Murine Solid Tumor Models Research Articles

Local delivery of accutox® synergises with immune-checkpoint inhibitors at disrupting tumor growth

BackgroundThe Accum® platform was initially designed to accumulate biomedicines in target cells by inducing endosomal-to-cytosol escape. Interestingly however, the use of unconjugated Accum® was observed to trigger cell death in a variety of cancer cell lines; a property further exploited in the development of Accum®-based anti-cancer therapies. Despite the impressive pro-killing abilities of the parent molecule, some cancer cell lines exhibited resistance. This prompted us to test additional Accum® variants, which led to the identification of the AccuTOX® molecule.MethodsA series of flow-cytometry and cell-based assays were used to assess the pro-killing properties of AccuTOX® along with its ability to trigger the production of reactive oxygen species (ROS), endosomal breaks and antigen presentation. RNA-seq was also conducted to pinpoint the most prominent processes modulated by AccuTOX® treatment in EL4 T-cell lymphoma. Finally, the therapeutic potency of intratumorally-injected AccuTOX® was evaluated in three different murine solid tumor models (EL4, E0771 and B16) both as a monotherapy or in combination with three immune-checkpoint inhibitors (ICI).ResultsIn total, 7 Accum® variants were screened for their ability to induce complete cell death in 3 murine (EL4, B16 and E0771) and 3 human (MBA-MD-468, A549, and H460) cancer cell lines of different origins. The selected compound (hereafter refereed to as AccuTOX®) displayed an improved killing efficiency (~ 5.5 fold compared to the parental Accum®), while retaining its ability to trigger immunogenic cell death, ROS production, and endosomal breaks. Moreover, transcriptomic analysis revealed that low dose AccuTOX® enhances H2-Kb cell surface expression as well as antigen presentation in cancer cells. The net outcome culminates in impaired T-cell lymphoma, breast cancer and melanoma growth in vivo especially when combined with anti-CD47, anti-CTLA-4 or anti-PD-1 depending on the animal model.ConclusionsAccuTOX® exhibits enhanced cancer killing properties, retains all the innate characteristics displayed by the parental Accum® molecule, and synergizes with various ICI in controlling tumor growth. These observations will certainly pave the path to continue the clinical development of this lead compound against multiple solid tumor indications.Graphical

Abstract 6336: Unleashing CAR T cell activity against osteosarcoma using tumor specific RNA-based vaccination

Abstract Background: Metastatic osteosarcoma (OSA) has a dismal prognosis despite novel targeted therapies, including CAR T cells. Unleashing CAR T cell therapy against poorly immunogenic cancers requires new technologies that activate the tumor microenvironment (TME), while inducing CAR T cell migration, proliferation, and persistence into tumor to sustain tumor-specific immunity. We have developed tumor RNA-loaded nanoparticle (CD70-encoding RNA-NP) vaccines to overcome CD70-specific CAR T cell limitations in OSA. Methods: Using two murine solid tumor models (K7M2 osteosarcoma and B16F0 melanoma, in Balb/c and C57Bl/6 background) highly metastatic to lungs, we implant K7M2 and B16F0 intravenously (i.v.). We subsequently generated NPs complexed with RNA encoding for CD70 antigen and control vaccine (GFP RNA). RNA-NPs are administered intravenously beginning on day 5 post-tumor implantation and day 7, 14 & 21. Mouse CD70-specific CAR T cells are manufactured after activation of T cells with CD3 and CD28 antibodies for 2 days. CAR T cells are ready for use in downstream experiments 2 days after pMSGV8-mCAR transduction and cells were injected (i.v.) on day 6. Results: In preliminary data, we show that intravenous administration of CD70-encoding RNA-NPs rapidly recruit CD70-directed CAR T cells into tumor stroma unlocking their activity in two immunocompetent aggressive lung metastatic tumor models. Interestingly, regression of CD70-expressing tumors associates with CAR T cell mobilization out of peripheral blood and into reticuloendothelial (RES) tissues (i.e., lung, and spleen), a phenomenon that correlates with upregulation of vaccine-induced type I interferon (IFN) and activation of CCR2 pathway. In addition to the CAR T-mediated antitumor killing, an antigen spreading phenomenon was observed. The long-term survival benefit (150 days) provided by this approach reached 70-80% in both models compared with 0-10% of control groups. Conclusions: Based on these findings, we hypothesize that tumor trafficking and persistence of CD70 CAR T cells depend on coordinated cytokine/chemokine signaling that can be ignited through intravenous RNA-NP vaccination. RNA-NPs in combination with CAR T cells prolong long-term survival of mice bearing these aggressive tumor models which was not only mediated by CAR T cells but also by activation of endogenous adaptive immunity. Future studies include the evaluation of this approach in large animal models prior to translation into first-in-human clinical trials. Citation Format: Arnav Barpujari, Rui Liu, Elizabeth Ogando-Rivas, Xiaojie Ma, Haipeng Taoa, Hector Gomez, Duane Mitchell, Jianping Huang, Elias Sayour, Paul Castillo. Unleashing CAR T cell activity against osteosarcoma using tumor specific RNA-based vaccination [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6336.

Abstract 2357: T cell receptor beta chain-directed Tri-specific antibody molecules retarget V-beta T cell subsets to tumors to promote potent and durable antitumor activity

Abstract Background We have previously shown that novel bifunctional “STAR” antibody-fusion molecules comprising germline β chain TCR-targeting antibodies fused to costimulatory cytokines co-engage the TCR and T cell cytokine receptors, thereby promoting activation and expansion of subsets of T cells expressing distinct germline variable β (Vβ) chain TCRs. Here we describe a new class of T cell engager (TriSTAR) that utilizes a tri-specific format comprising the STAR TCR β chain antibody-IL-2 fusion with an additional Fab arm targeting tumor-associated antigens (TAA). By design, TriSTAR molecules promote the activation and expansion of Vβ T cell subsets that adopt a novel memory phenotype and are subsequently re-targeted to tumors expressing specific TAAs. TriSTAR molecules induce potent and durable anti-tumor activity which in refractory murine solid tumor models appear to be superior to established anti-CD3 T cell engager (TCE) formats. Methods Using the gp75 (Tryp1) melanoma antigen as a model TAA, gp75-targeting TriSTARs were tested in vivo in the B16F10 syngeneic mouse tumor model, compared with a conventional gp75-targeting CD3 bispecific TCE. Cured mice were re-challenged with tumors to assess the potential of TriSTARs to induce immunological memory responses. The pharmacodynamics and pharmacokinetics of gp75-targeting TriSTARs and anti-CD3 TCEs were assessed in blood and tumor using flow cytometry, NanoString and other immunoassays. This work was further extended to testing human TAA-targeting TriSTARs using in vitro human cytotoxicity assays and a transgenic MC38 model. Results In the B16F10 murine melanoma model, gp75-targeting TriSTARs induced potent anti-tumor responses that were superior to control constructs and gp75-targeting CD3 TCEs. The superior anti-tumor activity of the gp75-targeting TriSTARs was attributed to a significant increase and accumulation of CD8+, CD25+ and Granzyme B+ T cells in tumors that was due to the expansion of the target Vβ T cell subset. Further immune profiling by flow cytometry and NanoString in tumors and blood isolated from treated B16F10 mice highlighted significant remodeling of tumor-infiltrating lymphocytes (TILs) and other immunological changes associated with immune activation. These observations with murine gp75-targeting TriSTAR molecules were broadly confirmed with TriSTARs targeting human TAAs in both human in vitro studies and human TAA transgenic mouse tumor models. Conclusions Vβ TCR-targeting TriSTAR molecules represent a new class of T cell engager with the potential to deliver improved anti-tumor activity compared with existing anti-CD3 T cell engagers in immunologically “cold” solid tumors with low levels of T cell infiltration and antigen presentation and may therefore offer a new therapeutic approach for the treatment of patients with refractory solid tumors. Citation Format: Madan Katragadda, Jacques Moisan, Gurkan Guntas, Manuel Sequeria, Roya Servattalab, Jessica Lowry, Robert Ruidera, Kent Nybakken, Erik Gerson, Ke Liu, Kevin Chin, Zhen Su, Andrew Bayliffe. T cell receptor beta chain-directed Tri-specific antibody molecules retarget V-beta T cell subsets to tumors to promote potent and durable antitumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2357.

A T cell receptor β chain-directed antibody fusion molecule activates and expands subsets of T cells to promote antitumor activity.

Despite the success of programmed cell death-1 (PD-1) and PD-1 ligand (PD-L1) inhibitors in treating solid tumors, only a proportion of patients respond. Here, we describe a first-in-class bifunctional therapeutic molecule, STAR0602, that comprises an antibody targeting germline Vβ6 and Vβ10 T cell receptors (TCRs) fused to human interleukin-2 (IL-2) and simultaneously engages a nonclonal mode of TCR activation with costimulation to promote activation and expansion of αβ T cell subsets expressing distinct variable β (Vβ) TCR chains. In solution, STAR0602 binds IL-2 receptors in cis with Vβ6/Vβ10 TCRs on the same T cell, promoting expansion of human Vβ6 and Vβ10 CD4+ and CD8+ T cells that acquire an atypical central memory phenotype. Monotherapy with a mouse surrogate molecule induced durable tumor regression across six murine solid tumor models, including several refractory to anti-PD-1. Analysis of murine tumor-infiltrating lymphocyte (TIL) transcriptomes revealed that expanded Vβ T cells acquired a distinct effector memory phenotype with suppression of genes associated with T cell exhaustion and TCR signaling repression. Sequencing of TIL TCRs also revealed an increased T cell repertoire diversity within targeted Vβ T cell subsets, suggesting clonal revival of tumor T cell responses. These immunological and antitumor effects in mice were recapitulated in studies of STAR0602 in nonhuman primates and human ex vivo models, wherein STAR0602 boosted human antigen-specific T cell responses and killing of tumor organoids. Thus, STAR0602 represents a distinct class of T cell-activating molecules with the potential to deliver enhanced antitumor activity in checkpoint inhibitor-refractory settings.

Cooperative Armoring of CAR and TCR T Cells by T Cell-Restricted IL15 and IL21 Universally Enhances Solid Tumor Efficacy.

Chimeric antigen receptor (CAR) and T-cell receptor (TCR) T-cell therapies are effective in a subset of patients with solid tumors, but new approaches are needed to universally improve patient outcomes. Here, we developed a technology to leverage the cooperative effects of IL15 and IL21, two common cytokine-receptor gamma chain family members with distinct, pleiotropic effects on T cells and other lymphocytes, to enhance the efficacy of adoptive T cells. We designed vectors that induce the constitutive expression of either membrane-tethered IL15, IL21, or IL15/IL21. We used clinically relevant preclinical models of transgenic CARs and TCRs against pediatric and adult solid tumors to determine the effect of the membrane-tethered cytokines on engineered T cells for human administration. We found that self-delivery of these cytokines by CAR or TCR T cells prevents functional exhaustion by repeated stimulation and limits the emergence of dysfunctional natural killer (NK)-like T cells. Across different preclinical murine solid tumor models, we observed enhanced regression with each individual cytokine but the greatest antitumor efficacy when T cells were armored with both. The coexpression of membrane-tethered IL15 and IL21 represents a technology to enhance the resilience and function of engineered T cells against solid tumors and could be applicable to multiple therapy platforms and diseases. See related commentary by Ruffin et al., p. 1431.

Targeting regulatory T cells by E7777 enhances CD8 T-cell-mediated anti-tumor activity and extends survival benefit of anti-PD-1 in solid tumor models.

Regulatory T cell (Treg)-targeting cancer immunotherapy aims to transiently deplete Treg cells in the tumor microenvironment, without affecting effector T cells (Teff), thus both enhancing anti-tumor activity and avoiding autoimmunity. This study evaluated whether adding E7777 (a new formulation of denileukin diftitox [DD]) improved the efficacy of anti-PD-1 antibody therapy. DD is a recombinant protein containing the hydrophobic and catalytic portions of diphtheria toxin fused to full-length human IL-2. E7777 has the same amino acid sequence and brief circulatory half-life as DD, but with greater purity and potency. Subcutaneous syngeneic murine solid tumor models (colon cancer CT-26 and liver cancer H22) were used to evaluate safety, efficacy, and overall survival with E7777 and anti-PD-1 antibodies, each administered as monotherapy or in concurrent or sequential combination. In Experiment 1, treatments were compared to assess anti-tumor activity at various time points, with tumors excised and dissociated and tumor leukocytes characterized. In Experiment 2, tumor growth, response, and overall survival were characterized for 100 days following a 3-week treatment. E7777 administered in combination with anti-PD-1 led to significantly increased anti-tumor activity and durable, extended overall survival compared to either treatment alone. In both tumor models, the Treg cell infiltration induced by anti-PD-1 treatment was counterbalanced by co-treatment with E7777, suggesting potential synergistic activity. Combination therapy showed the most favorable results. Treatment with E7777 was safe and well-tolerated. Combined E7777 and anti-PD-1 therapy was well tolerated and more effective than monotherapy with either drug.

Inducible expression of interleukin-12 augments the efficacy of affinity-tuned chimeric antigen receptors in murine solid tumor models

The limited number of targetable tumor-specific antigens and the immunosuppressive nature of the microenvironment within solid malignancies represent major barriers to the success of chimeric antigen receptor (CAR)-T cell therapies. Here, using epithelial cell adhesion molecule (EpCAM) as a model antigen, we used alanine scanning of the complementarity-determining region to fine-tune CAR affinity. This allowed us to identify CARs that could spare primary epithelial cells while still effectively targeting EpCAMhigh tumors. Although affinity-tuned CARs showed suboptimal antitumor activity in vivo, we found that inducible secretion of interleukin-12 (IL-12), under the control of the NFAT promoter, can restore CAR activity to levels close to that of the parental CAR. This strategy was further validated with another affinity-tuned CAR specific for intercellular adhesion molecule-1 (ICAM-1). Only in affinity-tuned CAR-T cells was NFAT activity stringently controlled and restricted to tumors expressing the antigen of interest at high levels. Our study demonstrates the feasibility of specifically gearing CAR-T cells towards recognition of solid tumors by combining inducible IL-12 expression and affinity-tuned CAR.

Abstract 3466: Microbial neoantigen vectors for cancer immunotherapy

Abstract Cancers employ a variety of immunosuppressive and evasive mechanisms to avoid detection by the immune system and prevent tumor neoantigen-mediated elimination. In recent years, tumor-specific neoantigen vaccines have shown significant promise—promoting tumor regression and increasing survival benefit in certain malignancies by therapeutically directing the immune system to target previously protected tumor specific antigens. However, despite encouraging preclinical data, objective and durable responses have remained elusive—highlighting a requirement for improved therapeutic platforms that permit delivery and targeting of diverse sets of tumor antigens to enhance the efficacy of cancer vaccines. Through synthetic biology, we have engineered living microbial vectors harboring constructs encoding synthetic arrays of tumor-specific neoantigens that specifically colonize tumors following intravenous injection and deliver high levels of neoantigen cargo—both extracellularly (within the tumor microenvironment) and intracellularly (to the cytosol of tumor-resident phagocytes). These engineered vectors robustly activate innate immune cascades and effectively prime tumor neoantigen-directed T cell responses to promote tumor regression and survival in advanced murine solid tumor and lung metastasis models. Extensive remodeling of the tumor immune microenvironment accompanies therapeutic efficacy, with increased activation of tumor neoantigen-specific CD4+ and CD8+ T cells, broadened stimulation of tumor-infiltrating B and T cells, and reduced numbers of intratumoral myeloid cells exhibiting immunosuppressive phenotypes. This work provides a foundational platform for the development of a new class of next-generation tumor vaccines, leveraging the advantages of living medicines to deliver diverse arrays of tumor-specific neoantigens within the optimal immunostimulatory context to induce potent anti-tumor immunity. Citation Format: Andrew A. Redenti, Tal Danino, Nicholas Arpaia. Microbial neoantigen vectors for cancer immunotherapy [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 3466.

1,2,4-Amino-triazine derivatives as pyruvate dehydrogenase kinase inhibitors: Synthesis and pharmacological evaluation

Among the different hallmarks of cancer, deregulation of cellular metabolism turned out to be an essential mechanism in promoting cancer resistance and progression. The pyruvate dehydrogenase kinases (PDKs) are well known as key regulators in cells metabolic process and their activity was found to be overexpressed in different metabolic alerted types of cancer, including the high aggressive pancreatic ductal adenocarcinoma (PDAC). To date few PDK inhibitors have been reported, and the different molecules developed are characterized by structural chemical diversity. In an attempt to find novel classes of potential PDK inhibitors, the molecular hybridization approach, which combine two or more active scaffolds in a single structure, was employed. Herein we report the synthesis and the pharmacological evaluation of the novel hybrid molecules, characterized by the fusion of three different pharmacophoric sub-units such as 1,2,4-amino triazines, 7-azaindoles and indoles, in a single structure. The synthesized derivatives demonstrated a promising ability in hampering the enzymatic activity of PDK1 and 4, further confirmed by docking studies. Interestingly, these derivatives retained a strong antiproliferative activity against pancreatic cancer cells either in 2D and 3D models. Mechanistic studies in highly aggressive PDAC cells confirmed their ability to hamper PDK axis and to induce cancer cell death by apoptosis. Moreover, in vivo translational studies in a murine syngeneic solid tumor model confirmed the ability of the most representative compounds to target the PDK system and highlight the ability to reduce the tumor growth without inducing substantial body weight changes in the treated mice.

A 31-plex panel for high-dimensional single-cell analysis of murine preclinical models of solid tumors by imaging mass cytometry.

Currently, the study of resistance mechanisms and disease progression in cancer relies on the capacity to analyze tumors as a complex ecosystem of healthy and malignant cells. Therefore, one of the current challenges is to decipher the intra-tumor heterogeneity and especially the spatial distribution and interactions of the different cellular actors within the tumor. Preclinical mouse models are widely used to extend our understanding of the tumor microenvironment (TME). Such models are becoming more sophisticated and allow investigating questions that cannot be addressed in clinical studies. Indeed, besides studying the tumor cell interactions within their environment, mouse models allow evaluating the efficacy of new drugs and delivery approaches, treatment posology, and toxicity. Spatially resolved analyses of the intra-tumor heterogeneity require global approaches to identify and localize a large number of different cell types. For this purpose, imaging mass cytometry (IMC) is a major asset in the field of human immuno-oncology. However, the paucity of validated IMC panels to study TME in pre-clinical mouse models remains a critical obstacle to translational or basic research in oncology. Here, we validated a panel of 31 markers for studying at the single-cell level the TME and the immune landscape for discovering/characterizing cells with complex phenotypes and the interactions shaping the tumor ecosystem in mouse models.

Siremadlin Monotherapy and in Combination with Donor Lymphocyte Infusion for Patients with Acute Myeloid Leukemia Post Allogeneic Stem Cell Transplantation Who Are in Complete Remission but at High Risk for Relapse

Background: Relapse post allogeneic stem cell transplantation (SCT) remains the main cause of treatment failure (~40%) for patients (pts) with acute myeloid leukemia (AML). Many pts who ultimately relapse post SCT do so within the first year post transplant, and earlier relapse is associated with poorer survival. Therapeutics that prevent early relapse for these pts are urgently needed. Early cellular or pharmacologic intervention or maintenance strategies that enhance the graft-versus-leukemia (GvL) effect after SCT are of renewed interest as they have the potential to reduce relapse risk post SCT. Siremadlin (HDM201) is a novel investigational MDM2 inhibitor with single-agent, anti-AML activity. MDM2 inhibitors, including siremadlin, possess potent immunomodulatory effects in murine solid tumor and AML models. MDM2 inhibition has been shown to synergize with the allogeneic immune response, increase the vulnerability of AML cells to allogeneic donor T cells, and promote the cytotoxicity and longevity of allogeneic donor T cells in murine models. As such, the post-SCT setting is ideal for investigating the immunomodulatory effects of MDM2 inhibition on enhancing the GvL effect (Ho et al. Blood. 2022). Here we describe the design of a Phase Ib/II, open-label study of siremadlin as monotherapy and in combination with donor lymphocyte infusion (DLI) in pts with AML post SCT who are in complete remission (CR) but at high risk for relapse based on the presence of pre-SCT risk factors (NCT05447663). Study Design: The trial is not yet recruiting but aims to enroll ~38 adult pts with AML who underwent first SCT and are currently between Day 60-120 post SCT. Prior to a patient's SCT, they must have a risk factor that puts them at high risk for relapse, including AML in first CR with adverse risk genetics per 2017 European LeukemiaNet risk stratification, therapy-related AML, or secondary AML; or AML in second or greater CR. The SCT must be from an 8/8 human leukocyte antigen (HLA) matched related or unrelated donor at HLA-A, -B, -C, -DRB1 loci using unmanipulated/T-cell replete bone marrow or peripheral blood stem cells as a stem cell source. Post SCT, pts must have achieved CR or CR with incomplete hematologic recovery (CRi) with no evidence of hematologic relapse. Exclusion criteria include prior exposure to an MDM inhibitor, active graft-vs-host disease (GvHD), past history of grade ≥III acute GvHD (aGvHD) or moderate/severe chronic GvHD (cGVHD), recipient of SCT from a haploidentical family donor or cord blood transplant, and prior systemic AML-directed treatments given at any time after SCT (including DLI). This study contains 2 parts, Part 1 is a dose confirmation of siremadlin monotherapy and will enroll ~12 pts. Siremadlin will be tested at a maximum of 3 dose levels with a starting dose of 30 mg/d on Days 1-5 of a 28-day cycle, dose level +1 at 40 mg/d and dose level -1 at 20 mg/d. The safety and tolerability of these dose levels will be assessed to determine the siremadlin recommended dose (RD) for Part 2. Pts in Part 1 of the study will receive siremadlin monotherapy for the entire duration of the treatment and will not receive DLI. Subsequently, ~26 pts will be enrolled in Part 2 and proceed through a priming phase with siremadlin monotherapy at the RD, followed by a combination phase of siremadlin + DLI, and subsequently a maintenance phase with siremadlin monotherapy (Figure). Primary safety endpoints of the study are incidence of dose-limiting toxicities (DLTs) in Part 1 and time to DLT in Part 2. The primary efficacy endpoint is the proportion of pts in Part 2 who are alive and maintained CR/CRi without hematologic relapse ≥6 months after the start of study treatment. Secondary endpoints include number of pts in Part 1 who are alive and maintained CR/CRi without hematologic relapse ≥6 months after the start of study treatment at the RD for Part 2, time to first documented hematologic relapse or death, incidence of relapse at 1 year and 2 years post study treatment, safety, GvHD-free/relapse-free survival, incidence of and time to treatment-emergent grade ≥III aGvHD or moderate/severe cGvHD, and pharmacokinetics. Conclusions: This Phase Ib/II study investigates siremadlin monotherapy and siremadlin in combination with DLI in pts with AML in remission but at high risk of relapse post SCT. The study will evaluate the unique immunomodulatory potential of siremadlin to reduce risk of relapse in this population with high unmet need. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

PD-L1/TLR7 dual-targeting nanobody-drug conjugate mediates potent tumor regression via elevating tumor immunogenicity in a host-expressed PD-L1 bias-dependent way

BackgroundVarious tumors are insensitive to immune checkpoint blockade (ICB) therapy. Toll-like receptors (TLRs) establish the link between innate and adaptive immunity, which can assist T-cell activation and serve as promising...

AML-142 Patients at High Risk of Relapse Post-Transplant: A Phase 1 Study Design With a Novel Treatment Strategy Using the Estimand Framework

The risk of disease relapse in patients allografted for acute myeloid leukemia (AML) is high and remains the main cause of transplant failure. A potent graft-versus-leukemia (GvL) effect is exerted post-stem cell transplantation (SCT); strategies that may augment a GvL effect have the potential to reduce relapse risk post SCT. Siremadlin/HDM201 is a novel investigational MDM2 inhibitor with single-agent (SA) anti-AML activity. MDM2 inhibitors possess potent immunomodulatory effects (IME) in murine solid tumor and AML models. The post-allogeneic SCT setting is ideal for investigating IME of MDM2 inhibition on the GvL effect. To present a study design assessing the safety and preliminary efficacy of treatment with monotherapy siremadlin and in combination with donor lymphocyte infusions (DLIs) for AML post SCT using the estimand framework (ICH E9 [R1] addendum). Phase Ib/II proof-of-concept. AML patients in CR post SCT at high risk for relapse based on pre-SCT risk factors. The estimand framework mandates definition of experimental intervention according to the clinical question. Due to the risk of GvHD with DLI early post SCT, siremadlin will first be given to eligible patients as priming monotherapy to prevent early relapse; patients who tolerate siremadlin with no evidence of GvHD are eligible to start siremadlin/DLI. Post combination, patients may continue maintenance SA siremadlin, which intends to prolong/enhance the GvL reaction to eradicate residual leukemic blasts. According to their treatment journey, patients may receive only part of the intervention's sequence (e.g., siremadlin priming monotherapy patients not eligible for DLI combination will continue priming). To determine 1) the siremadlin recommended dose (RD) across all parts of the treatment strategy and 2) preliminary efficacy regardless of the treatment parts received. Siremadlin RD will be determined using a Bayesian logistic regression model to guide dose finding of siremadlin during priming/Cycle 1 and a Bayesian time-to-DLT model to assess the siremadlin combination dose. Efficacy will be assessed by the proportion of patients remaining in CR ≥6 months. The estimand framework guided the design of an AML clinical study post SCT to conceptualize the assessment of safety and efficacy of the treatment strategy, accounting for different patient journeys.

In vitro and in vivo anticancer potential and molecular targets of the new colchicine analog IIIM-067

ObjectiveThe current study evaluated various new colchicine analogs for their anticancer activity and to study the primary mechanism of apoptosis and in vivo antitumor activity of the analogs with selective anticancer properties and minimal toxicity to normal cells. MethodsSulforhodamine B (SRB) assay was used to screen various colchicine analogs for their in vitro cytotoxicity. The effect of N-[(7S)-1,2,3-trimethoxy-9-oxo-10-(pyrrolidine-1-yl)5,6,7,9-tetrahydrobenzo[a] heptalene-7-yl] acetamide (IIIM-067) on clonogenicity, apoptotic induction, and invasiveness of A549 cells was determined using a clonogenic assay, scratch assay, and staining with 4′,6-diamidino-2-phenylindole (DAPI) and annexin V/propidium iodide. Mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) levels were observed using fluorescence microscopy. Western blot analysis was used to quantify expression of proteins involved in apoptosis, cell cycle, and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling. Pharmacokinetic and in vivo efficacy studies against Ehrlich ascites carcinoma (EAC) and Ehrlich solid tumor models were conducted using Swiss albino mice. ResultsIIIM-067 showed potent cytotoxicity and better selectivity than all other colchicine analogs screened in this study. The selective activity of IIIM-067 toward A549 cells was higher among other cancer cell lines, with a selectivity index (SI) value of 2.28. IIIM-067 demonstrated concentration- and time-dependent cytotoxicity against A549 cells with half-maximal inhibitory concentration values of 0.207, 0.150 and 0.106 μmol/L at 24, 48 and 72 h, respectively. It also had reduced toxicity to normal cells (SI > 1) than the parent compound colchicine (SI = 1). IIIM-067 reduced the clonogenic ability of A549 cells in a dose-dependent manner. IIIM-067 enhanced ROS production from 24.6% at 0.05 μmol/L to 82.1% at 0.4 μmol/L and substantially decreased the MMP (100% in control to 5.6% at 0.4 μmol/L). The annexin V-FITC assay demonstrated 78% apoptosis at 0.4 μmol/L. IIIM-067 significantly (P < 0.5) induced the expression of various intrinsic apoptotic pathway proteins, and it differentially regulated the PI3K/AKT/mTOR signaling pathway. Furthermore, IIIM-067 exhibited remarkable in vivo anticancer activity against the murine EAC model, with tumor growth inhibition (TGI) of 67.0% at a dose of 6 mg/kg (i.p.) and a reduced mortality compared to colchicine. IIIM-067 also effectively inhibited the tumor growth in the murine solid tumor model with TGI rates of 48.10%, 55.68% and 44.00% at doses of 5 mg/kg (i.p.), 6 mg/kg (i.p.) and 7 mg/kg (p.o.), respectively. ConclusionIIIM-067 exhibited significant anticancer activity with reduced toxicity both in vitro and in vivo and is a promising anticancer candidate. However, further studies are required in clinical settings to fully understand its potential.

Enhancing adoptive T cell therapy for solid tumor with cell-surface anchored immune checkpoint inhibitor nanogels

The efficacy of Adoptive Cell Therapy (ACT) for solid tumor is still mediocre. This is mainly because tumor cells can hijack ACT T cells' immune checkpoint pathways to exert immunosuppression in the tumor microenvironment. Immune Checkpoint Inhibitors such as anti-PD-1 (aPD1) can counter the immunosuppression, but the synergizing effects of aPD1 to ACT was still not satisfactory. Here we demonstrate an approach to safely anchor aPD1-formed nanogels onto T cell surface via bio-orthogonal click chemistry before adoptive transfer. The spatial-temporal co-existence of aPD1 with ACT T cells and the responsive drug release significantly improved the treatment outcome of ACT in murine solid tumor model. The average tumor weight of the group treated by cell-surface anchored aPD1 was only 18 % of the group treated by equivalent dose of free aPD1 and T cells. The technology can be broadly applicable in ACTs employing natural or Chimeric Antigen Receptor (CAR) T cells.

P583: PATIENTS AT HIGH RISK OF RELAPSE POST-TRANSPLANT: A PHASE 1 STUDY DESIGN WITH A NOVEL TREATMENT STRATEGY USING THE ESTIMAND FRAMEWORK

Background: The risk of disease relapse in patients (pts) allografted for acute myeloid leukemia (AML) is high and remains the main cause of transplant failure. The great majority of pts destined to relapse post-allogeneic stem cell transplant (SCT) will do so within the first 12 months and survival after early relapse is dismal. Therefore, strategies to prevent relapse are urgently required. A potent graft-versus-leukemia (GvL) effect is exerted after SCT, and strategies that may augment a GVL effect have the potential to reduce relapse risk post-SCT. Siremadlin (HDM201) is a novel investigational MDM2 inhibitor with single-agent (SA) anti-AML activity. MDM2 inhibitors, including siremadlin, possess potent immunomodulatory effects (IME) in murine solid tumor and AML models. The post-allogeneic SCT setting is ideal for investigating the IME of MDM2 inhibition on enhancing GvL effect. Here we present a phase Ib/II proof-of-concept study designed to assess safety and preliminary efficacy of a treatment strategy with siremadlin as monotherapy and, subsequently, in combination with donor lymphocyte infusions (DLIs) for AML in post-SCT setting using the estimand framework (ICH E9 (R1) addendum). Aims: The objective of the planned treatment strategy is to enhance GvL post-SCT by siremadlin as monotherapy and in combination with DLIs. Due to the risk of Graft-vs-Host Disease (GvHD) with DLI early after SCT, siremadlin will be given as priming monotherapy to prevent early relapse prior to starting siremadlin/DLI combination therapy. SA siremadlin maintenance after the combination therapy is intended to prolong and enhance the GvL reaction to eradicate residual leukemic blasts. Methods: The estimand framework mandates a careful definition of the experimental intervention according to the clinical question of interest. In this trial, the experimental intervention follows a treatment strategy: Patients (pts) with AML in complete remission (CR) post-SCT but at high risk for relapse based on pre-SCT risk factors will start with siremadlin priming monotherapy. Pts who tolerate the siremadlin dose with no evidence of GvHD are eligible to start siremadlin/DLI combination therapy. After combination therapy, pts may continue with SA siremadlin maintenance. Depending on the treatment journey, pts may receive only a part of the intervention’s sequence (e.g., pts in siremadlin priming monotherapy who are not eligible for DLI combination will continue the priming part). The clinical questions addressed in this trial are to determine the recommended dose (RD) of siremadlin across all parts of the treatment strategy and to assess preliminary efficacy regardless of the parts of the treatment strategy pts received. The doses of interest were preselected based on SA studies and in pts who received siremadlin after SCT. Results: The proposed approach is tailored to investigate safety and efficacy of the treatment strategy. The RD of siremadlin will be determined over 2 parts: A Bayesian logistic regression model will guide dose finding of siremadlin during priming (Cycle 1) and a Bayesian time-to-DLT model will assess the siremadlin dose for combination. Efficacy will be assessed by proportion of pts remaining in CR for at least 6 months. Image:Summary/Conclusion: In clinical practice, treatment decisions follow a strategy. The estimand framework guided the design of a clinical study in AML post SCT to conceptualize the assessment of a treatment strategy. Accordingly, safety and efficacy of the treatment strategy as a whole will be assessed taking into account different patient journeys.

Abstract 4182: Intratumoral treatment with VAX014 facilitates in situ immunization leading to systemic antitumor responses that enhance systemic immune checkpoint blockade

Abstract Intratumorally (i.t.) delivered immunotherapies stimulate in situ immunization by promoting local tumor inflammation, immune cell recruitment, and the generation of systemic antitumor lymphocytic responses. Clinical studies indicate combining i.t. immunotherapy with systemic immune checkpoint blockade (ICB) enhances durable complete response rates (CRR) in treated and distal untreated tumors (abscopal effect), although the number of i.t. therapies tested in this setting remains is limited. The purpose of this study was to evaluate and characterize the immunotherapeutic activity of VAX014, a novel clinical-stage bacterial minicell-based oncolytic agent, following i.t. delivery in two syngeneic murine solid tumor models. The nonimmunogenic B16F10 melanoma and immunogenic MB49 urothelial carcinoma models were used to establish single agent activity of VAX014 in mice bearing a single intradermal (i.d.) tumor, followed by an evaluation of synergy between VAX014 and ICB combinations (CTLA-4 ± PD-1) in mice bearing dual bilateral i.d. B16F10 tumors where only one tumor is treated with VAX014.Weekly i.t. treatment with VAX014 led to significant CD8+ T cell mediated antitumor activity in single i.d. tumor models. A significant reduction in tumor growth rate was observed in all mice (p&amp;lt; 0.0001, 38/38 for B16F10; p&amp;lt; 0.0001, 12/12 for MB49) which ultimately resulted in a durable CRR in 58.0% and 100% in the B16F10 and MB49 models, respectively. B16F10 tumors actively responding to i.t. treatment with VAX014 had a significant increase in CD8+ T cells (p= 0.0004) with a concomitant decrease in CD11b+ myeloid cells (p= 0.039) even after a single i.t. dose of VAX014. VAX014-mediated antitumor activity was specific to the treated tumor type as determined by cytotoxic T lymphocyte assay against the treated tumor cell line versus haplotype matched control target cells (p= 0.0015). Using a bilateral variation of each model, VAX014 exhibited a significant abscopal effect in the immunogenic MB49 model (22.0% CRR and ↓ tumor growth rate in untreated tumors, p= 0.009), an effect that was less pronounced in the nonimmunogenic B16F10 model. Addition of systemic CTLA-4 ICB significantly enhanced abscopal activity in the bilateral B16F10 model whereas PD-1 ICB did not. Remarkably, tripartite combination of VAX014 with CTLA-4/PD-1 ICB resulted in a significant reduction in treated (p&amp;lt; 0.0001) and distal untreated B16F10 tumors (p&amp;lt; 0.0012) as well as an enhanced survival advantage versus CTLA-4/PD-1 ICB combination (p= 0.03). Survival was associated with an improved CRR of both tumors (50.0% vs. 20.0%).In conclusion, these preclinical data provide scientific rationale and set the foundation for i.t. treatment with VAX014 in patients with injectable tumors refractory to standard treatment options and may be particularly effective when combined with systemic ICB. Citation Format: Shingo Tsuji, Katherine A. Reil, Kinsey L. Nelson, Kathleen L. McGuire, Matthew J. Giacalone. Intratumoral treatment with VAX014 facilitates in situ immunization leading to systemic antitumor responses that enhance systemic immune checkpoint blockade [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 4182.

Antitumor activity of T cells expressing a novel anti-folate receptor alpha (FOLR1) costimulatory antigen receptor (CoStAR) in a human xenograft murine solid tumor model and implications for in-human studies.

2535 Background: ITIL-306 is an autologous tumor-infiltrating lymphocyte (TIL) therapy that integrates T-cell receptor (TCR)-specific antigen recognition (Signal 1) with robust costimulation via the novel CoStAR transgene upon engagement with FOLR1 (Signal 2; Sukumaran, et al. JITC. 2021;9:198). Here, we assessed IL-2 independent effector function by anti-FOLR1 CoStAR T cells in vitro and evaluated activity in a novel, more representative murine solid tumor model. Methods: For in vitro studies, healthy donor T cells were manufactured to express anti-FOLR1 CoStAR or left nontransduced (NTD). Product cells were stimulated with a single (Day [D]0; ±IL-2) or serial (D0, 7, 14, 21; no IL-2) addition of target cells expressing membrane-anchored OKT3 (Signal 1) and FOLR1 (Signal 2). T-cell activation and proliferation were measured. For in vivo studies, the carcinoembryonic antigen (CEA)-positive (Signal 1) H508 cell line was engineered to express FOLR1 (Signal 2) and injected into 6 mice/group (D–21). NTD or healthy donor T cells expressing HLA-A*02-restricted anti-CEA TCR only, anti-FOLR1 CoStAR only, or dual TCR+CoStAR were given intravenously (D0), and compared with PBS control. Tumor growth, survival, and T-cell expansion were assessed in 2 donors to D96. Results: After single and serial stimulation, only anti-FOLR1 CoStAR T cells showed sustained proliferation without exogenous IL-2. PD-1 positivity was low ( &lt; 10%) on anti-FOLR1 T cells up to D24. In vivo, T-cell persistence on D14 was increased in dual TCR+CoStAR vs anti-CEA TCR only mice ( P&lt;.001) and all other groups ( P&lt;.01-.001). Tumor growth up to D58 was significantly lower in dual TCR+CoStAR ( &lt;.5 cm3) vs all other groups (each &gt; 2 cm3; P&lt;.0001). Survival was significantly longer in dual TCR+CoStAR ( P&lt;.01) vs all other groups; only dual TCR+CoStAR mice were alive at D58, with 5/6 alive at D96. The second donor showed similar tumor control irrespective of exogenous IL-2 in responder mice, with &gt; 50% alive at D96, and robust T-cell expansion in dual TCR+CoStAR mice. Conclusions: When combined with TCR-specific binding, CoStAR significantly enhanced T-cell proliferation, persistence, and antitumor activity in vivo vs TCR alone, resulting in tumor control and prolonged survival. Effects were not observed with CoStAR alone, underscoring that signaling through CoStAR alone does not induce T-cell effector function. The sustained proliferation of anti-FOLR1 CoStAR T cells without exogenous IL-2 support in vitro and in vivo supports a clinical TIL regimen free of high-dose IL-2. These results suggest that CoStAR may improve the clinical performance and benefit/risk profile of TILs, whereby fewer toxicities are expected with the removal of post-TIL IL-2 support. This will be explored in an upcoming first-in-human clinical study with ITIL-306.

Tumor-induced, TCR-independent upregulation of A2AR expression in effector CD8+ T cells

Abstract The tumor microenvironment (TME) is characterized by high levels of extracellular ATP (eATP) and adenosine, which are sensed by purinergic receptors. CD8+ T cells express two important receptors that bind these ligands: the extracellular receptor P2RX7 binds eATP and is generally immunostimulatory, while the adenosine receptor A2AR promotes immunosuppression. However, the role of the TME itself in the T cell expression of purinergic receptors is not clear. We used in vitro co-cultures of tumor cells and CD8+ T cells, and specifically found that A2AR is significantly upregulated after activation, in a tumor cell number-correlated manner. These tumor-induced changes in A2AR expression are independent of TCR:MHC contact, which was evidenced by co-cultures using transwells to physically separate tumor cells and T cells, as well as distinct combination of transgenic T cells and tumors. In vivo murine solid tumor models confirmed that tumor-infiltrating CD8+ T cells upregulate A2AR. As previously found by our group, P2RX7 expression is limited to a small percentage of tumor-infiltrating CD8+ T cells. Interestingly, we identified that these few P2RX7-expressing tumor-infiltrating CD8+ T cells are enriched for high A2AR expression – suggesting that co-expression of these two receptors is favored in T cells exposed to tumors. Overall, our findings suggest that TME exposure – likely through TCR-independent factors – directly promote the expression of A2AR in CD8+ T cells. We are currently investigating the nature of the tumor-derived factor(s) promoting this upregulation, as well as the possible role of A2AR/P2RX7 co-expressing tumor-infiltrating CD8+ T cells as effector antitumor cells with longer intratumoral lifespan. Supported by grants from NIH (R00 AI139381, NIAID).

Siremadlin (HDM201) Is Well Tolerated and Demonstrates Clinical Activity in Patients with Acute Myeloid Leukemia Who Have Relapsed after Allogeneic Stem Cell Transplantation: A Subset Analysis of Safety and Preliminary Efficacy

Siremadlin (HDM201) Is Well Tolerated and Demonstrates Clinical Activity in Patients with Acute Myeloid Leukemia Who Have Relapsed after Allogeneic Stem Cell Transplantation: A Subset Analysis of Safety and Preliminary Efficacy