Figure S5. CD137L-/- and anti-CD137L-injected mice show lower antitumor immunity than CD137-/- or anti-CD137-injected mice.

A. Expression of CD137 in intratumoral microphages. Intratumoral macrophages were stained with FITC-conjugated anti-CD45, PE-conjugated anti-CD137, PercpCy5-conjugated anti-Gr-1, APC-conjugated anti-F4/80, and APCCy7-conjugated anti-CD11b antibodies. CD137 expression in CD45+Gr-1-CD11b+F4/80+ cells is shown by FACS histograms. Filled: isotype control; unfilled, CD137 staining. B. Expression of CD137 or CD137L in CT26 tumor cells isolated from tumor masses.

Figure S6. Depletion of NK cells does not affect tumor growth, intratumoral macrophage differentiation and generation of Tc1 cells within tumors.

Background A population of MR1-restricted T cells (MR1T) which responds to tumor-associated antigens has been studied. The MR1T can kill tumor cells from various tissue origins across the MHC barriers, but inert to non-cancerous cells.1 These T cells appears to have a potential to be a safe anti-cancer therapeutics overcoming the HLA-restriction of conventional αβ T cells.2 We have developed Panck T cells, pan-cancer-killing CD8+ T cells, to examine their potential as a therapeutic in human trials. Methods We induced anti-cancer MR1T by stimulating peripheral T cells with cancer cells that were prepared to over express MR1 but not express HLA. MR1T cells with anti-cancer activities were purified and subjected to a rapid expansion in a large scale. These cells were phenotyped, assayed for cancer killing activities in both in vitro, and in vivo. Finally, the TCR sequences of the Panck T cells were analyzed to characterize TCR clones associated with the anti-cancer activities of Panck T cells. Results We established a method of isolating and expanding MR1-restricted Panck T cells at high purity, and confirmed that these cells showed cytotoxicity, IFN-γ and TNF responses to various target cancer cells. They showed strong anti-cancer activities in animal models of various hematologic and solid cancers. We isolated high purity of CD8+ 4-1BB+ T cells except CD8+ TCRva7.2+ T cells, which are MAIT cells after primary induction of Panck T. Final products have characteristics of phenotype, usually CD69hi, CD161- TCRvα7.2-, CD62Lmed, CD45RO+, CD57-, and PD-1-, and have shown anti-cancer activities against multiple cancers in both in vitro, and in vivo animal models. Expression of a representative TCR on T cells mediated anti-cancer effects against multiple cancers comparable to the Panck T itself. Conclusions In conclusion, we have developed and standardized the isolation and expansion method of Panck T, which showed a strong anti-cancer activity to various types of cancers regardless of their HLA types. References Crowther MD, Dolton G, Legut M, Caillaud ME, Lloyd A, Attaf M, Galloway SAE, Rius C, Farrell CP, Szomolay B, Ager A, Parker AL, Fuller A, Donia M, McCluskey J, Rossjohn J, Svane IM, Phillips JD, Sewell AK. Genome-wide CRISPR-Cas9 screening reveals ubiquitous T cell cancer targeting via the monomorphic MHC class I-related protein MR1. Nat Immunol. 2020;21(2):178–185. Lepore M, Kalinichenko A, Calogero S, Kumar P, Paleja B, Schmaler M, Narang V, Zolezzi F, Poidinger M, Mori L, De Libero G. Functionally diverse human T cells recognize non-microbial antigens presented by MR1. Elife. 2017;6:e24476. Ethics Approval Approval has been granted by the Institutional Review Board (IRB) for human-derived material research (NCC2020-0295)

Bone marrow cells of WT or CD137-/- mice were cultured in plates coated with human IgG1 or CD137-Fc¬ in the presence of murine Flt3L for 9 d. IFN-gamma and GM-CSF were added, and cells were cultured for an additional 2 d to polarize differentiated DCs toward CD103+ subsets. CD103+ DCs (middle panel) were gated from CD11c+F4/80- cells (upper panel). Macrophages are categorized as F4/80+CD11b+CD11c+ or F4/80+CD11b+CD11c- cells (upper and lower panels).

Figure S1. CD137-/- and anti-CD137-injected mice show systemic antitumor

e14516 Background: V-set and immunoglobulin domain-containing 4 (VSIG4) is one of the B7 family-related proteins that includes PD-L1, VISTA, and CTLA4 ligand. VSIG4 overexpression is correlated with poor prognosis in patients with ovarian cancer, lung cancer, gastric cancer, high-grade glioma, and multiple myeloma. VSIG4 is also known to function as a receptor for complements and the negative regulation of T cell proliferation. Although VSIG4 is highly expressed in tissue-resident macrophages and tumor-associated macrophages (TAM), its role in the tumor microenvironment (TME) is not fully elucidated. Methods: We previously developed a therapeutic antibody, EU103, in a humanized form of a VSIG4-specific antibody. Most importantly, we demonstrated that EU103 directly acts on the tumor-associated macrophages (TAM) and mechanistically induces repolarization of TAM to tumor killing M1 macrophages and blocks VSIG4-mediated T cell suppression, eventually leading to CD8 + T cell proliferation and tumor suppression in a PBMC-humanized human lung cancer mouse model. Results: Expanding our previous findings with the therapeutic potential of EU103 in a non-small cell lung carcinoma (NSCLC), here, we also proved its therapeutic effect in human ovarian cancer. Firstly, TAMs isolated from ovarian cancer patients express high levels of VSIG4 with immunosuppressive M2 macrophage phenotype. Secondly, EU103 treatment of the TAMs in in vitro studies induces M2-to-M1 conversion and activation of T cells in the ascites leading to ovarian tumor cell killing in a dose-dependent manner. Thirdly, in vivo therapeutic efficacy of EU103 in ovarian cancer, especially through M2-to-M1 conversion, is also verified in both CD34 + cell and peripheral blood mononuclear cell (PBMC) humanized mouse models. Finally, we validate the results above by treating ovarian cancer patient-derived ascites with EU103. Conclusions: Almost 300,000 women are diagnosed with ovarian cancer worldwide each year, and ovarian cancer ranks fifth in cancer deaths among women. In the United States alone, we expect approximately 20,000 new diagnoses and more than 12,000 deaths for this year. Given our findings in this study and clinical significance, we propose here the therapeutic potential of EU103, previously developed by our proprietary antibody discovery and engineering technology, in ovarian cancer.

Background V-set and immunoglobulin domain-containing 4 (VSIG4) is one of the B7 family-related proteins that includes PD-L1, VISTA, and CTLA4 ligand. VSIG4 overexpression is correlated with poor prognosis in patients with ovarian cancer, lung cancer, gastric cancer, high-grade glioma, and multiple myeloma. VSIG4 is also known to function as a receptor for complements and the negative regulation of T cell proliferation. Although VSIG4 is highly expressed in tissue-resident macrophages and tumor-associated macrophages (TAM), its role in the tumor microenvironment (TME) is not fully elucidated. Methods In vivo model Generation PBMC-humanized mouse model shows a relative enrichment of human T cells but generally not enough numbers of human monocyte/macrophage. Therefore, a strategy of adding M2 macrophages was used in the tumor site. Human PBMC were injected i.v. into the NSG mice to humanize. Results We previously developed a therapeutic antibody, EU103, in a humanized form of a VSIG4-specific antibody. Most importantly, we demonstrated that EU103 directly acts on the tumor-associated macrophages (TAM) and mechanistically induces repolarization of TAM to tumor killing M1 macrophages and blocking VSIG4-mediated T cell suppression, eventually leading to CD8+ T cell proliferation and tumor suppression in a PBMC-humanized human lung cancer mouse model. Expanding our previous findings with the therapeutic potential of EU103 in a non-small cell lung carcinoma (NSCLC), here, we also proved its therapeutic effect in human ovarian cancer. Firstly, TAMs isolated from ovarian cancer patients express high levels of VSIG4 with immunosuppressive M2 macrophage phenotype. Secondly, EU103 treatment of the TAMs in in vitro studies induces M2-to-M1 conversion and activation of T cells in the ascites leading to ovarian tumor cell killing in a dose-dependent manner. Thirdly, in vivo therapeutic efficacy of EU103 in ovarian cancer, especially through M2-to-M1 conversion, is also verified in both CD34+ cell and peripheral blood mononuclear cell (PBMC). humanized mouse models. Finally, we validate the results above by treating ovarian cancer patient-driven ascites with EU103. Conclusions Almost 300,000 women are diagnosed with ovarian cancer worldwide each year, and ovarian cancer ranks fifth in cancer deaths among women. In the United States alone, we expect approximately 20,000 new diagnoses and more than 12,000 deaths for this year. Given our findings in this study and clinical significance, we propose here the therapeutic potential of EU103, previously developed by our proprietary antibody discovery and engineering technology, in ovarian cancer.

The interaction between regulatory T (Treg) cells and self-reactive T cells is a crucial mechanism for maintaining immune tolerance. In this study, we investigated the cross-activation of Treg cells by self-antigens and its impact on self-reactive CD8+ T cell responses, with a focus on the P53 signaling pathway. We discovered that major histocompatibility complex (MHC) I-restricted self-peptides not only activated CD8+ T cells but also induced the delayed proliferation of Treg cells. Following HLA-A*0201-restricted Melan-A-specific (pMelan) CD8+ T cells, we observed the direct expansion of Treg cells and concurrent suppression of pMelan+CD8+ T cell proliferation upon stimulation with Melan-A peptide. Transcriptome analysis revealed no significant alterations in specific signaling pathways in pMelan+CD8+ T cells that were co-cultured with activated Treg cells. However, there was a noticeable upregulation of genes involved in P53 accumulation, a critical regulator of cell survival and apoptosis. Consistent with such observation, the blockade of P53 induced a continuous proliferation of pMelan+CD8+ T cells. The concurrent stimulation of Treg cells through self-reactive TCRs by self-antigens provides insights into the immune system's ability to control activated self-reactive CD8+ T cells as part of peripheral tolerance, highlighting the intricate interplay between Treg cells and CD8+ T cells and implicating therapeutic interventions in autoimmune diseases and cancer immunotherapy.

Supplementary Figures 1-6 from Mechanisms Involved in Synergistic Anticancer Immunity of Anti-4-1BB and Anti-CD4 Therapy

Figure S2. CD137-/- and anti-CD137-injected mice show enhanced antitumor immunity to EL-4 lymphoma and Renca renal carcinoma.

Figure S3. Increase in intratumoral CD103+ DCs and Tc1 cells in CD137-/- and anti-CD137-injected mice challenged with Renca tumor cells.

Supplementary Fig. from Mechanisms involved in synergistic anticancer effects of anti-4-1BB and cyclophosphamide therapy

Over the last decade, immunotherapy has revolutionized the way we treat cancer primarily by boosting the body’s own immune system to help fight cancer. In particular, an engineered T cell therapy, namely chimeric antigen receptor (CAR) T cell therapy, is a more aggressive way to modify T cells to recognize cancer cells and destroy them. Although these CAR-T therapies have shown remarkable clinical responses in patients with hematological malignancies, their efficacy in solid tumor treatment has been disappointing due to many challenges: lack of tumor-specific antigen targets, loss of the CAR T-cell persistence, the inability of CAR-T cells to effectively infiltrate into solid tumors, toxicities of cytokine release syndrome and neurologic toxicity, and the immunosuppressive tumor microenvironment (TME). In this regard, we report here that we have successfully developed a CAR-T cell therapy, referred to as EU307, to treat one type of solid tumor, hepatocellular carcinoma (HCC). EU307 is a fourth-generation CAR-T therapy that targets the HCC-specific tumor antigen of glypican-3 (GPC3), and also secretes IL-18 which results in autocrine co-stimulation of CAR-T cells and reprogramming of the TME into a tumor-killing environment. Through a sophistically optimized manufacturing process in our good manufacturing practices (GMP) facility, we are able to manufacture CAR-T cells with stem cell memory (TSCM) and central memory (TCM) phenotypes. Functionally, EU307, when infused with as little as 0.1 × 106 total cells per animal, showed superior in vivo persistence and antitumor immunity in an HCC tumor-bearing mouse model. Furthermore, a single dose toxicity study in the same disease mouse model determined the no observed adverse effect level (NOAEL) as 5.0 × 105 total cells per male and 1.0 × 106 total cells per female. Taken together, our studies demonstrate that we have a developed a novel fourth-generation CAR-T therapy to treat HCC, a solid tumor that expresses a high-level of tumor-specific GPC3, by overcoming previously limiting factors in the development of CAR-T therapies against solid tumors. Citation Format: Joseph H. Jeong, Young-Kyun Chang, You-Yeon Kang, Jeong-Yun Lee, Sae-Rom Lee, Yun-Kyung Lee, Young-Ho Kim, Byoung S. Kwon. IL-18 secreting chimeric antigen receptor T cells targeting glypican-3show superior persistence and antitumor immunity against hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB090.

BACKGROUND AIMS:Human telomerase reverse transcriptase (hTERT) is an attractive target for anti-cancer therapies. We developed an effective method for generating hTERT-specific CD8+ T cells (hTERT-induced natural T cells [TERTiNTs]) using peripheral blood mononuclear cells (PBMCs) from patients with solid cancers and investigated their feasibility and safety. METHODS:This was a single-center phase 1 trial using a 3 + 3 dose escalation design to evaluate six dose levels of TERTiNTs. PBMCs from each patient were screened using an hTERT peptide panel to select those that stimulated CD8+ T cells. The four most stimulatory peptides were used to produce autologous CD8+ T cells from patients refractory or intolerant to standard therapies. Eligible patients received a single intravenous infusion of TERTiNTs at different dose levels (4 × 108 cells/m2, 8 × 108 cells/m2 and 16 × 108 cells/m2). Pre-conditioning chemotherapy, including cyclophosphamide alone or in combination with fludarabine, was administered to induce lymphodepletion. RESULTS:From January 2014 to October 2019, a total of 24 patients with a median of three prior lines of therapy were enrolled. The most common adverse events were lymphopenia (79.2%), nausea (58.3%) and neutropenia (54.2%), mostly caused by pre-conditioning chemotherapy. The TERTiNT infusion was well tolerated, and dose-limiting toxicities were not observed. None of the patients showed objective responses. Seven patients (30.4%) achieved stable disease with a median progression-free survival of 3.9 months (range, 3.2-11.3). At the highest dose level (16 × 108 cells/m2), four of five patients showed disease stabilization. CONCLUSIONS:The generation of TERTiNTs was feasible and safe and provided an interesting disease control rate in heavily pre-treated cancer patients.

Supplementary Figures 1-9 from Combination Therapy with Cisplatin and Anti–4-1BB: Synergistic Anticancer Effects and Amelioration of Cisplatin-Induced Nephrotoxicity

Background V-set and immunoglobulin domain-containing 4 (VSIG4) is a B7 family-related protein which includes PDL1, VISTA and CTLA4 ligand. The known functions of VSIG4 are a receptor for complement to inhibit complement activity and negative regulation of proliferating T cell. Although VSIG4 is highly expressed in tissue-resident macrophage and tumor-associated macrophage (TAM), the mechanism of the signal transduction in tumor microenvironment (TME) is not fully elucidated. Methods Conversion assay CD14 monocytes from PBMC were differentiated to M0 macrophages by rhM-CSF, and subsequently to M2 macrophages with rhIL-4/rhIL-13. The conversion of M2 macrophages to M1 was carried out by incubating the macrophages with LPS/rhIFN-gamma or EU103. In vivo model Generation PBMC-humanized mouse model shows a relative enrichment of human T cells but generally not enough numbers of human monocyte/macrophage. Therefore, a strategy of adding M2 macrophages was used in the tumor site. Human PBMC were injected i.v. into the NSG mice to humanize. To establish a lung cancer orthotopic model, A549-luci lung cancer cells were injected i.v. with differentiated M2 macrophages into the naïve NSG mice, followed by PBMC infusion. The degree of humanization was determined by measuring the ratio of human CD45 cells in the mouse PBMC. Tumor size was measured using IVIS twice a week. Results Here we investigate the pivotal roles of anti-VSIG4 therapeutic antibody, EU103 in TAM. EU103 engagement altered a broad range of type II macrophage (M2) transcriptome which was related to immune-tolerance and increased the proliferation of cytotoxic CD8 T cells. EU103 induced phosphorylation of JNK and p38MAPK and in consequence, decreased the expression of M2 marker CD14, CSF-1R and CD163 while type I macrophage (M1) marker CD86 and CD40 were increased. In the PBMC-based humanized mouse, human orthotopic lung cancer model, stimulation of VSIG4 suppressed tumor growth and promoted infiltration of IFNgamma-producing CD8 T cells into tumor tissue. Furthermore, treatment of EU103 showed synergistic anti-cancer activity with an anti-PD-L1 therapy. Conclusions Our finding suggests that EU103 directly act on TAM and induce repolarization of TAM to tumor suppressive M1 macrophages, leading to a CD8 T cell proliferation and tumor suppression. Since the main cause of Immune Checkpoint Inhibitor (ICI) resistance is an immune-suppressive TME, EU103 could be an option for ICI-resistant patients, and synergistic anti-tumor effect could be expected with a combination with ICIs. Overall, targeting the VSIG4 would be an advantageous approach in cancer therapeutics.

Introduction Although PD-1/L1 mAb has demonstrated clinical benefits in certain cancer types, low response rate and resistance remain the main challenges for the application of these immune checkpoint inhibitors (ICIs). 4-1BB is a co-stimulator molecule expressed in T cells, which could enhance T cell proliferation and activation. Herein, the synergetic antitumor effect and underlying mechanism of 4-1BB agonist combined with PD-1/PD-L1 blockade were determined in B-cell lymphoma (BCL). Methods Subcutaneous transplantation BCL tumor models and metastasis models were established to evaluate the therapeutic effect of PD-L1 antibody and/or 4-1BB agonist in vivo. For the mechanistic study, RNA-seq was applied to analyze the tumor microenvironment and immune-related signal pathway after combination treatment. The level of IFN-γ, perforin, and granzyme B were determined by ELISA and Real-time PCR assays, while tumor-infiltrating T cells were measured by flow cytometry and immunohistochemical analysis. CD4/CD8 specific antibodies were employed to deplete the related T cells to investigate the role CD4+ and CD8+ T cells played in combination treatment. Results Our results showed that combining anti-PD-L1 ICI and 4-1BB agonists elicited regression of BCL and significantly extended the survival of mice compared to either monotherapy. Co-targeting PD-L1 and 4-1BB preferentially promoted intratumoral cytotoxic lymphocyte infiltration and remodeled their function. RNA-sequence analysis uncovered a series of up-regulated genes related to the activation and proliferation of cytotoxic T lymphocytes, further characterized by increased cytokines including IFN-γ, granzyme B, and perforin. Furthermore, depleting CD8+ T cells not CD4+ T cells totally abrogated the antitumor efficacy, indicating the crucial function of the CD8+ T cell subset in the combination therapy. Discussion In summary, our findings demonstrated that 4-1BB agonistic antibody intensified the antitumor immunity of anti-PD-1/PD-L1 ICI via promoting CD8+ T cell infiltration and activation, providing a novel therapeutic strategy to BCL.