Immune checkpoint blockade is a powerful weapon in the fight against cancer, but for most patients, checkpoint blockade alone is not enough, and standards of care like chemotherapy are still the norm. In a recent paper published in Cancer Immunology Research, Ariyan et al. sought to enhance the efficacy of CTLA-4 checkpoint blockade in poorly immunogenic tumors by combining it with melphalan, a common chemotherapeutic agent that could act as an in vivo vaccine by inducing cell death and inflammation at the tumor site.
Melphalan is known to induce tumor cell apoptosis and the release of inflammatory cytokines, and in preclinical experiments, Ariyan et al. explored the possibility that it could also have an immune effect. The researchers applied melphalan to B16 melanoma cells in vitro and found that it altered two key immune parameters: it increased tumor cell expression of MHC class I molecules by 2.5 fold and increased PD-L1 expression by 7 fold, indicating that melphalan could increase the antitumor response to immunotherapy.
Next, in mice with B16 melanoma, the researchers intratumorally administered melphalan and intraperitoneally administered anti-CTLA-4. While neither agent was effective as a monotherapy, the combination treatment improved survival and protected mice long term against cancer rechallenge. Immune cell phenotyping revealed that the combination therapy enriched the tumor microenvironment for effector T cells and reduced the number of T regulatory (Treg) cells, increasing the CD8/Treg ratio. They also showed that long-term anti-tumor immunity was dependent on host expression of IFNγ. Similar results were seen combining CTLA-4 blockade with gemcitabine chemotherapy in the TRAMP2 murine model of prostate cancer.
The preclinical efficacy of combining melphalan with anti-CTLA-4 prompted the initiation of a Phase II clinical trial in 26 patients with advanced, recurrent melanoma. Patients were treated locally with melphalan via isolated limb infusion (ILI), a technique that involves creating a closed circuit in an extremity in order to locally deliver chemotherapy and avoid systemic toxicity. Patients were then treated with their first dose of ipilimumab to block CTLA-4; patients went on to receive, on average, 3 out of 4 planned doses of ipilimumab.
Of the 26 patients,16 (62%) had complete responses and 6 (23%) had partial responses in the limb, for an impressive response rate of 85%. At one year, 58% of patients remained progression-free. With a median follow up of 3 years, median survival and median progression-free survival (PFS) were not reached. Patients achieved local and distal responses to the combination therapy, and the two patients who entered the study with stage IV disease showed complete responses lasting at least two years. Importantly, the researchers observed no increase in limb toxicity with the addition of ipilimumab over ILI with melaphan alone, however, most patients did have immune-related adverse events including diarrhea, colitis, pneumonia, and long-term adrenal insufficiency.
To better understand the immune reactions in patients, Ariyan et al. analyzed gene expression of tumors before and after each therapy. After ILI, researchers noted statistically significant upregulation of genes related to innate and adaptive immune responses, chemotaxis, costimulatory ligands and receptors, and MHC class I and class II molecules. The addition of ipilimumab induced upregulation of ICOS and genes related to cytotoxic T cell-effector molecules, including granzymes, IFNγ, and perforins. The researchers also measured circulating cytokines and found that the inflammatory cytokines IL8 and IL6 increased after ILI with melphalan, while IFNγ, IL17, and TNF increased following additional treatment with ipilimumab. Immune phenotyping showed an increase in proliferating CD4+ and CD8+ T cells as well as an increase in ICOS+CD4+ T cells in the blood after combination therapy. Tumor biopsies showed increased infiltration of CD4+ and CD8+ T cells and an increase in PD-L1 in the tumor microenvironment following therapy. Importantly, T cell frequencies were low in all patients before therapy and increased most in patients who achieved PFS for a year or more.
Overall, the results of preclinical and clinical research by Ariyan et aI. support the idea that local treatment with melphalan induces an inflammatory tumor microenvironment that supports an enhanced response to CTLA-4 blockade, which mobilizes cytotoxic effectors. This combination treatment offers a dramatic clinical improvement over CTLA-4 blockade or ILI alone, and the high PFS rate encourages further studies combining CTLA-4 blockade with strategies that prime adaptive immunity.
by Lauren Hitchings