Attorney Docket No.07039-2280WO1 / 2023-199 METHODS AND MATERIALS FOR TREATING CANCER CROSS-REFERENCE TO RELATED APPLICATIONS T
his application claims the benefit of U.S. Patent Application Serial No. 63/605,766, filed on December 4, 2023. The disclosure of the prior application is considered part of, and is incorporated by reference in, the disclosure of this application. SEQUENCE LISTING This application contains a Sequence Listing that has been submitted electronically as an XML file named “07039-2280WO1_SL.xml.” The XML file, created on November 22, 2024, is 34,554 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety. TECHNICAL FIELD This document relates to methods and materials for treating a mammal having cancer. For example, this document provides antigen receptors (e.g., chimeric antigen receptors (CARs)) that can target a programmed death-ligand 1 (PD-L1) polypeptide. In some cases, immune cells (e.g., lymphocytes such as tumor-infiltrating lymphocytes (TILs), T cells, natural killer (NK) cells, macrophages, and neutrophils) engineered to express an antigen receptor (e.g., a CAR) that can target a PD-L1 polypeptide can be administered to a mammal (e.g., a human) having cancer to treat the mammal. BACKGROUND C
AR T cell therapy has revolutionized treatment for hematological malignancies; however, treatment with CAR T cells is less effective against solid tumors. For example, the solid tumor stroma negatively impacts the homing of peripheral CAR T cells into the tumor micro-environment (TME), and the hostile immunosuppressive TME can drive
CAR T cells to functional exhaustion (Jafarzadeh et al., Front. Immunol., 11:702 (2020)). Among the several active exhaustion molecules expressed by the T cell, PD-1/PD-L1
signaling was most relevant in the TME (Pauken et al., Trends Immunol., 36(4):265-76 (2015)). For example, when a programmed cell death protein 1 (PD-1) polypeptide
Attorney Docket No.07039-2280WO1 / 2023-199 located on the surface of a T cell or NK cell interacts with a PD-L1 polypeptide on a tumor cell or on a tumor-associated cell, the immune cell can be functionally weakened or even inactivated. SUMMARY This document provides methods and materials for generating immune cells (e.g., lymphocytes such as TILs, T cells, NK cells, macrophages, and neutrophils) expressing (e.g., engineered to express) an antigen receptor (e.g., a CAR) that can target a PD-L1 polypeptide. In some cases, antigen receptors (e.g., CARs) that can target a PD-L1 polypeptide can include, in an extracellular to intracellular orientation, (a) an antigen- binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains. In some cases, an immune cell (e.g., a lymphocyte such as a TIL, T cell, or NK cell) engineered to express an antigen receptor (e.g., a CAR) that can target a PD-L1 polypeptide can include (e.g., can be engineered to include) nucleic acid encoding the antigen receptor (e.g., a CAR) that can target the PD-L1 polypeptide such that the antigen receptor is expressed by the immune cell (e.g., T cell). This document also provides methods and materials for using immune cells (e.g., lymphocytes such as TILs, T cells, NK cells, macrophages, and neutrophils) expressing (e.g., engineered to express) an antigen receptor (e.g., a CAR) that can target a PD-L1 polypeptide. In some cases, the immune cells (e.g., lymphocytes such as TILs, T cells, NK cells, macrophages, and neutrophils) expressing (e.g., engineered to express) an antigen receptor (e.g., a CAR) that can target a PD-L1 polypeptide can be administered (e.g., in an adoptive cell therapy) to a mammal (e.g., a human) having cancer to treat the mammal. As demonstrated herein, immune cells (e.g., lymphocytes such as TILs, T cells, NK cells, macrophages, and neutrophils) expressing an antigen receptor (e.g., a CAR) that includes: (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains can target (e.g., target and destroy) cancer cells expressing a PD-L1 polypeptide. For example, when immune cells (e.g., lymphocytes such as TILs, T cells, NK cells, macrophages, and neutrophils)
Attorney Docket No.07039-2280WO1 / 2023-199 expressing a CAR, which includes: (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains, bind a cell expressing a PD-L1 polypeptide, the immune cells (e.g., lymphocytes such as TILs, T cells, NK cells, macrophages, and neutrophils) can be activated. In some cases, the activated immune cells (e.g., lymphocytes such as TILs, T cells, NK cells, macrophages, and neutrophils) can infiltrate the tumor microenvironment. In some cases, the activated immune cells (e.g., lymphocytes such as TILs, T cells, NK cells, macrophages, and neutrophils) can retain a functional ability when bound to a PD-L1 polypeptide. Having the ability to target cells expressing a PD-L1 polypeptide using the antigen receptors (e.g., CARs) provided herein (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) provides a unique and unrealized opportunity to treat cancers such as one or more solid tumors that include cancer cells expressing a PD- L1 polypeptide and/or one or more solid tumors that have tumor-associated cells expressing a PD-L1 polypeptide. For example, immune cells (e.g., lymphocytes such as TILs, T cells, NK cells, macrophages, and neutrophils) expressing (e.g., engineered to express) an antigen receptor (e.g., a CAR) provided herein (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) can be used to convert a signal that normally weakens immune cells (e.g., allowing PD-L1 positive cancer cells to escape immune detection) to one that stimulates immune cells to regain their metabolic health, to reinstate their intrinsic activation potential, and/or to increase or restore their cytolytic capacity (e.g., such that the immune cells can target and destroy PD-L1 positive cancer cells). In general, one aspect of this document features antigen receptors having the ability to bind to a PD-L1 polypeptide, where the antigen receptor comprises a contiguous amino acid sequence comprising, in an extracellular to intracellular orientation when expressed by a cell: (a) antigen-binding domain that binds a PD-L1 polypeptide, where the antigen binding domain comprises (i) followed by (ii) or comprises (ii) followed by (i), where (i) comprises a heavy chain variable domain or
Attorney Docket No.07039-2280WO1 / 2023-199 region comprising the amino acid sequences set forth in SEQ ID NO:4 (or SEQ ID NO:4 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO:5 (or SEQ ID NO:5 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO:6 (or SEQ ID NO:6 with one, two, or three amino acid additions, deletions, or substitutions), and where (ii) comprises a light chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO:8 (or SEQ ID NO:8 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO:9 (or SEQ ID NO:9 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO:10 (or SEQ ID NO:10 with one, two, or three amino acid additions, deletions, or substitutions); (b) a transmembrane domain; and (c) a signaling domain. The antigen receptor can have the ability to bind to SEQ ID NO:1. The heavy chain variable domain or region can include an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO:3. The heavy chain variable domain or region can comprise, consist essentially of, or consist of an amino acid sequence having the amino acid sequence set forth in SEQ ID NO:3. The light chain variable domain or region can include an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO:7. The light chain variable domain or region can comprise, consist essentially of, or consist of an amino acid sequence having the amino acid sequence set forth in SEQ ID NO:7. The antigen receptor can include (i) followed by (ii). The antigen receptor can include (ii) followed by (i). The antigen receptor can include SEQ ID NO:3 followed by SEQ ID NO:7. The antigen receptor can include SEQ ID NO:7 followed by SEQ ID NO:3. The antigen- binding domain can be a monoclonal antibody. The antigen-binding domain can be an
scFv antibody. The transmembrane domain can be a CD3 transmembrane domain, a CD4 transmembrane domain, a CD8 transmembrane domain, a CD28 transmembrane domain, or a 4-1BB transmembrane domain. The signaling domain can be a CD3 intracellular signaling domain, a CD27 intracellular signaling domain, a CD28 intracellular signaling domain, an OX40 (CD134) intracellular signaling domain, a 4- 1BB (CD137) intracellular signaling domain, a CD278 intracellular signaling domain, a DAP10 intracellular signaling domain, a DAP12 intracellular signaling domain, a
Attorney Docket No.07039-2280WO1 / 2023-199 NKG2D intracellular signaling domain, a CD244 intracellular signaling domain, or a
CD266 intracellular signaling domain. In another aspect, this document features nucleic acid constructs encoding an antigen receptor having the ability to bind to a PD-L1 polypeptide, where the antigen receptor comprises a contiguous amino acid sequence comprising, in an extracellular to intracellular orientation when expressed by a cell: (a) antigen-binding domain that binds a PD-L1 polypeptide, where the antigen binding domain comprises (i) followed by (ii) or comprises (ii) followed by (i), where (i) comprises a heavy chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO:4 (or SEQ ID NO:4 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO:5 (or SEQ ID NO:5 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO:6 (or SEQ ID NO:6 with one, two, or three amino acid additions, deletions, or substitutions), and where (ii) comprises a light chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO:8 (or SEQ ID NO:8 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO:9 (or SEQ ID NO:9 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO:10 (or SEQ ID NO:10 with one, two, or three amino acid additions, deletions, or substitutions); (b) a transmembrane domain; and (c) a signaling domain. The nucleic acid construct can be in the form of a viral vector. The viral vector can be a lentiviral vector. The antigen receptor can have the ability to bind to SEQ ID NO:1. The heavy chain variable domain or region can include an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO:3. The heavy chain variable domain or region can comprise, consist essentially of, or consist of an amino acid sequence having the amino acid sequence set forth in SEQ ID NO:3. The light chain variable domain or region can include an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO:7. The light chain variable domain or region can comprise, consist essentially of, or consist of an amino acid sequence having the amino acid sequence set forth in SEQ ID NO:7. The antigen receptor can include (i) followed by (ii). The antigen receptor can include (ii) followed by (i). The antigen receptor can include SEQ ID NO:3 followed by SEQ ID
Attorney Docket No.07039-2280WO1 / 2023-199 NO:7. The antigen receptor can include SEQ ID NO:7 followed by SEQ ID NO:3. The antigen-binding domain can be a monoclonal antibody. The antigen-binding domain can
be an scFv antibody. The transmembrane domain can be a CD3 transmembrane domain, a CD4 transmembrane domain, a CD8 transmembrane domain, a CD28 transmembrane domain, or a 4-1BB transmembrane domain. The signaling domain can be a CD3 intracellular signaling domain, a CD27 intracellular signaling domain, a CD28 intracellular signaling domain, an OX40 (CD134) intracellular signaling domain, a 4- 1BB (CD137) intracellular signaling domain, a CD278 intracellular signaling domain, a DAP10 intracellular signaling domain, a DAP12 intracellular signaling domain, a NKG2D intracellular signaling domain, a CD244 intracellular signaling domain, or a
CD266 intracellular signaling domain. In another aspect, this document features immune cells including a nucleic acid encoding an antigen receptor having the ability to bind to a PD-L1 polypeptide, where the antigen receptor comprises a contiguous amino acid sequence comprising, in an extracellular to intracellular orientation when expressed by a cell: (a) antigen-binding domain that binds a PD-L1 polypeptide, where the antigen binding domain comprises (i) followed by (ii) or comprises (ii) followed by (i), where (i) comprises a heavy chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO:4 (or SEQ ID NO:4 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO:5 (or SEQ ID NO:5 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO:6 (or SEQ ID NO:6 with one, two, or three amino acid additions, deletions, or substitutions), and where (ii) comprises a light chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO:8 (or SEQ ID NO:8 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO:9 (or SEQ ID NO:9 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO:10 (or SEQ ID NO:10 with one, two, or three amino acid additions, deletions, or substitutions); (b) a transmembrane domain; and (c) a signaling domain, where the immune cell expresses the antigen receptor. The immune cell can be a human immune cell. The immune cell can be a TIL, a T cell, a NK cell, a macrophage, or a neutrophil. The antigen receptor can have the ability
Attorney Docket No.07039-2280WO1 / 2023-199 to bind to SEQ ID NO:1. The heavy chain variable domain or region can include an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO:3. The heavy chain variable domain or region can comprise, consist essentially of, or consist of an amino acid sequence having the amino acid sequence set forth in SEQ ID NO:3. The light chain variable domain or region can include an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO:7. The light chain variable domain or region can comprise, consist essentially of, or consist of an amino acid sequence having the amino acid sequence set forth in SEQ ID NO:7. The antigen receptor can include (i) followed by (ii). The antigen receptor can include (ii) followed by (i). The antigen receptor can include SEQ ID NO:3 followed by SEQ ID NO:7. The antigen receptor can include SEQ ID NO:7 followed by SEQ ID NO:3. The antigen-binding domain can be a monoclonal antibody. The antigen- binding domain can be an scFv antibody. The transmembrane domain can be a CD3
transmembrane domain, a CD4 transmembrane domain, a CD8 transmembrane domain, a CD28 transmembrane domain, or a 4-1BB transmembrane domain. The signaling
domain can be a CD3 intracellular signaling domain, a CD27 intracellular signaling domain, a CD28 intracellular signaling domain, an OX40 (CD134) intracellular signaling domain, a 4-1BB (CD137) intracellular signaling domain, a CD278 intracellular signaling domain, a DAP10 intracellular signaling domain, a DAP12 intracellular signaling domain, a NKG2D intracellular signaling domain, a CD244 intracellular signaling
domain, or a CD266 intracellular signaling domain. In another aspect, this document features methods for treating a mammal having cancer. The methods can include, or consist essentially of, administering, to a mammal having cancer, an immune cell including a nucleic acid encoding an antigen receptor having the ability to bind to a PD-L1 polypeptide, where the antigen receptor comprises a contiguous amino acid sequence comprising, in an extracellular to intracellular orientation when expressed by a cell: (a) antigen-binding domain that binds a PD-L1 polypeptide, where the antigen binding domain comprises (i) followed by (ii) or comprises (ii) followed by (i), where (i) comprises a heavy chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO:4 (or SEQ ID NO:4
Attorney Docket No.07039-2280WO1 / 2023-199 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO:5 (or SEQ ID NO:5 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO:6 (or SEQ ID NO:6 with one, two, or three amino acid additions, deletions, or substitutions), and where (ii) comprises a light chain variable domain or region comprising the amino acid sequences set forth in SEQ ID NO:8 (or SEQ ID NO:8 with one, two, or three amino acid additions, deletions, or substitutions), SEQ ID NO:9 (or SEQ ID NO:9 with one, two, or three amino acid additions, deletions, or substitutions), and SEQ ID NO:10 (or SEQ ID NO:10 with one, two, or three amino acid additions, deletions, or substitutions); (b) a transmembrane domain; and (c) a signaling domain, where the cancer comprises a cancer cell expressing a PD-L1 polypeptide. The mammal can be human. The cancer can include one or more solid tumors. The cancer can be a breast cancer, a glioblastoma, a lung cancer, a melanoma, a bile duct cancer, an ovarian cancer, or a lymphoma. The immune cell can be autologous to the mammal. The antigen receptor can have the ability to bind to SEQ ID NO:1. The heavy chain variable domain or region can include an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO:3. The heavy chain variable domain or region can comprise, consist essentially of, or consist of an amino acid sequence having the amino acid sequence set forth in SEQ ID NO:3. The light chain variable domain or region can include an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO:7. The light chain variable domain or region can comprise, consist essentially of, or consist of an amino acid sequence having the amino acid sequence set forth in SEQ ID NO:7. The antigen receptor can include (i) followed by (ii). The antigen receptor can include (ii) followed by (i). The antigen receptor can include SEQ ID NO:3 followed by SEQ ID NO:7. The antigen receptor can include SEQ ID NO:7 followed by SEQ ID NO:3. The antigen- binding domain can be a monoclonal antibody. The antigen-binding domain can be an
scFv antibody. The transmembrane domain can be a CD3 transmembrane domain, a CD4 transmembrane domain, a CD8 transmembrane domain, a CD28 transmembrane domain, or a 4-1BB transmembrane domain. The signaling domain can be a CD3 intracellular signaling domain, a CD27 intracellular signaling domain, a CD28
Attorney Docket No.07039-2280WO1 / 2023-199 intracellular signaling domain, an OX40 (CD134) intracellular signaling domain, a 4- 1BB (CD137) intracellular signaling domain, a CD278 intracellular signaling domain, a DAP10 intracellular signaling domain, a DAP12 intracellular signaling domain, a NKG2D intracellular signaling domain, a CD244 intracellular signaling domain, or a
CD266 intracellular signaling domain. The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. D
ESCRIPTION O
F D
RAWINGS Figures 1A – 1D. CAR-T cells expressing an exemplary CAR (also referred to as MC9999) containing (a) an antigen-binding domain that binds a PD-L1 polypeptide and includes SEQ ID NOs:4-6 and 8-10, (b) a hinge, (c) transmembrane domain, and (c) one or more signaling domains showed antigen specific cytotoxicity against a PD-L1 overexpressing human breast cancer cell line (MDA-MB-231 PD-L1 OE) with a PD-L1 knock out variant ( MDA-MB-231-PD-L1 KO) as a negative comparator. Non-CAR T- cells (non-transduced T cells from the same donor) were used as a T-cell alloreactivity control. Both CD8 and CD4 MC9999 CAR T-cells exhibited cytolytic activity in response to MDA-MD-231 PD-L1 OE, as evidenced by T-cell degranulation and the presence of CD107a at the cell surface. The absence of cytolytic activity of the same CAR T-cell populations to MDA-MB-231-PD-L1 KO confirm the antigen-specific functionality (Figure 1A and Figure 1B). Granzyme B release was observed when MC9999 CAR T-cells were incubated with MDA-MB-231 PD-L1 OE but not when the targets cells were MDA-MB-231-PD-L1 KO (Figure 1C). Antigen-specific cytotoxicity of MC10029 CAR-T cells was also evaluated from the perspective of the target cells in a cellular impedance cytolysis assay using the xCELLigence
® Real Time Cell Analyzer. MDA-MB-231 PD-L1 OE or MDA-MB-231-PD-L1 KO target cells were incubated for 24 hours before MC9999 CAR T-cells or non-CAR-T cells were added. Cytolysis, as determined with the decrease in the cell index of target cells, was observed only when
Attorney Docket No.07039-2280WO1 / 2023-199 MC9999 CAR T-cells were incubated with the MDA-MB-231 OE cells; all other conditions showed continued intact target cells (Figure 1D). F
igures 2A – 2D. In vivo anti-tumor effects of MC9999 CAR T-cells in female NSG mice that received an intramammary injection of tumor cells were evaluated. The mice were challenged with either luciferase labeled-MDA-MB-231 PD-L1 OE or luciferase labeled-MDA-MB-231-PD-L1 KO tumor cells. After seven days, the tumor- bearing mice (n =5 per group) were assigned to one of three treatment groups: MC9999 CAR T-cells, non-CAR T-cells, or PBS. Tumor progression in the mice and survival following treatment were monitored by bioluminescence imaging (Figure 2A). Three Kaplan-Meier plots were generated to compare the differences in treatment-associated survival rates between the antigen-positive tumor challenge group (MDA-MB-231 PD- L1 OE) and the antigen-deficient tumor challenge group (MDA-MB-231-PD-L1 KO). Treatment groups were as follows: MC9999 CAR T-cells (Figure 2B), non-CAR T-cells (Figure 2C), and PBS (Figure 2D). MC9999 CAR T-cell treatment resulted in a marked disappearance of the established tumors and long-term survival in mice challenged with MDA-MB-231 PD-L1 OE tumors, while no such effects were observed in mice MDA- MB-231-PD-L1 KO tumors, which conclusively supports the antigen-specific, in vivo antitumor effect of this novel CAR T-cell therapy. Figures 3A – 3F. Antigen-specific cytotoxicity of MC9999 CAR T-cells was evaluated across three solid tumor models. Antigen-specific cytotoxicity was observed when MC9999 CAR T-cells were exposed to Calu-1, but not to PD-L1 KO Calu-1 cells (Figure 3A). The release of granzyme B by MC9999 CAR T-cells in response to PD-L1- positive Calu-1 cells confirmed the antigen-specific cytotoxicity (Figure 3B). Antigen- specific T-cell degranulation was evident when MC9999 CAR T-cells were incubated with SH-4 cells engineered to overexpress PD-L1 (SH-4 PD-L1 OE), but not with SH-4 cells engineered to knock-out PD-L1 expression (SH-4 PD-L1 KO) (Figure 3C). An absence of granzyme B release was noted when the CAR T-cells were exposed to SH-4
PD-L1 KO target cells (Figure 3D). In vitro assays demonstrated specific cytotoxicity of MC9999 CAR T-cells against LN229 cells engineered to overexpress PD-L1 (LN229 PD-L1 OE), evidenced by T-cell degranulation (Figure 3E) and granzyme B release
Attorney Docket No.07039-2280WO1 / 2023-199 (Figure 3F), whereas T-cell activities were absent against LN229 cells engineered to knock-out PD-L1 expression (LN229 PD-L1 KO). Figures 4A – 4C. Patient-derived tumor cell lines retain the tumor characteristics of the original patient, and likely the clinical response to treatment, making these primary tumor cell lines critical in translational medicine. A technical platform for generating patient-derived tumor cell lines from surgically resected GBM tumors was established. QNS120 and QNS712 represent patient-derived GBM cell lines with high PD-L1 expression (Figure 4A). MC9999 CAR T-cells exhibited antigen-specific cytotoxicity against QNS120 and QNS712, as observed through T-cell degranulation (Figure 4B) and granzyme B release upon interaction with these primary GBM tumor cells (Figure 4C). To ensure the antigen-specificity of the CAR T-cell activities, a pair of PD-L1-positive (MDA-MB-231 PD-L1 OE) and PD-L1-negative (MDA-MB-231 PD-L1 KO) controls were included. Non-CAR T-cells derived from the same donor did not show efficacy against any of the target cells. The utilization of these patient-derived GBM cell lines confirmed the cytotoxicity of MC9999 CAR T-cells against primary tumor cells. F
igures 5A and 5B. In vivo anti-tumor effects of MC9999 CAR T-cells were evaluated using a LN229 GBM tumor model. Mice were challenged with an intracranial injection of luciferase-expressing LN229 PD-L1 OE cells. Treatment with MC9999 CAR T-cells was administered intravenously via tail vein on Day 7 and Day 14 following tumor challenge (arrows). Bioluminescence images tracking tumor development revealed significant tumor reduction in mice treated with the MC9999 CAR T-cells (Figure 5A). CAR T-cell therapy substantially extended overall survival. While both PBS and non- CAR T-cell treated mice did not exceed 70 days, mice treated with MC9999 CAR T-cells survived until the termination of the study on day 150 (Figure 5B). These findings
support the therapeutic potential of MC9999 CAR T-cells in eradicating established in vivo tumors. Figures 6A – 6D. Microglials residing within the tumor microenvironment represent a subset of TAMs in GBM, contributing to tumor growth and immune suppression. To confirm that immunosuppressive cells are targets for MC9999 CAR T- cells, the expression of PD-L1 was first queried in a microglial cell line, HMC3 (Figure
Attorney Docket No.07039-2280WO1 / 2023-199 6A). Cytotoxicity of MC9999 CAR T-cells against HMC3 cells was readily observed, evident through CAR T-cell degranulation (Figure 6B) and granzyme B release (Figure 6C). Alloreactivity was not noted as these T-cell activities were not detected when non- CAR T-cells were incubated with HMC3 cells. Using an impedance assay the cytolytic effects of MC9999 CAR T-cells on HMC3 were demonstrated. Adding MC9999 CAR T- cells resulted in a dramatic decrease in cell index, indicating disruption of the HMC3 cell monolayer and direct killing of the HMC3 cells (Figure 6D). Figures 7A – 7H. Immunosuppressive macrophages including TAMs typically exhibit M2 phenotype within the tumor microenvironment. To model this population, monocyte-derived M2 macrophages (MDM-M2) were generated. Immunophenotypic characterization confirmed the expression of CD163 and CD209 cell surface markers on MDM-M2 (Figure 7A). Surface expression of PD-L1 was also detected on these M2 macrophages, distinguishing themselves from their CD14+ monocyte precursor (Figure 7B). Expression of PD-L1 marks M2 macrophages as a target for MC9999 CAR T-cells, while the CD14+ monocytes that lack PD-L1 expression evade the cytotoxic effects of MC9999 CAR T-cells (Figure 7C). This PD-L1 targeted-cytotoxicity was further confirmed with the significant release of granzyme B by MC9999 CAR T-cells upon interaction with M2 macrophages (Figure 7D). Additionally, as observed with the HMC3 cells, MC9999 CAR T-cells disrupted the M2 macrophage monolayer in an impedance assay, showing direct killing of the M2 macrophages (Figure 7E). The cytotoxicity of MC9999 CAR T-cells on primary TAMs isolated from a surgically resected GBM tumor was tested. The immunophenotypic characterization verified the presence of a CD163+CD209+ double positive TAM population within the tumor (Figure 7F). Gated on this population, PD-L1 expression was identified on these immunosuppressive cells (Figure 7G). CAR T-cell degranulation was observed against TAMs (Figure 7H), underscoring the potential of MC9999 CAR T-cell therapy in targeting TAMs and subsequently mitigating immunosuppression within the tumor microenvironment of solid tumors. Figures 8A – 8D. Recognizing that T cell fitness in cancer patients may be compromised, the cytotoxicity of patient-derived MC9999 CAR T-cells against PD-L1-
Attorney Docket No.07039-2280WO1 / 2023-199 expressing target cells was evaluated. MC9999 CAR T-cells were engineered using peripheral blood T cells obtained from three patients with GBM. The GBM patient- derived MC9999 CAR T-cells were evaluated for the CAR T-cell specific characteristics of identity (Figure 8A, CD3 staining) and potency (Figure 8A, tEGFR staining). All three batches of patient-derived MC9999 CAR T-cells satisfied the quality control criteria (identity >70% and potency >10%). Antigen specific cytotoxicity of these patient-derived MC9999 CAR T-cells was evaluated the against two GBM patient-derived tumor cell lines (QNS120 and QNS712), as well as HMC3, using a degranulation assay (Figure 8B). Corresponding non-CAR T-cells were included as a negative control. Similar degranulation was observed between patient 1 and patient 2 as well as among the three target cell lines used. Patient 3 showed comparable degranulation among the three target cell lines but less activity compared to Patients 1 and 2. In a separate measure of activity, the release of cytotoxic granules and cytokines by patient-derived MC9999 CAR T-cells
were evaluated. Granzyme A, granzyme B, IFN , and perforin were significantly detected in all cases when the CAR T-cells were incubated with target cells (Figure 8C). Variations in the released granules/cytokines were observed among the MC9999 CAR T- cell derived from different patients. The CAR T-cells from Patients1 and Patient 2
released greater amounts of cytotoxic granules and IFN than Patient 3, which is consistent with the degranulation results. This difference was particularly observed when the CAR T-cells were stimulated by QNS 712. Despite anticipated individual variations in CAR T-cell cytotoxicity, all three batches of patient-derived CAR T-cells were validated for their functionality, which supports the feasibility of engineering qualified and functional MC9999 CAR T-cells for patient applications. Figure 9. A schematic map of an exemplary construct that can encode a MC9999 CAR. Figures 10A-10H. MC9999 CAR T cells displayed high specificity for PD-L1 expressing cells. Figure 10A) LN229 PD-L1-OE and KO cell lines were incubated with MC9999 CAR T cells and non-CAR T cells from the same healthy donor. Degranulation assay shows CD107a is highly expressed on CD8+ MC9999 CAR T cells when incubate with LN229 PD-L1 OE cells. Figure 10B) Granzyme B granule secretion is significantly
Attorney Docket No.07039-2280WO1 / 2023-199 increased when MC9999 CAR T cells are co-cultured with GBM cells expressing the target antigen (n=3; P<0.0001). Figure 10C) After T cell addition (orange arrow), only LN229 PD-L1-OE cells show a decrease in cell index, demonstrating the specific effect of MC9999 CAR T cells on target cells expressing PD-L1 (n=3 per condition). Figure 10D) Bioluminescence-based imaging of tumor cell specificity shows that tumors were depleted without recurrence in the MC9999 CAR T-treated group after 150 days, whereas tumor developed in PBS or non-CAR T groups. Figures 10E and 10F) Decreased bioluminescence (Figure 10E) and increased overall survival (Figure 10F) were observed in MC9999 CAR T- treated groups, but not in non-CAR T or PBS control groups (n=16 per group, P<0.001). Figure 10G) multi-Immunohistochemistry (mIHC) analysis of residual antigen expression in tissue showed proliferative tumor cells expressing PD-L1 and nestin in control groups but not in CAR T treatment group. Figure 10H) PD-L1 expression showing % of remaining tumor cells was significantly lower in CAR T
treatment group at endpoint (n=4 per group, P<0.05). Scale bars: 1 mm, magnification 50 µm. Figures 11A-11K. Intracranial delivery of MC9999 CAR T cells exhibited high
efficiency in GBM patient derived tumor models in vitro and in vivo. Figure 11A) T1WI Magnetic Resonance Image (MRI) of patient QNS108 showing the GBM and PD-L1 expression in tumor tissue. Figure 11B) schematic representation of the experimental design using healthy donor T cells for CAR T production and patient derived GBM (QNS108) engineered to develop an orthotopic xenograft model for intracranial therapeutic treatment. Figure 11C) MC9999 CAR T cells, but not non-CAR T cells, co- cultured QNS108with show CD107a membrane expression. Figure 11D) This activation
was further validated by granzyme B release (n=3; P<0.0001). Figure 11E) MC9999 CAR T cells efficiently eradicated patient derived tumor xenografts (QNS108) in vivo. intracranial CAR T cell infusion was performed on day 14 (orange arrow) after tumor burden confirmation. Figure 11F) Tumors were significantly reduced in the CAR T group
compared to non-CAR T and PBS controls (n=8 per group; P<0.0001) using bioluminescence fold change. Figure 11G) survival was also significantly increased in the
CAR T treated group (n=8 per group; P<0.0001). Figure 11H) analysis of the residual
Attorney Docket No.07039-2280WO1 / 2023-199 tumor cell population (N=5 per group) showing PD-L1, Nestin and Ki67 expression in tumor cells in the control groups but not in CAR T treated animals. Figures 11I-11K) Quantification shows significant increase in tumor cells (Figure 11I; PD-L1) (Kruskall-
Wallis P PBS vs MC9999 CAR T=0.01; P non-CAR T vs MC9999 CAR T=0.05), proliferation (Figure 11J; Ki67) (Kruskall-Wallis P PBS vs MC9999 CAR T=0.05; P non-CAR T vs MC9999 CAR T=0.05), and stemness (Figure 11K; Nestin) (Kruskall-
Wallis P PBS vs MC9999 CAR T=0.01; P non-CAR T vs MC9999 CAR T=0.05)in control groups compared to MC9999 CAR T group. Scale bars: 1 mm, magnification 50 µm. Figures 12A-12P. Single-cell RNA sequencing (scRNA-seq) revealed that MC
9999 CAR T treatment activates interferon-related pathways in GBM models in vivo. Figure 12A) Schematic representation of the experimental approach depicting CAR T cell intratumoral infusion (N= 4 per group) after tumor burden confirmation (14 days). Animals were sacrificed 24 hours after treatment and tissue was harvested for scRNA- seq. Figure 12B) Analysis revealed twelve distinct clusters including eleven mice brain populations and one human cluster comprised of T cells and GBM cells (orange square). Figure 12C) Human cell cluster was reclassified into two subclusters: Immune cell markers (CD6, CD53 and TRAC) shown expression in cluster 0, whereas GBM markers (MIA, S100B and MDK) were expressed in clusters 1 and 2. Figure 12D) Re-clustering showed less tumor cells in MC9999 CAR T treated animals compared to non-CAR T control. Figure 12E) Differentially Expressed Gene (DEG) analysis identified upregulated genes (orange) and downregulated genes(purple) in CAR T vs. non-CAR T cells. Figure 12F) Pathway analysis comparing CAR T to non-CAR T cells displayed activation of cytokine and interferon mediated cytotoxicity in T cells (orange rectangles). Figure 12G) CTSS and PLAAT4 (highlighted in orange rectangles) were observed to be the most upregulated genes in GBM cells. Figure 12H) GBM cells activated pathways in response to interferon signaling and antigen presentation (orange rectangle). Figures 12I- 12L. Validation of the most upregulated genes (CTSS and PLAAT4) in GBM in response to CAR T treatment, using CGGA database, showed a significant correlation with genes related to CD4+ Th1 including IL12R (Figure 12I; N=1011 glioma patients; R=0.62);
Attorney Docket No.07039-2280WO1 / 2023-199 IFNGR (Figure 12J; N= 1018 glioma patients; R=0.79); TNF (Figure 12K; N= 66 glioma patients; R=0.46); IL15 (Figure 12L; N=1010 glioma patients; R=0.51) and the top upregulated gene, CTSS. Figures 12M-12P. Similarly, CD8+ cytotoxicity-related genes including GZMB (Figure 12M; N= 649 glioma patients; R=0.41); GZMA (Figure 12N; N= 651 glioma patients; R=0.46); PRF1 (Figure 12O; N= 646 glioma patients; R=0.51); LAMP1 (Figure 12P; N=651 glioma patients; R=0.35) correlated strongly with PLAAT4, the second most upregulated gene. Figures 13A-13E. Patient derived CAR T cells showed robust activation in response to the presence of their autologous tumor cells. Figure 13A) T1WI Magnetic Resonance Image (MRI) of patients QNS985 and QNS986 showing GBMs and PD-L1 expression in tumor cells. Figures 13B-13F) Brain tumor initiating cells (BTICs) from these patients were isolated and co-cultured with their corresponding autologous MC9999 CAR T and non-CAR T cells (control) to assess cytotoxicity. Figure 13B) Activation of autologous CAR T cells by autologous CAR T cells by autologous BTICs was observed, indicated by increased CD107a expression in the CAR T group in both patients. Figure 13C) Granzyme B release was significantly elevated in the CAR T group for both
patients (N=3 per patient; P<0.001). Figures 13D and 13E) IFN secretion (Figure 13D) and perforin release (Figure 13E) from patient QNS985 showed statistically significant increases in the CAR-T group (N=3; P<0.0001 and P<0.001, respectively). Figure 13E) Representative images of CAR T cells co-cultured with autologous QNS986 tumor cells over time. Apoptotic tumor cells (orange arrows) became visibly apparent 24 hours after co-culture in the CAR T group, but not in the non-CAR T control group. Scale bars: 100 µm. Figures 14A-14C. Patient derived MC9999 CAR T cells effectively targeted autologous M2 macrophages and TAMs. Monocyte-derived M2 macrophages from patient blood samples (N=3; patients QNS 924, QNS 1065, and QNS 1070) or TAMs isolated from tumor tissues (N=2; patients QNS 1065 and QNS 1070) were incubated with autologous MC9999 CAR T cells to assess cytotoxicity. Figure 14A) CD107a expression increased in CD8+ CAR T cells for the two patients. Figure 14B) Granzyme
B, Granzyme A, and IFN release were significantly elevated in CAR T group on M2 for
Attorney Docket No.07039-2280WO1 / 2023-199 patient QNS1070 (N=3 per experiment; P<0.001). Figure 14C) Granzyme B, Granzyme
A, and IFN release were significantly elevated in CAR T group on autologous TAMs from patients QNS1065 and 1070 (N=3 per experiment; P<0.001). Figures 15A-15H. PD-L1 is a safe and effective target for anti-PD-L1 CAR T intracranial delivery and cytotoxic effects of astrocytoma patient-derived PD-L1 CAR T cells on autologous M2 macrophages. Figure 15A) Representative mIHC staining of high-grade glioma patents including Grade 4 astrocytoma, primary and matched recurrent GBM show PD-L1 expression in tumor cells and TAMs (white arrows). Some GFAP+ cells (tumor cells) express PD-L1 (yellow arrow). Most astrocytes do not express PD-L1 (orange arrows). No Neurons (NeuN+ cells) or Oligodendrocytes (Olig2+) cells co- express PD-L1. Figure 15B) Quantification of PD-L1 in high grade glioma patients (N=4 per condition) show all these tumors present PD-L1 in tumor cells (Figure 15C) and TAMs (Figure 15D). Figure 15E) Quantification and validation of PD-L1-positive cells and mRNA levels in a larger cohort (CGGA) indicate significantly higher PD-L1 mRNA in primary and recurrent glioblastomas compared to grade 4 astrocytoma (N=598; P<0.001, P<0.0001). Figures 15F-H15. Cytotoxicity of astrocytoma patient-derived PD- L1 CAR T cells on autologous M2 macrophages confirmed by degranulation and Granzyme B ELISA assays. Scale bars: whole section: 1 mm, magnification: 25 µm. Figures 16A-16B. PD-L1 expression in GBM cell lines. Figure 16A) High levels of PD-L1 expression were observed in LN229 PD-L1 overexpressing (OE) cells (right panel) and in engineered GBM patient-derived QNS108 cells (left panel), whereas no PD-L1 expression was detected in LN229 PD-L1 knockout (KO) cells (middle panel). Figure 16B) Native PD-L1 expression was detected in GBM patient-derived cell lines QNS985 and QNS986. Figures 17A-17B. H&E staining of orthotopic mice models at experimental endpoint. Figure 17A) Representative section of mice tumors from LN229 cell injection showing the whole section and the striatum (area of tumor implantation). Figure 17B) Representative section of mice tumors from patient derived QNS108 cell injection showing the whole section and the striatum (area of tumor implantation).
Attorney Docket No.07039-2280WO1 / 2023-199 Figures 18A-18L. Analysis and validation of scRNA seq. Figure 18A) Number of GBM cells found 24 hours after CAR T/non-CAR T cell intratumoral infusion. N=4 mice per group. Figure 18B) number of human T cells found 24 hours after CAR T/non-CAR T cell intratumoral infusion. N=4 mice per group. Validation of the most upregulated genes in GBM in response to CAR T treatment using CGGA database (human mRNA expression) showed significant correlation between genes related to CD4+ Th1 including Figure 18C) IL12R (N=1011 glioma patients ; R=0.27); Figure 18D) IFNGR (N= 1018 glioma patients; R=0.31); Figure 18E) TNF (N= 66 glioma patients ; R=0.18); Figure 18F) IL15 (N=1010 glioma patients ; R=0.51) and PLAAT4 (second highest upregulated gene). There was no correlation between CD8+ genes related to cytotoxicity including Figure 18G) GZMB (N= 649 glioma patients; R=0.019); Figure 18H) GZMA (N= 651 glioma patients; R=0.002); Figure 18I) PRF1 (N= 646 glioma patients; R=0.001); Figure 18J) LAMP1 (N=651 glioma patients; R=-0.30) and CTSS (highest upregulated gene). Showing that human GBM cells activate the same subset of genes in response to T cell activation (CD4+-Th1 T cell activation). Figure 18K) CTSS mRNA fold change in low grade gliomas vs. high grade gliomas showing an increase in higher grade tumors. Figure 18L) PLAAT4 mRNA fold change in low grade gliomas vs. high grade gliomas showing an increase in higher grade tumors. Figures 19A-19G. Analysis of T cells after infusion. Figure 19A) Mouse brain sections showing CD4+ T-cells in the meninges of MC9999 CAR T treated mice after 20 weeks (endpoint). Figure 19B) Mouse brain sections showing CD4+ and CD8+ T cells in the tumor and meninges of Non-CAR T treated mice after 11 weeks (endpoint). Figures 19C-19D) Volcano plots showing the upregulated and downregulated genes in CD4+ T (Figure 19C) and CD8+ T cells (Figure 19D) in CAR T vs. Non-CAR T treated group 24 hours after infusion. Figures 19E-19G pathway analysis of genes upregulated in CD4+ T cells (Figure 19E), downregulated in CD4+ T cells (Figure 19F), and upregulated in CD8 + T cells (Figure 19G). Figures 20A-20C. Timelapse video of CAR-T cell -mediated killing of autologous GBM cells (QNS986) Figure 20A) incubated with MC9999 CAR T cells; Figure 20B) Incubated with Non-CAR T cells; and Figure 20C) Tumor cells only (no T cell addition).
Attorney Docket No.07039-2280WO1 / 2023-199 Figure 21. Immunophenotypic Characterization and PD-L1 Expression in M2 Macrophages and TAMs from GBM Patients. Macrophages were identified using CD163 and CD209 as surface markers to define a double-positive population, followed by analysis of PD-L1 expression within this subset. High PD-L1 expression was observed in M2 macrophages from samples QNS1065, QNS1070, and QNS871, as well as in TAMs from QNS1065 and QNS1070. Figures 22A-22F. Analysis of brain reorganization after CAR T infusion. Figure 22A) Analysis of microglia and astrocytes of PBS, Non-CAR T and MC9999 CAR T cells at experimental endpoint has observed astrocytosis in the ipsilateral hemisphere including white matter and gray matter after tumor resolution. Figure 22B) Quantification of Iba1-expressing cells in the MC9999 CAR T treated mice (N=5) contralateral vs. ipsilateral showing no differences, whereas Figure 22C) GFAP-expressing cells in MC9999 CAR T-treated mice (N=5), comparing contralateral and ipsilateral sides, showing significant differences. Figure 22D) individual analysis of the MC9999 treated mice brains showed astrocytosis in all the mice. Figure 22E) single cell cluster analysis detected early myelinating oligodendrocytes (BCAS1+) as the largest population of cells 24 hours after CAR T infusion. Figure 22F) pathways upregulated in early myelinating oligodendrocytes showing activation of pathways related to regeneration and remyelination. D
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ESCRIPTION This document provides methods and materials for generating immune cells (e.g., lymphocytes such as TILs, T cells, NK cells, macrophages, and neutrophils) expressing (e.g., engineered to express) an antigen receptor (e.g., a CAR) that can target a PD-L1 polypeptide. In some cases, antigen receptors (e.g., CARs) that can target a PD-L1 polypeptide can include (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains. In some cases, an immune cell (e.g., a lymphocyte such as a TIL, T cell, or NK cell) engineered to express an antigen receptor (e.g., a CAR) that can target a PD-L1 polypeptide can include (e.g., can be engineered to include) nucleic acid encoding the antigen receptor (e.g., a CAR)
Attorney Docket No.07039-2280WO1 / 2023-199 that can target the PD-L1 polypeptide such that the antigen receptor is expressed by the immune cell. This document also provides methods and materials for using immune cells (e.g., lymphocytes such as TILs, T cells, NK cells, macrophages, and neutrophils) expressing (e.g., engineered to express) an antigen receptor (e.g., a CAR) that can target a PD-L1 polypeptide. In some cases, the immune cells (e.g., lymphocytes such as TILs, T cells, NK cells, macrophages, and neutrophils) expressing (e.g., engineered to express) an antigen receptor (e.g., a CAR) that can target a PD-L1 polypeptide can be administered (e.g., in an adoptive cell therapy) to a mammal (e.g., a human) having cancer (e.g., breast cancer) to treat the mammal. Immune cells (e.g., lymphocytes such as TILs, T cells, NK cells, macrophages, and neutrophils) can express an endogenous PD-1 polypeptide that can target and bind to cells (e.g., cancer cells) expressing a PD-L1 polypeptide on its surface. In the tumor microenvironment (TME), such a PD-1/PD-L1 interaction can weaken or inactivate immune cells allowing cancer cells to escape immune detection. Immune cells (e.g., lymphocytes such as TILs, T cells, NK cells, macrophages, and neutrophils) expressing (e.g., engineered to express) an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) can target and bind to a cell (e.g., a cancer cell) expressing a PD-L1 polypeptide on its surface, and can retain immune cell functions (e.g., can retain the ability to target and destroy a cell such as a cancer cell expressing a PD-L1 polypeptide on its surface). An antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) can target any appropriate PD-L1 polypeptide. Examples of PD-L1 polypeptides that can be targeted by an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) include, without limitation, PD-L1 polypeptides having the amino acid sequences set forth in National
Attorney Docket No.07039-2280WO1 / 2023-199 Center for Biotechnology Information (NCBI) GenBank
® Accession No. NP_001300958.1, Accession No.NP_001254635.1, and Accession No.NP_054862.1. In some cases, the targeted PD-L1 polypeptide can be a human PD-L1 polypeptide. For example, an antigen receptor provided herein can target a polypeptide comprising, consisting essentially of, or consisting of the PD-L1 amino acid set forth in SEQ ID NO:1 (see, e.g., Example 2). An antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) can include any appropriate antigen-binding domain that binds a PD-L1 polypeptide. In some cases, an antigen-binding domain that binds a PD-L1 polypeptide can include an antibody or a fragment thereof that targets a PD-L1 polypeptide. Examples of antigen-binding domains include, without limitation, an antigen-binding fragment (Fab), a variable region of an antibody heavy (VH) chain, a variable region of a light (VL) chain, a single chain variable fragment (scFv), and a PD-1 polypeptide. For example, an antigen-binding domain that targets a PD-L1 polypeptide can include a PD-1 polypeptide set forth in SEQ ID NO:2 (see, e.g., Example 2). An antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) can be any appropriate type of antigen receptor. In some cases, an antigen receptor can be a heterologous antigen receptor. In some cases, an antigen receptor can be a CAR. The term “chimeric antigen receptor” as used herein refers to a chimeric polypeptide that is designed to include an optional signal peptide, an antigen binding domain, an optional hinge, a transmembrane domain, and one or more intracellular signaling domains. As described herein, the antigen binding domain of a CAR provided herein can be designed to bind to a PD-L1 polypeptide (e.g., a human PD-L1 polypeptide). For example, a CAR provided herein can be designed to include the components of an antibody, antigen binding fragment, and/or antibody domain described herein (e.g., a combination of CDRs) as an antigen binding domain provided that that
Attorney Docket No.07039-2280WO1 / 2023-199 antigen binding domain has the ability to bind to a PD-L1 polypeptide (e.g., a human PD- L1 polypeptide). In some examples, a CAR provided herein can be designed to include an antigen binding domain that includes two sets of three CDRs (e.g., CDR1, CDR2, and CDR3 of a heavy chain and CDR1, CDR2, and CDR3 of a light chain) of an antigen binding fragment provided herein (e.g., SEQ ID NOs:4-6 and 8-10). In some cases, an antigen binding domain of a CAR targeting an ENPP1 polypeptide can be designed to include a VH domain described herein or a scFv antibody described herein. In some cases, an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) can include (i) a heavy chain variable domain having a CDR1 having the amino acid sequence set forth in SEQ ID NO:4 (or a variant of SEQ ID NO:4 with one, two, three, or four amino acid modifications), a CDR2 having the amino acid sequence set forth in SEQ ID NO:5 (or a variant of SEQ ID NO:5 with one, two, three, or four amino acid modifications), and a CDR3 having the amino acid sequence set forth in SEQ ID NO:6 (or a variant of SEQ ID NO:6 with one, two, three, or four amino acid modifications); and/or (ii) a light chain variable domain having a CDR1 having the amino acid sequence set forth in SEQ ID NO:8 (or a variant of SEQ ID NO:8 with one, two, three, or four amino acid modifications), a CDR2 having the amino acid sequence set forth in SEQ ID NO:9 (or a variant of SEQ ID NO:9 with one, two, three, or four amino acid modifications), and a CDR3 having the amino acid sequence set forth SEQ ID NO:10 (or a variant of SEQ ID NO:10 with one, two, three, or four amino acid modifications). Examples of such an antigen binding fragment having these CDRs and the ability to bind to a PD-L1 polypeptide (e.g., a human PD-L1 polypeptide) include, without limitation, the variable domains set forth in Example 3. In some cases, an antigen receptor provided herein having the ability to bind to a PD-L1 polypeptide (e.g., a human PD-L1 polypeptide) and having (a) a heavy chain variable domain having a CDR1 having the amino acid sequence set forth in SEQ ID NO:4 (or a variant of SEQ ID NO:4 with one, two, three, or four amino acid modifications), a CDR2 having the amino acid sequence set forth in SEQ ID NO:5 (or a
Attorney Docket No.07039-2280WO1 / 2023-199 variant of SEQ ID NO:5 with one, two, three, or four amino acid modifications), and a CDR3 having the amino acid sequence set forth in SEQ ID NO:6 (or a variant of SEQ ID NO:6 with one, two, three, or four amino acid modifications) and/or (b) a light chain variable domain having a CDR1 having the amino acid sequence set forth in SEQ ID NO:8 (or a variant of SEQ ID NO:8 with one, two, three, or four amino acid modifications), a CDR2 having the amino acid sequence set forth in SEQ ID NO:9 (or a variant of SEQ ID NO:9 with one, two, three, or four amino acid modifications), and a CDR3 having the amino acid sequence set forth SEQ ID NO:10 (or a variant of SEQ ID NO:10 with one, two, three, or four amino acid modifications) can include any appropriate framework regions. For example, an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) can include (a) a heavy chain variable domain that includes a framework region 1 having the amino acid sequence set forth in SEQ ID NO:11 (or a variant of SEQ ID NO:11 with one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid modifications), a framework region 2 having the amino acid sequence set forth in SEQ ID NO:12 (or a variant of SEQ ID NO:12 with one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid modifications), a framework region 3 having the amino acid sequence set forth in SEQ ID NO:13 (or a variant of SEQ ID NO:13 with one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid modifications), and a framework region 4 having the amino acid sequence set forth in SEQ ID NO:14 (or a variant of SEQ ID NO:14 with one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid modifications) and/or (b) a light chain variable domain that includes a framework region 1 having the amino acid sequence set forth in SEQ ID NO:15 (or a variant of SEQ ID NO:15 with one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid modifications), a framework region 2 having the amino acid sequence set forth in SEQ ID NO:16 (or a variant of SEQ ID NO:16 with one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid modifications), a framework region 3 having the amino acid sequence set forth in SEQ ID NO:17 (or a variant of SEQ ID NO:17 with one, two, three, four, five, six, seven, eight, nine, ten, or
Attorney Docket No.07039-2280WO1 / 2023-199 more amino acid modifications), and a framework region 4 having the amino acid sequence set forth in SEQ ID NO:18 (or a variant of SEQ ID NO:18 with one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid modifications). In some cases, an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) having any of the CDRs set forth in Example 3 can be designed to include framework regions as set forth in Example 3 or can be designed to include one or more framework regions from another antibody, antibody fragment, or antibody domain. For example, a scFv can be designed to include the six CDRs set forth in Example 3 and the framework regions set forth in Example 3. In some cases, a scFv can be designed to include the six CDRs set forth in Example 3 and the framework regions set forth in Example 3 except that one or more of the framework regions can be replaced with one or more framework regions from another antibody, antibody fragment, or antibody domain. In some cases, an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) can include (a) a heavy chain variable domain that includes an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO:3 and/or (b) a light chain variable domain that includes an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO:7. For example, an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) can include (a) a heavy chain variable domain that includes an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity to the amino acid sequence set forth in SEQ ID NO:3 and/or (b) a light chain variable domain that includes an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity to the amino acid sequence set forth in SEQ ID NO:7. In some cases, an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including
Attorney Docket No.07039-2280WO1 / 2023-199 (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) can include (a) a heavy chain variable domain that includes an amino acid sequence having 100 percent identity to the amino acid sequence set forth in SEQ ID NO:3 and/or (b) a light chain variable domain that includes an amino acid sequence having 100 percent identity to the amino acid sequence set forth in SEQ ID NO:7. In some cases, an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) can include (a) a heavy chain variable domain that includes an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO:3, provided that the heavy chain variable domain includes the amino acid sequences set forth in SEQ ID NOs:4, 5, and 6, and/or (b) a light chain variable domain that includes an amino acid sequence having at least 90 percent identity to the amino acid sequence set forth in SEQ ID NO:7, provided that the light chain variable domain includes the amino acid sequences set forth in SEQ ID NOs:8, 9, and 10. For example, an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) can include (a) a heavy chain variable domain that includes an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity to the amino acid sequence set forth in SEQ ID NO:3, provided that the heavy chain variable domain includes the amino acid sequences set forth in SEQ ID NOs:4, 5, and 6, and/or (b) a light chain variable domain that includes an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity to the amino acid sequence set forth in SEQ ID NO:7, provided that the light chain variable domain includes the amino acid sequences set forth in SEQ ID NOs:8, 9, and 10. In some cases, an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling
Attorney Docket No.07039-2280WO1 / 2023-199 domains) can include (a) a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO:3 or the amino acid set forth in SEQ ID NO:3 with one, two, three, four, five, six, seven, eight, nine, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and/or amino acid additions) and/or (b) a light chain variable domain that includes the amino acid sequence set forth in SEQ ID NO:7 or the amino acid set forth in SEQ ID NO:7 with one, two, three, four, five, six, seven, eight, nine, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and/or amino acid additions). For example, an antibody or antigen binding fragment provided herein can have the ability to bind to a PD-L1 polypeptide (e.g., a human PD-L1 polypeptide), can include a heavy chain variable domain having the amino acid sequence set forth in SEQ ID NO:3 with one, two, three, four, five, six, seven, eight, nine, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and/or amino acid additions), provided that the heavy chain variable domain includes the amino acid sequences set forth in SEQ ID NOs:4, 5, and 6, and can include a light chain variable domain having the amino acid sequence set forth in SEQ ID NO:7 with one, two, three, four, five, six, seven, eight, nine, or 10 amino acid modifications (e.g., amino acid substitutions, amino acid deletions, and/or amino acid additions), provided that the light chain variable domain includes the amino acid sequences set forth in SEQ ID NOs:8, 9, and 10. In some cases, an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) can include (a) a heavy chain variable domain comprising (i) a CDR1 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:4, (ii) a CDR2 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:5, and (iii) a CDR3 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:6, and/or (b) a light chain variable domain comprising (i) a CDR1 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:8, (ii) a CDR2 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ
Attorney Docket No.07039-2280WO1 / 2023-199 ID NO:9, and (iii) a CDR3 that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:10. As used herein, a “CDR1 that consists essentially of the amino acid sequence set forth in SEQ ID NO:4” is a CDR1 that has zero, one, or two amino acid substitutions within SEQ ID NO:4, that has zero, one, two, three, four, or five amino acid residues directly preceding SEQ ID NO:4, and/or that has zero, one, two, three, four, or five amino acid residues directly following SEQ ID NO:4, provided that the antigen receptor maintains its basic ability to bind to a PD-L1 polypeptide (e.g., a human PD-L1 polypeptide). As used herein, a “CDR2 that consists essentially of the amino acid sequence set forth in SEQ ID NO:5” is a CDR2 that has zero, one, or two amino acid substitutions within SEQ ID NO:5, that has zero, one, two, three, four, or five amino acid residues directly preceding SEQ ID NO:5, and/or that has zero, one, two, three, four, or five amino acid residues directly following SEQ ID NO:5, provided that the antigen receptor maintains its basic ability to bind to a PD-L1 polypeptide (e.g., a human PD-L1 polypeptide). As used herein, a “CDR3 that consists essentially of the amino acid sequence set forth in SEQ ID NO:6” is a CDR3 that has zero, one, or two amino acid substitutions within SEQ ID NO:6, that has zero, one, two, three, four, or five amino acid residues directly preceding SEQ ID NO:6, and/or that has zero, one, two, three, four, or five amino acid residues directly following SEQ ID NO:6, provided that the antigen receptor maintains its basic ability to bind to a PD-L1 polypeptide (e.g., a human PD-L1 polypeptide). As used herein, a “CDR1 that consists essentially of the amino acid sequence set forth in SEQ ID NO:8” is a CDR1 that has zero, one, or two amino acid substitutions within SEQ ID NO:8, that has zero, one, two, three, four, or five amino acid residues directly preceding SEQ ID NO:8, and/or that has zero, one, two, three, four, or five amino acid residues directly following SEQ ID NO:8, provided that the antigen receptor maintains its basic ability to bind to a PD-L1 polypeptide (e.g., a human PD-L1 polypeptide).
Attorney Docket No.07039-2280WO1 / 2023-199 As used herein, a “CDR2 that consists essentially of the amino acid sequence set forth in SEQ ID NO:9” is a CDR2 that has zero, one, or two amino acid substitutions within SEQ ID NO:9, that has zero, one, two, three, four, or five amino acid residues directly preceding SEQ ID NO:9, and/or that has zero, one, two, three, four, or five amino acid residues directly following SEQ ID NO:9, provided that the antigen receptor maintains its basic ability to bind to a PD-L1 polypeptide (e.g., a human PD-L1 polypeptide). As used herein, a “CDR3 that consists essentially of the amino acid sequence set forth in SEQ ID NO:10” is a CDR3 that has zero, one, or two amino acid substitutions within SEQ ID NO:10, that has zero, one, two, three, four, or five amino acid residues directly preceding SEQ ID NO:10, and/or that has zero, one, two, three, four, or five amino acid residues directly following SEQ ID NO:10, provided that the antigen receptor maintains its basic ability to bind to a PD-L1 polypeptide (e.g., a human PD-L1 polypeptide). When designing a single chain antibody (e.g., a scFv) having a heavy chain variable domain and a light chain variable domain, the two regions can be directly connected or can be connected using any appropriate linker sequence. For example, a heavy chain variable domain having the CDRs of SEQ ID NOs:4-6 can be directly connected to a light chain variable domain having the CDRs of SEQ ID NOs:8-10, respectively, via a linker sequence. Examples of linker sequences that can be used to connect a heavy chain variable domain and a light chain variable domain to create a scFv include, without limitation, those linkers set forth in Example 6. As indicated herein, the amino acid sequences described herein can include amino acid modifications (e.g., the articulated number of amino acid modifications). Such amino acid modifications can include, without limitation, amino acid substitutions, amino acid deletions, amino acid additions, and combinations. In some cases, an amino acid modification can be made to improve the binding and/or contact with an antigen and/or to improve a functional activity of an antigen receptor provided herein that can target a PD- L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling
Attorney Docket No.07039-2280WO1 / 2023-199 domains). In some cases, an amino acid substitution within an articulated sequence identifier can be a conservative amino acid substitution. For example, conservative amino acid substitutions can be made by substituting one amino acid residue for another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains can include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non- polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In some cases, an amino acid substitution within an articulated sequence identifier can be a non-conservative amino acid substitution. Non-conservative amino acid substitutions can be made by substituting one amino acid residue for another amino acid residue having a dissimilar side chain. Examples of non-conservative substitutions include, without limitation, substituting (a) a hydrophilic residue (e.g., serine or threonine) for a hydrophobic residue (e.g., leucine, isoleucine, phenylalanine, valine, or alanine); (b) a cysteine or proline for any other residue; (c) a residue having a basic side chain (e.g., lysine, arginine, or histidine) for a residue having an acidic side chain (e.g.,
aspartic acid or glutamic acid); and (d) a residue having a bulky side chain (e.g., phenylalanine) for glycine or other residue having a small side chain. Methods for generating an amino acid sequence variant (e.g., an amino acid sequence that includes one or more modifications with respect to an articulated sequence identifier) can include site-specific mutagenesis or random mutagenesis (e.g., by PCR) of
a nucleic acid encoding the antibody or fragment thereof. See, for example, Zoller, Curr. Opin. Biotechnol.3: 348-354 (1992). Both naturally occurring and non-naturally occurring amino acids (e.g., artificially-derivatized amino acids) can be used to generate an amino acid sequence variant provided herein. An antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) can include any
Attorney Docket No.07039-2280WO1 / 2023-199 appropriate transmembrane domain. Examples of transmembrane domains that can be included in an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains)
include, without limitation, CD3 transmembrane domains, CD4 transmembrane domains, CD8 transmembrane domains, CD28 transmembrane domains, and 4-1BB transmembrane domains. For example, an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) can include a transmembrane domain that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:22 (see, e.g., Example 4). In some cases, a transmembrane domain that is included in an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) can be designed to include a transmembrane domain that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in SEQ ID NO:22 (see, e.g., Example 4) with one, two, three, four, five, six, seven, eight, nine, or ten amino acid deletions, additions, substitutions, or combinations thereof. In some cases, a transmembrane domain that is included in an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) can be designed to include a transmembrane domain that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in SEQ ID NO:22 (see, e.g., Example 4) with two or less, three or less, four or less, five or less, six or less, seven or less, eight or less, nine or less, or ten or less amino acid deletions, additions, substitutions, or combinations thereof. An antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) can include any
Attorney Docket No.07039-2280WO1 / 2023-199 appropriate one or more signaling domains. For example, an antigen receptor provided herein can be designed to include one, two, three, four, or five signaling domains. When an antigen receptor provided herein includes more than one (e.g., two, three, four, or five) signaling domains, the antigen receptor can include any appropriate combination of signaling domains. In some cases, an antigen receptor provided herein can be designed to include one or more signaling domains normally found within an immune cell (e.g., a lymphocyte such as a TIL, a T cell, or a NK cell). In some cases, a signaling domain that can be included in an antigen receptor provided herein that can target a PD-L1 polypeptide can be from an adaptor polypeptide. Examples of signaling domains that can be included in an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains)
include, without limitation, CD3 intracellular signaling domains, CD27 intracellular signaling domains, CD28 intracellular signaling domains, OX40 (CD134) intracellular signaling domains, 4-1BB (CD137) intracellular signaling domains, CD278 intracellular signaling domains, DAP10 intracellular signaling domains, DAP12 intracellular signaling domains, NKG2D intracellular signaling domains, CD244 intracellular signaling
domains, and CD266 intracellular signaling domains. In some cases, an antigen receptor provided herein can be designed to be a first generation CAR having a CD3 signaling domain. In some cases, an antigen receptor provided herein can be designed to be a
second generation CAR having a CD28 signaling domain followed by a CD3 signaling domain. In some cases, an antigen receptor provided herein can be designed to be a third generation CAR having (a) a CD28 signaling domain followed by (b) a CD27 signaling domain, an OX40 signaling domains, or a 4-1BB signaling domain followed by (c) a
CD3 signaling domain. In some cases, an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) can include a NKG2D transmembrane domain followed by a DAP10 signaling domain followed by a 4-1BB signaling domain followed by a CD3 signaling domain. For example, an antigen receptor provided herein that can target a PD-
Attorney Docket No.07039-2280WO1 / 2023-199 L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) can be designed to include at least one intracellular signaling domain that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in SEQ ID NO:24 (see, e.g., Example 5). In some cases, an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) can be designed to include at least one intracellular signaling domain that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in SEQ ID NO:24 (see, e.g., Example 5) with one, two, three, four, five, six, seven, eight, nine, or ten amino acid deletions, additions, substitutions, or combinations thereof, provided that that intracellular signaling domain has at least some activity to activate intracellular signaling. In some cases, an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) can be designed to include at least one intracellular signaling domain that comprises, consists essentially of, or consists of one of the amino acid sequences set forth in SEQ ID NO:24 (see, e.g., Example 5) with two or less, three or less, four or less, five or less, six or less, seven or less, eight or less, nine or less, or ten or less amino acid deletions, additions, substitutions, or combinations thereof, provided that that intracellular signaling domain has at least some activity to activate intracellular signaling. In some cases, an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) can be designed to include a hinge. Any appropriate hinge can be used to design a CAR described herein. Examples of hinges that can be used to make a CAR described herein include, without limitation, Ig-derived hinges (e.g., an IgG1-derived hinge, an IgG2-derived hinge, or an IgG4-derived hinge), Ig-derived hinges containing a CD2 domain and a CD3 domain, Ig-derived hinges containing a CD2 domain and lacking a
Attorney Docket No.07039-2280WO1 / 2023-199 CD3 domain, Ig-derived hinges containing a CD3 domain and lacking a CD2 domain, Ig-
derived hinges lacking a CD2 domain and lacking a CD3 domain, CD8 -derived hinges, CD28-derived hinges, CD3 -derived hinges, and CD4-derived hinges. An antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) can be designed to include a hinge of any appropriate length. For example, an antigen receptor provided herein can be designed to include a hinge that is from about 3 to about 75 (e.g., from about 3 to about 65, from about 3 to about 50, from about 5 to about 75, from about 10 to about 75, from about 5 to about 50, from about 10 to about 50, from about 10 to about 40, or from about 10 to about 30) amino acid residues in length. In some cases, a linker sequence can be used as a hinge to make an antigen receptor described herein. In some cases, an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) can be designed to include a hinge that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:26 (see, e.g., Example 6). In some cases, an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) can be designed to include a hinge that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:26 (see, e.g., Example 6) with one, two, three, four, five, six, seven, eight, nine, or ten amino acid deletions, additions, substitutions, or combinations thereof. In some cases, an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) can be designed to include a hinge that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:26 (see, e.g., Example 6) with two or less, three or less, four or less, five or less, six or less, seven or less, eight
Attorney Docket No.07039-2280WO1 / 2023-199 or less, nine or less, or ten or less amino acid deletions, additions, substitutions, or combinations thereof. In some cases, an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) can include one or more additional components. Examples of additional components that can be included in an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) include, without limitation, detectable markers, suicide switches, and surface markers that aid in enrichment. When an antigen receptor (e.g., a CAR) provided herein includes a detectable marker, the detectable marker can be any appropriate detectable marker. Examples of detectable markers that can be included in an antigen receptor (e.g., a CAR) provided herein include, without limitation, non-functional polypeptides (e.g., truncated epidermal growth factor receptor (tEGFR) polypeptides), bioluminescent polypeptides (e.g., luciferase polypeptides), fluorescent polypeptides (e.g., green fluorescent polypeptides (GFPs)), truncated nerve growth factor receptor polypeptides, signaling deficient CD20 polypeptides, and self-cleaving polypeptides. For example, an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) can include a tEGFR marker polypeptide that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:28 (see, e.g., Example 7). Also provided herein are nucleic acid molecules encoding an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains). In some cases, a nucleic acid that can encode an antigen receptor (e.g., a CAR) that can target a PD-L1 polypeptide provided herein (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD- L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains)
Attorney Docket No.07039-2280WO1 / 2023-199 can be in the form of a vector (e.g., a viral vector or a non-viral vector). When a vector including nucleic acids encoding an antigen receptor (e.g., a CAR) that can target a PD- L1 polypeptide is a viral vector, any appropriate viral vector can be used. A viral vector can be derived from a positive-strand virus or a negative-strand virus. A viral vector can be derived from a virus with a DNA genome or an RNA genome. In some cases, a viral vector can be a chimeric viral vector. In some cases, a viral vector can infect dividing cells. In some cases, a viral vector can be replication-defective. In some cases, a viral vector can infect non-dividing cells. Examples virus-based vectors that can include nucleic acid encoding an antigen receptor (e.g., a CAR) that can target a PD-L1 polypeptide include, without limitation, lentiviral vectors, retroviral vectors, and adeno- associated viral vectors. When a vector including nucleic acid encoding an antigen receptor (e.g., a CAR) that can target a PD-L1 polypeptide is a non-viral vector, any appropriate non-viral vector can be used. In some cases, a non-viral vector can be an expression plasmid (e.g., a cDNA expression vector). In some cases, a nucleic acid construct provided herein can encode an antigen receptor (e.g., a CAR) that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) and can include (a) a nucleic acid sequence that encodes an antigen-binding domain that binds a PD-L1 polypeptide (e.g., the nucleic acid set forth in SEQ ID NO:20 and/or the nucleic acid set forth in SEQ ID NO:21), (b) a nucleic acid sequence that encodes a transmembrane domain such as a CD4 transmembrane domain (e.g., the nucleic acid sequence set forth in SEQ ID NO:23), and (c) a nucleic acid sequence that encodes one or more signaling domains such as a 4-1BB signaling domain (e.g., the nucleic acid sequence set forth in SEQ ID NO:25). In cases where an antigen receptor (e.g., a CAR) that can target a PD-L1 polypeptide provided herein (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) includes a detectable marker, a nucleic acid construct that can encode an antigen receptor provided herein that can target a PD-L1 polypeptide can
Attorney Docket No.07039-2280WO1 / 2023-199 include a nucleic acid sequence that encodes the detectable marker. For example, a nucleic acid sequence encoding a detectable marker can be included in a nucleic acid construct such that an antigen receptor (e.g., a CAR) provided herein that can target a PD-L1 polypeptide and that includes the detectable marker is expressed. An example of a nucleic acid sequence encoding a detectable marker is set forth in SEQ ID NO:29. In some cases, a detectable marker included in an antigen receptor (e.g., a CAR) that can target a PD-L1 polypeptide provided herein (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) can include a cleavage peptide. For example, a nucleic acid sequence encoding a cleavage peptide can be included in a nucleic acid construct such that an antigen receptor (e.g., a CAR) provided herein that can target a PD-L1 polypeptide and that includes the detectable marker fused to a cleavage peptide. An example of a cleavage peptide can include, without limitation, a T2A peptide. Also provided herein are immune cells (e.g., lymphocytes such as TILs, T cells, NK cells, macrophages, and neutrophils) expressing (e.g., engineered to express) an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains). An immune cell engineered to express an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) can be any appropriate type of immune cell. A T cell can be a naïve T cell. Examples of immune cells that can be engineered to express an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) include, without limitation, lymphocytes such as TILs, T cells (e.g., peripheral T cells and pluripotent stem cell-derived T cells such induced pluripotent stem cell-derived T cells), and NK cells (e.g., pluripotent stem cell- derived NK cells such as induced pluripotent stem cell-derived NK cells). In some cases,
Attorney Docket No.07039-2280WO1 / 2023-199 one or more immune cells designed to express an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) can be immune cells that were obtained from (e.g., are autologous to) a mammal (e.g., a mammal having cancer) that is to be treated with those immune cells designed to express an antigen receptor provided herein that can target a PD-L1 polypeptide. For example, T cells can be obtained from a mammal to be treated with the materials and method described herein. For example, NK cells can be obtained from a mammal to be treated with the materials and method described herein. Any appropriate method can be used to express an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) on an immune cell (e.g., a lymphocyte such as a TIL, a T cell, or a NK cell). For example, nucleic acid encoding an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) can be introduced into an immune cell. In some cases, nucleic acid encoding an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) can be introduced into an immune cell by transduction (e.g., viral transduction using a viral vector such as a lentiviral vector) or transfection. In some cases, nucleic acids encoding an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling
domains) can be introduced ex vivo into one or more immune cells. For example, ex vivo engineering of immune cells can include transducing isolated immune cells with a lentiviral vector encoding an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling
Attorney Docket No.07039-2280WO1 / 2023-199
domains). In cases where immune cells were engineered ex vivo to express an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains), the immune cells can be obtained from any appropriate source (e.g., a mammal such as the mammal to be treated or a donor mammal, or a cell line). This document also provides methods and materials involved in treating cancer. For example, one or more immune cells (e.g., lymphocytes such as TILs, T cells, NK cells, macrophages, and neutrophils) expressing (e.g., engineered to express) an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) can be administered (e.g., in an adoptive cell therapy such as a CAR T cell therapy) to a mammal (e.g., a human) having cancer (e.g., breast cancer) to treat the mammal. In some cases, administering one or more immune cells (e.g., lymphocytes such as TILs, T cells, NK cells, macrophages, and neutrophils) expressing (e.g., engineered to express) an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) to a mammal (e.g., a human) having cancer (e.g., breast cancer) can be effective to activate both NK cells and T cells within the mammal. Any appropriate mammal (e.g., a human) having cancer (e.g., thyroid cancer) can be treated as described herein. Examples of mammals that can be treated as described herein include, without limitation, humans, primates (such as monkeys), dogs, cats, horses, cows, pigs, sheep, mice, and rats. In some cases, a mammal that can be treated as described herein can be a non-human mammal that contains nucleic acid encoding a human PD-L1 polypeptide such that the non-human mammal contains cells (e.g., cancer cells) expressing the encoded PD-L1 polypeptide (e.g., a PD-L1 transgenic non-human mammal such as a PD-L1 transgenic mouse). In some cases, a human having cancer (e.g., breast cancer) can be treated with one or more immune cells (e.g., lymphocytes such as
Attorney Docket No.07039-2280WO1 / 2023-199 TILs, T cells, NK cells, macrophages, and neutrophils) expressing (e.g., engineered to express) an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains). For example, a human having cancer (e.g., breast cancer) can be administered one or more immune cells (e.g., lymphocytes such as TILs, T cells, NK cells, macrophages, and neutrophils) expressing (e.g., engineered to express) an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) in an adoptive T cell therapy such as a CAR T cell therapy using the methods and materials described herein. When treating a mammal (e.g., a human) having cancer as described herein, the cancer can be any appropriate type of cancer. In some cases, a cancer treated as described herein can include one or more solid tumors. In some cases, a cancer treated as described herein can be a blood cancer. In some cases, a cancer treated as described herein can be a primary cancer. In some cases, a cancer treated as described herein can be a metastatic cancer. In some cases, a cancer treated as described herein can be a refractory cancer. In some cases, a cancer treated as described herein can be a relapsed cancer. In some cases, a cancer treated as described herein can express a PD-L1 polypeptide. Examples of cancers that can be treated as described herein include, without limitation, breast cancers (e.g., triple negative breast cancers), glioblastomas (e.g., adult glioblastomas and pediatric glioblastomas), lung cancers (e.g., non-small-cell lung cancers (NSCLCs)), melanomas, bile duct cancers (e.g., cholangiocarcinomas), ovarian cancers, and lymphomas. In some cases, the methods described herein can include identifying a mammal (e.g., a human) as having cancer (e.g., breast cancer). Any appropriate method can be used to identify a mammal having cancer. For example, imaging techniques and biopsy techniques can be used to identify mammals (e.g., humans) having cancer. Any appropriate amount (e.g., number) of immune cells (e.g., lymphocytes such as TILs, T cells, NK cells, macrophages, and neutrophils) expressing (e.g., engineered to
Attorney Docket No.07039-2280WO1 / 2023-199 express) an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) can be administered (e.g., in an adoptive cell therapy such as a CAR T cell therapy) to a mammal (e.g., a human) having cancer (e.g., breast cancer). In some cases, from about 0.25 million CAR T cells per kg body weight of the mammal (cells/kg) to about 14 million CAR T cells/kg (e.g., from about 0.25 million to about 12 million, from about 0.25 million to about 10 million, from about 0.25 million to about 8 million, from about 0.25 million to about 5 million, from about 0.25 million to about 3 million, from about 0.25 million to about 1 million, from about 1 million to about 14 million, from about 3 million to about 14 million, from about 5 million to about 14 million, from about 8 million to about 14 million, from about 10 million to about 14 million, from about 12 million to about 14 million, from about 1 million to about 12 million, from about 3 million to about 10 million, from about 5 million to about 8 million, from about 1 million to about 3 million, from about 2 million to about 4 million, from about 3 million to about 5 million, from about 4 million to about 6 million, from about 5 million to about 7 million, from about 6 million to about 8 million, from about 7 million to about 9 million, from about 8 million to about 10 million, from about 9 million to about 11 million, from about 10 million to about 12 million, or from about 11 million to about 13 million CAR T cells/kg) expressing (e.g., engineered to express) an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) can be administered to a mammal having cancer to treat the mammal. For example, a mammal having cancer can be administered a composition including from about 0.25 million CAR T cells/kg to about 14 million CAR T cells/kg. In some cases, from about 15 million CAR T cells to about 850 million CAR T cells (e.g., from about 15 million to about 750 million, from about 15 million to about 500 million, from about 15 million to about 250 million, from about 15 million to about 100 million, from about 15 million to about 75 million, from about 15 million to about 50 million, from about 15 million to about 25 million, from about 25 million to about 850
Attorney Docket No.07039-2280WO1 / 2023-199 million, from about 50 million to about 850 million, from about 75 million to about 850 million, from about 100 million to about 850 million, from about 250 million to about 850 million, from about 500 million to about 850 million, from about 750 million to about 850 million, from about 25 million to about 750 million, from about 50 million to about 500 million, from about 75 million to about 250 million, from about 50 million to about 150 million, from about 150 million to about 250 million, from about 250 million to about 500 million, or from about 500 million to about 750 million CAR T cells) expressing (e.g., engineered to express) an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) can be administered to a mammal having cancer to treat the mammal. For example, a mammal having cancer can be administered a composition including from about 15 million CAR T cells to about 850 million CAR T cells. In some cases, from about 0.25 million CAR NK cells per kg body weight of the mammal (cells/kg) to about 14 million CAR NK cells/kg (e.g., from about 0.25 million to about 12 million, from about 0.25 million to about 10 million, from about 0.25 million to about 8 million, from about 0.25 million to about 5 million, from about 0.25 million to about 3 million, from about 0.25 million to about 1 million, from about 1 million to about 14 million, from about 3 million to about 14 million, from about 5 million to about 14 million, from about 8 million to about 14 million, from about 10 million to about 14 million, from about 12 million to about 14 million, from about 1 million to about 12 million, from about 3 million to about 10 million, from about 5 million to about 8 million, from about 1 million to about 3 million, from about 2 million to about 4 million, from about 3 million to about 5 million, from about 4 million to about 6 million, from about 5 million to about 7 million, from about 6 million to about 8 million, from about 7 million to about 9 million, from about 8 million to about 10 million, from about 9 million to about 11 million, from about 10 million to about 12 million, or from about 11 million to about 13 million CAR NK cells/kg) expressing (e.g., engineered to express) an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a
Attorney Docket No.07039-2280WO1 / 2023-199 transmembrane domain, and (c) one or more signaling domains) can be administered to a mammal having cancer to treat the mammal. For example, a mammal having cancer can be administered a composition including from about 0.25 million CAR NK cells/kg to about 14 million CAR NK cells/kg. In some cases, from about 15 million CAR NK cells to about 850 million CAR NK cells (e.g., from about 15 million to about 750 million, from about 15 million to about 500 million, from about 15 million to about 250 million, from about 15 million to about 100 million, from about 15 million to about 75 million, from about 15 million to about 50 million, from about 15 million to about 25 million, from about 25 million to about 850 million, from about 50 million to about 850 million, from about 75 million to about 850 million, from about 100 million to about 850 million, from about 250 million to about 850 million, from about 500 million to about 850 million, from about 750 million to about 850 million, from about 25 million to about 750 million, from about 50 million to about 500 million, from about 75 million to about 250 million, from about 50 million to about 150 million, from about 150 million to about 250 million, from about 250 million to about 500 million, or from about 500 million to about 750 million CAR NK cells) expressing (e.g., engineered to express) an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen- binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) can be administered to a mammal having cancer to treat the mammal. For example, a mammal having cancer can be administered a composition including from about 15 million CAR NK cells to about 850 million CAR NK cells. A mammal (e.g., a human) having cancer (e.g., thyroid cancer) can be administered one or more immune cells (e.g., lymphocytes such as TILs, T cells, NK cells, macrophages, and neutrophils) expressing (e.g., engineered to express) an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) using any appropriate method. For example, one or more immune cells (e.g., lymphocytes such as TILs, T cells, NK cells, macrophages, and neutrophils) expressing (e.g., engineered to express) an
Attorney Docket No.07039-2280WO1 / 2023-199 antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) can be used in an adoptive T cell therapy (e.g., a CAR T cell therapy) to treat a mammal having cancer. In some cases, methods of treating a mammal having cancer as described herein can reduce the number of cancer cells (e.g., cancer cells expressing a PD-L1 polypeptide) within a mammal. In some cases, methods of treating a mammal having cancer as described herein can reduce the size of one or more tumors (e.g., tumors expressing a PD-L1 polypeptide) within a mammal. In some cases, one or more immune cells (e.g., lymphocytes such as TILs, T cells, NK cells, macrophages, and neutrophils) expressing (e.g., engineered to express) an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having cancer such as a cancer containing cancer cells that express a PD-L1 polypeptide) to reduce the size of the cancer present within a mammal. For example, the materials and methods described herein can be used to reduce the number of cancer cells present within a mammal having cancer by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. For example, the materials and methods described herein can be used to reduce the size (e.g., volume) of one or more tumors present within a mammal having cancer by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. In some cases, one or more immune cells (e.g., lymphocytes such as TILs, T cells, NK cells, macrophages, and neutrophils) expressing (e.g., engineered to express) an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having cancer such as a cancer containing cancer cells that express a PD-L1 polypeptide) to improve survival of the mammal. For example, disease-free survival (e.g., relapse-free survival) can be improved
Attorney Docket No.07039-2280WO1 / 2023-199 using the materials and methods described herein. For example, progression-free survival can be improved using the materials and methods described herein. In some cases, the materials and methods described herein can be used to improve the survival of a mammal having cancer by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. In some cases, one or more immune cells (e.g., lymphocytes such as TILs, T cells, NK cells, macrophages, and neutrophils) expressing (e.g., engineered to express) an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having cancer such as a cancer containing cancer cells that express a PD-L1 polypeptide) to increase the number of tumor-infiltrating lymphocytes (e.g., T cells present in within the tumor microenvironment of a cancer) within the mammal. For example, the materials and methods described herein can be used to increase the number of tumor-infiltrating lymphocytes within a mammal having cancer by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. In some cases, a mammal (e.g., a human) having cancer (e.g., a breast cancer) can be administered a single administration of one or more immune cells (e.g., lymphocytes such as TILs, T cells, NK cells, macrophages, and neutrophils) expressing (e.g., engineered to express) an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains). For example, one or more immune cells (e.g., lymphocytes such as TILs, T cells, NK cells, macrophages, and neutrophils) expressing (e.g., engineered to express) an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) can be administered to a mammal having cancer (e.g., a cancer containing one or more cancer cells expressing a PD-L1 polypeptide) once.
Attorney Docket No.07039-2280WO1 / 2023-199 In some cases, a mammal (e.g., a human) having cancer (e.g., a breast cancer) can be administered more than one (e.g., two, three, four, five, or more) administrations of one or more immune cells (e.g., lymphocytes such as TILs, T cells, NK cells, macrophages, and neutrophils) expressing (e.g., engineered to express) an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains). For example, one or more immune cells (e.g., lymphocytes such as TILs, T cells, NK cells, macrophages, and neutrophils) expressing (e.g., engineered to express) an antigen receptor (e.g., a CAR) that can target a PD-L1 polypeptide can be administered to a mammal having cancer (e.g., a cancer containing one or more cancer cells expressing a PD-L1 polypeptide) multiple times (e.g., over a period of time ranging from days to weeks to months). In some cases, one or more immune cells (e.g., lymphocytes such as TILs, T cells, NK cells, macrophages, and neutrophils) expressing (e.g., engineered to express) an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) can be formulated into a composition (e.g., a pharmaceutically acceptable composition) for administration to a mammal having a cancer (e.g., a cancer containing one or more cancer cells expressing a PD-L1 polypeptide). For example, immune cells (e.g., lymphocytes such as TILs, T cells, NK cells, macrophages, and neutrophils) expressing (e.g., engineered to express) an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) can be formulated together with one or more pharmaceutically acceptable carriers (additives), excipients, and/or diluents. In some cases, a pharmaceutically acceptable carrier, excipient, or diluent can be a naturally occurring pharmaceutically acceptable carrier, excipient, or diluent. In some cases, a pharmaceutically acceptable carrier, excipient, or diluent can be a non- naturally occurring (e.g., an artificial or synthetic) pharmaceutically acceptable carrier, excipient, or diluent. Examples of pharmaceutically acceptable carriers, excipients, and
Attorney Docket No.07039-2280WO1 / 2023-199 diluents that can be used in a composition described herein include, without limitation, serum proteins (e.g., human serum albumin), water, and salts or electrolytes (e.g., phosphate salts, saline, protamine sulfate, and DMSO). A composition containing one or more immune cells (e.g., lymphocytes such as TILs, T cells, NK cells, macrophages, and neutrophils) expressing (e.g., engineered to express) an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) can be designed for parenteral (including subcutaneous, intramuscular, intravenous, and intradermal) or intratumoral administration. Compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions that can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient. A composition containing one or more immune cells (e.g., lymphocytes such as TILs, T cells, NK cells, macrophages, and neutrophils) expressing (e.g., engineered to express) an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) can be administered using any appropriate technique and to any appropriate location. A composition including one or more immune cells (e.g., lymphocytes such as TILs, T cells, NK cells, macrophages, and neutrophils) expressing (e.g., engineered to express) an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) can be administered locally or systemically. For example, a composition provided herein can be administered locally by intratumoral administration (e.g., injection into tumors) or by administration into biological spaces infiltrated by tumors (e.g. intraspinal administration, intracerebellar administration, intraperitoneal administration and/or pleural administration). For example, a composition provided herein can be administered systemically by intravenous administration (e.g., injection or infusion) to a mammal (e.g., a human).
Attorney Docket No.07039-2280WO1 / 2023-199 In some cases, one or more immune cells (e.g., lymphocytes such as TILs, T cells, NK cells, macrophages, and neutrophils) expressing (e.g., engineered to express) an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) can be the sole active ingredient administered to a mammal (e.g., a human) having cancer to treat the cancer. For example, a composition including one or more immune cells (e.g., lymphocytes such as TILs, T cells, NK cells, macrophages, and neutrophils) expressing (e.g., engineered to express) an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) can include the one or more engineered immune cells provided herein as the sole active agent to treat a mammal (e.g., a human) having cancer. In some cases, one or more immune cells (e.g., lymphocytes such as TILs, T cells, NK cells, macrophages, and neutrophils) expressing (e.g., engineered to express) an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) can be administered to a mammal having cancer as a combination therapy with one or more additional agents and/or therapies used to treat a cancer. In some cases, an anti-cancer agent can be a chemotherapeutic agent. In some cases, an anti-cancer agent can be an immunotherapeutic agent. In some cases, an anti-cancer agent can be an immune- checkpoint inhibitor. Examples of anti-cancer agents include, without limitation, trametinib, dabrafenib, binimetinib, selumntinib, vemurafenib, encorafenib, cobimetinib, busulfan, cisplatin, carboplatin, paclitaxel, docetaxel, nab-paclitaxel, altretamine, capecitabine, cyclophosphamide, etoposide (vp-16), gemcitabine, ifosfamide, irinotecan (cpt-11), liposomal doxorubicin, melphalan, pemetrexed, topotecan, vinorelbine, goserelin, leuprolide, tamoxifen, letrozole, anastrozole, exemestane, bevacizumab, olaparib, rucaparib, niraparib, ipilimumab (e.g., YERVOY
®), pembrolizumab (e.g., KEYTRUDA
®), nivolumab (e.g., OPDIVO
®) and any combinations thereof. In cases
Attorney Docket No.07039-2280WO1 / 2023-199 where one or more immune cells (e.g., lymphocytes such as TILs, T cells, NK cells, macrophages, and neutrophils) expressing (e.g., engineered to express) an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) are used with one or more additional agents treat a cancer, the one or more additional agents can be administered at the same time (e.g., in a single composition) or independently. In some cases, one or more immune cells (e.g., lymphocytes such as TILs, T cells, NK cells, macrophages, and neutrophils) expressing (e.g., engineered to express) an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) can be administered first, and the one or more additional agents administered second, or vice versa. Examples of therapies that can be used to treat cancer include, without limitation, surgery, radiation therapy, carbon ion therapy, and proton therapy. In cases where one or more immune cells (e.g., lymphocytes such as TILs, T cells, NK cells, macrophages, and neutrophils) expressing (e.g., engineered to express) an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen- binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) are used in combination with one or more additional therapies used to treat cancer, the one or more additional therapies can be performed at the same time or independently of the administration of one or more immune cells (e.g., lymphocytes such as TILs, T cells, NK cells, macrophages, and neutrophils) expressing (e.g., engineered to express) an antigen receptor provided herein that can target a PD-L1 polypeptide. For example, the one or more immune cells (e.g., lymphocytes such as TILs, T cells, NK cells, macrophages, and neutrophils) expressing (e.g., engineered to express) an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) can be administered before, during, and/or after the one or more additional therapies are performed.
Attorney Docket No.07039-2280WO1 / 2023-199 In certain instances, a cancer within a mammal can be monitored to evaluate the effectiveness of the cancer treatment. Any appropriate method can be used to determine whether or not a mammal having cancer is treated. For example, imaging techniques or laboratory assays can be used to assess the number of cancer cells and/or the size of a tumor present within a mammal. For example, imaging techniques or laboratory assays can be used to assess the location of cancer cells and/or a tumor present within a mammal. In some cases, one or more immune cells (e.g., lymphocytes such as TILs, T cells, NK cells, macrophages, and neutrophils) expressing (e.g., engineered to express) an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) can be used (e.g., in an adoptive T cell therapy such as a CAR T cell therapy) to treat a mammal having a disease or disorder other than cancer. For example, one or more T cells (e.g., CAR T cells) expressing (e.g., engineered to express) an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) can be used to treat one or more diseases associated with the expansion of PD-L1 positive cells. Examples of PD-L1 positive cells that can be targeted by one or more immune cells (e.g., lymphocytes such as TILs, T cells, NK cells, macrophages, and neutrophils) expressing (e.g., engineered to express) an antigen receptor provided herein that can target a PD-L1 polypeptide (e.g., an antigen receptor including (a) an antigen-binding domain that binds a PD-L1 polypeptide, (b) a transmembrane domain, and (c) one or more signaling domains) include, without limitation, myeloid suppressor cells (MDSCs), tumor associated macrophages (TAMs), tumor-associated cells (e.g., tumor-associated endothelial cells, tumor-associated fibroblasts, tumor-associated T regulatory cells, and tumor-associated dendritic cells). The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
Attorney Docket No.07039-2280WO1 / 2023-199 EXAMPLES Example 1: CAR T-cell Therapy targeting PD-L1 positive cancer This Example describes the design and characterization of CARs that can bind to a PD-L1 polypeptide. Antigen specific cytotoxicity of anti-PD-L1 CAR T-cells A MC9999 CAR was designed and generated based on a heavy chain variable region and a light chain variable region of a humanized anti-PD-L1 monoclonal antibody, and the following elements in tandem: IgG4 hinge, CD28 transmembrane domain, CD28
costimulatory domain, CD3 , and a truncated epidermal growth factor receptor (tEGFR) polypeptide. Antigen specific cytotoxicity of MC9999 CAR T-cells was confirmed against a PD-L1 overexpressing cell line (MDA-MB-231 PD-L1 OE) and a PD-L1 knock out cell line (MDA-MB-231-PD-L1 KO) as a negative control. Non-CAR T-cells (non- transduced T cells from the same donor) were also used as negative control. The antigen- specific cytotoxic properties of MC9999 CAR T-cells were measured by a CD107a degranulation assay by gating either CD4 or CD8 T cells to highlight the activity of these specific MC9999 CAR T-cell populations (Figure 1A and Figure 1B). Granzyme B release into the supernatant was observed when MC9999 CAR-T cells were incubated with MDA-MB-231 PD-L1 OE, but not when MC9999 CAR-T cells were incubated with MDA-MB-231-PD-L1 KO (Figure 1C, right bars). The corresponding non-CAR T-cell controls showed non-significant granzyme B release (Figure 1C, left bar). Antigen- specific cytotoxicity of MC9999 CAR-T cells was evaluated from the perspective of the target cells in a cellular impedance cytolysis assay. MDA-MB-231 PD-L1 OE or MDA- MB-231-PD-L1 KO target cells were cultured for 24 hours before MC9999 CAR T-cells or non-CAR-T cells were added. Cytolysis, as determined by the decrease in the cell index of target cells, was observed only when MC9999 CAR T-cells were incubated with the MDA-MB-231 OE cells; all other conditions showed continued intact target cells (Figure 1D). In vivo anti-tumor effects of MC9999 CAR T-cells were evaluated in a mouse model in which female NOD scid gamma (NSG) mice received an intermammary injection of
Attorney Docket No.07039-2280WO1 / 2023-199 either luciferase labeled-MDA-MB-231-OE cells or luciferase labeled-MDA-MB-231- KO cells. After 7 days, mice that had established tumors were intravenously injected with one of the three treatments: PBS, non-CAR T-cells, or MC9999 CAR T-cells (n =5 per group). Following the treatment, tumor progression was monitored by bioluminescence imaging and long-term survival of the mice was also monitored (Figure 2A). Only mice treated with MC9999 CAR-T cells after injecting with the luciferase labeled-MDA-MB- 231 PD-L1 OE (Figure 2B) showed effective tumor regression and long-term survival compared to mice that received non-CAR T-cells (Figure 2C) or PBS (Figure 2D). MC9999 CAR T-cells show antigen-specific cytotoxicity against various solid tumors. The antigen-specific cytotoxicity of MC9999 CAR T-cells was evaluated across three solid tumor models. Calu-1, a non-small-cell lung cancer (NSCLC) cell line, stably expresses PD-L1. A PD-L1 knockout version of Calu-1 was generated as a control to validate antigen-specific functionality of the CAR T-cells. In a CD107a degranulation assay, antigen-specific cytotoxicity was observed when MC9999 CAR T-cells were exposed to Calu-1, but not when MC9999 CAR T-cells were exposed to PD-L1 KO Calu-1 cells (Figure 3A). The release of granzyme B by MC9999 CAR T-cells exclusively in response to PD-L1-positive Calu-1 cells further confirmed the antigen- specific cytotoxicity (Figure 3B). No T-cell cytotoxicity was observed when non-CAR-T cells were exposed to either target cell line. SH-4, representing a metastatic melanoma model, was engineered to generate two cell lines, SH-4 PD-L1 OE (PD-L1 overexpressed) and SH-4 PD-L1 KO (PD-L1 knockout), that were used as target cells to investigate antigen-specific responses of the MC9999 CAR T-cells against melanoma. Antigen-specific T-cell degranulation was evident when MC9999 CAR T-cells were incubated with SH-4 PD-L1 OE, but not when MC9999 CAR T-cells were incubated with SH-4 PD-L1 KO cells (Figure 3C). Non-CAR T-cells from the same donor did not show activity against either target cell line. Further confirmation of antigen-specific cytotoxicity included that MC9999 CAR T-cells exhibited significant release of granzyme B when interacted with SH-4 PD-L1 OE cells.
Attorney Docket No.07039-2280WO1 / 2023-199 Conversely, the absence of granzyme B release was noted when the CAR T-cells were exposed to SH-4 PD-L1 KO target cells (Figure 3D). Another tumor used was LN229, a glioblastoma (GBM) cell line, which was also modified to create antigen-positive LN229 PD-L1 OE and antigen-negative LN229 PD-
L1 KO variants. The in vitro assays consistently demonstrated specific cytotoxicity of MC9999 CAR T-cells against LN229 PD-L1 OE cells, evidenced by T-cell degranulation (Figure 3E) and granzyme B release (Figure 3F), whereas T-cell activities were absent against LN229 PD-L1 KO cells. Collectively, these results suggest antigen-specific cytotoxicity of MC9999 CAR T-cells against PD-L1-positive solid tumors. MC9999 CAR T-cells show antigen-specific cytotoxicity against patient-derived GBM cell lines. The PD-L1 expression was evaluated and confirmed in two patient-derived GBM cell lines, QNS120 and QNS712 (Figure 4A). MC9999 CAR T-cells exhibited antigen specific cytotoxicity against QNS120 and QNS712 as determined in a CD107a degranulation assay (Figure 4B) and a granzyme B assay (Figure 4C). The MC9999 CAR T-cells showed significant efficacy against QNS120 and QNS712, as well as MDA-MB- 231 PD-L1 OE (positive control) but did not show significant efficacy when target cells were absent or when tested against the negative control (MDA-MB-231 PD-L1 KO). Non-CAR T-cells generated from the same donor did not show efficacy against any of the target cells. Significant amounts of granzyme B were detected in the supernatant of cultures of MC9999 CAR T-cells and QNS120 or QNS712. No significant amount of granzyme B was detected when non-CAR T-cells were cocultured with the target cells or cultured without the target cells. MC9999 CAR T-cells show anti-tumor effects against intracranially engrafted GBM tumor. T
he in vivo anti-tumor effects of MC9999 CAR T-cells against the LN229 PD-L1 OE cell line was evaluated in a mouse model (Figure 5A). Mice (n=5 per group) were challenged with an intracranial injection of luciferase-expressing LN229 PD-L1 OE cells (0.5 x 10
6 cells). Treatments were administered by a tail vein IV injection on day 7 and
Attorney Docket No.07039-2280WO1 / 2023-199
day 14 (black arrows). Each injection was either MC9999 CAR T-cells, or non-CAR T cells from the same donor, or PBS (tumor control). Bioluminescence images of the NSG mice showed significant tumor reduction in mice treated with the MC9999 CAR T-cells (Figure 5A). Kaplan-Meier plots showed that both PBS and non-CAR-T cell treated groups did not survive past day 70, whereas MC9999 CAR T-cell treated mice were tumor-free and survived until the study was terminated on day 140 (Figure 5B). MC9999 CAR T-cells show antigen-specific cytotoxicity against PD-L1 expressing cell types that modulate the tumor microenvironment of solid tumors. To test whether the immunosuppressive cells were targeted by the MC9999 CAR T-cells, microglial cell line HMC3 was analyzed for PD-L1 expression (Figure 6A). Antigen specific cytotoxicity of MC9999 CAR-T cells against the HMC3 cells was measured using a degranulation assay (Figure 6B) and a granzyme B release assay (Figure 6C). Significant activity was noted only when MC9999 CAR T-cells and HMC3 were cocultured and not when non-CAR T-cells were cultured with the HMC3 cells. An impedance assay was used to directly monitor the cytotoxicity of MC9999 CAR T-cells against the HMC3 cells. HMC3 cells were grown in culture for 24 hours, and then T cells were added. Upon addition of MC9999 CAR T-cells, a dramatic decrease in cell index (a measure of adherent cell integrity) was observed indicating that the MC9999 CAR T- cells had disrupted the HMC3 cell monolayer, showing direct killing of the HMC3 cells (Figure 6D). M2 macrophages were derived from the CD14+ monocytes found in peripheral blood mononuclear cells (PBMCs) to mimic tumor-associated macrophages (TAMs; immunosuppressive cells within the tumor microenvironment). The M2 macrophage phenotype was identified by gating CD163
+CD209
+ cells (Figure 7A). The surface expression of PD-L1 appears on M2 macrophages as they differentiate from CD14+ monocytes (Figure 7B) making them a target for MC9999 CAR T-cells (Figure 7C). The CD14
+ monocytes lacked PD-L1 expression, and thus served as a negative control and evaded the cytotoxic effects of MC9999 CAR T-cells. PD-L1 targeted cytotoxicity was further confirmed by higher levels of granzyme B released into the supernatant when the
Attorney Docket No.07039-2280WO1 / 2023-199 M2 macrophages were incubated with MC9999 CAR T-cells as compared to the non- CAR T cell control (Figure 7D). Additionally, as observed with the HMC3 cells, MC9999 CAR T-cells disrupted the M2 macrophage monolayer, showing direct killing of the M2 macrophages (Figure 7E). The cytotoxicity of MC9999 CAR T-cells against primary TAMs isolated from a GBM tumor was also examined. Phenotypic characterization demonstrated a CD163
+CD209
+ population of cells that matched the phenotype of a TAMs (Figure 7F). The TAM population exhibited surface expression of PD-L1 (Figure 7G). The degranulation assay showed MC9999 CAR-T cell mediated antigen specific cytotoxicity against the TAMs (Figure 7H). Patient-derived MC9999 CAR T-cells target PD-L1-expressing tumor and TME cells To examine the effects of patient derived CAR T-cells against the PD-L1 positive tumors, three batches of patient-derived MC9999 CAR T-cells were generated from peripheral blood collected from three patients with GBM. Generation of CAR T-cells from the patient PBMCs was confirmed by characterization of CD3
+ T cell population (Figure 8A, top panels). Potency of these CAR T cells was determined by EGFR detection (Figure 8A, bottom panels). The antigen specific cytotoxicity of these three batches of GBM patient-derived MC9999 CAR T-cells was evaluated against two GBM patient-derived tumor cell lines (QNS120 and QNS712) as well as HMC3 (Figure 8B). Non-CAR T-cells were included as a negative control. Similar degranulation was observed between patient 1 and patient 2 as well as between the three target cell lines that were used. Patient 3 showed similar degranulation as the other three target cell lines, but less activity compared to patients 1 and 2. Following the incubation of MC9999 CAR T-cells with the target cells,
supernatants were analyzed for cytokine release. Granzyme A, granzyme B, IFN , and perforin were detected in all cases where MC9999 CAR T-cells were incubated with the target cells (Figure 8C and 8D). For all subjects, the incubation of MC9999 CAR T-cells with QNS712 resulted in a robust release of all four cytokines. The release of cytokines
Attorney Docket No.07039-2280WO1 / 2023-199 from MC9999 CAR T-cells from each patient was similar to the release of cytokines observed from QNS120 and HMC cell lines. Example 2: Exemplary Polypeptide Sequences human PD-L1 polypeptide (SEQ ID NO:1) MRIFAVFIFM TYWHLLNAFT VTVPKDLYVV EYGSNMTIEC KFPVEKQLDL AALIVYWEME DKNIIQFVHG EEDLKVQHSS YRQRARLLKD QLSLGNAALQ ITDVKLQDAG VYRCMISYGG ADYKRITVKV NAPYNKINQR ILVVDPVTSE HELTCQAEGY PKAEVIWTSS DHQVLSGKTT TTNSKREEKL FNVTSTLRIN TTTNEIFYCT FRRLDPEENH TAELVIPGNI LNVSIKICLT LSPST human PD-1 polypeptide (SEQ ID NO:2) MQIPQAPWPV VWAVLQLGWR PGWFLDSPDR PWNPPTFSPA LLVVTEGDNA TFTCSFSNTS ESFVLNWYRM SPSNQTDKLA AFPEDRSQPG QDCRFRVTQL PNGRDFHMSV VRARRNDSGT YLCGAISLAP KAQIKESLRA ELRVTERRAE VPTAHPSPSP RPAGQFQTLV VGVVGGLLGS LVLLVWVLAV ICSRAARGTI GARRTGQPLK EDPSAVPVFS VDYGELDFQW REKTPEPPVP CVPEQTEYAT IVFPSGMGTS SPARRGSADG PRSAQPLRPE DGHCSWPL Example 3: Exemplary Variable Domains That Can Bind a PD-L1 Polypeptide This Example provides the amino acid sequences of the heavy chain variable domain and the light chain variable domain of an scFv that can bind a PD-L1 polypeptide. The CDRs and framework sequences of each also are provided and delineated. Heavy chain variable domain (SEQ ID NO:3) QVQLVQSGAEVKKPGSSVKVSCKASGYTFSNYWIEWVRQAPGQGLEWMGEILPGGGNPN YNEKFKGRVTFTADTSTNTAYMDLSSLRSDDTAVYYCARERAVDSWGQGTLVTVSS
Attorney Docket No.07039-2280WO1 / 2023-199 Framework Region 1 of heavy chain variable domain: QVQLVQSGAEVKKPGSSVKVSCKAS (SEQ ID NO:11) CDR1 of heavy chain variable domain: GYTFSNYW (SEQ ID NO:4) Framework Region 2 of heavy chain variable domain: IEWVRQAPGQGLEWMGE (SEQ ID NO:12) CDR2 of heavy chain variable domain: ILPGGGNP (SEQ ID NO:5) Framework Region 3 of heavy chain variable domain: YNEKFKGRVTFTADTSTNTAYMDLSSLRSDDTAVYYC (SEQ ID NO:13) CDR3 of heavy chain variable domain: ARERAVDS (SEQ ID NO:6) Framework Region 4 of heavy chain variable domain: WGQGTLVTVSS (SEQ ID NO:14) Nucleic acid sequence (SEQ ID NO:20) encoding SEQ ID NO:3: CAGGTTCAGCTGGTACAGTCTGGAGCTGAAGTAAAGAAGCCTGGGAGTTCAG TGAAGGTATCCTGCAAGGCTAGTGGCTACACATTCAGTAACTACTGGATAGA GTGGGTAAGACAGGCACCTGGACAAGGCCTTGAGTGGATGGGAGAGATTTTA CCTGGAGGTGGTAATCCTAACTACAATGAGAAGTTCAAGGGCAGAGTTACAT TCACTGCAGATACATCCACAAACACAGCCTACATGGATCTCAGCAGCCTGAG ATCTGATGACACTGCCGTCTATTACTGTGCAAGGGAGAGGGCTGTGGACTCC TGGGGTCAAGGAACCCTAGTCACCGTCTCCTCA
Attorney Docket No.07039-2280WO1 / 2023-199 Light chain variable domain (SEQ ID NO:7) DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKSPKLLIYYTSSLRSGVP SRFSGSGSGTDFSLTISSLQPEDFATYYCQQYSKLPWTFGGGTKVEIK Framework Region 1 of light chain variable domain: DIQMTQSPSSLSASVGDRVTITCSAS (SEQ ID NO:15) CDR1 of light chain variable domain: QDISNY (SEQ ID NO:8) Framework Region 2 of light chain variable domain: LNWYQQKPGKSPKLLIY (SEQ ID NO:16) CDR2 of light chain variable domain:
YTS (SEQ ID NO:9) Framework Region 3 of light chain variable domain: SLRSGVPSRFSGSGSGTDFSLTISSLQPEDFATYYC (SEQ ID NO:17) CDR3 of light chain variable domain: QQYSKLPWT (SEQ ID NO:10) Framework Region 4 of light chain variable domain:
FGGGTKVEIK (SEQ ID NO:18) Nucleic acid sequence (SEQ ID NO:21) encoding SEQ ID NO:7: GATATCCAGATGACACAGAGTCCATCCTCCCTGTCTGCCTCTGTTGGAGACAG AGTCACCATCACTTGCAGTGCAAGTCAGGACATTAGCAATTATTTAAACTGGT
Attorney Docket No.07039-2280WO1 / 2023-199 ATCAGCAAAAACCAGGTAAAAGTCCTAAACTCCTGATCTATTACACATCCAG TTTACGCTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGGACAGAT TTTTCTCTCACCATCAGCAGCCTGCAACCTGAAGATTTTGCCACTTACTATTGT CAGCAGTATAGTAAGCTTCCGTGGACGTTCGGTGGAGGCACCAAGGTAGAAA TCAAA Example 4: Exemplary Transmembrane Domains This Example provides the amino acid sequences of exemplary transmembrane domains that can be used to design a CAR. Amino acid sequence of an exemplary CD4 TM (SEQ ID NO:22) MALIVLGGVAGLLLFIGLGIFF Nucleic acid sequence (SEQ ID NO:23) encoding SEQ ID NO:22: ATGGCCCTGATCGTGCTGGGCGGAGTGGCCGGACTGCTGCTGTTTATCGGCCTGGGCAT CTTCTTC (SEQ ID NO:23) Example 5: Exemplary Signaling Domains This Example provides the amino acid sequences of exemplary signaling domains that can be used to design a CAR. Amino acid sequence of an exemplary 4-1BB costimulatory domain (SEQ ID NO:24) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGGG Nucleic acid sequence (SEQ ID NO:25) encoding SEQ ID NO:24: AAGCGGGGCAGAAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGCGGCCCGTGCA GACCACCCAGGAAGAGGACGGCTGCAGCTGCCGGTTCCCCGAGGAAGAGGAAGGCGGCT GCGAGCTGGGAGGCGGC (SEQ ID NO:25)
Attorney Docket No.07039-2280WO1 / 2023-199 Example 6: Exemplary Hinge and/or Linker Sequences This Example provides exemplary linker amino acid sequences that can be used to link a heavy chain variable domain and a light chain variable domain together to form a scFv. These amino acid sequences are also exemplary hinges that can be used to design a CAR. Amino acid sequence of an exemplary IgG4 hinge (SEQ ID NO:26) ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNW YVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:26) Nucleic acid sequence (SEQ ID NO:27) encoding SEQ ID NO:26: GAGTCTAAGTACGGCCCTCCCTGCCCCCCCTGCCCAGCCCCTGAATTTGAGGGCGGACC CAGCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCG AGGTAACCTGCGTGGTGGTGGACGTGTCCCAGGAAGATCCCGAGGTCCAGTTCAATTGG TACGTGGACGGCGTGGAAGTGCACAATGCCAAGACCAAGCCCAGAGAGGAACAGTTCCA AAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCA AAGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCCAGCAGCATCGAGAAAACCATC AGCAAGGCCAAGGGCCAGCCCCGCGAGCCCCAGGTGTACACACTGCCCCCCAGCCAGGA AGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTCAAGGGCTTCTACCCCAGCG ATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCC CCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACTCCCGGCTGACCGTGGACAAGAG CCGGTGGCAGGAAGGCAACGTCTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACC ACTACACCCAGAAGTCTCTGAGCCTGAGCCTGGGCAAG (SEQ ID NO:27) Example 7: Exemplary Marker Sequences This Example provides the amino acid sequences of exemplary detectable markers that can be used to design a CAR.
Attorney Docket No.07039-2280WO1 / 2023-199 Amino acid sequence of an exemplary tEGFR (SEQ ID NO:28) MLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLH ILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGR TKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKT KIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPR EFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTL VWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIG LFM (SEQ ID NO:28) Nucleic acid sequence (SEQ ID NO:29) encoding SEQ ID NO:28: ATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCTCCT GATCCCACGCAAAGTGTGTAACGGAATAGGTATTGGTGAATTTAAAGACTCACTCTCCA TAAATGCTACGAATATTAAACACTTCAAAAACTGCACCTCCATCAGTGGCGATCTCCAC ATCCTGCCGGTGGCATTTAGGGGTGACTCCTTCACACATACTCCTCCTCTGGATCCACA GGAACTGGATATTCTGAAAACCGTAAAGGAAATCACAGGGTTTTTGCTGATTCAGGCTT GGCCTGAAAACAGGACGGACCTCCATGCCTTTGAGAACCTAGAAATCATACGCGGCAGG ACCAAGCAACATGGTCAGTTTTCTCTTGCAGTCGTCAGCCTGAACATAACATCCTTGGG ATTACGCTCCCTCAAGGAGATAAGTGATGGAGATGTGATAATTTCAGGAAACAAAAATT TGTGCTATGCAAATACAATAAACTGGAAAAAACTGTTTGGGACCTCCGGTCAGAAAACC AAAATTATAAGCAACAGAGGTGAAAACAGCTGCAAGGCCACAGGCCAGGTCTGCCATGC CTTGTGCTCCCCCGAGGGCTGCTGGGGCCCGGAGCCCAGGGACTGCGTCTCTTGCCGGA ATGTCAGCCGAGGCAGGGAATGCGTGGACAAGTGCAACCTTCTGGAGGGTGAGCCAAGG GAGTTTGTGGAGAACTCTGAGTGCATACAGTGCCACCCAGAGTGCCTGCCTCAGGCCAT GAACATCACCTGCACAGGACGGGGACCAGACAACTGTATCCAGTGTGCCCACTACATTG ACGGCCCCCACTGCGTCAAGACCTGCCCGGCAGGAGTCATGGGAGAAAACAACACCCTG GTCTGGAAGTACGCAGACGCCGGCCATGTGTGCCACCTGTGCCATCCAAACTGCACCTA CGGATGCACTGGGCCAGGTCTTGAAGGCTGTCCAACGAATGGGCCTAAGATCCCGTCCA TCGCCACTGGGATGGTGGGGGCCCTCCTCTTGCTGCTGGTGGTGGCCCTGGGGATCGGC CTCTTCATG (SEQ ID NO:29) Example 8: Exemplary CAR That Can Target a PD-L1 Polypeptide This Example provides an amino acid sequence of a CAR (MC9999) designed to include a scFv created using the CDRs of the SEQ ID NOs:4-6 and 8-10. The various components of this CAR (e.g., domains and linkers) are provided and delineated.
Attorney Docket No.07039-2280WO1 / 2023-199 Exemplary CAR structure:
signal peptide + scFv including the VH and VL of Example 3 + hinge/linker + CD4 Transmembrane domain + 4-1BB Intracellular Signaling Domain + CD3 Intracellular Signaling Domain + T2A + tEGFR Amino acid sequence of a MC9999 CAR (SEQ ID NO:30) MLLLVTSLLLCELPHPAFLLIPQVQLVQSGAEVKKPGSSVKVSCKASGYTFSNYWIEWV RQAPGQGLEWMGEILPGGGNPNYNEKFKGRVTFTADTSTNTAYMDLSSLRSDDTAVYYC ARERAVDSWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSA SQDISNYLNWYQQKPGKSPKLLIYYTSSLRSGVPSRFSGSGSGTDFSLTISSLQPEDFA TYYCQQYSKLPWTFGGGTKVEIKESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDW LNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEA LHNHYTQKSLSLSLGKMALIVLGGVAGLLLFIGLGIFFKRGRKKLLYIFKQPFMRPVQT TQEEDGCSCRFPEEEEGGCELGGGRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVL DKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLS TATKDTYDALHMQALPPRLEGGGEGRGSLLTCGDVEENPGPRMLLLVTSLLLCELPHPA FLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPL DPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNIT SLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQV CHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLP QAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPN CTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM Nucleic acid sequence (SEQ ID NO:31) encoding SEQ ID NO:30: ATGCTGCTGCTCGTGACATCTCTGCTGCTGTGCGAGCTGCCCCACCCCGCCTTTCTGCT GATTCCTCAGGTTCAGCTGGTACAGTCTGGAGCTGAAGTAAAGAAGCCTGGGAGTTCAG TGAAGGTATCCTGCAAGGCTAGTGGCTACACATTCAGTAACTACTGGATAGAGTGGGTA AGACAGGCACCTGGACAAGGCCTTGAGTGGATGGGAGAGATTTTACCTGGAGGTGGTAA TCCTAACTACAATGAGAAGTTCAAGGGCAGAGTTACATTCACTGCAGATACATCCACAA ACACAGCCTACATGGATCTCAGCAGCCTGAGATCTGATGACACTGCCGTCTATTACTGT GCAAGGGAGAGGGCTGTGGACTCCTGGGGTCAAGGAACCCTAGTCACCGTCTCCTCAGG TGGAGGCGGTTCAGGTGGCGGCGGTTCGGGCGGTGGCGGCTCTGATATCCAGATGACAC AGAGTCCATCCTCCCTGTCTGCCTCTGTTGGAGACAGAGTCACCATCACTTGCAGTGCA AGTCAGGACATTAGCAATTATTTAAACTGGTATCAGCAAAAACCAGGTAAAAGTCCTAA ACTCCTGATCTATTACACATCCAGTTTACGCTCAGGAGTCCCATCAAGGTTCAGTGGCA GTGGGTCTGGGACAGATTTTTCTCTCACCATCAGCAGCCTGCAACCTGAAGATTTTGCC
Attorney Docket No.07039-2280WO1 / 2023-199 ACTTACTATTGTCAGCAGTATAGTAAGCTTCCGTGGACGTTCGGTGGAGGCACCAAGGT AGAAATCAAAGAGTCTAAGTACGGCCCTCCCTGCCCCCCCTGCCCAGCCCCTGAATTTG AGGGCGGACCCAGCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGC CGGACCCCCGAGGTAACCTGCGTGGTGGTGGACGTGTCCCAGGAAGATCCCGAGGTCCA GTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAATGCCAAGACCAAGCCCAGAGAGG AACAGTTCCAAAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGG CTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCCAGCAGCATCGA GAAAACCATCAGCAAGGCCAAGGGCCAGCCCCGCGAGCCCCAGGTGTACACACTGCCCC CCAGCCAGGAAGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTCAAGGGCTTC TACCCCAGCGATATCGCCGTGGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACAA GACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACTCCCGGCTGACCG TGGACAAGAGCCGGTGGCAGGAAGGCAACGTCTTCAGCTGCAGCGTGATGCACGAGGCC CTGCACAACCACTACACCCAGAAGTCTCTGAGCCTGAGCCTGGGCAAGATGGCCCTGAT CGTGCTGGGCGGAGTGGCCGGACTGCTGCTGTTTATCGGCCTGGGCATCTTCTTCAAGC GGGGCAGAAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGCGGCCCGTGCAGACC ACCCAGGAAGAGGACGGCTGCAGCTGCCGGTTCCCCGAGGAAGAGGAAGGCGGCTGCGA GCTGGGAGGCGGCAGAGTGAAGTTCAGCCGGTCCGCCGACGCCCCTGCCTACCAGCAGG GCCAGAACCAGCTGTACAACGAGCTGAACCTGGGCAGGCGGGAGGAATACGACGTGCTG GACAAGCGGAGAGGCCGGGACCCTGAGATGGGCGGCAAGCCCAGGCGGAAGAACCCTCA GGAAGGCCTGTATAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCG GCATGAAGGGCGAGCGGCGGAGGGGCAAGGGCCACGACGGCCTGTACCAGGGCCTGAGC ACCGCCACCAAGGATACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCAAGGCTCGA GGGCGGCGGAGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCG GCCCTAGGATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCA TTCCTCCTGATCCCACGCAAAGTGTGTAACGGAATAGGTATTGGTGAATTTAAAGACTC ACTCTCCATAAATGCTACGAATATTAAACACTTCAAAAACTGCACCTCCATCAGTGGCG ATCTCCACATCCTGCCGGTGGCATTTAGGGGTGACTCCTTCACACATACTCCTCCTCTG GATCCACAGGAACTGGATATTCTGAAAACCGTAAAGGAAATCACAGGGTTTTTGCTGAT TCAGGCTTGGCCTGAAAACAGGACGGACCTCCATGCCTTTGAGAACCTAGAAATCATAC GCGGCAGGACCAAGCAACATGGTCAGTTTTCTCTTGCAGTCGTCAGCCTGAACATAACA TCCTTGGGATTACGCTCCCTCAAGGAGATAAGTGATGGAGATGTGATAATTTCAGGAAA CAAAAATTTGTGCTATGCAAATACAATAAACTGGAAAAAACTGTTTGGGACCTCCGGTC AGAAAACCAAAATTATAAGCAACAGAGGTGAAAACAGCTGCAAGGCCACAGGCCAGGTC TGCCATGCCTTGTGCTCCCCCGAGGGCTGCTGGGGCCCGGAGCCCAGGGACTGCGTCTC TTGCCGGAATGTCAGCCGAGGCAGGGAATGCGTGGACAAGTGCAACCTTCTGGAGGGTG AGCCAAGGGAGTTTGTGGAGAACTCTGAGTGCATACAGTGCCACCCAGAGTGCCTGCCT CAGGCCATGAACATCACCTGCACAGGACGGGGACCAGACAACTGTATCCAGTGTGCCCA CTACATTGACGGCCCCCACTGCGTCAAGACCTGCCCGGCAGGAGTCATGGGAGAAAACA ACACCCTGGTCTGGAAGTACGCAGACGCCGGCCATGTGTGCCACCTGTGCCATCCAAAC TGCACCTACGGATGCACTGGGCCAGGTCTTGAAGGCTGTCCAACGAATGGGCCTAAGAT
Attorney Docket No.07039-2280WO1 / 2023-199 CCCGTCCATCGCCACTGGGATGGTGGGGGCCCTCCTCTTGCTGCTGGTGGTGGCCCTGG GGATCGGCCTCTTCATGTGA Example 9: Single CAR-Dual target: Intracranial Delivery of Anti-PD-L1 CAR T Cells Effectively Eradicates Glioma and Immunosuppressive Cells in the Tumor Microenvironment This Example describes the use of MC9999 CAR T cells to treat PD-L1-positive GBM. RESULTS: Anti-PD-L1 (MC9999 CAR-T) cells display high antigen specificity on PD-L1 expressing GBM tumor in vitro and in vivo. To validate MC9999 CAR T-cells specific cytotoxicity towards PD-L1- expressing targets in a GBM model, the human glioma cell line LN229 was utilized. This cell line was genetically modified to either overexpress PD-L1 (LN229 PD-L1 OE, Fig. 16A) or knock out PD-L1 (LN229 PD-L1 KO) via CRISPR. Healthy donor-derived MC9999 CAR T cells or non-CAR T cells were co- cultured with these target cells. CD107a expression, a marker of immune cell activation and cytotoxic degranulation, was significantly increased (from 1.43% in non-CAR T to 28.1% in MC9999 CAR T cells) in response to LN229 PD-L1 OE, but not in PD-L1 KO or control groups (Fig.10A). Further confirmation of T cell cytotoxicity was achieved by measuring granzyme B release, which was significantly higher in the MC9999 CAR T treated with LN229 PD-L1 OE cells (N=3, P<0.0001) (Fig.10B). Additionally, an impedance-based cytotoxicity assay demonstrated direct cytotoxic effects of MC9999 CAR T cells on target cell populations, with a sharp reduction in the cell index (inversely related to impedance) observed exclusively in the LN229 PD-L1 OE cells upon addition of MC9999 CAR T cells (Fig.10C). N
ext, the efficacy of MC9999 CAR T cells was evaluated in vivo using an orthotopic GBM xenograft model. LN229 PD-L1 OE cells were further transduced with a luciferase reporter for tumor tracking. Two weeks after tumor implantation and
Attorney Docket No.07039-2280WO1 / 2023-199 confirmation of tumor burden in all animals, a single intratumoral injection of MC9999 CAR T cells, non-CAR T cells from the same donor, or PBS (as a control) was administered. Tumor eradication was observed within two weeks in the MC9999 CAR T treated group, with no tumor recurrence detected during a 150-day follow-up period. In
contrast, tumor growth continued in both control groups (N=16 per group, P<0.0001) (Fig.10D, 10E) resulting in significantly extended survival for MC9999 CAR T- treated mice (Fig.10F). At the experimental endpoint, the brains of the treated animals were analyzed to assess the persistence of tumor cells. Multi-immunohistochemical (m-IHC) analysis revealed a significant reduction in human PD-L1-positive tumor cells in the MC9999
CAR T treated group compared to both control groups (N=5 per group, P<0.05) (Fig. 10G, 10H; Fig.17). Samples were also stained for human Nestin, a tumor marker that could indicate antigen (PD-L1) loss. No residual tumor cells were detected in the MC9999 CAR T-treated animals, suggesting complete tumor eradication without antigen escape. These findings provide preclinical evidence that MC9999 CAR T cells effectively and specifically target PD-L1-expressing GBM. Anti-PD-L1 (MC9999 CAR-T) cells exhibit high anti-tumor efficacy in GBM patient derived tumor cells in vitro and in vivo. After confirming antigen specificity, the anti-tumor efficacy of MC9999 CAR T cells was evaluated in patient-derived primary GBM models. Brain tumor-initiating cells (BTICs) were isolated from a GBM (QNS108) patient at the time of surgical resection and were cultured (Fig.11A; Table 2). The QNS108 BTIC line was transduced to stably overexpress PD-L1 (~98.9%, Fig.16A), luciferase, and GFP (QNS108-PD-L1-GFP-Luc
(Fig. 11B). This engineered cell line was used as a GBM for in vitro and in vivo studies. To assess MC9999 CAR T cells activation by QNS108 GBM cells a CD107a degranulation assay was performed and it showed 22.7% CD107a- positive cells in CAR T group compared to 1.69% in non-CAR T group (Fig.11C). This activation was further confirmed by a significant increase in granzyme B release from MC9999 CAR T cells upon incubation with QNS108 GBM cells (N=3, P<0.0001) (Fig.11D).
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ollowing this in vitro validation, the anti-tumor efficacy was tested in vivo using an orthotopic GBM xenograft model with patient-derived QNS108-PD-L1-GFP-Luc cells implanted in NSG mice brains. Fourteen days post-tumor implantation and confirmation of tumor burden, the animals were treated with a single intracranial injection of MC9999 CAR T cells, non-CAR T cells from the same donor (allogeneic control), or PBS as a vehicle control (N=8 per group). Remarkably, tumors were completely eradicated within two weeks after infusion in the MC9999 CAR T-treated group, with no recurrence detected in the 135-day observation period. In contrast, tumor size continued to grow significantly in both control groups (non-CAR T and PBS) (N=8 per group, P<0.0001) until reaching the humane endpoint (Fig.11E, 11F). significantly prolonged survival was observed in the CAR T-treated group (N=8 per group, P<0.0001) (Fig.11G). Mice brains were harvested at experimental endpoint, antigen persistence was assessed by measuring human PD-L1 expression in all groups. Tumor cells in the MC9999 CAR T-treated group were significantly reduced compared to both control groups (N=5 per group, P CAR T vs. PBS <0.01, P CAR T vs. non-CAR T <0.05) (Fig. 11H, I; Fig.17). Additionally, the proliferation (Fig.11J, N=5 per group, P CAR T vs. PBS <0.05, P CAR T vs. non-CAR T <0.05) and stemness (Fig.11K, N=5 per group, P CAR T vs. PBS <0.01, P CAR T vs. non-CAR T <0.05) of tumor cells were significantly reduced in the MC9999 CAR T-treated group. These findings emphasize the potential of MC9999 CAR T cells as a promising therapeutic option for patient-derived GBM models. scRNA-seq reveals that MC9999 CD4+ and CD8+ CAR T cells elicit an IFN mediated apoptosis in patient-derived GBM cells in vivo. To gain insights into the molecular pathways driving the robust response of MC9999 CAR T cells against GBM, scRNA-seq was performed on an orthotopic xenograft model using LN229 PD-L1-OE cells. Fourteen days post-implantation, after confirming tumor burden, a single intratumoral infusion of MC9999 CAR T cells or non- CAR T cells (control) was administered (N=4 mice per group). To capture both tumor
Attorney Docket No.07039-2280WO1 / 2023-199 and T cell populations, the animals were sacrificed 24 hours post-infusion, the tumor regions were harvested, and scRNA-seq and analysis was conducted (Fig.12A). The clustering analysis identified twelve distinct populations, with the majority (11/12 clusters) representing mouse brain cells, while one smaller cluster comprised human GBM and T cells (Fig.12B). This cluster was then separated into two subpopulations using markers specific to human immune cells (CD6, CD53, CD38) and GBM cells (MIA, S100B, MDK) (Fig.12C). Notably, the number of GBM cells was reduced in the MC9999 CAR T-treated group (Fig.18A) after only 24 hours, while the T cell population remained stable across both MC9999 CAR T and non-CAR T-treated groups (Fig.18B). In total, 10 GBM cells from the MC9999 CAR T group, 52 GBM cells from the non-CAR T group, 34 MC9999 CAR T cells, and 49 non-CAR T cells were analyzed (Fig.12D). Differential gene expression analysis revealed numerous genes upregulated and downregulated in MC9999 CAR T cells compared to non-CAR T cells (Fig.12E). Pathway analysis showed that the upregulated genes were predominantly associated with
cytokine and interferon / signaling pathways (Fig. 12F). A deeper dive into T cell subpopulations revealed that CD4+ T cells were the primary activators of these pathways (Fig.18C, 18E), while CD8+ T cells exhibited activation of pathways related to interleukin signaling and transmembrane transport, reflecting their role in degranulation and cytotoxic activity (Fig.18D, 18G). Conversely, downregulated pathways (those upregulated in non-CAR T cells or downregulated in CAR T cells) in CD4+ T cells were related to migration and anaerobic metabolism, processes typically associated with T cell exhaustion (Fig.18F). Interestingly, in GBM cells treated with MC9999 CAR T cells, DEG analysis revealed activation of interferon and TNF signaling pathways, particularly
type II interferon ( / from CD4+ T cells and from CD8+ T cells) and antigen processing for presentation (Fig.12G, 12H). These results suggest that the GBM cells responded to the T cell attack by activating genes involved in immune signaling, which likely led to their demise.
Attorney Docket No.07039-2280WO1 / 2023-199 Single cell-RNA seq data validation in datasets from glioma patients. To validate these findings in human glioma, data from the CGGA database were utilized to correlate the top two upregulated genes in GBM following MC9999 CAR T treatment with genes exclusively expressed by CD4+ Th1 and CD8+ T cells, both of which are known for their tumor-killing capabilities. First, the expression of (Cathepsin S) CTSS [AQ2] in GBM cells was correlated with the expression of active CD4+ Th1 markers, including IL12R (R=0.69, Fig.12I), IFNGR (R=0.79, Fig.12J), TNF (R=0.46, Fig.12K), and IL15 (R=0.51, Fig.12L). Next, the expression of (Phospholipase A and Acyltransferase 4) PLAAT4 was correlated with CD8+ T cell activation genes, including GZMB (R=0.41, Fig.12M), GZMA (R=0.46, Fig.12N), PRF1 (R=0.46, Fig.12O), and LAMP1 (R=0.35, Fig.12P). Additional correlations Were also performed between active CD4+ Th1 genes and PLAAT4 (Fig.19A-19D), showing strong correlation, while CD8+ activation genes did not correlate with CTSS (Fig.19E-19H). These correlations suggest a similar mechanism of response in human glioma cells when in contact with active T cells. Both CTSS and PLAAT4 demonstrated higher expression in HGG compared to lower-grade gliomas (Fig.19I, 19J), further underscoring their relevance in the GBM response to MC9999 CAR T cell therapy. These results highlight the activation of immune-related pathways in GBM cells under attack by MC9999 CAR T cells, which is critical for their eradication. GBM patient derived Anti-PD-L1 (MC9999 CAR-T) cells exhibit robust cytotoxicity when incubated with their autologous tumor cells. Given that local and systemic T cells are typically dysfunctional in glioma, and the fact that CAR T cell therapies for intracranial tumors require autologous T cells, the cytotoxic efficacy of MC9999 CAR T cells generated from GBM patient-derived T cells against their autologous BTICs were assessed. For this, both BTICs and T cells were isolated from two GBM patients (QNS985 and QNS986) (Fig.13A, Table 2). CAR T cells and non-CAR T cells were manufactured from their peripheral blood, with both products passing quality control analysis (Table 1). Following the same protocol,
Attorney Docket No.07039-2280WO1 / 2023-199 autologous BTICs were isolated as target cells for the function assessment of patient- derived MC9999 CAR T cells. IHC and flow cytometry staining confirmed positive PD- L1 expression in these BTICs (Fig.13A and Figure 10B). Table 1. QC analysis for patient derived CAR T cells.
Attorney Docket No.07039-2280WO1 / 2023-199 The degranulation assay showed a significant increase in CD107a membrane expression on MC9999 CAR T cells in response to autologous BTICs compared to non- CAR T cells, with a 30.9% CD107 increased in QNS985 and a 52.46% increase in QNS986 (Fig.13B). This activation was further validated by elevated Granzyme B release in the MC9999 CAR T-treated cells in both patient samples (Fig.13C) (N=3, P<0.001). Additionally, patient QNS985 showed significant increases in IFN- (Fig. 13D, P<0.0001) and perforin A (Fig.13E, P<0.001) in cytokine release analysis. To further evaluate the direct cytotoxic effects of patient-derived CAR T cells on autologous BTICs, time-lapse acquisition and analysis were conducted. BTICs from patient QNS986 were co-incubated with MC9999 CAR T or non-CAR T cells for 72 hours, capturing real-time imaging of cell proliferation and death (Fig.13F). Apoptotic bodies were observed in GBM cells treated with MC9999 CAR T cells as early as six hours after treatment, while tumor cells in the non-CAR T group continued to grow without any detected apoptotic bodies (Fig.13F, Fig.20). These findings demonstrate that even T cells from GBM patients, which may have impaired functionality as patients have cancer and are on high steroid dosages that are known to impair the immune system, can be engineered into potent MC9999 CAR T cells. More importantly, these CAR T cells exhibit strong cytotoxic activity against autologous tumor cells, highlighting their potential for effective personalized glioma therapy. GBM patient derived Anti-PD-L1 (MC9999 CAR-T) cells effectively target autologous immune suppressive populations in the tumor microenvironment. After demonstrating the direct anti-tumor efficacy of MC9999 CAR-T derived from both healthy donors and GBM patients targeting GBM tumor cell lines and autologous primary tumor BTICs, further studies were conducted to evaluate their efficacy on immunosuppressive cells within the TME. Specifically, autologous M2 phenotype macrophages derived from GBM patients QNS924, QNS1065 and QNS1070 (Table 2), as well as TAMs from the tumor tissues of patients QNS1065 and QNS1070,
Attorney Docket No.07039-2280WO1 / 2023-199 were utilized to investigate the efficacy of autologous patient derived MC9999 CAR T cells on immunosuppressive cells within the TME. CAR-T products from GBM patients QNS924, QNS1065 and QNS1070 achieved the quality control standards (Table 1). Immunophenotyping confirmed that the target macrophages were CD163 and CD209 double-positive with PD-L1 expression (Fig.21). Cytotoxic degranulation assay demonstrated significant increases in CD107a expression in MC9999 CAR-T cells against M2 macrophages - 26.11% in QNS924 and 31.52% in QNS1065 compared to non-CAR T groups (Fig.14A). Additionally, MC9999 CAR-T cells targeting M2, and TAMs induced substantial cytokine release, including Granzyme A, Granzyme B, and IFN- (Fig. 14B-14D, P<0.001). These findings demonstrate that MC9999 CAR T cells derived from patients with brain cancer can effectively target not only GBM cells but also the immunosuppressive components of the TME, enhancing their therapeutic potential. PD-L1 expression on high-grade glioma patient tissue and cytotoxic effects of astrocytoma patient-derived PD-L1 CAR T cells To assess the safety of anti-PD-L1 CAR T cells for intracranial delivery in humans, co-staining for neurons, glia (astrocytes and oligodendrocytes), tumor cells, blood vessels, and microglia markers with PD-L1 was performed (Fig.15A, Table 2)). The results showed that PD-L1 expression was restricted to Nestin/GFAP-positive tumor cells and Iba1-positive macrophages/microglia within the tumor, with no expression detected in neurons, astrocytes, oligodendrocytes, or blood vessels. This indicates that anti-PD-L1 CAR T therapy could be safely delivered to the brain without affecting normal cell populations. The cohort comprised 13 patient samples, including 4 matched primary and recurrent GBM cases and 5 grade 4 astrocytomas (AS). PD-L1 expression was notably high across all glioma samples (Fig.15B), with 14.45% in primary GBM, 21.42% in recurrent GBM, and 63.02% in grade 4 AS. Most of the PD-L1-positive cells co-localized with Nestin (Fig.15C): 7.25% in primary GBM, 10.29% in recurrent GBM, and 37.59% in grade 4 AS, while the remainder co-expressed Iba1 (Fig.15D): 7.2% in primary GBM,
Attorney Docket No.07039-2280WO1 / 2023-199 11.13% in recurrent GBM, and 25.42% in grade 4 AS. Further analysis of CGGA data confirmed significantly higher PD-L1 mRNA expression in GBM and grade 4 AS (Fig. 15E) (P for grade 4 AS vs. primary GBM <0.0001, P for grade 4 AS vs. recurrent GBM <0.001), supporting PD-L1 as a safe and viable therapeutic target in glioma patients. To evaluate long-term safety, mice tissue bearing QNS108 patient-derived tumors were analyzed at the experimental endpoint. CD4+ T cells were observed in the meninges of CAR T-treated mice even 5 months after treatment, without any histologically detectable damage to the brain (Fig.22A). Staining revealed glial scar formation with abundant astrocytes and invariable number of macrophages in the treated hemisphere vs the contralateral hemisphere (Fig.22B-22D), indicating effective tumor resolution with normal brain repair. Single-cell data from early stages further supported the tumor eradication (Fig.22E). In MC9999 CAR T-treated animals, early myelinating oligodendrocytes and oligodendrocyte precursor cells (OPCs) were among the most abundant populations, activating pathways (i.e., regulation of synapse organization, regulation of transmembrane protein, monocarboxylic acid biosynthesis process, etc.) related to regeneration (Fig.22E-22F). These results demonstrate that MC9999 CAR T therapy is not only safe for intracranial use in our preclinical model but also supports normal brain healing following tumor eradication. These results demonstrate that MC9999 CAR T therapy is not only safe for intracranial use in our preclinical model but also supports normal brain healing following tumor eradication. To further test the efficacy of our CAR T product in different glioma types, MC9999 CAR T cells were generated from a grade 3 AS patient (QNS871, Supplementary Table 2). High PD-L1 expression of autologous M2 (Fig.21) served as target immunosuppressive cell models. Given the unavailability of autologous tumor cells, LN229 PD-L1 OE and KO cells were used as target tumor cells. CAR T treatment increased CD107a expression by 38.44% against LN229 PD-L1 OE and a 23.27% against autologous M2 macrophages (Fig.15F), this finding was supported by an increase in granzyme B release (Fig.15G-15H).
Attorney Docket No.07039-2280WO1 / 2023-199 METHODS: T cell isolation. For healthy volunteer donors’ peripheral blood mononuclear cells (PBMCs) were collected via leukapheresis using leukocyte reduction system (LRS) cones. To isolate naïve and memory T cell (Tn/mem) from PBMCs a three-step procedure, involving negative selection of both CD14 and CD25, followed by positive selection of CD62L, using CD14, CD25, and CD62L microbeads (Miltenyi Biotech) was used. GBM patient blood was collected. Briefly, PBMCs were first isolated, then a Pan T cell isolation kit (Miltenyi Biotec) was used to isolate T cells. CAR T cell generation. MC9999 CAR T cells we engineered using a high affinity PD-L1 scFv (Bansal et al., Nucl. Med. Biol., 100-101:4–11 (2021)), a hinge region with a CD4 transmembrane domain, as well as 4-1BB and CD3 intracellular signaling domains, complemented with a truncated EGFR. The CAR cDNA was integrated into pHIV.7 lentiviral vector. To ensure efficient lentivirus production, 293FT cells were utilized, followed by concentration and titer determination using Jurkat cells. Tn/mem or Pan-T cell populations, isolated from PBMCs, were divided into two aliquots for generating non- CAR T-cells as control and another for generating CAR T-cells. GBM cell isolation and engineering. Patient tumor tissue was collected and Brain Tumor Initiating Cells (BTICs) were generated as described elsewhere (Luo et al., Molecular Therapy: Oncology, 32:200891 (2024); and Galli et al., Cancer Res., 64:7011–7021 (2004)). To engineer LN229, QNS 108, and QNS 986), were transduced with GFP luciferase (GFP-luc) and PD-L1 lentivirus, respectively. LN229 cells were engineered with PD-L1 overexpression (OE) and knockout (KO) variants as detailed in Luo et al. (Molecular Therapy: Oncology, 32:200891 (2024)).
Attorney Docket No.07039-2280WO1 / 2023-199 Degranulation assay. CAR T cells were incubated with target cells at a 2:1 E:T ratio in RPMI 1640 with GolgiStop™ and CD107a APC antibody for 6 hours. Cells were then stained with CD3, CD4, CD8, and EGFR antibodies and analyzed using an Attune or Fortessa flow cytometer, with non-CAR T cells as negative controls. Granule release ELISA assays. CAR T-cells and target cells were co-incubated for 72 hours at an E:T ratio of 4:1, the supernatant was collected and evaluated for granule protein release as described in 53. Impedance-based tumor cell killing assay (xCELLigence). To investigate the direct killing of CAR-T cells on tumor cells, impedance-based tumor cell killing assay were performed as described in Luo et al. (Molecular Therapy: Oncology, 32:200891 (2024)). Orthotopic xenograft model, intracranial CAR T infusion, and analysis. NOD SCID gamma (NSG) mice (8-12 weeks old- female for LN229 and male for QNS108 matching the sex of the patient from which we derived the cell line) received 3×105 cells (LN229-PDL1-OE-GFP-luc)/5×105 cells (QNS 108-PD-L1-OE-GFP-luc) intracranially in the striatum. Mice were randomized into three test groups (MC9999 CAR T, non-CAR T, and PBS) test groups (16 mice per group in LN229, 8 mice per group in QNS 108). Two weeks after tumor implantation and after tumor burden confirmation using IVIS® bioluminescence imaging system, mice received 2×106 T cells (MC9999 CAR T or non-CAR T) or PBS intratumorally. Tumor burden was quantified weekly by bioluminescent signal intensity. Animals were sacrificed by ketamine-xylazine overdose, followed by intracardiac infusion of 0.9% NaCl and 4% paraformaldehyde. Brains were harvested and kept in the same fixative solution overnight, then they were rinsed with PBS and embedded in paraffin following standard histology procedures. Tumor growth was calculated by fold change in bioluminescence intensity. Survival data were presented and reported in Kaplan–Meier plots. Statistical analysis for survival was performed using Log-rank analysis (Mantel-Cox), for changes in tumor growth
Attorney Docket No.07039-2280WO1 / 2023-199 (bioluminescence-fold change) Kruskall-Wallis was used. All analyses were performed on PRISM-GraphPad 10. Single cell RNA-sequencing in vivo experimental procedure. Briefly, the same experiment as described above was performed with an orthotopic xenograft model using LN229PD-L1-OE-GFP-luc (N=8 mice). Two weeks after tumor implantation, tumor burden was confirmed and 2x106 T cells (MC9999 CAR T (N=4) or non-CAR T (N=4)) were intratumorally injected in 5 L of PBS.24 hours after T cell infusion, the animals were sacrificed by ketamine-xylazine overdose and intracardiac perfused with 0.9% NaCl for 5 minutes. Brains were harvested, tumor area were isolated, and the tissue was dissociated into single cell suspensions using Tumor Dissociation Kit (Miltenyi) and gentleMACS Octo Dissociator (Milteny Biotech) following manufacturer’s instructions. Then, the number of cells was quantified and cells were assessed using 10X Genomics Chromium Next GEM Single Cell 3' Kit v3.1 according to manufacturer’s instructions targeting a recovery of 1x104 cells at a concentration of 1.2x103 cells/µL). Single cell RNA-sequencing processing and data analysis The single cell sequence preprocessing was performed using the standard 10X Genomics Cell Ranger Single Cell Software Suite (v.8.0.0). Briefly, raw sequencing data were demultiplexed and aligned to both the mouse transcriptome mm10 and human transcriptome GRCh38 to capture both mouse and human genes. Ambient RNA was removed in each sample using the DecontX algorithm. For quality assurance, cells with less than 200 or more than 5000 unique genes were removed from downstream anlaysis. Cell filtration, QC and normalization were performed using standard Seurat package procedures (v.5.1.0). Cell clustering was performed using the Harmony algorithm and visualized using Uniform Manifold Approximation and Projection (UMAP). Cell clusters were annotated into unique cell types using classical cell type marker genes. Genes in each cell type were tested for differential expression between the CART and non-CART groups using the MAST model. Pathway analysis of differentially expressed genes from each cell type was performed using Metascape (v3.5).
Attorney Docket No.07039-2280WO1 / 2023-199 Human data validation for single cell RNA sequencing. To validate the top two upregulated genes found in the GBM cells of MC9999 CAR T treated animals (CTSS and PLAAT4) CGGA mRNA expression data for genes related to T CD4+-Th1 cells (IL12R, IFNGR, TNF, and IL15) and for genes related to T CD8+ cells (GZMB. GZMA, PRF1, LAMP1) were used for all types of diffuse gliomas according to 2021 WHO classification (Oligodendroglioma, Anaplastic Oligodendroglioma, Astrocytoma, Anaplastic Astrocytoma, and Glioblastoma) and their expression was correlated to CTSS and PLAAT4 using linear correlation and Spearman r regression in PRISM, GraphPad 10. Human and rodent tissue staining and quantification. For histological tissue staining Akoya Opal 6-plex kit we used. Briefly, the paraffin in the tissue was dissolved following standard histological procedures, the tissue was rinsed twice in ddH2O and fixed for 20 minutes in 3% non-buffered formalin. Microwave antigen retrieval was performed prior to every cycle using the AR buffer (pH 6 or 9) recommended by primary antibody manufacturer. For every cycle non-specific binding was blocked for 10 minutes in primary diluent (Akoya), the primary antibody was incubated for 1 hour, rinsed with TBS-T, incubated with HRP secondary for 10 minutes, rinsed with TBS-T, and incubated with the corresponding opal for 10 minutes. For Opal 780, DIG-TSA was incubated at 1:100 for 1 hour before Opal 780 incubation. Three different panels were performed (1:150 mouse anti-nestin (Millipore-SIGMA), 1:750 rabbit anti-PD-L1 (abcam), 1:200 rabbit anti-PD-1 (abcam), 1:500 rabbit anti CD4 (abcam), 1:150 rabbit anti-Ki67 (ThermoFisher), 1:250 rabbit anti-CD8 (abcam), 1:300 rabbit-anti Olig2 (Millipore-SIGMA), 1:200 rabbit anti-NeuN (Cell Signaling Technology), 1:500 rabbit anti-GFAP (Dako), 1:4000 rabbit anti-Iba1 (abcam), and 1:250 rabbit anti-vimentin (abcam)). For quantification QuPath 0.5.1 object classification plugin was used. Statistical analysis was performed using % of positive cells for each marker, multiple comparisons were done using Kruskall Wallis and Dunn’s multiple comparisons in GraphPad PRISM 10.
Attorney Docket No.07039-2280WO1 / 2023-199 Timelapse acquisition and analysis For autologous patient GBM-T cell experiment 5x103 QNS986-GFP-Luc cells were seeded.12 hours after, 2.5x104 T cells (MC9999 CAR T or non-CAR T) were added at E:T ratio: 5:1 and a timelapse experiment was performed for 72 hours in Livecyte. Images were taken every 5 minutes. Livecyte analysis software was used to obtain the videos. Monocyte derived M2 macrophages from patient blood and TAMs isolation from tumor tissue Monocyte derived M2 macrophages from GBM patient blood samples were cultured using the procedures described above. CD14-positive monocytes were isolated from PBMCs using CD14 microbeads (Miltenyi) and differentiated into macrophages with M-CSF over 7 days. M2 polarization was induced on day 7 with Interleukin 4 and M-CSF for 48-72 hours, after which cells were harvested and analyzed for phenotype and PD-L1 expression via Fortessa flow cytometry. TAMs were isolated from GBM patient tumor tissue and their immunophenotype and PD-L1 expression were analyzed using Fortessa flow cytometry (BD). Example 10: Treating Cancer T cells (e.g., CAR T cells) engineered to express an antigen receptor (e.g., a CAR) that can target a PD-L1 polypeptide are administered to a human identified as having a cancer (e.g., a breast cancer) including PD-L1+ cancer cells. The T cells (e.g., CAR T cells) engineered to express an antigen receptor (e.g., a CAR) that can target a PD-L1 polypeptide are administered using intravenous or intratumoral injection. After the administration of the one or more T cells (e.g., one or more CAR T cells) expressing (e.g., engineered to express) an antigen receptor (e.g., a CAR) that can target a PD-L1 polypeptide the number of cancer cells (e.g., PD-L1+ cancer cells) within the human is reduced. After the administration of the one or more T cells (e.g., one or more CAR T cells) expressing (e.g., engineered to express) an antigen receptor (e.g., a CAR) that can target a PD-L1 polypeptide, the size of one or more solid tumors (e.g., one or more solid tumors including PD-L1+ cancer cells) within the human is reduced.
Attorney Docket No.07039-2280WO1 / 2023-199 Example 11: Treating Cancer T cells are obtained from a human identified as having a cancer (e.g., a breast cancer) including PD-L1+ cancer cells. Nucleic acid encoding an antigen receptor (e.g., a CAR) that can target a PD-L1 polypeptide is introduced into the T cell by transduction (e.g., viral transduction using a retroviral vector such as a lentiviral vector) or transfection such that the T cell expresses the antigen receptor that can target a PD-L1 polypeptide. The T cells engineered to express an antigen receptor (e.g., a CAR) that can target a PD- L1 polypeptide are administered back into the human using intravenous or intratumoral injection. After the administration of the one or more T cells (e.g., one or more CAR T cells) expressing (e.g., engineered to express) an antigen receptor (e.g., a CAR) that can target a PD-L1 polypeptide the number of cancer cells (e.g., PD-L1+ cancer cells) within the human is reduced. After the administration of the one or more T cells (e.g., one or more CAR T cells) expressing (e.g., engineered to express) an antigen receptor (e.g., a CAR) that can target a PD-L1 polypeptide, the size of one or more solid tumors (e.g., one or more solid tumors including PD-L1+ cancer cells) within the human is reduced. OTHER EMBODIMENTS It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.