Immunotherapies have achieved clinical success in a variety of cancers, but there is still much to be desired in terms of consistent and robust antitumor immunity. In their paper published recently in Molecular Cancer Therapeutics, Oberst et al. propose targeting the TNF receptor superfamily co-stimulatory receptor OX40. Their engineered OX40 ligand (OX40L) Fc fusion protein aims to expand tumor-specific T cell populations, and/or overcome the immunosuppressive tumor microenvironment (TME) in cancer patients.

MEDI6383, manufactured by MedImmune, is a recombinant human OX40L IgG4P Fc fusion protein composed of three distinct parts: human OX40L extracellular receptor binding domain (RBD), coiled coil domain from TRAF2, and human IgG4 fragment crystallizable (Fc) gamma domain. Mimicking the natural OX40L structure, the OX40L RBD forms homo-trimers, which are stabilized by the TRAF2 coiled coil domains. The IgG4 Fc domains of the fusion protein associate to form a hexameric complex and aid in Fcγ receptor (FcγR) clustering.

After the recombinant protein was produced, binding studies using activated CD4⁺ T cells indicated that MEDI6383 potently bound activated primate CD4⁺ T cells from cynomolgus macaques, rhesus macaques, and humans. The researchers then tested the ability of MEDI6383 to induce OX40 signaling in T cells with an in vitro bioluminescence assay. Human Jurkat reporter cells were engineered to express OX40 on their surface and infected with lentivirus to cause expression of luciferase from an NFκB-mediated promoter when OX40 was stimulated. The team found moderate NFκB-mediated gene expression upon the addition of MEDI6383 to the Jurkat cells, suggesting that low levels of OX40 clustering on the Jurkat reporter cells were mediated by soluble hexameric MEDI6383.

Next, HEK293 cells were modified to express FcγRs (CD32A[FcγRIIA], CD64[FcγRI], or CD16[FcγRIII]) to determine whether clustering between the Fc domains of MEDI6383 and the FcγRs would alter activity. Co-incubation of reporter cells with MEDI6383 and CD32A- or CD64-expressing HEK293 cells caused significantly increased expression of NFκB over incubation of reporter cells with MEDI6383 alone, signifying the importance of clustering in activity levels of the ligand. Further clustering experiments were performed using Raji tumor B cells or CD45 immune cells from human primary renal or lung tumors, where it was determined that MEDI6383 activity was again significantly enhanced when clustered.

To determine the functional requirements for activation of primary human T cells, CD3⁺ T cells isolated from healthy donors and activated with PHA-L and IL-2 to upregulate OX40 expression were plated with different concentrations of immobilized or free MEDI6383 and suboptimal levels of plate-bound anti-human CD3. Results demonstrated that immobilized MEDI6383, together with anti-CD3, induced the release of T cell cytokines and upregulated surface activation markers and T cell proliferation when cultured with activated T cells. Further experiments demonstrated that both TCR stimulation and clustering are necessary for MEDI6383 to elicit a biological response. A final in vitro experiment demonstrated that MEDI6383 was able to overcome the immunosuppressive TME; natural regulatory T cells (nTregs) were unable to inhibit effector T cell proliferation when MEDI6383 was present.

Due to incompatibility between human OX40L and mouse OX40, in vivo experiments relied on human tumor/T cell admixed tumor models in immunodeficient (NOD/SCID) mice. A375 melanoma cells were combined with OX40-expressing human CD4⁺ T cells and CD8⁺ T cells alloreactive to A375 melanoma cells, and then implanted into NOD/SCID mice. Mice were then treated with either an isotype-matched antibody or various doses of MEDI6383, which showed that MEDI6383 caused significant tumor growth inhibition, dependent on the activity of T cells.

Moving their animal work evolutionarily closer to humans, the researchers turned to rhesus monkeys and showed that intravenous administration of 3 injections of MEDI6383 every other day transiently increased the absolute number of circulating CD4⁺ and CD8⁺ T cell populations. Ki67⁺ staining, indicative of proliferation, showed that CD4⁺ memory cells were most robustly stimulated, CD8+ memory cells were not as robustly stimulated, and naive cells were not induced to proliferate. The most notable increase was in CD4⁺ central memory T cells. MEDI6383 treatment also modestly increased the population of Ki67⁺ B cells. ICOS⁺ staining, indicative of T cell activation, revealed an increase in activation in memory CD4⁺ and CD8⁺ T cell populations, with a higher percentage of ICOS expression in the CD4⁺ T cell subset. MEDI6383 also increased the percentage of PD-1⁺ cells, indicating its potential for combination therapy with anti-PD-1 or anti-PD-L1.

The MEDI6383 OX40-ligand fusion protein represents a novel molecular engineering approach to stimulation of TNFR family members, which may complement the blockade of inhibitory checkpoint receptors and thus, holds promise in mono- or combination therapy. Researchers have begun testing MEDI6383 in patients with advanced solid malignancies.

by Brynn Vessey