Integrated BioTherapeutics (United States)

Abstract Background: In many hyperproliferative disorders including cancer, aberrant stem cells are the root cause of the pathology. Acute myeloid leukemia (AML) is an especially aggressive form of cancer that continues to resist the application of novel treatment modalities. While single-antigen-targeted CAR T cells have demonstrated efficacy in treating B cell malignancies, in AML, commonly targeted antigens are co-expressed on normal hematopoietic lineages causing on-target, off-tumor toxicities. Targeting antigen-pairs through NOT gated CAR T cell designs offers a strategy for increased safety by improving discrimination between tumor and healthy cells. Here, we describe a system for genetically engineering cytotoxic T cells to confer protection of hematopoietic stem cells (HSCs), while directing cytotoxicity against AML. This system uses the Tmod NOT gate that targets activator antigens (A-Ags) with a CAR, and blocker antigens (B-Ags) with an inhibitory receptor based on LIR-1. The candidate therapeutic relies on CD33 and CLL-1 to target AML cells, but incorporates an inhibitory receptor triggered by CD90 to protect healthy HSCs. Methods: To discover novel target antigen profiles, we leveraged both single-cell and bulk RNA-Seq publicly available datasets from human hematopoietic cell populations to generate an integrated transcriptomic dataset and identified antigen A- and B-Ags where: 1) the Ags are expressed on the cell surface; 2) A-Ags have high and consistent expression on primary AML and AML cell lines; 3) B-Ags have minimal to no expression on AML cells and high expression on HSCs that may express the A-Ags. Subsequently, lead target antigens identified by mRNA expression were confirmed via cell-surface expression analysis of AML samples. Results: The CD33-CLL1 | CD90 Tmod construct was selected based on its optimal Ag-expression profile. Comprehensive binder discovery campaigns using mouse immunization, hybridoma screening, and phage display were performed. CD33-CLL1 | CD90 Tmod constructs were screened in Jurkat and primary T cell assays to determine potency and selectivity against target cell lines and primary HSCs. One specific Tmod construct demonstrated impressive sensitivity for both activation and blocking (EC50 and IC50 ∼ 1,000 molecules/cell). Studies in primary T cells confirmed the potency and selectivity of the construct using HL-60 target cells engineered with varying amounts of CD90 surface expression. The potency and selectivity windows were in the range observed previously with a wide variety of Tmod constructs (1). Finally, the CD33-CLL1 | CD90 Tmod constructs were tested in co-cultures with HSCs and shown significant protection (∼50%). Conclusions: We developed a CD33-CLL1 | CD90 Tmod construct with selective potency toward AML vs. healthy HSCs. The CD90 blocker was highly sensitive, a necessary attribute given the low but consistent levels of expression of CD90 in HSCs. (1) DiAndreth B et Al. Multi-targeted, NOT gated CAR-T cells as a strategy to protect normal lineages for blood cancer therapy. Front Immunol. 2025 Citation Format: Tanveer Gill, Vasantika Suryawanshi, Sara Martire, Michele Mcelvain, Aaron Winters, Breanna Luna, Diane Manry, John Welch, Alexander Kamb, Chawita Netirojjanakul, Michael Rettig, Ryan Elshimali. A CD90 NOT gate for cell therapies to treat AML and spare healthy hematopoietic stem cells [abstract]. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr A014.

Chimeric antigen receptor (CAR)-T cell therapy has transformed the outcomes of patients with haematological malignancies, yet its use is limited by labour-intensive manufacturing, constrained production capacity and variable clinical performance. In vivo CAR-T cell engineering, in which CAR-T cells are generated directly inside the patient's body, seeks to overcome these challenges by eliminating the need for ex vivo cell processing and complex logistics, as well as improve clinical performance. Recent advances in virology, RNA medicines and nanotechnology have catalysed a radical overhaul of this approach, which uses targeted delivery systems such as lentiviral vectors and lipid nanoparticles to introduce CAR-encoding genetic material into endogenous T cells. Early clinical studies have shown efficient transduction, sustained CAR expression and initial signs of antitumour activity, establishing proof of concept. This Review explores the underlying technologies - including RNA delivered by lipid nanoparticles and engineered viral vectors - and discusses how they are being adapted to develop more broadly applicable, scalable, safe and effective CAR-T cell therapies. By removing the need for ex vivo manipulation and chemotherapeutic conditioning, this strategy could enable the wider application of CAR-T cell therapies not just to blood cancers but to autoimmune diseases for which ex vivo CAR-T cell therapies have shown strong promise, such as systemic lupus erythematosus.

TPS2677 Background: Despite the success in hematologic malignancies, CAR T therapies face significant challenges in solid tumors due to the lack of tumor-specific targets that distinguish cancer from normal cells. EGFR plays a critical role in oncogenesis across several cancers and is often upregulated (TCGA 2022). While monoclonal antibodies targeting EGFR have demonstrated efficacy, these approaches are often limited by on-target, off-tumor toxicities, such as skin rash, which constrains dose escalation and efficacy (Macdonald, et al. J Am Acad Dermatol . 2015). A2B395 is an allogeneic, logic-gated, EGFR-targeted Tmod CAR T therapy designed to address these limitations and provide a convenient and consistent off-the-shelf option. This therapy incorporates 2 CARs: an activator targeting EGFR, and a blocker targeting HLA-A*02. The activator recognizes EGFR on both tumor and normal cells, while the blocker inhibits CAR T activity against normal cells with preserved HLA expression and decreases the risk for graft-versus-host disease (Hamburger, et al. Mol Immunol . 2020). To address potential host-vs-graft response, an shRNA expression module targeting B2M is included in the Tmod construct, which significantly reduces major histocompatibility complex class I levels and subsequent host immune response (DiAndreth, et al. Clin Immunol. 2022). Importantly, the Tmod system is modular and adaptable to multiple targets. Initial data on autologous Tmod CAR T therapy suggest reduced off-tumor toxicity and encouraging clinical efficacy (Grierson, et al. SITC 2024. Abstract 588). A2B395 represents a novel approach for EGFR-expressing solid tumors with HLA-A*02 LOH. Methods: DENALI-1 (NCT06682793) is a phase 1/2, open-label, nonrandomized study evaluating the safety and efficacy of A2B395 in adults. Patients are enrolled through BASECAMP-1 (NCT04981119), a master prescreening study that identifies patients with HLA LOH at any time in the course of their disease via next-generation sequencing (Tempus AI, Inc.). Key inclusion criteria include histologically confirmed recurrent unresectable, locally advanced, or metastatic cancers associated with EGFR expression, including colorectal, non-small cell lung, squamous cell head and neck, triple negative breast, and renal cell cancers. Patients must have received ≥1 line of prior therapy, such as a checkpoint inhibitor, molecular targeted therapy, or chemotherapy. The primary objective of phase 1 is to evaluate safety, tolerability, and the recommended phase 2 dose (RP2D) using a Bayesian optimal interval design for dose escalation. The dose-expansion phase will confirm RP2D and collect biomarker data. Phase 2 will assess overall response rate per RECIST v1.1. Clinical trial information: NCT06682793 .