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Chimeric antigen receptor T cells (CAR-Ts) have demonstrated remarkable efficacy in leukemia and lymphomas but limited responses in solid tumors. We conducted a phase I trial (NCT02107963) of GD2 CAR-Ts (GD2-CAR.OX40.28.z.ICD9), demonstrating feasibility and safety of administering GD2 CAR-Ts in children and young adults with neuroblastoma and, for the first time, osteosarcoma. 15 patients aged 8-28 years were enrolled on four dose levels, of which 13 patients were infused. No dose-limiting toxicities were observed, and administration of up to 1x107 GD2-CAR-T/kg was feasible and safe for children and young adults with neuroblastoma and osteosarcoma. At Day 28 following GD2 CAR-T infusion, 23.1% (3/13) of evaluable patients had progressive disease and 76.9% (10/13) had stable disease (SD). 3/10 SD patients remained stable at 60 days post-infusion of GD2 CAR-T, but all patients eventually progressed. Since a major barrier to CAR-T efficacy is inadequate CAR-T expansion, we evaluated CAR-T levels and found that patients stratified into good and poor expander groups, observed across dose levels and associated with pro-inflammatory cytokine signatures in patients. To understand the immune cell contributors to CAR-T expansion, patient pre-treatment apheresis, CAR-T product, and post-infusion samples were evaluated by high-dimensional proteomic (CyTOF), transcriptomic (RNAseq), and epigenetic (ATACseq) analyses. In patient apheresis, good CAR-T expansion associated with more open chromatin and with both proteomic and transcriptomic enrichment of naïve T cells, while poor CAR-T expansion associated with increased levels of T effector memory (TEMRA) cells and enrichment of myeloid derived suppressor cell (MDSC) transcriptomic signatures. CAR-T products across patients, regardless of CAR-T expansion, demonstrated increased T cell activation proteomic signatures, with enhanced exhaustion transcriptomic signatures in poor expanders compared to good. The most robust cellular correlate to good CAR-T expansion was a population of CXCR3-expressing monocytes in pre-treatment apheresis. Interestingly, this CXCR3+ monocyte population reduced in post-infusion timepoints of good expanders, resembling levels found in poor expanders. Our findings were validated in TARGET-OS patient data in The Cancer Genome Atlas, where high CXCR3 expression was found to be associated with survival benefit in osteosarcoma patients. CXCR3 has been extensively studied on T cells, but its function on myeloid populations is yet to be fully explored. These results are the first to demonstrate that the peripheral immune environment prior to CAR-T administration may effectively predict and modulate CAR-T expansion in patients. Citation Format: Tara Murty, Sabina Kaczanowska, Ahmad Alimadadi, Cristina Contreras, Caroline Duault, Priyanka Balasubrahmanyan, Warren Reynolds, Norma Gutierrez, Reema Baskar, Catherine Wu, Franziska Michor, Jennifer Altreuter, Yang Liu, Aashna Jhaveri, Vandon Duong, Hima Anbunathan, Radim Moravec, Joyce Hong, Roshni Biswas, Stephen Van Nostrand, James Lindsay, Mina Pichavant, Elena Sotillo, Bita Sahaf, Sean Bendall, Holden Maecker, Steven Highfill, David Stroncek, Melinda Merchant, John Glod, Catherine Hedrick, Crystal Mackall, Sneha Ramakrishna, Rosandra Kaplan. Immune determinants of CAR-T expansion in solid tumor patients receiving GD2 CAR-T cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2142.

Chimeric antigen receptor T-cells (CART) are active in relapsed/refractory (r/r) B-cell acute lymphoblastic leukemia (B-ALL), but relapse remains a substantial challenge. Reinfusion with the same CART product (CART2) in patients with suboptimal response or antigen positive relapse following first infusion (CART1) represents a potential treatment strategy, though early experiences suggest limited efficacy of CART2 with CD19 targeting. We report on our experience with CART2 across a host of novel CAR T-cell trials. This was a retrospective review of children and young adults with B-ALL who received reinfusion with an anti-CD19, anti-CD22, or anti-CD19/22 CART construct on one of 3 CAR T-cells trials at the National Cancer Institute (NCT01593696, NCT02315612, NCT0344839) between July 2012 and January 2021. All patients received lymphodepletion (LD) pre-CART (standard LD: 75 mg/m2 fludarabine, 900 mg/m2 cyclophosphamide; or intensified LD: 120 mg/m2 fludarabine, 1200 mg/m2 cyclophosphamide). Primary objectives were to describe response to and toxicity of CART2. Indication for CART2, impact of LD intensity, and CAR T-cell expansion and leukemia antigen expression between CART infusions was additionally evaluated. Eighteen patients proceeded to CART2 due to persistent (n=7) or relapsed antigen positive disease (n=11) following CART1. Seven of 18 (38.9%) demonstrated objective response (responders) to CART2: 5 achieved a minimal residual disease (MRD) negative CR, 1 had persistent MRD level disease, and 1 showed a partial remission, the latter with eradication of antigen positive disease and emergence of antigen negative B-ALL. Responders included four patients who had not achieved a CR with CART1. Limited cytokine release syndrome was seen following CART2. Peripheral blood CART1 expansion was higher than CART2 expansion (p=0.03). Emergence of antigen negative/dim B-ALL in 6 (33.3%) patients following CART2 contributed to lack of CR. Five of seven (71.4%) responders received intensified LD pre-CART2, which corresponded with higher CART2 expansion than in those receiving standard LD (p=0.029). Diminished CAR T-cell expansion and antigen downregulation/loss impeded robust responses to CART2. A subset of patients, however, may derive benefit from CART2 despite suboptimal response to CART1. Intensified LD may be one strategy to augment CART2 responses, though further study of factors associated with CART2 response, including serial monitoring of antigen expression, is warranted.

TPS624 Background: The mortality of hepatocellular carcinoma (HCC) is increasing worldwide, but outcome of systemic treatments in advanced HCC is suboptimal. Adoptive T-cell transfer therapy represents a promising field that exploits the ability of T-cells to recognize and eliminate their target. Targeting the tumor-associated antigen glypican- 3 (GPC3) through chimeric antigen receptors (CAR) engineered T cells is a mechanistically rational novel treatment for advanced HCC. This study aims to determine the dose and early signals of GPC3 targeted (CAR)-T cells in advanced GPC3 expressing HCC (NCT05003895). Methods: This phase I first in human dose escalation trial will study the safety and feasibility of CAR (hYP7)- T cells in advanced HCC patients expressing GPC3. Eligibility criteria includes advanced HCC, not candidates for curative interventions, progressed on first line systemic treatment, tumor GPC3 positivity of ³ 25% by IHC, Child-Pugh Class A, ³1 measurable lesion, ECOG 0 or 1, adequate organ and marrow function. ParticipantsÕ T cells collected through leukapheresis will be transduced with a lentivirus encoding the CAR construct to generate CAR expressing T cells. Patients receive a conditioning chemotherapy regimen of cyclophosphamide and fludarabine prior to the infusion of the GPC3 directed CAR-T cells (Table). The trial has a 4-level modified Fibonacci dose escalation with a minimum of 3 patients at each level and a 28-day interval between the first three patients. Response will be assessed by imaging every two months during the first year. Patients undergo close monitoring with safety assessments during the first year and are followed for life. The primary objective is to determine the MTD, DLT, safety and feasibility of anti-GPC3 CAR expressing T- cells in patients with GPC3-expressing advanced HCC. Secondary objectives include best overall response and overall survival. Exploratory objectives are multiple and include studies to evaluate the persistence and peak levels of anti-GPC3 CAR-T cells after infusion. Recruitment began in December 2021 and the two patients at dose level -1 has been treated; the planned sample size is 38 patients. Table 1 Dose Level Anti-GPC3 CAR-T Cyclophosphamide (mg/m2) Fludarabine (mg/m2) Level -1 0.3x106 CAR-T per kg bw 200 30 Level 1 0.3x106 CAR-T per kg bw 300 30 Level 2 1x106 CAR-T per kg bw 300 30 Level 3 3x106 CAR-T per kg bw 300 30. Clinical trial information: NCT05003895 .

Rhabdomyosarcoma (RMS) is an aggressive soft tissue sarcoma originating from skeletal muscle in children and adolescent young adults. Despite multi-modal aggressive therapies, relapsed, refractory or metastatic rhabdomyosarcoma remains a lethal disease with no significant improvement in outcome over decades of clinical trials. Therefore novel therapies are needed. FGFR4 is a developmentally regulated cell surface receptor tyrosine kinase that is overexpressed in RMS when compared with normal tissues, and mutationally activated in about 7.5% of RMS. Recently we showed that PAX3-FOXO1 establishes a super-enhancer in the FGFR4 genomic locus driving its high expression in fusion positive RMS. CAR T-cell therapy is effective in treating refractory and relapsed B-cell leukemia and lymphoma, with three CARs targeting CD19 approved by the FDA. Multiple CART trials are currently underway for solid tumors. Since FGFR4 is a cell surface protein, we hypothesized that FGFR4 will provide a rational target for immunotherapy in RMS. We confirmed by immunohistochemistry staining, western analysis, and Meso Scale Discovery that FGFR4 protein is highly differentially expressed in RMS samples. We developed a murine anti-FGFR4 antibody, 3A11, by immunizing mouse with FGFR4-IG fusion protein. 3A11 showed high affinity and specificity of binding to FGFR4. We then developed a second-generation CAR using the VL and VH domain of 3A11 antibody and found that the scFvFc retained its specificity and high affinity at nanomolar range. Human T cells transduced with 3A11 CAR construct were found to be highly potent at inducing IFN-γ, TNF-α, IL-2 and cytotoxicity when the FGFR4-CART was co-cultured with RMS cells, but not with RMS cells with FGFR4 knocked out or FGFR4 negative cells. 3A11 CART incubated with human primary cells obtained from liver, kidney, heart, and pancreas, did not elicit a cytokine response, indicating a low potential for “on-target off-tumor” toxicity. In vivo testing also found that 3A11 CART eliminated RMS cells in both murine xenograft metastatic and localized subcutaneous models. Therefore we have developed a CART targeting FGFR4 that shows high potency for treating RMS. A phase 1 FGFR4-CART clinical trial is planned for children and adolescent young adults with relapsed/refractory rhabdomyosarcoma. Citation Format: Adam Tai Chi Cheuk, Meijie Tian, Nityashree Shivaprasad, Steven Highfill, David Milewski, G Tom Brown, Peter Azorsa, Dina Schneider, Berkley Gryder, Jun S Wei, Young Kwok Song, Hsien-Chao Chou, Jerry Wu, Joon-Yong Chung, Brian Belyea, Corinne Linardic, Stephen M Hewitt, Boro Dropulic, Rimas Orentas, Javed Khan. Potent antitumor activity of a FGFR4 CAR-T in rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB213.

TPS2678 Background: T cell receptor (TCR)-T cell therapy is an emerging cancer treatment strategy. Thus far, demonstration of clinical activity has been limited to a subset of solid tumors including melanoma, synovial cell sarcoma and HPV-associated cancer. It is estimated that metastatic epithelial cancers are responsible for approximately 90% of cancer deaths in the United States. The estimated 600,000 cancer deaths each year is driven largely by lung adeno- and squamous cell carcinoma and invasive breast cancer, which account for approximately 30% of all cancer-related deaths. Kita-Kyushu Lung Cancer Antigen 1 (KK-LC-1) is a cancer germline antigen with expression restricted to germ cells in adults and certain epithelial cancers including lung, breast, gastric and cervical. We identified a KK-LC-1 TCR from the tumor-infiltrating lymphocytes (TIL) of a patient with cervical cancer who had a complete tumor response to TIL therapy. The KK-LC-1 TCR was the most dominate clonotype in the infused TIL product and persisted in the peripheral blood following infusion, suggesting that it may have contributed to cancer regression in this patient. Methods: We are conducting a phase I cell dose escalation trial to test the safety and efficacy of KK-LC-1 TCR-T cell therapy in patients with metastatic KK-LC-1 positive epithelial cancer. Patients receive a lymphocyte depleting conditioning regimen followed by a one-time infusion of genetically engineered T cells expressing the KK-LC-1 TCR (KK-LC-1 TCR-T cells) and high-dose systemic aldesleukin. KK-LC-1 positivity is determined by RNAscope assay measuring the percentage of cancer cells expressing CT83 (gene encoding KK-LC-1) with a percentage of 25 or greater considered positive. Main inclusion criteria include HLA-A*01:01 allele, prior first-line therapy, ECOG of 0 or 1 and adequate organ and hematologic function. Main exclusion criteria include active major medical illness of the cardiovascular, respiratory or immune system, primary or secondary immunodeficiency and autoimmune disease. Participants will be entered in sequential dose levels and receive escalating doses of cells beginning at 1x108 and ending with 6x1010. Dose-limiting toxicities will be assessed during the first 30 days of cell infusion. The primary objective is to determine the maximally tolerated dose of KK-LC-1 TCR-T cells. Exploratory objectives include assessing the safety and efficacy of KK-LC-1 TCR-T cells and to conduct immunologic studies to understand and improve the administered treatment. Clinical trial information: NCT05035407.

While CD19-directed chimeric antigen receptor (CAR) T cells can induce remission in patients with B cell acute lymphoblastic leukemia (ALL), a large subset relapse with CD19- disease. Like CD19, CD22 is broadly expressed by B-lineage cells and thus serves as an alternative immunotherapy target in ALL. Here we present the composite outcomes of two pilot clinical trials ( NCT02588456 and NCT02650414 ) of T cells bearing a 4-1BB-based, CD22-targeting CAR in patients with relapsed or refractory ALL. The primary end point of these studies was to assess safety, and the secondary end point was antileukemic efficacy. We observed unexpectedly low response rates, prompting us to perform detailed interrogation of the responsible CAR biology. We found that shortening of the amino acid linker connecting the variable heavy and light chains of the CAR antigen-binding domain drove receptor homodimerization and antigen-independent signaling. In contrast to CD28-based CARs, autonomously signaling 4-1BB-based CARs demonstrated enhanced immune synapse formation, activation of pro-inflammatory genes and superior effector function. We validated this association between autonomous signaling and enhanced function in several CAR constructs and, on the basis of these observations, designed a new short-linker CD22 single-chain variable fragment for clinical evaluation. Our findings both suggest that tonic 4-1BB-based signaling is beneficial to CAR function and demonstrate the utility of bedside-to-bench-to-bedside translation in the design and implementation of CAR T cell therapies.

This paper discusses the analysis and design for the reconstruction of the Massachusetts Bay Transit Authority (MBTA) Aquarium subway station in downtown Boston. This unusual design includes rebuilding and extension of the Blue Line rapid transit station, and construction of the new I-93 Central Artery highway tunnel directly above it. All construction work will be performed while the existing I-93 viaduct remains in service overhead, the MBTA subway operations are ongoing, and surface street vehicular and pedestrian traffic is maintained.

Genetically engineered T-cells expressing chimeric antigen receptors directly target cell surface antigens on tumor cells. CARs have shown remarkable activity in clinical trials for hematologic malignancies but remain unproven for the treatment of solid tumors. The disialoganglioside GD2 is considered a tumor-associated antigen for neuroblastoma and we have found that GD2 is expressed on pediatric sarcomas. Analysis of primary human tumor tissue samples taken from 18/18 patients with osteosarcoma and alveolar rhabdomyosarcoma (2/15) demonstrate robust expression of GD2 on the cell surface. Based on this finding, we developed assays to test the ability of GD2-CAR T-cells to target human sarcomas. We find that GD2-CAR T-cells specifically lyse GD2-expressing solid tumors in vitro even at low E:T, but fail to induce a response in vivo. In studying these xenografted tumors we discovered an expansion of murine CD11b+Gr1+ MDSC that inhibit human T-cell responses in vitro. This lead us to adopt a combinatorial therapeutic strategy in which we inhibited the suppressive potential of MDSC by administration of all-trans retinoic acid followed by the infusion of GD2-specific CAR T-cells. This resulted in significant improvements in overall survival and tumor growth. The results presented here suggest that ATRA coadministration with CAR T-cell therapy for solid tumors may yield a beneficial additive effect through the modulation of myeloid derived suppressor cells.

In addition to its effect to limit the number of cell division cycles and the accumulation of memory T cells, recent data suggest NFATc2 may be required to enable development and function of regulatory T cells (Tregs). Here, we verified loss of NFATc2 relieved intrinsic negative regulation: effector T cells (CD4+ /CD25-) from NFATc2 knockout (KO) mice showed more facile in vitro proliferation than effector T cells from WT or heterozygous mice and, in vivo had significantly more activated (CD69+ ) and memory phenotype (CD44hi ) T cells. In contrast, we found no differences in the number of Tregs (CD4+ /CD25+ /FoxP3+ ) between NFATc2 KO and WT mice. However, NFATc2 KO Tregs were less able to functionally suppress NFATc2 KO and WT effector cells in vitro. In contrast to NFATc2 KO Tregs, WT Tregs effectively suppressed proliferation of NFATc2 KO T cells, indicating that NFATc2 KO Tregs are hypofunctional. The reduced suppressive capacity of NFATc2 KO vs WT Tregs was not due to global T cell hyperproliferation since Tregs from Tob1 KO mice, that also have hyperproliferative T cells, were more potent suppressors than Tregs from WTs or NFATc2 KOs. The functional deficit of NFATc2 Tregs could not be replicated by treating WT Tregs with the NFAT inhibitor, cyclosporin A. Instead, Treg function was directly correlated with reduced expression of CTLA4 in NFATc2 vs WT Tregs. Our data support the premise that NFATc2 is required for Treg function, possibly by controlling CTLA4 expression.