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Design and characterization of a protein superagonist of IL-15 fused with IL-15R? and a high-affinity T cell receptor.

ABSTRACT: To avoid high systemic doses, strategies involving antigen-specific delivery of cytokine via linked antibodies or antibody fragments have been used. Targeting cancer-associated peptides presented by major histocompatibility complex (MHC) molecules (pepMHC) increases the number of potential target antigens and takes advantage of cross-presentation on tumor stroma and in draining lymph nodes. Here, we use a soluble, high-affinity single-chain T cell receptor V?-V? (scTv), to deliver cytokines to intracellular tumor-associated antigens presented as pepMHC. As typical wild-type T cell receptors (TCRs) exhibit low affinity (K(d) = 1-100 ?M or more), we used an engineered TCR, m33, that binds its antigenic peptide SIYRYYGL (SIY) bound to the murine class I major histocompatability complex protein H2-K(b) (SIY/K(b) ) with nanomolar affinity (K(d) = 30 nM). We generated constructs consisting of m33 scTv fused to murine interleukin 2 (IL-2), interleukin 15 (IL-15), or IL-15/IL-15R? (IL-15 linked to IL-15R? sushi domain, called "superfusion"). The fusions were purified with good yields and bound specifically to SIY/K(b) with high affinity. Proper cytokine folding and binding were confirmed, and the fusions were capable of stimulating proliferation of cytokine-dependent cells, both when added directly and when presented in trans, bound to cells with the target pepMHC. The m33 superfusion was particularly potent and stable and represents a promising design for targeted antitumor immunomodulation.

SUBMITTER: Stone JD

PROVIDER: S-EPMC3514595 | biostudies-literature | 2012 Nov-Dec

REPOSITORIES: biostudies-literature

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Design and characterization of a protein superagonist of IL-15 fused with IL-15Rα and a high-affinity T cell receptor.

Stone Jennifer D JD Chervin Adam S AS Schreiber Hans H Kranz David M DM

Biotechnology progress 20121018 6

To avoid high systemic doses, strategies involving antigen-specific delivery of cytokine via linked antibodies or antibody fragments have been used. Targeting cancer-associated peptides presented by major histocompatibility complex (MHC) molecules (pepMHC) increases the number of potential target antigens and takes advantage of cross-presentation on tumor stroma and in draining lymph nodes. Here, we use a soluble, high-affinity single-chain T cell receptor Vα-Vβ (scTv), to deliver cytokines to i ...[more]

PMID: 22961781

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Project description:BackgroundImmunotherapy targeting PD-1/PD-L1 fails to induce clinical responses in most patients with solid cancers. N-803, formerly ALT-803, is an IL-15 superagonist mutant and dimeric IL-15RαSushi-Fc fusion protein complex that enhances CD8+ T and NK cell expansion and function and exhibits anti-tumor efficacy in preclinical models. Previous in vitro studies have shown that IL-15 increases PD-L1 expression, a negative regulator of CD8+ T and NK cell function. Most reported preclinical studies administered N-803 intraperitoneally not subcutaneously, the current clinical route of administration. N-803 is now being evaluated clinically in combination with PD-1/PD-L1 inhibitors. However, the mechanism of action has not been fully elucidated. Here, we examined the anti-tumor efficacy and immunomodulatory effects of combining N-803 with an anti-PD-L1 antibody in preclinical models of solid carcinomas refractory to anti-PD-L1 or N-803.MethodsSubcutaneous N-803 and an anti-PD-L1 monoclonal antibody were administered as monotherapy or in combination to 4T1 triple negative breast and MC38-CEA colon tumor-bearing mice. Anti-tumor efficacy was evaluated, and a comprehensive analysis of the immune-mediated effects of each therapy was performed on the primary tumor, lung as a site of metastasis, and spleen.ResultsWe demonstrate that N-803 treatment increased PD-L1 expression on immune cells in vivo, supporting the combination of N-803 and anti-PD-L1. N-803 plus anti-PD-L1 was well-tolerated, reduced 4T1 lung metastasis and MC38-CEA tumor burden, and increased survival as compared to N-803 and anti-PD-L1 monotherapies. Efficacy of the combination therapy was dependent on both CD8+ T and NK cells and was associated with increased numbers of these activated immune cells in the lung and spleen. Most alterations to NK and CD8+ T cell phenotype and number were driven by N-803. However, the addition of anti-PD-L1 to N-803 significantly enhanced CD8+ T cell effector function versus N-803 and anti-PD-L1 monotherapies, as indicated by increased Granzyme B and IFNγ production, at the site of metastasis and in the periphery. Increased CD8+ T cell effector function correlated with higher serum IFNγ levels, without related toxicities, and enhanced anti-tumor efficacy of the N-803 plus anti-PD-L1 combination versus either monotherapy.ConclusionsWe provide novel insight into the mechanism of action of N-803 plus anti-PD-L1 combination and offer preclinical proof of concept supporting clinical use of N-803 in combination with checkpoint inhibitors, including for patients non- and/or minimally responsive to either monotherapy.

Dataset Information

Design and characterization of a protein superagonist of IL-15 fused with IL-15R? and a high-affinity T cell receptor.

Publications

Design and characterization of a protein superagonist of IL-15 fused with IL-15Rα and a high-affinity T cell receptor.

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