INHIBITORY CHIMERIC ANTIGEN RECEPTORS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001]This application is a § 371 filing of PCT/IB2015/058650 filed Nov. 9, 2015, which claims the benefit of U.S. Provisional Application No. 62/078,927 filed Nov. 12, 2014, and U.S. Provisional Application No. 62/081,960 filed Nov. 19, 2014, both of which are hereby incorporated by reference in their entireties.

SEQUENCE LISTING

[0002]The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Nov. 7, 2019, is named ALGN-002_02US_333466-2020_SL.txt and is 998,701 bytes in size.

FIELD OF THE INVENTION

[0003]The invention relates to negative T-cell signal inducing chimeric antigen receptor (N-CAR or ICAR) and to T-cells comprising such N-CAR as well as a positive T-cell signal inducing CAR (P-CAR) as well as their use in therapy.

BACKGROUND

[0004]T-cell therapies based on redirected T-cell targeting using chimeric antigen receptor (CAR) are beginning to show great promise in the clinic, particularly in the oncology setting (see Hutchinson L., Nat Rev Clin Oncol. 2014 Oct. 28; Lee D W et al., Lancet. 2014 Oct. 10. pii: S0140-6736(14)61403-3 or Grupp S A et al., N Engl J Med. 2013 Apr. 18; 368(16):1509-18). Given the growing enthusiasm of the field, there is a significant effort being made to identify appropriate targets for CAR T-cell therapy. Given the potency of such therapeutics, the field's ability to identify novel targets for such therapy is hindered by concerns about on-target off-tissue (meaning off-tumor) activity. Such events not only mitigate efficacy but also present tremendous safety challenges as demonstrated by recent clinical events (see Morgan R A et al, Mol Ther. 2010 April; 18(4):843-51; Morgan R A et al., J Immunother. 2013 February; 36(2):133-51 or Linette G P et al., Blood 2013 Aug. 8; 122(6):863-71). Clinical approaches to mitigate these safety concerns while effective also act directly or indirectly on the infused CAR T-cell therapeutic entities.

[0005]In order to address these safety issues pertaining to on-target off-tissue activity of CAR T-cells, and expand the target space amenable to this mode of therapeutics, there is growing emphasis in creating logic gates to modulate T-cell signaling (see Federov V D et al., Sci Transl Med. 2013 Dec. 11; 5(215):215ra172).

[0006]One such approach involves using a NOT gate, wherein the T-cell expresses two or more CARs on its cell surface. CARs that provide positive T-cell signals (P-CARs) bind to tumor antigens to enable T-cell activation and/or proliferation and/or cytokine secretion, and/or cytotoxicity mediated by CD3zeta or other immunoreceptor tyrosine-based activation motif (ITAM) containing motifs; while CARs that provide a negative T-cell signal (N-CARs) bind to the off-tissue antigens and attenuate or abrogate the positive signals.

[0007]Therefore under the on-tissue (on-tumor) scenario the T-cell only receives the P-CAR signal and subsequent activation and cytotoxicity and in the off-tissue (off-tumor) scenario the T-cell receives both the P-CAR and N-CAR signals, whereby the latter attenuates or terminates downstream signaling leading to impaired or no activation and cytotoxicity.

[0008]Therefore, there is a need for negative or inhibitory CAR (N-CAR) that can be used to generate a negative signal suitable to prevent off target activation of P-CAR T-cells (T-cells comprising a P-CAR). It would be an additional benefit if such negative signal is short-termed, reversible and sufficient to attenuate or prevent on-target off-tissue activity of CAR T-cells comprising such N-CAR.

DETAILED DESCRIPTION OF THE INVENTION

[0009]General Techniques

[0010]The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. I. Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds., 1993-1998) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds., Harwood Academic Publishers, 1995).

Definitions

[0011]The following terms, unless otherwise indicated, shall be understood to have the following meanings: the term “isolated molecule” as referring to a molecule (where the molecule is, for example, a polypeptide, a polynucleotide, or an antibody) that by virtue of its origin or source of derivation (1) is not associated with naturally associated components that accompany it in its native state, (2) is substantially free of other molecules from the same source, e.g., species, cell from which it is expressed, library, etc., (3) is expressed by a cell from a different species, or (4) does not occur in nature. Thus, a molecule that is chemically synthesized, or expressed in a cellular system different from the system from which it naturally originates, will be “isolated” from its naturally associated components. A molecule also may be rendered substantially free of naturally associated components by isolation, using purification techniques well known in the art. Molecule purity or homogeneity may be assayed by a number of means well known in the art. For example, the purity of a polypeptide sample may be assayed using polyacrylamide gel electrophoresis and staining of the gel to visualize the polypeptide using techniques well known in the art. For certain purposes, higher resolution may be provided by using HPLC or other means well known in the art for purification.

[0012]An “antibody” is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule. As used herein, the term encompasses not only intact polyclonal or monoclonal antibodies, but also, unless otherwise specified, any antigen binding portion thereof that competes with the antibody for specific binding, fusion proteins comprising an antigen binding portion, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site. Antigen binding portions include, for example, Fab, Fab′, F(ab′)2, Fd, Fv, domain antibodies (dAbs, e.g., shark and camelid antibodies), fragments including complementarity determining regions (CDRs), single chain variable fragment antibodies (scFv), maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide. An antibody includes an antibody of any class, such as IgG, IgA, or IgM (or sub-class thereof), and the antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant region of its heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy-chain constant regions that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0013]A “variable region” of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination. As known in the art, the variable regions of the heavy and light chains each consist of four framework regions (FRs) connected by three complementarity determining regions (CDRs) also known as hypervariable regions, and contribute to the formation of the antigen binding site of antibodies. If variants of a subject variable region are desired, particularly with substitution in amino acid residues outside of a CDR region (e.g., in the framework region), appropriate amino acid substitution, preferably, conservative amino acid substitution, can be identified by comparing the subject variable region to the variable regions of other antibodies which contain CDR1 and CDR2 sequences in the same canonincal class as the subject variable region (Chothia and Lesk, J Mol Biol 196(4): 901-917, 1987).

[0014]In certain embodiments, definitive delineation of a CDR and identification of residues comprising the binding site of an antibody is accomplished by solving the structure of the antibody and/or solving the structure of the antibody-ligand complex. In certain embodiments, that can be accomplished by any of a variety of techniques known to those skilled in the art, such as X-ray crystallography. In certain embodiments, various methods of analysis can be employed to identify or approximate the CDR regions. In certain embodiments, various methods of analysis can be employed to identify or approximate the CDR regions. Examples of such methods include, but are not limited to, the Kabat definition, the Chothia definition, the AbM definition, the contact definition, and the conformational definition.

[0015]The Kabat definition is a standard for numbering the residues in an antibody and is typically used to identify CDR regions. See, e.g., Johnson & Wu, 2000, Nucleic Acids Res., 28: 214-8. The Chothia definition is similar to the Kabat definition, but the Chothia definition takes into account positions of certain structural loop regions. See, e.g., Chothia et al., 1986, J. Mol. Biol., 196: 901-17; Chothia et al., 1989, Nature, 342: 877-83. The AbM definition uses an integrated suite of computer programs produced by Oxford Molecular Group that model antibody structure. See, e.g., Martin et al., 1989, Proc Natl Acad Sci (USA), 86:9268-9272; “AbM™, A Computer Program for Modeling Variable Regions of Antibodies,” Oxford, UK; Oxford Molecular, Ltd. The AbM definition models the tertiary structure of an antibody from positive sequence using a combination of knowledge databases and ab initio methods, such as those described by Samudrala et al., 1999, “Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach,” in PROTEINS, Structure, Function and Genetics Suppl., 3:194-198. The contact definition is based on an analysis of the available complex crystal structures. See, e.g., MacCallum et al., 1996, J. Mol. Biol., 5:732-45. In another approach, referred to herein as the “conformational definition” of CDRs, the positions of the CDRs may be identified as the residues that make enthalpic contributions to antigen binding. See, e.g., Makabe et al., 2008, Journal of Biological Chemistry, 283:1156-1166. Still other CDR boundary definitions may not strictly follow one of the above approaches, but will nonetheless overlap with at least a portion of the Kabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues do not significantly impact antigen binding. As used herein, a CDR may refer to CDRs defined by any approach known in the art, including combinations of approaches. The methods used herein may utilize CDRs defined according to any of these approaches. For any given embodiment containing more than one CDR, the CDRs may be defined in accordance with any of Kabat, Chothia, extended, AbM, contact, and/or conformational definitions.

[0016]As known in the art, a “constant region” of an antibody refers to the constant region of the antibody light chain or the constant region of the antibody heavy chain, either alone or in combination.

[0017]As used herein, “monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler and Milstein, 1975, Nature 256:495, or may be made by recombinant DNA methods such as described in U.S. Pat. No. 4,816,567. The monoclonal antibodies may also be isolated from phage libraries generated using the techniques described in McCafferty et al., 1990, Nature 348:552-554, for example. As used herein, “humanized” antibody refers to forms of non-human (e.g. murine) antibodies that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab′, F(ab′)2 or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. Preferably, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a CDR of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. The humanized antibody may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences, but are included to further refine and optimize antibody performance.

[0018]A “human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen binding residues.

[0019]The term “chimeric antibody” is intended to refer to antibodies in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.

[0020]The term “epitope” refers to that portion of a molecule capable of being recognized by and bound by an antibody at one or more of the antibody's antigen-binding regions. Epitopes often consist of a surface grouping of molecules such as amino acids or sugar side chains and have specific three-dimensional structural characteristics as well as specific charge characteristics. In some embodiments, the epitope can be a protein epitope. Protein epitopes can be linear or conformational. In a linear epitope, all of the points of interaction between the protein and the interacting molecule (such as an antibody) occur linearly along the positive amino acid sequence of the protein. A “nonlinear epitope” or “conformational epitope” comprises noncontiguous polypeptides (or amino acids) within the antigenic protein to which an antibody specific to the epitope binds. The term “antigenic epitope” as used herein, is defined as a portion of an antigen to which an antibody can specifically bind as determined by any method well known in the art, for example, by conventional immunoassays. Once a desired epitope on an antigen is determined, it is possible to generate antibodies to that epitope, e.g., using the techniques described in the present specification. Alternatively, during the discovery process, the generation and characterization of antibodies may elucidate information about desirable epitopes. From this information, it is then possible to competitively screen antibodies for binding to the same epitope. An approach to achieve this is to conduct competition and cross-competition studies to find antibodies that compete or cross-compete with one another for binding to the antigen.

[0021]The term “signaling domain” refers to the functional portion of a protein which acts by transmitting information within the cell to regulate cellular activity via defined signaling pathways by generating second messengers or functioning as effectors by responding to such messengers.

[0022]The term “off-tissue antigen” (or off-tumor antigen) refers to an antigen which is present on non-tumor tissue and not present on the tumor of interest (tumor to be treated by the cells of the invention comprising a P-CAR directed to a tumor antigen and a N-CAR directed to an off-tissue antigen), or only present on the tumor of interest at much lower levels compared to levels of tumor antigen (i.e. the antigen present on the tumor of interest and targeted by the P-CAR).

[0023]The term “anti-tumor effect” refers to a biological effect which can be manifested by various means, including but not limited to, e.g., a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, decrease in tumor cell proliferation, decrease in tumor cell survival, or amelioration of various physiological symptoms associated with the cancerous condition. An “anti-tumor effect” can also be manifested by the ability of the cells of the invention in prevention of the occurrence of tumor in the first place.

[0024]The term “autologous” refers to any material derived from the same individual to whom it is later to be re-introduced into the individual.

[0025]The term “allogeneic” refers to any material derived from a different animal of the same species as the individual to whom the material is introduced. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical. In some aspects, allogeneic material from individuals of the same species may be sufficiently unlike genetically to interact antigenically

[0026]The term “xenogeneic” refers to a graft derived from an animal of a different species.

[0027]The term “cancer” refers to a disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers are described herein and include but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cell cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer and the like.

[0028]The term “conservative sequence modifications” refers to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody or antibody fragment containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody or antibody fragment of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within a CAR of the invention can be replaced with other amino acid residues from the same side chain family and the altered CAR can be tested using the functional assays described herein.

[0029]In some embodiments, the “fragment” of a sequence of amino acids is shorter than said sequence of amino acid. In some embodiments, the fragment of a sequence of amino acids is at least 1%, 5% 10%, 20%, 40%, 50%, 60%, 70%, 80% or 90% shorter than said sequence of amino acids. In some embodiments, the fragment of a sequence of amino acids is shorter by at least 1, 5, 10, 20, 50, 100, 200, 300 amino acids as compared to said sequence of amino acids.

[0030]Unless otherwise specified, the left to right orientation of amino acid sequences or formula representing amino acid sequences are disclosed using the conventional left to right orientation N-Term to C-term.

[0031]N-terminal flanking region of a domain refers to the sequence of amino acid which is directly adjacent to the N-terminal amino acid of said domain. C-terminal flanking region of a domain refers to the sequence of amino acid which is directly adjacent to the C-terminal amino acid of said domain. For example, in the sequence seq1-ITIM-seq2, seq1 is the N-terminal flanking region of the ITIM intracellular domain and seq2 N-terminal flanking region of the ITIM intracellular domain. In another example, the naturally occurring N-terminal flanking region of ITIM.*ITSM intracellular domains is the sequence of amino acid which is directly adjacent to the N-terminal amino acid of the ITIM motif of the ITIM.*ITSM intracellular domain. In another example, the naturally occurring C-terminal flanking region of ITIM.*ITSM intracellular domain is the sequence of amino acid which is directly adjacent to the C-terminal amino acid of the ITSM motif of the ITIM.*ITSM intracellular domain.

[0032]In another example, the naturally occurring N-terminal flanking region of an ITIM only intracellular domain is the sequence of amino acid which is directly adjacent to the N-terminal amino acid of the ITIM of the ITIM only intracellular domain. In another example, the naturally occurring C-terminal flanking region of an ITIM only intracellular domain is the sequence of amino acid which is directly adjacent to the C-terminal amino acid of the ITIM of the ITIM only intracellular domain.

[0033]In another example, the naturally occurring N-terminal flanking region of an ITSM only intracellular domain is the sequence of amino acid which is directly adjacent to the N-terminal amino acid of the ITSM of the ITSM only intracellular domain. In another example, the naturally occurring C-terminal flanking region of an ITSM only intracellular domain is the sequence of amino acid which is directly adjacent to the C-terminal amino acid of the ITSM of the ITSM only intracellular domain.

[0034]The term “stimulation,” refers to a positive 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-β, and/or reorganization of cytoskeletal structures, and the like.

[0035]The term “antigen presenting cell” or “APC” refers to an immune system cell such as an accessory cell (e.g., a B-cell, a dendritic cell, and the like) that displays a foreign antigen complexed with major histocompatibility complexes (MHC's) on its surface. T-cells may recognize these complexes using their T-cell receptors (TCRs). APCs process antigens and present them to T-cells.

[0036]An “intracellular signaling domain,” as the term is used herein, refers to an intracellular portion of a molecule.

[0037]The term “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 other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene, cDNA, or RNA, 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.

[0038]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 a RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).

[0039]The term “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.

[0040]The term “endogenous” refers to any material from or produced inside an organism, cell, tissue or system.

[0041]The term “exogenous” refers to any material introduced from or produced outside an organism, cell, tissue or system.

[0042]The term “expression” refers to the transcription and/or translation of a particular nucleotide sequence driven by a promoter.

[0043]The term “transfer vector” refers to 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 “transfer vector” includes an autonomously replicating plasmid or a virus. The term should also be construed to further include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, a polylysine compound, liposome, and the like. Examples of viral transfer vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.

[0044]The term “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, including cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0045]The term “lentivirus” 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 most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses.

[0046]The term “lentiviral vector” refers to a vector derived from at least a portion of a lentivirus genome, including especially a self-inactivating lentiviral vector as provided in Milone et al., Mol. Ther. 17(8): 1453-1464 (2009). Other examples of lentivirus vectors that may be used in the clinic, include but are not limited to, e.g., the LENTIVECTOR® gene delivery technology from Oxford BioMedica, the LENTIMAX™ vector system from Lentigen and the like. Nonclinical types of lentiviral vectors are also available and would be known to one skilled in the art.

[0047]The term “homologous” or “identity” refers to the subunit sequence identity between two polymeric molecules, e.g., between two nucleic acid molecules, such as, two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomeric subunit; e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous or identical at that position. The homology between two sequences is a direct function of the number of matching or homologous positions; e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two sequences are homologous, the two sequences are 50% homologous; if 90% of the positions (e.g., 9 of 10), are matched or homologous, the two sequences are 90% homologous.

[0048]The term “operably linked” or “transcriptional control” refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences can be contiguous with each other and, e.g., where necessary to join two protein coding regions, are in the same reading frame.

[0049]The terms “polypeptide”, “oligopeptide”, “peptide” and “protein” are used interchangeably herein to refer to chains of amino acids of any length. The chain may be linear or branched, it may comprise modified amino acids, and/or may be interrupted by non-amino acids. The terms also encompass an amino acid chain that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. It is understood that the polypeptides can occur as single chains or associated chains.

[0050]As known in the art, “polynucleotide,” or “nucleic acid,” as used interchangeably herein, refer to chains of nucleotides of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a chain by DNA or RNA polymerase. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modification to the nucleotide structure may be imparted before or after assembly of the chain. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications include, for example, “caps”, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide(s). Further, any of the hydroxyl groups ordinarily present in the sugars may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or may be conjugated to solid supports. The 5′ and 3′ terminal OH can be phosphorylated or substituted with amines or organic capping group moieties of from 1 to 20 carbon atoms. Other hydroxyls may also be derivatized to standard protecting groups. Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars that are generally known in the art, including, for example, 2′-O-methyl-, 2′-O-allyl, 2′-fluoro- or 2′-azido-ribose, carbocyclic sugar analogs, alpha- or beta-anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs and abasic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P(O)S (“thioate”), P(S)S (“dithioate”), (O)NR₂ (“amidate”), P(O)R, P(O)OR′, CO or CH₂ (“formacetal”), in which each R or R′ is independently H or substituted or unsubstituted alkyl (1-20 C) optionally containing an ether (—O—) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.

[0051]An antibody that “preferentially binds” or “specifically binds” (used interchangeably herein) to an epitope is a term well understood in the art, and methods to determine such specific or preferential binding are also well known in the art. A molecule is said to exhibit “specific binding” or “preferential binding” if it reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with a particular cell or substance than it does with alternative cells or substances. An antibody “specifically binds” or “preferentially binds” to a target if it binds with greater affinity, avidity, more readily, and/or with greater duration than it binds to other substances.

[0052]A “host cell” includes an individual cell or cell culture that can be or has been a recipient for vector(s) for incorporation of polynucleotide inserts. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell includes cells transfected in vivo with a polynucleotide(s) of this invention.

[0053]The term “compete”, as used herein with regard to an antibody, means that a first antibody, or an antigen-binding portion thereof, binds to an epitope in a manner sufficiently similar to the binding of a second antibody, or an antigen-binding portion thereof, such that the result of binding of the first antibody with its cognate epitope is detectably decreased in the presence of the second antibody compared to the binding of the first antibody in the absence of the second antibody. The alternative, where the binding of the second antibody to its epitope is also detectably decreased in the presence of the first antibody, can, but need not be the case. That is, a first antibody can inhibit the binding of a second antibody to its epitope without that second antibody inhibiting the binding of the first antibody to its respective epitope. However, where each antibody detectably inhibits the binding of the other antibody with its cognate epitope or ligand, whether to the same, greater, or lesser extent, the antibodies are said to “cross-compete” with each other for binding of their respective epitope(s). Both competing and cross-competing antibodies are encompassed by the present invention. Regardless of the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, conformational change, or binding to a common epitope, or portion thereof), the skilled artisan would appreciate, based upon the teachings provided herein, that such competing and/or cross-competing antibodies are encompassed and can be useful for the methods disclosed herein.

[0054]As used herein, “treatment” is an approach for obtaining beneficial or desired clinical results.

[0055]As used herein, an “effective dosage” or “effective amount” of drug, compound, or pharmaceutical composition is an amount sufficient to effect any one or more beneficial or desired results. For prophylactic use, beneficial or desired results include eliminating or reducing the risk, lessening the severity, or delaying the outset of the disease, including biochemical, histological and/or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes presenting during development of the disease. For therapeutic use, beneficial or desired results include clinical results such as reducing incidence or amelioration of one or more symptoms of various diseases or conditions (such as, for example without limitation, renal cell, gastric, head and neck, lung, ovarian, and pancreatic cancers), decreasing the dose of other medications required to treat the disease, enhancing the effect of another medication, and/or delaying the progression of the disease. An effective dosage can be administered in one or more administrations. For purposes of this invention, an effective dosage of drug, compound, or pharmaceutical composition is an amount sufficient to accomplish prophylactic or therapeutic treatment either directly or indirectly. As is understood in the clinical context, an effective dosage of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an “effective dosage” may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable result may be or is achieved.

[0056]An “individual” or a “subject” is a mammal, more preferably, a human. Mammals also include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, mice and rats.

[0057]As used herein, “vector” means a construct, which is capable of delivering, and, preferably, expressing, one or more gene(s) or sequence(s) of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.

[0058]As used herein, “expression control sequence” means a nucleic acid sequence that directs transcription of a nucleic acid. An expression control sequence can be a promoter, such as a constitutive or an inducible promoter, or an enhancer. The expression control sequence is operably linked to the nucleic acid sequence to be transcribed.

[0059]The term “promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence.

[0060]The term “promoter/regulatory sequence” refers to a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter/regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product. The promoter/regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner.

[0061]The term “constitutive” promoter refers to a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.

[0062]The term “inducible” promoter refers to a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only when an inducer which corresponds to the promoter is present in the cell.

[0063]The term “tissue-specific” promoter refers to a nucleotide sequence which, when operably linked with a polynucleotide encodes or specified by a gene, causes the gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.

[0064]The term “flexible polypeptide linker” or “linker” as used in the context of a scFv refers to a peptide linker that consists of amino acids such as glycine and/or serine residues used alone or in combination, to link variable heavy and variable light chain regions together. In one embodiment, the flexible polypeptide linker is a Glycine/Serine linker and comprises the amino acid sequence (Gly-Gly-Gly-Ser)_n (SEQ ID NO: 2030) or (Gly-Gly-Gly-Gly-Ser)_n (SEQ ID NO: 2031), where n is a positive integer equal to or greater than 1. For example, n=1, n=2, n=3, n=4, n=5, n=6, n=7, n=8, n=9 and n=10. In one embodiment, the flexible polypeptide linkers include, but are not limited to, (Gly₄Ser)₄ (SEQ ID NO: 2032) or (Gly₄Ser)₃ (SEQ ID NO: 2033). In another embodiment, the linkers include multiple repeats of (Gly_xSer)_n, where x=1, 2, 3, 4 or 5 and n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NO: 2034), such as multiple repeat of (GlySer), (Gly₂Ser) or (Gly₅Ser) (SEQ ID NO: 2035). Also included within the scope of the invention are linkers described in WO2012/138475, incorporated herein by reference.

[0065]As used herein, a 5′ cap (also termed an RNA cap, an RNA 7-methylguanosine cap or an RNA m G cap) is a modified guanine nucleotide that has been added to the “front” or 5′ end of a eukaryotic messenger RNA shortly after the start of transcription. The 5′ cap consists of a terminal group which is linked to the first transcribed nucleotide. Its presence is critical for recognition by the ribosome and protection from RNases. Cap addition is coupled to transcription, and occurs co-transcriptionally, such that each influences the other. Shortly after the start of transcription, the 5′ end of the mRNA being synthesized is bound by a cap-synthesizing complex associated with RNA polymerase. This enzymatic complex catalyzes the chemical reactions that are required for mRNA capping. Synthesis proceeds as a multi-step biochemical reaction. The capping moiety can be modified to modulate functionality of mRNA such as its stability or efficiency of translation.

[0066]As used herein, “in vitro transcribed RNA” refers to RNA, preferably mRNA, that has been synthesized in vitro. Generally, the in vitro transcribed RNA is generated from an in vitro transcription vector. The in vitro transcription vector comprises a template that is used to generate the in vitro transcribed RNA.

[0067]As used herein, a “poly(A)” is a series of adenosines attached by polyadenylation to the mRNA. In the preferred embodiment of a construct for transient expression, the polyA is between 50 and 5000 (SEQ ID NO: 2036), preferably greater than 64, more preferably greater than 100, most preferably greater than 300 or 400. poly(A) sequences can be modified chemically or enzymatically to modulate mRNA functionality such as localization, stability or efficiency of translation.

[0068]As used herein, “polyadenylation” refers to the covalent linkage of a polyadenylyl moiety, or its modified variant, to a messenger RNA molecule. In eukaryotic organisms, most messenger RNA (mRNA) molecules are polyadenylated at the 3′ end. The 3′ poly(A) tail is a long sequence of adenine nucleotides (often several hundred) added to the pre-mRNA through the action of an enzyme, polyadenylate polymerase. In higher eukaryotes, the poly(A) tail is added onto transcripts that contain a specific sequence, the polyadenylation signal. The poly(A) tail and the protein bound to it aid in protecting mRNA from degradation by exonucleases. Polyadenylation is also important for transcription termination, export of the mRNA from the nucleus, and translation. Polyadenylation occurs in the nucleus immediately after transcription of DNA into RNA, but additionally can also occur later in the cytoplasm. After transcription has been terminated, the mRNA chain is cleaved through the action of an endonuclease complex associated with RNA polymerase. The cleavage site is usually characterized by the presence of the base sequence AAUAAA near the cleavage site. After the mRNA has been cleaved, adenosine residues are added to the free 3′ end at the cleavage site.

[0069]As used herein, “transient” refers to expression of a non-integrated transgene for a period of hours, days or weeks, wherein the period of time of expression is less than the period of time for expression of the gene if integrated into the genome or contained within a stable plasmid replicon in the host cell.

[0070]The term “signal transduction pathway” refers to the biochemical relationship between a variety of signal transduction molecules that play a role in the transmission of a signal from one portion of a cell to another portion of a cell. The phrase “cell surface receptor” includes molecules and complexes of molecules capable of receiving a signal and transmitting signal across the membrane of a cell.

[0071]Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X.” Numeric ranges are inclusive of the numbers defining the range. It is understood that wherever embodiments are described herein with the language “comprising,” otherwise analogous embodiments described in terms of “consisting of” and/or “consisting essentially of” are also provided.

[0072]Where aspects or embodiments of the invention are described in terms of a Markush group or other grouping of alternatives, the present invention encompasses not only the entire group listed as a whole, but each member of the group individually and all possible subgroups of the main group, but also the main group absent one or more of the group members. The present invention also envisages the explicit exclusion of one or more of any of the group members in the claimed invention.

[0073]Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control. Throughout this specification and claims, the word “comprise,” or variations such as “comprises” or “comprising” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Any example(s) following the term “e.g.” or “for example” is not meant to be exhaustive or limiting.

[0074]Exemplary methods and materials are described herein, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. The materials, methods, and examples are illustrative only and not intended to be limiting.

DESCRIPTION OF FIGURES

[0075]FIGS. 1 and 2 show the dual cell surface expression of P-CAR1 and various N-CARs assessed by multicolor flow cytometry in transduced NFAT-luciferase reporter Jurkat cells.

[0076]FIGS. 3 and 4 show the dual cell surface expression of P-CAR1 and various N-CARs assessed by multicolor flow cytometry in transduced NFkB-luciferase reporter Jurkat cells.

[0077]In FIGS. 1 to 4, P-CAR expression was detected using a recombinant human CD19-mouse IgG Fc fusion protein followed by APC-conjugated F(ab′)2 goat anti-mouse Fcγ (shown on x axis), and N-CAR expression was detected with a biotinylated recombinant human PSMA-human IgG1 Fc fusion protein followed by PE-conjugated streptavidin (y axis).

[0078]FIGS. 5A, 5B and 5C show the inhibitory effect of various N-CARs on P-CAR1 induced T cell activation. Control ΔPD1- or test N-CAR-transduced luciferase reporter Jurkat cells expressing P-CAR1 were incubated with either CD19-expressing AAPCs or dual CD19+PSMA-expressing AAPCs, and luciferase activity was assessed 16 h later. Data are expressed as a ratio of the mean RLU from co-culture with CD19+PSMA AAPCs/CD19 AAPCs. n=6 replicates per sample; data shown are the means+/−95% CI). FIGS. 5A/5C and 5B show results using NFAT-luciferase reporter and NFkB-luciferase reporter Jurkat cells, respectively.

[0079]FIGS. 6 and 7 show the dual cell surface expression of P-CAR2 and N-CARs listed in Table 10 assessed by multicolor flow cytometry in transduced NFAT-luciferase reporter Jurkat cells. FIGS. 8 and 9 show the dual cell surface expression of P-CAR2 and N-CARs listed in Table 10 assessed by multicolor flow cytometry in transduced NFkB-luciferase reporter Jurkat cells. In FIGS. 6 to 9, P-CAR expression was detected using a recombinant human CD19-mouse IgG Fc fusion protein followed by APC-conjugated F(ab′)2 goat anti-mouse Fcγ (shown on x axis), and N-CAR expression was detected with a biotinylated recombinant human PSMA-human IgG1 Fc fusion protein followed by PE-conjugated streptavidin (y axis).

[0080]FIGS. 10A and 10B show the inhibitory effect of various N-CARs on P-CAR2 induced T cell activation. Control ΔPD1- or test N-CAR-transduced luciferase reporter Jurkat cells expressing P-CAR2 were incubated with either CD19-expressing or dual PSMA/CD19-expressing AAPCs, and luciferase activity was assessed 16 h later. Data are expressed as a ratio of the mean RLU from co-culture with CD19+PSMA AAPCs/CD19 AAPCs. n=6 replicates per sample; data shown are the means+/−95% CI. FIGS. 10A and 10B show results using NFAT-luciferase reporter and NFkB-luciferase reporter Jurkat cells, respectively.

DETAILED DESCRIPTION

[0081]The invention relates to a negative signal (or inhibitory) chimeric antigen receptor (N-CAR) comprising

[0082]an extracellular domain comprising an antigen binding domain,

[0083]a transmembrane domain, and,

[0084]an intracellular domain

[0085]wherein the intracellular domain comprises an Immunoreceptor Tyrosine-based Switch Motif ITSM, wherein said ITSM is a sequence of amino acid TX₁YX₂X₃X₄ (SEQ ID NO: 2049),

[0086]wherein

[0087]X₁ is an amino acid,

[0088]X₂ is an amino acid,

[0089]X₃ is an amino acid, and,

[0090]X₄ is V or I.

[0091]In some embodiments the term amino acid refers to a natural amino acid. In some embodiments, the term amino acid refer to an amino acid selected from glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, serine, threonine, tyrosine, cysteine, methionine, lysine, arginine, histidine, tryptophan, aspartic acid, glutamic acid, asparagine or glutamine.

[0092]In some embodiments, when the extracellular domain is a scFv against PSMA, then the intracellular domain is not the intracellular domain of human PD-1.

[0093]In some embodiments, the intracellular domain is not the intracellular domain of human PD-1.

[0094]In some embodiments, the intracellular domain is not the intracellular domain of human BTLA.

[0095]In some embodiments, the intracellular domain is not the intracellular domain of human CD244.

[0096]In some embodiments, the intracellular domain is not SEQ ID No 2000, SEQ ID No 2001 or SEQ ID No 2002.

[0097]In some embodiments, the extracellular domain does not bind to PMSA.

[0098]In some embodiments, the intracellular domain does not comprise the full intracellular domain of PD-1.

[0099]In some embodiments, the ITSM is not TEYATI (SEQ ID NO: 937).

[0100]The intracellular domain or region of the N-CAR includes an inhibitory intracellular signaling domain. An inhibitory intracellular signaling domain is generally responsible for inactivation of the signal from a positive intracellular signaling domain from a P-CAR on the same immune cell in which the N-CAR has been introduced, thereby blocking activation of a normal effector function of the immune cell. The term “effector function” refers to a specialized function of a cell. Effector function of a T-cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines.

[0101]Intracellular Domain of the N-CAR

[0102]In some embodiments, the intracellular domain comprises the following sequence:

[0103]((L1-ITIM-L2)ⁿ-(L3-ITSM-L4)^m)^p, wherein

[0104]n is 0, 1 or an integer greater than 1;

[0105]m is 1 or an integer greater than 1;

[0106]p is 1 or an integer greater than 1;

[0121]X₅ is S, V, I or L,

[0122]X₆ is an amino acid,

[0123]X₇ is an amino acid,

[0124]X₈ is an amino acid, and,

[0125]X₉ is V, I or L, and,

[0126]the ITSM is the sequence TX₁YX₂X₃X₄ (SEQ ID NO: 2049), wherein

[0127]X₁ is an amino acid,

[0128]X₂ is an amino acid,

[0129]X₃ is an amino acid, and,

[0130]X₄ is V or I,

[0131]or a variant thereof.

[0132]In some embodiments, the known inhibitory receptor refers to an inhibitory receptor comprising an extracellular domain, a transmembrane domain and an intracellular domain which do not comprise any ITIM or ITSM and which provides a negative signal able to reduce the activation signal provided by the TCR/CD3 complex in a T-cell.

[0133]In some embodiments, the known inhibitory receptor refers to an inhibitory receptor comprising an extracellular domain, a transmembrane domain and an intracellular domain which provide a negative signal able to reduce the activation signal provided by the TCR/CD3 complex in a T-cell.

[0134]In some embodiments, the known inhibitory receptor is selected from CTLA4, LAG3 HAVCR2 (TIM3), KIR2DL2, LILRB1, TIGIT, CEACAM1, CSF1R, CD5, CD96, CD22 and LAIR1. In a preferred embodiment, the known inhibitory receptor is KIR2DL2.

[0135]ITIM.*ITSM intracellular domain refers to a domain comprising one ITIM and one ITSM. ITSM only intracellular domain refers to a domain comprising one ITSM and no ITIM. ITIM only intracellular domain refers to a domain comprising one ITIM and no ITSM.

[0136]When one or more of n, m or p are greater than 1, each occurrence of L1, L2, L3, L4, ITIM and ITSM is selected independently from the other. For example, the intracellular domain of the N-CAR may comprise several ITSM having different sequences.

• • (b) a naturally occurring N-terminal flanking region of ITIM.*ITSM intracellular domains selected from

• • (c) a naturally occurring intracellular domain from a known inhibitory receptor selected from the sequences shown in table 2, wherein said intracellular domain is N-terminally flanking to a sequence in (b) above; and
  • (d) a non-naturally occurring sequence comprising between 1 and 500 amino acids.

• • (f) a naturally occurring N-terminal flanking region of ITSM only intracellular domains selected from;

• • (g) a naturally occurring intracellular domain between ITIM and ITSM from proteins that have ITIM.*ITSM motif selected from;

• • (h) a naturally occurring intracellular domain from known inhibitory receptors selected from the sequences shown in table 2 wherein said intracellular domain is N-terminally flanking to a sequence in (f) or (g) above; and
  • (i) a non-naturally occurring sequence comprising between 1 and 500 amino acids.

• • (k) a naturally occurring C-terminal flanking region of ITSM only intracellular domain selected from

• • (l) a naturally occurring intracellular domain from a known inhibitory receptor selected from the sequences shown in table 2 wherein said intracellular domain is C-terminally flanking to a sequence in (j) or (k) above; and
  • (m) a non-naturally occurring sequence comprising between 1 and 500 amino acids.

[0153]In some embodiments the intracellular domain comprises the sequence (L3-ITSM-L4)^m (i.e, n is 0 and p is 1).

[0154]In some embodiments, the intracellular domain comprises the sequence L3-ITSM-L4 (i.e, n is 0, m is 1 and p is 1).

[0155]In some embodiments, the intracellular domain comprises the sequence L3-ITSM-L4-L3-ITSM-L4 (i.e, n is 0, m is 2 and p is 1).

[0156]In some embodiments, the intracellular domain comprises the following sequence:

[0157]((L1-ITIM-L2)ⁿ-(L3-ITSM-L4)^m)^p, wherein

[0158]n is 0;

[0159]m is 1;

[0160]p is 1;

[0167]the ITSM is the sequence TX₁YX₂X₃X₄ (SEQ ID NO: 2049), wherein

[0168]X₁ is an amino acid,

[0169]X₂ is an amino acid,

[0170]X₃ is an amino acid, and,

[0171]X₄ is V or I,

[0172]or a variant thereof.

[0173]In some embodiments, the intracellular domain comprises the following sequence:

[0174]((L1-ITIM-L2)ⁿ-(L3-ITSM-L4)^m)^p, wherein

[0175]n is 0;

[0176]m is 1;

[0177]p is 1;

[0178]L3 is selected from

and L4 comprises one sequence selected from the group consisting of

• • (k)

• • and optionally
  • (l) a naturally occurring intracellular domain from a known inhibitory receptor such as any of the sequences shown in table 2 or a fragment thereof wherein said intracellular domain is C-terminally flanking to a sequence in (k) above;

[0182]and the ITSM is the sequence TX₁YX₂X₃X₄ (SEQ ID NO: 2049), wherein

[0183]X₁ is an amino acid,

[0184]X₂ is an amino acid,

[0185]X₃ is an amino acid, and,

[0186]X₄ is V or I,

[0187]or a variant thereof.

[0188]In some embodiments, the intracellular domain comprises the following sequence:

[0189]((L1-ITIM-L2)ⁿ-(L3-ITSM-L4)^m)^p, wherein

[0190]n is 0;

[0191]m is 1;

[0192]p is 1;

[0193]L3 is selected from

• • and optionally
  • (l) a naturally occurring intracellular domain from a known inhibitory receptor selected from the sequences shown in table 2 or a fragment thereof wherein said intracellular domain is C-terminally flanking to a sequence in (k) above;

[0198]and the ITSM is the sequence TX₁YX₂X₃X₄ (SEQ ID NO: 2049), wherein

[0199]X₁ is an amino acid,

[0200]X₂ is an amino acid,

[0201]X₃ is an amino acid, and,

[0202]X₄ is V or I,

[0203]or a variant thereof.

[0204]In some embodiments, the intracellular domain comprises the following sequence:

[0205]((L1-ITIM-L2)ⁿ-(L3-ITSM-L4)^m)^p, wherein

[0206]n is 0;

[0207]m is 1;

[0208]p is 1;

[0209]L3 is selected from

• • and
  • (l) a naturally occurring intracellular domain from a known inhibitory receptor selected from the sequences shown in table 2 or a fragment thereof wherein said intracellular domain is C-terminally flanking to a sequence in (k) above;

[0214]and the ITSM is the sequence TX₁YX₂X₃X₄ (SEQ ID NO: 2049), wherein

[0215]X₁ is an amino acid,

[0216]X₂ is an amino acid,

[0217]X₃ is an amino acid, and,

[0218]X₄ is V or I,

[0219]or a variant thereof.

[0220]In some embodiments, the intracellular domain comprises the following sequence:

[0221]((L1-ITIM-L2)ⁿ-(L3-ITSM-L4)^m)^p, wherein

[0222]n is 0;

[0223]m is 1;

[0224]p is 1;

[0225]L3 is selected from

[0231]and the ITSM is the sequence TX₁YX₂X₃X₄ (SEQ ID NO: 2049), wherein

[0232]X₁ is an amino acid,

[0233]X₂ is an amino acid,

[0234]X₃ is an amino acid, and,

[0235]X₄ is V or I,

[0236]or a variant thereof.

[0237]In some embodiments, the intracellular domain comprises the following sequence:

[0238]((L1-ITIM-L2)ⁿ-(L3-ITSM-L4)^m)^p, wherein

[0239]n is 0;

[0240]m is 1;

[0241]p is 1 or 2;

[0246]the ITSM is the sequence TX₁YX₂X₃X₄ (SEQ ID NO: 2049), wherein

[0247]X₁ is an amino acid,

[0248]X₂ is an amino acid,

[0249]X₃ is an amino acid, and,

[0250]X₄ is V or I.

[0251]In some embodiments, the intracellular domain comprises the sequence (L1-ITIM-L2-L3-ITSM-L4)^p wherein

[0252]p is 1, 2, 3, 4 or 5;

[0253]L1 is a naturally occurring N-terminal flanking region of an ITIM only intracellular domain or a fragment thereof such as, for example, any of the sequences shown in Table 3 below or a fragment thereof;

[0254]L2 is absent;

[0255]L3 is a naturally occurring intracellular domain between ITIM and ITSM from proteins that have ITIM.*ITSM motif or a fragment thereof such as, for example, any of the sequences shown in Table 5 below or a fragment thereof;

[0256]L4 is a naturally occurring C-terminal flanking region of an ITIM.*ITSM intracellular domain or a fragment thereof such as, for example, any of the sequences shown in Table 7 below or a fragment thereof; or a naturally occurring C-terminal flanking region of ITSM only intracellular domain or a fragment thereof such as, for example, any of the sequences shown in Table 8 below or a fragment thereof.

[0257]In some embodiments, the intracellular domain comprises the sequence (L1-ITIM-L2-L3-ITSM-L4)^p wherein

[0258]p is 1, 2, 3, 4 or 5;

[0259]L1 is a naturally occurring N-terminal flanking region of ITIM only intracellular domains selected from the following sequences;

[0260]L2 is absent;

[0261]L3 is a naturally occurring intracellular domain between ITIM and ITSM from proteins that have ITIM.*ITSM motif selected from the following sequences:

[0262]L4 is a naturally occurring C-terminal flanking region of ITIM.*ITSM intracellular domains selected from the following sequences:

• • or a naturally occurring C-terminal flanking region of ITSM only intracellular domains selected from the following sequences:

or a variant thereof.

[0264]In some embodiments, the non-naturally occurring sequence of (d), (i) and (m) comprises between 1 and 500 amino acids, preferably 1 to 400, 1 to 300, 1 to 200, 1 to 100, 10 to 100, 10 to 80, 10 to 60, 10 to 40, 100 to 200, 100 to 300 or 100 to 400.

[0265]In some embodiments, the non-naturally occurring sequence of (d) or (i) is a Glycine/Serine linker (Gly_xSer)_n where x=1, 2, 3, 4 or 5 and n is 1 to 100 (SEQ ID NO: 2037). Preferably the Glycine/Serine linker comprises the amino acid sequence (Gly-Gly-Gly-Ser)_n (SEQ ID NO: 2030) or (Gly-Gly-Gly-Gly-Ser)_n (SEQ ID NO: 2031), where n is a positive integer equal to or greater than 1, preferably between 1 to 100, 1 to 80, 1 to 50, 1 to 20 or 1 to 10. For example, n=1, n=2, n=3, n=4, n=5, n=6, n=7, n=8, n=9 and n=10. In one embodiment, the glycine/serine linkers include, but are not limited to, (Gly₄Ser)₄ (SEQ ID NO: 2032) or (Gly₄Ser)₃ (SEQ ID NO: 2033).

[0266]In some embodiments, X₁ is E, V or I.

[0267]In some embodiments, X₁ is E.

[0268]In some embodiments, X₂ is S or A.

[0269]In some embodiments, X₂ is A.

[0270]In some embodiments, X₃ is E, S, T, Q or V.

[0271]In some embodiments, X₃ is E.

[0272]In some embodiments, X₃ is T.

[0273]In some embodiments, X₂ is I.

[0274]In some embodiments, X₅ is L, V or I.

[0275]In some embodiments, X₅ is L.

[0276]In some embodiments, X₅ is V.

[0277]In some embodiments, X₅ is I.

[0278]In some embodiments, X₆ is A, H, Q, T, D, V, L or E.

[0279]In some embodiments, X₆ is H.

[0280]In some embodiments, X₆ is D.

[0281]In some embodiments, X₇ is A, G, T, V or E.

[0282]In some embodiments, X₇ is A.

[0283]In some embodiments, X₇ is G.

[0284]In some embodiments, X₈ is V, S, D or E.

[0285]In some embodiments, X₈ is S or E.

[0286]In some embodiments, X₈ is E.

[0287]In some embodiments, X₉ is L or V.

[0288]In some embodiments, X₉ is L.

[0289]In some embodiments, X₅ is L or V, X₈ is E and X₉ is L.

[0290]In some embodiments, the ITSM, or at least one of the ITSMs when several ITSMs are present in the intracellular domain, is selected from SEQ ID No 926 to SEQ ID No 1015 (see below table).

[0291]In some embodiments, the ITSM, or at least one of the ITSMs when several ITSMs are present in the intracellular domain is TEYASI (SEQ ID NO: 936).

[0292]In some embodiments, the ITSM, or at least one of the ITSMs when several ITSMs are present in the intracellular domain is TEYSEI (SEQ ID NO: 940).

[0293]In some embodiments, the ITSM, or at least one of the ITSMs when several ITSMs are present in the intracellular domain is TVYSEV (SEQ ID NO: 1011).

[0294]In some embodiments, the ITSM, or at least one of the ITSMs when several ITSMs are present in the intracellular domain is TEYSTI (SEQ ID NO: 942).

[0295]In some embodiments, the ITIM, or at least one of the ITIMs when several ITIMs are present in the intracellular domain is selected from SEQ ID No 1016 to SEQ ID NO: 1100, SEQ ID NO: 2047, and SEQ ID NO: 1102 to SEQ ID 1998 (see below table).

[0296]In some embodiments, the ITIM, or at least one of the ITIMs when several ITIMs are present in the intracellular domain is selected from LSYRSL (SEQ ID NO: 1496), LPYYDL (SEQ ID NO: 1378), LLYSRL (SEQ ID NO: 1334), LIYTLL (SEQ ID NO: 1283), LLYADL (SEQ ID NO: 1303), ISYTTL (SEQ ID NO: 1116), VTYSAL (SEQ ID NO: 1982), IHYSEL (SEQ ID NO: 1059), VDYVIL (SEQ ID NO: 1832), LHYASL (SEQ ID NO: 1218), LDYDYL (SEQ ID NO: 1174), VDYDFL (SEQ ID NO: 1817), VTYSTL (SEQ ID NO: 1983), IIYSEV (SEQ ID NO: 1065), LEYLCL (SEQ ID NO: 1186), VLYGQL (SEQ ID NO: 1901), VPYTPL (SEQ ID NO: 1926), ISYPML (SEQ ID NO: 1115), VSYTNL (SEQ ID NO: 1965), LLYEMV (SEQ ID NO: 1016), VDYNLV (SEQ ID NO: 1825), ITYFAL (SEQ ID NO: 1017), VHYQSV (SEQ ID NO: 1859), VPYVMV (SEQ ID NO: 1929), IPYRTV (SEQ ID NO: 1089), IAYSLL (SEQ ID NO: 1026), VCYGRL (SEQ ID NO: 1813), LKYLYL (SEQ ID NO: 1294), LLYEHV (SEQ ID NO: 1307), ITYSLL (SEQ ID NO: 1125), VLYSEL (SEQ ID NO: 1905), IWYNIL (SEQ ID NO: 1140), ISYKGL (SEQ ID NO: 1018), IDYYNL (SEQ ID NO: 1035), LEYLQL (SEQ ID NO: 1189), LKYRGL (SEQ ID NO: 1301), VLYASV (SEQ ID NO: 1893), LQYLSL (SEQ ID NO: 1386), LFYRHL (SEQ ID NO: 1201), VQYKAV (SEQ ID NO: 1931), LSYSSL (SEQ ID NO: 1499), LSYTKV (SEQ ID NO: 1501), VQYSTV (SEQ ID NO: 1936), VKYNPV (SEQ ID NO: 1882), VVYSEV (SEQ ID NO: 1992), LEYVSV (SEQ ID NO: 1192), LAYHTV (SEQ ID NO: 1019), VQYLRL (SEQ ID NO: 1020), VTYTQL (SEQ ID NO: 1985), IVYTEL (SEQ ID NO: 1136), IVYAEL (SEQ ID NO: 1126), VTYAQL (SEQ ID NO: 1974), ILYTEL (SEQ ID NO: 1080), ITYAAV (SEQ ID NO: 1117), VIYIDV (SEQ ID NO: 1866), VTYAEV (SEQ ID NO: 1971), VTYAPV (SEQ ID NO: 1973), VTYAKV (SEQ ID NO: 1972), VTYARL (SEQ ID NO: 2038), ILYHTV (SEQ ID NO: 1076), VLYAML (SEQ ID NO: 1892), VIYAQL (SEQ ID NO: 1861), LVYENL (SEQ ID NO: 1527), LCYADL (SEQ ID NO: 1159), ISYASL (SEQ ID NO: 1108), LTYVLL (SEQ ID NO: 1021), VTYVNL (SEQ ID NO: 1986), VRYSIV (SEQ ID NO: 1022), VFYRQV (SEQ ID NO: 1845), LKYMEV (SEQ ID NO: 1295), VDYGEL (SEQ ID NO: 1820), LSYMDL (SEQ ID NO: 1487), VLYTAV (SEQ ID NO: 1907), VQYTEV (SEQ ID NO: 1937), IVYASL (SEQ ID NO: 1128), VEYLEV (SEQ ID NO: 1838), LEYVDL (SEQ ID NO: 1191), ITYADL (SEQ ID NO: 1118), LTYADL (SEQ ID NO: 1505), VIYENV (SEQ ID NO: 1863), LAYYTV (SEQ ID NO: 1158), VSYSAV (SEQ ID NO: 1960), LVYDKL (SEQ ID NO: 1525), LNYMVL (SEQ ID NO: 1356), LNYACL (SEQ ID NO: 1351), LDYINV (SEQ ID NO: 1177), LHYATL (SEQ ID NO: 1221), LHYAVL (SEQ ID NO: 1222), IQYAPL (SEQ ID NO: 1093), IQYASL (SEQ ID NO: 1094), LLYLLL (SEQ ID NO: 1023), VVYSQV (SEQ ID NO: 1993), VIYSSV (SEQ ID NO: 1873), VVYYRV (SEQ ID NO: 2039), VPYVEL (SEQ ID NO: 1928), LDYDKL (SEQ ID NO: 1173), LSYPVL (SEQ ID NO: 1492), VAYSQV (SEQ ID NO: 1810), LFYWDV (SEQ ID NO: 1203), LIYSQV (SEQ ID NO: 2040), or LDYEFL (SEQ ID NO: 1176).

[0297]In some embodiments, p is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In some embodiments, p is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, p is 1, 2, 3, 4 or 5. In some embodiments, p is 1. In some embodiments, p is 2. In some embodiments, p is 3. In some embodiments, p is 4. In some embodiments, p is 5.

[0298]In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, n is 1, 2, 3, 4 or 5. In some embodiments, n is 0. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3.

[0299]In some embodiments, m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In some embodiments, m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, m is 1, 2, 3, 4 or 5. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, m is 5.

[0300]In some embodiments, n is 1 and m is 1.

[0301]In some embodiments, n is 1 and m is 1 and p is 1 to 10.

[0302]In some embodiments, n is 1 and m is 1 and p is 1.

[0303]In some embodiments, n is 0 and m is 1 and p is 1 to 20.

[0304]In some embodiments, n is 0, m is 1 to 6 and p is 1.

[0305]In some embodiments, n is 0, m is 1 and p is 1.

[0306]In some embodiments, n is 0, m is 2 and p is 1.

[0307]In some embodiments, n is 0, m is 3 and p is 1.

[0308]In some embodiments, n is 0, m is 4 and p is 1.

[0309]In some embodiments, n is 0, m is 5 and p is 1.

[0310]In some embodiments, n is 0, m is 6 and p is 1.

[0311]In some embodiments, n is 0, m is 1 to 6 and p is 1 and ITSM is TEYATI (SEQ ID NO: 937).

[0312]In some embodiments, n is 0, m is 1 to 6 and p is 1 and ITSM is TEYSEI (SEQ ID NO: 940).

[0313]In some embodiments, n is 0, m is 1 to 6 and p is 1 and ITSM is TVYSEV (SEQ ID NO: 1011).

[0314]In some embodiments, n is 1, m is 1 and p is 1 to 5.

[0315]In some embodiments, n is 1, m is 1 and p is 1.

[0316]In some embodiments, n is 1, m is 1 and p is 2.

[0317]In some embodiments, n is 1, m is 1 and p is 3.

[0318]In some embodiments, n is 1, m is 1 and p is 4.

[0319]In some embodiments, n is 1, m is 1 and p is 5.

[0320]In some embodiments, n is 1, m is 1 and p is 1 to 5 and ITIM is VDYGEL (SEQ ID NO: 1820) and ITSM is TEYATI (SEQ ID NO: 937).

[0321]In some embodiments, n is 1, m is 1 and p is 1 to 5 and ITIM is LX₆YAX₈L (SEQ ID NO: 2041) wherein X₆ is selected from H or Q and X₈ is V or S, and ITSM is TEYSEI (SEQ ID NO: 940).

[0322]In some embodiments, n is 1, m is 1 and p is 1 to 5 and ITIM is LX₆YAX₈L (SEQ ID NO: 2041) wherein X₆ is selected from H or Q and X₈ is V or S, and ITSM is TEYASI (SEQ ID NO: 936).

[0323]In some embodiments, n is 1, m is 1 and p is 1 to 5 and ITIM is LX₆YAX₈L (SEQ ID NO: 2041) wherein X₆ is selected from H or Q and X₈ is V or S, and ITSM is TVYSEV (SEQ ID NO: 1011).

[0324]In some embodiments, the intracellular domain comprises several ITSMs having the same amino acid sequence.

[0325]In some embodiments, the intracellular domain comprises several ITSMs having different amino acid sequences.

[0326]In some embodiments, the intracellular domain comprises several ITIMs having the same amino acid sequence.

[0327]In some embodiments, the intracellular domain comprises several ITIMs having different amino acid sequences.

[0328]In some embodiments, the intracellular domain of the NCAR is selected from SEQ ID No 2000, SEQ ID No 2001, SEQ ID No 2002, SEQ ID No 2003, SEQ ID No 2004, SEQ ID No 2005, SEQ ID No 2006, SEQ ID No 2007, SEQ ID No 2008, SEQ ID No 2009, SEQ ID No 2010, SEQ ID No 2011, SEQ ID No 2012, SEQ ID No 2013, SEQ ID No 2014, SEQ ID No 2015, SEQ ID No 2016 and SEQ ID No 2017.