PDF file - 73K, Similar expression of Granzyme B and perforin in dgk-deficient mesoCAR T cells

Supplementary Table from Characterization of INCB086550: A Potent and Novel Small-Molecule PD-L1 Inhibitor

Reagents for immunohistochemistry studies.

Mesothelin-specific CD3ζ CARs and SS1-KIRS2/Dap12 show comparable antigen-specific in vitro cytotoxicity toward EM-meso cells.

FAP-specific KIR-CAR/Dap12 T cells induce bone marrow hypocellularity and loss of body weight.

Supplementary Figures S1-S4 from Expression of a Functional CCR2 Receptor Enhances Tumor Localization and Tumor Eradication by Retargeted Human T cells Expressing a Mesothelin-Specific Chimeric Antibody Receptor

PDF file - 52K, MesoCAR-transduced dgkzeta-deficient T cells demonstrate resistance to multiple inhibitory stimuli

Figure legends for supplemental data.

PDF file - 1196K, Supplementary Figure 1. Phenotypic analysis of FAP+ stromal cells in untreated TC1 flank tumors in C57BL/6 mice. Supplementary Figure 2. Depletion of FAP+ cells. Supplementary Figure 3. Persistence and antitumor activities of FAP-CAR T cells employing either human 4-1BB (73.3-hBBz) or mouse CD28 (73.3-m28z) co-stimulatory domain in mice and their in vitro activity. Supplementary Figure 4. Deletion of DGKzeta enhanced effector functions and in vivo persistence of FAP-CAR T cells. Supplementary Figure 5. Body weight of FAP-CAR T cells-treated mice remained constant or increased. Supplementary Figure 6. Histology of bone marrows and pancreas following treatment with FAP-CAR T cells. Supplementary Figure 7. Activation-induced cell death of FAP-CAR T cells. Supplementary Figure 8. Differential FAP expression on tumor and pancreatic FAP+ stromal cells. Supplementary Figure 9. mAb 73.3 and mAb FAP5 are specific for distinct epitopes of murine FAP expressed by fibroblasts.

SS1-KIRS2/DAP12 T cells mediate robust anti-tumor activity in vivo.

SS1-KIRS2 does not affect endogenous TCR expression.

Transforming growth factor-β (TGFβ) signaling is common in many solid tumors and is initiated by binding of the high affinity canonical ligands TGFβ1, 2, οr 3 to TGFβR2, which forms a heteromeric receptor complex with TGFβR1 (Derynck et al, Nature Review Clinical Oncology 2020). Activation of the pathway results in potent suppression of immune cell-mediated anti-tumor immunity and has been reported to predict poor response to PD-(L)1 targeted therapy in patients (Mariathasan et al, Nature 2018; Kieffer et al, Cancer Discovery 2020). However, TGFβ drug development has been hampered by the occurrence of adverse events. INCA33890 is a dual PD-1 and TGFβR2 binding bispecific Biclonics® antibody, developed to antagonize the TGFβ signaling pathway specifically in cells co-expressing PD-1 and TGFBR2. Additionally, it potently antagonizes the PD-1 axis independently of TGFβR2 co-expression. The cell-selective action of INCA33890 was designed to mitigate risks of the known adverse effects associated with TGFβ-pathway inhibition in tissues requiring active TGFβ signaling. ΙΝCΑ33890 mediates its specificity through a PD-1 binding arm with a >10-fold higher affinity relative to the TGFβR2 binding arm. Consistent with this profile, in isogenic Jurkat cells expressing TGFβR2 ± PD-1, INCA33890 potently inhibits TGFβ1-induced pSMAD activation in a PD-1-correlated manner. Additionally, in two independent PD-1 reporter assays, INCA33890 inhibited SHP recruitment and enhanced NFAT activation with a potency within an order of magnitude to that of pembrolizumab. In mixed lymphocyte reaction assays with exhausted primary human T-cells, INCA33890 was found to induce a similar level of anti-tumor cytokine production as the combination of pembrolizumab and an anti-TGFβR2 antagonist mAb. Treatment of primary ovarian ascites with INCA33890 ex vivo induced IFNγ production in all donors tested, while pembrolizumab had no activity. Similarly, in human CD34+ cell-engrafted NSG mice, INCA33890 significantly inhibited the growth of human MDA-MB-231 and A375 subcutaneous xenograft tumors, whereas pembrolizumab or an anti-TGFβR2 antibody had little or no monotherapy activity. INCA33890 had a balanced pharmacokinetic and potency profile and was well tolerated in NHPs at exposures required for pharmacodynamic and tumor growth inhibiting activity in rodents. Encouragingly, there was no evidence of adverse effects in NHPs due to TGFβ-pathway blockade. Collectively, these results provide compelling data for an effective and specific approach to simultaneously antagonizing TGFβ and PD-1 signaling in tumors. Clinical development of INCA33890 in checkpoint inhibitor-resistant and other cancers has been initiated. Citation Format: Liang-Chuan S. Wang, Rinse Klooster, Ashwini Kulkarni, Amaya Garcia de Vinuesa, Maxim Soloviev, Linda JA Hendriks, Lu Huo, Michael Weber, Arpita Mondal, Yonghong Zhao, Shane Harvey, Xin He, Hong Chang, April Horsey, Alla Volgina, Yue Zhang, Veethika Pandey, Yan-Ou Yang, Jonathan Rios-Doria, Evgeniy Eruslanov, Daniel J. Powell, Steven M. Albelda, John de Kruif, Horacio Nastri, Cecile Geuijen, Patrick A. Mayes. INCA33890, a novel PD-1×TGFꞵR2 bispecific antibody conditionally antagonizes TGFꞵ signaling in primary immune cells co-expressing PD-1 [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 2936.

Supplementary Figure 1 from CCL2 Blockade Augments Cancer Immunotherapy

Cytotoxicity induced by SS1-KIRS2, but not receptor expression at the cell surface, depends upon DAP12 co-expression.

Abstract Altering the immunosuppressive microenvironment that exists within a tumor will likely be necessary for cancer vaccines to trigger an effective antitumor response. Monocyte chemoattractant proteins (such as CCL2) are produced by many tumors and have both direct and indirect immunoinhibitory effects. We hypothesized that CCL2 blockade would reduce immunosuppression and augment vaccine immunotherapy. Anti-murine CCL2/CCL12 monoclonal antibodies were administered in three immunotherapy models: one aimed at the human papillomavirus E7 antigen expressed by a non–small cell lung cancer (NSCLC) line, one targeted to mesothelin expressed by a mesothelioma cell line, and one using an adenovirus-expressing IFN-α to treat a nonimmunogenic NSCLC line. We evaluated the effect of the combination treatment on tumor growth and assessed the mechanism of these changes by evaluating cytotoxic T cells, immunosuppressive cells, and the tumor microenvironment. Administration of anti-CCL2/CCL12 antibodies along with the vaccines markedly augmented efficacy with enhanced reduction in tumor volume and cures of approximately half of the tumors. The combined treatment generated more total intratumoral CD8+ T cells that were more activated and more antitumor antigen–specific, as measured by tetramer evaluation. Another important potential mechanism was reduction in intratumoral T regulatory cells. CCL2 seems to be a key proximal cytokine mediating immunosuppression in tumors. Its blockade augments CD8+ T-cell immune response to tumors elicited by vaccines via multifactorial mechanisms. These observations suggest that combining CCL2 neutralization with vaccines should be considered in future immunotherapy trials. Cancer Res; 70(1); 109–18

Effect of TIL isolation treatment on CAR expression in T cells.

PDF file - 103K, Enhanced expression of FasL and TRAIL in dgk-deficient mesoCAR T cells

Alteration of tumor structure by FAP-CAR T cells in murine PDAs.