Methods of central nervous system administration of compositions comprising a cell therapy and dimethyl sulfoxide

Abstract

This disclosure describes, in part, methods of administering a pharmaceutical composition comprising a cell therapy and DMSO to the central nervous system. These methods are in part based on the surprising discovery that administering a pharmaceutical composition comprising a cell therapy and DMSO into the central nervous system did not induce DMSO-associated measurable neurotoxicity in human patients.

Description

[0001] METHODS OF CENTRAL NERVOUS SYSTEM ADMINISTRATION OF COMPOSITIONS COMPRISING A CELL THERAPY AND DIMETHYL SULFOXIDE

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit under 35 U.S.C. §119(e) to U.S. Provisional Application Nos. 63 / 587,626, filed October 3, 2023, entitled “Methods of Central Nervous System Administration of Compositions Comprising a Cell Therapy and Dimethyl Sulfoxide”, and 63 / 600,569, filed November 17, 2023, entitled “Methods of Central Nervous System Administration of Compositions Comprising a Cell Therapy and Dimethyl Sulfoxide”, 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 (M105370049WO00-SEQ-ARM.xml; Size: 38,419 bytes; and Date of Creation: October 1, 2024) is herein incorporated by reference in its entirety.

[0006] BACKGROUND

[0007] Cell therapies (e.g., CAR-T cell therapies) have revolutionized treatment of certain types of cancers like leukemia and lymphoma but have had relatively little success in treating solid tumors including brain tumors. Cell therapies that are for human administration are generally prepared at a specialized facility then the cells are frozen in cryopreservatives (e.g., dimethyl sulfoxide (DMSO)) for transport to a medical facility where the cell therapy is administered to the patient. Many cryopreservatives, including DMSO, are known to be toxic to humans, but are needed to maintain cell integrity during transport to the medical facility. Upon arrival at the medical facility, cell therapies are typically thawed and administered to the patient intravenously. Intravenous administration dilutes the cryopreservatives in the blood stream and in turn decreases cryopreservative toxicity to an acceptable level. Additionally, cryopreservatives like DMSO can be removed from the blood stream by the lungs and exhaled by the patient, which may further decrease DMSO toxicity.

[0008] SUMMARY

[0009] The instant application addresses a central challenge in administering cell therapies into the central nervous system (e.g., by intraventricular infusion), namely cryopreservatives (like DMSO) that are needed in cell therapy formulations, but are neurotoxic and are not significantly diluted after administration (compared to intravenous administration into the blood). Akel S et al., Cytotherapy. 2023 Sep 6:S1465-3249(23)01035-6. For example, a recent study reported a pediatric patient who developed seizures and brain abnormalities after DMSO-cryopreserved stem cell intravenous administration. Ataseven E et al. Journal of pediatric hematology / oncology 39.5 (2017): e297-e299. This indicates that cell therapy administration into the human brain is expected to be neurotoxic. In another study, DMSO administration in mice produced widespread cell apoptosis in the brain. Hanslick JL Neurobiol Dis. 2009 Apr;34(l): 1-10. Because of this, the art teaches that cell therapies administered to the human central nervous system should be washed to remove DMSO prior to administration. Akel et al. 2023. However, washing has known disadvantages including potentially changing the composition of the cell therapy such that it (1) does not meet regulatory requirements or (2) requires additional measurements of cell therapy viability to meet regulatory requirements.

[0010] Surprisingly, the inventors discovered that pharmaceutical compositions comprising cell therapies and DMSO can be administered into the human brain without causing noticeable neurotoxicity. Specifically, in some embodiments, the inventors determined that diluting the cell therapy formulation comprising about 5% DMSO by volume (v / v) by about 20-fold prior to administration into the central nervous system was sufficient to reduce and / or eliminate DMSO neurotoxicity in human patients. This is further discussed in the examples.

[0011] In some aspects, this disclosure describes a method of administering a cell therapy to a human subject, the method comprising administering a pharmaceutical composition that comprises the cell therapy and dimethyl sulfoxide (DMSO) into the subject’s central nervous system.

[0012] In some aspects, this disclosure describes a method of administering a cell therapy to a subject, the method comprising: (i) obtaining a cell therapy formulation comprising the cell therapy and DMSO; (ii) diluting the cell therapy formulation to produce a pharmaceutical composition comprising less than or equal to 0.5% DMSO (v / v); and (iii) administering the pharmaceutical composition into the subject’s central nervous system. In some embodiments, the method further comprises thawing the cell therapy formulation prior to diluting the cell therapy formulation.

[0013] In some embodiments, administering comprises administering the pharmaceutical composition into the subject’s brain. In some embodiments, diluting the cell therapy comprises diluting by 20-fold to 40-fold (v / v). In some embodiments, administering the pharmaceutical composition comprises intracranial administering. In some embodiments, administering the pharmaceutical composition comprises intraventricular administering. In some embodiments, administering the pharmaceutical composition comprises administering into cerebral spinal fluid of the subject. In some embodiments, administering further comprising administering into the cerebral spinal fluid of the spine of the subject. In some embodiments, administering the pharmaceutical composition comprises intrathecal administering. In some embodiments, administering comprises administering to a tumor in the central nervous system of the subject. In some embodiments, administering comprises administering into a tumor in the central nervous system of the subject.

[0014] In some embodiments, the pharmaceutical composition comprises less than 0.5% by volume (v / v) DMSO. In some embodiments, the pharmaceutical composition comprises less than 0.25% (v / v) DMSO. In some embodiments, the method does not comprise washing the cell therapy to remove DMSO. In some embodiments, the pharmaceutical composition comprises more than 0.025% (v / v) DMSO. In some embodiments, the pharmaceutical composition comprises 0.025-0.5% (v / v) DMSO. In some embodiments, the pharmaceutical composition comprises 0.03-0.5% (v / v) DMSO. In some embodiments, the pharmaceutical composition comprises 0.05-0.25% (v / v) DMSO. In some embodiments, the pharmaceutical composition comprises 0.1-0.3% (v / v) DMSO. In some embodiments, the pharmaceutical composition comprises 0.12-.0.25% (v / v) DMSO.

[0015] In some embodiments, the pharmaceutical composition comprises an excipient. In some embodiments, the excipient comprises human serum albumin. In some embodiments, the pharmaceutical composition comprises a cryopreservative. In some embodiments, the cryopreservative comprises CryoStor® 10 (used interchangeably with CryoStor® CS10). In some embodiments, the cryopreservative is DMSO. In some embodiments, the pharmaceutical composition comprises Elliott’s B solution. In some embodiments, diluting comprises diluting using Elliott’s B solution. In some embodiments, the pharmaceutical composition is 5 mL to 15 mL in volume. In some embodiments, the pharmaceutical composition is 10 mL in volume.

[0016] In some embodiments, the cell therapy comprises an immune cell therapy. In some embodiments, the immune cell therapy comprises a T cell therapy or a natural killer (NK) cell therapy. In some embodiments, the T cell therapy comprises a CAR-T cell therapy. In some embodiments, the CAR-T cell therapy comprises a polynucleotide encoding a CAR comprising an antigen binding domain that binds to EGFRvIII.

[0017] In some embodiments, the 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: 2, CDR-H2 comprises SEQ ID NO: 3, and CDR-H3 comprises SEQ ID NO: 4, and (ii) a VL domain comprising three CDRs (CDR-L1, CDR-L2, and CDR-L3), wherein CDR-L1 comprises SEQ ID NO: 5, CDR-L2 comprises SEQ ID NO: 6, and CDR-L3 comprises SEQ ID NO: 7. In some embodiments, the CAR comprises an amino acid sequence of SEQ ID NO: 11.

[0018] In some embodiments, the CAR-T cell therapy comprises a polynucleotide encoding a T cell engaging antibody molecule (TEAM). In some embodiments, the CAR-T cell therapy comprises a polynucleotide encoding a T cell engaging antibody molecule (TEAM) comprising an anti-EGFR antibody and an anti-CD3 antibody.

[0019] In some embodiments, the anti-EGFR antibody comprises: (i) a VH domain comprising three complementarity determining regions (CDR-H1, CDR-H2, and CDR-H3), wherein CDR-H1 comprises SEQ ID NO: 26, CDR-H2 comprises SEQ ID NO: 27, and CDR-H3 comprises SEQ ID NO: 28, and (ii) a VL domain comprising three CDRs (CDR-L1, CDR-L2, and CDR-L3), wherein CDR-L1 comprises SEQ ID NO: 29, CDR-L2 comprises SEQ ID NO: 30, and CDR-L3 comprises SEQ ID NO: 31.

[0020] In some embodiments, the anti-CD3 antibody comprises: (i) a VH domain comprising three complementarity determining regions (CDR-H1, CDR-H2, and CDR-H3), wherein CDR-H1 comprises SEQ ID NO: 32, CDR-H2 comprises SEQ ID NO: 33, and CDR-H3 comprises SEQ ID NO: 34, and (ii) a VL domain comprising three CDRs (CDR-L1, CDR- L2, and CDR-L3), wherein CDR-L1 comprises SEQ ID NO: 35, CDR-L2 comprises SEQ ID NO: 36, and CDR-L3 comprises SEQ ID NO: 37.

[0021] In some embodiments, the TEAM comprises an amino acid sequence of SEQ ID NO: 38.

[0022] In some embodiments, the cell therapy in an autologous cell therapy.

[0023] BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0025] FIGs. 1A-1C relate to a CAR- TEAM construct and expression of said construct in cells. FIG. 1A shows a schematic of a CAR- TEAM encoding molecule with a truncated CD 19 (tCD19) molecule which was used as a surface marker for transduction. FIG. IB shows representative flow cytometric analysis on the cell product from Patient 1, with gating for CAR-positive and TEAM-positive cells among all CD3+T cells. FIG. 1C shows representative gating for TEAM-positive cells among CAR-positive and CAR-negative T-cell populations.

[0026] FIGs. 2A-2E correspond to treatment events and outcome related to Patient 1. FIG. 2A summarizes the timeline of events for Patient 1 while enrolled in the Phase I clinical study. FIG. 2B shows axial contrast-enhanced Tl-weighted images (which enhances signal of fatty tissue) for Patient 1 immediately post-resection (left), pre-infusion (center), and postinfusion (right). Numbers in each panel indicate days relative to infusion (day 0). FIG. 2C shows EGFRvIII and EGFR copy number over time, derived from extracellular vesicle (EV) RNA present in CSF. FIG. 2D shows respective copy numbers isolated from peripheral blood EV RNA at pre- and post-treatment time points, and FIG. 2E shows post-hoc analysis of EGFR copy number by fluorescence in situ hybridization (FISH).

[0027] FIGs. 3A-3C relate to treatment workflow and treatment outcomes for Patients 2 and 3. FIG. 3A shows a modified workflow used for Patient 2 and 3, which enabled concomitant craniotomy, tissue sampling and Ommaya placement during a single surgery. FIG. 3B shows longitudinal radiographic assessment after CAR- TEAM cell infusion of Patient 2. FIG. 3C shows pre- and post-infusion axial Tl-weighted post-contrast images for Patient 3, demonstrating tumor regression at D +5 following treatment. Numbers in each panel indicate days relative to infusion (day 0).

[0028] FIGs. 4A-4B related to temperature response in Patients 1-3 following CAR- TEAM cell infusion. FIG. 4A shows temperature curves for all patients which peaked within 2 days of infusion and normalized over time, and FIG. 4B shows management of body temperature via treatment with the interleukin-1 receptor (IL-1R) antagonist anakinra.

[0029] FIG. 5 shows expression of inflammatory markers C-reactive protein (CRP) and ferritin in cerebrospinal fluid of Patients 1-3 following CAR- TEAM cell infusion.

[0030] FIG. 6 shows counts and differentials of cells in cerebrospinal fluid of Patients 1-3 following infusion of CAR-TEAM cells. FIGs. 7A-7C relate to the presence CAR-positive and TEAM-positive cells and inflammatory cytokines in the cerebrospinal fluid (CSF) and peripheral blood of Patients 1-3. FIG. 7A shows quantification by vector copy number (VCN) and flow cytometry of CARpositive and TEAM-positive T cells in all three patients, which temporarily persisted in the CSF after infusion but declined by week 4. FIG. 7B shows the presence of inflammatory cytokines in the CSF of patients over the same period. FIG. 7C shows detection of CARpositive and TEAM-positive cells in the peripheral blood, which was transient.

[0031] DETAILED DESCRIPTION

[0032] This application, in part, is related to the surprising discovery that pharmaceutical compositions comprising cell therapies and DMSO can be administered to the human central nervous system without resulting in noticeable DMSO induced neurotoxicity. In some aspects, this disclosure describes a method of administering a cell therapy to a human subject, the method comprising administering a pharmaceutical composition that comprises the cell therapy and dimethyl sulfoxide (DMSO) into the subject’s central nervous system. In some embodiments, this disclosure describes a method of administering a cell therapy to a subject, the method comprising: (i) obtaining a cell therapy formulation comprising the cell therapy and DMSO; (ii) diluting the cell therapy formulation to produce a pharmaceutical composition comprising less than or equal to 0.5% DMSO (v / v); and (iii) administering the pharmaceutical composition into the subject’s central nervous system.

[0033] Administering

[0034] “Administering”, as used herein, refers to contacting a subject (e.g., a central nervous system of a subject) with a cell therapy (e.g., a pharmaceutical composition). In some embodiments, administering comprises injecting or infusing a pharmaceutical composition 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.

[0035] 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.

[0036] “Central nervous system”, as used herein, refers to 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 space between membranes of the meninges (e.g., the subarachnoid space), and brain or spinal tumors of the central nervous system (e.g., glioblastoma).

[0037] In some embodiments, administering a pharmaceutical composition into the central nervous system comprises intracranial administering. In some embodiments, administering the pharmaceutical composition into the central nervous system comprises intraventricular administering. In some embodiments, administering the pharmaceutical composition into the central nervous system comprises administering into cerebral spinal fluid of the subject. In some embodiments, administering further comprises administering into the cerebral spinal fluid of the spine of the subject. In some embodiments, administering the pharmaceutical composition into the central nervous system comprises intrathecal administering. In some embodiments, administering the pharmaceutical composition into the central nervous system comprises administering to a tumor in the central nervous system of the subject. In some embodiments, administering the pharmaceutical composition into the central nervous system comprises administering into a tumor in the central nervous system of the subject. In some embodiments, administering the pharmaceutical composition into the central nervous system comprises administering using an Ommaya Reservoir.

[0038] Pharmaceutical Compositions

[0039] In some embodiments, the method described herein comprises administering a pharmaceutical composition into a subject’s central nervous system. A “pharmaceutical composition”, as used herein, refers to a composition comprising an active agent (e.g., a cell therapy as described herein) and a pharmaceutically acceptable excipient for administration into the central nervous system. Pharmaceutically acceptable excipients for administration into the central nervous system include, but are not limited to, human serum albumin HSA), Elliott’s B Solution, preservative-free saline, autologous CSF, or a combination thereof. In some embodiments, a pharmaceutical composition may comprise a cell therapy, Elliott’s B Solution, and HSA. In some embodiments, a cell formulation described herein may be diluted with Elliott’s B solution, preservative-free saline, autologous CSF or a combination thereof to produce a pharmaceutical composition. In some embodiments, the pharmaceutical composition further comprises a cryopreservative (e.g., DMSO).

[0040] In some embodiments, the pharmaceutical composition comprises a cryopreservative. “A cryopreservative”, as used herein, refers to a composition that when mixed with a cell therapy (e.g., CAR-T cells) prior to freezing decreases cell death as compared to freezing the cell therapy without using a cryopreservative. Cryopreservatives for cell therapies are known in the art, e.g., as described in Murray, K.A., Gibson, M.I. Nat Rev Chem 6, 579-593 (2022). doi. org / 10.1038 / s41570-022-00407-4. In some embodiments, the cytoprotective agent comprises glycerol. In some embodiments, the cryopreservative comprises DMSO. In some embodiments, the cryopreservative comprises CryoStor® 10. In some embodiments, the cryopreservative has been reported to have adverse side effects upon administration (e.g., upon central nervous system administration).

[0041] In some embodiments, the method comprises administering into a subject’s central nervous system a pharmaceutical composition comprising DMSO and a cell therapy. In some embodiments, the pharmaceutical composition comprises less than 0.5% by volume (v / v) DMSO (e.g., less than 0.5% (v / v) DMSO, less than 0.35% (v / v) DMSO, less than 0.30% (v / v) DMSO, less than 0.25% (v / v) DMSO, less than 0.20% (v / v) DMSO, less than 0.15% (v / v) DMSO, less than 0.10% (v / v) DMSO, or less than 0.05% (v / v) DMSO). In some embodiments, the pharmaceutical composition comprises more than 0.01% (v / v) DMSO (e.g., more than 0.02% (v / v) DMSO or more than 0.025% (v / v) DMSO), and less 0.5% (v / v) DMSO (e.g., less than 0.40% (v / v) DMSO, less than 0.35% (v / v) DMSO, less than 0.30% (v / v) DMSO, less than 0.25% (v / v) DMSO, less than 0.20% (v / v) DMSO, less than 0.15% (v / v) DMSO, less than 0.10% (v / v) DMSO, or less than 0.05% (v / v) DMSO). In some embodiments, the pharmaceutical composition comprises less than 0.25% (v / v) DMSO. In some embodiments, the pharmaceutical composition comprises at least 0.01% (v / v) DMSO (e.g., at least, 0.01% (v / v) DMSO, at least 0.025% (v / v) DMSO, at least 0.05% (v / v) DMSO, at least 0.1% (v / v) DMSO, at least 0.15% (v / v) DMSO, at least 0.25% (v / v) DMSO, least 0.3% (v / v) DMSO, at least 0.35% (v / v) DMSO, least 0.4% (v / v) DMSO, at least 0.45% (v / v) DMSO), but no more than 0.5% (v / v) DMSO.

[0042] In some embodiments, the method comprises administering into a subject’s central nervous system a pharmaceutical composition comprising a cell therapy and 0.025-0.25% (v / v) DMSO. In some embodiments, the method comprises administering into a subject’s central nervous system a pharmaceutical composition comprising a cell therapy and 0.026- 0.25% (v / v) DMSO. In some embodiments, the method comprises administering into a subject’s central nervous system a pharmaceutical composition comprising a cell therapy and 0.027-0.25% (v / v) DMSO. In some embodiments, the method comprises administering into a subject’s central nervous system a pharmaceutical composition comprising a cell therapy and 0.028-0.25% (v / v) DMSO. In some embodiments, the method comprises administering into a subject’s central nervous system a pharmaceutical composition comprising a cell therapy and 0.029-0.25% (v / v) DMSO. In some embodiments, the method comprises administering into a subject’s central nervous system a pharmaceutical composition comprising a cell therapy and 0.030-0.25% (v / v) DMSO. In some embodiments, the method comprises administering into a subject’s central nervous system a pharmaceutical composition comprising a cell therapy and 0.040-0.25% (v / v) DMSO. In some embodiments, the method comprises administering into a subject’s central nervous system a pharmaceutical composition comprising a cell therapy and 0.050-0.25% (v / v) DMSO. In some embodiments, the method comprises administering into a subject’s central nervous system a pharmaceutical composition comprising a cell therapy and 0.075-0.25% (v / v) DMSO. In some embodiments, the method comprises administering into a subject’s central nervous system a pharmaceutical composition comprising a cell therapy and 0.1-0.25% (v / v) DMSO. In some embodiments, the method comprises administering into a subject’s central nervous system a pharmaceutical composition comprising a cell therapy and 0.1-0.25% (v / v) DMSO. In some embodiments, the method comprises administering into a subject’s central nervous system a pharmaceutical composition comprising a cell therapy and 0.12-0.25% (v / v) DMSO. In some embodiments, the method comprises administering into a subject’s central nervous system a pharmaceutical composition comprising a cell therapy and 0.125-0.25% (v / v) DMSO. In some embodiments, the method comprises administering into a subject’s central nervous system a pharmaceutical composition comprising a cell therapy and 0.05-0.30% (v / v) DMSO. In some embodiments, the method comprises administering into a subject’s central nervous system a pharmaceutical composition comprising a cell therapy and 0.1-0.30% (v / v) DMSO. In some embodiments, the method comprises administering into a subject’s central nervous system a pharmaceutical composition comprising a cell therapy and 0.05-0.25% (v / v) DMSO. In some embodiments, the method comprises administering into a subject’s central nervous system a pharmaceutical composition comprising a cell therapy and 0.1-0.25% (v / v) DMSO. In some embodiments, the method comprises administering into a subject’s central nervous system a pharmaceutical composition comprising a cell therapy and 0.12-0.25% (v / v) DMSO. In some embodiments, the method comprises administering into a subject’s central nervous system a pharmaceutical composition comprising a cell therapy and 0.125-0.25% (v / v) DMSO.

[0043] In some embodiments, the method comprises administering to a subject’s central nervous system a pharmaceutical composition comprising a cell therapy and 0.025-0.50% (v / v) DMSO. In some embodiments, the method comprises administering to a subject’s central nervous system a pharmaceutical composition comprising a cell therapy and 0.026- 0.50% (v / v) DMSO. In some embodiments, the method comprises administering to a subject’s central nervous system a pharmaceutical composition comprising a cell therapy and 0.027-0.50% (v / v) DMSO. In some embodiments, the method comprises administering to a subject’s central nervous system a pharmaceutical composition comprising a cell therapy and 0.028-0.50% (v / v) DMSO. In some embodiments, the method comprises administering to a subject’s central nervous system a pharmaceutical composition comprising a cell therapy and 0.029-0.50% (v / v) DMSO. In some embodiments, the method comprises administering to a subject’s central nervous system a pharmaceutical composition comprising a cell therapy and 0.030-0.50% (v / v) DMSO. In some embodiments, the method comprises administering to a subject’s central nervous system a pharmaceutical composition comprising a cell therapy and 0.040-0.50% (v / v) DMSO. In some embodiments, the method comprises administering to a subject’s central nervous system a pharmaceutical composition comprising a cell therapy and 0.050-0.50% (v / v) DMSO. In some embodiments, the method comprises administering to a subject’s central nervous system a pharmaceutical composition comprising a cell therapy and 0.075-0.50% (v / v) DMSO. In some embodiments, the method comprises administering to a subject’s central nervous system a pharmaceutical composition comprising a cell therapy and 0.1-0.50% (v / v) DMSO. In some embodiments, the method comprises administering to a subject’s central nervous system a pharmaceutical composition comprising a cell therapy and 0.1-0.50% (v / v) DMSO. In some embodiments, the method comprises administering to a subject’s central nervous system a pharmaceutical composition comprising a cell therapy and 0.12-0.50% (v / v) DMSO. In some embodiments, the method comprises administering to a subject’s central nervous system a pharmaceutical composition comprising a cell therapy and 0.125-0.50% (v / v) DMSO.

[0044] In some embodiments, the pharmaceutical composition comprises 10 million to 80 million cells of the cell therapy. In some embodiments, the pharmaceutical composition comprises 7 million to 13 million cells of the cell therapy. In some embodiments, the pharmaceutical composition comprises 10 million + / - 30% cells of the cell therapy.

[0045] In some embodiments, the pharmaceutical composition comprises Elliott’s B solution, a cell therapy, human serum albumin, and 0.1-0.30% (v / v) DMSO. In some embodiments, the pharmaceutical composition comprises 94-98% (v / v) Elliott’s B solution, a cell therapy, 0.3-0.6% (w / v) human serum albumin, and 0.1-0.25% (v / v) DMSO. In some embodiments, the pharmaceutical composition comprises Elliott’s B solution, a cell therapy, human serum albumin, and 0.1-0.30% (v / v) DMSO. In some embodiments, the pharmaceutical composition comprises 94-98% (v / v) Elliott’s B solution, a cell therapy, 0.3-0.6% (w / v) human serum albumin, and 0.05-0.25% (v / v) DMSO. In some embodiments, the pharmaceutical composition comprises 94-98% (v / v) Elliott’s B solution, a cell therapy, 0.3- 0.6% (w / v) human serum albumin, and less than 0.25% (v / v) DMSO.

[0046] In some embodiments, the pharmaceutical composition comprises 94-98% (v / v) Elliott’s B solution, 10 million + / - 30% cells of the cell therapy per mL of the pharmaceutical composition, 0.3-0.6% (w / v) human serum albumin, and 0.1-0.25% (v / v) DMSO. In some embodiments, the pharmaceutical composition comprises Elliott’s B solution, 10 million + / - 30% cells of the cell therapy per mL of the pharmaceutical composition, human serum albumin, and 0.1-0.30% (v / v) DMSO. In some embodiments, the pharmaceutical composition comprises 94-98% (v / v) Elliott’s B solution, 10 million + / - 30% cells of the cell therapy per mL of the pharmaceutical composition, 0.3-0.6% (w / v) human serum albumin, and 0.05-0.25% (v / v) DMSO. In some embodiments, the pharmaceutical composition comprises 94-98% (v / v) Elliott’s B solution, 10 million + / - 30% cells of the cell therapy per mL of the pharmaceutical composition, 0.3-0.6% (w / v) human serum albumin, and less than 0.25% (v / v) DMSO.

[0047] In some embodiments, the pharmaceutical composition is 5-15 mL, 7-13, 8-12, or 9- 11 mL in volume. In some embodiments, the pharmaceutical composition is less than or equal to 15 mL in volume. In some embodiments, the pharmaceutical composition is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mL in volume. In some embodiments, the pharmaceutical composition is 9-11 mL in volume. In some embodiments, the pharmaceutical composition is 10 mL in volume.

[0048] In some embodiments, a method of administering a cell therapy to a subject comprises producing the pharmaceutical composition. In some embodiments, the method comprises obtaining a cell therapy formulation and diluting the cell therapy formulation to produce a pharmaceutically composition. The cell therapy formulation may comprise the cell therapy, a cryopreservative (e.g., comprising DMSO) and an excipient (e.g., human serum albumin). In some embodiments, the method comprises obtaining a cell therapy formulation that was prepared at a separate facility, frozen, and sent to the location of patient administration (e.g., a hospital). In some embodiments, the method comprises thawing the frozen cell formulation and then diluting the thawed cell formulation using a pharmaceutically acceptable excipient (e.g., Elliott’s B Solution) for central nervous system administration. In some embodiments, the method comprises diluting the cell therapy formulation by at least 15-fold (e.g., at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, or at least 50-fold). In some embodiments, the method comprises diluting the cell therapy formulation by at least 20-fold. In some embodiments, the method comprises diluting the cell therapy formulation by 10-50 fold. In some embodiments, the method comprises diluting the cell therapy formulation by 20-40 fold. In some embodiments, the cell therapy formulation is about 200-600 pL (e.g., about 300 pL), and is diluted using about 10 mL of pharmaceutically acceptable excipient for central nervous system administration (e.g., Elliott’s B solution). In some embodiments, after diluting, the method comprises administering the pharmaceutical composition into the central nervous system of the subject (e.g., as described herein).

[0049] Cell Therapy

[0050] A “cell therapy” refers to one or more cells that are administered to a subject for the purpose of treating a disease, a disorder, or a syndrome of the subject. In some embodiments, a cell therapy comprises cells that have been modified to comprise a therapeutic molecule (e.g., cell transfected with a polynucleotide encoding a chimeric antigen receptor (CAR)) and cells that have not been modified to comprise a therapeutic molecule (e.g., cells that were not transfected with a polynucleotide encoding a CAR). For example, 30% of cells in a cell therapy may comprise a polynucleotide encoding a CAR and 70% cells in a cell therapy may not comprise a polynucleotide encoding a CAR. In some embodiments, the cell therapy comprises a T cell therapy, a chimeric antigen receptor (CAR)-T cell therapy, an engineered t-cell receptor (TCR) therapy, a natural killer (NK) cell therapy, or a CAR-NK cell therapy. In some embodiments, the cell therapy comprises stem-cell therapy. In some embodiments, the cell therapy comprises non-stem cell therapy. In some embodiments, the cell therapy comprises an autologous cell therapy. In some embodiments, the cell therapy comprises an allogeneic cell therapy. In some embodiments, the cell therapy comprises unicellular cell therapy. In some embodiments, the cell therapy comprises multicellular cell therapy. In some embodiments, the cell therapy comprises an immune cell (e.g., a T cell, a NK cell or a macrophage) that has been modified to bind to a diseased cell (e.g., a cancer cell). In some embodiments, the cells of the cell therapy secrete a therapeutic protein (e.g., a TEAM. For example, cell may secrete a bispecific antibody that binds to a T cell and a cancer cell. In some embodiments, the cell therapy comprises CAR-T cells (see section Chimeric antigen receptor (CAR) T cells for additional details about CAR-T cells).

[0051] In some embodiments, the cell therapy comprises one or more cells administered to treat a disease. In some embodiments, the disease is a cancer. In some embodiments, the disease is a central nervous system disease. In some embodiments the cancer is a central nervous system cancer. In some embodiments, the central nervous system cancer is a low- grade infiltrative astrocytoma, oligodendroglioma, anaplastic glioma, glioblastoma (e.g., glioblastoma multiforme (GBM)), medulloblastoma, supratentorial primitive neuroectodermal tumor, and secondary brain metastases. In some embodiments, the cancer is astrocytoma, oligodendroglioma, glioblastoma (e.g., glioblastoma multiforme (GBM)), diffuse astrocytoma, ganglioglioma, dysembryoplastic neuroepithelial tumor, glioneuronal tumor, gangliocytoma, multinodular and vacuolating neuronal tumor, neurocytoma, ependymoma, papilloma, carcinoma, medulloblastoma, rhabdoid tumor, cribriform neuroepithelial tumor, embryonal tumor, CNS neuroblastoma, pineal tumors, cranial and paraspinal nerve tumors, or meningioma. In some embodiments, the disease is glioblastoma.

[0052] In some embodiments, the cell therapy comprises one or more cells administered to treat a disorder. In some embodiments, the disorder is Parkinson's disease, multiple sclerosis, ALS, or stroke. In some embodiments, the disorder is an autoimmune disorder. In some embodiments, the autoimmune disorder is autoimmune encephalitis, autoimmune-related epilepsy, central nervous system (CNS) vasculitis, Hashimoto’s encephalopathy (steroid- responsive encephalopathy), neuromyelitis optica, optic neuritis, neurosarcoidosis, NeuroBehcet’s disease or cerebral lupus.

[0053] Chimeric Antigen Receptor (CAR) T cells

[0054] In some aspects, the method comprises administering a CAR-T cell into the central nervous system of a subject. As used herein, a "CAR-T cell" or "CAR-T" refers to a T cell that expresses a CAR. 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. 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.

[0055] 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.

[0056] A CAR places a chimeric 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 chimeric 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 chimeric antigen binding domain includes a ligand that specifically binds an antigen expressed on a cell to be targeted for a T cell response.

[0057] In some embodiments, the CAR excludes 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.

[0058] Antigen Binding Domain

[0059] As used herein, the term "antigen binding domain" refers to a polypeptide found on the outside of the cell that is sufficient to facilitate binding to a target. The extracellular target binding domain will specifically bind to its binding partner, i.e., the target. As non-limiting examples, the antigen binding domain can include an antigen domain of an antibody or antibody reagent, or 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.

[0060] 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. To target Tregs, antibody reagents can be targeted against, e.g., Glycoprotein A Repetitions Predominant (GARP), latency-associated peptide (LAP), CD25, CTLA-4, ICOS, TNFR2, GITR, 0X40, 4-1BB, and LAG-3.

[0061] In some embodiments, the CAR comprises an antigen binding domain that binds to an antigen expressed on a central nervous system cancer or tumor. For example, in some embodiments, the CAR comprises an antigen binding domain that binds to EGFRvIII, IL13Ra2, HER2, B7-H3, B7-H1, CD147, GD2, CAIX, CD70, CSPG4, EphAl, TROP2, or Survivin.

[0062] In some embodiments, the CAR comprises an antigen binding domain that binds to EGFRvIII. In some embodiments, the 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: 2, CDR-H2 comprises SEQ ID NO: 3, and CORED comprises SEQ ID NO: 4, and (ii) a VL domain comprising three CDRs (CDR-L1, CDR- L2, and CDR-L3), wherein CDR-L1 comprises SEQ ID NO: 5, CDR-L2 comprises SEQ ID NO: 6, and CDR-L3 comprises SEQ ID NO: 7. In some embodiments, the antigen binding domains comprises an amino acid sequence of SEQ ID NO: 8.

[0063] Hinge and Transmembrane Domains

[0064] In some embodiments, the CAR polypeptide further comprises a transmembrane domain, e.g., a hinge / transmembrane domain, which joins the 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 antigen binding 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 domain. In some embodiments, the CAR comprises a polynucleotide encoding a 4 IBB intracellular domain.

[0065] 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.

[0066] As used herein, "transmembrane domain" (TM domain) refers to the portion of the CAR that fuses the extracellular binding portion, in some embodiments 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.

[0067] 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.

[0068] 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 (LIGHTR), SLAMF7, NKp80 (KLRFI), CD 160, CD19, IL2R beta, IL2R gamma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD l id, ITGAE, CD 103, ITGAL, CDl la, LFA-1, IT GAM, CD 11b, ITGAX, CD 11c,

[0069] ITGB1, CD29, ITGB2, CD 18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD 160 (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.

[0070] 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: 9, 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.

[0071] 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.

[0072] In some embodiments, the CD8 hinge and transmembrane sequence comprises the amino acid sequence of SEQ ID NO: 9. In some embodiments, the CD8 hinge and 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: 9. Co-stimulatory Domains

[0073] 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 "costimulatory domain" refers to an intracellular signaling domain of a co-stimulatory molecule. Co-stimulatory molecules are cell surface molecules other than antigen receptors or Fe 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 4-1BB, CD27, CD28, or 0X40. In some embodiments, a 4-1BB co- stimulatory domain can be used (see, e.g., below and SEQ ID NO: 14, or variants thereof). In some embodiments, a CD28 co-stimulatory domain can be used (see, e.g., below and SEQ ID NO: 15, 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 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP 10, LAT, NKD2C SLP76, TRIM, and ZAP70. In some embodiments, the intracellular domain is the intracellular domain of 4-1 BB. 4-1 BB (CD137; TNFRS9) is an activation induced costimulatory molecule, and is an important regulator of immune responses.

[0074] 4-1BB is a membrane receptor protein, also known as CD137, which is a member of the tumor necrosis factor (TNF) receptor superfamily. 4- IBB is expressed on activated T lymphocytes. 4-1BB 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.

[0075] 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 corresponds to an amino acid sequence selected from SEQ ID NO: 14; or includes a sequence selected from SEQ ID NO: 14; or includes at least 75%, at least 80%, at least 85%, 35 at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to a sequence selected from SEQ ID NO: 14. In some embodiments, the co-stimulatory domain comprises the co-stimulatory domain of a CD28. In some embodiments, the CD28 co-stimulatory domain corresponds to an amino acid sequence selected from SEQ ID NO: 15; or includes a sequence selected from SEQ ID NO: 15; or includes at least 75%, at least 80%, at least 85%, 35 at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to a sequence selected from SEQ ID NO: 15.

[0076] Intracellular Signaling Domains

[0077] In some embodiments, the CAR comprises a polynucleotide encoding a CD3zeta (CD3z) intracellular signaling domain (e.g., SEQ ID NO: 16 or a variant thereof).

[0078] 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 / antigen binding domains(s) render the receptor sensitive to signaling activation when the chimeric target / antigen binding domain binds the target / antigen on the surface of a targeted cell.

[0079] With respect to intracellular signaling domains, so-called "first-generation" CARs include those that solely provide CD3-zeta signals upon antigen binding. So-called "second- generation" CARs include those that provide both co-stimulation (e.g., CD28 or CD 137) and activation (CD3-zeta) 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-zeta). In various embodiments, the CAR is selected to have high affinity or avidity for the target / antigen - for example, antibody-derived target or antigen binding 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.

[0080] 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; (see, e.g., below). Additional non-limiting examples of immunoreceptor tyrosine-based activation motif

[0081] (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.

[0082] 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 CD3delta chain, and two CD3-epsilon chains.

[0083] These chains associate with a molecule known as the T cell receptor (TCR) and the CD3-zeta to generate an activation signal in T lymphocytes. A complete TCR complex includes a TCR, CD3-zeta, and the complete CD3 complex.

[0084] 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 IT AM from CD3-zeta, including variants of CD3-zeta such as IT AM-mutated CD3- zeta, CD3-eta, or CD3 -theta. In some embodiments of any aspect, the ITAM includes three motifs of ITAM of CD3-zeta (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-zeta sequence includes the sequence of a CD3-zeta as set forth in the sequences provided herein, e.g., a CD3-zeta sequence of SEQ ID NO: 16, or variants thereof.

[0085] For example, a CAR polypeptide described herein includes the intracellular signaling domain of CD3-zeta. In some embodiments, the CD3-zeta intracellular signaling domain corresponds to an amino acid sequence of SEQ ID NO: 16 or includes a sequence of SEQ ID NO: 16; or includes a sequence with at least 75%, 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 a sequence of SEQ ID NOs: 16.

[0086] 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.

[0087] 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.

[0088] Signal Peptide

[0089] 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: 1 or including an amino acid sequence having at least 75%, 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: 1. 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: 1 or an IgK signal peptide of SEQ ID NO: 17.

[0090] Linker Domain

[0091] 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 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: 18) and Gly4Ser (G4S) linkers such as (G4S)3 (GGGGSGGGGSGGGGS (SEQ ID NO: 19)) and (G4S)4 (GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 20)); the linker sequence of GSTSGSGKPGSGEGSTKG (SEQ ID NO: 21) 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: 22) 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.

[0092] Furthermore, linkers may be cleavable or non-cleavable. Examples of cleavable linkers include 2A linkers (e.g., P2A (SEQ ID NO: 23) and T2A (SEQ ID NO: 24), 2A-like linkers or functional equivalents thereof and combinations thereof.

[0093] 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).

[0094] Full CARs

[0095] In some embodiments, the CAR comprises an antigen binding domain that binds to EGFRvIII, IL13Ra2, HER2, B7-H3, B7-H1, CD147, GD2, CAIX, CD70, CSPG4, EphAl, TROP2, or Survivin.

[0096] In some embodiments, the CAR comprises a polynucleotide encoding an antigen binding domain that binds to EGFRvIII, a CD8alpha hinge / transmembrane, a 4 IBB costimulatory domain, and a CD3zeta signaling domain. In some embodiments, the CAR polypeptide comprises an amino acid sequence with at least 75%, 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 greater sequence identity to a sequence selected from any one of SEQ ID NOs: 10-11. In some embodiments, the CAR comprises an amino acid sequence of any one of SEQ ID NOs: 10-11.

[0097] T cell engaging molecules (TEAMs)

[0098] 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™). By "T cell engaging molecules," "TEAM antibody constructs," or TEAMs" is meant polypeptides that each comprise tandemly linked single-chain variable fragments (scFvs). Optionally, the scFvs are linked by a linker (e.g., a glycine-rich linker). One scFv of the TEAM binds to the T cell receptor (TCR) (e.g., to the CD3£ 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 may 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.

[0099] In some embodiments, the CAR-T cells described herein secrete TEAMs. In some embodiments, the TEAM comprises a secretion tag (e.g., an IgK signal peptide). The TEAMs may, e.g., stimulate the CART cell itself, or operate in a paracrine fashion by redirecting nonspecific bystander T cells against tumors and therefore enhance the anti-tumor effects of CART cell immunotherapy. CAR-T 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.

[0100] 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: 32, CDR-H2 comprises SEQ ID NO: 33, and CDR-H3 comprises SEQ ID NO: 34, and (ii) a VL domain comprising three CDRs (CDR-L1, CDR-L2, and CDR-L3), wherein CDR-L1 comprises SEQ ID NO: 35, CDR-L2 comprises SEQ ID NO: 36, and CDR-L3 comprises SEQ ID NO: 37. In some embodiments, the anti-CD3 scFv comprises the amino acid sequence of SEQ ID NO: 13 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: 13.

[0101] 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: 26, CDR-H2 comprises SEQ ID NO: 27, and CDR-H3 comprises SEQ ID NO: 28, and (ii) a VL domain comprising three CDRs (CDR-L1, CDR-L2, and CDR-L3), wherein CDR-L1 comprises SEQ ID NO: 29, CDR-L2 comprises SEQ ID NO: 30, and CDR-L3 comprises SEQ ID NO: 31. In some embodiments, the anti-EGFR scFv comprises the amino acid sequence of SEQ ID NO: 12 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: 12.

[0102] In some embodiments, the CAR and TEAM are encoded on the same polypeptide. In some embodiments, the CAR and TEAM are encoded on the same polypeptide are separated by a linker domain as described above. In some embodiments, the linker domain in cleavable. In some embodiments, the CAR is an anti-EGFRvIII CAR and the TEAM is a EGFR and CD3 TEAM. In some embodiments, the CAR and TEAM polypeptide comprises an amino acid sequence with at least 75%, 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 greater sequence identity of SEQ ID NO: 39. In some embodiments, the CAR and team polypeptide comprises an amino acid sequence SEQ ID NO: 39. In some embodiments, the CAR and team polypeptide consists of an amino acid sequence SEQ ID NO: 39.

[0103] Sequences

[0104]

[0105] EXAMPLES

[0106] Example 1. Administration of cell therapy comprising DMSO into the central nervous system.

[0107] This example shows that cell therapies comprising DMSO can be administered into the brain without resulting in DMSO related neurotoxicity. DMSO related neurotoxicity events include transient paresthesias, altered mental status, syncope, stroke, seizures, and death. These events can occur during intravenous administration before administration of the cell therapy is complete and after administration until DMSO leaves the body. In the central nervous system, DMSO related toxicity is to occur within the first eight hours after administration of DMSO to the central nervous system because the cerebral spinal fluid is replaced in the central nervous system about every 8 hours, which in turn removes the vast majority of DMSO. Huff, Trevor, et al. "Neuroanatomy, cerebrospinal fluid." (2017).

[0108] A cell therapy comprising DMSO and CAR-T cells was injected into the intraventricular space of multiple human patients diagnosed with glioblastoma. The CAR-T cells had an EGFRvIII binding CAR and secreted an EGFR-CD3 binding TEAM (see SEQ ID NO: 39). A pharmaceutical composition for administrations was prepared from the cell therapy formulation, which had about 5% (v / v) DMSO, 1 x 107+ / - 30% CAR-T cells, and was about 300-500 pLs in volume. The following protocol was used for each patient. After thawing, the cell therapy formulation was diluted to about 10 mL using Elliott’s B solution to produce a pharmaceutical composition that was ready for administration to the patient. This dilution reduced the DMSO concentration to less than 0.25% (v / v). The pharmaceutical composition was then administered intraventricularly using an Ommaya reservoir. No neurotoxicity events were recorded in any of the patients during the 8 hours after administration indicating that cell therapy pharmaceutical formulations having less than 0.25% (v / v) DMSO can be administered to the central nervous system without resulting in neurotoxicity.

[0109] Including DMSO in a cell therapy administered to the brain had not previously been done for fear of DMSO neurotoxicity. This fear resulted in protocols that required multiple washing steps. These lengthy protocols extended the processing time and potential loss of viability and total number of cells in the cell therapy. However, this protocol, the dilution of DMSO, without requiring cell counting or washing steps, allows for much more rapid administration of the cell therapy to the patient after being thawed. This reduces labor costs, potential cell loss and processing errors.

[0110] Example 2. Rapid regression of recurrent glioblastoma after a single infusion of CAR-T EGFR-CD3 TEAM cells.

[0111] Glioblastoma (GBM) is the most aggressive primary brain tumor and the prognosis for recurrent disease is exceedingly poor with no effective treatment options. Chimeric antigen receptor (CAR) T cells represent a promising approach to cancer, as demonstrated by their proven utility for refractory lymphoid malignancies, where they have become the standard-of-care. However, the use of CAR-T cells in solid tumors like GBM has been limited to date, largely due to the targeting of a single antigen in a heterogeneous disease, as well as heavy intratumoral infiltration with suppressive regulatory T cells (Tregs) that impede immune responses. To address these barriers, provided herein is an engineered T-cell product that targets epidermal growth factor receptor variant III (EGFRvIII) via a second-generation CAR while also secreting bispecific antibodies known as T-cell engaging antibody molecules (TEAMs) against wild-type epidermal growth factor receptor (EGFR) (“CAR- TEAM cells”), which is not expressed in the normal brain but is nearly always expressed in GBM. TEAMs secreted by CAR-TEAM cells act locally at the site of CAR T-cell cognate antigen engagement to treat heterogeneous tumors in preclinical models, and that these molecules have the capacity to redirect even Tregs, converting them into cytotoxic killers. Based on these data, a first-in-human, Phase I clinical trial to evaluate the safety of CAR-TEAM cells in patients with recurrent or newly diagnosed GBM was opened and is further described below. Methods

[0112] Study Oversight and Design

[0113] This investigation was approved by the Dana-Farber Harvard Cancer Center Institutional Review Board. An external data and safety monitoring board oversaw the conduct of the study. This was a non-randomized, open label, single-site Phase 1 study.

[0114] Three patients (referred to as Patient 1, Patient 2, and Patient 3) were enrolled in a Safety Run-In Arm, in which subjects with recurrent EGFRvIII-positive GBM were treated with 10 x 106CAR-TEAM cells per infusion, administered via Ommaya reservoir.

[0115] Molecular Testing

[0116] Various methods for determining EGFRvIII expression and EGFR amplification, as well as methods to isolate RNA from extracellular vesicles, were used in the course of the trial. Molecular alterations in patient specimens, e.g., EGFRvIII expression and EGFR amplification, were assessed by routine clinical assays. These assays included targeted variant and fusion next generation sequencing panels utilizing anchored multiplex PCR-based chemistry, and in some instances additional sequencing of targeted amplicons on a complementary platform (Oncomine Precision Assay, Ion Torrent Genexus). EGFR copy number status was assessed routinely via sequencing pipelines and in some instances confirmed by a routine clinical fluorescence in-situ hybridization (FISH) assay.

[0117] Manufacture of CAR-TEAM T Cells

[0118] Autologous T cells were obtained by leukapheresis and transduced with the CARTEAM lentiviral vector containing an anti-EGFRvIII single-chain variable fragment and an intracellular 4-1BB costimulatory domain linked to CD3-zeta. An EGFR-targeted TEAM was also included in the same vector following a ribosomal skip element.

[0119] A third transgene coding for a truncated CD 19 (tCD19) molecule was used as a surface marker for transduction (FIG. 1 A). Detection of the TEAM bound to T cells was performed using biotinylated human EGFR (FIGs. 1B-1C). Details regarding the release criteria for the cell product are shown in Table 1 below. CAR- TEAM cells were formulated at a target dose of 10 * 106CAR-positive cells per vial post-thaw. Cells were thawed, resuspended, and transferred to a syringe under sterile conditions prior to infusion.

[0120] Toxicity Analysis

[0121] Patients were monitored for toxicity throughout the duration of the study. Adverse events were graded according to the Common Toxicity Criteria for Adverse Events version 5.0 (CTCAEv5). Dose-limiting toxicity (DLT) was defined as events at least possibly related to the investigational product, not attributed to disease progression, and: any related CTCAEv5 adverse effects grade 4 or greater, or any related CTCAEv5 grade adverse events that did not resolve to grade 2 or lower within 2 weeks, or any grade 3 or 4 cytokine release syndrome (CRS) or immune-effector cell-associated neurotoxicity syndrome (ICANS) that did not resolve to grade 2 or lower within 3 weeks.

[0122] Results

[0123] Patients Included in the Case Series

[0124] Between March 2023 and July 2023 three patients with recurrent glioblastoma were enrolled in the Phase I study at Massachusetts General Hospital.

[0125] Patient 1

[0126] A 74-year-old right-handed man (Patient 1) presented with a one-week history of headache and confusion, found on imaging to have an enhancing mass in the left insula. Patient 1 underwent craniotomy which revealed a diagnosis of GBM, isocitrate dehydrogenase (IDH) wild-type and 06-methylguanine-DNA methyltransferase (MGMT) methylated. The tumor was positive for EGFRvIII and EGFR gene copy number gain. Patient 1 received standard-of-care therapy consisting of radiation and temozolomide chemotherapy. Twelve months after diagnosis, magnetic resonance imaging (MRI) suggested disease recurrence. A repeat craniotomy was performed, and the recurrent tumor was confirmed to be EGFRvIII-positive (FIG. 2A). Patient 1 was subsequently enrolled in the Phase I clinical study. A pre-treatment interval MRI was obtained which showed active progression (FIG. 2B, center). Patient 1 underwent placement of a ventriculostomy catheter with Ommaya reservoir. CAR- TEAM cells (10 x 106CAR-positive T cells) were subsequently administered via the catheter device on post-operative day 1. An MRI obtained on day +1 after this single infusion exhibited rapid regression of the tumor (FIG. 2B, right). This remained stable by Response Assessment in Neuro-Oncology (RANO) criteria on additional MRIs obtained through 31 days. The patient received a second infusion of 10 x 106CAR-TEAM cells on day +37 (FIG. 2A).

[0127] Liquid biopsy was performed on extracellular vesicle (EV) RNA derived from cerebrospinal fluid (CSF) and peripheral blood at several time points throughout each treatment cycle. EGFRvIII and EGFR copy numbers were elevated during cycle 1 and decreased over time, eventually becoming undetectable in post-treatment CSF (FIG. 2C). EV RNA from peripheral blood samples obtained at pre- and post-study timepoints similarly demonstrated decrease in copy number of both EGFRvIII and EGFR (FIG. 2D). The patient returned to the operating room on day +72 for biopsy after interval MRI revealed progression. Consistent with liquid biopsy, next generation sequencing (NGS) on tumor tissue was also negative for both EGFRvIII and EGFR copy number alterations. However, post hoc assessment by FISH showed maintained EGFR copy number gain (ratio 3.5) (FIG. 2E).

[0128] Patient 2

[0129] A 72-year-old right-handed man (Patient 2) presented with difficulty reading and was found on MRI to have a left-sided, contrast-enhancing mass in the posterior temporal lobe. Patient 2 underwent craniotomy for tumor resection with pathology confirming GBM, which was EGFRvIII-positive, IDH wild-type and MGMT methylated. The patient was treated with standard-of-care radiation and temozolomide chemotherapy, as well as tumor treating fields. Twenty months after initial diagnosis, surveillance imaging demonstrated evidence of progression. Patient 2 was then enrolled in the Phase I clinical study based on EGFRvIII status at initial diagnosis, according to a new schematic workflow (FIG. 3 A), which had been modified to enable concomitant craniotomy, tissue sampling and Ommaya placement during a single surgery. Molecular histopathological analysis at the time of repeat craniotomy was consistent with recurrent GBM, positive for EGFRvIII but negative for EGFR copy number gain. Patient 2 was discharged and readmitted one week later for infusion. The patient received 10 x 106CAR- TEAM cells via intraventricular catheter. On day +2, an MRI was performed which showed a decrease in cross sectional area of the tumor by 18.5%, which on day +69 had further decreased by 60.7% from pre-infusion baseline (FIG. 3B).

[0130] Patient 3

[0131] A 57-year-old right-handed woman (Patient 3) presented with several weeks of wordfinding difficulty and concern for seizure, found on MRI to have a left parietal, contrastenhancing mass. The patient underwent craniotomy and tumor resection, with pathology revealing GBM, IDH wild-type and MGMT unmethylated. The tumor was positive for EGFRvIII expression and EGFR gene copy number gain. Patient 3 was treated with radiation and temozolomide chemotherapy, which was dose-reduced due to the development of thrombocytopenia. By six months after diagnosis, interval imaging demonstrated evidence of recurrent disease. The patient was enrolled in the Phase I clinical study (FIG. 3 A) and underwent repeat craniotomy for open biopsy and implantation of a ventricular catheter with Ommaya reservoir. Tissue analysis at the time of surgery for recurrence demonstrated loss of EGFRvIII expression and absence of EGFR gene amplification on NGS, which was confirmed by FISH. A pre-infusion MRI was performed which redemonstrated extensive recurrent disease burden (FIG. 3C, left). The patient was discharged and readmitted two weeks later for infusion of 10 x 106CAR- TEAM cells via intraventricular catheter. An MRI obtained at day +5 after this single infusion exhibited near-complete tumor regression (FIG. 3C, right), despite apparent lack of EGFRvIII expression or wild-type gene amplification immediately prior to treatment.

[0132] Safety and Adverse Events

[0133] In this Safety Run-In cohort, there were no associated DLTs. Grade 3 events that were at least possibly attributable to the investigational product included Grade 3 encephalopathy for 3 days (Patient 1, probably related) and Grade 3 fatigue for 8 days (Patient 3, related). One death occurred following progression of disease, 63 days after the patient came off study (Patient 1); the cause of death was gastrointestinal perforation while receiving bevacizumab and dexamethasone. This event was not attributed to CAR- TEAM cell infusion. Patients 2 and 3 developed transient pulmonary nodules and ground-glass opacities on chest CT on work-up of cyclic fevers. These were otherwise asymptomatic and resolved spontaneously on interval imaging within 4 to 6 weeks. Adverse events are shown in Tables 2-7 below. Table 2. Adverse Event Log: Patient 1.

[0134] SAE = serious adverse event; PACs / PVCs = premature atrial contractions / premature ventricular contractions; CRS = cytokine release syndrome; ALT = alanine transaminase; AST = aspartate aminotransferase; SIADH = syndrome of inappropriate antidiuretic hormone secretion; UTI = urinary tract infection.

[0135] Table 3. Adverse Event Summary: Patient 1 Table 4. Adverse Event Log: Patient 2

[0136] RUE = right upper extremity 1; BLE = bilateral lower extremity

[0137] Table 5. Adverse Event Summary: Patient 2 Table 6. Adverse Event Log: Patient 3.

[0138] Table 7. Adverse Event Summary: Patient 3

[0139] Correlative Data

[0140] All patients exhibited fevers which peaked by day +2 after infusion (FIG. 4A).

[0141] Intravenous therapy with the interleukin- 1 receptor antagonist, anakinra (100 mg Q6H), was intermittently used in the management of fevers (FIG. 4B). Systemic inflammatory markers increased and peaked in the second and third weeks after infusion, returning to near baseline by day +30 (FIG. 5A). None of the patients required steroids during the acute post-treatment phase or for any therapy-related indication. Cytopathological analysis of the CSF demonstrated elevated total nucleated cell count immediately after infusion, which decayed exponentially by D +7 and then gradually over time. The CSF cell differential was initially of a neutrophilic predominance, before shifting to a primarily lymphocytic population over the 30 days after infusion (FIG. 6). Quantification by vector copy number (VCN) and flow cytometry indicated that CAR-positive and TEAM-positive T cells temporarily persisted in the CSF after infusion but declined by week 4 (FIG. 7A), which corresponded with normalization of the inflammatory cytokine milieu in the CSF compartment over the same period (FIG. 7B). CAR- TEAM cells were detected in the peripheral blood between 2 to 3 weeks after infusion but their presence was transient (FIG. 7C).

[0142] Discussion

[0143] Provided herein is early phase I, first-in-human experience with intraventricular delivery of CAR- TEAM cells in patients with recurrent GBM. Here, dramatic radiographic responses were observed in multiple patients within days after a single intraventricular infusion of dualtargeting CAR-TEAM cells. Liquid biopsy is reported as a correlative study for cellular therapy in patients with GBM. Despite successful translation of CAR-T cells for hematological malignancies, the utility of this strategy for solid tumors like GBM remains unproven. Previously described CAR-T cells and bi specific antibodies for patients with GBM have targeted only single antigens and have thus been limited by tumor heterogeneity, the emergence of antigen-loss, and eventual immune escape. The experience with CAR- TEAM cells described in this Example provides proof-of-principle of targeting multiple surface antigens simultaneously using CAR-T cells and confirms EGFRvIII as a suitable immunotherapeutic target for GBM. Moreover, the secreted bispecific antibody was also safe, demonstrating viability of locally targeting antigens like wild-type EGFR with this approach, despite widespread expression in systemic tissues. This Example shows that antitumor CAR- mediated responses can be achieved in patients with GBM, even in the setting of advanced, intraparenchymal cerebral disease. This contrasts with a prior report of a complete response observed in the setting of recurrent leptomeningeal disease (LMD) treated with repeated intracranial infusions (16 infusions) of monospecific interleukin- 13 receptor alpha-2 (IL- 13Ra2) CAR-T cells. Here, the results demonstrate that intraventricularly administered, “living drugs” like CAR- TEAM cells also have the capacity to access and mediate activity against infiltrative, parenchymal GBM. Notably, CAR- TEAM cells showed signs of antitumor activity in the absence of EGFRvIII expression (Patient 3). Certainly, interpretation of these data remains subject to the sensitivity of corresponding assays that determine EGFRvIII expression, as well as inherent limitations of tissue sampling in a heterogeneous disease. However, the potential efficacy of CAR- TEAM cells in the setting of EGFRvIII- negative glioma recapitulates prior observations of CAR-independent, TEAM-mediated antitumor effects from our preclinical studies.

Claims

CLAIMS1. A method of administering a cell therapy to a human subject, the method comprising administering a pharmaceutical composition that comprises the cell therapy and dimethyl sulfoxide (DMSO) into the subject’s central nervous system.

2. A method of administering a cell therapy to a subject, the method comprising:(i) obtaining a cell therapy formulation comprising the cell therapy and DMSO;(ii) diluting the cell therapy formulation to produce a pharmaceutical composition comprising less than or equal to 0.5% DMSO (v / v); and(iii) administering the pharmaceutical composition into the subject’s central nervous system.

3. The method of claim 1 or claim 2, further comprising thawing the cell therapy formulation prior to diluting the cell therapy formulation.

4. The method of any one of claims 1-3, wherein administering comprises administering the pharmaceutical composition into the subject’s brain.

5. The method of any one of claims 2-4, wherein diluting the cell therapy comprises diluting by 20-fold to 40-fold (v / v).

6. The method of any one of claims 1-4, wherein administering the pharmaceutical composition comprises intracranial administering.

7. The method of any one of claims 1-6, wherein administering the pharmaceutical composition comprises intraventricular administering.

8. The method of claim 1 or claim 2, wherein administering the pharmaceutical composition comprises administering into cerebral spinal fluid of the subject.

9. The method of claim 8, wherein administering further comprising administering into the cerebral spinal fluid of the spine of the subject.

10. The method of claim 1 or claim 2, wherein administering the pharmaceutical composition comprises intrathecal administering.

11. The method of any one of claims 1-10, wherein administering comprises administering to a tumor in the central nervous system of the subject.

12. The method of any one of claims 1-11, wherein administering comprises administering into a tumor in the central nervous system of the subject.

13. The method of any one of claims 1-9, wherein the pharmaceutical composition comprises less than 0.5% by volume (v / v) DMSO.

14. The method of any one of claims 1-9, wherein the pharmaceutical composition comprises less than 0.25% (v / v) DMSO.

15. The method of any one of claims 1-14, wherein the method does not comprise washing the cell therapy to remove DMSO.

16. The method of any one of claims 1-9, wherein the pharmaceutical composition comprises more than 0.025% DMSO.

17. The method of any one of claims 1-9, wherein the pharmaceutical composition comprises 0.025-0.5% (v / v) DMSO.

18. The method of any one of claims 1-9, wherein the pharmaceutical composition comprises 0.03-0.5% (v / v) DMSO.

19. The method of any one of claims 1-9, wherein the pharmaceutical composition comprises 0.05-0.25% (v / v) DMSO.

20. The method any one of claims 1-9, wherein the pharmaceutical composition comprises 0.1-0.3% (v / v) DMSO.

21. The method any one of claims 1-9, wherein the pharmaceutical composition comprises 0.12-.0.25% (v / v) DMSO.

22. The method of any one of claim 1-21, wherein the pharmaceutical composition comprises an excipient.

23. The method of claim 22, wherein the excipient comprises human serum albumin.

24. The method of any one of claims 1-23, wherein the pharmaceutical composition comprises a cryopreservative.

25. The method of claim 24, wherein the cryopreservative comprises CryoStor® 10.

26. The method of claim 24, wherein the cryopreservative is DMSO.

27. The method of any one of claims 1-26, wherein the pharmaceutical composition comprises Elliott’s B solution.

28. The method of any one of claims 2-26, wherein diluting comprises diluting using Elliott’s B solution.

29. The method of any one of claims 1-27, wherein the pharmaceutical composition is 5 mL to 15 mL in volume.

30. The method of any one of claims 1-29, wherein the pharmaceutical composition is 10 mL in volume.

31. The method of any one of claims 1-30, wherein the cell therapy comprises an immune cell therapy.

32. The method of claim 31, where the immune cell therapy comprises a T cell therapy or a natural killer (NK) cell therapy.

33. The method of claim 32, wherein the T cell therapy comprises a CAR-T cell therapy.

34. The method of claim 33, wherein the CAR-T cell therapy comprises a polynucleotide encoding a CAR comprising an antigen binding domain that binds to EGFRvIII.

35. The method of claim 34, wherein the 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: 2, CDR-H2 comprises SEQ ID NO: 3, and CDR-H3 comprises SEQ ID NO: 4, and(ii) a VL domain comprising three CDRs (CDR-L1, CDR-L2, and CDR-L3), wherein CDR-L1 comprises SEQ ID NO: 5, CDR-L2 comprises SEQ ID NO: 6, and CDR-L3 comprises SEQ ID NO: 7.

36. The method of claim 34 or claim 35, wherein the CAR comprises an amino acid sequence of SEQ ID NO: 11.

37. The method of any one of claims 34-36, wherein the CAR-T cell therapy comprises a polynucleotide encoding a T cell engaging antibody molecule (TEAM).

38. The method of any one of claims 34-36, wherein the CAR-T cell therapy comprises a polynucleotide encoding a T cell engaging antibody molecule (TEAM) comprising an anti- EGFR antibody and an anti-CD3 antibody.

39. The method of claim 38, wherein the anti-EGFR antibody comprises:(i) a VH domain comprising three complementarity determining regions (CDR-H1, CDR-H2, and CDR-H3), wherein CDR-H1 comprises SEQ ID NO: 26, CDR-H2 comprises SEQ ID NO: 27, and CDR-H3 comprises SEQ ID NO: 28, and(ii) a VL domain comprising three CDRs (CDR-L1, CDR-L2, and CDR-L3), wherein CDR-L1 comprises SEQ ID NO: 29, CDR-L2 comprises SEQ ID NO: 30, and CDR- L3 comprises SEQ ID NO: 31.

40. The method of claim 38 or claim 39, wherein the anti-CD3 antibody comprises:(i) a VH domain comprising three complementarity determining regions (CDR-H1, CDR-H2, and CDR-H3), wherein CDR-H1 comprises SEQ ID NO: 32, CDR-H2 comprises SEQ ID NO: 33, and CDR-H3 comprises SEQ ID NO: 34, and(ii) a VL domain comprising three CDRs (CDR-L1, CDR-L2, and CDR-L3), wherein CDR-L1 comprises SEQ ID NO: 35, CDR-L2 comprises SEQ ID NO: 36, and CDR- L3 comprises SEQ ID NO: 37.

41. The method of any one of claims 37-40, wherein the TEAM comprises an amino acid sequence of SEQ ID NO: 38.

42. The method of any one of claims 1-41, wherein the cell therapy in an autologous cell therapy.

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