4-1BBL is a member of the tumor necrosis factor (TNF) superfamily and is the ligand for the TNFR superfamily receptor, 4-1BB. 4-1BB plays an immunomodulatory role in T cells and NK cells, and agonists of this receptor have garnered strong attention as potential immunotherapy agents. Broadly speaking, the structural features of TNF superfamily members, their receptors, and ligand-receptor complexes are similar. However, a published crystal structure of human 4-1BBL suggests that it may be unique in this regard, exhibiting a three-bladed propeller-like trimer assembly that is distinctly different from that observed in other family members. This unusual structure also suggests that the human 4-1BB/4-1BBL complex may be structurally unique within the TNF/TNFR superfamily, but to date no structural data have been reported. Here we report the crystal structure of the human 4-1BB/4-1BBL complex at 2.4-Å resolution. In this structure, 4-1BBL does not adopt the unusual trimer assembly previously reported, but instead forms a canonical bell-shaped trimer typical of other TNF superfamily members. The structure of 4-1BB is also largely canonical as is the 4-1BB/4-1BBL complex. Mutational data support the 4-1BBL structure reported here as being biologically relevant, suggesting that the previously reported structure is not. Together, the data presented here offer insight into structure/function relationships in the 4-1BB/4-1BBL system and improve our structural understanding of the TNF/TNFR superfamily more broadly.

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Fusion of proteins to the Fc region of IgG is widely used to express cellular receptors and other extracellular proteins, but cleavage of the fusion partner is sometimes required for downstream applications. Immunoglobulin G-degrading enzyme of Streptococcus pyogenes (IdeS) is a protease with exquisite specificity for human IgG, and it can also cleave Fc-fusion proteins at a single site in the Nterminal region of the CH2 domain. However, the site of IdeS cleavage results in the disulfide-linked hinge region partitioning with the released protein, complicating downstream usage of the cleaved product. To tailor the Fc fragment for release of partner proteins by IdeS treatment, we investigated the effect of deleting regions of IgG-derived sequence that are upstream of the cleavage site. Elimination of the IgGderived hinge sequence along with several residues of the CH2 domain had negligible effects on expression and purity of the fusion protein, while retaining efficient processing by IdeS. An optimal Fc fragment comprising residues 235-447 of the human IgG1 heavy chain sufficed for efficient production of fusion proteins and minimized the amount of residual Ig-derived sequence on the cleavage product following IdeS treatment. Pairing of this truncated Fc fragment with IdeS cleavage enables highly specific cleavage of Fc-fusion proteins, thus eliminating the need to engineer extraneous cleavage sequences. This system should be helpful for producing Fc-fusion proteins requiring downstream cleavage, particularly those that are sensitive to internal miscleavage if treated with alternative proteases.Abbreviations: EPOR, erythropoietin receptor; Fc, fragment crystallizable; IdeS, Immunoglobulin G-degrading enzyme of Streptococcus pyogenes; mAb, monoclonal antibody; PBS, phosphate-buffered saline; SEC, size exclusion chromatography; scFv, single chain variable fragment; TRAILR2, TNF-related apoptosis inducing ligand receptor 2

Genetically engineered medicines such as chimeric antigen receptor (CAR) T cells have great potential to be the next pillar of medical therapy beyond chemo- and traditional biologic therapies. To develop genetic medicines, new methods to understand their pharmacokinetics (PK) in humans are crucial. It is not feasible to perform traditional PK analysis for “living drugs”, because the genes themselves (in the form of DNA or RNA), are not typically responsible for the therapeutic effect. Rather, the protein products of the genes or the cells harboring the engineered genes are the actuators, and thus cannot be measured using standard HPLC or ligand binding immunoassays for PK analysis. We used a positron emission tomography (PET) reporter gene or “imaging tag” based on the intracellular bacterial enzyme dihydrofolate reductase (eDHFR) that can be paired with radiolabeled versions of trimethoprim (TMP). In this work, we evaluate the potential for immunogenicity using primary human cells and assays geared to assess low affinity and rare T cell clones that may react to eDHFR. We used overlapping pools of 15-mer eDHFR peptides and found that across 9 patients, there was little reactivity compared to EBV and CMV peptide controls. Further, the relative strength of reactivity to the eDHFR peptides was less than that of the viral peptides. Next, we showed that eDHFR iTag harboring CAR T cells were functionally comparable to unlabeled CAR T cells in vitro, and demonstrated strong, selective [18F]-TMP uptake in the eDHFR-expressing CAR T cells. Finally, using a glypican 3 (GPC3) CAR T rodent model, we performed a feasibility study to non-invasively track proliferation in antigen-harboring xenograft tumors over time with ex vivo correlation to anti-CD3 immunohistochemistry. These data demonstrate the potential for non-invasive monitoring of CAR T cells using PET imaging and translational applicability of DHFR/TMP radiotracers. Citation Format: Mark A Sellmyer, Iris K Lee, Kyle Kuszpit, Jyoti Roy, Alex Alfaro, Virginie Ory, Lily Cheng, Daniel Sutton, Emily Bosco, Christine Fazenbaker, Shabazz Novarra, Ryan Gilbreth, Nick Tschernia, Deborah Berry, Xiaoru Chen, Yuling Wu, Ryan Wong. Evaluation of eDHFR/iTag PET reporter gene immunogenicity and application in GPC3 CAR T cells [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr A37.