Single variable domains from the T cell receptor β chain function as mono- and bifunctional CARs and TCRs

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.

components of large-molecule and cell-therapy applications, constraints potentially rooted in their basic structure and function. Such structural complexity and increased size may hamper the practical development of next-generation bifunctional therapeutics.
The appeal of bifunctional targeting molecules, coupled with packaging limitations of gene-transfer vectors such as lenti-and retroviruses, raises the question of whether TCRs can function as a single binding domain; e.g., as a Vβ devoid of its normal partner, the α variable domain (Vα). To our knowledge, no instance of such a streamlined TCR has been reported, though shark species have a variety of novel TCRs that are distinct from mammalian orthologs 8,9 . If such a simplified TCR variable domain that retains specificity and functional activity against particular pMHCs were possible, it could open a path to engineering smaller, more complex binding capabilities with svd TCRs.
Though svd TCRs are an attractive concept, it is not evident that such stripped-down binding domains can be derived easily from mammalian TCRs. The three-dimensional structure of TCRs reveals a complex where the TCR Vα and β chains pack together, covering a large (>200 Å 2 ) hydrophobic surface. Removal of the Vα domain exposes this surface to destabilizing solvent interactions. By analogy with protein engineering efforts on antibodies conducted over the past decades, such dramatic changes in structure require significant adjustments, either by design or selection, to accommodate the single-variable domain format 10,11 . Despite the challenges, antibody VH-only domains are increasingly popular for generation of innovative clinical molecules with multi-specific binding properties 12,13 .
Here we demonstrate that svd TCRs can be constructed from a Vβ domain with no additional sequence engineering within the framework and constant regions of the β chain. These svd TCRs express stably on the surface of mammalian cells, including T cells. They bind pMHC tetramers selectively and appear to trigger T cells in much the same manner as full TCRs. Moreover, they function in tandem as CAR and TCR bifunctional proteins. Such Vβ-only domains provide a powerful basis for next-generation engineered T cell therapeutics and shed light on the mechanisms that dictate signaling sensitivity of TCRs and CARs.

Results
Identification of β-only pMHc binding domains. To identify peptide-specific pMHC-binding molecules, V(D)J recombination in cell culture was used to generate sequence diversity in the TCR Vβ domain while keeping the Vα constant in a controlled fashion in HEK293 cells (see patent WO2017/091905 for details about the HuTARG TM platform used to generate the TCR diversity). Over 100 million TCR-expressing cells were sorted for TCRs specific to the HLA-A*02-01 allele in complex with NY-ESO-1 9 V variant peptide (SLLMWITQV) or MAGE-A3 peptide (FLWGPRALV) using pMHC multivalent tetramers. Note that C-terminal to the Cα/β domains, the library construct encoded the human CD3ζ transmembrane and cytoplasmic domains fused to the β chain to facilitate its expression in HEK293 cells, and subsequent analysis. Specific pMHC-binding cells were enriched over multiple rounds and purified from the library. During the process, α chain expression was inadvertently lost and β chains that endow specific epitope-binding in the absence of a second TCR variable domain were recovered. Such β-only clones were used as the basis of the studies described below.

β-only domains express stably and show selective pMHc binding in cAR and tcR formats.
Several clones of β-only domains selective for either HLA-A2/NY-ESO-1 peptide or HLA-A2/MAGE-A3 peptide were recovered from the HuTARG ™ sort and sequenced. Among these, 3 and 4 unique CDR3 sequences were identified for NY-ESO-1-and MAGE-A3-selective binders, respectively (Supplementary Table S1). Interestingly, all 7 β idiotypes for both pMHC targets utilize the TRBV5-8*01 segment, suggesting that there are structural properties of this Vβ segment that facilitate stability of a single domain in the absence of its normal Vα partner (see below). Furthermore, the average CDR3 loop length was 16.3 amino acids (range 12-20), ~2 amino acids longer than the average observed among a set of human TCR Vβ sequences 14 . These idiotypes were characterized for their ability to express in HEK293T cells and bind pMHC targets by flow cytometry. The TCR βF1 (8A3) antibody was used to detect surface-expressing TCRβ chains. This antibody does not stain the native form of the TCR 15 , allowing detection of the TCRβ chain in CAR formats only. pMHC tetramers were generated by conjugating biotin-labeled, peptide-containing MHC Class I A*02:01 with fluorophore-labeled streptavidin in a tetramer format. All idiotypes were expressed stably on the cell surface with various tetramer-binding abilities ( Supplementary Fig. S1). The strongest binders, idiotype #2 for a NY-ESO-1-targeted Vβ-only domain and idiotype #5 for a MAGE-A3-targeted domain, were selected for further studies.
To assess the specificity and versatility of these Vβ-only domains, the NY-ESO-1-and MAGE-A3-binding idiotypes (#2 and #5, respectively) were formatted as TCRαβs (referred to as svd TCR) and CARs (referred to as Vβ-only CAR). For the TCRαβ format, a generic α chain that is abundantly expressed in humans was used as a surrogate (TRAV41*01/J49*01). For the CAR format, Vβs were linked to the β chain constant region (Cβ), which in turn was fused to a CD3ζ transmembrane and intracellular signaling domain (first-generation CAR) or a CD28 transmembrane region and CD28 and CD3ζ intracellular signaling domains (second-generation CAR) (Fig. 1a). For comparison we used the previously reported NY-ESO-1-targeted TCR clone 1G4 16 and MAGE-A3-targeted mouse TCR 17 , as well as CARs with scFvs derived from reformatted antibody surface display (HuTARG TM ) libraries. These constructs were transfected into HEK293T cells to assess expression and binding specificity by flow cytometry. Mixtures of 7 off-target pMHCs with different HLA allele and unrelated peptides were used to generate negative-control tetramer. Flow cytometry data showed that both NY-ESO-1-and MAGE-A3-targeted Vβ-only CARs are stably expressed and selectively bind to target tetramers in HEK293T cells. svd TCRs also express and show specificity for the pMHC target, but with significantly lower binding (Fig. 1b).
To examine expression and function of Vβ-only CARs and svd TCRs in a more natural setting, the constructs were transfected into the Jurkat human T cell line. Additional CAR constructs with the Vβ-Cβ domains linked to CD28 transmembrane region with CD28, 4-1BB and CD3ζ intracellular signaling domains (third-generation CAR) The TRB5-8 Vβ segment encodes a sequence that may more readily form a single functional binding domain. Based on the crystal structure of a Vβ−5 TCR complexed with MAGE-A3 pMHC, we modeled one of the MAGE-A3 Vβ-only domains described here (idiotype #5) and the results matched the expectation that some adjustments to the polypeptide backbone are required (Fig. 2a) 18 . A molecular dynamics simulation of the TRB5-8 Vβ-only structure revealed that the interface with Vα is at least partly covered by a collapse of the CDR3 loop over its hydrophobic surface to accommodate the loss of Vα (Fig. 2b). In addition, modeling two other TCRs (6AT6 and 6AVG) 19 that contain V segments from other families, suggests that at least some other Vβ domains require more radical changes to form a thermodynamically stable structure absent the Vα domain, compared to Vβ5−8 (Fig. 2c,d). Specifically, the crystal structure of a TRBV28-encoded domain shows nearly 80% more hydrophobic surface area exposed, compared to the Vβ5-8-only domain. Presumably such sequences have a more challenging route to formation of a stable single globular domain.
Vβ-only domains in cAR formats are functionally active. To assess whether target binding by Vβ-only domains can lead to functional activity, engineered Jurkat cells that expressed an NFAT-driven luciferase reporter gene were transfected with Vβ-only constructs. Serially diluted NY-ESO-1 and MAGE-A3 peptides were loaded onto T2 cells (HLA-A2+) 16 hours prior to co-culturing with transfected Jurkat effector cells. Population-averaged NFAT signal was measured by luminescence after 6 hours of co-culture. Peptide-titration curves showed that both NY-ESO-1 and MAGE-A3 Vβ-only CARs trigger peptide-concentration-dependent NFAT activation (Fig. 3). The activation is specific to its respective target peptides ( Supplementary Fig. S2). First-generation CAR formats showed about two-fold less sensitivity than second-and third-generation CARs with both binders based on the EC 50 (Supplementary Table S2). Compared to control third-generation CARs with affinity-matured scFv binders, third-generation CARs with Vβ domains were ~100 × (NY-ESO-1) and ~1000 × (MAGE-A3) less sensitive (Supplementary Table S2).
The MAGE-A3 Vβ-only CARs showed elevated background NFAT signaling (Fig. 3). However, this feature does not appear to be inherent to all Vβ-only CAR constructs as the other three idiotypes of MAGE-A3 Vβ-only modules showed lower tonic signal than the scFv CAR ( Supplementary Fig. S3). To explore the cellular localization and aggregation profile of the Vβ-only CARs, transfected Jurkat cells were fixed and stained with cognate target pMHC tetramers in the presence or absence of nonionic detergent for permeabilization ( Supplementary  Fig. S4). No obvious difference in surface expression or intracellular aggregation among constructs with high and low backgrounds was observed.
To identify the minimal module that is required for epitope binding of Vβ-only CAR constructs, CAR vectors were generated where the Cβ domain is replaced with a CD8 hinge ( Supplementary Fig. S5). Peptide-concentration-dependent NFAT signal was observed with these constructs with only modest decrease in sensitivity, indicating that Cβ is not necessary for the Vβ-only binding ability ( Supplementary Fig. S5).
Cells transfected with svd TCR did not signal, consistent with the lack of tetramer binding observed in these cells.   α and β gene cluster that prevents surface expression of the endogenous TCR complex. Because TCR α and β are not expressed, other subunits of the TCR complex, namely 4 CD3 proteins, are expressed but unable to assemble on the surface 20 . Therefore, CD3Ɛ staining was used as a readout for expression and pairing of the transfected TCRα and TCRβ constructs on the cell surface. As previously reported, the NY-ESO-1 control TCR is expressed on the surface of SupT1 cells. Moreover, the Vβ-only complex also are positive for CD3Ɛ staining, suggesting that the complex integrates the β and α subunits. However, only the control TCR showed tetramer binding, consistent with the observation in transfected Jurkat cells (Fig. 4b).
Because we observed pMHC tetramer binding of Vβ-only domains in CAR formats but not as a TCR complex, we hypothesized that the TCR Vα may hinder the Vβ-only domains' ability to bind targets. To test this hypothesis, the Vα domain of the α chain was deleted and co-expressed with Vβ chains in SUP-T1 cells. These cells showed tetramer binding, confirming that the Vα indeed hinders epitope binding of Vβ-only domains (Fig. 4a,b). Furthermore, Jurkat cells transfected with the β and Vα-deficient chains revealed T2/peptide-dependent activation, demonstrating that the svd TCRs when their normal Vα partner is removed but the remainder of the alpha chain is retained (Fig. 4c). Despite this functional activity, tetramer binding was not observed by flow cytometry in transfected Jurkat cells, suggesting that the NFAT reporter system is more sensitive than tetramer binding as we have observed previously.
Attempts to trim the Cα domain further resulted in complete loss of tetramer binding of the modified TCRs in SUP-T1 cells and corresponding loss of functional activity in Jurkat cells ( Supplementary Fig. S6). This result implies that an intact Cα domain is necessary to form a proper surrogate, and a minimal α polypeptide with only a transmembrane domain is not compatible with Vβ-only domain function, probably because of inefficient TCR complex formation absent the Cα domain, likely caused in part by exposed hydrophobic surfaces at the Cα/β interface. www.nature.com/scientificreports www.nature.com/scientificreports/ comparison of signaling sensitivities of Vβ-only domains in cAR and tcR formats. Our system permits direct comparison of the same ligand-binding domains on a CAR and TCR. Interestingly, the sensitivity of the two formats were similar, which suggests that the ligand-binding domain-its affinity and geometry-is the main determinant of sensitivity rather than the details of the signaling mechanism that differ between the CAR and TCR. This observation is consistent with studies where we grafted scFv against the same pMHC targets onto the control NY-ESO-1 TCR, modified to accept the scFvs by removing the Vα and Vβ domains. These hybrid TCRs have sensitivities in Jurkat signaling assays that are nearly identical to the CARs from which they are derived, nearly 100x less sensitive compared to the parental TCR ( Supplementary Fig. S7). These observations suggest that, at least in a controlled system where NFAT activation in Jurkat cells is the measure of sensitivity, the ligand-binding domain dictates the signaling sensitivity.
Bispecific Vβ-only domains function in cAR and tcR formats. Recent clinical trials have revealed that current adoptive T-cell therapy is susceptible to antigen escape by tumor cells 21,22 . T cells that recognize multiple antigens offer a prospective safeguard against this problem. Bispecific receptors have been explored and developed in the context of CARs 23,24 . However, TCRs with a capacity to target two different antigens have not been reported to our knowledge. Given the small and versatile binding properties of Vβ-only domains, we suspected they might function in a more complicated format as a bifunctional CAR or TCR.
To test this idea, bispecific Vβ-only CARs targeting NY-ESO-1 and MAGE-A3 pMHCs were generated in a second-generation CAR architecture with Cβ and the two Vβ domains connected in tandem via a (G 4 S) 3 GG flexible linker. Jurkat cells transfected with these constructs showed detectable binding of both NY-ESO-1 and MAGE-A3 tetramers by flow cytometry (Fig. 5a). Consistently, in the NFAT-luciferase reporter assay, bispecific CARs functioned in response to both NY-ESO-1-and MAGE-A3-peptide-loaded T2 cells with only modest declines in their individual sensitivities (Fig. 5b). The binding domain positioned further from the membrane (i.e., at the N-terminus) showed a slight decrease in sensitivity while the one directly attached to Cβ showed no change compared to the monospecific CARs.
To examine if Vβ-only domains could function as bispecific TCRs, we constructed variants in both cis and trans configurations (i.e., in tandem on the β chain, or with one Vβ domain on each chain, Cα and Cβ (Fig. 5c). As a control, Vβ fused on the TCRα chain was also expressed with a TCRβ chain that lacked a Vβ domain (i.e., Cβ only). This construct showed no function in the Jurkat NFAT-luciferase reporter assay, indicating that Vβ domains moved to the α-chain abolishes its function (Fig. 5d). Therefore, the trans bispecific svd TCRs showed functional activity only against the pMHC target of the binder fused on the β-chain (Fig. 5d). Cis bispecific svd TCRs were generated by connecting two Vβ domains in tandem via a (G 4 S) 3 GG flexible linker and expressing this construct with a surrogate TCRα chain with the Vα deleted. To our surprise, bifunctional cis svd TCRs with NY-ESO-1 binder on the N-terminus followed by MAGE-A3 binder N-terminal to Cβ showed both NY-ESO-1 and MAGE-A3 peptide-dependent signaling in Jurkat cells (Fig. 5d). Cis Vβ domains in the other orientation with the MAGE-A3 binder at the N-terminus also showed functional activity against both target peptides, although the magnitude of the signal (Emax) with MAGE-A3 peptide was reduced. The EC 50 in assays with peptides loaded on T2 was similar for both constructs, compared to the sensitivities of monospecific parental versions of the constructs (Supplementary Table S2). We also tested if there was interaction detectable at a functional level between the two pMHC ligands when supplied to Jurkat cell expressing bispecific constructs. Nothing beyond a potentially additive effect was observed using the analytical methods of Bliss and Loewe independence 25,26 . primary t cells expressing Vβ-only constructs have cytotoxic activity. To evaluate the effect of Vβ-only domain constructs on T cell activity, primary T cells were transduced with lentivirus and expression was confirmed by NY-ESO-1 or MAGE-A3 tetramer staining (Fig. 6a). CAR constructs expressed much better than TCR constructs, most likely due to the mispairing of the introduced TCR chains to endogenous TCR chains. Transduced T cells were used in an IncuCyte cell killing assay that enables visualization of target and effector cells by microscopy at 37 °C over time. A375 cells that stably express nuclear locating GFP were loaded with 10 μM NY-ESO-1 or MAGE-A3 peptides and co-cultured with transduced T cells at 1:1 ratios. T cell number was adjusted according to the transduction percentage measured by tetramer staining.
For NY-ESO-1 binders, T cells expressing the benchmark TCR showed the most potent cytotoxic activity, followed by T cells expressing the scFv-CAR, and the Vβ-only domain constructs in CAR and TCR formats which had similar killing activities (Fig. 6b). IFNγ measured in the supernatant of the co-culture at 24 hours showed a similar trend (Fig. 6c). K562 cells that overexpress single chain NY-ESO-1-β 2 m-HLA-A2 trimer 27 and GFP were also used as target cells in the real-time killing assay. In this situation where the antigen is presented abundantly, all 4 NY-ESO-1-targeted constructs showed similar killing activities (Supplementary Fig. S8).
T cells expressing the MAGE-A3 benchmark TCR and scFv-CARs only showed mild cytotoxic activities while the Vβ-only-CAR and svd TCR triggered more robust killing (Fig. 6b). However, these Vβ-only-CAR and svd TCR cells also showed weak cytotoxicity toward K562 cells without any MAGE-A3 peptide ( Supplementary  Fig. S8), suggesting that these constructs likely trigger ligand-independent apoptosis of target cells. This is consistent with the high background NFAT signal observed in Jurkat cells transfected with the MAGE-A3 Vβ-only-CAR and svd TCR (Fig. 4c).

Discussion
We have created Vβ-only domains that express, specifically recognize cognate pMHC ligands, and function robustly in Jurkat and primary T cells. The generality of this effect is suggested by the isolation and characterization of multiple binders against two different pMHC targets. (2019) 9:17291 | https://doi.org/10.1038/s41598-019-53756-4 www.nature.com/scientificreports www.nature.com/scientificreports/ In a TCR format, the β chain utilize a surrogate α chain that lacks a Vα segment and forms activation-competent TCRs complexed with the six CD3 subunits. We presume the requirement for a surrogate α subunit relates to the formation of a complex in the membrane with other CD3 subunits that comprise a functional TCR. Sequential N-terminal deletions demonstrate that the globular Cα domain is necessary for full activity of the svd TCR. In contrast, the presence of a Vα domain on the α subunit, opposite the single Vβ polypeptide, has no impact on surface expression, but interferes with ligand-binding, presumably via steric hindrance of the pMHC ligand to its cognate Vβ binding site.
Selectivity of the svd TCRs compares favorably with other TCRs and antibodies that bind pMHCs. They retain the HLA restriction for which they were selected. Thus, the fact that evolution has favored a two-domain ligand-binding structure does not necessarily preclude the creation of selective single-domain binders, a feature that also applies to the antibody gene family 28 . Rather, it may be that a repertoire composed of two chains generates necessary diversity through the agency of combinatorial association. www.nature.com/scientificreports www.nature.com/scientificreports/ In addition, the ligand-binding module can be grafted onto CAR signaling constructs where it functions independent of a TCR α chain. Both ligand-binding domains examined most closely (one NY-ESO-1 and one MAGE-A3 binder) are compatible with a variety of CAR formats that incorporate different CAR components (Figs. 1,3). Interestingly, the CAR versions of the Vβ-only ligand-binding domains have similar sensitivity to their TCR cousins. This result suggests that the properties of the ligand-binding domain determine functional sensitivity, regardless of the format; i.e., it is not the detailed structure of the signaling components, but rather, the binding properties of the receptor that dictate sensitivity, at least in the Jurkat NFAT-activation assay. This conclusion is strengthened by the observation that scFv sequences derived from antibodies generated against the same pMHC targets (NY-ESO-1 and MAGE-A3), when grafted onto TCR subunits, yield hybrid TCRs that are nearly identical in sensitivity to CAR constructs that harbor the same scFvs ( Supplementary Fig. S6). Thus, with two different pMHC targets and two independent origins of ligand-binding domains, one pair derived from TCRs and the other from antibodies, the sensitivity uniformly tracks with the ligand-binding domain, not the type of signaling molecule. These results again suggest that TCRs may acquire their striking sensitivity not by their highly evolved, multi-subunit mechanism, but rather through the process of selection in the body. It is possible that both α and β chains need to interact with the cognate MHC to deliver a sensitive signal. However, analysis of previously reported TCR-pMHC structures and amino acid sequences suggest that there are multiple instances where CDR3α makes no contact with the target pMHC whereas CDR3β contact is always present 29 . For TCRs isolated and studied in vitro by investigators, their extraordinary potency may be further explained by additional functional selection through growth on antigen-positive substrates or picking rare antigen-responsive T cells clones among hundreds of thousands of T cells 16,17 .
Our observations run contrary to the conclusion of Harris et al. who engineered two human TCR Vα/β pairs directed at either a WT-1 or MART-1 pMHC 30 . When these affinity-matured binders were tested as either reconstructed TCRs or CARs, the TCR versions were 10-100x more sensitive in both cases. The reasons for the discrepancy remain to be established, but include the possibilities that: (i) The Vα/β interaction is a critical part of the TCR signaling mechanism, and therefore the Vβ-only TCR is crippled with respect to TCR sensitivity because it lacks Vα; (ii) IL-2 expression used by Harris et al. as a readout favors the TCR signaling mechanism compared to NFAT activation measured in the Jurkat assay conducted by us; (iii) their use of a murine T cell hybridoma line and murine primary T cells as effectors with a human T2 lymphoid line as stimulus creates an interspecies www.nature.com/scientificreports www.nature.com/scientificreports/ activation scenario that somehow biases sensitivity toward TCRs; or, (iii) optimal TCR function follows from TCRs that are subjected to strong functional selection; if they originate from physical binding selections like the ones studied here, they lack the extraordinary sensitivity characteristic of some functionally-selected TCRs and, instead, display sensitivities equivalent to their CAR counterpart.
Signaling behavior of the Vβ-only CARs and svd TCRs are similar in many respect to TCRs regarding the shape of the dose/response curve (Fig. 4). However, they are less sensitive than the most highly selected or optimized TCRs, which are typically obtained from patients' functionally selected T cells, or otherwise optimized for activity 16,17 , including the benchmark NY-ESO-1 TCR used in our experiments. The MAGE-A3-targeting Vβ-only module showed high background (tonic signal) and a narrow activation window in Jurkat cells (Figs. 3, 4c). Note that Vβ-only modules used in this study are not optimized. Sensitivity, tonic signaling, and activation windows are properties that can be optimized by screening larger numbers of variants (unpublished data). For example, the other three idiotypes (ID #1, 2, 4) of MAGE-A3 Vβ-only modules show much lower tonic signal than ID #5 ( Supplementary  Fig. S3), which we initially chose for further study due to its strongest tetramer binding ability (Supplementary Fig. S1). Based on a broad range of studies with TCRs, we believe efforts to optimize binding of Vβ-only-modules would produce more potent receptors [31][32][33][34][35] . In other words, there is nothing obvious in a Vβ-only ligand binding domain that precludes it from mimicking a natural TCR in all its fundamental attributes. However, we acknowledge that further validation and optimization will be needed to prove the utility of this technology beyond its academic interest.
Considerable effort has been directed at antibodies to engineer single-variable domains that avoid the requirement for a second chain. The goal is to make the ligand-binding function smaller and more easily shuttled from one molecular setting to another. The existence of natural single-domain antibodies, notably from the family Camelidae (including camels and llamas) and class Chondrichthyes (including sharks), has pointed the way toward engineering similar fully human domains. These small phylogenetically distant single-domain antibodies represent a significant variation of the classic immunoglobulin fold in which the surface that is typically involved in the interaction with a light chain variable domain is altered and covered by an extension of the CDR3 loop, resulting in a more compact, convex globular structure [36][37][38] . Efforts to engineer antibody structures to produce single-domain (e.g., heavy-chain only) variable domains, have required the introduction of substantial amino acid variation and structural change that create molecules more similar to the camelid single-domain molecules, compared to the two-chain structures from which they derive 39,40 .
Although we have not elucidated the atomic structure of the Vβ-only domains described here, we strongly suspect that they also involve considerable deviation from the two-chain TCR. Notably, all the Vβ-only domains identified use the Vβ5-8 variable gene segment. This bias is not explained by a skewed TCR input library because TRB-5-8, the V gene segment involved, was present at roughly the expected frequency in the HuTARG TM library prior to induced TCR rearrangement (~1% of total V segments; data not shown). Its frequency was increased about 6-fold among cells that express surface TCRs, consistent with the view that it may more readily form stable globular domains. This possibility is supported by modeling (Fig. 2).
Vβ-only domains provide an opportunity to create bifunctional TCR ligand-binding domains that are smaller and more portable-potentially very useful in the context of CAR-T and TCR-T engineering. In particular, investigators have created bifunctional CARs that incorporate scFvs in tandem to direct engineered T cells at two separate antigens; e.g., CD19 and CD20 23,24 . The hypothesis is that such dual-targeted receptors will pose additional obstacles for tumor relapse, as the tumor must escape two rather than one T-cell targeting mechanisms. Such approaches have also been proposed for CARs, but the technical route toward TCR bifunctionality is less clear.
We have taken a significant step toward this goal by demonstrating that Vβ-only domains can be linked in tandem with only modest decline in their individual sensitivities to generate small, bifunctional single-Vβ-based CARs and TCRs. In principle, deployment of suitable TCR Vβs directed at different pMHCs permits construction of hybrid molecules activated by both pMHCs in a single engineered T cell, without either the problems associated with mispairing of multiple transferred subunits or the size limits imposed by current viral gene transfer vectors.
Interestingly, the bifunctional TCRs display improved functional selectivity compared to the individual binding domains tested on their own (Fig. 5b,d). Particularly the MAGE-A3 svd TCR exhibits some evidence of off-target activation when exposed to HLA-A2-positive T2 cells. However, in the bifunctional format this background nearly disappears. Though we do not have a simple explanation for this change, it suggests that the bifunctional format is at least compatible with production of stable, well-behaved surface receptors that bind ligands specifically. this study. HEK-293T and A375 cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Jurkat and SUP-T1 cells were maintained in RPMI supplemented with 10% heat-inactivated FBS and 1% penicillin/streptomycin. T2 cells were maintained in IMDM supplemented with 20% FBS and 1% penicillin/streptomycin. K562 cells engineered to stably express single chain NY-ESO-1β 2 m-HLA-A2 trimer 27 and GFP were generated and maintained in RPMI supplemented with 10% heat-inactivated FBS and 1% penicillin/streptomycin. Cells were incubated at 37 °C in 5% CO 2 . plasmid construction. All constructs, except for the recovered Vβ-only pool from the HuTARG TM platform (Patent WO2017/091905), were generated by Golden Gate assembly into the pLenti backbone, which has an EF1α promoter. NY-ESO-1 and MAGE-A3 scFvs on the control CARs were obtained from binder screens using the HuTARG TM platform. Sequences for the control NY-ESO-1 and MAGE-A3 TCRs were obtained from patents US8143376B2 and W02012054825A1, respectively. (2019) 9:17291 | https://doi.org/10.1038/s41598-019-53756-4 www.nature.com/scientificreports www.nature.com/scientificreports/ HutARG tM . See patent WO2017/091905 for details. Briefly, HEK293.2sus (ATTC CRL15733 ™ ) cells were engineered to contain a library of all combinations of unrearranged V, D and J segments of the β chain TCR locus. Because the host cells are constructed to contain a single loxP integration site, each cell post transfection of the library contains at most a single integrated copy of one of the roughly 1,800 combinations of V-D-J (64 × 2 × 14) gene segments, each segment is flanked by a recombination signal sequence (RSS). When triggered to rearrange by addition of an exogenous inducer that activates RAG1 expression, the RSSs mediate intramolecular site-specific recombination such that the population of cells produce a large repertoire of TCRβ chains that are further subjected to drug selection for in-frame recombinants; the TCRβ chain is expressed as a fusion with the puromycin resistance gene. The cells also contain a surrogate single alpha chain for pairing and surface expression. Homology model and hydrophobic patch calculation. 3D structural models were generated in Prime homology modeling suite (Schrödinger, LLC) using the crystal structure of the MAGE-A3 TCR (TRAV21/ TRBV5-1 family; PDBID: 5BRZ) as the template. All structures were prepared using the Schrödinger Protein Preparation Wizard 41 before further calculations.

Methods
Hydrophobic patches on the surface of the homology model and crystal structures were calculated and visualized using the Protein Surface Analysis tool as implemented in Bioluminate (Schrödinger, LLC) 42 .
Molecular dynamics simulation. The system was solvated using the Desmond System Builder (Schrödinger, LLC). Sodium ions were added to neutralize the system. The OPLS3e force field was utilized for all calculations 43 . The system equilibrated using the relaxation protocol as implemented in the Schrödinger package. Molecular Dynamics simulations were performed in the NPT ensemble at 300 K and 1 atm pressure using Desmond (D.E. Shaw Research). The reversible reference system propagation algorithm (RESPA) multiple time step approach was used with a time step of 2 psec and long-ranged electrostatic interactions were computed every 6 psec. 44 . Van der Waals and short-range electrostatic interactions were cut off at 9 A° and smooth particle mesh Ewald (PME) method 45 was employed for calculation of long range electrostatic interactions. The temperature was controlled using a Nose-Hoover chain thermostat 46 and the pressure was controlled using the Martyna-Tobias-Klein barostat 47 . Three simulations of 100 ns each were performed using the last frame as the initial coordinates on a Quadro P5000 GPU card with coordinates saved every 1.2 ps for subsequent analysis.
nfAt Luciferase assay. NY-ESO-1 peptide (SLLMWITQV), a variant of the native peptide where cysteine at position 9 is mutated to Valine 48 , and MAGE-A3 peptide (FLWGPRALV) were synthesized by Genscript (Piscataway, NJ). Target peptides were serially diluted 3-fold starting at 100 μM and loaded onto 10,000 T2 cells in RPMI plus 1% BSA and 0.1% penicillin/streptomycin. Eighteen hours later, 12,000 transfected Jurkat cells encoding a NFAT-luciferase reporter were resuspended in RPMI/10% FBS/0.1% penicillin/streptomycin and added to the peptide-loaded T2 cells. The co-culture was incubated for 6 hours at 37 °C. One-step luciferase assay system (BPS Cat. No. 60690) was used according to manufacturer's instructions to read luminescence on a microplate luminometer at 100 ms. Each experiment was done in duplicate. primary t-cell transduction and activation. Peripheral blood mononuclear cells (PBMCs) and lentivirus were obtained from Alstem. PBMCs were activated with human T-cell TransAct ™ according to manufacturer's instructions in X-vivo media. For the transduction, lentivirus was added directly to the cells at a MOI of 10. Transduced primary T-cells were expanded in X-vivo medium with 1% human serum, 0.1% penicillin/streptomycin, 10 ng/ml IL-15, and 10 ng/ml IL-21. Cells were quiesced for 48 hours without stimulation or cytokines prior to assays. cytotoxicity and ifnγ assay. A375 cells were transfected with nuclear locating GFP (IncuCyte NucLight Green) and selected for stable integration. Five thousand A375 cells were seeded with 10 μM of NY-ESO-1 or MAGE-A3 peptides. After 18 hours, cells were treated with 10 μg/ml of mitomycin C for 1 hour and washed three times. Five thousand transduced T-cells (adjusted for transduction percentage measured by tetramer staining) were added and monitored using the IncuCyte S3 live-cell analysis system (Essen Bioscience) at 37 °C and 5% CO 2 . Images were captured every two hours for 42 hours using a 10X objective. Green area at each time point was normalized to time zero to measure loss of live target cells. Experiments were done in duplicate.
Supernatants were harvested at the 24 hour time-point of the cytotoxicity assay co-culture. IFNγ levels were measured using the BD Cytometric bead array system according to manufacturer's instruction.