Potency-tuned CD70 ligands, targeted chimeric antigen receptors (CARS) and methods for cancer

Abstract

CD70 binding proteins or ligands, particularly CD27 polypeptides, and particularly variant CD27 extracellular binding domain polypeptides, are provided wherein the CD27 amino acid sequence is altered. The CD27 polypeptides and / or domains thereof, particularly the extracellular binding domains thereof are useful in the diagnosis and treatment of various conditions, including in cancer and immunotherapuetics. The CD27 polypeptides and / or domains thereof, particularly the extracellular binding domains thereof may also be used in lymphoid cell-mediated, including T cell-mediated, therapy, in chimeric antigen receptor (CAR) therapy, and / or in therapy in combination with chemotherapeutics, radiation therapy, immune modulators, cancer vaccines, cancer antigens, or anti-cancer agents. Particular variant CD27 polypeptides, extracellular domains, and novel CAR constructs are provided.

Description

POTENCY-TUNED CD70 LIGANDS, TARGETED CHIMERIC ANTIGEN RECEPTORS (CARS) AND METHODS FOR CANCERSEQUENCE LISTING

[0001] The instant application contains a Sequence Listing, conforming to the rules of WIPO Standard ST.26, submitted in XML format which was filed electronically by EFS-web via PatentCenter and is hereby incorporated by reference in its entirety. Said XML format Sequence Listing, created on September 24„ 2024, is named “2745-8P __24SEP2024_ST26.xml” and is 80,621 bytes in size.FIELD OF THE INVENTION

[0002] The present invention relates to CD70 binding proteins or ligands, particularly CD27 polypeptides, and particularly variant CD27 extracellular binding domain polypeptides, wherein the CD27 amino acid sequence is altered. The CD27 polypeptides and / or domains thereof, particularly the extracellular binding domains thereof are useful in the diagnosis and treatment of various conditions, including in cancer and immunotherapeutics. The CD27 polypeptides and / or domains thereof, particularly the extracellular binding domains thereof may also be used in lymphoid cell-mediated, including T cell-mediated, therapy, in chimeric antigen receptor (CAR) therapy, and / or in therapy in combination with chemotherapeutics, radiation therapy, immune modulators, cancer vaccines, cancer antigens, or anti-cancer agents.BACKGROUND OF THE INVENTION

[0003] Chimeric antigen receptor (CAR) T cell therapies have broadly changed management and treatment outcomes for patients with aggressive B cell malignancies and multiple myeloma. However, only two target antigens are currently explored in commercially approved products, CD 19 and B cell maturation antigen (BCMA), limiting the spectrum of application of CAR T cell therapies to B and plasma cell malignancies, or to autoimmune diseases. Extending the CAR-T paradigm to aggressive myeloid malignancies such as acute myeloid leukemia (AML) for example or to solid tumors has been hampered for several reasons, including; 1) the paucity of ideal target antigens that are selectively expressed on tumor cells but not on essential healthy tissues; 2) the complexity and clonal heterogeneity of the malignancies; 3) the poorly characterized impact of the tumor immune microenvironment on adoptively transferred CAR T cells; and 4) the manufacturing challenge when producing CAR T cells from autologous lymphocytes in an aggressive leukaemia with circulating blasts.

[0004] An emerging target that is widely and stably expressed in a variety of cancers is CD70, a member of the tumor necrosis factor (TNF) superfamily (1). CD70 is a co-stimulatory molecule with limited expression on normal tissues that is restricted to a subset of hematopoietic cells, namelyactivated T and B lymphocytes and a subset of dendritic cells, but absent on normal hematopoietic stem cells and maturating myeloid cells (2, 3). Therefore, litle to no on-target off-tumor toxicities on healthy haematopoiesis and other normal tissues is to be expected by targeting CD70. Indeed, the monoclonal antibody cusatuzumab targeting CD70 has demonstrated safety and some efficacy in clinical trials for AML (4). In patients with relapsed / refractory clear cell renal cell carcinoma (ccRCC), a recent clinical trial reported safety and some efficacy with an allogeneic off-the-shelf CAR T cell product targeting CD70 and no dose limiting toxicities (5). These data suggest that CD70 can be safely targeted in humans with monoclonal antibodies or CAR T cells.

[0005] CD27, a member of the TNF receptor superfamily is constitutively expressed on naive T-cells, memory B-cell and T cell populations, NK-cells, and hematopoietic stem cells (HSCs) and progenitor cells. CD70 (CD27L), the only ligand for CD27, is a tightly regulated transmembrane glycoprotein expressed on both B and T lymphocytes and antigen presenting cells (APCs). Upon binding of CD70, CD27 is bound to TNF receptor-associated factors (TRAFs) leading to intracellular signaling which potentiates survival and activation of T, B, and natural killer (NK) cells. The interaction of CD27-CD70 is tightly regulated to prevent overexpression and subsequent excessive lymphocyte activation. In oncology, CD70 is aberrantly expressed on malignant cells without (solid tumors) or with CD27 coexpression (hematological malignancies), facilitating immune evasion through the tumor microenvironment (TME) and tumor progression.

[0006] CARs targeting CD70 have been developed, either based on the natural ligand CD27 or single chain variable fragments (scFv) derived from CD70-targeted monoclonal antibodies (6-13). Recent data suggested that CD27-based CARs were more potent than scFv-based CARs targeting CD70 (12). However, CD27-based CARs may have issues with natural cleavage of CD27 incorporated in the CAR construct thus limiting CAR activity (12, 13). Engineering out the cleavage region of CD27 was proposed as a solution and resulted in more stable CAR cell surface expression and CAR T cell function in preclinical models (13). CD19 CAR T cell optimizations have demonstrated that the affinity of the scFv in the CAR construct can have a decisive impact on both CAR T cell potency and the associated toxicity profile in patients and that there is no clear correlation between binding affinity and CAR T cell potency (14). However, such optimizations have not been performed in natural ligand-based CARs and the impact of modulating the CD27-CD70 binding interface on CAR T cell potency has not been investigated. Modifying CD27 to provide a potency-tuned CD27 as a CD70 ligand, such as with enhanced CD70 affinity or improved binding and stability of CD70 binding, provides an improved CD70 ligand suitable for numerous applications in immunotherapeutics, including in hematological cancers or malignancies, tumors, and graft versus host disease. Further, CD70 targeted CARs with an optimized assembly of components including a designed enhanced CD27-CD70 binding interface are useful and needed for improved and effective cancer immunotherapy. The present invention addresses such unmet needs in the field and particularly with regard to CD27 and CD70 binding.

[0007] The citation of references herein shall not be construed as an admission that such is prior art to the present invention.SUMMARY OF THE INVENTION

[0008] In a general aspect, the present invention provides novel CD70 binding proteins, particularly CD27 polypeptides which are altered in amino acid sequence from native or wild-type human CD27 polypeptide sequence. The novel, variant CD27 polypeptides, particularly the variant CD27 extracellular domains, are capable of binding CD70. In an aspect, the variant CD27 polypeptides, particularly the variant CD27 extracellular domains, demonstrate stabilized or increased binding to CD70. In an aspect, the variant CD27 peptides, particularly the variant CD27 extracellular domains, demonstrate higher potency activity upon CD70 binding. In an aspect, the variant CD27 peptides, particularly the variant CD27 extracellular domains, are capable of stimulating IL-2 upon CD70 binding. In an aspect, the mutant CD27 peptides, particularly the CD27 extracellular domains, demonstrate increased or greated IL-2 stimulation and / or expression upon CD70 binding in comparison with wild type, native, or unmutated CD27.

[0009] In some aspects, the mutant CD27 peptide(s), particularly the CD27 extracellular domain(s), can be used in isolation, for example to characterize, identify, locate or tag CD70, including CD70 expressed on cells, such as lymphocytes. In some aspects, the mutant CD27 peptide(s) or the mutant CD27 extracellular domain(s) can be combined with other proteins or protein domains. In some aspects, the mutant CD27 peptide(s) or the mutant CD27 extracellular domain(s) can be fused with other proteins or protein domains or covalently attached with other proteins or protein domains. In aspects, the mutant CD27 polypeptides, particularly the mutant CD27 extracellular domains, are conjugated with other proteins or domains.

[0010] In some embodiments, the mutant CD27 peptide(s) or the mutant CD27 extracellular domain(s) is conjugated to another compound. In some embodiments, the mutant CD27 peptide(s) or the mutant CD27 extracellular domain(s) is conjugated to a half-life extending moiety. In some embodiments, the binding protein is conjugated to a polymer (e.g., PEG). In some embodiments, the binding protein is conjugated to a cytotoxic agent. In some embodiments, the mutant CD27 peptide(s) or the mutant CD27 extracellular domain(s) is conjugated to an immunomodulatory compound, protein or domain. In some embodiments, the mutant CD27 peptide(s) or the mutant CD27 extracellular domain(s) is conjugated to or combined with an immune stimulator or immune activator, such as an interleukin, such as IL-2, or an interferon, such as IFN-y.

[0011] In accordance with some aspects of the invention, CD27 mutant variants altered at one or more amino acids selected from F71, S72, D74, H76, R78, H80, E82, H86, R85, S89, QI 11, KI 15, El 19, D121, in the CD27 extracellular domain sequence are provided. These mutant locations are shown below (in bold and underlined) in the native human CD27 extracellular domain sequence corresponding to amino acids 22-124 (SEQ ID NO: 1):MARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAA 60QCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTEC 120DPLPNPSLTARSSQALS PHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPP 180QRSLCSSDFIRILVIFSGMFLVFTLAGALFLHQRRKYRSNKGESPVEPAEPCHYSCPREE 240EGSTIPIQEDYRKPEPACSP260

[0012] In an aspect, a potency-tuned and variant CD27 affinity ligand is altered at one or more amino acids selected from F71, S72, D74, H80, H86, R85, S89, QI 11, KI 15, El 19, D121, in the CD27 extracellular domain sequence. In an aspect, a potency-tuned and variant CD27 affinity ligand is altered at one or more amino acids selected from S72, H86, QI 11, KI 15, F71, D74, H80, R85, S89, El 19, and D121 in the CD27 extracellular domain sequence. In an aspect, a potency-tuned and variant CD27 affinity ligand is altered at two or more amino acids selected from F71, S72, D74, H80, H86, R85, S89, QI 1 1, KI 15, El 19, D121, in the CD27 extracellular domain sequence. In an aspect, a potency-tuned and variant CD27 affinity ligand is altered at two or more amino acids selected from amino acids selected from S72, H86, QI 11, KI 15, F71, D74, H80, R85, S89, El 19, and D121 in the CD27 extracellular domain sequence. In an aspect, a potency-tuned and variant CD27 affinity ligand is altered at two amino acids selected from F71, S72, D74, H80, H86, R85, S89, QI 11, KI 15, El 19, DI21, in the CD27 extracellular domain sequence. In an aspect, a potency-tuned and variant CD27 affinity ligand is altered at two amino acids selected from S72, H86, QI 11, KI 15, F71, D74, H80, R85, S89, El 19, and DI 21 in the CD27 extracellular domain sequence. In an aspect, a potency-tuned and variant CD27 affinity ligand is altered at three or more amino acids selected from F71, S72, D74, H80, H86, R85, S89, QI 1 1, KI 15, El 19, D121, in the CD27 extracellular domain sequence. In an aspect, a potency- tuned and variant CD27 affinity ligand is altered at three or more amino acids selected from S72, H86, QI 1 1, KI 15, F71, D74, H80, R85, S89, El 19, and D121 in the CD27 extracellular domain sequence. In an aspect, a potency-tuned and variant CD27 affinity ligand is altered at three amino acids selected from F71, S72, D74, H80, H86, R85, S89, QI 11, KI 15, El 19, D121, in the CD27 extracellular domain sequence. In an aspect, a potency-tuned and variant CD27 affinity ligand is altered at three amino acids selected from S72, H86, QI 11, KI 15, F71, D74, H80, R85, S89, El 19, and D121 in the CD27 extracellular domain sequence. In a preferred aspect, a potency-tuned and variant CD27 affinity ligand is altered at one or more amino acids selected from S72, H86, Q 1 1 1 and K 115 in the CD27 extracellular domain sequence. In a preferred aspect, a potency-tuned and variant CD27 affinity ligand is altered at two or more amino acids selected from S72, H86, QI 11 and KI 15 in the CD27 extracellular domain sequence. In a preferred aspect, a potency-tuned and variant CD27 affinity ligand is altered at three or more amino acids selected from S72, H86, QI 11 and KI 15 in the CD27 extracellular domain sequence.

[0013] In accordance with the invention, a variant extracellular binding domain of CD27 is provided comprising the sequence set out as follows (SEQ ID NO: 45):PAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFXiPDHHTRPHCES CR X2CNSGLLVRNCTITANAECACRNGWX3CRDX4ECTECDPLPWherein Xi is A or NX2is V, F or YX3is TX4 is E, D, Q or A

[0014] In another embodiment, a variant extracellular binding domains of CD27 is provided comprising the sequence set out as follows (SEQ ID NO: 46):

[0015] In another embodiment, a variant extracellular binding domain of CD27 is provided, having one or more amino acid substitution, comprising the sequence set out as follows (SEQ ID NO: 47):and wherein the variant extracellular binding domain of CD27 does not correspond in sequence to human CD27 extracellular binding domain of SEQ ID NO:2:PAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCES CRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLP

[0016] In some aspects, the mutant potency tuned CD27 extracellular domain is selected from a peptide sequence as set out in one or more of SEQ ID NO:s 13-47. In some aspects, the mutant potency tuned CD27 extracellular domain is selected from a peptide sequence as set out in one of SEQ ID NO:s 13-47. In some aspects, the mutant potency tuned CD27 extracellular domain is selected from a peptide sequence as set out in one of SEQ ID NO:s 16-47. In some aspects, the mutant potency tuned CD27 extracellular domain is selected from a peptide sequence as set out in any of SEQ ID NO:s 16-44. In preferred aspects, the mutant potency tuned CD27 extracellular domain is selected from mutant TQ14 (S72A) (SEQ ID NO: 18), TQ29 (H86V) (SEQ ID NO:3l), TQ37 (S72N-H86V) (SEQ ID NO:35), TQ44 (H86V-Q111T-K115E) (SEQ ID NO:41) and TQ49 (S72A-H86V) (SEQ IDNO:44). In preferred aspects, the mutant potency tuned CD27 extracellular domain is selected from mutant TQ37 (S72N-H86V) (SEQ ID NO:35), TQ44 (H86V-Q1 11T-K115E) (SEQ ID N0:41) and TQ49 (S72A-H86V) (SEQ ID NO:44). In some aspects, the mutant potency tuned CD27 extracellular domain is selected from mutant TQ14 (S72A) (SEQ ID NO:18) and TQ49 (S72A-H86V) (SEQ ID NO:44). In some aspects, the mutant potency tuned CD27 extracellular domain is selected from mutant TQM (S72A) (SEQ ID NO:18), TQ37 (S72N-H86V) (SEQ ID NO:35), TQ44 (H86V-Q111T-K.115E) (SEQ ID NO:41 ) and TQ49 (S72A-H86 V) (SEQ ID NO:44). In other aspects, the the mutant potency tuned CD27 extracellular domain is mutant TQ37 (S72N-H86V) (SEQ ID NO:35) or TQ49 (S72A-H86V) (SEQ ID NO:44).

[0017] Variant CD27 affinity ligand sequences, including full length CD27 variant sequences, which include or otherwise comprise a variant CD27 extracellular domain sequence are provided herein. CD27 variant sequences comprising a variant extracellular domain sequence as provided herein and further comprising a CD27 transmembrane or endodomain are also contemplated and included herein. These variant CD27 sequences may comprise one or more of the variant extracellular domains set out and described herein, along with the CD27 transmembrane and end domain, such as the transmembrane and endo domains corresponding to SEQ ID NO: 3 (amino acids 193-260 of human CD27).

[0018] In another aspect, the present invention provides a composition comprising the binding protein of the invention, particularly the mutant CD27 peptide or the mutant CD27 extracellular domain and a pharmaceutically acceptable carrier.

[0019] In another aspect, the present invention provides a cell comprising the binding protein of the invention particularly the mutant CD27 peptide or the mutant CD27 extracellular domain.

[0020] In relevant aspects of the invention, the binding proteins of the invention, particularly the mutant CD27 peptide or the mutant CD27 extracellular domain, can be incorporated into chimeric antigen receptors (CARs) to produce CAR expressing cells for targeting the CD27 ligand CD70 or CD70-expressing cells. Chimeric antigen receptors (CARs) are hybrid molecules comprising an antigen-targeting moiety, followed by a linker, transmembrane (TM) domain, and an intracellular domain comprising various endodomains (EDs) involved in T-cell activation. In accordance with the invention, CARs or other antigen targeting molecules contemplated herein target CD70, whereby CD70 is the target antigen CD70, and particularly wherein the antigen-targeting moiety is the mutant CD27 extracellular domain or CD70 binding domain. CARs provided herein may include the ED of CD3- zeta only (as a first signalling domain), or CARs may have one or more co-stimulatory EDs (or second signalling domains), respectively, such as CD28 and 4-1BB.

[0021] Thus, in another aspect, the present invention provides a CAR comprising particularly the mutant CD27 extracellular domain of the invention, a transmembrane domain, and an intracellular domain.

[0022] In some embodiments, the intracellular domain comprises a primary signalling domain and a costimulatory domain. In an embodiment, the intracellular domain comprises two or morecostimulatory domains. In an alternative embodiment, the CAR does not comprise a costimulatory domain.

[0023] In some embodiments, the primary signalling domain comprises a CD3 zeta (CD3Q, CD3 gamma (CD3y), CD3 delta, CD3 epsilon, common FcR gamma (FCER1G), FcR beta (Fc Epsilon Rib), CD79a, CD79b, Fcgamma Rlla, DAP10, or DAP12 primary signalling domain.

[0024] In some embodiments, the primary signalling domain comprises a CDS zeta (CD3Q signalling domain. In some embodiments, the primary signalling domain comprises an amino acid sequence which is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% or about 100% identical to the sequence set forth in SEQ ID NO:50.

[0025] In some embodiments, the costimulatory domain comprises a CD28, 4- IBB (CD 137), 0X40, CD27, CD30, CD40, CD134, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD 160, CD 19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLAl, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDlld, ITGAE, CD 103, ITGAL, CDlla, LFA-1, ITGAM, CDllb, ITGAX, CDlIc, ITGB1, CD29, ITGB2, CD 18, LFA-1, ITGB7, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD 160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, or TNFR2 costimulatoiy domain.

[0026] In some embodiments, the costimulatory domain is a CD28 costimulatory domain. In some embodiments, the costimulatory domain is a 41BB costimulatory domain.

[0027] In some embodiments, the the transmembrane domain comprises a CD28, CDS epsilon, CD3 zeta, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD 137, CD 154, KIRDS2, 0X40, CD2, CD27, LFA-1 (CDS la, CD 18), ICOS (CD278), 4- IBB (CD 137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (K.LRF 1 ), CD 160, CD 19, IL2R beta, IL2R gamma, IL7Ra, ITGAI, VLAl, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDlld, ITGAE, CD103, ITGAL, CDlla, LFA-1, ITGAM, CDllb, ITGAX, CDllc, ITGB1, CD29, ITGB2, CD 18, LFA-1, ITGB7, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD 160 (BY55), PSGL1, CDIOO (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, NKG2C, or TNFR2 transmembrane domain, or the alpha, beta or zeta chain of the T-cell receptor.

[0028] In some embodiments, the transmembrane domain is a CD27 transmembrane domain. In other embodiments, the transmembrane domain comprises an amino acid sequence which is at leastabout 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% or is about 100% identical to the sequence set forth in SEQ ID NOG.

[0029] In some embodiments, the antigen-binding domain, particularly the CD27 extracellular domain, is connected to the transmembrane domain by a hinge region. In some embodiments, the hinge region comprises a CD8 hinge, an IgG hinge, an IgD hinge, a CD28 hinge, a KIR2DS2 hinge, or a glycine-serine linker. In certain embodiments, the IgG hinge is from IgG 1, IgG2, IgG3, IgG4, IgMl, IgM2, IgAl, IgA2, IgD, IgE, or a chimera thereof. In certain embodiments, the hinge domain comprises the CHI, CH2, CH3 and / or hinge domain of the immunoglobulin. In certain embodiments, the hinge domain is a fragment of the immunoglobulin hinge domain.

[0030] In some embodiments, the hinge region comprises a CD8 alpha hinge region. In some embodiments, the hinge region comprises a sequence of amino acids which is at least 70% or at least 80% or at least 90%, at least 95% or is about 100% identical to SEQ ID NO:49.

[0031] In some embodiments, the CAR further comprises a leader sequence or a signal sequence. In some embodiments, the leader sequence is a human leader sequence. In some embodiments, the leader sequence is a signal sequence and is a human signal sequence. In some embodiments, the leader sequence is an N-terminal leader sequence. In some embodiments, the leader sequence or signal sequence is an immunoglobulin signal sequence. In some embodiments, the leader sequence comprises a sequence of amino acids which is at least 70% or at least 80% or at least 90%, at least 95% or is about 100% identical to SEQ ID NO:49. Other suitable leader sequences or signal sequences will be known by those skilled in the art. In some embodiments, the leader sequence is cleaved from the CAR during cellular processing and localization of the CAR to the cellular membrane when expressed in a cell.

[0032] In some embodiments, the CAR comprises:(a) a signal sequence;(b) a CD27 binding domain (CD27 extracellular domain amino acids 22-124) selected from SEQ ID NOs:I3-47;(c) a hinge sequence;(d) an intracellular or transmembrane domain; and(e) a signalling domain.

[0033] In some embodiments, the CAR comprises:(a) a signal sequence;(b) a CD27 binding domain (CD27 extracellular domain amino acids 22-124) selected from SEQ ID NO: 18, SEQ ID NOG 1 , SEQ ID NO:35, SEQ ID NO:41 and SEQ ID NO:44;;(c) a hinge sequence;(d) an intracellular or transmembrane domain; and(e) a signalling domain.

[0034] In some embodiments, the CAR comprises:(a) a signal sequence;(b) a CD27 binding domain (CD27 extracellular domain amino acids 22-124) selected from SEQ ID NO:44, SEQ ID NO:35 and SEQ ID NO:41 ;(c) a hinge sequence;(d) an intracellular or transmembrane domain; and(e) a signalling domain.

[0035] In some embodiments, the CAR comprises:(a) a signal sequence (such as immunoglobulin sequence) MEFGLSWLFLVAILKGVQC (SEQ ID NO: 48);(b) a CD27 binding domain (CD27 extracellular domain amino acids 22-124) selected from SEQ ID NOs: 13-47;(c) a hinge sequence (such as CD8a hinge sequence)TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDI (SEQ ID NO:49)(d) an intracellular or transmembrane domain (such as the CD27 transmembrane and endodomains):LVIFSGMFLVFTLAGALFLHQRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEP ACSP (SEQ ID NOG); and(e) a signalling domain (such as CD3^ chain sequence):RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYN ELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:50).

[0036] In some embodiments, the CAR comprises:(a) a signal sequence (such as immunoglobulin sequence) MEFGLSWLFLVAILKGVQC (SEQ ID NO: 48);(b) a CD27 binding domain (CD27 extracellular domain amino acids 22-124) selected from SEQ ID NO: 18, SEQ ID NOG 1, SEQ ID NO:35, SEQ ID NO:41 and SEQ ID NO:44;(c) a hinge sequence (such as CD8a hinge sequence)TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDI (SEQ ID NO:49)(d) an intracellular or transmembrane domain (such as the CD27 transmembrane and endodomains):LVIFSGMFLVFTLAGALFLHQRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP (SEQ ID NOG); and(e) a signalling domain (such as CD3C, chain sequence):RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYN ELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:50).

[0037] In some embodiments, the CAR comprises:(a) a signal sequence (such as immunoglobulin sequence) MEFGLSWLFLVAILKGVQC (SEQ ID NO: 48);(b) a CD27 binding domain (CD27 extracellular domain amino acids 22-124) selected from SEQ ID NO:44, SEQ ID NO:35 and SEQ ID NO:41 ;(c) a hinge sequence (such as CD8a hinge sequence)TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDI (SEQ ID NO:49)(d) an intracellular or transmembrane domain (such as the CD27 transmembrane and endodomains):LVIFSGMFLVFTLAGALFLHQRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP (SEQ ID NOD); and(e) a signalling domain (such as CD3^ chain sequence):RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYN ELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:50).

[0038] In some embodiments, the CAR comprises more than one antigen-binding domain. In some embodiments, the CAR comprises a mutant CD27 binding domain as provided herein and another distinct antigen-binding domain. Thus, in some embodiments, the CAR is a multi-specific CAR comprising two or more antigen-binding domains, wherein one of the antigen-binding domains comprises a mutant CD27 binding or extracellular domain as provided herein. The two or more antigenbinding domains may bind to the same or different targets.

[0039] In another aspect, the present invention provides a nucleic acid encoding a binding protein of the invention, particularly a mutant CD27 polypeptide, particularly a mutant CD27 extracellular domain polypeptide, or a CAR of the invention. In an embodiment, the nucleic acid comprises a nucleotide sequence that is codon optimized for expression in human cells.

[0040] In an embodiment, the nucleic acid comprises a nucleotide sequence which is capable of encoding a mutant CD27 extracellular domain polypeptide as set out in any of SEQ ID NOs: 13-47 or a polypeptide at least about 90%, or at least about 95% identical to any one of SEQ ID NOs: 13-47. In an embodiment, the nucleic acid comprises a nucleotide sequence which is capable of encoding a mutant CD27 extracellular domain polypeptide as set out in any of SEQ ID NOs: 13-47. In an embodiment, the nucleic acid comprises a nucleotide sequence which is capable of encoding a mutant CD27 extracellular domain polypeptide as set out in any of SEQ ID NOs: 16-47. In an embodiment, the nucleic acid comprises a nucleotide sequence which is capable of encoding a mutant CD27 extracellular domain polypeptide as set out in any of SEQ ID NOs: 16-44.

[0041] In addition to the CAR construct, the CAR may further comprise an accessory gene that encodes an accessory peptide. Examples of accessory genes can include a transduced host cellselection marker, an in vivo tracking marker, a cytokine, a suicide gene, or some other functional gene. In some embodiments, the accessory gene is a fluorescent protein, such as, but not limited to, green fluorescent protein (GFP), yellow fluorescent protein (YFP), or cyan fluorescent protein (CFP). Nonlimiting examples of classes of accessory genes that can be used to increase the effector function of CAR containing host cells, include i) secretable cytokines (e.g., but not limited to, IL-7, IL-12, IL-15, IL-18), ii) membrane bound cytokines (e.g., but not limited to, IL-15), iii) chimeric cytokine receptors ( e.g., but not limited to, IL-2 / IL-7, IL-4 / IL-7), iv) constitutive active cytokine recep-tors (e.g., but not limited to, C7R), v) dominant negative receptors (DNR; e.g., but not limited to TGFRII DNR), vi) ligands of co stimulatory molecules (e.g., but not limited to, CD80, 4-1BBL), vii) antibodies, including fragments thereof and bispecific antibodies ( e.g., but not limited to, bispecific T-cell engagers (BiTEs)), or vii) a second CAR.

[0042] In another aspect, the present invention provides a vector comprising the nucleic acid of the invention. In an embodiment, the vector is a DNA vector, an RNA vector, a plasmid, a lentivirus vector, adenovirus or adeno-associated virus vector, or a retrovirus vector. In some embodiments, the nucleic acid of the invention is operably linked to a promoter in the vector.

[0043] In another aspect, the present invention provides a cell comprising the binding protein of the invention, the mutant CD27 extracellular domain polypeptide, the CAR of the invention, the nucleic acid of the invention, or the vector of the invention. In a similar aspect, the present invention provides a cell expressing the binding protein, the mutant CD27 extracellular domain polypeptide, or CAR of the invention.

[0044] In some embodiments, the cell is an immune effector cell. In some embodiments, the immune effector cell is a T cell or an NK cell. In some embodiments, the immune effector cell is a CD8+ T cell. In some embodiments, the immune effector cell is a CD4+ T cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell.

[0045] In one embodiment, the cell described herein can further comprise a second (or more) CAR, e.g., a second CAR that includes a different antigen-binding domain, e.g., to the same target (i.e., CD70) or a different target. In one embodiment, the second CAR includes an antigen-binding domain which binds to a target expressed on the same cancer cell type as the target of the first CAR.

[0046] In another aspect, the present invention provides a composition comprising the nucleic acid of the invention, the vector of the invention, or the cell of the invention and a pharmaceutically acceptable carrier.

[0047] In another aspect, the present invention provides a method of making a CAR- expressing cell, comprising introducing the nucleic acid of the invention or the vector of the invention, into a cell, under conditions such that the CAR is expressed.

[0048] In certain embodiments, the method further comprises expanding the population of cells after the nucleic acid molecule encoding a CAR has been introduced.

[0049] In another aspect, the present invention provides a method of treating a subject having a cancer associated with expression of CD70, the method comprising administering to the subject the binding protein of the invention, the composition of the invention, the CAR of the invention, or the cell of the invention. In another aspect, the present invention provides a method of treating a subject having a cancer expressing CD70, the method comprising administering to the subject the binding protein of the invention, the composition of the invention, the CAR of the invention, or the cell of the invention.

[0050] In another aspect, the present invention provides a method of reducing or delaying progression of cancer in a subject having a cancer associated with expression of CD70, the method comprising administering to the subject the binding protein of the invention, the composition of the invention, the CAR of the invention, or the cell of the invention. In another aspect, the present invention provides a method of reducing or delaying progression of a cancer in a subject having a cancer expressing CD70, the method comprising administering to the subject the binding protein of the invention, the composition of the invention, the CAR of the invention, or the cell of the invention.

[0051] In an aspect, the present invention provides a method of treating a subject having a solid cancer expressing CD70, the method comprising administering to the subject the binding protein of the invention, the composition of the invention, the CAR of the invention, or the cell of the invention.

[0052] In another aspect, the present invention provides a method of reducing or delaying progression of a solid cancer in a subject having a cancer expressing CD70, the method comprising administering to the subject the binding protein of the invention, the composition of the invention, the CAR of the invention, or the cell of the invention.

[0053] In a related aspect, the present invention provides the binding protein of the invention, particularly the mutant CD27 polypeptide, particularly the mutant CD27 extracellular domain polypeptide, the composition of the invention, the CAR of the invention, or the cell of the invention, for use in the treatment of a cancer associated with expression of CD70. In a related aspect, the present invention provides the binding protein of the invention, particularly the mutant CD27 polypeptide, particularly the mutant CD27 extracellular domain polypeptide, the composition of the invention, the CAR of the invention, or the cell of the invention, for use in the treatment of a disease associated with altered expression or overexpression of CD70.

[0054] In another related aspect, the present invention provides use of the binding protein of the invention, particularly the mutant CD27 polypeptide, particularly the mutant CD27 extracellular domain polypeptide, the composition of the invention, the CAR of the invention, or the cell of the invention in the manufacture of a medicament for the treatment of a cancer associated with expression of CD70. In another related aspect, the present invention provides use of the binding protein of the invention, particularly the mutant CD27 polypeptide, particularly the mutant CD27 extracellular domain polypeptide, the composition of the invention, the CAR of the invention, or the cell of theinvention in the manufacture of a medicament for the treatment of a cancer associated with altered expression or overexpression of CD70.

[0055] In another aspect, the present invention provides use of the binding protein of the invention, particularly the mutant CD27 polypeptide, particularly the mutant CD27 extracellular domain polypeptide, the composition of the invention, the CAR of the invention, or the cell of the invention in the manufacture of a medicament for the treatment of cancer. In an aspect, the present invention provides use of the binding protein of the invention, particularly the mutant CD27 polypeptide, particularly the mutant CD27 extracellular domain polypeptide, the composition of the invention, the CAR of the invention, or the cell of the invention in the manufacture of a medicament for the treatment of a cancer expressing CD70. In an aspect, the present invention provides use of the binding protein of the invention, particularly the mutant CD27 polypeptide, particularly the mutant CD27 extracellular domain polypeptide, the composition of the invention, the CAR of the invention, or the cell of the invention in the manufacture of a medicament for the treatment of leukemia, lymphoid leukemia, multiple myeloma, B cell lymphoma, T cell lymphoma, glioblastoma, or renal cell carcinoma. In an embodiment, the present invention provides use of the binding protein of the invention, particularly the mutant CD27 polypeptide, particularly the mutant CD27 extracellular domain polypeptide, the composition of the invention, the CAR of the invention, or the cell of the invention in the treatment or or in the manufacture of a medicament for treatment of cancer, particularly of a cancer, including a solid cancer, expressing CD70, wherein the cancer is lung cancer, renal cancer, pancreatic cancer, ovarian cancer, glioma, head and neck cancer, osteosarcoma or breast cancer. In an embodiment, the cancer is lung cancer, renal cancer, pancreatic cancer, or ovarian cancer. In some embodiments, the cancer is a myeloid cancer. In some embodiments, the cancer is acute myeloid leukemia (AML). In an embodiment, the cancer is adult AML. In some embodiments, the cancer is pediatric AML.

[0056] Examples of cancers that express CD70 include, without limitation, acute lymphoblastic leukemias (B and T ceil origins), B cell lymphomas, bladder cancer, breast invasive carcinoma, cervical cancer, cholangiocarcinoma, colorectal cancer, diffuse large B-cell lymphoma (DLBC), Esophagus, glioblastoma (GBM), head and neck cancer, low-grade gliomas (LGG), liver cancer, lung adeno cancer, melanoma, mesothelioma, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, stomach cancer, T cell lymphomas, testicular germ cell cancer, thymoma, thyroid cancer, uterine cancer, uveal melanoma, clear cell renal cell carcinoma (ccRCC), chromophobe renal cell carcinoma, papillary renal cell carcinoma (pRCC), acute myeloid leukemia (AML), and adenoid cystic carcinoma (ACC). In some embodiments, the cancer is a myeloid cancer. In some embodiments, the cancer is acute myeloid leukemia. Cancers include adult cancers and pediatric cancers, and thus cancers in adults or in children. In an embodiment, the cancer is acute myeloid leukemia (AML). In an embodiment, the cancer is pediatric AML. In an embodiment, the subject is a child with AML. In some embodiments, the cancer is lung adeno cancer or adenocarcinoma of the lung. In some embodiments, the cancer is non-small cell lung cancer (NSCLC).

[0057] In some embodiments, the cancer is a solid tumor cancer. In some embodiments, the cancer is a solid tumor cancer expressing CD70. In an embodiment, the cancer is lung cancer, renal cancer, pancreatic cancer, ovarian cancer, glioma, head and neck cancer, osteosarcoma or breast cancer. In an embodiment, the cancer is lung cancer, renal cancer, pancreatic cancer, or ovarian cancer. In some embodiments, the cancer is lung cancer and is adenocarcinoma of the lung. In some embodiments, the cancer is lung cancer and is non-small cell lung cancer (NSCLC).

[0058] Examples of a disease associated with altered expression or overexpression of CD70 include Graft-versus-host disease (GVHD).

[0059] In some embodiments, a population of cells of the invention are administered. In some embodiments, the cells administered to the subject are allogenic cells or autologous cells. In some embodiments, the cells lack or have low expression of a functional T cell receptor (TCR) or a functional human leukocyte antigen (HLA). The phrase “low expression”, in this context, refers to an amount of expression that is insufficient to affect cell signalling, e.g., an amount of expression of a TCR which is insufficient to activate the cell in the presence of its antigen. Advantageously, such cells are specific for the antigen recognized by the CAR.

[0060] In some embodiments, the treatment comprises administering an agent that increases the efficacy of the cells. In some embodiments, the agent is a protein phosphatase inhibitor, a kinase inhibitor, a cytokine, an inhibitor of an immune inhibitory molecule, or an agent that decreases the level or activity of T regulatory cells.

[0061] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In other embodiments, the subject is a non-human mammal.

[0062] In another aspect, the present invention provides a method of detecting a cell expressingCD70, the method comprising contacting the binding protein mutant CD27 or mutant CD27 extracellular domain of the invention with a sample comprising the cell and detecting binding between the binding protein and CD70.

[0063] In a related aspect, the invention provides a method of diagnosing a subject with a cancer associated with expression of CD70, the method comprising a) obtaining a sample comprising cells from the subject; b) determining whether the cells express CD70 by contacting the sample with the binding protein of the invention and detecting binding between the binding protein and CD70; and c) diagnosing the subject with cancer associated with expression of CD70 if binding is detected in step b).

[0064] The diagnostic utility of the present invention extends to the use of the mutant CD27 binding peptides, particularly the mutant CD27 extracellular domain peptides, of the present invention in assays to characterize tumors or cellular samples or to screen for tumors or cancer, including in vitro and in vivo diagnostic assays. Peptides or polypeptides of the invention may carry a detectable or functionallabel. They may carry a radioactive label, such as the isotopes3H,14C,32P,35S,36C1,5 ICr,57Co,5SCo,59Fe,90Y,1211,124I,l25I,13 lI,11‘In,l l7Lu,211At,l98Au,67Cu,225Ac,213Bi, "Tc andl86Re. In an aspect, the label may be an enzyme, including wherein detection may be accomplished by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques known in the art.

[0065] Conjugates or fusion proteins of the present invention, wherein the mutant CD27 binding peptides, particularly the mutant CD27 extracellular domain peptides, of the present invention are conjugated or atached to other molecules or agents further include, but are not limited to binding members conjugated to a chemical ablation agent, toxin, immunomodulator, cytokine, cytotoxic agent, chemotherapeutic agent or drug.

[0066] The present invention includes an assay system which may be prepared in the form of a test kit for the quantitative analysis of the extent of the presence of, for instance, CD70. The system or test kit may comprise a labeled component prepared by one of the radioactive and / or enzymatic techniques discussed herein, coupling a label to the mutant CD27 binding peptides, particularly the mutant CD27 extracellular domain peptides, of the present invention, and one or more additional immunochemical reagents, at least one of which is a free or immobilized components to be determined or their binding partner(s).

[0067] Other objects and advantages will become apparent to those skilled in the art from a review of the ensuing detailed description, which proceeds with reference to the following illustrative drawings, and the attendant claims.BRlhMtr^'RIP I (< )\ ( )1 THE DRAWINGS

[0068] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0069] Figure 1 A-1E. Generation of CD27 ligand-based CAR constructs targeting CD70.(A) Schematic illustrating the interaction between CD70 and CD27. (B) Design of potential CD27 ligand-based CAR consisting of CD27 extracellular domain (ECD), hinge and transmembrane (TM) domains fused with different co-stimulatory and CD3^ endo-domains. (C) A simplified representation of the CD70 CAR constructs examined in this study. CD27-A (A): non-signaling control CAR, CD27- C, (Q: first-generation CAR with CD3^ signaling domain, CD27-28(j (28Q: second-generation CAR with CD28 and CD3(j endo-domains, CD27-BBij (BBQ: second-generation CAR with 41BB and CD3£ endo- domains, CD27-27£ (27Q: second-generation CAR containing full-length CD27 and CD3(j signaling domains. Truncated (non-signaling) CD271 was co-expressed as a selectable reporter gene and the entire transgene cassette was inserted into the SFG retroviral vector backbone. (D) Representative FACS plot showing the transduction efficiency of 27^ CAR construct, as assessed by %CD271+, 7 dayspost-transduction. The proportion of CD4+and CD8+T cells expressing 27^ CAR was shown in comparison to activated but non-transduced T cells (NT). (E) Transduction efficiencies for each CD70 CAR construct (A: 67±18%, £ 69±17%, 28£ 64±19%, BB£: 70±17%, 27^: 62±24%), n=6 donors, mean±SD).

[0070] Figure 2A-2D. In vitro anti-tumor activity of CD70 CAR-T cells. (A) Illustration of serial co-culture rechallenge assay. Tumor cells (MOLM-13.GFP-ffLuc) and CAR-T cells are cocultured in replicate wells at an effector-to-target (E:T) ratio of 1:1 on day 0. Flow cytometric analysis is conducted 3-4 days later to quantify the number of tumor and T cells left in the well. If <20% tumor cells are left, a kill was considered as complete, and the remaining replicate wells were rechallenged with fresh tumor cells. The same procedure was repeated for a total of 6 rechallenges. (B) Representative plot showing the readout on the day of FACS analysis. The proportion of MOLM- 13.GFP-ffLuc (CD33+CD3 ) and T cells (CD33'CD3+) among the leftover cells in a co-culture with non-signaling A control or full-length 27C, CAR-T cells, is shown respectively. (C) The number of kills observed per donor for each CD70 CAR construct was translated into a Kaplan Meier curve to depict the probability of tumor control upon repeated exposure to target antigen, n=6 donors. Table: statistical significance obtained from log-rank test. (D) T cell expansion curve of each CAR construct in coculture, n=6 donors, mean±SD, Table: statistical significance obtained from unpaired t-test with Welch’s correction on area under the curve (AUC) of T cell expansion. Significance levels: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: not significant.

[0071] Figure 3A-3F. In vivo anti-tumor activity of retroviral CD70 CAR-T cells. (A) Schematic of the experimental setup to investigate the anti-tumor efficacy of retroviral CD70 CAR-T cells in a MOLM-13.GFP-ffLuc NSG xenograft mouse model. (B) Images of tumor growth in each individual mouse in untreated and treated groups, as measured by bioluminescence imaging (BLI), n=5 mice per group, 1 of 2 representative experiments is shown. Grey scale 1x105to IxlO7p / sec / cm2. (C) Quantification of total flux (p / sec) for the back of each mouse in each respective group. (D) Kaplan Meier survival analysis with log-rank (Mantel-Cox) test, n=7-13 mice per group, summary of a total of 3 independent experiments. Significance levels: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: not significant. (E) Persistence of CD70 CAR-T cells, as measured by the frequency of mice with detectable huCD3+huCD45+T cells in various organs, on the day of sacrifice. BM: bone marrow, PB: peripheral blood, SP: spleen. (F) Left: summary of BLI from the back of mice treated with a low dose (0.5x106) of BB£ and 27£ CAR-T cells. Right: area under the curve (AUC) analysis of total flux, as shown in the left panel, from day -6 to day 17. n=2-4 mice per group, mean±SD, Welch’s t-test on AUC. Significance level: ***p<0.001.

[0072] Figure 4A-4E. In vitro characterization of hinge optimized CD70 CARs expressed in a self-inactivating ientiviral vector. (A) Pictogram depicting the vector constructs of hinge modified CD70 CARs. Endogenous CD27 hinge and transmembrane domains were replaced withcounterparts derived from CD8a and paired with various endo-domains. A truncated human EGFR (huEGFRt) gene was co-expressed as a selectable marker and the entire transgene cassette was encoded into a self-inactivating lentiviral vector backbone. (B) Top panel: transduction efficiency of hinge modified CAR-T cells on the day of harvest (D IO), as measured by %EGFR+(27^: 70±8%, CD8H&TM-41BBQ 76±5%, CD8H&TM-CD27£: 78±4%, CD8H-CD27^: 80±4%). Bottom panel: vector copy number analysis was performed in parallel to correlate the number of transgene copies present in each CAR-T cell (27^: 3.5±0.5, CD8H&TM-41 BB^: 3.9±0.5, CD8H&TM-CD27£ 4.2±0.7, CD8H-CD27£: 4±0.7), n=3 donors, mean±SD. (C). Top panel: the number of MOLM-13.GFP-ffLuc tumor cells left at each kill of a serial rechallenge assay where hinge modified CD70 CAR-T cells were co-cultured with target cells at an E:T ratio of 1 : 1. Lower panel: Kaplan Meier analysis of probability of tumor killing with log-rank (Mantel-Cox) test, n=3 donors, mean±SD. Significance level: *p<0.05, ns: not significant. (D) Effector cytokine production of hinge modified CD70 CAR-T cells after 21 hours of co-culture in the presence or absence of tumor cells. Top panel: IFNy. Lower panel: IL-2, n=3 donors, mean±SD, 2way ANOVA with Tukey’s multiple comparison test. Significance level: ***p<0.001 , ns: not significant. (E) Spontaneous proliferation of CD70 CAR-T cells plated in the absence of exogenous cytokines, monitored over a timespan of 8 days. Recombinant human IL-2 (50 lU / mL) was added to a separate 27£ well as a positive control, n=3 donors, mean±SD.

[0073] Figure 5A-5D. CD8H-CD27£ CAR efficiently suppressed tumor outgrowth in an AML xenograft model. (A) Schematic of in vivo CD8H-CD27£ CAR-T cell dose titration experiment to identify the suboptimal dosage that produces a partial anti-tumor response in the MOLM-13.GFP- ffLuc xenograft model. (B) Images of tumor growth in each individual mouse in control group (either untreated or infused with non-transduced T cells) and groups treated with IxlO6, 2xl06, 3xl06or 5xl06CD8H-CD27^ CAR-T cells, as measured by bioluminescence imaging (BLI), n=4-5 mice per group. Grey scale IxlO4to IxlO6p / sec / cm2. (C) Summary of BLI from the belly of mice over time, n=4-5 mice per group, mean±SD. (D) Overall survival as analyzed by Kaplan Meier curve with log-rank (Mantel-Cox) test, n=4-5 mice per group. Significance levels: *p<0.05, **p<0.01.

[0074] Figure 6A-6D. CD8H-CD27^ CAR displayed superior efficacy than CD8H&TM- 41 BB^ CAR in vivo. (A) Graphic of in vivo efficacy study performed to compare the potency of CD8H- CD27^ and previously published CD8H&TM-41BB^ CAR-T cell in a MOLM-13.GFP-ffLuc xenograft model. (B) Images of tumor growth in each individual mouse of the control groups and groups treated with a limiting dose of 1.5x106CAR-T cells, as measured by bioluminescence imaging (BLI), n=5 mice per group. Grey scale IxlO4to IxlO6p / sec / cm2. (C) Quantification of total flux (p / sec) for the belly of each mouse in each respective treatment group. Bottom panel: area under the curve (AUC) analysis of total flux for each group, shown in the panels above, from day 5 to day 22. n=5 mice per group, Welch’st-test on AUC, mean±SD. Significance levels: **p<0.01. (D) Kaplan Meier analysis with log-rank (Mantel-Cox) test, n=5 mice per group. Significance levels: *p<0.05.

[0075] Figure 7A-7D. CD8H-CD27£ CAR with potent anti-leukemic function in an adult AML PDX model. (A) Quantification of surface CD70 antigen density on the AML cell line MOLM- 13.GFP-ffLuc and PDX DFAM68555 cells (MOLM-13.GFP-ffLuc: 3173 molecules per cell, DFAM68555: 1359 molecules per cell). (B) Diagram depicting in vivo dose titration of CD8H-CD27^ CAR-T cells in mice engrafted with 2xl06DFAM68555 cells. (C) Representative FACS plot showing the gating strategy used to monitor the presence of leukemic cells (CD33+CD70+) and CAR-T cells (CD3+EGFR+) in the peripheral blood of mice. (D) Quantification of DFAM68555 tumor cells and CD8H-CD27ij CAR-T cells detected in blood of mice from untreated and treated groups (dosage of IxlO6, 2xl06, 3xl06or 5x10scells) over time, n=2 mice in tumor only and non-transduced groups, n=4 in treated groups, mean±SD.

[0076] Figure 8A-8B. Optimization of CD27-CD70 binding interface by computational design. (A) CD27 predominantly form dimers on the T-cell surface (blue). Up to three can interact simultaneously with the CD70 trimer on the tumor cell (red). Based on the resolved crystal structure of CD27:CD70 co-stimulatory complex, we applied a joint deep-learning and physics-based approach to redesign the CD27:CD70 interface. Our protocol (see methods) selected a total of 32 designs for further testing. The single-point mutations and their impact on interfacial energy are highlighted as filled spheres (on grey scale) from least stabilizing (white) to most stabilizing (black). (B) The selected mutations of interest were introduced into the extracellular domain (ECD) of CD8H-CD27lj CAR construct via PCR mutagenesis for downstream functional characterizations.

[0077] Figure 9A-9D. In vitro profiling of CD8H-CD27^ CAR variants with optimized binding interface. (A) Left panel: transduction efficiency of CD8H-CD27^ CAR muteins containing point mutations predicted to modulate the CD70-CD27 interaction, on day of harvest (D10). Destabilizing variants: TQ9, TQ10, TQ11. Mildly stabilizing variants: TQ12, TQ13, TQM (CD8H- CD27£: 80±8%, TQ9: 83±5%, TQ10: 79±6%, TQ11 : 89±1.5%, TQ12: 82±6%, TQ13: 8O±8%, TQM: 81±3%). Right panel: number of copies of transgene per CAR-T cell (CD8H-CD27Q 4.2±0.7, TQ9: 4.5±0.3, TQ10: 4.4±0.5, TQ11 : 4.9±0.5, TQ12: 4.5±0.3, TQ13: 4.1±0.5, TQM: 4.2±0.2), n=4 donors, mean±SD. (B) Quantification of MOLM-13.GFP-ffLuc tumor cells left at each kill of a serial rechallenge assay where CD8H-CD27(J mutant CAR-T cells were co-cultured with MOLM-13.GFP- ffLuc target cells at an E:T ratio of 1 :1. n=4 donors, mean±SEM. (C) Probability of tumor killing as delineated by Kaplan Meier analysis with log-rank (Mantel-Cox) test, n=4 donors. Top panel: destabilizing variants. Lower panel: mildly stabilizing variants. Significance levels: *p<0.05, ns: not significant. (D) Cytokine secretion by affinity-modulated CD8H-CD27^ CAR variants was analyzed insupernatants harvested 21 hours post co-culture with MOLM-13.GFP-ffLuc tumor cells. Top panel: IFN-y. Lower panel: IL-2, n=4 donors, mean±SEM. Significance levels: *p<0.05, ns: not significant.

[0078] Figure 10A-10D. In vivo characterization of affinity-modulated CD8H-CD27£ CAR variants in an AML xenograft model. (A) Schematic of experimental setup where mice bearing 5xl03MOLM-13.GFP-ffLuc cells were infused with respective CAR variants at a partial response dose of 1.5xl06cells. (B) Images of tumor growth in individual mice of control groups and treated groups, as measured by bioluminescence imaging (BLI), n=5 mice per group. Grey scale 1x104to IxlO6p / sec / cm2. (C) Summary of BLI from the belly of mice over time, n=5 mice per group, mean±SD. (D) Kaplan Meier survival analysis of mice treated with destabilizing variants (top panel) and mildly stabilizing variants (lower panel), with log-rank (Mantel-Cox) test, n=5 mice per group. Significance levels: *p<0.05, **p<0.01, ns: not significant.

[0079] Figure 11 A-l IB. In vitro functional screen of CD70 CAR variants with single and combinatorial stabilizing mutations in the binding interface. (A) Quantification of MOLM-13.GFP- ffLuc tumor cells left (%GFP+) at each kill of a serial rechallenge assay, where 16 CD8H-CD27^ CAR single point mutant CAR-T cells (TQ17-TQ32, as listed in Table 2) were co-cultured with target cells at an E:T ratio of 1 :1. Best-performing mildly stabilizing point mutant TQ14 identified from previous run was included here as a benchmark for comparison, n=l donor. (B) The remaining 10 CD8H-CD27£ CAR variants containing double or triple mutations in the CD27 extracellular binding domain (TQ29, TQ37-TQ40, TQ42-TQ46, as listed in Table 2) were similarly co-cultured with MOLM-13.GFP-ffLuc cells at an E:T ratio of 1 :4. The amount of remaining tumor cells at each kill is represented here as %GFP+, n=l donor. Mutants that displayed similar or beter killing capacity to the CD8H-CD27^ parental CAR and variant TQ14 were shortlisted for further in-depth functional characterization.

[0080] Figure 12A-12C. Functional delineation of shortlisted CD8H-CD27^ affinity variants in vitro showed improved tumor suppression and IL-2 production. (A) Based on the results obtained from previous screens, 4 best-performing mutants (TQM, TQ29, TQ37, TQ44) and 1 new double mutant TQ49 were further evaluated in a serial co-culture rechallenge assay. CAR-T cells were plated with MOLM-13.GFP-ffLuc target cells at an decreased E:T ratio of 1:4 and the probability of tumor killing was illustrated with a Kaplan Meier curve with log-rank (Mantel-Cox) test. n=7 donors for non-transduced, CD8H-CD27^ and TQM CAR constructs, n=6 donors for TQ29 and TQ44 CAR, n=5 donors for TQ37 and TQ49 CAR. Significance level: *p<0.05. (B) The corresponding T cell expansion curves of each affinity-modulated CAR construct in the serial co-culture assay over time. (C) Quantification of the amount of effector cytokines IFNy (top panel) and IL-2 (lower panel) secreted by the mutant CAR-T cells 3 days post co-culture with MOLM-13.GFP-ffLuc tumor cells. n=6 donors for non-transduced, CD8H-CD27^, TQM, TQ29 and TQ44 CAR constructs, n= 5 donors for TQ37CAR, n=4 donors for TQ49 CAR, median, 2way ANOVA with Dunnett’s multiple comparisons test. Significance levels: *p<0.05, **p<0.01, ***p<0.001, ns: not significant.

[0081] Figure 13A-13E. Double mutant TQ49 exhibited enhanced anti-leukemic activity and long-term persistence in vivo. (A) Schematic of experimental set up to assess the anti-tumor efficacy of top-performing CD8H-CD27^ affinity variants in a MOLM-13.GFP-ffLuc xenograft model. All mice were rechallenged with a single dose of 5xl03tumor cells on day 34 post-treatment. (B) Quantification of total flux (p / sec) of each mouse (belly view) treated with the corresponding variants over time. Arrows indicate the point of tumor rechallenge. (C) Area under the curve (AUC) analyses of total flux for each group at early timepoint (left; day -1 to day 18) and post-rechallenge (right; day 35 to day 60), Welch One-Way ANOVA with Dunnett’s multiple comparison test on AUC, mean±SD. Significance levels: *p<0.05, ***p<0.001, ns: not significant. (D) Overall survival of mice as portrayed by Kaplan Meier curve with log-rank (Mantel-Cox) test. Data representative of 2-3 independent experiments with n=14 mice for tumor only control, n=12 for CD8H-CD27C,, n=13 for TQM, TQ37, TQ44, n=8 for TQ29 and n=9 for TQ49. (E) The presence of tumor (% huCD45+CD33+GFP+) and human T cells (% huCD45+huCD3+) were assessed in the peripheral blood, bone marrow (BM) and spleen of all surviving mice harvested at the study endpoint (D70). n=4 mice for tumor only control, n=6 mice for CD8H-CD27^, TQM and TQ44, n=5 for TQ29, n=7 mice for TQ37 and n=4 mice for TQ49. Some spleen samples were excluded due to poor quality after processing.

[0082] Figure 14A-14E. Binding interface optimized TQ49 CAR-T cells were efficient in vivo against a pediatric AML PDX model. (A) Schematic of experimental set up to examine the antileukemic activity of TQ49 CAR-T cells against pediatric AML PDX model CBAM44728. (B) Quantification of tumor (huCD45+CD33+) and human T-cells (huCD45+CD3+) present in the peripheral blood (PB) of each mouse treated with non-transduced T-cells (5xl06cells) or various doses (IxlO6, 2xl06, 5x106) of TQ49 CAR-T cells. (C) Assessment of tumor and human T-cells present in the bone marrow (BM) and spleen of mice at the point of euthanasia (D32 for non-transduced, IxlO6and 2xl06doses; D56 for 5xl06dose). n=4 mice per group, median, Welch One-Way ANOVA with Dunnet’s multiple comparison test. Significance levels: *p<0.05, ***p<0.001. (D) Overall survival of mice as depicted by Kaplan Meier curve with log-rank (Mantel-Cox) test. Significance levels: *p<0.05, **p<0.01, ***p<0.001. (E) Memory profile of CAR-T cells detected in the PB, BM and spleen of each mouse treated with 5x106TQ49 CAR-T cells, at the experimental endpoint (D56). Effector memory (EM), central memory (CM), naive and effector memory re-expressing CD45RA (EMRA) T-cells were identified based on the surface expression of CD45RO and CD62L markers.

[0083] Figure 15A-15B. Surface CD70 antigen density on a panel of solid tumor cell lines. (A) Histogram illustrating the expression of CD70 detected on the surface of non-small cell lung cancer (A549), clear ceil renal carcinoma (ACHN), pancreatic carcinoma (PANC-1) and ovarian carcinoma (SK-OV-3) cell lines. AML cell lines HL-60.GFP-ffLuc (CD70') and MOLM-13.GFP-ffLuc (CD70+)were used as negative and positive staining controls, respectively. (B) Quantification of surface CD70 molecules on each of the above-mentioned cell lines: MOLM- 13. GFP-ffLuc (3940 molecules per cell), A549 (558 molecules per cell), ACHN (10037 molecules per cell), PANC-1 (27318 molecules per cell) and SK-OV-3 (52680 molecules per cell).

[0084] Figure 16A-16F. Double mutant TQ49 exhibited robust anti-tumor activity against ovarian cancer SK-OV-3 both in vitro and in vivo. (A) Transduction efficiency of TQ49 CAR-T cells on the day of harvest (D9), as measured by %EGFR+(69±13%). n=4 donors, mean±SD. (B) SK-OV-3. GFP-ffLuc cells were co-cultured with non-transduced or TQ49 CAR-T cells at the indicated E:T ratios for 96 hours, and the number of tumor cells left was quantified via flow cytometry. The counts were normalized to corresponding wells co-cultured with non-transduced T-cells to obtain the % cell lysis. (C) Experimental set up to evaluate the in vivo efficacy of TQ49 in a SK-OV-3.GFP- ffLuc xenograft model. (D) Images of tumor growth in each individual mouse treated with nontransduced (3xl06cells) or TQ49 CAR-T cells (dosages of IxlO6and 3xl06cells), as measured by bioluminescence imaging (BLI). n=5 mice per group, grey scale IxlO5to IxlO7p / sec / cm2. (E) Quantification of average total flux (p / sec) for the flank of mice in each respective treatment groups over time.#Saturation threshold reached in the non-transduced group. (F) Area under the curve (AUC) analyses of total flux as shown in (E), from day 0 to day 18. n=5 mice per group, Welch One-Way ANOVA with Dunnett’s multiple comparison test on AUC, mean±SD. Significance level: ***p<0.001 .

[0085] Figure 17A-17D. TQ49 demonstrated tumor suppression capacity against systemic CD70-low non-small cell lung cancer A549 in vivo. (A) Illustration of in vivo set up to assess the activity of TQ49 CAR-T cells in a systemic A549.GFP-ffLuc xenograft model. (B) Images of bioluminescence imaging (BLI) to track the tumor growth in each individual mouse treated with nontransduced (5xl06cells) orTQ49 CAR-T cells (dosages of IxlO6and 5xl06cells). n=5 mice per group, grey scale IxlO4to IxlO6p / sec / cm2. (C) Quantification of average total flux (p / sec) for the belly view of mice in each respective treatment groups over time. (D) Area under the curve (AUC) analyses of total flux as depicted in (C), from day 0 to day 11. n=5 mice per group, Welch One-Way ANOVA with Dunnett’s multiple comparison test on AUC, mean±SD. Significance level: ***p<0.001.DETAILED DESCRIPTION

[0086] In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook et al, "Molecular Cloning: A Laboratory Manual" (1989); "Current Protocols in Molecular Biology" Volumes I-III [Ausubel, R. M., ed. (1994)]; "Cell Biology: A Laboratory Handbook" Volumes I-III [J. E. Celis, ed. (1994))]; "Current Protocols in Immunology" Volumes I-III [Coligan, J. E., ed. (1994)]; "Oligonucleotide Synthesis" (M.J. Gait ed. 1984); "Nucleic Acid Hybridization" [B.D. Hames & S.J. Higgins eds. (1985)]; "Transcription AndTranslation" [B.D. Hames & S.J. Higgins, eds. (1984)]; "Animal Cell Culture" [R.I. Freshney, ed. (1986)]; "Immobilized Cells And Enzymes" [IRL Press, (1986)]; B. Perbal, "A Practical Guide To Molecular Cloning" (1984).

[0087] Therefore, if appearing herein, the following terms shall have the definitions set out below.A. TERMINOLOGY

[0088] The term “specific binding member” describes a member of a pair of molecules which have binding specificity for one another. The members of a specific binding pair may be naturally derived or wholly or partially synthetically produced. One member of the pair of molecules has an area on its surface, or a cavity, which specifically binds to and is therefore complementary to a particular spatial and polar organisation of the other member of the pair of molecules. Thus the members of the pair have the property of binding specifically to each other. Examples of types of specific binding pairs are antigen-antibody, biotin-avidin, hortnone-hormone receptor, protein-ligand, receptor-ligand, enzyme-substrate. This application is concerned with protein-ligand and receptor-ligand reactions.

[0089] The term “antibody” describes an immunoglobulin whether natural or partly or wholly synthetically produced. The term also covers any polypeptide or protein having a binding domain which is, or is homologous to, an antibody binding domain. CDR grafted antibodies are also contemplated by this term. An "antibody" is any immunoglobulin, including antibodies and fragments thereof, that binds a specific epitope. The term encompasses polyclonal, monoclonal, and chimeric antibodies, the last mentioned described in further detail in U.S. Patent Nos. 4,816,397 and 4,816,567. The term “antibody(ies)” includes a wild type immunoglobulin (Ig) molecule, generally comprising four full length polypeptide chains, two heavy (H) chains and two light (L) chains, or an equivalent Ig homologue thereof (e.g., a camelid nanobody, which comprises only a heavy chain); including full length functional mutants, variants, or derivatives thereof, which retain the essential epitope binding features of an Ig molecule, and including dual specific, bispecific, multispecific, and dual variable domain antibodies; Immunoglobulin molecules can be of any class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), or subclass (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2). Also included within the meaning of the term “antibody” are any “antibody fragment”.

[0090] An “antibody fragment” means a molecule comprising at least one polypeptide chain that is not full length, including (i) a Fab fragment, which is a monovalent fragment consisting of the variable light (VL), variable heavy (VH), constant light (CL) and constant heavy 1 (CHI) domains; (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a heavy chain portion of an Fab (Fd) fragment, which consists of the VH and CHI domains; (iv) a variable fragment (Fv), which consists of the VL and VH domains of a single arm of an antibody, (v) a domain antibody (dAb) fragment, which comprises a single variable domain (Ward, E.S. et al., Nature 341, 544-546 (1989)); (vi) a camelid antibody; (vii) an isolated complementaritydetermining region (CDR); (viii) a Single Chain Fv Fragment wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site (Bird et al, Science, 242, 423-426, 1988; Huston et al, PNAS USA, 85, 5879-5883, 1988); (ix) a diabody, which is a bivalent, bispecific antibody in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementarity domains of another chain and creating two antigen binding sites (WO94 / 13804; P. Holliger et al Proc. Natl. Acad. Sci. USA 90 6444-6448, (1993)); and (x) a linear antibody, which comprises a pair of tandem Fv segments (VH- CH1-VH-CH1) which, together with complementarity light chain polypeptides, form a pair of antigen binding regions; (xi) multivalent antibody fragments (scFv dimers, trimers and / or tetramers (Power and Hudson, J Immunol. Methods 242: 193-204 9 (2000)); (xii) a minibody, which is a bivalent molecule comprised of scFv fused to constant immunoglobulin domains, CH3 or CH4, wherein the constant CH3 or CH4 domains serve as dimerization domains (Olafsen T et al (2004) Prot Eng Des Sei 17(4):315- 323; Hollinger P and Hudson PJ (2005) Nature Biotech 23(9): 1126-1 136); and (xiii) other non-full length portions of heavy and / or light chains, or mutants, variants, or derivatives thereof, alone or in any combination.

[0091] Chimeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are included.

[0092] The term “adjuvant(s)” describes a substance, compound, agent or material useful for improving an immune response or immune cell or component stimulation, and may in some instances be combined with any particular antigen in an immunological, pharmaceutical or vaccine composition. Adjuvants can be used to increase the amount of antibody and effector T cells produced and to reduce the quantity of antigen or immune stimulant or modulator and the frequency of injection. Although some antigens are administered without an adjuvant, there are many antigens that lack sufficient immunogenicity to stimulate a useful immune response in the absence of an effective adjuvant. Adjuvants also improve the immune response from "self-sufficient" antigens, in that the immune response obtained may be increased or the amount of antigen administered may be reduced.

[0093] The term “specific” may be used to refer to the situation in which one member of a specific binding pair will not show any significant binding to molecules other than its specific binding partners). The term is also applicable where e.g. an antigen binding domain is specific for a particular epitope which is carried by a number of antigens, in which case the specific binding member carrying the antigen binding domain will be able to bind to the various antigens carrying the epitope.

[0094] The term “comprise” generally used in the sense of include, that is to say permitting the presence of one or more features or components.

[0095] The term “consisting essentially of’ refers to a product, particularly a peptide sequence, of a defined number of residues which is not covalently attached to a larger product. In the case of thepeptide of the invention referred to above, those of skill in the art will appreciate that minor modifications to the N- or C- terminal of the peptide may however be contemplated, such as the chemical modification of the terminal to add a protecting group or the like, e.g. the amidation of the C- terminus.

[0096] As used herein, "pg" means picogram, "ng" means nanogram, "ug" or "jig" mean microgram, "mg" means milligram, "ul" or "pl" mean microliter, "ml" means milliliter, "1" means liter.

[0097] The amino acid residues described herein are preferred to be in the "L" isomeric form. However, residues in the "D" isomeric form can be substituted for any L-amino acid residue, as long as the desired functional property of immunoglobulin-binding is retained by the polypeptide. NH2 refers to the free amino group present at the amino terminus of a polypeptide. COOH refers to the free carboxy group present at the carboxy terminus of a polypeptide.

[0098] It should be noted that all amino-acid residue sequences are represented herein by formulae whose left and right orientation is in the conventional direction of amino-terminus to carboxyterminus. Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino-acid residues. The above Table is presented to correlate the three-letter and one-letter notations which may appear alternately herein.

[0099] A "replicon" is any genetic element (e.g., plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo; i.e., capable of replication under its own control.[000100] A "vector" is a replicon, such as plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment.[000101] A "DNA molecule" refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in its either single stranded form, or a double-stranded helix. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes. In discussing the structure of particular double-stranded DNA molecules, sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA).[000102] An "origin of replication" refers to those DNA sequences that participate in DNA synthesis.[000103] A DNA "coding sequence" is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic(e.g., mammalian) DNA, and even synthetic DNA sequences, A polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.[000104] Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell.[000105] A "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. For purposes of defining the present invention, the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site (conveniently defined by mapping with nuclease S 1 ), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. Eukaryotic promoters will often, but not always, contain "TATA” boxes and "CAT" boxes. Prokaryotic promoters contain Shine-Dalgamo sequences in addition to the -10 and -35 consensus sequences.[000106] An "expression control sequence" is a DNA sequence that controls and regulates the transcription and translation of another DNA sequence. A coding sequence is "under the control" of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then translated into the protein encoded by the coding sequence. [000107] A "signal sequence" can be included before the coding sequence. This sequence encodes a signal peptide, N-terminal to the polypeptide, that communicates to the host cell to direct the polypeptide to the cell surface or secrete the polypeptide into the media, and this signal peptide is clipped off by the host cell before the protein leaves the cell. Signal sequences can be found associated with a variety of proteins native to prokaryotes and eukaryotes.[000108] The term "oligonucleotide," as used herein in referring to the probe of the present invention, is defined as a molecule comprised of two or more ribonucleotides, preferably more than three. Its exact size will depend upon many factors which, in tum, depend upon the ultimate function and use of the oligonucleotide.[000109] The term "primer" as used herein refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand, is induced, i.e., in the presence of nucleotides and an inducing agent such as a DNA polymerase and at a suitable temperature and pH. The primer may be either single-stranded or double-stranded and must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent. The exact length of the primer will depend upon many factors, including temperature, source of primer and use of the method. For example, for diagnostic applications, depending on the complexity of the target sequence, theoligonucleotide primer typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides.[000110] The primers herein are selected to be "substantially" complementary to different strands of a particular target DNA sequence. This means that the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be atached to the 5' end of the primer, with the remainder of the primer sequence being complementary to the strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence of the strand to hybridize therewith and thereby form the template for the synthesis of the extension product.[000111] As used herein, the terms "restriction endonucleases" and "restriction enzymes" refer to bacterial enzymes, each of which cut double-stranded DNA at or near a specific nucleotide sequence.[000112] A cell has been "transformed" by exogenous or heterologous DNA when such DNA has been introduced inside the cell. The transforming DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell. In prokaryotes, yeast, and mammalian cells for example, the transforming DNA may be maintained on an episomal element such as a plasmid. With respect to eukaryotic cells, a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA. A "clone" is a population of cells derived from a single cell or common ancestor by mitosis. A "cell line" is a clone of a primary cell that is capable of stable growth in vitro for many generations.[000113] Two DNA sequences are "substantially homologous" when at least about 75% (preferably at least about 80%, and most preferably at least about 90 or 95%) of the nucleotides match over the defined length of the DNA sequences. Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art.[000114] It should be appreciated that also within the scope of the present invention are DNA sequences encoding specific binding members (antibodies) of the invention which code for e.g. an antibody having amino acid sequence as provided in Figure(s) 10, I I, 12, 13 or 26, or comprising the CDR domain region sequences set out herein or in Figure(s) 7, 8, 10, 1 1, 12, 13 or 26, but which are degenerate thereto. By "degenerate to" is meant that a different three-letter codon is used to specify a particular amino acid. It is well known in the art that the following codons can be used interchangeably to code for each specific amino acid:Phenylalanine (Phe or F) UUU or UUCLeucine (Leu or L) UUA or UUG or CUU or CUC or CUA or CUGIsoleucine (lie or I) AUU or AUC or AUAMethionine (Met or M) AUGValine (Vai or V) GUU or GUC of GUA or GUG Serine (Ser or S) UCU or UCC or UCA or UCG or AGU or AGC Proline (Pro or P) CCU or CCC or CCA or CCG Threonine (Thr or T) ACU or ACC or ACA or ACG Alanine (Ala or A) GCU or GCG or GCA or GCG Tyrosine UAU or UAC HistidineCAU or CAC Glutamine (Gin or Q) CAA or CAGAsparagine (Asn or N) AAU or AACLysine (Lys or K) AAA or AAGAspartic Acid (Asp or D) GAU or GACGlutamic Acid (Glu or E) GAA or GAGCysteine (Cys or C) UGU or UGCArginine (Arg or R) CGU or CGC or CGA or CGG or AGA or AGGGlycine (Gly or G) GGU or GGC or GGA or GGGTryptophan (Trp or W) UGGTermination codon UAA (ochre) or UAG (amber) or UGA (opal)[000115] It should be understood that the codons specified above are for RNA sequences. The corresponding codons for DNA have a T substituted for U.[000116] Mutations can be made in the sequences encoding the amino acids, antibody fragments, CDR region sequences set out in Figure(s) 7, 8, 10, 11, 12, 13 and / or 26 and in the CDR sequences SEQ ID NOs: 27, 64, 65, 28, 29, 75, 9, 67, 68, 69, 70, 30, 72, 31, 14, 73, such that a particular codon is changed to a codon which codes for a different amino acid. Such a mutation is generally made by making the fewest nucleotide changes possible. A substitution mutation of this sort can be made to change an amino acid in the resulting protein in a non-conservative manner (for example, by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to another grouping) or in a conservative manner (for example, by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to the same grouping). Such a conservative change generally leads to less change in the structure and function of the resulting protein. A non-conservative change is more likely to alter the structure, activity or function of the resulting protein. The present invention includes sequences containing amino acid changes and substitutions, including conservative changes, which do not significantly alter the activity or binding characteristics of the resulting protein.[000117] The following is one example of various groupings of amino acids:Alanine, Valine, Leucine, Isoleucine, Proline, Phenylalanine, Tryptophan, MethionineAniino adds with R groupsGlycine, Serine, Threonine, Cysteine, Tyrosine, Asparagine, Glutamine(negatively charged at Ph 6.0)Aspartic acid, Glutamic acidBasic amino acids (positively charged at pH 6.0)Lysine, Arginine, Histidine (at pH 6.0)[000118] Another grouping may be those amino acids with phenyl groups:Phenylalanine, Tryptophan, Tyrosine[000119] Another grouping may be according to molecular weight (i.e., size of R groups):Glycine 75 Alanine 89Serine 105 Proline 115Valine 117 Threonine 119Cysteine 121 Leucine 131Isoleucine 131 Asparagine 132Aspartic acid 133 Glutamine 146Lysine 146 Glutamic acid 147Methionine 149 Histidine (at pH 6.0) 155Phenylalanine 165 Arginine 174Tyrosine 181 Tryptophan 204[000120] Particularly preferred substitutions are:- Lys for Arg and vice versa such that a positive charge may be maintained;- Glu for Asp and vice versa such that a negative charge may be maintained;- Ser for Thr such that a free -OH can be maintained; and- Gin for Asn such that a free NHz can be maintained.[000121] Exemplary and preferred conservative amino acid substitutions include any of: glutamine (Q) for glutamic acid (E) and vice versa; leucine (L) for valine (V) and vice versa; serine (S) for threonine (T) and vice versa; isoleucine (I) for valine (V) and vice versa; lysine (K) for glutamine (Q) and vice versa; isoleucine (I) for methionine (M) and vice versa; serine (S) for asparagine (N) and vice versa; leucine (L) for methionine (M) and vice versa; lysine (L) for glutamic acid (E) and vice versa; alanine (A) for serine (S) and vice versa; tyrosine (Y) for phenylalanine (F) and vice versa; glutamic acid (E) for aspartic acid (D) and vice versa; leucine (L) for isoleucine (I) and vice versa; lysine (K) for arginine (R) and vice versa.[000122] Amino acid substitutions may also be introduced to substitute an amino acid with a particularly preferable property. For example, a Cys may be introduced a potential site for disulfide bridges with another Cys. A His may be introduced as a particularly "catalytic" site (z.e., His can act as an acid or base and is the most common amino acid in biochemical catalysis). Pro may be introduced because of its particularly planar structure, which induces P-tums in the protein's structure.[000123] A "heterologous" region of the DNA construct is an identifiable segment of DNA within a larger DNA molecule that is not found in association with the larger molecule in nature. Thus, when the heterologous region encodes a mammalian gene, the gene will usually be flanked by DNA that does not flank the mammalian genomic DNA in the genome of the source organism. Another example of a heterologous coding sequence is a construct where the coding sequence itself is not found in nature (e.g., a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the native gene). Allelic variations or naturally-occurring mutational events do not give rise to a heterologous region of DNA as defined herein.[000124] A DNA sequence is "operatively linked" to an expression control sequence when the expression control sequence controls and regulates the transcription and translation of that DNA sequence. The term "operatively linked" includes having an appropriate start signal (e.g., ATG) in front of the DNA sequence to be expressed and maintaining the correct reading frame to permit expression of the DNA sequence under the control of the expression control sequence and production of the desired product encoded by the DNA sequence. If a gene that one desires to insert into a recombinant DNA molecule does not contain an appropriate start signal, such a start signal can be inserted in front of the gene.[000125] The term "agent" means any molecule, including polypeptides, antibodies, polynucleotides, chemical compounds and small molecules. In particular the term agent includes compounds such as test compounds or drug candidate compounds.[000126] The term "agonist" refers to a ligand that stimulates the receptor the ligand binds to in the broadest sense.[000127] The term "assay" means any process used to measure a specific property of a compound. A "screening assay" means a process used to characterize or select compounds based upon their activity from a collection of compounds.[000128] The term "preventing" or "prevention" refers to a reduction in risk of acquiring or developing a disease or disorder (i.e., causing at least one of the clinical symptoms of the disease not to develop) in a subject that may be exposed to a disease-causing agent, or predisposed to the disease in advance of disease onset.[000129] The term "prophylaxis" is related to and encompassed in the term ‘prevention’, and refers to a measure or procedure the purpose of which is to prevent, rather than to treat or cure a disease. Nonlimiting examples of prophylactic measures may include the administration of vaccines; theadministration of low molecular weight heparin to hospital patients at risk for thrombosis due, for example, to immobilization; and the administration of an anti-malarial agent such as chloroquine, in advance of a visit to a geographical region where malaria is endemic or the risk of contracting malaria is high.[000130] "Therapeutically effective amount" means that amount of a drug, compound, antimicrobial, antibody, or pharmaceutical agent that will elicit the biological or medical response of a subject that is being sought by a medical doctor or other clinician. In particular, with regard to gram-positive bacterial infections and growth of gram-positive bacteria, the term “effective amount” is intended to include an effective amount of a compound or agent that will bring about a biologically meaningful decrease in the amount of or extent of tumor regression and or increase in length of a subject’s survival or period disease-free or in remission. The phrase "therapeutically effective amount" is used herein to mean an amount sufficient to prevent, and preferably reduce by at least about 30 percent, more preferably by at least 50 percent, most preferably by at least 90 percent, a clinically significant change in the growth or amount of tumor size, or enhanced survival or disease-free period by at least about 30 percent, more preferably by at least 50 percent, most preferably by at least 90 percent.[000131] The term "treating" or "treatment" of any disease or infection refers, in one embodiment, to ameliorating the disease or infection (i.e., arresting the disease or growth of the infectious agent or bacteria or reducing the manifestation, extent or severity of at least one of the clinical symptoms thereof). In another embodiment "treating" or "treatment" refers to ameliorating at least one physical parameter, which may not be discernible by the subject. In yet another embodiment, "treating" or "treatment" refers to modulating the disease or infection, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both. In a further embodiment, "treating" or "treatment" relates to slowing the progression of a disease or reducing an infection.[000132] The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.[000133] As used herein, "pg" means picogram, "ng" means nanogram, "ug" or "pg" mean microgram, "mg" means milligram, "ul" or "pl" mean microliter, "ml" means milliliter, "1" means liter.B. DETAILED DISCLOSURE.[000134] The invention provides novel CD70 binding proteins, particularly CD27 polypeptides which are altered in amino acid sequence from native or wild-type CD27 polypeptide sequence, particularly native or wild-type human CD27 polypeptide sequence. The novel, variant or mutant CD27 polypeptides, particularly the variant or mutant CD27 extracellular domains, are capable of binding CD70. In some particular aspects, the variant or mutant CD27 polypeptides, particularly the variant ormutant CD27 extracellular domains, demonstrate stabilized or increased binding to CD70 and / or demonstrate higher potency activity upon CD70 binding, such as modulation of an immune response, such as stimulating IL-2 upon CD70 binding. In an aspect, the variant or mutant CD27 peptides, particularly the CD27 extracellular domains, demonstrate increased or greated IL-2 stimulation and / or expression upon CD70 binding in comparison with wild type, native, or unmutated CD27.[000135] The CD27 protein has extracellular, transmembrane, and cytoplasmic domains. CD27, a member of the TNF receptor superfamily is constitutively expressed on naive T-cells, memory B-cell and T cell populations, NK-cells, and hematopoietic stem cells (HSCs) and progenitor cells. CD70 is a cytokine that contains a cytoplasmic, transmembrane, and extracellular domains. The extracellular domain of CD70 is a ligand for CD27. CD70 (CD27L), the only ligand for CD27, is a tightly regulated transmembrane glycoprotein expressed on both B and T lymphocytes and antigen presenting cells (APCs). In some embodiments, the CD70 binding domain is located within the extracellular signaling domain of CD27.[000136] CD70 is expressed in numerous cancers, for example, bladder cancer, breast invasive carcinoma, cervical cancer, cholangiocarcinoma, colorectal cancer, diffuse large B-cell lymphoma (DLBC), Esophagus, glioblastoma (GBM), head and neck cancer, low-grade gliomas (LGG), liver cancer, lung adeno cancer, melanoma, mesothelioma, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, stomach cancer, testicular germ cell cancer, thymoma, thyroid cancer, uterine cancer, uveal melanoma, clear cell renal cell carcinoma (ccRCC), chromophobe renal cell carcinoma, papillary renal cell car-cinoma (pRCC), acute myeloid leukemia, and adenoid cystic carcinoma (ACC). In some embodiments, the cancer is a myeloid cancer. In some embodiments, the cancer is acute myeloid leukemia.[000137] The full length human CD27 amino acid sequence of 260 amino acids (corresponding to Uniprot P26842) is provided below (SEQ ID NO: 1) and includes an endogenous signal peptide sequence, extracellular domain (ECD), hinge region (H), transmembrane domain (TM) and endodomain (Endo).MARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAA 60QCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTEC 120DPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPP 180QRSLCSSDFIRILVIFSGMFLVFTLAGALFLHQRRKYRSNKGESPVEPAEPCHYSCPREE 240EGSTIPIQEDYRKPEPACS P260[000138] The extracellular domain amino acids 22-124 are as follows (SEQ ID NO:2):PAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCES CRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLP[000139] The transmembrane and endo domains correspond to amino acids 193-260 as follows (SEQ ID NO:3):LVIFSGMFLVFTLAGALFLHQRRKYRSNKGES PVEPAEPCHYSCPREEEGSTIPIQEDYRKP EPACSP[000140] In accordance with the invention, mutant or variant CD27 polypeptides are provided which are altered at one or more amino acid in the native CD27 sequence, particularly the native human CD27 amino acid sequence (SEQ ID NO:1)[000141] Certain exemplary mutant or variant CD27 polypeptides / peptides are provided that are altered at one or more amino acids selected from F71, S72, D74, H76, R78, H80, E82, H86, R85, S89, QI 11, KI 15, El 19, D121, in the CD27 extracellular domain sequence. These mutant locations are shown below (in bold and underlined) in the native human CD27 extracellular domain sequence corresponding to amino acids 22-124 (SEQ ID NO:1): MARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAA 60QCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTEC 120DPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPP 180QRSLCSSDFIRILVIFSGMFLVFTLAGALFLHQRRKYRSNKGESPVEPAEPCHYSCPREE 240EGSTIPIQEDYRKPEPACSP260[000142] Various sequences, proteins, peptide, ligand binding and domains are described and provided herein. Certain protein sequences are show in the following table. For exemplary CD70 ligand CD27 variants TQ9 through TQ49, mutated amino acids are shown on bold and underlined. The extracellular domain, corresponding to amino acids 22-124 (using the human CD27 Uniprot P26842 sequence numbering), are shown for each of the CD27 variants.[000143] Initially evaluated CD27 mutant variants were altered at one or more amino acids selected from F71, S72, D74, H76, R78, H80, E82, H86, R85, S89, QI 11, KI 15, El 19, D 121, in the CD27 extracellular domain sequence. These mutant locations are shown below (in bold and underlined) in the native human CD27 extracellular domain sequence corresponding to amino acids 22-124 (SEQ ID NO:1):MARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAA 60QCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTEC 120DPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPP 180QRSLCSSDFIRILVIFSGMFLVFTLAGALFLHQRRKYRSNKGESPVEPAEPCHYSCPREE 240EGSTIPIQEDYRKPEPACSP260[000144] In an aspect, a potency-tuned and variant CD27 affinity ligand is altered at one or more amino acids selected from F71, S72, D74, H80, H86, R85, S89, QI 11, KI 15, El 19, D121, in the CD27 extracellular domain sequence. In an aspect, a potency-tuned and variant CD27 affinity ligand is altered at two or more amino acids selected from F71, S72, D74, H80, H86, R85, S89, QI 11, KI 15, El 19, D 121, in the CD27 extracellular domain sequence. In an aspect, a potency-tuned and variant CD27 affinity ligand is altered at two amino acids selected from F71, S72, D74, H80, H86, R85, S89, QI 11, KI 15, El 19, D121, in the CD27 extracellular domain sequence. In an aspect, a potency-tuned and variant CD27 affinity ligand is altered at three or more amino acids selected from F71, S72, D74, H80, H86, R85, S89, QI 11, KI 15, El 19, D121, in the CD27 extracellular domain sequence. In an aspect, a potency-tuned and variant CD27 affinity ligand is altered at three amino acids selected from F71, S72, D74, H80, H86, R85, S89, QI 1 1, KI 15, El 19, D121, in the CD27 extracellular domain sequence. In a preferred aspect, a potency-tuned and variant CD27 affinity ligand is altered at three or more amino acids selected from S72, H86, QI 1 1 and KI 15 in the CD27 extracellular domain sequence.[000145] In accordance with the invention, a variant extracellular binding domain of CD27 is provided comprising the sequence set out as follows (SEQ ID NO: 45):PAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFXiPDHHTRPHCES CR X2CNSGLLVRNCTITANAECACRNGWX3CRDX4ECTECDPLPWherein Xi is A or NX2is V, F or YX3is TX4 is E, D, Q or A[000146] In another embodiment, a variant extracellular binding domains of CD27 is provided comprising the sequence set out as follows (SEQ ID NO: 46):[000147] In another embodiment, a variant extracellular binding domain of CD27 is provided, having one or more amino acid substitution, comprising the sequence set out as follows (SEQ ID NO: 47):and wherein the variant extracellular binding domain of CD27 does not correspond in sequence to human CD27 extracellular binding domain of SEQ ID NO:2:PAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCES CRHCNSGL'LVRNCTITANAECACRNGWQCRDKECTECDPLP[000148] In preferred aspects, the mutant potency tuned CD27 extracellular domain is selected from mutant TQ14 (S72A) (SEQ ID NO.T8), TQ29 (H86V) (SEQ ID NOG l), TQ37 (S72N-H86V) (SEQ ID NO:35), TQ44 (H86V-Q1 11T-K115E) (SEQ ID NO:41) and TQ49 (S72A-H86V) (SEQ ID NO:44).[000149] Variant CD27 affinity ligand sequences, including full length CD27 variant sequences, which include or otherwise comprise a variant CD27 extracellular domain sequence are provided herein. CD27 variant sequences comprising a variant extracellular domain sequence as provided herein, and further comprising a CD27 transmembrane or endodomain are also contemplated and included herein. These variant CD27 sequences may comprise one or more of the variant extracellular domains set out and described herein, along with the CD27 transmembrane and end domain, such as the transmembrane and endo domains corresponding to SEQ ID NO: 3 (amino acids 193-260 of human CD27).[000150] The mutant CD27 peptide(s), particularly the CD27 extracellular domain(s), can be used in isolation, for example to characterize, identify, locate or tag CD70, including CD70 expressed on cells, such as lymphocytes. The mutant CD27 peptide(s) or the mutant CD27 extracellular domain(s) can be combined with other proteins or protein domains, or can be fused with other proteins or protein domains or covalently attached with other proteins or protein domains. In aspects, the mutant CD27 polypeptides, particularly the mutant CD27 extracellular domains, are conjugated with other proteins or domains.[000151] In some embodiments, the mutant CD27 peptide(s) or the mutant CD27 extracellular domain(s) is conjugated to another compound, for example being conjugated to a half-life extending moiety. In some embodiments, the binding protein is conjugated to a polymer (e.g., PEG). In some embodiments, the binding protein is conjugated to a cytotoxic agent. In some embodiments, the mutant CD27 peptide(s) or the mutant CD27 extracellular domain(s) is conjugated to an immunomodulatory compound, protein or domain. In some embodiments, the mutant CD27 peptide(s) or the mutant CD27 extracellular domain(s) is conjugated to or combined with an immune stimulator or immune activator, such as an interleukin, such as IL-2, or an interferon, such as IFN-y.[000152] In aspects of the invention, the mutant CD27 peptide(s) or the mutant CD27 extracellular domain(s) are included or incorporated in immunotherapeutic constructs or components or compounds, which are effective and active by virtue of the CD27 binding, interaction or modulation of its ligand and binding molecule CD70.[000153] Such immunotherapeutic constructs or components or compounds include but are not limited to entities which are designed and utilized specifically to alter immunological responses and / or immune cell responses. These include stimulating or inhibit an immune or immune cell response, activating immune cells against an antigen or other cell, such as a cancer antigen or cancer or tumor cell.[000154] Immunotherapeutic approaches utilizing the mutant CD27 peptide(s) or the mutant CD27 extracellular domain(s) provided include but are not limited to protein or cell therapies such as chimeric antigen receptors (CARs), bispecific T cell engagers (BiTEs), which utilize protein constructs or cells expressing the protein constructs to modulate the immune system, immune response, anti-cancer response, etc. Other approaches utilize the mutant CD27 peptide(s) or the mutant CD27 extracellular domain(s) is conjugated to another compound, such as a cytotoxic agent, immunomodulatory agent.[000155] Chimeric antigen receptors (CARs)[000156] The term "chimeric antigen receptor" or "CAR" refers to a polypeptide or set of polypeptides, which when in an immune effector cell, provides the cell with specificity for a target cell, for example a cancer cell, and with intracellular signal generation.[000157] A CAR cell-surface receptor provided herein comprises an extracellular target-binding domain (e.g., a CD70 binding domain, such as a mutant CD27 extracellular domain or binding domain), a transmembrane domain, and an intracellular domain or endodomain, comprising a signalling domain and optionally at least one co-stimulatory signaling domain (referred to also as intracellular (IC) domain herein), all in a combination that is not naturally found together on a single protein. This particularly includes receptors wherein the extracellular domain and the cytoplasmic or intracellular domain are not naturally found together on a single receptor protein. The chimeric antigen receptors of the present invention are intended primarily for use with lymphocytes such as T cells and natural killer (NK) cells.[000158] Chimeric antigen receptors (CARs) are hybrid molecules comprising an antigentargeting moiety or binding domain, followed by a linker, transmembrane (TM) domain, and various endodomains (EDs) involved in T-cell activation. CARs may include a single endodomain, such as the ED of CD3-zeta only, to signal T cell activation. CARs also have one or more additional EDs or costimulatory EDs, as well such as CD28 and 4-1BB, to provide additional T cell signalling.[000159] The CARs described and provided herein comprise at least an extracellular binding domain comprising the CD27 binding domain of the invention, as well as a transmembrane domain and an intracellular domain (also referred to as a “cytoplasmic signalling domain” or "an intracellular signalling domain").[000160] In some embodiments, the CAR further comprises one or more additional polypeptide sequences. Exemplary additional polypeptide sequences include, but are not limited to, signal sequences, epitope tags, and polypeptides that produce a detectable signal.[000161] In some embodiments, the CAR further comprises a leader sequence, such as a signal sequence. As used herein the term “leader sequence” refers to a sequence of amino acids which, when fused to a CAR sequence, aids localization of the CAR to a cell membrane of a cell expressing the CAR. In some embodiments, the leader sequence is an N-terminal leader sequence. In some embodiments, the leader sequence is an N-terminal signal sequence. In some embodiments, the leader sequence is a signal sequence and is an immunoglobulin signal sequence. In some embodiments, the leader sequence comprises a sequence of amino acids which is at least 70% or at least 80% or at least 90% or at least 95% identical to SEQ ID NO: 48. In some embodiments, the leader sequence is cleaved from the CAR during cellular processing and localization of the CAR to the cellular membrane when expressed in a cell.[000162] In some embodiments, the CAR domains are in the same polypeptide chain (e.g., comprise a chimeric fusion protein). In some embodiments, the domains are contiguous with each other. In some embodiments, the domains are not contiguous with each other, e.g., are in different polypeptide chains, such as a split CAR. In some embodiments, the different polypeptide chains include a dimerization switch that, upon the presence of a dimerization molecule, can couple the polypeptides to one another, e.g., can couple an antigen or ligand-binding domain to an intracellular signalling domain. [000163] Target Binding Domain[000164] The binding domain comprises a CD27 binding domain as described and provided herein. The CD27 binding domain particularly comprises a mutant CD27 extracellular domain comprising amino acids 22-124 of CD27, wherein one or more amino acid is modified or substituted. The CD27 extracellular domain comprising amino acids 22-124 of CD27 (SEQ ID NO:2) is set out as follows:PAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCE SCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLP (SEQ ID NO:2)[000165] In particular aspects, the mutant CD27 extracellular domain (CD27 extracellular domain amino acids 22-124) is selected from SEQ ID NOs:13-47.[000166] Transmembrane Domain[000167] In various embodiments, a CAR can be designed to comprise a transmembrane domain that is attached to the extracellular domain of the CAR, particularly the mutant CD27 extracellular domain, for positioning of the CAR in a cell membrane. A transmembrane domain can include one or more additional amino acids adjacent to the transmembrane region, e.g., one or more amino acid associated with the extracellular region of the protein from which the transmembrane was derived (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids of the extracellular region) and / or one or more additional amino acids associated with the intracellular region of the protein from which the transmembrane protein is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids of the intracellular region). In one embodiment, the transmembrane domain is one that is associated with one of the other domains of the CAR e.g., in one embodiment, the transmembrane domain may be from the same protein that the primary signalling domain, costimulatory domain or the hinge region is derived from. In another embodiment, the transmembrane domain is not derived from the same protein that any other domain of the CAR is derived from.[000168] In some instances, 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, e.g., to minimize interactions with other members of the receptor complex. In one embodiment, the transmembrane domain is capable of homodimerization with another CAR on the cell surface of a CAR-expressing cell. In another embodiment, the amino acid sequence of the transmembrane domain may be modified or substituted so as to minimize interactions with the binding domains of the native binding partner present in the same CAR-expressing cell.[000169] The transmembrane domain may be derived either from a natural or from a recombinant source. In one embodiment the transmembrane domain is capable of signalling to the intracellular domain(s) whenever the CAR has bound to its target. A transmembrane domain of particular use in this invention may include at least the transmembrane region(s) of the alpha, beta or zeta chain of the T-cell receptor, TNFR2, CD28, CD27, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. In some embodiments, a transmembrane domain may include at least the transmembrane region(s) of, e.g., KIRDS2, 0X40, CD2, CD27, LFA-1 (CD I la, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD 160, CD 19, IL2R beta, IL2R gamma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE, CD 103, ITGAL, CD1 la, LFA-1, ITGAM, CD1 lb, ITGAX, CD1 1c, ITGB1, CD29, ITGB2, CD 18, LFA-1, ITGB7, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM,Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, LylO8), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKG2D, NKG2C, or a functional variant thereof.[000170] In some instances, the transmembrane domain can be atached to the extracellular region of the CAR, e.g., the antigen-binding domain of the CAR, via a hinge, such as a hinge from a human protein. As used herein, the term “hinge region” or “hinge domain” refers to any suitable amino acid sequence positioned between the transmembrane domain and the antigen binding domain or CD27 extracellular domain. Suitable hinge sequences are available and known in the art. For example, in one embodiment, the hinge can be a human Ig (immunoglobulin) hinge (such as an IgG4 hinge, an IgD hinge), a GS linker (e.g., a GS linker described herein), a KIR2DS2 hinge or a CD8 alpha hinge. In one embodiment, the hinge or spacer comprises an IgG4 hinge. In one embodiment, the hinge region comprises an IgD hinge. In another embodiment, the hinge comprises a CD8 alpha hinge region. In one such embodiment, the hinge domain comprises a sequence of amino acids that is at least 80%, 90%, 95% identical or is 100% identical to SEQ ID NO:49.[000171] In some embodiments, the transmembrane domain is derived from CD27. In embodiments, the transmembrane domain is derived from CD27 and comprises amino acids corresponding to amino acids 193-260, or a portion thereof. In embodiments, the encoded transmembrane domain comprises an amino acid sequence of SEQ ID NO:3, or a sequence with at least 80% identity, 90% identity, or 95% identity thereof.[000172] Optionally, a short oligo- or polypeptide linker, between 2 and 10 amino acids in length may form the linkage between the transmembrane domain and the cytoplasmic region of the CAR. A glycine-serine doublet provides a particularly suitable linker. For example, in one embodiment, the linker comprises the amino acid sequence of GGGGSGGGGS. In one embodiment, the hinge or spacer comprises a KIR2DS2 hinge.[000173] Intracellular Domain[000174] The intracellular domain of the CAR is generally responsible for activation of at least one of the normal effector functions of the immune cell in which the CAR has been introduced. 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. Thus the term "intracellular domain" refers to the portion of a CAR which transduces the effector function signal and directs the cell to perform a specialized function. To the extent that a truncated portion of the intracellular domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal. The term intracellular domain is meant to include any truncated portion of the intracellular domain sufficient to transduce the effector function signal.[000175] Intracellular domains for use in the CAR of the invention include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transductionfollowing antigen receptor engagement, as well as any derivative or variant of these sequences and any recombinant sequence that has the same functional capability.[000176] It is known that signals generated through the TCR alone are insufficient for full activation of the T cell and that a secondary and / or costimulatory signal is also required. Thus, T cell activation can be said to be mediated by two distinct classes of cytoplasmic signalling sequences: those that initiate antigen-dependent primary activation through the TCR (referred to herein as a “primary signalling domain”) and those that act in an antigen-independent manner to provide a secondary or costimulatory signal (referred to herein as a “costimulatory domain”).[000177] As used herein, the phrase “primary signalling domain” refers to a domain present in the intracellular domain of a CAR, which is capable of inducing, or inhbiting, antigen-dependent primary activation signalling in a cell expressing the CAR. For example, primary activiation signalling such as that initiated by, for instance, binding of a TCR / CD3 complex with an MHC molecule loaded with peptide, and which leads to mediation of a T cell response, such as proliferation, activation, differentiation, and the like. In this regard, a primary signalling domain regulates primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way.[000178] Primary signalling domains that are of use in the invention include those of CD3 zeta (CD3Q, common FcR gamma (FCERJG), Fc gamma Rlla, FcR beta (Fc Epsilon Rib), CDS gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP 10, and DAP 12, or a functional variant thereof. In one embodiment, a CAR of the invention comprises an intracellular signalling domain, e.g., a primary signalling domain of CD3-zeta, or a functional variant thereof. In one embodiment, a CAR of the invention comprises a primary signalling domain of CD3-zeta (SEQ ID NO:50).[000179] In one embodiment, a primary signalling domain comprises a modified primary signalling domain, e.g., a mutated signaling domain which has altered (e.g., increased or decreased) activity as compared to the native domain.[000180] The intracellular domain of the CAR can comprise the primary signalling domain by itself or it can be combined with another intracellular signalling domain(s) useful in the context of a CAR of the invention. For example, in some embodiments, the intracellular domain of the CAR comprises a primary signalling domain and a costimulatory domain.[000181] A “costimulatory domain” refers to a portion of the CAR which is derived from an intracellular domain of a costimulatory molecule and which is capable of providing a costimulatory signal in a cell expressing the CAR upon antigen binding. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligands that is required for an efficient response of lymphocytes to an antigen. Examples of such molecules include TNFR2, CD27, CD28, 4- IBB (CD137), 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83, and the like. For example, CD27 costimulation has been demonstrated to enhance expansion, effector function, andsurvival of human CAR-T cells in vitro and augments human T cell persistence and antitumour activity in vivo (Song et al., 2012). Further examples of such costimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD 160, CD 19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDlld, ITGAE, CD 103, ITGAL, CDlla, LFA- 1, ITGAM, CD1 lb, ITGAX, CDllc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), NK.G2D, CEACAM1, CRTAM, Ly9 (CD229), CD 160 (BY55), PSGL1, CD 100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, and CD 19a.[000182] The primary signalling domain and the costimulatory domain within the cytoplasmic portion of the CAR of the invention may be linked to each other in a random or specified order. Optionally, a short oligo- or polypeptide linker, for example, between 2 and 10 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) in length may form the linkage between intracellular signalling sequence. In one embodiment, a glycine- serine doublet can be used as a suitable linker. In one embodiment, a single amino acid, e.g., an alanine, a glycine, can be used as a suitable linker.[000183] In one embodiment, the intracellular domain is designed to comprise the signalling domain of CD3-zeta and the signalling domain of CD28. In one embodiment, the intracellular domain is designed to comprise the signalling domain of CD3-zeta, the signalling domain of CD28, and the signalling domain of 4-1 BB, or a functional variant thereof.[000184] In some embodiments, different linker sequences may be used between the different domains of the CAR, e.g., a (GGGS)n linker, wherein n is 1-20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20). In some embodiments, the linker is (GGGS)?. In some embodiments, the CAR comprises additional sequences from CD27, e.g., the stalk and hinge region of CD27, between the extracellular target binding domain and the transmembrane region. In some embodiments, the CAR does not comprise additional sequences from CD27, e.g., the stalk and hinge region of the between the extracellular target binding domain and the transmembrane region.[000185] In some embodiments, the CAR comprises:(a) a signal sequence;(b) a CD27 binding domain (CD27 extracellular domain amino acids 22-124) selected from SEQ ID NOs: 13-47;(c) a hinge sequence;(d) an intracellular or transmembrane domain; and(e) a signalling domain.[000186] In some embodiments, the CAR comprises:(a) a signal sequence (such as immunoglobulin sequence) MEFGLSWLFLVAILKGVQC (SEQ ID NO: 48);(b) a CD27 binding domain (CD27 extracellular domain amino acids 22-124) selected from SEQ ID NOs: 13-47;(c) a hinge sequence (such as CD8a hinge sequence)TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDI (SEQ ID NO:49)(d) an intracellular or transmembrane domain (such as the CD27 transmembrane and endodomains):LVIFSGMFLVFTLAGALFLHQRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEP ACSP (SEQ ID NOG); and(e) a signalling domain (such as CD3^ chain sequence):RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYN ELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:50).[000187] In some optimized embodiments, the CD70 targeted CARs provided herein comprise the following components:A signal sequence (such as immunoglobulin sequence):MEFGLSWLFLVAILKGVQC (SEQ ID NO:48)A CD27 binding domain (CD27 extracellular domain amino acids 22-124):SCR} ICNJiC^ RNCTI MNAFCACRNGV QC RDKTC 1 ECDPLP (SEQ ID NOG)A hinge sequence (such as CD8a hinge sequence):TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDI (SEQ ID NO:49)An intracellular or transmembrane domain (such as the CD27 transmembrane and endo-domains):LVIFSGMFLVFTLAGALFLHQRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP (SEQ ID NOG)And a signalling domain(such as CD3^ chain sequence):RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ IDNO:50)[000188] In particular embodiments, the designed CD27-CD70 binding interface corresponds to one or more of the variant CD27 binding sequences described and provided herein. In particular embodiments, one or more of the variant CD27 binding domain sequences, such as those provided in SEQ ID NOs: 13-44, particularly the variant CD27 binding domain sequences provided in SEQ ID NOs: 16-44, particularly the variant CD27 binding domain sequences set out in any of SEQ ID NOs: 18, 31, 35, 41 and 44, are utilized as the CD27 binding domain (extracellular domain) in a CARconstruct. One skilled in the art will recognize that the hinge region sequence and / or the signalling domain sequence can be replaced with an alternative relevant candidate sequence, provided that the function and activity of the CAR is suitably retained and maintained.[000189] The invention provides a novel set of CD70 targeted CARs with an optimized assembly of components comprising of a designed CD27-CD70 binding interface, a CD8a hinge, CD27 transmembrane and endo-domains, and the CD3^ chain.[000190] The designed CAR components are based on the CAR designated herein as CD8H- CD27^, which corresponds to SEQ ID NO: 1 1 as shown below. An exemplary suitable signal sequence is shown in bold and italics; the CD27 binding domain (extracellular domain) is shown underlined; the CD8a hinge sequence is next; and the CD27 transmembrane and endo-domains is provided in bold; followed by the CD3^ chain sequence.TTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDILVIFSGMFLVFTLAGALF LHQRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSPRVKFSRSADAP AYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEA YSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:11)[000191] In some embodiments, the CAR is a multi-specific (e.g., a bispecific or a trispecific) CAR. Protocols for generating bispecific or heterodimeric binding molecules are known in the art; including but not limited to, for example, as described in US 5731168; WO 09 / 089004, WO 06 / 106905 and WO 2010 / 129304; WO 07 / 110205; WO 08 / 119353, WO 2011 / 131746, and WO 2013 / 060867; US5273743; US5534254; US5582996; US5591828; US5635602; US5637481 ; US5837242; US5837821 ; US5844094; US5864019 and US5869620.[000192] A regulatable CAR (RCAR) is a CAR for which activity can be controlled is desirable to optimize the safety and efficacy of a CAR therapy. There are many ways CAR activities can be regulated. For example, inducible apoptosis using, e.g., a caspase fused to a dimerization domain, can be used as a safety switch in the CAR therapy of the instant invention. In some embodiments, the CAR of the invention is a split CAR. The split CAR approach is described in more detail in publications WO2014 / 055442 and WO2014 / 055657.[000193] Once a CAR described herein is constructed, various assays can be used to evaluate the activity of the molecule, such as but not limited to, the ability to expand T cells following target binding, sustain T cell expansion in the absence of re- stimulation, and anti-cancer activities in appropriate in vitro and animal models. Assays to evaluate the effects of CARs of the present invention are known and available to one skilled in the art and are described herein, including in the examples hereof.[000194] In vitro expansion of CAR-T cells following antigen stimulation can be measured by flow cytometry. Animal models can also be used to measure a CAR’s activity. For example, a xenograft model such as that utilized herein can be utilized. Assessment of cell proliferation and cytokine production has been previously described and is shown and utilized in the examples provided herein.Imaging technologies can be used to evaluate specific trafficking and proliferation of CARs in tumorbearing animal models.[000195] Bispecific T cell Engagers (BiTEs)[000196] A BiTE generally refers to a single polypeptide chain molecule that has two antigen binding sites, one of which binds to an immune effector cell antigen (e.g., CD3) and the second of which binds to an antigen present on the surface of a target cell, e.g., CD70. When both targets are engaged, the BiTE molecule forms a bridge between the cytotoxic T cell and the tumor cell, which enables the T cell to recognize the tumor cell and fight it through an infusion of toxic molecules. The tumor-binding arm of the molecule can be altered to create different BiTE constructs that target different types of cancer. BiTEs are typically produced as recombinant, glycosylated proteins secreted by higher eukaryotic cell lines. Accordingly, in another embodiment of this invention, the protein of the invention is a BiTE. The CD70 binding moiety in the BiTE comprises a mutant CD27 extracellular domain as provided herein.[000197] In some embodiments, the binding protein of the invention is conjugated to another compound. Conjugates could help extend half-life or impart other biological acti vites. Methods for conjugation of the binding protein (mutant CD27) will be apparent to the skilled person and / or described herein. All forms and methods of conjugation (i.e., binding) are contemplated by the present invention, including, for example, direct conjugation between the binding protein (mutant CD27) and another compound / moiety as described herein or indirect binding (e.g., by virtue of a linker between the binding protein and the other compound / moiety). In one embodiment, the conjugate is formed by a chemical conjugation (e.g., by an amine bond or disulphide bond) or by genetic fusion.[000198] In one embodiment, the disclosure provides a fusion protein comprising the binding protein of the invention (mutant CD27) and the other compound. For example, the other compound can be positioned at the N-terminus of the protein, C-terminus of the protein or any combination thereof. In one embodiment, the binding protein is conjugated to the other compound via a linker. For example, the linker can be a peptide linker. In one embodiment, the linker is a flexible linker. A “flexible” linker is an amino acid sequence which does not have a fixed structure (secondary or tertiary structure) in solution. Such a flexible linker is therefore free to adopt a variety of conformations. Flexible linkers suitable for use in the present invention are known in the art. The linker may comprise any amino acid sequence that does not substantially hinder interaction of the binding region with its target. Preferred amino acid residues for flexible linker sequences include, but are not limited to, glycine, alanine, serine, threonine proline, lysine, arginine, glutamine and glutamic acid.[000199] The linker sequences between the binding regions preferably comprise five or more amino acid residues. The flexible linker sequences according to the present invention consist of 5 or more residues, preferably, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or 25 or 30 or moreresidues. In an embodiment of the invention, the flexible linker sequences consist of 5, 7, 10, 13 or 16 or 30 residues.[000200] Exemplary compounds that can be conjugated to a binding protein of the invention may be selected from human serum albumin or functional fragment thereof, an immunoglobulin Fc region or functional fragment thereof, afamin, alpha-fetoprotein, vitamin D binding protein, antibody fragments that bind to albumin and polymers.[000201] In an embodiment, the binding protein is conjugated to a cytotoxic agent. Cytotoxic agents include any agent that is detrimental to the growth, viability or propagation of cells. Examples of suitable cytotoxic agents and chemotherapeutic agents that can be conjugated to binding proteins in accordance with this aspect of the invention include, e.g., l-(2chloroethyl)-l,2-dimethanesulfonyl hydrazide, 1 ,8-dihydroxy-bicyclo[7.3. l]trideca-4,9-diene-2, 6-diyne- 13-one, 1 -dehydrotestosterone, 5- fluorouracil, 6-mercaptopurine, 6-thioguanine, 9-amino camptothecin, actinomycin D, amanitins, aminopterin, anguidine, anthracycline, anthramycin (AMC), auristatins, bleomycin, busulfan, butyric acid, calicheamicins, camptothecin, carminomycins, carmustine, cemadotins, cisplatin, colchicin, combretastatins, cyclophosphamide, cytarabine, cytochalasin B, dactinomycin, daunorubicin, decarbazine, diacetoxypentyldoxorubicin, dibromomannitol, dihydroxy anthracin dione, disorazoles, dolastatin, doxorubicin, duocarmycin, echinomycins, eleutherobins, emetine, epothilones, esperamicin, estramustines, ethidium bromide, etoposide, fluorouracils, geldanamycins, gramicidin D, glucocorticoids, irinotecans, leptomycins, leurosines, lidocaine, lomustine (CCNU), maytansinoids, mechlorethamine, melphalan, mercatopurines, methopterins, methotrexate, mithramycin, mitomycin, mitoxantrone, N8-acetyl spermidine, podophyllotoxins, procaine, propranolol, pteridines, puromycin, pyrrolobenzodiazepines (PDBs), rhizoxins, streptozotocin, tallysomycins, taxol, tenoposide, tetracaine, thioepa chlorambucil, tomaymycins, topotecans, tubulysin, vinblastine, vincristine, vindesine, vinorelbines, and derivatives of any of the foregoing. Other cytotoxic agents known in the art are contemplated within the scope of the present invention, including, e.g., protein toxins such ricin, C. difficile toxin, pseudomonas exotoxin, ricin, diphtheria toxin, botulinum toxin, bryodin, saporin, pokeweed toxins (i.e., phytolaccatoxin and phytolaccigenin).[000202] Potent anti-tumor or anti-cancer immune response requires modulating multiple arms of host immune response and targeting pathways that contributes to tumor cell growth and survival. Combining agents that modulate immune response and arrest tumor growth and progression can generate anticancer immunity and arrest tumor growth to improve clinical outcomes (Vanneman, M (2012) Nature Reviews Cancer (12):237-251 ). Thus, in an aspect of the invention the mutant CD27 polypeptides, mutant CD27 extracellular domain peptides, mutant CD27- based CARs may be administered alone or in combination with other treatments, therapeutics or agents, either simultaneously or sequentially dependent upon the condition to be treated. Immune modulators may be included in a composition with mutant CD27 polypeptides, mutant CD27 extracellular domain peptides,mutant CD27- based CARs or administered with and / or administered at a different time to enhance immune modulation and / or cancer therapy, including immune therapies directed against cancer. An immune modulator may be an adjuvant. Applicable immune modulators include IDO, TDO (Flatten M (2012) Cancer Research 72(21):5435-40), a-galactosyl ceramide and analogs thereof such as threitolceramide (ThrCer) and ThrCer 6, TLR ligands such as poly I:C (TLR3), MPL (TLR4), imiquimod (TLR7), R848 (TLR8) or CpG (TLR9), iCOS, CTLA-4, PD1, PD1 ligand, 0X40 and 0X40 ligand, Lag3, GITR, GITR ligand interleukins, tumor necrosis factor (TNF) or other growth factors, colony stimulating factors, T cell modulators including modulators of CD8+T cells, cytokines or hormones which stimulate the immune response or reduction or elimination of cancer cells or tumors. Additional immunmodulators are small molecules, antagonist antibodies or agonist antibodies targeting the applicable immune modulators including IDO, TDO, Toll like receptor family or iCOS, CTLA-4, PD1, PD1 ligand, 0X40 and 0X40 ligand, interleukins, tumor necrosis factor (TNF) or other growth factors, colony stimulating factors, T cell modulators including modulators of CD8+T cells, cytokines which stimulate the immune response or reduction or elimination of cancer cells or tumors.[000203] Additional immune modulators, including TLR ligands such as poly I:C (TLR3), MPL (TLR4), imiquimod (TLR7), R848 (TLR8) or CpG (TLR9) can be used in combination with mutant CD27 polypeptides, mutant CD27 extracellular domain peptides, mutant CD27- based CARs to produce an enhanced immune stimulation and resulting protection from conditions in which it is desirable for the immune system to respond effectively such as infectious disease or cancer.[000204] Labeled, such as radiolabelled, mutant CD27 polypeptides or mutant CD27 extracellular domain peptides are useful in in vitro diagnostics techniques and in in vivo radioimaging techniques and in radioimmunotherapy. In the instance of in vivo imaging, the mutant CD27 polypeptides, mutant CD27 extracellular domain peptides of the present invention may be conjugated to an imaging agent rather than a radioisotope(s), including but not limited to a magnetic resonance image enhancing agent, wherein for instance a peptide / polypeptide molecule is loaded with a large number of paramagnetic ions through chelating groups. Examples of chelating groups include EDTA, porphyrins, polyamines crown ethers and polyoximes. Examples of paramagnetic ions include gadolinium, iron, manganese, rhenium, europium, lanthanium, holmium and ferbium. In a further aspect of the invention, radiolabelled mutant CD27 polypeptides, mutant CD27 extracellular domain peptides, particularly radioimmunoconjugates, are useful in radioimmunotherapy, particularly as radiolabelled antibodies for cancer therapy. In a still further aspect, the radiolabelled mutant CD27 polypeptides, mutant CD27 extracellular domain peptides are useful in radioimmuno-guided surgery techniques, wherein they can identify and indicate the presence and / or location of cancer cells, precancerous cells, tumor cells, and hyperproliferative ceils, prior to, during or following surgery to remove such cells.[000205] Immunoconjugates or fusion proteins of the present invention, wherein the mutant CD27 polypeptides, mutant CD27 extracellular domain peptides of the present invention are conjugated oratached to other molecules or agents further include, but are not limited to, CD27 peptides conjugated to a chemical ablation agent, toxin, immunomodulator, cytokine, cytotoxic agent, chemotherapeutic agent or drug.[000206] Adoptive cell transfer (ACT) is emerging as a new pillar in cancer therapy, based on collecting and using patients' own immune cells to treat their cancer. There are several types of ACT, including TILs, TCRs, and CARs (Haanen et al. (2018) J Immunother Cancer 474:449-461). One approach uses immune cells that have penetrated the environment in and around the tumor, known as tumor-infiltrating lymphocytes (TILs). Another approach to ACT involves engineering patients' T cells to express a specific T-cell receptor (TCR) to recognize tumor cell antigens (Mackall et al (2019) Nature Medicine 25:1341-1355).[000207] T-cells modified to express chimeric antigen receptor (CAR) and administered alone have been subject to supression within the hostile tumor microenvironment. As a way of a non-limiting example, further modification of these cells to express secretable scFvs (eg, PD- 1 , PDL- 1 , or CTL A-4) (known as Armored CARs) have improved anti-tumor function due to their ability to modulate the tumor microenvironment and resist suppressive factors (for example as described in US Patent 10,124,023 and Brentjens et al (2018) Nat Biotechnol 36(9):847-856). In another embodiment, the mutant CD27 polypeptides, mutant CD27 extracellular domain peptides of the invention could be used in Adoptive Cell Therapy (ACT) where the mutant CD27 would be genetically introduced into T-cells, preferably but not limited to, tumor infiltrating lymphocytes (TILs), isolated from cancer patients, and then such T-cells would be expanded and delivered back into the patients whereby the T-cells would target the tumor and express and secrete the mutant CD27, in the local tumor microenvironment to counter the immunosuppressive environment there.[000208] The mutant CD27 polypeptides, mutant CD27 extracellular domain peptides of the invention could also be used in Adoptive Cell Therapy (ACT) where the mutant CD27 polypeptides, mutant CD27 extracellular domain peptides would be genetically introduced into T-cells isolated from cancer patients, and then such T-cells would be expanded and delivered back into a patient whereby the T-cells would target the tumor and express the mutant CD27 polypeptides, mutant CD27 extracellular domain peptides in the local tumor microenvironment to counter the immonusuppressive environment there. Preferably the T-cells used would be tumor infiltrating lymphocytes (TILs).[000209] Mutant CD27 polypeptides, mutant CD27 extracellular domain peptides, mutant CD27- based CARs, fusion or conjugated proteins comprising mutant CD27 polypeptides, mutant CD27 extracellular domain peptides of the present invention may be administered to a patient in need of treatment via any suitable route, including by injection, including intreperitoneally, intramuscularly, subcutaneous, intravenous, into the bloodstream or CSF, or directly into the site of the tumor or by intratumoral administration or intratumoral injection. The precise dose will depend upon a number of factors, including whether for diagnosis or for treatment, the size and location of the tumor, the precisenature of the mutant CD27-based peptide, conjugate or fusion, and the nature of the detectable or functional label attached thereto as applicable. Treatments may be repeated at daily, twice-weekly, weekly or monthly intervals, at the discretion of the physician.[000210] Mutant CD27 polypeptides, mutant CD27 extracellular domain peptides, mutant CD27- based conjugates or fusion proteins of the present invention will usually be administered in the form of a pharmaceutical composition, which may comprise at least one component in addition to the mutant CD27 polypeptides, mutant CD27 extracellular domain peptides, mutant CD27-based conjugates or fusion proteins. Thus pharmaceutical compositions according to the present invention, and for use in accordance with the present invention, may comprise, in addition to active ingredient, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. intravenous, or by deposition at a tumor site. [000211] A composition of the present invention may be administered alone or in combination with other treatments, therapeutics or agents, either simultaneously or sequentially dependent upon the condition to be treated. In addition, the present invention contemplates and includes compositions comprising the mutant CD27 polypeptides, mutant CD27 extracellular domain peptides, mutant CD27-based conjugates or fusion proteins herein described and other agents or therapeutics such as anti-cancer agents or therapeutics, anti-mitotic agents, apoptotic agents or antibodies, or immune modulators, or small molecule inhibitors to immune modulators. More generally these anti-cancer agents may be tyrosine kinase inhibitors or phosphorylation cascade inhibitors, post-translational modulators, cell growth or division inhibitors (e.g. anti-mitotics), inhibitors or signal transduction inhibitors. Other treatments or therapeutics may include the administration of suitable doses of pain relief drugs such as non-steroidal anti-inflammatory drugs (e.g. aspirin, paracetamol, ibuprofen or ketoprofen) or opiates such as morphine, or anti-emetics. In addition, the composition may be administered with immune modulators, such as a-galactosyl ceramide, interleukins, tumor necrosis factor (TNF) or other growth factors, colony stimulating factors, cytokines or hormones which stimulate the immune response and reduction or elimination of cancer cells or tumors. The composition may be administered with an immune modulator such as an adjuvant. The composition may also be administered with, or may include combinations along with anti-cancer or immune cell antibodies such as immunomodulatory antibodies or anti-tumor antigen antibodies. In an aspect, the composition is administered in combination with an anti-tumor antigen antibody.[000212] The present invention also includes Mutant CD27 polypeptides, mutant CD27 extracellular domain peptides which are covalently attached to or otherwise associated with other molecules or agents. These other molecules or agents include, but are not limited to, molecules (including antibodies or antibody fragments) with distinct recognition characteristics, toxins, ligands,and chemotherapeutic agents.[000213] Mutant CD27 polypeptides, mutant CD27 extracellular domain peptides, mutant CD27- based conjugates or fusion proteins of the present invention can be administered at suitable doses. In instances wherein cells expressing mutant CD27 polypeptides, mutant CD27 extracellular domain peptides, or mutant CD27-based conjugates or fusion proteins are administered the cells can be administered at doses ranging from 1 x 104to IxlO8In some aspects, cells, such as mutant CD27 CAR-T cells, are administered at between IxlO5to IxlO7cells. In some aspects, cells, such as mutant CD27 CAR-T cells, are administered at between IxlO6to IxlO7cells. In aspects, cell doses may be administered at IxlO6, 2xl06, 3x106, 4xl06, 5xl06, 6xl06, 7xl06, 8xl06or 9xl06cells. In some aspects, cells, such as mutant CD27 CAR-T cells, are administered at between 2xl06to 8xl06cells. In some aspects, cells, such as mutant CD27 CAR-T cells, are administered at between 2xl06to 6xl06cells. In some aspects, cells, such as mutant CD27 CAR-T cells, are administered at between 3xl06to 6xl06cells. In some aspects, cells, such as mutant CD27 CAR-T cells, are administered at between 4xl06to 6xl06cells. In some aspects, cells, such as mutant CD27 CAR-T cells, are administered at about 4xl06to 6xl06cells. In some aspects, cells, such as mutant CD27 CAR-T cells, are administered at about 5xl06cells.[000214] Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may comprise a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. For intravenous, injection, or injection at the site of affliction, the active ingredient may be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and / or other additives may be included, as required.[000215] A composition may be administered alone or in combination with other treatments, therapeutics or agents, either simultaneously or sequentially dependent upon the condition to be treated. In addition, the present invention contemplates and includes compositions comprising the binding protein of the invention and other agents or therapeutics such as anti-cancer agents or therapeutics, hormones, anti-mitotic agents, anti-apoptotic agents, antibodies, or immune modulators. More generally these anti-cancer agents may be but are not limited to tyrosine kinase inhibitors or phosphorylation cascade inhibitors, post-translational modulators, cell growth or division inhibitors (e.g. anti-mitotics), or signal transduction inhibitors. Other treatments or therapeutics may include the administration of suitable doses of pain relief drugs such as non-steroidal anti-inflammatory drugs (e.g. aspirin, paracetamol, ibuprofen or ketoprofen) or opiates such as morphine, or anti-emetics. Thecomposition can be administered in combination (either sequentially (z.e. before or after) or simultaneously) with tyrosine kinase inhibitors (including, but not limited to AG1478 and ZD1839, STI571, OSI-774, SU-6668), doxorubicin, temozolomide, cisplatin, carboplatin, nitrosoureas, procarbazine, vincristine, hydroxyurea, 5-fluoruracil, cytosine arabinoside, cyclophosphamide, epipodophyl lotoxin, carmustine, lomustine, and / or other chemotherapeutic agents. Thus, these agents may be specific anti-cancer agents, or immune cell response modulators or may be more general anticancer and anti-neoplastic agents such as doxorubicin, cisplatin, temozolomide, nitrosoureas, procarbazine, vincristine, hydroxyurea, 5-fluoruracil, cytosine arabinoside, cyclophosphamide, epipodophyllotoxin, carmustine, or lomustine. In addition, the composition may be administered with hormones such as dexamethasone, immune modulators, such as interleukins, tumor necrosis factor (TNF) or other growth factors, colony stimulating factors, cytokines, agonist or antagonist antibodies to regulators of immune response which stimulate, enhance, or derepress the immune response and reduction or elimination of cancer cells or tumors. The composition may also be administered with, or may include combinations along with anti-tumor antigen antibodies.[000216] The presence of CD27 ligand CD70 in cells or CD70 responsive cells or CD27 / CD70 responsive genes or proteins can be ascertained by the usual in vitro or in vivo immunological procedures applicable to such determinations. A number of useful procedures are known. The procedures and their application are all familiar to those skilled in the art and accordingly may be utilized within the scope of the present invention.[000217] The present invention further provides an isolated nucleic acid encoding a Mutant CD27 polypeptides, mutant CD27 extracellular domain peptides, mutant CD27-based conjugates or fusion proteins, mutant CD27-based CAR of the present invention. Nucleic acid includes DNA and RNA. In a preferred aspect, the present invention provides a nucleic acid which codes for a polypeptide of the invention as defined above, including a polypeptide as set out in SEQ ID NOs: 13-47.[000218] The present invention also provides constructs in the form of plasmids, vectors, transcription or expression cassettes which comprise at least one polynucleotide as above. The present invention also provides a recombinant host cell which comprises one or more constructs as above. Expression may conveniently be achieved by culturing under appropriate conditions recombinant host cells containing the nucleic acid. Following production by expression a binding protein may be isolated and / or purified using any suitable technique, then used as appropriate.[000219] Systems for cloning and expression of a polypeptide in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, yeast and baculovirus systems. Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Vectors may be plasmids, viral e.g. 'phage, or phagemid, as appropriate.[000220] Thus, a further aspect of the present invention provides a host cell containing nucleic acid as disclosed herein. A still further aspect provides a method comprising introducing such nucleic acid into a host cell. The introduction may employ any available technique. The introduction may be followed by causing or allowing expression from the nucleic acid, e.g. by culturing host cells under conditions for expression of the gene. The present invention also provides a method which comprises using a construct as stated above in an expression system in order to express a binding member polypeptide. Another feature of this invention is the expression of the DNA sequences disclosed herein. As is well known in the art, DNA sequences may be expressed by operatively linking them to an expression control sequence in an appropriate expression vector and employing that expression vector to transform an appropriate unicellular host. A wide variety of host / expression vector combinations may be employed in expressing the DNA sequences of this invention.[000221] It will be understood that not all vectors, expression control sequences and hosts will function equally well to express the DNA sequences of this invention. Neither will all hosts function equally well with the same expression system. However, one skilled in the art will be able to select the proper vectors, expression control sequences, and hosts without undue experimentation to accomplish the desired expression without departing from the scope of this invention.[000222] The invention may be better understood by reference to the following non-limiting Examples, which are provided as exemplary of the invention. The following examples are presented in order to more fully illustrate the preferred embodiments of the invention and should in no way be construed, however, as limiting the broad scope of the invention.EXAMPLE 1[000223] Chimeric antigen receptor (CAR) T cell therapies have broadly changed management and treatment outcomes for patients with aggressive B cell malignancies and multiple myeloma. However, only two target antigens are currently explored in commercially approved products, CD 19 and B cell maturation antigen (BCMA), limiting the spectrum of application of CAR T cell therapies to B and plasma cell malignancies, or to autoimmune diseases. Extending the CAR-T paradigm to aggressive myeloid malignancies such as acute myeloid leukemia (AML) for example or to solid tumors has been hampered for several reasons, including; 1) the paucity of ideal target antigens that are selectively expressed on tumor cells but not on essential healthy tissues; 2) the complexity and clonal heterogeneity of the malignancies; 3) the poorly characterized impact of the tumor immune microenvironment on adoptively transferred CAR T cells; and 4) the manufacturing challenge when producing CAR T cells from autologous lymphocytes in an aggressive leukaemia with circulating blasts.[000224] An interesting and emerging target that is widely and stably expressed in a variety of cancers is CD70, a member of the tumor necrosis factor (TNF) superfamily (1). CD70 is a co-stimulatory molecule with limited expression on normal tissues that is restricted to a subset of hematopoietic cells, namely activated T and B lymphocytes and a subset of dendritic cells, but absent on normal hematopoietic stem cells and maturating myeloid cells (2, 3). Therefore, little to no on-target off-tumor toxicities on healthy haematopoiesis and other normal tissues is to be expected by targeting CD70. Indeed, the monoclonal antibody cusatuzumab targeting CD70 has demonstrated safety and some efficacy in clinical trials for AML (4). In patients with relapsed / refractory clear cell renal cell carcinoma (ccRCC), a recent clinical trial reported safety and some efficacy with an allogeneic off-the- shelf CAR T cell product targeting CD70 and no dose limiting toxicities (5). These data suggest that CD70 can be safely targeted in humans with monoclonal antibodies or CAR T cells.[000225] CARs targeting CD70 have already been developed for over a decade, either based on the natural ligand CD27 or single chain variable fragments (scFv) derived from monoclonal antibodies (6-13). Recent data suggested that CD27-based CARs were more potent than scFv-based CARs targeting CD70 (12). However, CD27-based CARs may have issues with natural cleavage of CD27 incorporated in the CAR construct and thus limiting CAR activity (12, 13). Engineering out the cleavage region of CD27 was proposed as a solution and resulted in more stable CAR cell surface expression and CAR T cell function in preclinical models (13). We previously learnt from CD 19 CAR T cell optimizations that the affinity of the scFv in the CAR construct can have a decisive impact on both CAR T cell potency and the associated toxicity profile in patients and that there is no clear correlation between binding affinity and CAR T cell potency (14). However, such optimizations have not been performed in natural ligand based CARs and the impact of modulating the CD27-CD70 binding interface on CAR T cell potency has not been investigated.[000226] Here we present a novel set of CD70 targeted CARs with an optimized assembly of components consisting of a designed CD27-CD70 binding interface, a CD8a hinge, CD27 transmembrane and endo-domains, and the CD3^ chain. Candidate sequences for each component were screened and selected against previously benchmarked CARs with the goal to enhance potency. The binding interface optimization was achieved with computational design of the CD27 sequence and single, double or triple point mutants were screened for enhanced anti-tumor activity using in vitro cocultures and in vivo in mouse xenograft models. In particular, we were able to engineer a wide range of binding properties and identify key enhancer mutations using an unprecedented combination of Artificial Intelligence and physics-based computational techniques.[000227] Materials and Methods[000228] Cell lines and maintenance. HEK293T and MOLM-I3 cell lines were purchased from the American Type Culture Collection (ATCC). HEK293T cells were maintained in DMEM with GlutaMAX (ThermoFisher Scientific, #31966) supplemented with 10% FBS (ThermoFisher Scientific, #10270) and 1% penicillin-streptomycin (ThermoFisher Scientific, #15140122). MOLM-13 cells were cultured in RPMI-1640 with GlutaMAX (ThermoFisher Scientific, #61870) supplemented with 20%FBS and 1% penicillin-streptomycin. The PG-13 retroviral producer cell line for the generation of retroviral particles encoding green fluorescent protein and firefly luciferase (GFP-ffLuc) was kindly provided by Dr. Stephen Gottschalk, Baylor College of Medicine. MOLM-13 cells were retrovirally transduced to express GFP-ffLuc and sorted to >98% purity (MOLM-13. GFP-ffLuc). Patient-derived acute myeloid leukemia (AML) xenograft (PDX) cells (DFAM68555) were purchased from the Dana- Faber Cancer Institute.[000229] Peripheral blood mononuclear cells from healthy humaiiJonors, Buffy coats from anonymized healthy volunteer blood donors were purchased from the Center of Interregional Blood Transfusion SRK Bern, Switzerland. Peripheral blood mononuclear cells (PBMCs) were isolated via density gradient centrifugation using Lymphoprep (Stemcell Technologies, 07851) and cryopreserved in freezing media comprised of 10% DMSO (Sigma Aldrich, #D8418), 40% FBS, and 50% RPMI- 1640. The cryovials were stored in liquid nitrogen until use.[000230]The sequence of human CD27, the natural ligand of CD70, was obtained from Uniprot (Uniprot ID: P26842). The endogenous signal peptide was predicted with Signal IP-4.0 webtool(cbs.dtu.dk / services / SignalP) and replaced with an immunoglobulin-derived signal peptide sequence (MEFGLSWLFLVAILKGVQC) to ensure efficient shuttling of the CAR transgene to the cell surface. The human CD27 amino acid sequence (amino acids 22-260) was fused to the immunoglobulin-derived signal peptide sequence, translated into nucleotide sequence, and then codon optimized and synthesized by GeneArt (ThermoFisher Scientific). Retroviral vectors encoding for non-signaling control CAR CD27-A (A), first generation CD27XJ (Q, second generation CARs CD27-28£ (28Q, CD27-BB^ (BBQ and CD27-27^ (27Q were constructed using In-Fusion HD Cloning Kit (Takara, #638933) following manufacturer’s instructions. Primers for In-Fusion cloning were designed using S Biotech LLC). Amplicons of interest were amplified by high-fidelity PCR (Clo Premix, #639298). CD27 was amplified from amino acids 22 to 212 to generate A, constructs, and from amino acids 22 to 260 to generate the full-length 27^ constructcostimulatory endo-domains and CD3£ chain were amplified from previously generated vectors (15). The p-SFG retroviral backbone containing an IRES-ACD271 selectable marker gene was linearized using Pmel and Xhol restriction enzymes. PCR fragments were purified from an agarose gel using the QIAquick Gel Extraction Kit (Qiagen, #28706X4). PCR products of interest were assembled in the linearized vector using the In-Fusion enzyme mix to generate the constructs of interest and transformed into Stellar competent cells (Takara, #636763). DNA plasmids were purified with QIAprep spin Miniprep Kit (Promega, #A1223) and transgene sequences were verified through Sanger sequencing(Microsynth).[000231] Production of retrovi ral supernatant and T celljrajisdyctjon, HEK293T cells were cotransfected with a RDF plasmid encoding the RD1 14 envelope, a PegPam plasmid encoding gag-poland the SFG retroviral plasmid encoding gene of interest, using GeneJuice transfection reagent (Merck Milipore, #70967-3) following manufacturer’s instructions. Retroviral supernatants were harvested after 48 hours and 72 hours of culture, filtered with a 0.45 pM filter (Sarsdedt, #83.1826), snap-frozen and then stored at -80 °C until use. Cryopreserved PBMCs were thawed and cultured in GlutaMAX- containing RPMI-1640 media supplemented with 10% FBS and 1% penicillin-streptomycin (herein known as R10 medium), in the presence of 10 ng / mL each of recombinant human IL-7 (Miltenyi Biotec, #130-095-765) and IL- 15 (Miltenyi Biotec, #130-095-362). Cells were activated for 72 hours on nontissue culture treated 24-well plates coated with anti-CD3 (Biolegend, #317347) and anti-CD28 (Biolegend, #302934) monoclonal antibodies at 1 ug / mL each. On the day of transduction, ImL of retroviral supernatant was added to each well of a 24-well plate pre-coated with 7 ug / mL of RetroNectin (Takara Bio, #T100B) and centrifuged at 2000 xg for 1 hour at room temperature. Retroviral supernatants were removed and 0.3x106activated T cells were added per well in 2mL of cytokine containing R10 medium. The plate was centrifuged at 1000 xg for 10 minutes and then incubated at 37 °C with 5% CO2. Three days later, T cells were harvested and replated in fresh cytokine supplemented R10 media at a density of IxlO6cells per ml for expansion.[000232] Geng-MioLLoL lemjw<Xl220. hinge_aiiOjfflnit> JJiodutatei CAR xarianls_. with optimized binding interface. Using the 27£ CAR as a template, the endogenous CD27 hinge and transmembrane domains were replaced with corresponding domains derived from human CD8a (Uniprot ID: P01732-1). The CD8a hinge and transmembrane (CD8aH&TM) containing fragments were paired with either 4 IBB or CD27 endo-domains and CD3^ chain to build CD8aH&TM-41BBt, and CD8aH&TM-CD27^, respectively. For the CD8aH-CD27ij variant, only the CD27 hinge domain but not the TM domain was replaced. All the hinge-modified variants, together with the parental 27£ CAR construct, were cloned into a self-inactivating (SIN) lenti viral backbone containing a EFla core promoter (EFl aS) previously shown to drive efficient transcription of transgenes (16). A truncated human EGFR (huEGFRt) gene was co-expressed in the vector as a selection marker through a furin linker and T2A sequence. To generate affinity variants with optimized binding interface, PCR mutagenesis was performed using CD8aH-CD27^ as the template. Briefly, primers were designed and used to introduce mutations of interest into the CD27 extracellular domain. Overlapping PCR was conducted to join the new binding domains with other parts of the CAR transgene cassette. All PCR amplification steps were performed using ROD One™ PCR master mix (Toyobo, #M0494L). The DNA sequences of all mutein plasmids were validated via Sanger sequencing (Microsynth).[000233] Prodi Il of lentiviral partides. Along with the EFla transfer plasmids containing our CAR transgenes, HEK293T were co-transfected with helper plasmids pCMV-Gag pol, pRSV-Rev and pCMV-VSV-G using TurboFect (ThermoFisher Scientific, #R0531). Supernatants containing viral particles were harvested 48 hours post-transfection and filtered through a 0.45 pM filter. To concentrate the lentiviral particles, filtered supernatants were ultracentrifuged at 24,000 xg for 2 hours at 4 °C(Beckman Coulter, Avanti JXN-30) and the pellets were resuspended at 1 / 10ththe starting volume. The virus particles were then aliquoted, snap- frozen and kept at -80 °C for long-term storage.[000234] Small-scale production of GMP-like CAR-T cells. On Day 0, CD4+and CD8+T cells were individually isolated from frozen PBMCs using EasySep™ negative selection kits (Stemcell Technologies, #17952 and #17953). The two T cell populations were plated at a ratio of 1 : 1 to achieve a final density of 5xl05cells in each well of a 48-well cell culture plate. T cells were activated using MACS® GMP T Cell TransAct™ (Miltenyi Biotec, #170-076-156) and maintained in PRIME-XV T- cell CDM (Irvine Scientific, #91154) supplemented with 10 ng / mL of recombinant human IL-7, 10 ng / mL of IL-15, and 30 ng / mL of IL-21 (Miltenyi Biotec, #130-095-784). At 19 hours post-activation, T cells were transduced with lenti viral particles containing CAR constructs at a multiplicity of infection (MOI) of 1.5. LentiBOOST (Sirion Biotech, #SB-P-LV-101) was added at a dilution of 1 :600 to enhance the transduction efficiency. The proportion of CAR-T cells (% EGFR+) was quantified via flow cytometry days 4 or 5, and again on the day of harvest (days 10 or 1 1).[000235] Vector copy number analysis. Cell pellets of CAR-T cells were harvested on days 10 or 11 and genomic DNA was isolated using DNeasy Blood & Tissue Kit (Qiagen, #69504) following manufacturer’s directions. A region of the CAR transgene was amplified with a set of primers that bind to the huEGFRt marker gene (5’ -AGGCTCCCTGCTGACTTG- 3’ (SEQ ID NO:51), 5’ - CAGCAGAAAGGCAGGATG- 3’ (SEQ ID NO:52)). A probe (5’ AGATGTGGAAGAGAACCCCGGACCT- 3’ (SEQ ID NO:53)) that specifically targets the T2A sequence was used to quantify the CAR transgene copy number, on a QIAcuity Digital PCR System (Qiagen). The number of copies of housekeeping gene RPP30 was assessed in parallel for the normalization of transgene copy number per CAR-T cell using the following equation:CAR-T (copies / pL) x 200;- %EGFR+RPP30 (copies / pL)[000236] Flow cytometry. Cells were stained with antibody cocktails for 30 minutes in the dark at 4 °C and washed with lx PBS buffer (ThermoFisher Scientific, #10010023) containing 2.5% FBS and 2mM EDTA (ThermoFisher Scientific, #15575). Cell pellets were resuspended in buffer and DAPI (Biolegend, #422801) was added at a dilution of 1:100 before acquisition on a LSR II flow cytometer (BD Biosciences). For quantification of surface CD70 antigen density, Quantibrite™ PE fluorescence quantification kit (BD Biosciences, #340495) was used following manufacturer’s instructions. Data acquisition was performed using FACSDiva software version 9.0 or higher while data analysis was conducted with FlowJo software version 10.9. or higher (Tree Star Inc.). The list of antibodies used, conjugated with various fluorochromes, are listed (Table 1).[000237] Sei ial co-culture rechallengc assay. MOLM-13.GFP-ffLuc cells were co-cultured with IxlO5CAR-T cells in 6 replicates at an effector to target (E:T) ratio of 1, unless specified otherwise in the figures. The cells were plated in 48-well cell culture plates, using cytokine-free RIO medium. The number of DAPI negative live tumor and T cells in each well of the co-culture were quantified three to four days later using CountBright absolute counting beads (ThermoFisher Scientific, #C36950) on a flow cytometer. T cells were stained with anti-CD3, -CD4 and -CD8 antibodies while tumor cells were identified through its GFP expression. A kill is considered when >80% of the tumor cells were eliminated at the point of analysis. Fresh MOLM-13.GFP-ffLuc cells were then added to the remaining replicates to rechallenge the CAR-T cells.[000238] Cytokine production assay Supernatants were harvested 21 hours or 3 days post- coculture with MOLM-13.GFP-ffLuc cells and stored at -80 °C. The levels of IFNy and IL-2 present in the co-culture supernatants were quantified using a U-PLEX multiplex assay (Meso Scale Diagnostics, #K15227N) according to manufacturer’s instructions. The plate was measured using MESO QuickPlex SQ 120MM instrument (Meso Scale Diagnostics) and the raw data obtained was analyzed on the Discovery Workbench 4.0 software.[000239] (. ell uro I i feral fen a-^ax . Hinge-modified CAR-T cells were harvested and washed once with R10 medium to remove any traces of exogenous cytokines. Cell pellets were resuspended in fresh cytokine-free R10 medium, counted and plated at a starting density of 6xl05cells / mL into each well of a 24-well cell culture plate. A total of 2mL was seeded into each well. At regular intervals (days 4 and 8), wells were resuspended, and aliquots were taken to perform a live cell count using 0.4% Trypan Blue solution (Thermofisher Scientific, #15250061), on a LUNA- FX7™ Automated Cell Counter (Logos Biosystems). As a positive control, recombinant human IL-2 (Miltenyi Biotec, #130-097-748) was supplemented at 50 lU / mL to a separate well containing 27^ CAR-T cells.[000240] Optimization of the CD2"-CD'ftl binding interface bx computational design.[000241] Design principles: All residues in CD27 within 5 A of the CD70 interface based on the crystal structure of the complex (PDB: 7KX0) (17) were defined as designable. Mutations at these sites were then selected to enhance binding strength to CD70 through a combination of physics and deep learning-based algorithms, namely via the Multi-State Design (MSD) (18) protocol in Rosetta ( 19), and ProteinMPNN (20).[000242] Rosetta Multi-Stale Design. We considered two states, a bound and an unbound, where the former relates to the entire complex, and the latter treats the CD27 and CD70 trimer as individual monomers. Our objective function for design, E, v / as derived to decrease the energy of the bound state versus the unbound without destabilizing either state within a margin of +10 Roseta Energy Units (REU) to avoid selecting mutations leading to protein unfolding. Specifically, to avoid a destabilizing runoff effect, we include a barrier via the third term in the following:[000243] Where Mi is the energy of the bound state during any design stage, M2 the unbound energy, M,gand M^ are the corresponding energies of the WT. a is a constant that modulates the strength of the barrier (benchmarked and set to 10). The form of the barrier ensures convexity in the potential energy surface. For the genetic algorithm in MSD, we set the population size to 100, the number of generations to 500, and the fraction by recombination to 0.02. Based on the 8000000 generated sequences from 200 independent trajectories, we extracted all mutations associated with a decrease in the overall energy of the bound state versus the unbound when compared with the WT.[000244] ProteinMPNN: We provided ProteinMPNN with the scaffold of the CD27-70 complex and generated 10000 sequences in total, at which point the sequence generation reached convergence, i.e. beyond 10000 no new sequences were produced. All mutations that occurred >1% of the time in any of the 10000 sequences were extracted for the next stage.[000245] Assessment of desjgns. The combined set of designs from MSD and ProteinMPNN resulted in 131 individual single-point mutations. Each mutation was assessed by first mutating the WT CD27 using Rosetta, then redocked 10000 times onto CD70 with RosettaDock (21) to facilitate any backbone shifts needed to optimise binding. We also examined the top 10 designs from ProteinMPNN with this approach based on their prevalence, each featuring ~15 single-point mutations. After clustering the output structural models, we assessed the energies of the cluster centers for each design in terms of total complex energy and interfacial energy (22). Final designs were selected for experimental testing if they satisfied the following conditions: 1) the change in the normalized interfacial energy is either negative vs. the WT or only mildly destabilizing (within +1.5 REU). The interfacial energies are normalized to the buried solvent accessible surface area at the interface. 2) there are no known pathogenic consequences associated with the mutation (23). 3) the proposed mutation is biophysically relevant, e.g. enhanced Hydrogen bonding, improved van der Waals packing, and salt bridge formation. The top 10 designs from ProteinMPNN failed in this first category.[000246] Animal experimentation and mouse models. All animal studies were conducted in accordance with a protocol approved by the Veterinary Authority of the Swiss Canton of Vaud (authorization VD3390 to Caroline Arber). NOD-scid IL2Rgammanu” (NSG) mice (The Jackson Laboratory strain #005557) orNSG-SGM3 mice (The Jackson Laboratory, strain #013062), expressing human IL3, CSF2 and KITLG, were purchased from The Jackson Laboratory and housed and bred at the animal facility of the Faculty of Biology and Medicine of the University of Lausanne (UNIL) and the Centre Hospitaller Universitaire Vaudois (CHUV). Animals were 6-8 weeks old at the start of the experiments and were infused with leukemia cells intravenously (tail-vein) as illustrated for each individual experiment. For the MOLM-13.GFP-ffLuc AML xenograft model, 5xl03leukemia cells were engrafted and CAR-T cells were injected intravenously (tail-vein) 7-8 days later. Leukemicprogression was monitored once or twice weekly via bioluminescence imaging (BLI) using the IVIS Lumina II In Vivo Imaging System (IVIS, Revvity) upon administration of IVISbrite D-luciferin substrate solution (Perkin Elmer, #122799). All mice were imaged one day prior to CAR-T cell treatment and randomized based on the intensity of bioluminescence signal.[000247] For the adult AML patient-derived xenograft (PDX) model, 2xl06DFAM68555 cells were infused intravenously (tail-vein), followed by various doses of CD8aH&TM-CD27^ CAR-T cells a week later. Peripheral blood was collected through weekly bleeding via the tail-vein and analyzed for the presence of leukemia and CAR-T cells on a flow cytometer. The antibodies used for these analyses are listed in Table 1. All mice in the PDX model were randomized based on body weight.Table 1. List of commercial antibodies used for flow cytometric analyses.[000248] Statistical analysis. All data were plotted and analyzed using GraphPad Prism software version 9 or higher (Dotmatics). Statistical tests used for each dataset are described in the respective figures. Briefly, for continuous variables, comparisons were made by t-test. The area under the curve (AUC) comparisons were analyzed with unpaired t-test with Welch’s correction when appropriate. Probability of killing in serial co-culture rechallenge assays and survival analyses of animal studies were analyzed by Kaplan Meier method with the log-rank (Mantel-Cox) test to assess significance.[000249] RESULTS[000250] Generation of CD27 ligand-based CAR-T cells to target CD70 with retroviral vector.[000251] CD27 can be leveraged to generate ligand-based CARs directed against CD70 overexpressed on malignant cells (Figure 1A) (6-9, 13). For this purpose, we generated a panel of retroviral vectors encoding different CAR constructs composed of the extracellular and transmembranedomains derived from CD27 coupled with different co-stimulatory endo-domains and CD3C, (Figure IB). The constructs included a non-signalling control CD27-A (A), a first-generation CAR CD27-£ (Q, second-generation CARs with either a CD28 (CD27-28Q 28Q or 4 IBB (CD27-BBQ BBQ costimulatory domain, and a full-length construct containing all components of the endogenous CD27 protein (CD27-27^, 27Q (Figure 1C). In all constructs, the endogenous CD27 signal peptide was replaced with a signal peptide sequence derived from an immunoglobulin sequence. In addition, a truncated CD271 sequence was co-expressed after an IRES sequence to serve as a selectable marker. Transduction of activated T cells derived from healthy donor peripheral blood mononuclear cells (PBMCs) was successful, with detectable CD271 expression across all constructs (Figures ID and IE), suggesting that all five CAR variants were efficiently expressed on the surface of human CD4+ and CD8+ T cells.[000252] In vitro anti-leukemic function of CD70 CAR-T cells.[000253] To investigate the anti-leukemic function of the CARs described in Figure 1, CD70 CAR-T cells were co-cultured with CD70 positive MOLM-13.GFP-ffLuc cells in a sequential coculture rechallenge assay (Figure 2A), performing a total of six rechallenges, with phenotyping and quantification of remaining leukemic cells (CD33+) and T cells (CD3+) in culture three to four days after each rechallenge (Figure 2B, representative FACS plots). A total of six independent donors were analysed in this assay and the probability of tumor killing was analysed by Kaplan-Meier analysis and log-rank Mantel Cox test (Figure 2C). As expected, control A CAR-T cells did not kill MOLM-13 cells. 27 Q and BB^ CAR-T cells most efficiently controlled tumor growth upon repetitive rechallenge and performed significantly beter than Q and 28^ CAR-T cells. The enhanced killing capacity of 27^ and BB^ CAR-T cells was also accompanied by significantly better CAR-T cell expansion during sequential challenge (Figure 2D).[000254] In vivo anti-leukemic function of CD70 CAR-T cells.[000255] We next evaluated the in vivo anti-leukemic function and persistence of CAR-T cells in NSG-SGM3 mice engrafted with MOLM-13.GFP-ffLuc cells (Figure 3 A). Mice that received control A CAR-T cells or no treatment rapidly progressed and reached the predefined humane endpoint criteria within the first 4 weeks. Mice treated with 5xl06Q 28Q BB^ or 27 CAR-T cells all responded to the treatment and their survival was significantly prolonged compared to controls (Figures 3B, 3C, 3D). With 92% overall survival, mice infused with 27^ CAR-T cells had the best survival of all groups. At the end of the experiment, peripheral blood (PB), spleen (SP) and bone marrow (BM) were analysed for the presence of human CD3+T cells in surviving mice. Survivors infused with 27Q or Q CAR-T cells had the highest percentage of long-term CAR-T cell persistence compared to the other groups (Figure 3E). In addition, 27^ CAR-T cells infused at a suboptimal dosage of 0.5x106cells per mouse provided significantly better tumor control than BB£ CAR-T cells (Figure 3F). Based on these findings, the 27CJ CAR design was retained for the next optimization steps.[000256] CAR optimization with a CD8a hinge region and expression in a self-inactivating lentiviral vector.[000257] The natural cleavage of CD27 into its soluble form (sCD27) may potentially limit the efficacy of CD27 ligand-based CARs (13). Thus, we generated three hinge-modified variants by replacing the CD27 native hinge regions with alternative sequences derived from CD8a. The constructs evaluated used CD8a hinge (CD8H) and transmembrane (TM) domains paired with either the 4 IBB (CD8H&TM-41BBQ (as previously described) (13) or the CD27 co-stimulatory domain (CD8H&TM- CD27Q, or a CD8a hinge only (CD8H-CD27Q retaining the CD27 TM and co-stimulatory domains (Figure 4A). All constructs with the CD8a hinge domain were herein abbreviated and illustrated as CD8H in the figures. A truncated human EGFR sequence (huEGFRt) was incorporated after a 2A sequence as a selectable marker and a potential “safety switch” for cetuximab-mediated CAR-T cell depletion in case of unwanted toxicities (24). All hinge-modified constructs were efficiently transduced into activated T cells, achieving a stable level of EGFRt+cells at a MOI of 1.5 (Figure 4B). In parallel, vector copy number analysis was performed to correlate transduction efficiency by flow cytometry to 3.2-4.8 copies of transgene per CAR-T cell.[000258] We next evaluated the anti-leukemic activity of hinge-modified CAR-T cells in a sequential co-culture stress test in vitro. For CD8H&TM-CD27^ and CD8H&TM-41BB<^ CAR-T cells, outgrowth of MOLM-13.GFP-ffLuc tumor cells was observed after the second and third rechallenge, respectively (Figure 4C). On the other hand, both theand CD8H-CD27^ CAR-T cells could perform up to 4 consecutive kills. These data indicate that constructs with CD27 TM and co-stimulatory domains significantly outperformed those containing the CD8a TM paired with either 4 IBB or CD27 co-stimulatory endo-domains.[000259] Interferon-y (IFN-y) and interleukin-2 (IL-2) secretion from CAR-T cells with or without MOLM-13 challenge was analysed in co-culture supernatants. CD8H-CD27^ CAR-T cells produced significantly higher amounts of both IFN-y and IL-2 than 27^ CAR-T cells (Figure 4D). In contrast, CD8H&TM-CD27^ CAR-T cells secreted very low levels of IL-2, in line with the poor cytotoxic capacity of this construct in the serial rechallenge co-culture assay. The production of cytokines by all CAR-T cells evaluated was also strictly antigen-dependent as only very low background levels of IFN-y and IL-2 could be detected in the absence of MOLM-13 target cells (Figure 4D). Moreover, no spontaneous growth of T cells was seen across all variants when cultured in the absence of exogenous cytokines (Figure 4E).[000260] Taken together, our hinge-modified CD70 CARs were devoid of tonic signalling activity and the CD8H-CD27^ variant stood out for its efficient tumor elimination and improved cytokine production in vitro.[000261] Potent in vivo anti-leukemic activity with CD8H-CD27^ CAR-T cells in both AML xenograft and PDX models.[000262] To characterize the in vivo potency of CD8H-CD27^ CAR-T cells, we performed a dose-response study with CD8H-CD27i^ CAR-T cells in mice systemically engrafted with MOLM- 13,GFP-ffLuc cells (Figure 5 A). Mice in all treatment groups responded rapidly compared to the control group, but only mice infused with 2, 3 or 5xl06CAR-T cells achieved sustained complete responses (Figures 5B and 5C). Although mice in the IxlO6treatment group eventually experienced leukemia recurrence, tumor progression was delayed as compared to the control groups. The overall survival of mice was significantly improved across all treatment groups and all mice infused with 2, 3 and 5xl06CAR-T cells remained leukemia-free and survived until the experimental endpoint at 3 months (Figure 5D). Mice in the 5x106group developed graft- vers us-host disease (GvHD) on day 64 which led to early termination of that treatment group.[000263] In a separate MOLM-13.GFP-ffLuc xenograft experiment, we directly compared the anti-tumor activity of our CD8H-CD27(j with the previously published CD8H&TM-41BB^ CAR T cells (13) (Figure 6A). Using a limiting dose of 1.5xl06CAR-T cells per mouse, we found that CD8H&TM-41BB^ CAR-T cells were barely able to control tumor growth as compared to the tumor only or non-transduced control groups (Figures 6B and 6C). Leukemia growth was only delayed by about 8 days before the majority of mice (4 / 5) rapidly progressed and succumbed to the disease (Figure 6D). In stark contrast, CD8H-CD27<^ CAR T cells significantly suppressed tumor growth and all mice (5 / 5) survived and remained tumor-free until the study endpoint (Figures 6C and 6D). This clearly demonstrated the superior potency of our CD8H-CD27^ over the CD8H&TM-41BB^ CAR construct. [000264] We next probed the efficacy of CD8H-CD27^ CAR construct against an adult AML patient-derived xenograft (PDX) model (DFAM68555) with low CD70 target antigen density. Via flow cytometric quantification, we detected 1360 CD70 molecules on the surface of each DFAM6855 cell, 2.3-fold lower than the 3173 molecules found on each MOLM-13 cell (Figure 7A). To test the in vivo anti-leukemic function, DFAM68555-bearing NSG mice were infused with various doses of CD8H- CD27i^ CAR-T cells and bled weekly for monitoring (Figure 7B). By day 20, DFAM68555 cells (CD33+CD70+) had expanded exponentially in the control groups and mice had to be sacrificed due to high tumor burden (Figures 7C and 7D). Conversely, no circulating malignant cells could be detected in the peripheral blood of mice from all CD8H-CD27dj CAR-T cell treated groups. Complete survival was seen until the experimental endpoint. Furthermore, we observed a dose-dependent expansion of CAR-T cells (CD3+EGFR+) in the peripheral blood during the first few weeks after DFAM68555 challenge, which then gradually contracted towards the later timepoints (Figure 7D).[000265] Collectively, these in vivo studies demonstrated the robust competency of our CD8H- CD27C, CAR-T cells in eliminating leukemic cells in both CD70 antigen high and low models of AML. [000266] Modulating CAR-T cell potency by optimizing the CD27-CD70 binding interface.[000267] While affinity tuning has been widely explored in scFv-based CARs and shown to have an impact on both their safety and efficacy profiles, little has been investigated on the optimization of target binding interfaces in the context of natural ligand-based CARs. Hence, we developed a combined physics- and Al-based design approach to identify critical residues and corresponding mutations in the CD27 extracellular domain predicted to modulate the strength of CD70-CD27 binding interface (Methods) (Figure 8A).[000268] With our design pipeline, we generated 36 single point mutant designs that satisfied our criteria, of which 22 were selected for experimental validation, spanning a wide range of binding energies from mildly destabilising (< +1.5 REU versus WT) to strongly stabilising ( — 3 REU) when compared to WT (Figure 8A). Both destabilizing and stabilizing variants were tested as it remained unknown whether the binding strength of the native CD27-CD70 interface was weaker or stronger than the value required for optimal CAR docking and subsequent killing of tumor cells.[000269] Promising point mutants from preliminary in vitro validation were then grouped as double and triple point mutations and reassessed using the same design-docking approach described (Methods), providing 64 total additional designs. From here, 10 spanning a significant dynamic range of interfacial energies were taken forward for downstream validation. Finally, all the selected single, double and triple mutations were applied to our lead CAR construct CD8H-CD27^ to examine the ultimate impact on CAR-T cell - target cell interaction (Figures 8A and 8B, Table 2). All the variants were encoded into our lentiviral vector for functional characterization in primary human T cells.TABLE 2[000270] Mildly stabilizing affinity variant TQ14 exhibited comparable efficacy but better cytokine profiles.[000271] To investigate the ensuing functional differences between stabilizing and destabilizing point mutations, activated T cells were lentivirally transduced to achieve a comparable transgene copy number across all CD8H-CD27^ affinity variants (Figure 9 A). By subjecting the CAR-T cells to an in vitro serial co-culture rechallenge assay with MOLM-13, we observed that the majority of the variants, whether stabilizing or destabilizing, failed to suppress tumor growth beyond the second challenge (Figures 9B and 9C). Particularly, destabilizing variant TQ11 could not eliminate any tumor cells, alike the non-transduced control. Among the six mutants tested, mildly stabilizing variant TQ14 achieved comparable killing capacity to CD8H-CD27^ parental construct. Neither IFN-y nor IL-2 effector cytokines were found to be secreted by TQ11 when co-cultured with MOLM-13, further corroborating that this variant is likely non-functional (Figure 9D). In contrast, TQM produced significantly higher amounts of IL-2 than parental CD8H-CD27^ CAR-T cells upon target engagement. [000272] To substantiate the in vitro findings, the anti-tumor potency of all variants, except the non-functional TQ11, was tested in the MOLM-13. GFP-ffLuc xenograft model in vivo (Figure 10A). At a limiting CAR-T cell dose of 1.5x106cells per mouse, tumor-bearing mice treated with destabilizing variants TQ9 and TQ10 experienced rapid progression (Figures 10B and 10C). Overall survival was poor as all mice succumbed to the disease within 30 days, akin to the control groups (Figure 10D). Similarly in mice infused with stabilizing CAR variants TQ12 and TQ13, suppression of tumoroutgrowth and overall survival was found to be significantly worse than that of CD8H-CD27<^ parental group. Consistent with our in vitro data, only TQM had a comparable performance with CD8H-CD27^ parental CAR construct.[000273] Taken together, these results suggested that modulating the binding interface towards a destabilizing conformation distinctly impacted the downstream effector functions of CAR-T cell in a negative manner.[000274] Top five affinity-modulated variants exhibited enhanced anti-tumor efficacy and IL-2 secretion in vitro.[000275] Since destabilizing mutations have been shown to abate CAR-T cell function, we focused on evaluating the remaining single, double and triple mutant variants predicted to stabilize the CD70-CD27 interaction. As a quick preliminary screen to identify potential candidates, variants were co-cultured with CD70-expressing MOLM-13 cells in a serial rechallenge assay. Mutants with a similar or better tumor suppression profile than parental CD8aH-CD27^ CAR were shortlisted and further examined (Figures 1IA and 11B). Following multiple rounds of selection, four top affinity variants (TQM, TQ29, TQ37, TQ44), together with an additional double mutant (TQ49), were selected for further in-depth functional characterizations.[000276] At an increased E:T ratio of 1 :4, all five variants showed better capacity to withstand rechallenges of fresh MOLM-13 cells when compared with CD8H-CD27^ parental CAR construct (Figure 12 A). This difference was most prominent in TQM, TQ44 and TQ49 albeit only TQM was found to be significantly different in a log-rank Mantel Cox test. The expansion of T cells was observed to be invariable across all CAR constructs, following a typical phase of exponential proliferation upon initial antigen encounter and a subsequent slow contraction phase with time (Figure 12B).[000277] In terms of effector cytokine production in the presence of target antigen, all CAR-T cells responded with high amounts of IFN-y production and no significant difference could be observed between the affinity variants and CD8H-CD27<^ parental CAR-T cells (Figure 12C). On the other hand, T cells bearing the five affinity-modulated CD70 CAR constructs secreted strikingly higher amounts of IL-2 compared to the CD8H-CD27^ parental CAR-T cells. Those higher levels of IL2 likely contribute to the improved ability of the variant CAR-T cells to cope with serial tumor cell rechallenge (Figure 12A).[000278] Double mutant TQ49 demonstrated superior anti-leukemic activity and long-term persistence in a MOLM-13 xenograft model in vivo.[000279] In order to single out the most potent variant among the five candidates, we compared the in vivo efficacy of the CAR variants in the MOLM-13.GFP-ffLuc xenograft model, at a reduced dose of IxlO6CAR-T cells per mouse. All surviving mice were rechallenged with a single dose of MOLM-13.GFP-ffLuc cells on day 34 to probe for functional CAR-T cell persistence in vivo (Figure 13A). In accordance with previous dose titration experiments, only a partial response was observed inmice treated with parental CD8H-CD27^ CAR-T cells (Figure 13B). TQ29 was found to be the least efficacious in suppressing leukemic outgrowth as tumor readily escaped and progressed in 5 / 8 mice. In the TQM and TQ37 groups, a rather slow anti-leukemic response was seen, with one early death in the TQ37 and one death after leukemia rechallenge in the TQM group. The same trend held true for TQ44. The double mutant TQ49 (S72A-H86V) however, substantially suppressed tumor growth at both early timepoints and post-rechallenge (4 / 4 mice evaluated). In the summary of the bioluminescent signal intensity over time using an area under the curve (AUC) analysis, treatment with TQ49 (S72A+H86V) CAR-T cells most potently suppressed the tumor signal compared to all other constructs, including the single mutants TQM (S72A) and TQ29 (H86V) at both early (AUC from Days 3 to 17) and late timepoints after rechallenge (AUC from Days 35 to 60). All mice in the TQ49 group survived and remained tumor-free until end of study (Figure 13C). Collectively, these data hinted towards a probable synergistic effect of S72A and H86V point mutations in favouring the CD70-CD27 interaction, leading to a superior anti-tumor efficacy and long-term survival in mice.[000280] Finally, we performed organ analysis at study endpoint (D70 post-treatment) to inspect the persistence of our CD8H-CD27(J CAR affinity variants in vivo and assess leukemia clearance. We harvested peripheral blood, bone marrow (BM) and spleen of surviving mice and probed for the presence of MOLM-13.GFP-ffLuc tumor cells (huCD45+CD33+GFP+), human T cells (huCD45+huCD3+) and CAR-T cells (huCD45+huCD3+EGFR+), respectively. Leukemic cells were only present in untreated mice post-tumor rechallenge and absent in all those treated with our CD70 CAR-T cells, indicating an efficient clearance in the major homing sites of malignant cells (Figure 13D). Reasonable quantities of human T cells were also detectable in all organs of treated mice, with much lower amounts in the BM than in the blood and spleen. The vast majority of the cells detected were indeed CAR-T cells, as >80% of these huCD45+huCD3+T cells also expressed the transgene reporter marker EGFR.[000281] Taken together, the data substantiated the potent anti-leukemic function and long-term persistence of our affinity-modulated CD8H-CD27<^ CAR-T cells in vivo.[000282] DISCUSSION[000283] Here we developed a novel set of CARs targeting CD70, a tumor associated antigen that is overexpressed in a variety of cancers including lymphoid and myeloid hematologic malignancies and solid tumors, and is an emerging target for clinical development of CAR T cells in various indications (1). The antigen recognition domain of our CARs is based on CD27, the natural ligand of CD70, linked to a non-cleavable hinge derived from CD8a, the transmembrane and co-stimulatory endodomain of CD27 and the CD3^ signalling domain. With a combination of in vitro and in vivo experiments targeting AML cell lines and patient derived xenografts, we selected the most potent construct compared to previously published CD70 CARs. We then optimized the CD27-CD70 binding interface by computational design using physics and deep learning-based algorithms and introducedallosteric mutations which further optimized the CAR. Another round of combined in vitro and in vivo experiments targeting AML allowed us to identify several novel CAR constructs with unique sequence and functional features and with enhanced in vivo potency.[000284] REFERENCES1. Flieswasser T, Van den Eynde A, Van Audenaerde J, De Waele J, Lardon F, Riether C, et al. The CD70-CD27 axis in oncology: the new kids on the block. J Exp Clin Cancer Res. 2022;41 ( 1 ): 12.2. Bowman MR, Crimmins MA, Yetz-Aldape J, Kriz R, Kelleher K, Herrmann S. The cloning of CD70 and its identification as the ligand for CD27. J Immunol. 1994; 152(4): 1756-61.3. Nolte MA, van Olffen RW, van Gisbergen KP, van Lier RA. Timing and tuning of CD27-CD70 interactions: the impact of signal strength in setting the balance between adaptive responses and immunopathology. Immunol Rev. 2009;229(l):216-31.4. Riether C, Pabst T, Hopner S, Bacher U, Hinterbrandner M, Banz Y, et al. Targeting CD70 with cusatuzumab eliminates acute myeloid leukemia stem cells in patients treated with hypomethylating agents. Nat Med. 2020;26(9): 1459-67.5. Pal SK, Tran B, Haanen J, Hurwitz ME, Sacher A, TannirNM, et al. CD70-Targeted Allogeneic CAR T-Cell Therapy for Advanced Clear Cell Renal Cell Carcinoma. Cancer Discov. 2024:OF1-OF14.6. Shaffer DR, Savoldo B, Yi Z, Chow KK, Kakarla S, Spencer DM, et al. T cells redirected against CD70 for the immunotherapy of CD70-positive malignancies. Blood. 2011 ; 117( 16):4304- 14.7. Wang QJ, Yu Z, Hanada KI, Patel K, Kleiner D, Restifo NP, et al. Preclinical Evaluation of Chimeric Antigen Receptors Targeting CD70-Expressing Cancers. Clin Cancer Res. 2017;23(9):2267- 76.8. Jin L, Ge H, Long Y, Yang C, Chang YE, Mu L, et al. CD70, a novel target of CAR T-cell therapy for gliomas. Neuro Oncol. 2018;20( 1 ):55-65.9. Park YP, Jin L, Bennett KB, Wang D, Fredenburg KM, Tseng JE, et al. CD70 as a target for chimeric antigen receptor T cells in head and neck squamous cell carcinoma. Oral Oncol. 2018;78: 145- 50.10. Tu S, Zhou X, Guo Z, Huang R, Yue C, He Y, et al. CD 19 and CD70 Dual-Target Chimeric Antigen Receptor T-Cell Therapy for the Treatment of Relapsed and Refractory Primary Central Nervous System Diffuse Large B-Cell Lymphoma. Front Oncol. 2019;9: 1350.11 . Yang M, Tang X, Zhang Z, Gu L, Wei H, Zhao S, et al. Tandem CAR-T ceils targeting CD70 and B7-H3 exhibit potent preclinical activity against multiple solid tumors. Theranostics. 2020;10(17):7622-34.12. Sauer T, Parikh K, Sharma S, Omer B, Sedloev D, Chen Q, et al. CD70-specific CAR T cells have potent activity against acute myeloid leukemia without HSC toxicity. Blood. 2021 ; 138(4):318-30.13. Leick MB, Silva H, Scarfo I, Larson R, Choi BD, Bouffard AA, et al. Non-cleavable hinge enhances avidity and expansion of CAR-T cells for acute myeloid leukemia. Cancer Cell. 2022;40(5):494-508 e5.14. Michelozzi IM, Gomez-Castaneda E, Pohle RVC, Cardoso Rodriguez F, Sufi J, Puigdevall Costa P, et al. Activation priming and cytokine polyfunctionality modulate the enhanced functionality of low-affinity CD 19 CAR T cells. Blood Adv. 2023 ;7(9): 1725-38.15. Camviel N, Wolf B, Croce G, Gfeller D, Zoete V, ArberC. Both APRIL and antibody-fragment- based CAR T cells for myeloma induce BCMA downmodulation by trogocytosis and internalization. J Immunother Cancer. 2022;10(l 1).16. Zychlinski D, Schambach A, Modlich U, Maetzig T, Meyer J, Grassman E, et al. Physiological promoters reduce the genotoxic risk of integrating gene vectors. Mol Then 2008; 16(4):718-25.17. Liu W, Maben Z, Wang C, Lindquist KC, Li M, Rayannavar V, et al. Structural delineation and phase-dependent activation of the costimulatory CD27:CD70 complex. J Biol Chem. 2021 ;297(4): 101102.18. Leaver-Fay A, Jacak R, Stranges PB, Kuhlman B. A generic program for multistate protein design. PLoS One. 2011 ;6(7):e20937.19. Leaver-Fay A, Tyka M, Lewis SM, Lange OF, Thompson J, Jacak R, et al. ROSETTA3: an object-oriented software suite for the simulation and design of macromolecules. Methods Enzymol. 2011;487:545-74.20. Dauparas J, Anishchenko I, Bennett N, Bai H, Ragotte RJ, Milles LF, et al. Robust deep learning-based protein sequence design using ProteinMPNN. Science. 2022;378(6615):49-56.21 . Marze NA, Roy Burman SS, Sheffler W, Gray JJ. Efficient flexible backbone protein-protein docking for challenging targets. Bioinformatics. 2018;34(20):3461-9.22. Stranges PB, Kuhlman B. A comparison of successful and failed protein interface designs highlights the challenges of designing buried hydrogen bonds. Protein Sci. 2013;22( l):74-82.23. Landrum MJ, Lee JM, Riley GR, Jang W, Rubinstein WS, Church DM, et al. ClinVar: public archive of relationships among sequence variation and human phenotype. Nucleic Acids Res. 2014;42(Database issue):D980-5.24. Johnson A J, Wei J, Rosser JM, Kunkele A, Chang CA, Reid AN, et al. Rationally Designed Transgene-Encoded Cell-Surface Polypeptide Tag for Multiplexed Programming of CAR T-cell Synthetic Outputs. Cancer Immunol Res. 2021 ;9(9): 1047-60.EXAMPLE 2[000286] Off- target screening:[000287] T cells expressing the TQ49 CAR were screened for potential off-target binding against a library of 6500 human proteins expressed on HEK cells (Retrogenix, Charles River) and binding was compared to non-transduced cells. No significant unspecific off-target interactions were identified for the TQ49 CAR T cells in this assay.[000288] A series of various single, double, triple, and multiple point mutations were collated for continued evaluation as variant CD27 affinity binders. A listing of single, double, triple, and multiple point mutations in the CD27 extracellular domain are provided below. The amino acid numbering is based on human CD27 sequence (Uniprot P26842) (SEQ ID NO:1) numbering shown as follows (the extracellular domain of amino acids 22-124 is depicted in bold):MARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAA 60QCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTEC 120DPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPP 180QRSLCSSDFIRILVIFSGMFLVFTLAGALFLHQRRKYRSNKGESPVEPAEPCHYSCPREE 240EGSTIPIQEDYRKPEPACS P260[000289] Various single and combined point mutations are provided below (single, double and triple mutations detailed in Example 1 shown in bold)Single point Double point Triple point Multiple point mutations mutations mutations mutationsF48Y, F71Y, D74S, R78L, H80Y, E82Y, S83P, R85T, H86V, N88D, Q111T, R113L, K115E,D63A F71Y_H80Y F71Y H80Y H86V E116T, E119Q, D121 LF48Y, F71Y, H80Y, S83A, R85T, H86V, N88D,D63S F71Y_S72N F71Y_H80Y_R85T Q111T,R113L, K115E, E116T, E119Q, D121 LF48Y, F71Y, S72N, D74S, R78L, H80Y, E82F,D63W H80Y_R85T F71Y_H86V_Q111T S83P, R85T, H86V, N88D, Q111T, R113L, K115E,E116T, E119Q, D121 LF48Y, F71Y, D74G, R78L, H80Y, E82Y, S83P,D74A H86A S72V F71 Y_R85T_Q111T R85T, H86V, N88D, Q111T, R113L, K115E,E116T, E119Q, D121LF48Y, F71Y, S72N, D74G, R78L, H80Y, E82F,S83P, R85T, H86V, N88D, Q111T, R113L, K115E,D74G H86I_S72G F71Y_S89N_Q111T E116T, E119Q, D121LF48Y, F71Y, D74G, R78L, H80Y, E82F, S83P,D74S H86L S72I H80Y_H86V_S89N R85T, H86V, N88D, Q111T, R113L, K115E, E116T,E119Q, D121LF48Y, F71Y, S72N, D74S, R78L, H80Y, E82Y,D74T H86V S72A H80Y_R85T_S89N S83P, R85T, H86V, N88D, Q111T, R113L, K115E,E116T, E119Q, D121 LF48Y, F71Y, D74G, R78L, H80Y, S83A, R85T,D121 I R85T_Q111T H86V_Q111T_K115E H86V, N88D, Q111T, R113L, K115E, E116T,E119Q, D121LF48Y, F71Y, S72N, D74G, R78L, H80Y, E82Y,S83P, R85T, H86V, N88D, Q111T, R113L, K115E,D121L S72A_H86L R85T_S89N_Q111T E116T, E119Q, D121 LF48Y, F71Y, S72N, H76S, H80Y, S83A, R85T,D121V S72N_H86V S72N_H80Y_Q111T H86V, N88D, Q111T, R113L, K115E, E116T,E119Q, D121LD121A S72VJH86V S72N_H86V_Q111T D121G F71Y_H86V S72N_R85T_Q111T D121F F71Y_S89N S72N_S89N_Q111T E82D H80Y_S89N F71Y_H80Y_K115E E82F H86G_S72G F71Y_H80Y_S89N E82I H86I.S72I F71Y_H86V_S89N E82L H86L_S72L F71Y_R85T_S89N E82V R85TJH86V H80Y_H86V_K115E E82Y R85T_S89N H80Y_Q111T_K115EE116M S72GJH86V H80Y_S89N_K115E E116S S72N_K115E H86V_S89N_K115E E116T S89N_K115E S72N_H80Y_H86V E119D F71Y_K115E S72N_H80Y_S89N E119H H80YJH86V S72N_H86V_S89N E119K H86A_S72G S72N_R85T_S89N E119N H86G.S72I S89N_Q111T_K115E E119Q H86I_S72L F71Y_H80Y_Q111T E119R H86L_S72V F71Y_H86V_K115E E119M Q111T_K115E F71Y_Q111T_K115E F48Y S72AJH86A F71Y_S89N_K115E F71E S72IJH86V H80YJH86V.Q111T F71IVI S72N_Q111T H80Y_R85T_Q111T F71Y S89N_Q111T H80Y_S89N_Q111T G68L F71Y_Q111T H86V_S89N_Q111T G68I H80Y_K115E S72N_H80Y_K115E G68A H86A.S72I S72N_H86V_K115E H76A H86G~S72L S72N_Q111T_K115E H76D H86I_S72V S72N_S89N_K115E H76N H86V_K115E H76Q R85T_H86V H76R S72A_H86G H76S S72L_H86V H76T S72N_R85TH80A F71Y_R85T H80F H80Y_Q111T H80S H86A_S72L H80W H86G_S72V H80Y H86L .S72G H86F H86V_Q111T H86I R85T_K115E H86V S72A H86I H86Y S72N_H80Y H86A S72N_S89N H86LH86G I66V I66L I66AK115A K115D K115E K115HK115I K115L K115N K115Q K115S K115F K115Y N88D N88E N101 D N101Q N101E N101AP79G P79R P79S P79T P79A P79Y Q111G Q1111 Q111K Q111L Q111M Q111R Q111T Q111VR78G R78L R78A R78K R78I R85L R85T R113E R113L R113I S70A S70T S72AS72N S72V S72G S72I S72L S83A S83P S83Q S89A S89G S89K S89N S89RT77GT77A T77V T77I T77LT118A T118G T1181 T118R T118S T118V T118LV69G V69I V69A V69LEXAMPLE 3[000290] Several candidate mutant CD27 polypeptides were further evaluated. In particular, variants TQI4 (S72A) (SEQ ID NO: 18), TQ29 (H86V) (SEQ ID NOG 1), TQ37 (S72N- H86V) (SEQ ID NO:35), TQ44 (H86V-Q111T-K115E) (SEQ ID NO:41) and TQ49 (S72A-H86V) (SEQ ID NO:44) were assessed in additional experiments.[000291] MATERIALS AND METHODS[000292] Cell lines and maintenance. HEK293T, SK-OV-3, A549, HL-60 and MOLM-13 cell lines were purchased from the American Type Culture Collection (ATCC). HEK293T, SK-OV-3 and A549 cells were maintained in DMEM with GlutaMAX (ThermoFisher Scientific, #31966) supplemented with 10% FBS (ThermoFisher Scientific, #10270) and 1% penicillin-streptomycin (ThermoFisher Scientific, #15140122). MOLM-13 and HL-60 cells were cultured in RPMI-1640 with GlutaMAX (ThermoFisher Scientific, #61870) supplemented with 10% FBS and 1% penicillinstreptomycin. The PG-13 retroviral producer cell line for the generation of retroviral particles encoding green fluorescent protein and firefly luciferase (GFP-ffLuc) was kindly provided by Dr. Stephen Gotschalk, Baylor College of Medicine. SK-OV-3, A549, HL-60 and MOLM-13 cells were retro virally transduced to express GFP-ffLuc and sorted to >98% purity to generate SK-OV-3.GFP-ffLuc, A549.GFP-ffLuc, HL-60.GFP-ffLuc and MOLM-13. GFP-ffLuc, correspondingly. Patient-derived acute myeloid leukemia (AML) xenograft (PDX) cells DFAM-68555 and CBAM-44728 were purchased from the Dana-Faber Cancer Institute, Center for Patient Derived Models, Collection of Hematologic PDX models.[000293] Cytotoxicity assay of ovarian cancer. SK-OV-3. GFP-ffLuc cells were harvested with Accutase and 1.5xl04cells were plated in 100 uL in 96-well flat bottom plates for 5 hours to allow attachment. Based on the transduction efficiency, CAR-T cells were adjusted to the indicated E:T ratios and plated at 100 uL using assay medium (DMEM supplemented with 10% FBS and 1% penicillinstreptomycin). Four days post-coculture, the number of tumor cells (GFP+) in each well was quantifiedusing CountBright™ absolute counting beads (ThermoFisher Scientific, #C36950) on a flow cytometer. The specific cell lysis induced by CAR-T cells was calculated using the following equation: / number of GFP+cells in presence of CAR — T cells \1 _ ( - - -J. - £ - — - — — U 100%\number of GFP+cells in presence of non — transduced cells / [000294] Surface CD70 quantification. Adherent cells were detached and harvested with Accutase (Innovative Cell Technologies Inc., #AT-104). 1x105tumor cells were stained with PE-anti- human CD70 antibody (Biolegend, #355104). Background signals, based on HL-60.GFP-ffLuc, were first subtracted from live-gated, CD70+populations. The surface target antigen densities were then quantified using BD Quantibrite™ Phycoerythrin Fluorescence Quantitation Kit (BD Biosciences, #340495) as per manufacturer’s instructions.[000295] Flow cytometry. Cells were stained with antibody cocktails for 30 minutes in the dark at 4 °C and washed with lx PBS buffer (ThermoFisher Scientific, #10010023) containing 2% FBS and 2mM EDTA (ThermoFisher Scientific, #15575). Cell pellets were resuspended in wash buffer supplemented with DAPI (Biolegend, #422801) a dilution of 1 : 100 before acquisition on a LSR II flow cytometer (BD Biosciences). Data acquisition was performed using FACSDiva software version 9.0 or higher while data analyses were conducted with FlowJo software version 10.9. or higher (Tree Star Inc.). The list of antibodies used, conjugated with various fluorochromes, is listed in Table 3.[000296] Animal experimentation and mouse models. All animal studies were conducted in accordance with a protocol approved by the Veterinary Authority of the Swiss Canton of Vaud (authorization VD3390 to Caroline Arber). NOD-sc / d IL2Rgammanu11(NSG) (The Jackson Laboratory strain #005557), NSG-SGM3 (The Jackson Laboratory, strain #013062) andNSG-QUAD (The Jackson Laboratory, strain #028657) mice were purchased from The Jackson Laboratory. All mice were housed and bred at the animal facility of the Faculty of Biology and Medicine of the University of Lausanne (UNIL) and the Centre Hospitalier Universitaire Vaudois (CHUV). All animals were between 6 to 10 weeks old at the start of the experiment and were handled as illustrated for each individual experiment. Animal well-being was monitored with predefined criteria and score sheets 3 times per week. Mouse survival was determined using humane endpoint criteria.[000297] For the MOLM-13.GFP-ffLuc AML xenograft model, 5xl03leukemia cells were infused intravenously (i.v.) via tail-vein, followed by CAR-T cells 7-8 days post-engraftment. Leukemic progression was monitored once or twice weekly via bioluminescence imaging (BLI) using the IVIS Lumina II In Vivo Imaging System (IVIS, Revvity) upon administration of IVISbrite D-luciferin substrate solution (Perkin Elmer, #122799). All mice were imaged one day prior to CAR-T cell treatment and randomized based on the intensity of bioluminescence signal.[000298] For the adult AML patient-derived xenograft (PDX) model, 2xl06DFAM68555 ceils were infused i.v., followed by various doses of CD8aH&TM-CD27£ CAR-T cells a week later. For the pediatric PDX model, 2xl06CBAM44728cells were engrafted for 13 days before infusing various doses of TQ49 CAR-T cells. In both cases, peripheral blood was collected through weekly bleeding via thetail-vein and analyzed for the presence of leukemia and CAR-T cells on a flow cytometer. The antibodies used for these analyses are listed in Table 3. All mice in the PDX models were randomized based on body weight.[000299] For the ovarian cancer xenograft model, IxlO6SK-OV-3.GFP-ffLuc cells were engrafted subcutaneously (s.c.) for 3 days prior to the infusion of TQ49 CAR-T cells via tail-vein. For the non-small cell lung cancer xenograft model, 2xl06A549.GFP-ffLuc cells were engrafted intravenously for 4 days before CAR-T cell treatment. For both models, tumor progression was tracked once or twice weekly via BLI imaging. All mice were imaged on the day of CAR-T cell infusion and randomized according to the intensity of the BLI signal.[000300] Table 3. List of commercial antibodies used for flow cytometric analyses.[000301] RESULTS[000302] Double mutant TQ49 (S72A-H86V) demonstrated superior anti-leukemic activity and long-term persistence in a MOLM-13 xenograft model in vivo.[000303] To single out the most potent variant among certain top candidates, we compared the in vivo efficacy of the CAR variants in the MOLM-13.GFP-ffLuc xenograft model, at a reduced dose of 1x106CAR-T cells per mouse. All surviving mice were rechallenged with a single dose of MOLM- 13. GFP-ffLuc cells on day 34 to probe for functional CAR-T cell persistence in vivo (Figure 13 A). In accordance with previous dose titration experiments, only a partial response was observed in mice treated with parental CD8H-CD27^ CAR-T cells (Figure 13B). Among the variants, TQ29 (S72A-H86V) was found to be the least efficacious in suppressing leukemic outgrowth as tumor readily escaped and progressed in 5 / 8 mice. This was followed by TQ37 (S72N-H86V) and TQ44 (H86V- Q111T-K115E), where disease progression was observed in 5 / 13 and 4 / 13 mice, respectively.[000304] Between the single mutant TQM (S72A) and the double mutant TQ49 (S72A-H86V), the latter displayed a better tumor suppression profile. Although death of one mouse was observed for TQ49 at early time-point, it was due to extramedullary disease. In the summary of the bioluminescent signal intensity over time using an area under the curve (AUC) analysis, treatment with TQ49 CAR-T cells substantially suppressed the tumor signal post-rechallenge (AUC from Days 35 to 60) (Figure 13C). In contrast to TQM, all mice in the TQ49 group that were subjected to rechallenge survived and remained disease-free until the end of the study (Figure 13D). Collectively, these data hinted towards a probable synergistic effect of S72A and H86V point mutations in favouring the CD70-CD27 interaction, leading to a superior anti-tumor efficacy and long-term survival in mice.[000305] Finally, we performed organ analyses at the study endpoint (Day 78) to inspect the persistence of our CD8H-CD27^ CAR affinity variants and leukemia clearance in vivo. We harvested the peripheral blood, bone marrow (BM) and spleen of surviving mice and probed for the presence of MOLM-13.GFP-ffLuc tumor cells (huCD45+CD33+GFP+), human T-cells (huCD45+huCD3+) and CAR-T cells (huCD45+huCD3+EGFR+), respectively. Leukemic cells were only detected in untreated mice post-tumor rechallenge and absent in all those treated with our CD70 CAR-T cells, indicating an efficient clearance from the major homing sites of malignant cells (Figure 13E). Reasonable quantities of human T-cells were also present in all organs of treated mice, with much lower amounts in the BM than that of the blood and spleen. The majority of the T-cells detected were indeed CAR-T cells, as >80% of these huCD45+huCD3+cells also expressed the transgene reporter marker EGFR.[000306] Taken together, the data substantiates the potent anti-leukemic function and long-term persistence of our affinity-modulated CD8H-CD27^ CAR-T cells in vivo. Among the four variants examined, TQ49 was selected for further downstream evaluations.[000307] Binding interface optimized TQ49 (S72A-H86V) proved in vivo efficacy against a pediatric AML relapse PDX model CBAM44728.[000308] Following the substantial functional characterizations against adult AML models, we next investigated the efficacy ofTQ49 in an aggressive pediatric AML PDX model CBAM44728. NSG- QUAD mice with constitutive secretion of human cytokines GM-CSF, M-CSF 1 , IL-3 and Steel factor (SCF) were used to support the engraftment of leukemic cells prior to treatment with various doses of TQ49 CAR-T cells (Figure 14A). Analyses of the peripheral blood (PB) revealed an exponential growth of tumor cells (huCD45+CD33+) in mice treated with non-transduced and IxlO6TQ49 CAR-T cells (Figure 14B), suggesting that this dose was too low to be efficacious. At 2xl06dosage, 1 / 4 mice experienced rapid progression (>60% tumor cells in blood) while 3 / 4 mice had a reduced tumor load (<20% tumor cells in blood), indicative of a partial response. In contrast, leukemic outgrowth wascompletely suppressed in all mice infused with 5xl06TQ49 CAR-T cells. This dose response effect correlated well with the expansion profile of human T-ceiis in the blood (huCD45+CD3+), where 20- 40% T-cells was detectable in the 5xl06group but only less than 10% in the IxlO6and 2xl06groups (Figure 14B).[000309] At the experimental endpoints (D32 for non-transduced, IxlO6and 2xl06dosages; D56 for 5x106dosage), the amount of tumor and human T-cells present in the major homing organs were analyzed. In accordance with the findings in blood, high tumor load and negligible T-cells were detected in both the bone marrow (BM) and spleen of mice in the non-transduced and IxlO6dosage groups (Figure 14C). Although tumor growth was largely suppressed in the spleen of mice treated with 2xI06cells, the BM remained burdened, contributing to disease progression. On the other hand, both organs were thoroughly cleared of malignant cells with 5xl06TQ49 CAR-T cells, along with a significantly increased proportion of human T-cells detectable. All mice in this group remained tumor-free and survived until the study endpoint (Figure 14D). In addition, we examined the differentiation profile of the CAR-T cells retrieved from the organs of these mice in complete remission. We found that human CAR-T cells in PB and spleen largely exhibited a central memory phenotype (CM; CD45RO+CD62L+) whereas those in the bone marrow were more towards an effector memory (EM; CD45RO+CD62L") phenotype (Figure 14E).[000310] TQ49 (S72A-H86V) CAR-T cells cleared human ovarian carcinoma ceil line SK- OV-3 both in vitro and in vivo.[000311] In addition to hematological malignancies, CD70 over-expression has also been reported on a wide variety of solid tumors. To validate that, we examined the CD70 cell surface expression on a panel of different solid tumor cell lines including non-small cell lung cancer (A549), clear cell renal carcinoma (ACHN), pancreatic carcinoma (PANC-1) and ovarian carcinoma (SK.-OV- 3) (Figure 15A). Using a flow cytometry-based quantification, A549 was found to have the lowest CD70 expression (558 molecules / cell) (Figure 15B). This was 7-fold lower than that of the AML cell line MOLM-I3 (3940 molecules / cell). In contrast, SK-OV-3 (52680 molecules / cell) had 13-fold higher CD70 expression than MOLM-13, making it the highest among the panel. The target antigen density of ACHN (10037 molecules / cell) and PANC-1 (27318 molecules / cell) were found to be in between A549 and SK-OV-3. Based on the expression profile, we selected A549 and SK-OV-3 as a start to investigate the efficacy of our lead candidate TQ49 in both a CD70-high and CD70-low solid tumor model.[000312] We first performed an in vitro cytotoxicity assay where SK-OV-3.GFP-ffLuc target cells were co-cultured with TQ49 transgenic cells (Figure 16A) at various effector to target (E:T) ratios. At an E:T ratio of 1 :2, a close to complete target cell lysis (90±6%) was seen (Figure 16B). As the E:T ratio decreased to 1 : 16, a modest level of cell lysis could still be observed (28±9%). The data showedthat our TQ49 CAR-T cells could elicit strong cytotoxic effect even when the target cells were in large excess.[000313] To validate the in vitro findings, we next evaluated the potency of TQ49 in a xenograft model in vivo. Mice were engrafted with SK-OV-3.GFP-ffLuc cells subcutaneously and infused with either 1 x 106or 3x 106TQ49 CAR-T cells (Figu re 16C). A strikingly rapid and potent clearance of tumor cells was observed in mice from the 3x106dosage group between day 3 to day 9, with all mice achieving remission by day 12 (Figures 16D and 16E). This kinetics was slower in the lower dose group (IxlO6cells), where tumor regression most drastically occurred after day 9. Nevertheless, all mice in the IxlO6group were free of the disease by day 18. The robust clearance of ovarian carcinoma SK-OV-3 cells by TQ49 was also reflected in the AUC analysis where a significant reduction in bioluminescent signal intensity was observed for both doses when compared to the non-transduced control group (Figure 16F). Together, the data clearly demonstrated the potent anti-tumor activity of our affinity-modulated CD70 CAR against ovarian cancer both in vitro and in vivo.[000314] TQ49 (S72A-H86V) CAR-T cells suppressed the growth of CD70-low A549 lung cancer cells in vivo.[000315] Finally, we probed the efficacy of our TQ49 CAR-T cells against the CD70-low A549 NSCLC model in vivo. Mice were engrafted with A549.GFP-ffLuc intravenously and CAR-T cells were infused when a high tumor burden was established in the lungs (Figure 17A). At a low dose of IxlO6TQ49 CAR-T cells, a partial elimination of A549 cells was seen at early time points between days 0 to 7 (Figure 17B). However, this dose was inadequate to suppress tumor growth completely as an increase in bioluminescent signal intensity was observed after day 7 (Figure 17C). At an increased dose of 5xl06CAR-T cells, a close to complete eradication of malignant cells from the lungs of the mice was seen. AUC analysis of the signal intensity also acknowledged that although tumor cells were not fully eliminated, there was a significant suppression of its growth in the presence of IxlO6TQ49 cells as compared to the non-transduced control group (Figure 17D). As a result, disease progression was delayed. This data showcased the potent anti-tumor capacity of our TQ49 CAR-T cells against A549 tumors with extremely low cell surface CD70 expression.[000316] These studies further demonstrate efficacy of mutant CD27 polypeptides, particularly when incorporated in a CAR system. CAR-T cells comprising mutant CD27 polypeptide, including particularly that of TQ49 variant (S72A-H86V), are effective in adult and pediatric AML models and in solid tumor models including ovarian cancer and non-small cell lung cancer (NSCLC). Disease progression is delayed at lower doses (IxlO6CAR T-cells) and eradication of malignant cells is observed at higher doses (such as 5xl06CAR-T cells). The studies extend the application of the CAR system with mutant CD27 polypeptide to pediatric cancel models particularly pediatric AML. Further,extension and efficacy in solid tumors is demonstrated, even in vivo against tumors with very low antigen densities for CD70.[000317] This invention may be embodied in other forms or carried out in other ways without departing from the spirit or essential characteristics thereof. The present disclosure is therefore to be considered as in all aspects illustrated and not restrictive, the scope of the invention being indicated by the appended Claims, and all changes which come within the meaning and range of equivalency are intended to be embraced therein.[000318] Various references are cited throughout this Specification, each of which is incorporated herein by reference in its entirety.

Claims

W11AT . IS CLA1MED IS:

1. A potency tuned mutant CD27 polypeptide, wherein one or more amino acid is altered in the native CD27 extracellular domain sequence corresponding to amino acids 22-124 of SEQ ID NO:1 : MARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAA 60 QCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTEC 120 DPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPP 180 QRSLCSSDFIRILVIFSGMFLVFTLAGALFLHQRRKYRSNKGESPVEPAEPCHYSCPREE 240 EGSTI PIQEDYRKPEPACSP 2602. The mutant CD27 polypeptide of claim 1, wherein the polypeptide demonstrates stabilized or increased binding to CD70, higher potency activity upon CD70 binding, and / or increased or greater IL-2 stimulation and / or expression upon CD70 binding in comparison with native or unmutated CD27.

3. The mutant CD27 polypeptide of claim 1 or 2, wherein the polypeptide is altered at one or more amino acids selected from S72, H86, QI 11, KI 15, F71, D74, H80, R85, S89, El 19, and D121 in the CD27 extracellular domain sequence.

4. The mutant CD27 polypeptide of claim 3, wherein the polypeptide is altered at two or more amino acids selected from S72, H86, QI 1 1, KI 15, F7I, D74, H80, R85, S89, EH9, and D121 in the CD27 extracellular domain sequence.

5. The mutant CD27 polypeptide of claim 3, wherein the polypeptide is altered at two or more amino acids, or at three or more amino acids, selected from S72, H86, QI 11, KI 15, F71, D74, H80, R85, S89, El 19, and D121 in the CD27 extracellular domain sequence.

6. The mutant CD27 polypeptide of any claims 1-3, wherein the polypeptide comprises a variant extracellular binding domain comprising the sequence set out in SEQ ID NO: 45:PAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFXiPDHHTRPHCESCR X2CNSGLLVRNCTITANAECACRNGWX3CRDX4ECTECDPLP wherein Xi is A or NX2is V, F or YX3is TX4 is E, D, Q or A.

7. The mutant CD27 polypeptide of any claims 1-3, wherein the polypeptide comprises a variant extracellular binding domain comprising the sequence set out in SEQ ID NO: 46): PAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCI PGVSFXiPDHHTRPHCESCR X2CNSGLLVRNCTITANAECACRNGWX3CRDX4ECTECDPLPwherein8. The mutant CD27 polypeptide of any claims 1-3, wherein the polypeptide comprises a variant extracellular binding domain comprising the sequence set out in SEQ ID NO: 47:and wherein the variant extracellular binding domain of CD27 does not correspond in sequence to human CD27 extracellular binding domain of SEQ ID NOG:PAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCI PGVSFS PDHHTRPHCESCRHCNS GLLVRNCTITANAECACRNGWQCRDKECTECDPLP .

9. The mutant CD27 polypeptide of any claims 1-3, wherein the polypeptide comprises a variant CD27 extracellular domain selected from a peptide sequence as set out any of SEQ ID NO:s 13-47.

10. The mutant CD27 polypeptide of any of claims 1-3 or 9, wherein the polypeptide comprises a variant extracellular domain sequence selected from mutant TQ14 (S72A) (SEQ ID NO: 18), TQ29 (H86V) (SEQ ID NOG 1), TQ37 (S72N-H86V) (SEQ ID NO:35), TQ44 (H86V-Q11 1T-K1 15E) (SEQ ID NO:41) and TQ49 (S72A-H86V) (SEQ ID NO:44).1 1 . The mutant CD27 polypeptide of any of claims 1-10, wherein the polypeptide consists of a mutant CD27 extracellular domain comprising amino acids corresponding to amino acids 22-124 of SEQ ID NO: 1, wherein at least one amino acid is altered.

12. The mutant CD27 polypeptide of claim 11, wherein the extracellular domain is selected from a peptide sequence as set out any of SEQ ID NO:s 13-47.

13. The mutant CD27 polypeptide of any of claims 1-12, wherein the polypeptide further comprises the CD27 transmembrane and endo domain corresponding to amino acids 193-260 of human CD27, such as the transmembrane and endo domains corresponding to SEQ ID NO: 3.

14. The mutant CD27 polypeptide of any of claims 1-12, wherein the mutant CD27 peptide or the mutant CD27 extracellular domain is conjugated to another compound, polymer, cytotoxic agent, immunomodulatory compound, protein domain, immune modulator or immune activator.

15. A composition comprising the mutant CD27 polypeptide of any of claims 1-14 and a pharmaceutically acceptable carrier.

16. A cell, particularly an immune cell, comprising the mutant CD27 polypeptide of any of claims 1-14.

17. A chimeric antigen receptor (CAR) or bispecific T cell engager (BiTE) comprising the mutant CD27 polypeptide of any of claims 1-14 for targeting the CD27 ligand CD70 or CD70-expressing cells.

18. The CAR of claim 17, wherein the CAR comprises:(a) a leader sequence or signal sequence;(b) a mutant CD27 extracellular domain;(c) a hinge sequence;(d) a transmembrane domain; and(e) an intracellular or signalling domain.

19. The CAR of claim 18, wherein the CAR comprises:(a) a leader sequence or signal sequence;(b) a mutant CD27 extracellular domain selected from SEQ ID NOs: 13-47;(c) a hinge sequence;(d) a transmembrane domain; and(e) an intracellular or signalling domain.

20. The CAR of claim 18 or 19, wherein:(a) the signal sequence is a human immunoglobulin sequence;(b) the mutant CD27 extracellular domain sequence is selected from SEQ ID NO: 13-47;(c) the hinge sequence is selected from a CD8 hinge, an IgG hinge, an IgD hinge, a CD28 hinge, a KIR2DS2 hinge, or a glycine-serine linker;(d) the transmembrane domain is selected from CD27, CD28, CD3 epsilon, CD3 zeta, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, 0X40, CD2, CD27, LFA-1 (CD1 la, CD18), ICOS (CD278), 4-1BB (CD 137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (K.LRF1), CD 160, CD 19, IL2R beta, IL2R gamma, IL7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDlld, ITGAE, CD103, ITGAL, CDlla, LFA-1, ITGAM, CDllb, ITGAX, CDllc, ITGB1, CD29, ITGB2, CD 18, LFA-1, ITGB7, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1,CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, NKG2C, or TNFR2 transmembrane domain, or the alpha, beta or zeta chain of the T-cell receptor; and / or(e) the intracellular or signalling domain comprises a primary signalling domain selected from a CD3 zeta (CD3Q, CD3 gamma (CD3y), CDS delta, CD3 epsilon, common FcR gamma (FCER1G), FcR beta (Fc Epsilon Rib), CD79a, CD79b, Fcgamma Rlla, DAP 10, or DAP 12 signalling domain; or the intracellular or signalling domain comprises a primary signalling domain and a costimulatory domain wherein the primary signalling domain is selected from a CD3 zeta (CD3Q, CD3 gamma (CD3y), CD3 delta, CD3 epsilon, common FcR gamma (FCER1G), FcR beta (Fc Epsilon Rib), CD79a, CD79b, Fcgamma Rlla, DAP 10, or DAP 12 signalling domain, and the costimulatory domain is selected from CD28, 4- IBB (CD 137), 0X40, CD27, CD30, CD40, CD 134, PD-1, ICOS, lymphocyte function-associated antigen- 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFI), CD 160, CD 19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLAl, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDlld, ITGAE, CD 103, ITGAL, CDlla, LFA-1, ITGAM, CDllb, ITGAX, CDllc, ITGB1, CD29, ITGB2, CD 18, LFA- 1, ITGB7, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD 160 (BY55), PSGL1, CD 100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, or TNFR2 costimulatory domain.21 . The CAR of claim 18, 19 or 20, wherein:(a) the signal sequence is a human immunoglobulin sequence comprising MEFGLSWLFLVAILKGVQC (SEQ ID NO: 48);(b) the mutant CD27 extraceiiuiar domain sequence is selected from SEQ ID NO: 13-47;(c) the hinge sequence is a CD8a hinge sequence comprising TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDI (SEQ ID NO:49)(d) the transmembrane domain sequence is a CD27 sequence comprisingLVIFSGMFLVFTLAGALFLHQRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEP ACSP (SEQ ID NOG); and(e) the intracellular or signalling domain is a CD3^ chain sequence comprising RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDK.RRGRDPEMGGKPRRKNPQEGLYN ELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:50).

22. The CAR of claim 18, 19, 20 or 21 comprising:(a) a signal sequence MEFGLSWLFLVAILKGVQC (SEQ ID NO: 48);(b) a mutant CD27 extracellular domain comprising amino acids selected from SEQ ID NO: 18, SEQ ID NOG 1, SEQ ID NO:35, SEQ ID NO:41 and SEQ ID NO:44;(c) a CD8a hinge sequence comprisingTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDI (SEQ ID NO:49)(d) a CD27 transmembrane domain sequence comprisingLVIFSGMFLVFTLAGALFLHQRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEP ACSP (SEQ ID NOG); and(e) a CD3<^ signalling domain sequence comprisingRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYN ELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:50).

23. A cell, particularly an immune cell, comprising the CD27 CAR or BiTE of any of claims 17- 22.

24. A nucleic acid encoding a mutant CD27 polypeptide or mutant CD27 extracellular domain of any of claims 1-14, or a CAR or BiTE of any of claims 17-22.

25. A vector comprising the nucleic acid of claim 24.

26. A cell, particularly an immune effector cell, comprising the nucleic acid of claim 24 or the vector of claim 25.

27. A method of treating a subject having a cancer or disease associated with expression of CD70, the method comprising administering to the subject mutant CD27 polypeptide or mutant CD27 extracellular domain of any of claims 1-14, the composition of claim 15, the cell of claim 16, the CAR or BiTE of any of claims 17-22, the cell of claim 23 or the cell of claim 26.

28. The method of claim 27, wherein the cancer is selected from leukemia, lymphoid leukemia, multiple myeloma, B cell lymphoma, T cell lymphoma, glioblastoma, or renal cell carcinoma.

29. The method of claim 27, wherein the cancer is selected from acute lymphoblastic leukemias (B and T cell origins), B cell lymphomas, bladder cancer, breast invasive carcinoma, cervical cancer, cholangiocarcinoma, colorectal cancer, diffuse large B-cell lymphoma (DLBC), Esophagus, glioblastoma (GBM), head and neck cancer, low-grade gliomas (LGG), liver cancer, lung adenocancer, melanoma, mesothelioma, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, stomach cancer, testicular germ cell cancer, thymoma, thyroid cancer, uterine cancer, uveal melanoma, clear cell renal cell carcinoma (ccRCC), chromophobe renal cell carcinoma, papillary renal cell carcinoma (pRCC), acute myeloid leukemia, and adenoid cystic carcinoma (ACC) bladder cancer, breast invasive carcinoma, cervical cancer, cholangiocarcinoma, colorectal cancer, diffuse large B-cell lymphoma (DLBC), Esophagus, glioblastoma (GBM), head and neck cancer, low-grade gliomas (LGG), liver cancer, lung adeno cancer, melanoma, mesothelioma, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, stomach cancer, T cell lumphomas, testicular germ cell cancer, thymoma, thyroid cancer, uterine cancer, uveal melanoma, clear cell renal cell carcinoma (ccRCC), chromophobe renal cell carcinoma, papillary renal cell carcinoma (pRCC), acute myeloid leukemia (AML), and adenoid cystic carcinoma (ACC).

30. The method of claim 27, wherein the disease is Graft-versus-host disease (GVHD).

Images