Erskine, Courtney L.

Abstract Purpose: CD4 T-cell immunity is associated with improved survival in patients with breast cancer. We previously reported a phase I trial evaluating a folate receptor alpha (FRα) vaccine administered with cyclophosphamide (CP), demonstrating excellent safety and immunogenicity. To simplify the vaccine strategy for ease of use, we report here the results of a randomized phase II trial evaluating a lower vaccine dose alongside the original dose, administered with or without CP pretreatment. Patients and Methods: Patients with triple-negative breast cancer who completed all systemic and local therapies were assigned to receive either low-dose (825 μg) or high-dose (2.5 mg) vaccine peptides plus GM-CSF in six 4-week cycles, with or without CP prior to vaccination. Safety was monitored, and immunogenicity was evaluated before and after vaccination. Results: A total of 80 patients were dosed with vaccine, and 58 patients were evaluable for immunogenicity. Vaccination was well tolerated and elicited immunity in 83% of patients. Levels of FRα-specific T cells were high, persistent, and comparable with T-cell levels against tetanus toxoid. No differences in immunity were observed between the two doses. CP pretreatment also did not affect the immune responses. Recurrences occurred in eight evaluable patients and were unrelated to the treatment arm. No patients died during the study because of their disease. Conclusions: The reduced-dose FRα vaccine is as effective as the original dose and does not require CP to generate maximal immunity. These findings may inform the design of trials testing efficacy in triple-negative breast cancer or other cancers.

6061 Background: PDS0101 is a T cell stimulating immunotherapy (therapeutic vaccine) targeting HPV16. The combination of PDS0101 + pembrolizumab (5 cycles) has shown durable clinical responses in HPV-positive recurrent/metastatic HNSCC. MC200710 is a window of opportunity study in patients with HPV-associated oropharyngeal squamous cell carcinoma (HPV+ OPSCC). This prospective phase II trial utilized 2 cycles of neoadjuvant PDS0101 aloneor in combination with pembrolizumab prior to surgical resection or chemoradiotherapy (CRT). Herein, we report the initial results, including primary endpoint of ctDNA response. Methods: Between June 2022 and September 2024, 20 patients (10 per Arm) with locally advanced HPV+ OPSCC were enrolled in a sequential alternating design. Arm A received 2 cycles of PDS0101 and Arm B received 2 cycles of PDS0101 and pembrolizumab with all patients undergoing subsequent surgery or CRT. Assessments were done at baseline, post cycle 1, and post cycle 2 (prior to surgery or CRT). The coprimary endpoint of ctDNA response was defined as a ≥50% decline in ctDNA post cycle 2 compared to baseline as quantified using NavDx (TTMV fragments/mL). Radiologic objective response rate (ORR) was assessed as per RECIST 1.1. Toxicity through the neoadjuvant period was assessed using CTCAE criteria and recurrence rates following surgery or CRT are reported. Results: Patients were similar between arms: male 90%, median age 61 years, cT1/T2 70%, cN1 65%, and no smoking history 65%. All patients completed both cycles of therapy with 13 (65%) undergoing primary operative management and 7 CRT (35%). The most common toxicity was injection site reaction 85% grade 1, 15% grade 2, 0% grade 3, consistent with prior studies. One patient experienced grade 2 pneumonitis during the neoadjuvant window (Arm B). There was one grade 3 toxicity (5%) possibly attributable to study intervention with one patient experiencing transient hepatitis requiring hospitalization (Arm A). Zero of 10 patients in Arm A had a ≥50% decline in ctDNA from baseline while 5 of 10 patients (50%) met this primary endpoint in ARM B (p=0.03). After Cycle 2, based on RECIST 1.1, no Arm A patients had a partial response (PR), with 7 having stable disease (SD); Arm B had 2 patients with PR and 8 with SD. With median follow up of 6 months, 2 patients in Arm A recurred and 0 patients in Arm B. Conclusions: The combination of PDS0101 and pembrolizumab met the trial’s primary endpoint of ctDNA response and shows promise for further evaluation. ctDNA can be used to assess early response and future studies could use ctDNA to adapt neoadjuvant therapy. Based on these findings, a neoadjuvant dose optimization study in HPV16+ oropharyngeal carcinoma is warranted and evaluation of PDS0101 and pembrolizumab in comparison to pembrolizumab alone. Clinical trial information: NCT05232851 .

Background Neoadjuvant treatment has become standard for patients with high-risk operable stage III melanoma, but the optimal regimen is unknown. Targeted therapy approaches yield high pathological response rates, while immunotherapy regimens show favorable recurrence-free survival (RFS). NeoACTIVATE was designed to address whether a neoadjuvant combination of both targeted therapy and immunotherapy might leverage the benefits of each. Methods We tested neoadjuvant treatment with 12 weeks of vemurafenib, cobimetinib, and atezolizumab for patients with BRAF-mutated (BRAFm) melanoma (cohort A) and cobimetinib and atezolizumab for patients with BRAF-wild-type (BRAFwt) melanoma (cohort B), regimens which we have shown generate a substantial major pathological response. After therapeutic lymph node dissection, patients received 24 weeks of adjuvant atezolizumab. Here, we report survival outcomes and their association with biomarkers assayed among the gut microbiome and peripheral blood immune subsets. Results With 49 months median follow-up, the median RFS was not reached for cohort A and was 40.8 months for cohort B. At 24 months after operation, 2 of 14 cohort A patients and 4 of 13 cohort B patients had experienced distant relapse. Key findings from correlative analyses included diversity, taxonomic and functional metagenomic gut microbiome signals associated with distant metastasis-free survival at 2 years. Notably, we observed a strong correlation between low microbial arginine biosynthesis (required for T-cell activation and effector function) and early distant recurrence (p=0.0005), which correlated with taxonomic differential abundance findings. Peripheral blood immune monitoring revealed increased double-positive (CD4+CD8+) T cells in patients with early recurrence. Conclusions Neoadjuvant treatment with cobimetinib and atezolizumab±vemurafenib was associated with a low rate of distant metastasis in patients with high-risk stage III melanoma. Freedom from early distant metastasis was highly associated with taxonomic differences in gut microbiome structure and with functional pathway alterations known to modulate T cell immunity. Identification of predictive biomarkers will permit optimization of neoadjuvant therapy regimens for individual patients. Trial registration number NCT03554083.

The optimal detection strategies for effective convalescent immunity after SARS-CoV-2 infection and vaccination remain unclear. The objective of this study was to characterize convalescent immunity targeting the SARS-CoV-2 spike protein using a multiparametric approach. At the beginning of the pandemic, we recruited 30 unvaccinated convalescent donors who had previously been infected with COVID-19 and 7 unexposed asymptomatic controls. Peripheral blood mononuclear cells (PBMCs) were obtained from leukapheresis cones. The humoral immune response was assessed by measuring serum anti-SARS-CoV-2 spike S1 subunit IgG via semiquantitative ELISA, and T-cell immunity against S1 and S2 subunits were studied via IFN-γ enzyme-linked immunosorbent spot (ELISpot) and flow cytometric (FC) activation-induced marker (AIM) assays and the assessment of cytotoxic CD8+ T-cell function (in the subset of HLA-A2-positive patients). No single immunoassay was sufficient in identifying anti-spike convalescent immunity among all patients. There was no consistent correlation between adaptive humoral and cellular anti-spike responses. Our data indicate that the magnitude of anti-spike convalescent humoral and cellular immunity is highly heterogeneous and highlights the need for using multiple assays to comprehensively measure SARS-CoV-2 convalescent immunity. These observations might have implications for COVID-19 surveillance, and the determination of optimal vaccination strategies for emerging variants. Further studies are needed to determine the optimal assessment of adaptive humoral and cellular immunity following SARS-CoV-2 infection, especially in the context of emerging variants and unclear vaccination schedules.

Abstract Optimal detection strategies for effective convalescent immunity after SARS-CoV-2 infection and vaccination remain unclear. The objective of this study was to characterize convalescent immunity targeting the SARS-CoV-2 spike protein using a multiparametric approach. At the beginning of the pandemic, between April 23, 2020, to May 11, 2020, we recruited 30 COVID-19 unvaccinated convalescent donors and 7 unexposed asymptomatic donors. Peripheral blood mononuclear cells (PBMCs) were obtained from leukapheresis cones. The humoral immune response was assessed by measuring serum anti-SARS-CoV-2 spike S1 subunit IgG semiquantitative ELISA and T cell immunity against S1 and S2 subunits were studied by IFN-γ Enzyme-Linked Immune absorbent Spot (ELISpot), flow cytometric (FC) activation-induced marker (AIM) assays and the assessment of cytotoxic CD8+ T-cell function (in the subset of HLA-A2 positive patients). No single immunoassay was sufficient in identifying anti-spike convalescent immunity among all patients. There was no consistent correlation between adaptive humoral and cellular anti-spike responses. Our data indicate that the magnitude of anti-spike convalescent humoral and cellular immunity is highly heterogeneous and highlights the need for using multiple assays to comprehensively measure SARS-CoV-2 convalescent immunity. These observations might have implications for COVID-19 surveillance, and optimal vaccination strategies for emerging variants. Further studies are needed to determine the optimal assessment of adaptive humoral and cellular immunity following SARS-CoV-2 infection, especially in the context of emerging variants and unclear vaccination schedules.

Abstract Rationale Candida albicans (CA) is ubiquitous and individuals are often immunized against it. However, skin testing reactivity with CA-antigens (CAAg) have been variable during latent tuberculosis infection (LTBI) screening. We hypothesized that the use of a CAAg in an ELISPOT assay would provide a positive control to study tuberculosis (TB)-antigen specific immune responses. Objective To test an ELISPOT with TB-antigens and control conditions, including a purified protein derivative of CA, CAAg, that differentiates subjects with LTBI from unexposed subjects. Methods An ELISPOT that detects interferon-γ from PBMCs (2×105 cells) after 2-day incubation with TB antigens, controls, including CAAg, and co-stimulatory antibodies. We studied two independent cohorts of subjects with LTBI diagnosis based on TST and IGRA results per clinical guidelines. Results One-hundred thirty-nine subjects were studied in 2010–12, including 27 patients with LTBI and 21 IGRA(−) unexposed subjects. ELISPOT with TB-antigens but also CAAg - nil (78 [21 – 182] vs. 9 [1 – 50] cfu) differentiated LTBI subjects from unexposed ones (P<0.005). A second cohort of 76 subjects were studied in 2015–17, including 14 patients with LTBI and 24 unexposed controls, which showed similar statistical results. Positive control (SEAB/antiCD3) did not differentiate these study groups. ELISPOT results with CAAg correlated with results of ELISPOT with TB antigens in all subjects (P<0.001), and in the LTBI and unexposed groups independently. Conclusion An ELISPOT assay that includes CAAg revealed immune reactivity that is prevalent in LTBI. The immune subsets that react to both CA and mycobacterial antigens are unclear, but may be relevant for both TB and candida immunity.

Abstract HER2 is a prime target for cancer therapeutics due to its high overexpression in tumors. Advanced clinical trials, in breast and prostate cancer, investigating the use of the HLA-A2-binding HER2 peptide p369-377 (E75) as a vaccine are ongoing. In the present study, we investigated the proteasomal processing of p369-377 and other epitopes from HER2. We did not detect processing of p369-377 by either the proteasome or immunoproteasome in vitro, suggesting that tumor cells do not naturally process this epitope from HER2 for presentation on HLA-A2. However, other HER2 peptides were found to be consistently processed by the proteasome and immunoproteasome. One newly identified processed peptide, HER2 p373-382, was found to bind HLA-A2 stronger than p369-377, despite algorithms that predicted p369-377 as a stronger binder. Cytotoxic T lymphocytes (CTL) specific for p373-382 recognized target cells pulsed with either p373-382 or p369-377. CTL specific for p373-382 also lysed HER2+HLA-A2+ human breast cancer cells more efficiently than p369-377-specific CTL. These results suggest that cancer cells are naturally processing and presenting p373-382. Furthermore, our results suggest that T cells generated against p369-377 in previous studies may have been cross-reacting with p373-382. Thus, the newly identified p373-382 peptide may be a more appropriate candidate for vaccines with this new evidence that it is processed and presented by HLA-A2 for recognition by HER2-specific CTL.

Most gene-based severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines are nonreplicating vectors. They deliver the gene or messenger RNA to the cell to express the spike protein but do not replicate to amplify antigen production. This study tested the utility of replication in a vaccine by comparing replication-defective adenovirus (RD-Ad) and replicating single-cycle adenovirus (SC-Ad) vaccines that express the SARS-CoV-2 spike protein. SC-Ad produced 100 times more spike protein than RD-Ad and generated significantly higher antibodies against the spike protein than RD-Ad after single immunization of Ad-permissive hamsters. SC-Ad–generated antibodies climbed over 14 weeks after single immunization and persisted for more than 10 months. When the hamsters were challenged 10.5 months after single immunization, a single intranasal or intramuscular immunization with SC-Ad-Spike reduced SARS-CoV-2 viral loads and damage in the lungs and preserved body weight better than vaccination with RD-Ad-Spike. This demonstrates the utility of harnessing replication in vaccines to amplify protection against infectious diseases.