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S.Figures 1-5 and S.Table 1

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 G12V- or 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 cross-reactivity 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.

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.

BackgroundMesothelin (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.MethodsT 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.ResultsThe 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.ConclusionsThe 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.

Immune cells that are engineered with receptors to integrate signals from multiple antigens offer a promising route to achieve the elusive property of therapeutic selectivity in cancer patients. Several types of multi-signal integrators have been described, among them mechanisms that pair activating and inhibitory receptors which are termed NOT gates by analogy to logical operations performed by machines. Here we review one such NOT-gated signal integrator called the Tmod system which is being developed for patients with solid tumors. Coupled with rigorous selection for patients with defined lesions in their tumor genomes (loss of heterozygosity), the Tmod approach presents an unusual opportunity to create truly selective therapies for certain cancer patients. Several of these agents are advancing toward the clinic, supported by a large body of quantitative preclinical data.

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.

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.

Here we address questions of comparative TCR and CAR function: How different are CARs from TCRs with regard to antigen sensitivity, selectivity, co-stimulation dependence, checkpoint control, cytokine sensitivity, and antigen-dependent exhaustion (defined as loss of cytotoxicity and proliferative capacity over time). We used optimized CARs and TCRs matched to the same target pMHCs, allowing direct comparison of these important parameters. We describe a large-scale, systematic comparison of 17 CARs and 5 TCRs directed at 5 different pMHC targets in both acute and chronic settings in vitro. Surprisingly, we find little fundamental difference between CARs and TCRs with respect to their relative sensitivity to known regulators of T cell activation, including co-stimulation, checkpoint, and cytokine control. Though TCRs often emerge from human blood directly as potent, selective receptors, CARs must be heavily optimized to attain these properties for pMHC targets. Nonetheless, when iteratively improved, CARs can achieve levels of sensitivity and selectivity similar to their vaunted counterparts. Thus, other than signaling sensitivity (and perhaps selectivity), there may be little to distinguish CARs from TCRs in the context of engineered-cell therapy. CARs appear similar to TCRs with regard to immune control, undermining the notion that CARs can, of themselves, overcome regulators that limit T cell activity. Nevertheless, CARs tolerate much more sequence variability within their structural components and this, coupled with their capability to bind different antigen classes, smaller size and subunit simplicity, confers advantages that may foster a leading role for CARs in the next generation of cell therapeutics. We believe that the major conclusions reached here should instruct thinking about the boundaries around therapeutic designs for CARs, which appear to be very wide, and those around differentiation from TCRs, which appear to be much narrower. Citation Format: Xueyin Wang, Aaron D. Martin, Kathleen Negri, Michele McElvain, Julyun Oh, Ming-Lun Wu, Wen-Hua Lee, Agnes Hamburger, Han Xu, Alexander Kamb. CARs and TCRs: Myth and reality [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1540.

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.

The exocyst is an evolutionarily conserved multiprotein complex required for the targeting and docking of post-Golgi vesicles to the plasma membrane. Through its interactions with a variety of proteins, including small GTPases, the exocyst is thought to integrate signals from the cell and signal that vesicles arriving at the plasma membrane are ready for fusion. Here we describe the three-dimensional crystal structure of one of the components of the exocyst, Exo70p, from Saccharomyces cerevisiae at 3.5A resolution. Exo70p binds the small GTPase Rho3p in a GTP-dependent manner with an equilibrium dissociation constant of approximately 70 microM. Exo70p is an extended rod approximately 155 angstroms in length composed principally of alpha helices, and is a novel fold. The structure provides a first view of the Exo70 protein family and provides a framework to study the molecular function of this exocyst component.

The production of the alphavirus virion is a multistep event requiring the assembly of the nucleocapsid core in the cytoplasm and the maturation of the glycoproteins in the endoplasmic reticulum and the Golgi apparatus. These components associate during the budding process to produce the mature virion. The nucleocapsid proteins of Sindbis virus and Ross River virus have been produced in a T7-based Escherichia coli expression system and purified. In the presence of single-stranded but not double-stranded nucleic acid, the proteins oligomerize in vitro into core-like particles which resemble the native viral nucleocapsid cores. Despite their similarities, Sindbis virus and Ross River virus capsid proteins do not form mixed core-like particles. Truncated forms of the Sindbis capsid protein were used to establish amino acid requirements for assembly. A capsid protein starting at residue 19 [CP(19–264)] was fully competent for in vitro assembly, whereas proteins with further N-terminal truncations could not support assembly. However, a capsid protein starting at residue 32 or 81 was able to incorporate into particles in the presence of CP(19–264) or could inhibit assembly if its molar ratio relative to CP(19–264) was greater than 1:1. This system provides a basis for the molecular dissection of alphavirus core assembly.

The mucosal regions of the body are responsible for defense against environmental pathogens. Particularly in the lumen of the gut, antibody-mediated immune responses are critical for preventing invasion by pathogens. In this chapter, we review structural studies that have illuminated various aspects of mucosal immunity. Crystal structures of IgA1-Fc and IgA-binding fragments of the polymeric immunoglobulin receptor and FcαRI, combined with models of intact IgA and IgM from solution scattering studies, reveal potential mechanisms for immune exclusion and induction of inflammatory responses. Other recent structures yield insights into bacterial mechanisms for evasion of the host immune response.

Ferritins are iron storage proteins made of 24 subunits forming a hollow spherical shell. Vertebrate ferritins contain varying ratios of heavy (H) and light (L) chains; however, known ferritin structures include only one type of chain and have octahedral symmetry. Here, we report the 1.9 Å structure of a secreted insect ferritin from Trichoplusia ni, which reveals equal numbers of H and L chains arranged with tetrahedral symmetry. The H/L-chain interface includes complementary features responsible for ordered assembly of the subunits. The H chain contains a ferroxidase active site resembling that of vertebrate H chains with an endogenous, bound iron atom. The L chain lacks the residues that form a putative iron core nucleation site in vertebrate L chains. Instead, a possible nucleation site is observed at the L chain 3-fold pore. The structure also reveals inter- and intrasubunit disulfide bonds, mostly in the extended N-terminal regions unique to insect ferritins. The symmetrical arrangement of H and L chains and the disulfide crosslinks reflect adaptations of insect ferritin to its role as a secreted protein.

During human immunodeficiency virus entry, gp41 undergoes a series of conformational changes that induce membrane fusion. Immediately prior to fusion, gp41 exists in a prehairpin intermediate in which the N- and C-peptide regions of gp41 are exposed. Rearrangement of this intermediate into a six-helix bundle composed of a trimeric coiled coil from the N-peptide region (N-trimer) surrounded by three peptides from the C-peptide region provides the driving force for membrane fusion, whereas prevention of six-helix bundle formation inhibits viral entry. Because of its central role in mediating viral entry, the N-trimer region of gp41 is a key vaccine target. Extensive efforts to discover potent and broadly neutralizing antibodies (Abs) against the N-trimer region have, thus far, been unsuccessful. In this study, we attached a potent C-peptide inhibitor that binds to the N-trimer region to cargo proteins of various sizes to examine the steric accessibility of the N-trimer during fusion. These inhibitors show a progressive loss of potency with increasing cargo size. Extension of the cargo/C-peptide linker partially restores inhibitory potency. These results demonstrate that the human immunodeficiency virus defends its critical hairpin-forming machinery by steric exclusion of large proteins and may explain the current dearth of neutralizing Abs against the N-trimer. In contrast, previous results suggest the C-peptide region is freely accessible during fusion, demonstrating that the N- and C-peptide regions are in structurally distinct environments. Based on these results, we also propose new strategies for the generation of neutralizing Abs that overcome this steric block.

Streptococcus pneumoniae (pneumococcus) remains a significant health threat worldwide, especially to the young and old. While some of the biomolecules involved in pneumococcal pathogenesis are known and understood in mechanistic terms, little is known about the molecular details of bacterium/host interactions. We report here the solution structure of the 'repeated' adhesion domains (domains R1 and R2) of the principal pneumococcal adhesin, choline binding protein A (CbpA). Further, we provide insights into the mechanism by which CbpA binds its human receptor, polymeric immunoglobulin receptor (pIgR). The R domains, comprised of 12 imperfect copies of the leucine zipper heptad motif, adopt a unique 3-alpha-helix, raft-like structure. Each pair of alpha-helices is antiparallel and conserved residues in the loop between Helices 1 and 2 exhibit a novel 'tyrosine fork' structure that is involved in binding pIgR. This and other structural features that we show are conserved in most pneumococcal strains appear to generally play an important role in bacterial adhesion to pIgR. Interestingly, pneumococcus is the only bacterium known to adhere to and invade human cells by binding to pIgR.