Chimeric Antigen Receptor

Abstract

Provided herein are chimeric antigen receptors (CARs) that include a spacer sequence that provides intercellular distance between a cell expressing the CAR and a target cell. Embodiments are provided in which the extracellular elements of the CAR include a spacer sequence, such that when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 17 nm to about 25 nm, including the endpoints.

Description

[Technical field]

[0001] Field of Disclosure [1] The present disclosure relates to chimeric antigen receptors (CARs) comprising spacer domains, compositions comprising CARs, and sequences encoding CARs, and methods of use. cross reference [2] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 277,984, filed November 10, 2021, and U.S. Provisional Patent Application No. 63 / 307,612, filed February 7, 2022, each of which is incorporated by reference in its entirety. [Background technology]

[0002] [3] Chimeric antigen receptors (CARs) are synthetic receptors that can be used to confer engineered specificity to immune cells. The use of CARs to redirect T cells to recognize target cells is a promising approach to cancer immunotherapy. CAR design has focused on identifying antigen-binding domains and designing intracellular signaling modules to activate T cell effector functions. The present disclosure provides CARs incorporating spacers, compositions and methods for the use of CARs to achieve superior CAR activity and, therefore, superior efficacy for cell therapy in which cells express these CARs on their surface. Incorporation by Reference [4] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Summary of the Invention

[0003] [5] The present disclosure provides a chimeric antigen receptor (CAR) comprising: (a) an extracellular element comprising (1) a binding element that specifically binds to a target and (2) a spacer sequence; (b) a transmembrane element; and (c) an intracellular element, wherein when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 17 nm to about 25 nm, including the endpoints. In some embodiments, when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 17 nm to about 22 nm. In some embodiments, when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 17 nm to about 23 nm. In some embodiments, when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 18 nm to about 22 nm. In some embodiments, when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 20 nm. In some embodiments, the target comprises an epitope. In some embodiments, the target comprises a linear epitope, a continuous epitope, a discontinuous epitope, and / or a conformational epitope. In some embodiments, the target comprises an antigen. In some embodiments, the target comprises an amino acid sequence. In some embodiments, the target does not comprise an amino acid.

[0004] [6] (a) In some embodiments of the CARs of the present disclosure, including those that include (1) a binding element that specifically binds to a target and (2) an extracellular element that includes a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 17 nm to about 25 nm, including the endpoints, and the spacer sequence includes a length of about 2 nanometers (nm) to about 20 nm, including the endpoints. In some embodiments, the spacer sequence includes a length of about 3 nm to 20 nm, including the endpoints. In some embodiments, the spacer sequence includes a length of about 4 nm to 20 nm, including the endpoints.

[0005] [7] (a) In some embodiments of CARs of the disclosure, including those that comprise (1) a binding element that specifically binds to a target and (2) an extracellular element comprising a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, when the CAR is expressed on the surface of a cell, the intercellular distance between a CAR-expressing cell and a target-expressing cell is, in each case, inclusive of the endpoints, about 17 nm to about 18 nm, about 18 nm to about 19 nm, about 19 nm to about 20 nm, about 20 nm to about 21 nm, about 21 nm to about 22 nm, about 22 nm to about 23 nm, about 23 nm to about 24 nm, or about 24 nm to about 25 nm.

[0006] [8] (a) In some embodiments of CARs of the disclosure, including those that comprise (1) a binding element that specifically binds to a target and (2) an extracellular element that comprises a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, when the CAR is expressed on the surface of a cell, the intercellular distance between a CAR-expressing cell and a target-expressing cell is, in each case, from about 17 nm to about 25 nm, from about 18 nm to about 24 nm, from about 19 nm to about 23 nm, from about 20 nm to about 22 nm, or from about 21 nm to about 23 nm, including the endpoints.

[0007] [9] (a) In some embodiments of CARs of the disclosure, including those that comprise (1) an extracellular element comprising a binding element that specifically binds to a target and (2) a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is, in each case, about 17 nm, about 18 nm, about 19 nm, about 20 nm, about 21 nm, about 22 nm, about 23 nm, about 24 nm, or about 25 nm.

[0008]

[10] (a) In some embodiments of CARs of the disclosure, including those that comprise (1) a binding element that specifically binds to a target and (2) an extracellular element that comprises a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is, in each case, 17 nm, 18 nm, 19 nm, 20 nm, 21 nm, 22 nm, 23 nm, 24 nm, or 25 nm.

[0009]

[11] (a) In some embodiments of CARs of the disclosure, including those that comprise (1) a binding element that specifically binds to a target and (2) an extracellular element comprising a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, when the CAR is expressed on the surface of a cell, the intercellular distance between a CAR-expressing cell and a target-expressing cell is, in each case, inclusive of the endpoints, about 17 nm to about 19 nm, about 18 nm to about 20 nm, about 19 nm to about 21 nm, about 20 nm to about 22 nm, about 21 nm to about 23 nm, about 22 nm to about 24 nm, or about 23 nm to about 25 nm.

[0010]

[12] In some embodiments of the CARs of the present disclosure, including those that include (a) an extracellular element that includes (1) a binding element that specifically binds to a target and (2) a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 17 nm to about 25 nm, including the endpoints, and the spacer sequence includes a linker sequence. In some embodiments, the linker includes a glycine-serine (GS) linker. In some embodiments, the linker sequence includes GGGSG (SEQ ID NO: 77).

[0011]

[13] In some embodiments of the CARs of the present disclosure, including those that include (a) an extracellular element that includes (1) a binding element that specifically binds to a target and (2) a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 17 nm to about 25 nm, including the endpoints, and the extracellular element includes a linker sequence. In some embodiments, the linker includes a glycine-serine (GS) linker. In some embodiments, the linker sequence includes GGGSG (SEQ ID NO: 77).

[0012]

[14] (a) In some embodiments of the CARs of the present disclosure, including those that include (1) a binding element that specifically binds to a target and (2) an extracellular element that includes a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 17 nm to about 25 nm, including the endpoints, and the intracellular element includes a linker sequence. In some embodiments, the linker includes a glycine-serine (GS) linker. In some embodiments, the linker sequence includes GGGSG (SEQ ID NO: 77).

[0013]

[15] (a) In some embodiments of the CARs of the disclosure, including those that include (1) a binding element that specifically binds to a target and (2) an extracellular element that includes a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 17 nm to about 25 nm, including the endpoints, and the spacer sequence includes one or more sequences selected from the group consisting of SEQ ID NOs: 1-44. In some embodiments, the spacer sequence includes any one of the sequences of SEQ ID NOs: 1-44.

[0014]

[16] The present disclosure provides a nucleic acid sequence encoding a CAR of the present disclosure or any element thereof. In some embodiments, the nucleic acid sequence further comprises one or more control sequences. In some embodiments, the control sequence comprises a human sequence. In some embodiments, the control sequence comprises a sequence isolated or derived from a human sequence. In some embodiments, the control sequence comprises a sequence isolated or derived from a mammalian sequence. In some embodiments, the nucleic acid sequence further comprises at least one repeat of the control sequence. In some embodiments, the nucleic acid sequence further comprises at least two repeats of the control sequence and a linking sequence located between the two repeats. In some embodiments, the one or more control sequences include, but are not limited to, sequences that encode, comprise, or consist of a response element, promoter, enhancer, repressor, insulator, silencer, intron, exon, untranslated region (UTR), 5'UTR, 3'UTR, post-translational control sequence (PRE), or any minimal functional sequence thereof. In some embodiments, the response element comprises an inducible response element. In some embodiments, the promoter comprises an inducible promoter. Alternatively, or additionally, the promoter comprises a minimal promoter, hi some embodiments, the PRE comprises a woodchuck PRE (WPRE).

[0015]

[17] The present disclosure provides a vector comprising a nucleic acid sequence of the present disclosure, including a nucleic acid sequence encoding a CAR of the present disclosure. In some embodiments, the vector further comprises one or more control sequences of the present disclosure. In some embodiments, the control sequence comprises a human sequence. In some embodiments, the control sequence comprises a sequence isolated or derived from a human sequence. In some embodiments, the control sequence comprises a sequence isolated or derived from a mammalian sequence. In some embodiments, the nucleic acid sequence further comprises at least one repeat of the control sequence. In some embodiments, the nucleic acid sequence further comprises at least two repeats of the control sequence, and a linking sequence located between the two repeats. In some embodiments, the one or more control sequences include, but are not limited to, sequences that encode, comprise, or consist of a response element, promoter, enhancer, repressor, insulator, silencer, intron, exon, untranslated region (UTR), 5'UTR, 3'UTR, post-translational control sequence (PRE), or any minimal functional sequence thereof. In some embodiments, the response element comprises an inducible response element. In some embodiments, the promoter comprises an inducible promoter. Alternatively or additionally, the promoter comprises a minimal promoter. In some embodiments, the PRE comprises a woodchuck PRE (WPRE). In some embodiments, the vector further comprises one or more integration sequences, including, but not limited to, terminal repeats, inverted terminal repeats, long terminal repeats, sequences homologous to a target sequence, sequences that may promote homologous recombination, transposable elements, and / or transposon sequences. In some embodiments, the vector further comprises one or more inverted terminal repeats (ITRs). In some embodiments, the vector further comprises a sequence encoding a selection element. In some embodiments, the vector further comprises a polycistronic sequence or a self-cleaving sequence. In some embodiments, the vector further comprises a polycistronic sequence or a self-cleaving sequence, the vector further comprises a sequence encoding a selection element. In some embodiments, the vector further comprises a polycistronic sequence or a self-cleaving sequence, the vector further comprises a safety switch.In some embodiments, the vector further comprises a polycistronic sequence or a self-cleaving sequence, the vector further comprises a second therapeutically effective agent. In some embodiments, the self-cleaving sequence comprises a 2A self-cleaving sequence. In some embodiments, the 2A self-cleaving sequence includes a foot and mouth disease virus (F2A) sequence, an equine rhinitis A virus (E2A) sequence, a porcine teschovirus-1 2A (P2A) sequence, or a Thosea asigna virus 2 (T2A) sequence. In some embodiments, the 2A self-cleaving sequence comprises GDVEXNPGP (SEQ ID NO: 80). In some embodiments, the P2A self-cleaving sequence comprises ATNFSLLKQAGDVEENPGP (SEQ ID NO: 81). In some embodiments, the T2A self-cleaving sequence comprises EGRGSLLTLGDVEENPGP (SEQ ID NO: 83). In some embodiments, the E2A self-cleaving sequence comprises QCTNYALLKLAGDVESNPGP (SEQ ID NO: 84).

[0016]

[18] The present disclosure provides compositions comprising a CAR of the present disclosure or any element thereof. In some embodiments, the CAR of the present disclosure comprises a "first generation" CAR. In some embodiments, the CAR of the present disclosure comprises an extracellular element, a transmembrane element, and an intracellular element, where the intracellular element comprises a CD3 signaling element. In some embodiments, the CAR of the present disclosure comprises a "second generation" CAR. In some embodiments, the CAR of the present disclosure comprises an extracellular element, a transmembrane element, and an intracellular element, where the intracellular element comprises a CD28 signaling element and a CD3 signaling element. In some embodiments, the CAR of the present disclosure comprises a "third generation" CAR. In some embodiments, the CAR of the present disclosure comprises an extracellular element, a transmembrane element, and an intracellular element, where the intracellular element comprises a CD28 signaling element, a 41BB signaling element, and a CD3 signaling element. In some embodiments, the CAR of the present disclosure comprises a "fourth generation" CAR. In some embodiments, the CAR of the present disclosure comprises an extracellular element, a transmembrane element, and an intracellular element, wherein the intracellular element comprises a CD28 signaling element, a 41BB signaling element, an inducible element, and a CD3 signaling element. In some embodiments, the inducible element responds to CAR activation by inducing the expression of the transgenic sequence. In some embodiments of the CAR of the present disclosure, the extracellular domain comprises a binding element, wherein the binding element comprises an scFv. In all embodiments of the CAR of the present disclosure, the extracellular element comprises a spacer sequence.

[0017]

[19] The present disclosure provides a CAR that, when expressed in a T cell, transforms the modified cell into a TRUCK, i.e., T-cell Redirected for Universal Cytokine-mediated Killing. In some embodiments, the CAR comprises a "fourth generation" CAR. In some embodiments, the CAR of the present disclosure comprises an extracellular element, a transmembrane element, and an intracellular element, where the intracellular element comprises a CD28 signaling element, a 41BB signaling element, an inducible element, and a CD3 signaling element. In some embodiments, the inducible element responds to CAR activation by inducing expression of the transgenic sequence. In some embodiments, the inducible sequence encodes a cytokine.

[0018]

[20] The present disclosure provides compositions comprising the nucleic acids of the present disclosure.

[21] The present disclosure provides a composition comprising a vector of the present disclosure. In some embodiments, the vector is an expression vector capable of expressing a nucleic acid sequence of the present disclosure in a cell of the present disclosure. In some embodiments, the expression vector is capable of expressing a nucleic acid sequence of the present disclosure in a mammalian cell. In some embodiments, the expression vector is capable of expressing a nucleic acid sequence of the present disclosure in a human cell. In some embodiments, the expression vector comprises a plasmid. In some embodiments, the vector is a delivery vector capable of introducing a nucleic acid sequence, amino acid sequence, or composition of the present disclosure into a cell of the present disclosure or a subject of the present disclosure. In some embodiments, the delivery vector comprises a viral vector. In some embodiments, the delivery vector comprises a non-viral vector.

[0019]

[22] The present disclosure provides a cell comprising a CAR of the present disclosure. In some embodiments, the cell is an immune cell or a precursor thereof. In some embodiments, the immune cell or precursor is a mammalian cell. In some embodiments, the immune cell or precursor is a human cell. In some embodiments, the immune cell or precursor is a stem cell, an adult stem cell, a bone marrow cell, a hematopoietic stem cell (HSC), an induced pluripotent stem cell (iPSC), a dedifferentiated cell, or a transdifferentiated cell. In some embodiments, the stem cell is not a human embryonic stem cell. In some embodiments, the cell is a primary cell, a cultured cell, an immortalized cell, or a cell isolated or derived from a cell line. In some embodiments, the immune cell is a T cell, a B cell, a natural killer (NK) cell, or a macrophage. In some embodiments, the T cell is a memory T cell, an effector T cell, a helper T cell, or a natural killer (NK) T cell. In some embodiments, the T cell is an alpha beta (αβ) effector T cell or a gamma delta (γδ) T cell. In some embodiments, the T cells are unstimulated T cells or naive T cells. In some embodiments, the T cells are stimulated T cells, stem cell memory T cells (TSCM), immune memory cells with naive phenotype (TMNP), or effector T cells. In some embodiments, the memory T cells are stem cell memory T cells, immune memory cells with naive phenotype (TMNP), central memory T cells (TCM), effector memory T cells (TEM), or residual memory T cells (TRM). In some embodiments, the memory T cells are stimulated cells that have been contacted with one or more antigens or have had one or more contacts with the same antigen, and the memory T cells are stem cell memory T cells, immune memory cells with naive phenotype (TMNP), central memory T cells (TCM), effector memory T cells (TEM), or residual memory T cells (TRM).

[0020]

[23] The present disclosure provides a cell comprising a nucleic acid of the present disclosure.

[24] The present disclosure provides a cell comprising the vector of the present disclosure.

[25] The present disclosure provides a composition comprising a cell of the present disclosure.

[0021]

[26] The present disclosure provides a pharmaceutical composition comprising a CAR of the present disclosure and a pharma- ceutically acceptable carrier.

[27] The present disclosure provides a pharmaceutical composition comprising the nucleic acid of claim 23 and a pharma- ceutically acceptable carrier.

[0022]

[28] The present disclosure provides a pharmaceutical composition comprising a vector of the present disclosure and a pharma- ceutically acceptable carrier.

[29] The present disclosure provides a pharmaceutical composition comprising a cell of the present disclosure and a pharma- ceutically acceptable carrier.

[0023]

[30] In some embodiments of the present disclosure, the pharmaceutical composition is formulated for systemic administration to a subject of the present disclosure. In some embodiments, the pharmaceutical composition is formulated for systemic administration to a subject by intravenous injection or infusion. In some embodiments, the pharmaceutical composition is formulated for local administration to a subject of the present disclosure. In some embodiments, the pharmaceutical composition is formulated for local administration to a subject by intratumoral injection or infusion. In some embodiments, the pharmaceutical acceptable carrier is a sterile solution containing one or more agents, individually or in combination, to maintain or improve the viability of the cell therapy, reduce immunogenicity, reduce discomfort at the subject's injection site, and / or maintain one or more of physiological pH, cell density, and viscosity.

[0024]

[31] The present disclosure provides the use of the CAR of the present disclosure, the nucleic acid of the present disclosure, the vector of the present disclosure, any one of the cells of the present disclosure, or the pharmaceutical composition of the present disclosure for the manufacture of a medicament. In some embodiments, the medicament can treat or is suitable for treating a disease or disorder of the present disclosure. In some embodiments, the medicament is formulated for use in treating a disease or disorder of the present disclosure. In some embodiments, the CAR selectively targets an antigen or epitope associated with a disease or disorder as a causative agent of the disease or disorder, a regulator of a causative agent of the disease or disorder, a biomarker of the disease or disorder, an indication of the level of severity of the disease or disorder, and / or an indication of the prognosis of the disease or disorder.

[0025]

[32] The present disclosure provides the use of the CAR of the present disclosure, the nucleic acid of the present disclosure, the vector of the present disclosure, any one of the cells of the present disclosure, or the pharmaceutical composition of the present disclosure for the treatment or prevention of a disease or disorder. In some embodiments, the disease or disorder comprises cancer. In some embodiments, the CAR selectively targets an antigen or epitope associated with a disease or disorder as a causative agent of the disease or disorder, a regulator of a causative agent of the disease or disorder, a biomarker of the disease or disorder, an indication of the level of severity of the disease or disorder, and / or an indication of the prognosis of the disease or disorder.

[0026]

[33] The present disclosure provides a method of treating a disease or disorder, comprising administering to a subject a therapeutically effective amount of a CAR of the present disclosure, a nucleic acid of the present disclosure, a vector of the present disclosure, a cell of any one of the present disclosures, or a pharmaceutical composition of the present disclosure, whereby the severity of a sign or symptom of the disease or disorder is reduced, thereby treating the disease or disorder. In some embodiments, the disease or disorder comprises cancer. In some embodiments, the CAR selectively targets an antigen or epitope associated with the disease or disorder as a causative agent of the disease or disorder, a regulator of a causative agent of the disease or disorder, a biomarker of the disease or disorder, an indication of the level of severity of the disease or disorder, and / or an indication of the prognosis of the disease or disorder.

[0027]

[34] The present disclosure provides a method of preventing a disease or disorder, comprising administering to a subject a therapeutically effective amount of a CAR of the present disclosure, a nucleic acid of the present disclosure, a vector of the present disclosure, a cell of any one of the present disclosures, or a pharmaceutical composition of the present disclosure, wherein the onset or recurrence of a sign or symptom of the disease or disorder is delayed or inhibited, thereby preventing the disease or disorder. In some embodiments, the disease or disorder comprises cancer. In some embodiments, the CAR selectively targets an antigen or epitope associated with the disease or disorder as a causative agent of the disease or disorder, a regulator of a causative agent of the disease or disorder, a biomarker of the disease or disorder, an indication of the level of severity of the disease or disorder, and / or an indication of the prognosis of the disease or disorder. [Brief description of the drawings]

[0028] [Figure 1]

[35] Figure 1 is a schematic diagram of an exemplary CAR of the present disclosure, which in some embodiments comprises an extracellular element (which in some embodiments comprises a binding element and a spacer element), a transmembrane element, and an intracellular element (optionally comprising a costimulatory element and one or more signaling elements). [Diagram 2]

[36] Figure 2 is a schematic showing exemplary spatial relationships between epitopes on the surface of a target cell and binding elements on the surface of a CAR T cell, including illustrations of exemplary measurements of intercellular distance. [Diagram 3]

[37] Figure 3 is a schematic diagram of an exemplary CAR construct of the present disclosure, showing, in some embodiments, the organization of the different components. [Figure 4]

[38] Figure 4 is a schematic diagram of an exemplary HA-specific CAR construct of the present disclosure. [Diagram 5]

[39] Figure 5 is a schematic diagram of an exemplary HA-epitope construct showing an exemplary configuration of the separate elements of the HA-epitope construct. [Figure 6]

[40] Figure 6 is a schematic diagram of an exemplary HA-epitope construct of the present disclosure. [Figure 7A]

[41] Figure 7A is a plot showing the percentage of transduced target cells as measured by expression of the transduction marker GFP on live A549-NLR cells. [Figure 7B]

[42] Figure 7B is a plot showing the percentage of HA tag surface expression on transduced GFP+ cells. The data show that the expressed epitope is detectable on A549-NLR target cells. [Figure 7C]

[43] Figure 7C is a plot showing the median fluorescence intensity (MFI) for bound anti-HA antibody on live transduced cells (GRP+ cells). [Figure 8-1]

[44] Figures 8A and 8B are plots showing the percentage of transduced cells as measured by surface expression of EGFR-derived transduction markers on live primary T cells in donor 1 and donor 2, respectively. [Figure 8-2]

[45] Figures 8C and 8D are plots showing median fluorescence intensity (MFI) as a measure of transduction efficiency for bound HA-Fc protein on live transduced cells (EGFRt+ cells) in donor 1 and donor 2, respectively. [Figure 9A]

[46] Figures 9A, 9B, 9C, and 9D are a series of plots showing the effect of the indicated spacers on anti-HA scFv-induced cytotoxicity in the presence of NucLight Red-labeled A549 target cells expressing the membrane-proximal HA target epitope in donor 1 (A549-HA target 1). [Figure 9B] Same as above. [Figure 9C] Same as above. [Figure 9D] Same as above. [Figure 10A]

[47] Figures 10A, 10B, 10C, and 10D are a series of plots showing the effect of the indicated spacers on anti-HA scFv-induced cytotoxicity in the presence of NucLight Red-labeled A549 target cells expressing the membrane-proximal HA target epitope in donor 2 (A549-HA target 1). [Figure 10B] Same as above. [Figure 10C] Same as above. [Figure 10D] Same as above. [Figure 11A]

[48] ​​Figures 11A, 11B, 11C, and 11D are a series of plots showing the effect of the indicated spacers on anti-HA scFv-induced cytotoxicity in the presence of NucLight Red-labeled A549 target cells expressing the HA target epitope at intermediate membrane distances in donor 1 (A549-HA target 6). [Figure 11B] Same as above. [Figure 11C] Same as above. [Figure 11D] Same as above. [Figure 12A]

[49] Figures 12A, 12B, 12C, and 12D are a series of plots showing the effect of the indicated spacers on anti-HA scFv-induced cytotoxicity in the presence of NucLight Red-labeled A549 target cells expressing the HA target epitope at intermediate membrane distances in donor 2 (A549-HA target 6). [Figure 12B] Same as above. [Figure 12C] Same as above. [Figure 12D] Same as above. [Figure 13A]

[50] Figures 13A, 13B, 13C, and 13D are a series of plots showing the effect of the indicated spacers on anti-HA scFv-induced cytotoxicity in the presence of NucLight Red-labeled A549 target cells expressing the membrane-distal HA target epitope in donor 1 (A549-HA target 16). [Figure 13B] Same as above. [Figure 13C] Same as above. [Figure 13D] Same as above. [Figure 14A]

[51] Figures 14A, 14B, 14C, and 14D are a series of plots showing the effect of the indicated spacers on anti-HA scFv-induced cytotoxicity in the presence of NucLight Red-labeled A549 target cells expressing the membrane-distal HA target epitope in donor 2 (A549-HA target 16). [Figure 14B] Same as above. [Figure 14C] Same as above. [Figure 14D] Same as above. [Figure 15A]

[52] Figures 15A, 15B, 15C, and 15D are a series of plots showing the effect of the indicated spacers on anti-HA scFv-induced cytotoxicity in the presence of NucLight Red-labeled A549 target cells that do not bear the HA target epitope. [Figure 15B] Same as above. [Figure 15C] Same as above. [Figure 15D] Same as above. [Figure 16]

[53] Figures 16A and 16B are plots showing IFN-γ secretion profiles on HA CAR-T cells from donor 1 and donor 2, respectively, expressing the indicated spacers in the absence of target cells. [Figure 17A]

[54] Figure 17A is a series of scatter plots summarizing the effect of systematically varying the HA epitope intermembrane distance with respect to optimal anti-HA CAR spacer length on target-dependent cytokine secretion and killing AUC for donor 1 CAR T cells co-cultured with A549-HA target cells (i.e., target cells 1 to 4, see Table 2). [Figure 17B]

[55] Figure 17B is a series of scatter plots summarizing the effect of systematically varying the HA epitope intermembrane distance with respect to optimal anti-HA CAR spacer length on target-dependent cytokine secretion and killing AUC for donor 2 CAR T cells co-cultured with A549-HA target cells (i.e., target cells 1 to 4, see Table 2). [Figure 18A]

[56] Figure 18A is a series of scatter plots summarizing the effect of systematically varying the HA epitope intermembrane distance with respect to optimal anti-HA CAR spacer length on target-dependent cytokine secretion and killing AUC for donor 1 CAR T cells co-cultured with A549-HA target cells (i.e., target cells 5 to 8, see Table 2). [Figure 18B]

[57] Figure 18B is a series of scatter plots summarizing the effect of systematically varying the HA epitope intermembrane distance with respect to optimal anti-HA CAR spacer length on target-dependent cytokine secretion and killing AUC for donor 2 CAR T cells co-cultured with A549-HA target cells (i.e., target cells 5 to 8, see Table 2). [Figure 19A]

[58] Figure 19A is a series of scatter plots summarizing the effect of systematically varying the HA epitope intermembrane distance with respect to optimal anti-HA CAR spacer length on target-dependent cytokine secretion and killing AUC for donor 1 CAR T cells co-cultured with A549-HA target cells (i.e., target cells 9 to 12, see Table 2). [Figure 19B]

[59] Figure 19B is a series of scatter plots summarizing the effect of systematically varying the HA epitope intermembrane distance with respect to optimal anti-HA CAR spacer length on target-dependent cytokine secretion and killing AUC for donor 2 CAR T cells co-cultured with A549-HA target cells (i.e., target cells 9 to 12, see Table 2). [Figure 20A]

[60] Figure 20A is a series of scatter plots summarizing the effect of systematically varying the HA epitope intermembrane distance with respect to optimal anti-HA CAR spacer length on target-dependent cytokine secretion and killing AUC for donor 1 CAR T cells co-cultured with A549-HA target cells (i.e., target cells 13 to 16, see Table 2). [Figure 20B]

[61] Figure 20B is a series of scatter plots summarizing the effect of systematically varying the HA epitope intermembrane distance with respect to optimal anti-HA CAR spacer length on target-dependent cytokine secretion and killing AUC for donor 2 CAR T cells co-cultured with A549-HA target cells (i.e., target cells 13 to 16, see Table 2). [Figure 21A]

[62] Figure 21A is a pair of overlaid scatter plots summarizing the effect of cell-cell distance on the AUC calculated from IncuCyte killing curves and IFN-γ secretion in donor 1 and donor 2, respectively, measured 24 hours after co-culture. [Figure 21B]

[63] Figure 21B is a pair of overlaid scatter plots summarizing the effect of intercellular distance on IFN-γ secretion in donor 1 and donor 2, respectively. [Figure 22A]

[64] Figure 22A is a series of plots showing IFN-γ secretion levels for CAR spacer CD27_1 and spacer 1 expressed in donor 1 and donor 2 paired with each target cell line (i.e., targets 1 to 16). [Figure 22B]

[65] Figure 22B is a series of plots showing IFN-γ secretion levels for CAR spacer ICOS and spacer 29 expressed in donor 1 and donor 2 paired with each target cell line (i.e., targets 1 to 16). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029]

[66] The present disclosure demonstrates that the optimal intercellular distance between the CAR-expressing cells and the antigen-expressing cells of the immune synapse is greater than was expected prior to the present disclosure. There is an upper limit of about 15 nm for the functional intercellular distance between the TCR-expressing cells and the antigen-expressing cells of the immune synapse. In contrast, the present disclosure demonstrates that the CAR optimally stimulates the cells expressing it when the intercellular distance between the CAR-expressing cells and the antigen-expressing cells of the immune synapse is about 17 nm to about 25 nm. Within this range, the present disclosure demonstrates that the CAR optimally stimulates the cells expressing it when the intercellular distance between the CAR-expressing cells and the antigen-expressing cells of the immune synapse is about 20 nm. The intercellular distance of the immune synapse with the CAR-expressing cells is a variable that achieves better activation or stimulation via the CAR. In some embodiments, the spacer of the CAR of the present disclosure that achieves an intercellular distance of about 17 nm to about 25 nm between the CAR-expressing cells and the antigen-expressing cells of the immune synapse results in a better optimally activated CAR-expressing cells of the present disclosure. In some embodiments, spacers of the CARs of the present disclosure that achieve an intercellular distance of about 20 nm between the CAR-expressing cells and the antigen-expressing cells of the immune synapse result in better optimally activated CAR-expressing cells of the present disclosure.

[0030]

[67] The present disclosure provides the physical length of each spacer sequence described by the present disclosure. The physical length of a spacer of a given sequence can refer to the length of the spacer protein when expressed by a cell of the present disclosure. In some embodiments, two given spacers, each having the same number of amino acids, may have different physical lengths due to the properties of the amino acids for each spacer sequence, the secondary structure of the spacer, and / or the interaction of the expressed and folded protein with its environment, including additional proteins fused to it. In some embodiments, the physical length of a spacer of a given sequence when expressed by a cell of the present disclosure is about the same or approximately the same as the physical length of the spacer when expressed by any cell under conditions suitable for transcribing the nucleic acid sequence, conditions suitable for translating the nucleic acid into amino acids, conditions suitable for folding the amino acid sequence into a functional protein, and / or conditions suitable for forming covalent or non-covalent bonds.

[0031]

[68] The present disclosure provides a chimeric antigen receptor (CAR) comprising: (a) an extracellular element comprising (i) a binding element that specifically binds to a target and (ii) a spacer sequence; (b) a transmembrane element; and (c) an intracellular element, wherein when the CAR is expressed on the surface of a cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 17 nm to about 25 nm, including the endpoints. In some embodiments, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 20 nm. In some embodiments, the intercellular distance between the CAR-expressing cell and the target-expressing cell is 20 nm. It is unexpected that the length of the spacer sequence has been discovered as a variable that results in better CAR signaling efficiency, for example, when expressed on the surface of a cell and when in contact with an antigen to which it can specifically bind. Based on previous studies of the immune synapse, which involves presentation of a target by the major histocompatibility complex (MHC) and signaling through the T cell receptor (TCR), one of skill in the art would readily recognize that an intercellular distance of 14-15 nm at the synapse provides effective TCR signaling. However, the present disclosure provides empirical evidence showing that when an immune synapse is formed by MHC presenting an antigen to a CAR, the optimal intercellular distance at the immune synapse is about 17 nm to about 25 nm, with the efficacy peak occurring at about 20 nm. The CARs of the present disclosure are designed to include spacer sequences to achieve an intercellular distance of about 17 nm to about 25 nm, preferably about 20 nm, at this MHC-CAR synapse.

[0032]

[69] The following table shows the relationship between target distance from the target-expressing cell, the length of the spacer sequence and intercellular distance.

[70]

[0033] [Table A-1]

[0034] [Table A-2]

[0035]

Table A-3

[0036]

Table A-4

[0037]

Table A-5

[0038]

[71]

[0039]

Table B-1

[0040]

Table B-2

[0041]

Table B-3

[0042]

[72]

[0043]

Table C-1

[0044]

Table C-2

[0045]

Table C-3

[0046] [Table C-4]

[0047] [Table C-5]

[0048] [Table C-6]

[0049] [Table C-7]

[0050]

[73] The present disclosure provides a chimeric antigen receptor (CAR) comprising: (a) an extracellular element comprising (i) a binding element that specifically binds to a target and (ii) a spacer sequence; (b) a transmembrane element; and (c) an intracellular element, wherein when the CAR is expressed on the surface of a cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 17 nm to about 25 nm, including the endpoints. The design of the CAR of the present disclosure is based on studies provided herein, the results of which demonstrate that when a CAR includes a spacer of a given length sufficient to achieve an intercellular distance of about 17 nm to about 25 nm, the CAR activity is superior when compared to a CAR having a spacer that does not provide this intercellular distance, or when compared to a CAR lacking the spacer sequence. The outcome effect variables of a spacer length sufficient to achieve an intercellular distance in the range of about 17 nm to about 25 nm have not been elucidated prior to the present disclosure. Additionally, because the CARs of the present disclosure can include binding elements (e.g., scFvs, monoclonal antibodies, protein scaffolds) of various lengths and formats, and targets can have various sizes and distances from the cells expressing them, the present disclosure provides detailed guidance on the selection of spacer sequences to create CARs that achieve intercellular distances of about 17 nm to about 25 nm for each binding element and target.

[0051] Chimeric antigen receptors (CARs)

[74] The present disclosure provides a CAR comprising (a) an extracellular element comprising (i) a binding element that specifically binds to a target and (ii) a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, wherein when the CAR is expressed on the surface of a cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 17 nm to about 25 nm, including the endpoints. In some embodiments, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 20 nm.

[0052]

[75] In some embodiments of the CAR of the present disclosure, the CAR can comprise one or more human sequences. In some embodiments, the CAR can comprise one or more sequences isolated or derived from a human sequence. In some embodiments, the one or more sequences derived from a human sequence can have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or any percentage therebetween identity to a naturally occurring human sequence. In some embodiments, the one or more sequences derived from a human sequence can have one or more synthetic amino acids. In some embodiments, the CAR can comprise one or more sequences modified to be covalently or non-covalently linked to a synthetic moiety that includes a synthetic amino acid. In some embodiments, the CAR can comprise one or more sequences that are not both present in the naturally occurring sequence or do not function in the same endogenous pathway, making the CAR a chimeric receptor. In some embodiments, the CAR can comprise one or more sequences isolated or derived from a non-human sequence. In some embodiments, one or more sequences derived from a non-human sequence can have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or any percentage therebetween, identity to a naturally occurring non-human sequence.

[0053]

[76] (a) In some embodiments of the CARs of the present disclosure, including those that include (1) a binding element that specifically binds to a target and (2) an extracellular element that includes a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 17 nm to about 25 nm, including the endpoints, and the binding element includes an antigen recognition domain. In some embodiments, the binding element includes a first antigen recognition domain and a second antigen recognition domain. In some embodiments, the binding element or antigen recognition domain includes an antibody (including, but not limited to, a monoclonal antibody), a protein scaffold, an antibody mimic, or an antigen-binding sequence thereof. In some embodiments, the binding element or antigen recognition domain includes one or more of a monoclonal antibody, a single domain antibody, a domain antibody, a VH or VHH, a monobody, or a single chain variable fragment (scFv).

[0054]

[77] (a) In some embodiments of the CARs of the present disclosure, including those that include (1) a binding element that specifically binds to a target and (2) an extracellular element that includes a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 17 nm to about 25 nm, including the endpoints, and the binding element includes an antigen recognition domain. In some embodiments, the transmembrane element includes a sequence isolated or derived from the sequence of a CD4 protein. In some embodiments, the transmembrane element includes a sequence isolated or derived from the sequence of a CD8 protein. In some embodiments, the transmembrane element includes a sequence isolated or derived from the sequence of a CD28 protein. In some embodiments, the CD28 protein is 1 MLRLLLALNL FPSIQVTGNK ILVKQSPMLV AYDNAVNLSC KYSYNLFSRE FRASLHKGLD 61 SAVEVCVVYG NYSQQLQVYS KTGFNCDGKL GNESVTFYLQ NLYVNQTDIY FCKIEVMYPP 121 PYLDNEKSNG TIIHVKGKHL CPSPLFPGPS KPFWVLVVVG GVLACYSLLV TVAFIIFWVR 181 SKRSRLLHSD YMNMTPRRPG PTRKHYQPYA PPRDFAAYRS (SEQ ID NO: 45 and UniProtKB Accession No. P10747-1). In some embodiments, the transmembrane element comprises a sequence isolated or derived from the sequence of a CD28 protein. In some embodiments, the CD28 protein comprises the amino acid sequence of 1 MLRLLLALNL FPSIQVTGKH LCPSPLFPGP SKPFWVLVVV GGVLACYSLL VTVAFIIFWV 61 RSKRSRLLHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYR S (SEQ ID NO: 46 and UniProtKB Accession No. P10747-2). In some embodiments, the CD28 protein comprises the amino acid sequence of 1 MLRLLLALNL FPSIQVTGNK ILVKQSPMLV AYDNAVNLSY NEKSNGTIIH VKGKHLCPSP 61 LFPGPSKPFW VLVVVGGVLA CYSLLVTVAF IIFWVRSKRS RLLHSDYMNM TPRRPGPTRK 121 HYQPYAPPRD FAAYRS (SEQ ID NO: 47 and UniProtKB Accession No. P10747-3). In some embodiments, the CD28 protein comprises the amino acid sequence of 1 MLRLLLALNL FPSIQVTGNK ILVKQSPMLV AYDNAVNLSW KHLCPSPLFP GPSKPFWVLV 61 VVGGVLACYS LLVTVAFIIF WVRSKRSRLL HSDYMNMTPR RPGPTRKHYQ PYAPPRDFAA 121 YRS (SEQ ID NO: 48 and UniProtKB Accession No. P10747-4). In some embodiments, the CD28 protein comprises the amino acid sequence of 1 MLRLLLALNL FPSIQVTGNK ILVKQSPMLV AYDNAVNLSY NEKSNGTIIH VKGEE (SEQ ID NO: 49 and UniProtKB Accession No. P10747-5). In some embodiments, the CD28 protein comprises the amino acid sequence of 1 MLRLLLALNL FPSIQVTGNK ILVKQSPMLV AYDNAVNLSY NEKSNGTIIH VKGKHLCPSP 61 LFPGPSKPYA PPRDFAAYRS (SEQ ID NO:50 and UniProtKB Accession No. P10747-6). In some embodiments, the CD28 protein comprises the amino acid sequence of 1 MPCGLSALIM CPKGMVAVVV AVDDGDSQAL AGNKILVKQS PMLVAYDNAV NLSCKYSYNL 61 FSREFRASLH KGLDSAVEVC VVYGNYSQQL QVYSKTGFNC DGKLGNESVT FYLQNLYVNQ 121 TDIYFCKIEV MYPPPYLDNE KSNGTIIHVK GKHLCPSPLF PGPSKPFWVL VVVGGVLACY 181 SLLVTVAFII FWVRSKRSRL LHSDYMNMTP RRPGPTRKHY QPYAPPRDFA AYRS (SEQ ID NO:51 and UniProtKB Accession No. P10747-7). In some embodiments, the CD28 protein comprises the amino acid sequence of FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO:52).

[0055]

[78] (a) In some embodiments of the CARs of the disclosure, including those that comprise (1) an extracellular element comprising a binding element that specifically binds to a target and (2) a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 17 nm to about 25 nm, including the endpoints, and the intracellular element comprises a costimulatory element that comprises a sequence isolated from or derived from one or more of a TNF receptor protein, an immunoglobulin-like protein, a cytokine receptor, an integrin, a signaling lymphocyte activation molecule (SLAM) protein, and an activating natural killer cell receptor. In some embodiments, the intracellular element comprises a costimulatory element comprising a sequence isolated or derived from one or more of CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, and MyD88.

[0056]

[79] (a) In some embodiments of the CARs of the present disclosure, including those that include (1) an extracellular element that includes a binding element that specifically binds to a target and (2) a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 17 nm to about 25 nm, including the endpoints, and the intracellular element includes a 4-1BB costimulatory element. In some embodiments, the 4-1BB costimulatory element includes 1 MGNSCYNIVA TLLLVLNFER TRSLQDPCSN CPAGTFCDNN RNQICSPCPP NSFSSAGGQR 61 TCDICRQCKG VFRTRKECSS TSNAECDCTP GFHCLGAGCS MCEQDCKQGQ ELTKKGCKDC 121 CFGTFNDQKR GICRPWTNCS LDGKSVLVNG TKERDVVCGP SPADLSPGAS SVTPPAPARE 181 PGHSPQIISF FLALTSTALL FLLFFLTLRF SVVKRGRKKL LYIFKQPFMR PVQTTQEEDG 241 CSCRFPEEEE GGCEL (SEQ ID NO:53 and UniProtKB Accession No. Q07011-1). In some embodiments, the 4-1BB costimulatory element comprises the sequence of KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO:54).

[0057]

[80] In some embodiments of the CARs of the present disclosure, including those that include (a) (1) a binding element that specifically binds to a target and (2) an extracellular element that includes a spacer sequence; (b) a transmembrane element; and (c) an intracellular element, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 17 nm to about 25 nm, including the endpoints, when the CAR is expressed on the surface of the cell, and the intracellular element includes a CD28 costimulatory element. In some embodiments, the CD28 costimulatory element includes a sequence of SEQ ID NOs: 45-52. In some embodiments, the CD28 costimulatory element includes a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or any percentage therebetween identity to SEQ ID NOs: 45-52.

[0058]

[81] (a) In some embodiments of the CARs of the present disclosure, including those that include (1) an extracellular element that includes a binding element that specifically binds to a target and (2) a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 17 nm to about 25 nm, including the endpoints, and the intracellular element includes a CD27 costimulatory element. In some embodiments, the CD27 costimulatory element is 1 MARPHPWWLC VLGTLVGLSA TPAPKSCPER HYWAQGKLCC QMCEPGTFLV KDCDQHRKAA 61 QCDPCIPGVS FSPDHHTRPH CESCRHCNSG LLVRNCTITA NAECACRNGW QCRDKECTEC 121 DPLPNPSLTA RSSQALSPHP QPTHLPYVSE MLEARTAGHM QTLADFRQLP ARTLSTHWPP 181 QRSLCSSDFI RILVIFSGMF LVFTLAGALF LHQRRKYRSN KGESPVEPAE PCHYSCPREE 241 EGSTIPIQED YRKPEPACSP (SEQ ID NO:55 and UniProt accession number P26842-1).

[0059]

[82] (a) In some embodiments of the CARs of the present disclosure, including those that include (1) an extracellular element that includes a binding element that specifically binds to a target and (2) a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 17 nm to about 25 nm, including the endpoints, and the intracellular element includes a CD3 zeta signaling element. In some embodiments, the CD3 zeta signaling element includes 1 MKWKALFTAA ILQAQLPITE AQSFGLLDPK LCYLLDGILF IYGVILTALF LRVKFSRSAD 61 APAYQQGQNQ LYNELNLGRR EEYDVLDKRR GRDPEMGGKP QRRKNPQEGL YNELQKDKMA 121 EAYSEIGMKG ERRRGKGHDG LYQGLSTATK DTYDALHMQA LPPR (SEQ ID NO: 76 and UniProtKB Accession No. P20963-1). In some embodiments, the CD3 zeta signaling element comprises the sequence of: 1 MKWKALFTAA ILQAQLPITE AQSFGLLDPK LCYLLDGILF IYGVILTALF LRVKFSRSAD 61 APAYQQGQNQ LYNELNLGRR EEYDVLDKRR GRDPEMGGKP RRKNPQEGLY NELQKDKMAE 121 AYSEIGMKGE RRRGKGHDGL YQGLSTATKD TYDALHMQAL PPR (SEQ ID NO:56 and UniProtKB Accession No. P20963-3). In some embodiments, the CD3 zeta signaling element comprises the sequence of: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:57) Contains an array of.

[0060]

[83] (a) In some embodiments of the CARs of the present disclosure, including those that include an extracellular element that includes (1) a binding element that specifically binds to a target and (2) a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 17 nm to about 25 nm, including the endpoints, and the intracellular element includes a 4-1BB costimulatory element of the present disclosure and a CD3 zeta signaling element of the present disclosure. In some embodiments, the intracellular element further includes a transduction marker. In some embodiments, the transduction marker includes an EGFR-derived marker. In some embodiments, the EGFR-derived marker includes a truncated EGFR (EGFRt). In some embodiments, the intracellular element further includes a sequence that includes a self-cleaving peptide. In some embodiments, the sequence that includes a self-cleaving peptide is located between the transduction marker and either the 4-1BB costimulatory element of the present disclosure or the CD3 zeta signaling element of the present disclosure.

[0061]

[84] (a) In some embodiments of the CARs of the disclosure, including those that include (1) a binding element that specifically binds to a target and (2) an extracellular element that includes a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 17 nm to about 25 nm, including the endpoints, and the binding element or antigen recognition domain specifically binds to the target, or any portion thereof, isolated from or derived from the extracellular antigen. In some embodiments, the target, or any portion thereof, isolated from or derived from the extracellular antigen, is present or expressed on the surface of the cell.

[0062]

[85] (a) In some embodiments of the CARs of the present disclosure, including those that include (1) a binding element that specifically binds to a target and (2) an extracellular element that includes a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 17 nm to about 25 nm, including the endpoints, and the target-expressing cell is in vivo, in vitro, or ex vivo. In some embodiments, the target-expressing cell includes one or more modifications. In some embodiments, the target-expressing cell is genetically modified.

[0063]

[86] In some embodiments of the CARs of the present disclosure, the CAR comprises a chimeric polypeptide comprising: (i) a chimeric fusion protein comprising an extracellular antigen recognition element; (ii) a transmembrane element; (iii) an intracellular signaling element comprising a functional signaling element derived from a stimulatory molecule; and (iv) a spacer linking the extracellular antigen recognition element to the transmembrane element.

[0064]

[87] In some embodiments of the CARs of the present disclosure, the CAR comprises a chimeric polypeptide comprising: (i) a chimeric fusion protein comprising an extracellular antigen recognition element; (ii) a transmembrane element; (iii) an intracellular signaling element comprising a functional signaling element derived from a costimulatory molecule and a functional signaling element derived from a stimulatory molecule; and (iv) a spacer linking the extracellular antigen recognition element to the transmembrane element.

[0065]

[88] In some embodiments of the CARs of the present disclosure, the CAR comprises a chimeric polypeptide comprising: (i) an extracellular antigen recognition element; (ii) a transmembrane element; and an intracellular signaling element comprising two functional signaling elements derived from one or more costimulatory molecules and a functional signaling element derived from a stimulatory molecule; and (iii) a spacer linking the extracellular antigen recognition element to the transmembrane element.

[0066]

[89] In some embodiments of the CARs of the present disclosure, the CAR comprises a chimeric (i) extracellular antigen recognition element, (ii) a transmembrane element, (iii) an intracellular signaling element comprising at least two functional signaling elements derived from one or more costimulatory molecules and a functional signaling element derived from a stimulatory molecule, and (iv) a spacer linking the extracellular antigen recognition element to the transmembrane element.

[0067]

[90] In some embodiments of the CAR of the present disclosure, the CAR comprises an optional leader sequence. In some embodiments, the leader sequence is located at the amino terminus (N-terminus) of the CAR. In some embodiments, the CAR comprises a fusion protein. In some embodiments, the leader sequence is located at the N-terminus of the CAR fusion protein. In some embodiments, the leader sequence is located at the N-terminus of the extracellular element of the CAR. In some embodiments, a cleavable sequence is located between the leader sequence and the CAR or between the leader sequence and the extracellular element of the CAR. In some embodiments, the cleavable sequence comprises a self-cleaving peptide. In some embodiments, the leader sequence is cleaved from the CAR during cellular processing. In some embodiments, cleavage of the leader sequence induces localization of the CAR to the cell membrane.

[0068]

[91] In some embodiments of the CAR of the present disclosure, the CAR comprises a fusion protein. In some embodiments, the fusion protein comprises a leader sequence. In some embodiments, a cleavable sequence is located between the leader sequence and the CAR. In some embodiments, the cleavable sequence comprises a self-cleaving peptide. In some embodiments, the leader sequence is cleaved from the CAR during cellular processing. In some embodiments, cleavage of the leader sequence induces localization of the CAR to the cell membrane.

[0069]

[92] (a) In some embodiments of CARs of the disclosure, including those that comprise (1) a binding element that specifically binds to a target and (2) an extracellular element comprising a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, the binding element specifically binds to a target, the target sequence is between about 3 nm and about 4 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell, the spacer sequence has a length of about 14 nm to about 19 nm, and when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 17 nm to 23 nm, including the endpoints. In some embodiments of CARs of the disclosure, including those that comprise (a) (1) a binding element that specifically binds to a target and (2) an extracellular element comprising a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, the binding element specifically binds to a target, the target sequence is between about 3 nm to about 4 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell, the spacer sequence has a length of about 14 nm to about 19 nm, the spacer sequence comprises any one of SEQ ID NOs: 6, 36, 43, 31, or 7, and when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 17 nm to 23 nm, including the endpoints.

[0070]

[93] (a) In some embodiments of CARs of the disclosure, including those that comprise (1) a binding element that specifically binds to a target and (2) an extracellular element comprising a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, the binding element specifically binds to a target, the target sequence is between about 5 nm and about 6 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell, the spacer sequence has a length of about 12 nm to about 17 nm, and when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 17 nm to 23 nm, including the endpoints. In some embodiments of CARs of the disclosure, including those that comprise (a) (1) a binding element that specifically binds to a target and (2) an extracellular element comprising a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, the binding element specifically binds to a target, the target sequence is between about 5 nm to about 6 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell, the spacer sequence has a length of about 12 nm to about 17 nm, the spacer sequence comprises any one of SEQ ID NOs: 36, 43, 31, or 7, and when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 17 nm to 23 nm, including the endpoints.

[0071]

[94] (a) In some embodiments of CARs of the disclosure, including those that comprise (1) a binding element that specifically binds to a target and (2) an extracellular element comprising a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, the binding element specifically binds to a target, the target sequence is between about 6 nm and about 7 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell, the spacer sequence has a length of about 11 nm to about 16 nm, and when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 17 nm to 23 nm, including the endpoints. In some embodiments of CARs of the disclosure, including those that comprise (a) (1) a binding element that specifically binds to a target and (2) an extracellular element comprising a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, the binding element specifically binds to a target, the target sequence is between about 6 nm to about 7 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell, the spacer sequence has a length of about 11 nm to about 16 nm, the spacer sequence comprises any one of SEQ ID NOs: 36, 43, 31, 7, 14, 27, or 40, and when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 17 nm to 23 nm, including the endpoints.

[0072]

[95] (a) In some embodiments of CARs of the disclosure, including those that comprise (1) a binding element that specifically binds to a target and (2) an extracellular element comprising a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, the binding element specifically binds to a target, the target sequence is between about 7 nm and about 8 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell, the spacer sequence has a length of about 10 nm to about 15 nm, and when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 17 nm to 23 nm, including the endpoints. In some embodiments of CARs of the disclosure, including those that comprise (a) (1) a binding element that specifically binds to a target and (2) an extracellular element comprising a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, the binding element specifically binds to a target, the target sequence is between about 7 nm to about 8 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell, the spacer sequence has a length of about 10 nm to about 15 nm, and the spacer sequence comprises any one of SEQ ID NOs: 43, 31, 7, 14, 27, 40, 32, or 42, and when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 17 nm to 23 nm, including the endpoints.

[0073]

[96] (a) In some embodiments of CARs of the disclosure, including those that comprise (1) a binding element that specifically binds to a target and (2) an extracellular element comprising a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, the binding element specifically binds to a target, the target sequence is between about 8 nm and about 9 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell, the spacer sequence has a length of about 9 nm to about 14 nm, and when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 17 nm to 23 nm, including the endpoints. In some embodiments of CARs of the disclosure, including those that comprise (a) (1) a binding element that specifically binds to a target and (2) an extracellular element comprising a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, the binding element specifically binds to a target, the target sequence is between about 8 nm to about 9 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell, the spacer sequence has a length of about 9 nm to about 14 nm, and the spacer sequence comprises any one of SEQ ID NOs: 43, 31, 7, 14, 27, 40, 32, 42, or 8, and when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 17 nm to 23 nm, including the endpoints.

[0074]

[97] (a) In some embodiments of CARs of the disclosure, including those that comprise (1) a binding element that specifically binds to a target and (2) an extracellular element comprising a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, the binding element specifically binds to a target, the target sequence is between about 9 nm and about 10 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell, the spacer sequence has a length of about 8 nm to about 13 nm, and when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 17 nm to 23 nm, including the endpoints. In some embodiments of CARs of the disclosure, including those that comprise (a) (1) a binding element that specifically binds to a target and (2) an extracellular element comprising a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, the binding element specifically binds to a target, the target sequence is between about 9 nm to about 10 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell, the spacer sequence has a length of about 8 nm to about 13 nm, the spacer sequence comprises any one of SEQ ID NOs: 14, 27, 40, 32, 42, 8, or 23, and when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 17 nm to 23 nm, including the endpoints.

[0075]

[98] (a) In some embodiments of CARs of the disclosure, including those that comprise (1) a binding element that specifically binds to a target and (2) an extracellular element comprising a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, the binding element specifically binds to a target, the target sequence is between about 10 nm and about 11 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell, the spacer sequence has a length of about 7 nm to about 12 nm, and when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 17 nm to 23 nm, including the endpoints. In some embodiments of CARs of the disclosure, including those that comprise (a) (1) a binding element that specifically binds to a target and (2) an extracellular element comprising a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, the binding element specifically binds to a target, the target sequence is between about 10 nm to about 11 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell, the spacer sequence has a length of about 7 nm to about 12 nm, and the spacer sequence comprises any one of SEQ ID NOs: 14, 27, 40, 32, 42, 8, 23, 9, 10, 24, 11, 3, or 18, and when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 17 nm to 23 nm, including the endpoints.

[0076]

[99] (a) In some embodiments of CARs of the disclosure, including those that comprise (1) a binding element that specifically binds to a target and (2) an extracellular element comprising a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, the binding element specifically binds to a target, the target sequence is between about 11 nm to about 12 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell, the spacer sequence has a length of about 6 nm to about 11 nm, and the spacer sequence comprises any one of SEQ ID NOs: 32, 42, 8, 23, 9, 10, 24, 11, 3, 18, 25, 38, 21, 29, 37, or 39, and when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 17 nm to 23 nm, including the endpoints. In some embodiments of CARs of the disclosure, including those that comprise (a) (1) a binding element that specifically binds to a target and (2) an extracellular element comprising a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, the binding element specifically binds to a target, the target sequence is between about 11 nm to about 12 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell, the spacer sequence has a length of about 6 nm to about 11 nm, and the spacer sequence comprises any one of SEQ ID NOs: 32, 42, 8, 23, 9, 10, 24, 11, 3, 18, 25, 38, 21, 29, 37, or 39, and when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 17 nm to 23 nm, including the endpoints.

[0077]

[0100] In some embodiments of the CARs of the disclosure, including those that comprise (a) (1) a binding element that specifically binds to a target and (2) an extracellular element comprising a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, the binding element specifically binds to a target, the target sequence is between about 12 nm to about 13 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell, the spacer sequence has a length of about 5 nm to about 10 nm, and the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 17 nm to 23 nm, including the endpoints, when the CAR is expressed on the surface of the cell. In some embodiments of CARs of the disclosure, including those that comprise (a) (1) a binding element that specifically binds to a target and (2) an extracellular element comprising a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, the binding element specifically binds to a target, and when the target sequence is expressed on the surface of the target cell, the target sequence is between about 12 nm to about 13 nm from the surface of the target cell, the spacer sequence has a length of about 5 nm to about 10 nm, and the spacer sequence comprises any one of SEQ ID NOs: 32, 42, 8, 23, 9, 10, 24, 11, 3, 18, 25, 38, 21, 29, 37, 39, 2, 15, 16, 26, 28, 1, or 17, and when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 17 nm to 23 nm, including the endpoints.

[0078]

[0101] In some embodiments of CARs of the disclosure, including those that comprise (a) (1) a binding element that specifically binds to a target and (2) an extracellular element comprising a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, the binding element specifically binds to a target, the target sequence is between about 13 nm to about 14 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell, the spacer sequence has a length of about 4 nm to about 9 nm, and when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 17 nm to 23 nm, including the endpoints. In some embodiments of CARs of the disclosure, including those that comprise (a) (1) a binding element that specifically binds to a target and (2) an extracellular element comprising a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, the binding element specifically binds to a target, and when the target sequence is expressed on the surface of the target cell, the target sequence is between about 13 nm to about 14 nm from the surface of the target cell, the spacer sequence has a length of about 4 nm to about 9 nm, and the spacer sequence comprises the sequence of any one of SEQ ID NOs: 23, 9, 10, 24, 11, 3, 18, 25, 38, 21, 29, 37, 39, 2, 15, 16, 26, 28, 1, 17, 12, 13, 19, 20, or 44, and when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 17 nm to 23 nm, including the endpoints.

[0079]

[0102] In some embodiments of the CARs of the disclosure, including those that comprise (a) (1) a binding element that specifically binds to a target and (2) an extracellular element comprising a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, the binding element specifically binds to the target, the target sequence is between about 14 nm to about 15 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell, the spacer sequence has a length of about 3 nm to about 8 nm, and when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 17 nm to 23 nm, including the endpoints. In some embodiments of CARs of the disclosure, including those that comprise (a) (1) a binding element that specifically binds to a target and (2) an extracellular element comprising a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, the binding element specifically binds to a target, and when the target sequence is expressed on the surface of the target cell, the target sequence is between about 14 nm to about 15 nm from the surface of the target cell, the spacer sequence has a length of about 3 nm to about 8 nm, and the spacer sequence comprises any one of SEQ ID NOs: 9, 10, 24, 11, 3, 18, 25, 38, 21, 29, 37, 39, 2, 15, 16, 26, 28, 1, 17, 12, 13, 19, 20, or 44, and when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 17 nm to 23 nm, including the endpoints.

[0080]

[0103] In some embodiments of CARs of the disclosure, including those that comprise (a) (1) a binding element that specifically binds to a target and (2) an extracellular element comprising a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, the binding element specifically binds to a target, the target sequence is between about 15 nm to about 16 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell, the spacer sequence has a length of about 2 nm to about 7 nm, and when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 17 nm to 23 nm, including the endpoints. In some embodiments of CARs of the disclosure, including those that comprise (a) (1) a binding element that specifically binds to a target and (2) an extracellular element comprising a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, the binding element specifically binds to a target, the target sequence is between about 15 nm to about 16 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell, the spacer sequence has a length of about 2 nm to about 7 nm, and the spacer sequence comprises any one of SEQ ID NOs: 25, 38, 21, 29, 37, 39, 2, 15, 16, 26, 28, 1, 17, 12, 13, 19, 20, or 44, and when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 17 nm to 23 nm, including the endpoints.

[0081]

[0104] In some embodiments of the CARs of the disclosure, including those that comprise (a) (1) a binding element that specifically binds to a target and (2) an extracellular element comprising a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, the binding element specifically binds to a target, the target sequence is between about 16 nm to about 17 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell, the spacer sequence has a length of about 1 nm to about 6 nm, and when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 17 nm to 23 nm, including the endpoints. In some embodiments of CARs of the disclosure, including those that comprise (a) (1) a binding element that specifically binds to a target and (2) an extracellular element comprising a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, the binding element specifically binds to a target, the target sequence is between about 16 nm to about 17 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell, the spacer sequence has a length of about 1 nm to about 6 nm, and the spacer sequence comprises any one of SEQ ID NOs: 2, 15, 16, 26, 28, 1, 17, 12, 13, 19, 20, or 44, and when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 17 nm to 23 nm, including the endpoints.

[0082]

[0105] In some embodiments of CARs of the disclosure, including those that comprise (a) (1) a binding element that specifically binds to a target and (2) an extracellular element comprising a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, the binding element specifically binds to a target, the target sequence is between about 3 nm to about 4 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell, the spacer sequence has a length of about 15 nm to about 18 nm, and when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 18 nm to 22 nm, including the endpoints. In some embodiments of CARs of the disclosure, including those that comprise (a) (1) a binding element that specifically binds to a target and (2) an extracellular element comprising a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, the binding element specifically binds to a target, the target sequence is between about 3 nm to about 4 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell, the spacer sequence has a length of about 15 nm to about 18 nm, the spacer sequence comprises the sequence of SEQ ID NO: 36, and when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 18 nm to 22 nm, including the endpoints.

[0083]

[0106] In some embodiments of the CARs of the disclosure, including those that comprise (a) (1) a binding element that specifically binds to a target and (2) an extracellular element comprising a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, the binding element specifically binds to a target, the target sequence is between about 5 nm to about 6 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell, the spacer sequence has a length of about 13 nm to about 16 nm, and when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 18 nm to 22 nm, including the endpoints. In some embodiments of the CARs of the disclosure, including those that comprise (a) (1) a binding element that specifically binds to a target and (2) an extracellular element comprising a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, the binding element specifically binds to a target, the target sequence is between about 5 nm to about 6 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell, the spacer sequence has a length of about 13 nm to about 16 nm, the spacer sequence comprises any one of SEQ ID NOs: 36, 43, 31, or 7, and when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 18 nm to 22 nm, including the endpoints.

[0084]

[0107] In some embodiments of the CARs of the disclosure, including those that comprise (a) (1) a binding element that specifically binds to a target and (2) an extracellular element comprising a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, the binding element specifically binds to a target, the target sequence is between about 6 nm to about 7 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell, the spacer sequence has a length of about 12 nm to about 15 nm, and the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 18 nm to 22 nm, including the endpoints, when the CAR is expressed on the surface of the cell. In some embodiments of the CARs of the disclosure, including those that comprise (a) (1) a binding element that specifically binds to a target and (2) an extracellular element comprising a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, the binding element specifically binds to a target, the target sequence is between about 6 nm to about 7 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell, the spacer sequence has a length of about 12 nm to about 15 nm, the spacer sequence comprises the sequence of any one of SEQ ID NOs: 43, 31, or 7, and when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 18 nm to 22 nm, including the endpoints.

[0085]

[0108] In some embodiments of CARs of the disclosure, including those that comprise (a) (1) a binding element that specifically binds to a target and (2) an extracellular element comprising a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, the binding element specifically binds to a target, the target sequence is between about 7 nm to about 8 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell, the spacer sequence has a length of about 11 nm to about 14 nm, and when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 18 nm to 22 nm, including the endpoints. In some embodiments of the CARs of the disclosure, including those that comprise (a) (1) a binding element that specifically binds to a target and (2) an extracellular element comprising a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, the binding element specifically binds to a target, the target sequence is between about 7 nm to about 8 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell, the spacer sequence has a length of about 11 nm to about 14 nm, and the spacer sequence comprises any one of SEQ ID NOs: 43, 31, 7, 14, 27, or 40, and when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 18 nm to 22 nm, including the endpoints.

[0086]

[0109] In some embodiments of CARs of the disclosure, including those that comprise (a) (1) a binding element that specifically binds to a target and (2) an extracellular element comprising a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, the binding element specifically binds to a target, the target sequence is between about 8 nm to about 9 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell, the spacer sequence has a length of about 10 nm to about 13 nm, and the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 18 nm to 22 nm, including the endpoints, when the CAR is expressed on the surface of the cell. In some embodiments of the CARs of the disclosure, including those that comprise (a) (1) a binding element that specifically binds to a target and (2) an extracellular element comprising a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, the binding element specifically binds to a target, the target sequence is between about 8 nm to about 9 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell, the spacer sequence has a length of about 10 nm to about 13 nm, the spacer sequence comprises any one of SEQ ID NOs: 14, 27, 40, 32, or 42, and when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 18 nm to 22 nm, including the endpoints.

[0087]

[0110] In some embodiments of CARs of the disclosure, including those that comprise (a) (1) a binding element that specifically binds to a target and (2) an extracellular element comprising a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, the binding element specifically binds to a target, the target sequence is between about 9 nm to about 10 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell, the spacer sequence has a length of about 9 nm to about 12 nm, and when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 18 nm to 22 nm, including the endpoints. In some embodiments of CARs of the disclosure, including those that comprise (a) (1) a binding element that specifically binds to a target and (2) an extracellular element comprising a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, the binding element specifically binds to a target, the target sequence is between about 9 nm to about 10 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell, the spacer sequence has a length of about 9 nm to about 12 nm, the spacer sequence comprises any one of SEQ ID NOs: 14, 27, 40, 32, 42, 8, or 23, and when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 18 nm to 22 nm, including the endpoints.

[0088]

[0111] In some embodiments of CARs of the disclosure, including those that comprise (a) (1) a binding element that specifically binds to a target and (2) an extracellular element comprising a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, the binding element specifically binds to a target, the target sequence is between about 10 nm to about 11 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell, the spacer sequence has a length of about 8 nm to about 11 nm, and when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 18 nm to 22 nm, including the endpoints. In some embodiments of CARs of the disclosure, including those that comprise (a) (1) a binding element that specifically binds to a target and (2) an extracellular element comprising a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, the binding element specifically binds to a target, the target sequence is between about 10 nm to about 11 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell, the spacer sequence has a length of about 8 nm to about 11 nm, the spacer sequence comprises any one of SEQ ID NOs: 32, 42, 8, or 23, and when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 18 nm to 22 nm, including the endpoints.

[0089]

[0112] In some embodiments of CARs of the disclosure, including those that comprise (a) (1) a binding element that specifically binds to a target and (2) an extracellular element comprising a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, the binding element specifically binds to a target, the target sequence is between about 11 nm to about 12 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell, the spacer sequence has a length of about 7 nm to about 10 nm, and the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 18 nm to 22 nm, including the endpoints, when the CAR is expressed on the surface of the cell. In some embodiments of CARs of the disclosure, including those that comprise (a) (1) a binding element that specifically binds to a target and (2) an extracellular element comprising a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, the binding element specifically binds to a target, the target sequence is between about 11 nm to about 12 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell, the spacer sequence has a length of about 7 nm to about 10 nm, and the spacer sequence comprises any one of SEQ ID NOs: 8, 23, 9, 10, 24, 11, 3, or 18, and when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 18 nm to 22 nm, including the endpoints.

[0090]

[0113] In some embodiments of the CARs of the disclosure, including those that comprise (a) (1) a binding element that specifically binds to a target and (2) an extracellular element comprising a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, the binding element specifically binds to the target, the target sequence is between about 12 nm to about 13 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell, the spacer sequence has a length of about 6 nm to about 9 nm, and when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 18 nm to 22 nm, including the endpoints. In some embodiments of CARs of the disclosure, including those that comprise (a) (1) a binding element that specifically binds to a target and (2) an extracellular element comprising a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, the binding element specifically binds to a target, the target sequence is between about 12 nm to about 13 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell, the spacer sequence has a length of about 6 nm to about 9 nm, and the spacer sequence comprises any one of SEQ ID NOs: 23, 9, 10, 24, 11, 3, 18, 25, 38, 21, 29, 37, or 39, and when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 18 nm to 22 nm, including the endpoints.

[0091]

[0114] In some embodiments of CARs of the disclosure, including those that comprise (a) (1) a binding element that specifically binds to a target and (2) an extracellular element comprising a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, the binding element specifically binds to a target, the target sequence is between about 13 nm to about 14 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell, the spacer sequence has a length of about 5 nm to about 8 nm, and the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 18 nm to 22 nm, including the endpoints, when the CAR is expressed on the surface of the cell. In some embodiments of CARs of the disclosure, including those that comprise (a) (1) a binding element that specifically binds to a target and (2) an extracellular element comprising a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, the binding element specifically binds to a target, the target sequence is between about 13 nm to about 14 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell, the spacer sequence has a length of about 5 nm to about 8 nm, and the spacer sequence comprises any one of SEQ ID NOs: 9, 10, 24, 11, 3, 18, 25, 38, 21, 29, 37, 39, 2, 15, 16, 26, 28, or 17, and when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 18 nm to 22 nm, including the endpoints.

[0092]

[0115] In some embodiments of CARs of the disclosure, including those that comprise (a) (1) a binding element that specifically binds to a target and (2) an extracellular element comprising a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, the binding element specifically binds to a target, the target sequence is between about 14 nm to about 15 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell, the spacer sequence has a length of about 4 nm to about 7 nm, and the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 18 nm to 22 nm, including the endpoints, when the CAR is expressed on the surface of the cell. In some embodiments of CARs of the disclosure, including those that comprise (a) (1) a binding element that specifically binds to a target and (2) an extracellular element comprising a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, the binding element specifically binds to a target, the target sequence is between about 14 nm to about 15 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell, the spacer sequence has a length of about 4 nm to about 7 nm, and the spacer sequence comprises any one of SEQ ID NOs: 25, 38, 21, 29, 37, 39, 2, 15, 16, 26, 28, 17, 12, 13, 19, 20, or 44, and when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 18 nm to 22 nm, including the endpoints.

[0093]

[0116] In some embodiments of CARs of the disclosure, including those that comprise (a) (1) a binding element that specifically binds to a target and (2) an extracellular element comprising a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, the binding element specifically binds to a target, the target sequence is between about 15 nm to about 16 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell, the spacer sequence has a length of about 3 nm to about 6 nm, and when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 18 nm to 22 nm, including the endpoints. In some embodiments of CARs of the disclosure, including those that comprise (a) (1) a binding element that specifically binds to a target and (2) an extracellular element comprising a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, the binding element specifically binds to a target, the target sequence is between about 15 nm to about 16 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell, the spacer sequence has a length of about 3 nm to about 6 nm, and the spacer sequence comprises any one of SEQ ID NOs: 2, 15, 16, 26, 28, 17, 12, 13, 19, 20, or 44, and when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 18 nm to 22 nm, including the endpoints.

[0094]

[0117] In some embodiments of CARs of the disclosure, including those that comprise (a) (1) a binding element that specifically binds to a target and (2) an extracellular element comprising a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, the binding element specifically binds to a target, the target sequence is between about 16 nm to about 17 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell, the spacer sequence has a length of about 2 nm to about 5 nm, and the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 18 nm to 22 nm, including the endpoints, when the CAR is expressed on the surface of the cell. In some embodiments of CARs of the disclosure, including those that comprise (a) (1) a binding element that specifically binds to a target and (2) an extracellular element comprising a spacer sequence, (b) a transmembrane element, and (c) an intracellular element, the binding element specifically binds to a target, the target sequence is between about 16 nm to about 17 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell, the spacer sequence has a length of about 2 nm to about 5 nm, and the spacer sequence comprises any one of SEQ ID NOs: 12, 13, 19, 20, or 44, and when the CAR is expressed on the surface of the cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 18 nm to 22 nm, including the endpoints.

[0095] Spacer

[0118] The present disclosure provides spacer sequences for each CAR-epitope binding pair to achieve an intercellular distance of about 17 nm to about 25 nm or 17 nm to 25 nm. In some embodiments, the present disclosure provides a set of spacer sequences for each CAR-epitope binding pair to achieve an intercellular distance of about 17 nm to about 25 nm or 17 nm to 25 nm.

[0096]

[0119] The present disclosure provides spacer sequences for each CAR-epitope binding pair to achieve an intercellular distance of about 17 nm to about 22 nm or 17 nm to 22 nm. In some embodiments, the present disclosure provides a set of spacer sequences for each CAR-epitope binding pair to achieve an intercellular distance of about 17 nm to about 22 nm or 17 nm to 22 nm.

[0097]

[0120] The present disclosure provides spacer sequences for each CAR-epitope binding pair to achieve an intercellular distance of about 20 nm or 20 nm. In some embodiments, the present disclosure provides a set of spacer sequences for each CAR-epitope binding pair to achieve an intercellular distance of about 20 nm or 20 nm.

[0098]

[0121] In some embodiments, the set of test spacers includes a first test spacer and a second test spacer. In some embodiments, the set of spacers includes at least one spacer comprising a sequence of SEQ ID NO: 1-44. In some embodiments, the set of spacers includes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, or 44 spacers comprising a sequence of SEQ ID NO: 1-44. In some embodiments, the set of spacers includes one each of the sequences of SEQ ID NO: 1-44.

[0099]

[0122] In some embodiments of the spacer sequences of the present disclosure, the spacer sequences comprise one or more of distinct sequences, distinct lengths, distinct secondary shapes, distinct tertiary shapes, distinct flexibility, distinct charge profiles, distinct hydrophobicity, distinct hydrophilicity, and distinct immunogenicity.

[0100]

[0123] In some embodiments of the spacer sequences of the present disclosure, the spacer sequences comprise distinct lengths.

[0124] In some embodiments of the spacer sequences of the present disclosure, the spacer sequence comprises a length of about 2 nm to about 20 nm. In some embodiments, the spacer sequence comprises a length of about 3 nm to about 20 nm. In some embodiments, the spacer sequence comprises a length of about 4 nm to about 20 nm.

[0101]

[0125] In some embodiments of the spacer sequence of the present disclosure, the spacer sequence comprises a sequence of SEQ ID NO: 1-44. In some embodiments, the spacer sequence comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence of SEQ ID NO: 1-44.

[0102]

[0126] In some embodiments of the spacer sequence of the present disclosure, the spacer sequence is derived from one or more of human immunoglobulin (Ig) proteins, the hinge region of an immunoglobulin protein, or the extracellular domain of a protein. In some embodiments of the spacer sequence of the present disclosure, the spacer sequence is engineered de novo. In some embodiments of the spacer sequence of the present disclosure, the spacer sequence comprises less than 50% identity to any known human sequence.

[0103]

[0127] In some embodiments of the spacer sequence of the present disclosure, the spacer sequence comprises a linker sequence. In some embodiments, the linker sequence comprises or encodes a flexible linker. In some embodiments, the linker sequence comprises a glycine-serine (GS) linker. In some embodiments, the linker sequence comprises the sequence GGGSG (SEQ ID NO: 77).

[0104]

[0128] In some embodiments of the spacer sequence of the present disclosure, the CAR comprises a linker sequence. In some embodiments, the linker sequence comprises or encodes a flexible linker. In some embodiments, the linker sequence comprises a glycine-serine (GS) linker. In some embodiments, the linker sequence comprises the sequence GGGSG (SEQ ID NO: 77). In some embodiments, the linker sequence is located between the binding element and the spacer sequence.

[0105]

[0129] Table 1 provides exemplary spacer sequences of the disclosure. In some embodiments, a CAR of the disclosure comprises one or more spacer sequences of Table 1. In some embodiments, a CAR of the disclosure comprises one or more spacer sequences of the disclosure (including those provided in Table 1), where the spacer sequence comprises one or more of a deletion, insertion, substitution, inversion, truncation, or modification. In some embodiments, the spacer sequence comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 80%, 95%, 97%, 99% identity to one or more of SEQ ID NOs: 1-44. In some embodiments, the spacer sequence comprises at least one non-naturally occurring residue. In some embodiments, the substitution comprises the replacement of one first amino acid with a second amino acid, the first amino acid and the second amino acid having one or more of polarity, side chain length, or hydrophobicity. For example, in some embodiments, a cysteine ​​(C) is replaced by a serine (S).

[0106]

[0130]

[0107] [Table 1-1]

[0108] [Table 1-2]

[0109] [Table 1-3]

[0110]

[0131]

[0111] [Table 2]

[0112] How to choose a spacer

[0132] The present disclosure provides methods for designing spacer sequences for each CAR-epitope binding pair to achieve an intercellular distance of about 17 nm to about 25 nm.

[0113]

[0133] In some embodiments of the method for designing a spacer sequence of the present disclosure, the method includes (a) expressing a CAR that specifically binds to a target in a first cell, where the CAR comprises a test spacer sequence, (b) contacting the first cell with a second cell, where the second cell expresses the target, (c) measuring the intercellular distance between the first cell and the second cell, and (d) selecting the test spacer when the intercellular distance is between about 17 nm and about 25 nm. In some embodiments, the set of test spacers includes the test spacer.

[0114]

[0134] In some embodiments of the method for designing spacer sequences of the present disclosure, the cell expressing the CAR is an immune cell. In some embodiments, the immune cell is a natural killer (NK) cell, a lymphocyte, a B cell and / or a T cell. In some embodiments, the immune cell is a progenitor cell. In some embodiments, the immune cell is a hematopoietic stem cell (HSC). In some embodiments, the immune cell is a differentiated cell, optionally differentiated from an induced pluripotent stem cell (iPSC).

[0115]

[0135] In some embodiments of the method for designing spacer sequences of the present disclosure, the cell expressing the target is an antigen-presenting cell (APC). In some embodiments, the (APC) is a monocyte, a dendritic cell, a macrophage, a B cell, or a target cell of the present disclosure. In some embodiments, the APC is modified to express the target. In some embodiments, the APC is not modified to express the target. In some embodiments, the target comprises a naturally occurring antigen. In some embodiments, the target comprises a cancer antigen. In some embodiments, the target comprises a synthetic antigen or a neo-antigen. In some embodiments, the target comprises a target spacer sequence or an epitope spacer sequence. In some embodiments, the target spacer sequence or the epitope spacer sequence mimics the extension of the target from the cell expressing the target during an in vitro assay, depending on the presentation of the target or epitope, for example, as part of the MHC. Exemplary target spacers or epitope spacers are provided in Table 2 of the present disclosure.

[0116]

[0136] In some embodiments of the method for designing spacer sequences of the present disclosure, the target-expressing cell is a primary cell. In some embodiments, the primary cell is isolated or derived from a biological sample. In some embodiments, the biological sample is obtained from a subject. In some embodiments, the primary cell is modified. In some embodiments, the primary cell is not modified.

[0117]

[0137] In some embodiments of the methods for designing spacer sequences of the present disclosure, the cells expressing the target are cultured cells. In some embodiments, the cultured cells are isolated or derived from a biological sample. In some embodiments, the biological sample is obtained from a subject. In some embodiments, the cultured cells are modified. In some embodiments, the cultured cells are immortalized. In some embodiments, the cultured cells are modified to express the target. In some embodiments, expression of the target by the cultured cells is modified. In some embodiments, expression of the target by the cultured cells is increased, decreased, or made inducible.

[0118]

[0138] In some embodiments of the method for designing a spacer sequence of the present disclosure, the method includes: (a) expressing in a first cell a first CAR that specifically binds to a target, the CAR comprising a first test spacer sequence; (b) expressing in a second cell a second CAR that specifically binds to a target, the CAR comprising a second test spacer sequence; (c) contacting the first cell and the second cell with a third cell, the third cell expressing the target; (d) measuring the intercellular distance between the first cell and the third cell, measuring the intercellular distance between the second cell and the third cell, and (d) selecting a first test article when the intercellular distance is about 17 nm to about 25 nm, or selecting a second test article when the intercellular distance is about 17 nm to about 25 nm. In some embodiments, the set of test spacers includes a first test spacer and a second test spacer. In some embodiments, the set of spacers includes at least one spacer comprising a sequence of SEQ ID NO: 1 to 44. In some embodiments, the set of spacers includes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, or 44 spacers comprising a sequence of SEQ ID NO: 1 to 44. In some embodiments, the set of spacers includes each one of the sequences of SEQ ID NO: 1 to 44.

[0119]

[0139] In some embodiments of the method for designing a spacer sequence of the present disclosure, the method includes a step of selecting a test spacer when the intercellular distance is about 17 nm to about 22 nm. In some embodiments, the method includes a step of selecting a first test object when the intercellular distance is about 17 nm to about 22 nm.

[0120]

[0140] In some embodiments of the method for designing a spacer sequence of the present disclosure, the method includes selecting a test spacer when the intercellular distance is about 20 nm. In some embodiments, the method includes selecting a first test object when the intercellular distance is about 20 nm.

[0121]

[0141] In some embodiments of the methods for designing spacer sequences of the present disclosure, the first test spacer and the second test spacer comprise one or more of distinct sequences, distinct lengths, distinct secondary shapes, distinct tertiary shapes, distinct flexibility, distinct charge profiles, distinct hydrophobicity, distinct hydrophilicity, and distinct immunogenicity.

[0122]

[0142] In some embodiments of the methods for designing spacer sequences of the present disclosure, the first test spacer and the second test spacer comprise distinct lengths.

[0143] In some embodiments of the methods for designing spacer sequences of the present disclosure, the test spacer comprises a length of about 2 nm to about 20 nm. In some embodiments, the test spacer comprises a length of about 3 nm to about 19 nm. In some embodiments, the test spacer comprises a length of about 4 nm to about 17 nm.

[0123]

[0144] In some embodiments of the methods for designing spacer sequences of the present disclosure, the first test spacer or the second test spacer comprises a sequence of SEQ ID NOs: 1-44. In some embodiments of the methods for designing spacer sequences of the present disclosure, the first test spacer or the second test spacer comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence of SEQ ID NOs: 1-44.

[0124]

[0145] In some embodiments of the methods for designing spacer sequences of the present disclosure, the method includes: (a) expressing a target on the surface of a target cell, where the target comprises a linear epitope, and where the linear epitope is presented at various distances from the surface of the target cell; (b) contacting the target cell with a CAR-expressing cell, where the CAR selectively binds to the target, and where the CAR comprises a test spacer sequence; and (c) measuring the intercellular distance between a first cell and a second cell; and (d) selecting the test spacer sequence when the intercellular distance is between about 17 nm and about 25 nm.

[0125]

[0146] In some embodiments of the methods for designing spacer sequences of the present disclosure, the methods include varying the length of the spacer sequence within the CAR and the spacer sequence within the target to perform pairwise analyses of T cell function, including, but not limited to, measuring target cell lysis and measuring cytokine production.

[0126]

[0147] Figure 2 is a diagram illustrating the intercellular distance between an exemplary HA epitope (target) on the surface of a target cell and the binding element of a CAR expressed on the surface of a CAR T cell. In this example, the intercellular distance D is the sum of A+B+C, where distance A is the contribution of the CAR spacer (including the GS linker) to the intercellular distance, distance B is the contribution of the epitope binding element that binds to the epitope to the intercellular distance, and distance C is the contribution of the epitope spacer, which also includes the GS linker, to the intercellular distance.

[0127]

[0148] The spacer design method of the present disclosure can include experimentally estimating or determining the intercellular distance during binding interaction, and optionally can estimate, determine, verify, or confirm empirical data by modeling or computer modeling.The spacer design method of the present disclosure can include various empirically measured distances, for example, distances determined by X-ray crystallography studies.

[0128]

[0149] The spacer sequences of the present disclosure can be used to generate one or more libraries of spacer sequences. The spacer library of the present disclosure can include a spacer sequence suitable for use with a CAR that specifically binds to a specific target, for example, a plurality of spacer sequences that, when incorporated into a target-specific CAR, result in an intercellular distance between a CAR-expressing cell and a target-expressing cell of about 17 nm to about 25 nm. In some embodiments, the spacer library of the present disclosure can include a spacer sequence suitable for use with a CAR that specifically binds to a specific target, for example, a plurality of spacer sequences that, when incorporated into a target-specific CAR, result in an intercellular distance between a CAR-expressing cell and a target-expressing cell of about 17 nm to about 22 nm. In some embodiments, the spacer library of the present disclosure can include a spacer sequence suitable for use with a CAR that specifically binds to a specific target, for example, a plurality of spacer sequences that, when incorporated into a target-specific CAR, result in an intercellular distance between a CAR-expressing cell and a target-expressing cell of about 20 nm.

[0129] cell

[0150] The present disclosure provides cells comprising one or more of a CAR of this disclosure, an sequence encoding a CAR of this disclosure, a vector encoding a CAR of this disclosure, and a composition of this disclosure (including a pharmaceutical composition of this disclosure).

[0130]

[0151] In some embodiments of the present disclosure, the cell of the compositions and methods of the present disclosure is a human cell. In some embodiments, the cell of the compositions and methods of the present disclosure is a non-human cell. In some embodiments, the cell of the compositions and methods of the present disclosure is a mammalian cell, a non-human primate cell, a rodent cell, a rat cell, a mouse cell, or a hybridoma thereof.

[0131]

[0152] In some embodiments of the present disclosure, the cells of the compositions and methods of the present disclosure are immune cells. In some embodiments, the immune cells are natural killer (NK) cells, lymphocytes, B cells and / or T cells. In some embodiments, the immune cells are progenitor cells. In some embodiments, the immune cells are hematopoietic stem cells (HSCs). In some embodiments, the immune cells are differentiated cells, optionally differentiated from induced pluripotent stem cells (iPSCs).

[0132]

[0153] In some embodiments of the present disclosure, the cell of the compositions and methods of the present disclosure is an antigen-presenting cell (APC). In some embodiments, the APC of the present disclosure includes, but is not limited to, a monocyte, a dendritic cell, a macrophage, a B cell, or a target cell of the present disclosure. In some embodiments, the APC is modified to express a target. In some embodiments, the APC is not modified to express a target. In some embodiments, the target comprises a naturally occurring antigen. In some embodiments, the target comprises a cancer antigen. In some embodiments, the target comprises a synthetic antigen or a neo-antigen. In some embodiments, the target comprises a target spacer sequence or an epitope spacer sequence. In some embodiments, the target spacer sequence or the epitope spacer sequence mimics the extension of the target from a cell expressing the target during an in vitro assay, depending on the presentation of the target or epitope, for example, as part of the MHC. Exemplary target or epitope spacers are provided in Table 2 of the present disclosure.

[0133]

[0154] In some embodiments of the present disclosure, the cells of the compositions and methods of the present disclosure are primary cells. In some embodiments, the primary cells are isolated or derived from a biological sample. In some embodiments, the biological sample is obtained from a subject. In some embodiments, the primary cells are modified. In some embodiments, the primary cells are not modified.

[0134]

[0155] In some embodiments of the present disclosure, the cells of the compositions and methods of the present disclosure are cultured cells. In some embodiments, the cultured cells are isolated or derived from a biological sample. In some embodiments, the biological sample is obtained from a subject. In some embodiments, the cultured cells are modified. In some embodiments, the cultured cells are immortalized. In some embodiments, the cultured cells are modified to express a target. In some embodiments, expression of the target by the cultured cells is modified. In some embodiments, expression of the target by the cultured cells is increased, decreased, or made inducible.

[0135]

[0156] In some embodiments of the present disclosure, the cells of the compositions and methods of the present disclosure are disease cells. In some embodiments, the disease cells are isolated or derived from a biological sample. In some embodiments, the biological sample is obtained from a subject. In some embodiments, the biological sample is a biopsy sample. In some embodiments, the disease cells are isolated or derived from a tumor. In some embodiments, the tumor is a malignant tumor. In some embodiments, the disease cells are cancerous, transformed or malignant cells.

[0136]

[0157] In some embodiments of the present disclosure, the cell of the compositions and methods of the present disclosure is a modified cell. In some embodiments, the modified cell expresses a CAR of the present disclosure. In some embodiments, the modified cell is a human cell. In some embodiments, the modified cell is a human T cell or a human NK cell. In some embodiments, the modified human T cell or the modified human NK cell comprises a CAR comprising a spacer sequence of SEQ ID NO: 1-44.

[0137]

[0158] The present disclosure provides amino acid and nucleic acid sequences encoding chimeric antigen receptors (CARs). In some embodiments, the dataset comprises a library of amino acid or nucleic acid sequences for use in empirical evaluation of spacer selection models. In some embodiments, the dataset comprises a library of amino acid or nucleic acid sequences encoding one or more spacers for use in empirical evaluation of spacer selection models. The sequences of the dataset can be physically selected from the library for assembly into amino acid or nucleic acid sequences encoding spacers of the present disclosure. The sequences of the dataset can be physically selected from the library for assembly into amino acid or nucleic acid sequences encoding CARs of the present disclosure. The sequences of the dataset can be physically selected from the library for assembly into amino acid or nucleic acid sequences encoding targets of the present disclosure, particularly for use in the spacer selection methods of the present disclosure. The sequences can be physically selected using a variety of techniques, for example, manually, microfluidically, or robotically selected.

[0138] target

[0159] In some embodiments of the CAR of the present disclosure, the CAR selectively and specifically binds to a target. In some embodiments, the target is expressed on a target cell of the present disclosure. In some embodiments, the target comprises an antigen. In some embodiments, the target comprises an epitope. In some embodiments, the epitope is linear. In some embodiments, the epitope is non-linear. In some embodiments, the epitope is conformational or discontinuous.

[0139]

[0160] In some embodiments of the CARs of the present disclosure, the CAR selectively and specifically binds to a target, hi some embodiments, the target comprises one or more of a protein, carbohydrate, or glycolipid molecule.

[0140]

[0161] In some embodiments of the CAR of the present disclosure, the CAR selectively and specifically binds to a target. In some embodiments, the target comprises an antigen. In some embodiments, the target comprises a tumor antigen or a marker of the tumor microenvironment (TME). In some embodiments, the target comprises a cell type marker or a biomarker.

[0141]

[0162] In some embodiments of the CAR of the present disclosure, the CAR selectively and specifically binds to a target expressed on a cancer cell. In some embodiments, the cancer cell is isolated or derived from one or more of the following cancers: acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adolescent cancer, adrenocortical carcinoma AIDS-related cancer (including, but not limited to, Kaposi's sarcoma (soft tissue sarcoma), AIDS-related lymphoma (lymphoma), and primary CNS lymphoma (lymphoma)), anal cancer, appendix cancer (also gastrointestinal carcinoid tumors), astrocytoma, atypical teratoid / rhabdoid tumor, central nervous system cancer, basal cell carcinoma of the skin (also skin cancer), bile duct cancer, bladder cancer, bone cancer (including Ewing's sarcoma and osteosarcoma and malignant fibrous histiocytoma), brain tumor (also brain cancer), breast cancer, bronchial tumor (lung (also non-Hodgkin's lymphoma), Burkitt's lymphoma (also non-Hodgkin's lymphoma), carcinoid tumors (also gastrointestinal tumors), cancer of unknown primary, cardiac (heart) tumors, central nervous system cancers, atypical teratoid / rhabdoid tumors (brain cancer), medulloblastoma and other CNS embryonal tumors (brain cancer), germ cell tumors (brain cancer), primary CNS lymphoma, cervical cancer, childhood cancer, rare cancers of childhood, cholangiocarcinoma (also bile duct cancer), chordoma (bone cancer), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myeloproliferative neoplasms, colorectal Intestinal Cancer, Craniopharyngioma (brain cancer), Cutaneous T-cell Lymphoma (also lymphoma and mycosis fungoides and Sézary syndrome), Ductal Carcinoma in Situ (DCIS) (also breast cancer), Embryonal Tumors, Medulloblastoma and other Central Nervous System (brain cancer), Endometrial Cancer (uterine cancer), Ependymoma (brain cancer), Esophageal Cancer, Esthesioneuroblastoma (head and neck cancer), Ewing's Sarcoma (bone cancer), Extracranial Germ Cell Tumors, Extragonadal Germ Cell Tumors, Eye Cancer, Intraocular Melanoma, Retinoblastoma, Fallopian Tube Cancer, Gallbladder Cancer, Gastric (Stomac) Cancer h)) Cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST) (soft tissue sarcoma), germ cell tumor, pediatric central nervous system germ cell tumor (brain cancer), pediatric extracranial germ cell tumor, extragonadal germ cell tumor, ovarian germ cell tumor, testicular cancer, gestational trophoblastic disease, hairy cell leukemia, head and neck cancer, cardiac tumor, hepatocellular (liver) cancer, histiocytosis (Langerhans cells), Hodgkin's lymphoma, hypopharyngeal cancer (head and neck cancer), intraocular melanoma, pancreatic islet cell tumor, pancreatic neuroendocrine tumor, Kaposi's sarcoma (soft tissue sarcoma),Renal (renal cell) cancer, Langerhans cell histiocytosis, Laryngeal cancer (head and neck cancer), Leukemia, Lip and oral cavity cancer (head and neck cancer), Liver cancer, Lung cancer (non-small cell, small cell, pleuropulmonary blastoma, and tracheobronchial tumors), Lymphoma, Male breast cancer, Melanoma, Intraocular (eye) melanoma, Merkel cell carcinoma (skin cancer), Mesothelioma (malignant), Metastatic carcinoma, Metastatic squamous cell carcinoma of the neck of unknown primary (head and neck cancer), Midline carcinoma with NUT gene alteration, Oral cancer (head and neck cancer), Multiple endocrine neoplasia syndrome, Multiple myeloma / plasma cell neoplasm, Mycosis fungoides (lymphoma), Myelodysplastic syndrome, Myelodysplasia / bone Myeloproliferative neoplasms, myeloid leukemia, chronic (CML), myeloid leukemia, acute (AML), myeloproliferative neoplasms, chronic, nasal and paranasal sinus cancer (head and neck cancer), nasopharyngeal cancer (head and neck cancer), neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer, oral cavity cancer, lip and oral cavity cancer and oropharyngeal cancer (head and neck cancer), treatment of osteosarcoma and undifferentiated pleomorphic sarcoma of bone, ovarian cancer, pancreatic cancer, pancreatic neuroendocrine tumors (islet cell tumors), papillomatosis (pediatric larynx), paraganglioma, paranasal sinus and nasal cancer (head and neck cancer), parathyroid cancer, penile cancer, pharyngeal cancer (head and neck cancer), pheochromocytoma, pituitary tumor tumors, plasma cell neoplasms / multiple myeloma, pleuropulmonary blastoma (lung cancer), pregnancy and breast cancer, primary central nervous system (CNS) lymphoma, primary peritoneal cancer, prostate cancer, rare cancers of childhood, rectal cancer, recurrent cancer, renal cell (kidney) cancer, retinoblastoma, rhabdomyosarcoma, childhood (soft tissue sarcoma), salivary gland cancer (head and neck cancer), sarcoma, childhood rhabdomyosarcoma (soft tissue sarcoma), childhood vascular tumor (soft tissue sarcoma), Ewing's sarcoma (bone cancer), Kaposi's sarcoma (soft tissue sarcoma), osteosarcoma (bone cancer), soft tissue sarcoma, uterine sarcoma, Sezary syndrome (lymphoma), skin cancer, small cell lung cancer, small intestine cancer, soft tissue Osteosarcoma, Squamous cell carcinoma of the skin (skin cancer), Unknown primary and metastatic squamous cell carcinoma of the cervix (head and neck cancer), Stomach (gastric) cancer, T-cell lymphoma (also cutaneous lymphoma, mycosis fungoides and Sézary syndrome), Testicular cancer, Pharyngeal cancer (head and neck cancer), Nasopharyngeal cancer, Oropharyngeal cancer, Hypopharyngeal cancer, Thymoma and thymic carcinoma, Thyroid cancer, Tracheobronchial tumor (lung cancer), Transitional cell carcinoma of the renal pelvis and ureter (renal (renal cell) cancer), Unknown primary cancer, ureter and renal pelvis, Transitional cell carcinoma (renal (renal cell) cancer, urethral cancer, uterine cancer (also endometrium), Uterine sarcoma, Vaginal cancer,Vascular tumors (soft tissue sarcomas), vulvar cancer, Wilms tumor and other childhood kidney tumors, and young adult cancers.

[0142] Joint elements

[0163] In some embodiments of the CARs of the present disclosure, the CAR comprises an extracellular element. In some embodiments of the CARs of the present disclosure, the extracellular element comprises a binding element. In some embodiments of the CARs of the present disclosure, the extracellular element comprises at least one binding element. In some embodiments of the CARs of the present disclosure, the extracellular element comprises one or more binding elements. In some embodiments of the CARs of the present disclosure, the extracellular element comprises a bispecific binding element. In some embodiments of the CARs of the present disclosure, the extracellular element comprises a trispecific binding element. In some embodiments of the CARs of the present disclosure, the extracellular element comprises a multispecific binding element.

[0143]

[0164] The exemplary binding element of the present disclosure can take any form.The exemplary binding element of the present disclosure selectively and specifically binds to the target of the present disclosure.In some embodiments, the binding element comprises one or more modifications to enhance binding affinity, selectivity or specificity when compared with unmodified binding element.

[0144]

[0165] Exemplary binding elements of the present disclosure can take any form. Exemplary binding elements of the present disclosure selectively and specifically bind to targets of the present disclosure. In some embodiments, the binding element comprises a protein scaffold. In some embodiments, the protein scaffold comprises one or more sequences isolated from or derived from human fibronectin protein. In some embodiments, the protein scaffold comprises one or more modifications to enhance binding affinity, selectivity or specificity when compared to unmodified protein scaffold. In some embodiments, the binding element or protein scaffold comprises a monobody.

[0145]

[0166] Exemplary binding members of the present disclosure can take any form. Exemplary binding members of the present disclosure selectively and specifically bind to a target of the present disclosure. In some embodiments, the binding member comprises an antibody or a functional fragment thereof. In some embodiments, the antibody comprises a monoclonal antibody. In some embodiments, the antibody comprises a single domain or domain antibody. In some embodiments, the antibody comprises a camelid antibody. In some embodiments, an antibody fragment comprises a molecule other than a complete antibody, including a portion of a complete antibody that binds to the antigen to which the complete antibody binds. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, single domain antibodies, e.g., sdAb(V L or V H (any of the above), Camelidae VHH domains, as well as multispecific antibodies formed from antibody fragments.

[0146]

[0167] Exemplary binding members of the present disclosure can take any form. Exemplary binding members of the present disclosure selectively and specifically bind to targets of the present disclosure. In some embodiments, the binding member comprises an antibody mimic or a functional fragment thereof.

[0147]

[0168] Exemplary binding elements of the present disclosure can take any form. Exemplary binding elements of the present disclosure selectively and specifically bind to targets of the present disclosure. In some embodiments, the binding element comprises a single chain variable fragment (scFv). In some embodiments of the present disclosure, the scFv comprises a variable region of a heavy chain (VH) and a variable region of a light chain (VL) linked by a flexible peptide linker.

[0148]

[0169] Exemplary binding members of the present disclosure can take any form. Exemplary binding members of the present disclosure selectively and specifically bind to targets of the present disclosure. In some embodiments, the binding member comprises a fusion protein.

[0149]

[0170] A binding member of the present disclosure specifically and / or selectively binds to a target of the present disclosure. A multispecific binding member of the present disclosure specifically and / or selectively binds to two or more targets of the present disclosure.

[0150]

[0171] In some embodiments of the present disclosure, the target comprises an epitope, hi some embodiments, the target comprises a linear epitope, a continuous epitope, a discontinuous epitope, and / or a conformational epitope.

[0151]

[0172] In some embodiments of the present disclosure, the target comprises an antigen.

[0173] In some embodiments of the present disclosure, the target comprises an amino acid sequence. In some embodiments, the target does not comprise an amino acid.

[0152] Method of preparation: Polynucleotide

[0174] The present disclosure provides a method for producing the polynucleotide of the present disclosure, for example, the DNA vector of the present disclosure and its partial components, as well as the packaging vector and plasmid of the present disclosure.Standard molecular biology techniques can be used to construct the polynucleotide of the present disclosure.Polynucleotide can be chemically synthesized.

[0153] Method of production: Packaged viral capsid

[0175] The present disclosure includes a method for producing a viral capsid containing the polynucleotide of the present disclosure.Generally, the viral capsid of the present disclosure can be produced by providing a packaging polynucleotide of the present disclosure to a cell.The packaging polynucleotide can be provided to a packaging cell as a plasmid.The packaging cell can be cultured to produce the viral capsid containing the polynucleotide of the present disclosure.Preferably, the packaged viral capsid is replication-incompetent.

[0154]

[0176] A variety of commercially available kits are suitable for producing packaged viral capsids of the present disclosure, including MISSION® Lentiviral Packaging Mix (available from Millipore Sigma) and LV-Max Lentiviral Packaging Mix (available from ThermoFisher Scientific).

[0155]

[0177] The viral capsids produced by the packaging cells can be purified for use in downstream processes, such as delivery to cells for use in the production of polypeptides, delivery to cells for use in cell-based therapy, or delivery to a subject for gene therapy methods. Purification can include treatment to eliminate contaminants from the host cells or culture medium. Purification steps can include steps based on the physical and / or chemical characteristics of the plasmid. Chemical characteristics can include, for example, hydrophilic-hydrophobic. Physical characteristics can include, for example, size. Examples of particle size-based purification strategies include density gradient ultracentrifugation, ultrafiltration, precipitation, two-phase extraction systems, and size exclusion chromatography. In some cases, precipitation can be used in conjunction with centrifugation, for example, using polyethylene glycol, ammonium sulfate, or calcium phosphate. In some cases, aqueous two-phase separation systems using PEG, dextran, or polyvinyl alcohol can be used. In some cases, membrane-based tangential flow filtration techniques are used, examples of which include ultrafiltration, diafiltration, and microfiltration. In other embodiments, chromatographic means can be used to purify the viral capsids. In yet other embodiments, immunoaffinity methods can be used to capture capsids using monoclonal antibodies with specificity for the relevant capsid. See Morenweiser, R., "Downstream processing of viral vectors and vaccines", Gene Therapy (2005) 12, S103-S110 (2005), the entire disclosure of which is incorporated herein by reference.

[0156]

[0178] Examples of suitable viral capsids include, but are not limited to, adenovirus, retrovirus, lentivirus, Sendai virus vectors, baculovirus, Epstein-Barr virus, papovavirus, vaccinia virus, herpes simplex virus, and adeno-associated virus (AAV).

[0157] Method of preparation: Cells

[0179] The present disclosure provides methods of making modified cells to express a CAR of the present disclosure.

[0158]

[0180] In certain embodiments, the present disclosure provides methods for producing therapeutic cells for use in treating a subject in need of cell therapy. In one aspect, the present disclosure provides methods for generating or preparing therapeutic cells expressing a CAR.

[0159]

[0181] In certain embodiments, the polynucleotide of the present disclosure is maintained as an extrachromosomal polynucleotide in host cell.In certain embodiments, the polynucleotide of the present disclosure is present in a vector (e.g., an expression vector) in host cell.In certain embodiments, the polynucleotide of the present disclosure or a subset or partial component thereof is integrated into the chromosome of host cell.

[0160]

[0182] A variety of methods can be used to introduce an expression vector encoding a polynucleotide of the present disclosure into a cell to generate a cell of the present disclosure. See, for example, Green et al., Molecular cloning: A laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press (2014).

[0161]

[0183] The polynucleotide of the present disclosure can be used to create or modify host cells using methods known in the art for modifying polynucleotides.Examples include targeted homologous recombination (e.g., "hit-and-run", "double replacement"), site-specific recombinase (e.g., Cre recombinase and Flp recombinase), PB transposase (e.g., Sleeping Beauty, piggyBac, To12 or Frog Prince), genome editing by engineered nuclease (e.g., meganuclease, zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN) and CRISPR / Cas system), and genome editing using recombinant adeno-associated virus (rAAV) platform.The agent for introducing nucleic acid changes into the gene of interest can be designed using publicly available sources or can be obtained commercially from Transposagen, Addgene and Sangamo Biosciences. The vectors of the present disclosure can utilize these methods to integrate the polynucleotides of the present disclosure into a host genome. The polynucleotides and vectors of the present disclosure can include polynucleotides that encode polypeptides necessary to carry out these methods for integrating the polynucleotides of the present disclosure into a host genome.

[0162]

[0184] A variety of approaches suitable for integrating a polynucleotide into a host cell genome are known in the art, including random integration or site-specific integration (e.g., "landing pad" approaches); see, e.g., Zhao, M. et al. (2018) Appl. Microbiol. Biotechnol. 102:6105-6117; Lee, JS et al. (2015) Sci. Rep. 5:8572; and Gaidukov, L. et al. (2018) Nucleic Acids Res. 46:4072-4086. Vectors of the present disclosure can utilize these methods to integrate a polynucleotide of the present disclosure into a host genome. Vectors of the present disclosure can include polynucleotides encoding polypeptides necessary for carrying out these methods to integrate a polynucleotide of the present disclosure into a host genome.

[0163]

[0185] Host cells can be cultured using methods and compositions known in the art. Examples of commercially available media suitable for culturing the host cells of the present disclosure include Ham's F10 (Sigma), Minimum Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma).

[0164]

[0186] The culture medium may be supplemented as necessary with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN™ drugs), trace elements (usually defined as inorganic compounds present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Other necessary supplements may also be included at appropriate concentrations that would be known to one of skill in the art. Culture conditions such as temperature, pH, etc. will be apparent to one of skill in the art.

[0165] Pharmaceutical Compositions

[0187] The present disclosure provides a pharmaceutical composition comprising a CAR of the present disclosure, a sequence comprising or encoding a CAR of the present disclosure, a vector comprising a sequence encoding a CAR of the present disclosure, or a cell comprising one or more of these in combination with a pharma- ceutically acceptable carrier.

[0166]

[0188] In some embodiments of the pharmaceutical compositions of the present disclosure, the pharma- ceutically acceptable carrier comprises sterile saline or a salt solution that balances or mimics the physiological conditions of the subject's bloodstream or the local conditions of the tumor microenvironment.

[0167]

[0189] In some embodiments of the pharmaceutical compositions of the present disclosure, the pharma- ceutically acceptable carrier comprises an adjuvant for modulation of the subject's immune system or immune response.

[0190] In some embodiments of the pharmaceutical compositions of the present disclosure, the pharma- ceutically acceptable carrier comprises an agent to stabilize or prevent the degradation of an amino acid or nucleic acid sequence in the composition.

[0168]

[0191] In some embodiments of the pharmaceutical compositions of the present disclosure, the pharma- ceutically acceptable carrier comprises an agent for optimizing the solubility, stability, tonicity, or viscosity of the composition. In some embodiments, the pharma-ceutically acceptable carrier comprises one or more of a bulking agent, a surfactant, and a chelating agent.

[0169]

[0192] In some embodiments of the pharmaceutical compositions of the present disclosure, the pharma- ceutically acceptable carrier comprises an agent for adjusting the pH of the pharmaceutical composition prior to administration to a subject, hi some embodiments, the pharma- ceutically acceptable carrier comprises a buffering agent.

[0170]

[0193] In some embodiments of the pharmaceutical compositions of the present disclosure, the pharma- ceutically acceptable carrier comprises an agent for maintaining the viability or enhancing the activity of the cells or any component of the cells of the composition. In some embodiments, the pharma-ceutically acceptable carrier comprises an immunostimulatory cytokine, including, but not limited to, IL-2, IL-7, IL-12 and / or IL-15. In some embodiments, the pharma-ceutically acceptable carrier comprises a chemokine.

[0171]

[0194] In some embodiments of the pharmaceutical composition of the present disclosure, the pharma- ceutically acceptable carrier comprises one or more of liposomes, viral capsids, micelles, polymersomes, or nanoparticles.In some embodiments of the pharmaceutical composition of the present disclosure, the pharma- ceutically acceptable carrier comprises allogeneic cells.In some embodiments of the pharmaceutical composition of the present disclosure, the pharma- ceutically acceptable carrier comprises allogeneic T cells.

[0172]

[0195] In some embodiments of the pharmaceutical composition of the present disclosure, the pharma-ceutically acceptable carrier comprises one or more pharma-ceutically acceptable salts.Pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines, alkali or organic salts of acidic residues such as carboxylic acids.Pharmaceutically acceptable salts include, for example, the conventional non-toxic salts or quaternary ammonium salts of parent compounds formed from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include salts derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, and the like, and salts prepared from organic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isethionic acid, and the like. For example, the CAR of the present disclosure can be modulated by small molecules that can be provided as pharma- ceutically acceptable salts.

[0173] Treatment method

[0196] The present disclosure provides for the use of a CAR of the present disclosure, a nucleic acid of the present disclosure, a vector of the present disclosure, a cell of the present disclosure, or a pharmaceutical composition of the present disclosure for the manufacture of a medicament.

[0174]

[0197] The present disclosure provides the use of the CAR of the present disclosure, the nucleic acid of the present disclosure, the vector of the present disclosure, the cell of the present disclosure, or the pharmaceutical composition of the present disclosure for the treatment or prevention of a disease or disorder. In some embodiments, the disease or disorder comprises cancer. In some embodiments, the cancer arises in the circulating blood or lymphatic fluid (liquid tumors). In some embodiments, the cancer arises in a sheet of cells or in a non-fluid organ (solid tumors). In some embodiments, the cancer is a metastatic cancer. In some embodiments, the cancer comprises a cancer described in the present disclosure.

[0175]

[0198] The present disclosure provides a method of treating a disease or disorder, comprising administering to a subject a therapeutically effective amount of a CAR of the present disclosure, a nucleic acid of the present disclosure, a vector of the present disclosure, a cell of the present disclosure, or a pharmaceutical composition of the present disclosure, whereby the severity of a sign or symptom of the disease or disorder is reduced, thereby treating the disease or disorder. In some embodiments, the disease or disorder comprises cancer. In some embodiments, the cancer arises in the circulating blood or lymphatic fluid (liquid tumors). In some embodiments, the cancer arises in a sheet of cells or in a non-fluid organ (solid tumors). In some embodiments, the cancer is a metastatic cancer. In some embodiments, the cancer comprises a cancer described in the present disclosure.

[0176]

[0199] In some embodiments of the present disclosure, the symptom of a disease or disorder is an objective indicator of the disease or disorder.The symptom, or even the change in severity of the symptom, can be measured by objective means, including but not limited to physical examination, body scan (MRI, cat scan, CT scan, X-ray), biopsy (liquid or solid), genetic testing, metabolite analysis, blood analysis, urine analysis, etc.The symptom may be interpreted by a medical professional.

[0177]

[0200] In some embodiments of the present disclosure, the symptoms of disease or disorder are subjective experiences of the subject's indicators of disease or disorder.Changes in symptoms, and even severity of symptoms, can be measured by subjective means, including but not limited to the comparative state from before the onset of disease to the present, or before treatment to after treatment.Exemplary conditions that can be examined include, but are not limited to, pain level, energy (or fatigue), mobility, nausea, appetite, and specific factors related to the type of cancer (e.g., local swelling or organ function).

[0178]

[0201] The present disclosure provides a method for preventing a disease or disorder, comprising administering to a subject a therapeutically effective amount of a CAR of the present disclosure, a nucleic acid of the present disclosure, a vector of the present disclosure, a cell of the present disclosure, or a pharmaceutical composition of the present disclosure, whereby the onset or recurrence of a sign or symptom of the disease or disorder is delayed or inhibited, thereby preventing the disease or disorder. In some embodiments, the disease or disorder comprises cancer. In some embodiments, the cancer arises in the circulating blood or lymphatic fluid (liquid tumors). In some embodiments, the cancer arises in a sheet of cells or in a non-fluid organ (solid tumors). In some embodiments, the cancer is a metastatic cancer. In some embodiments, the cancer comprises a cancer described in the present disclosure.

[0179]

[0202] In some embodiments of the present disclosure, the onset or recurrence of a disease or disorder sign or symptom is delayed if the onset or recurrence occurs after an expected period (e.g., prognosis based on known or average timing across similar populations). In some embodiments of the present disclosure, the onset or recurrence of a cancer sign or symptom is delayed if the subject's expected cancer-free survival is 3 years and the subject has been cancer-free for more than 3 years. In some embodiments of the present disclosure, the onset or recurrence of a disease or disorder sign or symptom is inhibited if the sign of the disease or disorder under consideration (which may be disease or condition specific) is no longer detectable. In some embodiments of the present disclosure, the onset or recurrence of a liquid tumor sign or symptom is inhibited if cancer cells are no longer detectable in the circulating blood or lymph of the subject.

[0180]

[0203] The present disclosure provides a method of treating a subject in need of cell therapy. The method can include administering to the subject a therapeutically effective amount of a composition comprising a CAR T cell of the present disclosure.

[0181]

[0204] In some embodiments, the method includes (i) administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising therapeutic cells encoding a therapeutic polypeptide product of interest (e.g., a CAR) and a pharma- ceutically acceptable carrier, (ii) monitoring the effectiveness of the treatment, and (iii) repeating (i) and (ii) as necessary. In some embodiments, the CAR can be administered with a pharma- ceutically acceptable carrier.

[0182]

[0205] In some embodiments, the present disclosure provides a method for treating cancer, e.g., tumor, in a subject in need of cancer treatment. The method can include administering to a subject with cancer a therapeutically effective amount of a pharmaceutical composition comprising a therapeutic cell encoding a CAR of the present disclosure. The CAR can be administered with a pharmaceutically acceptable carrier.

[0183]

[0206] Exemplary cancers that can be treated with the pharmaceutical compositions disclosed herein include, but are not limited to, melanoma, lymphoma, sarcoma, and cancers of the colon, kidney, stomach, bladder, brain (e.g., glioma, glioblastoma, astrocytoma, medulloblastoma), prostate, bladder, rectum, esophagus, pancreas, liver, lung, breast, uterus, cervix, ovary, blood (e.g., acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, Burkitt's lymphoma, EBV-induced B-cell lymphoma).

[0184]

[0207] In some embodiments, autologous cells are administered to the subject.

[0208] In some embodiments, allogeneic cells are administered to the subject.

[0209] The subject of the present disclosure may be of any age. In some embodiments, the subject of the methods of the present disclosure is an adult. In some embodiments, the adult is between 18 and 80 years old. In some embodiments, the subject of the methods of the present disclosure is an elderly person. In some embodiments, the elderly person is at least 80 years old. In some embodiments, the subject of the methods of the present disclosure is an adolescent. In some embodiments, the adolescent is between 12 and 18 years old. In some embodiments, the subject of the methods of the present disclosure is a child. In some embodiments, the child is between 5 and 11 years old. In some embodiments, the subject of the methods of the present disclosure is an infant. In some embodiments, the infant is between 2 and 5 years old. In some embodiments, the subject of the methods of the present disclosure is an infant. In some embodiments, the infant is less than 2 years old.

[0185]

[0210] A subject of the present disclosure can have one or two X chromosomes. A subject of the present disclosure can have a Y chromosome.

[0211] The subject of the present disclosure may be diagnosed with a disease or disorder before the method of the present disclosure is started.In some embodiments, the subject of the present disclosure is at risk of developing the disease or disorder of the present disclosure.In some embodiments, the subject is identified as "at risk" based on one or more of genetic testing, family history, workplace or home exposure, and health level, which cumulatively provide an assessment of risk.

[0186]

[0212] The subject of the present disclosure may be provided with a first medical intervention for treating or preventing a disease or disorder, which may be partially effective or ineffective.In some embodiments, the subject relapses after being provided with the first medical intervention, and thus the subject requires treatment by the method of the present disclosure.

[0187]

[0213] The subject of the present disclosure may be identified as not responding to existing treatment before the method of the present disclosure is started.In some embodiments, the subject of the present disclosure may be determined not responding to standard treatment based on one or more of gene or biomarker testing, family history, health level, and past experience with specific intervention, which cumulatively provide an assessment of responsiveness to treatment.

[0188]

[0214] The subject of the present disclosure can undergo a second medical intervention, either simultaneously or sequentially with the compositions and methods of the present disclosure.

[0215] In some embodiments, the subject of the present disclosure is a human. In some embodiments, the subject of the present disclosure is a non-human primate. In some embodiments, the subject of the present disclosure is a mammal. In some embodiments, the subject of the present disclosure is a horse, cow, sheep, dog, cat, pig, chicken, guinea pig, rodent, rat, or mouse.

[0189] nucleotide

[0216] "Nucleic acid", particularly DNA or RNA molecules, refers only to the primary and secondary structure of the molecule and is not limited to any particular tertiary form. Thus, the term includes double-stranded DNA found in, among others, linear or circular DNA molecules (e.g., restriction fragments), plasmids, supercoiled DNA, and chromosomes. When discussing the structure of a particular double-stranded DNA molecule, the sequence is provided according to the usual convention of writing the sequence from left to right in a 5' to 3' direction along the non-transcribed strand of DNA (i.e., the strand with sequence homologous to messenger RNA or mRNA). Unless otherwise indicated, all nucleic acid and nucleotide sequences are written from left to right in a 5' to 3' orientation.

[0190]

[0217] Nucleotides are referred to by their commonly known one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission: thus, "A" stands for adenine, "C" stands for cytosine, "G" stands for guanine, "T" stands for thymine, and "U" stands for uracil.

[0191]

[0218] "Polynucleotide" refers to a polymer of nucleotides of any length or type, including ribonucleotides, deoxyribonucleotides, their analogs, or mixtures thereof. The term refers to the primary structure of the molecule. The term includes double- and single-stranded nucleic acids, including deoxyribonucleic acid ("DNA") and ribonucleic acid ("RNA"). It also includes modified forms, for example, by alkylation and / or capping, as well as unmodified forms of polynucleotides.

[0192]

[0219] In some embodiments, the polynucleotide comprises DNA, for example DNA inserted into a vector. In some embodiments, the polynucleotide comprises mRNA. In some embodiments, the mRNA is synthetic mRNA. In some embodiments, the synthetic mRNA comprises at least one non-natural nucleobase. In some embodiments, all nucleobases of a certain class are replaced with non-natural nucleobases (e.g., all uridines in a polynucleotide can be replaced with non-natural nucleobases, for example 5-methoxyuridine).

[0193]

[0220] "Expression vector" refers to a plasmid, virus, or other nucleic acid designed for polypeptide expression in a cell. A vector or construct is used to introduce a gene into a host cell, whereby the vector interacts with a polymerase in the cell to express a protein encoded in the vector / construct. An expression vector may be present extrachromosomally in the cell or integrated into the chromosome. An expression vector may contain additional sequences that make the vector suitable for replication and integration in prokaryotes, eukaryotes, or preferably both (e.g., shuttle vectors). A polynucleotide of the present disclosure may be provided as a component of an expression vector.

[0194]

[0221] "Cloning vector" refers to a plasmid, virus, or other nucleic acid designed to generate copies of a polynucleotide. A cloning vector can contain transcription and translation initiation sequences, transcription and translation termination sequences, and a polyadenylation signal. Such constructs typically include a 5'LTR, a tRNA binding site, a packaging signal, an origin of second strand DNA synthesis, and a 3'LTR or a portion thereof. The polynucleotides of the present disclosure can be provided as components of a cloning vector that can be used to generate the polynucleotides of the present disclosure.

[0195]

[0222] "Promoter" refers to a nucleotide sequence that indicates where transcription of a gene begins and in what direction transcription continues.

[0223] "Encode" or the like refers to the inherent property that a particular sequence of nucleotides in a polynucleotide (e.g., a gene, cDNA, or mRNA) serves as a template for the synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA, and mRNA) or a defined sequence of amino acids. Thus, a gene, cDNA, or RNA encodes a protein when transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding and non-coding strands can be referred to as encoding the protein or other product of that gene or cDNA.

[0196]

[0224] Unless otherwise specified, a nucleotide sequence that "encodes an amino acid sequence," e.g., a polynucleotide that "encodes" a chimeric polypeptide as defined hereinafter in this disclosure, includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence.

[0197] Polypeptides

[0225] Amino acids may be referred to by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Amino acid residues are abbreviated as follows, with the abbreviation shown in parentheses: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0198]

[0226] Amino acid sequences are written left to right in amino to carboxy orientation.

[0227] "Polypeptide" is used in its broadest sense to refer to a sequence of amino acid subunits. In some embodiments, a "peptide" can be 50 amino acids or less in length, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length. "Polypeptide" refers to proteins, polypeptides, and peptides of any length, size, structure, or function. "Polypeptide," "peptide," and "protein" are used interchangeably to refer to polymers of amino acids of any length.

[0199]

[0228] Polypeptides can include naturally or synthetically produced or modified amino acids, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. This definition also includes, for example, polypeptides in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids (including, for example, synthetic amino acids such as homocysteine, ornithine, p-acetylphenylalanine, D-amino acids, and creatine), as well as other modifications known in the art. Polypeptides also include the aforementioned gene products, homologs, orthologs, paralogs, fragments, and other equivalents, variants, and analogs. A polypeptide can be a single polypeptide or a multi-molecular complex, such as a dimer, trimer, or tetramer. A polypeptide also includes single-chain or multi-chain polypeptides. Disulfide bonds may be present in multi-chain polypeptides. The described polypeptides can be chemically synthesized or recombinantly expressed.

[0200]

[0229] The polypeptides of the present disclosure may include additional residues or moieties at the N-terminus, C-terminus, within the polypeptide, or combinations thereof, and these additional residues or moieties are not included in determining the percent identity of the polypeptides of the present disclosure relative to the reference polypeptide. Such residues may be any residues suitable for the intended use, including, but not limited to, tags.

[0201]

[0230] "Tags" can include, for example, detectable moieties (e.g., fluorescent proteins, antibody epitope tags, etc.), therapeutic agents, purification tags (such as His tags), linkers, ligands suitable for purification purposes, ligands for effecting localization of a polypeptide, and / or peptide domains that add functionality to a polypeptide.

[0202]

[0231] "Chimeric polypeptide" refers to any polypeptide comprising a first amino acid sequence from a first source covalently or non-covalently bound to a second amino acid sequence from a second source, where the first and second sources are not identical. The non-identical first and second sources may include two different biological entities, or two different proteins from the same biological entity, or a biological entity and a non-biological entity. A chimeric protein may, for example, include a protein from at least two different biological sources. A biological source may include any non-synthetically produced nucleic acid or amino acid sequence (e.g., a genomic or cDNA sequence, a plasmid or viral vector, a natural virion, or a mutant or analog of any of the above). A synthetic source may include a protein or nucleic acid sequence that is chemically produced rather than by a biological system (e.g., solid-phase synthesis of an amino acid sequence). A chimeric protein may also include a protein from at least two different synthetic sources, or a protein from at least one biological source and at least one synthetic source. Chimeric proteins can also include those in which a first amino acid sequence from a first source is covalently or non-covalently linked to a nucleic acid from any source, or to a small organic or inorganic molecule from any source. Chimeric proteins can include a linker molecule between the first amino acid sequence and the second amino acid sequence, or between the first amino acid sequence and the nucleic acid, or between the first amino acid sequence and the small organic or inorganic molecule.

[0203]

[0232] A "fragment" of a polypeptide, or a "truncated polypeptide" refers to an amino acid sequence of a polypeptide that is shorter than the naturally occurring sequence. Compared to a naturally occurring polypeptide, a fragment may be missing the N-terminus and / or C-terminus, or any part of the polypeptide may be missing. Thus, a fragment does not necessarily have to only have N-terminus and / or C-terminus amino acids missing. A polypeptide that has internal amino acids missing with respect to a naturally occurring sequence is also considered a fragment. The polypeptide components of the present disclosure can be provided as fragments or truncated polypeptides.

[0204]

[0233] "Functional fragment" refers to a polypeptide fragment that retains the function of the polypeptide. Thus, in some embodiments, a functional fragment of a biologically active peptide, e.g., an enzyme, retains the ability to catalyze a biological action, e.g., has a catalytic domain of an enzyme. The polypeptides of the present disclosure may include or be provided as functional fragments.

[0205]

[0234] "Functional variant" refers to a modified form of a polypeptide, fragment, or member of a class of polypeptides that maintains the function of the polypeptide. The polypeptide components of the present disclosure may be provided as functional variants.

[0206]

[0235] "Amino acid substitution" refers to replacing an amino acid residue present in a parent or reference sequence (e.g., a wild-type sequence) with another amino acid residue. The amino acid can be replaced, for example, via chemical peptide synthesis or through recombinant methods. For example, replacing an amino acid residue with an alternative amino acid residue can be performed by replacing a codon that codes for a first amino acid with a codon that codes for a second amino acid. The polypeptides of the present disclosure can include amino acid substitutions.

[0207]

[0236] "Conservative amino acid substitution" refers to the replacement of one amino acid residue with an amino acid residue that has a chemically similar side chain. The art defines a family of amino acid residues that have similar side chains, including acidic side chains (e.g., aspartic acid, glutamic acid), basic side chains (e.g., lysine, arginine, histidine), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).Therefore, when an amino acid in a polypeptide is replaced with another amino acid from the same side chain family, the substitution is considered to be conservative. In another embodiment, stretches of amino acids can be conservatively replaced with chemically similar stretches that differ in the order and / or composition of side chain family members. Various polypeptide components of the disclosure may be provided with conservative amino acid substitutions.

[0208]

[0237] "Non-conservative amino acid substitutions" include (i) a residue having a positively charged side chain (e.g., Arg, His, or Lys) is substituted for or replaced by a negatively charged residue (e.g., Glu or Asp), (ii) a hydrophilic residue (e.g., Ser or Thr) is substituted for or replaced by a hydrophobic residue (e.g., Ala, Leu, Ile, Phe, or Val), (iii) a cysteine ​​or proline is substituted for or replaced by any other residue, or (iv) a residue having a bulky hydrophobic or aromatic side chain (e.g., Val, His, Ile, or Trp) is substituted for or replaced by one having a smaller side chain (e.g., Ala or Ser) or no side chain (e.g., Gly). Various polypeptide components of the disclosure may be provided with non-conservative amino acid substitutions. The possibility that one of the above non-conservative substitutions can change the functional properties of a protein also correlates with the position of the substitution relative to the functionally important regions of the protein, and therefore some non-conservative substitutions have little or no effect on biological properties. Various polypeptide components of the present disclosure may be provided with non-conservative amino acid substitutions that do not significantly change the function of the altered component in some cases. Various polypeptide components of the present disclosure may be provided with non-conservative amino acid substitutions that change the function of the altered component in some cases by enhancing or reducing the function.

[0209] Sequence identity or similarity

[0238] "Conserved" refers to a nucleotide or amino acid residue of a polynucleotide sequence or a polypeptide sequence that is present without change in the same position of two or more sequences being compared. A conserved nucleotide or amino acid is one that is more conserved between related sequences than a nucleotide or amino acid that is present elsewhere in the sequence. In some embodiments, two or more sequences are said to be "conserved" if they are at least about 30% identical, at least about 35% identical, at least about 40% identical, at least about 45% identical, at least about 50% identical, at least about 55%, at least about 60% identical, at least about 65% identical, at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, or at least about 99% identical to each other. Sequence conservation can apply to the entire length of a polynucleotide or polypeptide, or to a portion, region, or feature thereof.

[0210]

[0239] In some embodiments, two or more sequences are said to be "fully conserved" or "identical" if they are 100% identical to each other. In some embodiments, two or more sequences are said to be "highly conserved" if they are at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, or at least about 99% identical to each other.

[0211]

[0240] "Homology" refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules and / or polypeptide molecules. Homology includes both identity and similarity.

[0241] In some embodiments, polymer molecules are considered to be "homologous" to one another if at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% of the monomers in the molecule are identical (exactly the same monomers) or similar (conservative substitutions). "Homologous" necessarily refers to a comparison between at least two polynucleotide or polypeptide sequences. In various aspects, the present disclosure includes polynucleotides and polypeptides that are homologous to the polynucleotides and polypeptides described herein.

[0212]

[0242] "Identity" refers to the overall monomer conservation between polymer molecules, e.g., between polypeptide or polynucleotide molecules. "Identical" without any additional modifiers, e.g., protein A is identical to protein B, means that the sequences are 100% identical (100% sequence identity). Describing two sequences as, e.g., "70% identical" is equivalent to describing them as having, e.g., "70% sequence identity."

[0213]

[0243] When a position in the first sequence is occupied by the same amino acid as the corresponding position in the second sequence, the molecules are identical at that position.The percent identity between two sequences is a function of the number of identical positions shared by sequences, taking into account the number of gaps and the length of each gap that needs to be introduced for optimal alignment of the two sequences.Comparing sequences between two sequences and determining percent identity can be achieved using mathematical algorithms.

[0214]

[0244] In some embodiments, the percent identity (%ID) of a first amino acid (or nucleic acid) sequence to a second amino acid (or nucleic acid) sequence is calculated as %ID=100(Y / Z), where Y is the number of amino acid (or nucleic acid base) residues scored as identical matches in an alignment of the first and second sequences (aligned by visual inspection or by a specific sequence alignment program) and Z is the total number of residues in the second sequence. If the length of the first sequence is longer than the second sequence, the percent identity of the first sequence to the second sequence will be higher than the percent identity of the second sequence to the first sequence.

[0215]

[0245] For example, the percent identity calculation of two polypeptide sequences can be performed by aligning the two sequences for optimal comparison.For example, gaps can be introduced into one or both of the first and second polypeptide sequences for optimal alignment, and non-identical sequences can be ignored for comparison purposes.In some embodiments, the length of the sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence.Then, the amino acids at corresponding amino acid positions are compared.

[0216]

[0246] The generation of sequence alignment for calculating percent sequence identity is not limited to the two-way sequence comparison carried out by only primary sequence data.Sequence alignment can be generated by integrating sequence data with data from heterogeneous sources, such as structural data (e.g., crystallographic protein structure), functional data (e.g., mutation location), or phylogenetic data.Software can be used to integrate heterogeneous data to generate multiple sequence alignment, one example of which is T-Coffee, available at www.tcoffee.org, or available, for example, from the European Bioinformatics Institute (EBI) website at ebi.ac.uk / Tools / psa.The final alignment used to calculate percent sequence identity can be automatically or manually curated.

[0217]

[0247] Software for alignment of polypeptide and nucleotide sequences is available. One program used to determine percent sequence identity is bl2seq, which is part of the BLAST suite of programs available from the BLAST website (blast.ncbi.nlm.nih.gov) of the U.S. government's National Center for Biotechnology Information. Bl2seq uses either the BLASTN or BLASTP algorithm to perform comparisons. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water, or Matcher, which are part of the EMBOSS bioinformatics suite of programs, also available from EBI. Sequence alignment can be performed using methods known in the art, such as MAFFT, Clustal (ClustalW, Clustal X, or Clustal Omega), MUSCLE. Different regions within a single polynucleotide or polypeptide target sequence that align with a polynucleotide or polypeptide reference sequence can each have their own percent sequence identity. Please note that percent sequence identity values ​​are rounded to the nearest tenth, e.g., a value between 80.11 and 80.14 would be rounded to 80.1, a value between 80.15 and 80.19 would be rounded to 80.2, etc. Also note that length values ​​are always integers.

[0218]

[0248] "Similarity" refers to the overall relatedness between polymer molecules, e.g., between polynucleotide molecules and / or between polypeptide molecules. Calculation of percent similarity of polymer molecules to each other can be performed in the same manner as calculation of percent identity, except that the calculation of percent similarity takes into account conservative substitutions. The percentage of similarity depends on the comparison measure used, e.g., whether amino acids are compared according to, e.g., their evolutionary proximity, charge, volume, flexibility, polarity, hydrophobicity, aromaticity, isoelectric point, antigenicity, or a combination thereof.

[0219] cell therapy

[0249] In some embodiments of the present disclosure, hematopoietic cells are cells that arise from hematopoietic stem cells, including, but not limited to, myeloid progenitor cells, lymphoid progenitor cells, megakaryocytes, erythrocytes, mast cells, myeloblasts, basophils, neutrophils, eosinophils, macrophages, platelets, monocytes, natural killer cells, T lymphocytes, B lymphocytes, and plasma cells.

[0220]

[0250] In some embodiments of the present disclosure, T lymphocytes or T cells refer to hematopoietic cells that normally occur in the thymus. T lymphocytes or T cells of the present disclosure include, but are not limited to, natural killer T cells, regulatory T cells, helper T cells, cytotoxic T cells, memory T cells, gamma delta T cells, and mucosal invariant T cells.

[0221]

[0251] In some embodiments of the present disclosure, autologous cells are cells obtained from the same individual to which they can be administered as a therapy (the cells are autologous to the subject). Autologous cells of the present disclosure include, but are not limited to, hematopoietic cells and stem cells, e.g., hematopoietic stem cells.

[0222]

[0252] In some embodiments of the present disclosure, the allogeneic cells are cells obtained from an individual who is not the intended recipient of the cells as a therapy (the cells are allogeneic to the subject). The allogeneic cells of the present disclosure can be selected from a donor who is immunologically compatible with the subject of the method of the present disclosure. The allogeneic cells of the present disclosure can be modified to generate "universal" allogeneic cells suitable for administration to any subject without unintended immunogenicity. The allogeneic cells of the present disclosure include, but are not limited to, hematopoietic cells and stem cells, e.g., hematopoietic stem cells.

[0223] General Terms

[0253] The terms "a," "an," and "the" include their plural forms unless the context indicates otherwise.

[0224]

[0254] "And" is used interchangeably with "or" unless otherwise noted.

[0255] "And / or" should be interpreted as a specific disclosure of each of the two specified features or components, with or without the other. Thus, "and / or" used in phrases such as "A and / or B" includes "A and B," "A or B," "A" (single), and "B" (single). Similarly, "and / or" used in phrases such as "A, B, and / or C" is intended to cover each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (single); B (single); and C (single).

[0225]

[0256] "About" means approximately, roughly, around, or within the region. When "about" is used with a numerical range, it modifies that range by expanding the boundaries above and below the stated numerical values. "About" can modify numerical values ​​above and below the stated value by variance. As used throughout this disclosure, any numerical value disclosed in the context of "about" is also intended to be described in the absence of the term "about." For example, the term "about 17 nm" is also intended to disclose the term "17 nm."

[0226]

[0257] Numeric ranges include the numerical values ​​that define the range. When a range of values ​​is described, each intervening integer value and each fraction between the upper and lower recited limits of the range is specifically disclosed, as well as each subrange between such values. The upper and lower limits of any range can be independently included or excluded from the range, and each range in which either limit is included, neither limit is included, or both limits are included is also included within the scope of this disclosure. Thus, a range is understood to be a shorthand for all values ​​within the range, including the recited endpoints.

[0227]

[0258] Where values ​​are explicitly recited, it should be understood that equivalent values ​​that are approximately the same quantity or amount as the recited value (or that produce a substantially similar result by a substantially similar mechanism) are also within the scope of the disclosure.

[0228]

[0259] Where a combination is disclosed, each subcombination of the elements of that combination is also specifically disclosed and is within the scope of the disclosure. Conversely, where different elements or groups of elements are individually disclosed, combinations of those elements are also disclosed.

[0229]

[0260] Where any element of the disclosure is disclosed as having multiple alternatives, instances of that disclosure in which each alternative is excluded alone, or in any combination with the other alternatives, are also disclosed, and two or more elements of the disclosure may have such an exclusion, and all combinations of elements with such exclusions are disclosed herein.

[0230]

[0261] Unless the context clearly dictates otherwise, throughout the description and claims, the words "include," "including," and the like are meant to be interpreted in the sense of "including, but not limited to."

[0231]

[0262] Each singular or plural word also includes the plural and the singular.

[0263] The words "above" and "below" and words of similar import refer to this application as a whole and not to any particular portions of this application.

[0232]

[0264] A "set" includes a set of one or more elements or objects. A "subset" of a set includes any number of elements or objects from the set, from one to all of the elements of the set.

[0233]

[0265] Headings are included throughout this disclosure for reference and to aid in locating the various sections. These headings are not intended to limit the scope of the concepts described therein. Such concepts may have applicability throughout this specification.

[0234]

[0266] Although the present disclosure has been described in some detail by way of illustration and example, for purposes of clarity and understanding, certain changes and modifications may be implemented. References to "the present disclosure" and the like are intended to refer to any of the various embodiments or aspects of the present disclosure and are not intended to limit the present disclosure to a single embodiment or aspect.

[0235]

[0267] The descriptions and examples should not be construed as limiting the scope of the disclosure to the embodiments and examples described herein, but rather as including all modifications and alternatives falling within the true scope and spirit of the disclosure.

[0236] Device

[0268] The present disclosure provides devices comprising a CAR of this disclosure, a sequence comprising or encoding a CAR of this disclosure (including a spacer sequence), a vector comprising a sequence encoding a CAR of this disclosure (including a spacer sequence), a cell of this disclosure comprising any one or more of the foregoing, a composition of this disclosure comprising any one or more of the foregoing, and a pharmaceutical composition of this disclosure comprising any one or more of the foregoing.

[0237]

[0269] Exemplary devices of the present disclosure include, but are not limited to, syringes, needles, and vials. kit

[0270] The present disclosure provides a kit or article of manufacture that includes one or more of the following: CAR, sequences that include or code for CAR. The kit can include, for example, components for delivery of polynucleotides to cells or subjects. The kit can include, for example, components for delivery of cells, such as the CAR T cells of the present disclosure, to subjects. The polynucleotides can be provided in the kit as part of an expression vector configured for expression in T cells. In certain embodiments, the kit or article of manufacture further includes instructions for using a set of polynucleotides to transform cells to express a gene of interest to produce a CAR, as in the case of the present disclosure. In certain embodiments, the kit or article of manufacture further includes instructions for using the cells to treat a subject. EXAMPLES

[0238] Example 1: Functional benefits of spacer sequence selection for CAR efficacy

[0271] This study describes the influence of different Ig-derived or extracellular domain-derived spacers on the induction of antigen-directed cytokine release and efficacy. Anti-HA scFv (clone 2E2) was used as the CAR binder, and the HA peptide (YPYDVPDYA) was used as a model linear epitope that can be systematically presented at different distances from the membrane of the target cell. This approach allowed for the systematic evaluation of the optimal intercellular distance of CAR T cells.

[0239]

[0272] In this study, an in vitro screening strategy was used to evaluate the effect of CAR spacer length on induction of antigen-directed cytokine release and efficacy. The screening strategy included: (i) altering the distance of the epitope from the surface of the target cell; (ii) altering the distance of the corresponding epitope binder ("binder") from the surface of the CAR T cell; (iii) To evaluate pairwise combinations of target cells and CAR T cells for basic functional parameters (e.g., target cell lysis and cytokine production).

[0240]

[0273] A lentiviral construct was made using a bicistronic expression construct, in which the following coding sequences were linked in frame and under the control of the MND promoter: (i)HA-specific CAR (ii) the P2A self-cleaving peptide, and (iii) EGFRt transduction marker (i.e., a truncated EGFR having only domains III and IV and the transmembrane domain). FIG. 3 shows the organization of the components of the construct.

[0241]

[0274] The HA-specific CARs used in this study included spacer sequences derived from the human immunoglobulin (Ig) hinge region or extracellular protein domain (ECD) attached to a consensus Gly-Ser (GS) linker sequence.

[0242]

[0275] To analyze the effect of spacer length on CAR T cell function, the spacer length was varied while keeping other components in the HA-specific CAR constant, e.g., the antigen binding element was an anti-HA scFv (clone 2E2), the transmembrane element was derived from the CD28 protein transmembrane domain, and the intercellular element was derived from the 4-1BB protein costimulatory domain and CD3 zeta signaling domain.

[0243]

[0276] Figure 4 shows an example of an HA-specific CAR construct used in this study.

[0277] The sequences and lengths of the spacers used in this study are shown in Table 1. Spacer sequences are identified by spacer numbers 1-31 or other designated names. The total length of the CAR spacer is given in angstroms, which includes both the linker and spacer sequences.

[0244]

[0278] Human primary T cells were obtained from two normal donors, Donor 1 and Donor 2. The HA-specific CAR was introduced into T cells from Donor 1 and Donor 2 using a lentiviral construct encoding a bicistronic expression cassette.

[0245]

[0279] FIG. 5 presents an exemplary HA-epitope construct showing the composition of the various elements that were used in the context of this example.

[0280] The HA-epitope expression constructs used in this study contained a spacer sequence derived from human immunoglobulin (Ig) proteins linked to a common GS linker sequence. To systematically vary the distance of the HA-epitope from the surface of A549 target cells, the length of the spacer sequence was varied while keeping constant the other components in the expression construct, namely, the mini-FLAG tag sequence (to determine HA-epitope surface expression), the HA epitope sequence, the transmembrane domain from the CD28 protein, the intracellular linker sequence, and the eGFP transduction marker. Figure 6 is a schematic diagram of an example of an HA-epitope construct used in this study.

[0246]

[0281] The sequences and lengths of the epitope spacers used in this study are shown in Table 2. Target epitopes are identified by target epitope numbers 1-16, where 1 is assigned to the shortest epitope spacer length (i.e., the target epitope is considered closest to the membrane of the target cell) and 16 is assigned to the longest epitope length (i.e., the target epitope is considered furthest from the membrane of the target cell). The total length of the epitope spacer is given in angstroms, which includes both the linker and spacer sequences.

[0247]

[0282] A549-NLR cells were used as the target cell background for expression of the HA-epitope constructs. Sixteen A549-NLR target cell lines were generated, each expressing the HA epitope at a different distance from the surface of the cell membrane.

[0248]

[0283] In the functional assays described below, the 16 A549-NLR target cell lines are designated as Target 1, Target 2, Target 3, etc., based on the target epitope number shown in Table 2. For example, Target 1 expresses the most membrane proximal HA epitope, Target 6 expresses an HA epitope at an intermediate membrane distance, and Target 16 expresses the most membrane distal HA epitope.

[0249]

[0284] Functional assays correlate CAR T cell-mediated target cell killing and target-dependent cytokine secretion to assess the efficacy of pairwise combinations of HA epitope-targeted cells and anti-HA CAR T cells.

[0250]

[0285] Area under the curve (AUC) values ​​were calculated by integrating the area under the normalized target cell killing curve: the lower the AUC value, the more effective the killing mediated by the CAR T cells.

[0251]

[0286] The transduction efficiency of A549-NLR cells with different length-adjusted HA-epitope constructs was assessed by the percentage of transduction markers (GFP+), HA surface expression (%HATag+), and median mean fluorescence intensity (MFI).

[0252]

[0287] Figure 7A is a plot showing the percentage of transduced target cells as measured by expression of the transduction marker GFP on live A549-NLR cells. Transduced cells were monitored during expansion through the fourth (P4) and fifth (P5) passages. The data show that we were able to generate 16 A549-NLR target cell lines, each expressing a length-regulated HA epitope.

[0253]

[0288] Figure 7B is a plot showing the percentage of HA tag surface expression on transduced GFP+ cells. The data show that the expressed epitope is detectable on A549-NLR target cells.

[0254]

[0289] Figure 7C is a plot showing the median fluorescence intensity (MFI) for bound anti-HA antibodies on live transduced cells (GRP+ cells). The data show that there is some variation between cell lines in terms of surface expression of the HA epitope.

[0255]

[0290] The transduction efficiency of donor 1 and donor 2 cells with the different anti-HA CAR constructs was assessed by the percentage of transduced marker (EGFRt+) and median mean fluorescence intensity (MFI).

[0256]

[0291] Figures 8A and 8B are plots showing the percentage of transduced cells measured by surface expression of the transduction marker EGFRt on live primary T cells in donor 1 and donor 2, respectively. The anti-HA CARs used are listed on the x-axis (see Table 1). The cutoff for the functional assay was set to less than 20% and is shown by the dashed line. Now referring to Figure 8A, the anti-HA CAR constructs that were not fully transduced for donor 1 were the constructs with spacer 14 and spacer OX40. These two constructs were omitted from the subsequent functional assay experiments. Now referring to Figure 8B, all anti-HA CAR constructs were fully transduced for donor 2 and were included in the subsequent functional assay experiments.

[0257]

[0292] Figures 8C and 8D are plots showing median fluorescence intensity (MFI) as a measure of transduction efficiency for bound HA-Fc protein on live transduced cells (EGFRt+ cells) in donor 1 and donor 2, respectively.

[0258]

[0293] A systematic screening strategy was used to demonstrate the relationship between T cell efficacy and the intercellular distance between pairwise combinations of CAR T cells and target cells. Target cell killing and target-dependent cytokine production were used as indicators of CAR T cell efficacy.

[0259]

[0294] Figures 9A, 9B, 9C, and 9D show the effect of the indicated spacers on anti-HA scFv-induced cytotoxicity in the presence of NucLight Red-labeled A549 target cells expressing the most membrane-proximal HA target epitope in donor 1 (A549-HA target 1).

[0260]

[0295] Referring now to Figure 9A, this is a plot showing the area under the curve (AUC) calculated for the IncuCyte killing curves of each CAR variant with the indicated spacer. Non-transduced primary T cells are shown as "NTC". The smaller the AUC value, the more effective the killing of the anti-HA CAR T cells. In this example, donor 1 CAR T cells expressing spacers 6 (spacer length 187.2 Å) and 7 (spacer length 133.2 Å) had the smallest AUC value, indicating greater killing of A549-HA target 1, which expresses the most membrane-proximal HA epitope.

[0261]

[0296] Now, referring to Figure 9B and Figure 9C, which are bar graphs showing the concentration of IFN-γ and IL-2 in the co-culture medium after 24 hours of co-culture, respectively. Cytokine secretion is directly correlated with target cell killing. For example, donor 1 CAR T cells expressing spacers 6 and 7, which showed the best killing effect (see Figure 9A), also showed the highest level of cytokine secretion.

[0262]

[0297] Reference is now made to FIG. 9D, which is a scatter plot showing the effect of the indicated spacer lengths on target-dependent cytokine secretion and killing AUC for anti-HA CAR-T cells when co-cultured with A549-HA target 1. Normalized AUC is on the y-axis and spacer length is on the x-axis. In this plot, dot size represents IFN-γ, with larger dot size corresponding to higher levels of IFN-γ secretion. The data shows, for example, that the optimal spacer length for killing membrane-proximal target 1 is approximately 130 Å, which correlates with the highest levels of IFNγ secretion. The data shows a U-shaped curve, indicating that for a given target, as one moves away from the optimal spacer length, either left or right on the x-axis, killing efficacy and cytokine secretion begin to decline (i.e., data points begin to increase for AUC and dot size for IFNγ decreases).

[0263]

[0298] Figures 10A, 10B, 10C, and 10D show the effect of the indicated spacers on anti-HA scFv-induced cytotoxicity in the presence of NucLight Red-labeled A549 target cells expressing the most membrane-proximal HA target epitope (A549-HA target 1) in donor 2. Figure 10A is a plot showing the area under the curve (AUC) calculated for the IncuCyte killing curves of each CAR variant with the indicated spacers. Non-transduced primary T cells are shown as "NTC". Figures 10B and 10C are bar graphs showing the concentrations of IFN-γ and IL-2, respectively, in the co-culture medium after 24 hours of co-culture. Figure 10D is a scatter plot summarizing the effect of the indicated spacer lengths on target-dependent cytokine secretion and killing AUC for anti-HA CAR-T cells when co-cultured with A549-HA target 1. Dot size represents IFN-γ, with larger dot size indicating higher levels of IFN-γ secretion.

[0264]

[0299] Figures 11A, 11B, 11C, and 11D show the effect of the indicated spacers on anti-HA scFv-induced cytotoxicity in the presence of NucLight Red-labeled A549 target cells expressing the HA target epitope at intermediate membrane distances in donor 1 (A549-HA target 6). Figure 11A is a plot showing the area under the curve (AUC) calculated for the IncuCyte killing curves of each CAR variant with the indicated spacers. Non-transduced primary T cells are shown as "NTC". Figures 11B and 11C are bar graphs showing the respective concentrations of IFN-γ and IL-2 in the co-culture medium after 24 hours of co-culture. Figure 11D is a scatter plot summarizing the effect of the indicated spacer lengths on target-dependent cytokine secretion and killing AUC for anti-HA CAR-T cells when co-cultured with A549-HA target 6. Dot size represents IFN-γ, with larger dot size indicating higher levels of IFN-γ secretion.

[0265]

[0300] Figures 12A, 12B, 12C, and 12D show the effect of the indicated spacers on anti-HA scFv-induced cytotoxicity in the presence of NucLight Red-labeled A549 target cells expressing the HA target epitope at intermediate membrane distances in donor 2 (A549-HA target 6). Figure 12A is a plot showing the area under the curve (AUC) calculated for the IncuCyte killing curves of each CAR variant with the indicated spacers. Non-transduced primary T cells are shown as "NTC". Figures 12B and 12C are bar graphs showing the respective concentrations of IFN-γ and IL-2 in the co-culture medium after 24 hours of co-culture. Figure 12D is a scatter plot showing the effect of the indicated spacer length on target-dependent cytokine secretion and killing AUC for anti-HA CAR-T cells when co-cultured with A549-HA target 6. Dot size represents IFN-γ, with larger dot size indicating higher levels of IFN-γ secretion.

[0266]

[0301] Figures 13A, 13B, 13C, and 13D show the effect of the indicated spacers on anti-HA scFv-induced cytotoxicity in the presence of NucLight Red-labeled A549 target cells expressing the most membrane-distal HA target epitope in donor 1 (A549-HA target 16). Figure 13A is a plot showing the area under the curve (AUC) calculated for the IncuCyte killing curves of each CAR variant with the indicated spacers. Non-transduced primary T cells are shown as "NTC". Figures 13B and 13C are bar graphs showing the concentrations of IFN-γ and IL-2, respectively, in the co-culture medium after 24 hours of co-culture. Figure 13D is a scatter plot summarizing the effect of the indicated spacer lengths on target-dependent cytokine secretion and killing AUC for anti-HA CAR-T cells when co-cultured with A549-HA target 16. The dot size represents IFN-γ; the larger the dot size, the higher the level of IFN-γ secretion.

[0267]

[0302] Figures 14A, 14B, 14C, and 14D show the effect of the indicated spacers on anti-HA scFv-induced cytotoxicity in the presence of NucLight Red-labeled A549 target cells expressing the most membrane-distal HA target epitope (A549-HA target 16) in donor 2. Figure 14A is a plot showing the area under the curve (AUC) calculated for the IncuCyte killing curves of each CAR variant with the indicated spacers. Non-transduced primary T cells are shown as "NTC". Figures 14B and 14C are bar graphs showing the concentrations of IFN-γ and IL-2, respectively, in the co-culture medium after 24 hours of co-culture. Figure 14D is a scatter plot summarizing the effect of the indicated spacer lengths on target-dependent cytokine secretion and killing AUC for anti-HA CAR-T cells when co-cultured with A549-HA target 16. The dot size represents IFN-γ; the larger the dot size, the higher the level of IFN-γ secretion.

[0268]

[0303] Figures 15A, 15B, 15C, and 15D show the effect of the indicated spacers on anti-HA scFv-induced cytotoxicity in the presence of NucLight Red-labeled A549 target cells that do not have the HA target epitope (i.e., antigen-negative wild-type A549 cells = target 17). Referring now to Figure 15A, this is a plot showing IncuCyte kinetic killing curves (NucLight Red signal over time) for each CAR variant from donor 1 with the indicated spacers. Non-transduced primary T cells are shown as "NTC". Figure 15B is a bar graph showing the concentration of IFN-γ in the co-culture medium after 24 hours of co-culture from donor 1. The data show that full-length 41BB and full-length CD27 ECD spacers exhibit increased IFN-γ production and cytotoxic activity against antigen-negative A549 cells.

[0269]

[0304] Referring now to Figure 15C, which is a plot showing IncuCyte kinetic killing curves (NucLight Red signal over time) for each CAR variant from donor 2 with the indicated spacer. Non-transduced primary T cells are shown as "NTC". Figure 15D is a bar graph showing the concentration of IFN-γ in the co-culture medium after 24 hours of co-culture from donor 2. The data show that the intermediate spacer, full-length 41BB and full-length CD27 ECD spacers exhibit increased IFN-γ production and cytotoxic activity against antigen-negative A549 cells.

[0270]

[0305] Figures 16A and 16B are plots showing IFN-γ secretion profiles on HA CAR-T cells from donor 1 and donor 2, respectively, expressing the indicated spacers in the absence of target cells. Spacers that show increased cytokine production in the absence of target cells are considered to exhibit sustained signaling.

[0271]

[0306] Figures 17-20 are scatter plots summarizing the effect of systematically varying HA epitope intermembrane distance for optimal anti-HA CAR spacer length on target-dependent cytokine secretion and killing AUC for donor 1 and donor 2 CAR T cells co-cultured with A549-HA target cells (i.e., target cells 1 to 16, see Table 2). Normalized AUC is on the y-axis and spacer length is on the x-axis. Black dots represent IFN-γ, with larger dot size corresponding to higher levels of IFN-γ secretion. Blue dots represent IL-2, with higher blue intensity corresponding to higher levels of IL-2 secretion. Figures 17A, 18A, 19A, and 20A show HA CAR-T from donor 1 co-cultured with A549 target cells expressing HA epitopes at increasing distances from the target cell membrane. Figure 17B, Figure 18B, Figure 19B, and Figure 20B show HA CAR-T from donor 2 co-cultured with A549 target cells expressing HA epitopes at increasing distances from the target cell membrane. The data show that as the target epitope distance from the target cell surface increases (i.e., targets 1 to 16, see Table 2), the preferred spacer length decreases. For example, for target 16, which has the longest epitope distance (see plot 20B), only the shortest CAR spacer mediates sufficient cytokine secretion and target cell killing. The data also show that the changes in IL-2 and IFN-γ secretion follow a similar trend as the changes in target epitope distance.

[0272]

[0307] As described above with reference to Figure 2, the intercellular distance can be estimated by the combined length of the CAR spacer (including the GS linker) and the scFv-epitope binding element that binds the epitope. For the HA-epitope and anti-HA binding elements used in this study, the HA-epitope C-term relative to the scFv C-term is 36.5 Å as measured from its crystal structure. The length of the CAR spacer is known and is shown in Table 1. The length of the HA-epitope spacer is also known and is shown in Table 2.

[0273]

[0308] FIG. 21A shows a scatter plot summarizing the effect of cell-cell distance on AUC calculated from IncuCyte killing curves and IFN-γ secretion in donor 1 and donor 2, respectively, measured after 24 hours of co-culture. Normalized AUC is on the y-axis and combined spacer length is on the x-axis. Dot size represents IFN-γ levels, with larger dot size corresponding to higher levels of IFN-γ secretion. The scatter plot summarizing data from over 700 data points, i.e., over 700 pairwise combinations of CAR spacers (n=42, see Table 1) and epitope spacers (n=16, see Table 2). FIG. 21B shows a scatter plot summarizing the effect of cell-cell distance on IFN-γ secretion in donor 1 and donor 2, respectively. Now referring to FIG. 21A and FIG. 21B, the data show that the highest killing efficacy and cytotoxicity are observed when the total length is approximately equal to 200 Å. The results indicate that optimal interaction between CAR T cells and target cells occurs at a synaptic distance of approximately 200 Å.

[0274]

[0309] FIG. 22A is a series of plots showing IFN-γ secretion levels for CAR spacer CD27_1 and spacer 1 expressed in donor 1 and donor 2 paired with each target cell line (i.e., targets 1 to 16). Spacer CD27_1 has a length of about 324 Å, which is longer than the optimal intercellular distance of about 200 Å for CAR T cells. Spacer 1, which has a length of about 50 Å, is used as a reference control. The data show that for any given target 1 to 16, the too-long spacer CD27_1 results in submaximal IFN-γ levels, likely due to interactions of the spacer with other proteins on the surface of the target cell. In contrast, spacer 1 shows comparable IFN-γ secretion levels relative to the distance of the epitope from the surface of the target cell.

[0275]

[0310] Figure 22B is a series of plots showing IFN-γ secretion levels for CAR spacers ICOS and spacer 29 expressed in donor 1 and donor 2 paired with each target cell line (i.e., targets 1 to 16). Both ICOS and spacer 29 have a length of about 112 Å, which is considered a suitable length for a spacer, i.e., neither too short nor too long. The data show that both ICOS and spacer 29 result in IFN-γ levels that are about 10-fold lower than the reference spacer 1 (see Figure 22A), which may indicate that sequence composition independent of spacer length influences the performance of these CAR T cells.

Claims

1. (a)(i) a binding member that specifically binds to a target; and (ii) spacer sequence Extracellular elements, including (b) a transmembrane element, and (c) Intracellular elements A chimeric antigen receptor (CAR) comprising: A CAR, wherein when the CAR is expressed on the surface of a cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 17 nanometers (nm) to about 25 nm, including the endpoints.

2. The CAR according to claim 1, wherein, when the CAR is expressed on the surface of a cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 17 nm to about 22 nm, preferably about 20 nm.

3. The CAR according to claim 1 or 2, wherein the spacer sequence has a length of about 2 nm to about 20 nm including the endpoints.

4. The CAR according to claim 1, wherein the spacer sequence comprises a linker sequence, preferably the linker sequence comprises a glycine-serine (GS) linker, more preferably the linker sequence comprises GGGSG (SEQ ID NO: 77).

5. 2. The CAR of claim 1, wherein the spacer sequence comprises one or more sequences selected from the group consisting of SEQ ID NOs: 1-44, optionally the spacer sequence comprises one or more of a deletion, insertion, substitution, inversion, truncation, or alteration, optionally the spacer sequence comprises at least one non-naturally occurring residue; and optionally the substitution comprises replacement of a first amino acid with a second amino acid, wherein the first amino acid and the second amino acid have one or more of polarity, side chain length, or hydrophobicity.

6. 2. The CAR of claim 1, wherein the spacer sequence comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% identity to one or more of SEQ ID NOs: 1-44; optionally, the spacer sequence comprises one or more of a deletion, insertion, substitution, inversion, truncation or modification; optionally, the spacer sequence comprises at least one non-naturally occurring residue; and optionally, the substitution comprises replacement of a first amino acid with a second amino acid, wherein the first amino acid and the second amino acid have one or more of polarity, side chain length, or hydrophobicity.

7. 2. The CAR of claim 1, wherein the binding element comprises an antigen recognition domain, optionally wherein the binding element comprises a first antigen recognition domain and a second antigen recognition domain, and optionally wherein the binding element or the antigen recognition domain comprises an antibody, a protein scaffold, an antibody mimetic, or an antigen-binding sequence thereof.

8. 2. The CAR of claim 1, wherein the transmembrane element comprises a sequence isolated from or derived from the sequence of a CD28 protein; optionally, the intracellular element comprises one or more of a costimulatory element and a CD3-zeta signaling element; optionally, the binding element or antigen recognition domain specifically binds to a target, or any portion thereof, isolated from or derived from an extracellular antigen; and optionally, the target, or any portion thereof, isolated from or derived from an extracellular antigen, is present or expressed on the surface of a cell.

9. 2. The CAR of claim 1, wherein the target-expressing cells are in vivo, in vitro, or ex vivo, optionally the target-expressing cells comprise one or more modifications, and optionally the target-expressing cells are genetically modified.

10. (a) the binding member specifically binds to the target, and the target sequence is between about 3 nm and about 4 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell; the spacer sequence has a length of about 14 nm to about 19 nm; and the spacer sequence comprises the sequence of any one of SEQ ID NOs: 6, 36, 43, 31, or 7; (b) the binding member specifically binds to the target, and the target sequence is between about 5 nm and about 6 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell; the spacer sequence has a length of about 12 nm to about 17 nm; and the spacer sequence comprises the sequence of any one of SEQ ID NOs: 36, 43, 31, or 7; (c) the binding member specifically binds to the target, and the target sequence is between about 6 nm and about 7 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell; the spacer sequence has a length of about 11 nm to about 16 nm; and the spacer sequence comprises the sequence of any one of SEQ ID NOs: 36, 43, 31, 7, 14, 27, or 40; (d) the binding member specifically binds to the target, and the target sequence is between about 7 nm and about 8 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell; the spacer sequence has a length of about 10 nm to about 15 nm; and the spacer sequence comprises the sequence of any one of SEQ ID NOs: 43, 31, 7, 14, 27, 40, 32, or 42; (e) the binding member specifically binds to the target, and the target sequence is between about 8 nm and about 9 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell; the spacer sequence has a length of about 9 nm to about 14 nm; and the spacer sequence comprises the sequence of any one of SEQ ID NOs: 43, 31, 7, 14, 27, 40, 32, 42, or 8; (f) the binding member specifically binds to the target, and the target sequence is between about 9 nm and about 10 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell; the spacer sequence has a length of about 8 nm to about 13 nm; and the spacer sequence comprises the sequence of any one of SEQ ID NOs: 14, 27, 40, 32, 42, 8, or 23; (g) the binding member specifically binds to the target, and the target sequence is between about 10 nm and about 11 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell; the spacer sequence has a length of about 7 nm to about 12 nm; and the spacer sequence comprises the sequence of any one of SEQ ID NOs: 14, 27, 40, 32, 42, 8, 23, 9, 10, 24, 11, 3, or 18; (h) the binding member specifically binds to the target, and the target sequence is between about 11 nm and about 12 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell; the spacer sequence has a length of about 6 nm to about 11 nm; and the spacer sequence comprises the sequence of any one of SEQ ID NOs: 32, 42, 8, 23, 9, 10, 24, 11, 3, 18, 25, 38, 21, 29, 37, or 39; (i) the binding member specifically binds to the target, and the target sequence is between about 12 nm and about 13 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell; the spacer sequence has a length of about 5 nm to about 10 nm; and the spacer sequence comprises the sequence of any one of SEQ ID NOs: 32, 42, 8, 23, 9, 10, 24, 11, 3, 18, 25, 38, 21, 29, 37, 39, 2, 15, 16, 26, 28, 1, or 17; (j) the binding member specifically binds to the target, and the target sequence is between about 13 nm and about 14 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell; the spacer sequence has a length of about 4 nm to about 9 nm; and the spacer sequence comprises the sequence of any one of SEQ ID NOs: 23, 9, 10, 24, 11, 3, 18, 25, 38, 21, 29, 37, 39, 2, 15, 16, 26, 28, 1, 17, 12, 13, 19, 20, or 44; (k) the binding member specifically binds to the target, and the target sequence is between about 14 nm and about 15 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell; the spacer sequence has a length of about 3 nm to about 8 nm; and the spacer sequence comprises the sequence of any one of SEQ ID NOs: 9, 10, 24, 11, 3, 18, 25, 38, 21, 29, 37, 39, 2, 15, 16, 26, 28, 1, 17, 12, 13, 19, 20, or 44; (l) the binding member specifically binds to the target, and the target sequence is between about 15 nm and about 16 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell; the spacer sequence has a length of about 2 nm to about 7 nm; and the spacer sequence comprises the sequence of any one of SEQ ID NOs: 25, 38, 21, 29, 37, 39, 2, 15, 16, 26, 28, 1, 17, 12, 13, 19, 20, or 44; (m) the binding member specifically binds to the target, and the target sequence is between about 16 nm and about 17 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell; the spacer sequence has a length of about 1 nm to about 6 nm; and the spacer sequence comprises the sequence of any one of SEQ ID NOs: 2, 15, 16, 26, 28, 1, 17, 12, 13, 19, 20, or 44; The CAR according to claim 1, wherein, when the CAR is expressed on the surface of a cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 17 nm to 23 nm including endpoints.

11. (a) the binding member specifically binds to the target, and the target sequence is between about 3 nm and about 4 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell; the spacer sequence has a length of about 15 nm to about 18 nm; and the spacer sequence comprises the sequence of SEQ ID NO: 36; (b) the binding member specifically binds to the target, and the target sequence is between about 5 nm and about 6 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell; the spacer sequence has a length of about 13 nm to about 16 nm; and the spacer sequence comprises the sequence of any one of SEQ ID NOs: 36, 43, 31, or 7; (c) the binding member specifically binds to the target, and the target sequence is between about 6 nm and about 7 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell; the spacer sequence has a length of about 12 nm to about 15 nm; and the spacer sequence comprises the sequence of any one of SEQ ID NOs: 43, 31, or 7; (d) the binding member specifically binds to the target, and the target sequence is between about 7 nm and about 8 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell; the spacer sequence has a length of about 11 nm to about 14 nm; and the spacer sequence comprises the sequence of any one of SEQ ID NOs: 43, 31, 7, 14, 27, or 40; (e) the binding member specifically binds to the target, and the target sequence is between about 8 nm and about 9 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell; the spacer sequence has a length of about 10 nm to about 13 nm; and the spacer sequence comprises the sequence of any one of SEQ ID NOs: 14, 27, 40, 32, or 42; (f) the binding member specifically binds to the target, and the target sequence is between about 9 nm and about 10 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell; the spacer sequence has a length of about 9 nm to about 12 nm; and the spacer sequence comprises the sequence of any one of SEQ ID NOs: 14, 27, 40, 32, 42, 8, or 23; (g) the binding member specifically binds to the target, and the target sequence is between about 10 nm and about 11 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell; the spacer sequence has a length of about 8 nm to about 11 nm; and the spacer sequence comprises the sequence of any one of SEQ ID NOs: 32, 42, 8, or 23; (h) the binding member specifically binds to the target, and the target sequence is between about 11 nm and about 12 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell; the spacer sequence has a length of about 7 nm to about 10 nm; and the spacer sequence comprises the sequence of any one of SEQ ID NOs: 8, 23, 9, 10, 24, 11, 3, or 18; (i) the binding member specifically binds to the target, and the target sequence is between about 12 nm and about 13 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell; the spacer sequence has a length of about 6 nm to about 9 nm; and the spacer sequence comprises the sequence of any one of SEQ ID NOs: 23, 9, 10, 24, 11, 3, 18, 25, 38, 21, 29, 37, or 39; (j) the binding member specifically binds to the target, and the target sequence is between about 13 nm and about 14 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell; the spacer sequence has a length of about 5 nm to about 8 nm; and the spacer sequence comprises the sequence of any one of SEQ ID NOs: 9, 10, 24, 11, 3, 18, 25, 38, 21, 29, 37, 39, 2, 15, 16, 26, 28, or 17; (k) the binding member specifically binds to the target, and the target sequence is between about 14 nm and about 15 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell; the spacer sequence has a length of about 4 nm to about 7 nm; and the spacer sequence comprises the sequence of any one of SEQ ID NOs: 25, 38, 21, 29, 37, 39, 2, 15, 16, 26, 28, 17, 12, 13, 19, 20, or 44; (l) the binding member specifically binds to the target, and the target sequence is between about 15 nm and about 16 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell; the spacer sequence has a length of about 3 nm to about 6 nm; and the spacer sequence comprises the sequence of any one of SEQ ID NOs: 2, 15, 16, 26, 28, 17, 12, 13, 19, 20, or 44; (m) the binding member specifically binds to the target, and the target sequence is between about 16 nm and about 17 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell; the spacer sequence has a length of about 2 nm to about 5 nm; and the spacer sequence comprises the sequence of any one of SEQ ID NOs: 12, 13, 19, 20, or 44; The CAR according to claim 1, wherein, when the CAR is expressed on the surface of a cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 18 nm to 22 nm including endpoints.

12. (a) the binding member specifically binds to the target, and the target sequence is between about 3 nm and about 4 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell; the spacer sequence has a length of about 14 nm to about 21 nm; and the spacer sequence comprises the sequence of SEQ ID NO: 5, 6, 30, 33, 36, or 41; (b) the binding member specifically binds to the target, and the target sequence is between about 5 nm and about 6 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell; the spacer sequence has a length of about 12 nm to about 19 nm; and the spacer sequence comprises the sequence of any one of SEQ ID NOs: 6, 7, 31, 36, or 43; (c) the binding member specifically binds to the target, and the target sequence is between about 6 nm and about 7 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell; the spacer sequence has a length of about 11 nm to about 18 nm; and the spacer sequence comprises the sequence of any one of SEQ ID NOs: 7, 14, 27, 31, 36, 40, or 43; (d) the binding member specifically binds to the target, and the target sequence is between about 7 nm and about 8 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell; the spacer sequence has a length of about 10 nm to about 17 nm; and the spacer sequence comprises the sequence of any one of SEQ ID NOs: 7, 14, 27, 31, 32, 36, 40, 42, or 43; (e) the binding member specifically binds to the target, and the target sequence is between about 8 nm and about 9 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell; the spacer sequence has a length of about 9 nm to about 16 nm; and the spacer sequence comprises the sequence of any one of SEQ ID NOs: 7, 8, 14, 27, 31, 32, 36, 40, 42, or 43; (f) the binding member specifically binds to the target, and the target sequence is between about 9 nm and about 10 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell; the spacer sequence has a length of about 8 nm to about 15 nm; and the spacer sequence comprises the sequence of any one of SEQ ID NOs: 7, 8, 14, 23, 27, 31, 32, 40, 42, or 43; (g) the binding member specifically binds to the target, and the target sequence is between about 10 nm and about 11 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell; the spacer sequence has a length of about 7 nm to about 14 nm; and the spacer sequence comprises the sequence of any one of SEQ ID NOs: 3, 8, 9, 10, 11, 14, 18, 23, 24, 27, 32, 40, or 42; (h) the binding member specifically binds to the target, and the target sequence is between about 11 nm and about 12 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell; the spacer sequence has a length of about 6 nm to about 13 nm; and the spacer sequence comprises the sequence of any one of SEQ ID NOs: 3, 8, 9, 10, 11, 14, 18, 21, 23, 24, 25, 27, 29, 32, 37, 38, 39, 40, or 42; (i) the binding member specifically binds to the target, and when the target sequence is expressed on the surface of the target cell, the target sequence is between about 12 nm and about 13 nm from the surface of the target cell; the spacer sequence has a length of about 5 nm to about 12 nm; and the spacer sequence comprises the sequence of any one of SEQ ID NOs: 1, 2, 3, 6, 7, 8, 9, 10, 11, 14, 15, 16, 17, 18, 21, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 36, 37, 38, 39, 40, 41, 42, or 43; (j) the binding member specifically binds to the target, and when the target sequence is expressed on the surface of the target cell, the target sequence is between about 13 nm and about 14 nm from the surface of the target cell; the spacer sequence has a length of about 4 nm to about 11 nm; and the spacer sequence comprises the sequence of any one of SEQ ID NOs: 1, 2, 3, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, 23, 24, 25, 26, 28, 29, 32, 37, 38, 39, 42, or 44; (k) the binding member specifically binds to the target, and when the target sequence is expressed on the surface of the target cell, the target sequence is between about 14 nm and about 15 nm from the surface of the target cell; the spacer sequence has a length of about 3 nm to about 10 nm; and the spacer sequence comprises the sequence of any one of SEQ ID NOs: 1, 2, 3, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, 23, 24, 25, 26, 28, 29, 37, 38, 39, or 44; (l) the binding member specifically binds to the target, and when the target sequence is expressed on the surface of the target cell, the target sequence is between about 15 nm and about 16 nm from the surface of the target cell; the spacer sequence has a length of about 2 nm to about 9 nm; and the spacer sequence comprises the sequence of any one of SEQ ID NOs: 1, 2, 3, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, 23, 24, 25, 26, 28, 29, 37, 38, 39, or 44; (m) the binding member specifically binds to the target, and the target sequence is between about 16 nm and about 17 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell; the spacer sequence has a length of about 1 nm to about 8 nm; and the spacer sequence comprises the sequence of any one of SEQ ID NOs: 1, 2, 3, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 28, 29, 37, 38, 39, or 44; (n) the binding member specifically binds to the target, and the target sequence is between about 18 nm and about 19 nm from the surface of the target cell when the target sequence is expressed on the surface of the target cell; the spacer sequence has a length of about 1 nm to about 6 nm; and the spacer sequence comprises the sequence of any one of SEQ ID NOs: 1, 2, 12, 13, 15, 16, 17, 19, 20, 26, 28, 29, 37, 38, 39, or 44; (o) where the binding member specifically binds to the target and the target sequence is expressed on the surface of the target cell wherein the target sequence is between about 19 nm and about 20 nm from the surface of the target cell; the spacer sequence has a length of about 1 nm to about 5 nm; and the spacer sequence comprises any one of SEQ ID NOs: 12, 13, 19, 20, or 44; The CAR according to claim 1, wherein, when the CAR is expressed on the surface of a cell, the intercellular distance between the CAR-expressing cell and the target-expressing cell is about 18 nm to 22 nm including endpoints.

13. 13. The CAR of any one of claims 10 to 12, wherein the spacer sequence comprises one or more of a deletion, insertion, substitution, inversion, truncation or alteration; optionally, the spacer sequence comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% identity to one or more of SEQ ID NOs: 1-44; optionally, the spacer sequence comprises at least one non-naturally occurring residue; and optionally, the substitution comprises replacement of a first amino acid with a second amino acid, wherein the first amino acid and the second amino acid have one or more of polarity, side chain length, or hydrophobicity.

14. A nucleic acid sequence encoding the CAR of claim 1 or any element thereof.

15. A vector comprising the nucleic acid of claim 14.

16. A cell comprising the CAR described in claim 1, the nucleic acid sequence described in claim 14, or the vector described in claim 15.

17. A composition comprising the CAR described in claim 1, the nucleic acid described in claim 14, the vector described in claim 15, the cell described in claim 16 or any of their elements.

18. A pharmaceutical composition comprising the CAR described in claim 1, the nucleic acid described in claim 14, or the vector described in claim 15, and a pharmaceutically acceptable carrier.

19. A pharmaceutical composition according to claim 18 for use in the treatment or prevention of a disease or disorder, optionally wherein the disease or disorder comprises cancer.

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