Using rituximab to prevent car-t or team immunity

Abstract

This disclosure includes methods for treating cancer in a subject, the method comprising administering an anti-CD20 antibody and an immune cell expressing a therapeutic polypeptide (e.g., a TEAM) to the subject.

Description

[0001] USING RITUXIMAB TO PREVENT CAR-T OR TEAM IMMUNITY

[0002] RELATED APPLICATIONS

[0003] This application claims benefit under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 63 / 734,369, filed December 16, 2024, entitled “Using Rituximab to Prevent CAR-T or TEAM Immunity” and U.S. Provisional Application No. 63 / 768,005, filed March 6, 2025, entitled “Using Rituximab to Prevent CAR-T or TEAM Immunity”, the entire contents of each of which are incorporated herein by reference.

[0004] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0005] The contents of the electronic sequence listing (M105370057WO00-SEQ-ARM.xml; Size: 93,848 bytes; and Date of Creation: December 4, 2025) are herein incorporated by reference in their entirety.

[0006] FEDERALLY SPONSORED RESEARCH

[0007] This invention was made with government support under 1R01CA294071-01A1 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0008] BACKGROUND

[0009] Cell therapies are used to treat many diseases, including some cancers. Cell therapies typically work by modifying an immune cell to express a receptor that binds to target cancer cells and / or secrete a polypeptide (e.g., a cancer killing polypeptide or a T-cell engaging antibody molecule).

[0010] SUMMARY

[0011] A central challenge in cell therapy (e.g., CAR-T cell therapy) is reducing subject immune response against the cell therapy. A host immune response can decrease the efficacy of the cell therapy over time, and can in result in adverse side effects in the subject, like cytokine storm. Some cell therapies work by engineering the cells of the cell therapy (e.g., T cells) to secrete a therapeutic polypeptide (e.g., a T-cell-engaging antibody molecule (TEAM)). TEAMs (also known as bispecific T cell engagers) bind to T cells and cancer cells, which can mediate killing of the cancer cell by T cells. In some aspects, T cells can be engineered to secrete a therapeutic polypeptide (e.g., a TEAM) and express a chimeric antigen receptor (CAR). The CAR

[0012] #14707301 v1 comprises an extracellular antigen binding domain, which can provide an orthogonal way for the T cell to bind to the cancer.

[0013] In some cases, the subject’s adaptive immune system generates antibodies against the therapeutic polypeptide (e.g., the TEAM), which disrupts the therapeutic efficacy of the TEAM and the cell therapy. This disclosure provide evidence that such an adaptive immune response is occurring in human patients treated with CAR3-TEAM-E (a CAR-T cell comprising an EGFRvIII binding CAR and secreting an EGFR-CD3 binding TEAM), which is used to treat brain cancer.

[0014] Disclosed herein are methods of decreasing the host immune response against cell therapies that secrete therapeutic polypeptides by administering to the subject an anti-CD20 antibody (e.g., rituximab). Without being bound to theory, the anti-CD20 antibody is expected to kill the subject’s endogenous B cells, which in turn decreases the subject’s ability to produce antibodies against the TEAM. Additionally disclosed herein are methods of treating a subject having a cancer by administering an immune cell that secretes a therapeutic polypeptide (e.g., a TEAM), and an anti-CD20 antibody.

[0015] In some aspects, this disclosure provides a method of treating cancer in a subject, the method comprising administering to the subject: (a) an anti-CD20 antibody; and (b) an immune cell comprising a first polynucleotide encoding a therapeutic polypeptide, wherein the immune cell secretes the therapeutic polypeptide.

[0016] In some embodiments, the anti-CD20 antibody is rituximab. In some embodiments, administering the rituximab comprises administering 300-500 mg / m2of rituximab. In some embodiments, administering the rituximab comprises administering 375 mg / m2of rituximab. In some embodiments, the method comprises administering the anti-CD20 antibody to the subject prior to administering the immune cell. In some embodiments, the method comprises administering the anti-CD20 antibody to the subject after administering the immune cell. In some embodiments, the immune cell does not express CD20 or a fragment of CD20. In some embodiments, the method further comprises lymphodepleting the subject. In some embodiments, the method comprises, in order: administering an anti-CD20 antibody to the subject; lymphodepleting the subject; and administering the immune cell to the subject.

[0017] In some embodiments, the anti-CD20 antibody is administered to the subject 3-15 days before lymphodepleting the subject. In some embodiments, the anti-CD20 antibody is administered to the subject 5-10 days before lymphodepleting the subject. In some embodiments, lymphodepleting the subject occurs at least 5 days before administering the immune cell to the subject.

[0018] #14707301 v1 In some embodiments, lymphodepleting the subject occurs 5, 4 and 3 days before administering the immune cell to the subject. In some embodiments, the immune cell is a T cell. In some embodiments, the therapeutic polypeptide comprises an antibody that binds to a cancer antigen that is expressed by cancer cells of the subject. In some embodiments, the therapeutic polypeptide comprises a signal peptide. In some embodiments, the therapeutic polypeptide is a bispecific antibody. In some embodiments, the bispecific antibody is a T-cell-engaging antibody molecule (TEAM). In some embodiments, the bispecific antibody comprises a first binding domain that binds to CD3.

[0019] In some embodiments, the first binding domain that binds to CD3 comprises: (a) a variable heavy (VH) domain comprising three complementarity determining regions (CDR-H1, CDR-H2, and CDR-H3), wherein CDR-H1 comprises SEQ ID NO: 16, CDR-H2 comprises SEQ ID NO: 17, and CDR-H3 comprises SEQ ID NO: 18; and (b) a variable light (VL) domain comprising three CDRs (CDR-L1, CDR-L2, and CDR-L3), wherein CDR-L1 comprises SEQ ID NO: 19, CDR-L2 comprises SEQ ID NO: 20, and CDR-L3 comprises SEQ ID NO: 21.

[0020] In some embodiments, the bispecific antibody comprises a second binding domain that binds to a cancer antigen. In some embodiments, the cancer antigen is CD70, CD33, FAP, mesothelin, EGFR, or EGFRvIII. In some embodiments, the second binding domain binds to a cancer antigen that is expressed by cancer cells of the subject. In some embodiments, the cancer antigen is EGFR.

[0021] In some embodiments, the second binding domain comprises: (a) a VH domain comprising three complementarity determining regions (CDR-H1, CDR-H2, and CDR-H3), wherein CDR-H1 comprises SEQ ID NO: 9, CDR-H2 comprises SEQ ID NO: 10, and CDR-H3 comprises SEQ ID NO: 11, and (b) a VL domain comprising three CDRs (CDR-L1, CDR-L2, and CDR-L3), wherein CDR-L1 comprises SEQ ID NO: 12, CDR-L2 comprises SEQ ID NO: 13, and CDR-L3 comprises SEQ ID NO: 14.

[0022] In some embodiments, the immune cell comprises a second polynucleotide encoding a chimeric antigen receptor (CAR). In some embodiments, the CAR comprises an extracellular antigen binding domain. In some embodiments, the extracellular antigen binding domain binds to a cancer antigen. In some embodiments, the cancer antigen is CD70, CD33, FAP, mesothelin, EGFR, or EGFRvIII. In some embodiments, the extracellular antigen binding domain binds to a cancer antigen that is expressed by cancer cells of the subject. In some embodiments, the extracellular antigen binding domain binds to EGFRvIII.

[0023] #14707301 v1 In some embodiments, the first polynucleotide encodes a TEAM comprising and CD3 binding domain and an EGFR binding domain; and second polynucleotide encodes a CAR comprising an extracellular antigen binding domain that binds to EGFRvIII.

[0024] In some embodiments, the extracellular antigen binding domain comprises a monoclonal antibody, a fragment antigen binding (Fab) fragment, F(ab')2 fragment, Fv fragment, and / or a single-chain variable fragments (scFv).

[0025] In some embodiments, the extracellular antigen binding domain comprises: (a) a VH domain comprising three complementarity determining regions (CDR-H1, CDR-H2, and CDR- H3), wherein CDR-H1 comprises SEQ ID NO: 1, CDR-H2 comprises SEQ ID NO: 2, and CDR- H3 comprises SEQ ID NO: 3, and (b) a VL domain comprising three CDRs (CDR-L1, CDR-L2, and CDR-L3), wherein CDR-L1 comprises SEQ ID NO: 4, CDR-L2 comprises SEQ ID NO: 5, and CDR-L3 comprises SEQ ID NO: 6.

[0026] In some embodiments, the first polynucleotide encodes a TEAM comprising and CD3 binding domain and a CD33 binding domain; and the CAR comprises an extracellular antigen binding domain that binds to CD70.

[0027] In some embodiments, the first polynucleotide encodes a TEAM comprising and CD3 binding domain and a FAP binding domain; and the CAR comprises an extracellular antigen binding domain that binds to mesothelin.

[0028] In some embodiments, the CAR comprises: a transmembrane domain; and an intracellular signaling domain.

[0029] In some embodiments, the transmembrane domain is a transmembrane domain of an alpha chain of an immune cell receptor, beta chain of an immune cell receptor, zeta chain of an immune cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, 0X40, CD2, CD27, LFA-1 (CDlla, CD18), ICOS (CD278), 4-1BB (CD137), 4-1BBL, GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFI), CD160, CD19, IL2R beta, IL2R gamma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDlld, ITGAE, CD103, ITGAL, CDlla, LFA-1, ITGAM, CDllb, ITGAX, CDllc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C.

[0030] #14707301 v1 In some embodiments, the transmembrane domain is a CD8 transmembrane domain. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain from 4- IBB, CD27, CD28, 0X4, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (0X40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM, and / or ZAP70. In some embodiments, the intracellular signaling domain comprises a CD28 intracellular signaling domain. In some embodiments, the intracellular signaling domain further comprises a TCR-zeta, FcR-gamma, FcR-beta, CD3- gamma, CD3-theta, CD3-sigma, CD3-eta, CD3-epsilon, CD3-zeta, CD22, CD79a, CD79b, and / or CD66d.

[0031] In some embodiments, the intracellular signaling domain comprises a CD28 intracellular signaling domain and a CD3-zeta intracellular signaling domain. In some embodiments, the extracellular antigen binding domain further comprises a leader sequence. In some embodiments, the CAR comprises an amino acid sequence having at least 85% identity to SEQ ID NO: 8. In some embodiments, the CAR comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 8. In some embodiments, the CAR comprises an amino acid sequence of SEQ ID NO: 8. In some embodiments, the first polynucleotide and the second polynucleotide are part of the same polynucleotide.

[0032] In some embodiments, the CAR and the TEAM are encoded as a polyprotein. In some embodiments, the polyprotein comprises a cleavable peptide between the CAR and the TEAM. In some embodiments, the polyprotein comprises an amino acid sequence having at least 85% identity to SEQ ID NO: 37. In some embodiments, the polyprotein comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 37. In some embodiments, the polyprotein comprises an amino acid sequence of SEQ ID NO: 37.

[0033] In some embodiments, the cancer is glioblastoma. In some embodiments, this disclosure provides a method of treating glioblastoma in a subject, the method comprising administering to the subject, in order: (a) rituximab; and (b) a T cell comprising: a first polynucleotide encoding a TEAM comprising an EGFR binding domain and a CD3 binding domain; and a second polynucleotide encoding a CAR comprising an extracellular antigen binding domain that binds to EGFRvIII.

[0034] In some embodiments, the method further comprises lymphodepleting the subject. In some embodiments, lymphodepleting the subject comprises lymphodepleting prior to T cell administration. In some embodiments, the first polynucleotide and the second polynucleotide are part of the same polynucleotide. In some embodiments, the TEAM comprises an amino acid

[0035] #14707301 v1 sequence of SEQ ID NO: 23. In some embodiments, the CAR comprises an amino acid sequence of SEQ ID NO: 8. In some embodiments, the CAR and the TEAM are encoded as a polyprotein. In some embodiments, the polyprotein comprises an amino acid sequence of SEQ ID NO: 37.

[0036] In some embodiments, administering the rituximab comprises administering 300-500 mg / m2rituximab to the subject. In some embodiments, administering the rituximab comprises administering 375 mg / m2rituximab to the subject. In some embodiments, administering the immune cell comprises administering into the subject’s central nervous system.

[0037] In some embodiments, the glioblastoma is EGFR positive. In some embodiments, the glioblastoma is EGFRvIII positive. In some embodiments, the glioblastoma is not EGFRvIII positive.

[0038] In some embodiments, this disclosure provides a method of decreasing antibody production against an immune cell therapy in a subject, the method comprising: (a) administering an anti-CD20 antibody to the subject; and (b) administering the immune cell therapy to the subject, wherein immune cells of the immune cell therapy comprise a first polynucleotide encoding a therapeutic polypeptide, wherein the immune cell secretes the therapeutic polypeptide.

[0039] In some embodiments, this disclosure provides a method of decreasing antibody production against an immune cell therapy in a subject having a solid tumor, the method comprising: (a) administering an anti-CD20 antibody to the subject; and (b) administering an immune cell therapy to the subject, wherein immune cells of the immune cell therapy comprise: a first polynucleotide encoding a therapeutic polypeptide; and / or a second polynucleotide encoding a chimeric antigen receptor (CAR) that comprises an extracellular binding domain that binds to an antigen of the solid tumor, wherein decreasing antibody production comprises decreasing antibody production against the therapeutic polypeptide and / or the CAR.

[0040] In some embodiments, this disclosure provides a method of treating a subject having a solid tumor, the method comprising: (a) administering an anti-CD20 antibody to the subject; and (b) administering an immune cell therapy to the subject, wherein immune cells of the immune cell therapy comprise: a first polynucleotide encoding a therapeutic polypeptide; and / or a second polynucleotide encoding a chimeric antigen receptor (CAR) that comprises an extracellular binding domain that binds to an antigen of the solid tumor, wherein treating the subject includes decreasing antibody production against the therapeutic polypeptide and / or the CAR.

[0041] In some embodiments, administering the anti-CD20 antibody comprises administering rituximab. In some embodiments, the method further comprises lymphodepleting the subject.

[0042] #14707301 v1 In some embodiments, lymphodepleting the subject and administering the anti-CD20 antibody are performed before administering the immune cell therapy.

[0043] In some embodiments, the antibody production against the therapeutic polypeptide and / or the CAR is reduced by at least 50%, 60%, 70% , 80%, 90%, or 100% compared to an otherwise identical method not comprising administering the anti-CD20 antibody to the subject. In some embodiments, the antibody production against the therapeutic polypeptide and / or the CAR is reduced for at least 1 day, 5 days, 10 days, 20 days, 30 days, 50 days, 60 days, 70 days, 80 days or more following administration of the anti-CD20 antibody to the subject,

[0044] BRIEF DESCRIPTION OF DRAWINGS

[0045] FIG. 1 shows measurements of serum and cerebral spinal fluid (CSF) production of a antibodies against a TEAM in a human subject who received CAR-T cell therapy. The CAR-T cells were engineered to express an EGFRvIII binding CAR and secrete an EGFR-CD3 binding TEAM (CARv3-TEAM-3). Lymphodepleting (LD) chemo was not administered to these subjects.

[0046] FIG. 2 shows measurements of serum and cerebral spinal fluid (CSF) production of a antibodies against a TEAM in a human subject who received CAR-T cell therapy. The CAR-T cells were engineered to express an EGFRvIII binding CAR and secrete an EGFR-CD3 binding TEAM (CARv3-TEAM-3). LD chemo was administered to these subjects.

[0047] FIG. 3 shows an exemplary treatment timeline for a subject having glioblastoma.

[0048] FIGs. 4A-4C relate to the number of CAR+ and CAR+ / TEAM+ cells in cerebral spinal fluid (CSF) of rituximab-treated patients after multiple infusions. FIG. 4A, FIG. 4B, and FIG. 4C each represent a different patient.

[0049] FIGs. 5A-5C relate to the number of CAR+ and CAR+ / TEAM+ cells in the peripheral blood (PB) of rituximab-treated patients after multiple infusions. FIG. 5A, FIG. 5B, and FIG. 5C each represent a different patient.

[0050] FIGs. 6A-6C relate to anti-TEAM and anti-CAR IgG antibodies after multiple infusions in patients pre-treated with rituximab. FIG. 6A, FIG. 6B, and FIG. 6C each represent a different patient.

[0051] DETAILED DESCRIPTION

[0052] In some aspects, this disclosure provides a method of treating cancer in a subject, the method comprising administering to the subject: (a) an anti-CD20 antibody; and (b) an immune cell comprising a first polynucleotide encoding a therapeutic polypeptide.

[0053] #14707301 v1 A “antibody” as described herein includes full size antibodies (e.g., a monoclonal antibody) and antigen binding fragments thereof (e.g., a scFv). In some embodiments, the antibody is monoclonal antibody. In some embodiments, the antibody is a human antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is an IgA, IgD, IgG, IgE, and IgM antibody. In some embodiments, the antibody is an antigen binding fragment thereof (of an antibody). In some embodiments, the antibody comprises a variable heavy (VH) domain that comprises three complementary determining regions. In some embodiments, the antibody comprises a variable light (VL) domain that comprises three complementary determining regions. In some embodiments, the antibody comprises a VH domain and a VL domain. In some embodiments, an antigen binding fragment comprises a Fab fragment, F(ab')2 fragment, Fv fragments, single chain variable fragments (scFv), or single-domain antibody. In some embodiments, the antibody is a VHH (also called a NANOBODY). In some embodiments, the antibody is camelid antibody.

[0054] Anti-CD20 Antibody

[0055] An “anti-CD20 antibody” refers to an antibody that binds to CD20 or a fragment thereof. The anti-CD20 antibody may be a full size antibody or an antigen binding fragment thereof. In some embodiments, the anti-CD20 antibody is rituximab. In some embodiments, the anti-CD20 antibody is Obinutuzumab. In some embodiments, the anti-CD20 antibody is Ofatumumab.

[0056] In some embodiments, the method comprises administering an anti-CD20 antibody to a subject having cancer. The anti-CD20 antibody may be administered in any suitable way (e.g., patient transarterially, intratumorally, intranodally, intraperitoneally, intrathecally, intramedullary, intravenously, intraventricularly, subcutaneously or orally). In some embodiments, the anti-CD20 antibody is administered to the subject intravenously.

[0057] In some embodiments, the method comprises administering the anti-CD20 antibody prior to administering the immune cells. In some embodiments, the method comprises administering the anti-CD20 antibody (e.g., rituximab) to the subject 2-20, 3-15, 3-12, 4-11, or 5-10 days prior to administering the immune cells. In some embodiments, the method comprises administering the anti-CD20 antibody to the subject 5-10 days prior to administering the immune cells. In some embodiments, the method comprises administering the anti-CD20 antibody to the subject 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and / or 20 days prior to administering the immune cells. In some embodiments, administering the anti-CD20 antibody comprises administering a single dose of the anti-CD20 antibody (e.g., rituximab). In some embodiments,

[0058] #14707301 v1 administering the anti-CD20 antibody (e.g., rituximab) comprises administering at least 1 dose (e.g., at least 2 doses, at least 3 doses, at least 4 doses, or at least 5 doses) of the anti-CD20 antibody to the subject.

[0059] In some embodiments, administering the anti-CD20 antibody to the subject comprises administering rituximab to the subject. In some embodiments, administering rituximab comprises administering 300-1500 mg / m2, 300-1000 mg / m2, 300-500 mg / m2, 350-450 mg / m2, 350-400 mg / m2, or 370-380 mg / m2of rituximab. In some embodiments, administering rituximab comprises administering 375 mg / m2of rituximab. In some embodiments, administering rituximab comprises administering rituximab weekly (e.g., administering about 375 mg / m2of rituximab weekly). In some embodiments, administering rituximab comprises administering rituximab (e.g., about 375 mg / m2of rituximab) weekly for up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 weeks. In some embodiments, administering rituximab comprises administering about 375 mg / m2of rituximab weekly for 4 weeks. In some embodiments, administering rituximab comprises administering rituximab (e.g., about 1000 mg / m2of rituximab) every two weeks. In some embodiments, administering rituximab (e.g., about 1000 mg / m2of rituximab) comprises administering rituximab every two weeks for up to 1, 2, 3, 4, 5 or 6 months. In some embodiments, administering rituximab (e.g., about 1000 mg / m2of rituximab) comprises administering rituximab every two weeks 1 month. In some embodiments, administering rituximab comprises administering 375 mg / m2of rituximab intravenously to the subject. In some embodiments, administering rituximab comprises administering 375 mg / m2of rituximab intravenously to the subject prior to administering the immune cells. In some embodiments, administering rituximab comprises administering 375 mg / m2of rituximab intravenously to the subject 5-10 days prior to administering the immune cells.

[0060] In some embodiments, the method comprises administering an anti-CD20 antibody to the subject after administering the immune cells (e.g., administering rituximab (e.g., 1000 mg / m2of rituximab) 4-6 months after administering the immune cells). In some embodiments, the method comprises administering an anti-CD20 antibody to the subject after administering the immune cells (e.g., administering rituximab (e.g., 1000 mg / m2of rituximab) every 4-6 months after administering the immune cells). In some embodiments, an anti-CD20 antibody may be administered to the subject if anti-TEAM antibodies are found in the subject (e.g., in the subject’s blood or CSF) and / or if subsequent administration of the immune cells is therapeutically ineffective.

[0061] #14707301 v1 In some embodiments, the method comprises administering an anti-CD20 antibody to the subject before administering the immune cells and after administering the immune cells to the subject.

[0062] Immune Cells

[0063] In some embodiments, a method of treating cancer in a subject comprises administering an immune cell comprising a first polynucleotide encoding a therapeutic polypeptide. In some embodiments, a method of treating cancer in a subject comprises administering to the subject: (a) an anti-CD20 antibody; and (b) an immune cell comprising a first polynucleotide encoding a therapeutic polypeptide.

[0064] An “immune cell” as used herein refers to cell that is part of the immune system (e.g., of a subject) and helps the body fight infections and other diseases. In some embodiments, the immune cell is a lymphocyte (e.g., a T cell). In some embodiments, the immune cell is a natural killer (NK) cell. In some embodiments, the immune cell does not express CD20 or a fragment thereof. CD20 immune cell expression may be detected in any suitable way (e.g., using an anti- CD20 antibody).

[0065] In some embodiments, generating an immune cell (e.g., a plurality of T cells) comprises obtaining an immune cell from the subject and modifying the immune cell to comprise the first polynucleotide and / or the second polynucleotide described herein.

[0066] In some embodiments, the immune cell comprises a first polynucleotide encoding a therapeutic polypeptide. The first polynucleotide may be any suitable polynucleotide. In some embodiments, the first polynucleotide is a plasmid or vector (e.g., as described herein). In some embodiments, the first polynucleotide is incorporated into the genome of the immune cell. In some embodiments, the first polynucleotide is operably linked to a promoter.

[0067] A “therapeutic polypeptide” refers to a peptide that can be used for treating a cancer. In some embodiments, the therapeutic polypeptide is an antibody. In some embodiments, the antibody is a cancer antigen binding antibody. In some embodiments, the antibody is a bispecific antibody (e.g., with two cancer antigen binding domains, or with an immune cell binding domain and a cancer antigen binding domain). In some embodiments, the therapeutic polypeptide is a TEAM (e.g., as described herein). In some embodiments, the TEAM comprises a CD3 binding domain. In some embodiments, the TEAM comprises a CD3 binding domain and a cancer antigen binding domain. In some embodiments, the TEAM comprises a CD3 binding domain and a CD33 binding domain. In some embodiments, the TEAM comprises a CD3 binding domain and a FAP binding domain. In some embodiments, the therapeutic

[0068] #14707301 v1 polypeptide comprises a signal tag. Signal tags can cause the therapeutic polypeptide to be secreted by a cell (e.g., an immune cell). In some embodiments, the signal peptide is an IgK signal peptide or a CD8 signal peptide. In some embodiments, the signal peptide is an IgK signal peptide. “Secrete” and “secretion” as used herein includes the release of a product (e.g., a therapeutic protein) from a cell (e.g., an immune cell). For example, secretion may involve transport of the product across the cell membrane of a cell to the outside of the cell.

[0069] In some embodiments, the immune cell comprises a second polynucleotide encoding chimeric antigen receptor. In some embodiments, the first polynucleotide and the second polynucleotide are part of the same polynucleotide. In some embodiments, the first polynucleotide and the second polynucleotide collectively encode a polyprotein comprising a therapeutic polypeptide and a CAR. In some embodiments, the first polynucleotide and the second polynucleotide collectively encode a polyprotein comprising a therapeutic polypeptide, a cleavage polypeptide (e.g., a 2A self-cleaving peptide) and a CAR. In some embodiments, the first polynucleotide and the second polynucleotide collectively encode a polyprotein comprising, from N-terminal to C-Terminal, a therapeutic polypeptide, a cleavage polypeptide (e.g., a 2A self-cleaving peptide) and a CAR. In some embodiments, the first polynucleotide and the second polynucleotide collectively encode a polyprotein comprising, from N-terminal to C- Terminal, a CAR, a cleavage polypeptide (e.g., a 2 A self-cleaving peptide) and a therapeutic polypeptide. In some embodiments, the first polynucleotide and the second polynucleotide are encoded on the same vector (e.g., an expression vector).

[0070] Chimeric Antigen Receptor (CAR) T cells

[0071] In some aspects, methods provided by this disclosure comprise administering an immune cell (e.g., a T cell) comprising a first nucleic acid encoding a therapeutic polypeptide to a subject. In some embodiments, the immune cell comprises a second polynucleotide encoding a CAR. The second polynucleotide may be any suitable polynucleotide. In some embodiments, the second polynucleotide is a plasmid or vector described herein. In some embodiments, the first polynucleotide and the second polynucleotide are the same polynucleotide (e.g., included in the same plasmid or vector). In some embodiments, the first polynucleotide and the second polynucleotide are operably linked to different promoters. In some embodiments, the first polynucleotide and the second polynucleotide are operably linked to the same promoter (e.g., the first polynucleotide and the second polynucleotide collective encode a fusion protein of the therapeutic polypeptide and CAR, or an IRES is located between the first polynucleotide and the second polynucleotide).

[0072] #14707301 v1 In some embodiments, the T cell is a CAR T cell. As used herein, a “CAR-T cell” or “CAR-T” refers to a T cell that expresses a CAR. In some embodiments, when expressed in a T cell, CARs have the ability to redirect T cell specificity and reactivity toward a selected target in a non-MHC-restricted manner, exploiting the antigen binding properties of monoclonal antibodies. In some embodiments, the non-MHC-restricted antigen recognition gives T cells expressing CARs the ability to recognize an antigen independent of antigen processing, thus bypassing a major mechanism of tumor escape.

[0073] The terms “chimeric antigen receptor” or “CAR” or “CARs”, as used herein, refer to engineered T cell receptors, which graft a ligand or antigen specificity onto T cells (for example, naive T cells, central memory T cells, effector memory T cells or combinations thereof). CARs are also known as artificial T cell receptors, chimeric T cell receptors or chimeric immunoreceptors.

[0074] In some embodiments, a CAR places an extracellular antigen binding domain that specifically binds a target, e.g., a polypeptide, expressed on the surface of a cell to be targeted for a T cell response onto a construct including a transmembrane domain and intracellular domain(s) of a T cell receptor molecule. In some embodiments, the extracellular antigen binding domain includes the antigen domain(s) of an antibody reagent that specifically binds an antigen expressed on a cell to be targeted for a T cell response. In some embodiments, the extracellular antigen binding domain includes a ligand that specifically binds an antigen expressed on a cell to be targeted for a T cell response.

[0075] In some embodiments, the CAR includes a CD8 signal peptide as described herein. As can be determined by those of skill in the art, various functionally similar or equivalent components of these CARs can be swapped or substituted with one another, as well as other similar or functionally equivalent components known in the art or listed herein.

[0076] Extracellular Antigen Binding Domain

[0077] In some embodiments, a CAR comprises an extracellular domain. In some embodiments, the extracellular domain is an extracellular antigen binding domain. As used herein, the term “extracellular antigen binding domain” used in the context of a CAR refers to a polypeptide found on the outside of the cell that is sufficient to facilitate binding of a CAR to a target (e.g., EGFRvIII). In some embodiments, the extracellular antigen binding domain comprises an antibody. The extracellular antigen binding domain may specifically bind to its binding partner, i.e., the target. As non-limiting examples, the extracellular antigen binding domain can include an antigen domain of an antibody or an antigen binding fragment thereof, or

[0078] #14707301 v1 a ligand, which recognizes and binds with a cognate binding partner protein. In this context, a ligand is a molecule that binds specifically to a portion of a protein and / or receptor. The cognate binding partner of a ligand useful in the methods and compositions described herein can generally be found on the surface of a cell. Ligand:cognate partner binding can result in the alteration of the ligand-bearing receptor, or activate a physiological response, for example, the activation of a signaling pathway. In some embodiments, the ligand can be non-native to the genome. In some embodiments, the ligand has a conserved function across at least two species.

[0079] Any cell-surface moiety can be targeted by a CAR. In some embodiments, the target will be a cell-surface polypeptide that may be differentially or preferentially expressed on a cell that one wishes to target for a T cell response. In some embodiments, the CAR comprises an extracellular antigen binding domain that binds to an antigen expressed on cancer cell and / or tumor cell. In some embodiments, the CAR comprises an extracellular antigen binding domain that binds to an extracellular antigen expressed on a central nervous system cancer or tumor. In some embodiments, the CAR comprises an extracellular antigen binding domain that binds to EGFRvIII. In some embodiments, the CAR comprises an extracellular antigen binding domain that binds to CD70. In some embodiments, the CAR comprises an extracellular antigen binding domain that binds to mesothelin.

[0080] In some embodiments, the CAR comprises an extracellular antigen binding domain that binds to EGFRvIII. “EGFRvIII” is a variant of EGFR that is known to be associated with glioblastoma (GBM). About 30% of GBM cells are positive for EGFRvIII. EGFRvIII may comprise a mutation that leads to deletion of exon 2-7 of EGFR, as described in Gan HK FEBS J. 2013 Nov;280(21):5350-70. In some embodiments, an EGFRvIII extracellular antigen binding domain comprises (i) a VH domain comprising three complementarity determining regions (CDR-H1, CDR-H2, and CDR-H3), wherein CDR-H1 comprises SEQ ID NO: 1, CDR- H2 comprises SEQ ID NO: 2, and CDR-H3 comprises SEQ ID NO: 3, and (ii) a VL domain comprising three CDRs (CDR-L1, CDR-L2, and CDR-L3), wherein CDR-L1 comprises SEQ ID NO: 4, CDR-L2 comprises SEQ ID NO: 5, and CDR-L3 comprises SEQ ID NO: 6. In some embodiments, the extracellular antigen binding domains comprises an amino acid sequence of SEQ ID NO: 7.

[0081] In some embodiments, the method comprises administering to the subject a T cell comprising a mesothelin binding CAR. In some embodiments, a mesothelin extracellular antigen binding domain comprises (i) a VH domain comprising three complementarity determining regions (CDR-H1, CDR-H2, and CDR-H3), wherein CDR-H1 comprises SEQ ID NO: 44, CDR-H2 comprises SEQ ID NO: 45, and CDR-H3 comprises SEQ ID NO: 46, and (ii)

[0082] #14707301 v1 a VL domain comprising three CDRs (CDR-L1, CDR-L2, and CDR-L3), wherein CDR-L1 comprises SEQ ID NO: 47, CDR-L2 comprises SEQ ID NO: 48, and CDR-L3 comprises SEQ ID NO: 49. In some embodiments, the mesothelin extracellular antigen binding domain comprises a VH of SEQ ID NO: 42 and a VL of SEQ ID NO: 43.

[0083] In some embodiments, a mesothelin extracellular antigen binding domain comprises (i) a VH domain comprising three complementarity determining regions (CDR-H1, CDR-H2, and CDR-H3), wherein CDR-H1 comprises SEQ ID NO: 59, CDR-H2 comprises SEQ ID NO: 60, and CDR-H3 comprises SEQ ID NO: 61, and (ii) a VL domain comprising three CDRs (CDR- Ll, CDR-L2, and CDR-L3), wherein CDR-L1 comprises SEQ ID NO: 47, CDR-L2 comprises SEQ ID NO: 62, and CDR-L3 comprises SEQ ID NO: 63. In some embodiments, the extracellular antigen binding domains comprises a VH of SEQ ID NO: 65 and a VL of SEQ ID NO: 66.

[0084] In some embodiments, the method comprises administering to the subject a T cell comprising a CD70 binding CAR. In some embodiments, a CD70 extracellular antigen binding domain comprises a CD27 fragment that binds to CD70 (e.g., SEQ ID NO: 75), or a sequence having at least 85% (e.g., at least 90%, at least 95%, at least 98%, or at least 99%) identity to SEQ ID NO: 75 and binds to CD70.

[0085] Hinge and Transmembrane Domains

[0086] In some embodiments, the CAR polypeptide further comprises a transmembrane domain, e.g., a hinge / transmembrane domain, which joins the extracellular domain (e.g., the extracellular antigen binding domain) to the intracellular signaling domain. The binding domain of the CAR is, in some embodiments, followed by one or more “hinge domains,” which plays a role in positioning the extracellular domain away from the effector cell surface to enable proper cell / cell contact, antigen binding and activation. A CAR may include one or more hinge domains between the binding domain and the transmembrane domain (TM). The hinge domain may be derived either from a natural, synthetic, semi-synthetic, or recombinant source. The hinge domain can include the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region. Illustrative hinge domains suitable for use in the CARs described herein include the hinge region derived from the extracellular regions of type 1 membrane proteins such as CD8 (e.g., CD8alpha), CD4, CD28, 4-1BB, and CD7, which may be wild-type hinge regions from these molecules or may be altered. In some embodiments, the CAR comprises polynucleotide encoding CD8alpha hinge / transmembrane

[0087] #14707301 v1 domain. In some embodiments, the CAR comprises a polynucleotide encoding a 4 IBB intracellular domain.

[0088] In some embodiments, the hinge region is derived from the hinge region of an immunoglobulin like protein (e.g., IgA, IgD, IgE, IgG, or IgM), CD28, or CD8. In some embodiments, the hinge domain includes a CD8a hinge region.

[0089] As used herein, “transmembrane domain” (TM domain) refers to the portion of the CAR that fuses the extracellular domain, In some embodiments, (e.g., via a hinge domain), to the intracellular portion (e.g., the costimulatory domain and intracellular signaling domain) and anchors the CAR to the plasma membrane of the immune effector cell. The transmembrane domain is a generally hydrophobic region of the CAR, which crosses the plasma membrane of a cell. The TM domain can be the transmembrane region or fragment thereof of a transmembrane protein (for example a Type I transmembrane protein or other transmembrane protein), an artificial hydrophobic sequence, or a combination thereof. While specific examples are provided herein and used herein, other transmembrane domains will be apparent to those of skill in the art and can be used in connection with alternate embodiments of the technology. A selected transmembrane region or fragment thereof would preferably not interfere with the intended function of the CAR.

[0090] As used in relation to a transmembrane domain of a protein or polypeptide, “fragment thereof’ refers to a portion of a transmembrane domain that is sufficient to anchor or attach a protein to a cell surface.

[0091] In some embodiments, the transmembrane domain or fragment thereof of the CAR described herein includes a transmembrane domain selected from the transmembrane domain of an alpha, beta or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, 0X40, CD2, CD27, LFA-1 (CDl la, CD18), ICOS (CD278), 4-1BB (CD137), 4-1BBL, GITR, CD40, BAFFR, HVEM (EIGHTR), SEAMF7, NKp80 (KERFI), CD160, CD19, IE2R beta, IE2R gamma, IE7R a, ITGA1, VEA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VEA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAE, CDl la, LFA-1, ITGAM, CDl lb, ITGAX, CDl lc, ITGB1, CD29, ITGB2, CD 18, EFA-1, ITGB7, TNFR2, DNAM1 (CD226), SEAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD 160 (BY55), PSGE1, CD100 (SEMA4D), SEAMF6 (NTB-A, Lyl08), SEAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C.

[0092] #14707301 v1 As used herein, a “hinge / transmembrane domain” refers to a domain including both a hinge domain and a transmembrane domain. For example, a hinge / transmembrane domain can be derived from the hinge / transmembrane domain of CD8, CD28, CD7, or 4-1BB. In some embodiments, the hinge / transmembrane domain of a CAR or fragment thereof is derived from or includes the hinge / transmembrane domain of CD8 (e.g., SEQ ID NO: 24, or variants thereof). CD8 is an antigen preferentially found on the cell surface of cytotoxic T lymphocytes. CD8 mediates cell-cell interactions within the immune system, and acts as a T cell co-receptor. CD8 consists of an alpha (CD8alpha or CD8a) and beta (CD813 or CD8b) chain. CD8a sequences are known for a number of species, e.g., human CD8a, (NCBI Gene ID: 925) polypeptide (e.g., NCBI Ref Seq NP 001139345.1) and mRNA (e.g., NCBI Ref Seq NM_ 000002.12). CD8 can refer to human CD8, including naturally occurring variants, molecules, and alleles thereof. In some embodiments of any of the aspects, e.g., in veterinary applications, CD8 can refer to the CD8 of, e.g., dog, cat, cow, horse, pig, and the like.

[0093] Homologs and / or orthologs of human CD8 are readily identified for such species by one of skill in the art, e.g., using the NCBI ortholog search function or searching available sequence data for a given species for sequence similar to a reference CD8 sequence.

[0094] In some embodiments, the CD8 hinge / transmembrane sequence comprises the amino acid sequence of SEQ ID NO: 24. In some embodiments, the CD8 hinge / transmembrane sequence comprises the amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to the sequence of SEQ ID NO: 24.

[0095] Co-stimulatory Domains

[0096] Each CAR described herein optionally includes the intracellular domain of one or more co- stimulatory molecule or co- stimulatory domain. As used herein, the term “co-stimulatory domain” refers to an intracellular signaling domain of a co-stimulatory molecule. Co-stimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that provide a second signal required for efficient activation and function of T lymphocytes upon binding to antigen. The co-stimulatory domain can be, for example, the co-stimulatory domain of 4- IBB, CD27, CD28, or 0X40. In some embodiments, a 4- IBB co-stimulatory domain can be used (see, e.g., below and SEQ ID NO: 25, or variants thereof). In some embodiments, a CD28 co- stimulatory domain can be used (see, e.g., below and SEQ ID NO: 26, or variants thereof). Additional illustrative examples of such co-stimulatory domains include CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (0X40), CD137 (4-1BB), CD150

[0097] #14707301 v1 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD- Ll), CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM, and ZAP70. In some embodiments, the intracellular domain is the intracellular domain of 4-1 BB. 4-1 BB (CD 137; TNFRS9) is an activation induced costimulatory molecule and is an important regulator of immune responses.

[0098] 4- IBB is a membrane receptor protein, also known as CD 137, which is a member of the tumor necrosis factor (TNF) receptor superfamily. 4- IBB is expressed on activated T lymphocytes. 4- IBB sequences are known for a number of species, e.g., human 4-1 BB, also known as TNFRSF9 (NCBI Gene 25 ID: 3604) and mRNA (NCBI Reference Sequence: NM_001561.5). 4-1BB can refer to human 4-1BB, including naturally occurring variants, molecules, and alleles thereof. In some embodiments of any of the aspects, e.g., in veterinary applications, 4-1BB can refer to the 4-1BB of, e.g., dog, cat, cow, horse, pig, and the like. Homologs and / or orthologs of human 4- IBB are readily identified for such species by one of skill in the art, e.g., using the NCBI ortholog search function or searching available sequence data for a given species for sequence similar to a reference 4- IBB sequence.

[0099] In some embodiments, the co- stimulatory domain comprises the co-stimulatory domain of a 4- IBB. In some embodiments, the 4- IBB co-stimulatory domain comprises the amino acid sequence of SEQ ID NO: 25. In some embodiments, the co-stimulatory domain comprises the co-stimulatory domain of CD28. In some embodiments, the CD28 co-stimulatory domain comprises the amino acid sequence of SEQ ID NO: 26.

[0100] Intracellular Signaling Domains

[0101] In some embodiments, the CAR comprises a polynucleotide encoding a CD3zeta (CD3Q intracellular signaling domain (e.g., SEQ ID NO: 27 or a variant thereof).

[0102] The properties of the intracellular signaling domain(s) of the CAR can vary as known in the art and as disclosed herein, but the chimeric target / extracellular antigen binding domains(s) render the receptor sensitive to signaling activation when the chimeric target / extracellular antigen binding domain binds the target / antigen on the surface of a targeted cell.

[0103] With respect to intracellular signaling domains, so-called “first-generation” CARs include those that solely provide CD3(^ signals upon antigen binding. So-called “second- generation” CARs include those that provide both co- stimulation (e.g., CD28 or CD137) and activation (CD3Q domains, and so-called “third-generation” CARs include those that provide multiple costimulatory (e.g., CD28 and CD137) domains and activation (signaling) domains (e.g., CD3 . In various embodiments, the CAR is selected to have high affinity or avidity for the target / antigen - for example, antibody-derived target or extracellular antigen binding

[0104] #14707301 v1 domains will generally have higher affinity and / or avidity for the target antigen than would a naturally occurring T cell receptor. This property, combined with the high specificity one can select for an antibody provides highly specific T cell targeting by CAR-T cells.

[0105] CARs as described herein include an intracellular signaling domain. An “intracellular signaling domain” refers to the part of a CAR polypeptide that participates in transducing the message of effective CAR binding to a target antigen into the interior of the immune effector cell to elicit effector cell function, e.g., activation, cytokine production, proliferation and cytotoxic activity, including the release of cytotoxic factors to the CAR-bound target cell, or other cellular responses elicited following antigen binding to the extracellular CAR domain. In some embodiments, the intracellular signaling domain is from CD3-zeta (CD3Q (see, e.g., below). Additional non-limiting examples of immunoreceptor tyrosine-based activation motif (ITAM)-containing intracellular signaling domains that are of particular use in the technology include those derived from TCR-zeta, FcR-gamma, FcR-beta, CD3-gamma, CD3-theta, CD3- sigma, CD3-eta, CD3-epsilon, CD3-zeta, CD22, CD79a, CD79b, and CD66d.

[0106] CD3 is a T cell co-receptor that facilitates T lymphocyte activation when simultaneously engaged with the appropriate co-stimulation (e.g., binding of a co- stimulatory molecule). A CD3 complex consists of 4 distinct chains; mammalian CD3 consists of a CD3-gamma chain, a CD3- delta chain, and two CD3-epsilon chains.

[0107] These chains associate with a molecule known as the T cell receptor (TCR) and the CD3(^ to generate an activation signal in T lymphocytes. A complete TCR complex includes a TCR, CD3(^, and the complete CD3 complex.

[0108] In some embodiments of any aspect, a CAR polypeptide described herein includes an intracellular signaling domain that includes an Immunoreceptor Tyrosine-based Activation Motif or ITAM from CD3(^, including variants of CD3(^ such as IT AM-mutated CD3(^, CD3-eta, or CD3-theta. In some embodiments of any aspect, the ITAM includes three motifs of ITAM of CD3(^ (ITAM3). In some embodiments of any aspect, the three motifs of ITAM of CD3-zeta are not mutated and, therefore, include native or wild-type sequences. In some embodiments, the CD3(^ sequence includes the sequence of a CD3(^ as set forth in the sequences provided herein, e.g., a CD3(^ sequence of SEQ ID NO: 27, or variants thereof.

[0109] In some embodiments, the intracellular signaling domain comprises the intracellular signaling domain of 4- IBB. In some embodiments, the 4- IBB intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 25. In some embodiments, the intracellular signaling domain comprises the intracellular signaling domain of CD28. In some embodiments, the CD28 intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 26.

[0110] #14707301 v1 In some embodiments, an intracellular signaling domain comprises a 4- IBB intracellular signaling domain (e.g., comprising the amino acid sequence of SEQ ID NO: 25) and a CD3(^ intracellular signaling domain (e.g., comprising the amino acid sequence of SEQ ID NO: 27). In some embodiments, an intracellular signaling domain comprises a CD28 intracellular signaling domain (e.g., comprising the amino acid sequence of SEQ ID NO: 26) and a CD3(^ intracellular signaling domain (e.g., comprising the amino acid sequence of SEQ ID NO: 27).

[0111] Individual CAR and other construct components as described herein can be used with one another and swapped in and out of various constructs described herein, as can be determined by those of skill in the art. Each of these components can include or consist of any of the corresponding sequences set forth herein, or variants thereof.

[0112] Additional description of CARs and CAR-T cells can be found in Maus et al. , Blood. 123:2624-2635, 2014; Reardon et al., Neuro-Oncology 16:1441-1458, 2014; Hoyos et al., Haematologica. 97:1622, 2012; Byrd et al., J. Clin. Oncol. 32:3039-3047, 2014; Maher et al., Cancer Res. 69:4559-4562, 2009; and Tamada et al., Clin. Cancer Res. 18:6436-6445, 2012; each of which is incorporated by reference herein in its entirety.

[0113] Signal Peptide

[0114] As used herein, a “signal peptide” or “signal sequence” refers to a peptide at the N- terminus of a newly synthesized protein that serves to direct a nascent protein into the endoplasmic reticulum. In some embodiments, a CAR polypeptide as described herein includes a signal peptide. Signal peptides can be derived from any protein that has an extracellular domain or is secreted. A CAR polypeptide as described herein may include any signal peptides known in the art. In some embodiments, the CAR polypeptide includes a CD8 signal peptide, e.g., a CD8 signal peptide corresponding to the amino acid sequence of SEQ ID NO: 28. In some embodiments, a CAR polypeptide described herein may optionally exclude one of the signal peptides described herein, e.g., a CD8 signal peptide of SEQ ID NO: 28 or an IgK signal peptide of SEQ ID NO: 29.

[0115] Linker Domain

[0116] In some embodiments, the CAR further includes a linker domain. As used herein, “linker domain” refers to an oligo- or polypeptide region from about 2 to 100 amino acids in length, which links together any of the domains / regions of the CAR as described herein. In some embodiment, linkers can include or be composed of flexible residues such as glycine and serine so that the adjacent protein domains are free to move relative to one another. Linker

[0117] #14707301 v1 sequences useful for the invention can be from 2 to 100 amino acids, 5 to 50 amino acids, 10 to 15 amino acids, 15 to 20 amino acids, or 18 to 20 amino acids in length, and include any suitable linkers known in the art. For instance, linker sequences useful for the invention include, but are not limited to, glycine / serine linkers, e.g., GGGSGGGSGGGS (SEQ ID NO: 30) and Gly4Ser (G4S) (SEQ ID NO: 91) linkers such as (G4S)3 (GGGGSGGGGSGGGGS (SEQ ID NO: 31)) and (G4S)4 (GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 32)); the linker sequence of GSTSGSGKPGSGEGSTKG (SEQ ID NO: 33) as described by Whitlow et al., Protein Eng. 6(8):989-95, 1993, the contents of which are incorporated herein by reference in its entirety; the linker sequence of GGSSRSSSSGGGGSGGGG (SEQ ID NO: 34) as described by Andris- Widhopf et al., Cold Spring Harb. Protoc. 2011 (9), 2011, the contents of which are incorporated herein by reference in its entirety; as well as linker sequences with added functionalities, e.g., an epitope tag or an encoding sequence containing Cre-Lox recombination site as described by Sblattero et al., Nat. Biotechnol. 18( l):75-80, 2000, the contents of which are incorporated herein by reference in its entirety. Longer linkers may be used when it is desirable to ensure that two adjacent domains do not sterically interfere with one another.

[0118] Furthermore, linkers may be cleavable or non-cleavable. Examples of cleavable linkers include 2A linkers (e.g., P2A (SEQ ID NO: 35) and T2A (SEQ ID NO: 36)), 2A-like linkers or functional equivalents thereof and combinations thereof.

[0119] In various examples, linkers having sequences as set forth herein, or variants thereof, are used. It is to be understood that the indication of a particular linker in a construct in a particular location does not mean that only that linker can be used there. Rather, different linker sequences (e.g., P2A, E2A, F2A, and T2A) can be swapped with one another (e.g., in the context of the constructs of the present invention), as can be determined by those of skill in the art. In some embodiments, the linker region is T2A derived from Thosea asigna virus. Non-limiting examples of linkers that can be used in this technology include T2A, P2A, E2A, BmCPV2A, and BmlFV2A. Linkers such as these can be used in the context of polyproteins, such as those described below. For example, they can be used to separate a CAR component of a polyprotein from a therapeutic agent (e.g., an antibody, such as a scFv, single domain antibody (e.g., a camelid antibody), or a bispecific antibody (e.g., a TEAM)) component of a polyprotein (see below).

[0120] In some embodiments, the CAR-T cells described herein are generated from autologous T cells. Autologous T cells are derived from the subject and are advantageous in comparison to allogeneic T cells in that they minimize or abolish the risk of immune rejection or graft-versus- host disease.

[0121] #14707301 v1 In some embodiments, a polynucleotide comprises nucleic acids encoding an amino acid sequence comprising an epidermal growth factor receptor (EGFR) binding chimeric antigen receptor (EGFRvIII CAR) comprising an extracellular antigen binding domain that comprises: a variable heavy chain (VH) complimentary determining region (CDR) 1 of SEQ ID NO: 1, a VH CDR2 of SEQ ID NO: 2, a VH CDR3 of SEQ ID NO: 3, a variable light chain (VL) CDR1 of SEQ ID NO: 4, a VL CDR2 of SEQ ID NO: 5, and a VL CDR3 of SEQ ID NO: 6.

[0122] In some embodiments, this disclosure describes a polypeptide comprising amino acids of an epidermal growth factor receptor (EGFR) binding chimeric antigen receptor (EGFRvIII CAR) comprising an extracellular antigen binding domain that comprises: a variable heavy chain (VH) complimentary determining region (CDR) 1 of SEQ ID NO: 1, a VH CDR2 of SEQ ID NO: 2, a VH CDR3 of SEQ ID NO: 3, a variable light chain (VL) CDR1 of SEQ ID NO: 4, a VL CDR2 of SEQ ID NO: 5, and a VL CDR3 of SEQ ID NO: 6.

[0123] T Cell Engaging Antibody Molecules (TEAMs)

[0124] In some embodiments, the CAR-T cells described herein express a T cell engaging molecule (TEAM) (also referred to in the literature as bispecific T cell engagers or BiTEs™) (“CAR-TEAM cells”). By “T cell engaging molecules,” “TEAM antibody constructs,” or “TEAMs” is meant polypeptides that each comprise tandemly linked antibodies (e.g., linked single-chain variable fragments (scFvs)) where a first antibody binds to a T cell (e.g., CD3) and a second antibody binds to a cancer / tumor antigen (e.g., EGFR). Optionally, the scFvs are linked by a linker (e.g., a glycine-rich linker). In some embodiments, the linker is a non-cleavable linker. In some embodiments, the linker is a linker described herein. One scFv of the TEAM binds to the T cell receptor (TCR) (e.g., to the CD3e subunit) and the other binds to a target antigen (e.g., a tumor antigen). Such molecules can target T cells by binding to a T cell antigen (e.g., by binding CD3) as well as a target antigen, e.g., a tumor antigen. The TEAMs can be used to augment the T cell response in, e.g., the tumor microenvironment. The two components of a TEAM can also be connected in either orientation, e.g., with the anti-CD3 component N- terminal to the anti-target antigen component, or vice versa. The anti-CD3 component or the anti-target antigen component of the TEAM may include any of the antibody reagents described herein.

[0125] In some embodiments, the immune cells (e.g., CAR-TEAM cells) described herein secrete TEAMs. In some embodiments, the TEAM comprises a secretion tag (e.g., an IgK signal peptide). The TEAMs may stimulate the CAR-TEAM cell itself, or operate in a paracrine fashion by redirecting nonspecific bystander T cells against tumors and therefore enhance the

[0126] #14707301 v1 anti-tumor effects of CAR-T cell immunotherapy. CAR-TEAM cell-mediated TEAM secretion may allow for the reduction of risk of undesired TEAM activity in systemic tissues by directing TEAM secretion to the tumor microenvironment. Exemplary TEAM constructs are provided below; however, TEAMs other than those described herein may also be useful for the CAR T cells and methods of the disclosure.

[0127] In some embodiments, the anti-CD3 scFv of any of the TEAMs described herein may be arranged in the VH-VL orientation, or in the VL-VH orientation. In some embodiments, the anti-CD3 scFv comprises (i) a VH domain comprising three complementarity determining regions (CDR-H1, CDR-H2, and CDR-H3), wherein CDR-H1 comprises SEQ ID NO: 16, CDR-H2 comprises SEQ ID NO: 17, and CDR-H3 comprises SEQ ID NO: 18, and (ii) a VL domain comprising three CDRs (CDR-L1, CDR-L2, and CDR-L3), wherein CDR-L1 comprises SEQ ID NO: 19, CDR-L2 comprises SEQ ID NO: 20, and CDR-L3 comprises SEQ ID NO: 21. In some embodiments, the anti-CD3 scFv comprises the amino acid sequence of SEQ ID NO: 22 or an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to the amino acid sequence of SEQ ID NO: 22.

[0128] In some embodiments, the anti-EGFR scFv of any of the TEAMs described herein may be arranged in the VH-VL orientation, or in the VL-VH orientation. In some embodiments, the anti-EGFR scFv comprises (i) a VH domain comprising three complementarity determining regions (CDR-H1, CDR-H2, and CDR-H3), wherein CDR-H1 comprises SEQ ID NO: 9, CDR- H2 comprises SEQ ID NO: 10, and CDR-H3 comprises SEQ ID NO: 11, and (ii) a VL domain comprising three CDRs (CDR-L1, CDR-L2, and CDR-L3), wherein CDR-L1 comprises SEQ ID NO: 12, CDR-L2 comprises SEQ ID NO: 13, and CDR-L3 comprises SEQ ID NO: 14. In some embodiments, the anti-EGFR scFv comprises the amino acid sequence of SEQ ID NO: 15 or an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to the amino acid sequence of SEQ ID NO: 15. In some embodiments, the anti-EGFR / anti-CD3 team comprises the amino acid sequence of SEQ ID NO: 23 or an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater sequence identity to the amino acid sequence of SEQ ID NO: 23.

[0129] In some embodiments, an anti-FAP antibody (e.g., scFv) of any of the TEAMs described herein may be arranged in the VH-VL orientation, or in the VL-VH orientation. In some embodiments, the anti-FAP antibody comprises a VH of SEQ ID NO: 54 and / or a VL of SEQ ID NO: 55 or a conservatively modified variant thereof that maintains binding to FAP. In some

[0130] #14707301 v1 embodiments, the anti-FAP antibody comprises the amino acid sequence of SEQ ID NO: 56 or an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater sequence identity to the amino acid sequence of SEQ ID NO: 56. In some embodiments, the TEAM comprises the anti- FAP antibody further comprises an anti-CD3 antibody (e.g., as described herein).

[0131] In some embodiments, an anti-CD33 antibody (e.g., scFv) of any of the TEAMs described herein may be arranged in the VH-VL orientation, or in the VL-VH orientation. In some embodiments, the anti-CD33 antibody comprises a VH of SEQ ID NO: 86 and / or a VL of SEQ ID NO: 86, or a conservatively modified variant thereof that maintains binding to CD33. In some embodiments, the FAP-CD3 binding TEAM comprises the amino acid sequence of SEQ ID NO: 83 or SEQ ID NO: 84 or an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater sequence identity to the amino acid sequence of SEQ ID NO: 83 or SEQ ID NO: 84.

[0132] Truncated CD 19

[0133] “Truncated CD 19 (tCD19)” refers to a fragment B-lymphocyte antigen CD 19 protein (e.g., Uniprot ID P15391 as October 10, 2023). A “fragment” of a protein (e.g., CD19) has at least one amino acid missing relative to the wildtype protein. In some embodiments, the truncated CD 19 does not comprise a n-terminal fragment of CD 19. In some embodiments, the truncated CD 19 does not comprise a c-terminal fragment of CD 19. In some embodiments, the truncated CD19 comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO: 38. In some embodiments, the truncated CD19 comprises an amino acid sequence of SEQ ID NO: 38. In some embodiments, the truncated CD19 consists an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO: 38. In some embodiments, the truncated CD19 consists of an amino acid sequence of SEQ ID NO: 38. In some embodiments, the truncated CD19 comprises an amino acid sequence of SEQ ID NO: 38. and does not comprise the remainer of CD 19 outside of the fragment.

[0134] In some embodiments, the truncated CD 19 may be used for detection a CAR-T cell. For example, the CAR-T cell may comprise an amino acid sequence encoding the truncated CD 19; truncated CD 19 may be expressed on the surface of the CAR-T cell and be detected using an antibody that is specific to truncated CD 19.

[0135] CARMeso-TEAM-FAP

[0136] #14707301 v1 In some embodiments, this disclosure provide a method of treating a mesothelin expressing cancer in a subject, the method comprises administering an anti-CD20 antibody and an immune cell (e.g., a CAR-T cell) that comprises a first polynucleotide encoding a fibroblast activation protein (FAP)-binding TEAM (e.g., a CD3 and FAP binding TEAM) and a second polynucleotide encoding a mesothelin binding CAR. In some embodiments, the mesothelin binding CAR comprises a mesothelin extracellular antigen binding domain, a CD8 hinge and transmembrane domain, a 4- IBB co-stimulatory domains, and a CD3-zeta domain. In some embodiments, the anti-FAP scFv of any of the TEAMs described herein may be arranged in the VH-VL orientation, or in the VL-VH orientation. In some embodiments, the first polynucleotide and the second polynucleotide are encoded on the same polynucleotide and collectively encode a polyprotein comprising the mesothelin binding CAR and the FAP binding team. In some embodiments, the polyprotein comprises an amino acid sequence having at least 80% identity (e.g., at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity) to SEQ ID NO: 41. In some embodiments, the polyprotein comprises an amino acid sequence of SEQ ID NO: 41. In some embodiments, the polyprotein comprises a CAR comprising an amino acid sequence having at least 80% identity (e.g., at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity) to any one of SEQ ID NOs: 50-53 and a TEAM comprising an amino acid sequence having at least 80% identity (e.g., at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity) to SEQ ID NO: 56. In some embodiments, the polyprotein comprises a CAR comprising an amino acid sequence of any one of SEQ ID NOs: 50-53 and a TEAM comprising an amino acid sequence of SEQ ID NO: 56.

[0137] CARCD70-TEAM-CD33

[0138] In some embodiments, this disclosure provide a method of treating a CD70 and / or CD33 expressing cancer in a subject, the method comprises administering an anti-CD20 antibody and an immune cell (e.g., a CAR-T cell) that comprises a first polynucleotide encoding a CD33- binding TEAM (e.g., a CD3 and CD33 binding TEAM) and a second polynucleotide encoding a CD70 binding CAR. In some embodiments, the CD70 CAR comprises a truncated CD27 extracellular antigen binding domain, a CD8 hinge / transmembrane domain, a 4-lBBz costimulatory domains, and a CD3-zeta intracellular signaling domain. In some embodiments, the anti-CD33 scFv of any of the TEAMs described herein may be arranged in the VH-VL orientation, or in the VL-VH orientation. In some embodiments, the first polynucleotide and the second polynucleotide are encoded on the same polynucleotide and collectively encode a

[0139] #14707301 v1 polyprotein comprising the CD70 binding CAR and the CD33 binding team. In some embodiments, the polyprotein comprises an amino acid sequence having at least 80% identity (e.g., at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity) to SEQ ID NO: 88-90. In some embodiments, the polyprotein comprises an amino acid sequence of SEQ ID NO: 88-90. In some embodiments, the polyprotein comprises a CAR comprises an amino acid sequence having at least 80% identity (e.g., at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity) to any one of SEQ ID NOs: 68-73, and a TEAM comprising an amino acid sequence having at least 80% identity (e.g., at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% identity) to any one of SEQ ID NOs: 83-84. In some embodiments, the polyprotein comprises a CAR comprising an amino acid sequence of any one of SEQ ID NOs: 68-73 and a TEAM comprising an amino acid sequence of any one of SEQ ID NOs: 83-84.

[0140] CARV3-TEAM-E

[0141] In some aspects, this disclosure describes method of treating glioblastoma in a subject, the method comprising administering an anti-CD20 antibody (e.g., rituximab) and an immune cell comprising a first polynucleotide encoding an EGFR binding TEAM. In some embodiment, the immune cell comprises a second polynucleotide encoding a EGFRvIII binding CAR. In some embodiments, the EGFRvIII binding CAR comprises, from n-terminal to c-terminal, a CD8 leader sequence, an EGFRvIII extracellular antigen binding domain (e.g., an EGFRvIII scFv), a CD8 hinge, a CD8 transmembrane domain, a 4- IBB co-stimulatory domain, a CD3-zeta intracellular signaling domain, a P2A peptide, an IgK signal peptide, an anti-EGFR antibody (e.g., a cetuximab scFv), an anti-CD3 antibody (e.g., scFv), a T2A peptide, and a truncated CD19 (e.g., of SEQ ID NO: 38).

[0142] In some aspects, this disclosure describes a polynucleotide comprising nucleic acids encoding an EGFR- VIII binding CAR, and EGFR-binding and CD3-binding TEAM. In some embodiments, a polynucleotide comprising nucleic acids encoding, an amino acid sequence from n-terminal to c-terminal encoding: a CD8 leader sequence, an EGFRvIII extracellular antigen binding domain (e.g., an EGFRvIII scFv), a CD8 hinge, a CD8 transmembrane domain, a 4- IBB co-stimulatory domain, a CD3-zeta intracellular signaling domain, a P2A peptide, an IgK signal peptide, an anti-EGFR antibody (e.g., a cetuximab scFv), an anti-CD3 antibody (e.g., scFv), a T2A peptide, and a truncated CD19 (e.g., of SEQ ID NO: 38).

[0143] #14707301 v1 In some embodiments, this disclosure describes a polypeptide comprising from n- terminal to c-terminal encoding: a CD8 leader sequence, an EGFRvIII extracellular antigen binding domain (e.g., an EGFRvIII scFv), a CD8 hinge, a CD8 transmembrane domain, a 4- IBB co- stimulatory domain, a CD3-zeta intracellular signaling domain, a P2A peptide, an IgK signal peptide, an anti-EGFR antibody (e.g., a cetuximab scFv), an anti-CD3 antibody (e.g., scFv), a T2A peptide, and a truncated CD19 (e.g., of SEQ ID NO: 38).

[0144] In some embodiments, this disclosure describes a polynucleotide comprising nucleic acids encoding an amino acid sequence comprising: (i) an epidermal growth factor receptor (EGFR) binding chimeric antigen receptor (EGFRvIII CAR) comprising an extracellular antigen binding domain that comprises: a variable heavy chain (VH) complimentary determining region (CDR) 1 of SEQ ID NO: 1, a VH CDR2 of SEQ ID NO: 2, a VH CDR3 of SEQ ID NO: 3, a variable light chain (VL) CDR1 of SEQ ID NO: 4, a VL CDR2 of SEQ ID NO: 5, and a VL CDR3 of SEQ ID NO: 6; and (ii) a T cell engaging antibody molecule (TEAM) comprising: (a) an EGFR binding domain comprising a variable heavy chain (VH) complimentary determining region (CDR) 1 of SEQ ID NO: 9, a VH CDR2 of SEQ ID NO: 10, a VH CDR3 of SEQ ID NO: 11, a variable light chain (VL) CDR1 of SEQ ID NO: 12, a VL CDR2 of SEQ ID NO: 13, and a VL CDR3 of SEQ ID NO: 14; (b) a CD3 binding domain comprising a variable heavy chain (VH) complimentary determining region (CDR) 1 of SEQ ID NO: 16, a VH CDR2 of SEQ ID NO: 17, a VH CDR3 of SEQ ID NO: 18, a variable light chain (VL) CDR1 of SEQ ID NO: 19, a VL CDR2 of SEQ ID NO: 20, and a VL CDR3 of SEQ ID NO: 21; and optionally (iii) a truncated CD 19 domain (tCD19) comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 38.

[0145] In some embodiments, this disclosure describes a polynucleotide comprising nucleic acids encoding an amino acid sequence comprising: (i) an epidermal growth factor receptor (EGFR) binding chimeric antigen receptor (EGFRvIII CAR) comprising an extracellular antigen binding domain that comprises: a variable heavy chain (VH) complimentary determining region (CDR) 1 of SEQ ID NO: 1, a VH CDR2 of SEQ ID NO: 2, a VH CDR3 of SEQ ID NO: 3, a variable light chain (VL) CDR1 of SEQ ID NO: 4, a VL CDR2 of SEQ ID NO: 5, and a VL CDR3 of SEQ ID NO: 6; and (ii) a T cell engaging antibody molecule (TEAM) comprising: (a) an EGFR binding domain comprising a variable heavy chain (VH) complimentary determining region (CDR) 1 of SEQ ID NO: 9, a VH CDR2 of SEQ ID NO: 10, a VH CDR3 of SEQ ID NO: 11, a variable light chain (VL) CDR1 of SEQ ID NO: 12, a VL CDR2 of SEQ ID NO: 13, and a VL CDR3 of SEQ ID NO: 14; (b) a CD3 binding domain comprising a variable heavy chain (VH) complimentary determining region (CDR) 1 of SEQ ID NO: 16, a VH CDR2 of SEQ ID

[0146] #14707301 v1 NO: 17, a VH CDR3 of SEQ ID NO: 18, a variable light chain (VL) CDR1 of SEQ ID NO: 19, a VL CDR2 of SEQ ID NO: 20, and a VL CDR3 of SEQ ID NO: 21; and optionally (iii) a truncated CD19 domain (tCD19) comprising an amino acid sequence of SEQ ID NO: 38.

[0147] In some embodiments, this disclosure describes a polypeptide comprising: (i) an epidermal growth factor receptor (EGFR) binding chimeric antigen receptor (EGFRvIII CAR) comprising an extracellular antigen binding domain that comprises: a variable heavy chain (VH) complimentary determining region (CDR) 1 of SEQ ID NO: 1, a VH CDR2 of SEQ ID NO: 2, a VH CDR3 of SEQ ID NO: 3, a variable light chain (VL) CDR1 of SEQ ID NO: 4, a VL CDR2 of SEQ ID NO: 5, and a VL CDR3 of SEQ ID NO: 6; and (ii) a T cell engaging antibody molecule (TEAM) comprising: (a) an EGFR binding domain comprising a variable heavy chain (VH) complimentary determining region (CDR) 1 of SEQ ID NO: 9, a VH CDR2 of SEQ ID NO: 10, a VH CDR3 of SEQ ID NO: 11, a variable light chain (VL) CDR1 of SEQ ID NO: 12, a VL CDR2 of SEQ ID NO: 13, and a VL CDR3 of SEQ ID NO: 14; (b) a CD3 binding domain comprising a variable heavy chain (VH) complimentary determining region (CDR) 1 of SEQ ID NO: 16, a VH CDR2 of SEQ ID NO: 17, a VH CDR3 of SEQ ID NO: 18, a variable light chain (VL) CDR1 of SEQ ID NO: 19, a VL CDR2 of SEQ ID NO: 20, and a VL CDR3 of SEQ ID NO: 21; and (iii) optionally a truncated CD19 domain (tCD19) comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 38.

[0148] In some embodiments, this disclosure describes a polypeptide comprising: (i) an epidermal growth factor receptor (EGFR) binding chimeric antigen receptor (EGFRvIII CAR) comprising an extracellular antigen binding domain that comprises: a variable heavy chain (VH) complimentary determining region (CDR) 1 of SEQ ID NO: 1, a VH CDR2 of SEQ ID NO: 2, a VH CDR3 of SEQ ID NO: 3, a variable light chain (VL) CDR1 of SEQ ID NO: 4, a VL CDR2 of SEQ ID NO: 5, and a VL CDR3 of SEQ ID NO: 6; and (ii) a T cell engaging antibody molecule (TEAM) comprising: (a) an EGFR binding domain comprising a variable heavy chain (VH) complimentary determining region (CDR) 1 of SEQ ID NO: 9, a VH CDR2 of SEQ ID NO: 10, a VH CDR3 of SEQ ID NO: 11, a variable light chain (VL) CDR1 of SEQ ID NO: 12, a VL CDR2 of SEQ ID NO: 13, and a VL CDR3 of SEQ ID NO: 14; (b) a CD3 binding domain comprising a variable heavy chain (VH) complimentary determining region (CDR) 1 of SEQ ID NO: 16, a VH CDR2 of SEQ ID NO: 17, a VH CDR3 of SEQ ID NO: 18, a variable light chain (VL) CDR1 of SEQ ID NO: 19, a VL CDR2 of SEQ ID NO: 20, and a VL CDR3 of SEQ ID NO: 21; and optionally (iii) a truncated CD19 domain (tCD19) comprising an amino acid sequence of SEQ ID NO: 38.

[0149] #14707301 v1 In some embodiments, a method comprises administering to a subject having glioblastoma (e.g., EGFR positive glioblastoma) a CAR-T cell comprising: an EGFRvIII- binding CAR (e.g., SEQ ID NO: 8) and an EGFR-CD3 binding TEAM (e.g., SEQ ID NO: 23). In some embodiments, the method further comprises administering to the subject an anti-CD20 antibody (e.g., rituximab). In some embodiments, the CAR T cell comprises a polynucleotide encoding SEQ ID NO: 37. In some embodiments, the method comprises administering 6.0 x 106to 1.6 x 107of the CAR-T cells to the subject. In some embodiments, the method of treating GBM in a subject comprises administering 7.0 x 106to 1.3 x 107of the CAR-T cells to the subject. In some embodiments, the method comprises administering 8.0 x 106 to 1.2 x 107of the CAR-T cells to the subject. In some embodiments, the method comprises administering 9.0 x 106to 1.1 x 107of the CAR-T cells to the subject. In some embodiments, the method comprises administering 1.0 x 107of the CAR-T cells to the subject. In some embodiments, the method comprises administering the CAR-T cells to the subject up to 6 times (e.g., up to 5 times, up to 4 times, up to 3 times, or up to 2 times). In some embodiments, the method comprises administering the CAR-T cells to the subject 1, 2, 3, 4, 5, 6, 7, or 8 times. In some embodiments, the method comprises administering the CAR-T cells into the central nervous system of the subject (e.g., intraventricular infusion).

[0150] In some embodiments, a method comprises treating glioblastoma in a subject, the method comprising administering about 10 x 106CAR-T cells into the central nervous system of the subject (e.g. intraventricular administration) up to 6 times (e.g., 1, 2, 3, 4, 5, or 6 times), wherein the CAR-T cells comprises a first polynucleotide that encodes an EGFR-CD3 binding TEAM (e.g., SEQ ID NO: 23) and a second polynucleotide that encodes an EGFRvIII binding CAR (e.g., SEQ ID NO 8). In some embodiments, the EGFR-CD3 binding TEAM and the EGFRvIII binding CAR form a polyprotein (e.g., SEQ ID NO: 37). In some embodiments, the method further comprises administering an anti-CD20 antibody (e.g., rituximab) to the subject. In some embodiments, the anti-CD20 antibody is administered prior to administering the CAR-T cells (e.g., 5-10 days prior to administering the CAR-T cells). In some embodiments, the anti- CD20 antibody is administered after administering the CAR-T cells. In some embodiments, the method further comprises administering LD chemo to the subject. In some embodiments, the LD chemo is administered after administering the rituximab but before administering the CAR- T cells (e.g., on days 5, 4, and 3before administering the CAR-T cells). In some embodiments, the LD chemo is administered before administering the rituximab and before administering the CAR-T cells (e.g., on days 5, 4, and 3 before administering the CAR-T cells). In some

[0151] #14707301 v1 embodiments, the method comprises administering LD chemo and rituximab before administering the CAR-T cells.

[0152] In some embodiments, an immune cell is a T cell. In some embodiments, an immune cell is an autologous T cell. In some embodiments, an immune cell is an allogeneic T cell.

[0153] Vectors

[0154] In some aspects, this application discloses a vector (e.g., an expression vector) comprising the polynucleotide described herein. A “vector,” refers to a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells. As used herein, a vector can be viral or non- viral. The term “vector” encompasses any genetic element that is capable of replication when associated with the proper control elements and that can transfer gene sequences to cells. A vector can include, but is not limited to, a cloning vector, an expression vector, a plasmid, phage, transposon, cosmid, artificial chromosome, virus, virion, etc. In some embodiments, a vector may comprise a polynucleotide described herein (e.g., a polynucleotide comprising a polynucleic acid sequence encoding a VEGF binding protein and / or a CAR).

[0155] As used herein, the term “expression vector” may refer to a vector that directs expression of an RNA or polypeptide from sequences linked to transcriptional regulatory sequences on the vector. The sequences expressed will often, but not necessarily, be heterologous to the cell. An expression vector may comprise additional elements, for example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example, in human cells for expression and in a prokaryotic host for cloning and amplification. The term “expression” refers to the cellular processes involved in producing RNA and proteins and as appropriate, secreting proteins, including where applicable, but not limited to, for example, transcription, transcript processing, translation and protein folding, modification and processing. “Expression products” include RNA transcribed from a gene, and polypeptides obtained by translation of mRNA transcribed from a gene.

[0156] As used herein, the term “viral vector” may refer to a nucleic acid vector construct that includes at least one element of viral origin and has the capacity to be packaged into a viral vector particle. The viral vector can contain a nucleic acid encoding a polypeptide as described herein in place of non-essential viral genes. The vector and / or particle may be utilized for the purpose of transferring nucleic acids into cells either in vitro or in vivo. Numerous forms of viral vectors are known in the art.

[0157] #14707301 v1 By “recombinant vector” may be a vector that includes a heterologous nucleic acid sequence or “transgene” that is capable of expression in vivo. It should be understood that the vectors described herein can, in some embodiments, be combined with other suitable compositions and therapies. In some embodiments, the vector is episomal. The use of a suitable episomal vector provides a means of maintaining the nucleotide of interest in the subject in high copy number extra-chromosomal DNA thereby eliminating potential effects of chromosomal integration.

[0158] Treating Cancer

[0159] “Treating”, “treatment” and “treat” as used herein include improving and / or ameliorating one or more of the signs or symptoms of a condition (e.g., cancer). In some embodiments, treating comprises inducing a desired response, e.g., by at least 10% following treatment according to the methods described herein. Treatment efficacy can be assessed, for example, by measuring a marker, indicator, symptom, and / or the incidence of a cancer treated according to the methods described herein or any other measurable parameter appropriate. Treatment according to the methods described herein can reduce levels of a marker or symptom of a cancer, e.g., by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80 % or at least 90% or more. Treatment efficacy can also be measured by a failure of an individual to worsen as assessed by hospitalization, or need for medical interventions (i.e., progression of the disease is halted). Methods of measuring these indicators are known to those of skill in the art and / or are described herein. Treatment includes any treatment of cancer in an individual or an animal (some nonlimiting examples include a human or an animal) and includes: (1) inhibiting the cancer, e.g., preventing a worsening of symptoms (e.g., pain or inflammation); or (2) relieving the severity of the cancer, e.g., causing regression of symptoms. An effective amount for the treatment of a cancer means that amount which, when administered to a subject in need thereof, is sufficient to result in effective treatment as that term is defined herein, for cancer. Efficacy of an agent can be determined by assessing physical indicators of mesothelin-expressing cancer or desired response. It is well within the ability of one skilled in the art to monitor efficacy of administration and / or treatment by measuring any one of such parameters, or any combination of parameters. Efficacy of a given approach can be assessed in animal models of a condition described herein. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant change in a marker is observed.

[0160] #14707301 v1 “Cancer” as used herein can refer to a hyperproliferation of cells whose unique trait, loss of normal cellular control, results in unregulated growth, lack of differentiation, local tissue invasion, and / or metastasis. In some embodiments, treating a cancer comprises treating a solid tumor. A “solid tumor” includes an abnormal mass of tissue. The abnormal mass of tissue may be benign of cancerous. In some embodiments, the solid tumor is a cancer. In some embodiments, treating a solid tumor comprises treating a brain cancer, a lung cancer, a breast cancer, a thyroid cancer, a skin cancer, a head or neck cancer, a liver cancer, a kidney cancer, a colon cancer, a testicular cancer, an ovarian cancer, or a uterine cancer. In some embodiments, treating a brain cancer comprises treating glioblastoma. In some embodiments, treating glioblastoma comprises treating EGFRvIII positive glioblastoma. In some embodiments, treating glioblastoma comprises treating EGFRvIII negative glioblastoma. In some embodiments, treating glioblastoma comprises treating EGFR positive glioblastoma. In some embodiments, treating glioblastoma comprises treating EGFRvIII positive and EGFR positive glioblastoma. In some embodiments, treating glioblastoma comprises treating EGFRvIII negative and EGFR positive glioblastoma. In some embodiments, treating a cancer comprises treating a liquid cancer. In some embodiments, treating a liquid cancer comprises treating a leukemia. In some embodiments, treating a liquid cancer comprises treating a lymphoma.

[0161] In some embodiments, the method comprises treating a mesothelin expressing cancer (e.g., pancreatic cancer, a lung cancer, an ovarian cancer, endometrial cancer, biliary cancer, gastric cancer, or mesothelioma). In some embodiments, the method comprises treating a CD70 expressing cancer (e.g., bladder cancer, breast invasive carcinoma, cervical cancer, cholangiocarcinoma, colorectal cancer, diffuse large B-cell lymphoma (DLBC), Esophagus, glioblastoma (GBM), head and neck cancer, low-grade gliomas (LGG), liver cancer, lung adeno cancer, melanoma, mesothelioma, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, stomach cancer, testicular germ cell cancer, thymoma, thyroid cancer, uterine cancer, uveal melanoma, clear cell renal cell carcinoma (ccRCC), chromophobe renal cell carcinoma, papillary renal cell carcinoma (pRCC), acute myeloid leukemia, and adenoid cystic carcinoma (ACC).

[0162] As used herein, a “subject” means a human or animal. Usually, the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include, for example, chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, e.g., rhesus. Rodents include, for example, mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include, for example, cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and

[0163] #14707301 v1 fish, e.g., trout, catfish and salmon. In some embodiments, the subject is a mammal, e.g., a primate, e.g., a human. The terms, “individual,” “patient,” and “subject” are used interchangeably herein. Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of disease, e.g., cancer. A subject can be male or female. In some embodiments, the subject is a human subject.

[0164] A subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment (e.g., diagnosed cancer) or one or more complications related to such a condition, and optionally, have already undergone treatment for the condition or the one or more complications related to the condition.

[0165] Alternatively, a subject can also be one who has not been previously diagnosed as having such condition or related complications. For example, a subject can be one who exhibits one or more risk factors for the condition or one or more complications related to the condition or a subject who does not exhibit risk factors. In some embodiments, the subject has been previously treated using a different cancer therapy (e.g., chemotherapy and / or radiation).

[0166] In some embodiments, the methods described herein include administering an effective amount of the immune cells described herein and / or an effective amount of an anti-CD20 antibody.

[0167] The term “effective amount” as includes the amount active immune cell administered to a subject. Described herein needed to treat at least one or more symptom of the cancer and relates to a sufficient amount of the cell preparation or composition to provide the desired effect. The term “therapeutically effective amount” therefore refers to an amount of activated immune cells (e.g., activated CAR-T cells) described herein that is sufficient to provide a particular anticondition effect when administered to a typical subject. An effective amount as used herein, in various contexts, would also include an amount sufficient to delay the development of a symptom cancer, alter the course of the cancer (for example but not limited to, slowing the progression of the cancer), or reverse a symptom of a cancer. Thus, it is not generally practicable to specify an exact “effective amount.” However, for any given case, an appropriate “effective amount” can be determined by one of ordinary skill in the art using only routine experimentation.

[0168] In some embodiments, the methods of treating a subject having a cancer described herein comprises administering an immune cell described herein via intravenous administration. A variety of means for administering the compositions described herein to subjects are known to

[0169] #14707301 v1 those of skill in the art. In some embodiments, the compositions described herein are administered systemically or locally. In a preferred embodiment, the compositions described herein are administered intravenously. In another embodiment, the compositions described herein are administered at the site of a tumor. In some embodiments, the methods of treating a subject having a cancer described herein comprises administering an immune cell described herein via intravascular administration. In some embodiments, the methods of treating a subject having a cancer described herein comprises administering an immune cell described herein via intraperitoneal administration.

[0170] The immune cells described herein can be administered to a patient transarterially, intratumorally, intranodally, intraperitoneally, intrathecally, intramedullary, or orally. In some embodiments, the compositions of the immune cells (e.g., the CAR-T cells) may be injected directly into a tumor, lymph node, or site of infection. In some embodiments, the compositions described herein are administered into a body cavity or body fluid (e.g., ascites, pleural fluid, peritoneal fluid, or cerebrospinal fluid).

[0171] In some embodiments, administering comprises injecting or infusing a pharmaceutical composition (e.g., comprising an immune cell and a pharmaceutically acceptable excipient) into a subject. In some embodiments, injecting or infusion includes passing the pharmaceutical composition through a filter. In some embodiments, injecting or infusion does not include passing the pharmaceutical composition through a filter. In some embodiments, administering comprises contacting the central nervous system of a subject with the cell therapy. In some embodiments, administering comprises administering the pharmaceutical composition into the subject’s central nervous system.

[0172] The word “into” when used in the context of administering to a specific location (e.g., administering the pharmaceutical composition into the subject’s central nervous system) refers to direct administration of a therapy (e.g., a cell therapy or a pharmaceutical composition) to that specific location. For example, administering a pharmaceutical composition into the central nervous system of the subject includes injection or infusion of the pharmaceutical composition into the cerebral spinal fluid of the subject (which is part of the central nervous system), but does not include intravenous infusion or oral administration of the pharmaceutical composition even if the pharmaceutical composition eventually crosses the blood-brain barrier and enters the cerebral spinal fluid.

[0173] “Central nervous system”, as used herein, refers the biological matter (e.g., fluids, tissues, organs, tumors, etc.) within the blood brain barrier including the blood brain barrier, the brain, the spinal cord, the cerebral spinal fluid (CSF), the meninges, the ventricles, the space

[0174] #14707301 v1 between membranes of the meninges (e.g., the subarachnoid space), and brain or spinal tumors of the central nervous system (e.g., GBM).

[0175] In some embodiments, subjects may undergo leukapheresis, wherein leukocytes are collected, enriched, or depleted ex vivo to select and / or isolate the immune cells of interest, e.g., T cells. The isolated immune cells may be engineered to comprise the first polynucleotide and / or second polynucleotide described herein. T cell isolates can be expanded by contact with an artificial APC (aAPC), e.g., an aAPC expressing anti-CD28 and anti-CD3 CDRs, and treated such that one or more CAR constructs or engineered immune cell receptor constructs of the technology may be introduced, thereby creating a CAR-T cell or an engineered immune cell receptor T cell. Subjects in need thereof can subsequently undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation. Following or concurrent with the transplant, subjects can receive an infusion of the expanded CAR-T cells. In some embodiment, expanded cells are administered before or following surgery.

[0176] In some embodiments, lymphodepletion is performed on a subject prior to administering one or more immune cell as described herein. In some embodiments, lymphodepletion is performed on a subject after administering the anti-CD20 antibody and prior to administering one or more immune cell as described herein. In some embodiments, lymphodepletion is performed on a subject currently with administering the anti-CD20 antibody and prior to administering one or more immune cell as described herein. In such embodiments, the lymphodepletion can include administering one or more of bendamustine, melphalan, survivin, cyclophosphamide (e.g., 200 mg / m2 to 400 mg / m2 (e.g., 300 mg / m2) of cyclophosphamide), and fludarabine (e.g., 20 mg / m2 to 40 mg / m2 e.g., (30 mg / m2) of Fludarabine). For example, lymphodepletion may be accomplished by administering a combination of cyclophosphamide and fludarabine. In some embodiments, lymphodepletion comprises administering cyclophosphamide. In some embodiments, lymphodepletion comprises administering fludarabine. The dosage of the above treatments to be administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The scaling of dosages for human administration can be performed according to art-accepted practices. In such embodiments, the lymphodepletion comprising lymphodepletion with low dose chemotherapy.

[0177] In some embodiments, a single treatment regimen is required. In others, administration of one or more subsequent doses or treatment regimens can be performed. For example, after treatment biweekly for three months, treatment can be repeated once per month, for six months or a year or longer. In some embodiments, no additional treatments are administered following the initial treatment.

[0178] #14707301 v1 In some embodiments, the immune cells comprising the first polynucleotide and / or second polynucleotide described herein are administered as a monotherapy, i.e., another treatment for the condition is not concurrently administered to the subject. If necessary, immune cells compositions can also be administered multiple times at these dosages. The cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. Med. 30 319:1676, 1988).

[0179] In some embodiments, this disclosure describes a method of treating a subject having EGFR and / or EGFRvIII expressing cancer, the method comprising (i) administering (e.g., intravenously) an anti-CD20 antibody to the subject (e.g., rituximab) and administering to the central nervous system of a subject CAR-T cells (e.g., 6.0 x 106to 1.4 x 107CAR-T cells) comprising a polynucleotide encoding an EGFRvIII binding CAR and an EGFR-CD3 binding TEAM.

[0180] In some embodiments, this disclosure describes a method of treating a subject having glioblastoma multiforme (GBM) (e.g., an EGFR and / or EGFRvIII expressing cancer), the method comprising (i) administering (e.g., intravenously) an anti-CD20 antibody to the subject (e.g., rituximab) and administering to the central nervous system of a subject CAR-T cells (e.g., 6.0 x 106to 1.4 x 107CAR-T cells) comprising a polynucleotide encoding an EGFRvIII binding CAR and an EGFR-CD3 binding TEAM.

[0181] In some embodiments, this disclosure describes a method of treating a subject having mesothelin and / or FAP expressing cancer, the method comprising (i) administering (e.g., intravenously) an anti-CD20 antibody to the subject (e.g., rituximab) and administering to the subject CAR-T cells (e.g., 6.0 x 106to 1.4 x 107CAR-T cells) comprising a polynucleotide encoding an mesothelin binding CAR and an FAP-CD3 binding TEAM.

[0182] In some embodiments, this disclosure describes a method of treating a subject having pancreatic cancer, a lung cancer, an ovarian cancer, endometrial cancer, biliary cancer, gastric cancer, or mesothelioma, the method comprising (i) administering (e.g., intravenously) an anti- CD20 antibody to the subject (e.g., rituximab) and administering to the subject CAR-T cells (e.g., 6.0 x 106to 1.4 x 107CAR-T cells) comprising a polynucleotide encoding an mesothelin binding CAR and an FAP-CD3 binding TEAM.

[0183] In some embodiments, this disclosure describes a method of treating a CD70 expressing cancer (e.g., bladder cancer, breast invasive carcinoma, cervical cancer, cholangiocarcinoma, colorectal cancer, diffuse large B-cell lymphoma (DLBC), Esophagus, glioblastoma (GBM), head and neck cancer, low-grade gliomas (LGG), liver cancer, lung adeno cancer, melanoma, mesothelioma, ovarian cancer, pancreatic cancer, prostate cancer, sarcoma, stomach cancer,

[0184] #14707301 v1 testicular germ cell cancer, thymoma, thyroid cancer, uterine cancer, uveal melanoma, clear cell renal cell carcinoma (ccRCC), chromophobe renal cell carcinoma, papillary renal cell carcinoma (pRCC), acute myeloid leukemia, and adenoid cystic carcinoma (ACC) (Pan-Cancer Atlas 2018)), the method comprising (i) administering (e.g., intravenously) an anti-CD20 antibody to the subject (e.g., rituximab) and administering to the subject CAR-T cells (e.g., 6.0 x 106to 1.4 x 107CAR-T cells) comprising a polynucleotide encoding an CD70 binding CAR and an CD33- CD3 binding TEAM.

[0185] In some embodiments, this disclosure describes a method of treating a subject having CD70 and / or CD33 expressing cancer (e.g., a blood cancer), the method comprising (i) administering (e.g., intravenously) an anti-CD20 antibody to the subject (e.g., rituximab) and administering to the subject CAR-T cells (e.g., 6.0 x 106to 1.4 x 107CAR-T cells) comprising a polynucleotide encoding an CD70 binding CAR and an CD33-CD3 binding TEAM.

[0186] Decreasing Immune Response

[0187] In some aspects, this disclosure provide a method of decreasing the immune response (e.g., the adaptive immune response) of a subject to a therapeutic polypeptide (e.g., as described herein) that is secreted by an immune cell (e.g., as described herein) in the subject, the method comprising administering an anti-CD20 antibody and the immune cell (e.g., a CAR-T cell expressing and / or secreting a TEAM) to the subject. In some embodiments, the method comprises administering the anti-CD20 antibody before administering the immune cell. In some embodiments, the method comprises administering the anti-CD20 antibody after administering the immune cell. Decreases in the immune response may be determined in any suitable means including by comparing the immune response in the subject to an immune response of a subject who was not administered the anti-CD20 antibody. Metrics for measuring the immune response include, but are not limited to, measuring anti-therapeutic polypeptide antibodies (e.g., anti- TEAM antibodies) in the patient after administering the immune cells. In some embodiments, decreasing the immune response of a subject to a therapeutic peptide comprises decreasing anti- therapeutic peptide antibody amount in the patient (e.g., the blood or CSF of the patient) by at least 10%, (e.g., at least 25%, at least 50%, at least 100%, at least 250%, at least 500%, or at least 1000%).

[0188] In some embodiments, the antibody production against the therapeutic polypeptide and / or the CAR is reduced by at least 10%, 15%, 20%, 25%, 50%, 60%, 70% , 80%, 90%, or 100% compared to an otherwise identical method not comprising administering the anti-CD20 antibody to the subject. In some embodiments, the antibody production against the therapeutic

[0189] #14707301 v1 polypeptide and / or the CAR is reduced for at least 1 day, 2 days, 3 days, 4 days, 5 days, 10 days, 20 days, 30 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 110 days, 120 days, 130 days, 140 days, 150 days or more following administration of the anti-CD20 antibody to the subject.

[0190] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0191] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

[0192] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0193] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A,

[0194] #14707301 v1 and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0195] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively.

[0196] Sequences

[0197] #14707301 v1

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[0200] #14707301 v1

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[0202] #14707301 v1

[0203]

[0204] #14707301 v1

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[0206] #14707301 v1

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[0208] #14707301 v1

[0209] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms. The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

[0210] #14707301 v1 The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0211] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0212] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively.

[0213] #14707301 v1 EXAMPLES

[0214] Example 1: Intraventricular CARv3-TEAM-E T Cells for Patients with GBM

[0215] This Example describes a method to prevent anti-CAR and / or anti-TEAM (T-cell engaging antibody molecule) immunity in patients. In one of the human clinical trials, it was discovered that anti-CAR / TEAM immune responses occur and likely limit the feasibility of redosing patients with the same CAR / TEAM treatment. This Example describes treating patients with rituximab, an anti-CD20 monoclonal antibody, to prevent this immunity by depleting B cells that cause this immunity.

[0216] Two of the subjects in the safety arm of the CARv3-TEAM-3 clinical trial where retreated with CARv3-TEAM-3 when they had disease progression (patients 1 and 3). In both of these patients, it was found that the second infusion was clinically silent, unlike the first infusion. Whereas the first infusion was followed by fever, encephalopathy, and improvements in disease based on MRI scans, the second infusion was not associated with any symptoms or signs or imaging changes: no fever, no change in mental status, and no changes in imaging. Clinically, this appears to be most consistent with rapid rejection of the CARv3-TEAM-E investigational product. This was the initial rationale for adding lymphodepleting chemotherapy.

[0217] Now, with the addition of lymphodepleting chemotherapy (LDC), CAR T cell persistence has been improved to approximately 30-40 days post-infusion. However, in certain patients, a rapid decline in the number of CAR T cells in the cerebrospinal fluid (CSF) has been observed approximately 30-50 days post-infusion. In exploratory analyses, an assay was developed to measure patient antibodies to the TEAM molecule, a critical component of the CARv3-TEAM-3 product. Antibodies to the TEAM molecule coinciding with the decrease in CAR T cell numbers were detected, indicating that immune-mediated rejection is a major factor limiting CAR T cell persistence. These antibodies are present at especially high levels in the serum and CSF samples of the two patients who were re-treated without any lymphodepleting chemotherapy, and it was noticed that the antibodies also become detectable in patients who have received LDC prior to CARv3-TEAM-3 (see FIGs. 1-2). To address this challenge and enhance the durability of CAR T cell responses, we administer rituximab as part of the conditioning regimen for CARv3-TEAM-3 (FIG. 3). Rituximab is expected to effectively deplete the B cell population responsible for producing antibodies against the TEAM molecule, thereby mitigating the immune response and preventing rejection of the CAR T cells.

[0218] #14707301 v1 Example 2. Treatment with Rituximab Results in CARv3-TEAM-E T Cell Detection After Multiple Infusions

[0219] The number of CAR+ and TEAM+ cells in the cerebral spinal fluid (CSF) and peripheral blood (PB) of three rituximab-treated patients receiving CARv3-TEAM-3 treatment was evaluated after multiple infusions. CAR+ and CAR+ / TEAM+ cells were detected in the CSF after every re-infusion, which was not enhanced by treatment with rituximab (FIGs. 4A-4C). Additionally, CAR+ and CAR+ / TEAM+ cells were detected in the PB of 1 out of the 3 patients treated with rituximab (FIGs. 5A-5C). Importantly, in patients pre-treated with rituximab, no anti-TEAM or anti-CAR IgG antibodies were detected (FIGs. 6A-6C). Taken together, the data suggest that treatment with rituximab can abrogate antibody responses against CAR and TEAM proteins, allowing for re-dosing of patients with multiple CARv3-TEAM-E treatments.

[0220] #14707301 v1

Claims

CLAIMSWhat is claimed is:

1. A method of treating cancer in a subject, the method comprising administering to the subject:(a) an anti-CD20 antibody; and(b) an immune cell comprising a first polynucleotide encoding a therapeutic polypeptide, wherein the immune cell secretes the therapeutic polypeptide.

2. The method of claim 1, wherein the anti-CD20 antibody is rituximab.

3. The method of claim 2, wherein administering the rituximab comprises administering 300-500 mg / m2of rituximab.

4. The method of claim 3, wherein administering the rituximab comprises administering 375 mg / m2of rituximab.

5. The method of any one of claims 1-4, comprising administering the anti-CD20 antibody to the subject prior to administering the immune cell.

6. The method of any one of claims 1-5, comprising administering the anti-CD20 antibody to the subject after administering the immune cell.

7. The method of any one of claims 1-6, wherein the immune cell does not express CD20 or a fragment of CD20.

8. The method of any one of claims 1-7, further comprising lymphodepleting the subject.

9. The method of any one of claims 1-8, comprising, in sequential order: administering the anti-CD20 antibody to the subject; lymphodepleting the subject; and administering the immune cell to the subject.#14707301 v110. The method of claim 9, wherein the anti-CD20 antibody is administered to the subject 3- 15 days before lymphodepleting the subject.

11. The method of claim 10, wherein the anti-CD20 antibody is administered to the subject 5-10 days before lymphodepleting the subject.

12. The method of any one of claims 9-11, wherein lymphodepleting the subject occurs at least 5 days before administering the immune cell to the subject.

13. The method of claim 12, wherein lymphodepleting the subject occurs 5, 4 and 3 days before administering the immune cell to the subject.

14. The method of any one of claims 1-13, wherein the immune cell is a T cell.

15. The method of any one of claims 1-14, wherein the therapeutic polypeptide comprises an antibody that binds to a cancer antigen that is expressed by cancer cells of the subject.

16. The method of any one of claims 1-15, wherein the therapeutic polypeptide comprises a signal peptide.

17. The method of any one of claims 1-16, wherein the therapeutic polypeptide is a bispecific antibody.

18. The method of claim 17, wherein the bispecific antibody is a T-cell-engaging antibody molecule (TEAM).

19. The method of claim 17 or claim 18, wherein the bispecific antibody comprises a first binding domain that binds to CD3.

20. The method of claim 19, wherein the first binding domain that binds to CD3 comprises:(a) a variable heavy (VH) domain comprising three complementarity determining regions (CDR-H1, CDR-H2, and CDR-H3), wherein CDR-H1 comprises SEQ ID NO: 16, CDR-H2 comprises SEQ ID NO: 17, and CDR-H3 comprises SEQ ID NO: 18; and#14707301 v1(b) a variable light (VL) domain comprising three CDRs (CDR-L1, CDR-L2, and CDR- L3), wherein CDR-L1 comprises SEQ ID NO: 19, CDR-L2 comprises SEQ ID NO: 20, and CDR-L3 comprises SEQ ID NO: 21.

21. The method of any one of claims 17-20, wherein the bispecific antibody comprises a second binding domain that binds to a cancer antigen.

22. The method of claim 21, wherein the cancer antigen is CD70, CD33, FAP, mesothelin, EGFR, or EGFRvIII.

23. The method of claim 21 or 22, wherein the second binding domain binds to a cancer antigen that is expressed by cancer cells of the subject.

24. The method of claim 22 or 23, wherein the cancer antigen is EGFR.

25. The method of claim 24, wherein the second binding domain comprises:(a) a VH domain comprising three complementarity determining regions (CDR-H1, CDR-H2, and CDR-H3), wherein CDR-H1 comprises SEQ ID NO: 9, CDR-H2 comprises SEQ ID NO: 10, and CDR-H3 comprises SEQ ID NO: 11, and(b) a VL domain comprising three CDRs (CDR-L1, CDR-L2, and CDR-L3), wherein CDR-L1 comprises SEQ ID NO: 12, CDR-L2 comprises SEQ ID NO: 13, and CDR-L3 comprises SEQ ID NO: 14.

26. The method of any one of claim 1-25, wherein the immune cell comprises a second polynucleotide encoding a chimeric antigen receptor (CAR).

27. The method of claim 26, wherein the CAR comprises an extracellular antigen binding domain.

28. The method of claim 27, wherein the extracellular antigen binding domain binds to a cancer antigen.

29. The method of claim 27, wherein the cancer antigen is CD70, CD33, FAP, mesothelin,EGFR, or EGFRvIII.#14707301 v130. The method of any one of claims 27-29, wherein the extracellular antigen binding domain binds to a cancer antigen that is expressed by cancer cells of the subject.

31. The method of any one of claims 27-30, wherein the extracellular antigen binding domain binds to EGFRvIII.

32. The method of any one of claims 26-31, wherein the first polynucleotide encodes a TEAM comprising a CD3 binding domain and an EGFR binding domain; and the second polynucleotide encodes a CAR comprising an extracellular antigen binding domain that binds to EGFRvIII.

33. The method of any one of claims 27-32, wherein the extracellular antigen binding domain comprises a monoclonal antibody, a fragment antigen binding (Fab) fragment, F(ab')2 fragment, Fv fragment, and / or a single-chain variable fragments (scFv).

34. The method of any one of claims 32-33, wherein the extracellular antigen binding domain comprises:(a) a VH domain comprising three complementarity determining regions (CDR-H1, CDR-H2, and CDR-H3), wherein CDR-H1 comprises SEQ ID NO: 1, CDR-H2 comprises SEQ ID NO: 2, and CDR-H3 comprises SEQ ID NO: 3, and(b) a VL domain comprising three CDRs (CDR-L1, CDR-L2, and CDR-L3), wherein CDR-L1 comprises SEQ ID NO: 4, CDR-L2 comprises SEQ ID NO: 5, and CDR-L3 comprises SEQ ID NO: 6.

35. The method of any one of claims 26-30, wherein the first polynucleotide encodes a TEAM comprising and CD3 binding domain and a CD33 binding domain; and the CAR comprises an extracellular antigen binding domain that binds to CD70.

36. The method of any one of claims 26-30, wherein the first polynucleotide encodes a TEAM comprising and CD3 binding domain and a FAP binding domain; and the CAR comprises an extracellular antigen binding domain that binds to mesothelin.

37. The method of any one of claims 26-36, wherein the CAR comprises:#14707301 v1a transmembrane domain; and an intracellular signaling domain.

38. The method of claim 37, wherein the transmembrane domain is a transmembrane domain of an alpha chain of an immune cell receptor, beta chain of an immune cell receptor, zeta chain of an immune cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, 0X40, CD2, CD27, LFA-1 (CDlla, CD18), ICOS (CD278), 4-1BB (CD137), 4-1BBL, GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFI), CD160, CD19, IL2R beta, IL2R gamma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDlld, ITGAE, CD103, ITGAL, CDlla, LFA-1, ITGAM, CDllb, ITGAX, CDllc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, LylO8), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C.

39. The method of claim 38, wherein the transmembrane domain is a CD8 transmembrane domain.

40. The method of any one of claims 37-39, wherein the intracellular signaling domain comprises an intracellular signaling domain from 4- IBB, CD27, CD28, 0X4, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (0X40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM, and / or ZAP70.

41. The method of claim 40, wherein the intracellular signaling domain comprises a CD28 intracellular signaling domain.

42. The method of any one of claims 37-41, wherein the intracellular signaling domain further comprises an intracellular signaling domain from TCR-zeta, FcR-gamma, FcR-beta, CD3-gamma, CD3-theta, CD3-sigma, CD3-eta, CD3-epsilon, CD3-zeta, CD22, CD79a, CD79b, and / or CD66d.#14707301 v143. The method of claim 42, wherein the intracellular signaling domain comprises a CD28 intracellular signaling domain and a CD3-zeta intracellular signaling domain.

44. The method of any one of claims 27-43, wherein the extracellular antigen binding domain further comprises a leader sequence.

45. The method of any one of claims 26-44, wherein the CAR comprises an amino acid sequence having at least 85% identity to SEQ ID NO: 8.

46. The method of claim 45, wherein the CAR comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 8.

47. The method of claim 45, wherein the CAR comprises an amino acid sequence of SEQ ID NO: 8.

48. The method of any one of claims 26-47, wherein the first polynucleotide and the second polynucleotide are part of the same polynucleotide.

49. The method of claim 48, wherein the CAR and the TEAM are encoded as a polyprotein.

50. The method of claim 49, wherein the polyprotein comprises a cleavable peptide between the CAR and the TEAM.

51. The method of claim 50, wherein the polyprotein comprises an amino acid sequence having at least 85% identity to SEQ ID NO: 37.

52. The method of claim 50, wherein the polyprotein comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 37.

53. The method of claim 52, wherein the polyprotein comprises an amino acid sequence of SEQ ID NO: 37.

54. The method of any one of claims 1-53, wherein the cancer is glioblastoma.#14707301 v155. A method of treating glioblastoma in a subject, the method comprising administering to the subject, in order:(a) rituximab; and(b) a T cell comprising: a first polynucleotide encoding a TEAM comprising an EGFR binding domain and a CD3 binding domain; and a second polynucleotide encoding a CAR comprising an extracellular antigen binding domain that binds to EGFRvIII.

56. The method of claim 55, further comprising lymphodepleting the subject.

57. The method of claim 56, wherein lymphodepleting the subject comprises lymphodepleting prior to T cell administration.

58. The method of any one of claims 55-57, wherein the first polynucleotide and the second polynucleotide are comprised by a single nucleic acid.

59. The method of any one of claims 55-58, wherein the TEAM comprises an amino acid sequence of SEQ ID NO: 23.

60. The method of any one of claims 55-59, wherein the CAR comprises an amino acid sequence of SEQ ID NO: 8.

61. The method of any one of claims 55-60, wherein the CAR and the TEAM are encoded as a polyprotein.

62. The method of any one of claims 55-61, wherein the polyprotein comprises an amino acid sequence of SEQ ID NO: 37.

63. The method of any one of claims 55-62, wherein administering the rituximab comprises administering 300-500 mg / m2rituximab to the subject.

64. The method of claim 63, wherein administering the rituximab comprises administering 375 mg / m2rituximab to the subject.#14707301 v165. The method of any one of claims 1-64, wherein administering the immune cell comprises administering into the subject’s central nervous system.

66. The method of any one of claims 54-65, wherein the glioblastoma is EGFR positive.

67. The method of any one of claims 54-66, wherein the glioblastoma is EGFRvIII positive.

68. The method of any one of claims 54-66, wherein the glioblastoma is not EGFRvIII positive.

69. A method of decreasing antibody production against an immune cell therapy in a subject, the method comprising:(a) administering an anti-CD20 antibody to the subject; and(b) administering the immune cell therapy to the subject, wherein immune cells of the immune cell therapy comprise a first polynucleotide encoding a therapeutic polypeptide, wherein the immune cell secretes the therapeutic polypeptide.

70. A method of decreasing antibody production against an immune cell therapy in a subject having a solid tumor, the method comprising:(a) administering an anti-CD20 antibody to the subject; and(b) administering the immune cell therapy to the subject, wherein immune cells of the immune cell therapy comprise: a first polynucleotide encoding a therapeutic polypeptide; and / or a second polynucleotide encoding a chimeric antigen receptor (CAR) that comprises an extracellular binding domain that binds to an antigen of the solid tumor, wherein decreasing antibody production comprises decreasing antibody production against the therapeutic polypeptide and / or the CAR.

71. A method of treating a subject having a solid tumor, the method comprising:(a) administering an anti-CD20 antibody to the subject; and(b) administering an immune cell therapy to the subject, wherein immune cells of the immune cell therapy comprise: a first polynucleotide encoding a therapeutic polypeptide; and / or#14707301 v1a second polynucleotide encoding a chimeric antigen receptor (CAR) that comprises an extracellular binding domain that binds to an antigen of the solid tumor, wherein treating the subject includes decreasing antibody production against the therapeutic polypeptide and / or the CAR.

72. The method of any one of claims 69-71, wherein administering the anti-CD20 antibody comprises administering rituximab.

73. The method of any one of claims 69-72, further comprising lymphodepleting the subject.

74. The method of claim 73, wherein lymphodepleting the subject and administering the anti-CD20 antibody are performed before administering the immune cell therapy.

75. The method of any one of claims 69-74, wherein the antibody production against the therapeutic polypeptide and / or the CAR is reduced by at least 50%, 60%, 70% , 80%, 90%, or 100% compared to an otherwise identical method not comprising administering the anti-CD20 antibody to the subject.

76. The method of claim 75, wherein the antibody production against the therapeutic polypeptide and / or the CAR is reduced for at least 1 day, 5 days, 10 days, 20 days, 30 days, 50 days, 60 days, 70 days, 80 days or more following administration of the anti-CD20 antibody to the subject.#14707301 v1

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