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S3. Serial quantification of serum IL6 from a patient with prolonged subjectove myalgia post IT injection of mRNA c-Met CAR T cells.

S1. Clinical trial schema. S2. Representative immunohistochemistry (IHC). S3. Histology and IHC of tumor tissue pre and post intratumoral injection of RNA CAR T c- Met from one patient. S4. Histology and IHC of tumor tissue pre and post intratumoral injection with lidocaine (1 mL) and RNA CAR T c-Met (1 mL) from another patient. S5. Characterization of CD68+ tumor infiltrated immune cells using multiplex IHC analyses as described (1) in pre and post IT injection of RNA CAR T c-Met tumor tissue sections from two patients.

Chimeric antigen receptors (CAR) are synthetic molecules that provide new specificities to T cells. Although successful in treatment of hematologic malignancies, CAR T cells are ineffective for solid tumors to date. We found that the cell-surface molecule c-Met was expressed in ∼50% of breast tumors, prompting the construction of a CAR T cell specific for c-Met, which halted tumor growth in immune-incompetent mice with tumor xenografts. We then evaluated the safety and feasibility of treating metastatic breast cancer with intratumoral administration of mRNA-transfected c-Met-CAR T cells in a phase 0 clinical trial (NCT01837602). Introducing the CAR construct via mRNA ensured safety by limiting the nontumor cell effects (on-target/off-tumor) of targeting c-Met. Patients with metastatic breast cancer with accessible cutaneous or lymph node metastases received a single intratumoral injection of 3 × 107 or 3 × 108 cells. CAR T mRNA was detectable in peripheral blood and in the injected tumor tissues after intratumoral injection in 2 and 4 patients, respectively. mRNA c-Met-CAR T cell injections were well tolerated, as none of the patients had study drug–related adverse effects greater than grade 1. Tumors treated with intratumoral injected mRNA c-Met-CAR T cells were excised and analyzed by immunohistochemistry, revealing extensive tumor necrosis at the injection site, cellular debris, loss of c-Met immunoreactivity, all surrounded by macrophages at the leading edges and within necrotic zones. We conclude that intratumoral injections of mRNA c-Met-CAR T cells are well tolerated and evoke an inflammatory response within tumors. Cancer Immunol Res; 5(12); 1152–61. ©2017 AACR.

SummaryGlioblastoma (GBM) treatment includes maximal safe resection of the core and MRI contrast-enhancing (CE) tumor. Complete resection of the infiltrative non-contrast-enhancing (NCE) tumor rim is rarely achieved. We established a safe, semi-automated workflow for spatially-registered sampling of MRI-defined GBM regions in 19 patients with downstream analysis and biobanking, enabling studies of NCE, wherefrom recurrence/progression typically occurs. Immunophenotyping revealed underrepresentation of myeloid cell subsets and CD8+ T cells in the NCE. While NCE T cells phenotypically and functionally resembled those in matching CE tumor, subsets of activated (CD69hi) effector memory CD8+ T cells were overrepresented. Contrarily, CD25hiTregs and other subsets were underrepresented. Overall, our study demonstrated that MRI-guided, spatially-registered, intraoperative immunosampling is feasible as part of routine GBM surgery. Further elucidation of the shared and spatially distinct microenvironmental biology of GBM will enable development of therapeutic approaches targeting the NCE infiltrative tumor to decrease GBM recurrence.

OBJECTIVE: Heterogeneity has hampered sepsis trials, and sub-phenotyping may assist with enrichment strategies. However, biomarker-based strategies are difficult to operationalize. Four sub-phenotypes defined by distinct temperature trajectories in the first 72 hours have been reported in adult sepsis. Given the distinct epidemiology of pediatric sepsis, the existence and relevance of temperature trajectory-defined sub-phenotypes in children is unknown. We aimed to classify septic children into de novo sub-phenotypes derived from temperature trajectories in the first 72 hours, and compare cytokine, immune function, and immunometabolic markers across subgroups. METHODS: This was a secondary analysis of a prospective cohort of 191 critically ill septic children recruited from a single academic pediatric intensive care unit. We performed group-based trajectory modeling using temperatures over the first 72 hours of sepsis to identify latent profiles. We then used mixed effects regression to determine if temperature trajectory-defined sub-phenotypes were associated with cytokine levels, immune function, and mitochondrial respiration. RESULTS: We identified four temperature trajectory-defined sub-phenotypes: hypothermic, normothermic, hyperthermic fast-resolvers, and hyperthermic slow-resolvers. Hypothermic patients were less often previously healthy and exhibited lower levels of pro- and anti-inflammatory cytokines and chemokines. Hospital mortality did not differ between hypothermic children (17%) and other sub-phenotypes (3 to 11%; p = 0.26). CONCLUSIONS: Critically ill septic children can be categorized into temperature trajectory-defined sub-phenotypes that parallel adult sepsis. Hypothermic children exhibit a blunted cytokine and chemokine profile. Group-based trajectory modeling has utility for identifying subtypes of clinical syndromes by incorporating readily available longitudinal data, rather than relying on inputs from a single timepoint.

Chimeric antigen receptor (CAR) T cell therapies targeting CD19 and CD22 have been successful for treating B cell cancers, but CAR T cells targeting non–B cell cancers remain unsuccessful. We propose that rather than being strictly a side effect of therapy, the large number of CAR interactions with normal B cells may be a key contributor to clinical CAR T cell responses.

Chimeric antigen receptor (CAR) T-cell therapy has achieved remarkable milestones in the treatment of B-cell malignancies. However, cancer cells frequently survive CAR T-cell killing in a large cohort of patients. Relapse oftentimes is associated with antigen loss. In this issue, Im and colleagues report a new mechanism of leukemic-cell resistance to anti-CD19 CAR T cells: Leukemic cells can enable a B-cell activation and germinal center reaction signature, which causes CD19 transcriptional downregulation and survival from CAR exposure. See related article by Im et al., p. 1055 (5).

Along with the clinical success of immuno-oncology drugs and cellular therapies, T-cell biology has attracted considerable attention in the immunology community. Long-term immunity, traditionally analyzed in the context of infection, is increasingly studied in cancer. Many signaling pathways, transcription factors, and metabolic regulators have been shown to participate in the formation of memory T cells. There is increasing evidence that the signal transducer and activator of transcription-3 (STAT3) signaling pathway is crucial for the formation of long-term T-cell immunity capable of efficient recall responses. In this review, we summarize what is currently known about STAT3 role in the context of memory T-cell formation and antitumor immunity.

Background: The efficacy of anti-CD19 Chimeric Antigen Receptor (CAR)-T cells in treating relapsed/refractory chronic lymphoid leukemia has proven effective, but only in a small group of cases. Our group previously reported that TET2 disruption promoted the therapeutic efficacy of CART19 cells via the enrichment of the CD8+ central-memory (Tcm) subsets. TET2 is a tumor suppressor; hence, the permanent ablation via genome engineering could result in oncogenic transformation. Since TET2 utilizes alpha-ketoglutarate as a cofactor in the removal of methylgroups from cytosine residues in DNA, the competitive inhibition of TET2 could prove safer. We here explore this strategy by expressing an R132H variant of isocitrate dehydrogenase-I (IDH1), which generates millimolar quantities of D-2-hydroxyglutarate, a competitive inhibitor of TET2. Methods: We generated a lentiviral construct carrying the full-length wildtype or mutant IDH1 open reading frame aminoterminal of a fully human, 4-1BB/CD3z-signaling CAR19, separated by a 2A element. A cell-surface expressed marker carrying the epitopes for CD20 and CD34 was added for CAR-T cells detection and purification. The above construct was transduced into healthy donors' T lymphocytes by a lentiviral transfer system. Sorted engineered cells were subsequently stimulated with artificial antigen presenting cells (aAPCs) expressing the target antigen. Cell growth was monitored and flow cytometry immunophenotyping was performed at the given time points upon several rounds of restimulation. Results: Western blot assay showed IDH1 R132H neomorph only expressed in the CAR-T cells carrying mutant IDH1. The proliferation of IDH1 neomorph-expressing CAR-T cells was similar to the IDH1 wildtype group in this repeat stimulation assay. Similar results were obtained with the anti-CD3 expressing APCs. However, CAR-T cells harboring IDH1 R132H contained a remarkable enrichment of CD8+ anti-CD19 CAR T cells after four rounds of tumor stimulations compared with the control groups. To correct for donor-to-donor variability in the proportion of CD4+ and CD8+ T cells at the start of the experiment, we seeded engineered T cells at a 2:1 ratio. The selective elevation of CD8+ proportion was only observed in CAR-T cells with IDH1 R132H. We further found CAR-T cells having mutant IDH1 displayed an increasing enrichment of CD8+ Tcm (CD45RO+CD27+) subsets after repetitive tumor challenges. In contrast, the other two groups showed a stable or reducing percentage of CD8+ Tcm. Conclusion: The above findings suggested that the introduction of IDH1[R132H] in CAR-T cells could lead to an increase of CD8+ frequency, as well as CD8+ Tcm proportion upon CAR signaling activation. Meanwhile, this introduction didn't make CAR-T cells grow wildly, indicating it is a safe strategy with the potential for further preclinical and clinical studies. Citation Format: Jie Xu, Yaroslav Kaminskiy, Jan Joseph Melenhorst. Chimeric antigen receptor T cells carrying IDH1 neomorph increase CD8 positive central memory proportion without interfering with cell growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1507.