CXCR3 isoforms to improve recombinant receptor trafficking

Abstract

Artificial expression constructs encoding a CXCR3 isoform and a recombinant receptor are described. The artificial expression constructs include the CXCR3 isoform: CXCR3-A, CXCR3-B, or CXCR3-alt, and are used to enhance the tumor trafficking of cells engineered to express the artificial expression construct. The artificial expression constructs and engineered cells disclosed herein can be used in the treatment of B7-H3-expressing cancers, such as diffuse intrinsic pontine glioma.

Description

CXCR3 ISOFORMS TO IMPROVE RECOMBINANT RECEPTOR TRAFFICKINGCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 569,626 filed March 25, 2024 and U.S. Provisional Patent Application No. 63 / 653,023 filed May 29, 2024, both of which are incorporated herein by reference in its entirety as if fully set forth herein.REFERENCE TO SEQUENCE LISTING

[0002] The Sequence Listing associated with this application is provided in XML format in lieu of a paper copy and is hereby incorporated by reference into the specification. The name of the file containing the Sequence Listing is 3EG8067.XML. The file is 145,496 bytes, was created on March 25, 2025 and is being submitted electronically via Patent Center.FIELD OF THE DISCLOSURE

[0003] The current disclosure provides artificial expression constructs encoding a CXCR3-A isoform and a recombinant receptor. Engineered cells expressing the artificial expression constructs disclosed herein, target and kill cancer antigen-expressing cells, and upon target binding, further promote engineered cell migration to a tumor site. The artificial expression constructs and engineered cells disclosed herein can be used in the treatment of B7-H3- expressing cancers, such as diffuse intrinsic pontine glioma.BACKGROUND OF THE DISCLOSURE

[0004] Diffuse intrinsic pontine glioma (DI PG) is an aggressive type of childhood cancer that forms in the brainstem. It is universally fatal with a median survival of less than 1 year from diagnosis and it affects 400 children per year in the United States.

[0005] 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.

[0006] 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 thatallow 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.

[0007] Although CAR T cells have had substantial success in treating some cancers, challenges in the treatment of solid tumors remain. One major challenge is in trafficking CAR-T cells to solid tumors.SUMMARY OF THE DISCLOSURE

[0008] The shortcomings of chimeric antigen receptor (CAR) T cell therapy in solid tumors necessitates the need to better traffic engineered cells to antigen-expressing cells or tumor sites. The current disclosure provides artificial expression constructs encoding a CXCR3-A isoform and a recombinant receptor. Engineered cells expressing the artificial expression constructs disclosed herein target and kill cancer antigen-expressing cells and, upon target binding, further promote engineered cell migration to the tumor site. In particular embodiments, recombinant receptors, expressed by engineered cells, bind and kill cancer antigen-expressing cells and upon binding, release a chemokine. Furthermore, in particular embodiments, cancer antigen-expressing cells release the chemokine in response to cytokines released by the stimulated recombinant receptor. In particular embodiments, the chemokine receptor, CXCR3-A, expressed by the engineered cell, detects and promotes cell migration toward the increasing concentration gradient of the chemokine, thereby trafficking the engineered cells to the cancer antigen-expressing cells. In particular embodiments, the chemokine includes CXCL9, CXCL10, or CXCL11 .

[0009] In particular embodiments, an artificial expression construct includes (i) a sequence encoding CXCR3-A, and (ii) a sequence encoding a recombinant receptor including a cancer antigen binding domain. In particular embodiments, the recombinant receptor includes a CAR. In particular embodiments, the cancer antigen binding domain is a B7-H3 binding domain. In particular embodiments, the B7-H3 binding domain includes an scFv including the sequence of SEQ ID NO: 33 or SEQ ID NO: 35. In particular embodiments, the recombinant receptor includes an extracellular component including the B7-H3 binding domain, an intracellular component, and a transmembrane domain linking the extracellular component to the intracellular component. Inparticular embodiments, the intracellular component includes a CD3z activation domain and a 4- 1 BB costimulatory domain. In particular embodiments, the extracellular component includes a spacer. In particular embodiments, the transmembrane domain includes a CD28 transmembrane domain. In particular embodiments, the artificial expression construct further includes a sequence encoding a self-cleaving polypeptide. In particular embodiments, the self-cleaving polypeptide includes a 2A peptide from Thosea asigna virus (T2A).

[0010] In particular embodiments, an artificial expression construct encoding CXCR3-A and an artificial expression construct encoding a cancer antigen binding recombinant receptor are in the same vector. In particular embodiments, an artificial expression construct encoding CXCR3-A and an artificial expression construct encoding a cancer antigen binding recombinant receptor are in different vectors.

[0011] The artificial expression constructs and engineered cells disclosed herein can be used in the treatment of, for example, B7-H3-expressing cancers, such as diffuse intrinsic pontine glioma, diffuse midline glioma, glioblastoma, prostate cancer, renal cell carcinoma, urothelial cell carcinoma, ovarian cancer, osteosarcoma, neuroblastoma, mesothelioma, colorectal cancer, gastric cancer, breast cancer, small cell lung cancer, non-small-cell lung cancer (NSCLC), and pancreatic cancer.BRIEF DESCRIPTION OF THE FIGURES

[0012] 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.

[0013] FIG. 1. Schematic of recombinant receptor interactions with a target antigen-expressing cancer cell.

[0014] FIG. 2. CXCL10 upregulation has been detected in patients’ CSF post-CAR T cell injections. CXCR3 is the chemokine receptor for CXCL10, CXCL9, and CXCL11. A chemoattractant gradient can be used to traffic cells to a tumor site using the chemokine receptor CXCR3 to drive migration toward increasing chemoattractant concentration. A list of example chemokine receptors and their respective chemokines are shown.

[0015] FIGs. 3A, 3B. (3A) CXCR3 has three isoforms due to alternative splicing. (3B) Interactions among CXCR3 isoforms and the ligands CXCL9, CXCL10, and CXCL11.

[0016] FIGs. 4A-4H. B7-H3-targeting chimeric antigen receptor (CAR) T cells can kill patient- derived diffuse intrinsic pontine glioma (DIPG) cells and produce CXCL10, which can induce chemotactic migration of CAR T cells that heterogeneously upregulate CXCR3. (4A) B7-H3expression on primary DI PG cells by flow cytometry with isotype controls. (4B) Relative cell viability of PBT-22FH or PBT-29FH cells after co-cultured with non-transduced (NTD) or CAR T cells at the indicated effectontarget (E:T) ratios for 24 hours. (4C) Concentrations of CXCL9, CXCL10, and CXCL11 by ELISA in co-cultures of NTD T or CAR T cells with PBT-22FH or PBT- 29FH target cells at E:T of 1 :1 for 24 hours. (4D) CXCR3 expression on quiescent T cells, anti- CD3 / CD28 beads-stimulated T cells, and generated CAR T cells by flow cytometry. (4E) Percentage of CAR T cells that migrated from insert wells through 5-pm pores to bottom wells with CXCL10 at the indicated time points. (4F) Relative cell viability of PBT-29FH cells after cocultured with CXCLIO-induced migrated CAR T cells for 24 hours. CAR T cells were seeded on insert wells to migrate through 5-pm pores to bottom wells with CXCL10 for 2 hours before removal of the insert wells. (4G) Relative cell viability of PBT-29FH cells after cultured with CXCL10 for 24 hours. (4H) Relative cell viability of PBT-29FH cells after directly co-cultured with CAR T cells at E:T of 1 :1 in the presence of CXCL10 for 24 hours. Cell viability was normalized to the vehicle control without T cells in (4B, 4F, 4G and 4H). p values were determined by two- way ANOVA with Sidak’s multiple comparisons test (4B) or one-way ANOVA with Dunnett’s multiple comparisons test (4C, 4E, 4F, 4G and 4H) using data from three or more independent experiments. Significance was compared with NTD T cells at each E:T ratio in (4B). Results are presented as means ± standard deviation (SD) in (4B, 4C, 4E, 4F, 4G and 4H). ns, not significant; *p < 0.05; **p < 0.01 ; ***p < 0.001 ; ****p < 0.0001.

[0017] FIGs. 5A-5F. CXCR3-A-modified CAR T cells show enhanced migration toward CXCL10. (5A) Illustration of constructs to generate B7-H3-targeting CAR T cells that overexpress various isoforms of CXCR3. (5B and 5C) CAR, EGFRt transduction marker, and CXCR3 expression on unmodified and CXCR3-modifed CAR T cells by flow cytometry. gMFI, geometric mean fluorescent intensity. (5D) Relative expression of CXCR3 isoforms in unmodified or CXCR3- modifed CAR T cells by RT-qPCR. The data were normalized to [3-actin control then nominally multiplied by 108for data display. (5E and 5F) Percentage of CAR T cells that migrated from insert wells to bottom wells with CXCL10 or CXCL11 after 2 hours, p values were determined by two- way ANOVA with Dunnett’s multiple comparisons test (5E and 5F) using data from three or more independent experiments. Results are presented as means ± SD in (5E and 5F). ns, not significant; *p < 0.05; **p < 0.01 ; ***p < 0.001.

[0018] FIGs. 6A-6F. CXCR3-A-modified CAR T cells show similar phenotypes as unmodified CAR T cells across three T cell donors. (6A) Fold expansion by CAR T cells during production. (6B) Percentage of CAR expression on unmodified and CXCR3-A-modifed CAR T cells by flow cytometry. (6C) Geometric mean fluorescent intensity of (gMFI) of CXCR3 on unmodified andCXCR3-A-modifed CAR T cells by flow cytometry. (6D) Percentage of CD4 and CD8 T cell subset of generated CAR T cells. (6E) Percentage of naTve-like (CD62L+ / CD45RA+), central memory (CD62L+ / CD45RA-), effector memory (CD62L- / CD45RA-), and effector (CD62L- / CD45RA+) subset of generated CAR T cells. (6F) Percentage of the exhaustion markers PD1 , TIM3, and LAG3 expression on generated CAR T cells, p values were determined by randomized block (matching the data for each donor) one-way ANOVA with Tukey’s multiple comparisons test (6A, 6D, 6E, and 6F) or paired t test (6B and 6C) using data from three different T cell donors. Results are presented as means ± SD. ns, not significant, *p < 0.05.

[0019] FIGs. 7A-7G. CXCR3-A-modified CAR T cells show enhanced migration and efficacy against DIPG across multiple T cell donors in response to CXCR3 ligands (7A) Relative cell viability of PBT-22FH or PBT-29FH cells after co-cultured with CAR T cells at the indicated E:T ratios for 24 hours. (7B) Concentrations of IL-2, IFN-y, and TNF by ELISA in co-cultures of CAR T cells with PBT-22FH or PBT-29FH target cells at E:T of 10:1 for 24 hours. (7C) Percentage of CAR T cells that migrated from insert wells to bottom wells with vehicle, 300 ng / mL CXCL9, 100 ng / mL CXCL10, or 300 ng / mL CXCL11 after 2 hours (7D) Relative cell viability of PBT-29FH cells after co-cultured with migrated CAR T cells for 24 hours. CAR T cells were seeded on insert wells to migrate to bottom wells with vehicle, 300 ng / mL CXCL9, 100 ng / mL CXCL10, or 300 ng / mL CXCL11 for 2 hours before removal of the insert wells. (7E) Concentrations of IL-2, IFN-y, and TNF by ELISA in cultures from (7D) after 24 hours. (7F) Relative cell viability of PBT-29FH cells after directly co-cultured with CAR T cells at E:T of 1 :1 in the presence of vehicle, 300 ng / mL CXCL9, 100 ng / mL CXCL10, or 300 ng / mL CXCL11 for 24 hours. (7G) Concentrations of IL-2, IFN-y, and TNF by ELISA in cultures from (7F) after 24 hours. Cell viability was normalized to the vehicle control without T cells in (7A, 7D, and 7F). p values were determined by randomized block (matching the data for each donor) two-way ANOVA with Tukey’s multiple comparisons test (7A), randomized block one-way ANOVA with Dunnett’s multiple comparisons test (7B), or randomized block two-way ANOVA with Sidak’s multiple comparisons test (7C, 7D, 7E, 7F, and 7G) using data from three different T cell donors. Results are presented as means ± SD. ns, not significant; *p < 0.05; **p < 0.01 ; ***p < 0.001 ; ****p < 0.0001.

[0020] FIGs. 8A-8C. Paracrine chemokines can lead to enhanced efficacy by CXCR3-A-modified CAR T cells against DIPG. (8A) Relative cell viability of PBT-29FH cells after co-cultured with migrated CAR T cells for 24 hours. CAR T cells were seeded on insert wells to migrate for 2 hours to bottom wells with conditioned medium (CM) from antigen-stimulated CAR T cell culture, before removal of the insert wells. (8B) Relative cell viability of PBT-29FH cells after co-cultured with continuously migrating CAR T cells for 24 hours. CAR T cells were seeded on insert wells tomigrate for 24 hours to bottom wells without chemoattractant. (8C) Concentrations of CXCL10 by ELISA at the indicated time points in cultures of DI PG cells with added IFN-y at the indicated doses, p values were determined by randomized block (matching the data for each donor) oneway ANOVA with Dunnett’s multiple comparisons test (8A) or paired t test (8B) using data from three different T cell donors. Results are presented as means ± SD in (8A and 8B). *p < 0.05.

[0021] FIG. 9. Paracrine chemokines can lead to enhanced efficacy by CXCR3-A-modified CAR T cells against DIPG. Relative cell viability of PBT-29FH cells after co-cultured with continuously migrating CAR T cells for 24 hours. CAR T cells were seeded on insert wells to migrate for 24 hours to bottom wells without chemoattractant, p values were determined by unpaired t test using data from three independent experiments with CAR T cells of the same donor. Results are presented as means ± SD. ***p < 0.001.

[0022] FIGs. 10A, 10B. CXCR3-A-modified CAR T cells show enhanced trafficking to the tumor site in vivo against an orthotopic DI PG model via intracerebroventricular injection. (10A) Illustration of the experimental plan. (10B) Representative images of H&E staining for the tumors and immunohistochemistry (IHC) staining for human CD3 around the corresponding tumor areas from the mouse brains collected on day 1 and day 2 (additional images shown in FIG. 11).

[0023] FIG. 11. CXCR3-A-modified CAR T cells show enhanced trafficking to the tumor site post intracerebroventricular delivery in an orthotopic DIPG (SU-DIPG-13) model. Representative images of Hematoxylin-Eosin (H&E) staining for the tumors and IHC staining for human CD3, CD4, and CD8 around the corresponding tumor areas from the mouse brains collected on day 1 and day 2, as illustrated in FIG. 10.

[0024] FIG. 12. CAR T cells show lack of trafficking to the mouse brain post intravenous delivery in an orthotopic DIPG (SU-DIPG-13) model. Representative images of H&E staining for the tumors and IHC staining for human CD3, CD4, and CD8 around the corresponding tumor areas from the mouse brains collected on day 1 and day 2, as illustrated in FIG. 10.

[0025] FIG. 13. IHC staining for human Ki67 and B7-H3 confirms the tumor areas. Representative images of IHC staining for human Ki67 and B7-H3 around the focused areas in FIGs. 11 and 12.

[0026] FIGs. 14A-14F. CXCR3-A-modified CAR T cells show enhanced therapeutic efficacy when intracerebroventricularly delivered in an orthotopic DIPG (SU-DIPG-13) model. (14A) Bioluminescence imaging (BLI) images of mice at the indicated imaging time points. (14B and 14C) Log of bioluminescent total flux from SU-DIPG-13 tumor cells in each mouse on day 7. (14D) Bioluminescent total flux from SU-DIPG-13 tumor cells at the indicated time points. (14E) Kaplan-Meier plot for mouse survival. (14F) Mouse weight at the indicated time points. Results are presented as means ± SD in (14B and 14C). p values were determined by one-way ANOVAwith Tukey’s multiple comparisons test (14B and 14C) or log-rank (Mantel-Cox) test (14E). ns, not significant; *p < 0.05; **p < 0.01 ; ***p < 0.001 ; ****p < 0.0001.

[0027] FIG. 15. H&E and IHC staining for human Ki67 and B7-H3 suggests tumor antigen escape. Representative images of H&E and IHC staining for human Ki67 and B7-H3 from the mouse brain after relapse post intracerebroventricular CAR-3A T cell treatment in FIG. 14.

[0028] FIGs. 16A-16E. CXCR3-A-modified CAR T cells of another T cell donor show enhanced therapeutic efficacy when intracerebroventricularly delivered in a second orthotopic DIPG model. (16A) Illustration of the experimental plan. (16B) Log of bioluminescent total flux from PBT-27FH tumor cells in each mouse on day 7. (16C) Bioluminescent total flux from PBT-27FH tumor cells at the indicated time points. (16D) Kaplan-Meier plot for mouse overall survival that includes nontumor related causes of death. (16E) Mouse weight at the indicated time points. Results are presented as means ± SD in (16B). p values were determined by one-way ANOVA with Tukey’s multiple comparisons test (16B) or log-rank (Mantel-Cox) test (16D). ns, not significant; **p < 0.01.

[0029] FIG. 17. CXCR3-A-modified CAR T cells of another T cell donor show enhanced therapeutic efficacy when intracerebroventricularly delivered in the second orthotopic DI PG (PBT- 27FH) model. BLI images of mice at the indicated imaging time points from the study shown in FIGs. 16A-16E. tNon-tumor related causes of death and dates. Particularly, causes of death include vaginal prolapse at day 182 (left) and bacterial infection at day 193 (right). The scale bar indicates Luminescence (x107).

[0030] FIG. 18. H&E and IHC staining for human Ki67 and B7-H3 confirms absence of tumor. Representative images of H&E and IHC staining for human Ki67 and B7-H3 from the brains of the two mice in the CAR-3A T cell group of FIG. 16A-16E, who were euthanized due to non-tumor related causes in the absence of tumor BLI signal.

[0031] FIGs. 19A-19F. Schematic of the proposed working model. (19A and 19B) Some of the intracerebroventricularly delivered CAR T cells encounter tumor and get stimulated by the tumor antigen, leading to some tumor cell death. (19C and 19D) The stimulated CAR T cells produce chemokines such as CXCL10, which can recruit additional CAR T cells that endogenously express CXCR3. DI PG tumor cells also express CXCL10 in response to IFN-y released by stimulated CAR T cells. (19E) By engineering the CAR T cells to overexpress CXCR3-A, more CAR T cells respond and migrate to the tumor site. (19F) This effect in turn results in more CXCL10 production, followed by more CAR T cell recruitment through positive feedback, which lead to more tumor cell death, thus improving the trafficking and efficacy of CAR T cell therapies for DI PG.

[0032] FIG. 20. Sequences supporting the disclosure including CXCR3-A coding sequence (SEQ ID NO: 3); CXCR3-B coding sequence (SEQ ID NO: 5); CXCR3alt coding sequence (SEQ ID NO: 7); Human B7H3-specific scFv (SEQ ID NO: 33); Human B7H3-specific scFv coding sequence (SEQ ID NO: 34); Human B7H3-specific scFv (SEQ ID NO: 35) ; Human B7H3-specific scFv coding sequence (SEQ ID NO: 36); EF1 promoter (SEQ ID NO: 37); lgG4 hinge (SEQ ID NO: 38); lgG4 hinge coding sequence (SEQ ID NOS: 39-42); lgG4-CH2 domain (SEQ ID NO: 43); lgG4-CH2 domain coding sequence (SEQ ID NOs: 44 and 45); lgG4 CH3 domain (SEQ ID NO: 46); lgG4 CH3 domain coding sequence (SEQ ID NOS: 47-49); CD28 Transmembrane Domain (SEQ ID NOs: 50-52); CD28TM coding sequence (SEQ ID NO: 53-56); 4-1 BB signaling domain (SEQ ID NOs: 57-59); 4-1 BB signaling domain coding sequence (SEQ ID NOs: 60-62); CD3 signaling domain (SEQ ID NOs: 63-65); CD3 signaling domain coding sequence (SEQ ID NOs: 66 and 67); Thoseaasigna Virus 2A (T2A) Peptide (SEQ ID NOs: 68 and 69); T2A coding sequence (SEQ ID NOs: 70 and 71); Porcine Teschovirus-1 2a (P2A) Peptide (SEQ ID NO: 72); Equine Rhinitis A Virus (ERAV) 2A (E2A) Peptide (SEQ ID NO: 73); Foot-And-Mouth Disease Virus 2A (F2A) Peptide (SEQ ID NO: 74); EGFRt transduction marker (SEQ ID NO: 75); EGFRt transduction marker coding sequence (SEQ ID NO: 76); WPRE (SEQ ID NO: 77); WPRE coding sequence (SEQ ID NO: 78); CAR amino acid sequence (SEQ ID NO: 79) ; CAR coding sequence (SEQ ID NO: 80); CAR -T2A-EGFRt-T2A-CXCR3A coding sequence (SEQ ID NO: 81); CAR - T2A-EGFRt-T2A-CXCR3B coding sequence (SEQ ID NO: 82); CAR -T2A-EGFRt-T2A-CXCR3- alt coding sequence (SEQ ID NO: 83); CAR with CXCR3A and Regulatory Elements coding sequence (SEQ ID NO: 84); CAR with CXCR3B and Regulatory Elements coding sequence (SEQ ID NO: 85); and CAR with CXCR3alt and Regulatory Elements coding sequence (SEQ ID NO: 86).DETAILED DESCRIPTION

[0033] For many years, the chosen treatments for cancer were surgery, chemotherapy, and / or radiation therapy. In recent years, more targeted therapies have emerged to 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 particular antigens on their cellular surfaces and these antigens have provided targets for successful therapeutics.

[0034] An example of these targeted therapies include recombinant receptor therapy, such as chimeric antigen receptor (CAR) therapy, engineered T cell receptor (eTCR) therapy, and hybrids thereof. Recombinant receptor therapy involves engineering immune cells to express syntheticreceptors designed to target cells, such as cancer cells, for destruction. Although recombinant receptor therapies have had substantial success in treating various hematological malignancies, challenges remain, for example, in the treatment of solid tumors. One reason that recombinant receptor therapies may be less successful in the treatment of solid tumors is that they may not sufficiently traffic to the tumor site.

[0035] A key to successful targeted recombinant receptor therapy is in the choice of the target antigen. An ideal target antigen can be immunogenic, play a critical role in proliferation and / or differentiation of immune cells, is expressed only on the surface of all malignant cells and malignant stem cells, and / or at least a large portion of patients should test positive for the target antigen (Cheever, et al., 2009. Clin. Cancer Res. 15(17): 5323-8337).

[0036] B7-H3, also known as CD276, is a member of the B7 family of immune cell modulating molecules. Its expression is inhibited in normal cells by the microRNA, miR-29 while it is highly expressed by a wide variety of cancer cells, such as diffuse intrinsic pontine glioma (DIPG) cells, diffuse midline glioma (DMG) cells, glioblastoma cells, neuroblastoma cells, melanoma cells, renal cell carcinoma cells, prostate cancer cells, colorectal cancer cells, pancreatic cancer cells, osteosarcoma cells, urothelial cell carcinoma cells, mesothelioma cells, breast cancer cells, ovarian cancer cells, small cell lung cancer cells and non-small-cell lung cancer (NSCLC) cells. B7-H3 is involved in helping cancer cells evade the surveillance of cytotoxic T-cells and natural killer cells.

[0037] In particular embodiments, B7-H3 [Homo sapiens] includes the sequence:MLRRRGSPGMGVHVGAALGALWFCLTGALEVQVPEDPWALVGTDATLCCSFSPEPGFSLAQ LNLIWQLTDTKQLVHSFAEGQDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSFTCFVS IRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYQGYPEAEVFWQDGQGVPLTG NVTTSQMANEQGLFDVHSILRVVLGANGTYSCLVRNPVLQQDAHSSVTITPQRSPTGAVEVQV PEDPWALVGTDATLRCSFSPEPGFSLAQLNLIWQLTDTKQLVHSFTEGRDQGSAYANRTALF PDLLAQGNASLRLQRVRVADEGSFTCFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGD TVTITCSSYRGYPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHSVLRVVLGANGTYSCL VRNPVLQQDAHGSVTITGQPMTFPPEALWVTVGLSVCLIALLVALAFVCWRKIKQSCEEENAGA EDQDGEGEGSKTALQPLKHSDSKEDDGQEIA (SEQ ID NO: 1).

[0038] In particular embodiments, engineered cells include a recombinant receptor with a B7-H3 binding domain.

[0039] The effects of an immune cell therapy, such as CAR T cell therapy, can be augmented by further engineering the immune cell’s ability to traffic or migrate to the tumor site of interest. In particular embodiments, artificial expression constructs include a chemokine receptor to augmentthe engineered immune cell’s trafficking to the tumor site. In particular embodiments, the chemokine receptor is a CXCR3-A isoform. CXCR3, also referred to as G protein-coupled receptor 9 (GPR9) and CD183, is a CXC chemokine receptor largely expressed on activated T lymphocytes, NK cells, and some epithelial cells. CXCR3 includes three isoforms in humans: CXCR3-A, CXCR3-B, and CXCR3-alternative (CXCR3-alt). CXCR3-A binds CXCL9, CXCL10, and CXCL11; CXCR-B binds to CXCL4, CXCL9, CXCL10, and CXCL11 ; and CXCR3-alt binds to CXCL9 and CXCL11 .

[0040] In particular embodiments, CXCR3-A includes the sequence:MVLEVSDHQVLNDAEVAALLENFSSSYDYGENESDSCCTSPPCPQDFSLNFDRAFLPALYSLL FLLGLLGNGAVAAVLLSRRTALSSTDTFLLHLAVADTLLVLTLPLWAVDAAVQWVFGSGLCKVA GALFNINFYAGALLLACISFDRYLNIVHATQLYRRGPPARVTLTCLAVWGLCLLFALPDFIFLSAH HDERLNATHCQYNFPQVGRTALRVLQLVAGFLLPLLVMAYCYAHILAVLLVSRGQRRLRAMRL VVVVVVAFALCWTPYHLVVLVDILMDLGALARNCGRESRVDVAKSVTSGLGYMHCCLNPLLYA FVGVKFRERMWMLLLRLGCPNQRGLQRQPSSSRRDSSWSETSEASYSGL (SEQ ID NO: 2). In particular embodiments, CXCR3-A is encoded by the sequence of SEQ ID NO: 3.

[0041] In particular embodiments, CXCR3-B includes the sequence:MELRKYGPGRLAGTVIGGAAQSKSQTKSDSITKEFLPGLYTAPSSPFPPSQVSDHQVLNDAEV AALLENFSSSYDYGENESDSCCTSPPCPQDFSLNFDRAFLPALYSLLFLLGLLGNGAVAAVLLS RRTALSSTDTFLLHLAVADTLLVLTLPLWAVDAAVQWVFGSGLCKVAGALFNINFYAGALLLACI SFDRYLNIVHATQLYRRGPPARVTLTCLAVWGLCLLFALPDFIFLSAHHDERLNATHCQYNFPQ VGRTALRVLQLVAGFLLPLLVMAYCYAHILAVLLVSRGQRRLRAMRLVVWVVAFALCWTPYHL VVLVDILMDLGALARNCGRESRVDVAKSVTSGLGYMHCCLNPLLYAFVGVKFRERMWMLLLRL GCPNQRGLQRQPSSSRRDSSWSETSEASYSGL (SEQ ID NO: 4). In particular embodiments, CXCR3-B is encoded by the sequence of SEQ ID NO: 5.

[0042] In particular embodiments, CXCR3-alt includes the sequence:MVLEVSDHQVLNDAEVAALLENFSSSYDYGENESDSCCTSPPCPQDFSLNFDRAFLPALYSLL FLLGLLGNGAVAAVLLSRRTALSSTDTFLLHLAVADTLLVLTLPLWAVDAAVQWVFGSGLCKVA GALFNINFYAGALLLACISFDRYLNIVHATQLYRRGPPARVTLTCLAVWGLCLLFALPDFIFLSAH HDERLNATHCQYNFPQGSSSGSGCGCCSCAWAAPTREGSRGSHRLPAGIHPGLRPQRPPTR ACEAGIRAPLSPTV (SEQ ID NO: 6). In particular embodiments, CXCR3-alt is encoded by the sequence of SEQ ID NO: 7.

[0043] The current disclosure provides artificial expression constructs encoding a CXCR3-A isoform and a recombinant receptor for the treatment of cancer. Engineered cells expressing the artificial expression constructs disclosed herein target and kill cancer antigen-expressing cellsand, upon target binding, further promote engineered cell migration to a tumor site. In particular embodiments, recombinant receptors, expressed by engineered cells, bind and kill B7-H3- expressing cells and upon binding, release a chemokine. Furthermore, in particular embodiments, B7-H3-expressing cells release the chemokine in response to cytokines released by the stimulated recombinant receptor. In particular embodiments, the chemokine receptor, CXCR3-A, expressed by the engineered cell, detects and promotes migration toward an increasing concentration gradient of the chemokine, thereby trafficking other engineered cells to the site of antigen-stimulated engineered immune cell action. In particular embodiments, the chemokine includes CXCL9, CXCL10, or CXCL11. The artificial expression constructs and engineered cells disclosed herein can be used in the treatment of antigen-expressing cancers, such as B7-H3- expressing cancers.

[0044] In particular embodiments, CAR, expressed by engineered cells, bind and kill B7-H3- expressing cells and upon binding, release CXCL10. Furthermore, in particular embodiments, B7- H3-expressing cells release the CXCL10 in response to IFN-y released by the stimulated CAR- expressing cells. In particular embodiments, the chemokine receptor, CXCR3-A, expressed by CAR-expressing cell, detects and promotes cell migration toward the increasing concentration gradient of CXCL10, thereby trafficking the engineered cells to the B7-H3-expressing cells.

[0045] A “B7-H 3- related disorder” is one where diseased or infected cells within a subject express B7-H3, such that B7-H3 provides an antigen for the targeted delivery of therapeutic treatments. In these disorders, B7-H3 should be preferentially-expressed by the diseased or infected cells such that on-target / off-site side effects are minimized or eliminated.

[0046] Diseased cells expressing B7-H3 are cells targeted for destruction by a treatment described herein. Diseased cells expressing B7-H3 include, for example, diffuse intrinsic pontine glioma (DI PG) cells, diffuse midline glioma (DMG) cells, glioblastoma cells, prostate cancer cells, renal cell carcinoma cells, urothelial cell carcinoma cells, ovarian cancer cells, osteosarcoma cells, neuroblastoma cells, mesothelioma cells, colorectal cancer cells, gastric cancer cells, breast cancer cells, small cell lung cancer cells, non-small-cell lung cancer (NSCLC) cells, and pancreatic cancer cells.

[0047] In particular embodiments, an artificial expression construct includes a sequence encoding a chemokine receptor. In particular embodiments, an artificial expression construct includes a sequence encoding a recombinant receptor including a B7-H3 binding domain. In particular embodiments, an artificial expression construct includes (i) a sequence encoding a chemokine receptor, and (ii) a sequence encoding a recombinant receptor including a B7-H3 binding domain. In particular embodiments, the chemokine receptor includes a CXCR3 isoform selected fromCXCR3-A, CXCR3-B, and CXCR3-alt. In particular embodiments, the chemokine receptor isoform includes CXCR3-A. In particular embodiments, the B7-H3 binding domain includes an scFv including the sequence of SEQ ID NO: 33 or SEQ ID NO: 35. In particular embodiments, the recombinant receptor includes an extracellular component including the B7-H3 binding domain, an intracellular component, and a transmembrane domain linking the extracellular component to the intracellular component. In particular embodiments, the recombinant receptor is a CAR. In particular embodiments, the intracellular component includes a CD3z activation domain and a 4- 1 BB costimulatory domain. In particular embodiments, the extracellular domain includes a spacer. In particular embodiments, the transmembrane domain includes a CD28 transmembrane domain. In particular embodiments, the artificial expression construct further includes a sequence encoding a self-cleaving polypeptide. In particular embodiments, the self-cleaving polypeptide includes a 2A peptide from Thosea asigna virus (T2A).

[0048] In particular embodiments, a genetically modified cell expresses a recombinant receptor and a chemokine receptor, wherein the genetically modified cell expresses the chemokine receptor at an elevated level compared to endogenous chemokine receptor expression of a wild type cell.

[0049] Aspects of the current disclosure are now described in more supporting detail as follows: (I) Immune Cells; (II) Cell Sample Collection and Cell Enrichment; (III) Genetic Engineering Techniques; (IV) Recombinant Receptors; (I -A) Binding Domains; (I -B) Spacers; (IV-C) Transmembrane Domains; (IV-D) Intracellular Effector Domains; (IV-E) Linkers; (V) Control Features Including Tag Cassettes, Transduction Markers, Selection Cassettes, and / or Suicide Switches; (VI) Characterization of Genetically Engineered Cells; (VII) Cell Activating Culture Conditions; (VIII) Ex Vivo Manufactured Cell Formulations; (IX) Targeted Viral Vectors & Nanoparticles for In Vivo Cell Modification; (X) Methods of Use; (XI) Kits; (XII) Exemplary Embodiments; (XIII) Experimental Example; and (XIV) Closing Paragraphs. These headings are provided for organizational purposes only and do not limit the scope or interpretation of the disclosure.

[0050] (I) Immune Cells. The present disclosure describes cells genetically modified to express a recombinant receptor (e.g., CAR) and CXCR3. Genetically modified cells can include T-cells, B cells, natural killer (NK) cells, NK-T cells, monocytes / macrophages, lymphocytes, hematopoietic stem cells (HSCs), hematopoietic progenitor cells (HPC), and / or a mixture of HSC and HPC (i.e., HSPC). In particular embodiments, genetically modified cells include T-cells.

[0051] Several different subsets of T-cells have been discovered, each with a distinct function. For example, a majority of T-cells have a T-cell receptor (TCR) existing as a complex of severalproteins. The actual T-cell receptor is composed of two separate peptide chains, which are produced from the independent T-cell receptor alpha and beta (TCRa and TCRP) genes and are called a- and [3-TCR chains.

[0052] y8 T-cells represent a small subset of T-cells that possess a distinct T-cell receptor (TCR) on their surface. In y5 T-cells, the TCR is made up of one y-chain and one 5-chain. This group of T-cells is much less common (2% of total T-cells) than the a[3 T-cells.

[0053] CD3 is expressed on all mature T cells. Activated T-cells express 4-1 BB (CD137), CD69, and CD25. CD5 and transferrin receptor are also expressed on T-cells.

[0054] T-cells can further be classified into helper cells (CD4+ T-cells) and cytotoxic T-cells (CTLs, CD8+ T-cells), which include cytolytic T-cells. T helper cells assist other white blood cells in immunologic processes, including maturation of B cells into plasma cells and activation of cytotoxic T-cells and macrophages, among other functions. These cells are also known as CD4+ T-cells because they express the CD4 protein on their surface. Helper T-cells become activated when they are presented with peptide antigens by MHC class II molecules that are expressed on the surface of antigen presenting cells (APCs). Once activated, they divide rapidly and secrete small proteins called cytokines that regulate or assist in the active immune response.

[0055] Cytotoxic T-cells destroy virally infected cells and tumor cells and are also implicated in transplant rejection. These cells are also known as CD8+ T-cells because they express the CD8 glycoprotein on their surface. These cells recognize their targets by binding to antigen associated with MHC class I, which is present on the surface of nearly every cell of the body.

[0056] "Central memory" T-cells (or "TCM") refers to an antigen experienced CTL that expresses CD62L or CCR7 and CD45RO on the surface thereof and does not express or has decreased expression of CD45RA as compared to naive cells. In particular embodiments, central memory cells are positive for expression of CD62L, CCR7, CD25, CD127, CD45RO, and CD95, and have decreased expression of CD45RA as compared to naive cells.

[0057] "Effector memory" T-cell (or "TEM") refers to an antigen experienced T-cell that does not express or has decreased expression of CD62L on the surface thereof as compared to central memory cells and does not express or has decreased expression of CD45RA as compared to a naive cell. In particular embodiments, effector memory cells are negative for expression of CD62L and CCR7, compared to naive cells or central memory cells, and have variable expression of CD28 and CD45RA.

[0058] "Naive" T-cells refers to a non-antigen experienced T cell that expresses CD62L and CD45RA and does not express CD45RO as compared to central or effector memory cells. In particular embodiments, naive CD8+ T lymphocytes are characterized by the expression ofphenotypic markers of naive T-cells including CD62L, CCR7, CD28, CD127, and CD45RA.

[0059] Natural killer cells (also known as NK cells, K cells, and killer cells) are activated in response to interferons or macrophage-derived cytokines. They serve to contain viral infections while the adaptive immune response is generating antigen-specific cytotoxic T cells that can clear the infection. NK cells express CD8, CD16 and CD56 but do not express CD3.

[0060] NK cells include NK-T cells. NK-T cells are a specialized population of T cells that express a semi invariant T cell receptor (TCR ab) and surface antigens typically associated with natural killer cells. NK-T cells contribute to antibacterial and antiviral immune responses and promote tumor-related immunosurveillance or immunosuppression. Like natural killer cells, NK-T cells can also induce perforin-, Fas-, and TNF-related cytotoxicity. Activated NK-T cells are capable of producing IFN-y and IL-4. In particular embodiments, NK-T cells are CD3+ / CD56+.

[0061] Macrophages (and their precursors, monocytes) reside in every tissue of the body (in certain instances as microglia, Kupffer cells and osteoclasts) where they engulf apoptotic cells, pathogens and other non-self-components. Monocytes / macrophages express CD11b, F4 / 80; CD68; CD11c; IL-4Ra; and / or CD163.

[0062] Immature dendritic cells (i.e., pre-activation) engulf antigens and other non-self- components in the periphery and subsequently, in activated form, migrate to T-cell areas of lymphoid tissues where they provide antigen presentation to T cells. Dendritic cells express CD1a, CD1b, CD1c, CD1d, CD21 , CD35, CD39, CD40, CD86, CD101 , CD148, CD209, and DEC-205.

[0063] Hematopoietic Stem / Progenitor Cells or HSPC refer to a combination of hematopoietic stem cells and hematopoietic progenitor cells.

[0064] Hematopoietic stem cells refer to undifferentiated hematopoietic cells that are capable of self-renewal either in vivo, essentially unlimited propagation in vitro, and capable of differentiation to all other hematopoietic cell types.

[0065] A hematopoietic progenitor cell is a cell derived from hematopoietic stem cells or fetal tissue that is capable of further differentiation into mature cell types. In certain embodiments, hematopoietic progenitor cells are CD24|0Lin_CD117+hematopoietic progenitor cells. HPC can differentiate into (i) myeloid progenitor cells which ultimately give rise to monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, or dendritic cells; or (ii) lymphoid progenitor cells which ultimately give rise to T-cells, B-cells, and NK-cells.

[0066] HSPC can be positive for a specific marker expressed in increased levels on HSPC relative to other types of hematopoietic cells. For example, such markers include CD34, CD43, CD45RO, CD45RA, CD59, CD90, CD109, CD117, CD133, CD166, HLA DR, or a combinationthereof. Also, the HSPC can be negative for an expressed marker relative to other types of hematopoietic cells. For example, such markers include Lin, CD38, or a combination thereof. Preferably, the HSPC are CD34+cells.

[0067] A statement that a cell or population of cells is "positive" for or expressing a particular marker refers to the detectable presence on or in the cell of the particular marker. When referring to a surface marker, the term can refer to the presence of surface expression as detected by flow cytometry, for example, by staining with an antibody that binds to the marker and detecting said antibody, wherein the staining is detectable by flow cytometry at a level substantially above the staining detected carrying out the same procedure with an isotype-matched control under otherwise identical conditions and / or at a level substantially similar to that for cell known to be positive for the marker, and / or at a level substantially higher than that for a cell known to be negative for the marker.

[0068] A statement that a cell or population of cells is "negative" for a particular marker or lacks expression of a marker refers to the absence of substantial detectable presence on or in the cell of a particular marker. When referring to a surface marker, the term can refer to the absence of surface expression as detected by flow cytometry, for example, by staining with an antibody that binds to the marker and detecting said antibody, wherein the staining is not detected by flow cytometry at a level substantially above the staining detected carrying out the same procedure with an isotype-matched control under otherwise identical conditions, and / or at a level substantially lower than that for cell known to be positive for the marker, and / or at a level substantially similar as compared to that for a cell known to be negative for the marker.

[0069] Cells to be genetically modified according to the teachings of the current disclosure can be patient-derived cells (autologous) or allogeneic when appropriate, and can also be in vivo or ex vivo.

[0070] (II) Cell Sample Collection and Cell Enrichment. Methods of sample collection and enrichment are known by those skilled in the art. In particular embodiments, cells are derived from humans, for example a patient to be treated. Cells can be derived from cell lines. The cells in some embodiments are obtained from a xenogeneic source, for example, from mouse, rat, nonhuman primate, or pig. In particular embodiments, cells are derived from humans, for example a patient to be treated.

[0071] In some embodiments, T cells are derived or isolated from samples such as whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone,prostate, cervix, testes, ovaries, tonsil, or other organ, and / or cells derived therefrom. In particular embodiments, cells from the circulating blood of a subject are obtained, e.g., by apheresis or leukapheresis. The samples, in particular embodiments, contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, HSC, HPC, HSPC, red blood cells, and / or platelets, and in some aspects contains cells other than red blood cells and platelets and further processing is necessary. In particular embodiments, T cells are derived from PBMCs.

[0072] In some embodiments, blood cells collected from a subject are washed, e.g., to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In particular embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the wash solution lacks calcium and / or magnesium and / or many or all divalent cations. Washing can be accomplished using a semi-automated "flow-through" centrifuge (for example, the Cobe 2991 cell processor, Baxter) according to the manufacturer's instructions. Tangential flow filtration (TFF) can also be performed. In particular embodiments, cells can be re-suspended in a variety of biocompatible buffers after washing, such as, Ca++ / Mg++ free PBS.

[0073] The isolation can include one or more of various cell preparation and separation steps, including separation based on one or more properties, such as size, density, sensitivity or resistance to particular reagents, and / or affinity, e.g., immunoaffinity, to antibodies or other binding partners. In particular embodiments, the isolation is carried out using the same apparatus or equipment sequentially in a single process stream and / or simultaneously. In particular embodiments, the isolation, culture, and / or engineering of the different populations is carried out from the same starting composition or material, such as from the same sample.

[0074] In particular embodiments, a sample can be enriched for T cells by using density-based cell separation methods and related methods. For example, white blood cells can be separated from other cell types in the peripheral blood by lysing red blood cells and centrifuging the sample through a Percoll or Ficoll gradient.

[0075] In particular embodiments, a bulk T cell population can be used that has not been enriched for a particular T cell type. In particular embodiments, a selected T cell type can be enriched for and / or isolated based on cell-marker based positive and / or negative selection. In positive selection, cells having bound cellular markers are retained for further use. In negative selection, cells not bound by a capture agent, such as an antibody to a cellular marker are retained for further use. In some examples, both fractions can be retained for a further use. In particular embodiments, CD4+ and / or CD8+ T cells are enriched from PBMCs.

[0076] The separation need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection of or enrichment for cells of a particular type refers to increasing the number or percentage of such cells but need not result in a complete absence of cells not expressing the marker. Likewise, negative selection, removal, or depletion of cells of a particular type refers to decreasing the number or percentage of such cells but need not result in a complete removal of all such cells.

[0077] In some examples, multiple rounds of separation steps are carried out, where the positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection.

[0078] In some embodiments, an antibody or binding domain for a cellular marker is bound to a solid support or matrix, such as a magnetic bead or paramagnetic bead, to allow for separation of cells for positive and / or negative selection. For example, in some embodiments, the cells and cell populations are separated or isolated using immunomagnetic (or affinity magnetic) separation techniques (reviewed in Methods in Molecular Medicine, vol. 58: Metastasis Research Protocols, Vol. 2: Cell Behavior In Vitro and In Vivo, p 17-25 Edited by: S. A. Brooks and U. Schumacher © Humana Press Inc., Totowa, NJ); see also US 4,452,773; US 4,795,698; US 5,200,084; and EP 452342.

[0079] In some embodiments, affinity-based selection is via magnetic-activated cell sorting (MACS) (Miltenyi Biotec, Auburn, CA). MACS systems are capable of high-purity selection of cells having magnetized particles attached thereto. In certain embodiments, MACS operates in a mode wherein the non-target and target species are sequentially eluted after the application of the external magnetic field. That is, the cells attached to magnetized particles are held in place while the unattached species are eluted. Then, after this first elution step is completed, the species that were trapped in the magnetic field and were prevented from being eluted are freed in some manner such that they can be eluted and recovered. In certain embodiments, the non-target cells are labelled and depleted from the heterogeneous population of cells.

[0080] In some embodiments, a cell population described herein is collected and enriched (or depleted) via flow cytometry, in which cells stained for multiple cell surface markers are carried in a fluidic stream. In some embodiments, a cell population described herein is collected and enriched (or depleted) via preparative scale (FACS)-sorting. In certain embodiments, a cell population described herein is collected and enriched (or depleted) by use of microelectromechanical systems (MEMS) chips in combination with a FACS-based detection system (see, e.g., WO 2010 / 033140, Cho et al. (2010) Lab Chip 10, 1567-1573; and Godin et al. (2008) J Biophoton. 1(5):355 — 376). In both cases, cells can be labeled with multiple markers,allowing for the isolation of well-defined cell subsets at high purity.

[0081] Cell-markers for different T cell subpopulations are described above. In particular embodiments, specific subpopulations of T cells, such as cells positive or expressing high levels of one or more surface markers, e.g., CCR7, CD45RO, CD8, CD27, CD28, CD62L, CD127, CD4, and / or CD45RA T cells, are isolated by positive or negative selection techniques.

[0082] CD3+, CD28+ T cells can be positively selected for and expanded using anti-CD3 / anti- CD28 conjugated magnetic beads (e.g., DYNABEADS® (Life Technologies AS, Norway) M-450 CD3 / CD28 T Cell Expander).

[0083] In particular embodiments, a CD8+ or CD4+ selection step is used to separate CD4+ helper and CD8+ cytotoxic T cells. Such CD8+ and CD4+ populations can be further sorted into sub-populations by positive or negative selection for markers expressed or expressed to a relatively higher degree on one or more naive, memory, and / or effector T cell subpopulations.

[0084] In some embodiments, enrichment for central memory T (TCM) cells is carried out. In particular embodiments, memory T cells are present in both CD62L subsets of CD8+ peripheral blood lymphocytes. PBMC can be enriched for or depleted of CD62L, CD8 and / or CD62L+CD8+ fractions, such as by using anti-CD8 and anti-CD62L antibodies.

[0085] In some embodiments, the enrichment for central memory T (TCM) cells is based on positive or high surface expression of CCR7, CD45RO, CD27, CD62L, CD28, CD3, and / or CD127; in some aspects, it is based on negative selection for cells expressing or highly expressing CD45RA and / or granzyme B. In some aspects, isolation of a CD8+ population enriched for TCM cells is carried out by depletion of cells expressing CD4, CD14, CD45RA, and positive selection or enrichment for cells expressing CCR7, CD45RO, and / or CD62L. In one aspect, enrichment for central memory T (TCM) cells is carried out starting with a negative fraction of cells selected based on CD4 expression, which is subjected to a negative selection based on expression of CD14 and CD45RA, and a positive selection based on CD62L. Such selections in some aspects are carried out simultaneously and in other aspects are carried out sequentially, in either order. In some aspects, the same CD4 expression-based selection step used in preparing the CD8+ cell population or subpopulation, also is used to generate the CD4+ cell population or sub-population, such that both the positive and negative fractions from the CD4-based separation are retained, optionally following one or more further positive or negative selection steps.

[0086] Other cell types can be enriched based on known marker profiles and techniques. For example, CD34+ HSC, HSP, and HSPC can be enriched using anti-CD34 antibodies directly or indirectly conjugated to magnetic particles in connection with a magnetic cell separator, for example, the CliniMACS® Cell Separation System (Miltenyi Biotec, Bergisch Gladbach,Germany).

[0087] (III) Genetic Engineering Techniques. In particular embodiments, cell populations are genetically modified or genetically engineered to express an anti-B7-H3 recombinant receptor and a CXCR3 isoform.

[0088] Desired genes (e.g., genes encoding a recombinant receptor and / or CXCR3) or artificial expression constructs disclosed herein can be introduced into cells by any method known in the art, including transfection, electroporation, microinjection, lipofection, calcium phosphate mediated transfection, infection with a viral or bacteriophage vector including the gene sequences, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, in vivo nanoparticle-mediated delivery, etc. Numerous techniques are known in the art for the introduction of foreign genes into cells (see e.g., Loeffler and Behr, 1993, Meth. Enzymol. 217:599-618; Cohen, et al., 1993, Meth. Enzymol. 217:618-644; Cline, 1985, Pharmac. Ther. 29:69-92) and may be used, provided that the necessary developmental and physiological functions of the recipient cells are not unduly disrupted. The technique can provide for the stable transfer of the gene to the cell, so that the gene is expressible by the cell and, in certain instances, preferably heritable and expressible by its cell progeny.

[0089] The term “gene” refers to a nucleic acid sequence (used interchangeably with polynucleotide or nucleotide sequence) that encodes a recombinant receptor and / or CXCR3 as described herein. This definition includes various sequence polymorphisms, mutations, and / or sequence variants wherein such alterations do not substantially affect the function of the encoded recombinant receptor or CXCR3. The term “gene” may include not only coding sequences but also regulatory regions such as promoters, enhancers, and termination regions. The term further can include all introns and other DNA sequences spliced from an mRNA transcript, along with variants resulting from alternative splice sites. Gene sequences encoding the molecule can be DNA or RNA that directs the expression of the open reading frame(s) within the artificial expression construct. These nucleic acid sequences may be a DNA strand sequence that is transcribed into RNA or an RNA sequence that is translated into protein. The nucleic acid sequences include both the full-length nucleic acid sequences as well as non-full-length sequences derived from the full-length protein. The sequences can also include degenerate codons of the native sequence or sequences that may be introduced to provide codon preference in a specific cell type. Portions of complete gene sequences are referenced throughout the disclosure as is understood by one of ordinary skill in the art. Herein, the term “gene” and “artificial expression construct” are used interchangeably.

[0090] Gene sequences encoding a recombinant receptor and / or CXCR3 are provided hereinand can also be readily prepared by synthetic or recombinant methods from the relevant amino acid sequences and other description provided herein. In particular embodiments, the gene sequence encoding any of these sequences can also have one or more restriction enzyme sites at the 5' and / or 3' ends of the coding sequence in order to provide for easy excision and replacement of the gene sequence encoding the sequence with another gene sequence encoding a different sequence. In particular embodiments, the gene sequence encoding the sequences can be codon optimized for expression in mammalian cells.

[0091] "Encoding” refers to the property of specific sequences of nucleotides in a gene, such as a cDNA, or an mRNA, to serve as templates for the synthesis of other macromolecules such as a defined sequence of amino acids. Thus, a gene codes for a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. A "gene sequence encoding a protein" includes all nucleotide sequences that are degenerate versions of each other and that code for the same amino acid sequence or amino acid sequences of substantially similar form and function.

[0092] Polynucleotide gene sequences encoding more than one portion of an expressed recombinant receptor and / or CXCR3 can be operably linked to each other and relevant regulatory sequences. For example, there can be a functional linkage between a regulatory sequence and an exogenous nucleic acid sequence resulting in expression of the latter. For another example, a first nucleic acid sequence can be operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary or helpful, join coding regions, into the same reading frame.

[0093] Promoters can include general promoters, tissue-specific promoters, cell-specific promoters, and / or promoters specific for the cytoplasm. Promoters may include strong promoters, weak promoters, constitutive expression promoters (i.e. , constitutive promoters), and / or inducible promoters. Constitutive promoters are promoters that allow for the continual transcription of its associated gene or genes.

[0094] Particular examples of constitutive promoters include human elongation factor la (EF1a) promoter, myeloproliferative sarcoma virus (MND), cytomegalovirus (CMV), simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well ashuman gene promoters such as the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. In particular embodiments, the artificial expression construct is under the control of an EF1 promoter.

[0095] A "vector" is a nucleic acid molecule that is capable of transporting another nucleic acid. Vectors may be, e.g., plasmids (DNA plasmids or RNA plasmids), transposon-based systems, cosmids, bacterial artificial chromosomes, viruses, or phage. An "expression vector" is a vector that is capable of directing the expression of a protein encoded by one or more genes carried by the vector when it is present in the appropriate environment.

[0096] "Lentivirus" refers to a genus of retroviruses that are capable of infecting dividing and nondividing cells. Several examples of lentiviruses include HIV (human immunodeficiency virus: including HIV type 1, and HIV type 2); equine infectious anemia virus; feline immunodeficiency virus (FIV); bovine immune deficiency virus (BIV); and simian immunodeficiency virus (SIV).

[0097] A lentiviral vector is a vector derived from at least a portion of a lentivirus genome, including especially a self-inactivating lentiviral vector as provided in Milone et ah, Mol. Ther. 17(8): 1453-1464 (2009). Other examples of lentivirus vectors that may be used in the clinic, include: the LENTIVECTOR® gene delivery technology from Oxford BioMedica, the LENTIMAX™ vector system from Lentigen and the like. Nonclinical types of lentiviral vectors are also available and would be known to one skilled in the art. In particular embodiments, cells are genetically engineered to express a recombinant receptor and / or CXCR3 using a lentivirus or lentiviral vector.

[0098] "Retroviruses" are viruses having an RNA genome. "Gammaretrovirus" refers to a genus of the retroviridae family. Exemplary gammaretroviruses include mouse stem cell virus, murine leukemia virus, feline leukemia virus, feline sarcoma virus, and avian reticuloendotheliosis viruses.

[0099] Retroviral vectors (see Miller, et al., 1993, Meth. Enzymol. 217:581-599) can be used. In such embodiments, the gene to be expressed is cloned into the retroviral vector for its delivery into cells. In particular embodiments, a retroviral vector includes all of the cis-acting sequences necessary for the packaging and integration of the viral genome, i.e. , (a) a long terminal repeat (LTR), or portions thereof, at each end of the vector; (b) primer binding sites for negative and positive strand DNA synthesis; and (c) a packaging signal, necessary for the incorporation of genomic RNA into virions. More detail about retroviral vectors can be found in Boesen, et al., 1994, Biotherapy 6:291-302; Clowes, et al., 1994, J. Clin. Invest. 93:644-651 ; Kiem, et al., 1994, Blood 83:1467-1473; Salmons and Gunzberg, 1993, Human Gene Therapy 4:129-141 ; and Grossman and Wilson, 1993, Curr. Opin. in Genetics and Devel. 3:110-114. Adenoviruses, adeno-associated viruses (AAV) and alphaviruses can also be used. See Kozarsky and Wilson,1993, Current Opinion in Genetics and Development 3:499-503, Rosenfeld, et al., 1991 , Science 252:431-434; Rosenfeld, et al., 1992, Cell 68:143-155; Mastrangeli, et al., 1993, J. Clin. Invest. 91 :225-234; Walsh, et al., 1993, Proc. Soc. Exp. Bioi. Med. 204:289-300; and Lundstrom, 1999, J. Recept. Signal Transduct. Res. 19: 673-686. Other methods of gene delivery include use of mammalian artificial chromosomes (Vos, 1998, Curr. Op. Genet. Dev. 8:351-359); liposomes (Tarahovsky and Ivanitsky, 1998, Biochemistry (Mose) 63:607-618); ribozymes (Branch and Klotman, 1998, Exp. Nephrol. 6:78-83); and triplex DNA (Chan and Glazer, 1997, J. Mol. Med.75:267-282).

[0100] There are a large number of available viral vectors suitable within the current disclosure, including those identified for human gene therapy applications (see Pfeifer and Verma, 2001 , Ann. Rev. Genomics Hum. Genet. 2:177). Methods of using retroviral and lentiviral viral vectors and packaging cells for transducing mammalian host cells with viral particles including transgenes are described in, e.g., US 8,119,772; Walchli, et al., 2011 , PLoS One 6:327930; Zhao, et al., 2005, J. Immunol. 174:4415; Engels, et al., 2003, Hum. Gene Ther. 14:1155; Frecha, et al., 2010, Mol. Ther. 18:1748; and Verhoeyen, et al., 2009, Methods Mol. Biol. 506:97. Retroviral and lentiviral vector constructs and expression systems are also commercially available.

[0101] Targeted genetic engineering approaches may also be utilized. The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas (CRISPR-associated protein) nuclease system is an engineered nuclease system used for genetic engineering that is based on a bacterial system. Information regarding CRISPR-Cas systems and components thereof are described in, for example, US8697359, US8771945, US8795965, US8865406, US8871445, US8889356, US8889418, US8895308, US8906616, US8932814, US8945839, US8993233 andUS8999641 and applications related thereto; and WO2014 / 018423, WO2014 / 093595, WO2014 / 093622, WO2014 / 093635, WO2014 / 093655, WO2014 / 093661, WO2014 / 093694, WO2014 / 093701 , WO2014 / 093709, WO2014 / 093712, WO2014 / 093718, WO2014 / 145599, WO2014 / 204723, WO2014 / 204724, WO2014 / 204725, WO2014 / 204726, WO2014 / 204727, WO2014 / 204728, WO2014 / 204729, WO2015 / 065964, WO2015 / 089351, WO2015 / 089354, WO2015 / 089364, WO2015 / 089419, WO2015 / 089427, WO2015 / 089462, WO2015 / 089465, WO2015 / 089473 and WO2015 / 089486, W02016205711 , WO2017 / 106657, WO2017 / 127807 and applications related thereto.

[0102] Particular embodiments utilize zinc finger nucleases (ZFNs) as gene editing agents. ZFNs are a class of site-specific nucleases engineered to bind and cleave DNA at specific positions. ZFNs are used to introduce double stranded breaks (DSBs) at a specific site in a DNA sequence which enables the ZFNs to target unique sequences within a genome in a variety of different cells.A zinc finger is a domain of 30 amino acids within the zinc finger binding domain whose structure is stabilized through coordination of a zinc ion. Examples of zinc fingers include C2H2 zinc fingers, C3H zinc fingers, and C4 zinc fingers. A designed zinc finger domain is a domain not occurring in nature whose design / composition results principally from rational criteria, e.g., application of substitution rules and computerized algorithms for processing information in a database storing information of existing ZFP designs and binding data. A well-known example of a ZFN is a fusion of the Fokl nuclease with a zinc finger DNA binding domain. For additional information regarding ZFNs and ZFNs useful within the teachings of the current disclosure, see, e.g., US 6,534,261 ; US 6,607,882; US 6,746,838; US 6,794, 136; US 6,824,978; 6,866,997; US 6,933,113; 6,979,539; US 7,013,219; US 7,030,215; US 7,220,719; US 7,241 ,573; US 7,241 ,574; US 7,585,849; US 7,595,376; US 6,903,185; US 6,479,626; US 2003 / 0232410 and US 2009 / 0203140 as well as Gaj et al., Nat Methods, 2012, 9(8):805-7; Ramirez et al., Nucl Acids Res, 2012, 40(12):5560-8; Kim et al., Genome Res, 2012, 22(7): 1327-33; Urnov et al., Nature Reviews Genetics, 2010, 11 :636-646; Miller, et al. Nature biotechnology 25, 778-785 (2007); Bibikova, et al. Science 300, 764 (2003); Bibikova, et al. Genetics 161 , 1169-1175 (2002); Wolfe, et al. Annual review of biophysics and biomolecular structure 29, 183-212 (2000); Kim, et al. Proceedings of the National Academy of Sciences of the United States of America 93, 1156-1160 (1996); and Miller, et al. The EMBO journal 4, 1609-1614 (1985).

[0103] Particular embodiments can use transcription activator like effector nucleases (TALENs) as gene editing agents. TALENs refer to fusion proteins including a transcription activator-like effector (TALE) DNA binding protein and a DNA cleavage domain. TALENs are used to edit genes and genomes by inducing double DSBs in the DNA, which induce repair mechanisms in cells. Generally, two TALENs must bind and flank each side of the target DNA site for the DNA cleavage domain to dimerize and induce a DSB. For additional information regarding TALENs, see US 8,440,431 ; US 8,440,432; US 8,450,471 ; US 8,586,363; and US 8,697,853; as well as Joung and Sander, Nat Rev Mol Cell Biol, 2013, 14(l):49-55; Beurdeley et al., Nat Commun, 2013, 4: 1762; Scharenberg et al., Curr Gene Ther, 2013, 13(4):291-303; Gaj et al., Nat Methods, 2012, 9(8):805-7; Miller, et al. Nature biotechnology 29, 143-148 (2011); Christian, et al. Genetics 186, 757-761 (2010); Boch, et al. Science 326, 1509-1512 (2009); and Moscou, & Bogdanove, Science 326, 1501 (2009).

[0104] Particular embodiments can utilize MegaTALs as gene editing agents. MegaTALs have a sc rare-cleaving nuclease structure in which a TALE is fused with the DNA cleavage domain of a meganuclease. Meganucleases, also known as homing endonucleases, are single peptide chains that have both DNA recognition and nuclease function in the same domain. In contrast to theTALEN, the megaTAL only requires the delivery of a single peptide chain for functional activity.

[0105] Particular embodiments can use transposon-based systems as gene editing agents to mediate the integration of an artificial expression construct into cells. Generally, such methods will involve introducing into cells (i) a first vector encoding a transposase (or a transposase polypeptide) and (ii) a second vector encoding a desired genetic element that is flanked by transposon repeats. Transposons or transposable elements include a (short) nucleic acid sequence with terminal repeat sequences upstream and downstream thereof and encode enzymes that facilitate the excision and insertion of the nucleic acid into target DNA sequences.

[0106] Several transposon / transposase systems have been adapted for genetic insertions of heterologous DNA sequences. Examples of such transposases include sleeping beauty (“SB”, e.g., derived from the genome of salmonid fish); piggyback (e.g., derived from lepidopteran cells and / or the Myotis lucifugus); mariner (e.g., derived from Drosophila); frog prince (e.g., derived from Rana pipiens); Toll ; Tol2 (e.g., derived from medaka fish); TcBuster (e.g., derived from the red flour beetle Tribolium castaneum), Helraiser, Himarl , Passport, Minos, Ac / Ds, PIF, Harbinger, Harbinger3-DR, HSmarl , and spinON. Transposases and transposon systems are further described in U.S. Pat. Nos. 6,489,458; 7,148,203; 8,227,432; and 9,228,180.

[0107] (IV) Recombinant Receptors. In particular embodiments, a recombinant receptor is or includes a binding domain that binds a target antigen, wherein the recombinant receptor is expressed by a cell following the artificial introduction of a nucleic acid encoding the recombinant receptor into the cell. The recombinant receptor can be, e.g., a CAR, an engineered T cell receptor (eTCR), or a hybrid thereof.

[0108] As described previously, CAR include several distinct subcomponents that allow genetically modified cells to recognize and kill unwanted cells, such as cancer cells. The subcomponents include at least an extracellular component and an intracellular component. The extracellular component includes a binding domain that specifically binds a target antigen that is preferentially present on the surface of diseased cells or within the immediate area thereof. When the binding domain binds such antigens, the intracellular component activates the genetically modified cell to destroy the bound diseased 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 target antigen. Each of these subcomponents is described in more detail in the following section (IV) subheadings.

[0109] Many considerations associated with CAR apply to eTCR as well. eTCR disclosed herein include a binding domain that binds a target antigen (e.g., an scFv) linked to the Co and / or Cp chains of a TCR. A TCR is a heterodimeric fusion protein that typically includes an a and p chain. Each chain includes a variable region (Va and P) and a constant region (Ca and CP). In particular embodiments, an eTCR does not include the native TCR variable region but does include the native TCR constant region. In particular embodiments, the eTCR includes an scFv as the variable region of either the a or p chain. In particular embodiments, the eTCR includes an scFv as the variable region of both the a and p chain.

[0110] (IV-A) Binding Domains. In certain examples, the current disclosure provides binding domains for use in a recombinant receptor 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.

[0111] 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).

[0112] 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)); Chothia (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 Kabat and 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.

[0113] 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 ABodyBuilder2.

[0114] In particular embodiments, a B7-H3 binding domain includes a CDR set provided herein. A CDR set refers to 3 heavy chain CDRs and 3 light chain CDRs that together result in binding to B7-H3.

[0115] Table 1. B7-H3 Binding Domain CDR Sequences.

[0116] In particular embodiments, the B7-H3 binding domain includes a variable heavy chain including the sequence:EVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSDSSAIYYAD TVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCGRGRENIYYGSRLDYWGQGTTVTVSS (SEQ ID NO: 29) and a variable light chain including the sequence:DIQLTQSPSFLSASVGDRVTITCKASQNVDTNVAWYQQKPGKAPKALIYSASYRYSGVPSRFS GSGSGTDFTLTISSLQPEDFATYYCQQYNNYPFTFGQGTKLEIK (SEQ ID NO: 30).

[0117] In particular embodiments, the B7-H3 binding domain includes a variable heavy chain encoded by the sequence:GAAGTCCAGCTGGTCGAGAGCGGAGGAGGACTGGTGCAGCCTGGAGGATCACTGAGACT GAGCTGCGCCGCTTCCGGATTCACCTTTAGCTCCTTCGGCATGCACTGGGTGAGGCAGGC ACCAGGAAAAGGCCTGGAGTGGGTCGCTTACATCTCTAGTGACTCAAGCGCCATCTACTAT GCAGATACCGTGAAAGGCAGGTTTACAATCAGTCGCGACAACGCTAAGAATTCCCTGTATC TGCAGATGAACTCTCTGCGCGACGAGGATACAGCAGTCTACTATTGCGGGCGGGGAAGAGAAAATATCTACTATGGAAGCCGACTGGACTACTGGGGACAGGGAACCACAGTGACAGTCT CCTCT (SEQ ID NO: 31) and a variable light chain encoded by the sequence: GATATCCAGCTGACTCAGAGCCCCTCCTTCCTGTCTGCCAGTGTGGGCGACAGGGTCACT ATTACCTGTAAGGCATCCCAGAACGTGGATACCAATGTCGCCTGGTACCAGCAGAAGCCC GGGAAAGCACCTAAGGCCCTGATCTATTCAGCCAGCTACCGATATTCTGGCGTGCCAAGT CGGTTCTCCGGATCTGGCAGTGGGACTGACTTTACACTGACTATTAGTTCACTGCAGCCCGAAGATTTTGCTACCTACTATTGTCAGCAGTACAATAACTACCCATTCACCTTCGGACAGGGG ACAAAACTGGAAATCAAA (SEQ ID NO: 32).

[0118] 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 linkerorientations 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. In particular embodiments, the binding domain that binds B7-H3 is an scFv. In particular embodiments, the B7-H3 binding domain is derived from the scFv, MGA271.

[0119] In particular embodiments, the scFv that binds B7-H3 includes the sequence of SEQ ID NO: 33 or SEQ ID NO: 35.

[0120] In particular embodiments, the scFv that binds B7-H3 is encoded by the sequence of SEQ ID NO: 34 or SEQ ID NO: 36.

[0121] Any binding domain that binds B7-H3 can be used. Commercially available antibodies that bind B7-H3 include: 6A1, 7-517, MIH35, RM335, M3.2D7, 1 E7D1 , EPNCIR122, SP265, RM2041 , SP206, EPR20115, BLR026F, and MM0104-20J12. Binding domains can be derived from the CDRs of these antibodies.

[0122] 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.

[0123] 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 592,106 and EP 519,596; Padlan, 1991, Molecular Immunology, 28(4 / 5):489-498; Studnicka et al., 1994, Protein Engineering, 7(6):805-814; and Roguska et al., 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 et al., 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).

[0124] 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.

[0125] 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 orboth 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 still specifically bind its target with an affinity similar to the wild type binding domain.

[0126] 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.

[0127] Additional cancer antigens that can be bound by recombinant receptors disclosed herein include CD19, CD20, CD22, ROR1, CD33, WT-1 , CD123, BCMA, PSMA, PSCA, HER2, ERBB2, CD133, CAIX, GD2, CEA, and mesothelin.

[0128] (I -B) Spacers. Spacers are used to create appropriate distances and / or flexibility from other recombinant receptor sub-components. As indicated, in particular embodiments, the length of a spacer is customized for binding targeted antigen-expressing cells and mediating destruction. In particular embodiments, a spacer length can be selected based upon the location of a cellular marker antigen epitope, affinity of a binding domain for the antigen epitope, and / or the ability of the recombinant receptor to mediate cell destruction following target antigen binding.

[0129] Spacers typically include those having 5 to 270 amino acids, 5 to 200 amino acids, 5 to 150 amino acids, 5 to 100 amino acids, 5 to 50 amino acids, or 10 to 25 amino acids.

[0130] In particular embodiments, a spacer is 5 amino acids, 8 amino acids, 10 amino acids, 12 amino acids, 14 amino acids, 20 amino acids, 21 amino acids, 26 amino acids, 27 amino acids, 45 amino acids, 50 amino acids, or 75 amino acids. These lengths qualify as short spacers.

[0131] In particular embodiments, a spacer is 76 amino acids, 90 amino acids, 100 amino acids, 110 amino acids, 120 amino acids, 125 amino acids, 128 amino acids, 131 amino acids, 135 amino acids, 140 amino acids, 150 amino acids, 160 amino acids, 170 amino acids, or 179 amino acids. These lengths qualify as intermediate spacers.

[0132] In particular embodiments, a spacer is 180 amino acids, 190 amino acids, 200 amino acids, 210 amino acids, 212 amino acids, 214 amino acids, 216 amino acids, 218 amino acids, 220 amino acids, 228 amino acids, 230 amino acids, 240 amino acids, 250 amino acids, 260 amino acids, or 270 amino acids. These lengths qualify as long spacers.

[0133] Exemplary spacers include all or a portion of an immunoglobulin hinge region. An immunoglobulin hinge region may be a wild-type immunoglobulin hinge region or an altered wildtype immunoglobulin hinge region. In certain embodiments, an immunoglobulin hinge region is ahuman immunoglobulin hinge region. As used herein, a “wild type immunoglobulin hinge region” refers to a naturally occurring upper and middle hinge amino acid sequences interposed between and connecting the CH1 and CH2 domains (for IgG, IgA, and IgD) or interposed between and connecting the CH1 and CH3 domains (for IgE and IgM) found in the heavy chain of an antibody.

[0134] An immunoglobulin hinge region may be an IgG, IgA, IgD, IgE, or IgM hinge region. An IgG hinge region may be an I gG 1 , lgG2, 1 gG3, or lgG4 hinge region. Sequences from IgG 1 , lgG2, lgG3, lgG4 or IgD can be used alone or in combination with all or a portion of a CH2 region; all or a portion of a CH3 region; or all or a portion of a CH2 region and all or a portion of a CH3 region.

[0135] In particular embodiments, the spacer is a short spacer including an lgG4 hinge region. In particular embodiments, the spacer is an intermediate (or medium) spacer including an lgG4 hinge region and an lgG4 CH3 region. In particular embodiments, the spacer is a long spacer including an lgG4 hinge region, an lgG4 CH2 region, and an lgG4 CH3 region.

[0136] Other examples of hinge regions that can be used in a recombinant receptor described herein include the hinge region present in the extracellular regions of type 1 membrane proteins, such as CD8a, CD4, CD28 and CD7.

[0137] In particular embodiments, a spacer includes a hinge region that includes a type II C-lectin interdomain (stalk) region or a cluster of differentiation (CD) molecule stalk region. A “stalk region” of a type II C-lectin or CD molecule refers to the portion of the extracellular domain (ECD) of the type II C-lectin or CD molecule that is located between the C-type lectin-like domain (CTLD; e.g., similar to CTLD of natural killer cell receptors) and the hydrophobic portion (transmembrane domain). For example, the ECD of human CD94 (GenBank Accession No. AAC50291.1) corresponds to amino acid residues 34-179, but the CTLD corresponds to amino acid residues 61-176, so the stalk region of the human CD94 molecule includes amino acid residues 34-60, which are located between the hydrophobic portion (transmembrane domain) and CTLD (see Boyington et al., Immunity 10:15, 1999; for descriptions of other stalk regions, see also Beavil et al., Proc. Nat'l. Acad. Sci. USA 89:153, 1992; and Figdor et al., Nat. Rev. Immunol. 2:11 , 2002). These type II C-lectin or CD molecules may also have junction amino acids (described below) between the stalk region and the transmembrane region or the CTLD. In another example, the 233 amino acid human NKG2A protein (GenBank Accession No. P26715.1) has a hydrophobic portion (transmembrane domain) ranging from amino acids 71-93 and an ECD ranging from amino acids 94-233. The CTLD includes amino acids 119-231 and the stalk region includes amino acids 99-116, which may be flanked by additional junction amino acids. Other type II C- lectin or CD molecules, as well as their extracellular ligand-binding domains, stalk regions, and CTLDs are known in the art (see, e.g., GenBank Accession Nos. NP 001993.2; AAH07037.1 ; NP001773.1; AAL65234.1 ; CAA04925.1 ; for the sequences of human CD23, CD69, CD72, NKG2A, and NKG2D and their descriptions, respectively).

[0138] (IV-C) Transmembrane Domains. As indicated, transmembrane domains within a recombinant receptor serve to functionally and / or physically connect the extracellular component and intracellular component through the cell membrane. The transmembrane domain can anchor the recombinant receptor in the modified cell’s membrane.

[0139] The transmembrane domain can be derived either from a natural and / or a synthetic source. When the source is natural, the transmembrane domain can be derived from any membrane-bound or transmembrane protein. Transmembrane domains can include at least the transmembrane region(s) of the a, p or chain of a T-cell receptor, CD28, CD27, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22; CD33, CD37, CD64, CD80, CD86, CD134, CD137 CD154, Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9. In particular embodiments, a transmembrane domain may include at least the transmembrane region(s) of, e.g., KIRDS2, 0X40, CD2, CD27, LFA-1 (CD 11a, CD18), ICOS (CD278), 4-1 BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2Rp, IL2Ry, IL7R a, ITGA1 , VLA1 , CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, GDI Id, ITGAE, CD103, ITGAL, CDI la, ITGAM, CDI lb, ITGAX, GDI Ic, ITGB1, CD29, ITGB2, CD18, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1 , CRT AM, Ly9(CD229), , PSGL1, CD100 (SEMA4D), SLAMF6 (NTB- A, LylOS), SLAM (SLAMF1 , CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKG2D, or NKG2C. In particular embodiments, a variety of human hinges can be employed as well including the human Ig (immunoglobulin) hinge (e.g., an lgG4 hinge, an IgD hinge), a GS linker (e.g., a GS linker described herein), a KIR2DS2 hinge or a CD8a hinge. In particular embodiments, the recombinant receptor includes a CD28 transmembrane domain. It has been shown that a CD28 transmembrane domain reduces the antigen-threshold for second- generation 4-1 BB CAR T cell activation.

[0140] In particular embodiments, a transmembrane domain has 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.

[0141] 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 recombinant receptor (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 recombinant receptor (e.g., up to 15amino acids of the intracellular components). In one aspect, the transmembrane domain is from the same protein that the signaling domain, co-stimulatory domain or the hinge domain is derived from. In another aspect, the transmembrane domain is not derived from the same protein that any other domain of the recombinant receptor is derived from. In some instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other unintended members of the receptor complex. In particular embodiments, the transmembrane domain is encoded by the nucleic acid sequence encoding the CD28 transmembrane domain (SEQ ID NOs: 53, 54, 55, or 56). In particular embodiments, the transmembrane domain includes the amino acid sequence of the CD28 transmembrane domain (SEQ ID NOs: 50, 51 , or 52).

[0142] (IV-D) Intracellular Effector Domains. The intracellular effector domains of a recombinant receptor are responsible for activation of the cell in which the recombinant receptor is expressed. The term “effector domain” is thus meant to include any portion of the intracellular domain sufficient to transduce an activation signal. An effector domain can directly or indirectly promote a biological or physiological response in a cell when receiving the appropriate signal. In certain embodiments, an effector domain is part of a protein or protein complex that receives a signal when bound, or it binds directly to a target molecule, which triggers a signal from the effector domain. An effector domain may directly promote a cellular response when it contains one or more signaling domains or motifs, such as an immunoreceptor tyrosine-based activation motif (ITAM). In other embodiments, an effector domain will indirectly promote a cellular response by associating with one or more other proteins that directly promote a cellular response, such as costimulatory domains.

[0143] Effector domains can provide for activation of at least one function of a modified cell upon binding to the cellular marker expressed by a cancer cell. Activation of the modified cell can include one or more of differentiation, proliferation and / or activation or other effector functions. In particular embodiments, an effector domain can include an intracellular signaling component including a T cell receptor and a co-stimulatory domain which can include the cytoplasmic sequence from co-receptor or co-stimulatory molecule.

[0144] 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), CARD11, CD3y, CD35, CD3c, CD3 , CD27, CD28, CD79A, CD79B, DAP10, FcRa, FcR (FcsRIb), FcRy, Fyn, HVEM (LIGHTR), ICOS, LAG3, LAT, Lek,LRP, NKG2D, N0TCH1 , pTa, PTCH2, 0X40, ROR2, Ryk, SLAMF1 , Slp76, TCRa, TCR , TRIM, Wnt, Zap70, or any combination thereof. In particular embodiments, exemplary effector domains include signaling and co-stimulatory domains selected from: CD86, FcyRlla, DAP12, CD30, CD40, PD-1 , lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7- H3, a ligand that specifically binds with CD83, CDS, ICAM-1, GITR, BAFFR, SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8a, CD8 , I L2R , 1 L2Ry, 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, NKp46, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9. In particular embodiments, the effector domain includes a CD3£ signaling domain.

[0145] 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 FcRy (FCER1G), FcyRlla, FcRfJ (FCE Rib), DAP10, and DAP12. In particular embodiments, variants of CD3 retain at least one, two, three, or all ITAM regions.

[0146] In particular embodiments, an effector domain includes a cytoplasmic portion that associates with a cytoplasmic signaling protein, wherein the cytoplasmic signaling protein is a lymphocyte receptor or signaling domain thereof, a protein including a plurality of ITAMs, a costimulatory domain, or any combination thereof.

[0147] Additional examples of intracellular signaling components include the cytoplasmic sequences of the CD3 chain, and / or co- receptors that act in concert to initiate signal transduction following binding domain engagement.

[0148] 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 (CD 137), 0X40, CD30, CD40, PD-1 , ICOS, lymphocyte function- associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83. For example, CD27 co-stimulation has been demonstrated to enhance expansion, effector function, and survival of human CAR T cells in vitro and augments human T cell persistence and anti-cancer activity in vivo (Song et al. Blood. 2012; 119(3):696-706). Further examples of such co-stimulatory domain molecules include CDS, ICAM-1 , GITR, BAFFR, HVEM(LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8a, CD8p, IL2Rp, IL2Ry, IL7Ra, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDIId, ITGAE, CD103, ITGAL, CDIIa, ITGAM, CDI lb, ITGAX, CDIIc, ITGBI, CD29, ITGB2, CD18, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), NKG2D, CEACAM1, CRTAM, Ly9 (CD229), PSGL1 , CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, LylOS), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, and CD19a. In particular embodiments, the costimulatory domain includes a 4-1 BB signaling domain.

[0149] In particular embodiments, the intracellular signaling components include CD3^ (SEQ ID NO: 63, 64, or 65) and / or 4-1 BB (SEQ ID NOs: 57, 58, or 59). In particular embodiments, the intracellular signaling components include a CD3 (encoded by, for example, SEQ ID NOs: 66 or 67) and / or a portion of the 4-1 BB (encoded by, for example, SEQ ID NO: 60, 61 , or 62).

[0150] In particular embodiments, the intracellular signaling component includes all or a portion of the signaling domain of CD3 and all or a portion of the signaling domain of 4-1 BB.

[0151] Intracellular components may also include one or more of a protein of a Wnt signaling pathway (e.g., LRP, Ryk, or ROR2), NOTCH signaling pathway (e.g., NOTCH1 , NOTCH2, NOTCH3, or NOTCH4), Hedgehog signaling pathway (e.g., PTCH or SMO), receptor tyrosine kinases (RTKs) (e.g., epidermal growth factor (EGF) receptor family, fibroblast growth factor (FGF) receptor family, hepatocyte growth factor (HGF) receptor family, insulin receptor (IR) family, platelet-derived growth factor (PDGF) receptor family, vascular endothelial growth factor (VEGF) receptor family, tropomycin receptor kinase (Trk) receptor family, ephrin (Eph) receptor family, AXL receptor family, leukocyte tyrosine kinase (LTK) receptor family, tyrosine kinase with immunoglobulin-like and EGF-like domains 1 (TIE) receptor family, receptor tyrosine kinase-like orphan (ROR) receptor family, discoidin domain (DDR) receptor family, rearranged during transfection (RET) receptor family, tyrosine- protein kinase-like (PTK7) receptor family, related to receptor tyrosine kinase (RYK) receptor family, or muscle specific kinase (MuSK) receptor family); G-protein-coupled receptors, GPCRs (Frizzled or Smoothened); serine / threonine kinase receptors (BMPR or TGFR); or cytokine receptors (IL1R, IL2R, IL7R, or IL15R).

[0152] (IV-E) Linkers. As used herein, a linker can include a chemical moiety that serves to connect two other subcomponents of a molecule. Some linkers serve no purpose other than to link components while many linkers serve an additional purpose. Linkers can, for example, link VL and VH of antibody derived binding domains of scFvs and serve as junction amino acids between subcomponent portions of a recombinant receptor. In particular embodiments, the recombinant receptor includes a linker.

[0153] Linkers can be flexible, rigid, or semi-rigid, depending on the desired function of the linker. Linkers can include junction amino acids. 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 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: 87), (Gly3Ser)n(Gly4Ser)n (SEQ ID NO: 88), (Gly3Ser)n(Gly2Ser)n (SEQ ID NO: 89), or (Gly3Ser)n(Gly4Ser)i (SEQ ID NO: 90). In particular embodiments, the linker is (Gly4Ser)4(SEQ ID NO: 91), (Gly4Ser)3(SEQ ID NO: 92), (Gly4Ser)2(SEQ ID NO: 93), (Gly4Ser)i (SEQ ID NO: 94), (Gly3Ser)2(SEQ ID NO: 95), (Gly3Ser)i (SEQ ID NO: 96), (Gly2Ser)2(SEQ ID NO: 97) or (Gly2Ser)i, GGSGGGSGGSG (SEQ ID NO: 98), GGSGGGSGSG (SEQ ID NO: 99), or GGSGGGSG (SEQ ID NO: 100).

[0154] In particular embodiments, a linker region is (GGGGS)n (SEQ ID NO: 87) wherein n is an integer including, 1 , 2, 3, 4, 5, 6, 7, 8, 9, or more. In particular embodiments, a linker includes the Whitlow linker (GSTSGSGKPGSGEGSTKG (SEQ ID NO: 101)). In particular embodiments, the linker is (EAAAK)n (SEQ ID NO: 102) wherein n is an integer including 1, 2, 3, 4, 5, 6, 7, 8, 9, or more.

[0155] In some situations, flexible linkers may be incapable of maintaining a distance or positioning of a component needed for a particular use. In these instances, rigid or semi-rigid linkers may be useful. Examples of rigid or semi-rigid linkers include proline-rich linkers. In particular embodiments, a proline-rich linker is a peptide sequence having more proline residues than would be expected based on chance alone. In particular embodiments, a proline-rich linker is one having at least 30%, at least 35%, at least 36%, at least 39%, at least 40%, at least 48%, at least 50%, or at least 51 % proline residues. Particular examples of proline-rich linkers include fragments of proline-rich salivary proteins (PRPs).

[0156] Linkers can be susceptible to cleavage (cleavable linker), such as, acid-induced cleavage, photo-induced cleavage, peptidase-induced cleavage, esterase- induced cleavage, and disulfide bond cleavage. Alternatively, linkers can be substantially resistant to cleavage (e.g., stable linker or noncleavable linker). In some aspects, the linker is a procharged linker, a hydrophilic linker, or a dicarboxylic acid-based linker.

[0157] Junction amino acids can be a linker which can be used to connect sequences when the distance provided by a spacer is not needed and / or wanted. For example, junction amino acids can be short amino acid sequences that can be used to connect co-stimulatory intracellularsignaling components. In particular embodiments, junction amino acids are 9 amino acids or less (e g., 2, 3, 4, 5, 6, 7, 8, or 9 amino acids). In particular embodiments, a glycine-serine doublet can be used as a suitable junction amino acid linker. In particular embodiments, a single amino acid, e.g., an alanine, a glycine, can be used as a suitable junction amino acid.

[0158] (V) Control Features Including Tag Cassettes, Transduction Markers, Selection Cassettes, and / or Suicide Switches. In particular embodiments, artificial expression constructs can encode one or more tag cassettes and / or transduction markers. Tag cassettes and transduction markers can be used to activate, promote proliferation of, detect, enrich for, isolate, track, deplete and / or eliminate genetically modified cells in vitro, in vivo and / or ex vivo. "Tag cassette" refers to a unique synthetic peptide sequence affixed to, fused to, or that is part of an expressed molecule (e.g., recombinant receptor or chemokine receptor), to which a cognate binding molecule (e.g., ligand, antibody, or other binding partner) is capable of specifically binding where the binding property can be used to activate, promote proliferation of, detect, enrich for, isolate, track, deplete and / or eliminate the tagged protein and / or cells expressing the tagged protein. Transduction markers can serve the same purposes but are derived from naturally occurring molecules and are often expressed using a skipping element that separates the transduction marker from the rest of the expressed molecule.

[0159] Tag cassettes that bind cognate binding molecules include, for example, His tag (HHHHHH; SEQ ID NO: 103), Flag tag (DYKDDDDK; SEQ ID NO: 104), Xpress tag (DLYDDDDK; SEQ ID NO: 105), Avi tag (GLNDIFEAQKIEWHE; SEQ ID NO: 106), Calmodulin tag (KRRWKKNFIAVSAANRFKKISSSGAL; SEQ ID NO: 107), Polyglutamate tag, HA tag (YPYDVPDYA; SEQ ID NO: 108), Myc tag (EQKLISEEDL; SEQ ID NO: 109), Strep tag (which refers the original STREP® tag (WRHPQFGG; SEQ ID NO: 110), STREP® tag II (WSHPQFEK SEQ ID NO: 111 (IBA Institut fur Bioanalytik, Germany); see, e.g., US 7,981 ,632), Softag 1 (SLAELLNAGLGGS; SEQ ID NO: 112), Softag 3 (TQDPSRVG; SEQ ID NO: 113), and V5 tag (GKPIPNPLLGLDST; SEQ ID NO: 114).

[0160] Conjugate binding molecules that specifically bind tag cassette sequences disclosed herein are commercially available. For example, His tag antibodies are commercially available from suppliers including Life Technologies, Pierce Antibodies, and GenScript. Flag tag antibodies are commercially available from suppliers including Pierce Antibodies, GenScript, and Sigma- Aldrich. Xpress tag antibodies are commercially available from suppliers including Pierce Antibodies, Life Technologies and GenScript. Avi tag antibodies are commercially available from suppliers including Pierce Antibodies, IsBio, and Genecopoeia. Calmodulin tag antibodies are commercially available from suppliers including Santa Cruz Biotechnology, Abeam, and PierceAntibodies. HA tag antibodies are commercially available from suppliers including Pierce Antibodies, Cell Signal and Abeam. Myc tag antibodies are commercially available from suppliers including Santa Cruz Biotechnology, Abeam, and Cell Signal. Strep tag antibodies are commercially available from suppliers including Abeam, Iba, and Qiagen.

[0161] Transduction markers may be selected from at least one of a truncated CD19 (tCD19; see Budde et al., Blood 122: 1660, 2013); a truncated human EGFR (tEGFR or EGFRt; see Wang et al., Blood 118: 1255, 2011); an ECD 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, cells are genetically modified to express EGFRt.

[0162] 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 retain desired cells.

[0163] 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.

[0164] In particular embodiments, the selection cassette includes DHFRdm. In particular embodiments, the method does not require a selection cassette to acquire highly purified cell populations.

[0165] 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., AnalBiochem. 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 p-globin, etc.).

[0166] In particular embodiments, artificial expression constructs can include a polynucleotide that encodes a self-cleaving polypeptide, wherein the polynucleotide encoding the self-cleaving polypeptide is located between the polynucleotide encoding the recombinant receptor and a polynucleotide encoding a transduction marker (e.g., EGFRt). In particular embodiments, the polynucleotide encoding the self-cleaving polypeptide is located between the polynucleotide encoding a transduction marker and a polynucleotide encoding a CXCR3. In particular embodiments, the polynucleotide encoding the self-cleaving polypeptide is located between the polynucleotide encoding a transduction marker and a polynucleotide encoding a selection cassette (e.g., DHFRdm).

[0167] 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). Further exemplary nucleic acid and amino acid sequences of 2A peptides are set forth in, for example, Kim et al. (PLOS One 6:e18556 (2011). In particular embodiments, cells are genetically modified to include a self-cleaving polypeptide. In particular embodiments, the selfcleaving polypeptide includes T2A. In particular embodiments, the sequence encoding the selfcleaving polypeptide is between the sequence encoding the recombinant receptor and the sequence encoding the transduction marker. In particular embodiments, the sequence encoding the self-cleaving polypeptide is between the sequence encoding the recombinant receptor and the sequence encoding the chemokine receptor. In particular embodiments, the sequence encoding the self-cleaving polypeptide is between the sequence encoding the transduction marker and the sequence encoding the chemokine receptor.

[0168] 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 skipping element. For example, an artificial expression construct can have one, two, three, four or five tag cassettes and / or one, two, three, four, or five transduction markers could also be expressed and / or one, two, three, four or five selection cassettes. 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 cognatepairs are described in US 13 / 463,247.

[0169] One advantage of including at least one sequence encoding a control feature in an artificial expression construct is that cells expressing the artificial expression construct administered to a subject 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 a recombinant receptor (e.g., CAR) 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 anti-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 anti-EGFRt / anti-CD3 bispecific scFv, or an anti-EGFRt CAR T cell may be used.

[0170] In particular embodiments, a polynucleotide encoding an iCaspase9 construct (iCasp9) may be inserted into an artificial expression construct as a suicide switch.

[0171] In certain embodiments, modified cells expressing an artificial expression construct may be detected or tracked in vivo by using antibodies that bind with specificity to a control feature (e.g., anti-Tag antibodies), or by other cognate binding molecules that specifically bind the control feature, which binding partners for the control feature are conjugated to a fluorescent dye, radiotracer, iron-oxide nanoparticle or other imaging agent known in the art for detection by X-ray, CT-scan, MRI-scan, PET-scan, ultrasound, flow-cytometry, near infrared imaging systems, or other imaging modalities (see, e.g., Yu, et al., Theranostics 2:3, 2012).

[0172] Thus, modified cells expressing at least one control feature can be, e.g., more readily identified, isolated, sorted, induced to proliferate, tracked, and / or eliminated as compared to a modified cell without a tag cassette.

[0173] (VI) Characterization of Genetically Engineered Cells. In particular embodiments, the engineered cells can be assessed for surface expression of the recombinant receptor and / or CXCR3. In particular embodiments, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the engineered cells express a detectable level of the recombinant receptor and / or CXCR3 (e.g., CXCR3-A).

[0174] Surface protein expression can be determined by flow cytometry using methods known in the art. By labeling a population of cells with an element that targets the desired cell surface marker (e.g., an antibody) and is tagged with a fluorescent molecule, flow cytometry can be used to quantify the portion of the population that is positive for the surface marker, as well as the levelof surface marker expression.

[0175] Genomic incorporation of a recombinant receptor and / or CXCR3 within engineered cells can be determined by digital droplet PCR (ddPCR). Digital PCR enables quantification of DNA concentration in a sample. Digital PCR is performed by fractionating a mixture of a PCR reaction (e.g., containing a sample of nucleic acid molecules and copies of a PCR probe) such that some fractions contain no PCR probe copy, while other fractions contain one or more PCR probe copies. A PCR amplification of the fractions is performed and the fractions are analyzed for a PCR reaction. A fraction containing one or more probes and one or more target DNA molecules yields a positive end-point, while a fraction containing no PCR probe yields a negative end-point. The fraction of positive reactions is then fitted to a Poisson distribution to determine the absolute copy number of target DNA molecules per given volume of the unfractionated sample (i.e., copies per microliter of sample) (see Hindson, B. et al., (2011) Anal Chem. 83:8604-8610). Digital droplet PCR is a variation of digital PCR wherein a sample of nucleic acids is fractionated into droplets using a water-oil emulsion. PCR amplification is performed on the droplets collectively, whereupon a fluidics system is used to separate the droplets and provide analysis of each individual droplet. For one skilled in the art, ddPCR is used to provide an absolute quantification of DNA in a sample, to perform a copy number variation analysis, or to assess efficiency of genomic edits.

[0176] Engineered cells can also be assessed for cytokine-independent growth. Engineered cells are expected to grow in the presence of stimulatory cytokines (e.g., IL-2, IL-7). Growth in the absence of cytokines is an indicator of tumorigenic potential. In particular embodiments, engineered cells are grown for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, or 20 days in either the presence or in the absence of one or more stimulatory cytokines (e.g., IL- 2, IL-7). In particular embodiments, proliferation is assessed by cell count and viability using conventional methods (e.g., flow cytometry, microscopy, optical density, metabolic activity). In particular embodiments, proliferation is assessed starting on day 1 , day 2, day 3, day 4, day 5, day 6. In particular embodiments, proliferation is assessed every 1 day, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every 7 days, or every 8 days. In particular embodiments, growth in the absence of cytokines is assessed at the end of a growth period. In some particular embodiments, engineered cells with no growth in the absence of cytokines is defined as lacking tumorigenic potential. In particular embodiments, no growth is defined as an expansion of the population that is less than 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1 , 1.2, 1.3, 1.4, or 1.5-fold between the end of the growth period relative to the beginning of the growthperiod. In particular embodiments, the engineered cells do not proliferate in the absence of cytokine stimulation, growth factor stimulation, or antigen stimulation.

[0177] (VII) Cell Activating Culture Conditions. Cell populations can be incubated in a cultureinitiating composition to expand cell populations. The incubation can be carried out in a culture vessel, such as a bag, cell culture plate, flask, chamber, chromatography column, cross-linked gel, cross-linked polymer, column, culture dish, hollow fiber, microtiter plate, silica-coated glass plate, tube, tubing set, well, vial, or other container for culture or cultivating cells.

[0178] In particular embodiments, the cell population can be incubated in the culture-initiating composition before or after genetic engineering the cell populations. In particular embodiments, the incubation can be carried out for 1 day to 6 days, 1 day to 5 days, 1 day to 4 days, 1 day to 3 days, 1 day to 2 days, or 1 day before genetically engineering the cell populations. In particular embodiments, the incubation can be carried out for 1 day to 6 days, 1 day to 5 days, 1 day to 4 days, 1 day to 3 days, 1 day to 2 days, or 1 day after genetically engineering the cell populations. In particular embodiments, the incubation can be carried out at the same time as genetically engineering the cell populations.

[0179] Culture conditions can include one or more of particular media, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and / or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells.

[0180] In some aspects, incubation is carried out in accordance with techniques such as those described in US 6,040,1 77, Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakura et al. (2012) Blood.1 :72-82, and / or Wang et al. (2012) J Immunother. 35(9): 689-701.

[0181] Exemplary culture media for culturing T cells include (i) RPMI supplemented with non- essential amino acids, sodium pyruvate, and penicillin / streptomycin; (ii) RPMI with HEPES, 5- 15% human serum, 1-3% L-Glutamine, 0.5-1.5% penicillin / streptomycin, and 0.25x10-4 - 0.75x10-4 M p-MercaptoEthanol; (iii) RPMI-1640 supplemented with 10% fetal bovine serum (FBS), 2mM L-glutamine, 10mM HEPES, 100 U / ml penicillin and 100 m / mL streptomycin; (iv) DMEM medium supplemented with 10% FBS, 2mM L-glutamine, 10mM HEPES, 100 U / ml penicillin and 100 m / mL streptomycin; and (v) X-Vivo 15 medium (Lonza, Walkersville, MD) supplemented with 5% human AB serum (Gemcell, West Sacramento, CA), 1% HEPES (Gibco, Grand Island, NY), 1% Pen-Strep (Gibco), 1% GlutaMax (Gibco), and 2% N-acetyl cysteine (Sigma-Aldrich, St. Louis, MO). T cell culture media are also commercially available from Hyclone (Logan, UT). Additional T cell activating components that can be added to such culture media are described in more detail below.

[0182] In some embodiments, the T cells are expanded by adding to the culture-initiating composition feeder cells, such as non-dividing peripheral blood mononuclear cells (PBMC), (e.g., such that the resulting population of cells contains at least 5, 10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in the initial population to be expanded); and incubating the culture (e.g., for a time sufficient to expand the numbers of T cells). In some aspects, the non-dividing feeder cells can include gamma-irradiated PBMC feeder cells. In some embodiments, the PBMC are irradiated with gamma rays in the range of 3000 to 3600 rads to prevent cell division. In some aspects, the feeder cells are added to culture medium prior to the addition of the populations of T cells.

[0183] Optionally, the incubation may further include adding non-dividing EBV-transformed lymphoblastoid cells (LCL) as feeder cells. LCL can be irradiated with gamma rays in the range of 6000 to 10,000 rads. The LCL feeder cells in some aspects is provided in any suitable amount, such as a ratio of LCL feeder cells to initial T lymphocytes of at least 10: 1 .

[0184] In some embodiments, the stimulating conditions include temperature suitable for the growth of human T lymphocytes, for example, at least 25°C, at least 30°C, or 37°C.

[0185] The activating culture conditions for T cells include conditions whereby T cells of the culture-initiating composition proliferate or expand. T cell activating conditions can include one or more cytokines, for example, interleukin (IL)-2, IL-7, IL-15 and / or IL-21. IL-2 can be included at a range of 10 - 100 ng / ml (e.g., 40, 50, or 60 ng / ml). IL-7, IL-15, and / or IL-21 can be individually included at a range of 0.1 - 50 ng / ml (e.g., 5, 10, or 15 ng / ml). Particular embodiments utilize IL- 2 at 50 ng / ml. Particular embodiments utilize, IL-7, IL-15 and IL-21 individually included at 10 ng / ml.

[0186] In particular embodiments, T cell activating culture conditions can include T cell stimulating epitopes. T cell stimulating epitopes include CD3, CD27, CD2, CD4, CD5, CD7, CD8, CD28, CD30, CD40, CD56, CD83, CD90, CD95, 4-1 BB (CD 137), B7-H3, CTLA-4, Frizzled-1 (FZD1), FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, HVEM, ICOS, IL-1 R, LAT, LFA- 1 , LIGHT, MHCI, MHCII, NKG2D, 0X40, ROR2 and RTK.

[0187] CD3 is a primary signal transduction element of T cell receptors. As indicated previously, CD3 is expressed on all mature T cells. In particular embodiments, the CD3 stimulating molecule (i.e., CD3 binding domain) can be derived from the OKT3 antibody (see US 5,929,212; US 4,361 ,549; ATCC® CRL-8001 ™; and Arakawa et al., J. Biochem. 120, 657-662 (1996)), the 20G6-F3 antibody, the 4B4-D7 antibody, the 4E7-C9, or the 18F5-H10 antibody.

[0188] In particular embodiments, CD3 stimulating molecules can be included within culture media at a concentration of at least 0.25 or 0.5 ng / ml or at a concentration of 2.5 - 10 pg / ml.Particular embodiments utilize a CD3 stimulating molecule (e.g., OKT3) at 5 pg / ml.

[0189] In particular embodiments, activating molecules associated with avi-tags can be biotinylated and bound to streptavidin beads. This approach can be used to create, for example, a removable T cell epitope stimulating activation system.

[0190] An exemplary binding domain for CD28 can include or be derived from TGN1412, CD80, CD86 or the 9D7 antibody. Additional antibodies that bind CD28 include 9.3, KOLT-2, 15E8, 248.23.2, EX5.3D10, and CD28.3 (deposited as a synthetic single chain Fv construct under GenBank Accession No. AF451974.1 ; see also Vanhove et al., BLOOD, 15 Jul. 2003, Vol. 102, No. 2, pages 564-570). Further, 1YJD provides a crystal structure of human CD28 in complex with the Fab fragment of a mitogenic antibody (5.11A1). In particular embodiments, antibodies that do not compete with 9D7 are selected.

[0191] 4-1 BB binding domains can be derived from LOB12, lgG2a, LOB12.3, or lgG1 as described in Taraban et al. Eur J Immunol. 2002 December; 32(12):3617-27. In particular embodiments a 4-1 BB binding domain is derived from a monoclonal antibody described in US 9,382,328. Additional 4-1 BB binding domains are described in US 6,569,997, US 6,303,121 , and Mittler et al. Immunol Res. 2004; 29(1 -3): 197-208.

[0192] 0X40 (CD134) and / or ICOS activation may also be used. 0X40 binding domains are described in US20100196359, US 20150307617, WO 2015 / 153513, WO2013 / 038191 and Melero et al. Clin Cancer Res. 2013 Mar. 1 ; 19(5): 1044-53. Exemplary binding domains that can bind and activate ICOS are described in e.g., US20080279851 and Deng et al. Hybrid Hybridomics. 2004 June; 23(3): 176-82.

[0193] When in soluble form, T-cell activating agents can be coupled with another molecule, such as polyethylene glycol (PEG) molecule. Any suitable PEG molecule can be used. Typically, PEG molecules up to a molecular weight of 1000 Da are soluble in water or culture media. In some cases, such PEG based reagent can be prepared using commercially available activated PEG molecules (for example, PEG-NHS derivatives available from NOF North America Corporation, Irvine, Calif., USA, or activated PEG derivatives available from Creative PEGWorks, Chapel Hills, N.C., USA).

[0194] In particular embodiments, cell stimulating agents are immobilized on a solid phase within the culture media. In particular embodiments, the solid phase is a surface of the culture vessel (e.g., bag, cell culture plate, chamber, chromatography column, cross-linked gel, cross-linked polymer, column, culture dish, hollow fiber, microtiter plate, silica-coated glass plate, tube, tubing set, well, vial, other structure or container for culture or cultivation of cells).

[0195] In particular embodiments, a solid phase can be added to a culture media. Such solidphases can include, for example, beads, hollow fibers, resins, membranes, and polymers.

[0196] Exemplary beads include magnetic beads, polymeric beads, and resin beads (e.g., Strep- Tactin® Sepharose, Strep-Tactin® Superflow, and Strep-Tactin® MacroPrep IBA GmbH, Gottingen)). Anti-CD3 / anti-CD28 beads are commercially available reagents for T cell expansion (Invitrogen). These beads are uniform, 4.5 pm superparamagnetic, sterile, non-pyrogenic polystyrene beads coated with a mixture of affinity purified monoclonal antibodies against the CD3 and CD28 cell surface molecules on human T cells. Hollow fibers are available from TerumoBCT Inc. (Lakewood, Colo., USA). Resins include metal affinity chromatography (IMAC) resins (e.g., TALON® resins (Westburg, Leusden)). Membranes include paper as well as the membrane substrate of a chromatography matrix (e.g., a nitrocellulose membrane or a polyvinylidene difluoride (PVDF) membrane).

[0197] Exemplary polymers include polysaccharides, such as polysaccharide matrices. Such matrices include agarose gels (e.g., Superflow™ agarose or a Sepharose® material such as Superflow™ Sepharose® that are commercially available in different bead and pore sizes) or a gel of crosslinked dextran(s). A further illustrative example is a particulate cross-linked agarose matrix, to which dextran is covalently bonded, that is commercially available (in various bead sizes and with various pore sizes) as Sephadex® or Superdex®, both available from GE Healthcare.

[0198] Synthetic polymers that may be used include polyacrylamide, polymethacrylate, a copolymer of polysaccharide and agarose (e.g. a polyacrylamide / agarose composite) or a polysaccharide and N,N'-methylenebisacrylamide. An example of a copolymer of a dextran andN,N'-methylenebisacrylamide is the Sephacryl® (Pharmacia Fine Chemicals, Inc., Piscataway, NJ) series of materials.

[0199] Particular embodiments may utilize silica particles coupled to a synthetic or to a natural polymer, such as polysaccharide grafted silica, polyvinylpyrrolidone grafted silica, polyethylene oxide grafted silica, poly(2-hydroxyethylaspartamide) silica and poly(N-isopropylacrylamide) grafted silica.

[0200] Cell activating agents can be immobilized to solid phases through covalent bonds or can be reversibly immobilized through non-covalent attachments.

[0201] In particular embodiments, a T-cell activating culture media includes a FACS-sorted T cell population cultured within RPMI with HEPES, 5-15% human serum, 1-3% L-Glutamine, 0.5-1.5% Pen / strep, 0.25x10'4- 0.75x10'4M p-MercaptoEthanol, with IL-7, IL-15 and IL-21 individually included at 5-15 (e.g., 10) ng / ml. The culture is carried out on a flat-bottom well plate with 0.1-O.5x106plated cells / well. On Day 3 post activation cells are transferred to a tissue culture (TC)- treated plate.

[0202] In particular embodiments, a T-cell activating culture media includes a FACS-sorted CD8+ T population cultured within RPMI with HEPES, 10% human serum, 2% L-Glutamine, 1% Pen / strep, 0.5x1 O’4M [3-MercaptoEthanol, with IL-7, IL-15 and IL-21 individually included at 5-15 (e.g., 10) ng / ml. The culture is carried out on a flat-bottom non-tissue culture-treated 96 / 48-well plate with 0.1 -0.5x106plated cells / well. On Day 3 post activation cells are transferred to TC- treated plate.

[0203] Culture conditions for HSC / HSP can include expansion with a Notch agonist (see, e.g., US 7,399,633; US 5,780,300; US 5,648,464; US 5,849,869; and US 5,856,441 and growth factors present in the culture condition as follows: 25-300 ng / ml SCF, 25-300 ng / ml Flt-3L, 25-100 ng / ml TPO, 25-100 ng / ml IL-6 and 10 ng / ml IL-3. In more specific embodiments, 50, 100, or 200 ng / ml SCF; 50, 100, or 200 ng / ml of Flt-3L; 50 or 100 ng / ml TPO; 50 or 100 ng / ml IL-6; and 10 ng / ml IL-3 can be used.

[0204] (VIII) Ex Vivo Manufactured Cell Formulations. In particular embodiments, genetically modified cells can be harvested from a culture medium and washed and concentrated into a carrier in a therapeutically-effective amount. Exemplary carriers include saline, buffered saline, physiological saline, water, Hanks' solution, Ringer's solution, Normosol-R (Abbott Labs), PLASMA-LYTE A® (Baxter Laboratories, Inc., Morton Grove, IL), and combinations thereof.

[0205] In particular embodiments, carriers can be supplemented with human serum albumin (HSA) or other human serum components or fetal bovine serum. In particular embodiments, a carrier for infusion includes buffered saline with 5% HSA or dextrose. Additional isotonic agents include polyhydric sugar alcohols including trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, or mannitol.

[0206] Carriers can include buffering agents, such as citrate buffers, succinate buffers, tartrate buffers, fumarate buffers, gluconate buffers, oxalate buffers, lactate buffers, acetate buffers, phosphate buffers, histidine buffers, and / or trimethylamine salts.

[0207] Stabilizers refer to a broad category of excipients which can range in function from a bulking agent to an additive which helps to prevent cell adherence to container walls. Typical stabilizers can include polyhydric sugar alcohols; amino acids, such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, and threonine; organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol, and cyclitols, such as inositol; PEG; amino acid polymers; sulfur-containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, alpha-monothioglycerol, and sodium thiosulfate; low molecular weight polypeptides (i.e., <10 residues); proteins such as HSA, bovine serum albumin, gelatin orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides such as xylose, mannose, fructose and glucose; disaccharides such as lactose, maltose and sucrose; trisaccharides such as raffinose, and polysaccharides such as dextran.

[0208] Where necessary or beneficial, formulations can include a local anesthetic such as lidocaine to ease pain at a site of injection.

[0209] Exemplary preservatives include phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, octadecyldimethylbenzyl ammonium chloride, benzalkonium halides, hexamethonium chloride, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, and 3-pentanol.

[0210] Therapeutically effective amounts of cells within formulations can be greater than 102cells, greater than 103cells, greater than 104cells, greater than 105cells, greater than 106cells, greater than 107cells, greater than 108cells, greater than 109cells, greater than 1010cells, or greater than 1011.

[0211] In formulations disclosed herein, cells are generally in a volume of a liter or less, 500 ml or less, 250 ml or less or 100 ml or less. Hence the density of administered cells is typically greater than 104cells / ml, 107cells / ml or 108cells / ml.

[0212] In particular embodiments, formulations can include at least one genetically modified cell type (e.g., modified T cells, NK cells, or stem cells). Formulations can include different types of genetically-modified cells (e.g., T cells, NK cells, and / or stem cells in combination).

[0213] Different types of genetically-modified cells or cell subsets (e.g., modified T cells, NK cells, and / or stem cells) can be provided in different ratios e.g., a 1 :1 :1 ratio, 2:1 :1 ratio, 1 :2:1 ratio, 1 :1 :2 ratio, 5:1 :1 ratio, 1 :5:1 ratio, 1 :1:5 ratio, 10:1 :1 ratio, 1 :10:1 ratio, 1 :1 :10 ratio, 2:2:1 ratio, 1 :2:2 ratio, 2:1 :2 ratio, 5:5:1 ratio, 1:5:5 ratio, 5:1 :5 ratio, 10:10:1 ratio, 1:10:10 ratio, 10:1 :10 ratio, etc. These ratios can also apply to numbers of cells expressing the same or different expressed molecule (e.g., recombinant receptor and / or chemokine receptor) components. If only two of the cell types are combined or only 2 combinations of expressed molecule components are included within a formulation, the ratio can include any 2-number combination that can be created from the 3 number combinations provided above. In embodiments, the combined cell populations are tested for efficacy and / or cell proliferation in vitro, in vivo and / or ex vivo, and the ratio of cells that provides for efficacy and / or proliferation of cells is selected. Particular embodiments include genetically-modified cells expressing different isoforms of CXCR3.

[0214] In particular embodiments, cells expressing a recombinant receptor to cells expressing a recombinant receptor and chemokine receptor can be provided in different ratios e.g., a 1 :2 ratio, a 1 :3 ratio, a 1 :4 ratio, a 1 :5 ratio, a 1 :6 ratio, a 1 :7 ratio, a 1 :10 ratio, etc. In particularembodiments, cells expressing a chemokine receptor to cells expressing a recombinant receptor and chemokine receptor can be provided in different ratios e.g., a 1 :2 ratio, a 1 :3 ratio, a 1 :4 ratio, a 1 :5 ratio, a 1 :6 ratio, a 1 :7 ratio, a 1 :10 ratio, etc. In particular embodiments, cells expressing a chemokine receptor to cells expressing a recombinant receptor to cells expressing a recombinant receptor and chemokine receptor can be provided in different ratios e.g., a 1 :1 :1 ratio, 2:1:1 ratio, 1 :2:1 ratio, 1 :1 :2 ratio, 5:1 :1 ratio, 1 :5:1 ratio, 1 :1 :5 ratio, 10:1 :1 ratio, 1:10:1 ratio, 1 :1 :10 ratio, 2:2:1 ratio, 1 :2:2 ratio, 2:1 :2 ratio, 5:5:1 ratio, 1:5:5 ratio, 5:1 :5 ratio, 10:10:1 ratio, 1 :10:10 ratio, 10:1:10 ratio, etc.

[0215] The cell-based formulations disclosed herein can be prepared for administration by, e.g., injection, infusion, perfusion, or lavage. The formulations can further be formulated for intravenous (IV), intracerebroventricular (ICV), intradermal, intraarterial, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, intrathecal, intratumoral, intramuscular, intravesicular, bone marrow, and / or subcutaneous injection. In particular embodiments, compositions and / or formulations are administered by ICV injection. In particular embodiments, compositions and / or formulations are administered by IV injection.

[0216] (IX) Targeted Viral Vectors & Nanoparticles for In Vivo Cell Modification. Targeted viral vectors and / or nanoparticles can also be used to genetically-modify immune cells in vivo or ex vivo. Viral vectors that can be used to deliver artificial expression constructs (encoding recombinant receptor and / or CXCR3) to cells are described elsewhere herein, and numerous targeted (e.g., pseudotyped) viral vectors are known in the art.

[0217] Exemplary cell-targeted nanoparticles include a cell targeting ligand (e.g., CD3, CD4, CD8, CD34) on the surface of the nanoparticle wherein the cell targeting ligand results in selective uptake of the nanoparticle by a selected cell type. The nanoparticle then delivers gene modifying components that result in expression of the recombinant receptor and CXCR3.

[0218] Exemplary nanoparticles include liposomes (microscopic vesicles including at least one concentric lipid bilayer surrounding an aqueous core), liposomal nanoparticles (a liposome structure used to encapsulate another smaller nanoparticle within its core); and lipid nanoparticles (liposome-like structures that lack the continuous lipid bilayer characteristic of liposomes). Other polymer-based nanoparticles can also be used as well as porous nanoparticles constructed from any material capable of forming a porous network. Exemplary materials include metals, transition metals and metalloids (e.g., lithium, magnesium, zinc, aluminum and silica).

[0219] Therapeutically effective amounts of vectors and / or nanoparticles within formulations can range from 0.1 to 5 pg / kg or from 0.5 to 1 pg / kg. In other examples, a dose can include 1 pg / kg, 30 pg / kg, 90 pg / kg, 150 pg / kg, 500 pg / kg, 750 pg / kg, 0.1 to 5 mg / kg or from 0.5 to 1 mg / kg. Inother examples, a dose can include 1 mg / kg, 10 mg / kg, 30 mg / kg, 50 mg / kg, 70 mg / kg, 100 mg / kg, 300 mg / kg, 500 mg / kg, 700 mg / kg, 1000 mg / kg or more.

[0220] (X) Methods of Use. Methods disclosed herein include treating subjects (humans, nonhuman primates, veterinary animals (dogs, cats, reptiles, birds, etc.) livestock (horses, cattle, goats, pigs, chickens, etc.) and research animals (monkeys, rats, mice, fish, etc.)) with formulations disclosed herein. Treating subjects includes delivering therapeutically effective amounts. Therapeutically effective amounts include those that provide effective amounts, prophylactic treatments and / or therapeutic treatments.

[0221] In particular embodiments, the subject is a pediatric patient. In particular embodiments, a pediatric patient is 0 to 2 years of age. In particular embodiments, a pediatric patient is 0 or 1 years of age. In particular embodiments, the subject is a child. In particular embodiments, a child is 2 to 12 years of age. In particular embodiments, a child is 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 years of age. In particular embodiments, the subject is an adolescent. In particular embodiments, an adolescent is 12 to 18 years of age. In particular embodiments, an adolescent is 12, 13, 14, 15, 16, or 17 years of age. In particular embodiments, the subject is an adult. In particular embodiments, an adult is over 18 years of age. In particular embodiments, an adult is 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 years of age.

[0222] An "effective amount" is the amount of a formulation necessary to result in a desired physiological change in the subject. For example, an effective amount can provide an immunogenic anti-cancer effect. Effective amounts are often administered for research purposes. Effective amounts disclosed herein can cause a statistically significant effect in an animal model or in vitro assay relevant to the assessment of a cancer’s development or progression. An immunogenic formulation can be provided in an effective amount, wherein the effective amount stimulates an immune response.

[0223] A "prophylactic treatment" includes a treatment administered to a subject who does not display signs or symptoms of a cancer or displays only early signs or symptoms of a cancer such that treatment is administered for the purpose of diminishing or decreasing the risk of developing the cancer further. Thus, a prophylactic treatment functions as a preventative treatment against a B7-H3-expressing cancer. In particular embodiments, prophylactic treatments reduce, delay, or prevent metastasis from a primary a cancer tumor site from occurring.

[0224] A "therapeutic treatment" includes a treatment administered to a subject who displays symptoms or signs of a cancer and is administered to the subject 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 sideeffects of the cancer.

[0225] 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.

[0226] 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 subject life, reduced cancer-associated pain, and / or reduced relapse or re-occurrence of cancer following treatment.

[0227] 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.

[0228] In particular embodiments, therapeutically effective amounts induce an immune response. The immune response can be against a B7-H3-expressing cancer cell.

[0229] 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).

[0230] Examples of B7-H3-related disorders that can be treated with formulations and / or compositions disclosed herein include diffuse intrinsic pontine glioma (DIPG), diffuse midline glioma (DMG), glioblastoma, prostate cancer, renal cell carcinoma, urothelial cell carcinoma, ovarian cancer, osteosarcoma, neuroblastoma, mesothelioma, colorectal cancer, gastric cancer, breast cancer, small cell lung cancer, non-small-cell lung cancer (NSCLC), and pancreatic cancer.

[0231] 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 drooping parts of face, increase in strength in extremities, or decrease in headaches.

[0232] 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.

[0233] For administration, therapeutically effective amounts (also referred to herein as doses) can be initially estimated based on results from in vitro assays and / or animal model studies. Such information can be used to more accurately determine useful doses in subjects of interest. The actual dose amount administered to a particular subject can be determined by a physician, veterinarian or researcher taking into account parameters such as physical and physiological factors including target, body weight, severity of condition, type of cancer, stage of cancer, previous or concurrent therapeutic interventions, idiopathy of the subject and route of administration.

[0234] Therapeutically effective amounts of cell-based formulations can include 104to 109cells / kg body weight, or 103to 1011cells / kg body weight. Therapeutically effective amounts to administer can include greater than 102cells, greater than 103cells, greater than 104cells, greater than 105cells, greater than 106cells, greater than 107cells, greater than 108cells, greater than 109cells, greater than 1010cells, or greater than 1011.

[0235] Therapeutically effective amounts of vectors and / or nanoparticles within formulations and / or compositions can range from 0.1 to 5 pg / kg or from 0.5 to 1 pg / kg. In other examples, a dose can include 1 pg / kg, 30 pg / kg, 90 pg / kg, 150 pg / kg, 500 pg / kg, 750 pg / kg, 0.1 to 5 mg / kg or from 0.5 to 1 mg / kg. In other examples, a dose can include 1 mg / kg, 10 mg / kg, 30 mg / kg, 50 mg / kg, 70 mg / kg, 100 mg / kg, 300 mg / kg, 500 mg / kg, 700 mg / kg, 1000 mg / kg or more.

[0236] Therapeutically effective amounts can be achieved by administering single or multiple doses during the course of a treatment regimen (e.g., daily, every other day, every 3 days, every 4 days, every 5 days, every 6 days, weekly, every 2 weeks, every 3 weeks, monthly, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months or yearly). In particular embodiments, the treatment protocol may be dictated by a clinical trial protocol or an FDA- approved treatment protocol.

[0237] Therapeutically effective amounts can be administered by, e.g., injection, infusion, perfusion, or lavage. Routes of administration can include bolus intravenous (IV), intracerebroventricular (ICV), intradermal, intraarterial, intraparenteral, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, topical, intrathecal,intratumoral, intramuscular, intravesicular, and / or subcutaneous administration. In particular embodiments, therapeutically effective amounts are administered by ICV injection. In particular embodiments, therapeutically effective amounts are administered by IV injection.

[0238] In certain embodiments, formulations and / or compositions 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 chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAM PATH, anti-CD3 antibodies or other antibody therapies, cytoxin, fludaribine, cyclosporin, FK506, rapamycin, mycoplienolic acid, steroids, FR901228, cytokines, and irradiation.

[0239] In certain embodiments, formulations and / or compositions 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.

[0240] 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 to 1 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.

[0241] In additional embodiments, the compositions and / or 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 anti-inflammatory 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.

[0242] In certain embodiments, the compositions and / or 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; glycoprotein 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-alpha or TNF-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.

[0243] (XI) Kits. The current disclosure also includes kits. Kits can include various components to practice methods disclosed herein. For example, depending on the aspect of the methodspracticed, kits could include one or more of nucleic acids encoding a recombinant receptor and / or CXCR3 disclosed herein; nucleic acids encoding a recombinant receptor; nucleic acids encoding second-generation 4-1 BB chimeric antigen receptors (CAR); lentiviral artificial expression construct; a nucleic acid encoding a CXCR3 isoform; a protein or encoding sequence as set forth in FIG. 20; a nucleic acid encoding an scFv; a nucleic acid encoding a VL; a nucleic acid encoding a VH; a nucleic acid encoding a transmembrane domain; a nucleic acid encoding EGFRt; cells (e.g., immune cells, T-cells, CD4 T cells, CD8 T cells, B cells, natural killer (NK) cells, NK-T cells, monocytes / macrophages, lymphocytes, hematopoietic stem cells (HSCs), hematopoietic progenitor cells (HPC), a mixture of HSC and HPC (i.e., HSPC), untransduced T cells, and / or anti-B7-H3 CAR T cells expressing CXCR3); cell lines (e.g., A172, SW1088, H4, U118-MG, U87- MG, PBT-22FH, DIPG tumor cells (PBT-29FH), glioma cancer cell lines); tissue samples (e.g., peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, or other organ, and / or cells derived therefrom); genetic expression components (e.g., genes for expression provided by vectors (e.g., lentiviral vector, retroviral vector), CRISPR components, ZFNs, TALENs, MegaTALs, targeted viral vectors and / or nanoparticles); cell formulation or activation components (e.g., saline, buffered saline, phosphate buffered saline (PBS); biocompatible buffers such as, Ca++ / Mg++ free PBS; physiological saline, water, Hanks' solution, Ringer's solution, T cell stimulating epitopes (e.g., anti-CD3 / anti-CD28 conjugated beads; OKT3, TGN1412), culture-initiating compositions, RPMI, non-essential amino acids, sodium pyruvate, penicillin / streptomycin, non-dividing EBV-transformed lymphoblastoid cells (LCL), IL-21 , human serum albumin (HSA) or other human serum components orfetai bovine serum, dextrose, Stabilizers, preservatives); combination therapy components (e.g., local anesthetics, chemotherapeutic agents, immunosuppressive agents, anti-inflammatory agents); an antibody tagged with a fluorescent molecule; PCR amplification sequences; cytokines (e.g., IL-2, IL-7, IL-15, IL-21); culture vessels; reference levels, animal models; primer pairs; GAPDH; IFN-y enzyme-linked immunosorbent assay (ELISA); culture plates; etc.

[0244] The Exemplary Embodiments and Example below are included to demonstrate particular embodiments of the disclosure. Those of ordinary skill in the art will 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.

[0245] (XII) Exemplary Embodiments.1. An artificial expression construct including (i) a sequence encoding a CXCR3 isoformselected from CXCR3-A, CXCR3-B, or CXCR3-alt; and (ii) a sequence encoding a cancer antigen-binding recombinant receptor. The artificial expression construct of embodiment 1 , further including a promoter. The artificial expression construct of embodiment 2, wherein the promoter includes an EF1 promoter. The artificial expression construct of any of embodiments 1-3, wherein the CXCR3-A includes the sequence of SEQ ID NO: 2 or a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 2. The artificial expression construct of any of embodiments 1-4, wherein the CXCR3-A is encoded by the sequence of SEQ ID NO: 3 or a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 3. The artificial expression construct of any of embodiments 1-3, wherein the CXCR3-B includes the sequence of SEQ ID NO: 4 or a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 4. The artificial expression construct of any of embodiments 1-3 or 6, wherein the CXCR3-B is encoded by the sequence of SEQ ID NO: 5 or a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 5. The artificial expression construct of any of embodiments 1-3, wherein the CXCR3-alt includes the sequence of SEQ ID NO: 6 or a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 6. The artificial expression construct of any of embodiments 1-3 or 8, wherein the CXCR3- alt is encoded by the sequence of SEQ ID NO: 7 or a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 7. The artificial expression construct of any of embodiments 1-9, wherein the cancer antigenbinding recombinant receptor includes, when expressed by a cell, an extracellular component including a cancer antigen-binding domain; an intracellular component; and a transmembrane domain linking the extracellular component to the intracellular component. The artificial expression construct of any of embodiments 1-10, wherein the cancer antigen-binding recombinant receptor includes a chimeric antigen receptor (CAR), an engineered T cell receptor (eTCR), or a hybrid thereof. The artificial expression construct of embodiments 10 or 11 , wherein the cancer antigenbinding domain includes a B7-H3 binding domain. The artificial expression construct of embodiment 12, wherein the B7-H3 binding domainincludes a complementarity determining region (CDR) set including a variable heavy chain including a CDR heavy (H)1 , a CDRH2, and a CDRH3 and a variable light chain including a CDR light (L)1 , CDRL2, and CDRL3, wherein: the CDRH1 includes the sequence of SEQ ID NO: 8, the CDRH2 includes the sequence of SEQ ID NO: 9, the CDRH3 includes the sequence of SEQ ID NO: 10, the CDRL1 includes the sequence of SEQ ID NO: 11, the CDRL2 includes the sequence of SEQ ID NO: 12, and the CDRL3 includes the sequence of SEQ ID NO: 13, according to Kabat; the CDRH1 includes the sequence of SEQ ID NO: 14, the CDRH2 includes the sequence of SEQ ID NO: 15, the CDRH3 includes the sequence of SEQ ID NO: 10, the CDRL1 includes the sequence of SEQ ID NO: 11, the CDRL2 includes the sequence of SEQ ID NO: 12, and the CDRL3 includes the sequence of SEQ ID NO: 13, according to Chothia; the CDRH1 includes the sequence of SEQ ID NO: 16, the CDRH2 includes the sequence of SEQ ID NO: 17, the CDRH3 includes the sequence of SEQ ID NO: 18, the CDRL1 includes the sequence of SEQ ID NO: 19, the CDRL2 includes the sequence as SAS, and the CDRL3 includes the sequence of SEQ ID NO: 13, according to IMGT; the CDRH1 includes the sequence of SEQ ID NO: 20, the CDRH2 includes the sequence of SEQ ID NO: 21 , the CDRH3 includes the sequence of SEQ ID NO: 18, the CDRL1 includes the sequence of SEQ ID NO: 11, the CDRL2 includes the sequence of SEQ ID NO: 22, and the CDRL3 includes the sequence of SEQ ID NO: 13, according to North; or the CDRH1 includes the sequence of SEQ ID NO: 23, the CDRH2 includes the sequence of SEQ ID NO: 24, the CDRH3 includes the sequence of SEQ ID NO: 25, the CDRL1 includes the sequence of SEQ ID NO: 26, the CDRL2 includes the sequence of SEQ ID NO: 27, and the CDRL3 includes the sequence of SEQ ID NO: 28, according to Contact. The artificial expression construct of embodiments 12 or 13, wherein the B7-H3 binding domain includes a variable heavy chain including a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 29 and a variable light chain including a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 30. The artificial expression construct of any of embodiments 12-14, wherein the B7-H3 binding domain includes a variable heavy chain including the sequence of SEQ ID NO: 29 and a variable light chain including the sequence of SEQ ID NO: 30. The artificial expression construct of any of embodiments 12-15, wherein the B7-H3 binding domain includes a variable heavy chain encoded by a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 31 and a variable light chain encoded by a sequence having at least 95% sequence identity to the sequence of SEQID NO: 32. The artificial expression construct of any of embodiments 12-16, wherein the B7-H3 binding domain includes a variable heavy chain encoded by the sequence of SEQ ID NO: 31 and a variable light chain encoded by the sequence of SEQ ID NO: 32. The artificial expression construct of any of embodiments 12-17, wherein the B7-H3 binding domain includes an scFv. The artificial expression construct of embodiment 18, wherein the scFv includes a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 33 or SEQ ID NO: 35. The artificial expression construct of any of embodiments 18 or 19, wherein the scFv includes the sequence of SEQ ID NO: 33 or SEQ ID NO: 35. The artificial expression construct of any of embodiments 18-20, wherein the scFv is encoded by a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 34 or SEQ ID NO: 36. The artificial expression construct of any of embodiments 18-21, wherein the scFv is encoded by the sequence of SEQ ID NO: 34 or SEQ ID NO: 36. The artificial expression construct of any of embodiments 10-22, wherein the extracellular component further includes a spacer. The artificial expression construct of embodiment 23, wherein the spacer includes an lgG4 hinge and lgG4 CH3 domain. The artificial expression construct of embodiment 24, wherein the lgG4 hinge includes the sequence of SEQ ID NO: 38 or a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 38. The artificial expression construct of embodiments 24 or 25, wherein the lgG4 hinge is encoded by the sequence of any of SEQ ID NOs: 39-42 or a sequence having at least 95% sequence identity to the sequence of any of SEQ ID NOs: 39-42. The artificial expression construct of any of embodiments 24-26, wherein the lgG4 CH3 domain includes the sequence of SEQ ID NO: 46 or a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 46. The artificial expression construct of any of embodiments 24-27, wherein the lgG4 CH3 domain is encoded by the sequence of any of SEQ ID NOs: 47-49 or a sequence having at least 95% sequence identity to the sequence of any of SEQ ID NOs: 47-49. The artificial expression construct of any of embodiments 10-28, wherein the intracellular component includes all or a portion of a CD3 signaling domain; all or a portion of a 4-1 BBsignaling domain; or all or a portion of a CD3£ signaling domain and all or a portion of a 4-1 BB signaling domain. The artificial expression construct of any of embodiments 10-29, wherein the intracellular component includes all or a portion of a CD3^ signaling domain and all or a portion of a 4- 1 BB signaling domain. The artificial expression construct of embodiments 29 or 30, wherein the 4-1 BB signaling domain includes a sequence of any of SEQ ID NOs: 57, 58, or 59; or a sequence having at least 95% sequence identity to the sequence of any of SEQ ID NOs: 57, 58, or 59. The artificial expression construct of any of embodiments 29-31 , wherein the 4-1 BB signaling domain is encoded by a sequence of any of SEQ ID NOs: 60, 61 , or 62; or a sequence having at least 95% sequence identity to the sequence of any of SEQ ID NOs: 60, 61 , or 62. The artificial expression construct of any of embodiments 29-32, wherein the CD3 signaling domain includes a sequence of any of SEQ ID NOs: 63, 64, or 65; or a sequence having at least 95% sequence identity to the sequence of any of SEQ ID NOs: 63, 64, or 65. The artificial expression construct of any of embodiments 29-33, wherein the CD3 signaling domain is encoded by a sequence of SEQ ID NO: 66 or SEQ ID NO: 67; or a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 66 or SEQ ID NO: 67. The artificial expression construct of any of embodiments 10-34, wherein the transmembrane domain includes a CD28 transmembrane domain. The artificial expression construct of embodiment 35, wherein the CD28 transmembrane domain includes a sequence of any of SEQ ID NOs: 50, 51 , or 52; or a sequence having at least 95% sequence identity to the sequence of any of SEQ ID NOs: 50, 51, or 52. The artificial expression construct of embodiments 35 or 36, wherein the CD28 transmembrane domain is encoded by a sequence of any of SEQ ID NOs: 53-56; or a sequence having at least 95% sequence identity to the sequence of any of SEQ ID NOs: 53-56. The artificial expression construct of any of embodiments 10-37, wherein the cancer antigen-binding domain includes the sequence of SEQ ID NO: 33, the intracellular component includes a CD3 signaling domain and a 4-1 BB signaling domain, and the transmembrane domain includes a CD28 transmembrane domain. The artificial expression construct of any of embodiments 1-38, wherein the cancerantigen-binding recombinant receptor includes the sequence of SEQ ID NO: 79 or a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 79. The artificial expression construct of any of embodiments 1-39, wherein the cancer antigen-binding recombinant receptor is encoded by the sequence of SEQ ID NO: 80 or a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 80. The artificial expression construct of any of embodiments 1-40, further including a selfcleaving polypeptide. The artificial expression construct of embodiment 41 , wherein the self-cleaving polypeptide is a porcine teschovirus-1 (P2A) self-cleaving polypeptide, Thosea asigna virus (T2A) self-cleaving polypeptide, equine rhinitis A virus (E2A) self-cleaving polypeptide, foot-and-mouth disease virus (F2A) self-cleaving polypeptide. The artificial expression construct of embodiments 41 or 42, wherein the self-cleaving polypeptide is a T2A self-cleaving polypeptide. The artificial expression construct of any of embodiments 1-43, further including a transduction marker. The artificial expression construct of embodiment 44, wherein the transduction marker includes a truncated epidermal growth factor receptor (EGFRt). The artificial expression construct of embodiment 45, wherein the EGFRt includes the sequence of SEQ ID NO: 75 or a sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 75. The artificial expression construct of embodiments 45 or 46, wherein the EGFRt is encoded by the sequence of SEQ ID NO: 76 or a sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 76. The artificial expression construct of any of embodiments 41-47, further including a second self-cleaving polypeptide. The artificial expression construct of embodiment 48, wherein the second self-cleaving polypeptide is P2A self-cleaving polypeptide, T2A self-cleaving polypeptide, E2A selfcleaving polypeptide, F2A self-cleaving polypeptide. The artificial expression construct of embodiments 48 or 49, wherein the self-cleaving polypeptide is a T2A self-cleaving polypeptide. The artificial expression construct of any of embodiments 1-50, including the sequence of any of SEQ ID NOs: 81 , 82, 83, 84, 85, or 86; or a sequence having at least 90% or at least 95% sequence identity to the sequence of any of SEQ ID NOs: 81 , 82, 83, 84, 85, or 86.The artificial expression construct of any of embodiments 1-51 , including the sequence of SEQ ID NO: 84 or a sequence having at least 90% or at least 95% sequence identity to the sequence of SEQ ID NO: 84. The artificial expression construct of any of embodiments 1-51 , including the sequence of SEQ ID NO: 85 or a sequence having at least 90% or at least 95% sequence identity to the sequence of SEQ ID NO: 85. The artificial expression construct of any of embodiments 1-51 , including the sequence of SEQ ID NO: 86 or a sequence having at least 90% or at least 95% sequence identity to the sequence of SEQ ID NO: 86. A system including a first sequence encoding a cancer antigen-binding recombinant receptor and a second sequence encoding a CXCR3 isoform selected from CXCR3-A, CXCR3-B, or CXCR3-alt. The system of embodiment 55, wherein the first sequence and second sequence are on a same artificial expression construct. The system of embodiment 55, wherein the first sequence and second sequence are on different artificial expression constructs. The system of any of embodiments 55-57, wherein the first sequence and second sequence are encapsulated within a same nanoparticle. The system of any of embodiments 55-58, wherein the first sequence and second sequence are encapsulated in different nanoparticles. The system of any of embodiments 55-59, wherein the first sequence and second sequence are introduced into a same cell. The system of any of embodiments 55-60, wherein the first sequence encodes the sequence of SEQ ID NO: 79 or a sequence having at least 90% or at least 95% sequence identity to the sequence of SEQ ID NO: 79. The system of any of embodiments 55-61 , wherein the first sequence includes the sequence of SEQ ID NO: 80 or a sequence having at least 90% or at least 95% sequence identity to the sequence of SEQ ID NO: 80. The system of any of embodiments 55-62, wherein the second sequence encodes the sequence of any of SEQ ID NOs: 2, 4, or 6; or a sequence having at least 90% or at least 95% sequence identity to the sequence of any of SEQ ID NOs: 2, 4, or 6. The system of any of embodiments 55-63, wherein the second sequence includes the sequence of any of SEQ ID NOs: 3, 5, or 7; or a sequence having at least 90% or at least 95% sequence identity to the sequence of any of SEQ ID NOs: 3, 5, or 7.The system of any of embodiments 55-64, including a sequence of any of SEQ ID NOs: 81 , 82, 83, 84, 85, or 86; or a sequence having at least 90% or at least 95% sequence identity to the sequence of any of SEQ ID NOs: 81 , 82, 83, 84, 85, or 86. A nanoparticle encapsulating the artificial expression construct of any of embodiments 1- 53, or the system of any of embodiments 55-65. A cell genetically modified to express the artificial expression construct of any of embodiments 1-54 or the system of any of embodiments 55-65. The cell of embodiment 67, wherein the cell is an autologous cell or an allogeneic cell in reference to a subject. The cell of embodiments 67 or 68, wherein the cell is in vivo or ex vivo. The cell of any of embodiments 67-69, wherein the cell is an immune cell. The cell of embodiment 70, wherein the immune cell is a lymphocyte. The cell of embodiments 70 or 71 , wherein the immune cell is a T cell, B-cell, natural killer (NK) cell, NK-T cell, a monocyte, or a macrophage. The cell of any of embodiments 70-72, wherein the immune cell is a T cell selected from a CD3+ T cell, a CD4+ T cell, a CD8+ T cell, a central memory T cell, an effector memory T cell, and / or a naive T cell. The cell of any of embodiments 70-73, wherein the immune cell is a CD8+ T cell. The cell of any of embodiments 70-73, wherein the immune cell is a CD4+ T cell. A population of cells genetically modified to express the artificial expression construct of any of embodiments 1-54 or the system of any of embodiments 55-65. The population of cells of embodiment 76, wherein the population of cells includes autologous cells or allogeneic cells in reference to a subject. The population of cells of embodiments 76 or 77, wherein the population is in vivo or ex vivo. The population of cells of any of embodiments 76-78, wherein the cell is an immune cell. The population of cells of embodiment 79, wherein the immune cell is a lymphocyte. The population of cells of embodiments 79 or 80, wherein the immune cell is a T cell, 13- cell, natural killer (NK) cell, NK-T cell, a monocyte, or a macrophage. The population of cells of any of embodiments 76-81 , wherein the population includes CD4+ T cells and / or CD8+ T cells. A formulation including (i) cells genetically modified to express the artificial expression construct of any of embodiments 1 -54 or the system of any of embodiments 55-65 and (ii) a pharmaceutically acceptable carrier.84. A method of genetically modifying an immune cell including contacting the immune cell with the artificial expression construct of any of embodiments 1-54 or the system of any of embodiments 55-65.85. A method of treating a B7H3-expressing cancer in a subject in need thereof including administering a therapeutically effective amount of an artificial expression construct of any of embodiments 1-54 and / or a system of any of embodiments 55-65 and / or a nanoparticle of embodiment 66, and / or a cell of any of embodiments 67-75 and / or a formulation of embodiment 83 to the subject thereby treating the cancer antigen-expressing cancer in the subject in need thereof.86. The method of embodiment 85, wherein the cancer antigen-expressing cancer includes B7-H3-expressing cancer.87. The method of embodiment 86, wherein the B7H3-expressing cancer includes a diffuse intrinsic pontine glioma, a diffuse midline glioma, a glioblastoma, a prostate cancer, a renal cell carcinoma, a urothelial cell carcinoma, an ovarian cancer, an osteosarcoma, a neuroblastoma, a mesothelioma, a colorectal cancer, a gastric cancer, a breast cancer, a small cell lung cancer, a non-small-cell lung cancer (NSCLC), or a pancreatic cancer.88. The method of embodiments 86 or 87, wherein the B7H3-expressing cancer includes a diffuse intrinsic pontine glioma or a diffuse midline glioma.89. The method of any of embodiments 86-88, wherein the subject is a pediatric patient.90. The method of any of embodiments 86-89, wherein the administering includes injecting a therapeutically effective amount to a living subject.91. The method of embodiment 90, wherein the injecting includes injecting intracerebroventricularly, intravenously, intradermally, intraarterially, intraparenterally, intranodally, intralymphatically, intravesically, intraperitoneally, intralesionally, intraprostatically, intravaginally, intrarectally, topically, intrathecally, intratumorally, intramuscularly, or subcutaneously.92. The method of embodiments 90 or 91 , wherein the injecting includes intracerebroventricular injection.93. The method of embodiment 90, wherein the injecting includes intravenous injection.

[0246] (XIII) Experimental Example 1. Engineered CXCR3-A Expression Enhances the Trafficking and Efficacy of Intracerebroventricularly Delivered B7-H3-targeting CAR T Cells against Diffuse Intrinsic Pontine Glioma.

[0247] Abstract. Diffuse intrinsic pontine glioma (DIPG) is an aggressive and universally fatal brainstem tumor that desperately needs more effective treatments. CAR T cell therapies for DI PGhave shown clinical evidence of tolerability and some activity, yet the benefit was not universal. As insufficient CAR T cell trafficking to the tumor is a major obstacle in solid tumors such as DIPG, the clinically tested anti-B7-H3-CAR T cells were engineered to overexpress the chemokine receptor CXCR3-A, which directs cell migration against the concentration gradient of its ligands, including CXCL10. The trials showed evidence that some CAR T cells would encounter tumor and produce CXCL10 following antigen-stimulation. Therefore, it was hypothesized that this CXCL10 will attract substantially more CXCR3-A-modified anti-B7-H3-CAR T cells to the DI PG tumor site, compared to unmodified CAR T cells. More CXCR3-A-modified CAR T cells engaging the tumor would lead to more CXCL10 production, which can further amplify CAR T-cell recruitment through positive feedback, thus improving the efficacy of CAR T cell therapy against DIPG. Here, it is demonstrated that compared to unmodified anti-B7-H3-CAR T cells, CAR T cells engineered to overexpress CXCR3-A migrate more efficiently towards the CXCR3 ligands, including CXCL10. In vivo, intracerebroventricularly delivered CXCR3-A-modified CAR T cells show significantly improved trafficking and efficacy in orthotopic DI PG xenograft mouse models. Overall, the data support the potential for engineering CXCR3-A expression in CAR T cells as a novel approach to enhancing CAR T cell trafficking and efficacy against DI PG.

[0248] Introduction. Central nervous system (CNS) tumors have surpassed leukemia as the leading cause of fatality among children with cancer (Siegel et al., CA Cancer J Clin 73, 17-48, 2023). Among the pediatric CNS tumors, diffuse intrinsic pontine glioma (DIPG) is particularly devastating, affecting 400 children per year in the US at a median age of 7 (Cooney et al., Neuro Oncol 19, 1279-1280, 2017; and Vitanza et al., Curr Treat Options Neurol 21 , 37, 2019). DIPG is an aggressive and universally fatal brainstem tumor, for which surgical excision is not feasible, chemotherapy is largely ineffective, and focal radiation is the only standard treatment option providing a palliative benefit as most patients die within a year. Therefore, more effective treatments against DI PG are desperately needed.

[0249] Chimeric antigen receptor (CAR) T cell therapies targeting various antigens have been developed to treat pediatric CNS tumors including DI PG. However, despite evidence of feasibility, tolerability, and some CAR T cell activity in phase 1 clinical trials, the overall efficacy remains limited (Ronsley et al., Cancer Metastasis Rev 43(4): 1205-1216, 2024). Among the challenges (Uslu and June, Nat Biotechnol, 2024; and Ferreras et al., Cells 10, 2021), insufficient CAR T cell trafficking to the tumor has been a major obstacle to successful CAR T cell treatment against solid tumors such as DIPG. Therefore, to address this issue, the anti-B7-H3-CAR T cells, which have been clinically studied and well tolerated in the phase 1 trial (CT04185038) (Vitanza et al., Cancer Discov 13, 114-131 , 2023 (“Vitanza 1”); and Vitanza et al., Nat Med, 2025 (“Vitanza 2”)), wereengineered to overexpress the chemokine receptor CXCR3 to promote CAR T cell trafficking and improve the efficacy of CAR T cell therapy against DI PG.

[0250] CXCR3 is a chemokine receptor that interacts with the chemokines CXCL10, CXCL9, and CXCL11 and directs cell migration against the concentration gradient of these ligands (Karin et al., Front Immunol 11 , 976, 2020). Given that CXCLIO is produced by activated T cells (Biddison et al., J Immunol 158, 3046-3053, 1997) and that CXCL10 is upregulated in the cerebrospinal fluid (CSF) of pediatric CNS tumor patients following intracranial CAR T cell treatment (Vitanza 1 and Vitanza 2), the interaction between CXCL10 and CXCR3 could be leveraged to enhance CAR T cell trafficking to the tumor by overexpressing CXCR3 in CAR T cells. In addition, with more CXCR3-modified CAR T cell trafficking to the tumor, more CXCL10 would be produced, further amplifying CAR T cell recruitment through positive feedback. Therefore, this new iteration of CAR T cell technology could be more effective in treating children with DI PG.

[0251] Many efforts to promote CAR T cell trafficking to solid tumors have been devoted to capitalizing on the chemokines upregulated by the tumor types of interest. For example, it was shown that glioblastoma (GBM) cell lines express CXCL8 post-irradiation and CAR T cells engineered to express the receptors for CXCL8, can migrate more effectively to the irradiated tumor in mouse brain by intravenous (IV) injection (Jin et al., Nat Commun 10, 4016, 2019). In contrast, the strategy described in this example aims to take advantage of the chemokines expressed by antigen-stimulated CAR T cells such as CXCL10, given the clinical evidence of CXCL10 upregulation following CAR T cell doses (Vitanza 1; Vitanza2; and Vitanza et al., Nat Med 27, 1544-1552, 2021 (“Vitanza 3”)). While endogenous CXCR3 is expressed on activated T cells (Loetscher et al., J Exp Med 184, 963-969, 1996), here whether uniformly overexpressing CXCR3 could further sensitize CAR T cells to migrate toward CXCL10 is investigated.

[0252] In this example, it is demonstrated that anti-B7-H3-CAR T cells engineered to overexpress the CXCR3 isoform CXCR3-A can migrate more efficiently toward the CXCR3 ligands such as CXCL10 in vitro, in comparison to the standard anti-B7-H3-CAR T cells. In vivo, CXCR3-A- modified CAR T cells show enhanced trafficking to the tumor sites post intracerebroventricular (ICV) delivery and result in improved efficacy in treating orthotopic DI PG xenograft mouse models. Overall, the data support the potential for engineering CXCR3-A overexpression to enhance CAR T cell trafficking to the tumor and improve the efficacy of CAR T cell therapy for DIPG. This is the first investigation of the trafficking of chemokine receptor-modified CAR T cells via ICV delivery, which is clinically significant for CNS tumors.

[0253] Results. B7-H3-targeting CAR T cells can kill patient-derived DI PG cells and produce CXCL10. Several CAR T cell therapies with various antigen specificities have been developed(Vitanza 1 ; Vitanza 2; Vitanza 3; and Ravanpay et al., Oncotarget 10, 7080-7095, 2019) and clinically investigated in a series of first-in-human phase 1 trials at Seattle Children’s Research Institute for pediatric CNS tumors using intracranial delivery (Vitanza et al., Neoplasia 36, 100870, 2023; and Vitanza et al., J Hematol Oncol Pharm 14, 148-154, 2024). This example focuses on the B7-H3-targeting CAR T cells, which have demonstrated clinical tolerability after intracerebroventricular (ICV) delivery to children with DI PG (Vitanza 1 ; and Vitanza 2). B7-H3 (CD276) is a member of the B7 family of immunoregulatory proteins and is highly expressed on DIPG tumor cells (Haydar et al., Neuro Oncol 23, 999-1011, 2021 ; and Majzner et al., Clin Cancer Res 25, 2560-2574, 2019), including the treatment-naive biopsy-derived DIPG cells (Biery et al., J Exp Neurol 1 , 158-167, 2020) (PBT-22FH, PBT-27FH, and PBT-29FH) and an autopsy-derived DIPG cell line (SU-DIPG-13) used in this example (FIG. 4A). The anti-B7-H3-CAR T cells kill the primary DIPG cells when co-cultured in vitro (FIG. 4B) and produce CXCL9 and CXCL10 post stimulation, although CXCL11 was not significantly released (FIG. 4C).

[0254] Manufactured CAR T cells heterogeneously express CXCR3 and can migrate toward CXCL10. CXCR3 is upregulated by activated T cells (Loetscher et al., J Exp Med 184, 963-969, 1996), and since T cell activation is the first step to generate CAR T cells among widely used protocols, manufactured CAR T cells are expected to display endogenous expression CXCR3. To confirm this, T cells were isolated from a healthy donors’ peripheral blood mononuclear cells (PBMC), activated with clinically used anti-CD3 / CD28 beads, and generated CAR T cells, which resulted in a heterogeneous increase of CXCR3 surface expression (FIG. 4D). To test CAR T cell migration efficiency in vitro, standard anti-B7-H3-CAR T cells were used to establish a trans-well chemotaxis assay. CAR T cells were seeded in the insert wells that allow them to migrate through a membrane with 5-p.m pores into the bottom wells with chemoattractant, and the number of CAR T cells in the bottom wells were counted and normalized to the seeded cell number. As the CAR T cells had endogenous CXCR3 expression, they were able to migrate toward CXCL10 in a dosedependent manner within 2 hours (FIG. 4E). A trans-well killing assay was further established, in which CAR T cells were permitted to migrate for 2 hours then co-cultured overnight with seeded DIPG cells in the bottom wells. As CXCL10 could induce CAR T cell migration, significantly more DIPG cells were killed when seeded with CXCL10 (FIG. 4F). Given that CXCL10 neither directly inhibited DIPG tumor viability (FIG. 4G) nor affected CAR T cell cytotoxicity against DI PG cells (FIG. 4H) in the control experiments, the reduced tumor viability in the cultures with CXCL10 in FIG. 4F reflects CXCL10-induced CAR T cell migration.

[0255] CXCR3-A-modified CAR T cells show enhanced migration toward CXCL10. Genetic constructs were developed that allow co-expression of the anti-B7-H3-CAR, a truncatedepidermal growth factor receptor (EGFRt) transduction marker, and a CXCR3 isoform (FIG. 5A) in order to test whether engineering CAR T cells to overexpress CXCR3 can further sensitize CAR T cells to migrate toward CXCL10. CXCR3 has three different isoforms in human due to alternative splicing (Reynders et al., Cells 8, 2019), namely CXCR3-A, CXCR3-B, and CXCR3- alt, and anti-B7-H3-CAR T cells overexpressing each of these isoforms were generated. While the CAR and EGFRt marker genes have been consistently transduced and expressed on the surface across the CAR T cell groups (FIG. 5B), the commercially available antibodies for CXCR3 could only detect elevated CXCR3 expression on the CXCR3-A-modified CAR T cells (CAR-3A) (FIG. 5C), but not on the CAR T cells engineered to overexpress CXCR3-B (CAR-3B) or CXCR3- alt (CAR-3alt) in comparison to the unmodified CAR T cells (CAR-only). Therefore, RT-qPCR was performed to evaluate the relative RNA expression of each CXCR3 isoform. The data suggest that CXCR3-A was the dominant isoform in the CAR-only T cells (FIG. 5D, left panel), and CXCR3-A expression was exponentially increased in the CAR-3A T cells, while CXCR3-B and CXCR3-alt were specifically overexpressed in the CAR-3B and CAR-3alt T cells respectively (FIG. 5D, right panel). Of note, the primer pairs to detect CXCR3-A had unavoidable overlaps with the CXCR3-B and CXCR3-alt sequences, which accounted for the artifacts of elevated CXCR3- A expression in the CAR-3B and CAR-3alt T cells.

[0256] To compare the migration efficiency of all the CXCR3-modified CAR T cells, the trans-well chemotaxis assay was performed with CXCL10. Compared to the CAR-only T cells, the CAR-3A T cells showed significantly enhanced migration toward CXCL10; however, this was not observed with the CAR-3B or CAR-3alt T cells (FIG. 5E). Based on the literature, it is controversial whether CXCR3-B promotes cell migration, and CXCR3-alt has been described to primarily interact with CXCL11 among the CXCR3 ligands (Reynders et al., Cells 8, 2019; and Korniejewska et al., Immunology 132, 503-515, 2011). To test the activity of the overexpressed CXCR3-alt in the CAR- Salt T cells, the CAR T cell migration toward CXCL11 was compared, and both the CAR-3A and CAR-3alt T cell groups migrated more efficiently when induced by CXCL11 (FIG. 5F).

[0257] CXCR3-A-modified CAR T cells show enhanced migration and efficacy against DIPG across multiple T cell donors in response to CXCR3 ligands. To validate the results across multiple T cell donors, the CXCR3-A-modified and unmodified anti-B7-H3-CAR T cells were generated with isolated T cells from three different PBMC donors. Despite donor variability, there was no significant difference between the CAR-3A and CAR-only T cells in terms of the fold expansion during production (FIG. 6A), the CAR transduction rate (FIG. 6B), the proportion of CD4 and CD8 T cells (FIG. 6D), the distribution of T cell phenotypes (FIG. 6E), as well as the expression of T cell exhaustion markers including PD1 , TIM3, and LAG3 (FIG. 6F), while theCXCR3 expression was elevated on the CAR-3A T cells (FIG. 6C) across the three donors. Furthermore, the CAR-3A and CAR-only T cells had similar cytotoxicity and cytokine production when they were directly co-cultured with the patient-derived DI PG cells in vitro (FIGs. 7 A, 7B).

[0258] Next, the migration efficiency of the CAR-3A and CAR-only T cells were compared from the three T cell donors toward CXCR3 ligands including CXCL9, CXCL10, and CXCL11. In transwell chemotaxis assays, the CAR-3A T cells showed enhanced migration toward all three ligands of CXCR3 (FIG. 7C). The trans-well killing assay was then performed using DI PG cells cultured with CXCL9, CXCL10, or CXCL11. As shown in FIG. 7D, despite some variations among the donors, when the migration was induced by CXCL9, CXCL10, or CXCL11 , the CAR-3A T cells consistently resulted in more DI PG tumor death, when compared to the CAR-only T cells. This enhanced efficacy was corroborated by the increased cytokines produced by the migrated CAR- 3A T cells (FIG. 7E). As the data showed that the presence of CXCL9, CXCL10, or CXCLU would not specifically enhance the cytotoxicity or cytokine production of the CAR-3A T cells when they were directly co-cultured with the DIPG cells in the control experiments (FIGs. 7F, 7G), it was confirmed that the enhanced efficacy by the CAR-3A T cells in the trans-well killing assays (FIGs. 7D, 7E) should be attributed to their more efficient migration toward the CXCR3 ligands.

[0259] Paracrine chemokines can lead to enhanced efficacy by CXCR3-A-modified CAR T cells against DI PG. As antigen-stimulated CAR T cells would make CXCL9 and CXCL10 (FIG. 4C), it was investigated if the chemokines produced by CAR T cells from co-culturing with DI PG cells would be sufficient to induce CAR T cell migration in trans-well killing assays. CAR T cell- conditioned medium was first prepared by stimulating the anti-B7-H3-CAR T cells with DIPG cells and harvesting the supernatant after 24 hours, then the conditioned medium was applied in a trans-well killing assay. As shown in FIG. 8A, while the CAR T cell conditioned medium had some direct inhibitory effect on the DI PG cells, it induced the CAR-3A T cells to migrate and kill DI PG cells, as evidenced by the significantly more reduced tumor viability, which was also substantially lower (though not statistically significant) in comparison to the CAR-only T cell group.

[0260] Next, it was tested whether the CAR-3A T cells would demonstrate enhanced efficacy in a long-term trans-well killing assay without exogenously added chemoattractant, in which seeded CAR T cells were given 24 hours to continuously migrate from the insert wells to the bottom wells with only DI PG cells (FIG. 8B). Since some CAR T cells would pass through the insert pores in the absence of exogenous chemoattractant (FIG. 4E), they would be stimulated by the DI PG cells and release chemokines including CXCL9 and CXCL10 in the bottom wells, which should attract more CAR T cells to actively migrate and in turn lead to more chemokine production as positive feedback in the following hours. Given that the CAR-3A T cells were more efficient migratingtoward CXCL9 and CXCL10 than the CAR-only T cells, more DI PG cells were killed by the migrated CAR-3A T cells via the amplified positive feedback loop. Indeed, the CAR-3A T cells consistently outperformed the CAR-only T cells in this assay, either in repeated experiments using CAR T cells of the same donor (FIG. 9A) or across all three T cell donors (FIG. 8B).

[0261] Moreover, whether DIPG tumors would contribute to the paracrine CXCL10 was investigated, as CXCL10 is inducible by IFN-y (Luster and Ravetch, J Exp Med 166, 1084-1097, 1987), a major cytokine produced by stimulated CAR T cells (FIG. 7B). As demonstrated in FIG. 8C, while the primary DIPG cells would not make CXCL10 in the absence of IFN-y, substantial amount of CXCL10 was produced by the DIPG cells when cultured with IFN-y. Therefore, the production of CXCL10 can be reinforced by DI PG tumors in response to IFN-y when treated by CAR T cells.

[0262] CXCR3-A-modified CAR T cells show enhanced trafficking and therapeutic efficacy when delivered intracerebroventricularly in orthotopic DI PG models. Based on the encouraging in vitro data, the effect of engineering CXCR3-A in CAR T cell against orthotopic DI PG mouse models was investigated. First, luciferase-labeled SU-DIPG-13 cells were intracranially implanted in NSG mice, which is an aggressive and well-studied DIPG in vivo model (Mount et al., Nat Med 24, 572- 579, 2018). As illustrated in FIG. 10A, after 7 days, the mice were treated with the CAR-3A, CAR- only, or non-transduced (NTD) T cells of the same donor via either IV or ICV injection, as both routes have been used in clinical studies for pediatric CNS tumor patients. To compare CAR T cell trafficking from the periphery via IV or from the CSF via ICV to the tumor, mice were sacrificed from each treatment group at 24-hour and 48-hour time points to harvest the mouse brains. The immunohistochemistry (IHC) staining for human CD3, CD4, and CD8 indicated increased presence of the CAR-3A T cells around the tumor sites, compared with the CAR-only or NTD T cells, at both time points post-delivery to the mouse CSF through ICV injection (representative images shown in FIG. 10B; additional images shown in FIG. 11). In contrast, there was lack of T cell trafficking to the brain through IV administration during the first 48 hours (FIG. 12). The tumor areas were defined based on H&E staining (FIGs. 10B, 11 , and 12) and confirmed by IHC staining for human Ki67 and B7-H3 (FIG. 13).

[0263] To compare the therapeutic efficacy of the CAR-3A and CAR-only T cells, bioluminescent imaging (BLI) was performed weekly for the remaining mice to measure the signals from the DI PG tumors. As SU-DIPG-13 cells could metastasize to the spinal cord (FIG. 14A), the BLI signals were quantified from the whole body of the mice in this experiment. When the CAR T cells were locoregionally delivered via ICV injection, the CAR-3A T cells reduced the tumor signals to the background level within 7 days (FIG. 14C) and maintained complete tumor regression (FIG. 14D).In contrast, although the ICV administered CAR-only T cells significantly inhibited the tumor growth compared to the NTD T cells (FIG. 14C), rapid rebound of tumor signals from either the brain or the spinal cord was observed in 4 out of 5 mice from this group (FIGs. 14A, 14D), which led to their fatality based on protocol-defined euthanasia criteria (FIG. 14E).

[0264] To be noted, prior to the end of this study, one mouse treated with ICV injected CAR-3A T cells met the euthanasia criteria without apparent tumor signal from previous BLI images (FIG. 14A). Hematoxylin-Eosin (H&E) and IHC staining for the mouse brain showed presence of xenograft tumor (human Ki67+) yet with low expression of human B7-H3 (FIG. 15), suggesting tumor antigen escape as a resistance mechanism under the selective pressure by the CAR-3A T cells. The fact that this coincided with loss of luciferase expression indicated that the tumor relapse was derived from a limited number of double-negative tumor cells, which could be addressed by CAR T cells with multiple specificities such as Seattle Children’s Research Institute’s ongoing trial (NCT05768880).

[0265] On the other hand, neither CAR T cell groups demonstrated efficacy when delivered systemically through IV injection (FIGs. 14A, 14B, 14D, and 14E), which was most likely due to the insufficient trafficking of CAR T cells from the periphery to the brain (FIG. 12).

[0266] Mouse weight loss and neurologic symptoms were correlated with tumor progression (FIG. 14F), and there was no evident toxicity by the CAR-3A T cells in comparison with the CAR-only T cells.

[0267] To validate these findings, the safety and efficacy of the CAR-3A T cells was evaluated in a second DIPG in vivo model and T cells from another donor were used. In brief, luciferase- labeled PBT-27FH cells were intracranially engrafted in NSG mice for 35 days before T cell administration through ICV injection (FIG. 16A). The BLI signals from the mouse brain suggested substantial decrease in tumor burden by the CAR-3A T cells within 7 days of treatment (FIG. 16B), indicating efficient tumor engagement by the CAR-3A T cells, while the tumor signals remained above baseline in most mice treated with the CAR-only T cells. Despite the extended time for the tumor to progress in this model (FIGs. 16C and 17), the mice treated with the CAR-3A T cells had survival advantage compared to the mice in the NTD or CAR-only T cell groups (FIG. 16D), though the comparison to the latter did not reach statistical significance due to the circumstance that two mice in the CAR-3A T cell group without tumor BLI signal were euthanized due to non-tumor related causes (vaginal prolapse, day 182; bacterial infection, day 193); IHC staining for human Ki67 and B7-H3 confirmed absence of tumor in these brains (FIG. 18).

[0268] There was no weight loss (FIG. 16E) or sign of illness such as lethargy, hunching posture at rest, or hair loss exhibited by the mice in relation to the treatment of the CAR-3A T cells,supporting the safety of this CAR T cell engineering approach.

[0269] Discussion. As DIPG is universally fatal and responsible for 25,000 years of lost potential life each year in the US alone (Cooney et al., Neuro Oncol 19, 1279-1280, 2017; and Vitanza et al., Curr Treat Options Neurol 21, 37, 2019), more effective treatments are desperately needed. Here, a new iteration of CAR T cell therapy was developed by engineering anti-B7-H3-CAR T cells to overexpress CXCR3-A. Furthermore, compared to the standard anti-B7-H3-CAR T cell therapy, the CXCR3-A-modified CAR T cells migrated more efficiently toward the CXCR3 ligands including CXCL10 and showed enhanced trafficking and therapeutic efficacy against the orthotopic DI PG mouse models by intracerebroventricular delivery. Because CXCL10 is produced by activated CAR T cells as well as by DI PG cells in response to IFN-y from activated CAR T cells, the CXCL10 could attract more CXCR3-A-modified CAR T cells to migrate to the tumor, which would result in a positive feedback loop by amplifying the CXCL10 production and further recruitment of CAR T cells, thus leading to improved therapeutic efficacy against DIPG (FIG. 19).

[0270] It is worth noting that while the tumor microenvironment (TME) of DIPG has been described as immunologically inert (Lin et al., Acta Neuropathol Commun 6, 51 , 2018; and Lieberman et al., Neuro Oncol 21, 83-94, 2019), other immune cells in the CNS such as microglia could be attracted and in turn contribute to the chemokine and cytokine production in the TME following CAR T cell administration, which may reinforce a pro-inflammatory environment and further recruit other anti-tumor immune cells. However, the interactions between CAR T cells and other immune cell types need to be studied in immuno-competent syngeneic DIPG mouse models with murine CAR T cells, which has its own limitations and is out of the scope of this manuscript.

[0271] Clinical data with the standard anti-B7-H3-CAR T cells indicated some CAR T cell activity and elevated CXCL10 in the CSF of patients with DI PG (Vitanza 1 and Vitanza 2), which hypothetically may be sufficient to initiate the positive feedback loop for recruiting the CXCR3-A- modified CAR T cells. Nevertheless, the approach described herein could further synergize with strategies that promote the production of CXCR3 ligands from the tumor cells prior to CAR T cell administration, to increase the initial tumor engagement by the CXCR3-A-modified CAR T cells. For example, oncolytic virus encoding a CXCL11 gene has been preclinically developed (Wang et al., Mol Ther 31 , 134-153, 2023), which could target tumor cells to express CXCL11 and potentially attract more CXCR3-A-modified CAR T cells immediately after the injection. In addition, since DI PG cells would make CXCL10 in response to IFN-y, oncolytic virus encoding an IFN-y gene may provide a similar effect for enhancing the trafficking of the CXCR3-A-modified CAR T cells as a combination therapy for DI PG.

[0272] Preclinical evidence, including data in this Example, suggests the superiority oflocoregional CAR T cell delivery to the CNS over systemic dosing in treating CNS tumor models (Brown et al., Mol Ther 26, 31-44, 2018; Theruvath et al., Nat Med 26, 712-719, 2020; and Donovan et al., Nat Med 26, 720-731 , 2020), and CAR T cell clinical trials for CNS tumors have demonstrated feasibility and tolerability of injecting CAR T cells to the CNS of patients (Ronsley et al., Cancer Metastasis Rev 43(4): 1205-1216, 2024; and Vitanza et al., Neoplasia 36, 100870, 2023). This is the first demonstration that CAR T cell trafficking to the CNS tumor can be enhanced by engineering chemokine receptor expression in the CAR T cells when they are ICV delivered.

[0273] While the example focuses on enhancing the anti-B7-H3-CAR T cell therapy for DI PG, this concept of CXCR3-A-modified CAR T cells is readily applicable to CAR T cell therapies targeting other types of solid tumor for enhanced CAR T cell trafficking and efficacy as well, provided with modifications of the CAR specificities for suitable tumor antigens.

[0274] Of note, the role of CXCR3-B in cell migration has been conflicting based on the current literature (Reynders et al., Cells 8, 2019; and Korniejewska et al., Immunology 132, 503-515, 2011). As CXCR3 only has one isoform in mouse, which is the counterpart for CXCR3-A in human, CXCR3-B as well as CXCR3-alt have not been well studied. Here, the data suggested that CXCR3-B overexpression did not significantly increase T cell migration toward CXCL10 or CXCL11 (FIGs. 5E, 5F).

[0275] Enhanced trafficking and efficacy by the CXCR3-A-modified CAR T cells was shown despite the fact that the unmodified CAR T cells upregulate CXCR3 expression secondary to the activation step in the commonly adopted production process (FIG. 4D). However, as new CAR T cell production protocols are developed, some of which either omit the activation step to preserve the sternness of the CAR T cells (Ghassemi et al., Nat Biomed Eng 6, 118-128, 2022) or generate CAR T cells through inducible pluripotent stem cells (iPSCs) for potential off-the-shelf applications (Mishra and Kalyuzhny, Cells 13, 2024), in these cases, engineering the CAR T cells to overexpress CXCR3 may be even more essential to promote CAR T cell trafficking and efficacy, as CAR T cells generated by these methods would have less endogenous CXCR3 expression.

[0276] Safety concerns derived from the engineering of CXCR3-A in the anti-B7-H3-CAR T cells were not observed in the xenograft mouse models.

[0277] Materials and Methods.

[0278] Primary human DI PG cell culture. Human cell cultures were generated with informed consent in compliance with institutional review board approval at Seattle Children’s Hospital (#14449). PBT-22FH, PBT-27FH, and PBT-29FH were biopsy derived at diagnosis. For PBT- 22FH, PBT-27FH, and PBT-29FH, tumor tissue was obtained at Seattle Children’s Hospital, and cell cultures were created at Fred Hutchinson Cancer Research Center as previously described(Biery et al., J Exp Neurol 1, 158-167, 2020). SU-DIPG-13 was autopsy-derived and generously donated by Dr. Michael Monje (Stanford University). Cells were modified by lentiviral transduction followed by blasticidin selection (Fisher Scientific, #A1113903) to steadily express firefly luciferase. Cells were maintained in NeuroCult NS-A Basal Medium with NS-A Proliferation Supplement (Stemcell Technologies, #5751), 1x Antibiotic / Antimycotic (ThermoFisher Scientific, #15-240-062), 40 ng / mL epidermal growth factor (PeproTech, #AF-100-15), and 40 ng / mL fibroblast growth factor (PeproTech, #AF-100-18B). All cell culture models were validated by DNA fingerprinting and routinely tested to be mycoplasma negative.

[0279] CAR constructs. The medium-spacer human B7-H3-specific CAR construct has previously been described (Vitanza 1). In brief, the CAR includes a scFv derived from the MGA 271 monoclonal antibody, a medium-length spacer of lgG4-hinge-CH3, a CD28 transmembrane domain, a 4-1 BB co-stimulatory domain, and a CD3 signaling domain, and the CAR sequence was appended to a T2A ribosomal skip sequence followed by a truncated EGFR (EGFRt) marker gene, which was appended to a second T2A sequence followed by the methotrexate-resistant human DHFR mutein gene, the last of which was not utilized in this paper. For the CAR constructs co-expressing a CXCR3 isoform, the human DHFR mutein gene was replaced with the open reading frame sequence of CXCR3-A, CXCR3-B, or CXCR3-alt (synthesized by Genewiz).

[0280] Lentiviral vector preparation. Lentiviral vectors were produced by transfecting Lenti-X 293- T cells (Takara, #632180). In brief, 293-T cells were seeded in T150 flasks that were pre-coated with collagen (Millipore Sigma, #C3867-1VL) and cultured in DMEM medium (Gibco, #11960044) supplemented 10% fetal bovine serum (Gibco, #A5256701), 10 mM HEPES (Gibco, #15630080), 100 U / mL penicillin / streptomycin (Gibco, #15140122), and 1x GlutaMax (Gibco, #35050061) to grow to 80% confluence, after which the cells were incubated briefly with a cocktail of 3 mL of Opti-MEM (Gibco, #31985070) with 90 pL Lipofectamine 3000 transfection reagent (Invitrogen, # L3000015) that was gently mixed with 15 pg of the vector plasmid of interest and the packaging plasmids of 18 pg of psPAX2 (Addgene, #12260) and 7 pg of pCMV-VSV-G (Addgene, #8454). Additional culture medium was added thereafter. After culturing the transfected 293-T cells for 24 h, supernatants that contained lentiviral vectors were collected and filtered through 0.45-mm syringe filters (Millipore Sigma, #SLHP033RS), then centrifuged at 18,000 x g for 20 h at 4 °C. The pellets of lentiviral vectors were resuspended in 2 mL of culture medium, aliquoted, and stored at -80 °C.

[0281] Generation of human CAR T cells. Bulk human T cells were isolated from peripheral blood mononuclear cells of healthy donors (Bloodworks Northwest) by negative selection using EasySep Human T Cell Isolation Kit (StemCell, #17951) and maintained in RPMI 1640 medium(Gibco, #21870076) supplemented 10% fetal bovine serum (Gibco, #A5256701), 10 mM HEPES (Gibco, #15630080), 100 U / mL penicillin / streptomycin (Gibco, #15140122), GlutaMax (Gibco, #35050061), 5 ng / mL recombinant human IL-7 (Biolegend, # 581904), and 0.5 ng / mL recombinant human IL15 (Biolegend, # 570304). Following isolation, T cells were stimulated with anti-CD3 / CD28 Dynabeads (Gibco, #40203D) in the medium at 1 x 106 cells / mL for 24 h and transduced at a multiplicity of infection of 3, after which the transduced T cells were enumerated by a Vi-CELL BLU counter (Beckman Coulter) every other day and fed with the medium to 0.8 x 106cells / mL density. After the expansion, the cells were cryopreserved in CryoStor CS10 (StemCell, #07930) until further use. To resuscitate cryopreserved cells, the cells were quickly thawed with pre-warmed medium and pelleted by centrifugation, then cultured in the medium overnight to recover, and the viable cell number was decided by a Vi-CELL BLU counter (Beckman Coulter) before being used for functional assays.

[0282] RT-qPCR. The RNA was extracted from cells using RNeasy Plus Mini Kit (QIAGEN, #74134) and converted to cDNA with High-Capacity RNA-to-cDNA kit (AppliedBiosystems, #4387406). The cDNA was then set up with TB Green (Takara, #RR42LR) and a primer pair (Integrated DNA Technologies) in four replicate wells for qPCR with Light Cycler 480 II (Roche). The expression of the gene of interest was normalized to the expression of actin, then multiplied by a nominal factor for data display.

[0283] Flow cytometry. For flow cytometry, the antibodies used included mouse-anti-human B7- H3-PE (BioLegend, clone MIH42, #351004), mouse-anti-human CXCR3-FITC (BioLegend, clone G025H7, #353704), mouse-anti-human EGFR-PE (BioLegend, clone AY13, #352904), mouse- anti-human CD4-PE (BioLegend, clone OKT4, #317410), mouse-anti-human CD8-APC (BioLegend, clone RPA-T8, #301049), mouse-anti-human CD45RA-PE (BioLegend, clone H 1100, #304108), mouse-anti-human CD62L-BV421 (BioLegend, clone DREG-56, #304828), mouse- anti-human PD1-APC (BioLegend, clone A17188B, #621610), mouse-anti-human TIM3-APC (BioLegend, clone A18087E, #364804), mouse-anti-human LAG3-APC (BioLegend, clone 7H2C65, #369212), and biotinylated Protein L (GenScript, #M00097) followed by streptavidin-PE (BioLegend, #405204) secondary staining to detect the B7-H3-specific CAR expression. For flow cytometry analysis, cells were sampled and washed with PBS (Gibco, #10010023), incubated with antibodies in PBS on ice in darkness for 30 min, washed again with PBS twice, followed by secondary staining with additional wash steps when necessary, then evaluated on a LSR Fortessa (BD Biosciences) or Novocyte 3000 (Agilent) flow cytometer. The cell-surface expression of the proteins of interest was analyzed by FlowJo (Tree Star).

[0284] Luciferase assay. The primary DIPG cell lines used for luciferase assays were modified toexpress firefly luciferase. D-Luciferin (Revvity, #122799-5) substrate solution was added to the cell culture at the indicated time points for each assay and incubated at room temperature for 15 min before quantifying the bioluminescent signal from each well with a SpectraMax i D5 (Molecular Devices). All the in vitro viability assays were performed three times or with T cells from three different donors, and the bioluminescent signals were normalized to the vehicle-treated control wells as 100% viability.

[0285] ELISA. ELISA assays were performed with DuoSet ELISA kits (R&D Systems) to detect CXCL9 (#DY392), CXCL10 (#DY266), CXCL11 (#DY672), IL-2 (#DY202), TNF (#DY210), and IFN-y (#DY285B) concentrations in the collected supernatants per the manufacturer’s instructions.

[0286] Trans-well chemotaxis assay. Trans-well chemotaxis assays were performed with Transwell 24-well Plate with 5.0 pm Pore Polycarbonate Membrane Insert (Corning, #3421) to assess the migration efficiency of CAR T cells toward chemoattractant. In brief, blank medium (vehicle) or medium with chemoattractant was added to the bottom wells, then the same number of CAR T cells was seeded on the insert wells, which allow active T cell migration through the pores to the bottom wells. After the indicated length of incubation, the number of migrated CAR T cells in each bottom well was counted in duplicates with Vi-CELL BLU counter (Beckman Coulter). All the trans-well chemotaxis assays were performed at least three times or with T cells from three different donors, and percent migration was calculated as the migrated cell number over the seeded cell number.

[0287] Trans-well killing assay. Trans-well killing assays were performed with Transwell 24-well Plate with 5.0 pm Pore Polycarbonate Membrane Insert (Corning, #3421). In brief, luciferase- labeled DI PG tumor cells were seeded in the bottom wells, then blank medium (vehicle) or medium with chemoattractant was added to the bottom wells. The same number of CAR T cells was seeded on the insert wells, which allow active T cell migration through the pores to the bottom wells. After 2-hour incubation, the insert wells were removed, and the plate was further incubated overnight to allow the migrated CAR T cells to kill the seeded DI PG tumor cells. At 24-hour time point, luciferase assay was performed as described above to quantify the viability of the seeded DIPG tumor cells. All the trans-well killings assays were performed three times or with T cells from three donors, and the bioluminescent signals were normalized to the vehicle-treated control wells as 100% viability.

[0288] The chemoattractants used in this study include recombinant human CXCL9 (#ab283902), CXCL10 (#ab280332), and CXCL11 (#ab283901) from Abeam, as well as conditioned media harvested from co-cultures of CAR T cells and DI PG tumor cells.

[0289] Mouse experiments. All mouse experiments were conducted in accordance with the lACUC-approved protocol ACUC00669 at Seattle Children’s Research Institute. For the orthotopic xenograft model with SU-DIPG-13, eight-week-old male NSG mice (Jackson Laboratories) were intracranially injected with 50,000 SU-DIPG-13 cells in 2 pL PBS (Cytiva, #SH30256.LS) using a stereotactic surgical setup with the coordinates of 0.8 mm lateral and 1.5 mm posterior from lambda, and 2.5mm deep. Seven days after tumor implantation, 2 x 106CAR T cells in 5 pL PBS were injected into the lateral ventricle of brain with the coordinates of 1 .2 mm lateral and 0.6 mm posterior from bregma, and 2.0mm deep, or intravenously in 200 pL PBS. Mice were anesthetized by intraperitoneal injected cocktail of Ketamine (Patterson Veterinary Supply, #07-894-8462; 100mg / kg animal) and Xylazine (Patterson Veterinary Supply, #07-808- 1947; 8mg / kg animal) for the surgeries and given the painkiller of buprenorphine (Wedgewood Connect, #BUPREN-INJ010VC; 1 mg / kg animal, extended release) by the veterinary staff before the surgeries. Mice from each treatment group were euthanized at 24-hour and 48-hour posttreatment time points to collect their brains, which were used for immunohistochemistry (IHC) analysis to evaluate the migration of T cells toward the tumor site as describe below. For the rest of the mice in each treatment group, bioluminescent imaging (BLI) was performed weekly to measure the tumor signals, for which mice were anesthetized with isoflurane (Patterson Veterinary Supply, #07-890-8115), subcutaneously injected with D-luciferin (Revvity, #122799-5) substrate solution and imaged with IVIS Spectrum Imaging System (Perkin Elmer) after 15 min. Mouse weight was recorded on a weekly basis with routine health checks to monitor any sign of illness such as weight loss, hair loss, cranial distension, lethargy, and hunching posture at rest. Mice with severe symptoms were euthanized based on protocol-defined criteria, followed by collection of brains, livers, and spleens for IHC analysis.

[0290] For the experiment with PBT-27FH, gender-balanced NSG mice were intracranially injected with 350,000 PBT-27FH cells in 2 pL PBS using a stereotactic surgical setup with the same coordinates as described above. 35 days after tumor implantation, 1 x 105CAR T cells in 3 pL PBS were injected into the lateral ventricle of the brain with the coordinates as described above. BLI was performed to measure the tumor signals. Mouse weight was recorded with routine health checks to monitor any sign of illness, and mice with severe symptoms were euthanized based on protocol-defined criteria, followed by collection of brains, livers, and spleens for IHC analysis if needed.

[0291] Tissue processing, IHC, and histopathologic analysis. Mouse brains were fixed in 10% neutral buffered formalin (Fisher Scientific, #57-35) for 24-72 hours, then transferred into 70% ethanol for long-term storage. Wet tissues were submitted to the Microscopy and HistopathologyCoLab at Seattle Children’s Research Institute for paraffin embedding. Formalin-fixed paraffin- embedded blocks were then submitted to the Experimental Histopathology lab at Fred Hutchinson Cancer Research Center for further processing. Tissue sections were cut 4-5 pm thick for Hematoxylin-Eosin (H&E) stains and IHC. IHC stains included human CD3 (Fisher Scientific, #RM9107S, 1 :300), human CD4 (Millipore Sigma, #104R-26, 1:100), human CD8 (Agilent, #M7103, 1:200), human B7H3 (Abeam, #ab219648, 1:5000), human Ki67 (Cell Signaling, #12202, 1 :1000), and Albumin (Genetex; #GTX102419, 1 :2000) with a hematoxylin counterstain. H&E and IHC images were produced with an Aperio AT slide scanner.

[0292] Statistical analysis. The statistical analyses were performed as noted in the figure legends using Prism 10 (GraphPad) to determine the p values. Significance is reported if p < 0.05 (marked by an asterisk).

[0293] Illustrations. The illustrations and schematics in this manuscript were created in https: / / BioRender.com.

[0294] (XIV) 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.

[0295] 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.

[0296] 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; Group 8 (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.

[0297] 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).

[0298] 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.

[0299] As detailed in US 4,554,101 , the following hydrophilicity values have been assigned to amino 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.

[0300] As outlined above, amino acid substitutions may be based on the relative similarity of theamino 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.

[0301] 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.

[0302] “% 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 and Griffin, eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (Von Heijne, ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov and Devereux 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 (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wisconsin); BLASTP, BLASTN, BLASTX (Altschul, et al., 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, 111-20. 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.

[0303] 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.

[0304] Functional variants include one or more residue additions or substitutions that do not substantially impact the physiological effects of the protein. Functional fragments include one or more deletions or truncations that do not substantially impact the physiological effects of the protein. A lack of substantial impact can be confirmed by observing experimentally comparable results in an activation study or a binding study. Functional variants and functional fragments of intracellular domains (e.g., intracellular signaling components) transmit activation or inhibition signals comparable to a wild-type reference when in the activated state of the current disclosure. Functional variants and functional fragments of binding domains bind their cognate antigen or ligand at a level comparable to a wild-type reference.

[0305] In particular embodiments, a protein or peptide can be derived from or based on a another protein or peptide 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 protein or peptide disclosed herein. An insertion, deletion or substitution may be anywhere in theprotein or peptide, including at the amino- or carboxy-terminus or both ends of this region.

[0306] "Binds" refers to an association of a binding domain (of, for example, a recombinant receptor binding domain) to its cognate binding molecule with an affinity or Ka(i.e., an equilibrium association constant of 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 Ka of at least 107M-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 10-13M). 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 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. 51 :660; and U.S. Patent Nos. 5,283,173, 5,468,614, or the equivalent).

[0307] 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).

[0308] 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, butis 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 reduction in the killing of B7-H3-expressing cells as assessed according to experimental methods described herein.

[0309] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such 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.

[0310] 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.

[0311] 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 indicatedherein, 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.

[0312] Groupings of alternative elements or embodiments of the invention disclosed herein are not 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.

[0313] 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.

[0314] 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.

[0315] 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.

[0316] 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 thecause 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.

[0317] 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 examples or 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 et al., Oxford University Press, Oxford, 2006).

Claims

CLAIMSWhat is claimed is:

1. An artificial expression construct comprising (i) a sequence encoding CXCR3-A and (ii) a sequence encoding a B7-H3 binding chimeric antigen receptor (CAR).

2. An artificial expression construct comprising (i) a sequence encoding a CXCR3 isoform selected from CXCR3-A, CXCR3-B, or CXCR3-alt; and (ii) a sequence encoding a cancer antigen-binding recombinant receptor.

3. The artificial expression construct of claim 2, further comprising a promoter.

4. The artificial expression construct of claim 3, wherein the promoter comprises an EF1 promoter.

5. The artificial expression construct of claim 2, wherein the CXCR3-A comprises the sequence of SEQ ID NO: 2 or a sequence having at least 95% sequence identity to the full length of SEQ ID NO: 2.

6. The artificial expression construct of claim 2, wherein the CXCR3-A is encoded by the sequence of SEQ ID NO: 3 or a sequence having at least 95% sequence identity to the full length of SEQ ID NO: 3.

7. The artificial expression construct of claim 2, wherein the cancer antigen-binding recombinant receptor comprises, when expressed by a cell, an extracellular component comprising a cancer antigen binding domain; an intracellular component; and a transmembrane domain linking the extracellular component to the intracellular component.

8. The artificial expression construct of claim 2, wherein the cancer antigen-binding recombinant receptor comprises a chimeric antigen receptor (CAR), an engineered T cell receptor (eTCR), or a hybrid thereof.

9. The artificial expression construct of claim 2, wherein the cancer antigen-binding recombinant receptor comprises a B7-H3 binding domain.

10. The artificial expression construct of claim 9, wherein the B7-H3 binding domain comprises a complementarity determining region (CDR) set comprising a variable heavy chain comprising a CDR heavy (H)1, a CDRH2, and a CDRH3 and a variable light chain comprising a CDR light (L)1 , CDRL2, and CDRL3, wherein: the CDRH1 comprises the sequence of SEQ ID NO: 8, the CDRH2 comprises the sequence of SEQ ID NO: 9, the CDRH3 comprises the sequence of SEQ ID NO: 10, the CDRL1 comprises the sequence of SEQ ID NO: 11 , the CDRL2 comprises the sequence of SEQ ID NO: 12, and the CDRL3 comprises the sequence of SEQ ID NO: 13, accordingto Kabat; the CDRH1 comprises the sequence of SEQ ID NO: 14, the CDRH2 comprises the sequence of SEQ I D NO: 15, the CDRH3 comprises the sequence of SEQ I D NO: 10, the CDRL1 comprises the sequence of SEQ ID NO: 11, the CDRL2 comprises the sequence of SEQ ID NO: 12, and the CDRL3 comprises the sequence of SEQ ID NO: 13, according to Chothia; the CDRH1 comprises the sequence of SEQ ID NO: 16, the CDRH2 comprises the sequence of SEQ I D NO: 17, the CDRH3 comprises the sequence of SEQ I D NO: 18, the CDRL1 comprises the sequence of SEQ ID NO: 19, the CDRL2 comprises the sequence as SAS, and the CDRL3 comprises the sequence of SEQ ID NO: 13, according to IMGT; the CDRH1 comprises the sequence of SEQ ID NO: 20, the CDRH2 comprises the sequence of SEQ ID NO: 21 , the CDRH3 comprises the sequence of SEQ ID NO: 18, the CDRL1 comprises the sequence of SEQ ID NO: 11, the CDRL2 comprises the sequence of SEQ ID NO: 22, and the CDRL3 comprises the sequence of SEQ ID NO: 13, according to North; or the CDRH1 comprises the sequence of SEQ ID NO: 23, the CDRH2 comprises the sequence of SEQ ID NO: 24, the CDRH3 comprises the sequence of SEQ ID NO: 25, the CDRL1 comprises the sequence of SEQ ID NO: 26, the CDRL2 comprises the sequence of SEQ I D NO: 27, and the CDRL3 comprises the sequence of SEQ I D NO: 28, according to Contact.

11. The artificial expression construct of claim 9, wherein the B7-H3 binding domain comprises a variable heavy chain comprising a sequence having at least 95% sequence identity to the full length of SEQ ID NO: 29 and a variable light chain comprising a sequence having at least 95% sequence identity to the full length of SEQ ID NO: 30.

12. The artificial expression construct of claim 9, wherein the B7-H3 binding domain comprises a variable heavy chain comprising the sequence of SEQ ID NO: 29 and a variable light chain comprising the sequence of SEQ ID NO: 30.

13. The artificial expression construct of claim 9, wherein the B7-H3 binding domain comprises a variable heavy chain encoded by a sequence having at least 95% sequence identity to the full length of SEQ ID NO: 31 and a variable light chain encoded by a sequence having at least 95% sequence identity to the full length of SEQ ID NO: 32.

14. The artificial expression construct of claim 9, wherein the B7-H3 binding domain comprises a variable heavy chain encoded by the sequence of SEQ ID NO: 31 and avariable light chain encoded by the sequence of SEQ ID NO: 32.

15. The artificial expression construct of claim 9, wherein the B7-H3 binding domain comprises an scFv.

16. The artificial expression construct of claim 15, wherein the scFv comprises a sequence having at least 95% sequence identity to the full length of SEQ ID NO: 33 or SEQ ID NO:35.

17. The artificial expression construct of claim 15, wherein the scFv comprises the sequence of SEQ ID NO: 33 or SEQ ID NO: 35.

18. The artificial expression construct of claim 15, wherein the scFv is encoded by a sequence having at least 95% sequence identity to the full length of SEQ ID NO: 34 or SEQ ID NO:36.

19. The artificial expression construct of claim 15, wherein the scFv is encoded by the sequence of SEQ ID NO: 34 or SEQ ID NO: 36.

20. The artificial expression construct of claim 7, wherein the extracellular component further comprises a spacer.

21. The artificial expression construct of claim 20, wherein the spacer comprises an lgG4 hinge and lgG4 CH3 domain.

22. The artificial expression construct of claim 7, wherein the intracellular component comprises all or a portion of a CD3 signaling domain; all or a portion of a 4-1 BB signaling domain; or all or a portion of a CD3 signaling domain and all or a portion of a 4-1 BB signaling domain.

23. The artificial expression construct of claim 7, wherein the intracellular component comprises all or a portion of a CD3 signaling domain and all or a portion of a 4-1 BB signaling domain.

24. The artificial expression construct of claim 7, wherein the transmembrane domain comprises a CD28 transmembrane domain.

25. The artificial expression construct of claim 9, wherein the B7-H3 binding domain comprises the sequence of SEQ ID NO: 33, the intracellular component comprises a CD3 signaling domain and a 4-1 BB signaling domain, and the transmembrane domain comprises a CD28 transmembrane domain.

26. The artificial expression construct of claim 9, wherein the B7-H3 binding recombinant receptor comprises the sequence of SEQ ID NO: 79 or a sequence having at least 95% sequence identity to the full length of SEQ ID NO: 79.

27. The artificial expression construct of claim 9, wherein the B7-H3 binding recombinantreceptor is encoded by the sequence of SEQ ID NO: 80 or a sequence having at least 95% sequence identity to the full length of SEQ ID NO: 80.

28. The artificial expression construct of claim 2, further comprising a self-cleaving polypeptide.

29. The artificial expression construct of claim 28, wherein the self-cleaving polypeptide is a porcine teschovirus-1 (P2A) self-cleaving polypeptide, Thosea asigna virus (T2A) selfcleaving polypeptide, equine rhinitis A virus (E2A) self-cleaving polypeptide, foot-and- mouth disease virus (F2A) self-cleaving polypeptide.

30. The artificial expression construct of claim 28, wherein the self-cleaving polypeptide is a T2A self-cleaving polypeptide.

31. The artificial expression construct of claim 2, further comprising a transduction marker.

32. The artificial expression construct of claim 31 , wherein the transduction marker comprises a truncated epidermal growth factor receptor (EGFRt).

33. The artificial expression construct of claim 28, further comprising a second self-cleaving polypeptide.

34. The artificial expression construct of claim 33, wherein the second self-cleaving polypeptide is P2A self-cleaving polypeptide, T2A self-cleaving polypeptide, E2A selfcleaving polypeptide, F2A self-cleaving polypeptide.

35. The artificial expression construct of claim 34, wherein the self-cleaving polypeptide is a T2A self-cleaving polypeptide.

36. The artificial expression construct of claim 2, comprising the sequence of any of SEQ ID NOs: 81 , 82, 83, 84, 85, or 86; or a sequence having at least 90% or at least 95% sequence identity to the full length of any of SEQ ID NOs: 81 , 82, 83, 84, 85, or 86.

37. The artificial expression construct of claim 2, comprising the sequence of SEQ ID NO: 84 or a sequence having at least 90% or at least 95% sequence identity to the full length of SEQ ID NO: 84.

38. The artificial expression construct of claim 2, comprising the sequence of SEQ ID NO: 85 or a sequence having at least 90% or at least 95% sequence identity to the sequence of SEQ ID NO: 85.

39. The artificial expression construct of claim 2, comprising the sequence of SEQ ID NO: 86 or a sequence having at least 90% or at least 95% sequence identity to the full length of SEQ ID NO: 86.

40. A system comprising a first sequence encoding a B7-H3 binding recombinant receptor and a second sequence encoding a CXCR3 isoform selected from CXCR3-A, CXCR3-B,or CXCR3-alt.

41. The system of claim 40, wherein the first sequence and second sequence are on a same artificial expression construct.

42. The system of claim 40, wherein the first sequence and second sequence are on different artificial expression constructs.

43. The system of claim 40, wherein the first sequence and second sequence are encapsulated within a same nanoparticle.

44. The system of claim 40, wherein the first sequence and second sequence are encapsulated in different nanoparticles.

45. The system of claim 40, wherein the first sequence and second sequence are introduced into a same cell.

46. The system of claim 40, wherein the first sequence encodes the sequence of SEQ ID NO: 79 or a sequence having at least 90% or at least 95% sequence identity to the full length of SEQ ID NO: 79.

47. The system of claim 40, wherein the first sequence comprises the sequence of SEQ ID NO: 80 or a sequence having at least 90% or at least 95% sequence identity to the full length of SEQ ID NO: 80.

48. The system of claim 40, wherein the second sequence encodes the sequence of any of SEQ ID NOs: 2, 4, or 6; or a sequence having at least 90% or at least 95% sequence identity to the full length of any of SEQ ID NOs: 2, 4, or 6.

49. The system of claim 40, wherein the second sequence comprises the sequence of any of SEQ ID NOs: 3, 5, or 7; or a sequence having at least 90% or at least 95% sequence identity to the full length of any of SEQ ID NOs: 3, 5, or 7.

50. The system of claim 40, comprising the sequence of any of SEQ ID NOs: 81 , 82, 83, 84, 85, or 86; or a sequence having at least 90% or at least 95% sequence identity to the full length of any of SEQ ID NOs: 81, 82, 83, 84, 85, or 86.

51. A nanoparticle encapsulating the artificial expression construct of claim 2, or the system of claim 40.

52. A cell genetically modified to express the artificial expression construct of claim 2 or the system of claim 40.

53. The cell of claim 52, wherein the cell is an autologous cell or an allogeneic cell in reference to a subject.

54. The cell of claim 52, wherein the cell is in vivo or ex vivo.

55. The cell of claim 52, wherein the cell is an immune cell.

56. The cell of claim 55, wherein the immune cell is a lymphocyte.

57. The cell of claim 55, wherein the immune cell is a T cell, B-cell, natural killer (NK) cell, NK- T cell, a monocyte, or a macrophage.

58. The cell of claim 55, wherein the immune cell is a T cell selected from a CD3+ T cell, aCD4+ T cell, a CD8+ T cell, a central memory T cell, an effector memory T cell, and / or a naive T cell.

59. The cell of claim 55, wherein the immune cell is a CD8+ T cell.

60. The cell of claim 55, wherein the immune cell is a CD4+ T cell.

61. A population of cells genetically modified to express the artificial expression construct of claim 2 or the system of claim 40.

62. The population of cells of claim 61, wherein the population of cells comprises autologous cells or allogeneic cells in reference to a subject.

63. The population of cells of claim 61 , wherein the population is in vivo or ex vivo.

64. The population of cells of claim 61 , wherein the cell is an immune cell.

65. The population of cells of claim 64, wherein the immune cell is a lymphocyte.

66. The population of cells of claim 64, wherein the immune cell is a T cell, B-cell, natural killer (NK) cell, NK-T cell, a monocyte, or a macrophage.

67. The population of cells of claim 66, wherein the population comprises CD4+ T cells and / or CD8+ T cells.

68. A formulation comprising (i) cells genetically modified to express the artificial expression construct of claim 2 or the system of claim 40 and (ii) a pharmaceutically acceptable carrier.

69. A method of genetically modifying an immune cell comprising contacting the immune cell with the artificial expression construct of claim 2 or the system of claim 40.

70. A method of treating a cancer antigen-expressing cancer in a subject in need thereof comprising administering a therapeutically effective amount of the artificial expression construct of claim 2, the system of claim 40, the nanoparticle of claim 50, the cell of claim 51 , or the formulation of claim 68 to the subject, thereby treating the cancer antigenexpressing cancer in the subject in need thereof.

71. The method of claim 70, wherein the cancer antigen-expressing cancer comprises a B7- H3-expressing cancer.

72. The method of claim 71 , wherein the B7-H3-expressing cancer comprises a diffuse intrinsic pontine glioma, a diffuse midline glioma, a glioblastoma, a prostate cancer, a renal cell carcinoma, a urothelial cell carcinoma, an ovarian cancer, an osteosarcoma, aneuroblastoma, a mesothelioma, a colorectal cancer, a gastric cancer, a breast cancer, a small cell lung cancer, a non-small-cell lung cancer (NSCLC), or a pancreatic cancer.

73. The method of claim 71, wherein the B7-H3-expressing cancer comprises a diffuse intrinsic pontine glioma or a diffuse midline glioma.

74. The method of claim 70, wherein the subject is a pediatric patient.

75. The method of claim 70, wherein the administering comprises injecting a therapeutically effective amount to a living subject.

76. The method of claim 75, wherein the injecting comprises injecting intracerebroventricularly, intravenously, intradermally, intraarterially, intraparenterally, intranodally, intralymphatically, intravesically, intraperitoneally, intralesionally, intraprostatically, intravaginally, intrarectally, topically, intrathecally, intratumorally, intramuscularly, or subcutaneously.

77. The method of claim 75, wherein the injecting comprises intracerebroventricular injection.

78. The method of claim 75, wherein the injecting comprises intravenous injection.

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