇ -actm. Maxima SYBRGreen qPCR Master Mix (Thermo Scientific, Waltham, MA) and gene specific primers were used: ?-actin F
• T-cell differentiation genes T-bet, BLIMP 1 , Eomes and BCL6 were previously described. Primers were purchased from Integrated DNA Technologies (Coralville, IA).
• PDL1 and PDL2 on RMA and T cells and of PD 1 on T cells was tested using flow cytometry.
• Cells were stained with allophycocyanin-labelled anti-PDLl (clone 10F.9G2), phycoerythrin-labelled anti-PDL2 (clone TY25), or phycoerythrin-labelled anti-PD l (clone RMP1 -30) antibodies or isotype controls.
• wtPD l or chPD l T cells (2 x 10 3 cells/well) were stimulated with RMA cells (2 x
• the chPD l , wtPD I and non-transduced T cells (10 5 ) were cultured with RMA cells ⁇ ⁇ 0 y ) or medium in a round-bottom 96-well plate. After 24 hr, cell-free supernatants were tested for the presence of interferon-y (IFN-y), tumor necrosis factor-a (TNF-oc), granulocyte- macrophage colony-stimulating factor (GM-CSF), IL-2 and IL-10 by ELISA according to the manufacturer's instructions (BioLegend). Cytokine and chemokine secretion was also measured in cell-free supernatants using mouse T helper cytokine and mouse
• chPD l , wtPD I and non-transduced T cells 10 5 were cultured with RMA at various effector to target ratios (E : T 25 : 1 , 5 : 1 , and 1 : 1 ). Specific lysis was measured after 5 hr using a lactate dehydrogenase cytotoxicity assay kit (Pierce, Waltham, MA) according to the manufacturer's instructions.
• T cells were pre-incubated at 37° for 2 hr with anti-PD l monoclonal antibodies (clone RMP 1 -14, 20 ⁇ tg/ml, Low Endotoxin, Azide-Free LEAF purified, BioLegend) or isotype control monoclonal antibodies before addition of target cells.
• anti-PD l monoclonal antibodies clone RMP 1 -14, 20 ⁇ tg/ml, Low Endotoxin, Azide-Free LEAF purified, BioLegend
• isotype control monoclonal antibodies before addition of target cells.
• mice with genetically modified T cells Treatment of mice with genetically modified T cells
• RMA and RMA-GFP cells were grown in complete RPMI-1640.
• RMA-GFP cells (2 x 10 6 ) were injected intravenously into B6 mice.
• mice were administered one dose of wtPD I or chPD l -modified T cells (5 x 10 6 ) intravenously 2 days or 5 days after tumor injection, or two doses of T cells 5 and 8 days after tumor injection.
• spleens and lymph nodes were collected 13 days after tumor injection.
• RMA-bearing mice were treated 5 days after tumor cell injection with congenic
• Ly5.1 + chPD l -Dap lO or chPD l -CD28 T cells (5 x 10 6 ) intravenously and mice were killed 1 , 3, 7, 10, 14 or 18 days after T-cell injection.
• Spleen and lymph node cells were incubated with FcR block and mouse y-globulin (Jackson ImmunoResearch, West Grove, PA) to prevent non-specific binding, and stained with phycoerythrin-conjugated anti-CD3 and
• chPDl T cells secrete pro-inflammatory cytokines and lyse PDL-expressing RMA cells in a PD1 -dependent manner
• a chPDl receptor was created by fusing the extracellular region of the PD 1 receptor with the intracellular regions of the Dap 10 co-stimulatory receptor and CO (, (Fig. l a).
• a wtPD l receptor consisting of the extracellular and transmembrane domains of the PD 1 receptor was also created as a control.
• the chPD l and wtPD l receptors were successfully expressed in activated murine T cells as shown by an increased cell surface expression of the PD 1 receptor compared with non-transduced, activated T cells (Fig. l b). Both wtPD l and chPD l T cells consisted of a mix of activated CD4 + (- 10%) and CD8 + (-90%) T cells.
• RMA murine lymphoma cell line
• the expression of PDLl and PDL2 was measured.
• RMA cells expressed cell surface PDLl and PDL2, as determined by flow cytometry (Fig. I d).
• RT-PCR for PD l ligands was also performed, and RMA cells expressed mRNA for PDLl and PDL2.
• the chPD l T cells lysed RMA cells significantly more than T cells expressing a wtPD l receptor or
• T cells secrete pro-inflammatory cytokines to enhance antitumor immunity. 15 - 30 Compared with non-transduced or wtPD l T cells, chPD l T cells secreted significant amounts of pro-inflammatory cytokines IFN-)', TNF-a, GM-CSF and IL-2 but did not secrete anti-inflammatory cytokine IL- 10 when cultured with RMA cells. (Fig. 2). Together, these data show that RMA cells expressed PD 1 ligands and that expression of the chPD l receptor induced pro-inflammatory cytokine secretion and lysis of the RMA murine lymphoma cell line.
• chPD l T cells When injected intravenously into mice, RMA tumor cells traffic to the spleen and lymph nodes; hence, this model recapitulates features of human lymphoma in syngeneic, immunocompetent mice. 31 Therefore, the potential of using chPD l T cells in vivo as a therapy for lymphoma was investigated. Because ligands for PD 1 may also be expressed on healthy tissues, the safety of chPD l T cells was first tested. The chPD l T cells did not lyse or secrete IFN-y when cultured with splenocytes, liver cells or lung cells isolated from a naive mouse.
• mice did not show any adverse symptoms or increased levels of serum IFN-y, suggesting that chPDl T cells did not target healthy tissues.
• lymphoma-bearing mice were treated with a single dose of chPD l T cells 2 days after tumor cell injection and tumor burden was measured in the spleen and lymph nodes (Fig. 3a).
• RMA tumor burden was significantly decreased in mice treated with chPD l T cells.
• the tumor burden in mice treated with PBS or wtPD l T cells was not significantly different, indicating that wtPD l T cells did not decrease tumor burden.
• mice were treated with wtPD 1 or chPD 1 T cells 5 days after tumor cell injection.
• Treatment with chPD l T cells significantly reduced these established tumors, although there was a low yet detectable level of tumor cells in the spleens and lymph nodes of the chPD l T-cell-treated mice (Fig. 3b).
• Fig. 3b As previous studies have shown that multiple treatments with CAR T cells enhance anti-tumor efficacy, tumor-bearing mice were injected with two treatments of wtPD l or chPD l T cells 5 and 8 days after tumor cell injection.
• mice treated with two doses of chPD l T cells had undetectable tumor levels of tumor cells (Fig. 3c ).
• mice treated with wtPD l T cells that succumbed to tumors by day 20 after tumor cell injection mice treated with two doses of chPD l T cells had a significant increase in survival and there was long-term, tumor-free survival in 70% of lymphoma-bearing mice (Fig. 3d).
• chPD l T-cell treatment of established lymphoma increased survival
• multiple doses of chPD l T cells led to long-term survival in tumor-bearing mice.
• chPDl-DaplO T cells secrete increased levels of pro-inflammatory cytokines and decreased levels of anti-inflammatory cytokines compared with chPDl-CD28 T cells
• co-stimulatory domains in CARs enhances T-cell anti-tumor effector functions and each co-stimulatory receptor has a unique effect on T cells. 15, 16 Therefore to compare the inclusion of the Dap 10 domain with another co-stimulatory receptor, a chPD l receptor was made that contained the cytoplasmic domain of CD28 instead of the Dap 10 cytoplasmic domain (Fig. l ).
• One effector function that often differs between co-stimulatory receptors is their ability to induce cytokine secretion. 26"28 Therefore, the secretion of pro- and anti-inflammatory cytokines by chPD l -Dap l O and chPD l -CD28 T cells was compared.
• chPD l -Dap l O T cells secreted higher amounts of pro-inflammatory cytokines TNF-cc, GM-CSF, IL- 17 and IL-21 . Comparatively,
• chPD l -CD28 T cells secreted more IL-2 and T helper type 2/anti-inflammatory cytokines IL-5 and IL-10 (Fig. 4a).
• the chPD l -Dap l O and chPD l -CD28 T cells also secreted similar amounts of inflammatory chemokines regulated on activation, normal T cell expressed and secreted (RANTES) macrophage inflammatory proteins l and 1 ⁇ .
• RANTES normal T cell expressed and secreted
• CD28-containing CARs often induce an effector memory or effector cell phenotype and do not live as long in vivo whereas CARs that induce a central memory phenotype usually persist longer in vivo and often have stronger anti-tumor efficacy.
• chPDl-DaplO T cells expressed cell surface markers associated with a central memory phenotype (CD127 h ⁇ CD62L hi , KLRGl' 0 ) and chPDl-CD28 T cells expressed effector memory phenotype markers (CD127' 0 , CD62L' 0 , KLRGl hl ) (Fig.5b).
• CD127 h ⁇ CD62L hi , KLRGl' 0 chPDl-CD28 T cells expressed effector memory phenotype markers (CD127' 0 , CD62L' 0 , KLRGl hl )
• chPDl-DaplO and chPDl-CD28 receptors induce different T-cell phenotypes, which could contribute to altered in vivo anti-tumor efficacy.
• Treatment with chPDl-DaplO T cells leads to a greater reduction in tumor burden and increased survival of RMA-GFP -bearing mice
• lymphoma-bearing mice were treated with two doses of wtPDl, chPDl-DaplO or chPDl-CD28 T cells.
• Treatment with chPDl-DaplO or chPDl-CD28 T cells significantly reduced tumor burden, but chPDl-DaplO T cells reduced tumor burden significantly more than chPD 1 -CD28 T cells (Fig.6a).
• chPD 1 -Dap 10 T cells led to long-term, tumor-free survival in a higher percentage of mice (66% of mice) compared with mice treated with chPDl-CD28 T cells (14% of mice) (Fig.6b).
• One potential factor that contributed to the enhanced anti-tumor efficacy of chPDl-DaplO T cells was their increased in vivo persistence in the spleens and lymph nodes of lymphoma-bearing mice (Fig.6c).
• Ly5.1 + chPDl-DaplO T cells were still detectable by flow cytometry in the spleen and lymph nodes 14 days after T-cell injection, whereas the chPDl-CD28 T cells were not detected after day 10.
• chPDl T cells can reduce tumor burden and increase survival in this mouse model of lymphoma and that inclusion of a Dap 10 co-stimulatory domain has enhanced in vivo therapeutic efficacy compared with a
• CD28-containing chPDl receptor CD28-containing chPDl receptor
• the chPDl -expressing T cells secreted pro-inflammatory cytokines and lysed PDL-expressing tumor cells and also reduced tumor burden and increased tumor-free survival in lymphoma-bearing mice.
• the chPD l receptor containing a Dap 10 co-stimulatory domain was functionally superior compared with the chPD l receptor containing a CD28 co-stimulatory domain.
• PD 1 -CD28 switch receptor which replaces the cytoplasmic domain of PD 1 with the cytoplasmic domain of CD28, has been shown to prevent T-cell inhibition. 37"40
• the PD 1 -CD28 switch receptor When co-expressed with a tumor-specific T-cell receptor or CAR, the PD 1 -CD28 switch receptor induces T-cell activation as shown by extracellular signal regulated kinase phosphorylation, cytokine secretion, proliferation, granzyme B expression and enhanced anti-tumor function.
• CAR T-cell clinical trials are using second-generation CARs consisting of CD3 ⁇ and CD28 or 4- lBB co-stimulatory domains. 1 - 3 - 4 - > 5 - ' 6 ⁇ 33 ⁇ 41
• One difference that is observed between CAR T cells with CD28 or 4- I BB signalling domains is that inclusion of 4- I BB induces a central memory phenotype, and these T cells persist longer in vivo, and have stronger anti-tumor efficacy whereas T cells with a CD28-CAR induce an effector memory or effector cell phenotype and do not live as long in vivo.
• NKG2D/Dap l 0 Stimulation of NKG2D/Dap l 0 has recently been shown to induce a central memory phenotype in murine effector CD8 cells in part due to differential activation of mTOR. 29 Interestingly, mTOR activates specific metabolic pathways in T cells such as aerobic glycolysis and compared with CD28 co-stimulation, activation through NKG2D/Dap l 0 shows weaker activation of mTOR. 29 43 Hence, the induction of mTOR activation, metabolism and cell differentiation are likely key characteristics in CAR T-cell success.
• cytokine release syndrome 44 - 43 This may include elevation of cytokines including IFN-)', GM-CSF, IL- 10 and IL-6 following CAR T-cell infusion and the dramatic increase in cytokines generally correlates with expansion and activation of adoptively transferred cells.
• the challenge may lie in selecting the appropriate CAR design to mitigate or prevent uncontrolled inflammation without hindering the antitumor efficacy of T cells.
• Tumor-bearing mice receiving chPD l T cells did not show any adverse effects following treatment and survived long-term; however, the degree of cytokine release syndrome severity is probably dictated by disease burden at the time of infusion. 44 47
• chPD l -Dap l O T cells may be combined with agents that prevent cytokine release syndrome, such as IL-6R blockade.
• anti-inflammatory cytokines has been shown to not only inhibit CAR T-cell efficacy but also to induce chronic toxicity in some studies. 49 Therefore, without being bound by theory, the decrease in IL- 10 secretion from chPD l -Dap lO T cells may contribute to their enhanced in vivo anti-tumor efficacy.
• chPD l receptor was developed that induces strong antitumor T-cell responses and induction of long-term, tumor-free survival in an immunocompetent mouse model of lymphoma.
• the strong induction of pro-inflammatory cytokines induced by inclusion of a Dapl O co-stimulatory receptor may be beneficial for anti-tumor therapy.
• EXAMPLE 2 Human T cells expressing a chimeric-PD l -Dapl O receptor as an
• chimeric antigen receptors consisting of signaling domains fused to receptors that recognize tumor antigens can be created and expressed in T cells.
• CAR chimeric antigen receptors
• one receptor that is a target for a new chimeric antigen receptor is PD l because the ligands for the PD l receptor are expressed on many cancer types.
• chPD l human chimeric PD l receptor
• the Dap l O costimulatory domain was also included in the chPD l receptor as discussed in Example 1.
• the nucleic acid sequence of the CAR is presented in SEQ ID NO: 2 and the amino acid sequence is presented in SEQ ID NO: 3.
• SEQ ID NO: 3 The nucleic acid sequence of the CAR is presented in SEQ ID NO: 3.
• the chPD l receptor was successfully expressed on the surface of human T cells and expression of chPD l T cells induced significant tumor cell lysis of all tumor cell lines and secreted pro-inflammatory (IFNy, TNFa, IL-2, GM-CSF, IL- 17, and IL-21 ) cytokines. Therefore, adoptive transfer of chPD 1 - Dap l O T cells represents a novel therapeutic strategy to treat multiple types of cancer.
• Pardoll DM The blockade of immune checkpoints in cancer immunotherapy. Nat Rev CancerlOM; 12:252-64.
• NKG2D is a
• Tumor PD-L 1 co-stimulates primary human CD8 + cytotoxic T cells modified to express a PD 1 :CD28 chimeric receptor.
• Mol ImmunollO M 51:263-72.
• Immunotherapy 201 5:677-81 .

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