Chimeric antigen receptors targeted to CD70

Abstract

This disclosure relates to compositions comprising and methods making and using T cells expressing a chimeric antigen receptor (CAR) targeted to CD70 via CD27.

Description

[0001] Attorney Docket No.: 40056-0110WO1 / COH Ref.: TEC 25-002

[0002] CHIMERIC ANTIGEN RECEPTORS TARGETED TO CD70

[0003] CLAIM OF PRIORITY

[0004] This application claims the benefit of U. S. Provisional Application Serial No. 63 / 770,320, filed on March 11, 2025. The entire contents of the foregoing are incorporated herein by reference.

[0005] BACKGROUND

[0006] CD70, a member of the tumor necrosis factor receptor (TNFR) family, is expressed on activated T cells, B cells, natural killer (NK) cells, and mature dendritic cells (DCs) after antigen encounter. It is broadly expressed in various tumors, including hematological malignancies and solid tumors such as glioblastoma multiforme (GBM) and renal cell carcinoma (RCC), with minimal expression on healthy tissues. This makes CD70 an attractive target for chimeric antigen receptor T-cell (CAR T-cell) therapy.

[0007] CD70 contributes to tumor progression by maintaining cancer stem cells (CSCs), promoting epithelial-to-mesenchymal transition (EMT), and facilitating tumor migration. It also plays an immune-suppressive role by inducing apoptosis of CD8+ T cells, promoting T-cell exhaustion, depleting NK cells, and recruiting or activating regulatory T cells (Tregs) and tumor-associated macrophages (TAMs), particularly the M2 phenotype (CD163+ or CD69+ TAM-M2). Studies have demonstrated the feasibility and safety of CD70-targeting CAR T-cell therapy in models such as GBM. Additionally, therapies targeting CD70's only ligand, CD27, through CD27-ligand CAR approaches, have shown promise in hematological malignancies

[0008] SUMMARY

[0009] Described herein are chimeric antigen receptors targeted to CD70 (CD70 CAR). The studies described in the Examples assess various CAR components for their impact on various activities, including, tumor cell killing, CAR expansion and induction of fratricide in order to design a CD70 CAR with the ability to be therapeutically beneficial.

[0010] The studies described in the Examples demonstrate, among other things, that certain CD70-targeted CARs with a cleavable hinge (spacer) between the CD70 binding domain and theAttorney Docket No.: 40056-0110WO1 / COH Ref.: TEC 25-002

[0011] transmembrane domain can outperform certain CD70 targeted CARs without a cleavable hinge in terms of cytotoxicity against two solid tumor models (GBM and RCC). They also show that certain CD70-targeted CARs with a cleavable hinge exhibit cytotoxicity that is comparable to certain ScFv-based CARs in vivo and in vitro rechallenge assays, but with significantly reduced fratricide. Moreover, CD70 knockout had minimal impact on the efficacy of certain CD70-targeted CARs with a cleavable hinge. These findings suggest that when expressed in T cells that express CD70, certain CD70-targeted CARs with a cleavable hinge may have a therapeutic advantage compared to CD70 CAR that lack a cleavable hinge.

[0012] Described herein is a nucleic acid molecule comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR) comprising or consisting of: a CD70 binding domain comprising or consisting of: (a) the amino acid sequence:

[0013] ATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHC ESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQ

[0014] (SEQ ID NO: 43) or (b) a scFv targeted to CD70; a spacer domain; a transmembrane domain; a costimulatory domain; and a CD3 signaling domain. In certain embodiments a linker is present between two or more adjacent domains. In other embodiments, the linkers are joined directly to the adjacent domain (i.e., there are no interdomain linkers). In certain embodiments, the CAR does not comprise a CD27 intracellular domain (e.g., the CAR does not comprise the costimulatory domain of CD27),

[0015] In various embodiments: the CD70 binding domain comprises or consists of the amino acid sequence:

[0016] ATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHC ESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQ

[0017] (SEQ ID NO: 43); and the spacer domain comprises or consists of an amino acid sequence selected from the group consisting of: SEQ ID NOs: 1-13 and 36, the transmembrane domain comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 14-22 and 37-39; the costimulatory domain comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 31-35, and the CD3 signaling domain comprises or consists of an amino sequence selected from the group consisting of SEQ ID NOs: 23-30.

[0018] In various embodiments: the spacer comprises or consists of the amino acid sequence of SEQ ID NO: 36; the transmembrane domain comprises or consists of an amino acid sequenceAttorney Docket No.: 40056-0110WO1 / COH Ref.: TEC 25-002

[0019] selected from the group consisting of: SEQ ID NOs: 15-20 and 37; the transmembrane domain comprises or consists of the amino acid sequence of: SEQ ID NO: 17; and costimulatory domain comprises KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 33);

[0020] In various embodiments: the spacer domain comprises or consists of the amino acid sequence of SEQ ID NO: 36, the transmembrane domain comprises or consist of an amino acid sequence selected from the group consisting of SEQ ID NOs: 14-22 and 37-39; the costimulatory domain comprises or consists of the amino acid sequence of SEQ ID NO: 33, and the CD3 signaling domain comprises a sequence selected from the group consisting of SEQ ID NOs: 23-30.

[0021] In various embodiments: the CD3 signaling domain comprises or consists of the amino acid sequence of SEQ ID NO: 23; the sequence encoding the CAR is immediately preceded by a nucleic acid sequence encoding a signal sequence; the signal sequence comprises or consists of the amino acid sequence of SEQ ID NO: 55; the CAR comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 45-54; the CAR comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 56-68; the CAR comprises or consists of the amino acid sequence of SEQ ID NO: 53; an interdomain linker of 1-5 amino acids is located between two or more adjacent domains; an interdomain linker of 1-5 amino acids is located between the costimulatory domain and the CD3 signaling domain; the interdomain linker comprises glycine or alanine; the interdomain linker comprises only glycine or alanine; and the interdomain linker comprises only glycine or only alanine.

[0022] Also described herein is a population of T cells harboring the nucleic acid molecule of any of the forgoing claims. In various embodiments: the T cell does not express CD70; the T cell has been genetically modified to knock out CD70 expression; the T cell has been genetically modified to knock out CD70 coding sequence; and at least 50%, 70% or 90% of the T cells in the T cell population comprise memory T cells, naive T cells or central memory T cells.

[0023] Also described is a method of treating a patient having renal cell carcinoma, comprising administering a therapeutically effect amount of a population of T cells described herein.

[0024] Also described is a method of treating a patient suffering from a cancer expressing CD70, comprising administering a therapeutically effect amount of a population of T cells described herein.Attorney Docket No.: 40056-0110WO1 / COH Ref.: TEC 25-002

[0025] Also described is a method of treating a patient having glioblastoma, comprising administering a therapeutically effect amount of a population of T cells described herein.

[0026] In various embodiment of the treatment methods, the cells are administered intravenously or intraventricularly.

[0027] In various embodiments, the method further comprises administering decitabine or azacytidine.

[0028] Described herein is a nucleic acid molecule comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR) comprising: (i) a CD70 binding domain comprising or consisting of the amino acid sequence:

[0029] ATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHC ESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQ

[0030] (huCD27L: SEQ ID NO: 43) or an svFV targeted to CD70; (ii) a cleavable hinge domain comprising or consisting of the amino acid sequence:

[0031] PTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIR (CD27h: SEQ ID NO: 36) or a spacer; (iii) a transmembrane domain; (iv) a costimulatory domain; and (v) a CD3 signaling domain.

[0032] In various instances: the transmembrane domain comprises a sequence selected from the group consisting of SEQ ID NOs: 14-22, A, B and C; the costimulatory domain comprises a sequence selected from the group consisting of SEQ ID NOs: 31-35, and the CD3 signaling domain comprises a sequence selected from the group consisting of SEQ ID NOs: 23-30; the transmembrane domain selected from the group consisting of: a CD4 transmembrane domain, a CD8 transmembrane domain, a CD28 transmembrane domain, and a CD3 transmembrane domain, a CD99 transmembrane domain, a CD27 transmembrane domain, and a CD8-LYC transmembrane domain; the costimulatory domain comprises KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 33); an interdomain linker of 1-5 glycine is located between the costimulatory domain and the CD3 signaling domain.

[0033] In various instances the CAR comprises an amino acid sequence selected from:

[0034] huCD27L-CD27tm-41BB-CD3z (C1) ATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHC ESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTAttorney Docket No.: 40056-0110WO1 / COH Ref.: TEC 25-002

[0035] HLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVFTLAGA LFLHKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGGGRVKFSRSADAPAYQ QGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIG MKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRLEGGGEGRGSLLTCGDVEENPGPR

[0036] (SEQ ID NO: 45);

[0037] huCD27L-CD8h3 (AKP 2516-2524 Del)-CD28tm (M2660-2662 Del)-41BB-CD3z(CO) (C2) ATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHC ESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQTTT PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLWVGGVLACYSLLVTVAFI IFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGGGRVKFSRSADAPAYQ QGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIG MKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 46)

[0038] huCD27L-IgG4(HL-CH3)-CD28tm-41BB-CD3z(CO) (C3) ATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHC ESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQES KYGPPCPPCPGGGSSGGGSGGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLS LGKFWVLWVGGVLACYSLLVTVAFIIFVWKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPE EEEGGCELGGGRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0039] (SEQ ID NO: 47)

[0040] huCD27L-CD28tm (M 2666-2668Del)-41BB-CD3z(CO) (C4) ATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHC ESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPT HLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRFWVLWVGGVLACYS LLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGGGRVKFSRS ADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMA EAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 48);

[0041] huCD27L-CD8h-CD8tm3-41BB-CD3z(CO) (C5) ATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHC ESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQTTT PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGGGRVKFSRSADAPAYQQGQ NQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKG ERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRLEGGGEGRGSLLTCGDVEENPGPR (SEQ ID NO: 49);Attorney Docket No.: 40056-0110WO1 / COH Ref.: TEC 25-002

[0042] huCD27L-IgG4(L235E,N297Q)-CD28tm-41BB-CD3z(CO) (C6) ATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHC ESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQES KYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFQSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREP QVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSR LTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKFWVLVVGGVLACYSLLVTVAFIIFVW VKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGGGRVKFSRSADAPAYQQG QNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMK GERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 50);

[0043] huCD70ScFv (vl-vh-14)-IgG4(HL-CH3)-CD28tm-41BB-CD3z(CO) (C7) QIQLVQSGPELKKPGETVKISCKASGYIFTDYSMHWVKQAPGKGLKWMGWINTETGESTYADD FKGRFAFSLETSASTAYLQINNLKNEDTATYFCAKSFYANYDWHFDVWGAGTTVTVSSGSTSG GGSGGGSGGGGSSQIVLTQSPAIMSASPGEKVTITCSATSSVSYMHWFQQKPGTSPKLWIYST SNLASGVPARFSGSGSGTSYSLTISRMEAEDAATYYCQQRTSFPPTFGGGTKLEIKESKYGPPC PPCPGGGSSGGGSGGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKMFWVLVVGGVLACYSLLVTVAFIIFVWKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEG GCELGGGRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNP QEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 51);

[0044] huCD27L-CD99tm-41BB-CD3z (C8) ATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHC ESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPT HLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRAPGVIPGIVGAWVAV AGAISSFIAKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGGGRVKFSRSAD APAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEA YSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 52);

[0045] huCD27L-CD4tm-41 BB-CD3z(CO) (C9) ATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHC ESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPT HLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRMALIVLGGVAGLLLFI GLGIFFKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGGGRVKFSRSADAPA YQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSE IGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 53); andAttorney Docket No.: 40056-0110WO1 / COH Ref.: TEC 25-002

[0046] huCD27L-CD8tm-41 BB-CD3z(CO) (C10) ATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHC ESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPT HLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRIYIWAPLAGTCGVLLL SLVITKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGGGRVKFSRSADAPAY QQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEI GMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 54).

[0047] Also described in a population of T cells harboring the nucleic acid molecule described herein. In various instances, T cell does not express CD70; T cell has been genetically modified to knock out the CD70 gene; and T cell does express CD70.

[0048] Described herein is method of treating a patient having glioblastoma, comprising administering a therapeutically effect amount of the population of T cells described here, e.g., by intraventricular administration.

[0049] Also described is a method of treating renal cell carcinoma, comprising administering a therapeutically effect amount of the population of T cells described herein

[0050] The methods of treatment can include comprising administering decitabine or azacytidine to the patient.

[0051] DESCRIPTION OF DRAWINGS

[0052] FIG. 1: Schematic depiction of certain CAR constructs.

[0053] FIG 2: Cleavable CAR T cells exhibit enhanced cytotoxicity against CD70-expressing kidney tumor cells. Cleavable CAR T cells (C1 and C4) demonstrated superior cytotoxicity compared to non-cleavable CAR T cells against the 786-0 kidney tumor cell line. The CD70 ko CD70-targeted CAR T cells were co-cultured with 786-0 cells at a 1:1 effector-to-target (E: T) ratio for 100 hours. Cytotoxicity was measured by real-time cell impedance.

[0054] FIG. 3: Safety assessment of CD70-targeting ligand-based CAR T cells. Non-cleavable ligand-based CAR T cells were assessed for potential off-target cytotoxicity. Neither cleavable nor non-cleavable ligand-based CAR T cells exhibited cytotoxicity against CD70 knockout 786-O tumor cel. The CD70 Ko CAR T cells were co-cultured with 786-0 cells at a 1:4 effector-to-target (E: T) ratio for 100 hours. Cytotoxicity was measured by real-time cell impedance using an xCELLigence system.Attorney Docket No.: 40056-0110WO1 / COH Ref.: TEC 25-002

[0055] FIG 4: C4 and C7 CD70-targeting CAR T cells exhibit potent and comparable in vitro killing kinetics in a tumor rechallenge assay. CD70-expressing 786-0 renal cell carcinoma cells were co-cultured with CD70-targeting CAR T cells (C2 and C7, CD70 knockout) from donor HD707 at a 1:1 effector-to-target (E: T) ratio. CAR T cells were repeatedly challenged with fresh tumor cells four times to assess their capacity for sustained cytotoxic activity. Tumor cell killing was assessed over time by measuring cell impedance using an xCELLigence system.

[0056] FIG 5: C4 and C7 CD70-targeting CAR T cells exhibit potent and comparable in vitro killing kinetics in a tumor rechallenge assay. CD70-expressing 786-0 renal cell carcinoma cells were co-cultured with CD70-targeting CAR T cells (C2 and C7, CD70 knockout) from donor HD704 at a 1:1 effector-to-target (E: T) ratio. CAR T cells were repeatedly challenged with fresh tumor cells four times to assess their capacity for sustained cytotoxic activity. Tumor cell killing was assessed over time by measuring cell impedance using an xCELLigence system.

[0057] FIG 6: C4 and C7 CD70-targeting CAR T cells (CD70 knockout) exhibit robust anti-tumor activity in vivo. To assess the therapeutic efficacy of CD70-targeting CAR T cells, 786-0 renal cell carcinoma cells (4e6 cells) were injected intraperitoneally (i.p.) into NSG mice.

[0058] Subsequently, CD70-targeting CAR T cells (2e6 cells) were administered i.p. Tumor progression was evaluated via bioluminescence imaging on days 7, 18, 30, and 66 post-CAR T cell infusion. Both C4 and C7 CAR T cells demonstrated potent anti-tumor activity, with C4 exhibiting a slightly slower onset of tumor control compared to C7 but achieving comparable overall efficacy.

[0059] FIG 7: Cleavable CARs (C1 and C4) demonstrate superior cytotoxicity compared to non-cleavable CARs against GBM cells in vitro. Cytotoxicity was measured using the xCELLigence system to assess the function of CD70 knockout (KO) CAR T cells. The effector-to-target (E: T) ratio was set at 1:8, with CD70 KO CAR T cells as effectors and WT GBM cells as targets. svFv is C7,

[0060] FIG 8: CD70-targeting CAR T cells other than C7 exhibit no off-target cytotoxicity against CD70 KO GBM. Cytotoxicity was assessed using the xCELLigence system. The effector-to-target (E: T) ratio was set at 1:1, with CD70 KO CAR T cells as effectors and CD70 KO GBM cells as targets.

[0061] FIG 9: C7 and C4 CAR T cells demonstrated the highest activation and killing potency. CD70-targeting CAR T cells were assessed for activation markers (CD69 and 4-1BB) and intracellular IFNy production after 4 hours, correlating with their cytotoxicity against CD70 WTAttorney Docket No.: 40056-0110WO1 / COH Ref.: TEC 25-002

[0062] GBM cells (PBT030). The experiment was conducted at a 1:1 effector-to-target (E: T) ratio, using CD70 KO CAR T cells as effectors and the wild-type GBM cell line (PBT030) as targets.

[0063] FIG 10: C7 and C4 CAR T cells exhibit the highest proliferative capacity in a cytotoxicity assay. CD70-targeting CAR T cell proliferation was assessed after a 5-day cytotoxicity assay conducted at an effector-to-target (E: T) ratio of 1:8. CD70 knockout (KO) CAR T cells were used as effectors against the wild-type GBM cell line (PBT030).

[0064] FIG 11: C7 CAR T Cells exhibit characteristics of fratricide-induced exhaustion. C7 CAR-expressing T cells exhibited reappearance of the CD70+ population in the CAR T cell monoculture, likely due to fratricide and subsequent exhaustion. CD70 WT CAR T cells were generated using CD3 / CD28 bead activation at a 2:1 cell-to-bead ratio and virus transduction (MOI=1).

[0065] FIG 12: Differential CART cell proliferation during manufacturing. Non-cleavable ligand CARs (C4 and C7) demonstrated better proliferation compared to cleavable and scFv-based CAR T cells during CAR T cell production, suggesting possibly reduced fratricide in the former. CAR T cells (CD70 wild type) were generated via CD3 / CD28 bead stimulation (1:1 bead-to-cell ratio).

[0066] FIG 13: Fratricide by scFv-based CAR T cells but reduced toxicity by ligand-based CAR T cells. The C7 (scFv-based) CAR T cells demonstrated significantly higher cytotoxicity against mock T cells (CD70+) compared to C1 and C4 (ligand-based) CAR T cells. Effector CD70-knockout CAR T cells were co-cultured with target mock T cells at a 1:2 effectontarget (E: T) ratio. CD107a degranulation and intracellular INFg were measured after 6 hours of co-culture, and secreted IFNy production was measured at 18 hours using ELISA.

[0067] FIG 14: CD70 Knockout Differentially Regulates the Phenotype of scFv CAR T Cells During CAR T Cell Production. A) CAR T cell proliferation during production (over one month) utilized TransAct for T cell activation. CD70+ population fluctuated during CAR T production possible because of fratricide. B) CD70 deficiency did not alter the T cell subset distribution in ligand-based CAR T cells, with both wild-type and knockout CAR T cells maintaining a predominantly stem cell / central memory (Tscm / Tcm) phenotype. Conversely, in scFv-based CAR T cells, CD70 knockout resulted in a shift from the more effector (Teff)-like phenotype observed in wild-type scFv-based CAR T cells-potentially due to fratricide- towards a more prominent Tscm / Tcm phenotype in the CD70-ko CAR T cells, likely by preventing fratricideAttorney Docket No.: 40056-0110WO1 / COH Ref.: TEC 25-002

[0068] FIG 15: CD70 expression on scFv-based CARs mediates fratricide, impacting CAR T cell expansion and CAR+ cell frequency. (A) CD70-knockout (KO) scFv-based CAR T cells (C7) exhibited superior proliferation compared to CD70-wildtype (WT) C7 CAR T cells, likely due to fratricide in the latter. Ligand-based CAR T cells (C4) showed the most robust expansion. CD70 disruption augmented scFv-based CAR T cell growth more than ligand-based CARs during manufacturing. (B) CAR T cell (CD19+ population) frequency was diminished in WT C7 CAR T cells due to fratricide; CD70 ko restored it. CAR+ cell frequency remained stable in both WT and KO ligand-based CAR T cells (C4), indicating minimal fratricide. CAR T cells were generated via TransAct activation and CD19+ population measured at the end of CAR T cell production (day 14).

[0069] FIG 16: Fratricide diminishes the cytotoxic potency of scFv-based CAR T cells. (A) CD70 knockout (KO) in scFv-based CAR T cells (C7) enhances their killing efficacy, whereas it has no discernible effect on ligand-based CAR T cells (C4). (B) CAR T cells do not exhibit non-specific killing against CD70-deficient tumor cells. Effector cells (WT or CD70 KO) were co-cultured with target cells (CD70+ and CD70 KO 786-0 renal cell carcinoma cell line) at an effector:target (E: T) ratio of 1:4, and target cell proliferation was monitored using real-time impedance-based cytotoxicity assays (xCELLigence).

[0070] FIG 17: Fratricide diminishes the cytotoxic potency specifically in scFv-based CAR T cells. CD70 knockout (KO) in scFv-based CAR T cells (C7) significantly enhances their cytotoxic efficacy, as measured by IFNy secretion, compared to ligand-based CAR T cells (C4). Effector cells (WT or CD70 KO) were co-cultured with target cells (CD70+ and CD70 KO 786-0 renal cell carcinoma cell line) at an effector: target (E: T) ratio of 2:1. IFNy levels were measured by ELISA after 21 hours of co-culture.

[0071] FIG 18: Schematic depiction of certain CAR constructs

[0072] FIG 19: TC9 and C7 CAR T cell exhibited highest cytotoxicity against GBM cells. (A) Realtime cell analysis of patient-derived GBM tumor cells (PBT030) co-cultured with CD70-targeting CAR T cells (CD70 ko) at an effector-to-target (E: T) ratio of 1:8. Tumor cell viability was monitored over time by cell index measurements in xCelligence assay.

[0073] FIG 20: C9 and C7 CAR T cells demonstrate enhanced degranulation upon co-culture with GBM cells. The CD70-targeting CAR T cells (CD70 ko) were co-cultured with GBM cells for 4 hours at effector-to-target (E: T) ratio of 1:1. The percentage of CD107a+ (degranulationAttorney Docket No.: 40056-0110WO1 / COH Ref.: TEC 25-002

[0074] marker) CAR T cells within the CD19+ population was assessed by flow cytometry. Statistical significance was determined using two-way ANOVA and is indicated as ** *P < 0.05, **P < 0.01, ***P < 0.001, ****p < 0.0001**. Error bars represent standard deviation of 4 replicates.

[0075] FIG 21: C7 and C9 CAR T cells showed enhanced activation upon co-culture with GBM cells. The CD70-targeting CAR T cells (CD70 ko) co-cultured with GBM cells for 18 hours at E=T = 1:1. The percentage of 4-1BB+, CD69+, and CD25+ CAR T cells within the CD19+ population was assessed by flow cytometry. Statistical significance was determined using Two way ANOVA and is indicated as ** *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001**. "ns" denotes not significant. Error bars represent standard deviation of four replicates.

[0076] FIG 22: C9 and C7 CAR T-cell exhibited show potent, specific cytotoxicity against RCC cells, sparing CD70 KO tumor cells. Real-time xCELLigence assay of 786-0 RCC tumor cells (WT and CD70 -ko) co-cultured with CD70-targeting CAR T cells (CD70 KO) at effector-to-target (E: T) ratios of 1:8 and 1:1. Tumor cell viability was monitored over time by cell index measurements.

[0077] FIG 23: C9 and C7 CAR T cells demonstrate enhanced degranulation upon co-culture with RCC cells. The CD70-targeting CAR T cells (CD70 ko) were co-cultured with RCC cell line (786_o) for 4 hours at effector-to-target (E: T) ratio of 1:1. The percentage of CD107a+ (degranulation marker) CAR T cells within the CD19+ population was assessed by flow cytometry. The secreted cytokines (IL2, INFg) measured by ELISA. Statistical significance was determined using two-way ANOVA and is indicated as ** *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001**. Error bars represent standard deviation of 4 replicates.

[0078] FIG 24: C7 and C9 CAR T cells showed enhanced activation upon co-culture with GBM cells. The CD70-targeting CAR T cells (CD70 ko) co-cultured with GBM cells for 18 hours at E=T = 1:1. The percentage of 4-1BB+, CD69+, and CD25+ CAR T cells within the CD19+ population was assessed by flow cytometry. Statistical significance was determined using Two way ANOVA and is indicated as ** *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001**. "ns" denotes not significant. Error bars represent standard deviation of four replicates.

[0079] FIG 25: Self-CAR activation drives T cell exhaustion, and differentiation. Exhaustion markers (TIGIT, TIM3, and LAG3) and sternness (CD62L / CD45RO) were analyzed during the CAR T cell manufacturing process using flow cytometry. Increased exhaustion and a more differentiated T cell phenotype were observed in CD70 wild-type (WT) CAR T cells compared toAttorney Docket No.: 40056-0110WO1 / COH Ref.: TEC 25-002

[0080] CD70 knockout (KO) CAR T cells, suggesting that self-activation contributes to exhaustion and differentiation in the potent CD70-targeting CAR T cells.

[0081] FIG 26: Both ligand-based and ScFv-based CARs demonstrated fratricide, with reduced fratricide observed in the ligand-based CAR. Fratricide was assessed by detecting CD107a degranulation, cytokine secretion (IL-2 and IFN-y) measured via ELISA, and the expression of activation markers (CD25, CD69, and 4-1BB) analyzed using flow cytometry. Statistical significance was determined using two-way ANOVA and is indicated as ** *P < 0.05, **P < 0.01, ***P < 0.001, ****p < 0.0001**. "ns" denotes not significant. Error bars represent standard deviation of four replicates.

[0082] FIG 27: CD70 knockout (KO) enhances the efficacy of the ligand-based CAR (C9) and ScFv-based CAR (C7) in GBM models, underscoring the necessity of CD70 KO in CD70-targeting CAR T cells. CD70 wild-type (WT) and CD70 KO CAR T cells were co-cultured with patient-derived GBM cell lines (PBT030) at E: T= 1:8 ratio, and cytotoxicity was evaluated using cell impedance analysis (xCELLigence assay).

[0083] FIG 28: CD70 knockout (KO) enhances the efficacy of the ligand-based CAR (C9) and ScFv-based CAR (C7) in RCC models. CD70 wild-type (WT) and CD70 KO CAR T cells were co-cultured with RCC cell lines (786_o) at E: T= 1:8 ratio, and cytotoxicity was evaluated using cell impedance analysis (xCELLigence assay).

[0084] FIG 29: Truncation or sequence modification in the intracellular domain of human CD70 impacts protein expression and its binding affinity to the CD27 ligand. Various human CD70 variants were transduced into Jurkat cells. A) The expression of CD70 variants was monitored over one month. Certain variants, such as truncated CD70 and double-flagged CD70, exhibited expression issues. B) The chimeric and flagged CD70 variants showed reduced binding affinity to CD27 (CD70’s ligand).

[0085] FIG. 30: Transmembrane (TM) optimization enhances the antitumor potency of ligandbased CD70 CAR T cells. (A) Representative brightfield images of RCC (786-0) and GBM (PBT030) tumor cells following co-culture with the indicated CAR T-cell products. RCC images were acquired on day 3 (E: T = 1:8), and GBM images were acquired on day 6 (E: T = 1:18). Conditions include tumor-only control, un-transduced T cells (UT), first-generation ligand CAR (C4), TM-optimized ligand CAR (C9), and scFv-based CAR (C7). Representative fields show enhanced tumor clearance with the TM-optimized C9 CAR compared to C4. (B) QuantificationAttorney Docket No.: 40056-0110WO1 / COH Ref.: TEC 25-002

[0086] of cytotoxicity against GBM at 100 hours across decreasing effector-to-target (E: T) ratios (1:2, 1:8, 1:16, and 1:32). TM optimization significantly improved the killing potency of the ligand CAR (C9 versus C4), enabling C9 CAR T cells to achieve cytotoxicity comparable to the scFv-based CAR (C7), particularly at lower E: T ratios. (C) Cytotoxicity against RCC at 200 hours across multiple E: T ratios. Consistent with GBM results, TM-optimized C9 CAR T cells demonstrate enhanced antitumor activity relative to C4 and maintain tumor killing comparable to the scFv-based CAR at reduced effector cell numbers. Data represent mean ± SEM of replicate wells.

[0087] FIG 31: C9 CAR-KO T Cells Demonstrate Superior Anti-Tumor Efficacy and Proliferative Capacity Over C4 and C7 Variants in RCC Models. (A) Real-Time Impedance-Based Cytotoxicity Assay (xCELLigence). RCC tumor cell growth and lysis were monitored via electrical impedance, expressed as Cell Index, at an Effector-to-Target (E: T) ratio of 1:8. Both C9 and C7 CAR-KO constructs achieved rapid and complete tumor clearance within 100 hours, whereas C4 CAR-KO exhibited significantly delayed and incomplete cytotoxicity compared to the Untransduced (UT-KO) control. (B-C) Expression of inhibitory receptors (B) LAG3 and (C) PD-1 was assessed in %CD19+T cells over 6 days. C7 CAR-KO cells showed the highest upregulation of both exhaustion markers by Day 6, suggesting that its potent initial cytotoxicity is coupled with a more rapid progression toward functional exhaustion compared to the C9 variant. (D-E) T Cell Proliferation and Persistence. C9 CAR-KO demonstrated the most robust CAR T cell expansion, peaking at Day 4, while C7 CAR-KO showed markedly lower proliferative capacity and persistence despite its high initial killing rate in the impedance assay.

[0088] FIG. 32: Ligand-based (C8) and scFv-based (C6) CD70 CAR T cells mediate efficient cytotoxicity against low-CD70-expressing tumors with distinct activation profiles. (A) Generation and characterization of CD70-modified tumor cells. The BGM cell line (PBT030) was first engineered to knock out CD70 and subsequently reconstituted to express CD70 at graded levels, including a low-expression population (MFI = 610), as determined by flow cytometry. (B) Tumor cell quantification following co-culture with CAR T cells at a 1:1 effector-to-target (E: T) ratio, demonstrating effective tumor clearance by both ligand-based (C4, C8) and scFv-based (C6) CART cells. (C) IFN-y secretion in co-culture supernatants measured after the cytotoxicity assay, reflecting functional activation of CART cells upon antigen engagement. (D) Frequency (%) of CD70-positive cells among residual tumor cells following co-culture. (E) Mean fluorescence intensity (MFI) of CD70 on residual tumor cells after CAR T-cell treatment. (F) Surface expression of the activation marker 4-1BB on CAR T cells following the killing assay.Attorney Docket No.: 40056-0110WO1 / COH Ref.: TEC 25-002

[0089] (G) Comparative functional analysis across increasing tumor antigen densities, showing that ligand-based CAR T cells achieve cytotoxicity comparable to scFv-based CARs while exhibiting reduced activation and more consistent persistence, even at higher CD70 expression levels.

[0090] FIG. 33: Fratricide reduces the cytotoxic potency of the CD70 CAR T cells. (A-B) Representative microscopic images of GFP+target cell lines (RCC and GBM) co-cultured with either CD70 wild-type (WT) or CD70 knockout (KO) CAR T cells (From Two different donor HD704 and HD707). (B) Baseline CD70 expression levels in 786-0 (RCC) and PBT030 (GBM) cell lines. (D-E) CD70 knockout (KO) in both ligand-based CAR T cells (C4 and C9) and scFv-based CAR T cells (C7) enhances cytotoxic activity in GBM models (D) and the RCC model (E). The greater difference in cytotoxic efficacy between WT and KO CAR T cells observed in the RCC model compared to GBM is likely due to the higher CD70 expression in RCC cells (panel B), which may promote increased CAR T-cell activation and subsequent fratricide.

[0091] Effector cells (WT or CD70 KO CAR T cells) were co-cultured with CD70+ target cells (786-0 renal cell carcinoma or PBT030 GBM cells) at an effector-to-target (E: T) ratio of 1:8. Target cell proliferation was monitored using a real-time impedance-based cytotoxicity assay (xCELLigence).

[0092] FIG. 34: CD70 CARs Exhibit Robust Trans-Binding and Cellular Aggregation Following CD70 Transduction. (A) Flow cytometric analysis of Jurkat cells 24 hours prior to CD70 transduction (DO) confirms high surface expression of IL13Ra2 (as a control CAR), C4, C9, and C7 CAR constructs (t CD19 marker). B) Confocal microscopy (LSM880) was used to visualize cellular aggregation 48 hours post-transduction with CD70 lenti virus. C4, C9, and C7 CAR variants show extensive trans-binding (T cell-T cell interaction) characterized by large multicellular clusters, whereas Mock and IL13 CAR controls maintain a disparate, nonaggregated morphology. Jurkat cells were transduced at DO, followed by a 6-hour incubation before dilution with media containing 100nM Dasatinib to modulate signaling. Half-media changes with 2x Dasatinib were performed at Day 1 and Day 2 to maintain concentrations without disturbing cell clusters prior to imaging.

[0093] FIG 35: CD70 Masking via CAR Cis-Binding. Cis-binding of CAR to its cognate antigen CD70 on the same cell surface results in ligand masking, a process demonstrated here in non-cytotoxic cell models (GBM and Jurkat cells). To isolate this binding mechanism from effector functions, Jurkat and GBM cell lines were engineered to express CD70 and subsequently transduced with truncated CAR constructs lacking the CD3z signaling domain. (A) FlowAttorney Docket No.: 40056-0110WO1 / COH Ref.: TEC 25-002

[0094] cytometric analysis confirms the successful engineering of Jurkat cells to stably express different level of CD70. (B) A Jurkat line with moderate CD70 expression (LS2) was selected for secondary transduction with truncated CARs. Surface expression of tEGFR serves as a surrogate marker to demonstrate successful CAR transduction. (C) Following CAR expression, surface staining for CD70 was significantly reduced or abolished, demonstrating that the truncated CAR effectively masks the CD70 ligand in cis. D-E) Similar to the Jurkat model, a CD70 knocked out GBM line (D) was engineered to express intermediate and high levels of CD70 and subsequently transduced with truncated CARs, as confirmed by tEGFR expression (E). (F) Comparison of ligand masking reveals that the ligand-based CAR (C9) results in partial CD70 masking, whereas the ScFv-based CAR (C7) achieves complete masking, suggesting higher cis-binding affinity or more efficient steric blocking by the ScFv format.

[0095] FIG 36: Comparative Activation and Species Cross-Reactivity of CD70-Targeting CAR T Cells. A-B) Flow cytometric evaluation of activation markers including 4-1BB (A) and CD25 (B) on T cells 72 hours post-stimulation. Cells were cultured on plates coated with human or mouse recombinant CD70 at 0.75 or 5 ug / mL. While the ligand-based CARs (C4 and C9) exhibit comparable activation and significant cross-reactivity between human and mouse CD70, the scFv-based C7 CAR demonstrates potent, human-specific activation with negligible reactivity toward the mouse ortholog. C) Representative histograms showing CFSE dilution in CAR T cells 72 hours post-co-culture. The scFv-based C7 CAR displays a robust, dose-dependent proliferative shift exclusively in response to human (H) CD70. In contrast, the ligand-based CARs (C4 and C9) maintain a cross-reactive profile, showing similar CFSE dilution patterns when exposed to either human or mouse (M) CD70, albeit with lower overall proliferative capacity compared to the C7 / human CD70 interaction.

[0096] FIG 37: Heterogeneous CD70 Expression in GBM and Its Impact on CAR T Cell Efficacy.

[0097] A) Representative Immunohistochemical (IHC) micrographs of patient-derived glioblastoma (GBM) samples and healthy brain tissue. GBM specimens exhibit significant inter-patient heterogeneity, ranging from high CD70 immunoreactivity to low / absent expression (middle). Conversely, healthy brain tissue shows no detectable CD70 expression, suggesting a favorable therapeutic target for GBM model. B) Linear regression analysis correlating residual tumor cell count with CD70 Mean Fluorescence Intensity (MFI). All tested constructs (C4, C9, and C7 CAR-KO) demonstrate a strong inverse correlation between antigen density and tumor survival.Attorney Docket No.: 40056-0110WO1 / COH Ref.: TEC 25-002

[0098] These data conclude that CD70 expression levels directly impact CAR T cell efficacy, where higher MFI values result in significantly enhanced tumor clearance across all CAR variants.

[0099] FIG 38: Decitabine (DAC) induces higher CD70 expression than Azacytidine (AZA) in patient-derived glioblastoma (GBM) tumor cell lines without compromising cell viability.

[0100] DAC-induced CD70 expression increased with increasing concentration, peaking at 4 hours post-treatment. DAC and AZA were freshly prepared before each assay by dissolving in DMSO and added to patient-derived GBM tumor cell lines (pbt030 and pbt206) and T cells (from two different donors). Cells were treated with the drugs for either two or four consecutive days. Cell viability and CD70 expression were assessed at 24 and 48 hours post-treatment.

[0101] FIG 39: Sustained CD70 induction by decitabine (DAC) in some GBM Cells. A) PBT030 cells treated with DAC exhibited sustained elevated CD70 expression from Day 2 through Day 19 post-treatment. PBT206 cells showed transient CD70 induction peaking on Day 6, but with markedly lower levels compared to PBT030. CD70 did not induced in T cells with DAC. B) representative FACS data of DAC-induced CD70. Patient-derived Glioblastoma (GBM) cells (PBT030 and PBT206) and T cells (two donors) were treated with FDA-approved DAC concentration (0.64 μM) DAC or DMSO as a control for 4 constitutive days (after washing out from the last day of treatment). CD70 expression and viability were assessed at different time point from 6 hours to day 19thpost last day of treatment. The experiment was performed with three technical replicates (n=3).

[0102] FIG. 40: DAC-induced CD70 expression persists for 4 days in vivo in a GBM model. GBM tumor-bearing NSG mice were treated with the FDA-approved dose of decitabine (DAC; 6.62 mg / kg), and CD70 expression levels were assessed at 24 and 48 hours post-treatment. DAC treatment significantly increased CD70 expression and mean fluorescence intensity (MFI), with the elevated levels maintained for up to 4 days following treatment.

[0103] FIG. 41: Decitabine (DAC) Enhances CD70 Expression and Sensitizes GBM to CAR T Cell Cytotoxicity. A) Flow cytometric analysis of Renal Cell Carcinoma (RCC) and Glioblastoma (GBM) cell lines 4 days post-treatment with a constitutive FDA-equivalent dose of DAC (0,65uM). Histograms and demonstrate a significant increase in CD70 Mean Fluorescence Intensity (MFI) in both RCC (from 22,478 to 29,194) and GBM (from 6,270 to 12,098) cells, while maintaining high cell viability. B) Cytotoxicity assay (140h) measuring the Cell Index of PBT030 GBM cells treated with CAR T cells (C4, C9, or C7) in the presence or absence ofAttorney Docket No.: 40056-0110WO1 / COH Ref.: TEC 25-002

[0104] DAC. While DAC alone reduces tumor cell index, the combination therapy significantly improves tumor clearance. Notably, the C4 CAR, which exhibits lower baseline potency, shows the most marked improvement in killing efficacy when antigen density is increased via DAC. In contrast, the more potent C9 and C7 CARs achieve near-complete tumor eradication, concluding that pharmacologically increasing antigen density via DAC is a viable strategy to enhance the killing potency of the CAR T cell constructs specifically in a heterogeneous Tumor model (GBM).

[0105] DETAILED DESCRIPTION

[0106] I. Chimeric Antigen Receptors

[0107] A chimeric antigen receptor (CAR) refers to an artificial immune cell receptor that is engineered to recognize and bind to a surface antigen. AT cell that expresses a CAR polypeptide is referred to as a CAR T cell. CAR have the ability to redirect T-cell specificity and reactivity toward a selected target in a non-MHC-restricted manner. The non-MHC-restricted antigen recognition gives CAR T cells the ability to recognize an antigen independent of antigen processing, thereby bypassing a major mechanism of tumor escape. A CAR can also be expressed by other immune effector cells, including but not limited to natural killer CAR (“NK CAR”) and directed NK cell killing to cells expressing the target of the CAR.

[0108] There are various generations of CARs, each of which contains different components. First generation CARs join a target binding domain, generally an antibody-derived scFv, to the CD3 intracellular signaling domain of the T cell receptor through a spacer region (also called a hinge domain) and a transmembrane domain. Second generation CARs incorporate a co-stimulatory domain (e.g., CD28, 4-BB, or ICOS) to supply a co-stimulatory signal. Some CAR contain two co-stimulatory domains (e.g., a combination of CD27, CD28, 4-1BB, ICOS, or 0X40) fused with the TCR CD3 chain.

[0109] There can be a spacer between the co-stimulatory domain and the CD3 domain, but this is optional. A CAR is often fused to a signal peptide at the N-terminus for surface expression. In some cases, the CAR can be co-expressed with a polypeptide that can serve as marker, for example a truncated EGFR receptor lacking signaling function or a truncated CD19 receptor lacking signaling function.

[0110] The following CD70 targeted CAR were used in the examples:Attorney Docket No.: 40056-0110WO1 / COH Ref.: TEC 25-002

[0111] huCD27L-CD27tm-41BB-CD3z (C1) ATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHC ESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPT HLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVFTLAGA LFLHKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGGGRVKFSRSADAPAYQ QGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIG MKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRLEGGGEGRGSLLTCGDVEENPGPR

[0112] (SEQ ID NO: 45);

[0113] huCD27L-CD8h3 (AKP 2516-2524 Del)-CD28tm (M2660-2662 Del)-41BB-CD3z(CO) (C2) ATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHC ESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQTTT PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLWVGGVLACYSLLVTVAFI IFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGGGRVKFSRSADAPAYQ QGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIG MKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 46)

[0114] huCD27L-IgG4(HL-CH3)-CD28tm-41BB-CD3z(CO) (C3) ATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHC ESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQES KYGPPCPPCPGGGSSGGGSGGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLS LGKFWVLWVGGVLACYSLLVTVAFIIFVWKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPE EEEGGCELGGGRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0115] (SEQ ID NO: 47)

[0116] huCD27L-CD28tm (M 2666-2668Del)-41BB-CD3z(CO) (C4) ATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHC ESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPT HLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRFWVLWVGGVLACYS LLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGGGRVKFSRS ADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMA EAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 48);

[0117] huCD27L-CD8h-CD8tm3-41BB-CD3z(CO) (C5) ATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHC ESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQTTT PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCAttorney Docket No.: 40056-0110WO1 / COH Ref.: TEC 25-002

[0118] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGGGRVKFSRSADAPAYQQGQ NQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKG ERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRLEGGGEGRGSLLTCGDVEENPGPR (SEQ ID NO: 49);

[0119] huCD27L-IgG4(L235E,N297Q)-CD28tm-41BB-CD3z(CO) (C6) ATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHC ESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQES KYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFQSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREP QVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSR LTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKFWVLVVGGVLACYSLLVTVAFIIFVW VKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGGGRVKFSRSADAPAYQQG QNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMK GERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 50);

[0120] huCD70ScFv (vl-vh-14)-IgG4(HL-CH3)-CD28tm-41BB-CD3z(CO) (C7) QIQLVQSGPELKKPGETVKISCKASGYIFTDYSMHWVKQAPGKGLKWMGWINTETGESTYADD FKGRFAFSLETSASTAYLQINNLKNEDTATYFCAKSFYANYDWHFDVWGAGTTVTVSSGSTSG GGSGGGSGGGGSSQIVLTQSPAIMSASPGEKVTITCSATSSVSYMHWFQQKPGTSPKLWIYST SNLASGVPARFSGSGSGTSYSLTISRMEAEDAATYYCQQRTSFPPTFGGGTKLEIKESKYGPPC PPCPGGGSSGGGSGGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKMFWVLVVGGVLACYSLLVTVAFIIFVWKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEG GCELGGGRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNP QEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 51);

[0121] huCD27L-CD99tm-41BB-CD3z (C8) ATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHC ESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPT HLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRAPGVIPGIVGAWVAV AGAISSFIAKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGGGRVKFSRSAD APAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEA YSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 52);

[0122] huCD27L-CD4tm-41 BB-CD3z(CO) (C9) ATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHC ESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPT HLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRMALIVLGGVAGLLLFI

[0123]

[0124] Attorney Docket No.: 40056-0110WO1 / COH Ref.: TEC 25-002

[0125] GLGIFFKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGGGRVKFSRSADAPA YQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSE IGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 53); and

[0126] huCD27L-CD8tm-41 BB-CD3z(CO) (C10) ATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHC ESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPT HLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRIYIWAPLAGTCGVLLL SLVITKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGGGRVKFSRSADAPAY QQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEI GMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 54).

[0127] C9 and C9 Variants

[0128] C9 includes a CD27 domain (underline) that binds CD70, followed by a CD27 hinge domain (no underline), a CD4 transmembrane domain (double underline). 41-BB co-stimulatory domain (bold), GGG linker (heavy underline), and a CD3 domain (italics).

[0129] ATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHC ESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPT HLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRMALIVLGGVAGLLLFI GLGIFFKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGGGRVKFSRSADAP AYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYS EIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 53).

[0130] C9 variants include the following:

[0131] C9A, which lacks the GGG linker:

[0132] ATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHC ESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPT HLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRMALIVLGGVAGLLLFI GLGIFFKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQ GQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGM KGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 56);

[0133] C9B, which has a CD8 TM (CD8tm (SEQ ID NO: 18) double underline):

[0134] ATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHC ESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPT HLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRIYIWAPLAGTCGVLLL SLVITKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGGGRVKFSRSADAPAY QQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEI GMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 57);Attorney Docket No.: 40056-0110WO1 / COH Ref.: TEC 25-002

[0135] C9C, which has a variant CD8 TM (CD8tm2 (SEQ ID NO: 19) double underline):

[0136] ATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHC ESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPT HLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRIYIWAPLAGTCGVLLL SLVITLYKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGGGRWCFSRS D P AYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYS EIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 58);

[0137] C9D, which has a variant CD8 TM (CD8tm3 (SEQ ID NO: 20 or 38) double underline) ATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHC ESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPT HLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRIYIWAPLAGTCGVLLL SLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGGGRWCFSRS D PAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAY SEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 59);

[0138] C9E, which has a CD28 TM (SEQ ID NO: 15) double underline):

[0139] ATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHC ESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPT HLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRMALIVLGGVAGLLLFI GLGIFFKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGGGF? WCFSRS D P AYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYS EIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 60);

[0140] C9F, which has a variant CD28 TM (CD28(M) SEQ ID NO: 16) double underline):

[0141] ATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHC ESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPT HLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIR

[0142]

[0143] MFWVLWVGGVLACY SLLVTVAFIIFVWKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGGGRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDK MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 61);

[0144] C9G, which has a variant CD27 TM (SEQ ID NO: 37) double underline):

[0145] ATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHC ESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPT HLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVFTLAGA LFLHKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGGGRWCFSRS D P Y QQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEI GMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 62);

[0146] C9H, which has a CD8 TM (CD8tm (SEQ ID NO: 18) double underline) and no GGG linker: ATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHC ESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPT HLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRIYIWAPLAGTCGVLLL SLVITKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRWCFSRS D P YQQGAttorney Docket No.: 40056-0110WO1 / COH Ref.: TEC 25-002

[0147] QNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMK GERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 63).

[0148] C9I, which has a variant CD8 TM (CD8tm2 (SEQ ID NO: 19) double underline) and no GGG linker:

[0149] ATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHC ESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPT HLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRIYIWAPLAGTCGVLLL SLVITLYKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRWCFSRS D P YQ QGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIG MKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 64);

[0150] C9J, which has a variant CD8 TM (CD8tm3 (SEQ ID NO: 20 or 38) double underline) and no GGG linker:

[0151] ATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHC ESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPT HLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRIYIWAPLAGTCGVLLL SLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRWCFSRS D P Y QQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEI GMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 65);

[0152] C9K, which has a CD28 TM (SEQ ID NO: 15) double underline) and no GGG linker.

[0153] ATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHC ESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPT HLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRMALIVLGGVAGLLLFI GLGIFFKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRWCFSRS D P YQQ GQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGM KGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 66);

[0154] C9L, which has a variant CD28 TM (CD28(M) SEQ ID NO: 16) double underline) and no GGG linker:

[0155] ATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHC ESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPT HLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRMFWVLWVGGVLACY SLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEA YSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 67); and C9M, which has a variant CD27 TM (SEQ ID NO: 37) double underline) and no GGG linker: ATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHC ESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPT HLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVFTLAGA LFLHKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRWCFSRS D P YQQG QNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMK GERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 68).Attorney Docket No.: 40056-0110WO1 / COH Ref.: TEC 25-002

[0156] (a) Extracellular Binding Domain

[0157] Certain of the CD70 targeted CAR used an extracellular portion of mature human CD27. This portion includes a CD70 binding domain (which acts as the targeting domain) and a hinge, the latter of which can include a cleavage site. For the sake of convenience CAR that include this hinge are referred to a cleavable CAR.

[0158] CD27 - CD70 Binding Domain (huCD27L) ATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHH TRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSS QALSPHPQ (SEQ ID NO: 43)

[0159] scFv- CD70 Binding Domain (41D12) QIQLVQSGPELKKPGETVKISCKASGYIFTDYSMHVWKQAPGKGLKWMGWINTETGES TYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCAKSFYANYDWHFDVWGAGTTV TVSSGSTSGGGSGGGSGGGGSSQIVLTQSPAIMSASPGEKVTITCSATSSVSYMHWFQ QKPGTSPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISRMEAEDAATYYCQQRTSFPP TFGGGTKLEIK (SEQ ID NO: 44)

[0160] (b) Spacer Domain

[0161] The CAR or polypeptide described herein can include a spacer domain located between the targeting domain and the transmembrane domain. The spacer region can function to provide flexibility to the CAR, or domains thereof, or to prevent steric hindrance of the CAR, or domains thereof. A variety of different spacers can be used. Some of them include at least portion of a human Fc region, for example a hinge portion of a human Fc region or a CH3 domain or variants thereof. Table 1 below provides various spacer domains that can be used in the CARs described herein.

[0162] Table 1: Examples of Spacer Domains

[0163] Name Length Sequence

[0164] a3 3 aa AAA

[0165] linker 10 aa GGGSSGGGSG (SEQ ID NO: 1)

[0166] lgG4 hinge (S228P) 12 aa ESKYGPPCPPCP (SEQ ID NO: 2)

[0167]

[0168] Attorney Docket No.: 40056-0110WO1 / COH Ref.: TEC 25-002

[0169] lgG4 hinge 12 aa ESKYGPPCPSCP (SEQ ID NO: 3)

[0170] lgG4 hinge (S228P) + 22 aa ESKYGPPCPPCPGGGSSGGGSG (SEQ ID NO: 4) linker

[0171] lgG4 hinge (S228P) + 22 aa ESKYGPPCPSCPGGGSSGGGSG (SEQ ID NO: 5) linker

[0172] CD28 hinge 39 aa IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 6)

[0173] CD8 hinge-48aa 48 aa AKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVH TRGLDFACD (SEQ ID NO: 7)

[0174] CD8 hinge-45aa 45aa TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRG LDFACD (SEQ ID NO: 8)

[0175] lgG4(HL-CH3) 129 aa ESKYGPPCPPCPGGGSSGGGSGGQPREPQVYTLPPS QEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY

[0176] Also called lgG4(HL- KTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVM ACH2) HEALHNHYTQKSLSLSLGK (SEQ ID NO: 9)

[0177] (includes S228P in

[0178] hinge)

[0179] lgG4(HL-CH3) 129 aa ESKYGPPCPSCPGGGSSGGGSGGQPREPQVYTLPPS QEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY

[0180] Also called lgG4(HL- KTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVM ACH2) HEALHNHYTQKSLSLSLGK (SEQ ID NO:10)

[0181] (includes S228P in

[0182] hinge)

[0183] lgG4(L235E, N297Q) 229 aa ESKYGPPCPSCPAPEFEGGPSVFLFPPKPKDTLMISRT PEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPRE EQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPS SIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLS LGK (SEQ ID NO: 11)

[0184] lgG4(S228P, 229 aa ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRT L235E, N297Q) PEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPRE EQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPS SIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLS LGK (SEQ ID NO: 12)

[0185] lgG4(CH3) 107 aa GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKS

[0186] Also called lgG4(ACH2)

[0187]

[0188] Attorney Docket No.: 40056-0110WO1 / COH Ref.: TEC 25-002

[0189] RWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 13)

[0190] CD27 hinge PTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHW PPQRSLCSSDFIR (SEQ ID NO: 36)

[0191] Cleavable

[0192]

[0193] Some spacer domains include all or part of an immunoglobulin (e.g., lgG1, lgG2, lgG3, lgG4) hinge region, i.e., the sequence that falls between the CH1 and CH2 domains of an immunoglobulin, e.g., an lgG4 Fc hinge or a CD8 hinge. Some spacer domains include an immunoglobulin CH3 domain (called CH3 or ACH2) or both a CH3 domain and a CH2 domain. The immunoglobulin derived sequences can include one or more amino acid modifications, for example, 1, 2, 3, 4 or 5 substitutions, e.g., substitutions that reduce off-target binding.

[0194] In some cases, the spacer has 1, 2, 3, 4, or 5 single amino acid changes (e.g., conservative changes) compared to any of SEQ ID NOs 1-13. In some cases, the lgG4 Fc hinge / linker region that is mutated at two positions (L235E; N297Q) in a manner that reduces binding by Fc receptors (FcRs).

[0195] (c) Transmembrane Domain

[0196] The CAR disclosed herein can contain a transmembrane domain, which can be a hydrophobic alpha helix that spans the membrane. As used herein, a transmembrane domain refers to any protein structure that is thermodynamically stable in a cell membrane, preferably a eukaryotic cell membrane.

[0197] Useful transmembrane domains include a CD4 transmembrane domain having the sequence: MALIVLGGVAGLLLFIGLGIFF (SEQ ID NO: 17) and a CD28 TM having the sequence FVWLVVVGGVLACYSLLVTVAFIIFVW (SEQ ID NO: 15) or MFWVLWVGGVLACYSLLVTVAFIIFVW (SEQ ID NO:16). Other transmembrane domains can be used including those shown in Table 2.

[0198] Table 2: Examples of Transmembrane Domains

[0199] Name Accession Length Sequence

[0200] CD3z J04132.1 21 aa LCYLLDGILFIYGVILTALFL (SEQ ID NO: 14)

[0201]

[0202] Attorney Docket No.: 40056-0110WO1 / COH Ref.: TEC 25-002

[0203] CD28 NM_006139 27aa FWVLVWGGVLACYSLLVTVAFIIFWV (SEQ ID

[0204] NO: 15)

[0205] CD28(M) NM_006139 28aa MFVWLVWGGVLACYSLLVTVAFIIFVW (SEQ ID NO: 16)

[0206] CD4 M35160 22aa MALIVLGGVAGLLLFIGLGIFF (SEQ ID NO: 17) CD8tm NM_001768 21 aa IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 18) CD8tm2 NM_001768 23aa IYIWAPLAGTCGVLLLSLVITLY (SEQ ID NO: 19) CD8tm3 NM_001768 24aa IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 20) 4-1BB NM_001561 27aa IISFFLALTSTALLFLLFF LTLRFSW (SEQ ID NO:

[0207] 21)

[0208] NKG2D NM_007360 21 aa PFFFCCFIAVAMGIRFIIMVA (SEQ ID NO: 22)

[0209] CD27 ILVIFSGMFLVFTLAGALFLH (SEQ ID NO: 37)

[0210] CD8-LYC IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 38) CD99 APGVIPGIVGAWVAVAGAISSFIA (SEQ ID NO: 39)

[0211]

[0212] (d) Intracellular Signaling Domains

[0213] Any of the CAR constructs described herein contain one or more intracellular signaling domains (e.g., CD3, and optionally one or more co-stimulatory domains), which are the functional end of the receptor. Following antigen recognition, receptors cluster and a signal is transmitted to the cell.

[0214] CD3 is the cytoplasmic signaling domain of the T cell receptor complex. CD3 contains three immunoreceptor tyrosine-based activation motifs (ITAMs), which transmit an activation signal to the T cell after the T cell is engaged with a cognate antigen. In some cases, CD3 provides a primary T cell activation signal but not a fully competent activation signal, which requires a costimulatory signal.

[0215] Accordingly, the CAR polypeptides disclosed herein may further comprise one or more costimulatory signaling domains in addition to CD3. For example, the co-stimulatory domain CD28 and / or 4-1BB can be used to transmit a proliferative / survival signal together with the primary signaling mediated by CD3.Attorney Docket No.: 40056-0110WO1 / COH Ref.: TEC 25-002

[0216] The co-stimulatory domain(s) are located between the transmembrane domain and the CD3 signaling domain. Table 3 includes examples of suitable co-stimulatory domains together with the sequence of the CD3 signaling domain.

[0217] Table 3: CD3 Domain and Examples of Co-stimulatory Domains

[0218] Name Accession Length Sequence

[0219] CD3C J04132.1 113 aa RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVL DKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAE AYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALH MQALPPR (SEQ ID NO: 23)

[0220] ITAMS 1-3 underlined

[0221] CD3C 113 aa RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVL DKRRGRDPEMGGKPRRKNPQEGLFNELQKDKMAEA

[0222] Variant FSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHM 1xx QALPPR (SEQ ID NO: 24)

[0223] CD3C 113 aa RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVL DKRRGRDPEMGGKPRRKNPQEGLFNELQKDKMAEA variant FSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM QALPPR (SEQ ID NO: 25)

[0224] CD3C 113 aa RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVL DKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAE

[0225] variant AYSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALH MQALPPR (SEQ ID NO: 26)

[0226] CD3C 113 aa RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVL DKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAE

[0227] variant AFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALH MQALPPR (SEQ ID NO: 27)

[0228] CD3C 113 aa RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVL DKRRGRDPEMGGKPRRKNPQEGLFNELQKDKMAEA variant YSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHM QALPPR (SEQ ID NO: 28)

[0229] CD3C 113 aa RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVL DKRRGRDPEMGGKPRRKNPQEGLFNELQKDKMAEA variant FSEIGMKGERRRGKGHDGLYQGLSTATKDTFDALHM QALPPR (SEQ ID NO:29)

[0230] CD3C 113 aa RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVL DKRRGRDPEMGGKPRRKNPQEGLFNELQKDKMAEA variant FSEIGMKGERRRGKGHDGLFQGLSTATKDTYDALHM QALPPR (SEQ ID NO:30)

[0231]

[0232] Attorney Docket No.: 40056-0110WO1 / COH Ref.: TEC 25-002

[0233] CD28 NM_006139 42aa RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRD FAAYRS (SEQ ID NO: 31)

[0234] CD28gg* NM_006139 42aa RSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRD FAAYRS (SEQ ID NO: 32)

[0235] 41BB NM_001561 42 aa KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEE EGGCEL (SEQ ID NO: 33)

[0236] 0X40 NM_003327 42 aa ALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAH STLAKI (SEQ ID NO:34)

[0237] 2B4 NM_016382 120 aa WRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQE QTFPGGGSTIYSMIQSQSSAPTSQEPAYTLYSLIQPSR KSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLSRK ELENFDVYS (SEQ ID NO: 35)

[0238]

[0239] The CD3 signaling domain can include an amino acid sequence that is at least 90%, at least 95%, at least 98% identical to SEQ ID NO: 23. In such instances, the CD3 signaling domain has 1, 2, 3, 4, or 5 amino acid changes (preferably conservative substitutions) compared to SEQ ID NO: 23. In other examples, the CD3 signaling domain is SEQ ID NO: 23.

[0240] For the CAR described herein, a variant CD3 (1xx) comprising SEQ ID NO: 24 is a preferred alternative CD3 domain. For the CAR described herein having a 41 BB costimulatory domain, a CD28 co-stimulatory domain (SEQ ID NO: 31 or 32) is preferred alternative co-stimulatory domain.

[0241] The CAR described herein can include a co-stimulatory domain depicted in Table 3 or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications, a CD28 co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications, a 4-1BB co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications and an 0X40 co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications. In certain embodiments, a 4-1BB co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications is present in the CAR polypeptides described herein.

[0242] A CAR can include two co-stimulatory domains, for example, a CD28 co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions) and a 4-1BB co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions). The 1-5 (e.g., 1 or 2) amino acid modifications can be substitutions. The co-stimulatory domain can be amino terminal to the CD3 signaling domain and a short linkerAttorney Docket No.: 40056-0110WO1 / COH Ref.: TEC 25-002

[0243] consisting of 2 - 10, e.g., 3 amino acids (e.g., GGG) can be positioned between the costimulatory domain and the CD3 signaling domain.

[0244] The CAR can be produced using a vector in which the CAR open reading frame is followed by a T2A ribosome skip sequence and a truncated EGFR (EGFRt), which lacks the cytoplasmic signaling tail, ora truncated CD19R (also called CD19t). In this arrangement, co-expression of EGFRt or CD19t provides an inert, non-immunogenic surface marker that allows for accurate measurement of gene modified cells, and enables positive selection of gene-modified cells, as well as efficient cell tracking of the therapeutic T cells in vivo following adoptive transfer.

[0245] Efficiently controlling proliferation to avoid cytokine storm and off-target toxicity is an important hurdle for the success of T cell immunotherapy. The EGFRt or the CD19t incorporated in the CAR lentiviral vector can act as suicide gene to ablate the CAR+ T cells in cases of treatment-related toxicity.

[0246] The CD3 signaling domain can be followed by a ribosomal skip sequence (e.g., LEGGGEGRGSLLTCGDVEENPGPR; SEQ ID NO: 40) and a truncated EGFR having a sequence that is at least 90%, at least 95%, at least 98% identical to or identical to:

[0247] LVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFT HTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAWSLNITSLG LRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEG CWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGP DNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPT NGPKIPSIATGMVGALLLLLWALGIGLFM (SEQ ID NO: 41). In some cases, the truncated EGFR has 1, 2, 3, 4 of 5 amino acid changes (preferably conservative) compared to SEQ ID NO: 41.

[0248] Alternatively the CD3 signaling domain can be followed by a T2A ribosomal skip sequence (e.g., LEGGGEGRGSLLTCGDVEENPGPR; SEQ ID NO: 40) and a truncated CD19 receptor (also called CD19t) having a sequence that is at least 90%, at least 95%, at least 98% identical to or identical to:

[0249] MPPPRLLFFLLFLTPMEVRPEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPFLKL SLGLPGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNV SDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCVPPRDSLNQSLSQDL TMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLLLPRAttorney Docket No.: 40056-0110WO1 / COH Ref.: TEC 25-002

[0250] ATAQDAGKYYCHRGNLTMSFHLEITARPVLWHWLLRTGGWKVSAVTLAYLIFCLCSLVGILHLQR ALVLRRKR (SEQ ID NO: 42).

[0251] The CAR described herein can be produced by any means known in the art, though preferably it is produced using recombinant DNA techniques. Nucleic acids encoding the several regions of the chimeric receptor can be prepared and assembled into a complete coding sequence by standard techniques of molecular cloning known in the art (genomic library screening, overlapping PCR, primer-assisted ligation, site-directed mutagenesis, etc.) as is convenient. The resulting coding region is preferably inserted into an expression vector and used to transform a suitable expression host cell line, preferably a T lymphocyte, and most preferably an autologous T lymphocyte.

[0252] Various T cell subsets isolated from the patient can be transduced with a vector for CAR or polypeptide expression. Central memory T cells are one useful T cell subset. Central memory T cell can be isolated from peripheral blood mononuclear cells (PBMC) by selecting for CD45RO+ / CD62L+ cells, using, for example, the CliniMACS® device to immunomagnetically select cells expressing the desired receptors. The cells enriched for central memory T cells can be activated with anti-CD3 / CD28, transduced with, for example, a lentiviral vector that directs the expression of a CAR or as well as a non-immunogenic surface marker for in vivo detection, ablation, and potential ex vivo selection. The activated / genetically modified central memory T cells can be expanded in vitro with IL-2 / IL-15 and then cryopreserved. Additional methods of preparing CART cells can be found in PCT / US2016 / 043392.

[0253] Methods for preparing useful T cell populations are described in, for example, WO 2017 / 015490 and WO 2018 / 102761. In some cases, it may be useful to use natural killer (NK) cells, e.g., allogenic NK cells derived from peripheral blood or cord blood. In other cases, NK cells can be derived from human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs).

[0254] In some embodiments, described herein is a composition comprising the iPSC-derived CAR T cells or CAR NK cells. In some embodiments, a composition comprising iPSC-derived CAR T cells or CAR NK cells has enhanced therapeutic properties. In some embodiments, the iPSC-derived CAR T cells or CAR NK cells demonstrate enhanced functional activity including potent cytokine production, cytotoxicity and cytostatic inhibition of tumor growth, e.g., as activity that reduces the amount of tumor load.Attorney Docket No.: 40056-0110WO1 / COH Ref.: TEC 25-002

[0255] The CAR can be transiently expressed in a T cell population by an mRNA encoding the CAR. The mRNA can be introduced into the T cells by electroporation (Wiesinger et al. 2019 Cancers (Basel) 11:1198).

[0256] Useful cell types for expression of the CAR described herein include helper T cells, cytotoxic T cells, memory T cells, naive T cells, regulatory T cells, natural killer T cells and combinations thereof.

[0257] II. Treatment of Patients

[0258] (a) Subjects

[0259] The CD70 targeted CAR T cells described herein can be used to treat patients suffering from a solid tumor, for example, glioblastoma or renal cell carcinoma, low-grade glioma, renal cancers, ovarian cancer, myeloid leukemia, EGFR mutant tumors (e.g., EGFR mutant tumors with acquired tyrosine kinase inhibitor resistance.

[0260] (b) Administration

[0261] An effective amount of a therapy (e.g., immune cells expressing a CAR described herein) can be administered to a subject (e.g., a human) in need of the treatment via any suitable route (e.g., administered locally or systemically to a subject). Suitable modes of administration include injection, infusion, instillation, or ingestion. Injection includes, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intraventricular, intradermal, intraperitoneal, subcutaneous injection, and infusion.

[0262] An effective amount refers to the amount of each active agent required to confer therapeutic effect on the subject, either alone or in combination with one or more other active agents.

[0263] Effective amounts vary, as recognized by those skilled in the art, depending on the condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size, gender and weight, the duration of treatment, the nature of concurrent therapy, if any, the specific route of administration and like factors. A therapeutic effect on the subject can be determined as the amelioration of one symptom associated with the condition being treated or a reduction of size or number of cancer cells in the subject (e.g., 1%, 2%, 3%, 4%, or 5% decrease in size or number of cancer cells).Attorney Docket No.: 40056-0110WO1 / COH Ref.: TEC 25-002

[0264] EXAMPLES

[0265] Example 1: CD70 Targeted CAR

[0266] A variety of CD70 targeted CAR were created. Each was expressed by a lentiviral vector. In each case, the mature CAR sequence was preceded by a signal peptide (MLLLVTSLLLCELPHPAFLLIP; SEQ ID NO: 55) and followed by a truncated CD19 receptor (SEQ ID NO: 42) that was joined to the CAR sequence by a T2A skip sequence (SEQ ID NO: 40). Each had a 41 BB co-stimulatory domain (SEQ ID NO: 33) and a CD3 signaling domain (SEQ ID NO: 23), which were separated by a GGG linker.

[0267] Certain of the CD70 targeted CAR used an extracellular portion of mature human CD27. This portion includes a CD70 binding domain (which acts as the targeting domain) and a hinge, the latter of which includes a cleavage site. For the sake of convenience CAR that include this hinge are referred to a cleavable CAR.

[0268] CD27 - CD70 Binding Domain (huCD27L) ATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHH TRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSS QALSPHPQ (SEQ ID NO: 43)

[0269] CD27 Cleavable Hinge (CD27h) PTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIR (SEQ ID NO: 36)

[0270] In some CAR, the CD70 binding domain of CD27 was present, but the cleavable hinge was replaced by an alternative hinge such as a CD8 hinge (SEQ ID NO: 8), IgG4(L235E,N297Q) (SEQ ID NO: 11) or IgG4(HL-ΔCH2) (SEQ ID NO: 10). For the sake of convenience these are referred to as non-cleavable hinges and CAR that include them are referred to as non-cleavable CAR.

[0271] The cleavable CD70 CAR are: C1 (SEQ ID NO: 45), 04 (SEQ ID NO: 48) and 09 (SEQ ID NO: 53).

[0272] The non-cleavable CD70 CAR are: C2 (SEQ ID NO: 46), 03 (SEQ ID NO: 47), 05 (SEQ ID NO: 49), 06 (SEQ ID NO: 50), 07 (SEQ ID NO: 51), 08 (SEQ ID NO: 52) and C10 (SEQ ID NO: 54)Attorney Docket No.: 40056-0110WO1 / COH Ref.: TEC 25-002

[0273] Certain of these CD70 CAR are depicted schematically in FIG 1.

[0274] Example 2: Characterization of CD70 Targeted CAR

[0275] As shown in FIG 2, cleavable CAR T cells exhibit enhanced cytotoxicity against CD70-expressing kidney tumor cells. Cleavable CAR T cells (C1 and C4) demonstrated superior cytotoxicity compared to non-cleavable CAR T cells against the 786-0 kidney tumor cell line. The CD70 ko CD70-targeted CAR T cells were co-cultured with 786-0 cells at a 1:1 effector-to-target (E: T) ratio for 100 hours. Cytotoxicity was measured by real-time cell impedance.

[0276] Non-cleavable ligand-based CAR T cells were assessed for potential off-target cytotoxicity. Neither cleavable nor non-cleavable ligand-based CAR T cells exhibited cytotoxicity against CD70 knockout 786-0 tumor cell. The CD70 Ko CAR T cells were co-cultured with 786-0 cells at a 1:4 effector-to-target (E: T) ratio for 100 hours (FIG 3). Cytotoxicity was measured by realtime cell impedance using an xCELLigence system.

[0277] As shown in FIG 4, C4 and C7 CD70-targeting CAR T cells exhibit potent and comparable in vitro killing kinetics in a tumor rechallenge assay. CD70-expressing 786-O renal cell carcinoma cells were co-cultured with CD70-targeting CAR T cells (C2 and C7, CD70 knockout) from donor HD707 at a 1:1 effector-to-target (E: T) ratio. CAR T cells were repeatedly challenged with fresh tumor cells four times to assess their capacity for sustained cytotoxic activity. Tumor cell killing was assessed over time by measuring cell impedance using an xCELLigence system.

[0278] As shown in FIG 5, C4 and C7 CD70-targeting CAR T cells exhibit potent and comparable in vitro killing kinetics in a tumor rechallenge assay. CD70-expressing 786-O renal cell carcinoma cells were co-cultured with CD70-targeting CAR T cells (C2 and C7, CD70 knockout) from donor HD704 at a 1:1 effector-to-target (E: T) ratio. CAR T cells were repeatedly challenged with fresh tumor cells four times to assess their capacity for sustained cytotoxic activity. Tumor cell killing was assessed over time by measuring cell impedance using an xCELLigence system.

[0279] To assess the therapeutic efficacy of CD70-targeting CAR T cells, 786-O renal cell carcinoma cells (4e6 cells) were injected intraperitoneally (i.p.) into NSG mice. Subsequently, CD70-targeting CAR T cells (2e6 cells) were administered i.p. Tumor progression was evaluated via bioluminescence imaging on days 7, 18, 30, and 66 post-CAR T cell infusion. Both C4 and C7 CAR T cells demonstrated potent anti-tumor activity, with C4 exhibiting a slightly slower onset of tumor control compared to C7 but achieving comparable overall efficacy (FIG 6)Attorney Docket No.: 40056-0110WO1 / COH Ref.: TEC 25-002

[0280] As shown in FIG 7, cleavable CARs (C1 and C4) demonstrate superior cytotoxicity compared to non-cleavable CARs against GBM cells in vitro. Cytotoxicity was measured using the xCELLigence system to assess the function of CD70 knockout (KO) CAR T cells. The effector-to-target (E: T) ratio was set at 1:8, with CD70 KO CAR T cells as effectors and WT GBM cells as targets.

[0281] As shown in FIG 8, CD70-targeting CAR T cells other than C7, which uses an scFv for targeting, exhibit no off-target cytotoxicity against CD70 KO GBM. Cytotoxicity was assessed using the xCELLigence system. The effector-to-target (E: T) ratio was set at 1: 1, with CD70 KO CAR T cells as effectors and CD70 KO GBM cells as targets.

[0282] CD70-targeting CAR T cells were assessed for activation markers (CD69 and 4-1BB) and intracellular IFNy production after 4 hours, correlating with their cytotoxicity against CD70 WT GBM cells (PBT030) (FIG 9). The experiment was conducted at a 1:1 effector-to-target (E: T) ratio, using CD70 KO CAR T cells as effectors and the wild-type GBM cell line (PBT030) as targets. C7 and C4 CAR T cells demonstrated the highest activation and killing potency.

[0283] CD70-targeting CAR T cell proliferation was assessed after a 5-day cytotoxicity assay conducted at an effector-to-target (E: T) ratio of 1:8. CD70 knockout (KO) CAR T cells were used as effectors against the wild-type GBM cell line (PBT030). As shown in FIG 10, C7 and C4 CAR T cells exhibit the highest proliferative capacity in a cytotoxicity assay.

[0284] C7 CAR-expressing T cells exhibited reappearance of the CD70+ population in the CAR T cell monoculture, likely due to fratricide and subsequent exhaustion (FIG 11). CD70 WT CAR T cells were generated using CD3 / CD28 bead activation at a 2:1 cell-to-bead ratio and virus transduction (MOI=1).

[0285] Non-cleavable ligand CARs (C4 and C7) demonstrated better proliferation compared to cleavable and scFv-based CAR T cells during CAR T cell production, suggesting possibly reduced fratricide in the former (FIG 12). CAR T cells (CD70 wild type) were generated via CD3 / CD28 bead stimulation (1:1 bead-to-cell ratio).

[0286] The C7 (scFv-based) CAR T cells demonstrated significantly higher cytotoxicity against mock T cells (CD70+) compared to C1 and C4 (ligand-based) CAR T cells (FIG 13). Effector CD70-knockout CAR T cells were co-cultured with target mock T cells at a 1:2 effectontarget (E: T)Attorney Docket No.: 40056-0110WO1 / COH Ref.: TEC 25-002

[0287] ratio. CD107a degranulation and intracellular INFg were measured after 6 hours of co-culture, and secreted IFNy production was measured at 18 hours using ELISA.

[0288] As shown in FIG 14, CD70 Knockout Differentially Regulates the Phenotype of scFv CAR T Cells During CAR T Cell Production. CAR T cell proliferation during production (over one month) utilized TransAct for T cell activation. CD70+ population fluctuated during CAR T production possible because of fratricide (Panel A). CD70 deficiency did not alter the T cell subset distribution in ligand-based CAR T cells, with both wild-type and knockout CAR T cells maintaining a predominantly stem cell / central memory (Tscm / Tcm) phenotype (Panel B).

[0289] Conversely, in scFv-based CAR T cells, CD70 knockout resulted in a shift from the more effector (Teff)-like phenotype observed in wild-type scFv-based CAR T cells-potentially due to fratricide- towards a more prominent Tscm / Tcm phenotype in the CD70-ko CAR T cells, likely by preventing fratricide

[0290] As shown in FIG 15, CD70 expression on scFv-based CARs mediates fratricide, impacting CAR T cell expansion and CAR+ cell frequency. (A) CD70-knockout (KO) scFv-based CAR T cells (C7) exhibited superior proliferation compared to CD70-wildtype (WT) C7 CAR T cells, likely due to fratricide in the latter. Ligand-based CAR T cells (C4) showed the most robust expansion. CD70 disruption augmented scFv-based CAR T cell growth more than ligand-based CARs during manufacturing. (B) CAR T cell (CD19+ population) frequency was diminished in WT C7 CAR T cells due to fratricide; CD70 ko restored it. CAR+ cell frequency remained stable in both WT and KO ligand-based CAR T cells (C4), indicating minimal fratricide. CAR T cells were generated via TransAct activation and CD19+ population measured at the end of CAR T cell production (day 14).

[0291] As shown in FIG 16, fratricide diminishes the cytotoxic potency of scFv-based CAR T cells. CD70 knockout (KO) in scFv-based CAR T cells (C7) enhances their killing efficacy, whereas it has no discernible effect on ligand-based CAR T cells (C4) (panel A). CAR T cells do not exhibit non-specific killing against CD70-deficient tumor cells. Effector cells (WT or CD70 KO) were cocultured with target cells (CD70+ and CD70 KO 786-0 renal cell carcinoma cell line) at an effector:target (E: T) ratio of 1:4, and target cell proliferation was monitored using real-time impedance-based cytotoxicity assays (xCELLigence) (Panel B).

[0292] As shown in FIG 17, fratricide diminishes the cytotoxic potency specifically in scFv-based CAR T cells. CD70 knockout (KO) in scFv-based CAR T cells (C7) significantly enhances theirAttorney Docket No.: 40056-0110W01 / COH Ref.: TEC 25-002

[0293] cytotoxic efficacy, as measured by IFNy secretion, compared to ligand-based CAR T cells (C4). Effector cells (WT or CD70 KO) were co-cultured with target cells (CD70+ and CD70 KO 786-0 renal cell carcinoma cell line) at an effector: target (E: T) ratio of 2:1. IFNy levels were measured by ELISA after 21 hours of co-culture.

[0294] Example 3: Additional CD70 Targeted CAR

[0295] Additional CD70 CAR were generated to further optimize function. These are shown schematically in FIG 18. These CD70 are similar to C4, but have alternative transmembrane domains.

[0296] C8 (huCD27L-CD99tm-41 BB-Zeta) ATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHC ESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPT HLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRAPGVIPGIVGAWVAV AGAISSFIAKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGGGRVKFSRSAD APAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEA YSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0297] C9 (huCD27L-CD4tm-41 BB-Zeta) ATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHC ESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPT

[0298]

[0299] HLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRMALIVLGGVAGLLLFI GLGIFFKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGGGRVKFSRSADAPA YQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSE IGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0300] C10 (huCD27L-CD8tm-41 BB-Zeta) ATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHC ESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPT HLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRIYIWAPLAGTCGVLLL SLVITKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGGGRVKFSRSADAPAY QQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEI GMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0301] Example 4: Characterization of Additional CD70 Targeted CAR

[0302] As shown in FIG 19, C9 and C7 CAR T cell exhibited highest cytotoxicity against GBM cells. Real-time cell analysis of patient-derived GBM tumor cells (PBT030) co-cultured with CD70-Attorney Docket No.: 40056-0110WO1 / COH Ref.: TEC 25-002

[0303] targeting CAR T cells (CD70 KO) at an effector-to-target (E: T) ratio of 1:8. Tumor cell viability was monitored over time by cell index measurements in xCelligence assay.

[0304] As shown in FIG 20, C9 and C7 CAR T cells demonstrate enhanced degranulation upon coculture with GBM cells. The CD70-targeting CAR T cells (CD70 ko) were co-cultured with GBM cells for 4 hours at effector-to-target (E: T) ratio of 1:1. The percentage of CD107a+ (degranulation marker) CAR T cells within the CD19+ population was assessed by flow cytometry. Statistical significance was determined using two-way ANOVA and is indicated as ** *P < 0.05, **P < 0.01, ***P < 0.001, ****p < 0.0001**. Error bars represent standard deviation of 4 replicates.

[0305] As shown in FIG 21, C7 and C9 CAR T cells showed enhanced activation upon co-culture with GBM cells. The CD70-targeting CAR T cells (CD70 ko) co-cultured with GBM cells for 18 hours at E=T = 1:1. The percentage of 4-1BB+, CD69+, and CD25+ CAR T cells within the CD19+ population was assessed by flow cytometry. Statistical significance was determined using Two-way ANOVA and is indicated as ** *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001**. "ns" denotes not significant. Error bars represent standard deviation of four replicates.

[0306] As shown in FIG 22, C9 and C7 CAR T-cell exhibited show potent, specific cytotoxicity against RCC cells, sparing CD70 KO tumor cells. Real-time xCELLigence assay of 786-0 RCC tumor cells (WT and CD70 -ko) co-cultured with CD70-targeting CAR T cells (CD70 KO) at effector-to-target (E: T) ratios of 1:8 and 1:1. Tumor cell viability was monitored over time by cell index measurements.

[0307] As shown in FIG 23, C9 and C7 CAR T cells demonstrate enhanced degranulation upon coculture with RCC cells. The CD70-targeting CAR T cells (CD70 ko) were co-cultured with RCC cell line (786_o) for 4 hours at effector-to-target (E: T) ratio of 1:1. The percentage of CD107a+ (degranulation marker) CAR T cells within the CD19+ population was assessed by flow cytometry. The secreted cytokines (IL2, INFg) measured by ELISA. Statistical significance was determined using two-way ANOVA and is indicated as ** *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001**. Error bars represent standard deviation of 4 replicates.

[0308] As shown in FIG 24, C7 and C9 CAR T cells show enhanced activation upon co-culture with GBM cells. The CD70-targeting CAR T cells (CD70 ko) co-cultured with GBM cells for 18 hours at E=T = 1:1. The percentage of 4-1BB+, CD69+, and CD25+ CAR T cells within the CD19+ population was assessed by flow cytometry. Statistical significance was determined using TwoAttorney Docket No.: 40056-0110WO1 / COH Ref.: TEC 25-002

[0309] way ANOVA and is indicated as ** *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001**. "ns" denotes not significant. Error bars represent standard deviation of four replicates.

[0310] As shown in FIG 25, self-CAR activation drives T cell exhaustion, and differentiation. Exhaustion markers (TIGIT, TIM3, and LAG3) and sternness (CD62L / CD45RO) were analyzed during the CAR T cell manufacturing process using flow cytometry. Increased exhaustion and a more differentiated T cell phenotype were observed in CD70 wild-type (WT) CAR T cells compared to CD70 knockout (KO) CAR T cells, suggesting that self-activation contributes to exhaustion and differentiation in the potent CD70-targeting CAR T cells.

[0311] As shown in FIG 26, both ligand-based and ScFv-based CARs demonstrated fratricide, with reduced fratricide observed in the ligand-based CAR. Fratricide was assessed by detecting CD107a degranulation, cytokine secretion (IL-2 and IFN-y) measured via ELISA, and the expression of activation markers (CD25, CD69, and 4-1BB) analyzed using flow cytometry. Statistical significance was determined using two-way ANOVA and is indicated as ** *P < 0.05, **P < 0.01, ***P < 0.001, ****p < 0.0001**. "ns" denotes not significant. Error bars represent standard deviation of four replicates.

[0312] As shown in FIG 27, CD70 knockout (KO) enhances the efficacy of the ligand-based CAR (C9) and ScFv-based CAR (C7) in GBM models, underscoring the necessity of CD70 KO in CD70-targeting CAR T cells. CD70 wild-type (WT) and CD70 KO CAR T cells were co-cultured with patient-derived GBM cell lines (PBT030) at E: T= 1:8 ratio, and cytotoxicity was evaluated using cell impedance analysis (xCELLigence assay).

[0313] As shown in FIG 28, CD70 knockout (KO) enhances the efficacy of the ligand-based CAR (C9) and ScFv-based CAR (C7) in RCC models. CD70 wild-type (WT) and CD70 KO CAR T cells were co-cultured with RCC cell lines (786_o) at E: T= 1:8 ratio, and cytotoxicity was evaluated using cell impedance analysis (xCELLigence assay).

[0314] As shown in FIG 29, truncation or sequence modification in the intracellular domain of human CD70 impacts protein expression and its binding affinity to the CD27 ligand. Various human CD70 variants were transduced into Jurkat cells. A) The expression of CD70 variants was monitored over one month. Certain variants, such as truncated CD70 and double-flagged CD70, exhibited expression issues. B) The chimeric and flagged CD70 variants showed reduced binding affinity to CD27 (CD70’s ligand).Attorney Docket No.: 40056-0110WO1 / COH Ref.: TEC 25-002

[0315] Example 4: Transmembrane domain modification

[0316] As shown in FIG 30, transmembrane selection can enhance the antitumor potency of ligandbased CD70 CAR T cells. (A) Representative brightfield images of RCC (786-0) and GBM (PBT030) tumor cells following co-culture with the indicated CAR T-cell products. RCC images were acquired on day 3 (E: T = 1:8), and GBM images were acquired on day 6 (E: T = 1:18). Conditions include tumor-only control, un-transduced T cells (UT), first-generation ligand CAR (C4), TM-optimized ligand CAR (C9), and scFv-based CAR (C7). Representative fields show enhanced tumor clearance with the TM-optimized C9 CAR compared to C4. (B) Quantification of cytotoxicity against GBM at 100 hours across decreasing effector-to-target (E: T) ratios (1:2, 1:8, 1:16, and 1:32). TM optimization significantly improved the killing potency of the ligand CAR (C9 versus C4), enabling C9 CAR T cells to achieve cytotoxicity comparable to the scFv-based CAR (C7), particularly at lower E: T ratios. (C) Cytotoxicity against RCC at 200 hours across multiple E: T ratios. Consistent with GBM results, TM-optimized C9 CAR T cells demonstrate enhanced antitumor activity relative to C4 and maintain tumor killing comparable to the scFv-based CAR at reduced effector cell numbers. Data represent mean ± SEM of replicate wells.

[0317] Example 5: Characterization of activation and species cross-reactivity of certain CD70 CAR

[0318] As shown in FIG. 36, the scFv-based C7 CAR displays a robust, dose-dependent proliferative shift exclusively in response to human (H) CD70. In contrast, the ligand-based CARs (C4 and C9) maintain a cross-reactive profile, showing similar CFSE dilution patterns when exposed to either human or mouse (M) CD70, albeit with lower overall proliferative capacity compared to the C7 / human CD70 interaction. A-B) Flow cytometric evaluation of activation markers including 4-1BB (A) and CD25 (B) on T cells 72 hours post-stimulation. Cells were cultured on plates coated with human or mouse recombinant CD70 at 0.75 or 5 ug / mL. While the ligand-based CARs (C4 and C9) exhibit comparable activation and significant cross-reactivity between human and mouse CD70, the scFv-based C7 CAR demonstrates potent, human-specific activation with negligible reactivity toward the mouse ortholog. C) Representative histograms showing CFSE dilution in CAR T cells 72 hours post-co-culture.

[0319] Example 6: Impact of CD70 expression on CD70 CAR T cells

[0320] As shown in FIG 31, knockout of CD70 in CAR T cells can improve anti-tumor efficacy and proliferative capacity in RCC Models. (A) Real-Time Impedance-Based Cytotoxicity AssayAttorney Docket No.: 40056-0110WO1 / COH Ref.: TEC 25-002

[0321] (xCELLigence). RCC tumor cell growth and lysis were monitored via electrical impedance, expressed as Cell Index, at an Effector-to-Target (E: T) ratio of 1:8. Both C9 and C7 CAR-KO constructs achieved rapid and complete tumor clearance within 100 hours, whereas C4 CAR-KO exhibited significantly delayed and incomplete cytotoxicity compared to the Untransduced (UT-KO) control. (B-C) Expression of inhibitory receptors (B) LAG3 and (C) PD-1 was assessed in %CD19+T cells over 6 days. C7 CAR-KO cells showed the highest upregulation of both exhaustion markers by Day 6, suggesting that its potent initial cytotoxicity is coupled with a more rapid progression toward functional exhaustion compared to the C9 variant. (D-E) T Cell Proliferation and Persistence. C9 CAR-KO demonstrated the most robust CAR T cell expansion, peaking at Day 4, while C7 CAR-KO showed markedly lower proliferative capacity and persistence despite its high initial killing rate in the impedance assay.

[0322] As shown in FIG. 33, fratricide reduces the cytotoxic potency of the CD70 CAR T cells. (A-B) Representative microscopic images of GFP+target cell lines (RCC and GBM) co-cultured with either CD70 wild-type (WT) or CD70 knockout (KO) CAR T cells (From Two different donor HD704 and HD707). (B) Baseline CD70 expression levels in 786-0 (RCC) and PBT030 (GBM) cell lines. (D-E) CD70 knockout (KO) in both ligand-based CAR T cells (C4 and C9) and scFv-based CAR T cells (C7) enhances cytotoxic activity in GBM models (D) and the RCC model (E). The greater difference in cytotoxic efficacy between WT and KO CAR T cells observed in the RCC model compared to GBM is likely due to the higher CD70 expression in RCC cells (panel B), which may promote increased CAR T-cell activation and subsequent fratricide.

[0323] Effector cells (WT or CD70 KO CAR T cells) were co-cultured with CD70+ target cells (786-0 renal cell carcinoma or PBT030 GBM cells) at an effector-to-target (E: T) ratio of 1:8. Target cell proliferation was monitored using a real-time impedance-based cytotoxicity assay (xCELLigence).

[0324] As shown in FIG. 34, CD70 CARs can exhibit trans-binding and cellular aggregation following CD70 CAR transduction. (A) Flow cytometric analysis of Jurkat cells 24 hours prior to CD70 transduction (DO) confirms high surface expression of IL13Ra2 (as a control CAR), C4, C9, and C7 CAR constructs (tCD19 marker). B) Confocal microscopy (LSM880) was used to visualize cellular aggregation 48 hours post-transduction with CD70 lenti virus. C4, C9, and C7 CAR variants show extensive trans-binding (T cell-T cell interaction) characterized by large multicellular clusters, whereas Mock and IL13 CAR controls maintain a disparate, nonaggregated morphology. Jurkat cells were transduced at DO, followed by a 6-hour incubationAttorney Docket No.: 40056-0110WO1 / COH Ref.: TEC 25-002

[0325] before dilution with media containing 100nM Dasatinib to modulate signaling. Half-media changes with 2x Dasatinib were performed at Day 1 and Day 2 to maintain concentrations without disturbing cell clusters prior to imaging.

[0326] As shown in FIG 35, cis-binding of a CD70 CAR to its cognate antigen CD70 on the same cell surface results in ligand masking, a process demonstrated here in non-cytotoxic cell models (GBM and Jurkat cells). To isolate this binding mechanism from effector functions, Jurkat and GBM cell lines were engineered to express CD70 and subsequently transduced with truncated CAR constructs lacking the CD3z signaling domain. (A) Flow cytometric analysis confirms the successful engineering of Jurkat cells to stably express different level of CD70. (B) A Jurkat line with moderate CD70 expression (LS2) was selected for secondary transduction with truncated CARs. Surface expression of tEGFR serves as a surrogate marker to demonstrate successful CAR transduction. (C) Following CAR expression, surface staining for CD70 was significantly reduced or abolished, demonstrating that the truncated CAR effectively masks the CD70 ligand in cis. D-E) Similar to the Jurkat model, a CD70 knocked out GBM line (D) was engineered to express intermediate and high levels of CD70 and subsequently transduced with truncated CARs, as confirmed by tEGFR expression (E). (F) Comparison of ligand masking reveals that the ligand-based CAR (C9) results in partial CD70 masking, whereas the ScFv-based CAR (C7) achieves complete masking, suggesting higher cis-binding affinity or more efficient steric blocking by the ScFv format.

[0327] Example 7: Ligand-based CD70 CAR and scFv-based CD70 CAR can mediate toxicity against low-CD70-expressing tumors

[0328] As shown in FIG. 32, ligand-based (C8) CD70 CAR T cells and scFv-based (C6) CD70 CAR T cells can mediate efficient cytotoxicity against low-CD70-expressing tumors with distinct activation profiles. (A) Generation and characterization of CD70-modified tumor cells. The BGM cell line (PBT030) was first engineered to knock out CD70 and subsequently reconstituted to express CD70 at graded levels, including a low-expression population (MFI = 610), as determined by flow cytometry. (B) Tumor cell quantification following co-culture with CAR T cells at a 1:1 effector-to-target (E: T) ratio, demonstrating effective tumor clearance by both ligand-based (C4,C8) and scFv-based (C6) CAR T cells. (C) IFN-y secretion in co-culture supernatants measured after the cytotoxicity assay, reflecting functional activation of CAR T cells upon antigen engagement. (D) Frequency (%) of CD70-positive cells among residual tumor cells following co-culture. (E) Mean fluorescence intensity (MFI) of CD70 on residual tumor cells afterAttorney Docket No.: 40056-0110WO1 / COH Ref.: TEC 25-002

[0329] CAR T-cell treatment. (F) Surface expression of the activation marker 4-1BB on CAR T cells following the killing assay. (G) Comparative functional analysis across increasing tumor antigen densities, showing that ligand-based CAR T cells achieve cytotoxicity comparable to scFv-based CARs while exhibiting reduced activation and more consistent persistence, even at higher CD70 expression levels.

[0330] Example 8: Influence of CD70 expression on tumor cells

[0331] As shown in FIG 37, heterogeneous CD70 expression in glioblastoma (GBM) can impact CD70 CAR T cell efficacy. A) Representative Immunohistochemical (IHC) micrographs of patient-derived glioblastoma (GBM) samples and healthy brain tissue. GBM specimens exhibit significant inter-patient heterogeneity, ranging from high CD70 immunoreactivity to low / absent expression (middle). Conversely, healthy brain tissue shows no detectable CD70 expression, suggesting a favorable therapeutic target for GBM model. B) Linear regression analysis correlating residual tumor cell count with CD70 Mean Fluorescence Intensity (MFI). All tested constructs (C4, C9, and C7 CAR-KO) demonstrate a strong inverse correlation between antigen density and tumor survival. These data conclude that CD70 expression levels directly impact CAR T cell efficacy, where higher MFI values result in significantly enhanced tumor clearance across all CAR variants.

[0332] As shown in FIG 38, decitabine (DAC) induces higher CD70 expression than azacytidine (AZA) in patient-derived glioblastoma tumor cell lines without compromising cell viability. DAC-induced CD70 expression increased with increasing concentration, peaking at 4 hours post-treatment. DAC and AZA were freshly prepared before each assay by dissolving in DMSO and added to patient-derived GBM tumor cell lines (pbt030 and pbt206) and T cells (from two different donors). Cells were treated with the drugs for either two or four consecutive days. Cell viability and CD70 expression were assessed at 24 and 48 hours post-treatment.

[0333] As shown in FIG 39, decitabine can sustain CD70 expression in some glioblastoma cells lines for an extended period. A) PBT030 cells treated with DAC exhibited sustained elevated CD70 expression from Day 2 through Day 19 post-treatment. PBT206 cells showed transient CD70 induction peaking on Day 6, but with markedly lower levels compared to PBT030. CD70 did not induced in T cells with DAC. B) representative FACS data of DAC-induced CD70. Patient-derived Glioblastoma (GBM) cells (PBT030 and PBT206) and T cells (two donors) were treated with FDA-approved DAC concentration (0.64 μM) DAC or DMSO as a control for 4 constitutiveAttorney Docket No.: 40056-0110WO1 / COH Ref.: TEC 25-002

[0334] days (after washing out from the last day of treatment). CD70 expression and viability were assessed at different time point from 6 hours to day 19thpost last day of treatment. The experiment was performed with three technical replicates (n=3).

[0335] As shown in FIG 40, decitabine-induced CD70 expression can persist for 4 days in vivo in a glioblastoma model. Glioblastoma tumor-bearing NSG mice were treated with the FDA-approved dose of decitabine (DAC; 6.62 mg / kg), and CD70 expression levels were assessed at 24 and 48 hours post-treatment. DAC treatment significantly increased CD70 expression and mean fluorescence intensity (MFI), with the elevated levels maintained for up to 4 days following treatment.

[0336] As shown in FIG 41, decitabine can enhance CD70 expression and sensitize glioblastoma to CAR T Cell Cytotoxicity. A) Flow cytometric analysis of Renal Cell Carcinoma (RCC) and Glioblastoma (GBM) cell lines 4 days post-treatment with a constitutive FDA-equivalent dose of DAC (0,65uM). Histograms and demonstrate a significant increase in CD70 Mean Fluorescence Intensity (MFI) in both RCC (from 22,478 to 29,194) and GBM (from 6,270 to 12,098) cells, while maintaining high cell viability. B) Cytotoxicity assay (140h) measuring the Cell Index of PBT030 GBM cells treated with CAR T cells (C4, C9, or C7) in the presence or absence of DAC. While DAC alone reduces tumor cell index, the combination therapy significantly improves tumor clearance. Notably, the C4 CAR, which exhibits lower baseline potency, shows the most marked improvement in killing efficacy when antigen density is increased via DAC. In contrast, the more potent C9 and C7 CARs achieve near-complete tumor eradication, concluding that pharmacologically increasing antigen density via DAC is a viable strategy to enhance the killing potency of the CAR T cell constructs specifically in a heterogeneous Tumor model (GBM).

Claims

Attorney Docket No.: 40056-0110WO1 / COH Ref.: TEC 25-002What is clamed is:

1. A nucleic acid molecule comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR) comprising:a CD70 binding domain comprising or consisting of: (a) the amino acid sequence:ATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHC ESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQ(SEQ ID NO: 43) or (b) a scFv targeted to CD70;a spacer domain;a transmembrane domain;a costimulatory domain; anda CD3 signaling domain.

2. The nucleic acid molecule of claim 1, wherein the CD70 binding domain comprises or consists of the amino acid sequence:ATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHC ESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQ(SEQ ID NO: 43).

3. The nucleic acid molecule of claim 1, wherein the spacer domain comprises or consists of an amino acid sequence selected from the group consisting of: SEQ ID NOs: 1-13 and 36, the transmembrane domain comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 14-22 and 37-39; the costimulatory domain comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 31-35, and the CD3 signaling domain comprises or consists of an amino sequence selected from the group consisting of SEQ ID NOs: 23-30.Attorney Docket No.: 40056-0110WO1 / COH Ref.: TEC 25-0024. The nucleic acid molecule of claim 1 or claim 3 wherein the spacer comprises or consists of the amino acid sequence of SEQ ID NO: 36.

5. The nucleic acid molecule of claim 1 or claim 2, wherein the transmembrane domain comprises or consists of an amino acid sequence selected from the group consisting of: SEQ ID NOs: 15-20 and 37.

6. The nucleic acid molecule of claim 1 or claim 2, wherein the transmembrane domain comprises or consists of the amino acid sequence of: SEQ ID NO: 17.

7. The nucleic acid molecule of any of claims 1-3, wherein the costimulatory domain comprises KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 33)8. The nucleic acid molecule of claim 1 or claim 2, wherein the spacer domain comprises or consists of the amino acid sequence of SEQ ID NO: 36, the transmembrane domain comprises or consist of an amino acid sequence selected from the group consisting of SEQ ID NOs: 14-22 and 37-39; the costimulatory domain comprises or consists of the amino acid sequence of SEQ ID NO: 33, and the CD3 signaling domain comprises a sequence selected from the group consisting of SEQ ID NOs: 23-30.

9. The nucleic acid molecule of claim 1, 2 or 8, wherein the CD3 signaling domain comprises or consists of the amino acid sequence of SEQ ID NO: 23.

10. The nucleic acid molecule of any of the forgoing claims wherein the sequence encoding the CAR is immediately preceded by a nucleic acid sequence encoding a signal sequence.

11. The nucleic acid molecule of claim 10, wherein the signal sequence comprises or consists of the amino acid sequence of SEQ ID NO: 55.Attorney Docket No.: 40056-0110WO1 / COH Ref.: TEC 25-00212. The nucleic acid molecule of claim 1, wherein the CAR comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 45-54.

13. The nucleic acid molecule of claim 1, wherein the CAR comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 56-68.

14. The nucleic acid molecule of claim 1, wherein the CAR comprises or consists of the amino acid sequence of SEQ ID NO: 53.

15. The nucleic acid molecule of any of claims 1-11, wherein an interdomain linker of 1-5 amino acids is located between two or more adjacent domains.

16. The nucleic acid molecule of any of claims 1-11, wherein an interdomain linker of 1-5 amino acids is located between the costimulatory domain and the CD3 signaling domain.

17. The nucleic acid molecule of claim 15 or claim 16, wherein the interdomain linker comprises glycine or alanine.

18. The nucleic acid molecule of claim 17, wherein the interdomain linker comprises only glycine or alanine.

19. The nucleic acid molecule of claim 18, wherein the interdomain linker comprises only glycine or only alanine.

20. A population of T cells harboring the nucleic acid molecule of any of the forgoing claims.Attorney Docket No.: 40056-0110WO1 / COH Ref.: TEC 25-00221. The population of T cells of claim 20, wherein the T cell does not express CD70.

22. The population of T cells of claim 20, wherein the T cell has been genetically modified to knock out CD70 expression.

23. The population of T cells of claim 20, wherein the T cell has been genetically modified to knock out CD70 coding sequence.

24. The population of T cells of any of claims 20-23 wherein at least 50%, 70% or 90% of the T cells comprise memory T cells, naive T cells or central memory T cells.

25. A method of treating a patient having renal cell carcinoma, comprising administering a therapeutically effect amount of the population of T cells of any of claims 17-20.

26. A method of treating a patient having glioblastoma, comprising administering a therapeutically effect amount of the population of T cells of any of claims 17-20.

27. The method of claim 22, wherein the T cells are administered intraventricularly.

28. A method of treating a patient suffering from a cancer expressing CD70, comprising administering a therapeutically effect amount of the population of T cells of any of claims 17-20.

29. The method of any of claim 21-24 further comprising administering decitabine or azacytidine.Attorney Docket No.: 40056-0110WO1 / COH Ref.: TEC 25-00230. The nucleic acid molecule of any of claims 1-19, wherein the chimeric antigen receptor does not comprise a CD27 intracellular domain.

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