Instructor, Stanford Institutes of Medicine
Enhanced safety and efficacy of protease-regulated CAR-T cell receptors.
Regulatable CAR platforms could circumvent toxicities associated with CAR-T therapy, but existing systems have shortcomings including leakiness and attenuated activity. Here, we present SNIP CARs, a protease-based platform for regulating CAR activity using an FDA-approved small molecule. Design iterations yielded CAR-T cells that manifest full functional capacity with drug and no leaky activity in the absence of drug. In numerous models, SNIP CAR-T cells were more potent than constitutive CAR-T cells and showed diminished T cell exhaustion and greater stemness. In a ROR1-based CAR lethality model, drug cessation following toxicity onset reversed toxicity, thereby credentialing the platform as a safety switch. In the same model, reduced drug dosing opened a therapeutic window that resulted in tumor eradication in the absence of toxicity. SNIP CARs enable remote tuning of CAR activity, which provides solutions to safety and efficacy barriers that are currently limiting progress in using CAR-T cells to treat solid tumors.
View details for DOI 10.1016/j.cell.2022.03.041
View details for PubMedID 35483375
Anti-GD2 synergizes with CD47 blockade to mediate tumor eradication.
The disialoganglioside GD2 is overexpressed on several solid tumors, and monoclonal antibodies targeting GD2 have substantially improved outcomes for children with high-risk neuroblastoma. However, approximately 40% of patients with neuroblastoma still relapse, and anti-GD2 has not mediated significant clinical activity in any other GD2+ malignancy. Macrophages are important mediators of anti-tumor immunity, but tumors resist macrophage phagocytosis through expression of the checkpoint molecule CD47, a so-called 'Don't eat me' signal. In this study, we establish potent synergy for the combination of anti-GD2 and anti-CD47 in syngeneic and xenograft mouse models of neuroblastoma, where the combination eradicates tumors, as well as osteosarcoma and small-cell lung cancer, where the combination significantly reduces tumor burden and extends survival. This synergy is driven by two GD2-specific factors that reorient the balance of macrophage activity. Ligation of GD2 on tumor cells (a) causes upregulation of surface calreticulin, a pro-phagocytic 'Eat me' signal that primes cells for removal and (b) interrupts the interaction of GD2 with its newly identified ligand, the inhibitory immunoreceptor Siglec-7. This work credentials the combination of anti-GD2 and anti-CD47 for clinical translation and suggests that CD47 blockade will be most efficacious in combination with monoclonal antibodies that alter additional pro- and anti-phagocytic signals within the tumor microenvironment.
View details for DOI 10.1038/s41591-021-01625-x
View details for PubMedID 35027753
- GPC2-CAR T cells tuned for low antigen density mediate potent activity against neuroblastoma without toxicity CANCER CELL 2022; 40 (1): 53-+
GPC2-CAR Tcells tuned for low antigen density mediate potent activity against neuroblastoma without toxicity.
Pediatric cancers often mimic fetal tissues and express proteins normally silenced postnatally that could serve as immune targets. We developed Tcells expressing chimeric antigen receptors (CARs) targeting glypican-2 (GPC2), a fetal antigen expressed on neuroblastoma (NB) and several other solid tumors. CARs engineered using standard designs control NBs with transgenic GPC2 overexpression, but not those expressing clinically relevant GPC2 site density (5,000 molecules/cell, range 1-6* 103). Iterative engineering of transmembrane (TM) and co-stimulatory domains plus overexpression of c-Jun lowered the GPC2-CAR antigen density threshold, enabling potent and durable eradication of NBs expressing clinically relevant GPC2 antigen density, without toxicity. These studies highlight the critical interplay between CAR design and antigen density threshold, demonstrate potent efficacy and safety of a lead GPC2-CAR candidate suitable for clinical testing, and credential oncofetal antigens as a promising class of targets for CAR Tcell therapy of solid tumors.
View details for DOI 10.1016/j.ccell.2021.12.005
View details for PubMedID 34971569
Potent activity of CAR T cells targeting the oncofetal protein GPC2 engineered to recognize low antigen density in neuroblastoma.
AMER ASSOC CANCER RESEARCH. 2021
View details for Web of Science ID 000680263502187
Transient rest restores functionality in exhausted CAR-T cells through epigenetic remodeling.
Science (New York, N.Y.)
2021; 372 (6537)
T cell exhaustion limits immune responses against cancer and is a major cause of resistance to chimeric antigen receptor (CAR)-T cell therapeutics. Using murine xenograft models and an in vitro model wherein tonic CAR signaling induces hallmark features of exhaustion, we tested the effect of transient cessation of receptor signaling, or rest, on the development and maintenance of exhaustion. Induction of rest through enforced down-regulation of the CAR protein using a drug-regulatable system or treatment with the multikinase inhibitor dasatinib resulted in the acquisition of a memory-like phenotype, global transcriptional and epigenetic reprogramming, and restored antitumor functionality in exhausted CAR-T cells. This work demonstrates that rest can enhance CAR-T cell efficacy by preventing or reversing exhaustion, and it challenges the notion that exhaustion is an epigenetically fixed state.
View details for DOI 10.1126/science.aba1786
View details for PubMedID 33795428
Glypican-2 targeted CAR T cells designed to effectively eradicate endogenous site density solid tumors in the absence of toxicity
AMER ASSOC CANCER RESEARCH. 2020: 46
View details for Web of Science ID 000551367400054
Locoregionally administered B7H3-targeting CAR T cells mediate potent antitumor effects in atypical teratoid/rhabdoid tumor
AMER ASSOC CANCER RESEARCH. 2020: 33
View details for Web of Science ID 000551367400032
GD2 is a macrophage checkpoint molecule and combined GD2/CD47 blockade results in synergistic effects and tumor clearance in xenograft models of neuroblastoma and osteosarcoma
AMER ASSOC CANCER RESEARCH. 2020: 35
View details for Web of Science ID 000551367400035
Tuning the Antigen Density Requirement for CAR T Cell Activity.
Insufficient reactivity against cells with low antigen density has emerged as an important cause of CAR resistance. Little is known about factors that modulate the threshold for antigen recognition. We demonstrate that CD19 CAR activity is dependent upon antigen density and the CAR construct in axicabtagene-ciloleucel (CD19-CD28z) outperforms that in tisagenlecleucel (CD19-4-1BBz) against antigen low tumors. Enhancing signal strength by including additional ITAMs in the CAR enables recognition of low antigen density cells, while ITAM deletions blunt signal and increase the antigen density threshold. Further, replacement of the CD8 hinge-transmembrane (H/T) region of a 4-1BBz CAR with a CD28-H/T lowers the threshold for CAR reactivity despite identical signaling molecules. CARs incorporating a CD28-H/T demonstrate a more stable and efficient immunological synapse. Precise design of CARs can tune the threshold for antigen recognition and endow 4-1BBz-CARs with enhanced capacity to recognize antigen low targets while retaining a superior capacity for persistence.
View details for DOI 10.1158/2159-8290.CD-19-0945
View details for PubMedID 32193224
Locoregionally administered B7-H3-targeted CAR T cells for treatment of atypical teratoid/rhabdoid tumors.
Atypical teratoid/rhabdoid tumors (ATRTs) typically arise in the central nervous system (CNS) of children under 3 years of age. Despite intensive multimodal therapy (surgery, chemotherapy and, if age permits, radiotherapy), median survival is 17 months1,2. We show that ATRTs robustly express B7-H3/CD276 that does not result from the inactivating mutations in SMARCB1 (refs. 3,4), which drive oncogenesis in ATRT, but requires residual SWItch/Sucrose Non-Fermentable (SWI/SNF) activity mediated by BRG1/SMARCA4. Consistent with the embryonic origin of ATRT5,6, B7-H3 is highly expressed on the prenatal, but not postnatal, brain. B7-H3.BB.z-chimeric antigen receptor (CAR) T cells administered intracerebroventricularly or intratumorally mediate potent antitumor effects against cerebral ATRT xenografts in mice, with faster kinetics, greater potency and reduced systemic levels of inflammatory cytokines compared to CAR T cells administered intravenously. CAR T cells administered ICV also traffic from the CNS into the periphery; following clearance of ATRT xenografts, B7-H3.BB.z-CAR T cells administered intracerebroventricularly or intravenously mediate antigen-specific protection from tumor rechallenge, both in the brain and periphery. These results identify B7-H3 as a compelling therapeutic target for this largely incurable pediatric tumor and demonstrate important advantages of locoregional compared to systemic delivery of CAR T cells for the treatment of CNS malignancies.
View details for DOI 10.1038/s41591-020-0821-8
View details for PubMedID 32341579
- CAR T Cells Targeting B7-H3, a Pan-Cancer Antigen, Demonstrate Potent Preclinical Activity Against Pediatric Solid Tumors and Brain Tumors CLINICAL CANCER RESEARCH 2019; 25 (8): 2560–74
- LOCALLY ADMINISTERED CART CELLS DEMONSTRATE MOST FAVORABLE ROUTE OF ADMINISTRATION IN A MODEL OF ATRT OXFORD UNIV PRESS INC. 2019: 94–95
CAR T cells targeting B7-H3, a Pan-Cancer Antigen, Demonstrate Potent Preclinical Activity Against Pediatric Solid Tumors and Brain Tumors.
Clinical cancer research : an official journal of the American Association for Cancer Research
PURPOSE: Patients with relapsed pediatric solid tumors and CNS malignancies have few therapeutic options and frequently die of their disease. Chimeric antigen receptor (CAR) T cells have shown tremendous success in treating relapsed pediatric acute lymphoblastic leukemia, but this has not yet translated to treating solid tumors. This is partially due to a paucity of differentially expressed cell surface molecules on solid tumors that can be safely targeted. Here, we present B7-H3 (CD276) as a putative target for CAR T cell therapy of pediatric solid tumors, including those arising in the central nervous system.EXPERIMENTAL DESIGN: We developed a novel B7-H3 CAR whose binder is derived from a monoclonal antibody that has been shown to preferentially bind tumor tissues and has been safely used in humans in early phase clinical trials. We tested B7-H3 CAR T cells in a variety of pediatric cancer models.RESULTS: B7-H3 CAR T cells mediate significant anti-tumor activity in vivo, causing regression of established solid tumors in xenograft models including osteosarcoma, medulloblastoma, and Ewing sarcoma. We demonstrate that B7-H3 CAR T cell efficacy is largely dependent upon high surface target antigen density on tumor tissues and that activity is greatly diminished against target cells that express low levels of antigen, thus providing a possible therapeutic window despite low-level normal tissue expression of B7-H3.CONCLUSIONS: B7-H3 CAR T cells could represent an exciting therapeutic option for patients with certain lethal relapsed or refractory pediatric malignancies which should be tested in carefully designed clinical trials.
View details for PubMedID 30655315
Pregnancy-Associated Plasma Protein-A (PAPP-A) in Ewing Sarcoma: Role in Tumor Growth and Immune Evasion.
Journal of the National Cancer Institute
Ewing sarcoma (EWS) manifests one of the lowest somatic mutation rates of any cancer, leading to a scarcity of druggable mutations and neoantigens. Immunotherapeutics targeting differentially expressed cell surface antigens could provide therapeutic benefit for such tumors. Pregnancy-associated plasma protein A (PAPP-A) is a cell membrane-associated proteinase produced by the placenta that promotes fetal growth by inducing insulinlike growth factor (IGF) signaling.By comparing RNA expression of cell surface proteins in EWS (n = 120) versus normal tissues (n = 42), we comprehensively characterized the surfaceome of EWS to identify highly differentially expressed molecules. Using CRISPR/Cas-9 and anti-PAPP-A antibodies, we investigated biological roles for PAPP-A in EWS in vitro and in vivo in NSG xenograft models and performed RNA-sequencing on PAPPA knockout clones (n = 5) and controls (n = 3). All statistical tests were two-sided.EWS surfaceome analysis identified 11 highly differentially overexpressed genes, with PAPPA ranking second in differential expression. In EWS cell lines, genetic knockout of PAPPA and treatment with anti-PAPP-A antibodies revealed an essential survival role by regulating local IGF-1 bioavailability. MAb-mediated PAPPA inhibition diminished EWS growth in orthotopic xenografts (leg area mm2 at day 49 IgG2a control (CTRL) [n = 14], mean = 397.0, SD = 86.1 vs anti-PAPP-A [n = 14], mean = 311.7, SD = 155.0; P = .03; median OS anti-PAPP-A = 52.5 days, 95% CI = 46.0 to 63.0 days vs IgG2a = 45.0 days, 95% CI = 42.0 to 52.0 days; P = .02) and improved the efficacy of anti-IGF-1R treatment (leg area mm2 at day 49 anti-PAPP-A + anti-IGF-1R [n = 15], mean = 217.9, SD = 148.5 vs IgG2a-CTRL; P < .001; median OS anti-PAPP-A + anti-IGF1R = 63.0 days, 95% CI = 52.0 to 67.0 days vs IgG2a-CTRL; P < .001). Unexpectedly, PAPPA knockout in EWS cell lines induced interferon (IFN)-response genes, including proteins associated with antigen processing/presentation. Consistently, gene expression profiles in PAPPA-low EWS tumors were enriched for immune response pathways.This work provides a comprehensive characterization of the surfaceome of EWS, credentials PAPP-A as a highly differentially expressed therapeutic target, and discovers a novel link between IGF-1 signaling and immune evasion in cancer, thus implicating shared mechanisms of immune evasion between EWS and the placenta.
View details for DOI 10.1093/jnci/djy209
View details for PubMedID 30698726
CHECKPOINT MOLECULE B7-H3 IS HIGHLY EXPRESSED ON ATYPICAL RHABDOID TERATOID TUMOR (ATRT) AND IS A PROMISING CANDIDATE FOR CAR T CELL THERAPY
OXFORD UNIV PRESS INC. 2018: 33
View details for Web of Science ID 000438339000025
CAR T CELLS TARGETING B7-H3, A PAN-CANCER ANTIGEN, DEMONSTRATE POTENT PRECLINICAL ACTIVITY AGAINST PEDIATRIC SOLID TUMORS AND BRAIN TUMORS
View details for Web of Science ID 000428851200005
Harnessing the Immunotherapy Revolution for the Treatment of Childhood Cancers
2017; 31 (4): 476-485
Cancer immunotherapies can be classified into agents that amplify natural immune responses (e.g., checkpoint inhibitors) versus synthetic immunotherapies designed to initiate new responses (e.g., monoclonal antibodies [mAbs], chimeric antigen receptors [CARs]). Checkpoint inhibitors mediate unprecedented benefit in some adult cancers, but have not demonstrated significant activity in pediatric cancers, likely due their paucity of neoantigens. In contrast, synthetic immunotherapies such as mAbs and CAR T cells demonstrate impressive effects against childhood cancers. Intense efforts are underway to enhance the effectiveness of pediatric cancer immunotherapies through improved engineering of synthetic immunotherapies and by combining these with agents designed to amplify immune responses.
View details for DOI 10.1016/j.ccell.2017.03.002
View details for Web of Science ID 000398670600005
View details for PubMedID 28366678
Identification of GPC2 as an Oncoprotein and Candidate Immunotherapeutic Target in High-Risk Neuroblastoma.
2017; 32 (3): 295–309.e12
We developed an RNA-sequencing-based pipeline to discover differentially expressed cell-surface molecules in neuroblastoma that meet criteria for optimal immunotherapeutic target safety and efficacy. Here, we show that GPC2 is a strong candidate immunotherapeutic target in this childhood cancer. We demonstrate high GPC2 expression in neuroblastoma due to MYCN transcriptional activation and/or somatic gain of the GPC2 locus. We confirm GPC2 to be highly expressed on most neuroblastomas, but not detectable at appreciable levels in normal childhood tissues. In addition, we demonstrate that GPC2 is required for neuroblastoma proliferation. Finally, we develop a GPC2-directed antibody-drug conjugate that is potently cytotoxic to GPC2-expressing neuroblastoma cells. Collectively, these findings validate GPC2 as a non-mutated neuroblastoma oncoprotein and candidate immunotherapeutic target.
View details for PubMedID 28898695
View details for PubMedCentralID PMC5600520