Facile repurposing of peptide-MHC-restricted antibodies for cancer immunotherapy.

Monoclonal antibodies (Abs) that recognize major histocompatability complex (MHC)-presented tumor antigens in a manner similar to T cell receptors (TCRs) have great potential as cancer immunotherapeutics. However, isolation of 'TCR-mimic' (TCRm) Abs is laborious because Abs have not evolved the structurally nuanced peptide-MHC restriction of αß-TCRs. Here, we present a strategy for rapid isolation of highly peptide-specific and 'MHC-restricted' Abs by re-engineering preselected Abs that engage peptide-MHC in a manner structurally similar to that of conventional αß-TCRs. We created structure-based libraries focused on the peptide-interacting residues of TCRm Ab complementarity-determining region (CDR) loops, and rapidly generated MHC-restricted Abs to both mouse and human tumor antigens that specifically killed target cells when formatted as IgG, bispecific T cell engager (BiTE) and chimeric antigen receptor-T (CAR-T). Crystallographic analysis of one selected pMHC-restricted Ab revealed highly peptide-specific recognition, validating the engineering strategy. This approach can yield tumor antigen-specific antibodies in several weeks, potentially enabling rapid clinical translation.

Autoimmunity-associated T cell receptors recognize HLA-B*27-bound peptides.

Human leucocyte antigen B*27 (HLA-B*27) is strongly associated with inflammatory diseases of the spine and pelvis (for example, ankylosing spondylitis (AS)) and the eye (that is, acute anterior uveitis (AAU))1. How HLA-B*27 facilitates disease remains unknown, but one possible mechanism could involve presentation of pathogenic peptides to CD8+ T cells. Here we isolated orphan T cell receptors (TCRs) expressing a disease-associated public ß-chain variable region-complementary-determining region 3ß (BV9-CDR3ß) motif2-4 from blood and synovial fluid T cells from individuals with AS and from the eye in individuals with AAU. These TCRs showed consistent α-chain variable region (AV21) chain pairing and were clonally expanded in the joint and eye. We used HLA-B*27:05 yeast display peptide libraries to identify shared self-peptides and microbial peptides that activated the AS- and AAU-derived TCRs. Structural analysis revealed that TCR cross-reactivity for peptide-MHC was rooted in a shared binding motif present in both self-antigens and microbial antigens that engages the BV9-CDR3ß TCRs. These findings support the hypothesis that microbial antigens and self-antigens could play a pathogenic role in HLA-B*27-associated disease.

Immune receptor inhibition through enforced phosphatase recruitment.

Antibodies that antagonize extracellular receptor-ligand interactions are used as therapeutic agents for many diseases to inhibit signalling by cell-surface receptors1. However, this approach does not directly prevent intracellular signalling, such as through tonic or sustained signalling after ligand engagement. Here we present an alternative approach for attenuating cell-surface receptor signalling, termed receptor inhibition by phosphatase recruitment (RIPR). This approach compels cis-ligation of cell-surface receptors containing ITAM, ITIM or ITSM tyrosine phosphorylation motifs to the promiscuous cell-surface phosphatase CD452,3, which results in the direct intracellular dephosphorylation of tyrosine residues on the receptor target. As an example, we found that tonic signalling by the programmed cell death-1 receptor (PD-1) results in residual suppression of T cell activation, but is not inhibited by ligand-antagonist antibodies. We engineered a PD-1 molecule, which we denote RIPR-PD1, that induces cross-linking of PD-1 to CD45 and inhibits both tonic and ligand-activated signalling. RIPR-PD1 demonstrated enhanced inhibition of checkpoint blockade compared with ligand blocking by anti-PD1 antibodies, and increased therapeutic efficacy over anti-PD1 in mouse tumour models. We also show that the RIPR strategy extends to other immune-receptor targets that contain activating or inhibitory ITIM, ITSM or ITAM motifs; for example, inhibition of the macrophage SIRPα 'don't eat me' signal with a SIRPα-CD45 RIPR molecule potentiates antibody-dependent cellular phagocytosis beyond that of SIRPα blockade alone. RIPR represents a general strategy for direct attenuation of signalling by kinase-activated cell-surface receptors.

Discovery of surrogate agonists for visceral fat Treg cells that modulate metabolic indices in vivo.

T regulatory (Treg) cells play vital roles in modulating immunity and tissue homeostasis. Their actions depend on TCR recognition of peptide-MHC molecules; yet the degree of peptide specificity of Treg-cell function, and whether Treg ligands can be used to manipulate Treg cell biology are unknown. Here, we developed an Ab-peptide library that enabled unbiased screening of peptides recognized by a bona fide murine Treg cell clone isolated from the visceral adipose tissue (VAT), and identified surrogate agonist peptides, with differing affinities and signaling potencies. The VAT-Treg cells expanded in vivo by one of the surrogate agonists preserved the typical VAT-Treg transcriptional programs. Immunization with this surrogate, especially when coupled with blockade of TNFα signaling, expanded VAT-Treg cells, resulting in protection from inflammation and improved metabolic indices, including promotion of insulin sensitivity. These studies suggest that antigen-specific targeting of VAT-localized Treg cells could eventually be a strategy for improving metabolic disease.

A strategy for the selection of monovalent antibodies that span protein dimer interfaces.

Ligand-induced dimerization is the predominant mechanism through which secreted proteins activate cell surface receptors to transmit essential biological signals. Cytokines are a large class of soluble proteins that dimerize transmembrane receptors into precise signaling topologies, but there is a need for alternative, engineerable ligand scaffolds that specifically recognize and stabilize these protein interactions. Recombinant antibodies can potentially serve as robust and versatile platforms for cytokine complex stabilization, and their specificity allows for tunable modulation of dimerization equilibrium. Here, we devised an evolutionary strategy to isolate monovalent antibody fragments that bridge together two different receptor subunits in a cytokine-receptor complex, precisely as the receptors are disposed in their natural signaling orientations. To do this, we screened a naive antibody library against a stabilized ligand-receptor ternary complex that acted as a "molecular cast" of the natural receptor dimer conformation. Our selections elicited "stapler" single-chain variable fragments (scFvs) of antibodies that specifically engage the interleukin-4 receptor heterodimer. The 3.1 Å resolution crystal structure of one such stapler revealed that, as intended, this scFv recognizes a composite epitope between the two receptors as they are positioned in the complex. Extending our approach, we evolved a stapler scFv that specifically binds to and stabilizes the interface between the interleukin-2 cytokine and one of its receptor subunits, leading to a 15-fold enhancement in interaction affinity. This demonstration that scFvs can be selected to recognize epitopes that span protein interfaces presents new opportunities to engineer structurally defined antibodies for a broad range of research and therapeutic applications.

Engineering a Single-Agent Cytokine/Antibody Fusion That Selectively Expands Regulatory T Cells for Autoimmune Disease Therapy.

IL-2 has been used to treat diseases ranging from cancer to autoimmune disorders, but its concurrent immunostimulatory and immunosuppressive effects hinder efficacy. IL-2 orchestrates immune cell function through activation of a high-affinity heterotrimeric receptor (composed of IL-2Rα, IL-2Rß, and common γ [γc]). IL-2Rα, which is highly expressed on regulatory T (TReg) cells, regulates IL-2 sensitivity. Previous studies have shown that complexation of IL-2 with the JES6-1 Ab preferentially biases cytokine activity toward TReg cells through a unique mechanism whereby IL-2 is exchanged from the Ab to IL-2Rα. However, clinical adoption of a mixed Ab/cytokine complex regimen is limited by stoichiometry and stability concerns. In this study, through structure-guided design, we engineered a single agent fusion of the IL-2 cytokine and JES6-1 Ab that, despite being covalently linked, preserves IL-2 exchange, selectively stimulating TReg expansion and exhibiting superior disease control to the mixed IL-2/JES6-1 complex in a mouse colitis model. These studies provide an engineering blueprint for resolving a major barrier to the implementation of functionally similar IL-2/Ab complexes for treatment of human disease.