Treatment of glioblastoma using car-t cell therapy

Abstract

The present disclosure relates to methods of treating glioblastoma multiforme (GBM) using EGFRvIII-binding chimeric antigen receptor (CAR) T cells and EGFR-binding T cell engaging molecules (TEAMS) administered to the central nervous system, and methods of detecting CAR expression on cells based on tCD19 cell surface expression.

Description

[0001] Related applications

[0002] This application is filed pursuant to 35 USC §119(e) with U.S. Provisional Application No. 63 / 590,108 entitled "Chimeric Antigen Receptor (CAR) Polynucleotides Encoding Truncated CD19"; U.S. Provisional Application No. 63 / 590,141 entitled "Treating Glioblastoma Using CAR-TCell Therapy"; U.S. Provisional Application No. 63 / 600,552 entitled "Chimeric Antigen Receptor (CAR) Polynucleotides Encoding Truncated CD19"; and U.S. Provisional Application No. 63 / 600,552 entitled "Treating Glioblastoma Using CAR-TCell Therapy"; and U.S. Provisional Application No. 63 / 600,552 entitled "Treating Glioblastoma Using CAR-TCell Therapy". The rights of 63 / 600,620, the entire contents of which are incorporated herein by reference.

[0003] Reference to electronic sequence listing

[0004] The contents of the electronic serial number (M105370046WO00-SEQ-ARM.xml; size: 39,747 bytes; and creation date: October 9, 2024) are incorporated herein by reference in their entirety. Background Technology

[0005] Cell-based gene therapy shows great promise in treating diseases such as hematological malignancies. For example, chimeric antigen receptor-T cells (CAR-T cells), a cell-based gene therapy for cancer, have achieved great success in treating hematological malignancies. Autologous CAR-T cells are prepared by collecting a patient's T cells and genetically modifying them to express a chimeric antigen receptor (CAR). This CAR endows the T cells with new specificity: recognizing tumor surface antigens via CAR activates the T cells and initiates tumor killing and CAR-T cell proliferation. While this therapy has transformed the treatment landscape for patients with hematological malignancies, patients with solid tumors have not yet truly benefited from CAR-T cell therapy. Summary of the Invention

[0006] Glioblastoma multiforme (GBM) is an aggressive and highly malignant brain cancer originating from glial cells. It is characterized by its rapid growth, invasive nature, and resistance to standard treatments. GBM typically presents with neurological symptoms, and despite advances in treatment, its poor prognosis makes it one of the most challenging cancers to treat. While chimeric antigen receptor (CAR) T-cell therapy has shown great promise in treating hematologic malignancies, its application in GBM treatment has proven challenging, partly due to the fact that known CAR-T cell administration and dosing regimens involve treating hematologic malignancies or solid tumors outside the central nervous system. The tissues of the central nervous system are protected by the blood-brain barrier, which prevents intravenously administered CAR-T cells from infiltrating tumor sites in the brain or spinal cord. In addition, the space available in the central nervous system and the volume of cerebrospinal fluid (CSF) are limited compared to circulating blood, and therefore the dose of CAR-T cells, the volume of the CAR-T cell-containing composition administered, and the administration rate may not necessarily be expected to be consistent with routine procedures in intravenous CAR-T cell therapy.

[0007] Therefore, in some aspects, this disclosure provides a method for treating glioblastoma multiforme (GBM) in a subject, the method comprising administering 6.0 × 10 6 Up to 1.4×10 7 A CAR-T cell is administered to the central nervous system of the subject, wherein the CAR-T cell contains a polynucleotide encoding the following: (a) a CAR containing an extracellular binding domain that binds to EGFRvIII; and (b) a T cell engaging antibody molecule (TEAM) containing an anti-EGFR antibody and an anti-CD3 antibody.

[0008] In some implementations, the method includes applying 7.0 × 10⁻⁶ to the object. 6 Up to 1.3×10 7 One CAR-T cell. In some implementations, the method includes administering 8.0 × 10⁸ CAR-T cells to the subject. 6 Up to 1.2×10 7 9.0 × 10⁶ CAR-T cells. In some implementations, the method includes administering 9.0 × 10⁶ CAR-T cells to the subject. 6 Up to 1.1×10 7 One CAR-T cell. In some implementations, the method includes administering 1.0 × 10⁶ CAR-T cells to the subject. 7 One CAR-T cell.

[0009] In some embodiments of the methods described herein, administering CAR-T cells includes administering CAR-T cells into the brain of a subject. In some embodiments, administering CAR-T cells includes intracranial administration. In some embodiments, administering CAR-T cells includes intraventricular administration. In some embodiments, administering CAR-T cells includes administering them into the cerebrospinal fluid of a subject. In some embodiments, administering CAR-T cells includes intrathecal administration. In some embodiments, administering CAR-T cells includes administering them to a tumor in the central nervous system of a subject. In some embodiments, administering CAR-T cells includes administering them to a tumor in the central nervous system of a subject.

[0010] In some embodiments, the CAR-T cells are in a solution of 5 to 15 mL. In some embodiments, the solution contains Elliott's B solution. In some embodiments, administration of the CAR-T cells includes administration at a rate not exceeding 2 mL / min. In some embodiments, administration of the CAR-T cells includes administration at a rate of 1 to 2 mL / min.

[0011] In some embodiments, the extracellular binding domain comprises: (a) a VH domain containing three complementarity-determining regions (CDR-H1, CDR-H2, and CDR-H3), wherein CDR-H1 contains SEQ ID NO: 1, CDR-H2 contains SEQ ID NO: 2, and CDR-H3 contains SEQ ID NO: 3; and (b) a VL domain containing three CDRs (CDR-L1, CDR-L2, and CDR-L3), wherein CDR-L1 contains SEQ ID NO: 4, CDR-L2 contains SEQ ID NO: 5, and CDR-L3 contains SEQ ID NO: 6. In some embodiments, the extracellular domain is scFv. In some embodiments, scFv contains the amino acid sequence of SEQ ID NO: 7. In some embodiments, CAR contains the amino acid sequence of SEQ ID NO: 8.

[0012] In some embodiments, the anti-EGFR antibody comprises: (a) a VH domain containing three complementarity-determining regions (CDR-H1, CDR-H2, and CDR-H3), wherein CDR-H1 contains SEQ ID NO: 9, CDR-H2 contains SEQ ID NO: 10, and CDR-H3 contains SEQ ID NO: 11; and (b) a VL domain containing three CDRs (CDR-L1, CDR-L2, and CDR-L3), wherein CDR-L1 contains SEQ ID NO: 12, CDR-L2 contains SEQ ID NO: 13, and CDR-L3 contains SEQ ID NO: 14. In some embodiments, the anti-EGFR antibody is an scFv. In some embodiments, the scFv contains the amino acid sequence of SEQ ID NO: 15.

[0013] In some embodiments, the anti-CD3 antibody comprises: (a) a VH domain containing three complementarity-determining regions (CDR-H1, CDR-H2, and CDR-H3), wherein CDR-H1 contains SEQ ID NO: 16, CDR-H2 contains SEQ ID NO: 17, and CDR-H3 contains SEQ ID NO: 18; and (b) a VL domain containing three CDRs (CDR-L1, CDR-L2, and CDR-L3), wherein CDR-L1 contains SEQ ID NO: 19, CDR-L2 contains SEQ ID NO: 20, and CDR-L3 contains SEQ ID NO: 21. In some embodiments, the anti-CD3 antibody is an scFv. In some embodiments, the scFv contains the amino acid sequence of SEQ ID NO: 22.

[0014] In some implementations, TEAM comprises the amino acid sequence of SEQ ID NO: 23.

[0015] In some embodiments, GBM contains a polynucleotide encoding EGFRvIII. In some embodiments, GBM expresses EGFRvIII. In some embodiments, GBM does not express EGFRvIII. In some embodiments, GBM expresses both EGFRvIII and EGFR. In some embodiments, GBM expresses EGFR. In some embodiments, GBM contains a gene duplication of EGFR.

[0016] In some embodiments, the subject has previously received radiation therapy. In some embodiments, the subject has recurrent GBM. In some embodiments, at the time of administration, the subject has not received alkylation therapy or immunotherapy within the past 4 weeks. In some embodiments, the subject's GBM is unmethylated with O-6-methylguanine-DNA methyltransferase (MGMT). In some embodiments, the subject has not previously been treated for GBM. In some embodiments, the subject's GBM contains a polynucleotide encoding EGFRvIII, and the subject's GBM is unmethylated with MGMT.

[0017] In some embodiments of the method described herein, the method further includes administering an antihistamine to the subject. In some embodiments, the antihistamine is Benadryl. In some embodiments, 25 mg of Benadryl is administered to the subject. In some embodiments, Benadryl is administered prior to CAR-T cell administration.

[0018] In some embodiments of the method described herein, the method further includes administering acetaminophen to the subject. In some embodiments, 650 mg of acetaminophen is administered to the subject. In some embodiments, acetaminophen is administered prior to CAR-T cell administration.

[0019] In some embodiments of the method described herein, the method further includes generating CAR-T cells using autologous T cells.

[0020] In some embodiments of the method described herein, the method includes administering a single dose of CAR-T cells to a subject. In some embodiments, the method described herein includes administering six doses of CAR-T cells to a subject. In some embodiments, the method includes administering CAR-T cells to a subject every four to six weeks. In some embodiments, the method includes administering CAR-T cells every four to six weeks for a total of four administrations.

[0021] In some embodiments of the methods described herein, the methods include administering lymphodepleting (LD) chemotherapy to the subject. In some embodiments, administering LD chemotherapy includes administering cyclophosphamide and / or fludarabine. In some embodiments, the methods include administering approximately 300 mg / m² of cyclophosphamide. In some embodiments, the methods include administering approximately 25 mg / m² of fludarabine. In some embodiments, LD chemotherapy is administered prior to CAR-T cell administration. In some embodiments, cyclophosphamide and fludarabine are administered daily for three consecutive days prior to CAR-T cell administration. In some embodiments, cyclophosphamide and / or fludarabine are administered 5, 4, and 3 days prior to CAR-T cell administration. In some embodiments, cyclophosphamide and / or fludarabine are administered 7, 6, and 5 days prior to CAR-T cell administration.

[0022] In some aspects, this disclosure describes polynucleotides comprising nucleic acids encoding amino acid sequences comprising: (i) an epidermal growth factor receptor (EGFR)-binding chimeric antigen receptor (EGFRvIIICAR) comprising an antigen-binding domain comprising: the variable heavy chain (VH) complementarity-determining region (CDR) 1 of SEQ ID NO: 1, the VH CDR2 of SEQ ID NO: 2, the VH CDR3 of SEQ ID NO: 3, the variable light chain (VL) CDR1 of SEQ ID NO: 4, the VL CDR2 of SEQ ID NO: 5, and the VL CDR3 of SEQ ID NO: 6; and (ii) a T cell conjugating antibody molecule (TEAM) comprising: (a) an EGFR-binding domain comprising the variable heavy chain (VH) complementarity-determining region (CDR) 1 of SEQ ID NO: 9, the VH CDR2 of SEQ ID NO: 10, the VH CDR3 of SEQ ID NO: 11, and the EGFR CDR3 of SEQ ID NO: 9. (a) SEQ ID NO: 12, VL CDR1, SEQ ID NO: 13, and VL CDR3 of SEQ ID NO: 14; (b) a CD3 binding domain comprising SEQ ID NO: 16, VH CDR1, SEQ ID NO: 17, VH CDR3, SEQ ID NO: 19, VL CDR1, SEQ ID NO: 20, and VL CDR3 of SEQ ID NO: 21; and (iii) a truncated CD19 domain (tCD19) comprising an amino acid sequence having at least 95% identity with SEQ ID NO: 38.

[0023] In some embodiments, the antigen-binding domain comprises scFv. In some embodiments, the antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, the EGFRvIII CAR comprises a CD8 hinge / transmembrane domain. In some embodiments, the EGFRvIII CAR comprises a 4-1BB intracellular signaling domain. In some embodiments, the EGFRvIII CAR comprises a CD3z signaling domain. In some embodiments, the polynucleotide further comprises an amino acid sequence encoding a 2A self-cleaving peptide, which is encoded between the EGFRvIII CAR and TEAM. In some embodiments, the polynucleotide further comprises an amino acid sequence encoding a 2A self-cleaving peptide, which is encoded between TEAM and tCD19. In some embodiments, the 2A self-cleaving peptide comprises the amino acid sequence of either SEQ ID NO: 35 or SEQ ID NO: 36. In some embodiments, TEAM comprises a peptide linker encoded between the EGFR-binding domain and the CD3-binding domain. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 30 to 34 or 36. In some embodiments, TEAM comprises an IgK leader sequence.

[0024] In some embodiments, the EGFR-binding domain comprises the amino acid sequence of SEQ ID NO: 15. In some embodiments, the CD3-binding domain comprises the amino acid sequence of SEQ ID NO: 22. In some embodiments, tCD19 comprises the amino acid sequence of SEQ ID NO: 38. In some embodiments, the polynucleotide comprises the amino acid sequence of SEQ ID NO: 37.

[0025] In some aspects, this disclosure describes cells comprising the polynucleotides described herein. In some embodiments, the cells are T cells. In some embodiments, tCD19 is expressed on the surface of the cells.

[0026] In some respects, this disclosure describes polypeptides encoded by the polynucleotides described herein.

[0027] In some aspects, this disclosure describes a method for detecting the cells described herein, the method comprising: contacting the cells with a tCD19 binding agent; and detecting the tCD19 binding protein. In some embodiments, the tCD19 binding agent comprises the tCD19 binding protein. In some embodiments, the tCD19 binding protein comprises an anti-tCD19 antibody. In some embodiments, the anti-tCD19 antibody is an antibody fragment. In some embodiments, the anti-tCD19 antibody is CD19-PE. In some embodiments, the anti-tCD19 antibody has a higher binding affinity to tCD19 than to CD19. In some embodiments, the tCD19 binding protein further comprises a fluorescent molecule or a radioactive molecule. In some embodiments, the method further comprises contacting the tCD19 binding protein with a secondary antibody comprising the fluorescent molecule or the radioactive molecule. In some embodiments, the detection comprises detecting the fluorescent molecule or the radioactive molecule. In some embodiments, detecting the fluorescent molecule or the radioactive molecule comprises detection using microscopy, Western blotting, or flow cytometry. Attached Figure Description

[0028] The following figures form part of this specification and are included to further illustrate certain aspects of this disclosure, which can be better understood by referring to one or more of these figures in combination with the detailed description of some specific embodiments given herein.

[0029] Figures 1A to 1B The CAR-TEAM construct and its proposed mechanism of action are shown. Figure 1A The EGFRvIII-in-CAR and anti-EGFR and anti-CD3 TEAM components are shown. Figure 1B CAR-T cells with EGFRvIII-binding CARs are shown, which secrete TEAM cells that bind EGFR and CD3 (“CAR-TEAM cells”).

[0030] Figure 2 It is Figures 1A to 1B A schematic diagram of CAR-TEAM cell therapy for glioblastoma multiforme (GBM) is shown, illustrating the process from tumor resection (1) to CAR-T cell infusion (5).

[0031] Figure 3The image shows the reduction in tumor volume resulting from CAR-TEAM cell therapy in three human patients (right) and the patient response types over time (left). Complete response: all enhancing disease disappeared for ≥ 4 weeks; Partial response: the sum of the biperpendicular diameters of enhancing disease decreased by ≥ 50% for ≥ 4 weeks; Stable disease: not meeting the criteria for complete response, partial response, or disease progression, and no new lesions appeared; Disease progression: the sum of the biperpendicular diameters of enhancing disease increased by ≥ 25%.

[0032] Figures 4A to 4D This corresponds to the presence of CAR-TEAM cells and TEAM-binding cells in the patient's blood and CSF. Figures 4A to 4B The patient's CSF was shown. Figure 4A (see above) and blood ( Figure 4B The absolute number of CAR-TEAM cells in the above figure, and in CSF ( Figure 4A (see image below) and blood ( Figure 4B The percentage of CAR-TEAM cells in the total cells (see figure below). Figures 4C to 4D The patient's CSF was shown. Figure 4C (see above) and blood ( Figure 4D The absolute number of TEAM-bound cells in the above figure, and in CSF ( Figure 4C (see image below) and blood ( Figure 4D The percentage of TEAM-bound cells in the total number of cells (see figure below).

[0033] Figure 5 The levels of cytokine release in patients after CAR-T cell administration are shown. Specifically, the concentrations of IFNγ, IL-6, and TNFα in serum CSF over time are presented in three patients.

[0034] Figures 6A to 6C This relates to the CAR-TEAM construct and its expression in cells. Figure 6A A schematic diagram of a CAR-TEAM encoded molecule with a truncated CD19 (tCD19) molecule is shown, which is used as a surface marker for transduction. Figure 6B This paper presents a representative flow cytometry analysis of cell products from patient 1, in which all CD3 + T cells are gated to CAR-positive and TEAM-positive cells. Figure 6C Representative gating of TEAM-positive cells is shown in CAR-positive and CAR-negative T cell populations.

[0035] Figures 7A to 7E Corresponding to the treatment events and outcomes related to Patient 1. Figure 7AThe event timeline for Patient 1, who participated in the Phase I clinical trial, was summarized. Figure 7B The image shows axial contrast-enhanced T1-weighted images of patient 1 immediately after resection (left), before infusion (middle), and after infusion (right), which enhance the signal of adipose tissue. The numbers in each image represent the number of days relative to the infusion (day 0). Figure 7C The copy numbers of EGFRvIII and EGFR derived from extracellular vesicle (EV) RNA present in CSF over time are shown. Figure 7D The corresponding copy numbers of EV RNA isolated from peripheral blood at time points before and after treatment are shown, and Figure 7E The post-hoc analysis of EGFR copy number by fluorescence in situ hybridization (FISH) is shown.

[0036] Figures 8A to 8C Treatment workflow and outcomes for patients 2 and 3. Figure 8A An improved workflow for patients 2 and 3 is shown, which enables craniotomy, tissue sampling, and Ommaya placement to be performed simultaneously in a single procedure. Figure 8B The longitudinal radiographic assessment of patient 2 following CAR-TEAM cell infusion is shown. Figure 8C The images show axial T1-weighted angiography of patient 3 before and after infusion, demonstrating tumor regression at D+5 post-treatment. The numbers in each figure represent the number of days relative to the infusion (day 0).

[0037] Figures 9A to 9B This involves the temperature response in patients 1 to 3 following CAR-TEAM cell infusion. Figure 9A Temperature curves for all patients are shown, peaking within 2 days of infusion and normalizing over time. Figure 9B Temperature management was demonstrated by treatment with the interleukin-1 receptor (IL-1R) antagonist anakinra.

[0038] Figure 10 The expression of inflammatory markers C-reactive protein (CRP) and ferritin in the cerebrospinal fluid of patients 1 to 3 after CAR-TEAM cell infusion is shown.

[0039] Figure 11 The cell counts and differentials in the cerebrospinal fluid of patients 1 to 3 after CAR-TEAM cell infusion are shown.

[0040] Figures 12A to 12C The presence of CAR-positive and TEAM-positive cells and inflammatory cytokines in the cerebrospinal fluid (CSF) and peripheral blood of patients 1 to 3 was investigated. Figure 12A The quantification of CAR-positive and TEAM-positive T cells in all three patients by vector copy number (VCN) and flow cytometry is shown. These cells were temporarily present in the CSF after infusion but decreased by week 4. Figure 12B The presence of inflammatory cytokines in the patient's CSF during the same period was shown. Figure 12C The detection of CAR-positive and TEAM-positive cells in peripheral blood is shown, and it is transient.

[0041] Figure 13 The flow cytometry measurements of the T cell population are shown to determine the percentage of cells expressing CAR based on truncated CD19 expression. Detailed Implementation

[0042] This application relates in part to a method for treating glioblastoma multiforme (GBM) by administering chimeric antigen receptor (CAR) T cells to a subject's central nervous system (CNS). CAR-T cell therapy for GBM has proven challenging due to factors such as administration. Intravenous administration and dosage of CAR-T cells are known in the art and have been successfully used clinically to treat hematologic malignancies. However, in the case of brain cancers (e.g., GBM), the blood-brain barrier impairs the delivery of CAR-T cells to the tumor site. Therefore, a method for directly administering a specific amount of CAR-T cells to a subject's central nervous system to treat GBM is proposed herein.

[0043] In some aspects, this disclosure describes polynucleotides comprising nucleic acids encoding an EGFRvIII-binding CAR, an EGFR-binding and CD3-binding TEAM, and a truncated CD19 (tCD19) domain. In some embodiments, this disclosure describes polypeptides comprising amino acids encoding an EGFRvIII-binding CAR, an EGFR-binding and CD3-binding TEAM, and a truncated CD19 domain. In some embodiments, tCD19 expression indicates EGFRvIII CAR expression.

[0044] Glioblastoma multiforme (GBM)

[0045] Glioblastoma multiforme (GBM) is a highly aggressive and malignant form of brain cancer originating from glial cells in the brain. Its rapid and invasive growth makes treatment challenging. GBM typically causes neurological symptoms such as headache, seizures, and cognitive impairment, and is associated with poor prognosis. Treatment usually involves a combination of surgical resection, radiation therapy, and chemotherapy; however, the disease is known to be resistant to standard treatments, and most patients face a limited life expectancy after diagnosis.

[0046] In some aspects, this disclosure provides a method for treating GBM in a subject by administering GBM-targeting CAR-T cells to the central nervous system of the subject. In some embodiments, in some aspects, the CAR-T cells described in the methods herein comprise a polynucleotide encoding a CAR and a T-cell conjugating antibody molecule (“CAR-TEAM cells”), said CAR binding to epidermal growth factor variant III (EGFRvIII), said T-cell conjugating antibody molecule comprising an anti-epidermal growth factor (EGFR) antibody and an anti-CD3 antibody. In GBM, EGFR mutations, particularly EGFRvIII mutations, are associated with EGFR amplification and overexpression, which promote tumor growth and lead to increased expression of EGFR protein on the surface of GBM cells.

[0047] In some embodiments, GBMs expressing EGFRvIII have detectable amounts of EGFRvIII on their cell surface. In some embodiments, GBMs expressing EGFRvIII express detectable amounts of EGFRvIII RNA. In some embodiments, GBM EGFRvIII expression is determined by measuring EGFRvIII protein or RNA expression in the GBM. In some embodiments, EGFRvIII expression is measured by measuring EGFRvIII cell surface expression on the GBM (e.g., by biopsy of the GBM tumor and subsequent immunostaining with an EGFRvIII antibody). In some embodiments, EGFRvIII expression is determined by measuring EGFRvIII mRNA (e.g., by biopsy of the GBM tumor and rtPCR). In some embodiments, the GBM contains a polynucleotide encoding EGFRvIII. In some embodiments, the cells of the GBM contain a polynucleotide encoding EGFRvIII. In some embodiments, the cells of the GBM express EGFRvIII. In some embodiments, the GBM expresses both EGFRvIII and EGFR. In some embodiments, the cells of the GBM express both EGFRvIII and EGFR. In some embodiments, GBM expresses EGFR. In some embodiments, GBM cells express EGFR. In some embodiments, GBM cells contain EGFR gene duplication. In some embodiments, GBM contains EGFR gene duplication.

[0048] In some embodiments, GBM does not express EGFRvIII, as determined by measuring EGFRvIII protein or RNA expression in GBM. In some embodiments, GBM initially expresses EGFRvIII, but subsequently loses EGFRvIII expression. In some embodiments, GBM that does not express EGFRvIII does not have a detectable amount of EGFRvIII on the GBM cell surface. In some embodiments, GBM that does not express EGFRvIII does not express a detectable amount of EGFRvIII RNA.

[0049] In some embodiments, the target GBM is O-6-methylguanine-DNA methyltransferase (MGMT) unmethylated. MGMT-unmethylated GBM is a subgroup of GBM in which the MGMT gene is not methylated. The MGMT gene encodes a DNA repair enzyme that plays a crucial role in repairing DNA damage caused by alkylating agents, including certain chemotherapeutic agents. When the MGMT gene is methylated, it is silenced, and the enzyme it encodes is no longer produced. Therefore, GBM cells have a lower ability to repair DNA damage, making them more sensitive to chemotherapy. Thus, MGMT-unmethylated GBM is resistant to chemotherapy. In some embodiments, the target GBM contains a polynucleotide encoding EGFRvIII, and the target GBM is MGMT-unmethylated.

[0050] Radiation therapy is often a key component of standard treatment for GBM, which is known to have a high relapse rate. In some embodiments, the subject has previously received radiation therapy. In some embodiments, the subject has recurrent GBM. In some embodiments, the subject has not received alkylation therapy (e.g., chemotherapy) or immunotherapy within the past 4 weeks when CAR-T cells are administered according to the methods described herein. In some embodiments, the subject has not previously been treated for GBM.

[0051] object

[0052] As used herein, “object” means either a person or an animal. Generally, an animal is a vertebrate, such as a primate, rodent, domesticated animal, or hunting animal. In some embodiments, the object is a mammal, such as a primate, for example, a human. The terms “individual,” “patient,” and “object” are used interchangeably herein. Preferably, the object is a mammal. A mammal can be a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow, but is not limited to these examples. Mammals other than humans can be advantageously used as objects representing animal models of diseases (e.g., cancer). The object can be male or female.

[0053] The subject may be a subject who has been previously diagnosed or identified as having or having a condition requiring treatment (e.g., being diagnosed with GBM) or one or more complications associated with such a condition (e.g., GBM), and optionally, the subject has already undergone treatment for the condition (e.g., GBM) or one or more complications associated with the condition.

[0054] Alternatively, the subject can be someone who has not previously been diagnosed with such a condition (e.g., GBM) or related complications. For example, the subject could be someone exhibiting the condition or one or more risk factors for one or more complications related to the condition, or someone who does not exhibit any risk factors.

[0055] "Individuals in need" for treatment of a specific condition can be those who have the condition (e.g., GBM), have been diagnosed with the condition (e.g., GBM), or are at risk of developing the condition (e.g., GBM).

[0056] application

[0057] As used herein, “application” means bringing a subject (e.g., the subject’s central nervous system) into contact with a cell therapy (e.g., a pharmaceutical composition). In some embodiments, application includes injecting or infusing the pharmaceutical composition into the subject. In some embodiments, injection or infusion includes passing the pharmaceutical composition through a filter. In some embodiments, injection or infusion does not include passing the pharmaceutical composition through a filter. In some embodiments, application includes bringing the subject’s central nervous system into contact with the cell therapy. In some embodiments, application includes administering the pharmaceutical composition into the subject’s central nervous system.

[0058] When used in the context of administration to a specific location, the phrase "to..." (e.g., administering a pharmaceutical composition to a subject's central nervous system) refers to the direct application of a treatment (e.g., cell therapy or a pharmaceutical composition) to that location. For example, administering a pharmaceutical composition to a subject's central nervous system includes injecting or infusing the pharmaceutical composition into the subject's cerebrospinal fluid (which is part of the central nervous system), but does not include intravenous infusion or oral administration of the pharmaceutical composition, even if the pharmaceutical composition eventually crosses the blood-brain barrier and enters the cerebrospinal fluid.

[0059] As used in this article, "central nervous system" refers to biological substances (e.g., fluids, tissues, organs, tumors, etc.) within the blood-brain barrier, including the blood-brain barrier, brain, spinal cord, cerebrospinal fluid (CSF), meninges, ventricles, spaces between the membranes of the meninges (e.g., subarachnoid space), and brain or spinal cord tumors of the central nervous system (e.g., GBM).

[0060] "Approximately" refers to + / - 5% of the quoted number. For example, approximately 10 million T cells means 9.5 million to 11.5 million T cells.

[0061] The key finding provided in this disclosure is the appropriate dosage of CAR-T cells administered to the central nervous system of a subject. As stated above, CAR-T cell therapy, particularly in hematologic malignancies, is known in the art regarding intravenous administration of CAR-T cells. Given the restrictive nature of the central nervous system and the limited volume of CSF, the dosages and administration regimens known in the art for CAR-T cells (e.g., those for hematologic malignancies) are unsuitable for the treatment of GBM.

[0062] In short, CAR-T cells are generated by transducing immune cells obtained from a subject in vitro with one or more polynucleotides encoding CAR, followed by cell expansion and eventual infusion back into the subject. Typically, cell transduction does not result in 100% CAR expression in the transduced cell population. In fact, transduction efficiency can vary widely, leading to approximately 30% fluctuation in the number of CAR-T cells administered to the subject.

[0063] Some aspects of this disclosure provide a method for treating glioblastoma multiforme (GBM) in subjects, the method comprising inserting 6.0 × 10 6 Up to 1.4×10 7 A CAR-T cell is administered to the central nervous system of a subject, wherein the CAR-T cell contains a polynucleotide encoding: (a) a CAR containing an extracellular binding domain that binds to EGFRvIII; and (b) a T cell conjugating antibody molecule (TEAM) containing an anti-EGFR antibody and an anti-CD3 antibody. In some embodiments, the CAR-T cell contains SEQ ID NO: 37.

[0064] In some implementations, the method of treating GBM in a subject includes administering 3.0 × 10⁻⁶ ppm to the subject. 6 Up to 7.5×10 6 CAR-T cells (e.g., 3.0 × 10⁶) 6 Up to 7.5×10 6 3.5×10 6 Up to 7.5×10 6 4.0×10 6 Up to 7.5×10 6 4.5×10 6 Up to 7.5×10 6 5.0×10 6 Up to 7.5×10 6 5.5×10 6 Up to 7.5×10 6 6.0×10 6 Up to 7.5×10 6 6.5×10 6 Up to 7.5×10 6 7.0×10 6 Up to 7.5×10 6 Or 7.5×10 6 (3.5 × 10 CAR T cells). In some implementations, the method of treating GBM in a subject includes administering 3.5 × 10 CAR T cells to the subject. 6 Up to 8.0×10 6 CAR-T cells (e.g., 3.5 × 10⁶) 6 Up to 8.0×106 4.0×10 6 Up to 8.0×10 6 4.5×10 6 Up to 8.0×10 6 5.0×10 6 Up to 8.0×10 6 5.5×10 6 Up to 8.0×10 6 6.0×10 6 Up to 8.0×10 6 6.5×10 6 Up to 8.0×10 6 7.0×10 6 Up to 8.0×10 6 7.5×10 6 Up to 8.0×10 6 Or 8.0×10 6 (4.0 × 10 CAR T cells). In some implementations, the method of treating GBM in a subject includes administering 4.0 × 10 CAR T cells to the subject. 6 Up to 8.5×10 6 CAR-T cells (e.g., 4.0 × 10⁶) 6 Up to 8.5×10 6 4.5×10 6 Up to 8.5×10 6 5.0×10 6 Up to 8.5×10 6 5.5×10 6 Up to 8.5×10 6 6.0×10 6 Up to 8.5×10 6 6.5×10 6 Up to 8.5×10 6 7.0×10 6 Up to 8.5×10 6 7.5×10 6 Up to 8.5×10 6 8.0×10 6 Up to 8.5×10 6 Or 8.5×10 6 (4.5 × 10 CAR T cells). In some implementations, the method of treating GBM in a subject includes administering 4.5 × 10 CAR T cells to the subject. 6 Up to 9.0×10 6 CAR-T cells (e.g., 4.5 × 10⁶) 6 Up to 9.0×10 6 5.0×10 6 Up to 9.0×10 6 5.5×106 Up to 9.0×10 6 6.0×10 6 Up to 9.0×10 6 6.5×10 6 Up to 9.0×10 6 7.0×10 6 Up to 9.0×10 6 7.5×10 6 Up to 9.0×10 6 8.0×10 6 Up to 9.0×10 6 8.5×10 6 Up to 9.0×10 6 Or 9.0×10 6 (5.0 × 10 CAR T cells). In some implementations, the method of treating GBM in a subject includes administering 5.0 × 10 CAR T cells to the subject. 6 Up to 9.5×10 6 CAR-T cells (e.g., 5.0 × 10⁶) 6 Up to 9.5×10 6 5.5×10 6 Up to 9.5×10 6 6.0×10 6 Up to 9.5×10 6 6.5×10 6 Up to 9.5×10 6 7.0×10 6 Up to 9.5×10 6 7.5×10 6 Up to 9.5×10 6 8.0×10 6 Up to 9.5×10 6 8.5×10 6 Up to 9.5×10 6 9.0×10 6 Up to 9.5×10 6 Or 9.5×10 6 (5.5 × 10 CAR T cells). In some implementations, the method of treating GBM in a subject includes administering 5.5 × 10 CAR T cells to the subject. 6 Up to 1.0×10 7 CAR-T cells (e.g., 5.5 × 10⁶) 6 Up to 1.0×10 7 6.0×10 6 Up to 1.0×10 7 6.5×10 6 Up to 1.0×10 7 7.0×10 6 Up to 1.0×107 7.5×10 6 Up to 1.0×10 7 8.0×10 6 Up to 1.0×10 7 8.5×10 6 Up to 1.0×10 7 9.0×10 6 Up to 1.0×10 7 9.5×10 6 Up to 1.0×10 7 Or 1.0×10 7 (6.0 × 10 CAR T cells). In some implementations, the method of treating GBM in a subject includes administering 6.0 × 10 CAR T cells to the subject. 6 Up to 1.5×10 7 CAR-T cells (e.g., 6.0 × 10⁶) 6 Up to 1.5×10 7 6.5×10 6 Up to 1.5×10 7 7.0×10 6 Up to 1.5×10 7 7.5×10 6 Up to 1.5×10 7 8.0×10 6 Up to 1.5×10 7 8.5×10 6 Up to 1.5×10 7 9.0×10 6 Up to 1.5×10 7 9.5×10 6 Up to 1.5×10 7 1.0×10 7 Up to 1.5×10 7 Or 1.5×10 7 (6.5 × 10 CAR T cells). In some implementations, the method of treating GBM in a subject includes administering 6.5 × 10 CAR T cells to the subject. 6 Up to 2.0×10 7 CAR-T cells (e.g., 6.5 × 10⁶) 6 Up to 2.0×10 7 7.0×10 6 Up to 2.0×10 7 7.5×10 6 Up to 2.0×10 7 8.0×10 6 Up to 2.0×10 7 8.5×10 6 Up to 2.0×10 7 9.0×106 Up to 2.0×10 7 9.5×10 6 Up to 2.0×10 7 1.0×10 7 Up to 2.0×10 7 1.5×10 7 Up to 2.0×10 7 Or 2.0×10 7 (7.0 × 10 CAR T cells). In some implementations, the method of treating GBM in a subject includes administering 7.0 × 10 CAR T cells to the subject. 6 Up to 2.5×10 7 CAR-T cells (e.g., 7.0 × 10⁶) 6 Up to 2.5×10 7 7.5×10 6 Up to 2.5×10 7 8.0×10 6 Up to 2.5×10 7 8.5×10 6 Up to 2.5×10 7 9.0×10 6 Up to 2.5×10 7 9.5×10 6 Up to 2.5×10 7 1.0×10 7 Up to 2.5×10 7 1.5×10 7 Up to 2.5×10 7 2.0×10 7 Up to 2.5×10 7 Or 2.5×10 7 (7.5 × 10⁶ CAR T cells). In some implementations, the method of treating GBM in a subject includes administering 7.5 × 10⁶ CAR T cells to the subject. 6 Up to 3.0×10 7 CAR-T cells (e.g., 7.5 × 10⁶) 6 Up to 3.0×10 7 8.0×10 6 Up to 3.0×10 7 8.5×10 6 Up to 3.0×10 7 9.0×10 6 Up to 3.0×10 7 9.5×10 6 Up to 3.0×10 7 1.0×10 7 Up to 3.0×10 7 1.5×10 7 Up to 3.0×107 2.0×10 7 Up to 3.0×10 7 2.5×10 7 Up to 3.0×10 7 Or 3.0×10 7 (8.0 × 10 CAR T cells). In some implementations, the method of treating GBM in a subject includes administering 8.0 × 10 CAR T cells to the subject. 6 Up to 3.5×10 7 CAR-T cells (e.g., 8.0 × 10⁶) 6 Up to 3.5×10 7 8.5×10 6 Up to 3.5×10 7 9.0×10 6 Up to 3.5×10 7 9.5×10 6 Up to 3.5×10 7 1.0×10 7 Up to 3.5×10 7 1.5×10 7 Up to 3.5×10 7 2.0×10 7 Up to 3.5×10 7 2.5×10 7 Up to 3.5×10 7 3.0×10 7 Up to 3.5×10 7 Or 3.5×10 7 (8.5 × 10 CAR T cells). In some implementations, the method of treating GBM in a subject includes administering 8.5 × 10 CAR T cells to the subject. 6 Up to 4.0×10 7 CAR-T cells (e.g., 8.5 × 10⁶) 6 Up to 4.0×10 7 9.0×10 6 Up to 4.0×10 7 9.5×10 6 Up to 4.0×10 7 1.0×10 7 Up to 4.0×10 7 1.5×10 7 Up to 4.0×10 7 2.0×10 7 Up to 4.0×10 7 2.5×10 7 Up to 4.0×10 7 3.0×10 7 Up to 4.0×10 7 3.5×107 Up to 4.0×10 7 Or 4.0×10 7 (9.0 × 10 CAR T cells). In some implementations, the method of treating GBM in a subject includes administering 9.0 × 10 CAR T cells to the subject. 6 Up to 4.5×10 7 CAR-T cells (e.g., 9.0 × 10⁶) 6 Up to 4.5×10 7 9.5×10 6 Up to 4.5×10 7 1.0×10 7 Up to 4.5×10 7 1.5×10 7 Up to 4.5×10 7 2.0×10 7 Up to 4.5×10 7 2.5×10 7 Up to 4.5×10 7 3.0×10 7 Up to 4.5×10 7 3.5×10 7 Up to 4.5×10 7 4.0×10 7 Up to 4.5×10 7 Or 4.5×10 7 CAR T cells).

[0065] In some implementations, the method of treating GBM in a subject includes administering 6.0 × 10⁻⁶ ppm to the subject. 6 Up to 1.6×10 7 7.0 × 10⁶ CAR-T cells. In some implementations, the method of treating GBM in a subject includes administering 7.0 × 10⁶ CAR-T cells to the subject. 6 Up to 1.3×10 7 10 CAR-T cells. In some embodiments, the method includes administering 8.0 × 10⁸ CAR-T cells to the subject. 6 Up to 1.2×10 7 9.0 × 10⁶ CAR-T cells. In some embodiments, the method includes administering 9.0 × 10⁶ CAR-T cells to the subject. 6 Up to 1.1×10 7 1.0 × 10⁶ CAR-T cells. In some embodiments, the method includes administering 1.0 × 10⁶ CAR-T cells to the subject. 7 One CAR-T cell.

[0066] In some implementations, the method of treating GBM in a subject includes administering at least 3.0 × 10⁻⁶ ppm to the subject. 6One CAR-T cell. In some implementations, the method of treating GBM in a subject includes administering at least 3.5 × 10⁶ CAR-T cells to the subject. 6 One CAR-T cell. In some implementations, the method of treating GBM in a subject includes administering at least 4.0 × 10⁶ CAR-T cells to the subject. 6 One CAR-T cell. In some implementations, the method of treating GBM in a subject includes administering at least 4.5 × 10⁶ CAR-T cells to the subject. 6 One CAR-T cell. In some implementations, the method of treating GBM in a subject includes administering at least 5.0 × 10⁶ CAR-T cells to the subject. 6 One CAR-T cell. In some implementations, the method of treating GBM in a subject includes administering at least 5.5 × 10⁶ CAR-T cells to the subject. 6 One CAR-T cell. In some implementations, the method of treating GBM in a subject includes administering at least 6.0 × 10⁶ CAR-T cells to the subject. 6 One CAR-T cell. In some implementations, the method of treating GBM in a subject includes administering at least 6.5 × 10⁶ CAR-T cells to the subject. 6 One CAR-T cell. In some implementations, the method of treating GBM in a subject includes administering at least 7.0 × 10⁶ CAR-T cells to the subject. 6 One CAR-T cell. In some implementations, the method of treating GBM in a subject includes administering at least 7.5 × 10⁶ CAR-T cells to the subject. 6 One CAR-T cell. In some implementations, the method of treating GBM in a subject includes administering at least 8.0 × 10⁸ CAR-T cells to the subject. 6 One CAR-T cell. In some implementations, the method of treating GBM in a subject includes administering at least 8.5 × 10⁸ CAR-T cells to the subject. 6 One CAR-T cell. In some implementations, the method of treating GBM in a subject includes administering at least 9.0 × 10⁶ CAR-T cells to the subject. 6 One CAR-T cell.

[0067] In some embodiments of the methods provided herein, administration includes administration to the brain of the subject. In some embodiments, administration includes intracranial administration. In some embodiments, administration includes intraventricular administration. In some embodiments, administration includes administration to the cerebrospinal fluid of the subject. In some embodiments, administration includes intrathecal administration. In some embodiments, administration includes administration to a tumor in the central nervous system of the subject. In some embodiments, administration includes administration to a tumor in the central nervous system of the subject.

[0068] An important factor in administering the composition to the central nervous system is the administration rate, because the space within the central nervous system and the volume of CSF are significantly smaller than the volume of circulating blood in the periphery. In some embodiments, administering CAR-T cells includes administering CAR-T cells at a rate not exceeding 1 mL / min. In some embodiments, administering CAR-T cells includes administering CAR-T cells at a rate not exceeding 2 mL / min. In some embodiments, administering CAR-T cells includes administering CAR-T cells at a rate not exceeding 3 mL / min. In some embodiments, administering CAR-T cells includes administering CAR-T cells at a rate of 0.1 to 1 mL / min. In some embodiments, administering CAR-T cells includes administering CAR-T cells at a rate of 0.5 to 1.5 mL / min. In some embodiments, administering CAR-T cells includes administering CAR-T cells at a rate of 1 to 2 mL / min. In some embodiments, administering CAR-T cells includes administering CAR-T cells at a rate of 1.5 to 2.5 mL / min. In some embodiments, administering CAR-T cells includes administering CAR-T cells at a rate of 2 to 3 mL / min. In some embodiments, administering CAR-T cells includes administering CAR-T cells at a rate of 2.5 to 3.5 mL / min.

[0069] In some embodiments, CAR-T cells are in a solution of 5 to 15 mL. In some embodiments, CAR-T cells are in a solution of 1 to 5 mL. In some embodiments, CAR-T cells are in a solution of 2 to 7 mL. In some embodiments, CAR-T cells are in a solution of 5 to 10 mL. In some embodiments, CAR-T cells are in a solution of 7 to 12 mL. In some embodiments, CAR-T cells are in a solution of 10 to 15 mL. In some embodiments, CAR-T cells are in a solution of 12 to 17 mL. In some embodiments, CAR-T cells are in a solution of 15 to 20 mL. In some embodiments, CAR-T cells are in a solution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mL. In some embodiments, CAR-T cells are in a solution of 9 to 11 mL. In some embodiments, CAR-T cells are in a solution of 10 mL. In some embodiments, CAR-T cells are in a solution of no more than 30 mL. In some implementations, the solution contains Elliott's B solution.

[0070] CAR-T cell therapy can be administered to a subject once or multiple times, depending on the subject's response to treatment and combination therapy. In some embodiments, the method described herein includes administering a single dose of CAR-T cells to the subject. In some embodiments, the method described herein includes administering six doses of CAR-T cells to the subject. In some embodiments, the method described herein includes administering CAR-T cells to the subject weekly for six weeks. In some embodiments, the method described herein includes administering CAR-T cells to the subject at least monthly for four to six months. In some embodiments, the method described herein includes administering CAR-T cells to the subject monthly for four months. In some embodiments, the method described herein includes administering CAR-T cells to the subject every four to six weeks for 16 weeks. In some embodiments, the method includes administering CAR-T cells to the subject every three to seven weeks. In some embodiments, the method includes administering CAR-T cells to the subject every two, three, four, five, or seven weeks. In some embodiments, the method includes administering CAR-T cells to the subject every four weeks. In some embodiments, the method includes administering CAR-T cells to the subject every five weeks. In some embodiments, the method includes administering CAR-T cells to the subject every 6 weeks. In some embodiments, the method includes administering CAR-T cells to the subject every 4 weeks for up to 16 weeks. In some embodiments, the method includes administering CAR-T cells to the subject every 5 weeks for up to 16 weeks. In some embodiments, the method includes administering CAR-T cells to the subject every 6 weeks for up to 16 weeks. In some embodiments, the method includes administering CAR-T cells to the subject every 4 weeks for up to 14 to 18 weeks. In some embodiments, the method includes administering CAR-T cells to the subject every 5 weeks for up to 14 to 18 weeks. In some embodiments, the method includes administering CAR-T cells to the subject every 6 weeks for up to 14 to 18 weeks.

[0071] Administering CAR-T cells to a subject can lead to potential side effects, including inflammation and fever. Therefore, it is beneficial to consider administering medications that can improve such symptoms. As a combination therapy, the methods for treating GBM described herein may also include administering an antihistamine to the subject. In some embodiments, the antihistamine is benzodiazepine. In some embodiments, 25 mg of benzodiazepine is administered to the subject. In some embodiments, benzodiazepine is administered prior to CAR-T cell administration. In some embodiments, the methods described herein also include administering acetaminophen to the subject. In some embodiments, 650 mg of acetaminophen is administered to the subject. In some embodiments, acetaminophen is administered prior to CAR-T cell administration.

[0072] efficacy and treatment

[0073] The efficacy of activated CAR-T cells containing the CAR peptides described herein in, for example, treating the conditions described herein, or inducing responses as described herein (e.g., reduction of cancer cells), can be determined by a skilled clinician. However, treatment is considered “effective” as used herein if, following treatment according to the methods described herein, one or more of the signs or symptoms of the conditions described herein change in a beneficial manner, other clinically acceptable symptoms improve or even lessen, or the desired response is induced (e.g., at least 10%). Efficacy can be assessed, for example, by measuring biomarkers, indicators, symptoms, and / or incidence rates of the conditions treated according to the methods described herein, or any other suitable measurable parameter.

[0074] Treatment according to the methods described herein can reduce the level of disease markers or symptoms, for example, by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% or more.

[0075] Efficacy can also be measured by the absence of deterioration (i.e., cessation of disease progression) in an individual assessed for hospitalization or the need for medical intervention. Methods for measuring these indicators are known to those skilled in the art and / or described herein. Treatment includes any treatment of a disease in an individual or animal (some non-limiting examples include humans or animals) and includes: (1) suppressing the disease, for example, preventing the worsening of symptoms (e.g., pain or inflammation); or (2) reducing the severity of the disease, for example, causing symptom resolution. An effective amount for treating a disease means an amount sufficient to achieve effective treatment of the disease (as defined herein) when administered to an individual in need. The efficacy of a drug can be determined by assessing physical indicators of the symptoms or desired response. Monitoring the efficacy of administration and / or treatment by measuring any one or any combination of such parameters is entirely within the capabilities of those skilled in the art. The efficacy of a given method can be assessed in animal models of the diseases described herein. When using experimental animal models, the efficacy of treatment is indicated when a statistically significant change in a biomarker is observed.

[0076] In assessing the efficacy of treatments for GBM, numerous criteria were evaluated, including: changes in tumor volume and mass; expansion and survival of CAR-T cells in cerebrospinal fluid (CSF) and blood (e.g., measured by flow cytometry and / or vector copy number); presence of TEAM-binding cells in CSF; cytokine response in CSF and blood (e.g., inflammatory cytokines such as interleukin (IL)-6, interferon-γ (IFNγ), tumor necrosis factor α (TNFα), etc.); and overall response (complete response, partial response, disease stabilization, or progression; see [link to relevant documentation]). Figure 3 (1) No progress survival; and total survival.

[0077] Chimeric antigen receptor (CAR) T cells

[0078] In some aspects, the methods provided in this disclosure include administering CAR-T cells to the central nervous system of a subject. As used herein, “CAR-T cells” or “CAR-T” refers to T cells expressing a CAR. When expressed in T cells, a CAR has the ability to redirect T cell specificity and responsiveness to a selected target in a non-MHC-restricted manner by utilizing the antigen-binding properties of a monoclonal antibody. This non-MHC-restricted antigen recognition confers the ability of CAR-expressing T cells to recognize antigens independently of antigen processing, thereby circumventing key mechanisms of tumor escape.

[0079] As used in this article, the terms "chimeric antigen receptor," "CAR," or "CARs" refer to a modified T-cell receptor on which a ligand or antigen is specifically implanted onto T cells (e.g., naive T cells, central memory T cells, effector memory T cells, or combinations thereof). CARs are also known as artificial T-cell receptors, chimeric T-cell receptors, or chimeric immune receptors.

[0080] CARs place a chimeric antigen-binding domain on a construct comprising a transmembrane domain and an intracellular domain of a T-cell receptor molecule, the chimeric antigen-binding domain specifically binding to a target (e.g., a peptide) expressed on the cell surface targeted by a T-cell response. In some embodiments, the chimeric antigen-binding domain comprises an antigen domain of an antibody reagent that specifically binds to an antigen expressed on cells targeted by a T-cell response. In some embodiments, the chimeric antigen-binding domain comprises a ligand that specifically binds to an antigen expressed on cells targeted by a T-cell response.

[0081] In some embodiments, the CAR does not include the CD8 signaling peptide described herein. As those skilled in the art will determine, various functionally similar or equivalent components of these CARs, as well as other similar or functionally equivalent components known in the art or listed herein, may be interchanged or substituted for each other.

[0082] Antigen-binding domain

[0083] As used herein, the term "antigen-binding domain" refers to a polypeptide present outside the cell sufficient to facilitate the binding of a CAR to a target (e.g., EGFRvIII). The extracellular target-binding domain will specifically bind to its binding partner (i.e., the target). As a non-limiting example, the antigen-binding domain may comprise the antigen domain of an antibody or antibody reagent, or a ligand, which recognizes and binds to a homologous binding partner protein. In this context, a ligand is a molecule that specifically binds to a portion of a protein and / or receptor. Homologous binding partners of ligands that can be used in the methods and compositions described herein are typically present on the cell surface. Ligand: Homologous partner binding can lead to alterations in the receptor carrying the ligand, or activation of a physiological response, such as activation of a signaling pathway. In some embodiments, the ligand may be non-natural with respect to the genome. In some embodiments, the ligand has a conserved function in at least two species.

[0084] CARs can target any cell surface region. In some embodiments, the target will be a cell surface polypeptide that is differentially or preferentially expressed on the cell to which a T cell response is desired. In some embodiments, the CAR includes an antigen-binding domain that binds to antigens expressed on central nervous system cancers or tumors. In some embodiments, the CAR includes an antigen-binding domain that binds to EGFRvIII.

[0085] In some implementations, the CAR includes an antigen-binding domain that binds to EGFRvIII. EGFRvIII is a known variant of EGFR associated with glioblastoma (GBM). Approximately 30% of GBM cells are positive for EGFRvIII. EGFRvIII may contain mutations that result in the deletion of exons 2 through 7 of EGFR, as described in Gan HK FEBS J. 2013 Nov;280(21):5350-70. In some embodiments, the antigen-binding domain comprises (i) a VH domain containing three complementarity-determining regions (CDR-H1, CDR-H2, and CDR-H3), wherein CDR-H1 contains SEQ ID NO: 1, CDR-H2 contains SEQ ID NO: 2, and CDR-H3 contains SEQ ID NO: 3; and (ii) a VL domain containing three CDRs (CDR-L1, CDR-L2, and CDR-L3), wherein CDR-L1 contains SEQ ID NO: 4, CDR-L2 contains SEQ ID NO: 5, and CDR-L3 contains SEQ ID NO: 6. In some embodiments, the antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 7.

[0086] Hinges and transmembrane domains

[0087] In some embodiments, the CAR peptide further comprises a transmembrane domain, such as a hinge / transmembrane domain, which connects the antigen-binding domain to the intracellular signaling domain. In some embodiments, the binding domain of the CAR is followed by one or more "hinge domains" that function in positioning the antigen-binding domain away from the effector cell surface to achieve proper cell / cell contact, antigen binding, and activation. The CAR may include one or more hinge domains between the binding domain and the transmembrane domain (TM). The hinge domain may be derived from natural, synthetic, semi-synthetic, or recombinant sources. The hinge domain may contain an amino acid sequence of a naturally occurring immunoglobulin hinge region or a modified immunoglobulin hinge region. Exemplary hinge domains suitable for use in the CAR described herein include hinge regions derived from the extracellular regions of type 1 membrane proteins (e.g., CD8 (e.g., CD8α), CD4, CD28, 4-1BB, and CD7), which may be wild-type hinge regions from these molecules or may be modified hinge regions. In some embodiments, the CAR contains a polynucleotide encoding a CD8α hinge / transmembrane domain. In some embodiments, the CAR contains a polynucleotide encoding a 41BB intracellular domain.

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

[0089] As used herein, a “transmembrane domain” (TM domain) refers to a portion of the CAR that fuses an extracellular binding portion (via a hinge domain in some embodiments) with an intracellular portion (e.g., a co-stimulatory domain and an intracellular signaling domain) and anchors the CAR to the plasma membrane of an immune effector cell. The transmembrane domain is typically a hydrophobic region of the CAR that crosses the cytoplasmic membrane. The TM domain can be a transmembrane region or fragment of a transmembrane protein (e.g., type I transmembrane protein or other transmembrane proteins), an artificial hydrophobic sequence, or a combination thereof. While specific examples are provided and used herein, other transmembrane domains will be apparent to those skilled in the art and can be used in combination with alternative embodiments of the present technology. The selected transmembrane region or fragment thereof preferably does not interfere with the intended function of the CAR.

[0090] The term "fragment of" as used for transmembrane domains of proteins or peptides refers to the portion of the transmembrane domain sufficient to anchor or attach the protein to the cell surface.

[0091] In some embodiments, the transmembrane domain or fragment thereof of the CAR described herein includes a transmembrane domain selected from the following transmembrane domains: α, β, or ζ chains of T cell receptors; CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154; KIRDS2, OX40, CD2, CD27; LFA-1 (CD11a, CD18); ICOS (CD278); 4-1BB (CD137); 4-1BBL; GITR; CD40; BAFFR; HVEM (LIGHTR); SLAMF7; NKp80 (KLRFI); CD160; CD19; IL2Rβ; IL2Rγ; IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITG AX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD15 0, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D and / or NKG2C.

[0092] As used herein, "hinge / transmembrane domain" refers to a domain that contains both a hinge domain and a transmembrane domain. For example, the hinge / transmembrane domain may be derived from the hinge / transmembrane domain of CD8, CD28, CD7, or 4-1BB. In some embodiments, the hinge / transmembrane domain of a CAR, or a fragment thereof, is derived from or contains a hinge / transmembrane domain of CD8 (e.g., SEQ ID NO: 24, or a variant thereof). CD8 is an antigen preferentially present on the cell surface of cytotoxic T lymphocytes. CD8 mediates cell-cell interactions within the immune system and acts as a T cell co-receptor. CD8 consists of an α chain (CD8α or CD8a) and a β chain (CD813 or CD8b). CD8a sequences are known in many species, such as the human CD8a (NCBI gene ID: 925) polypeptide (e.g., NCBI Ref Seq NP 001139345.1) and mRNA (e.g., NCBI Ref Seq NM_ 000002.12). CD8 can refer to human CD8, including its naturally occurring variants, molecules, and alleles. In some implementations of either aspect, such as in veterinary applications, CD8 can refer to, for example, the CD8 of dogs, cats, cattle, horses, pigs, etc.

[0093] For such species, homologs and / or orthologs of human CD8 are readily identified by those skilled in the art, for example, by using the NCBI ortholog search function or by searching for sequences similar to a reference CD8 sequence in the available sequence data for a given species.

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

[0095] Co-stimulatory domain

[0096] Each CAR described herein optionally includes an intracellular domain or co-stimulatory domain of one or more co-stimulatory molecules. As used herein, the term "co-stimulatory domain" refers to the intracellular signaling domain of a co-stimulatory molecule. A co-stimulatory molecule is a cell surface molecule other than an antigen receptor or Fc receptor that provides a secondary signal required for the efficient activation and function of T lymphocytes upon binding to an antigen. A co-stimulatory domain may be, for example, a 4-1BB, CD27, CD28, or OX40 co-stimulatory domain. In some embodiments, a 4-1BB co-stimulatory domain may be used (see, for example, hereinafter and SEQ ID NO: 25, or a variant thereof). In some embodiments, a CD28 co-stimulatory domain may be used (see, for example, hereinafter and SEQ ID NO: 26, or a variant thereof). Other illustrative examples of such costimulatory domains include CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2C, SLP76, TRIM, and ZAP70. In some embodiments, the intracellular domain is the intracellular domain of 4-1BB. 4-1BB (CD137; TNFRS9) is an activation-inducing costimulatory molecule and an important regulator of the immune response.

[0097] 4-1BB is a membrane receptor protein, also known as CD137, and is a member of the tumor necrosis factor (TNF) receptor superfamily. 4-1BB is expressed on activated T lymphocytes. Sequences of 4-1BB are known in many species, such as human 4-1BB, also known as TNFRSF9 (NCBI Gene 25 ID: 3604) and its mRNA (NCBI Reference Sequence: NM_001561.5). 4-1BB can refer to human 4-1BB, including its naturally occurring variants, molecules, and alleles. In some embodiments of either aspect, such as in veterinary applications, 4-1BB can refer to, for example, 4-1BB in dogs, cats, cattle, horses, pigs, etc. Homologs and / or orthologs of human 4-1BB for such species are readily identifiable by those skilled in the art, for example, by using the NCBI ortholog search function or by searching available sequence data for a given species for sequences similar to a reference 4-1BB sequence.

[0098] In some embodiments, the co-stimulatory domain comprises a 4-1BB co-stimulatory domain. In some embodiments, the 4-1BB co-stimulatory domain corresponds to an amino acid sequence selected from SEQ ID NO: 25. In some embodiments, the co-stimulatory domain comprises a CD28 co-stimulatory domain. In some embodiments, the CD28 co-stimulatory domain corresponds to an amino acid sequence selected from SEQ ID NO: 26.

[0099] Intracellular signal transduction domains

[0100] In some implementations, the CAR contains a polynucleotide encoding the CD3zeta (CD3ζ) intracellular signaling domain (e.g., SEQ ID NO: 27 or a variant thereof).

[0101] The properties of the intracellular signal transduction domain of a CAR can vary as are known in the art and disclosed herein, but when the chimeric target / antigen binding domain binds to a target / antigen on the surface of a target cell, the chimeric target / antigen binding domain sensitizes the receptor to signal transduction activation.

[0102] Regarding intracellular signal transduction domains, so-called "first-generation" CARs include those that provide only CD3ζ signaling upon antigen binding. So-called "second-generation" CARs include those that provide both co-stimulatory (e.g., CD28 or CD137) and activating (CD3ζ) domains, and so-called "third-generation" CARs include those that provide multiple co-stimulatory (e.g., CD28 and CD137) domains and activating (signal transduction) domains (e.g., CD3ζ). In various implementations, CARs with high affinity or affinity for the target / antigen are selected—for example, antibody-derived target or antigen-binding domains typically have higher affinity and / or affinity for the target antigen compared to naturally occurring T-cell receptors. This characteristic, combined with the high specificity of the antibodies available for selection, provides highly specific T-cell targeting of CAR-T cells.

[0103] The CARs described herein include intracellular signal transduction domains. An “intracellular signal transduction domain” refers to a portion of the CAR polypeptide that participates in transducing information about an effective CAR bound to a target antigen into the interior of immune effector cells to elicit effector cell functions, such as activation, cytokine production, proliferation, and cytotoxic activity, including the release of cytotoxic factors to the CAR-bound target cell, or to elicit other cellular responses following the binding of the antigen to the extracellular CAR domain. In some embodiments, the intracellular signal transduction domain is derived from CD3-zeta (CD3ζ) (see, for example, below). Further non-limiting examples of intracellular signal transduction domains comprising immunoreceptor tyrosine-based activation motifs (ITAMs) that are particularly useful in this art include those derived from TCR-ζ, FcR-γ, FcR-β, CD3-γ, CD3-θ, CD3-σ, CD3-η, CD3-ε, CD3-ζ, CD22, CD79a, CD79b, and CD66d.

[0104] CD3 is a T cell co-receptor that promotes T lymphocyte activation when it binds to an appropriate co-stimulus (e.g., a co-stimulatory molecule). The CD3 complex consists of four distinct chains; mammalian CD3 consists of one CD3-γ chain, one CD3-δ chain, and two CD3-ε chains.

[0105] These chains associate with molecules called T-cell receptors (TCRs) and CD3ζ to generate activation signals in T lymphocytes. The complete TCR complex consists of TCR, CD3ζ, and the complete CD3 complex.

[0106] In some embodiments of any aspect, the CAR peptide described herein comprises an intracellular signaling domain containing an immune receptor tyrosine-based activation motif or ITAM derived from CD3ζ, wherein CD3ζ comprises variants of CD3ζ, such as ITAM-mutated CD3ζ, CD3-η, or CD3-θ. In some embodiments of any aspect, the ITAM comprises the three motifs of the ITAM of CD3ζ (ITAM3). In some embodiments of any aspect, the three motifs of the ITAM of CD3-ζ are not mutated and therefore comprise natural or wild-type sequences. In some embodiments, the CD3ζ sequence comprises a CD3ζ sequence listed in the sequences provided herein, such as the CD3ζ sequence of SEQ ID NO: 27 or a variant thereof.

[0107] As can be determined by those skilled in the art, the individual CARs and other construct components described herein can be used together and swapped in and out of the various constructs described herein. Each of these components may contain any corresponding sequence or variant thereof listed herein or may consist of any corresponding sequence or variant thereof listed herein.

[0108] Further descriptions of CAR and CAR-T cells can be found in Maus et al., Blood 123:2624-2635, 2014; Reardon et al., Neuro-Oncology 16:1441-1458, 2014; Hoyos et al., Haematologica 97:1622, 2012; Byrd et al., J. Clin. Oncol. 32:3039-3047, 2014; Maher et al., Cancer Res69:4559-4562, 2009; and Tamada et al., Clin. Cancer Res. 18:6436-6445, 2012; each of which is incorporated herein by reference in its entirety.

[0109] signal peptide

[0110] As used herein, "signal peptide" or "signal sequence" refers to a peptide at the N-terminus of a newly synthesized protein that directs the nascent protein into the endoplasmic reticulum. In some embodiments, the CAR polypeptide described herein comprises a signal peptide. The signal peptide may be derived from any protein having an extracellular domain or a secreted protein. The CAR polypeptide described herein may comprise any signal peptide known in the art. In some embodiments, the CAR polypeptide comprises a CD8 signal peptide, such as the CD8 signal peptide corresponding to the amino acid sequence of SEQ ID NO: 28. In some embodiments, the CAR polypeptide described herein may optionally exclude one of the signal peptides described herein, such as the CD8 signal peptide of SEQ ID NO: 28 or the IgK signal peptide of SEQ ID NO: 29.

[0111] Joint structural domain

[0112] In some embodiments, the CAR also includes a linker domain. As used herein, "linker domain" refers to an oligopeptide or polypeptide region of about 2 to 100 amino acids in length that links together any domains / regions of the CAR described herein. In some embodiments, the linker may comprise or be composed of flexible residues (e.g., glycine and serine) such that adjacent protein domains are free to move relative to each other. Linker sequences usable in this invention can be 2 to 100 amino acids in length, 5 to 50 amino acids, 10 to 15 amino acids, 15 to 20 amino acids, or 18 to 20 amino acids, and include any suitable linker known in the art. For example, linker sequences usable in this invention include, but are not limited to, glycine / serine linkers, such as... and Gly4Ser (G4S) connector, for example and ; The linker sequences, such as those described by Whitlow et al., Protein Eng. 6(8):989-95, 1993, are incorporated herein by reference in their entirety; Connector sequences, such as those described in Andris-Widhopf et al., ColdSpring Harb. Protoc. 2011 (9), 2011, are incorporated herein by reference in their entirety; and connector sequences with additional functionality, such as epitope tags or coding sequences containing Cre-Lox recombination sites, such as those described in Sblattero et al., Nat. Biotechnol. 18(1):75-80, 2000, are incorporated herein by reference in their entirety. Longer connectors may be used when it is desirable to ensure that two adjacent domains do not interfere with each other spatially.

[0113] Furthermore, the connector may be cuttable or non-cuttable. Examples of cuttable connectors include 2A connectors (e.g., P2A (SEQ ID NO: 35) and T2A (SEQ ID NO: 36)), 2A-type connectors, or their functional equivalents and combinations thereof.

[0114] In several instances, adapters having sequences listed herein or variants thereof are used. It should be understood that the identification of a particular adapter in a construct at a particular location does not imply that only that adapter is usable therein. Rather, as those skilled in the art can determine, different adapter sequences (e.g., P2A, E2A, F2A, and T2A) can be interchanged (e.g., in the context of the constructs of this invention). In some embodiments, the adapter region is T2A derived from the *Thosea asigna* virus. Non-limiting examples of adapters that can be used in this technology include T2A, P2A, E2A, BmCPV2A, and BmlFV2A. These adapters can be used, for example, in the case of polyproteins, such as those described below. For example, they can be used to separate a polyprotein CAR component from a polyprotein therapeutic component (e.g., an antibody, such as scFv, a single-domain antibody (e.g., a camelid antibody), or a bispecific antibody (e.g., TEAM)) (see below).

[0115] In some implementations, the CAR-T cells described herein are generated from autologous T cells. Autologous T cells are derived from the recipient and, compared to allogeneic T cells, have the advantage of minimizing or eliminating the risk of immune rejection or graft-versus-host disease.

[0116] In some embodiments, the polynucleotide comprises a nucleic acid encoding an amino acid sequence containing an epidermal growth factor receptor (EGFR)-binding chimeric antigen receptor (EGFRvIII CAR), said EGFR-binding chimeric antigen receptor comprising an antigen-binding domain:

[0117] The complementary determination region (CDR) 1 of the variable heavy chain (VH) in SEQ ID NO: 1, the VH CDR 2 of SEQ ID NO: 2, the VH CDR 3 of SEQ ID NO: 3, the variable light chain (VL) CDR 1 of SEQ ID NO: 4, the VL CDR 2 of SEQ ID NO: 5, and the VL CDR 3 of SEQ ID NO: 6.

[0118] In some embodiments, this disclosure describes a polypeptide comprising amino acids of an epidermal growth factor receptor (EGFR)-binding chimeric antigen receptor (EGFRvIII CAR), the EGFR-binding chimeric antigen receptor comprising an antigen-binding domain containing: a variable heavy chain (VH) complementarity-determining region (CDR) 1 of SEQ ID NO: 1, a VH CDR 2 of SEQ ID NO: 2, a VH CDR 3 of SEQ ID NO: 3, a variable light chain (VL) CDR 1 of SEQ ID NO: 4, a VL CDR 2 of SEQ ID NO: 5, and a VL CDR 3 of SEQ ID NO: 6.

[0119] T-cell conjugation antibody molecules (TEAM)

[0120] In some embodiments, the CAR-T cells described herein express T cell conjugating molecules (TEAMs) (also referred to in the literature as bispecific T cell conjugates or BiTEs™) (“CAR-TEAM cells”). “T cell conjugating molecule,” “TEAM antibody construct,” or “TEAM” means a polypeptide, each comprising tandemly linked single-chain variable fragments (scFvs). Optionally, the scFvs are linked via a linker (e.g., a glycine-rich linker). One scFv of the TEAM binds to a T cell receptor (TCR) (e.g., to the CD3ε subunit), and the other binds to a target antigen (e.g., a tumor antigen). Such a molecule can target T cells by binding to both the T cell antigen (e.g., by binding to CD3) and the target antigen (e.g., a tumor antigen). The TEAM can be used to enhance T cell responses, for example, in the tumor microenvironment. The two components of the TEAM can also be linked in either direction, for example, with the anti-CD3 component located at the N-terminus of the anti-target antigen component, or vice versa. The anti-CD3 component or the anti-target antigen component of the TEAM may contain any antibody reagent described herein.

[0121] In some embodiments, the CAR-TEAM cells described herein secrete a TEAM. In some embodiments, the TEAM contains a secretion tag (e.g., an IgK signal peptide). The TEAM can act paracrinely, for example, by stimulating the CAR-TEAM cells themselves or by redirecting nonspecific bystander T cells to the tumor, thereby enhancing the antitumor effect of CAR-T cell immunotherapy. CAR-TEAM cell-mediated TEAM secretion can reduce the risk of undesirable TEAM activity in systemic tissues by directing TEAM secretion to the tumor microenvironment. Exemplary TEAM constructs are provided below; however, TEAMs other than those described herein may also be used in the CAR T cells and methods of this disclosure.

[0122] In some embodiments, the anti-CD3 scFv of any TEAM described herein may be arranged in a VH-VL orientation or a VL-VH orientation. In some embodiments, the anti-CD3 scFv comprises (i) a VH domain containing three complementarity-determining regions (CDR-H1, CDR-H2, and CDR-H3), wherein CDR-H1 contains SEQ ID NO: 16, CDR-H2 contains SEQ ID NO: 17, and CDR-H3 contains SEQ ID NO: 18; and (ii) a VL domain containing three CDRs (CDR-L1, CDR-L2, and CDR-L3), wherein CDR-L1 contains SEQ ID NO: 19, CDR-L2 contains SEQ ID NO: 20, and CDR-L3 contains SEQ ID NO: 21. In some embodiments, the anti-CD3 scFv comprises the amino acid sequence of SEQ ID NO: 22 or an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with the amino acid sequence of SEQ ID NO: 22.

[0123] In some embodiments, the anti-EGFR scFv of any TEAM described herein may be arranged in a VH-VL orientation or a VL-VH orientation. In some embodiments, the anti-EGFR scFv comprises (i) a VH domain containing three complementarity-determining regions (CDR-H1, CDR-H2, and CDR-H3), wherein CDR-H1 contains SEQ ID NO: 9, CDR-H2 contains SEQ ID NO: 10, and CDR-H3 contains SEQ ID NO: 11; and (ii) a VL domain containing three CDRs (CDR-L1, CDR-L2, and CDR-L3), wherein CDR-L1 contains SEQ ID NO: 12, CDR-L2 contains SEQ ID NO: 13, and CDR-L3 contains SEQ ID NO: 14. In some embodiments, the anti-EGFR scFv comprises the amino acid sequence of SEQ ID NO: 15 or an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with the amino acid sequence of SEQ ID NO: 15. In some embodiments, the anti-EGFR / anti-CD3team comprises the amino acid sequence of SEQ ID NO: 23 or an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with the amino acid sequence of SEQ ID NO: 23.

[0124] Shortened CD19

[0125] "Truncation of CD19 (tCD19)" refers to a fragment of the B lymphocyte antigen CD19 protein (e.g., Uniprot IDP15391 as of October 10, 2023). The "fragment" of the protein (e.g., CD19) is missing at least one amino acid relative to the wild-type protein. In some embodiments, truncated CD19 does not contain the n-terminal fragment of CD19. In some embodiments, truncated CD19 does not contain the c-terminal fragment of CD19. In some embodiments, truncated CD19 contains an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with SEQ ID NO: 38. In some embodiments, truncated CD19 contains the amino acid sequence of SEQ ID NO: 38. In some embodiments, truncated CD19 consists of an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with SEQ ID NO: 38. In some embodiments, the truncated CD19 consists of the amino acid sequence of SEQ ID NO: 38. In some embodiments, the truncated CD19 contains the amino acid sequence of SEQ ID NO: 38 and does not contain the rest of the CD19 segment.

[0126] In some implementations, truncated CD19 can be used to detect CAR-T cells. For example, CAR-T cells may contain an amino acid sequence encoding truncated CD19; truncated CD19 may be expressed on the surface of CAR-T cells, and truncated CD19 may be detected using antibodies specific to truncated CD19.

[0127] CAR-TEAM-tCD19

[0128] In some aspects, this disclosure describes a polynucleotide comprising a nucleic acid encoding an EGFR-VIII binding CAR, an EGFR-binding and CD3-binding TEAM, and a truncated CD19. In some embodiments, the EGFR-VIII binding CAR comprises, from the n-terminus to the c-terminus, a CD8 leader sequence, an EGFRvIII antigen-binding domain (e.g., EGFRvIII scFv), a CD8 hinge, a CD8 transmembrane domain, a 4-1BB co-stimulatory domain, a CD3-ζ intracellular signal transduction domain, a P2A peptide, an IgK leader, an anti-EGFR antibody (e.g., cetuximab scFv), an anti-CD3 antibody (e.g., scFv), a T2A peptide, and a truncated CD19 (e.g., the truncated CD19 of SEQ ID NO: 38).

[0129] In some aspects, this disclosure describes polynucleotides comprising nucleic acids encoding EGFR-VIII-binding CARs, EGFR-binding and CD3-binding TEAMs, and truncated CD19. In some embodiments, the polynucleotide comprises a nucleic acid encoding an amino acid sequence from the n-terminus to the c-terminus encoding: a CD8 leader sequence, an EGFRvIII antigen-binding domain (e.g., EGFRvIII scFv), a CD8 hinge, a CD8 transmembrane domain, a 4-1BB co-stimulatory domain, a CD3-ζ intracellular signal transduction domain, a P2A peptide, an IgK leader, an anti-EGFR antibody (e.g., cetuximab scFv), an anti-CD3 antibody (e.g., scFv), a T2A peptide, and truncated CD19 (e.g., truncated CD19 of SEQ ID NO: 38).

[0130] In some embodiments, this disclosure describes a polypeptide encoding the following from the n-terminus to the c-terminus: a CD8 leader sequence, an EGFRvIII antigen-binding domain (e.g., EGFRvIII scFv), a CD8 hinge, a CD8 transmembrane domain, a 4-1BB co-stimulatory domain, a CD3-ζ intracellular signal transduction domain, a P2A peptide, an IgK leader, an anti-EGFR antibody (e.g., cetuximab scFv), an anti-CD3 antibody (e.g., scFv), a T2A peptide, and a truncated CD19 (e.g., the truncated CD19 of SEQ ID NO: 38).

[0131] In some embodiments, this disclosure describes a polynucleotide comprising a nucleic acid encoding an amino acid sequence comprising: (i) an epidermal growth factor receptor (EGFR) binding chimeric antigen receptor (EGFRvIII CAR) comprising an antigen-binding domain containing: a variable heavy chain (VH) complementarity-determining region (CDR) 1 of SEQ ID NO: 1, a VH CDR2 of SEQ ID NO: 2, a VH CDR3 of SEQ ID NO: 3, a variable light chain (VL) CDR1 of SEQ ID NO: 4, a VL CDR2 of SEQ ID NO: 5, and a VL CDR3 of SEQ ID NO: 6; and (ii) a T cell conjugating antibody molecule (TEAM) comprising: (a) an EGFR binding domain comprising a variable heavy chain (VH) complementarity-determining region (CDR) 1 of SEQ ID NO: 9, a VH CDR2 of SEQ ID NO: 10, a VH CDR3 of SEQ ID NO: 11, and a VH CDR3 of SEQ ID NO: 9; (a) SEQ ID NO: 12, VL CDR1, SEQ ID NO: 13, and VL CDR3 of SEQ ID NO: 14; (b) CD3 binding domain comprising SEQ ID NO: 16, VH complementarity-determining region (CDR) 1, SEQ ID NO: 17, VH CDR2, SEQ ID NO: 18, SEQ ID NO: 19, VL CDR1, SEQ ID NO: 20, and VL CDR3 of SEQ ID NO: 21; and (iii) truncated CD19 domain (tCD19) comprising an amino acid sequence having at least 95% identity with SEQ ID NO: 38.

[0132] In some embodiments, this disclosure describes a polynucleotide comprising a nucleic acid encoding an amino acid sequence comprising: (i) an epidermal growth factor receptor (EGFR) binding chimeric antigen receptor (EGFRvIII CAR) comprising an antigen-binding domain containing: a variable heavy chain (VH) complementarity-determining region (CDR) 1 of SEQ ID NO: 1, a VH CDR2 of SEQ ID NO: 2, a VH CDR3 of SEQ ID NO: 3, a variable light chain (VL) CDR1 of SEQ ID NO: 4, a VL CDR2 of SEQ ID NO: 5, and a VL CDR3 of SEQ ID NO: 6; and (ii) a T cell conjugating antibody molecule (TEAM) comprising: (a) an EGFR binding domain comprising a variable heavy chain (VH) complementarity-determining region (CDR) 1 of SEQ ID NO: 9, a VH CDR2 of SEQ ID NO: 10, a VH CDR3 of SEQ ID NO: 11, and a VH CDR3 of SEQ ID NO: 9; (a) SEQ ID NO: 12, VL CDR1, SEQ ID NO: 13, and VL CDR3 of SEQ ID NO: 14; (b) CD3 binding domain comprising SEQ ID NO: 16, VH complementarity determinant region (CDR) 1, SEQ ID NO: 17, VH CDR2, SEQ ID NO: 18, SEQ ID NO: 19, VL CDR1, SEQ ID NO: 20, and VL CDR3 of SEQ ID NO: 21; and (iii) truncated CD19 domain (tCD19) comprising the amino acid sequence of SEQ ID NO: 38.

[0133] In some embodiments, this disclosure describes a polypeptide comprising: (i) an epidermal growth factor receptor (EGFR) binding chimeric antigen receptor (EGFRvIII CAR) comprising an antigen-binding domain including: a variable heavy chain (VH) complementarity-determining region (CDR) 1 of SEQ ID NO: 1, a VH CDR2 of SEQ ID NO: 2, a VHCDR3 of SEQ ID NO: 3, a variable light chain (VL) CDR1 of SEQ ID NO: 4, a VL CDR2 of SEQ ID NO: 5, and a VLCDR3 of SEQ ID NO: 6; and (ii) a T cell conjugating antibody molecule (TEAM) comprising: (a) an EGFR binding domain including a variable heavy chain (VH) complementarity-determining region (CDR) 1 of SEQ ID NO: 9, a VH CDR2 of SEQ ID NO: 10, a VHCDR3 of SEQ ID NO: 11, a variable light chain (VL) CDR1 of SEQ ID NO: 12, and a VL CDR3 of SEQ ID NO: 13. (a) CDR2 and VL CDR3 of SEQ ID NO: 14; (b) CD3 binding domain comprising the variable heavy chain (VH) complementarity-determining region (CDR) 1 of SEQ ID NO: 16, VH CDR2 of SEQ ID NO: 17, VH CDR3 of SEQ ID NO: 18, variable light chain (VL) CDR1 of SEQ ID NO: 19, VL CDR2 of SEQ ID NO: 20 and VL CDR3 of SEQ ID NO: 21; and (iii) truncated CD19 domain (tCD19) comprising an amino acid sequence having at least 95% identity with SEQ ID NO: 38.

[0134] In some embodiments, this disclosure describes a polypeptide comprising: (i) an epidermal growth factor receptor (EGFR) binding chimeric antigen receptor (EGFRvIII CAR) comprising an antigen-binding domain including: a variable heavy chain (VH) complementarity-determining region (CDR) 1 of SEQ ID NO: 1, a VH CDR2 of SEQ ID NO: 2, a VHCDR3 of SEQ ID NO: 3, a variable light chain (VL) CDR1 of SEQ ID NO: 4, a VL CDR2 of SEQ ID NO: 5, and a VLCDR3 of SEQ ID NO: 6; and (ii) a T cell conjugating antibody molecule (TEAM) comprising: (a) an EGFR binding domain including a variable heavy chain (VH) complementarity-determining region (CDR) 1 of SEQ ID NO: 9, a VH CDR2 of SEQ ID NO: 10, a VHCDR3 of SEQ ID NO: 11, a variable light chain (VL) CDR1 of SEQ ID NO: 12, and a VL CDR3 of SEQ ID NO: 13. (a) CDR2 and VL CDR3 of SEQ ID NO: 14; (b) CD3 binding domain comprising the variable heavy chain (VH) complementarity-determining region (CDR) 1 of SEQ ID NO: 16, VH CDR2 of SEQ ID NO: 17, VH CDR3 of SEQ ID NO: 18, variable light chain (VL) CDR1 of SEQ ID NO: 19, VL CDR2 of SEQ ID NO: 20 and VL CDR3 of SEQ ID NO: 21; and (iii) truncated CD19 domain (tCD19) comprising the amino acid sequence of SEQ ID NO: 38.

[0135] In some aspects, this application discloses vectors (e.g., expression vectors) comprising the polynucleotides described herein. “Vector” refers to a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells. Vectors used herein can be viral or non-viral. The term “vector” encompasses any genetic element capable of replicating and transferring a gene sequence into a cell when associated with a suitable control element. Vectors may include, but are not limited to, cloning vectors, expression vectors, plasmids, bacteriophages, transposons, kinases, artificial chromosomes, viruses, virions, etc. In some embodiments, the vector may comprise the polynucleotides described herein (e.g., polynucleotides comprising polynucleotide sequences encoding VEGF-binding proteins and / or CARs).

[0136] As used herein, the term "expression vector" can refer to a vector that directs the expression of RNA or polypeptides by a sequence linked to a transcriptional regulatory sequence on the vector. The expressed sequence is typically, but not necessarily, heterologous to the cell. Expression vectors may contain additional elements; for example, they may have two replication systems, allowing them to be maintained in two organisms, such as for expression in human cells and for cloning and amplification in a prokaryotic host. The term "expression" refers to the cellular processes involved in the production of RNA and proteins, and, where appropriate, the secretion of proteins, including, where applicable, transcription, transcript processing, translation, and protein folding, modification, and processing. "Expression products" include RNA transcribed from a gene, and polypeptides obtained by translating mRNA transcribed from a gene.

[0137] As used herein, the term "viral vector" can refer to a nucleic acid vector construct that contains at least one element of viral origin and has the ability to be packaged into viral vector particles. Viral vectors may contain nucleic acids encoding polypeptides as described herein in place of non-essential viral genes. Vectors and / or particles can be used for the purpose of transferring nucleic acids into cells in vitro or in vivo. Many forms of viral vectors are known in the art.

[0138] A “recombinant vector” can be a vector containing a heterologous nucleic acid sequence or “transgenic” capable of being expressed in vivo. It should be understood that in some embodiments, the vectors described herein can be combined with other suitable compositions and treatments. In some embodiments, the vector is episomal. The use of suitable episomal vectors provides a means of maintaining the target nucleotide in the subject within high copy numbers of extrachromosomal DNA, thereby eliminating the potential effects of chromosomal integration.

[0139] In some aspects, this disclosure describes cells comprising the CAR-TEAM-tCD19 polynucleotide or polypeptide described herein. In some embodiments, the cells are eukaryotic cells. In some embodiments, the cells are prokaryotic cells. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are immune cells. In some embodiments, the cells are T cells. In some embodiments, the cells are autologous T cells. In some embodiments, the cells are allogeneic T cells. In some embodiments, tCD19 is expressed on the cell surface (e.g., tCD19 is bound to the cell membrane, and at least a portion of tCD19 is on the outer surface of the cell membrane).

[0140] Detection of CAR-T cells expressing truncated CD19

[0141] In some aspects, this disclosure describes a method for detecting cells expressing truncated CD19 (e.g., CAR-T cells described herein), the method comprising contacting the cells with a tCD19 binder (e.g., a tCD19-binding protein); and detecting the tCD19 binder. A tCD19 binder is a molecule that selectively binds to CD19 or tCD19. This molecule can be a protein, peptide, small molecule, or polynucleotide that binds to CD19 or tCD19. In some embodiments, the tCD19 binder comprises a tCD19-binding protein. In some embodiments, the tCD19-binding protein is an antibody that binds to tCD19. In some embodiments, the antibody specifically binds to truncated CD19 rather than full-length CD19. In some embodiments, the antibody is an antibody fragment (e.g., an antigen-binding fragment (Fab), Fab', or F(ab')2, a variable fragment (Fv), or a single-chain variable fragment (scFv)). In some embodiments, the antibody is conjugated to a detectable moiety (e.g., a fluorescent molecule or a radioactive molecule). In some embodiments, the fluorescent molecule is a fluorophore. In some embodiments, the fluorophore is selected from DyLight, Alexa Fluor, Pacific Blue, Alexa Fluor, FITC, DyLight, PE, APC, Alexa Fluor, DyLight, PerCP, and Alexa Fluor. In some embodiments, the tCD19 antibody is CD19-PE, which is commercially available from several suppliers, including INVITROGEN (catalog number 12-0199-42, as of October 9, 2023). In some implementations, the radioactive molecule is selected from actinium-225 (225Ac), astatine-211 (211At), bismuth-213 (213Bi), indium-111 (111In), iodine-123 (123I), iodine-124 (124I), iodine-131 (131I), lead-212 (212Pb), lutetium-177 (177Lu), technetium-99m (99mTc), copper-64 (64Cu), gallium-68 (68Ga), yttrium-86 (86Y), yttrium-90 (90Y), and zirconium-89 (89Zr).

[0142] In some embodiments, the method includes detecting CAR-T cells expressing tCD19 by contacting CAR-T cells with a fluorophore-conjugated tCD19 binding protein (e.g., an anti-tCD19 antibody conjugated to a fluorophore), and detecting CAR-T cells with a fluorophore using flow cytometry. In some embodiments, the tCD19 antibody is a CD19 antibody. In some embodiments, the tCD19 antibody is CD19-PE, which is commercially available from several vendors, including INVITROGEN (catalog number 12-0199-42, as of October 9, 2023). In some embodiments, the method includes using flow cytometry to count the number of fluorophore-positive CAR-T cells. In some embodiments, the method includes using microscopy (e.g., fluorescence microscopy) to count the number of fluorophore-positive CAR-T cells.

[0143] In some embodiments, the method includes using Western blotting to detect CAR-T cells expressing tCD19. In some embodiments, the method includes, as part of Western blotting, detecting CAR-T cells expressing tCD19 by contacting CAR-T cells with a tCD19-binding protein containing a fluorescent molecule (e.g., CD19-PE) or a tCD19-binding protein containing a radioactive molecule, and subsequently detecting the amount of fluorescence or radioactivity. In some embodiments, the method includes detecting CAR-T cells expressing tCD19 by contacting CAR-T cells with a tCD19-binding protein and subsequently with a secondary antibody binding to the tCD19-binding protein, wherein the secondary antibody contains a detectable portion (e.g., which is fluorescent or radioactive).

[0144] In some implementations, the CAR-T cells described herein (e.g., CAR-T cells containing EGFRvIII-binding CAR, EGFR-binding CD3-binding TEAM, and truncated CD19 (tCD19) domains) can be used to treat glioblastoma multiforme (GBM) in humans.

[0145] Applying CAR / TEAM building blocks

[0146] In some embodiments, the CAR and TEAM are encoded on the same polypeptide (e.g., an EGFRvIII-binding CAR / EGFR-CD3 TEAM construct, also known as a CAR-TEAM construct). In some embodiments, the CAR and TEAM are encoded on the same polypeptide and separated by a linker domain as described above. In some embodiments, the linker domain comprises the amino acid sequence of SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, or SEQ ID NO:34. In some embodiments, the linker domain is non-cleavable. In some embodiments, the linker domain comprises the amino acid sequence of SEQ ID NO:35. In some embodiments, the linker domain comprises the amino acid sequence of SEQ ID NO:36.

[0147] In some implementations, the CAR is an anti-EGFRvIII binding CAR, and the TEAM is an EGFR-CD3 TEAM. In some implementations, the EGFRvIII binding CAR includes an EGFRvIII antigen-binding domain, a CD3ζ signaling domain, a CD8 hinge / transmembrane domain, and a 4-1BB co-stimulatory domain.

[0148] In some embodiments, the EGFRvIII-binding CAR and EGFR-CD3 TEAM peptide (also known as the CAR-TEAM peptide) comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with SEQ ID NO: 37. In some embodiments, the EGFRvIII-binding CAR and EGFR-CD3 TEAM peptide comprises the amino acid sequence SEQ ID NO: 37. In some embodiments, the EGFRvIII-binding CAR and EGFR-CD3 TEAM peptide is composed of the amino acid sequence SEQ ID NO: 37. In some embodiments, this disclosure provides polynucleotides encoding an EGFRvIII-binding CAR and an EGFR-CD3 TEAM, wherein the EGFRvIII-binding CAR has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with SEQ ID NO: 8, and the EGFR-CD3 TEAM has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with SEQ ID NO: 23. In some embodiments, the polynucleotide encodes an EGFRvIII-binding CAR comprising the amino acid sequence of SEQ ID NO: 8 and an EGFR-CD3 TEAM comprising the amino acid sequence of SEQ ID NO: 23. In some embodiments, the polynucleotide encodes an EGFRvIII CAR and an EGFR-CD3 TEAM, wherein the EGFRvIII CAR has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with SEQ ID NO: 8, and the EGFR-CD3 TEAM has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with SEQ ID NO: 23.In some embodiments, the polynucleotide encodes an EGFRvIII CAR containing the amino acid sequence of SEQ ID NO: 8 and an EGFR-CD3 TEAM construct containing the amino acid sequence of SEQ ID NO: 23.

[0149] In some embodiments, this disclosure describes a method for treating a subject with glioblastoma multiforme, the method comprising administering 6.0 × 10⁶ ppm of the drug to the subject's central nervous system. 6 Up to 1.4×10 7 Each CAR-T cell contains a polynucleotide encoding EGFRvIII CAR and EGFR-CD3 TEAM. In some embodiments, the CAR-T cell contains a polynucleotide encoding the amino acid sequence of SEQ ID NO: 37.

[0150] In some embodiments, the method described herein includes administering 7.0 × 10⁻⁶ ppm to the central nervous system of the subject. 6 Up to 1.3×10 7 Each CAR-T cell contains a polynucleotide encoding the amino acid sequence SEQ ID NO: 37.

[0151] In some embodiments, the method described herein includes administering 8.0 × 10⁸ ppm to the central nervous system of the subject. 6 Up to 1.2×10 7 Each CAR-T cell contains a polynucleotide encoding the amino acid sequence SEQ ID NO: 37.

[0152] In some implementations, the method described herein includes administering 9.0 × 10⁻⁶ ppm to the central nervous system of the subject. 6 Up to 1.1×10 7 Each CAR-T cell contains a polynucleotide encoding the amino acid sequence SEQ ID NO: 37.

[0153] In some embodiments, the method described herein includes administering 1.0 × 10⁻⁶ ppm to the central nervous system of the subject. 7 Each CAR-T cell contains a polynucleotide encoding the amino acid sequence SEQ ID NO: 37.

[0154] In some implementations, the method for treating glioblastoma multiforme described herein includes applying 7.0 × 10⁻⁶ mol / L granules. 6 Up to 1.3×10 7A CAR-T cell is administered to a subject’s central nervous system (e.g., into a CSF), the CAR-T cell comprising a polynucleotide encoding the amino acid sequence SEQ ID NO: 37, wherein the CAR-T cell is in a volume of 5 to 15 milliliters (mL), and wherein the CAR-T cell is administered at a rate not exceeding 2 mL / min.

[0155] In some implementations, the method for treating glioblastoma multiforme described herein includes applying 7.0 × 10⁻⁶ mol / L granules. 6 Up to 1.3×10 7 A CAR-T cell is administered to a subject’s central nervous system (e.g., into a CSF), the CAR-T cell comprising a polynucleotide encoding the amino acid sequence SEQ ID NO: 37, wherein the CAR-T cell is in a volume of about 10 ml, and wherein the CAR-T cell is administered at a rate not exceeding 2 mL / min.

[0156] In some implementations, the method for treating glioblastoma multiforme described herein includes applying 7.0 × 10⁻⁶ mol / L granules. 6 Up to 1.3×10 7 A CAR-T cell is administered to a subject’s central nervous system (e.g., into a CSF), the CAR-T cell comprising a polynucleotide encoding the amino acid sequence SEQ ID NO: 37, wherein the CAR-T cell is in a volume of about 10 ml (mL), and wherein the CAR-T cell is administered at a rate not exceeding 1 to 2 mL / min.

[0157] In some implementations, the method for treating glioblastoma multiforme described herein includes applying 7.0 × 10⁻⁶ mol / L granules. 6 Up to 1.3×10 7 One CAR-T cell is administered to the central nervous system of a subject (e.g., into CSF), the CAR-T cell comprising a polynucleotide encoding the amino acid sequence SEQ ID NO: 37, wherein the CAR-T cell is in a volume of approximately 10 mL, wherein the CAR-T cell is administered at a rate not exceeding 1 to 2 mL / min, wherein 7.0 × 10⁻⁶ cells are administered every 4 to 6 weeks. 6 Up to 1.3×10 7 One CAR-T cell, lasting up to 20 weeks.

[0158] In some implementations, the method for treating glioblastoma multiforme described herein includes applying 7.0 × 10⁻⁶ mol / L granules. 6 Up to 1.3×10 7One CAR-T cell is administered to the central nervous system of a subject (e.g., into CSF), the CAR-T cell comprising a polynucleotide encoding the amino acid sequence SEQ ID NO: 37, wherein the CAR-T cell is in a volume of approximately 10 mL, wherein the CAR-T cell is administered at a rate not exceeding 1 to 2 mL / min, wherein 7.0 × 10⁻⁶ cells are administered every 4 to 6 weeks. 6 Up to 1.3×10 7 Each CAR-T cell can last up to 16 weeks.

[0159] In some implementations, the method for treating glioblastoma multiforme described herein includes applying 7.0 × 10⁻⁶ mol / L granules. 6 Up to 1.3×10 7 One CAR-T cell is administered to the central nervous system of a subject (e.g., into CSF), the CAR-T cell comprising a polynucleotide encoding the amino acid sequence SEQ ID NO: 37, wherein the CAR-T cell is in a volume of approximately 10 mL, wherein the CAR-T cell is administered at a rate not exceeding 1 to 2 mL / min, wherein 7.0 × 10⁻⁶ cells are administered every 4 to 6 weeks. 6 Up to 1.3×10 7 Each CAR-T cell can last up to 16 weeks.

[0160] In some implementations, the method for treating glioblastoma multiforme described herein includes administering 1.0 × 10¹² ppm of the drug to the central nervous system of the subject. 7 Each CAR-T cell contains a polynucleotide encoding an EGFRvIII-binding CAR and an EGFR-CD3 TEAM of SEQ ID NO: 23, wherein the CAR-T cells are administered at a rate of 1 to 2 mL / min.

[0161] In some embodiments, the method described herein includes administering 1.0 × 10⁻⁶ ppm to the central nervous system of the subject. 7 CAR-T cells, wherein the CAR-T cells comprise a polynucleotide encoding the amino acid sequence SEQ ID NO: 37, wherein the CAR-T cells are administered in six doses at a rate of 1 to 2 mL / min. In some embodiments, the method herein includes administering 1.0 × 10⁻⁶ CAR-T cells to the central nervous system of a subject. 7 Each CAR-T cell contains a polynucleotide encoding the amino acid sequence SEQ ID NO: 37, wherein the CAR-T cells are administered at a rate of 1 to 2 mL / min at a frequency of once every 4 to 6 weeks for 16 weeks.

[0162] In some embodiments, the method described herein includes administering 1.0 × 10⁻⁶ ppm to the central nervous system of the subject. 7 Each CAR-T cell contains a polynucleotide encoding EGFRvIII-binding CAR and EGFR-CD3TEAM (SEQ ID NO: 23), wherein the CAR-T cells are administered to the subject's cerebrospinal fluid at a rate of 1 to 2 mL / min.

[0163] In some embodiments, the method described herein includes administering 1.0 × 10⁻⁶ ppm to the central nervous system of the subject. 7 Each CAR-T cell contains a polynucleotide encoding EGFRvIII-binding CAR and EGFR-CD3TEAM (SEQ ID NO: 23), wherein the CAR-T cells are administered intraventricularly to the subject at a rate of 1 to 2 mL / min. In some embodiments, the method described herein includes administering 1.0 × 10⁻⁶ CAR-T cells to the central nervous system of the subject. 7 Each CAR-T cell contains a polynucleotide encoding an EGFRvIII-binding CAR and an EGFR-CD3 TEAM of SEQ ID NO: 23, wherein the CAR-T cells are administered to the subject intracerebrospinally at a rate of 1 to 2 mL / min.

[0164] In some embodiments, this disclosure describes a method of administering lymphodepletion (LD) chemotherapy to a subject. Without being bound by theory, LD chemotherapy is expected to enhance CAR-T cell efficacy. In some embodiments, LD chemotherapy includes the administration of cyclophosphamide and fludarabine. In some embodiments, the method includes the administration of about 200 mg / m² cyclophosphamide, about 300 mg / m² cyclophosphamide, about 400 mg / m² cyclophosphamide, or about 500 mg / m² cyclophosphamide. In some embodiments, the method includes the administration of about 300 mg / m² cyclophosphamide. In some embodiments, the method includes the administration of 300 mg / m² cyclophosphamide. In some embodiments, the method includes the administration of about 25 mg / m² fludarabine. In some embodiments, the method includes the administration of 25 mg / m² fludarabine. In some embodiments, LD chemotherapy is administered prior to CAR-T cell administration. In some embodiments, cyclophosphamide and fludarabine are administered daily for three consecutive days prior to CAR-T cell administration. In some embodiments, cyclophosphamide and fludarabine are administered for three consecutive days, from 10 days to 1 day prior to CAR-T cell administration (including endpoint values). In some embodiments, cyclophosphamide and fludarabine are administered for three consecutive days, from 7 days to 5 days prior to CAR-T cell administration (including endpoint values). In some embodiments, cyclophosphamide and fludarabine are administered for three consecutive days, from 8 days to 6 days prior to CAR-T cell administration (including endpoint values). In some embodiments, cyclophosphamide and fludarabine are administered for three consecutive days, from 7 days to 5 days prior to CAR-T cell administration (including endpoint values). In some embodiments, cyclophosphamide and fludarabine are administered for three consecutive days, from 6 days to 4 days prior to CAR-T cell administration (including endpoint values). In some embodiments, cyclophosphamide and fludarabine are administered on days -5, -4, and -3 prior to CAR-T cell administration.

[0165] Polynucleotides

[0166] In some aspects, the methods for treating glioblastoma multiforme (GBM) in a subject, as provided in this disclosure, include administering CAR-T cells to the central nervous system of the subject, said CAR-T cells comprising a polynucleotide (“CAR-TEAM cell”) encoding a CAR and a T-cell conjugating antibody molecule (TEAM), said CAR comprising an extracellular binding domain that binds EGFRvIII, said T-cell conjugating antibody molecule comprising an anti-EGFR antibody and an anti-CD3 antibody. The terms “polynucleotide” and “nucleic acid molecule” are used interchangeably herein to refer to a polymer of nucleosides. Typically, polynucleotides consist of nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine) naturally present in DNA or RNA linked by phosphodiester bonds. However, the term encompasses molecules comprising nucleosides or nucleoside analogues containing chemically or biologically modified bases, modified backbones, etc., whether present in naturally occurring nucleic acids, and such molecules may be preferred for certain applications. When this application relates to polynucleotides, it should be understood that both DNA and RNA are provided, and in each case both single-stranded and double-stranded forms are provided (as well as complements to each single-stranded molecule). As used herein, “polynucleotide sequence” can refer to the polynucleotide substance itself and / or to sequence information that biochemically characterizes a particular nucleic acid (i.e., consecutive letters used as base abbreviations). In some embodiments, the nucleic acid molecule is a heterologous nucleic acid molecule. As used herein, the term “heterologous nucleic acid molecule” refers to a nucleic acid molecule that is not naturally present in a given cell or a nucleic acid sequence that has been engineered into a cell. For example, a heterologous nucleic acid molecule can be a nucleic acid molecule encoding a gene engineered into a cell (e.g., by plasmid, vector, or some other method). Unless otherwise stated, polynucleotide sequences shown herein are shown in a 5' to 3' orientation. In some embodiments, the methods for treating glioblastoma provided herein describe CAR-T cells (“CAR-TEAM cells”) comprising a polynucleotide encoding an EGFRvIII-binding CAR and a T-cell conjugating antibody molecule (TEAM), said T-cell conjugating antibody molecule comprising an anti-EGFR antibody and an anti-CD3 antibody (EGFR-CD3 TEAM). In some embodiments, the polynucleotide comprises a region encoding an EGFRvIII-binding CAR and a region encoding an EGFR-CD3 TEAM. In some embodiments, the polynucleotide comprises a region encoding an EGFRvIII-binding CAR and a region encoding an EGFR-CD3 TEAM from 5' to 3'. In some embodiments, the polynucleotide comprises a region encoding an EGFR-CD3 TEAM and a region encoding an EGFRvIII-binding CAR from 5' to 3'.In some embodiments, the region encoding the EGFRvIII-binding CAR and the region encoding the EGFR-CD3 TEAM are operatively linked to different promoters. In some embodiments, the region encoding the EGFRvIII-binding CAR and the region encoding the EGFR-CD3 TEAM are operatively linked to the same promoter. In some embodiments, a polynucleotide encodes a cleavable linker (e.g., the 2A peptide described herein) between the EGFRvIII-binding CAR and the EGFR-CD3 TEAM.

[0167] sequence

[0168]

[0169] Example

[0170] Example 1. Recruitment and treatment plan for subjects with glioblastoma multiforme (GBM)

[0171] Glioblastoma multiforme (GBM) is a challenging cancer to treat due to its invasive nature and high recurrence rate. Initial treatment for GBM typically involves removing as much of the tumor as possible. To identify potential candidates for treatment with chimeric antigen receptor (CAR)-T cells (which are specific for EGFRvIII and secrete T cell-conjugating antibody molecules (TEAMs) that bind to EGFR and CD3) (CAR-TEAM cells), the resected tumor tissue was analyzed to determine EGFR genetic status. Based on EGFR status and tumor recurrence rate, three patients were selected to receive CAR-TEAM cell therapy. After recruitment, patients underwent leukoablation to isolate T cells for modification, followed by additional resection of tumor recurrence and implantation of an Ommaya reservoir, a shunt that provides a direct channel to cerebrospinal fluid (CSF). CAR-TEAM cells were prepared from isolated patient cells (genetic construction see [link to genetic construction]). Figure 1A See the diagram for protein expression. Figure 1B The patient then received 1×10 7 Autologous CAR-TEAM cells were directly infused into the central nervous system (CNS). Figure 2 ).

[0172] Example 2. Outcomes of CAR-TEAM cell therapy

[0173] To date, CAR-T cell therapy for GBM has not achieved the same success as observed in hematologic malignancies. This is largely because the administration of CAR-T cells involves large-volume intravascular delivery, which does not allow CAR-T cells to infiltrate CNS tumor sites due to the blood-brain barrier. Furthermore, large-volume delivery is also unsuitable for the CNS due to its restrictive nature.

[0174] As described in Example 1, patients selected to undergo CAR-T cell therapy received 10 × 10 CAR-T cells in 10 mL via an Omaye reservoir implanted during a second resection. 6 CAR-TEAM cells were directly infused into the CNS. To determine whether the volume and dose administered to the CNS were effective, patients were monitored to assess treatment outcomes and changes in tumor size relative to pre-infusion size. Figure 3 Patient 3 showed potential pseudoprogression at approximately 30 days post-infusion, with tumor size decreasing by approximately 40% compared to baseline; Patient 2 showed partial response at approximately 70 days post-infusion, with tumor size decreasing by 60%; and Patient 1 showed rapid tumor regression 1 day post-infusion, demonstrating the effectiveness of CAR-T cell therapy despite significant changes in the number and volume of CAR-TEAM cells administered. Evaluation of patients 2 and 3 is ongoing.

[0175] Example 3. Persistence of CAR-T cells and TEAM-binding T cells after infusion

[0176] Key elements of CAR-TEAM cell therapy are the persistence of CAR-T cells and the binding of secreted TEAM, both of which are affected by the dosage and method of administration. CSF and blood samples from patients selected in Example 1 were measured to determine the percentage and absolute number of CAR-TEAM cells up to 35 days after infusion. Figures 4A to 4B CAR-TEAM cells were observed in CSF and persisted for approximately 5 weeks, with a brief migration to the periphery approximately 2 to 3 weeks after infusion. TEAM-binding T cells were also measured in CSF and blood, and were found to persist in CSF for approximately 4 weeks. Unlike CAR-TEAM cells, they were rarely detected in peripheral blood. Figures 4C to 4D Furthermore, these results indicate that altering the dose and volume applied to the CNS does not impair CAR-TEAM cell survival or TEAM activity.

[0177] Example 4. Inflammatory response following CAR-TEAM cell infusion

[0178] Although CAR-TEAM cells and TEAM-binding T cells were primarily localized in the CSF after infusion, the presence of inflammatory cytokines interleukin (IL)-6, interferon-γ (IFNγ), and tumor necrosis factor-α (TNFα), as well as inflammatory markers C-reactive protein and ferritin, was also measured in the CSF and blood of patients 1, 2, and 3 to determine whether the immune response induced by CAR-T cell administration was local or systemic. Within one week of CAR-TEAM cell infusion, the presence of inflammatory cytokines in the CSF peaked, but did not fluctuate in serum, confirming that the response was localized in the CNS and was not systemic.

[0179] Example 5. EGFRvIII expression was not shown to be essential for CAR-TEAM cell cancer killing.

[0180] Patient 3's GBM tumor initially expressed EGFRvIII, but in subsequent biopsies prior to CAR-TEAM cell administration, it did not. Nevertheless, after CAR-TEAM cell administration, Patient 3's GBM tumor volume decreased by approximately 30%, which was greater than the 20% reduction in Patient 1's tumor, despite Patient 1's GBM tumor expressing EGFRvIII. Figure 3 In summary, these results suggest that CAR-TEAM cells may be useful for treating GBM in patients without EGFRvIII expression.

[0181] Example 6. CAR-TEAM cells have minimal EGFR-CD3 TEAM side effects.

[0182] One concern with CAR-TEAM cell administration is the potential toxicity of EGFR-CD3 TEAM, as EGFR is expressed in multiple tissues, including the skin and lungs. However, only patient 1 experienced a skin reaction, a mild rash lasting approximately one day. Patient 1 did not exhibit any harmful lung changes. Patients 2 and 3 showed some transient pulmonary nodules and ground-glass opacities in their lungs, but were asymptomatic. In patient 2, by day 55, the pulmonary nodules showed a reduction in size, and the ground-glass opacity regressed. In patient 3, by day 36, there was significant improvement in the pulmonary nodules. Overall, these results suggest that CAR-TEAM cell administration to the central nervous system has little or no toxicity to EGFR-expressing tissues such as the skin and lungs. Interestingly, Figure 4B The results showed that the CAR-TEAM cells eventually entered the bloodstream, therefore the fact that the CAR-TEAM cells were confined to the central nervous system alone cannot explain this lack of toxicity. Additionally, all patients experienced fever, which peaked two days after infusion and was controlled with intermittent treatment using the interleukin-1 receptor (IL-1R) antagonist anakinin.

[0183] Example 7. Rapid regression of recurrent glioblastoma after a single infusion of CAR-T EGFR-CD3 TEAM cells.

[0184] Glioblastoma (GBM) is the most aggressive primary brain tumor, with a poor prognosis due to recurrent disease and no effective treatment options. Chimeric antigen receptor (CAR) T cells represent a promising approach for cancer, as demonstrated by their proven efficacy in refractory lymphomas, where they have become standard of care. However, the use of CAR-T cells in solid tumors such as GBM has been limited to date, primarily due to targeting only a single antigen in heterogeneous disease and the large infiltration of inhibitory regulatory T cells (Tregs) within the tumor, which impedes the immune response. To address these obstacles, this paper presents a modified T cell product that targets epidermal growth factor receptor variant III (EGFRvIII) via a second-generation CAR while simultaneously secreting a bispecific antibody against wild-type epidermal growth factor receptor (EGFR) (called a T cell conjugating antibody molecule) (TEAM) (“CAR-TEAM cells”), which is not expressed in the normal brain but is almost always expressed in GBM. CAR-TEAM cells secrete TEAM that locally acts on the binding sites of CAR T cell homologous antigens to treat heterogeneous tumors in preclinical models, and these molecules can redirect even Tregs, converting them into cytotoxic killers. Based on these data, a Phase I clinical trial in humans was initiated to evaluate the safety of CAR-TEAM cells in patients with relapsed or newly diagnosed GBM, and is further described below.

[0185] method

[0186] Research supervision and design

[0187] This study has been approved by the Institutional Review Board of Dana-Farber Harvard Cancer Center. An external Data and Safety Monitoring Board oversaw the implementation of the study. This is a non-randomized, open-label, single-center phase 1 study.

[0188] Three patients (referred to as Patient 1, Patient 2, and Patient 3) were recruited into the Safety Run-InArm group, in which subjects with recurrent EGFRvIII-positive GBM received 10 × 10 infusions per dose. 6 Treatment with CAR-TEAM cells is administered via an Omaje reservoir.

[0189] Molecular testing

[0190] During the trials, various methods were used to determine EGFRvIII expression and EGFR amplification, as well as to isolate RNA from extracellular vesicles. Molecular alterations in patient samples, such as EGFRvIII expression and EGFR amplification, were assessed using routine clinical assays. These assays included targeted variant and fusion next-generation sequencing panels using chemical methods based on anchored multiplex PCR, and in some cases, additional sequencing of targeted amplicones on complementary platforms (Oncomine Precision Assay, Ion Torrent Genexus). EGFR copy number status was routinely assessed via sequencing procedures and, in some cases, confirmed by routine clinical fluorescence in situ hybridization (FISH) assays.

[0191] Preparation of CAR-TEAM T cells

[0192] Autologous T cells were obtained through leukocyte removal and transduced using a CAR-TEAM lentiviral vector containing an anti-EGFRvIII single-stranded variable fragment and an intracellular 4-1BB co-stimulatory domain linked to CD3-ζ. An EGFR-targeted TEAM was also included in the same vector, following a ribosomal jumping element.

[0193] The third transgene encoding the truncated CD19 (tCD19) molecule was used as a surface marker for transduction. Figure 6A ). Detection of TEAM binding to T cells using biotinylated human EGFR ( Figures 6B to 6C Details regarding the cell product release criteria are shown in Table 1 below. (Based on 10 × 10⁻⁶ cells after thawing). 6 CAR-TEAM cells are formulated to a target dose of CAR-positive cells / vial. Prior to infusion, the cells are thawed, resuspended, and transferred to the syringe under sterile conditions.

[0194] Table 1. Data at the end of CAR-TEAM cell production.

[0195]

[0196] Toxicity analysis

[0197] Toxicity monitoring was conducted on patients throughout the study. Adverse events were graded according to the Common Toxicity Criteria for Adverse Events version 5.0 (CTCAEv5). Dose-limiting toxicity (DLT) was defined as an event that was at least likely related to the study product and not attributable to disease progression, and: any associated grade 4 or higher CTCAEv5 adverse event, or any associated CTCAEv5 adverse event that did not resolve to grade 2 or lower within 2 weeks, or any grade 3 or 4 cytokine release syndrome (CRS) or immune-effector cell-associated neurotoxicity syndrome (ICANS) that did not resolve to grade 2 or lower within 3 weeks.

[0198] result

[0199] Patients included in the case series

[0200] From March to July 2023, three patients with recurrent glioblastoma participated in a Phase I study at Massachusetts General Hospital.

[0201] Patient 1

[0202] A 74-year-old right-handed man (Patient 1) presented with a one-week history of headache and confusion. Imaging revealed an enhancing mass in the left insula. Patient 1 underwent craniotomy, which revealed a diagnosis of GBM, wild-type isocitrate dehydrogenase (IDH) and methylated O6-methylguanine-DNA methyltransferase (MGMT). The tumor was positive for EGFRvIII and EGFR gene copy number increase. Patient 1 received standard care consisting of radiation therapy and temozolomide chemotherapy. Twelve months after diagnosis, magnetic resonance imaging (MRI) suggested disease recurrence. A second craniotomy was performed, and the recurrent tumor was confirmed to be EGFRvIII positive. Figure 7A Patient 1 was then recruited into a Phase I clinical trial.

[0203] Pre-treatment inter-epidemic MRI was obtained, which showed active progression ( Figure 7B (middle). Patient 1 underwent ventriculostomy with placement of a catheter containing an Omaye reservoir. CAR-TEAM cells (10 × 10⁻⁶) were then administered via the catheter device on postoperative day 1. 6 CAR-positive T cells). MRI obtained on day +1 after this single infusion showed rapid tumor regression ( Figure 7B (Right). On subsequent MRI scans obtained within 31 days, the patient remained stable according to the Response Assessment in Neuro-Oncology (RANO) criteria. The patient received a second infusion of 10 × 10⁻⁶ on day +37. 6 CAR-TEAM cells ( Figure 7A ).

[0204] Liquid biopsies were performed on extracellular vesicle (EV) RNA derived from cerebrospinal fluid (CSF) and peripheral blood at several time points within each treatment cycle. EGFRvIII and EGFR copy numbers increased during cycle 1 and decreased over time, eventually becoming undetectable in post-treatment CSF. Figure 7C EVRNA from peripheral blood samples obtained before and after the study similarly showed decreased copy numbers of both EGFRvIII and EGFR. Figure 7D Following progression revealed by inter-interval MRI, the patient returned to the operating room for a biopsy on day +72. Consistent with the liquid biopsy, next-generation sequencing (NGS) of the tumor tissue was negative for both EGFRvIII and EGFR copy number alterations. However, post-hoc evaluation by FISH showed a maintained EGFR copy number increase (ratio 3.5). Figure 7E ).

[0205] Patient 2

[0206] A 72-year-old right-handed male (Patient 2) presented with dyslexia and a contrast-enhancing mass was found in the posterior left temporal lobe on MRI. Patient 2 underwent craniotomy for tumor resection, and pathology confirmed GBM, which was EGFRvIII positive, IDH wild-type, and MGMT methylated. The patient was treated with standard care radiation therapy and temozolomide chemotherapy, as well as a tumor treatment field. Twenty months after the initial diagnosis, monitoring imaging showed evidence of progression. Then, based on the EGFRvIII status at the time of initial diagnosis, according to a new schematic workflow ( Figure 8APatient 2 was recruited for a Phase I clinical trial. The workflow had been improved to allow for simultaneous craniotomy, tissue sampling, and Omaye placement during a single procedure. Molecular histopathological analysis at the time of re-craniostomy was consistent with recurrent GBM, showing EGFRvIII positivity but negative for EGFR copy number increase. Patient 2 was discharged and readmitted one week later for infusion. The patient received 10 × 10 oz Omaye via an intraventricular catheter. 6 CAR-TEAM cells. On day +2, an MRI showed a 18.5% reduction in tumor cross-sectional area, and on day +69, a further 60.7% reduction compared to pre-infusion baseline. Figure 8B ).

[0207] Patient 3

[0208] A 57-year-old right-handed woman (Patient 3) presented with several weeks of difficulty finding words and concerns about seizures. MRI revealed a contrast-enhancing mass in her left parietal lobe. The patient underwent craniotomy and tumor resection, and pathology revealed GBM, wild-type IDH, and unmethylated MGMT. The tumor was positive for EGFRvIII expression and increased EGFR gene copy number. Patient 3 was treated with radiation therapy and temozolomide chemotherapy, with dose reduction due to thrombocytopenia. Six months after diagnosis, inter-episode imaging showed evidence of disease relapse. This patient was recruited for a Phase I clinical trial (… Figure 8A The patient underwent a second craniotomy for open biopsy and implantation of a ventricular catheter with an Omaye reservoir. Histological analysis at the time of recurrence surgery showed loss of EGFRvIII expression, and no EGFR gene amplification was observed on NGS, confirmed by FISH. Pre-infusion MRI again revealed a broad recurrent disease burden. Figure 8C (Left). The patient was discharged and readmitted two weeks later, receiving 10×10 mmol / L intravenous catheter infusion. 6 One CAR-TEAM cell. MRI obtained on day +5 after this single infusion showed near-complete tumor regression. Figure 8C (right), despite a significant lack of EGFRvIII expression or wild-type gene amplification prior to treatment.

[0209] Safety and adverse events

[0210] In this safety adjustment cohort, no related DLTs were observed. Grade 3 events at least attributable to the study product included Grade 3 encephalopathy lasting 3 days (Patient 1, possibly related) and Grade 3 fatigue lasting 8 days (Patient 3, related). One death occurred after disease progression, 63 days after the patient left the study (Patient 1); the cause of death was gastrointestinal perforation during administration of bevacizumab and dexamethasone. This event was not attributable to CAR-TEAM cell infusion. Patients 2 and 3 presented with transient pulmonary nodules and ground-glass opacities on chest CT scans during periodic fever examinations. These were otherwise asymptomatic and resolved spontaneously within 4 to 6 weeks on inter-episode imaging. Adverse events are shown in Tables 2 through 7 below.

[0211] Table 2. Adverse event record: Patient 1.

[0212]

[0213] SAE = Serious adverse event; PAC / PVC = Atrial premature contraction / Ventricular premature contraction; CRS = Cytokine release syndrome; ALT = Alanine aminotransferase; AST = Aspartate aminotransferase; SIADH = Inappropriate secretion of antidiuretic hormone syndrome; UTI = Urinary tract infection.

[0214] Table 3. Summary of Adverse Events: Patient 1

[0215]

[0216] Table 4. Adverse Event Records: Patient 2

[0217]

[0218] RUE = Right upper limb 1; BLE = Both lower limbs

[0219] Table 5. Summary of Adverse Events: Patient 2

[0220]

[0221] Table 6. Adverse event record: Patient 3.

[0222]

[0223] Table 7. Summary of Adverse Events: Patient 3

[0224]

[0225] Relevant data

[0226] All patients presented with fever, which peaked on day +2 after the infusion. Figure 9A In fever management, intermittent intravenous treatment with the interleukin-1 receptor antagonist anaraxine (100 mg Q6H) is administered. Figure 9B Systemic inflammatory markers increased and peaked in the second and third weeks after infusion, returning to near baseline by day +30. Figure 10 A). No patients required steroids during the post-acute treatment phase or for any treatment-related indication. Cytopathological analysis of CSF showed an immediate increase in total nucleated cell count after infusion, followed by an exponential decline by day +7, and then a gradual decline over time. The CSF cell differential was initially predominantly neutrophilic, then shifted to a predominantly lymphocyte population within 30 days after infusion. Figure 11 Quantitative analysis using vector copy number (VCN) and flow cytometry showed that CAR-positive and TEAM-positive T cells were transiently present in CSF after infusion, but decreased by week 4. Figure 12A This corresponds to the normalization of the inflammatory cytokine environment in the CSF compartment during the same period. Figure 12B CAR-TEAM cells were detected in peripheral blood 2 to 3 weeks after infusion, but their presence was transient. Figure 12C ).

[0227] discuss

[0228] This article presents an early-phase I, first-in-human experience with intraventricular delivery of CAR-TEAM cells in patients with relapsed GBM. Significant radiographic responses were observed in multiple patients within days following a single intraventricular infusion of dual-targeting CAR-TEAM cells. Liquid biopsy has been reported as a relevant study for cell therapy in patients with GBM. Although CAR-T cells have been successfully converted for hematologic malignancies, the efficacy of this strategy for solid tumors such as GBM remains unproven. Previously described CAR-T cells and bispecific antibodies for patients with GBM target only a single antigen and are therefore limited by tumor heterogeneity, the occurrence of antigen loss, and eventual immune escape. The experience with CAR-TEAM cells described in this example provides a proof-of-principle for using CAR-T cells to simultaneously target multiple surface antigens and confirms EGFRvIII as a suitable immunotherapeutic target for GBM. Furthermore, the secreted bispecific antibody was safe, demonstrating the feasibility of locally targeting antigens such as wild-type EGFR using this method, even when it is widely expressed in systemic tissues. This example demonstrates that antitumor CAR-mediated responses can be achieved in patients with GBM, even in the context of advanced, intraparenchymal disease. This contrasts with a previous report that observed a complete response in patients with recurrent leptomeningeal disease (LMD) treated with repeated intracranial infusions (16 infusions) of monospecific interleukin-13 receptor α-2 (IL-13Rα2) CAR-T cells. Here, the results indicate that intraventricularly administered "live drugs" (such as CAR-TEAM cells) can also enter infiltrative parenchymal GBM and mediate activity against it. Notably, CAR-TEAM cells showed signs of antitumor activity in the absence of EGFRvIII expression (patient 3). Of course, the interpretation of these data remains limited by the sensitivity of the corresponding assays for determining EGFRvIII expression and the inherent limitations of tissue sampling in heterogeneous diseases. However, the potential efficacy of CAR-TEAM cells in the context of EGFRvIII-negative gliomas reproduced the CAR-independent, TEAM-mediated antitumor activity previously observed in the inventors' preclinical studies.

[0229] Example 8. Detection of CAR-T cells expressing truncated CD19

[0230] A crucial aspect of CAR-T cell therapy is developing methods for detecting CAR-T cells. This is useful, for example, in preparing CAR-T cell therapy for administration to patients. CAR-T cells are prepared by transfecting T cells (usually autologous T cells) with a CAR expression vector, in this case, encoding an EGFRvIII-binding CAR and anti-EGFR and anti-CD3 TEAM. A truncated CD19 can be added to the vector, specifically as a component of the CAR-TEAM protein product, to determine CAR expression (Figure 1). This is achieved by encoding a 2A cleavage site between CAR-TEAM and tCD19. Because CAR and tCD19 reside on the same promoter, tCD19 expression also indicates CAR expression and that the vector has been successfully transfected into the cells.

[0231] tCD19 can be detected using anti-tCD19 antibodies that bind to a fluorophore. Here, an anti-tCD19 antibody conjugated to a PE fluorophore (CD19-PE) is used to detect tCD19, which indicates CAR expression (…). Figure 13 The antibody conjugate was then incubated with T cells that had been transfected with the vector. After incubation, the T cells were analyzed by flow cytometry, and the number of PE fluorescent T cells indicated the number of successfully transduced CAR-T cells and indicated EGFRvIII binding to CAR.

Claims

1. A method for treating glioblastoma multiforme (GBM) in a subject, the method comprising administering 6.0 × 10 6 Up to 1.4×10 7 One CAR-T cell is administered to the central nervous system of the subject, wherein the CAR-T cell contains a polynucleotide encoding the following: (a) A CAR containing an extracellular binding domain that binds to EGFRvIII; and (b) T cell conjugating antibody molecules (TEAM) containing anti-EGFR antibody and anti-CD3 antibody.

2. The method of claim 1, comprising applying 7.0 × 10⁻⁶ to the object. 6 Up to 1.3×10 7 The CAR-T cells mentioned above.

3. The method of claim 1, further comprising applying 8.0 × 10⁸ mg / L to the object. 6 Up to 1.2×10 7 The CAR-T cells mentioned above.

4. The method of claim 1, comprising applying 9.0 × 10⁻⁶ to the object. 6 Up to 1.1×10 7 The CAR-T cells mentioned above.

5. The method of claim 1, comprising applying 1.0 × 10⁻⁶ to the object. 7 The CAR-T cells mentioned above.

6. The method of any one of claims 1 to 5, wherein administering the CAR-T cells comprises administering the CAR-T cells into the brain of the subject.

7. The method of any one of claims 1 to 5, wherein administering the CAR-T cells comprises intracranial administration.

8. The method of any one of claims 1 to 5, wherein administering the CAR-T cells comprises intraventricular administration.

9. The method of any one of claims 1 to 5, wherein administering the CAR-T cells comprises administering them to the cerebrospinal fluid of the subject.

10. The method of any one of claims 1 to 5, wherein administering the CAR-T cells comprises intrathecal administration.

11. The method of any one of claims 1 to 5, wherein administering the CAR-T cells comprises administering them to a tumor in the central nervous system of the subject.

12. The method of any one of claims 1 to 5, wherein administering the CAR-T cells comprises administering them to a tumor in the central nervous system of the subject.

13. The method of any one of claims 1 to 12, wherein the CAR-T cells are in a solution of 5 to 15 mL.

14. The method of claim 13, wherein the solution comprises Elliott's B solution.

15. The method of claim 13 or claim 14, wherein administering the CAR-T cells comprises administering the CAR-T cells at a rate not exceeding 2 mL / min.

16. The method of claim 15, wherein administering the CAR-T cells comprises administering the CAR-T cells at a rate of 1 to 2 mL / min.

17. The method of any one of claims 1 to 16, wherein the extracellular binding domain comprises: (a) A VH domain comprising three complementary determinant regions (CDR-H1, CDR-H2, and CDR-H3), wherein CDR-H1 contains SEQ ID NO: 1, CDR-H2 contains SEQ ID NO: 2, and CDR-H3 contains SEQ ID NO: 3; and (b) A VL domain containing three CDRs (CDR-L1, CDR-L2 and CDR-L3), wherein CDR-L1 contains SEQ ID NO:4, CDR-L2 contains SEQ ID NO:5 and CDR-L3 contains SEQ ID NO:

6.

18. The method of claim 17, wherein the extracellular domain is scFv.

19. The method of claim 18, wherein the scFv comprises the amino acid sequence of SEQ ID NO:

7.

20. The method of any one of claims 1 to 17, wherein the CAR comprises the amino acid sequence of SEQ ID NO:

8.

21. The method of any one of claims 1 to 20, wherein the anti-EGFR antibody comprises: (a) A VH domain comprising three complementary determinant regions (CDR-H1, CDR-H2, and CDR-H3), wherein CDR-H1 contains SEQ ID NO: 9, CDR-H2 contains SEQ ID NO: 10, and CDR-H3 contains SEQ ID NO: 11; and (b) A VL domain containing three CDRs (CDR-L1, CDR-L2 and CDR-L3), wherein CDR-L1 contains SEQ ID NO:12, CDR-L2 contains SEQ ID NO:13 and CDR-L3 contains SEQ ID NO:

14.

22. The method of claim 21, wherein the anti-EGFR antibody is scFv.

23. The method of claim 22, wherein the scFv comprises the amino acid sequence of SEQ ID NO:

15.

24. The method of any one of claims 1 to 23, wherein the anti-CD3 antibody comprises: (a) A VH domain comprising three complementary determinant regions (CDR-H1, CDR-H2, and CDR-H3), wherein CDR-H1 contains SEQ ID NO: 16, CDR-H2 contains SEQ ID NO: 17, and CDR-H3 contains SEQ ID NO: 18; and (b) A VL domain containing three CDRs (CDR-L1, CDR-L2 and CDR-L3), wherein CDR-L1 contains SEQ ID NO:19, CDR-L2 contains SEQ ID NO:20 and CDR-L3 contains SEQ ID NO:

21.

25. The method of claim 24, wherein the anti-CD3 antibody is scFv.

26. The method of claim 25, wherein the scFv comprises the amino acid sequence of SEQ ID NO:

22.

27. The method of any one of claims 1 to 26, wherein the TEAM comprises the amino acid sequence of SEQ ID NO:

23.

28. The method of any one of claims 1 to 27, wherein the GBM comprises a polynucleotide encoding EGFRvIII.

29. The method of claim 28, wherein the GBM expresses EGFRvIII.

30. The method of any one of claims 1 to 28, wherein the GBM does not express EGFRvIII.

31. The method of any one of claims 1 to 28, wherein the GBM expresses EGFRvIII and EGFR.

32. The method of any one of claims 1 to 31, wherein the GBM expresses EGFR.

33. The method of claim 32, wherein the GBM comprises an EGFR gene duplication.

34. The method of any one of claims 1 to 33, wherein the subject has previously received radiation therapy.

35. The method of any one of claims 1 to 34, wherein the subject suffers from recurrent GBM.

36. The method of any one of claims 1 to 35, wherein at the time of administration, the subject has not received alkylation treatment or immunotherapy within the past 4 weeks.

37. The method of any one of claims 1 to 36, wherein the GBM of the object is unmethylated 6-methylguanine-DNA methyltransferase (MGMT).

38. The method of any one of claims 1 to 31, wherein the subject has not previously been treated for GBM.

39. The method of any one of claims 1 to 38, wherein the GBM of the object comprises a polynucleotide encoding EGFRvIII, and the GBM of the object is MGMT unmethylated.

40. The method of any one of claims 1 to 39, further comprising administering an antihistamine to the subject.

41. The method of claim 40, wherein the antihistamine is benzodiazepine.

42. The method of claim 41, wherein 25 mg of benzodiazepine is administered to the subject.

43. The method of claim 41 or 42, wherein the benzodiazepine is administered prior to the administration of the CAR-T cells.

44. The method of any one of claims 1 to 43, further comprising administering acetaminophen to the object.

45. The method of claim 44, wherein 650 mg of acetaminophen is administered to the subject.

46. ​​The method of claim 44 or claim 45, wherein the acetaminophen is administered prior to the administration of the CAR-T cells.

47. The method of any one of claims 1 to 46, further comprising generating CAR-T cells using autologous T cells.

48. The method of any one of claims 1 to 47, comprising administering a single dose of the CAR-T cells to the subject.

49. The method of any one of claims 1 to 47, comprising administering six doses of the CAR-T cells to the subject.

50. The method of claim 49, comprising administering the CAR-T cells to the subject every 4 to 6 weeks.

51. The method of any one of claims 1 to 47, comprising administering the CAR-T cells once every 4 to 6 weeks, for a total of 4 administrations.

52. The method of any one of claims 1 to 51, further comprising administering lymphatic depletion (LD) chemotherapy to the subject.

53. The method of claim 52, wherein the administration of the LD chemotherapy comprises the administration of cyclophosphamide and / or fludarabine.

54. The method of claim 52, comprising administering about 300 mg / m2 of cyclophosphamide.

55. The method of claims 53 and 54, comprising administering about 25 mg / m2 of fludarabine.

56. The method of any one of claims 52 to 55, wherein the LD chemotherapy is administered prior to the administration of the CAR-T cells.

57. The method of any one of claims 53 to 56, wherein the cyclophosphamide and / or the fludarabine are administered daily for three consecutive days prior to CAR-T cell administration.

58. The method of claim 57, wherein the cyclophosphamide and / or the fludarabine are administered 5 days, 4 days, and 3 days prior to CAR-T cell administration.

59. The method of claim 57, wherein the cyclophosphamide and / or the fludarabine are administered 7, 6, and 5 days prior to CAR-T cell administration.

60. A polynucleotide comprising a nucleic acid encoding an amino acid sequence, the amino acid sequence comprising: (i) An epidermal growth factor receptor (EGFR) binding chimeric antigen receptor (EGFRvIII CAR) comprising an antigen-binding domain, wherein the antigen-binding domain comprises: The complementary determinant region (CDR) 1 of the variable heavy chain (VH) in SEQ ID NO: 1, the VH CDR 2 of SEQ ID NO: 2, the VH CDR 3 of SEQ ID NO: 3, the variable light chain (VL) CDR 1 of SEQ ID NO: 4, the VL CDR 2 of SEQ ID NO: 5, and the VL CDR 3 of SEQ ID NO: 6; and (ii) T cell conjugating antibody molecules (TEAM), which include: (a) An EGFR-binding domain comprising the variable heavy chain (VH) complementarity determination region (CDR) 1 of SEQ ID NO: 9, the VH CDR 2 of SEQ ID NO: 10, the VH CDR 3 of SEQ ID NO: 11, the variable light chain (VL) CDR 1 of SEQ ID NO: 12, the VL CDR 2 of SEQ ID NO: 13, and the VL CDR 3 of SEQ ID NO: 14; (b) A CD3-binding domain comprising the variable heavy chain (VH) complementarity determination region (CDR) 1 of SEQ ID NO: 16, the VH CDR 2 of SEQ ID NO: 17, the VH CDR 3 of SEQ ID NO: 18, the variable light chain (VL) CDR 1 of SEQ ID NO: 19, the VL CDR 2 of SEQ ID NO: 20, and the VL CDR 3 of SEQ ID NO: 21; and (iii) A truncated CD19 domain (tCD19) containing an amino acid sequence that is at least 95% identical to SEQ ID NO:

38.

61. The polynucleotide of claim 60, wherein the antigen-binding domain comprises scFv.

62. The polynucleotide of claim 61, wherein the antigen-binding domain comprises the amino acid sequence of SEQ ID NO:

7.

63. The polynucleotide of claim 60, wherein the EGFRvIII CAR comprises a CD8 hinge / transmembrane domain.

64. The polynucleotide of claim 60 or claim 63, wherein the EGFRvIII CAR comprises a 4-1BB intracellular signal transduction domain.

65. The polynucleotide of any one of claims 60 to 64, wherein the EGFRvIII CAR comprises a CD3z signaling domain.

66. The polynucleotide of any one of claims 60 to 65, further comprising an amino acid sequence encoding a 2A self-cleaving peptide, said 2A self-cleaving peptide being encoded between said EGFRvIII CAR and said TEAM.

67. The polynucleotide of any one of claims 60 to 66, further comprising an amino acid sequence encoding a 2A self-cleaving peptide, said 2A self-cleaving peptide being encoded between said TEAM and said tCD19.

68. The polynucleotide of claim 66 or claim 67, wherein the 2A self-cleaving peptide comprises the amino acid sequence of any one of SEQ ID NO:35 or SEQ ID NO:

36.

69. The polynucleotide of any one of claims 60 to 68, wherein the TEAM comprises a peptide linker encoded between the EGFR-binding domain and the CD3-binding domain.

70. The polynucleotide of claim 69, wherein the linker comprises the amino acid sequence of SEQ ID NO: 30 to 34 or 36.

71. The polynucleotide of any one of claims 60 to 70, wherein the TEAM comprises an IgK leader sequence.

72. The polynucleotide of any one of claims 60 to 71, wherein the EGFR-binding domain comprises the amino acid sequence of SEQ ID NO:

15.

73. The polynucleotide of any one of claims 60 to 72, wherein the CD3 binding domain comprises the amino acid sequence of SEQ ID NO:

22.

74. The polynucleotide of any one of claims 60 to 73, wherein the tCD19 comprises the amino acid sequence of SEQ ID NO:

38.

75. The polynucleotide of any one of claims 60 to 74, comprising the amino acid sequence of SEQ ID NO:

37.

76. A cell comprising any one of claims 60 to 75.

77. The cell of claim 76, wherein the cell is a T cell.

78. The cell of claim 76 or claim 77, wherein tCD19 is expressed on the surface of the cell.

79. A polypeptide encoded by any one of claims 60 to 75.

80. A method for detecting cells according to any one of claims 78 to 79, the method comprising: Contact the cells with the tCD19 binding agent; as well as Detection of tCD19 binding protein.

81. The method of claim 80, wherein the tCD19 binder comprises a tCD19 binding protein.

82. The method of claim 81, wherein the tCD19 binding protein comprises an anti-tCD19 antibody.

83. The method of claim 82, wherein the anti-tCD19 antibody is an antibody fragment.

84. The method of claim 82 or claim 83, wherein the anti-tCD19 antibody is CD19-PE.

85. The method of any one of claims 82 to 84, wherein the anti-tCD19 antibody has a higher binding affinity to tCD19 than it has a higher binding affinity to CD19.

86. The method of any one of claims 80 to 85, wherein the tCD19 binder further comprises a fluorescent molecule or a radioactive molecule.

87. The method of any one of claims 80 to 85, further comprising contacting the tCD19 binder with a secondary antibody containing fluorescent or radioactive molecules.

88. The method of claim 86 or claim 87, wherein the detection comprises detecting the fluorescent molecule or the radioactive molecule.

89. The method of claim 88, wherein detecting the fluorescent or radioactive molecule comprises using microscopy, Western blotting, or flow cytometry.

Images