Cell therapies for solid tumors are associated with unique challenges compared with liquid tumors, such as lower access to tumor tissues that express target antigen. Antigen (signal 1) is the ignition and fuel for T cell responses and is necessary to induce chimeric antigen receptor (CAR) T-cell activation and expansion. Efforts to boost cell therapy activity have focused on improving the sensitivity of the CAR ligand-binding domains and enhancing the CAR through added intracellular domains from other signaling proteins (eg, CD28 and 4-1BB), thus boosting sensitivity and persistence while preserving antigen dependence. Little effort, other than vaccination,1 has been expended to enhance signal 1 (antigen), which may be essential to optimize responses in solid tumors. We sought to create a signal 1 booster that mimics an antigen stimulus with a small molecule that triggers signaling by the CAR or T-cell receptor (TCR).
Methods
Expression constructs were designed to control tonic signaling of T cells engineered with CARs and TCRs. These constructs contained FK506-binding protein (FKBP), ligand-binding domains that mediate protein multimerization in the presence of the small molecule rimiducid.2
Results
A variety of constructs, including fusions to LAT or to CD3E, were shown to produce the desired effects on tonic signaling (figure 1). In the absence of rimiducid, these constructs produced only small elevations of tonic signaling in Jurkat or primary T cells. The addition of rimiducid induced dose-dependent increases in tonic signaling 2- to 10-fold. The constructs also enhanced tonic signaling in the context of TmodTM, a dual-receptor NOT gate based on the LIR-1 inhibitory receptor.3
Conclusions
Signal 1 boosters that mimic an antigen stimulus with a small molecule that triggers signaling by the CAR or TCR were generated. The signal 1 mimetics allow tuned stimulation, which could improve the quality and performance of the T cell product in patients. Critically, they bypass the need for antigen exposure in the patient's blood, a key difference between blood and solid tumor therapy.
References
Mackensen A, et al. Nat Med. 2023;29(11):2844–2853. National center for biotechnology information. PubChem Compound Summary for CID 16135625, Rimiducid. https://pubchem.ncbi.nlm.nih.gov/compound/Rimiducid. Accessed June 24, 2024. Hamburger AE, et al. Mol Immunol. 2020;128:298–310.
DNA double-strand breaks (DSBs) are cytotoxic lesions that must be accurately repaired to maintain genome stability. Replication protein A (RPA) plays an important role in homology-dependent repair of DSBs by protecting the single-stranded DNA (ssDNA) intermediates formed by end resection and by facilitating Rad51 loading. We found that hypomorphic mutants of RFA1 that support intra-chromosomal homologous recombination are profoundly defective for repair processes involving long tracts of DNA synthesis, in particular break-induced replication (BIR). The BIR defects of the rfa1 mutants could be partially suppressed by eliminating the Sgs1-Dna2 resection pathway, suggesting that Dna2 nuclease attacks the ssDNA formed during end resection when not fully protected by RPA. Overexpression of Rad51 was also found to suppress the rfa1 BIR defects. We suggest that Rad51 binding to the ssDNA formed by excessive end resection and during D-loop migration can partially compensate for dysfunctional RPA.
The yeast Mre11-Rad50-Xrs2 (MRX) complex has structural, signaling, and catalytic functions in the response to DNA damage. Xrs2, the eukaryotic-specific component of the complex, is required for nuclear import of Mre11 and Rad50 and to recruit the Tel1 kinase to damage sites. We show that nuclear-localized MR complex (Mre11-NLS) catalyzes homology-dependent repair without Xrs2, but MR cannot activate Tel1, and it fails to tether DSBs, resulting in sensitivity to genotoxins, replisome instability, and increased gross chromosome rearrangements (GCRs). Fusing the Tel1 interaction domain from Xrs2 to Mre11-NLS is sufficient to restore telomere elongation and Tel1 signaling to Xrs2-deficient cells. Furthermore, Tel1 stabilizes Mre11-DNA association, and this stabilization function becomes important for DNA damage resistance in the absence of Xrs2. Enforcing Tel1 recruitment to the nuclear MR complex fully rescues end tethering and stalled replication fork stability, and suppresses GCRs, highlighting important roles for Xrs2 and Tel1 to ensure optimal MR activity.
Abstract 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.
Significance Chromosomal double-strand breaks (DSBs) are cytotoxic forms of DNA damage that must be accurately repaired to maintain genome integrity. The conserved Mre11–Rad50–Xrs2/NBS1 nuclease/ATPase complex plays an important role in repair by functioning as a damage sensor and by regulation of DNA end processing to ensure repair by the most appropriate mechanism. Yeast Sae2 is known to function with Mre11 to process DNA ends, but its precise role is poorly understood. Here we show that it is the failure to remove Mre11 from DNA ends, leading to persistent DNA damage signaling and cell cycle arrest, that causes sensitivity of Sae2-deficient cells to DNA damaging agents.
The Mre11-Rad50-Xrs2/Nbs1 (MRX/N) complex orchestrates the cellular response to DSBs through its structural, enzymatic, and signaling roles. Xrs2/Nbs1 is essential for nuclear translocation of Mre11, but its role as a component of the complex is not well defined. Here, we demonstrate that nuclear localization of Mre11 (Mre11-NLS) is able to bypass several functions of Xrs2, including DNA end resection, meiosis, hairpin resolution, and cellular resistance to clastogens. Using purified components, we show that the MR complex has equivalent activity to MRX in cleavage of protein-blocked DNA ends. Although Xrs2 physically interacts with Sae2, we found that end resection in its absence remains Sae2 dependent in vivo and in vitro. MRE11-NLS was unable to rescue the xrs2Δ defects in Tel1/ATM kinase signaling and non-homologous end joining, consistent with the role of Xrs2 as a chaperone and adaptor protein coordinating interactions between the MR complex and other repair proteins.
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-E6 29–38 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.
