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

Abstract

The present disclosure describes, in part, methods of administering a pharmaceutical composition comprising a cell therapy and DMSO to the central nervous system. These methods are based, in part, on the unexpected finding that administration of a pharmaceutical composition comprising a cell therapy and DMSO into the central nervous system does not induce DMSO-related measurable neurotoxicity in human patients.

Description

[0001] Related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 587,626, filed October 3, 2023, entitled “Methods of Central Nervous System Administration of Compositions Comprising a Cell Therapy and Dimethyl Sulfoxide,” and U.S. Provisional Application No. 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 which are incorporated herein by reference.

[0003] Reference to electronic sequence listing

[0004] The contents of the electronic serial number (M105370049WO00-SEQ-ARM.xml; size: 38,419 bytes; and creation date: October 1, 2024) are incorporated herein by reference in their entirety. Background Technology

[0005] Cell therapy (e.g., CAR-T cell therapy) has revolutionized the treatment of certain types of cancer, such as leukemia and lymphoma, but its success in treating solid tumors, including brain tumors, has been relatively limited. Cell therapies for human administration are typically prepared in specialized facilities, and the cells are then frozen in cryoprotectants (e.g., dimethyl sulfoxide (DMSO)) for transport to the medical facility where the cell therapy will be administered to the patient. Many cryoprotectants, including DMSO, are known to be toxic to humans, but maintaining cell integrity is crucial during transport to the medical facility. Upon arrival at the medical facility, the cell therapy is typically thawed and administered intravenously to the patient. Intravenous administration dilutes the cryoprotectant in the bloodstream, thereby reducing its toxicity to acceptable levels. Additionally, cryoprotectants such as DMSO can be removed from the bloodstream through the lungs and exhaled by the patient, which further reduces DMSO toxicity. Summary of the Invention

[0006] This application addresses a core challenge in administering cell therapy to the central nervous system (e.g., via intraventricular infusion) by using cryoprotectants (such as DMSO), which are required in cell therapy formulations but are neurotoxic and are not significantly diluted after administration (compared to intravenous administration into the bloodstream). Akel S et al., Cytotherapy. 2023 Sep 6:S1465-3249(23)01035-6. For example, recent studies have reported seizures and brain abnormalities in pediatric patients following intravenous administration of DMSO-crystallized stem cells. Ataseven E et al. Journal of pediatric hematology / oncology 39.5 (2017): e297-e299. This suggests that cell therapy administered to the human brain is expected to be neurotoxic. In another study, administration of DMSO in mice produced widespread apoptosis in the brain. Hanslick JL Neurobiol Dis. 2009 Apr;34(1):1-10. Therefore, existing techniques teach 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 drawbacks, including the potential to alter the composition of the cell therapy, making it (1) unacceptable to regulatory requirements, or (2) requiring additional measurement of cell therapy viability to meet regulatory requirements.

[0007] Surprisingly, the inventors have discovered that pharmaceutical compositions comprising cell therapy and DMSO can be administered to the human brain without causing significant neurotoxicity. Specifically, in some embodiments, the inventors have determined that diluting a cell therapy formulation comprising about 5% DMSO (v / v) by volume by about 20 times before administration to the central nervous system is sufficient to reduce and / or eliminate DMSO neurotoxicity in human patients. This is further discussed in the examples.

[0008] In some aspects, this disclosure describes a method of administering cell therapy to a human subject, the method comprising administering a pharmaceutical composition comprising cell therapy and dimethyl sulfoxide (DMSO) to the central nervous system of the subject.

[0009] In some aspects, this disclosure describes a method of administering cell therapy to a subject, the method comprising: (i) obtaining a cell therapy formulation comprising 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 to the central nervous system of the subject. In some embodiments, the method further includes thawing the cell therapy formulation prior to diluting it.

[0010] In some embodiments, administration includes administering the pharmaceutical composition to the brain of a subject. In some embodiments, diluting the cell therapy includes diluting it 20 to 40 times (v / v). In some embodiments, administration of the pharmaceutical composition includes intracranial administration. In some embodiments, administration of the pharmaceutical composition includes intraventricular administration. In some embodiments, administration of the pharmaceutical composition includes administration to the cerebrospinal fluid of a subject. In some embodiments, administration further includes administration to the spinal cerebrospinal fluid of a subject. In some embodiments, administration of the pharmaceutical composition includes intrathecal administration. In some embodiments, administration includes administration to a tumor in the central nervous system of a subject. In some embodiments, administration includes administration to a tumor in the central nervous system of a subject.

[0011] In some embodiments, the pharmaceutical composition contains less than 0.5% DMSO by volume (v / v). In some embodiments, the pharmaceutical composition contains less than 0.25% (v / v) DMSO. In some embodiments, the method does not include cell washing therapy to remove DMSO. In some embodiments, the pharmaceutical composition contains more than 0.025% (v / v) DMSO. In some embodiments, the pharmaceutical composition contains 0.025% to 0.5% (v / v) DMSO. In some embodiments, the pharmaceutical composition contains 0.03% to 0.5% (v / v) DMSO. In some embodiments, the pharmaceutical composition contains 0.05% to 0.25% (v / v) DMSO. In some embodiments, the pharmaceutical composition contains 0.1% to 0.3% (v / v) DMSO. In some embodiments, the pharmaceutical composition contains 0.12% to 0.25% (v / v) DMSO.

[0012] 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 cryoprotectant. In some embodiments, the cryoprotectant comprises CryoStor ® 10 (with CryoStor) ® (CS10 can be used interchangeably). In some embodiments, the cryoprotectant is DMSO. In some embodiments, the pharmaceutical composition comprises Elliott's B solution. In some embodiments, dilution includes dilution using Elliott's B solution. In some embodiments, the volume of the pharmaceutical composition is 5 mL to 15 mL. In some embodiments, the volume of the pharmaceutical composition is 10 mL.

[0013] In some embodiments, the cell therapy comprises immune cell therapy. In some embodiments, the immune cell therapy comprises T cell therapy or natural killer (NK) cell therapy. In some embodiments, the T cell therapy comprises CAR-T cell therapy. In some embodiments, the CAR-T cell therapy comprises a polynucleotide encoding a CAR, wherein the CAR comprises an antigen-binding domain that binds to EGFRvIII.

[0014] In some embodiments, the antigen-binding domain comprises: (i) a VH domain containing three complementarity-determining regions (CDR-H1, CDR-H2, and CDR-H3), wherein CDR-H1 contains SEQ ID NO: 2, CDR-H2 contains SEQ ID NO: 3, and CDR-H3 contains SEQ ID NO: 4; and (ii) a VL domain containing three CDRs (CDR-L1, CDR-L2, and CDR-L3), wherein CDR-L1 contains SEQ ID NO: 5, CDR-L2 contains SEQ ID NO: 6, and CDR-L3 contains SEQ ID NO: 7. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 11.

[0015] 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), which includes an anti-EGFR antibody and an anti-CD3 antibody.

[0016] 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.

[0017] 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.

[0018] In some implementations, the TEAM comprises the amino acid sequence of SEQ ID NO: 38.

[0019] In some implementations, the cell therapy is autologous cell therapy. Attached Figure Description

[0020] The following figures form part of this specification and are included to further illustrate certain aspects of this disclosure, which can be better understood by referring to one or more of these figures in combination with the detailed description of some specific embodiments given herein.

[0021] Figures 1A to 1C This relates to the CAR-TEAM construct and its expression in cells. Figure 1A A schematic diagram of a molecule encoding CAR-TEAM with a truncated CD19 (tCD19) molecule is shown, which is used as a surface marker for transduction. Figure 1B Representative flow cytometry analysis of cell products from patient 1 is shown, in which all CD3 + Gating of CAR-positive and TEAM-positive cells in T cells. Figure 1C Representative gating of TEAM-positive cells is shown in CAR-positive and CAR-negative T cell populations.

[0022] Figures 2A to 2E Corresponding to the treatment events and outcomes related to Patient 1. Figure 2A The timeline of events for Patient 1 when they were recruited into the Phase I clinical trial is summarized. Figure 2B The image shows axial contrast-enhanced T1-weighted images of patient 1 immediately after resection (left), before infusion (middle), and after infusion (right), which enhance the signal of adipose tissue. The numbers in each image represent the number of days relative to the infusion (day 0). Figure 2CThe copy numbers of EGFRvIII and EGFR derived from extracellular vesicle (EV) RNA present in CSF over time are shown. Figure 2D The copy numbers isolated from peripheral blood EV RNA at pre- and post-treatment time points are shown, and Figure 2E The post-hoc analysis of EGFR copy number by fluorescence in situ hybridization (FISH) is shown.

[0023] Figures 3A to 3C Treatment workflow and outcomes for patients 2 and 3. Figure 3A The modified workflow for patients 2 and 3 is shown, which enables craniotomy, tissue sampling, and Ommaya placement to be performed in a single procedure. Figure 3B The longitudinal radiographic assessment following CAR-TEAM cell infusion in patient 2 is shown. Figure 3C The images shown are axial T1-weighted contrast images of patient 3 before and after infusion, showing tumor regression at D+5 post-treatment. The numbers in each figure represent the number of days relative to the infusion (day 0).

[0024] Figures 4A to 4B This involves the temperature response in patients 1 to 3 following CAR-TEAM cell infusion. Figure 4A Temperature curves for all patients are shown, peaking within 2 days of infusion and normalizing over time. Figure 4B The study demonstrated the control of body temperature through treatment with the interleukin-1 receptor (IL-1R) antagonist anakinra.

[0025] Figure 5 The expression of inflammatory markers C-reactive protein (CRP) and ferritin in the cerebrospinal fluid of patients 1 to 3 after CAR-TEAM cell infusion is shown.

[0026] Figure 6 The cell counts and differences in cerebrospinal fluid of patients 1 to 3 after CAR-TEAM cell infusion are shown.

[0027] Figures 7A to 7C The presence of CAR-positive and TEAM-positive cells and inflammatory cytokines in the cerebrospinal fluid (CSF) and peripheral blood of patients 1 to 3 was investigated. Figure 7AThe quantification of CAR-positive and TEAM-positive T cells by vector copy number (VCN) and flow cytometry in all three patients is shown. These CAR-positive and TEAM-positive T cells were transiently present in CSF after infusion but decreased at week 4. Figure 7B The presence of inflammatory cytokines in the patient's CSF during the same period was shown. Figure 7C The detection of CAR-positive and TEAM-positive cells (which are transient) in peripheral blood is shown. Detailed Implementation

[0028] This application relates in part to the unexpected discovery that pharmaceutical compositions comprising cell therapy and DMSO can be administered to the human central nervous system without causing significant DMSO-induced neurotoxicity. In some aspects, this disclosure describes a method of administering cell therapy to a human subject, the method comprising administering a pharmaceutical composition comprising cell therapy and dimethyl sulfoxide (DMSO) to the central nervous system of the subject. In some embodiments, this disclosure describes a method of administering cell therapy to a subject, the method comprising: (i) obtaining a cell therapy formulation comprising 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 to the central nervous system of the subject.

[0029] application

[0030] As used herein, “application” means bringing a subject (e.g., the subject’s central nervous system) into contact with a cell therapy (e.g., a pharmaceutical composition). In some embodiments, application includes injecting or infusing the pharmaceutical composition into the subject. In some embodiments, injection or infusion includes passing the pharmaceutical composition through a filter. In some embodiments, injection or infusion does not include passing the pharmaceutical composition through a filter. In some embodiments, application includes bringing the subject’s central nervous system into contact with the cell therapy. In some embodiments, application includes administering the pharmaceutical composition into the subject’s central nervous system.

[0031] When used in the context of administration to a specific location (e.g., administering a pharmaceutical composition to a subject's central nervous system), the term "to" refers to the direct application of a treatment (e.g., cell therapy or a pharmaceutical composition) to that specific location. For example, administering a pharmaceutical composition to a subject's central nervous system includes injecting or infusing the pharmaceutical composition into the subject's cerebrospinal fluid (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 cerebrospinal fluid.

[0032] As used in this article, "central nervous system" refers to biological substances within the blood-brain barrier (e.g., fluids, tissues, organs, tumors, etc.), including the blood-brain barrier, brain, spinal cord, cerebrospinal fluid (CSF), meninges, the space between the membranes of the meninges (e.g., subarachnoid space), and brain or spinal cord tumors of the central nervous system (e.g., glioblastoma).

[0033] In some embodiments, administration of the pharmaceutical composition to the central nervous system includes intracranial administration. In some embodiments, administration of the pharmaceutical composition to the central nervous system includes intraventricular administration. In some embodiments, administration of the pharmaceutical composition to the central nervous system includes administration to the cerebrospinal fluid of the subject. In some embodiments, administration also includes administration to the spinal cerebrospinal fluid of the subject. In some embodiments, administration of the pharmaceutical composition to the central nervous system includes intrathecal administration. In some embodiments, administration of the pharmaceutical composition to the central nervous system includes administration to a tumor in the central nervous system of the subject. In some embodiments, administration of the pharmaceutical composition to the central nervous system includes administration to a tumor in the central nervous system of the subject. In some embodiments, administration of the pharmaceutical composition to the central nervous system includes administration using an Ommaya Reservoir.

[0034] Pharmaceutical Composition

[0035] In some embodiments, the methods described herein include administering a pharmaceutical composition to the central nervous system of a subject. As used herein, "pharmaceutical composition" refers to a composition comprising an active agent (e.g., cell therapy as described herein) and a pharmaceutically acceptable excipient for administration to the central nervous system. Pharmaceutically acceptable excipients for administration to the central nervous system include, but are not limited to, human serum albumin (HSA), Elliott's B solution, preservative-free saline, autologous CSF, or combinations thereof. In some embodiments, the pharmaceutical composition may comprise cell therapy, Elliott's B solution, and HSA. In some embodiments, the cell preparations described herein may be diluted with Elliott's B solution, preservative-free saline, autologous CSF, or combinations thereof to produce the pharmaceutical composition. In some embodiments, the pharmaceutical composition also comprises a cryoprotectant (e.g., DMSO).

[0036] In some embodiments, the pharmaceutical composition comprises a cryoprotectant. As used herein, "cryoprotectant" means a composition that, when mixed with cell therapy (e.g., CAR-T cells) prior to freezing, reduces cell death compared to freezing cell therapy without a cryoprotectant. Cryoprotectants for cell therapy are known in the art, for example, as described in Murray, KA, Gibson, MI Nat Rev Chem 6, 579-593 (2022). doi.org / 10.1038 / s41570-022-00407-4. In some embodiments, the cell protectant comprises glycerol. In some embodiments, the cryoprotectant comprises DMSO. In some embodiments, the cryoprotectant comprises CryoStor. ® 10. In some implementations, cryoprotectants have been reported to have adverse side effects when administered (e.g., when administered to the central nervous system).

[0037] In some embodiments, the method includes administering a pharmaceutical composition comprising DMSO and cell therapy to the central nervous system of a subject. In some embodiments, the pharmaceutical composition comprises less than 0.5% by volume (v / v) of DMSO (e.g., less than 0.5% (v / v) of DMSO, less than 0.35% (v / v) of DMSO, less than 0.30% (v / v) of DMSO, less than 0.25% (v / v) of DMSO, less than 0.20% (v / v) of DMSO, less than 0.15% (v / v) of DMSO, less than 0.10% (v / v) of DMSO, or less than 0.05% (v / v) of DMSO). In some embodiments, the pharmaceutical composition comprises more than 0.01% (v / v) DMSO (e.g., more than 0.02% (v / v) or more than 0.025% (v / v) DMSO) and less than 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) of 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, at least 0.3% (v / v) DMSO, at least 0.35% (v / v) DMSO, at least 0.4% (v / v) DMSO, at least 0.45% (v / v) DMSO), but not more than 0.5% (v / v) of DMSO.

[0038] In some embodiments, the method includes administering a pharmaceutical composition comprising cell therapy and 0.025% to 0.25% (v / v) DMSO to the central nervous system of a subject. In some embodiments, the method includes administering a pharmaceutical composition comprising cell therapy and 0.026% to 0.25% (v / v) DMSO to the central nervous system of a subject. In some embodiments, the method includes administering a pharmaceutical composition comprising cell therapy and 0.027% to 0.25% (v / v) DMSO to the central nervous system of a subject. In some embodiments, the method includes administering a pharmaceutical composition comprising cell therapy and 0.028% to 0.25% (v / v) DMSO to the central nervous system of a subject. In some embodiments, the method includes administering a pharmaceutical composition comprising cell therapy and 0.029% to 0.25% (v / v) DMSO to the central nervous system of a subject. In some embodiments, the method includes administering a pharmaceutical composition comprising cell therapy and 0.030% to 0.25% (v / v) DMSO to the central nervous system of a subject. In some embodiments, the method includes administering a pharmaceutical composition comprising cell therapy and 0.040% to 0.25% (v / v) DMSO to the central nervous system of a subject. In some embodiments, the method includes administering a pharmaceutical composition comprising cell therapy and 0.050% to 0.25% (v / v) DMSO to the central nervous system of a subject. In some embodiments, the method includes administering a pharmaceutical composition comprising cell therapy and 0.075% to 0.25% (v / v) DMSO to the central nervous system of a subject. In some embodiments, the method includes administering a pharmaceutical composition comprising cell therapy and 0.1% to 0.25% (v / v) DMSO to the central nervous system of a subject. In some embodiments, the method includes administering a pharmaceutical composition comprising cell therapy and 0.1% to 0.25% (v / v) DMSO to the central nervous system of a subject. In some embodiments, the method includes administering a pharmaceutical composition comprising cell therapy and 0.12% to 0.25% (v / v) DMSO to the central nervous system of a subject. In some embodiments, the method includes administering a pharmaceutical composition comprising cell therapy and 0.125% to 0.25% (v / v) DMSO to the central nervous system of a subject. In some embodiments, the method includes administering a pharmaceutical composition comprising cell therapy and 0.05% to 0.30% (v / v) DMSO to the central nervous system of a subject. In some embodiments, the method includes administering a pharmaceutical composition comprising cell therapy and 0.1% to 0.30% (v / v) DMSO to the central nervous system of a subject. In some embodiments, the method includes administering a pharmaceutical composition comprising cell therapy and 0.05% to 0.25% (v / v) DMSO to the central nervous system of a subject.In some embodiments, the method includes administering a pharmaceutical composition comprising cell therapy and 0.1% to 0.25% (v / v) DMSO to the central nervous system of a subject. In some embodiments, the method includes administering a pharmaceutical composition comprising cell therapy and 0.12% to 0.25% (v / v) DMSO to the central nervous system of a subject. In some embodiments, the method includes administering a pharmaceutical composition comprising cell therapy and 0.125% to 0.25% (v / v) DMSO to the central nervous system of a subject.

[0039] In some embodiments, the method includes administering a pharmaceutical composition comprising cell therapy and 0.025% to 0.50% (v / v) DMSO to the central nervous system of a subject. In some embodiments, the method includes administering a pharmaceutical composition comprising cell therapy and 0.026% to 0.50% (v / v) DMSO to the central nervous system of a subject. In some embodiments, the method includes administering a pharmaceutical composition comprising cell therapy and 0.027% to 0.50% (v / v) DMSO to the central nervous system of a subject. In some embodiments, the method includes administering a pharmaceutical composition comprising cell therapy and 0.028% to 0.50% (v / v) DMSO to the central nervous system of a subject. In some embodiments, the method includes administering a pharmaceutical composition comprising cell therapy and 0.029% to 0.50% (v / v) DMSO to the central nervous system of a subject. In some embodiments, the method includes administering a pharmaceutical composition comprising cell therapy and 0.030% to 0.50% (v / v) DMSO to the central nervous system of a subject. In some embodiments, the method includes administering a pharmaceutical composition comprising cell therapy and 0.040% to 0.50% (v / v) DMSO to the central nervous system of a subject. In some embodiments, the method includes administering a pharmaceutical composition comprising cell therapy and 0.050% to 0.50% (v / v) DMSO to the central nervous system of a subject. In some embodiments, the method includes administering a pharmaceutical composition comprising cell therapy and 0.075% to 0.50% (v / v) DMSO to the central nervous system of a subject. In some embodiments, the method includes administering a pharmaceutical composition comprising cell therapy and 0.1% to 0.50% (v / v) DMSO to the central nervous system of a subject. In some embodiments, the method includes administering a pharmaceutical composition comprising cell therapy and 0.1% to 0.50% (v / v) DMSO to the central nervous system of a subject. In some embodiments, the method includes administering a pharmaceutical composition comprising cell therapy and 0.12% to 0.50% (v / v) DMSO to the central nervous system of a subject. In some embodiments, the method includes administering a pharmaceutical composition comprising cell therapy and 0.125% to 0.50% (v / v) DMSO to the central nervous system of the subject.

[0040] In some embodiments, the pharmaceutical composition comprises 10 million to 80 million therapeutic cells. In some embodiments, the pharmaceutical composition comprises 7 million to 13 million therapeutic cells. In some embodiments, the pharmaceutical composition comprises 10 million ± 30% therapeutic cells.

[0041] In some embodiments, the pharmaceutical composition comprises Elliott's B solution, cell therapy, human serum albumin, and 0.1% to 0.30% (v / v) DMSO. In some embodiments, the pharmaceutical composition comprises 94% to 98% (v / v) Elliott's B solution, cell therapy, 0.3% to 0.6% (w / v) human serum albumin, and 0.1% to 0.25% (v / v) DMSO. In some embodiments, the pharmaceutical composition comprises Elliott's B solution, cell therapy, human serum albumin, and 0.1% to 0.30% (v / v) DMSO. In some embodiments, the pharmaceutical composition comprises 94% to 98% (v / v) Elliott's B solution, cell therapy, 0.3% to 0.6% (w / v) human serum albumin, and 0.05% to 0.25% (v / v) DMSO. In some embodiments, the pharmaceutical composition comprises 94% to 98% (v / v) Elliott's B solution, cell therapy, 0.3% to 0.6% (w / v) human serum albumin, and less than 0.25% (v / v) DMSO.

[0042] In some embodiments, the pharmaceutical composition comprises 94% to 98% (v / v) Elliott's B solution, 10 million ± 30% therapeutic cells per mL of the pharmaceutical composition, 0.3% to 0.6% (w / v) human serum albumin, and 0.1% to 0.25% (v / v) DMSO. In some embodiments, the pharmaceutical composition comprises Elliott's B solution, 10 million ± 30% therapeutic cells per mL of the pharmaceutical composition, human serum albumin, and 0.1% to 0.30% (v / v) DMSO. In some embodiments, the pharmaceutical composition comprises 94% to 98% (v / v) Elliott's B solution, 10 million ± 30% therapeutic cells per mL of the pharmaceutical composition, 0.3% to 0.6% (w / v) human serum albumin, and 0.05% to 0.25% (v / v) DMSO. In some embodiments, the pharmaceutical composition comprises 94% to 98% (v / v) Elliott's B solution, 10 million ± 30% therapeutic cells per mL of pharmaceutical composition, 0.3% to 0.6% (w / v) human serum albumin, and less than 0.25% (v / v) DMSO.

[0043] In some embodiments, the volume of the pharmaceutical composition is 5 to 15 mL, 7 to 13, 8 to 12, or 9 to 11 mL. In some embodiments, the volume of the pharmaceutical composition is less than or equal to 15 mL. In some embodiments, the volume of the pharmaceutical composition is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mL. In some embodiments, the volume of the pharmaceutical composition is 9 to 11 mL. In some embodiments, the volume of the pharmaceutical composition is 10 mL.

[0044] In some embodiments, the method of administering cell therapy to a subject includes generating a pharmaceutical composition. In some embodiments, the method includes obtaining a cell therapy formulation and diluting the cell therapy formulation to generate a pharmaceutical composition. The cell therapy formulation may comprise a cell therapy agent, a cryoprotectant (e.g., containing DMSO), and an excipient (e.g., human serum albumin). In some embodiments, the method includes obtaining a cell therapy formulation prepared in a separate facility, frozen, and delivered to the location of patient administration (e.g., a hospital). In some embodiments, the method includes thawing a frozen cell formulation and subsequently diluting the thawed cell formulation with a pharmaceutically acceptable excipient for central nervous system administration (e.g., Elliott's B solution). In some embodiments, the method includes diluting the cell therapy formulation 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 includes diluting the cell therapy formulation at least 20-fold. In some embodiments, the method includes diluting the cell therapy formulation 10 to 50-fold. In some embodiments, the method includes diluting the cell therapy formulation 20 to 40 times. In some embodiments, the cell therapy formulation is about 200 to 600 µL (e.g., about 300 µL) and diluted with about 10 mL of a pharmaceutically acceptable excipient for central nervous system administration (e.g., Elliott's B solution). In some embodiments, after dilution, the method includes administering the pharmaceutical composition to the central nervous system of a subject (e.g., as described herein).

[0045] Cell therapy

[0046] "Cell therapy" refers to the application of one or more types of cells to a subject for the purpose of treating a disease, symptom, or syndrome. In some embodiments, cell therapy comprises cells modified to contain therapeutic molecules (e.g., cells transfected with a polynucleotide encoding a chimeric antigen receptor (CAR)) and cells not modified to contain therapeutic molecules (e.g., cells not transfected with a polynucleotide encoding a CAR). For example, 30% of the cells in cell therapy may contain a polynucleotide encoding a CAR, and 70% of the cells in cell therapy may not contain a polynucleotide encoding a CAR.

[0047] In some embodiments, cell therapy includes T-cell therapy, chimeric antigen receptor (CAR)-T-cell therapy, engineered T-cell receptor (TCR) therapy, natural killer (NK) cell therapy, or CAR-NK cell therapy. In some embodiments, cell therapy includes stem cell therapy. In some embodiments, cell therapy includes non-stem cell therapy. In some embodiments, cell therapy includes autologous cell therapy. In some embodiments, cell therapy includes allogeneic cell therapy. In some embodiments, cell therapy includes single-cell therapy. In some embodiments, cell therapy includes multi-cell therapy. In some embodiments, cell therapy includes immune cells (e.g., T cells, NK cells, or macrophages) modified to bind to diseased cells (e.g., cancer cells). In some embodiments, the cells in cell therapy secrete therapeutic proteins (e.g., TEAM). For example, the cells may secrete bispecific antibodies that bind to both T cells and cancer cells. In some embodiments, cell therapy includes CAR-T cells (see the chimeric antigen receptor (CAR) T-cell section for further details on CAR-T cells).

[0048] In some embodiments, cell therapy comprises one or more types of cells administered to treat a disease. In some embodiments, the disease is 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 invasive 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, dysembryonic neuroepithelial tumor, glial neuronal tumor, gangliocytoma, multinodular and vacuolar neuronal tumor, neurocytoma, ependymoma, papilloma, carcinoma, medulloblastoma, rhabdomyosarcoma, cribriform neuroepithelial tumor, embryonal tumor, CNS neuroblastoma, pineal gland tumor, cranial nerve and paravertebral nerve tumor, or meningioma. In some embodiments, the disease is glioblastoma.

[0049] In some embodiments, the cell therapy comprises one or more types of cells administered to treat a condition. In some embodiments, the condition is Parkinson's disease, multiple sclerosis, ALS, or stroke. In some embodiments, the condition is an autoimmune disease. In some embodiments, the autoimmune disease is autoimmune encephalitis, autoimmune-related epilepsy, central nervous system (CNS) vasculitis, Hashimoto's encephalopathy (steroid-responsive encephalopathy), neuromyelitis optica, optic neuritis, neurosarcoma, neuro-Behcet's disease, or cerebral lupus.

[0050] Chimeric antigen receptor (CAR) T cells

[0051] In some aspects, the method involves administering CAR-T cells to the central nervous system of a subject. As used herein, “CAR-T cells” or “CAR-T” refers to T cells expressing a CAR. When expressed in T cells, CARs possess the ability to redirect T cell specificity and reactivity to a selected target in a non-MHC-restricted manner by utilizing the antigen-binding properties of monoclonal antibodies. This non-MHC-restricted antigen recognition confers the ability of CAR-expressing T cells to recognize antigens without antigen processing, thereby bypassing key mechanisms of tumor escape.

[0052] As used in this article, the term "chimeric antigen receptor" or "CAR" refers to an engineered T cell receptor on which a ligand or antigen is specifically grafted onto T cells (e.g., 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 immune receptors.

[0053] CARs place a chimeric antigen-binding domain on a construct comprising a transmembrane domain and an intracellular domain of a T-cell receptor molecule, the chimeric antigen-binding domain specifically binding to a target (e.g., a peptide) expressed on the cell surface targeted by a T-cell response. In some embodiments, the chimeric antigen-binding domain comprises an antigen domain of an antibody reagent that specifically binds to an antigen expressed on cells targeted by a T-cell response. In some embodiments, the chimeric antigen-binding domain comprises a ligand that specifically binds to an antigen expressed on cells targeted by a T-cell response.

[0054] In some implementations, the CAR does not contain the CD8 signal peptide described herein. As those skilled in the art will determine, various functionally similar or equivalent components of these CARs may be interchanged or substituted with each other, and with other similar or functionally equivalent components known in the art or listed herein.

[0055] Antigen-binding domain

[0056] As used herein, the term "antigen-binding domain" refers to a polypeptide present outside the cell sufficient to facilitate binding to a target. Extracellular target-binding domains will specifically bind to their binding chaperone (i.e., the target). As some non-limiting examples, an antigen-binding domain may comprise the antigen domain of an antibody or antibody reagent or ligand that recognizes and binds to a homologous binding chaperone protein. In this context, a ligand is a molecule that specifically binds to a portion of a protein and / or receptor. Homologous binding chaperones of ligands used in the methods and compositions described herein are typically present on the cell surface. Ligand: Homologous chaperone binding can lead to alterations in the receptor with the ligand, or activation of physiological responses, such as activation of signaling pathways. In some embodiments, the ligand may be non-natural with respect to the genome. In some embodiments, the ligand has a conserved function in at least two species.

[0057] CARs can target any cell surface region. In some implementations, the target will be a cell surface peptide that is differentially or preferentially expressed on the cell to which the T cell response is desired. To target Tregs, antibody reagents can target, for example, glycoprotein A repetitions predominant (GARP), latency-associated peptide (LAP), CD25, CTLA-4, ICOS, TNFR2, GITR, OX40, 4-1BB, and LAG-3.

[0058] In some embodiments, the CAR includes an antigen-binding domain that binds to an antigen expressed on a cancer or tumor of the central nervous system. For example, in some embodiments, the CAR includes an antigen-binding domain that binds to EGFRvIII, IL13Ra2, HER2, B7-H3, B7-H1, CD147, GD2, CAIX, CD70, CSPG4, EphA1, TROP2, or a survival protein.

[0059] In some embodiments, the CAR includes an antigen-binding domain that binds to EGFRvIII. In some embodiments, the antigen-binding domain includes (i) a VH domain containing three complementarity-determining regions (CDR-H1, CDR-H2, and CDR-H3), wherein CDR-H1 contains SEQ ID NO: 2, CDR-H2 contains SEQ ID NO: 3, and CDR-H3 contains SEQ ID NO: 4, and (ii) a VL domain containing three CDRs (CDR-L1, CDR-L2, and CDR-L3), wherein CDR-L1 contains SEQ ID NO: 5, CDR-L2 contains SEQ ID NO: 6, and CDR-L3 contains SEQ ID NO: 7. In some embodiments, the antigen-binding domain includes the amino acid sequence of SEQ ID NO: 8.

[0060] Hinges and transmembrane domains

[0061] In some embodiments, the CAR peptide further comprises a transmembrane domain, such as a hinge / transmembrane domain, which connects the antigen-binding domain to an intracellular signaling domain. In some embodiments, the binding domain of the CAR is followed by one or more hinge domains that function in positioning the antigen-binding domain away from the effector cell surface to achieve proper cell / cell contact, antigen binding, and activation. The CAR may include one or more hinge domains between the binding domain and the transmembrane domain (TM). The hinge domain may be derived from natural, synthetic, semi-synthetic, or recombinant sources. The hinge domain may contain an amino acid sequence of a naturally occurring immunoglobulin hinge region or a modified immunoglobulin hinge region. Exemplary hinge domains suitable for use in the CARs described herein include hinge regions derived from the extracellular regions of type 1 membrane proteins (e.g., CD8 (e.g., CD8α), CD4, CD28, 4-1BB, and CD7), which may be wild-type hinge regions from these molecules or may be modified hinge regions. In some embodiments, the CAR contains a polynucleotide encoding a CD8α hinge / transmembrane domain. In some embodiments, the CAR contains a polynucleotide encoding a 41BB intracellular domain.

[0062] 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 the CD8a hinge region.

[0063] As used herein, a “transmembrane domain” (TM domain) refers to a portion of the CAR that fuses an extracellular binding portion (via a hinge domain in some embodiments) with an intracellular portion (e.g., a co-stimulatory domain and an intracellular signaling domain) and anchors the CAR to the plasma membrane of an immune effector cell. The transmembrane domain is typically a hydrophobic region of the CAR that crosses the cytoplasmic membrane. The TM domain can be a transmembrane region or fragment of a transmembrane protein (e.g., type I transmembrane protein or other transmembrane proteins), an artificial hydrophobic sequence, or a combination thereof. While specific examples are provided and used herein, other transmembrane domains will be apparent to those skilled in the art and can be used in combination with alternative embodiments of the present technology. The selected transmembrane region or fragment thereof preferably does not interfere with the intended function of the CAR.

[0064] The term "fragment of" used in relation to the transmembrane domains of proteins or peptides refers to a portion of the transmembrane domain sufficient to anchor or attach the protein to the cell surface.

[0065] In some embodiments, the transmembrane domain or fragment thereof of the CAR described herein includes a transmembrane domain selected from the following transmembrane domains: α, β, or ζ chains of T cell receptors; CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154; KIRDS2, OX40, CD2, CD27; LFA-1 (CD11a, CD18); ICOS (CD278); 4-1BB (CD137); 4-1BBL; GITR; CD40; BAFFR; HVEM (LIGHTR); SLAMF7; NKp80 (KLRFI); CD160; CD19; IL2Rβ; IL2Rγ; IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c,

[0066] 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.

[0067] As used herein, "hinge / transmembrane domain" refers to a domain that contains both a hinge domain and a transmembrane domain. For example, the hinge / transmembrane domain may 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 contains the hinge / transmembrane domain of CD8 (e.g., SEQ ID NO: 9 or a variant thereof). CD8 is an antigen preferentially present 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 α chain (CD8α or CD8a) and a β chain (CD813 or CD8b). CD8a sequences are known in many species, such as the 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 its naturally occurring variants, molecules, and alleles. In some implementations of either aspect, such as in veterinary applications, CD8 can refer to, for example, the CD8 of dogs, cats, cattle, horses, pigs, etc.

[0068] For such species, those skilled in the art can easily identify human CD8 homologs and / or orthologs, for example, by using the NCBI ortholog search function or by searching for sequences similar to a reference CD8 sequence in the available sequence data for a given species.

[0069] 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 an 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 with the sequence of SEQ ID NO: 9.

[0070] Co-stimulatory domain

[0071] Each CAR described herein optionally includes an intracellular domain or co-stimulatory domain of one or more co-stimulatory molecules. As used herein, the term "co-stimulatory domain" refers to the 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 the effective activation and function of T lymphocytes after T lymphocytes bind to antigens. Co-stimulatory domains can be, for example, 4-1BB, CD27, CD28, or OX40 co-stimulatory domains. In some embodiments, the 4-1BB co-stimulatory domain may be used (see, for example, below and SEQ ID NO: 14 or a variant thereof). In some embodiments, the CD28 co-stimulatory domain may be used (see, for example, below and SEQ ID NO: 15 or a variant thereof). Other illustrative examples of such co-stimulatory molecules include: CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), 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 ZAP70. In some embodiments, the intracellular domain is the intracellular domain of 4-1BB. 4-1BB (CD137; TNFRS9) is an activation-inducing co-stimulatory molecule and an important regulator in the immune response.

[0072] 4-1BB is a membrane receptor protein, also known as CD137, and is a member of the tumor necrosis factor (TNF) receptor superfamily. 4-1BB is expressed on activated T lymphocytes. Sequences of 4-1BB are known in many species, such as human 4-1BB, also known as TNFRSF9 (NCBI Gene 25 ID: 3604) and its mRNA (NCBI reference sequence: NM_001561.5). 4-1BB can refer to human 4-1BB, including its naturally occurring variants, molecules, and alleles. In some embodiments of either aspect, such as in veterinary applications, 4-1BB can refer to, for example, 4-1BB in dogs, cats, cattle, horses, pigs, etc. For such species, those skilled in the art can readily identify homologs and / or orthologs of human 4-1BB, for example, using the NCBI ortholog search function or searching for sequences similar to a reference 4-1BB sequence in the available sequence data for a given species.

[0073] In some embodiments, the co-stimulatory domain comprises a 4-1BB co-stimulatory domain. In some embodiments, the 4-1BB co-stimulatory domain corresponds to an amino acid sequence selected from SEQ ID NO: 14; or comprises a sequence selected from SEQ ID NO: 14; or comprises a 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 at least 100% sequence identity with a sequence selected from SEQ ID NO: 14.

[0074] In some embodiments, the co-stimulatory domain comprises the co-stimulatory domain of CD28. In some embodiments, the CD28 co-stimulatory domain corresponds to an amino acid sequence selected from SEQ ID NO: 15; or comprises a sequence selected from SEQ ID NO: 15; or comprises a 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 at least 100% sequence identity with a sequence selected from SEQ ID NO: 15.

[0075] Intracellular signal transduction domains

[0076] In some implementations, the CAR contains a polynucleotide encoding a CD3ζ (CD3z) intracellular signaling domain (e.g., SEQ ID NO: 16 or a variant thereof).

[0077] The properties of the intracellular signal transduction domain of a CAR can vary as are known in the art and disclosed herein, but when the chimeric target / antigen binding domain binds to a target / antigen on the surface of a target cell, the chimeric target / antigen binding domain sensitizes the receptor to signal transduction activation.

[0078] Regarding intracellular signal transduction domains, so-called "first-generation" CARs include those that provide only CD3-ζ signaling upon antigen binding. So-called "second-generation" CARs include those that provide both co-stimulatory (e.g., CD28 or CD137) and activating (CD3-ζ) domains, and so-called "third-generation" CARs include those that provide multiple co-stimulatory (e.g., CD28 and CD137) domains and activating (signal transduction) domains (e.g., CD3-ζ). In various implementations, CARs with high affinity or affinity for the target / antigen are selected—for example, antibody-derived target or antigen-binding domains typically have higher affinity and / or affinity for the target antigen compared to naturally occurring T cell receptors. This characteristic, combined with the high specificity of the antibodies available for selection, provides highly specific T cell targeting for CAR-T cells.

[0079] The CARs described herein include intracellular signal transduction domains. An “intracellular signal transduction domain” refers to a portion of the CAR polypeptide that participates in transducing effective CAR information bound to a target antigen into the interior of immune effector cells to elicit effector cell functions, such as activation, cytokine production, proliferation, and cytotoxic activity, including the release of cytotoxic factors to the CAR-bound target cell, or the eliciting of other cellular responses following the binding of the antigen to the extracellular CAR domain. In some embodiments, the intracellular signal transduction domain is derived from CD3-ζ; (see, for example, below). Other non-limiting examples of intracellular signal transduction domains comprising immunoreceptor tyrosine-based activation motifs (ITAMs) that are particularly useful in this art include intracellular signal transduction domains derived from TCR-ζ, FcR-γ, FcR-β, CD3-γ, CD3-θ, CD3-σ, CD3-η, CD3-ε, CD3-ζ, CD22, CD79a, CD79b, and CD66d.

[0080] CD3 is a T cell co-receptor that promotes T lymphocyte activation when it binds to an appropriate co-stimulus (e.g., to a co-stimulatory molecule). The CD3 complex consists of four distinct chains; mammalian CD3 consists of one CD3-γ chain, one CD3δ chain, and two CD3-ε chains.

[0081] These chains associate with CD3-ζ and molecules known as the T cell receptor (TCR) to generate activation signals in T lymphocytes. The complete TCR complex consists of the TCR, CD3-ζ, and the complete CD3 complex.

[0082] In some embodiments of any aspect, the CAR peptide described herein comprises an intracellular signaling domain containing an immune receptor tyrosine-based activation motif or an ITAM derived from CD3-ζ, wherein CD3-ζ includes variants of CD3-ζ, such as ITAM-mutated CD3-ζ, CD3-η, or CD3-θ. In some embodiments of any aspect, the ITAM comprises the three motifs of the CD3-ζ ITAM (ITAM3). In some embodiments of any aspect, the three motifs of the CD3-ζ ITAM are not mutated and therefore comprise natural or wild-type sequences. In some embodiments, the CD3-ζ sequence comprises the CD3-ζ sequence shown in the sequences provided herein, such as the CD3-ζ sequence of SEQ ID NO: 16 or a variant thereof.

[0083] For example, the CAR peptide described herein contains an intracellular signal transduction domain of CD3-ζ. In some embodiments, the CD3-ζ intracellular signal transduction domain corresponds to the amino acid sequence of SEQ ID NO: 16 or contains a sequence of SEQ ID NO: 16; or contains a 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 with the sequence of SEQ ID NO: 16.

[0084] As can be determined by those skilled in the art, the individual CARs and other construct components described herein can be used together and interchanged with and substituted into the various constructs described herein. Each of these components may comprise any corresponding sequence or variant thereof shown herein.

[0085] Further descriptions of CAR 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 these is incorporated herein by reference in its entirety.

[0086] signal peptide

[0087] As used herein, “signal peptide” or “signal sequence” refers to a peptide at the N-terminus of a newly synthesized protein that directs the nascent protein into the endoplasmic reticulum. In some embodiments, the CAR polypeptide described herein comprises a signal peptide. The signal peptide may be derived from any protein having an extracellular domain or a secreted protein. The CAR polypeptide described herein may comprise any signal peptide known in the art. In some embodiments, the CAR polypeptide comprises a CD8 signal peptide, such as the amino acid sequence corresponding to SEQ ID NO: 1 or a CD8 signal peptide comprising 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 with the sequence of SEQ ID NO: 1. In some embodiments, the CAR polypeptide described herein may optionally not comprise one of the signal peptides described herein, such as the CD8 signal peptide of SEQ ID NO: 1 or the IgK signal peptide of SEQ ID NO: 17.

[0088] Joint structural domain

[0089] In some embodiments, the CAR also includes a linker domain. As used herein, "linker domain" refers to an oligopeptide or polypeptide region of about 2 to 100 amino acids in length that links together any domains / regions of the CAR described herein. In some embodiments, the linker may comprise or be composed of flexible residues (e.g., glycine and serine) such that adjacent protein domains are free to move relative to each other. Linker sequences usable in this invention can be 2 to 100 amino acids in length, 5 to 50 amino acids, 10 to 15 amino acids, 15 to 20 amino acids, or 18 to 20 amino acids, and may include any suitable linker known in the art. For example, linker sequences usable in this invention include, but are not limited to, glycine / serine linkers, for example… (SEQ ID NO: 18) and Gly4Ser (G4S) connectors, such as (G4S)3 ( (SEQ ID NO: 19) and (G4S)4 ( (SEQ ID NO: 20)); as described by Whitlow et al., Protein Eng. 6(8):989-95 (the contents of which are incorporated herein by reference in their entirety). (SEQ ID NO: 21); as described by Andris-Widhopf et al., Cold Spring Harb. Protoc. 2011 (9), 2011 (the contents of which are incorporated herein by reference in their entirety). (SEQ ID NO: 22); and adapter sequences with additional functionality, such as epitope tags or coding sequences containing Cre-Lox recombination sites as described by Sblattero et al., Nat. Biotechnol. 18(1):75-80, 2000 (the contents of which are incorporated herein by reference in their entirety). Longer adapters can be used when it is desirable to ensure that two adjacent domains do not interfere with each other spatially.

[0090] Furthermore, the connector can be cuttable or non-cuttable. Some examples of cuttable connectors include 2A connectors (e.g., P2A (SEQ ID NO: 23) and T2A (SEQ ID NO: 24), 2A-type connectors or their functional equivalents, and combinations thereof.

[0091] In several instances, adapters having the sequences shown herein or variants thereof are used. It should be understood that the identification of a particular adapter in a construct at a specific location does not imply that only that adapter is usable therein. Rather, as those skilled in the art can determine, different adapter sequences (e.g., P2A, E2A, F2A, and T2A) can be interchanged (e.g., in the context of the constructs of this invention). In some embodiments, the adapter region is T2A derived from the virus *Thoseaasigna*. Some non-limiting examples of adapters that can be used in this technology include T2A, P2A, E2A, BmCPV2A, and BmlFV2A. These adapters can be used, for example, in the case of multi-proteins, such as the adapters described below. For example, they can be used to separate the CAR component of a multi-protein from the therapeutic agent (e.g., antibody, such as scFv, single-domain antibody (e.g., camelid antibody), or bispecific antibody (e.g., TEAM)) component in the multi-protein (see below).

[0092] Full CAR

[0093] In some implementations, the CAR includes an antigen-binding domain that binds to EGFRvIII, IL13Ra2, HER2, B7-H3, B7-H1, CD147, GD2, CAIX, CD70, CSPG4, EphA1, TROP2, or a surviving protein.

[0094] In some embodiments, the CAR comprises a polynucleotide encoding an antigen-binding domain that binds to EGFRvIII, a CD8α hinge / transmembrane domain, a 41BB co-stimulatory domain, and a CD3ζ signaling domain. In some embodiments, the CAR peptide comprises 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 higher sequence identity with a sequence selected from any one of SEQ ID NO: 10 to 11. In some embodiments, the CAR comprises an amino acid sequence from any one of SEQ ID NO: 10 to 11.

[0095] T-cell conjugation molecules (TEAM)

[0096] In some embodiments, the CAR-T cells described herein express a T-cell conjugating molecule (TEAM) (also referred to in the literature as a bispecific T-cell conjugate or BiTE™). “T-cell conjugating molecule,” “TEAM antibody construct,” or “TEAM” means a polypeptide, each comprising tandemly linked single-chain variable fragments (scFvs). Optionally, the scFvs are linked via a linker (e.g., a glycine-rich linker). One scFv of the TEAM binds to a T-cell receptor (TCR) (e.g., to a CD3 subunit), and the other binds to a target antigen (e.g., a tumor antigen). Such a molecule can target T cells by binding to both the T-cell antigen (e.g., by binding to CD3) and the target antigen (e.g., a tumor antigen). The TEAM can be used to enhance T-cell responses, for example, in the tumor microenvironment. The two components of the TEAM can also be linked in either orientation, for example, the anti-CD3 component at the N-terminus of the anti-target antigen component, or vice versa. The anti-CD3 component or the anti-target antigen component of the TEAM may contain any antibody reagent described herein.

[0097] In some embodiments, the CAR-T cells described herein secrete a TEAM. In some embodiments, the TEAM comprises a secretion tag (e.g., an IgK signal peptide). The TEAM may act paracrinely, for example, by stimulating the CAR-T cells themselves, or by redirecting nonspecific bystander T cells to the tumor, thereby enhancing the antitumor effect of CAR-T cell immunotherapy. CAR-T cell-mediated TEAM secretion may allow for a reduction in the risk of undesirable TEAM activity in systemic tissues by directing TEAM secretion into the tumor microenvironment. Exemplary TEAM constructs are provided below; however, TEAMs other than those described herein may also be used in the CAR T cells and methods of this disclosure.

[0098] In some embodiments, the anti-CD3 scFv of any TEAM described herein may be arranged in a VH-VL orientation or a VL-VH orientation. In some embodiments, the anti-CD3 scFv comprises (i) a VH domain containing three complementarity-determining regions (CDR-H1, CDR-H2, and CDR-H3), wherein CDR-H1 contains SEQ ID NO: 32, CDR-H2 contains SEQ ID NO: 33, and CDR-H3 contains SEQ ID NO: 34, and (ii) a VL domain containing three CDRs (CDR-L1, CDR-L2, and CDR-L3), wherein CDR-L1 contains SEQ ID NO: 35, CDR-L2 contains SEQ ID NO: 36, and CDR-L3 contains 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 higher sequence identity with the amino acid sequence of SEQ ID NO: 13.

[0099] In some embodiments, the anti-EGFR scFv of any TEAM described herein may be arranged in a VH-VL orientation or a VL-VH orientation. In some embodiments, the anti-EGFR scFv comprises (i) a VH domain containing three complementarity-determining regions (CDR-H1, CDR-H2, and CDR-H3), wherein CDR-H1 contains SEQ ID NO: 26, CDR-H2 contains SEQ ID NO: 27, and CDR-H3 contains SEQ ID NO: 28, and (ii) a VL domain containing three CDRs (CDR-L1, CDR-L2, and CDR-L3), wherein CDR-L1 contains SEQ ID NO: 29, CDR-L2 contains SEQ ID NO: 30, and CDR-L3 contains 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 higher sequence identity with the amino acid sequence of SEQ ID NO: 12.

[0100] In some embodiments, the CAR and TEAM encode the same polypeptide. In some embodiments, the CAR and TEAM encoded on the same polypeptide are separated by a linker domain as described above. In some embodiments, the linker domain is cleavable. In some embodiments, the CAR is an anti-EGFRvIII CAR, and the TEAM is an EGFR and CD3 TEAM. In some embodiments, the CAR and TEAM polypeptides comprise 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 higher sequence identity with SEQ ID NO: 39. In some embodiments, the CAR and team polypeptides comprise the amino acid sequence of SEQ ID NO: 39. In some embodiments, the CAR and team polypeptides consist of the amino acid sequence of SEQ ID NO: 39.

[0101] sequence

[0102]

[0103] Example

[0104] Example 1. Administering cell therapy containing DMSO to the central nervous system.

[0105] This embodiment demonstrates that cell therapy containing DMSO can be administered to the brain without causing DMSO-related neurotoxicity. DMSO-related neurotoxicity events include transient sensory abnormalities, altered mental status, syncope, stroke, seizures, and death. These events can occur during intravenous administration before the completion of cell therapy administration and after administration until the DMSO leaves the body. In the central nervous system, DMSO-related toxicity will occur within the first eight hours after administration of DMSO to the central nervous system because cerebrospinal fluid is replaced approximately every eight hours in the central nervous system, thus clearing away the vast majority of DMSO. Huff, Trevor, et al., “Neuroanatomy, cerebrospinal fluid.” (2017).

[0106] Cell therapy comprising DMSO and CAR-T cells was injected into the intraventricular space of multiple patients diagnosed with glioblastoma. The CAR-T cells possessed EGFRvIII-binding CARs and secreted EGFR-CD3-binding TEAMs (see SEQ ID NO: 39). The pharmaceutical composition for administration was prepared from a cell therapy formulation containing approximately 5% (v / v) DMSO, 1 × 10⁻⁶ CARs. 7 ± 30% CAR-T cells, with a volume of approximately 300 to 500 µL. The following protocol was used for each patient. After thawing, the cell therapy formulation was diluted with Elliott's B solution to approximately 10 mL to produce a drug composition ready for administration to the patient. This dilution reduced the DMSO concentration to less than 0.25% (v / v). The drug composition was then administered intraventrally using an Omayer reservoir.

[0107] No neurotoxic events were recorded in any patient during the 8-hour period following administration, indicating that cell therapy formulations with less than 0.25% (v / v) DMSO can be administered to the central nervous system without causing neurotoxicity.

[0108] Due to concerns about the neurotoxicity of DMSO, it was not previously included in cell therapies administered to the brain. This concern led to protocols requiring multiple washing steps. These lengthy protocols increased processing time and could potentially result in a loss of cell count and viability during cell therapy. However, this protocol (diluting DMSO, eliminating the need for cell counting or washing steps) allows for faster administration of cell therapy to patients after thawing. This reduces labor costs, potential cell loss, and processing errors.

[0109] Example 2. Rapid regression of recurrent glioblastoma after a single infusion of CAR-T EGFR-CD3 TEAM cells

[0110] Glioblastoma (GBM) is the most aggressive primary brain tumor, and recurrent disease has a very poor prognosis with no effective treatment options. Chimeric antigen receptor (CAR) T cells represent a promising approach to cancer, as evidenced by their proven efficacy in refractory lymphomas, in which they have become standard of care. However, the use of CAR-T cells in solid tumors such as GBM has been limited to date, primarily due to targeting a single antigen in heterogeneous disease and the large intratumoral infiltration of inhibitory regulatory T cells (Tregs) that impedes the immune response. To address these obstacles, this paper presents engineered T cell products that target epidermal growth factor receptor variant III (EGFRvIII) via a second-generation CAR while simultaneously secreting a bispecific antibody against wild-type epidermal growth factor receptor (EGFR) (called T cell conjugating antibody molecules (TEAM)) (“CAR-TEAM cells”), which is not expressed in the normal brain but is almost always expressed in GBM. TEAM secreted by CAR-TEAM cells acts locally at the CAR T cell homologous antigen binding site to treat heterogeneous tumors in preclinical models, and these molecules have the ability to redirect even Tregs, transforming them into cytotoxic killers. Based on these data, a first-in-human, phase I clinical trial evaluating the safety of CAR-TEAM cells in patients with relapsed or newly diagnosed GBM was initiated, and is further described below.

[0111] method

[0112] Research supervision and design

[0113] This study was approved by the Dana-Farber Harvard Cancer Center Institutional Review Board. An external data and safety monitoring committee oversaw the conduct of this study. This is a non-randomized, open-label, single-center phase I study.

[0114] Three patients (referred to as Patient 1, Patient 2, and Patient 3) were recruited into the Safety Run-InArm group, among whom those with recurrent EGFRvIII-positive GBM were treated with 10 × 10 6 CAR-TEAM cell / infusion (administered via Omaye reservoir) therapy.

[0115] Molecular testing

[0116] Multiple methods were used to determine EGFRvIII expression and EGFR amplification, as well as methods for isolating RNA from extracellular vesicles, during the experiments. Molecular alterations in patient samples, such as EGFRvIII expression and EGFR amplification, were assessed using routine clinical assays. These assays included targeted variant and fusion next-generation sequencing using chemical methods based on anchored multiplex PCR, and in some cases, additional sequencing of targeted amplicones on a complementary platform (Oncomine Precision Assay, Ion TorrentGenexus). EGFR copy number status was routinely assessed via the sequencing pipeline and, in some cases, determined using routine clinical fluorescence in situ hybridization (FISH) assays.

[0117] Preparation of CAR-TEAM T cells

[0118] Autologous T cells were obtained through leukoablation and transduced using a CAR-TEAM lentiviral vector containing an anti-EGFRvIII single-stranded variable fragment and an intracellular 4-1BB co-stimulatory domain linked to CD3-ζ. Following a ribosomal jumping element, an EGFR-targeting TEAM was also included in the same vector.

[0119] The third transgene encoding the truncated CD19 (tCD19) molecule was used as a surface marker for transduction. Figure 1A ). Detection of TEAM binding to T cells using biotinylated human EGFR ( Figures 1B to 1C Details regarding the cell product release criteria are shown in Table 1 below. CAR-TEAM cells were thawed and then stored at 10 × 10⁻⁶ cells / day. 6 Prepare the target dose for each CAR-positive cell / vial. Thaw, resuspend, and transfer the cells to a syringe under aseptic conditions, followed by infusion.

[0120] Table 1. Production completion data for CAR-TEAM.

[0121]

[0122] Toxicity analysis

[0123] Toxicity monitoring was conducted on patients throughout the study duration. Adverse events were graded according to the Common Toxicity Criteria for Adverse Events version 5.0 (CTCAEv5). Dose-limiting toxicity (DLT) was defined as an event that was at least likely related to the study product, not attributable to disease progression, and which of the following was true: any grade 4 or higher related CTCAEv5 adverse event, or any related CTCAEv5 adverse event 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.

[0124] result

[0125] Patients included in the case series

[0126] Between March and July 2023, three patients with recurrent glioblastoma were recruited at Massachusetts General Hospital for a Phase I study.

[0127] Patient 1

[0128] A 74-year-old right-handed male (Patient 1) presented with a one-week history of headache and confusion, and imaging revealed an enhancing mass in the left insula. Patient 1 underwent craniotomy, which revealed a diagnosis of GBM, with wild-type isocitrate dehydrogenase (IDH) and O6-methylguanine-DNA methyltransferase (MGMT) methylation. The tumor was EGFRvIII positive and EGFR gene copy number increased. Patient 1 received standard care consisting of radiation and temozolomide chemotherapy. Twelve months after diagnosis, magnetic resonance imaging (MRI) suggested disease recurrence. Repeat craniotomy was performed, and the recurrent tumor was confirmed to be EGFRvIII positive. Figure 2A Patient 1 was subsequently recruited into a Phase I clinical trial.

[0129] A pre-treatment MRI was obtained, which showed active progression ( Figure 2BPatient 1 underwent ventriculostomy with placement of a catheter containing an Omaye reservoir. CAR-TEAM cells (10 × 10⁻⁶ cells) were subsequently administered via the catheter device on postoperative day 1. 6 CAR-positive T cells). MRI obtained on day +1 following this single infusion showed rapid tumor regression ( Figure 2B (Right). On a subsequent MRI obtained within 31 days, this remained stable according to the Response Assessment in Neuro-Oncology (RANO) criteria. The patient received a second 10 × 10 MRI on day +37. 6 Infusion of CAR-TEAM cells ( Figure 2A ).

[0130] Liquid biopsies were performed on extracellular vesicle (EV) RNA derived from cerebrospinal fluid (CSF) and peripheral blood at several time points during each treatment cycle. EGFRvIII and EGFR copy numbers increased during cycle 1 and decreased over time, eventually becoming undetectable in post-treatment CSF. Figure 2C EV RNA from peripheral blood samples obtained before and after the study also showed decreased copy numbers of both EGFRvIII and EGFR. Figure 2D The patient returned to the operating room for a biopsy on day +72 after progression was shown on intermittent MRI. Consistent with the liquid biopsy, next-generation sequencing (NGS) of the tumor tissue was negative for both EGFRvIII and EGFR copy number alterations. However, post-hoc evaluation by FISH showed a continued increase in EGFR copy number (ratio 3.5). Figure 2E ).

[0131] Patient 2

[0132] A 72-year-old right-handed male (Patient 2) presented with dyslexia and an MRI revealed a contrast-enhancing mass in the left posterior temporal lobe. Patient 2 underwent craniotomy for tumor resection, and pathology confirmed GBM, which was EGFRvIII positive, IDH wild-type, and MGMT methylated. The patient was treated with standard-care radiation and temozolomide chemotherapy, as well as a tumor treatment field. Twenty months after the initial diagnosis, monitoring imaging showed evidence of progression. Then, according to a new schematic workflow ( Figure 3APatient 2 was recruited into a Phase I clinical trial based on their EGFRvIII status at initial diagnosis. The workflow had been modified to allow for craniotomy, tissue sampling, and Omaye placement during a single procedure. Molecular histopathological analysis at the time of repeat craniotomy was consistent with recurrent GBM, showing EGFRvIII positivity but negative for EGFR copy number increase. Patient 2 was discharged and readmitted one week later for infusion. The patient received 10 × 10 oz. via intraventricular catheter. 6 CAR-TEAM cells. On day +2, an MRI showed a 18.5% reduction in tumor cross-sectional area, and on day +69, a further 60.7% reduction from pre-infusion baseline. Figure 3B ).

[0133] Patient 3

[0134] A 57-year-old right-handed woman (Patient 3) presented with word-finding dysphagia for several weeks and was concerned about seizures. MRI revealed a contrast-enhancing mass in the left parietal lobe. The patient underwent craniotomy and tumor resection, and pathology showed unmethylated GBM, IDH wild-type, and MGMT. The tumor was EGFRvIII expressed and positive for increased EGFR gene copy number. Patient 3 was treated with radiation and temozolomide chemotherapy, with dose reduction due to thrombocytopenia. Six months after diagnosis, mitral imaging showed evidence of recurrent disease. The patient was recruited for a Phase I clinical trial (… Figure 3A The patient underwent repeated craniotomies for open biopsy and implantation of a ventricular catheter with an Omaye reservoir. Histological analysis at the time of recurrence surgery showed loss of EGFRvIII expression and EGFR gene amplification deletion on NGS, as determined by FISH. Pre-infusion MRI was performed, which again showed extensive recurrent disease burden. Figure 3C (Left). The patient was discharged and readmitted two weeks later for intraventricular catheter infusion of 10 × 10 6 One CAR-TEAM cell. MRI obtained on day +5 after a single infusion showed almost complete tumor regression ( Figure 3C (right), although there was no EGFRvIII expression or wild-type gene amplification before treatment.

[0135] Safety and adverse events

[0136] No related DLTs were reported in this safety induction cohort. Grade 3 events at least attributable to the study product included Grade 3 encephalopathy lasting 3 days (Patient 1, likely related) and Grade 3 fatigue lasting 8 days (Patient 3, related). One death occurred after disease progression, 63 days after the patient withdrew from the study (Patient 1); the cause of death was gastrointestinal perforation during bevacizumab and dexamethasone administration. This event was not attributable to CAR-TEAM cell infusion. Patients 2 and 3 presented with transient pulmonary nodules and ground-glass opacities on chest CT during periodic febrile examinations. These were otherwise asymptomatic and spontaneously resolved on septal imaging within 4 to 6 weeks. Adverse events are shown in Tables 2 through 7 below.

[0137] Table 2. Adverse Event Log: Patient 1.

[0138]

[0139] SAE = Serious adverse event; PAC / PVC = Atrial premature contraction / ventricular premature contraction; CRS = Cytokine release syndrome; ALT = Alanine aminotransferase; AST = Aspartate aminotransferase; SIADH = Inappropriate secretion of antidiuretic hormone syndrome; UTI = Urinary tract infection.

[0140] Table 3. Summary of Adverse Events: Patient 1

[0141]

[0142] Table 4. Adverse Event Log: Patient 2

[0143]

[0144] RUE = Right upper limb 1; BLE = Both lower limbs

[0145] Table 5. Summary of Adverse Events: Patient 2

[0146]

[0147] Table 6. Adverse Event Log: Patient 3.

[0148]

[0149] Table 7. Summary of Adverse Events: Patient 3

[0150]

[0151] Relevant data

[0152] All patients presented with fever, which peaked on day +2 after infusion. Figure 4A Intravenous treatment with the interleukin-1 receptor antagonist anaraxin (100 mg Q6H) was intermittently used for fever management. Figure 4B Systemic inflammatory markers were elevated, peaking in the second and third weeks after infusion, and returning to near baseline by day +30. Figure 5 A). No patients required steroids during the post-acute treatment phase or for any treatment-related indication. Cytopathological analysis of CSF showed an immediate increase in total nucleated cell count after infusion, which decreased exponentially on day +7 and then gradually declined over time. The cytovariance of CSF was initially neutrophil-dominated, then shifted to a predominantly lymphocyte population within 30 days after infusion. Figure 6 Quantitative analysis by vector copy number (VCN) and flow cytometry showed that CAR-positive and TEAM-positive T cells were transiently present in CSF after infusion, but decreased by week 4. Figure 7A This corresponds to the normalization of the inflammatory cytokine environment in the CSF compartment during the same period. Figure 7B CAR-TEAM cells were detected in peripheral blood 2 to 3 weeks after infusion, but their presence was transient. Figure 7C ).

[0153] discuss

[0154] This article presents an early-stage, first-in-human experience of intraventricular delivery of CAR-TEAM cells in patients with relapsed GBM. Significant radiographic responses were observed in multiple patients within days following a single intraventricular infusion of dual-targeting CAR-TEAM cells. Liquid biopsies have been reported as a relevant study of cell therapy in patients with GBM. While CAR-T cells have been successfully converted for hematologic malignancies, the efficacy of this strategy for solid tumors such as GBM remains unproven. Previously described CAR-T cells and bispecific antibodies for patients with GBM target only a single antigen and are therefore limited by tumor heterogeneity, the occurrence of antigen loss, and eventual immune escape. The experience with CAR-TEAM cells described in this example provides a proof-of-principle for using CAR-T cells to simultaneously target multiple surface antigens and identifies EGFRvIII as a suitable immunotherapeutic target for GBM. Furthermore, the secreted bispecific antibody was safe, demonstrating the feasibility of locally targeting antigens such as wild-type EGFR using this method, despite its widespread expression in systemic tissues. This example demonstrates that an antitumor CAR-mediated response can be achieved even in patients with GBM, particularly in cases of advanced, intraparenchymal brain disease. This contrasts with a previous report that observed a complete response in relapsed leptomeningeal disease (LMD) treated with repeated intracranial infusions (16 infusions) of monospecific interleukin-13 receptor alpha-2 (IL-13Rα2) CAR-T cells. Here, the results indicate that intraventricularly administered “live drugs” such as CAR-TEAM cells also possess the ability to enter infiltrative parenchymal GBM and mediate activity against it. Notably, CAR-TEAM cells showed signs of antitumor activity in the absence of EGFRvIII expression (patient 3). Of course, the interpretation of these data remains limited by the sensitivity of the corresponding assays for determining EGFRvIII expression and the inherent limitations of tissue sampling in heterogeneous diseases. However, the potential efficacy of CAR-TEAM cells in EGFRvIII-negative gliomas replicates our previous observations of CAR-independent, TEAM-mediated antitumor activity in our preclinical studies.

Claims

1. A method of administering cell therapy to a human subject, the method comprising administering a pharmaceutical composition comprising the cell therapy and dimethyl sulfoxide (DMSO) to the central nervous system of the subject.

2. A method for administering cell therapy to a subject, the method comprising: (i) Obtaining a cell therapy formulation comprising the cell therapy and DMSO; (ii) Dilute the cell therapy preparation to produce a pharmaceutical composition containing less than or equal to 0.5% DMSO (v / v); and (iii) The pharmaceutical composition is administered to the central nervous system of the subject.

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

4. The method of any one of claims 1 to 3, wherein administration comprises administering the pharmaceutical composition to the brain of the subject.

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

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

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

8. The method of claim 1 or claim 2, wherein administering the pharmaceutical composition comprises administering it to the cerebrospinal fluid of the subject.

9. The method of claim 8, wherein the application further comprises applying it to the spinal cerebrospinal fluid of the subject.

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

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

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

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

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

15. The method of any one of claims 1 to 14, wherein the method does not include washing the cells to remove DMSO.

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

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

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

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

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

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

22. The method of any one of claims 1 to 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 to 23, wherein the pharmaceutical composition comprises a cryoprotectant.

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

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

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

28. The method of any one of claims 2 to 26, wherein dilution comprises dilution using Elliott's B solution.

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

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

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

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

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

34. The method of claim 33, wherein the CAR-T cell therapy comprises a polynucleotide encoding a CAR, the 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 complementary determinant regions (CDR-H1, CDR-H2, and CDR-H3), wherein CDR-H1 contains SEQ ID NO: 2, CDR-H2 contains SEQ ID NO: 3, and CDR-H3 contains SEQ ID NO: 4, and (ii) A VL domain containing three CDRs (CDR-L1, CDR-L2 and CDR-L3), wherein CDR-L1 contains SEQ ID NO: 5, CDR-L2 contains SEQ ID NO: 6 and CDR-L3 contains SEQ ID NO:

7.

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

11.

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

38. The method of any one of claims 34 to 36, wherein the CAR-T cell therapy comprises a polynucleotide encoding a T-cell conjugating antibody molecule (TEAM), the T-cell conjugating 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 complementary determinant regions (CDR-H1, CDR-H2, and CDR-H3), wherein CDR-H1 contains SEQ ID NO: 26, CDR-H2 contains SEQ ID NO: 27, and CDR-H3 contains SEQ ID NO: 28, and (ii) A VL domain containing three CDRs (CDR-L1, CDR-L2 and CDR-L3), wherein CDR-L1 contains SEQ ID NO: 29, CDR-L2 contains SEQ ID NO: 30 and CDR-L3 contains 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 complementary determinant regions (CDR-H1, CDR-H2, and CDR-H3), wherein CDR-H1 contains SEQ ID NO: 32, CDR-H2 contains SEQ ID NO: 33, and CDR-H3 contains SEQ ID NO: 34, and (ii) A VL domain containing three CDRs (CDR-L1, CDR-L2 and CDR-L3), wherein CDR-L1 contains SEQ ID NO: 35, CDR-L2 contains SEQ ID NO: 36 and CDR-L3 contains SEQ ID NO:

37.

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

38.

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

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