WO2025080994A1

WO2025080994A1 - Chimeric antigen receptor (car)-t cell engaging antibody molecule (team) formulations and pharmaceutical compositions

Info

Publication number: WO2025080994A1
Application number: PCT/US2024/050985
Authority: WO
Inventors: Heather L. DALEY; Marcela V. Maus; Bryan CHOI
Original assignee: General Hospital Corp; Dana Farber Cancer Institute Inc
Current assignee: General Hospital Corp; Dana Farber Cancer Institute Inc
Priority date: 2023-10-13
Filing date: 2024-10-11
Publication date: 2025-04-17
Anticipated expiration: 2026-04-13

Abstract

Disclosed herein, in part, are chimeric antigen receptor (CAR)-T cell engaging antibody molecule (TEAM)-T cell formulations (e g., for storage of CAR-T cells prior to administration) and CAR-TEAM-T cell pharmaceutical compositions for administration to a subject's central nervous system.

Description

CHIMERIC ANTIGEN RECEPTOR (CAR)-T CELL ENGAGING ANTIBODY MOLECULE (TEAM) FORMULATIONS AND PHARMACEUTICAL COMPOSITIONS

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) to U.S. Provisional Application 63/590,128, filed October 13, 2023, entitled “Chimeric Antigen Receptor (CAR)-T Cell Engaging Antibody Molecule (TEAM) Formulations and Pharmaceutical Compositions” and U.S. Provisional Application 63/600,564, filed November 17, 2023, entitled “Chimeric Antigen Receptor (CAR)-T Cell Engaging Antibody Molecule (TEAM) Formulations and Pharmaceutical Compositions”, the entire contents of each of which is incorporated herein by reference.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing (M105370047WO00-SEQ-ARM.xml; Size: 38,959 bytes; and Date of Creation: October 8, 2024) is herein incorporated by reference in its entirety.

BACKGROUND

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. CAR-T cells that are for human administration are generally prepared at a specialized facility then the CAR-T cells are frozen in a formulation for transport to a medical facility where the cell therapy is administered to the patient. The formulation generally comprises a cryoprotect to protect the CAR-T cells during freezing. However, CAR-T cell cryoprotects commonly included in formulations are known to be neurotoxic.

SUMMARY

A general challenge in preparing a CAR-T cell formulation is producing a formulation that maintains the CAR-T cell therapy efficacy after the CAR-T cells are made but before administration to the patient (e.g., while frozen during shipping), and is also compatible with administration to patients. This is particularly challenging for administering CAR-T cell into the central nervous system (e.g., injection into the cerebral spinal fluid), for which a clinical best practice has not been established. One strategy for doing this is developing a formulation for stable storage of CAR-T cells, and then washing away any potentially toxic components of the formulation prior to administration to the patient. However, this can result in CAR-T cell death and may require additional quality control steps when administering the CAR-T cells to patients (e g., measuring the CAR-T cells in the washed formulation to determine their quantity and activity). Alternatively, the inventors of instant application identified a formulation comprising cryoprotectant that maintains sufficient CAR-T cell therapy efficacy, and that can be diluted to reduce cryoprotectant concentration and in turn decrease or eliminate adverse side effects from the cryoprotectant (e.g., during administration of the CAR-T cells to the central nervous system). Herein, this diluted formulation is referred to as a pharmaceutical composition. One aspect of this disclosure describes a formulation developed to decrease the volume of the formulation while increasing the number of T cells in the formulation. Decreasing the volume of the formulation can decrease the cryoprotect in the formulation, and thus decrease the percentage of cryoprotect in the pharmaceutical composition and decrease the amount of diluent needed to dilute the formulation. This is an important consideration in central nervous system administration given the relatively small volume of the central nervous system and the known toxicity of cryoprotectants on the central nervous system. However, decreasing the volume of the formulation and increasing the concentration of CAR-T cells was found to decrease CAR-T cell viability, and thus, many iterations of the formulation and pharmaceutical composition were needed to result in a formulation that can be diluted (instead of washed) and the corresponding pharmaceutical composition maintains sufficient cell viability for patient administration (about 1 x 10⁷ CAR-T cells in the pharmaceutical composition +/- 30%). The pharmaceutical compositions were administered into the central nervous system of human patients and treated glioblastoma multiform.

In some aspects, this disclosure describes, a formulation comprising: (i) 20 million to 250 million T cells per milliliter (mL) of the formulation, wherein at least 1% of the T cells are chimeric antigen receptor (CAR)-T cells comprising a polynucleotide encoding: (a) a CAR comprising an antigen binding domain that binds to EGFRvIII; and (b) a T cell engaging antibody molecule (TEAM) that comprises an anti-EGFR antibody and an anti-CD3 antibody; (ii) 10-15% weight by volume (w/v) Human Serum Albumin; and (iii) 2.5-7.5% volume by volume (v/v) dimethyl sulfoxide (DMSO). In some embodiment, the formulation comprises 30-200 million of the CAR-T cells per mL of the formulation. In some embodiment, the formulation comprises 50-150 million of the CAR-T cells per mL of the formulation. In some embodiment, the formulation comprises 30-200 million of the CAR-T cells per mL of the formulation. In some embodiment, the formulation comprises about 200 million of the CAR-T cells per mL of the formulation. In some embodiment, the formulation comprises 200 million of the CAR-T cells per mL of the formulation. In some embodiment, the formulation comprises about 12.5% (w/v) Human Serum Albumin. In some embodiment, the formulation comprises a 12.5% (w/v) Human Serum Albumin.

In some embodiment, the formulation comprises about 5% (v/v) DMSO. In some embodiment, the formulation comprises 5% (v/v) DMSO. In some embodiment, the formulation further comprises a cryopreservative solution comprising the DMSO. In some embodiment, the cryopreservative solution is CRYSTOR 10 (CS-10).

In some embodiment, 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: 1, CDR-H2 comprises SEQ ID NO: 2, and CDR-H3 comprises SEQ ID NO: 3, and (ii) a VL domain comprising three CDRs (CDR-L1, CDR-L2, and CDR-L3), wherein CDR-L1 comprises SEQ ID NO: 4, CDR-L2 comprises SEQ ID NO: 5, and CDR-L3 comprises SEQ ID NO: 6. In some embodiment, the CAR comprises an amino acid sequence of SEQ ID NO: 39.

In some embodiment, 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: 9, CDR-H2 comprises SEQ ID NO: 10, and CDR-H3 comprises SEQ ID NO: 11, and (ii) a VL domain comprising three CDRs (CDR-L1, CDR-L2, and CDR- L3), wherein CDR-L1 comprises SEQ ID NO: 12, CDR-L2 comprises SEQ ID NO: 13, and CDR-L3 comprises SEQ ID NO: 14.

In some embodiment, 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: 16, CDR-H2 comprises SEQ ID NO: 17, and CDR-H3 comprises SEQ ID NO: 18, and (ii) a VL domain comprising three CDRs (CDR-L1, CDR-L2, and CDR-L3), wherein CDR-L1 comprises SEQ ID NO: 19, CDR-L2 comprises SEQ ID NO: 20, and CDR-L3 comprises SEQ ID NO: 21.

In some embodiment, the TEAM comprises an amino acid sequence of SEQ ID NO: 23.

In some embodiments, a formulation comprises: (i) about 200 million CAR-T cells per mL of the formulation, wherein the chimeric antigen receptor (CAR)-T cells comprise a polynucleotide encoding: (a) a CAR comprising an amino acid sequence of SEQ ID NO: 39; and (b) a T cell engaging antibody molecule (TEAM) comprising an amino acid sequence of SEQ ID NO: 23;

(ii) 12.5% (w/v) Human Albumin; and (iii) 50% (v/v) CS-10.

In some embodiment, the polynucleotide encoding the CAR and the TEAM comprises nucleic acids encoding an amino acid sequence of SEQ ID NO: 37. In some embodiment, the formulation is frozen. In some embodiment, after thawing the cells, 50-70% of the CAR-T cells are positive for a CAR comprising an antigen binding domain that binds to EGFRvIII and the TEAM comprising an EGFR antibody and an anti-CD3 antibody; and (b) a T cell engaging antibody molecule (TEAM) comprising an anti-EGFR antibody and an anti-CD3 antibody. In some embodiment, the formulation volume is about 200 pL.

In some aspects, this disclosure describes a pharmaceutical composition comprising: (i) about 200-500 pL of a formulation described herein; and (ii) about 10 mL of Eliotts B Solution. In some embodiments, the pharmaceutical composition comprises about 30 million of the CAR- T cells. In some embodiments, 50-70% of the CAR-T cells are positive for (a) the CAR comprising an antigen binding domain that binds to EGFRvIII and (b) the TEAM comprising an anti-EGFR antibody and an anti-CD3 antibody. In some embodiments, the composition is about 10 mL in volume.

In some aspects, this disclosure describes A pharmaceutical composition comprising: (i) 7-13 million chimeric antigen receptor (CAR)-T cells comprising a polynucleotide encoding: (a) a CAR comprising an antigen binding domain that binds to EGFRvIII; and (b) a T cell engaging antibody molecule (TEAM) that comprises an anti-EGFR antibody and an anti-CD3 antibody; (ii) 0.3-0.6% (w/v) Human Serum Albumin; (iii) less than 0.25% volume by volume (v/v) dimethyl sulfoxide (DMSO); and (iv) about 90% Elliott’s B solution. In some embodiments, the CAR-T cells of the formulation comprise a polynucleotide encoding an amino acid sequence of SEQ ID NO: 37. In some embodiments, the pharmaceutical composition has a volume of about 10 mL. BRIEF DESCRIPTION OF THE DRAWINGS

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.

FIGs. 1A-1B show a CAR TEAM construct and the proposed mechanism of action thereof in treating glioblastoma in the central nervous system. FIG. 1A depicts the components of the EGFRvIII binding CAR and the anti-EGFR and anti-CD3 TEAM. FIG. IB depicts a CAR-TEAM-T cell with an EGFRvIII binding CAR that secretes a TEAM that binds EGFR and CD3.

FIGs. 2A-2C relates to a CAR-TEAM construct and expression of said construct in cells. FIG. 2A 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. 2B shows representative flow cytometric analysis on the cell product from Patient 1, with gating for CARpositive and TEAM-positive cells among all CD3⁺ T cells. FIG. 2C shows representative gating for TEAM-positive cells among CAR-positive and CAR-negative T-cell populations.

FIGs. 3A-3E correspond to treatment events and outcome related to Patient 1. FIG. 3A summarizes the timeline of events for Patient 1 while enrolled in the Phase I clinical study. FIG. 3B shows axial contrast-enhanced T1 -weighted images (which enhances signal of fatty tissue) for Patient 1 immediately post-resection (left), pre-infusion (center), and post-infusion (right). Numbers in each panel indicate days relative to infusion (day 0). FIG. 3C shows EGFRvIII and EGFR copy number over time, derived from extracellular vesicle (EV) RNA present in CSF. FIG. 3D shows respective copy numbers isolated from peripheral blood EV RNA at pre- and post-treatment time points, and FIG. 3E shows post-hoc analysis of EGFR copy number by fluorescence in situ hybridization (FISH).

FIGs. 4A-4C relate to treatment workflow and treatment outcomes for Patients 2 and 3. FIG. 4A shows a modified workflow used for Patient 2 and 3, which enabled concomitant craniotomy, tissue sampling and Ommaya placement during a single surgery. FIG. 4B shows longitudinal radiographic assessment after CAR-TEAM cell infusion of Patient 2. FIG. 4C shows pre- and post-infusion axial T1 -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).

FIGs. 5A-5B related to temperature response in Patients 1-3 following CAR- TEAM cell infusion. FIG. 5A shows temperature curves for all patients which peaked within 2 days of infusion and normalized over time, and FIG. 5B shows management of body temperature via treatment with the interleukin-1 receptor (IL-1R) antagonist anakinra.

FIG. 6 shows expression of inflammatory markers C-reactive protein (CRP) and ferritin in cerebrospinal fluid of Patients 1-3 following CAR-TEAM cell infusion.

FIG. 7 shows counts and differentials of cells in cerebrospinal fluid of Patients 1-3 following infusion of CAR-TEAM cells.

FIGs. 8A-8C 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. 8A 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. 8B shows the presence of inflammatory cytokines in the CSF of patients over the same period. FIG. 8C shows detection of CAR-positive and TEAM- positive cells in the peripheral blood, which was transient.

DETAILED DESCRIPTION

Formulations

In some aspects, this disclosure describes a formulation comprising: (i) 20 million to 250 million T cells per milliliter (mL) of the formulation, wherein at least 1% of the T cells are chimeric antigen receptor (CAR)-T cells comprising a polynucleotide encoding: (a) a CAR comprising an antigen binding domain that binds to EGFRvIII; and (b) a T cell engaging antibody molecule (TEAM) that comprises an anti-EGFR antibody and an anti-CD3 antibody; (ii) 10-15% weight by volume (w/v) Human Serum Albumin; and (iii) 2.5-7.5% volume by volume (v/v) dimethyl sulfoxide (DMSO).

A “formulation” refers to a composition that comprises a therapeutic (e.g., CAR-T cells), an excipient (e g., Human Serum Albumin), water, and a cryopreservative (e.g., DMSO). In some embodiments, the formulation is a liquid. In some embodiments, when cooled, the formulation becomes a solid. In some embodiments, the formulation is designed to decrease CAR-T cell death during freezing, storage, and shipping of the formulation as compared to CAR- T cells that are in standard CAR-T cell media.

In some embodiments, the formulation comprises 20 million to 250 million T cells per milliliter (mL) of the formulation. In some embodiments, the formulation comprises 20-200 million, 20-150 million, 20-100 million, or 20-50 million T cells per milliliter (mL) of the formulation. In some embodiments, the formulation comprises 30-200 million, 30-150 million, 30-100 million, or 30-50 million T cells per milliliter (mL) of the formulation. In some embodiments, the formulation comprises 50-200 million, 50-150 million, or 50-100 million per milliliter (mL) of the formulation. In some embodiments, the formulation comprises 100-200 million or 100-150 million T cells per milliliter (mL) of the formulation. In some embodiments, the formulation comprises about 20 million, about 30 million, about 40 million, about 50 million, about 75 million, about 100 million, about 125 million, about 150 million, about 175 million, about 200 million, or about 250 million T cells per milliliter (mL) of the formulation.

“About” refers to +/- 5% of the number referenced. For example, about 100 million T cells refers to 95 million to 105 million T cells.

In some embodiments, at least 1% (e.g., at least 2%, at least 3%, at least 4%, at least 5%, at least 7.5%, at least 10%, at least 20%>, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%) of the T cells of the formulation are the CAR-T cells. In some embodiments, 100% of the T cells of the formulation are the CAR-T cells. In some embodiments, 1-5%, 1-10%, 1-25%, 1-50%, 1-75%, 1-90%, 1-95%, 1-99% of the T cells of the formulation are the CAR-T cells. In some embodiments, 3-5%, 3-10%, 3-25%, 3-50%, 3-75%, 3-90%, 3-95%, 3-99% of the T cells of the formulation are the CAR-T cells. In some embodiments, 5-10%, 5-25%, 5-50%, 5-75%, 5-90%, 5-95%, 5-99% of the T cells of the formulation are the CAR-T cells. In some embodiments, 10- 25%, 10-50%, 10-75%, 10-90%, 10-95%, 10-99% of the T cells of the formulation are the CAR- T cells. In some embodiments, 25-50%, 25-75%, 25-90%, 25-95%, 25-99% of the T cells of the formulation are the CAR-T cells. In some embodiments, 50-75%, 50-90%, 50-95%, 50-99% of the T cells of the formulation are the CAR-T cells. In some embodiments about 1%, about 2%, about 3%, about 4%, about 5%, about 7.5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, or about 99%) of the T cells of the formulation are the CAR-T cells. In some embodiments, 100% of the T cells of the formulation are the CAR-T cells.

In some embodiments, the number of T cells in the formulation is determined based on the desired number of CAR-T cells in the formulation (e.g., based on subsequent administration to a patient). For example, in some embodiments, the desired number of CAR-T cells in the formulation is about 30 million; therefore, if 20% of the T cells in the formulation are CAR-T cells then the formulation would comprise about 150 million T cells. In some embodiments, the number of CAR-T cells in the formulation in 20 million to 40 million. In some embodiments, the number of CAR-T cells in the formulation in 25 million to 35 million. In some embodiments, the number of CAR-T cells in the formulation is about 30 million.

In some embodiments, the CAR-T cells of the formulation comprise a CAR comprising an antigen binding domain that binds to EGFRvIII; and (b) a T cell engaging antibody molecule (TEAM) that comprises an anti-EGFR antibody and an anti-CD3 antibody. The CAR and the TEAM are described throughout this disclosure including in the section below entitled CARTEAM polynucleotides.

In some embodiments, the formulation comprises 20-40 million CAR-T cells. In some embodiments, the formulation comprises 25-35 million CAR-T cells. In some embodiments, the formulation comprises about 30 million CAR-T cells. In some embodiments, the formulation comprises 30 million +/- 30% CAR-T cells.

In some embodiments, the formulation comprises an excipient (e g., Human Serum Albumin (HSA)). Human serum albumin in cell therapy formulations is described in Van der Walle et al., Pharmaceutics 13.8 (2021): 1317. Without being bound the theory, including Human Serum Albumin in a formulation comprising CAR-T cells is expected to increase CAR-T cells viability. Human serum albumin may be obtained from numerous manufacturers including CSL BEHRING AG, which sells ALBURX 25 (25% HSA); AKRON Biotech, which sells cGMP HSA 25% solution), and NOVA BIOLOGICS, which sells 25% HSA solution. In some embodiments, the Human Serum Albumin in the formulation is UNII:ZIF514RVZR (precision.fda.gov/uniisearch/srs/unii/zif514rvzr, accessed October 12, 2023).

In some embodiments, the formulation comprises 10-15% weight by volume (w/v) Human Serum Albumin. “Weight by volume (w/v)” percentage refers to the fraction of (1) the number of grams of a substance (e.g., HSA) in a liquid (e.g., a solution or mixture), and (2) the volume in mL of the liquid after the substance has been added to the liquid (e.g., after the HSA has been dissolved by the liquid). For example, 25% HSA refers to a solution made in part by combining 25 grams of HSA with enough of a solution to bring the total volume of HSA + solution to 100 mL. In another example, if the 25% (w/v) HSA is diluted 10-fold when producing a formulation (e.g., 1 mL of 25% (w/v) HSA is combined with 9 mL of a liquid (e.g., including other components of the formulation like the CRYOSTOR 10 and the cells) then the HSA in the formulation would be 2.5% (w/v).

In some embodiments, the formulation comprises 5%-20% (w/v) HSA. In some embodiments, the formulation comprises 10%- 15% (w/v) HSA. In some embodiments, the formulation comprises 11%-13% (w/v) HSA. In some embodiments, the formulation comprises about 10%, about 11%, about 12%, about 13%, about 14%, or about 15% (w/v) HSA. In some embodiments, the formulation comprises about 12.5% (w/v) HSA. In some embodiments, the formulation comprises 12.5% (w/v) HSA.

In some embodiments, the formulation comprises a cryopreservative (e.g., dimethyl sulfoxide (DMSO)). Without being bound to theory, cryopreservatives, like DMSO, reduce ice formulation and in turn reduce damage to CAR-T cells caused by ice formulation when the CAR-T cells are stored in freezing temperatures. In some embodiments, the formulation comprises 1 %- 10% DMSO volume by volume (v/v). “Volume by volume” percentage refers to the percentage, by volume, of a given component of a liquid in the total volume of the liquid. For example, a formulation comprising 1 mL of DMSO, and 9 mL of water would comprise 10% (v/v) DMSO. In some embodiments, the formulation comprises 2.5%-7.5% (v/v) DMSO. In some embodiments, the formulation comprises 3%-7% (v/v) DMSO. In some embodiments, the formulation comprises 4%-6% (v/v) DMSO. In some embodiments, the formulation comprises about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%, (v/v) DMSO. In some embodiments, the formulation comprises about 5% (v/v) DMSO. In some embodiments, the formulation comprises 5% (v/v) DMSO. In some embodiments, the formulation comprises about 50% (v/v) CRYOSTOR-IO. CRYSTOR-10 is a cryopreservative that comprises 10% (v/v) DMSO.

In some embodiments, the formulation comprises about 20 million T cells per milliliter (mL) of the formulation, wherein at least 1% of the T cells are chimeric antigen receptor (CAR)- T cells comprising a polynucleotide encoding an amino acid of SEQ ID NO: 37; (ii) 10-15% weight by volume (w/v) Human Serum Albumin; and (iii) 2.5-7.5% volume by volume (v/v) dimethyl sulfoxide (DMSO).

In some embodiments, the formulation comprises about 30 million T cells per milliliter (mL) of the formulation, wherein at least 1% of the T cells are chimeric antigen receptor (CAR)- T cells comprising a polynucleotide encoding an amino acid sequence of SEQ ID NO: 37; 10- 15% weight by volume (w/v) Human Serum Albumin; and 2.5-7.5% volume by volume (v/v) dimethyl sulfoxide (DMSO).

In some embodiments, the formulation comprises about 40 million T cells per milliliter (mL) of the formulation, wherein at least 1% of the T cells are chimeric antigen receptor (CAR)- T cells comprising a polynucleotide encoding an amino acid sequence of SEQ ID NO: 37; 10- 15% weight by volume (w/v) Human Serum Albumin; and 2.5-7.5% volume by volume (v/v) dimethyl sulfoxide (DMSO).

In some embodiments, the formulation comprises about 50 million T cells per milliliter (mL) of the formulation, wherein at least 1% of the T cells are chimeric antigen receptor (CAR)- T cells comprising a polynucleotide encoding an amino acid sequence of SEQ ID NO: 37; 10- 15% weight by volume (w/v) Human Serum Albumin; and 2.5-7.5% volume by volume (v/v) dimethyl sulfoxide (DMSO).

In some embodiments, the formulation comprises about 75 million T cells per milliliter (mL) of the formulation, wherein at least 1% of the T cells are chimeric antigen receptor (CAR)- T cells comprising a polynucleotide encoding an amino acid sequence of SEQ ID NO: 37; 10- 15% weight by volume (w/v) Human Serum Albumin; and 2.5-7.5% volume by volume (v/v) dimethyl sulfoxide (DMSO).

In some embodiments, the formulation comprises about 100 million T cells per milliliter (mL) of the formulation, wherein at least 1% of the T cells are chimeric antigen receptor (CAR)- T cells comprising a polynucleotide encoding an amino acid sequence of SEQ ID NO: 37; 10- 15% weight by volume (w/v) Human Serum Albumin; and 2.5-7.5% volume by volume (v/v) dimethyl sulfoxide (DMSO).

In some embodiments, the formulation comprises about 125 million T cells per milliliter (mL) of the formulation, wherein at least 1% of the T cells are chimeric antigen receptor (CAR)- T cells comprising a polynucleotide encoding an amino acid sequence of SEQ ID NO: 37; 10- 15% weight by volume (w/v) Human Serum Albumin; and 2.5-7.5% volume by volume (v/v) dimethyl sulfoxide (DMSO).

In some embodiments, the formulation comprises about 150 million T cells per milliliter (mL) of the formulation, wherein at least 1% of the T cells are chimeric antigen receptor (CAR)- T cells comprising a polynucleotide encoding an amino acid sequence of SEQ ID NO: 37; 10- 15% weight by volume (w/v) Human Serum Albumin; and 2.5-7.5% volume by volume (v/v) dimethyl sulfoxide (DMSO).

In some embodiments, the formulation comprises about 175 million T cells per milliliter (mL) of the formulation, wherein at least 1% of the T cells are chimeric antigen receptor (CAR)- T cells comprising a polynucleotide encoding an amino acid sequence of SEQ ID NO: 37; 10- 15% weight by volume (w/v) Human Serum Albumin; and 2.5-7.5% volume by volume (v/v) dimethyl sulfoxide (DMSO).

In some embodiments, the formulation comprises about 200 million T cells per milliliter (mL) of the formulation, wherein at least 1% of the T cells are chimeric antigen receptor (CAR)- T cells comprising a polynucleotide encoding an amino acid sequence of SEQ ID NO: 37; 10- 15% weight by volume (w/v) Human Serum Albumin; and 2.5-7.5% volume by volume (v/v) dimethyl sulfoxide (DMSO).

In some embodiments, the formulation comprises about 225 million T cells per milliliter (mL) of the formulation, wherein at least 1% of the T cells are chimeric antigen receptor (CAR)- T cells comprising a polynucleotide encoding an amino acid sequence of SEQ ID NO: 37; 10- 15% weight by volume (w/v) Human Serum Albumin; and 2.5-7.5% volume by volume (v/v) dimethyl sulfoxide (DMSO).

In some embodiments, the formulation comprises about 250 million T cells per milliliter (mL) of the formulation, wherein at least 1% of the T cells are chimeric antigen receptor (CAR)- T cells comprising a polynucleotide encoding an amino acid sequence of SEQ ID NO: 37; 10- 15% weight by volume (w/v) Human Serum Albumin; and 2.5-7.5% volume by volume (v/v) dimethyl sulfoxide (DMSO).

In some embodiments, the formulation comprises about 20 million T cells per milliliter (mL) of the formulation, wherein at least 1% of the T cells are chimeric antigen receptor (CAR)- T cells comprising a polynucleotide encoding an amino acid of SEQ ID NO: 37; about 12.5% weight by volume (w/v) Human Serum Albumin; and about 5% volume by volume (v/v) dimethyl sulfoxide (DMSO).

In some embodiments, the formulation comprises about 30 million T cells per milliliter (mL) of the formulation, wherein at least 1% of the T cells are chimeric antigen receptor (CAR)- T cells comprising a polynucleotide encoding an amino acid sequence of SEQ ID NO: 37; about 12.5% weight by volume (w/v) Human Serum Albumin; and about 5% volume by volume (v/v) dimethyl sulfoxide (DMSO).

In some embodiments, the formulation comprises about 40 million T cells per milliliter (mL) of the formulation, wherein at least 1% of the T cells are chimeric antigen receptor (CAR)- T cells comprising a polynucleotide encoding an amino acid sequence of SEQ ID NO: 37; about 12.5% weight by volume (w/v) Human Serum Albumin; and about 5% volume by volume (v/v) dimethyl sulfoxide (DMSO).

In some embodiments, the formulation comprises about 50 million T cells per milliliter (mL) of the formulation, wherein at least 1% of the T cells are chimeric antigen receptor (CAR)- T cells comprising a polynucleotide encoding an amino acid sequence of SEQ ID NO: 37; about 12.5% weight by volume (w/v) Human Serum Albumin; and about 5% volume by volume (v/v) dimethyl sulfoxide (DMSO).

In some embodiments, the formulation comprises about 75 million T cells per milliliter (mL) of the formulation, wherein at least 1% of the T cells are chimeric antigen receptor (CAR)- T cells comprising a polynucleotide encoding an amino acid sequence of SEQ ID NO: 37; about 12.5% weight by volume (w/v) Human Serum Albumin; and about 5% volume by volume (v/v) dimethyl sulfoxide (DMSO).

In some embodiments, the formulation comprises about 100 million T cells per milliliter (mL) of the formulation, wherein at least 1% of the T cells are chimeric antigen receptor (CAR)- T cells comprising a polynucleotide encoding an amino acid sequence of SEQ ID NO: 37; about 12.5% weight by volume (w/v) Human Serum Albumin; and about 5% volume by volume (v/v) dimethyl sulfoxide (DMSO).

In some embodiments, the formulation comprises about 125 million T cells per milliliter (mL) of the formulation, wherein at least 1% of the T cells are chimeric antigen receptor (CAR)- T cells comprising a polynucleotide encoding an amino acid sequence of SEQ ID NO: 37; about 12.5% weight by volume (w/v) Human Serum Albumin; and about 5% volume by volume (v/v) dimethyl sulfoxide (DMSO).

In some embodiments, the formulation comprises about 150 million T cells per milliliter (mL) of the formulation, wherein at least 1% of the T cells are chimeric antigen receptor (CAR)- T cells comprising a polynucleotide encoding an amino acid sequence of SEQ ID NO: 37; about 12.5% weight by volume (w/v) Human Serum Albumin; and about 5% volume by volume (v/v) dimethyl sulfoxide (DMSO).

In some embodiments, the formulation comprises about 175 million T cells per milliliter (mL) of the formulation, wherein at least 1% of the T cells are chimeric antigen receptor (CAR)- T cells comprising a polynucleotide encoding an amino acid sequence of SEQ ID NO: 37; about 12.5% weight by volume (w/v) Human Serum Albumin; and about 5% volume by volume (v/v) dimethyl sulfoxide (DMSO).

In some embodiments, the formulation comprises about 200 million T cells per milliliter (mL) of the formulation, wherein at least 1% of the T cells are chimeric antigen receptor (CAR)- T cells comprising a polynucleotide encoding an amino acid sequence of SEQ ID NO: 37; about 12.5% weight by volume (w/v) Human Serum Albumin; and about 5% volume by volume (v/v) dimethyl sulfoxide (DMSO).

In some embodiments, the formulation comprises about 225 million T cells per milliliter (mL) of the formulation, wherein at least 1% of the T cells are chimeric antigen receptor (CAR)- T cells comprising a polynucleotide encoding an amino acid sequence of SEQ ID NO: 37; about 12.5% weight by volume (w/v) Human Serum Albumin; and about 5% volume by volume (v/v) dimethyl sulfoxide (DMSO).

In some embodiments, the formulation comprises about 250 million T cells per milliliter (mL) of the formulation, wherein at least 1% of the T cells are chimeric antigen receptor (CAR)- T cells comprising a polynucleotide encoding an amino acid sequence of SEQ ID NO: 37; about 12.5% weight by volume (w/v) Human Serum Albumin; and about 5% volume by volume (v/v) dimethyl sulfoxide (DMSO).

In some embodiments, the formulation comprises about 250 million T cells per milliliter (mL) of the formulation, wherein at least 5% of the T cells are chimeric antigen receptor (CAR)- T cells comprising a polynucleotide encoding an amino acid sequence of SEQ ID NO: 37; about 12.5% weight by volume (w/v) Human Serum Albumin; and about 5% volume by volume (v/v) dimethyl sulfoxide (DMSO).

In some embodiments, the formulation comprises about 250 million T cells per milliliter (mL) of the formulation, wherein at least 10% of the T cells are chimeric antigen receptor (CAR)-T cells comprising a polynucleotide encoding an amino acid sequence of SEQ ID NO: 37; about 12.5% weight by volume (w/v) Human Serum Albumin; and about 5% volume by volume (v/v) dimethyl sulfoxide (DMSO).

In some embodiments, the formulation comprises about 30 million T cells per milliliter (mL) of the formulation, about 30 million of the T cells are chimeric antigen receptor (CAR)-T cells comprising a polynucleotide encoding an amino acid sequence of SEQ ID NO: 37; about 12.5% weight by volume (w/v) Human Serum Albumin; and about 5% volume by volume (v/v) dimethyl sulfoxide (DMSO).

In some embodiments, the formulation comprises about 40 million T cells per milliliter (mL) of the formulation, about 30 million of the T cells are chimeric antigen receptor (CAR)-T cells comprising a polynucleotide encoding an amino acid sequence of SEQ ID NO: 37; about 12.5% weight by volume (w/v) Human Serum Albumin; and about 5% volume by volume (v/v) dimethyl sulfoxide (DMSO).

In some embodiments, the formulation comprises about 50 million T cells per milliliter (mL) of the formulation, about 30 million of the T cells are chimeric antigen receptor (CAR)-T cells comprising a polynucleotide encoding an amino acid sequence of SEQ ID NO: 37; about 12.5% weight by volume (w/v) Human Serum Albumin; and about 5% volume by volume (v/v) dimethyl sulfoxide (DMSO).

In some embodiments, the formulation comprises about 75 million T cells per milliliter (mL) of the formulation, about 30 million of the T cells are chimeric antigen receptor (CAR)-T cells comprising a polynucleotide encoding an amino acid sequence of SEQ ID NO: 37; about 12.5% weight by volume (w/v) Human Serum Albumin; and about 5% volume by volume (v/v) dimethyl sulfoxide (DMSO).

In some embodiments, the formulation comprises about 100 million T cells per milliliter (mL) of the formulation, about 30 million of the T cells are chimeric antigen receptor (CAR)-T cells comprising a polynucleotide encoding an amino acid sequence of SEQ ID NO: 37; about 12.5% weight by volume (w/v) Human Serum Albumin; and about 5% volume by volume (v/v) dimethyl sulfoxide (DMSO).

In some embodiments, the formulation comprises about 200 million T cells per milliliter (mL) of the formulation, about 30 million of the T cells are chimeric antigen receptor (CAR)-T cells comprising a polynucleotide encoding an amino acid sequence of SEQ ID NO: 37; about 12.5% weight by volume (w/v) Human Serum Albumin; and about 5% volume by volume (v/v) dimethyl sulfoxide (DMSO).

In some embodiments, the formulation comprises about 225 million T cells per milliliter (mL) of the formulation, about 30 million of the T cells are chimeric antigen receptor (CAR)-T cells comprising a polynucleotide encoding an amino acid sequence of SEQ ID NO: 37; about 12.5% weight by volume (w/v) Human Serum Albumin; and about 5% volume by volume (v/v) dimethyl sulfoxide (DMSO).

In some embodiments, the formulation comprises about 250 million T cells per milliliter (mL) of the formulation, about 30 million of the T cells are chimeric antigen receptor (CAR)-T cells comprising a polynucleotide encoding an amino acid sequence of SEQ ID NO: 37; about 12.5% weight by volume (w/v) Human Serum Albumin; and about 5% volume by volume (v/v) dimethyl sulfoxide (DMSO).

In some embodiments, the formulation comprises about 275 million T cells per milliliter (mL) of the formulation, about 30 million of the T cells are chimeric antigen receptor (CAR)-T cells comprising a polynucleotide encoding an amino acid sequence of SEQ ID NO: 37; about 12.5% weight by volume (w/v) Human Serum Albumin; and about 5% volume by volume (v/v) dimethyl sulfoxide (DMSO).

In some embodiments, the formulation comprises about 300 million T cells per milliliter (mL) of the formulation, about 30 million of the T cells are chimeric antigen receptor (CAR)-T cells comprising a polynucleotide encoding an amino acid sequence of SEQ ID NO: 37; about 12.5% weight by volume (w/v) Human Serum Albumin; and about 5% volume by volume (v/v) dimethyl sulfoxide (DMSO).

In some embodiments, the formulation is 0.1-1 mL in volume. In some embodiments, the formulation is 0.2-0.8 mL in volume. In some embodiments, the formulation is 0.3-0.7 mL in volume. In some embodiments, the formulation is 0.4-0.6 mL in volume. In some embodiments, the formulation is 0.2-0.5 mL in volume. In some embodiments, the formulation is about 0.2, about 0.3, about 0.4, or about 0.5 mb in volume. In some embodiments, the formulation is 0.2, 0.3, 0.4, or 0.5 mL in volume.

In some embodiments, the formulation comprises about 30 million chimeric antigen receptor (CAR)-T cells comprising a polynucleotide encoding an amino acid sequence of SEQ ID NO: 37; about 12.5% weight by volume (w/v) Human Serum Albumin; and about 5% volume by volume (v/v) dimethyl sulfoxide (DMSO).

In some embodiments, the formulation comprises about 30 million chimeric antigen receptor (CAR)-T cells comprising a polynucleotide encoding an amino acid sequence of SEQ ID NO: 37; about 12.5% weight by volume (w/v) Human Serum Albumin; and about 5% volume by volume (v/v) dimethyl sulfoxide (DMSO), wherein the formulation is about 0.2-0.5 mL in volume.

In some embodiments, the formulation comprises about 6.0 x 10⁶ to 1.4 x 10⁷ million chimeric antigen receptor (CAR)-T cells comprising a polynucleotide encoding an amino acid sequence of SEQ ID NO: 37; about 12.5% weight by volume (w/v) Human Serum Albumin; and about 5% volume by volume (v/v) dimethyl sulfoxide (DMSO), wherein the formulation is about 0.2-0.5 mL in volume.

In some embodiments, after being frozen and thawed, the formulation comprises about 6.0 x 10⁶ to 1.4 x 10⁷ million chimeric antigen receptor (CAR)-T cells comprising a polynucleotide encoding an amino acid sequence of SEQ ID NO: 37; about 12.5% weight by volume (w/v) Human Serum Albumin; and about 5% volume by volume (v/v) dimethyl sulfoxide (DMSO), wherein the formulation is about 0.2-0.5 mL in volume.

Pharmaceutical Compositions

In some aspects, this disclosure describes a pharmaceutical composition comprising 0.2- 0.5 mL of a formulation described herein and pharmaceutically acceptable diluent (e.g., Elliott’s B solution). In some embodiments, the pharmaceutical composition comprises 0.2-0.5 mL of a formulation described herein and about 10 mL of Elliott’s B solution (e g., enough Elliott’s B solution to bring the volume of the pharmaceutical composition to about 10 mL. A “pharmaceutical composition” refers to a composition comprising a formulation (as described herein) that has been diluted with a pharmaceutically acceptable dilutant (e.g., with Elliott’s B solution). A pharmaceutical composition may be produced for the purpose of administering the therapeutic agent of the formulation (e.g., the CAR-T cells) to a patient. In some embodiments, the pharmaceutical composition comprises a formulation that has been diluted by 20-40 fold using pharmaceutically acceptable dilutant (e.g., Elliott’s B solution) to reduce to concentration of a DMSO by 20-40 fold. In some embodiments, the pharmaceutical composition comprises a formulation that has been diluted by about 20 fold, about 25 fold, about 30 fold, about 35 fold, or about 40 fold using pharmaceutically acceptable dilutant (e.g., Elliott’s B solution). A pharmaceutically acceptable dilutant refers to a liquid that is compatible for administration to a subject (e.g., a human subject), and is also compatible with the formulation. In some embodiments, the pharmaceutically acceptable dilutant is Elliott’s B solution, preservative free saline, or autologous cerebral spinal fluid.

In some embodiments, the pharmaceutical composition comprises 20 million to 300 million T cells and 5 million to 40 million CAR-T cells. In some embodiments, the pharmaceutical composition comprises 50 million to 250 million T cells and 5 million to 15 million CAR-T cells. In some embodiments, the pharmaceutical composition comprises 100 million to about 200 million T cells and 7 million to 13 million CAR-T cells. In some embodiments, the pharmaceutical composition comprises 150 million to about 200 million T cells and 7 million to 13 million CAR-T cells, n some embodiments, the pharmaceutical composition comprises about 200 million T cells and 7 million to 13 million CAR-T cells.

In some embodiments, the pharmaceutical composition comprises 0.2%-0.7% (w/v) HSA. In some embodiments, the pharmaceutical composition comprises 0.3%-0.6% (w/v) HSA. In some embodiments, the pharmaceutical composition comprises about 0.2% (w/v) HSA, about 0.3% (w/v) HSA, about 0.4% (w/v) HSA, about 0.5% (w/v) HSA, about 0.6% (w/v) HSA, or about 0.7% (w/v) HSA.

In some embodiments, the pharmaceutical composition comprises less than 0.30% (v/v) DMSO (e.g., 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 0.5% (v/v) DMSO). In some embodiments, the pharmaceutical composition comprises less than 0.25% (v/v) DMSO. In some embodiments, the pharmaceutical composition comprises 0.10%-0.25% (v/v) DMSO. In some embodiments, the pharmaceutical composition comprises about 0.12%-0.25% (v/v) DMSO.

In some embodiments, the pharmaceutical composition comprises about 85% (v/v), about 90% (v/v), about 95% (v/v), about 98% (v/v), or about 99% (v/v) Elliott’s B solution. In some embodiments, the pharmaceutical composition comprises about 90% (v/v) Elliot’s B solution. In some embodiments, the pharmaceutical composition comprises at least 85%, at least 90% (v/v), at least 95% (v/v), at least 98% (v/v), or at least 99% (v/v) Elliott’s B solution. In some embodiments, the pharmaceutical composition comprises enough Elliott’s B solution to dilute the formulation by 20-40 fold. In some embodiments, the pharmaceutical composition comprises about 10 mL of Elliott’s B solution.

In some embodiments, the pharmaceutical composition comprises: (i) 20-40 million chimeric antigen receptor (CAR)-T cells comprising a polynucleotide encoding: (a) a CAR comprising an antigen binding domain that binds to EGFRvIII; and (b) a T cell engaging antibody molecule (TEAM) that comprises an anti-EGFR antibody and an anti-CD3 antibody; (ii) 0.2-0.7% weight by volume (w/v) Human Serum Albumin; (iii) less than 0.25% volume by volume (v/v) dimethyl sulfoxide (DMSO); and (iv) about 90% Elliott’s B solution.

In some embodiments, the pharmaceutical composition comprises: (i) 20-40 million chimeric antigen receptor (CAR)-T cells comprising a polynucleotide encoding: (a) a CAR comprising an antigen binding domain that binds to EGFRvIII; and (b) a T cell engaging antibody molecule (TEAM) that comprises an anti-EGFR antibody and an anti-CD3 antibody;

(ii) 0.3-0.6% weight by volume (w/v) Human Serum Albumin; (iii) 0.1-0.25% volume by volume (v/v) dimethyl sulfoxide (DMSO); and (iv) about 90% Elliott’s B solution.

(ii) 0.3-0.6% weight by volume (w/v) Human Serum Albumin; (iii) less than 0.25% volume by volume (v/v) dimethyl sulfoxide (DMSO); and (iv) about 90% Elliott’s B solution.

In some embodiments, the pharmaceutical composition comprises: (i) 25-35 million chimeric antigen receptor (CAR)-T cells comprising a polynucleotide encoding: (a) a CAR comprising an antigen binding domain that binds to EGFRvIII; and (b) a T cell engaging antibody molecule (TEAM) that comprises an anti-EGFR antibody and an anti-CD3 antibody;

(ii) 0.2-0.7% weight by volume (w/v) Human Serum Albumin; (iii) less than 0.25% volume by volume (v/v) dimethyl sulfoxide (DMSO); and (iv) about 90% Elliott’s B solution. In some embodiments, the pharmaceutical composition comprises: (i) 25-35 million chimeric antigen receptor (CAR)-T cells comprising a polynucleotide encoding: (a) a CAR comprising an antigen binding domain that binds to EGFRvIII; and (b) a T cell engaging antibody molecule (TEAM) that comprises an anti-EGFR antibody and an anti-CD3 antibody; (ii) 0.3-0.6% weight by volume (w/v) Human Serum Albumin; (iii) 0.1-0.25% volume by volume (v/v) dimethyl sulfoxide (DMSO); and (iv) about 90% Elliott’s B solution.

In some embodiments, the pharmaceutical composition comprises: (i) 25-35 million chimeric antigen receptor (CAR)-T cells comprising a polynucleotide encoding: (a) a CAR comprising an antigen binding domain that binds to EGFRvIII; and (b) a T cell engaging antibody molecule (TEAM) that comprises an anti-EGFR antibody and an anti-CD3 antibody; (ii) 0.3-0.6% weight by volume (w/v) Human Serum Albumin; (iii) less than 0.25% volume by volume (v/v) dimethyl sulfoxide (DMSO); (iv) about 90% Elliott’s B solution.

In some embodiments, the pharmaceutical composition comprises: (i) about 30 million chimeric antigen receptor (CAR)-T cells comprising a polynucleotide encoding: (a) a CAR comprising an antigen binding domain that binds to EGFRvIII; and (b) a T cell engaging antibody molecule (TEAM) that comprises an anti-EGFR antibody and an anti-CD3 antibody; (ii) 0.2-0.7% weight by volume (w/v) Human Serum Albumin; (iii) less than 0.25% volume by volume (v/v) dimethyl sulfoxide (DMSO); and (iv) about 90% Elliott’s B solution.

In some embodiments, the pharmaceutical composition comprises: (i) about 30 million chimeric antigen receptor (CAR)-T cells comprising a polynucleotide encoding: (a) a CAR comprising an antigen binding domain that binds to EGFRvIII; and (b) a T cell engaging antibody molecule (TEAM) that comprises an anti-EGFR antibody and an anti-CD3 antibody; (ii) 0.3-0.6% weight by volume (w/v) Human Serum Albumin; (iii) 0.1-0.25% volume by volume (v/v) dimethyl sulfoxide (DMSO); and (iv) about 90% Elliott’s B solution.

In some embodiments, the pharmaceutical composition comprises: (i) about 30 million chimeric antigen receptor (CAR)-T cells comprising a polynucleotide encoding: (a) a CAR comprising an antigen binding domain that binds to EGFRvIII; and (b) a T cell engaging antibody molecule (TEAM) that comprises an anti-EGFR antibody and an anti-CD3 antibody; (ii) 0.3-0.6% weight by volume (w/v) Human Serum Albumin; (iii) less than 0.25% volume by volume (v/v) dimethyl sulfoxide (DMSO); and (iv) about 90% Elliott’s B solution. In some embodiments, the pharmaceutical composition comprises: (i) 20-40 million chimeric antigen receptor (CAR)-T cells comprising a polynucleotide encoding an amino acid sequence of SEQ ID NO: 37; (ii) 0.2-0.7% weight by volume (w/v) Human Serum Albumin; (iii) less than 0.25% volume by volume (v/v) dimethyl sulfoxide (DMSO); and (iv) about 90% Elliott’s B solution.

In some embodiments, the pharmaceutical composition comprises: (i) 20-40 million chimeric antigen receptor (CAR)-T cells comprising a polynucleotide encoding a polynucleotide encoding an amino acid sequence of SEQ ID NO: 37; (ii) 0.3-0.6% weight by volume (w/v) Human Serum Albumin; (iii) 0.1-0.25% volume by volume (v/v) dimethyl sulfoxide (DMSO); and (iv) about 90% Elliott’s B solution.

In some embodiments, the pharmaceutical composition comprises: (i) 20-40 million chimeric antigen receptor (CAR)-T cells comprising a polynucleotide encoding a polynucleotide encoding an amino acid sequence of SEQ ID NO: 37; (ii) 0.3-0.6% weight by volume (w/v) Human Serum Albumin; (iii) less than 0.25% volume by volume (v/v) dimethyl sulfoxide (DMSO); and (iv) about 90% Elliott’s B solution.

In some embodiments, the pharmaceutical composition comprises: (i) 25-35 million chimeric antigen receptor (CAR)-T cells comprising a polynucleotide encoding a polynucleotide encoding an amino acid sequence of SEQ ID NO: 37; (ii) 0.2-0.7% weight by volume (w/v) Human Serum Albumin; (iii) less than 0.25% volume by volume (v/v) dimethyl sulfoxide (DMSO); and (iv) about 90% Elliott’s B solution.

In some embodiments, the pharmaceutical composition comprises: (i) 25-35 million chimeric antigen receptor (CAR)-T cells comprising a polynucleotide encoding a polynucleotide encoding an amino acid sequence of SEQ ID NO: 37; (ii) 0.3-0.6% weight by volume (w/v) Human Serum Albumin; (iii) 0.1-0.25% volume by volume (v/v) dimethyl sulfoxide (DMSO); and (iv) about 90% Elliott’s B solution.

In some embodiments, the pharmaceutical composition comprises: (i) 25-35 million chimeric antigen receptor (CAR)-T cells comprising a polynucleotide encoding a polynucleotide encoding an amino acid sequence of SEQ ID NO: 37; (ii) 0.3-0.6% weight by volume (w/v) Human Serum Albumin; (iii) less than 0.25% volume by volume (v/v) dimethyl sulfoxide (DMSO); (iv) about 90% Elliott’s B solution. In some embodiments, the pharmaceutical composition comprises: (i) about 30 million chimeric antigen receptor (CAR)-T cells comprising a polynucleotide encoding a polynucleotide encoding an amino acid sequence of SEQ ID NO: 37; (ii) 0.2-0.7% weight by volume (w/v) Human Serum Albumin; (iii) less than 0.25% volume by volume (v/v) dimethyl sulfoxide (DMSO); and (iv) about 90% Elliott’s B solution.

In some embodiments, the pharmaceutical composition comprises: (i) about 30 million chimeric antigen receptor (CAR)-T cells comprising a polynucleotide encoding a polynucleotide encoding an amino acid sequence of SEQ ID NO: 37; (ii) 0.3-0.6% weight by volume (w/v) Human Serum Albumin; (iii) 0.1-0.25% volume by volume (v/v) dimethyl sulfoxide (DMSO); and (iv) about 90% Elliott’s B solution.

In some embodiments, the pharmaceutical composition comprises: (i) about 30 million chimeric antigen receptor (CAR)-T cells comprising a polynucleotide encoding a polynucleotide encoding an amino acid sequence of SEQ ID NO: 37; (ii) 0.3-0.6% weight by volume (w/v) Human Serum Albumin; (iii) less than 0.25% volume by volume (v/v) dimethyl sulfoxide (DMSO); and (iv) about 90% Elliott’s B solution.

In some embodiments, the formulation is frozen and thawed prior to being diluted and becoming a pharmaceutical composition. This freezing and thawing may result in a decrease in the number of CAR-T cells that are positive for CAR and TEAM expression. For example, the number of CAR-T cells that are positive for CAR and TEAM expression may decrease by 30- 70% (e.g., about 40%) as compared to at the last measurement before the formulation is made.

In some embodiments, the pharmaceutical composition comprises: (i) 5-15 million chimeric antigen receptor (CAR)-T cells comprising a polynucleotide encoding: (a) a CAR comprising an antigen binding domain that binds to EGFRvIII; and (b) a T cell engaging antibody molecule (TEAM) that comprises an anti-EGFR antibody and an anti-CD3 antibody; (ii) 0.2-0.7% weight by volume (w/v) Human Serum Albumin; (iii) less than 0.25% volume by volume (v/v) dimethyl sulfoxide (DMSO); and (iv) about 90% Elliott’s B solution.

In some embodiments, the pharmaceutical composition comprises: (i) 7-13 million chimeric antigen receptor (CAR)-T cells comprising a polynucleotide encoding: (a) a CAR comprising an antigen binding domain that binds to EGFRvIII; and (b) a T cell engaging antibody molecule (TEAM) that comprises an anti-EGFR antibody and an anti-CD3 antibody; (ii) 0.2-0.7% weight by volume (w/v) Human Serum Albumin; (iii) less than 0.25% volume by volume (v/v) dimethyl sulfoxide (DMSO); and (iv) about 90% Elliott’s B solution.

In some embodiments, the pharmaceutical composition comprises: (i) 5-15 million chimeric antigen receptor (CAR)-T cells comprising a polynucleotide encoding: (a) a CAR comprising an antigen binding domain that binds to EGFRvIII; and (b) a T cell engaging antibody molecule (TEAM) that comprises an anti-EGFR antibody and an anti-CD3 antibody; (ii) 0.3-0.6% weight by volume (w/v) Human Serum Albumin; (iii) 0.1-0.25% volume by volume (v/v) dimethyl sulfoxide (DMSO); and (iv) about 90% Elliott’s B solution.

In some embodiments, the pharmaceutical composition comprises: (i) 7-13 million chimeric antigen receptor (CAR)-T cells comprising a polynucleotide encoding: (a) a CAR comprising an antigen binding domain that binds to EGFRvIII; and (b) a T cell engaging antibody molecule (TEAM) that comprises an anti-EGFR antibody and an anti-CD3 antibody; (ii) 0.3-0.6% weight by volume (w/v) Human Serum Albumin; (iii) 0.1-0.25% volume by volume (v/v) dimethyl sulfoxide (DMSO); (iv) about 90% Elliott’s B solution.

In some embodiments, the pharmaceutical composition comprises: (i) 5-15 million chimeric antigen receptor (CAR)-T cells comprising a polynucleotide encoding: (a) a CAR comprising an antigen binding domain that binds to EGFRvIII; and (b) a T cell engaging antibody molecule (TEAM) that comprises an anti-EGFR antibody and an anti-CD3 antibody; (ii) 0.3-0.6% weight by volume (w/v) Human Serum Albumin; (iii) less than 0.25% volume by volume (v/v) dimethyl sulfoxide (DMSO); and (iv) about 90% Elliott’s B solution.

In some embodiments, the pharmaceutical composition comprises: (i) 7-13 million chimeric antigen receptor (CAR)-T cells comprising a polynucleotide encoding: (a) a CAR comprising an antigen binding domain that binds to EGFRvIII; and (b) a T cell engaging antibody molecule (TEAM) that comprises an anti-EGFR antibody and an anti-CD3 antibody; (ii) 0.3-0.6% weight by volume (w/v) Human Serum Albumin; (iii) less than 0.25% volume by volume (v/v) dimethyl sulfoxide (DMSO); and (iv) about 90% Elliott’s B solution.

In some embodiments, the pharmaceutical composition comprises: (i) 5-15 million chimeric antigen receptor (CAR)-T cells comprising a polynucleotide encoding a polynucleotide encoding an amino acid sequence of SEQ ID NO: 37; (ii) 0.2-0.7% weight by volume (w/v) Human Serum Albumin; (iii) less than 0.25% volume by volume (v/v) dimethyl sulfoxide (DMSO); and (iv) about 90% Elliott’s B solution. In some embodiments, the pharmaceutical composition comprises: (i) 7-13 million chimeric antigen receptor (CAR)-T cells comprising a polynucleotide encoding a polynucleotide encoding an amino acid sequence of SEQ ID NO: 37; (ii) 0.2-0.7% weight by volume (w/v) Human Serum Albumin; (iii) less than 0.25% volume by volume (v/v) dimethyl sulfoxide (DMSO); and (iv) about 90% Elliott’s B solution.

In some embodiments, the pharmaceutical composition comprises: (i) 5-15 million chimeric antigen receptor (CAR)-T cells comprising a polynucleotide encoding a polynucleotide encoding an amino acid sequence of SEQ ID NO: 37; (ii) 0.3-0.6% weight by volume (w/v) Human Serum Albumin; (iii) 0.1-0.25% volume by volume (v/v) dimethyl sulfoxide (DMSO); and (iv) about 90% Elliott’s B solution.

In some embodiments, the pharmaceutical composition comprises: (i) 7-13 million chimeric antigen receptor (CAR)-T cells comprising a polynucleotide encoding a polynucleotide encoding an amino acid sequence of SEQ ID NO: 37; (ii) 0.3-0.6% weight by volume (w/v) Human Serum Albumin; (iii) 0.1-0.25% volume by volume (v/v) dimethyl sulfoxide (DMSO); (iv) about 90% Elliott’s B solution.

In some embodiments, the pharmaceutical composition comprises: (i) 5-15 million chimeric antigen receptor (CAR)-T cells comprising a polynucleotide encoding a polynucleotide encoding an amino acid sequence of SEQ ID NO: 37; (ii) 0.3-0.6% weight by volume (w/v) Human Serum Albumin; (iii) less than 0.25% volume by volume (v/v) dimethyl sulfoxide (DMSO); and (iv) about 90% Elliott’s B solution.

In some embodiments, the pharmaceutical composition comprises: (i) 7-13 million chimeric antigen receptor (CAR)-T cells comprising a polynucleotide encoding a polynucleotide encoding an amino acid sequence of SEQ ID NO: 37; (ii) 0.3-0.6% weight by volume (w/v) Human Serum Albumin; (iii) less than 0.25% volume by volume (v/v) dimethyl sulfoxide (DMSO); and (iv) about 90% Elliott’s B solution.

In some embodiments, the pharmaceutical composition comprises: (i) 7-13 million chimeric antigen receptor (CAR)-T cells comprising a polynucleotide encoding a polynucleotide encoding an amino acid sequence of SEQ ID NO: 37; (ii) 0.3-0.6% weight by volume (w/v) Human Serum Albumin; (iii) less than 0.25% volume by volume (v/v) dimethyl sulfoxide (DMSO); and (iv) about 90% Elliott’s B solution; wherein the pharmaceutical composition has a total volume of 5-15 mb. In some embodiments, the pharmaceutical composition comprises: (i) 7-13 million chimeric antigen receptor (CAR)-T cells comprising a polynucleotide encoding a polynucleotide encoding an amino acid sequence of SEQ ID NO: 37; (ii) 0.3-0.6% weight by volume (w/v) Human Serum Albumin; (iii) less than 0.25% volume by volume (v/v) dimethyl sulfoxide (DMSO); and (iv) about 90% Elliott’s B solution; wherein the pharmaceutical composition has a total volume of about 10 mL.

In some embodiments, the pharmaceutical composition comprises: (i) 7-13 million chimeric antigen receptor (CAR)-T cells comprising a polynucleotide encoding a polynucleotide encoding an amino acid sequence of SEQ ID NO: 37; (ii) 0.3-0.6% weight by volume (w/v) Human Serum Albumin; (iii) l%-3% volume by volume (v/v) CRYOSTOR-IO; and (iv) about 90% Elliott’s B solution; wherein the pharmaceutical composition has a total volume of about 10 mL.

In some embodiments, the pharmaceutical composition comprises: (i) 7-13 million chimeric antigen receptor (CAR)-T cells comprising a polynucleotide encoding a polynucleotide encoding an amino acid sequence of SEQ ID NO: 37; (ii) 0.3-0.6% weight by volume (w/v) Human Serum Albumin; (iii) 1.25%-2.5% volume by volume (v/v) CRYOSTOR-IO; and (iv) about 90% Elliott’s B solution; wherein the pharmaceutical composition has a total volume of about 10 mL.

CAR-TEAM polynucleotides

In some aspects, the formulations described herein comprise CAR-T cells. In some embodiments, the CAR-T cells comprise a polynucleotide encoding (1) a CAR comprising an antigen binding domain and (2) a T cell engaging antibody molecule (TEAM) that comprises an anti-EGFR antibody and an anti-CD3 antibody.

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. 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.

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.

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.

Antigen Binding Domain

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

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

Hinge and Transmembrane Domains

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. 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.

As used herein, "transmembrane domain" (TM domain) refers to the portion of the CAR that fuses the antigen 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.

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.

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, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD 137, CD 154, 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, CD 19, IL2R beta, IL2R gamma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, CDl la, LFA-1, ITGAM, CDl lb, ITGAX, CDl lc, 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 1 As used herein, a "hinge/transmembrane domain" refers to a domain including both a hinge domain and a transmembrane domain. For example, a hinge/transmembrane domain can be derived from the hinge/transmembrane domain of CD8, CD28, CD7, or 4-1BB. In some embodiments, the hinge/transmembrane domain of a CAR or fragment thereof is derived from or includes the hinge/transmembrane domain of CD8 (e.g., SEQ ID NO: 24, or variants thereof). CD8 is an antigen preferentially found on the cell surface of cytotoxic T lymphocytes. CD8 mediates cell-cell interactions within the immune system, and acts as a T cell co-receptor. CD8 consists of an alpha (CD8alpha or CD8a) and beta (CD813 or CD8b) chain. CD8a sequences are known for a number of species, e.g., human CD8a, (NCBI Gene ID: 925) polypeptide (e.g., NCBI Ref Seq NP 001139345.1) and mRNA (e.g., NCBI Ref Seq NM_ 000002.12). CD8 can refer to human CD8, including naturally occurring variants, molecules, and alleles thereof. In some embodiments of any of the aspects, e.g., in veterinary applications, CD8 can refer to the CD8 of, e.g., dog, cat, cow, horse, pig, and the like.

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.

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

Co-stimulatory Domains

Each CAR described herein optionally includes the intracellular domain of one or more co-stimulatory molecule or co-stimulatory domain. As used herein, the term "co-stimulatory domain" refers to an intracellular signaling domain of a co-stimulatory molecule. Co-stimulatory molecules are cell surface molecules other than antigen receptors or 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- IBB, CD27, CD28, or 0X40. In some embodiments, a 4- IBB co-stimulatory domain can be used (see, e.g., below and SEQ ID NO: 25, or variants thereof). In some embodiments, a CD28 co- stimulatory domain can be used (see, e.g., below and SEQ ID NO: 26, or variants thereof). Additional illustrative examples of such co-stimulatory domains include CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (0X40), CD137 (4-1BB), CD150 (SLAMF1), CD 152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD- Ll), 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.

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.

In some embodiments, the co-stimulatory domain comprises the co-stimulatory domain of a 4-1BB. In some embodiments, the 4-1BB co-stimulatory domain corresponds to an amino acid sequence selected from SEQ ID NO: 25. 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: 26.

Intracellular Signaling Domains

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

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/ anti gen 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. With respect to intracellular signaling domains, so-called "first-generation" CARs include those that solely provide CD3(^ signals upon antigen binding. So-called "second-generation" CARs include those that provide both co-stimulation (e.g., CD28 or CD 137) and activation (CD3Q domains, and so-called "third-generation" CARs include those that provide multiple costimulatory (e.g., CD28 and CD137) domains and activation (signaling) domains (e g., CD3Q. 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.

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 ; (see, e.g., below). Additional non-limiting examples of immunoreceptor tyrosine-based activation motif (ITAM)-containing intracellular signaling domains that are of particular use in the technology include those derived from TCR-zeta, FcR-gamma, FcR-beta, CD3-gamma, CD3-theta, CD3-sigma, CD3-eta, CD3- epsilon, CD3-zeta, CD22, CD79a, CD79b, and CD66d.

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

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

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

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.

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.

Signal Peptide

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

Linker Domain

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: 30) and Gly4Ser (G4S) linkers such as (G4S)3 (GGGGSGGGGSGGGGS (SEQ ID NO: 31)) and (G4S)4 (GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 32)); the linker sequence of GSTSGSGKPGSGEGSTKG (SEQ ID NO: 33) as described by Whitlow et al., Protein Eng. 6(8):989-95, 1993, the contents of which are incorporated herein by reference in its entirety; the linker sequence of GGSSRSSSSGGGGSGGGG (SEQ ID NO: 34) as described by Andris- Widhopf et al ., Cold Spring Harb. Protoc. 2011 (9), 2011, the contents of which are incorporated herein by reference in its entirety; as well as linker sequences with added functionalities, e.g., an epitope tag or an encoding sequence containing Cre-Lox recombination site as described by Sblattero et al., Nat. Biotechnol. 18(l):75-80, 2000, the contents of which are incorporated herein by reference in its entirety. Longer linkers may be used when it is desirable to ensure that two adjacent domains do not sterically interfere with one another.

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

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 bi specific antibody (e.g., a TEAM)) component of a polyprotein (see below).

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

T cell engaging antibody molecules (TEAMs)

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 CD3e subunit) and the other binds to a target antigen (e.g., a tumor antigen). Such molecules can target T cells by binding to a T cell antigen (e g., by binding CD3) as well as a target antigen, e.g., a tumor antigen. The TEAMs can be used to augment the T cell response in, e.g., the tumor microenvironment. The two components of a TEAM can 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.

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

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

In some aspects, this disclosure describes a polynucleotide comprising nucleic acids encoding an EGFRvIII binding CAR, and EGFR-binding and CD3 -binding TEAM, and a truncated CD 19. In some embodiments, the polynucleotide comprises nucleic acids encoding, from n-terminal to c-terminal of a polypeptide, a CD8 leader sequence, an EGFRvIII antigen binding domain (e g., an EGFRvIII scFv), a CD8 hinge, a CD8 transmembrane domain, a 4-1BB co- stimulatory domain, a CD3-zeta intracellular signaling domain, a P2A peptide, an IgK leader, an anti-EGFR antibody (e.g., a cetuximab scFv), an anti-CD3 antibody (e.g., scFv), a T2A peptide, and a truncated CD19 (e.g., of SEQ ID NO: 38). In some embodiments, the formulation comprises a CAR-T cell comprising SEQ ID NO: 37. In some embodiments, the formulation comprises a CAR-T cell comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO: 37.

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

In some aspects, this application discloses a vector (e.g., an expression vector) comprising the polynucleotide described herein. A "vector," refers to a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells. As used herein, a vector can be viral or non-viral. The term "vector" encompasses any genetic element that is capable of replication when associated with the proper control elements and that can transfer gene sequences to cells. A vector can include, but is not limited to, a cloning vector, an expression vector, a plasmid, phage, transposon, cosmid, artificial chromosome, virus, virion, etc. In some embodiments, a vector may comprise a polynucleotide described herein (e.g., a polynucleotide comprising a polynucleic acid sequence encoding a VEGF binding protein and/or a CAR). As used herein, the term "expression vector" may refer to a vector that directs expression of an RNA or polypeptide from sequences linked to transcriptional regulatory sequences on the vector. The sequences expressed will often, but not necessarily, be heterologous to the cell. An expression vector may comprise additional elements, for example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example, in human cells for expression and in a prokaryotic host for cloning and amplification. The term "expression" refers to the cellular processes involved in producing RNA and proteins and as appropriate, secreting proteins, including where applicable, but not limited to, for example, transcription, transcript processing, translation and protein folding, modification and processing. "Expression products" include RNA transcribed from a gene, and polypeptides obtained by translation of mRNA transcribed from a gene.

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

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

In some aspects, this disclosure described a cell comprising a CAR-TEAM-tCD19 polynucleotide or polypeptide as described herein. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is an immune cell. In some embodiments, the cell is a T cell. In some embodiments, the cell is an autologous T cell. In some embodiments, the cell is an allogeneic T cell. In some embodiments, the tCD19 is expressed on the surface of the cells (e.g., tCD19 is bound to the cell membrane and at least a portion of tCD19 is on the exterior of the cell membrane).

Sequences

EXAMPLES

Example 1 : Developing CAR-T cell formulations and pharmaceutical compositions. One challenge in administering CAR-T cells into the brain is preparing the CAR-T cells in a pharmaceutical composition that is compatible with the central nervous system and is in a volume that is a relatively small (compared to intravenous administration). This is challenging because typically CAR-T cell formulations comprise DMSO (a cryoprotectant), which is needed to maintain CAR-T cell viability during storage and transit, but which is also known to be neurotoxic. Neurotoxicity is observed even in current CAR-T cell therapies, which are administered to the blood. To address this problem, the inventors iterated through multiple different formulations and pharmaceutical compositions in order find a formulation that retained acceptable CAR-T cell viability for patient administration, and that could be modified into a pharmaceutical composition that also had acceptable CAR-T cell viability for patient administration and that also had minimal neurotoxicity. These experiments were performed with T cells that had been transfected with a construct that has an anti-EGFRvIII binding CAR and an anti-EGFR / anti-CD3 TEAM (i.e., SEQ ID NO: 37) (FIG. 1).

A first formulation was tested that had about 1.0 x 10⁷ total T cells (including CAR-T cells), 12.5% percent HSA and 5% percent DMSO and was about 4 mL in volume. After freezing (which is typical for CAR-T cell transport to the patient), the T cells were thawed and washed with cold PlasmaLyte A. The CAR-T cells were stable (e.g., maintained sufficient viability) after thawing but freezing and washing the cells resulted in a large and unacceptable loss in viability.

A second formulation was tested that had about 1.0 x 10⁷ total T cells (including CAR-T cells), 12.5% percent HSA and 5% percent DMSO and was about 4 mL in volume. After freezing, the T cells were (i) thawed in cold DMEM+HSAand washed with cold DMEM, or (ii) thawed with Elliott’s B solution and washed once or twice with room temperature Elliott’s B solution. The CAR-T cells were stable (e.g., maintained sufficient viability) after thawing but freezing and washing the cells DMEM in a large and unacceptable loss in viability. Washing the CAR-T cells with room temperature Elliott’s B solution improved viability compared to DMEM and PlasmaLyte A, but recovery after the wash was still poor.

A third formulation was tested that had about 1.0 x 10⁸ total T cells, 12.5% percent HSA and 5% precent DMSO and was about 0.3 mL in volume. Instead of thawing and washing the T cells, the T cells were diluted in Elliott’s B solution to form the pharmaceutical composition. This resulted in an increase in CAR-T cell viability as compared to washing.

Additional formulations were tested increasing the total number of T cells (which include CAR-T cells) in the formulation to about 2 x 10⁸ or about 3 x 10⁸ T cells, all in about 0.2-0.5 mL of formulation. Then the T cells were diluted with about 10 mL of Elliott’s B solution. Results indicate that CAR-T cell viability decreases as the number of T cells in the formulation approaches 3 x 10⁸ T cells.

Formulations comprising about 30 million CAR-T cells (comprising SEQ ID NO: 37), about 12.5% HSA, and about 5% DMSO with a total volume of about 300 pL were prepared. Prior to administration to the ventricular space of human patients, the formulations were thawed and diluted to 10 mL of total volume using Elliott’s B solution. The pharmaceutical compositions had the expected number of CAR-T cells for administration (about 10 million). Some CAR-T cell loss is expected due to freezing. The pharmaceutical compositions were administered to patients without showing any DMSO-associated neurotoxicity, which was expected to occur within 8 hours of administration given the CSF completely turns over about every 8 hours.

Overall, these results demonstrate that this formulation and pharmaceutical composition can maintain CAR-T cell viability and the pharmaceutical composition is compatible with administration to the human central nervous system.

Example 2: Treating Glioblastoma using a CAR-TEAM

Glioblastoma multiforme (GBM) is a challenging cancer to treat due to its infiltrative nature and high rate of recurrence. To date, treatment of GBM with CAR-T cell therapy has not resulted in the same success as observed with CAR-T cell therapy in blood cancers.

To address this challenge, three human patients were treated with autologous EGFRvIII CAR-T EGFR-CD3 TEAM tCD19 CAR-T cells by administration to the patient’s CSF. The autologous EGFRvIII CAR-T EGFR-CD3 TEAM tCD19 CAR-T cells for each patient were prepared in separate formulations each comprising (i) about 3.0 x 10⁷ EGFRvIII CAR-T EGFR- CD3 TEAM cells, (ii) 12.5% HSA, and 50% CRYOSTOR 10 (5% DMSO) in a total volume of about 200-500 pL. Prior to administration to each patient, the formulation was thawed and diluted to a total volume of 10 mL using Elliott’s B solution to produce a pharmaceutical composition. As a result of the freezing, thawing, and diluting of the CAR-T cells, the pharmaceutical compositions had 1 x 10⁷ +/- 30% EGFRvIII CAR-T EGFR-CD3 TEAM cells.

The pharmaceutical composition was infused directly into the CNS via the Ommaya reservoir implanted during secondary resection. Patient 3 showed potential pseudoprogression with approximately 40% reduction in tumor size compared to baseline at approximately 30 days post-infusion, while Patient 2 showed a partial response at approximately 70 days post-infusion and a 60% decrease in tumor size, demonstrating the effectiveness of the CAR-T cell therapy despite the dramatically altered number of CAR-T cells and volume of administration. Patient 1 died of complications that are thought to be caused by an adverse side effect to a different drug. Evaluation is ongoing for patients 2 and 3. Example 3. Rapid regression of recurrent glioblastoma after a single infusion of CAR-T EGFR- CD3 TEAM cells.

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 CARTEAM 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

Study Oversight and Design

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.

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 10⁶ CAR- TEAM cells per infusion, administered via Ommaya reservoir.

Molecular Testing 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.

Manufacture of CAR-TEAM T Cells

Autologous T cells were obtained by leukapheresis and transduced with the CAR- TEAM 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.

A third transgene coding for a truncated CD 19 (tCD19) molecule was used as a surface marker for transduction (FIG. 2A). Detection of the TEAM bound to T cells was performed using biotinylated human EGFR (FIGs. 2B-2C). 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 x 10⁶ CAR-positive cells per vial post-thaw. Cells were thawed, resuspended, and transferred to a syringe under sterile conditions prior to infusion.

Toxicity Analysis

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.

Results

Patients Included in the Case Series

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

Patient 1

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. 3A). Patient 1 was subsequently enrolled in the Phase I clinical study.

A pre-treatment interval MRI was obtained which showed active progression (FIG. 3B, center). Patient 1 underwent placement of a ventriculostomy catheter with Ommaya reservoir. CAR-TEAM cells (10 x 10⁶ CAR-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. 3B, 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 io⁶ CAR-TEAM cells on day +37 (FIG. 3A).

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. 3C). 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. 3D). 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. 3E).

Patient 2

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. 4A), 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 10⁶ CARTEAM 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. 4B).

Patient 3

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. 4A) 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. 4C, left). The patient was discharged and readmitted two weeks later for infusion of 10 x io⁶ CAR-TEAM cells via intraventricular catheter. An MRI obtained at day +5 after this single infusion exhibited near-complete tumor regression (FIG. 4C, right), despite apparent lack of EGFRvIII expression or wild-type gene amplification immediately prior to treatment.

Safety and Adverse Events

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.

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.

Table 3. Adverse Event Summary: Patient 1

Table 4. Adverse Event Log: Patient 2

RUE = right upper extremity 1; BLE = bilateral lower extremity

Table 5. Adverse Event Summary: Patient 2

Table 6. Adverse Event Log: Patient 3.

Table 7. Adverse Event Summary: Patient 3

Correlative Data

All patients exhibited fevers which peaked by day +2 after infusion (FIG. 5A).

Intravenous therapy with the interleukin- 1 receptor antagonist, anakinra (100 mg Q6H), was intermittently used in the management of fevers (FIG. 5B). Systemic inflammatory markers increased and peaked in the second and third weeks after infusion, returning to near baseline by day +30 (FIG. 6A). 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. 7). 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. 8A), which corresponded with normalization of the inflammatory cytokine milieu in the CSF compartment over the same period (FIG. 8B). CAR-TEAM cells were detected in the peripheral blood between 2 to 3 weeks after infusion but their presence was transient (FIG. 8C).

Discussion

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 bispecific 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

1. A formulation comprising:

(i) 20 million to 250 million T cells per milliliter (mL) of the formulation, wherein at least 1% of the T cells are chimeric antigen receptor (CAR)-T cells comprising a polynucleotide encoding: (a) a CAR comprising an antigen binding domain that binds to EGFRvIII; and (b) a T cell engaging antibody molecule (TEAM) that comprises an anti-EGFR antibody and an anti- CD3 antibody;

(ii) 10-15% weight by volume (w/v) Human Serum Albumin; and

(iii) 2.5-7.5% volume by volume (v/v) dimethyl sulfoxide (DMSO).

2. The formulation of claim 1 comprising 30-200 million of the CAR-T cells per mL of the formulation.

3. The formulation of claim 1 comprising 50-150 million of the CAR-T cells per mL of the formulation.

4. The formulation of claim 1 comprising 30-200 million of the CAR-T cells per mL of the formulation.

5. The formulation of claim 1 comprising about 200 million of the CAR-T cells per mL of the formulation.

6. The formulation of claim 1 comprising 200 million of the CAR-T cells per mL of the formulation.

7. The formulation of any one of claims 1-6 comprising about 12.5% (w/v) Human Serum Albumin.

8. The formulation of any one of claims 1-6 comprising a 12.5% (w/v) Human Serum Albumin.

9. The formulation of any one of claims 1-8 comprising about 5% (v/v) DMSO.

10. The formulation of any one of claims 1-8 comprising 5% (v/v) DMSO.

11. The formulation of any one of claims 1-10, further comprising a cryopreservative solution comprising the DMSO.

12. The formulation of claim 11, wherein the cryopreservative solution is CRYSTOR 10 (CS- 10).

13. The formulation of any one of claims 1-12, 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: 1, CDR-H2 comprises SEQ ID NO: 2, and CDR-H3 comprises SEQ ID NO: 3, and

(ii) a VL domain comprising three CDRs (CDR-L1, CDR-L2, and CDR-L3), wherein CDR-L1 comprises SEQ ID NO: 4, CDR-L2 comprises SEQ ID NO: 5, and CDR-L3 comprises SEQ ID NO: 6.

14. The formulation of any one of claims 1-13, wherein the CAR comprises an amino acid sequence of SEQ ID NO: 39.

15. The formulation of any one of claims 1-14, 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: 9, CDR-H2 comprises SEQ ID NO: 10, and CDR-H3 comprises SEQ ID NO: 11, and

(ii) a VL domain comprising three CDRs (CDR-L1, CDR-L2, and CDR-L3), wherein CDR-L1 comprises SEQ ID NO: 12, CDR-L2 comprises SEQ ID NO: 13, and CDR-L3 comprises SEQ ID NO: 14.

16. The formulation of any one of claims 1-15, 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: 16, CDR-H2 comprises SEQ ID NO: 17, and CDR-H3 comprises SEQ ID NO: 18, and

(ii) a VL domain comprising three CDRs (CDR-L1, CDR-L2, and CDR-L3), wherein CDR-L1 comprises SEQ ID NO: 19, CDR-L2 comprises SEQ ID NO: 20, and CDR-L3 comprises SEQ ID NO: 21.

17. The formulation of any one of claims 1-16, wherein the TEAM comprises an amino acid sequence of SEQ ID NO: 23.

18. A formul ati on compri si ng :

(i) about 200 million CAR-T cells per mL of the formulation, wherein the chimeric antigen receptor (CAR)-T cells comprise a polynucleotide encoding: (a) a CAR comprising an amino acid sequence of SEQ ID NO: 39; and (b) a T cell engaging antibody molecule (TEAM) comprising an amino acid sequence of SEQ ID NO: 23;

(ii) 12.5% (w/v) Human Albumin; and

(iii) 50% (v/v) CS-10.

19. The formulation of any one of claims 1-18, wherein the polynucleotide encoding the CAR and the TEAM comprises nucleic acids encoding an amino acid sequence of SEQ ID NO: 37.

20. The formulation of any one of claims 1-19, wherein the formulation is frozen.

21. The formulation of claim 20, wherein, after thawing the cells, 50-70% of the CAR-T cells are positive for a CAR comprising an antigen binding domain that binds to EGFRvIII and the TEAM comprising an EGFR antibody and an anti-CD3 antibody; and (b) a T cell engaging antibody molecule (TEAM) comprising an anti-EGFR antibody and an anti-CD3 antibody.

22. The formulation of any one of claims 1-21, wherein the formulation volume is about 200 pL.

23. A pharmaceutical composition comprising:

(i) about 200-500 pL of the formulation of any one of claims 1-20; and

(ii) about 10 mL of Eliotts B Solution.

24. The pharmaceutical composition of claim 23, comprising about 30 million of the CAR-T cells.

25. The pharmaceutical composition of claim 23 or claim 24, wherein 50-70% of the CAR-T cells are positive for (a) the CAR comprising an antigen binding domain that binds to EGFRvIII and (b) the TEAM comprising an anti-EGFR antibody and an anti-CD3 antibody.

26. The pharmaceutical composition of claim 24 or 25, wherein the composition is about 10 mL in volume.

27. A pharmaceutical composition comprising:

(i) 7-13 million chimeric antigen receptor (CAR)-T cells comprising a polynucleotide encoding: (a) a CAR comprising an antigen binding domain that binds to EGFRvIII; and (b) a T cell engaging antibody molecule (TEAM) that comprises an anti-EGFR antibody and an anti- CD3 antibody;

(ii) 0.3-0.6% (w/v) Human Serum Albumin;

(iii) less than 0.25% volume by volume (v/v) dimethyl sulfoxide (DMSO); and

(iv) about 90% Elliott’s B solution.

28. The pharmaceutical composition of claim 27, wherein the CAR-T cells comprise a polynucleotide encoding an amino acid sequence of SEQ ID NO: 37.

29. The pharmaceutical composition of claim 27 or claim 28 having a volume of about 10 mL.