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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.

Though TCRs have been subject to limited engineering in the context of therapeutic design and optimization, they are used largely as found in nature. On the other hand, CARs are artificial, composed of different segments of proteins that function in the immune system. This characteristic raises the possibility of altered response to immune regulatory stimuli. Here we describe a large-scale, systematic comparison of CARs and TCRs across 5 different pMHC targets, with a total of 19 constructs examined in vitro. These functional measurements include CAR- and TCR-mediated activation, proliferation, and cytotoxicity in both acute and chronic settings. Surprisingly, we find no consistent difference between CARs and TCRs as receptor classes with respect to their relative sensitivity to major regulators of T cell activation: PD-L1, CD80/86 and IL-2. 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 and compared head to head in functional tests, CARs appear remarkably similar to TCRs with respect to immune modulation.

Supplemental Digital Content is available in the text., 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.

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.

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.

Cell therapy is an emerging therapeutic modality with the power to exploit new cancer targets and potentially achieve positive outcomes for patients with few other options. Like all synthetic treatments, cell therapy has the risk of toxicity via unpredicted off-target behavior. We describe an empirical method to model off-tumor, off-target reactivity of receptors used for investigational T cell therapies. This approach utilizes an optimal panel of diverse human cell-lines to capture the large majority of protein-coding gene expression in adult human tissues. We apply this cell-line set to test Jurkat and primary T cells engineered with a dual-signal integrator, called Tmod

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.

Cell therapy using T cell receptors (TCRs) and chimeric antigen receptors (CARs) represents a new wave of immunotherapies garnering considerable attention and investment. Further progress in this area of medicine depends in part on improving the functional capabilities of the engineered components, while maintaining the overall size of recombinant constructs to ensure their compatibility with existing gene delivery vehicles. We describe a single-variable-domain TCR (svd TCR) that utilizes only the variable domain of the β chain (Vβ). This Vβ module not only works in TCR and CAR formats, but also can be used to create single-chain bispecific CARs and TCRs. Comparison of individual ligand-binding Vβ domains in different formats suggests that the lone Vβ sequence controls the sensitivity and a major part of the specificity of the CAR or TCR construct, regardless of signaling format, in Jurkat and primary T cells.

A disulfide-linked imidazoline nitroxide side chain (V1) has a similar and highly constrained internal motion at diverse topological sites in a protein, unlike that for the disulfide-linked pyrroline nitroxide side chain (R1) widely used in site directed spin labeling EPR. Crystal structures of V1 at two positions in a helix of T4 Lysozyme and quantum mechanical calculations suggest the source of the constraints as intra-side chain interactions of the disulfide sulfur atoms with both the protein backbone and the 3-nitrogen in the imidazoline ring. These interactions apparently limit the conformation of the side chain to one of only three possible rotamers, two of which are observed in the crystal structure. An inter-spin distance measurement in frozen solution using double electron–electron resonance (DEER) gives a value essentially identical to that determined from the crystal structure of the protein containing two copies of V1, indicating that lattice forces do not dictate the rotamers observed. Collectively, the results suggest the possibility of predetermining a unique rotamer of V1 in helical structures. In general, the reduced rotameric space of V1 compared to R1 should simplify interpretation of inter-spin distance information in terms of protein structure, while the highly constrained internal motion is expected to extend the dynamic range for characterizing large amplitude nanosecond backbone fluctuations.

Molecular dynamics simulations of a dioleoylphosphocholine (DOPC) lipid bilayer were performed to explore its mechanosensitivity. Variations in the bilayer properties, such as area per lipid, volume, thickness, hydration depth (HD), hydration thickness (HT), lateral diffusion coefficient, and changes in lipid structural order were computed in the membrane tension range 0 to 15 dyn/cm. We determined that an increase in membrane tension results in a decrease in the bilayer thickness and HD of ~ 5% and ~ 5.7% respectively, whereas area per lipid, volume, and HT/HD increased by 6.8%, 2.4%, and 5% respectively. The changes in lipid conformation and orientation were characterized using orientational (S2) and deuterium (SCD) order parameters. Upon increase of membrane tension both order parameters indicated an increase in lipid disorder by 10–20%, mostly in the tail end region of the hydrophobic chains. The effect of membrane tension on lipid lateral diffusion in the DOPC bilayer was analyzed on three different time scales corresponding to inertial motion, anomalous diffusion and normal diffusion. The results showed that lateral diffusion of lipid molecules is anomalous in nature due to the non-exponential distribution of waiting times. The anomalous and normal diffusion coefficients increased by 20% and 52% when the membrane tension changed from 0 to 15 dyn/cm, respectively. In conclusion, our studies showed that membrane tension causes relatively significant changes in the area per lipid, volume, polarity, membrane thickness, and fluidity of the membrane suggesting multiple mechanisms by which mechanical perturbation of the membrane could trigger mechanosensitive response in cells.

In site directed spin labeling, a nitroxide side chain is introduced at a selected site in a protein; the most commonly used is a disulfide-linked side chain designated R1. The electron paramagnetic resonance (EPR) spectra of R1, and the interspin distance between pairs of R1 residues as determined by dipolar EPR spectroscopy, encode a wealth of information on the protein structure and dynamics. However, extracting this information requires structural and dynamical models of the R1 side chain, that is, the favored rotamers, the intraresidue interactions that stabilize them, and the internal modes of motion. X-ray crystal structures of R1 in proteins have revealed a set of preferred rotamers in the crystal lattice. To identify the intraresidue interactions that stabilize particular rotamers of R1 in the absence of interactions with nearby side chains in a helix, and to evaluate models for the internal motion of the side chain, quantum mechanical calculations were performed on a relevant fragment of R1 in a 10-residue α-helix. Relative rotamer energies were determined in the gas phase, and solvation energies were estimated from a continuum solvent model that includes both electrostatic and hydrophobic contributions. The results identified preferred rotamers that are in agreement with the X-ray crystallographic studies. The rotamers are apparently stabilized by intraresidue sulfur-backbone interactions, suggesting that the preferred rotamers may be the same at all solvent-exposed helix sites.

It has been postulated that the hydrophobic loop of actin (residues 262-274) swings out and inserts into the opposite strand in the filament, stabilizing the filament structure. Here, we analyzed the hydrophobic loop dynamics utilizing four mutants that have cysteine residues introduced at a single location along the yeast actin loop. Lateral, copper-catalyzed disulfide cross-linking of the mutant cysteine residues to the native C374 in the neighboring strand within the filament was fastest for S265C, followed by V266C, L267C, and then L269C. Site-directed spin labeling (SDSL) studies revealed that C265 lies closest to C374 within the filament, followed by C266, C267, and then C269. These results are not predicted by the Holmes extended loop model of F-actin. Furthermore, we find that disulfide cross-linking destroys L267C and L269C filaments; only small filaments are observed via electron microscopy. Conversely, phalloidin protects the L267C and L269C filaments and inhibits their disulfide cross-linking. Combined, our data indicate that, in solution, the loop resides predominantly in a "parked" position within the filament but is able to dynamically populate other conformational states which stabilize or destabilize the filament. Such states may be exploited within a cell by filament-stabilizing and -destabilizing factors.

According to the original Holmes model of F-actin structure, the hydrophobic loop 262-274 stabilizes the actin filament by inserting into a pocket formed at the interface between two protomers on the opposing strand. Using a yeast actin triple mutant, L180C/L269C/C374A [(LC)(2)CA], we showed previously that locking the hydrophobic loop to the G-actin surface by a disulfide bridge prevents filament formation. We report here that the hydrophobic loop is mobile in F- as well as in G-actin, fluctuating between the extended and parked conformations. Copper-catalyzed, brief air oxidation of (LC)(2)CA F-actin on electron microscopy grids resulted in the severing of thin filaments and their conversion to amorphous aggregates. Disulfide, bis(methanethiosulfonate) (MTS), and dibromobimane (DBB) cross-linking reactions proceeded in solution at a faster rate with G- than with F-actin. Cross-linking of C180 to C269 by DBB (4.4 A) in either G- or F-actin resulted in shorter and less stable filaments. The cross-linking with a longer MTS-6 reagent (9.6 A) did not impair actin polymerization or filament structure. Myosin subfragment 1 (S1) and tropomyosin inhibited the disulfide cross-linking of phalloidin-stabilized F-actin. Electron paramagnetic resonance measurements with nitroxide spin-labeled actin revealed strong spin-spin coupling and a similar mean interspin distance ( approximately 10 A) in G- and in F-actin, with a broader distance distribution in G-actin. These results show loop 262-274 fluctuations in G- and F-actin and correlate loop dynamics with actin filament formation and stability.

The dipole potential of lipid bilayer membrane controls the difference in permeability of the membrane to oppositely charged ions. We have combined molecular dynamics (MD) simulations and experimental studies to determine changes in electric field and electrostatic potential of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipid bilayer in response to applied membrane tension. MD simulations based on CHARMM36 force field showed that electrostatic potential of DOPC bilayer decreases by ~ 45 mV in the physiologically relevant range of membrane tension values (0 to 15 dyn/cm). The electrostatic field exhibits a peak (~ 0.8 × 109 V/m) near the water/lipid interface which shifts by 0.9 A towards the bilayer center at 15 dyn/cm. Maximum membrane tension of 15 dyn/cm caused 6.4% increase in area per lipid, 4.7% decrease in bilayer thickness and 1.4% increase in the volume of the bilayer. Dipole-potential sensitive fluorescent probes were used to detect membrane tension induced changes in DOPC vesicles exposed to osmotic stress. Experiments confirmed that dipole potential of DOPC bilayer decreases at higher membrane tensions. These results are suggestive of a potentially new mechanosensing mechanism by which mechanically induced structural changes in the lipid bilayer membrane could modulate the function of membrane proteins by altering electrostatic interactions and energetics of protein conformational states.

International audience; Cofilin is a major cytoskeletal protein that binds to both monomeric actin (G-actin) and polymeric actin (F-actin) and is involved in microfilament dynamics. Although an atomic structure of the G-actin-cofilin complex does not exist, models of the complex have been built using molecular dynamics simulations, structural homology considerations, and synchrotron radiolytic footprinting data. The hydrophobic cleft between actin subdomains 1 and 3 and, alternatively, the cleft between actin subdomains 1 and 2 have been proposed as possible high-affinity cofilin binding sites. In this study, the proposed binding of cofilin to the subdomain 1/subdomain 3 region on G-actin has been probed using site-directed mutagenesis, fluorescence labeling, and chemical cross-linking, with yeast actin mutants containing single reactive cysteines in the actin hydrophobic cleft and with cofilin mutants carrying reactive cysteines in the regions predicted to bind to G-actin. Mass spectrometry analysis of the cross-linked complex revealed that cysteine 345 in subdomain 1 of mutant G-actin was cross-linked to native cysteine 62 on cofilin. A cofilin mutant that carried a cysteine substitution in the alpha 3-helix (residue 95) formed a cross-link with residue 144 in actin subdomain 3. Distance constraints imposed by these cross-links provide experimental evidence for cofilin binding between actin subdomains 1 and 3 and fit a corresponding docking-based structure of the complex. The cross-linking of the N-terminal region of recombinant yeast cofilin to actin residues 346 and 374 with dithio-bis-maleimidoethane (12.4 A) and via disulfide bond formation was also documented. This set of cross-linking data confirms the important role of the N-terminal segment of cofilin in interactions with G-actin.