Compromised antigen binding and signaling interfere with bispecific CD19 and CD79a chimeric antigen receptor function.

Therapy with CD19-directed chimeric antigen receptor (CAR) T cells has transformed the treatment of advanced B-cell malignancies. However, loss of or low antigen expression can enable tumor escape and limit the duration of responses achieved with CAR T-cell therapy. Engineering bispecific CAR T cells that target 2 tumor antigens could overcome antigen-negative escape.

A split, conditionally active mimetic of IL-2 reduces the toxicity of systemic cytokine therapy.

The therapeutic potential of recombinant cytokines has been limited by the severe side effects of systemic administration. We describe a strategy to reduce the dose-limiting toxicities of monomeric cytokines by designing two components that require colocalization for activity and that can be independently targeted to restrict activity to cells expressing two surface markers. We demonstrate the approach with a previously designed mimetic of cytokines interleukin-2 and interleukin-15-Neoleukin-2/15 (Neo-2/15)-both for trans-activating immune cells surrounding targeted tumor cells and for cis-activating directly targeted immune cells.

NOTCH1 signaling during CD4+ T-cell activation alters transcription factor networks and enhances antigen responsiveness.

Adoptive transfer of T cells expressing chimeric antigen receptors (CAR-T) effectively treats refractory hematologic malignancies in a subset of patients but can be limited by poor T-cell expansion and persistence in vivo. Less differentiated T-cell states correlate with the capacity of CAR-T to proliferate and mediate antitumor responses, and interventions that limit tumor-specific T-cell differentiation during ex vivo manufacturing enhance efficacy. NOTCH signaling is involved in fate decisions across diverse cell lineages and in memory CD8+ T cells was reported to upregulate the transcription factor FOXM1, attenuate differentiation, and enhance proliferation and antitumor efficacy in vivo.

Comparative analysis of TCR and CAR signaling informs CAR designs with superior antigen sensitivity and in vivo function.

Chimeric antigen receptor (CAR)-modified T cell therapy is effective in treating lymphomas, leukemias, and multiple myeloma in which the tumor cells express high amounts of target antigen. However, achieving durable remission for these hematological malignancies and extending CAR T cell therapy to patients with solid tumors will require receptors that can recognize and eliminate tumor cells with a low density of target antigen. Although CARs were designed to mimic T cell receptor (TCR) signaling, TCRs are at least 100-fold more sensitive to antigen.

De novo design of potent and selective mimics of IL-2 and IL-15.

We describe a de novo computational approach for designing proteins that recapitulate the binding sites of natural cytokines, but are otherwise unrelated in topology or amino acid sequence. We use this strategy to design mimics of the central immune cytokine interleukin-2 (IL-2) that bind to the IL-2 receptor βγ heterodimer (IL-2Rβγ) but have no binding site for IL-2Rα (also called CD25) or IL-15Rα (also known as CD215). The designs are hyper-stable, bind human and mouse IL-2Rβγ with higher affinity than the natural cytokines, and elicit downstream cell signalling independently of IL-2Rα and IL-15Rα.

A Highly Diverse and Functional Naïve Ubiquitin Variant Library for Generation of Intracellular Affinity Reagents.

We report the design, construction, and validation of a highly diverse phage-displayed naïve ubiquitin variant (Ubv) library. We first conducted a mutation tolerance scan of 27 residues and confirmed that 24 of these could be substituted by chemically diverse amino acids without compromising the display of Ubvs on phage. Subsequently, we constructed a library containing 6.