Figure S1. Overall survival by treatment arm

Figure S4. Recurrence-free survival by treatment arm

Deidentified Participant Data File for Long-Term Outcomes of the Mel43 Clinical Trial

All treatment-related adverse events

Figure S3. Overall survival by selected participant characteristics.

Figure S2. Overall survival by duration of institutional follow-up.

PURPOSE:This randomized phase I/II clinical trial (NCT03617328) was designed to test whether administration of systemic agonistic anti-CD27 antibody (varlilumab) concurrent with a melanoma vaccine is safe and enhances vaccine immunogenicity. PATIENTS AND METHODS:Adults with definitively treated, high-risk melanoma were randomized to receive a shared antigen vaccine targeting CD4+ T cells (6 melanoma helper peptides; 6MHP) either with (arm A) or without (arm B) systemic varlilumab over 11 weeks. A final vaccine was given at week 25 to assess memory response. RESULTS:Thirty-three participants were treated at two centers. After enrolling 17 participants, the dose-limiting toxicity (DLT) rate with vaccine alone required protocol revision to limit local toxicity. Overall, DLT rates for arms A and B were 6% (1/17) and 31% (5/16), respectively. Twenty (61%) participants had CD4+ T-cell responses ex vivo. Persistent responses were detected in two (2/14, 14%) on arm A and one (1/16, 6%) on arm B. Memory response was detected in two (2/13, 15%) on arm A and four (4/16, 25%) on arm B. On arm A, there was a significant reduction in circulating CD4+ T cells. Four-year disease-free survival rates for arms A and B were 20% [95% confidence interval (CI), 6%-67%] and 69% (95% CI, 49%-96%), respectively. CONCLUSIONS:No synergistic toxicity was observed with combination treatment. Similar durability in immunogenicity was found with or without varlilumab. Depletion of circulating CD4+ T cells with varlilumab may have abrogated benefits of inducing tumor-cognate CD4+ T cells with vaccination. Induction of memory responses supports further work to optimize shared antigen vaccines in combination with other immunotherapies. SIGNIFICANCE:Combination treatment with 6MHP vaccination and varlilumab was safe but did not improve CD4+ T-cell response rates. Circulating CD4+ T cells were reduced with varlilumab, and clinical outcome was improved without varlilumab. This study demonstrates induction of memory responses after shared antigen vaccination; however, CD27 agonism did not enhance durability of response. Depletion of CD4+ T cells after varlilumab may have interfered with the beneficial effects of vaccination.

Representativeness of study participants

PURPOSE:We report the long-term clinical outcomes of a multicenter, randomized phase II trial (NCT00089193) that tested immunogenicity of a vaccine composed of 12 class I MHC-restricted melanoma peptides (12MP), with or without granulocyte-macrophage colony-stimulating factor (GM-CSF) as an adjuvant and administered at one or two sites in patients with resected high-risk melanoma. PATIENTS AND METHODS:Participants were randomized to one of four treatment arms: 12MP at one site (arm A), 12MP + GM-CSF at one site (arm B), 12MP at two sites (arm C), and 12MP + GM-CSF at two sites (arm D). The trial was powered to detect differences in immunogenicity by vaccine groups defined by GM-CSF status (arms B + D vs. A + C) and vaccine sites (arms A + B vs. C + D). For this analysis, overall survival (OS) and recurrence-free survival (RFS) were evaluated by these vaccine groups. RESULTS:All eligible participants (n = 121) were evaluated. The median follow-up was 5.6 years. No significant differences in RFS or OS were observed by GM-CSF status. Participants vaccinated at two sites compared with one had significantly improved RFS [hazard ratio (HR), 0.59; 95% confidence interval (CI), 0.38-0.93; P = 0.02] and a trend to improved OS (HR, 0.64; 95% CI, 0.39-1.06; P = 0.08). On landmark multivariable analysis, two-site vaccination was the only significant predictor of RFS (HR, 0.55; 95% CI, 0.34-0.88; P = 0.01) after adjusting for CD8+ T-cell response and other prognostic factors. CONCLUSIONS:These results challenge the use of GM-CSF as a local vaccine adjuvant and support two-site vaccination. Future work to characterize the locoregional immune response to cancer vaccination at the injection site and vaccine-draining lymph nodes is warranted.

Figure S5. Recurrence-free survival by duration of institutional follow-up.

Changes in absolute number of circulating Tregs over time

Immune response rates by protocol version

Changes in circulating Tregs as a percentage of CD4+ T cells over time

Table S1. Representativeness of study participants.

Table S5. Stratified multivariable analysis for recurrence-free survival of the intention-to-treat population

Table S6. Stratified multivariable analysis for recurrence-free survival of the intention-to-treat population by randomized arm.

Table S2. Participant characteristics by enrolling institution.

Table S3. Immune response rates by treatment arm.

Trial design and protocol amendment for safety. A and B, Trial schema of study interventions (A) before and (B) after protocol amendment for exceeding the DLT threshold. Vaccines included poly-ICLC in the adjuvant at all time points except for week 25, represented by the dashed arrow. Blood draws were obtained prior to vaccination and/or varlilumab therapy. C, CONSORT flow diagram. ID, intradermal; IV, intravenous; SQ, subcutaneous.

Treatment-related adverse events after protocol amendment