CHIMERIC CO-STIMULATORY PROTEINS COMPRISING MUTANT INTRACELLULAR DOMAINS WITH INCREASED EXPRESSION

BACKGROUND

While adoptive cell therapies show efficacy in cancer treatment, the effectiveness of these therapies can be further improved through genetic engineering of T cells for better expansion and persistence. T cells require functionally non-overlapping co-stimulatory signals from CD28 family and tumor necrosis factor receptor (TNFR) family along with antigen triggered TCR signaling to promote full-fledged activation and persistent proliferation. In developing gene-engineered T cell therapeutics, there is a need to introduce chimeric T cell co-stimulatory molecules that can be locally activated upon T cell engaging with pathological antigens to potently augment T cell activation for increased therapeutic efficacy. Currently, the second generation of chimeric co-stimulatory molecules incorporating one co-stimulatory signaling domain from proteins of either CD28 family or TNFR family has been widely adopted in CAR T cell therapy. Chimeric antigen receptors (CARs) integrating one co-stimulatory signaling domain augment T cell function through both activating and co-stimulatory signals, thus resulting in enhanced anti-tumor potency and T cell persistence. Given that the capacity for T cell to expand depends on the structural design, the current second generation co-stimulatory proteins may not be optimal for induction of a durable tumor remissions. Thus, there is a desired effort to develop third-generation chimeric molecules combining two co-stimulatory signaling domains from CD28 family and TNFR family members to further enhance T cell therapeutic potential, capitalizing on non-overlapping functions of the two families of co-stimulatory molecules. In addition, such third-generation chimeric co-stimulatory molecules can be integrated into TCR T therapy where T cell activation remains suboptimal due to insufficient co-stimulatory signals during activation of exogenously expressed TCRs by antigens. However, existing recombinant DNA strategies often suffer from reduced cell surface expression of the chimeric proteins combining two co-stimulatory signaling domains, preventing realization of the functional potential of the chimeric proteins. The present application addresses such needs. The present application discloses third-generation chimeric T cell co-stimulatory molecules that incorporate two signaling domains from CD28 and TNFR families and express at significantly improved levels than what have been conventionally reported for enhanced T cell functions, and methods of making the co-stimulatory molecules.

SUMMARY

Provided herein are novel chimeric co-stimulatory intracellular domains. The chimeric co-stimulatory intracellular domains provided herein comprise: (a) a first signaling domain that is based on the intracellular signaling domain of a CD28 family protein; and (b) at least a second signaling domain that comprises a mutant intracellular signaling domain of a tumor necrosis factor receptor (TNFR) family protein.

In some embodiments, the first signaling domain that is based on the intracellular signaling domain of a CD28 family proteins is selected from a CD28 protein, ICOS protein or a combination thereof. In some embodiments, the at least second signaling domain is based on a mutant of the intracellular signaling domain of a TNFR family protein selected from CD137 (4-1BB) and CD134 (OX-40).

The chimeric co-stimulatory intracellular domains provided herein comprise: (a) a first signaling domain that is based on the intracellular signaling domain of a CD28 protein, ICOS protein or a combination thereof; and (b) at least a second signaling domain that comprises a mutant CD137 (4-1BB) intracellular domain or a mutant CD134 (OX-40) intracellular domain. In some embodiments, the mutant CD137 (4-1BB) intracellular domain or the mutant CD134 (OX-40) intracellular domain comprises a deletion, an insertion or a substitution of one or more amino acids in the membrane proximal portion of the CD137 or CD134 intracellular domain. Without being bound by theory, in some embodiments, the one or more amino acids in the membrane proximal portion can be ubiquitination sites involved in the ubiquitination and degradation of the CD137 or CD134 protein.

Also disclosed herein is a functionally optimized intracellular co-stimulatory domain for use in novel adoptive cell therapy, optionally in combination with cell-intrinsic immune checkpoint inhibitory receptors or immune-stimulatory receptors or portions thereof, developed to treat human diseases and disorders, including hematological and solid tumors. Also disclosed herein is a functionally optimized intracellular co-stimulatory domain for use in combination with a T cell receptor (TCR), e.g. an endogenous TCR or an affinity enhanced TCR targeting a tumor-associated antigen. Optionally, the intracellular co-stimulatory domain is used in combination with a second component (e.g., a cell surface receptor or portion thereof) that directs migration of an immune cell to bind to a target tissue or cell or induces activation and/or proliferation of an immune cell, such as a PD-1 switch receptor (PD-1 based co-stimulatory molecule), that can increase T cell functionality in tumors, such as a PD-L1/PD-L2-expressing tumor. Also disclosed herein is a therapy that utilizes the PD-1 checkpoint blockade in a cell-intrinsic fashion, which simultaneously minimizes autoimmune side effects and provides increased on-tumor functionality. The present application discloses recombinant T cell co-stimulatory receptors (RTCRs) based on T cell co-receptors or chimeric antigen receptors (CARs) comprising a functionally optimized intracellular co-stimulatory domain of the present application. The present application also discloses T cell co-receptors comprising a functionally optimized intracellular co-stimulatory domain and a PD-1 extracellular domain (i.e., PD-1 switch receptors or PD-1 based co-stimulatory molecules). The present application also discloses CD19 and B cell maturation Ag (BCMA) based CARs comprising a functionally optimized intracellular co-stimulatory domain that promotes CD19 and BCMA binding mediated T cell activation, proliferation, and tumor killing. The RTCRs disclosed in the present application can be used for evaluation of checkpoint targets, safety screening, and for development of pre-clinical animal models to evaluate the effectiveness of the combination of the functionally optimized intracellular co-stimulatory domain of the present application with any TCRs or CARs. Additional cell-intrinsic immune checkpoint inhibitors with the efficacious TCRs are also developed.

The present disclosure provides a recombinant T cell co-stimulatory receptor (RTCR), comprising: (a) an extracellular domain; (b) a transmembrane domain; and (c) a chimeric intracellular domain comprising a first and at least a second signal transduction domains, wherein the first and the at least second signal transduction domains are non-identical; and wherein the at least second signal transduction domain comprises a mutant intracellular signaling domain of a tumor necrosis factor receptor (TNFR) family protein.

The present disclosure provides a recombinant T cell co-stimulatory receptor (RTCR), comprising: (a) an extracellular domain; (b) a transmembrane domain; and (c) a chimeric intracellular domain comprising a first and at least a second signal transduction domains, wherein the first and the at least second signal transduction domains are non-identical; and wherein the at least second signal transduction domain comprises a mutant CD137 (4-1BB) intracellular domain or a mutant CD134 (OX-40) intracellular domain.

The present disclosure also provides a nucleic acid encoding the RTCR disclosed herein. The present disclosure also provides a vector comprising the nucleic acid disclosed herein. The present disclosure also provides a cell comprising the nucleic acid or the vector disclosed herein.

The present disclosure also provides a composition comprising the RTCR disclosed herein. The present disclosure also provides a composition comprising the nucleic acid encoding the RTCR disclosed herein. The present disclosure also provides a composition comprising the vector comprising the nucleic acid disclosed herein. The present disclosure also provides a composition comprising the cell disclosed herein. The present disclosure also provides a composition comprising the modified T cell disclosed herein.

The present disclosure also provides a composition comprising a population of cells, wherein the population comprises a plurality of the cell comprising the nucleic acid encoding or a vector comprising the nucleic acid encoding the RTCR disclosed herein. The present disclosure also provides a composition comprising a population of cells, wherein the population comprises a plurality of the modified T cell disclosed herein.

The present disclosure provides a method of producing a plurality of modified T cells, wherein the method comprises: a) providing a plurality of primary T cells disclosed herein; b) providing a composition comprising the RTCR disclosed herein, the nucleic acid encoding the RTCR disclosed herein, or the vector comprising the nucleic acid encoding the RTCR disclosed herein; and c) introducing into the plurality of primary T cells of (a) the composition of (b), to produce a plurality of modified T cells under conditions that stably express the RTCR within the plurality of modified T cells. In some embodiments, the method of producing a plurality of modified T cells disclosed herein, further comprises a step of modifying an endogenous sequence encoding an endogenous T cell Receptor (TCR), wherein the modification reduces or eliminates a level of expression or activity of the endogenous TCR. In some embodiments, the method of producing a plurality of modified T cells disclosed herein, further comprises a step of modifying an endogenous sequence, wherein the modification reduces or eliminates a level of expression or activity of a major histocompatibility complex (MHC) class I (MHC-I). In some embodiments, the method of producing a plurality of modified T cells disclosed herein, further comprises: d) maintaining the plurality of modified T cells in a suitable cell culture media; and e) either: i) cryopreserving the plurality of modified T cells in a suitable cell freezing media; or ii) preparing the plurality of modified T cells for administering to a subject suffering from a disease or disorder.

The present disclosure also provides a method of treating a disease or disorder, comprising administering to a subject in need thereof a therapeutically effective number of the cell comprising the nucleic acid encoding or the vector comprising the nucleic acid encoding the RTCR disclosed herein, a therapeutically effective number of any one of the modified T cell disclosed herein, a therapeutically effective amount of any one of the compositions disclosed herein, or a therapeutically effective number of the plurality of modified T cells produced by the method disclosed herein.

The present disclosure also provides a chimeric co-stimulatory intracellular protein (CIP) comprising a first and at least a second signal transduction domains, wherein the first and the at least second signal transduction domains are non-identical; and wherein the at least second signal transduction domain comprises a mutant intracellular signaling domain of a tumor necrosis factor receptor (TNFR) family protein.

The present disclosure also provides a chimeric co-stimulatory intracellular protein (CIP) comprising a first and at least a second signal transduction domains, wherein the first and the at least second signal transduction domains are non-identical; and wherein the at least second signal transduction domain comprises a mutant CD137 (4-1BB) intracellular domain or a mutant CD134 (OX-40) intracellular domain.

Throughout the specification the term “comprising,” or variations such as “comprises” or “comprise,” will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

Throughout the specification the term “signal domain”, “signaling domain”, and “signal transduction domain”, are used interchangeably, unless the context dictates otherwise.

While the disclosure has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the disclosure, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. Genbank and NCBI submissions indicated by accession number cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference.

DESCRIPTION OF THE FIGURES

FIG. 1 depicts alignment of the intracellular tails of TNF receptor superfamily members that are used in the T cell co-stimulatory molecules of the present application. Membrane-proximal poly-basic regions are italicized. Potential PI3K binding sites are bold and underlined. TRAF1/2 binding motifs: major motif Px(Q/E)E and minor motif Px(Q/E)x, are underlined. Potential ubiquitination sites are in bold.

FIGS. 2A-2B depict the modular design of 2^ndand 3^rdgeneration co-stimulatory molecules. FIG. 2A depicts modular design of co-stimulatory molecules denoting the signal peptide, extracellular domain, transmembrane domain, and intracellular signaling domain. FIG. 2B depicts structures and sequences of first signal transduction domains: ICOS, CD28 and ICOS intracellular domain with a portion of CD28 domain inserted. Regions and known binding partners of the ICOS and CD28 intracellular domain with specific binding function are indicated. The amino acid/nucleic acid sequences of the co-stimulatory molecules and the intracellular domains are as indicated.

FIG. 3 depicts the combinations of extracellular effector domains and intracellular signaling domains of the present application.

FIGS. 4A-4B depict that deletion of the N-terminal section of the 4-1BB signaling domain, including the polybasic domain and lysine residues, rescues the expression of the co-stimulatory molecules. FIG. 4A depicts expression of human PD1 and huEGFRt on the surface of T-lymphocytes following lentiviral transduction with the indicated construct. FIG. 4B depicts the normalized PD1 surface expression on huEGFRt-expressing cells expressing different co-stimulatory molecules with ICOS or CD28 based chimeric intracellular domains comprising wild type or truncated 4-1BB domains or OX-40 domains, as indicated. The amino acid/nucleic acid sequences of the chimeric intracellular domains are as indicated.

FIGS. 5A-5D depict cytokine production and proliferation of T cells expressing different co-stimulatory molecules with ICOS based chimeric intracellular domains comprising wild type or truncated 4-1BB or OX-40 domains. FIGS. 5A-5C depict that antibody-mediated crosslinking of co-stimulatory molecules increases T cell cytokine production in-vitro. IL-2 (FIG. 5 A), TNF (FIG. 5 B), and IFNγ (FIG. 5 C) were measured by bead-based multiplex assay in culture supernatants following 18 hr stimulation of T cells transduced with the indicated constructs with plate-bound anti-PD1 [2 μg/mL] and the indicated amount of plate-bound anti-CD3. The x-axis indicates the different co-stimulatory molecules and the y-axis indicates the amount of cytokine produced expressed as absorbance unit (A.U.). FIG. 5D depicts proliferation of T cells stimulated with the indicated plate-bound antibodies for 96 hrs. The amino acid/nucleic acid sequences of the chimeric intracellular domains are as indicated.

FIGS. 6A-6C depict that PD-L1 engagement of co-stimulatory molecules increases T cell cytokine production in-vitro. FIG. 6A depicts IL-2 (upper panel), IFNγ (middle panel), and TNF (lower panel) measured by bead-based multiplex assay in culture supernatants following 18 hr stimulation of T cells transduced with the indicated constructs. The x-axis indicates amount of anti-CD3 antibody (μg/ml) and the y-axis indicates cytokine production as percentage of control. FIGS. 6B-6C depict IL-2 (upper panels), IFNγ (middle panel), and TNF (lower panel) production by T cells transduced with the indicated co-stimulatory molecules comprising CD28 intracellular domain (FIG. 6B, upper, middle and lower panels) and ICOS intracellular domain (FIG. 6C, upper, middle and lower panels), respectively, and stimulated with the indicated concentration of soluble anti-CD3 antibody in the presence of K562 cells expressing HLA-A2 (left panels) or HLA-A2 and PD-L1 (right panels). The x-axis indicates amount of anti-CD3 antibody (μg/ml) and the y-axis indicates cytokine production as percentage of control. The amino acid/nucleic acid sequences of the co-stimulatory molecules are as indicated.

FIGS. 7A-7C depict that PD-L1 engagement of co-stimulatory molecules increases T cell cytotoxicity and proliferation in-vitro. T cells expressing PD-1 constructs, as indicated, and K562 cells were mixed and stimulated as in FIG. 6. FIGS. 7A and 7B depict the number of remaining K562 cells (upper panel) and number of T cells (lower panel) evaluated by flow cytometry, after 96 hours of stimulation with the indicated concentration of soluble anti-CD3 antibody in the presence of K562 cells expressing HLA-A2 (left panels) or HLA-A2 and PD-L1 (right panels). FIG. 7C depicts proliferation of T cells expressing the various PD1 constructs co-cultured with K562 cells expressing HLA-A2 (indicated by “X”) or HLA-A2 and PD-L1 (indicated by “*”) and 0.3 μg/ml of anti-CD3, as measured by shift in Cell Trace violet dilution as indicated on x-axis. FIG. 7D is a graph depicting target cell (K562) numbers remaining evaluated by flow cytometry, after 96 hours post stimulation with T cells expressing co-stimulatory molecules, in presence of increasing amounts anti-CD3 antibody (μg/ml), as indicated. FIG. 7E is a graph depicting number of T cells evaluated by flow cytometry, after 96 hours post stimulation with T cells expressing co-stimulatory molecules, in presence of increasing amounts anti-CD3 antibody (μg/ml), as indicated. The x-axis indicates amount of CD3 antibody (μg/ml) and the y-axis indicates number of cells. The amino acid/nucleic acid sequences of the co-stimulatory molecules are as indicated.

FIG. 8 depicts that engagement of co-stimulatory molecules increases T cell proliferation in-vitro. T cells were stimulated for 96 hrs on plate-bound antibodies with 2 μg/mL anti-PD1 and the concentration of anti-CD3 [mg/mL] (indicated by “*”) or only anti-CD3 (indicated by “X”), as indicated on y-axis, and proliferation of T cells expressing the various PD1 constructs as measured by shift in crystal violet tracing as indicated on x-axis. The amino acid/nucleic acid sequences of the co-stimulatory molecules are as indicated.

FIGS. 9A-9C depict the effect of mutation of the polybasic and lysine residues on expression or function of co-stimulatory molecules incorporating ICOS and 4-1BB signaling domains. FIG. 9A is a series of flow cytometry plots depicting proliferation of T-cells expressing either a wild type PD1 receptor (indicated by “*”) or the different PD1 based co-stimulatory molecules (indicated by “X”), as indicated by labeling at top of each plot. T-cells expressing endogenous PD-1 were used as control (line with no indication). FIG. 9B is a graph depicting PD-1 expression (expressed as a fold increase from endogenous levels) from the FACS plots in FIG. 9A. FIG. 9C are graphs depicting cytokine production (IL-2, left panels; IFNy, middle panels; and TNF, right panels) (y-axis) by T cells expressing different co-stimulatory molecules, as indicated, responding to K562 cells (top row) and K562-PDL1 expressing cells (middle row), when stimulated with the indicated concentration of anti-CD3 (x-axis). The difference between the level of cytokine production between T cells responding to K562 cells and K562-PDL1 expressing cells, is depicted in the graphs in the bottom row. FIG. 9D are graphs depicting proliferation of T cells 96 hr post culturing with K562 cells (left graph) or K562 cells expressing PD-L1 (middle graph), when stimulated with the indicated concentration of anti-CD3 (x-axis). The amino acid/nucleic acid sequences of the co-stimulatory molecules are as indicated.

FIGS. 10A-10D depicts the expression and function of co-stimulatory molecules incorporating ICOS and OX-40 signaling domains. FIG. 10A is a series of flow cytometry plots depicting proliferation of T-cells expressing either a wild type PD1 receptor (indicated by “*”) or the different PD1-switch receptors (PD1 based costimulatory molecules) (indicated by “X”), as indicated by labeling at top of each plot. T-cells expressing endogenous PD-1 were used as control (no indication). FIG. 10B is a graph depicting PD-1 expression (fold of endogenous expression) from the FACS plots in FIG. 10A. FIG. 10C are graphs depicting cytokine production (IL-2, left panels; IFNy, middle panels; and TNF, right panels) (y-axis) by T cells expressing different co-stimulatory molecules, as indicated, responding to K562 cells (top row) and K562-PDL1 expressing cells (middle row), when stimulated with the indicated concentration of anti-CD3 (x-axis). The difference between the level of cytokine production between T cells responding to K562 cells and K562-PDL1 expressing cells, is depicted in the graphs in the bottom row. FIG. 10D are graphs depicting T Cell proliferation 96 hr post culturing with K562 cells (left graph) or K562 cells expressing PD-L1 (middle graph), when stimulated with the indicated concentration of anti-CD3 (x-axis). The difference between T cell proliferation in the presence or absence of PD-L1 on the target cells is depicted in the right-most graph. The amino acid/nucleic acid sequences of the co-stimulatory molecules are as indicated.

FIGS. 11A-11B depict that engagement of co-stimulatory molecules increases T cell conjugation with PD-L1 expressing cells. FIG. 11A depicts flow cytometry gating strategy of a 30-minute conjugation of CFSE-labelled T cells with CTV-labelled K562 targets. FIG. 11B depicts quantification of results from two experiments shown in FIG. 11A and normalized to control conjugations. The amino acid/nucleic acid sequences of the co-stimulatory molecules, with and without a signaling peptide, are as indicated.

FIGS. 12A-12C depict increase in T cell proliferation and function upon engagement of co-stimulatory molecules with PD-L1 expressing cells. FIG. 12A are flow cytometry plots depicting surface expression of PD-1 and TCR β chain, in T cells expressing either a wild type HLA-A2/NY-ESO-1 specific TCRs or mutant NY-ESO TCR as indicated, with (lower middle and right plots) and without (upper middle and right plots) a co-stimulatory molecule construct comprising an ICOS_4-1BB (truncated) signaling domain (PD-1_ICOS_BBt), as indicated, when co-cultured with K562 cells), 72 hrs after lentiviral transfection. FIG. 12B are graphs depicting IL-2 (top graph) and IFNγ (bottom graph) production T cells expressing NY-ESO-1/PD1 based co-stimulatory molecule combinations, as indicated, when co-cultured with A375-tumor cells that express HLA-A2 and antigen, at T cell: A375 cell ratio as indicated in x-axis. FIG. 12C is a graph depicting dose dependent killing of A375 cells by T cells expressing the indicated wild type NY-ESO TCR or mutant, high affinity (HA) NY-ESO TCR, as indicated with/without a co-stimulatory molecules construct comprising an ICOS_4-1BB (truncated) signaling domain (PD-1_ICOS_BBt), as indicated. The x-axis depicts the dose (T cell: A375 cell ratio) and percentage of total input A375 cells surviving. The amino acid/nucleic acid sequences of the co-stimulatory molecules, with and without a signaling peptide, are as indicated.

FIGS. 13A-13B depict that mutations of 3^rdgeneration tails increase surface expression of CD-19 CAR receptors on transduced primary T cells. FIG. 13A depicts histograms of CD-19Fc binding to the untransduced T cells (marked by x) or T cells transduced with the indicated constructs (marked by *). FIG. 13B depicts MFI measurements of histograms shown in FIG. 13A, normalized to FMC63scFV_BB_Z. The amino acid/nucleic acid sequences of the co-stimulatory molecules are as indicated.

FIGS. 14A-14C depict that modified 3^rdgeneration tails increase cytokine production and tumor killing. In-vitro killing of CD19-positive cells by CAR-transduced primary T cells is shown. FIG. 14A depicts residual cell number of B cell line (Nalm6 cells), after a 96-hr co-culture with CAR-T cells expressing CD28-based (left panel) and ICOS-based (right panel) 2^ndgeneration and 3^rdgeneration receptors. FIG. 14B depicts residual cell number of B cell line (Raji cells), after a 96-hr co-culture with CAR-T cells expressing CD28-based (left panel) and ICOS-based (right panel) 2^ndgeneration and 3^rd-generation receptors. The y-axis depicts number of remaining CD19-positive cells corresponding to the ratio of T cells to CD19-positive cell indicated on x-axis. FIG. 14C depicts 18 hr-IFNγ production, as indicated on the y-axis, by T cells expressing 2^ndgeneration and 3^rdgeneration, CD28-based receptors (left panel) and ICOS-based receptors (right panel), in response to incubation with CD19-positive B cells for 18 hours, at T cell: target cell ratio, as indicated on x-axis. The amino acid/nucleic acid sequences of the co-stimulatory molecules are as indicated.

FIG. 15A-15E depict modified 3^rdgeneration signaling domains increase CD19-CAR function in-vitro, compared to original 3^rdgeneration signaling domains. FIG. 15A is a graph depicting cumulative T cell numbers (indicative of T cell proliferation) (y-axis) of T-cells expressing: a) CD28-based 2^ndand 3^rdgeneration receptors (left panel); and b) ICOS-based and 3^rd-generation receptors (right panel), as indicated, over repeated stimulations with Nalm6 B cells, as indicated on x-axis. FIG. 15B is a graph depicting cumulative T cell numbers (indicative of T cell proliferation) (y-axis) of T-cells expressing: a) CD28-based 2^ndand 3^rdgeneration receptors (left panel); and b) ICOS-based 2^ndand 3^rdgeneration receptors (right panel), as indicated, over repeated stimulations with RAJI B cells, as indicated on x-axis. FIG. 15C is a graph depicting cumulative target cell (Nalm6) numbers (indicative of target cell killing) (y-axis) of T-cells expressing: a) CD28-based 2^ndand 3^rdgeneration receptors (left panel); and b) ICOS-based 2^ndand 3^rdgeneration receptors (right panel), as indicated, over repeated stimulations with Nalm6 B cells, as indicated on x-axis. FIG. 15D is a graph depicting cumulative target cell (Raji) numbers (indicative of target cell killing) (y-axis) of T-cells expressing: a) CD28-based 2^ndand 3^rdgeneration receptors (left panel); and b) ICOS-based 2^ndand 3^rdgeneration receptors (right panel), as indicated, over repeated stimulations with Raji B cells, as indicated on x-axis. FIG. 15E is a series of flow cytometry plots depicting Tim3 and PD-1 expression on CAR-T cells, as indicated, at time zero or after 5 consecutive stimulations, with RAJI B cell targets. The amino acid/nucleic acid sequences of the co-stimulatory molecules are as indicated.

FIGS. 16A-16E depicts modified 3^rd-generation signaling domains increase BCMA-CAR function in-vitro, compared to original 3^rd-generation sequences. FIG. 16A depicts flow cytometry histograms of BCMA-Fc binding to untransduced T cells (indicated by “X”) or T cells transduced with the indicated BCMA CAR-T receptor comprising CD28-based and ICOS based-2^ndgeneration and 3^rdgeneration co-stimulatory molecules (indicated by “*”), as indicated. FIG. 16 B is a graph depicting BCMA-Fc binding (MFI) (x-axis) by from the transduced T cells of the FACS plots in FIG. 16A. FIG. 16C is a set of graphs depicting cumulative T cell numbers (indicative of T cell proliferation) (y-axis) of T-cells expressing: a) CD28-based 2^ndand 3^rdgeneration receptors (left panel); and b) ICOS-based 2^ndand 3^rdgeneration receptors (right panel), as indicated, over repeated stimulations with RPMI-8226 multiple myeloma target cells. FIG. 16D is a graph depicting cumulative target cell (RPMI-8226 cells) numbers (indicative of target cell killing) (y-axis) of T-cells expressing: a) CD28-based 2^ndand 3^rdgeneration receptors (left panel); and b) ICOS-based 2^ndand 3^rdgeneration receptors (right panel), as indicated, over repeated stimulations with RPMI-8226 multiple myeloma target cells, as indicated on x-axis. FIG. 16E is a set of graphs depicting cytokine production (IL-2, left panel, TNF, middle panel and IFNγ, right panel) (y-axis) with CAR-T cells transduced with the indicated BCMA CAR constructs comprising CD28-based or ICOS-based co-stimulatory molecules, as indicated, incubated at the indicated effector to target ratio indicated on x-axis, for 18 hrs. The amino acid/nucleic acid sequences of the co-stimulatory molecules are as indicated.

DETAILED DESCRIPTION

The CD28 family proteins have a single extracellular immunoglobulin variable-like (IgV) domain followed by a short cytoplasmic tail. Members of the CD28 family proteins include CD28, CD28H, inducible costimulator (ICOS), cytotoxic T lymphocyte-associated antigen 4 (CTLA-4, CD152), program death-1 (PD-1), and B- and T-lymphocyte attenuator (BTLA). CD28, CD28H and ICOS are co-stimulatory proteins that are expressed on T cells that promote activation, high levels of cytokine/chemokine expression, resistance to apoptosis, and proliferation of T cells.

The Tumor Necrosis Factor Receptor (TNFR) family proteins includes TNFR1 (tumor necrosis factor receptor 1/TNFRSF1A), TNFR2 (tumor necrosis factor receptor 2/TNFRSF1B), lymphotoxin β receptor/TNFRSF3, OX40/TNFRSF4, CD40/TNFRSF5, Fas/TNFRSF6, decoy receptor 3/TNFRSF6B, CD27/TNFRSF7, CD30/TNFRSF8, 4-1BB/TNFRSF9, DR4 (death receptor 4/TNFRSF10A), DR5 (death receptor 5/TNFRSF10B), decoy receptor 1/TNFRSF10C, decoy receptor 2/TNFRSF10D, RANK (receptor activator of NF-kappa B/TNFRSF11A), OPG (osteoprotegerin/TNFRSF11B), DR3 (death receptor 3/TNFRSF25), TWEAK receptor/TNFRSF12A, TACI/TNFRSF13B, BAFF-R (BAFF receptor/TNFRSF13C), HVEM (herpes virus entry mediator/TNFRSF14), nerve growth factor receptor/TNFRSF16, BCMA (B cell maturation antigen/TNFRSF17, GITR (glucocorticoid-induced TNF receptor/TNFRSF18), TAJ (toxicity and JNK inducer/TNFRSF19), RELT/TNFRSF19L, DR6 (death receptor 6/TNFRSF21), TNFRSF22, TNFRSF23, ectodysplasin A2 isoform receptor/TNFRS27 and ectodysplasin 1-anhidrotic receptor. Interactions between tumor necrosis factor superfamily (TNFSF) ligands and TNF receptor superfamily (TNFRSF) receptors provide the co-stimulatory signals that control the survival, proliferation, differentiation, and effector function of immune cells. Depending upon the specific intracellular signal induced by TNFRSF members, they can be categorized into three groups—death domain (DD)-containing receptors, decoy receptors, and TNF receptor-associated factor (TRAF)-binding receptors. Some TNFRSFs such as TNFR-1, Fas, DR3, DR4, DR5, and DR6, contain their own DDs and/or interact with other cytoplasmic DD-containing adaptor molecules. Some other TNFRSFs, such as TNFR-2, CD27, CD30, CD40, glucocorticoid-induced TNFR family-related gene (GITR), Fn1, lymphotoxin beta-receptor (LTβR), OX40, receptor activator of NF-κB (RANK), and XEDAR, lack a DD and contain motifs with four to six amino acids called TRAF-interacting motifs (TIMs) which recruits TRAF proteins. TRAF proteins are adaptor molecules that activate multiple downstream signaling pathways such as NF-κB, Janus kinase (JNK), ERK, p38MAPK, and PI3K that help in cell survival, proliferation, and cytokine production. In some embodiments, the first signaling domain that is based on the intracellular signaling domain of a CD28 family protein is selected from a CD28 protein, ICOS protein or a combination thereof. In some embodiments, the at least second signaling domain is based on a mutant of the intracellular signaling domain of a TNFR family protein is selected from CD137 (4-1BB) and CD134 (OX-40).

Provided herein are novel chimeric co-stimulatory intracellular domains based on the third-generation co-stimulatory domains of the present application. Reduced surface expression is a major hindrance in the development of chimeric co-stimulatory proteins for therapeutic purposes. The present disclosure provides novel chimeric co-stimulatory intracellular domains generated through mutations in the third-generation co-stimulatory domains of the present application that are both highly expressed and highly functional compared to the current second-generation and third-generation chimeric receptors that are effective in inducing costimulation. The chimeric co-stimulatory intracellular domains provided herein comprise: (a) a first signaling domain that is based on the intracellular signaling domain of a CD28 protein, ICOS protein or a combination thereof; and (b) at least a second signaling domain that is a mutant CD137 (4-1BB) intracellular domain or a mutant CD134 (OX-40) intracellular domain. In some embodiments, the mutant CD137 (4-1BB) intracellular domain or the mutant CD134 (OX-40) intracellular domain comprises a deletion, an insertion or a substitution of one or more amino acids in the membrane proximal portion of the CD137 or CD134 intracellular domain. In some embodiments, the one or more amino acids in the membrane proximal portion are ubiquitination sites involved in the ubiquitination and degradation of the CD137 or CD134 protein. In some embodiments, the mutant CD137 (4-1BB) intracellular domain or a mutant CD134 (OX-40) intracellular domain comprises substitution or deletion of one or more lysine residues in the membrane proximal portion of the CD137 or CD134 intracellular domain. In some embodiments, the lysine residues are ubiquitination sites involved in the ubiquitination and degradation of the CD137 or CD134 protein. In some embodiments, the chimeric co-stimulatory intracellular domains provided herein further comprise a third signaling domain. In some embodiments, the third signaling domain can be based on a CD3 signaling domain.

In some embodiments, the novel co-stimulatory intracellular domain of the present application can be combined or fused in frame with the extracellular domain of any known co-stimulatory protein, a cell intrinsic immune checkpoint inhibitor, a chimeric antigen receptor, an antibody or a portion thereof, a ligand or a receptor thereof, a cytokine or a receptor thereof, a chemokine or a receptor thereof or a complement receptor, to form a functional recombinant T cell co-stimulatory receptor (RTCR). In some embodiments, the RTCR can be expressed in a cell in combination with another T cell receptor (TCR), chimeric antigen receptor or co-stimulatory protein. A RTCR comprising the novel co-stimulatory intracellular domain disclosed herein, when co-expressed with a TCR in a T cell, significantly increases the cell surface expression of the RTCR, and/or cell proliferation, activation, persistence, cytokine production and/or effector function of the T cell, as compared to a second-generation co-stimulatory receptor.

A highly efficacious adoptive cell therapeutic targeting a shared and safe tumor associated antigen and comprising a cell-intrinsic inhibitor of T cell exhaustion able to withstand the suppressive tumor microenvironment is described in the present application. An exemplary chimeric molecule expressing the extracellular domain of PD-1 and a functionally optimized chimeric intracellular co-stimulatory domain are disclosed herein. Modified T cells expressing the chimeric molecule of the present disclosure are generated to show the efficacy of the chimeric molecule in enhancing T cell stimulation, activation and proliferation. Both molecules are expressed on the same T cell, creating a TCR-T product that responds robustly to tumor cells expressing both the cognate MHC/peptide complex and high levels of PD-L1//PD-L2.

For exemplification, a cell-intrinsic inhibitor of T cell exhaustion is developed by co-expression of third generation chimeric PD-1 receptors combined with T cell receptors targeting tumor associated or specific antigens to enhance the efficacy of T cell mediated killing of tumor cells. The 3^rdgeneration chimeric receptors disclosed herein can be used in combination with any endogenous or modified T cell receptors as well as with chimeric artificial receptors (CARs). The results herein show that the 3^rd-generation co-stimulatory molecules disclosed herein produces T cells with high physiological avidity and persistent proliferative potential, while negating negative signaling by PD-1, delivering instead co-stimulatory signals in a PD-L1 rich environment. The novel co-stimulatory molecule can be co-expressed with a tumor associated antigen (TAA) specific TCR and used to target PD-L1/PD-L2 and the TAA expressing tumors. This demonstrates that the synergistic effect between the TCR activation and co-stimulatory molecule significantly increases the therapeutic window and generate a potentially more effective candidate for clinical investigation. In the disclosure described below, the design of the third-generation chimeric proteins is systematically optimized, to further validate in vitro the improved anti-cancer effectiveness, and to investigate the in vivo anti-tumor efficacy. Co-stimulatory molecules incorporating the extracellular domains of PD-1 with the intracellular domains of CD28, ICOS, CD134, and CD137 alone and in various combinations are generated. These sequences are optimized for surface expression and functionality by incorporating key mutations/deletions within the signaling domain of the chimeric receptors, focusing on the junction between CD28 and TNF-receptor family signaling domains. The functionality of these receptors is tested based on surface expression, in-vitro signaling, in-vitro T cell conjugation, cytokine production, proliferation, and cytotoxicity using a combination of soluble and plate-bound antibody stimulations and K562 target cells expressing PD-L1 or A375 tumor cells.

The disclosure herein provides an approach in which the TCR-T product co-expresses a chimeric co-stimulatory molecule alongside a recombinant TAA-specific TCR or an endogenous TCR. This approach allows for the targeting of the tumor associated antigen with simultaneous antagonization of checkpoint inhibition and delivery of co-stimulatory signals to the transfused T cell product. This approach not only results in a much-improved product, but also help to develop a universal function-boosting platform for additional TCR-T products.

The key technical challenge hindering the clinical adoption of 3^rd-generation CARs combining the two domains has been the abnormally low expression of chimeric proteins at the cell surface and associated diminished functionality (Zhao, 2015, Guedan, 2018). This has held true in co-stimulatory molecules where the combination of the CD28 and CD137 signaling domains resulted in a poorly expressed and non-functional receptor (Ankri, 2013). Disclosed herein is a switch receptor/co-stimulatory molecule based on the ICOS/CD137 signaling domain and optimized for surface expression that is a significant improvement over past trials using the CD28 signaling domain alone, mediating both increased effector function and persistence of adoptively transferred cells. Results described herein show that the 3^rd-generation co-stimulatory molecule disclosed herein produces T cells with high physiological avidity and persistent proliferation potential, while negating negative signaling by PD-1, delivering instead a co-stimulatory signal in a PD-L1 rich environment. The novel switch receptor/co-stimulatory molecule disclosed herein can be co-expressed with an endogenous TCR or a TAA specific TCR and used to target PD-L1/PD-L2 expressing tumors. This demonstrates that the synergistic effect between the TCR activation and co-stimulatory molecule significantly increases the therapeutic window for a potentially more effective candidate for clinical investigation.

Tumor associated antigens and tumor specific antigens allow for the immunological targeting of the tumor with relatively minimal risk of off-tumor, on-target side effects. Tumor cells can upregulate these antigens which can then be targeted by the human immune response or ACT. The disclosure herein combines a co-stimulatory molecule based on 3^rd-generation CARs that exhibits superior functionality to CD28-based receptors with a new affinity enhanced TCR targeting TAAs to generate a TCR-T product that resists the suppressive function of the TME.

In some embodiments, the mutant intracellular signaling domain of a TNFR family protein is any one of a mutant CD137 (4-1BB) intracellular domain or a mutant CD134 (OX-40) intracellular domain.

The present disclosure provides a recombinant T cell co-stimulatory receptor (RTCR), comprising: (a) an extracellular domain; (b) a transmembrane domain; and (c) a chimeric intracellular domain comprising a first and at least a second signal transduction domains, wherein the first and the at least second signal transduction domains are non-identical; and wherein the at least second signal transduction domain comprises a mutant CD137 (4-1BB) intracellular domain or a mutant CD134 (OX-40) intracellular domain.

Unless indicated otherwise, the terms “co-stimulatory molecule”, “costimulatory molecule”, “co stimulatory molecule”, “co-stimulatory protein”, “costimulatory protein”, “co stimulatory protein”, “co-stimulatory receptor”, “costimulatory receptor” “co stimulatory receptor” and “switch receptor” are used interchangeably, to refer to the recombinant T cell co-stimulatory receptors (RTCRs) comprising the novel chimeric co-stimulatory intracellular domains of the present application. These terms may be used in combination with terms such as “recombinant T cell”, “recombinant”, “chimeric T cell”, and “chimeric”, to refer to the RTCRs of the present application.

As described herein, “a recombinant T cell co-stimulatory receptor” or “switch receptor” of the present disclosure is a “costimulatory molecule” “co-stimulatory receptor” or “co-stimulatory protein” generated by operably linking an extracellular domain to an intracellular chimeric intracellular protein of the present disclosure.

“CD137” as described herein is a member of the tumor necrosis factor (TNF) receptor family, and also referred to as 4-1BB, CD137, tumor necrosis factor receptor superfamily member 9 (TNFRSF9) and induced by lymphocyte activation (ILA). As described herein, the terms “CD137”, “4-1BB”, “4-1BB wt”, “4-1BB wild type”, “BB”, “BB wt” and “BB wild type” are used interchangeably throughout, for example, when describing constructs or co-stimulatory molecules of the present application, unless otherwise indicated.

In some embodiments, the CD137 intracellular domain can be from a mammalian CD137. In some embodiments, the mammalian CD137 can be a human CD137, a mouse CD137, a rat CD137 or a monkey CD137. In some embodiments, the CD137 intracellular domain can be from a human CD137, or an isoform or a variant thereof, comprising an amino acid sequence identical to any one of the human CD137 amino acid sequence according to GenBank Accession Nos: U03397, AAA62478, NP_001552, Q07011, AAH06196 and XP_006710681. In some embodiments, the CD137 intracellular domain can be from a mouse CD137, or an isoform or a variant thereof, comprising an amino acid sequence identical to any one of the mouse CD137 amino acid sequence according to GenBank Accession Nos: NP_001070977.1, NP_001070976.1, NP_035742.1, NP_033430.1, P20334.1, XP_011248530.1, XP_011248530.1, ABI30213.1, BAE32724.1 and AAH28507.1. In some embodiments, the CD137 intracellular domain can be from a rat CD137, or an isoform or a variant thereof, comprising an amino acid sequence identical to any one of the rat CD137 amino acid sequence according to GenBank Accession Nos: NP_852049.1, NP_001020944.1, BAD99404.1, XP_008762504.1, XP_006239534.1, EDL81196.1, AAH97483.1, EHB16663.1, EHB16663.1, KF038282.1, XP_010618177.1, XP_029414155.1, XP_029414154.1, XP_021099219.1 and XP_012888584.1. In some embodiments, the CD137 intracellular domain can be from a monkey CD137, or an isoform or a variant thereof, comprising an amino acid sequence identical to any one of the monkey CD137 amino acid sequence according to GenBank Accession Nos: ABY47575.1, ABI30212.1, ABY47577.1, ABY47576.1 and ABY47578.1.

In some embodiments, the CD137 intracellular domain, as described herein, comprises an amino acid sequence starting from the amino acid position 214 to the last amino acid at the C-terminal end of the amino acid sequence of the human CD137 protein, described herein. In some embodiments, the CD137 intracellular domain, as described herein, comprises an amino acid sequence starting from the amino acid position 215 to the last amino acid at the C-terminal end of the amino acid sequence of the mouse CD137 protein, described herein.

In some embodiments, the mutant CD137 intracellular domain described herein is from any one of the CD137 proteins as described herein, comprising one or more mutation(s), wherein the mutation can be addition/insertion, deletion/truncation or substitution/replacement of one or more amino acids within the amino acid sequence of the CD137 protein. In some embodiments, the mutant CD137 intracellular domain described herein is any one of the CD137 intracellular domain sequences, as described herein, comprising one or more mutation(s), wherein the mutation can be addition/insertion, deletion/truncation or substitution/replacement of one or more amino acids within the amino acid sequence of the CD137 intracellular domain. In some embodiments, the mutant CD137 intracellular domain described herein is a CD137 intracellular domain as described herein, comprising a deletion or substitution of one or more amino acids within the amino acid sequence of the CD137 intracellular domain that can be targets for ubiquitination. In some embodiments, the mutant CD137 intracellular domain described herein is a CD137 protein as described herein, comprising a deletion or substitution, of one or more lysine residues within the amino acid sequence of the CD137 intracellular domain that can be targets for ubiquitination. In some embodiments, the mutant CD137 intracellular domain described herein is a CD137 protein as described herein, comprising a deletion or substitution, of one, two, three or four lysine residues within the amino acid sequence of the CD137 intracellular domain that can be targets for ubiquitination. In some embodiments, the lysine residues within the amino acid sequence of the CD137 intracellular domain described herein, that can be deleted or substituted are at amino acid positions 214, 218, 219 and/or 225 of the CD137 intracellular domain.

In some embodiments, the mutant CD137 intracellular domain can be a truncated CD137 intracellular domain. A truncated CD137 intracellular domain as described herein can be any one of the CD137 proteins described herein, in which a continuous stretch of more than one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, twenty, twenty-five, fifty, hundred, two hundred or more amino acids are deleted from the N-terminus the CD137 protein as described herein. A truncated CD137 intracellular domain as described herein can be any one of the CD137 intracellular domain sequences described herein, in which a continuous stretch of more than one, two, three, four, five, six, seven, eight, nine, ten or more amino acids are deleted from the N-terminus the CD137 intracellular domain as described herein. In some embodiments, the amino acids deleted from the N-terminus the CD137 intracellular domain includes one or more proximal polybasic amino acids of the CD137 intracellular domain.

In some embodiments, the mutant CD137 intracellular domain can be a truncated CD137 intracellular domain. In some embodiments, the truncated CD137 intracellular domain comprises an amino acid sequence according to amino acid position 13 to amino acid position 42 of the CD137 intracellular domain, of the present disclosure. In some embodiments, the truncated CD137 intracellular domain comprises a deletion of a continuous stretch of one, two, three, four, five, six, seven, eight, nine, ten or more amino acids from the N-terminus of the CD137 intracellular domain, of the present disclosure. In some embodiments, the truncated CD137 intracellular domain comprises a deletion of one, two, three, four, five, six, seven, eight, nine, ten or more amino acids from amino acid position 1 to amino acid position 12 of the N-terminus of the CD137 intracellular domain, of the present disclosure. In some embodiments, the truncated CD137 intracellular domain comprises a deletion of amino acid position 1 to amino acid position 12 of the N-terminus of the CD137 intracellular domain, of the present disclosure. In some embodiments, the CD137 intracellular domain comprises an amino acid sequence according to SEQ ID NO: 1.

In some embodiments, the truncated CD137 intracellular domain comprises an amino acid sequence according to SEQ ID NO: 3. In some embodiments, the truncated CD137 intracellular domain comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 3.

A truncated CD137 intracellular domain as described herein, is referred to as “truncated CD137”, “CD137t”, “truncated 4-1BB”, “4-1BBt”, “truncated BB” or “BBt” interchangeably throughout, for example, when describing constructs or co-stimulatory molecules of the present application, unless otherwise indicated. In some embodiments, the mutant CD137 intracellular domain comprises a deletion of one, two, three or four lysine residue(s) from amino acid position 1 to amino acid position 12 of the N-terminus of the CD137 intracellular domain, of the present disclosure. In some embodiments, the mutant CD137 intracellular domain comprises one or more lysine mutation(s) from amino acid position 1 to amino acid position 12 of the N-terminus of the CD137 intracellular domain, of the present disclosure. In some embodiments, the mutant CD137 intracellular domain comprises one or more lysine mutation(s) at amino acid positions selected from amino acid positions 1, 5, 6 and 12 of the N-terminus of the CD137 intracellular domain, of the present disclosure. In some embodiments, the one or more lysine mutation(s) are lysine to alanine mutations. In some embodiments, the CD137 intracellular domain comprises an amino acid sequence according to SEQ ID NO: 1.

In some embodiments, the mutant CD137 intracellular domain comprises a deletion of one or more proximal basic amino acids from amino acid position 1 to amino acid position 12 of the N-terminus of the CD137 intracellular domain, of the present disclosure. In some embodiments, the mutant CD137 intracellular domain comprises one or more proximal basic amino acid mutation(s) from amino acid position 1 to amino acid position 12 of the N-terminus of the CD137 intracellular domain, of the present disclosure. In some embodiments, the mutant CD137 intracellular domain comprises one or more proximal basic amino acid mutation(s) at amino acid positions selected from amino acid positions 1, 2, 3, 4, 5 and 6 of the N-terminus of the CD137 intracellular domain, of the present disclosure. In some embodiments, the mutant CD137 intracellular domain comprising one or more proximal basic amino acid mutation(s), of the present disclosure, further comprises a lysine mutation at amino acid position 12 of the N-terminus of the CD137 intracellular domain, of the present disclosure. In some embodiments, the lysine mutation is a lysine to alanine mutation. In some embodiments, the CD137 intracellular domain comprises an amino acid sequence according to SEQ ID NO: 1.

“CD134” as described herein is a member of the tumor necrosis factor (TNF) receptor family, and also referred to as OX-40, ACT35, IMD16, TXGP1L and tumor necrosis factor receptor superfamily member 4 (TNFRSF4). As described herein, the terms “CD134”, “OX-40”, “OX40”, “OX-40 wild type”, “OX-40 wt”, “OX40 wild type”, “OX40 wt”, “40”, “40 wild type” and “40 wt” are used interchangeably throughout, for example, when describing constructs or co-stimulatory molecules of the present application, unless otherwise indicated.

In some embodiments, the CD134 intracellular domain can be from a mammalian CD134. In some embodiments, the mammalian CD134 can be a human CD134, a mouse CD134, a rat CD134 or a monkey CD134. In some embodiments, the CD134 intracellular domain can be from a human CD134, or an isoform or a variant thereof, comprising an amino acid sequence identical to any one of the human CD134 amino acid sequence according to GenBank Accession Nos: NP_003318, AA105071, AA105073, XP_016857721.1, XP_016857720.1, XP_011540377.1, XP_011540379.1, XP_011540378.1, XP_011540376.1, P43489.1, NP_001284491.1, NP_003317.1, EAW56278.1 and CAB96543.1. In some embodiments, the CD134 intracellular domain can be from a mouse CD134, or an isoform or a variant thereof, comprising an amino acid sequence identical to any one of the mouse CD134 amino acid sequence according to GenBank Accession Nos: NP_035789.1, AAI39267.1, AAI39240.1, NP_033478.1, XP_006538787.3, P47741.1, EDL15067.1, CAA79772.1, CAA59476.1, XP_021017102.2, and XP_021056714.1. In some embodiments, the CD134 intracellular domain can be from a rat CD134, or an isoform or a variant thereof, comprising an amino acid sequence identical to any one of the rat CD134 amino acid sequence according to GenBank Accession Nos: NP_035789.1, NP_037181.1, P15725.1, EDL81353.1, CAB96543.1, and CAA34897.1. In some embodiments, the CD134 intracellular domain can be from a monkey CD134, or an isoform or a variant thereof, comprising an amino acid sequence identical to any one of the monkey CD134 amino acid sequence according to GenBank Accession Nos: XP_010375483.1, XP_001090870.1, XP_021523144.1, XP_017750744.1, XP_003939714.1, XP_026313229.1, XP_026313228.1, XP_003890998.2, XP_025242473.1, XP_011768627.1, XP_005545179.1, XP_011886513.1, XP_011886512.1, XP_011857387.1 and XP_011811769.1.

In some embodiments, the CD134 intracellular domain, as described herein, comprises an amino acid sequence starting from amino acid position 241 to the last amino acid at the C-terminal end of the amino acid sequence of any one of the human CD134 protein, described herein. In some embodiments, the CD134 intracellular domain, as described herein, comprises an amino acid sequence starting from the amino acid position 236 to the last amino acid at the C-terminal of the amino acid sequence of the mouse CD134 protein, described herein.

In some embodiments, the mutant CD134 intracellular domain described herein is from any one of the CD134 proteins as described herein, comprising one or more mutation(s), wherein the mutation can be addition/insertion, deletion/truncation or substitution/replacement of one or more amino acids within the amino acid sequence of the CD134 protein. In some embodiments, the mutant CD134 intracellular domain described herein, is any one of the CD134 intracellular domain sequences as described herein, comprising one or more mutation(s), wherein the mutation can be addition/insertion, deletion/truncation or substitution/replacement of one or more amino acids within the amino acid sequence of the CD134 intracellular domain. In some embodiments, the mutant CD134 intracellular domain described herein is a CD134 intracellular domain as described herein, comprising a deletion or substitution of one or more amino acids within the amino acid sequence of the CD134 intracellular domain that can be targets for ubiquitination. In some embodiments, the mutant CD134 intracellular domain described herein is a CD134 protein as described herein, comprising a deletion or substitution, of one or more lysine residues within the amino acid sequence of the CD134 intracellular domain that can be targets for ubiquitination. In some embodiments, the mutant CD134 intracellular domain described herein is a CD134 protein as described herein, comprising a deletion or substitution, of one or two lysine residues within the amino acid sequence of the CD134 intracellular domain that can be targets for ubiquitination. In some embodiments, the lysine residues within the amino acid sequence of the CD134 intracellular domain described herein, that can be deleted or substituted are at amino acid positions 252 and/or 276 of the CD134 intracellular domain.

In some embodiments, the mutant CD134 intracellular domain can be a truncated CD134 intracellular domain. A truncated CD134 intracellular domain as described herein can be any one of the CD134 proteins described herein, in which a continuous stretch of more than one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, twenty, twenty-five, fifty, hundred, two hundred or more amino acids are deleted from the N-terminus the CD137 protein as described herein. A truncated CD134 intracellular domain as described herein can be any one of the CD134 intracellular domain sequences described herein, in which a continuous stretch of more than one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen or more amino acids are deleted from the N-terminus the CD134 intracellular domain as described herein. In some embodiments, the amino acids deleted from the N-terminus the CD134 intracellular domain includes one or more proximal polybasic amino acids of the CD134 intracellular domain.

In some embodiments, the truncated CD134 intracellular domain comprises an amino acid sequence according to amino acid position 15 to amino acid position 37 of a CD134 intracellular domain, of the present disclosure. In some embodiments, the truncated CD134 intracellular domain comprises a deletion of a continuous stretch of one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen or more amino acids from the N-terminus of the CD134 intracellular domain, of the present disclosure. In some embodiments, the truncated CD134 intracellular domain comprises a deletion of one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen or more amino acids from amino acid position 1 to amino acid position 14 of the N-terminus of the CD134 intracellular domain, of the present disclosure. In some embodiments, the truncated CD137 intracellular domain comprises a deletion of amino acid position 1 to amino acid position 14 of the N-terminus of the CD134 intracellular domain, of the present disclosure. In some embodiments, the CD134 intracellular domain comprises an amino acid sequence according to SEQ ID NO: 4.

In some embodiments, the mutant CD134 intracellular domain comprises an amino acid sequence according to SEQ ID NO: 6. In some embodiments, the mutant CD134 intracellular domain comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 6.

A truncated CD134 intracellular domain as described herein, is referred to as “truncated CD134”, “CD134t”, “truncated OX-40”, “truncated OX40”, “OX-40t”, “OX40t” and “40t” are used interchangeably throughout, for example, when describing constructs or co-stimulatory molecules of the present application, unless otherwise indicated.

In some embodiments, the mutant CD134 intracellular domain comprises a deletion of a lysine residue from amino acid position 1 to amino acid position 14 of the N-terminus of the CD134 intracellular domain, of the present disclosure. In some embodiments, the mutant CD134 intracellular domain comprises a lysine mutation at amino acid position 12 of the N-terminus of the CD134 intracellular domain, of the present disclosure. In some embodiments, the lysine mutation is a lysine to alanine mutation. In some embodiments, the CD134 intracellular domain comprises an amino acid sequence according to SEQ ID NO: 4.

In some embodiments, the mutant CD134 intracellular domain comprises a deletion of one or more proximal basic amino acids from amino acid position 1 to amino acid position 14 of the N-terminus of the CD134 intracellular domain, of the present disclosure. In some embodiments, the mutant CD134 intracellular domain comprises one or more proximal basic amino acid mutation(s) from amino acid position 1 to amino acid position 14 of the N-terminus of the CD134 intracellular domain, of the present disclosure. In some embodiments, the mutant CD134 intracellular domain comprises one or more proximal basic amino acid mutation(s) at amino acid positions selected from amino acid positions 1, 2, and 5 of the N-terminus of the CD134 intracellular domain, of the present disclosure. In some embodiments, the mutant CD137 intracellular domain further comprises a lysine mutation at amino acid position 12 of the N-terminus of the CD137 intracellular domain, of the present disclosure. In some embodiments, the CD134 intracellular domain comprises an amino acid sequence according to SEQ ID NO: 4.

TABLE 1

Amino acid sequences of second signal

transduction domains of RTCR (CD137/4-1BB and

CD134/OX-40 intracellular signaling domain).

Human CD137/4-1BB

KRGRKKLLYIFKQPFMRPVQTTQEED

(SEQ ID NO: 1)
GCSCRFPEEEEGGCEL

Mouse CD137/4-1BB

RKKFPHIFKQPFKKTTGAAQEEDACS

(SEQ ID NO: 2)
CRCPQEEEGGGGGYEL

truncated/mutated
QPFMRPVQTTQEEDGCSCRFPEEEEG

CD137/4-1BB
GCEL

(SEQ ID NO: 3)

Human CD134/OX-40

RRDQRLPPDAHKPPGGGSFRTPIQEE

(SEQ ID NO: 4)
QADAHSTLAKI

Mouse CD134

RKAWRLPNTPKPCWGNSFRTPIQEEH

(SEQ ID NO: 5)
TDAHFTLAKI

truncated/mutated
GGGSFRTPIQEEQADAHSTLA

CD134/OX-40

(SEQ ID NO: 6)

Human CD27

QRRKYRSNKGESPVEPAEPCHYSCPR

(SEQ ID NO: 7)

EEEGSTIPIQEDYRKPEPACSP

Human GITR
QLGLHIWQLRSQCMWPRETQLLLEVP

(SEQ ID NO: 8)
PSTEDARSCQFPEEERGERSAEEKGR

LGDLWV

Membrane-proximal poly-basic regions are italicized. Potential PI3K binding sites are bold and underlined. TRAF1/2 binding motifs, major motif Px(Q/E)E and minor motifs Px(Q/E)x, are highlighted in underlined. Potential ubiquitination sites are in bold.

In some embodiments, the chimeric intracellular domain comprises a first signal transduction domain derived from a protein of the CD28 family. In some embodiments, the first signal transduction domain derived from any one of CD28, CD28H, ICOS or a combination thereof.

In some embodiments, the chimeric intracellular domain comprises a first signal transduction domain derived from ICOS protein.

The “ICOS protein” as described herein is an inducible T cell co-stimulatory protein, also referred to as AILIM, CD278, CCLP, CRP-1, H4, Ly115 and CVID1. In some embodiments, the ICOS intracellular domain can be from a mammalian ICOS. In some embodiments, the mammalian ICOS can be a human ICOS, a mouse ICOS, a rat ICOS or a monkey ICOS. In some embodiments, the ICOS intracellular domain can be from a human ICOS, or an isoform or a variant thereof, comprising an amino acid sequence identical to any one of the human ICOS amino acid sequence according to GenBank Accession Nos: AAH28006.1, NP_036224.1, AIC51287.1, AIC60036.1, NP_036224.1, Q9Y6W8.1, EAW70357.1, EAW70356.1, EAW70355.1, AAL40934.1, AAL40933.1, CAC06612.1, AAX93073.1, AAM00909.1, AAH28210.1 and CAD59742.1. In some embodiments, the ICOS intracellular domain can be from a mouse ICOS, or an isoform or a variant thereof, comprising an amino acid sequence identical to any one of the mouse ICOS amino acid sequence according to GenBank Accession Nos: NP_059508.2, Q9WVS0.2, EDL00161.1, CAM13242.1, CAM13241.1, CAB71153.1, AAG48732.1, AAH34852.1, XP_006496203.1, XP_006496202.1, XP_006496201.1, ACX50464.1, ACX50463.1, AAH28006.1, XP_021052880.1, XP_029334968.1 and XP_021030282.1. In some embodiments, the ICOS intracellular domain can be from a rat ICOS, or an isoform or a variant thereof, comprising an amino acid sequence identical to any one of the rat ICOS amino acid sequence according to GenBank Accession Nos: NP_072132.1, Q9R1T7.1, XP_008765358.1, XP_006245100.1, XP_006245099.1, EDL98922.1, EDL98921.1, XP_038940099.1, XP_032755449.1, XP_017457364.1, XP_006256324.1, XP_006256323.1, XP_006256322.1, XP_029425757.1, XP_029425757.1, XP_021119236.1, XP_012929934.1, XP_012867370.1 and XP_012867363.1. In some embodiments, the ICOS intracellular domain can be from a monkey ICOS, or an isoform or a variant thereof, comprising an amino acid sequence identical to any one of the monkey ICOS amino acid sequence according to GenBank Accession Nos: XP_007964137.1, NP_001253918.1, XP_010350939.1, XP_012301785.1, XP_012301784.1, XP_017739861.1, XP_010334714.1, XP_003925677.1, AFH29328.1, XP_008997520.1, XP_023075107.1, XP_023075099.1, XP_021779593.1, XP_003907887.1, XP_025260988.1, XP_025260987.1, XP_025260986.1, XP_011716287.1, XP_011716285.1, XP_005574075.1, XP_011903009.1, XP_011805288.1, XP_011805287.1, XP_011847867.1, XP_011847866.1, XP_017392362.1, XP_033086489.1, XP_032134414.1, XP_032134413.1, and XP_017802331.1.

In some embodiments, the human ICOS intracellular domain as described herein, comprises an amino acid sequence from amino acid position 133 to the last amino acid at the C-terminus of the amino acid sequence of the human ICOS protein, described herein. In some embodiments, the human ICOS intracellular domain as described herein, comprises an amino acid sequence from an amino acid position at one, two, three, four, five, six, seven, eight, nine, ten or more amino acids N-terminus to the amino acid position 133, to the last amino acid at the C-terminus of the amino acid sequence of the human ICOS protein, described herein. In some embodiments, the human ICOS intracellular domain as described herein, comprises an amino acid sequence from amino acid position 133 to an amino acid position at one, two, three, four, five, six, seven, eight, nine, ten or more amino acids N-terminus to the last amino acid at the C-terminus of the amino acid sequence of the human ICOS protein, described herein. In some embodiments, the human ICOS intracellular domain as described herein, comprises an amino acid sequence from an amino acid position at one, two, three, four, five, six, seven, eight, nine, ten or more amino acids N-terminus to the amino acid position 133, to an amino acid position at one, two, three, four, five, six, seven, eight, nine, ten or more amino acids N-terminus to the last amino acid at the C-terminus of the amino acid sequence of the human ICOS protein, described herein.

In some embodiments, the human ICOS(28) intracellular domain as described herein, comprises a portion of the ICOS domain amino acid sequence from amino acid position 133 to amino acid position 183, and a portion of the ICOS domain amino acid sequence from amino acid position 184 to the last amino acid at the C-terminus of the amino acid sequence of the human ICOS protein, described herein. In some embodiments, the human ICOS(28) intracellular domain as described herein, comprises a portion of the ICOS domain amino acid sequence from an amino acid position at one, two, three, four, five, six, seven, eight, nine, ten or more amino acids N-terminus to the amino acid position 133, to amino acid position 183 of the human ICOS protein, described herein. In some embodiments, the human ICOS(28) intracellular domain as described herein, comprises a portion of the ICOS domain amino acid sequence from amino acid position 133, to an amino acid position at one, two, three, four, five, six, seven, eight, nine, ten or more amino acids C-terminus to the amino acid position 183 of the human ICOS protein, described herein. In some embodiments, the human ICOS(28) intracellular domain as described herein, comprises a portion of the ICOS domain amino acid sequence from an amino acid position at one, two, three, four, five, six, seven, eight, nine, ten or more amino acids N-terminus to the amino acid position 133, to an amino acid position at one, two, three, four, five, six, seven, eight, nine, ten or more amino acids C-terminus to the amino acid position 183 of the human ICOS protein, described herein.

The “CD28 protein”, also referred to as Tp44, is a constitutively expressed receptor for CD80 (B7.1) and CD86 (B7.2) proteins on naïve T cells and is important for T cell activation. In some embodiments, the CD28 intracellular domain can be from a mammalian CD28. In some embodiments, the mammalian CD28 can be a human CD28, a mouse CD28, a rat CD28 or a monkey CD28. In some embodiments, the CD28 intracellular domain can be from a human CD28, or an isoform or a variant thereof, comprising an amino acid sequence identical to any one of the human CD28 amino acid sequence according to GenBank Accession Nos: P10747.1, NP_001230007.1, NP_001230006.1, NP_006130.1, EAW70350.1, EAW70349.1, EAW70348.1, EAW70347.1, AIC48451.1, CAC29237.1, AAA51945.1, AAA51944.1, AAL40931.1, AAF33794.1, AAF33793.1, AAF33792.1, XP_011510499.1, XP_011510497.1, XP_011510496.1, AAI12086.1, AAH93698.1, ABK41938.1, AAY24123.1, CAD57003.1 and AAA60581. In some embodiments, the CD28 intracellular domain can be from a mouse CD28, or an isoform or a variant thereof, comprising an amino acid sequence identical to any one of the mouse CD28 amino acid sequence according to GenBank Accession Nos: AAA37396.1, NP_031668.3, P31041.2, AAH64058.1, EDL00156.1, CAM13249.1, XP_036012281.1, XP_021054806.1, XP_021027481.1, XP_036015651.1, and XP_030104805. In some embodiments, the CD28 intracellular domain can be from a rat CD28, or an isoform or a variant thereof, comprising an amino acid sequence identical to any one of the rat CD28 amino acid sequence according to GenBank Accession Nos: CAA39003.1, NP_037253.2, P31042.1, XP_008765300.1, EDL98926.1, XP_032755445.1, XP_034354910.1, XP_019061859.2, XP_008844474.1, XP_004851403.1 and XP_012865504.1. In some embodiments, the CD28 intracellular domain can be from a monkey CD28, or an isoform or a variant thereof, comprising an amino acid sequence identical to any one of the monkey CD28 amino acid sequence according to GenBank Accession Nos: ABH06891.1, ABH08508.1, ABH06892.1, ABH08509.1, ABQ09493.1, NP_001274262.1, NP_001036106.2, ABG77998.1, ABG77997.1 and XP_0149662071.

The “CD28H protein”, also referred to CD28 homolog, transmembrane and immunoglobulin domain-containing protein 2, has co-stimulatory activity in T cells by binding to B7H7. CD28H was initially described as a molecule involved in cell-cell interaction, cell migration, and angiogenesis of epithelial and endothelial cells (7, 8). CD28H has a single extracellular immunoglobulin domain followed by a transmembrane domain and a 110 amino acid-long cytoplasmic region. In some embodiments, the CD28 intracellular domain can be from a mammalian CD28H. In some embodiments, the mammalian CD28 can be a human CD28H, a mouse CD28H, a rat CD28H or a monkey CD28H. In some embodiments, the CD28H intracellular domain can be from a human CD28H, or an isoform or a variant thereof, comprising an amino acid sequence identical to any one of the human CD28H amino acid sequence according to GenBank Accession Nos: NP_001295161.1, NP_001162597.1, Q96BF3.2, XP_024307127.1 and XP_0168817731.

In some embodiments, the human CD28 intracellular domain as described herein, comprises an amino acid sequence from amino acid position 145 to the last amino acid at the C-terminus of the amino acid sequence of the human CD28 protein, described herein. In some embodiments, a portion of the human CD28 intracellular domain as described herein, can comprise an amino acid sequence from about amino acid position 195 to about amino acid position 212 of the amino acid sequence of the human CD28 protein, described herein. In some embodiments, a portion of the human CD28 intracellular domain as described herein, can comprises an amino acid sequence from one, two, three, four, five, six, seven, eight, nine or 10 or more amino acid amino acid position N-terminus to amino acid position 195 to one, two, three, four, five, six, seven, eight, nine or 10 or more amino acid amino acid position C-terminus amino acid position 220 of the amino acid sequence of the human CD28 protein, described herein.

In some embodiments, the first signal transduction domain derived from ICOS comprises an amino acid sequence according to SEQ ID NO: 9. In some embodiments, the first signal transduction domain derived from ICOS comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 9.

In some embodiments, the chimeric intracellular domain comprises a first signal transduction domain comprising a portion of a CD28 intracellular domain combined with an ICOS protein (ICOS (28) domain) according to SEQ ID NO: 9. In some embodiments, the ICOS (28) domain comprises the portion of CD28 intracellular domain inserted N-terminal to the PI-3K binding site of the ICOS protein according to SEQ ID NO: 9. In some embodiments, the ICOS (28) domain comprises the portion of CD28 inserted at 1, 2, 3, 4 or 5 amino acid position N-terminal to the PI-3K binding site of the ICOS protein according to SEQ ID NO: 9. In some embodiments, the ICOS(28) domain comprises the portion of CD28 inserted C-terminal to the PI-3K binding site of the ICOS protein according to SEQ ID NO: 9. In some embodiments, the ICOS(28) domain comprises the portion of CD28 inserted at 1, 2, 3, 4 or 5 amino acid position C-terminal to the PI-3K binding site of the ICOS protein according to SEQ ID NO: 9.

In some embodiments, the portion of CD28 is inserted at any amino acid position before amino acid position 48 within an ICOS protein of amino acid sequence according to SEQ ID NO: 9. In some embodiments, the portion of CD28 is inserted at any amino acid position between amino acid position 1 and amino acid position 48, within an ICOS protein of amino acid sequence according to SEQ ID NO: 9. In some embodiments, the portion of CD28 is inserted between the amino acid position 47 and amino acid position 48 of an ICOS protein with the amino acid sequence according to SEQ ID NO: 9. In some embodiments, the portion of CD28 is inserted between the amino acid position 46 and amino acid position 47 of an ICOS protein with the amino acid sequence according to SEQ ID NO: 9. In some embodiments, the portion of CD28 is inserted between the amino acid position 45 and amino acid position 46 of an ICOS protein with the amino acid sequence according to SEQ ID NO: 9. In some embodiments, the portion of CD28 is inserted between the amino acid position 44 and amino acid position 45 of an ICOS protein with the amino acid sequence according to SEQ ID NO: 9. In some embodiments, the portion of CD28 is inserted between the amino acid position 43 and amino acid position 44 of an ICOS protein with the amino acid sequence according to SEQ ID NO: 9.

In some embodiments, the portion of CD28 is inserted at any position after amino acid position 51 within an ICOS protein of amino acid sequence according to SEQ ID NO: 9. In some embodiments, the portion of CD28 is inserted at any amino acid position between amino acid position 51 and amino acid position 67, within an ICOS protein of amino acid sequence according to SEQ ID NO: 9. In some embodiments, the portion of CD28 is inserted between the amino acid position 51 and amino acid position 52 of an ICOS protein with the amino acid sequence according to SEQ ID NO: 9. In some embodiments, the portion of CD28 is inserted between the amino acid position 53 and amino acid position 54 of an ICOS protein with the amino acid sequence according to SEQ ID NO: 9. In some embodiments, the portion of CD28 is inserted between the amino acid position 54 and amino acid position 55 of an ICOS protein with the amino acid sequence according to SEQ ID NO: 9. In some embodiments, the portion of CD28 is inserted between the amino acid position 56 and amino acid position 57 of an ICOS protein with the amino acid sequence according to SEQ ID NO: 9. In some embodiments, the portion of CD28 is inserted between the amino acid position 57 and amino acid position 58 of an ICOS protein with the amino acid sequence according to SEQ ID NO: 9.

In some embodiments, the portion of CD28 of the ICOS(28) domain disclosed herein comprises an amino acid sequence according to amino acid position 51 to amino acid position 68 of a CD28 signaling domain according to SEQ ID NO: 10. In some embodiments, the portion of CD28 of the ICOS(28) domain disclosed herein comprises an amino acid sequence according to amino acid position 51 to amino acid position 76 of a full length CD28 signaling domain according to SEQ ID NO: 10. In some embodiments, the portion of CD28 of the ICOS(28) domain disclosed herein comprises an amino acid sequence according to amino acid position 45 to amino acid position 68 of a CD28 signaling domain according to SEQ ID NO: 10. In some embodiments, the portion of CD28 inserted within the ICOS(28) domain comprises a PRRP motif. In some embodiments, the portion of CD28 inserted within the ICOS(28) domain comprises an amino acid sequence according to SEQ ID NO: 11. In some embodiments, the portion of CD28 inserted within the ICOS(28) domain comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 11.

In some embodiments, the ICOS(28) domain comprises an amino acid sequence according to SEQ ID NO: 12. In some embodiments, the ICOS(28) comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 12.

In some embodiments, the chimeric intracellular domain comprises a first signal transduction domain derived from CD28. In some embodiments, the first signal transduction domain derived from CD28 comprises an amino acid sequence according to SEQ ID NO: 10. In some embodiments, the first signal transduction domain derived from CD28 comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 10.

A signal transduction domain derived from CD28 as described herein, is referred to as “CD28” or “28”, interchangeably throughout.

TABLE 2

Amino acid sequences of first intracellular signaling domains.

ICOS signaling domain (SEQ ID NO: 9) (Other name: ICOS): Stalk (underlined), TM (regular

font), intracellular domain (IC) (bold) and PI-3K binding site (bold and underlined)

SQLCCQLKFWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPNGEYMFMRAVNT

AKKSRLTDVTL

CD28 Transmembrane_CD28 Signaling Domain (Other names: CD28 or 28) (SEQ ID NO:

10): CD28 Stalk (underlined), TM (regular font), intracellular domain (IC) (bold), PI3K

regulatory subunit binding, GRB2, GADS association domain (bold and dotted-underlined),

ITK interaction site (bold and double-underlined, GRB2, GADS, LCK interaction site (bold

and dash-underlined)

embedded image

CD28 fragment (Other names: mini-CD28 or (28)) (SEQ ID NO: 11): PRRP motif in bold

TPRRPGPTRKHYQPYAPP

ICOS (28) domain (Other name: ICOS (mini-CD28)) (SEQ ID NO: 12): intracellular domain

(italicized), TBK1 binding site (TRAF-like motif) (italicized and dotted-underlined), PI-3K

regulatory subunit binding site (italicized and underlined), CD28 portion (bold), distal domain

(italicized and dash-underlined)

embedded image

In some embodiments, the chimeric intracellular domain comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 13. In some embodiments, the chimeric intracellular domain comprises an amino acid sequence according to any one of SEQ ID NOs: 14-17. In some embodiments, the chimeric intracellular domain comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to any one of SEQ ID NOs: 14-17. In some embodiments, the chimeric intracellular domain comprises an amino acid sequence according to SEQ ID NO: 14. In some embodiments, the chimeric intracellular domain comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 14. In some embodiments, the chimeric intracellular domain comprises an amino acid sequence according to SEQ ID NO: 15. In some embodiments, the chimeric intracellular domain comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 15. In some embodiments, the chimeric intracellular domain comprises an amino acid sequence according to SEQ ID NO: 16. In some embodiments, the chimeric intracellular domain comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 16. In some embodiments, the chimeric intracellular domain comprises an amino acid sequence according to SEQ ID NO: 17. In some embodiments, the chimeric intracellular domain comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 17.

In some embodiments, the chimeric intracellular domain comprises an amino acid sequence according to any one of SEQ ID NOs: 120-129. In some embodiments, the chimeric intracellular domain comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to any one of SEQ ID NOs: 120-129. In some embodiments, the chimeric intracellular domain comprises an amino acid sequence according to SEQ ID NO: 120. In some embodiments, the chimeric intracellular domain comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 120. In some embodiments, the chimeric intracellular domain comprises an amino acid sequence according to SEQ ID NO: 121. In some embodiments, the chimeric intracellular domain comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 121. In some embodiments, the chimeric intracellular domain comprises an amino acid sequence according to SEQ ID NO: 122. In some embodiments, the chimeric intracellular domain comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 122.

In some embodiments, the chimeric intracellular domain comprises an amino acid sequence according to SEQ ID NO: 123. In some embodiments, the chimeric intracellular domain comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 123. In some embodiments, the chimeric intracellular domain comprises an amino acid sequence according to SEQ ID NO: 124. In some embodiments, the chimeric intracellular domain comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 124. In some embodiments, the chimeric intracellular domain comprises an amino acid sequence according to SEQ ID NO: 125. In some embodiments, the chimeric intracellular domain comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 125. In some embodiments, the chimeric intracellular domain comprises an amino acid sequence according to SEQ ID NO: 126. In some embodiments, the chimeric intracellular domain comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 126. In some embodiments, the chimeric intracellular domain comprises an amino acid sequence according to SEQ ID NO: 127. In some embodiments, the chimeric intracellular domain comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 127. In some embodiments, the chimeric intracellular domain comprises an amino acid sequence according to SEQ ID NO: 128.

TABLE 3

Amino acid sequences of

chimeric intracellular domains of RTCR.

ICOS Transmembrane_ICOS Signaling Domain_4-1BB Signaling Domain (other names:

ICOS-4-1BB (CD137) intracellular domain, ICOS-137; ICOS_137; ICOS137; ICOS_BB;

ICOS-BB; ICOSBB; ICOSBBwt; ICOS_BBwt or ICOS_BB wild type)(SEQ ID NO: 13):

ICOS sequence underlined and 4-1BB (BB) domain in normal font

SQLCCQLKFWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPNGEYMFMRAVNTA

KKSRLTDVTLKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

ICOS Transmembrane_ICOS Signaling Domain_Truncated 4-1BB Signaling Domain (other

names: ICOS-truncated 4-1BB (CD137) intracellular domain; ICOS_137t; ICOS137t; ICOS-

137t; ICOS_BBt; ICOSBBt or ICOS-BBt)(SEQ ID NO: 14): ICOS sequence underlined and

mutated/truncated 4-1BB (BBt) domain in normal font

SQLCCQLKFWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPNGEYMFMRAVNTA

KKSRLTDVTLQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

ICOS Transmembrane_ICOS Signaling Domain_Truncated OX-40 Signaling Domain (other

names: ICOS-truncated OX-40 (CD134) intracellular domain, ICOS_OX40t; ICOS-OX40t;

ICOS_40t; ICOS40t or ICOS-40t (SEQ ID NO: 15): ICOS sequence underlined and

mutated/truncated OX-40 (OX40t, 40t) domain in normal font

SQLCCQLKFWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPNGEYMFMRAVNTA

KKSRLTDVTLGGGSFRTPIQEEQADAHSTLA

ICOS Transmembrane_ICOS Signaling Domain (mini-CD28)_Truncated 4-1BB Signaling

Domain (other names: ICOS(28)-truncated 4-1BB (BBt) intracellular domain, ICOS(28)_BBt;

ICOS(28)BBt; ICOS(28)-BBt; ICOS(28)_4-1BBt; ICOS(28)4-1BBt or ICOS(28)-4-1BBt)

(SEQ ID NO: 16): ICOS sequence underlined, CD28 portion in bold and BBt domain in

normal font

SQLCCQLKFWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPNGEYMFM
TPRRPGP

TRKHYQPYAPP
RAVNTAKKSRLTDVTLQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

ICOS Transmembrane_ICOS Signaling Domain (mini-CD28)_Truncated OX-40 Signaling

Domain (other names: ICOS(28)-truncated OX-40; ICOS(28)-OX40t; ICOS(28)_OX40t;

ICOS(28)OX40t; ICOS (28)-40t; ICOS (28)_40t or ICOS(28)40t)(CD134) intracellular

domain (SEQ ID NO: 17): ICOS sequence underlined, CD28 portion in bold and OX40 (OX-

40t, 40t) domain in normal font

SQLCCQLKFWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPNGEYMFM
TPRRPGP

TRKHYQPYAPP
RAVNTAKKSRLTDVTLGGGSFRTPIQEEQADAHSTLA

ICOS(28) (shortened ICOS(28)) (SEQ ID NO: 109): ICOS Transmembrane_ICOS Signaling

Domain (mini-CD28)

CWLTKKKYSSSVHDPNGEYMFMTPRRPGPTRKHYQPYAPPRAVNTAKKSRLTDVTL

CD28 Transmembrane_CD28 Signaling Domain_4-1BB Signaling Domain (other names:

CD28_BBwt signaling domain; CD28_BB; CD28BB; CD28-BB; 28_BBwt; 28BB; 28_BB or

28-BB) (SEQ NO: 120): CD28 Transmembrane domain_CD28 Signaling Domain_4-1BB

Signaling Domain

LFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPT

RKHYQPYAPPRDFAAYRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGC

EL

CD28 Transmembrane_CD28 Signaling Domain_truncated 4-1BB Signaling Domain (other

names: CD28_BBt signaling domain; CD28BBt; CD28-BBt; 28_BBt; 28BB; or 28-BBt) (SEQ

ID NO: 121):

LFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPT

RKHYQPYAPPRDFAAYRSQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

CD28 Transmembrane_CD28 Signaling Domain_truncated OX-40 Signaling Domain (other

names: CD28_OX40t signaling domain; 28-OX40t; 28-40t; 28_OX40t; 28_40t; 28OX40t or

2840t) (SEQ ID NO: 122):

LFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPT

RKHYQPYAPPRDFAAYRSGGGSFRTPIQEEQADAHSTLA

Transmembrane_ICOS Signaling Domain_4-1BB Signaling Domain with mutated polybasic

region (other names: ICOS_BB(xPB); ICOSBB(xPB) or ICOS-BB(xPB)) (SEQ ID NO: 123):

ICOS

SQLCCQLKFWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPNGEYMFMRAVNTA

KKSRLTDVTLAAGAAALLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

ICOS Transmembrane_ICOS Signaling Domain_4-1BB Signaling Domain with mutated

lysines (Other names: ICOS_BB(xUB); ICOS-BB(xUB) or ICOSBB(xUB))((SEQ ID NO:

124):

SQLCCQLKFWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPNGEYMFMRAVNTA

KKSRLTDVTLKRGRKKLLYIFAQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

ICOS Transmembrane_ICOS Signaling Domain_4-1BB Signaling Domain with mutated

lysines and polybasic regions (Other names: ICOS_BB(xPB xUB); ICOS-BB(xPB xUB) or

ICOSBB(xPB xUB))(SEQ ID NO: 125):

SQLCCQLKFWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPNGEYMFMRAVNTA

KKSRLTDVTLAAGAAALLYIFAQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

ICOS Transmembrane_ICOS Signaling Domain_OX-40 Signaling Domain (Other names:

ICOS_OX40wt; ICOS-40; ICOS_40 wild type; ICOS_40wt; ICOS_40wt; ICOS-40wt; ICOS-

OX40wt; ICOSOX40wt; ICOS40 or ICOS40wt) (SEQ ID NO: 126):

SQLCCQLKFWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPNGEYMFMRAVNTA

KKSRLTDVTLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI

ICOS Transmembrane_ICOS Signaling Domain_OX-40 Signaling Domain with mutated

polybasic region (other names: ICOS_OX40(xPB); ICOS_40(xPB); ICOS-40(xPB);

ICOS40(xPB); ICOS-OX40(xPB); or ICOSOX40(xPB))(SEQ ID NO: 127):

SQLCCQLKFWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPNGEYMFMRAVNTA

KKSRLTDVTLAADQALPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI

ICOS Transmembrane_ICOS Signaling Domain_OX-40 Signaling Domain with mutated

lysine residues (other names: ICOS_OX40(xUB) ICOS_40(xUB); ICOS-40(xUB);

ICOS40(xUB); ICOS-OX40(xUB); or ICOSOX40(xUB))(SEQ ID NO: 128):

SQLCCQLKFWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPNGEYMFMRAVNTA

KKSRLTDVTLRRDQRLPPDAHAPPGGGSFRTPIQEEQADAHSTLAAI

ICOS Transmembrane_ICOS Signaling Domain_OX-40 Signaling Domain with mutated

lysine residues and polybasic regions (other names: ICOS_OX40(xPBxUB)

ICOS_40(xPBxUB); ICOS-40(xPBxUB); ICOS40(xPBxUB); ICOS-OX40(xPBxUB); or

ICOSOX40(xPBxUB))(SEQ ID NO: 129):

SQLCCQLKFWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPNGEYMFMRAVNTA

KKSRLTDVTLAADQALPPDAHAPPGGGSFRTPIQEEQADAHSTLAAI

ICOS intracellular domain (SEQ ID NO: 147)

CWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL

CD28 intracellular domain (SEQ ID NO: 193)

RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

In some embodiments, the chimeric intracellular domain further comprises a third signal transduction domain. In some embodiments, the third signal transduction domain is derived from any one of a CD3 signaling domain, a CD2 signaling domain, or an interleukin 2 receptor binding (IL-2RB) protein signaling domain. In some embodiments, the CD3 signaling domain is derived form a CD3ζ or a CD3ε domain or a combination thereof.

In some embodiments, the chimeric intracellular domain further comprises a third signal transduction domain derived from a CD3ζ protein. In some embodiments, the third signal transduction domain of the chimeric intracellular domain is a CD3ζ protein of amino acid sequence according to SEQ ID NO: 18. In some embodiments, the third signal transduction domain of the chimeric intracellular domain is a CD3ζ comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to according to SEQ ID NOs: 18.

Human CD3 ζ full length (CD3Z full length)

(SEQ ID NO: 18)

MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYG

VILTALFLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVL

DKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMK

GERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

In some embodiments, the third signal transduction domain of the chimeric intracellular domain is a CD3 signaling domain comprising an amino acid sequence according to any one of SEQ ID NOs: 45, 46, 47 and 48. In some embodiments, the third signal transduction domain of the chimeric intracellular domain is a CD3 comprising an amino acid sequence according to SEQ ID NO: 45. In some embodiments, the third signal transduction domain of the chimeric intracellular domain is a CD3 comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 45. In some embodiments, the third signal transduction domain of the chimeric intracellular domain is a truncated CD3ζ comprising an amino acid sequence according to SEQ ID NO: 46. In some embodiments, the third signal transduction domain of the chimeric intracellular domain is a truncated CD3ζ comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 46. In some embodiments, the third signal transduction domain of the chimeric intracellular domain is a truncated CD3ε comprising an amino acid sequence according to SEQ ID NO: 47. In some embodiments, the third signal transduction domain of the chimeric intracellular domain is a truncated CD3ε comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 47. In some embodiments, the third signal transduction domain of the chimeric intracellular domain is a combination of a CD3ε and a truncated CD3ζ domains (CD3ζε domain). In some embodiments, the third signal transduction domain of the chimeric intracellular domain is a CD3ζε comprising an amino acid sequence according to SEQ ID NO: 48. In some embodiments, the third signal transduction domain of the chimeric intracellular domain is a CD3ζε comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 48.

In some embodiments, the third signal transduction domain of the chimeric intracellular domain is a mutant CD2 signaling domain. In some embodiments, the third signal transduction domain of the chimeric intracellular domain is a truncated CD2 signaling domain. In some embodiments, the third signal transduction domain of the chimeric intracellular domain is a truncated CD2 signaling domain comprising an amino acid sequence according to SEQ ID NO: 49. In some embodiments, the third signal transduction domain of the chimeric intracellular domain is a truncated CD2 signaling domain comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NOs: 49.

In some embodiments, the third signal transduction domain of the chimeric intracellular domain is an IL-2RB protein signaling domain comprising an amino acid sequence according to SEQ ID NO: 50. In some embodiments, the third signal transduction domain of the chimeric intracellular domain is an IL-2RB protein signaling domain comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NOs: 50.

In some embodiments, the chimeric intracellular domain further comprises a fourth signal transduction domain. In some embodiments, the fourth signal transduction domain is derived from any one of a CD3 signaling domain, a CD2 signaling domain or an interleukin 2 receptor binding (IL-2RB) protein signaling domain or a combination thereof, wherein the third and the fourth signal transduction domain are not identical.

In some embodiments, the fourth signal transduction domain of the chimeric intracellular domain is a CD3 signaling domain comprising an amino acid sequence according to any one of SEQ ID NOs: 45, 46, 47 and 48. In some embodiments, the fourth signal transduction domain of the chimeric intracellular domain is a CD3ζ comprising an amino acid sequence according to SEQ ID NO: 45. In some embodiments, the fourth signal transduction domain of the chimeric intracellular domain is a CD3ζ comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 45. In some embodiments, the fourth signal transduction domain of the chimeric intracellular domain is a truncated CD3ζ comprising an amino acid sequence according to SEQ ID NO: 46. In some embodiments, the fourth signal transduction domain of the chimeric intracellular domain is a truncated CD3ζ comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 46. In some embodiments, the fourth signal transduction domain of the chimeric intracellular domain is a truncated CD3ε comprising an amino acid sequence according to SEQ ID NO: 47. In some embodiments, the fourth signal transduction domain of the chimeric intracellular domain is a truncated CD3ε comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 47. In some embodiments, the fourth signal transduction domain of the chimeric intracellular domain is a combination of a CD3ε and a truncated CD3ζ domains (CD3ζε domain). In some embodiments, the fourth signal transduction domain of the chimeric intracellular domain is a CD3ε comprising an amino acid sequence according to SEQ ID NO: 48. In some embodiments, the fourth signal transduction domain of the chimeric intracellular domain is a CD3ζε comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 48.

In some embodiments, the fourth signal transduction domain of the chimeric intracellular domain is a mutant CD2 signaling domain. In some embodiments, the fourth signal transduction domain of the chimeric intracellular domain is a truncated CD2 signaling domain. In some embodiments, the fourth signal transduction domain of the chimeric intracellular domain is a truncated CD2 signaling domain comprising an amino acid sequence according to SEQ ID NO: 49. In some embodiments, the fourth signal transduction domain of the chimeric intracellular domain is a truncated CD2 signaling domain comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NOs: 49.

In some embodiments, the fourth signal transduction domain of the chimeric intracellular domain is an IL-2RB protein signaling domain comprising an amino acid sequence according to SEQ ID NO: 50. In some embodiments, the fourth signal transduction domain of the chimeric intracellular domain is an IL-2RB protein signaling domain comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NOs: 50.

The terms T cell, T-cell, t cell, t-cell, and T lymphocyte can be used interchangeably in the present disclosure.

In some embodiments, the extracellular domain comprises a protein or a portion thereof that binds to a target to induce activation and/or proliferation of an immune cell. In some embodiments, the extracellular domain comprises any one of: a) a component of a T cell Receptor (TCR) complex; b) a component of a chimeric antigen receptor (CAR); c) a component of a T cell co-receptor, wherein the T cell co-receptor is a T cell co-stimulatory protein or T cell inhibitory protein; d) a ligand that binds to a cell surface receptor or a component thereof; e) a component of a cytokine receptor; e) a component of a chemokine receptor; g) a component of an integrin receptor; h) a component of an endothelial cell surface protein receptor or a fragment thereof; i) a component of a neuronal guidance protein receptor; and f) a component of a complement receptor. In some embodiments, the component of the T cell co-receptor or the CAR is a component of PD1, CD28, CD2, OX-40, ICOS, CTLA-4, CD28, CD3, CD4, CD8, CD40L, Lag-3, Tim-3, or TIGIT, or a combination thereof. In some embodiments, the ligand or component of the T cell co-receptor or CAR binds to CD19, B cell maturation Ag (BCMA), PD-L1, PD-L2, IL-10, a proliferation-inducing ligand (APRIL), BAFF, OX-40L, ICOS-L, B7-1, B7-2, CD40, CD58, CD59, nectin, CD155, or CD112, or a combination thereof. In some embodiments, the cytokine receptor binds to IL-10, IL-27, TGF-β, IL-12, IL-1, IL-2, IL-4, IL-5, IFN-γ, or IFN-α/β, or a combination thereof. In some embodiments, the component of the complement receptor is a component of a single C3aR, C5aR, CD46/MCP, CD55, CD97, or DAF, or a combination thereof.

In some embodiments, the extracellular domain comprises an amino acid sequence of a component of any one of: a) a chemokine receptor; b) a cytokine receptor; c) a ligand for a cell surface receptor; d) an integrin receptor; e) a cell adhesion molecule or a receptor thereof; f) an endothelial cell surface protein receptor or a fragment thereof; g) a complement receptor; and h) a neuronal guidance protein receptor. In some embodiments, the extracellular domain comprises an amino acid sequence of a component of any one of epithelial growth factor receptor (EGFR), vascular-endothelial growth factor (VEGFR), chemokine receptor (CCR) 4, CCR5, CCR7, CCR10, Lymphocyte function-associated antigen-1 (LFA-1), leukocyte-specific β2 integrins (αLβ2, αMβ2, αXβ2, αDβ2), β7 integrins (α4β7 and αEβ7), extracellular matrix (ECM)-binding β1 integrins (α1-α6β1), L-selectin, or sialyl Lewis^x.

In some embodiments, the extracellular domain is a protein, a peptide, a glycoprotein, an antibody or a fragment thereof. In some embodiments, the antibody or fragment thereof is a Fab fragment, a F(ab)₂fragment, a diabody, a nanobody, a sdAb, Fv, a VHH fragment, or a single chain Fv fragment.

In some embodiments, the extracellular domains comprises two or more binding sites for targeting two or more non-identical target antigens. In some embodiments, the extracellular domains comprises two or more binding sites for targeting two or more non-identical sites on a target antigen. In some embodiments, the extracellular domain comprises two antigen binding domains or fragments of a bispecific antibody. In some embodiments, the extracellular domain comprises a F(ab)₂fragment of a bispecific antibody. In some embodiments, the extracellular domain comprises two or more antigen binding domains or fragments of a multi-specific antibody.

In some embodiments, the extracellular domain binds to a target that is a tumor antigen, a pathogen associated protein, or an antigen associated with the disease or disorder that is a cancer, an autoimmune disease or disorder, an infectious disease, an inflammatory disease, a renal disease or disorder, a lung disease or disorder, a liver disease or disorder, a cardiovascular disease or disorder, a neurodegenerative disorder or disorder, or a metabolic disorder or disorder.

In some embodiments, the tumor antigen is any one of a tumor associated antigen (TAA), a tumor secreted antigen (TSA) or an unconventional antigen (UCA). In some embodiments, the TAA is any one of a cancer germline antigen (CGA), a Human endogenous retroviruses (HERVs), tissue differentiation antigen (TDA) and overexpressed tumor antigen. In some embodiments, the TSA is derived from any one of a mosaic single nucleotide variations (mSNVs), a insertion-deletion mutations (INDELs), gene fusions and viral oncoproteins. In some embodiments, the UCA is derived from non-coding regions of the genome or from coding regions of the genome. In some embodiments, the UCA is derived from aberrant transcription, translation, or post-translational modifications.

In some embodiments, the TAA is associated with a solid tumor or cancer or a hematologic cancer. In some embodiments, the TAA is associated with a solid tumor or cancer is selected from a sarcoma, a carcinoma or a lymphoma that manifests as, leads to, or is associated with a solid tumor.

In some embodiments, the TAA is associated with a sarcoma that is a soft tissue sarcoma or a bone sarcoma (osteosarcoma). In some embodiments, the TAA is associated with a sarcoma selected from vesicular rhabdomyosarcoma, vesicular soft tissue sarcoma, ameloblastoma, angiosarcoma, chondrosarcoma, chordoma, bright tissue sarcoma, dedifferentiated liposarcoma, Hyperplastic small round cell tumor of connective tissue, embryonic rhabdomyosarcoma, epithelioid fibrosarcoma, epithelioid hemangioendothelioma, epithelioid sarcoma; sensitive neuroblastoma (esthesioneuroblastoma), Ewing sarcoma, extrarenal rhabdomyosarcoma, extraosseous myxoid chondrosarcoma, extraosseous osteosarcoma, fibrosarcoma, giant cell tumor, hemangiopericytoma, infantile fibrosarcoma, inflammatory myofibroblastoma, Kaposi sarcoma, bone smooth muscle sarcoma, liposarcoma, osteosarcoma, malignant fibrous histiocytoma (WE), malignant fibrous histiocytoma (WE), malignant mesenchymal tumor, malignant peripheral nerve sheath tumor, mesenchymal chondrosarcoma, myxoid liposarcoma, myxoid inflammatory fibroblastic sarcoma, multiple tumors with perivascular epithelioid cell differentiation, osteosarcoma, extraperiosteal osteosarcoma, tumors with perivascular epithelial cell differentiation, periosteum osteosarcoma, polymorphic liposarcoma, polymorphic rhabdomyosarcoma, PNET/extraosseous Ewing's tumor, rhabdomyosarcoma, small cell osteosarcoma, single fibroids, synovial sarcoma or capillary dilated osteosarcoma.

In some embodiments, the TAA is associated with a carcinoma selected from basal cell carcinoma, squamous cell carcinoma, renal cell carcinoma, ductal carcinoma in situ (DCIS), invasive ductal carcinoma or adenocarcinoma. In some embodiments, the TAA is associated with a carcinoma selected from adenosquamous carcinoma, anaplastic carcinoma, large cell carcinoma, colorectal carcinoma, pancreatic carcinoma, nasopharyngeal carcinoma or small cell carcinoma.

In some embodiments, the TAA is associated with a solid tumor or cancer selected from anal cancer, appendix cancer; cholangiocarcinoma (i.e., biliary tract cancer), breast cancer, bladder cancer, brain tumor, breast cancer, cervical cancer, colon cancer, colorectal cancer, colon polyp, unidentified primary cancer (cup), esophagus cancer, eye cancer, tubal cancer, kidney cancer, liver cancer, lung cancer, medulloblastoma, melanoma, oral cancer, ovarian cancer, prostate cancer, pancreatic cancer, gastric cancer, testicular cancer, laryngeal cancer, thyroid cancer, uterine cancer, vaginal cancer, or vulvar cancer.

In some embodiments, the breast cancer is an invasive breast duct cancer, carcinoma in situ of the duct, invasive lobular carcinoma or lobular carcinoma in situ. In some embodiments, the pancreatic cancer is adenocarcinoma or islet cell carcinoma. In some embodiments, the colorectal cancer is adenocarcinoma. In some embodiments, colonic polyps are associated with familial adenomatous polyposis. In some embodiments, the bladder cancer is transitional cell bladder cancer, squamous cell bladder cancer, or adenocarcinoma. In some embodiments, the lung cancer is non-small cell lung cancer. In some embodiments, the non-small cell lung cancer is adenocarcinoma, squamous cell lung cancer, or large cell lung cancer. In some embodiments, the non-small cell lung cancer is large cell lung cancer. In some embodiments, the lung cancer is small cell lung cancer. In some embodiments, the prostate cancer is adenocarcinoma or small cell carcinoma. In some embodiments, the ovarian cancer is epithelial ovarian cancer. In some embodiments, the cholangiocarcinoma is proximal cholangiocarcinoma or distal cholangiocarcinoma.

In some embodiments, the TAA is associated with any one of the hematological cancer selected from a leukemia, a myeloma or a lymphoma. In some embodiments, the TAA is associated with a leukemia selected from acute leukemia, acute lymphoblastic leukemia (ALL), acute lymphocytic leukemia, a B cell, T cell or FAB ALL, acute myeloid leukemia (AML), acute myelogenous leukemia, chronic myelocytic leukemia (CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia, acute promyelocytic leukemia (APL), mixed-lineage leukemia (MLL) or myelodysplastic syndrome (MDS).

In some embodiments, the TAA is associated with a myeloma that is a multiple myeloma. In some embodiments, the TAA is associated with a multiple myeloma selected from the hyperdiploid (HMM) or the non-hyperdiploid or hypodiploid subtypes of multiple myeloma. In some embodiments, the TAA is associated with a multiple myeloma selected from light chain myeloma, non-secretory myeloma, solitary plasmacytoma, extramedullary plasmacytoma, monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), immunoglobulin D (IgD) myeloma or, immunoglobulin E (IgE) myeloma.

In some embodiments, the TAA is associated with a lymphoma that is a Hodgkin's lymphoma or a non-Hodgkin's lymphoma. In some embodiments, the TAA is associated with a non-Hodgkin's lymphoma. In some embodiments, the TAA is associated with a non-Hodgkin's lymphoma selected from a Small lymphocytic lymphoma (SLL), Lymphoplasmacytic lymphoma, Diffuse large cell lymphoma, Follicle center cell lymphoma, Burkitt's lymphoma, Burkitt-like lymphoma, Mantle cell lymphoma or Marginal zone B-cell lymphoma. In some embodiments, the TAA is associated with a lymphoma that is a Hodgkin's lymphoma. In some embodiments, the TAA is associated with a Hodgkin's lymphoma selected from nodular sclerosis classical Hodgkin lymphoma, lymphocyte-rich classical Hodgkin lymphoma or lymphocyte-depleted classical Hodgkin lymphoma.

In some embodiments, the TAA is associated with a cancer that is any one of acute leukemia, acute lymphoblastic leukemia (ALL), acute lymphocytic leukemia, B cell, T cell or FAB ALL, acute myeloid leukemia (AML), acute myelogenous leukemia, chronic myelocytic leukemia (CIVIL), chronic lymphocytic leukemia (CLL), hairy cell leukemia, myelodysplastic syndrome (MDS), Hodgkin's lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, colorectal carcinoma, pancreatic carcinoma, nasopharyngeal carcinoma, malignant histiocytosis, paraneoplastic syndrome/hypercalcemia of malignancy, bladder cancer, breast cancer, colorectal cancer, endometrial cancer, head cancer, neck cancer, hereditary nonpolyposis cancer, liver cancer, lung cancer, non-small cell lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, testicular cancer, adenocarcinomas, sarcomas, malignant melanoma, and hemangioma.

In some embodiments, the extracellular domain binds to a TAA selected from kallikrein 4, papillomavirus binding factor (PBF), preferentially expressed antigen of melanoma (PRAME), Wilms' tumor-I (WTI), Hydroxysteroid Dehydrogenase Like I (HSDLI), mesothelin, cancer testis antigen (NY-ESO-1), carcinoembryonic antigen (CEA), p53, human epidermal growth factor receptor 2/neuro receptor tyrosine kinase (Her2/Neu), carcinoma-associated epithelial cell adhesion molecule (EpCAM), ovarian and uterine carcinoma antigen (CAI25), folate receptor a, sperm protein 17, tumor-associated differentially expressed gene-12 (TADG-12), mucin-16 (MUC-16), LI cell adhesion molecule (LICAM), mannan-MUC-1, Human endogenous retrovirus K (HERV-K-MEL), Kita-kyushu lung cancer antigen-I (KK-LC-1), human cancer/testis antigen (KM-HN-1), cancer testis antigen (LAGE-I), melanoma antigen-A1 (MAGE-A1), Sperm surface zona pellucida binding protein (Spl 7), Synovial Sarcoma, X Breakpoint 4 (SSX-4), Transient axonal glycoprotein-1 (TAG-I), Transient axonal glycoprotein-2 (TAG-2), Enabled Homolog (ENAH), mammoglobin-A, NY-BR-I, Breast Cancer Antigen, (BAGE-1), B melanoma antigen, melanoma antigen-A1 (MAGE-A1), melanoma antigen-A2 (MAGE-A2), mucin k, synovial sarcoma, X breakpoint 2 (SSX-2), Taxol-resistance-associated gene-3 (TRAG-3), Avian Myelocytomatosis Viral Oncogene (c-myc), cyclin B 1, mucin I (MUC I), p62, survivin, lymphocyte common antigen (CD45), DickkopfWNT Signaling Pathway Inhibitor I (DKKI), telomerase, Kirsten rat sarcoma viral oncogene homolog (K-ras), G250, intestinal carboxyl esterase, alpha-fetoprotein, Macrophage Colony-Stimulating Factor (M-CSF), Prostate-specific membrane antigen (PSMA), caspase 5 (CASP-5), Cytochrome C Oxidase Assembly Factor I Homolog (COA-1), 0-linked β-N-acetylglucosamine transferase (OGT), Osteosarcoma Amplified 9, Endoplasmic Reticulum Lectin (OS-9), Transforming Growth Factor Beta Receptor 2 (TGF-betaRll), murine leukemia glycoprotein 70 (gp70), Calcitonin Related Polypeptide Alpha (CALCA), Programmed cell death 1 ligand 1 (CD274), Mouse Double Minute 2Homolog (mdm-2), alpha-actinin-4, elongation factor 2, Malic Enzyme 1 (MEI), Nuclear Transcription Factor Y Subunit C (NFYC), G Antigen 1,3 (GAGE-1,3), melanoma antigen-A6 (MAGE-A6), cancer testis antigen XAGE-lb, six transmembrane epithelial antigen of the prostate 1 (STEAP1), PAP, prostate specific antigen (PSA), Fibroblast Growth Factor 5 (FGF5), heat shock protein hsp70-2, melanoma antigen-A9 (MAGE-A9), Arg-specific ADP-ribosyltransferase family C (ARTC1), B-Raf Proto-Oncogene (B-RAF), Serine/Threonine Kinase, beta-catenin, Cell Division Cycle 27 homolog (Cdc27), cyclin dependent kinase 4 (CDK4), cyclin dependent kinase 12 (CDK12), Cyclin Dependent Kinase Inhibitor 2A (CDKN2A), Casein Kinase 1 Alpha 1 (CSNK1A1), Fibronectin 1 (FN1), Gruwih Anest Specific 7 (GAS7), Glycoprotein nonmetastatic melanoma protein B (GPNMB), HAUS Augmin Like Complex Subunit 3 (HAUS3), LDLR-fucosyltransferase, Melanoma Antigen Recognized By T cells 2 (MART2), myostatin (MSTN), Melanoma Associated Antigen (Mutated) 1 (MUM-1-2-3), Poly(A) polymerase gamma (neo-PAP), myosin class I, Protein phosphatase 1 regulatory subunit 3B (PPP1R3B), Peroxiredoxin-5 (PRDX5), Receptor-type tyrosine-protein phosphatase kappa (PTPRK), Transforming protein N-Ras (N-ras), retinoblastoma-associated factor 600 (RBAF600), sirtuin-2 (SIRT2), SNRPD1, triosephosphate isomerase, Ocular Albinism Type 1 Protein (OAl), member RAS oncogene family (RAB38), Tyrosinase related protein 1-2 (TRP-1-2), Melanoma Antigen Gp75 (gp75), tyrosinase, Melan-A (MART-1), Glycoprotein 100 melanoma antigen (gplOO), N-acetylglucosaminyltransferase V gene (GnTVf), Lymphocyte Antigen 6 Complex Locus K (LY6K), melanoma antigen-AlO (MAGE-AlO), melanoma antigen-Al2 (MAGE-Al2), melanoma antigen-C2 (MAGE-C2), melanoma antigen NA88-A, Taxol-resistant-associated protein 3 (TRAG-3), BDZ binding kinase (pbk), caspase 8 (CASP-8), sarcoma antigen 1 (SAGE), Breakpoint Cluster Region-Abelson oncogene (BCR-ABL), fusion protein in leukemia, dek-can, Elongation Factor Tu GTP Binding Domain Containing 2 (EFTUD2), ETS Variant gene 6/acute myeloid leukemia fusion protein (ETV6-AML1), FMS-like tyrosine kinase-3 internal tandem duplications (FLT3-ITD), cyclin-Al, Fibronectin Type III Domain Containing 3B (FDNC3B) promyelocytic leukemia/retinoic acid receptor alpha fusion protein (pml-RARalpha), melanoma antigen-Cl (MAGE-Cl), membrane protein alternative spliced isoform (D393-CD20), melanoma antigen-A4 (MAGE-A4), or melanoma antigen-A3 (MAGE-A3).

In some embodiments, the autoimmune condition or disorder is any one of Type 1 Diabetes, rheumatoid arthritis (RA), systemic lupus erythematosis (SLE), multiple sclerosis (MS), celiac disease, sjÖgren syndrome, polymyalgia rheumatica, ankylosing spondylitis, alopecia areata, vasculitis and temporal arteritis. In some embodiments, the tumor associated antigen (TAA) associated with the autoimmune condition or disorder is derived from any one of Carboxypeptidase H, Chromogranin A, Glutamate decarboxylase, Imogen-38, Insulin, Insulinoma antigen-2 and 2β, Islet-specific glucose-6-phosphatase catalytic subunit related protein (IGRP), Proinsulin, α-enolase, Aquaporin-4, β-arrestin, Myelin basic protein, Myelin oligodendrocytic glycoprotein, Proteolipid protein, S100-β, Citrullinated protein, Collagen II, Heat shock proteins, Human cartilage glycoprotein, Double-stranded DNA, La antigen, Nucleosomal histones and ribonucleoproteins (snRNP), Phospholipid-β-2 glycoprotein I complex, Poly(ADP-ribose) polymerase, and Sm antigens of U-1 small ribonucleoprotein complex.

In some embodiments, the pathogen associated antigen is an antigen from a bacterial, a fungal or a parasitic protein or fragment thereof. In some embodiments, the pathogen associated antigen is associated with HIV infection, human Cytomegalovirus infection, Hepatitis B infection, Hepatitis C infection, Ebola virus infection, Dengue, Yellow fever, Listeriosis, Tuberculosis, Cholera, Malaria, Leishmaniasis, or Trypanosoma infection, or a combination thereof.

In some embodiments, the neurodegenerative disorder or condition is any one of Alzheimer's disease (AD) and other dementias, Parkinson's disease (PD) and PD-related disorders, Prion disease, Motor neurone diseases (MND), Huntington's disease (HD), Spinocerebellar ataxia (SCA) or Spinal muscular atrophy (SMA). In some embodiments, the antigen associated with the neurodegenerative disorder or condition is any one of Amyloid (Ab), tau, alpha-synuclein (α-syn), mHTT or prion PrP^scor a combination thereof.

In some embodiments, the extracellular domain binds to a target with a binding affinity of 1 fM to 100 μM. In some embodiments, the extracellular domain binds to a target with a binding affinity of 1 pM to 100 μM. In some embodiments, the extracellular domain binds to a target with a binding affinity of 1 pM to 10 pM. In some embodiments, the extracellular domain binds to a target with a binding affinity of 10 pM to 50 pM. In some embodiments, the extracellular domain binds to a target with a binding affinity of 10 pM to 100 pM. In some embodiments, the extracellular domain binds to a target with a binding affinity of 100 pM to 500 pM. In some embodiments, the extracellular domain binds to a target with a binding affinity of 500 pM to 1 nM. In some embodiments, the extracellular domain binds to a target with a binding affinity of 1 nM to 10 nM. In some embodiments, the extracellular domain binds to a target with a binding affinity of 10 nM to 100 nM. In some embodiments, the extracellular domain binds to a target with a binding affinity of 100 nM to 500 nM. In some embodiments, the extracellular domain binds to a target with a binding affinity of 500 nM to 1 μM. In some embodiments, the extracellular domain binds to a target with a binding affinity of 1 μM to 10 μM. In some embodiments, the extracellular domain binds to a target with a binding affinity of 1 μM to 5 μM. In some embodiments, the extracellular domain binds to a target with a binding affinity of 5 μM to 7.5 μM. In some embodiments, the extracellular domain binds to a target with a binding affinity of 7.5 μM to 10 μM.

In some embodiments, the extracellular domain comprises a signal peptide at the N-terminus. In some embodiments, the signal peptide can be derived from a surface expressing protein or a secretory protein. In some embodiments, the signal peptide can be derived from Preprolactin, HIV pre-Env, HCV polyprotein, CB virus polyprotein, Pestivirus polyprotein, Precalreticulin, pre-VSV-G, HLA class I histocompatibility antigen or PD-1 signal peptide (PD-1 SP), interleukin 12 (IL12), GM-CSF or CD8 alpha chain (CD8a). In some embodiments, the signal peptide is PD-1 signal peptide (PD-1 SP). In some embodiments, the signal peptide is a HLA class I histocompatibility antigen or a portion thereof. In some embodiments, the extracellular domain is derived from PD1. In some embodiments, the extracellular domain comprises the amino acid sequence from position 1 to 163 of the amino acid sequence according to any one of SEQ ID NOs: 19-21. In some embodiments, the extracellular domain comprises the amino acid sequence from position 1 to 163 of the amino acid sequence according to SEQ ID NOs: 19. In some embodiments, the extracellular domain comprises the amino acid sequence from position 1 to 163 of the amino acid sequence according to SEQ ID NOs: 20. In some embodiments, the extracellular domain comprises the amino acid sequence from position 1 to 163 of the amino acid sequence according to SEQ ID NOs: 21.

In some embodiments, the extracellular domain comprises the amino acid sequence according to any one of SEQ ID NOs: 22-23. In some embodiments, the extracellular domain comprises the amino acid sequence according to SEQ ID NOs: 22. In some embodiments, the extracellular domain comprises the amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NOs: 22. In some embodiments, the extracellular domain comprises the amino acid sequence according to SEQ ID NOs: 23. In some embodiments, the extracellular domain comprises the amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NOs: 23.

TABLE 4

Amino acid sequences of PD1 extracellular

domains of RTCR PD1

(PD1 Signal Peptide_PD1 Extracellular_PD1

Transmembrane_PD1 Intracellular)

(other names: PD1 wt (human-wild type);

PD1:WT; PD-1; PD-1 wt; PD-1 wild type; PD1;

PD1wt or PD1 wild type) (SEQ ID NO: 19):

Signal Peptide (italicized), Extracellular

domain (IG-like V domain in bold and stalk

in bold and underlined), Trans Membrane

(underlined) and Intracellular domain

in double underline

MQIPQAPWPVFWAVLQLGWRPGWFLDSPDRPWNP

PTFSPALLVVTEGDNATFTCSFSNTSESFVLNWY

RMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPN

GRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKE

SLRAELRVT
custom-character

VGVVGGLLGSLVLLVWVLAVI

CSRAARGTIGARRTGQPLKEDPSAVPVFSVDY

GELDFQWREKTPEPPVPCVPEQTEYATIV

FPSGMGTSSPARRGSADGPRSAQPLRPED

GHCSWPL

PD1 Signal Peptide_PD1 Extracellular PD1

Transmembrane (Other name: PD-1 truncated)

(SEQ ID NO: 20): PD1 Signal Peptide

(italicized), Extracellular domain

(IG-like V domain in bold and stalk

in bold and underlined), Trans Membrane

(underlined) and Intracellular tail

in double underline

MQIPQAPWPVFWAVLQLGWRPGW
FLDSPDRPWNP

PTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYR

MSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGR

DFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLR

AELRVT
custom-character

VGVVGGLLGSLVLLVWVLAVI
CSR

HLA-A2 Signal Peptide_PD1 Extracellular PD1

Transmembrane (PD1-TLs; HLASP-Truncated,

PD1-TLs, PD1:TLs) (SEQ ID NO: 21):

HLA-A2 Signal Peptide (italicized),

Extracellular domain (IG-like V domain

in bold and stalk in bold and underlined),

Trans Membrane (underlined) and Intracellular

tail in double underline

MAVMAPRTLVLLLSGALALTQTWA
FLDSPDRPWNP

PTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYR

MSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGR

DFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLR

AELRVT
custom-character

VVGVVGGLLGSLVLLVWVLAVI
CSR

PD-1 (extracellular domain) (SEQ ID NO: 22):

Signal Peptide (italicized), Extracellular

domain (IG-like V domain in bold and stalk

in bold and underlined)

MQIPQAPWPVFWAVLQLGWRPGWFLDSPDRPWNP

PTFSPALLVVTEGDNATFTCSFSNTSESFVLNWY

RMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPN

GRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKE

SLRAELRVT
custom-character

PD-1 (HLA A2-Signal Peptide extracellular

domain) (other name: PD-1 (HLA A2-SP

extracellular domain) (SEQ ID NO: 23):

Signal Peptide (italicized), Extracellular

domain (IG-like V domain in bold and

stalk in bold and underlined)

MAVMAPRTLVLLLSGALALTQTWA
FLDSPDRPWNP

PTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYR

MSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNG

RDFHMSVVRARRNDSGTYLCGAISLAPKAQIKES

LRAELRVT
custom-character

In some embodiments, the RTCR disclosed herein comprises the amino acid sequence according to any one of SEQ ID NOs: 24-44 and 130-132.

In some embodiments, the RTCR disclosed herein comprises the amino acid sequence according to SEQ ID NO: 24. In some embodiments, the RTCR disclosed herein comprises the amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 24.

In some embodiments, the RTCR disclosed herein comprises the amino acid sequence according to SEQ ID NO: 25. In some embodiments, the RTCR disclosed herein comprises the amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 25.

In some embodiments, the RTCR disclosed herein comprises the amino acid sequence according to SEQ ID NO: 26. In some embodiments, the RTCR disclosed herein comprises the amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 26.

In some embodiments, the RTCR disclosed herein comprises the amino acid sequence according to SEQ ID NO: 27. In some embodiments, the RTCR disclosed herein comprises the amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 27.

In some embodiments, the RTCR disclosed herein comprises the amino acid sequence according to SEQ ID NO: 28. In some embodiments, the RTCR disclosed herein comprises the amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 28.

In some embodiments, the RTCR disclosed herein comprises the amino acid sequence according to SEQ ID NO: 29. In some embodiments, the RTCR disclosed herein comprises the amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 29.

In some embodiments, the RTCR disclosed herein comprises the amino acid sequence according to SEQ ID NO: 30. In some embodiments, the RTCR disclosed herein comprises the amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 30.

In some embodiments, the extracellular domain of the RTCR disclosed herein comprises the amino acid sequence according to SEQ ID NO: 31. In some embodiments, the extracellular domain of the RTCR disclosed herein comprises the amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 31.

In some embodiments, the RTCR disclosed herein comprises the amino acid sequence according to SEQ ID NO: 32. In some embodiments, the RTCR disclosed herein comprises the amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 32.

In some embodiments, the RTCR disclosed herein comprises the amino acid sequence according to SEQ ID NO: 33. In some embodiments, the RTCR disclosed herein comprises the amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 33.

In some embodiments, the RTCR disclosed herein comprises the amino acid sequence according to SEQ ID NO: 34. In some embodiments, the RTCR disclosed herein comprises the amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 34.

In some embodiments, the RTCR disclosed herein comprises the amino acid sequence according to SEQ ID NO: 35. In some embodiments, the RTCR disclosed herein comprises the amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 35.

In some embodiments, the RTCR disclosed herein comprises the amino acid sequence according to SEQ ID NO: 36. In some embodiments, the RTCR disclosed herein comprises the amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 36.

In some embodiments, the RTCR disclosed herein comprises the amino acid sequence according to SEQ ID NO: 37. In some embodiments, the RTCR disclosed herein comprises the amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 37.

In some embodiments, the RTCR disclosed herein comprises the amino acid sequence according to SEQ ID NO: 38. In some embodiments, the RTCR disclosed herein comprises the amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 38.

In some embodiments, the RTCR disclosed herein comprises the amino acid sequence according to SEQ ID NO: 39. In some embodiments, the RTCR disclosed herein comprises the amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 39.

In some embodiments, the RTCR disclosed herein comprises the amino acid sequence according to SEQ ID NO: 40. In some embodiments, the RTCR disclosed herein comprises the amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 40.

In some embodiments, the RTCR disclosed herein comprises the amino acid sequence according to SEQ ID NO: 41. In some embodiments, the RTCR disclosed herein comprises the amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 41.

In some embodiments, the RTCR disclosed herein comprises the amino acid sequence according to SEQ ID NO: 42. In some embodiments, the RTCR disclosed herein comprises the amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 42.

In some embodiments, the RTCR disclosed herein comprises the amino acid sequence according to SEQ ID NO: 43. In some embodiments, the RTCR disclosed herein comprises the amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 43.

In some embodiments, the RTCR disclosed herein comprises the amino acid sequence according to SEQ ID NO: 44. In some embodiments, the RTCR disclosed herein comprises the amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 44.

In some embodiments, the RTCR disclosed herein comprises the amino acid sequence according to SEQ ID NO: 130. In some embodiments, the RTCR disclosed herein comprises the amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 130.

In some embodiments, the RTCR disclosed herein comprises the amino acid sequence according to SEQ ID NO: 131. In some embodiments, the RTCR disclosed herein comprises the amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 131.

In some embodiments, the RTCR disclosed herein comprises the amino acid sequence according to SEQ ID NO: 132. In some embodiments, the RTCR disclosed herein comprises the amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 132.

TABLE 5

Amino acid sequences of PD1 based recombinant T cell co-stimulatory receptor

(RTCR).

HLA-A2 Signal Peptide_PD1 Extracellular_CD28 Transmembrane_CD28 Signaling Domain

(Other names: PD-1-CD28 Domain Swap; HLA A2-SP-PD-1_28; HLA A2-SP-PD-1_CD28

DS; HLA A2-SP-PD-1_CD28; PD1_CD28 or PD1:CD28 or PD_28) (SEQ ID NO: 24):

HLA-A2 Signal Peptide (italicized), PD 1 extracellular domain (IG-like V domain in bold and

stalk in bold and underlined), CD28 Transmembrane (underlined) and Intracellular domain in

double underline; and CD28 signal domain: Stalk (underlined and italicized), transmembrane

domain (double underlined), intracellular domain (IC) (dashed underlined) (SEQ ID NO: 10)

MAVMAPRTLVLLLSGALALTQTWAFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFS

NTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVV

embedded image

HLA-A2 Signal Peptide_PD1 Extracellular_ICOS Transmembrane_ICOS Signaling Domain

(Other names: PD-1-ICOS Domain Swap; HLASP-PD-1-ICOS DS; HLA A2-SP-PD-1_ICOS;

PD-1-ICOS; PD-1 :ICOS; PD1_ICOS) (SEQ ID NO: 25): HLA-A2 Signal Peptide (italicized),

PD 1 extracellular domain (IG-like V domain in bold and stalk in bold and underlined), ICOS

Transmembrane (underlined) and Intracellular domain in double underline; and ICOS domain:

Stalk (underlined and italicized), transmembrane domain (double underlined), intracellular

domain (IC) (dashed underlined)

MAVMAPRTLVLLLSGALALTQTWAFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFS

NTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVV

embedded image

HLA-A2 Signal Peptide_PD1 Extracellular_ICOS Transmembrane_ICOS Signaling

Domain_4-1BB Signaling Domain (other names: PD1 HLASP-ICOS-4-1BB; HLA A2-SP-

PD-1_ICOS BBwt; HLA A2-SP-PD-1_ICOS_BB; HLA A2-SP-PD-1_ICOS CD137; HLA

A2-SP-PD-1_ICOS CD137 wt; PD1_ICOS BBwt; PD1:ICOSBBwt; PD1:ICOSBB) (SEQ ID

NO: 26): PD 1 extracellular domain in regular font; ICOS domain underlined; and 4-1BB

domain (CD137 signaling domain, wild type) in bold

MAVMAPRTLVLLLSGALALTQTWAFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSF

SNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRA

RRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPASQLCCQLKF

WLPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVT

L
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

HLA-A2 Signal Peptide_PD1 Extracellular_ICOS Transmembrane_ICOS Signaling

Domain Truncated 4-1BB Signaling Domain

(Other names: PD1 HLASP-ICOS-truncated 4-1BB; HLA A2-SP-PD-1-ICOS BBt; HLA A2-

SP-PD-1_ICOS BBt. HLA A2-SP-PD-1_ICOS truncated CD137; HLA A2-SP-PD-

1_ICOS truncated CD137 wt; PD1_ICOS BBt; PD1;ICOSBBt) (SEQ ID NO: 27): PD 1

extracellular domain in regular font; ICOS domain underlined; and truncated 4-1BB (truncated

CD137 signaling domain in bold

MAVMAPRTLVLLLSGALALTQTWAFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSF

SNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRA

RRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPASQLCCQLKF

WLPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVT

L
QPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

HLA-A2 Signal Peptide_PD1 Extracellular_ICOS Transmembrane_ICOS Signaling

Domain Truncated OX-40 Signaling Domain

(Other names: PD1 HLASP-ICOS-truncated OX40; HLA A2-SP-PD-1_ICOS OX40t, HLA

A2-SP-PD-1_ICOS_40t; HLA A2-SP-PD-1_ICOS truncated CD134; PD_ICOS_OX40t;

PD1:ICOS40t) (SEQ ID NO: 28): PD 1 extracellular domain in regular font; ICOS domain

underlined; and truncated OX40 (truncated CD134 signaling domain) in bold

MAVMAPRTLVLLLSGALALTQTWAFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSF

SNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRA

RRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPASQLCCQLKF

WLPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVT

L
GGGSFRTPIQEEQADAHSTLA

HLA-A2 Signal Peptide_PD1 Extracellular_ICOS Transmembrane_ICOS Signaling Domain

(mini-CD28)_Truncated 4-1BB Signaling Domain (Other names: PD1_ICOS(28) BBt;

PD1_ICOS(28)_truncated CD137; PD1:ICOS(28)-BBt or PD1:ICOS(28)BBt) (SEQ ID NO:

29): PD 1 extracellular domain in regular font; ICOS (28) domain: ICOS portions are

underlined and inserted CD28 portion bold and underline; and truncated CD137 domain in

bold

MAVMAPRTLVLLLSGALALTQTWAFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSF

SNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRA

RRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPASQLCCQLKF

WLPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPNGEYMFMTPRRPGPTRKHYQPY

embedded image

HLA-A2 Signal Peptide_PD1 Extracellular_ICOS Transmembrane_ICOS Signaling Domain

(mini-CD28)_Truncated OX-40 Signaling Domain (Other names: HLASP-ICOS DS-

CD28(PRRP)-mutated CD134; PD_ICOS(28) OX40t; PD_ICOS(28)_40t;

PD1_ICOS(28) truncated CD134; PD1:ICOS(28)-OX40t; PD1:ICOS(28)OX40t;

PD1:ICOS(28)-40t or PD1:ICOS(28)40t) (SEQ ID NO: 30): PD 1 extracellular domain in

regular font; ICOS (28) domain: ICOS portions are underlined and inserted CD28 portion bold

and underline; and truncated CD134 domain in bold

MAVMAPRTLVLLLSGALALTQTWAFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSF

SNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRA

RRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPASQLCCQLKF

embedded image

PD1 Extracellular (without HLA A2 Signal Peptide) (SEQ ID NO: 31)

FLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAA

FPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLR

AELRVTERRAEVPTAHPSPSPRPA

PD1 extracellular domain without signal peptide-CD28 domain swap (DS) (Other name:

PD_28) (SEQ ID NO: 32)

FLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAA

FPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLR

AELRVTERRAEVPTAHPSPSPRPALFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWV

RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

PD1 extracellular domain without signal peptide-CD28 DS-CD137 domain (Other name:

PD1_28_BBwt) (SEQ ID NO: 33)

FLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAA

FPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLR

AELRVTERRAEVPTAHPSPSPRPALFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWV

RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYIFKQPFM

RPVQTTQEEDGCSCRFPEEEEGGCEL

PD1 extracellular domain without signal peptide-CD28 DS-truncated CD137 domain (Other

name: PD1_28_BBt) (SEQ ID NO: 34)

FLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAA

FPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLR

AELRVTERRAEVPTAHPSPSPRPALFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWV

RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSQPFMRPVQTTQEEDGC

SCRFPEEEEGGCEL

PD1 extracellular domain without signal peptide-CD28 DS-truncated CD134 domain (Other

name: PD1_28_OX40t) (SEQ ID NO: 35)

FLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAA

FPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLR

AELRVTERRAEVPTAHPSPSPRPALFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWV

RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSGGGSFRTPIQEEQADA

HSTLA

PD1 extracellular domain without signal peptide-ICOS DS (Other name: PD1_ICOS) (SEQ ID

NO: 36)

FLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAA

FPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLR

AELRVTERRAEVPTARPSPSPRPASQLCCQLKFWLPIGCAAFVVVCILGCILICWLTKK

KYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL

PD1 extracellular domain without signal peptide-ICOS DS-CD137 (Other name:

PD1_ICOS_BBwt) (SEQ ID NO: 37)

FLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAA

FPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLR

AELRVTERRAEVPTAHPSPSPRPASQLCCQLKFWLPIGCAAFVVVCILGCILICWLTKK

KYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTLKRGRKKLLYIFKQPFMRPVQTTQEE

DGCSCRFPEEEEGGCEL

PD1 extracellular domain without signal peptide-ICOS DS-truncated CD137 (Other name:

PD1_ICOS_BBt) (SEQ ID NO: 38)

FLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAA

FPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLR

AELRVTERRAEVPTARPSPSPRPASQLCCQLKFWLPIGCAAFVVVCILGCILICWLTKK

KYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTLQPFMRPVQTTQEEDGCSCRFPEEEE

GGCEL

PD1 extracellular domain without signal peptide-ICOS DS-truncated CD134 (Other name:

PD1_ICOS OX40t (SEQ ID NO: 39)

FLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAA

FPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLR

AELRVTERRAEVPTARPSPSPRPASQLCCQLKFWLPIGCAAFVVVCILGCILICWLTKK

KYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTLGGGSFRTPIQEEQADAHSTLA

PD1 extracellular domain without signal peptide-ICOS DS-CD28(PRRP)-truncated CD137)

(Other name: PD1_ICOS(28)_BBt (SEQ ID NO: 40)

FLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAA

FPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLR

AELRVTERRAEVPTARPSPSPRPASQLCCQLKFWLPIGCAAFVVVCILGCILICWLTKK

KYSSSVHDPNGEYMFMTPRRPGPTRKHYQPYAPPRAVNTAKKSRLTDVTLQPFMRPV

QTTQEEDGCSCRFPEEEEGGCEL

PD1 extracellular domain without signal peptide-ICOS DS-CD28(PRRP)-truncated CD134

(Other name: PD1_ICOS(28)_OX40t) (SEQ ID NO: 41)

FLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAA

FPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLR

AELRVTERRAEVPTARPSPSPRPASQLCCQLKFWLPIGCAAFVVVCILGCILICWLTKK

KYSSSVHDPNGEYMFMTPRRPGPTRKHYQPYAPPRAVNTAKKSRLTDVTLGGGSFRT

PIQEEQADAHSTLA

PD1 extracellular domain without signal peptide-ICOS DS-truncated CD137 domain-truncated

CD2 signaling domain (Other name: PD1_ICOS_BBt:CD2t) (SEQ ID NO: 42)

FLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAA

FPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLR

AELRVTERRAEVPTARPSPSPRPASQLCCQLKFWLPIGCAAFVVVCILGCILICWLTKK

KYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTLQPFMRPVQTTQEEDGCSCRFPEEEE

GGCELQNPATSQHPPPPPGHRSQAPSHRPPPPGHRVQHQPQKRPPAPSGTQVHQQKGP

PLPRPRVQPKPPHGAAENSLSPSSN

PD1 extracellular domain without signal peptide-ICOS DS-truncated CD137 domain-truncated

CD2 signaling domain (Other name: PD1_ICOS_BBt:IL2RB(YLRQ)) (SEQ ID NO: 43)

FLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAA

FPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLR

AELRVTERRAEVPTARPSPSPRPASQLCCQLKFWLPIGCAAFVVVCILGCILICWLTKK

KYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTLQPFMRPVQTTQEEDGCSCRFPEEEE

GGCELNCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGL

APEISPLEVLERDKVTQLLPLNTDAYLSLQELQGQDPTHLVSYLRQWVVIPPPLSSPGP

QAS

PD1 extracellular domain without signal peptide-ICOS DS-truncated CD137 domain-truncated

CD2 signaling domain-IL-2 receptor binding (IL2RB)(YLRQ) protein (Other name:

PD1_ICOS_BBt_CD2t_IL2RB(YLRQ) (SEQ ID NO: 44)

FLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAA

FPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLR

AELRVTERRAEVPTARPSPSPRPASQLCCQLKFWLPIGCAAFVVVCILGCILICWLTKK

KYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTLQPFMRPVQTTQEEDGCSCRFPEEEE

GGCELQNPATSQHPPPPPGHRSQAPSHRPPPPGHRVQHQPQKRPPAPSGTQVHQQKGP

PLPRPRVQPKPPHGAAENSLSPSSNNCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGG

DVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLPLNTDAYLSLQELQGQDPTH

LVSYLRQWVVIPPPLSSPGPQAS

HLA-A2 Signal Peptide_PD1 Extracellular_CD28 Transmembrane_CD28 Signaling

Domain 4-1BB Signaling Domain (Other names: PD1_28_BBwt; PD1_28_BB;

PD1_CD28_BB; PD1_CD28_BB wt; PD1_CD28_CD137; PD1:28BB; PD1:28BB wt;

PD1:28-BB or PD1:28-BB wt) (SEQ ID NO: 130)

MAVMAPRTLVLLLSGALALTQTWAFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSF

SNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRA

RRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPALFPGPSKPF

WVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPY

APPRDFAAYRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

HLA-A2 Signal Peptide_PD1 Extracellular_CD28 Transmembrane_CD28 Signaling

Domain_Truncated 4-1BB Signaling Domain (Other names: PD1_28_BBt; PD1_CD28_BBt;

PD1_CD28_truncated CD137; PD1:28BBt; or PD1:28-BBt) (SEQ ID NO: 131)

MAVMAPRTLVLLLSGALALTQTWAFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSF

SNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRA

RRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPALFPGPSKPF

WVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPY

APPRDFAAYRSQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

HLA-A2 Signal Peptide_PD1 Extracellular_CD28 Transmembrane_CD28 Signaling

Domain_Truncated OX-40 Signaling Domain (Other names: PD1_28_OX40t; PD1_28_40t;

PD_CD28_OX40t; PD_CD28_40t; PD-1_CD28_truncated CD134; PD1:2840t;

PD1:28OX40t; PD1:20-OX40t or PD1:28-40t) (SEQ ID NO: 132): PD1 Extracellular (with

HLA-A2 Signal Peptide)_CD28 Transmembrane_CD28 Signaling Domain_Truncated OX-40

Signaling Domain

MAVMAPRTLVLLLSGALALTQTWAFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSF

SNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRA

RRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPALFPGPSKPF

WVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPY

APPRDFAAYRSGGGSFRTPIQEEQADAHSTLA

TABLE 6

Amino acid sequences of third and fourth

signaling domains of RTCR

Human CD3 ζ, intracellular signaling

domain (Other names: CD3Z (intracellular

Signaling Domain);

CD3Z) (SEQ ID NO: 45)

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRR

GRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMK

GERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

Human CD3 Z signaling domain truncated

(CD3 Z truncated domain) (Other names:

Human CD3 ζ signaling domain

truncated Z; CD3 ζ truncated

domain; CD3Zt or Zt)

(SEQ ID NO: 46)

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRR

GRDPEMGGK

Human CD3 E signaling domain truncated

(CD3 E truncated domain) (Other name: Human

CD3 ϵ signaling domain truncated;

CD3 ϵ truncated domain; CD3Et or

Et (SEQ ID NO: 47)

PVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRD

LYSGLNQRRI

Human CD3 ZE signaling domain (CD3 ZE

domain) (Other name: Human CD3 ζϵ signaling

domain; CD3 ζϵ domain; CD3ZE or ZE)

(SEQ ID NO: 48)

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRR

GRDPEMGGKPVTRGAGAGGRQRGQNKERPPPVPNPDYEP

IRKGQRDLYSGLNQRRI

CD2 truncated Signaling Domain (Other name:

CD2 or 2) (SEQ ID NO: 49)

QNPATSQHPPPPPGHRSQAPSHRPPPPGHRVQHQPQKRPPA

PSGTQVHQQKGPPLPRPRVQPKPPHGAAENSLSPSSN

IL-2 receptor binding (IL2RB) protein

Signaling Domain (YLRQ shown in bold)

(Other name: IL2RB(YLRQ)

(SEQ ID NO: 50)

NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQK

WLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLPL

NTDAYLSLQELQGQDPTHLVSYLRQWVVIPPPLSSPG

PQAS

In some embodiments, the extracellular domain is derived from CD19 binding protein. In some embodiments, the CD19 binding protein is a CD19 binding chimeric antigen receptor (CAR). In some embodiments, the extracellular domain comprises the amino acid sequence according to SEQ ID NO: 51. In some embodiments, the extracellular domain comprises the amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 51.

In some embodiments, the CD19 binding chimeric antigen receptor comprises the amino acid sequence according to any one of SEQ ID NOs: 52-69. In some embodiments, the CD19 binding chimeric antigen receptor comprises the amino acid sequence according to SEQ ID NO: 52. In some embodiments, the CD19 binding chimeric antigen receptor comprises the amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 52.

In some embodiments, the CD19 binding chimeric antigen receptor comprises the amino acid sequence according to SEQ ID NO: 53. In some embodiments, the CD19 binding chimeric antigen receptor comprises the amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 53.

In some embodiments, the CD19 binding chimeric antigen receptor comprises the amino acid sequence according to SEQ ID NO: 54. In some embodiments, the CD19 binding chimeric antigen receptor comprises the amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 54.

In some embodiments, the CD19 binding chimeric antigen receptor comprises the amino acid sequence according to SEQ ID NO: 55. In some embodiments, the CD19 binding chimeric antigen receptor comprises the amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 55.

In some embodiments, the CD19 binding chimeric antigen receptor comprises the amino acid sequence according to SEQ ID NO: 56. In some embodiments, the CD19 binding chimeric antigen receptor comprises the amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 56.

In some embodiments, the CD19 binding chimeric antigen receptor comprises the amino acid sequence according to SEQ ID NO: 57. In some embodiments, the CD19 binding chimeric antigen receptor comprises the amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 57.

In some embodiments, the CD19 binding chimeric antigen receptor comprises the amino acid sequence according to SEQ ID NO: 58. In some embodiments, the CD19 binding chimeric antigen receptor comprises the amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 58.

In some embodiments, the CD19 binding chimeric antigen receptor comprises the amino acid sequence according to SEQ ID NO: 59. In some embodiments, the CD19 binding chimeric antigen receptor comprises the amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 59.

In some embodiments, the CD19 binding chimeric antigen receptor comprises the amino acid sequence according to SEQ ID NO: 60. In some embodiments, the CD19 binding chimeric antigen receptor comprises the amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 60.

In some embodiments, the CD19 binding chimeric antigen receptor comprises the amino acid sequence according to SEQ ID NO: 61. In some embodiments, the CD19 binding chimeric antigen receptor comprises the amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 61.

In some embodiments, the CD19 binding chimeric antigen receptor comprises the amino acid sequence according to any one of SEQ ID NO: 62. In some embodiments, the CD19 binding chimeric antigen receptor comprises the amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 62.

In some embodiments, the CD19 binding chimeric antigen receptor comprises the amino acid sequence according to SEQ ID NO: 63. In some embodiments, the CD19 binding chimeric antigen receptor comprises the amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 63.

In some embodiments, the CD19 binding chimeric antigen receptor comprises the amino acid sequence according to SEQ ID NO: 64. In some embodiments, the CD19 binding chimeric antigen receptor comprises the amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 64.

In some embodiments, the CD19 binding chimeric antigen receptor comprises the amino acid sequence according to SEQ ID NO: 65. In some embodiments, the CD19 binding chimeric antigen receptor comprises the amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 65.

In some embodiments, the CD19 binding chimeric antigen receptor comprises the amino acid sequence according to SEQ ID NO: 66. In some embodiments, the CD19 binding chimeric antigen receptor comprises the amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 66.

In some embodiments, the CD19 binding chimeric antigen receptor comprises the amino acid sequence according to SEQ ID NO: 67. In some embodiments, the CD19 binding chimeric antigen receptor comprises the amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 67.

In some embodiments, the CD19 binding chimeric antigen receptor comprises the amino acid sequence according to SEQ ID NO: 68. In some embodiments, the CD19 binding chimeric antigen receptor comprises the amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 68.

In some embodiments, the CD19 binding chimeric antigen receptor comprises the amino acid sequence according to SEQ ID NO: 69. In some embodiments, the CD19 binding chimeric antigen receptor comprises the amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 69.

TABLE 7

Amino acid sequences related to

CD19 CAR based RTCR.

CD19 binding extracellular domain, FMC63scFV

(Other name: CD19) (SEQ ID NO: 51):

CD8a leader/signal peptide (bold,

SEQ ID NO: 117) and CD8a Hinge

(underlined, SEQ ID NO: 118)

[FMC63 scFV (CD8a Leader_Light Chain_

Linker_Heavy Chain_CD8a Hinge)]

MALPVTALLLPLALLLHAARPDIQMTQTTSSLSAS

LGDRVTISCRASQDISKYLNAVYQQKPDGTVKLLI

YHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDI

ATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSG

GGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPD

YGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSR

LTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYY

GGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIA

SQPLSLRPEACRPAAGGAVHTRGLDFAC

CD19 (FMC63 scFV) CD8a Transmembrane 4-1BB

Signaling_CD3Z chimeric antigen receptor

(CAR) (Other names: FMC63scFV_BB_Z;

CD19_BB_Z; CD19_BBwt_Z; CD19_CD137_Z;

CD19-BBZ; or CD19:BBZ) (SEQ ID NO: 52):

CD19 binding extracellular

domain (underlined)-CD137 intracellular

domain-CD3ζ signaling domain

MALPVTALLLPLALLLHAARPDIQMTQTTSSLSAS

LGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIY

HTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIA

TYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGG

GGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDY

GVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRL

TIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYG

GSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIAS

QPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPL

AGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRP

VQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAP

AYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMG

GKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGER

RRGKGHDGLYQGLSTATKDTYDALHMQALPPR

CD19 (FMC63 scFV)_CD28 Transmembrane

CD28 Signaling_CD3Z CAR (Other names:

FMC63scFV_28_Z; CD19_28_Z; CD19_CD28_Z;

CD19 28Z or CD19-28Z or CD19:28Z)

(SEQ ID NO: 53):

CD19 binding extracellular domain

(underlined)-CD28 DS-CD3ζ signaling

domain

MALPVTALLLPLALLLHAARPDIQMTQTTSSLSAS

LGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIY

HTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIA

TYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGG

GGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDY

GVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRL

TIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYG

GSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIAS

QPLSLRPEACRPAAGGAVHTRGLDFACLFPGPSKP

FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLL

HSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRV

KFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDK

RRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAY

SEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM

QALPPR

CD19 (FMC63 scFV)_CD28 Transmembrane_

CD28 Signaling 4-1BB Signaling_CD3Z CAR

(Other names: FMC63scFV_28_BBwt_Z;

CD19_CD28_BB_Z; CD19_28_BBwt_Z; CD19-

28BBwt_Z; CD19-28BBZ; or CD19:28BBZ)

(SEQ ID NO: 54):

[CD19 binding extracellular

domain (underlined)-CD28 DS-CD137

intracellular domain-CD3t signaling domain

MALPVTALLLPLALLLHAARPDIQMTQTTSSLSAS

LGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIY

HTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIA

TYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGG

GGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDY

GVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRL

TIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYG

GSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIAS

QPLSLRPEACRPAAGGAVHTRGLDFACLFPGPSKP

FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLL

HSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKR

GRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEE

GGCELRVKFSRSADAPAYQQGQNQLYNELNLGRRE

EYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQK

DKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKD

TYDALHMQALPPR

CD19 (FMC63 scFV)_CD28 Transmembrane

CD28 Signaling 4-1BB truncated

Signaling_CD3Z CAR (Other names:

FMC63scFV_28_BBt_Z; CD19_CD28_BBt_Z;

CD19_28_BBt_Z; CD19-28BBt_Z; CD19-28BBtZ

or CD19:28BBtZ) (SEQ ID NO: 55): []

CD19 binding extracellular domain

(underlined)-CD28 DS-truncated CD137

intracellular domain-CD3

signaling domain

MALPVTALLLPLALLLHAARPDIQMTQTTSSLSAS

LGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIY

HTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIA

TYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGG

GGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDY

GVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRL

TIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYG

GSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIAS

QPLSLRPEACRPAAGGAVHTRGLDFACLFPGPSKP

FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLL

HSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSQP

FMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRS

ADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRD

PEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGM

KGERRRGKGHDGLYQGLSTATKDTYDALHMQALPP

R

CD19 (FMC63 scFV)_CD28 Transmembrane CD28

Signaling OX-40 Truncated Signaling_CD3Z

CAR (Other names: FMC63scFV_28_OX40t_Z;

CD19_CD28_OX40t_Z; CD19_28_OX40t_Z;

CD19_28_OX40tZ; CD19_28_40t_Z;

CD19_28_40tZ; CD19-2840tZ or

CD19:2840tZ) (SEQ ID NO: 56):

CD19 binding extracellular domain

(underlined)-CD28 DS-

truncated CD134 intracellular

domain-CD3ζ signaling domain

MALPVTALLLPLALLLHAARPDIQMTQTTSSLSAS

LGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIY

HTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIA

TYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGG

GGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDY

GVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRL

TIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYG

GSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIAS

QPLSLRPEACRPAAGGAVHTRGLDFACLFPGPSKP

FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLL

HSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSGG

GSFRTPIQEEQADAHSTLARVKFSRSADAPAYQQG

QNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQR

RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKG

HDGLYQGLSTATKDTYDALHMQALPPR

CD19 (FMC63 scFV) ICOS Transmembrane

ICOS Signaling_CD3Z CAR (Other names:

FMC63scFV_ICOS_Z; CD19_ICOS_Z;

CD19_ICOSZ; CD19-ICOSZ or CD19:ICOSZ)

(SEQ ID NO: 57): [(CD19 binding

extracellular domain (underlined)-

ICOS DS-CD3ζ signaling domain)

MALPVTALLLPLALLLHAARPDIQMTQTTSSLSAS

LGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIY

HTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIA

TYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGG

GGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDY

GVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRL

TIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYG

GSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIAS

QPLSLRPEACRPAAGGAVHTRGLDFACSQLCCQLK

FWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHD

PNGEYMFMRAVNTAKKSRLTDVTLRVKFSRSADAP

AYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMG

GKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGER

RRGKGHDGLYQGLSTATKDTYDALHMQALPPR

CD19 (FMC63 scFV) ICOS Transmembrane

ICOS Signaling 4-1BB Signaling_CD3Z

CAR (Other names:

FMC63scFV_ICOS_BBwt_Z;

CD19_ICOS_BB_Z; CD19_ICOS_BBwt_Z;

CD19-ICOSBBwt_Z; CD19-ICOSBBZ or

CD19:ICOSBBZ) (SEQ ID NO: 58): []

CD19 binding extracellular domain

(underlined)-ICOS DS-CD137

intracellular domain-

CD3ζ signaling domain

MALPVTALLLPLALLLHAARPDIQMTQTTSSLSAS

LGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIY

HTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIA

TYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGG

GGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDY

GVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRL

TIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYG

GSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIAS

QPLSLRPEACRPAAGGAVHTRGLDFACSQLCCQLK

FWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHD

PNGEYMFMRAVNTAKKSRLTDVTLKRGRKKLLYIF

KQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKF

SRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRR

GRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSE

IGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQA

LPPR

CD19 (FMC 63 scFV)_ICOS Transmembrane

ICOS Signaling 4-1BB Truncated

Signaling_CD3Z CAR (Other names:

FMC63scFV_ICOS_BBt_Z; CD19_ICOS_BBt_Z;

CD19-ICOSBBtZ or CD19:ICOSBBtZ)

(SEQ ID NO: 59): []

CD19 binding extracellular

domain (underlined)-ICOS DS-truncated

CD137 intracellular domain-CD3ζ

signaling domain

MALPVTALLLPLALLLHAARPDIQMTQTTSSLSAS

LGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIY

HTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIA

TYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGG

GGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDY

GVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRL

TIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYG

GSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIAS

QPLSLRPEACRPAAGGAVHTRGLDFACSQLCCQLK

FWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHD

PNGEYMFMRAVNTAKKSRLTDVTLQPFMRPVQTTQ

EEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQG

QNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQR

RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKG

HDGLYQGLSTATKDTYDALHMQALPPR

CD19 (FMC 63 scFV) ICOS Transmembrane

ICOS Signaling OX-40 Truncated

Signaling_CD3Z CAR (Other names:

FMC63scFV_ICOS_OX40t_Z;

CD19_ICOS_OX40t_Z;

CD19_ICOS_40t_Z; CD19_ICOS_OX40tZ;

CD19_ICOS_40tZ; CD19-ICOS4OtZ or

CD19:ICOS4OtZ) (SEQ ID NO: 60):

CD19 binding extracellular domain

(underlined)-ICOS

DS-truncated CD134 intracellular

domain-CD3ζ signaling domain

MALPVTALLLPLALLLHAARPDIQMTQTTSSLSAS

LGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIY

HTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIA

TYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGG

GGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDY

GVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRL

TIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYG

GSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIAS

QPLSLRPEACRPAAGGAVHTRGLDFACSQLCCQLK

FWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHD

PNGEYMFMRAVNTAKKSRLTDVTLGGGSFRTPIQE

EQADAHSTLARVKFSRSADAPAYQQGQNQLYNELN

LGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLY

NELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLS

TATKDTYDALHMQALPPR

CD19 (FMC63 scFV)_ICOS_Transmembrane_

ICOS Signaling (mini CD28 Signaling)_CD3Z

CAR (Other names: FMC63scFV_ICOS(28)_Z;

CD19_ICOS(28)_Z; CD19-ICOS(28)_Z;

CD19-ICOS(28)Z or CD19:ICOS(28)Z)

(SEQ ID NO: 61): []

CD19 binding extracellular

domain (underlined)-ICOS DS-CD28(PRRP)

-CD3ζ signaling domain)

MALPVTALLLPLALLLHAARPDIQMTQTTSSLSAS

LGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIY

HTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIA

TYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGG

GGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDY

GVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRL

TIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYG

GSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIAS

QPLSLRPEACRPAAGGAVHTRGLDFACSQLCCQLK

FWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHD

PNGEYMFMTPRRPGPTRKHYQPYAPPRAVNTAKKS

RLTDVTLRVKFSRSADAPAYQQGQNQLYNELNLGR

REEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNEL

QKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTAT

KDTYDALHMQALPPR

CD19 (FMC63 scFV) ICOS Transmembrane

ICOS Signaling (mini CD28 Signaling)

4-1BB Truncated Signaling_CD3Z CAR

(Other names: FMC63scFV_ICOS(28)_BBt_Z;

CD19_ICOS(28)_BBt_Z; CD19_ICOS(28)_BBtZ;

CD19-ICOS(28)BBtZ or

CD19:ICOS(28)BBtZ) (SEQ ID NO: 62):

(CD19 binding extracellular domain

(underlined)-ICOS DS-CD28(PRRP)-

truncated CD137 intracellular

domain-CD3ζ signaling domain)

MALPVTALLLPLALLLHAARPDIQMTQTTSSLSAS

LGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIY

HTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIA

TYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGG

GGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDY

GVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRL

TIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYG

GSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIAS

QPLSLRPEACRPAAGGAVHTRGLDFACSQLCCQLK

FWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHD

PNGEYMFMTPRRPGPTRKHYQPYAPPRAVNTAKKS

RLTDVTLQPFMRPVQTTQEEDGCSCRFPEEEEGGC

ELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYD

VLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKM

AEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD

ALHMQALPPR

CD19 (FMC 63 scFV)_ICOS_Transmembrane_

ICOS Signaling (mini CD28 Signaling)_

OX-40 Truncated Signaling_CD3Z CAR

(Other names: FMC63scFV_ICOS(28)_OX40t_Z

CD19_ICOS(28)_OX40t_Z; CD19_ICOS(28)_OX40tZ;

CD19_IC05(28)_40t_Z; CD19_ICOS(28)_40tZ;

CD19-ICOS(28)40tZ or CD19:ICOS(28)40tZ)

(SEQ ID NO: 63):

CD19 binding extracellular domain

(underlined)-ICOS DS-CD28(PRRP)-truncated

CD134 intracellular domain-CD3ζ

signaling domain

MALPVTALLLPLALLLHAARPDIQMTQTTSSLSAS

LGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIY

HTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIA

TYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGG

GGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDY

GVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRL

TIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYG

GSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIAS

QPLSLRPEACRPAAGGAVHTRGLDFACSQLCCQLK

FWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHD

PNGEYMFMTPRRPGPTRKHYQPYAPPRAVNTAKKS

RLTDVTLGGGSFRTPIQEEQADAHSTLARVKFSRS

ADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRD

PEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGM

KGERRRGKGHDGLYQGLSTATKDTYDALHMQALPP

R

CD19_ICOSBBt_Zt CAR (CD19 binding

extracellular domain-ICOS DS-

truncated CD137 intracellular

domain-CD3Ztruncated domain)

CAR (Other names:

FMC63scFV_ICOS_BBt_Zt;

CD19_ICOS_BBt_Zt; CD19-ICOSBBtZt

or CD19:ICOSBBtZt) (SEQ ID NO: 64):

CD19 binding extracellular domain

(underlined)-ICOS DS-truncated

CD137 intracellular domain-CD3ζ

truncated domain

MALPVTALLLPLALLLHAARPDIQMTQTTSSLSAS

LGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIY

HTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIA

TYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGG

GGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDY

GVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRL

TIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYG

GSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIAS

QPLSLRPEACRPAAGGAVHTRGLDFACSQLCCQLK

FWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHD

PNGEYMFMRAVNTAKKSRLTDVTLQPFMRPVQTTQ

EEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQG

QNQLYNELNLGRREEYDVLDKRRGRDPEMGGK

CD19_ICOSBBt_ZE_CAR (CD19 binding

extracellular domain-ICOS DS-

truncated CD137 intracellular

domain-CD3ZE domain) (Other

names: FMC63scFV_ICOS_BBt_ZE; CD19

CD19-ICOSBBtZE ICOS_BBt_ZE; or

CD19:ICOSBBtZE) (SEQ ID NO: 65):

CD19 binding extracellular domain

(underlined)-ICOS DS-truncated

CD137 intracellular domain-

CD3ζ domain

MALPVTALLLPLALLLHAARPDIQMTQTTSSLSAS

LGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIY

HTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIA

TYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGG

GGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDY

GVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRL

TIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYG

GSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIAS

QPLSLRPEACRPAAGGAVHTRGLDFACSQLCCQLK

FWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHD

PNGEYMFMRAVNTAKKSRLTDVTLQPFMRPVQTTQ

EEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQG

QNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPVT

RGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDL

YSGLNQRRI

CD19_ICOSBBt:CD2t_Z CAR (CD19 binding

extracellular domain-ICOS DS-truncated

CD137 intracellular domain-CD2 truncated

Signaling Domain-CD3 domain) CAR (Other

names: FMC63scFV_ICOS_BBt_CD2tZ; CD19_

ICOS_BBt_CD2t_Z; CD19_ICOS_BBt CD2tZ;

CD19_ICOS_BBtCD2tZ; CD19-ICOSBBtCD2tZ or

CD19:ICOSBBtCD2tZ) (SEQ ID NO: 66):

CD19 binding extracellular domain

(underlined)-ICOS DS-truncated CD137

intracellular domain-CD2 truncated

Signaling Domain-CD3ζ domain

MALPVTALLLPLALLLHAARPDIQMTQTTSSLSAS

LGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIY

HTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIA

TYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGG

GGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDY

GVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRL

TIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYG

GSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIAS

QPLSLRPEACRPAAGGAVHTRGLDFACSQLCCQLK

FWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHD

PNGEYMFMRAVNTAKKSRLTDVTLQPFMRPVQTTQ

EEDGCSCRFPEEEEGGCELQNPATSQUPPPPPGHR

SQAPSHRPPPPGHRVQHQPQKRPPAPSGTQVHQQK

GPPLPRPRVQPKPPHGAAENSLSPSSNRVKFSRSA

DAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDP

EMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMK

GERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

CD19_ICOSBBt:CD2t ZE CAR (CD19 binding

extracellular domain-ICOS DS-truncated

CD137 intracellular domain-CD2 truncated

Signaling Domain-CD3ZE domain) (Other

names: FMC63scFV_ICOS_BBt_CD2tZE CAR CD19_

ICOS_BBt_CD2tZE; CD19_ICOS_BBt_CD2t_ZE;

CD19_ICOS_BBt_2tZE; CD19_ICOS_BBt_2t_ZE;

CD19-ICOSBBtCD2tZE; CD19-ICOSBBt2tZE;

CD19:ICOSBBtCD2tZE or CD19:ICOSBBt2tZE)

(SEQ ID NO: 67):

CD19 binding extracellular domain

(underlined)-ICOS DS-truncated

CD137 intracellular domain-CD2

truncated Signaling Domain-CD3ζϵ domain

MALPVTALLLPLALLLHAARPDIQMTQTTSSLSAS

LGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIY

HTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIA

TYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGG

GGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDY

GVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRL

TIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYG

GSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIAS

QPLSLRPEACRPAAGGAVHTRGLDFACSQLCCQLK

FWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHD

PNGEYMFMRAVNTAKKSRLTDVTLQPFMRPVQTTQ

EEDGCSCRFPEEEEGGCELQNPATSQUPPPPPGHR

SQAPSHRPPPPGHRVQHQPQKRPPAPSGTQVHQQK

GPPLPRPRVQPKPPHGAAENSLSPSSNRVKFSRSA

DAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDP

EMGGKPVTRGAGAGGRQRGQNKERPPPVPNPDYEP

IRKGQRDLYSGLNYRHQRRI

CD19_ICOSBBt:IL2RB(YLRQ)_Z CAR (CD19

binding extracellular domain-ICOS DS-

truncated CD137 intracellular domain-

IL2RB(YLRQ) domain-CD3Z domain)

(Other names: FMC63scFV_ICOS_BBt_IL2RB

(YLRQ)_Z_CAR_CD19_ICOS_BBt_IL2RB(YLRQ)Z;

CD19_ICOS_BBt_IL2RB(YLRQ)_Z; CD19_ICOS_

BBtIL2RB(YLRQ)Z; CD19-ICOSBBtIL2RB(YLRQ)Z

or CD191COSBBtIL2RB(YLRQ)Z)

(SEQ ID NO: 68): CD19

binding extracellular domain

underlined)-ICOS DS-truncated CD137

intracellular domain-IL2RB(YLRQ)

domain-CD3ζ domain)

MALPVTALLLPLALLLHAARPDIQMTQTTSSLSAS

LGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIY

HTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIA

TYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGG

GGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDY

GVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRL

TIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYG

GSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIAS

QPLSLRPEACRPAAGGAVHTRGLDFACSQLCCQLK

FWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHD

PNGEYMFMRAVNTAKKSRLTDVTLQPFMRPVQTTQ

EEDGCSCRFPEEEEGGCELNCRNTGPWLKKVLKCN

TPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPG

GLAPEISPLEVLERDKVTQLLPLNTDAYLSLQELQ

GQDPTHLVRVKFSRSADAPAYQQGQNQLYNELNLG

RREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNE

LQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTA

TKDTYDAYRHQALPPR

CD19_ICOSBBt:IL2RB(YLRQ)_ZE CAR (CD19

binding extracellular domain-ICOS DS-

truncated CD137 intracellular domain-

IL2RB(YLRQ) domain-CD3ZE domain) CAR

(Other names: FMC63scFV_ICOS_BBt

IL2RB(YLRQ)_ZE_CD19_COS_BBt_IL2RB

(YLRQ)_ZE; CD19_ICOS_BBtIL2RB(YLRQ)ZE;

CD19-ICOSBBtIL2RB(YLRQ)ZE or

CD191COSBBtIL2RB(YLRQ)ZE)

(SEQ ID NO: 69): CD19 binding

extracellular domain

(underlined)-ICOS DS-truncated CD137

intracellular domain-IL2RB(YLRQ)

domain-CD3ζϵ domain

MALPVTALLLPLALLLHAARPDIQMTQTTSSLSAS

LGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIY

HTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIA

TYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGG

GGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDY

GVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRL

TIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYG

GSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIAS

QPLSLRPEACRPAAGGAVHTRGLDFACSQLCCQLK

FWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHD

PNGEYMFMRAVNTAKKSRLTDVTLQPFMRPVQTTQ

EEDGCSCRFPEEEEGGCELNCRNTGPWLKKVLKCN

TPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPG

GLAPEISPLEVLERDKVTQLLPLNTDAYLSLQELQ

GQDPTHLVRVKFSRSADAPAYQQGQNQLYNELNLG

RREEYDVLDKRRGRDPEMGGKPVTRGAGAGGRQRG

QNKERPPPVPNPDYEPIRKGQRDLYSGLNYRHQRR

I

In some embodiments, the extracellular domain comprises a hinge region. In some embodiments, the hinge region is derived from CD8, PD-1, CD28, ICOS, or IgG. In some embodiments, the transmembrane domain of the RTCR disclosed herein, is derived from CD8, PD1, CD28, ICOS, or IgG.

The present disclosure also provides a nucleic acid encoding the RTCR disclosed herein. In some embodiments, the nucleic acid encoding the RTCR disclosed herein is according to SEQ ID NO: 75-86 and 92-110. In some embodiments, the nucleic acid disclosed herein comprises a nucleic acid sequence encoding a chimeric intracellular domain. In some embodiments, the RTCR disclosed herein is for expression in a T cell, wherein the T cell co-expresses at least one of the endogenous co-stimulatory molecules CD28, CD2, OX-40, ICOS, CD28, CD3, CD4, CD8 and CD40L or a combination thereof.

The present disclosure also provides a vector comprising the nucleic acid disclosed herein. In some embodiments, the vector disclosed herein is any one of a viral vector, a plasmid, a cosmid, a yeast artificial chromosome, a bacterial artificial chromosome or a transposon/transposase system. In some embodiments, the viral vector is an adeno-viral vector or a lentiviral vector. In some embodiments, the vector is a lentiviral vector.

The present disclosure also provides a cell comprising the nucleic acid or the vector disclosed herein. In some embodiments, the cell disclosed herein is a modified T cell. In some embodiments, the modified T cell is an allogenic T cell. In some embodiments, the modified T cell is an autologous T cell. In some embodiments, the modified T cell is any one of a naïve T cell, an early memory T cell, a stem cell-like T cell, a stem memory T cell (T_SCM), a central memory T cell (T_CM) and a regulatory T cell (T_reg).

In some embodiments, the extracellular domain is a B cell maturation Ag (BCMA) binding protein. In some embodiments, the BCMA binding protein is a BCMA specific T cell receptor (TCR). In some embodiments, the BCMA binding protein is a BCMA specific chimeric antigen receptor (CAR). In some embodiments, the BCMA binding chimeric antigen receptor comprises the amino acid sequence according to any one of: SEQ ID NOs: 137-146.

In some embodiments, the BCMA binding chimeric antigen receptor comprises the amino acid sequence according to SEQ ID NO: 137. In some embodiments, the BCMA binding chimeric antigen receptor comprises the amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 137.

In some embodiments, the BCMA binding chimeric antigen receptor comprises the amino acid sequence according to SEQ ID NO: 138. In some embodiments, the BCMA binding chimeric antigen receptor comprises the amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 138.

In some embodiments, the BCMA binding chimeric antigen receptor comprises the amino acid sequence according to SEQ ID NO: 139. In some embodiments, the BCMA binding chimeric antigen receptor comprises the amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 139.

In some embodiments, the BCMA binding chimeric antigen receptor comprises the amino acid sequence according to SEQ ID NO: 140. In some embodiments, the BCMA binding chimeric antigen receptor comprises the amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 140.

In some embodiments, the BCMA binding chimeric antigen receptor comprises the amino acid sequence according to SEQ ID NO: 141. In some embodiments, the BCMA binding chimeric antigen receptor comprises the amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 141.

In some embodiments, the BCMA binding chimeric antigen receptor comprises the amino acid sequence according to SEQ ID NO: 142. In some embodiments, the BCMA binding chimeric antigen receptor comprises the amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 142.

In some embodiments, the BCMA binding chimeric antigen receptor comprises the amino acid sequence according to SEQ ID NO: 143. In some embodiments, the BCMA binding chimeric antigen receptor comprises the amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 143.

In some embodiments, the BCMA binding chimeric antigen receptor comprises the amino acid sequence according to SEQ ID NO: 144. In some embodiments, the BCMA binding chimeric antigen receptor comprises the amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 144.

In some embodiments, the BCMA binding chimeric antigen receptor comprises the amino acid sequence according to SEQ ID NO: 145. In some embodiments, the BCMA binding chimeric antigen receptor comprises the amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 145.

In some embodiments, the BCMA binding chimeric antigen receptor comprises the amino acid sequence according to SEQ ID NO: 146. In some embodiments, the BCMA binding chimeric antigen receptor comprises the amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 146.

TABLE 8

Amino acid sequences of BCMA specific chimeric antigen

receptors (CAR) and BCMA specific CAR-based RTCR.

11-D5-3 scFv (CD8a Signal Peptide_11-D5-3 scFv, mouse_CD8a_

Hinge_BB_Z) (SEQ ID NO: 133):

CD8a Signal Peptide (bold, SEQ ID NO: 117)

11-D5-3 scFv, mouse (italics)_CD8a Hinge (bold,

SEQ ID NO: 118)_BB_Z)

MALPVTALLLPLALLLHAARP
DIVLTQSPPSLAMSLGKRATISCRASESVTILGSH

LIHWYQQKPGQPPTLLIQLASNVQTGVPARFSGSGSRTDFTLTIDPVEEDDVAVYYC

LQSRTIPRTFGGGTKLEIKGSTSGSGKPGSGEGSTKGQIQLVQSGPELKKPGETVKI

SCKASGYTFTDYSINWVKRAPGKGLKWMGWINTETREPAYAYDFRGRFAFSLETSAS

TAYLQINNLKYEDTATYFCALDYSYAMDYWGQGTSVTVSS
TTTPAPRPPTPAPTIAS

QPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRK

KLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQL

YNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGM

KGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

FHVH33 HVV (CD8a Signal Peptide_FHVH33 HVV_CD8a Hinge_BB_Z)

(SEQ ID NO: 134):

(CD8a Signal Peptide (bold, SEQ ID NO: 117)_FHVH33 HVV

(italicized)_CD8a Hinge

(bold, SEQ ID NO: 118)_BB_Z)

MALPVTALLLPLALLLHAARP
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVR

QAPGKGLEWVSSISGSGDYIYYADSVKGRFTISRDISKNTLYLQMNSLRAEDTAVYYCA

KEGTGANSSLADYRGQGTLVTVSSFVPVFLPAKP
TTTPAPRPPTPAPTIASQPLSLRPE

ACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNKRGRKKLLYIF

KQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGR

REEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRG

KGHDGLYQGLSTATKDTYDALHMQALPPR

BCAR003 HVV (CD8a Signal Peptide BCAR003 HVV_CD8a Hinge_BB_Z)

(SEQ ID NO: 135): CD8a Signal Peptide (Bold)_BCAR003 HVV

(italicized)_CD8a Hinge (Bold)_BB_Z

MALPVTALLLPLALLLHAARP
QVKLEESGGGLVQAGRSLRLSCAASEHTFSSHVMGWF

RQAPGKERESVAVIGWRDISTSYADSVKGRFTISRDNAKKTLYLQMNSLKPEDTAVYY

CAARRIDAADFDSWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSAVQLVESGG

GLVQAGDSLRLTCTASGRAFSTYFMAWFRQAPGKEREFVAGIAWSGGSTAYADSVKGR

FTISRDNAKNTVYLQMNSLKSEDTAVYYCASRGIEVEEFGAWGQGTQVTVSS
TSTTTP

APRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLS

LVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSAD

APAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDK

MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

GSI5022 HVV (CD8a Signal Peptide_G515022 HVV_CD8a Hinge_BB_Z)

(SEQ ID NO: 136): CD8a Signal Peptide (bold, SEQ ID NO: 117)

G5I5022 HVV (italicized)_CD8a Hinge

(bold, SEQ ID NO: 118)_BB_Z

MALPVTALLLPLALLLHAARP
QVKLEESGGGLVQAGRSLRLSCAASEHTFSSHVMGWF

RQAPGKERESVAVIGWRDISTSYADSVKGRFTISRDNAKKTLYLQMNSLKPEDTAVYY

CAARRIDAADFDSWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLR

LSCAASGRTFTMGWFRQAPGKEREFVAAISLSPTLAYYAESVKGRFTISRDNAKNTVV

LQMNSLKPEDTALYYCAADRKSVMSIRPDYWGQGTQVTVSSTSTTTPAPRPPTPAPTI

ASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGR

KKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQL

YNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMK

GERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

BCMAsdAb1 HVV (CD8a Signal Peptide anti-BCMAsdAB1 HHV_CD8a

Hinge) (SEQ ID NO: 137): CD8a Signal Peptide (bold,

SEQ ID NO: 117)_anti-BCMAsdAB1 HHV (italicized)_

CD8a Hinge (bold, SEQ ID NO: 118)

MALPVTALLLPLALLLHAARP
EVQLQASGGGLAQPGGSLRLSCAASGRTFSTYFMAWF

RQPPGKGLEYVGGIRWSDGVPHYADSVKGRFTISRDNAKNTVYLQMNSLRAEDTAVYF

CASRGIADGSDFGSYGQGTQVTVSS
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGG

AVHTRGLDFAC

BCMAsdAb1 HVV CD8a Transmembrane 4-1BB Signaling_CD3Z

(Other names: BCMAsdAb1_BB_Z or BCMAsdAb1-BBZ)

(SEQ ID NO: 138): BCMAsdAb1 HVV (bold, SEQ ID NO: 137)_

CD8a_Transmembrane_4-1BB Signaling_CD3Z

MALPVTALLLPLALLLHAARPEVQLQASGGGLAQPGGSLRLSCAASGRTFSTYF

MAWFRQPPGKGLEYVGGIRWSDGVPHYADSVKGRFTISRDNAKNTVYLQMNSLR

AEDTAVYFCASRGIADGSDFGSYGQGTQVTVSSTTTPAPRPPTPAPTIASQPLS

LRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKK

LLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQN

QLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAY

SEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

BCMAsdAb1 HVV_CD28 Transmembrane_CD28 Signaling_CD3Z

(Other name: BCMAsdAb1_28_Z; BCMAsdAb1-28Z)

(SEQ ID NO: 139): BCMAsdAb1 HVV (bold, SEQ ID NO: 137)

CD28 Transmembrane_CD28 Signaling_CD3Z

MALPVTALLLPLALLLHAARPEVQLQASGGGLAQPGGSLRLSCAASGRTFSTYF

MAWFRQPPGKGLEYVGGIRWSDGVPHYADSVKGRFTISRDNAKNTVYLQMNSLR

AEDTAVYFCASRGIADGSDFGSYGQGTQVTVSSTTTPAPRPPTPAPTIASQPLS

LRPEACRPAAGGAVHTRGLDFACLFPGPSKPFWVLVVVGGVLACYSLLVTVAFI

IFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSAD

GAPAYQQQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNEL

QKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

BCMAsdAb1 HVV_CD28 Transmembrane CD28 Signaling 4-1BB

Signaling_CD3Z (Other 28 BBZ.

names: BCMAsdAb1_28_BBwt_Z; BCMAsdAb1, 28_BB_Z_BCMAsdAb1,

BCMAsdAb1-28_BBz; or BCMAsdAb1-28BBZ)

(SEQ ID NO: 140): BCMAsdAb1 HVV (bold, SEQ ID NO: 137)_

CD28 Transmembrane_CD28 Signaling 4-1BB Signaling_CD3Z

MALPVTALLLPLALLLHAARPEVQLQASGGGLAQPGGSLRLSCAASGRTFSTYF

MAWFRQPPGKGLEYVGGIRWSDGVPHYADSVKGRFTISRDNAKNTVYLQMNSLR

AEDTAVYFCASRGIADGSDFGSYGQGTQVTVSSTTTPAPRPPTPAPTIASQPLS

LRPEACRPAAGGAVHTRGLDFACLFPGPSKPFWVLVVVGGVLACYSLLVTVAFI

IFWLVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKLL

YIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQL

YNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSE

IGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

BCMAsdAb1 HVV_CD28 Transmembrane CD28 Signaling

4-1BB truncated Signaling_CD3Z (Other names:

BCMAsdAb1_28_BBt_Z; BCMAsdAb1_28_BBtZ

BCMAsdAb1-28 BBtZ; or BCMAsdAb1-28BBtZ)

(SEQ ID NO: 141): BCMAsdAb1 HVV (bold, SEQ ID NO: 137)_

CD28 Transmembrane_CD28 Signaling_4-1BB truncated

Signaling_CD3Z

MALPVTALLLPLALLLHAARPEVQLQASGGGLAQPGGSLRLSCAASGRTFSTYF

MAWFRQPPGKGLEYVGGIRWSDGVPHYADSVKGRFTISRDNAKNTVYLQMNSLR

AEDTAVYFCASRGIADGSDFGSYGQGTQVTVSSTTTPAPRPPTPAPTIASQPLS

LRPEACRPAAGGAVHTRGLDFACLFPGPSKPFWVLVVVGGVLACYSLLVTVAFI

IFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSQPFMRPVQT

TQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYD

VLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGH

DGLYQGLSTATKDTYDALHMQALPPR

BCMAsdAb1 HVV_CD28 Transmembrane CD28 Signaling

OX-40 Truncated Signaling_CD3Z (Other names: BCMAsdAb1

28_OX40t_Z; BCMAsdAb1_28_OX40tZ;

BCMAsdAb1_28_40t_Z; BCMAsdAb1_28_40tZ BCMAsdAb1-28

40tZ; or BCMAsdAb1-2840tZ) (SEQ ID NO: 142):

BCMAsdAb1 HVV (bold, SEQ ID NO: 137)_CD28

Transmembrane_CD28 Signaling_OX-40 Truncated

Signaling_CD3Z

MALPVTALLLPLALLLHAARPEVQLQASGGGLAQPGGSLRLSCAASGRTFSTYF

MAWFRQPPGKGLEYVGGIRWSDGVPHYADSVKGRFTISRDNAKNTVYLQMNSLR

AEDTAVYFCASRGIADGSDFGSYGQGTQVTVSSTTTPAPRPPTPAPTIASQPLS

LRPEACRPAAGGAVHTRGLDFACLFPGPSKPFWVLVVVGGVLACYSLLVTVAFI

IFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSGGGSFRTPI

QEEQADAHSTLARVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRD

PEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLST

ATKDTYDALHMQALPPR

BCMAsdAb1 HVV_ICOS Transmembrane_ICOS Signaling_

CD3Z (Other names: BCMAsdAb1_ICOS_Z; BCMAsdAb1-ICOSZ;

or BCMAsdAb1-ICOSZ) (SEQ ID NO: 143): BCMAsdAb1 HVV (bold,

SEQ ID NO: 137)_ICOS Transmembrane_ICOS Signaling_CD3Z

MALPVTALLLPLALLLHAARPEVQLQASGGGLAQPGGSLRLSCAASGRTFSTYF

MAWFRQPPGKGLEYVGGIRWSDGVPHYADSVKGRFTISRDNAKNTVYLQMNSLR

AEDTAVYFCASRGIADGSDFGSYGQGTQVTVSSTTTPAPRPPTPAPTIASQPLS

LRPEACRPAAGGAVHTRGLDFACSQLCCQLKFWLPIGCAAFVVVCILGCILICW

LTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTLRVKFSRSADAPAYQQGQN

QLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAY

SEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

BCMAsdAb1 HVV_ICOS Transmembrane_ICOS Signaling 4-1BB

Signaling_CD3Z (Other names: BCMAsdAb1 ICOS_BBwt_Z;

BCMAsdAb1_ICOS_BB_Z; BCMAsdAb1_ICOS_BBZ; BCMAsdAb1-28_BBZ;

or BCMAsdAb1-ICOSBBZ) (SEQ ID NO: 144): BCMAsdAb 1 HVV

(bold, SEQ ID NO: 137)_ICOS Transmembrane_ICOS Signaling_

4-1BB Signaling_CD3Z

MALPVTALLLPLALLLHAARPEVQLQASGGGLAQPGGSLRLSCAASGRTFSTYF

MAWFRQPPGKGLEYVGGIRWSDGVPHYADSVKGRFTISRDNAKNTVYLQMNSLR

AEDTAVYFCASRGIADGSDFGSYGQGTQVTVSSTTTPAPRPPTPAPTIASQPLS

LRPEACRPAAGGAVHTRGLDFACSQLCCQLKFWLPIGCAAFVVVCILGCILICW

LTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTLKRGRKKLLYIFKQPFMRP

VQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRRE

EYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRG

KGHDGLYQGLSTATKDTYDALHMQALPPR

BCMAsdAb1 HVV_ICOS Transmembrane_ICOS Signaling 4-1BB

Truncated Signaling_CD3Z (Other names: BCMAsdAb1 ICOS_BBt_Z;

BCMAsdAb1 ICOS_BBtZ; BCMAsdAb 1-ICOS_BBtZ; or BCMAsdAb

1-ICOSBBtz) (SEQ ID NO: 145): BCMAsdAb1 HVV (bold,

SEQ ID NO: 137)_ICOS Transmembrane_ICOS Signaling

4-1BB Truncated Signaling_CD3Z

MALPVTALLLPLALLLHAARPEVQLQASGGGLAQPGGSLRLSCAASGRTFSTYF

MAWFRQPPGKGLEYVGGIRWSDGVPHYADSVKGRFTISRDNAKNTVYLQMNSLR

AEDTAVYFCASRGIADGSDFGSYGQGTQVTVSSTTTPAPRPPTPAPTIASQPLS

LRPEACRPAAGGAVHTRGLDFACSQLCCQLKFWLPIGCAAFVVVCILGCILICW

LTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTLQPFMRPVQTTQEEDGCSC

RFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRD

PEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLST

ATKDTYDALHMQALPPR

BCMAsdAb1 HVV_ICOS Transmembrane_ICOS Signaling_OX-40

Truncated Signaling_CD3Z

(Other names: BCMAsdAb1_ICOS_OX40t_Z;

BCMAsdAb1_ICOS_OX40tZ; BCMAsdAb1_ICOS_40t_Z;

BCMAsdAb1_ICOS_40tZ; BCMAsdAb1-ICOS_40tZ; or

BCMAsdAb1_ICOS4OtZ) (SEQ ID NO: 146): BCMAsdAb1

HVV (bold, SEQ ID NO: 137)_Transmembrane_ICOS

Signaling_OX-40 Truncated Signaling_CD3Z

MALPVTALLLPLALLLHAARPEVQLQASGGGLAQPGGSLRLSCAASGRTFSTYF

MAWFRQPPGKGLEYVGGIRWSDGVPHYADSVKGRFTISRDNAKNTVYLQMNSLR

AEDTAVYFCASRGIADGSDFGSYGQGTQVTVSSTTTPAPRPPTPAPTIASQPLS

LRPEACRPAAGGAVHTRGLDFACSQLCCQLKFWLPIGCAAFVVVCILGCILICW

LTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTLGGGSFRTPIQEEQADAHS

TLARVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQR

RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDAL

HMQALPPR

TABLE 9

Nucleic acid sequences of intracellular signaling

domains, extracellular domains of RTCRs and RTCRs.

CD2 truncated Signaling Domain (SEQ ID NO: 70)

CAGAATCCTGCCACCTCTCAGCACCCTCCACCTCCACCTGGACAC

AGATCTCAGGCCCCATCTCACAGACCTCCACCACCTGGTCATCGG

GTGCAGCATCAGCCCCAGAAAAGACCTCCTGCTCCTAGCGGAACA

CAGGTGCACCAGCAAAAGGGACCTCCACTGCCTAGACCTAGAGTG

CAGCCTAAGCCTCCTCATGGCGCTGCCGAGAATAGCCTGTCTCCT

AGCAGCAAC

IL2RB(YLRQ) Signaling Domain (SEQ ID NO: 71)

AATTGCAGAAACACAGGCCCCTGGCTGAAGAAAGTGCTGAAGTGC

AACACCCCTGATCCGAGCAAGTTCTTTAGCCAGCTGAGCAGCGAG

CATGGCGGCGACGTTCAGAAATGGCTGTCTAGCCCATTTCCTAGC

AGCAGCTTCAGCCCTGGTGGACTGGCCCCTGAGATTAGCCCTCTG

GAAGTGCTGGAACGGGACAAAGTGACCCAGCTGCTGCCCCTGAAT

ACCGACGCTTACCTGAGCCTGCAAGAGCTGCAAGGACAGGACCCT

ACACACCTGGTGTCCTACCTGAGACAGTGGGTCGTGATCCCTCCA

CCTCTCTCTAGTCCTGGACCTCAGGCCTCT

PD1 (PD1 Signal Peptide_PD1 Extracellular PD1

Transmembrane_PD1 Intracellular) PD-1

(Other name: PD1-wt or PD1 (SEQ ID NO: 72)

ATGCAGATTCCTCAAGCTCCTTGGCCTGTCGTGTGGGCCGTTCTG

CAACTTGGATGGCGGCCTGGCTGGTTCCTGGACTCTCCTGACAGA

CCCTGGAATCCTCCAACATTCAGCCCCGCTCTGCTGGTGGTTACC

GAGGGCGATAATGCCACCTTCACCTGTAGCTTCAGCAACACCAGC

GAGAGCTTCGTGCTGAACTGGTACAGAATGAGCCCCAGCAACCAG

ACCGACAAGCTGGCCGCCTTTCCTGAGGATAGATCTCAGCCCGGC

CAGGACTGCCGGTTCAGAGTTACACAGCTGCCCAACGGCCGGGAC

TTCCACATGTCTGTCGTCCGGGCCAGAAGAAACGACAGCGGCACA

TATCTGTGCGGCGCCATTTCTCTGGCCCCTAAGGCTCAGATCAAA

GAGAGCCTGAGAGCCGAGCTGAGAGTGACAGAAAGACGGGCCGAA

GTGCCCACAGCTCACCCTTCACCTTCTCCAAGACCTGCCGGCCAG

TTCCAGACACTGGTCGTGGGAGTTGTTGGCGGACTGCTGGGATCT

CTGGTGCTGCTTGTTTGGGTGCTCGCCGTGATCTGTAGCAGAGCC

GCCAGAGGAACAATCGGCGCCAGAAGGACAGGCCAGCCTCTGAAA

GAGGATCCCTCTGCTGTCCCCGTGTTCAGCGTGGACTATGGCGAG

CTGGATTTCCAGTGGCGGGAAAAGACACCCGAGCCTCCAGTGCCT

TGTGTGCCTGAGCAGACAGAGTACGCCACCATCGTGTTCCCTAGC

GGCATGGGCACATCTAGCCCTGCCAGAAGAGGATCTGCCGACGGA

CCTAGATCTGCCCAGCCTCTCAGACCTGAGGATGGCCACTGTTCT

TGGCCTCTT

PD1 Extracellular (Other name: PD1)

(SEQ ID NO: 73)

TTTCTGGACAGCCCCGACAGACCCTGGAATCCTCCTACATTCAGC

CCCGCTCTGCTGGTGGTTACCGAGGGCGATAATGCCACCTTCACC

TGTAGCTTCAGCAACACCAGCGAGAGCTTCGTGCTGAACTGGTAC

AGAATGAGCCCCAGCAACCAGACCGACAAGCTGGCCGCCTTTCCT

GAGGATAGATCTCAGCCCGGCCAGGACTGCCGGTTCAGAGTTACA

CAGCTGCCCAACGGCCGGGACTTCCACATGTCTGTCGTTCGGGCC

AGAAGAAACGACAGCGGCACATATCTGTGCGGCGCCATTTCTCTG

GCCCCTAAGGCTCAGATCAAAGAGAGCCTGAGAGCCGAGCTGAGA

GTGACAGAAAGACGGGCCGAAGTGCCCACAGCTCACCCTTCACCT

TCTCCAAGACCTGCT

PD-1 (extracellular domain without signal

peptide)-CD28 domain swap (DS)

(SEQ ID NO: 74)

TTTCTGGACAGCCCCGACAGACCCTGGAATCCTCCTACATTCAGC

CCCGCTCTGCTGGTGGTTACCGAGGGCGATAATGCCACCTTCACC

TGTAGCTTCAGCAACACCAGCGAGAGCTTCGTGCTGAACTGGTAC

AGAATGAGCCCCAGCAACCAGACCGACAAGCTGGCCGCCTTTCCT

GAGGATAGATCTCAGCCCGGCCAGGACTGCCGGTTCAGAGTTACA

CAGCTGCCCAACGGCCGGGACTTCCACATGTCTGTCGTTCGGGCC

AGAAGAAACGACAGCGGCACATATCTGTGCGGCGCCATTTCTCTG

GCCCCTAAGGCTCAGATCAAAGAGAGCCTGAGAGCCGAGCTGAGA

GTGACAGAAAGACGGGCCGAAGTGCCCACAGCTCACCCTTCACCT

TCTCCAAGACCTGCTCTGTTCCCCGGACCTAGCAAGCCCTTTTGG

GTGCTCGTTGTTGTTGGCGGCGTGCTGGCCTGTTATAGCCTGCTG

GTTACCGTGGCCTTCATCATCTTTTGGGTCCGAAGCAAGCGGAGC

CGGCTGCTGCACAGCGACTACATGAACATGACCCCTAGACGGCCC

GGACCAACCAGAAAGCACTACCAGCCTTACGCTCCTCCTAGAGAC

TTCGCCGCCTACAGATCT

PD-1 (extracellular domain without signal

peptide)-CD28 DS-CD137 domain

(SEQ ID NO: 75)

TTTCTGGACAGCCCCGACAGACCCTGGAATCCTCCTACATTCAGC

CCCGCTCTGCTGGTGGTTACCGAGGGCGATAATGCCACCTTCACC

TGTAGCTTCAGCAACACCAGCGAGAGCTTCGTGCTGAACTGGTAC

AGAATGAGCCCCAGCAACCAGACCGACAAGCTGGCCGCCTTTCCT

GAGGATAGATCTCAGCCCGGCCAGGACTGCCGGTTCAGAGTTACA

CAGCTGCCCAACGGCCGGGACTTCCACATGTCTGTCGTTCGGGCC

AGAAGAAACGACAGCGGCACATATCTGTGCGGCGCCATTTCTCTG

GCCCCTAAGGCTCAGATCAAAGAGAGCCTGAGAGCCGAGCTGAGA

GTGACAGAAAGACGGGCCGAAGTGCCCACAGCTCACCCTTCACCT

TCTCCAAGACCTGCTCTGTTCCCCGGACCTAGCAAGCCCTTTTGG

GTGCTCGTTGTTGTTGGCGGCGTGCTGGCCTGTTATAGCCTGCTG

GTTACCGTGGCCTTCATCATCTTTTGGGTCCGAAGCAAGCGGAGC

CGGCTGCTGCACAGCGACTACATGAACATGACCCCTAGACGGCCC

GGACCAACCAGAAAGCACTACCAGCCTTACGCTCCTCCTAGAGAC

TTCGCCGCCTACAGATCCAAGCGGGGCAGAAAGAAGCTGCTGTAC

ATCTTCAAGCAGCCCTTCATGCGGCCCGTGCAGACCACACAAGAG

GAAGATGGCTGCTCCTGTCGGTTCCCCGAGGAAGAAGAAGGCGGT

TGCGAACTG

PD-1 (extracellular domain without signal

peptide)-CD28 DS-truncated CD137 domain

(SEQ ID NO: 76)

TTTCTGGACAGCCCCGACAGACCCTGGAATCCTCCTACATTCAGC

CCCGCTCTGCTGGTGGTTACCGAGGGCGATAATGCCACCTTCACC

TGTAGCTTCAGCAACACCAGCGAGAGCTTCGTGCTGAACTGGTAC

AGAATGAGCCCCAGCAACCAGACCGACAAGCTGGCCGCCTTTCCT

GAGGATAGATCTCAGCCCGGCCAGGACTGCCGGTTCAGAGTTACA

CAGCTGCCCAACGGCCGGGACTTCCACATGTCTGTCGTTCGGGCC

AGAAGAAACGACAGCGGCACATATCTGTGCGGCGCCATTTCTCTG

GCCCCTAAGGCTCAGATCAAAGAGAGCCTGAGAGCCGAGCTGAGA

GTGACAGAAAGACGGGCCGAAGTGCCCACAGCTCACCCTTCACCT

TCTCCAAGACCTGCTCTGTTCCCCGGACCTAGCAAGCCCTTTTGG

GTGCTCGTTGTTGTTGGCGGCGTGCTGGCCTGTTATAGCCTGCTG

GTTACCGTGGCCTTCATCATCTTTTGGGTCCGAAGCAAGCGGAGC

CGGCTGCTGCACAGCGACTACATGAACATGACCCCTAGACGGCCC

GGACCAACCAGAAAGCACTACCAGCCTTACGCTCCTCCTAGAGAC

TTCGCCGCCTACAGATCCCAGCCTTTCATGAGGCCTGTGCAGACC

ACACAAGAAGAGGACGGCTGCTCCTGTCGGTTCCCCGAGGAAGAG

GAAGGCGGTTGCGAACTT

PD-1 (extracellular domain without signal

peptide)-CD28 DS-truncated CD134 domain

(SEQ ID NO: 77)

TTTCTGGACAGCCCCGACAGACCCTGGAATCCTCCTACATTCAGC

CCCGCTCTGCTGGTGGTTACCGAGGGCGATAATGCCACCTTCACC

TGTAGCTTCAGCAACACCAGCGAGAGCTTCGTGCTGAACTGGTAC

AGAATGAGCCCCAGCAACCAGACCGACAAGCTGGCCGCCTTTCCT

GAGGATAGATCTCAGCCCGGCCAGGACTGCCGGTTCAGAGTTACA

CAGCTGCCCAACGGCCGGGACTTCCACATGTCTGTCGTTCGGGCC

AGAAGAAACGACAGCGGCACATATCTGTGCGGCGCCATTTCTCTG

GCCCCTAAGGCTCAGATCAAAGAGAGCCTGAGAGCCGAGCTGAGA

GTGACAGAAAGACGGGCCGAAGTGCCCACAGCTCACCCTTCACCT

TCTCCAAGACCTGCTCTGTTCCCCGGACCTAGCAAGCCCTTTTGG

GTGCTCGTTGTTGTTGGCGGCGTGCTGGCCTGTTATAGCCTGCTG

GTTACCGTGGCCTTCATCATCTTTTGGGTCCGAAGCAAGCGGAGC

CGGCTGCTGCACAGCGACTACATGAACATGACCCCTAGACGGCCC

GGACCAACCAGAAAGCACTACCAGCCTTACGCTCCTCCTAGAGAC

TTCGCCGCCTACAGATCTGGCGGCGGAAGCTTCAGAACCCCTATC

CAAGAGGAACAGGCCGACGCTCACTCTACACTGGCT

PD-1 (extracellular domain without signal

peptide)-ICOS DS (SEQ ID NO: 78)

TTTCTGGACAGCCCCGACAGACCCTGGAATCCTCCTACATTCAGC

CCCGCTCTGCTGGTGGTTACCGAGGGCGATAATGCCACCTTCACC

TGTAGCTTCAGCAACACCAGCGAGAGCTTCGTGCTGAACTGGTAC

AGAATGAGCCCCAGCAACCAGACCGACAAGCTGGCCGCCTTTCCT

GAGGATAGATCTCAGCCCGGCCAGGACTGCCGGTTCAGAGTTACA

CAGCTGCCCAACGGCCGGGACTTCCACATGTCTGTCGTTCGGGCC

AGAAGAAACGACAGCGGCACATATCTGTGCGGCGCCATTTCTCTG

GCCCCTAAGGCTCAGATCAAAGAGAGCCTGAGAGCCGAGCTGAGA

GTGACAGAAAGACGGGCCGAAGTGCCCACAGCTCACCCTTCACCT

TCTCCAAGACCTGCCAGCCAGCTGTGCTGCCAGCTGAAGTTTTGG

CTGCCTATCGGCTGTGCCGCCTTCGTGGTTGTGTGTATCCTGGGC

TGCATCCTGATCTGCTGGCTGACCAAGAAAAAGTACAGCAGCTCC

GTGCACGACCCCAACGGCGAGTACATGTTCATGAGAGCCGTGAAC

ACCGCCAAGAAGTCCAGACTGACCGACGTGACACTT

PD-1 (extracellular domain without signal

peptide)-ICOS DS-truncated CD137

(SEQ ID NO: 79)

TTTCTGGACAGCCCCGACAGACCCTGGAATCCTCCTACATTCAGCC

CCGCTCTGCTGGTGGTTACCGAGGGCGATAATGCCACCTTCACCTG

TAGCTTCAGCAACACCAGCGAGAGCTTCGTGCTGAACTGGTACAG

AATGAGCCCCAGCAACCAGACCGACAAGCTGGCCGCCTTTCCTGA

GGATAGATCTCAGCCCGGCCAGGACTGCCGGTTCAGAGTTACACA

GCTGCCCAACGGCCGGGACTTCCACATGTCTGTCGTTCGGGCCAG

AAGAAACGACAGCGGCACATATCTGTGCGGCGCCATTTCTCTGGC

CCCTAAGGCTCAGATCAAAGAGAGCCTGAGAGCCGAGCTGAGAGT

GACAGAAAGACGGGCCGAAGTGCCCACAGCTCACCCTTCACCTTC

TCCAAGACCTGCCAGCCAGCTGTGCTGCCAGCTGAAGTTTTGGCT

GCCTATCGGCTGTGCCGCCTTCGTGGTTGTGTGTATCCTGGGCTG

CATCCTGATCTGCTGGCTGACCAAGAAAAAGTACAGCAGCTCCGT

GCACGACCCCAACGGCGAGTACATGTTCATGAGAGCCGTGAACAC

CGCCAAGAAGTCCAGACTGACCGACGTGACCCTGAAGCGGGGCAG

AAAGAAACTGCTGTACATCTTCAAGCAGCCCTTCATGCGGCCCGT

GCAGACCACACAAGAGGAAGATGGCTGCTCCTGCAGATTCCCCGA

GGAAGAAGAAGGCGGCTGCGAACTT

PD-1 (extracellular domain without signal

peptide)-ICOS DS-truncated CD137

(SEQ ID NO: 80)

TTTCTGGACAGCCCCGACAGACCCTGGAATCCTCCTACATTCAGC

CCCGCTCTGCTGGTGGTTACCGAGGGCGATAATGCCACCTTCACC

TGTAGCTTCAGCAACACCAGCGAGAGCTTCGTGCTGAACTGGTAC

AGAATGAGCCCCAGCAACCAGACCGACAAGCTGGCCGCCTTTCCT

GAGGATAGATCTCAGCCCGGCCAGGACTGCCGGTTCAGAGTTACA

CAGCTGCCCAACGGCCGGGACTTCCACATGTCTGTCGTTCGGGCC

AGAAGAAACGACAGCGGCACATATCTGTGCGGCGCCATTTCTCTG

GCCCCTAAGGCTCAGATCAAAGAGAGCCTGAGAGCCGAGCTGAGA

GTGACAGAAAGACGGGCCGAAGTGCCCACAGCTCACCCTTCACCT

TCTCCAAGACCTGCCAGCCAGCTGTGCTGCCAGCTGAAGTTTTGG

CTGCCTATCGGCTGTGCCGCCTTCGTGGTTGTGTGTATCCTGGGC

TGCATCCTGATCTGCTGGCTGACCAAGAAAAAGTACAGCAGCTCC

GTGCACGACCCCAACGGCGAGTACATGTTCATGAGAGCCGTGAAC

ACCGCCAAGAAGTCCAGACTGACCGACGTGACACTTCAGCCTTTC

ATGAGGCCCGTGCAGACCACACAAGAAGAGGACGGCTGCTCCTGC

AGATTCCCCGAGGAAGAGGAAGGCGGTTGCGAACTT

PD-1 (extracellular domain without signal

peptide)-ICOS DS-truncated CD134

(SEQ ID NO: 81)

TTTCTGGACAGCCCCGACAGACCCTGGAATCCTCCTACATTCAGC

CCCGCTCTGCTGGTGGTTACCGAGGGCGATAATGCCACCTTCACC

TGTAGCTTCAGCAACACCAGCGAGAGCTTCGTGCTGAACTGGTAC

AGAATGAGCCCCAGCAACCAGACCGACAAGCTGGCCGCCTTTCCT

GAGGATAGATCTCAGCCCGGCCAGGACTGCCGGTTCAGAGTTACA

CAGCTGCCCAACGGCCGGGACTTCCACATGTCTGTCGTTCGGGCC

AGAAGAAACGACAGCGGCACATATCTGTGCGGCGCCATTTCTCTG

GCCCCTAAGGCTCAGATCAAAGAGAGCCTGAGAGCCGAGCTGAGA

GTGACAGAAAGACGGGCCGAAGTGCCCACAGCTCACCCTTCACCT

TCTCCAAGACCTGCCAGCCAGCTGTGCTGCCAGCTGAAGTTTTGG

CTGCCTATCGGCTGTGCCGCCTTCGTGGTTGTGTGTATCCTGGGC

TGCATCCTGATCTGCTGGCTGACCAAGAAAAAGTACAGCAGCTCC

GTGCACGACCCCAACGGCGAGTACATGTTCATGAGAGCCGTGAAC

ACCGCCAAGAAGTCCAGACTGACCGACGTGACACTTGGCGGCGGA

AGCTTTAGAACCCCTATCCAAGAGGAACAGGCCGACGCTCACTCT

ACACTGGCT

PD1 (extracellular domain without signal

peptide)-ICOS DS-CD28(PRRP)-truncated CD137)

(SEQ ID NO: 82)

TTTCTGGACAGCCCCGACAGACCCTGGAATCCTCCTACATTCAGC

CCCGCTCTGCTGGTGGTTACCGAGGGCGATAATGCCACCTTCACC

TGTAGCTTCAGCAACACCAGCGAGAGCTTCGTGCTGAACTGGTAC

AGAATGAGCCCCAGCAACCAGACCGACAAGCTGGCCGCCTTTCCT

GAGGATAGATCTCAGCCCGGCCAGGACTGCCGGTTCAGAGTTACA

CAGCTGCCCAACGGCCGGGACTTCCACATGTCTGTCGTTCGGGCC

AGAAGAAACGACAGCGGCACATATCTGTGCGGCGCCATTTCTCTG

GCCCCTAAGGCTCAGATCAAAGAGAGCCTGAGAGCCGAGCTGAGA

GTGACAGAAAGACGGGCCGAAGTGCCCACAGCTCACCCTTCACCT

TCTCCAAGACCTGCCAGCCAGCTGTGCTGCCAGCTGAAGTTTTGG

CTGCCTATCGGCTGTGCCGCCTTCGTGGTTGTGTGTATCCTGGGC

TGCATCCTGATCTGCTGGCTGACCAAGAAAAAGTACAGCAGCTCC

GTGCACGACCCCAACGGCGAGTACATGTTCATGACCCCTAGAAGG

CCTGGACCTACCAGAAAGCACTACCAGCCTTACGCTCCTCCTAGA

GCCGTGAACACCGCCAAGAAGTCCAGACTGACCGACGTGACACTT

CAGCCTTTCATGAGGCCCGTGCAGACCACACAAGAAGAGGACGGC

TGCTCCTGCAGATTCCCCGAGGAAGAGGAAGGCGGTTGCGAACTT

PD1 (extracellular domain without signal

peptide)-ICOS DS-CD28(PRRP)-truncated CD134)

(SEQ ID NO: 83)

TTTCTGGACAGCCCCGACAGACCCTGGAATCCTCCTACATTCAGC

CCCGCTCTGCTGGTGGTTACCGAGGGCGATAATGCCACCTTCACC

TGTAGCTTCAGCAACACCAGCGAGAGCTTCGTGCTGAACTGGTAC

AGAATGAGCCCCAGCAACCAGACCGACAAGCTGGCCGCCTTTCCT

GAGGATAGATCTCAGCCCGGCCAGGACTGCCGGTTCAGAGTTACA

CAGCTGCCCAACGGCCGGGACTTCCACATGTCTGTCGTTCGGGCC

AGAAGAAACGACAGCGGCACATATCTGTGCGGCGCCATTTCTCTG

GCCCCTAAGGCTCAGATCAAAGAGAGCCTGAGAGCCGAGCTGAGA

GTGACAGAAAGACGGGCCGAAGTGCCCACAGCTCACCCTTCACCT

TCTCCAAGACCTGCCAGCCAGCTGTGCTGCCAGCTGAAGTTTTGG

CTGCCTATCGGCTGTGCCGCCTTCGTGGTTGTGTGTATCCTGGGC

TGCATCCTGATCTGCTGGCTGACCAAGAAAAAGTACAGCAGCTCC

GTGCACGACCCCAACGGCGAGTACATGTTCATGACCCCTAGAAGG

CCTGGACCTACCAGAAAGCACTACCAGCCTTACGCTCCTCCTAGA

GCCGTGAACACCGCCAAGAAGTCCAGACTGACCGACGTGACACTT

GGCGGCGGAAGCTTTAGAACCCCTATCCAAGAGGAACAGGCCGAC

GCTCACTCTACACTGGCT

PD1:ICOSBBt:CD2t (PD1 extracellular domain

without signal peptide)-ICOS DS-truncated

CD137 domain-truncated CD2 signaling domain)

(SEQ ID NO: 84)

TTTCTGGACAGCCCCGACAGACCCTGGAATCCTCCTACATTCAGC

CCCGCTCTGCTGGTGGTTACCGAGGGCGATAATGCCACCTTCACC

TGTAGCTTCAGCAACACCAGCGAGAGCTTCGTGCTGAACTGGTAC

AGAATGAGCCCCAGCAACCAGACCGACAAGCTGGCCGCCTTTCCT

GAGGATAGATCTCAGCCCGGCCAGGACTGCCGGTTCAGAGTTACA

CAGCTGCCCAACGGCCGGGACTTCCACATGTCTGTCGTTCGGGCC

AGAAGAAACGACAGCGGCACATATCTGTGCGGCGCCATTTCTCTG

GCCCCTAAGGCTCAGATCAAAGAGAGCCTGAGAGCCGAGCTGAGA

GTGACAGAAAGACGGGCCGAAGTGCCCACAGCTCACCCTTCACCT

TCTCCAAGACCTGCCAGCCAGCTGTGCTGCCAGCTGAAGTTTTGG

CTGCCTATCGGCTGTGCCGCCTTCGTGGTTGTGTGTATCCTGGGC

TGCATCCTGATCTGCTGGCTGACCAAGAAAAAGTACAGCAGCTCC

GTGCACGACCCCAACGGCGAGTACATGTTCATGAGAGCCGTGAAC

ACCGCCAAGAAGTCCAGACTGACCGACGTGACCCTCCAGCCTTTC

ATGAGGCCTGTGCAGACCACACAAGAAGAGGACGGCTGCTCCTGT

CGGTTCCCCGAGGAAGAGGAAGGCGGTTGCGAACTCCAGAATCCT

GCCACCTCTCAGCACCCTCCACCTCCACCTGGACACAGATCTCAG

GCCCCATCTCACAGACCTCCACCACCTGGTCATCGGGTGCAGCAT

CAGCCCCAGAAAAGACCTCCTGCTCCTAGCGGAACACAGGTGCAC

CAGCAAAAGGGACCTCCACTGCCTAGACCTAGAGTGCAGCCTAAG

CCTCCTCATGGCGCTGCCGAGAATAGCCTGTCTCCTAGCAGCAAC

PD1 extracellular domain without signal

peptide-ICOS DS-truncated CD134 domain-

truncated CD2 signaling domain

(SEQ ID NO: 85)

TTTCTGGACAGCCCCGACAGACCCTGGAATCCTCCTACATTCAGC

CCCGCTCTGCTGGTGGTTACCGAGGGCGATAATGCCACCTTCACC

TGTAGCTTCAGCAACACCAGCGAGAGCTTCGTGCTGAACTGGTAC

AGAATGAGCCCCAGCAACCAGACCGACAAGCTGGCCGCCTTTCCT

GAGGATAGATCTCAGCCCGGCCAGGACTGCCGGTTCAGAGTTACA

CAGCTGCCCAACGGCCGGGACTTCCACATGTCTGTCGTTCGGGCC

AGAAGAAACGACAGCGGCACATATCTGTGCGGCGCCATTTCTCTG

GCCCCTAAGGCTCAGATCAAAGAGAGCCTGAGAGCCGAGCTGAGA

GTGACAGAAAGACGGGCCGAAGTGCCCACAGCTCACCCTTCACCT

TCTCCAAGACCTGCCAGCCAGCTGTGCTGCCAGCTGAAGTTTTGG

CTGCCTATCGGCTGTGCCGCCTTCGTGGTTGTGTGTATCCTGGGC

TGCATCCTGATCTGCTGGCTGACCAAGAAAAAGTACAGCAGCTCC

GTGCACGACCCCAACGGCGAGTACATGTTCATGAGAGCCGTGAAC

ACCGCCAAGAAGTCCAGACTGACCGACGTGACCCTCCAGCCTTTC

ATGAGGCCTGTGCAGACCACACAAGAAGAGGACGGCTGCTCCTGT

CGGTTCCCCGAGGAAGAGGAAGGCGGCTGCGAACTGAATTGCAGA

AACACAGGCCCCTGGCTGAAGAAAGTGCTGAAGTGCAACACCCCT

GATCCGAGCAAGTTCTTTAGCCAGCTGAGCAGCGAGCATGGCGGC

GACGTTCAGAAATGGCTGTCTAGCCCATTTCCTAGCAGCAGCTTC

AGCCCTGGTGGACTGGCCCCTGAGATTAGCCCTCTGGAAGTGCTG

GAACGGGACAAAGTGACCCAGCTGCTGCCCCTGAATACCGACGCT

TACCTGAGCCTGCAAGAGCTGCAAGGACAGGACCCTACACACCTG

GTGTCCTACCTGAGACAGTGGGTCGTGATCCCTCCACCTCTCTCT

AGTCCTGGACCTCAGGCCTCT

PD1 extracellular domain without signal

peptide)-ICOS DS-truncated CD137 domain-

truncated CD2 signaling domain-IL-2 receptor

binding (IL2RB)(YLRQ) (SEQ ID NO: 86)

TTTCTGGACAGCCCCGACAGACCCTGGAATCCTCCTACATTCAGC

CCCGCTCTGCTGGTGGTTACCGAGGGCGATAATGCCACCTTCACC

TGTAGCTTCAGCAACACCAGCGAGAGCTTCGTGCTGAACTGGTAC

AGAATGAGCCCCAGCAACCAGACCGACAAGCTGGCCGCCTTTCCT

GAGGATAGATCTCAGCCCGGCCAGGACTGCCGGTTCAGAGTTACA

CAGCTGCCCAACGGCCGGGACTTCCACATGTCTGTCGTTCGGGCC

AGAAGAAACGACAGCGGCACATATCTGTGCGGCGCCATTTCTCTG

GCCCCTAAGGCTCAGATCAAAGAGAGCCTGAGAGCCGAGCTGAGA

GTGACAGAAAGACGGGCCGAAGTGCCCACAGCTCACCCTTCACCT

TCTCCAAGACCTGCCAGCCAGCTGTGCTGCCAGCTGAAGTTTTGG

CTGCCTATCGGCTGTGCCGCCTTCGTGGTTGTGTGTATCCTGGGC

TGCATCCTGATCTGCTGGCTGACCAAGAAAAAGTACAGCAGCTCC

GTGCACGACCCCAACGGCGAGTACATGTTCATGAGAGCCGTGAAC

ACCGCCAAGAAGTCCAGACTGACCGACGTGACCCTCCAGCCTTTC

ATGAGGCCTGTGCAGACCACACAAGAAGAGGACGGCTGCTCCTGT

CGGTTCCCCGAGGAAGAGGAAGGCGGTTGCGAACTCCAGAATCCT

GCCACCTCTCAGCACCCTCCACCTCCACCTGGACACAGATCTCAG

GCCCCATCTCACAGACCTCCACCACCTGGTCATCGGGTGCAGCAT

CAGCCCCAGAAAAGACCTCCTGCTCCTAGCGGAACACAGGTGCAC

CAGCAAAAGGGACCTCCACTGCCTAGACCTAGAGTGCAGCCTAAG

CCTCCTCATGGCGCTGCCGAGAATAGCCTGTCTCCTAGCAGCAAC

AACTGCCGCAACACAGGCCCCTGGCTGAAGAAAGTGCTGAAGTGC

AACACCCCTGATCCGAGCAAGTTCTTTAGCCAGCTGAGCAGCGAG

CATGGCGGCGACGTTCAGAAATGGCTGTCTAGCCCATTTCCAAGC

AGCAGCTTCAGCCCTGGTGGACTGGCCCCTGAGATTAGCCCTCTG

GAAGTGCTGGAACGGGACAAAGTGACCCAGCTGCTGCCCCTGAAT

ACCGACGCTTACCTGAGCCTGCAAGAGCTGCAAGGACAGGACCCT

ACACACCTGGTGTCCTACCTGAGACAGTGGGTCGTGATCCCACCT

CCTTTGAGCAGTCCAGGACCTCAGGCCTCT

CD3Z (intracellular Signaling Domain (Human

CD3ζ signaling domain) (SEQ ID NO: 87)

AGAGTGAAGTTCAGCAGATCCGCCGACGCTCCTGCCTATCAGCAG

GGCCAAAACCAGCTGTACAACGAGCTGAACCTGGGGAGAAGAGAA

GAGTACGACGTGCTGGACAAGCGGAGAGGCAGAGATCCTGAAATG

GGCGGCAAGCCCCAGCGGAGAAAGAATCCTCAAGAGGGCCTGTAT

AATGAGCTGCAAAAGGACAAGATGGCCGAGGCCTACAGCGAGATC

GGAATGAAGGGCGAGCGCAGAAGAGGCAAGGGACACGATGGACTG

TACCAGGGCCTGAGCACCGCCACCAAGGATACCTATGATGCCCTG

CACATGCAGGCCCTGCCTCCAAGA

Human CD3 Z signaling domain truncated

(CD3 ζ truncated domain)

(Other name: Human CD3ζ signaling domain

truncated; CD3 truncated domain)

(SEQ ID NO: 88)

AGAGTGAAGTTCAGCAGATCCGCCGACGCTCCTGCCTATCAGCAG

GGCCAAAACCAGCTGTACAACGAGCTGAACCTGGGGAGAAGAGAA

GAGTACGACGTGCTGGACAAGCGGAGAGGCAGAGATCCTGAAATG

GGCGGCAAA

Human CD3 E signaling domain truncated

(CD3 E truncated domain (Human

CD3 ϵ signaling domain truncated

(CD3 ϵ truncated domain) (SEQ ID NO: 89)

CCTGTGACTAGAGGTGCTGGTGCTGGCGGCAGACAGAGAGGCCAG

AACAAAGAAAGACCTCCTCCTGTGCCTAATCCTGACTACGAGCCC

ATCCGGAAGGGCCAGAGAGATCTGTACAGCGGCCTGAACCAGCGG

AGAATC

Human CD3 ZE signaling domain (CD3 ζϵ domain)

(Human CD3ζϵ signaling domain (CD3 domain)

(SEQ ID NO: 90)

AGAGTGAAGTTCAGCAGATCCGCCGACGCTCCTGCCTATCAGCAG

GGCCAAAACCAGCTGTACAACGAGCTGAACCTGGGGAGAAGAGAA

GAGTACGACGTGCTGGACAAGCGGAGAGGCAGAGATCCTGAAATG

GGCGGCAAGCCTGTGACTAGAGGTGCTGGTGCTGGCGGCAGACAG

AGAGGCCAGAACAAAGAAAGACCTCCTCCTGTGCCTAATCCTGAC

TACGAGCCCATCCGGAAGGGCCAGAGAGATCTGTACAGCGGCCTG

AACCAGCGGAGAATC

FMC63 scFV (CD8a Leader_Light_Chain_Linker_

Heavy_Chain_CD8a Hinge) (CD19 binding

extracellular domain) (SEQ ID NO: 91)

ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTT

CTGCATGCCGCCAGACCTGACATCCAGATGACCCAGACAACCAGC

AGCCTGTCTGCCAGCCTGGGCGATAGAGTGACCATCAGCTGTAGA

GCCAGCCAGGACATCAGCAAGTACCTGAACTGGTATCAGCAGAAA

CCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCAGACTG

CACAGCGGCGTGCCAAGCAGATTTTCTGGCAGCGGCTCTGGCACC

GACTACAGCCTGACCATCTCCAACCTGGAACAAGAGGATATCGCT

ACCTACTTCTGCCAGCAAGGCAACACCCTGCCTTACACCTTTGGC

GGAGGCACCAAGCTGGAAATCACAGGCGGCGGAGGAAGCGGAGGC

GGAGGATCTGGTGGTGGTGGATCTGAAGTGAAACTGCAAGAGTCT

GGCCCTGGCCTGGTGGCCCCATCTCAATCTCTGAGCGTGACCTGT

ACCGTCAGCGGAGTGTCCCTGCCTGATTATGGCGTGTCCTGGATC

CGGCAGCCTCCTAGAAAAGGCCTGGAATGGCTGGGCGTGATCTGG

GGCAGCGAGACAACCTACTACAACAGCGCCCTGAAGTCCCGGCTG

ACCATCATCAAGGACAACTCCAAGAGCCAGGTGTTCCTGAAGATG

AACAGCCTCCAGACCGACGACACCGCCATCTACTATTGCGCCAAG

CACTACTACTACGGCGGCAGCTACGCCATGGATTATTGGGGCCAG

GGCACCAGCGTGACCGTGTCTAGTACAACAACCCCTGCTCCTCGG

CCTCCTACACCAGCTCCTACAATTGCCAGCCAGCCACTGTCTCTG

AGGCCCGAAGCTTGTAGACCTGCTGCTGGCGGAGCCGTGCATACA

AGAGGACTGGATTTCGCCTGC

CD19 (FMC63 scFV)_CD8a Transmembrane_4-1BB

Signaling_CD3Z (Other names: FMC63scFV_BB_Z

chimeric antigen receptor (CAR), CD19_BB_Z)

(SEQ ID NO: 92)

ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTT

CTGCATGCCGCCAGACCTGACATCCAGATGACCCAGACAACCAGC

AGCCTGTCTGCCAGCCTGGGCGATAGAGTGACCATCAGCTGTAGA

GCCAGCCAGGACATCAGCAAGTACCTGAACTGGTATCAGCAGAAA

CCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCAGACTG

CACAGCGGCGTGCCAAGCAGATTTTCTGGCAGCGGCTCTGGCACC

GACTACAGCCTGACCATCTCCAACCTGGAACAAGAGGATATCGCT

ACCTACTTCTGCCAGCAAGGCAACACCCTGCCTTACACCTTTGGC

GGAGGCACCAAGCTGGAAATCACAGGCGGCGGAGGAAGCGGAGGC

GGAGGATCTGGTGGTGGTGGATCTGAAGTGAAACTGCAAGAGTCT

GGCCCTGGCCTGGTGGCCCCATCTCAATCTCTGAGCGTGACCTGT

ACCGTCAGCGGAGTGTCCCTGCCTGATTATGGCGTGTCCTGGATC

CGGCAGCCTCCTAGAAAAGGCCTGGAATGGCTGGGCGTGATCTGG

GGCAGCGAGACAACCTACTACAACAGCGCCCTGAAGTCCCGGCTG

ACCATCATCAAGGACAACTCCAAGAGCCAGGTGTTCCTGAAGATG

AACAGCCTCCAGACCGACGACACCGCCATCTACTATTGCGCCAAG

CACTACTACTACGGCGGCAGCTACGCCATGGATTATTGGGGCCAG

GGCACCAGCGTGACCGTGTCTAGTACAACAACCCCTGCTCCTCGG

CCTCCTACACCAGCTCCTACAATTGCCAGCCAGCCACTGTCTCTG

AGGCCCGAAGCTTGTAGACCTGCTGCTGGCGGAGCCGTGCATACA

AGAGGACTGGATTTCGCCTGCGACATCTACATCTGGGCCCCTCTG

GCTGGAACATGTGGCGTGCTGCTGCTGAGCCTGGTCATCACCCTG

TATTGCAAGCGGGGCAGAAAGAAACTGCTCTACATCTTCAAGCAG

CCCTTCATGCGGCCCGTGCAGACCACACAAGAGGAAGATGGCTGC

TCCTGTCGGTTCCCCGAGGAAGAAGAAGGCGGCTGCGAGCTGAGA

GTGAAGTTCAGCAGATCCGCCGACGCTCCTGCCTATCAGCAGGGC

CAAAACCAGCTGTACAACGAGCTGAACCTGGGGAGAAGAGAAGAG

TACGACGTGCTGGACAAGCGGAGAGGCAGAGATCCTGAAATGGGC

GGCAAGCCCCAGCGGAGAAAGAATCCTCAAGAGGGCCTGTATAAT

GAGCTGCAAAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGA

ATGAAGGGCGAGCGCAGAAGAGGCAAGGGACACGATGGACTGTAC

CAGGGCCTGAGCACCGCCACCAAGGATACCTATGATGCCCTGCAC

ATGCAGGCCCTGCCTCCAAGATGA

CD19 (FMC63 scFV) CD28 Transmembrane CD28

Signaling_CD3Z (Other names: FMC63scFV_28_Z CAR,

CD19_28_Z) (SEQ ID NO: 93)

ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTT

CTGCATGCCGCCAGACCTGACATCCAGATGACCCAGACAACCAGC

AGCCTGTCTGCCAGCCTGGGCGATAGAGTGACCATCAGCTGTAGA

GCCAGCCAGGACATCAGCAAGTACCTGAACTGGTATCAGCAGAAA

CCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCAGACTG

CACAGCGGCGTGCCAAGCAGATTTTCTGGCAGCGGCTCTGGCACC

GACTACAGCCTGACCATCTCCAACCTGGAACAAGAGGATATCGCT

ACCTACTTCTGCCAGCAAGGCAACACCCTGCCTTACACCTTTGGC

GGAGGCACCAAGCTGGAAATCACAGGCGGCGGAGGAAGCGGAGGC

GGAGGATCTGGTGGTGGTGGATCTGAAGTGAAACTGCAAGAGTCT

GGCCCTGGCCTGGTGGCCCCATCTCAATCTCTGAGCGTGACCTGT

ACCGTCAGCGGAGTGTCCCTGCCTGATTATGGCGTGTCCTGGATC

CGGCAGCCTCCTAGAAAAGGCCTGGAATGGCTGGGCGTGATCTGG

GGCAGCGAGACAACCTACTACAACAGCGCCCTGAAGTCCCGGCTG

ACCATCATCAAGGACAACTCCAAGAGCCAGGTGTTCCTGAAGATG

AACAGCCTCCAGACCGACGACACCGCCATCTACTATTGCGCCAAG

CACTACTACTACGGCGGCAGCTACGCCATGGATTATTGGGGCCAG

GGCACCAGCGTGACCGTGTCTAGTACAACAACCCCTGCTCCTCGG

CCTCCTACACCAGCTCCTACAATTGCCAGCCAGCCACTGTCTCTG

AGGCCCGAAGCTTGTAGACCTGCTGCTGGCGGAGCCGTGCATACA

AGAGGACTGGATTTCGCCTGCCTGTTTCCCGGACCTAGCAAGCCT

TTCTGGGTGCTCGTTGTTGTTGGCGGCGTGCTGGCCTGTTACAGC

CTGCTGGTTACCGTGGCCTTCATCATCTTTTGGGTCCGAAGCAAG

CGGAGCCGGCTGCTGCACAGCGACTACATGAACATGACCCCTAGA

CGGCCCGGACCAACCAGAAAGCACTACCAGCCTTACGCTCCTCCT

AGAGACTTCGCCGCCTACAGATCTAGAGTGAAGTTCAGCAGATCC

GCCGACGCTCCTGCCTATCAGCAGGGCCAAAACCAGCTGTACAAC

GAGCTGAACCTGGGGAGAAGAGAAGAGTACGACGTGCTGGACAAG

CGGAGAGGCAGAGATCCTGAAATGGGCGGCAAGCCCCAGCGGAGA

AAGAATCCTCAAGAGGGCCTGTATAATGAGCTGCAAAAGGACAAG

ATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGCAGA

AGAGGCAAGGGACACGATGGACTGTACCAGGGCCTGAGCACCGCC

ACCAAGGATACCTATGATGCCCTGCACATGCAGGCCCTGCCTCCA

AGATGA

CD19 (FMC63 scFV)_CD28 Transmembrane_CD28

Signaling_4-1BB Signaling_CD3Z

(Other names: FMC63scFV_28_BBwt_Z CAR,

CD19_28_BBwt_Z) (SEQ ID NO: 94)

ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTT

CTGCATGCCGCCAGACCTGACATCCAGATGACCCAGACAACCAGC

AGCCTGTCTGCCAGCCTGGGCGATAGAGTGACCATCAGCTGTAGA

GCCAGCCAGGACATCAGCAAGTACCTGAACTGGTATCAGCAGAAA

CCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCAGACTG

CACAGCGGCGTGCCAAGCAGATTTTCTGGCAGCGGCTCTGGCACC

GACTACAGCCTGACCATCTCCAACCTGGAACAAGAGGATATCGCT

ACCTACTTCTGCCAGCAAGGCAACACCCTGCCTTACACCTTTGGC

GGAGGCACCAAGCTGGAAATCACAGGCGGCGGAGGAAGCGGAGGC

GGAGGATCTGGTGGTGGTGGATCTGAAGTGAAACTGCAAGAGTCT

GGCCCTGGCCTGGTGGCCCCATCTCAATCTCTGAGCGTGACCTGT

ACCGTCAGCGGAGTGTCCCTGCCTGATTATGGCGTGTCCTGGATC

CGGCAGCCTCCTAGAAAAGGCCTGGAATGGCTGGGCGTGATCTGG

GGCAGCGAGACAACCTACTACAACAGCGCCCTGAAGTCCCGGCTG

ACCATCATCAAGGACAACTCCAAGAGCCAGGTGTTCCTGAAGATG

AACAGCCTCCAGACCGACGACACCGCCATCTACTATTGCGCCAAG

CACTACTACTACGGCGGCAGCTACGCCATGGATTATTGGGGCCAG

GGCACCAGCGTGACCGTGTCTAGTACAACAACCCCTGCTCCTCGG

CCTCCTACACCAGCTCCTACAATTGCCAGCCAGCCACTGTCTCTG

AGGCCCGAAGCTTGTAGACCTGCTGCTGGCGGAGCCGTGCATACA

AGAGGACTGGATTTCGCCTGCCTGTTTCCCGGACCTAGCAAGCCT

TTCTGGGTGCTCGTTGTTGTTGGCGGCGTGCTGGCCTGTTACAGC

CTGCTGGTTACCGTGGCCTTCATCATCTTTTGGGTCCGAAGCAAG

CGGAGCCGGCTGCTGCACAGCGACTACATGAACATGACCCCTAGA

CGGCCCGGACCAACCAGAAAGCACTACCAGCCTTACGCTCCTCCT

AGAGACTTCGCCGCCTACAGATCCAAGCGGGGCAGAAAGAAGCTG

CTGTACATCTTCAAGCAGCCCTTCATGCGGCCCGTGCAGACCACA

CAAGAGGAAGATGGCTGCTCCTGTCGGTTCCCCGAGGAAGAAGAA

GGCGGTTGCGAACTGAGAGTGAAGTTCAGCAGATCCGCCGACGCT

CCTGCCTATCAGCAGGGCCAAAACCAGCTGTACAACGAGCTGAAC

CTGGGGAGAAGAGAAGAGTACGACGTGCTGGACAAGCGGAGAGGC

AGAGATCCTGAAATGGGCGGCAAGCCCCAGCGGAGAAAGAATCCT

CAAGAGGGCCTGTATAATGAGCTGCAAAAGGACAAGATGGCCGAG

GCCTACAGCGAGATCGGAATGAAGGGCGAGCGCAGAAGAGGCAAG

GGACACGATGGACTGTACCAGGGCCTGAGCACCGCCACCAAGGAT

ACCTATGATGCCCTGCACATGCAGGCCCTGCCTCCAAGATGA

CD19 (FMC63 scFV) CD28 Transmembrane CD28

Signaling_4-1BB truncated Signaling_CD3Z

(Other names: FMC63scFV_28_BBt_Z CAR,

CD19_28_BBt_Z (SEQ ID NO: 95)

ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTT

CTGCATGCCGCCAGACCTGACATCCAGATGACCCAGACAACCAGC

AGCCTGTCTGCCAGCCTGGGCGATAGAGTGACCATCAGCTGTAGA

GCCAGCCAGGACATCAGCAAGTACCTGAACTGGTATCAGCAGAAA

CCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCAGACTG

CACAGCGGCGTGCCAAGCAGATTTTCTGGCAGCGGCTCTGGCACC

GACTACAGCCTGACCATCTCCAACCTGGAACAAGAGGATATCGCT

ACCTACTTCTGCCAGCAAGGCAACACCCTGCCTTACACCTTTGGC

GGAGGCACCAAGCTGGAAATCACAGGCGGCGGAGGAAGCGGAGGC

GGAGGATCTGGTGGTGGTGGATCTGAAGTGAAACTGCAAGAGTCT

GGCCCTGGCCTGGTGGCCCCATCTCAATCTCTGAGCGTGACCTGT

ACCGTCAGCGGAGTGTCCCTGCCTGATTATGGCGTGTCCTGGATC

CGGCAGCCTCCTAGAAAAGGCCTGGAATGGCTGGGCGTGATCTGG

GGCAGCGAGACAACCTACTACAACAGCGCCCTGAAGTCCCGGCTG

ACCATCATCAAGGACAACTCCAAGAGCCAGGTGTTCCTGAAGATG

AACAGCCTCCAGACCGACGACACCGCCATCTACTATTGCGCCAAG

CACTACTACTACGGCGGCAGCTACGCCATGGATTATTGGGGCCAG

GGCACCAGCGTGACCGTGTCTAGTACAACAACCCCTGCTCCTCGG

CCTCCTACACCAGCTCCTACAATTGCCAGCCAGCCACTGTCTCTG

AGGCCCGAAGCTTGTAGACCTGCTGCTGGCGGAGCCGTGCATACA

AGAGGACTGGATTTCGCCTGCCTGTTTCCCGGACCTAGCAAGCCT

TTCTGGGTGCTCGTTGTTGTTGGCGGCGTGCTGGCCTGTTACAGC

CTGCTGGTTACCGTGGCCTTCATCATCTTTTGGGTCCGAAGCAAG

CGGAGCCGGCTGCTGCACAGCGACTACATGAACATGACCCCTAGA

CGGCCCGGACCAACCAGAAAGCACTACCAGCCTTACGCTCCTCCT

AGAGACTTCGCCGCCTACAGATCCCAGCCTTTCATGAGGCCTGTG

CAGACCACACAAGAAGAGGACGGCTGCTCCTGTCGGTTCCCCGAG

GAAGAGGAAGGCGGTTGCGAACTTAGAGTGAAGTTCAGCAGATCC

GCCGACGCTCCTGCCTATCAGCAGGGCCAAAACCAGCTGTACAAC

GAGCTGAACCTGGGGAGAAGAGAAGAGTACGACGTGCTGGACAAG

CGGAGAGGCAGAGATCCTGAAATGGGCGGCAAGCCCCAGCGGAGA

AAGAATCCTCAAGAGGGCCTGTATAATGAGCTGCAAAAGGACAAG

ATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGCAGA

AGAGGCAAGGGACACGATGGACTGTACCAGGGCCTGAGCACCGCC

ACCAAGGATACCTATGATGCCCTGCACATGCAGGCCCTGCCTCCA

AGATGA

CD19 (FMC_63_scFV)_CD28_Transmembrane CD28

Signaling_OX-40 Truncated Signaling_CD3Z

(Other names: FMC63scFV_28_OX40t_Z CAR,

CD19_28_OX40t_Z) (SEQ ID NO: 96)

ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTT

CTGCATGCCGCCAGACCTGACATCCAGATGACCCAGACAACCAGC

AGCCTGTCTGCCAGCCTGGGCGATAGAGTGACCATCAGCTGTAGA

GCCAGCCAGGACATCAGCAAGTACCTGAACTGGTATCAGCAGAAA

CCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCAGACTG

CACAGCGGCGTGCCAAGCAGATTTTCTGGCAGCGGCTCTGGCACC

GACTACAGCCTGACCATCTCCAACCTGGAACAAGAGGATATCGCT

ACCTACTTCTGCCAGCAAGGCAACACCCTGCCTTACACCTTTGGC

GGAGGCACCAAGCTGGAAATCACAGGCGGCGGAGGAAGCGGAGGC

GGAGGATCTGGTGGTGGTGGATCTGAAGTGAAACTGCAAGAGTCT

GGCCCTGGCCTGGTGGCCCCATCTCAATCTCTGAGCGTGACCTGT

ACCGTCAGCGGAGTGTCCCTGCCTGATTATGGCGTGTCCTGGATC

CGGCAGCCTCCTAGAAAAGGCCTGGAATGGCTGGGCGTGATCTGG

GGCAGCGAGACAACCTACTACAACAGCGCCCTGAAGTCCCGGCTG

ACCATCATCAAGGACAACTCCAAGAGCCAGGTGTTCCTGAAGATG

AACAGCCTCCAGACCGACGACACCGCCATCTACTATTGCGCCAAG

CACTACTACTACGGCGGCAGCTACGCCATGGATTATTGGGGCCAG

GGCACCAGCGTGACCGTGTCTAGTACAACAACCCCTGCTCCTCGG

CCTCCTACACCAGCTCCTACAATTGCCAGCCAGCCACTGTCTCTG

AGGCCCGAAGCTTGTAGACCTGCTGCTGGCGGAGCCGTGCATACA

AGAGGACTGGATTTCGCCTGCCTGTTTCCCGGACCTAGCAAGCCT

TTCTGGGTGCTCGTTGTTGTTGGCGGCGTGCTGGCCTGTTACAGC

CTGCTGGTTACCGTGGCCTTCATCATCTTTTGGGTCCGAAGCAAG

CGGAGCCGGCTGCTGCACAGCGACTACATGAACATGACCCCTAGA

CGGCCCGGACCAACCAGAAAGCACTACCAGCCTTACGCTCCTCCT

AGAGACTTCGCCGCCTACAGATCTGGCGGCGGAAGCTTCAGAACC

CCTATCCAAGAGGAACAGGCCGACGCTCACTCTACACTGGCTAGA

GTGAAGTTCAGCAGATCCGCCGACGCTCCTGCCTATCAGCAGGGC

CAAAACCAGCTGTACAACGAGCTGAACCTGGGGAGAAGAGAAGAG

TACGACGTGCTGGACAAGCGGAGAGGCAGAGATCCTGAAATGGGC

GGCAAGCCCCAGCGGAGAAAGAATCCTCAAGAGGGCCTGTATAAT

GAGCTGCAAAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGA

ATGAAGGGCGAGCGCAGAAGAGGCAAGGGACACGATGGACTGTAC

CAGGGCCTGAGCACCGCCACCAAGGATACCTATGATGCCCTGCAC

ATGCAGGCCCTGCCTCCAAGATGA

CD19 (FMC63scFV)_ICOS Transmembrane_ICOS_

Signaling_CD3Z (Other names:

FMC63scFV_ICOS_Z CAR, CD19_ICOS_Z

(SEQ ID NO: 97)

ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTT

CTGCATGCCGCCAGACCTGACATCCAGATGACCCAGACAACCAGC

AGCCTGTCTGCCAGCCTGGGCGATAGAGTGACCATCAGCTGTAGA

GCCAGCCAGGACATCAGCAAGTACCTGAACTGGTATCAGCAGAAA

CCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCAGACTG

CACAGCGGCGTGCCAAGCAGATTTTCTGGCAGCGGCTCTGGCACC

GACTACAGCCTGACCATCTCCAACCTGGAACAAGAGGATATCGCT

ACCTACTTCTGCCAGCAAGGCAACACCCTGCCTTACACCTTTGGC

GGAGGCACCAAGCTGGAAATCACAGGCGGCGGAGGAAGCGGAGGC

GGAGGATCTGGTGGTGGTGGATCTGAAGTGAAACTGCAAGAGTCT

GGCCCTGGCCTGGTGGCCCCATCTCAATCTCTGAGCGTGACCTGT

ACCGTCAGCGGAGTGTCCCTGCCTGATTATGGCGTGTCCTGGATC

CGGCAGCCTCCTAGAAAAGGCCTGGAATGGCTGGGCGTGATCTGG

GGCAGCGAGACAACCTACTACAACAGCGCCCTGAAGTCCCGGCTG

ACCATCATCAAGGACAACTCCAAGAGCCAGGTGTTCCTGAAGATG

AACAGCCTCCAGACCGACGACACCGCCATCTACTATTGCGCCAAG

CACTACTACTACGGCGGCAGCTACGCCATGGATTATTGGGGCCAG

GGCACCAGCGTGACCGTGTCTAGTACAACAACCCCTGCTCCTCGG

CCTCCTACACCAGCTCCTACAATTGCCAGCCAGCCACTGTCTCTG

AGGCCCGAAGCTTGTAGACCTGCTGCTGGCGGAGCCGTGCATACA

AGAGGACTGGATTTCGCCTGCAGCCAGCTGTGCTGCCAGCTGAAG

TTCTGGCTGCCTATTGGCTGCGCCGCCTTCGTGGTTGTGTGTATC

CTGGGCTGCATCCTGATCTGCTGGCTGACCAAGAAAAAGTACAGC

AGCAGCGTGCACGACCCCAACGGCGAGTACATGTTCATGAGAGCC

GTGAACACCGCCAAGAAGTCCAGACTGACCGACGTGACACTGAGA

GTGAAGTTCAGCAGATCCGCCGACGCTCCTGCCTATCAGCAGGGC

CAAAACCAGCTGTACAACGAGCTGAACCTGGGGAGAAGAGAAGAG

TACGACGTGCTGGACAAGCGGAGAGGCAGAGATCCTGAAATGGGC

GGCAAGCCCCAGCGGAGAAAGAATCCTCAAGAGGGCCTGTATAAT

GAGCTGCAAAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGA

ATGAAGGGCGAGCGCAGAAGAGGCAAGGGACACGATGGACTGTAC

CAGGGCCTGAGCACCGCCACCAAGGATACCTATGATGCCCTGCAC

ATGCAGGCCCTGCCTCCAAGATGA

CD19 (FMC63scFV)_ICOS_Transmembrane_ICOS_

Signaling_4-1BB Signaling_CD3Z

(Other names: FMC63scFV_ICOS_BBwt_Z CAR,

CD19_ICOS_BBwt_Z) (SEQ ID NO: 98)

ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTT

CTGCATGCCGCCAGACCTGACATCCAGATGACCCAGACAACCAGC

AGCCTGTCTGCCAGCCTGGGCGATAGAGTGACCATCAGCTGTAGA

GCCAGCCAGGACATCAGCAAGTACCTGAACTGGTATCAGCAGAAA

CCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCAGACTG

CACAGCGGCGTGCCAAGCAGATTTTCTGGCAGCGGCTCTGGCACC

GACTACAGCCTGACCATCTCCAACCTGGAACAAGAGGATATCGCT

ACCTACTTCTGCCAGCAAGGCAACACCCTGCCTTACACCTTTGGC

GGAGGCACCAAGCTGGAAATCACAGGCGGCGGAGGAAGCGGAGGC

GGAGGATCTGGTGGTGGTGGATCTGAAGTGAAACTGCAAGAGTCT

GGCCCTGGCCTGGTGGCCCCATCTCAATCTCTGAGCGTGACCTGT

ACCGTCAGCGGAGTGTCCCTGCCTGATTATGGCGTGTCCTGGATC

CGGCAGCCTCCTAGAAAAGGCCTGGAATGGCTGGGCGTGATCTGG

GGCAGCGAGACAACCTACTACAACAGCGCCCTGAAGTCCCGGCTG

ACCATCATCAAGGACAACTCCAAGAGCCAGGTGTTCCTGAAGATG

AACAGCCTCCAGACCGACGACACCGCCATCTACTATTGCGCCAAG

CACTACTACTACGGCGGCAGCTACGCCATGGATTATTGGGGCCAG

GGCACCAGCGTGACCGTGTCTAGTACAACAACCCCTGCTCCTCGG

CCTCCTACACCAGCTCCTACAATTGCCAGCCAGCCACTGTCTCTG

AGGCCCGAAGCTTGTAGACCTGCTGCTGGCGGAGCCGTGCATACA

AGAGGACTGGATTTCGCCTGCAGCCAGCTGTGCTGCCAGCTGAAG

TTCTGGCTGCCTATTGGCTGCGCCGCCTTCGTGGTTGTGTGTATC

CTGGGCTGCATCCTGATCTGCTGGCTGACCAAGAAAAAGTACAGC

AGCAGCGTGCACGACCCCAACGGCGAGTACATGTTCATGAGAGCC

GTGAACACCGCCAAGAAGTCCAGACTGACCGACGTGACCCTGAAG

CGGGGCAGAAAGAAGCTGCTGTATATCTTCAAGCAGCCCTTCATG

CGGCCCGTGCAGACCACACAAGAGGAAGATGGCTGCTCCTGTCGG

TTCCCCGAGGAAGAAGAAGGCGGTTGCGAACTGAGAGTGAAGTTC

AGCAGATCCGCCGACGCTCCTGCCTATCAGCAGGGCCAAAACCAG

CTGTACAACGAGCTGAACCTGGGGAGAAGAGAAGAGTACGACGTG

CTGGACAAGCGGAGAGGCAGAGATCCTGAAATGGGCGGCAAGCCC

CAGCGGAGAAAGAATCCTCAAGAGGGCCTGTATAATGAGCTGCAA

AAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGC

GAGCGCAGAAGAGGCAAGGGACACGATGGACTGTACCAGGGCCTG

AGCACCGCCACCAAGGATACCTATGATGCCCTGCACATGCAGGCC

CTGCCTCCAAGATGA

CD19 (FMC_63_scFV)_ICOS_Transmembrane_ICOS_

Signaling_4-1BB Truncated Signaling_CD3Z

(Other names: FMC63scFV_ICOS_BBt_Z

CAR, CD19_ICOS_BBt_Z (SEQ ID NO: 99)

ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTT

CTGCATGCCGCCAGACCTGACATCCAGATGACCCAGACAACCAGC

AGCCTGTCTGCCAGCCTGGGCGATAGAGTGACCATCAGCTGTAGA

GCCAGCCAGGACATCAGCAAGTACCTGAACTGGTATCAGCAGAAA

CCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCAGACTG

CACAGCGGCGTGCCAAGCAGATTTTCTGGCAGCGGCTCTGGCACC

GACTACAGCCTGACCATCTCCAACCTGGAACAAGAGGATATCGCT

ACCTACTTCTGCCAGCAAGGCAACACCCTGCCTTACACCTTTGGC

GGAGGCACCAAGCTGGAAATCACAGGCGGCGGAGGAAGCGGAGGC

GGAGGATCTGGTGGTGGTGGATCTGAAGTGAAACTGCAAGAGTCT

GGCCCTGGCCTGGTGGCCCCATCTCAATCTCTGAGCGTGACCTGT

ACCGTCAGCGGAGTGTCCCTGCCTGATTATGGCGTGTCCTGGATC

CGGCAGCCTCCTAGAAAAGGCCTGGAATGGCTGGGCGTGATCTGG

GGCAGCGAGACAACCTACTACAACAGCGCCCTGAAGTCCCGGCTG

ACCATCATCAAGGACAACTCCAAGAGCCAGGTGTTCCTGAAGATG

AACAGCCTCCAGACCGACGACACCGCCATCTACTATTGCGCCAAG

CACTACTACTACGGCGGCAGCTACGCCATGGATTATTGGGGCCAG

GGCACCAGCGTGACCGTGTCTAGTACAACAACCCCTGCTCCTCGG

CCTCCTACACCAGCTCCTACAATTGCCAGCCAGCCACTGTCTCTG

AGGCCCGAAGCTTGTAGACCTGCTGCTGGCGGAGCCGTGCATACA

AGAGGACTGGATTTCGCCTGCAGCCAGCTGTGCTGCCAGCTGAAG

TTCTGGCTGCCTATTGGCTGCGCCGCCTTCGTGGTTGTGTGTATC

CTGGGCTGCATCCTGATCTGCTGGCTGACCAAGAAAAAGTACAGC

AGCAGCGTGCACGACCCCAACGGCGAGTACATGTTCATGAGAGCC

GTGAACACCGCCAAGAAGTCCAGACTGACCGACGTGACCCTCCAG

CCTTTCATGAGGCCTGTGCAGACCACACAAGAAGAGGACGGCTGC

TCCTGTCGGTTCCCCGAGGAAGAGGAAGGCGGTTGCGAACTTAGA

GTGAAGTTCAGCAGATCCGCCGACGCTCCTGCCTATCAGCAGGGC

CAAAACCAGCTGTACAACGAGCTGAACCTGGGGAGAAGAGAAGAG

TACGACGTGCTGGACAAGCGGAGAGGCAGAGATCCTGAAATGGGC

GGCAAGCCCCAGCGGAGAAAGAATCCTCAAGAGGGCCTGTATAAT

GAGCTGCAAAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGA

ATGAAGGGCGAGCGCAGAAGAGGCAAGGGACACGATGGACTGTAC

CAGGGCCTGAGCACCGCCACCAAGGATACCTATGATGCCCTGCAC

ATGCAGGCCCTGCCTCCAAGATGA

CD19 (FMC63 scFV)_ICOS Transmembrane_ICOS_

Signaling_OX-40 Truncated

Signaling_CD3Z (Other names: FMC63scFV_

ICOS_OX40t_Z CAR, CD19_ICOS_OX40t_Z

(SEQ ID NO: 100)

ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTT

CTGCATGCCGCCAGACCTGACATCCAGATGACCCAGACAACCAGC

AGCCTGTCTGCCAGCCTGGGCGATAGAGTGACCATCAGCTGTAGA

GCCAGCCAGGACATCAGCAAGTACCTGAACTGGTATCAGCAGAAA

CCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCAGACTG

CACAGCGGCGTGCCAAGCAGATTTTCTGGCAGCGGCTCTGGCACC

GACTACAGCCTGACCATCTCCAACCTGGAACAAGAGGATATCGCT

ACCTACTTCTGCCAGCAAGGCAACACCCTGCCTTACACCTTTGGC

GGAGGCACCAAGCTGGAAATCACAGGCGGCGGAGGAAGCGGAGGC

GGAGGATCTGGTGGTGGTGGATCTGAAGTGAAACTGCAAGAGTCT

GGCCCTGGCCTGGTGGCCCCATCTCAATCTCTGAGCGTGACCTGT

ACCGTCAGCGGAGTGTCCCTGCCTGATTATGGCGTGTCCTGGATC

CGGCAGCCTCCTAGAAAAGGCCTGGAATGGCTGGGCGTGATCTGG

GGCAGCGAGACAACCTACTACAACAGCGCCCTGAAGTCCCGGCTG

ACCATCATCAAGGACAACTCCAAGAGCCAGGTGTTCCTGAAGATG

AACAGCCTCCAGACCGACGACACCGCCATCTACTATTGCGCCAAG

CACTACTACTACGGCGGCAGCTACGCCATGGATTATTGGGGCCAG

GGCACCAGCGTGACCGTGTCTAGTACAACAACCCCTGCTCCTCGG

CCTCCTACACCAGCTCCTACAATTGCCAGCCAGCCACTGTCTCTG

AGGCCCGAAGCTTGTAGACCTGCTGCTGGCGGAGCCGTGCATACA

AGAGGACTGGATTTCGCCTGCAGCCAGCTGTGCTGCCAGCTGAAG

TTCTGGCTGCCTATTGGCTGCGCCGCCTTCGTGGTTGTGTGTATC

CTGGGCTGCATCCTGATCTGCTGGCTGACCAAGAAAAAGTACAGC

AGCAGCGTGCACGACCCCAACGGCGAGTACATGTTCATGAGAGCC

GTGAACACCGCCAAGAAGTCCAGACTGACCGACGTGACACTCGGC

GGAGGCAGCTTTAGAACCCCTATCCAAGAGGAACAGGCCGACGCT

CACTCTACACTGGCTAGAGTGAAGTTCAGCAGATCCGCCGACGCT

CCTGCCTATCAGCAGGGCCAAAACCAGCTGTACAACGAGCTGAAC

CTGGGGAGAAGAGAAGAGTACGACGTGCTGGACAAGCGGAGAGGC

AGAGATCCTGAAATGGGCGGCAAGCCCCAGCGGAGAAAGAATCCT

CAAGAGGGCCTGTATAATGAGCTGCAAAAGGACAAGATGGCCGAG

GCCTACAGCGAGATCGGAATGAAGGGCGAGCGCAGAAGAGGCAAG

GGACACGATGGACTGTACCAGGGCCTGAGCACCGCCACCAAGGAT

ACCTATGATGCCCTGCACATGCAGGCCCTGCCTCCAAGATGA

CD19 (FMC63scFV)_ICOS_Transmembrane_ICOS

Signaling_(mini CD28 Signaling)_CD3Z

(Other names: FMC63scFV_ICOS(28)_Z CAR,

CD19_ICOS(28)_Z (SEQ ID NO: 101)

ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTT

CTGCATGCCGCCAGACCTGACATCCAGATGACCCAGACAACCAGC

AGCCTGTCTGCCAGCCTGGGCGATAGAGTGACCATCAGCTGTAGA

GCCAGCCAGGACATCAGCAAGTACCTGAACTGGTATCAGCAGAAA

CCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCAGACTG

CACAGCGGCGTGCCAAGCAGATTTTCTGGCAGCGGCTCTGGCACC

GACTACAGCCTGACCATCTCCAACCTGGAACAAGAGGATATCGCT

ACCTACTTCTGCCAGCAAGGCAACACCCTGCCTTACACCTTTGGC

GGAGGCACCAAGCTGGAAATCACAGGCGGCGGAGGAAGCGGAGGC

GGAGGATCTGGTGGTGGTGGATCTGAAGTGAAACTGCAAGAGTCT

GGCCCTGGCCTGGTGGCCCCATCTCAATCTCTGAGCGTGACCTGT

ACCGTCAGCGGAGTGTCCCTGCCTGATTATGGCGTGTCCTGGATC

CGGCAGCCTCCTAGAAAAGGCCTGGAATGGCTGGGCGTGATCTGG

GGCAGCGAGACAACCTACTACAACAGCGCCCTGAAGTCCCGGCTG

ACCATCATCAAGGACAACTCCAAGAGCCAGGTGTTCCTGAAGATG

AACAGCCTCCAGACCGACGACACCGCCATCTACTATTGCGCCAAG

CACTACTACTACGGCGGCAGCTACGCCATGGATTATTGGGGCCAG

GGCACCAGCGTGACCGTGTCTAGTACAACAACCCCTGCTCCTCGG

CCTCCTACACCAGCTCCTACAATTGCCAGCCAGCCACTGTCTCTG

AGGCCCGAAGCTTGTAGACCTGCTGCTGGCGGAGCCGTGCATACA

AGAGGACTGGATTTCGCCTGCAGCCAGCTGTGCTGCCAGCTGAAG

TTCTGGCTGCCTATTGGCTGCGCCGCCTTCGTGGTTGTGTGTATC

CTGGGCTGCATCCTGATCTGCTGGCTGACCAAGAAAAAGTACAGC

AGCAGCGTGCACGACCCCAACGGCGAGTACATGTTCATGACCCCT

AGACGGCCCGGACCTACCAGAAAGCACTACCAGCCTTACGCTCCT

CCTCGGGCCGTGAACACAGCCAAGAAAAGCAGACTGACCGACGTG

ACCCTGAGAGTGAAGTTCAGCAGATCCGCCGACGCTCCTGCCTAT

CAGCAGGGCCAAAACCAGCTGTACAACGAGCTGAACCTGGGGAGA

AGAGAAGAGTACGACGTGCTGGACAAGCGGAGAGGCAGAGATCCT

GAAATGGGCGGCAAGCCCCAGCGGAGAAAGAATCCTCAAGAGGGC

CTGTATAATGAGCTGCAAAAGGACAAGATGGCCGAGGCCTACAGC

GAGATCGGAATGAAGGGCGAGCGCAGAAGAGGCAAGGGACACGAT

GGACTGTACCAGGGCCTGAGCACCGCCACCAAGGATACCTATGAT

GCCCTGCACATGCAGGCCCTGCCTCCAAGATGA

CD19 (FMC63scFV)_ICOS_Transmembrane_ICOS_

Signaling (mini CD28 Signaling)_4-1BB

Truncated Signaling_CD3Z (Other names:

FMC63scFV_ICOS(28)_BBt_Z CAR,

CD19_ICOS(28)_BBt_Z) (SEQ ID NO: 102)

ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTT

CTGCATGCCGCCAGACCTGACATCCAGATGACCCAGACAACCAGC

AGCCTGTCTGCCAGCCTGGGCGATAGAGTGACCATCAGCTGTAGA

GCCAGCCAGGACATCAGCAAGTACCTGAACTGGTATCAGCAGAAA

CCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCAGACTG

CACAGCGGCGTGCCAAGCAGATTTTCTGGCAGCGGCTCTGGCACC

GACTACAGCCTGACCATCTCCAACCTGGAACAAGAGGATATCGCT

ACCTACTTCTGCCAGCAAGGCAACACCCTGCCTTACACCTTTGGC

GGAGGCACCAAGCTGGAAATCACAGGCGGCGGAGGAAGCGGAGGC

GGAGGATCTGGTGGTGGTGGATCTGAAGTGAAACTGCAAGAGTCT

GGCCCTGGCCTGGTGGCCCCATCTCAATCTCTGAGCGTGACCTGT

ACCGTCAGCGGAGTGTCCCTGCCTGATTATGGCGTGTCCTGGATC

CGGCAGCCTCCTAGAAAAGGCCTGGAATGGCTGGGCGTGATCTGG

GGCAGCGAGACAACCTACTACAACAGCGCCCTGAAGTCCCGGCTG

ACCATCATCAAGGACAACTCCAAGAGCCAGGTGTTCCTGAAGATG

AACAGCCTCCAGACCGACGACACCGCCATCTACTATTGCGCCAAG

CACTACTACTACGGCGGCAGCTACGCCATGGATTATTGGGGCCAG

GGCACCAGCGTGACCGTGTCTAGTACAACAACCCCTGCTCCTCGG

CCTCCTACACCAGCTCCTACAATTGCCAGCCAGCCACTGTCTCTG

AGGCCCGAAGCTTGTAGACCTGCTGCTGGCGGAGCCGTGCATACA

AGAGGACTGGATTTCGCCTGCAGCCAGCTGTGCTGCCAGCTGAAG

TTCTGGCTGCCTATTGGCTGCGCCGCCTTCGTGGTTGTGTGTATC

CTGGGCTGCATCCTGATCTGCTGGCTGACCAAGAAAAAGTACAGC

AGCAGCGTGCACGACCCCAACGGCGAGTACATGTTCATGACCCCT

AGACGGCCCGGACCTACCAGAAAGCACTACCAGCCTTACGCTCCT

CCTCGGGCCGTGAACACAGCCAAGAAAAGCAGACTGACCGACGTG

ACCCTCCAGCCTTTCATGAGGCCTGTGCAGACCACACAAGAAGAG

GACGGCTGCTCCTGTCGGTTCCCCGAGGAAGAGGAAGGCGGTTGC

GAACTTAGAGTGAAGTTCAGCAGATCCGCCGACGCTCCTGCCTAT

CAGCAGGGCCAAAACCAGCTGTACAACGAGCTGAACCTGGGGAGA

AGAGAAGAGTACGACGTGCTGGACAAGCGGAGAGGCAGAGATCCT

GAAATGGGCGGCAAGCCCCAGCGGAGAAAGAATCCTCAAGAGGGC

CTGTATAATGAGCTGCAAAAGGACAAGATGGCCGAGGCCTACAGC

GAGATCGGAATGAAGGGCGAGCGCAGAAGAGGCAAGGGACACGAT

GGACTGTACCAGGGCCTGAGCACCGCCACCAAGGATACCTATGAT

GCCCTGCACATGCAGGCCCTGCCTCCAAGATGA

CD19 (FMC63 scFV)_ICOS_Transmembrane_ICOS

Signaling_(mini CD28 Signaling)_OX-40

Truncated Signaling_CD3Z (Other names:

FMC63scFV_ICOS(28)_40t_Z CAR,

CD19_ICOS(28)_40t_Z) (SEQ ID NO: 103)

ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTT

CTGCATGCCGCCAGACCTGACATCCAGATGACCCAGACAACCAGC

AGCCTGTCTGCCAGCCTGGGCGATAGAGTGACCATCAGCTGTAGA

GCCAGCCAGGACATCAGCAAGTACCTGAACTGGTATCAGCAGAAA

CCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCAGACTG

CACAGCGGCGTGCCAAGCAGATTTTCTGGCAGCGGCTCTGGCACC

GACTACAGCCTGACCATCTCCAACCTGGAACAAGAGGATATCGCT

ACCTACTTCTGCCAGCAAGGCAACACCCTGCCTTACACCTTTGGC

GGAGGCACCAAGCTGGAAATCACAGGCGGCGGAGGAAGCGGAGGC

GGAGGATCTGGTGGTGGTGGATCTGAAGTGAAACTGCAAGAGTCT

GGCCCTGGCCTGGTGGCCCCATCTCAATCTCTGAGCGTGACCTGT

ACCGTCAGCGGAGTGTCCCTGCCTGATTATGGCGTGTCCTGGATC

CGGCAGCCTCCTAGAAAAGGCCTGGAATGGCTGGGCGTGATCTGG

GGCAGCGAGACAACCTACTACAACAGCGCCCTGAAGTCCCGGCTG

ACCATCATCAAGGACAACTCCAAGAGCCAGGTGTTCCTGAAGATG

AACAGCCTCCAGACCGACGACACCGCCATCTACTATTGCGCCAAG

CACTACTACTACGGCGGCAGCTACGCCATGGATTATTGGGGCCAG

GGCACCAGCGTGACCGTGTCTAGTACAACAACCCCTGCTCCTCGG

CCTCCTACACCAGCTCCTACAATTGCCAGCCAGCCACTGTCTCTG

AGGCCCGAAGCTTGTAGACCTGCTGCTGGCGGAGCCGTGCATACA

AGAGGACTGGATTTCGCCTGCAGCCAGCTGTGCTGCCAGCTGAAG

TTCTGGCTGCCTATTGGCTGCGCCGCCTTCGTGGTTGTGTGTATC

CTGGGCTGCATCCTGATCTGCTGGCTGACCAAGAAAAAGTACAGC

AGCAGCGTGCACGACCCCAACGGCGAGTACATGTTCATGACCCCT

AGACGGCCCGGACCTACCAGAAAGCACTACCAGCCTTACGCTCCT

CCTCGGGCCGTGAACACAGCCAAGAAAAGCAGACTGACCGACGTG

ACACTCGGCGGAGGCAGCTTTAGAACCCCTATCCAAGAGGAACAG

GCCGACGCTCACTCTACACTGGCTAGAGTGAAGTTCAGCAGATCC

GCCGACGCTCCTGCCTATCAGCAGGGCCAAAACCAGCTGTACAAC

GAGCTGAACCTGGGGAGAAGAGAAGAGTACGACGTGCTGGACAAG

CGGAGAGGCAGAGATCCTGAAATGGGCGGCAAGCCCCAGCGGAGA

AAGAATCCTCAAGAGGGCCTGTATAATGAGCTGCAAAAGGACAAG

ATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGCAGA

AGAGGCAAGGGACACGATGGACTGTACCAGGGCCTGAGCACCGCC

ACCAAGGATACCTATGATGCCCTGCACATGCAGGCCCTGCCTCCA

AGATGA

CD19_ICOSBBt_Zt CAR (CD19 binding

extracellular domain-ICOS DS-truncated CD137

intracellular domain-CD3Ztruncated domain)

(Other names: FMC63scFV_ICOS_BBt_Zt

CAR, CD19_ICOS_BBt_Zt (SEQ ID NO: 104)

ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTT

CTGCATGCCGCCAGACCTGACATCCAGATGACCCAGACAACCAGC

AGCCTGTCTGCCAGCCTGGGCGATAGAGTGACCATCAGCTGTAGA

GCCAGCCAGGACATCAGCAAGTACCTGAACTGGTATCAGCAGAAA

CCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCAGACTG

CACAGCGGCGTGCCAAGCAGATTTTCTGGCAGCGGCTCTGGCACC

GACTACAGCCTGACCATCTCCAACCTGGAACAAGAGGATATCGCT

ACCTACTTCTGCCAGCAAGGCAACACCCTGCCTTACACCTTTGGC

GGAGGCACCAAGCTGGAAATCACAGGCGGCGGAGGAAGCGGAGGC

GGAGGATCTGGTGGTGGTGGATCTGAAGTGAAACTGCAAGAGTCT

GGCCCTGGCCTGGTGGCCCCATCTCAATCTCTGAGCGTGACCTGT

ACCGTCAGCGGAGTGTCCCTGCCTGATTATGGCGTGTCCTGGATC

CGGCAGCCTCCTAGAAAAGGCCTGGAATGGCTGGGCGTGATCTGG

GGCAGCGAGACAACCTACTACAACAGCGCCCTGAAGTCCCGGCTG

ACCATCATCAAGGACAACTCCAAGAGCCAGGTGTTCCTGAAGATG

AACAGCCTCCAGACCGACGACACCGCCATCTACTATTGCGCCAAG

CACTACTACTACGGCGGCAGCTACGCCATGGATTATTGGGGCCAG

GGCACCAGCGTGACCGTGTCTAGTACAACAACCCCTGCTCCTCGG

CCTCCTACACCAGCTCCTACAATTGCCAGCCAGCCACTGTCTCTG

AGGCCCGAAGCTTGTAGACCTGCTGCTGGCGGAGCCGTGCATACA

AGAGGACTGGATTTCGCCTGCAGCCAGCTGTGCTGTCAGCTGAAG

TTCTGGCTGCCTATCGGCTGCGCCGCCTTTGTGGTTGTGTGTATC

CTGGGCTGCATCCTGATCTGCTGGCTGACCAAGAAAAAGTACAGC

AGCAGCGTGCACGACCCCAACGGCGAGTACATGTTCATGAGAGCC

GTGAACACCGCCAAGAAGTCCAGACTGACCGACGTGACCCTCCAG

CCTTTCATGAGGCCTGTGCAGACCACACAAGAAGAGGACGGCTGC

TCCTGTCGGTTCCCCGAGGAAGAGGAAGGCGGTTGCGAGCTGAGA

GTGAAGTTCAGCAGATCCGCCGACGCTCCTGCCTATCAGCAGGGC

CAAAACCAGCTGTACAACGAGCTGAACCTGGGGAGAAGAGAAGAG

TACGACGTGCTGGACAAGCGGAGAGGCAGAGATCCTGAAATGGGC

GGCAAATGA

CD19_ICOSBBt_ZE CAR (CD19 binding extracellular

domain-ICOS DS-truncated CD137

intracellular domain-CD3ZE domain)

(Other names: FMC63scFV_ICOS_BBt_ZE CAR,

CD19_ICOS_BBt_ZE (SEQ ID NO: 105)

ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTT

CTGCATGCCGCCAGACCTGACATCCAGATGACCCAGACAACCAGC

AGCCTGTCTGCCAGCCTGGGCGATAGAGTGACCATCAGCTGTAGA

GCCAGCCAGGACATCAGCAAGTACCTGAACTGGTATCAGCAGAAA

CCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCAGACTG

CACAGCGGCGTGCCAAGCAGATTTTCTGGCAGCGGCTCTGGCACC

GACTACAGCCTGACCATCTCCAACCTGGAACAAGAGGATATCGCT

ACCTACTTCTGCCAGCAAGGCAACACCCTGCCTTACACCTTTGGC

GGAGGCACCAAGCTGGAAATCACAGGCGGCGGAGGAAGCGGAGGC

GGAGGATCTGGTGGTGGTGGATCTGAAGTGAAACTGCAAGAGTCT

GGCCCTGGCCTGGTGGCCCCATCTCAATCTCTGAGCGTGACCTGT

ACCGTCAGCGGAGTGTCCCTGCCTGATTATGGCGTGTCCTGGATC

CGGCAGCCTCCTAGAAAAGGCCTGGAATGGCTGGGCGTGATCTGG

GGCAGCGAGACAACCTACTACAACAGCGCCCTGAAGTCCCGGCTG

ACCATCATCAAGGACAACTCCAAGAGCCAGGTGTTCCTGAAGATG

AACAGCCTCCAGACCGACGACACCGCCATCTACTATTGCGCCAAG

CACTACTACTACGGCGGCAGCTACGCCATGGATTATTGGGGCCAG

GGCACCAGCGTGACCGTGTCTAGTACAACAACCCCTGCTCCTCGG

CCTCCTACACCAGCTCCTACAATTGCCAGCCAGCCACTGTCTCTG

AGGCCCGAAGCTTGTAGACCTGCTGCTGGCGGAGCCGTGCATACA

AGAGGACTGGATTTCGCCTGCAGCCAGCTGTGCTGTCAGCTGAAG

TTCTGGCTGCCTATCGGCTGCGCCGCCTTTGTGGTTGTGTGTATC

CTGGGCTGCATCCTGATCTGCTGGCTGACCAAGAAAAAGTACAGC

AGCAGCGTGCACGACCCCAACGGCGAGTACATGTTCATGAGAGCC

GTGAACACCGCCAAGAAGTCCAGACTGACCGACGTGACCCTCCAG

CCTTTCATGAGGCCTGTGCAGACCACACAAGAAGAGGACGGCTGC

TCCTGTCGGTTCCCCGAGGAAGAGGAAGGCGGTTGCGAGCTGAGA

GTGAAGTTCAGCAGATCCGCCGACGCTCCTGCCTATCAGCAGGGC

CAAAACCAGCTGTACAACGAGCTGAACCTGGGGAGAAGAGAAGAG

TACGACGTGCTGGACAAGCGGAGAGGCAGAGATCCTGAAATGGGC

GGCAAGCCTGTGACTAGAGGTGCTGGTGCTGGCGGCAGACAGAGA

GGCCAGAACAAAGAAAGACCTCCTCCTGTGCCTAATCCTGACTAC

GAGCCCATCCGGAAGGGCCAGAGAGATCTGTACAGCGGCCTGAAC

CAGCGGAGAATCTGA

CD19_ICOSBBt:CD2t_Z CAR (CD19 binding

extracellular domain-ICOS DS-truncated

CD137 intracellular domain-CD2 truncated

Signaling Domain-CD3 domain) (Other names:

FMC63scFV_ICOS_BBt_CD2tZ CAR, CD19_ICOS_

BBt_CD2tZ (SEQ ID NO: 106)

ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTT

CTGCATGCCGCCAGACCTGACATCCAGATGACCCAGACAACCAGC

AGCCTGTCTGCCAGCCTGGGCGATAGAGTGACCATCAGCTGTAGA

GCCAGCCAGGACATCAGCAAGTACCTGAACTGGTATCAGCAGAAA

CCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCAGACTG

CACAGCGGCGTGCCAAGCAGATTTTCTGGCAGCGGCTCTGGCACC

GACTACAGCCTGACCATCTCCAACCTGGAACAAGAGGATATCGCT

ACCTACTTCTGCCAGCAAGGCAACACCCTGCCTTACACCTTTGGC

GGAGGCACCAAGCTGGAAATCACAGGCGGCGGAGGAAGCGGAGGC

GGAGGATCTGGTGGTGGTGGATCTGAAGTGAAACTGCAAGAGTCT

GGCCCTGGCCTGGTGGCCCCATCTCAATCTCTGAGCGTGACCTGT

ACCGTCAGCGGAGTGTCCCTGCCTGATTATGGCGTGTCCTGGATC

CGGCAGCCTCCTAGAAAAGGCCTGGAATGGCTGGGCGTGATCTGG

GGCAGCGAGACAACCTACTACAACAGCGCCCTGAAGTCCCGGCTG

ACCATCATCAAGGACAACTCCAAGAGCCAGGTGTTCCTGAAGATG

AACAGCCTCCAGACCGACGACACCGCCATCTACTATTGCGCCAAG

CACTACTACTACGGCGGCAGCTACGCCATGGATTATTGGGGCCAG

GGCACCAGCGTGACCGTGTCTAGTACAACAACCCCTGCTCCTCGG

CCTCCTACACCAGCTCCTACAATTGCCAGCCAGCCACTGTCTCTG

AGGCCCGAAGCTTGTAGACCTGCTGCTGGCGGAGCCGTGCATACA

AGAGGACTGGATTTCGCCTGCAGCCAGCTGTGCTGTCAGCTGAAG

TTCTGGCTGCCTATCGGCTGCGCCGCCTTTGTGGTTGTGTGTATC

CTGGGCTGCATCCTGATCTGCTGGCTGACCAAGAAAAAGTACAGC

AGCAGCGTGCACGACCCCAACGGCGAGTACATGTTCATGAGAGCC

GTGAACACCGCCAAGAAGTCCAGACTGACCGACGTGACCCTCCAG

CCTTTCATGAGGCCTGTGCAGACCACACAAGAAGAGGACGGCTGC

TCCTGTCGGTTCCCCGAGGAAGAGGAAGGCGGTTGCGAACTCCAG

AATCCTGCCACCTCTCAGCACCCTCCACCTCCACCTGGACACAGA

TCTCAGGCTCCTAGCCACAGACCTCCACCACCTGGTCATAGAGTG

CAGCACCAGCCTCAGAAGAGGCCTCCTGCTCCTTCTGGAACACAG

GTGCACCAGCAAAAGGGCCCTCCACTGCCTAGACCTAGGGTGCAG

CCTAAACCTCCTCATGGCGCCGCTGAGAACTCTCTGAGCCCCAGC

AGCAACAGAGTGAAGTTCAGCAGATCCGCCGACGCTCCTGCCTAT

CAGCAGGGCCAAAACCAGCTGTACAACGAGCTGAACCTGGGGAGA

AGAGAAGAGTACGACGTGCTGGACAAGCGGAGAGGCAGAGATCCT

GAAATGGGCGGCAAGCCCCAGCGGAGAAAGAATCCTCAAGAGGGC

CTGTATAATGAGCTGCAAAAGGACAAGATGGCCGAGGCCTACAGC

GAGATCGGAATGAAGGGCGAGCGCAGAAGAGGAAAGGGACACGAC

GGACTGTACCAGGGCCTGAGCACCGCCACAAAGGATACCTATGAC

GCCCTGCACATGCAGGCCCTGCCTCCAAGATGA

CD19_ICOSBBt:CD2t_ZE CAR (CD19 binding

extracellular domain-ICOS DS-truncated

CD137 intracellular domain-CD2 truncated

Signaling Domain-CD3ZE domain) (Other

names: FMC63scFV_ICOS_BBt_CD2tZE CAR,

CD19_ICOS_BBt_CD2tZE) (SEQ ID NO: 107)

ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTT

CTGCATGCCGCCAGACCTGACATCCAGATGACCCAGACAACCAGC

AGCCTGTCTGCCAGCCTGGGCGATAGAGTGACCATCAGCTGTAGA

GCCAGCCAGGACATCAGCAAGTACCTGAACTGGTATCAGCAGAAA

CCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCAGACTG

CACAGCGGCGTGCCAAGCAGATTTTCTGGCAGCGGCTCTGGCACC

GACTACAGCCTGACCATCTCCAACCTGGAACAAGAGGATATCGCT

ACCTACTTCTGCCAGCAAGGCAACACCCTGCCTTACACCTTTGGC

GGAGGCACCAAGCTGGAAATCACAGGCGGCGGAGGAAGCGGAGGC

GGAGGATCTGGTGGTGGTGGATCTGAAGTGAAACTGCAAGAGTCT

GGCCCTGGCCTGGTGGCCCCATCTCAATCTCTGAGCGTGACCTGT

ACCGTCAGCGGAGTGTCCCTGCCTGATTATGGCGTGTCCTGGATC

CGGCAGCCTCCTAGAAAAGGCCTGGAATGGCTGGGCGTGATCTGG

GGCAGCGAGACAACCTACTACAACAGCGCCCTGAAGTCCCGGCTG

ACCATCATCAAGGACAACTCCAAGAGCCAGGTGTTCCTGAAGATG

AACAGCCTCCAGACCGACGACACCGCCATCTACTATTGCGCCAAG

CACTACTACTACGGCGGCAGCTACGCCATGGATTATTGGGGCCAG

GGCACCAGCGTGACCGTGTCTAGTACAACAACCCCTGCTCCTCGG

CCTCCTACACCAGCTCCTACAATTGCCAGCCAGCCACTGTCTCTG

AGGCCCGAAGCTTGTAGACCTGCTGCTGGCGGAGCCGTGCATACA

AGAGGACTGGATTTCGCCTGCAGCCAGCTGTGCTGTCAGCTGAAG

TTCTGGCTGCCTATCGGCTGCGCCGCCTTTGTGGTTGTGTGTATC

CTGGGCTGCATCCTGATCTGCTGGCTGACCAAGAAAAAGTACAGC

AGCAGCGTGCACGACCCCAACGGCGAGTACATGTTCATGAGAGCC

GTGAACACCGCCAAGAAGTCCAGACTGACCGACGTGACCCTCCAG

CCTTTCATGAGGCCTGTGCAGACCACACAAGAAGAGGACGGCTGC

TCCTGTCGGTTCCCCGAGGAAGAGGAAGGCGGTTGCGAACTCCAG

AATCCTGCCACCTCTCAGCACCCTCCACCTCCACCTGGACACAGA

TCTCAGGCTCCTAGCCACAGACCTCCACCACCTGGTCATAGAGTG

CAGCACCAGCCTCAGAAGAGGCCTCCTGCTCCTTCTGGAACACAG

GTGCACCAGCAAAAGGGCCCTCCACTGCCTAGACCTAGGGTGCAG

CCTAAACCTCCTCATGGCGCCGCTGAGAACTCTCTGAGCCCCAGC

AGCAACAGAGTGAAGTTCAGCAGATCCGCCGACGCTCCTGCCTAT

CAGCAGGGCCAAAACCAGCTGTACAACGAGCTGAACCTGGGGAGA

AGAGAAGAGTACGACGTGCTGGACAAGCGGAGAGGCAGAGATCCT

GAAATGGGCGGCAAGCCTGTGACTAGAGGTGCTGGCGCTGGTGGA

AGGCAGAGAGGCCAGAACAAAGAACGGCCTCCTCCTGTGCCTAAT

CCTGACTACGAGCCCATCCGGAAGGGCCAGAGAGATCTGTACAGC

GGCCTGAACTACAGACACCAGCGGAGAATCTGA

CD19_ICOSBBt:IL2RB(YLRQ)_ZCAR (CD19 binding

extracellular domain-ICOS DS-truncated CD137

intracellular domain-IL2RB(YLRQ) domain-CD3Z

domain) (Other names: FMC63scFV_ICOS_BBt_IL2RB

(YLRQ)Z CAR, CD19_ICOS_BBt_IL2RB(YLRQ)Z

(SEQ ID NO: 108)

ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTT

CTGCATGCCGCCAGACCTGACATCCAGATGACCCAGACAACCAGC

AGCCTGTCTGCCAGCCTGGGCGATAGAGTGACCATCAGCTGTAGA

GCCAGCCAGGACATCAGCAAGTACCTGAACTGGTATCAGCAGAAA

CCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCAGACTG

CACAGCGGCGTGCCAAGCAGATTTTCTGGCAGCGGCTCTGGCACC

GACTACAGCCTGACCATCTCCAACCTGGAACAAGAGGATATCGCT

ACCTACTTCTGCCAGCAAGGCAACACCCTGCCTTACACCTTTGGC

GGAGGCACCAAGCTGGAAATCACAGGCGGCGGAGGAAGCGGAGGC

GGAGGATCTGGTGGTGGTGGATCTGAAGTGAAACTGCAAGAGTCT

GGCCCTGGCCTGGTGGCCCCATCTCAATCTCTGAGCGTGACCTGT

ACCGTCAGCGGAGTGTCCCTGCCTGATTATGGCGTGTCCTGGATC

CGGCAGCCTCCTAGAAAAGGCCTGGAATGGCTGGGCGTGATCTGG

GGCAGCGAGACAACCTACTACAACAGCGCCCTGAAGTCCCGGCTG

ACCATCATCAAGGACAACTCCAAGAGCCAGGTGTTCCTGAAGATG

AACAGCCTCCAGACCGACGACACCGCCATCTACTATTGCGCCAAG

CACTACTACTACGGCGGCAGCTACGCCATGGATTATTGGGGCCAG

GGCACCAGCGTGACCGTGTCTAGTACAACAACCCCTGCTCCTCGG

CCTCCTACACCAGCTCCTACAATTGCCAGCCAGCCACTGTCTCTG

AGGCCCGAAGCTTGTAGACCTGCTGCTGGCGGAGCCGTGCATACA

AGAGGACTGGATTTCGCCTGCAGCCAGCTGTGCTGTCAGCTGAAG

TTCTGGCTGCCTATCGGCTGCGCCGCCTTTGTGGTTGTGTGTATC

CTGGGCTGCATCCTGATCTGCTGGCTGACCAAGAAAAAGTACAGC

AGCAGCGTGCACGACCCCAACGGCGAGTACATGTTCATGAGAGCC

GTGAACACCGCCAAGAAGTCCAGACTGACCGACGTGACCCTCCAG

CCTTTCATGAGGCCTGTGCAGACCACACAAGAAGAGGACGGCTGC

TCCTGTCGGTTCCCCGAGGAAGAGGAAGGCGGCTGCGAACTGAAT

TGCAGAAACACAGGCCCCTGGCTGAAGAAAGTGCTGAAGTGCAAC

ACCCCTGATCCGAGCAAGTTTTTCAGCCAGCTGAGCAGCGAGCAT

GGCGGCGACGTTCAGAAATGGCTGTCTAGCCCATTTCCTAGCTCC

AGCTTCAGCCCTGGTGGACTGGCCCCTGAGATTAGCCCTCTGGAA

GTGCTGGAACGGGACAAAGTGACCCAGCTGCTGCCCCTGAATACC

GACGCCTACCTGAGCCTGCAAGAGCTGCAAGGCCAGGATCCTACA

CACCTCGTGCGCGTGAAGTTCAGCAGATCCGCTGATGCCCCTGCC

TATCAGCAGGGCCAAAACCAGCTGTACAACGAGCTGAACCTGGGG

AGAAGAGAAGAGTACGACGTGCTGGACAAGCGGAGAGGCAGAGAT

CCTGAAATGGGCGGCAAGCCCCAGCGGAGAAAGAATCCTCAAGAG

GGCCTGTATAATGAGCTGCAAAAGGACAAGATGGCCGAGGCCTAC

AGCGAGATCGGAATGAAGGGCGAGCGCAGAAGAGGCAAGGGACAC

GATGGACTGTACCAGGGCCTGAGCACCGCCACCAAGGATACCTAC

GATGCCTACAGACACCAGGCTCTGCCACCTAGATGA

CD19_ICOSBBt:IL2RB(YLRQ)_ZE_CAR (CD19 binding

extracellular domain-ICOS DS-truncated CD137

intracellular domain-IL2RB(YLRQ) domain-CD3ZE

domain (Other names: FMC63scFV_ICOS_BBt_IL2RB

(YLRQ)_ZE CAR) (SEQ ID NO: 110)

ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTT

CTGCATGCCGCCAGACCTGACATCCAGATGACCCAGACAACCAGC

AGCCTGTCTGCCAGCCTGGGCGATAGAGTGACCATCAGCTGTAGA

GCCAGCCAGGACATCAGCAAGTACCTGAACTGGTATCAGCAGAAA

CCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCAGACTG

CACAGCGGCGTGCCAAGCAGATTTTCTGGCAGCGGCTCTGGCACC

GACTACAGCCTGACCATCTCCAACCTGGAACAAGAGGATATCGCT

ACCTACTTCTGCCAGCAAGGCAACACCCTGCCTTACACCTTTGGC

GGAGGCACCAAGCTGGAAATCACAGGCGGCGGAGGAAGCGGAGGC

GGAGGATCTGGTGGTGGTGGATCTGAAGTGAAACTGCAAGAGTCT

GGCCCTGGCCTGGTGGCCCCATCTCAATCTCTGAGCGTGACCTGT

ACCGTCAGCGGAGTGTCCCTGCCTGATTATGGCGTGTCCTGGATC

CGGCAGCCTCCTAGAAAAGGCCTGGAATGGCTGGGCGTGATCTGG

GGCAGCGAGACAACCTACTACAACAGCGCCCTGAAGTCCCGGCTG

ACCATCATCAAGGACAACTCCAAGAGCCAGGTGTTCCTGAAGATG

AACAGCCTCCAGACCGACGACACCGCCATCTACTATTGCGCCAAG

CACTACTACTACGGCGGCAGCTACGCCATGGATTATTGGGGCCAG

GGCACCAGCGTGACCGTGTCTAGTACAACAACCCCTGCTCCTCGG

CCTCCTACACCAGCTCCTACAATTGCCAGCCAGCCACTGTCTCTG

AGGCCCGAAGCTTGTAGACCTGCTGCTGGCGGAGCCGTGCATACA

AGAGGACTGGATTTCGCCTGCAGCCAGCTGTGCTGTCAGCTGAAG

TTCTGGCTGCCTATCGGCTGCGCCGCCTTTGTGGTTGTGTGTATC

CTGGGCTGCATCCTGATCTGCTGGCTGACCAAGAAAAAGTACAGC

AGCAGCGTGCACGACCCCAACGGCGAGTACATGTTCATGAGAGCC

GTGAACACCGCCAAGAAGTCCAGACTGACCGACGTGACCCTCCAG

CCTTTCATGAGGCCTGTGCAGACCACACAAGAAGAGGACGGCTGC

TCCTGTCGGTTCCCCGAGGAAGAGGAAGGCGGCTGCGAACTGAAT

TGCAGAAACACAGGCCCCTGGCTGAAGAAAGTGCTGAAGTGCAAC

ACCCCTGATCCGAGCAAGTTTTTCAGCCAGCTGAGCAGCGAGCAT

GGCGGCGACGTTCAGAAATGGCTGTCTAGCCCATTTCCTAGCTCC

AGCTTCAGCCCTGGTGGACTGGCCCCTGAGATTAGCCCTCTGGAA

GTGCTGGAACGGGACAAAGTGACCCAGCTGCTGCCCCTGAATACC

GACGCCTACCTGAGCCTGCAAGAGCTGCAAGGCCAGGATCCTACA

CACCTCGTGCGCGTGAAGTTCAGCAGATCCGCTGATGCCCCTGCC

TATCAGCAGGGCCAAAACCAGCTGTACAACGAGCTGAACCTGGGG

AGAAGAGAAGAGTACGACGTGCTGGACAAGCGGAGAGGCAGAGAT

CCTGAAATGGGCGGCAAGCCTGTGACTAGAGGTGCTGGTGCTGGC

GGCAGACAGAGAGGCCAGAACAAAGAAAGACCTCCTCCTGTGCCT

AATCCTGACTACGAGCCCATCCGGAAGGGCCAGAGAGATCTGTAC

AGCGGCCTGAACTACAGACACCAGCGGAGAATCTGA

CD28 BB signaling domain: CD28 signaling

domain_4-1BB Signaling Domain

(SEQ ID NO: 151)

CTGTTCCCCGGACCTAGCAAGCCCTTTTGGGTGCTCGTTGTTGTT

GGCGGCGTGCTGGCCTGTTATAGCCTGCTGGTTACCGTGGCCTTC

ATCATCTTTTGGGTCCGAAGCAAGCGGAGCCGGCTGCTGCACAGC

GACTACATGAACATGACCCCTAGACGGCCCGGACCAACCAGAAAG

CACTACCAGCCTTACGCTCCTCCTAGAGACTTCGCCGCCTACAGA

TCCAAGCGGGGCAGAAAGAAGCTGCTGTACATCTTCAAGCAGCCC

TTCATGCGGCCCGTGCAGACCACACAAGAGGAAGATGGCTGCTCC

TGTCGGTTCCCCGAGGAAGAAGAAGGCGGTTGCGAACTG

CD28 Signaling Domain truncated 4-1BB Signaling

Domain (Other name: CD28_BBt):

(SEQ ID NO: 152)

CTGTTCCCCGGACCTAGCAAGCCCTTTTGGGTGCTCGTTGTTGTT

GGCGGCGTGCTGGCCTGTTATAGCCTGCTGGTTACCGTGGCCTTC

ATCATCTTTTGGGTCCGAAGCAAGCGGAGCCGGCTGCTGCACAGC

GACTACATGAACATGACCCCTAGACGGCCCGGACCAACCAGAAAG

CACTACCAGCCTTACGCTCCTCCTAGAGACTTCGCCGCCTACAGA

TCCCAGCCTTTCATGAGGCCTGTGCAGACCACACAAGAAGAGGAC

GGCTGCTCCTGTCGGTTCCCCGAGGAAGAGGAAGGCGGTTGCGAA

CTT

CD28 Signaling Domain_truncated OX-40 Signaling

Domain (Other name: CD28_OX40t)

(SEQ ID NO: 153)

CTGTTCCCCGGACCTAGCAAGCCCTTTTGGGTGCTCGTTGTTGTT

GGCGGCGTGCTGGCCTGTTATAGCCTGCTGGTTACCGTGGCCTTC

ATCATCTTTTGGGTCCGAAGCAAGCGGAGCCGGCTGCTGCACAGC

GACTACATGAACATGACCCCTAGACGGCCCGGACCAACCAGAAAG

CACTACCAGCCTTACGCTCCTCCTAGAGACTTCGCCGCCTACAGA

TCTGGCGGCGGAAGCTTCAGAACCCCTATCCAAGAGGAACAGGCC

GACGCTCACTCTACACTGGCT

ICOS Transmembrane_ICOS Signaling Domain

(SEQ ID NO: 154)

AGCCAGCTGTGCTGCCAGCTGAAGTTTTGGCTGCCTATCGGCTGT

GCCGCCTTCGTGGTTGTGTGTATCCTGGGCTGCATCCTGATCTGC

TGGCTGACCAAGAAAAAGTACAGCAGCTCCGTGCACGACCCCAAC

GGCGAGTACATGTTCATGAGAGCCGTGAACACCGCCAAGAAGTCC

AGACTGACCGACGTGACACTT

ICOS Transmembrane_ICOS Signaling Domain 4-1BB

Signaling Domain (SEQ ID NO: 155)

AGCCAGCTGTGCTGCCAGCTGAAGTTTTGGCTGCCTATCGGCTGT

GCCGCCTTCGTGGTTGTGTGTATCCTGGGCTGCATCCTGATCTGC

TGGCTGACCAAGAAAAAGTACAGCAGCTCCGTGCACGACCCCAAC

GGCGAGTACATGTTCATGAGAGCCGTGAACACCGCCAAGAAGTCC

AGACTGACCGACGTGACCCTGAAGCGGGGCAGAAAGAAACTGCTG

TACATCTTCAAGCAGCCCTTCATGCGGCCCGTGCAGACCACACAA

GAGGAAGATGGCTGCTCCTGCAGATTCCCCGAGGAAGAAGAAGGC

GGCTGCGAACTT

ICOS Transmembrane_ICOS Signaling Domain_

Truncated 4-1BB Signaling Domain

(SEQ ID NO: 156)

AGCCAGCTGTGCTGCCAGCTGAAGTTTTGGCTGCCTATCGGCTGT

GCCGCCTTCGTGGTTGTGTGTATCCTGGGCTGCATCCTGATCTGC

TGGCTGACCAAGAAAAAGTACAGCAGCTCCGTGCACGACCCCAAC

GGCGAGTACATGTTCATGAGAGCCGTGAACACCGCCAAGAAGTCC

AGACTGACCGACGTGACACTTCAGCCTTTCATGAGGCCCGTGCAG

ACCACACAAGAAGAGGACGGCTGCTCCTGCAGATTCCCCGAGGAA

GAGGAAGGCGGTTGCGAACTT

ICOS Transmembrane_ICOS Signaling Domain_

Truncated OX-40 Signaling Domain

(ICOS_OX40t) (SEQ ID NO: 157)

AGCCAGCTGTGCTGCCAGCTGAAGTTTTGGCTGCCTATCGGCTGT

GCCGCCTTCGTGGTTGTGTGTATCCTGGGCTGCATCCTGATCTGC

TGGCTGACCAAGAAAAAGTACAGCAGCTCCGTGCACGACCCCAAC

GGCGAGTACATGTTCATGAGAGCCGTGAACACCGCCAAGAAGTCC

AGACTGACCGACGTGACACTTGGCGGCGGAAGCTTTAGAACCCCT

ATCCAAGAGGAACAGGCCGACGCTCACTCTACACTGGCT

ICOS Transmembrane_ICOS Signaling Domain

(mini-CD28)_Truncated 4-1BB Signaling

Domain (ICOS(28)_BBt) (SEQ ID NO: 158)

AGCCAGCTGTGCTGCCAGCTGAAGTTTTGGCTGCCTATCGGCTGT

GCCGCCTTCGTGGTTGTGTGTATCCTGGGCTGCATCCTGATCTGC

TGGCTGACCAAGAAAAAGTACAGCAGCTCCGTGCACGACCCCAAC

GGCGAGTACATGTTCATGACCCCTAGAAGGCCTGGACCTACCAGA

AAGCACTACCAGCCTTACGCTCCTCCTAGAGCCGTGAACACCGCC

AAGAAGTCCAGACTGACCGACGTGACACTTCAGCCTTTCATGAGG

CCCGTGCAGACCACACAAGAAGAGGACGGCTGCTCCTGCAGATTC

CCCGAGGAAGAGGAAGGCGGTTGCGAACTT

ICOS(28)_truncated OX-40 (CD134) intracellular

domain (ICOS(28)_OX40t) (SEQ ID NO: 159)

AGCCAGCTGTGCTGCCAGCTGAAGTTTTGGCTGCCTATCGGCTGT

GCCGCCTTCGTGGTTGTGTGTATCCTGGGCTGCATCCTGATCTGC

TGGCTGACCAAGAAAAAGTACAGCAGCTCCGTGCACGACCCCAAC

GGCGAGTACATGTTCATGACCCCTAGAAGGCCTGGACCTACCAGA

AAGCACTACCAGCCTTACGCTCCTCCTAGAGCCGTGAACACCGCC

AAGAAGTCCAGACTGACCGACGTGACACTTGGCGGCGGAAGCTTT

AGAACCCCTATCCAAGAGGAACAGGCCGACGCTCACTCTACACTG

GCT

ICOS Transmembrane_ICOS Signaling Domain_

4-1BB Signaling Domain with mutated

polybasic region (ICOS_BB(xPB))

(SEQ ID NO: 160)

AGCCAGCTGTGCTGCCAGCTGAAGTTTTGGCTGCCTATCGGCTGT

GCCGCCTTCGTGGTTGTGTGTATCCTGGGCTGCATCCTGATCTGC

TGGCTGACCAAGAAAAAGTACAGCAGCTCCGTGCACGACCCCAAC

GGCGAGTACATGTTCATGAGAGCCGTGAACACCGCCAAGAAGTCC

AGACTGACCGACGTGACCCTGGCTGCCGGCGCTGCAGCTCTGCTG

TACATCTTCAAGCAGCCCTTCATGCGGCCCGTGCAGACCACACAA

GAGGAAGATGGCTGCTCCTGCAGATTCCCCGAGGAAGAAGAAGGC

GGCTGCGAACTT

ICOS Transmembrane_ICOS Signaling Domain_

4-1BB Signaling Domain with mutated

lysines (ICOS_BB(xUb)) (SEQ ID NO: 161)

AGCCAGCTGTGCTGCCAGCTGAAGTTTTGGCTGCCTATCGGCTGT

GCCGCCTTCGTGGTTGTGTGTATCCTGGGCTGCATCCTGATCTGC

TGGCTGACCAAGAAAAAGTACAGCAGCTCCGTGCACGACCCCAAC

GGCGAGTACATGTTCATGAGAGCCGTGAACACCGCCAAGAAGTCC

AGACTGACCGACGTGACCCTGAAGCGGGGCAGAAAGAAACTGCTG

TACATCTTCGCACAGCCCTTCATGCGGCCCGTGCAGACCACACAA

GAGGAAGATGGCTGCTCCTGCAGATTCCCCGAGGAAGAAGAAGGC

GGCTGCGAACTT

ICOS Transmembrane_ICOS Signaling Domain 4-1BB

Signaling Domain with mutated lysines and

polybasic regions (ICOS_BB(xPBxUb))

(SEQ ID NO: 162)

AGCCAGCTGTGCTGCCAGCTGAAGTTTTGGCTGCCTATCGGCTGT

GCCGCCTTCGTGGTTGTGTGTATCCTGGGCTGCATCCTGATCTGC

TGGCTGACCAAGAAAAAGTACAGCAGCTCCGTGCACGACCCCAAC

GGCGAGTACATGTTCATGAGAGCCGTGAACACCGCCAAGAAGTCC

AGACTGACCGACGTGACCCTGGCTGCCGGCGCTGCAGCTCTGCTG

TACATCTTCGCACAGCCCTTCATGCGGCCCGTGCAGACCACACAA

GAGGAAGATGGCTGCTCCTGCAGATTCCCCGAGGAAGAAGAAGGC

GGCTGCGAACTT

ICOS Transmembrane_ICOS Signaling Domain Wild_

Type OX-40 Signaling Domain (ICOS_40)

(SEQ ID NO: 163)

AGCCAGCTGTGCTGCCAGCTGAAGTTTTGGCTGCCTATCGGCTGT

GCCGCCTTCGTGGTTGTGTGTATCCTGGGCTGCATCCTGATCTGC

TGGCTGACCAAGAAAAAGTACAGCAGCTCCGTGCACGACCCCAAC

GGCGAGTACATGTTCATGAGAGCCGTGAACACCGCCAAGAAGTCC

AGACTGACCGACGTGACACTTAGGAGAGACCAGCGTCTGCCACCA

GATGCACATAAGCCACCTGGCGGCGGAAGCTTTAGAACCCCTATC

CAAGAGGAACAGGCCGACGCTCACTCTACACTGGCTAAAATC

ICOS Transmembrane_ICOS Signaling Domain_OX-40

Signaling Domain with mutated

polybasic region (ICOS_40(xPB)

(SEQ ID NO: 164)

AGCCAGCTGTGCTGCCAGCTGAAGTTTTGGCTGCCTATCGGCTGT

GCCGCCTTCGTGGTTGTGTGTATCCTGGGCTGCATCCTGATCTGC

TGGCTGACCAAGAAAAAGTACAGCAGCTCCGTGCACGACCCCAAC

GGCGAGTACATGTTCATGAGAGCCGTGAACACCGCCAAGAAGTCC

AGACTGACCGACGTGACACTTGCCGCGGACCAGGCCCTGCCACCA

GATGCACATAAGCCACCTGGCGGCGGAAGCTTTAGAACCCCTATC

CAAGAGGAACAGGCCGACGCTCACTCTACACTGGCTAAAATC

ICOS Transmembrane_ICOS Signaling Domain_OX-40

Signaling Domain with mutated

lysine residues (ICOS_40(xUb)):

(SEQ ID NO: 165)

AGCCAGCTGTGCTGCCAGCTGAAGTTTTGGCTGCCTATCGGCTGT

GCCGCCTTCGTGGTTGTGTGTATCCTGGGCTGCATCCTGATCTGC

TGGCTGACCAAGAAAAAGTACAGCAGCTCCGTGCACGACCCCAAC

GGCGAGTACATGTTCATGAGAGCCGTGAACACCGCCAAGAAGTCC

AGACTGACCGACGTGACACTTAGGAGAGACCAGCGTCTGCCACCA

GATGCACATGCACCACCTGGCGGCGGAAGCTTTAGAACCCCTATC

CAAGAGGAACAGGCCGACGCTCACTCTACACTGGCTGCAATC

ICOS Transmembrane_ICOS Signaling Domain

OX-40_Signaling Domain with mutated

lysine residues and polybasic regions

(ICOS_40(xPBxUb)): (SEQ ID NO: 166)

AGCCAGCTGTGCTGCCAGCTGAAGTTTTGGCTGCCTATCGGCTGT

GCCGCCTTCGTGGTTGTGTGTATCCTGGGCTGCATCCTGATCTGC

TGGCTGACCAAGAAAAAGTACAGCAGCTCCGTGCACGACCCCAAC

GGCGAGTACATGTTCATGAGAGCCGTGAACACCGCCAAGAAGTCC

AGACTGACCGACGTGACACTTGCCGCGGACCAGGCCCTGCCACCA

GATGCACATGCACCACCTGGCGGCGGAAGCTTTAGAACCCCTATC

CAAGAGGAACAGGCCGACGCTCACTCTACACTGGCTGCAATC

PD1 Extracellular (SEQ ID NO: 167)

TTCCTGGACTCTCCTGACAGACCCTGGAATCCTCCAACATTCAGC

CCCGCTCTGCTGGTGGTTACCGAGGGCGATAATGCCACCTTCACC

TGTAGCTTCAGCAACACCAGCGAGAGCTTCGTGCTGAACTGGTAC

AGAATGAGCCCCAGCAACCAGACCGACAAGCTGGCCGCCTTTCCT

GAGGATAGATCTCAGCCCGGCCAGGACTGCCGGTTCAGAGTTACA

CAGCTGCCCAACGGCCGGGACTTCCACATGTCTGTCGTCCGGGCC

AGAAGAAACGACAGCGGCACATATCTGTGCGGCGCCATTTCTCTG

GCCCCTAAGGCTCAGATCAAAGAGAGCCTGAGAGCCGAGCTGAGA

GTGACAGAAAGACGGGCCGAAGTGCCCACAGCTCACCCTTCACCT

TCTCCAAGACCTGCC

HLA-A2 Signal Peptide_PD1 Extracellular_PD1

Transmembrane (PD1 (HLASP-Truncated;

PD1_TLs) (SEQ ID NO: 168)

ATGGCTGTGATGGCTCCTAGAACACTGGTGCTGCTGCTGTCTGGT

GCCCTGGCTCTGACTCAGACATGGGCCTTTCTGGACAGCCCCGAC

AGACCCTGGAATCCTCCTACATTCAGCCCCGCTCTGCTGGTGGTT

ACCGAGGGCGATAATGCCACCTTCACCTGTAGCTTCAGCAACACC

AGCGAGAGCTTCGTGCTGAACTGGTACAGAATGAGCCCCAGCAAC

CAGACCGACAAGCTGGCCGCCTTTCCTGAGGATAGATCTCAGCCC

GGCCAGGACTGCCGGTTCAGAGTTACACAGCTGCCCAACGGCCGG

GACTTCCACATGTCTGTCGTTCGGGCCAGAAGAAACGACAGCGGC

ACATATCTGTGCGGCGCCATTTCTCTGGCCCCTAAGGCTCAGATC

AAAGAGAGCCTGAGAGCCGAGCTGAGAGTGACAGAAAGACGGGCC

GAAGTGCCCACAGCTCACCCTTCACCTTCTCCAAGACCTGCCGGC

CAGTTCCAGACACTGGTCGTGGGAGTTGTTGGCGGCCTGCTGGGA

TCTCTGGTTCTGCTTGTTTGGGTGCTCGCCGTGATCTGCTCTAGA

HLA-A2 Signal Peptide_PD1 Extracellular_CD28

Transmembrane CD28 Signaling Domain

(HLASP CD28 DS or PD1_CD28 or PD_28)

(SEQ ID NO: 169)

ATGGCTGTGATGGCTCCTAGAACACTGGTGCTGCTGCTGTCTGGT

GCCCTGGCTCTGACTCAGACATGGGCCTTTCTGGACAGCCCCGAC

AGACCCTGGAATCCTCCTACATTCAGCCCCGCTCTGCTGGTGGTT

ACCGAGGGCGATAATGCCACCTTCACCTGTAGCTTCAGCAACACC

AGCGAGAGCTTCGTGCTGAACTGGTACAGAATGAGCCCCAGCAAC

CAGACCGACAAGCTGGCCGCCTTTCCTGAGGATAGATCTCAGCCC

GGCCAGGACTGCCGGTTCAGAGTTACACAGCTGCCCAACGGCCGG

GACTTCCACATGTCTGTCGTTCGGGCCAGAAGAAACGACAGCGGC

ACATATCTGTGCGGCGCCATTTCTCTGGCCCCTAAGGCTCAGATC

AAAGAGAGCCTGAGAGCCGAGCTGAGAGTGACAGAAAGACGGGCC

GAAGTGCCCACAGCTCACCCTTCACCTTCTCCAAGACCTGCTCTG

TTCCCCGGACCTAGCAAGCCCTTTTGGGTGCTCGTTGTTGTTGGC

GGCGTGCTGGCCTGTTATAGCCTGCTGGTTACCGTGGCCTTCATC

ATCTTTTGGGTCCGAAGCAAGCGGAGCCGGCTGCTGCACAGCGAC

TACATGAACATGACCCCTAGACGGCCCGGACCAACCAGAAAGCAC

TACCAGCCTTACGCTCCTCCTAGAGACTTCGCCGCCTACAGATCT

HLA-A2 Signal Peptide_PD1 Extracellular_CD28

Transmembrane_CD28 Signaling Domain_

4-1BB Signaling Domain (PD1_28_BBwt)

(SEQ ID NO: 170)

ATGGCTGTGATGGCCCCTAGAACACTGGTGCTGCTGCTGTCTGGT

GCCCTGGCTCTGACTCAGACATGGGCCTTTCTGGACAGCCCCGAC

AGACCCTGGAATCCTCCTACATTCAGCCCCGCTCTGCTGGTGGTT

ACCGAGGGCGATAATGCCACCTTCACCTGTAGCTTCAGCAACACC

AGCGAGAGCTTCGTGCTGAACTGGTACAGAATGAGCCCCAGCAAC

CAGACCGACAAGCTGGCCGCCTTTCCTGAGGATAGATCTCAGCCC

GGCCAGGACTGCCGGTTCAGAGTTACACAGCTGCCCAACGGCCGG

GACTTCCACATGTCTGTCGTTCGGGCCAGAAGAAACGACAGCGGC

ACATATCTGTGCGGCGCCATTTCTCTGGCCCCTAAGGCTCAGATC

AAAGAGAGCCTGAGAGCCGAGCTGAGAGTGACAGAAAGACGGGCC

GAAGTGCCCACAGCTCACCCTTCACCTTCTCCAAGACCTGCTCTG

TTCCCCGGACCTAGCAAGCCCTTTTGGGTGCTCGTTGTTGTTGGC

GGCGTGCTGGCCTGTTATAGCCTGCTGGTTACCGTGGCCTTCATC

ATCTTTTGGGTCCGAAGCAAGCGGAGCCGGCTGCTGCACAGCGAC

TACATGAACATGACCCCTAGACGGCCCGGACCAACCAGAAAGCAC

TACCAGCCTTACGCTCCTCCTAGAGACTTCGCCGCCTACAGATCC

AAGCGGGGCAGAAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTC

ATGCGGCCCGTGCAGACCACACAAGAGGAAGATGGCTGCTCCTGT

CGGTTCCCCGAGGAAGAAGAAGGCGGTTGCGAACTG

HLA-A2 Signal Peptide_PD1 Extracellular_CD28

Transmembrane_CD28 Signaling

Domain_Truncated 4-1BB Signaling Domain

(PD1_28_BBt) (SEQ ID NO: 171)

ATGGCTGTGATGGCCCCTAGAACACTGGTGCTGCTGCTGTCTGGT

GCCCTGGCTCTGACTCAGACATGGGCCTTTCTGGACAGCCCCGAC

AGACCCTGGAATCCTCCTACATTCAGCCCCGCTCTGCTGGTGGTT

ACCGAGGGCGATAATGCCACCTTCACCTGTAGCTTCAGCAACACC

AGCGAGAGCTTCGTGCTGAACTGGTACAGAATGAGCCCCAGCAAC

CAGACCGACAAGCTGGCCGCCTTTCCTGAGGATAGATCTCAGCCC

GGCCAGGACTGCCGGTTCAGAGTTACACAGCTGCCCAACGGCCGG

GACTTCCACATGTCTGTCGTTCGGGCCAGAAGAAACGACAGCGGC

ACATATCTGTGCGGCGCCATTTCTCTGGCCCCTAAGGCTCAGATC

AAAGAGAGCCTGAGAGCCGAGCTGAGAGTGACAGAAAGACGGGCC

GAAGTGCCCACAGCTCACCCTTCACCTTCTCCAAGACCTGCTCTG

TTCCCCGGACCTAGCAAGCCCTTTTGGGTGCTCGTTGTTGTTGGC

GGCGTGCTGGCCTGTTATAGCCTGCTGGTTACCGTGGCCTTCATC

ATCTTTTGGGTCCGAAGCAAGCGGAGCCGGCTGCTGCACAGCGAC

TACATGAACATGACCCCTAGACGGCCCGGACCAACCAGAAAGCAC

TACCAGCCTTACGCTCCTCCTAGAGACTTCGCCGCCTACAGATCC

CAGCCTTTCATGAGGCCTGTGCAGACCACACAAGAAGAGGACGGC

TGCTCCTGTCGGTTCCCCGAGGAAGAGGAAGGCGGTTGCGAACTT

HLA-A2 Signal Peptide_PD1 Extracellular_CD28

Transmembrane_CD28 Signaling Domain Truncated

OX-40 Signaling Domain (PD1_28_OX40t)

(SEQ ID NO: 172)

ATGGCTGTGATGGCCCCTAGAACACTGGTGCTGCTGCTGTCTGGT

GCCCTGGCTCTGACTCAGACATGGGCCTTTCTGGACAGCCCCGAC

AGACCCTGGAATCCTCCTACATTCAGCCCCGCTCTGCTGGTGGTT

ACCGAGGGCGATAATGCCACCTTCACCTGTAGCTTCAGCAACACC

AGCGAGAGCTTCGTGCTGAACTGGTACAGAATGAGCCCCAGCAAC

CAGACCGACAAGCTGGCCGCCTTTCCTGAGGATAGATCTCAGCCC

GGCCAGGACTGCCGGTTCAGAGTTACACAGCTGCCCAACGGCCGG

GACTTCCACATGTCTGTCGTTCGGGCCAGAAGAAACGACAGCGGC

ACATATCTGTGCGGCGCCATTTCTCTGGCCCCTAAGGCTCAGATC

AAAGAGAGCCTGAGAGCCGAGCTGAGAGTGACAGAAAGACGGGCC

GAAGTGCCCACAGCTCACCCTTCACCTTCTCCAAGACCTGCTCTG

TTCCCCGGACCTAGCAAGCCCTTTTGGGTGCTCGTTGTTGTTGGC

GGCGTGCTGGCCTGTTATAGCCTGCTGGTTACCGTGGCCTTCATC

ATCTTTTGGGTCCGAAGCAAGCGGAGCCGGCTGCTGCACAGCGAC

TACATGAACATGACCCCTAGACGGCCCGGACCAACCAGAAAGCAC

TACCAGCCTTACGCTCCTCCTAGAGACTTCGCCGCCTACAGATCT

GGCGGCGGAAGCTTCAGAACCCCTATCCAAGAGGAACAGGCCGAC

GCTCACTCTACACTGGCT

HLA-A2 Signal Peptide_PD1 Extracellular_ICOS

intracellular domain (PD1_ICOS)

(SEQ ID NO: 173)

ATGGCTGTGATGGCTCCTAGAACACTGGTGCTGCTGCTGTCTGGT

GCCCTGGCTCTGACTCAGACATGGGCCTTTCTGGACAGCCCCGAC

AGACCCTGGAATCCTCCTACATTCAGCCCCGCTCTGCTGGTGGTT

ACCGAGGGCGATAATGCCACCTTCACCTGTAGCTTCAGCAACACC

AGCGAGAGCTTCGTGCTGAACTGGTACAGAATGAGCCCCAGCAAC

CAGACCGACAAGCTGGCCGCCTTTCCTGAGGATAGATCTCAGCCC

GGCCAGGACTGCCGGTTCAGAGTTACACAGCTGCCCAACGGCCGG

GACTTCCACATGTCTGTCGTTCGGGCCAGAAGAAACGACAGCGGC

ACATATCTGTGCGGCGCCATTTCTCTGGCCCCTAAGGCTCAGATC

AAAGAGAGCCTGAGAGCCGAGCTGAGAGTGACAGAAAGACGGGCC

GAAGTGCCCACAGCTCACCCTTCACCTTCTCCAAGACCTGCCAGC

CAGCTGTGCTGCCAGCTGAAGTTTTGGCTGCCTATCGGCTGTGCC

GCCTTCGTGGTTGTGTGTATCCTGGGCTGCATCCTGATCTGCTGG

CTGACCAAGAAAAAGTACAGCAGCTCCGTGCACGACCCCAACGGC

GAGTACATGTTCATGAGAGCCGTGAACACCGCCAAGAAGTCCAGA

CTGACCGACGTGACACTT

HLA-A2 Signal Peptide_PD1 Extracellular_ICOS

DS_CD137 (PD1_ICOS_BBwt)

(SEQ ID NO: 174)

ATGGCTGTGATGGCTCCTAGAACACTGGTGCTGCTGCTGTCTGGT

GCCCTGGCTCTGACTCAGACATGGGCCTTTCTGGACAGCCCCGAC

AGACCCTGGAATCCTCCTACATTCAGCCCCGCTCTGCTGGTGGTT

ACCGAGGGCGATAATGCCACCTTCACCTGTAGCTTCAGCAACACC

AGCGAGAGCTTCGTGCTGAACTGGTACAGAATGAGCCCCAGCAAC

CAGACCGACAAGCTGGCCGCCTTTCCTGAGGATAGATCTCAGCCC

GGCCAGGACTGCCGGTTCAGAGTTACACAGCTGCCCAACGGCCGG

GACTTCCACATGTCTGTCGTTCGGGCCAGAAGAAACGACAGCGGC

ACATATCTGTGCGGCGCCATTTCTCTGGCCCCTAAGGCTCAGATC

AAAGAGAGCCTGAGAGCCGAGCTGAGAGTGACAGAAAGACGGGCC

GAAGTGCCCACAGCTCACCCTTCACCTTCTCCAAGACCTGCCAGC

CAGCTGTGCTGCCAGCTGAAGTTTTGGCTGCCTATCGGCTGTGCC

GCCTTCGTGGTTGTGTGTATCCTGGGCTGCATCCTGATCTGCTGG

CTGACCAAGAAAAAGTACAGCAGCTCCGTGCACGACCCCAACGGC

GAGTACATGTTCATGAGAGCCGTGAACACCGCCAAGAAGTCCAGA

CTGACCGACGTGACCCTGAAGCGGGGCAGAAAGAAACTGCTGTAC

ATCTTCAAGCAGCCCTTCATGCGGCCCGTGCAGACCACACAAGAG

GAAGATGGCTGCTCCTGCAGATTCCCCGAGGAAGAAGAAGGCGGC

TGCGAACTT

HLA-A2 Signal Peptide_PD1 Extracellular_ICOS

truncated CD137 (PD1_ICOS_BBt)

(SEQ ID NO: 175)

ATGGCTGTGATGGCTCCTAGAACACTGGTGCTGCTGCTGTCTGGT

GCCCTGGCTCTGACTCAGACATGGGCCTTTCTGGACAGCCCCGAC

AGACCCTGGAATCCTCCTACATTCAGCCCCGCTCTGCTGGTGGTT

ACCGAGGGCGATAATGCCACCTTCACCTGTAGCTTCAGCAACACC

AGCGAGAGCTTCGTGCTGAACTGGTACAGAATGAGCCCCAGCAAC

CAGACCGACAAGCTGGCCGCCTTTCCTGAGGATAGATCTCAGCCC

GGCCAGGACTGCCGGTTCAGAGTTACACAGCTGCCCAACGGCCGG

GACTTCCACATGTCTGTCGTTCGGGCCAGAAGAAACGACAGCGGC

ACATATCTGTGCGGCGCCATTTCTCTGGCCCCTAAGGCTCAGATC

AAAGAGAGCCTGAGAGCCGAGCTGAGAGTGACAGAAAGACGGGCC

GAAGTGCCCACAGCTCACCCTTCACCTTCTCCAAGACCTGCCAGC

CAGCTGTGCTGCCAGCTGAAGTTTTGGCTGCCTATCGGCTGTGCC

GCCTTCGTGGTTGTGTGTATCCTGGGCTGCATCCTGATCTGCTGG

CTGACCAAGAAAAAGTACAGCAGCTCCGTGCACGACCCCAACGGC

GAGTACATGTTCATGAGAGCCGTGAACACCGCCAAGAAGTCCAGA

CTGACCGACGTGACACTTCAGCCTTTCATGAGGCCCGTGCAGACC

ACACAAGAAGAGGACGGCTGCTCCTGCAGATTCCCCGAGGAAGAG

GAAGGCGGTTGCGAACTT

HLA-A2 Signal Peptide_PD1 Extracellular_ICOS

truncated CD134 (PD1_ICOS_OX40t)

(SEQ ID NO: 176)

ATGGCTGTGATGGCTCCTAGAACACTGGTGCTGCTGCTGTCTGGT

GCCCTGGCTCTGACTCAGACATGGGCCTTTCTGGACAGCCCCGAC

AGACCCTGGAATCCTCCTACATTCAGCCCCGCTCTGCTGGTGGTT

ACCGAGGGCGATAATGCCACCTTCACCTGTAGCTTCAGCAACACC

AGCGAGAGCTTCGTGCTGAACTGGTACAGAATGAGCCCCAGCAAC

CAGACCGACAAGCTGGCCGCCTTTCCTGAGGATAGATCTCAGCCC

GGCCAGGACTGCCGGTTCAGAGTTACACAGCTGCCCAACGGCCGG

GACTTCCACATGTCTGTCGTTCGGGCCAGAAGAAACGACAGCGGC

ACATATCTGTGCGGCGCCATTTCTCTGGCCCCTAAGGCTCAGATC

AAAGAGAGCCTGAGAGCCGAGCTGAGAGTGACAGAAAGACGGGCC

GAAGTGCCCACAGCTCACCCTTCACCTTCTCCAAGACCTGCCAGC

CAGCTGTGCTGCCAGCTGAAGTTTTGGCTGCCTATCGGCTGTGCC

GCCTTCGTGGTTGTGTGTATCCTGGGCTGCATCCTGATCTGCTGG

CTGACCAAGAAAAAGTACAGCAGCTCCGTGCACGACCCCAACGGC

GAGTACATGTTCATGAGAGCCGTGAACACCGCCAAGAAGTCCAGA

CTGACCGACGTGACACTTGGCGGCGGAAGCTTTAGAACCCCTATC

CAAGAGGAACAGGCCGACGCTCACTCTACACTGGCT

HLA-A2 Signal Peptide_PD1 Extracellular_ICOS

Transmembrane IC OS Signaling Domain

(mini-CD28)_Truncated 4-1BB Signaling

(PD1_ICOS(28)_BBt)

(SEQ ID NO: 177)

ATGGCTGTGATGGCTCCTAGAACACTGGTGCTGCTGCTGTCTGGT

GCCCTGGCTCTGACTCAGACATGGGCCTTTCTGGACAGCCCCGAC

AGACCCTGGAATCCTCCTACATTCAGCCCCGCTCTGCTGGTGGTT

ACCGAGGGCGATAATGCCACCTTCACCTGTAGCTTCAGCAACACC

AGCGAGAGCTTCGTGCTGAACTGGTACAGAATGAGCCCCAGCAAC

CAGACCGACAAGCTGGCCGCCTTTCCTGAGGATAGATCTCAGCCC

GGCCAGGACTGCCGGTTCAGAGTTACACAGCTGCCCAACGGCCGG

GACTTCCACATGTCTGTCGTTCGGGCCAGAAGAAACGACAGCGGC

ACATATCTGTGCGGCGCCATTTCTCTGGCCCCTAAGGCTCAGATC

AAAGAGAGCCTGAGAGCCGAGCTGAGAGTGACAGAAAGACGGGCC

GAAGTGCCCACAGCTCACCCTTCACCTTCTCCAAGACCTGCCAGC

CAGCTGTGCTGCCAGCTGAAGTTTTGGCTGCCTATCGGCTGTGCC

GCCTTCGTGGTTGTGTGTATCCTGGGCTGCATCCTGATCTGCTGG

CTGACCAAGAAAAAGTACAGCAGCTCCGTGCACGACCCCAACGGC

GAGTACATGTTCATGACCCCTAGAAGGCCTGGACCTACCAGAAAG

CACTACCAGCCTTACGCTCCTCCTAGAGCCGTGAACACCGCCAAG

AAGTCCAGACTGACCGACGTGACACTTCAGCCTTTCATGAGGCCC

GTGCAGACCACACAAGAAGAGGACGGCTGCTCCTGCAGATTCCCC

GAGGAAGAGGAAGGCGGTTGCGAACTT

HLA-A2 Signal Peptide_PD1 Extracellular_ICOS

Transmembrane_ICOS Signaling Domain

(mini-CD28)_Truncated OX-40 Signaling Domain

(PD1 (HLASP-ICOS DS-CD28(PRRP)-

mutated CD134) (PD1_ICOS_OX40t, PD1:ICOS40t)

(SEQ ID NO: 178)

ATGGCTGTGATGGCTCCTAGAACACTGGTGCTGCTGCTGTCTGGT

GCCCTGGCTCTGACTCAGACATGGGCCTTTCTGGACAGCCCCGAC

AGACCCTGGAATCCTCCTACATTCAGCCCCGCTCTGCTGGTGGTT

ACCGAGGGCGATAATGCCACCTTCACCTGTAGCTTCAGCAACACC

AGCGAGAGCTTCGTGCTGAACTGGTACAGAATGAGCCCCAGCAAC

CAGACCGACAAGCTGGCCGCCTTTCCTGAGGATAGATCTCAGCCC

GGCCAGGACTGCCGGTTCAGAGTTACACAGCTGCCCAACGGCCGG

GACTTCCACATGTCTGTCGTTCGGGCCAGAAGAAACGACAGCGGC

ACATATCTGTGCGGCGCCATTTCTCTGGCCCCTAAGGCTCAGATC

AAAGAGAGCCTGAGAGCCGAGCTGAGAGTGACAGAAAGACGGGCC

GAAGTGCCCACAGCTCACCCTTCACCTTCTCCAAGACCTGCCAGC

CAGCTGTGCTGCCAGCTGAAGTTTTGGCTGCCTATCGGCTGTGCC

GCCTTCGTGGTTGTGTGTATCCTGGGCTGCATCCTGATCTGCTGG

CTGACCAAGAAAAAGTACAGCAGCTCCGTGCACGACCCCAACGGC

GAGTACATGTTCATGACCCCTAGAAGGCCTGGACCTACCAGAAAG

CACTACCAGCCTTACGCTCCTCCTAGAGCCGTGAACACCGCCAAG

AAGTCCAGACTGACCGACGTGACACTTGGCGGCGGAAGCTTTAGA

ACCCCTATCCAAGAGGAACAGGCCGACGCTCACTCTACACTGGCT

11-D5-3 scFv (CD8a Signal Peptide_11-D5-3

scFv, mouse_CD8a Hinge_BBZ)

(SEQ ID NO: 179)

ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTT

CTGCATGCCGCCAGACCTGACATCGTGCTGACACAGTCTCCACCT

AGCCTGGCCATGAGCCTGGGAAAGAGAGCCACCATCAGCTGTAGA

GCCAGCGAGAGCGTGACAATCCTGGGCTCTCACCTGATCCACTGG

TATCAGCAGAAGCCCGGCCAGCCTCCTACACTGCTGATTCAGCTG

GCCTCCAATGTGCAGACAGGCGTGCCAGCCAGATTTTCTGGCAGC

GGCAGCAGAACCGACTTCACCCTGACAATCGACCCCGTGGAAGAG

GACGATGTGGCCGTGTACTACTGCCTCCAGAGCCGGACAATCCCC

AGAACATTTGGCGGAGGCACCAAGCTGGAAATCAAGGGCAGCACA

AGCGGCTCTGGCAAGCCTGGATCTGGCGAGGGATCTACCAAGGGA

CAGATCCAGCTGGTGCAGTCTGGCCCCGAGCTGAAGAAACCTGGC

GAGACAGTGAAGATCAGCTGCAAGGCCAGCGGCTACACCTTCACC

GACTACAGCATCAACTGGGTCAAGAGAGCCCCTGGCAAGGGCCTG

AAATGGATGGGCTGGATCAACACCGAAACCAGAGAGCCCGCCTAC

GCCTACGACTTCAGAGGCAGATTCGCCTTCAGCCTGGAAACCAGC

GCCAGCACAGCCTACCTCCAGATCAACAACCTGAAGTACGAGGAC

ACCGCCACCTACTTTTGCGCCCTGGATTACAGCTACGCCATGGAC

TATTGGGGCCAGGGCACAAGCGTGACCGTGTCCTCTACAACAACC

CCTGCTCCTCGGCCTCCAACACCAGCTCCTACAATTGCCTCTCAG

CCCCTGTCTCTGAGGCCCGAAGCTTGTAGACCTGCTGCTGGCGGA

GCCGTGCATACAAGAGGACTGGATTTCGCCTGCGACATCTACATC

TGGGCCCCTCTGGCTGGAACATGTGGCGTGCTGCTGCTGAGCCTG

GTCATCACCCTGTACTGCAAGCGGGGCAGAAAGAAGCTGCTGTAC

ATCTTCAAGCAGCCCTTCATGCGGCCCGTGCAGACCACACAAGAG

GAAGATGGCTGCTCCTGTCGGTTCCCTGAGGAAGAAGAAGGCGGC

TGCGAGCTGAGAGTGAAGTTCAGCAGATCCGCCGACGCTCCTGCC

TATCAGCAGGGACAGAATCAGCTCTACAACGAGCTGAACCTGGGG

CGCAGAGAAGAGTACGACGTGCTGGACAAGAGAAGAGGCAGGGAC

CCTGAGATGGGCGGAAAGCCCCAGAGAAGAAAGAACCCTCAAGAG

GGCCTGTACAATGAGCTGCAAAAGGACAAGATGGCCGAGGCCTAC

AGCGAGATCGGAATGAAGGGCGAACGCAGAAGAGGAAAGGGCCAC

GACGGACTGTATCAGGGCCTGAGCACAGCCACCAAGGACACCTAT

GATGCCCTGCACATGCAGGCCCTGCCTCCAAGA

FHVH33 HVV (CD8a Signal Peptide_FHVH33 HVV_

CD8a Hinge_BBZ)

(SEQ ID NO: 180)

ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTT

CTGCATGCCGCTAGACCTGAGGTGCAGCTGCTTGAATCTGGCGGA

GGACTGGTTCAGCCTGGCGGATCTCTGAGACTGTCTTGTGCCGCC

AGCGGCTTCACCTTTAGCAGCTACGCCATGAGCTGGGTCCGACAG

GCTCCTGGCAAAGGCCTTGAATGGGTGTCCAGCATCAGCGGCAGC

GGCGACTACATCTACTACGCCGATAGCGTGAAGGGCAGATTCACC

ATCAGCCGGGACATCAGCAAGAACACCCTGTACCTCCAGATGAAC

AGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCAAAGAA

GGCACCGGCGCCAATAGCAGCCTGGCCGATTATAGAGGCCAGGGC

ACACTGGTCACCGTGTCCAGTTTCGTGCCTGTGTTCCTGCCTGCC

AAGCCTACCACAACACCCGCTCCTAGACCTCCAACACCAGCTCCA

ACAATCGCCAGCCAGCCTCTGTCTCTGAGGCCAGAAGCTTGTAGA

CCTGCTGCTGGCGGAGCCGTGCATACAAGAGGACTGGATTTCGCC

TGCGACATCTACATCTGGGCCCCTCTGGCTGGAACATGTGGCGTG

CTGCTGCTGAGCCTGGTCATCACACTGTACTGCAACCACCGGAAC

AAGCGGGGCAGAAAGAAGCTGCTGTACATCTTTAAGCAGCCCTTC

ATGCGGCCCGTGCAGACCACACAAGAGGAAGATGGCTGCTCCTGT

CGGTTCCCCGAGGAAGAAGAAGGCGGCTGCGAGCTGAGAGTGAAG

TTCAGCAGATCCGCAGACGCCCCTGCCTATCAGCAGGGACAGAAC

CAGCTGTACAACGAGCTGAACCTGGGGAGAAGAGAAGAGTACGAC

GTGCTGGACAAGCGGAGAGGCAGAGATCCTGAGATGGGCGGAAAG

CCCCAGCGGAGAAAGAATCCTCAAGAGGGCCTGTATAATGAGCTG

CAAAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAG

GGCGAGCGCAGAAGAGGCAAGGGACACGATGGACTGTACCAGGGC

CTGAGCACCGCCACCAAGGATACCTATGATGCCCTGCACATGCAG

GCCCTGCCTCCAAGA

BCAR003 HVV (CD8a Signal Peptide_BCAR003 HVV_

CD8a Hinge_BBZ)

(SEQ ID NO: 181)

ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTT

CTGCATGCCGCTAGACCCCAAGTGAAGCTGGAAGAGTCTGGCGGA

GGACTGGTGCAGGCTGGCAGATCTCTGAGACTGTCTTGTGCCGCC

AGCGAGCACACCTTTAGCTCTCACGTGATGGGCTGGTTCAGACAG

GCCCCTGGCAAAGAAAGGGAAAGCGTGGCCGTTATCGGCTGGCGG

GATATCAGCACAAGCTACGCCGATAGCGTGAAGGGCAGATTCACC

ATCAGCCGGGACAACGCCAAGAAAACCCTGTACCTCCAGATGAAC

AGCCTGAAGCCTGAGGACACCGCCGTGTACTACTGCGCCGCCAGA

AGAATTGACGCCGCCGACTTTGATTCTTGGGGCCAGGGAACCCAA

GTGACCGTTTCTAGCGGAGGCGGTGGAAGTGGCGGCGGTGGATCA

GGTGGTGGTGGATCTGGTGGCGGAGGAAGCGGCGGAGGCGGATCT

GCTGTTCAGCTTGTTGAATCAGGTGGCGGCCTGGTTCAGGCCGGG

GATTCTCTTAGACTGACCTGCACAGCCAGCGGCAGAGCCTTCAGC

ACCTACTTCATGGCCTGGTTTCGGCAGGCTCCCGGAAAAGAACGG

GAATTTGTGGCCGGAATCGCTTGGAGCGGAGGCTCTACAGCCTAT

GCCGATTCCGTGAAAGGCCGGTTTACCATCAGCAGAGATAATGCC

AAAAACACGGTGTACCTGCAAATGAACTCTCTGAAGTCCGAGGAT

ACGGCCGTCTATTACTGTGCCAGCAGAGGCATCGAGGTGGAAGAG

TTTGGAGCCTGGGGACAGGGCACACAAGTCACAGTGTCTAGCACC

AGCACCACCACACCAGCTCCTAGACCTCCAACTCCTGCTCCTACA

ATCGCCAGCCAGCCTCTGTCTCTGAGGCCAGAAGCTTGTAGACCT

GCTGCTGGCGGAGCCGTGCATACAAGAGGACTGGATTTCGCCTGC

GACATCTACATCTGGGCCCCTCTGGCTGGAACATGTGGCGTGCTG

CTGCTGAGCCTGGTCATCACCCTGTACTGCAAGCGGGGCAGAAAG

AAGCTGCTGTACATCTTCAAGCAGCCCTTCATGCGGCCCGTGCAG

ACCACACAAGAGGAAGATGGCTGCTCCTGTCGGTTCCCCGAGGAA

GAAGAAGGCGGCTGCGAGCTGAGAGTGAAGTTCAGCAGAAGTGCC

GACGCTCCCGCCTATCAGCAGGGACAGAACCAGCTGTACAACGAG

CTGAACCTGGGGAGAAGAGAAGAGTACGACGTGCTGGACAAGCGG

AGAGGCAGAGATCCTGAGATGGGCGGAAAGCCCCAGCGGAGAAAG

AATCCTCAAGAGGGCCTGTATAATGAGCTGCAAAAGGACAAGATG

GCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGCAGAAGA

GGCAAGGGACACGATGGACTGTATCAGGGCCTGAGCACCGCCACC

AAGGATACCTATGATGCCCTGCACATGCAGGCCCTGCCTCCAAGA

GSI5022 HVV (CD8a Signal Peptide_G515022 HVV_

CD8a Hinge_BBZ) (SEQ ID NO: 182)

ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTT

CTGCATGCCGCTAGACCCCAAGTGAAGCTGGAAGAGTCTGGCGGA

GGACTGGTGCAGGCTGGCAGATCTCTGAGACTGTCTTGTGCCGCC

AGCGAGCACACCTTTAGCTCTCACGTGATGGGCTGGTTCAGACAG

GCCCCTGGCAAAGAAAGGGAAAGCGTGGCCGTTATCGGCTGGCGG

GATATCAGCACAAGCTACGCCGATAGCGTGAAGGGCAGATTCACC

ATCAGCCGGGACAACGCCAAGAAAACCCTGTACCTCCAGATGAAC

AGCCTGAAGCCTGAGGACACCGCCGTGTACTACTGCGCCGCCAGA

AGAATTGACGCCGCCGACTTTGATTCTTGGGGCCAGGGAACCCAA

GTGACCGTTTCTAGCGGAGGCGGTGGAAGTGGCGGCGGTGGATCA

GGTGGTGGTGGATCTGAAGTGCAGCTGGTGGAATCAGGCGGCGGT

CTTGTTCAAGCCGGTGGTTCACTGAGACTGAGCTGTGCCGCTTCC

GGCAGAACCTTTACCATGGGATGGTTTAGGCAGGCTCCAGGGAAA

GAACGCGAGTTCGTGGCCGCCATTTCTCTGTCTCCAACACTGGCC

TACTACGCCGAGTCCGTGAAAGGCCGGTTCACAATCTCCAGAGAT

AATGCCAAGAACACCGTGGTGCTGCAAATGAACTCCCTGAAGCCA

GAAGATACAGCCCTGTATTACTGTGCCGCCGACCGGAAGTCCGTG

ATGAGCATCAGACCTGACTACTGGGGACAGGGCACACAAGTCACA

GTGTCCAGCACCAGCACCACAACACCCGCTCCTAGACCTCCAACA

CCAGCTCCAACAATCGCCAGCCAGCCTCTGTCTCTGAGGCCAGAA

GCTTGTAGACCTGCTGCTGGCGGAGCCGTGCATACAAGAGGACTG

GATTTCGCCTGCGACATCTACATCTGGGCCCCTCTGGCTGGAACA

TGTGGCGTGCTGCTGCTGAGCCTGGTCATCACCCTGTACTGCAAG

CGGGGCAGAAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATG

CGGCCCGTGCAGACCACACAAGAGGAAGATGGCTGCTCCTGTCGG

TTCCCCGAGGAAGAAGAAGGCGGCTGCGAGCTGAGAGTGAAGTTC

AGCAGATCCGCCGACGCTCCTGCCTATCAGCAGGGACAGAACCAG

CTGTACAACGAGCTGAACCTGGGGAGAAGAGAAGAGTACGACGTG

CTGGATAAGCGGAGAGGCAGAGATCCTGAGATGGGCGGAAAGCCC

CAGCGGAGAAAGAATCCTCAAGAGGGCCTGTATAATGAGCTGCAA

AAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGC

GAGCGCAGAAGAGGCAAGGGACACGATGGACTGTATCAGGGCCTG

AGCACCGCCACCAAGGATACCTATGATGCCCTGCACATGCAGGCC

CTGCCTCCAAGA

BCMAsdAb1 HHV (CD8a Signal Peptide_

anti-BCMAsdAb1 HHV_CD8a Hinge)

(SEQ ID NO: 183)

ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTT

CTGCATGCCGCTAGACCTGAAGTGCAGTTGCAGGCTTCTGGCGGA

GGACTTGCTCAACCTGGCGGAAGCCTGAGACTGTCTTGTGCCGCC

TCTGGCAGAACCTTCAGCACCTACTTCATGGCCTGGTTCAGACAG

CCTCCTGGCAAAGGCCTGGAATACGTTGGCGGAATCCGTTGGAGT

GATGGCGTGCCACACTACGCCGATAGCGTGAAGGGCAGATTCACC

ATCAGCCGGGACAACGCCAAGAACACCGTGTACCTCCAGATGAAC

AGCCTGAGAGCCGAGGATACCGCCGTGTACTTCTGTGCCAGCAGA

GGAATCGCCGACGGCAGCGATTTTGGCTCTTATGGCCAGGGCACC

CAAGTGACCGTGTCCAGCACAACAACCCCTGCTCCTAGACCTCCT

ACACCAGCTCCTACAATCGCCAGCCAGCCTCTGTCTCTGAGGCCA

GAGGCTTGTAGACCTGCTGCTGGCGGAGCCGTGCATACAAGAGGA

CTGGATTTCGCCTGC

BCMAsdAb1 HHV CD8a Transmembrane_4-1BB

Signaling_CD3Z (BCMAsdAb1 HHV_

BB_z) (SEQ ID NO: 184)

ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTT

CTGCATGCCGCTAGACCTGAAGTGCAGTTGCAGGCTTCTGGCGGA

GGACTTGCTCAACCTGGCGGAAGCCTGAGACTGTCTTGTGCCGCC

TCTGGCAGAACCTTCAGCACCTACTTCATGGCCTGGTTCAGACAG

CCTCCTGGCAAAGGCCTGGAATACGTTGGCGGAATCCGTTGGAGT

GATGGCGTGCCACACTACGCCGATAGCGTGAAGGGCAGATTCACC

ATCAGCCGGGACAACGCCAAGAACACCGTGTACCTCCAGATGAAC

AGCCTGAGAGCCGAGGATACCGCCGTGTACTTCTGTGCCAGCAGA

GGAATCGCCGACGGCAGCGATTTTGGCTCTTATGGCCAGGGCACC

CAAGTGACCGTGTCCAGCACAACAACCCCTGCTCCTAGACCTCCT

ACACCAGCTCCTACAATCGCCAGCCAGCCTCTGTCTCTGAGGCCA

GAGGCTTGTAGACCTGCTGCTGGCGGAGCCGTGCATACAAGAGGA

CTGGATTTCGCCTGCGACATCTACATCTGGGCCCCTCTGGCTGGA

ACATGTGGCGTGCTGCTGCTGAGCCTGGTCATCACCCTGTATTGC

AAGCGGGGCAGAAAGAAACTGCTCTACATCTTCAAGCAGCCCTTC

ATGCGGCCCGTGCAGACCACACAAGAGGAAGATGGCTGCTCCTGT

CGGTTCCCCGAGGAAGAAGAAGGCGGCTGCGAGCTGAGAGTGAAG

TTCAGCAGATCCGCCGACGCTCCTGCCTATCAGCAGGGCCAAAAC

CAGCTGTACAACGAGCTGAACCTGGGGAGAAGAGAAGAGTACGAC

GTGCTGGACAAGCGGAGAGGCAGAGATCCTGAAATGGGCGGCAAG

CCCCAGCGGAGAAAGAATCCTCAAGAGGGCCTGTATAATGAGCTG

CAAAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAG

GGCGAGCGCAGAAGAGGCAAGGGACACGATGGACTGTACCAGGGC

CTGAGCACCGCCACCAAGGATACCTATGATGCCCTGCACATGCAG

GCCCTGCCTCCAAGA

BCMAsdAb1 HHV CD28 Transmembrane CD28

Signaling_CD3Z (BCMAsdAb1 HHV_

28_Z) (SEQ ID NO: 185)

ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTT

CTGCATGCCGCTAGACCTGAAGTGCAGTTGCAGGCTTCTGGCGGA

GGACTTGCTCAACCTGGCGGAAGCCTGAGACTGTCTTGTGCCGCC

TCTGGCAGAACCTTCAGCACCTACTTCATGGCCTGGTTCAGACAG

CCTCCTGGCAAAGGCCTGGAATACGTTGGCGGAATCCGTTGGAGT

GATGGCGTGCCACACTACGCCGATAGCGTGAAGGGCAGATTCACC

ATCAGCCGGGACAACGCCAAGAACACCGTGTACCTCCAGATGAAC

AGCCTGAGAGCCGAGGATACCGCCGTGTACTTCTGTGCCAGCAGA

GGAATCGCCGACGGCAGCGATTTTGGCTCTTATGGCCAGGGCACC

CAAGTGACCGTGTCCAGCACAACAACCCCTGCTCCTAGACCTCCT

ACACCAGCTCCTACAATCGCCAGCCAGCCTCTGTCTCTGAGGCCA

GAGGCTTGTAGACCTGCTGCTGGCGGAGCCGTGCATACAAGAGGA

CTGGATTTCGCCTGCCTGTTTCCCGGACCTAGCAAGCCTTTCTGG

GTGCTCGTTGTTGTTGGCGGCGTGCTGGCCTGTTACAGCCTGCTG

GTTACCGTGGCCTTCATCATCTTTTGGGTCCGAAGCAAGCGGAGC

CGGCTGCTGCACAGCGACTACATGAACATGACCCCTAGACGGCCC

GGACCAACCAGAAAGCACTACCAGCCTTACGCTCCTCCTAGAGAC

TTCGCCGCCTACAGATCTAGAGTGAAGTTCAGCAGATCCGCCGAC

GCTCCTGCCTATCAGCAGGGCCAAAACCAGCTGTACAACGAGCTG

AACCTGGGGAGAAGAGAAGAGTACGACGTGCTGGACAAGCGGAGA

GGCAGAGATCCTGAAATGGGCGGCAAGCCCCAGCGGAGAAAGAAT

CCTCAAGAGGGCCTGTATAATGAGCTGCAAAAGGACAAGATGGCC

GAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGCAGAAGAGGC

AAGGGACACGATGGACTGTACCAGGGCCTGAGCACCGCCACCAAG

GATACCTATGATGCCCTGCACATGCAGGCCCTGCCTCCAAGA

BCMAsdAb1 HHV_CD28 Transmembrane_CD28

Signaling_4-1BB Signaling_CD3Z

(BCMAsdAb1 HHV_28_BBwt_z) (SEQ ID NO: 186)

ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTT

CTGCATGCCGCTAGACCTGAAGTGCAGTTGCAGGCTTCTGGCGGA

GGACTTGCTCAACCTGGCGGAAGCCTGAGACTGTCTTGTGCCGCC

TCTGGCAGAACCTTCAGCACCTACTTCATGGCCTGGTTCAGACAG

CCTCCTGGCAAAGGCCTGGAATACGTTGGCGGAATCCGTTGGAGT

GATGGCGTGCCACACTACGCCGATAGCGTGAAGGGCAGATTCACC

ATCAGCCGGGACAACGCCAAGAACACCGTGTACCTCCAGATGAAC

AGCCTGAGAGCCGAGGATACCGCCGTGTACTTCTGTGCCAGCAGA

GGAATCGCCGACGGCAGCGATTTTGGCTCTTATGGCCAGGGCACC

CAAGTGACCGTGTCCAGCACAACAACCCCTGCTCCTAGACCTCCT

ACACCAGCTCCTACAATCGCCAGCCAGCCTCTGTCTCTGAGGCCA

GAGGCTTGTAGACCTGCTGCTGGCGGAGCCGTGCATACAAGAGGA

CTGGATTTCGCCTGCCTGTTTCCCGGACCTAGCAAGCCTTTCTGG

GTGCTCGTTGTTGTTGGCGGCGTGCTGGCCTGTTACAGCCTGCTG

GTTACCGTGGCCTTCATCATCTTTTGGGTCCGAAGCAAGCGGAGC

CGGCTGCTGCACAGCGACTACATGAACATGACCCCTAGACGGCCC

GGACCAACCAGAAAGCACTACCAGCCTTACGCTCCTCCTAGAGAC

TTCGCCGCCTACAGATCCAAGCGGGGCAGAAAGAAGCTGCTGTAC

ATCTTCAAGCAGCCCTTCATGCGGCCCGTGCAGACCACACAAGAG

GAAGATGGCTGCTCCTGTCGGTTCCCCGAGGAAGAAGAAGGCGGT

TGCGAACTGAGAGTGAAGTTCAGCAGATCCGCCGACGCTCCTGCC

TATCAGCAGGGCCAAAACCAGCTGTACAACGAGCTGAACCTGGGG

AGAAGAGAAGAGTACGACGTGCTGGACAAGCGGAGAGGCAGAGAT

CCTGAAATGGGCGGCAAGCCCCAGCGGAGAAAGAATCCTCAAGAG

GGCCTGTATAATGAGCTGCAAAAGGACAAGATGGCCGAGGCCTAC

AGCGAGATCGGAATGAAGGGCGAGCGCAGAAGAGGCAAGGGACAC

GATGGACTGTACCAGGGCCTGAGCACCGCCACCAAGGATACCTAT

GATGCCCTGCACATGCAGGCCCTGCCTCCAAGA

BCMAsdAb1 HHV_CD28 Transmembrane CD28

Signaling 4-1BB truncated

Signaling_CD3Z BCMAsdAb1 HHV_28_BBt_z

(SEQ ID NO: 187)

ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTT

CTGCATGCCGCTAGACCTGAAGTGCAGTTGCAGGCTTCTGGCGGA

GGACTTGCTCAACCTGGCGGAAGCCTGAGACTGTCTTGTGCCGCC

TCTGGCAGAACCTTCAGCACCTACTTCATGGCCTGGTTCAGACAG

CCTCCTGGCAAAGGCCTGGAATACGTTGGCGGAATCCGTTGGAGT

GATGGCGTGCCACACTACGCCGATAGCGTGAAGGGCAGATTCACC

ATCAGCCGGGACAACGCCAAGAACACCGTGTACCTCCAGATGAAC

AGCCTGAGAGCCGAGGATACCGCCGTGTACTTCTGTGCCAGCAGA

GGAATCGCCGACGGCAGCGATTTTGGCTCTTATGGCCAGGGCACC

CAAGTGACCGTGTCCAGCACAACAACCCCTGCTCCTAGACCTCCT

ACACCAGCTCCTACAATCGCCAGCCAGCCTCTGTCTCTGAGGCCA

GAGGCTTGTAGACCTGCTGCTGGCGGAGCCGTGCATACAAGAGGA

CTGGATTTCGCCTGCCTGTTTCCCGGACCTAGCAAGCCTTTCTGG

GTGCTCGTTGTTGTTGGCGGCGTGCTGGCCTGTTACAGCCTGCTG

GTTACCGTGGCCTTCATCATCTTTTGGGTCCGAAGCAAGCGGAGC

CGGCTGCTGCACAGCGACTACATGAACATGACCCCTAGACGGCCC

GGACCAACCAGAAAGCACTACCAGCCTTACGCTCCTCCTAGAGAC

TTCGCCGCCTACAGATCCCAGCCTTTCATGAGGCCTGTGCAGACC

ACACAAGAAGAGGACGGCTGCTCCTGTCGGTTCCCCGAGGAAGAG

GAAGGCGGTTGCGAACTTAGAGTGAAGTTCAGCAGATCCGCCGAC

GCTCCTGCCTATCAGCAGGGCCAAAACCAGCTGTACAACGAGCTG

AACCTGGGGAGAAGAGAAGAGTACGACGTGCTGGACAAGCGGAGA

GGCAGAGATCCTGAAATGGGCGGCAAGCCCCAGCGGAGAAAGAAT

CCTCAAGAGGGCCTGTATAATGAGCTGCAAAAGGACAAGATGGCC

GAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGCAGAAGAGGC

AAGGGACACGATGGACTGTACCAGGGCCTGAGCACCGCCACCAAG

GATACCTATGATGCCCTGCACATGCAGGCCCTGCCTCCAAGA

BCMAsdAb1 HHV CD28 Transmembrane CD28

Signaling_OX-40 Truncated

Signaling_CD3Z (BCMAsdAb1 HHV_28_OX40t_z)

(SEQ ID NO: 188)

ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTT

CTGCATGCCGCTAGACCTGAAGTGCAGTTGCAGGCTTCTGGCGGA

GGACTTGCTCAACCTGGCGGAAGCCTGAGACTGTCTTGTGCCGCC

TCTGGCAGAACCTTCAGCACCTACTTCATGGCCTGGTTCAGACAG

CCTCCTGGCAAAGGCCTGGAATACGTTGGCGGAATCCGTTGGAGT

GATGGCGTGCCACACTACGCCGATAGCGTGAAGGGCAGATTCACC

ATCAGCCGGGACAACGCCAAGAACACCGTGTACCTCCAGATGAAC

AGCCTGAGAGCCGAGGATACCGCCGTGTACTTCTGTGCCAGCAGA

GGAATCGCCGACGGCAGCGATTTTGGCTCTTATGGCCAGGGCACC

CAAGTGACCGTGTCCAGCACAACAACCCCTGCTCCTAGACCTCCT

ACACCAGCTCCTACAATCGCCAGCCAGCCTCTGTCTCTGAGGCCA

GAGGCTTGTAGACCTGCTGCTGGCGGAGCCGTGCATACAAGAGGA

CTGGATTTCGCCTGCCTGTTTCCCGGACCTAGCAAGCCTTTCTGG

GTGCTCGTTGTTGTTGGCGGCGTGCTGGCCTGTTACAGCCTGCTG

GTTACCGTGGCCTTCATCATCTTTTGGGTCCGAAGCAAGCGGAGC

CGGCTGCTGCACAGCGACTACATGAACATGACCCCTAGACGGCCC

GGACCAACCAGAAAGCACTACCAGCCTTACGCTCCTCCTAGAGAC

TTCGCCGCCTACAGATCTGGCGGCGGAAGCTTCAGAACCCCTATC

CAAGAGGAACAGGCCGACGCTCACTCTACACTGGCTAGAGTGAAG

TTCAGCAGATCCGCCGACGCTCCTGCCTATCAGCAGGGCCAAAAC

CAGCTGTACAACGAGCTGAACCTGGGGAGAAGAGAAGAGTACGAC

GTGCTGGACAAGCGGAGAGGCAGAGATCCTGAAATGGGCGGCAAG

CCCCAGCGGAGAAAGAATCCTCAAGAGGGCCTGTATAATGAGCTG

CAAAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAG

GGCGAGCGCAGAAGAGGCAAGGGACACGATGGACTGTACCAGGGC

CTGAGCACCGCCACCAAGGATACCTATGATGCCCTGCACATGCAG

GCCCTGCCTCCAAGA

BCMAsdAb1 HHV_ICOS Transmembrane_ICOS

Signaling_CD3Z (BCMAsdAb1 HHV_

ICOS_Z) (SEQ ID NO: 189)

ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTT

CTGCATGCCGCTAGACCTGAAGTGCAGTTGCAGGCTTCTGGCGGA

GGACTTGCTCAACCTGGCGGAAGCCTGAGACTGTCTTGTGCCGCC

TCTGGCAGAACCTTCAGCACCTACTTCATGGCCTGGTTCAGACAG

CCTCCTGGCAAAGGCCTGGAATACGTTGGCGGAATCCGTTGGAGT

GATGGCGTGCCACACTACGCCGATAGCGTGAAGGGCAGATTCACC

ATCAGCCGGGACAACGCCAAGAACACCGTGTACCTCCAGATGAAC

AGCCTGAGAGCCGAGGATACCGCCGTGTACTTCTGTGCCAGCAGA

GGAATCGCCGACGGCAGCGATTTTGGCTCTTATGGCCAGGGCACC

CAAGTGACCGTGTCCAGCACAACAACCCCTGCTCCTAGACCTCCT

ACACCAGCTCCTACAATCGCCAGCCAGCCTCTGTCTCTGAGGCCA

GAGGCTTGTAGACCTGCTGCTGGCGGAGCCGTGCATACAAGAGGA

CTGGATTTCGCCTGCAGCCAGCTGTGCTGCCAGCTGAAGTTCTGG

CTGCCTATTGGCTGCGCCGCCTTCGTGGTTGTGTGTATCCTGGGC

TGCATCCTGATCTGCTGGCTGACCAAGAAAAAGTACAGCAGCAGC

GTGCACGACCCCAACGGCGAGTACATGTTCATGAGAGCCGTGAAC

ACCGCCAAGAAGTCCAGACTGACCGACGTGACACTGAGAGTGAAG

TTCAGCAGATCCGCCGACGCTCCTGCCTATCAGCAGGGCCAAAAC

CAGCTGTACAACGAGCTGAACCTGGGGAGAAGAGAAGAGTACGAC

GTGCTGGACAAGCGGAGAGGCAGAGATCCTGAAATGGGCGGCAAG

CCCCAGCGGAGAAAGAATCCTCAAGAGGGCCTGTATAATGAGCTG

CAAAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAG

GGCGAGCGCAGAAGAGGCAAGGGACACGATGGACTGTACCAGGGC

CTGAGCACCGCCACCAAGGATACCTATGATGCCCTGCACATGCAG

GCCCTGCCTCCAAGA

BCMAsdAb1 HHV_ICOS Transmembrane_ICOS

Signaling_4-1BB Signaling_CD3Z

(BCMAsdAb1 HHV_ICOS_BBwt_z)

(SEQ ID NO: 190)

ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTT

CTGCATGCCGCTAGACCTGAAGTGCAGTTGCAGGCTTCTGGCGGA

GGACTTGCTCAACCTGGCGGAAGCCTGAGACTGTCTTGTGCCGCC

TCTGGCAGAACCTTCAGCACCTACTTCATGGCCTGGTTCAGACAG

CCTCCTGGCAAAGGCCTGGAATACGTTGGCGGAATCCGTTGGAGT

GATGGCGTGCCACACTACGCCGATAGCGTGAAGGGCAGATTCACC

ATCAGCCGGGACAACGCCAAGAACACCGTGTACCTCCAGATGAAC

AGCCTGAGAGCCGAGGATACCGCCGTGTACTTCTGTGCCAGCAGA

GGAATCGCCGACGGCAGCGATTTTGGCTCTTATGGCCAGGGCACC

CAAGTGACCGTGTCCAGCACAACAACCCCTGCTCCTAGACCTCCT

ACACCAGCTCCTACAATCGCCAGCCAGCCTCTGTCTCTGAGGCCA

GAGGCTTGTAGACCTGCTGCTGGCGGAGCCGTGCATACAAGAGGA

CTGGATTTCGCCTGCAGCCAGCTGTGCTGCCAGCTGAAGTTCTGG

CTGCCTATTGGCTGCGCCGCCTTCGTGGTTGTGTGTATCCTGGGC

TGCATCCTGATCTGCTGGCTGACCAAGAAAAAGTACAGCAGCAGC

GTGCACGACCCCAACGGCGAGTACATGTTCATGAGAGCCGTGAAC

ACCGCCAAGAAGTCCAGACTGACCGACGTGACCCTGAAGCGGGGC

AGAAAGAAGCTGCTGTATATCTTCAAGCAGCCCTTCATGCGGCCC

GTGCAGACCACACAAGAGGAAGATGGCTGCTCCTGTCGGTTCCCC

GAGGAAGAAGAAGGCGGTTGCGAACTGAGAGTGAAGTTCAGCAGA

TCCGCCGACGCTCCTGCCTATCAGCAGGGCCAAAACCAGCTGTAC

AACGAGCTGAACCTGGGGAGAAGAGAAGAGTACGACGTGCTGGAC

AAGCGGAGAGGCAGAGATCCTGAAATGGGCGGCAAGCCCCAGCGG

AGAAAGAATCCTCAAGAGGGCCTGTATAATGAGCTGCAAAAGGAC

AAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAGGGCGAGCGC

AGAAGAGGCAAGGGACACGATGGACTGTACCAGGGCCTGAGCACC

GCCACCAAGGATACCTATGATGCCCTGCACATGCAGGCCCTGCCT

CCAAGA

BCMAsdAb1_ICOS Transmembrane_ICOS Signaling

4-1BB Truncated Signaling_CD3Z

(BCMAsdAb1 HHV_ICOS_BBt_z) (SEQ ID NO: 191)

ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTT

CTGCATGCCGCTAGACCTGAAGTGCAGTTGCAGGCTTCTGGCGGA

GGACTTGCTCAACCTGGCGGAAGCCTGAGACTGTCTTGTGCCGCC

TCTGGCAGAACCTTCAGCACCTACTTCATGGCCTGGTTCAGACAG

CCTCCTGGCAAAGGCCTGGAATACGTTGGCGGAATCCGTTGGAGT

GATGGCGTGCCACACTACGCCGATAGCGTGAAGGGCAGATTCACC

ATCAGCCGGGACAACGCCAAGAACACCGTGTACCTCCAGATGAAC

AGCCTGAGAGCCGAGGATACCGCCGTGTACTTCTGTGCCAGCAGA

GGAATCGCCGACGGCAGCGATTTTGGCTCTTATGGCCAGGGCACC

CAAGTGACCGTGTCCAGCACAACAACCCCTGCTCCTAGACCTCCT

ACACCAGCTCCTACAATCGCCAGCCAGCCTCTGTCTCTGAGGCCA

GAGGCTTGTAGACCTGCTGCTGGCGGAGCCGTGCATACAAGAGGA

CTGGATTTCGCCTGCAGCCAGCTGTGCTGCCAGCTGAAGTTCTGG

CTGCCTATTGGCTGCGCCGCCTTCGTGGTTGTGTGTATCCTGGGC

TGCATCCTGATCTGCTGGCTGACCAAGAAAAAGTACAGCAGCAGC

GTGCACGACCCCAACGGCGAGTACATGTTCATGAGAGCCGTGAAC

ACCGCCAAGAAGTCCAGACTGACCGACGTGACCCTCCAGCCTTTC

ATGAGGCCTGTGCAGACCACACAAGAAGAGGACGGCTGCTCCTGT

CGGTTCCCCGAGGAAGAGGAAGGCGGTTGCGAACTTAGAGTGAAG

TTCAGCAGATCCGCCGACGCTCCTGCCTATCAGCAGGGCCAAAAC

CAGCTGTACAACGAGCTGAACCTGGGGAGAAGAGAAGAGTACGAC

GTGCTGGACAAGCGGAGAGGCAGAGATCCTGAAATGGGCGGCAAG

CCCCAGCGGAGAAAGAATCCTCAAGAGGGCCTGTATAATGAGCTG

CAAAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGAATGAAG

GGCGAGCGCAGAAGAGGCAAGGGACACGATGGACTGTACCAGGGC

CTGAGCACCGCCACCAAGGATACCTATGATGCCCTGCACATGCAG

GCCCTGCCTCCAAGA

BCMAsdAb1_ICOS Transmembrane_ICOS Signaling

OX-40 Truncated Signaling_CD3Z

(BCMAsdAb1 HHV_ICOS_OX40t_z)

(SEQ ID NO: 192)

ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTT

CTGCATGCCGCTAGACCTGAAGTGCAGTTGCAGGCTTCTGGCGGA

GGACTTGCTCAACCTGGCGGAAGCCTGAGACTGTCTTGTGCCGCC

TCTGGCAGAACCTTCAGCACCTACTTCATGGCCTGGTTCAGACAG

CCTCCTGGCAAAGGCCTGGAATACGTTGGCGGAATCCGTTGGAGT

GATGGCGTGCCACACTACGCCGATAGCGTGAAGGGCAGATTCACC

ATCAGCCGGGACAACGCCAAGAACACCGTGTACCTCCAGATGAAC

AGCCTGAGAGCCGAGGATACCGCCGTGTACTTCTGTGCCAGCAGA

GGAATCGCCGACGGCAGCGATTTTGGCTCTTATGGCCAGGGCACC

CAAGTGACCGTGTCCAGCACAACAACCCCTGCTCCTAGACCTCCT

ACACCAGCTCCTACAATCGCCAGCCAGCCTCTGTCTCTGAGGCCA

GAGGCTTGTAGACCTGCTGCTGGCGGAGCCGTGCATACAAGAGGA

CTGGATTTCGCCTGCAGCCAGCTGTGCTGCCAGCTGAAGTTCTGG

CTGCCTATTGGCTGCGCCGCCTTCGTGGTTGTGTGTATCCTGGGC

TGCATCCTGATCTGCTGGCTGACCAAGAAAAAGTACAGCAGCAGC

GTGCACGACCCCAACGGCGAGTACATGTTCATGAGAGCCGTGAAC

ACCGCCAAGAAGTCCAGACTGACCGACGTGACACTCGGCGGAGGC

AGCTTTAGAACCCCTATCCAAGAGGAACAGGCCGACGCTCACTCT

ACACTGGCTAGAGTGAAGTTCAGCAGATCCGCCGACGCTCCTGCC

TATCAGCAGGGCCAAAACCAGCTGTACAACGAGCTGAACCTGGGG

AGAAGAGAAGAGTACGACGTGCTGGACAAGCGGAGAGGCAGAGAT

CCTGAAATGGGCGGCAAGCCCCAGCGGAGAAAGAATCCTCAAGAG

GGCCTGTATAATGAGCTGCAAAAGGACAAGATGGCCGAGGCCTAC

AGCGAGATCGGAATGAAGGGCGAGCGCAGAAGAGGCAAGGGACAC

GATGGACTGTACCAGGGCCTGAGCACCGCCACCAAGGATACCTAT

GATGCCCTGCACATGCAGGCCCTGCCTCCAAGA

CD28 signaling domain: CD28 transmembrane_CD28

signaling domain (SEQ ID NO: 194)

CTGTTCCCCGGACCTAGCAAGCCCTTTTGGGTGCTCGTTGTTGTT

GGCGGCGTGCTGGCCTGTTATAGCCTGCTGGTTACCGTGGCCTTC

ATCATCTTTTGGGTCCGAAGCAAGCGGAGCCGGCTGCTGCACAGC

GACTACATGAACATGACCCCTAGACGGCCCGGACCAACCAGAAAG

CACTACCAGCCTTACGCTCCTCCTAGAGACTTCGCCGCCTACAGA

TCT

Human CD137/4-1BB (SEQ ID NO: 195)

AAGCGGGGCAGAAAGAAACTGCTGTACATCTTCAAGCAGCCCTTC

ATGCGGCCCGTGCAGACCACACAAGAGGAAGATGGCTGCTCCTGC

AGATTCCCCGAGGAAGAAGAAGGCGGCTGCGAACTT

Human CD134/0X40 (SEQ ID NO: 196)

AGGAGAGACCAGCGTCTGCCACCAGATGCACATAAGCCACCTGGC

GGCGGAAGCTTTAGAACCCCTATCCAAGAGGAACAGGCCGACGCT

CACTCTACACTGGCTAAAATC

truncated/mutated CD137/4-1BB (SEQ ID NO: 197)

CAGCCTTTCATGAGGCCCGTGCAGACCACACAAGAAGAGGACGGC

TGCTCCTGCAGATTCCCCGAGGAAGAGGAAGGCGGTTGCGAACTT

Truncated/mutated CD134/0X40 (SEQ ID NO: 198)

GGCGGCGGAAGCTTTAGAACCCCTATCCAAGAGGAACAGGCCGAC

GCTCACTCTACACTGGCT

In some embodiments, the cell disclosed herein further comprises a sequence encoding an artificial antigen receptor, a therapeutic polypeptide, an immune cell modulatory protein, or a combination thereof. In some embodiments, the artificial antigen receptor comprises a chimeric antigen receptor (CAR). In some embodiments, the artificial antigen receptor comprises a recombinant T cell receptor (rTCR). In some embodiments, the artificial antigen receptor comprises an enhanced affinity TCR. In some embodiments, the artificial antigen receptor binds to a tumor associated antigen (TAA), a pathogen associated protein, or an antigen associated with the disease or disorder is a cancer, an autoimmune disease or disorder, an infectious disease, an inflammatory disease, a renal disease or disorder, a lung disease or disorder, a liver disease or disorder a neurodegenerative disorder or disorder, or a metabolic disorder or disorder.

In some embodiments, the artificial antigen receptor binds to a TAA associated with a solid tumor or a hematologic cancer. In some embodiments, artificial antigen receptor binds to a TAA associated with a cancer selected from any one of leukemia, acute leukemia, acute lymphoblastic leukemia (ALL), acute lymphocytic leukemia, B cell, T cell or FAB ALL, acute myeloid leukemia (AML), acute myelogenous leukemia, chronic myelocytic leukemia (CIVIL), chronic lymphocytic leukemia (CLL), hairy cell leukemia, myelodysplastic syndrome (MDS), a lymphoma, Hodgkin's disease, a malignant lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, multiple myeloma, Kaposi's sarcoma, colorectal carcinoma, pancreatic carcinoma, nasopharyngeal carcinoma, malignant histiocytosis, paraneoplastic syndrome/hypercalcemia of malignancy, solid tumors, bladder cancer, breast cancer, colorectal cancer, endometrial cancer, head cancer, neck cancer, hereditary nonpolyposis cancer, Hodgkin's lymphoma, liver cancer, lung cancer, non-small cell lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, testicular cancer, adenocarcinomas, sarcomas, malignant melanoma, and hemangioma.

In some embodiments, the artificial antigen receptor binds to a TAA selected from kallikrein 4, papillomavirus binding factor (PBF), preferentially expressed antigen of melanoma (PRAME), Wilms' tumor-I (WTI), Hydroxysteroid Dehydrogenase Like I (HSDLI), mesothelin, cancer testis antigen (NY-ESO-1), carcinoembryonic antigen (CEA), p53, human epidermal growth factor receptor 2/neuro receptor tyrosine kinase (Her2/Neu), carcinoma-associated epithelial cell adhesion molecule (EpCAM), ovarian and uterine carcinoma antigen (CAI25), folate receptor a, sperm protein 17, tumor-associated differentially expressed gene-12 (TADG-12), mucin-16 (MUC-16), LI cell adhesion molecule (LICAM), mannan-MUC-1, Human endogenous retrovirus K (HERV-K-MEL), Kita-kyushu lung cancer antigen-I (KK-LC-1), human cancer/testis antigen (KM-HN-1), cancer testis antigen (LAGE-I), melanoma antigen-Al (MAGE-A1), Sperm surface zona pellucida binding protein (Spl 7), Synovial Sarcoma, X Breakpoint 4 (SSX-4), Transient axonal glycoprotein-1 (TAG-I), Transient axonal glycoprotein-2 (TAG-2), Enabled Homolog (ENAH), mammoglobin-A, NY-BR-I, Breast Cancer Antigen, (BAGE-1), B melanoma antigen, melanoma antigen-Al (MAGE-Al), melanoma antigen-A2 (MAGE-A2), mucin k, synovial sarcoma, X breakpoint 2 (SSX-2), Taxol-resistance-associated gene-3 (TRAG-3), Avian Myelocytomatosis Viral Oncogene (c-myc), cyclin B 1, mucin I (MUC I), p62, survivin, lymphocyte common antigen (CD45), DickkopfWNT Signaling Pathway Inhibitor I (DKKI), telomerase, Kirsten rat sarcoma viral oncogene homolog (K-ras), G250, intestinal carboxyl esterase, alpha-fetoprotein, Macrophage Colony-Stimulating Factor (M-CSF), Prostate-specific membrane antigen (PSMA), caspase 5 (CASP-5), Cytochrome C Oxidase Assembly Factor I Homolog (COA-1), 0-linked β-N-acetylglucosamine transferase (OGT), Osteosarcoma Amplified 9, Endoplasmic Reticulum Lectin (OS-9), Transforming Growth Factor Beta Receptor 2 (TGF-betaRII), murine leukemia glycoprotein 70 (gp70), Calcitonin Related Polypeptide Alpha (CALCA), Programmed cell death 1 ligand 1 (CD274), Mouse Double Minute 2Homolog (mdm-2), alpha-actinin-4, elongation factor 2, Malic Enzyme 1 (MEI), Nuclear Transcription Factor Y Subunit C (NFYC), G Antigen 1,3 (GAGE-1,3), melanoma antigen-A6 (MAGE-A6), cancer testis antigen XAGE-lb, six transmembrane epithelial antigen of the prostate 1 (STEAPl), PAP, prostate specific antigen (PSA), Fibroblast Growth Factor 5 (FGF5), heat shock protein hsp70-2, melanoma antigen-A9 (MAGE-A9), Arg-specific ADP-ribosyltransferase family C (ARTCl), B-Raf Proto-Oncogene (B-RAF), Serine/Threonine Kinase, beta-catenin, Cell Division Cycle 27 homolog (Cdc27), cyclin dependent kinase 4 (CDK4), cyclin dependent kinase 12 (CDK12), Cyclin Dependent Kinase Inhibitor 2A (CDKN2A), Casein Kinase 1 Alpha 1 (CSNK1A1), Fibronectin 1 (FNl), Gruwih Anest Specific 7 (GAS7), Glycoprotein nonmetastatic melanoma protein B (GPNMB), HAUS Augmin Like Complex Subunit 3 (HAUS3), LDLR-fucosyltransferase, Melanoma Antigen Recognized By T cells 2 (MART2), myostatin (MSTN), Melanoma Associated Antigen (Mutated) 1 (MUM-1-2-3), Poly(A) polymerase gamma (neo-PAP), myosin class I, Protein phosphatase 1 regulatory subunit 3B (PPP1R3B), Peroxiredoxin-5 (PRDX5), Receptor-type tyrosine-protein phosphatase kappa (PTPRK), Transforming protein N-Ras (N-ras), retinoblastoma-associated factor 600 (RBAF600), sirtuin-2 (SIRT2), SNRPD1, triosephosphate isomerase, Ocular Albinism Type 1 Protein (OAl), member RAS oncogene family (RAB38), Tyrosinase related protein 1-2 (TRP-1-2), Melanoma Antigen Gp75 (gp75), tyrosinase, Melan-A (MART-1), Glycoprotein 100 melanoma antigen (gp100), N-acetylglucosaminyltransferase V gene (GnTVf), Lymphocyte Antigen 6 Complex Locus K (LY6K), melanoma antigen-AlO (MAGE-AlO), melanoma antigen-Al2 (MAGE-Al2), melanoma antigen-C2 (MAGE-C2), melanoma antigen NA88-A, Taxol-resistant-associated protein 3 (TRAG-3), BDZ binding kinase (pbk), caspase 8 (CASP-8), sarcoma antigen 1 (SAGE), Breakpoint Cluster Region-Abelson oncogene (BCR-ABL), fusion protein in leukemia, dek-can, Elongation Factor Tu GTP Binding Domain Containing 2 (EFTUD2), ETS Variant gene 6/acute myeloid leukemia fusion protein (ETV6-AML1), FMS-like tyrosine kinase-3 internal tandem duplications (FLT3-ITD), cyclin-Al, Fibronectin Type III Domain Containing 3B (FDNC3B) promyelocytic leukemia/retinoic acid receptor alpha fusion protein (pml-RARalpha), melanoma antigen-Cl (MAGE-Cl), membrane protein alternative spliced isoform (D393-CD20), melanoma antigen-A4 (MAGE-A4), and melanoma antigen-A3 (MAGE-A3).

In some embodiments, the artificial antigen receptor binds to an antigen associated with an autoimmune condition or disorder selected from any one of Type 1 Diabetes, rheumatoid arthritis (RA), systemic lupus erythematosis (SLE), or multiple sclerosis (MS). In some embodiments, the artificial antigen receptor binds to an antigen associated with an autoimmune condition or disorder selected from any one of Carboxypeptidase H, Chromogranin A, Glutamate decarboxylase, Imogen-38, Insulin, Insulinoma antigen-2 and 2β, Islet-specific glucose-6-phosphatase catalytic subunit related protein (IGRP), Proinsulin, α-enolase, Aquaporin-4, β-arrestin, Myelin basic protein, Myelin oligodendrocytic glycoprotein, Proteolipid protein, S100-β, Citrullinated protein, Collagen II, Heat shock proteins, Human cartilage glycoprotein, Double-stranded DNA, La antigen, Nucleosomal histones and ribonucleoproteins (snRNP), Phospholipid-β-2 glycoprotein I complex, Poly(ADP-ribose) polymerase, Sm antigens of U-1 small ribonucleoprotein complex.

In some embodiments, the artificial antigen receptor binds to a pathogen associated antigen from a bacterial, a fungal or a parasitic protein or fragment thereof. In some embodiments, the artificial antigen receptor binds to an antigen associated with HIV infection, human Cytomegalovirus infection, Hepatitis B infection, Hepatitis C infection, Ebola virus infection, Dengue, Yellow fever, Listeriosis, Tuberculosis, Cholera, Malaria, Leishmaniasis, or Trypanosoma infection, or a combination thereof.

In some embodiments, the artificial antigen receptor binds to an antigen associated with a neurodegenerative disorder or condition selected from Alzheimer's disease (AD) and other dementias, Parkinson's disease (PD) and PD-related disorders, Prion disease, Motor neurone diseases (MND), Huntington's disease (HD), Spinocerebellar ataxia (SCA) or Spinal muscular atrophy (SMA). In some embodiments, the antigen associated with the neurodegenerative disorder or condition is any one of Amyloid β (Aβ), tau, alpha-synuclein (α-syn), mHTT or prion PrPsc or a combination thereof.

In some embodiments, the therapeutic polypeptide is a cytokine, a cytokine receptor, a chemokine, a chemokine receptor, an immunogenic polypeptide, or a cell surface protein that binds to a target on the surface of another cell. In some embodiments, the immune cell modulatory protein is a cytokine, a chemokine, a transcription factor, a protein kinase, a protease, a component or an adaptor protein of a cell signaling pathway.

In some embodiments, the cell disclosed herein expresses the RTCR disclosed herein. In some embodiments, the cell disclosed herein expresses the RTCR disclosed herein stably or transiently. In some embodiments, the cell disclosed herein expresses the RTCR disclosed herein stably. In some embodiments, the cell disclosed herein expresses the RTCR disclosed herein transiently.

In some embodiments, the cell disclosed herein co-expresses at least one of the endogenous co-stimulatory molecules CD28, CD2, OX-40, ICOS, CD28, CD3, CD4, CD8 and CD40L or a combination thereof.

The present disclosure also provides a modified T lymphocyte (T cell), comprising: (a) a modification of an endogenous sequence encoding a T cell Receptor (TCR), wherein the modification reduces or eliminates a level of expression or activity of the TCR or; and (b) a recombinant T cell co-stimulatory receptor (RTCR) disclosed herein. In some embodiments, the modification of an endogenous sequence encoding a T cell Receptor (TCR) is done using a nucleic acid modifying system. In some embodiments, the nucleic acid modifying system is one or more of a CRISPR/Cas protein, a Transcription Activator-Like Effector Nuclease (TALEN), a Zinc Finger Nuclease (ZFN), and an endonuclease. In some embodiments, the modification of an endogenous sequence encoding a T cell Receptor (TCR) is done by nonhomologous end joining repair. In some embodiments, the nonhomologous end joining repair is generated by zinc finger nuclease, introduced into the cell by physical means, viral vector, or non-viral vector. In some embodiments, the nonhomologous end joining repair is generated by TALE nuclease, introduced into the cell by physical means, viral vector, or non-viral vector. In some embodiments, the modification of an endogenous sequence encoding a T cell Receptor (TCR) reduces or eliminates a level of expression of the alpha chain of the TCR. In some embodiments, the modification of an endogenous sequence encoding a T cell Receptor (TCR) reduces or eliminates a level of expression of beta chain of the TCR. In some embodiments, the modification of an endogenous sequence encoding a T cell Receptor (TCR) reduces or eliminates a level of expression of both the alpha chain and the beta chain TCR alpha chain.

In some embodiments, the modified T cell disclosed herein co-expresses at least one of the endogenous co-stimulatory molecules CD28, CD2, OX-40, ICOS, CD28, CD3, CD4, CD8 and CD40L or a combination thereof.

In some embodiments, the method disclosed herein further comprises a modification of an endogenous sequence encoding a component of major histocompatibility complex (MHC) class I (MHC-I), wherein the modification reduces or eliminates a level of expression or activity of the MHC-I. In some embodiments, the modification reduces or eliminates the expression or activity of β2-macroglobulin.

The present disclosure also provides a method of producing a plurality of modified T cells, wherein the method comprises: a) providing a plurality of primary T cells disclosed herein; b) providing a composition comprising the RTCR disclosed herein, the nucleic acid encoding the RTCR disclosed herein, or the vector comprising the nucleic acid encoding the RTCR disclosed herein; and c) introducing into the plurality of primary T cells of (a) the composition of (b), to produce a plurality of modified T cells under conditions that stably express the RTCR within the plurality of modified T cells. In some embodiments, the method of producing a plurality of modified T cells disclosed herein, further comprises a step of modifying an endogenous sequence encoding an endogenous T cell Receptor (TCR), wherein the modification reduces or eliminates a level of expression or activity of the endogenous TCR. In some embodiments, the method of producing a plurality of modified T cells disclosed herein, further comprises a step of modifying an endogenous sequence, wherein the modification reduces or eliminates a level of expression or activity of a major histocompatibility complex (MHC) class I (MHC-I).

In some embodiments, the modifying an endogenous sequence encoding a T cell Receptor (TCR) is done using a nucleic acid modifying system. In some embodiments, the modifying an endogenous sequence that reduces or eliminates a level of expression or activity of is done using a nucleic acid modifying system. In some embodiments, the nucleic acid modifying system is a one or more of a CRISPR/Cas protein, a Transcription Activator-Like Effector Nuclease (TALEN), a Zinc Finger Nuclease (ZFN), and an endonuclease. In some embodiments, the modifying an endogenous sequence is done by nonhomologous end joining repair. In some embodiments, the nonhomologous end joining repair is generated by zinc finger nuclease, introduced into the cell by physical means, viral vector, or non-viral vector. In some embodiments, the nonhomologous end joining repair is generated by TALE nuclease, introduced into the cell by physical means, viral vector, or non-viral vector. In some embodiments, the modifying an endogenous sequence encoding a T cell Receptor (TCR) reduces or eliminates a level of expression of the alpha chain of the TCR. In some embodiments, the modifying an endogenous sequence encoding a T cell Receptor (TCR) reduces or eliminates a level of expression of beta chain of the TCR. In some embodiments, the modifying an endogenous sequence encoding a T cell Receptor (TCR) reduces or eliminates a level of expression of both the alpha chain and the beta chain TCR alpha chain.

In some embodiments, the modifying an endogenous sequence that reduces or eliminates a level of expression or activity of a major histocompatibility complex (MHC) class I (MHC-I), wherein the modifying of an endogenous sequence reduces or eliminates a level of expression or activity of the MHC-I. In some embodiments, the modifying of an endogenous sequence reduces or eliminates the expression or activity of β2-macroglobulin.

In some embodiments, the method of producing a plurality of modified T cells disclosed herein, further comprises: d) maintaining or expanding the plurality of modified T cells in a suitable cell culture media; and e) either: i) cyropreserving the plurality of modified T cells in a suitable cell freezing media; or ii) preparing the plurality of modified T cells for administering to a subject suffering from a disease or disorder.

The compositions comprising the cells or modified T cells of the disclosure, and the plurality of modified T cells produced by the methods of the disclosure, intended for administration to a subject may be required to meet one or more “release criteria” that indicate that the composition is safe and efficacious for formulation as a pharmaceutical product and/or administration to a subject. Release criteria may include a requirement that a composition of the disclosure (e.g., a cell or modified T cell of the disclosure) comprises a particular percentage of cells or modified T cells expressing the RTCR of the disclosure on their cell surface. The expansion process should be continued until a specific criterion has been met (e.g., achieving a certain total number of cells or modified T cells of the disclosure or a certain percentage of total number of cells or modified T cells expressing the RTCR of the disclosure).

Certain criterion signal a point at which the expansion process should end. For example, cells should be formulated, reactivated, or cryopreserved once they reach a cell size of 300fL (otherwise, cells reaching a size above this threshold may start to die). Cryopreservation immediately once a population of cells reaches an average cell size of less than 300 fL may yield better cell recovery upon thawing and culture because the cells haven't yet reached a fully quiescent state prior to cryopreservation (a fully quiescent size is approximately 180 fL). Prior to expansion, T cells of the disclosure may have a cell size of about 180 fL, but may more than quadruple their cell size to approximately 900 fL at 3 days post-expansion. Over the next 6-12 days, the population of T cells will slowly decrease cell size to full quiescence at 180 fL.

A process for preparing a cell population for formulation may include, but is not limited to the steps of, concentrating the cells of the cell population, washing the cells, and/or further selection of the cells via drug resistance or magnetic bead sorting against a particular surface-expressed marker. A process for preparing a cell population for formulation may further include a sorting step to ensure the safety and purity of the final product. For example, if a tumor cell from a patient has been used to stimulate a modified T cell of the disclosure or that have been modified in order to stimulate a modified T cell of the disclosure that is being prepared for formulation, it is critical that no tumor cells from the patient are included in the final product.

In some embodiments, the cell disclosed herein, or the modified T cell disclosed herein, expresses on the cell surface the RTCR comprising a mutant CD137 or a mutant CD134 intracellular signaling domain disclosed herein, at a level that is at least about 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10× or 20×, more as compared to the level of expression of a co-stimulatory molecule comprising a wild type CD137 or a wild type CD134 intracellular domain, respectively.

Pharmaceutical Composition or Formulation

In some embodiments, the compositions disclosed herein, and the population of modified T cells produced using the methods disclosed herein, is in the form of a pharmaceutical formulation (or composition). In some embodiments, the pharmaceutical formulation disclosed herein comprises a pharmaceutically acceptable carrier. A pharmaceutical formulation of the disclosure may be distributed into bags for infusion, cryopreservation, and/or storage.

A pharmaceutical formulation of the disclosure may be cryopreserved using a standard protocol and, optionally, an infusible cryopreservation medium. For example, a DMSO free cryopreservant (e.g. CryoSOfree™ DMSO-free Cryopreservation Medium) may be used to reduce freezing-related toxicity. A cryopreserved pharmaceutical formulation of the disclosure may be stored for infusion to a patient at a later date. An effective treatment may require multiple administrations of a pharmaceutical formulation of the disclosure and, therefore, pharmaceutical formulations may be packaged in pre-aliquoted “doses” that may be stored frozen but separated for thawing of individual doses.

A pharmaceutical formulation of the disclosure may be stored at room temperature. An effective treatment may require multiple administrations of a pharmaceutical formulation of the disclosure and, therefore, pharmaceutical formulations may be packaged in pre-aliquoted “doses” that may be stored together but separated for administration of individual doses.

A pharmaceutical formulation of the disclosure may be archived for subsequent re-expansion and/or selection for generation of additional doses to the same patient in the case of an allogenic therapy who may need an administration at a future date following, for example, a remission and relapse of a condition.

As noted above, the disclosure provides for stable formulations, which preferably comprise a phosphate buffer with saline or a chosen salt, as well as preserved solutions and formulations containing a preservative as well as multi-use preserved formulations suitable for pharmaceutical or veterinary use, comprising at least one modified cell in a pharmaceutically acceptable formulation. Preserved formulations contain at least one known preservative or optionally selected from the group consisting of at least one phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride (e.g., hexahydrate), alkylparaben (methyl, ethyl, propyl, butyl and the like), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and thimerosal, polymers, or mixtures thereof in an aqueous diluent. Any suitable concentration or mixture can be used as known in the art, such as about 0.0015%, or any range, value, or fraction therein. Non-limiting examples include, no preservative, about 0.1-2% m-cresol (e.g., 0.2, 0.3. 0.4, 0.5, 0.9, 1.0%), about 0.1-3% benzyl alcohol (e.g., 0.5, 0.9, 1.1, 1.5, 1.9, 2.0, 2.5%), about 0.001-0.5% thimerosal (e.g., 0.005, 0.01), about 0.001-2.0% phenol (e.g., 0.05, 0.25, 0.28, 0.5, 0.9, 1.0%), 0.0005-1.0% alkylparaben(s) (e.g., 0.00075, 0.0009, 0.001, 0.002, 0.005, 0.0075, 0.009, 0.01, 0.02, 0.05, 0.075, 0.09, 0.1, 0.2, 0.3, 0.5, 0.75, 0.9, 1.0%), and the like.

As noted above, the disclosure provides an article of manufacture, comprising packaging material and at least one vial comprising a solution of at least one modified cell with the prescribed buffers and/or preservatives, optionally in an aqueous diluent, wherein said packaging material comprises a label that indicates that such solution can be held over a period of 1, 2, 3, 4, 5, 6, 9, 12, 18, 20, 24, 30, 36, 40, 48, 54, 60, 66, 72 hours or greater.

The articles of manufacture of the present disclosure are useful for administration over a period ranging from immediate to twenty-four hours or greater. Accordingly, the presently claimed articles of manufacture offer significant advantages to the patient. Formulations of the disclosure can optionally be safely stored at temperatures of from about 2° C. to about 40° C. and retain the biological activity of the protein for extended periods of time, thus allowing a package label indicating that the solution can be held and/or used over a period of 6, 12, 18, 24, 36, 48, 72, or 96 hours or greater.

The products of the present disclosure include packaging material. The packaging material provides, in addition to the information required by the regulatory agencies, the conditions under which the product can be used.

The present disclosure also provided a method of treating a disease or disorder, comprising administering to a subject in need thereof a therapeutically effective number of the cell comprising the nucleic acid encoding or the vector comprising the nucleic acid encoding the RTCR disclosed herein, a therapeutically effective number of any one of the modified T cell disclosed herein, a therapeutically effective amount of any one of the compositions disclosed herein, or a therapeutically effective number of the plurality of modified T cells produced by the method disclosed herein.

In some embodiments, the subject is a mammal. In some embodiments, the mammal is any one of a human, a primate, a rodent, a canine, a feline, an ungulate, an equine and a porcine. In some embodiments, the mammal is a human. In some embodiments, the disease or disorder is any one of a cancer, an autoimmune disorder, an infectious disease, an inflammatory disease or condition, a renal disease or disorder, a lung disease or disorder, a liver disease or disorder, a cardiovascular system disease or disorder, a neurodegenerative disorder or condition, or a metabolic disorder or condition. In some embodiments, the cancer is a solid tumor or a hematologic cancer. In some embodiments, the infectious disease is caused by a bacteria, a virus, a fungi, a protozoa, or a parasite. In some embodiments, the neurodegenerative disorder or condition is any one of Alzheimer's disease (AD) and other dementias, Parkinson's disease (PD) and PD-related disorders, Prion disease, Motor neurone diseases (MND), Huntington's disease (HD), Spinocerebellar ataxia (SCA) or Spinal muscular atrophy (SMA).

The present disclosure provides a chimeric co-stimulatory intracellular protein (CIP) comprising a first and at least a second signal transduction domains, wherein the first and the at least second signal transduction domains are non-identical; and wherein the at least second signal transduction domain comprises a mutant intracellular signaling domain of a tumor necrosis factor receptor (TNFR) family protein.

The present disclosure also provides a chimeric co-stimulatory intracellular protein (CIP) comprising a first and at least a second signal transduction domains, wherein the first and the at least second signal transduction domains are non-identical; and wherein the at least second signal transduction domain comprises a mutant CD137 (4-1BB) intracellular domain or a mutant CD134 (OX-40) intracellular domain.

In some embodiments of the CIP disclosed herein, the mutant intracellular signaling domain of a TNFR family protein is any one of a mutant CD137 (4-1BB) intracellular domain or a mutant CD134 (OX-40) intracellular domain. In some embodiments, the CIP further comprises a transmembrane domain. In some embodiments of the CIP disclosed herein, the mutant CD137 intracellular domain is a truncated CD137 intracellular domain.

In some embodiments of the CIP disclosed herein, the truncated CD137 intracellular domain comprises an amino acid sequence according to amino acid position 13 to amino acid position 42 of the CD137 intracellular domain, of the present disclosure. In some embodiments of the CIP disclosed herein, the truncated CD137 intracellular domain comprises a deletion of a continuous stretch of one, two, three, four, five, six, seven, eight, nine, ten or more amino acids from the N-terminus the CD137 intracellular domain, of the present disclosure. In some embodiments of the CIP disclosed herein, the truncated CD137 intracellular domain comprises a deletion of one, two, three, four, five, six, seven, eight, nine, ten or more amino acids from amino acid position 1 to amino acid position 12 of the N-terminus of the CD137 intracellular domain, of the present disclosure. In some embodiments of the CIP disclosed herein, the CD137 intracellular domain of the present disclosure comprises an amino acid sequence according to SEQ ID NO: 1.

In some embodiments of the CIP disclosed herein, the truncated CD137 intracellular domain comprises an amino acid sequence according to SEQ ID NO: 3.

In some embodiments of the CIP disclosed herein, the mutant CD137 intracellular domain comprises a deletion of one, two, three or four lysine residue(s) from amino acid position 1 to amino acid position 12 of the N-terminus of the CD137 intracellular domain, of the present disclosure. In some embodiments of the CIP disclosed herein, the mutant CD137 intracellular domain comprises one or more lysine mutation(s) from amino acid position 1 to amino acid position 12 of the N-terminus of the CD137 intracellular domain, of the present disclosure. In some embodiments of the CIP disclosed herein, the mutant CD137 intracellular domain comprises one or more lysine mutation(s) at amino acid positions selected from amino acid positions 1, 5, 6 and 12 of the N-terminus of the CD137 intracellular domain, of the present disclosure.

In some embodiments of the CIP disclosed herein, the mutant CD137 intracellular domain comprises a deletion of one or more proximal basic amino acids from amino acid position 1 to amino acid position 12 of the N-terminus of the CD137 intracellular domain, of the present disclosure. In some embodiments of the CIP disclosed herein, the mutant CD137 intracellular domain comprises one or more proximal basic amino acid mutation(s) from amino acid position 1 to amino acid position 12 of the N-terminus of the CD137 intracellular domain, of the present disclosure. In some embodiments of the CIP disclosed herein, the mutant CD137 intracellular domain comprises one or more proximal basic amino acid mutation(s) at amino acid positions selected from amino acid positions 1, 2, 3, 4, 5 and 6 of the N-terminus of the CD137 intracellular domain, of the present disclosure. In some embodiments of the CIP disclosed herein, the mutant CD137 intracellular domain further comprises a lysine mutation at amino acid position 12 of the N-terminus of the CD137 intracellular domain, of the present disclosure.

In some embodiments of the CIP disclosed herein, the mutant CD134 intracellular domain is a truncated CD134 intracellular domain. In some embodiments of the CIP disclosed herein, the truncated CD134 intracellular domain comprises an amino acid sequence according to amino acid position 15 to amino acid position 37 of the CD134 intracellular domain, of the present disclosure. In some embodiments of the CIP disclosed herein, the truncated CD134 intracellular domain comprise a deletion of a continuous stretch of one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen or more amino acids from the N-terminus of the CD134 intracellular domain, of the present disclosure. In some embodiments of the CIP disclosed herein, the truncated CD134 intracellular domain comprises a deletion of one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen or more amino acids from amino acid position 1 to amino acid position 14 of the N-terminus of the CD134 intracellular domain, of the present disclosure.

In some embodiments of the CIP disclosed herein, the truncated CD134 intracellular domain comprises an amino acid sequence according to SEQ ID NO: 6.

In some embodiments of the CIP disclosed herein, the mutant CD134 intracellular domain comprises a deletion of a lysine residue from amino acid position 1 to amino acid position 14 of the N-terminus of the CD134 intracellular domain, of the present disclosure. In some embodiments of the CIP disclosed herein, the mutant CD134 intracellular domain comprises a lysine mutation at amino acid position 12 of the N-terminus of the CD134 intracellular domain, of the present disclosure.

In some embodiments of the CIP disclosed herein, the mutant CD134 intracellular domain comprises a deletion of one or more proximal basic amino acids from amino acid position 1 to amino acid position 14 of the N-terminus of the CD134 intracellular domain, of the present disclosure. In some embodiments of the CIP disclosed herein, the mutant CD134 intracellular domain comprises one or more proximal basic amino acid mutation(s) from amino acid position 1 to amino acid position 14 of the N-terminus of the CD134 intracellular domain. In some embodiments of the CIP disclosed herein, the mutant CD134 intracellular domain comprises one or more proximal basic amino acid mutation(s) at amino acid positions selected from amino acid positions 1, 2, and 5 of the N-terminus of the CD134 intracellular domain. In some embodiments of the CIP disclosed herein, the mutant CD134 intracellular domain further comprises a lysine mutation at amino acid position 12 of the N-terminus of the CD134 intracellular domain

In some embodiments, the CIP disclosed herein comprises a first signal transduction domain derived from a protein of the CD28 family. In some embodiments, the CIP disclosed herein comprises a first signal transduction domain derived from any one of CD28, CD28H, ICOS or a combination thereof.

In some embodiments, the CIP disclosed herein comprises a first signal transduction domain derived from ICOS. In some embodiments, the first signal transduction domain derived from ICOS comprises an amino acid sequence according to SEQ ID NO: 9.

In some embodiments, the CIP disclosed herein comprises a first signal transduction domain comprising a portion of a CD28 intracellular domain combined with an ICOS domain according to SEQ ID NO: 9. In some embodiments of the CIP disclosed herein, the first signal transduction domain comprises an amino acid sequence according to any one of SEQ ID NOs: 12 or 109. In some embodiments, the CIP disclosed herein comprises a first signal transduction domain derived from CD28. In some embodiments of the CIP disclosed herein, the first signal transduction domain derived from CD28 comprises an amino acid sequence according to SEQ ID NO: 10. In some embodiments of the CIP disclosed herein, the first signal transduction domain derived from CD28 comprises an amino acid sequence according to any one of SEQ ID NOs: 121-122. In some embodiments, the CIP comprises an amino acid sequence according to any one of SEQ ID NOs: 14-17.

In some embodiments, the CIP disclosed herein further comprises a third signal transduction domain. In some embodiments, the CIP disclosed herein further comprises a third signal transduction domain derived from any one of a CD3 signaling domain, a CD2 signaling domain or an interleukin 2 receptor binding (IL-2RB) protein signaling domain or a combination thereof. In some embodiments, the CD3 signaling domain of the CIP disclosed herein is derived form a CD3ζ or a CD3ε domain or a combination thereof. In some embodiments, the CD3 signaling domain of the CIP disclosed herein is a CD3 domain comprising an amino acid sequence according to any one of SEQ ID NOs: 18, 45, 46, 47 and 48.

In some embodiments, the third signal transduction domain of the CIP disclosed herein is a CD3ζ domain comprising an amino acid sequence having according to SEQ ID NO: 18. In some embodiments, the third signal transduction domain of the CIP disclosed herein is a CD3 domain comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to according to SEQ ID NO: 18. In some embodiments, the third signal transduction domain of the CIP disclosed herein is a CD3ζ domain comprising an amino acid sequence having according to SEQ ID NO: 45. In some embodiments, the third signal transduction domain of the CIP disclosed herein is a CD3ζ domain comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to according to SEQ ID NO: 45. In some embodiments, the third signal transduction domain of the CIP disclosed herein is a truncated CD3ζ domain comprising an amino acid sequence having according to SEQ ID NO: 46. In some embodiments, the third signal transduction domain of the CIP disclosed herein, the third signal transduction domain of the CIP disclosed herein is a truncated CD3 domain comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to according to SEQ ID NO: 46. In some embodiments, the third signal transduction domain of the CIP disclosed herein is a CD3ε domain comprising an amino acid sequence according to SEQ ID NO: 47. In some embodiments, the third signal transduction domain of the CIP disclosed herein is a CD3ε domain comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 47. In some embodiments, the third signal transduction domain of the CIP disclosed herein is a combination of a CD3ε and a truncated CD3ζ domains (CD3ζε domain). In some embodiments, the third signal transduction domain of the CIP disclosed herein is a CD3ζε domain comprising an amino acid sequence according to SEQ ID NO: 48. In some embodiments, the third signal transduction domain of the CIP disclosed herein is a CD3ζε domain comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 48.

In some embodiments, the third signal transduction domain of the CIP disclosed herein is a CD2 signaling domain. In some embodiments, the third signal transduction domain of the CIP disclosed herein is a mutant CD2 signaling domain. In some embodiments, the mutant CD2 signaling domain is a truncated CD2 signaling domain. In some embodiments, the third signal transduction domain of the CIP disclosed herein is a CD2 signaling domain comprising an amino acid sequence according to SEQ ID NO: 49. In some embodiments, the third signal transduction domain of the CIP disclosed herein is a CD2 signaling domain comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 49.

In some embodiments, the third signal transduction domain of the CIP disclosed herein, is an IL-2RB protein signaling domain comprising an amino acid sequence according to SEQ ID NO: 50. In some embodiments, the third signal transduction domain of the CIP disclosed herein is an IL-2RB protein signaling domain comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 50.

In some embodiments, the CIP disclosed herein further comprises a fourth signal transduction domain. In some embodiments, the CIP disclosed herein further comprises a fourth signal transduction domain derived from any one of a CD3 signaling domain, a CD2 signaling domain or an interleukin 2 receptor binding (IL-2RB) protein signaling domain or a combination thereof, wherein the third and the fourth signal transduction domain are not identical. In some embodiments, the fourth signal transduction domain of the CIP disclosed herein, is derived form a CD3ζ or a CD3ε domain or a combination thereof. In some embodiments, the fourth signal transduction domain of the CIP disclosed herein is a CD3 domain comprising an amino acid sequence according to any one of SEQ ID NOs: 18, 45, 46, 47 and 48.

In some embodiments, the fourth signal transduction domain of the CIP disclosed herein is a CD3ζ domain comprising an amino acid sequence having according to SEQ ID NO: 18. In some embodiments, the fourth signal transduction domain of the CIP disclosed herein is a CD3ζ domain comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to according to SEQ ID NO: 18. In some embodiments, the fourth signal transduction domain of the CIP disclosed herein is a CD3ζ domain comprising an amino acid sequence having according to SEQ ID NO: 45. In some embodiments, the fourth signal transduction domain of the CIP disclosed herein is a CD3ζ domain comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to according to SEQ ID NO: 45. In some embodiments, the fourth signal transduction domain of the CIP disclosed herein is a truncated CD3ζ domain comprising an amino acid sequence having according to SEQ ID NO: 46. In some embodiments, the third signal transduction domain of the CIP disclosed herein, the fourth signal transduction domain of the CIP disclosed herein is a truncated CD3ζ domain comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to according to SEQ ID NO: 46. In some embodiments, the fourth signal transduction domain of the CIP disclosed herein is a CD3ε domain comprising an amino acid sequence according to SEQ ID NO: 47. In some embodiments, the fourth signal transduction domain of the CIP disclosed herein is a CD3ε domain comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 47. In some embodiments, the fourth signal transduction domain of the CIP disclosed herein is a combination of a CD3ε and a truncated CD3ζ domains (CD3ζε domain). In some embodiments, the fourth signal transduction domain of the CIP disclosed herein is a CD3ζε domain comprising an amino acid sequence according to SEQ ID NOs: 48. In some embodiments, the fourth signal transduction domain of the CIP disclosed herein is a CD3ζε domain comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 48.

In some embodiments, the fourth signal transduction domain of the CIP disclosed herein is a CD2 signaling domain. In some embodiments, the fourth signal transduction domain of the CIP disclosed herein is a mutant CD2 signaling domain. In some embodiments, the mutant CD2 signaling domain is a truncated CD2 signaling domain. In some embodiments, the fourth signal transduction domain of the CIP disclosed herein is a CD2 signaling domain comprising an amino acid sequence according to SEQ ID NO: 49. In some embodiments, the fourth signal transduction domain of the CIP disclosed herein is a CD2 signaling domain comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 49.

In some embodiments, the fourth signal transduction domain of the CIP disclosed herein, is an IL-2RB protein signaling domain comprising an amino acid sequence according to SEQ ID NO: 50. In some embodiments, the fourth signal transduction domain of the CIP disclosed herein is an IL-2RB protein signaling domain comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to SEQ ID NO: 50.

In some embodiments, the CIP disclosed herein is for expression in a T cell, wherein the T cell co-expresses at least one of the endogenous co-stimulatory molecules CD28, CD2, OX-40, ICOS, CD28, CD3, CD4, CD8 and CD40L or a combination thereof.

In some embodiments, the CIP disclosed herein, is co-expressed with a T cell receptor (TCR) in a T cell. In some embodiments, the TCR is an endogenous TCR. In some embodiments, the TCR is an artificial TCR. In some embodiments, the artificial TCR is an affinity enhanced TCR. In some embodiments, the CIP when co-expressed with a TCR in a T cell provides a second activation signal for inducing activation and proliferation of the T cell, wherein the first activation signal is provided by antigen binding by the TCR.

In some embodiments, the CIP disclosed herein, is expressed in a T cell as a component of an artificial receptor for a target. In some embodiments, the artificial receptor is a chimeric antigen receptor (CAR), a receptor for a ligand or a component thereof, an antibody or a fragment thereof. In some embodiments, the CIP disclosed herein, is expressed as a component of a CAR. In some embodiments, the CIP disclosed herein, is expressed as a component of an antibody or a fragment thereof. In some embodiments, the antibody or a fragment thereof is a Fab fragment, a F(ab)2 fragment, a diabody, a nanobody, a sdAb, a Fv, a VHH fragment, or a single chain Fv fragment. In some embodiments, the CIP expressed as a component of an artificial receptor in a T cell, as disclosed herein induces activation and proliferation of the T cell upon target binding by the artificial receptor.

The term “about” or “approximately” can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. In some embodiments, “about” or “approximately” can be understood as within 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. In some embodiments, “about” or “approximately” can be understood as within 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. In some embodiments, “about” or “approximately” can be understood as within 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value.

The following examples are provided to better illustrate the present disclosure and are not to be interpreted as limiting the scope of the disclosure. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the disclosure. One skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the disclosure.

EXAMPLES
Materials and Methods
Media and Cell Lines

DMEM was supplemented with Penn/Strep/Glutamine, 20 mM HEPES, 10 μg/mL Gentamycin and 10% FBS to make complete DMEM. RPMI was supplemented with Penn/Strep/Glutamine, 20 mM HEPES, 10 μg/mL Gentamycin, 10% FBS, and 50 uM 2-ME to make complete RPMI. T cell growth media was made by supplementing complete RPMI with 50 ng/ml IL2, 10 ng/ml IL7, and 10 ng/mL IL15 (Peprotech). X-Vivo15 was supplemented with 1% Human Serum, 20 mM HEPES, Penn/Strep/Glutamine, and 10 μg/mL Gentamycin to make Cytokine Media. Human PBMCs were purchased from iSpecimen and cultured in complete RPMI. 293FT were purchased from Invitrogen. K562 and A375 cells were purchased from ATCC and cultured in complete DMEM.

Plasmids and Cloning A lentiviral plasmid containing the PGK promoter driving a truncated human EGFR receptor (huEGFRt) followed by the MSCV promoter driving GFP and a subsequent WPRE sequence was ordered from vector builder. Co-stimulatory molecules followed by a P2A sequence were ordered as a single gene block (Invitrogen) and placed in frame with the huEGFRt sequence using NEB builder homology-based recombination. CAR and TCR sequences were constructed from 3 gene block fragments (Invitrogen) and cloned with NEB builder downstream of the MSCV promoter following GFP excision. PD-L1_P2A and HLA-A2 were cloned in frame with the huEGFRt and in place of GFP, respectively.

P2A amino acid sequence

(SEQ ID NO: 111)

GSGATNFSLLKQAGDVEENPGP

Human EGFRt amino acid sequence (Other name:

huEGFRt (AA112))

(SEQ ID NO: 112)

MLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIK

HFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITG

FLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLG

LRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRG

ENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNL

LEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYID

GPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGL

EGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM

HLA-A2 signal peptide

(SEQ ID NO: 113)

MAVMAPRTLVLLLSGALALTQTWA

huGMCSF Signal Peptide, amino acid sequence

(SEQ ID NO: 119)

MLLLVTSLLLCELPHPAFLLIP

P2A nucleic acid sequence

(SEQ ID NO: 114)

GGATCCGGCGCCACCAATTTCAGCCTGCTGAAACAGGCTGGCGACG

TGGAAGAGAACCCTGGACCT

Human EGFRt nucleic acid sequence

(SEQ ID NO: 115)

ATGCTGCTGCTGGTTACATCTCTGCTGCTGTGCGAGCTGCCCCATC

CTGCCTTTCTGCTGATCCCCAGAAAAGTGTGCAACGGCATCGGCAT

CGGAGAGTTCAAGGACAGCCTGAGCATCAACGCCACCAACATCAAG

CACTTCAAGAACTGCACCAGCATCAGCGGCGACCTGCACATTCTGC

CTGTGGCCTTTAGAGGCGACAGCTTCACCCACACACCTCCACTGGA

CCCTCAAGAGCTGGACATCCTGAAAACCGTGAAAGAGATCACCGGA

TTTCTGTTGATCCAGGCTTGGCCCGAGAACCGGACAGATCTGCACG

CCTTCGAGAACCTGGAAATCATCAGAGGCCGGACCAAGCAGCACGG

CCAGTTTTCTCTGGCTGTGGTGTCCCTGAACATCACCAGCCTGGGC

CTGAGAAGCCTGAAAGAAATCAGCGACGGCGACGTGATCATCTCCG

GCAACAAGAACCTGTGCTACGCCAACACCATCAACTGGAAGAAGCT

GTTCGGCACCAGCGGCCAGAAAACAAAGATCATCAGCAACCGGGGC

GAGAACAGCTGCAAGGCTACAGGCCAAGTGTGCCACGCTCTGTGTA

GCCCTGAAGGCTGTTGGGGACCCGAGCCTAGAGATTGCGTGTCCTG

TCGGAATGTGTCCCGGGGCAGAGAATGCGTGGACAAGTGCAATCTG

CTGGAAGGCGAGCCCCGCGAGTTCGTGGAAAACAGCGAGTGCATCC

AGTGTCACCCCGAGTGTCTGCCCCAGGCCATGAACATTACCTGTAC

CGGCAGAGGCCCCGACAACTGTATTCAGTGCGCCCACTACATCGAC

GGCCCTCACTGCGTGAAAACATGTCCTGCTGGCGTGATGGGAGAGA

ACAACACCCTCGTGTGGAAGTATGCCGACGCCGGACATGTGTGCCA

CCTGTGTCACCCTAATTGCACCTACGGCTGTACAGGCCCTGGCCTG

GAAGGCTGTCCAACAAACGGACCTAAGATCCCCTCTATCGCCACCG

GCATGGTTGGAGCCCTGCTGCTTCTGCTGGTGGTGGCCCTTGGAAT

CGGCCTGTTCATGTGA

HLA-A2 signal peptide nucleic acid sequence

(SEQ ID NO: 116)

ATGGCTGTGATGGCCCCTAGAACACTGGTGCTGCTGCTGTCTGGTG

CCCTGGCTCTGACTCAGACATGGGCC

CD8a signal peptide nucleic acid sequence

(SEQ ID NO: 148)

ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTTC

TGCATGCCGCTAGACCT

CD8a Hinge

(SEQ ID NO: 149)

ACCACCACCCCCGCCCCCAGACCCCCCACCCCCGCCCCCACCATCG

CCAGCCAGCCCCTGAGCCTGAGACCCGAGGCCTGCAGACCCGCCGC

CGGCGGCGCCGTGCACACCAGAGGCCTGGACTTCGCCTGC

huGMCSF Signal Peptide, nucleic acid sequence

(SEQ ID NO: 150)

ATGCTGCTGCTGGTTACATCTCTGCTGCTGTGCGAGCTGCCCCATC

CTGCCTTTCTGCTGATCCCC

Lentiviral Production and Preparation of Retronectin Plates

VSV pseudotyped lentivirus was produced in 6 well plates. In brief, 293FT were seeded the night before or the day of at 0.9×10⁶or 1.4×10⁶cells/well, respectively. Once the cells had adhered and reached at least 80% confluency a mix of lentiviral plasmid, packaging vector (psPAX2) and VSV-G envelope expressing plasmid (PMD2.G) were transfected using lipofectamine 3000 (Invitrogen), according to the manufacturer's protocol. After 18 hrs, the media was replaced with 3mLs of fresh DMEM. Viral supernatants were harvested 48 hrs following changing the media and spun down at 1500RPM to remove 293FT cell/debris. Retronectin was coated on 24 well non-tissue culture treated plates at 20 m/well in PBS−/− for 2 hrs at 37° C. or overnight at 4° C. Retronectin was removed and washed once with PBS prior to addition of lentiviral SN (2mLs). The plate was then spun at 1500 G for 90 minutes at 32° C. to concentrate viral particles onto the retronectin. Lentiviral SN was removed immediately prior to transduction of primary T cells or tumor cells. Alternatively, T cells were transduced with polybrene at 8 ug/mL with a spinfection of 800 G for 2 hrs at 32° C.

T Cell Culture, Transduction, and Isolation

Human PBMCs were activated in T cell growth media with CD3/28 microbeads (Invitrogen) in complete RPMI (100 ul beads/50×10⁶PBMCs). 48 hrs after activation, activated PBMCs were transferred to Lentiviral-coated Retronectin plates for 48 hrs before being transferred to 6 well plates containing fresh T cell growth media. After an additional 24 hrs in culture cell transduction was determined by flow cytometry and transduced cells were enriched based on huEGFRt expression. To isolate cells based on EGFR expression, T cells cultures were collected and activation beads removed. Cells were then stained in 1:100 anti-EGFR-APC antibody in MACS buffer at 4° C. for 30 minutes. Cells were then washed and incubated with anti-APC microbeads (Miltenyi) for 15-30 minutes at 4° C. Unbound microbeads were then removed by centrifugation and huEGFRt cells were isolated by positive selection on mini-macs columns. Cells were eluted from the mini-MACS columns and put back into culture in T cell growth media and used within 2 weeks for experiments. To create stable cell lines, cells were collected and transduced as with primary T cells. EGFR selection was performed twice, two weeks apart.

T Cell Stimulation

In the case where T cells were stimulated with plate bound antibodies, maxisorp Flat-bottom plates (Invitrogen) were coated with the indicated amount of anti-human CD3 antibody (HIT3a-Biolegend) in PBS−/− for 2 hrs at 37° C. Plates were washed twice in basal RPMI before use. For K562 stimulation K562 cells were collected and resuspended in Cytokine media and aliquoted to U-bottom plates. Similarly, A375 cells were plated 1 day prior to the addition of T cells in DMEM in 96 well flat-bottom plates. The media was exchanged prior to the addition of cognate T cells. Following EGFR+ selection, T cells were collected, counted, and resuspended at the appropriate concentration in Cytokine media and distributed to antibody or APC-bearing wells. For K562 experiments anti-CD3 (HIT3a/Biolegend) was added at the indicated dose following 1-2 hrs of K562/T cell interaction at 37° C. In the case where T cell proliferation was to be tracked, T cells were labelled with Violet Tracking Dye (CTV) according to Biolegend's protocol prior to the addition to stimulatory plates. Supernatant was collected at 18-36 hr post stimulation to assess cytokine secretion and proliferation/T cell killing was assessed following 96 hrs of stimulation.

Cytokine Multiplex Assay

Following collection of T cell supernatants cytokines were measured with the Legendplex Multi-Analyte Flow Assay Kit foe human Th or Th1 cytokines (Biolegend). Manufactures protocol was followed with the following exceptions: 75 uL T cell SN was used to measure cytokines and 2 uL of each reagent was used/well. Secreted cytokines were measured by flow cytometry and the values were normalized to the maximal response of the control group in order to combine and analyze multiple experiments and normalize for variability between experiments and donors.

Conjugation Assays

To assess conjugation of T cells to target cells, T cells were labelled with CFSE (Biolegend) and K562_HLA-A2 or K562_HLA-A2_PD-L1 cells were labelled with CTV according to manufacturer's protocols. T cells and APCs were mixed in a 1:2 ratio and briefly centrifuged in a 1.5 mL eppendorf tube to encourage conjugation. Cell pellets were incubated at 37° C. for 30 minutes and then cell pellets were gently resuspended by repeat pipetting (20×) with a p200 and a cut-off pipette tip and assessed immediately by flow cytometry.

Flow Cytometry

Cells were collected and washed in MACS Buffer (PBS−/−, 1% FBS, 1 mM EDTA) before being stained in MACS buffer containing relevant antibodies. Anti-EGFR-APC, anti-mouse TCRbeta-FITC, anti-human PD1-PE, anti-CD3 APC-Cy7, anti-CD8 PE-Cy7 were all purchased from biolegend. Following addition of antibodies, cells were stained for 30-60 minutes at 4° C. For the detection of CD-19 CAR expression cells were incubated with CD-19Fc recombinant protein in MACS buffer at 1 μg/mL for 30 minutes at RT. Cells were then washed and incubated with anti-human FC antibody at 1:100 dilution. Cells were then washed 3× in MACS buffer and analyzed on an Acea NovoCyte flow cytometer. Cells were collected at constant volume, allowing for accurate cell counts to be obtained.

Example 1: Design of Co-Stimulatory Molecules Comprising Chimeric Intracellular Signaling Domains

The disclosure herein provides the design of the co-stimulatory molecules comprising intracellular signaling domains comprising or derived from CD137/4-1BB or CD134/OX-40 receptors as depicted in FIG. 1. Examination of the sequence of the CD137 family of cytoplasmic tails (FIG. 1) showed a common membrane-proximal polybasic domain as well as several lysine residues that could serve as ubiquitination sites, as well as the TRAF binding domain that serves to activate the NF-κB signaling pathway following receptor ligation. Without being bound by the theories, the conserved lysine residues may function as ubiquitination sites that could control the ubiquitination and degradation CD134/CD137 and that the disrupted location of the CD137 or CD134 cytoplasmic tail in the potential CD28/ICOS-CD137 CAR or CD28/ICOS-CD134 CAR receptors, respectively, could be affecting the localization or half-life of the resulting molecule, through either the poly-basic domain or the conserved lysine residues. New fusion domains of ICOS/CD28 intracellular domain and the cytoplasmic domains of CD137 or OX-40 lacking the polybasic sequence and the conserved lysine residues, as well as their wild-type (WT) counterparts were generated (FIGS. 2A and 2B). A portion of the cytoplasmic domain of CD28 responsible for the binding of Lck and Vav3 to possible enhance stimulation was also included. The extracellular domain of PD-1 was used, creating either dominant-negative (DN) version by omitting the intracellular tail or an inhibitory-switch receptor that would change a negative regulatory signal into a positive one, thus providing a cell-intrinsic PD-1 blockade. The cytoplasmic tail, i.e., intracellular co-stimulatory domain, described herein can be expanded through the use of cytoplasmic tails of other signaling proteins of interest to create new CAR receptors or different inhibitory-switch receptors, or express other immune-modulatory extracellular domains, as detailed in FIG. 3.

Example 2: Generation and Testing of the In-Vitro Functionality of Checkpoint Co-Stimulatory Molecules

The disclosure herein provides the design of the co-stimulatory molecules and validation of their effect on function of a high affinity TCR. The co-stimulatory molecules described herein were designed as depicted in FIGS. 2A and 2B. In all recombinant receptors, the PD1 signal peptide (SP) was exchanged for the signal peptide from HLA-A2, which increases the surface expression of the receptor. As controls, the PD1-WT and a truncated PD-1 lacking the ITIM-containing intracellular tail (TLs) were included. Two second-generation receptors, containing the transmembrane and intracellular domains of CD28 or ICOS were included as 2^nd-generation control receptors. Additionally, fifteen 3^rd-generation receptors containing the transmembrane and intracellular domains derived from ICOS and/or CD28 linked to an intracellular signaling domain of a TNF-Receptor super family member were generated. The first contained the intracellular domain of wild type CD137/4-1BB (HLA A2-SP_PD1_ICOS_BB: SEQ ID NO: 26), while the second and third contained either the CD137/4-1BB domain or the CD134/OX-40 intracellular domain with key mutations incorporated to increase surface expression (HLA A2-SP_PD1_ICOS_BBt: SEQ ID NO: 27 and HLA A2-SP_PD1_ICOS_OX40t: SEQ ID NO: 28, respectively). Further 3^rdgeneration receptors described herein contain a chimeric intracellular domain comprising a portion of CD28 intracellular domain inserted within an ICOS intracellular domain that is further linked to either the mutated CD137 (ICOS4BBt) or mutated CD134/OX40 (ICOS-OX40t) domains (HLA A2-SP_PD1_ICOS(28) BBt: SEQ ID NO: 29 and HLA A2-SP_PD1_ICOS(28)_OX40t: SEQ ID NO: 30). Two more 3^rdgeneration receptors were created as described herein containing a CD28 intracellular domain linked to either mutated CD137 (28_BBt) or mutated CD134/OX40 (28_OX40t) domains (HLA A2-SP_PD1_28_BBt: SEQ ID NO: 131 and HLA A2-SP_PD_28_OX40t: SEQ ID NO: 132) (FIG. 2A). These vectors were cloned into a lentiviral vector and fused by a self-cleaving peptide to a truncated huEGFR receptor (huEGFRt) for tracking transduced cells and magnetic selection. When these receptors were expressed by lentiviral transduction into primary T cells, each receptor expresses significantly over endogenous PD-1 levels (FIG. 4A). While the 2^ndgeneration co-stimulatory molecules were well expressed, the inclusion of the CD137 (4-1BB) intracellular domain resulted in a considerable decrease in surface expression of the recombinant receptor. The disclosure herein shows that inclusion of the mutated intracellular domains, which maintain the TRAF-binding domains, rescues the surface expression of these optimized 3^rdgeneration receptors. The surface expression of huEGFRt and co-stimulatory molecules demonstrates the increased expression of the truncated CD137 (4-1BB) design (ICOS_BBt) compared to the non-truncated version (ICOS_BBwt). Similar mutations in the cytoplasmic domain of CD134 (OX-40) also resulted in high surface expression of the co-stimulatory molecules (FIG. 4B).

Following transduction, T cells were isolated based on the expression of huEGFRt, to >90% purity, and used in restimulation experiments. The results disclosed herein demonstrate that, in-vitro, engagement of co-stimulatory molecule enhanced T cell cytokine production and proliferation, especially at lower doses of anti-CD3 antibody (FIGS. 5A-5D). To make a more physiological system, either HLA-A2 alone or HLA-A2 alongside PD-L1 were overexpressed on K562 cells and incubated with the co-stimulatory receptor-transduced T cells and the indicated dose of anti-CD3. Incubation with K562: PD-L1 cells reduced the amount of secreted cytokine, especially with T cells overexpressing PD1_WT (FIG. 6A). While expression of either PD1_TLs or PD1_ICOS_BBwt did little to affect the secretion of IL-2, TNF, or IFNγ, the expression of PD1_28 or PD1_ICOS increased effector cytokine secretion 3-4 fold over GFP control in the presence of PD-L1 expressing K562 cells. Notably, both PD1_ICOS_BBt and PD1_ICOS_OX40t co-stimulatory molecules further improved on this effect, increasing effector cytokine secretion 2 to 3-fold over PD1_ICOS expressing cells in the presence of PD-L1 (FIG. 6C). The expression of PD1_28, PD1_28_BBt and PD1_28_OX40t resulted in comparable effector cytokine secretion (FIG. 6B). None of the constructs were constitutively active and had minimal effect on cytokine secretion in the absence of PD-L1 or anti-CD3, indicating the necessity of both PD-1 and antigen to initiate a T cell response. Fitting with the cytokine data, T cells expressing a) PD1_28, PD1_28_BBt and PD1_28_OX40t (FIG. 7A, lower panels, and FIG. 7C), and b) PD1_ICOS_BBt and PD1_ICOS_OX40t (FIG. 7B, lower panels, and FIG. 7C), proliferated best in response to K562 cells expressing PD-L1 and were best able to kill PD-L1 expressing K562 cells (FIGS. 7A-7B, upper panels). Fitting with the cytokine data, T cells expressing PD1_ICOS_BBt and PD1_ICOS_OX40t proliferated best in response to K562 cells expressing PD-L1 and were best able to kill PD-L1 expressing K562 cells (FIG. 7C-7E). Again, this response required both anti-CD3 and PD-L1 expression. The co-stimulatory molecules demonstrated co-stimulatory ability as their expression increased T cell proliferation when cells were stimulated on 96-well plates coated with anti-CD3 and anti-PD1 (FIG. 8).

The effect of the receptors with mutation of the polybasic and lysine residues is less than the PD1_ICOS_BBt co-stimulatory molecule, in terms of both surface expression of the co-stimulatory molecule (FIGS. 9A-9B), effector cytokine production in response to stimulation with anti-CD3 antibody (FIG. 9C), and T cell proliferation in response to stimulation with target cells expressing PD-L1 (FIG. 9D). The PD1_ICOS_OX40t receptor (with truncated OX40 intracellular domain) had an effect comparable to that of the wild type PD1_ICOS_OX40wt receptor (comprising wild type OX40 intracellular domain), in terms of both surface expression of the co-stimulatory molecule (FIGS. 10A-10B), effector cytokine production in response to stimulation with anti-CD3 antibody (FIG. 10C), and T cell proliferation in response to stimulation with target cells expressing PD-L1 (FIG. 10D).

Further, the ICOS-based co-stimulatory molecules encouraged T cell: PD-L1 expressing (PD-L1+) target cell interaction in a flow-based conjugation assay, suggesting that these receptors encourage prolonged T cell—APC interactions while scanning for cognate antigen, a useful property when scanning for low-abundance antigen in the TME (FIGS. 11A-11B).

The disclosure herein shows that the co-stimulatory molecules based on the modified 3^rd-generation intracellular signaling domain disclosed herein are superior to currently existing PD1_28 co-stimulatory molecules in enhancing T cell effector function when responding to a PD-L1+ target cell. This includes increased T cell proliferation, cytokine secretion, and target cell killing. The 3^rd-generation intracellular signaling domain disclosed herein can be successfully combined with TCR-T therapy targeting TAAs.

Example 3: In-Vitro Preclinical Studies

Described herein are T cells expressing specific HLA-A2/NY-ESO specific TCRs and co-stimulatory molecules comprising ICOS and mutated CD137 signaling domains, that increase expression of the co-stimulatory molecule on T cell surface (FIG. 12A), effector cytokine production (FIG. 12B), and killing of target cells expressing NY-ESO (FIG. 12C), as compared to T cells expressing the specific HLA-A2/NY-ESO specific TCRs alone.

Described herein are CD-19 CAR constructs comprising the modified 3^rd-generation intracellular signaling domains disclosed herein. The CD-19 (FMC63scFV) CARs with 3^rd-generation intracellular signaling constructs described herein include constructs comprising the intracellular chimeric domains: CD28-CD137-CD3ζ (28_BBwt_z), CD28-CD137mutant-CD3ζ (28_BBt_z), CD28-CD134mutant-CD3ζ (28_OX40t_Z), ICOS-CD137-CD3ζ (ICOS_BB_z), ICOS-CD137mutant-CD3ζ (ICOS_BBt_z), and ICOS-CD134mutant-CD3ζ (ICOS_OX40t_z). Also, provided are CD-19 CARs with a 3^rd-generation intracellular signaling construct with a portion of CD28 inserted within the ICOS domain: ICOS(28)-CD137-CD3ζ (ICOS(28)_BBwt_z), ICOS(28)-CD137mutant-CD3ζ (ICOS(28)_BBt_z), and ICOS(28)-CD134mutant-CD3ζ(ICOS(28)_OX40t_z). Second-generation constructs comprising CD137-CD3ζ (BBwt_z), CD28-CD3ζ (28_z) and ICOS-CD3ζ (ICOS_z) are used as controls. Similar to the study described herein using the PD-1 3^rd-generation intracellular signaling constructs, the CD19 CAR constructs with the CD137 and CD134 mutants domains showed higher expression as compared to the corresponding constructs with wild type CD137 and CD134 domains (28_BBwt_z and ICOS_BBwt_z, respectively) (FIGS. 13A-13B). In-Vitro studies described herein show increased killing of CD19 expressing cells (CD19+) (FIGS. 14A-14B and FIGS. 15C-15D), increased effector cytokine production (FIG. 14C, right panels) and increased T cell proliferation and persistence (FIGS. 15A-15B and 15E), by primary T cells transduced with the 3^rdgeneration CD28 based and ICOS based CD19 CARs.

The disclosure also shows that expression of CD28 based receptors comprising a mutated CD134/CD137 signaling domains and ICOS based receptors comprising a mutated CD134/CD137 signaling domains, increased binding of BCMA specific T cells (BCMA CAR T cells) to the target antigen (BCMA-Fc) (FIGS. 16A-16B). Also, the disclosure shows that expression of the CD28 based receptors comprising a mutated CD134/CD137 signaling domains and ICOS based receptors comprising a mutated CD134/CD137 signaling domains, increased proliferation and effector cytokine production (FIGS. 16C and 16E), and target cell killing (FIG. 16D) by the BCMA specific T cells in response to myeloma cell line expressing BCMA.

	Number	Date	Country
	63133494	Jan 2021	US
	63163171	Mar 2021	US

CHIMERIC CO-STIMULATORY PROTEINS COMPRISING MUTANT INTRACELLULAR DOMAINS WITH INCREASED EXPRESSION

Information

Publication Number

Date Filed

Date Published

Inventors

Original Assignees

CPC

International Classifications

Abstract

Description

Claims

CROSS-REFERENCE TO RELATED APPLICATIONS

Provisional Applications (2)