Abstract Introduction MyD88 is a scaffold protein that plays a key role in propagating innate immune signals. Recent work has shown that a chemically induced dimerization (CID) platform comprising MyD88 fused to the intracellular domain of CD40 can enhance CAR-T cell fitness via multiple mechanisms. However, this system was associated with toxicity in the clinic due to excessive pro-inflammatory signaling. We sought to better understand the mechanistic levers by which engineered MyD88 assembles and propagates pro-inflammatory signals, with the goal of developing next-generation variants with improved safety profiles. Methods Using structural analysis and bioinformatics, a series of engineered MyD88 fusion proteins were designed, cloned, and expressed in Jurkat cells and primary human T cells in combination with the Tmod™ NOT logic-gated CAR-T platform. Effects on NF-kB induction, cytokine production and cell expansion were evaluated. Results Consistent with previous reports, we observe that Myd88-based CID systems synergize with TCR signaling pathways in human T cells. We demonstrate that engineered MyD88 assembly can be modulated by either raising or lowering the stability of an initial dimeric “seed.” We then characterize how the integration of either natural or synthetic TRAF recruitment motif(s) impart distinct cytokine profiles, enabling the construction of variants with reduced pro-inflammatory signaling. Conclusion Our results shed light on the mechanisms underlying inducible Myddosome assembly and offer insight into why MyD88 is particularly amenable to CID. We show that functional outputs can be modulated via altering assembly thresholds as well as tuning signaling motifs, two parameters which help to define design goals for next-generation immunotherapies integrating innate and adaptive signals. Funding Source n/a Topic Categories Vaccines and Immunotherapy (VAC)

IntroductionDespite advances in treatment of blood cancers, several—including acute myeloid leukemia (AML)—continue to be recalcitrant. Cell therapies based on chimeric antigen receptors (CARs) have emerged as promising approaches for blood cancers. However, current CAR-T treatments suffer from on-target, off-tumor toxicity, because most familiar blood cancer targets are also expressed in normal lineages. In addition, they face the common problem of relapse due to target-antigen loss. Cell therapeutics engineered to integrate more than one signal, often called logic-gated cells, can in principle achieve greater selectivity for tumors.MethodsWe applied such a technology, a NOT gated system called Tmod™ that is being developed to treat solid-tumor patients, to the problem of therapeutic selectivity for blood cancer cells.ResultsHere we show that Tmod cells can be designed to target 2-4 antigens to provide different practical and conceptual options for a blood cancer therapy: (i) mono- and bispecific activating receptors that target CD33, a well-known AML antigen expressed on the majority of AML tumors (as well as healthy myeloid cells) and CD43 (SPN), an antigen expressed on many hematopoietic cancers (and normal blood lineages); and (ii) mono- and bispecific inhibitory receptors that target CD16b (FCGR3B) and CLEC9A, antigens expressed on key normal blood cells but not on most blood cancers.DiscussionThese results further demonstrate the robust modularity of the Tmod system and generalize the Tmod approach beyond solid tumors.

AML relapse after a stem cell transplant is associated with dismal outcomes and no current FDA-approved therapeutic options. Cell therapy for AML has been challenging due to a lack of antigens distinguishing malignant from normal hematopoiesis, the potential for antigen escape due to intratumor antigen heterogeneity, and poor T cell function in lymphocytes from AML patients. To overcome these problems, we focus on opportunities in patients who have received human leukocyte antigen (HLA)-A*02 mismatched transplants. We have developed a Tmod HLA-A*02 NOT gate (blocker) that prevents CAR T killing of HLA-A*02 positive cells. In patients who are HLA-A*02 negative and receive an HLA-A*02 positive stem cell transplant, the blocker distinguishes tumor (HLA-A*02 negative) and normal (HLA-A*02 positive) hematopoiesis. A similar Tmod NOT gate has been safe in an ongoing solid tumor clinical trial (NCT05736731) (Grierson, et al. SITC. 2024). The blocker is paired with a dual CD33/CLL-1 activator to prevent antigen escape in donor-derived HLA-A*02 positive T cells to mitigate AML patient T-cell dysfunction. Additional blockers targeting HLA-A*03, etc can expand therapeutic opportunity to patients who have received other HLA-mismatch transplants. CAR constructs were designed and screened in Jurkat and primary T cell assays. The 50% effective and inhibitory concentration (EC50 and IC50) results were compared to monospecific CAR constructs using benchmark activators based on single-chain variable fragments (scFvs) from M26 (CLL-1) and lintuzumab (CD33) using wild-type A*02-negative and A*02-positive engineered AML cell lines. 111 tandem CD33/CLL-1 activator CAR constructs were evaluated in Jurkat cells and 23 in conjunction with HLA-A*02 blocker. Six tandem constructs were prioritized for evaluation in primary T cells, identifying one (20I21-1A4 mBAAsh) with IC50 potency similar to monospecific CAR constructs, and selectivity observed between HLA-A*02 positive vs. HLA-A*02 negative cells. Selective protection also was observed in HLA-A*02 positive primary hematopoietic stem cells compared with CAR constructs without the blocker. We have developed a cell therapy strategy to leverage HLA mismatched stem cell transplants using a Tmod HLA-A*02 blocker. This approach could be applied to patients who have relapsed after a mismatched transplant or integrated into a stem cell transplant to prevent relapse. Pre-clinical data of this design demonstrates selectivity between HLA-A*02 positive and HLA-A*02 negative cells, and dual antigen activity addresses antigen escape. Future studies will interrogate the impact of membrane-tethered cytokines to augment potency and xenografts studies to further define activity. Diane Manry, Breanna L. Luna, Aaron G. Winters, Aaron D. Flynn, Tanveer K. Gill, Michele Mcelvain, Alaia Sima, Jason Wang, Alexandre Zampieri, Mark Daris, Jingli Zhang, Sara Martire, Breanna DiAndreth, Chawita Netirojjanakul, Talar Tokatlian, Alexander Kamb, John S. Welch. A2 Biotherapeutics, Inc, Agoura Hills, CA. A logic-gated chimeric antigen receptor T cell (CAR T) for post-transplant acute myeloid leukemia (AML) that distinguishes donor and recipient hematopoiesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 818.

Clinical applications of CAR-T cells are limited by the scarcity of tumor-specific targets and are often afflicted with the same on-target/off-tumor toxicities that plague other cancer treatments. A new promising strategy to enforce tumor selectivity is the use of logic-gated, two-receptor systems. One well-described application is termed Tmod™, which originally utilized a blocking inhibitory receptor directed towards HLA-I target antigens to create a protective NOT gate. Here we show that the function of Tmod blockers targeting non-HLA-I antigens is dependent on the height of the blocker antigen and is generally compatible with small, membrane-proximal targets. We compensate for this apparent limitation by incorporating modular hinge units to artificially extend or retract the ligand-binding domains relative to the effector cell surface, thereby modulating Tmod activator and blocker function. By accounting for structural differences between activator and blocker targets, we developed a set of simple geometric parameters for Tmod receptor design that enables targeting of blocker antigens beyond HLA-I, thereby broadening the applications of logic-gated cell therapies.

S.Figures 1-5 and S.Table 1

We set out to examine the behavior in preclinical models of a dual-receptor cell therapy approach (Tmod࣪ system) designed to exploit instances of somatic LOH in cancer that can be readily identified through molecular diagnostics. We utilized quantitative pharmacologic assays in vitro and dual-flank efficacy/selectivity models in vivo to test the activity of a variety of constructs comprised of: (i) CAR “activators” that target tumor-associated antigens (TAAs) such as CEA, MSLN and others; and, (ii) LIR-1-based “blockers” that target HLA class I proteins. We generated substantial datasets around multiple targets, including well known tumor-associated antigens (TAAs). In the presence of the HLA class I blocker antigen, Tmod cells were OFF within a wide range of activator antigen expression levels on the “normal” target cells. They were ON when tumor cells expressed the TAA without the HLA blocker antigen, and killed tumor cells selectively even in mixed tumor-normal cell populations. The Tmod system transformed imperfect TAAs into true tumor-selective targets, where the activity of the engineered T cells is controlled by the presence/absence of the HLA blocker antigen. The system was sufficiently modular to use with autologous and allogeneic products. Several of these Tmod constructs are advancing to the clinic where a robust genetic selection tool is being used to screen patients for treatment and match them with a suitable Tmod therapy. In summary, Tmod is a robust system with quantitative pharmacologic behavior in preclinical models consistent with efficacy and safety across a range of targets. Citation Format: Han Xu, Agnes Hamburger, Yuta Ando, Grace Asuelime, Kristian Bolanos, Mark Daris, Breanna DiAndreth, Maria C. Imun, Wen-Hua Lee, Chuck Z. Li, Breanna Luna, Diane Manry, Aaron D. Martin, Michele E. McElvain, Jee-Young Mock, Martin Naradikian, Mark L. Sandberg, Julyun Oh, Sanam Shafaattalab, Shruti Sharma, Talar Tokatlian, Xueyin Wang, Lu Min Wong, Alexander Kamb. The modular Tmod dual_receptor system exploits tumor deletions to achieve robust tumor-selective cytotoxicity [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 2751.

Background Mesothelin (MSLN) is a classic tumor-associated antigen that is expressed in lung cancer and many other solid tumors. However, MSLN is also expressed in normal mesothelium which creates a significant risk of serious inflammation for MSLN-directed therapeutics. We have developed a dual-receptor (Tmod™) system that exploits the difference between tumor and normal tissue in a subset of patients with defined heterozygous gene loss (LOH) in their tumors. Methods T cells engineered with the MSLN CAR Tmod construct described here contain (1) a novel MSLN-activated CAR and (2) an HLA-A*02-gated inhibitory receptor (blocker). A*02 binding is intended to override T-cell cytotoxicity, even in the presence of MSLN. The Tmod system is designed to treat heterozygous HLA class I patients, selected for HLA LOH. When A*02 is absent from tumors selected for LOH, the MSLN Tmod cells are predicted to mediate potent killing of the MSLN(+)A*02(−) malignant cells. Results The sensitivity of the MSLN Tmod cells is comparable with a benchmark MSLN CAR-T that was active but toxic in the clinic. Unlike MSLN CAR-T cells, the Tmod system robustly protects surrogate “normal” cells even in mixed-cell populations in vitro and in a xenograft model. The MSLN CAR can also be paired with other HLA class I blockers, supporting extension of the approach to patients beyond A*02 heterozygotes. Conclusions The Tmod mechanism exemplified by the MSLN CAR Tmod construct provides an alternative route to leverage solid-tumor antigens such as MSLN in safer, more effective ways than previously possible.

Neoantigens are among the most intriguing potential immuno-oncology targets because, unlike many cancer targets that are expressed on normal tissues, they are by definition restricted to cancer cells. Medicines directed at common neoantigens such as mutant KRAS are especially interesting because they may offer the convenience and cost of an off-the-shelf therapy. However, all common KRAS mutations produce proteins that differ from the wild type at a single amino acid, creating challenges for molecular discrimination. We have undertaken an effort to optimize single-chain variable fragments (scFv) against peptide/major histocompatibility antigen complexes composed of HLA-A*11 and either G12Vor G12D-mutant KRAS peptides. These scFvs could in principle be used in chimeric antigen receptor (CAR) T-cell therapies for selected patients whose tumors bear either of these mutations. Here we show that optimization of such CARs involves a trade-off between potency and selectivity.We further show that targeting this family without high selectivity engenders risks of crossreactivity against other members of the G-protein family to which KRAS belongs. Significance:We report an effort to generate high potency, selective CARs directed at mutant KRAS peptides. Although the heavily optimized CARs maintain high selectivity against wild-type KRAS, they lose selectivity against other KRAS-related peptides derived from human proteins. To our knowledge, this work is the first to examine the trade-off between potency and selectivity with regard to KRAS pMHC-directed CARs, illustrating the challenge to achieve both sufficient potency and high selectivity.

BackgroundMesothelin (MSLN) and carcinoembryonic antigen (CEA) are classic tumor-associated antigens that are expressed in many solid tumors including the majority of lung, colorectal and pancreatic cancers. However, both MSLN and CEA are also expressed in vital normal organs. This normal expression creates risk of serious inflammation for CEA- or MSLN-directed therapeutics. To date all active CEA- or MSLN-targeted investigational therapeutics have been toxic when administered systemically.MethodsWe have developed a safety mechanism to protect normal tissues without abrogating sensitivity of cytotoxic T cells directed at MLSN(+) or CEA(+) tumors in a subset of patients with defined loss of heterozygosity (LOH) in their tumors (figure 1). This dual-receptor (Tmod< sup >TM) system exploits common LOH at the HLA locus in cancer cells, allowing T cells to recognize the difference between tumor and normal tissue.1 2 T cells engineered with specific Tmod constructs contain: (i) a MSLN- or CEA-activated CAR; and, (ii) an inhibitory receptor gated by HLA-A*02. HLA-A*02 binding blocks T cell cytotoxicity, even in the presence of MSLN or CEA. The Tmod system is designed to treat heterozygous HLA class I patients, selected for HLA LOH. When HLA-A*02 is absent from tumors selected for LOH, the CARs are predicted to mediate potent killing of the A*02(-) malignant cells.ResultsThe Tmod system robustly protects surrogate normal cells even in mixed-cell populations in vitro while mediating robust cytotoxicity of tumor cells in xenograft models (see example in figure 2). The MSLN CAR can also be paired with other blockers, supporting scalability of the approach to patients beyond HLA-A*02 heterozygotes.Abstract 122 Figure 1Illustration of the Tmod T cell engaging with tumor cells with somatic loss of HLA-A*02 and with normal cells.Abstract 122 Figure 2Bioluminescence measurements show the average difference between the size of the MSLN(+)A*02(+) ‘normal’ graft compared to the MSLN(+)A*02(-) tumor graft on the two flanks of mice after T cell infusion. Both tumor and normal grafts are destroyed by CAR-Ts (CAR-3 and M5 benchmark) while the MSLN Tmod cells kill the tumor but not the normal graft.ConclusionsThe Tmod mechanism may provide an alternative route to leverage solid-tumor antigens such as MSLN and CEA in safer, more effective ways than previously possible.ReferencesHamburger AE, DiAndreth B, Cui J, et al. Engineered T cells directed at tumors with defined allelic loss. Mol Immunol 2020;128:298–310.Hwang MS, Mog BJ, Douglass J, et al. Targeting loss of heterozygosity for cancer-specific immunotherapy. Proc Natl Acad Sci U S A 2021;118(12):e2022410118.

We designed variant human TCRs composed of the full-length TCRα/β or extracellular and transmembrane domains of the associated CD3 subunits fused to polypeptides derived from proteins thought to either enhance or inhibit normal T cell function. First, we showed that the C termini of both the TCR α- and β-chains can accommodate specific additional sequences, without abrogating complex formation or acute sensitivity of the receptor. Replacement of ITAMs with ITIM-containing intracellular domains inverted the TCR signal (i.e., created a ligand-dependent inhibitory receptor). The normal signaling function of the CD3 complex was transferable to the TCR by eliminating all CD3 ITAMs and grafting three to six ITAMs onto the C termini of the α/β-chains, with no effect on acute sensitivity. The observation that TCR variants of such diverse C-terminal composition can fold and function as signaling receptors demonstrates substantial structural and functional malleability of TCRs. These results add to knowledge about TCR structure-function with regard to acute signaling and may provide a route to use TCRs in different ways for T cell therapy.

Next-generation T-cell therapies will likely continue to utilize T-cell receptors (TCRs) and chimeric antigen receptors (CARs) because each receptor type has advantages. TCRs often possess exceptional properties even when tested unmodified from patients' T cells. CARs are generally less sensitive, possibly because their ligand-binding domains are grafted from antibodies selected for binding affinity or avidity and not broadly optimized for a functional response. Because of the disconnect between binding and function among these receptor types, the ultimate potential of CARs optimized for sensitivity and selectivity is not clear. Here, we focus on a thoroughly studied immuno-oncology target, the HLA-A*02/HPV-E629-38 complex, and show that CARs can be optimized by a combination of high-throughput binding screens and low-throughput functional assays to have comparable activity to clinical TCRs in acute assays in vitro. These results provide a case study for the challenges and opportunities of optimizing high-performing CARs, especially in the context of targets utilized naturally by TCRs.

Inhibitors that block the programmed cell death-1 (PD-1) pathway can potentiate endogenous antitumor immunity and have markedly improved cancer survival rates across a broad range of indications. However, these treatments work for only a minority of patients. The efficacy of anti-PD-1 inhibitors may be extended by cytokines, however, the incorporation of cytokines into therapeutic regimens has significant challenges. In their natural form when administered as recombinant proteins, cytokine treatments are often associated with low response rates. Most cytokines have a short half-life which limits their exposure and efficacy. In addition, cytokines can activate counterregulatory pathways, in the case of immune-potentiating cytokines this can lead to immune suppression and thereby diminish their potential efficacy. Improving the drug-like properties of natural cytokines using protein engineering can yield synthetic cytokines with improved bioavailability and tissue targeting, allowing for enhanced efficacy and reduced off-target effects. Using structure guided engineering we have designed a novel class of antibody-cytokine fusion proteins consisting of a PD-1 targeting antibody fused together with an interleukin-21 (IL-21) cytokine mutein. Our bifunctional fusion proteins can block PD-1/programmed death-ligand 1 (PD-L1) interaction whilst simultaneously delivering IL-21 cytokine to PD-1 expressing T cells. Targeted delivery of IL-21 can improve T cell function in a manner that is superior to anti-PD-1 monotherapy. Fusion of engineered IL-21 variants to anti-PD1 antibodies can improve the drug-like properties of IL-21 cytokine leading to improved cytokine serum half-life allowing for less frequent dosing. In addition, we show that targeted delivery of IL-21 can minimize any potential detrimental effect on local antigen-presenting cells. A highly attenuated IL-21 mutein variant (R9E:R76A) fused to a PD-1 antibody provides protection in a humanized mouse model of cancer that is refractory to anti-PD-1 monotherapy. Collectively, our preclinical data demonstrate that this approach may improve upon and extend the utility of anti-PD-1 therapeutics currently in the clinic.

Recombinant adeno-associated virus (rAAV) vectors are a leading gene delivery platform, but vector manufacturing remains a challenge. New methods are needed to increase rAAV yields and reduce costs. Past efforts to improve rAAV production have focused on optimizing a single variable at a time, but this approach does not account for the interactions of multiple factors that contribute to vector generation. Here, we utilized a design-of-experiment (DOE) methodology to optimize rAAV production in a HEK293T suspension cell system. We simultaneously varied the transgene, packaging, and helper plasmid ratios, the total DNA concentration, and the cell density to systematically evaluate the impact of each variable across 52 conditions. The results revealed a unique set of parameters with a lower concentration of transgene plasmid, a higher concentration of packaging plasmid, and a higher cell density than previously described protocols. Using this DOE-optimized protocol, we achieved unpurified yields approaching 3 x 10(14) viral genomes (VGs)/L of cell culture. Additionally, we incorporated polyethylene glycol (PEG)-based virus precipitation, pH-mediated protein removal, and affinity chromatography to our downstream processing, enabling average purified yields of >1 x 10(14) VGs/L for rAAV-EGFPs across 13 serotypes and capsid variants.

BACKGROUND AIMS:To investigate the feasibility of using CD4 + T cells genetically modified to express an allo-HLA directed CAR and FOXP3 to suppress T cell proliferation and cytokine secretion in GvHD.METHODS:Human CD4+ T cells from A*02:01 negative donors were transduced to express A*02 CAR and FOXP3 and co-cultured in mixed lymphocyte reaction assays to demonstrate T cell suppression. A*02- CAR/FOXP CD4+ T cells were then injected into mice engrafted with allogeneic T cells in a GvHD mouse model.RESULTS:CD4+ T cells genetically modified to express allo-HLA-directed CAR and FOXP3 proliferate rapidly, downregulate CD127 and interferon-γ, express high CD25 and Helios and convert to a stable antigen-dependent suppressive phenotype. In mixed lymphocyte reaction assays, these cells potently suppressed T-cell proliferation and secreted IL-10. In a graft-versus-host disease model, A*02-CAR/FOXP3 CD4+ T cells outperformed polyclonal Tregs by reducing liver and lung inflammation, inhibiting pro-inflammatory cytokine production and limiting grafted CD3+ T-cell expansion.CONCLUSIONS:CD4 + T cells expressing allo-antigen directed HLA-specific CAR and FOXP3 act as potent, specific and stable suppressors of inflammation that out-perform their Treg counterparts both in vitro and in vivo.

Background Recurrent cancer-specific targets are rare. Given the pace of genomic research over the past three decades, few are likely to lie yet undiscovered. In 2013 an innovative MAGE-A3-directed cancer therapeutic of great potential value was terminated in the clinic because of neurotoxicity.1 The safety problems were hypothesized to originate from off-target TCR activity against a closely related MAGE-A12 peptide. Methods A combination of published and new data led us to test this hypothesis with current technology, including RNA hybridization in situ and further analysis of the clinical TCR's specificity to MAGE-A12 and other antigens. Results We find that a key prediction of the MAGE-A12 toxicity hypothesis, the existence of rare, high-MAGE-A12-expressing cells in the brain, is not supported by the data. Our results imply that an alternative related peptide from the EPS8L2 protein is more likely responsible for the toxicity. Therefore, it may be valuable to reconsider MAGE-A3 as a cancer target using HLA-A*02-restricted-TCRs or CARs. As a step in this direction, we isolated MAGE-A3 pMHC-directed CARs, targeting the same peptide as the clinical TCR. These CARs have high selectivity, and avoid cross-reaction with the EPS8L2 peptide that represents a significant risk for MAGE-A3-targeted therapeutics. Conclusions Given the qualities of MAGE-A3 as an onco-testis antigen widely expressed in tumors and largely absent from normal adult tissues, our findings suggest that MAGE-A3 may deserve further consideration as a cancer target. We have identified CARs with selectivity profiles consistent with a cell therapeutic directed against HLA-A*02-positive, MAGE-A3-expressing cancers. The relative merits of TCRs and CARs for this target will be discussed. Reference Morgan RA, Chinnasamy N, Abate-Daga D, Gros A, Robbins PF, Zheng Z, Dudley ME, Feldman SA, Yang JC, Sherry RM, et al. Cancer regression and neurological toxicity following anti-MAGE-A3 TCR gene therapy. J Immunother 2013;36:133–151, doi:10.1097/CJI.0b013e3182829903.

Background: Chimeric Antigen Receptor (CAR) T-cell therapy is a proven, powerful clinical modality. However, it is still limited by the fundamental obstacle of cancer therapy: discriminating cancer from normal cells. Current FDA-approved CAR T-cell therapies eliminate normal B cells, leaving patients with B cell aplasia, hypogammaglobulinemia, and susceptible to infection. HLA-Class I loss of heterozygosity (LOH) occurs at an average frequency of ~13% among cancers and specifically ~13% in DLBCL (Broad Institute TCGA database). These losses are irreversible and distinguish the cancer from normal cells. To exploit LOH at the HLA locus, we target the remaining allelic product in tumors with LOH. We evaluated a novel AND NOT Boolean logic gate CAR T module (Tmod) T-cell system to target HLA-A*02 (A2) LOH in lymphoma using both in vitro and in vivo models. Methods: To model tumor cells that have lost A2 via LOH, we used CD19+ Raji lymphoma tumor cells. To model the corresponding "normal" cells, we established CD19+ Raji cells stably expressing A2 (CD19+/A2+ Raji). We then engineered human primary T cells to express a modular signal-integration circuit designed to be activated only by CD19+ lymphoma that do not express A2 (CD19+/A2- Raji). Each primary Tmod CAR T cell expresses both a CD19 activator (A) module using a CD19-targeting 3rd generation CAR, and a separate A2-targeting blocker (B) module using a novel A2-targeting inhibitory receptor. Human primary Tmod CAR T cells were engineered to co-express the A/B modules. First, T cells were stimulated via CD3/CD28 activation, followed by A/B module lentivirus transduction, and enriched for the B module. In vitro Tmod CAR T cells were evaluated for selective killing of CD19+/A2- Raji compared with CD19+/A2+ Raji. For in vivo proof of concept, both CD19+/A2- Raji and CD19+/A2+ Raji cell lines were injected and established into flanks of immunocompromised NGS mice and challenged with adoptive transfer of engineered human primary Tmod CAR T cells. Results: Engineered primary Tmod CAR T cells selectively killed CD19+/A2- Raji and spared CD19+/A2+ Raji (Figure 1). Tmod CAR T cells reversibly cycled from a state of non-killing, "block", to cytotoxicity and back, depending on the CD19+/A2- Raji vs. CD19+/A2+ Raji cells to which they were exposed. Importantly, primary Tmod CAR T cells selectively eliminated only the CD19+/A2- Raji cells in mixed cultures. In vivo, Tmod CAR T cells selectively eradicated CD19+/A2- Raji. More importantly, Tmod CAR T cells did not eradicate CD19+/A2+ Raji in vivo. Conclusions: CD19-targeting Tmod CAR T cells demonstrated robust and selective killing, distinguishing Raji lymphoma lines, one with A2 (CD19+/A2+) and one without (CD19+/A2-), both in vitro and in vivo. A critical requirement for Tmod CAR T-cell therapy is to determine reversibility and lack of anergy in the kill-"block"-kill and "block"-kill-"block" scenarios. This result demonstrates that Tmod CAR T cells do not terminally differentiate into one state (blockade or activation), but rather can switch back and forth as they integrate signals from "normal" and tumor cells. Furthermore, because Tmod CAR T cells can selectively target malignant B cells, it may increase the clinical therapeutic window for CAR T. Tmod CAR T cells may provide a powerful system to address hematologic malignancies and solid tumors with HLA-Class I LOH. Disclosures Hamburger: A2 Biotherapeutics: Current Employment, Current equity holder in private company. DiAndreth:A2 Biotherapeutics: Current Employment. Daris:A2 Biotherapeutics: Current Employment, Current equity holder in private company. Munguia:A2 Biotherapeutics: Current Employment, Current equity holder in private company. Deshmukh:A2 Biotherapeutics: Current Employment. Mock:A2 Biotherapeutics: Current Employment, Current equity holder in private company. Asuelime:A2 Biotherapeutics: Current Employment, Current equity holder in private company. Lim:A2 Biotherapeutics: Current Employment, Current equity holder in private company. Kreke:A2 Biotherapeutics: Current Employment, Current equity holder in private company; Gilead: Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months. Tokatlian:A2 Biotherapeutics: Current Employment, Current equity holder in private company. Maloney:A2 Biotherapeutics: Consultancy, Current equity holder in publicly-traded company, Honoraria; Bioline Rx: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Genentech: Consultancy, Honoraria; Gilead Science: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Juno Therapeutics: Consultancy, Honoraria, Patents & Royalties, Research Funding. Go:A2 Biotherapeutics: Current Employment, Current equity holder in private company; Amgen: Current equity holder in publicly-traded company; Allogene: Divested equity in a private or publicly-traded company in the past 24 months; Gilead: Current equity holder in publicly-traded company; Illumina: Divested equity in a private or publicly-traded company in the past 24 months. Kamb:A2 Biotherapeutics: Current Employment, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

We describe an approach to cancer therapy based on exploitation of common losses of genetic material in tumor cells (loss of heterozygosity) (Basilion et al., 1999; Beroukhim et al., 2010). This therapeutic concept addresses the fundamental problem of discrimination between tumor and normal cells and can be applied in principle to the large majority of tumors. It utilizes modular activator/blocker elements that integrate signals related to the presence and absence of ligands displayed on the cell surface (Fedorov et al., 2013). We show that the targeting system works robustly in vitro and in a mouse cancer model where absence of the HLA-A*02 allele releases a brake on engineered T cells activated by the CD19 surface antigen. This therapeutic approach potentially opens a route toward a large, new source of cancer targets.

Chimeric antigen receptors (CARs) and their parent signaling molecule, the T cell receptor (TCR), are fascinating proteins of increasing relevance to disease therapy. Here we use a collection of 1221 pMHC-directed CAR constructs representing 10 pMHC targets to study aspects of CAR structure-activity relationships (SAR), with particular focus on the extracellular and transmembrane structural components. These experiments that involve pMHC targets whose number/cell can be manipulated by peptide dosing in vitro enable systematic analysis of the SAR of CARs in carefully controlled experimental situations (Harris and Kranz, 2016). We find that CARs tolerate a wide range of structural variation, with the ligand-binding domains (LBDs) dominating the SAR of CAR antigen sensitivity. Notwithstanding the critical role of the LBD, CAR antigen-binding on the cell surface, measured by pMHC tetramer staining, is not an effective predictor of functional sensitivity. These results have important implications for the design and testing of CARs aimed toward the clinic.

In 2013, an innovative MAGE-A3-directed cancer therapeutic of great potential value was terminated in the clinic because of neurotoxicity. The safety problems were hypothesized to originate from off-target T-cell receptor activity against a closely related MAGE-A12 peptide. A combination of published and new data led us to test this hypothesis with current technology. Our results call into question MAGE-A12 as the source of the neurotoxicity. Rather, the data imply that an alternative related peptide from EPS8L2 may be responsible. Given the qualities of MAGE-A3 as an onco-testis antigen widely expressed in tumors and largely absent from normal adult tissues, these findings suggest that MAGE-A3 may deserve further consideration as a cancer target. As a step in this direction, the authors isolated 2 MAGE-A3 peptide-major histocompatibility complex-directed chimeric antigen receptors, 1 targeting the same peptide as the clinical T-cell receptor. Both chimeric antigen receptors have improved selectivity over the EPS8L2 peptide that represents a significant risk for MAGE-A3-targeted therapeutics, showing that there may be other options for MAGE-A3 cell therapy.