WO2025101807A1 - Treatment of gliomas with chimeric antigen receptor t cells - Google Patents

Abstract

Methods are provided for treatment of brain cancers with repeated administration of CART cells on a schedule that reflects the patient's inflammatory response to the introduced cells.

Description

TREATMENT OF GLIOMAS WITH CHIMERIC ANTIGEN RECEPTOR T CELLS

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. provisional application No. 63/547,645, which was filed November 7, 2023, entitled “TREATMENT OF GLIOMAS WITH CHIMERIC ANTIGEN RECEPTOR T CELLS,” and is incorporated by reference in its entirety.

SEQUENCE LISTING

[0002] The Sequence Listing filed herewith has the filename STFD-016-PCT Sequence Listing.xml, was created on November 7, 2024, has a file size of 36,580 bytes, and is incorporated herein by reference in its entirety.

BACKGROUND

[0003] Glioblastoma Multiforme (GBM) is an aggressive, high-grade primary brain tumor that arises from the glial cells of the central nervous system. GBM is the most common malignant primary brain tumor in adults (48.3%), with over 10,000 new cases diagnosed each year in the United States alone. Current standard-of-care treatment for GBM involves a multi-modal approach of safe maximal resection followed by radiation concurrent with temozolomide chemotherapy, followed by monthly adjuvant temozolomide chemotherapy with electric tumortreating fields. While there have been improvements in short-term survival due in large part to improvements in the standard of care treatment at initial GBM diagnosis, the long-term prognosis for GBM remains grim with an estimated 5-year survival rate of 6.8% and 10-year survival rate of <1%.

[0004] The diffuse and infiltrative nature of GBM renders complete resection impossible and recurrence a virtual certainty. On average, time to GBM progression or recurrence occurs 6 months after resection. Following progression or recurrence, current therapeutic options include systemic chemotherapy with lomustine or bevacizumab, radiotherapy or further surgical intervention. While many of these approaches can provide symptomatic relief, none have definitively demonstrated benefit in terms of overall survival. [0005] Improving overall survival in GBM is contingent upon the development of novel and effective therapies for recurrent disease. As immunotherapy has produced revolutionary advances in the treatment of many cancers, there has been significant interest in harnessing immune-based therapies in the treatment of GBM. While immune checkpoint inhibition has demonstrated impressive activity in many cancers, it has not yet demonstrated consistent activity in GBM.

SUMMARY

[0006] In some embodiments, the disclosure relates to a method of treating a high-grade glioma in a subject in need thereof. In some embodiments, the method comprises, after surgical resection of cancer cells in the brain of the subject: (i) administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of a plurality of T cells comprising a nucleic acid molecule encoding a chimeric antigen receptor (CAR) molecule that binds to cells expressing B7-H3 or a functional variant thereof; and, subsequently; and (ii) administering a first dose of pharmaceutical composition comprising a therapeutically effective amount of bevacizumab. In some embodiments, the dose of bevacizumab is less than or equal to about 7.5 mg/kg, wherein the bevacizumab is administered no less than about two weeks after administering the plurality of autologous T cells. In some embodiments, the T cells are delivered in a first and a second fraction; the first fraction administered intracerebroventricularly and the second fraction administered locoregionally at a site of surgical resection. In some embodiments, the T cells are autologous T cells from the subject. In some embodiments, the therapeutically effective amount of a plurality of T cells is administered at a dose of from about 1 x 10⁶ cells to about 100 x 10⁶ cells. In some embodiments, the therapeutically effective amount of a plurality of T cells is administered at a dose of from about 15 x 10⁶ cells to about 35 x 10⁶ cells. In some embodiments, the therapeutically effective amount of a plurality of T cells is administered at a dose of about 25 x 10⁶ cells. In some embodiments, the autologous T cells are administered a total of at least about 2 times over a period of from about 8 to about 12 weeks. In some embodiments, the step of administering the autologous T cells is repeated no less than about two weeks after the first dose of bevacizumab. In some embodiments, the method further comprises: (iii) monitoring the individual for inflammation following step (ii); and (iv) determining when inflammation is at a predetermined level of inflammation appropriate for repeating administration of the T cells. In some embodiments, the dose of bevacizumab is from about 3 mg/kg to about 7.5 mg/kg In some embodiments, the dose of bevacizumab is about 3 mg/kg and is administered no less than about 2 weeks after the first dose of autologous T cells. In some embodiments, the pre-determined level of inflammation appropriate for repeating administration of the autologous T cells is determined by evaluation of the subject. In some embodiments, the subject exhibits an absence of herniation or midline shift upon magnetic resonance imaging (MRI) of the brain of the subject and/or the subject is free of clinical symptoms of intracerebral edema. In some embodiments, the subject is human. In some embodiments, the subject is from about 1 year to about 17 years old. In some embodiments, the glioma is a glioblastoma, gliosarcoma, glioblastoma with oligodendroglial features, glioblastoma multiforme, or glioblastoma with PNET features. In some embodiments, the step of administering bevacizumab is repeated from about 2 to about 10 times before a successive dose of autologous T cells. In some embodiments, the T cells are delivered in a first and a second fraction sequentially or substantially contemporaneously; the first fraction administered intracerebroventricularly and the second fraction administered locoregionally at a site of surgical resection. In some embodiments, the first fraction and the second fraction comprise substantially equal doses of cells and are administered a total of about 2 to about 6 times about every four weeks. In some embodiments, the first fraction is from about 10 x 10⁶ cells to about 15 x IO⁶ cells; and the second fraction is from about 10 x IO⁶ cells to about 15 x 10⁶ cells. In some embodiments, the first fraction is about 12.5 x 10⁶ cells; and the second fraction is about 12.5 x 10⁶ cells.

[0007] In some embodiments, the disclosure relates to a method of treating inflammation in a subject in need thereof. In some embodiments, the method comprises (i) after surgical resection of glioma in the brain of the subject, administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of a plurality of autologous T cells comprising a nucleic acid molecule encoding a chimeric antigen receptor (CAR) molecule that binds to cells expressing B7-H3 or a functional variant thereof; and, subsequently (ii) administering a first dose. In some embodiments, the dose of bevacizumab is less than or equal to about 7.5 mg/kg. In some embodiments, the bevacizumab is administered no less than about two weeks after administering the plurality of autologous T cells. In some embodiments, the method further comprises (iii) monitoring inflammation in the subject. In some methods, the method further comprises (iv) repeating step (i) after the determining when inflammation is at a pre-determined level of inflammation appropriate for repeating administration of the autologous T cells. In some embodiments, the autologous T cells are delivered in a first and a second fraction. In some embodiments, the first fraction is administered intracerebroventricularly and the second fraction is administered locoregionally at a site of surgical resection. In some embodiments, a pre-determined level of inflammation appropriate for repeating administration of the autologous T cells is determined by evaluation of the subject. In some embodiments, subject exhibits an absence of herniation or midline shift upon magnetic resonance imaging (MRI) of the brain of the subject and/or the subject is free of clinical symptoms of intracerebral edema. In some embodiments, the T cells are autologous T cells from the subject. In some embodiments, the therapeutically effective amount of a plurality of T cells is administered at a dose of from about 1 x 10⁶ cells to about 100 x 10⁶ cells. In some embodiments, the therapeutically effective amount of a plurality of T cells is administered at a dose of from about 15 x 10⁶ cells to about 35 x 10⁶ cells. In some embodiments, the therapeutically effective amount of a plurality of T cells is administered at a dose of about 25 x 10⁶ cells. In some embodiments, the autologous T cells are administered a total of at least about 2 times over a period of from about 8 to about 12 weeks. In some embodiments, the step of administering the autologous T cells is repeated no less than about two weeks after the first dose of bevacizumab. In some embodiments, the dose of bevacizumab is from about 3 mg/kg to about 7.5 mg/kg. In some embodiments, the dose of bevacizumab is about 3 mg/kg and is administered no less than about 2 weeks after the first dose of autologous T cells. In some embodiments, the pre-determined level of inflammation appropriate for repeating administration of the autologous T cells is determined by evaluation of the subject, wherein the subject exhibits an absence of herniation or midline shift upon magnetic resonance imaging (MRI) of the brain of the subject and/or the subject is free of clinical symptoms of intracerebral edema. In some embodiments, the subject is human. In some embodiments, the subject is from about 1 year to about 17 years old. In some embodiments, the glioma is a glioblastoma, gliosarcoma, glioblastoma with oligodendroglial features, glioblastoma multiforme, or glioblastoma with PNET features. In some embodiments, the step of administering bevacizumab is repeated from about 2 to about 10 times before a successive dose of autologous T cells. In some embodiments, the T cells are delivered in a first and a second fraction sequentially or substantially contemporaneously; the first fraction administered intracerebroventricularly and the second fraction administered locoregionally at a site of surgical resection. In some embodiments, the first fraction and the second fraction comprise substantially equal doses of cells and are administered a total of about 2 to about 6 times about every four weeks.

[0008] In some embodiments, methods are provided for treatment of glioma brain tumors, which methods can provide for a significant decrease in tumor volume. The methods of embodiments herein may provide for increased overall survival of the individual being treated.

[0009] In an embodiment, and individual is treated for a high grade glioma by administration of multiple doses of chimeric antigen receptor (CAR) T cells specific for B7H3 antigen (CD276), wherein the method comprises obtaining a population of cells comprising CD3+ T cells from the individual; generating a population of transduced CART cells by a highly efficient manufacturing process; and treating the individual within a period of from about 11 to about 20 days following obtention of the CD3⁺ T cells, by a method comprising: (a) administering to the individual a dose of at least about 5 to about 100 xlO⁶ of the CART cells; (b) monitoring the individual for inflammation following step (a); (c) determining when inflammation is at a pre-determined level appropriate for additional CART cell administration; and (d) repeating steps (a) to (c) for a total of at least 4 rounds of treatment. In some embodiments steps (a) to (c) are repeated for a total of at least 6 rounds of treatment.

[0010] In some embodiments, the patient is treated with maximal safe surgical resection of the glioma. CAR T treatment can induce an inflammatory response and if the entire tumor is left in place, the volumetric constraints may result in injury and the need for anti-inflammatory intervention. By resecting the tumor, the volume available to alleviate pressure from inflammatory response to CAR T infusion is increased. Administration of the CART cells may be initiated within about 24 to about 48 hours following surgery.

[0011] In some embodiments, treatment is performed in the absence of testing the individual cancer for a confirmation of CD276 expression in order to avoid the exclusion of qualified patients in the event of a false negative or ambiguous B7H3 assessment.

[0012] In some embodiments, treatment is performed in the absence of lymphodepleting chemotherapy immediately prior to the CART- Cell treatment, thereby sparing the patient from additional stress. [0013] In some embodiments, delivery of the CART cells is by an intraventricular or intracavitary route, which optimizes provision of therapeutic cells at the tumor site, and can reduce counterproductive systemic inflammatory responses.

[0014] In some embodiments, inflammation in the individual is addressed by administration of an effective dose of an anti-VEGF agent, including, without limitation Bevacizumab. The anti-VEGF treatment provides non-steroidal inflammatory relief that is not cytolytic. In some embodiments throughout treatment the individual is kept to low or no steroid treatment, usually less than or equal to about 4 mg dexamethasone per day, or an equivalent thereof.

[0015] In some embodiments, during treatment the individual is assessed for inflammatory responses following an infusion of CART cells. In some embodiments assessment utilizes magnetic resonance imaging. In some embodiments, MRI is combined with dynamic susceptibility contrast (DSC) perfusion. Normal contrast imaging of the tumor can be confounded by the inflammatory response that follows each treatment. By combining standard imaging with DSC perfusion MRI, cerebral blood flow can be used to monitor tumor progression or regression. Monitoring inflammation following CART cell infusion allows the delivery of multiple doses of the CART cells on a schedule that is appropriate for the patient, by delivering subsequent cell doses when the patient is at a pre-determined level of inflammation resolution from the prior dose. Cells may be administered monthly, e.g. at about 28 days, where the timing is varied to be shorter or longer depending on the inflammation status. In some embodiments, a second dose of CART cells is administered at 28 or more days following the initial dose.

[0016] Inflammatory markers useful in evaluating a patient’s fitness for additional cell doses may include features in MRI/DSC imaging, assessment of inflammation may also comprise determining the level and presence of hematologic toxicities, for example cytopenias including anemia, thrombocytopenia, lymphopenia, neutropenia and white blood cell decrease, coagulopathy, fever, neurotoxicity, hepatic function, and the like, for example see Gatto et al. (2023) Front. Oncol., Sec. Cancer Immunity and Immunotherapy, vol 13 doi.org/10.3389/fonc.2023.1206983.

[0017] In some embodiments, administration of a CART cell is combined with chemotherapy, with tumor specific antibodies, with radiation therapy, with an immuno-oncology agent such as a checkpoint inhibitor, agonist of an immune response protein, and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The disclosure is best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not to-scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures.

[0019] FIG. 1 A illustrates a B7-H3 CAR T Cell protocol overview.

[0020] FIG. IB illustrates a B7H3 CAR Construct (1491 bp), Majzner et al. (2019) Clin Cancer Res. 2019;25: 2560-2574.

[0021] FIGS. 2A through 2F illustrates On Target Specificity. MRI Brain of subject 001 with left frontal GBM on Day 37 following CAR T infusion showing significant inflammation (A-C) which resolved after Bevacizumab therapy (D-F). (A) Large volume of contrast enhancement around the resection cavity, with corresponding (B) low cerebral blood volume and fractional tumor burden on dynamic susceptibility contrast perfusion imaging (blue voxels). (C) Extensive surrounding edema. (D) Resolution of contrast enhancement and (E) perfusion signal following Bevacizumab therapy, as well as (F) improved edema. Signs and symptoms of local inflammation are noted following the first cycle of CAR T administration in the intracerebral parenchyma surrounding the surgical resection cavity. No off-target inflammation was seen in other organ systems, contralateral cerebral hemisphere, or outside of the immediate vicinity of the original tumor resection. The demonstrated on target specificity of B7-H3CART product to the location of expected residual GBM disease and trafficking of the CAR T product to anticipated loco-regional GBM cell infiltration.

[0022] FIGS. 3A and 3B illustrate Loco-regional Inflammation. (A) Large volume parenchymal enhancement in the left temporo-occipital lobe, with ependymal enhancement along the margin of the right lateral ventricle (white arrow). (B) Resolution of right ventricular ependymal enhancement (in the absence of intervening steroids or bevacizumab) consistent with resolving inflammation.

[0023] FIGS. 4A and 4B illustrate Intraventricular and Intratumoral Administration. Two Ommaya catheters placed during surgery: one in the right frontal tumor bed and the second in the left frontal ventricle to allow for administration of CAR T treatment. (A) Right frontal approach Ommaya Reservoir tip in the resection cavity (left arrow). Left frontal approach ventricular catheter tracking through the brain (right arrow). (B) Tip of the left frontal approach ventricular catheter in the frontal horn of the left lateral ventricle (arrow).

[0024] FIGS. 5 A through 5D illustrates Surgical Intervention of Recurrent GBM prior to CAR T treatment. MRIs (A) Contrast enhancing mass at the site of the original tumor (not shown) with (B) elevated blood flow on arterial spin labeling perfusion imaging (white arrow); imaging features are consistent with recurrent GBM. (C) Expected post-surgical changes after resection of recurrent tumor, without residual contrast enhancement nor (D) elevated blood flow to suggest residual tumor.

[0025] FIGS. 6A through 6C illustrate Clinical Course Highlighting Efficacy of CAR T. MRI Brain of patient 002 at three time points during the course of CAR T treatment: (A) Pre-CAR T and Pre-surgery; (B) Pre-CAR T and Post-surgery, and (C) Post-CAR T and Post-surgery. Apotent efficacy signal of B7-H3CART is evidenced by changes in glioblastoma tumor mass pre- and post- CART treatment.

[0026] FIGS. 7A through 7C illustrates Correlative studies in B7H3 CAR T cell trial to date A. Samples are collected from CD4/CD8 enriched sample, CAR-T product, cerebrospinal fluid (CSF) and peripheral blood of patients throughout the B7H3 CAR-T treatment course. B. By flow cytometry, B7H3 CAR T cells have been identified in peripheral blood and CSF of patients throughout the treatment course. C. Peak% B7H3 CART cells of CD3+ T cells.

[0027] FIG. 8 illustrates clinical trial schema.

[0028] FIG. 9 illustrates results of a method herein.

[0029] FIG. 10 illustrate s/p CAR T s/p 5^th (top) and s/p 6^th CAR T infusion results.

[0030] FIG. 11 illustrates on-target edema and treatment-related enhancement.

DETAILED DESCRIPTION

[0031] Before the present active agents and methods are described, it is to be understood that this invention is not limited to the particular methodology, products, apparatus and factors described, as such methods, apparatus and formulations may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of other embodiments.

[0032] As used herein the singular forms “a”, “and”, and “the” include plural referents unless the context clearly dictates otherwise. For example, reference to “a cell” includes a plurality of such cells and reference to “the culture” includes reference to one or more cultures and equivalents thereof known to those skilled in the art, and so forth. Also, reference to “a drug candidate” refers to one or mixtures of such candidates, and reference to “the method” includes reference to equivalent steps and methods known to those skilled in the art, and so forth.

[0033] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications mentioned herein are incorporated herein by reference in their entireties for the purpose of describing and disclosing devices, formulations and methodologies which are described in the publication and which might be used in connection with the presently described invention.

[0034] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically 20 disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a range such as 95-99% identity, includes something with 95%, 96%, 97%, 98% or 99% sequence identity, and includes subranges such as 96-99%, 96-98%, 96- 97%, 97-99%, 97-98% and 98-99% sequence identity. This applies regardless of the breadth of the range.

[0035] In the following description, numerous specific details are set forth to provide a more thorough understanding of the present invention. However, it will be apparent to one of skill in the art that the present embodiments may be practiced without one or more of these specific details. In other instances, well-known features and procedures well known to those skilled in the art have not been described in order to avoid obscuring the invention.

[0036] Generally, conventional methods of protein synthesis, recombinant cell culture and protein isolation, and recombinant DNA techniques within the skill of the art are employed in the present invention. Such techniques are explained fully in the literature, see, e.g., Maniatis, Fritsch & Sambrook, Molecular Cloning: A Laboratory Manual (1982); Sambrook, Russell and Sambrook, Molecular Cloning: A Laboratory Manual (2001); Harlow, Lane and Harlow, Using Antibodies: A Laboratory Manual: Portable Protocol No. I, Cold Spring Harbor Laboratory (1998); and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory; (1988).

Definitions

[0037] The term “a” and “an” refers to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0038] The term “about” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, ±10%, ±5%,, ±1%, or ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods. For recitation of numeric ranges herein, each intervening number therebetween with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers

7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6,9, and 7.0 are explicitly contemplated.

[0039] “Pharmaceutically acceptable excipient” means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients can be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous. [0040] As used herein, the term “pharmaceutically acceptable salt” or “esters” refers to those salts or esters which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of subjects without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in theart. For example, Berge et al. describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66: 1-19. Such salts include salts that can be formed where acidic protons present in the compounds are capable of reacting with inorganic or organic bases. Suitable inorganic salts include those formed with the alkali metals, e.g. sodium and potassium, magnesium, calcium, and aluminum. Suitable organic salts include those formed with organic bases such as the amine bases, e.g. , ethanolamine, diethanolamine, triethanolamine, tromethamine, N methylglucamine, and the like. Such salts also include acid addition salts formed with inorganic acids (e.g., hydrochloric and hydrobromic acids) and organic acids (e.g., acetic acid, citric acid, maleic acid, and the alkane- and arene-sulfonic acids such as methanesulfonic acid and benzenesulfonic acid). Pharmaceutically acceptable esters include esters formed from carboxy, sulfonyloxy, and phosphonoxy groups present in the compounds, e.g., Ci-6 alkyl esters. When there are two acidic groups present, a pharmaceutically acceptable salt or ester can be a mono-acid- mono-salt or ester or a di-salt or ester; and similarly where there are more than two acidic groups present, some or all of such groups can be salified or esterified. Compounds named in this invention can be present in unsalified or unesterified form, or in salified and/or esterified form, and the naming of such compounds is intended to include both the original (unsalified and unesterified) compound and its pharmaceutically acceptable salts and esters. Also, certain compounds named in this invention may be present in more than one stereoisomeric form, and the naming of such compounds is intended to include all single stereoisomers and all mixtures (whether racemic or otherwise) of such stereoisomers.

[0041] The terms “pharmaceutically acceptable”, “physiologically tolerable” and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to or upon a human without the production of undesirable physiological effects to a degree that would prohibit administration of the composition. [0042] The term “Chimeric Antigen Receptor,” a “CAR,” or a “CAR molecule” refers to a set of polypeptides, typically two in the simplest embodiments, which when in an immune effector cell, provides the cell with specificity for a target cell and with intracellular signal generation. In some embodiments, a CAR comprises at least an extracellular antigen binding domain, a transmembrane domain and a cytoplasmic signaling domain (also referred to herein as “an intracellular signaling domain”) comprising a functional signaling domain derived from a stimulatory molecule and/or costimulatory molecule as defined below. In some embodiments, the set of polypeptides are contiguous with each other, e.g., are in the same polypeptide chain, e.g., comprise a chimeric fusion protein. In some embodiments, the set of polypeptides are not contiguous with each other, e.g., are in different polypeptide chains. In some embodiments, the set of polypeptides include a dimerization switch that, upon the presence of a dimerization molecule, can couple the polypeptides to one another, e.g., can couple an antigen binding domain to an intracellular signaling domain.

[0043] In some embodiments, the stimulatory molecule is the zeta chain associated with the T cell receptor complex.

[0044] In some embodiments, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule as defined below.

[0045] In some embodiments, the costimulatory molecule is chosen from the costimulatory molecules described herein, e.g., 4-IBB (i.e., CD137), CD27 and/or CD28. In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule.

[0046] In some embodiments, the CAR comprises an optional leader sequence at the aminoterminus (N-ter) of the CAR fusion protein. In some embodiments, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen binding domain, wherein the leader sequence is optionally cleaved from the antigen binding domain (e.g, a scFv) during cellular processing and localization of the CAR to the cellular membrane.

[0047] As used herein, the term “binding domain” refers to a protein, e.g., an immunoglobulin chain or fragment thereof, comprising at least one immunoglobulin variable domain sequence. The term “binding domain” (also referred to herein as “antibody molecule”) encompasses antibodies and antibody fragments. In some embodiments an antibody molecule is a multispecific antibody molecule, e.g., it comprises a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence of the plurality has binding specificity for a first epitope and a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope. In some embodiments, a multispecific antibody molecule is a bispecific antibody molecule. A bispecific antibody has specificity for no more than two antigens. A bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence which has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope.

[0048] The term “antibody fragment” refers to at least one portion of an antibody, that retains the ability to specifically interact with (e.g., by binding, steric hinderance, stabilizing/destabilizing, spatial distribution) an epitope of an antigen. Examples of antibody fragments include, but are not limited to, Fab, Fab’, F(ab’)2, Fv fragments, scFv antibody fragments, disulfide-linkedFvs (sdFv), a Fd fragment consisting of the VH and CHI domains, linear antibodies, single domain antibodies such as sdAb (either VL or VH), camelid VHH domains, multi-specific antibodies formed from antibody fragments such as a bivalent fragment comprising two Fab fragments linked by a disulfide brudge at the hinge region, and an isolated CDR or other epitope binding fragments of an antibody. An antigen binding fragment can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger and Hudson, Nature Biotechnology 23: 1126-1136, 2005). Antigen binding fragments can also be grafted into scaffolds based on polypeptides such as a fibronectin type III (Fn3) (see U.S. Patent No.: 6,703,199, which describes fibronectin polypeptide minibodies). The term “scFv” refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked, e.g., via a synthetic linker, e.g., a short flexible polypeptide linker, and capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specified, as used herein an scFv may have the VL and VH variable regions in either order, e.g., with respect to the N- terminal and C-terminal ends of the polypeptide, the scFv may comprise VL-linker-VH or may comprise VH-linker-VL.

[0049] The term “complementarity determining region” or “CDR,” as used herein, refers to the sequences of amino acids within antibody variable regions which confer antigen specificity and binding affinity. The precise amino acid sequence boundaries of a given CDR can be determined using any of a number of well-known schemes, including those described by Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5^th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (“Kabat” numbering scheme), Al-Lazikani et al., (1997) JMB 273, 927-948 (“Chothia” numbering scheme) and ImMunoGenTics (IMGT) numbering (Lefranc, M.-P, The Immunologist, 7, 132-136 (1999); Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55- 77 (2003) (“IMGT” numbering scheme). For example, for classic formats, under Kabat, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1 ), 50- 65 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Under Chothia, the CDR amino acids in the VH are numbered 26-32 (HCDR1 ), 52- 56 (HCDR2), and 95-102 (HCDR3); and the amino acid residues in VL are numbered 26-32 (LCDR1 ), 50-52 (LCDR2), and 91-96 (LCDR3). By combining the CDR definitions of both Kabat and Chothia, the CDRs consist of amino acid residues 26-35 (HCDR1 ), 50-65 (HCDR2), and 95-102 (HCDR3) in human VH and amino acid residues 24-34 (LCDR1 ), 50-56 (LCDR2), and 89-97 (LCDR3) in human VL. Under IMGT, the CDR amino acid residues in the VH are numbered approximately 26-35 (CDR1), 51-57 (CDR2) and 93-102 (CDR3), and the CDR amino acid residues in the VL are numbered approximately 27-32 (CDR1), 50-52 (CDR2), and 89-97 (CDR3) (numbering according to “IMGT”). Under IMGT, the CDR regions of an antibody can be determined using the program IMGT/DomainGap Align.

[0050] The portion of the CAR of some embodiments herein comprises an antibody or antibody fragment thereof may exist in a variety of forms where the antigen binding domain is expressed as part of a contiguous polypeptide chain including, for example, a single domain antibody fragment (sdAb), a single chain antibody (scFv), a humanized antibody, or bispecific antibody (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al.,

1988, Science 242:423-426). In some embodiments, the antigen binding domain of a CAR composition of the invention comprises an antibody fragment. In some embodiments, the CAR comprises an antibody fragment that comprises a scFv.

[0051] The term “antibody heavy chain” or refers to the larger of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations, and which normally determines the class to which the antibody belongs.

[0052] The term “antibody light chain,” refers to the smaller of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations. Kappa (x) and lambda (1-) light chains refer to the two major antibody light chain isotypes.

[0053] The term “recombinant antibody” refers to an antibody which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage or yeast expression system. The term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the

DNA or amino acid sequence has been obtained using recombinant DNA or amino acid sequence technology which is available and well known in the art.

[0054] The term “antigen” or “Ag” refers to a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises a nucleic acid sequence or a partial nucleic acid sequence encoding a protein that elicits an immune response therefore encodes an “antigen” as that term is used herein.

Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full length nucleic acid sequence of a gene. Present embodiments include, but are not limited to, the use of partial nucleic acid sequences of more than one gene and that these nucleic acid sequences are arranged in various combinations to encode polypeptides that elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated, synthesized, or can be derived from a biological sample, or might be macromolecule besides a polypeptide. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a fluid with other biological components.

[0055] The term “combination” refers to either a fixed combination in one dosage unit form, or a combined administration where a compound of the present disclosure and a combination partner (e.g. a cell, a CAR T cell, another drug as explained below, also referred to as “therapeutic agent” or “co-agent”) may be administered independently at the same time or separately within time intervals, especially where these time intervals allow that the combination partners show a cooperative, e.g. synergistic effect. The single components may be packaged in a kit or separately. One or both of the components (e.g., powders or liquids) may be reconstituted or diluted to a desired dose prior to administration. The terms “co-administration” or “combined administration” or the like as utilized herein are meant to encompass administration of the selected combination partner to a single subject in need thereof (e.g. a patient), and are intended to include treatment regimens in which the agents are not necessarily administered by the same route of administration or at the same time. The term “pharmaceutical combination” as used herein means a product that results from the mixing or combining of more than one therapeutic agent and includes both fixed and non-fixed combinations of the therapeutic agents. The term “fixed combination” means that the therapeutic agents, e.g. a compound of the present disclosure and a combination partner, are both administered to a patient simultaneously in the form of a single entity or dosage. The term “non- fixed combination” means that the therapeutic agents, e.g. a compound of the present disclosure and a combination partner, are both administered to a patient as separate entities either simultaneously, concurrently or sequentially with no specific time limits, wherein such administration provides therapeutically effective levels of the two compounds in the body of the patient. The latter also applies to cocktail therapy, e.g. the administration of three or more therapeutic agent.

[0056] “Dosage unit” refers to physically discrete units suited as unitary dosages for the particular individual to be treated. Each unit can contain a predetermined quantity of active compound(s) calculated to produce the desired therapeutic effect(s) in association with the required pharmaceutical carrier. The specification for the dosage unit forms can be dictated by (a) the unique characteristics of the active compound(s) and the particular therapeutic effect(s) to be achieved, and (b) the limitations inherent in the art of compounding such active compound(s).

[0057] The term “cancer” refers to a disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. The terms “tumor” and “cancer” are used interchangeably herein, e.g., both terms encompass solid and liquid tumors. As used herein, the term “cancer” or “tumor” includes premalignant, as well as malignant cancers and tumors.

[0058] As used herein, unless otherwise specified, the terms “prevent,” “preventing” and “prevention” refer to an action that occurs before the subject begins to suffer from the condition, or relapse of the condition. Prevention need not result in a complete prevention of the condition; partial prevention or reduction of the condition or a symptom of the condition, or reduction of the risk of developing the condition, is encompassed by this term.

[0059] Administered “in combination”, as used herein, means that two (or more) different treatments are delivered to the subject during the course of the subject’s affliction with the disorder, e.g. , the two or more treatments are delivered after the subj ect has been diagnosed with the di sorder and before the disorder has been cured or eliminated or treatment has ceased for other reasons. In some embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as “simultaneous” or “concurrent delivery”. In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments of either case, the treatment is more effective because of combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment, or the analogous situation is seen with the first treatment. In some embodiments, delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other. The effect of the two treatments can be partially additive, wholly additive, or greater than additive. The delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered. In some embodiments, a CAR-expressing cell is administered at a dose and/or dosing schedule described herein, and an anti-inflammatory agent is administered at a dose and/or dosing schedule described herein.

[0060] “Derived from” as that term is used herein, indicates a relationship between a first and a second molecule. It generally refers to structural similarity between the first molecule and a second molecule and does not connote or include a process or source limitation on a first molecule that is derived from a second molecule. For example, in the case of an intracellular signaling domain that is derived from a CD3zeta molecule, the intracellular signaling domain retains sufficient CD3zeta structure such that is has the required function, namely, the ability to generate a signal under the appropriate conditions. It does not connote or include a limitation to a particular process of producing the intracellular signaling domain, e.g., it does not mean that, to provide the intracellular signaling domain, one must start with a CD3zeta sequence and delete unwanted sequence, or impose mutations, to arrive at the intracellular signaling domain.

[0061] The term “conservative sequence modifications” refers to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody or antibody fragment containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody or antibody fragment of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g, aspartic acid, glutamic acid), uncharged polar side chains (e.g, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within a CAR of embodiments herein can be replaced with other amino acid residues from the same side chain family and the altered CAR can be tested using the functional assays described herein. [0062] The term “stimulation,” refers to a primary response induced by binding of a stimulatory molecule e.g., a TCR/CD3 complex or CAR) with its cognate ligand (or tumor antigen in the case of a CAR) thereby mediating a signal transduction event, such as, but not limited to, signal transduction via the TCR/CD3 complex or signal transduction via the appropriate NK receptor or signaling domains of the CAR. Stimulation can mediate altered expression of certain molecules.

[0063] The term “stimulatory molecule,” refers to a molecule expressed by an immune cell, e.g., T cell, NK cell, or B cell, that provides the cytoplasmic signaling sequence(s) that regulates activation of the immune cell in a stimulatory way for at least some embodiment of the immune cell signaling pathway. In some embodiments, the signal is a primary signal that is initiated by, for instance, binding of a TCR/CD3 complex with an MHC molecule loaded with peptide, and which leads to mediation of a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like. A primary cytoplasmic signaling sequence (also referred to as a “primary signaling domain”) that acts in a stimulatory manner may contain a signaling motif which is known as immunoreceptor tyrosine-based activation motif or ITAM. Examples of an ITAM containing cytoplasmic signaling sequence that is of particular use in the invention includes, but is not limited to, those derived from CD3 zeta, common FcR gamma (FCERI G), Fc gamma Rlla, FcR beta (Fc Epsilon RI b), CD3 gamma, CD3 delta , CD3 epsilon, CD79a, CD79b, DAPIO, and DAP 12. In a specific CAR of the invention, the intracellular signaling domain in any one or more CARS of the invention comprises an intracellular signaling sequence, e.g., a primary signaling sequence of CD3-zeta. In a specific CAR of the invention, the primary signaling sequence of CD3- zeta is the sequence, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape and the like.

[0064] The term “antigen presenting cell” or “APC” refers to an immune system cell such as an accessory cell e.g., a B-cell, a dendritic cell, and the like) that displays a foreign antigen complexed with major histocompatibility complexes (MHC’s) on its surface. T-cells may recognize these complexes using their T-cell receptors (TCRs). APCs process antigens and present them to T-cells.

[0065] “Immune effector cell,” as that term is used herein, refers to a cell that is involved in an immune response, e.g., in the promotion of an immune effector response. Examples of immune effector cells include T cells, e.g., alpha/beta T cells and gamma/delta T cells, B cells, natural killer (NK) cells, natural killer T (NK-T) cells, mast cells, and myeloid-derived phagocytes.

[0066] An “intracellular signaling domain,” as the term is used herein, refers to an intracellular portion of a molecule. The intracellular signaling domain generates a signal that promotes an immune effector function of the CAR containing cell, e.g., a CART cell or CAR-expressing NK cell. Examples of immune effector function, e.g., in a CART cell or CAR-expressing NK cell, include cytolytic activity and helper activity, including the secretion of cytokines.

[0067] In some embodiments, the intracellular signaling domain can comprise a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from the molecules responsible for primary stimulation, or antigen dependent simulation. In some embodiments, the intracellular signaling domain can comprise a costimulatory intracellular domain. Exemplary costimulatory intracellular signaling domains include those derived from molecules responsible for costimulatory signals, or antigen independent stimulation. For example, in the case of a CART, a primary intracellular signaling domain can comprise a cytoplasmic sequence of a T cell receptor, and a costimulatory intracellular signaling domain can comprise cytoplasmic sequence from co-receptor or costimulatory molecule.

[0068] A primary intracellular signaling domain can comprise a signaling motif which is known as an immunoreceptor tyrosine-based activation motif or ITAM. Examples of ITAM-containing primary cytoplasmic signaling sequences include, but are not limited to, those derived from CD3 zeta, common FcR gamma (FCER1 G), Fc gamma Rlla, FcR beta (Fc Epsilon R1 b), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP1O and DAP12.

[0069] The terms “inflammation is at a pre-determined level of inflammation appropriate for repeating administration of the autologous T cells” is defined as the level of inflammation in a subject where there is no or substantially reduced brain herniation as compared to the degree of brain herniation before a treatment. “Brain herniation”, in some embodiments, is defined as a movement of the brain or localized brain tissue due to pressure in the skull. In some embodiments, brain herniation is caused by neurologically symptomatic intracranial brain edema or swelling confirmed by MRI brain or CT head imaging findings. In some embodiments, the edema or swelling is caused by a tumor and/or general inflammation of tissues surrounding an area of the brain resected due to the presence of tumor. In some embodiments, the swelling is caused, at least partially, by CAR-T therapy in the brain after a step of administration of thecells disclosed herein. In some embodiments, the general inflammation is caused by surgical resection of a tumor. In some embodiments, the brain herniation is transtentorial, subfalcine, central, upward, and/or tonsilar. In some embodiments, the subject exhibits neurologic symptoms of brain herniation including one or more symptoms of: high blood pressure, decreased heart rate, headache, weakness, loss of consciousness, loss of brainstem reflexes, respiratory arrest, vomiting, gait instability, altered mental status, and nausea, relative to a control or control image of the subject (e.g. an mgae of the subject’s brain before treatment began).

[0070] The term “zeta” or alternatively “zeta chain”, “CD3-zeta” or “TCR-zeta” is defined as the protein provided as GenBan Acc. No. BAG36664. 1, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape and the like, and a “zeta stimulatory domain” or alternatively a “CD3-zeta stimulatory domain” or a “TCR-zeta stimulatory domain” is defined as the amino acid residues from the cytoplasmic domain of the zeta chain or functional derivative thereof, that are sufficient to functionally transmit an initial signal necessary for T cell activation. In one embodiment the cytoplasmic domain of zeta comprises residues 52 through 164 of GenBank Acc. No. BAG36664. 1 or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape and the like, that are functional orthologs thereof.

[0071] The term “costimulatory molecule” refers to the cognate binding partner on a T cell that specifically binds with a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that contribute to an efficient immune response. Costimulatory molecules include, but are not limited to an MHC class I molecule, TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, 64ignalling lymphocytic activation molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, 0X40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1(CDI la/CD18), 4-IBB (CD137), B7-H3, CDS, ICAM-1, ICOS 5 (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRFI), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLAI, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDI Id, ITGAE, CD 103, ITGAL, CDI la, LFA-1, ITGAM, CDI lb, ITGAX, CDI le, ITGBI, CD29, ITGB2, CD 18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAMI (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAMI, CRTAM, Ly9 (CD229), CD 160 (BY55), PSGLI, CDI00 (SEMA4D), CD69, SLAMF6 (NTB-A, LylO8), SLAM (SLAMFI, CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD 19a, and a ligand that specifically binds with CD83.

[0072] A costimulatory intracellular signaling domain can be the intracellular portion of a costimulatory molecule. A costimulatory molecule can be represented in the following protein families: TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), and activating NK cell receptors. Examples of such molecules include CD27, CD28, 4-IBB (CD137), 0X40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, ICAM-1, lymphocyte function-associated antigen-I (LFA-1), CD2, 25CSD, CD7, CD287, LIGHT, NKG2C, NKG2D, SLAMF7, NKp80, NKp30, NKp44, NKp46, CD160, B7-H3, and a ligand that specifically binds with CD83, and the like.

[0073] The intracellular signaling domain can comprise the entire intracellular portion, or the entire native intracellular signaling domain, of the molecule from which it is derived, or a functional fragment or derivative thereof.

[0074] The term “4-IBB” refers to a member of the TNFR superfamily with an amino acid sequence provided as GenBank Acc. No. AAA62478.2, or the equivalent residues from a nonhuman species, e.g., mouse, rodent, monkey, ape and the like; and a “4-IBB costimulatory domain” is defined as amino acid residues 214-255 of GenBank Accession No. AAA62478.2, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape and the like.

[0075] The term “encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene, cDNA, or RNA, encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleic acid sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0076] “Membrane anchor” or “membrane tethering domain”, as that term is used herein, refers to a polypeptide or moiety, e.g., a myristoyl group, sufficient to anchor an extracellular or intracellular domain to the plasma membrane.

[0077] The term “bioequivalent” refers to an amount of an agent other than the reference compound required to produce an effect equivalent to the effect produced by the reference dose or reference amount of the reference compound.

[0078] Unless otherwise specified, a “nucleic acid sequence encoding an amino acid sequence” includes all nucleic acid sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleic acid sequence that encodes a protein or a RNAmay also include introns to the extent that the nucleic acid sequence encoding the protein may in some version contain an intron(s).

[0079] The term “effective amount” or “therapeutically effective amount” are used interchangeably herein, and refer to an amount of a compound, formulation, material, or composition, as described herein effective to achieve a particular biological result. In some embodiments, “therapeutically effective amount” means the amount that, when administered to a subject for treating a disease, is sufficient to effect treatment for that disease.

[0080] The term “therapeutic” as used herein means a treatment. A therapeutic effect is obtained by reduction, suppression, remission, or eradication of a disease state

[0081] The term “prophylaxis” as used herein means the prevention of or protective treatment for a disease or disease state.

[0082] A subject “responds” to treatment if a parameter of a pathology in the subject is retarded or reduced by a detectable amount, e.g., about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more as determined by any appropriate measure, e.g., by symptoms. In one example, a subject responds to treatment if the subject experiences a life expectancy extended by about 5%, 10%, 20%, 30%, 40%, 50% or more beyond the life expectancy predicted if no treatment is administered. In another example, a subject responds to treatment, if the subject has an increased disease-free survival, overall survival or increased time to progression. Several methods can be used to determine if a patient responds to a treatment including, for example, criteria provided by NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines®). For example, in the context of BALL, a complete response or complete responder, may involve one or more of: < 5% BM blast, >1000 neutrophil/ANC (/pL). >100,000 platelets (/pL) with no circulating blasts or extramedullary disease (no lymphadenopathy, splenomegaly, skin/gum infdtration/testicular mass/CNS involvement), Trilineage hematopoiesis, and no recurrence for 4 weeks. A partial responder may involve one or more of >50% reduction in BM blast, >1000 neutrophil/ANC (/pL). >100,000 platelets (/pL). A non-responder can show disease progression, e.g.,> 25% in BM blasts. In some embodiments, a complete responder is defined as having 7% or greater CD27+ CD45RO- cells in the CD8+ population. In some embodiments, the percent of CAR+ cells at pre- harvest levels distinguish responders (e.g., complete responders and partial responders) from non-responders (NR).

[0083] The term “relapse” as used herein refers to reappearance of a pathology after an initial period of responsiveness. The initial period of responsiveness may involve the level of symptoms or pathology falling below a certain threshold, e.g, below 20%, 1%, 10%, 5%, 4%, 3%, 2%, or 1%. The reappearance may involve the level symptoms or pathology rising above a certain threshold, e.g., above 20%, 1%, 10%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, the initial period of 30 responsiveness lasts at least 1, 2, 3, 4, 5, or 6 days; at least 1, 2, 3, or 4 weeks; at least 1, 2, 3, 4, 6, 8, 10, or 12 months; or at least 1, 2, 3, 4, or 5 years.

[0084] The term “endogenous” refers to any material from or produced inside an organism, cell, tissue or system.

[0085] The term “exogenous” refers to any material introduced from or produced outside an organism, cell, tissue or system.

[0086] The term “expression” refers to the transcription and/or translation of a particular nucleic acid sequence driven by a promoter.

[0087] The term “transfer vector” refers to a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell.

[0088] Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “transfer vector” includes an autonomously replicating plasmid or a virus. The term should also be construed to further include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, a polylysine compound, liposome, and the like. Examples of viral transfer vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.

[0089] The term “expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleic acid sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system.

Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno- associated viruses) that incorporate the recombinant polynucleotide.

[0090] The term “lentivirus” refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses.

[0091] The term “lentiviral vector” refers to a vector derived from at least a portion of a lentivirus genome, including especially a self-inactivating lentiviral vector as provided in Milone et al., Mol. Ther. 17(8): 1453-1464 (2009). Other examples of lentivirus vectors that may be used in the clinic, include but are not limited to, e.g., the LENTIVECTOR® gene delivery technology from Oxford BioMedica, the LENTIMAX™ vector system from Lentigen and the like. Nonclinical types of lentiviral vectors are also available and would be known to one skilled in the art.

[0092] The “percent identity” of two polynucleotide or two polypeptide sequences is determined by comparing the sequences using the GAP computer program (a part of the GCG Wisconsin Package, version 10.3 (Accelrys, San Diego, Calif.)) using its default parameters. “Identical” or “identity” as used herein in the context of two or more nucleic acids or amino acid sequences, may mean that the sequences have a specified percentage of residues that are the same over a specified region. The percentage may be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which the identical residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to yield the percentage of sequence identity. In cases where the two sequences are of different lengths or the alignment produces one or more staggered ends and the specified region of comparison includes only a single sequence, the residues of single sequence are included in the denominator but not the numerator of the calculation. When comparing DNAand RNA, thymine (T) and uracil (U) may be considered equivalent. Identity may he performed manually or by using a computer sequence algorithm such as BLAST or BLAST 2.0. Briefly, the BLAST algorithm, which stands for Basic Local Alignment Search Tool is suitable for determining sequence similarity. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov). This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length Win the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension for the word hits in each direction are halted when: 1) the cumulative alignment score falls off by the quantity X from its maximum achieved value; 2) the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or 3) the end of either sequence is reached. The Blast algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The Blast program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff et al., Proc. Natl. Acad. Sci. USA, 1992, 89, 10915-10919, which is incorporated herein by reference in its entirety) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands. The BLAST algorithm (Karlin et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 5873- 5787, which is incorporated herein by reference in its entirety) and Gapped BLAST perform a statistical analysis of the similarity between two sequences. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide sequences or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to another if the smallest sum probability in comparison of the test nucleic acid to the other nucleic acid is less than about 1, less than about 0.1, less than about 0.01, and less than about 0.001. Two single- stranded polynucleotides are “the complement” of each other if their sequences can be aligned in an anti-parallel orientation such that every nucleotide in one polynucleotide is opposite its complementary nucleotide in the other polynucleotide, without the introduction of gaps, and without unpaired nucleotides at the 5’ or the 3’ end of either sequence. A polynucleotide is “complementary” to another polynucleotide if the two polynucleotides can hybridize to one another under moderately stringent conditions. Thus, a polynucleotide can be complementary to another polynucleotide without being its complement.

[0093] The term “homologous” or “identity” refers to the subunit sequence identity between two polymeric molecules, e.g., between two nucleic acid molecules, such as, two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomeric subunit; e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous or identical at that position. The homology between two sequences is a direct function of the number of matching or homologous positions; e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two sequences are homologous, the two sequences are 50% homologous; if 90% of the positions (e.g., 9 of I 0), are matched or homologous, the two sequences are 90% homologous. [0094] “Humanized” forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab’, F(ab’)2 or other antigen-binding subsequences of antibodies), which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies and antibody fragments thereof are human immunoglobulins (recipient antibody or antibody fragment) in which residues from a complementarity-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, a humanized antib ody/antibody fragment can comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications can further refine and optimize antibody or antibody fragment performance. In general, the humanized antibody or antibody fragment thereof will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or a significant portion of the FR regions are those of a human immunoglobulin sequence. The humanized antibody or antibody fragment can also comprise at least a portion of an immunoglobulin constant region(Fe), typically that of a human immunoglobulin. For further details, see Jones et al., Nature, 321 : 522-525, 1986; Reichmann et al., Nature, 332: 323-329, 1988; Presta, Curr. Op. Struct. Biol., 2:593-596, 1992. [0095] “Fully human” refers to an immunoglobulin, such as an antibody or antibody fragment, where the whole molecule is of human origin or consists of an amino acid sequence identical to a human form of the antibody or immunoglobulin.

[0096] “Murine” refers to mice or rats. For example, a murine antibody or fragment thereof contains the sequence of an antibody or fragment thereof that is isolated from a murine animal, e.g., mouse or rat.

[0097] The term “isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0098] In the context of the present disclosure, the following abbreviations for the commonly occurring nucleic acid bases are used. “A” refers to adenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine.

[0099] The term “operably linked” or “transcriptional control” refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences can be contiguous with each other and, e.g., where necessary to join two protein coding regions, are in the same reading frame.

[0100] The term “parenteral” administration of an immunogenic composition includes, e.g., subcutaneous (s.c.), intravenous (i .v.), intramuscular (i.m.), or intrasternal injection, intratumoral, or infusion techniques. [0101 ] The term “nucleic acid” or “polynucleotide” refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementarity sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0102] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. A polypeptide includes a natural peptide, a recombinant peptide, or a combination thereof.

[0103] The term “promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence.

[0104] The term “promoter/regulatory sequence” refers to a nucleic acid sequence that is required for expression of a gene product operably linked to the promoter/regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product. The promoter/regulatory sequence may, for example, be one that expresses the gene product in a tissue specific manner.

[0105] As used herein, the terms “treat”, “treatment” and “treating” refer to the reduction or amelioration of the progression, severity and/or duration of a proliferative disorder, or the amelioration of one or more symptoms (preferably, one or more discernible symptoms) of a proliferative disorder resulting from the administration of one or more therapies (e.g., one or more therapeutic agents; e.g., a CAR of embodiments herein and an anti inflammatory agent of embodiments herein). In specific embodiments, the terms “treat”, “treatment” and “treating” refer to the amelioration of at least one measurable physical parameter of a proliferative disorder, such as growth of a tumor, not necessarily discernible by the patient. In some embodiments, the terms “treat”, “treatment” and “treating” -refer to the inhibition of the progression of a proliferative disorder, either physically by, e.g., stabilization of a discernible symptom, physiologically by, e.g., stabilization of a physical parameter, or both.

[0106] The term “signal transduction pathway” refers to the biochemical relationship between a variety of signal transduction molecules that play a role in the transmission of a signal from one portion of a cell to another portion of a cell. The phrase “cell surface receptor” includes molecules and complexes of molecules capable of receiving a signal and transmitting signal across the membrane of a cell.

[0107] The term, a “substantially purified” cell refers to a cell that is essentially free of other cell types. A substantially purified cell also refers to a cell which has been separated from other cell types with which it is normally associated in its naturally occurring state. In some instances, a population of substantially purified cells refers to a homogenous population of cells. In other instances, this term refers simply to cell that have been separated from the cells with which they are naturally associated in their natural state. In some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.

[0108] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a mammal being assessed for treatment and/or being treated. In some embodiments, the mammal is a human. The terms “subject,” “individual,” and “patient” encompass, without limitation, individuals having cancer. Subjects may be human, but also include other mammals, particularly those mammals useful as laboratory models for human disease, e.g. mouse, rat, etc. Humans, e.g. less than about 18 years of age, are of interest as subjects herein.

[0109] The term “sample” with respect to a patient encompasses blood and other liquid samples of biological origin, solid tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom and the progeny thereof. The definition also includes samples that have been manipulated in any way after their procurement, such as by treatment with reagents; washed; or enrichment for certain cell populations, such as cancer cells. The definition also includes sample that have been enriched for particular types of molecules, e.g., nucleic acids, polypeptides, etc. The term “biological sample” encompasses a clinical sample, and also includes tissue obtained by surgical resection, tissue obtained by biopsy, cells in culture, cell supernatants, cell lysates, tissue samples, organs, bone marrow, blood, plasma, serum, and the like. A “biological sample” includes a sample obtained from a patient’s cancer cell, e.g., a sample comprising polynucleotides and/or polypeptides that is obtained from a patient’s cancer cell (e.g., a cell lysate or other cell extract comprising polynucleotides and/or polypeptides); and a sample comprising cancer cells from a patient. Abiological sample comprising a cancer cell from a patient can also include non-cancerous cells.

[0110] The terms “cancer,” “neoplasm,” and “tumor” are used interchangeably herein to refer to cells which exhibit autonomous, unregulated growth, such that they exhibit an aberrant growth phenotype characterized by a significant loss of control over cell proliferation. Cells of interest for detection, analysis, or treatment in the present application include malignant, pre-metastatic, metastatic, and non-metastatic cells. The phrase “cancer burden” refers to the quantum of cancer cells or cancer volume in a subject. Reducing cancer burden accordingly refers to reducing the number of cancer cells or the cancer volume in a subject. The term “cancer cell” as used herein refers to any cell that is a cancer cell or is derived from a cancer cell e.g. clone of a cancer cell.

[0111] The “pathology” of cancer includes all phenomena that compromise the well-being of the patient. This includes, without limitation, abnormal or uncontrollable cell growth, metastasis, interference with the normal functioning of neighboring cells, release of cytokines or other secretory products at abnormal levels, suppression or aggravation of inflammatory or immunological response, neoplasia, premalignancy, malignancy, invasion of surrounding or distant tissues or organs, such as lymph nodes, etc. [0112] As used herein, the terms “cancer recurrence” and “tumor recurrence,” and grammatical variants thereof, refer to further growth of neoplastic or cancerous cells after diagnosis of cancer. Particularly, recurrence may occur when further cancerous cell growth occurs in the cancerous tissue. “Tumor spread,” similarly, occurs when the cells of a tumor disseminate into local or distant tissues and organs; therefore tumor spread encompasses tumor metastasis. “Tumor invasion” occurs when the tumor growth spread out locally to compromise the function of involved tissues by compression, destruction, or prevention of normal organ function.

[0113] The term “diagnosis” is used herein to refer to the identification of a molecular or pathological state, disease or condition, such as the identification of a molecular subtype of breast cancer, prostate cancer, or other type of cancer.

[0114] The term “prognosis” is used herein to refer to the prediction of the likelihood of cancer- attributable death or progression, including recurrence, metastatic spread, and drug resistance, of a neoplastic disease, such as ovarian cancer. The term “prediction” is used herein to refer to the act of foretelling or estimating, based on observation, experience, or scientific reasoning. In one example, a physician may predict the likelihood that a patient will survive, following surgical removal of a primary tumor and/or chemotherapy for a certain period of time without cancer recurrence.

[0115] As used herein, the terms “treatment,” “treating,” and the like, refer to administering an agent, or carrying out a procedure, for the purposes of obtaining an effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of effecting a partial or complete cure for a disease and/or symptoms of the disease. “Treatment,” as used herein, may include treatment of a tumor in a mammal, particularly in a human, and includes: (a) preventing the disease or a symptom of a disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it (e.g., including diseases that may be associated with or caused by a primary disease; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.

[0116] Treating may refer to any indicia of success in the treatment or amelioration or prevention of an cancer, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the disease condition more tolerable to the patient; slowing in the rate of degeneration or decline; or making the final point of degeneration less debilitating. The treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of an examination by a physician. Accordingly, the term “treating” includes the administration of the compounds or agents of the present invention to prevent or delay, to alleviate, or to arrest or inhibit development of the symptoms or conditions associated with cancer or other diseases. The term “therapeutic effect” refers to the reduction, elimination, or prevention of the disease, symptoms of the disease, or side effects of the disease in the subject.

[0117] “In combination with”, “combination therapy” and “combination products” refer, in certain embodiments, to the concurrent administration to a patient of a first therapeutic and the compounds as used herein. When administered in combination, each component can be administered at the same time or sequentially in any order at different points in time. Thus, each component can be administered separately but sufficiently closely in time so as to provide the desired therapeutic effect.

[0118] “Concomitant administration” of a cancer therapeutic drug, ESA or tumor-directed antibody with a pharmaceutical composition of the present invention means administration with the high affinity reagent at such time that both the drug, ESA or antibody and the composition of the present invention will have a therapeutic effect. Such concomitant administration may involve concurrent (i.e. at the same time), prior, or subsequent administration of the drug, ESA or antibody with respect to the administration of a compound of the invention. A person of ordinary skill in the art would have no difficulty determining the appropriate timing, sequence and dosages of administration for particular drugs and compositions of the present invention.

[0119] As used herein, endpoints for treatment will be given a meaning as known in the art and as used by the Food and Drug Administration.

[0120] Overall survival is defined as the time from randomization until death from any cause, and is measured in the intent-to-treat population. Survival is considered the most reliable cancer endpoint, and when studies can be conducted to adequately assess survival, it is usually the preferred endpoint. This endpoint is precise and easy to measure, documented by the date of death. Bias is not a factor in endpoint measurement. Survival improvement should be analyzed as a risk-benefit analysis to assess clinical benefit. Overall survival can be evaluated in randomized controlled studies. Demonstration of a statistically significant improvement in overall survival can be considered to be clinically significant if the toxicity profile is acceptable, and has often supported new drug approval. Abenefit of the methods of the invention can include increased overall survival of patients.

[0121] Endpoints that are based on tumor assessments include DFS, ORR, TTP, PFS, and time-to- treatment failure (TTF). The collection and analysis of data on these time-dependent endpoints are based on indirect assessments, calculations, and estimates (e.g., tumor measurements). Disease- Free Survival (DFS) is defined as the time from randomization until recurrence of tumor or death from any cause. The most frequent use of this endpoint is in the adjuvant setting after definitive therapy. DFS also can be an important endpoint when a large percentage of patients achieve complete responses with chemotherapy,

[0122] Objective Response Rate. ORR is defined as the proportion of patients with tumor size reduction of a predefined amount and for a minimum time period. Response duration usually is measured from the time of initial response until documented tumor progression. Generally, the FDA has defined ORR as the sum of partial responses plus complete responses. When defined in this manner, ORR is a direct measure of drug antitumor activity, which can be evaluated in a singlearm study.

[0123] Time to Progression and Progression-Free Survival. TTP and PFS have served as primary endpoints for drug approval. TTP is defined as the time from randomization until objective tumor progression; TTP does not include deaths. PFS is defined as the time from randomization until objective tumor progression or death. The precise definition of tumor progression is important and should be carefully detailed in the protocol.

[0124] As used herein, the term “correlates,” or “correlates with,” and like terms, refers to a statistical association between instances of two events, where events include numbers, data sets, and the like. For example, when the events involve numbers, a positive correlation (also referred to herein as a “direct correlation”) means that as one increases, the other increases as well. A negative correlation (also referred to herein as an “inverse correlation”) means that as one increases, the other decreases.

[0125] [0126] As used herein, the term “metastasis” refers to the growth of a cancerous tumor in an organ or body part, which is not directly connected to the organ of the original cancerous tumor. Metastasis will be understood to include micrometastasis, which is the presence of an undetectable amount of cancerous cells in an organ or body part which is not directly connected to the organ of the original cancerous tumor. Metastasis can also be defined as several steps of a process, such as the departure of cancer cells from an original tumor site, and migration and/or invasion of cancer cells to other parts of the body.

[0127] Brain tumors. Glioma is a general term used to describe primary brain tumors, and is classified according to their presumed cell of origin. These include astrocytic tumors (astrocytoma, anaplastic astrocytoma and glioblastoma), oligodendrogliomas, ependymomas, and mixed gliomas. They are the most commonly occurring tumors of the central nervous system (CNS), which account for almost 80% of all malignant primary tumors of brain. These cell gliomas are further classified to low grade, atypical, and high-grade tumors based on cell morphology, mitotic activities, and molecular marker. The World Health Organization (WHO) grading system utilizes molecular markers that have shown to have significant prognostic and therapeutic implications.

[0128] Astrocytomas originate from astrocytes and can be encapsulated, preserving clear borders between normal and tumor cells, or infiltrative, indicating advanced grade. Low grades are common in children while high grades are common in young adults and older patients.

[0129] Oligodendrogliomas originate from oligodendrocyte cells. These are less infiltrating than astrocytomas and are common in adults.

[0130] Ependymomas originate from ependymal cells which are found lining the ventricular cavities and the central canal of the spinal cord. These are common in the pediatric patient population.

[0131] Glioblastoma multiforme is the most malignant and frequently occurring type of primary astrocytomas, which accounts for more than 60% of all brain tumors in adults. Despite the variety of modern therapies against GBM, it is still a deadly disease with extremely poor prognosis.

Patients usually have a median survival of approximately 14 to 15 months from the diagnosis.

[0132] The current international standard for the nomenclature and diagnosis of gliomas is

WHO (World Health Organization) classification. It classifies gliomas into grade I to IV on the basis of level of malignancy that is determined by the histopathological criteria. Grade I gliomas relate to lesions that have low proliferative potential and can be cured by surgical procedure, whereas grade II to IV gliomas are highly malignant and invasive. Glioblastoma multiforme is the most aggressive, invasive and undifferentiated type of tumor and has been designated Grade IV by WHO.

[0133] The most frequent location for GBM is cerebral hemispheres; with 95% of these tumors arise in supratentorial region, while only few percent of tumors occur in cerebellum, brainstem and spinal cord.

[0134] Macroscopically GBM is quite heterogeneous featuring multifocal hemorrhage, necrosis, and cystic and gelatinous areas. A characteristic feature of GBM is the variation in gross appearance of the tumor from one region to the other. Some of the regions as a result of tissue necrosis appear as soft and yellow in colour, whereas some of the tumor areas are firm and white and some regions show marked cystic degeneration and hemorrhage. The tumor usually is represented by a single, relatively large, irregular shaped lesion which usually arises in the white matter.

[0135] The WHO classification system has subtyped malignant gliomas on the basis of their histological and immunohistochemical similarity to putative cell of origin. Grading has been done according to the histological features related to biological aggressiveness i.e. necrosis, mitotic figures, and vascular endothelial hyperplasia. Based on clinical characteristic GBM can be subdivided into primary and secondary GBMs. Primary GBMs arise de novo without clinical and histological evidences of precursor lesion. Secondary GBMs progress slowly from preexisting lower-grade astrocytoma.

[0136] Imaging techniques carried out on individuals suspected of having brain tumors include invasive procedures such as catheter angiography and non-invasive tests such as computed tomography (CT) and magnetic resonance imaging (MRI) scans which are more routinely used for the purpose of visualising the tumors. The gold standard imaging technique used is MR scans due to their superior soft tissue contrast, which allows the complexity and the heterogeneity of the tumor lesion to be better visualized than a CT scan. Hypointense lesions are seen on Tl-weighted

MR scans, whereas hyperintense lesions are visualized on proton density weighted and T2- weighted images. Usual findings on a MR scan enhanced with gadolinium of patients with malignant gliomas shows a central area of necrosis, surrounded by white matter edema. Single photon emission computed tomography (SPECT) and positron emission tomography (PET) are also used.

[0137] Currently, GBM treatment is challenging, and treatment has limited success. Surgery is the principal component of standard care. The extent of surgical resection depends upon the site and eloquence of the brain area involved, however GBM is locally very invasive tumor and relapses are common. Radiotherapy finds use as well, particularly brachytherapy and stereotactic radiosurgery. Temozolomide is the only standard chemotherapy for patients with GBM, as adjuvant or concomitant with radiotherapy. Carboplatin, oxaliplatin, etoposide and irinotecan are among the second line drugs for patients who do not respond. Procarbazine, vincristine and CCNU are also used. Other approaches include anti-angiogenic agents like anti-VEGF monoclonal antibodies (Bevacizumab), anti-FGF antibodies, monoclonal antibodies targeting EGFR (Erlotinib and Gefitinib) and tyrosine kinase inhibitors.

[0138] Dynamic susceptibility contrast (DSC) MR perfusion relies on the susceptibility induced signal loss on T2* -weighted sequences that results from a bolus of gadolinium-based contrast passing through a capillary bed. The most commonly calculated parameters are rCBV, rCBF, and MTT. This technique is sometimes referred to as dynamic susceptibility contrast-enhanced MR perfusion. DSC perfusion exploits the regional susceptibility-induced signal loss caused by paramagnetic contrast agents, such as commonly used gadolinium-based compounds, on T2- weighted images. Although this technique can be performed with both T2 (e.g. spin echo) and T2* (e g. gradient-echo echo-planar) sequences, the former requires higher doses of contrast, which is why T2* techniques are more commonly employed. A bolus of gadolinium-containing contrast is injected intravenously and rapid repeated imaging of the tissue (most commonly brain) is performed during the first pass. This leads to a series of images with the signal in each voxel representing intrinsic tissue T2/T2* signal attenuated by susceptibility-induced signal loss proportional to the amount of contrast primarily in the microvasculature. After image acquisition, a region’s signal is interrogated over the time-course of the perfusion sequence, generating a signal intensity-time curve, from which various parameters can be calculated (e.g. rCBV, rCBF, MTT). These values can then be used to create color maps of regional perfusion.

[0139] The DSC-MRI contrast mechanism is based upon compartmentalization of paramagnetic GBCA that establishes magnetic susceptibility difference between the intra- and extravascular space, creating magnetic field gradients. Protons lose phase coherence as they diffuse through the transient, spatially varying gradients, yielding signal attenuation dependent upon physiological factors, including vessel or compartment size and proton diffusion rate, and experimental factors, including pulse sequence parameters and contrast agent concentration. Although this behavior can be solved analytically for limited regimes, this phenomenon has been most generally studied using Monte Carlo numerical methods that quantify the relationship between change in relaxation rate and the physiological and experimental parameters. These simulations yield the vessel size-dependence relationships for GRE (AR2*) and SE (AR2) change in relaxation rate, with AR2* plateauing for large diameter vessels, and AR2 peaking for capillarysized vessels. These relationships are qualitatively independent of vessel geometry.

[0140] Inflammation. Toxicity associated with CAR-T cell therapy can include cytokine release syndrome (CRS), which typically occurs within 1-2 weeks of dosing. Patients with CRS present a variety of symptoms such as high fever, chills, fatigue, hypotension, headache, tachycardia, dyspnea, respiratory insufficiency, capillary leak and even and even life-threatening multi-organ failure. CRS is due to the release of several inflammatory cytokines such as IL-6, interferon gamma (IFN-y), IL-1, IL-2, IL-10. Circulating levels of these cytokines can be monitored for safety. Blood concentration of IL-6 have been correlated to the development of grade >4 neurotoxicity in the first six days after CAR-T cell infusion.

[0141] Neurological symptoms associated with CAR-T cell therapy have been recorded as ICANS (Immune effector Cell-Associated Neurotoxicity Syndrome) and include symptoms such as headaches, confusion, agitation, seizures, tremors, trouble regarding speaking and understanding, aphasia, cranial nerve abnormalities and visual hallucination. The most significant risk factor for ICANS is the presence of antecedent severe CRS and its severity is closely related to the severity of CRS. It has been proposed that high blood concentrations of inflammatory cytokines such as IL-1, IL-6, IL-2, IL-15, IL-10, tumor necrosis factor (TNF-a), interferon-gamma (INF-y, granulocyte-macrophage colony-stimulating factor and the chemokines CXCL8 and CCL2) accumulate in the blood, activate endothelial cells, alter the permeability of the BBB and spread in the brain parenchyma, activating the resident microglia. [0142] Neurotoxicity typically occurs within 4-10 days of CAR-T cell infusion, but incidence rates, rate of progression and clinical presentation are variable based on the CAR-T product received. An ICANS score can be used to monitor levels of inflammation. Accurately establishing ICANS grading may be needed to manage neurotoxicity as best as possible. There are many grading score systems to measure ICANS. The American Society for Transplantation and Cellular Therapy (ASTCT) has proposed the ASTCT Consensus Grading for Cytokine Release Syndrome and Neurologic Toxicity Associated with Immune Effector Cells in order to standardize CRS and ICANS grading. The ASTCT grading scale for ICANS uses a tool called the Immune Effector Cell-Associated Encephalopathy (ICE) score. ICE is a score based on five parameters: orientation (year, months, city, hospital), attention (ability to count backwards from 100 to 10), naming (ability to name three simple objects), ability to follow simple commands, and handwriting (ability to write a standard sentence). Each parameter of ICE score gives points to the patient and it is used to determine the grading of ICANS. ICE score can be determined on all CAR-T cell therapy patients at baseline, after infusion, and is administered a minimum of twice daily or more frequently if ICANS is suspected. ICANS’ grading can be determined by combining ICE score with other parameters, including conscious level, presence of seizures, motor disturbance and presence of cerebral edema.

[0143] TIAN (tumor inflammation-associated neurotoxicity) is an on-tumor, on-target neurotoxicity syndrome, distinct from ICANS, observed in CNS tumors treated with CAR-T cell therapies. Its symptom spectrum varies from headache or fever to fatal hydrocephalus. The severity of symptoms is determined by several factors, including the neuroanatomical location of the tumor and the specific CAR-T target. While ICANS is a global neurological dysfunction leading to seizures, decreased level of consciousness or speaking/movement disorders, TIAN manifests with specific regional symptoms, linked to the site of the tumor and to local inflammation, without signs of widespread neuronal suffering. Within TIAN’s clinical manifestations, it is possible to identify two different syndromes, type 1 TIAN and type 2 TIAN. Type 1 TIAN reflects peritumoral therapy- related inflammation and is the result of increased intracranial pressure associated with obstruction of CSF flow which can potentially lead to herniation. Type 2 TIAN consists of a neuronal dysfunction related to local inflammation without CSF obstruction/hydrocephalus and, clinically, it manifests with a worsening of pre-existing neurological symptoms. Precautions can include CSF drainage via Ommaya device.

[0144] In some embodiments, symptoms related to neurotoxicity have no radiological equivalent in MRI. Only in cases of very severe neurotoxicity, abnormal MRI imaging patterns have been described, particularly microhemorrhages, white matter changes, cerebral edema, and/or diffuse leptomeningeal enhancement. When MRI detectable changes are present, the risk of more severe neurotoxicity associated with an unfavorable outcome is greater.

[0145] Other serious side effects of CAR T-cell therapy may include allergic reactions, changes in plasma electrolyte levels (potassium, sodium or phosphorous), anemia, leukopenia and neutropenia resulting in an increased risk of infections, fatigue or bleeding.

[0146] Toxicity may be evaluated according to the criteria in Table 1, Adverse Event Monitoring and Treatment Guidelines. In some embodiments, inflammation is at a level appropriate for additional CART cell administration when each of CRS, ICANS and TIAN toxi cities are at grade 1 or lower toxicity. In some embodiments inflammation is at a level appropriate for additional CART cell administration when any one of CRS, ICANS and TIAN toxicities are at grade 1 or lower toxicity. In some embodiments inflammation is at a level appropriate for additional CART cell administration when each of CRS, ICANS and TIAN toxicities are at grade 2 or lower toxicity. In some embodiments inflammation is at a level appropriate for additional CART cell administration when any one of CRS, ICANS and TIAN toxicities are at grade 2 or lower toxicity. In some embodiments inflammation is at a level appropriate for additional CART cell administration when each of CRS, ICANS and TIAN toxicities are at not higher than grade 3 toxicity. In some embodiments inflammation is at a level appropriate for additional CART cell administration when any one of CRS, ICANS and TIAN toxicities is at not higher than grade 3 toxicity.

[0147] In some embodiments, the disclosure relates to a method of treating TIAN, CRS and/or ICANS in a subject in need thereof comprising administering to the subject a therapeutically effective dose of CAR-T cells comprising B7-H3 and a therapeutically effective dose of a VEGF inhibitor, such as bevacizumab or a pharmaceutically acceptable salt thereof, wherein the therapeutically effect dose of the VEGF inhibitor is less than or equal to about 7.5 mg/kg and wherein the subject has been diagnosed with and treated for glioma. In some embodiments, the method comprises (i) after surgical resection of glioma in the brain of the subject, administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of a plurality of autologous T cells comprising a nucleic acid molecule encoding a chimeric antigen receptor (CAR) molecule that binds to cells expressing B7-H3 or a functional variant thereof; and, subsequently

(ii) administering a first dose of pharmaceutical composition comprising a therapeutically effective amount of bevacizumab; wherein the dose of bevacizumab is less than or equal to about 7.5 mg/kg, wherein the bevacizumab is administered no less than about two weeks after administering the plurality of autologous T cells; (iii) monitoring inflammation in the subject; and (iv) repeating step (i) after the determining when inflammation is at a pre-determined level of inflammation appropriate for repeating administration of the autologous T cells; wherein the autologous T cells are delivered in a first and a second fraction; the first fraction administered intracerebroventricularly and the second fraction administered locoregionally at a site of surgical resection. In some embodiments, the pre-determined level of inflammation appropriate for repeating administration of the autologous T cells is determined by physical examination of the subject and an MRI.

Table 1

[0148] B7H3. The B7 family proteins are a type of integral membrane proteins found on activated antigen-presenting cells and consists of structurally related cell-surface protein ligands that bind to receptors on lymphocytes. B7.1 (CD80) and B7.2 (CD86) are the two major types of B7 proteins, but currently, there are other proteins grouped in the B7 family, including inducible costimulator ligand (ICOS-L), and co-inhibitory programmed death- 1 ligand (PD-L1), programmed death-2 ligand (PD-L2), B7-H3, and B7-H4.

[0149] The B7 family produces a costimulatory or a coinhibitory signal to enhance or decrease the activity of the MHC-TCR signal between the antigen presenting cells (APC) and the T cells. Interaction of B7-family members with costimulatory receptors augments immune responses while interaction with coinhibitory receptors attenuates immune responses.

[0150] B7-H3 shares 20-27% amino acid identity with other B7 family members. It is a type-I transmembrane protein that primarily functions as a negative immunoregulatory protein, and is overexpressed in various human tumor tissues. The basic structure (21g form) of B7-H3 contains a single pair of IgV-like and IgC-like immunoglobulin domains, a transmembrane region, and a short highly diverse cytoplasmic tail. The dominantly expressed form of human 4IgB7- H3 contains tandemly duplicated VC domains with four Ig-like domains. Human B7-H3 has two isoforms (2IgB7-H3 and 4IgB7-H3). Serine and arginine-rich splicing factor 3 (SRSF3) involves the splicing of B7-H3 by directly binding to its exon 4 and/or 6. B7-H3 crystallized as an unusual dimer arising from the exchange of the G strands in the IgV domains of partner molecules, which indicates the dynamic nature and plasticity of the immunoglobulin fold.

[0151] B7-H3 has been observed to be expressed in different cellular compartments and different cancer types may have different B7-H3 localization profiles. Several immunostaining results show B7-H3 was expressed on the cell membrane and in cytoplasm of tumor tissues. B7-H3 is overexpressed in tumor tissues while its expression is low in normal tissues. B7-H3 overexpression and its negative correlation with patient survival has been reported in various malignancies. Additionally, B7-H3 is expressed in immune cells; monocytes, dendritic cells, myeloid derived suppresser cells (MDSCs), neutrophils, macrophages, B cells, and activated T cells. Furthermore, B7-H3 is also expressed in normal tissues and body fluids at very low levels, including epithelial cells, pleural effusion, anterior pituitary progenitor cells and human serum.

[0152] Anti-B7H3 antibodies useful in CART cell constructs have been described, and find use in the methods of the disclosure. Li et al. (2023) Cell Death Discov. 147 disclose B7-H3 scFv sequence based on 8H9 clone and constructed the second-generation B7-H3 CAR containing CD8a transmembrane region, CD28 intracellular costimulatory domain, and CD3^ intracellular signaling domain after confirming that B7-H3 is highly expressed in prostate cancer. Zhang et al. (2020) Mol Ther Oncolytics. 17: 180-189 disclose a B7-H3-specific mAb (mAb-J42), and scFv derived from mAb-J42 used for B7-H3 -targeted CAR-T Cells. Nehama et al. (2019) E Biomedicine 47:33-43 discloses B7-H3.CAR cassettes generated using the single chain variable fragment (scFv) from the anti-B7-H3 376.96 mAb. In some embodiments, the anti-B7-H3 scFv is derived from antibodies MGA271, 376.96, 8H9, or humanized 8H9, disclosed in WO 2021/207171. WO2017044699A1 (Mackall), discloses the antibodies MGA271 (CD276.MG), CD276.N1, CD276.N2, CD276.N3, CD276.N4, and CD276.N5.

[0153] In some embodiments, the anti-B7-H3 scFv is derived from antibody MGA271. In some embodiments, the anti-B7-H3 scFv derived from antibody MGA271 comprises a heavy chain variable region (VH) comprising an amino acid sequence of SEQ ID NO: 1, or a functional variant thereof having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereof. In some embodiments, the nucleotide sequence encoding the anti-B7- H3 heavy chain variable region (VH) comprises the sequence of SEQ ID NO: 2, or a functional variant thereof having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereof. In some embodiments, the anti-B7-H3 scFv derived from antibody MGA271 comprises a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NO: 3, or a functional variant thereof having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereof. In some embodiments, the nucleotide sequence encoding the anti-B7-H3 light chain variable region (VL) comprises the sequence of SEQ ID NO: 4, or a functional variant thereof having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereof. In some embodiments, the anti-B7-H3 scFv derived from antibody MGA271 comprises a linker sequence between the VH and the VL, said linker sequence comprising an amino acid sequence of SEQ ID NO: 5, or a functional variant thereof having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereof. In some embodiments, the nucleotide sequence encoding the linker sequence comprises the sequence of SEQ ID NO: 6, or a functional variant thereof having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereof. In some embodiments, anti-B7-H3 scFv derived from antibody MGA271 comprises the amino acid sequence of SEQ ID NO: 7, or a functional variant thereof having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereof. In some embodiments, the nucleotide sequence encoding the anti-B7- H3 scFv derived from antibody MGA271 comprises the sequence SEQ ID NO: 8, or a functional variant thereof having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereof.

[0154] In some embodiments, the anti-B7-H3 scFv is derived from antibody 376.96. In some embodiments, the anti-B7-H3 scFv derived from antibody 376.96 comprises a heavy chain variable region (VH) comprising an amino acid sequence of SEQ ID NO: 9, or a functional variant thereof having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereof. In some embodiments, the nucleotide sequence encoding the anti-B7- H3 heavy chain variable region (VH) comprises the sequence of SEQ ID NO: 10, or a functional variant thereof having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereof. In some embodiments, the anti-B7-H3 scFv derived from antibody 376.96 comprises a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NO: 11, or a functional variant thereof having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereof. In some embodiments, the nucleotide sequence encoding the anti-B7-H3 light chain variable region (VL) comprises the sequence of SEQ ID NO: 12, or a functional variant thereof having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereof. In some embodiments, the anti-B7-H3 scFv derived from antibody 376.96 comprises a linker sequence between the VH and the VL, said linker sequence comprising an amino acid sequence of SEQ ID NO: 13, or a functional variant thereof having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereof. In some embodiments, the nucleotide sequence encoding the linker sequence comprises the sequence of SEQ ID NO: 14, or a functional variant thereof having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereof. In some embodiments, anti-B7-H3 scFv derived from antibody 376.96 comprises the amino acid sequence of SEQ ID NO: 15, or a functional variant thereof having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereof. In some embodiments, the nucleotide sequence encoding the anti-B7- H3 scFv derived from antibody 376.96 comprises the sequence SEQ ID NO: 16, or a functional variant thereof having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereof.

[0155] In some embodiments, the anti-B7-H3 scFv is derived from antibody 8H9. In some embodiments, the anti-B7-H3 scFv derived from antibody 8H9 comprises a heavy chain variable region (VH) comprising an amino acid sequence of SEQ ID NO: 17, or a functional variant thereof having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about

95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereof. In some embodiments, the nucleotide sequence encoding the anti-B7-H3 heavy chain variable region (VH) comprises the sequence of SEQ ID NO: 18, or a functional variant thereof having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereof. In some embodiments, the anti-B7-H3 scFv derived from antibody 8H9 comprises a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NO: 19, or a functional variant thereof having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereof. In some embodiments, the nucleotide sequence encoding the anti-B7-H3 light chain variable region (VL) comprises the sequence of SEQ ID NO: 20, or a functional variant thereof having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereof. In some embodiments, the anti-B7-H3 scFv derived from antibody 8H9 comprises a linker sequence between the VH and the VL, said linker sequence comprising an amino acid sequence of SEQ ID NO: 21, or a functional variant thereof having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereof. In some embodiments, the nucleotide sequence encoding the linker sequence comprises the sequence of SEQ ID NO: 22, or a functional variant thereof having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereof. In some embodiments, anti-B7-H3 scFv derived from antibody 8H9 comprises the amino acid sequence of SEQ ID NO: 23, or a functional variant thereof having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereof. In some embodiments, the nucleotide sequence encoding the anti-B7- H3 scFv derived from antibody 8H9 comprises the sequence SEQ ID NO: 24, or a functional variant thereof having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereof.

[0156] In some embodiments, a B7-H3 CAR herein comprises a transmembrane region that is derived from CD8a, CD28, CD8, CD4, Ot)3z, CD40, CD134 (OX-40), or CD7. In some embodiments, the transmembrane region is derived from CD8OL In some embodiments, the CD8a transmembrane region comprises the amino acid sequence of SEQ ID NO: 25, or a functional variant thereof having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereof. In some embodiments, the nucleotide sequence encoding the CD8a transmembrane region comprises the sequence of SEQ ID NO: 26, or a functional variant thereof having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereof. In some embodiments, the transmembrane region is derived from CD28. In some embodiments, the CD28 transmembrane region comprises the amino acid sequence of SEQ ID NO: 27, or a functional variant thereof having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereof. In some embodiments, the nucleotide sequence encoding the CD28 transmembrane region comprises the sequence of SEQ ID NO: 28, or a functional variant thereof having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereof.

[0157] In some embodiments, a B7-H3 CAR herein comprises an intracellular co-stimulatory domain derived from CD28, 4- IBB, and CD3 zeta (Q.

[0158] In some embodiments, a B7-H3 CAR herein comprises an intracellular signaling domain derived from CD28, 4-1BB, and CD3 zeta (Q.

[0159] In some embodiments, the extracellular target binding domain further comprises a hinge domain between the B7-H3-binding moiety and the transmembrane domain. The hinge domain may be derived from all or part of naturally occurring molecules, such as from all or part of the extracellular region of CD8, CD4, or CD28, or from all or part of an antibody constant region. In some embodiments, the hinge domain may be a synthetic sequence that corresponds to a naturally occurring hinge domain sequence or may be an entirely synthetic hinge domain sequence. Non limiting examples of linker domains which may be used in accordance to the invention include a part of human CD8a, partial extracellular domain of CD28, FcyRllla receptor, IgG, IgM, IgA, IgD, IgE, an Ig hinge, or functional fragment thereof. The hinge may be mutated to prevent Fc receptor binding. The hinge domain can be derived from CD8a stalk, CD28, or IgGl. In certain embodiments, the hinge domain is derived from CD8a stalk. In various embodiments, the hinge domain is derived from CD28. The hinge domain can provide flexibility and accessibility between the B7-H3-binding moiety and the transmembrane domain. In some embodiments, the hinge domain comprises up to 300 amino acids, from 10 to 100 amino acids, or from 25 to 50 amino acids. In some embodiments, the hinge domain comprises

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 29), or a function variant having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereof.

[0160] In some embodiments a CAR herein can be of any length, i.e., can comprise any number of amino acids, provided that the polypeptides, proteins, or CARs retain their biological activity, e.g., the ability to specifically bind to antigen, detect diseased cells in a mammal, or treat or prevent disease in a mammal, etc, For example, the CAR can be about 50 to about 5000 ammo acids long, such as 50, 70, 75, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more amino acids in length.

[0161] In some embodiments, the antigen binding domain may comprise a light chain variable region and/or a heavy chain variable region. In some embodiments, the heavy chain variable region comprises a complementarity determining region (CDR) 1 region, a CDR2 region, and a CDR3 region. In some embodiments, the antigen binding domain may comprise one or more of a heavy chain CDR1 region comprising the amino acid sequence of SEQ ID NO: 30 or a function variant having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereof; a heavy chain CDR2 region comprising the amino acid sequence of SEQ ID NO: 32 or a function variant having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereof; and a heavy chain CDR3 region comprising the amino acid sequence of SEQ ID NO: 34 or a function variant having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereof. , In some embodiments, the heavy chain comprises all of the amino acid sequences of SEQ ID NOS: 30, 32, or 34 or a function variant having respective sequence identity of at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% thereof. In some embodiments, the antigen binding domain may comprise one or more of a heavy chain CDR1 region comprising an amino acid sequence encoded by SEQ ID NO: 31 or a function variant having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereof; a heavy chain CDR2 region comprising an amino acid sequence encoded by of SEQ ID NO: 33 or a function variant having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereof; and a heavy chain CDR3 region comprising an amino acid sequence encoded by SEQ ID NO: 35 or a function variant having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereof. , In some embodiments, the heavy chain comprises all of the amino acid sequences encoded by SEQ ID NOS: 31, 33, 35 or a function variant having respective sequence identity of at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% thereof.

[0162] Conditioning regimen. The methods of the disclosure are generally performed in the absence of a lymphodepleting conditioning regimen. Such a conditioning regimen may suppress the recipient’s immune system and deplete endogenous immune cells. The intensity of conventional conditioning regimens can vary significantly. See, for example, Bacigalupo et al. (2009) Biol Blood Marrow Transplant. 15(12): 1628-1633, herein specifically incorpoirated by reference.

[0163] Direct delivery to the brain. CART cells are generally delivered directly to the brain in the methods of the disclosure. In some embodiments the cells are delivered by intraventricular or intratumoral CNS administration. Examples of methods for local delivery, that is, delivery to the site of the tumor, include, e.g. through an Ommaya reservoir, e.g. for intrathecal delivery (see e.g. US Patent Nos. 5,222,982 and 5385582, incorporated herein by reference); by bolus injection, e.g. by a syringe, e g. intracranially; by continuous infusion, e.g. by cannulation, e.g. with convection (see e.g. US Application No. 20070254842, incorporated here by reference); or by implanting a device upon which the cells have been reversibly affixed (see e.g. US Application Nos. 20080081064 and 20090196903, incorporated herein by reference). [0164] Anti -VEGF agents. In some embodiments, inflammation in the individual is addressed by administration of an effective dose of an anti-VEGF agent, including, without limitation Bevacizumab, which can be administered at a conventional dosage. The anti-VEGF treatment provides non-steroidal inflammatory relief that is not cytolytic. In some embodiments throughout treatment the individual is kept to low or no steroid treatment, usually less than or equal to 4 mg dexamethasone per day, or an equivalent thereof. An anti-VEGF agent, in some embodiments, is an “anti-VEGF antibody,” which is an antibody that binds to VEGF with sufficient affinity and specificity. An anti-VEGF antibody, in some embodiments, is a monoclonal antibody that binds to the same epitope as the monoclonal anti-VEGF antibody A4.6.1 produced by hybridoma ATCC HB 10709.

[0165] In some embodiments, the effective dose of a VEG inhibitor is determined based on the specific tissue, rate of release from the implant, size of the implant, and the like. In some embodiments, the effective dose is empirically determined by one of skill in the art. In some embodiments, the dose provides for biological activity equivalent to 1 pg, 10 pg, 100 pg, 1 mg, 5 mg, 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 250 mg, 500 mg, 750 mg, or 1 g of soluble VEGF receptor. In some embodiments, the dose provides for biological activity equivalent to about 1 pg, about 10 pg, about 100 pg, about 1 mg, about 5 mg, about 10 mg, about 25 mg, about 50 mg, about 75 mg, about 100 mg, about 250 mg, about 500 mg, about 750 mg, or 1 g of soluble VEGF receptor. In some embodiments, the dose provides for biological activity equivalent to from about 1 pg soluble VEGF receptor to about 10 pg, about 100 pg, about 1 mg, about 5 mg, about 10 mg, about 25 mg, about 50 mg, about 75 mg, about 100 mg, about 250 mg, about 500 mg, about 750 mg, or 1 g of soluble VEGF receptor. In some embodiments, the dose provides for biological activity equivalent to from about 10 pg soluble VEGF receptor to about 100 pg, about 1 mg, about 5 mg, about 10 mg, about 25 mg, about 50 mg, about 75 mg, about 100 mg, about 250 mg, about 500 mg, about 750 mg, or 1 g of soluble VEGF receptor. In some embodiments, the dose provides for biological activity equivalent to from about 100 pg soluble VEGF receptor to about 1 mg, about 5 mg, about 10 mg, about 25 mg, about 50 mg, about 75 mg, about 100 mg, about 250 mg, about 500 mg, about 750 mg, or 1 g of soluble VEGF receptor. In some embodiments, the dose provides for biological activity equivalent to from about 1 mg soluble VEGF receptor to about 5 mg, about 10 mg, about 25 mg, about 50 mg, about 75 mg, about 100 mg, about 250 mg, about 500 mg, about 750 mg, or 1 g of soluble VEGF receptor. In some embodiments, the dose provides for biological activity equivalent to from about 5 mg soluble VEGF receptor to about 10 mg, about 25 mg, about 50 mg, about 75 mg, about 100 mg, about 250 mg, about 500 mg, about 750 mg, or 1 g of soluble VEGF receptor. In some embodiments, the dose provides for biological activity equivalent to from about 10 mg soluble VEGF receptor to about 25 mg, about 50 mg, about 75 mg, about 100 mg, about 250 mg, about 500 mg, about 750 mg, or 1 g of soluble VEGF receptor. In some embodiments, the dose provides for biological activity equivalent to from about 25 mg soluble VEGF receptor to about 50 mg, about 75 mg, about 100 mg, about 250 mg, about 500 mg, about 750 mg, or 1 g of soluble VEGF receptor. In some embodiments, the dose provides for biological activity equivalent to from about 50 mg soluble VEGF receptor to about 75 mg, about 100 mg, about 250 mg, about 500 mg, about 750 mg, or 1 g of soluble VEGF receptor. In some embodiments, the dose provides for biological activity equivalent to from about 75 mg soluble VEGF receptor to about 100 mg, about 250 mg, about 500 mg, about 750 mg, or 1 g of soluble VEGF receptor. In some embodiments, the dose provides for biological activity equivalent to from about 100 mg soluble VEGF receptor to about 250 mg, about 500 mg, about 750 mg, or 1 g of soluble VEGF receptor. In some embodiments, the dose provides for biological activity equivalent to from about 250 mg soluble VEGF receptor to about 500 mg, about 750 mg, or 1 g of soluble VEGF receptor. In some embodiments, the dose provides for biological activity equivalent to from about 500 mg soluble VEGF receptor to about 750 mg, or 1 g of soluble VEGF receptor. In some embodiments, the dose provides for biological activity equivalent to from about 750 mg soluble VEGF receptor to 1 g of soluble VEGF receptor. In some embodiments, the dose is administered at a single time point, e.g. as a single implant; or may be fractionated, e.g. delivered in a microneedle configuration. In some embodiments, the dose is administered, once, two, three time, 4 times, 5 times, 10 times, or more as required to achieve the desired effect, and administration may be daily, every 2 days, every 3 days, every 4 days, weekly, bi-weekly, monthly, or more.

In addition to bevacizumab, the following are useful specific VEGF inhibitors, and some embodiments comprise any one or more of the following ant- VEGF inhibitors: bevacizumab, ABT-869 (Abbott) including formulations for oral administration and closely related VEGF inhibitors; AEE-788 (Novartis) (also called AE-788 and NVP-AEE-788, among others) including formulations for oral administration and closely related VEGF inhibitors; AG-13736 (Pfizer) (also called AG-013736) including formulations for oral administration and closely related VEGF inhibitors; AG-028262 (Pfizer) and closely related VEGF inhibitors; Angiostatin (EntreMed) (also called CAS Registry Number 86090-08-6, Kl-4, and rhuAngiostatin, among others) and closely related inhibitors as described in, among others, US Patent Nos. 5,792,825 and 6,025,688 , particularly in parts pertaining to Angiostatin and closely related VEGF inhibitors, their structures and properties, and methods for making and using them; Avastin™ (Genentech) (also called bevacizumab, R-435, rhuMAB-VEGF, and CAS Registry Number 216974-75-3, among others) and closely related VEGF inhibitors; AVE-8062 (Ajinomoto Co. and Sanofi-aventis) (also called AC-7700 and combretastatin A4 analog, among others), and closely related VEGF inhibitors; AZD-2171 (AstraZeneca) and closely related VEGF inhibitors; Nexavar® (Bayer AG and Onyx) (also called CAS Registry Number 284461-73-0, BAY-43-9006, raf kinase inhibitor, sorafenib, sorafenib analogs, and IDDBCP 150446, among others) and closely related VEGF inhibitors; BMS-387032 (Sunesis and Bristol-Myers Squibb) (also called SNS-032 and CAS Registry Number 345627-80-7, among others) and closely related VEGF inhibitors; CEP-7055 (Cephalon and Sanofi-aventis) (also called CEP-11981 and SSR-106462, among others) and closely related VEGF inhibitors; CHIR-258 (Chiron) (also called CAS Registry Number 405169-16-6, GFKI, and GFKI-258, among others) and closely related VEGF inhibitors; CP-547632 (OSI Pharmaceuticals and Pfizer) (also called CAS Registry Number 252003-65-9, among others) and closely related VEGF inhibitors such as, for instance, CP-564959; E-7080 (Eisai Co.) (also called CAS Registry Number 417716-92-8 and ER-203492-00, among others) and closely related VEGF inhibitors; 786034 (GlaxoSmithKline) and closely related VEGF inhibitors; GW-654652 (GlaxoSmithKline) and closely related indazolylpyrimidine Kdr inhibitors; IMC-1C11 (ImClone) (also called DC-101 and c-plCl 1, among others) and closely related VEGF inhibitors; KRN-951 (Kirin Brewery Co.) and other closely related quinoline-urea VEGF inhibitors; PKC-412 (Novartis) (also called CAS Registry Number 120685-11-2, benzoylstaurosporine, CGP -41251, midostaurin, and STI-412, among others) and closely related VEGF inhibitors; PTK-787 (Novartis and Schering) (also called CAS Registry Numbers 212141-54- 3 and 212142-18-2, PTK/ZK, PTK-787/ZK- 222584, ZK-22584, VEGF-TKI, VEGF-RKI, PTK-787A, DE-00268, CGP-79787, CGP- 79787D, vatalanib, ZK-222584, among others) and closely related anilinophthalazine derivative VEGF inhibitors; SU11248 (Sugen and Pfizer) (also called SU-11248, SU-011248, SU-11248J, Sutent®, and sunitinib malate, among others) and closely related VEGF inhibitors; SU-5416 (Sugen and Pfizer/Pharmacia) (also called CAS Registry Number 194413-58-6, semaxanib, 204005-46-9, among others) and closely related VEGF inhibitors; SU-6668 (Sugen and Taiho) (also called CAS Registry Number 252916-29-3, SU-006668, and TSU-68, among others) and closely related VEGF inhibitors as described in, among others, WO-09948868 , WO-09961422 , and WO-00038519 , particularly in parts pertaining to SU-6668 and closely related VEGF inhibitors, their structures and properties, and methods for making and using them; VEGF Trap (Regeneron and Sanofi-aventis) (also called AVE-0005 and Systemic VEGF Trap, among others) and closely related VEGF inhibitors as described in, among others, WO-2004110490 , particularly in parts pertaining to VEGF Trap and closely related VEGF inhibitors, their structures and properties, and methods for making and using them; Thalidomide (Celgene) (also called CAS Registry Number 50-35-1, Synovir, Thalidomide Pharmion, and Thalomid, among others) and closely related VEGF inhibitors; XL-647 (Exelixis) (also called EXEL-7647, among others) and closely related VEGF inhibitors; XL-999 (Exelixis) (also called EXEL-0999, among others) and closely related VEGF inhibitors; XL-880 (Exelixis) (also called EXEL-2880, among others) and closely related VEGF inhibitors; ZD-6474 (AstraZeneca) (also called CAS Registry Number 443913-73-3, Zactima, and AZD-6474, among others) and closely related anilinoquinazoline VEGF inhibitors; and ZK-304709 (Schering) (also called CDK inhibitors (indirubin derivatives), ZK-CDK, MTGI, and multi -target tumor growth inhibitor, among others) and other closely related compounds including the indirubin derivative VEGF inhibitors described in WO-00234717 , WO- 02074742 , WG-02100401 , WO-00244148 , WO-02096888 , WO-03029223 , WG-02092079 , and WO-02094814 , particularly in parts pertinent to these and closely related VEGF inhibitors, their structures and properties, and methods for making and using them. See also Liu, Y. et al. “Recent progress on vascular endothelial growth factor receptor inhibitors with dual targeting capabilities for tumor therapy” (2022) J. Hematology & Oncology 15:89- and Cardones, A.R. and Banez, L.L. (2006) Curr. Pharm. Des. 12(3): 387-94, both of which are incorporated herein by reference as if fully set forth. Some embodiments comprise one or more VEGF inhibitor or pharmaceutically acceptable salt thereof, from any of the above cited references or a derivative thereof, wherein the derivative retains anti-VEGF activity. A derivative thereof, in some embodiments, comprises conservative amino acid substitutions when the VEGF inhibitor comprises an amino acid sequence; e.g., in bevacizumab. A derivative, in some embodiments, comprises alteration, deletion, or replacement of a substituent on a chemical structure. The VEGF inhibitors sunitinib (Formula I), sorafenib (Formula II), and pazopanib (Formula III) comprise the following structures:

[0166] CAR T cells have been modified to surface express a chimeric antigen receptor (a CAR- Treg cell). As used herein, the terms “chimeric antigen receptor T-cell” and “CAR-Treg cell” are used interchangeably to refer to a T-cell that has been recombinantly modified to express a CAR. In some embodiments, the CAR T cell is T cell comprising a chimeric antigen receptor for B7-H3. As used herein, the terms “chimeric antigen receptor” and “CAR” are used interchangeably to refer to a polyprotein comprising multiple functional domains arranged from amino to carboxy terminus in the sequence: (a) an antigen binding domain, or targeting domain, (b) a transmembrane domain (TD); (c) one or more cytoplasmic signaling domains (CSDs) wherein the foregoing domains (a) - (c) may optionally be linked by one or more spacer domains. The CAR may also further comprise a signal peptide sequence which is conventionally removed during post-translational processing and presentation of the CAR on the cell surface. CARs useful in the practice of the present invention are prepared in accordance with principles well known in the art. See e.g., Eshhaar et al. United States Patent No. 7,741,465 Bl issued June 22, 2010; Sadelain, et al (2013) Cancer Discovery 3(4):388-398; Jensen and Riddell (2015) Current Opinions in Immunology 33:9-15; Gross, et al. (1989) PNAS(USA) 86(24): 10024-10028; Curran, et al. (2012) J Gene Med 14(6):405-15. CAR-T cell therapy products have been approved for commercial use.

[0167] Generally, the targeting domain is specific for B7-H3, and may utilize an scFv as discussed herein. An ScFv is a polypeptide comprised of the variable regions of the immunoglobulin heavy and light chain of an antibody covalently connected by a peptide linker (Bird, et al. (1988) Science 242:423-426; Huston, et al. (1988) PNAS(USA) 85:5879-5883; S-z Hu, et al. (1996) Cancer Research, 56, 3055-3061. The generation of ScFvs based on monoclonal antibody sequences is well known in the art.

[0168] In some embodiments, a linker polypeptide molecule is optionally incorporated into the CAR between the antigen binding domain and the transmembrane domain to facilitate antigen binding. Moritz and Groner (1995) Gene Therapy 2(8) 539-546. In one embodiment, the linker is the hinge region from an immunoglobulin, e.g. the hinge from any one of IgGl, IgG2a, IgG2b, IgG3, IgG4, particularly the human protein sequences. Alternatives include the CH2CH3 region of immunoglobulin and portions of CD3. In those instances where the ABD is an scFv, an IgG hinge is effective. [0169] CARs useful in the practice of the present invention further comprise a transmembrane domain joining the targeting domain to the intracellular cytoplasmic domain of the CAR. The transmembrane domain is comprised of any polypeptide sequence which is thermodynamically stable in a eukaryotic cell membrane. The transmembrane spanning domain may be derived from the transmembrane domain of a naturally occurring membrane spanning protein or may be synthetic. In designing synthetic transmembrane domains, amino acids favoring alpha-helical structures are preferred. Transmembrane domains useful in construction of CARs are comprised of approximately 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 22, 23, or 24 amino acids favoring the formation having an alpha-helical secondary structure. Amino acids having a to favor alpha-helical conformations are well known in the art. See, e. Pace, et al. (1998) Biophysical Journal 75: 422- 427. Amino acids that are particularly favored in alpha helical conformations include methionine, alanine, leucine, glutamate, and lysine. In some embodiments, the CAR transmembrane domain may be derived from the transmembrane domain from type I membrane spanning proteins, such as CD3i CD4, CD8, CD28, etc.

[0170] The cytoplasmic domain of the CAR polypeptide comprises one or more intracellular signal domains. In one embodiment, the intracellular signal domains comprise the cytoplasmic sequences of the T-cell receptor (TCR) and co-receptors that initiate signal transduction following antigen receptor engagement and functional derivatives and sub-fragments thereof. A cytoplasmic signaling domain, such as those derived from the T cell receptor ^-chain, is employed as part of the CAR in order to produce stimulatory signals for T lymphocyte proliferation and effector function following engagement of the chimeric receptor with the target antigen. Examples of cytoplasmic signaling domains include but are not limited to the cytoplasmic domain of CD27, the cytoplasmic domain S of CD28, the cytoplasmic domain of CD137 (also referred to as 4-1BB and TNFRSF9), the cytoplasmic domain of CD278 (also referred to as ICOS), pl 10a, , or 8 catalytic subunit of PI3 kinase, the human CD3 (j- chain, cytoplasmic domain of CD134 (also referred to as 0X40 and TNFRSF4), FcsRly and 0 chains, MB1 (Iga) chain, B29 (Ig0) chain, etc.), CD3 polypeptides (8, A and s), syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lek, Fyn, Lyn, etc.) and other molecules involved in T-cell transduction, such as CD2, CD5 and CD28. [0171 ] In some embodiments, the CAR may also provide a co- stimulatory domain. The term “costimulatory domain”, refers to a stimulatory domain, typically an endodomain, of a CAR that provides a secondary non-specific activation mechanism through which a primary specific stimulation is propagated. The co-stimulatory domain refers to the portion of the CAR which enhances the proliferation, survival or development of memory cells. Examples of co-stimulation include antigen nonspecific T cell co-stimulation following antigen specific signaling through the T cell receptor and antigen nonspecific B cell co-stimulation following signaling through the B cell receptor. Co-stimulation, e.g., T cell co-stimulation, and the factors involved have been described in Chen & Flies. (2013) Nat Rev Immunol 13(4):227-42. In some embodiments of the present disclosure, the CSD comprises one or more of members of the TNFR superfamily, CD28, CD137 (4-1BB), CD134 (0X40), DaplO, CD27, CD2, CD5, ICAM-1, LFA-1 (CDlla/CD18), Lek, TNFR-I, TNFR-II, Fas, CD30, CD40 or combinations thereof.

[0172] Typically, the chimeric antigen receptor T-cells (CAR-T cells) are T-cells which have been recombinantly modified by transduction with an expression vector encoding a CAR construct. T- cells useful in the preparation of CAR-T cells contemplated herein include naive T-cells, central memory T-cells, effector memory T-cells or combination thereof. In some embodiments the T cells are autologous CD3⁺ cells obtained from a patient.

CAR-T cell manufacture

[0173] T cells for engineering as described above are collected from a subject or a donor may be separated from a mixture of cells by techniques that enrich for desired cells, or may be engineered and cultured without separation. An appropriate solution may be used for dispersion or suspension. Such solution will generally be a balanced salt solution, e.g. normal saline, PBS, Hank’s balanced salt solution, etc., conveniently supplemented with fetal calf serum or other naturally occurring factors, in conjunction with an acceptable buffer at low concentration, generally from about 5 to about 25 mM. Convenient buffers include HEPES, phosphate buffers, lactate buffers, etc. Techniques for affinity separation may include magnetic separation, using antibody-coated magnetic beads, affinity chromatography, cytotoxic agents joined to a monoclonal antibody or used in conjunction with a monoclonal antibody, e.g., complement and cytotoxins, or other convenient technique. Any technique may be employed which is not unduly detrimental to the viability of the selected cells.

[0174] The separated cells may be collected in any appropriate medium that maintains the viability of the cells, usually having a cushion of serum at the bottom of the collection tube. Various media are commercially available and may be used according to the nature of the cells, including dMEM, HBSS, dPBS, RPMI, Iscove’s medium, etc., frequently supplemented with fetal calf serum (FCS). The collected and optionally enriched cell population may be used immediately for genetic modification, or may be frozen at liquid nitrogen temperatures and stored, being thawed and capable of being reused. The cells will usually be stored in 10% DMSO, 50% FCS, 40% RPMI 1640 medium.

[0175] The T cells are, in some embodiments, expanded in a highly efficient process, e.g. where sufficient numbers of cells are generated for at least 4, at least 5, at least 6 doses of from about 5 to about lOOxlO⁶ cells, e.g. where at least 10⁸, 10⁹, 10¹⁰ engineered cells are generated in the initial culture period. Ex vivo expansion of the population of genetically engineered T cells may be performed in the presence of a tyrosine kinase inhibitors (e.g., dasatinib, ponatinib) (e.g., Src family kinase inhibitors) (e.g., Lek inhibitors), resulting in genetically engineered T cells that are resistant and/or less prone to T cell exhaustion. See, for example, WO2018183842A1, herein specifically incorporated by reference. In some embodiments, the therapeutically effective dose of T cells comprises from about 1 x 10⁶ cells to about 30 x 10⁶ cells. In some embodiments, the therapeutically effective dose of T cells comprises from about 5 x 10⁶ cells to about 30 x 10⁶ cells. In some embodiments, the therapeutically effective dose of T cells comprises from about 10 x 10⁶ cells to about 30 x 10⁶ cells. In some embodiments, the therapeutically effective dose of T cells comprises from about 15 x 10⁶ cells to about 30 x 10⁶ cells. In some embodiments, the therapeutically effective dose of T cells comprises from about 1 x 10⁶ cells to about 25 x 10⁶ cells. In some embodiments, the therapeutically effective dose of T cells comprises from about 1 x 10⁶ cells to about 20 x 10⁶ cells. In some embodiments, the therapeutically effective dose of T cells comprises from about 1 x 10⁶ cells to about 15 x 10⁶ cells. In some embodiments, the therapeutically effective dose of T cells comprises from about 10 x 10⁶ cells to about 30 x 10⁶ cells. [0176] In some embodiments, the VEGF inhibitor is pazopanib and the VEGF inhibitor is administered at about 800 mg daily for about 1, 2, 3, 4, 5, or 6 or more; or is sorafenib and administered at about 400 mg twice daily for about 1, 2, 3, 4, 5, or 6 or more days; or is sunitinib and is administered at about 37.5 mg daily for about 1, 2, 3, 4, 5, or 6 or more days.

Methods of Use

[0177] Methods are provided for treatment of glioma brain tumors, which methods can provide for a significant decrease in tumor volume. The methods of the disclosure may provide for increased overall survival of the individual being treated.

[0178] In an embodiment, an individual is treated for a high grade glioma by administration of multiple doses of chimeric antigen receptor (CAR) T cells specific for B7H3 antigen (CD276), wherein the method comprises obtaining a population of cells comprising CD3+ T cells from the individual; generating a population of transduced CART cells by a highly efficient manufacturing process; and treating the individual within a period of from 11-20 days following obtention of the CD3⁺ T cells, by a method comprising: (a) administering to the individual a dose of at least about 5 to about lOOxlO⁶ of the CART cells; (b) monitoring the individual for inflammation following step (a); (c) determining when inflammation is at a pre-determined level appropriate for additional CART cell administration; and (d) repeating steps (a) to (c) for a total of at least 2, 3, or 4 rounds of treatment. In some embodiments steps (a) to (c) are repeated for a total of at least 6 rounds of treatment, and between each step (a), the subject is dosed with an anti-VEGF agent at a dose of from about 2.5 to about 7.5 milligrams of agent per kilogram of the subject.

[0179] In some embodiments, the patient is treated with maximal safe surgical resection of the glioma. CAR T treatment can induce an inflammatory response and if the entire tumor is left in place, the volumetric constraints may result in injury and the need for anti-inflammatory intervention. By resecting the tumor, the volume available to alleviate pressure from inflammatory response to CAR T infusion is increased. Administration of the CART cells may be initiated within about 24 to about 48 hours following surgery.

[0180] In some embodiments, treatment is performed in the absence of testing the individual cancer for a confirmation of CD276 expression in order to avoid the exclusion of qualified patients in the event of a false negative or ambiguous B7H3 assessment. [0181 ] In an embodiment, an individual is treated for a high grade glioma by (a) administering to the individual a dose of from about 5 to about lOOxlO⁶ of the CAR-T cells, in one or two fractions; (b) monitoring the individual for inflammation following step (a); (c) administering a therapeutically effective dose of bevacizumab, a derivative thereof, a tautomer thereof or a pharmaceutically acceptable salt thereof at a dose of from about 2 to about 7.5 milligrams per kilogram of weight of the subject, (d) determining when inflammation is at a pre-determined level appropriate for additional CART cell administration; and (e) repeating steps (a) for a total of at least 2, 3, 4, 5 or 6 times, wherein, before each repeated step (a), step (c) is also re-administered.

[0182] In some embodiments steps (a) to (c) are repeated for a total of at least 6 rounds of treatment, and between each treatment, the subject is dosed with bevacizumab at a dose of from about 2.5 to about 7.5 milligrams of agent per kilogram of the subject. In some embodiments, the step of determining when inflammation is at a pre-determined level appropriate for additional CART cell administration comprises performing a physical examination of the patient, evaluating the presence or absence of neurological symptoms of brain herniation during a physical examination, performing an MRI or CT scan to image the brain of the subject. In some embodiments, a pre-determined level appropriate for additional CART cell administration, is the lack brain herniation or reduction of brain herniation to less than about 1 millimeter as determined by an MRI or CT scan of the brain of the subject. In some embodiments, a pre-determined level appropriate for additional CART cell administration, is the lack brain herniation or reduction of brain herniation to less than about 0.9, 0.8, 0.7, 0.6, or 0.5 millimeters as determined by an MRI or CT scan of the brain of the subject.

[0183] The also disclosure relates to a method of treating a high-grade glioma in a subject in need thereof comprising, after surgical resection of cancer cells in the brain of the subject:

(i) administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of a plurality of T cells comprising a nucleic acid molecule encoding a chimeric antigen receptor (CAR) molecule that binds to cells expressing B7-H3 or a functional variant thereof; and, subsequently;

(ii) administering a first dose of pharmaceutical composition comprising a therapeutically effective amount of bevacizumab; wherein the dose of bevacizumab is less than or equal to about 7.5 mg/kg, wherein the bevacizumab is administered no less than about two weeks after administering the plurality of autologous T cells; wherein the T cells are delivered in a first and a second fraction; the first fraction administered intracerebroventricularly and the second fraction administered locoregionally at a site of surgical resection; and wherein step (i) is repeated only after determining when inflammation from step (i) is at a pre-determined level appropriate for additional CART cell administration. In some embodiments, the pre-determined level of inflammation comprises is the absence of brain herniation of the subject or reduction of brain herniation of the subject to less than about 1.0, 0.9, 0.8, 0.7, 0.6, or 0.5 millimeters as determined by an MRI or CT scan of the brain of the subject. In some embodiments, the method further comprises a step of determining whether the subject has brain herniation and the step comprises performing a physical examination of the patient, evaluating the presence or absence of neurological symptoms of brain herniation during a physical examination, and/or performing an MRI or CT scan to image the brain of the subject. In some embodiments, a pre-determined level appropriate for a repeated step CART cell administration, is the lack brain herniation or reduction of brain herniation to less than about 1 millimeter as determined by an MRI or CT scan of the brain of the subject. In some embodiments, a pre-determined level appropriate for additional CART cell administration is the lack brain herniation or reduction of brain herniation to less than about 0.9, 0.8, 0.7, 0.6, or 0.5 millimeters as determined by an MRI or CT scan of the brain of the subject. In some embodiments, the method is further free of administration of steroids. In some embodiments, the method is free of steroid dosages of about 2.5 mg or more. In some embodiments, the steps (i) and (ii) are performed from about 4 to about 6 times. In some embodiments, the steps (i) and (ii) are performed from about 4 to about 6 times in a 6 month period. In some embodiments, the steps (i) and (ii) are performed from about 4 to about 6 times in a time period of from about 4 to about 6 month period. In some embodiments, the steps (i) and (ii) are performed from about 4 to about 6 times in a time period of from about 4 to about 6 month period after surgical resection of a tumor. In some embodiments, treatment is performed in the absence of lymphodepleting chemotherapy immediately prior to the CART-Cell treatment, thereby sparing the patient from additional stress.

[0184] Delivery of the CART cells is generally by an intraventricular or intracavitary route, which optimizes provision of therapeutic cells at the tumor site, and can reduce counterproductive systemic inflammatory responses. [0185] In some embodiments, inflammation in the individual is addressed by administration of an effective dose of an anti-VEGF agent, including without limitation, Bevacizumab. The anti-VEGF treatment provides non-steroidal inflammatory relief that is not cytolytic. In some embodiments throughout treatment the individual is kept to low or free of steroid treatment, usually less than or equal to 4 mg dexamethasone per day, or an equivalent thereof.

[0186] In some embodiments, the disclosure relates to a method of treating high grade glioma in an individual by administration of autologous chimeric antigen receptor (CAR) T cells. In some embodiments, the method comprises obtaining a population of cells comprising CD3+ T cells from the individual; generating a population of transduced CART cells; and treating the individual within a period of from 11-20 days following obtention of the CD3+ T cells. In some embodiment the treating following obtention of the CD3+ T cells comprises (a) administering to the individual a dose of from about 5 to about lOOxlO⁶ of the CART cells. In some embodiments, the method further comprises (b) monitoring the individual for inflammation following step (a). In some embodiments, the method further comprises (c) determining when inflammation is at a predetermined level appropriate for additional CART cell administration; and (d) repeating steps (a) to (c) for a total of at least 4 rounds of treatment. In some embodiments, at least 6 rounds of treatment are performed.

[0187] In some embodiments, the treating is performed in the absence of a prior lymphodepleting conditioning regimen to the individual. In some embodiments, the chimeric antigen receptor is specific for human B7H3. In some embodiments, the individual is treated within 48 hours prior to step (a) by maximal safe resection of tumor mass. In some embodiments, treatment is performed in the absence of testing the individual cancer for a confirmation of CD276 expression. In some embodiments, step (a) is performed by one or both of infusion by an intraventricular or intracavitary route. In some embodiments, the individual is treated for inflammation with an effective dose of an anti-VEGF agent. In some embodiments, the anti-VEGF agent is

Bevacizumab. In some embodiments, the individual is treated with an equivalent of not more than

4 mg dexamethasone per day. In some embodiments, the individual is assessed for inflammatory responses following an infusion of CART cells by MRI. In some embodiments, MRI is combined with dynamic susceptibility contrast (DSC) perfusion. In some embodiments, step (b) monitors indicia of cytokine release syndrome (CRS). In some embodiments, step (b) monitors indicia of immune effector cell-associated neurotoxicity syndrome. In some embodiments, step (b) monitors indicia of tumor inflammation-associated neurotoxicity. In some embodiments, CART cells are generated by a highly efficient manufacturing process. In some embodiments, the glioma is a glioblastoma, gliosarcoma, glioblastoma with oligodendroglial features, or glioblastoma with PNET features.

[0188] The disclosure also relates to a method of treating inflammation in a subject with glioma in need thereof comprising:

(i) after surgical resection of glioma in the brain of the subject, administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of a plurality of autologous T cells comprising a nucleic acid molecule encoding a chimeric antigen receptor (CAR) molecule that binds to cells expressing B7-H3 or a functional variant thereof; and, subsequently

(ii) administering a first dose of pharmaceutical composition comprising a therapeutically effective amount of bevacizumab; wherein the dose of bevacizumab is less than or equal to about 7.5 mg/kg, wherein the bevacizumab is administered no less than about two weeks after administering the plurality of autologous T cells; (iii) monitoring inflammation in the subject; and

(iv) repeating step (i) after the determining when inflammation is at a pre-determined level of inflammation appropriate for repeating administration of the autologous T cells; wherein the autologous T cells are delivered in a first and a second fraction; the first fraction administered intracerebroventricularly and the second fraction administered locoregionally at a site of surgical resection. In some embodiments, the method further comprises a step of determining whether the subject has brain herniation and the step comprises performing a physical examination of the patient, evaluating the presence or absence of neurological symptoms of brain herniation during a physical examination, and/or performing an MRI or CT scan to image the brain of the subject. In some embodiments, a pre-determined level appropriate for a repeated step CART cell administration is the lack brain herniation or reduction of brain herniation (as compared to a control or an image of the brain taken before step (i)), to at about or less than about 1 millimeter as determined by an MRI or CT scan of the brain of the subject. In some embodiments, a predetermined level appropriate for additional CART cell administration is the lack brain herniation or reduction of brain herniation to less than about 0.9, 0.8, 0.7, 0.6, or 0.5 millimeters as determined by an MRI or CT scan of the brain of the subject and compared to an image of the brain of the subject prior to step (i) or compared to an image of a brain of a control. In some embodiments, the method is further free of administration of steroids. In some embodiments, the method is free of steroid dosages of about 4mg, or about 2.5 mg or more. In some embodiments, the steps (i) and (ii) are performed from about 4 to about 6 times. In some embodiments, the steps (i) and (ii) are performed from about 4 to about 6 times in a 6 month period. In some embodiments, the steps (i) and (ii) are performed from about 4 to about 6 times in a time period of from about 4 to about 6 month period. In some embodiments, the steps (i) and (ii) are performed from about 4 to about 6 times in a time period of from about 4 to about 6 month period after surgical resection of a tumor. In some embodiments, treatment is performed in the absence of lymphodepleting chemotherapy immediately prior to the CART-Cell treatment, thereby sparing the patient from additional stress.

EXAMPLES

Example 1

[0189] B7H3 expression is found in many cancers, including gliomas, and is believed to contribute to invasiveness and poor outcomes. Here we provide data demonstrating: a significant unmet need for novel therapeutics for the treatment of gliomas, including isocitrate dehydrogenase (IDH) wildtype GBM; B7-H3 is highly overexpressed within the adult brain of glioma patients and is correlated with poor prognosis; and antitumor activity of the B7H3.BB.Z-CAR against numerous B7H3+ tumors, including GBM.

[0190] The clinical trial utilizes locoregional administration without lymphodepleting therapy for CAR T treatment in CNS malignancy. In this clinical trial, therapy comprises administration of an autologous CAR T cell product that uses a scFV targeting B7-H3, a CD8 hinge/transmembrane domain, the CD3(^ signaling endo-domain and a 4-1BB costimulatory signaling endo-domain. [0191] ICV administration of B7-H3CART at Dose Level 1 (10 x 10⁶ B7-H3CART+ cells (± 20%)) demonstrated clinical and radiographic antitumor effects as well as a robust inflammatory response within the local brain parenchyma without significant systemic adverse effects. The toxicities experienced have been expected based on preclinical and clinical data, as well as effectively managed through intensive monitoring and supportive care. None of the patients treated at DL1 experienced a dose limiting toxicity. A trial overview is as follows. The trial was an open label, non-randomized, single site Phase I study to test the manufacturing feasibility and safety of locoregional (LR) administration of B7-H3CART into the central nervous system of adult subjects with recurrent IDH wild-type GBM using a standard 3+3 dose escalation design. Patients had resectable disease with a Karnofsky Performance score of greater than or equal to 60. Steroides were limited to less than or equal to 4 mg of decadron daily. B7-H3CART were administered locoregionally (Intracerebroventricular and Intratumoral) at a doses levels -1, 1, 2, 3, and 4 of 5 x 10⁶, 10 x 10⁶, 25 x 10⁶, 50 x 10⁶, and 100 x 10⁶ cells, respectively. Primary outcome measure were number of succefully manufactured products (B7-3CART) that met minimum dose levels, and maximum tolerated dose (MTD) or recommended phase 2 dose (RP2D). Secondary outcome measures were cumulative safety of B7-H3CART, immunotherapy response assessment in neuro-oncology (iRANO) in subjects with recurrent IDH wild-type GBM, time to progression (TTP), median overall survival (OS), and percentage of subjects able to receive at least three (3) doses of B7-H3CART.

[0192] All 10 x 10^ B7-H3CART+ cells were infused in a single infusion via two Ommaya catheters, one in the ventricle and one in the resection cavity. A debulking procedure is performed in advance to optimize the effectiveness of the CAR. We have seen target activity in our first 3 patients and have established manufacturing feasibility in all 3 patients.

[0193]

Protocol

[0194] A schematic of the protocol is shown in FIG. 8. Non-mobilized autologous PBMC are obtained by leukapheresis in all enrolled subjects and transduced with Efla-CAR276 lentiviral vector. Cryopreserved PBMC stored from participation in other institutional cell therapy or cell collection studies may be used to generate the cellular. In brief, cryopreserved PBMC undergo selection, activation, transduction with lentiviral vector, co-culture with dasatinib and protamine sulfate, expansion and formulation in a GMP Facility using the Miltenyi CliniMACS Prodigy® system for the manufacture of B7-H3CART cells.

[0195] Subjects undergo standard of care maximal surgical resection/debulking of their tumor at which time an ICV catheter is placed. In some instances, the tumor bed may not communicate directly with the CSF, in which case a second catheter (i.e. Ommaya catheter or equivalent catheter device with reservoir) is placed intratumorally, at the neurosurgeon’s discretion, based on assessment of disease location, presence of multiple tumors, leptomeningeal disease, etc. Subjects undergo a postoperative brain MRI within 72 hr. after resection.

[0196] Subjects who meet cell infusion eligibility have the first dose of B7-H3CART cells infused within 60 hours of the postoperative brain MRI. Corticosteroid doses must be < 4 mg/day. The total dose/infusion of B7-H3CART is a flat dose of CAR⁺ transduced T cells. Four dose levels are tested:

[0197] A 28-day safety evaluation window follows the first infusion of B7-H3C ART. Subjects who continue to meet cell infusion eligibility receive up to six (6) doses of B7-H3CART administered every 28 days (-7 / +14 days). The dose schedule is delayed if the subject needs more time to recover from toxicity or to receive interval bevacizumab or surgical intervention. The second dose cannot be administered before Day 28 in order to allow completion of the DLT assessment period whereas doses 3, 4, 5, and 6 can be administered every 28 days (-7 / +14 days). MRI scans for disease assessment will occur every 2 months or prior to each B7-H3CART infusion, whenever possible.

[0198] All subjects who received at least one infusion are evaluable for toxicity assessment. The safety evaluation period for decision of dose escalation is 28 days after the date of first infusion of LR B7-H3CART. Adverse events that are at least possibly related to the investigational agent (B7-H3CART) with onset within the first 28 days following B7-H3CART infusion will be considered DLTs for determination of MTD. [0199] Hematologic toxicities are common after CAR T cell therapies therefore, the following criteria will be considered: Subjects with preexisting history of abnormal counts (cytopenias including anemia, thrombocytopenia, lymphopenia, neutropenia and white blood cell decreased), or preexisting coagulopathy, will not be evaluable for hematological toxicity Subjects evaluable for hematologic toxicity: Any Grade 4 neutropenia or thrombocytopenia lasting > 14 days despite best supportive care is a DLT. Among non-hematologic toxicities area any Grade 5 toxicity, CRS toxicity Grade 4 in severity, or Grade 3 in severity for greater than 7 days, Grade 3 or greater fever lasting > 14 days, grade 4 infection uncontrolled for > 7 days, grade 3 or greater infusion reactions lasting more than 24 hours despite standard supportive care, grade 4 neurotoxicity, (life threatening, urgent intervention needed) for greater than 96 hours, any new Grade 3 neurotoxicity (not present at baseline) lasting longer than 21 days, any other Grade 3 or greater, non- hematological toxicity lasting longer than 72 hours which is possibly, probably, or definitely attributed to B7- H3CART, and occurring within 28 days of investigational product administration will be considered a DLT with the following exceptions, Grade 3 neurotoxicity; Grade 3 diarrhea that resolves to < Grade 2 within 4 days; Grade 3 or greater isolated elevations in laboratory values will not be considered DLT unless they result in any one of the following: Discontinuation from the study therapy; medically significant requiring hospitalization or prolongation of hospitalization; judged by the Investigator to be of significant clinical impact, Hepatic function test (e g. transaminase, alkaline phosphatase, bilirubin or other liver function test) elevation < lOx ULN, provided there is resolution to < Grade 2 or baseline within 14 days.

Results

[0200] Development and Optimization ofB7-H3CART Construct. The B7H3.BB.z-CAR construct to be tested in the proposed trial is shown in FIG. 1. We utilized the scFv from the MGA271 mAb, which has demonstrated safety as a naked monoclonal as well as an antibody- drug conjugate. Iterative engineering revealed that a B7H3. BB.z-CAR incorporated into the MGA271 scFv, a CD8 hinge/transmembrane and CD3 zeta and 4- IBB signaling endodomains mediated specific and potent activity against pediatric sarcomas and pediatric brain tumors in preclinical models after intravenous administration. [0201 ] Clinical Effectiveness in Patients Treated with Locoregional B7-H3CART at Stanford. Three subjects were enrolled and treated on Dose Level 1 (DL1): locoregionally administration of 10 x 10⁶ B7-H3CART/kg. Cells were successfully manufactured and met release criteria for all 3 patients. Important conclusions resulting from our experience caring for the first 3 patients on study are summarized here.

[0202] On Target Specificity. Our experience treating subject 001 with left frontal GBM was notable for signs and symptoms of inflammation following the first cycle of CAR T administration in the local intracerebral parenchyma surrounding the surgical resection cavity (Figure 12). This local inflammation was accompanied by symptoms including headache, worsening aphasia, mild right facial droop, and intermittent fevers. These symptoms were treated with anti-inflammatory agents including anakinra, acetaminophen, and bevacizumab with eventual resolution. Importantly, there was no off-target inflammation seen in other organ systems, contralateral cerebral hemisphere, or outside of the immediate vicinity of the original tumor resection. This shows demonstrated on target specificity of our CAR T product to the location of expected residual GBM disease and trafficking of the CAR T product to anticipated loco-regional GBM cell infiltration.

[0203] Loco-regional CNS Inflammation'. Subject 002 experienced signs and symptoms of loco- regional CNS inflammation after each of first 3 cycles of CAR T treatment including intermittent fevers, worsening of neurologic deficits such as aphasia, headaches, and altered mental status with eventual resolution following concomitant medication administration. Radiographic evidence of this inflammatory response was seen as ependymal enhancement of the contralateral ventricle on brain MRI (FIG. 5) which resolved on follow up imaging.

[0204] Intratumoral and Intraventricular CAR T administration'. Subject 003 had two Ommaya catheters placed during surgery, one in the right frontal tumor bed and the second in the left frontal ventricle (FIG. 3, FIG. 4) to allow for administration of CAR T treatment. This patient had gross total resection of her tumor and a sufficient resection cavity that successfully allowed for split administration of her CAR T dose between the catheter left at the site of resection and the intraventricular space.

[0205] In summary, ICV administration of B7- H3CART at DL1 demonstrated clinical and radiographic antitumor effects (FIG. 5) as well as a robust inflammatory response within the local brain parenchyma without significant systemic adverse effects. The toxicities experienced have been expected based on preclinical and clinical data, as well as effectively managed through intensive monitoring and supportive care. None of the patients treated at DL1 experienced a dose limiting toxicity.

[0206] Our clinical experience to date demonstrates the efficacy of CAR T therapy. FIG. 7 includes images from patient 002 at key time points throughout the clinical course of therapy demonstrating the potent efficacy signal of this therapeutic and shows the changes in glioblastoma tumor mass pre- and post-CART treatment. This therapeutic provides for on-target, anti-tumor efficacy. Given the infiltrating nature of glioblastoma tumors, CAR T offers the potential to home to their target tumor cells with great promise of preventing further recurrences. Dose escalation using locoregional administration is continuing.

[0207] Correlative Analyses'. To address contributors to durable CAR T cell responses, we have developed a robust correlative platform to understand the biology of CAR T cells throughout the patient treatment course. Using this infrastructure, we have been able to characterize CAR T cell expansion in patient peripheral blood, define immune activation signatures throughout the treatment course in peripheral blood and CSF, and identify immune suppressive contributors to poor CAR T cell responses. These findings have been central to our ability to iterate and improve upon the clinical responses seen in our ongoing CAR T cell clinical trials.

[0208] Based on this experience, we have incorporated a parallel robust and extensive correlative platform to evaluate patient B7H3 CAR T cell biology throughout the treatment course. This includes obtaining peripheral blood and cerebrospinal fluid (CSF) samples throughout CAR-T treatments (FIG. 7A). Using these samples from patients treated on the trial so far, we have been able to demonstrate CAR T cells in peripheral blood following B7H3 CAR T cell administration, with peak levels varying by patient but more pronounced in CSF compared to peripheral blood

(FIGS. 7B and 7C). In our first three treated patients, we have collected 30 CSF samples, of which

23 have provided adequate sample for single-cell RNA-sequencing. These samples are evaluated for T cell and immune suppressive contributors to B7H3 CAR T cell functionality in patients. The correlative studies identify the contributors to CAR T cell activity in patients, including tumor-intrinsic and T cell-intrinsic contributors, From the tumor side, the correlatives assess the relationship between antigen modulation and patient response. If the antigen is not present after treatment, recurrence reflects tumor heterogeneity and may require targeting additional antigens or incorporate proteins to induce endogenous immune responses (e.g. checkpoint inhibitors). Alternatively, if there is recurrence and the antigen is not modulated, the limitation in activity can stem from the CAR T cells themselves.

Methods

[0209] Manufacturing B7-H3C ART'. For the generation ofB7-H3CART drug substance, apheresis is sent from BMT-CTF to the manufacturing site. On Day 0, subject CD4/CD8 T cells are enriched from autologous apheresis products by immunomagnetic selection using fully cGMP compliant reagents on the CliniMACS Prodigy device. The enriched cells are activated in the CentriCult unit of the CliniMACS Prodigy with Trans Act T Cell Reagent and cultured overnight in TexMACS medium supplemented with 3% HABS and IL-7 and IL-15. Following overnight culture, on Day 1 the activated T cells are fed with additional TexMACS medium and undergo an additional overnight culture. On Day 2, enriched T cells are transduced with the Efla-CAR276 lentiviral vector, with QC sampling for viability and cell counts during the culture process. On Day 3, transduced T cells undergo a culture wash to remove Transact activation agent and are supplemented with 0.05 mg/ml Dasatinib Solution. A second dose of 0.05 mg/ml Dasatinib Solution is added to the CentriCult unit on Day 5 of the culture process. A sample is taken for analysis of cell count and CAR T % and FACS analysis of phenotype from Day 6 QC sampling, which is used to determine if sufficient CAR+ T cells are available for both QC release testing and treatment of the subject at the proposed dose. If the number of CAR+ T cells is deemed sufficient to produce a full subject dose, the culture will proceed to harvest on Day 7. If there are insufficient number of CAR+ T cells on Day 6 to proceed to harvest, the culture may be extended to Day 11, with two more cycles of culture wash and addition of Dasatinib Solution. Cells are harvested and washed using the CliniMACS Prodigy in formulation medium comprising of Plasmalyte A, pH 7.4 supplemented with 4% HSA in the CentriCult unit. Drug substance is then concentrated and formulated in final formulation medium, diluted 1: 1 with CryoStor CS10, transferred to the product bag and is referred to as the drug product. Released product will be delivered via courier to the BMT-CTF for infusion. [0210] The CAR T cells suspended in culture medium are considered the drug substance, and the final drug product is considered to be those harvested cells suspended for infusion in Plasmalyte- A, pH 7.4 supplemented with 4% HSA, diluted 1 : 1 with CryoStorlO.

[0211] Vector Manufacturing'. The manufacturing process includes transient transfection using LTI four plasmid system, clarification, Benzonase® treatment, tangential flow filtration, and column chromatography. After purification, the vector is concentrated, formulated in formulation buffer, sterile filtered, and aliquoted into 1 mb vials.

[0212] Administration. B7-H3CART is administered locoregionally (LR) via an Ommaya catheter or equivalent catheter device with reservoir. Most catheters are placed intracerebroventricularly (ICV). In the event there is limited communication between the ventricle/cerebrospinal (CSF) fluid and the tumor cavity post operatively, at the surgeon’s discretion, in consultation with the PI, an additional catheter is placed in the tumor bed (intratumoral or IT).

[0213] For preparation of drug product for LR administration, the cryopreserved B7-H3CART drug product is thawed and washed using cold 0.9% Sodium Chloride, USP supplemented with 3% human albumin (HSA) in order to remove the dimethyl sulfoxide (DMSO) used for cryopreservation. Once the post-thaw total CAR+ cell count is determined, an additional wash using cold 0.9% Sodium Chloride + 3% HSAand centrifugation is performed. Supernatant samples are submitted to Stanford University Hospital Clinical Microbiology Laboratory for sterility and gram stain testing. The cell solution is filtered through a 30 pm MACS SmartStrainer, and a sample visually inspected under microscope to ensure no foreign particles are present in the sample. The B7-H3CART cells are subsequently resuspended in cold 0.9% Sodium Chloride + 3% HSA to deliver the target dose in 3 mL (acceptable range: 1-5 mL). If the total CAR+ cell count is below the target dose, all remaining cells will be used for LR injection. The final dose (+ 20%) is transferred to a sterile syringe(s) and capped. The syringe(s) is labeled according to ISBT 128 labeling standards and stored at 2-8C until the gram stain results are available.

[0214] Subjects who meet cell infusion eligibility receive the first dose of B7-H3CART cells infused within 60 hours of the postoperative brain MRI. The total dose/infusion of B7-H3CART is a flat dose of CAR+ transduced T cells. B7- H3CART administration is expected to recur every 28 days, with up to a 2 -week delay in the scheduled infusion permitted. If the subject does not meet the eligibility criteria for infusion during this window, the dose will be skipped and subject will be reevaluated for infusion eligibility at the next scheduled dose, 48 days (-7 / 14 days) after the last administered dose.

[0215] Each publication cited in this specification is hereby incorporated by reference in its entirety for all purposes.

[0216] It is to be understood that this invention is not limited to the particular methodology, protocols, cell lines, animal species or genera, and reagents described, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention.

Claims

What is Claimed is:

1. A method of treating a high-grade glioma in a subject in need thereof comprising, after surgical resection of cancer cells in the brain of the subject:

(i) administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of a plurality of T cells comprising a nucleic acid molecule encoding a chimeric antigen receptor (CAR) molecule that binds to cells expressing B7-H3 or a functional variant thereof; and, subsequently;

(ii) administering a first dose of pharmaceutical composition comprising a therapeutically effective amount of bevacizumab; wherein the dose of bevacizumab is less than or equal to about 7.5 mg/kg, wherein the bevacizumab is administered no less than about two weeks after administering the plurality of autologous T cells; wherein the T cells are delivered in a first and a second fraction; the first fraction administered intracerebroventricularly and the second fraction administered locoregionally at a site of surgical resection.

2. The method of claim 1, wherein the T cells are autologous T cells from the subject.

3. The method of either of claims 1 or 2, wherein the therapeutically effective amount of a plurality of T cells is administered at a dose of from about 1 x 10⁶ cells to about 100 x 10⁶ cells.

4. The method of any of claims 1 through 3, wherein the therapeutically effective amount of a plurality of T cells is administered at a dose of from about 15 x 10⁶ cells to about 35 x 10⁶ cells.

5. The method of any of claims 1 through 4, wherein the therapeutically effective amount of a plurality of T cells is administered at a dose of about 25 x 10⁶ cells.

6. The method of claim 1, wherein the autologous T cells are administered a total of at least about 2 times over a period of from about 8 to about 12 weeks; and wherein the step of administering the autologous T cells is repeated no less than about two weeks after the first dose of bevacizumab.

7. The method of either claim 1 or claim 2, wherein the method further comprises: (iii) monitoring the individual for inflammation following step (ii); and (iv) determining when inflammation is at a pre-determined level of inflammation appropriate for repeating administration of the T cells.

8. The method of any of claims 1 through 3, wherein the dose of bevacizumab is from about 3 mg/kg to about 7.5 mg/kg.

9. The method of any of claims 1 through 4, wherein the dose of bevacizumab is about 3 mg/kg and is administered no less than about 2 weeks after the first dose of autologous T cells.

10. The method of claim 3, wherein a pre-determined level of inflammation appropriate for repeating administration of the autologous T cells is determined by evaluation of the subject, wherein the subject exhibits an absence of herniation or midline shift upon magnetic resonance imaging (MRI) of the brain of the subject and/or the subject is free of clinical symptoms of intracerebral edema.

11. The method of any of claims 1 through 6, wherein the subject is human.

12. The method of claim 7, wherein the subject is from about 1 year to about 17 years old.

13. The method of any of claims 1 through 8, wherein the glioma is a glioblastoma, gliosarcoma, glioblastoma with oligodendroglial features, glioblastoma multiforme, or glioblastoma with PNET features.

14. The method of claim 2, wherein the step of administering bevacizumab is repeated from about 2 to about 10 times before a successive dose of autologous T cells.

15. The method of any of claims 1 through 10, wherein the T cells are delivered in a first and a second fraction sequentially or substantially contemporaneously; the first fraction administered intracerebroventricularly and the second fraction administered locoregionally at a site of surgical resection.

16. The method of claim 13, wherein the first fraction and the second fraction comprise substantially equal doses of cells and are administered a total of about 2 to about 6 times about every four weeks.

17. The method of claim 1, wherein the first fraction is about 12.5 x 10⁶ cells; and the second fraction is about 12.5 x 10⁶ cells.

18. A method of treating inflammation in a subject in need thereof comprising:

(i) after surgical resection of glioma in the brain of the subject, administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of a plurality of autologous T cells comprising a nucleic acid molecule encoding a chimeric antigen receptor (CAR) molecule that binds to cells expressing B7-H3 or a functional variant thereof; and, subsequently

(ii) administering a first dose of pharmaceutical composition comprising a therapeutically effective amount of bevacizumab; wherein the dose of bevacizumab is less than or equal to about 7.5 mg/kg, wherein the bevacizumab is administered no less than about two weeks after administering the plurality of autologous T cells;

(iii) monitoring inflammation in the subject; and

(iv) repeating step (i) after the determining when inflammation is at a pre-determined level of inflammation appropriate for repeating administration of the autologous T cells; wherein the autologous T cells are delivered in a first and a second fraction; the first fraction administered intracerebroventricularly and the second fraction administered locoregionally at a site of surgical resection.

19. The method of claim 11, wherein a pre- determined level of inflammation appropriate for repeating administration of the autologous T cells is determined by evaluation of the subject, wherein the subject exhibits an absence of herniation or midline shift upon magnetic resonance imaging (MRI) of the brain of the subject and/or the subject is free of clinical symptoms of intracerebral edema.

20. The method of claim 18 or 19, wherein the T cells are autologous T cells from the subject.

21. The method of either of any claims 18 through 20, wherein the therapeutically effective amount of a plurality of T cells is administered at a dose of from about 1 x 10⁶ cells to about 100 x 10⁶ cells.

22. The method of any of claims 18 through 21, wherein the therapeutically effective amount of a plurality of T cells is administered at a dose of from about 15 x 10⁶ cells to about 35 x 10⁶ cells.

23. The method of any of claims 18 through 22, wherein the therapeutically effective amount of a plurality of T cells is administered at a dose of about 25 x 10⁶ cells.

24. The method of any of claims 18 through 23, wherein the autologous T cells are administered a total of at least about 2 times over a period of from about 8 to about 12 weeks; and wherein the step of administering the autologous T cells is repeated no less than about two weeks after the first dose of bevacizumab.

25. The method of any of claims 18 through 24, wherein the dose of bevacizumab is from about 3 mg/kg to about 7.5 mg/kg.

26. The method of any of claims 18 through 25, wherein the dose of bevacizumab is about 3 mg/kg and is administered no less than about 2 weeks after the first dose of autologous T cells.

27. The method of claim 19, wherein the pre-determined level of inflammation appropriate for repeating administration of the autologous T cells is determined by evaluation of the subject, wherein the subject exhibits an absence of herniation or midline shift upon magnetic resonance imaging (MRI) of the brain of the subject and/or the subject is free of clinical symptoms of intracerebral edema.

28. The method of any of claims 18 through 27, wherein the subject is human.

29. The method of claim 28, wherein the subject is from about 1 year to about 17 years old.

30. The method of any of claims 18 through 29, wherein the glioma is a glioblastoma, gliosarcoma, glioblastoma with oligodendroglial features, glioblastoma multiforme, or glioblastoma with PNET features.

31. The method of any of claims 18 through 30, wherein the step of administering bevacizumab is repeated from about 2 to about 10 times before a successive dose of autologous T cells.

32. The method of any of claims 18 through 31, wherein the T cells are delivered in a first and a second fraction sequentially or substantially contemporaneously; the first fraction administered intracerebroventricularly and the second fraction administered locoregionally at a site of surgical resection.

33. The method of any of claims 18 through claim 32, wherein the first fraction and the second fraction comprise substantially equal doses of cells and are administered a total of about 2 to about 6 times about every four weeks.