Methods and dosing regimens for b7-h3 chimeric antigen receptors in human central nervous system cancers

Abstract

Methods of administration and dosing regimens for intracranial administration of T cells to human patients having a B7-H3-expressing central nervous system cancer are provided. The intracranially administered T cells can be genetically modified to express a B7-H3 chimeric antigen receptors (CAR). The B7-H3-expressing central nervous system cancer can be diffuse intrinsic pontine glioma (DIPG).

Description

S281-6010PCT / SCRL544WOMETHODS AND DOSING REGIMENS FOR B7-H3 CHIMERIC ANTIGEN RECEPTORS IN HUMAN CENTRAL NERVOUS SYSTEM CANCERSFIELD OF THE DISCLOSURE

[0001] The current disclosure describes methods for intracranial dosing and administration of therapeutic T cells that express a B7-H3 chimeric antigen receptor (CAR) to human patients with a central nervous system (CNS) cancer. The CNS cancer can be diffuse intrinsic pontine glioma (DI PG) and the human patients can be pediatric or young adult patients.BACKGROUND OF THE DISCLOSURE

[0002] Diffuse intrinsic pontine glioma (DI PG) is an aggressive type of childhood cancer that forms in the brainstem. It has been universally fatal with a median survival of less than 1 year from diagnosis and it affects 300 children per year in the United States.

[0003] In recent years, more targeted therapies to treat cancer have been developed. Targeted therapies specifically target cancer cells for destruction by identifying and exploiting specific molecular and / or immunophenotypic changes seen primarily in those cells. For example, many cancer cells preferentially express markers on their cellular surfaces which may be used as targets for antibody-based therapeutics.

[0004] Significant progress has been made in genetically engineering cells of the immune system to target and kill unwanted cell types, such as cancer cells. Many of these immune cells are T cells that have been genetically engineered to express a recombinant receptor, for example, a chimeric antigen receptor (CAR). CAR are proteins including several distinct subcomponents that allow the genetically modified immune cells to recognize and kill targeted cell types. The subcomponents include at least an extracellular component and an intracellular component expressed as a single protein or in a form that assembles into a functional unit. The extracellular component includes a binding domain that specifically binds a marker (e.g., an antigen) that is preferentially present on the surface of unwanted cells, such as cancer cells. When the binding domain binds such markers, the intracellular component signals the immune cell to destroy the bound cell. CAR can additionally include a transmembrane domain that can link the extracellular component to the intracellular component.

[0005] Particular examples of CAR T cells bind and kill cancer cells expressing B7-H3.SUMMARY OF THE DISCLOSURE

[0006] The current disclosure describes methods for intracranial (e.g., intracerebroventricular (ICV)) dosing and administration of T cells that express a B7-H3 chimeric antigen receptor (CAR)S281-6010PCT / SCRL544WOto human patients having a central nervous system cancer. In particular embodiments, provided methods include intracerebroventricularly administering doses of T cells expressing a B7-H3 CAR to a human patient in need thereof. In particular embodiments, the doses are escalating doses that can range from 0.1x107to 10x107cells / dose. In particular embodiments, the doses are repeated doses of 50x106cells / dose. In particular embodiments, each dose is administered every 14 to 29 days. In particular embodiments, the human patient in need thereof is a pediatric or young adult patient having diffuse intrinsic pontine glioma (DIPG). In particular embodiments, the human patient is pre-progression when the dosing regimen begins.BRIEF DESCRIPTION OF THE FIGURES

[0007] Some of the drawings submitted herewith may be better understood in color. Applicant considers the color versions of the drawings as part of the original submission and reserves the right to present color images of the drawings in later proceedings.

[0008] FIG. 1. Demographics and characteristics of all patients enrolled on BrainChild-03 Arm C.

[0009] FIG. 2. Pathologic diagnostic information for all patients. N / A means none available;aThe human patient did not receive CAR T cells.bHistologic diagnosis not obtained. Pathology report lists, “atypical cells seen both intraoperatively and in the permanent sections are suggestive of an infiltrating glioma. The catheter was placed into the ventricular system, most often in one of the lateral ventricles.

[0010] FIG. 3. BrainChild-03 Arm C trial design. CONSORT diagram of BrainChild-03 Arm C.

[0011] FIGs. 4A-4C. Survival following ICV B7-H3 CAR T cells. (4A) Swimmer plot describing patient history from time of diagnosis through death or most recent follow up. For patients with multiple progressions or longstanding ongoing progression, only initial progression may be noted. (4B) Kaplan-Meier survival after the initial B7-H3 CAR T cell infusion for all treated patients. (4C) Kaplan-Meier survival stratified by prior progression status at time of initial CAR T infusion.

[0012] FIG. 5. Tolerability of ICV B7-H3 CAR T cells. Common Terminology Criteria for Adverse Events (CTCAE) grading of all observed toxicities during the dose limiting toxicity (DLT) observation constituting an increase from baseline. If the toxicity was not present pre-Course 1 Week 1 administration, then no baseline grade is shown.

[0013] FIGs. 6A, 6B. Neuroimaging after locoregional B7-H3 CAR T cell infusion. (6A) Longitudinal MRIs of S021. T2-weighted (coronal top row, axial middle row) and axial postcontrast T1-weighted (bottom row) MRI images focused on the pontine lesion at various time points (see column labels). At diagnosis (DX), the pons is expanded with non-enhancing T2 hyperintensity, prepontine cistern effacement (dashed arrow), and partial effacement of the fourth ventricle (asterisk). Following radiation (Post RT, 4 months prior to immunotherapy (IT)), theS281-6010PCT / SCRL544WOlesion is smaller, with reduced prepontine cistern effacement (dashed arrow), persistent nodular T2 hyperintensity in the left dorsal pons (solid arrow), and therapy-related cystic changes in the central pons (arrowhead). From the initiation of IT (Pre IT) to 30 months Post IT, the overall pons size remained stable while hyperintensity decreased. (6B) Longitudinal MRI of the pontine lesion of S053. Axial T2-weighted (top row) and axial post-contrast T1-weighted (bottom row) MRI images, focused on the pontine lesion at various time points (see column labels). At diagnosis (DX), the pons is enlarged (arrows) with diffuse T2 hyperintensity, heterogeneous enhancement, and partial effacement of the fourth ventricle (asterisk). Following radiation (Post RT, 2 months prior to IT), there is further expansion of the pons and increased T2 signal abnormality (arrows), with persistent enhancement. From Pre IT to 2 months Post IT, the pontine lesion is smaller (arrows). However, by 4 months Post IT, the lesion is larger, with a new region of enhancement (arrowhead). At 6- and 8-months Post IT, the lesion size, T2 signal abnormality, and enhancement are reduced.

[0014] FIG. 7. Intracranial Detection of chimeric antigen receptor (CAR) T Cells in cerebrospinal fluid (CSF) during the DLT period. Detection of B7-H3 CAR T cells in CSF pre- and post- infusion during the DLT period consists of 10 scheduled collections: Cr1.W1.Pre, Cr1.W1.Post, Cr1.W3.Pre, Cr1.W3.Post, Cr1.W4, Cr2.W1.Pre, Cr2.W1.Post, Cr2.W3.Pre, Cr2.W3.Post, Cr2.W4, which are defined as (where Cr: course; W: week; Pre: pre-infusion, and Post: postinfusion). The x-axis shows the sequence of these collections. Samples with lymphocytes count below the limit of quantitation (LOQ) were excluded from analysis. Shaded circles represent samples where B7-H3 CAR T cells were detected, defined by lymphocyte / EGFRt+% levels above the limit of detection (LOD) and a minimal count (>1) of detected EGFRt+ cells. The size of the circles denotes the percentage of EGFRt+ lymphocytes (Lymph / EGFRt+%) detected in the sample. Notably, S014 had no sample collected due to the placement of the shunt, S018 only received one dose, and S045 missed all post-infusion collection. The human patient numbers for each row (from top to bottom) are: DR4: S061, S060, S059, S057, S053, S049, S045, S044, S041, S039; DR3: S036, S034, S033; DR2: S031, S027, S024, S023, S021, S018, S012; and DR1: S014, S008, S006, and S005.

[0015] FIG. 8. CSF CAR T cell detection. Levels of CAR detection by EGFRt+ across all patients and across all dose regimens (DR1, DR2, DR3 and DR4) during the DLT period. Gating Strategy: Live> Singlets> Lymphocyte> CD3+> EGFRt+.

[0016] FIG. 9. CSF Flow cytometry data. Flow cytometric analysis of longitudinal CSF samples collected during Course 1 and Course 2. The data display CD3+T cells as a percentage of total lymphocytes and CD4+ / CD8+T cells as percentages of CD3+T cells.S281-6010PCT / SCRL544WO

[0017] FIG. 10. CSF cytokine data. Absolute concentrations and pre-infusion versus post-infusion changes for cytokines exhibiting at least a two-fold change and a false discovery rate (FDR)-adjusted p-value < 0.05 in a linear mixed model. The model combined data from Cr1.W1 (n = 12), Cr1.W3 (n = 11), Cr2.W1 (n = 11), and Cr2.W3 (n = 10), resulting in n = 44 pre-infusion and n = 44 post-infusion measurements for each cytokine that met quality criteria. Human patients were included as random intercepts, and pre / post-infusion status was included as a fixed effect. Analytes were measured in duplicate; those with coefficients of variation (CV) greater than 25% were excluded. Concentrations were log2-transformed for analysis, and fold changes are presented on a linear scale. Subsequent columns show fold changes and adjusted p-values for the separate and combined infusion time points.

[0018] FIGs. 11 A, 11 B. Chemokine and cytokine concentrations in CSF during the DLT period measured using the Meso Scale Discovery (MSD) assay. (11A) Volcano plot of all 53 cytokines tested. Labels indicate cytokines exhibiting at least a two-fold change and a false discovery rate (FDR)-adjusted p-value < 0.05 across all four course-week combinations. (11B) Forest plot for six cytokines that demonstrate distinctive pre- and post-infusion patterns with subsequent infusions: those trending with cumulative infusions (C-reactive protein [CRP], serum amyloid A [SAA], and thymus and activation-regulated chemokine [TARC]) and those consistently upregulated or downregulated after each infusion (interferon gamma [IFNy], CXC motif chemokine ligand 10 [CXCL10, also known as interferon gamma-induced protein 10 (IP-10)], and granulocytemacrophage colony-stimulating factor [GM-CSF]). Data are presented as the model estimate of the post-pre change on a log2scale + / - SEM. For differential analyses, only samples with matched pre- and post-infusion pairs at each Course (Cr) and Week (W) combination from the same patient were included. For the analysis presented in the volcano plot, the linear mixed model combined data from Cr1.W1 (n = 12), Cr1.W3 (n = 11), Cr2.W1 (n = 11), and Cr2.W3 (n = 10), resulting in 44 pre-infusion and 44 post-infusion measurements for each cytokine meeting quality filters. Human patients were included as random intercepts, and pre / post-infusion status was included as a fixed effect. Analytes were measured in duplicate; samples with signal coefficients of variation (CV) greater than 25% were excluded; and concentrations below the lowest limit of detection (LLOD) were considered undetectable (0 pg / mL). Data were log2-transformed for analysis, and fold changes are presented on the log2scale.

[0019] FIG. 12. Representative flow gating strategy for CAR T cell detection. Gating strategy used for flow-based CAR detection in CSF. Selection of the singlet, viable [CD36-] lymphocyte population was performed prior to T cell gating and examination of the EGFRt+ CAR T cells and CD4 / CD8 expression.S281-6010PCT / SCRL544WO

[0020] FIG. 13. Example 2 study design schema.DETAILED DESCRIPTION

[0021] Diffuse intrinsic pontine glioma (DI PG) is an aggressive type of childhood cancer that forms in the brainstem. It has been universally fatal with a median survival of less than 1 year from diagnosis and it affects 300 children per year in the United States.

[0022] In recent years, more targeted therapies to treat cancer have been developed. Targeted therapies specifically target cancer cells by identifying and exploiting specific molecular and / or immunophenotypic changes seen primarily in those cells. For example, many cancer cells preferentially express markers on their cellular surfaces which may be used as targets for antibody-based therapeutics.

[0023] Significant progress has been made in genetically engineering cells of the immune system to target and kill unwanted cell types, such as cancer cells. Many of these immune cells are T cells that have been genetically engineered to express a recombinant receptor, for example, a chimeric antigen receptor (CAR). CAR are proteins including several distinct subcomponents that allow the genetically modified immune cells to recognize and kill targeted cell types. The subcomponents include at least an extracellular component and an intracellular component expressed as a single protein or in a form that assembles into a functional unit. The extracellular component includes a binding domain that specifically binds a marker (e.g., an antigen) that is preferentially present on the surface of unwanted cells, such as cancer cells. When the binding domain binds such markers, the intracellular component signals the immune cell to destroy the bound cell. CAR can additionally include a transmembrane domain that can link the extracellular component to the intracellular component.

[0024] Although CAR T cells have had substantial success in treating some cancers, challenges in the treatment of solid tumors remain including potential systemic inflammation and toxicity, on-target off-tumor toxicity, and trafficking and infiltration issues. In order to develop effective CAR T cell therapies while controlling potential toxicity, administration protocols and dosing regimens must be carefully developed and assessed.

[0025] The current disclosure describes methods for intracranial (e.g., intracerebroventricular (ICV)) dosing and administration of T cells that express a B7-H3 CAR. Particular embodiments provide administering doses of T cells expressing a B7-H3 CAR to a human patient in need thereof. In particular embodiments, doses are escalating doses that can range from 0.1x107to 10x107T cells / dose. For example, a dosing regimen can include a first dose including 0.1x107T cells / dose or 1x107T cells / dose, a second dose including 2.5x107T cells / dose, a third dose including 5x107T cells / dose, and a fourth dose including 10x107T cells / dose. In particularS281-6010PCT / SCRL544WOembodiments, a dosing regimen can include a first dose including 0.1x107T cells / dose or 1x107T cells / dose, a second dose including 5x107T cells / dose, a third dose including 10x107T cells / dose, and a fourth dose including 10x107T cells / dose. In particular embodiments, a dosing regimen can include a first dose including 0.1x107T cells / dose or 1x107T cells / dose, a second dose including 2.5x107T cells / dose, a third dose including 5x107T cells / dose, and a fourth dose including 5x107T cells / dose. In particular embodiments, a dosing regimen can include a first dose including 0.1x107T cells / dose or 1x107T cells / dose, a second dose including 0.1x107T cells / dose or 1x107T cells / dose, a third dose including 2.5x107T cells / dose, and a fourth dose including 2.5x107T cells / dose. In particular embodiments, a dosing regimen can include a first dose including 0.1x107T cells / dose or 1x107T cells / dose for a plurality of doses.

[0026] In particular embodiments, the doses are repeated doses of 50x106cells / dose.

[0027] In particular embodiments, each dose is administered at least every 14 to at least every 28 days. In particular embodiments, each dose is administered at least every 14 days. In particular embodiments, each dose is administered at least every 14 days ±2 days. In particular embodiments, each dose is administered twice a month. In particular embodiments, the dosing regimen is administered for as long as it provides therapeutic benefit to the human patient or until the human patient experiences unmanageable adverse events.

[0028] In particular embodiments, the human patient in need thereof has a B7-H3-related disorder, such as diffuse intrinsic pontine glioma (DIPG). In particular embodiments, the cancer of the human patient is pre-progression at time of administering. Additional conditions that can be treated according to the current disclosure include diffuse midline glioma (DMG), H3K27m-altered DMG, high grade glioma (HGG), glioblastoma (GBM), neuroblastoma, craniopharyngioma, ependymoma, an embryonal tumor, medulloblastoma, pineoblastoma, an atypical teratoid / rhabdoid tumor (ATRT), and an embryonal not otherwise specified (NOS) tumor

[0029] Aspects of the current disclosure are now described in more supporting detail as follows: (i) Human Patients with a B7-H3-Expressing Central Nervous System Cancer; (ii) T cells & CAR; (iii) Methods of Use; (iv) Selection for Clinical Trial; (v) Exemplary Embodiments; (vi) Experimental Example; and (vii) Closing Paragraphs. These headings are provided for organizational purposes only and do not limit the scope or interpretation of the disclosure.

[0030] (i) Human Patients with a B7-H3-Expressing Central Nervous System Cancer. In particular embodiments, the dosing and administration regimens disclosed herein can be used to treat human patients with a B7-H3-expressing central nervous system cancer.

[0031] B7 Homolog 3 (B7-H3), also known as CD276, is a type I transmembrane protein encoded by chromosome 15 in humans. The extracellular domain is composed of two identical pairs of anS281-6010PCT / SCRI.544WOimmunoglobulin variable domain and an immunoglobulin constant domain in humans (4lgB7-H3 isoform) due to exon duplication. The intracellular tail of B7-H3 is short and has no known signaling motif. B7-H3 is universally expressed among species. A soluble form, cleaved from the surface by a matrix metallopeptidase MMP or produced through alternative splicing of the intron, is also detectable in human sera.

[0032] B7-H3 is induced on antigen presenting cells and plays an important role in the inhibition of T cell function. Importantly, B7-H3 is highly overexpressed on a wide range of human solid cancers and often correlates with both negative prognosis and poor clinical outcome in patients. B7-H3 is linked to inhibiting immune responses, tumor cell proliferation, metastasis, therapeutic resistance, and tumor cell metabolism. In particular embodiments, B7-H3 [Homo sapiens] includes the sequence:MLRRRGSPGMGVHVGAALGALWFCLTGALEVQVPEDPVVALVGTDATLCCSFSPEPGFSLAQ LNLIWQLTDTKQLVHSFAEGQDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSFTCFVS IRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYQGYPEAEVFWQDGQGVPLTG NVTTSQMANEQGLFDVHSILRVVLGANGTYSCLVRNPVLQQDAHSSVTITPQRSPTGAVEVQV PEDPWALVGTDATLRCSFSPEPGFSLAQLNLIWQLTDTKQLVHSFTEGRDQGSAYANRTALF PDLLAQGNASLRLQRVRVADEGSFTCFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGD TVTITCSSYRGYPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHSVLRVVLGANGTYSCL VRNPVLQQDAHGSVTITGQPMTFPPEALWVTVGLSVCLIALLVALAFVCWRKIKQSCEEENAGA EDQDGEGEGSKTALQPLKHSDSKEDDGQEIA (SEQ ID NO: 1).

[0033] The term "B7-H3-positive cell" refers to a cell that expresses B7-H3 on its surface. The term “B7-H3-positive cancer cell" refers to a cancer cell that expresses B7-H3 on its surface. In some embodiments, expression of B7-H3 on the cell surface is determined, for example, using antibodies to B7-H3 in a method such as immunohistochemistry, FACS, etc. Alternatively, B7-H3 mRNA expression is considered to correlate to B7-H3 expression on the cell surface and can be determined by, for example, in situ hybridization and / or RT-PCR (including quantitative RT-PCR).

[0034] A “B7-H3-related disorder” is one where diseased or cancerous cells within a human patient express B7-H3, such that B7-H3 provides an antigen for the targeted delivery of therapeutic treatments. In particular embodiments, B7-H3 is preferentially-expressed by the diseased or cancerous cells such that on-target / off-site side effects are minimized or eliminated.

[0035] Diseased cells expressing B7-H3 are cells targeted for destruction by a treatment described herein. In particular embodiments, a B7-H3-related disorder includes brain cancer. In particular embodiments, the brain cancer includes diffuse intrinsic pontine glioma (DI PG), glioblastoma (GBM), medulloblastoma, neuroblastoma, craniopharyngioma, or an atypicalS281-6010PCT / SCRL544WOteratoid / rhabdoid tumor. Diseased cells expressing B7-H3 include, for example, DIPG cells, diffuse midline glioma (DMG) cells, glioblastoma cells, and neuroblastoma cells.

[0036] Methods disclosed herein include treating human patients with formulations disclosed herein.

[0037] In particular embodiments, the human patient has been diagnosed with a B7-H3-expressing CNS cancer. Examples of B7-H3 expressing cancers include diffuse intrinsic pontine glioma (DIPG), diffuse midline glioma (DMG), H3K27m-altered DMG, high grade glioma (HGG), glioblastoma (GBM), neuroblastoma, craniopharyngioma, ependymoma, an embryonal tumor, medulloblastoma, pineoblastoma, an atypical teratoid / rhabdoid tumor (ATRT), and an embryonal not otherwise specified (NOS) tumor

[0038] In particular embodiments, the human patient is a baby, a child, a young adult, or an adult. In particular embodiments, a baby is under one year old. In particular embodiments, a child is between 1 year of age and 18 years of age. In particular embodiments, a child is 1 year old, 2 years old, 3 years old, 4 years old, 5 years old, 6 years old, 7 years old, 8 years old, 9 years old, 10 years old, 11 years old, 12 years old, 13 years old, 14 years old, 15 years old, 16 years old, or 17 years old. In particular embodiments, a young adult is 18 years old to 26 years old. In particular embodiments, the young adult is 18 years old, 19 years old, 20 years old, 21 years old, 22 years old, 23 years old, 24 years old, or 25 years old. In particular embodiments, the human patient is under 26 years old. In particular embodiments, an adult is over 26 years old.

[0039] In some cases, cancers described herein are classified as pre-progression or postprogression. Progression is defined as clinical or radiographic worsening of disease consistent with the historical course for children with the disorder (e.g., DIPG) as determined by a qualified treating health care provider (e.g., physician). Pre-progression refers to before a clinical or radiographic worsening. For example, a patient is pre-progression when the patient has been diagnosed with cancer and received a standard therapy (e.g., radiation) and has not had a subsequent progression of their cancer. Post-progression refers to after a clinical or radiographic worsening. For example, a patient is post-progression when the patient has been diagnosed with cancer and received a standard therapy (e.g., radiation) and has had a subsequent progression of their cancer. In particular embodiments, the human patient is pre-progression or postprogression. In particular embodiments, the human patient is pre-progression.

[0040] (ii) T cells & CAR. In particular embodiments, T cells are intracranially (e.g, ICV) administered to a human patient having a B7-H3 expressing CNS cancer. In particular embodiments, T cells include CD8+ T cells and / or CD4+ T cells. Markers for T cells and subsets thereof are known to those of skill in the art. In particular embodiments, T cells can be autologousS281-6010PCT / SCRL544WOto a human patient being treated. In particular embodiments, T cells are genetically modified to express a B7-H3 CAR.

[0041] Methods for genetic modification are known in the art and include contacting a cell with a viral vector or nanoparticle or targeted genetic engineering approaches such as the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas (CRISPR-associated protein) nuclease system, zinc finger nucleases (ZFNs), transcription activator like effector nucleases (TALENs), or base editors.

[0042] A CAR as described herein includes a binding domain that binds B7-H3. CAR include several distinct subcomponents that allow genetically modified cells (e.g., T cells) to recognize and kill B7-H3-expressing cells. The subcomponents include at least an extracellular component and an intracellular component. The extracellular component includes a binding domain that specifically binds a B7-H3 epitope that is preferentially present on the surface of cells or in the area thereof. When the binding domain binds such epitopes, the intracellular component activates the cell to destroy the bound cell. CAR additionally include a transmembrane domain that directly or indirectly links the extracellular component to the intracellular component, and other subcomponents that can increase the CAR’s function. For example, the inclusion of a spacer region and / or one or more linker sequences can allow the CAR to have additional conformational flexibility, often increasing the binding domain’s ability to bind the targeted epitope.

[0043] In certain examples, binding domains for use in a CAR are based on antibodies that bind B7-H3. Antibodies are one example of binding domains and include whole antibodies or binding fragments of an antibody, e.g., Fv, Fab, Fab', F(ab')2, and single chain (sc) forms and fragments thereof that specifically bind a cellular antigen (such as B7-H3). Antibodies or antigen binding fragments can include all or a portion of polyclonal antibodies, monoclonal antibodies, human antibodies, humanized antibodies, synthetic antibodies, non-human antibodies, recombinant antibodies, chimeric antibodies, bispecific antibodies, mini bodies, and linear antibodies.

[0044] Antibodies are produced from two genes, a heavy chain gene and a light chain gene. Generally, an antibody includes two identical copies of a heavy chain, and two identical copies of a light chain. Within a variable heavy chain and variable light chain, segments referred to as complementary determining regions (CDRs) dictate epitope binding. Each heavy chain has three CDRs (i.e., CDRH1, CDRH2, and CDRH3) and each light chain has three CDRs (i.e., CDRL1, CDRL2, and CDRL3). CDR regions are flanked by framework residues (FR).

[0045] The assignment of amino acids to each domain can be in accordance with Kabat numbering (Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (“Kabat” numbering scheme));S281-6010PCT / SCRL544WOChothia (Al-Lazikani et al., (1997) JMB 273, 927-948 (“Chothia” numbering scheme)), Martin (Abinandan et al., Mol Immunol. 45:3832-3839 (2008), “Analysis and improvements to Kabatand structurally correct numbering of antibody variable domains”), Gelfand, Contact (MacCallum et al., J. Mol. Biol. 262:732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding site topography,” J. Mol. Biol. 262, 732-745.” (Contact numbering scheme)), IMGT (Lefranc et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol, 2003 January; 27(1):55-77 (“IMGT” numbering scheme)), AHo (Honegger and Pluckthun, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J Mol Biol, 2001 Jun. 8; 309(3):657-70, (AHo numbering scheme)), North (North et al., J Mol Biol. 406(2) :228-256 (2011), “A new clustering of antibody CDR loop conformations”), or other numbering schemes.

[0046] The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignments, while the Chothia scheme is based on structural information. Numbering for both the Kabat and Chothia schemes is based upon the most common antibody region sequence lengths, with insertions accommodated by insertion letters, for example, "30a," and deletions appearing in some antibodies. The two schemes place certain insertions and deletions ("indels") at different positions, resulting in differential numbering. The Contact scheme is based on analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme. In particular embodiments, the antibody CDR sequences disclosed herein are according to Kabat numbering. North numbering uses longer sequences in the structural analysis of the conformations of CDR loops. CDR residues can be identified using software programs such as ABodyBuilder.

[0047] In particular embodiments, a B7-H3 binding domain includes a CDRH1 having the sequence SFGMH (SEQ ID NO: 2), a CDRH2 having the sequence YISSDSSAIYYADTVKG (SEQ ID NO: 3), a CDRH3 having the sequence GRENIYYGSRLDY (SEQ ID NO: 4), a CDRL1 having the sequence KASQNVDTNVA (SEQ ID NO: 5), a CDRL2 having the sequence SASYRYS (SEQ ID NO: 6), and a CDRL3 having the sequence QQYNNYPFT (SEQ ID NO: 7), according to Kabat. CDRs for Chothia, IMGT, North, and Contact can be determined by those with skill in the art.

[0048] In particular embodiments, the B7-H3 binding domain includes a variable heavy chain including the sequence:EVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSDSSAIYYAD TVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCGRGRENIYYGSRLDYWGQGTTVTVSS(SEQ ID NO: 8) and a variable light chain including the sequence:S281-6010PCT / SCRL544WODIQLTQSPSFLSASVGDRVTITCKASQNVDTNVAWYQQKPGKAPKALIYSASYRYSGVPSRFS GSGSGTDFTLTISSLQPEDFATYYCQQYNNYPFTFGQGTKLEIK (SEQ ID NO: 9).

[0049] In particular embodiments, the B7-H3 binding domain includes an scFv (humanized MGA271, Enoblituzumab) having the sequence:EVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSDSSAIYYAD TVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCGRGRENIYYGSRLDYWGQGTTVTVSSGG GGSGGGGSGGGGSDIQLTQSPSFLSASVGDRVTITCKASQNVDTNVAWYQQKPGKAPKALIY SASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNNYPFTFGQGTKLEIK (SEQ ID NO: 10).

[0050] In particular embodiments, a B7-H3 binding domain includes a CDRH1 having the sequence FGMH (SEQ ID NO: 11), a CDRH2 having the sequence YISSDSSAIYYADTVK (SEQ ID NO: 12), a CDRH3 having the sequence GRENIYYGSRLDY (SEQ ID NO: 13), a CDRL1 having the sequence KASQNVDTNVA (SEQ ID NO: 14), a CDRL2 having the sequence SASYRYS (SEQ ID NO: 15), and a CDRL3 having the sequence QQYNNYPFT (SEQ ID NO: 16).

[0051] In particular embodiments, the B7-H3 binding domain includes a variable heavy chain including the sequence:DVQLVESGGGLVQPGGSRKLSCAASGFTFSSFGMHWVRQAPEKGLEWVAYISSDSSAIYYAD TVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCGRGRENIYYGSRLDYVVGQGTTLTVSS(SEQ ID NO: 17) and a variable light chain including the sequence:DIAMTQSQKFMSTSVGDRVSVTCKASQNVDTNVAWYQQKPGQSPKALIYSASYRYSGVPDRF TGSGSGTDFTLTINNVQSEDLAEYFCQQYNNYPFTFGSGTKLEIK (SEQ ID NO: 18).

[0052] In particular embodiments, a B7-H3 binding domain includes a CDRH1 having the sequence GFTFSSFG (SEQ ID NO: 19), a CDRH2 having the sequence ISSDSSAI (SEQ ID NO: 20), a CDRH3 having the sequence GRGRENIYYGSRLDYW(SEQ ID NO: 21), a CDRL1 having the sequence QNVDTN (SEQ ID NO: 22), a CDRL2 having the sequence SASY (SEQ ID NO: 23), and a CDRL3 having the sequence QQYNNYPFT (SEQ ID NO: 24).

[0053] In particular embodiments, the B7-H3 binding domain includes a variable heavy chain including the sequence:EVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSDSSAIYYAD TVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCGRGRENIYYGSRLDYWGQGTTVTV (SEQ ID NO: 25) and a variable light chain including the sequence:DIQLTQSPSFLSASVGDRVTITCKASQNVDTNVAWYQQKPGKAPKALIYSASYRYSGVPSRFS GSGSGTDFTLTISSLQPEDFATYYCQQYNNYPFTFGQGTKLEIK (SEQ ID NO: 26).

[0054] In particular embodiments the B7-H3 binding domain is derived from a commerciallyS281-6010PCT / SCRL544WOavailable B7-H3 antibody such as 6A1, 7-517, MIH35, RM335, 23GB5035, M3.2D7, 1E7D1, BLR026F, or PD00-36.

[0055] scFvs can be prepared according to methods known in the art (see, for example, Bird et al., (1988) Science 242:423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). ScFv molecules can be produced by linking VH and VL regions of an antibody together using flexible polypeptide linkers. If a short polypeptide linker is employed (e.g., between 5-10 amino acids) intrachain folding is prevented. Interchain folding is also required to bring the two variable regions together to form a functional epitope binding site. For examples of linker orientations and sizes see, e.g., Hollinger et al., 1993 Proc Natl Acad. Sci. U.S.A. 90:6444-6448, US 2005 / 0100543, US 2005 / 0175606, US 2007 / 0014794, and W02006 / 020258 and W02007 / 024715. More particularly, linker sequences that are used to connect the VL and VH of an scFv are generally five to 35 amino acids in length. In particular embodiments, a VL-VH linker includes from five to 35, ten to 30 amino acids or from 15 to 25 amino acids. Variation in the linker length may retain or enhance activity, giving rise to superior efficacy in activity studies. scFv are commonly used as the binding domains of a recombinant receptor. In particular embodiments, the recombinant receptor includes a binding domain that binds B7-H3.

[0056] Other binding fragments, such as Fv, Fab, Fab', F(ab')2, can also be used within the recombinant receptor disclosed herein. Additional examples of antibody-based binding domain formats for use in a recombinant receptor include scFv-based grababodies and soluble VH domain antibodies. These antibodies form binding regions using heavy chain variable regions. See, for example, Jespers et al., Nat. Biotechnol. 22:1161, 2004; Cortez-Retamozo et al., Cancer Res. 64:2853, 2004; Baral et al., Nature Med. 12:580, 2006; and Barthelemy et al., J. Biol. Chem.283:3639, 2008.

[0057] In particular embodiments, the binding domain includes a humanized antibody or an engineered fragment thereof. In particular embodiments, a non-human antibody is humanized, where one or more amino acid residues of the antibody are modified to increase similarity to an antibody naturally produced in a human or fragment thereof. These nonhuman amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. As provided herein, humanized antibodies or antibody fragments include one or more CDRs from nonhuman immunoglobulin molecules and framework regions wherein the amino acid residues including the framework are derived completely or mostly from human germline. A humanized antibody can be produced using a variety of techniques known in the art, including CDR-grafting (see, e.g., European Patent No. EP 239,400; WO 91 / 09967; and US 5,225,539, US 5,530,101, and US 5,585,089), veneering or resurfacing (see, e.g., EP 592,106S281-6010PCT / SCRL544WOand EP 519,596; Padlan, 1991, Molecular Immunology, 28(4 / 5):489-498; Studnicka et al., 1994, Protein Engineering, 7(6):805-814; and Roguska etal., 1994, PNAS, 91:969-973), chain shuffling (see, e.g., US. 5,565,332), and techniques disclosed in, e.g., US 2005 / 0042664, US 2005 / 0048617, US 6,407,213, US 5,766,886, WO 9317105, Tan etal., J. Immunol., 169:1119-25 (2002), Caldas et al., Protein Eng., 13(5):353-60 (2000), Morea et al., Methods, 20(3):267-79 (2000), Baca et al., J. Biol. Chem., 272(16): 10678-84 (1997), Roguska et al., Protein Eng., 9(10):895-904 (1996), Couto et al., Cancer Res., 55 (23 Supp):5973s-5977s (1995), Couto et al., Cancer Res., 55(8):1717-22 (1995), Sandhu J S, Gene, 150(2):409-10 (1994), and Pedersen et al., J. Mol. Biol., 235(3):959-73 (1994). Often, framework residues in the framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, for example improve, target antigen binding. These framework substitutions are identified by methods well-known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for target antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., US 5,585,089; and Riechmann et al., 1988, Nature, 332:323).

[0058] In particular embodiments, a VL region in a binding domain of the present disclosure is derived from or based on a VL of an antibody disclosed herein and contains one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., conservative amino acid substitutions), or a combination of the above-noted changes, when compared with the VL of the antibody disclosed herein. An insertion, deletion or substitution may be anywhere in the VL region, including at the amino- or carboxy-terminus or both ends of this region, provided that each CDR includes zero changes or at most one, two, or three changes and provided a binding domain containing the modified VL region can still specifically bind its target with an affinity similar to the wild type binding domain.

[0059] In particular embodiments, a binding domain VH region of the present disclosure can be derived from or based on a VH of an antibody disclosed herein and can contain one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., conservative amino acid substitutions or non-conservative amino acid substitutions), or a combination of the above-noted changes, when compared with the VH of the antibody disclosed herein. An insertion, deletion or substitution may be anywhere in the VH region, including at the amino- or carboxy-terminus or both ends of this region, provided that each CDR includes zero changes or at most one, two, or three changes and provided a binding domain containing the modified VH region can stillS281-6010PCT / SCRL544WOspecifically bind its target with an affinity similar to the wild type binding domain.

[0060] In particular embodiments, a binding domain includes or is a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to an amino acid sequence of a light chain variable region (VL) or to a heavy chain variable region (VH), or both, wherein each CDR includes zero changes or at most one, two, or three changes, from an antibody disclosed herein or fragment or derivative thereof that binds to B7-H3.

[0061] In particular embodiments, an scFv includes a VH connected to a VL using a linker. As used herein, a linker can include any chemical moiety that is capable of linking any two components. Some linkers serve no purpose other than to link components while many linkers serve an additional purpose. Linkers can be flexible, rigid, or semi-rigid, depending on the desired function of the linker.

[0062] For example, in particular embodiments, linkers provide flexibility and room for conformational movement between different components of a recombinant receptor. Commonly used flexible linkers include linker sequence with the amino acids glycine and serine (Gly-Ser linkers). In particular embodiments, the linker sequence includes sets of glycine and serine repeats such as from one to ten repeats of (GlyxSery)n, wherein x and y are independently an integer from 0 to 10 provided that x and y are not both 0 and wherein n is an integer of 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10). Particular examples include (Gly4Ser)n(SEQ ID NO: 27), (Gly3Ser)n(Gly4Ser)n(SEQ ID NO: 28), (Gly3Ser)n(Gly2Ser)n (SEQ ID NO: 29), or (Gly3Ser)n(Gly4Ser)i (SEQ ID NO: 30). In particular embodiments, the linker is (Gly4Ser)4(SEQ ID NO: 31), (Gly4Ser)3(SEQ ID NO: 32), (Gly4Ser)2(SEQ ID NO: 33), (Gly4Ser)i (SEQ ID NO: 34), (Gly3Ser)2(SEQ ID NO: 35), (Gly3Ser)i (SEQ ID NO: 36), (Gly2Ser)2(SEQ ID NO: 37) or (Gly2Ser)i, GGSGGGSGGSG (SEQ ID NO: 38), GGSGGGSGSG (SEQ ID NO: 39), or GGSGGGSG (SEQ ID NO: 40). In particular embodiments, a linker includes (Gly4Ser)3(SEQ ID NO: 41). In particular embodiments, a linker is encoded by GGAGGAGGAGGAAGCGGAGGAGGAGGATCCGGAGGAGGCGGGTCT (SEQ ID NO: 42).

[0063] Recombinant receptors can additionally include spacer regions, transmembrane domains, intracellular effector domains, and tags. Artificial expression constructs that encode a recombinant receptor can also include a transduction marker, self-cleaving peptide, tag, and / or selection cassette.

[0064] Spacer regions are used to create appropriate distances and / or flexibility between subcomponents of a protein. Spacer regions typically include 10 to 250 amino acids, 10 to 200 amino acids, 10 to 150 amino acids, 10 to 100 amino acids, 10 to 50 amino acids, or 10 to 25 aminoS281-6010PCT / SCRL544WOacids. Exemplary spacer regions include all or a portion of an immunoglobulin hinge region.

[0065] Transmembrane domains typically have a three-dimensional structure that is thermodynamically stable in a cell membrane and generally ranges in length from 15 to 30 amino acids. The structure of a transmembrane domain can include an a helix, a p barrel, a p sheet, a P helix, or any combination thereof. Transmembrane domains can include at least the transmembrane region(s) of the a, p or £ chain of a T cell receptor, CD28, CD27, CD3, CD45, CD4, CD5, CD8, CD9, CD16, CD22; CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.

[0066] A transmembrane domain can include one or more additional amino acids adjacent to the transmembrane region, e.g., one or more amino acid within the extracellular region of the expressed protein (e.g., up to 15 amino acids of the extracellular region) and / or one or more additional amino acids within the intracellular region of the expressed protein (e.g., up to 15 amino acids of the intracellular components).

[0067] Intracellular effector domains activate the expressing cell when the binding domain binds the antigen. The term “effector domain” is thus meant to include any portion of the intracellular domain sufficient to transduce an activation signal.

[0068] An effector domain can include one, two, three or more intracellular signaling components (e.g., receptor signaling domains, cytoplasmic signaling sequences), co-stimulatory domains, or combinations thereof. Exemplary effector domains include signaling and stimulatory domains selected from: 4-1 BB (CD137), CD3y, CD36, CD3c, CD3 , CD27, CD28, DAP10, ICOS, LAG3, NKG2D, NOTCH1, 0X40, ROR2, SLAMF1, TCRa, TCRp, TRIM, Wnt, Zap70, or any combination thereof. In particular embodiments, exemplary effector domains include signaling and costimulatory domains selected from: CD86, FcyRlla, DAP12, CD30, CD40, PD-1, lymphocyte function-associated antigen-1 (LFA-1), LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CDS, ICAM-1, GITR, BAFFR, SLAMF7, NKp80 (KLRF1), CD127, CD19, CD4, CD8a, CD8P, IL2RP, IL2Ry, IL7Ra, ITGA4, VLA1, CD49a, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, GADS, PAG / Cbp, NKp44, NKp30, or NKp46.

[0069] Intracellular signaling component sequences that act in a stimulatory manner may include iTAMs. Examples of iTAMs including primary cytoplasmic signaling sequences include those derived from CD3y, CD36, CD3E, CD3 , CD5, CD22, CD66d, CD79a, CD79b, and common FcRyS281-6010PCT / SCRL544WO(FCER1G), FcyRlla, FcR[3 (Fee Rib), DAP10, and DAP12. In particular embodiments, variants of CD3 retain at least one, two, three, or all ITAM regions.

[0070] A co- stimulatory domain is a domain whose activation can be required for an efficient lymphocyte response to cellular marker binding. Some molecules are interchangeable as intracellular signaling components or co-stimulatory domains. Examples of costimulatory domains include CD27, CD28, 4-1 BB (CD137), 0X40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), NKG2C, and a ligand that specifically binds with CD83.

[0071] Transduction markers may be selected from, for example, at least one of a truncated CD19 (tCD19; see Budde et al., Blood 122: 1660, 2013); a truncated human EGFR (EGFRt; see Wang et al., Blood 118: 1255, 2011); a cell surface-localizing polypeptide tag based on truncated human HER2, designated Her2tG; an extracellular domain of human CD34; and / or RQR8 which combines target epitopes from CD34 (see Fehse et al, Mol. Therapy 1(5 Pt 1); 448-456, 2000) and CD20 antigens (see Philip et al, Blood 124: 1277-1278). In particular embodiments, an artificial expression construct encodes an EGFRt transduction marker.

[0072] In particular embodiments, a selection cassette provides for positive selection or negative selection of a desired cell population. Negative selection is when several cell types are removed, leaving the cell type of interest. Positive selection involves targeting the desired cell population to only retain desired cells.

[0073] A selection cassette can encode proteins that (a) confer resistance to antibiotics or other toxins, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli. Any number of selection systems may be used to recover transformed cells. In particular embodiments, a positive selection cassette includes resistance genes to neomycin, hygromycin, ampicillin, puromycin, phleomycin, zeomycin, blasticidin, or viomycin. In particular embodiments, a selection cassette includes the DHFR (dihydrofolate reductase) gene or DHFR double mutant (DHFRdm) gene providing resistance to methotrexate (MTX), the MGMT P140K gene responsible for the resistance to O6BG / BCNU, the HPRT (Hypoxanthine phosphoribosyl transferase) gene responsible for the transformation of specific bases present in the HAT selection medium (aminopterin, hypoxanthine, thymidine) or other genes for detoxification with respect to some drugs. In particular embodiments, the selection agent includes neomycin, hygromycin, puromycin, phleomycin, zeomycin, blasticidin, viomycin, ampicillin, O6BG / BCNU, MTX, tetracycline, aminopterin, hypoxanthine, thymidine kinase, DHFR, Gin synthetase, or ADA.

[0074] In particular embodiments, the selection cassette includes DHFRdm and the selection agent includes MTX. In particular embodiments, the method does not require a selection cassetteS281-6010PCT / SCRL544WOto acquire highly purified cell populations. In particular embodiments, an artificial expression construct encodes an DHFRdm selection cassette.

[0075] In particular embodiments, negative selection cassettes include a gene for transformation of a substrate present in the culture medium into a toxic substance for the cell that expresses the gene. These molecules include detoxification genes of diptheria toxin (DTA) (Yagi et al., Anal Biochem. 214(1):77-86, 1993; Yanagawa et al., Transgenic Res. 8(3):215-221, 1999), the kinase thymidine gene of the Herpes virus (HSV TK) sensitive to the presence of ganciclovir or FIAU. The HPRT gene may also be used as a negative selection by addition of 6-thioguanine (6TG) into the medium, and for all positive and negative selections, a poly A transcription termination sequence from different origins, the most classical being derived from SV40 poly A, or a eukaryotic gene poly A (bovine growth hormone, rabbit [3-globin, etc.).

[0076] In particular embodiments, artificial expression constructs can include a polynucleotide or skip sequence that encodes a self-cleaving polypeptide (or ribosomal skip sequence). In particular embodiments, the polynucleotide encoding the self-cleaving polypeptide is located between the transgene encoding an intracellular molecule and the polynucleotide encoding the control feature. In particular embodiments, the polynucleotide encoding the self-cleaving polypeptide is located between the polynucleotide encoding the recombinant receptor and a polynucleotide encoding a selection cassette. In particular embodiments, the polynucleotide encoding the self-cleaving polypeptide is located between the polynucleotide encoding the selection cassette and the polynucleotide encoding the transduction marker (e.g., EGFRt). Exemplary self-cleaving polypeptides include 2A peptide from porcine teschovirus-1 (P2A), Thosea asigna virus (T2A), equine rhinitis A virus (E2A), foot-and-mouth disease virus (F2A), or variants thereof. Further exemplary nucleic acid and amino acid sequences of 2A peptides are set forth in, for example, Kim et al. (PLOS One6:e18556 (2011). In particular embodiments, cells are genetically modified to include a self-cleaving polypeptide. In particular embodiments, the self-cleaving polypeptide includes T2A. In particular embodiments, a 2A sequence does not result in a 2A scar. In particular embodiments, a 2A sequence results in the addition of amino acids to proteins (also referred to as a 2A scar). A 2A sequence is a peptide sequence that induces ribosomal skipping during translation. Cleavage is triggered by ribosomal skipping of a peptide bond between proline and glycine within the 2A sequence. This results in the upstream protein having extra amino acids at the C terminal end, which has unknown impact on protein functionality.

[0077] Control features may be present in multiple copies in an artificial expression construct or can be expressed as distinct molecules with the use of a skip sequence. For example, an artificial expression construct can have one, two, three, four or five tag cassettes and / or one, two, three,S281-6010PCT / SCRL544WOfour, or five transduction markers could also be expressed. For example, embodiments can include an artificial expression construct having two Myc tag cassettes, or a His tag and an HA tag cassette, or a HA tag and a Softag 1 tag cassette, or a Myc tag and a SBP tag cassette. Exemplary transduction markers and cognate pairs are described in US 13 / 463,247.

[0078] One advantage of including at least one control feature in an artificial expression construct is that cells expressing the artificial expression construct administered to a human patient can be increased or depleted using the cognate binding molecule to a tag cassette. In certain embodiments, the present disclosure provides a method for depleting a modified cell expressing an artificial expression construct by using an antibody specific for the tag cassette, using a cognate binding molecule specific for the control feature, or by using a second modified cell expressing an artificial expression construct and having specificity for the control feature. Elimination of modified cells may be accomplished using depletion agents specific for a control feature. For example, if EGFRt is used, then an EGFRt binding domain (e.g., antibody, scFv) fused to or conjugated to a cell-toxic reagent (such as a toxin, radiometal) may be used, or an EGFRt I CD3 bispecific scFv, or an EGFRt CAR T cell may be used. Similarly, if Her2tG is used, then an Her2tG binding domain fused to or conjugated to a cell-toxic reagent may be used.

[0079] In particular embodiments, an artificial expression construct includes an EF1p promoter, a GM-CSFss signal peptide, a B7-H3 recombinant receptor, a ribosomal skip sequence (e.g., T2A), an EGFRt transduction marker, a ribosomal skip sequence (e.g., T2A or P2A), a selection cassette (e.g., DHFRdm), and a post regulatory control sequence (e.g., WPRE). In particular embodiments, a B7-H3 recombinant receptor includes a B7-H3 binding domain, a spacer, a CD28tm transmembrane domain, a 4-1 BB costimulatory domain, and a CD3 intracellular signaling domain. In particular embodiments, a B7-H3 recombinant receptor includes a B7-H3 binding domain (e.g., B7-H3 scFv), an lgG4-CH3 hinge spacer, a CD28tm transmembrane domain, a 4-1 BB costimulatory domain, and a CD3 intracellular signaling domain.

[0080] In particular embodiments, an artificial expression construct includes an EF1p promoter, a GM-CSFss signal peptide, a B7-H3 CAR, a T2A skip sequence, an EGFRt transduction marker, a T2A skip sequence, a DHFRdm selection cassette, and a WPRE post regulatory control element. In particular embodiments, a B7-H3 CAR includes a B7-H3 scFv, an lgG4-CH3 hinge spacer, a CD28tm transmembrane domain, a 4-1 BB costimulatory domain, and a CD3 intracellular signaling domain.

[0081] In particular embodiments, a B7-H3 CAR is as disclosed in WO 2020 / 047257.

[0082] (iii) Methods of Use. Methods disclosed herein include treating human patients with a B7-H3-expressing CNS cancer. Treating human patients includes delivering therapeutically effectiveS281-6010PCT / SCRL544WOamounts. Therapeutically effective amounts include those that provide effective amounts, prophylactic treatments and / or therapeutic treatments.

[0083] An "effective amount" is the amount of a formulation necessary to result in a desired physiological change in the patient. For example, an effective amount can provide an immunogenic anti-cancer effect. Effective amounts are often administered for research purposes. An immunogenic formulation can be provided in an effective amount, wherein the effective amount stimulates an immune response.

[0084] In particular embodiments, prophylactic treatments reduce, delay, or prevent metastasis from a primary CNS tumor site from occurring.

[0085] A "therapeutic treatment" includes a treatment administered to a human patient who displays symptoms or signs of a cancer and is administered to the human patient for the purpose of diminishing or eliminating those signs or symptoms of the cancer. The therapeutic treatment can reduce, control, or eliminate the presence or activity of the cancer and / or reduce control or eliminate side effects of the cancer.

[0086] Function as an effective amount, prophylactic treatment or therapeutic treatment are not mutually exclusive, and in particular embodiments, administered dosages may accomplish more than one treatment type.

[0087] In particular embodiments, therapeutically effective amounts provide anti-cancer effects. Anti-cancer effects include a decrease in the number of cancer cells, decrease in the number of metastases, a decrease in tumor volume, an increase in life expectancy, induced chemo- or radiosensitivity in cancer cells, inhibited angiogenesis near cancer cells, inhibited cancer cell proliferation, inhibited tumor growth, prevented or reduced metastases, prolonged human patient life, reduced cancer-associated pain, and / or reduced relapse or re-occurrence of cancer following treatment.

[0088] A "tumor" is a swelling or lesion formed by an abnormal growth of cells (called neoplastic cells or tumor cells). A "tumor cell" is an abnormal cell that grows by a rapid, uncontrolled cellular proliferation and continues to grow after the stimuli that initiated the new growth cease. Tumors show partial or complete lack of structural organization and functional coordination with the normal tissue, and usually form a distinct mass of tissue, which may be benign, pre-malignant or malignant.

[0089] In particular embodiments, anti-cancer effects are observed against DI PG, diffuse midline glioma (DMG), H3K27m-altered DMG, high grade glioma (HGG), glioblastoma (GBM), neuroblastoma, craniopharyngioma, ependymoma, an embryonal tumor, medulloblastoma, pineoblastoma, an atypical teratoid / rhabdoid tumor (ATRT), and / or an embryonal not otherwiseS281-6010PCT / SCRL544WOspecified (NOS) tumor

[0090] In particular embodiments, therapeutically effective amounts induce an immune response. The immune response can be against a B7-H3-expressing cancer cell.

[0091] In particular embodiments, therapeutically effective amounts provide anti-DIPG effects. Anti-DIPG effects include a decrease in slurred speech, decrease in odd eye movements, decrease in difficulty swallowing, improvement in balance, decrease in facial drooping, increase in strength in extremities, or decrease in headaches.

[0092] In particular embodiments, therapeutically effective amounts provide anti-DMG effects. Anti-DMG effects include an improvement in vision and eye movement, decrease in slurred speech, decrease in fatigue, decrease in vomiting, decrease in facial drooping, improvement in balance, decrease in weakness on one or both sides of the body, or decrease in trouble swallowing.

[0093] In some methods of the present disclosure, a human patient is not lymphodepleted before administration of a dose of CAR T cells.

[0094] Lymphodepletion usually includes a short course of chemotherapy to reduce the number of T cells in the immune system. A lymphodepletion regimen can include administration of one or more chemotherapeutic drugs selected from fludarabine, cyclophosphamide, busulfan, melphalan, etoposide, carmustine, bendamustine, thiotepa, cytarabine, and cladribine. In particular embodiments, lymphodepletion regimens include a combination of chemotherapy drugs. Oftentimes, a lymphodepletion regimen includes fludarabine and cyclophosphamide. In particular embodiments, T cells are administered in a dose. In some cases, each dose within a dose regimen has the same number of T cells. In other embodiments, each dose within a dose regimen has the different number of T cells (i.e., doses are not the same). In particular embodiments, each subsequent dose within a dose regimen is the same or greater than the previous dose. In particular embodiments, a dose includes 1x105- 10x1015T cells per dose. In particular embodiments, a dose includes 0.1x107T cells / dose - 10x107T cells per dose. In particular embodiments, a dose includes 0.2x107T cells / dose - 10x107T cells per dose. In particular embodiments, a dose includes 0.3x107T cells / dose - 10x107T cells per dose. In particular embodiments, a dose includes 0.4x107T cells / dose - 10x107T cells per dose. In particular embodiments, a dose includes 0.5x107T cells / dose - 10x107T cells per dose. In particular embodiments, a dose includes 0.6x107T cells / dose - 10x107T cells per dose. In particular embodiments, a dose includes 0.7x107T cells / dose - 10x107T cells per dose. In particular embodiments, a dose includes 0.8x107T cells / dose - 10x107T cells per dose. In particular embodiments, a dose includes 0.9x107T cells / dose - 10x107T cells per dose. InS281-6010PCT / SCRL544WOparticular embodiments, a dose includes 1x107- 10x107T cells per dose. In particular embodiments, a dose includes 0.1x107, 0.2x107, 0.3x107, 0.4x107, 0.5x107, 0.6x107, 0.7x107, 0.8x107, 0.9x107, 1x107, 1.5x107, 2x107, 2.5x107, 3x107, 4x107, 5x107, 6x107, 8x107, 9x107, or 10 x107T cells per dose. In particular embodiments, a dose includes 50x106T cells / dose.

[0095] In particular embodiments, a dose regimen includes at least one dose. In particular embodiments, a dose regimen includes as many doses as the human patient can handle before experiencing neurotoxicity, organ dysfunction, cytokine release syndrome, and / or on-target off-tumor toxicity. In particular embodiments, a dose regimen include 1-100, 1-80, 1-81, 1-82, 1-83, 1-84, 1-85, 1-90, 1-100, 1-110, 1-120, 1-130, 1-140, 1-150, 1-160, 1-15, or 1-4 doses. In particular embodiments, a dose regimen includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 doses of T cells. In particular embodiments, a dose regimen includes up to 15 doses of T cells. In particular embodiments, a dose regimen includes 15 doses of T cells. In particular embodiments, a dose regimen includes 81 doses of T cells. In particular embodiments, a dose regimen includes 82 doses of T cells. In particular embodiments, a dose regimen includes 83 doses of T cells. In particular embodiments, a dose regimen includes 84 doses of T cells. In particular embodiments, a dose regimen includes 85 doses of T cells. In particular embodiments, a dose regimen includes 86 doses of T cells. In particular embodiments, a dose regimen includes 87 doses of T cells. In particular embodiments, a dose regimen includes 88 doses of T cells. In particular embodiments, a dose regimen includes 89 doses of T cells. In particular embodiments, a dose regimen includes 90 doses of T cells.

[0096] In particular embodiments, a dose regimen includes a first dose, a second dose, a third dose, and a fourth dose. In particular embodiments, the first dose includes 0.1x107, 0.2x107, 0.3x107, 0.4x107, 0.5x107, 0.6x107, 0.7x107, 0.8x107, 0.9x107, or 1x107T cells per dose. In particular embodiments, the second dose includes 0.1x107, 0.2x107, 0.3x107, 0.4x107, 0.5x107, 0.6x107, 0.7x107, 0.8x107, 0.9x107, 1x107- 2x107T cells per dose. In particular embodiments, the third dose includes 0.1x107, 0.2x107, 0.3x107, 0.4x107, 0.5x107, 0.6x107, 0.7x107, 0.8x107, 0.9x107, 1x107- 5x107T cells per dose. In particular embodiments, the fourth dose includes 0.1x107, 0.2x107, 0.3x107, 0.4x107, 0.5x107, 0.6x107, 0.7x107, 0.8x107, 0.9x107, 1x107- 10x107T cells per dose. In particular embodiments, a dose is administered every 14 days or twice a month. In particular embodiments, the dose regimen includes additional doses (e.g., a fifth dose, a sixth dose, a seventh dose, an eight dose, etc.).

[0097] Therapeutically effective amounts can be achieved by administering single or multiple doses during the course of a treatment regimen (e.g., every 11 days, every 12 days, every 13 days, every 14 days, every 15 days, every 16 days, every 17 days, every 18 days, every 19 days,S281-6010PCT / SCRL544WOevery 20 days, every 21 days, every 22 days, every 23 days, every 24 days, every 25 days, every 26 days, every 27 days, every 28 days, every 29 days, every 30 days, every 31 days, every 32 days, every 33 days, every 34 days, or every 35 days). In particular embodiments, the treatment protocol may be dictated by a clinical trial protocol or an FDA-approved treatment protocol. In particular embodiments, a dose is administered every 14 days. In particular embodiments, a dose is administered at least every 28 days. In particular embodiments, a dose is administered weekly.

[0098] In particular embodiments, the dosing regimen changes. For example, a dosing regimen can be divided into initiation, continuation, and long-term continuation phases. The initiation phase can include the first 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses. In particular embodiments, the initiation phase includes the first 3, 4, or 5 doses. The continuation phase follows the initiation phase and can include the next 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, or more doses. In particular embodiments, the continuation phase includes dose 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and / or 14. The long-term continuation phase follows the continuation phase and can include any number of doses to maintain or improve a patient’s condition. In particular embodiments, the long-term continuation phase can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more doses.

[0099] The initiation, continuation, and / or long-term continuation phases can included doses administered bi-weekly (twice a month), monthly, or any period of time described earlier. In particular embodiments, the initiation phase includes bi-weekly doses. In particular embodiments, the continuation phase includes doses every three weeks. In particular embodiments, the longterm continuation phase includes monthly doses. In particular embodiments, the long-term continuation phase includes a dose every three months.

[0100] Over the course of treatment, a human patient is administered a total or cumulative number of T cells. In particular embodiments, a human patient is administered 0.1x107, 0.2x107, 0.3x107, 0.4x107, 0.5x107, 0.6x107, 0.7x107, 0.8x107, 0.9x107, 1x107, 2x107, 3x107, 4x107, 5 x107, 6x107, 7x107, 8x107, 9x107, 1x108, 2x108, 3 x108, 4 x108, 5 x108, 6 x108, 7 x108, 7.5x108, 8 x108, 9 x108, 1 x109, 2 x109, 3 x109, 4 x109, 5 x109, 6 x109, 7 x109, 8 x109, 9 x109, or more T cells over the course of treatment.

[0101] Therapeutically effective amounts can be administered intracranially (e.g., ICV). In particular embodiments, therapeutically effective amounts are administered by ICV injection.

[0102] In certain embodiments, formulations (i.e., T cells ) are administered to a patient in conjunction with (e.g., before, simultaneously or following) any number of relevant treatment modalities. In particular embodiments, cells may be used in combination with radiation.

[0103] Radiation or radiotherapy refers to a cancer treatment that uses radiation to kill cancerS281-6010PCT / SCRL544WOcells and shrink tumors. It uses high doses of ionizing radiation, such as X-rays, gamma rays, or high-energy electrons to damage the DNA of cancer cells, preventing them from dividing and thereby killing the cancer cells. Radiation can include external beam radiation therapy, internal radiation therapy, or systemic radiation therapy.

[0104] External beam radiation therapy uses a machine that locally aims radiation at a cancer. External beam radiation can be used to shrink tumors to treat pain, trouble breathing, or loss of bowel or bladder control. In some embodiments, external beam radiation therapy includes three-dimensional conformal radiation therapy (3D-CRT), intensity modulated radiation therapy (IMRT), proton beam therapy, image-guided radiation therapy (IGRT), or stereotactic radiation therapy (SRT).

[0105] Internal radiation therapy is a treatment that places a source of radiation in the human patient’s body. The source of radiation can be a liquid or a solid. In some embodiments, the internal radiotherapy uses a permanent implant or a temporary internal radiotherapy, for example, a needle, tube, or applicator.

[0106] Systemic radiation therapy involves a radioactive substance that travels through the blood to tissues throughout the body. A systemic radiation therapy can use a radioactive iodine, or I-131.

[0107] Radiation can also include intraoperative radiation therapy (IORT), or radioimmunotherapy. Radiation can be applied once or several times.

[0108] In certain embodiments, formulations disclosed herein may be administered in conjunction with any number of chemotherapeutic agents. Examples of chemotherapeutic agents include alkylating agents; aziridines; ethylenimines and methylamelamines; nitrogen mustards; nitrosureas; antibiotics; anti-metabolites); folic acid analogues; purine analogs; pyrimidine analogs; androgens; anti-adrenals; folic acid replenishers; platinum analogs; topoisomerase inhibitors; retinoic acid derivatives; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and anti-androgens; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Combinations of chemotherapeutic agents are also administered where appropriate, including, CHOP, i.e., Cyclophosphamide (Cytoxan®), Doxorubicin (hydroxydoxorubicin), Vincristine (Oncovin®), and Prednisone.

[0109] In some embodiments, the chemotherapeutic agent is administered at the same time or within one week after the administration of the engineered cell or nucleic acid. In other embodiments, the chemotherapeutic agent is administered from 1 to 4 weeks or from 1 week toS281-6010PCT / SCRL544WO1 month, 1 week to 2 months, 1 week to 3 months, 1 week to 6 months, 1 week to 9 months, or 1 week to 12 months after the administration of the engineered cell or nucleic acid. In other embodiments, the chemotherapeutic agent is administered at least 1 month before administering the cell or nucleic acid. In some embodiments, the methods further include administering two or more chemotherapeutic agents.

[0110] In additional embodiments, the formulations disclosed herein can be administered with an anti-inflammatory agent. Anti-inflammatory agents or drugs include steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone), nonsteroidal antiinflammatory drugs (NSAIDS) including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF medications, cyclophosphamide and mycophenolate. Exemplary NSAIDs include ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors, and sialylates. Exemplary analgesics include acetaminophen, oxycodone, tramadol of proporxyphene hydrochloride. Exemplary glucocorticoids include cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone. Exemplary biological response modifiers include molecules directed against cell surface markers (e.g., CD4, CD5, etc.), cytokine inhibitors, such as the TNF antagonists, (e.g., etanercept (ENBREL®), adalimumab (HUMIRA®) and infliximab (REMICADE®), chemokine inhibitors and adhesion molecule inhibitors. The biological response modifiers include monoclonal antibodies as well as recombinant forms of molecules. Exemplary DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, Gold (oral (auranofm) and intramuscular) and minocycline. In particular embodiments, the anti-inflammatory agent or drug is provided at a limited amount. In particular embodiments, the amount of anti-inflammatory provided to a human patient does not exceed 25 mg / m2 / day, 20 mg / m2 / day, 15 mg / m2 / day, 10 mg / m2 / day, 7 mg / m2 / day, 5 mg / m2 / day, 4 mg / m2 / day, 3 mg / m2 / day, 2.5 mg / m2 / day, 2 mg / m2 / day, 1.5 mg / m2 / day, 1 mg / m2 / day, 0.9 mg / m2 / day, 0.8 mg / m2 / day, 0.7 mg / m2 / day, 0.6 mg / m2 / day, or 0.5 mg / m2 / day. In particular embodiments, the amount of dexamethasone provided to a human patient does not exceed 2.5 mg / m2 / day.

[0111] In certain embodiments, the formulations described herein are administered in conjunction with a cytokine. “Cytokine” as used herein is meant to refer to proteins released by one cell population that act on another cell as intercellular mediators. Examples of cytokines are lymphokines, monokines, and traditional polypeptide hormones. Included among the cytokines are growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin;S281-6010PCT / SCRL544WOglycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor (HGF); fibroblast growth factor (FGF); prolactin; placental lactogen; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors (NGFs) such as NGF-beta; platelet-growth factor; transforming growth factors (TGFs) such as TGFa and TGFP; insulin-like growth factor-1 and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-alpha, beta, and - gamma; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte- macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL- 1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-I I, IL-12; IL-15, a tumor necrosis factor such as TNF-alphaorTNF-beta; and other polypeptide factors including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture, and biologically active equivalents of the native sequence cytokines.

[0112] (iv) Selection for Clinical Trial. Particular embodiments disclosed herein include screening human patients for a clinical trial. In particular embodiments, a human patient is eligible for inclusion in a clinical trial if they meet inclusion criteria. Clinical trial inclusion criteria are the characteristics that potential participants must have to be included in the study and will depend on the goals and parameters of the clinical trial study.

[0113] The Lansky Play-Performance Scale (PPS) is a tool used to assess the functional status of children with cancer. A child with a score of 0 is unresponsive and a child with a score of 100 is fully active and normal. A child with a score of 50 lies around much of the day but gets dressed and participates in quiet play. In particular embodiments, a child is eligible for a clinical trial if they have a PPS greater than 0, a PPS greater than 10, a PPS greater than 20, a PPS greater than 30, a PPS greater than 40, a PPS greater than 50, a PPS greater than 60, a PPS greater than 70, a PPS greater than 80, or a PPS greater than 90. In particular embodiments, a child is eligible for a clinical trial if they have a PPS greater than 60.

[0114] The Karnofsky Performance Scale (KPS) is an assessment tool intended to assist clinicians and caretakers in gauging a patient's functional status and ability to carry out activities of daily living. A human patient with a score of 0 is considered dead, a human patient with a score of 50 requires considerable assistance and frequent medical care, and a human patient with a score of 100 is normal with no evidence of disease. In particular embodiments, a human patient is eligible for a clinical trial if they have a KPS greater than 0, a KPS greater than 10, a KPS greater than 20, a KPS greater than 30, a KPS greater than 40, a KPS greater than 50, a KPS greater than 60, a KPS greater than 70, a KPS greater than 80, or a KPS greater than 90. InS281-6010PCT / SCRL544WOparticular embodiments, a human patient is eligible for a clinical trial if they have a KPS greater than 60.

[0115] In some cases, clinical trial inclusion criteria may include life expectancy of the patient. The life expectancy of a human patient can be determined by stage of disease, biological age, response to current treatments, and other assessments and predictive models. In particular embodiments, a human patient is eligible for a clinical trial if the human patient has a life expectancy greater than 3 days, greater than 1 week, greater than 2 weeks, greater than 3 weeks, greater than 4 weeks, greater than 5 weeks, greater than 6 weeks, greater than 7 weeks, greater than 8 weeks, greater than 9 weeks, greater than 10 weeks, greater than 11 weeks, greater than 12 weeks, greater than 6 months, greater than 9 months, or greater than a year. In particular embodiments, a human patient is eligible for a clinical trial if the human patient has a life expectancy greater than or equal to 8 weeks.

[0116] In some cases, clinical trial inclusion criteria may require the human patient to have adequate organ function. Adequate organ function can be measured in different ways depending on the clinical trial and the patient’s condition. For example, organ function can be measured by blood tests. Examples of organs whose malfunctions can be visible in a blood test include your heart, thyroid, liver, or kidneys. In particular embodiments, organ function is measured by absolute lymphocyte count (ALC), absolute neutrophil count, hemoglobin, platelet count, creatinine count, bilirubin, and / or oxygen saturation.

[0117] In particular embodiments, a human patient is eligible for a clinical trial if the human patient has an ALC >10 cells / pL, >50 cells / pL, >75 cells / L, >100 cells / L, >150 cells / pL, >200 cells / pL, >250 cells / pL, >500 cells / pL, >1000 cells / pL, >2000 cells / pL, >3000 cells / pL, or >4000 cells / pL. In particular embodiments, a human patient is eligible for a clinical trial if the human patient has an ALC > 100 cells / pL.

[0118] In particular embodiments, a human patient is eligible for a clinical trial if the human patient has an absolute neutrophil count > 100 cells / pL, > 200 cells / pL > 300 cells / pL, > 400 cells / pL, > 500 cells / pL, > 600 cells / pL, > 700 cells / pL, > 800 cells / pL, > 900 cells / pL, > 1000 cells / pL, > 2500 cells / pL, > 3000 cells / pL, > 4000 cells / pL, > 5000 cells / pL, or > 6000 cells / pL. In particular embodiments, a human patient is eligible for a clinical trial if the human patient has an absolute neutrophil count > 500 cells / pL.

[0119] In particular embodiments, a human patient is eligible for a clinical trial if the human patient has a hemoglobin count > 1 g / dL, > 3 g / dL, > 5 g / dL, > 6 g / dL, > 7 g / dL, > 8 g / dL, > 9 g / dL, > 10 g / dL, > 11 g / dL, > 12 g / dL, or> 13 g / dL. In particular embodiments, a human patient is eligible for a clinical trial if the human patient has a hemoglobin count > 9 g / dL.S281-6010PCT / SCRL544WO

[0120] In particular embodiments, a human patient is eligible for a clinical trial if the human patient has a platelet count >50,000 / pL, >100,000 / pL, >120,000 / pL, >130,000 / pL, >140,000 / pL, >150,000 / pL, >200,000 / pL, >300,000 / pL, or >400,000 / p. In particular embodiments, a human patient is eligible for a clinical trial if the human patient has a platelet count > 100,000 / pL.

[0121] In particular embodiments, a human patient is eligible for a clinical trial if the human patient has a creatinine count < upper limit of normal (ULN). In particular embodiments, the ULN includes 0.1 mg / dL -2 mg / dL, 0.7 mg / dL -1.3 mg / dL, 0.6 mg / dL -1.1 mg / dL, or 0.3 mg / dL -0.7 mg / dL. In particular embodiments, the ULN depends on age.

[0122] In particular embodiments, a human patient is eligible for a clinical trial if the human patient has a total bilirubin < 3x ULN or conjugated bilirubin < 2 mg / dL.

[0123] In particular embodiments, a human patient is eligible for a clinical trial if the human patient has a blood oxygen saturation of > 70%, > 80%, > 90%%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, or >99%. In particular embodiments, a human patient is eligible for a clinical trial if the human patient has a blood oxygen saturation of > 90%.

[0124] In particular embodiments, the human patient is eligible for a clinical trial if the human patient has not received or been receiving an anti-inflammatory agent at a dose exceeding 25 mg / m2 / day, 20 mg / m2 / day, 15 mg / m2 / day, 10 mg / m2 / day, 7 mg / m2 / day, 5 mg / m2 / day, 4 mg / m2 / day, 3 mg / m2 / day, 2.5 mg / m2 / day, 2 mg / m2 / day, 1.5 mg / m2 / day, 1 mg / m2 / day, 0.9 mg / m2 / day, 0.8 mg / m2 / day, 0.7 mg / m2 / day, 0.6 mg / m2 / day, or 0.5 mg / m2 / day.. In particular embodiments, the human patient is eligible for a clinical trial if the human patient has not received or been receiving dexamethasone at a dose exceeding 2.5 mg / m2 / day.In particular embodiments, the human patient is eligible for a clinical trial if the human patient has received washout from prior therapies. Washout refers to a time since stopping a prior therapy. Washout times vary in length per therapy, and can be individually defined in clinical protocols. Washout is often determined based on a prior therapy’s half-life.

[0125] In particular embodiments, the human patient is eligible for a clinical trial if the human patient does not have encephalopathy. Encephalopathy is disease in which the functioning of the brain is affected by some agent or condition (such as viral infection or toxins in the blood). Examples of encephalopathy include hypertensive encephalopathy, anoxic encephalopathy, chronic traumatic encephalopathy (CTE), hepatic encephalopathy, Wernicke encephalopathy, metabolic encephalopathy, or toxic encephalopathy.

[0126] In particular embodiments, the human patient is ineligible for a clinical trial if the human patient is infected with a virus. In particular embodiments, the human patient is ineligible for a clinical trial if the human patient is infected with human immunodeficiency virus (HIV), hepatitis B,S281-6010PCT / SCRI.544WOhepatitis C, influenza, human papilloma virus, measles, polio, coronavirus, or enterovirus. In particular embodiments, the human patient is ineligible for a clinical trial if the human patient is infected with a HIV, hepatitis B, or hepatitis C.

[0127] The Exemplary Embodiments and Example below are included to demonstrate particular embodiments of the disclosure. Those of ordinary skill in the art should recognize in light of the present disclosure that many changes can be made to the specific embodiments disclosed herein and still obtain a like or similar result without departing from the spirit and scope of the disclosure.

[0128] (v) Exemplary Embodiments.1. A method of treating a human patient having a B7-H3-expressing central nervous system (CNS) cancer, the method including:administering T cells expressing a B7-H3-binding chimeric antigen receptor (CAR) to the CNS of the human patient,thereby treating the human patient having the B7-H3-expressing CNS cancer.2. The method of embodiment 1 , wherein the human patient is a child or young adult.3. The method of embodiment 1 or 2, wherein the human patient is under 26 years of age.4. The method embodiment 1 or 2, wherein the human patient is over one year of age. 5. The method of embodiment 1 or 2, wherein the human patient is between 2 and 22 years of age.6. The method of any of embodiments 1-5, wherein the human patient has received radiotherapy before the treating.7. The method of embodiment 6, wherein the radiotherapy is completed within 6 weeks of the administering.8. The method of any of embodiments 1-7, wherein the B7-H3-expressing CNS cancer has not progressed before the treating.9. The method of any of embodiments 1-8, wherein the B7-H3-expressing CNS cancer includes brain cancer or a spinal cord tumor.10. The method of embodiment 9, wherein the brain cancer includes diffuse intrinsic pontine glioma (DIPG), diffuse midline glioma (DMG), H3K27m-altered DMG, high grade glioma (HGG), glioblastoma (GBM), neuroblastoma, craniopharyngioma, ependymoma, an embryonal tumor, medulloblastoma, pineoblastoma, an atypical teratoid / rhabdoid tumor (ATRT), or an embryonal not otherwise specified (NOS) tumor.11. The method of any of embodiments 1-8, wherein the B7-H3-expressing CNS cancer includes DIPG.12. The method of any of embodiments 1-11, wherein the administering includes intracranialS281-6010PCT / SCRL544WOadministration.13. The method of any of embodiments 1-12, wherein the administering includes intratumoral administration.14. The method of any of embodiments 1-13, wherein the administering includes intrathecal administration.15. The method of embodiment 12, wherein intracranial administration includes intracerebroventricular (ICV) injection.16. The method of any of embodiments 1-15, wherein the treating further includes a dose regimen including a plurality of doses of T cells.17. The method of embodiment 16, wherein the dose regimen includes a first dose of T cells and a second dose of T cells.18. The method of embodiment 16, wherein the dose regimen includes a first dose of T cells, a second dose of T cells, and a third dose of T cells.19. The method of embodiment 16, wherein the dose regimen includes a first dose of T cells, a second dose of T cells, a third dose of T cells, and a fourth dose of T cells.20. The method of any of embodiments 16-19, wherein the dose regimen has 1-100 doses of T cells.21. The method of any of embodiments 16-19, wherein the dose regimen has 1-81 doses of T cells.22. The method of any of embodiments 16-19, wherein the dose regimen has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 doses of T cells.23. The method of any of embodiments 16-19, wherein the dose regimen has up to 15 doses of T cells.24. The method of any of embodiments 16-19, wherein the dose regimen has 15 doses of T cells.25. The method of any of embodiments 16-24, wherein each dose of the plurality of doses of T cells has 0.1x107- 10x107T cells per dose.26. The method of any of embodiments 16-24, wherein each dose of the plurality of doses of T cells has 50x106T cells per dose.27. The method of any of embodiments 16-24, wherein the dose regimen includes an escalating number of T cells per dose.28. The method of any of embodiments 17-27, wherein the first dose has 0.1-1x107T cells per dose.29. The method of any of embodiments 18-28, wherein the second dose has 0.1x107-2.5x107S281-6010PCT / SCRL544WOT cells per dose.30. The method of any of embodiments 19-29, wherein the third dose has 0.1x107- 5x107T cells per dose.31. The method of any of embodiments 20-30, wherein the fourth dose has 0.1x107- 10x107T cells per dose.32. The method of embodiment 16, wherein the dose regimen includes a first dose having 1x107T cells, a second dose having 1x107T cells, a third dose having 1x107T cells, and a fourth dose having 1x107T cells.33. The method of embodiment 16, wherein the dose regimen includes a first dose having 1x107T cells, a second dose having 1x107T cells, a third dose having 2.5x107T cells, and a fourth dose having 2.5x107T cells.34. The method of embodiment 16, wherein the dose regimen includes a first dose having 1x107T cells, a second dose having 2.5x107T cells, a third dose having 5x107T cells, and a fourth dose having 5x107T cells.35. The method of embodiment 16, wherein the dose regimen includes a first dose having 1x107T cells, a second dose having 2.5x107T cells, a third dose having 5x107T cells, and a fourth dose having 10x107T cells.36. The method of embodiment 16, wherein the dose regimen includes a first dose having 1x107T cells, a second dose having 5x107T cells, a third dose having 10x107T cells, and a fourth dose having 10x107T cells.37. The method of any of embodiments 16-36, wherein the dose regimen results in delivery of 1x107- 3x1010T cells over its course.38. The method of any of embodiments 16-36, wherein the dose regimen results in delivery of 1x107- 2.5x109T cells over its course.39. The method of any of embodiments 16-38, wherein the dose regimen includes delivery of a dose of T cells at least every 1-28 days for a period of time.40. The method of any of embodiments 16-38, wherein the dose regimen includes delivery of a dose of T cells at least every 14 days ±2 days for a period of time.41. The method of embodiment 40, wherein the period of time is 30 weeks.42. The method of any of embodiments 16-38, wherein the dose regimen includes delivery of a dose of T cells at least every 2 weeks for a period of time, followed by delivery of a dose of T cells at least once a month for a period of time.43. The method of any of embodiments 1-42, wherein the T cells include CD8+ T cells or CD4+ T cells.S281-6010PCT / SCRL544WO44. The method of any of embodiments 1-43, wherein the CAR includes an extracellular component linked to an intracellular component through a transmembrane domain, wherein the extracellular component includes a B7-H3 binding domain.45. The method of embodiment 44, wherein the intracellular component includes an effector domain including 4-1BB (CD137), CD3y, CD35, CD3E, CD3 , CD27, CD28, DAP10, ICOS, LAG3, NKG2D, NOTCH1, 0X40, ROR2, SLAMF1, TCRa, TCR , TRIM, Wnt, Zap70, or a combination thereof.46. The method of embodiment 44, wherein the intracellular component includes a 4-1 BB signaling domain and a CD3^ signaling domain.47. The method of any of embodiments 44-46, wherein the transmembrane domain includes a transmembrane region of: an a, p orchain of a T cell receptor; CD28; CD27; CD3; CD45; CD4; CD5; CD8; CD9; CD16; CD22; CD33; CD37; CD64; CD80; CD86; CD134; CD137; CD154; or a combination thereof.48. The method of any of embodiments 1-47, wherein the T cell is genetically modified to express the CAR by contacting the T cell with an artificial expression construct including a sequence encoding the CAR.49. The method of embodiment 48, wherein the artificial expression construct includes a viral vector.50. The method of embodiment 49, wherein the viral vector includes a lentiviral vector.51. The method of any of embodiments 48-50, wherein the artificial expression construct further encodes a skip sequence.52. The method of embodiment 51 , wherein the skip sequence includes a T2A skip sequence, a P2A skip sequence, an E2A skip sequence, or an F2A skip sequence.53. The method of any of embodiments 48-52, wherein the artificial expression construct further includes a sequence encoding a transduction marker.54. The method of embodiment 53, wherein the transduction marker includes a truncated EGFR (EGFRt).55. The method of any of embodiments 48-54, wherein the artificial expression construct further includes a sequence encoding a selection cassette.56. The method of embodiment 55, wherein the selection cassette includes a dihydrofolate reductase (DHFR)dm.57. The method of any of embodiments 1 -56, wherein the T cells are autologous to the human patient.58. The method of any of embodiments 1-57, wherein the human patient is notS281-6010PCT / SCRL544WOlymphodepleted at a time of the treating.59. The method of any of embodiments 1-57, wherein the patient is lymphodepleted at a time of the treating.60. The method of any of embodiments 1-59, further including screening human patients for eligibility in a clinical trial.61. The method of embodiment 60, wherein the screening includes determining that the human patient matches a set of inclusion criteria for the clinical trial.62. The method of embodiment 61 , wherein the set of inclusion criteria include that the human patient has a Lanksy or Karnofsky performance score greater than 60.63. The method of embodiment 61 or 62, wherein the set of inclusion criteria include that the human patient receives 2.5 mg / m2 / day of dexamethasone or less.64. The method of any of embodiments 61-63, wherein the set of inclusion criteria include that the human patient has an implanted CNS catheter.65. The method of any of embodiments 61-64, wherein the set of inclusion criteria include that the human patient undergoes a washout from previous therapies for the CNS cancer.66. The method of any of embodiments 61-65, wherein the set of inclusion criteria include that the human patient has a life expectancy greater than or equal to 8 weeks.67. The method of any of embodiments 61-66, wherein the set of inclusion criteria include that the human patient has adequate organ function.68. The method of embodiment 67, wherein adequate organ function includes an absolute lymphocyte count (ALC) > 100 cells / pL.69. The method of embodiment 67 or 68, wherein adequate organ function includes an absolute neutrophil count > 500 cells / pL.70. The method of embodiment 67 Or 68, wherein adequate organ function includes a hemoglobin count > 9 g / dL.71. The method of any of embodiments 67-70, wherein adequate organ function includes a platelet count > 100,000 / pL.72. The method of any of embodiments 67-71, wherein adequate organ function includes a creatinine count < upper limit of normal (ULN).73. The method of any of embodiments 67-72, wherein adequate organ function includes a total bilirubin < 3x ULN or conjugated bilirubin < 2 mg / dL.74. The method of any of embodiments 67-73, wherein adequate organ function includes an oxygen saturation > 90%.75. The method of any of embodiments 61-74, wherein the set of inclusion criteria include thatS281-6010PCT / SCRL544WOthe human patient does not have encephalopathy.76. The method of any of embodiments 61-75, wherein the set of inclusion criteria include that the human patient is not infected with a virus.77. The method of any of embodiments 61-76, wherein the set of inclusion criteria include that the human patient is not infected with human immunodeficiency virus (HIV), hepatitis B, or hepatitis C.78. A method for administering a dose regimen including:administering a first dose of T cells to the central nervous system (CNS) of a human patient having a B7-H3 expressing CNS cancer;administering a second dose of T cells to the CNS of the human patient at least 7 days after administering the first dose; andrepeating the administering of an effective dose of T cells to the CNS of the human patient at least every 7 days.79. The method of embodiment 78, wherein the B7-H3 expressing CNS cancer has not progressed at a time of administering the first dose.80. The method of embodiment 78 or 79, wherein the CNS cancer includes brain cancer or a spinal cord tumor.81. The method of embodiment 80, wherein the brain cancer includes diffuse intrinsic pontine glioma (DIPG), diffuse midline glioma (DMG), H3K27m-altered DMG, high grade glioma (HGG), glioblastoma (GBM), neuroblastoma, craniopharyngioma, ependymoma, an embryonal tumor, medulloblastoma, pineoblastoma, an atypical teratoid / rhabdoid tumor (ATRT), or an embryonal not otherwise specified (NOS) tumor.82. The method of embodiment 78, wherein the B7-H3-expressing CNS cancer includes DIPG.83. The method of any of embodiments 78-82, wherein the administering includes intracranial administration.84. The method of any of embodiments 78-83, wherein the administering includes intratumoral administration.85. The method of any of embodiments 78-84, wherein the administering includes intrathecal administration.86. The method of embodiment 83, wherein intracranial administration includes intracerebroventricular (ICV) injection.87. The method of any of embodiments 78-86, wherein the dose regimen includes a first dose of T cells, a second dose of T cells, and a third dose of T cells.S281-6010PCT / SCRL544WO88. The method of any of embodiments 78-86, wherein the dose regimen includes a first dose of T cells, a second dose of T cells, a third dose of T cells, and a fourth dose of T cells.89. The method of any of embodiments 78-86, wherein the dose regimen has upto 100 doses of T cells.90. The method of any of embodiments 78-86, wherein the dose regimen has up to 81 doses of T cells.91. The method of any of embodiments 78-86, wherein the dose regimen has up to 20 doses of T cells.92. The method of any of embodiments 78-86, wherein the dose regimen has up to 15 doses of T cells.93. The method of any of embodiments 78-86, wherein the dose regimen has 15 doses of T cells.94. The method of any of embodiments 78-93, wherein the dose regimen includes repeated doses of 50x106T cells per dose.95. The method of any of embodiments 78-93, wherein the dose regimen includes a same or an escalating number of T cells per dose.96. The method of any of embodiments 87-93, wherein the first dose has 0.1-1x107T cells per dose.97. The method of any of embodiments 88-93, wherein the second dose has 0.1 x107- 2.5x107T cells per dose.98. The method of any of embodiments 88-93, wherein the dose regimen includes a first dose having 1x107T cells, a second dose having 1x107- 2.5x107T cells, a third dose having 1x107-5x107T cells, and a fourth dose having 1x107- 10x107T cells.99. The method of any of embodiments 88-93, wherein the dose regimen includes a first dose having 1x107T cells, a second dose having 1x107T cells, a third dose having 1x107T cells, and a fourth dose having 1x107T cells.100. The method of any of embodiments 88-93, wherein the dose regimen includes a first dose having 1x107T cells, a second dose having 1x107T cells, a third dose having 2.5x107T cells, and a fourth dose having 2.5x107T cells.101. The method of any of embodiments 88-93, wherein the dose regimen includes a first dose having 1x107T cells, a second dose having 2.5x107T cells, a third dose having 5x107T cells, and a fourth dose having 5x107T cells.102. The method of any of embodiments 88-93, wherein the dose regimen includes a first dose having 1x107T cells, a second dose having 2.5x107T cells, a third dose having 5x107T cells, andS281-6010PCT / SCRL544WOa fourth dose having 10x107T cells.103. The method of any of embodiments 88-93, wherein the dose regimen includes a first dose having 1x107T cells, a second dose having 5x107T cells, a third dose having 10x107T cells, and a fourth dose having 10x107T cells.104. The method of any of embodiments 88-93, wherein the dose regimen results in intracranially administering 1x107- 3x1010T cells over its course.105. The method of any of embodiments 78-104, wherein the dose regimen results in intracranially administering 1x107- 2.5x109T cells over its course.106. The method of any of embodiments 78-105, wherein the dose regimen includes delivery of a dose of T cells at least every 1-28 days for a period of time.107. The method of any of embodiments 78-105, wherein the dose regimen includes delivery of a dose of T cells at least every 14 days ±2 days for a period of time.108. The method of any of embodiments 78-105, wherein the dose regimen includes delivery of a dose of T cells at least every 2 weeks for a period of time, followed by delivery of a dose of T cells at least every month for a period of time.109. The method of any of embodiments 78-108, wherein the T cells are genetically modified to express a chimeric antigen receptor (CAR) having an extracellular B7-H3 binding domain. 110. The method of any of embodiments 78-109, wherein the T cells include CD8+ T cells or CD4+ T cells.111. The method of embodiment 109 or 110, wherein the CAR includes the extracellular component linked to an intracellular component through a transmembrane domain.112. The method of embodiment 111, wherein the intracellular component includes an effector domain including 4-1BB (CD137), CD3y, CD35, CD3E, CD3 , CD27, CD28, DAP10, ICOS, LAG3, NKG2D, NOTCH1, 0X40, ROR2, SLAMF1, TCRa, TCR , TRIM, Wnt, Zap70, or a combination thereof.113. The method of embodiment 111, wherein the intracellular component includes a 4-1 BB signaling domain and a CD3 signaling domain.114. The method of any of embodiments 111-113, wherein the transmembrane domain includes a transmembrane region of: an a, p or chain of a T cell receptor; CD28; CD27; CD3; CD45; CD4; CD5; CD8; CD9; CD16; CD22; CD33; CD37; CD64; CD80; CD86; CD134; CD137; CD154; or a combination thereof.115. The method of any of embodiments 78-114, wherein the T cell is genetically modified to express a CAR by contacting the T cell with an artificial expression construct including a sequence encoding the CAR.S281-6010PCT / SCRL544WO116. The method of embodiment 115, wherein the artificial expression construct includes a viral vector.117. The method of embodiment 116, wherein the viral vector includes a lentiviral vector. 118. The method of any of embodiments 115-117, wherein the artificial expression construct further encodes a skip sequence.119. The method of embodiment 118, wherein the skip sequence includes a T2A skip sequence, a P2A skip sequence, an E2A skip sequence, or an F2A skip sequence.120. The method of any of embodiments 115-119, wherein the artificial expression construct further includes a sequence encoding a transduction marker.121. The method of embodiment 120, wherein the transduction marker includes a truncated EGFR (EGFRt).122. The method of any of embodiments 115-121, wherein the artificial expression construct further includes a sequence encoding a selection cassette.123. The method of embodiment 122, wherein the selection cassette includes a dihydrofolate reductase (DHFR)dm.124. The method of any of embodiments 78-123, wherein the T cells are autologous to the human patient.125. The method of any of embodiments 78-124, wherein the method does not include lymphodepletion.126. The method of any of embodiments 78-124, wherein the method includes lymphodepletion.

[0129] (vi) Experimental Example. Example 1. Intracerebroventricular B7-H3 targeting CAR T cells for diffuse intrinsic pontine glioma: a phase 1 trial.

[0130] Abstract. Diffuse intrinsic pontine glioma (DI PG) is a fatal central nervous system (CNS) tumor with a median survival of 11 months. As B7-H3 is expressed on pediatric CNS tumors, BrainChild-03, a single-center, dose-escalation phase 1 clinical trial of repetitive intracerebroventricular (ICV) B7-H3 targeting chimeric antigen receptor T cells (B7-H3 CAR T cells) for children with recurrent / refractory CNS tumors and DI PG was conducted. Here, Arm C restricted to patients with DIPG was reported on. The primary objectives to assess feasibility and tolerability were met. Secondary objectives included assessments of CAR T cell distribution and survival. Twenty-three patients with DIPG enrolled and 21 were treated with repeated ICV B7-H3 CAR T cells using intra-patient dose escalation dose regimens without prior lymphodepletion. Concurrent tumor-directed therapy, including re-irradiation, was not allowed while on protocol therapy. A total of 253 ICV doses were delivered and the highest planned dose regimen DR4,S281-6010PCT / SCRL544WOwhich escalated up to 10x107cells / dose, as the maximally tolerated dose regimen (MTDR) was established. Common adverse events included headache, fatigue, and fever. There was 1 DLT (dose limiting toxicity) (intratumoral hemorrhage) during DR2. For all treated patients (n=21), the median survival from their initial CAR T cell infusion was 10.7 months and the median survival from diagnosis was 19.8 months with 3 patients still alive at 44, 45, and 52 months from diagnosis. Ultimately, this completed first-in-human trial demonstrates that repetitive ICV dosing of B7-H3 CAR T cells in pediatric and young adult patients with DI PG is tolerable, even over repeated multiyear dosing, and may have clinical efficacy that warrants further investigation on a multi-site phase 2 trial. ClinicalTrials.gov Identifier: NCT04185038.

[0131] Introduction. Diffuse intrinsic pontine glioma (DIPG) is a fatal brainstem tumor responsible for 25,000 years of life lost each year in the United States (Cooney et al., Neuro-oncology 19, 1279-1280, 2017 ("Cooney 2017”); and Vitanza and Monje. Curr Treat Options Neurol 21, 37, 2019). Chimeric antigen receptor (CAR) T cells have shown significant survival benefits against pediatric leukemia (Gardner et al., Blood 129, 3322-3331, 2017; and Maude et al., N Engl J Med 371, 1507-1517, 2014), yet the clinical application of intracranial CAR T cells for children with DIPG is a nascent area of therapeutic development (Vitanza et al., Neoplasia 36, 100870, 2023; Vitanza et al., Cancer discovery 13, 114-131, 2023; Vitanza et al., J Hematol Oncol Pharm 14, 148-154, 2024; Lin et a!., J Clin Oncol, JC02302019, 2024; Monje et a!., Nature, 2024; and Wang et al., Res Sq, 2023).

[0132] The initial phase 1 clinical trials delivering intracranial CAR T cells (BrainChild-01 delivering HER2-specific CAR T cells [NCT03500991] (Vitanza et al., Nat Med 27, 1544-1552, 2021); BrainChild-02 delivering EGFR806-specific CAR T cells (Ravanpay et al., Oncotarget 10, 7080-7095, 2019) [NCT03638167]) excluded patients with DIPG. As B7-H3 (CD276) is expressed on DIPG (Haydar et al., Neuro-oncology 23, 999-1011, 2021 (“Haydar2021”); Majzner et al., Clin Cancer Res 25, 2560-2574, 2019 (“Majzner”); Zhou et al., Journal of neuro-oncology 111, 257-264, 2013 ("Zhou 2013”); and Maachani et al., Transl Oncol 13, 365-371, 2020), B7-H3 targeting CAR T cells (B7-H3 CAR T cells) were designed and their preclinical efficacy was established (Vitanza et al., Cancer discovery 13, 114-131, 2023). Next, BrainChild-03 (NCT04185038) was opened, delivering B7-H3 CAR T cells to children with recurrent or refractory central nervous system (CNS) tumors and DI PG, and published on the preliminary tolerability (Vitanza et al., Cancer discovery 13, 114-131, 2023).

[0133] Here, the completed Arm C of the first-in-human phase 1 clinical trial BrainChild-03 dedicated to children with DIPG was reported on. Patients were enrolled at any time following standard radiotherapy (including patients with disease progression and / or metastases). TheS281-6010PCT / SCRL544WOsafety of repetitive intracerebroventricularly (ICV) dosed B7-H3 CAR T cells up to 10x107cells / dose to children with DIPG, evidence of immune activation in the CNS, and the potential clinical benefit was demonstrated.

[0134] Results. Study Design and Patient Characteristics. This was a phase 1 study of repeatedly dosed ICV adoptive cell therapy with autologous CD4+and CD8+T cells lentivirally transduced to express a B7-H3-specific CAR to children and young adults with DI PG. The primary objectives were to assess the feasibility, safety, and tolerability of ICV B7-H3 CAR T cell therapy, while the secondary objectives included assessments of CAR T cell distribution and survival. Eligibility criteria included: age > 1 and < 26 years; a diagnosis of DI PG (diagnosed radiographically or via histopathologic confirmation of high-grade glioma or diffuse midline glioma H3K27M-altered [DMG]) at any timepoint following completion of standard radiation; presence of a CNS catheter; Lansky / Karnofsky performance of > 60; dexamethasone dose < 2.5 mg / m2 / day; protocol-defined washout from prior therapies; and protocol-defined laboratory evidence of adequate organ function (including an absolute lymphocyte count [ALC)] > 100 cells / pL).

[0135] The median age at enrollment of 6 years (range: 2 to 22) is consistent with the historical median age for this disease (Cooney 2017). Patients had Lansky / Karnofsky performance scores of 60 (2, 9%), 70 (2, 9%), 80 (7, 30%), and 90 (12, 52%). All patients received standard radiation therapy at diagnosis. The two enrolled patients who did not receive CAR T cells each experienced clinical progression during CAR T cell manufacturing and never met eligibility for CAR T cell infusion. Of the 21 treated patients, 12 (57%) received treatment after at least one tumor progression, while 9 patients (43%) were treated prior to any tumor progression. The primary objective of the feasibility to manufacture CAR T cells from a single apheresis was met for all enrollees. The baseline characteristics of the patients are provided in FIG. 1. Of the 21 treated patients, 17 met the molecular diagnosis of DMG either by tumor tissue (n=15) or cerebrospinal fluid (CSF) / plasma testing (n=2) (FIG. 2). One patient (S008) had a pontine anaplastic astrocytoma harboring an IDH1 mutation.

[0136] Treatment. BrainChild-03 Arm C delivered ICV B7-H3 CAR T cells without prior lymphodepletion every 14 days over 8 weeks during the DLT observation period (Courses 1 and 2). Concurrent tumor-directed therapy, including re-irradiation, was notallowed while on protocol therapy. As the optimal number of doses is unknown, patients - who met criteria for subsequent infusions beyond Course 2 (and for whom additional cryopreserved CAR T cell doses were available) - were eligible to continue receiving CAR T cell therapy beyond the DLT period with doses every 2-4 weeks. Patients were treated on four escalating dose regimens (DR; FIG. 3). In DR1, three evaluable patients received 1x107CAR T cells / dose for each dose. DR2, 3, and 4S281-6010PCT / SCRL544WOemployed an intra-patient dose escalation. In DR2, six evaluable patients received up to 2.5x107CAR T cells / dose (DR2); in DR3, three patients received up to 5x107CAR T cells / dose; and in DR4, six evaluable patients received up to 10x107CAR T cells / dose. DR2 expanded to six patients due to a DLT (intratumoral hemorrhage) and DR4 expanded to six patients for a planned statistical confirmation of the Maximally Tolerated Dose Regimen (MTDR). Three patients were not DLT evaluable (as they elected to pursue other tumor-directed therapy out of concern for progressive disease without having experienced a DLT) and were replaced as per the protocol.

[0137] For patients who began treatment prior to any progression (n=9), the median time from diagnosis to initial CAR T cell infusion was 6.6 months (range: 4.8-14.1; FIG. 4A). For patients who began treatment after progression (n=12), the median time from initial diagnosis to initial CAR T cell infusion was 10.3 months (range: 4.5-24.6). For all patients receiving at least one CAR T cell infusion (n=21), the median interval between enrollment and initial CAR T cell dose was 1.4 months (range 1.0-4.4). The 18 patients evaluable for DLT received 253 total doses (median: 9 / patient, range: 1-81) and remained on protocol therapy for a median of 5 months (range: 0.2-37.5).

[0138] Safety. All patients who received at least one CAR T infusion are included in the adverse event (AE) summary (n=21, FIG. 5). The most frequent AEs that were possibly, probably, or definitely attributable to CAR T therapy were headache (17 patients, 81%), nausea / vomiting (17 patients, 81%), fatigue (13 patients, 62%), and fever (12 patients, 57%). Most events were grade 1 or 2 with few patients experiencing grade 3 toxicities (two with headache, one with nausea, and one with vomiting). Only one patient developed hydrocephalus (grade 3). Fever was considered evidence of local immune activation rather than conventional systemic cytokine release syndrome (CRS). Immune effector cell-associated neurotoxicity syndrome (ICANS) was not observed. The sole DLT was an intratumoral hemorrhage (grade 4) occurring in a 3-year-old with progressive disease between enrollment and initial infusion. They received their first CAR T cell dose (DL1 of DR2), then had a stable neurologic exam and unchanged performance score without evidence of systemic inflammation (e.g. normal serum C-reactive protein [CRP], ferritin, D-dimer, IL-2, IL-6, and interferon-gamma [IFN-y]) for one week prior to the acute event. Following the hemorrhage, they required a PICU admission with intensive care followed by slow, limited neurologic improvement, but ultimately had fatal progression of their tumor.

[0139] Clinical Outcomes. Some clinical trials for DIPG mandate enrollment at diagnosis, while others allow patients to enroll anytime following standard radiation. The latter risks an immortalization bias in which some patients have a prolonged period prior to enrollment that constitutes a significant portion of their overall survival. Therefore, while the median survival fromS281-6010PCT / SCRL544WOinitial diagnosis was calculated, the survival from study enrollment (for all enrolled patients) and from initial CAR T cell infusion (for all treated patients) was also evaluated. Patients were followed from the time of enrollment, which occurred between August 2020 and April 2023, through treatment until death or censoring at their last follow-up visit prior to November 132024.

[0140] For all enrolled patients (n=23), the median survival from the time of study enrollment was 11.4 months (range: 2.7-48.8) and the median survival from diagnosis to death (or last contact for survivors) was 19.5 months (range: 6.5-52.5; FIG. 4A). For all treated patients (n=21), the median survival from their initial CAR T cell infusion was 10.7 months (range: 0.6-45.8; FIG. 4B) and the median time from diagnosis to death (or last contact for survivors) was 19.8 months with three patients still alive 44.6, 45.6, and 52.5 months from diagnosis (range: 6.5-52.5; FIG. 4B).

[0141] As it is unknown if intracranial cellular therapy delivered temporally near initial radiation may cause added toxicity or, conversely, be more efficacious when disease burden may be lowest, a post hoc analysis was performed comparing patients who enrolled pre progression (n=9) versus post progression (n=12). For patients who began CAR T treatment after progression (n=12), the median survival following their initial CAR T cell infusion was 9.4 months (range: 0.6-16.9; FIG.4C) and the median survival from diagnosis was 20.1 months (range: 6.5-36.2). For patients who began treatment prior to progression (n=9), the median survival following their initial CAR T cell infusion was 13.6 months (range: 2.8-45.8; FIG. 4C) and the median time from diagnosis to death (or last contact for survivors) was 19.5 months (range: 7.6-52.5). All three surviving patients were treated prior to progression. One surviving patient is in long term follow-up: S008 (pontine highgrade glioma with mutations in TP53 and IDH1 diagnosed at 22 years of age) left protocol therapy due to patient preference after 1 year of treatment. Two surviving patients remain on protocol therapy: S021 (pontine DMG with a H3F3A K27M mutation diagnosed at 2 years of age) has received 81 doses (3x109total CAR T cells) over 37.5 months and S027 (non-diagnostic biopsy at 5 years of age) has received 39 doses (9.5 x108total CAR T cells) over 31.5 months.

[0142] 18 patients received sufficient CAR T cell infusions to be evaluable for neuroimaging assessment. The best individual neuroimaging responses on MRI were 1 partial response (PR, 6%), 14 stable disease (SD, 78%), and 3 progressive disease (PD, 17%). 88.9% of patients evaluable for disease response who had progressed prior to treatment recorded a best neuroimaging response of stable disease (SD) or partial response (PR) after initiating protocol therapy. While S021 is not classified as an objective radiographic response, MRI reveals a sustained and ongoing decrease in pontine T2 hyperintensity (FIG. 6A). While S053 experienced a PR correlating with improvement in clinical symptoms, this patient ultimately had tumor progression at metastatic sites of disease 60 days later (FIG. 6B).S281-6010PCT / SCRL544WO

[0143] CAR T Cell Detection and Cytokine Analysis. A secondary objective was to assess B7-H3 CAR T cell distribution in the CSF. To complete this, serial CSF samples were collected from the CNS catheter pre- and post-CAR T cell infusions, then flow cytometry was performed to detect the EGFRt transduction tag. B7-H3 CAR T cells were detected via their EGFRt transduction tag in 38.1% (40 / 105) of CSF biospecimens in Courses 1 and 2 (FIGs. 7, 8, and 9) with 13 / 18 (72%) evaluable patients having detectable CAR T cells in at least one timepoint. The median peak CAR T cell detection was at the Course (Cr) 2 Week (W) 3 post-infusion timepoint. Overall, Course 2 timepoints demonstrated higher median detection than matched Course 1 timepoints, except Cr2.W3.Pre. Of the 96 peripheral blood samples collected, only two had detectable vector (S021: Cr2.W4: 264.4 copies / pg by qPCR; S039: Cr2.W1. Post 205.5 copies / pg by qPCR). Overall, this supports that while ICV delivered B7-H3 CAR T cells are consistently detected in CSF postinfusion, systemic circulation was rare, transient, and at low levels.

[0144] An exploratory objective of this study was to assess biomarkers indicative of CAR T cell activity. To achieve this, 53 cytokines associated with T cell function and immune microenvironment interactions were measured using a Meso Scale Discovery (MSD) assay. A total of 105 CSF samples during Courses 1 and 2 from 18 evaluable patients were analyzed using matched pre- and post-infusion biospecimens from the following time points: Cr1.W1 (n = 12), Cr1.W3 (n = 11), Cr2.W1 (n = 11), and Cr2.W3 (n = 10) (FIG. 10). Following the initial CAR T infusion (Cr1.W1), significant elevations were observed in the levels of CXC motif chemokine ligand 10 (CXCL10, also known as interferon gamma-induced protein 10 [IP-10], granulocytemacrophage colony-stimulating factor (GM-CSF), IFN-y, and thymus and activation-regulated chemokine (TARC) (FIG. 11). Interestingly, the inflammatory markers CRP and serum amyloid A (SAA) did not significantly increase after the first infusion but showed notable elevations following subsequent infusions (FIGs. 11 A, 11B). While GM-CSF and TARC displayed their most pronounced increases after the first infusion, CXCL10 and IFN-y levels consistently increased after each infusion. In aggregate, these findings support locoregional CAR T cell activation and cytotoxic activity.

[0145] Discussion. Results from the completed first-in-human phase 1 BrainChild-03 Arm C delivering repetitive ICV B7-H3 CAR T cells to children and young adults with DI PG was presented. While other groups have generated B7-H3 CAR T cells (Haydar 2021; Majzner 2019; Du et al., Cancer cell 35, 221-237 e228, 2019; Nehama et al., EBioMedicine 47, 33-43, 2019; Talbot et al., Front Immunol 12, 691741, 2021; Zhang et al., Mol Ther Oncolytics 17, 180-189, 2020; Tang et al., ClinTransI Immunology 9, e1137, 2020; and Tang etal., Signal Transduct Target Ther 6, 125, 2021) and the team had delivered them systemically for patients with solid tumors (Pinto et al., JS281-6010PCT / SCRL544WOClin Oncol, JCO2302229, 2024), here the first completed ICV B7-H3 CAR T cell trial is presented. Tolerability of repetitive ICV dosing up to 10x107cells, cumulatively as high as 3x109CAR T cells in one patient, is demonstrated.

[0146] Regarding tolerability, the single DLT occurred in a patient with progressive DIPG (unbiopsied) who received the lowest CAR T cell dose (1x107cells), then 1 week later developed intratumoral hemorrhage. While spontaneous tumor hemorrhage is a known natural risk (Broniscer et al., Cancer 106, 1364-1371, 2006) and this was a solitary event, hemorrhage deserves attention as it was also seen in a patient receiving GD2 CAR T cells (Monje et al., Nature, 2024).

[0147] One patient (S053) met radiographic criteria for PR. S021 - a patient with histone mutant tumor who has received 81 doses and remains on protocol therapy - has had near resolution of T2 hyperintensity. Considering the historical consistency of survival in patients with DIPG, survival amongst newly diagnosed patients represents the best metric of clinical success. The median survival of 19.8 months for all treated patients is superior to the historical median survival of 11.2 months (Cooney 2017) and the 9.4-month survival post initial CAR T cell infusion for patients enrolled after progression is superior to the <3 month historical progression to death (Cooney 2017).

[0148] Tumor biopsy was not required for enrollment because B7-H3 is expressed on most DIPG (Haydar 2021; Majzner 2019; Zhou 2013)To assess other metrics of CAR T cell activity, CSF cytokine levels were evaluated and signals of intracranial inflammation following CAR T cells were confirmed. It is clear cytokines related to CAR T cell trafficking, such as CXCL10 which was also identified in the prior publications (Vitanza etal., Cancer discovery 13, 114-131, 2023; and Vitanza et al., Nat Med 27, 1544-1552, 2021), are elevated following infusion. Notably, a recent study found a decrease in CXCL10 concentrations associated with timing of tumor progression (Monje et al., Nature, 2024).

[0149] This example used ICV dosing from inception, allowed enrollment of children receiving dexamethasone, and did not incorporate lymphodepletion. Notably, their median age was 15 and included spinal DMG, while the median age was 6 years on this example and patients with DI PG were treated. Ultimately, the example has demonstrated that repeatedly dosed ICV B7-H3 CAR T cells are tolerable and feasible for children and young adults with DIPG.

[0150] Methods. CAR T Cell Product. The B7-H3-specific CAR T cells (SCRI-CARB7-H3(s)) and good manufacturing process (GMP) were previously described (Vitanza et al., Cancer discovery 13, 114-131, 2023). Briefly, the second generation, 4-1BB:zetaCAR is appended to a T2A ribosomal skip sequence followed by a truncated EGFR (EGFRt) cell-surface tag (Ceppi et al.,S281-6010PCT / SCRI.544WOCancer Immunol Res 10, 856-870, 2022). A methotrexate-resistant human DHFR mutein (huDHFRFS; L22F, F31S) was appended to allow enrichment with methotrexate (MTX) ex vivo (Jonnalagadda et al., Gene Ther20, 853-860, 2013).

[0151] Objectives. The example’s primary objectives were to assess the feasibility, safety, and tolerability of intracerebroventricular (ICV) delivery of B7-H3 CAR T cells for children and young adults with DIPG, as well as defining the maximally tolerated phase 2 dose regimen (RP2DR). Feasibility was primarily assessed by the ability to generate sufficient product to receive all planned doses in Courses 1 and 2 from a single apheresis. Safety and tolerability were primarily assessed by history / physical exams, laboratory / radiographic evaluations, and Common Terminology Criteria for Adverse Events (CTCAE v5.0). A DLT was defined as an event which is possibly, probably, or definitely attributable to CAR T cells and occurs from initial CAR T cell infusion through 28 days following final CAR T cell infusion. A DLT included all > grade 3 CTCAE v5.0 toxicities except > grade 3 toxicities known to be related to CAR T cells including: grade 3 CRS that decreased to < grade 2 within 72 hours; > grade 3 hypotension, fever, and / or chills not controlled with medical intervention that decreased to < grade 2 within 72 hours; > grade 3 activated PTT, fibrinogen, and / or INR that were asymptomatic and resolved within 72 hours; > grade 3 hypoglycemia and / or electrolyte imbalance that were asymptomatic and resolved within 72 hours; > grade 3 nausea and / or vomiting that decreased to < grade 2 with 7 days; and grade 3 neurologic symptoms that decreased to < grade 2 within 21 days. DLTs included any toxicity lasting > 14 days preventing the patient from receiving subsequent doses in Courses 1 or 2. Patients were considered DR escalation-evaluable if evaluable for toxicity and counted in a dose escalation cohort. Radiologic response criteria used the sum of the two longest 2D perpendicular diameters to distinguish: stable disease, progressive disease (>25% increase), partial response (>50% decrease), and complete response.

[0152] Patients. Enrollment criteria for BrainChild-03 Arm C included: age > 1 and < 26 years; DIPG (diagnosed radiographically or via histopathology confirming high-grade glioma or DMG) at any timepoint following completion of standard radiation; ability to tolerate apheresis; presence of a CNS catheter; life expectancy > 8 weeks; Lansky / Karnofsky performance of > 60; study-determined washout from prior therapies; adequate organ function including absolute lymphocyte count (ALC) > 100 cells / pL, absolute neutrophil count > 500 cells / pL, hemoglobin > 9 g / dL, platelets > 100,000 / pL, creatinine < upper limit of normal (ULN), total bilirubin < 3x ULN or conjugated bilirubin < 2 mg / dL, an oxygen saturation > 90%, adequate neurologic function defined as stable deficits for > 1 week, < 2 anti-epileptic agents required, and no encephalopathy; negative virology for HIV, Hepatitis B or C; and use of contraception in patients of child-bearing age.S281-6010PCT / SCRL544WOExclusion criteria included: dexamethasone > 2.5 mg / m2 / day; severe cardiac dysfunction; primary immunodeficiency or bone marrow failure; impending CNS herniation; presence of > grade 3 dysphagia; another active malignancy; severe, active infection; active receipt of any anti-cancer therapy; or pregnancy / breastfeeding. Patients and / or their guardians provided written informed consent in accordance with local regulatory review.

[0153] Study Design and Treatment. BrainChild-03 began accrual on November 222019. Clinical data through November 13 2024 is included. The first reported patient was enrolled in August 2020 and the last reported patient was enrolled in April 2023. This study was conducted in accordance with FDA and International Conference on Harmonisation Guidelines for Good Clinical Practice, the Declaration of Helsinki, and applicable institutional review board requirements, including study protocol approval by the Seattle Children’s Institutional Review Board. BrainChild-03 Arm C patients underwent leukopheresis, CAR T cell manufacture, and infusions through their CNS catheter. Dose regimens (DR) other than DR1 (in which all doses were DL1) used an intra-patient dose level (DL) (FIG. 3). Dose escalation and de-escalation decisions were made using a modified 3 + 3 design. The MTDR was defined as the highest DR with at least six DLT-evaluable human patients whose cumulative DLT rate during Courses 1 and 2 was below 34%. The study was monitored by a Data Monitoring Committee (DMC). The BrainChild-03 protocol and list of amendments are available as supplementary information.

[0154] Requirements to receive CAR T cell infusions included: a CNS catheter (Vitanza et al., Neoplasia 36, 100870, 2023); > 5 days from surgery; evidence of disease; not breastfeeding / pregnant; meeting study-defined washout periods from bridging therapy; adequate study-defined organ function; no encephalopathy or uncontrolled seizure activity; compliance with prescribed anti-epileptic drug(s); no evidence of active severe infection; and no prior DLT. Beyond Course 2, patients were eligible to receive additional infusions at the previous maximum tolerated DL, if the above criteria were met and sufficient CAR T cells were available. Response was assessed following Course 2, and subsequent even-numbered courses, via MRI and CSF cytology. Following the initial DLT observation period, eligible patients could continue therapy (at least 1 dose every 28 days for an infinite number of courses). Tumor-directed therapy following discontinuation of protocol therapy was not collected as this was a phase 1 trial. Follow-up is ongoing, with patients monitored until death or for 15 years in long-term follow up.

[0155] Statistical Analysis. Sample size was based on the phase 1 3+3 design. The MTDR was defined as the highest DR with at least six DLT-evaluable human patients and a cumulative DLT rate during Courses 1 and 2 below 34%. Human patient demographics, clinical characteristics and CAR T manufacturing feasibility were summarized with descriptive statistics includingS281-6010PCT / SCRL544WOfrequencies and percentages for categorical data and medians with ranges for continuous variables. The lengths of intervals between diagnosis and enrollment, first CAR T infusion and death or end of follow up were described using ordinary median and range statistics. Kaplan-Meier analysis was used for evaluating survival time from first CAR T infusion. Overall survival (OS) was defined as time from first CAR T infusion to death. Surviving human patients were censored at most recent follow up. A supplemental intent-to-treat survival analysis starting from time of enrollment and including the human patients who did not receive any CAR T product was also done. SAS 9.4 (SAS Institute Inc., Cary, NC) software was used for analyses of clinical and time to event variables.

[0156] CSF Analysis. CSF processing: Patient CSF samples were collected via lumbar puncture or ventricular catheter and kept at 4°C until processing. The samples underwent serial centrifugation: first at 250xg for 10 minutes to remove cells, followed by a final centrifugation at 10,000xg for 10 minutes to remove any remaining debris. The cell-free supernatant was then aliquoted and cryopreserved at-80°C. CAR T cell expression was quantified by detecting EGFRt transduction tag using cetuximab custom-conjugated to allophycocyanin (BD Biosciences). Additionally, cells were stained with custom-biotinylated trastuzumab followed by streptavidin (BD Bioscience) for detection of a HER2 tag, which was not relevant to this trial. CAR T cells were identified as singlets / lymphocytes / viable cells and characterized by the phenotype CD3+ / EGFRt+ HER2- (with or without CD36-). The expression of CD4 and CD8 expression in both CAR+ and CAR- populations was evaluated. BD LSRFortessa was used. Representative flow gating strategies are provided in FIG. 12. Samples with lymphocytes count under the limit of quantitation (LOQ) requirement for the assay were excluded from reporting. CAR T cell detection status was determined by a combination of at least one detectable EGFRt+ cell count in the sample, as well as the level of Lymphocytes / EGFRt+% cell in the sample to be above the pre-defined limit of detection (LOD) for the assay. Electrochemiluminescence assays: Patient CSF samples were thawed and assessed for cytokines and chemokines using the V-PLEX Plus Human Biomarker 54-Plex Kit (MesoScale Diagnostics, Cat. No. K15248G), following the manufacturer's instructions. All CSF samples were diluted according to the manufacturer's recommendations, except for the analytes CXCL10 and MCP-1, which were diluted 50-fold for measurement. A precoated 96-well plate with capture antibody was used. The plate was blocked with MSD Blocker A for 1 hour at room temperature. After blocking, the plate was washed, and diluted standards and samples were added in duplicates to the respective wells and incubated overnight at 4°C with shaking. Following the overnight incubation, a proprietary SULFO-TAG conjugated detection antibody was added to the wells and incubated for 1-2 hours at room temperature. The plate was washed again,S281-6010PCT / SCRL544WOdeveloped using read buffer, and measured using the MESO QuickPlex SQ 120 instrument Data processing was conducted using MSD Discovery Workbench version 4.0.13 software after a final wash step. Standard curves were generated using a 5-parameter logistic model. The concentration of each analyte was extrapolated from the standard curve. Values below the lowest limit of detection (LLOD) were considered undetectable. Statistical analyses for correlative cytokine data were conducted in R, utilizing the Ime4, emmeans, and EnhancedVolcano packages (Wickham. ggplot2: Elegant Graphics for Data Analysis, Springer, 2016 ("Wickham 2016”); and Blighe et al., EnhancedVolcano: Publication-ready volcano plots with enhanced colouring and labeling. R package version 1.22.0. 2024 ("Blighe 2024”)). Cytokine levels were measured in duplicate, and those with a coefficient of variation exceeding 25% were excluded. Differential expression between pre- and post-infusion time points was assessed using linear mixed-effects models, with human patients included as random intercepts and Iog2-transformed cytokine expression values as the response variable. A single model was fitted to include all cytokines and time points, and linear contrasts were calculated to evaluate pre- versus postinfusion differences at the specified time points (Wickham 2016; and Blighe 2024).

[0157] Peripheral Blood CAR T Cell Detection. CAR T cell peripheral blood detection was assessed by qPCR quantification of the human 5-lipoxygenase-activating protein elongation factor-1 (FI.AP-EF1). Genomic DNA (gDNA) was extracted from mononuclear cells. The in vivo persistence of CAR T cells was evaluated through batched analysis. A standard curve for the transcript copy number was generated by amplifying serial dilutions of the plasmid epHIV7. The number of transgene copies per nanogram of gDNA input was then determined. Measurements were taken from distinct samples.

[0158] Example 2. Phase 2 Study of B7-H3 CAR T Cell Locoregional Immunotherapy for Diffuse Intrinsic Pontine Glioma (DIPG). Diffuse intrinsic pontine glioma (DIPG) is a primary high-grade brain tumor affecting over 300 children per year in the U.S. It has been uniformly fatal, and despite decades of clinical studies, no therapeutic advantage has been afforded to DI PG patients beyond that provided by palliative focal radiation therapy which extends median overall survival to 11 months from diagnosis. Once the disease begins to progress, children are often left with reirradiation as the only option to even marginally extend survival.

[0159] Study Design. This is a Phase 2, single-arm, multi-center clinical study to evaluate the efficacy and confirm safety of B7-H3 CAR T in children and young adult patients with DI PG who have only received standard of care focal radiation and have no clinical or radiological evidence of disease progression.

[0160] Enrolled patients will undergo baseline evaluation followed by leukapheresis to enableS281-6010PCT / SCRI.544WOgeneration of a B7-H3 CAR T product that will be fractionated for the use of repetitive ICV dosing.

[0161] The study will be monitored by a Data Safety Monitoring Board (DSMB), an independent committee with no affiliation to the protocol. DSMB composition and procedures will be documented in a charter.

[0162] Sixty patients will be enrolled to ensure that 50 are evaluable for the primary endpoint. Patients must receive at least 1 infusion of B7-H3 CAR T without disease progression to be evaluable for the primary endpoint.

[0163] Treatment Duration and End of Study. Patients will receive up to 15 ICV infusions (each 50 x 106CAR+ T cells) delivered every 14 days (+ / - 2 days) for an approximate total duration of 30 weeks.

[0164] Patients will receive B7-H3 CAR T until disease progression, unacceptable toxicity, or all doses of B7-H3 CAR T are completed.

[0165] If patients complete the initial dose regimen without disease progression or unacceptable toxicity, an additional retreatment regimen of up to 15 ICV infusions (each 50 x 106CAR+ T cells) delivered every 14 days (+ / - 2 days) may be delivered if additional doses are available, or after a repeat leukapheresis and generation of a second lot of B7-H3 CAR T.

[0166] Patients will be followed until death or study withdrawal.

[0167] Study Treatment. B7-H3 CAR T, consisting of autologous CD4+ and CD8+ T cells obtained from apheresis starting material that has undergone CD4 and CD8 immunomagnetic selection, activation on CD3xCD28 beads, transduction with a SIN lentiviral vector encoding a B7H3-specific second generation (41BB:zeta) CAR and human DHFRdm conferring methotrexate resistance, followed by expansion in recombinant gamma-c cytokines and methotrexate, is supplied as a cryopreserved single dose for bedside thaw.

[0168] Dose: B7-H3 CAR T will be drawn up in a sterile syringe and via needles access to the reservoir of an Ommaya shunt delivered as a slow push followed by a Normosol™ flush. Each single dose will consist of 50 x 106B7-H3 CAR+ T cells.

[0169] Inclusion Criteria: To be eligible to participate in this study, an individual must meet all the following criteria:1. Patients must be age > 1 and < 26 years.2. Patient must have localized DIPG (if tissue / s not surgically obtained, the tumor must be radiographically classic; if tissue / s surgically obtained, histopathology must be consistent with diffuse midline glioma H3K27M-altered (DMG) or a high-grade neuroglial tumor.3. Patient must have completed standard radiation therapy within 6 weeks of enrollment.S281-6010PCT / SCRL544WONo clinical or radiological evidence of disease progression during or following completion of standard of care focal radiation.Able to tolerate apheresis.CNS reservoir catheter, such as an Ommaya or Rickham catheter, present in the lateral ventricle.Life expectancy > 8 weeks.Lansky or Karnofsky score > 60. Patients who are unable to walk because of paralysis, but who are up in a wheelchair, will be considered ambulatory for the purposes of assessing performance status.Adequate hematologic values, all the following must be true:>>>>1Value not required to be met if participant has previously obtained apheresis product acceptable and available for manufacturing of CAR T cells2Patients receiving blood product transfusion(s) are acceptable as long as they are not determined to be transfusion refractoryAdequate renal function, as indicated by serum creatinine < the upper limit of normal (ULN), per the following:<<<<<>Adequate hepatic function as indicated by either of the following:a. Total bilirubin < 3 times ULN for age, ORb. Conjugated bilirubin < 2 mg / dL.Adequate respiratory function as indicated by BOTH of the following:S281-6010PCT / SCRL544WOa. Oxygen saturation > 90% on room air without supplemental oxygen or mechanical ventilation, ANDb. No dyspnea at rest.13. Adequate neurologic function as indicated by all of the following:a. Signs and symptoms of neurologic deficit must be stable for > 1 week prior to enrollment on a stable or tapering dose of dexamethasone, AND b. < 2 anti-epileptic agents are required to control seizure activity, AND c. No clinically evident encephalopathy.14. Virology negative within 3 months prior to enrollment, to include all of the following:a. Human Immunodeficiency Virus (HIV) antigen & antibody, ANDb. Hepatitis B surface antigen, ANDc. Hepatitis C antibody negative OR if antibody positive, Hepatitis C polymerase chain reaction (PCR) is negative.15. Patients of childbearing / fathering potential must agree to use highly effective contraception from the time of enrollment through 12 months following the last T cell infusion.16. Participants must abstain from alcohol, tobacco, and recreational drug use.Exclusion Criteria: An individual who meets any of the following criteria will be excluded from participation in this study:1. Previous tumor-directed therapy other than standard radiation, except for single-agent temozolomide during radiation.2. Receiving tumor-directed therapy on another clinical study.3. Presence of neurologic symptoms requiring increasing dexamethasone dose within two weeks of enrollment.4. Presence of > Grade 3 dysphagia.5. Presence of clinical and / or radiographic evidence of impending herniation.6. Presence of primary immunodeficiency / bone marrow failure syndrome.7. Presence of an active malignancy other than DI PG8. Presence of active severe infection, defined as either of the following:a. Positive blood culture within 48 hours of enrollment, ORb. Fever > 38.2°C AND clinical signs of infection within 48 hours of enrollment. 9. Pregnant or breastfeeding.10. Presence of any condition that, in the investigator's opinion, would prohibit the participant from undergoing treatment under this protocol.S281-6010PCT / SCRL544WO11. Patient and / or authorized legal representative unwilling to provide consent / assent for study participation, including participation in the 15-year follow-up period, which is required if CAR T cell therapy is administered.

[0170] Statistical Analysis. Primary Endpoint Analysis: The primary endpoint is overall survival, defined as the time from the date of diagnosis to the date of death due to any cause. Survival distributions will be estimated using the Kaplan-Meier method, and median survival times with corresponding 95% confidence intervals (Cis) will be reported. Patients who are alive at the time of analysis will be censored at the last known date of follow-up.

[0171] A comparison will be made between the overall survival of patients in this study and that of an external control group derived from the International DIPG / DMG Registry. The Registry cohort will be left-truncated to be consistent with B7-H3 CAR T to mitigate this bias and ensure accurate comparisons. A Cox proportional hazards regression model will be used to compare the hazard of death between the two groups, adjusting for key baseline covariates such as age, disease severity, and radiation history.

[0172] Missing Data: To address missing data, a multiple imputation approach will be applied where appropriate. Sensitivity analyses will assess the robustness of primary endpoint results under different missing data assumptions (i.e., missing completely at random, missing at random, and missing not at random).

[0173] Primary Objective: To evaluate the efficacy of ICV autologous B7-H3- CAR T cells in patients with DIPG, as measured by overall survival.

[0174] Primary Endpoint: Overall survival, in months, from date of diagnosis compared to a historical control of an equivalent patient population from the International DIPG / DMG Registry.

[0175] (vii) Closing Paragraphs. The nucleic acid and amino acid sequences provided herein are shown using letter abbreviations for nucleotide bases and amino acid residues, as defined in 37 C.F.R. §1.831-1.835 and set forth in WIPO Standard ST.26 (implemented on July 1, 2022). Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included in embodiments where it would be appropriate.

[0176] Variants of the sequences disclosed and referenced herein are also included. Guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological activity can be found using computer programs well known in the art, such as DNASTAR™ (Madison, Wisconsin) software. Preferably, amino acid changes in the protein variants disclosed herein are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. A conservative amino acid change involves substitution of one of a family of amino acids which are related in their side chains.S281-6010PCT / SCRI.544WO

[0177] In a peptide or protein, suitable conservative substitutions of amino acids are known to those of skill in this art and generally can be made without altering a biological activity of a resulting molecule. Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. Co., p. 224). Naturally occurring amino acids are generally divided into conservative substitution families as follows: Group 1: Alanine (Ala), Glycine (Gly), Serine (Ser), and Threonine (Thr); Group 2: (acidic): Aspartic acid (Asp), and Glutamic acid (Glu); Group 3: (acidic; also classified as polar, negatively charged residues and their amides): Asparagine (Asn), Glutamine (Gin), Asp, and Glu; Group 4: Gin and Asn; Group 5: (basic; also classified as polar, positively charged residues): Arginine (Arg), Lysine (Lys), and Histidine (His); Group 6 (large aliphatic, nonpolar residues): Isoleucine (lie), Leucine (Leu), Methionine (Met), Valine (Vai) and Cysteine (Cys); Group 7 (uncharged polar): Tyrosine (Tyr), Gly, Asn, Gin, Cys, Ser, and Thr; Group8 (large aromatic residues): Phenylalanine (Phe), Tryptophan (Trp), and Tyr; Group 9 (nonpolar): Proline (Pro), Ala, Vai, Leu, lie, Phe, Met, and Trp; Group 11 (aliphatic): Gly, Ala, Vai, Leu, and lie; Group 10 (small aliphatic, nonpolar or slightly polar residues): Ala, Ser, Thr, Pro, and Gly; and Group 12 (sulfur-containing): Met and Cys. Additional information can be found in Creighton (1984) Proteins, W.H. Freeman and Company.

[0178] In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982, J. Mol. Biol. 157(1), 105-32). Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982). These values are: lie (+4.5); Vai (+4.2); Leu (+3.8); Phe (+2.8); Cys (+2.5); Met (+1.9); Ala (+1.8); Gly (-0.4); Thr (-0.7); Ser (-0.8); Trp (-0.9); Tyr (-1.3); Pro (-1.6); His (-3.2); Glutamate (-3.5); Gin (-3.5); aspartate (-3.5); Asn (-3.5); Lys (-3.9); and Arg (-4.5).

[0179] It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e., still obtain a biological functionally equivalent protein. In making such changes, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity.

[0180] As detailed in US 4,554,101, the following hydrophilicity values have been assigned toS281-6010PCT / SCRL544WOamino acid residues: Arg (+3.0); Lys (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); Ser (+0.3); Asn (+0.2); Gin (+0.2); Gly (0); Thr (-0.4); Pro (-0.5+1); Ala (-0.5); His (-0.5); Cys (-1.0); Met (-1.3); Vai (-1.5); Leu (-1.8); lie (-1.8); Tyr (-2.3); Phe (-2.5); Trp (-3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0181] As outlined above, amino acid substitutions may be based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. As indicated elsewhere, variants of gene sequences can include codon optimized variants, sequence polymorphisms, splice variants, and / or mutations that do not affect the function of an encoded product to a statistically-significant degree.

[0182] Variants of the protein, nucleic acid, and gene sequences disclosed herein also include sequences with at least 70% sequence identity, 80% sequence identity, 85% sequence, 90% sequence identity, 95% sequence identity, 96% sequence identity, 97% sequence identity, 98% sequence identity, or 99% sequence identity to the protein, nucleic acid, or gene sequences disclosed herein.

[0183] “% sequence identity” refers to a relationship between two or more sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between protein, nucleic acid, or gene sequences as determined by the match between strings of such sequences. "Identity" (often referred to as "similarity") can be readily calculated by known methods, including those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, NY (1994); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (Von Heijne, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Oxford University Press, NY (1992). Methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR, Inc., Madison, Wisconsin). Multiple alignment of the sequences can also be performed using the Clustal method of alignment (Higgins and Sharp CABIOS, 5, 151-153 (1989) with default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Relevant programs also include the GCG suite of programs (WisconsinS281-6010PCT / SCRL544WOPackage Version 9.0, Genetics Computer Group (GCG), Madison, Wisconsin); BLASTP, BLASTN, BLASTX (Altschul, etal., J. Mol. Biol. 215:403-410 (1990); DNASTAR (DNASTAR, Inc., Madison, Wisconsin); and the FASTA program incorporating the Smith-Waterman algorithm (Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, HI-20. Editor(s): Suhai, Sandor. Publisher: Plenum, New York, N.Y.. Within the context of this disclosure it will be understood that where sequence analysis software is used for analysis, the results of the analysis are based on the "default values" of the program referenced. As used herein "default values" will mean any set of values or parameters, which originally load with the software when first initialized.

[0184] Variants also include nucleic acid molecules that hybridize under stringent hybridization conditions to a sequence disclosed herein and provide the same function as the reference sequence. Exemplary stringent hybridization conditions include an overnight incubation at 42 °C in a solution including 50% formamide, 5XSSC (750 mM NaCI, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5XDenhardt's solution, 10% dextran sulfate, and 20 pg / ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1XSSC at 50 °C. Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation of formamide concentration (lower percentages of formamide result in lowered stringency); salt conditions, or temperature. For example, moderately high stringency conditions include an overnight incubation at 37°C in a solution including 6XSSPE (20XSSPE=3M NaCI; 0.2M NaH2PO4; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 pg / ml salmon sperm blocking DNA; followed by washes at 50 °C with 1XSSPE, 0.1 % SDS. In addition, to achieve even lower stringency, washes performed following stringent hybridization can be done at higher salt concentrations (e.g. 5XSSC). Variations in the above conditions may be accomplished through the inclusion and / or substitution of alternate blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.

[0185] "Bbinds" refers to an association of a binding domain (of, for example, a CAR binding domain) to its cognate binding molecule with an affinity or Ka( / .e., an equilibrium association constantof a particular binding interaction with units of 1 / M) equal to or greater than 105M-1, while not significantly associating with any other molecules or components in a relevant environment sample. Binding domains may be classified as "high affinity" or "low affinity". In particular embodiments, "high affinity" binding domains refer to those binding domains with a Kaof at leastS281-6010PCT / SCRL544WO107M’1, at least 108M’1, at least 109M’1, at least 1010M-1, at least 1011M’1, at least 1012M-1, or at least 1013M’1. In particular embodiments, "low affinity" binding domains refer to those binding domains with a Kaof up to 107M’1, up to 106M’1, up to 105M’1. Alternatively, affinity may be defined as an equilibrium dissociation constant (Kd) of a particular binding interaction with units of M e.g., 10-5M to 1013M). In certain embodiments, a binding domain may have "enhanced affinity," which refers to a selected or engineered binding domains with stronger binding to a cognate binding molecule than a wild type (or parent) binding domain. For example, enhanced affinity may be due to a Ka (equilibrium association constant) for the cognate binding molecule that is higher than the reference binding domain or due to a Kd (dissociation constant) for the cognate binding molecule that is less than that of the reference binding domain, or due to an off-rate (KOff) for the cognate binding molecule that is less than that of the reference binding domain. A variety of assays are known for detecting binding domains that specifically bind a particular cognate binding molecule as well as determining binding affinities, such as Western blot, ELISA, and BIACORE® analysis (see also, e.g., Scatchard, et al., 1949, Ann. N. Y. Acad. Sci. 57:660; and U.S. Patent Nos. 5,283,173, 5,468,614, or the equivalent).

[0186] Unless otherwise indicated, the practice of the present disclosure can employ conventional techniques of immunology, molecular biology, microbiology, cell biology and recombinant DNA. These methods are described in the following publications. See, e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4th Edition (2012); F. M. Ausubel, et al. eds., Current Protocols in Molecular Biology, (2003); the series Methods In Enzymology (Academic Press, Inc.); Behlke, et al., Polymerase Chain Reaction: Theory and Technology (2019); Greenfield, ed. Antibodies, A Laboratory Manual, Second Edition (2014); and Capes-Davis and R. I. Freshney, eds. Freshney's Culture of Animal Cells 8th Edition (2021).

[0187] As will be understood by one of ordinary skill in the art, each embodiment disclosed herein can comprise, consist essentially of or consist of its particular stated element, step, ingredient or component. Thus, the terms “include” or “including” should be interpreted to recite: “comprise, consist of, or consist essentially of.” The transition term “comprise” or “comprises” means has, but is not limited to, and allows for the inclusion of unspecified elements, steps, ingredients, or components, even in major amounts. The transitional phrase “consisting of” excludes any element, step, ingredient or component not specified. The transition phrase “consisting essentially of limits the scope of the embodiment to the specified elements, steps, ingredients or components and to those that do not materially affect the embodiment. A material effect would cause a statistically significant decrease in human patient survival as compared to survival results described herein.

[0188] Unless otherwise indicated, all numbers expressing quantities of ingredients, propertiesS281-6010PCT / SCRL544WOsuch as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. When further clarity is required, the term “about” has the meaning reasonably ascribed to it by a person skilled in the art when used in conjunction with a stated numerical value or range, i.e. denoting somewhat more or somewhat less than the stated value or range, to within a range of ±20% of the stated value; ±19% of the stated value; ±18% of the stated value; ±17% of the stated value; ±16% of the stated value; ±15% of the stated value; ±14% of the stated value; ±13% of the stated value; ±12% of the stated value; ±11% of the stated value; ±10% of the stated value; ±9% of the stated value; ±8% of the stated value; ±7% of the stated value; ±6% of the stated value; ±5% of the stated value; ±4% of the stated value; ±3% of the stated value; ±2% of the stated value; or ±1% of the stated value.

[0189] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0190] The terms “a,” “an,” “the” and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0191] Groupings of alternative elements or embodiments of the invention disclosed herein areS281-6010PCT / SCRL544WOnot to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0192] Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0193] Furthermore, numerous references have been made to patents, printed publications, journal articles and other written text throughout this specification (referenced materials herein). Each of the referenced materials are individually incorporated herein by reference in their entirety for their referenced teaching.

[0194] In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described.

[0195] The particulars shown herein are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of various embodiments of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for the fundamental understanding of the invention, the description taken with the drawings and / or examples making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.

[0196] Definitions and explanations used in the present disclosure are meant and intended to be controlling in any future construction unless clearly and unambiguously modified in the examplesS281-6010PCT / SCRL544WOor when application of the meaning renders any construction meaningless or essentially meaningless. In cases where the construction of the term would render it meaningless or essentially meaningless, the definition should be taken from Webster's Dictionary, 3rd Edition or a dictionary known to those of ordinary skill in the art, such as the Oxford Dictionary of Biochemistry and Molecular Biology (Eds. Attwood T etal., Oxford University Press, Oxford, 2006).

Claims

S281-6010PCT / SCRL544WOCLAIMSWhat is claimed is:

1. A method of treating a human patient having a B7-H3-expressing central nervous system (CNS) cancer, the method comprising:administering T cells expressing a B7-H3-binding chimeric antigen receptor (CAR) to the CNS of the human patient,thereby treating the human patient having the B7-H3-expressing CNS cancer.

2. The method of claim 1 , wherein the human patient is a child or young adult.

3. The method of claim 1 , wherein the human patient is under 26 years of age.

4. The method of claim 1 , wherein the human patient is over one year of age.

5. The method of claim 1 , wherein the human patient is between 2 and 22 years of age.

6. The method of claim 1, wherein the human patient has received radiotherapy before the treating.

7. The method of claim 6, wherein the radiotherapy is completed within 6 weeks of the administering.

8. The method of claim 1 , wherein the B7-H3-expressing CNS cancer has not progressed before the treating.

9. The method of claim 1, wherein the B7-H3-expressing CNS cancer comprises brain cancer or a spinal cord tumor.

10. The method of claim 9, wherein the brain cancer comprises diffuse intrinsic pontine glioma (DIPG), diffuse midline glioma (DMG), H3K27m-altered DMG, high grade glioma (HGG), glioblastoma (GBM), neuroblastoma, craniopharyngioma, ependymoma, an embryonal tumor, medulloblastoma, pineoblastoma, an atypical teratoid / rhabdoid tumor (ATRT), or an embryonal not otherwise specified (NOS) tumor.

11. The method of claim 1, wherein the B7-H3-expressing CNS cancer comprises DI PG.

12. The method of claim 1, wherein the administering comprises intracranial administration.

13. The method of claim 1, wherein the administering comprises intratumoral administration.

14. The method of claim 1, wherein the administering comprises intrathecal administration.

15. The method of claim 12, wherein intracranial administration comprises intracerebroventricular (ICV) injection.

16. The method of claim 1 , wherein the treating further comprises a dose regimen comprising a plurality of doses of T cells.

17. The method of claim 16, wherein the dose regimen comprises a first dose of T cells and a second dose of T cells.S281-6010PCT / SCRL544WO18. The method of claim 16, wherein the dose regimen comprises a first dose of T cells, a second dose of T cells, and a third dose of T cells.

19. The method of claim 16, wherein the dose regimen comprises a first dose of T cells, a second dose of T cells, a third dose of T cells, and a fourth dose of T cells.

20. The method of claim 16, wherein the dose regimen has 1-100 doses of T cells.

21. The method of claim 16, wherein the dose regimen has 1-81 doses of T cells.

22. The method of claim 16, wherein the dose regimen has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 doses of T cells.

23. The method of claim 16, wherein the dose regimen has up to 15 doses of T cells.

24. The method of claim 16, wherein the dose regimen has 15 doses of T cells.

25. The method of claim 16, wherein each dose of the plurality of doses of T cells has 0.1x107- 10x107T cells per dose.

26. The method of claim 16, wherein each dose of the plurality of doses of T cells has 50x106T cells per dose.

27. The method of claim 16, wherein the dose regimen comprises an escalating number of T cells per dose.

28. The method of any of claims 17-19, wherein the first dose has 0.1-1x107T cells per dose.

29. The method of any of claims 17-19, wherein the second dose has 0.1x107- 2.5x107T cells per dose.

30. The method of claims 18 or 18, wherein the third dose has 0.1x107- 5x107T cells per dose.

31. The method of claim 19, wherein the fourth dose has 0.1x107- 10x107T cells per dose.

32. The method of claim 16, wherein the dose regimen comprises a first dose having 1x107T cells, a second dose having 1x107T cells, a third dose having 1x107T cells, and a fourth dose having 1x107T cells.

33. The method of claim 16, wherein the dose regimen comprises a first dose having 1x107T cells, a second dose having 1x107T cells, a third dose having 2.5x107T cells, and a fourth dose having 2.5x107T cells.

34. The method of claim 16, wherein the dose regimen comprises a first dose having 1x107T cells, a second dose having 2.5x107T cells, a third dose having 5x107T cells, and a fourth dose having 5x107T cells.

35. The method of claim 16, wherein the dose regimen comprises a first dose having 1x107T cells, a second dose having 2.5x107T cells, a third dose having 5x107T cells, and a fourth dose having 10x107T cells.S281-6010PCT / SCRL544WO36. The method of claim 16, wherein the dose regimen comprises a first dose having 1x107T cells, a second dose having 5x107T cells, a third dose having 10x107T cells, and a fourth dose having 10x107T cells.

37. The method of claim 16, wherein the dose regimen results in delivery of 1x107- 3x1010T cells over its course.

38. The method of claim 16, wherein the dose regimen results in delivery of 1x107- 2.5x109T cells over its course.

39. The method of claim 16, wherein the dose regimen comprises delivery of a dose of T cells at least every 1-28 days for a period of time.

40. The method of claim 16, wherein the dose regimen comprises delivery of a dose of T cells at least every 14 days ±2 days for a period of time.

41. The method of claim 40, wherein the period of time is 30 weeks.

42. The method of claim 16, wherein the dose regimen comprises delivery of a dose of T cells at least every 2 weeks for a period of time, followed by delivery of a dose of T cells at least once a month for a period of time.

43. The method of claim 1 , wherein the T cells comprise CD8+ T cells or CD4+ T cells.

44. The method of claim 1 , wherein the CAR comprises an extracellular component linked to an intracellular component through a transmembrane domain, wherein the extracellular component comprises a B7-H3 binding domain.

45. The method of claim 44, wherein the intracellular component comprises an effector domain comprising 4-1 BB (CD137), CD3y, CD36, CD3c, CD3 , CD27, CD28, DAP10, ICOS, LAG3, NKG2D, NOTCH1, 0X40, ROR2, SLAMF1, TCRa, TCR , TRIM, Wnt, Zap70, or a combination thereof.

46. The method of claim 44, wherein the intracellular component comprises a 4-1 BB signaling domain and a CD3£ signaling domain.

47. The method of claim 44, wherein the transmembrane domain comprises a transmembrane region of: an a, [3 or chain of a T cell receptor; CD28; CD27; CD3; CD45; CD4; CD5; CD8; CD9; CD16; CD22; CD33; CD37; CD64; CD80; CD86; CD134; CD137; CD154; or a combination thereof.

48. The method of claim 1, wherein the T cell is genetically modified to express the CAR by contacting the T cell with an artificial expression construct comprising a sequence encoding the CAR.

49. The method of claim 48, wherein the artificial expression construct comprises a viral vector.S281-6010PCT / SCRL544WO50. The method of claim 49, wherein the viral vector comprises a lentiviral vector.

51. The method of claim 48, wherein the artificial expression construct further encodes a skip sequence.

52. The method of claim 51, wherein the skip sequence comprises a T2A skip sequence, a P2A skip sequence, an E2A skip sequence, or an F2A skip sequence.

53. The method of claim 48, wherein the artificial expression construct further comprises a sequence encoding a transduction marker.

54. The method of claim 53, wherein the transduction marker comprises a truncated EGFR (EGFRt).

55. The method of claim 48, wherein the artificial expression construct further comprises a sequence encoding a selection cassette.

56. The method of claim 55, wherein the selection cassette comprises a dihydrofolate reductase (DHFR)dm.

57. The method of claim 1 , wherein the T cells are autologous to the human patient.

58. The method of claim 1, wherein the human patient is not lymphodepleted at a time of the treating.

59. The method of claim 1 , wherein the patient is lymphodepleted at a time of the treating.

60. The method of claim 1, further comprising screening human patients for eligibility in a clinical trial.

61. The method of claim 60, wherein the screening comprises determining that the human patient matches a set of inclusion criteria for the clinical trial.

62. The method of claim 61, wherein the set of inclusion criteria comprise that the human patient has a Lanksy or Karnofsky performance score greater than 60.

63. The method of claim 61, wherein the set of inclusion criteria comprise that the human patient receives 2.5 mg / m2 / day of dexamethasone or less.

64. The method of claim 61, wherein the set of inclusion criteria comprise that the human patient has an implanted CNS catheter.

65. The method of claim 61, wherein the set of inclusion criteria comprise that the human patient undergoes a washout from previous therapies for the CNS cancer.

66. The method of claim 61, wherein the set of inclusion criteria comprise that the human patient has a life expectancy greater than or equal to 8 weeks.

67. The method of claim 61, wherein the set of inclusion criteria comprise that the human patient has adequate organ function.

68. The method of claim 67, wherein adequate organ function comprises an absoluteS281-6010PCT / SCRL544WOlymphocyte count (ALC) > 100 cells / pL.

69. The method of claim 67, wherein adequate organ function comprises an absolute neutrophil count > 500 cells / pL.

70. The method of claim 67, wherein adequate organ function comprises a hemoglobin count > 9 g / dL.

71. The method of claim 67, wherein adequate organ function comprises a platelet count > 100,000 / pL.

72. The method of claim 67, wherein adequate organ function comprises a creatinine count < upper limit of normal (ULN).

73. The method of claim 67, wherein adequate organ function comprises a total bilirubin < 3x ULN or conjugated bilirubin < 2 mg / dL.

74. The method of claim 67, wherein adequate organ function comprises an oxygen saturation > 90%.

75. The method of claim 61, wherein the set of inclusion criteria comprise that the human patient does not have encephalopathy.

76. The method of claim 61, wherein the set of inclusion criteria comprise that the human patient is not infected with a virus.

77. The method of claim 61, wherein the set of inclusion criteria comprise that the human patient is not infected with human immunodeficiency virus (HIV), hepatitis B, or hepatitis C.

78. A method for administering a dose regimen comprising:administering a first dose of T cells to the central nervous system (CNS) of a human patient having a B7-H3 expressing CNS cancer;administering a second dose of T cells to the CNS of the human patient at least 7 days after administering the first dose; andrepeating the administering of an effective dose of T cells to the CNS of the human patient at least every 7 days.

79. The method of claim 78, wherein the B7-H3 expressing CNS cancer has not progressed at a time of administering the first dose.

80. The method of claim 78, wherein the CNS cancer comprises brain cancer or a spinal cord tumor.

81. The method of claim 80, wherein the brain cancer comprises diffuse intrinsic pontine glioma (DIPG), diffuse midline glioma (DMG), H3K27m-altered DMG, high grade glioma (HGG), glioblastoma (GBM), neuroblastoma, craniopharyngioma, ependymoma, an embryonal tumor, medulloblastoma, pineoblastoma, an atypical teratoid / rhabdoid tumor (ATRT), or an embryonalS281-6010PCT / SCRL544WOnot otherwise specified (NOS) tumor.

82. The method of claim 78, wherein the B7-H3-expressing CNS cancer comprises DIPG.

83. The method of claim 78, wherein the administering comprises intracranial administration.

84. The method of claim 78, wherein the administering comprises intratumoral administration.

85. The method of claim 78, wherein the administering comprises intrathecal administration.

86. The method of claim 83, wherein intracranial administration comprises intracerebroventricular (ICV) injection.

87. The method of claim 83, wherein the dose regimen comprises a first dose of T cells, a second dose of T cells, and a third dose of T cells.

88. The method of claim 83, wherein the dose regimen comprises a first dose of T cells, a second dose of T cells, a third dose of T cells, and a fourth dose of T cells.

89. The method of claim 83, wherein the dose regimen has up to 100 doses of T cells.

90. The method of claim 83, wherein the dose regimen has up to 81 doses of T cells.

91. The method of claim 83, wherein the dose regimen has up to 20 doses of T cells.

92. The method of claim 83, wherein the dose regimen has up to 15 doses of T cells.

93. The method of claim 83, wherein the dose regimen has 15 doses of T cells.

94. The method of claim 83, wherein the dose regimen comprises repeated doses of 50x106 T cells per dose.

95. The method of claim 83, wherein the dose regimen comprises a same or an escalating number of T cells per dose.

96. The method of any of claims 87-89, wherein the first dose has 0.1-1x107T cells per dose.

97. The method of any of claims 87-89, wherein the second dose has 0.1x107- 2.5x107T cells per dose.

98. The method of claim 87-89, wherein the dose regimen comprises a first dose having 1x107T cells, a second dose having 1x107- 2.5x107T cells, a third dose having 1x107- 5x107T cells, and a fourth dose having 1x107- 10x107T cells.

99. The method of claim 83, wherein the dose regimen comprises a first dose having 1x107T cells, a second dose having 1x107T cells, a third dose having 1x107T cells, and a fourth dose having 1x107T cells.

100. The method of claim 83, wherein the dose regimen comprises a first dose having 1x107T cells, a second dose having 1x107T cells, a third dose having 2.5x107T cells, and a fourth dose having 2.5x107T cells.

101. The method of claim 83, wherein the dose regimen comprises a first dose having 1x107T cells, a second dose having 2.5x107T cells, a third dose having 5x107T cells, and a fourth doseS281-6010PCT / SCRL544WOhaving 5x107T cells.

102. The method of claim 83, wherein the dose regimen comprises a first dose having 1x107T cells, a second dose having 2.5x107T cells, a third dose having 5x107T cells, and a fourth dose having 10x107T cells.

103. The method of claim 83, wherein the dose regimen comprises a first dose having 1x107T cells, a second dose having 5x107T cells, a third dose having 10x107T cells, and a fourth dose having 10x107T cells.

104. The method of claim 83, wherein the dose regimen results in intracranially administering 1x107- 3x1010T cells over its course.

105. The method of claim 83, wherein the dose regimen results in intracranially administering 1x107- 2.5x109T cells over its course.

106. The method of claim 78, wherein the dose regimen comprises delivery of a dose of T cells at least every 1-28 days for a period of time.

107. The method of claim 78, wherein the dose regimen comprises delivery of a dose of T cells at least every 14 days ±2 days for a period of time.

108. The method of claim 78, wherein the dose regimen comprises delivery of a dose of T cells at least every 2 weeks for a period of time, followed by delivery of a dose of T cells at least every month for a period of time.

109. The method of claim 78, wherein the T cells are genetically modified to express a chimeric antigen receptor (CAR) having an extracellular component comprising a B7-H3 binding domain.

110. The method of claim 78, wherein the T cells comprise CD8+ T cells or CD4+ T cells.

111. The method of claim 109, wherein the extracellular component is linked to an intracellular component of the CAR through a transmembrane domain.

112. The method of claim 111, wherein the intracellular component comprises an effector domain comprising 4-1 BB (CD137), CD3y, CD36, CD3E, CD3 , CD27, CD28, DAP10, ICOS, LAG3, NKG2D, NOTCH1, 0X40, ROR2, SLAMF1, TCRa, TCR , TRIM, Wnt, Zap70, or a combination thereof.

113. The method of claim 111, wherein the intracellular component comprises a 4-1 BB signaling domain and a CD3^ signaling domain.

114. The method of claim 111, wherein the transmembrane domain comprises a transmembrane region of: an a, p or chain of a T cell receptor; CD28; CD27; CD3; CD45; CD4; CD5; CD8; CD9; CD16; CD22; CD33; CD37; CD64; CD80; CD86; CD134; CD137; CD154; or a combination thereof.

115. The method of claim 78, wherein the T cell is genetically modified to express a CAR byS281-6010PCT / SCRL544WOcontacting the T cell with an artificial expression construct comprising a sequence encoding the CAR.

116. The method of claim 115, wherein the artificial expression construct comprises a viral vector.

117. The method of claim 116, wherein the viral vector comprises a lentiviral vector.

118. The method of claim 115, wherein the artificial expression construct further encodes a skip sequence.

119. The method of claim 118, wherein the skip sequence comprises a T2A skip sequence, a P2A skip sequence, an E2A skip sequence, or an F2A skip sequence.

120. The method of claim 115, wherein the artificial expression construct further comprises a sequence encoding a transduction marker.

121. The method of claim 120, wherein the transduction marker comprises a truncated EGFR (EGFRt).

122. The method of claim 115, wherein the artificial expression construct further comprises a sequence encoding a selection cassette.

123. The method of claim 122, wherein the selection cassette comprises a dihydrofolate reductase (DHFR)dm.

124. The method of claim 78, wherein the T cells are autologous to the human patient.

125. The method of claim 78, wherein the method does not include lymphodepletion.

126. The method of claim 78, wherein the method includes lymphodepletion.

Images