Structure-guided engineering of immunotherapies targeting TRBC1 and TRBC2 in T cell malignancies.

Peripheral T cell lymphomas are typically aggressive with a poor prognosis. Unlike other hematologic malignancies, the lack of target antigens to discriminate healthy from malignant cells limits the efficacy of immunotherapeutic approaches. The T cell receptor expresses one of two highly homologous chains [T cell receptor ß-chain constant (TRBC) domains 1 and 2] in a mutually exclusive manner, making it a promising target. Here we demonstrate specificity redirection by rational design using structure-guided computational biology to generate a TRBC2-specific antibody (KFN), complementing the antibody previously described by our laboratory with unique TRBC1 specificity (Jovi-1) in targeting broader spectrum of T cell malignancies clonally expressing either of the two chains. This permits generation of paired reagents (chimeric antigen receptor-T cells) specific for TRBC1 and TRBC2, with preclinical evidence to support their efficacy in T cell malignancies.

Designer Small-Molecule Control System Based on Minocycline-Induced Disruption of Protein-Protein Interaction.

A versatile, safe, and effective small-molecule control system is highly desirable for clinical cell therapy applications. Therefore, we developed a two-component small-molecule control system based on the disruption of protein-protein interactions using minocycline, an FDA-approved antibiotic with wide availability, excellent biodistribution, and low toxicity. The system comprises an anti-minocycline single-domain antibody (sdAb) and a minocycline-displaceable cyclic peptide. Here, we show how this versatile system can be applied to OFF-switch split CAR systems (MinoCAR) and universal CAR adaptors (MinoUniCAR) with reversible, transient, and dose-dependent suppression; to a tunable T cell activation module based on MyD88/CD40 signaling; to a controllable cellular payload secretion system based on IL12 KDEL retention; and as a cell/cell inducible junction. This work represents an important step forward in the development of a remote-controlled system to precisely control the timing, intensity, and safety of therapeutic interventions.

Exploration of T cell immune responses by expression of a dominant-negative SHP1 and SHP2.

SHP1 and SHP2 are SH2 domain-containing proteins which have inhibitory phosphatase activity when recruited to phosphorylated ITIMs and ITSMs on inhibitory immune receptors. Consequently, SHP1 and SHP2 are key proteins in the transmission of inhibitory signals within T cells, constituting an important point of convergence for diverse inhibitory receptors. Therefore, SHP1 and SHP2 inhibition may represent a strategy for preventing immunosuppression of T cells mediated by cancers hence improving immunotherapies directed against these malignancies. Both SHP1 and SHP2 contain dual SH2 domains responsible for localization to the endodomain of inhibitory receptors and a protein tyrosine phosphatase domain which dephosphorylates and thus inhibits key mediators of T cell activation. We explored the interaction of the isolated SH2 domains of SHP1 and SHP2 to inhibitory motifs from PD1 and identified strong binding of both SH2 domains from SHP2 and more moderate binding in the case of SHP1. We next explored whether a truncated form of SHP1/2 comprising only of SH2 domains (dSHP1/2) could act in a dominant negative fashion by preventing docking of the wild type proteins. When co-expressed with CARs we found that dSHP2 but not dSHP1 could alleviate immunosuppression mediated by PD1. We next explored the capacity of dSHP2 to bind with other inhibitory receptors and observed several potential interactions. In vivo we observed that the expression of PDL1 on tumor cells impaired the ability of CAR T cells to mediate tumor rejection and this effect was partially reversed by the co-expression of dSHP2 albeit at the cost of reduced CAR T cell proliferation. Modulation of SHP1 and SHP2 activity in engineered T cells through the expression of these truncated variants may enhance T cell activity and hence efficacy in the context of cancer immunotherapy.

Enhancing CAR T-cell Therapy Using Fab-Based Constitutively Heterodimeric Cytokine Receptors.

Adoptive T-cell therapy aims to achieve lasting tumor clearance, requiring enhanced engraftment and survival of the immune cells. Cytokines are paramount modulators of T-cell survival and proliferation. Cytokine receptors signal via ligand-induced dimerization, and this principle has been hijacked utilizing nonnative dimerization domains. A major limitation of current technologies resides in the absence of a module that recapitulates the natural cytokine receptor heterodimeric pairing. To circumvent this, we created a new engineered cytokine receptor able to constitutively recreate receptor-heterodimer utilizing the heterodimerization domain derived from the IgG1 antibody (dFab_CCR). We found that the signal delivered by the dFab_CCR-IL2 proficiently mimicked the cytokine receptor heterodimerization, with transcriptomic signatures like those obtained by activation of the native IL2 receptor. Moreover, we found that this dimerization structure was agnostic, efficiently activating signaling through four cytokine receptor families. Using a combination of in vivo and in vitro screening approaches, we characterized a library of 18 dFab_CCRs coexpressed with a clinically relevant solid tumor-specific GD2-specific chimeric antigen receptor (CAR). Based on this characterization, we suggest that the coexpression of either the common ß-chain GMCSF or the IL18 dFab_CCRs is optimal to improve CAR T-cell expansion, engraftment, and efficacy. Our results demonstrate how Fab dimerization is efficient and versatile in recapitulating a cytokine receptor heterodimerization signal. This module could be applied for the enhancement of adoptive T-cell therapies, as well as therapies based on other immune cell types. Furthermore, these results provide a choice of cytokine signal to incorporate with adoptive T-cell therapies.

Dual targeting of CD19 and CD22 against B-ALL using a novel high-sensitivity aCD22 CAR.

CAR T cells recognizing CD19 effectively treat relapsed and refractory B-ALL and DLBCL. However, CD19 loss is a frequent cause of relapse. Simultaneously targeting a second antigen, CD22, may decrease antigen escape, but is challenging: its density is approximately 10-fold less than CD19, and its large structure may hamper immune synapse formation. The characteristics of the optimal CD22 CAR are underexplored. We generated 12 distinct CD22 antibodies and tested CARs derived from them to identify a CAR based on the novel 9A8 antibody, which was sensitive to low CD22 density and lacked tonic signaling. We found no correlation between affinity or membrane proximity of recognition epitope within Ig domains 3-6 of CD22 with CART function. The optimal strategy for CD19/CD22 CART co-targeting is undetermined. Co-administration of CD19 and CD22 CARs is costly; single CARs targeting CD19 and CD22 are challenging to construct. The co-expression of two CARs has previously been achieved using bicistronic vectors. Here, we generated a dual CART product by co-transduction with 9A8-41BBζ and CAT-41BBζ (obe-cel), the previously described CD19 CAR. CAT/9A8 CART eliminated single- and double-positive target cells in vitro and eliminated CD19- tumors in vivo. CAT/9A8 CART is being tested in a phase I clinical study (NCT02443831).