Chimeric coreceptors

Abstract

The present invention relates to a chimeric coreceptor comprising an immunoglobulin variable-like domain. The present invention also relates to a combination of the chimeric coreceptor with an antigen receptor, to nucleic acids encoding the chimeric coreceptor, and to pharmaceutical compositions comprising the chimeric coreceptor or the combination, or the encoding nucleic acids. Further disclosed herein is their use in the treatment of a disease characterized by the expression of at least one tumor-associated antigen, and preferably cancer.

Description

[0001] CHIMERIC CORECEPTORS

[0002] The present invention relates to a chimeric coreceptor comprising an immunoglobulin variable-like domain. The present invention also relates to a combination of the chimeric coreceptor with an antigen receptor, to nucleic acids encoding the chimeric coreceptor, and to pharmaceutical compositions comprising the chimeric coreceptor or the combination, or the encoding nucleic acids. Further disclosed herein is their use in the treatment of a disease characterized by the expression of at least one tumor-associated antigen, and preferably cancer.

[0003] BACKGROUND OF THE INVENTION

[0004] Chimeric antigen receptor (CAR)-T cells represent an innovative immunotherapeutic approach and such therapeutics have shown tremendous potential for the treatment of cancer. For instance, CAR-T cells targeting the B cell-specific proteins CD 19 or CD22 induced complete remissions in more than 70% of patients suffering from relapsed or refractory (r / r) B cell acute lymphoblastic leukaemia (B-ALL), and in approximately 50% of patients with r / r large B cell lymphoma. CAR-T cells are generated from patient-derived autologous T cells genetically modified to express a CAR. After genetic modification and ex vivo expansion, CAR-T cells are reinfused into the patient where they bind to a specific structure exposed on the surface of tumor cells. The antigen specificity of CAR-T cells is provided by the CAR, which is an artificially constructed fusion protein containing an extracellular antigen-binding domain (e.g. a single chain variable fragment, scFv) derived from an antibody linked to T cell-derived transmembrane and intracellular signaling domains.

[0005] Since CARs exploit the antigen-binding properties of the parental monoclonal antibody, CAR-T cells are enabled to respond to native antigens expressed on the surface of tumor cells in a non-MHC restricted manner. Therefore, the mechanism of antigen-recognition and T cell activation is fundamentally different between CAR-T cells and conventional T cells, which respond exclusively to processed peptide antigens presented by MHC molecules. The CAR-dependent activation of the CAR-T cell elicits an immune response against the antigen-expressing cells resulting in T cell-mediated destruction of the tumor.

[0006] Early CAR designs comprised an antibody-derived scFv as antigen-recognition domain, fused through a hinge and transmembrane domain to the cytoplasmic tail of the TCR signalling component CD3^. T cells expressing such first-generation CARs could induce cytotoxicity toward antigen-positive tumor cells, however, they were unable to efficiently control tumor growth in vivo due to their poor persistence (Eshhar, Z, et al., 1993. PNAS, 90(2):720-724). T cell activation, proliferation and survival is greatly improved by co-engagement of the TCR and costimulatory receptors such as CD28 or 4- IBB. Therefore, current CAR designs incorporate one or more costimulatory domains in addition to an intracellular CD3^ T cell activation domain, which classify second- (one costimulatory domain) and third-generation (two costimulatory domains) CAR architectures. This design principle aims at combining signal 1 (from the TCR) and signal 2 (from a costimulatory-receptor) required to unleash full T cell activation, and indeed significantly improved in vivo persistence and clinical efficacy of CAR-T cells. In most cases, the costimulatory domains derive either from the CD28 receptor family (CD28, ICOS) or the tumor necrosis factor receptor (TNFR) family (4- IBB, CD27, 0X40), although second-generation CD28 / CD3(^ (28z) and 4- 1 BB / CD3(^ (4-lBBz) CARs are the most frequently used combinations in clinically tested cell products. Notably, all six currently FDA-approved CAR-T cell products use second- generation CAR designs, four of which contain 4-lBBz and two of which contain 28z signaling domains.

[0007] Growing practical experience with 4-lBBz- and 28z-CARs indicates some important functional differences between these two CAR designs, which affect the in vivo persistence of the CAR-T cells on the one hand, and the antigen- sensitivity on the other hand. 4-lBBz-CAR-T cells typically show enhanced in vivo persistence in mice and humans compared to 28z CAR-T cells, the latter of which rarely persist for more than one or two months in patients. In contrast, 28z- CARs outperform 4-lBBz-CARs in tumors with low antigen density. However, 28z-CARs also fail to clear tumor cells when target antigen density drops below 1,000 molecules per cell (Hamieh, M, et al., 2023. Cancer Discov 13(4):829-843).

[0008] In fact, antigen-low or antigen-loss escape has emerged as a major issue that impacts the durability of CAR-T cell therapy. For instance, clinical trials with anti-BCMA CAR-T cells in patients with multiple myeloma have demonstrated outgrowth of tumor cells expressing low levels of BCMA (Brudno, JN, et al., 2018. J Clin Oncol, (22):2267-2280; Cohen, AD, et al., 2019. J Clin Invest, 129(6):2210-2221). Furthermore, anti-CD22 CAR-T cells that utilize the m971 antigenrecognition domain showed highly efficient anti-tumor responses in preclinical models (Haso, W, et al., 2013. Blood, 121(7): 1165-1174) and induced complete response rates of more than 70% in B-AEE patients, but the majority of responding patients relapsed due to the outgrowth of tumor cells with reduced CD22 antigen density (Fry, TJ, et al., 2018. Nat Med, 24(l):20-28; Shah, NN, et al., 2020. J Clin Oncol. 38(17): 1938-1959). Preclinical modelling using engineered variants of the B-ALL tumor cell line NALM6 revealed that a reduction in CD22 site densities to approximately 1,800 molecules per cell was sufficient to escape curative anti-tumor responses by anti-CD22 CAR-T cells (Fry, TJ, et al., 2018). A similar insufficient reactivity of CAR-T cells, particularly of those with 4-lBBz signalling domains, against target cells with reduced antigen densities has also been shown for other specificities, including anti-CD19 and anti-GPC2 CARs that failed to eradicate tumor cells with about 2,000 CD 19 and 6,000 GPC2 molecules per cell, respectively (Majzner, RG, et al., 2020. Cancer Discov. 10(5):702-723; Heitzeneder, S, et al., 2022. Cancer Cell, 40(1): 53-69). These results indicate that antigen downmodulation to a few thousand molecules per cell is a general cause of resistance to CAR-T cells and indicate insufficient sensitivity of CARs to kill antigen-low target cells, in sharp contrast to the exquisite antigen sensitivity of endogenous TCRs, which require less than ten antigenic peptide-MHC complexes for killing (Irvine, DJ, et al., 2002. Nature 419:845-849; Purbhoo, MA, et al., 2004. Nat. Immunol. 5(5):524-30).

[0009] Besides decrease or loss of target antigen expression, other tumor- specific factors have been identified that drive resistance to CAR-T cells. For example, reduced or loss of CD58 (LFA-3) expression was found in a significant proportion of all subtypes of B-, T-, and NK-cell lymphomas, as well as in patients with acute myeloid leukemia (AML), melanoma and colon cancer (Younes, S, etal., 2023. Mod Pathol. 36(10): 100256). For patients with r / r large B cell lymphoma, low CD58 protein expression on tumor samples before treatment was shown to correlate with unresponsiveness to anti-CD19 CAR-T cells and disease progression, whereas high CD58 expression was associated with complete responses and survival (Romain, G, et al., 2022. J Clin Invest. 132(17):el59402). Since CD58 is the natural ligand for CD2 on T cells, the absence of CD2 costimulation in anti-CD19 CAR-T cells has been linked to immune resistance in lymphoma patients (Majzner, RG, et al., 2020. Blood 136(1): 53-54).

[0010] Another known mechanism for the inhibition of (CAR-) T cells is the PD-L1 / PD-1 immune checkpoint axis. T cells activated by antigen recognition induce immune responses such as the secretion of Thl cytokines, but also increase the expression of inhibitory receptors such as PD-1. The Thl cytokines (e.g. IFNy) in turn trigger the expression of the inhibitory ligand PD-L1 in tumor cells and other cell types in the tumor microenvironment. The interaction between PD-L1 and PD- 1 then leads to sustained inhibitory signaling in T cells, ultimately attenuating or terminating the therapeutic effect of T cells (Sharpe, AH, et al., 2018. Nat Rev Immunol. 18(3): 153- 167; Patsoukis N, et al., 2020. Sci Adv. 6(38):eabd2712). For example, PD-L1 was upregulated in relapsed B-ALL patients and in B-ALL patients refractory to blinatumomab, a CD19xCD3-bispecific T cellactivating molecule (Feucht J, et al., 2016. Oncotarget 7:76902-76919).

[0011] Engineering CAR-T cells to enhance their potency toward tumor cell expressing low target antigen densities also increases the likelihood of on-target / off-tumor toxicities. Thus, improving tumor specificity while minimizing reactivity to healthy tissue is another area where novel T cell engineering approaches are needed to improve the safety and applicability of CAR-T cell therapies. Novel strategies to render T cells specific for a tumor in the absence of a truly tumor-restricted antigen are essential to successfully target tumors beyond B cell malignancies, particularly solid tumors. Novel approaches apply Boolean logic gating to CAR-T cells, which is the integration of a combinatorial recognition of two antigens that are co-expressed on a tumor cell to trigger a productive T cell response, while the presence of either antigen alone is not sufficient for T cell activation. Examples for the combinatorial recognition of tumor cells include the antigens MUC1, HER2, or EpCAM, none of which is truly tumor specific, but are exclusively co-expressed on breast cancer cells. Several AND-gate CAR systems have been developed, including SynNotch (Roybal, KT, et al., 2016. Cell 167(2):419-432) and SPLIT (Kloss et al., 2013. Nat Biotechnol 31(1): 71-75) CAR-T cells, however, these have shortcomings in specificity and efficacy in vivo.

[0012] Therefore, there is a need for novel methods and tools to modulate T cell activation to overcome the limitations of the current therapies and increase their reach.

[0013] Thus, the present invention provides a chimeric coreceptor to enhance specific immune cell activation, and reduce unspecific antigen-independent immune cell activation.

[0014] SUMMARY OF THE INVENTION

[0015] The present inventors have discovered that a chimeric coreceptor comprising an IgV -like domain (IgVD) (also referred to as "chimeric IgVD coreceptor") can enhance the activation of CAR T cells and their sensitivity, particularly to tumor cells expressing low levels of the antigen recognized by the CAR and / or low or null levels of CD58 (Example 15, Table 6). Indeed, the inventors have shown that CD22 CAR T cells co-expressing a chimeric IgVD coreceptor, as disclosed herein, have enhanced immune response and killing activity against tumor cells expressing only about 400 or 40 CD22 molecules per cell, respectively, or lacking expression of CD58 (Examples 2, 3). Advantageously, co-expression of a chimeric CD8 or CD28 IgVD coreceptor increased the proportion of CD4+CD62L+ / CD45RA+ Tscm cells, suggesting that the chimeric CD8- and CD28-IgVD coreceptors may prevent non-specific CAR phosphorylation, tonic CAR signaling and T cell differentiation (Example 1, 9, and 11). Importantly, expression of chimeric IgVD coreceptors alone did not activate T cells (Examples 4). Co-expression of a chimeric CD 8 or CD28 IgVD coreceptor not only enhanced the cytotoxic activity of CAR T cells against tumor cells harboring resistance mechanisms such as low antigen and absent CD58 expression, but also significantly enhanced the antitumor responses of CAR-T cells against PD- Ll-positive tumor cells, even at low target antigen density or in the absence of CD58 expression (Example 5, 6, 7, 8). The chimeric CD8 or CD28 IgVD coreceptors were expressed at higher intensity on the CAR-T cell surface compared to the chimeric CD8 (Figure 43) or CD28 (Figures 23A-B, 38A-B) coreceptors without IgVD.

[0016] Accordingly, CD28 IgVD coreceptors induced improved anti-tumor responses of CAR T cells in vivo (Figures 35-37).

[0017] The increased expression of CD8 or CD28 IgVD coreceptor seems to be associated with the following technical advantages: more LCK molecules can bind to the chimeric CD 8 or CD28 IgVD coreceptor, which may reduce antigen-independent CD3<^ phosphorylation and tonic signaling in the CAR-T cells, and which in turn may be responsible for a higher proportion of Tscm cells in the CAR-T cell product; increased proportion of Tscm, improved T cell proliferation (Figures 27, 42) and improved persistence in vivo (Figures 35 -35B).

[0018] The increased proportion of Tscm induced by co-expression with a CD8 or CD28 IgVD is of great advantage for immuntherapy, since it is known that the improved longevity and proliferative potential, and the capacity to reconstitute a wide-ranging diversity of the T cell compartement make the Tscm cell type an ideal cell population to employ in CAR-T cell therapy (e.g. Gattinoni et al., 2017. Nature Medicine 23, 18-27).

[0019] Accordingly, in a first aspect the invention provides a chimeric coreceptor (CCR) subunit polypeptide comprising:

[0020] (a) an extracellular antigen -recognition domain 1 (ARD1) specifically binding an antigen 1 (AG1), (b) an immunoglobulin variable-like domain (IgVD),

[0021] (c) a hinge domain 1 (HD1),

[0022] (d) a transmembrane domain 1 (TMD1), and

[0023] (e) a cytosolic domain (CYTD):

[0024] (i) comprising a costimulatory domain (CSD), and

[0025] (ii) not comprising a cell activation domain, wherein the ARD1 is linked to the IgVD.

[0026] In a second aspect, the invention provides a chimeric coreceptor homodimer comprising two substantially identical chimeric coreceptor subunit polypeptides according to the first aspect or a chimeric coreceptor complex comprising a chimeric coreceptor subunit polypeptide according to the first aspect and a CD8a, CD8P or CD28 subunit.

[0027] In a third aspect, the invention provides a combination of: the chimeric coreceptor homodimer or chimeric coreceptor complex according to the second aspect; and a chimeric antigen receptor (CAR), an endogenous TCR (TCR) or a transgene TCR (tgTCR), wherein the CAR, the TCR or the tgTCR specifically binds an antigen 2 (AG2), and wherein AG1 and AG2 are two different antigens or are substantially the same antigen.

[0028] In a fourth aspect, the invention provides a nucleic acid construct (N-CCR) comprising a nucleic acid encoding the chimeric coreceptor subunit polypeptide according to the first aspect, or a nucleic acid construct (N-CCR-CAR) comprising a nucleic acid encoding the chimeric coreceptor subunit polypeptide according to the first aspect and a nucleic acid encoding a CAR subunit polypeptide, or two nucleic acid constructs (N-CCR, N-CAR), wherein the first nucleic acid construct (N- CCR) comprises a nucleic acid encoding the chimeric coreceptor subunit polypeptide according to the first aspect and the second nucleic acid construct (N-CAR) comprises a nucleic acid encoding a CAR, or a nucleic acid construct (N-CCR-tgTCR) comprising a nucleic acid encoding the chimeric coreceptor subunit polypeptide according to the first aspect and a nucleic acid encoding a tgTCR, or two nucleic acid constructs (N-CCR, N-tgTCR), wherein the first nucleic acid construct (N- CCR) comprises a nucleic acid encoding the chimeric coreceptor subunit polypeptide according to the first aspect and the second nucleic acid construct (N-CAR) comprises a nucleic acid encoding a tgTCR.

[0029] In a fifth aspect, the invention provides a vector or a kit of a first and second vector, wherein

[0030] (a) the vector comprises the nucleic acid construct (N-CCR), or the nucleic acid construct (N-CCR-CAR) or the nucleic acid construct (N-CCR-tgTCR) according to the fourth aspect;

[0031] (b) the first vector comprises the first nucleic acid construct (N-CCR) according to the fourth aspect, and the second vector comprises the second nucleic acid construct (N-CAR) according to the fourth aspect; or

[0032] (c) the first vector comprises the first nucleic acid construct (N-CCR) according to the fourth aspect, and the second vector comprises the second nucleic acid construct (N- tgTCR) according to the fourth aspect.

[0033] In a sixth aspect, the invention provides a recombinant cell comprising the nucleic acid construct (N-CCR), the nucleic acid construct (N-CCR-CAR), the two nucleic acid constructs (N- CCR, N-CAR), the nucleic acid construct (N-CCR-tgTCR) or the two nucleic acid constructs (N- CCR, N- tgTCR) according to the fourth aspect, or the vector or the kit of the first and second vector as defined in the fifth aspect, and optionally wherein the cell expresses the chimeric coreceptor subunit polypeptide or the combination according to third aspect, and optionally wherein AG1 and AG2 are two different antigens or are substantially the same antigen.

[0034] In a seventh aspect, the invention provides a pharmaceutical composition comprising the:

[0035] (i) the chimeric coreceptor subunit polypeptide according to the first aspect,

[0036] (ii) the combination according to the third aspect,

[0037] (iii) the nucleic acid construct (N-CCR), the nucleic acid construct (N-CCR-CAR), the two nucleic acid constructs (N-CCR, N-CAR), the nucleic acid construct (N-CCR-tgTCR) or the two nucleic acid constructs (N-CCR, N-tgTCR) according to the fourth aspect, or the vector or the kit of the first and second vector as defined in the fifth aspect, or

[0038] (iv) the recombinant cell according to the sixth aspect, and a pharmaceutically acceptable carrier, stabilizer and / or excipient.

[0039] In an eighth aspect, the invention provides a chimeric coreceptor subunit polypeptide according to the first aspect, a combination according to the third aspect, a nucleic acid construct (N-CCR), a nucleic acid construct (N-CCR-CAR), a two nucleic acid constructs (N-CCR, N-CAR), a nucleic acid construct (N-CCR-tgTCR) or two nucleic acid constructs (N-CCR, N-tgTCR) according to the fourth aspect, a vector or a kit of a first and second vector according to the fifth aspect, a recombinant cell according to the sixth aspect, or a pharmaceutical composition according to the seventh aspect, for use in medicine.

[0040] In a ninth aspect, the invention provides a chimeric coreceptor subunit polypeptide according to the first aspect, a combination according to the third aspect, a nucleic acid construct (N-CCR), a nucleic acid construct (N-CCR-CAR), a two nucleic acid constructs (N-CCR, N-CAR), a nucleic acid construct (N-CCR-tgTCR) or two nucleic acid constructs (N-CCR, N-tgTCR) according to the fourth aspect, a vector or a kit of a first and second vector according to the fifth aspect, a recombinant cell according to the sixth aspect, or a pharmaceutical composition according to the seventh aspect, for use in treating cancer, and wherein the cancer is preferably characterized by a cell-surface expression of at least one tumor-associated antigen selected from the group consisting of ALK, B7-H3 (CD276), BCMA, c-MET, CD3, CD5, CD19, CD20, CD22, CD23, CD30, CD33, CD38, CD44, CD44v6, CD52, CD70, CD79A, CD79B, CD123, CD138, CD171, CEA, Claudin-6, Claudin-18.2, CLL1, CXCR5, EGFR, EGFRvIII, EPH-receptor A2, IGLV3-21, IL-1RAP, GPC-2, GPC-3, HER2, ErbB3, ErbB4, FAP, FBP, AchR, Fr-a, GD2, GD3, HMW- MAA, IE-13Ra2, Kappa-EC, IGEV3-21-R110, Eewis Y, EMP1, EMP2A, Mesothelin, MAGE-4A, MUC1, MUC16, NKG2D Eigands, NCAM, NY-ESO1, Oncofetal antigen h5T4, PRAME, PSCA, PSMA, ROR1, TAG-72, VEGFR, GOLPH2, and SLAMF7, and / or wherein the cancer is preferably a B cell malignancy.

[0041] In a tenth aspect, the invention provides a CAR, a nucleic acid comprising a nucleic acid encoding a CAR, a vector comprising a nucleic acid encoding a CAR, or a cell comprising said nucleic acid or vector for use in the treatment of a cancer in combination with a chimeric coreceptor subunit polypeptide according to the first aspect, a chimeric coreceptor homodimer or a chimeric coreceptor complex according to the second aspect, a nucleic acid construct (N-CCR) according to the fourth aspect, or a vector comprising said N-CCR.

[0042] In an eleventh aspect, the invention provides a method of treating cancer in a subject in need thereof, comprising the step of administering to the subject an effective amount of:

[0043] (i) the chimeric coreceptor subunit polypeptide according to the first aspect,

[0044] (ii) the combination according to the third aspect,

[0045] (iii) the nucleic acid construct (N-CCR), the nucleic acid construct (N-CCR-CAR), the two nucleic acid constructs (N-CCR, N-CAR), the nucleic acid construct (N-CCR-tgTCR) or the two nucleic acid constructs (N-CCR, N-tgTCR) according to the fourth aspect, or the vector or the kit of the first and second vector as defined in the fifth aspect, or

[0046] (iv) the recombinant cell according to the sixth aspect,

[0047] (v) a pharmaceutical composition according to the seventh aspect, and wherein the cancer is preferably characterized by a cell-surface expression of at least one tumor- associated antigen selected from the group consisting of ALK, B7-H3 (CD276), BCMA, c-MET, CD3, CD5, CD19, CD20, CD22, CD23, CD30, CD33, CD38, CD44, CD44v6, CD52, CD70, CD79A, CD79B, CD123, CD138, CD171, CEA, Claudin-6, Claudin-18.2, CLL1, CXCR5, EGFR, EGFRvIII, EPH-receptor A2, IGLV3-21, IL-1RAP, GPC-2, GPC-3, HER2, ErbB3, ErbB4, FAP, FBP, AchR, Fr-a, GD2, GD3, HMW-MAA, IE-13Ra2, Kappa-EC, IGEV3-21-R110, Eewis Y, EMP1, EMP2A, Mesothelin, MAGE-4A, MUC1, MUC16, NKG2D Eigands, NCAM, NY-ES01, Oncofetal antigen h5T4, PRAME, PSCA, PSMA, R0R1, TAG-72, VEGFR, G0LPH2, and SLAMF7, and wherein the cancer is preferably a B cell malignancy.

[0048] Brief description of the drawings

[0049] In the following, the content of the figures comprised in this specification is described. In this context please also refer to the detailed description of the invention above and / or below.

[0050] Figure 1: A) illustrates the structure of a chimeric coreceptor comprising an IgVD domain according to the present invention; B) illustrates schematically the difference between an exemplary CAR and a chimeric CD8 IgVD coreceptor. The left drawing in Figure IB depicts a chimeric CD8 coreceptor polypeptide containing an antigen-recognition domain (in this example an anti-CD19 scFv, clone FMC63) fused to the hinge domain of the CD8a coreceptor polypeptide (control construct). The right drawing in Figure IB depicts a chimeric CD8 IgVD coreceptor containing an antigen-recognition domain fused to the IgV-like domain of the CD8a coreceptor. The chimeric CD8 coreceptors may be co-expressed with a second-generation CAR in a T cell (in this example an anti-CD22 CAR containing the m971 scFv). When expressed in a CD8 T cell, the chimeric CD8 IgVD coreceptor polypeptide may pair with an endogenous CD80 subunit polypeptide to form a monovalent chimeric CD8 coreceptor complex (Figure 1C). When expressed in a CD4 T cell, the chimeric CD 8 IgVD coreceptor polypeptide may undergo homotypic interactions to form a divalent chimeric CD8 coreceptor dimer (Figure ID). The chimeric CD8 IgVD coreceptor polypeptides contain CD8 or CD4 intracellular domains and lack a CD3(^ T cell activation domain, so that they can only act in trans to amplify CAR signalling, e.g. by facilitating the recruitment of LCK.

[0051] Figure 2 shows expression of exemplary anti-CD22 CAR (22-28hi / tm-BBz, SEQ ID NO: 87) and anti-CD19 chimeric CD8 coreceptors on the surface of transduced primary human T cells, as determined by flow cytometry. Chimeric CD8 coreceptors with or without an IgV-like domain showed equal cell-surface expression.

[0052] Figure 3 shows the differentiation status of CD4+ or CD8+ anti-CD22 CAR-T cells (CAR= 22-28hi / tm-BBz, SEQ ID NO: 87) co-expressing an anti-CD19 chimeric CD8 coreceptor, as determined by flow cytometric analysis of CD62L and CD45RA expression. CD4 and CD8 CAR- T cells co-expressing a chimeric CD8 coreceptor contained increased numbers of CD62L+CD45RA+ T cells representing undifferentiated stem cell-like memory T cells (Tscm) compared to conventional CAR-T cells or untransduced T cells.

[0053] Figure 4 shows expression of (A) CD22, (B) CD 19 and (C) CD58 proteins on the cellsurface of wild-type and engineered NALM6 cell lines. Quantification of CD22 or CD 19 molecules per cell was performed by flow cytometry using PE-labeled anti-CD22 and CD 19 antibodies and Quantibrite™ PE quantification beads (BD Biosciences) and the calculated numbers are depicted on the right of Figure 4 A and B.

[0054] Figure 5 shows expression of CD69 and CD25 activation markers on unstimulated anti- CD22 CAR-T cells, or CAR-T cells co-cultured with wild-type NALM6 cells, CD22 very-low or ultra-low NALM6 cells, or CD58 -deficient NALM6 cells at an effector-to-target ratio of 1:1 for 24 hours. Co-expression of an anti-CD19 chimeric CD8 IgVD coreceptor enhanced CAR-T cell activation in response to NALM6 tumor cells expressing reduced CD22 antigen densities or lacking CD58 expression, which more closely reflects the situation found in relapsing and / or nonresponding leukemia or lymphoma patients. In particular, the chimeric CD8 IgVD coreceptor outperformed the control construct lacking this domain. 22-BBz CAR = 22-28hi / tm-BBz, SEQ ID NO: 87.

[0055] Figure 6 shows the concentration of (A) IFNy, (B) IL-2, (C) TNFa and (D) GM-CSF in supernatants of anti-CD22 CAR-T cells co-expressing an anti-CD19 chimeric CD8 IgVD coreceptor co-cultured with the indicated tumor cell lines for 24 hours. The concentration of indicated cytokines were measured by a Bio-plex assay (Bio-Rad) in technical duplicates. Coexpression of a chimeric CD8 IgVD coreceptor enhanced secretion of effector cytokines by CAR- T cells in response to NALM6 tumor cells expressing reduced CD22 antigen densities or lacking CD58 expression. The chimeric CD8 IgVD coreceptor outperformed the control construct lacking this domain. 22-BBz CAR = 22-28hi / tm-BBz, SEQ ID NO: 87.

[0056] Figure 7 shows the cytotoxicity of anti-CD22 CAR-T cells with or without co-expressing an anti-CD19 chimeric CD8 IgVD coreceptor co-cultured with different NALM6 cell clones expressing varying amounts of CD22 molecules per cell, or lacking CD58 protein expression, at an effector-to-target ratio of 1:1 for 48 hours. Co-expression of a chimeric CD8 IgVD coreceptor enhanced killing of NALM6 tumor cells expressing reduced CD22 antigen densities or lacking CD58 expression. 22-BBz CAR = 22-28hi / tm-BBz, SEQ ID NO: 87.

[0057] Figure 8 shows expression of anti-CD22 CAR and anti-CD19 chimeric CD8 coreceptors on the surface of transduced primary human T cells, as determined by flow cytometry. Chimeric CD8 coreceptors having CD8 or CD4 intracellular domains, with or without an IgVD, showed equal cell-surface expression. 22-BBz CAR = 22-28hi / tm-BBz, SEQ ID NO: 87.

[0058] Figure 9 shows expression of CD69 and CD25 activation markers on unstimulated anti- CD22 CAR-T cells, or CAR-T cells co-cultured with wild-type, CD22 ultra- low, or CD58-deficient NALM6 cells at an effector-to-target ratio of 1:1 for 24 hours. Co-expression of an anti-CD19 chimeric CD8 IgVD coreceptor enhanced CAR-T cell activation in response to NALM6 tumor cells expressing reduced CD22 antigen densities or lacking CD58 expression. The chimeric CD8 IgVD coreceptors having a CD4 intracellular domain outperformed the chimeric CD 8 IgVD coreceptors with a CD8 intracellular domain and chimeric CD8 coreceptors lacking the IgVD. 22- BBz CAR = 22-28hi / tm-BBz, SEQ ID NO: 87.

[0059] Figure 10 shows the concentration of (A) IFNy, (B) IL-2 and (C) TNFa in supernatants of the indicated anti-CD22 CAR-T cell lines co-cultured with wild-type, CD22 ultra- low, or CD58- deficient NALM6 cells for 24 hours. CAR-T cells co-expressing an anti-CD19 chimeric CD8 IgVD coreceptor released more effector cytokines in response to NALM6 tumor cells expressing only 40 CD22 molecules per cell or lacking CD58 expression compared to conventional CAR-T cells. The chimeric CD 8 IgVD coreceptors having a CD4 intracellular domain outperformed the chimeric CD8 IgVD coreceptors with a CD8 intracellular domain and chimeric CD8 coreceptors lacking the IgVD. 22-BBz CAR = 22-28hi / tm-BBz, SEQ ID NO: 87.

[0060] Figure 11 shows the cytotoxicity of anti-CD22 CAR-T cells with or without co-expressing an anti-CD19 chimeric CD8 coreceptor against (A) wild-type, (B) CD22 ultra-low, and (C) CD58- deficient NALM6 cells. CAR-T cells co-expressing a chimeric CD8 IgVD coreceptor showed enhanced cytotoxicity against NALM6 tumor cells expressing only 40 CD22 molecules per cell or lacking CD58 expression, and chimeric CD8 IgVD coreceptors having a CD4 intracellular domain outperformed the chimeric CD8 IgVD coreceptors with a CD8 intracellular domain and chimeric CD8 coreceptors lacking the IgVD at low effector:target ratios. 22-BBz CAR = 22-28hi / tm-BBz, SEQ ID NO: 87.

[0061] Figure 12 shows expression of exemplary CAR and chimeric CD8 coreceptors with irrelevant specificity (gB protein of Herpes Simplex Virus, HSV) on the surface of transduced primary human T cells, as determined by flow cytometry. 22-BBz CAR = 22-28hi / tm-BBz, SEQ ID NO: 87.

[0062] Figure 13 shows expression of CD69 and CD25 activation markers on unstimulated CAR- T cells with or without co-expression of a chimeric CD8 IgVD coreceptors, or CAR-T cells cocultured with wild-type or CD58-deficient NALM6 cells at an effector-to-target ratio of 1:1 for 24 hours. Simultaneous engagement of both anti-CD22 CAR and anti-CD19 chimeric CD 8 IgVD coreceptors was required to enhance activation of CAR-T cells. 22-BBz CAR = 22-28hi / tm-BBz, SEQ ID NO: 87; HSV-BBz CAR = HSV-28hi / tm-BBz, SEQ ID NO: 89.

[0063] Figure 14 shows the concentration of (A) IFNy, (B) IL-2 and (C) TNFa in supernatants of the indicated CAR-T cell lines co-cultured with wild-type or CD58-deficient NALM6 cells for 24 hours. Simultaneous engagement of both anti-CD22 CAR and anti-CD19 chimeric CD 8 IgVD coreceptors was required to enhance the release of effector cytokines by CAR-T cells. 22-BBz CAR = 22-28hi / tm-BBz, SEQ ID NO: 87; HSV-BBz CAR = HSV-28hi / tm-BBz, SEQ ID NO: 89.

[0064] Figure 15 shows the cytotoxicity of indicated CAR-T cell lines co-expressing a chimeric CD8 IgVD coreceptor against (A) wild-type, CD22 ultra-low, and CD58-deficient NALM6 cells, at an effector:target ratio of 1:1, or (B) against Nalm6 cells at a low ratio of 1:5. Simultaneous engagement of both anti-CD22 CAR and anti-CD19 chimeric CD8 IgVD coreceptors was required for efficient elimination of tested tumor cell lines, whereas individual engagement of either the anti-CD22 CAR or the anti-CD19 chimeric CD8 IgVD coreceptor was unable to trigger killing of tumor cells at a low effectortarget ratio. 22-BBz CAR = 22-28hi / tm-BBz, SEQ ID NO: 87; HSV- BBz CAR = HSV-28hi / tm-BBz, SEQ ID NO: 89.

[0065] Figure 16 shows expression of CD69 and CD25 activation markers on unstimulated anti- CD22 CAR-T cells, or CAR-T cells co-cultured with primary autologous B cells at an effector-to- target ratio of 1:1 for 24 hours. Simultaneous engagement of both anti-CD22 CAR and anti-CD19 chimeric CD8 IgVD coreceptors was required to trigger activation of CAR-T cells, whereas individual engagement of the anti-CD19 chimeric CD8 IgVD coreceptor did not lead to CAR-T cell activation in the absence of CAR signalling. 22-BBz CAR = 22-28hi / tm-BBz, SEQ ID NO: 87; HSV-BBz CAR = HSV-28hi / tm-BBz, SEQ ID NO: 89.

[0066] Figure 17 shows the concentration of IFNy, IL-2 and TNFa in supernatants of CAR-T cells co-expressing an anti-CD19 chimeric CD8 IgVD coreceptor that were co-cultured with primary autologous B cells for 24 hours. Simultaneous engagement of both anti-CD22 CAR and anti-CD19 chimeric CD 8 IgVD coreceptors was required to induce the release of effector cytokines by CAR- T cells, whereas individual engagement of the anti-CD19 chimeric IgVD CD8 coreceptor did not lead to cytokine release in the absence of CAR signalling. 22-BBz CAR = 22-28hi / tm-BBz, SEQ ID NO: 87; HSV-BBz CAR = HSV-28hi / tm-BBz, SEQ ID NO: 89.

[0067] Figure 18 shows the cytotoxicity of CD4 or CD8 anti-CD22 CAR-T cells co-expressing an anti-CD19 chimeric CD8 coreceptor against (A) wild-type, (B) CD22 very-low, and (C) CD58- deficient NALM6 cells, at an effector: target ratio of 1:1. Co-expressing a chimeric CD8 IgVD coreceptor enhanced the cytotoxicity of both CD4 and CD8 CAR-T cells against tumor cells with reduced CD22 site densities or absent CD58 expression. 22-BBz CAR = 22-28hi / tm-BBz, SEQ ID NO: 87; HSV-BBz CAR = HSV-28hi / tm-BBz, SEQ ID NO: 89.

[0068] Figure 19 shows expression of CD22, CD58 and PD-L1 proteins on the cell- surface of wild-type and engineered NALM6 tumor cell lines, as determined by flow cytometry.

[0069] Figure 20 shows the concentration of (A) IFNy, (B) IL-2 and (C) TNFa in supernatants of anti-CD22 CAR-T cells with or without co-expressing an anti-CD19 chimeric CD8 coreceptor that were co-cultured with NALM6 cell lines overexpressing PD-L1 for 24 hours. CAR-T cells coexpressing a chimeric CD8 IgVD coreceptor showed enhanced secretion of effector cytokines when stimulated with PD-L1 expressing NALM6 cells with normal or reduced CD22 expression, or with normal or absent CD58 expression. The chimeric CD8 IgVD coreceptors outperformed the control coreceptors without this domain. 22-BBz CAR = 22-28hi / tm-BBz, SEQ ID NO: 87.

[0070] Figure 21 shows the cytotoxicity of anti-CD22 CAR-T cells co-expressing an anti-CD19 chimeric CD8 coreceptor against wild-type, CD22 very-low, and CD58-deficient NALM6 cells overexpressing PD-L1, at an effector:target ratio of (A) 1:1 or (B) 1:5. Co-expressing a chimeric CD8 IgVD coreceptor enhanced the cytotoxicity of CAR-T cells against PD-L1 positive tumor cells, even at reduced CD22 antigen densities or absent CD58 protein expression. 22-BBz CAR = 22-28hi / tm-BBz, SEQ ID NO: 87.

[0071] Figure 22 A) illustrates schematically the difference between an exemplary CAR and a chimeric CD28 coreceptor. The left drawing depicts a chimeric CD28 coreceptor polypeptide containing an antigen-recognition domain (in this example an anti-CD19 scFv, clone FMC63) fused to the hinge domain of the CD28 coreceptor polypeptide, which is used in the experiments as control. The right drawing depicts a chimeric CD28 IgVD coreceptor polypeptide containing an antigen -recognition domain fused to the IgV-like domain of the CD28 coreceptor. The chimeric CD28 coreceptors may be co-expressed with a second-generation CAR in a T cell (in this example an anti-CD22 CAR containing the m971 scFv), whereas the CAR contains hinge and transmembrane domains different from CD28 (in this example a CD8a hinge and transmembrane domain). B) shows interaction between a chimeric CD28 IgVD coreceptor polypeptide and a CD28 subunit polypeptide expressed on a cell surface.

[0072] Figure 23 shows expression of exemplary CAR and chimeric CD28 coreceptors on the surface of transduced primary human T cells, as determined by flow cytometry. (A) shows dot-plot graphs and (B) shows the median fluorescence intensities of CAR and chimeric CD28 coreceptor expression summarized from 3 measurements.

[0073] Figure 24 shows expression of CD69 and CD25 activation markers on anti-CD22 CAR-T cells co-cultured with wild-type or engineered NALM6 cell lines. Co-expressing a functional antiCD 19 chimeric CD28 IgVD coreceptor strongly enhanced CAR-T cell activation.

[0074] Figure 25 shows the concentration of (A) IFNy, (B) IL-2 and (C) TNFa in supernatants of anti-CD22 CAR-T cells with or without co-expressing an anti-CD19 chimeric CD28 coreceptor after a 24-hour co-culture with wild-type or engineered NALM6 cell lines. Co-expressing a functional chimeric CD28 IgVD coreceptor enhanced cytokine secretion by CAR-T cells in response to CD22-low and CD58-deficient tumor cell lines, even in the presence of PD-L1 expression.

[0075] Figure 26 shows the cytotoxicity of anti-CD22 CAR-T cells co-expressing an anti-CD19 chimeric CD28 coreceptor against wild-type and engineered NALM6 cell lines at an effector:target ratio of (A) 1:1 or (B) 1:5. Co-expressing a chimeric CD28 IgVD coreceptor enhanced the cytotoxicity of CAR-T cells against CD22-low and CD58-deficient tumor cells, even at high PD- L1 expression.

[0076] Figure 27 shows the proliferation of anti-CD22 CAR-T cells after 4 days of co-culture with wild-type, CD22-low or CD58-deficient NALM6 cell lines. Co-expressing a functional anti-CD19 chimeric CD28 IgVD coreceptor strongly enhanced CAR-induced proliferation of T cells, even at low CD22 densities or absent CD58 protein expression. Figure 28 shows the concentration of IFNy, IL-2 and TNFa in supernatants of anti-CD22 CAR-T cells with or without co-expressing an anti-CD19 chimeric CD28 coreceptor after a 24- hour co-culture with autologous primary B cells. Co-engagement of CAR and chimeric CD28 coreceptors enhanced cytokine secretion, whereas engagement of the full-length chimeric CD28 coreceptor having an IgV-like domain resulted in low cytokine secretion in the absence of concomitant CAR engagement.

[0077] Figure 29 shows the proliferation of anti-CD22 CAR-T cells after 4 days of co-culture with autologous primary B cells.

[0078] Figure 30 shows co-expression of a CD22-specific CAR and an anti-HSV chimeric CD28 coreceptor on the surface of transduced primary human T cells, as determined by flow cytometry.

[0079] Figure 31 shows expression of CD69 and CD25 activation markers on anti-CD22 CAR-T cells co-expressing either a CD 19- or a HSV-specific chimeric CD28 coreceptor, or without coexpressing a chimeric CD28 coreceptor, co-cultured with wild-type or CD22-low NALM6 cell lines. Co-expressing a chimeric CD28 coreceptor with irrelevant specificity inhibits CAR-mediated T cell activation.

[0080] Figure 32 shows the concentration of IFNy and IL-2 in supernatants of anti-CD22 CAR-T cells with or without co-expressing an anti-CD19 or anti-HSV chimeric CD28 coreceptor after a 24-hour co-culture with wild-type or CD22-low NALM6 cells. Cytokine secretion is severely impaired in CAR / chimeric CD28 coreceptor T cells in the absence of chimeric coreceptor engagement.

[0081] Figure 33 shows the cytotoxicity of anti-CD22 CAR-T cells with or without co-expressing an anti-CD19 or anti-HSV himeric CD28 coreceptor co-cultured with wild- type or CD22-low NALM6 cells for 72 hours. Cytotoxicity of CAR / chimeric CD28 coreceptor T cells in the absence of chimeric coreceptor engagement.

[0082] Figure 34 shows the phenotype of anti-CD22 CAR-T cells co-expressing an anti-CD19 chimeric CD28 coreceptor, as determined by flow cytometric analysis of CD62L and CD45RA expression for CAR+CD4+ and CAR+CD8+ T cells. CAR-T cells co-expressing a chimeric CD28 polypeptide with an IgV-like domain contained increased numbers of CD62L+CD45RA+ CD4 and CD8 T cells representing stem cell-like memory T cells (Tscm) compared to conventional CAR-T cells or CAR-T cells co-expressing a chimeric CD28 polypeptide lacking a IgV-like domain.

[0083] Figure 35 shows the absolute numbers of CAR-T cells circulating in the peripheral blood of mice bearing (A) CD221ow or (B) CD58-deficient leukemia 11 days after tumor inoculation. Anti-CD22 CAR-T cells co-expressing an anti-CD19 chimeric CD28 polypeptide expanded more efficiently in vivo compared to conventional 2nd generation anti-CD22 CAR-T cells, and the construct having an IgV-like domain (19-28IgV) was the most effective.

[0084] Figure 36 shows tumor progression of (A) CD22-low and (B) CD58-deficient leukemia in mice receiving anti-CD22 CAR-T cells or untransduced T cells, as determined by bioluminescence imaging (BLI). The top graph in (A) and (B) shows BLI imaging of mice, and the bottom graph shows total flux (p / s) of each group of mice from the top graph, respectively. Co-expression of an anti-CD19 chimeric CD28 polypeptide significantly enhanced antitumor activity of anti-CD22 CAR-T cells, whereas the construct having an IgV-like domain was significantly more effective in the CD58-deficient tumor setting as the counterpart lacking this domain.

[0085] Figure 37 shows Kaplan-Meier survival curves of mice bearing (A) CD22-low or (B) CD58-deficient leukemia and treated with anti-CD22 CAR-T cells or untransduced T cells. CAR- T cells co-expressing an anti-CD19 chimeric CD28 polypeptide significantly prolonged the survival compared to mice received conventional CAR-T cells. Significance was calculated by the Log-Rank (Mantel-Cox) test.

[0086] Figure 38 shows expression of anti-CD22 CAR and anti-CD22 chimeric CD28 polypeptides on the surface of transduced T cells. (A) shows dot-plot graphs of flow cytometric analyses, and graph in (B) shows quantification of median fluorescence intensities. The incorporation of an IgV-like domain greatly increased the expression of the chimeric CD28 polypeptide on the cell surface.

[0087] Figure 39 shows expression of CD69 and CD25 activation markers on anti-CD22 CAR-T cells with or without co-expressing a CD22-specific chimeric CD28 polypeptide after a 24-hour co-culture with wild-type or engineered NALM6 cell lines carrying CAR-T cell-resistance mechanisms. Chimeric CD28 polypeptides enhance CAR-mediated T cell activation when both receptor types recognize the same antigen (here CD22), and the construct with IgV-like domain can enhance T cell activation more effectively than the counterpart without this domain.

[0088] Figure 40 shows the concentration of (A) IFNy, (B) IL-2 and (C) TNFa in supernatants of anti-CD22 CAR-T cells with or without co-expressing an anti-CD22 chimeric CD28 coreceptor after a 24-hour co-culture with wild-type, CD22-low or CD58-deficient NALM6 cells. Chimeric CD28 polypeptides can enhance CAR-mediated release of effector cytokines when both receptor types recognize the same antigen (here CD22), and the construct with IgV-like domain can enhance cytokine secretion more effectively than the counterpart without this domain in response to antigen- low or CD58-deficient tumors.

[0089] Figure 41 shows the cytotoxicity of anti-CD22 CAR-T cells with or without co-expressing an anti-CD22 chimeric CD28 polypeptide against K562 (CD22- control), wild-type and engineered NALM6 cell lines carrying CAR-T cell-resistance mechanisms at an effector:target ratio of 1:1. The growth of GFP-positive tumor cells was monitored by an Incucyte live cell imaging system. Chimeric CD28 polypeptides can enhance CAR-mediated killing of tumor cells when both receptor types recognize the same antigen (here CD22).

[0090] Figure 42 shows the proliferation of anti-CD22 CAR-T cells with or without co-expressing a CD22-specific chimeric CD28 polypeptide after 4 days co-cultured with wild-type or engineered NALM6 cell lines carrying CAR-T cell-resistance mechanisms at an effector:target ratio of 1:1 in cytokine-free culture medium. The percentages indicate the proportion of T cells with 3 or more cell divisions. Chimeric CD28 polypeptides enhance CAR-mediated T cell proliferation when both receptor types recognize the same antigen (here CD22).

[0091] Figure 43 shows expression of anti-CD22 CAR and anti-CD22 chimeric CD8 polypeptides on the surface of transduced T cells. (A) shows dot-plot graphs of flow cytometric analyses, and graph in (B) shows quantification of median fluorescence intensities. The incorporation of an IgV- like domain greatly increased the expression of the chimeric CD8 polypeptides on the cell surface.

[0092] Figure 44 shows the concentration of (A) IFNy, (B) IL-2 and (C) TNFa in supernatants of anti-CD22 CAR-T cells with or without co-expressing an anti-CD22 chimeric CD8 coreceptor after a 24-hour co-culture with wild-type, CD22-low or CD58-deficient NALM6 cells. Chimeric CD8 polypeptides with CD8a-derived IgV-like and intracellular domains can enhance CAR-mediated release of effector cytokines when both receptor types recognize the same antigen (here CD22).

[0093] Figure 45 shows the cytotoxicity of anti-CD22 CAR T cells co-expressing anti-CD22 chimeric CD8 polypeptides. (A-I), (A-II) Cytotoxicity of anti-CD22 CAR-T cells with or without co-expressing an anti-CD22 chimeric CD8 polypeptide against K562 (CD22- control), wild-type and engineered NALM6 cell lines carrying CAR-T cell-resistance mechanisms at an effector: target ratio of 1:1. (B) Cytotoxicity of engineered T cells against PD-L1 negative and positive CD22-low NALM6 cells at an effector: target ratio of 1:5. The growth of GFP-positive tumor cells was monitored by an Incucyte live cell imaging system. Chimeric CD8 polypeptides with CD8a- derived IgV-like and intracellular domains can enhance CAR-mediated killing of tumor cells when both receptor types recognize the same antigen (here CD22), even at low antigen densities and low effector- target ratios.

[0094] Figure 46 shows the proliferation of anti-CD22 CAR-T cells with or without co-expressing a CD22-specific chimeric CD8 polypeptide after 4 days co-cultured with wild-type or engineered NALM6 cell lines carrying CAR-T cell-resistance mechanisms at an effector:target ratio of 1:1 in cytokine-free culture medium. The percentages indicate the proportion of T cells with 3 or more cell divisions. Chimeric CD8 polypeptides with CD8a-derived IgV-like and intracellular domains can enhance CAR-mediated T cell proliferation when both receptor types recognize the same antigen (here CD22), even at low antigen densities and expression of PD-L1 on tumor cells.

[0095] Nucleotide and amino acid sequences

[0096] The following Table 1 provides an overview over the sequences referred to herein.

[0097] Table 1: SEO ID NOs referred to in the application

[0098]

[0099]

[0100] DETAILED DESCRIPTION OF THE INVENTION

[0101] Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0102] Preferably, the terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, HGW, Nagel, B and Klbl, H eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland) and as described in "Pharmaceutical Substances: Syntheses, Patents, Applications" by Axel Kleemann and Jurgen Engel, Thieme Medical Publishing, 1999; the "Merck Index: An Encyclopedia of Chemicals, Drugs, and Biologicals", edited by Susan Budavari et al., CRC Press, 1996, and the United States Pharmacopeia-25 / National Formulary-20, published by the United States Pharmacopeial Convention, Inc., Rockville Md., 2001.

[0103] To practice the present invention, unless otherwise indicated, conventional methods of chemistry, biochemistry, and recombinant DNA techniques are employed which are explained in the literature in the field (cf., e.g., Molecular Cloning: A Laboratory Manual, 2ndEdition, Sambrook J et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0104] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated feature, integer or step or group of features, integers or steps but not the exclusion of any other feature, integer or step or group of integers or steps. As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents, unless the content clearly dictates otherwise. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it was individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as"), provided herein is intended merely to better illustrate the disclosure and does not pose a limitation on the scope of the claims. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the disclosure.

[0105] In the following passages different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0106] Various modifications and variations of the invention will be apparent to those skilled in the art without departing from the scope of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the relevant fields are intended to be covered by the present invention.

[0107] Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0108] Aspects of the invention and particular embodiments thereof

[0109] The invention relates to several aspects as set out above in the summary of the invention. These aspects comprise alternative embodiments and preferred embodiments, which are described below.

[0110] Chimeric coreceptors The present inventors have discovered that a chimeric coreceptor comprising an IgV -like domain (also referred to herein as "chimeric IgVD coreceptor" or "chimeric IgVD CCR") can enhance the activation of CAR T cells and their sensitivity to tumor cells.

[0111] Therefore, a first aspect of the invention provides a chimeric coreceptor (CCR) subunit polypeptide comprising:

[0112] (a) an extracellular antigen -recognition domain 1 (ARD1) specifically binding an antigen

[0113] 1 (AG1),

[0114] (b) an immunoglobulin variable-like domain (IgVD),

[0115] (c) a hinge domain 1 (HD1),

[0116] (d) a transmembrane domain 1 (TMD1), and

[0117] (e) a cytosolic domain (CYTD):

[0118] (i) comprising a costimulatory domain (CSD), and

[0119] (ii) not comprising a cell activation domain, wherein the ARD1 is linked to the IgVD.

[0120] The term "coreceptor" is used in the context of the present invention according to its traditional definition (Kdnig, R. Handbook of Cell Signaling 2ndEd., 2010, Vol. 3, pp. 2679-2688) to refer to a protein that associates with an antigen receptor, such as a CAR or a TCR, exerts complex regulatory effects on T cell activation, and is characterized by a temporal association with the CAR or the TCR that is triggered only by binding to its specific antigen expressed on a tumor cell. A coreceptor may comprise the following domains: an extracellular domain or ectodomain, a hinge domain, a transmembrane domain, and / or a cytoplasmic domain.

[0121] The term "chimeric coreceptor (CCR) subunit polypeptide", as used herein, describes a single amino acid chain comprising or consisting of the following structural elements in the order indicated: (a) an extracellular antigen -recognition domain 1 (ARD1) specifically binding an antigen 1 (AG1), (b) an immunoglobulin variable-like domain (IgVD) linked to ARD1, (c) a hinge domain 1 (HD1), (d) a transmembrane domain 1 (TMD1), and (e) a cytosolic domain (CYTD) (i) comprising a costimulatory domain (CSD), and (ii) not comprising a cell activation domain, wherein the cell activation domain preferably is a CD3zeta domain.

[0122] The "chimeric coreceptor (CCR) subunit polypeptide" as used in the context of the present invention does not comprise a TCR-recruitment domain, as is the case for the TAC disclosed by Helsen et al., 2018 (Helsen et al., 2018. Nat Commun. 9(l):3049). In addition, the "chimeric coreceptor (CCR) subunit polypeptide" as used in the context of the present invention does not comprise a primary intracellular signaling domain (PSD) or intracellular cell activation domain, such as a CD3<^ (CD3zeta) T cell activation domain or a Fc-epsilon-Receptor I-gamma (FceRIg) chain. Therefore, the "chimeric coreceptor (CCR) subunit polypeptide" is not able to transmit the primary signal (‘ signal- 1’) to induce immune cell activation, and has a structure different from a chimeric antigen receptor (CAR). Consequently, engagement of the ARD1 of the CCR to an AG1 does not induce immune cell activation in absence of a primary signal delivered by a TCR, tgTCR or CAR.

[0123] The terms “primary signaling domain” or “PSD” or “intracellular cell activation domain” are used interchangeably herein to refer to the intracellular activation domain, such as CD3(^ T cell activation domain or a Fc-epsilon-Receptor I-gamma activation domain, that transmits the primary signal (‘ signal- 1’) for T cell activation following antigen recognition. In particular, the CD3(^ T cell activation domain transmits the primary signal of the T cell receptor following TCR-mediated antigen recognition (see Thomas S. and Abken H. (2023) Frontiers in Immunology 13:1090959 and Weinkove R. el al. (2019) Clin. Trans. Immunol. E1049 for a review on signalling of CARs and CCR through CD3(^ (primary signal) and costimulatory domains (secondary signal)), or other primary signaling domains, such as the Fc-epsilon-Receptor I-gamma (FceRIg) chain.

[0124] The term "extracellular antigen-recognition domain 1 (ARD1) specifically binding an antigen 1", as used in the context of the present invention, refers to an extracellular domain capable of specifically binding a particular epitope or set of epitopes of the antigen 1 (AG1) present on a cell and preferably on a tumor cell.

[0125] The term "specifically binding", in the context of this invention, refers to the binding of an antigen-recognition domain or fragments thereof to a specific binding site of its target when the target comprises specific and non-specific binding sites. An antigen-recognition domain as comprised in the chimeric coreceptor of the present invention is considered to specifically bind if it binds stronger or enhanced to its target antigen than to one or more similar antigens.

[0126] The term "antigen" refers to an agent comprising an epitope against which an immune response is to be generated and / or is directed. The term "antigen" includes in particular proteins and peptides. According to the present invention, an antigen may comprise a naturally occurring antigen or a variant thereof, or a fragment of the naturally occurring antigen or variant thereof, wherein the antigen is expressed on the cell surface. Such naturally occurring antigens may include or be derived from allergens, viruses, bacteria, fungi, parasites and other infectious agents and pathogens, or an antigen may preferably be a tumor antigen. Preferably, an antigen in the context of the present invention is a molecule which, optionally after processing, induces an immune reaction, which is preferably specific for the antigen or cells expressing the antigen, preferably on the cell surface and preferably of malignant cells. Preferably, the term "antigen 1" or "antigen 2" refer to an antigen that has expression or reduced expression or low cell-surface expression on a cancer cell, i.e. a target cell.

[0127] An antigen expressed at an ultra-low (UL) density refers in the context of the present invention to an antigen expressed on a cell surface at a density lower than about 400 molecules / cell, or lower than about 40 molecules / cell. The antigen density can be measured by methods known in the state of the art, such as staining the cells with fluorescent antibodies specific for that antigen and acquiring the stained cells and quantification beads (e.g. Quantibrite, Becton Dickinson) by flow cytometry.

[0128] In some embodiments of the chimeric coreceptor subunit polypeptide of the first aspect,

[0129] (a) the ARD1 comprises or consists of:

[0130] (i) a mono-, bi- or multispecific scFv, a single domain antibody, or a single chain antibody-like protein scaffold, in particular an anticalin, DARPins, affibody, affimer, avimer, neutrophil gelatinase-associated lipocalin (NGAL) or evasin;

[0131] (ii) a ligand selected from the group comprising a cytokine, in particular IL-1, IL- 10, IL- 13, APRIL, GM-CSF, FLT3L; a growth factor, in particular TPO; an immunoglobulin superfamily protein, in particular adnectin; a tumor associated antigen (TA A) targeting chimeric peptides, in particular TIE; or any combination thereof;

[0132] (b) the IgVD comprises or consists of a CD8a IgVD, a CD8P IgVD, CD28 IgVD or variant thereof,

[0133] (c) the HD1 comprises or consists of a CD8a HD, a CD8P HD, or a CD28 HD, or dimerizing variant thereof,

[0134] (d) the TMD1 comprises or consists of a CD4, CD8a, CD8P, or CD28 TMD, or variant thereof, and / or

[0135] (e) the CSD comprises or consists of a CD4, CD8a, CD8P, CD28, CD2, CD27, CD40, CD278, 0X40, GITR, CD40L, CD30, CD44, or CD 146 intracellular signaling domain (ICD), or variant thereof. In some embodiments, the ARD1 is selected from the group comprising an anti-CD19, anti- CD5, anti-CD22, anti-CD38, anti-EGFR, anti-HER2, anti-Mesothelin, anti-IL-13Ra2, anti-PSMA, and anti-EPH-receptor-A2 antigen-recognition domain. In some preferred embodiments, the ARD1 is an scFv selected from the group comprising an anti-CD19, anti-CD5, anti-CD22, anti-CD38, anti-EGFR, anti-HER2, anti-Mesothelin, anti-IL-13Ra2, anti-PSMA, and anti-EPH-receptor-A2 scFv.

[0136] The term "antibody" in the context of the present invention refers to an immunoglobulin comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. Each VH and VL is composed of three complementarity determining regions (CDRs) and four framework regions (FRs), arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, and specifically for the VH: HFR1, HCDR1, HFR2, HCDR2, HFR3, HCDR3, HFR4, and for the VL: LFR1, LCDR1, LFR2, LCDR2, LFR3, LCDR3, LFR4. The variable regions of the heavy and light chains form a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. Antibodies may be intact immunoglobulins derived from natural sources or from recombinant sources and may be immunoreactive portions or fragments of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. The antigen recognizing domain ARD1 of the CCR of the present invention preferably comprises an antibody fragment such as a mono-, bi- or multispecific scFv or a single domain antibody, or a single chain antibody-like protein scaffold, and more preferably a monospecific scFv.

[0137] The term "scFv" (single-chain variable fragment) as used herein refers to an antibody fragment having the general structure:

[0138] VH-linker-VL or when referring to the CDRs (complementarity-determining regions) and FRs (framework region), the general structure:

[0139] HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4-Iinker-LFR1-LCDR1-LFR2-

[0140] LCDR2-LFR3-LCDR3-LFR4. A "bispecific scFv" refers to a fusion polypeptide comprising two scFvs from different antibodies or amino acid sequences from four different genes linked by a peptide linker. A "multispecific scFv" refers to a fusion polypeptide comprising more than two (e.g. three, four, or more) scFvs from different antibodies, or amino acid sequences from more than four different genes linked by a peptide linker.

[0141] The term "single-domain antibody" as used herein refers to a peptide chain of approximately 110 amino acids in length, comprising a variable domain (VH) of a heavy chain antibody or a common immunoglobulin G (IgG).

[0142] In some embodiments, the ARD1 comprises or consists of a mono-, bi- or multi- specific scFv. In preferred embodiments, the ARD1 comprises or consists of a mono-, bi- or multi- specific scFv, wherein the scFv is selected from the group comprising an anti-CD19 scFv, anti-CD5, anti- CD22, anti-CD38, anti-EGFR, anti-HER2, anti-Mesothelin, anti-IL-13Ra2, anti-PSMA, and anti- EPH-receptor-A2 scFv, and preferably is an anti-CD19 scFv. In more preferred embodiments, the ARD1 comprises or consists of a monospecific scFv (i.e. one scFv), preferably wherein the scFv is an anti-CD19 scFv.

[0143] In preferred embodiments, the ARD1 is an anti-CD19 ARD1 comprising a VH comprising or consisting of the amino acid sequence in SEQ ID NO: 27, or a variant or a fragment thereof having at least 95% sequence identity to SEQ ID NO: 27, and a VL comprising or consisting of the amino acid sequence in SEQ ID NO: 23, or a variant or a fragment thereof having at least 95% sequence identity to SEQ ID NO: 23 and optionally a linker sequence located between the VH and VL sequence, wherein the linker sequence preferably comprises or consists of the amino acid sequence in SEQ ID NO: 28, and wherein the ARD1 is preferably a scFv.

[0144] As used herein, the variant of an amino acid sequence of a VH or VL is to be understood as a functional variant that has identical functional properties to the parent amino acid sequence with respect to the ability to constitute the variable region that recognises and binds the target antigen AG1.

[0145] In preferred embodiments, the ARD1 is an anti-CD19 ARD1 comprising an HFR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 33, an HCDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 24, an HFR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 34, an HCDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 25, an HFR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 35, an HCDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 26, an HFR4 comprising or consisting of the amino acid sequence of SEQ ID NO: 36, optionally a linker comprising or consisting of the amino acid sequence of SEQ ID NO: 28, a LFR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 29, a LCDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 20, a LFR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 30, a LCDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 21, a LFR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 31, and a LCDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 22, and wherein the ARD1 is preferably a scFv.

[0146] In one embodiment of the present invention, the anti-CD19 ARD1 comprises a leader sequence preferably comprising or consisting of the amino acid sequence of SEQ ID NO: 19 (CD8a leader sequence).

[0147] In one embodiment of the present invention, the anti-CD19 ARD1 comprises

[0148] (1) a first variable domain comprising three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) of the light chain variable domain according to SEQ ID NO: 23, and

[0149] (2) a second variable domain comprising three heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) of the heavy chain variable domain according to SEQ ID NO: 27.

[0150] The positions of the CDRs and framework regions as defined herein are assigned according to Kabat, Chothia, or IMGT (Lefranc M.P., 1997. Immunology Today, 18, 509), in particular according to Kabat numbering, except for HCDR1, for which the combined Chothia / Kabat definition is applied. Thus, according to a preferred embodiment of the present invention, the numbering of the light and heavy chain variable regions described herein is according to Kabat. With the exception of HCDR1, when not otherwise specified, the antigen binding loops that were grafted onto the human framework regions as used herein are defined according to Kabat et al. 1991. (NIH Publication No. 91-3242, Bethesda). As residues H26 to H32 comprise the structural loop of HCDR1 (Chothia et al., 1989. Nature 342:877-883,), residues H26 to H35 were applied as HCDR1 according to the combined Kabat / Chothia definition of HCDR1. The CDRs and framework regions were thus defined according to Kabat, except for said HCDR1, for which the combined Chothia / Kabat definition was applied.

[0151] In preferred embodiments, the ARD1 is an anti-CD22 ARD1 comprising a VH comprising or consisting of the amino acid sequence in SEQ ID NO: 144, or a variant or a fragment thereof having at least 95% sequence identity to SEQ ID NO: 144, and a VL comprising or consisting of the amino acid sequence in SEQ ID NO: 140, or a variant or a fragment thereof having at least 95% sequence identity to SEQ ID NO: 140 and optionally a linker sequence located between the VH and VL sequence, wherein the linker sequence preferably comprises or consists of the amino acid sequence in SEQ ID NO: 10, and wherein the ARD1 is preferably a scFv.

[0152] In preferred embodiments, the ARD1 is an anti-CD22 ARD1 comprising an HFR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 149, an HCDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 141, an HFR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 150, an HCDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 142, an HFR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 151, an HCDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 143, an HFR4 comprising or consisting of the amino acid sequence of SEQ ID NO: 152, optionally a linker comprising or consisting of the amino acid sequence of SEQ ID NO: 10, a LFR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 145, a LCDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 137, a LFR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 146, a LCDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 138, a LFR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 147, and a LCDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 139, and wherein the ARD1 is preferably a scFv.

[0153] In one embodiment of the present invention, the anti-CD22 ARD1 comprises a leader sequence preferably comprising or consisting of the amino acid sequence of SEQ ID NO: 37 (IGHV3-23 leader sequence).

[0154] In one embodiment of the present invention, the anti-CD22 ARD1 comprises a leader sequence preferably comprising or consisting of the amino acid sequence of SEQ ID NO: 37 (IGHV3-23 leader sequence).

[0155] In one embodiment of the present invention, the anti-CD22 ARD1 comprises

[0156] (1) a first variable domain comprising three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) of the light chain variable domain according to SEQ ID NO: 140, and

[0157] (2) a second variable domain comprising three heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) of the heavy chain variable domain according to SEQ ID NO: 144. "Linker"

[0158] In the extracellular antigen-recognition domain 1 (ARD1) as described herein, the VH and VL are preferably connected by a flexible peptide linker having four or more amino acids. The term "linker" or "peptide linker" as used in the context of the present invention refers to an amino acid sequence which sterically separates two parts or moieties of a complex, e.g. two peptides, polypeptides or proteins. According to one embodiment, such linker comprises or consists of more than 4 amino acid residues, preferably of 4, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids, most preferably 15amino acid residues. Peptide linkers provide flexibility among the two moieties that are linked together. Flexibility is generally increased if the amino acids are small. Accordingly, flexible peptide linkers comprise an increased content of small amino acids, in particular of glycine, and / or hydrophilic amino acids such as serine, threonine, asparagine and glutamines. It is believed that peptide linkers inserted between the VH and VL allow the correct folding to form the antigen binding site.

[0159] According to a preferred embodiment, the linker connecting VH and VL of the ARD1 of the present invention comprises glycine (G) and serine (S) residues. More preferably, the linker preferably has the structure (G4S)Xwith x denoting any integer of between 1 and 6, such as G4S (SEQ ID NO: 125), (G4S)2(SEQ ID NO: 126), (G4S)3(SEQ ID NO: 10), (G4S)4(SEQ ID NO: 127), (G4S)S (SEQ ID NO: 128), and (G4S)e (SEQ ID NO: 129). Most preferably, linker has the amino acid sequence of SEQ ID NO: 10. A further preferred linker is the Whitlow linker L2 of SEQ ID NO: 28.

[0160] In some embodiments, the ARD1 is linked to the IgVD directly or by a linker. In some embodiments, the ARD1 is linked to the IgVD by a linker. The linker preferably has an amino acid sequence selected from the group comprising SEQ ID NO: 10, 125, 126, 127, 128 and 129, and more preferably SEQ ID NO: 10.

[0161] The term "single chain antibody-like protein scaffold" as used in the context of the invention refers to a small single-domain molecule (1-20 kDa) composed of two parts that are analogous to antibodies. In some embodiments, a "single chain antibody-like protein scaffold" is selected from the group comprising anticalin, DARPins (designed ankyrin repeat proteins), affibody, affimer, avimer, neutrophil gelatinase-associated lipocalin (NGAL) and evasin. Single chain antibody-like protein scaffolds are known in the state of the art and have been reviewed for example by Luo R., et al., 2022. RSC Chem. Biol., 3, 830-847. In some embodiments, the ARD1 comprises or consists of a mono-, bi- or multi- specific scFv, a single domain antibody, a single chain antibody-like protein scaffold, in particular an anticalin, DARPins, affibody, affimer, avimer, neutrophil gelatinase-associated lipocalin (NGAL) or evasin; or any combination thereof.

[0162] In some embodiments, the ARD1 comprises or consists of a ligand selected from the group comprising a cytokine, in particular IL-1, IL- 10, IL- 13, APRIL, GM-CSF, FLT3L; a growth factor, in particular TPO; an immunoglobulin superfamily protein, in particular adnectin; a tumor associated antigen (TAA) targeting chimeric peptides, in particular TIE; or any combination thereof.

[0163] The term “tumor associated antigen (TAA) targeting chimeric peptide” as used herein refers to a peptide that comprises one or more binding fragments of receptors or binding fragments of ligands or a binding peptide. The ligands and receptors from which the binding fragments are derived are preferably of human origin. The term “binding fragment” refers to that part of the receptor and ligand, respectively that is necessary and sufficient for the specific binding of the receptor to its ligand. A binding peptide refers to a peptide, typically 6 to 20 amino acids in length that has preferably been selected from a library of peptides for its ability to specifically bind to a given TAA. Such selection processes are well known in the art and may involve PEPSCAN approaches or phage display. To increase the affinity through avidity effects the TAA targeting chimeric peptide preferably comprises 2 to 10, i.e. 2, 3, 4, 5, 6, 7, 8, 9 or 10, binding fragments of a receptor or ligand or a binding peptide, which may be linked directly through peptide bonds or via a peptide linker. The use of peptide linkers is preferred, if the binding sites may otherwise not be available for binding of the TAA. Alternatively, or additionally the effects may be improved by including two or more different binding fragments of a receptor or ligand or a binding peptide that specifically bind to the same TAA or two TAA of the same family or the TAA and adjacent proteins. Such a TAA targeting chimeric peptide is also referred to as mixed TAA targeting chimeric peptide. An example of a mixed TAA targeting chimeric peptide is TIE, which is an Erbb targeting chimeric peptide in which the N-terminal seven amino acids from human transforming growth factor (TGF)-a have been fused to the C-terminal 48 amino acids of epidermal growth factor (EGF) (Davies DM, et al., 2012. Mol Med., 18(1), 565-76. Thus, a TAA targeting chimeric peptide may in particular be a TAA targeting chimeric ligand. The use of ligands such as cytokines, in particular IL-1, IL-10, IL-13, APRIL, GM-CSF, FLT3L; growth factors, in particular TPO; immunoglobulin superfamily proteins, in particular adnectin; and tumor associated antigen (TAA) targeting chimeric peptides, as binding moieties to target tumor cells and autoreactive B cells in autoimmune diseases is known in the state of the art and has been reviewed by Ramirez-Chacon A, et al., 2022. Front Immunol. 13:932559.

[0164] The terms "immunoglobulin variable-like domain (IgVD)" and “immunoglobulin V-set domain” are used interchangeably herein and, as used in the context of the present invention, refer to a domain having homology to an immunoglobulin (Ig) variable domain (Ig VH, VL) or to a TCR variable domain (TCR Va or V|3) for both sequence and structure (Chidyausiku TM et al., Nat Commun. 2022 Oct 3; 13(1):5661). An IgVD as used herein is part of the immunoglobulin superfamily (IgSF) and may comprise or consist of 7 to 11 beta strands, wherein the 7 to 11 beta strands are comprised in two sheets with Greek key beta-barrel topology. The general shape of Ig- like domains is well conserved, but they can differ significantly in their size, owing to high variability of the loops. An IgVD as used herein may comprise or consist of 70 to 110 amino acid residues (https: / / www.ebi.ac.uk / interpro / entry / profile / PS50835). Preferably, the IgVD is of human origin. The IgVD of a CCR may comprise the IgVD of a protein selected from the group consisting of T-cell co-receptors (e.g. CD8a, CD8P, CD28, CD2, CD4); checkpoint receptors (e.g. CD80, CD86, PD-1, CTLA-4, TIGIT, LAG-3, TIM-3, VSIG1, VSIG2, VSIG3, VSIG4, VSIG9, VISTA, CD112R, VSIG10, VSTM1-4); Lectins (e.g. Siglec-receptors, LAIR-1); myelin membrane adhesion molecules; junctional adhesion molecules (JAM) (e.g. CD111, CD 112); tyro sine-protein kinase receptors (e.g. Flt-1, VEGFR-2); and Fc-receptor / Fc-receptor-like family (e.g. FcaRI, FCRL5). In some embodiments, the "immunoglobulin variable-like domain (IgVD)" comprises or consists of a IgVD of a protein selected from the group consisting of AC AM; AC AN; ADAMTSL1; AGC1; AMICA1; BCAM; BCAN; BGP; BGPc; BT3.3; BTN1A1; BTN2A1; BTN2A2; BTN2A3; BTN3A1; BTN3A2; BTN3A3; BTNL2; BTNL3; BTNL8; BTNL9; C10orf54; Clorf32; C9orf94; CADM1; CADM2; CADM3; CADM4; CD2; CD28; CD226; CD274; CD276; CD300A; CD300C; CD300D; CD300E; CD300LB; CD300LF; CD300LG; CD33; CD3G; CD7; CD79A; CD79B; CD80; CD83; CD86; CD8A; CD8B; CD8B1; CD96; CEACAM1; CEACAM16; CEACAM19; CEACAM21; CEACAM3; CEACAM4; CEACAM5; CEACAM6; CEACAM7; CEACAM8; CHL1; CREA7-4; CRTAM; CSF1R; CTLA4; CXADR; ERMAP; ESAM; FUR; FCAMR; FCRL2; FKSG87; GLUDP5; GPA33; HAPLN1; HAPLN2; HAPLN3; HAPLN4; HAVCR1; HEPACAM; HHLA2; HSPG2; ICOSLG; IGHA1; IGHA2; IGHD; IGHG1; IGHG3; IGHM; IGHV1-69; IGHV4-31; IGHV7-81; IGKC; IGKV1-5; IGKV2- 24; IGLC6; IGLC1; IGLV2-14; IGLV3-21; IGLV3-25; IGLV4-3; IGLV5-52; IGLV6-57; IGSF11; IGSF2; IGSF3; IGSF6; IGSF8; IGSF9; IL18R1; IREM2; IREM3; JAM2; JAM3; KDR; KIRREL; KIRREL2; KIRREL3; LAG3; LOC253012; LOC402482; MAG; MGC33530; MOG; MPZ; MPZL1; MPZL2; MXRA8; MYBPC3; NCA; NCR2; NCR3; NPHS1; OBSL1; OPCML; PO; PDCD1; PIGR; PILRA; PILRB; PRODH2; PSG1; PSG10; PSG11; PSG-l ls; PSG2; PSG3; PSG4; PSG5; PSG6; PSG7; PSG8; PSG9; PTGFRN; PTPN1L; PVR; PVRL1; PVRL2; PVRL3; PVRL4; SCN2B; SCN3B; SCN4B; SEMA3D; SIGLEC1; SIGLEC10; SIGLEC11; SIGLEC12; SIGLEC14; SIGLEC15; SIGLEC6; SIGLEC7; SIGLEC8; SIGLEC9; SIRPA; SIRPB1; SIRPD; SIRPG; SISP1; SLAMF6; SLAMF7; TAPBPL; TCRA; TCRB; TIMD4; TCRA; TRAV20; TRBC1; TRBV19; TRBV3-1; TRBV5-4; TRBV7-2; TRDV2; TREM1; TREM2; TREML1; TREML2; TREML4; TRGV3; TRGV5; TRGV7; TRGV9; VCAM1; VCAN; VPREB1; VPREB3; VSIG1; VSIG2; VSIG4; VSIG9; VSIG10; VSTM1; VSTM2; VTCNE In some preferred embodiments, the "immunoglobulin variable-like domain (IgVD)" comprises or consists of a CD8a IgVD, a CD8P IgVD, CD28 IgVD or a variant thereof, preferably a functional variant thereof. A variant of CD8a IgVD or of a CD8P IgVD is more preferably a dimerizing variant. In more preferred embodiments, the immunoglobulin variable-like domain comprises or consists of an amino acid sequence selected from the group comprising SEQ ID NO: 62, 63 and 64 or a variant thereof, wherein the variant preferably has at least 80%, 81%, 82%, 83%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with SEQ ID NO: 62, 63 or 64. In preferred embodiments, the immunoglobulin variablelike domain comprises or consists of an amino acid sequence of SEQ ID NO: 62, or a variant thereof, preferably a dimerizing variant or dimerizing fragment thereof, wherein the variant has at least 80%, 81%, 82%, 83%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with SEQ ID NO: 62. In preferred embodiments, the immunoglobulin variable-like domain comprises or consists of an amino acid sequence of SEQ ID NO: 63, or a variant thereof, preferably a dimerizing variant or dimerizing fragment thereof, wherein the variant has at least 80%, 81%, 82%, 83%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with SEQ ID NO: 63. In preferred embodiments, the immunoglobulin variable-like domain comprises or consists of an amino acid sequence of SEQ ID NO: 64, or a variant thereof, wherein the variant has at least 80%, 81%, 82%, 83%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with SEQ ID NO: 64.

[0165] The term "dimerizing variant" of a domain (i.e. a parent domain) of chimeric coreceptor subunit polypeptide as used in the context of the invention refers to a functional variant of the parental domain which retains its ability to undergo homotypic or heterotypic interactions with the corresponding domain of a second chimeric coreceptor subunit polypeptide or of an endogenous coreceptor subunit, or which retains its ability to binds to a dimeric ligand thereby inducing dimerization of the chimeric coreceptor. In one embodiment, a "dimerizing variant" of an IgVD, HD1, TMD1, CYTD, ICD or CSD refers to a functional variant of the parental IgVD, HD1, TMD1, CYTD, ICD or CSD which retains its ability to undergo homotypic or heterotypic interactions with the corresponding IgVD, HD1, TMD1, CYTD, ICD or CSD of a second chimeric coreceptor subunit polypeptide or of a natural occurring coreceptor subunit. In particular, a dimerizing variant may preserve the one or more cysteines of the parent domain involved in forming disulphide bridges between two monomer polypeptides or the one or more amino acid motifs of the parent domain mediating non-covalent interations between the two monomer polypeptides (Wu et al., 2022, Structure 30, 803-812, Muller YD et al., 2021. Front. Immunol., Sec. Cancer Immunity and Immunotherapy, 12: 639818).

[0166] Additionally or alternatively, a "dimerizing variant" of a domain of a chimeric coreceptor subunit polypeptide as used in the context of the invention differs from the parental domain by one or more changes in its length or sequence as detailed in the section "Definitions and further embodiments". The term "dimerizing variant" comprises "fragment" or "derivative" of the parental domain.

[0167] The term "hinge domain" as used in the context of the invention refers to a region that imparts flexibility to the chimeric coreceptor and allows strong binding to the desired antigen. As used herein a "hinge", "hinge region" or "hinge domain", hereafter referred to simply as "hinge", are used interchangeably. The hinge domain of a chimeric coreceptor according to this invention is herein referred to as "hinge domain 1 " (HD 1), to distinguish from the hinge domain of a chimeric antigen receptor (CAR), which is referred to as HD2. As used herein, a hinge domain may comprise or consist of a HD of a costimulatory molecule as defined herein or of an immunoglobulin molecule. Methods to identify a hinge domain in an amino acid sequence are known to a person skilled in the art. For example, information on hinge domain sequences may be found in the respective protein entries in protein databases, such as Uniprot. In some embodiments, the HD1 comprises or consists of a HD of a molecule selected from the group consisting of CD8a, CD8P, CD28, IgD, IgGl, IgG2, IgG3, IgG4, CD4, CD2, CD27, CD40, CD278, 0X40, GITR, CD40L, CD30, and CD 146. In preferred embodiments, the HD1 comprises or consists of a CD8a HD, a CD8P HD, CD28 HD or a variant thereof, preferably a dimerizing variant thereof. In preferred embodiments, the HD1 comprises or consists of the amino acid sequence of SEQ ID NO: 65, 66, or 67, or a variant thereof, preferably a functional variant thereof, more preferably a dimerizing variant thereof, or a fragment thereof, wherein the variant preferably has at least 80%, 81%, 82%, 83%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with SEQ ID NO: 65, 66, or 67. In preferred embodiments, the HD1 comprises or consists of the amino acid sequence of SEQ ID NO: 65 or a variant thereof, preferably a functional variant thereof, more preferably a dimerizing variant thereof, or a fragment thereof, wherein the variant has at least 80%, 81%, 82%, 83%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with SEQ ID NO: 65. In preferred embodiments, the HD1 comprises or consists of the amino acid sequence of SEQ ID NO: 66 or a variant thereof, preferably a functional variant thereof, more preferably a dimerizing variant thereof, or a fragment thereof, wherein the variant has at least 80%, 81%, 82%, 83%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with SEQ ID NO: 66. In preferred embodiments, the HD1 comprises or consists of the amino acid sequence of SEQ ID NO: 67 or a variant thereof, preferably a functional variant thereof, more preferably a dimerizing variant thereof, or a fragment thereof, wherein the variant has at least 80%, 81%, 82%, 83%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with SEQ ID NO: 67.

[0168] The term “functional variant” of a hinge domain as used in the context of the invention refers to a variant of the parental domain which retains at least its function of imparting flexibility to the polypeptide.

[0169] The term "transmembrane domain" (TMD), as used in the context of the present invention refers to a domain having the function of 1) anchoring the polypeptide (i.e. the chimeric coreceptor or the chimeric antigen receptor) to the membrane of the respective cell, 2) mediation of homodimerization or heterodimerization (e.g. with endogenous receptors), and / or 3) activation of intracellular signalling pathways (e.g. by interaction with GP130 and STAT3 activation). The transmembrane domain of a chimeric coreceptor according to the invention is herein referred to as "transmembrane domain 1" (TMD1), to distinguish from the transmembrane domain of a CAR, which is referred to as TMD2. As used herein, a transmembrane domain may comprise or consist of a transmembrane domain of a costimulatory molecule as defined herein. As used herein, a transmembrane domain may comprise or consist of a transmembrane domain of a costimulatory molecule selected from the group consisting of CD4, CD8a, CD8P, CD28, CD2, CD27, CD40, CD278, 0X40, GITR, CD40L, CD30, CD44, and CD 146. Methods to identify a transmembrane domain in an amino acid sequence are known to a person skilled in the art. For example, information on transmembrane domain sequences may be found in the respective protein entries in protein databases, such as Uniprot. In some embodiments, the TMD1 comprises or consists of a TMD of a costimulatory molecule selected from the group consisting of CD4, CD8, CD8P, CD28, CD2, CD27, CD40, CD278, 0X40, GITR, CD40L, CD30, CD44, and CD 146. In preferred embodiments, the TMD1 comprises or consists of a CD4, CD8a, CD8P, CD28, CD44, or CD 146 TMD, or a variant thereof, preferably a functional variant thereof. In preferred embodiments, the TMD1 comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 68-71, or a variant or fragment thereof, preferably a functional variant thereof, wherein the variant preferably has at least 80%, 81%, 82%, 83%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with any one of SEQ ID NOs: 68-71. In preferred embodiments, the TMD1 comprises or consists of the amino acid sequence of SEQ ID NO: 68, or a variant or fragment thereof, preferably a functional variant thereof, , wherein the variant has at least 80%, 81%, 82%, 83%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with SEQ ID NO: 68. In preferred embodiments, the TMD1 comprises or consists of the amino acid sequence of SEQ ID NO: 69, or a variant or fragment thereof, preferably a functional variant thereof, more preferably a dimerizing variant thereof, wherein the variant has at least 80%, 81%, 82%, 83%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with SEQ ID NO: 69. In preferred embodiments, the TMD1 comprises or consists of the amino acid sequence of SEQ ID NO: 70, or a variant or fragment thereof, preferably a functional variant thereof, more preferably a dimerizing variant thereof, wherein the variant has at least 80%, 81%, 82%, 83%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with SEQ ID NO: 70. In preferred embodiments, the TMD1 comprises or consists of the amino acid sequence of SEQ ID NO: 71, or a variant or fragment thereof, preferably a functional variant thereof, more preferably a dimerizing variant thereof, wherein the variant has at least 80%, 81%, 82%, 83%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with SEQ ID NO: 71.

[0170] The term “functional variant” of a transmembrane domain as used in the context of the invention refers to a variant of the parental domain which retains at least its functions of 1) anchoring the polypeptide to the membrane of the cell, 2) mediation of homodimerization or / heterodimerization (e.g. with endogenous receptors), and / or 3) activation of intracellular signalling pathways (e.g. by interaction with GP130 and STAT3 activation).

[0171] The term "costimulatory domain" (CSD), as used in the context of the present invention refers to a domain having the function of augmenting immune cell activation induced by the primary ‘signal-1’, e.g. the CD3z (i.e. CD3zeta or CD3Q. Thus, the costimulatory domain provides ‘signal-2’ for immune cell (in particular T cell) activation (see Thomas S. and Abken H. (2023) Frontiers in Immunology 13:1090959 and Weinkove R. el al. (2019) Clin. Trans. Immunol. E1049 for a review on signalling of CARs and CCR through CD3(^ (signal-1) and costimulatory domains (signal-2)). In other words, a costimulatory domain as used herein refers to an intracellular domain of a costimulatory molecule that is able to transduce a signal (‘signal-2’) into immune cells to enhance a primary signal (‘signal-1’), e.g. a TCR CD3z-mediated signal. In particular, a costimulatory domain of a CCR as used herein refers to an intracellular domain of a costimulatory molecule that is able of augmenting immune cell activation induced by the primary ‘ signal- 1’ delivered by the CD3z of a TCR or of a CAR or tgTCR co-expressed with the CCR: Additionally, a costimulatory domain (CSD) as used in the context of the present invention is not capable of inducing immune cell activation upon engagement of the ARD1 of the CCR with the target antigen in absence of a primary signal (‘signal-1’).

[0172] The terms “intracellular signaling domain”, “intracellular signaling domain of a costimulatory molecule” and the abbreviations “ISD” and “ICD” are used interchangeably and as used herein refer to the intracellular signaling domain of a costimulatory molecule selected from the group consisting of an immunoglobulin superfamily molecule, a tumor necrosis factor receptor (TNFR) family molecule, a cell adhesion molecule, a MyD88-dependent Toll-like receptor (TLR), and a cytokine receptor. A costimulatory molecule as used herein interacts with its respective cognate intracellular signalling molecule leading to a functional response in the cell, e.g proliferation, cell adhesion, migration, spreading, and / or focal adhesion formation, with the effect of costimulating the primary signal of a PSD, e.g. CD3z or FceRIg, thus inducing for example cytokine production and proliferation. The cognate intracellular signalling molecule binding and / or interacting with the ISD is preferably selected from the group consisting of LCK, FYN, Interleukin- 2-inducible T cell kinase (ITK), vav guanine nucleotide exchange factor 1 (VAV1), C-terminal Src kinase (CSK), Phospholipase C gamma 1 and 2 (PLCyl, PLCy2), Lymphocyte cytosolic protein 2 (SLP-76), Janus Kinases (JAK1, JAK2, JAK3, TYK2), Signal transducer and activator of transcription 1-6 (STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, STAT6), TNF- associated factors (TRAFs), Phosphoinositide 3-Kinase (PI3K), Growth factor receptor-bound protein 2 (Grb2), GRB2-related adapter downstream of She (GADS), MyD88, TIR domain containing adapter protein (TIRAP), FERM (F for 4.1 protein, E for ezrin, R for radixin and M for moesin), ankyrin, dileucine motif, cytoskeleton association site, dihydrophobic basolateral targeting motif LV, and PDZ (PSD-95 / Dlg / ZO-l) binding site. Based on amino acid sequence alignment, the skilled person can determine ISDs in a given costimulatory molecule. Costimulatory molecules for immune cell activation have been described in the prior art, for example in Sharpe AH. 2009, Mechanisms of co stimulation. Immunol Rev 229(1):5-11; Chester C, et al., 2015. Natural Killer Cell Immunomodulation. Front Immunol 6:601.

[0173] A preferred costimulatory molecule as used herein is selected from the group consisting of: immunoglobulin superfamily (IgSF) costimulatory molecules, preferably CD2, CD28, CD278 (ICOS);

[0174] - tumor necrosis factor receptor (TNFR) family costimulatory molecules, preferably CD27, CD40, CD40L, CD134 (0X40), CD137 (4-1BB), or GITR (glucocorticoid-induced TNFR family related gene);

[0175] - cell adhesion molecules, preferably a cell adhesion molecule listed in Table 5 below, more preferably CD4, CD8a, CD8P, CD44, and CD 146;

[0176] - MyD 88 -dependent Toll-like receptors (TLR), preferably TLR2 and TLR4, or

[0177] - cytokine receptors, preferably IL-2RB, IL-2RG, IL-7RA, IL-9R and IL-21R.

[0178] The term “cell adhesion molecule (CAM)” as used herein refers to a cell surface protein involved in the binding of a cell with another cell or to the extracellular matrix (ECM), in the process of cell adhesion. Preferably, a cell adhesion molecule (CAM) as used herein is selected from the group consisting of integrins, immunoglobulin superfamily (IgSF), cadherins, and selectins. More preferably, a cell adhesion molecule (CAM) as used herein is selected from the group consisting of cell adhesion molecules of Table 5.

[0179] Table 5: Human cell adhesion molecules

[0180] In preferred embodiments, the costimulatory domain (CSD) comprises or consists of a CD4, CD8a, CD8P, CD28, CD2, CD27, CD40, CD278, 0X40, GITR, CD40L, CD30, CD44, or CD 146 intracellular signaling domain (ICD or ISD), or a variant thereof, preferably a functional variant thereof. In preferred embodiments, the CSD comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 72-85, or a variant thereof, preferably a functional variant thereof, or a fragment of said ICD or variant, wherein the variant preferably has at at least 80%, 81%, 82%, 83%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with SEQ ID NO: 72-85. In preferred embodiments, the CSD comprises or consists of SEQ ID NO: 72, or a variant thereof, preferably a functional variant thereof, wherein the variant preferably has at at least 80%, 81%, 82%, 83%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with SEQ ID NO: 72. In preferred embodiments, the CSD comprises or consists of SEQ ID NO: 73, or a variant thereof, preferably a functional variant thereof, wherein the variant preferably has at at least 80%, 81%, 82%, 83%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with SEQ ID NO: 73. In preferred embodiments, the CSD comprises or consists of SEQ ID NO: 74, or a variant thereof, preferably a functional variant thereof, wherein the variant preferably has at at least 80%, 81%, 82%, 83%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with SEQ ID NO: 74. In preferred embodiments, the CSD comprises or consists of SEQ ID NO: 76, or a variant thereof, preferably a functional variant thereof, wherein the variant preferably has at at least 80%, 81%, 82%, 83%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with SEQ ID NO: 76. In preferred embodiments, the CSD comprises or consists of SEQ ID NO: 77, or a variant thereof, preferably a functional variant thereof, more preferably a dimerizing or trimerizing variant thereof, wherein the variant preferably has at at least 80%, 81%, 82%, 83%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with SEQ ID NO: 77. In preferred embodiments, the CSD comprises or consists of SEQ ID NO: 78, or a variant thereof, preferably a functional variant thereof, more preferably a dimerizing or trimerizing variant thereof, wherein the variant preferably has at at least 80%, 81%, 82%, 83%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with SEQ ID NO: 78. In preferred embodiments, the CSD comprises or consists of SEQ ID NO: 79, or a variant thereof, preferably a functional variant thereof, more preferably a dimerizing or trimerizing variant thereof, wherein the variant preferably has at at least 80%, 81%, 82%, 83%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with SEQ ID NO: 79. In preferred embodiments, the CSD comprises or consists of SEQ ID NO: 80, or a variant thereof, preferably a functional variant thereof, more preferably a dimerizing or trimerizing variant thereof, wherein the variant preferably has at at least 80%, 81%, 82%, 83%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with SEQ ID NO: 80. In preferred embodiments, the CSD comprises or consists of SEQ ID NO: 80, or a variant thereof, preferably a functional variant thereof, wherein the variant preferably has at at least 80%, 81%, 82%, 83%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with SEQ ID NO: 80. In preferred embodiments, the CSD comprises or consists of SEQ ID NO: 81, or a variant thereof, preferably a functional variant thereof, more preferably a dimerizing variant thereof, wherein the variant preferably has at at least 80%, 81%, 82%, 83%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with SEQ ID NO: 81. In preferred embodiments, the CSD comprises or consists of SEQ ID NO: 82, or a variant thereof, preferably a functional variant thereof, more preferably a dimerizing variant thereof, wherein the variant preferably has at at least 80%, 81%, 82%, 83%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with SEQ ID NO: 82. In preferred embodiments, the CSD comprises or consists of SEQ ID NO: 83, or a variant thereof, preferably a functional variant thereof, more preferably a dimerizing or trimerizing variant thereof, wherein the variant preferably has at at least 80%, 81%, 82%, 83%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with SEQ ID NO: 83. In preferred embodiments, the CSD comprises or consists of SEQ ID NO: 84, or a variant thereof, preferably a functional variant thereof, more preferably a dimerizing or trimerizing variant thereof, wherein the variant preferably has at at least 80%, 81%, 82%, 83%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with SEQ ID NO: 84. In preferred embodiments, the CSD comprises or consists of SEQ ID NO: 85, or a variant thereof, preferably a functional variant thereof, more preferably a dimerizing or trimerizing variant thereof, wherein the variant preferably has at at least 80%, 81%, 82%, 83%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with SEQ ID NO: 85.

[0181] The term "trimerizing variant" of a domain of chimeric coreceptor subunit polypeptide as used in the context of the invention refers to a functional variant of the parental domain which retains its ability to undergo homotypic or heterotypic interactions with the corresponding domains of a second and / or a third chimeric coreceptor subunit polypeptide to form a trimer, or which retains its ability to binds to a trimeric ligand thereby inducing trimerization of the chimeric coreceptor.

[0182] A " trimerizing variant" of a domain of a chimeric coreceptor subunit polypeptide as used in the context of the invention differs from the parental domain by one or more changes in its length or sequence as detailed in the section "Definitions and further embodiments". The term "trimerizing variant" comprises "fragment" or "derivative" of the parental domain.

[0183] Since CARs contain well characterized T cell signalling components, it has been assumed that CARs signal in a similar way to conventional TCRs. In fact, both CARs and TCRs require CD3^ ITAM phosphorylation to initiate signalling, which is mediated by Src family kinases, the most important of which is LCK. LCK can be found either free in the cytosol, anchored to the plasma membrane though N-terminal palmitoylation and myristoylation, or associated with the intracellular tails of a coreceptor, such as a CD4 or CD8 coreceptor (Kim PW, el al., 2003. Science 301(5640): 1725-1728), and each of these LCK forms is capable of mediating phosphorylation of the CD3<^ ITAMs of the CAR or TCR. Engagement of TCR with peptide-MHC complexes also facilitates the recruitment of CD4 or CD8 coreceptors that bind to the same MHC molecule as the TCR, creating a positive feedback loop to recruit more LCKs, which phosphorylate more CD3 ITAMs.

[0184] As mentioned above, phosphorylation of CD3^ ITAMs is critical for the initiation of CAR activity, however, CARs do not recruit or interact with coreceptors comprising an LCK binding domain.

[0185] Therefore, the costimulatory domain of the chimeric coreceptor of the present invention preferably comprises an "intracellular signaling domain capable of binding LCK", which is a domain comprising one or more motifs capable of binding LCK, in order to increase the phosphorylation of the CD3^ ITAMs of the CAR or TCR co-expressed with the chimeric coreceptor.

[0186] The regulated recruitment of chimeric coreceptor- associated LCK to the CAR synapse in response to antigen binding may provide a technical solution to the general problems of (i) the antigen sensitivity of BBz-CARs, which often require thousands of antigen molecules per cell to activate T cell responses, and (ii) CARs that induce T cell exhaustion as a result of tonic signaling due to high basal CAR-CD3^ phosphorylation.

[0187] According to a preferred embodiment, the costimulatory domain of the chimeric coreceptor of the present invention preferably comprises an "intracellular signaling domain capable of binding LCK", wherein the at least one intracellular signaling domain capable of binding LCK comprises or consists of a CD4, CD8a, CD8P, CD28, CD2, CD44, or CD 146 intracellular signaling domain (ICD), which is capable of binding LCK, or a dimerizing and / or LCK binding variant thereof.

[0188] According to a preferred embodiment, the co stimulatory domain of the chimeric coreceptor of the present invention preferably comprises an "intracellular signaling domain capable of binding TRAP (TNFR-associated factors)", wherein the at least one intracellular signaling domain capable of binding TRAF comprises or consists of a CD27, CD40, CD278, GITR, CD40L, or CD30 intracellular signaling domain (ICD), which is capable of binding TRAF, or a dimerizing or trimerizing and / or TRAF binding variant thereof.

[0189] The term "CYTD not comprising a cell activation domain", as used in the context of the present invention means that the chimeric coreceptor (CCR) subunit polypeptide of the present invention does not comprise an intracellular cell activation domain, such as a CD3(^ (CD3zeta) T cell activation domain or a Fc-epsilon-Receptor I-gamma (FceRIg) chain, and thus is not capable of inducing immune cell activation upon engagement with the target antigen, i.e. AG1. In one embodiment, the chimeric coreceptor subunit polypeptide of the present invention comprises:

[0190] (a) an extracellular antigen-recognition domain 1 (ARD1) specifically binding an antigen 1 (AG1),

[0191] (b) an immunoglobulin variable-like domain (IgVD) comprising or consisting of a CD8a IgVD,

[0192] (c) a hinge domain 1 (HD1) comprising or consisting of a CD8a HD,

[0193] (d) a transmembrane domain 1 (TMD1) comprising or consisting of a CD8a TMD, and

[0194] (e) a cytosolic domain (CYTD):

[0195] (i) comprising a costimulatory domain (CSD), and

[0196] (ii) not comprising a cell activation domain, wherein the CSD comprises or consists of a CD8a ICD, and wherein the ARD1 is linked to the IgVD.

[0197] The chimeric coreceptor with the structure above is also referred to as generally "chimeric CD8 IgVD coreceptor" or CD8aIgV-CCR or [AGl]-8IgV, for example 19-8IgV. According to one embodiment, the amino acid sequence of 19-8IgV comprises or consists of the amino acid sequence of SEQ ID NO: 92, or a sequence being at least about 80%, 81%, 82%, 83%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0198] In one embodiment, the chimeric coreceptor subunit polypeptide of the present invention comprises:

[0199] (a) an ARD1 specifically binding an AG1,

[0200] (b) an IgVD comprising or consisting of a CD8a IgVD,

[0201] (c) a HD1 comprising or consisting of a CD8a HD,

[0202] (d) a TMD1 comprising or consisting of a CD8a TMD, and

[0203] (e) a CYTD:

[0204] (i) comprising a CSD, and

[0205] (ii) not comprising a cell activation domain, wherein the CSD comprises or consists of a CD4 ICD, and wherein the ARD1 is linked to the IgVD.

[0206] The chimeric coreceptor with the structure above is also referred to as generally "chimeric CD8 IgVD coreceptor" or CD8a.4IgV-CCR or [AGl]-8.4IgV, for example 19-8.4IgV. According to one embodiment, the amino acid sequence of 19-8.4IgV comprises or consists of the amino acid sequence of SEQ ID NO: 93, or a sequence being at least about 80%, 81%, 82%, 83%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0207] In one embodiment, the chimeric coreceptor subunit polypeptide of the present invention comprises:

[0208] (a) an ARD1 specifically binding an AG1,

[0209] (b) an IgVD comprising or consisting of a CD8P IgVD,

[0210] (c) a HD1 comprising or consisting of a CD8P HD,

[0211] (d) a TMD1 comprising or consisting of a CD8P TMD, and

[0212] (e) a CYTD:

[0213] (i) comprising CSD, and

[0214] (ii) not comprising a cell activation domain, wherein the CSD comprises or consists of a CD8P ICD, and wherein the ARD1 is linked to the IgVD.

[0215] The chimeric coreceptor with the structure above is also referred to as "chimeric CD8 IgVDcoreceptor" or CD8pigVD-CCR or [AGl]-8bIgV, for example 19-8bIgV.

[0216] In one embodiment, the chimeric coreceptor subunit polypeptide of the present invention comprises:

[0217] (a) an ARD1 specifically binding an AG1,

[0218] (b) an IgVD comprising or consisting of a CD28 IgVD,

[0219] (c) a HD1 comprising or consisting of a CD28 HD,

[0220] (d) a TMD1 comprising or consisting of a CD28 TMD, and

[0221] (e) a CYTD:

[0222] (i) comprising a CSD, and

[0223] (ii) not comprising a cell activation domain, wherein the CSD comprises or consists of a CD28 ICD, and wherein the ARD1 is linked to the IgVD.

[0224] The chimeric coreceptor with the structure above is also referred to as generally "chimeric CD28 IgVD coreceptor" or CD28IgV-CCR or [AGl]-28IgV, for example 19-28IgV. According to one embodiment, the amino acid sequence of 19-28IgV comprises or consists of the amino acid sequence of SEQ ID NO: 97, or a sequence being at least about 80%, 81%, 82%, 83%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0225] The Table 2 below describes the composition and nomenclature of the chimeric coreceptors disclosed herein. Table 2: Structure and nomenclature of the chimeric coreceptors

[0226] The chimeric IgVD coreceptor of the claimed invention aims to improve the properties of CARs known in the art by enhancing the activation of CAR-expressing immune cells (e.g. CAR-T cells) in response to target cells, e.g. tumor cells, even when the antigen recognized by the CAR (herein referred to as AG2) is expressed at a very low density on said target cells.

[0227] In one embodiment, the AG1 and / or the AG2 is a "tumor-associated antigen". The term "tumor antigen" or "tumor-associated antigen" refers to a constituent of cancer cells that may be derived from the cytoplasm, the cell surface and the cell nucleus. In particular, it refers to those antigens that are produced intracellularly or as surface antigens on tumor cells. A tumor antigen is typically expressed preferentially by cancer cells (e.g., it is expressed at higher levels in cancer cells than in non-cancer cells), and in some cases it is expressed exclusively by cancer cells. Exemplary tumor-associated antigens to be used in the present invention include, but are not limited to, those described in Table 3.

[0228] Table 3: Exemplary tumor-associated antigens

[0229] Therefore, in one embodiment of all aspects disclosed herein, the AG1 is a tumor-associated antigen selected from the group consisting of ALK, B7-H3 (CD276), BCMA, c-MET, CD3, CD5, CD19, CD20, CD22, CD23, CD30, CD33, CD38, CD44, CD44v6, CD52, CD70, CD79A, CD79B, CD123, CD138, CD171, CEA, Claudin-6, Claudin-18.2, CLL1, CXCR5, EGFR, EGFRvIII, EPH- receptor A2, IGLV3-21, IL-1RAP, GPC-2, GPC-3, HER2, ErbB3, ErbB4, FAP, FBP, AchR, Fr-a, GD2, GD3, HMW-MAA, IL-13Ra2, Kappa-EC, IGEV3-21-R110, Eewis Y, EMP1, EMP2A, Mesothelin, MAGE-4A, MUC1, MUC16, NKG2D Eigands, NCAM, NY-ESO1, Oncofetal antigen h5T4, PRAME, PSCA, PSMA, R0R1, TAG-72, VEGFR, GOLPH2, and SLAMF7, and preferably CD 19 or CD22. In one preferred embodiment of all aspects disclosed herein, the AG1 is a tumor- associated antigen selected from the group consisting of CD19, CD5, CD22, CD38, EGFR, HER2, Mesothelin, IL-13Ra2, PSMA, and EPH-receptor-A2. In one embodiment of all aspects disclosed herein, the AG2 is a tumor-associated antigen selected from the group consisting of ALK, B7-H3 (CD276), BCMA, c-MET, CD3, CD5, CD19, CD20, CD22, CD23, CD30, CD33, CD38, CD44, CD44v6, CD52, CD70, CD79A, CD79B, CD123, CD138, CD171, CEA, Claudin-6, Claudin-18.2, CLL1, CXCR5, EGFR, EGFRvIII, EPH-receptor A2, IGLV3-21, IL-1RAP, GPC-2, GPC-3, HER2, ErbB3, ErbB4, FAP, FBP, AchR, Fr-a, GD2, GD3, HMW-MAA, IL-13Ra2, Kappa-LC, IGLV3-21-R110, Lewis Y, LMP1, LMP2A, Mesothelin, MAGE-4A, MUC1, MUC16, NKG2D Ligands, NCAM, NY-ESO1, Oncofetal antigen h5T4, PRAME, PSCA, PSMA, R0R1, TAG-72, VEGFR, GOLPH2, and SLAMF7, and preferably CD19 or CD22.

[0230] In other words, in one embodiment of all aspects disclosed herein, the ARD1 is selected from the group consisting of anti-ALK, anti-B7-H3 (CD276), anti-BCMA, anti-c-MET, anti-CD3, anti-CD5, anti-CD19, anti-CD20, anti-CD22, anti-CD23, anti-CD30, anti-CD33, anti-CD38, anti- CD44, anti-CD44v6, anti-CD52, anti-CD70, anti-CD79A, anti-CD79B, anti-CD123, anti-CD138, anti-CD171, anti-CEA, anti-Claudin-6, anti-Claudin-18.2, anti-CLLl, anti-CXCR5, anti-EGFR, anti-EGFRvIII, anti-EPH-receptor A2, anti-IGLV3-21, anti-IGLV3-21-Rl 10, anti-IL-lRAP, anti- GPC-2, anti-GPC-3, anti-HER2, anti-ErbB3, anti-ErbB4, anti-FAP, anti-FBP, anti-AchR, anti- Fr- a, anti-GD2, anti-GD3, anti-HMW-MAA, anti-IE-13Ra2, anti-Kappa-EC, anti-IGEV3-21, anti- Eewis Y, anti-EMPl, anti-EMP2A, anti-Mesothelin, anti-MAGE-4A, anti-MUCl, anti-MUC16, anti-NKG2D Eigands, anti-NCAM, anti-NY-ESOl, anti-Oncofetal antigen h5T4, anti-PRAME, anti-PSCA, anti-PSMA, anti-RORl, anti-TAG-72, anti-VEGFR, anti-GOLPH2, and anti- SLAMF7. In other words, in one embodiment of all aspects disclosed herein, the ARD2 is selected from the group consisting of anti-ALK, anti-B7-H3 (CD276), anti-BCMA, anti-c-MET, anti-CD3, anti-CD5, anti-CD19, anti-CD20, anti-CD22, anti-CD23, anti-CD30, anti-CD33, anti-CD38, anti- CD44, anti-CD44v6, anti-CD52, anti-CD70, anti-CD79A, anti-CD79B, anti-CD123, anti-CD138, anti-CD171, anti-CEA, anti-Claudin-6, anti-Claudin-18.2, anti-CLLl, anti-CXCR5, anti-EGFR, anti-EGFRvIII, anti-EPH-receptor A2, anti-IGLV3-21, anti-IGLV3-21-Rl 10, anti-IL-lRAP, anti- GPC-2, anti-GPC-3, anti-HER2, anti-ErbB3, anti-ErbB4, anti-FAP, anti-FBP, anti-AchR, anti- Fr- a, anti-GD2, anti-GD3, anti-HMW-MAA, anti-IL-13Ra2, anti-Kappa-LC, anti-IGLV3-21, antiLewis Y, anti-LMPl, anti-LMP2A, anti-Mesothelin, anti-MAGE-4A, anti-MUCl, anti-MUCl 6, anti-NKG2D Ligands, anti-NCAM, anti-NY-ESOl, anti-Oncofetal antigen h5T4, anti-PRAME, anti-PSCA, anti-PSMA, anti-RORl, anti-TAG-72, anti-VEGFR, anti-GOLPH2, and anti- SLAMF7.

[0231] Definitions and embodiments described below, in particular under the headers 'Definitions' and 'Further embodiments of the invention' apply to chimeric coreceptor (CCR) subunit polypeptide of the first, aspect.

[0232] Chimeric coreceptor dimers and complex

[0233] The term "chimeric coreceptor (CCR) subunit polypeptide" refers to a single amino acid chain of the chimeric coreceptor as described herein. When expressed on a cell surface, a CCR subunit polypeptide tends to form a homodimer with a second chimeric coreceptor subunit polypeptide or a complex with an endogenous coreceptor subunit, e.g. a CD8a, CD8P or CD28 subunit, comprising a substantially identical IgVD, and / or HD and / or TMD and / or CSD. Homodimerization may be mediated by covalent interactions (e.g. formation of a disulphide bridge between cysteine residues) or by non-covalent interactions (e.g. hydrophobic interactions, van der Waals interactions, or electrostatic interactions) between the IgVD (if present), TMD, HD and / or CSD of two CCR monomers. Indeed, when expressed on cytotoxic CD8+T cells, a CD8a IgVD chimeric coreceptor subunit polypeptide, as defined above, may undergo heterotypic interactions with the IgVD of natural occurring CD 8 a or CD8P subunits, to form a monovalent chimeric coreceptor complex capable of binding to a defined and native target antigen exposed on the surface of a target cell (Figure 1C). When expressed on CD4+T helper cells, a CD8a IgVD chimeric coreceptor subunit polypeptide, as defined above, may prefer to undergo homotypic interactions with a second CD8a IgVD chimeric coreceptor subunit polypeptide to form a divalent homodimer containing two antigen-binding domains (Figure ID). In contrast, the chimeric CD8 coreceptor comprising an extracellular CD 8 a HD, but lacking the IgVD may preferably form divalent chimeric coreceptor homodimers in both CD8+and CD4+T cells, as it was shown for conventional CARs known in the art containing the same CD8a-derived hinge and transmembrane domains (Milone, MC, et al., 2009. Mol Ther 17(8): 1453-64; Chen, X, et al., 2022. J Magn Reson, 340, 107234).

[0234] Although only CD8a can bind LCK kinase in the intracellular signaling domain, the intracellular signaling domain of CD8P also plays an important role as it is palmitoylated in the intracellular signaling domain, which is important to localize the CD8 complex in lipid rafts where the coupling of CD8a with LCK occurs. Therefore, it can be assumed that a CD8a IgVD chimeric coreceptor can also form a complex with endogenous CD8P polypeptide, which is then associated in lipid rafts, so that CAR-T cells co-expressing CD8a IgVD chimeric coreceptor are more potent than CAR-T cells co-expressing CD8a chimeric coreceptor without IgVD.

[0235] When expressed on cells expressing a CD28 subunit on their surface, a CD28IgVD chimeric coreceptor subunit polypeptide, as defined above, may undergo heterotypic interactions with the HD and / or TMD of a natural occurring CD28 subunits, to form a monovalent chimeric CD28 coreceptor complex capable of binding to a defined and native target antigen exposed on the surface of a target cell (Figure 22). For example, a CAR comprising a CD28 TMD has been shown to form heterodimers with natural occurring CD28 (Muller YD, et al., 2021. Front Immunol. 12:639818). Alternatively, a CD28IgVD chimeric coreceptor subunit polypeptide, as defined above, when expressed on cells that may or may not express a CD28 subunit on the surface, can undergo homotypic interactions to form a divalent chimeric coreceptor homodimer containing two antigen-binding domains (Figure 22B).

[0236] Accordingly, in one embodiment of the second aspect, the present invention provides a chimeric coreceptor homodimer comprising two substantially identical chimeric coreceptor subunit polypeptides comprising: (a) an extracellular antigen-recognition domain 1 (ARD1) specifically binding an antigen 1 (AG1),

[0237] (b) an immunoglobulin variable-like domain (IgVD),

[0238] (c) a hinge domain 1 (HD1),

[0239] (d) a transmembrane domain 1 (TMD1), and

[0240] (e) a cytosolic domain (CYTD):

[0241] (i) comprising a costimulatory domain (CSD), and

[0242] (ii) not comprising a cell activation domain, wherein the ARD1 is linked to the IgVD.

[0243] In this context, "two substantially identical CCR subunit polypeptides'" means that the first CCR subunit polypeptide does not need to be 100% identical to the second CCR subunit polypeptide but can comprise amino acid substitutions and / or additions and / or deletions as defined herein. It is preferred that two substantially identical CCR subunit polypeptides show at least 60%, 65%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, such that the two substantially identical CCR subunit polypeptides have the same functional characteristics, such as but not limited to capacity to bind AG1, dimerization, interaction with the antigen receptor co-expressed by the recombinant cell, and recombinant cell activation.

[0244] In a further embodiment of the second aspect, the present invention provides a chimeric coreceptor complex comprising a chimeric coreceptor subunit polypeptide comprising:

[0245] (a) an extracellular antigen-recognition domain 1 (ARD1) specifically binding an antigen 1 (AG1),

[0246] (b) an immunoglobulin variable-like domain (IgVD),

[0247] (c) a hinge domain 1 (HD1),

[0248] (d) a transmembrane domain 1 (TMD1), and

[0249] (e) a cytosolic domain (CYTD):

[0250] (i) comprising a costimulatory domain (CSD), and

[0251] (ii) not comprising a cell activation domain, wherein the ARD1 is linked to the IgVD, and a CD8a, CD8P or CD28 subunit, wherein the chimeric coreceptor subunit polypeptide and the CD8a, CD8P or CD28 subunit preferably comprise a substantially identical IgVD, and / or HD and / or TMD and / or CSD. The term "chimeric coreceptor complex" refers to a protein complex comprising a chimeric coreceptor (CCR) subunit polypeptide as disclosed herein and a CD8a, CD8P or CD28 subunit, wherein the CCR subunit polypeptide and the CD8a, CD8P or CD28 subunit interact via the IgVDs and / or the HDs and / or the TMDs and / or the CSD of the two subunits. Preferably, the IgVDs and / or the HDs and / or the TMDs and / or the CSD of the two subunits interact by covalent interactions, e.g. formation of one or more disulphide bridges, or by non-covalent interactions.

[0252] The term "CD8a subunit" refers to the CD8a chain of the naturally occurring CD8 coreceptor. The term "CD8P subunit" refers to the CD8P chain of the naturally occurring CD8 coreceptor. The CD8 coreceptor forms a dimer, consisting of a pair of CD8a and CD8P chain, or a homodimer of two CD8a chains. The term "CD28 subunit" refers to one monomer of the naturally occurring CD28 coreceptor, which forms a homodimer of two CD28 subunits bound through a disulfide bond in the hinge region and by a dimerization motif in the transmembrane domain.

[0253] In this context, "substantially identical IgVD and / or HD and / or TMD and / or CSD" means that the polypeptide forming the IgVD and / or HD, TMD or CSD does not need to be 100% identical to the corresponding domain of the endogenous coreceptor subunit, but can comprise amino acid substitutions and / or additions and / or deletions as defined herein. It is preferred that a substantially identical IgVD and / or HD and / or TMD and / or CSD polypeptide shows at least 60%, 65%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the corresponding domain of the endogenous coreceptor subunit, such that the chimeric coreceptor and the endogenous subunit are able to dimerize.

[0254] In one embodiment of the second aspect, the invention provides a chimeric coreceptor homodimer comprising two substantially identical CD8aIgV-CCR subunit polypeptides. In one embodiment of the second aspect, the invention provides a chimeric coreceptor homodimer comprising two substantially identical CD8a.4IgV-CCR subunit polypeptides. In one embodiment of the second aspect, the invention provides a chimeric coreceptor homodimer comprising two substantially identical CD8pigV-CCR subunit polypeptides. In one embodiment of the second aspect, the invention provides a chimeric coreceptor homodimer comprising two substantially identical CD28IgV-CCR subunit polypeptides. In one embodiment of the second aspect, the invention provides a chimeric coreceptor complex comprising a CD8a IgV-CCR subunit polypeptide and a CD8a or CD8P subunit. In one embodiment of the second aspect, the invention provides a chimeric coreceptor complex comprising a CD8P IgV-CCR subunit polypeptide and a CD8a subunit. In one embodiment of the second aspect, the invention provides a chimeric coreceptor complex comprising a CD28IgV-CCR subunit polypeptide and a CD28 subunit.

[0255] Definitions given and embodiments described with respect to the first aspect apply also to the second aspect, in as far as they are applicable. Also, definitions and embodiments described below, in particular under the header 'Definitions' and 'Further embodiments of the invention' apply to the homodimer or complex of the second aspect.

[0256] Combination of chimeric coreceptors and CAR, TCR, or tgTCR

[0257] Co-expression of a chimeric coreceptor according to the present invention with a CAR, TCR or tgTCR has the advantages of (1) increasing the antigen- specific antitumor responses elicited by that CAR, TCR or tgTCR alone, (2) preventing antigen-independent CAR or TCR signaling.

[0258] In the chimeric coreceptor of the present invention, the ARD1 is able of binding to an AG1 on the surface of a target cell, wherein binding of the chimeric coreceptor to AG1 does not induce activation of a recombinant cell expressing the chimeric coreceptor. Differently, when the chimeric coreceptor of the present invention is co-expressed with a CAR, TCR, or a tgTCR comprising an ARD2 which binds an AG2 (an AG2 bound to an MHC in the case of a tgTCR) on the surface of a target cell, the binding of the CAR, TCR or tgTCR to the AG2 induces activation of the recombinant cell. Binding of the chimeric coreceptor to the AG1 may enhance the activation of the recombinant cell induced by binding of the CAR, TCR or tgTCR to the AG2. The chimeric coreceptor may attenuate the antigen-independent activation and differentiation of the recombinant cell induced by the CAR, TCR or tgTCR.

[0259] When chimeric coreceptors and CARs TCRs or tgTCRs bind to their cognate antigens AG1 and AG2, coreceptors and receptors relocate to the immunological synapse and come into proximity with each other. Proximal signaling molecules such as LCK bound to the cytosolic domains of the chimeric coreceptors can amplify the phosphorylation of the IT AM motifs present in the CD3<^ domains of adjacent CAR molecules or of the TCR complex, thereby enhancing CAR or TCR signaling, T cell activation and immune responses toward the target cells. The chimeric coreceptors of the present invention may be co-expressed with any CAR, TCR or tgTCR known in the art in primary human T cells or primary human NK cells.

[0260] In one embodiment of the third aspect, the invention provides a combination of: a chimeric coreceptor homodimer comprising two substantially identical chimeric coreceptor subunit polypeptides comprising:

[0261] (a) an extracellular antigen-recognition domain 1 (ARD1) specifically binding an antigen 1 (AG1),

[0262] (b) an immunoglobulin variable-like domain (IgVD),

[0263] (c) a hinge domain 1 (HD1),

[0264] (d) a transmembrane domain 1 (TMD1), and

[0265] (e) a cytosolic domain (CYTD):

[0266] (i) comprising a costimulatory domain (CSD), and

[0267] (ii) not comprising a cell activation domain, wherein the ARD1 is linked to the IgVD; and a chimeric antigen receptor (CAR), an endogenous TCR (TCR) or a transgene TCR (tgTCR), wherein the CAR, the TCR or the tgTCR specifically bind an antigen 2 (AG2), and wherein AG1 and AG2 are two different antigens or are substantially the same antigen.

[0268] Preferably, when AG1 and AG2 are substantially the same antigen, AG1 and AG2 are CD22.

[0269] The term "substantially the same antigen" means that the amino acid sequences of the two antigens do not need to be 100% identical but can differ due to amino acid substitutions and / or additions and / or deletions as defined herein. It is preferred that two amino acid sequences of AG1 and AG2 being "substantially the same antigen" show at least 60%, 65%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, such that the two antigens preserve the epitope or set of epitopes recognized by the ARD1 and ARD2.

[0270] Preferably, the AG1 and / or AG2 are independently selected from the group consisting of ALK, B7-H3 (CD276), BCMA, c-MET, CD3, CD5, CD19, CD20, CD22, CD23, CD30, CD33, CD38, CD44, CD44v6, CD52, CD70, CD79A, CD79B, CD123, CD138, CD171, CEA, Claudin- 6, Claudin-18.2, CLL1, CXCR5, EGFR, EGFRvIII, EPH-receptor A2, IGLV3-21, IL-1RAP, GPC- 2, GPC-3, HER2, ErbB3, ErbB4, FAP, FBP, AchR, Fr-a, GD2, GD3, HMW-MAA, IE-13Ra2, Kappa-EC, IGEV3-21, Eewis Y, EMP1, EMP2A, Mesothelin, MAGE-4A, MUC1, MUC16, NKG2D Eigands, NCAM, NY-ESO1, Oncofetal antigen h5T4, PRAME, PSCA, PSMA, ROR1, TAG-72, VEGFR, GOLPH2, and SLAMF7, preferably wherein AG2 and AG1 are selected from the group of pairs: CD22 and CD19; CD20 and CD5; CD79B and CD22; CD79B and CD5; LMP1 and CD19; LMP1 and EGFR; LMP2A and CD19; LMP2A and EGFR; EGFRvIII and EGFR; BCMA and CD38; IL-13Ra2 and EPH-receptor A2; IL-13Ra2 and EGFR; MUC1 and EGFR; MUC1 and HER2; MUC1 and EpCAM; IGLV3-21-R110 and IGLV3-21; PSMA and PCSA; R0R1 and CD5; Claudin-6 and Mesothelin; Claudin-18.2 and MUC-1; CD70 and CD33; IL-1RAP and CD70; and preferably AG1 and / or AG2 is CD19 or CD22, and more preferably AG1 is CD19 and AG2 is CD22.

[0271] In a further embodiment of the third aspect, the invention provides a combination of: a chimeric coreceptor complex comprising a chimeric coreceptor subunit polypeptide comprising:

[0272] (a) an extracellular antigen-recognition domain 1 (ARD1) specifically binding an antigen 1 (AG1),

[0273] (b) an immunoglobulin variable-like domain (IgVD),

[0274] (c) a hinge domain 1 (HD1),

[0275] (d) a transmembrane domain 1 (TMD1), and

[0276] (e) a cytosolic domain (CYTD):

[0277] (i) comprising a costimulatory domain (CSD), and

[0278] (ii) not comprising a cell activation domain, wherein the ARD1 is linked to the IgVD, and a CD8a, CD8P or CD28 subunit; and a CAR, a TCR or a tgTCR, wherein the CAR, the TCR or the tgTCR specifically bind an antigen 2 (AG2), and wherein AG1 and AG2 are two different antigens or are substantially the same antigen.

[0279] In some embodiments of the third aspect, AG1 and AG2 are different antigens expressed on the surface of a target cell. In some embodiments of the third aspect, AG1 and AG2 are substantially the same antigen expressed on the surface of a target cell, wherein the ARD1 and the antigen recognition domain 2 (ARD2) of the CAR, TCR or the tgTCR specifically bind two different epitopes of that same antigen. Preferably, ARD1 and ARD2 specifically bind two different epitopes of CD22.

[0280] In some embodiments of the third aspect, the invention provides a combination of: a chimeric coreceptor homodimer, wherein the chimeric coreceptor subunit polypeptide is a CD8aIgV-CCR; and a chimeric antigen receptor (CAR), an endogenous TCR (TCR) or a transgene TCR (tgTCR), and preferably a CAR; wherein the CAR, the TCR or the tgTCR specifically bind an antigen 2 (AG2), and wherein AG1 and AG2 are two different antigens or are substantially the same antigen.

[0281] In some embodiments of the third aspect, the invention provides a combination of: a chimeric coreceptor complex comprising a CD8aIgV-CCR and a CD8a or a CD8B subunit; and a chimeric antigen receptor (CAR), an endogenous TCR (TCR) or a transgene TCR (tgTCR), and preferably a CAR; wherein the CAR, the TCR or the tgTCR specifically bind an antigen 2 (AG2), and wherein AG1 and AG2 are two different antigens or are substantially the same antigen.

[0282] In some embodiments of the third aspect, the invention provides a combination of: a chimeric coreceptor homodimer, wherein the chimeric coreceptor subunit polypeptide is a CD8a.4IgV-CCR; and a CAR, an endogenous TCR or a tgTCR, and preferably a CAR; wherein the CAR, the TCR or the tgTCR specifically bind an antigen 2 (AG2), and wherein AG1 and AG2 are two different antigens or are substantially the same antigen.

[0283] In some embodiments of the third aspect, the invention provides a combination of: a chimeric coreceptor complex comprising a CD8a.4IgV-CCR and a CD8a or a CD8B subunit; and a CAR, an endogenous TCR or a tgTCR, and preferably a CAR; wherein the CAR, the TCR or the tgTCR specifically bind an antigen 2 (AG2), and wherein AG1 and AG2 are two different antigens or are substantially the same antigen.

[0284] In some embodiments of the third aspect, the invention provides a combination of: a chimeric coreceptor homodimer, wherein the chimeric coreceptor subunit polypeptide is a CD28IgV-CCR; and a CAR, an endogenous TCR or a tgTCR, and preferably a CAR; wherein the CAR, the TCR or the tgTCR specifically bind an antigen 2 (AG2), and wherein AG1 and AG2 are two different antigens or are substantially the same antigen.

[0285] In some embodiments of the third aspect, the invention provides a combination of: a chimeric coreceptor complex, comprising a CD28IgV-CCR and a CD28 subunit; and a CAR, an endogenous TCR or a tgTCR, and preferably a CAR; wherein the CAR, the TCR or the tgTCR specifically bind an antigen 2 (AG2), and wherein AG1 and AG2 are two different antigens or are substantially the same antigen.

[0286] In some embodiments of the third aspect, the invention provides a combination of: i) a chimeric coreceptor homodimer, wherein the chimeric coreceptor subunit polypeptide is a 19-8IgV, or a chimeric coreceptor complex, comprising a 19-8IgV subunit polypeptide and a CD8a or a CD8B subunit; and ii) an anti-CD22 CAR as described herein, preferably selected from 22-BBz and m971- Fc-BBz.

[0287] In some embodiments of the third aspect, the invention provides a combination of: i) a chimeric coreceptor homodimer, wherein the chimeric coreceptor subunit polypeptide is a 19-8.4IgV, or chimeric coreceptor complex, comprising a 19-8.4IgV subunit polypeptide and a CD8a or a CD8B subunit; and ii) an anti-CD22 CAR as described herein, preferably selected from 22-BBz and m971- Fc-BBz.

[0288] In some embodiments of the third aspect, the invention provides a combination of: i) a chimeric coreceptor homodimer, wherein the chimeric coreceptor subunit polypeptide is a 19-28IgV, or a chimeric coreceptor complex, comprising a 19-28IgV subunit polypeptide and a CD28 subunit; and ii) an anti-CD22 CAR as described herein, preferably selected from 22-BBz, and m971- Fc-BBz.

[0289] The term "chimeric antigen receptor (CAR)" as used in the context of the present invention, refers to a "chimeric antigen receptor (CAR)" as known in the art and in particular comprising:

[0290] (i) an extracellular antigen-recognition domain 2 (ARD2) specifically binding an antigen 2 (AG2),

[0291] (ii) a hinge domain 2 (HD2),

[0292] (iii) a transmembrane domain 2 (TMD2), and (iv) a 4- IBB intracellular signaling domain and / or a CD28 intracellular signaling domain, and

[0293] (v) a CD3zeta domain.

[0294] In some embodiments, the ARD2 is selected from the group consisting of anti-ALK, anti- B7-H3 (CD276), anti-BCMA, anti-c-MET, anti-CD3, anti-CD5, anti-CD19, anti-CD20, anti- CD22, anti-CD23, anti-CD30, anti-CD33, anti-CD38, anti-CD44, anti-CD44v6, anti-CD52, anti- CD70, anti-CD79A, anti-CD79B, anti-CD123, anti-CD138, anti-CD171, anti-CEA, anti-Claudin- 6, anti-Claudin-18.2, anti-CLLl, anti-CXCR5, anti-EGFR, anti-EGFRvIII, anti-EPH-receptor A2, anti-IGLV3-21, anti-IGLV3-21-R110, anti-IL-lRAP, anti-GPC-2, anti-GPC-3, anti-HER2, anti- ErbB3, anti-ErbB4, anti-FAP, anti-FBP, anti-AchR, anti- Fr-a, anti-GD2, anti-GD3, anti-HMW- MAA, anti-IE-13Ra2, anti-Kappa-EC, anti-IGEV3-21, anti-Eewis Y, anti-LMPl, anti-LMP2A, anti-Mesothelin, anti-MAGE-4A, anti-MUCl, anti-MUC16, anti-NKG2D Ligands, anti-NCAM, anti-NY-ESOl, anti- Oncofetal antigen h5T4, anti-PRAME, anti-PSCA, anti-PSMA, anti-RORl, anti-TAG-72, anti-VEGFR, anti-GOLPH2, and anti-SLAMF7.

[0295] In some preferred embodiments, the ARD2 is selected from the group comprising anti- EGFRvIII, anti-CD20, anti-CD79A / B, anti-BCMA, anti-MUCl, anti-IL-13Ra2, anti-Claudin-6, anti-Claudin-18.2, anti-PSMA, anti-IGLV3-21R110antigen-recognition domain. In some embodiments, the ARD2 is an scFv selected from the group comprising an anti-EGFRvIII, anti- CD20, anti-CD79A / B, anti-BCMA, anti-MUCl, anti-IL-13Ra2, anti-Claudin-6, anti-Claudin-18.2, anti-PSMA, anti-IGLV3-21R110 scFv. In preferred embodiments, the ARD2 is an anti-CD22 ARD2 comprising a VH comprising or consisting of the amino acid sequence in SEQ ID NO: 9, or a variant or a fragment thereof having at least 95% sequence identity to SEQ ID NO: 9, and a VL comprising or consisting of the amino acid sequence in SEQ ID NO: 5, or a variant or a fragment thereof having at least 95% sequence identity to SEQ ID NO: 5, and optionally a linker sequence located between the VH and VL sequence, wherein the linker preferably comprises or consists of the amino acid sequence in SEQ ID NO: 10, and wherein the ARD2 is preferably a scFv.

[0296] As used herein, a "variant" of an amino acid sequence of a VH or VL is to be understood as a functional variant that has identical functional properties to the parent amino acid sequence with respect to the ability to constitute the variable region that recognises and binds the target antigen AG2.

[0297] In preferred embodiments, the ARD2 is an anti-CD22 ARD2 comprising a HFR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 15, a HCDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 6, a HFR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 16, a HCDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 7, a HFR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 17, a HCDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 8, a HFR4 comprising or consisting of the amino acid sequence of SEQ ID NO: 18, optionally a linker comprising or consisting of the amino acid sequence of SEQ ID NO: 28, a LFR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 11, a LCDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 2, a LFR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 12, a LCDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 3, a LFR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 13, and a LCDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 4, and wherein the ARD1 is preferably a scFv.

[0298] In one embodiment of the present invention, the anti-CD22 ARD2 comprises a leader sequence preferably comprising or consisting of the amino acid sequence of SEQ ID NO: 1 (human CSF2Ra leader sequence).

[0299] Definitions given and embodiments described with respect to the linker comprised in the ARD1 of the chimeric coreceptor apply also to the linker comprised in the ARD2 of the chimeric antigen receptor, in as far as they are applicable.

[0300] In the combination of the third aspect of the present invention of a chimeric coreceptor and a CAR, a chimeric coreceptor subunit polypeptide may undergo heterotypic interactions with a CAR subunit polypeptide upon expression on a cell surface to form a complex comprising the chimeric coreceptor subunit polypeptide and the CAR subunit polypeptide. Such a complex may be capable of inducing an immune response in the cell upon binding of only AG1 and not AG2 to a target cell, such as a tumor cell. In particular, a chimeric coreceptor subunit polypeptide may undergo heterotypic interaction with a CAR subunit polypeptide upon expression on a cell surface if HD1 and HD2 are substantially identical and / or if TMD1 and TMD2 are substantially identical. Therefore, it is preferred that in the chimeric coreceptor and CAR of a combination according to the present invention, the HD1 is substantially different from HD2, and / or the TMD1 is substantially different from TMD2.

[0301] The wordings "HD1 is substantially different from HD2" and the "TMD1 is substantially different from TMD2" mean in the context of the invention that the two amino acid sequences of HD1 and HD2 or of TMD1 and TMD2, respectively, show less than 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 70%, 65%, or 60% sequence identity, such that the respective chimeric coreceptor and CAR cannot undergo heterotypic interactions.

[0302] Therefore, an embodiment of the present invention provides a combination of: i) a chimeric coreceptor homodimer comprising two substantially identical chimeric coreceptor subunit polypeptides according to the first aspect, or a chimeric coreceptor complex comprising a chimeric coreceptor subunit polypeptide according to the first aspect and a CD8a, CD8P or CD28 subunit; and ii)- a chimeric antigen receptor (CAR) comprising a hinge domain 2 (HD2) and a transmembrane domain 2 (TMD2), wherein the CAR specifically binds an antigen 2 (AG2), and wherein AG1 and AG2 are two different antigens or are substantially the same antigen, and wherein the HD1 is substantially different from the HD2, and / or wherein the TMD1 is substantially different from the TMD2.

[0303] In a preferred embodiment, the HD1 comprises or consists of a CD8a or CD8P HD, HD2 comprises or consists of a CD28 HD, TMD1 comprises or consists of a CD8a or CD8P HD, and TMD2 comprises or consists of a CD28 HD. In a preferred embodiment, the HD1 comprises or consists of a CD28 HD, HD2 comprises or consists of a CD8a or CD8P HD, TMD1 comprises or consists of a CD28 TMD, and TMD2 comprises or consists of a CD8a or CD8P TMD.

[0304] The term "TCR" (T cell receptor) as used in the context of the invention refers to a heterodimeric cell surface protein of the immunoglobulin superfamily that associates with the invariant CD3 dimers CD3ey, CD3c5, and CD3^ to form a TCR-CD3 complex. The TCR mediates recognition of antigenic peptides bound to MHC molecules (pMHC), while the CD3 molecules transduce activation signals to the T cell. TCRs consist of two main subunit a- and P- chains (aP- TCR) or y- and 5-chains (y5-TCR), which are twisted together and have quite distinct anatomical locations and functions. Both TCR chains possess a variable antigen binding region, an invariant extracellular constant region, and a TM domain. The antigen binding region binds a specific antigen (antigen 2, AG2, for the present invention) only when it is loaded on an MHC molecule (pMHC), in particular an MHCI or MHCII molecule. The term "transgenic TCR (tgTCR)" in the context of the present invention refers to an exogenous TCR that recognizes and binds to a specific antigen of interest loaded on an MHC molecule.

[0305] T cells expressing a transgenic T cell receptor (TCR T cells or tgTCR T cells) allow to generate sufficient numbers of T cells specific for a given tumor antigen in a short period of time, avoiding their exhaustion. The tgTCR is usually transduced into central memory T cells or T cells with stem cell characteristics, which may ensure better persistence and function upon transfer. tgTCR-engineered T cells are usually infused into cancer patients who have been rendered lymphopenic by chemotherapy or irradiation, which allows for efficient engraftment but inhibits immune suppression.

[0306] Definitions given and embodiments described with respect to the first and second aspect apply also to the third aspect, in as far as they are applicable. Also, definitions and embodiments described below, in particular under the header 'Definitions' and 'Further embodiments of the invention' apply to the combination of the third aspect.

[0307] Nucleic acids

[0308] In a fourth aspect, the invention provides a nucleic acid construct (N-CCR) comprising a nucleic acid encoding the chimeric coreceptor subunit polypeptide according to the first aspect, or a nucleic acid construct (N-CCR-CAR) comprising a nucleic acid encoding the chimeric coreceptor subunit polypeptide according to the first aspect and a nucleic acid encoding a CAR subunit polypeptide, or two nucleic acid constructs (N-CCR, N-CAR), wherein the first nucleic acid construct (N- CCR) comprises a nucleic acid encoding the chimeric coreceptor subunit polypeptide according to the first aspect and the second nucleic acid construct (N-CAR) comprises a nucleic acid encoding a CAR, or a nucleic acid construct (N-CCR-tgTCR) comprising a nucleic acid encoding the chimeric coreceptor subunit polypeptide according to the first aspect and a nucleic acid encoding a tgTCR, or two nucleic acid constructs (N-CCR, N-tgTCR), wherein the first nucleic acid construct (N- CCR) comprises a nucleic acid encoding the chimeric coreceptor subunit polypeptide according to the first aspect and the second nucleic acid construct (N-CAR) comprises a nucleic acid encoding a tgTCR. The term "nucleic acid", as used herein, includes DNA and RNA such as genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules. A nucleic acid may be single-stranded or double-stranded. RNA includes in vitro transcribed RNA (IVT RNA) or synthetic RNA. According to the invention, a nucleic acid is preferably an isolated nucleic acid.

[0309] The term "nucleic acid construct (N-CCR)" refers to a nucleic acid encoding the chimeric coreceptor subunit polypeptide according to the first aspect.

[0310] The term "nucleic acid construct (N-CCR-CAR)" refers to a nucleic acid bicistronic construct comprising a nucleic acid (N-CCR) encoding the chimeric coreceptor subunit polypeptide according to the first aspect and a nucleic acid encoding a CAR subunit polypeptide (N-CAR).

[0311] The term "nucleic acid construct (N-CCR-tgTCR)" refers to a nucleic acid bicistronic construct comprising a nucleic acid (N-CCR) encoding the chimeric coreceptor subunit polypeptide of the first aspect and a nucleic acid encoding a tgTCR subunit polypeptide (N- tgTCR).

[0312] According to one embodiment, the nucleic acid (N-CCR) comprises a nucleic acid sequence of nucleotides 1534 to 3024 of SEQ ID NO: 119 [19-8IgV], nucleotides 1534 to 3048 of SEQ ID NO: 120 [19-8.4IgV], nucleotides 1543 to 2997 of SEQ ID NO: 126 [19-28IgV], or a sequence being at least about 80%, 81%, 82%, 83%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0313] According to one embodiment, the nucleic acid encoding a CAR subunit polypeptide comprises the nucleic acid sequence of nucleotides 1 to 1464 of SEQ ID NO: 117 [22-28hi / tm- BBz], or of nucleotides 1 to 1473 of SEQ ID NO: 125 [22-8hi / tm-BBz], a sequence being at least about 80%, 81%, 82%, 83%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0314] According to one embodiment, the nucleic acid construct (N-CCR-CAR) comprises a nucleic acid sequence selected from SEQ ID NOs 114 - 119 and 121, or a sequence being at least about 80%, 81%, 82%, 83%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0315] The nucleic acid or nucleic acid construct encoding the chimeric coreceptor of the present invention may be administered to a patient in need thereof in naked form or in a carrier. Suitable carriers, e.g. lipid carriers include any substance or vehicle with which nucleic acids such as DNA or RNA can be associated, e.g. by forming complexes with the nucleic acid or by forming vesicles in which the nucleic acid is enclosed or encapsulated. This may result in increased stability of the nucleic acid compared to naked nucleic acid. In particular, the stability of the nucleic acid in the blood may be increased. For example, nanoparticulate RNA formulations with a defined particle size, such as lipoplexes of RNA and liposomes, e.g. lipoplexes comprising DOTMA and DOPE or DOTMA and cholesterol, can be used.

[0316] Nucleic acids according to the present invention, may be present alone or functionally linked to other nucleic acids, which may be homologous or heterologous.

[0317] In the nucleic acid constructs of the invention, the nucleic acid, as described herein, may be functionally linked to at least one expression control sequence, such as EFla.

[0318] A nucleic acid and an expression control sequence are "functionally" linked each another, if they are covalently linked to one another in such a way that expression or transcription of the nucleic acid is under the control or under the influence of the expression control sequence. When the nucleic acid is to be translated into a functional protein, then, with an expression control sequence functionally linked to a coding sequence, induction of the expression control sequence results in transcription of the nucleic acid, without causing a frame shift in the coding sequence or the coding sequence being unable to be translated into the desired protein or peptide.

[0319] The term "expression control sequence" or "expression control element" as used in the context of the invention comprises promoters (e.g. EFla promoter), ribosome binding sites, enhancers (e.g. a WPRE sequence), upstream activator sequences and other control elements which regulate transcription of of the nucleic acid(s) or translation of mRNA(s) and enhancer sequences or upstream activator sequences. The exact structure of expression control sequences may vary as a function of the species or cell type, but generally comprises 5 ’-untranscribed and 5’- and 3’- untranslated sequences which are involved in initiation of transcription and translation, respectively, such as TATA box, capping sequence, CAAT sequence, and the like. More specifically, 5 ’-untranslated expression control sequences comprise a promoter region which includes a promoter sequence for transcriptional control of the functionally linked nucleic acid.

[0320] Moreover, in the bicistronic constructs or in the nucleic acid constructs of the present invention, the first and second nucleic acids are separated by a sequence (such as P2A, E2A, F2A, T2A, and IRES, and preferably T2A) encoding a cleavage peptide that allows separation of the chimeric coreceptor polypeptides from the CAR or tgTCR polypeptides after protein translation.

[0321] Definitions given and embodiments described with respect to the first, second and third aspect apply also to the fourth aspect, in as far as they are applicable. Also, definitions and embodiments described below, in particular under the header 'Definitions' and 'Further embodiments of the invention' apply to the nucleic acids of the fourth aspect.

[0322] Vectors

[0323] The nucleic acids and nucleic acid constructs encoding the chimeric coreceptors and CARs according to the fourth aspect of the invention are preferably cloned into expression vectors, for example retroviral or lentiviral vectors, which are then transduced into mammalian cells.

[0324] In a fifth aspect, the invention provides a vector or a kit of a first and second vector, wherein

[0325] (a) the vector comprises the nucleic acid construct (N-CCR), or the nucleic acid construct (N-CCR-CAR) or the nucleic acid construct (N-CCR-tgTCR) according to the fourth aspect;

[0326] (b) the first vector comprises the first nucleic acid construct (N-CCR) according to the fourth aspect, and the second vector comprises the second nucleic acid construct (N- CAR) according to the fourth aspect; or

[0327] (c) the first vector comprises the first nucleic acid construct (N-CCR) according to the fourth aspect, and the second vector comprises the second nucleic acid construct (N- tgTCR) according to the fourth aspect.

[0328] The term "vector" as used herein includes any vectors known to the skilled person in the art, including plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as retroviral, in particular lentiviral, adenoviral, adeno-associated viral or baculoviral vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or Pl artificial chromosomes (PAC). Said vectors include expression as well as cloning vectors. Expression vectors comprise plasmids as well as viral vectors and generally contain a desired coding sequence and appropriate DNA sequences necessary for the expression of the operably linked coding sequence in a particular host organism (e.g., bacterial, yeast, plant, insect, or mammalian) or in in vitro expression systems. Cloning vectors are generally used to engineer and amplify a certain desired DNA fragment and may lack functional sequences needed for expression of the desired DNA fragments. In one embodiment of the present invention, the vector is a viral vector selected from the group comprising a retroviral vector, in particular a lentiviral vector, adenoviral vector, adeno-associated viral vector or baculoviral vector.

[0329] In one embodiment of the "kit of a first and second vector" of the fifth aspect, the first vector comprises the first nucleic acid construct N-CCR according to the fourth aspect, and the second vector comprises the second nucleic acid construct N-CAR according to the fourth aspect. In a further embodiment of the "kit of a first and second vector" of the fifth aspect, the first vector comprises the first nucleic acid construct N-CCR according to the fourth aspect, and the second vector comprises the second nucleic acid construct N- tgTCR according to the fourth aspect. The kit may further comprise reagents and written instructions for using the vector to transduce a cell, preferably an immune effector cell, and to express the CCR and the CAR or tgTCR in the cell, and to store the resulting recombinant cells.

[0330] As used herein, the "written instructions" include a protocol, publication, a recording, a diagram, or any other medium of expression which can be used to provide information about the vectors of the invention and their uses. The written instructions of the kit of the invention may, for example, be affixed to a container which contains the vectors of the invention or be shipped together with a container which contains the vectors. Alternatively, the written instructions may be shipped separately from the container with the intention that the written instructions and the vectors be used cooperatively by the recipient.

[0331] Definitions given and embodiments described with respect to the first, second, third and fourth aspect apply also to the fifth aspect, in as far as they are applicable. Also, definitions and embodiments described below, in particular under the header 'Definitions' and 'Further embodiments of the invention' apply to the vectors or kit of vectors of the fifth aspect.

[0332] Recombinant cell

[0333] The inventors have here shown that mammalian cells, and in particular immune effector cells such as T cells, express the chimeric coreceptor according to the present invention and optionally a CAR, at high levels on their surface after transduction with an expression vector comprising a nucleic acid encoding for the subunit polypeptide of that chimeric coreceptor, and optionally a nucleic acid encoding for the subunit polypeptide of that CAR (Figures 2, 8, 12, 23).

[0334] In the recombinant cell of the invention expressing the chimeric coreceptor according to the first aspect on the surface, the ARD1 is capable of binding an AG1 on the surface of a target cell, which binding does not induce activation of the recombinant cell. When the recombinant cell of the invention co-expresses an antigen receptor, such as a CAR, comprising an ARD2 that binds an AG2 on the surface of a target cell, binding of the antigen receptor to the AG2 induces activation of the recombinant cell. In this case, binding of the chimeric coreceptor to the AG1 can enhance the activation of the recombinant cell induced by binding of the antigen receptorto the AG2. In addition, the chimeric coreceptor can attenuate the antigen-independent activation and differentiation of the recombinant cell induced by the antigen receptor.

[0335] In one embodiment of the sixth aspect, the invention provides a recombinant cell comprising the nucleic acid construct (N-CCR) according to the fourth aspect, and optionally wherein the cell expresses the chimeric coreceptor subunit polypeptide according to first aspect or the combination according to the third aspect, and optionally wherein AG1 and AG2 are two different antigens or are substantially the same antigen. Preferably, when AG1 and AG2 are substantially the same antigen, AG1 and AG2 are CD22.

[0336] In one embodiment of the sixth aspect, the invention provides a recombinant cell comprising the nucleic acid construct (N-CCR-CAR) or the two nucleic acid constructs (N-CCR, N-CAR) according to the fourth aspect, and optionally wherein the cell expresses the combination according to the third aspect, and optionally wherein AG1 and AG2 are two different antigens or are substantially the same antigen. Preferably, when AG1 and AG2 are substantially the same antigen, AG1 and AG2 are CD22.

[0337] In one embodiment of the sixth aspect, the invention provides a recombinant cell comprising the nucleic acid construct (N-CCR-tgTCR) or the two nucleic acid constructs (N-CCR, N- tgTCR) according to the fourth aspect, and optionally wherein the cell expresses the combination according to the third aspect, and optionally wherein AG1 and AG2 are two different antigens or are substantially the same antigen.

[0338] In one embodiment of the sixth aspect, the invention provides a recombinant cell comprising the vector or the kit of a first and second vector as defined in the fifth aspect, and optionally wherein the cell expresses the chimeric coreceptor subunit polypeptide according to first aspect or the combination according to third aspect, and optionally wherein AG1 and AG2 are two different antigens or are substantially the same antigen.

[0339] The term "recombinant cell" in the context of the present invention means a cell produced by genetic engineering, which is thus not occurring naturally.

[0340] According to a preferred embodiment of the present invention, the recombinant cell is an immune effector cell. According to a more preferred embodiment of the present invention, the recombinant cell is an NK cell, an NKT cell, a macrophage, a monocyte, a dendritic cell, a tumorinfiltrating lymphocyte (TIL), or a T cell, preferably a CD8 positive T cell, a CD4 positive T cell, a regulatory T cell, or a cytotoxic T cell, and / or wherein the cell is autologous or allogeneic. The inventors have shown that co-expression of a chimeric CD8 IgVD coreceptor not only enhances the cytotoxic activity of CD8+CAR-T cells against tumor cells harboring resistance mechanisms (such as low antigen and absent CD58 expression), but also converts CD4+CAR-T cells into potent cytotoxic killer cells (Figure 18).

[0341] Thus, according to a preferred embodiment of the present invention, the recombinant cell is a T cell, more preferably a CD4+or a CD8+T cell. Upon activation, cytotoxic T cells, NK cells and NKT cells induce the destruction of target cells by releasing cytotoxins such as perforin, granzymes, and granulysin, or by inducing apoptosis (e.g. via Fas-Fas ligand interaction). The cytotoxic lymphocytes are preferably autologous, although heterologous or allogeneic cells may be used.

[0342] The present invention also provides a population of respective recombinant cells, preferably of respective immune effector cells described herein, which population may be a clonally expanded population. The recombinant immune effector cells or populations thereof provide for therapeutic or prophylactic immune effector function in an antigen-specific manner.

[0343] Preferably, the chimeric coreceptor of the invention is expressed on the cell surface of such a recombinant cell and in particular on the cell surface of such an immune effector cell.

[0344] The term "B cells" as used in the context of the invention refers to a subset of lymphocytes that produce antibody molecules that can be either secreted or inserted into the plasma membrane where they serve as part of B cell receptors.

[0345] The term "dendritic cell" (DC), as used in the context of the invention, refers to a subset of phagocytic cells belonging to the class of antigen presenting cells. In one embodiment, dendritic cells are derived from hematopoietic bone marrow progenitor cells.

[0346] The term "macrophage" as used in the context of the invention refers to a subset of phagocytic cells produced by the differentiation of monocytes.

[0347] The term "NK cell" or "natural killer cell" as used herein refers to a subset of peripheral blood lymphocytes defined by the expression of CD56 or CD16 and the absence of the T cell receptor. As provided herein, the NK cell may also be differentiated from a stem or progenitor cell.

[0348] The term "NKT cell" or "natural killer T cell" as used in the context of the invention refers to a subset of T cells that coexpress an aP T cell receptor, and also express different molecular markers typically associated with NK cells, such as NK1.1. CD Id-restricted NKT cells ("type 2 NKT") recognize lipids and glycolipids presented by CD Id molecules rather than by major histocompatibility complexes (MHCs). The terms "T cell" and "T lymphocyte" as used in the context of the invention are used interchangeably herein and include T helper cells (CD4+T cells) and cytotoxic T cells (CTLs, CD8+T cells), which include cytolytic T cells.

[0349] T cells may generally be prepared in vitro or ex vivo, using standard procedures. For example, T cells may be isolated from bone marrow, peripheral blood or a fraction of bone marrow or peripheral blood of a mammal, such as a patient, using a commercially available cell separation system. Alternatively, T cells may be derived from related or unrelated humans or non-human animals, cell lines or cultures. A sample comprising T cells may, for example, be peripheral blood mononuclear cells (PBMCs).

[0350] T cells to be used according to the invention may express an endogenous T cell receptor or may lack expression of an endogenous T cell receptor.

[0351] The chimeric coreceptor of the present invention enhances the effect of a chimeric antigen receptor or TCR, i.e. when e.g. present on an immune effector cell such as a T cell and recognizes the antigen on for example the surface of antigen presenting cells or diseased cells such as cancer cells, the immune effector cell is more strongly stimulated, primed and / or expanded or exerts effector functions of immune effector cells as described above.

[0352] The term "antigen-specific T cell" or similar terms relate to a T cell that, in particular when provided with an antigen receptor, recognizes the antigen to which the antigen receptor is targeted such as on the surface of antigen presenting cells or diseased cells such as cancer cells and preferably exerts effector functions of T cells as described above. T cells and other lymphoid cells are considered to be specific for an antigen if the cells kill target cells expressing the antigen. T cell specificity may be evaluated using any of a variety of standard techniques, for example synthesis of lymphokines (such as interferon-y (IFN-y), interleukin-2 (IL-2), andTNF-a), proliferation assay, chromium release assay.

[0353] The invention may involve introduction, e.g. transfection, of nucleic acids encoding the chimeric coreceptor subunit polypeptides of the present invention into cells such as immune effector cells or T cells in vitro or in vivo.

[0354] For purposes of the present invention, the term "transfection" refers to the introduction of a nucleic acid into a cell or the uptake of a nucleic acid by a cell, wherein the cell may be present in a subject, e.g., a patient. Thus, according to the present invention, a cell for transfection of a nucleic acid described herein can be present in vitro, ex-vivo, or in vivo, e.g. the cell can form part of an organ, a tissue and / or an organism of a patient. According to the invention, transfection can be transient or stable. For some applications of transfection, it is sufficient if the transfected genetic material is only transiently expressed. Since the nucleic acid introduced in the transfection process is usually not integrated into the nuclear genome, the foreign nucleic acid will be diluted through mitosis or degraded. Cells allowing episomal amplification of nucleic acids greatly reduce the rate of dilution. If it is desired that the transfected nucleic acid actually remains in the genome of the cell and its daughter cells, a stable transfection must occur. RNA can be transfected into cells for transiently expressing its coded protein.

[0355] A variety of methods may be used to introduce the CCR and optionally the antigen receptor constructs into T cells including non- viral-based DNA transfection, transposon-based systems and viral-based systems. Non-viral DNA transfection has low risk of insertional mutagenesis. Transposon-based systems can integrate transgenes more efficiently than plasmids that do not contain an integrating element. Viral-based systems include the use of y-retroviruses and lentiviral vectors. y-Retro viruses are relatively easy to produce, efficiently and permanently transduce T cells, and have been preliminarily shown to be safe from an integration standpoint in primary human T cells. Lentiviral vectors also efficiently and permanently transduce T cells, but are more expensive to produce. They are also potentially safer than retrovirus-based systems.

[0356] Alternatively, the CCR and optionally the antigen receptor nucleic acids may be transfected into cells by electroporation, lipofection and microinjection. The nucleic acids can be introduced into cells by electroporation. Electroporation or electro-permeabilization refers to a significant increase in the electrical conductivity and permeability of the cell plasma membrane caused by an externally applied electrical field. It is usually used in molecular biology as a way of introducing some substance into a cell. It is preferred that introduction of nucleic acid encoding a protein or peptide into cells results in expression of said protein or peptide on the cell surface.

[0357] For in vivo transfection a pharmaceutical composition according to the seventh aspect comprising nucleic acids encoding the chimeric coreceptors or the vectors comprising said nucleic acids may be used. A specific cell-targeted vector (e.g. a pseudotyped vector) that targets a nucleic acid to a specific cell such as a T cell may be administered to a patient resulting in targeted in vivo transfection.

[0358] Definitions given and embodiments described with respect to the first, second, third, fourth and fifth aspect apply also to the sixth aspect, in as far as they are applicable. Also, definitions and embodiments described below, in particular under the header 'Definitions' and 'Further embodiments of the invention' apply to the recombinant cell of the sixth aspect. Pharmaceutical composition

[0359] In one embodiment of the seventh aspect, the invention provides a pharmaceutical composition comprising the chimeric coreceptor subunit polypeptide according to the first aspect, and a pharmaceutically acceptable carrier, stabilizer and / or excipient.

[0360] In one embodiment of the seventh aspect, the invention provides a pharmaceutical composition comprising the combination according to the third aspect, and a pharmaceutically acceptable carrier, stabilizer and / or excipient.

[0361] In one embodiment of the seventh aspect, the invention provides a pharmaceutical composition comprising the nucleic acid construct (N-CCR), the nucleic acid construct (N-CCR- CAR), the two nucleic acid constructs (N-CCR, N-CAR), the nucleic acid construct (N-CCR- tgTCR) or the two nucleic acid constructs (N-CCR, N-tgTCR) according to the fourth aspect, or the vector or the kit of the first and second vector as defined in the fifth aspect, and a pharmaceutically acceptable carrier, stabilizer and / or excipient.

[0362] In one embodiment of the seventh aspect, the invention provides a pharmaceutical composition comprising the recombinant cell according to the sixth aspect, and a pharmaceutically acceptable carrier, stabilizer and / or excipient.

[0363] A pharmaceutical composition according to the present invention is preferably sterile and contains an effective amount of the antigen receptors, peptide chains, nucleic acids, recombinant cells, immune effector cells of the invention, as well as other compounds and agents described herein, and optionally further agents as discussed herein, to generate the desired reaction or the desired effect.

[0364] Pharmaceutical compositions are usually provided in a uniform dosage form and may be prepared in a manner known per se. A pharmaceutical composition may e.g. be in the form of a solution or suspension.

[0365] A pharmaceutical composition may comprise salts, buffer substances, preservatives, carriers, diluents and / or excipients in pharmaceutically acceptable form. The term "pharmaceutically acceptable" refers to the non-toxicity of a material which does not interact with the action of the active component of the pharmaceutical composition.

[0366] Salts which are not pharmaceutically acceptable may be used for preparing pharmaceutically acceptable salts and are included in the invention. Pharmaceutically acceptable salts of this kind comprise in a non-limiting way those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic acids, and the like. Pharmaceutically acceptable salts may also be prepared as alkali metal salts or alkaline earth metal salts, such as sodium salts, potassium salts or calcium salts.

[0367] Suitable buffer substances for use in a pharmaceutical composition include acetic acid in a salt, citric acid in a salt, boric acid in a salt and phosphoric acid in a salt.

[0368] Suitable preservatives for use in a pharmaceutical composition include benzalkonium chloride, chlorobutanol, paraben and thimerosal.

[0369] The term "carrier" refers to an organic or inorganic component, of a natural or synthetic nature, in which the active component is combined in order to facilitate, enhance or enable application. According to the invention, the term "carrier" also includes one or more compatible solid or liquid fillers, diluents or encapsulating substances, which are suitable for administration to a patient. Possible carrier substances for parenteral administration are e.g. sterile water, Ringer, Ringer lactate, sterile sodium chloride solutions, polyalkylene glycols, hydrogenated naphthalenes and, in particular, biocompatible lactide polymers, lactide / glycolide copolymers or polyoxyethylene / polyoxy- propylene copolymers.

[0370] The term "excipient" when used herein is intended to indicate all substances which may be present in a pharmaceutical composition and which are not active ingredients such as, e.g., binders, lubricants, thickeners, surface active agents, preservatives, emulsifiers, buffers, flavoring agents, or colorants.

[0371] The term "stabilizer" when used herein is intended to indicate a substance which may be present in a pharmaceutical composition to improve the stability and efficacy of the CCR or antigen receptor polypeptide or nucleic acid or recombinant cell or vector, e.g. by protecting them from degradation, improving bioavailability, and controlling the release over time. Possible stabilizer substances are e.g. polymers, cyclodextrins, and chelating agents.

[0372] The compositions described herein may be administered via any conventional route, such as by parenteral administration including by injection or infusion. Administration is preferably parenterally, e.g. intravenously, intraarterially, subcutaneously, intradermally or intramuscularly.

[0373] Compositions suitable for parenteral administration usually comprise a sterile aqueous or nonaqueous preparation of the active compound, which is preferably isotonic to the blood of the recipient. Examples of compatible carriers and solvents are Ringer solution and isotonic, slightly hypertonic or slightly hypotonic sodium chloride solutions. In addition, usually sterile, fixed oils can be used as solution or suspension medium. The pharmaceutical compositions described herein are preferably administered in effective amounts. An "effective amount" refers to the amount which achieves a desired reaction or a desired effect alone or together with further doses. In the case of treatment of a particular disease or of a particular condition, the desired reaction preferably relates to inhibition of the course of the disease. This comprises slowing down the progress of the disease and, in particular, interrupting or reversing the progress of the disease. The desired reaction in a treatment of a disease or of a condition may also be delay of the onset or a prevention of the onset of said disease or said condition.

[0374] An effective amount of a composition described herein will depend on the condition to be treated, the severeness of the disease, the individual parameters of the patient, including age, physiological condition, size and weight, the duration of treatment, the type of an accompanying therapy (if present), the specific route of administration and similar factors. Accordingly, the doses administered of the polypeptides, nucleic acids, cells or compositions described herein may depend on various of such parameters. In case a reaction in a patient is insufficient with an initial dose, higher doses (or effectively higher doses achieved by a different, more localized route of administration) may be used.

[0375] The compositions described herein can be administered to patients to treat or prevent a variety of disorders such as those described herein. Patients preferably include human patients having disorders that can be corrected or ameliorated by administering the agents and compositions described herein. This includes disorders involving cells characterized by expression of an antigen, in particular by expression of CD19, CD5, CD22, CD38, EGFR, HER2, Mesothelin, IL-13Ra2, PSMA, and EPH-receptor-A2, preferably of CD22.

[0376] For example, in one embodiment, the compositions described herein can be used to treat a patient with a cancer disease, such as a cancer as described herein characterized by the presence of cancer cells expressing a tumor-associated antigen.

[0377] The pharmaceutical composition of the invention may be administered together with supplementing immunity-enhancing substances such as one or more adjuvants and may comprise one or more immunity-enhancing substances to further increase its effectiveness, preferably to achieve a synergistic effect of immune-stimulation. The term "adjuvant" relates to compounds which prolongs or enhances or accelerates an immune response. Various mechanisms are possible in this respect, depending on the various types of adjuvants. For example, compounds which allow the maturation of the DC, e.g. lipopolysaccharides or CD40 ligand, form a first class of suitable adjuvants. Generally, any agent which influences the immune system of the type of a "danger signal" (LPS, GP96, dsRNA etc.) or cytokines, such as GM-CSF, can be used as an adjuvant which enables an immune response to be intensified and / or influenced in a controlled manner. CpG oligodeoxynucleotides can optionally also be used in this context, although their side effects which occur under certain circumstances, as explained above, are to be considered. Particularly preferred adjuvants are cytokines, such as monokines, lymphokines, interleukins or chemokines, e.g. IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL- 10, IL- 12, IFNa, IFNy, GM-CSF, LT-a, or growth factors, e.g. hGH. Further known adjuvants are aluminium hydroxide, Freund's adjuvant or oil such as Montanide®, most preferred Montanide® ISA51. Lipopeptides, such as Pam3Cys, are also suitable for use as adjuvants in the pharmaceutical composition of the present invention.

[0378] The pharmaceutical composition of the invention can be administered locally or systemically, preferably systemically.

[0379] The term "systemic administration" refers to the administration of an agent such that the agent is widely distributed in significant amounts throughout the body of an individual and develops a desired effect. For example, the agent may develop its desired effect in the blood and / or reach its desired site of action via the vascular system. Typical systemic routes of administration include administration by direct introduction of the drug into the vascular system, or oral, pulmonary, or intramuscular administration in which the drug is adsorbed, enters the vascular system, and is carried by the blood to the desired site(s) of action. Systemic administration by parenteral route is a particularly preferred route. The term "parenteral administration" refers to the administration of an agent such that the agent does not pass through the intestine. The term "parenteral administration" includes, but is not limited to, intravenous administration, subcutaneous administration, intradermal administration, or intraarterial administration.

[0380] Administration may also be carried out, for example, orally, intraperitoneally or intramuscularly.

[0381] The pharmaceutical compositions described herein may be used alone or in combination with conventional therapeutic regimens such as surgery, irradiation, chemotherapy and / or bone marrow transplantation (autologous, syngeneic, allogeneic or unrelated).

[0382] Definitions given and embodiments described with respect to the first, second, third, fourth fifth, and sixth aspect apply also to the seventh aspect, in as far as they are applicable. Also, definitions and embodiments described below, in particular under the header 'Definitions' and 'Further embodiments of the invention' apply to the pharmaceutical composition of the seventh aspect.

[0383] Uses

[0384] In one embodiment of the eighth aspect, the invention provides a chimeric coreceptor subunit polypeptide according to the first aspect, for use in medicine. In one embodiment of the eighth aspect, the invention provides a combination according to the third aspect, for use in medicine. In one embodiment of the eighth aspect, the invention provides a nucleic acid construct (N-CCR), a nucleic acid construct (N-CCR-CAR), a two nucleic acid constructs (N-CCR, N-CAR), a nucleic acid construct (N-CCR-tgTCR) or two nucleic acid constructs (N-CCR, N-tgTCR) according to the fourth aspect for use in medicine. In one embodiment of the eighth aspect, the invention provides a vector or a kit of a first and second vector according to the fifth aspect, for use in medicine. In one embodiment of the eighth aspect, the invention provides a recombinant cell according to the sixth aspect, for use in medicine. In one embodiment of the eighth aspect, the invention provides a pharmaceutical composition according to the seventh aspect, for use in medicine.

[0385] The term "use in medicine" as used in the context of the invention refers to the use in the prevention and / or treatment of a disease in a subject, such as a malignant tumor disease and preferably a B cell malignancy, or an autoimmune disease. The tumor disease is, for example, a tumor disease characterized by the expression of the antigen CD22, in a cancer or tumor cell of said subject. The autoimmune disease is, for example, an autoimmune disease characterized by the presence of autoreactive or hyperactive B cells.

[0386] With respect to the above mentioned medical applications of the chimeric coreceptors (as polypeptides, nucleic acids or vectorsjand other materials (recombinant cells, pharmaceutical compositionsjderived therefrom, the to be treated cancer can be any cancer characterized by a cellsurface expression of at least one tumor-associated antigen selected from the group consisting of ALK, B7-H3 (CD276), BCMA, c-MET, CD3, CD5, CD19, CD20, CD22, CD23, CD30, CD33, CD38, CD44, CD44v6, CD52, CD70, CD79A, CD79B, CD123, CD138, CD171, CEA, Claudin- 6, Claudin-18.2, CLL1, CXCR5, EGFR, EGFRvIII, EPH-receptor A2, IGLV3-21, IL-1RAP, GPC- 2, GPC-3, HER2, ErbB3, ErbB4, FAP, FBP, AchR, Fr-a, GD2, GD3, HMW-MAA, IE-13Ra2, Kappa-EC, IGEV3-21-R110, Eewis Y, EMP1, EMP2A, Mesothelin, MAGE-4A, MUC1, MUC16, NKG2D Eigands, NCAM, NY-ESO1, Oncofetal antigen h5T4, PRAME, PSCA, PSMA, ROR1, TAG-72, VEGFR, G0LPH2, and SLAMF7, and preferably the cancer is preferably a B cell malignancy.

[0387] In a preferred embodiment, the at least one tumor-associated antigen is CD22, and the cancer is selected from the group consisting of Non-Hodgkin lymphoma (NHL), including Diffuse large B-cell lymphoma (DLBCL), Follicular lymphoma, Mantle cell lymphoma, Burkitt lymphoma, and Marginal zone lymphoma; Chronic lymphocytic leukemia (CLL), B-cell acute lymphoblastic leukemia (B-ALL), Waldenstrom macroglobulinemia, Hairy cell leukemia.

[0388] In a preferred embodiment, to be treated disease is an autoimmune disease preferably selected from the group consisting of systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), multiple sclerosis (MS), systemic sclerosis (SSc), type 1 diabetes (T1D), myasthenia gravis (MG), goodpasture syndrome, autoimmune hemolytic anemia (AIHA), antiphospholipid syndrome (APS), and vasculitis, such as giant cell arteritis, granulomatosis with polyangiitis (GPA), and eosinophilic granulomatosis with polyangiitis (EGPA), wherein the autoimmune disease is characterized by the presence of autoreactive or hyperactive B cells.

[0389] The chimeric receptor subunit polypeptides, combinations, nucleic acids, nucleic acid constructs, vectors / kit of vectors, and pharmaceutical compositions of the invention are in particular for use in immunotherapy, preferably in adoptive T cell therapy. The administration of the chimeric receptor subunit polypeptides, combinations, nucleic acids, nucleic acid constructs, vectors / kit of vectors, and pharmaceutical compositions of the invention can, for example, involve the infusion of T cells of the invention into said patient. Preferably, such T cells are autologous T cells of the patient and in vitro transduced with a nucleic acid, nucleic acid construct, or vector of the present invention.

[0390] Definitions given and embodiments described with respect to the first, second, third, fourth fifth, sixth, and seventh aspect apply also to the eighth aspect, in as far as they are applicable. Also, definitions and embodiments described below, in particular under the header 'Definitions' and 'Further embodiments of the invention' apply to the items for use of the eighth aspect.

[0391] In one embodiment of the ninth aspect, the invention provides a chimeric coreceptor subunit polypeptide according to the first aspect for use in treating cancer, and wherein the cancer is preferably characterized by a cell-surface expression of at least one tumor-associated antigen selected from the group consisting of ALK, B7-H3 (CD276), BCMA, c-MET, CD3, CD5, CD19, CD20, CD22, CD23, CD30, CD33, CD38, CD44, CD44v6, CD52, CD70, CD79A, CD79B, CD123, CD138, CD171, CEA, Claudin-6, Claudin-18.2, CLL1, CXCR5, EGFR, EGFRvIII, EPH- receptor A2, IGLV3-21, IL-1RAP, GPC-2, GPC-3, HER2, ErbB3, ErbB4, FAP, FBP, AchR, Fr-a, GD2, GD3, HMW-MAA, IL-13Ra2, Kappa-EC, IGEV3-21-R110, Eewis Y, EMP1, EMP2A, Mesothelin, MAGE-4A, MUC1, MUC16, NKG2D Eigands, NCAM, NY-ES01, Oncofetal antigen h5T4, PRAME, PSCA, PSMA, R0R1, TAG-72, VEGFR, GOLPH2, and SLAMF7, and / or wherein the cancer is preferably a B cell malignancy.

[0392] In one embodiment of the ninth aspect, the invention provides a combination comprising:

[0393] I) a chimeric coreceptor homodimer comprising two substantially identical chimeric coreceptor subunit polypeptides according to the first aspect, or a chimeric coreceptor complex comprising a chimeric coreceptor subunit polypeptide according to the first aspect and a CD8a, CD8P or CD28 subunit;

[0394] II) a chimeric antigen receptor (CAR), an endogenous TCR (TCR) or a transgene TCR (tgTCR), wherein the CAR, the TCR or the tgTCR specifically bind an antigen 2 (AG2), and wherein AG1 and AG2 are two different antigens or are substantially the same antigen; for use in treating cancer, and wherein the cancer is preferably characterized by a cell-surface expression of at least one tumor-associated antigen selected from the group consisting of ALK, B7- H3 (CD276), BCMA, c-MET, CD3, CD5, CD19, CD20, CD22, CD23, CD30, CD33, CD38, CD44, CD44v6, CD52, CD70, CD79A, CD79B, CD123, CD138, CD171, CEA, Claudin-6, Claudin-18.2, CLL1, CXCR5, EGFR, EGFRvIII, EPH-receptor A2, IGLV3-21, IL-1RAP, GPC-2, GPC-3, HER2, ErbB3, ErbB4, FAP, FBP, AchR, Fr-a, GD2, GD3, HMW-MAA, IL-13Ra2, Kappa-LC, IGLV3-21-R110, Lewis Y, LMP1, LMP2A, Mesothelin, MAGE-4A, MUC1, MUC16, NKG2D Ligands, NCAM, NY-ESO1, Oncofetal antigen h5T4, PRAME, PSCA, PSMA, ROR1, TAG-72, VEGFR, GOLPH2, and SLAMF7, and / or wherein the cancer is preferably a B cell malignancy. Preferably, the AG2 comprises or consists of the tumor-associated antigen expresses on the surface of the cancer cell. Preferably, AG2 is CD22 and / or AG1 is CD19, or AG1 is CD22 and / or AG2 is CD 19.

[0395] In one embodiment of the ninth aspect, the invention provides a nucleic acid construct (N- CCR), a nucleic acid construct (N-CCR-CAR), a two nucleic acid constructs (N-CCR, N-CAR), a nucleic acid construct (N-CCR-tgTCR) or two nucleic acid constructs (N-CCR, N-tgTCR) according to the fourth aspect for use in treating cancer, and wherein the cancer is preferably characterized by a cell-surface expression of at least one tumor-associated antigen selected from the group consisting of ALK, B7-H3 (CD276), BCMA, c-MET, CD3, CD5, CD19, CD20, CD22, CD23, CD30, CD33, CD38, CD44, CD44v6, CD52, CD70, CD79A, CD79B, CD123, CD138, CD171, CEA, Claudin-6, Claudin-18.2, CLL1, CXCR5, EGFR, EGFRvIII, EPH-receptor A2, IGLV3-21, IL-1RAP, GPC-2, GPC-3, HER2, ErbB3, ErbB4, FAP, FBP, AchR, Fr-a, GD2, GD3, HMW-MAA, IL-13Ra2, Kappa-EC, IGEV3-21-R110, Eewis Y, EMP1, EMP2A, Mesothelin, MAGE-4A, MUC1, MUC16, NKG2D Eigands, NCAM, NY-ES01, Oncofetal antigen h5T4, PRAME, PSCA, PSMA, R0R1, TAG-72, VEGFR, G0LPH2, and SLAMF7, and / or wherein the cancer is preferably a B cell malignancy. Preferably, the AG2 comprises or consists of the tumor- associated antigen expresses on the surface of the cancer cell. Preferably, AG2 is CD22 and / or AG1 is CD19, or AG1 is CD22 and / or AG2 is CD19.

[0396] In one embodiment of the ninth aspect, the invention provides a vector or a kit of a first and second vector according to the fifth aspect for use in treating cancer, and wherein the cancer is preferably characterized by a cell-surface expression of at least one tumor-associated antigen selected from the group consisting of ALK, B7-H3 (CD276), BCMA, c-MET, CD3, CD5, CD19, CD20, CD22, CD23, CD30, CD33, CD38, CD44, CD44v6, CD52, CD70, CD79A, CD79B, CD123, CD138, CD171, CEA, Claudin-6, Claudin-18.2, CLL1, CXCR5, EGFR, EGFRvIII, EPH- receptor A2, IGLV3-21, IL-1RAP, GPC-2, GPC-3, HER2, ErbB3, ErbB4, FAP, FBP, AchR, Fr-a, GD2, GD3, HMW-MAA, IL-13Ra2, Kappa-LC, IGLV3-21-R110, Lewis Y, LMP1, LMP2A, Mesothelin, MAGE-4A, MUC1, MUC16, NKG2D Ligands, NCAM, NY-ESO1, Oncofetal antigen h5T4, PRAME, PSCA, PSMA, R0R1, TAG-72, VEGFR, G0LPH2, and SLAMF7, and / or wherein the cancer is preferably a B cell malignancy. Preferably, the AG2 comprises or consists of the tumor-associated antigen expresses on the surface of the cancer cell. Preferably, AG2 is CD22 and / or AG1 is CD19, or AG1 is CD22 and / or AG2 is CD19.

[0397] In one embodiment of the ninth aspect, the invention provides a recombinant cell according to the sixth aspect, for use in treating cancer, and wherein the cancer is preferably characterized by a cell-surface expression of at least one tumor-associated antigen selected from the group consisting of ALK, B7-H3 (CD276), BCMA, c-MET, CD3, CD5, CD19, CD20, CD22, CD23, CD30, CD33, CD38, CD44, CD44v6, CD52, CD70, CD79A, CD79B, CD123, CD138, CD171, CEA, Claudin-6, Claudin-18.2, CLL1, CXCR5, EGFR, EGFRvIII, EPH-receptor A2, IGLV3-21, IL-1RAP, GPC-2, GPC-3, HER2, ErbB3, ErbB4, FAP, FBP, AchR, Fr-a, GD2, GD3, HMW- MAA, IL-13Ra2, Kappa-LC, IGLV3-21-R110, Lewis Y, LMP1, LMP2A, Mesothelin, MAGE-4A, MUC1, MUC16, NKG2D Ligands, NCAM, NY-ESO1, Oncofetal antigen h5T4, PRAME, PSCA, PSMA, R0R1, TAG-72, VEGFR, GOLPH2, and SLAMF7, and / or wherein the cancer is preferably a B cell malignancy. Preferably, the AG2 comprises or consists of the tumor-associated antigen expresses on the surface of the cancer cell. Preferably, AG2 is CD22 and / or AG1 is CD 19, or AG1 is CD22 and / or AG2 is CD 19.

[0398] In one embodiment of the ninth aspect, the invention provides a pharmaceutical composition according to the seventh aspect, for use in treating cancer, and wherein the cancer is preferably characterized by a cell-surface expression of at least one tumor-associated antigen selected from the group consisting of ALK, B7-H3 (CD276), BCMA, c-MET, CD3, CD5, CD19, CD20, CD22, CD23, CD30, CD33, CD38, CD44, CD44v6, CD52, CD70, CD79A, CD79B, CD123, CD138, CD171, CEA, Claudin-6, Claudin-18.2, CLL1, CXCR5, EGFR, EGFRvIII, EPH- receptor A2, IGLV3-21, IL-1RAP, GPC-2, GPC-3, HER2, ErbB3, ErbB4, FAP, FBP, AchR, Fr-a, GD2, GD3, HMW-MAA, IL-13Ra2, Kappa-LC, IGLV3-21-R110, Lewis Y, LMP1, LMP2A, Mesothelin, MAGE-4A, MUC1, MUC16, NKG2D Ligands, NCAM, NY-ESO1, Oncofetal antigen h5T4, PRAME, PSCA, PSMA, R0R1, TAG-72, VEGFR, GOLPH2, and SLAMF7, and / or wherein the cancer is preferably a B cell malignancy. Preferably, the AG2 comprises or consists of the tumor-associated antigen expresses on the surface of the cancer cell. Preferably, AG2 is CD22 and / or AG1 is CD19, or AG1 is CD22 and / or AG2 is CD19.

[0399] More preferably, the chimeric receptors, combinations, nucleic acids, nucleic acid constructs, vectors / vectors kit, and pharmaceutical compositions of the invention described herein is for use in treating a cancer characterized by a cell-surface expression of at least one tumor- associated antigen, and wherein the at least one tumor-associated antigen has a density lower than about 1000 molecules / cell, about 400 molecules / cell, about 300 molecules / cell, about 200 molecules / cell, about 100 molecules / cell, or about 40 molecules / cell, and preferably lower than about 400 molecules / cell, and more preferably lower than about 40 molecules / cell.

[0400] More preferably, the chimeric receptors, combinations, nucleic acids, nucleic acid constructs, vectors / vectors kit, and pharmaceutical compositions of the invention described herein are for use in treating a cancer characterized by a cell-surface expression of at least one tumor- associated antigen, wherein the at least one tumor-associated antigen has a density lower than about 1000 molecules / cell, about 400 molecules / cell, about 300 molecules / cell, about 200 molecules / cell, about 100 molecules / cell, or about 40 molecules / cell, and preferably lower than about 400 molecules / cell, and more preferably lower than about 40 molecules / cell, and wherein the at least one tumor-associated antigen is CD22. Thus, it is preferred that the cancer is characterized by the presence of cancer cells expressing CD22. According to a particularly preferred embodiment, the cancer characterized by the presence of cancer cells expressing CD22 is a B cell malignancy.

[0401] Accordingly, the chimeric receptors, combinations, nucleic acids, nucleic acid constructs, vectors / vectors kit, and pharmaceutical compositions of the invention described herein can be used to treat a subject with a cancer. According to a preferred embodiment, the chimeric receptors, combinations, nucleic acids, nucleic acid constructs, vectors / vectors kit, and pharmaceutical compositions of the invention described herein can be used to treat a cancer characterized by a cellsurface expression of CD22 in a subject, for example a cancer characterized by the presence of cancer cells expressing CD22. The cancer associated with cell-surface CD22 expression is preferably a B cell malignancy.

[0402] The term "disease" refers to an abnormal condition that affects the body of an individual. A disease is often construed as a medical condition associated with specific symptoms and signs. A disease may be caused by factors originally from an external source, such as infectious disease, or it may be caused by internal dysfunctions, such as autoimmune diseases. In humans, "disease" is often used more broadly to refer to any condition that causes pain, dysfunction, distress, social problems, or death to the individual afflicted, or similar problems for those in contact with the individual. In this broader sense, it sometimes includes injuries, disabilities, disorders, syndromes, infections, isolated symptoms, deviant behaviors, and atypical variations of structure and function, while in other contexts and for other purposes these may be considered distinguishable categories. According to the invention, the term "disease" includes autoimmune diseases and cancer diseases, in particular those forms of cancer described herein. Any reference herein to cancer or particular forms of cancer also includes cancer metastasis thereof.

[0403] The term "autoimmune disease" refers to a disease caused by autoreactive B cells (producting autoantibodies) or T cells targeting autoantigen of a organ / tissue and thus causing its pathological and / or functional damage. Thus, in autoimmune diseases the autoreactive lymphocytes are the actual cause of the disease.

[0404] The term "cancer characterized by a cell-surface expression of an antigen" or similar expressions means according to the invention that the antigen is expressed in cancer cells of a diseased tissue or organ. Expression in cancer cells of a diseased tissue or organ may be increased compared to the state in a healthy tissue or organ. In one embodiment, expression is only found in a diseased tissue, while expression in a healthy tissue is not found, e.g. expression is repressed. According to the invention, diseases involving an antigen include autoimmune diseases and cancer diseases, wherein the disease-associated antigen is preferably an antigen of the autoreactive and hyperactive B cells or a tumor antigen, respectively. Preferably a disease involving an antigen preferably is a disease involving cells expressing an antigen, preferably on the cell surface.

[0405] The term "healthy" or "normal" refer to non-pathological conditions, and preferably means non- infected or non-cancerous.

[0406] The terms "cancer disease" or "cancer" refer to or describe the physiological condition in an individual that is typically characterized by unregulated cell growth. The term “cancer” according to the invention also comprises cancer metastases. Preferably, a "cancer disease" is characterized by cells expressing CD22. Preferably, a "cancer disease" is a B cell malignancy.

[0407] In one embodiment, a cancer disease is a malignant disease which is characterized by the properties of anaplasia, invasiveness, and metastasis. A malignant tumor may be contrasted with a non-cancerous benign tumor in that a malignancy is not self-limited in its growth, is capable of invading into adjacent tissues, and may be capable of spreading to distant tissues (metastasizing), while a benign tumor has none of those properties.

[0408] The term "B cell malignancy" refers to a condition characterized by B cells which malfunction, and become malignant so that they grow when the body doesn’t need them and reproduce at an abnormal rate. B-cell malignancies can broadly be categorised into Hodgkin and non-Hodgkin lymphomas. Exemplary B-cell malignancies are selected from the group comprising Chronic lymphocytic leukaemia (CLL), Mantle Cell Lymphoma (MCL), Waldenstrom’s Macroglobulinemia (WM), Diffuse Large B-cell lymphoma (DLBCL), and Follicular Lymphoma (FL).

[0409] According to the invention, the term "tumor" or "tumor disease" refers to a swelling or lesion formed by an abnormal growth of cells (called neoplastic cells or tumor cells). By "tumor cell" is meant an abnormal cell that grows by a rapid, uncontrolled cellular proliferation and continues to grow after the stimuli that initiated the new growth cease. Tumors show partial or complete lack of structural organization and functional coordination with the normal tissue, and usually form a distinct mass of tissue, which may be either benign, pre-malignant or malignant.

[0410] According to the invention, a "carcinoma" is a malignant tumor derived from epithelial cells. This group represents the most common cancers, including the common forms of breast, prostate, lung and colon cancer. Lymphoma and leukemia are malignancies derived from hematopoietic (blood-forming) cells.

[0411] The term "metastasis" as used herein refers to the spread of cancer cells from its original site to another part of the body. The formation of metastasis is a very complex process and depends on detachment of malignant cells from the primary tumor, invasion of the extracellular matrix, penetration of the endothelial basement membranes to enter the body cavity and vessels, and then, after being transported by the blood, infiltration of target organs. Finally, the growth of a new tumor at the target site depends on angiogenesis. In one embodiment, the term "metastasis" according to the invention relates to "distant metastasis" which relates to a metastasis which is remote from the primary tumor and the regional lymph node system. In one embodiment, the term “metastasis” according to the invention relates to lymph node metastasis.

[0412] The term "relapse" or "recurrence" as used herein refers to the condition that occurs when a person is affected again by a condition that affected him in the past. For example, if a patient has suffered from a tumor disease, has received a successful treatment of said disease and again develops said disease said newly developed disease may be considered as relapse or recurrence. A relapse or recurrence of a tumor also includes situations wherein a tumor occurs at a site different to the site of the original tumor as well as at the site of the original tumor. The original tumor for which the patient has received a treatment can be a primary tumor and the tumor at a site different to the site of the original tumor is a secondary or metastatic tumor.

[0413] Definitions given and embodiments described with respect to the first, second, third, fourth fifth, sixth, seventh, and eighth aspect apply also to the ninth aspect, in as far as they are applicable. Also, definitions and embodiments described below, in particular under the header 'Definitions' and 'Further embodiments of the invention' apply to the items for use of the ninth aspect.

[0414] In one embodiment of the tenth aspect, the invention provides a CAR for use in the treatment of a cancer in combination with a chimeric coreceptor subunit polypeptide according to the first aspect.

[0415] In one embodiment of the tenth aspect, the invention provides a CAR for use in the treatment of a cancer in combination with a chimeric coreceptor homodimer or a chimeric coreceptor complex according to the second aspect. In one embodiment of the tenth aspect, the invention provides a nucleic acid comprising a nucleic acid encoding a CAR, for use in the treatment of a cancer in combination with a nucleic acid construct (N-CCR) encoding the chimeric coreceptor subunit polypeptide according to the first aspect.

[0416] In one embodiment of the tenth aspect, the invention provides a vector comprising a nucleic acid encoding a CAR, for use in the treatment of a cancer in combination with a vector comprising a N-CCR encoding the chimeric coreceptor subunit polypeptide according to the first aspect.

[0417] In one embodiment of the tenth aspect, the invention provides a cell comprising a nucleic acid comprising a nucleic acid encoding a CAR or a vector comprising said nucleic acid, for use in the treatment of a cancer in combination with a nucleic acid construct (N-CCR) encoding the chimeric coreceptor subunit polypeptide according to the first aspect, or a vector comprising said N-CCR.

[0418] A further embodiment of the present invention refers to: the chimeric coreceptor subunit polypeptide according to the first aspect, the combination according to the third aspect, the nucleic acid construct (N-CCR-CAR), the two nucleic acid constructs (N-CCR, N- CAR), the nucleic acid construct (N-CCR-tgTCR) or the two nucleic acid constructs (N-CCR, N-tgTCR) according to the fourth aspect, or the vectors or the kit of the first and second vector as defined in the fifth aspect, the recombinant cell according to the sixth aspect, or a pharmaceutical composition according to the seventh aspect, for use in treating a cancer, wherein the CAR, the TCR or the tgTCR specifically bind an antigen 2 (AG2) of a target cell in the subject, and wherein the target cell of the subject comprises:

[0419] (A) reduced expression of AG2 and / or

[0420] (B) (i) reduced or abrogated expression or (ii) expression of a partial or full loss of function mutant of CD58, and / or

[0421] (C) expression of PD-L1. CD58 determination

[0422] In some embodiments, the expression of AG2, of CD58, of a partial or full loss of function mutant form of CD58 (e.g. a mutant that no longer effectively ligates CD2), and / or of PD-L1 by the target cells (e.g., tumor cells) is determined prior to using the present invention for treating a disease in a subject. The determination can be provided by an external laboratory.

[0423] The pretreatment expression of CD58 can be determined as CD58 H-score according to the method of Romain, et al. 2022 (Romain, G, et al. 2022. J Clin Invest. 132(17):el59402). In this method, CD58 expression is measured in tissue sections by immunohistochemistry using an antibody against CD58. The tissue sections are then analysed by standard microscopy by one or more pathologists (blinded to response data), and the percentage (10% increments) and intensity (mild, moderate, and intense) of tumor cells staining is evaluated. The CD58 expression is then determined as H score = [(% mild intensity x 1) + (% moderate intensity x 2) + (% intense intensity x 3)]). The cut-off level of the CD58 H score for efficacy of treatment of relapsed or refractory large B cell lymphomas with CD 19 CAR T cells was determined to be 80.

[0424] Accordingly, in a preferred embodiment of the present invention, reduced expression of CD58 (B)(i) means that the CD58 score is 80 or less than 80, as measured by immunohistochemistry.

[0425] The term "abrogated expression of CD58" means in the context of the invention that the expression is reduced to a level so low that CD58 cannot be detected in a sample of a target cell from the subject by using standard methods.

[0426] Moreover, the expression level of functional CD58 can be determined by standard methods, such as flow cytometry or mass cytometry, employing antibodies specific for a functional epitope of CD58 (Dengler, TJ, et al., 1992. Eur J Immunol 22(11):2809-17). In this case, reduced expression of CD58 means that the target cell expresses less than 20000, 15000, 10000, 5000, 4000, 3500, 3000, 2500, 2000, 1500, 1000, 500, 400, 300, 200, 100, 50, or 40 molecules per cell of CD58, or that less than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% of target cells express CD58.

[0427] The expression of a partial or full loss of function mutant of CD58 having reduced or abolished ability to activate CD2 on T cells in comparison to the wild-type form can be determined using a T cell activation assay, for example by measuring the amount of IL-2 and / or IFNy produced by a reference CAR-T cell in response to contact with a target cell that expresses the AG2 and CD58: these results can be calibrated against model target cells such as Nalm6 cells that express CD58 at different known levels (e.g., as determined by flow cytometry). This approach is useful for determining if the target cell expresses a partial or full loss of function mutant of CD58 that has impaired CD2- binding ability, as an equivalent to the expression level of fully functional CD58. Indeed, it has been shown that expression of a partial or full loss of function CD58 mutant can significantly reduce IFNy and IL-2 production in CAR T treatment, resulting in reduced efficacy of CAR T cell.

[0428] PD-L1 determination

[0429] PD-L1 (Programmed Death-Ligand 1) is a protein expressed on the surface of cells that play a crucial role in suppressing the immune system in many disease conditions including cancer. In immunotherapy sensitive cancers progression -free survival and overall survival have been shown to correlate with the expression level of PD-L1 on malignant cells, i.e. PD-L1 can be used as prognostic biomarker. As a consequence, the expression levels of PD-L1 in different cancer types may define eligibility for immunotherapeutic interventions.

[0430] The methodology for determining PD-L1 expression in cancer involves immunohistochemistry (IHC) with readouts being defined as TPS (Tumor Proportion Score) and CPS (Combined Positive Score) as describe by Fundytus, A, et al., 2021. Annals of Oncology 32(7): -833-835. TPS and CPS are evaluated by using clinically approved antibodies specifically binding to PD-L1 and by assessment of positively stained cells in tumor specimens by a pathologist.

[0431] TPS is calculated based on the percentage of viable tumor cells showing partial or complete membrane staining for PD-L1. TPS is calculated as:

[0432] (number of PD-L1 positive tumor cells / total number of viable tumor cells) xlOO.

[0433] TPS focuses exclusively on the tumor cells, and a specimen is typically considered to have PD-L1 expression if the TPS is at least 1%, i.e. if the TPS is 1% or more than 1%.

[0434] CPS is calculated by including both tumor cells and immune cells (lymphocytes and macrophages) that show PD-L1 staining. CPS is calculated as:

[0435] (Number of PD-L1 positive cells / total number of viable tumor cells) xlOO wherein the PD-L1 positive cells can be tumor cells, lymphocytes, macrophages.

[0436] A specimen is typically considered to have PD-L1 expression if CPS is at least 1 (i.e. the CPS is 1 or more than 1). CPS can sometimes exceed 100, but a maximum score is set at 100. The most commonly used antibodies in the assessment of PD-L1 expression through IHC for diagnostic and prognostic purposes in various cancers are described in the Table 4 below.

[0437] Table 4: anti PD-L1 antibodies

[0438] Definitions given and embodiments described with respect to the first, second, third, fourth fifth, sixth, seventh, eighth, and ninth aspect apply also to the tenth aspect, in as far as they are applicable. Also, definitions and embodiments described below, in particular under the header 'Definitions' and 'Further embodiments of the invention' apply to the combination and items for use of the tenth aspect.

[0439] Methods

[0440] The present invention is also directed to the use of the chimeric coreceptor, the combination, its peptide chains, the nucleic acids, the nucleic acid constructs, the vectors, the kit of vectors, the recombinant cell and / or the pharmaceutical composition as described herein in therapeutic methods. One of such uses comprises the production of antigen-specific immune cells coexpressing with the chimeric coreceptor of the invention, which can be administered to a patient for preventing a cancer that is characterized by expression of a tumor-associated antigen that can be bound by the CAR or TCR or tgTCR expressed by the antigen- specific immune cells. Preferably, the cancer is a B cell malignancy. In an eleventh aspect, the invention provides a method of treating cancer in a subject in need thereof, comprising the step of administering to the subject an effective amount of:

[0441] (i) the chimeric coreceptor subunit polypeptide according to the first aspect,

[0442] (ii) the combination according to the third aspect,

[0443] (iii) the nucleic acid construct (N-CCR), the nucleic acid construct (N-CCR-CAR), the two nucleic acid constructs (N-CCR, N-CAR), the nucleic acid construct (N-CCR- tgTCR) or the two nucleic acid constructs (N-CCR, N-tgTCR) according to the fourth aspect, or the vector or the kit of the first and second vector as defined in the fifth aspect, or

[0444] (iv) the recombinant cell according to the sixth aspect,

[0445] (v) a pharmaceutical composition according to the seventh aspect, and wherein the cancer is preferably characterized by a cell-surface expression of at least one tumor- associated antigen selected from the group consisting of ALK, B7-H3 (CD276), BCMA, c-MET, CD3, CD5, CD19, CD20, CD22, CD23, CD30, CD33, CD38, CD44, CD44v6, CD52, CD70, CD79A, CD79B, CD123, CD138, CD171, CEA, Claudin-6, Claudin-18.2, CLL1, CXCR5, EGFR, EGFRvIII, EPH-receptor A2, IGLV3-21, IL-1RAP, GPC-2, GPC-3, HER2, ErbB3, ErbB4, FAP, FBP, AchR, Fr-a, GD2, GD3, HMW-MAA, IE-13Ra2, Kappa-EC, IGEV3-21-R110, Eewis Y, EMP1, EMP2A, Mesothelin, MAGE-4A, MUC1, MUC16, NKG2D Eigands, NCAM, NY-ES01, Oncofetal antigen h5T4, PRAME, PSCA, PSMA, R0R1, TAG-72, VEGFR, G0LPH2, and SLAMF7, and wherein the cancer is preferably a B cell malignancy. Preferably, the AG1 and / or AG2 comprise or consist of the at least one tumor-associated antigen expressed on the surface of the cancer cell. Preferably AG1 and / or AG2 is CD 19 or CD22, more preferably AG1 is CD 19 and AG2 is CD22.

[0446] The term "treatment" or "therapeutic treatment" relates to any treatment which improves the health status and / or prolongs (increases) the lifespan of an individual. Said treatment may eliminate the disease in an individual, arrest, inhibit or slow the development of a disease in an individual, decrease the frequency or severity of symptoms in an individual, and / or decrease the recurrence in an individual who currently has or who previously has had a disease.

[0447] The terms "prophylactic treatment" or "preventive treatment" relate to any treatment that are intended to prevent a disease from occurring in an individual. The terms "prophylactic treatment" and "preventive treatment" are used herein interchangeably. The terms "individual" and "subject" are used herein interchangeably. They refer to human beings, non-human primates or other mammals (e.g. mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate) that can be afflicted with or are susceptible to a disease or disorder (e.g., cancer) but may or may not have the disease or disorder. In many embodiments, the individual is a human being. Unless otherwise stated, the terms "individual" and "subject" do not denote a particular age, and thus encompass adults, elderlies, children, and newborns. In preferred embodiments of the present invention, the "individual" or "subject" is a "patient". The term "patient" means a subject for treatment, in particular a diseased subject.

[0448] By "being at risk" or "in need of" is meant to refer to a subject, i.e. a patient, that is identified as having a higher than normal chance of developing a disease, in particular cancer, compared to the general population. In addition, a subject who has had, or who currently has, a disease, in particular cancer, is a subject who has an increased risk for developing a disease, as such a subject may continue to develop a disease. Subjects who currently have, or who have had, a cancer also have an increased risk for cancer metastases.

[0449] In the context of the present invention, terms such as "protect", "prevent", "prophylactic", "preventive", or "protective" relate to the prevention or treatment or both of the occurrence and / or the propagation of a disease in a subject and, in particular, to minimizing the chance that a subject will develop a disease or to delaying the development of a disease. For example, a person at risk for a tumor, as described above, would be a candidate for therapy to prevent a tumor.

[0450] A therapeutic administration of an immunotherapy, for example, a therapeutic administration of a chimeric coreceptor or combination of the invention, may lead to the inhibition of the progress / growth of the cancer. This comprises the deceleration of the progress / growth of the cancer, in particular a disruption of the progression of the cancer, which preferably leads to elimination of the cancer.

[0451] Immunotherapy may be performed using any of a variety of techniques, in which chimeric coreceptors provided herein preferably enhance the function of a cell to remove the diseased target cells from a patient. Such removal may take place as a result of enhancing or inducing an immune response in a patient specific for antigen or a cell expressing antigen.

[0452] In one embodiment, a method of treating cancer in a subject in need thereof comprises administering to the subject an effective amount of the chimeric coreceptor subunit polypeptide according to the first aspect. In particular, the ARD1 of the chimeric coreceptor subunit polypeptide is able to bind to an antigen that is associated with the cancer to be treated. According to a preferred embodiment, the cancer is characterized by a cell-surface expression of at least one tumor- associated antigen, and is preferably a B cell malignancy.

[0453] In one embodiment, a method of treating cancer in a subject in need thereof comprises administering to the subject an effective amount of the combination according to the third aspect. In particular, the AG1 bound by the ARD1 of the chimeric coreceptor subunit polypeptide and the AG2 bound by the ARD2 of the CAR or TCR comprise or consist of an antigen that is associated with the cancer to be treated. According to a preferred embodiment, the cancer is characterized by a cell-surface expression of at least one tumor-associated antigen, and preferably is a B cell malignancy. Therefore, the AG1 and / or AG2 preferably comprise or consist of the at least one tumor-associated antigen expressed on the surface of the cancer cell. Preferably AG1 and / or AG2 is CD 19 or CD22, more preferably AG1 is CD 19 and AG2 is CD22.

[0454] In one embodiment, a method of treating cancer in a subject in need thereof comprises administering to the subject an effective amount of the nucleic acid construct (N-CCR), the nucleic acid construct (N-CCR-CAR), the two nucleic acid constructs (N-CCR, N-CAR), the nucleic acid construct (N-CCR-tgTCR) or the two nucleic acid constructs (N-CCR, N-tgTCR) according to the fourth aspect, or the vector or the kit of the first and second vector as defined in the fifth aspect. In particular, the AG1 bound by the ARD1 of the chimeric coreceptor subunit polypeptide and the AG2 bound by the ARD2 of the CAR or TCR comprise or consist of an antigen that is associated with the cancer to be treated. According to a preferred embodiment, the cancer is characterized by a cell-surface expression of at least one tumor-associated antigen, and preferably is a B cell malignancy. Therefore, the AG1 and / or AG2 preferably comprise or consist of the at least one tumor-associated antigen expressed on the surface of the cancer cell. Preferably AG1 and / or AG2 is CD 19 or CD22, more preferably AG1 is CD 19 and AG2 is CD22.

[0455] In one embodiment, a method of treating cancer in a subject in need thereof comprises administering to the subject an effective amount of the recombinant cell according to the sixth aspect. In particular, the AG1 bound by the ARD1 of the chimeric coreceptor subunit polypeptide and the AG2 bound by the ARD2 of the CAR or TCR comprise or consist of an antigen that is associated with the cancer to be treated. According to a preferred embodiment, the cancer is characterized by a cell-surface expression of at least one tumor-associated antigen, and preferably is a B cell malignancy. Therefore, the AG1 and / or AG2 preferably comprise or consist of the at least one tumor-associated antigen expressed on the surface of the cancer cell. Preferably AG1 and / or AG2 is CD 19 or CD22, more preferably AG1 is CD 19 and AG2 is CD22.

[0456] In one embodiment, a method of treating cancer in a subject in need thereof comprises administering to the subject an effective amount of the recombinant cell according to the sixth aspect. In particular, the AG1 bound by the ARD1 of the pharmaceutical composition according to the seventh aspect and the AG2 bound by the ARD2 of the CAR or TCR comprise or consist of an antigen that is associated with the cancer to be treated. According to a preferred embodiment, the cancer is characterized by a cell-surface expression of at least one tumor- associated antigen, and preferably is a B cell malignancy. Therefore, the AG1 and / or AG2 preferably comprise or consist of the at least one tumor-associated antigen expressed on the surface of the cancer cell. Preferably AG1 and / or AG2 is CD 19 or CD22, more preferably AG1 is CD 19 and AG2 is CD22.

[0457] According to a preferred embodiment, the cancer is characterized by a cell-surface expression of at least one tumor-associated antigen, and preferably of at least one tumor-associated antigen having a density lower than about 1000 molecules / cell, about 400 molecules / cell, about 300 molecules / cell, about 200 molecules / cell, about 100 molecules / cell, or about 40 molecules / cell, and preferably lower than about 400 molecules / cell, and more preferably lower than about 40 molecules / cell.

[0458] The at least one tumor-associated antigen can be selected from the group consisting of B7- H3 (CD276), BCMA, CD3, CD5, CD19, CD20, CD22, CD23, CD30, CD33, CD38, CD44, CD44v6, CD52, CD70, CD79A, CD79B, CD123, CD138, CD171, CEA, Claudin-6, Claudin-18.2, CLL1, CXCR5, EGFR, EGFRvIII, EPH-receptor A2, IGLV3-21, GP-2, GP-40, HER2, ErbB3, ErbB4, FBP, AchR, Fr-a, GD2, GD3, HMW-MAA, IL13Ra2, Kappa-LC, IGLV3-21-R110, Lewis Y, Mesothelin, MUC1, MUC16, NKG2D Ligands, NCAM, NY-ESO1, Oncofetal antigen h5T4, PSCA, PSMA, ROR1, TAG-72, VEGFR, GOLPH2, and SLAMF7. A preferred tumor-associated antigen is selected from the group consisting of BCMA, CD22, CD79B, GOLPH2, EGFR / EGFRvIII, HER2, CEA, and GOLPH2. A more preferred tumor-associated antigen is selected from the group consisting of CD22, CD 19, HER2, and EGFR.

[0459] According to a preferred embodiment, the cancer is characterized by a cell-surface expression of CD22, and wherein CD22 preferably has a density lower than about 1000 molecules / cell, about 400 molecules / cell, about 300 molecules / cell, about 200 molecules / cell, about 100 molecules / cell, or about 40 molecules / cell, and preferably lower than about 400 molecules / cell, and more preferably lower than about 40 molecules / cell. In one embodiment of the method of the eleventh aspect, _the invention provides a method of treating a disease in a subject in need thereof, comprising the step of administering to the subject an effective amount of: the combination according to the third aspect, the nucleic acid construct (N-CCR-CAR), the two nucleic acid constructs (N-CCR, N- CAR), the nucleic acid construct (N-CCR-tgTCR) or the two nucleic acid constructs (N-CCR, N-tgTCR) according to the fourth aspect, or the vectors or the kit of the first and second vector as defined in the fifth aspect, the recombinant cell according to the sixth aspect, or a pharmaceutical composition according to the seventh aspect, wherein the CAR, the TCR or the tgTCR specifically bind an antigen 2 (AG2) of a target cell in the subject, and wherein the target cell of the subject comprises:

[0460] (A) reduced expression of AG2 and / or

[0461] (B) (i) reduced or abrogated expression or (ii) expression of a partial or full loss of function mutant of CD58, and / or

[0462] (C) expression of PD-L1.

[0463] In preferred embodiments, reduced expression of the AG2 (A) means that the target cell expresses less than 5000, 4000, 3500, 3000, 2500, 2000, 1500, 1000, 500, 400, 300, 200, 100, 50, or 40 molecules per cell of the AG2, and preferably less than 400 molecules per cell of the AG2, more preferably less than 40 molecules per cell of the AG2, as measured by flow cytometry using appropriate quantification beads.

[0464] In a further aspect, the invention provides a method of treating an autoimmune disease in a subject in need thereof, comprising the step of administering to the subject an effective amount of:

[0465] (i) the chimeric coreceptor subunit polypeptide according to the first aspect,

[0466] (ii) the combination according to the third aspect,

[0467] (iii) the nucleic acid construct (N-CCR), the nucleic acid construct (N-CCR-CAR), the two nucleic acid constructs (N-CCR, N-CAR), the nucleic acid construct (N-CCR- tgTCR) or the two nucleic acid constructs (N-CCR, N-tgTCR) according to the fourth aspect, or the vector or the kit of the first and second vector as defined in the fifth aspect, or

[0468] (iv) the recombinant cell according to the sixth aspect,

[0469] (v) a pharmaceutical composition according to the seventh aspect, and wherein the autoimmune disease is characterized by autoreactive B cells preferably expressing CD22, and optionally wherein the AG1 and / or AG2 is CD22.

[0470] Definitions given and embodiments described with respect to the first, second, third, fourth fifth, sixth, seventh, eighth, ninth and tenth aspect apply also to the eleventh aspect, in as far as they are applicable. Also, definitions and embodiments described below, in particular under the header 'Definitions' and 'Further embodiments of the invention' apply to the method of the eleventh aspect.

[0471] 'Further embodiments of the invention'

[0472] Adoptive cell therapies using genetically modified TCR-transgenic or CAR-T cells have shown efficacy against melanoma and B cell malignancies, respectively. However, their broad applicability, especially to solid tumors, is limited by the fact that there are only few truly tumourspecific antigens, i.e. not expressed by healthy cells, and to very low on-tumour expression of the target antigens. Therefore, CAR-based therapies are oft associated with ‘on-target, off tumor’ toxicity causing the concomitant targeting of healthy tissue. In some scenarios, unintentional targeting of healthy tissue can mediate unacceptable, or even life threatening, toxicity.

[0473] The inventors have here shown that these problems may be overcome by using a CCR as disclosed herein, in particular the CD28IgV CCR, and a CAR, wherein the CCR and the CAR must recognise two different antigens (i.e. AG1 and AG2) for full activation and target cell elimination (Example 10, Figures 30-33). Such a Boolean AND-gate targeting strategy requires minimal or absent T cell activation in the presence of either AG1 or AG2 alone, which was exemplarily shown for anti-CD22 CAR and anti-CD19 CD28IgV CCR-T cells in the present invention. The CD28IgV CCR might be particularly suitable for an AND-gate targeting strategy as it exhibits enhanced expression properties on T cells compared to CCRs known in the art lacking an IgV-like domain. This leads to advanced sequestration of proximal signaling proteins from the immunological synapse, making them unavailable for the initiation of TCR and / or CAR signaling when the CCR is not co-engaged with the CAR by binding of AG1 and AG2. In particular, the defined antigen combination of AG1 and AG2 is expressed only by tumors and not by healthy cells. Therefore, T cells expressing such a CCR and CAR combination allow selective tumor targeting whilst preserving healthy cells that express either of the targeted antigens in isolation.

[0474] In another embodiment, recognition of AG2 by the ARD of the CAR may still can trigger some T cell activation in the absence of CCR engagement by AG1. Such scenario eventually occurs e.g. when AG2 is expressed at very high densities or when the ARD2 has a very high affinity for AG2. In this situation, the selection of AG2 should be based on low expression levels on healthy tissues, thereby limiting CAR-T cell responses to AG2 expressing cells, whereas co-expression of AG1 on a given target cell (e.g. a tumor cell) helps to unleash full T cell activation and efficient immune responses following recognition of AG2. Such an IF-BETTER-gate is present when a CAR-engaging AG2 functions better in the presence of AG1, whereas AG1 is not mandatory for CAR-T cell function, as it has been shown for chimeric CD8IgV and chimeric CD28IgV polypeptides. An IF-BETTER-gate differs from the obligate dual-requirement of AG1 and AG2 in an AND-gate, but it is still capable of generating CAR-T cells that can modulate sensitivity to AG2 in the presence of AG1 and thus mediate T cell preference for target cells expressing AG1 (e.g. tumor cells), albeit with less stringency than an AND-gate.

[0475] 1. A combination comprising a chimeric coreceptor (CCR) and a chimeric antigen receptor (CAR), wherein the chimeric coreceptor comprises:

[0476] (a) an antigen-recognition domain (ARD1) specifically binding an antigen 1 (AG1),

[0477] (b) an IgVD;

[0478] (c) an HD 1; and / or

[0479] (d) a TMDl; wherein the chimeric coreceptor does not comprise a cell activation domain, wherein the CAR comprises: an ARD2 specifically binding an AG2, an HD2, and / or a TMD2, and wherein AG2 and AG1 are two substantially different tumor-associated antigens, and wherein AG2 and AG1 are not co-expressed on a healthy cell. The combination of a CCR and a CAR of Item 1, wherein HD1 and HD2 are substantially identical and / or TMD1 and TMD2 are substantially identical. The combination of a CCR and a CAR of Item 1, wherein HD1 and HD2 are substantially different and / or TMD1 and TMD2 are substantially different. The combination of a CCR and a CAR of any one of Items 1 to 3, wherein the chimeric coreceptor comprises:

[0480] (a) an ARD1 comprising or consisting of:

[0481] (i) a mono-, bi- or multispecific scFv, a single domain antibody, or a single chain antibody-like protein scaffold, in particular an anticalin, DARPins, affibody, affimer, avimer, neutrophil gelatinase-associated lipocalin (NGAL) or evasin;

[0482] (ii) a ligand selected from the group comprising a cytokine, in particular IL-1, IL- 10, IL- 13, APRIL, GM-CSF, FLT3L; a growth factor, in particular TPO; an immunoglobulin superfamily protein, in particular adnectin; a tumor associated antigen (TA A) targeting chimeric peptides, in particular TIE; or any combination thereof;

[0483] (b) an IgVD comprising or consisting of a CD8a IgVD, a CD8P IgVD, CD28 IgVD or variant thereof,

[0484] (c) an HD1 comprising or consisting of a CD8a HD, a CD8P HD, or a CD28 HD, or dimerizing variant thereof,

[0485] (d) a TMD1 comprising or consisting of a CD4, CD8a, CD8P, or CD28 TMD, or variant thereof, and / or

[0486] (e) a CSD comprising or consisting of a CD4, CD8a, CD8P, CD28, CD2, CD27, CD40, CD278, 0X40, GITR, CD40L, CD30, CD44, or CD 146 intracellular signaling domain (ICD), or a variant thereof. The combination of a CCR and a CAR of any one of Items 1 to 4, wherein the CAR further comprises: optionally a 4- IBB intracellular signaling domain and / or a CD28 intracellular signaling domain, and a CD3zeta domain. The combination of a CCR and a CAR of any one of Items 1 to 4, wherein the CAR does not comprise a 4- IBB intracellular signaling domain and / or a CD28 intracellular signaling domain. The combination of a CCR and a CAR of any one of Items 1-6, wherein:

[0487] (b) the IgVD comprises or consists of a CD8a IgVD,

[0488] (c) the HD1 comprises or consists of a CD8a HD,

[0489] (d) the TMD1 comprises or consists of a CD8a TMD, and

[0490] (e)the CSD comprises or consists of a CD8a or CD4 ICD; or wherein:

[0491] (b) the IgVD comprises or consists of a CD28 IgVD,

[0492] (c) the HD1 comprises or consists of a CD28 HD,

[0493] (d) the TMD1 comprises or consists of a CD28 TMD, and

[0494] (e) the CSD comprises or consists of a CD28 ICD. The combination of a CCR and a CAR of any one of Items 1-7, wherein the AG2 and / or AG1 are individually selected from the group of tumor-associated antigens consisting of ALK, B7-H3 (CD276), BCMA, c-MET, CD3, CD5, CD19, CD20, CD22, CD23, CD30, CD33, CD38, CD44, CD44v6, CD52, CD70, CD79A, CD79B, CD123, CD138, CD171, CEA, Claudin-6, Claudin-18.2, CLL1, CXCR5, EGFR, EGFRvIII, EPH-receptor A2, IGLV3-21, IL-1RAP, GPC-2, GPC-3, HER2, ErbB3, ErbB4, FAP, FBP, AchR, Fr-a, GD2, GD3, HMW- MAA, IE-13Ra2, Kappa-EC, IGEV3-21-R110, Eewis Y, EMP1, EMP2A, Mesothelin, MAGE-4A, MUC1, MUC16, NKG2D Eigands, NCAM, NY-ES01, Oncofetal antigen h5T4, PRAME, PSCA, PSMA, R0R1, TAG-72, VEGFR, G0LPH2, and SLAMF7, and preferably CD 19 or CD22. The combination of any one of Items 1-8, wherein the chimeric coreceptor is a chimeric coreceptor homodimer or wherein the chimeric coreceptor is a chimeric coreceptor complex comprising a chimeric coreceptor subunit polypeptide and a CD8a, CD8P or CD28 subunit. A nucleic acid composition comprising a first nucleic acid encoding a CCR subunit polypeptide (N-CCR) and a second nucleic acid encoding a CAR (N-CAR), wherein the chimeric coreceptor subunit polypeptide comprises:

[0495] (a) an ARD1 specifically binding an AG1,

[0496] (b) an IgVD;

[0497] (c) an HDl;

[0498] (d) a TMD1; and / or

[0499] (e) a CSD; wherein the chimeric coreceptor does not comprise a cell activation domain, wherein the CAR comprises: an ARD2 specifically binding an AG2, a hinge domain 2 (HD2), a transmembrane domain 2 (TMD2), optionally a 4- IBB intracellular signaling domain and / or a CD28 intracellular signaling domain, and / or a CD3zeta domain, and wherein AG2 and AG1 are two substantially different tumor-associated antigens, and wherein AG2 and AG1 are not co-expressed on a healthy cell. The nucleic acid composition according to Item 10, wherein HD1 and HD2 are substantially identical and / or TMD1 and TMD2 are substantially identical. The nucleic acid composition according to Item 10, wherein HD1 and HD2 are substantially different and / or TMD1 and TMD2 are substantially different. A recombinant cell expressing the combination of a CCR and a CAR of any one of Items 1- 9. A recombinant cell comprising the nucleic acid composition of any one of items 10-12. A recombinant cell comprising the nucleic acid composition of any one of items 10-12 and optionally expressing the combination of a CCR and a CAR of any one of Items 1-9. A pharmaceutical composition comprising:

[0500] (i) the combination of a CCR and a CAR of any one of Items 1-9,

[0501] (ii) the nucleic acid composition of any one of Items 10-12, or

[0502] (iii) the recombinant cell according to any one of Items 13-15, and a pharmaceutically acceptable carrier, stabilizer and / or excipient. A combination of a CCR and a CAR of any one of Items 1-9, a nucleic acid composition of any one of Items 10-12, a recombinant cell according to any one of Items 13-15, or a pharmaceutical composition according to Item 16, for use in medicine. A combination of a CCR and a CAR of any one of Items 1-9, a nucleic acid composition of any one of Items 10-12, a recombinant cell according to any one of Items 13-15, or a pharmaceutical composition according to Item 16, for use in treating cancer in a subject, and wherein the cancer is characterized by a cell-surface expression of AG2 and AG1. A combination of a CCR and a CAR of any one of Items 1-9, a nucleic acid composition of any one of Items 10-12, a recombinant cell according to any one of Items 13-15, or a pharmaceutical composition according to Item 16, for use in treating cancer in a subject, and wherein the cancer is characterized by a low cell-surface expression of AG2 and by a cellsurface expression of AG1, and wherein AG2 and AG1 are not co-expressed on a healthy cell of the subject, and optionally wherein AG2 is not expressed on a healthy cell of the subject, or wherein AG2 has low cell-surface expression on a healthy cell of the subject. A combination of a CCR and a CAR of any one of Items 1-9, a nucleic acid composition of any one of Items 10-12, a recombinant cell according to any one of Items 13-15, or a pharmaceutical composition according to Item 16, for use in treating cancer in a subject, and wherein the cancer is characterized by a cell-surface expression of AG2 and AG1, and wherein AG2 is not expressed on a healthy cell of the subject. A combination of a CCR and a CAR of any one of Items 1-9, a nucleic acid composition of any one of Items 10-12, a recombinant cell according to any one of Items 13-15, or a pharmaceutical composition according to Item 16, for use in treating cancer in a subject, and wherein AG2 and AG1 are not co-expressed on a healthy cell of the subject, and wherein the cancer is characterized by a cell-surface expression of AG2 and AG1, and wherein AG2 has low cell-surface expression on a healthy cell of the subject. The combination of a CCR and a CAR, the nucleic acid composition, the recombinant cell, or the pharmaceutical composition for use according to Items 18 or 19, wherein the cancer is characterized by a low cell-surface expression of AG2 and / or AG1. A method of treating cancer in a subject in need thereof, comprising the step of administering to the subject an effective amount of:

[0503] (i) the combination of a CCR and a CAR of any one of Items 1-9,

[0504] (ii) the nucleic acid composition of any one of Item 10-12, or

[0505] (iii) the recombinant cell according to any one of Item 13-15,

[0506] (iv) a pharmaceutical composition according to Item 16, and wherein the cancer is characterized by a cell-surface expression of AG2 and AG1 and wherein AG2 and AG1 are not co-expressed on a healthy cell of the subject. A method of treating cancer in a subject in need thereof, comprising the step of administering to the subject an effective amount of:

[0507] (i) the combination of a CCR and a CAR of any one of Items 1-9,

[0508] (ii) the nucleic acid composition of any one of Item 10-12, or

[0509] (iii) the recombinant cell according to any one of Item 13-15,

[0510] (iv) a pharmaceutical composition according to Item 16, and wherein the cancer is characterized by a low cell- surface expression of-AG2 and by cellsurface expression of AG1, and wherein AG2 and AG1 are not co-expressed on a healthy cell of the subject, and optionally: wherein AG2 is not expressed on a healthy cell of the subject, or wherein AG2 has low cell-surface expression on a healthy cell of the subject. A method of treating cancer in a subject in need thereof, comprising the step of administering to the subject an effective amount of:

[0511] (i) the combination of a CCR and a CAR of any one of Items 1-9,

[0512] (ii) the nucleic acid composition of any one of Item 10-12, or

[0513] (iii) the recombinant cell according to any one of Item 13-15,

[0514] (iv) a pharmaceutical composition according to Item 16, and wherein the cancer is characterized by a cell-surface expression of AG1 and AG2, and wherein AG2 and AG1 are not co-expressed on a healthy cell of the subject, and wherein AG2 is not expressed on a healthy cell of the subject. A method of treating cancer in a subject in need thereof, comprising the step of administering to the subject an effective amount of:

[0515] (i) the combination of a CCR and a CAR of any one of Items 1-9,

[0516] (ii) the nucleic acid composition of any one of Item 10-12, or

[0517] (iii) the recombinant cell according to any one of Item 13-15,

[0518] (iv) a pharmaceutical composition according to Item 16, and wherein the cancer is characterized by a cell-surface expression of AG1 and AG2, and wherein AG2 and AG1 are not co-expressed on a healthy cell of the subject, and wherein AG2 has low cell-surface expression on a healthy cell of the subject. A method of treating cancer in a subject in need thereof, comprising the step of administering to the subject an effective amount of:

[0519] (i) the combination of a CCR and a CAR of any one of Items 1-9,

[0520] (ii) the nucleic acid composition of any one of Item 10-12, or

[0521] (iii) the recombinant cell according to any one of Item 13-15,

[0522] (iv) a pharmaceutical composition according to Item 16, and wherein the cancer is characterized by a low cell-surface expression of-AG2 and / or AG1, and wherein AG2 and AG1 are not co-expressed on a healthy cell of the subject, and optionally: wherein AG2 is not expressed on a healthy cell of the subject, or wherein AG2 has low cell-surface expression on a healthy cell of the subject. 28. The combination of a CCR and a CAR, the nucleic acid composition, the recombinant cell, or the pharmaceutical composition for use according to Items 18 or 22, or the method according to any one of Items 23-27, wherein the tumor-associated antigen is selected from the group consisting of ALK, B7-H3 (CD276), BCMA, c-MET, CD3, CD5, CD 19, CD20, CD22, CD23, CD30, CD33, CD38, CD44, CD44v6, CD52, CD70, CD79A, CD79B, CD 123, CD138, CD171, CEA, Claudin-6, Claudin-18.2, CLL1, CXCR5, EGFR, EGFRvIII, EPH- receptor A2, IGLV3-21, IL-1RAP, GPC-2, GPC-3, HER2, ErbB3, ErbB4, FAP, FBP, AchR, Fr-a, GD2, GD3, HMW-MAA, IE-13Ra2, Kappa-EC, IGEV3-21-R110, Eewis Y, EMP1, EMP2A, Mesothelin, MAGE-4A, MUC1, MUC16, NKG2D Eigands, NCAM, NY-ESO1, Oncofetal antigen h5T4, PRAME, PSCA, PSMA, R0R1, TAG-72, VEGFR, GOLPH2, and SLAMF7, and wherein the cancer is preferably a B cell malignancy.

[0523] The wording “AG2 and AG1 are not co-expressed on a healthy cell” means that the expression of both AG2 and AG1 in the same healthy cell (e.g. in a sample comprising only healthy cells) is not found, whereas the single expression of only AG2 or AG1 may be found or not on that heathy cell (e.g. in a sample comprising healthy cells). In other words, “AG2 and AG1 are not coexpressed on a healthy cell” means that the double expression of AG2 and AG1 in the same healthy cell (e.g. in a sample comprising only healthy cells) is not found, whereas the single expression of only AG2 or AG1 may be found or not on that heathy cell (e.g. in a sample comprising healthy cells).

[0524] The wording “HD1 and HD2 are substantially different” means in the context of the invention that the two amino acid sequences of HD1 and HD2 show less than 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 70%, 65%, or 60% sequence identity, such that the respective chimeric coreceptor and CAR cannot undergo heterotypic interactions.

[0525] The wording “TMD1 and TMD2 are substantially different” means in the context of the invention that the two amino acid sequences of TMD1 and TMD2 show less than 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 70%, 65%, or 60% sequence identity, such that the respective chimeric coreceptor and CAR cannot undergo heterotypic interactions. The term "HD1 and HD2 are substantially identical" means in the context of the invention that the two amino acid sequences of HD1 and HD2 do not need to be 100% identical, but can comprise amino acid substitutions and / or additions and / or deletions as defined herein, wherein HD1 or HD2 can be considered as reference sequence. It is preferred that a substantially identical HD1 and HD2 amino acid sequences show at least 60%, 65%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, such that a chimeric coreceptor subunit polypeptide and a CAR subunit polypeptide are able to dimerize.

[0526] The term "TMD1 and TMD2 are substantially identical" means in the context of the invention that the two amino acid sequences of TMD1 and TMD2 do not need to be 100% identical, but can comprise amino acid substitutions and / or additions and / or deletions as defined herein, wherein TMD1 or TMD2 can be considered as reference sequence. It is preferred that a substantially identical TMD1 and TMD2 amino acid sequences show at least 60%, 65%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, such that a chimeric coreceptor subunit polypeptide and a CAR subunit polypeptide are able to dimerize.

[0527] In the context of the invention, "cancer is characterized by a cell-surface expression of AG2 and AG1" means that a cancer cell of the subject expresses more than 5000, 4000, 3500, 3000, 2500, 2000, 1500, 1000, 500, 400, 300, 200, 100, 50, or 40 molecules per cell of the AG2 and AG1, respectively. In this context, "cancer is characterized by a cell-surface expression of AG2" means that a cancer cell of the subject expresses more than 5000, 4000, 3500, 3000, 2500, 2000, 1500, 1000, 500, 400, 300, 200, 100, 50, or 40 molecules per cell of the AG2. In this context, "cancer is characterized by a cell-surface expression of AG1" means that a cancer cell of the subject expresses more than 5000, 4000, 3500, 3000, 2500, 2000, 1500, 1000, 500, 400, 300, 200, 100, 50, or 40 molecules per cell of the AG1. Expression of a given AG can be measured by various methods known in the art including fluorescent staining of the cancer cell with fluorescent binders, e.g. antibodies specifically binding to AG and flow cytometric analysis.

[0528] In the context of the invention, "cancer is characterized by a low cell-surface expression of AG2 and by a cell-surface expression of AG1 " means that a cancer cell of the subject expresses less than 5000, 4000, 3500, 3000, 2500, 2000, 1500, 1000, 500, 400, 300, 200, 100, 50, or 40 molecules per cell of the AG2, and more than 5000, 4000, 3500, 3000, 2500, 2000, 1500, 1000, 500, 400, 300, 200, 100, 50, or 40 molecules per cell of the AG1. In one embodiment, "cancer is characterized by a low cell-surface expression of AG2 and by a cell-surface expression of AG1" means that a cancer cell of the subject expresses less than 5000, 4000, 3500, 3000, 2500, 2000, 1500, 1000, 500, 400, 300, 200, 100, 50, or 40 molecules per cell of the AG2, and more than 5000 molecules per cell of the AG1. In one embodiment, "cancer is characterized by a low cellsurface expression of AG2 and by a cell-surface expression of AG1" means that a cancer cell of the subject expresses less than 5000, 4000, 3500, 3000, 2500, 2000, 1500, 1000, 500, 400, 300, 200, 100, 50, or 40 molecules per cell of the AG2, an more than 4000 molecules per cell of the AG1. In one embodiment, "cancer is characterized by a low cell-surface expression of AG2 and by a cell-surface expression of AG1" means that a cancer cell of the subject expresses less than 5000, 4000, 3500, 3000, 2500, 2000, 1500, 1000, 500, 400, 300, 200, 100, 50, or 40 molecules per cell of the AG2, and more than 3500 molecules per cell of the AG1. In one embodiment, "cancer is characterized by a low cell-surface expression of AG2 and by a cell-surface expression of AG1" means that a cancer cell of the subject expresses less than 5000, 4000, 3500, 3000, 2500, 2000, 1500, 1000, 500, 400, 300, 200, 100, 50, or 40 molecules per cell of the AG2, and more than 3000 molecules per cell of the AG1. In one embodiment, "cancer is characterized by a low cell-surface expression of AG2 and by a cell-surface expression of AG1" means that a cancer cell of the subject expresses less than 5000, 4000, 3500, 3000, 2500, 2000, 1500, 1000, 500, 400, 300, 200, 100, 50, or 40 molecules per cell of the AG2, and more than 2500 molecules per cell of the AG1. In one embodiment, "cancer is characterized by a low cell-surface expression of AG2 and by a cell-surface expression of AG1" means that a cancer cell of the subject expresses less than 5000, 4000, 3500, 3000, 2500, 2000, 1500, 1000, 500, 400, 300, 200, 100, 50, or 40 molecules per cell of the AG2, and more than 2000 molecules per cell of the AG1. In one embodiment, "cancer is characterized by a low cell-surface expression of AG2 and by a cell-surface expression of AG1" means that a cancer cell of the subject expresses less than 5000, 4000, 3500, 3000, 2500, 2000, 1500, 1000, 500, 400, 300, 200, 100, 50, or 40 molecules per cell of the AG2, and more than 1500 molecules per cell of the AG1. In one embodiment, "cancer is characterized by a low cell-surface expression of AG2 and by a cell-surface expression of AG1" means that a cancer cell of the subject expresses less than 5000, 4000, 3500, 3000, 2500, 2000, 1500, 1000, 500, 400, 300, 200, 100, 50, or 40 molecules per cell of the AG2, and more than 1000 molecules per cell of the AG1. In one embodiment, "cancer is characterized by a low cellsurface expression of AG2 and by a cell-surface expression of AG1" means that a cancer cell of the subject expresses less than 5000, 4000, 3500, 3000, 2500, 2000, 1500, 1000, 500, 400, 300, 200, 100, 50, or 40 molecules per cell of the AG2, and more than 500 molecules per cell of the AG1.

[0529] In the context of the invention, " cancer is characterized by a low cell-surface expression of AG2" means that a cancer cell of the subject expresses less than 5000, 4000, 3500, 3000, 2500, 2000, 1500, 1000, 500, 400, 300, 200, 100, 50, or 40 molecules per cell of the AG2. Expression of a given AG2 can be measured by various methods known in the art including fluorescent staining of the cancer cell with fluorescent binders, e.g. antibodies specifically binding to AG2 and, flow cytometric analysis.

[0530] Alternatively, "low cell-surface expression of AG2" means in the context of the invention that a cancer cell of the subject expresses 20%, 30%, 40%, 50%, 60%, 70%, 80%, preferably 90% less of AG2 than a cell that is of the same cell type as the cancer cell but healthy.

[0531] Alternatively, "AG2 has low cell-surface expression on a healthy cell" means in the context of the invention that the healthy cell expresses 20%, 30%, 40%, 50%, 60%, 70%, 80%, preferably 90% less of AG2 than a healthy cell that is of the same subject but of a different type or than a healthy cell of the same type but of a different subject.

[0532] In the context of the invention, "low cell-surface expression of AG1" means that the cancer cell expresses less than 5000, 4000, 3500, 3000, 2500, 2000, 1500, 1000, 500, 400, 300, 200, 100, 50, or 40 molecules per cell of the AG1. Expression of a given AGlcan be measured by various methods known in the art including fluorescent staining of the cancer cell with fluorescent binders, e.g. antibodies specifically binding to AGland, flow cytometric analysis.

[0533] Alternatively, "low cell-surface expression of AG1" means in the context of the invention that the cancer cell expresses 20%, 30%, 40%, 50%, 60%, 70%, 80%, preferably 90% less of AG1 than a cell that is of the same cell type as the cancer cell but healthy.

[0534] The expression of AG1 or AG2 in a healthy comparator cell can either be determined in a healthy cell of the subject to be treated or in a cell of the same cell type of a healthy subject. In the latter case, typically the expression of AG1 or AG2 has been determined as the average expression in a group of heathy subjects. Once this average expression level has been determined, this value is used in the calculation of the relative reduction of the expression of AG1 or AG2 on the cancer cell.

[0535] Definitions

[0536] The specification uses a variety of terms and phrases, which have certain meanings as defined below. Preferred meanings are to be construed as preferred embodiments of the aspects of the invention described herein. As such, they and also further embodiments described in the following can be combined with any embodiment of the aspects of the invention and in particular any preferred embodiment of the aspects of the invention described above.

[0537] The term "about" means approximately or nearly, and in the context of a numerical value or range set forth herein in one embodiment means ± 20%, ± 10%, ± 5%, or ± 3% of the numerical value or range recited or claimed.

[0538] The term "plurality" with reference to an object refers to a population of a certain number of said object. In certain embodiments, the term refers to a population of more than 10, 102, 103, 104, 105, 106, 107, 108, 109, IO10, 1011, 1012, 1013, 1014, 1015, 1016, 1017, 1018, 1019, IO20, 1021, 1022, or 1023or more.

[0539] The term "homologous" is used herein to describe a nucleic acid, a gene, a transgene or a protein derived from the same subject.

[0540] The term "heterologous" is used herein to describe a nucleic acid, a gene, a transgene or a protein derived from a source other than the subject.

[0541] The term "expression" is used in the context the invention in its most general meaning and comprises the production of RNA and / or peptides or proteins, e.g. by transcription and / or translation. With respect to RNA, the term "expression" or "translation" relates in particular to the production of peptides or proteins. It also comprises partial expression of nucleic acids. Moreover, expression can be transient or stable.

[0542] The term "aberrant expression" or "abnormal expression" of a certain protein means according to the invention that the expression is altered, e.g. increased or decreased, compared to a reference, e.g. in a subject not having a disease associated with the expression of the protein, e.g., a tumor associated antigen.

[0543] The term "fragment" with reference to an amino acid sequence (peptide or protein), relates to a part of an amino acid sequence, i.e. a sequence which represents the amino acid sequence shortened at the N-terminus and / or C-terminus. A fragment shortened at the C-terminus (N- terminal fragment) is obtainable e.g. by translation of a truncated open reading frame that lacks the 3'-end of the open reading frame. A fragment shortened at the N-terminus (C-terminal fragment) is obtainable e.g. by translation of a truncated open reading frame that lacks the 5'-end of the open reading frame, as long as the truncated open reading frame comprises a start codon that serves to initiate translation. A fragment of an amino acid sequence comprises e.g. at least 50 %, at least 60 %, at least 70 %, at least 80%, at least 90% of the amino acid residues from an amino acid sequence. A fragment of an amino acid sequence preferably comprises at least 6, in particular at least 8, at least 12, at least 15, at least 20, at least 30, at least 50, or at least 100 consecutive amino acids from an amino acid sequence.

[0544] By "parent polypeptide", "parent protein", "precursor polypeptide", or "precursor protein" as used in the context of the invention is meant an unmodified polypeptide that is subsequently modified to generate a variant. A parent polypeptide may be a wild type polypeptide, or a variant or engineered version of a wild type polypeptide.

[0545] By "wild type" or "WT" or "native" in the context of the invention is meant an amino acid sequence that is found in nature, including allelic variations. A wild type protein or polypeptide has an amino acid sequence that has not been intentionally modified.

[0546] In the context of the invention, the term 'chimeric coreceptor' refers to an engineered coreceptor that has fused domains from different native coreceptors.

[0547] By "variant" or "variant protein" or "variant polypeptide" in the context of the invention is meant a protein that differs from a wild type protein by virtue of at least one amino acid modification. The parent polypeptide may be a naturally occurring or wild type (WT) polypeptide, or may be a modified version of a wild type polypeptide.

[0548] For the purposes of the present disclosure, "variant" of an amino acid sequence (peptide, protein or polypeptide) comprises amino acid insertion variants, amino acid addition variants, amino acid deletion variants and / or amino acid substitution variants. The term "variant" includes all splice variants, posttranslationally modified variants, conformations, isoforms and species homologs, in particular those which are naturally expressed by cells. The term "variant" includes, in particular, fragments of an amino acid sequence.

[0549] Amino acid addition variants comprise amino- and / or carboxy-terminal fusions of one or more amino acids, such as 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, such as by removal of 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletions may be in any position of the protein. Amino acid substitution variants are characterized by at least one residue in the sequence being removed and another residue being inserted in its place. Preference is given to the modifications being in positions in the amino acid sequence which are not conserved between homologous proteins or peptides and / or to replacing amino acids with other ones having similar properties. Preferably, amino acid changes in peptide and protein variants are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. A conservative amino acid change involves substitution of one of a family of amino acids which are related in their side chains. In one embodiment, conservative amino acid substitutions include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

[0550] Preferably the degree of identity between a given amino acid sequence and an amino acid sequence which is a variant of said given amino acid sequence will be at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The degree of identity is given preferably for an amino acid region which is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of identity is given preferably for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acids, preferably continuous amino acids. In preferred embodiments, the degree of identity is given for the entire length of the reference amino acid sequence. The alignment for determining sequence sequence identity can be done with art known tools, preferably using the best sequence alignment, for example, using Align, using standard settings, preferably EMBOSS "needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5. The term "sequence identity" between two amino acid sequences indicates the percentage of amino acids that are identical between the sequences. The term "percentage identity" is intended to denote a percentage of amino acid residues which are identical between the two sequences to be compared, obtained after the best alignment, this percentage being purely statistical and the differences between the two sequences being distributed randomly and over their entire length. Sequence comparisons between two amino acid sequences are conventionally carried out by comparing these sequences after having aligned them optimally, said comparison being carried out by segment or by "window of comparison" in order to identify and compare local regions of sequence similarity. The optimal alignment of the sequences for comparison may be produced, besides manually, by means of the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, by means of the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, by means of the similarity search method of Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 85, 2444, or by means of computer programs which use these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TEASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). The percentage identity is calculated by determining the number of identical positions between the two sequences being compared, dividing this number by the number of positions compared and multiplying the result obtained by 100 so as to obtain the percentage identity between these two sequences.

[0551] Homologous amino acid sequences exhibit according to the disclosure at least 40%, in particular at least 50%, at least 60%, at least 70%, at least 80%, at least 90% and preferably at least 95%, at least 98 or at least 99% identity of the amino acid residues.

[0552] The amino acid sequence variants described herein may readily be prepared by the skilled person, for example, by recombinant DNA manipulation. The manipulation of DNA sequences for preparing peptides or proteins having substitutions, additions, insertions or deletions, is described in detail in Sambrook et al. (1989), for example. Lurthermore, the peptides and amino acid variants described herein may be readily prepared with the aid of known peptide synthesis techniques such as, for example, by solid phase synthesis and similar methods.

[0553] An amino acid sequence (peptide, protein or polypeptide) "derived from" a designated amino acid sequence (peptide, protein or polypeptide) refers to the origin of the first amino acid sequence. Preferably, the amino acid sequence which is derived from a particular amino acid sequence has an amino acid sequence that is identical, substantially identical or homologous to that particular sequence or a fragment thereof. Amino acid sequences derived from a particular amino acid sequence may be variants of that particular sequence or a fragment thereof.

[0554] The term "isolated" means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not "isolated", but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated". An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0555] The term "naturally occurring" as used in the context of the invention refers to the fact that an object can be found in nature. For example, a peptide or nucleic acid that is present in an organism (including viruses) and can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally occurring.

[0556] As used in the context of the invention, the term "endogenous" refers to any material derived from or produced within an organism, cell, tissue or system. As used herein, the term "exogenous" refers to any material introduced from or produced outside an organism, cell, tissue or system.

[0557] As used in the context of the invention, the terms "linked", "fused", or "fusion" are used interchangeably. These terms refer to the joining together of two or more elements or components or domains.

[0558] As used in the context of the invention, the terms "protein", "peptide", "polypeptide", "peptides" and "polypeptides" are used interchangeably throughout. These terms refer to both naturally occurring peptides, e.g. naturally occurring proteins and synthesized peptides that may include naturally or non-naturally occurring amino acids. Peptides can be also chemically modified by modifying a side chain or a free amino or carboxy-terminus of a natural or non-naturally occurring amino acid. This chemical modification includes the addition of further chemical moieties as well as the modification of functional groups in side chains of the amino acids, such as a glycosylation. A peptide is a polymer preferably having at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or at least 100 amino acids, most preferably at least 8 or at least 30 amino acids.

[0559] An "effective amount" or "therapeutically effective amount" is an amount of a therapeutic agent, such as a therapeutic protein, that is sufficient to achieve the intended purpose. In the case of treatment of a particular disease or of a particular condition, the desired reaction preferably relates to inhibition of the course of the disease. This comprises slowing down the progress of the disease and, in particular, interrupting or reversing the progress of the disease. The desired reaction in a treatment of a disease or of a condition may also be delay of the onset or a prevention of the onset of said disease or said condition. An effective amount of a recombinant cell or nucleic acid composition or pharmaceutical composition described herein will depend on the condition to be treated, the severeness of the disease, the individual parameters of the patient, including age, physiological condition, size and weight, the duration of treatment, the type of an accompanying therapy (if present), the specific route of administration and similar factors. Accordingly, the doses administered of the cells and compositions described herein may depend on various of such parameters. In case a reaction in a patient is insufficient with an initial dose, higher doses (or effectively higher doses achieved by a different, more localized route of administration) may be used. The effective amount in each individual case may be determined empirically by a skilled artisan in accordance with established methods of the art.

[0560] The terms "individual" and "subject" are used interchangeably herein to refer to a human or other mammal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse, or primate) that may be afflicted with or susceptible to a disease or disorder, but may or may not have the disease or disorder. In preferred embodiments, the subject is a human. Unless otherwise noted, the terms "individual" and "subject" do not denote a specific age and thus include adults, elderly, children, and neonates. In embodiments of the present disclosure, the "individual" or "subject" is a "patient". The term "patient" means an individual or subject to be treated, in particular a diseased individual or subject.

[0561] The term "immunotherapy" refers to the treatment of a disease or condition by inducing, or enhancing an immune response. The term "immunotherapy" includes antigen immunization or antigen vaccination. The terms "immunization" or "vaccination" describe the process of administering an antigen to an individual with the purpose of inducing an immune response, for example, for therapeutic or prophylactic reasons.

[0562] ***

[0563] The invention is described by way of the following examples which are to be construed as merely illustrative and not limiting the scope of the invention, which is limited only by the appended claims. EXAMPLES

[0564] Example 1: Generation of CAR-T cells co-expressing chimeric CD8 coreceptors

[0565] The inventors hypothesized that the low responsiveness of conventional CAR-T cells known in the art to tumor cells with low target antigen densities (e.g. less than 1,000 antigens per cell), or to tumor cells with low or absent CD58 protein, may be the result of insufficient or absent engagement of coreceptors on the CAR-T cells. To mimic CD8 coreceptor function to enhance the activation of CAR-T cells and their sensitivity toward antigen-low or CD58-deficient tumor cells, an antigen -recognition domain 1 (ARD1, e.g. a scFv) was recombinantly linked to an extracellular domain of the CD8-alpha chain using a linker sequence, such as G4S (SEQ ID NO: 131), (648)2 (SEQ ID NO: 132), (G4S)3(SEQ ID NO: 10) or (G4S)4(SEQ ID NO: 133). The scFv was linked to the N-terminus of the CD8a IgV-like domain (IgVD) (shown in Figure 1A, and IB on the right). Such scFv-CD8a fusion polypeptides, also referred to as chimeric CD8 IgVD coreceptors, contain an intracellular domain capable of binding LCK, which was derived either from CD8a (CD8a.8IgV-CCR), or from CD4 (CD8a.4IgV-CCR), the latter of which may have a higher capacity to bind LCK than the CD8a cytoplasmic domain (Stepanek, O, et al., 2014, Cell 159(2):333-45; Morch, AM, 2022 PNAS 119(49): e2213538119). The control chimeric CD8 coreceptor comprises an ARD1, such as a scFv, linked to the N-terminus of a CD8a hinge domain (shown in Figure IB on the left). When expressed in cytotoxic T cells expressing endogenous CD8, the chimeric CD8a IgVD coreceptors may undergo heterotypic interactions with the IgVD of endogenous CD8 subunits, particularly with the CD80 chain, to form a monovalent chimeric CD8 coreceptor complex capable of binding to a defined and native target antigen exposed on the surface of a target cell (Figure 1C). When expressed in T helper cells expressing endogenous CD4, the chimeric CD8a IgVD coreceptors may prefer to undergo homotypic interactions to form a divalent chimeric CD 8 coreceptor dimer containing two antigen-binding domains (Figure ID). In contrast, the chimeric CD8 coreceptors with an extracellular CD8a-hinge, but lacking a CD8a IgVD (not claimed in the present invention), may preferably form homodimers in CD8 and CD4 T cells, as it was shown for conventional CARs known in the art containing the same CD8a-derived hinge and transmembrane domains (Milone, MC, et al., 2009; Chen, X, et al., 2022). Bicistronic constructs were made for co-expression of an anti-CD22 CAR (designated as 22-BBz or 22-28hi / tm-BBz) containing the m971 scFv, CD28-derived hinge and transmembrane domains (abbreviated as 28hi / tm), and 4-1BB and CD3-zeta signalling domains, and anti-CD19 chimeric CD8 coreceptor polypeptides containing the FMC63 scFv (anti-CD19 scFv derived from the FMC63 mouse hybridoma), where the two coding sequences were separated by a T2A sequence. The constructs were as follows: 22-28hi / tm-BBz (CAR only, SEQ ID NO: 87), 22- 28hi / tm-BBz / 19-8 (anti-CD22 CAR co-expressed with an anti-CD19 chimeric CD8 coreceptor containing a CD8a intracellular domain (control construct), SEQ ID NO: 102 (aa), SEQ ID NO: 117 (na)), 22-28hi / tm-BBz / 19-8.4 (anti-CD22 CAR co-expressed with an anti-CD19 chimeric CD8 coreceptor containing a CD4 intracellular domain (control construct), SEQ ID NO: 103 (aa), SEQ ID NO: 118 (na)), 22-28hi / tm-BBz / 19-8IgV (anti-CD22 CAR co-expressed with an anti-CD19 chimeric CD8a IgVD coreceptor, SEQ ID NO: 104 (aa), SEQ ID NO: 119 (na)), and 22-28hi / tm- BBz / 19-8.4IgV (anti-CD22 CAR co-expressed with an anti-CD19 chimeric CD8a IgVD coreceptor containing a CD4 intracellular domain, SEQ ID NO: 105 (aa), SEQ ID NO: 120 (na)).

[0566] The chimeric CD8a IgVD coreceptors may be co-expressed with any CAR known in the art in primary human T cells. When chimeric CD8a IgVD coreceptors and CARs bind to their cognate antigen, which may be two different antigens expressed on a target cell (in this example CD22 and CD 19), they relocate to the immunological synapse and come into proximity with each other. Proximal signaling molecules such as LCK bound to the intracellular domains of the chimeric CD8a IgVD coreceptors can amplify the phosphorylation of the IT AM motifs present in the CD3<^ domains of adjacent CAR molecules, thereby enhancing CAR signaling, T cell activation and immune responses toward the target cells.

[0567] Preferably, the nucleic acid constructs encoding the CAR and chimeric CD8a IgVD coreceptors are cloned into a retroviral or lenti viral expression vector. In the examples presented herein, lentiviral transfer vectors were used to produce the genomic material packaged into the VSV-G pseudotyped lentiviral particles. Lentiviral transfer vector DNA was mixed with the three packaging components of VSV-G, gag / pol, and rev, in combination with polyethylenimine (PEI) reagent to transfect them together into HEK293T cells (ATCC® CRL-3216™). After 24 hours, the media were replaced with fresh media, and 24-38 hours later, the vector-containing media were collected, filtered and concentrated by ultracentrifugation or by centrifugation in Centricon Plus- 70 Centrifugal Filter Units (Merck, Darmstadt). The resulting lentiviral vectors were stored at - 80°C. The number of transducing units was determined by titration on HEK293T cells. Redirected CAR and chimeric CD8a IgVD coreceptor T cells were produced by activating fresh or thawed human T cells with TransAct (Miltenyi Biotech, Bergisch-Gladbach), or any other CD3 and CD28 agonistic reagents, for 48-72 hours, and then adding lentiviral vectors at a multiplicity of infection (MOI) of 5 to transduce T cells. These modified T cells were allowed to expand for further 9 to 12 days until they were cryopreserved for later analysis. In some experiments, the transduced T cells were enriched for CAR expression by magnetic sorting using biotinylated recombinant CD22 and anti-Biotin microbeads (Miltenyi Biotech, Bergisch-Gladbach). Before cryopreserving, the percentage of transduced T cells expressing CAR and chimeric CD8a IgVD coreceptor molecules was determined by flow cytometric analysis. To detect CAR and chimeric CD8a IgVD coreceptor expression, transduced T cells were stained with recombinant and fluorescently labelled antigens (e.g. APC-labelled recombinant human CD22-Fc, Creative Biomart, NY), or with fluorescently labelled ProteinL (ProteinL-APC, Cell Signaling Technology, MA), or with fluorescently labelled anti-idiotypic antibodies (anti-FMC63-PE, ACROBiosystems, Basel, Switzerland), or with fluorescently labelled anti-G4S or anti-Whitlow scFv-Linker antibodies (Cell Signaling Technology, MA). As a negative control to set the gates for positivity, untransduced (UTD) T cells were stained similarly. As shown in Figure 2, chimeric CD8 coreceptors with or without extracellular IgV-like domain were efficiently co-expressed with the CAR on the surface of primary human T cells in a 1:1 ratio. The CAR-T cells were phenotypic ally characterized by flow cytometry to test for the effect of co-expressing a CAR and a chimeric CD8 coreceptor on T cell differentiation. As shown in Figure 3, co-expression of a chimeric CD8 coreceptor in addition to a CAR did not result in increased T cell differentiation, but enhanced T cell sternness compared to conventional CD22 CAR-T cells, as determined by the increased proportion of CD4+CD62L+ / CD45RA+ T cells in the CAR / chimeric CD8 coreceptor-double positive population. A similar trend was observed for CD8+ T cells. Without being bound by any theory, this is believed to be due to sequestration of free or membrane -bound LCK molecules in unstimulated CAR-T cells, thereby preventing unspecific and basal CAR-CD3<^ phosphorylation, tonic CAR signalling and antigen-independent immune cell activation. Example 2: Antitumor responses of CAR-T cells co-expressing chimeric CD8 IgVD coreceptors

[0568] This experiment was performed to determine whether co-expression of a chimeric CD8 IgVD coreceptor could enhance the function of CAR-T cells against tumor cells expressing very- low levels of the target antigen of the CAR (i.e. AG2), or against tumor cells lacking CD58 expression. To test this, CAR-T cells were co-cultured with NALM6 cells (a pre-B cell acute lymphoblastic leukemia cell line) expressing varying amounts of CD22 on their cell-surface, or with CD58-deficient NALM6 cells. Parental NALM6 cells express approximately 4,000 CD22 and 24,000 CD 19 molecules per cell (Figure 4A and B), which represents the median amount of the CD22 antigen found on primary B-ALL tumor samples (Shah et al., (2015) Pediatr Blood Cancer, (6) 964-969). Furthermore, NALM6 cells are highly positive for CD58 protein expression. In patients with leukemia or lymphoma, tumor cells can downregulate expression of CD22 (or other target antigens), which results in decreased CAR-T cell activity using conventional CAR designs. Besides decrease or loss of target antigen expression, reduced or loss of expression of CD58 was found in a significant proportion of all subtypes of B-, T-, and NK-cell lymphomas, and which correlated with low response rates to anti-CD19 CAR-T cells.

[0569] To generate NALM6 cell lines with lower CD22 antigen density, the gene encoding for CD22 was disrupted using the CRISPR / Cas9 technology. The CD22-deficient NALM6 cells were then transduced with lentiviral vectors encoding human CD22. Subsequent single-cell cloning and analyses of CD22 expression level using Quantibrite Phycoerythrin (PE) Quantification Beads (Becton Dickinson) identified several NALM6 cell clones that displayed reduced CD22 antigen densities, including clones that expressed very-low (about 400 CD22 molecules per cell) and ultralow (about 40 CD22 molecules per cell) CD22 site densities, which both are below the sensitivity limit of second- or third- generation CARs known in the art (Figure 4A). CD 19 site densities were not affected by reduced CD22 expression and were comparable in all engineered NALM6 cell lines (Figure 4B). To obtain a NALM6 cell line lacking CD58 protein expression, the CD58 gene was disrupted by CRISPR / Cas9. Subsequent single-cell cloning identified a NALM6 cell clone that lacked CD58 protein expression (Figure 4C), and CD58-deficiency in this clone was confirmed by western-blotting and sequencing of genomic DNA (data not shown). To test if co-expressing an anti-CD19 chimeric CD8 IgVD coreceptor (19-8IgV) could enhance anti-CD22 CAR-T cell activation in response to tumor cells with these known resistance mechanisms, each of the CAR-T cell group (100,000 cells), prepared as described in Example 1, was incubated with wild-type NALM6 cells, or with NALM6 cell lines expressing 400 (NALM6- CD22VL) or 40 (NALM6-CD22UL) CD22 molecules per cells, or with CD58-deficient NALM6 cells in an effector: target ratio of 1:1 for 24 hours. The prior art chimeric CD8 coreceptor having the same structure as the chimeric CD8 IgVD coreceptor but lacking of the IgVD (19-8, 22- BBz / 19-8), was used as control. After the incubation period, the T cells were harvested and the expression of activation markers CD69 and CD25 was analyzed by flow cytometry. In unstimulated CAR-T cells the fraction of CD25 / CD69 positive was low in all CAR-T cell groups, indicating that the chimeric CD8 coreceptors could not induce CAR-T cell activation in the absence of antigen (Figure 5). Interestingly, basal T cell activation was a bit more reduced in CAR-T cells coexpressing a chimeric CD8 coreceptor than in conventional CAR-T cells or untransduced (UTD) T cells, supporting the notion that chimeric CD8 coreceptors are able to inhibit tonic CAR signaling. The co-culture with wild-type NALM6 tumor cells led to upregulation CD69 and CD25 expression in CAR-expressing T cells, and T cell activation was markedly improved by coexpression of an anti-CD19 chimeric CD8 coreceptors, wherein T cell activation was more improved by 19-8IgV than by 19-8 (Figure 5). As expected, the activation of T cells expressing the conventional CD22 CAR was substantially reduced in response to CD58 -deficient NALM6 cells or NALM6-CD22VL tumor cells expressing 400 CD22 molecules / cell, and was completely absent when co-cultured with NALM6-CD22VL tumor cells expressing 40 CD22 molecules / cell, consistent with available data from the literature (Fry, T, et al., 2018. Nat. Med 24, 20-28; Majzner RG, et al., Cancer Discov. 2020; Heitzeneder et al., Cancer Cell 2022). In contrast, co-expressing a chimeric CD8 coreceptors significantly enhanced CAR-T cell activation, even in the absence of CD58 expression on tumor cells or at ultra-low antigen densities, and CAR-T cells co-expressing the chimeric CD8 IgVD coreceptor with extracellular IgVD (19-8IgV) outperformed the CAR-T cells co-expressing the control chimeric CD8 coreceptor (19-8) lacking of the IgVD for all NALM6 cell lines.

[0570] To further characterize the effect of co-expressing a CAR and a chimeric CD8 IgVD coreceptor on immune responses toward antigen-low or CD58-deficient tumor cells, the CAR-T cells (100,000), prepared as described in Example 1, were co-cultured with an equal number of wild-type, CD22UL or CD58 -deficient NALM6 tumor cells for 24 hours, and the cell-free culture supernatants were analyzed for cytokine secretion by a Bio-Plex multiplex assay (Bio-Rad). As shown in Figure 6, CAR-T cells having a chimeric CD8 coreceptor outperformed conventional CAR-T cells in cytokine release against wild-type, CD22VL and CD58 -deficient NALM6 cells. Consistent with the data obtained in the T cell activation assay (Figure 5), CAR-T cells coexpressing a chimeric CD8 IgVD coreceptor (22-28hi / tm-BBz / 19-8IgV) outperformed 22- 28hi / tm-BBz / 19-8 CAR-T cells expressing the control coreceptor lacking the IgVD.

[0571] To measure the effect of co-expressing a chimeric CD8 IgVD coreceptor on the cytotoxicity of 22-28hi / tm-BBz CAR-T cells, each of the CAR-T cell lines (50,000 cells) was incubated with GFP-expressing wild-type, CD22VL, CD22UL or CD58-deficient NALM6 cells, at an effector:target ratio of 1:1 in 96-well plates, and the growth of tumor cells was monitored in an IncuCyte live cell imaging instrument (Sartorius) for 48 hours. As shown in Figure 7, all CAR-T cell lines killed wild-type NALM6 cells similarly, while only CAR-T cells co-expressing a chimeric CD8 coreptor were capable to efficiently eliminate NALM6 tumor cells with ultra-low CD22 densities or with absent CD58 protein expression.

[0572] Example 3: CAR-T cells co-expressing chimeric CD8 IgVD coreceptors with intracellular CD4 domain

[0573] This experiment was performed to test whether the inclusion of the intracellular CD4 domain instead of the intracellular CD8a domain influences the coreceptor function of the chimeric CD8 IgVD coreceptors. Bicistronic constructs 22-28hi / tm-BBz / 19-8 (control), 22-28hi / tm- BBz / 19-8IgV, 22-28hi / tm-BBz / 19-8.4 (control), and 22-28hi / tm-BBz / 19-8.4IgV were prepared and transduced into primary human T cells as described in Example 1. Resulting CAR-T cells were stained with fluorescently labeled anti-CD3, anti-FMC63 idiotype, and recombinant CD22 protein, and analyzed by flow cytometry. As shown in Figure 8, all tested CAR and chimeric CD8 coreceptors were efficiently expressed on the surface of primary T cells at comparable levels. To test for CAR- induced T cell activation, CAR-T cells (100,000) were co-cultured with equal number of wild-type, CD22UL or CD58-deficient NALM6 cells for 24 hours, and T cell activation was determined by quantification of CD69 and CD25 expression by flow cytometry. As shown in Figure 9, co-expressing a chimeric CD8 coreceptor enhanced the activation of 22-BBz CAR-T cells in response to wild-type, to CD22 ultra- low, and to CD58-deficient NALM6 tumor cells. In particular, CAR-T cells co-expressing the chimeric CD8 IgVD coreceptor performed better than those co-expressing the control coreceptor without this domain, and the replacement of the CD8 intracellular domain with the corresponding domain of CD4 domain further enhanced T cell activation.

[0574] As an additional read-out for T cell activation, CAR-T cells (100,000 per group) were cocultured with equal numbers of wild-type, CD22UL and CD58 -deficient NALM6 cells for 24 hours, and the cell-free culture supernatants were analyzed for cytokine secretion by a LEGENDPlex multiplex assay (BioLegend). As shown in Figure 10 A-C, co-expressing a chimeric CD8 coreceptors enhanced secretion of IFNy, IL-2 and TNFa by 22-BBz CAR-T cells in response to wild-type, CD22 ultra-low and CD58-deficient NALM6 tumor cells, and CAR-T cells co-expressing chimeric CD8 IgVD coreceptor outperformed those co-expressing the control chimeric CD8 coreceptors without this domain. Interestingly, the chimeric CD8 IgVD coreceptor with intracellular CD4 domain enhanced secretion of IFNy by 22-BBz CAR-T cells, while the secretion of IL-2 and TNFa was comparable between the CAR-T cell groups having a chimeric CD8 IgVD coreceptor with intracellular CD8 or CD4 domains.

[0575] To measure the cytotoxicity of CAR-T cells, each of the CAR-T cell groups (50,000 cells per group) were co-cultured with wild-type, CD22UL or CD58 -deficient NALM6 cells at effector:target ratios of 1:1 or 1:5, and the growth of GFP -positive tumor cells was monitored in an IncuCyte live cell imaging instrument (Sartorius) for 72 hours. As shown in Figure 11A, CAR- T cells with or without co-expressing a chimeric CD8 coreceptors eliminated wild-type NALM6 cells efficiently when co-cultured at effector: target ratios of 1:1, while CAR-T cells co-expressing a chimeric CD8 coreceptor performed better than conventional 22-BBz CAR-T cells at the low effector:target ratio of 1:5. As shown in Figure 11B, conventional CAR-T cells lost their ability to kill NALM6 cells having only about 400 CD22 molecules per cell, while CAR-T cells coexpressing a chimeric CD8 coreceptor maintained their cytotoxic activity at these ultra-low target antigen densities, and CAR-T cells co-expressing a chimeric CD8 IgVD coreceptor performed better than those co-expressing a chimeric CD8 coreceptor without IgVD. As shown in Figure 11C, conventional 22-BBz CAR-T cells could not efficiently kill CD58 NALM6, while CAR-T cells co-expressing a chimeric CD8 coreceptor showed cytotoxic activity toward both CD58+and CD58 tumor cells, and CAR-T cells having a chimeric CD8 IgVD coreceptor performed better than those having a chimeric CD8 coreceptor without this extracellular domain at a low effector :target ratio, meaning that they are more cytotoxic. Example 4: Chimeric CD8 coreceptors depend on CAR engagement to amplify T cell responses

[0576] This experiment was performed to study T cell responses following individual engagement of either the chimeric CD 8 IgVD coreceptor or the CAR by tumor cells. To mimic engagement of chimeric CD8 IgVD coreceptors in the absence of CAR engagement, CAR-T cells were prepared to express anti-CD19 chimeric CD8 IgVD coreceptors in combination with an anti-HSV-BBz CAR, which contains an antigen-binding domain specific for the gB protein of Herpes-Simplex- Virus (HSV) 1 and 2 (HSV-28hi / tm-BBz / 19-8IgV, SEQ ID NO: 106 (aa) and 121 (na), or HSV- 28hi / tm-BBz / 19-8.4IgV, SEQ ID NO: 107 (aa) and 122 (na)), as previously described. To mimic CAR engagement in the absence of chimeric CD8 IgVD coreceptorsengagement, CAR-T cells were prepared to co-express the anti-CD22-BBz CAR and an HSV-specific chimeric CD8 IgVD coreceptor (22-28hi / tm-BBz / HSV-8IgV, SEQ ID NO: 108 (aa) and 123 (na) or 22-28hi / tm- BBz / HSV-8.4IgV, SEQ ID NO: 109 (aa) and 124 (na)). The generated CAR-T cells were stained with fluorescently labelled anti-CD3, anti-FMC63 idiotype antibody, anti-HSV scFv idiotype antibody, and recombinant human CD22 protein, and analyzed by FACS. As shown in Figure 12, all CAR and chimeric CD8 IgVD coreceptor constructs were expressed at comparable levels on the surface of human T cells.

[0577] 22-28hi / tm-BBz, 22-28hi / tm-BBz / 19-8IgV, HSV-28hi / tm-BBz / 19-8IgV, 22-28hi / tm- BBz / HSV-8IgV, 22-28hi / tm-BBz / 19-8.4IgV, HSV-28hi / tm-BBz / 19-8.4IgV, and 22-28hi / tm- BBz / HSV-8.4IgV CAR-T cells (100,000) were incubated with wild-type or CD58-deficient NALM6 cells (100,000) for 24 hours. After the incubation period, CAR-T cells were harvested and CD69 and CD25 expression was analyzed by FACS. As shown in Figure 13, co-expression of the 19-8IgV chimeric CD8 IgVD coreceptor significantly enhanced 22-BBz CAR T cell activation in response to wild-type or CD58-deficient NALM6 cells compared to conventional 22-BBz CAR-T cells. In contrast, engagement of the 19-8IgV chimeric CD8 IgVD coreceptor did not result in activation of anti-HSV-BBz CAR-T cells in the absence of CAR engagement. Interestingly, CAR activation without simultaneous activation of the chimeric CD8 IgVD coreceptor resulted in reduced T cell activation compared to CAR-T cells that co-express a chimeric CD8 IgVD coreceptor. Similar results were obtained for CAR-T cells co-expressing a chimeric CD8 IgVD coreceptor with an intracellular CD4 domain. To test if engagement of the chimeric CD8 IgVD coreceptor induced cytokine release in the absence of CAR signaling, CAR-T cells (100,000) were co-cultured with wild-type or CD58- deficient NALM6 cells at a ratio of 1:1 for 24 hours, and the culture supernatants were analyzed for IFNy, IL-2 and TNFa release by flow cytometry using the LEGENDPlex multiplex kit (Bio Legend). As shown in Figure 14, stimulated 22-BBz CAR-T cells co-expressing an anti-CD19 chimeric CD8 IgVD coreceptor released higher amounts of effector cytokines compared to conventional 22-Bz CAR-T cells. Notably, individual engagement of 19-8IgV or 19-8.4IgV chimeric CD8 IgVD coreceptor in HSV-BBz CAR-T cells by NALM6 cells resulted in significantly reduced IFNy and TNFa cytokine secretion (Figure 14A and C) and almost undetectable levels of IL-2 (Figure 14B). When stimulated with CD58 -deficient NALM6 cells, only 22-28hi / tm-BBz / 19-8IgV and 22-28hi / tm-BBz / 19-8.4IgV CAR-T cells were capable to release IFNy and TNFa, while no cytokine secretion was detected following individual engagement of 19-8IgV or 19-8.4IgV chimeric CD8 IgVD coreceptors in HSV-28hi / tm-BBz CAR- T cells (in the absence of CAR engagement). In line with the results in Figure 13, individual CAR engagement in 22-28hi / tm-BBz / HSV-8IgV and 22-28hi / tm-BBz / HSV-8.4IgV CAR-T cells led to substantially less cytokine release compared to 22-28hi / tm-BBz CAR T cells that did not coexpress a chimeric CD8 IgVD coreceptor, indicating impaired CAR signaling in the absence of concomitant engagement of the chimeric CD 8 IgVD coreceptor.

[0578] To test if engagement of chimeric CD8 IgVD coreceptors promote CAR-T cell cytotoxicity toward target cells in the absence of CAR engagement, each of the CAR-T cell lines (50,000 cells) was incubated with GFP-expressing NALM6, NALM6-CD22UL or NALM6-CD58KO tumor cells at an effector:target ratio of 1:1 in 96-well plates, and the growth of GFP-positive tumor cells was monitored in an IncuCyte instrument (Sartorius) for 48 hours. As shown in Figure 15A, only simultaneous engagement of CAR and chimeric CD8 IgVD coreceptors in 22-28hi / tm-BBz / 19- 8IgV and 22-28hi / tm-BBz / 19-8.4IgV CAR-T cells resulted in efficient elimination of wild-type, CD22UL- or CD 58 -deficient NALM6 cells, whereas cytotoxicity was markedly reduced when either CAR or chimeric CD8 IgVD coreceptors were engaged individually in 22-28hi / tm- BBz / HSV-8IgV (and 22-28hi / tm-BBz / HSV-8.4IgV) or HSV-28hi / tm-BBz / 19-8IgV (and HSV- 28hi / tm-BBz / 19-8.4IgV) CAR-T cells, respectively. When co-cultured at a low E:T ratio, only 22- 28hi / tm-BBz / 19-8IgV or 22-28hi / tm-BBz / 19-8.4IgV were able to eliminate NALM6 cells, while 22-28hi / tm-BBz / HSV-8.4IgV and HSV-28hi / tm-BBz / 19-8.4IgV CAR-T cells were unable to inhibit tumor cell growth (Figure 15B). To test if engaged chimeric CD8 IgVD coreceptors enhance signaling of endogenous TCRs (e.g. because of a mismatch with allogenic MHC molecules presented on NALM6 cells), 22- 28hi / tm-BBz / 19-8IgV and HSV-28hi / tm-BBz / 19-8IgV CAR-T cells (100,000) were co-cultured with an equal number of autologous peripheral blood B cells, which express high CD22- and CD 19- antigen densities, for 24 hours in an incubator. After the incubation period, the T cells were harvested and stained for CAR expression and expression of CD69 and CD25 activation markers. As shown in Figure 16, co-engagement of both the CAR and the chimeric CD8 IgVD coreceptors were required to activate the T cells, because individual engagement of the 19-8IgV CCR did not lead to HSV-BBz CAR-T cell activation in the absence of CAR engagement.

[0579] In a separate experiment, CAR-T cells (100,000) were co-cultured with autologous peripheral blood B cells at an E:T ratio of 1 : 1 , or were left unstimulated, for 24 hours, and the cell- free culture supernatant was assayed for cytokine secretion by flow cytometry using a LEGENDPlex assay kit (Bio Legend). As shown in Figure 17, co-engagement of CAR and chimeric CD8 IgVD coreceptors induced secretion of IFNy, IL-2 and TNFa by 22-BBz / 19-8IgV CAR-T cells, while engagement of the 19-8IgV coreceptor did not induce cytokine secretion by HSV-BBz / 19-8IgV CAR-T cells in the absence of CAR engagement.

[0580] In summary, these results demonstrate that individual engagement of 19-8IgV or 19-8.4IgV chimeric CD 8 IgVD coreceptors does not result in T cell activation or T cell immune responses without concomitant CAR engagement, and that simultaneous activation of CAR and chimeric CD8 IgVD coreceptors is required to elicit a potent T cell response.

[0581] Example 5: Cytotoxicity of CD4 CAR-T cells co-expressing chimeric CD8 IgVD coreceptors

[0582] There are two major T cell compartments that are separated by the expression of the CD4 or CD8 coreceptor. The effector functions of CD4 T cells are thought to focus on the secretion of cytokines and the coordination of immune responses, while CD8 T cells have direct cytotoxic activity against target cells. This experiment was performed to investigate if the co-expression of a chimeric CD 8 IgVD coreceptor enhances the cytotoxic activity of CD4 CAR-T cells against wildtype, CD22VL or CD58 -deficient NALM6 tumor cells. To this end, 22-28hi / tm-BBz, 22-28hi / tm- BBz / 19-8, 22-28hi / tm-BBz / 19-8IgV, 22-28hi / tm-BBz / 19-8.4, and 22-28hi / tm-BBz / 19-8.4IgV CAR-T cells were prepared as described in Example 1, and CD4- and CD8-positive T cells were separated by MACS sorting (Miltenyi Biotech, Bergisch-Gladbach). The CD4 and CD8 CAR-T cell groups (50,000 cells) were separately co-cultured with GFP-expressing NALM6, NALM6- CD22VL or NALM6-CD58KO tumor cells at an effector:target ratio of 1:1 in 96-well plates, and the growth of GFP-positive tumor cells was monitored in an IncuCyte instrument (Sartorius) for 48 hours. As shown in Figure 18A, both CD4 and CD8 CAR-T cells were able to eliminate wildtype NALM6 cells and no difference between the CAR-T cell groups were detected. As shown in Figure 18B, the cytotoxic activity of conventional 22-28hi / tm-BBz CAR-T cells was generally reduced against NALM6-CD22VL tumor cells, and the CD4+ 22-28hi / tm-BBz CAR-T cells showed a lower cytotoxic activity than their CD8+ counterparts, as expected. Remarkably, coexpression of the 22-BBz CAR with one of the tested chimeric CD8 IgVD coreceptors enhanced the killing of NALM6-CD22VL tumor cells, and both CD4+ and CD8+ 22-28hi / tm-BBz CAR-T cells showed similar cytotoxic activity. As shown in Figure 18C, conventional CD8+ 22-28hi / tm- BBz CAR-T cells showed low cytotoxic activity and CD4+ 22-28hi / tm-BBz CAR-T cells showed no cytotoxicity at all against CD58 -deficient NALM6. In contrast, both the CD4 and CD8 compartments of 22-28hi / tm-BBz CAR-T cells co-expressing one of the chimeric CD8 IgVD coreceptors showed high cytotoxic activity against CD58 tumor cells as well. These results demonstrate that co-expression of a chimeric CD8 IgVD coreceptor not only enhances the cytotoxic activity of CD8+ CAR-T cells against tumor cells carrying resistance mechanisms (such as low antigen and absent CD58 expression), but also converts CD4+ CAR-T cells into potent cytotoxic killer cells.

[0583] Example 6: Antitumor responses of CAR-T cells co-expressing chimeric CD8 IgVD coreceptors against tumor cells expressing PD-L1

[0584] This experiment was performed to demonstrate the efficacy of CAR-T cells co-expressing a chimeric CD8 IgVD coreceptor against antigen-low or CD58-deficient tumor cells that additionally overexpress PD-L1.

[0585] Tumor cells often upregulate inhibitory receptors such as PD-L1, and patients with high PD-L1 expression are less susceptible to CAR-T cell therapy because the interaction between PD- L1 and PD-1 can dampen CAR-T cell activity, resulting in reduced tumor cell killing and limited efficacy of therapy. To investigate whether PD-L1 expression on B-ALL cells inhibits the response of CAR-T cells, wild-type, CD22VL and CD58 -deficient NALM6 cell lines were modified to overexpress PD-L1 by transduction with PD-L1 encoding lentiviral vectors, and the transduced cells were sorted by FACS for high PD-L1 expression. Figure 19 shows CD22, CD58 and PD-L1 expression on sorted NALM6 tumor cell lines.

[0586] 22-28hi / tm-BBz CAR-T cells with or without co-expression of a 19-8 or 19-8IgV CCR were prepared as described in Example 1. Each CAR-T cell group was incubated with 100,000 NALM6, NALM6-PD-L1 or NALM6-CD22UL / PD-L1 target cells in a 1 : 1 ratio for 24 hours. After the incubation period, the cell culture supernatants were harvested and analyzed for effector cytokine secretion by flow cytometry using a LEGENDPlex multiplex assay kit (Bio Legend). As shown in Figure 20, co-expression of a chimeric CD8 IgVD coreceptor increased cytokine secretion by 22-BBz CAR-T cells in response to PD-L1+ NALM6 cells, as compared to conventional anti-CD22 CAR-T cells, and, notably, the chimeric CD8 IgVD coreceptor(19-8IgV) performed significantly better than the counterpart without the IgVD (19-8). The differences in the antitumor response between the 22-28hi / tm-BBz, 22-28hi / tm-BBz / 19-8 and 22-28hi / tm-BBz / 19- 8IgV CAR-T cells became more evident when stimulated with CD22VL or CD58 -deficient NALM6 cells that additionally overexpressed PD-L1. Here, conventional 22-28hi / tm-BBz-CAR- T cells showed a weak cytokine response, while 22-28hi / tm-BBz / 19-8IgV-CAR-T cells showed a superior cytokine response and even stronger than the control 22-28hi / tm-BBz / 19-8-CAR-T cells.

[0587] To test if co-expressing a chimeric CD8 IgVD coreceptor enhanced the cytotoxic activity of CAR-T cells against PD-L1+ NALM6 tumor cell lines, each of the CAR-T cell lines (50,000 cells) was incubated with wild-type NALM6, NALM6-PD-L1, CD22VL NALM6-PD-L1 and CD58-deficient NALM6-PD-L1 cells at effector:target ratios of 1:1 or 1:5 in 96-well plates, and the growth of GFP-positive tumor cells was measured in an IncuCyte live-cell imaging system (Sartorius) for 48-72 hours. As shown in Figure 21A, conventional 22-28hi / tm-BBz CAR-T cells showed reduced efficacy against PD-Ll-positive NALM6 cells and had no cytotoxic activity against PD-Ll-positive NALM6 cells at very-low antigen levels (400 CD22 molecules per cell), or in the absence of CD58 protein expression. In contrast, CAR-T cells co-expressing a chimeric CD8 IgVD coreceptor showed efficient cytotoxic activity against NALM6-PD-L1 cells and outperformed conventional 22-28hi / tm-BBz CAR-T cells, even when the CAR antigen was expressed at very-low densities or in the absence of CD58 expression. At a 1 : 1 effector:target ratio, there was no detectable difference in cytotoxicity between the 22-28hi / tm-BBz CAR-T cells with 19-8 or 19-8IgV CCRs. However, as shown in Figure 21B, 22-BBz CAR-T cells having a chimeric CD8 IgVD coreceptor (22-28hi / tm-BBz / 19-8IgV) performed better than those co-expressing a chimeric CD8 coreceptor without the IgVD (22-28hi / tm-BBz / 19-8IgV) against wild-type NALM6 cells, PD-L1+ NALM6 cells and CD22VL / PD-L1+ NALM6 cells at a low effector: target ratio. In summary, this example demonstrates that co-expressing a chimeric CD8 IgVD coreceptor with extracellular IgVD and HD significantly enhances the antitumor responses of CAR-T cells against PD-L1 -positive tumor cells, even at a low target antigen density or in the absence of CD58 expression.

[0588] Example 7: Anti-CD19 chimeric CD28 coreceptors potently boost antitumor responses of anti-CD22 CAR-T cells

[0589] CD28 is another coreceptor expressed on T cells that can provide potent co-stimulatory signals required for T cell activation, cytokine production and proliferation. To mimic CD28 coreceptor function to enhance the activation of CAR-T cells and their responsiveness toward antigen-low or CD58-deficient tumor cells, chimeric CD28 coreceptors were created by functionally linking an antigen-recognition domain (in this example an anti-CD19 scFv, FMC63) to an extracellular portion of the CD28 coreceptor, either directly to the hinge domain (shown in Figure 22A on the left, SEQ ID NO: 98), or to the N-terminal end of the IgV-like domain (SEQ ID NO: 99) using a linker sequence, such as G4S (SEQ ID NO: 131), (648)2 (SEQ ID NO: 132), (648)3 (SEQ ID NO: 10), or (648)4 (SEQ ID NO: 133). (shown in Figure 22A on the right). Both chimeric CD28 coreceptor variants either form covalently linked homodimers, or heterodimers with endogenous CD28 subunit polypeptides by interactions in their hinge and transmembrane domains.

[0590] The anti-CD19 chimeric CD28 coreceptor were co-expressed with an exemplary second- generation anti-6D22 GAR, which contained hinge and transmembrane domains derived from CD8a instead from CD28 to avoid inter-chain pairings between CAR and chimeric CD28 polypeptides, in primary human T cells. The bicistronic constructs were as follows: 22-8hi / tm-BBz (anti-CD22 CAR having a CD8a hinge / transmembrane region, without a chimeric CD28 coreceptor, SEQ ID NO: 88), 22-8hi / tm-BBz / 19-28 (anti-CD22 CAR with an anti-CD19 chimeric CD28 coreceptor containing CD28-derived hinge, transmembrane and intracellular domains, SEQ ID NOs: 110 (aa) and 125 (na)), 22-8hi / tm-BBz / 19-28IgV (anti-CD22 CAR co-expressed with an anti-CD19 chimeric CD28 coreceptor having CD28-derived IgV-like, hinge, transmembrane and intracellular domains, SEQ ID NOs: 111 (aa) and 126 (na)), and 22-8hi / tm-BBz / 19-28IgVAic (anti- CD22 CAR co-expressed with an anti-CD19 chimeric CD28 coreceptor having CD28-derived IgV- like, hinge and transmembrane domains but lacking an intracellular domain, SEQ ID NOs: 112 (aa) and 127 (na)). In the HSV-8hi / tm-BBz / 19-28 (SEQ ID NOs: 113 (aa) and 128 (na)) and HSV- 8hi / tm-BBz / 19-28IgV (SEQ ID NOs: 114 (aa) and 129 (na)) constructs, the CD22-specific m971 scFv of the CAR was replaced by a scFv with irrelevant specificity (in this example specific for the HSV-gB protein) to study T cell responses induced by the chimeric CD28 coreceptor in the absence of CAR stimulation. T cells expressing these constructs were prepared by lentiviral transduction, as previously described. The expression of CAR and chimeric CD28 coreceptor was analyzed by flow cytometry using fluorescently labelled recombinant CD22 protein and anti- FMC63 or anti-HSV scFv anti-idiotypic antibodies. As shown in Figure 23A, transduced primary human T cells expressed all tested CAR and chimeric CD28 coreceptors on their surface. Interestingly, chimeric CD28 coreceptors having a IgV-like domain were more efficiently expressed (~8-fold higher) on the cell-surface of T cells compared to their counterparts lacking this domain (Figure 23B).

[0591] To test whether co-expression of an anti-CD19 chimeric CD28 IgVD coreceptor could enhance the activation of anti-CD22 CAR-T cells in response to tumor cells bearing known resistance mechanisms, each CAR-T cell group (100,000 cells) was incubated for 24 hours with an equal number of wild-type or engineered NALM6 cell lines with very-low CD22 antigen density (NALM6-CD22VL; -400 CD22 molecules / cell) or no CD58 protein expression, with or without PD-L1 expression. After the incubation period, the T cells were harvested and the expression of activation markers CD69 and CD25 was analyzed by flow cytometry. As shown in Figure 24, coexpression of a chimeric CD28 IgVD coreceptor enhanced the activation of CAR-T cells in response to wild-type NALM6 cells or to tumor cells that have low CD22 antigen density or lack CD58 expression, even in the presence of high PD-L1 expression. Notably, the chimeric CD28 IgVD coreceptor having a IgV-like domain performed better than the counterpart lacking this domain. The capability of chimeric CD28 IgVD coreceptors to enhance CAR-T cell activation in response to CD22-low or CD58-deficient tumor cells required the intracellular domain of CD28, since a truncated version lacking the intracellular domain failed to boost CAR-T cell activation in response to NALM6-CD22VL or -CD58KO tumor cells. To further study antitumor responses of anti-CD22 CAR-T cells co-expressing a 19-28, 19- 28IgV or 19-28IgVAic CCRs, each of the CAR-T cell group (100,000 cells) were co-cultured in a 1:1 ratio with wild-type or engineered NALM6 tumor cell lines for 24 hours, and the culture supernatants were analyzed for cytokine secretion by flow cytometry using a LEGENDPlex assay (Bio Legend). As shown in Figure 25, conventional anti-CD22 CAR-T cells secreted Thl cytokines (A) IFNy, (B) IL-2 and (C) TNFa in response to wild-type NALM6 cells, and cytokine secretion was enhanced when the CAR-T cells additionally co-expressed an anti-CD 19 chimeric CD28 IgVD coreceptor. Conventional CAR-T cells secreted lower levels of cytokines when the target cells lacked CD58 protein expression, and cytokine secretion was further reduced when PD-L1 was additionally present on the tumor cells, as expected. In contrast, CAR-T cells co-expressing a chimeric CD28 IgVD coreceptor showed an efficient cytokine response against CD58-deficient NALM6 cells, even in the presence of high PD-L1 expression on the tumor cells. Notably, CAR- T cells co-expressing the full-length 19-28IgV CCR secreted slightly higher amounts of IFNy and IL-2 in response to CD58 -deficient tumor cells compared to their counterparts expressing the truncated 19-28 without an ICD. When stimulated with CD22-low NALM6 cells, conventional anti-CD22 CAR-T cells failed to produce detectable amounts of effector cytokines, whereas coexpression of a chimeric CD28 IgVD coreceptor restored cytokine production against antigen-low tumor cells, even in the presence of PD-L1 expression. Importantly, the boost in responsiveness against antigen-low or CD 58 -deficient tumor cells required the CD28 signaling domain, since a chimeric CD28 IgVD coreceptor lacking the intracellular domain was not capable to boost cytokine secretion by CAR-T cells in response to such tumor cells.

[0592] To study the effect of co-expressing an anti-CD 19 chimeric CD28 IgVD coreceptor on the cytotoxicity of anti-CD22 CAR-T cells, each group of CAR-T cells was co-cultured with 50,000 wild-type or engineered NALM6 tumor cell lines, or CD19-CD22- K562 cells as negative control, at an effector:target ratio of 1:1 for 72 hours, and the growth of GFP -positive tumor cells was monitored in an Incucyte instrument (Sartorius). As shown in Figure 26A, conventional CAR-T cells were capable to efficiently kill wild-type NALM6 cells, but not CD22-negative K562 cells and CD22-low or CD58-deficient NALM6 cells. In contrast, CAR-T cells co-expressing a chimeric CD28 IgVD coreceptor efficiently killed wild-type, CD22-low and CD58-deficient NALM6 cells, even in the presence of PD-L1 expression.

[0593] To test whether anti-CD22 CAR-T cells co-expressing an anti-CD 19 chimeric CD28 IgVD coreceptor can eliminate CD22-low or CD58-deficient tumor cells with or without PD-L1 expression even at a low effector:target ratio, each of the CAR-T cell groups was co-cultured with the corresponding NALM6 cell lines at an effector:target ratio of 1:5. As shown in Figure 26B, conventional CAR-T cells showed low cytotoxicity against CD22-low or CD58-negative tumor cells, whereas CAR-T cells co-expressing a chimeric CD28 IgVD coreceptor showed an improved cytotoxic function and inhibited the outgrowth of CD22-low or CD58KO tumor cells, even in the presence of PD-L1 expression. Although co-expression of a signaling incompetent chimeric CD28 IgVD coreceptor could enhance the cytotoxic function of CAR-T cells, most presumably because of the higher avidity of the T cells toward the tumor cells mediated by the high-affinity anti-CD19 antigen-recognition domain, the intracellular signaling domain in the chimeric CD28 IgVD coreceptors was required to optimally boost the cytotoxic function of CAR-T cells against tumor cells that are resistant to conventional CAR-T cells.

[0594] Example 8: Chimeric CD28 coreceptors enhance the proliferation of CAR-T cells

[0595] Proliferation in response to antigen binding is an important feature of CAR-T cells and correlates with clinical response in patients. In order to measure proliferation, the CAR-T cells were labelled with the dye ce...

Claims

1. Claims1. A chimeric coreceptor subunit polypeptide comprising:(a) an extracellular antigen-recognition domain 1 (ARD1) specifically binding an antigen 1 (AG1),(b) an immunoglobulin variable-like domain (IgVD),(c) a hinge domain 1 (HD1),(d) a transmembrane domain 1 (TMD1), and(e) a cytosolic domain (CYTD):(i) comprising a costimulatory domain (CSD), and(ii) not comprising a cell activation domain, wherein the ARD1 is linked to the IgVD.

2. The chimeric coreceptor subunit polypeptide according to claim 1, wherein:(a) the ARD1 comprises or consists of:(i) a mono-, bi- or multispecific scFv, a single domain antibody, or a single chain antibody-like protein scaffold, in particular an anticalin, DARPins, affibody, affimer, avimer, neutrophil gelatinase-associated lipocalin (NGAL) or evasin;(ii) a ligand selected from the group comprising a cytokine, in particular IL-1, IL- 10, IL- 13, APRIL, GM-CSF, FLT3L; a growth factor, in particular TPO; an immunoglobulin superfamily protein, in particular adnectin; a tumor associated antigen (TA A) targeting chimeric peptides, in particular TIE; or any combination thereof;(b) the IgVD comprises or consists of a CD8a IgVD, a CD8P IgVD, CD28 IgVD or variant thereof,(c) the HD1 comprises or consists of a CD8a HD, a CD8P HD, or a CD28 HD, or dimerizing variant thereof,(d) the TMD1 comprises or consists of a CD4, CD8a, CD8P, or CD28 TMD, or variant thereof, and / or(e) the CSD comprises or consists of a CD4, CD8a, CD8P, CD28, CD2, CD27, CD40, CD278, 0X40, GITR, CD40L, CD30, CD44, or CD 146 intracellular signaling domain (ICD), or a variant thereof.

3. The chimeric coreceptor subunit polypeptide according to any one of claims 1 to 2, wherein:(a) the ARD1 comprises or consists of:(i) a mono-, bi- or multispecific scFv, a single domain antibody, or a single chain antibody-like protein scaffold, in particular a anticalin, DARPins, affibody, affimer, avimer, neutrophil gelatinase-associated lipocalin (NGAL) or evasin;(ii) a ligand selected from the group comprising, a cytokine, in particular IL-1, IL- 10, IL- 13, APRIL, GM-CSF, FLT3L; a growth factor, in particular TPO; an immunoglobulin superfamily protein, in particular adnectin; a tumor associated antigen (TA A) targeting chimeric peptides, in particular TIE; or any combination thereof;(b) the IgVD comprises or consists of a CD8a IgVD,(c) the HD1 comprises or consists of a CD8a HD,(d) the TMD1 comprises or consists of a CD8a TMD, and(e) the CSD comprises or consists of a CD8a or CD4 ICD.

4. The chimeric coreceptor subunit polypeptide according to any one of claims 1 to 2, wherein:(a) the ARD1 comprises or consists of:(i) a mono-, bi- or multispecific scFv, a single domain antibody, or a single chain antibody-like protein scaffold, in particular an anticalin, DARPins, affibody, affimer, avimer, neutrophil gelatinase-associated lipocalin (NGAL) or evasin;(ii) a ligand selected from the group comprising, a cytokine, in particular IL-1, IL- 10, IL- 13, APRIL, GM-CSF, FLT3L; a growth factor, in particular TPO; an immunoglobulin superfamily protein, in particular adnectin; a tumor associated antigen (TAA) targeting chimeric peptides, in particular TIE; or any combination thereof;(b) the IgVD comprises or consists of a CD28 IgVD,(c) the HD1 comprises or consists of a CD28 HD,(d) the TMD1 comprises or consists of a CD28 TMD, and(e) the CSD comprises or consists of a CD28 ICD.

5. The chimeric coreceptor subunit polypeptide according to any one of claims 1 to 4, wherein the AG1 is a tumor-associated antigen selected from the group consisting of ALK, B7-H3 (CD276), BCMA, c-MET, CD3, CD5, CD19, CD20, CD22, CD23, CD30, CD33, CD38, CD44, CD44v6, CD52, CD70, CD79A, CD79B, CD123, CD138, CD171, CEA, Claudin-6, Claudin-18.2, CLL1, CXCR5, EGFR, EGFRvIII, EPH-receptor A2, IGLV3-21, IL-1RAP, GPC-2, GPC-3, HER2, ErbB3, ErbB4, FAP, FBP, AchR, Fr-a, GD2, GD3, HMW-MAA, IL- 13Ra2, Kappa-EC, IGLV3-21-R110, Lewis Y, LMP1, LMP2A, Mesothelin, MAGE-4A, MUC1, MUC16, NKG2D Ligands, NCAM, NY-ES01, Oncofetal antigen h5T4, PRAME, PSCA, PSMA, R0R1, TAG-72, VEGFR, G0LPH2, and SLAMF7, and preferably CD19 or CD22.

6. A chimeric coreceptor homodimer comprising two substantially identical chimeric coreceptor subunit polypeptides according to any one of claims 1 to 5 or a chimeric coreceptor complex comprising a chimeric coreceptor subunit polypeptide according to any one of claims 1 to 5 and a CD8a, CD8P or CD28 subunit.

7. A combination of: the chimeric coreceptor homodimer or chimeric coreceptor complex according to claim 6; and a chimeric antigen receptor (CAR), an endogenous TCR (TCR) or a transgene TCR (tgTCR), wherein the CAR, the TCR or the tgTCR specifically bind an antigen 2 (AG2), and wherein AG1 and AG2 are two different antigens or are substantially the same antigen.

8. A nucleic acid construct (N-CCR) comprising a nucleic acid encoding the chimeric coreceptor subunit polypeptide according to any one of claims 1-5, or a nucleic acid construct (N-CCR-CAR) comprising a nucleic acid encoding the chimeric coreceptor subunit polypeptide according to any one of claims 1-5 and a nucleic acid encoding a CAR subunit polypeptide, or two nucleic acid constructs (N-CCR, N-CAR), wherein the first nucleic acid construct (N- CCR) comprises a nucleic acid encoding the chimeric coreceptor subunit polypeptideaccording to any one of claims 1-5 and the second nucleic acid construct (N-CAR) comprises a nucleic acid encoding a CAR, or a nucleic acid construct (N-CCR-tgTCR) comprising a nucleic acid encoding the chimeric coreceptor subunit polypeptide according to any one of claims 1-5 and a nucleic acid encoding a tgTCR, or two nucleic acid constructs (N-CCR, N-tgTCR), wherein the first nucleic acid construct (N- CCR) comprises a nucleic acid encoding the chimeric coreceptor subunit polypeptide according to any one of claims 1-5 and the second nucleic acid construct (N-CAR) comprises a nucleic acid encoding a tgTCR.

9. A vector or a kit of a first and second vector, wherein(a) the vector comprises the nucleic acid construct (N-CCR), or the nucleic acid construct (N-CCR-CAR) or the nucleic acid construct (N-CCR-tgTCR) according to claim 8;(b) the first vector comprises the first nucleic acid construct (N-CCR) according to claim 8, and the second vector comprises the second nucleic acid construct (N-CAR) according to claim 8; or(c) the first vector comprises the first nucleic acid construct (N-CCR) according to claim 8, and the second vector comprises the second nucleic acid construct (N- tgTCR) according to claim 8.

10. A recombinant cell comprising the nucleic acid construct (N-CCR), the nucleic acid construct (N-CCR-CAR), the two nucleic acid constructs (N-CCR, N-CAR), the nucleic acid construct (N-CCR-tgTCR) or the two nucleic acid constructs (N-CCR, N- tgTCR) according to claim 8, or the vector or the first and second vector as defined in clam 9, and optionally wherein the cell expresses the chimeric coreceptor subunit polypeptide or the combination according to claim 7, and optionally wherein AG1 and AG2 are two different antigens or are substantially the same antigen.

11. A pharmaceutical composition comprising the:(i) the chimeric coreceptor subunit polypeptide according to any one of claims 1 to 5,(ii) the combination according to claim 7,(iii) the nucleic acid construct (N-CCR), the nucleic acid construct (N-CCR-CAR), the two nucleic acid constructs (N-CCR, N-CAR), the nucleic acid construct (N-CCR-tgTCR) or the two nucleic acid constructs (N-CCR, N-tgTCR) according to claim 8, or the vector or the first and second vector as defined in clam 9, or(iv) the recombinant cell according to claim 10, and a pharmaceutically acceptable carrier, stabilizer and / or excipient.

12. A chimeric coreceptor subunit polypeptide according to any one of claims 1 to 5, a combination according to claim 7, a nucleic acid construct (N-CCR), a nucleic acid construct (N-CCR-CAR), a two nucleic acid constructs (N-CCR, N-CAR), a nucleic acid construct (N- CCR-tgTCR) or two nucleic acid constructs (N-CCR, N-tgTCR) according to claim 8, a vector or a first and second vector as defined in clam 9, a recombinant cell according to claim 10, or a pharmaceutical composition according to claim 11, for use in medicine.

13. A chimeric coreceptor subunit polypeptide according to any one of claims 1 to 5, a combination according to claim 7, a nucleic acid construct (N-CCR), a nucleic acid construct (N-CCR-CAR), two nucleic acid constructs (N-CCR, N-CAR), a nucleic acid construct (N- CCR-tgTCR) or two nucleic acid constructs (N-CCR, N-tgTCR) according to claim 8, a vector or a first and second vector as defined in clam 9, a recombinant cell according to claim 10, or a pharmaceutical composition according to claim 11, for use in treating cancer, and wherein the cancer is preferably characterized by a cell-surface expression of at least one tumor-associated antigen selected from the group consisting of ALK, B7-H3 (CD276), BCMA, c-MET, CD3, CD5, CD19, CD20, CD22, CD23, CD30, CD33, CD38, CD44, CD44v6, CD52, CD70, CD79A, CD79B, CD123, CD138, CD171, CEA, Claudin-6, Claudin-18.2, CLL1, CXCR5, EGFR, EGFRvIII, EPH-receptor A2, IGLV3-21, IL-1RAP, GPC-2, GPC-3, HER2, ErbB3, ErbB4, FAP, FBP, AchR, Fr-a, GD2, GD3, HMW-MAA, IE- 13Ra2, Kappa-EC, IGLV3-21-R110, Lewis Y, LMP1, LMP2A, Mesothelin, MAGE-4A, MUC1, MUC16, NKG2D Ligands, NCAM, NY-ESO1, Oncofetal antigen h5T4, PRAME, PSCA, PSMA, ROR1, TAG-72, VEGFR, GOLPH2, and SLAMF7, and / or wherein the cancer is preferably a B cell malignancy.

14. A CAR, a nucleic acid comprising a nucleic acid encoding a CAR, a vector comprising a nucleic acid encoding a CAR or a cell comprising said nucleic acid or vector for use in the treatment of a cancer in combination with a chimeric coreceptor subunit polypeptideaccording to any one of claims 1 to 5, a chimeric coreceptor homodimer or a chimeric coreceptor complex according to claim 6, a nucleic acid construct (N-CCR) according to claim 8, or a vector comprising said N-CCR.

15. A method of treating cancer in a subject in need thereof, comprising the step of administering to the subject an effective amount of:(i) the chimeric coreceptor subunit polypeptide according to any one of claims 1 to 5,(ii) the combination according to claim 7,(iii) the nucleic acid construct (N-CCR), the nucleic acid construct (N-CCR-CAR), the two nucleic acid constructs (N-CCR, N-CAR), the nucleic acid construct (N-CCR- tgTCR) or the two nucleic acid constructs (N-CCR, N-tgTCR) according to claim 8, or the vector or the first and second vector as defined in clam 9, or(iv) the recombinant cell according to claim 10,(v) a pharmaceutical composition according to claim 11, and wherein the cancer is preferably characterized by a cell-surface expression of at least one tumor-associated antigen selected from the group consisting of ALK, B7-H3 (CD276), BCMA, c-MET, CD3, CD5, CD19, CD20, CD22, CD23, CD30, CD33, CD38, CD44, CD44v6, CD52, CD70, CD79A, CD79B, CD123, CD138, CD171, CEA, Claudin-6, Claudin-18.2, CLL1, CXCR5, EGFR, EGFRvIII, EPH-receptor A2, IGLV3-21, IL-1RAP, GPC-2, GPC-3, HER2, ErbB3, ErbB4, FAP, FBP, AchR, Fr-a, GD2, GD3, HMW-MAA, IE- 13Ra2, Kappa-EC, IGLV3-21-R110, Lewis Y, LMP1, LMP2A, Mesothelin, MAGE-4A, MUC1, MUC16, NKG2D Ligands, NCAM, NY-ESO1, Oncofetal antigen h5T4, PRAME, PSCA, PSMA, ROR1, TAG-72, VEGFR, GOLPH2, and SLAMF7, and wherein the cancer is preferably a B cell malignancy.

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