IntroductionAlthough T-cell immunotherapies have been effective in the treatment of hematological malignancies, solid tumors have proven challenging due to the immunosuppressive microenvironment and lack of viable target antigens. The immune checkpoint ligand CD70, overexpressed in several solid tumors, yet with limited expression in healthy tissue, has emerged as a promising immunotherapeutic target.MethodThis study describes the generation and preclinical characterization of ADP-520, a high-affinity, fratricide-resistant, CD70-targeted T-cell receptor fusion construct (TRuC) T-cell therapy enhanced with constitutively expressed mbIL-15, a membrane-bound fusion protein comprising interleukin-15 (IL-15) linked to full-length IL-15 receptor-alpha. The phenotypic distribution, expansion and persistence of ADP-520 TRuC T cells were measured in vitro under autonomous and antigen-dependent conditions, with the contributions of TCR and IL-15 signaling pathways ascertained using inhibition assays. Chronic antigen stimulation was used to evaluate exhaustion-resistance, while anti-tumor potency was explored both in vitro and in vivo.ResultsADP-520 was found to have potent and antigen-specific activity against hematological and solid CD70-expressing tumors, without apparent fratricide or killing of bystander T cells despite CD70 expression by activated lymphocytes. Engineered co-expression of mbIL-15 augmented antigen-dependent expansion through pro-survival effects and enrichment of an early memory T-cell phenotype, thus enhancing tumor-autonomous, exogenous cytokine-free persistence and bolstering exhaustion resistance during chronic stimulation. mbIL-15 co-expression also enhanced intratumoral T-cell infiltration in vivo for potent and persistent antitumor efficacy.DiscussionThese findings characterize ADP-520 as a first-in-class, CD70-targeted, fratricide-resistant autologous TRuC T-cell therapy leveraging native TCR signaling combined with constitutive IL-15 signaling to impart T cells with enhanced persistence, tumor penetration, and antitumor efficacy. This makes ADP-520 a promising cell immunotherapy candidate for clinical development, with the potential to overcome hurdles intrinsic to the treatment of solid tumors.

Vitamin B 12 (cobalamin) is a high-value yet scarce cofactor required for various metabolic processes, making its efficient handling important for maintaining metabolic homeostasis. While the involvement of ATP:cob(I)alamin adenosyltransferases (MMAB) in the synthesis, delivery, and repair of 5'-deoxyadenosylcobalamin (AdoCbl) is well established, the kinetic mechanisms that regulate this process, particularly its negative cooperativity, remain poorly understood. Understanding these mechanisms is key to clarifying how MMAB efficiently uses AdoCbl, prevents resource wastage, and supports bacterial survival in nutrient-limited environments. Using single-molecule relative fluorescence (SRF) spectroscopy, we found that conformation-gated binding is the driving force behind MMAB's preference for AdoCbl over hydroxocobalamin and is the underlying mechanism for negative cooperativity. This mechanism significantly slows down the binding of the second equivalent of AdoCbl, favoring the singly bound state. Our findings indicate that MMAB predominantly binds a single AdoCbl, optimizing the AdoCbl loading to methylmalonyl-CoA mutase. Additionally, our SRF approach also serves as a tool to explore other cofactor interactions, such as those between riboswitches and cobalamin derivatives, to provide insights into regulatory mechanisms of cobalamin sensing and gene regulation, which are crucial for bacterial adaptation to changing nutrient conditions. Significance Statement:MMAB is important for B 12 -dependent propionate metabolism in bacteria. Our findings reveal that conformation-driven binding mechanism underlines the negative cooperativity of MMAB, as it favors the binding of the first AdoCbl while limiting further binding. The larger k on for the first site, combined with similar unbinding rates for both sites, could provide a solution for optimizing cobalamin handling and minimize unnecessary waste. Our single-molecule fluorescence approach offers a powerful tool for investigating other dynamic cofactor interactions, providing new insights into regulatory mechanisms in bacterial metabolism.

Vitamin B12 (cobalamin) is a high-value yet scarce cofactor critical for metabolic homeostasis, necessitating efficient handling mechanisms. ATP:cob(I)alamin adenosyltransferase (MMAB) plays a central role in synthesizing, delivering, and repairing 5'-deoxyadenosylcobalamin (AdoCbl), but the kinetic mechanisms regulating this process, including negative cooperativity, remain unclear. Using single-molecule relative fluorescence spectroscopy, we reveal that conformation-gated binding mechanism, involving a required structural rearrangement prior to the first cofactor association, dictates MMAB's interaction kinetics. This mechanism slows the association of a second AdoCbl, resulting in strong negative cooperativity, favoring the singly bound state, and optimizing AdoCbl handling. This gating mechanism, supported by direct observation of a kinetic intermediate, also contributes to MMAB's preferential handling of AdoCbl over hydroxocobalamin, highlighting MMAB's effective cofactor utilization, supporting bacterial survival in nutrient-limited environments. Furthermore, our approach offers a platform to study cofactor interactions, including cobalamin sensing and gene regulation, shedding light on bacterial adaptation to nutrient fluctuations.

T cells expressing a mesothelin (MSLN)-specific T cell receptor fusion construct (TRuC®), called TC-210, have demonstrated robust antitumor activity in preclinical models of mesothelioma, ovarian cancer, and lung cancer. However, they are susceptible to suppression by the programmed cell death protein 1 (PD-1)/programmed cell death protein ligand 1 (PD-L1) axis and lack intrinsic costimulatory signaling elements. To enhance the function of anti-MSLN TRuC-T cells, chimeric switch receptors (CSRs) have been designed to co-opt the immunosuppressive PD-1/PD-L1 axis and to deliver a CD28-mediated costimulatory signal. Here, we report that coexpression of the PD1-CD28 CSR in TRuC-T cells enhanced T cell receptor signaling, increased proinflammatory effector cytokines, decreased anti-inflammatory cytokines, and sustained effector function in the presence of PD-L1 when compared with TC-210. Anti-MSLN TRuC-T cells engineered to coexpress PD1-CD28 CSRs comprising the ectodomain of PD-1 and the intracellular domain of CD28 linked by the transmembrane domain of PD-1 were selected for integration into an anti-MSLN TRuC-T cell therapy product called TC-510. In vitro, TC-510 showed significant improvements in persistence and resistance to exhaustion upon chronic stimulation by tumor cells expressing MSLN and PD-L1 when compared with TC-210. In vivo, TC-510 showed a superior ability to provide durable protection following tumor rechallenge, versus TC-210. These data demonstrate that integration of a PD1-CD28 CSR into TRuC-T cells improves effector function, resistance to exhaustion, and prolongs persistence. Based on these findings, TC-510 is currently being evaluated in patients with MSLN-expressing solid tumors.

Background Gavo-cel is an autologous and HLA-independent T-Cell Receptor Fusion Construct (TRuCTM) T cell therapy that targets mesothelin-expressing tumors and is under Phase 2 evaluation for treatment-resistant MPM, NSCLC, cholangiocarcinoma, and ovarian cancer tumors (NCT03907852). Mesothelin (MSLN) is a 71 kDa GPI-anchored membrane protein that undergoes proteolytic membrane shedding to generate soluble mesothelin-related peptides (sMRPs) whose levels are correlated with tumor burden in MPM. sMRPs contain the juxtamembrane epitope of that is recognized by the MH1 binder domain of the gavo-cel TRuC and whose levels are correlated with tumor burden in MPM. Because shed MLSN has been implicated as a potential obstacle to successful anti-MSLN therapies, including cell therapies, we assessed the impact of soluble MSLN (sMSLN) on the function of gavo-cel and or allogeneic anti-MSLN TRuC, MH1gd. Methods In this study, we generated primary human TRuC-T cells modeling MSLN-targeting clinical agent gavo-cel or that express MH1gd TRuC, and then measured the impact of soluble MSLN on the in vitro activation, cytotoxicity, and cytokine response of gavo-cel or MH1gd during acute and chronic challenge with antigen-expressing tumor cells. We also evaluated whether sMRP in human serum impacts the in vitro cytotoxicity and cytokine response of gavo-cel in response to MSLN-expressing tumor cell lines. Results High, supraphysiological levels of the full-length shed domain of MSLN, sMSLN, does not impair, block, or disrupt the effector function of gavo-cel or MH1gd TRuC-T cells with respect to in vitro cytotoxicity or cytokine production. Furthermore, gavo-cel demonstrates potent efficacy in vivo in a tumor model characterized by circulating sMSLN. Conclusions Our data indicate that both gavo-cel and allogeneic MSLN-targeting TRuC-T cells are not susceptible to functional suppression by sMRPs, even at supraphysiological levels that far exceed those found in cancer patients.

Abstract Previously we have described the design and antitumor activity of T cell receptor fusion constructs (TRuC™) that tether an antibody-derived binder to one of the TCR subunits to achieve redirected T cell killing of tumor cells independent of HLA. Different from CAR-T cells, TRuCs are integrated into the full TCR complex and thus harness its full signaling capacity. The cell surface antigen CD70 represents a promising target for cancer immunotherapy for its selective overexpression in various hematological and solid tumor indications. Because the normal tissue expression of CD70 occurs on activated lymphocytes, including activated T cells, fratricide (self-killing) has been recognized as a significant challenge for CD70-targeted T cell therapies. To address this challenge, we discovered a diverse pool of fully human anti-CD70 scFv binders that were used to make TRuC-T cells and then functionally screened for fratricide-resistance in vitro. We successfully identified a CD70-targeted TRuC-T cell candidate that exhibits normal T cell expansion and an improved memory phenotype, clearly differentiating from fratricide-prone candidates, all while maintaining potent cytotoxicity and cytokine production against tumor cells expressing both low and high levels of CD70. In addition, our CD70-targeted TRuC-T cells showed significant anti-tumor efficacy in multiple xenograft mouse models with no evidence of in vivo fratricide. In summary, we have engineered a fratricide-resistant CD70-directed TRuC-T cell therapy that has the potential to treat a wide range of both hematologic and solid cancers. Citation Format: Jian Ding, Amy Watt, Erica Liu, Zieba Adam, Derrick McCarthy, Jessica Gierut, Brett Schrand, Michael Lofgren, Jason Lajoie, Vania Kenanova, Philippe Kiefer-Kwon, Holly Horton, Dario A. Gutierrez, Robert Hofmeister, Robert Tighe. Discovery and preclinical characterization of fratricide-resistant TRuC T-cells targeting CD70 [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 1528.

Abstract Cluster of differentiation 28 (CD28) and programmed death receptor 1 (PD-1) are members of the CD28 superfamily of co-receptors that have critical roles in the regulation of T cell-mediated immunity and inflammation. Ligation of CD28 synergizes with T cell receptor (TCR) signaling to enhance T cell activation through the PI3K-Akt pathway, while PD-1 ligation by its ligands (PD-L1/L2) sequesters critical mediators of signaling from the TCR complex, thereby shunting T cell activation and effector function. Thus, the expression of PD-L1/L2 in solid tumors may pose a significant barrier to anti-tumor immunity and the efficacy of adoptive T cell therapies (ACT). We have recently described a novel class of engineered T cells that integrate a T cell receptor fusion construct (TRuC®) into the natural TCR complex, thereby reprogramming the specificity of the T cell to recognize tumor surface antigen in a human leukocyte antigen (HLA)-independent fashion. TC-210 T cells expressing mesothelin (MSLN) specific TRuCs demonstrate robust anti-tumor immunity in preclinical models of mesothelioma, protecting mice from tumor re-challenge while inducing lower levels of inflammatory cytokine release when compared to a 2nd generation MSLN-targeted CAR T. Here, we show that co-expression of a PD-1:CD28 switch receptor comprising the PD-1 extracellular domain fused to the CD28 intracellular domain, enhances the activity of TC-210 T cells. When compared to TC-210 expressing only the TRuC, co-expression of PD1:CD28 was able to restore PD-L1 mediated inhibition of cytokine production and proliferation in co-culture with tumor cells. In vivo and molecular mechanistic studies are currently underway. Citation Format: Derrick P. McCarthy, Sarah Guyette, Michael Lofgren, Jyothi Sethuraman, Thamara DeSilva, Ahmar Aziz, Troy Patterson, Shruti Datari, Tiffany Chan, Philippe Kieffer-Kwon, Christopher J. Rold, Reshma Singh, Jian Ding, Holly Horton, R. Anthony Barnitz, Andrew Cornforth, Robert Tighe, Robert J. Hofmeister, Dario A. Gutierrez. A chimeric PD1-CD28 switch receptor enhances the activity of TRuC-T cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 893.

G-proteins regulate various processes ranging from DNA replication and protein synthesis to cytoskeletal dynamics and cofactor assimilation and serve as models for uncovering strategies deployed for allosteric signal transduction. MeaB is a multifunctional G-protein chaperone, which gates loading of the active 5 '-deoxyadenosylcobalamin cofactor onto methylmalonyl-CoA mutase (MCM) and precludes loading of inactive cofactor forms. MeaB also safeguards MCM, which uses radical chemistry, against inactivation and rescues MCM inactivated during catalytic turnover by using the GTP-binding energy to offload inactive cofactor. The conserved switch I and II signaling motifs used by G-proteins are predicted to mediate allosteric regulation in response to nucleotide binding and hydrolysis in MeaB. Herein, we targeted conserved residues in the MeaB switch I motif to interrogate the function of this loop. Unexpectedly, the switch I mutations had only modest effects on GTP binding and on GTPase activity and did not perturb stability of the MCM-MeaB complex. However, these mutations disrupted multiple MeaB chaperone functions, including cofactor editing, loading, and offloading. Hence, although residues in the switch I motif are not essential for catalysis, they are important for allosteric regulation. Furthermore, single-particle EM analysis revealed, for the first time, the overall architecture of the MCM MeaB complex, which exhibits a 2:1 stoichiometry. These EM studies also demonstrate that the complex exhibits considerable conformational flexibility. In conclusion, the switch I element does not significantly stabilize the MCM-MeaB complex or influence the affinity of MeaB for GTP but is required for transducing signals between MeaB and MCM.

MeaB is an accessory GTPase protein involved in the assembly, protection, and reactivation of 5'-deoxyadenosyl cobalamin-dependent methylmalonyl-CoA mutase (MCM). Mutations in the human ortholog of MeaB result in methylmalonic aciduria, an inborn error of metabolism. G-proteins typically utilize conserved switch I and II motifs for signaling to effector proteins via conformational changes elicited by nucleotide binding and hydrolysis. Our recent discovery that MeaB utilizes an unusual switch III region for bidirectional signaling with MCM raised questions about the roles of the switch I and II motifs in MeaB. In this study, we addressed the functions of conserved switch II residues by performing alanine-scanning mutagenesis. Our results demonstrate that the GTPase activity of MeaB is autoinhibited by switch II and that this loop is important for coupling nucleotide-sensitive conformational changes in switch III to elicit the multiple chaperone functions of MeaB. Furthermore, we report the structure of MeaB center dot GDP crystallized in the presence of AlFx- to form the putative transition state analog, GDP center dot AlF4-. The resulting crystal structure and its comparison with related G-proteins support the conclusion that the catalytic site of MeaB is incomplete in the absence of the GTPase-activating protein MCM and therefore unable to stabilize the transition state analog. Favoring an inactive conformation in the absence of the client MCM protein might represent a strategy for suppressing the intrinsic GTPase activity of MeaB in which the switch II loop plays an important role.

The reactivity of the cobalt-carbon bond in cobalamins is the key to their chemical versatility, supporting both methyl transfer and isomerization reactions. During evolution of higher eukaryotes that utilize vitamin B12, the high reactivity of the cofactor coupled with its low abundance pressured development of an efficient system for uptake, assimilation, and delivery of the cofactor to client B12-dependent enzymes. Although most proteins suspected to be involved in B12 trafficking were discovered by 2009, the recent identification of a new protein reveals that the quest for elucidating the intracellular B12 highway is still far from complete. Herein, we review the biochemistry of cobalamin trafficking.

Fidelity during cofactor assembly is essential for the proper functioning of metalloenzymes and is ensured by specific chaperones. MeaB, a G-protein chaperone for the coenzyme B-12-dependent radical enzyme methylmalonyl-CoA mutase (MCM), uses the energy of GTP binding, hydrolysis or both to regulate cofactor loading into MCM, protect MCM from inactivation and rescue MCM that is inactivated during turnover. Typically, G proteins signal to client proteins using the conformationally mobile switch I and II loops. Crystallographic snapshots of MeaB reported herein reveal a new switch III element that has substantial conformational plasticity. Using alanine-scanning mutagenesis, we demonstrate that the switch III motif is critical for bidirectional signal transmission of the GTPase-activating protein activity of MCM and the chaperone functions of MeaB in the MeaB-MCM complex. Mutations in the switch III loop identified in patients corrupt this interprotein communication and lead to methylmalonic aciduria, an inborn error of metabolism.

The ATP‐dependent cob(I)alamin adenosyltransferase (ATR) is a bifunctional enzyme that catalyzes a reductive adenosylation of cob(II)alamin and then ferries the product, adenosylcobalamin (AdoCbl or coenzyme B12), to methylmalonyl‐CoA mutase (MCM). Failure to assemble and preserve the MCM holoenzyme is incompatible with life. Thus, AdoCbl trafficking from ATR to MCM is tightly regulated. Detailed kinetic and structural, along with genetic analysis of patients with mutations to MCM suggest a mechanism of direct cofactor transfer from ATR to MCM. In this study, we have mimicked one such mutation involving a C‐terminal truncation and probed the putative role of this region in cofactor migration. Most strikingly, the affinity of the truncated protein for its product, AdoCbl, is greatly reduced and the negative cooperativity for its binding, exhibited by WT ATR, is lost. We show that this truncation leads to corruption of the underlying mechanism of ATP‐dependent cofactor transfer, and provide biological relevance for this mechanism of AdoCbl delivery and provide a biochemical basis for the mechanistic deficiency that precipitates disease in patients.

ATP-dependent cob(I)alamin adenosyltransferase (ATR) is a bifunctional protein: an enzyme that catalyzes the adenosylation of cob(I)alamin and an escort that delivers the product, adenosylcobalamin (AdoCbl or coenzyme B12), to methylmalonyl-CoA mutase (MCM), resulting in holoenzyme formation. Failure to assemble holo-MCM leads to methylmalonic aciduria. We have previously demonstrated that only 2 equiv of AdoCbl bind per homotrimer of ATR and that binding of ATP to the vacant active site triggers ejection of 1 equiv of AdoCbl from an adjacent site. In this study, we have mimicked in the Methylobacterium extorquens ATR, a C-terminal truncation mutation, D180X, described in a patient with methylmalonic aciduria, and characterized the associated biochemical penalties. We demonstrate that while kcat and KMCob(I) for D180X ATR are only modestly decreased (by 3- and 2-fold, respectively), affinity for the product, AdoCbl, is significantly diminished (400-fold), and the negative cooperativity associated with its binding is lost. We also demonstrate that the D180X mutation corrupts ATP-dependent cofactor ejection, which leads to transfer of AdoCbl from wild-type ATR to MCM. These results suggest that the pathogenicity of the corresponding human truncation mutant results from its inability to sequester AdoCbl for direct transfer to MCM. Instead, cofactor release into solution is predicted to reduce the capacity for holo-MCM formation, leading to disease.