Co-receptor chimeric antigen receptor

Abstract

The present invention relates to LCK-binding coreceptor chimeric antigen receptors (CoCARs), combinations of coreceptor chimeric antigen receptors and CARs, nucleic acid constructs encoding such CoCARs, recombinant cells engineered to express such CoCARs, and pharmaceutical compositions comprising the same. Preferably, the present invention further provides its use in the treatment of diseases characterized by the expression of tumor-associated antigens.

Description

[Technical Field]

[0001] The present invention relates to co-receptors-CARs (CoCARs), combinations of CoCARs and CARs, nucleic acids encoding such antigen receptors, recombinant cells engineered to express such CoCARs, and pharmaceutical compositions comprising the same. Preferably, its use in the treatment of diseases characterized by the expression of at least one tumor-associated antigen is further disclosed. [Background technology]

[0002] Chimeric antigen receptor (CAR)-T cells represent a groundbreaking immunotherapeutic approach that has shown immense potential for treating cancer. CAR-T cells are generated from patient-derived autologous T cells, which are genetically modified to express CARs. After ex vivo genetic modification and proliferation, CAR-T cells are reinjected into the patient, where they bind to specific structures exposed on the surface of tumor cells. The antigen specificity of CAR-T cells is provided by the CAR, an artificially constructed fusion protein containing an extracellular antigen-binding domain (e.g., single-chain variable fragment, scFv) derived from an antibody linked to T cell-derived transmembrane and intracellular signaling domains. Classical CARs consist of an antigen-specific single-chain antibody (scFv) fragment fused to a transmembrane and signaling domain, e.g., CD3ζ. When introduced into T cells, they are expressed as membrane-bound proteins and induce an immune response upon binding to their cognitive antigen (Eshhar et al., (1993) PNAS, (90) 720-724).

[0003] CARs utilize the antigen-binding properties of parental monoclonal antibodies, enabling T cells to respond to native antigens expressed on the surface of tumor cells in a non-MHC-restrictive manner. Therefore, the mechanism of CAR-T cell activation is fundamentally different from that of conventional T cells, which respond exclusively to processed peptide antigens presented by MHC molecules. CAR-dependent activation of CAR-T cells triggers an immune response against antigen-expressing cells, resulting in T cell-mediated destruction of tumors.

[0004] Current CAR-T cell therapies exhibit limited efficacy when the specific target antigens recognized by the CARs are expressed at low densities. Initially, tumor cells often express large amounts of tumor-associated antigens, but in the presence of therapeutic intervention, tumor cells can downregulate the amount of target antigens expressed on their surface, resulting in the CARs being unable to activate T cells and kill the tumor cells. In other scenarios, CARs can inhibit T cell function even when antigen densities are normal or high (e.g., CARs induce T cell exhaustion, thereby ultimately attenuating or terminating the therapeutic effect of T cells). Therefore, it is still necessary to modulate T cell activation to overcome these limitations and enhance the applicability of these therapies.

[0005] Early CAR designs consisted of an antibody single-chain variable fragment (scFv) as the antigen-recognition domain, which was fused to the cytoplasmic end of the TCR signaling component CD3ζ via a hinge and transmembrane domain. T cells expressing first-generation CARs could induce cytotoxicity towards antigen-positive tumor cells, but they could not efficiently control tumor growth in vivo due to insufficient persistence (Eshhar et al., (1993) PNAS, (90) 720-724). Current CAR designs incorporate one or more costimulatory domains in addition to the intracellular CD3ζ T cell activation domain, thereby classifying second-generation (one costimulatory domain) and third-generation (two costimulatory domain) CAR structures. The design principle is to combine signal 1 (from the TCR) and signal 2 (from the costimulatory receptor) necessary to release full T cell activation, which has indeed significantly improved in vivo persistence and clinical efficacy. In most cases, the co-stimulatory domain is derived from either the CD28 receptor family (CD28, ICOS) or the tumor necrosis factor receptor (TNFR) family (4-1BB, CD27, OX40), while CD28 / CD3ζ(28z) and 4-1BB / CD3ζ(BBz) second-generation 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 the BBz signaling domain and two contain the 28z domain.

[0006] Growing practical experience with BBz- and 28z-CARs has revealed fundamental functional differences between the two CAR designs, which affect the in vivo persistence of CAR-T cells and, conversely, antigen sensitivity. BBz-CAR-T cells typically exhibit enhanced in vivo growth and persistence in mice and humans compared to 28z CAR-T cells, the latter rarely persisting longer than one or two months in patients. In contrast, 28z-CARs outperform BBz-CARs in tumors with low antigen density, and 28z-CAR-T cells are less likely to develop low-antigen relapses compared to their BBz counterparts. Therefore, BBz-CARs need to be reprogrammed to enhance anti-tumor immune responses against low-antigen-density tumor cells while maintaining their unique persistence capabilities.

[0007] 28z-CARs typically induce more rapid and larger-scale activation of downstream signaling events, resulting from their association with the CD28 endodomain of the CAR in the Src-family kinase LCK (Salter et al., (2018) Sci Signal. 11(544)). LCK association promotes increased basal CAR-CD3ζ phosphorylation and stronger antigen-dependent T cell activation, which is likely the main reason for the high sensitivity observed for CD28-based anti-CD19 CAR-T cells to target cells with reduced antigen density (Hamieh et al., (2019) Nature, (568) 112-116). 28z-CARs induce glycolytic metabolism and an effector T cell-like phenotype, which translates to high effector function, but in exchange, they exhibit rapid exhaustion after repeated stimulation, induce activation-induced cell death, and have low in vivo persistence. In fact, 28z-CAR-T cells do not persist longer than 60 days after injection, and the disappearance of CAR-T cells is the primary cause of relapse (Cappell & Kochenderfer, (2021) Nat Rev Clin Oncol. (11) 715-727; Neelapu et al., (2017) N Engl J Med, (377) 2531-2544). In contrast, BBz-CAR induces weaker downstream signaling events after stimulation, because the 4-1BB end domain of the CAR promotes the recruitment of the THEMIS-SHP1 phosphatase complex to the CAR synapse, where SHP1 attenuates LCK-mediated phosphorylation of the CAR-CD3ζ signaling domain (Sun et al., (2020) Cancer Cell, (2) 216-225). Nevertheless, while 28z- and BBz-CARs induce comparable antitumor responses in target cells with high antigen expression, BBz-CAR-T cells exhibit superior proliferation and persistence capabilities in vivo.It has been demonstrated that incorporating the 4-1BB endodomain into CAR synapses induces fatty acid oxidation metabolism, increases mitochondrial neogenesis, and thereby significantly enhances respiratory capacity and differentiation into T cell memory-like phenotypes (Kawalekar et al, (2016) Immunity, (2) 380-390). Because the 4-1BB endodomain contains binding sites for classical and non-classical NFκB pathways and anti-apoptotic proteins such as TRAF1, TRAF2, and TRAF3, which activate the expression of BCL-2 and BCL-XL, BBz CAR-T cells are more resistant to exhaustion and activation-induced cell death (Li et al., (2018) JCI Insight, (18) 121322). Therefore, BBz-CAR-T cells exhibit sustained antitumor activity against encroaching tumors in vivo, sometimes lasting for months or even years in responsive patients (Melenhorst et al., (2022) Nature, (602) 503-509). A concurrent comparison of BBz- and 28z-anti-CD19 CAR-T cells in patients with B-cell lymphoma showed similar antitumor efficacy, however, the 28z-CAR product induced severe cytokine release syndrome (CRS) and neurotoxicity, which led to the conclusion of further evaluation of 28z-CAR-T cells (Ying et al., (2019) Mol Ther Oncolytics, (15) 60-68). Furthermore, compared to its 28z counterpart, BBz-anti-CD19 CAR-T cells demonstrated superior antitumor efficacy and fewer adverse events in patients with B-cell acute lymphoblastic leukemia (B-ALL) (Zhao et al, (2020) Mol Ther Oncolytics, (18) 272-281), showing the superior function of BBz-CAR in the treatment of hematological malignancies. However, relapse is common with BBz-CAR-T cells, mostly due to a mechanism called antigen escape, which refers to recurrent tumor cells that show a reduced number of target antigens or completely lose antigen expression.For example, a clinical trial using anti-BCMA BBz-CAR-T cells in patients with multiple myeloma demonstrated the proliferation of tumor cells expressing low levels of BCMA (Brudno et al., (2018) J Clin Oncol, (22) 2267-2280; Cohen et al., (2019) J Clin Invest, (6) 2210-2221). Furthermore, anti-CD22 BBz-CAR-T cells utilizing the m971 antigen recognition domain showed a highly efficient antitumor response in preclinical models (Haso et al, (2013) Blood, (7) 1165-1174), inducing a complete response rate of over 70% in B-ALL patients. However, the majority of responsive patients relapsed due to the proliferation of tumor cells with reduced CD22 antigen density (Fry et al., (2017) Nat Med, (1) 20-28; Shah et al., (2020) J Clin Oncol. (17) 1938-1959). Preclinical modeling using engineered mutants of the B-ALL tumor cell line Nalm6 showed that reducing the CD22 site density to approximately 1,800 molecules per cell was sufficient to blunt the radical antitumor response by anti-CD22 BBz-CAR-T cells (Fry et al., (2017) Nat Med, (1) 20-28). Similar insufficient responsiveness of BBz-CAR-T cells to target cells with reduced antigen density was also demonstrated for other specificities, including anti-CD19 and anti-GPC2 CARs, which failed to eradicate tumor cells with 2,053 CD19 and 6,000 GPC2 molecules per cell, respectively (Majzner et al., (2020) Cancer Discov. (5) 702-723; Heitzeneder et al, (2022) Cancer Cell, (1) 53-69). These results suggest that a reduction in antigen expression to several thousand molecules per cell is a common cause of resistance to BBz-CAR-T cells and indicates insufficient sensitivity of BBz-CAR to low-antigen target cells.Therefore, a solution to this problem may be available by remanipulating BBz-CAR to enhance its sensitivity while retaining its ability for expansion and persistence, potentially preventing recurrence of low-antigen tumors.

[0008] Because CARs incorporate well-characterized T cell signaling components, they have been thought to signal in a similar manner to conventional TCRs. Indeed, both CARs and TCRs require CD3ζ ITAM phosphorylation to initiate signaling, which is mediated by Src-family kinases, most importantly LCKs. LCKs can be found either free in the cytosol and fixed to the cell membrane by N-terminal palmitoylation and myristoylation, or associated with the intracellular tail of the CD4 or CD8 coreceptor via a "zinc-clasp" structure (Kim et al, (2003) Science, (5640) 1725-1728), and each of these LCK forms can mediate the phosphorylation of CD3 ITAM in the TCR. Engagement of the TCR with the peptide-MHC complex also facilitates the recruitment of CD4 or CD8 coreceptors that bind to the same MHC molecule as the TCR, creating a positive feedback loop that recruits more LCKs to phosphorylate more CD3 ITAM. Phosphorylated ITAM then acts as a docking site for the SH2 domain of ZAP-70, a kinase whose activation is enhanced primarily by phosphorylation by LCK bound to the CD4 or CD8 coreceptor. ZAP-70 then phosphorylates the adapter proteins LAT and SLP-76, which form a signaling scaffold to which downstream signaling proteins, such as phospholipase C(PLC)-γ and Grb2, are recruited, and this complex is called the signalosome. Activated PLC-γ catalyzes the formation of inositol-1,4,5-triphosphate (IP3) and diacylglycerol (DAG), second messengers, from the membrane lipid phosphatidylinositol-4,5-biphosphate. This leads to the activation of several signaling pathways that result in intracellular calcium mobilization and alterations in gene expression, proliferation, and differentiation.

[0009] CAR ligation also induces phosphorylation of CD3ζ ITAM, which is crucial for the initiation of CAR activity, although CAR is unable to engage CD4 or CD8 coreceptors. ZAP-70, SLP-76, and PLC-γ also become phosphorylated upon CAR ligation, and this phosphorylation is more robust in 28z-CAR-T cells than in BBz-CAR-T cells, due to the presence of an LCK recruitment site in the CD28 endodomain, similar to CD3ζ. However, the precise mechanisms by which LCK, ZAP-70, LAT, SLP-76, and other signaling proteins are recruited to CAR synapses remain unclear, especially considering that BBz-CAR does not contain an LCK binding site at all.

[0010] To design BBz-CARs with improved antigen sensitivity, it was investigated whether the addition of the intracellular tail of the CD3ε- subunit of the TCR to the CAR sequence improved CAR-T cell function. This was because the CD3 subunit contains an internal proline-rich sequence (PRS) and a receptor kinase (RK) motif, which promote the recruitment of kinases Nck and LCK, respectively, and synergistically promote initial CD3 ITAM phosphorylation in relation to the TCR (Borroto et al., (2014) J Immunol, (5) 2042-2053; Hartl et al., (2021) Cell, (4) 834). In fact, the addition of the CD3ε domain to BBz-CAR has had beneficial effects on T cell activation and antitumor immune responses in vitro and in vivo, improving antigen sensitivity (Hartl et al., (2020) Nat Immunol. (8) 902-913; Salter et al., (2021) Sci Signal. (697) 2606). Other studies have shown that CAR function may be improved by modifying the receptor scaffold to include cytokine signaling domains (Kagoya et al., (2018) Nat Med. (3) 352-359), a second CD3ζ endodomain, or altered hinge and transmembrane domains (Majzner et al., (2020) Cancer Discov. (5) 702-723).

[0011] Another study investigated whether overall overexpression of the kinase LCK in BBz-CAR-T cells could compensate for enhanced activity of the phosphatase SHP1 at CAR synapses, thereby enhancing basal CAR-CD3ζ phosphorylation and the function of BBz-CAR-expressing T cells. Indeed, LCK overexpression led to increased basal CAR-CD3ζ phosphorylation and intracellular calcium mobilization after antigen stimulation, and these LCK-BBz-CAR-T cells showed improved in vivo proliferation and tumor control compared to conventional BBz-CAR-T cells lacking LCK overexpression (Sun et al., (2020) Cancer Cell, (2) 216-225; WO2021087183). However, the study did not show whether LCK overexpression improved the sensitivity of BBz-CAR-T cells to low-antigen tumor cells, nor whether increased LCK-mediated basal CAR-CD3ζ phosphorylation ultimately leads to persistent CAR signaling and antigen-independent T cell exhaustion and T cell differentiation, which are common problems when using 28z-CAR constructs (Long et al., (2015) Nat Med, (6) 581-590).

[0012] Further studies have described the expression of a chimeric molecule called the T cell antigen coupler (TAC) (Helsen et al., (2018) Nat Commun, (1) 3049; WO2015117229), which is a chimeric receptor containing an antigen recognition domain, a TCR mobilization domain (anti-CD3ε scFv), and a co-receptor domain (hinge, transmembrane, and cytosolic regions from CD4 or CD8α). Rather than modifying the current CAR design, the TAC receptor was designed to engage both the co-receptor and the endogenously expressed TCR in response to binding to a single antigen, with the aim of replicating the structure of the TCR-co-receptor complex that activates the innate signaling pathway and T cell response. [Overview of the Initiative]

[0013] The present disclosure generally relates to immunotherapy for use, for example, in enhancing immunotherapy in the treatment of various conditions, such as diseases (e.g., cancer), and includes chimeric antigen receptors (CARs) containing antigen recognition domains fused to hinge, transmembrane, and T cell activation domains, as well as chimeric co-receptors (CoCARs) containing extracellular domains, hinge domains, transmembrane domains, and antigen recognition domains fused to (a) a cytosolic domain capable of binding LCK and / or (b) a cytosolic domain containing LCK, and recombinant cells containing such polypeptides or polynucleotides encoding such polypeptides, and to the development of pharmaceutical compositions containing the same. As described in more detail below, different CAR and CoCAR constructs have been prepared and have been found to have a significant effect of enhancing the activation of immune cells (e.g., T cells) that co-express 4-1BB-based second-generation CARs and CoCARs in response to tumor cells, even when the antigen is expressed at very low density, increasing their efficacy.

[0014] According to a first aspect, the present invention provides a co-receptor chimeric antigen receptor (CoCAR) comprising (i) a first extracellular antigen recognition domain, (ii) a first hinge domain, (iii) a first transmembrane domain, and (iv) at least one cytosolic domain containing (a) a domain capable of binding LCK and / or (b) LCK or a variant or fragment thereof.

[0015] In particular, (i) the first extracellular antigen recognition domain, (ii) the first hinge domain, (iii) the first transmembrane domain, and (iv) the at least one cytosolic domain are arranged from the N- to the C-terminus.

[0016] In particular, the co-receptor chimeric antigen receptor does not include a CD3 zeta domain.

[0017] According to one embodiment, the first antigen recognition domain is a scFv.

[0018] According to one embodiment, the first hinge domain is derived from CD4, CD8, CD28, or an IgG-Fc hinge domain.

[0019] According to another embodiment, the first transmembrane domain is derived from a CD4, CD8, CD28, or CD3 zeta transmembrane domain.

[0020] In yet another embodiment, (a) at least one cytosolic domain capable of binding to LCK is (1) an intracellular signaling domain or a variant or fragment thereof derived from the CD4, CD8α, CD3ε, CD28, CD44, or CD146 intracellular signaling domain, and / or (2) one or more motifs capable of binding to LCK.

[0021] In particular, the intracellular signaling domain (1) is derived from the CD4, CD8α, CD3ε, CD44, or CD146 intracellular signaling domain or its variants or fragments.

[0022] In particular, the intracellular signaling domain (1) is derived from the CD4, CD8α, CD28, CD44, or CD146 intracellular signaling domain or its variants or fragments.

[0023] In particular, the intracellular domain (1) is derived from the CD4, CD8α, CD44, or CD146 intracellular signaling domain or its variants or fragments.

[0024] In particular, the intracellular domain (1) is derived from the CD4 or CD8α intracellular signaling domain or its variants or fragments.

[0025] In particular, the motifs are derived from the CD4, CD8α, CD28, CD3ε, CD44, or CD146 intracellular signaling domains.

[0026] In particular, motifs that can bind to LCK originate from the intracellular signaling domains of CD4, CD8α, CD28, CD3ε, CD44, or CD146.

[0027] In particular, motifs capable of binding to LCK originate from the CD4, CD8α, CD3ε, CD44, or CD146 intracellular signaling domains or their variants or fragments.

[0028] In particular, motifs capable of binding to LCK originate from the CD4, CD8α, CD28, CD44, or CD146 intracellular signaling domains or their variants or fragments.

[0029] In particular, motifs capable of binding to LCK originate from the CD4, CD8α, CD44, or CD146 intracellular signaling domains or their variants or fragments.

[0030] In particular, LCK-binding motifs are derived from the CD28, CD3ε, CD44, or CD146 intracellular signaling domains.

[0031] In particular, motifs capable of binding to LCK originate from the CD4 or CD8α intracellular signaling domain or its variants or fragments.

[0032] In yet another embodiment, CoCAR is capable of specifically binding to antigens on target cells.

[0033] According to a second aspect, the present invention is (a) The present invention provides a combination comprising a CoCAR and (b) a chimeric antigen receptor (CAR).

[0034] According to one embodiment, CAR(b) comprises (i) a second extracellular antigen recognition domain, (ii) a second hinge region, (iii) a second transmembrane domain, (iv) a 4-1BB domain and / or a CD28 domain, and (v) a CD3 zeta domain.

[0035] In particular, in CAR, (i) a second extracellular antigen recognition domain, (ii) a second hinge region, (iii) a second transmembrane domain, (iv) a 4-1BB domain and / or a CD28 domain, and (v) a CD3 zeta domain are located from N to the C terminus.

[0036] According to a third aspect, the present invention provides nucleic acids (single and multiple) and nucleic acid constructs (single and multiple) that encode the CoCAR of the present invention or a combination of the present invention.

[0037] According to a fourth aspect, the present invention provides vectors or combinations of vectors comprising nucleic acids (single and multiple), nucleic acid constructs (single and / or multiple) of the present invention.

[0038] According to a fifth aspect, the present invention provides recombinant cells comprising the nucleic acid construct of the present invention.

[0039] According to one embodiment, the cells of the present invention are immune effector cells.

[0040] According to one embodiment, the cells of the present invention express the CoCAR of the present invention on their cell surface.

[0041] According to one embodiment, the cells of the present invention express the combination of the present invention on their cell surface.

[0042] In yet another embodiment, in the cells of the present invention, (i)CAR and (ii)CoCAR recognize different epitopes located within the same antigen expressed on the surface of the target cell.

[0043] In yet another embodiment, in the cells of the present invention, (i) CAR and (ii) CoCAR recognize different antigens expressed on the surface of target cells.

[0044] According to a sixth aspect, the present invention provides a pharmaceutical composition comprising (i) the CoCAR of the present invention, (ii) a combination of the present invention, (iii) a nucleic acid construct of the present invention, and / or (iv) recombinant cells and a pharmaceutically acceptable carrier of the present invention.

[0045] According to a seventh aspect, the present invention provides CoCAR, combinations, nucleic acid constructs, recombinant cells, or pharmaceutical compositions for use in pharmaceuticals.

[0046] According to an eighth aspect, the present invention provides CoCAR, combinations, nucleic acid constructs, recombinant cells, or pharmaceutical compositions for use in the treatment of cancer.

[0047] According to one embodiment, in the treatment of malignant tumors having cell surface expression, the CoCAR, combination, nucleic acid construct, recombinant cell, or pharmaceutical composition for use of the present invention is preferably 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 The antigens selected are from the group consisting of 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 ligand, NCAM, NY-ESO1, Carcinoembryonic antigen h5T4, PSCA, PSMA, ROR1, TAG-72, VEGFR, GOLPH2, and SLAMF7.

[0048] According to the ninth aspect, the present invention provides CoCARs, combinations, nucleic acid constructs, recombinant cells and / or pharmaceutical compositions for use in the treatment of B-cell malignancies.

[0049] According to a tenth aspect, the present invention provides a method for treating cancer in a subject requiring treatment, comprising the steps of administering to the subject an effective amount of (i) the CoCAR of the present invention, (ii) the combination of the present invention, (iii) the nucleic acid construct of the present invention and / or (iv) recombinant cells of the present invention.

[0050] According to one embodiment, cancer is preferably 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, HE A malignant tumor that is positive for at least one tumor-associated antigen selected from the group consisting of R2, ErbB3, ErbB4, FBP, AchR, Fr-a, GD2, GD3, HMW-MAA, IL13Ra2, Kappa-LC, IGLV3-21-R110, Lewis Y, Mesothelin, MUC1, MUC16, NKG2D ligand, NCAM, NY-ESO1, Carcinoembryonic antigen h5T4, PSCA, PSMA, ROR1, TAG-72, VEGFR, GOLPH2, and SLAMF7.

[0051] According to another embodiment, cancer is a B-cell malignant tumor.

[0052] Other features and advantages of the present invention will become apparent from the following detailed description, figures, and claims. [Brief explanation of the drawing]

[0053] [Figure 1A]Figure 1 shows the characteristics of T cells expressing anti-CD22 BBz-CAR, whether transduced (UTD) T cells or with or without LCK overexpression. (A) LCK co-expression with BBz-CAR increases basal CAR-CD3ζ phosphorylation, thereby potentially enhancing CAR-T cell activity. (B) T cell phenotypes were analyzed by flow cytometry 14 days after initial T cell activation, and T cell subsets were defined according to the expression of CAR, CD4, CD8, CCR7 (CD197), and CD45R0. CCR7+ / CD45R0- cells represent naive-like T cells, CCR7+ / CD45R0+ cells represent central memory T cells, CCR7- / CD45R0+ cells represent effector memory T cells, and CCR7- / CD45R0- cells represent effector T cells. These data suggest that overexpression of LCK along with BBz-CAR leads to effector memory and T cell differentiation into effector T cells. (C) shows ex vivo expansion and proliferation of T cells expressing the indicated construct. Untransduced T cells and T cells transduced with anti-CD22 BBz-CAR proliferate efficiently during the first 14 days after T cell activation by CD3 / CD28 agonists on day 0, but CAR-T cells overexpressing LCK show only insufficient expansion and proliferation capacity. [Figure 1B] Same as above. [Figure 1C] Same as above. [Figure 2A]Figure 2 illustrates the concept of the present invention involving the co-expression of BBz-CAR and CoCAR. CoCAR contains an antigen-recognition domain fused to the coreceptor, in this example, the hinge, transmembrane, and cytosolic domains of CD4. The antigen-recognition domains of BBz-CAR and the coreceptor-CAR recognize different epitopes present in either the same antigen expressed by the same target cell or in two different antigens. (A) In unstimulated T cells, the CAR and CoCAR molecules are spatially separated from each other, so that the LCK bound to CoCAR is unavailable for basal CAR-CD3z phosphorylation. (B) After binding to the cognitive antigen, BBz-CAR and the coreceptor-CAR relocalize and proximally approach each other, and the coreceptor-CAR recruits the coupled LCK to the BBz-CAR synapse, where it assists in CAR-CD3z phosphorylation. ZAP-70 binds to the phosphorylated ITAM of the CAR-CD3z domain, and the recruited ZAP-70 molecule is then efficiently phosphorylated by CoCAR-associated LCK. Activated ZAP-70 then phosphorylates its substrates, including LAT and SLP-76, which form signalosomes that activate various downstream signaling pathways. [Figure 2B] Same as above. [Figure 3A]Figure 3 shows the characteristics of T cells transduced to express anti-CD19 CoCAR or anti-CD22 BBz-CAR, or co-transduced with both lentiviral vectors to co-express BBz-CAR with CoCAR. (A) Schematic diagrams of CAR and CoCAR molecules. (B) Flow cytometry analysis shows cell surface expression of anti-CD22 BBz-CAR and anti-CD19 CoCAR molecules. (C) Ex vivo expansion and proliferation of T cells during the first 14 days after activation with anti-CD3 / CD28 agonist (TransAct, Miltenyi). (D) Quantification of CD22 receptor density on the cell surface of Nalm6 and Raji tumor cell lines using Quantibrite PE quantification beads (Becton Dickinson). (E) shows the percentage of CD69 / CD25 double-positive T cells expressing the indicated construct 24 hours after co-culture with the indicated target cell lines, as determined by flow cytometry analysis. (F) shows the amount of IFN-gamma produced by T cells expressing the expressed construct 24 hours after co-culture with the indicated target cell line, as determined by flow cytometry analysis using the LEGENDplex kit (BioLegend). [Figure 3B] Same as above. [Figure 3C] Same as above. [Figure 3D] Same as above. [Figure 3E] Same as above. [Figure 3F] Same as above. [Figure 4A]Figure 4 shows the antigen-dependent activity of transduced T cells expressing anti-CD22 CoCAR, anti-CD22 BBz-CAR, or BBz-CAR co-expressed with CoCAR from a bicistronic vector. (A) Schematic diagram of CAR and CoCAR molecules. (B) Flow cytometry analysis shows cell surface expression of anti-CD22 BBz-CAR and CoCAR molecules. (C) T cells were incubated in 96-well cell culture plates coated with recombinant human CD22 protein at the indicated concentrations. After 24 hours, the concentrations of effector cytokines in the cell culture supernatant were determined by flow cytometry using a LEGENDPlex multiplex assay (BioLegend). (D) shows the amounts of IFNγ, TNFα, and IL-2 produced by T cells expressing the construct shown, 24 hours after co-culture with CD22-positive Nalm6 cells (or unstimulated T cells), as determined by flow cytometry analysis using the LEGENDplex multiplex assay (BioLegend). (E) shows the CD22 site density on the surface of wild-type Nalm6 cells, CD22 knockout Nalm6 cells, and Nalm6 cell clone number 3 engineered to express very low CD22 antigen levels. CD22 site density was quantified using Quantibrite PE quantification beads (Becton Dickinson). (F) Transduced T cells (UTD) or T cells expressing the indicated construct were co-cultured with GFP-positive Nalm6 cell clone number 3 (expressing approximately 400 CD22 molecules per cell) in a T cell to target cell ratio of 1:10, and the proliferation of GFP-positive target cells was monitored for 2 days using an Incucyte instrument (Sartorius). [Figure 4B] Same as above. [Figure 4C] Same as above. [Figure 4D] Same as above. [Figure 4E] Same as above. [Figure 4F] Same as above. [Figure 5A]Figure 5 shows antigen-dependent and antigen-independent activation of transduced T cells expressing anti-CD22 CoCAR, anti-CD22 BBz-CAR, or BBz-CAR co-expressed with CoCAR from a bicistronic vector. (A) Expression of activation markers CD69 and CD25 is shown for unstimulated T cells and T cells stimulated for 24 hours using the CD22-negative myeloid cell line K562, or the CD22-positive B-ALL cell line Nalm6. (B) Expression of inhibitory receptors LAG-3, TIM-3, and PD-1 is shown in unstimulated T cells or T cells stimulated for 24 hours using the CD22-negative myeloid cell line K562, or the CD22-positive B-ALL cell line Nalm6. (C) Flow cytometry analysis of T cell subsets 14 days after initial T cell activation. T cell subsets are defined according to the expression of CD4, CD8, CD62L, and CD45RA. CD62L+ / CD45RA+ cells represent stem cell memory-like T cells, CD62L+ / CD45RA- cells represent central memory T cells, CD62L- / CD45RA- cells represent effector memory T cells, and CD62L- / CD45RA+ cells represent effector T cells. [Figure 5B] Same as above. [Figure 5C] Same as above. [Figure 6A] Figure 6 shows a CoCAR system using anti-EGFR-CAR and anti-CEA-CoCAR. (A) shows a schematic CAR / CoCAR design in immune cells. (B) shows flow cytometry measurements of receptor expression in transduced T cells. CAR expression was detected using a fluorescently labeled anti-IgG antibody, and CoCAR expression was detected with biotinylated protein L followed by streptavidin-PE. (C) shows increased expression of the exhaustion markers LAG-3 and TIM-3 three weeks after transduction in T cells expressing anti-EGFR-CAR alone, compared to T cells co-expressing anti-EGFR-CAR and anti-CEA-CoCAR. [Figure 6B] Same as above. [Figure 6C] Same as above. [Figures 7A-7B]Figure 7 shows the advantageous functional characteristics of anti-EGFR-CAR-T cells when co-expressing anti-CEA CoCAR. (A) shows low EGFR expression by MCF-7 compared to other cell lines (A-431, SKOV-3), as assessed by flow cytometry, and (B) is further quantified using Quantibrite PE quantification beads (Becton Dickinson). (C) shows increased IL-2 secretion by anti-EGFR-CAR-T cells co-expressing anti-CEA CoCAR compared to anti-EGFR-CAR alone, as measured after 1:1 co-culture with MCF-7 cells using the LEGENDplex assay. (D) monitors cell proliferation of MCF-7 in 1:1 co-culture with T cells expressing different CAR constructs using the Incucyte system (Sartorius). The graph clearly shows that T cells expressing anti-EGFR-CAR exhibit enhanced long-term cytolytic activity when co-expressing anti-CEA-CoCAR. This is further confirmed in (E), which shows induction of apoptosis in MCF-7 cells as measured using Annexin V-binding dye. [Figure 7C] Same as above. [Figure 7D] Same as above. [Figure 7E] Same as above. [Figure 8] Figure 8 schematically illustrates the differences between exemplary CARs and chimeric coreceptors (CoCARs). CoCARs lack the CD3ζ T cell activation domain and therefore act only in trans to amplify CARs. It should be noted that both CARs and CoCAR polypeptides can form covalently linked homodimers in T cells, due to the presence of cysteine ​​residues in the hinge domains of CD28 and CD8α, which form disulfide bonds. [Figure 9]Figure 9 shows the expression of exemplary CAR and CoCAR polypeptides on the surface of transduced primary human T cells, as determined by flow cytometry. Anti-CD19 CoCARs with an intracellular CD28 domain were efficiently expressed on the T cell surface, while CoCARs with intracellular CD2, CD3ε, CD44, or CD146 domains were only weakly visible on the T cell surface. [Figure 10] Figure 10 shows the expression of CD69 and CD25 activation markers in unstimulated CAR-T cells or wild-type or CD22UL cl.3 NALM6 cells and CAR-T cells co-cultured for 24 hours at an effector-to-target ratio of 1:1. CoCAR with an intracellular CD28 domain was the only polypeptide tested that significantly enhanced CAR-T cell activation in response to NALM6-CD22UL tumor cells expressing 400 CD22 antigens per cell. [Figure 11-1] Figure 11 shows the concentrations of (A) IFNγ, (B) IL-2, and (C) TNFα in the supernatant of the shown tumor cell line and the shown CAR-T cell line co-cultured for 24 hours. The concentrations of the shown cytokines were measured by flow cytometry using the LegendPlex assay kit (BioLegend). CoCAR with an intracellular CD28 domain was the only polypeptide tested that significantly enhanced effector cytokine secretion by CAR-T cells in a strictly antigen-dependent manner in response to NALM6-CD22UL tumor cells expressing 400 CD22 antigens per cell. [Figure 11-2] Same as above. [Figure 11-3] Same as above. [Figure 12]Figure 12 shows the cytotoxicity of shown CAR-T cell lines with or without co-expression of CoCAR, co-cultured for 18 hours with (A) NALM6 cells, (B) NALM6-CD22UL cl.3 cells, or (C) K562 cells at different effector-to-target ratios. CAR-T cells co-expressing CoCAR with an intracellular CD28 domain were superior to all tested CoCAR-T cells and conventional CAR-T cells against NALM6-CD22UL tumor cells expressing 400 CD22 antigens per cell. [Figure 13] Figure 13 schematically shows the co-expression of exemplary CAR and CoCAR polypeptides. In CoCAR polypeptides possessing intracellular CD44 and CD146 domains, the CD8α transmembrane domain was substituted with the CD44 or CD146 transmembrane domain, respectively. These version 2 CoCARs are called 19-44v2 and 19-146v2. [Figure 14] Figure 14 shows the expression of exemplary CAR and CoCAR polypeptides on the surface of transduced primary human T cells, as determined by flow cytometry. Modification of the transmembrane domain restored cell surface expression of CoCARs with intracellular CD44 and CD146 domains in transduced human T cells. [Figure 15] Figure 15 shows the differentiation state of CD4+ or CD8+ CAR-T cells expressing the indicated constructs, as determined by flow cytometry analysis of CD62L and CD45RA expression. CD62L+CD45RA+ T cells represent stem cell-like memory T cells (Tscm), CD62L+CD45RA- T cells represent central memory T cells (Tcm), CD62L-CD45RA- T cells represent effector memory T cells (Tem), and CD62L-CD45RA+ T cells represent effector T cells (Teff). CAR-T cells co-expressing 19-28, 19-44v2, or 19-146v2 CoCARs showed a less differentiated phenotype compared to conventional CAR-T cells or untransduced T cells. [Figure 16]Figure 16 shows the expression of (A) CD22 and (B) CD19 molecules on the cell surface of different NALM6 cell clones. The calculated number of CD22 or CD19 molecules per cell is shown on the right. Quantification of CD22 or CD19 molecules per cell was performed by flow cytometry using PE-labeled anti-CD22 and CD19 antibodies and Quantibrite® PE quantification beads (BD Biosciences). [Figure 17-1] Figure 17 shows the expression of CD69 and CD25 activation markers in unstimulated CAR-T cells or wild-type or CD22UL cl.3 NALM6 cells and CAR-T cells co-cultured for 24 hours at an effector-to-target ratio of 1:1. All tested CoCARs enhanced CAR-T cell activation in response to NALM6 cells, but CD28-based and CD44-based CoCARs were able to enhance anti-CD22 CAR-T cell activation in response to tumor cells expressing as few as 40 CD22 antigens per cell. [Figure 17-2] Same as above. [Figure 18] Figure 18 shows the cytotoxicity of different NALM6 cell clones expressing varying amounts of CD22 molecules per cell and the shown CAR-T cell lines co-cultured for 18 hours at different effector-versus-target ratios. CAR-T cells co-expressing 19-28, 19-44v2, or 19-146v2 CoCARs were superior to conventional CAR-T cells against NALM6-CD22UL tumor cells expressing very low CD22 antigen densities. [Figure 19]Figure 19 shows the concentrations of (A) IFNγ, (B) IL-2, and (C) TNFα in the supernatant of the shown tumor cell line and the shown CAR-T cell line co-cultured for 24 hours. The concentrations of the shown cytokines were measured by Bio-Plex using the Bio-Plex assay kit (Bio-Rad). CAR-T cells co-expressing 19-28, 19-44v2, or 19-146v2 CoCAR were superior to conventional CAR-T cells in effector cytokine release against CD22 very low NALM6 tumor cells, but CAR-T cells co-expressing 19-28 CoCAR secreted the highest amounts of cytokines in the CD22 very low tumor setting. [Figure 20] Figure 20 shows the expression of exemplary CoCAR and CAR polypeptides holding wild-type or mutated CAR-CD3ζ ITAM (indicated as xxx) on the surface of transduced primary human T cells, as determined by flow cytometry. Mutations in CAR-CD3ζ ITAM had no effect whatsoever on the cell surface expression of CAR or CoCAR polypeptides. [Figure 21] Figure 21 shows the expression of CD69 and CD25 activation markers in CAR-T cells co-cultured for 24 hours with unstimulated CAR-T cells or wild-type NALM6 cells in an effector-to-target ratio of 1:1. Engaged 19-28 CoCAR failed to enhance CAR-T cell activation in the absence of CAR signaling. [Figure 22] Figure 22 shows the cytotoxicity of indicated CAR-T cell lines containing wild-type or mutant ITAM cells co-cultured with NALM6 cells at an effector-to-target ratio of 1:1 for 48 hours. Proliferation of GFP-positive NALM6 cells, as measured by live-cell imaging in an IncuCyte instrument (Sartorius), is shown. Engaged 19-28 CoCAR failed to promote cytotoxicity of CAR-T cells in the absence of CAR signaling. [Figure 23]Figure 23 shows the concentrations of effector cytokines IFNγ, IL-2, and TNFα in the supernatant of the shown CAR-T cell lines co-cultured with NALM6 cells at an effector-to-target ratio of 1:1 for 24 hours. The concentrations of the shown cytokines were measured by flow cytometry using the LegendPlex assay kit (BioLegend). Engaged 19-28 CoCARs failed to promote cytokine release by CAR-T cells in the absence of CAR signaling. [Figure 24] Figure 24 shows the expression of exemplary CoCAR and CAR polypeptides with anti-HSV gB specificity on the surface of transduced primary human T cells, as determined by flow cytometry. [Figure 25] Figure 25 shows the expression of CD69 and CD25 activation markers in CAR-T cells co-cultured for 24 hours with unstimulated CAR-T cells or wild-type NALM6 cells in an effector-to-target ratio of 1:1. Engaged 19-44v2 and 19-146v2 CoCARs failed to promote CAR-T cell activation in the absence of CAR engagement. [Figure 26] Figure 26 shows the cytotoxicity of the indicated CAR-T cell lines co-cultured with NALM6 cells at an effector-to-target ratio of 1:1 for 48 hours. Proliferation of GFP-positive NALM6 cells, as measured by live-cell imaging in an IncuCyte instrument (Sartorius), is shown. Engaged 19-44v2 and 19-146v2 CoCARs failed to promote CAR-T cell cytotoxicity in the absence of CAR engagement. [Figure 27]Figure 27 shows the concentrations of effector cytokines (A) IFNγ, (B) IL-2, and (C) TNFα in the supernatant of the shown CAR-T cell lines co-cultured with NALM6 cells at an effector-to-target ratio of 1:1 for 24 hours. The concentrations of the shown cytokines were measured by flow cytometry using the LegendPlex assay kit (BioLegend). Engaged 19-44v2 and 19-146v2 CoCARs failed to promote cytokine release by CAR-T cells in the absence of CAR engagement. [Figure 28] Figure 28 shows the co-expression of exemplary CAR and CoCAR polypeptides targeting different epitopes on the same antigen CD22 on the surface of transduced primary human T cells, as determined by flow cytometry. [Figure 29] Figure 29 shows the concentrations of effector cytokines (A) IFNγ, (B) IL-2, and (C) TNFα in the supernatant of the shown T cell line co-expressing anti-CD22 CAR and CoCAR constructs, co-cultured for 24 hours with a CD22 ultra-low NALM6 cell line at an effector-to-target ratio of 1:1. The concentrations of the shown cytokines were measured by flow cytometry using the LegendPlex assay kit (BioLegend). Co-expression of CoCAR with CD28 or CD146 signaling domains enhanced the release of effector cytokines by CAR-T cells stimulated in low-antigen tumor cells. [Figure 30] Figure 30 shows the cytotoxicity of CAR / CoCAR-T cells targeting different epitopes on CD22 against parental cells and CD22 very low NALM6 cells. Anti-CD22 CAR / CoCAR-T cells with CD28- or CD146-CoCAR showed enhanced death of CD22 very low NALM6 cells compared to conventional anti-CD22 CAR-T cells and CAR / CoCAR-T cells with CD44-CoCAR. [Figure 31]Figure 31 shows the density of HER2 and EGFR molecules on the cell surface of SKOV-3 and MCF-7 cells. Quantification of HER2 and EGFR molecules per cell was performed by flow cytometry using PE-labeled anti-HER2 and anti-EGFR antibodies and Quantibrite® PE quantification beads (BD Biosciences). SKOV-3 cells express high levels of HER2 and EGFR molecules, while MCF-7 cells express significantly lower levels of these tumor-associated antigens. [Figure 32] Figure 32 shows the cell surface expression of exemplary CAR and CoCAR polypeptides targeting tumor-associated antigens HER2 and EGFR on solid tumor cells on transduced primary human T cells, as determined by flow cytometry. [Figure 33] Figure 33 shows the concentration of IL-2 in the supernatant of the shown CAR-T cell lines co-cultured for 24 hours with SKOV-3 or MCF-7 cells at an effector-to-target ratio of 1:1. The concentrations of the shown cytokines were measured by ELISA (ACROBiosystems). Co-expression of CoCAR enhanced IL-2 release by CAR-T cells stimulated with SKOV-3 or MCF-7 solid tumor cells expressing high or low densities of target antigens, respectively. [Figure 34] Figure 34 shows the cytotoxicity of the indicated CAR-T cell lines co-cultured for 72 hours with SKOV-3 (A) or MCF-7 (B) solid tumor cells at an effector-to-target ratio of 1:1. Proliferation of GFP-positive tumor cells, as measured by live-cell imaging in the IncuCyte instrument (Sartorius), is shown. Co-expression of 19-28, 19-44v2, or 19-146v2 CoCAR enhanced the cytotoxicity of CAR-T cells against SKOV-3 or MCF-7 solid tumor cells expressing high or low densities of target antigens, respectively. [Modes for carrying out the invention]

[0054] array Sequence anti-CD22 antigen recognition domain (humanized RFB4 scFv) Sequence ID 1 (IGHV3-23 reader sequence): MEFGLSWLFLVAILKGVQC Sequence ID 2 (VL CDR1):RASQDISNYLN Sequence ID 3 (VL CDR2): YTSILHS Sequence ID 4 (VL CDR3): QQGNTLPWT Sequence ID 5(VL): JPEG2026514993000001.jpg17161 (Bold CDR1-3, underlined framework regions 1, 2, 3, and 4, numbered from N-terminus to C-terminus (left to right)) Sequence ID 6 (VH CDR1): GFAFSIYDMS Sequence ID 7 (VH CDR2): YISSGGGTTYYPDTVKG Sequence ID 8 (VH CDR3): HSGYGSSYGVLFAY Sequence ID 9(VH): JPEG2026514993000002.jpg22161 (Bold CDR1-3, underlined framework areas 1, 2, 3, and 4, numbered from N-terminus to C-terminus (left to right)) Sequence ID 10 (Linker L1): GGGGSGGGGSGGGGS Sequence ID 11 (LFR1): DIQMTQSPSSLSASVGDRVTITC Sequence ID 12 (LFR2):WLQQKPGKAPKLLIY Sequence ID 13 (LFR3): GVPSRFSGSGSGTEFTLTISSLQPEDFATYYC Sequence ID 14(LFR4):FGQGTKLEIKR Sequence ID 15(HFR1):EVQLVESGGGLVQPGGSLRLSCAAS Sequence ID 16(HFR2):WVRQVPGKGLEWVS Sequence ID 17(HFR3):RFTISRDNSRNTLDLQMNSLRVEDTAVYYCA Sequence ID 18(HFR4):WGQGTLVTVSS

[0055] Sequence anti-CD22 antigen recognition domain (mouse LL2 scFv) Sequence ID 19 (Mouse IgH Reader Sequence): MERHWIFLFLLSVTAGVHS Sequence ID 20 (VL CDR1): QSVLYSANHKNY Sequence ID 21 (VL CDR2): WAS Sequence ID 22 (VL CDR3): HQYLSSWT Sequence ID 23(VL): JPEG2026514993000003.jpg17161 (Bold CDR1-3, underlined framework regions 1, 2, 3, and 4, numbered from N-terminus to C-terminus (left to right)) Sequence ID 24 (VH CDR1): GYTFTSYW Sequence ID 25 (VH CDR2): INPRNDYT Sequence ID 26 (VH CDR3): ARRDITTFY Sequence ID 27(VH): JPEG2026514993000004.jpg17161 (Bold CDR1-3, underlined framework regions 1, 2, 3, and 4, numbered from N-terminus to C-terminus (left to right)) Sequence ID 28 (Linker L1): GGGGSGGGGSGGGGS Sequence ID 29(LFR1):DIQLTQSPSSLAVSAGENVTMSCKSS Sequence ID 30(LFR2):LAWYQQKPGQSPKLLIY Sequence ID 31 (LFR3): TRESGVPDRFTGSGSGTDFTLTISRVQVEDLAIYYC Sequence ID 32 (LFR4): FGGGTKLEIK Sequence ID 33(HFR1):QVQLQQSGAELVKPGASVKMSCKAS Sequence ID 34(HFR2):LHWIKQRPGQGLEWIGY Sequence ID 35 (HFR3): EYNQKFKDKATLTADKSSSTAYMQLSSLTSEDSAVYYC Sequence ID 36(HFR4):WGQGTTLTVSS

[0056] Sequence anti-CD19 antigen recognition domain (mouse FMC63 scFv) Sequence ID 37 (CSF2Ra reader sequence): MLLLVTSLLLCELPHPAFLLIP Sequence ID 38 (VL CDR1): QDISKY Sequence ID 39 (VL CDR2): HTS Sequence ID 40 (VL CDR3): QQGNTLPYT Sequence ID 41(VL): JPEG2026514993000005.jpg17161 (Bold CDR1-3, underlined framework areas 1, 2, 3, and 4, numbered from N-terminus to C-terminus (left to right)) Sequence ID 42 (VH CDR1): GVSLPDYG Sequence ID 43 (VH CDR2): IWGSETT Sequence ID 44 (VH CDR3): AKHYYYGGSYAMDY Sequence ID 45(VH): JPEG2026514993000006.jpg22161 (Bold CDR1-3, underlined framework regions 1, 2, 3, and 4, numbered from N-terminus to C-terminus (left to right)) Sequence ID 46 (Whitlow Linker L2): GSTGSSGKPGSGEGSTKG Sequence ID 47(LFR1):DIQMTQTTSSLSASLGDRVTISCRAS Sequence ID 48(LFR2):LNWYQQKPDGTVKLLIY Sequence ID 49 (LFR3): RHLSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFC Sequence ID 50 (LFR4): FGGGTKLEIT Sequence ID 51(HFR1):EVKLQESGPGLVAPSQSLSVTCTVS Sequence ID 52(HFR2):VSWIRQPPRKGLEWLGV Sequence ID 53 (HFR3): YYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYC Sequence ID 54(HFR4):WGQGTSVTVSS

[0057] Sequence anti-CEA antigen recognition domain (human scFv) Sequence ID 55 (Mouse IGHV1-61 Reader Sequence): MGWSCIILFLVATATGVHS Sequence ID 56 (VL CDR1): SSVSYMH Sequence ID 57 (VL CDR2):LLIYSTSNLAS Sequence ID 58 (VL CDR3): HQWSSYP Sequence ID 59(VL): JPEG2026514993000007.jpg17161 (Bold CDR1-3, underlined framework regions 1, 2, 3, and 4; numbered from N-terminus to C-terminus (left to right)) Sequence ID 60 (VH CDR1): FTISSGYSWH Sequence ID 61 (VH CDR2): WIGYIQYSGITNY Sequence ID 62 (VH CDR3): AREDYDYHWYFDV Sequence ID 63(VH): JPEG2026514993000008.jpg22161 (Bold CDR1-3, underlined framework regions 1, 2, 3, and 4; numbered from N-terminus to C-terminus (left to right)) Sequence ID 64 (Whitlow Linker L2): GSTGSSGKPGSGEGSTKG Sequence ID 65(LFR1):DIQMTQSPSSLSASVGDRVTITCSTS Sequence ID 66(LFR2):WYQQKPGKAPR Sequence ID 67(LFR3): GVPSRFSGSGSGTDFTFTISSLQPEDIATYYC Sequence ID 68(LFR4):TFGQGTKVEIK Sequence ID 69(HFR1):QVQLQESGPGLVRPSQTLSLTCTVSG Sequence ID 70 (HFR2): WVRQPPGRGLE Sequence ID 71(HFR3):NPSLKSRVTMLVDTSKNQFSLRLSSVTAADTAVYYC Sequence ID 72(HFR4):WGQGSTVTVSS

[0058] CAR skeleton Sequence ID 73 (CD8a hinge): FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD Sequence ID 74 (CD8a transmembrane): IYIWAPLAGTCGVLLLSLVITLYCNHRN Sequence ID 75 (IgG1 hinge): EPKSPDKTHTCPPCP Sequence ID 76 (IgG1 CH2 domain): APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK Sequence ID 77 (IgG1 CH3 domain): GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence ID 78 (CD28 hinge): IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP Sequence ID 79 (CD28 transmembrane domain): FWVLVVVGGVLACYSLLVTVAFIIFWV Sequence ID 80 (4-1BB domain): RFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL Sequence ID 81 (CD3ζ domain): RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0059] CoCAR skeleton Sequence ID 82 (CD4 hinge): SGQVLLESNIKVLPTWSTPVQP Sequence ID 83 (CD4 transmembrane): MALIVLGGVAGLLLFIGLGIFF Sequence ID 84 (intracellular CD4): CVRCRHRRRQAERMSQIKRLLSEKKTCQCPHRFQKTCSPI Sequence ID 85 (CD8a hinge): TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD Sequence ID 86 (CD8a transmembrane): IYIWAPLAGTCGVLLLSLVIT Sequence ID 87 (intracellular CD8a): LYCNHRNRRRVCKCPRPVVKSGDKPSLSARYV Sequence ID 88(T2A):SGQVLLESNIKVLPTWSTPVQP

[0060] Sequence of an intracellular domain capable of binding to LCK, a motif capable of binding to LCK, or an intracellular domain containing LCK Sequence ID 84 (intracellular CD4): JPEG2026514993000009.jpg11161 Sequence ID 87 (intracellular CD8α): JPEG2026514993000010.jpg9129 Sequence ID 89 (CD4 and CD8α LCK binding motif): CxCP (x = any amino acid; Kim, 2003, Science) Sequence ID 90 (CD4 LCK combined motif): CQCP Sequence ID 91 (CD8a LCK binding motif): CKCP Sequence ID 92 (intracellular CD3ε): JPEG2026514993000011.jpg11161 Sequence ID 93 (CD3ε LCK bonded motif): RKx QRxxY(x = any amino acid, Hartl, 2020, Nat Immunol) Sequence ID 94 (CD3ε LCK binding motif): RKGQRDLY Sequence ID 95 (intracellular CD28 cells): JPEG2026514993000012.jpg11161 Sequence ID 96 (CD28 LCK bonded motif): RSKRSR (Dobbins, 2018, Sci Signal; Holdorf, 1999, J Exp Med) Sequence ID 97 (CD28 LCK bond motif): RRPGPTRK (Dobbins, 2018, Sci Signal; Holdorf, 1999, J Exp Med) Sequence ID 98 (CD28 LCK bond motif): PYAP P(Dobbins, 2018, Sci Signal; Holdorf, 1999, J Exp Med) Sequence ID 99 (intracellular CD44 cells): JPEG2026514993000013.jpg12161 Sequence ID 100 (CD44 LCK bonded motif): NSRRRCGQKKKLVINSGNGAVEDRKPSGLNG(Lefebvre, 2010, Mol Immunol) Sequence ID 101 (intracellular CD146 cells): JPEG2026514993000014.jpg12161 Sequence ID 102 (CD146 LCK binding motif): KKGKLPCRRSGKQEITLPPSRKSEL (Duan, J Clin Invest, 2021) Sequence ID 103 (LCK): MGCGCSSHPEDDWMENIDVCENCHYPIVPLDGKGTLLIRNGSEVRDPLVTYEGSNPPASPLQDNLVIALHSYEPSHDGDLGFEKGEQLRILEQSGEWWKAQSLTTGQEGFIPFNFVAKANSLEPEPW FFKNLSRKDAERQLLAPGNTHGSFLIRESESTAGSFSLSVRDFDQNQGEVVKHYKIRNLDNGGFYISPRITFPGLHELVRHYTNASDGLCTRLSRPCQTQKPQKPWWEDEWEVPRETLKLVERLGAG QFGEVWMGYYNGHTKVAVKSLKQGSMSPDAFLAEANLMKQLQHQRLVRLYAVVTQEPIYIITEYMENGSLVDFLKTPSGIKLTINKLLDMAAQIAEGMAFIEERNYIHRDLRAANILVSDTLSCKIA DFGLARLIEDNEYTAREGAKFPIKWTAPEAINYGTFTIKSDVWSFGILLTEIVTHGRIPYPGMTNPEVIQNLERGYRMVRPDNCPEELYQLMRLCWKERPEDRPTFDYLRSVLEDFFTATEGQYQPQP

[0061] Array CAR construct Sequence ID No. 104 (Humanized RFB4 scFv and CD8a-based anti-CD22 CAR): MEFGLSWLFLVAILKGVQCEVQLVESGGGLVQPGGSLRLSCAASGFAFSIYDMSWVRQVPGKGLEWVSYISSGGGTTYYPDTVKGRFTISRDNSRNTLDLQMNSLRVEDTAVYYCARHSGYGSSYGV LFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWLQQKPGKAPKLLIYYTSILHSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQGNTLPWTF GQGTKLEIKRAAAFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRFSVVKRGRKKLLYIFKQPFMRPVQTTQEED GCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Sequence ID No. 105 (Anti-CD22 CAR with humanized RFB4 scFv and IgG-Fc scaffold): MEFGLSWLFLVAILKGVQCEVQLVESGGGLVQPGGSLRLSCAASGFAFSIYDMSWVRQVPGKGLEWVSYISSGGGTTYYPDTVKGRFTISRDNSRNTLDLQMNSLRVEDTAVYYCARHSGYGSSYGVLFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWLQQKPGKAPKLLIYYTSILHSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKLEIKRAAAEPKSPDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKFWVLVVVGGVLACYSLLVTVAFIIFWVRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0062] CoCAR construct array Sequence number 106 (anti-CD19 CD4-CoCAR): MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSVAAAASGQVLLESNIKVLPTWSTPVQPMALIVLGGVAGLLLFIGLGIFFCVRCRHRRRQAERMSQIKRLLSEKKTCQCPHRFQKTCSPI Sequence number 107 (anti-CD22 CD4-CoCAR): MERHWIFLFLLSVTAGVHSQVQLQQSGAELVKPGASVKMSCKASGYTFTSYWLHWIKQRPGQGLEWIGYINPRNDYTEYNQKFKDKATLTADKSSSTAYMQLSSLTSEDSAVYYCARRDITTFYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLAVSAGENVTMSCKSSQSVLYSANHKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISRVQVEDLAIYYCHQYLSSWTFGGGTKLEIKAAASGQVLLESNIKVLPTWSTPVQPMALIVLGGVAGLLLFIGLGIFFCVRCRHRRRQAERMSQIKRLLSEKKTCQCPHRFQKTCSPI Sequence number 108 (anti-CEA CD4-CoCAR): MGWSCIILFLVATATGVHSDIQMTQSPSSLSASVGDRVTITCSTSSSVSYMHWYQQKPGKAPRLLIYSTSNLASGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCHQWSSYPTFGQGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVRPSQTLSLTCTVSGFTISSGYSWHWVRQPPGRGLEWIGYIQYSGITNYNPSLKSRVTMLVDTSKNQFSLRLSSVTAADTAVYYCAREDYDYHWYFDVWGQGSTVTVSSGAAASGQVLLESNIKVLPTWSTPVQPMALIVLGGVAGLLLFIGLGIFFCVRCRHRRRQAERMSQIKRLLSEKKTCQCPHRFQKTCSPI

[0063] Array Bi-Cistronic CAR-CoCAR Construct Sequence number 109 (anti-CD22-CAR-T2A-anti-CD22-CD4-CoCAR):

[0064] nucleic acid sequence Sequence ID No. 110 (Anti-CD22 CAR with humanized RFB4 scFv and CD8a skeleton) Sequence ID No. 111 (Anti-CD22CAR with humanized RFB4 scFv and IgG-Fc scaffold) Sequence ID 112 (Anti-CD19 CD4-CoCAR): Sequence ID 113 (Anti-CD22 CD4-CoCAR): Sequence ID 114 (Anti-CEA CD4-CoCAR): Layout number 115 (anti-CD22-CAR-T2A-anti-CD22-CD4-CoCAR):

[0065] Detailed description of the invention Although the present invention is described in detail below, it should be understood that the present invention is not limited thereto, as the specific methodologies, protocols, and reagents described herein may vary. It should also be understood that the terms used herein are solely for the purpose of describing specific embodiments and are not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art.

[0066] The elements of the present invention are described below. These elements are listed together with specific embodiments, but it should be understood that they can be combined in any way and in any number to create additional embodiments. The various examples and preferred embodiments described should not be construed as limiting the present invention to only the embodiments expressedly. This description should be understood as supporting and encompassing embodiments that combine the expressedly described embodiments with any number of disclosed and / or preferred elements. Furthermore, all any permutations and combinations of the elements described herein should be considered disclosed by this description unless the context otherwise indicates.

[0067] Preferably, terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", HGW Leuenberger, B. Nagel, and H. Koelbl, Eds., (1995) Helvetica Chimica Acta, CH-4010 Basel, Switzerland.

[0068] The implementation of this invention will, unless otherwise specified, utilize conventional methods of biochemistry, cell biology, immunology, and recombinant DNA techniques described in the literature of the art (e.g., Molecular Cloning: A Laboratory Manual, 2 nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0069] Throughout this specification and the following claims, unless the context requires otherwise, variations of the phrase “comprise,” “comprises,” and “comprising” mean that they include the described members, integers, or processes or groups of members, integers, or processes, but do not exclude any other members, integers, or processes or groups of members, integers, or processes, although in some embodiments such other members, integers, or processes or groups of members, integers, or processes may be excluded; that is, the subject matter is understood to consist of including the described members, integers, or processes or groups of members, integers, or processes. The terms “a,” “an,” and “the” and similar references used in the context of the described invention (particularly in the context of the claims) should be interpreted as encompassing both singular and plural, unless otherwise specified herein or unless the context clearly contradicts this. The enumeration of value ranges in this specification is intended simply as a simple way to refer individually to each distinct value that falls within the range. Unless otherwise specified herein, each individual value is incorporated herein as if it were listed separately.

[0070] The term “approximately” means roughly or nearly, and in the context of a number or range shown herein in one embodiment, it means ±20%, ±10%, ±5%, or ±3% of the number or range enumerated or claimed.

[0071] All methods described herein may be performed in any suitable order unless otherwise specified herein or unless explicitly contradicted by the context. The use of any examples or illustrative language provided herein (e.g., "such as") is intended solely to better illustrate the invention and does not limit the scope of the invention as otherwise claimed. The language herein should not be construed as indicating any unclaimed element essential for carrying out the invention.

[0072] Throughout this specification, several documents are referenced. Each of the documents referenced herein (including all patents, patent applications, scientific publications, manufacturer specifications, instructions, etc.), whether preceding or following, is incorporated herein by reference in its entirety. Nothing in this specification should be construed as an acknowledgment that the present invention has no prior rights to such disclosure arising from prior art.

[0073] The CAR-CoCAR concept according to the present invention is fundamentally different from previously published concepts in that it describes the co-expression of a conventional CAR containing an intracellular CD3ζ T cell activation domain and a co-receptor-based chimeric antigen receptor (CoCAR or "chimeric co-receptor") containing at least one intracellular domain capable of binding to LCK in a given immune effector cell (e.g., T cell or NK cell), for the purpose of modulating the recruitment of kinase LCK to CAR synapses by antigen binding. The regulated recruitment of LCK associated with CoCAR in response to antigen binding to CAR synapses may provide a technical solution to the common problem of antigen sensitivity of BBz-CARs, which often requires thousands of antigen molecules per cell to activate the T cell response, and the common problem of CARs that induce T cell exhaustion as a result of sustained signaling due to high basal CAR-CD3ζ phosphorylation. The exemplary results presented herein demonstrate that co-expression of a CAR that recognizes a first antigen and a CoCAR that recognizes a second antigen expressed on the same target cell (e.g., tumor cell) can enhance cell (e.g., CAR-T cell) activation, resulting in increased T cell effector function and antitumor efficacy (e.g., increased effector cytokine production and cytotoxicity). Furthermore, it was shown that co-expression of CAR and CoCAR can enhance CAR-T cell activity in response to tumor cells expressing extremely low antigen densities, resulting in increased T cell effector function and antitumor efficacy (e.g., increased effector cytokine production and cytotoxicity). Surprisingly, it was also found that immune cells (e.g., T cells) co-expressing CAR and CoCAR showed less T cell exhaustion (e.g., antigen-independent effector cytokine production and reduced expression of inhibitory receptors such as TIM-3 and LAG-3) compared to immune cells expressing conventional CARs.Therefore, co-expression of CAR and CoCAR may lower the T cell activation threshold, improve CAR function in immune cells, enhance proximal antigen-induced CAR signaling and thus improve immune cell function, while potentially reducing antigen-independent CAR-CD3ζ phosphorylation. Accordingly, we hypothesize that immune cells co-expressing CAR and CoCAR will produce a more potent clinical cell product with a reduced likelihood of relapse due to the proliferation of low-antigen tumor cells and / or termination of the CAR-T cell response due to T cell exhaustion.

[0074] To overcome these limitations and expand the scope of application of these therapeutics, there is an urgent unmet medical need for the development of novel CAR constructs with improved antitumor properties.

[0075] One object of the present invention is to provide a CoCAR capable of binding to LCK by improving the properties of CARs known in the art, by enhancing the activation and efficacy of immune cells (e.g., T cells) in response to tumor cells, even when antigens are expressed at extremely low densities. Another object of the present invention is to provide an immune response against affected cells expressing at least one tumor-associated antigen, and to treat diseases involving cells expressing at least one tumor-associated antigen, such as cancer. Preferably, the present invention includes the administration of antigen receptor-modified immune effector cells, such as T cells, targeted against affected cells expressing at least one tumor-associated antigen. Generally, cells expressing antigens on their surface can be targeted by immune effector cells that hold antigen receptors targeted to the antigen.

[0076] Examples of tumor-associated antigens to be used in the present invention are listed in Table 1, but are not limited to these.

[0077] [Table 1-1] [Table 1-2]

[0078] Those skilled in the art will understand that it is possible to design and generate the coreceptor-CAR (CoCAR) of the present invention, which includes a first extracellular antigen recognition domain that recognizes any tumor-associated antigen.

[0079] The term “antigen” refers to a substance containing an epitope to which an immune response should be generated and / or directed. Preferably, in the context of the present invention, an antigen is a molecule that, after processing, preferably, induces an immune response that is specific to the antigen or to a cell that preferably expresses the antigen on its cell surface. The term “antigen” includes proteins and peptides in particular. An antigen is preferably a product corresponding to or derived from a naturally occurring antigen. Such naturally occurring antigens may include, or may be derived from, allergens, viruses, bacteria, fungi, parasites and other infectious agents and pathogens, or an antigen may also be a tumor antigen. According to the present invention, an antigen preferably corresponds to a naturally occurring protein or a portion thereof expressed on the cell surface of a malignant cell.

[0080] In the context of this invention, ultra-low density expressed antigens refer to antigens expressed on the cell surface at densities of less than approximately 1000 molecules / cell, less than approximately 400 molecules / cell, less than approximately 300 molecules / cell, less than approximately 200 molecules / cell, less than approximately 100 molecules / cell, or less than approximately 40 molecules / cell, preferably less than approximately 400 molecules / cell, more preferably less than approximately 40 molecules / cell. Antigen density can be measured using state-of-the-art, well-known methods, such as staining cells with a fluorescent antibody specific to their antigen and obtaining stained cells and quantification beads (e.g., Quantibrite, Becton Dickinson) by flow cytometry.

[0081] The term “cell surface” is used according to its common meaning in the art and therefore includes the area outside the cell that is accessible for binding by proteins and other molecules. Antigens are expressed on the cell surface if they are located on the cell surface and are accessible for binding by antigen-binding molecules, such as antigen receptors or antigen-specific antibodies added to the cell. Antigens expressed on the cell surface may be membrane-bound proteins having an extracellular component recognized by antigen receptors. Antigen receptors are expressed on the cell surface if they are located on the cell surface and are accessible for binding by, for example, an antigen added to the cell (to which the antigen receptor is specific). Antigen receptors expressed on the cell surface may also be membrane-bound proteins having an extracellular component that recognizes antigens.

[0082] In the context of the present invention, the term "extracellular component" or "ectodomain" refers to a portion of a molecule, such as a protein, that faces the extracellular space of a cell and is preferably accessible from outside the cell by a binding molecule, such as an antibody, located outside the cell. Preferably, this term refers to one or more extracellular loops or domains or fragments thereof.

[0083] According to the present invention, antigens and in particular tumor-associated antigens are expressed in cells when the level of expression exceeds the detection limit and / or when the level of expression is high enough to allow binding by antigen-specific antibodies or antigen receptors added to the cells. Preferably, the antigens and in particular tumor-associated antigens expressed in cells are expressed or exposed, i.e., present on the surface of the cells, and therefore available for binding by antigen-specific molecules such as antibodies or antigen receptors added to the cells.

[0084] The term "tumor-associated antigen," as used herein, refers to an antigen expressed in and / or on the surface of tumor cells. In particular, tumor-associated antigens are specifically expressed in and / or on tumor cells.

[0085] According to the present invention, the term “antigen receptor” includes an engineered receptor that confers arbitrary specificity, for example, the specificity of a monoclonal antibody, to an immune effector cell such as a T cell. In this way, a large number of antigen-specific T cells can be generated for adoptive cell transfer. Thus, the antigen receptor may be present on a T cell, for example, in place of or in addition to the T cell's own T cell receptor. Such a T cell does not necessarily require the processing and presentation of an antigen for the recognition of a target cell, and rather, preferably, can recognize any antigen present on the target cell with specificity. Preferably, the antigen receptor is expressed on the surface of the cell. For the purposes of the present invention, a T cell containing an antigen receptor is included by the term “T cell” as used herein. Specifically, according to the present invention, the term “antigen receptor” includes an artificial receptor that includes a complex of domains that recognize a target structure (e.g., an antigen) on a target cell such as a cancer cell, i.e., bind to it (e.g., by binding of an antigen-binding site or antigen-binding domain to an antigen expressed on the surface of the target cell), and can confer specificity to an immune effector cell such as a T cell that expresses the antigen receptor on its cell surface. Preferably, recognition of the target structure by the antigen receptor leads to the activation of immune effector cells expressing the antigen receptor. According to the present invention, the term "antigen receptor" is preferably synonymous with the terms "chimeric antigen receptor (CAR)", "chimeric T cell receptor", and "artificial T cell receptor".

[0086] The term "coreceptor" is used in the context of this invention according to its traditional definition (Rolf Koenig, Handbook of Cell Signaling 2 nd (Ed., Vol. 3, p. 2679-2688, 2010), refers to a protein that associates with antigen receptors, such as CARs, exerts complex regulatory effects on T cell activation, and is characterized by transient association with CARs that is triggered only by binding to its specific antigen expressed on tumor cells.

[0087] The terms “coreceptor-CAR,” “coreceptor-CAR,” or “CoCAR,” as used herein, describe a chimeric antigen receptor comprising (a) a first extracellular antigen recognition domain and (b) a domain comprising at least one cytosolic domain and / or LCK-binding domain and / or LCK or a variant or fragment thereof. Domains (a) and (b) may be linked by a first hinge domain, e.g., a CD4, CD8, CD28, or IgG-Fc hinge domain and a first transmembrane domain, e.g., a CD4, CD8, CD28, or CD3ζ transmembrane domain. A “coreceptor-CAR,” as used in the context of the present invention, does not include a TCR mobilization domain, as in the case of a TAC disclosed by Helsen et al., (2018). Furthermore, a “coreceptor-CAR,” as used in the context of the present invention, does not include an intracellular cell activation domain, e.g., a CD3ζ (CD3 zeta) T cell activation domain. Therefore, the "co-receptor-CAR" cannot induce immune cell activation (Example 9), has a different structure from the chimeric antigen receptor (CAR), and is also called a "chimeric co-receptor" in the context of the present invention.

[0088] The term "target cell" refers to a cell that is the target of an immune response, such as a cellular immune response. Target cells include any undesirable cells, such as cancer cells. Target cells may preferably be cells that express target antigens present on their cell surface, in particular tumor-associated antigens.

[0089] In the context of this invention, the terms “antibody” or “immunoglobulin” refer to an antigen-binding polypeptide comprising two heavy chains linked to each other by disulfide bonds, each heavy chain linked to a light chain by a disulfide bond. There are two types of light chains: lambda (λ) and kappa (κ). There are five major heavy chain classes (or isotypes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE. Each chain contains distinct sequence domains. The light chain contains two domains or regions: a variable domain (VL) and a constant domain (CL). The heavy chain contains four domains: a variable domain (VH) and three constant domains (CH1, CH2, and CH3, collectively referred to as CH). The variable regions of both the light chain (VL) and the heavy chain (VH) determine the binding recognition and specificity to the antigen. The constant domains of the light chain (CL) and heavy chain (CH) give rise to important biological properties, such as antibody chain association, secretion, transplacental mobility, complement binding, and binding to the Fc receptor (FcR). The Fv fragment is the N-terminal portion of the Fab fragment of an immunoglobulin and consists of a variable region of one light chain and one heavy chain. Antibody specificity lies in the structural complementarity between the antibody-binding site or paratope and the antigenic determinant. The antibody-binding site is mainly composed of residues derived from the hypervariable or complementarity-determining region (CDR). Occasionally, residues derived from the non-hypervariable or FR may affect the entire domain structure and, consequently, the antigen-binding site. The CDR refers to the amino acid sequence that together defines the binding affinity and specificity of the innate Fv region of the innate immunoglobulin-binding site. Examples of antibodies or immunoglobulins include IgM, IgD, IgG, IgA, or IgE. The CDR of an antigen-binding polypeptide can be grafted onto an antibody, a bispecific antibody, or a multispecific antibody. For example, by knowing the amino acid sequences of the antibody, TCR, or antigen-binding polypeptide CDR of the present invention, a person skilled in the art can determine the framework region, for example, the antibody framework region or the TCR framework region. However, if the CDR is not provided, a person skilled in the art can first determine the CDR amino acid sequence based on the IMGT definition of the antibody, and then determine the amino acid sequence of the framework region.

[0090] In the context of this invention, the term "humanized antibody" refers to an antibody that is entirely or partially of non-human origin and has been modified, in particular to replace certain amino acids in the heavy and light chain framework regions, in order to avoid or minimize the immune response in humans. The constant domains of a humanized antibody are primarily human heavy and light chain domains. Methods for humanizing antibody sequences are known in the art; (Almagro & Fransson (2008) Front Biosci. 13: 1619-1633). One commonly used method is CDR transplantation or antibody reshaping, which involves transplanting the CDR sequence of a donor antibody, generally a mouse antibody, onto a framework scaffold of a human antibody of different specificity. Since CDR transplantation can reduce the binding specificity and affinity of the CDR-transplanted non-human antibody, and therefore its biological activity, a reverse mutation may be introduced at a selected position in the CDR-transplanted antibody to preserve the binding specificity and affinity of the parent antibody. While amino acid residues that are part of a CDR are usually left unchanged, in certain cases it may be desirable to modify individual CDR amino acid residues, for example, to remove glycosylation sites, deamidation sites, isomerization sites, or undesirable cysteine ​​residues. N-linked glycosylation occurs by the attachment of oligosaccharide chains to asparagine residues in the tripeptide sequences Asn-X-Ser or Asn-X-Thr (wherein X can be any amino acid except Pro). Removal of N-glycosylation sites can be achieved by mutating either the Asn or Ser / Thr residue to a different residue, particularly by conservative substitution. Deamidation of asparagine and glutamine residues can occur depending on factors such as pH and surface exposure. Asparagine residues are particularly sensitive to deamidation when they are mainly present in the sequence Asn-Gly, and less sensitive in other dipeptide sequences such as Asn-Ser. If such a deamidation site, particularly Asn-Gly, is present in the CDR sequence, it is therefore desirable to remove that site, or, usually, remove one of the residues involved by a conservative substitution.Substitutions in the CDR sequences for removing one of the relevant residues are also encompassed by the present invention.

[0091] According to the present invention, an antigen, particularly a tumor-associated antigen, can be recognized by the antigen receptor of the present invention via any antigen recognition domain or binding domain (also simply referred to as "domain" herein) capable of forming an antigen-binding site, such as the antigen-binding portion of an antibody. Specifically, the present invention utilizes antibody light and heavy chain variable domains that bind to tumor-associated antigens.

[0092] According to the present invention, the antigen receptor has a significant affinity for a predetermined target and can bind (target) to the predetermined target when binding to the predetermined target in a standard assay. "Affinity" or "binding affinity" can be measured by the equilibrium dissociation constant (K D ). Preferably, the term "significant affinity" refers to a dissociation constant (K -5 ) of 10 -6 M or less, 10 -7 M or less, 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, 10 -11 M or less or 10 -12 M or less for binding to a predetermined target.

[0093] The antigen receptor is capable of binding to a predetermined target and, on the other hand, is not (substantially) capable of binding to other targets, that is, has no significant affinity for other targets and does not significantly bind to other targets in a standard assay, and is specific for the predetermined target. Preferably, the antigen receptor is specific for the predetermined target when the affinity and binding to such other targets are not significantly exceeded by the affinity or binding to a protein that is irrelevant to the predetermined target, such as bovine serum albumin (BSA), casein, or human serum albumin (HSA). Preferably, the antigen receptor has a K D for binding to a non-specific target that is at least 10-fold, 100-fold, 10 3 -fold, 10​​4 double, 10 5 double or 10 6 K is twice as low D When binding to a target, it is specific to the predetermined target. For example, the K of the binding of the antigen receptor to a specific target. D However, 10 -7 If M is present, then K is bound to a non-specific target. D at least 10 -6 M, 10 -5 M, 10 -4 M, 10 -3 M, 10 -2 M or 10 -1 It's probably M.

[0094] The binding of the antigen receptor to the target can be empirically determined using any suitable method, such as those disclosed in Berzofsky et al., "Antibody-Antigen Interactions", Fundamental Immunology, Paul, WE, Ed., Raven Press New York, NY (1984), Kuby, Janis Immunology, WH Freeman and Company New York, NY (1992), and the methods described herein. Affinity can be readily determined using conventional techniques, for example, by equilibrium dialysis, by surface plasmon resonance using the BIAcore 2000 instrument using general procedures outlined by the manufacturer, by radioimmunoassay using radiolabeled target antigens, or by other methods known to those skilled in the art. Affinity data can be analyzed, for example, by the method of Scatchard et al., Ann NY Acad. ScL, 51:660 (1949). The measured affinity of a particular antibody-antigen interaction can vary when measured under different conditions, such as salt concentration and pH. Therefore, affinity and other antigen-binding parameters, such as K, can be used to determine the appropriate level of affinity. D ,I C 50 The measurement is preferably performed using standardized solutions of the antibody and antigen, as well as a standardized buffer.

[0095] The terms “immune effector cells” or “immunoreactive cells” in the context of this invention refer to cells that exert effector functions during an immune response. “Immune effector cells” are preferably capable of binding to antigens expressed on the cell surface, such as tumor-associated antigens, and mediating an immune response. For example, such cells secrete cytokines and / or chemokines to kill microorganisms, secrete antibodies, recognize infected or cancerous cells, and, as appropriate, eliminate such cells. For example, immunoreactive cells include T cells (cytotoxic T cells, helper T cells, tumor-infiltrating T cells), B cells, natural killer cells, neutrophils, macrophages, and dendritic cells. Preferably, in the context of this invention, “immune effector cells” refers to T cells, preferably CD4 + and / or CD8 + These are T cells. As used herein, the term “immune effector cells” also includes cells that can mature into immune cells (e.g., T cells, in particular helper T cells or cytolytic T cells) upon appropriate stimulation. Immune effector cells are CD34 + This includes hematopoietic stem cells, immature and mature T cells, and immature and mature B cells. Differentiation of T cell precursors into cytolytic T cells upon exposure to antigens is similar to clonal selection in the immune system.

[0096] Preferably, "immune effector cells" recognize antigens, such as tumor-associated antigens, with some specificity, especially when they are present on the surface of affected cells such as antigen-presenting cells or cancer cells. Preferably, such recognition allows the antigen-recognizing cells to be responsive or reactive. + If it is a T cell, then such responsiveness or reactivity is due to the release of cytokines and / or CD8 +This may include activation of lymphocytes (CTLs) and / or B cells. If the cells are CTLs, such responsiveness or reactivity may include, for example, the elimination of cells via apoptosis or perforin-mediated cytolysis, i.e., cells characterized by antigen expression. CTL responsiveness may include sustained calcium influx, cell division, production of cytokines such as IFN-γ, IL-2, and TNF-α, upregulation of activation markers such as CD25, CD44, and CD69, and specific cytolytic death of antigen-expressing target cells. CTL responsiveness may also be determined using artificial reporters that accurately exhibit CTL responsiveness. Such CTLs that recognize antigens and are responsive or reactive are also referred to herein as “antigen-responsive CTLs.”

[0097] In the context of this invention, the terms "specific binding" or "specificity" refer to the binding of an antigen-binding polypeptide or fragment thereof to a specific binding site on a target, where the target includes both specific and nonspecific binding sites. However, binding of polypeptides to closely related proteins may be unavoidable, and therefore, while the actual binding to the target may be specific, the antigen-binding polypeptide is considered nonspecific in relation to the intended target binding. Antigen-binding polypeptides, such as those contained in the chimeric antigen receptor of this invention, are considered to bind specifically if they bind to their target more strongly or enhancedly than one or more similar antigens.

[0098] "Lymphoid cells" are cells or precursor cells of such cells that, after appropriate modification, for example, after the transfer of a T cell receptor or antigen receptor, are capable of generating an immune response, such as a cellular immune response, and include lymphocytes, preferably T lymphocytes, lymphoblasts, and plasma cells. Lymphoid cells may be immunoreactive cells or immune effector cells as described herein. Preferred lymphoid cells include T cells that can be modified to express a T cell receptor or antigen receptor on their cell surface. For example, lymphoid cells lack endogenous expression of the T cell receptor.

[0099] The terms “T cell” and “T lymphocyte” are used synonymously herein and include helper T cells (CD4+ T cells) and cytotoxic T cells (CTLs, CD8+ T cells), which include cytolytic T cells. T cells belong to a group of white blood cells known as lymphocytes and play a central role in cellular immunity. They can be distinguished from other types of lymphocytes, such as B cells and natural killer cells, by the presence of a special receptor on their cell surface called the T cell receptor (TCR). The thymus is the main organ responsible for the maturation of T cells. Several different subsets of T cells have been discovered, each with distinct functions. Helper T cells assist other white blood cells in immunological processes, including the maturation of B cells into plasma cells and the activation of cytotoxic T cells and macrophages, among other functions. These cells are also known as CD4+ T cells because they express the CD4 protein on their surface.

[0100] Helper T cells are activated when peptide antigens are presented by MHC class II molecules expressed on the surface of antigen-presenting cells (APCs). Once activated, they rapidly divide and secrete small proteins called cytokines that modulate or assist the active immune response. Cytotoxic T cells destroy virus-infected cells and tumor cells and are also involved in transplant rejection. These cells are also known as CD8+ T cells because they express the CD8 glycoprotein on their surface. These cells recognize their targets by binding to antigens associated with MHC class I, which are present on the surface of almost all cells in the body.

[0101] Most T cells possess a T cell receptor (TCR), which exists as a complex of several proteins. The actual T cell receptor is generated from independent T cell receptor alpha and beta (TCRα and TCRβ) genes and consists of two distinct peptide chains called α- and β-TCR chains. γδ T cells (gamma delta T cells) represent a small subset of T cells that possess a distinct T cell receptor (TCR) on their surface. However, in γδ T cells, the TCR consists of one γ chain and one δ chain. This group of T cells is considerably smaller than αβ T cells (2% of total T cells). Each chain of the T cell receptor consists of two extracellular domains: a variable (V) region and a constant (C) region. The constant region is located proximal to the cell membrane, followed by a transmembrane region and a short cytoplasmic end, while the variable region binds to the peptide / MHC complex. For the purposes of the present invention, the term “the constant region or portion thereof of the T cell receptor chain” also includes, for example, the transmembrane region and cytoplasmic end that are naturally ligated to the constant region of the T cell receptor chain, when the constant region of the T cell receptor chain is followed (from the N-terminus to the C-terminus) by a transmembrane region and a cytoplasmic end.

[0102] All T cells originate from hematopoietic stem cells in the bone marrow. Hematopoietic progenitor cells derived from hematopoietic stem cells reside in the thymus, where they expand and proliferate through cell division, generating a large population of immature thymocytes. The earliest thymocytes do not express either CD4 or CD8 and are therefore classified as double-negative (CD4-CD8-) cells. As they progress through their development, they mature into double-positive thymocytes (CD4+CD8+) and eventually into single-positive (CD4+CD8- or CD4-CD8+) thymocytes, which are then released from the thymus into peripheral tissues.

[0103] T cells can generally be prepared in vitro or ex vivo using standard procedures. For example, T cells can be isolated from mammals, e.g., a patient's bone marrow, peripheral blood, or fractions of bone marrow or peripheral blood, using commercially available cell isolation systems. Alternatively, T cells may be derived from related or unrelated human, non-human animals, cell lines, or cultures. A sample containing T cells may be, for example, peripheral blood mononuclear cells (PBMCs).

[0104] The T cells to be used in accordance with the present invention may or may not express the endogenous T cell receptor.

[0105] The nucleic acid, such as RNA or DNA, that encodes CoCAR according to the present invention can be introduced into immune effector cells, specifically T cells or other cells with lytic ability, in particular lymphoid cells.

[0106] The antigen receptor of the present invention, when present on immune effector cells, such as T cells, recognizes antigens on the surface of affected cells, such as antigen-presenting cells or cancer cells, and as a result, the immune effector cells are stimulated, primed, and / or proliferate, or exert their effector function as described above.

[0107] The term “antigen-specific T cell” or similar terms particularly refers to T cells that, when an antigen receptor is provided, recognize an antigen targeted by an antigen receptor, such as on the surface of an antigen-presenting cell or a diseased cell such as a cancer cell, and preferably exert the effector function of the T cell as described above. T cells and other lymphoid cells are considered specific to an antigen if the cell kills the target cell expressing the antigen. T cell specificity can be assessed using one of a variety of standard techniques, such as a chromium release assay or a proliferation assay. Alternatively, the synthesis of lymphokines (e.g., interferon-γ) can be measured.

[0108] While clinical administration of CAR-T cells has been well-documented as highly effective, the frequent occurrence of class-specific severe and life-threatening adverse events poses a major challenge in the clinical management of patients treated with these novel immunotherapies. Major class-specific toxicities of these compounds include the development of so-called cytokine release syndrome (CRS), which clinically manifests as high fever, hypoxia, hypotension, tachycardia, tachypnea, chills, and subsequent major organ dysfunction. Neurological toxicity, known as immunoeffector cell-associated neurotoxicity syndrome (ICANS), may occur after CRS sedation and manifests as clinical signs of treatment-induced encephalopathy, including speech disorders, aphasia, somnolence, epileptic seizures, tremors, and cerebral edema. Clinical trials using CD19 CAR-T cells in adult ALL have reported fatal CRS / ICANS-related outcomes in 2% to 16% of cases (reviewed in Sheth and Gauthier, Bone Marrow Transplant 56(3):552-566, 2021). Regarding CD22 CAR-T cell therapy utilizing the m971 antigen recognition domain, 86% of treated patients developed CRS, and delayed-onset hemophagocytic lymphohistiocytosis / macrophage activation syndrome (HLH / MAS), presenting with fever, pancytopenia, and the possibility of subsequent multiple organ failure, has been reported additionally at a high frequency (38%) in patients who developed CRS after CAR-T cell infusion. High ferritin levels may indicate HLH / MAS and have therefore been suggested as a potential biomarker for detecting this condition (La Rosee et al., Blood 133(23):2465-2477, 2019). The development of CRS may correlate with treatment-induced inflammatory cytokine and chemokine release, and host myeloid cells have been suggested to play a significant role in the induction of CRS.Preclinical mouse models of CAR-T cell-induced CRS suggest that the secretion of pro-inflammatory cytokines, such as IL-1 and IL-6, by activated macrophages is a central mechanism in the pathophysiology of CRS (Giavridis et al., Nat Med 24(6):731-738, 2018; Norelli et al., Nat Med 24(6):739-748). As clinical trials have shown a higher rate of toxicity induced by CD28-containing CARs compared to CARs containing the 4-1BB costimulatory domain (Cappell & Kochenderfer, Nat Rev Clin Oncol, 2021), one important factor that may determine the morbidity and severity of CRS, HLH / MAS, and / or ICANS is the design of the CAR. The reason for this difference is still unclear, but broader activation and rapid proliferation of CD28-based CARs appear to correlate with higher toxicity in patients (Salter et al., (2018) Sci Signal. 11(544); Cappell & Kochenderfer, (2021) Nat Rev Clin Oncol. (11) 715-727). Increased signal intensity induced by CD28-based CARs is associated with constitutive association of LCKs with CAR synapses, where high basal CAR-CD3ζ phosphorylation and sustained CAR signaling are promoted (Salter et al., (2018) Sci Signal. 11(544); Sun et al., (2020) Cancer Cell, (2) 216-225). Highly persistent CAR signaling has also been described, for example, as anti-CD22 CARs utilizing the m971 antigen recognition domain, including CARs containing the 4-1BB / CD3ζ signaling domain (Singh et al., (2021) Nat Med. (5) 842-850), and in particular, such CAR-T cells have induced inflammatory toxicity in an unexpectedly large number of treated patients (Shah et al., (2020) J Clin Oncol. (17) 1938-1959).In summary, these findings demonstrate a correlation between the severity of treatment-related toxicity and CARs capable of inducing antigen-independent (sustained) CAR signaling. Sustained CAR signaling may be induced by high basal CAR-CD3ζ phosphorylation resulting from the constitutive association of LCK with CAR synapses, which can induce distinct functional states of immune cells (e.g., T cells) that support the development of toxicities such as CRS, ICANs, and / or HLH / MAS. Therefore, a further object of the present invention is to provide a CoCAR that improves the safety of CARs known in the art by reducing the constitutive association of LCK with CAR synapses and thereby preventing basal CAR-CD3ζ phosphorylation, sustained CAR signaling, and antigen-independent immune cell activation without impairing the activation and efficacy of immune cells (e.g., T cells) in response to tumor cells (Figure 2A).

[0109] The present invention provides, in particular, a polypeptide chain of such a CoCAR that determines the coreceptor-CAR (CoCAR) and the individual domains of the CoCAR, such as variable light and heavy domains, hinge domains or regions, transmembrane domains and one or more intracellular signaling domains of the antigen-recognition domain, including the CDR and framework region of the antigen-binding domain.

[0110] The CoCAR of the present invention comprises a first extracellular antigen recognition domain. As used herein, the “extracellular antigen recognition domain” is capable of specifically recognizing an antigen. The extracellular antigen recognition domain may comprise a heavy chain variable domain (VH) and a light chain variable domain (VL) having an antibody complementarity-determining region (CDR) and a framework region (FR) or fragment thereof, as defined herein. Preferably, the antigen recognition domain is as described herein. Preferably, the antigen recognition domain comprises VH and VL as defined herein as antigen-binding polypeptides.

[0111] The extracellular antigen recognition domain may include a light chain variable domain and a heavy chain variable domain, which form an antigen-binding polypeptide or antigen-binding site that specifically binds to tumor-associated tumor antigens as described herein.

[0112] The CAR to be combined with the CoCAR of the present invention comprises a second extracellular antigen recognition domain. The extracellular antigen recognition domain of the CAR may comprise a heavy chain variable domain (VH) and a light chain variable domain (VL), and have an antibody complementarity determining region (CDR) and a framework region (FR) or fragment thereof as defined herein. Preferably, the antigen recognition domain of the CAR is as described herein. Preferably, the antigen recognition domain of the CAR comprises VH and VL as defined herein as antigen-binding polypeptides.

[0113] The first and second antigen-recognition domains may be any antigen-recognition domains independently, as described herein. The first and second antigen-recognition domains may (i) recognize different epitopes located within the same antigen expressed on the surface of a target cell, or (ii) recognize the same antigen expressed on the surface of a target cell. More preferably, the first and second antigen-recognition domains may (i) recognize different epitopes located within the same antigen expressed on the surface of a target cell, or (ii) recognize the same antigen expressed on the surface of a target cell different antigens It can recognize tumor-specific or tumor-associated antigens. Therefore, both the first and second antigen-recognition domains recognize tumor-specific or tumor-associated antigens.

[0114] In the context of this invention, the terms "antigen-binding polypeptide" or "antigen-binding domain" refer to a polypeptide containing a paratope (or "antigen-binding site") that specifically binds to an antigen. Examples of antigen-binding polypeptides include, among others, antibodies or fragments thereof, or single-chain antibodies.

[0115] In the context of this invention, the term "variable domain" refers to a region of immunoglobulin, which is defined based on sequence homology, as is known to those skilled in the art. Typically, two variable domains form an antigen-binding site. Non-exclusive examples of such domains include the variable light chain domain (VL) contained in the antibody light chain, the variable heavy chain domain (VH) contained in the antibody heavy chain, the alpha variable domain (Vα) contained in the alpha chain of a T cell receptor (TCR) molecule, or the beta variable domain (Vβ) contained in the beta chain of a TCR.

[0116] In the context of this invention, the term "complementarity-determining region" (CDR) refers to discontinuous antigen-binding sites found within the variable domains of immunoglobulins, for example, in VH, VL, Vα, and Vβ. CDRs are from Lefranc et al. (2003) Developmental and Comparative Immunology 27:55; Kabat et al., J. Biol. Chem. 252:6609-66I6 (1977); Kabat et al., US Dept. of Health and Human Services, "Sequences of proteins of immunological interest", 1991; Chothia et al., J. Mol. Biol. I96:90I-917, 1987; and Contact annotations (for Contact annotations, see MacCallum et al., J. Mol. Biol. 262:732-745 (1996)); for AbM annotations, see Abhinandan and Martin, Mol. Immunol. (2008), 45(14):3832-9; IMGT (Lefranc MP. Unique database numbering system for immunogenetic analysis; Immunol As described in Today (1997) 18:509), the definitions, when compared to one another, include duplication or subsets of amino acid residues. Nevertheless, the application of either definition is intended to refer to the CDR of an antibody or a grafted antibody or its variants or fragments, and is intended to be within the scope of the terms as defined and used herein.

[0117] In the context of this invention, the terms "HCDR1" or "VH CDR1", "HCDR2" or "VH CDR2", "HCDR3" or "VH CDR3" refer to the first, second, and third CDRs in the heavy chain variable domain of an antigen-binding polypeptide, such as an antibody or a functional fragment thereof. As used herein, the terms "LCDR1" or "VL CDR1", "LCDR2" or "VL CDR2", "LCDR3" or "VL CDR3" refer to the first, second, and third CDRs in the light chain variable domain of an antigen-binding polypeptide, such as an antibody or a fragment thereof, respectively. As used herein, the terms "CDR1", "CDR2", and "CDR3" refer to the first, second, and third CDRs in the variable region of any chain of an antigen-binding polypeptide, such as an antibody or a functional fragment thereof, respectively.

[0118] The locations of the CDR and framework regions as defined herein are assigned according to Kabat or Chothia, and in particular according to Kabat numbering. Accordingly, according to a particularly preferred embodiment of the present invention, the numbering of the light chain and heavy chain variable regions described herein follows Kabat.

[0119] According to the present invention, the antigen recognition domain may be an anti-CD22 antigen recognition domain. The anti-CD22 antigen recognition domain may include LCDR1 of SEQ ID NO: 2, LCDR2 of SEQ ID NO: 3, and LCDR of SEQ ID NO: 4. The anti-CD22 antigen recognition domain may also include HCDR1 of SEQ ID NO: 6, HCDR2 of SEQ ID NO: 7, and HCDR3 of SEQ ID NO: 8. The anti-CD22 antigen recognition domain may include the IGHV3-23 reader sequence of SEQ ID NO: 1.

[0120] The VL domain anti-CD22 antigen recognition domain may include LFR1 of SEQ ID NO: 11, LFR2 of SEQ ID NO: 12, LFR3 of SEQ ID NO: 13, and LFR4 of SEQ ID NO: 14.

[0121] The VH domain anti-CD22 antigen recognition domain may include HFR1 of SEQ ID NO: 15, HFR2 of SEQ ID NO: 16, HFR3 of SEQ ID NO: 17, and HFR4 of SEQ ID NO: 18.

[0122] The anti-CD22 antigen recognition domain may include VL of sequence number 5.

[0123] The anti-CD22 antigen recognition domain may include VH of sequence number 9.

[0124] The anti-CD22 antigen recognition domain may include a linker sequence located between the VH and VL sequences, for example, between sequence numbers 5 and 9, e.g., sequence number 10.

[0125] According to the present invention, the antigen recognition domain may be an anti-CD22 antigen recognition domain (mouse LL2 scFv). The anti-CD22 antigen recognition domain may include LCDR1 of SEQ ID NO: 20, LCDR2 of SEQ ID NO: 21, and LCDR3 of SEQ ID NO: 22. The anti-CD22 antigen recognition domain may also include HCDR1 of SEQ ID NO: 24, HCDR2 of SEQ ID NO: 25, and HCDR3 of SEQ ID NO: 26. The anti-CD22 antigen recognition domain may include the mouse IgH reader sequence of SEQ ID NO: 19.

[0126] The VL domain anti-CD22 antigen recognition domain may include LFR1 of SEQ ID NO: 29, LFR2 of SEQ ID NO: 30, LFR3 of SEQ ID NO: 31, and LFR4 of SEQ ID NO: 32.

[0127] The VH domain anti-CD22 antigen recognition domain may include HFR1 of SEQ ID NO: 33, HFR2 of SEQ ID NO: 34, HFR3 of SEQ ID NO: 35, and HFR4 of SEQ ID NO: 36.

[0128] The anti-CD22 antigen recognition domain may include VL of sequence number 23.

[0129] The anti-CD22 antigen recognition domain may contain VH of sequence number 27.

[0130] The anti-CD22 antigen recognition domain may include a linker sequence located between the VH and VL sequences, for example, between sequence numbers 23 and 27, e.g., sequence number 28.

[0131] According to the present invention, the antigen recognition domain may be an anti-CD22 antigen recognition domain containing human m971 scFv. The anti-CD22 antigen recognition domain may include LCDR1 of SEQ ID NO: 117, LCDR2 of SEQ ID NO: 118, and LCDR3 of SEQ ID NO: 119. The anti-CD22 antigen recognition domain may also include HCDR1 of SEQ ID NO: 121, HCDR2 of SEQ ID NO: 122, and HCDR3 of SEQ ID NO: 123. The anti-CD22 antigen recognition domain may include the CSFR2a reader sequence of SEQ ID NO: 116.

[0132] The VL domain anti-CD22 antigen recognition domain may include LFR1 of SEQ ID NO: 126, LFR2 of SEQ ID NO: 127, LFR3 of SEQ ID NO: 128, and LFR4 of SEQ ID NO: 129.

[0133] The VH domain anti-CD22 antigen recognition domain may include HFR1 of SEQ ID NO: 130, HFR2 of SEQ ID NO: 131, HFR3 of SEQ ID NO: 132, and HFR4 of SEQ ID NO: 133.

[0134] The anti-CD22 antigen recognition domain may include the VL of sequence number 120.

[0135] The anti-CD22 antigen recognition domain may contain VH of sequence number 124.

[0136] The anti-CD22 antigen recognition domain may include a linker sequence located between the VH and VL sequences, for example, between sequence numbers 120 and 124, e.g., sequence number 125.

[0137] In one embodiment, the antigen recognition domain may be an anti-CD22 antigen recognition domain containing a humanized SGIII scFv.

[0138] According to the present invention, the antigen recognition domain may be an anti-CD19 antigen recognition domain. The anti-CD19 antigen recognition domain may include LCDR1 of SEQ ID NO: 38, LCDR2 of SEQ ID NO: 39, and LCDR3 of SEQ ID NO: 40. The anti-CD22 antigen recognition domain may also include HCDR1 of SEQ ID NO: 42, HCDR2 of SEQ ID NO: 43, and HCDR of SEQ ID NO: 44. The anti-CD19 antigen recognition domain may include the IGHV3-23 reader sequence of SEQ ID NO: 37.

[0139] The VL domain anti-CD19 antigen recognition domain may include LFR1 of SEQ ID NO: 47, LFR2 of SEQ ID NO: 48, LFR3 of SEQ ID NO: 49, and LFR4 of SEQ ID NO: 50.

[0140] The VH domain anti-CD19 antigen recognition domain may include HFR1 of SEQ ID NO: 51, HFR2 of SEQ ID NO: 52, HFR3 of SEQ ID NO: 53, and HFR4 of SEQ ID NO: 54.

[0141] The anti-CD19 antigen recognition domain may include the VL of sequence number 41.

[0142] The anti-CD19 antigen recognition domain may contain VH of sequence number 45.

[0143] The anti-CD19 antigen recognition domain may include a linker sequence located between the VH and VL sequences, for example, between sequence numbers 41 and 45, e.g., sequence number 46.

[0144] According to the present invention, the antigen recognition domain may be an anti-CEA antigen recognition domain. The anti-CEA antigen recognition domain may include LCDR1 of SEQ ID NO: 56, LCDR2 of SEQ ID NO: 57, and LCDR of SEQ ID NO: 58. The anti-CEA antigen recognition domain may also include HCDR1 of SEQ ID NO: 60, HCDR2 of SEQ ID NO: 61, and HCDR3 of SEQ ID NO: 62. The anti-CEA antigen recognition domain may include the leader sequence of SEQ ID NO: 55.

[0145] The VL domain anti-CEA antigen recognition domain may include LFR1 of SEQ ID NO: 65, LFR2 of SEQ ID NO: 66, LFR3 of SEQ ID NO: 67, and LFR4 of SEQ ID NO: 68.

[0146] The VH domain anti-CEA antigen recognition domain may include HFR1 of SEQ ID NO: 69, HFR2 of SEQ ID NO: 70, HFR3 of SEQ ID NO: 71, and HFR4 of SEQ ID NO: 72.

[0147] The anti-CEA antigen recognition domain may contain the VL of sequence number 59.

[0148] The anti-CEA antigen recognition domain may contain VH of sequence number 63.

[0149] The anti-CEA antigen recognition domain may include a linker sequence located between the VH and VL sequences, for example, between sequence numbers 59 and 63, e.g., sequence number 64.

[0150] According to the present invention, the antigen recognition domain may be an anti-EGFR antigen recognition domain. The anti-EGFR antigen recognition domain may include scFv derived from the anti-EGFR antibody cetuximab. The anti-EGFR antigen recognition domain may also include a fully human scFv derived from a phage display library selected for recombinant human EGFR protein. In particular, it will be understood by those skilled in the art that hypervariable and variable regions in the CDR sequence can be modified without losing their ability to bind to the target. For example, the CDR region may be identical or highly homologous to the CDR disclosed herein. "Highly homologous" means that 1 to 3, preferably 1 to 2 or 1 substitution may be made in the CDR.

[0151] In the context of this invention, the term “framework region” (FR) refers to all amino acid residues outside the CDR region within the variable domain of an antigen-binding polypeptide, such as an antibody or a fragment thereof. The framework region is generally a discontinuous amino acid sequence of about 100 to 120 amino acids in length and is intended to refer only to the amino acids outside the CDR. As used herein, the term “framework region” is intended to mean each domain of the framework separated by the CDR. “FR1-FR4” refers to framework region 1, which is the first N-terminal amino acid sequence of the variable domain, followed by FR2, FR3, and FR4, which are dispersed by CDR1, 2, and 3, respectively.

[0152] As used herein, the framework regions of the VL are referred to as LFR1, LFR2, LFR3, and LFR4, respectively. As used herein, the framework regions of the VL are referred to as HFR1, HFR2, HFR3, and HFR4, respectively.

[0153] According to the present invention, the antigen recognition domain of the present invention comprises a variant or fragment of the framework region as defined herein.

[0154] According to preferred embodiments of the present invention, the antigen recognition domain preferably comprises one or more light chain and heavy chain frameworks as defined herein. Preferably, the extracellular antigen recognition domain comprises at least one of light chain framework region 1 (LFR1), light chain framework region 2 (LFR2), light chain framework region 3 (LFR3), and light chain framework region 4 (LFR4), having at least 90% amino acid sequence identity with respect to the amino acid sequences of each framework region of the light chain variable region according to SEQ ID NOs. 5, 23, 41, or 59. Furthermore, or alternatively, the extracellular antigen recognition domain comprises at least heavy chain framework region 2 (HFR2), heavy chain framework region 3 (HFR3), and heavy chain framework region 4 (HFR4), having at least 90% amino acid sequence identity with respect to the amino acid sequences of each framework region of the heavy chain variable region according to SEQ ID NOs. 9, 27, 45, or 63.

[0155] According to a preferred embodiment, LFR1 comprises a sequence such as that shown in SEQ ID NO: 11 or a sequence that is at least 90% identical thereto. Preferably, LFR1 comprises Q at position 3, S at position 7, P at position 8, V at position 15, and / or T at position 22 of SEQ ID NO: 11.

[0156] According to a preferred embodiment, LFR2 comprises a sequence such as that shown in SEQ ID NO: 12 or a sequence that is at least 90% identical thereto. Preferably, LFR2 comprises L at position 2, P at position 6, G at position 7, K at position 8, A at position 9, P at position 10, L at position 12, and / or Y at position 15 of SEQ ID NO: 12.

[0157] According to a preferred embodiment, LFR3 comprises a sequence such as that shown in SEQ ID NO: 13 or a sequence that is at least 90% identical thereto. Preferably, LFR3 comprises E at position 14, F at position 15, T at position 16, S at position 21, Q at position 23, P at position 24 and / or Y at position 31 of SEQ ID NO: 13.

[0158] According to a preferred embodiment, LFR4 comprises a sequence such as that shown in SEQ ID NO: 14 or a sequence that is at least 90% identical thereto. Preferably, LFR4 contains Q at position 3 of SEQ ID NO: 14.

[0159] According to a preferred embodiment, HFR1 comprises a sequence such as that shown in SEQ ID NO: 15 or a sequence that is at least 90% identical thereto. Preferably, HFR1 comprises E at position 6, Q at position 13, and / or R at position 19 of SEQ ID NO: 15.

[0160] According to a preferred embodiment, HFR2 comprises a sequence such as that shown in SEQ ID NO: 16 or a sequence that is at least 90% identical thereto. Preferably, HFR2 comprises V at position 5, G at position 7, G at position 9, and / or S at position 14 of SEQ ID NO: 16.

[0161] According to a preferred embodiment, HFR3 comprises a sequence such as that shown in SEQ ID NO: 17 or a sequence that is at least 90% identical thereto. Preferably, HFR3 comprises S at position 9, R at position 10, D at position 14, N at position 18, R at position 21, V at position 22 and / or V at position 27 of SEQ ID NO: 17.

[0162] According to a preferred embodiment, HFR4 comprises a sequence such as that shown in SEQ ID NO: 18 or a sequence that is at least 90% identical thereto. Preferably, HFR4 comprises V at position 9 and / or S at position 11 of SEQ ID NO: 18.

[0163] It should be understood that the sequences of LFR1-4 (sequences 11-14) and HFR1-4 (sequences 15-18) are contained within the larger VL sequence (sequence 5) and the larger VH sequence (sequence 9), respectively. Therefore, the specific amino acids identified above for each individual LFR and HFR sequence are similarly contained within the larger VL and VH sequences, respectively, and those skilled in the art will understand that this involves different numbering when defining their positions within the larger sequences. For example, the sequence of LFR2 begins at position W35 in sequence 5 and ends at position Y49. Thus, position 2 in LFR2 of sequence 12 corresponds to position 36 in sequence 5, position 3 in LFR2 corresponds to position 37 in sequence 5, and so on.

[0164] According to a preferred embodiment, LFR1 comprises a sequence such as that shown in Sequence ID No. 29 or a sequence that is at least 90% identical thereto.

[0165] According to a preferred embodiment, LFR2 comprises a sequence such as that shown in Sequence ID No. 30 or a sequence that is at least 90% identical thereto.

[0166] According to a preferred embodiment, LFR3 comprises a sequence such as that shown in Sequence ID No. 31 or a sequence that is at least 90% identical thereto.

[0167] According to a preferred embodiment, LFR4 comprises a sequence such as that shown in Sequence ID No. 32 or a sequence that is at least 90% identical thereto.

[0168] According to a preferred embodiment, HFR1 comprises a sequence such as that shown in Sequence ID No. 33 or a sequence that is at least 90% identical thereto.

[0169] According to a preferred embodiment, HFR2 comprises a sequence such as that shown in Sequence ID No. 34 or a sequence that is at least 90% identical thereto.

[0170] According to a preferred embodiment, HFR3 comprises a sequence such as that shown in Sequence ID No. 35 or a sequence that is at least 90% identical thereto.

[0171] According to a preferred embodiment, HFR4 comprises a sequence such as that shown in Sequence ID No. 36 or a sequence that is at least 90% identical thereto.

[0172] It should be understood that the sequences of LFR1-4 (sequences 29-32) and HFR1-4 (sequences 33-36) are contained within the larger VL sequence (sequence 23) and the larger VH sequence (sequence 29), respectively. Therefore, the specific amino acids identified above for each individual LFR and HFR sequence are similarly contained within the larger VL and VH sequences, respectively, and those skilled in the art will understand that this involves different numbering when defining their positions within the larger sequences. For example, the sequence of LFR2 begins at position L41 in sequence 23 and ends at position Y57. Thus, position 2 in LFR2 of sequence 23 corresponds to position 42 in sequence 23, position 3 in LFR2 corresponds to position 43 in sequence 23, and so on.

[0173] According to a preferred embodiment, LFR1 comprises a sequence such as that shown in Sequence ID No. 47 or a sequence that is at least 90% identical thereto.

[0174] According to a preferred embodiment, LFR2 comprises a sequence such as that shown in Sequence ID No. 48 or a sequence that is at least 90% identical thereto.

[0175] According to a preferred embodiment, LFR3 comprises a sequence such as that shown in Sequence ID No. 49 or a sequence that is at least 90% identical thereto.

[0176] According to a preferred embodiment, LFR4 comprises a sequence such as that shown in Sequence ID No. 50 or a sequence that is at least 90% identical thereto.

[0177] According to a preferred embodiment, HFR1 comprises a sequence such as that shown in SEQ ID NO: 51 or a sequence that is at least 90% identical thereto.

[0178] According to a preferred embodiment, HFR2 comprises a sequence such as that shown in Sequence ID No. 52 or a sequence that is at least 90% identical thereto.

[0179] According to a preferred embodiment, HFR3 comprises a sequence such as that shown in Sequence ID No. 53 or a sequence that is at least 90% identical thereto.

[0180] According to a preferred embodiment, HFR4 comprises a sequence such as that shown in Sequence ID No. 54 or a sequence that is at least 90% identical thereto.

[0181] It should be understood that the sequences of LFR1-4 (sequences 47-50) and HFR1-4 (sequences 51-54) are contained within the larger VL sequence (sequence 41) and the larger VH sequence (sequence 45), respectively. Therefore, the specific amino acids identified above for each individual LFR and HFR sequence are similarly contained within the larger VL and VH sequences, respectively, and those skilled in the art will understand that this involves different numbering when defining their positions within the larger sequences. For example, the sequence of LFR2 begins at position L33 in sequence 41 and ends at position Y49. Thus, position 2 in LFR2 of sequence 41 corresponds to position 34 in sequence 41, position 3 in LFR2 corresponds to position 35 in sequence 41, and so on.

[0182] According to a preferred embodiment, LFR1 comprises a sequence such as that shown in Sequence ID No. 65 or a sequence that is at least 90% identical thereto.

[0183] According to a preferred embodiment, LFR2 comprises a sequence such as that shown in Sequence ID No. 66 or a sequence that is at least 90% identical thereto.

[0184] According to a preferred embodiment, LFR3 comprises a sequence as shown in SEQ ID NO: 67 or a sequence that is at least 90% identical thereto.

[0185] According to a preferred embodiment, LFR4 comprises a sequence as shown in SEQ ID NO: 68 or a sequence that is at least 90% identical thereto.

[0186] According to a preferred embodiment, HFR1 comprises a sequence as shown in SEQ ID NO: 69 or a sequence that is at least 90% identical thereto.

[0187] According to a preferred embodiment, HFR2 comprises a sequence as shown in SEQ ID NO: 70 or a sequence that is at least 90% identical thereto.

[0188] According to a preferred embodiment, HFR3 comprises a sequence as shown in SEQ ID NO: 71 or a sequence that is at least 90% identical thereto.

[0189] According to a preferred embodiment, HFR4 comprises a sequence as shown in SEQ ID NO: 72 or a sequence that is at least 90% identical thereto.

[0190] It should be understood that the sequences of LFR1-4 (SEQ ID NOs: 65-68) and the sequences of HFR1-4 (SEQ ID NOs: 69-72) are respectively included in the larger sequences of VL (SEQ ID NO: 59) and the larger sequences of VH (SEQ ID NO: 63). Thus, the specific amino acids identified above for the individual LFR and HFR sequences are similarly included in the larger sequences of VL and VH, and those skilled in the art understand that different numbering is involved when defining the positions in the larger sequences. For example, the sequence of LFR2 starts at position V34 and ends at position Y50 of SEQ ID NO: 59. Therefore, the 2nd position in LFR2 of SEQ ID NO: 59 corresponds to the 35th position of SEQ ID NO: 59, the 3rd position in LFR2 corresponds to the 36th position of SEQ ID NO: 59, and so on.

[0191] According to a preferred embodiment, LFR1 comprises a sequence as shown in SEQ ID NO: 126 or a sequence that is at least 90% identical thereto.

[0192] According to a preferred embodiment, LFR2 comprises a sequence as shown in SEQ ID NO: 127 or a sequence that is at least 90% identical thereto.

[0193] According to a preferred embodiment, LFR3 comprises a sequence as shown in SEQ ID NO: 128 or a sequence that is at least 90% identical thereto.

[0194] According to a preferred embodiment, LFR4 comprises a sequence as shown in SEQ ID NO: 129 or a sequence that is at least 90% identical thereto.

[0195] According to a preferred embodiment, HFR1 comprises a sequence as shown in SEQ ID NO: 130 or a sequence that is at least 90% identical thereto.

[0196] According to a preferred embodiment, HFR2 comprises a sequence as shown in SEQ ID NO: 131 or a sequence that is at least 90% identical thereto.

[0197] According to a preferred embodiment, HFR3 comprises a sequence as shown in SEQ ID NO: 132 or a sequence that is at least 90% identical thereto.

[0198] According to a preferred embodiment, HFR4 comprises a sequence as shown in SEQ ID NO: 133 or a sequence that is at least 90% identical thereto.

[0199] It should be understood that the sequences of LFR1-4 (SEQ ID NOs: 126-129) and the sequences of HFR1-4 (SEQ ID NOs: 130-133) are respectively included in the larger sequences of VL (SEQ ID NO: 120) and the larger sequences of VH (SEQ ID NO: 124).

[0200] The preferred locations in the framework regions LFR1-4 and HFR1-4, as shown in the above embodiments, are indicated according to their respective sequence numbers, but those skilled in the art will know that these locations, as shown above, differ according to the Kabat numbering of their respective variable heavy and light chain domains.

[0201] Exemplary frameworks and CDR sequences of the variable regions disclosed herein are shown, for example, in SEQ ID NOs. 5, 9, 23, 27, 41, 45, 59 and 63, 120 and 124. Except for HCDR1, the antigen-binding loops grafted onto the human framework regions were defined according to Kabat et al. (1991) Sequences of Proteins of Immunological Interest. (NIH Publication No. 91-3242, Bethesda). Since residues H26-H32 contain the structural loop of HCDR1 (Chothia et al, Nature 342:877-883, 1989), residues H26-H35 were applied as HCDR1 according to the combined Kabat / Chothia definition of HCDR1. Thus, except for HCDR1 to which the combined Chothia / Kabat definition was applied, the CDRs and framework regions were defined according to Kabat. Examples of the positions of LCDR1~LCDR3 and HCDR1~HCDR3 (sequences 5 and 9):

[0202] [Table 1-3]

[0203] As outlined above, the sequences identified herein as sequence numbers 2-4 and 11-14 together form a VL sequence as shown in sequence number 5, and similarly, sequence numbers 6-8 and 15-18 together form a VH sequence as shown in sequence number 9. Therefore, the positions in the sequence numbers differ according to the Kabat numbering of the respective variable heavy and light chain domains.

[0204] According to a preferred embodiment, the present invention provides an extracellular antigen recognition domain comprising a VL region as defined herein, comprising one or more of the following amino acid positions 3Q, 40P, 46L, 49Y, and / or 71Y. Furthermore, or alternatively, the extracellular antigen recognition domain of the present invention comprises a VH region as defined herein, comprising one or more of the following amino acid positions 6E, 40T, 79Y, and 84S.

[0205] In a further preferred embodiment, the extracellular antigen recognition domain of the present invention comprises amino acid 6E in VH and 3Q in VL. In a further preferred embodiment, the extracellular antigen recognition domain of the present invention comprises amino acid 6E in VH and 3Q, 40P, and 49Y in VL. In a further preferred embodiment, the extracellular antigen recognition domain of the present invention comprises amino acid 6E in VH and 3Q, 40P, 46L, and 49Y in VL. In a further preferred embodiment, the extracellular antigen recognition domain of the present invention comprises amino acids 6E and 79Y in VH and 3Q, 40P, 46L, 49Y, and 71Y in VL. In a further preferred embodiment, the extracellular antigen recognition domain of the present invention comprises amino acids 6E, 40T, 79Y, and 84S in VH and 3Q, 40P, 46L, 49Y, and 71Y in VL.

[0206] According to one preferred embodiment, the extracellular antigen recognition domain of the present invention comprises at least LFR2 of SEQ ID NO: 12 or a sequence at least 90% identical thereto, and LFR3 of SEQ ID NO: 13 or a sequence at least 90% identical thereto.

[0207] According to one preferred embodiment, the extracellular antigen recognition domain of the present invention comprises at least the HFR3 of SEQ ID NO: 17 or a sequence that is at least 90% identical thereto.

[0208] According to one preferred embodiment, the extracellular antigen recognition domain comprises at least LFR2 and / or LFR3 as defined herein. According to a further preferred embodiment, the extracellular antigen recognition domain comprises at least HFR2 and / or HFR3 as defined herein. According to a particularly preferred embodiment, the extracellular antigen recognition domain comprises at least LFR2 and LFR3, LFR2 and HFR2, LFR2 and HFR3, LFR3 and HFR2, LFR3 and HFR3, LFR2, LFR3 and HFR2, LFR2, LFR3 and HFR3, HFR2, HFR3 and LFR2, HFR2, HFR3 and LFR3 or LFR2, LFR3, HFR2 and HFR3 as defined herein.

[0209] According to a preferred embodiment, the extracellular antigen recognition domain of the present invention comprises LFR1 of SEQ ID NO: 11, LFR2 of SEQ ID NO: 12, LFR3 of SEQ ID NO: 13, and / or LFR4 of SEQ ID NO: 14.

[0210] According to a further preferred embodiment, the extracellular antigen recognition domain of the present invention comprises HFR1 of SEQ ID NO: 15, HFR2 of SEQ ID NO: 16, HFR3 of SEQ ID NO: 17 and / or LFR4 of SEQ ID NO: 18.

[0211] According to one particularly preferred embodiment, the extracellular antigen recognition domain of the present invention comprises LFR1 of SEQ ID NO: 11, LFR2 of SEQ ID NO: 12, LFR3 of SEQ ID NO: 13 and LFR4 of SEQ ID NO: 14 as well as HFR1 of SEQ ID NO: 15, HFR2 of SEQ ID NO: 16, HFR3 of SEQ ID NO: 17 and LFR4 of SEQ ID NO: 18.

[0212] According to a preferred embodiment, the extracellular antigen recognition domain of the present invention comprises LFR1 of SEQ ID NO: 29, LFR2 of SEQ ID NO: 30, LFR3 of SEQ ID NO: 31 and / or LFR4 of SEQ ID NO: 32.

[0213] In a more preferred embodiment, the extracellular antigen recognition domain of the present invention comprises HFR1 of SEQ ID NO: 33, HFR2 of SEQ ID NO: 34, HFR3 of SEQ ID NO: 35, and / or LFR4 of SEQ ID NO: 36.

[0214] According to one particularly preferred embodiment, the extracellular antigen recognition domain of the present invention includes LFR1 of SEQ ID NO: 29, LFR2 of SEQ ID NO: 30, LFR3 of SEQ ID NO: 31 and LFR4 of SEQ ID NO: 32, as well as HFR1 of SEQ ID NO: 33, HFR2 of SEQ ID NO: 34, HFR3 of SEQ ID NO: 35 and LFR4 of SEQ ID NO: 36.

[0215] According to a preferred embodiment, the extracellular antigen recognition domain of the present invention comprises LFR1 of SEQ ID NO: 47, LFR2 of SEQ ID NO: 48, LFR3 of SEQ ID NO: 49, and / or LFR4 of SEQ ID NO: 50.

[0216] In a more preferred embodiment, the extracellular antigen recognition domain of the present invention comprises HFR1 of SEQ ID NO: 51, HFR2 of SEQ ID NO: 52, HFR3 of SEQ ID NO: 53, and / or LFR4 of SEQ ID NO: 54.

[0217] According to one particularly preferred embodiment, the extracellular antigen recognition domain of the present invention includes LFR1 of SEQ ID NO: 47, LFR2 of SEQ ID NO: 48, LFR3 of SEQ ID NO: 49, and LFR4 of SEQ ID NO: 50, as well as HFR1 of SEQ ID NO: 51, HFR2 of SEQ ID NO: 52, HFR3 of SEQ ID NO: 53, and LFR4 of SEQ ID NO: 54.

[0218] According to a preferred embodiment, the extracellular antigen recognition domain of the present invention comprises LFR1 of SEQ ID NO: 65, LFR2 of SEQ ID NO: 66, LFR3 of SEQ ID NO: 67, and / or LFR4 of SEQ ID NO: 68.

[0219] In a more preferred embodiment, the extracellular antigen recognition domain of the present invention comprises HFR1 of SEQ ID NO: 69, HFR2 of SEQ ID NO: 70, HFR3 of SEQ ID NO: 71, and / or LFR4 of SEQ ID NO: 72.

[0220] According to one particularly preferred embodiment, the extracellular antigen recognition domain of the present invention includes LFR1 of SEQ ID NO: 65, LFR2 of SEQ ID NO: 66, LFR3 of SEQ ID NO: 67, and LFR4 of SEQ ID NO: 68, as well as HFR1 of SEQ ID NO: 69, HFR2 of SEQ ID NO: 70, HFR3 of SEQ ID NO: 71, and LFR4 of SEQ ID NO: 72.

[0221] According to a preferred embodiment, the extracellular antigen recognition domain of the present invention comprises LFR1 of SEQ ID NO: 126, LFR2 of SEQ ID NO: 127, LFR3 of SEQ ID NO: 128, and / or LFR4 of SEQ ID NO: 129.

[0222] In a more preferred embodiment, the extracellular antigen recognition domain of the present invention comprises HFR1 of SEQ ID NO: 130, HFR2 of SEQ ID NO: 131, HFR3 of SEQ ID NO: 132, and / or LFR4 of SEQ ID NO: 133.

[0223] According to one particularly preferred embodiment, the extracellular antigen recognition domain of the present invention includes LFR1 of SEQ ID NO: 126, LFR2 of SEQ ID NO: 127, LFR3 of SEQ ID NO: 128 and LFR4 of SEQ ID NO: 129, as well as HFR1 of SEQ ID NO: 130, HFR2 of SEQ ID NO: 131, HFR3 of SEQ ID NO: 132 and LFR4 of SEQ ID NO: 133.

[0224] According to a preferred embodiment of the present invention, the extracellular antigen recognition domain preferably comprises the amino acid sequences of SEQ ID NO: 5 and SEQ ID NO: 9, or variants or fragments thereof, wherein the variants or fragments maintain the CDR as defined above. Preferably, the variants or fragments have at least 80% sequence identity with SEQ ID NO: 5 and SEQ ID NO: 9, respectively.

[0225] According to a preferred embodiment of the present invention, the extracellular antigen recognition domain preferably comprises the amino acid sequences of SEQ ID NO: 23 and SEQ ID NO: 27 or a variant or fragment thereof, wherein the variant or fragment maintains the CDR as defined above. Preferably, the variant or fragment has at least 80% sequence identity with SEQ ID NO: 23 and SEQ ID NO: 27, respectively.

[0226] According to a preferred embodiment of the present invention, the extracellular antigen recognition domain preferably comprises the amino acid sequences of SEQ ID NO: 41 and SEQ ID NO: 45, or variants or fragments thereof, wherein the variants or fragments maintain the CDR as defined above. Preferably, the variants or fragments have at least 80% sequence identity with SEQ ID NO: 41 and SEQ ID NO: 45, respectively.

[0227] According to preferred embodiments of the present invention, the extracellular antigen recognition domain preferably comprises the amino acid sequences of SEQ ID NO: 59 and SEQ ID NO: 63, or variants or fragments thereof, wherein the variants or fragments maintain the CDR as defined above. Preferably, the variants or fragments have at least 80% sequence identity with SEQ ID NO: 59 and SEQ ID NO: 63, respectively. For the purposes of the present invention, the "variants(s)" of the amino acid sequence include amino acid insertion variants, amino acid addition variants, amino acid deletion variants, amino acid substitution variants, and any combination thereof.

[0228] For the purposes of the present invention, the "fragment (one or more)" of the amino acid sequence preferably includes a partial sequence of the amino acid sequence that exhibits the functional characteristics of the amino acid sequence.

[0229] Amino acid insertion mutants involve the insertion of one, two, or more amino acids into a specific amino acid sequence. In the case of amino acid sequence mutants with insertions, one or more amino acid residues are inserted into specific sites within the amino acid sequence, but random insertions are also possible, accompanied by appropriate screening of the resulting product.

[0230] Amino acid addition mutants include amino- and / or carboxy-terminal fusions of one or more amino acids, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 or more amino acids.

[0231] Amino acid deletion mutants are characterized by the removal of one or more amino acids from a sequence, for example, the removal of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 or more amino acids. The deletion can occur at any position in the protein.

[0232] Amino acid substitution mutants are characterized by at least one residue in the removed sequence and another residue inserted in its place. Modifications and / or replacement of amino acids at non-conserved positions in amino acid sequences between homologous proteins or peptides are preferred. Preferably, amino acid changes in protein mutants are conservative amino acid changes, i.e., substitutions of amino acids with similar or unconserved charges. Conservative amino acid changes involve substitution of one of the families of amino acids associated with its side chain. Naturally occurring amino acids are generally divided into four families: acidic (aspartic acid, glutamic acid), basic (lysine, arginine, histidine), nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and unconserved polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified together as aromatic amino acids.

[0233] In accordance with the present invention, the degree of similarity, preferably identity, between a given amino acid sequence and an amino acid sequence that is a variant of the given amino acid sequence is 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%. The degree of similarity or identity is preferably given for an amino acid region that is 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 total length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is preferably given 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 consecutive amino acids. In a preferred embodiment, the degree of similarity or identity is given for the entire length of the reference amino acid sequence. Alignment for determining sequence similarity, preferably sequence identity, can be performed using tools known in the art, preferably using best sequence alignment, for example, using Align with standard settings, preferably EMBOSS::needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5, or using the NCBI Blast Protein tool "blastp suite".

[0234] The term "sequence similarity" refers to the percentage of amino acids that are identical or represent a conserved amino acid substitution. "Sequence identity" between two amino acid sequences refers to the percentage of amino acids that are identical between the sequences.

[0235] The term "identity percentage" is intended to represent the percentage of amino acid residues that are identical between two sequences being compared. It is obtained after best alignment, and this percentage is purely statistical, as the differences between the two sequences are randomly distributed throughout the entire length. The identity percentage is calculated by determining the number of identical positions between the two sequences being compared, dividing this number by the number of positions being compared, and multiplying the result by 100 to obtain the identity percentage between these two sequences.

[0236] According to the present invention, variants, fragments, parts, portions, or derivatives of amino acid sequences, peptides, or proteins preferably have the functional properties of the amino acid sequence, peptide, or protein from which they are derived, i.e., are functionally equivalent. Those skilled in the art will understand that the sequence variants of peptides, domains, and regions described herein can be used without adversely affecting the present invention, and that the variants have the same or similar activity as the model domain. As described above, such variants will have 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% sequence identity with respect to the amino acid sequence of the domain from which they are derived. Preferably, such variants will have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to the amino acid sequence of the domain from which they originate. The variants, fragments, parts, portions, or derivatives of an amino acid sequence, peptide, or protein are preferably functionally equivalent, for example, immunologically equivalent, to the amino acid sequence, peptide, or protein from which they originate. In one embodiment, the functional property is the ability to bind to a tumor-associated antigen or to transmit a binding signal within an immune effector cell.

[0237] The term "derived from" means, according to the present invention, that a particular entity, in particular a particular sequence, exists in an object, in particular a living organism or molecule, from which it originates. In the case of an amino acid sequence, in particular a particularly specific sequence region, "derived from" means, in particular, that the relevant amino acid sequence originates from the amino acid sequence in which it exists.

[0238] According to one particularly preferred embodiment, the extracellular antigen recognition domain of the present invention comprises VL and VH domains as shown in SEQ ID NO: 5 and SEQ ID NO: 9.

[0239] According to one particularly preferred embodiment, the extracellular antigen recognition domain of the present invention comprises VL and VH domains as shown in SEQ ID NO: 23 and SEQ ID NO: 27.

[0240] According to one particularly preferred embodiment, the extracellular antigen recognition domain of the present invention comprises VL and VH domains as shown in SEQ ID NO: 41 and SEQ ID NO: 45.

[0241] According to one particularly preferred embodiment, the extracellular antigen recognition domain of the present invention comprises VL and VH domains as shown in SEQ ID NO: 59 and SEQ ID NO: 63.

[0242] According to one particularly preferred embodiment, the extracellular antigen recognition domain of the present invention comprises VL and VH domains as shown in SEQ ID NO: 120 and SEQ ID NO: 124.

[0243] In antigen-binding domains or antigen-binding polypeptides as described herein, the heavy chain variable region (VH) and the light chain variable region (VL) are preferably linked by a mobile peptide linker having 10 or more amino acids.

[0244] The terms “linker” or “peptide linker,” when used in the context of the present invention, refer to an amino acid sequence that sterically separates two parts or portions of a complex, for example, two peptides, polypeptides, or proteins. According to one embodiment, such a linker contains or consists of more than 10 amino acid residues, preferably 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids, most preferably 15 amino acid residues. A peptide linker provides mobility between the two parts linked together. Mobility generally increases when the amino acids are small. Therefore, a mobile peptide linker includes an increased content of small amino acids, in particular glycine and / or alanine and / or hydrophilic amino acids, such as serine, threonine, asparagine, and glutamine. A peptide linker inserted between two domains, for example, one or more amino acid residues, is thought to provide sufficient mobility, for example, between domains in a single-chain construct or between variable domains of light-chain and heavy-chain variable domains, enabling the formation of an antigen-binding site through correct folding. In the context of this invention, the linker is abbreviated as L1, L2, L3, L4, etc.

[0245] In a preferred embodiment, the linker L1 connecting the VH and VL of the CoCAR of the present invention comprises glycine and alanine residues. In a more preferred embodiment, the linker L1 connecting the VH and VL of the CoCAR of the present invention comprises glycine (G) and serine (S) residues. More preferably, the linker has a G4S configuration, meaning that in the linker, four glycine residues are followed by one alanine residue, and this configuration can be repeated several times, preferably between three and six times. In other words, the linker is preferably (G4S) xThe structure is (wherein x represents any integer between 3 and 6). Most preferably, linker L1 has the amino acid sequence GGGGSGGGGSGGGGS([G4S]3)(SEQ ID NO: 10) or GTSTGSGKPGSGEGSTKG(SEQ ID NO: 46).

[0246] According to a preferred embodiment, the CoCAR of the present invention includes at least one spacer region as defined herein.

[0247] According to a preferred embodiment, the CoCAR of the present invention comprises the following structural elements in the order shown: (i) preferably an extracellular antigen recognition domain as described herein, comprising a light chain variable domain and a heavy chain variable domain as described herein, separated by a linker as defined herein above; (ii) a first hinge region; (iii) a first transmembrane domain; and (iv) at least one cytosolic domain comprising (a) an LCK-binding and / or (b) an LCK or a variant or fragment thereof.

[0248] The CoCAR of the present invention preferably does not contain a CD3 zetadomain.

[0249] In the CoCAR of the present invention, at least one cytosolic domain preferably does not contain a CD3 zeta domain.

[0250] In one embodiment, the co-receptor-CAR (CoCAR) of the present invention is (i) First extracellular antigen recognition domain, (ii) The first hinge domain, (iii) the first transmembrane domain, and (iv) At least one cytosolic domain that can bind to the LCK Includes, CoCAR does not contain a CD3 zetadomain.

[0251] In one embodiment, the co-receptor-CAR (CoCAR) of the present invention is (i) First extracellular antigen recognition domain, (ii) The first hinge domain derived from CD8α, (iii) The first transmembrane domain derived from CD8α, and (iv) At least one cytosolic domain capable of binding to LCK derived from CD28 Includes, CoCAR does not contain a CD3 zetadomain.

[0252] The coreceptor-CAR having the above domain is also called [first antigen / scFv]-28.

[0253] In one embodiment, the co-receptor-CAR (CoCAR) of the present invention is (i) First extracellular antigen recognition domain, (ii) The first hinge domain derived from CD8α, (iii) The first transmembrane domain derived from CD8α, and (iv) At least one cytosolic domain that can bind to LCK derived from CD44 Includes, CoCAR does not contain a CD3 zetadomain.

[0254] The coreceptor-CAR having the above domain is also called [first antigen / scFv]-44.

[0255] In one embodiment, the co-receptor-CAR (CoCAR) of the present invention is (i) First extracellular antigen recognition domain, (ii) The first hinge domain derived from CD8α, (iii) A first transmembrane domain derived from CD44, and (iv) At least one cytosolic domain that can bind to LCK derived from CD44 Includes, CoCAR does not contain a CD3 zetadomain.

[0256] The coreceptor-CAR having the above domain is also called [first antigen / scFv]-44v2.

[0257] In one embodiment, the co-receptor-CAR (CoCAR) of the present invention is (i) First extracellular antigen recognition domain, (ii) The first hinge domain derived from CD8α, (iii) The first transmembrane domain derived from CD8α, and (iv) At least one cytosolic domain that can bind to LCK derived from CD146 Includes, CoCAR does not contain a CD3 zetadomain.

[0258] The coreceptor-CAR having the above domain is also called [first antigen / scFv]-146.

[0259] In one embodiment, the co-receptor-CAR (CoCAR) of the present invention is (i) First extracellular antigen recognition domain, (ii) The first hinge domain derived from CD8α, (iii) A first transmembrane domain derived from CD146, and (iv) At least one cytosolic domain that can bind to LCK derived from CD146 Includes, CoCAR does not contain a CD3 zetadomain.

[0260] Coreceptor-CARs possessing the above domains are also called CD146v2 CoCAR or [first antigen / scFv]-146v2.

[0261] In further embodiments, the co-receptor-CAR (CoCAR) of the present invention is (i) First extracellular antigen recognition domain, (ii) The first hinge domain derived from CD28, (iii) A first transmembrane domain derived from CD28, and (iv) At least one cytosolic domain derived from CD28 that can bind to LCK Includes, CoCAR does not contain a CD3 zetadomain.

[0262] The “spacer” or “hinge” region preferably imparts mobility to the domain forming the antigen-binding site, enabling strong binding to the desired antigen. As used herein, “hinge,” “hinge region,” or “hinge domain” are used synonymously and are simply referred to as “hinge.”

[0263] The first hinge region is preferably based on a CD4, CD8α, CD28, or IgG-Fc domain, and more preferably on a CD4 domain.

[0264] When used in the context of the present invention, the term "Fc domain" encompasses natural Fc domains and Fc domain variants and sequences as further defined herein. In the context of Fc variants and natural Fc molecules, the term "Fc domain" includes molecules in monomeric or polymeric form, whether digested from a whole antibody or generated by other means.

[0265] The term “natural Fc,” as used herein, refers to a molecule containing a sequence of non-antigen-binding fragments, whether in monomeric or polymeric form, obtained from the digestion of an antibody or produced by other means, which may also contain a hinge region. The original immunoglobulin source of natural Fc is, in particular, of human origin and may be an immunoglobulin, preferably one of IgG1, IgG2, or IgG4, most preferably IgG1. Natural Fc molecules consist of monomeric polypeptides, which can be linked into dimeric or polymeric forms by covalent (i.e., disulfide bonds) and non-covalent associations. The number of intermolecular disulfide bonds between monomeric subunits of a natural Fc molecule ranges from 1 to 4, depending on the class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, and IgGA2). An example of natural Fc is a disulfide-bonded dimer resulting from the papain digestion of IgG. The term "natural Fc" is a general term encompassing monomer, dimer, and polymer forms.

[0266] The present invention further provides a CoCAR comprising (i) preferably a first extracellular antigen recognition domain as described herein, including a light chain variable domain and a heavy chain variable domain, not limited to the VH and VL domains as described herein, separated by a linker as defined herein above; (ii) a first hinge region; (iii) a first transmembrane domain; and (iv) at least one cytosolic domain that is (a) bindable to LCK and / or (b) LCK or a variant or fragment thereof.

[0267] The CoCAR and / or CAR to be combined with the CoCAR of the present invention may include spacers selected independently of the spacers described herein.

[0268] As used herein, the spacer region may include any other sequence suitable for connecting the hinge region and extracellular antigen recognition domain with at least one cytosolic domain, as described herein.

[0269] The CoCAR of the present invention may include a spacer as described herein, which may include a first hinge.

[0270] The CAR to be combined with the CoCAR of the present invention may include a spacer as described herein, which may include a second hinge.

[0271] The first and second hinge regions may be selected independently from the hinge regions described herein. In particular, the first and second hinge regions may be different.

[0272] The spacer region used in the present invention preferably comprises a CH2 domain containing one or more amino acid mutations that reduce or prevent off-target interactions with host myeloid cells. In a particularly preferred embodiment, the CH2 domain comprises the amino acid sequence of SEQ ID NO: 76 or a dimerized variant thereof having preferably 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 a particularly preferred embodiment, the CH2 domain comprises a polypeptide having the amino acid sequence of SEQ ID NO: 76. Furthermore, or alternatively, the spacer region used in the present invention preferably comprises a CH2 domain containing one or more amino acid mutations that prevent glycosylation of the spacer region.

[0273] The spacer region used in the present invention may further include a CH3 domain. The CH3 domain is preferably derived from IgG1. More preferably, the CH3 domain is an IgG1 CH3 domain. In a particularly preferred embodiment, the CH3 domain includes the amino acid sequence of SEQ ID NO: 77 or a dimerized variant thereof having preferably 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 a particularly preferred embodiment, the CH3 domain includes a polypeptide having the amino acid sequence of SEQ ID NO: 77.

[0274] As used herein, the hinge domain may be any hinge domain known in the art. Those skilled in the art will be aware of suitable hinge domains. The hinge preferably comprises sequences selected from SEQ ID NOs: 73, 78, 82, and 85, and their variants and fragments.

[0275] Preferably, the hinge domain is a CD4 hinge domain. In a particularly preferred embodiment, the hinge includes the amino acid sequence of SEQ ID NO: 82 or a variant or fragment thereof having 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.

[0276] Preferably, the hinge domain is a CD8 hinge domain, more preferably a CD8α hinge domain. In a particularly preferred embodiment, the hinge includes the amino acid sequence of SEQ ID NO: 73 or 85, or a variant or fragment thereof having 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 or 85.

[0277] Preferably, the hinge domain is the CD28 hinge domain. In a particularly preferred embodiment, the hinge includes the amino acid sequence of SEQ ID NO: 78 or a variant or fragment thereof having 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.

[0278] Preferably, the hinge is derived from immunoglobulin. More preferably, the hinge is an IgG1-derived hinge. Even more preferably, the hinge is an IgG1 hinge. According to a particularly preferred embodiment, the hinge includes the amino acid sequence of SEQ ID NO: 75 or a variant or fragment thereof having 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: 75. According to a particularly preferred embodiment, the hinge includes a polypeptide having the amino acid sequence of SEQ ID NO: 75.

[0279] The CAR to be combined with the CoCAR of the present invention may include a second hinge as described herein. The second hinge preferably includes SEQ ID NO: 75 or a variant or fragment thereof.

[0280] The CAR to be combined with the CoCAR of the present invention may include a spacer as described herein, which may include a second hinge.

[0281] The second hinge includes a hinge derived from CD28, and more preferably the second hinge includes Sequence ID No. 78 or a variant or fragment thereof. It is also preferable that (1) the second hinge includes a hinge derived from CD28, particularly including Sequence ID No. 78 or a variant or fragment thereof, and (2) the CoCAR of the present invention does not include a hinge region derived from CD28, particularly including a hinge including Sequence ID No. 78 or a variant or fragment thereof. For example, (1) the CAR to be combined with the CoCAR of the present invention includes a second hinge domain derived from CD28 as described herein, particularly including a second hinge including Sequence ID No. 78, and (2) the CoCAR of the present invention includes a first hinge region derived from CD4, particularly including Sequence ID No. 82.

[0282] The second hinge includes a hinge derived from CD8, preferably CD8α, and it is particularly preferable that the second hinge includes SEQ ID NO: 73 or 85 or a variant or fragment thereof. (1) The second hinge includes a hinge derived from CD8, preferably CD8α, and it is particularly preferable that the second hinge includes SEQ ID NO: 73 or 85 or a variant or fragment thereof, and (2) It is also preferable that the CoCAR does not include a hinge derived from CD8, preferably CD8α, and particularly SEQ ID NO: 73 or 85 or a variant or fragment thereof. For example, (1) The CAR to be combined with the CoCAR of the present invention includes a second hinge including a hinge derived from CD8, preferably CD8α, and in particular the second hinge includes SEQ ID NO: 73 or 85 or a variant or fragment thereof, and (2) The CoCAR of the present invention includes a first hinge region derived from CD28, and in particular including SEQ ID NO: 78.

[0283] The CAR to be combined with the CoCAR of the present invention may include a spacer, the spacer comprising CH2 containing SEQ ID NO: 76 or a variant or fragment thereof, and / or CH3 containing SEQ ID NO: 77 or a variant or fragment thereof.

[0284] The CoCAR of the present invention may include a first transmembrane domain as described herein.

[0285] The CAR to be combined with the CoCAR of the present invention may include a second transmembrane domain as described herein.

[0286] The first and second transmembrane domains may be selected independently from the transmembrane domains described herein. In particular, the first and second transmembrane domains may be different.

[0287] As used herein, a transmembrane domain can be any transmembrane domain known in the art. Those skilled in the art will be familiar with suitable transmembrane domains.

[0288] The transmembrane domain works to fix the antigen receptor to the membrane of each cell. The transmembrane domain may be a hydrophobic alpha-helix that spans the membrane. Preferably, the transmembrane region is derived from CD28, more preferably from human CD28. Accordingly, according to a preferred embodiment of the present invention, the transmembrane region preferably comprises a polypeptide having the amino acid sequence of SEQ ID NO: 79 or a variant or fragment thereof having 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. According to a particularly preferred embodiment, the transmembrane region comprises a polypeptide having the amino acid sequence of SEQ ID NO: 79.

[0289] Preferably, the transmembrane region of the present invention is derived from CD4, more preferably from human CD4. Accordingly, according to a preferred embodiment of the present invention, the transmembrane region preferably comprises a polypeptide having the amino acid sequence of SEQ ID NO: 83 or a variant or fragment thereof having 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. According to a particularly preferred embodiment, the transmembrane region comprises a polypeptide having the amino acid sequence of SEQ ID NO: 83.

[0290] Preferably, the transmembrane region of the present invention is derived from CD8 or CD8α, more preferably from human CD8 or CD8α. Accordingly, according to a preferred embodiment of the CAR of the present invention, the transmembrane region preferably comprises a polypeptide having the amino acid sequence of SEQ ID NO: 74 or 86, or a variant or fragment thereof having 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. According to a particularly preferred embodiment, the transmembrane region comprises a polypeptide having the amino acid sequence of SEQ ID NO: 74 or 86.

[0291] In some embodiments, the transmembrane domain of the present invention is derived from CD44, more preferably from human CD44. Accordingly, according to preferred embodiments of the present invention, the transmembrane domain preferably comprises a polypeptide having the amino acid sequence of SEQ ID NO: 136 or a variant or fragment thereof having 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: 136. According to particularly preferred embodiments, the transmembrane domain comprises a polypeptide having the amino acid sequence of SEQ ID NO: 136.

[0292] In some embodiments, the transmembrane domain of the present invention is derived from CD146, more preferably from human CD146. Accordingly, according to preferred embodiments of the present invention, the transmembrane domain preferably comprises a polypeptide having the amino acid sequence of SEQ ID NO: 137 or a variant or fragment thereof having 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: 137. According to particularly preferred embodiments, the transmembrane domain comprises a polypeptide having the amino acid sequence of SEQ ID NO: 137.

[0293] "LCK" or "lymphocyte-specific protein tyrosine kinase" is known in the art. LCK can be endogenously expressed in T cells. In particular, LCK is non-covalently bound to an intracellular signaling domain capable of binding to LCK. As used herein, LCK can be recombinantly expressed in recombinant cells, for example, ligated to the transmembrane domain of CoCAR. For example, LCK may include SEQ ID NO: 103 or its variants or fragments.

[0294] As used herein, “LCK-binding cytosolic domain” or “LCK-binding intracellular signaling domain” is a domain containing one or more motifs that can bind to LCK. For example, an LCK-binding signaling domain may originate from the cytoplasmic end of the receptor. Examples of amino acid sequences containing one or more LCK-binding motifs include SEQ ID NOs: 84, 87, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, and 102. Preferred sequences containing one or more LCK-binding motifs are selected from SEQ ID NOs: 89, 90, 91, 93, 94, 96, 97, 98, 100, and 102.

[0295] According to a preferred embodiment, the LCK-binding intracellular signaling domain comprises at least one CD4, CD8α, CD28, CD3ε, CD44, and / or CD146 intracellular signaling domain or a variant or fragment thereof.

[0296] According to a preferred embodiment, at least one LCK-binding intracellular signaling domain comprises at least one motif derived from CD4, CD8α, CD28, CD3ε, CD44 and / or CD146 intracellular signaling domains that are LCK-binding.

[0297] According to a preferred embodiment, the at least one intracellular signaling domain capable of binding to LCK comprises at least one CD28, CD44, and / or CD146 intracellular signaling domain or a variant or fragment thereof.

[0298] According to a preferred embodiment, the LCK-binding intracellular signaling domain comprises at least one LCK-binding motif derived from the CD28, CD44, and / or CD146 intracellular signaling domains.

[0299] In a particularly preferred embodiment, the at least one intracellular signaling domain capable of binding to LCK comprises at least one CD4 intracellular signaling domain, preferably having the amino acid sequence of SEQ ID NO: 99 or a variant or fragment thereof having 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: 99.

[0300] In a particularly preferred embodiment, the at least one intracellular signaling domain capable of binding to LCK includes at least one CD8α intracellular signaling domain, preferably having the amino acid sequence of SEQ ID NO: 87 or a variant or fragment thereof having 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: 87, and at least one CD4 intracellular signaling domain.

[0301] In a particularly preferred embodiment, the at least one intracellular signaling domain capable of binding to LCK comprises at least one CD3ε intracellular signaling domain, preferably having the amino acid sequence of SEQ ID NO: 92 or a variant or fragment thereof having 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: 92.

[0302] In a particularly preferred embodiment, the at least one intracellular signaling domain capable of binding to LCK comprises at least one CD28 intracellular signaling domain, preferably having the amino acid sequence of SEQ ID NO: 95 or a variant or fragment thereof having 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: 95.

[0303] In a particularly preferred embodiment, the at least one intracellular signaling domain capable of binding to LCK comprises at least one CD44 intracellular signaling domain, preferably having the amino acid sequence of SEQ ID NO: 99 or a variant or fragment thereof having 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: 99.

[0304] In a particularly preferred embodiment, the at least one intracellular signaling domain capable of binding to LCK comprises at least one CD146 intracellular signaling domain, preferably having the amino acid sequence of SEQ ID NO: 101 or a variant or fragment thereof having 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: 101.

[0305] According to a preferred embodiment, at least one intracellular signaling domain of the CAR to be combined with the CoCAR of the present invention comprises at least one of a 4-1BB intracellular domain and a CD3ζ intracellular domain or a combination of both.

[0306] The 4-1BB intracellular signaling domain preferably includes the amino acid sequence of SEQ ID NO: 80 or a variant or fragment thereof having at least 80%, 81%, 82%, 83%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO: 80. According to a particularly preferred embodiment, the 4-1BB intracellular signaling domain includes a polypeptide having the amino acid sequence of SEQ ID NO: 80.

[0307] The CD3ζ intracellular signaling domain preferably includes the amino acid sequence of SEQ ID NO: 81 or a variant or fragment thereof having at least 80%, 81%, 82%, 83%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 81. In a particularly preferred embodiment, the CD3ζ intracellular signaling domain includes a polypeptide having the amino acid sequence of SEQ ID NO: 81.

[0308] At least one intracellular signaling domain of the CAR to be combined with the CoCAR of the present invention may also include a CD28 intracellular domain, which preferably includes or comprises a sequence such as that shown in SEQ ID NO: 95, or a variant or fragment thereof having at least 80%, 81%, 82%, 83%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 95.

[0309] The antigen recognition domain is preferably scFv.

[0310] The CoCARs of the present invention are preferably single-chain CoCARs (also referred to herein as "sc CoCARs"). Such single-chain CoCARs preferably essentially represent a single-chain variable fragment (scFv), which is a fusion protein of the variable regions of the heavy chain (VH) and light chain (VL) of an immunoglobulin, linked by a linker as defined herein. The linker may connect the N-terminus of the VH to the C-terminus of the VL, or vice versa. Divalent (or bivalent) single-chain variable fragments (di-scFv, bi-scFv) can be manipulated by linking two scFvs. This can be done by generating a single peptide chain using two VH and two VL regions, resulting in a tandem scFv.

[0311] The CAR to be combined with the CoCAR of the present invention is preferably a single-chain CAR (also referred to herein as "sc CAR"). Such a single-chain CAR preferably essentially represents a single-chain variable fragment (scFv), which is a fusion protein of the variable regions of the heavy chain (VH) and light chain (VL) of an immunoglobulin, linked by a linker as defined herein. The linker may connect the N-terminus of the VH to the C-terminus of the VL, or vice versa. A divalent (or bivalent) single-chain variable fragment (di-scFv, bi-scFv) can be manipulated by linking two scFvs. This can be done by generating a single peptide chain using two VH and two VL regions, resulting in a tandem scFv.

[0312] In addition to the single-chain form, other forms are also possible: for example, a Fab-like form or a dimeric form comprising two peptide chains, each containing a VH and VL as well as a hinge region, a transmembrane domain, and one or more intracellular signaling domains. In such a double-chain form, the signaling domain on one peptide chain preferably dimerizes with the signaling domain on the second chain, for example, via a disulfide crosslink.

[0313] While we do not wish to be limited to a specific mechanism of action, it is thought that the two peptide chains of the dimerized antigen receptor of the present invention, when expressed on the surface of immune cells, form a dimer at least through interactions (e.g., disulfide bonding) between the individual immune receptor signaling domains on the two chains.

[0314] The CAR to be combined with the CoCAR of the present invention includes a VL domain as shown in SEQ ID NO: 5 and a VH domain as shown in SEQ ID NO: 9, and includes VHCDR1~VHCDR3 and VLCDR1~VLCDR3 sequences as defined in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, a linker as shown in SEQ ID NO: 10, a CH2 domain as shown in SEQ ID NO: 76, a CH3 domain as shown in SEQ ID NO: 77 or a hinge domain as shown in SEQ ID NO: 73 or 75, a transmembrane domain as shown in SEQ ID NO: 74, a 4-1BB domain as shown in SEQ ID NO: 80 and a CD3ζ domain as shown in SEQ ID NO: 81.

[0315] Sequence ID 104 describes an anti-CD22 CAR having a humanized RFB4 scFv and CD8a skeleton, as encoded by Sequence ID 110.

[0316] Sequence ID 105 describes an anti-CD22 CAR having a humanized RFB4 scFv and IgG-Fc scaffold, as encoded by Sequence ID 111.

[0317] In the context of this invention, the term "IgG-Fc skeleton" is used to refer to the IgG1-Fc spacer (hinge-CH2-CH3) and the CD28 transmembrane domain.

[0318] In the context of this invention, the term "CD8a skeleton" is used to refer to the CD8a hinge and the CD8a transmembrane domain. In the context of this invention, the term "CD28 skeleton" is used to refer to the CD28 hinge and the CD28 transmembrane domain.

[0319] The CAR to be combined with the CoCAR of the present invention preferably includes an amino acid sequence selected from SEQ ID NOs: 104, 105, 134, 185, 186, and 187, or a variant or fragment thereof having at least 80%, 81%, 82%, 83%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NOs: 104, 105, 134, 185, 186, or 187.

[0320] The antigen receptor of the present invention or the peptide chain of the antigen receptor of the present invention may further include other domains, for example, additional domains that are involved in or enhance antigen binding, signal sequences for membrane-bound expression or secretion, and / or domains that provide improved dimerization.

[0321] Since the domains that form antigen-binding sites must be able to freely bind to antigens, it is understandable that the arrangement of these domains in a protein generally achieves the presentation of extracellular regions. Similarly, since the co-stimulatory domains and signaling domains work to induce the activity and proliferation of immune effector cells, the CoCAR and / or CAR to be combined with the CoCAR of the present invention preferably present these domains inside the cell.

[0322] The CoCAR of the present invention preferably includes additional elements such as a signal or leader peptide to ensure proper efflux of the fusion protein to the cell surface. The signal or leader peptide is a sequence or peptide that allows sufficient passage through the secretory pathway and expression on the cell surface, thereby enabling the antigen receptor to bind to an antigen present, for example, in the extracellular environment. Preferably, the leader peptide is cleavable and removed from the mature peptide chain. The signal or leader peptide is preferably selected with respect to the cell or organism from which the peptide chain is produced. According to a preferred embodiment, the CoCAR of the present invention includes a signal peptide selected from amino acid sequences such as those shown in SEQ ID NOs: 1, 19, 37, and 55, as well as variants and fragments thereof having at least 80%, 81%, 82%, 83%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NOs: 1, 19, 37, and 55.

[0323] The present invention further provides CoCARs comprising amino acid sequences selected from SEQ ID NOs: 106-109, 138-151, and 188-190, and variants and fragments thereof having at least 80%, 81%, 82%, 83%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NOs: 106-109, 138-151, and 188-190. Preferably, the CDR sequence of such a CoCAR is as defined herein. According to one embodiment of the present invention, the CoCAR comprises the amino acid sequences shown in SEQ ID NOs: 106-109, 138-151, and 188-190.

[0324] Sequence ID 106 describes the anti-CD19 CD4-CoCAR encoded by Sequence ID 112.

[0325] Sequence ID 107 describes the anti-CD22 CD4-CoCAR encoded by Sequence ID 113.

[0326] Sequence ID 108 describes the anti-CEA CD4-CoCAR encoded by Sequence ID 114.

[0327] Sequence ID 109 describes the bicistronic anti-CD22-CAR-T2A-anti-CD22-CD4-CoCAR encoded by Sequence ID 115.

[0328] The CoCAR of the present invention, in combination with a CAR, preferably not only efficiently induces an antigen-dependent antitumor response to the same extent as CARs known in the art, but also inhibits antigen-independent CAR signaling, myeloid cell activation, and pro-inflammatory cytokine production, thereby providing a remarkable efficacy and safety profile for the treatment of cancers such as B-cell malignancies.

[0329] In the CoCAR of the present invention, the first antigen-recognition domain may be capable of binding to a first antigen on the surface of a target cell, and the binding of the CoCAR to the first antigen does not induce activation of recombinant cells expressing the CoCAR. In particular, the CoCAR of the present invention may be co-expressed with a CAR containing a second antigen-recognition domain, and the second antigen-recognition domain may be capable of binding to a second antigen on the surface of a target cell, and the binding of the CAR to the second antigen induces activation of recombinant cells. The binding of the CoCAR to the first antigen may enhance the activation of recombinant cells induced by the binding of the CAR to the second antigen. The CoCAR may attenuate antigen-independent activation and differentiation of recombinant cells induced by the CAR. The CAR may be any CAR as described herein.

[0330] A further aspect of the present invention is a chimeric antigen receptor (CAR), in particular a CoCAR of the present invention in combination with a CAR as described herein. The CoCAR of the present invention can be combined with any CAR known to those skilled in the art.

[0331] In some embodiments, the combination of the present invention is (a) Anti-CD19 CoCAR, preferably selected from 19-28, 19-44, 19-44v2, 19-146, 19-146v2, as described herein. (b) Anti-CD22 CAR, preferably selected from 22-BBz and m971-Fc-BBz, as described herein. Includes, The combination is expressed on the cell surface.

[0332] In some embodiments, the combination of the present invention is (a) Anti-CD19 CoCAR, preferably selected from 19-28, 19-44, 19-44v2, 19-146, 19-146v2, as described herein. (b) The second antigen recognition domain preferably comprises or consists of SG-III scFv, LL2 scFv, or m971 scFv, as described herein, anti-CD22 CAR Includes, The combination is expressed on the cell surface.

[0333] In some embodiments, the combination of the present invention is (a) Anti-CEA CoCAR as described herein, (b) Anti-EGFR CAR as described herein Includes, The combination is expressed on the cell surface.

[0334] In some embodiments, the combination of the present invention is (a) Anti-EGFR CoCARs, preferably selected from EGFR-28, EGFR-44, EGFR-44v2, EGFR-146, and EGFR-146v2, as described herein. (b) Anti-HER2 CAR as described herein, preferably aHER2-BBz Includes, The combination is expressed on the cell surface.

[0335] In a further embodiment, the present invention provides nucleic acids (singular and plural), nucleic acid constructs, and / or nucleic acid constructs encoding the CoCAR of the present invention.

[0336] Nucleic acids as described herein may also encode CARs to be combined with the CoCARs of the present invention as described herein.

[0337] The term “nucleic acid,” as used herein, is intended to include DNA and RNA, e.g., genomic DNA, cDNA, mRNA, recombinant and chemically synthesized molecules. Nucleic acids may be single-stranded or double-stranded. RNA includes in vitro transcribed RNA (IVT RNA) or synthetic RNA. According to the present invention, nucleic acids are preferably isolated nucleic acids.

[0338] According to one embodiment, the nucleic acid or nucleic acid construct includes a nucleic acid sequence selected from SEQ ID NOs. 112-115, 152-166 or 198-207, or a sequence that is 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.

[0339] The nucleic acids or nucleic acid constructs encoding the antigen receptors of the present invention may be administered to patients requiring administration in a naked form or in a carrier. Each carrier, for example, a lipid carrier, comprises any substance or medium to which nucleic acids, such as DNA or RNA, can associate, for example, by forming a complex with the nucleic acid or by forming a vesicle in which the nucleic acid is encapsulated or contained. This may result in increased stability of the nucleic acid compared to naked nucleic acids. In particular, the stability of nucleic acids in blood may be increased. For example, nanoparticle-like RNA formulations having a defined particle size, lipoplexes from RNA and liposomes, for example, lipoplexes containing DOTMA and DOPE or DOTMA and cholesterol may be used.

[0340] The term "combination of first and second nucleic acids" refers to a nucleic acid bicistronic construct that codes for a combination of CAR and CoCAR, where the first nucleic acid codes for CoCAR(a) and the second nucleic acid codes for CAR(b).

[0341] In a bicistronic construct, or in a nucleic acid construct, the first and second nucleic acids are functionally linked to at least one expression regulatory sequence, such as EF1α.

[0342] In a bicistronic construct or a nucleic acid construct, the first and second nucleic acids are separated by a sequence encoding a cleavage peptide (such as P2A, E2A, F2A, T2A, and IRES, preferably T2A), which allows for the separation of CoCAR and CAR polypeptides after protein translation.

[0343] As used herein, the term “nanoparticles” means any particles having a diameter, typically less than 1,000 nanometers (nm), that makes the particles suitable for systemic administration, particularly parenteral administration, of nucleic acids. Preferably, nanoparticles have a diameter of less than 600 nm or less than 400 nm.

[0344] In the nucleic acid construct of the present invention, nucleic acids as described herein can be functionally linked to at least one expression regulatory sequence. The at least one expression regulatory sequence may be selected from promoters, ribosome binding sites, enhancers, and regulatory elements that regulate the transcription of nucleic acids or translation of mRNA(s), as well as enhancer sequences or upstream activator sequences.

[0345] Still further aspects of the present invention relate to vectors comprising nucleic acids or combinations of first and second nucleic acids as described herein, or nucleic acid constructs or combinations of nucleic acid constructs as described herein. The vectors and / or combinations of vectors of the present invention can be independently selected from vectors suitable for introducing antigen receptor constructs into T cells, such as lentiviruses, γ-retroviruses, and adeno-associated viruses.

[0346] The present invention also provides recombinant cells that express the nucleic acids or nucleic acid constructs of the present invention as defined herein.

[0347] In the context of this invention, the term "recombinant" means "produced by genetic engineering." Preferably, "recombinant object," such as recombinant cells, does not occur naturally in the context of this invention.

[0348] The term "naturally occurring," as used herein, means that a substance can be found in nature. For example, peptides or nucleic acids that are present in living organisms (including viruses), can be isolated from a natural source, and have not been intentionally modified by humans in a laboratory are considered naturally occurring.

[0349] The terms “cell” or “host cell” preferably refer to an intact cell, i.e., a cell having an intact membrane that does not release its normal intracellular components, such as enzymes, organelles, or genetic material. An intact cell is preferably a viable cell, i.e., a living cell capable of performing its normal metabolic functions. Preferably, the terms “cell” or “host cell” refer to any cell that can be transfected with exogenous nucleic acids. Preferably, a cell, when transfected with exogenous nucleic acids and transferred to a recipient, can express the nucleic acid in the recipient. The terms “cell” include bacterial cells and other useful cells, such as yeast cells, fungal cells, or mammalian cells. Suitable bacterial cells include Gram-negative strains, such as Escherichia coli, Proteus, and Pseudomonas, as well as Gram-positive bacterial strains, such as Bacillus, Streptomyces, Staphylococcus, and Lactococcus. Suitable fungal cells include cells from Trichoderma, Neurospora, and Aspergillus species. Suitable yeast cells include those from the genera Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris and Pichia methanolica), and Hansenula species. Suitable mammalian cells include, for example, CHO cells, BHK cells, HeLa cells, COS cells, and 293HEK cells. However, amphibian cells, insect cells, plant cells, and any other cells used in the art for heterologous protein expression can be used similarly.Mammalian cells, such as those derived from humans, mice, hamsters, pigs, goats, and primates, are particularly preferred for adoptive transfer. The cells may be derived from a number of tissue types and include primary cells and cell lines such as immune system cells, in particular antigen-presenting cells, e.g., dendritic cells and T cells, and stem cells, e.g., hematopoietic stem cells and mesenchymal stem cells and other cell types.

[0350] The present invention also provides recombinant cells expressing nucleic acids or nucleic acid constructs as defined herein. Recombinant cells may co-express CoCAR and BBz-CAR of the present invention, for example, BBz-CAR as described herein. CoCAR and BBz-CAR of the present invention may be co-expressed on the cell surface.

[0351] According to a preferred embodiment of the present invention, recombinant cells are immune effector cells. Immune effector cells are preferably, but not limited to, selected from the group consisting of T cells (cytotoxic T cells, helper T cells, tumor-infiltrating T cells), B cells, natural killer cells, neutrophils, macrophages, and dendritic cells. Preferably, in the context of the present invention, immune effector cells are T cells, preferably CD4 + and / or CD8 +The cells in question are T cells. Particularly preferred cells for use according to the present invention are T cells, and more preferably cytotoxic lymphocytes selected from cytotoxic T cells, natural killer (NK) cells, and lymphokine-activated killer (LAK) cells. When activated, each of these cytotoxic lymphocytes causes the destruction of target cells. For example, cytotoxic T cells cause the destruction of target cells by one or both of the following means: Firstly, when activated, T cells release cytotoxic substances such as perforin, granzyme, and granulysin. Perforin and granulysin create pores in the target cell, allowing granzyme to enter the cell and trigger a caspase cascade in the cytoplasm, which induces apoptosis (programmed cell death) of the cell. Secondly, apoptosis may be induced via Fas-Fas ligand interaction between the T cell and the target cell. The cytotoxic lymphocytes are preferably autologous cells, but heterogeneous or allogeneic cells may also be used.

[0352] In recombinant cells of the present invention expressing the CoCAR of the present invention on their surface, the first antigen-recognition domain may be able to bind to a first antigen on the surface of a target cell, and the binding of the CoCAR to the first antigen does not induce activation of the recombinant cell. The recombinant cells of the present invention may further express a CAR including a second antigen-recognition domain, and the second antigen-recognition domain may be able to bind to a second antigen on the surface of a target cell, and the binding of the CAR to the second antigen induces activation of the recombinant cell. In recombinant cells of the present invention, the binding of the CoCAR to the first antigen may enhance the activation of the recombinant cell induced by the binding of the CAR to the second antigen. In recombinant cells of the present invention, the CoCAR may attenuate antigen-independent activation and differentiation of the recombinant cell induced by the CAR. The CAR may be any CAR as described herein.

[0353] The present invention may include the introduction, i.e., transfection, of nucleic acids or CoCAR polypeptides encoding the present invention into respective (host) cells, such as T cells, in vitro or in vivo.

[0354] For the purposes of this invention, the term “transfection” includes the introduction of nucleic acids into cells or the uptake of nucleic acids by cells, where cells may be present in a subject, e.g., a patient. Therefore, according to this invention, cells for nucleic acid transfection described herein may be present in vitro or in vivo; for example, cells may form organs, tissues, and / or parts of an organism of a patient. According to this invention, transfection may be transient or stable. For some applications of transfection, it is sufficient that the transfected genetic material is only transiently expressed. Since nucleic acids introduced in the transfection process are not usually incorporated into the nuclear genome, the foreign nucleic acids are diluted or degraded via mitosis. Cells that enable episomal amplification of nucleic acids significantly reduce the dilution rate. If it is desired that the transfected nucleic acids actually exist in the genomes of cells and their daughter cells, then stable transfection must occur. RNA can be transfected into cells to transiently express the protein it encodes.

[0355] Any technique useful for introducing nucleic acids into cells, i.e., transferring or transfecting them, may be used. Preferably, nucleic acids such as DNA or RNA are transfected into cells by standard techniques. Such techniques include electroporation, lipofection, and microinjection. Nucleic acids can be introduced into cells by electroporation. Electroporation, or electropermeable treatment, is associated with a significant increase in the electrical conductivity and permeability of the cell membrane caused by an externally applied electric field. It is commonly used in molecular biology as a method for introducing some substance into cells. It is preferable that introducing nucleic acids encoding proteins or peptides into cells results in the expression of said proteins or peptides.

[0356] Various methods can be used to introduce antigen receptor constructs into T cells, including non-viral-based DNA transfection, transposon-based systems, and virus-based systems. Non-viral-based DNA transfection carries a low risk of insertional mutagenesis. Transposon-based systems can integrate the transgene more efficiently than plasmids that do not contain the integration element. Virus-based systems include the use of gamma-retroviruses and lentiviral vectors. Gamma-retroviruses are relatively easy to produce, efficiently and sustainably transduce T cells, and have been preliminaryly proven to be safe in terms of integration into primary human T cells. Lentiviral vectors also efficiently and sustainably transduce T cells, but are more expensive to produce. They are also potentially safer than retrovirus-based systems.

[0357] For in vivo cell transfection, a pharmaceutical composition containing a nucleic acid encoding an antigen receptor may be used. A delivery medium that targets the nucleic acid to specific cells, such as T cells, may be administered to the patient, resulting in transfection occurring in vivo.

[0358] Preferably, the cells of the present invention express the CoCAR of the present invention on their cell surface.

[0359] Preferably, the cells of the present invention comprise (i) a chimeric antigen receptor (CAR) and (ii) a nucleic acid and / or nucleic acid construct of the present invention. In particular, the CAR comprises a second extracellular antigen recognition domain, a second hinge region, a second transmembrane domain, a 4-1BB domain, and a CD3 zeta domain, as described herein. Specifically, the cells express (i) the CAR and (ii) the CoCAR on their cell surface. The CAR and CoCAR of the present invention can recognize different epitopes located within the same antigen expressed on the surface of a target cell. The CAR and CoCAR of the present invention can recognize different antigens expressed on the surface of a target cell.

[0360] Nucleic acids may be contained in vectors. The term “vector,” as used herein, includes plasmid vectors, cosmid vectors, phage vectors (e.g., lambda phage), viral vectors (e.g., adenovirus or baculovirus vectors), or artificial chromosome vectors (e.g., bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), or P1 artificial chromosomes (PACs)), and any other vector known to those skilled in the art. Such vectors include expression and cloning vectors. Expression vectors, including plasmids and viral vectors, generally contain a desired coding sequence and appropriate DNA sequences necessary for the expression of the coding sequence operably linked in a particular host organism (e.g., bacteria, yeast, plants, insects, or mammals) or in an in vitro expression system. Cloning vectors are generally used to manipulate and amplify a particular desired DNA fragment and may lack the functional sequences required for the expression of the desired DNA fragment.

[0361] In the context of the present invention, the term “transcription” refers to the process by which the genetic code in a DNA sequence is transcribed into RNA. The RNA can then be translated into a protein. According to the present invention, the term “transcription” includes “in vitro transcription,” which refers to the process by which RNA, particularly mRNA, is synthesized in vitro in a cell-free system, preferably using a suitable cell extract. Preferably, cloning vectors are used for the production of transcripts. These cloning vectors are generally called transcription vectors and, according to the present invention, are encompassed by the term “vector.”

[0362] In the context of this invention, the term "translation" refers to the process in cellular ribosomes in which a chain of messenger RNA directs the assembly of an amino acid sequence to produce a peptide or protein.

[0363] Nucleic acids may exist alone or in combination with other nucleic acids, which may be homologous or heterologous, according to the present invention. Nucleic acids may be functionally ligated to expression regulatory sequences, which may be homologous or heterologous with respect to the nucleic acid. The term “homologous” means that the nucleic acids are also functionally ligated in nature, and the term “heterologous” means that the nucleic acids are not functionally ligated in nature.

[0364] Nucleic acids and regulatory sequences are "functionally" linked to each other by covalent bonds in such a way that the expression or transcription of the nucleic acid is under the control or influence of the regulatory sequence. When the nucleic acid is to be translated into a functional protein, a regulatory sequence functionally linked to the coding sequence is used to induce transcription of the nucleic acid without causing a frameshift in the coding sequence or making translation from the coding sequence to the desired protein or peptide impossible.

[0365] The term “expression regulatory sequence” or “expression regulatory element” includes promoters, ribosome binding sites, enhancers, and other regulatory elements that regulate gene transcription or mRNA translation. In certain embodiments of the present invention, expression regulatory sequences may be modulated. The exact structure of an expression regulatory sequence may vary as a function of species or cell type, but generally includes 5'-non-transcriptional and 5'- and 3'-non-translating sequences involved in the initiation of transcription and translation, respectively, such as TATA boxes, capping sequences, and CAAT sequences. More specifically, a 5'-non-transcriptional expression regulatory sequence includes a promoter region containing a promoter sequence for the transcriptional regulation of functionally linked nucleic acids. An expression regulatory sequence may also include an enhancer sequence or an upstream activator sequence.

[0366] The term “expression” is used in the context of this invention in its most general sense, and includes, for example, the production of RNA and / or peptides or proteins by transcription and / or translation. With respect to RNA, the terms “expression” or “translation” particularly relate to the production of peptides or proteins. This also includes partial expression of nucleic acids. Furthermore, expression may be transient or stable. According to this invention, the term “expression” also includes “abnormal expression” or “not normal expression.”

[0367] "Abnormal expression" or "abnormal expression" means, according to the present invention, that the expression of a particular protein, for example, a tumor antigen, is altered, preferably increased, compared to the state in a subject without the disease associated with abnormal or abnormal expression. An increase in expression refers to an increase of at least 10%, particularly at least 20%, at least 50%, or at least 100%, or more.

[0368] According to a preferred embodiment, the immune effector cells include T cells (cytotoxic T cells, helper T cells, tumor-infiltrating T cells), B cells, natural killer cells, neutrophils, macrophages, and dendritic cells. Preferably, the "immune effector cells" are T cells, more preferably CD4 + and / or CD8 + These are T cells. When activated / stimulated, each of these cytotoxic lymphocytes causes the destruction of target cells. For example, cytotoxic T cells cause the destruction of target cells by one or both of the following means: Firstly, when activated, T cells release cytotoxic substances such as perforin, granzyme, and granulysin. Perforin and granulysin create pores in the target cell, allowing granzyme to enter the cell and trigger a caspase cascade in the cytoplasm, which induces apoptosis (programmed cell death) of the cell. Secondly, apoptosis can be induced via Fas-Fas ligand interaction between the T cell and the target tumor cell. T cells and other cytotoxic lymphocytes are preferably autologous cells, but heterogeneous or allogeneic cells may also be used according to the present invention.

[0369] Therefore, the antigen receptor of the present invention can replace the function of the T cell receptor as described above, and in particular can confer reactivity, such as cytolytic activity, to cells, such as T cells, as described above. However, in contrast to the binding of the T cell receptor to the antigen peptide-MHC complex, the antigen receptor binds to the antigen, especially when it is expressed on the cell surface.

[0370] The present invention also provides a population of recombinant cells, preferably the immunoeffector cells described herein, the population may be a clonally proliferated population. Recombinant immunoeffector cells or populations thereof provide therapeutic or prophylactic immunoeffector function in an antigen-specific manner.

[0371] Preferably, the chimeric antigen receptor of the present invention is expressed on the cell surface of such recombinant cells, particularly on the cell surface of such immune effector cells.

[0372] In a further embodiment, the present invention provides pharmaceutical compositions comprising the CoCAR of the present invention, combinations of the present invention, nucleic acids, combinations of nucleic acids, nucleic acid constructs, combinations of nucleic acid constructs, vectors, combinations of vectors of the present invention and / or recombinant cells of the present invention and pharmaceutically acceptable carriers.

[0373] A pharmaceutical composition according to the present invention (also simply called "composition") is preferably sterile and contains an effective amount of the antigen receptor, peptide chain, nucleic acid, recombinant cell, immune effector cell, and other compounds and agents described herein, and optionally further agents as discussed herein to produce a desired reaction or effect.

[0374] Pharmaceutical compositions are usually provided in a uniform dosage form and can be prepared by methods known to the public. Pharmaceutical compositions may be in the form of solutions or suspensions, for example.

[0375] Pharmaceutical compositions may contain salts, buffering agents, preservatives, carriers, diluents, and / or excipients in a pharmaceutically acceptable form. The term "pharmaceutically acceptable" means that the materials are non-toxic and do not interact with the action of the active ingredient of the pharmaceutical composition.

[0376] Pharmaceutically unacceptable salts may be used for the preparation of pharmaceutically acceptable salts and are included in the present invention. This type of pharmaceutically acceptable salt includes, but is not limited to, those prepared from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, citric acid, formic acid, malonic acid, succinic acid, etc. 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.

[0377] Suitable buffering substances for use in pharmaceutical compositions include acetates, citrates, borates, and phosphates.

[0378] Suitable preservatives for use in pharmaceutical compositions include benzalkonium chloride, chlorobutanol, parabens, and thimerosal.

[0379] The term "carrier" refers to an organic or inorganic component of natural or synthetic nature, which is combined with an active ingredient to facilitate, enhance, or enable its application. According to the present invention, the term "carrier" also includes one or more compatible solid or liquid fillers, diluents, or encapsulating materials suitable for administration to a patient.

[0380] Possible carriers for parenteral administration include, for example, sterile water, Ringer's solution, lactated Ringer's solution, sterile sodium chloride solution, polyalkylene glycol, hydrogenated naphthalene, and in particular, biocompatible lactide polymers, lactide / glycolide copolymers, or polyoxyethylene / polyoxypropylene copolymers.

[0381] The term “excipient,” as used herein, is intended to refer to all substances that are not active ingredients and may be present in a pharmaceutical composition, such as carriers, binders, lubricants, thickeners, surfactants, preservatives, emulsifiers, buffers, flavorings, or colorants.

[0382] The compositions described herein may be administered via any conventional route, for example, by parenteral administration including administration by injection or infusion. Administration is preferably parenteral, such as intravenous, intra-arterial, subcutaneous, intradermal, or intramuscular administration.

[0383] Compositions suitable for parenteral administration typically and preferably comprise a sterile aqueous or non-aqueous preparation of the active compound that is isotonic with respect to the recipient's blood. Examples of suitable carriers and solvents include Ringer's solution and isotonic, slightly hypertonic, or slightly hypotonic sodium chloride solutions. Furthermore, sterile fixed oil can often be used as a solution or suspension medium.

[0384] The pharmaceutical compositions described herein are preferably administered in an effective dose. “Effective dose” means the amount that achieves the desired response or effect alone or in combination with additional doses. In the case of treatment of a particular disease or condition, the desired response preferably relates to the inhibition of the disease process. This includes slowing the progression of the disease, in particular preventing or reversing its progression. The desired response in treatment of a disease or condition may also be the delay or prevention of the onset of the disease or condition.

[0385] The effective dose of the compositions described herein varies depending on the individual patient's parameters, including the condition to be treated, the severity of the disease, age, physiological state, size and weight, duration of treatment, type of ancillary therapy (if any), specific route of administration, and similar factors. Therefore, the dose administered of the drugs and compositions described herein may vary depending on such parameters. If the response in the patient is insufficient with the initial dose, a higher dose (or an effectively higher dose achieved by a different, more localized route of administration) may be used.

[0386] The compositions described herein may be administered to patients to treat or prevent a variety of disorders, including those described herein. Patients preferably include human patients with disorders that can be corrected or improved by administration of the agents and compositions described herein. These include disorders involving cells characterized by antigen expression, particularly CD22 expression.

[0387] For example, in one embodiment, the compositions described herein may be used to treat patients having cancer, such as those described herein characterized by the presence of cancer cells expressing tumor-associated antigens.

[0388] The pharmaceutical compositions described in accordance with the present invention may also be used for immunization or vaccination to prevent the diseases described herein.

[0389] The pharmaceutical compositions of the present invention may be administered together with adjuvants, for example, one or more immune enhancers, to further increase their efficacy, preferably to achieve a synergistic effect of immune stimulation, and may also contain one or more immune enhancers. The term “adjuvant” refers to a compound that prolongs, enhances, or accelerates the immune response. Depending on the type of adjuvant, there may be various mechanisms in this regard. For example, compounds that enable DC maturation, such as lipopolysaccharides or CD40 ligands, form a suitable adjuvant of the first class. In general, any drug that affects the immune system of the “danger signal” type (e.g., LPS, GP96, dsRNA) or cytokines such as GM-CSF can be used as adjuvants that enable the immune response to be enhanced and / or affected in a controlled manner. CpG oligodeoxyribonucleotides may be used in connection therewith, but their side effects that occur under certain circumstances as described above should be taken into consideration. Particularly preferred adjuvants include cytokines, such as monokines, lymphokines, interleukins, or chemokines, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IFNα, IFNγ, GM-CSF, LT-α, or growth factors, such as hGH. Further known adjuvants include aluminum hydroxide, Freund's adjuvant, or oil, such as Montanide®, with Montanide® ISA51 being the most preferred. Lipopeptides, such as Pam3Cys, are also suitable for use as adjuvants in the pharmaceutical compositions of the present invention.

[0390] The pharmaceutical composition of the present invention can be administered topically or systemically, preferably systemically.

[0391] The term "systemic administration" refers to the administration of a drug in such a way that it is widely distributed throughout the body of an individual in sufficient quantities to produce the desired effect. For example, a drug may produce its desired effect in the bloodstream and / or reach the desired site of action via the vascular system. Common systemic administration routes include administration by directly introducing the drug into the vascular system, or oral, pulmonary, or intramuscular administration, in which the drug is absorbed, enters the vascular system, and is transported to one or more desired sites of action.

[0392] Systemic administration via parenteral administration is a particularly preferred route. The term "parenteral administration" refers to the administration of a drug in a manner that does not involve the passage of the intestines. The term "parenteral administration" includes, but is not limited to, intravenous, subcutaneous, intradermal, or intra-arterial administration.

[0393] Administration can also be performed, for example, orally, intraperitoneally, or intramuscularly.

[0394] The compositions described herein may be used alone or in combination with conventional therapeutic regimens, such as surgery, radiation, chemotherapy, and / or bone marrow transplantation (autologous, allogeneic, allogeneic, or unrelated).

[0395] According to the present invention, CoCAR, its peptide chain, combination, nucleic acid, combination of nucleic acid, nucleic acid construct, combination of nucleic acid construct, vector, combination of vector, recombinant cell, and / or pharmaceutical composition described herein can be used in pharmaceuticals. According to preferred embodiments of the present invention, CoCAR, its peptide chain, combination, nucleic acid, combination of nucleic acid, nucleic acid construct, combination of nucleic acid construct, vector, combination of vector, recombinant cell, and / or pharmaceutical composition described herein is for use in the treatment of cancer. More preferably, CoCAR, its peptide chain, nucleic acid, combination of nucleic acid, nucleic acid construct, combination of nucleic acid construct, vector, combination of vector, recombinant cell, and / or pharmaceutical composition described herein is for use in the treatment of malignancies associated with the cell surface expression of at least one tumor-associated antigen.

[0396] Tumor-associated antigens include 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, and GP-2. The following antigens may be selected from the group consisting of 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 ligand, NCAM, NY-ESO1, Carcinoembryonic antigen h5T4, PSCA, PSMA, ROR1, TAG-72, VEGFR, GOLPH2, and SLAMF7. Preferred tumor-associated antigens are selected from the group consisting of BCMA, CD22, CD79B, GOLPH2, EGFR / EGFRvIII, HER2, CEA, and GOLPH2. More preferred tumor-associated antigens are selected from the group consisting of CD22, CD19, HER2, and EGFR.

[0397] More preferably, the CoCARs, their peptide chains, nucleic acids, combinations of nucleic acids, nucleic acid constructs, combinations of nucleic acid constructs, vectors, combinations of vectors, recombinant cells and / or pharmaceutical compositions described herein are for use in the treatment of malignancies associated with the cell surface expression of at least one tumor-associated antigen, wherein the at least one tumor-associated antigen has a density of less than about 1000 molecules / cell, less than about 400 molecules / cell, less than about 300 molecules / cell, less than about 200 molecules / cell, less than about 100 molecules / cell or less than about 40 molecules / cell, preferably less than about 400 molecules / cell, more preferably less than about 40 molecules / cell.

[0398] More preferably, the CoCARs, their peptide chains, nucleic acids, combinations of nucleic acids, nucleic acid constructs, combinations of nucleic acid constructs, vectors, combinations of vectors, recombinant cells and / or pharmaceutical compositions described herein are for use in the treatment of malignancies associated with the cell surface expression of at least one tumor-associated antigen, wherein the at least one tumor-associated antigen is selected from the group consisting of CD22, CD19, HER2 and EGFR, and the at least one tumor-associated antigen has a density of less than about 1000 molecules / cell, less than about 400 molecules / cell, less than about 300 molecules / cell, less than about 200 molecules / cell, less than about 100 molecules / cell or less than about 40 molecules / cell, preferably less than about 400 molecules / cell, more preferably less than 40 molecules / cell.

[0399] According to a preferred embodiment, the malignant tumor is a B-cell malignant tumor.

[0400] Preferably, the disease is characterized by the presence of affected cells that express CD22. In a particularly preferred embodiment, the malignant tumor associated with cell surface CD22 expression is a B-cell malignant tumor.

[0401] Accordingly, the CoCARs, their peptide chains, nucleic acids, nucleic acid combinations, nucleic acid constructs, combinations of nucleic acid constructs, vectors, combinations of vectors, recombinant cells and / or pharmaceutical compositions described herein may be used to treat subjects having a disease, particularly for the treatment of cancer. According to preferred embodiments, the CoCARs, their peptide chains, nucleic acids, nucleic acid combinations, nucleic acid constructs, combinations of nucleic acid constructs, vectors, combinations of vectors, recombinant cells and / or pharmaceutical compositions described herein may be used to treat malignancies associated with cell surface CD22 expression in subjects requiring treatment, for example, diseases characterized by the presence of affected cells expressing CD22. Malignancies associated with cell surface CD22 expression are preferably B-cell malignancies.

[0402] CoCARs, nucleic acids, nucleic acid combinations, nucleic acid constructs, combinations of nucleic acid constructs, vectors, vector combinations, recombinant cells and / or pharmaceutical compositions described herein may also be used for immunization or vaccination to prevent the diseases described herein.

[0403] The term “disease” refers to an abnormal condition affecting an individual’s body. Often, a disease is interpreted as a medical condition associated with specific symptoms and signs. Diseases can be caused by external factors, such as infectious diseases, or by internal dysfunctions, such as autoimmune diseases. In humans, “disease” is more often used broadly to refer to any condition that causes pain, impairment, distress, social problems, or death in the affected individual, or similar problems in those who come into contact with the individual. This broad sense may also include injury, disability, impairment, syndrome, infection, isolated symptoms, abnormal behavior, and atypical changes in structure and function, while in other contexts and for other purposes, these can be considered identifiable categories. Diseases usually affect an individual not only physically but also emotionally, as suffering from and living with multiple diseases can alter a person’s worldview and personality. According to this invention, the term “disease” includes infectious diseases and cancerous diseases, in particular the forms of cancer described herein. Any reference in this specification to cancer or any particular form of cancer also includes its metastasis.

[0404] The diseases to be treated according to the present invention are preferably diseases involving the expression of the CD22 antigen. “Antigen-related diseases,” “diseases associated with antigen expression or elevated expression,” or similar expressions mean, according to the present invention, that the antigen is expressed in cells of affected tissue or organ. Expression in cells of affected tissue or organ may be increased compared to the state in healthy tissue or organ. In one embodiment, expression is found only in affected tissue and not in healthy tissue, for example, expression is suppressed. According to the present invention, antigen-related diseases include infectious diseases and cancerous diseases, and the disease-related antigens are preferably antigens of infectious pathogens and tumor antigens, respectively. Preferably, antigen-related diseases are diseases involving cells that express the antigen, preferably cells that express it on the cell surface.

[0405] The terms "healthy" or "normal" refer to a non-pathological state, preferably meaning non-infectious or non-cancerous.

[0406] The term "cancer" or "cancer" usually refers to or describes a physiological condition in an individual characterized by unregulated cell proliferation. Examples of cancer include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias. More specifically, examples of such cancers include bone cancer, blood cancer, lung cancer, liver cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous melanoma or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, colon cancer, breast cancer, prostate cancer, uterine cancer, carcinomas of the genitals and reproductive organs, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, bladder cancer, kidney cancer, renal cell carcinoma, renal pelvis carcinoma, neoplasms of the central nervous system (CNS), neuroectodermal cancer, spinal axis tumor, glioma, meningioma, and pituitary adenoma. The term “cancer” also includes cancer metastasis according to the present invention. Preferably, “cancer disease” is characterized by cells expressing CD22.

[0407] In one embodiment, cancer is a malignant disease characterized by anaplastic, invasive, and metastatic properties. Malignant tumors can be contrasted with non-cancerous benign tumors in that malignant tumors are not self-limiting in their growth, can invade adjacent tissues, and may spread to distal tissues (metastasize), while benign tumors do not possess any of these properties.

[0408] According to the present invention, the terms “tumor” or “tumor disease” refer to a swelling or lesion formed by the abnormal proliferation of cells (called neoplastic cells or tumor cells). “Tumor cells” means abnormal cells that proliferate rapidly and uncontrolledly and continue to proliferate after the stimulus that initiated the new proliferation has ceased. Tumors exhibit a partial or complete absence of structural tissue and functional coordination with normal tissue, forming a distinct tissue mass that can be benign, premalignant, or malignant.

[0409] According to the present invention, "carcinoma" is a malignant tumor of epithelial cell origin. This group represents the most common cancers and includes common forms of breast cancer, prostate cancer, lung cancer, and colon cancer.

[0410] Adenocarcinoma is a type of cancer that develops in glandular tissue. This tissue is also part of a larger tissue category known as epithelial tissue. Epithelial tissue includes skin, glands, and various other tissues that line the inside of cavities and organs in the body. Epithelium is embryologically derived from the ectoderm, endoderm, and mesoderm. To be classified as adenocarcinoma, cells do not necessarily have to be part of a gland, as long as they have secretory properties. This form of carcinoma can occur in several higher mammals, including humans. Well-differentiated adenocarcinomas tend to resemble the glandular tissue from which they originate, but poorly differentiated ones may not. By staining cells obtained from a biopsy, a pathologist will determine whether the tumor is adenocarcinoma or some other type of cancer. Due to the ubiquity of glands in the body, adenocarcinoma can arise from numerous tissues in the body. Each gland may not secrete the same substance, but as long as it has an exocrine function to cells, it is considered glandular, and therefore its malignant form is named adenocarcinoma. Malignant adenocarcinoma often invades other tissues and metastasizes if given enough time. Ovarian adenocarcinoma is the most common type of ovarian carcinoma. It includes serous adenocarcinoma, mucosal adenocarcinoma, clear cell adenocarcinoma, and endometrioid adenocarcinoma.

[0411] Lymphoma and leukemia are malignant tumors that originate from hematopoietic (blood-forming) cells.

[0412] Blastomas, or blastocyte tumors, are tumors (usually malignant) that resemble immature or embryonic tissue. Many of these tumors are most common in children.

[0413] "Metastasis" refers to the spread of cancer cells from the original site to another part of the body. The formation of metastasis is an extremely complex process that depends on the detachment of malignant cells from the primary tumor, invasion of the extracellular matrix, permeation of the endothelial basement membrane to enter cavities and blood vessels in the body, and then invasion of the target organ after being transported by the blood. Finally, the growth of new tumors at the target site depends on angiogenesis. Tumor metastasis often occurs even after the removal of the primary tumor, as tumor cells or components remain and give rise to the possibility of metastasis. In one embodiment, the term "metastasis" according to the present invention refers to "distant metastasis," which is distant from the primary tumor and the local lymph node system. In one embodiment, the term "metastasis" according to the present invention refers to lymph node metastasis.

[0414] A relapse or recurrence occurs when a person is affected again by a condition that previously affected them. For example, if a patient has a tumor, has received treatment for the disease and it has been successful, and then the disease recurs, the newly recurring disease can be considered a relapse or recurrence. However, a relapse or recurrence of a tumor does not necessarily occur at the site of the original tumor. For example, if a patient has an ovarian tumor and has received successful treatment, a relapse or recurrence could be the appearance of an ovarian tumor or a tumor at a site other than the ovary. Tumor relapses or recurrences also include situations where the tumor occurs at a site different from the original tumor site, as well as at the site of the original tumor. The original tumor that the patient has received treatment for may be the primary tumor, and a tumor at a site different from the original tumor site may be a secondary or metastatic tumor.

[0415] The present invention also relates to the use of CoCARs, combinations thereof, their peptide chains, nucleic acids, combinations of nucleic acids, nucleic acid constructs, combinations of nucleic acid constructs, vectors, combinations of vectors, recombinant cells and / or pharmaceutical compositions as described herein in therapeutic and prophylactic methods. One such use is in the generation of antigen-specific immune cells, which can be administered to a patient for the prevention or treatment of a disease, characterized by the expression of tumor-associated antigens to which the antigen receptors of the present invention, expressed in immune cells, can bind. Preferably, the disease is cancer. Furthermore, the antigen receptors and related molecules of the present invention may also be used for the selective eradication of cells expressing tumor-associated antigens, and for immunization or vaccination against diseases in which tumor-associated antigens are expressed.

[0416] In one embodiment, a method for treating or preventing cancer in a subject or patient requiring treatment or prevention of cancer comprises administering an effective amount of the CoCAR of the present invention to the subject or patient. In particular, in the CoCAR, the antigen-binding site of the antigen receptor can bind to an antigen associated with the disease to be treated or prevented. According to a preferred embodiment, the cancer is a malignant tumor, preferably a B-cell malignant tumor, that is positive for at least one tumor-associated antigen.

[0417] In one embodiment, a method for treating or preventing cancer in a subject or patient requiring treatment or prevention of cancer comprises administering to the subject or patient an effective amount of nucleic acids, combinations of nucleic acids, nucleic acid constructs, combinations of nucleic acid constructs, vectors and / or combinations of vectors according to the present invention. According to a preferred embodiment, the cancer is a malignant tumor positive for at least one tumor-associated antigen, preferably a B-cell malignant tumor. According to a preferred embodiment, the cancer is a malignant tumor positive for at least one tumor-associated antigen having a density of at least one tumor-associated antigen, preferably less than about 1000 molecules / cell, less than about 400 molecules / cell, less than about 300 molecules / cell, less than about 200 molecules / cell, less than about 100 molecules / cell or less than about 40 molecules / cell, preferably less than about 400 molecules / cell, more preferably less than 40 molecules / cell, preferably at least one tumor-associated antigen selected from CD22, CD19, HER2 and EGFR.

[0418] In one embodiment, a method for treating or preventing cancer in a subject or patient requiring treatment or prevention of cancer comprises administering to the subject or patient an effective amount of recombinant cells or population of recombinant cells according to the present invention. According to a preferred embodiment, the cancer is a malignant tumor positive for at least one tumor-associated antigen, preferably a B-cell malignant tumor.

[0419] In one embodiment, a method for treating or preventing cancer in a subject or patient requiring treatment or prevention of cancer comprises administering to the subject or patient an effective amount of a pharmaceutical composition according to the present invention. According to a preferred embodiment, the cancer is a malignant tumor, preferably a B-cell malignant tumor, that is positive for at least one tumor-associated antigen.

[0420] The present invention also provides a method for immunizing or vaccinating against a disease associated with the expression of at least one tumor-associated antigen, the method comprising administering to a patient an effective amount of, as described herein, its peptide chain, nucleic acid, combination of nucleic acid, nucleic acid construct, combination of nucleic acid construct, vector and / or combination of vectors, recombinant cells and / or pharmaceutical composition, wherein the antigen-binding site of the antigen receptor can bind to the antigen associated with the disease (e.g., CD22).

[0421] At least one tumor-associated antigen is 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, The following antigens may be selected from the group consisting of 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 ligand, NCAM, NY-ESO1, Carcinoembryonic antigen h5T4, PSCA, PSMA, ROR1, TAG-72, VEGFR, GOLPH2, and SLAMF7. Preferred tumor-associated antigens are selected from the group consisting of BCMA, CD22, CD79B, GOLPH2, EGFR / EGFRvIII, HER2, CEA, and GOLPH2. More preferred tumor-associated antigens are selected from the group consisting of CD22, CD19, HER2, and EGFR.

[0422] According to a preferred embodiment, at least one tumor-associated antigen has a density of less than 1000 molecules / cell, less than 400 molecules / cell, less than 300 molecules / cell, less than 200 molecules / cell, less than 100 molecules / cell, or less than 40 molecules / cell, preferably less than 400 molecules / cell, more preferably less than 40 molecules / cell.

[0423] The term “treatment” or “therapeutic treatment” refers to any treatment that improves a health condition and / or extends (increases) the lifespan of an individual. Such treatment may eliminate a disease in an individual, stop, inhibit or slow the onset of a disease in an individual, reduce the frequency or severity of symptoms in an individual, and / or reduce recurrence in an individual that currently has or has previously had the disease.

[0424] The terms “preventive measures” or “preventive measures” refer to any measures intended to prevent a disease from occurring in an individual. The terms “preventive measures” and “preventive measures” are used synonymously herein.

[0425] The terms “individual” and “subject” are used synonymously herein. They refer to humans, non-human primates or other mammals (e.g., mice, rats, rabbits, dogs, cats, cattle, pigs, sheep, horses, or primates) that are capable of or 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. Unless otherwise indicated, the terms “individual” and “subject” do not represent a specific age and therefore encompass adults, the elderly, children and newborns. In preferred embodiments of the present invention, “individual” or “subject” is a “patient.” The term “patient” means the subject of treatment, in particular the subject of disease.

[0426] "At risk" or "needing" refers to subjects identified as having a higher-than-normal chance of developing a disease, particularly cancer, compared to the general population; i.e., patients. Furthermore, subjects who have had or currently have a disease, particularly cancer, are at increased risk of developing the disease because they may continue to develop it. Subjects who currently have or have had cancer are also at increased risk of cancer metastasis.

[0427] In the context of this invention, terms such as “protect,” “prevent,” “preventive,” “protective,” and “protective” relate to the prevention or treatment of the onset and / or transmission of disease in a subject, or both, particularly to minimizing the subject’s chances of developing the disease or delaying its onset. For example, a person at risk of the above-mentioned tumors would be a candidate for tumor-preventive therapy.

[0428] Prophylactic administration of immunotherapy, for example, prophylactic administration of the agent or composition of the present invention, preferably protects the recipient from the onset of disease. Therapeutic administration of immunotherapy, for example, therapeutic administration of the agent or composition of the present invention, may lead to inhibition of disease progression / proliferation. This includes slowing down disease progression / proliferation, in particular blocking disease progression, which preferably leads to the elimination of the disease.

[0429] Immunotherapy may be carried out using any of the various techniques which preferably function to remove antigen-expressing cells from a patient. Such removal may occur as a result of an enhanced or induced immune response in patients specific to the antigen or antigen-expressing cells.

[0430] The terms "immunization" or "vaccination" describe the process of treating a subject with the aim of inducing an immune response for therapeutic or preventive reasons.

[0431] In accordance with the above, the present invention preferably relates to the following items: 1. (i) The first extracellular antigen recognition domain, (ii) The first hinge domain, (iii) the first transmembrane domain, and (iv)(a)LCK-compatible and / or (b) At least one cytosolic domain containing LCK or a variant or fragment thereof Co-receptors including CARs (CoCARs). 2. A CoCAR conforming to item 1, where the first antigen recognition domain is scFv. 3. The first antigen recognition domain is 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, E A CoCAR that specifically recognizes at least one tumor-associated antigen selected from rbB3, ErbB4, FBP, AchR, Fr-a, GD2, GD3, HMW-MAA, IL13Ra2, Kappa-LC, IGLV3-21-R110, Lewis Y, Mesothelin, MUC1, MUC16, NKG2D ligand, NCAM, NY-ESO1, Carcinoembryonic antigen h5T4, PSCA, PSMA, ROR1, TAG-72, VEGFR, GOLPH2, and SLAMF7, conforming to item 1 or 2. 4. A CoCAR that meets Item 3, in which at least one tumor-associated antigen is selected from the group consisting of BCMA, CD22, CD79B, GOLPH2, EGFR / EGFRvIII, HER2, CEA, and GOLPH2. 5. The first antigen recognition domain is (a) Light chain variable domain (VL domain) including light chain complementarity determination region (LCDR)1 of SEQ ID NO: 2, LCDR2 of SEQ ID NO: 3, and LCDR of SEQ ID NO: 4, and (b) A CoCAR comprising a heavy chain variable domain (VH domain) including the heavy chain complementarity determining region (HCDR)1 of SEQ ID NO: 6, HCDR2 of SEQ ID NO: 7, and HCDR of SEQ ID NO: 8, wherein the light chain variable domain and the heavy chain variable domain specifically bind to CD22 and conform to one of items 1 to 4. 6. (a) One or more of the light chain framework regions 1 (LFR1), 2 (LFR2), 3 (LFR3), and 4 (LFR4) have at least 90% amino acid sequence identity with respect to the amino acid sequence of each framework region of the light chain variable region according to SEQ ID NO: 5, and / or (b) A CoCAR that conforms to any one of items 1 to 5, wherein one or more of the heavy chain framework regions 1 (HFR1), 2 (HFR2), 3 (HFR3), and 4 (HFR4) have at least 90% amino acid sequence identity with respect to the amino acid sequence of each framework region of the heavy chain variable region according to SEQ ID NO: 9. 7. LFR1 contains sequence number 11, LFR2 includes sequence number 12, LFR3 includes sequence number 13, LFR4 includes sequence number 14, HFR1 contains sequence number 15, HFR2 includes sequence number 16, HFR3 includes sequence number 17, and / / or HFR4 is a CoCAR that follows one of items 1-6, including sequence number 18. 8. The first antigen recognition domain is (a) Light chain variable domains including light chain complementarity determination region (LCDR) 1 of SEQ ID NO: 20, LCDR2 of SEQ ID NO: 21, and LCDR of SEQ ID NO: 22, and (b) A CoCAR that includes a heavy chain variable domain containing the heavy chain complementarity determining region (HCDR)1 of SEQ ID NO: 24, HCDR2 of SEQ ID NO: 25, and HCDR of SEQ ID NO: 26, and the light chain variable domain and the heavy chain variable domain specifically bind to CD22, conforming to any one of items 1 to 4. 9.(a) One or more of the light chain framework regions 1 (LFR1), 2 (LFR2), 3 (LFR3), and 4 (LFR4) having at least 90% amino acid sequence identity with respect to the amino acid sequence of each framework region of the light chain variable region according to Sequence ID No. 23, and / or (b) One or more of the following heavy chain framework regions: heavy chain framework region 1 (HFR1), heavy chain framework region 2 (HFR2), heavy chain framework region 3 (HFR3), and heavy chain framework region 4 (HFR4), having at least 90% amino acid sequence identity with respect to the amino acid sequence of each framework region of the heavy chain variable region according to Sequence ID No. 27. CoCAR that includes items 1-4 and one of items 8. 10. LFR1 contains sequence number 29, LFR2 includes sequence number 30, LFR3 includes sequence number 31, LFR4 includes sequence number 32, HFR1 includes sequence number 33, HFR2 includes sequence number 34, HFR3 includes sequence number 35, and / or HFR4 is a CoCAR that conforms to one of items 1-4, 8, and 9, including SEQ ID NO: 36. 11. The first antigen recognition domain is (a) Light chain variable domains including light chain complementarity determination region (LCDR)1 of SEQ ID NO: 38, LCDR2 of SEQ ID NO: 39, and LCDR of SEQ ID NO: 40, and (b) A CoCAR that includes a heavy chain variable domain containing the heavy chain complementarity determining region (HCDR)1 of SEQ ID NO: 42, HCDR2 of SEQ ID NO: 43, and HCDR of SEQ ID NO: 44, wherein the light chain variable domain and the heavy chain variable domain specifically bind to CD19, and conforms to any one of items 1 to 4. 12. (a) One or more of the light chain framework regions 1 (LFR1), 2 (LFR2), 3 (LFR3), and 4 (LFR4) having at least 90% amino acid sequence identity with respect to the amino acid sequence of each framework region of the light chain variable region according to Sequence ID No. 41, and / or (b) One or more of the following heavy chain framework regions: Heavy chain framework region 1 (HFR1), Heavy chain framework region 2 (HFR2), Heavy chain framework region 3 (HFR3), and Heavy chain framework region 4 (HFR4), having at least 90% amino acid sequence identity with respect to the amino acid sequence of each framework region of the heavy chain variable region according to Sequence ID No. 45. CoCAR that includes items 1-4 and one of item 11. 13. LFR1 contains sequence number 47, LFR2 includes sequence number 48, LFR3 contains sequence number 49, LFR4 contains sequence number 50, HFR1 contains sequence number 51, HFR2 includes sequence number 52, HFR3 includes sequence number 53, and / or HFR4 includes sequence number 54, CoCAR follows one of the following criteria: items 1-4, 11, or 12. 14. The first antigen recognition domain is (a) Light chain variable domains including light chain complementarity determining region (LCDR)1 of SEQ ID NO: 56, LCDR2 of SEQ ID NO: 57, and LCDR of SEQ ID NO: 58, and (b) A CoCAR that includes a heavy chain variable domain containing the heavy chain complementarity determining region (HCDR)1 of SEQ ID NO: 60, HCDR2 of SEQ ID NO: 61, and HCDR of SEQ ID NO: 62, wherein the light chain variable domain and the heavy chain variable domain specifically bind to CEA, and conform to any one of items 1 to 4. 15. (a) One or more of the light chain framework regions 1 (LFR1), 2 (LFR2), 3 (LFR3), and 4 (LFR4) having at least 90% amino acid sequence identity with respect to the amino acid sequence of each framework region of the light chain variable region according to Sequence ID No. 59, and / or (b) One or more of the following heavy chain framework regions: Heavy chain framework region 1 (HFR1), Heavy chain framework region 2 (HFR2), Heavy chain framework region 3 (HFR3), and Heavy chain framework region 4 (HFR4), having at least 90% amino acid sequence identity with respect to the amino acid sequence of each framework region of the heavy chain variable region according to Sequence ID No. 63. CoCAR must comply with any one of items 1-4 and 14, including the following: 16. LFR1 contains sequence number 65, LFR2 includes sequence number 66, LFR3 includes sequence number 67, LFR4 includes sequence number 68, HFR1 contains sequence number 69, HFR2 includes sequence number 70, HFR3 includes sequence number 71, and / or HFR4 is a CoCAR that follows any one of items 1-4, 14, and 15, including SEQ ID NO: 72. 17. In the first antigen recognition domain, the VH domain and VL domain are linked by a linker, according to one of items 5-16. 18. A CoCAR in accordance with item 17, in which the linker contains glycine and / or alanine residues. 19. A CoCAR according to item 17, in which the linker is selected from SEQ ID NO: 10, SEQ ID NO: 46 and its fragments and variants. 20. A CoCAR that is capable of specifically binding to an antigen on a target cell and conforms to any one of the above items. 21. A CoCAR that conforms to any one of items 1-20, wherein the first antigen-recognition domain is capable of binding to a first antigen on the surface of a target cell, and the binding of the CoCAR to the first antigen does not induce activation of recombinant cells expressing the CoCAR. 22. CoCAR according to item 21, wherein recombinant cells further express a CAR containing a second antigen-recognition domain, the second antigen-recognition domain being capable of binding to a second antigen on the surface of a target cell, and the binding of the CAR to the second antigen induces activation of the recombinant cell. 23. CoCAR binding to a first antigen enhances the activation of recombinant cells induced by the binding of CAR to a second antigen, according to item 22. 24. CoCARs that attenuate antigen-independent activation and differentiation of recombinant cells induced by CARs, in accordance with item 22 or 23. 25. A CoCAR conforming to any one of the above items, wherein the first hinge domain is derived from a CD4, CD8, CD28, or IgG-Fc domain. 26. A CoCAR conforming to any one of the above items, wherein the first hinge domain comprises a sequence selected from SEQ ID NOs. 73, 78, 82, and 85 and its variants and fragments. 27. A CoCAR conforming to any one of the above items, including at least one spacer area. 28. The spacer includes the first hinge domain, in accordance with item 27 of CoCAR. 29. A CoCAR conforming to any one of the above items, wherein the first transmembrane domain is derived from a CD4, CD8, CD28, or CD3 zeta transmembrane domain. 30. A CoCAR conforming to any one of the above items, wherein the first transmembrane domain comprises sequences selected from SEQ ID NOs: 74, 79, 83, and 86 and their variants and fragments, preferably SEQ ID NOs: 83 and 86 and their variants and fragments. 31. (a) At least one cytosolic domain capable of binding to LCK, (1) An intracellular signaling domain derived from the CD4, CD8α, CD28, CD3ε, CD44, or CD146 intracellular signaling domain or its variant or fragment, or (2) Including one or more motifs that can be attached to the LCK, A CoCAR that complies with any one of the above items. 32. A CoCAR conforming to item 31, wherein the intracellular signaling domain (1) is derived from the CD4, CD8α, CD3ε, CD44, or CD146 intracellular signaling domain or a variant or fragment thereof. 33. A CoCAR conforming to item 31, wherein the intracellular signaling domain (1) is derived from the CD4, CD8α, CD28, CD44, or CD146 intracellular signaling domain or a variant or fragment thereof. 34. A CoCAR whose intracellular domain (1) is derived from the CD4, CD8α, CD44, or CD146 intracellular signaling domain or a variant or fragment thereof, conforming to one of items 31-33. 35. A CoCAR whose intracellular domain (1) is derived from a CD4 or CD8α intracellular signaling domain or a variant or fragment thereof, and which follows one of items 31-34. 36. A CoCAR that follows one of items 31-35, in which a motif capable of binding to LCK originates from the intracellular signaling domains of CD4, CD8α, CD28, CD3ε, CD44, or CD146. 37. A CoCAR that follows any one of items 31-36, wherein the LCK-binding motif is derived from the CD4, CD8α, CD3ε, CD44, or CD146 intracellular signaling domain or its variant or fragment. 38. A CoCAR that follows any one of items 31-36, wherein the LCK-binding motif is derived from the CD4, CD8α, CD28, CD44, or CD146 intracellular signaling domain or its variant or fragment. 39. A CoCAR that follows any one of items 31-38, wherein the LCK-binding motif is derived from the CD4, CD8α, CD44, or CD146 intracellular signaling domain or its variant or fragment. 40. A CoCAR that follows one of items 31-39, wherein the LCK-binding motif originates from the CD4 or CD8α intracellular signaling domain or its variant or fragment. 41. A CoCAR conforming to any one of items 31-40 above, wherein the LCK-binding motif includes sequences selected from sequence numbers 84, 87, and 89-102 and their fragments and variants. 42. A CoCAR that conforms to any one of the above items, wherein at least one cytosolic domain contains an LCK. 43. A CoCAR that conforms to item 42, including sequence number 103 or a fragment or variant thereof. 44. A CoCAR conforming to any one of the above items, wherein at least one cytosolic domain does not contain a CD3 zeta domain. 45.(a) Any one of items 1-44 of CoCAR, and (b) Chimeric antigen receptor (CAR) A combination that includes this. 46. ​​Chimeric antigen receptor (b) (i) Second extracellular antigen recognition domain, (ii) Second hinge region, (iii) Second transmembrane domain, (iv) 4-1BB domain and / or CD28 domain, and (v) CD3 zeta domain 45 combinations of items, including [this item]. 47. In the chimeric antigen receptor (b), the 4-1BB domain is a combination of item 45 or 46, including SEQ ID NO: 80 or a fragment or variant thereof. 48. In a chimeric antigen receptor (b), the CD3 zeta domain is one of any combinations of items 45-47, including SEQ ID NO: 81 or a fragment or variant thereof. 49. In CAR(b), the second hinge includes a hinge derived from CD28, and in particular, the second hinge includes SEQ ID NO: 78 or a variant or fragment thereof, any one combination of items 45-48. 50. In CoCAR(a), the first hinge does not contain an array derived from CD28, in particular the first hinge does not contain sequence number 78, a combination of item 49. 51. Any combination of items 49-50, wherein in CAR(b), the second hinge includes a hinge derived from CD28, and in particular, the second hinge includes SEQ ID NO: 78 or a variant or fragment thereof, and CoCAR(a) includes a first hinge region derived from CD4, in particular, SEQ ID NO: 82 or a variant or fragment thereof. 52. Any combination of items 49 to 50, wherein in CAR(b), the second hinge includes a hinge derived from CD28, in particular the second hinge includes SEQ ID NO: 78 or a variant or fragment thereof, and CoCAR(a) includes a first hinge region derived from CD8, preferably CD8α, in particular the second hinge includes SEQ ID NO: 73 or 85 or a variant or fragment thereof. 53. In CAR(b), the second hinge comprises a hinge derived from CD8, preferably CD8α, and in particular, the second hinge comprises SEQ ID NO: 73 or 85 or a variant or fragment thereof, one combination of items 45 to 52. 54. In CoCAR(a), the first hinge is a combination of items 53, not including sequence numbers 73 or 85. 55. Any one combination of items 53 to 54, wherein in CAR(b), the second hinge includes a hinge derived from CD8, preferably CD8α, and in particular the second hinge includes SEQ ID NO: 73 or 85 or a variant or fragment thereof, and CoCAR(a) includes a first hinge region derived from CD4, in particular SEQ ID NO: 82. 56. In CAR(b), the second hinge comprises a hinge derived from CD8, preferably CD8α, and in particular the second hinge comprises SEQ ID NO: 73 or 85 or a variant or fragment thereof, and CoCAR(a) comprises a first hinge region derived from CD28, in particular SEQ ID NO: 78, any one combination of items 53 to 54. 57. A nucleic acid that codes for a CoCAR that follows any one of items 1-44 or any combination of items 45-56. 58. A combination of a first and a second nucleic acid that encodes one combination of any of items 45-56, wherein the first nucleic acid encodes CoCAR(a) and the second nucleic acid encodes CAR(b). 59. A nucleic acid construct comprising the nucleic acids of item 57 and / or the first and second nucleic acids of item 58. 60. A combination of a first and a second nucleic acid construct, comprising the combination of item 58, wherein the first nucleic acid construct comprises a first nucleic acid and the second nucleic acid construct comprises a second nucleic acid. 61. A nucleic acid construct of item 59 or a combination of nucleic acid constructs of item 60, wherein the nucleic acid and / or the first and second nucleic acids are functionally linked independently to at least one expression regulatory sequence. 62. A nucleic acid construct or combination of nucleic acid constructs of item 61, wherein at least one expression regulatory sequence is selected from promoters, ribosome binding sites, enhancers and regulatory elements, as well as enhancer sequences or upstream activator sequences, which regulate the transcription of nucleic acid(s) or translation of mRNA(s). 63. A vector containing the nucleic acid of item 57, the first and second nucleic acid combination of item 58, the nucleic acid construct of item 59, and / or a combination of any one of the nucleic acid constructs of items 60-62. 64. A combination of first and second vectors comprising a combination of nucleic acids from item 58 or a combination of any one nucleic acid construct from items 60-62, wherein the first vector comprises the first nucleic acid and / or the first nucleic acid construct, and the second vector comprises the second nucleic acid and / or the second nucleic acid construct. 65.(a) The vector is selected from lentiviruses, γ-retroviruses and adeno-associated viruses, and / or (b) In the combination, the vector is independently selected from lentivirus, gamma-retrovirus and adeno-associated virus. A vector of item 63 or a combination of the first and second vectors of item 64. 66. Recombinant cells containing nucleic acids, combinations of nucleic acids, nucleic acid constructs, combinations of nucleic acid constructs, vectors, and / or combinations of vectors, conforming to any one of items 57-65. 67. Recombinant cells of item 66, which are immune effector cells. 68. Recombinant cells of any one of items 66-67, expressing any one CoCAR from items 1-44 and / or any one combination from items 45-56 on the cell surface. 69. Recombinant cells of item 68, wherein the first antigen-recognition domain is capable of binding to a first antigen on the surface of the target cell, and the binding of CoCAR to the first antigen does not induce activation of the recombinant cell. 70. Recombinant cells expressing any one of items 66-69, wherein the CAR further expresses a CAR containing a second antigen recognition domain, the second antigen recognition domain is capable of binding to a second antigen on the surface of a target cell, and the binding of the CAR to the second antigen induces the activation of the recombinant cell. 71. Recombinant cells of item 70, in which the binding of CoCAR to the first antigen enhances the activation of recombinant cells induced by the binding of CAR to the second antigen. 72. Recombinant cells of item 70 or 71 in which CoCAR attenuates antigen-independent activation and differentiation of CAR-induced recombinant cells. 73. Recombinant cells from any one of items 66-72, in which (i) CAR and (ii) CoCAR recognize different epitopes located within the same antigen expressed on the surface of the target cell. 74. Recombinant cells from any one of items 66-73, in which (i) CAR and (ii) CoCAR recognize different antigens expressed on the surface of target cells. 75.(i) CoCAR that complies with any one of items 1-44, (ii) Any combination of items 45-56, (iii) Any one nucleic acid, combination of nucleic acids, nucleic acid construct, combination of nucleic acid constructs, vector and / or combination of vectors, and / or (iv) Recombinant cells that conform to any one of items 66-74 as well as pharmaceutically acceptable carriers A pharmaceutical composition containing the following: 76. CoCARs conforming to any one of items 1-44, any combination of items 45-56, any nucleic acid, nucleic acid combination, nucleic acid construct, combination of nucleic acid construct, vector, vector combination, recombinant cells conforming to any one of items 66-74, and / or pharmaceutical compositions conforming to item 75, for use in pharmaceuticals. 77. CoCARs conforming to any one of items 1-44, any combination of items 45-56, any nucleic acid, nucleic acid combination, nucleic acid construct, nucleic acid (nucleic) construct combination, vector, vector combination, recombinant cells conforming to any one of items 66-74, and / or pharmaceutical compositions conforming to item 75, for use in the treatment of cancer. 78. At least one tumor-associated antigen, preferably 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. CoCAR, combination, nucleic acid construct, recombinant cell or pharmaceutical composition for use in accordance with item 76 or 77 in the treatment of malignancies having cell surface expression of at least one tumor-associated antigen selected from the group consisting of GD2, GD3, HMW-MAA, IL13Ra2, Kappa-LC, IGLV3-21-R110, Lewis Y, Mesothelin, MUC1, MUC16, NKG2D ligand, NCAM, NY-ESO1, carcinoembryonic antigen h5T4, PSCA, PSMA, ROR1, TAG-72, VEGFR, GOLPH2, and SLAMF7. 79. A CoCAR, combination, nucleic acid construct, recombinant cell or pharmaceutical composition for use in accordance with any one of items 76-78, wherein at least one tumor-associated antigen is selected from the group consisting of BCMA, CD22, CD79B, GOLPH2, EGFR / EGFRvIII, HER2, CEA, and GOLPH2. 80. CoCAR, combination, nucleic acid construct, recombinant cell and / or pharmaceutical composition for use in accordance with any one of items 76-79 for use in the treatment of B-cell malignancies. 81. A method for treating cancer in a subject requiring treatment, (i) CoCAR that complies with any one of items 1-44, (ii) Any combination of items 45-56, (iii) Any one nucleic acid, combination of nucleic acids, nucleic acid construct, combination of nucleic acid constructs, vector and / or combination of vectors, and / or (iv) Recombinant cells that conform to any one of items 64-74 A method comprising the step of administering an effective amount of [the substance]. 82. Cancer is associated with 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, E A malignant tumor that is positive for at least one tumor-associated antigen selected from the group consisting of rbB4, FBP, AchR, Fr-a, GD2, GD3, HMW-MAA, IL13Ra2, Kappa-LC, IGLV3-21-R110, Lewis Y, Mesothelin, MUC1, MUC16, NKG2D ligand, NCAM, NY-ESO1, Carcinoembryonic antigen h5T4, PSCA, PSMA, ROR1, TAG-72, VEGFR, GOLPH2, and SLAMF7, according to item 81. 83. A method according to item 81 or 82, wherein at least one tumor-associated antigen is selected from the group consisting of BCMA, CD22, CD79B, GOLPH2, EGFR / EGFRvIII, HER2, CEA, and GOLPH2. 84. How does cancer become a B-cell malignant tumor according to any one of items 81-83? 85. (i) The first extracellular antigen recognition domain, (ii) The first hinge domain, (iii) the first transmembrane domain, and (iv) At least one cytosolic domain that can bind to the LCK Includes, A chimeric coreceptor is a CoCAR that does not contain a TCR mobilization domain and conforms to one of items 1-44. 86. Recombinant cells of item 71, wherein the second antigen has an antigen density of less than approximately 1000 molecules / cell, less than approximately 400 molecules / cell, less than approximately 300 molecules / cell, less than approximately 200 molecules / cell, less than approximately 100 molecules / cell, or less than approximately 40 molecules / cell, preferably less than approximately 400 molecules / cell, more preferably less than approximately 40 molecules / cell. 87. A CoCAR, combination, nucleic acid construct, recombinant cell or pharmaceutical composition for use in accordance with any one of items 76-79, wherein at least one tumor-associated antigen is selected from the group consisting of CD22, CD19, HER2 and EGFR, and / or at least one tumor-associated antigen has a density of less than about 1000 molecules / cell, less than about 400 molecules / cell, less than about 300 molecules / cell, less than about 200 molecules / cell, less than about 100 molecules / cell or less than about 40 molecules / cell, preferably less than about 400 molecules / cell. 88. A method according to any one of items 81 to 84, wherein at least one tumor-associated antigen is selected from the group consisting of CD22, CD19, HER2, and EGFR, and / or the at least one tumor-associated antigen has a density of less than about 1000 molecules / cell, less than about 400 molecules / cell, less than about 300 molecules / cell, less than about 200 molecules / cell, less than about 100 molecules / cell, or less than about 40 molecules / cell, preferably less than about 400 molecules / cell, more preferably less than 40 molecules / cell. 89. A CoCAR according to item 3, wherein at least one tumor-associated antigen is selected from the group consisting of CD19, CD22, HER2, and EGFR, and / or at least one tumor-associated antigen has a density of less than about 1000 molecules / cell, less than about 400 molecules / cell, less than about 300 molecules / cell, less than about 200 molecules / cell, less than about 100 molecules / cell, or less than about 40 molecules / cell, preferably less than about 400 molecules / cell. 90. The first antigen recognition domain, (a) Light chain variable domains (VL domains) including the light chain complementarity determination region LCDR1 of SEQ ID NO: 117, LCDR2 of SEQ ID NO: 118, and LCDR3 of SEQ ID NO: 119, and (b) A CoCAR that includes a heavy chain variable domain (VH domain) comprising the heavy chain complementarity determining region HCDR1 of SEQ ID NO: 121, HCDR2 of SEQ ID NO: 122, and HCDR3 of SEQ ID NO: 123, wherein the light chain variable domain and the heavy chain variable domain specifically bind to CD22, and conforms to one of items 1-4 or 89. 91.(a) One or more of LFR1, LFR2, LFR3, and LFR4 have at least 90% amino acid sequence identity with respect to the amino acid sequence of each framework region of the light chain variable region according to SEQ ID NO: 120, and / or (b) One or more of HFR1, HFR2, HFR3, and HFR4 have at least 90% amino acid sequence identity with respect to the amino acid sequence of each framework region of the heavy chain variable region according to SEQ ID NO: 124. CoCAR follows one of the following criteria: items 1-4 or 90. 92.LFR1 contains sequence number 126, LFR2 includes sequence number 127, LFR3 includes sequence number 128, LFR4 includes sequence number 129, HFR1 includes sequence number 130, HFR2 includes sequence number 131, HFR3 includes sequence number 132, and / or HFR4 includes sequence number 133, CoCAR follows either item 1-4 or item 90-91. 93. In the first antigen recognition domain, the VH domain and the VL domain are linked by a linker, the linker preferably comprising glycine and / or serine residues, according to any one of items 90-92, CoCAR. 94. A CoCAR according to item 93, in which the linker is selected from sequence numbers 10, 46, 125, or 176, or fragments and variants thereof. 95. A CoCAR that is specifically capable of binding to a first antigen on a target cell, whose first antigen-recognition domain is capable of binding to the first antigen on the surface of the target cell, and whose binding to the first antigen does not induce activation of recombinant cells expressing the CoCAR, according to any one of items 90-94. 96. A CoCAR that conforms to any one of the above conditions, wherein recombinant cells further express a CAR containing a second antigen-recognition domain, the second antigen-recognition domain is capable of binding to a second antigen on the surface of a target cell, and the binding of the CAR to the second antigen induces activation of the recombinant cell. 97. A CoCAR that conforms to any one of the above items, wherein the binding of the CoCAR to a first antigen enhances the activation of recombinant cells induced by the binding of the CAR to a second antigen, and / or the CoCAR attenuates antigen-independent activation and differentiation of recombinant cells induced by the CAR. 98. A CoCAR conforming to any one of the above items, wherein the first hinge domain is derived from a CD8 or CD28 hinge domain, and the first hinge domain preferably comprises a sequence selected from SEQ ID NOs: 73, 78 and its variants and fragments. 99. A CoCAR conforming to any one of the above items, wherein the first transmembrane domain is derived from the CD8, CD28, CD44, or 146 transmembrane domain, and the first transmembrane domain preferably comprises a sequence selected from SEQ ID NOs: 74, 79, 86, 136, 137 and its variants and fragments, and more preferably comprises a sequence selected from SEQ ID NOs: 79, 136, 137 and its variants and fragments. 100. At least one cytosolic domain that can bind to LCK (1) An intracellular signaling domain derived from the CD28, CD44, or CD146 intracellular signaling domain or its variant or fragment, or (2) Including one or more motifs that can be attached to the LCK, A CoCAR that complies with any one of the above items. 101. A CoCAR conforming to item 100, wherein the intracellular domain (1) is derived from the CD28 intracellular signaling domain or a variant or fragment thereof. 102. A CoCAR conforming to item 100, wherein the intracellular domain (1) is derived from the CD44 intracellular signaling domain or a variant or fragment thereof. 103. A CoCAR conforming to item 100, wherein the intracellular domain (1) is derived from the CD146 intracellular signaling domain or a variant or fragment thereof. 104. A CoCAR conforming to any one of the above items, wherein at least one cytosolic domain does not contain a CD3 zeta domain. 105. (i) The first extracellular antigen recognition domain, and A)(ii) The first hinge domain derived from the CD8α hinge domain, (iii) A first transmembrane domain derived from the CD8α transmembrane domain, and (iv) At least one cytosolic domain derived from the CD28 intracellular signaling domain, or B)(ii) The first hinge domain derived from the CD8α hinge domain, (iii) A first transmembrane domain derived from the CD8α transmembrane domain, and (iv) At least one cytosolic domain derived from the CD44 intracellular signaling domain, or C)(ii) The first hinge domain derived from the CD8α hinge domain, (iii) A first transmembrane domain derived from the CD44 transmembrane domain, and (iv) At least one cytosolic domain derived from the CD44 intracellular signaling domain, or D)(ii) The first hinge domain derived from the CD8α hinge domain, (iii) A first transmembrane domain derived from the CD8α transmembrane domain, and (iv) At least one cytosolic domain derived from the CD146 intracellular signaling domain; or E)(ii) The first hinge domain derived from the CD8α hinge domain, (iii) A first transmembrane domain derived from the CD146 transmembrane domain, and (iv) At least one cytosolic domain derived from the CD146 intracellular signaling domain, or F)(ii) The first hinge domain derived from the CD28 hinge domain, (iii) A first transmembrane domain derived from the CD28 transmembrane domain, and (iv) At least one cytosolic domain derived from the CD28 intracellular signaling domain A coreceptor-CAR (CoCAR) that contains a CD3 zeta domain but does not contain one. 106. A CoCAR that has an amino acid sequence containing any of sequence numbers 106-108, 138-144, 188-190 or a variant thereof, and which conforms to one of items 1-44 or 90-105. 107.(a) One of the CoCAR items 85 or 90-106, and (b) Chimeric antigen receptor (CAR) Includes, A combination of expressions on the cell surface. 108. Chimeric antigen receptor (b) (i) Second extracellular antigen recognition domain, (ii) Second hinge region, (iii) Second transmembrane domain, (iv) 4-1BB domain and / or CD28 domain, and (v) CD3 zeta domain A combination of 107 items, including [this item]. 109. A nucleic acid that codes a CoCAR or any combination of items 107 or 108 according to any one of the items above, and preferably comprises a nucleic acid sequence selected from sequence numbers 153-166, 201-207. 110. A combination of a first and second nucleic acid that codes for one combination of item 107 or 108, where the first nucleic acid codes for CoCAR(a) and the second nucleic acid codes for CAR(b). 111. A nucleic acid construct comprising the nucleic acids of item 109 and / or combinations of the first and second nucleic acids of item 110. 112. A combination of first and second nucleic acid constructs, including the combination of item 110, in which the first nucleic acid construct contains the first nucleic acid and the second nucleic acid construct contains the second nucleic acid. 113. A nucleic acid construct of item 111 or a combination of nucleic acid constructs of item 112, wherein the nucleic acid and / or the first and second nucleic acids are functionally linked independently to at least one expression regulatory sequence. 114. A nucleic acid construct or combination of nucleic acid constructs of item 113, wherein at least one expression regulatory sequence is selected from promoters, ribosome binding sites, enhancers, and regulatory elements that regulate the transcription of nucleic acids or translation of mRNA(s), as well as enhancer sequences or upstream activator sequences. 115. A vector containing the nucleic acid of item 109, the first and second nucleic acid combination of item 110, and one nucleic acid construct and / or combination of nucleic acid constructs from any of items 111-114. 116. A combination of first and second vectors comprising a combination of nucleic acids from item 110 or a combination of any one nucleic acid construct from items 112 to 114, wherein the first vector comprises a first nucleic acid and / or a first nucleic acid construct, and the second vector comprises a second nucleic acid and / or a second nucleic acid construct. 117.(a) The vector is selected from lentiviruses, γ-retroviruses and adeno-associated viruses, and / or (b) A combination of the vectors of item 115 or the first and second vectors of item 116, wherein the vectors are independently selected from lentiviruses, gamma-retroviruses, and adeno-associated viruses. 118. Recombinant cells comprising nucleic acids, combinations of nucleic acids, nucleic acid constructs, combinations of nucleic acid constructs, vectors and / or combinations of vectors, which conform to any one of items 109 to 117, preferably immunoeffector cells. 119. Recombinant cells of item 118 that express CoCAR and / or a combination thereof on the cell surface. 120. Recombinant cells of item 119, wherein the first antigen-recognition domain is capable of binding to a first antigen on the surface of the target cell, and the binding of CoCAR to the first antigen does not induce activation of the recombinant cell. 121. A recombinant cell from any one of items 118-120, further expressing a CAR containing a second antigen recognition domain, wherein the second antigen recognition domain is capable of binding to a second antigen on the surface of a target cell, and the binding of the CAR to the second antigen induces the activation of the recombinant cell. 122. The binding of CoCAR to the first antigen enhances the activation of recombinant cells induced by the binding of CAR to the second antigen, and / or CoCAR attenuates antigen-independent activation and differentiation of recombinant cells induced by CAR. Recombinant cells of item 121. 123. (i) CARs and (ii) CoCARs recognize different epitopes located within the same antigen expressed on the surface of target cells, or (i) CAR and (ii) CoCAR recognize different antigens expressed on the surface of target cells, one of any two recombinant cells from items 118–122. 124. Recombinant cells of any one of items 118-123, wherein the second antigen has an antigen density of less than approximately 1000 molecules / cell, less than approximately 400 molecules / cell, less than approximately 300 molecules / cell, less than approximately 200 molecules / cell, less than approximately 100 molecules / cell, or less than approximately 40 molecules / cell, preferably less than approximately 400 molecules / cell, more preferably less than approximately 40 molecules / cell. 125.(i) CoCAR that complies with either item 85 or 90-106, (ii) Any one combination of items 107-108, (iii) Any one nucleic acid, combination of nucleic acids, nucleic acid construct, combination of nucleic acid constructs, vector and / or combination of vectors, and / or (iv) One recombinant cell according to items 118-124 as well as pharmaceutically acceptable carriers A pharmaceutical composition containing the following: 126. CoCARs, any combination of any one of items 85 or 90-106, any combination of any one of items 107-108, any nucleic acid, any combination of nucleic acid, any one of items 109-117, nucleic acid constructs, combinations of nucleic acid constructs, vectors, any combination of vectors, recombinant cells, and / or pharmaceutical compositions, any one of items 118-124, for use in pharmaceuticals. 127. CoCARs in accordance with any one of items 85 or 90-106, any combination of any one of items 110-111, nucleic acids, combinations of nucleic acids, nucleic acid constructs, combinations of nucleic acid constructs, vectors, any combination of any one of items 109-117, recombinant cells in accordance with any one of items 118-124, and / or pharmaceutical compositions in accordance with item 125, for use in the treatment of cancer. 128. At least one tumor-associated antigen, preferably 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 CoCAR, combination, nucleic acid construct, recombinant cell or pharmaceutical composition for use in accordance with item 126 or 127 in the treatment of malignancies having cell surface expression of at least one tumor-associated antigen selected from the group consisting of IL13Ra2, kappa-LC, IGLV3-21-R110, Lewis Y, mesothelin, MUC1, MUC16, NKG2D ligand, NCAM, NY-ESO1, carcinoembryonic antigen h5T4, PSCA, PSMA, ROR1, TAG-72, VEGFR, GOLPH2, and SLAMF7, preferably from the group consisting of CD19, CD22, HER2, and EGFR. 129. CoCAR, combination, nucleic acid construct, recombinant cell or pharmaceutical composition for use in accordance with either item 126 or 127, for use in the treatment of B-cell malignancies. 130. A CoCAR, combination, nucleic acid construct, recombinant cell or pharmaceutical composition for use in accordance with any one of items 126-129, wherein at least one tumor-associated antigen has a density of less than about 1000 molecules / cell, less than about 400 molecules / cell, less than about 300 molecules / cell, less than about 200 molecules / cell, less than about 100 molecules / cell or less than about 40 molecules / cell, preferably less than about 400 molecules / cell. 131. A method for treating cancer in a person in need thereof, (i) CoCAR that complies with either item 85 or one of items 90-106, (ii) Any combination of items 107-108, (iii) Any one nucleic acid, combination of nucleic acids, nucleic acid construct, combination of nucleic acid constructs, vector and / or combination of vectors, and / or (iv) Recombinant cells that conform to any one of items 118-124 A method comprising the step of administering an effective amount of [the substance]. 132. Cancer, 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 A method according to item 131, wherein the malignant tumor is positive for at least one tumor-associated antigen selected from the group consisting of GD2, GD3, HMW-MAA, IL13Ra2, Kappa-LC, IGLV3-21-R110, Lewis Y, Mesothelin, MUC1, MUC16, NKG2D ligand, NCAM, NY-ESO1, carcinoembryonic antigen h5T4, PSCA, PSMA, ROR1, TAG-72, VEGFR, GOLPH2, and SLAMF7, preferably from the group consisting of CD19, CD22, HER2, and EGFR. 133. How does cancer follow any one of items 131-132, that it is a B-cell malignant tumor? 134. A method according to any one of items 131 to 133, wherein at least one tumor-associated antigen has a density of less than about 1000 molecules / cell, less than about 400 molecules / cell, less than about 300 molecules / cell, less than about 200 molecules / cell, less than about 100 molecules / cell, or less than about 40 molecules / cell, preferably less than about 400 molecules / cell, more preferably less than 40 molecules / cell.

[0432] [Table 2-1] [Table 2-2]

[0433] The concepts of the present invention will be described in more detail by the embodiments described below, which are for illustrative purposes only and are not intended to limit the scope of this disclosure or the claims. For the purposes of description and examples, further embodiments similarly included in the present invention can be utilized by those skilled in the art. [Examples]

[0434] The techniques and methods used herein are described herein or are known by themselves, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2 nd The procedure shall be carried out as described in the Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. All methods, including the use of kits and reagents, shall be carried out according to the manufacturer's instructions unless otherwise explicitly stated. [Example 1]

[0435] LCK overexpression leads to terminal differentiation and restricted proliferation of BBz-CAR-T cells (Comparative Example). In the development of this invention, we investigated whether co-expression of LCK with anti-CD22 CAR in primary human T cells could improve the function of CAR-T cells obtained by increasing basal CAR-CD3ζ phosphorylation (Figure 1A). For this purpose, primary human T cells were transduced with or without co-transduction of a lentiviral vector encoding an anti-CD22 CAR (SEQ ID NO: 105) containing a humanized antigen recognition domain (derived from RFB4 antibody) linked to an IgG1 Fc spacer, CD28 transmembrane, and 4-1BB-CD3 zeta (BBz) signaling domain, with or without co-transduction of a vector encoding full-length human LCK. Surprisingly, we found that co-expression of LCK with anti-CD22 CAR led to CD8 T cell depletion and preferential differentiation of CD4-positive T cells into an effector T cell-like phenotype after 14 days of culture. In contrast, T cells expressing the same anti-CD22 CAR contained a mixture of CD8-positive and CD4-positive T cells exhibiting a memory T cell-like phenotype (Figure 1B). Furthermore, BBz-CAR-T cells overexpressing LCK showed less viable proliferative capacity ex vivo compared to their counterparts expressing BBz-CAR alone or to CAR-negative T cells (Figure 1C). Thus, in contrast to previous studies (Sun et al., (2020) Cancer Cell, (2) 216-225; WO2021087183), these surprising results suggest that co-expression of BBz-CAR with LCK may induce unregulated, overly potent basal CAR-CD3ζ phosphorylation, which leads to enhanced antigen-independent persistent signaling, resulting in advanced T cell differentiation toward an effector phenotype and ultimately reduced proliferative capacity. These T cells tend to be depleted and generally exhibit limited long-term persistence in vivo, and are therefore likely to induce an inadequate therapeutic response in patients; thus, these characteristics are undesirable in the context of adoption therapy. [Example 2]

[0436] The concept involves co-expressing BBz-CAR and CoCAR to regulate LCK recruitment to CAR synapses through antigen binding. Enhancement of CAR-T cell function can be achieved by specifically regulating the recruitment of the kinase LCK to CAR synapses in response only to antigen-mediated CAR binding, and not in unstimulated T cells. This mechanism should not only prevent persistent CAR signaling but also improve the effectiveness of BBz-CAR-T cells against low-antigen tumor cells, which is hypothesized to be because LCK is recruited more efficiently to CAR synapses. To achieve this objective, we have developed the CoCAR concept, which is based on the simultaneous expression of two distinct chimeric receptor molecules in the same immune cell (e.g., T cell): the first receptor molecule consists of a conventional CAR known in the art that functions as a T cell activating receptor, while the second receptor molecule consists of a fusion between an antigen-recognition domain (e.g., scFv) and a co-receptor-derived domain (in this embodiment, a CD4-derived hinge, transmembrane, and cytosolic domain; hereafter referred to as CoCAR), which functions as a co-receptor to enhance the T cell response activated by the first receptor (CAR). The antigen-recognition domains of these two receptor molecules recognize different epitopes located either in the same antigen or in different antigens expressed on the surface of a given target cell. Since CoCAR contains a co-receptor hinge, transmembrane, and LCK-binding intracellular domain (in this embodiment, the CD4 intracellular domain contains an LCK-binding "zinc clasp" motif), it is assumed that CoCAR stably associates with the endogenous LCK molecule to exert its function. In unstimulated T cells, CAR and CoCAR are spatially separated, and therefore the CoCAR-coupled LCK is not available to mediate basal CAR-CD3ζ phosphorylation, sustained signaling, and antigen-independent T cell differentiation and exhaustion (Figure 2A). After binding to the cognitive antigen, CAR and co-receptor-CAR relocalize and proximally align, and the CoCAR-associated LCK is now available to induce CAR-CD3ζ ITAM phosphorylation and ZAP-70 recruitment.The ZAP-70 molecule bound to ITAM is then efficiently phosphorylated by CoCAR-associated LCK, and the activated ZAP-70 subsequently phosphorylates its substrates, including LAT and SLP-76, which can lead to more efficient assembly of the signalosome and activation of various downstream signaling pathways (Figure 2B). [Example 3]

[0437] Co-expression of a CD22-recognizing CAR and a CD19-recognizing CoCAR enhances CAR-T cell activity against B-cell tumor cells. Exemplary CAR and CoCAR molecules that recognize different extracellular antigens exposed on tumor cells were manipulated, and their function in activating primary human T cells expressing these CAR and CoCAR molecules was tested. Figure 3A shows an example of T cells co-expressing an anti-CD22 CAR (SEQ ID NO: 104) containing a humanized antigen-recognition domain (derived from an RFB4 monoclonal antibody), CD8α hinge region and transmembrane domain, and 4-1BB / CD3ζ signaling domain, with an anti-CD19 CoCAR (SEQ ID NO: 106) containing a clinically established FMC63 antigen-recognition domain linked to the CD4 hinge, transmembrane domain, and intracellular domain. Primary human T cells were transduced with lentiviral vectors to express either the anti-CD22 CAR or the anti-CD19 CoCAR, or both lentiviral vectors were co-transduced to express both molecules. Seven days after transduction, the transduced T cells were sorted and purified, and CAR and CoCAR expression was determined by flow cytometry. As shown in Figure 3B, transduced T cells efficiently expressed anti-CD22 CAR and anti-CD19 CoCAR molecules on their cell surface, as determined by their ability to specifically bind to fluorescently labeled soluble antigens. Importantly, co-expression of anti-CD22 CAR with anti-CD19 CoCAR did not attenuate T cell expansion and proliferation ex vivo (Figure 3C), as observed with anti-CD22 CAR-T cells co-expressing LCK (Figure 1C). To test CAR-induced T cell function, T cells were incubated with Nalm6 cells (derived from patients with B-cell acute lymphoblastic leukemia) or Raji cells (derived from patients with Burkitt lymphoma), which correspond to tumor cells expressing low (approximately 3,000 antigens per cell) and high (approximately 60,000 antigens per cell) CD22 antigen densities (Figure 3D). Both tumor cell lines expressed CD19 antigen at high densities (>50,000 molecules / cell; Haso et al, (2013) Blood, (7) 1165-1174).When T cells expressing anti-CD22 CAR were incubated with Nalm6 or Raji tumor cells, the CAR molecule activated the corresponding T cells, upregulating the expression of activation markers on their cell surface, such as CD69, CD25, or both, and secreting specific effector cytokines, including interferon-gamma (IFNγ) or IL-2. As shown in Figure 3E, this T cell activation by anti-CD22 CAR (represented by the co-expression of CD69 and CD25 molecules on the cell surface) was significantly enhanced when the T cells co-expressed anti-CD19 CoCAR, whereas the expression of anti-CD19 CoCAR alone had no effect on T cell activation. Similarly, co-incubation of anti-CD22 CAR-expressing T cells with Nalm6 or Raji tumor cells led to moderate IFNγ production, but co-expression of anti-CD22 CAR with anti-CD19 CoCAR significantly enhanced effector cytokine production (Figure 3F). These results indicate that T cells co-expressing a CAR that recognizes antigen 1 (CD22 in this example) along with a CD4-based CoCAR that recognizes antigen 2 (CD19 in this example) on the surface of target cells exhibit enhanced activity against target cells expressing high or low antigen densities compared to T cells expressing the CAR alone. [Example 4]

[0438] Co-expression of CARs and CoCARs that recognize different epitopes in the CD22 antigen enhances CAR-T cell activity against B-cell tumor cells. Cancer patients with CD19-positive B-cell tumors experience good response rates after treatment with CD19-specific immunotherapy (e.g., anti-CD19 CAR-T cells), but relapse in many patients is due to the proliferation of CD19-low or CD19-negative tumor cells. Such CD19-low or negative tumor cells maintain CD22 expression, and for this reason, we investigated whether T cells co-expressing CARs and CoCARs, both recognizing different epitopes in the CD22 antigen, could induce a superior antitumor response against tumor cells with low CD22 antigen density compared to T cells expressing anti-CD22 CAR alone. In this exemplary construct, the CAR contained a humanized anti-CD22 scFv (derived from RFB4 antibody) ligated to an IgG1-Fc spacer, CD28 transmembrane, and 4-1BB / CD3ζ signaling domain, and the CoCAR contained a mouse anti-CD22 scFv (derived from LL2 antibody) ligated to a CD4-derived hinge, transmembrane, and cytosolic domain. CAR (SEQ ID NO: 105) and CoCAR (SEQ ID NO: 107) are encoded in a single bicistronic lentiviral vector, which induces equimolar translation of both molecules in transduced cells. Primary human T cells were separately transduced with lentiviral vectors encoding CAR, CoCAR, or BBz-CAR-T2A-CoCAR, and the resulting T cells were incubated with fluorescently labeled CD22 protein to detect cell surface expression of CAR and CoCAR molecules. Flow cytometry analysis showed binding of the CD22 antigen to transduced T cells but not to untransduced T cells, demonstrating efficient cell surface expression of both anti-CD22 CAR and CoCAR molecules (Figure 4B). To compare the responsiveness between conventional anti-CD22 CAR-T cells and T cells co-expressing anti-CD22 CAR and CoCAR, T cells were incubated in cell culture plates coated with different concentrations of recombinant human CD22 antigen. After 24 hours, the concentrations of secreted effector cytokines were measured by flow cytometry using the LEGENDPlex human Th1 panel multiplex assay (BioLegend).The results showed that co-expression of anti-CD22 CoCAR significantly enhanced the activity of anti-CD22 CAR-T cells, and importantly, a smaller amount of coated antigen was sufficient to activate CAR-T cells when CoCAR was co-expressed (Figure 4C). Furthermore, stimulation using CD22-positive Nalm6 cells induced significantly higher levels of effector cytokine secretion from T cells expressing the anti-CD22 BBz-CAR-T2A-CoCAR construct (SEQ ID NO: 109) compared to conventional anti-CD22 CAR-T cells (Figure 4D). Notably, while conventional anti-CD22 CAR-T cells also secreted detectable amounts of IFN-gamma and TNF-alpha antigen-independently, possibly due to sustained signaling of the CAR, antigen-independent cytokine secretion was completely abolished with co-expression of CoCAR (Figure 4D).

[0439] Nalm6 cells express only about 3,000 CD22 molecules per cell (Figure 3D), which corresponds to the median amount of CD22 antigen found in primary B-ALL tumor cells (Shah et al., (2015) Pediatr Blood Cancer, (6) 964-969). Clinical studies have shown that a reduction in CD22 site density to <1,800 molecules per cell is a major cause of relapse after treatment with anti-CD22 CAR-T cells (Fry et al., (2017) Nat Med, (1) 20-28). To investigate whether co-expression of anti-CD22 CoCAR enhanced the sensitivity of anti-CD22 CAR-T cells to tumor cells with very low CD22 antigen expression, Nalm6 cells were engineered to express reduced CD22 antigen levels. To generate such cell lines, the CD22 gene was first disrupted using CRISPR / Cas9 technology, followed by transduction of the resulting CD22-deficient Nalm6 cells with a lentiviral vector encoding human CD22. Subsequently, single-cell cloning and analysis of CD22 expression density using Quantibrite Phycoerythrin (PE) quantification beads (Becton Dickinson) identified several Nalm6 cell clones exhibiting reduced CD22 antigen density, including clone number 3, which expressed only about 400 CD22 molecules per cell, which is well below the sensitivity limit of 4-1BB-based CARs known in the art (Figure 4E). The CAR-T2A-CoCAR co-expression system was then tested under this ultra-low antigen density experimental setting. Specifically, T cells expressing anti-CD22 CAR with or without co-expression of anti-CD22 CoCAR were incubated with Nalm6 cell clone 3, and the proliferation of GFP-positive tumor cells was monitored over a 48-hour period in the Incucyte system. As shown in Figure 4F, T cells expressing both molecules simultaneously were able to inhibit the proliferation of tumor cells expressing very low antigen densities, whereas conventional anti-CD22 CAR-T cells could not. Therefore, simultaneous expression of anti-CD22 CoCAR significantly improved sensitivity to anti-CD22 CAR.In summary, these examples demonstrate that co-expression of CoCARs significantly enhances the activity and sensitivity of T cells expressing anti-CD22 CARs against tumor cells expressing high, low, or very low antigen levels. [Example 5]

[0440] Co-expression of CoCAR reduces antigen-independent activation of T cells induced by conventional CARs. CARs may induce antigen-independent (sustained) signaling due to the aggregation tendency of some scFvs or due to high CAR expression levels, which may drive T cell differentiation and exhaustion, thereby limiting the antitumor activity of therapeutic cells (Long et al., (2015) Nat Med, (6) 581-590). The anti-CD22 CAR used in this invention may also induce sustained signaling, as indicated by the increased concentration of effector cytokines in the supernatant of unstimulated CAR-T cells (Figure 4D). Similarly, flow cytometry phenotypic analysis showed that unstimulated anti-CD22 CAR-T cells exhibited an activated phenotype, as indicated by increased expression of CD69 and CD25 activation markers, and 24-hour co-culture of anti-CD22 CAR-T cells with the CD22-negative myeloid cell line K562 further increased antigen-independent CAR-T cell activation (Figure 5A). Notably, co-expression of anti-CD22 CoCAR significantly reduced antigen-independent activation of T cells induced by anti-CD22 CAR, and T cells co-expressing CAR and CoCAR showed a considerably less activated phenotype than untransduced (CAR-negative) T cells. In contrast, stimulation with CD22-positive Nalm6 cells, with co-expression of CoCAR, significantly enhanced anti-CD22 CAR-T cell activation, as indicated by increased expression of CD69 and CD25 (Figure 5A), which is consistent with the effector cytokine secretion pattern shown in Figure 4D. In addition to activation markers, T cells expressing conventional anti-CD22 CARs exhibited increased levels of inhibitory receptors PD-1, LAG-3, and TIM-3 on their surface, regardless of antigenic stimulation, suggesting T cell exhaustion induced by sustained CAR signaling (Figure 5B). Importantly, co-expression of anti-CD22 CoCAR completely eliminated the upregulation of inhibitory receptors on the surface of anti-CD22 CAR-T cells, suggesting that co-expression of CoCAR may prevent T cell exhaustion induced by sustained CAR signaling (Figure 5B).Notably, when stimulated with CD22-positive tumor cells, T cells co-expressing CAR and CoCAR did not show enhanced expression of PD-1, LAG-3, and TIM-3 compared to conventional anti-CD22 CAR-T cells, but these T cells did show enhanced antigen-dependent activation and immune response. To investigate whether co-expression of CoCAR with CAR also inhibits T cell differentiation, T cells were analyzed by flow cytometry for the expression of differentiation markers including CD4, CD8, CD45RA, CD62L, and CD95. Indeed, T cells co-expressing anti-CD22 CAR and CoCAR contained a significantly higher fraction of CD4-positive cells with an undifferentiated stem cell memory-like phenotype (shown as CD45RA and CD62L double-positive cells) compared to T cells expressing anti-CD22 CAR alone or untransduced T cells. A similar trend was observed for CD8-positive T cells, suggesting that co-expression of CoCAR inhibits CAR-induced T cell differentiation. In summary, these results clearly demonstrate that co-expression of CoCAR with anti-CD22 CAR enhances the properties of therapeutic cells through multiple mechanisms, including inhibiting antigen-independent T cell exhaustion and differentiation while enhancing the immune response against antigen-expressing cells. [Example 6]

[0441] Co-expression of anti-CEA CoCAR enhances the antitumor activity of T cells expressing anti-EGFR CARs against low-antigen breast cancer cells. This example describes experiments conducted to investigate the effect of co-expressing CoCAR with CAR on enhancing T cell activation and function against low-antigen target cells of solid tumors. In the exemplary experiment, T cells were transduced to express an epidermal growth factor receptor (EGFR)-targeted CAR construct containing a human scFv linked to an IgG1-Fc spacer, CD28 transmembrane, and 4-1BB / CD3ζ signaling domain, with or without a CoCAR containing a carcinoembryonic antigen (CEA)-specific antigen recognition domain fused to a CD4-derived hinge, transmembrane, and intracellular domain (Figure 6A). Both the anti-EGFR CAR and the anti-CEA CoCAR (SEQ ID NO: 108) were expressed on the surface of the transduced T cells, as determined by flow cytometry (Figure 6B). Notably, three weeks after transduction, unstimulated anti-EGFR CAR-T cells were highly positive for LAG-3 and TIM-3 expression, indicating sustained signaling of the anti-EGFR CAR. However, T cells co-expressing anti-EGFR CARs with anti-CEA CoCARs showed significantly lower expression of these inhibitory receptors, indicating that CoCAR expression reduces sustained CAR signaling (Figure 6C), which is consistent with results observed for anti-CD22 CARs and CoCAR constructs (Figure 5B). EGFR expression levels were quantified in several target cell lines derived from solid tumors using Quantibrite Phycoerythrin (PE) quantification beads (Becton Dickinson). Figure 7A shows flow cytometry blots illustrating EGFR expression in exemplary human cell lines A-431 (squamous cell carcinoma), SKOV-3 (ovarian adenocarcinoma), and MCF-7 (breast carcinoma), which express extremely high (>1,300,000), high (>120,000), and low (<1,000) levels of EGFR molecules on their cell surface, respectively (Figure 7B).To investigate the activity of T cells expressing anti-EGFR CAR and anti-CEA CoCAR constructs against low-antigen solid tumor cells, T cells were co-cultured with MCF-7 cells that co-expressed low levels of EGFR and CEA antigens on their cell surface (Abdul Wahid et al, (2014) Molecular Oncology, (2) 337-350). After 24 hours of incubation, IL-2 production levels of these T cells were measured by flow cytometry using the LEGENDplex kit (BioLegend), demonstrating that co-expression of anti-CEA CoCAR significantly enhanced T cell activity induced by anti-EGFR CAR (Figure 7C). The ability of T cells to inhibit the proliferation of GFP-positive target cells was analyzed over an 84-hour period using the Incucyte instrument (Sartorius) (Figures 7D, E). As shown in Figure 7D, T cells expressing anti-EGFR CARs were able to moderately inhibit the proliferation of MCF-7 tumor cells compared to T cells expressing CEA-CoCARs or untransduced T cells. Notably, co-expression of anti-CEA CoCARs with anti-EGFR CARs significantly enhanced T cell activity against tumor cell proliferation (Figure 7D), based on their enhanced ability to kill tumor cells (Figure 7E). Taken together, these results indicate that the ability of co-expression of CoCARs to enhance CAR-T cell activity is not limited to specific CAR constructs. [Example 7]

[0442] Comparison of CoCAR constructs that possess a CD8α hinge and transmembrane domain, but also have different intracellular domains capable of binding to LCK. This experiment was conducted to determine the usefulness of LCK-binding intracellular domains in CoCAR for enhancing CAR-T cell activation and function towards low-antigen tumor cells. The CoCAR construct contained anti-CD19 scFv (clone FMC63, SEQ ID NOs. 37-54), CD8α-derived hinge and transmembrane domains, and intracellular domains derived from CD2, CD3ε, CD28, CD44, or CD146 (SEQ ID NOs. 138-142). Bi-cistronic constructs were prepared for the expression of anti-CD22 CAR (named 22-BBz) and CoCAR polypeptides containing m971 scFv, CD28-derived hinge and transmembrane domains, and 4-1BB and CD3-zeta signaling domains, with the coding sequences separated by a T2A sequence. The bicistronic constructs were as follows: 22-BBz (CAR only, SEQ ID NO: 152), 22-BBz / 19-2 (anti-CD22 CAR co-expressed with anti-CD19 CoCAR containing an intracellular CD2 domain, SEQ ID NO: 153), 22-BBz / 19-3ε (SEQ ID NO: 154), 22-BBz / 19-28 (SEQ ID NO: 155), 22-BBz / 19-44 (SEQ ID NO: 156), and 22-BBz / 19-146 (SEQ ID NO: 157). Figure 8 schematically illustrates the designs of the CAR and CoCAR constructs. None of the CoCAR constructs contained a CD3ζ activating domain and therefore acted only in trans to boost CAR. T cells co-expressing CAR and CoCAR were prepared as described, and the resulting T cells were stained with fluorescently labeled recombinant CD22 and anti-FMC63 idiotype antibodies, and CAR and CoCAR expression was determined by flow cytometry. As shown in Figure 9, CoCARs containing the CD28 intracellular domain were efficiently co-expressed with CARs on the surface of primary human T cells in a 1:1 ratio, as predicted. In contrast, cell surface expression of other CoCARs was substantially lower than that of 19-28 CoCARs, indicating that the intracellular domain plays a crucial role in CoCAR cell surface deposition. Nevertheless, none of the CoCAR constructs affected CAR expression.To investigate whether any CoCAR could enhance CAR-T cell activation in response to tumor cells expressing low CD22 antigen density, each of the CAR-T cell populations (100,000 cells) was incubated for 24 hours with wild-type NALM6 cells or with NALM6 cells expressing very low CD22 density (NALM6-CD22UL) in a 1:1 effector:target ratio. After the incubation period, T cells were harvested and stained for CAR expression and the expression of CD69 and CD25 activation markers. As shown in Figure 10, all CAR-T cell lines were efficiently activated by NALM6 cells expressing 4,000 CD22 molecules / cells, and the contribution of CoCAR was only detected faintly, if at all. In contrast, activation of T cells expressing conventional CD22 CARs was substantially low in response to NALM6-CD22UL tumor cells expressing 400 CD22 molecules / cells, and only CD28-based CoCARs were able to enhance CAR-T cell activation in this low-antigen tumor setting.

[0443] To further characterize the effect of co-expression of CAR and CoCAR on immune responses to low-antigen tumor cells, CAR-T cells (100,000 cells) were co-cultured for 24 hours with an equal number of wild-type or CD22UL NALM6 tumor cells, or with myeloid K562 tumor cells lacking CD19 and CD22 expression. The cell-free culture supernatant was analyzed by flow cytometry using the LEGENDPlex kit (BioLegend) for effector cytokine secretion. As shown in Figure 11, CAR-T cells with CoCAR containing the CD28 intracellular domain were superior to CAR-T cells co-expressing other CoCAR constructs and CAR-T cells without CoCAR in cytokine release against wild-type and CD22UL NALM6 cells, while being unresponsive to antigen-negative K562 cells. CD2-CoCAR had little effect on boosting cytokine release by CAR-T cells stimulated by NALM6-CD22UL cells, while CD44-CoCAR had no effect at all. CAR-T cells with CoCARs possessing CD3ε or CD146 intracellular domains also showed enhanced effector cytokine secretion depending on whether they were wild-type or CD22UL NALM6 cells, but this was antigen-independent, as CD22 / CD19-negative K562 cells were also stimulated to secrete cytokines by these two CAR-T cell lines.

[0444] To measure the effect of co-expression of CoCAR on the cytotoxicity of CD22 CAR-T cells, each CAR-T cell line (50,000 cells) was incubated in 96-well plates with firefly-luciferase-expressing wild-type or CD22UL NALM6 cells, or with K562 cells as a negative control, at different effector:target ratios. After 18 hours of co-culture, luciferase substrates (Bright-Glo, Promega) were added to each well, and luminescence was detected using a luminescence plate reader (Tecan). As shown in Figure 12, all CAR-T cell lines similarly killed wild-type NALM6 cells (Figure 12A), but only 22-BBz / 19-28 CAR-T cells efficiently eliminated NALM6-CD22UL tumor cells (Figure 12B). CAR-T cells co-expressing CoCAR with CD2, CD3ε, or CD146 intracellular domains showed slightly enhanced death of NALM6-CD22UL tumor cells compared to conventional CD22 CAR-T cells without CoCAR expression, and co-expression of CD44-CoCAR had no effect on the cytotoxicity of CD22 CAR-T cells. Significant death of K562 cells above background levels, as seen in untransduced (UTD) T cells, was not detected for any CAR-T cell line, despite induction of nonspecific cytokine release by 22-BBz / 19-3ε and 22-BBz / 19-146 CAR-T cells (Figure 11A-C) (Figure 12C). [Example 8]

[0445] Modification of the transmembrane domain rescued the cell surface expression and antigen-inducible functions of CoCARs containing CD44 or CD146 intracellular domains. CoCARs containing the CD44 or CD146 intracellular domain showed only weak cell surface expression in T cells compared to CoCARs with the CD28 signaling domain (Figure 9), which may explain their lack of or limited ability to enhance the responsiveness of CAR-T cells to low-antigen tumor cells (Figures 10-12). This experiment was conducted to investigate whether cell surface expression of CoCARs with the CD44 and CD146 intracellular domains could be rescued by including the CD44 and CD146 transmembrane domains (e.g., SEQ ID NOs. 136, 137) instead of the CD8a transmembrane domain. Figure 13 illustrates the domain arrangements of combinations 22-BBz / 19-44v2 and 22-BBz / 19-146v2 encoded by the bicistronic constructs of SEQ ID NOs. 165 and 166, respectively.

[0446] CAR-T cells were prepared as previously described, and the resulting T cells were stained with fluorescently labeled recombinant CD22 and anti-FMC63 idiotype antibodies. CAR and CoCAR expression was detected by flow cytometry. As shown in Figure 14, modification of the transmembrane domain restored cell surface expression of CoCARs containing the CD44 or CD146 intracellular domain in primary human T cells at a 1:1 ratio with CAR, as observed in 22-BBz / 19-28 CAR-T cells. To investigate the effect of co-expression of CAR and CoCAR on T cell differentiation, CAR-T cells were phenotypically characterized by flow cytometry. As shown in Figure 15, co-expression of CD28, CD44, or CD146 CoCAR in addition to CAR maintained T cell stem cell characteristics compared to conventional CD22 CAR-T cells, as determined by the increased proportion of CD4+CD62L+ / CD45RA+ T cells in the CAR / CoCAR-positive population. A similar trend was observed for CD8+ T cells. While I don't intend to be bound by any particular theory, this is likely due to the sequestering of free or membrane-bound LCK molecules in unstimulated CAR-T cells, thereby inhibiting nonspecific CAR-CD3ζ phosphorylation, sustained CAR signaling, and T cell differentiation.

[0447] To investigate whether 19-44v2 and 19-164v2 CoCARs could enhance 22-BBz CAR function in response to tumor cells expressing very low antigen densities, CAR-T cells were co-cultured with NALM6 tumor cells expressing variable amounts of CD22 on their cell surface. Wild-type NALM6 cells expressed approximately 4,000 CD22 molecules per cell, while the previously used NALM6-CD22UL clone 3 expressed approximately 400 CD22 molecules per cell. Subsequent functional assays included NALM6-CD22UL clone 1, which expressed 40 CD22 molecules per cell (Figure 16A), which is well below the sensitivity limit of conventional 4-1BBz- or 28z-CAR-T cells. Notably, CD19 expression was similar across different NALM6 clones and was not affected by CD22 expression (Figure 16B). Each of the CAR-T cell populations (100,000 cells) was incubated for 24 hours with wild-type NALM6 cells or with NALM6 cells expressing very low CD22 densities (NALM6-CD22UL cl.3 and cl.1) in a 1:1 effector:target ratio. After the incubation period, T cells were harvested and stained for CAR expression and the expression of CD69 and CD25 activation markers. As shown in Figure 17, all CAR-T cell lines were activated by wild-type NALM6 cells expressing 4,000 CD22 molecules / cell, but T cell activation was substantially enhanced by co-expression of any of the 19-28, 19-44v2, or 19-146v2 CoCARs. Interestingly, CoCAR expression reduced antigen-independent T cell activation mediated by persistent CAR signaling, which is consistent with the increased proportion of undifferentiated CD62L+ / CD45RA+ stem cell-like memory T cell subsets (Tscm) observed in CoCAR-expressing CAR-T cells (Figure 15). Stimulation in NALM6-CD22UL3 tumor cells led to weak activation of conventional CAR-T cells, while co-expression of either CoCAR enhanced T cell activation. CD28-based CoCARs were the most efficient polypeptides in enhancing CAR function, followed by CD44- and CD146-based CoCARs.A similar trend was observed when stimulated with NALM6-CD22UL1 tumor cells expressing only 40 CD22 molecules per cell: conventional 22-BBz CAR-T cells were unresponsive to tumor cells expressing such low antigen density, as predicted, while CD28-based CoCARs efficiently boosted CAR-T cell activation. 19-44v2 CoCARs also enhanced CAR-T cell activation in response to NALM6-22UL1 cells, albeit to a lesser extent than 19-28 CoCARs, but CD146-based CoCARs failed to boost CAR-T cell activation in response to 40 CD22 antigens per cell.

[0448] To investigate whether co-expression of CoCAR enhanced the cytotoxicity of CAR-T cells, each CAR-T cell line (50,000 cells) was incubated in 96-well plates with firefly-luciferase-expressing wild-type or CD22-low NALM6 cell lines at different effector:target ratios. After 18 hours of co-culture, luciferase substrates (Bright-Glo, Promega) were added to each well, and luminescence was detected using a luminescence plate reader (Tecan). As shown in Figure 18, all CAR-T cell lines efficiently killed wild-type NALM6 cells, but only the 22-BBz CAR-T cells co-expressing CoCAR were able to efficiently eliminate NALM6 tumor cells...

Claims

1. (i) First extracellular antigen recognition domain, (ii) The first hinge domain, (iii) The first transmembrane domain, and (iv) (a) Can bind to LCK and / or (b) At least one cytosolic domain comprising LCK or a variant or fragment thereof It includes, but does not include, CD3 zetadomain. Co-receptor-CAR (CoCAR).

2. The CoCAR according to claim 1, wherein the first antigen recognition domain is scFv.

3. The CoCAR according to any one of the claims, which is capable of specifically binding to an antigen on a target cell.

4. The CoCAR according to any one of the claims, wherein the first hinge domain is derived from a CD4, CD8, CD28, or IgG-Fc domain.

5. The CoCAR according to any one of the claims, wherein the first transmembrane domain is derived from a CD4, CD8, CD28, CD44, CD146, or CD3 zeta transmembrane domain.

6. (a) At least one cytosolic domain capable of binding to LCK, (1) An intracellular signaling domain derived from the CD4, CD8α, CD28, CD3ε, CD44, or CD146 intracellular signaling domain or its variant or fragment, or (2) Including one or more motifs that can be attached to LCK, The CoCAR according to any one of the above claims.

7. The CoCAR according to claim 6, wherein the motif that can bind to LCK is derived from the CD4, CD8α, CD28, CD3ε, CD44, or CD146 intracellular signaling domain.

8. (a) CoCAR according to any one of claims 1 to 7, and (b) Chimeric antigen receptor (CAR) The combination.

9. A nucleic acid, or a combination of a first and a second nucleic acid, (a) The nucleic acid codes a CoCAR as described in any one of claims 1 to 7 or a combination as described in claim 8, and (b) A nucleic acid or a combination of the first and second nucleic acids, wherein the combination codes for the combination described in claim 8, wherein the first nucleic acid codes for CoCAR(a) and the second nucleic acid codes for CAR(b).

10. A nucleic acid construct, or a combination of a first and a second nucleic acid construct, (a) The nucleic acid construct comprises the nucleic acid described in claim 9 or a combination of the first and second nucleic acids, wherein the nucleic acid and / or the first and second nucleic acids are functionally linked to at least one expression control sequence, and (b) A combination of first and second nucleic acid constructs, wherein the first nucleic acid construct comprises the first nucleic acid described in claim 9, and the second nucleic acid construct comprises the second nucleic acid described in claim 9, and the first and second nucleic acids are independently functionally linked to at least one expression control sequence. A nucleic acid construct or a combination of a first and second nucleic acid construct.

11. A vector, or a combination of a first and a second vector, (a) The vector comprises the nucleic acid described in claim 9 or a combination of the first and second nucleic acids and / or the nucleic acid construct described in claim 10 or a combination of nucleic acid constructs, and / or (b) In the combination described above, the first vector comprises the first nucleic acid and / or the first nucleic acid construct, and the second vector comprises the second nucleic acid and / or the second nucleic acid construct described in claims 9 and 10. A vector or a combination of the first and second vectors.

12. Recombinant cells comprising the nucleic acid construct according to claim 9.

13. Recombinant cells according to claim 12, which are immune effector cells.

14. Recombinant cells according to claim 12 or 13, which express CoCAR and / or the combination thereof on the cell surface.

15. (i) CAR and (ii) CoCAR recognize different epitopes located within the same antigen expressed on the surface of a target cell, according to any one of claims 12 to 14.

16. (i) CAR and (ii) CoCAR recognize different antigens expressed on the surface of target cells, according to any one of claims 12 to 14.

17. (i) CoCAR according to any one of claims 1 to 7, (ii) The combination described in claim 8, (iii) The nucleic acid, combination of nucleic acid, nucleic acid construct, combination of nucleic acid construct, vector and / or combination of vector according to any one of claims 9 to 11, and / or (iv) Recombinant cell according to any one of claims 12 to 16 as well as pharmaceutically acceptable carriers A pharmaceutical composition containing the following:

18. A CoCAR according to any one of claims 1 to 7, a combination according to claim 8, a nucleic acid according to any one of claims 9 to 11, a combination of nucleic acids, a nucleic acid construct, a combination of nucleic acid constructs, a vector, a combination of vectors, a recombinant cell according to any one of claims 12 to 16, and / or a pharmaceutical composition according to claim 17, for use in pharmaceuticals.

19. A CoCAR according to any one of claims 1 to 7, a combination according to claim 8, a nucleic acid according to any one of claims 9 to 11, a combination of nucleic acids, a nucleic acid construct, a combination of nucleic acid constructs, a vector, a combination of vectors, a recombinant cell according to any one of claims 12 to 16, and / or a pharmaceutical composition according to claim 17, for use in the treatment of cancer.

20. 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-L CoCAR, combination, nucleic acid, combination of nucleic acids, nucleic acid construct, combination of nucleic acid constructs, vector, combination of vectors, recombinant cells and / or pharmaceutical compositions according to claim 18 or 19, for the treatment of a malignant tumor having cell surface expression of at least one tumor-associated antigen selected from the group consisting of C, IGLV3-21-R110, Lewis Y, mesothelin, MUC1, MUC16, NKG2D ligand, NCAM, NY-ESO1, carcinoembryonic antigen h5T4, PSCA, PSMA, ROR1, TAG-72, VEGFR, GOLPH2 and SLAMF7.

21. CoCAR, combination, nucleic acid, combination of nucleic acids, nucleic acid construct, combination of nucleic acid constructs, vector, combination of vectors, recombinant cells and / or pharmaceutical compositions according to any one of claims 18 to 20, for use in the treatment of B-cell malignancies.

22. A method for treating cancer in a person who requires treatment for cancer, (i) CoCAR according to any one of claims 1 to 7, (ii) The combination described in claim 8, (iii) A nucleic acid, combination of nucleic acids, nucleic acid construct, combination of nucleic acid constructs, vector, combination of vectors, and / or according to any one of claims 9 to 11. (iv) Recombinant cell according to any one of claims 12 to 16 A method comprising the step of administering an effective amount of [a substance].

23. Cancer is associated with 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, and ErbB The method according to claim 22, wherein the malignant tumor is positive for at least one tumor-associated antigen selected from the group consisting of FBP, AchR, Fr-a, GD2, GD3, HMW-MAA, IL13Ra2, Kappa-LC, IGLV3-21-R110, Lewis Y, Mesothelin, MUC1, MUC16, NKG2D ligand, NCAM, NY-ESO1, carcinoembryonic antigen h5T4, PSCA, PSMA, ROR1, TAG-72, VEGFR, GOLPH2, and SLAMF7.

24. The method according to claim 22 or 23, wherein the cancer is a B-cell malignant tumor.

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