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NeuVax is a vaccine comprised of the HER2-derived MHC class I peptide E75 (nelipepimut-S, NPS) combined with GM-CSF. We completed a randomized trial of preoperative vaccination with NeuVax versus GM-CSF alone in patients with ductal carcinoma in situ (DCIS). The primary objective was to evaluate for NPS-specific cytotoxic T lymphocyte (CTL) responses. Patients with human leukocyte antigen (HLA)-A2-positive DCIS were enrolled and randomized 2:1 to NeuVax versus GM-CSF alone and received two inoculations prior to surgery. The number of NPS-specific CTL was measured pre-vaccination, at surgery, and 1 and 3 to 6 months post-operation by dextramer assay. Differences in CTL responses between groups and between pre-vaccination and 1-month post-operation were analyzed using a two-sample t test or Wilcoxon rank sum test. The incidence and severity of adverse events were compared between groups. Overall, 45 patients were registered; 20 patients were HLA-A2 negative, 7 declined participation, 1 withdrew, and 4 failed screening for other reasons. The remaining 13 were randomized to NeuVax (n = 9) or GM-CSF alone (n = 4). Vaccination was well-tolerated with similar treatment-related toxicity between groups with the majority (>89%) of adverse events being grade 1. The percentage of NPS-specific CTLs increased in both arms between baseline (pre-vaccination) and 1-month post-operation. The increase was numerically greater in the NPS+GM-CSF arm, but the difference was not statistically significant. NPS+GM-CSF is safe and well-tolerated when given preoperatively to patients with DCIS. In patients with HLA-A2-positive DCIS, two inoculations with NPS+GM-CSF can induce in vivo immunity and a continued antigen-specific T-cell response 1-month postsurgery. Prevention Relevance: This trial showed that vaccination of patients with HLA-A2-positive DCIS with NeuVax in the preoperative setting can induce a sustained antigen-specific T-cell response. This provides proof of principle that vaccination in the preoperative or adjuvant setting may stimulate an adaptive immune response that could potentially prevent disease recurrence.

NeuVax is a vaccine comprised of the HER2-derived MHC class I peptide E75 (nelipepimut-S, NPS) combined with GM-CSF. We completed a randomized trial of preoperative vaccination with NeuVax versus GM-CSF alone in patients with ductal carcinoma in situ (DCIS). The primary objective was to evaluate for NPS-specific cytotoxic T lymphocyte (CTL) responses. Patients with human leukocyte antigen (HLA)-A2-positive DCIS were enrolled and randomized 2:1 to NeuVax versus GM-CSF alone and received two inoculations prior to surgery. The number of NPS-specific CTL was measured pre-vaccination, at surgery, and 1 and 3 to 6 months post-operation by dextramer assay. Differences in CTL responses between groups and between pre-vaccination and 1-month post-operation were analyzed using a two-sample t test or Wilcoxon rank sum test. The incidence and severity of adverse events were compared between groups. Overall, 45 patients were registered; 20 patients were HLA-A2 negative, 7 declined participation, 1 withdrew, and 4 failed screening for other reasons. The remaining 13 were randomized to NeuVax (n = 9) or GM-CSF alone (n = 4). Vaccination was well-tolerated with similar treatment-related toxicity between groups with the majority (>89%) of adverse events being grade 1. The percentage of NPS-specific CTLs increased in both arms between baseline (pre-vaccination) and 1-month post-operation. The increase was numerically greater in the NPS+GM-CSF arm, but the difference was not statistically significant. NPS+GM-CSF is safe and well-tolerated when given preoperatively to patients with DCIS. In patients with HLA-A2-positive DCIS, two inoculations with NPS+GM-CSF can induce in vivo immunity and a continued antigen-specific T-cell response 1-month postsurgery.Prevention Relevance:This trial showed that vaccination of patients with HLA-A2-positive DCIS with NeuVax in the preoperative setting can induce a sustained antigen-specific T-cell response. This provides proof of principle that vaccination in the preoperative or adjuvant setting may stimulate an adaptive immune response that could potentially prevent disease recurrence.

Supplementary Table S5: HER2 expression in biopsy and resection specimens

Supplementary Fig. S2: Nelipepimut-S (NPS)-specific cytotoxic T lymphocyte (CTL) responses in patients receiving GM-CSF alone with corresponding HER2 changes

Supplemental File 1

No approved medical therapies prevent progression of low-grade prostate cancer. Rapamycin inhibits cell proliferation and augments immune responses, producing an antitumor effect. Encapsulated rapamycin (eRapa) incorporates rapamycin into a pH-sensitive polymer, ensuring consistent dosing. Here, we present results from a phase I trial evaluating the safety and tolerability of eRapa in patients with prostate cancer. Patients with Gleason ≤7 (3+4) disease (low and intermediate risk) under active surveillance were enrolled in a 3+3 study with three eRapa dosing cohorts (cohort 1, 0.5 mg/week; cohort 2, 1 mg/week; and cohort 3, 0.5 mg/day). Patients were treated for 3 months and followed for an additional 3 months to assess safety, pharmacokinetics, quality of life (QoL), immune response, and disease progression. Fourteen patients (cohort 1, n = 3; cohort 2, n = 3; and cohort 3, n = 8) were enrolled. In cohort 3, one dose-limiting toxicity (DLT; neutropenia) and two non-DLT grade 1–2 adverse events (AE) occurred that resulted in patient withdrawal. All AEs in cohorts 1 and 2 were grade 1. Peak serum rapamycin concentration was 7.1 ng/mL after a 1 mg dose. Stable trough levels (∼2 ng/mL) developed after 48–72 hours. Daily dosing mildly worsened QoL, although QoL recovered after treatment cessation in all categories, except fatigue. Weekly dosing increased naïve T-cell populations. Daily dosing increased central memory cell populations and exhaustion markers. No disease progression was observed. In conclusion, treatment with eRapa was safe and well-tolerated. Daily dosing produced higher frequencies of lower grade toxicities and transient worsening of QoL, while weekly dosing impacted immune response. Future studies will verify clinical benefit and long-term tolerability.Prevention Relevance: There is an unmet medical need for a well-tolerated treatment capable of delaying progression of newly diagnosed low-grade prostate cancer. This treatment would potentially obviate the need for future surgical intervention and improve the perception of active surveillance as a more acceptable option among this patient population.

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Background: SV-BR-1-GM is a GM-CSF secreting breast cancer cell line derived from a Grade II (moderately differentiated) breast tumor that also expresses HLA class I & II antigens and is able to function as an antigen-presenting cell. Irradiated SV-BR-1-GM is used in a regimen including pre-dose low-dose cyclophosphamide and post-dose injection of IFNα2b into the inoculation sites. The SV-BR-1-GM regimen has been used alone (“monotherapy”, ClinicalTrials.gov NCT03066947 - study completed) and in combination with checkpoint inhibitors (“combination”, ClinicalTrials.gov NCT03328026 - study ongoing). Here we report survival data for patients with advanced metastatic breast cancer (aMBC) treated with the SV-BR-1-GM regimen. Methods: 27 patients with refractory aMBC were treated with the SV-BR-1-GM regimen as monotherapy (cycles every 2 weeks x3 and then monthly). The combination study uses the SV-BR-1-GM regimen with PD-1 inhibitors (PD-1i) pembrolizumab or retifanlimab with cycles every 3 weeks (12 patients dosed to date). Here we report progression free survival (PFS) and overall survival (OS) for patients where that data was collected. Results: A total of 35 patients received the SV-BR-1-GM regimen. The SV-BR-1-GM regimen alone (monotherapy) was given to 27 and 12 received the regimen with a PD-1i checkpoint inhibitor (combination therapy): 4 subjects crossed over from monotherapy. Patients had been heavily pre-treated, median prior regimens = 5. Most patients were estrogen receptor and/or progesterone receptor positive, 18% were Her2/neu positive and 33% were triple negative. The treatment was generally safe and well tolerated. The disease control rate was 30% for the SV-BR-1-GM regimen alone and 33% for the combination with a PD-1i. Several patients had objective complete regression of selected metastases. Median progression free survival was 2.8 months for the SV-BR-1-GM regimen alone and 4.2 months for the PD-1i combination. Median overall survival was 7.0 months for the SV-BR-1-GM regimen alone (data available on 9 patients), and 12.0 months for the PD-1i combination (data available on 7 patients). Conclusions: The median OS compares favorably with published data regarding survival in third line trials (Kazmi Breast Cancer Res Treat. 2020 Aug 17). The protracted OS seen in some subjects suggests some patient subpopulations are more likely to derive clinical benefit. The SV-BR-1-GM regimen alone or in combination with a PD-1i, when administered to heavily pre-treated patients with aMBC, may have elicited effective immune responses in some patients. TablePatients by StudyCharacteristicSV-BR-1-GM Regimen Alone (n=27)SV-BR-1-GM Regimen + PD-1i (n=12)All Patients* (n=35)Age60 ± 1063 ± 1060 ± 10Mean Prior Systemic Regimens5 (range 0-12)6 (range 1-10)5 (range 0-12)% ER/PR +52%75%58%% Her2/neu +15%17%18%% Triple Negative36%25%33%Delayed-type Hypersensitivity81%91%82%Disease Control Rate30%33%29%Median (Range) Progression Free Survival (months)2.8 (0.4-7.4) (n=27)4.2 (0.8-9.4) (n=11)2.8 (0.4-9.4) (n=34)Median (Range) Overall Survival (months)7.0 (1-41) (n=9)12.0 (5.1-21.4) (n=7)10.2 (1-41) (n=14)• Note that 4 patients crossed over from the monotherapy study to the combination therapy study. Citation Format: William Williams, Shaker R Dakhil, Carmen Calfa, Jarrod P Holmes, Saveri Bhattacharya, Jason Lukas, Elizabeth Tan-Chui, George E Peoples, Vivek G Sunkari, Markus D Lacher, Charles L Wiseman. Overall survival following treatment with a modified whole tumor cell targeted immunotherapy in patients with advanced breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-14-02.

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Clinicopathologic characteristics of randomized triple-negative breast cancer patients

Purpose:Preclinical data provide evidence for synergism between HER2-targeted peptide vaccines and trastuzumab. The efficacy of this combination was evaluated in patients with HER2 low-expressing breast cancer in the adjuvant setting.Patients and Methods:A phase IIb, multicenter, randomized, single-blinded, controlled trial enrolled disease-free patients after standard therapy completion (NCT01570036). Eligible patients were HLA-A2, A3, A24, and/or A26+, and had HER2 IHC 1+/2+, FISH nonamplified breast cancer, that was node positive and/or hormone receptor–negative [triple-negative breast cancer (TNBC)]. Patients received trastuzumab for 1 year and were randomized to placebo (GM-CSF, control) or nelipepimut-S (NPS) with GM-CSF. Primary outcome was 24-month disease-free survival (DFS). Secondary outcomes were 36-month DFS, safety, and immunologic response.Results:Overall, 275 patients were randomized; 136 received NPS with GM-CSF, and 139 received placebo with GM-CSF. There were no clinicopathologic differences between groups. Concurrent trastuzumab and NPS with GM-CSF was safe with no additional overall or cardiac toxicity compared with control. At median follow-up of 25.7 (interquartile range, 18.4–32.7) months, estimated DFS did not significantly differ between NPS and control [HR, 0.62; 95% confidence interval (CI), 0.31–1.25; P = 0.18]. In a planned exploratory analysis of patients with TNBC, DFS was improved for NPS versus control (HR, 0.26; 95% CI, 0.08–0.81, P = 0.01).Conclusions:The combination of NPS with trastuzumab is safe. In HER2 low-expressing breast cancer, no significant difference in DFS was seen in the intention-to-treat analysis; however, significant clinical benefit was seen in patients with TNBC. These findings warrant further investigation in a phase III randomized trial.

The toxicity profiles were similar between the treatment arms. (A) There was no difference in the total number of graded adverse events (AE) between arms of the trial. (B) There was no significant difference in the maximum local and systemic toxicity experienced by each patient between arms of the trial.

Related Article from Overcoming Cancer Immune Tolerance and Escape

SKBR3 breast cancer cells shed HER2 after treatment with chemotherapy or irradiation.

Although HER2/neu–targeted cancer vaccines have shown initial promise in the adjuvant setting, a therapeutic vaccine remains elusive due to the tumor escape mechanisms of established cancer. As described by Seavey et al. in this issue of CCR, a Listeria-delivered vaccine may help overcome immune tolerance, leading to an effective therapeutic vaccine.

Early-phase clinical trials evaluating CD8+ T cell–eliciting, HER2-derived peptide vaccines administered to HER2+ breast cancer patients in the adjuvant setting suggest synergy between the vaccines and trastuzumab, the mAb targeting the HER2 protein. Among 60 patients enrolled in clinical trials evaluating the E75 + GM-CSF and GP2 + GM-CSF vaccines, there have been no recurrences in patients vaccinated after receiving trastuzumab as part of standard therapy in the per treatment analyses conducted after a median follow-up of greater than 34 months. Here, we describe a mechanism by which this synergy may occur. Flow cytometry showed that trastuzumab facilitated uptake of HER2 by dendritic cells (DC), which was mediated by the Fc receptor and was specific to trastuzumab. In vitro, increased HER2 uptake by DC increased cross-presentation of E75, the immunodominant epitope derived from the HER2 protein, an observation confirmed in two in vivo mouse models. This increased E75 cross-presentation, mediated by trastuzumab treatment, enabled more efficient expansion of E75-specific cytotoxic T cells (E75-CTL). These results demonstrate a mechanism by which trastuzumab links innate and adaptive immunity by facilitating activation of antigen-specific T cells. On the basis of these data, we conclude that HER2-positive breast cancer patients that have been treated with trastuzumab may experience a more robust antitumor immune response by restimulation of T cells with the E75 peptide vaccine, thereby accounting for the improved disease-free survival observed with combination therapy. Cancer Res; 77(19); 5374–83. ©2017 AACR.

Pancreatic ductal adenocarcinoma (PDAC) remains deadly despite advances in systemic therapies and surgical techniques. While there is increasing utilization of immune therapies across diverse cancer types, PDAC remains generally resistant to these treatments. We report a case of locally advanced PDAC treated with preoperative radiation and anti-PD-1 immunotherapy guided by preoperative PD-L1 tumor analysis. After 4 months of preoperative therapy, the patient was submitted to resection, demonstrating a near-complete pathologic response on final tumor analysis. We will discuss the relevant literature and current state of immunotherapeutics for PDAC.

Background Melanoma therapy has changed dramatically over the last decade with improvements in immunotherapy, yet many patients do not respond to current therapies. This novel vaccine strategy may prime a patient’s immune system against their tumor and work synergistically with immunotherapy against advanced-stage melanoma. Methods This was a prospective, randomized, double-blind, placebo-controlled, phase IIb trial of the tumor lysate, particle-loaded, dendritic cell (TLPLDC) vaccine administered to prevent recurrence in patients with resected stage III/IV melanoma. Patients were enrolled and randomized 2:1 to the TLPLDC vaccine or placebo (empty yeast cell wall particles and autologous dendritic cells). Both intention-to-treat (ITT) and per treatment (PT) analyses were predefined, with PT analysis including patients who remained disease-free through the primary vaccine/placebo series (6 months). Results A total of 144 patients were randomized (103 vaccine, 41 control). Therapy was well-tolerated with similar toxicity between treatment arms; one patient in each group experienced related serious adverse events. While disease-free survival (DFS) was not different between groups in ITT analysis, in PT analysis the vaccine group showed improved 24-month DFS (62.9% vs. 34.8%, p = 0.041). Conclusions This phase IIb trial of TLPLDC vaccine administered to patients with resected stage III/IV melanoma shows TLPLDC is well-tolerated and improves DFS in patients who complete the primary vaccine series. This suggests patients who do not recur early benefit from TLPLDC in preventing future recurrence from melanoma. A phase III trial of TLPLDC + checkpoint inhibitor versus checkpoint inhibitor alone in patients with advanced, surgically resected melanoma is under development. Trial Registration NCT02301611. Supplementary information The online version contains supplementary material available at (10.1245/s10434-021-09709-1).

Background: SV-BR-1-GM is a GM-CSF transfected breast cancer cell line, exceptional for having antigen-presenting capability and expressing both HLA I and II. The parent cell line, SV-BR-1, was derived from a patient with grade II (moderately differentiated) breast cancer. We report molecular characterization of SV-BR-1-GM, noting it retains features of a grade II tumor, and report enhanced disease control in patients with grade I or II breast cancer.Methods: SV-BR-1 and SV-BR-1-GM were characterized molecularly using RNAseq and proteomic analyses. We treated 23 evaluable patients with recurrent and/or metastatic breast cancer refractory to standard therapy. The SV-BR-1-GM regimen included cyclophosphamide 300 mg/m2 2-3d prior to intradermal injection of SV-BR-1-GM (20-40x106 cells divided into 4 sites) and IFNα into the inoculation sites (10,000 IU/site) about 48 and 96 hours subsequently. Cycles were q2 weeks x3 then qmo x 3 (clinical trial NCT03066947). Eleven patients were treated with the above regimen in combination with a PD-1 inhibitor (pembrolizumab or INCMGA00012) (clinical trial NCT03328026). Disease response was evaluated radiographically q3 mo and as clinically indicated. Results: To estimate the tumor grade represented by the SV-BR-1-GM cell line, we developed a score we refer to as Relative Molecular Grade (RMG). SV-BR-1-GM is most similar to the MDA-MB-468 cell line (RMG of 52.1), which was classified as Basal A phenotype. Basal A cancers are less aggressive than Basal B but more aggressive than Luminal, suggesting that SV-BR-1-GM may have retained features of a grade II breast cancer. We also noted that SV-BR-1-GM expresses both Class I (HLA-A, B & C) and Class II (HLA-DR and -DP) molecules, and that the HLA-DR expression is enhanced by treatment with IFNγ. SV-BR-1-GM expressed 31 genes which are overexpressed in breast cancer, 8 cancer-testis antigens and 3 genes expressed in breast tissue. In 30 patients treated with the SV-BR-1-GM regimen (19 with the SV-BR-1-GM regimen alone, 4 who began on the SV-BR-1-GM regimen and transitioned to combination with a PD-1i, and 7 with combination therapy alone) there were 7 with grade II breast cancer and 1 with grade I breast cancer (Table). These patients were heavily pre-treated with an average of 10 prior regimens. While only one patient with grade III cancer showed disease control, 75% of the patients with grade I or II tumors showed disease control. Patients remained on study for up to 259 days.Conclusions: SV-BR-1-GM appears to retain characteristics of a moderately differentiated breast cancer, expresses multiple potential tumor antigens, and can elicit disease control especially in patients with grade I and II breast cancer. TablePatients with Grade I/II TumorsCharacteristicSV-BR-1-GM Regimen Alone(n=6)SV-BR-1-GM Regimen + PD-1i(n=3)All Patients(n=8)Age64 ± 767 ± 465 ± 7Mean Prior Systemic Regimens6 (range 1-20)15 (range 14-15)10 (range 1-20)% ER/PR +80%100%86%% Her2/neu +0%33%14%% Triple Negative20%0%14%Delayed-type Hypersensitivity83%100%88%Disease Control Rate*67%100%75%Days on Study (Range)94 (32-181)189 (133-259)141 (32-259)•Includes CR, PR, SD (including minor responses and mixed responses) Citation Format: William Williams, Shaker R Dakhil, Carmen Calfa, Jarrod P Holmes, Saveri Bhattacharya, Jason Lukas, Elizabeth Tan-Chiu, George E Peoples, Vivek G Sunkari, Markus D Lacher, Charles L Wiseman. Response to a modified whole tumor cell targeted immunotherapy in patients with advanced breast cancer correlates with tumor grade [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-20.

HER2 is a prevalent growth factor in a variety of malignancies, most prominently breast cancer. Over-expression has been correlated with the poorest overall survival and has been the target of successful therapies such as trastuzumab. AE37 is a novel, HER2-directed vaccine based on the AE36 hybrid peptide (aa776-790), which is derived from the intracellular portion of the HER2 protein, and the core portion of the MHC Class II invariant chain (the Ii-Key peptide). This hybrid peptide is given with GM-CSF immunoadjuvant as the AE37 vaccine.This article describes in detail the preclinical science leading to the creation of the AE37 vaccine and examines use of this agent in multiple clinical trials for breast and prostate cancer. The safety profile of AE37 is discussed and opinions on the potential of the vaccine in breast and prostate cancer patient subsets along with other malignancies, are offered.Future trials utilizing the AE37 vaccine to treat other HER2-expressing malignancies are likely to see similar success, and this will be enhanced by combination immunotherapy. Ii-Key modification of other peptides of interest across oncology and virology could yield impressive results over the longer term.

Background Colorectal cancer (CRC) tumor microenvironment (TME) characteristics, such as tumor infiltrating lymphocyte (TIL) densities and PD-L1 status, are predictive of recurrence, disease-free survival, and overall survival. In many malignancies, TME characteristics are also predictive of response to immunotherapy. As window of opportunity studies using neoadjuvant immunotherapy become more common and treatment guidelines incorporate TME features, accurate assessment of the pre-treatment TME using the biopsy specimen is critical. However, no study has thoroughly evaluated the correlation between the TMEs of the biopsy and resection specimens. Methods We conducted a retrospective analysis of patients with stage I–III CRC with matched biopsy and resection specimens. CD3+, CD4+, CD8+, and FoxP3+ lymphocyte populations at the center of tumor (CT) and invasive margin (IM) and tumor PD-L1 status in the biopsy and resection specimens were evaluated. TIL populations were compared using Mann–Whitney U tests or Student’s t tests and correlated using Pearson r. Results CD3+ and CD4+ densities were significantly higher in the CT of the biopsy relative to the resection specimen Comparing biopsy and resection specimens, no TIL population at either the CT or IM had a correlation coefficient > 0.5. Determining PD-L1 status based on biopsy tissue resulted in a sensitivity of 37.1%, specificity of 81.4%, and accuracy of 61.5%. Conclusions These findings demonstrate significant discordance between the TME of the biopsy and resection specimens. Caution should be used when basing treatment decisions on pre-treatment endoscopic biopsy findings and when interpreting changes in the TME between pre-treatment biopsy and resection specimens after neoadjuvant therapy. Electronic supplementary material The online version of this article (10.1007/s00262-020-02784-5) contains supplementary material, which is available to authorized users.

Purpose: Preclinical data provide evidence for synergism between HER2-targeted peptide vaccines and trastuzumab. The efficacy of this combination was evaluated in patients with HER2 low-expressing breast cancer in the adjuvant setting. Patients and Methods: A phase IIb, multicenter, randomized, single-blinded, controlled trial enrolled disease-free patients after standard therapy completion (NCT01570036). Eligible patients were HLA-A2, A3, A24, and/or A26+, and had HER2 IHC 1+/2+, FISH nonamplified breast cancer, that was node positive and/or hormone receptor–negative [triple-negative breast cancer (TNBC)]. Patients received trastuzumab for 1 year and were randomized to placebo (GM-CSF, control) or nelipepimut-S (NPS) with GM-CSF. Primary outcome was 24-month disease-free survival (DFS). Secondary outcomes were 36-month DFS, safety, and immunologic response. Results: Overall, 275 patients were randomized; 136 received NPS with GM-CSF, and 139 received placebo with GM-CSF. There were no clinicopathologic differences between groups. Concurrent trastuzumab and NPS with GM-CSF was safe with no additional overall or cardiac toxicity compared with control. At median follow-up of 25.7 (interquartile range, 18.4–32.7) months, estimated DFS did not significantly differ between NPS and control [HR, 0.62; 95% confidence interval (CI), 0.31–1.25; P = 0.18]. In a planned exploratory analysis of patients with TNBC, DFS was improved for NPS versus control (HR, 0.26; 95% CI, 0.08–0.81, P = 0.01). Conclusions: The combination of NPS with trastuzumab is safe. In HER2 low-expressing breast cancer, no significant difference in DFS was seen in the intention-to-treat analysis; however, significant clinical benefit was seen in patients with TNBC. These findings warrant further investigation in a phase III randomized trial.

Purpose AE37 and GP2 are HER2 derived peptide vaccines. AE37 primarily elicits a CD4+ response while GP2 elicits a CD8+ response against the HER2 antigen. These peptides were tested in a large randomized trial to assess their ability to prevent recurrence in HER2 expressing breast cancer patients. The primary analyses found no difference in 5-year overall disease-free survival (DFS) but possible benefit in subgroups. Here, we present the final landmark analysis. Methods In this 4-arm, prospective, randomized, single-blinded, multi-center phase II trial, disease-free node positive and high-risk node negative breast cancer patients enrolled after standard of care therapy. Six monthly inoculations of vaccine (VG) vs. control (CG) were given as the primary vaccine series with 4 boosters at 6-month intervals. Demographic, safety, immunologic, and DFS data were evaluated. Results 456 patients were enrolled; 154 patients in the VG and 147 in CG for AE37, 89 patients in the VG and 91 in CG for GP2. The AE37 arm had no difference in DFS as compared to CG, but pre-specified exploratory subgroup analyses showed a trend towards benefit in advanced stage (p = 0.132, HR 0.573 CI 0.275–1.193), HER2 under-expression (p = 0.181, HR 0.756 CI 0.499–1.145), and triple-negative breast cancer (p = 0.266, HR 0.443 CI 0.114–1.717). In patients with both HER2 under-expression and advanced stage, there was significant benefit in the VG (p = 0.039, HR 0.375 CI 0.142–0.988) as compared to CG. The GP2 arm had no significant difference in DFS as compared to CG, but on subgroup analysis, HER2 positive patients had no recurrences with a trend toward improved DFS (p = 0.052) in VG as compared to CG. Conclusions This phase II trial reveals that AE37 and GP2 are safe and possibly associated with improved clinical outcomes of DFS in certain subgroups of breast cancer patients. With these findings, further evaluations are warranted of AE37 and GP2 vaccines given in combination and/or separately for specific subsets of breast cancer patients based on their disease biology.

Background: SV-BR-1-GM is a GM-CSF transfected breast cancer cell line, exceptional for having antigen-presenting capability expressing both HLA I and II. We report clinical efficacy, safety, and immunologic correlates of response from our initial Phase I/II trial and initial data from our trial of SV-BR1-GM in combination with immune checkpoint inhibitors. Methods: We enrolled patients with recurrent and/or metastatic breast cancer refractory to standard therapy. Patients received cyclophosphamide 300 mg/m2 2-3d prior to intradermal injection of SV-BR-1-GM (20-40 × 106 cells divided into 4 sites) and IFNα into the inoculation sites (10,000 IU/site) ~2 & 4 days subsequently. Cycles were q2 weeks x3 then qmo x 3. Adverse events (AE) were evaluated after each inoculation. Immunologic responses were measured by delayed type hypersensitivity (DTH) after each inoculation with humoral and cellular responses evaluated ~q3 mo. Disease response was evaluated radiographically q3 mo and as clinically indicated (clinical trial NCT03066947). A similar regimen was used with SV-BR-1-GM in combination with pembrolizumab (200 mg IV) with cycles every 3 weeks (Phase I/II study NCT03328026). Results: In Phase I/IIa (NCT03066947), 23 patients underwent 1 - 8 cycles of treatment. Tumor regression was seen in 3 patients, all of whom matched SV-BR-1-GM at least at one HLA allele. There were no related serious adverse events. The most common adverse event was minor local irritation at the inoculation site. Clinical data are shown in the table. A measurable DTH response was present in 21 patients. Of patients who developed a DTH response and had at least one HLA match, the tumor regression rate was 33% and for those with 2 HLA matches 67%. We saw evidence of antibody responses in 3 of 5 patients evaluated to date. Especially in responders after treatment, blood lymphocytes showed increased cytokine secretion (including ITAC, IFNγ, IL-6 & IL-8) following stimulation with antigens expressed in SV-BR-1-GM. 21/23 patients had expression of PD-L1 in identified circulating cancer-associated cells, and expression levels increased with treatment. Therefore, a combination study with pembrolizumab was initiated. Data on the first 6 patients shows that the regimen is clinically active and safe. One patient with a robust DTH response had evidence of tumor regression in liver metastases. This study is ongoing and is being modified to evaluate combination therapy with the PD-1 inhibitor INCMGA00012 and the IDO inhibitor epacadostat. Conclusions: SV-BR-1-GM appears to be safe and well-tolerated. Contrary to conventional wisdom, SV-BR-1-GM can produce regression of metastatic breast cancer correlating with an immunologic response and HLA matching. Combination therapy with checkpoint inhibitors is ongoing. Citation Format: William Williams, Shaker R Dakhil, Jarrod P Holmes, Saveri Bhattacharya, Carmen Calfa, Ajay Kundra, Daniel L Adams, Diane DaSilva, George E Peoples, Vivek Sunkari, Markus Lacher, Charles L Wiseman. Efficacy and safety of a modified whole tumor cell targeted immunotherapy in patients with advanced breast cancer alone and in combination with immune checkpoint inhibitors [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-09-08.

A randomized, double-blind, placebo-controlled phase 2b trial of the tumor lysate, particle-loaded, dendritic cell (TLPLDC) vaccine was conducted in patients with resected stage III/IV melanoma. Dendritic cells (DCs) were harvested with and without granulocyte-colony stimulating factor (G-CSF). This analysis investigates differences in clinical outcomes and RNA gene expression between DC harvest methods.The TLPLDC vaccine is created by loading autologous tumor lysate into yeast cell wall particles (YCWPs) and exposing them to phagocytosis by DCs. For DC harvest, patients had a direct blood draw or were pretreated with G-CSF before blood draw. Patients were randomized 2:1 to receive TLPLDC or placebo. Differences in disease-free survival (DFS) and overall survival (OS) were evaluated. RNA-seq analysis was performed on the total RNA of TLPLDC + G and TLPLDC vaccines to compare gene expression between groups.144 patients were randomized: 103 TLPLDC (47 TLPLDC/56 TLPLDC + G) and 41 placebo (19 placebo/22 placebo + G). Median follow-up was 27.0 months. Both 36-month DFS (55.8% vs. 24.4% vs. 30.0%, p = 0.010) and OS (94.2% vs. 69.8% vs. 70.9%, p = 0.024) were improved in TLPLDC compared to TLPLDC + G or placebo, respectively. When compared to TLPLDC + G vaccine, RNA-seq from TLPLDC vaccine showed upregulation of genes associated with DC maturation and downregulation of genes associated with DC suppression or immaturity.Patients receiving TLPLDC vaccine without G-CSF had improved OS and DFS. Outcomes remained similar between patients receiving TLPLDC + G and placebo. Direct DC harvest without G-CSF had higher expression of genes linked to DC maturation, likely improving clinical efficacy.

BackgroundThe tumor lysate, particle-loaded, dendritic cell (TLPLDC) vaccine is an autologous tumor vaccine that decreased recurrence in stage III/IV melanoma when granulocyte-colony stimulating factor (G-CSF) was not used to harvest the dendritic cells in a randomized phase 2B adjuvant trial.1The tumor lysate (TL) particle only (TLPO) vaccine utilizes a similar mechanism, but with autologous TL-loaded yeast cell wall particles; this eliminates the need for dendritic cell (DC) collection and ex-vivo loading and reduces production costs and time. The TLPO vaccine was compared to TLPLDC in an embedded bridging portion of the trial. Here, we examine 36-month outcomes of the ongoing randomized, double-blind phase 2 trial in patients (pts) with resected stage III/IV melanoma.MethodsPts were randomized 2:1 to receive TLPO or TLPLDC as a continuation of a previously established clinical trial comparing TLPLDC versus placebo. The TLPLDC group was analyzed separately based on use (or not) of G-CSF for collection of DC. Safety was measured by the Common Terminology Criteria for Adverse Events (CTCAE). Kaplan-Meier and log-rank analysis was used to compare 36-month disease-free survival (DFS) and overall survival (OS) in the intention-to-treat (ITT) main arms as well as pre-specified subgroups.ResultsA total of 187 pts were randomized with 41, 47, 56, and 43 pts enrolled in the placebo, TLPLDC without G-CSF (TLPLDC), TLPLDC with G-CSF (TLPLDC+G), and TLPO arm, respectively. Pts randomized to the TLPO arm were more likely to have stage IV melanoma (22.0% for placebo, 20.4% for TLPLDC and TLPLDC+G, and 44.2% for TLPO; p = 0.002) and to receive prior immunotherapy (36.6% for placebo, 39.8% for both TLPLDC and TLPLDC+G, and 83.7% for TLPO; p < 0.001). Grade 3+ adverse events were not significantly different between arms. In the ITT analysis, 36-month DFS was 30.0% for placebo, 55.8% for TLPLDC, 24.4% for TLPLDC+G, and 64.0% for TLPO (p < 0.001). OS at 36 months was 70.9% for placebo, 94.2% for TLPLDC, 69.8%% for TLPLDC+G, and 94.8% for TLPO (p = 0.011) (figure 1).Abstract 542 Figure 1Kaplan-Meier curves demonstrating DFS (A) and OS (B) between Placebo (n=41), TLPLDC (n=47), TLPLDC+G (n=56), and TLPO (n=43)ConclusionsThe TLPO and TLPLDC (without G-CSF) vaccines improved 36-month DFS and OS in this randomized phase 2 trial. The efficacy of the TLPO and TLPLDC vaccines will be confirmed in a phase III trial in resected Stage III/IV melanoma pts.Trial RegistrationNIH, clinicaltrials.gov, NCT02301611ReferencesO’Shea AE, Chick RC, Clifton GT, et al. The effect of pretreatment with G-CSF prior to dendritic cell collection during the phase IIb trial of an autologous DC-based vaccine for advanced, resectable melanoma. Presented at: Society for Immunotherapy of Cancer 35th Anniversary Annual Meeting & Preconference Programs (SITC 2020); November 11–14, 2020. Abstract 310. J Immunother Cancer. 2020;8(Suppl 3):A656–A959.Ethics ApprovalThe clinical trial protocol was approved by the Western Institutional Review Board (2014–1932). All participants provided informed consent prior to enrollment in the trial.

Rapamycin inhibits the mechanistic (formally mammalian) target of rapamycin (mTOR), an evolutionarily conserved intracellular kinase that influences activation of growth signaling pathways and immune responses to malignancy. Rapamycin has been found to have both immunosuppressant and immunostimulatory effects throughout the innate and adaptive responses based on the inhibition of mTOR signaling. While the immunosuppressant properties of rapamycin and mTOR inhibition explain rapamycin's success in the prevention of transplant rejection, the immunostimulatory characteristics are likely partially responsible for rapamycin's anti-neoplastic effects. The immunologic response to rapamycin is at least partially dependent on the dose and administration schedule, with lower doses inducing immunostimulation and intermittent dosing promoting immune function while limiting metabolic and immunosuppressant toxicities. In addition to its FDA-approved application in advanced malignancies, rapamycin may be effective as a chemopreventive agent, suspending progression of low-grade cancers, preventing invasive conversion of in situ malignancy, or delaying malignant transformation of established pre-malignant conditions.

Immunotherapy has revolutionized the treatment of melanoma, yet survival remains poor for patients with metastatic disease. The autologous tumor lysate, particle-loaded, dendritic cell (TLPLDC) vaccine has been shown to be safe adjuvant therapy for patients with resected stage III/IV melanoma who complete the primary vaccine series. Here, we describe an open-label trial of patients with metastatic melanoma treated with TLPLDC vaccine in addition to standard of care (SoC) therapies. The TLPLDC vaccine is created by loading autologous tumor lysate into yeast cell wall particles, which are phagocytosed by autologous dendritic cells ex vivo. Patients who recurred while enrolled in a phase IIb trial of adjuvant TLPLDC vaccine (crossover cohort) and patients with measurable metastatic melanoma cohort were offered TLPLDC vaccine along with SoC therapies. Tumor response was measured by RECIST 1.1 criteria. Overall survival (OS) and progression-free survival (PFS) were estimated by intention-to-treat analysis. Fifty-four patients were enrolled (28 in crossover cohort; 26 in metastatic melanoma cohort). The vaccine was well-tolerated with no grade ≥3 adverse events when given with SoC therapies to include checkpoint inhibitors, BRAF/MEK inhibitors, tyrosine kinase inhibitors, intralesional therapy and/or radiation. In the crossover arm, OS was 76.5% and PFS was 57.1% (median follow-up of 13.9 months). In the metastatic melanoma arm, OS was 85.7% and PFS was 52.2% (median follow-up 8.5 months). The TLPLDC vaccine is well-tolerated and safe in combination with SoC therapies. Future trials will determine the efficacy of TLPLDC in combination with SoC therapies in metastatic melanoma.

Background E39, an HLA‐A2‐restricted, immunogenic peptide derived from the folate‐binding protein (FBP), is overexpressed in multiple malignancies. We conducted a phase I/IIa trial of the E39 + GM‐CSF vaccine with booster inoculations of either E39 or E39′ (an attenuated version of E39) to prevent recurrences in disease‐free endometrial and ovarian cancer patients(pts). Here, we present the final 24‐month landmark analysis. Patients and methods HLA‐A2 + patients receiving E39 + GM‐CSF were included in the vaccine group (VG), and HLA‐A2‐ pts (or HLA‐A2 + patients refusing vaccine) were followed as the control group (CG). VG group received 6 monthly inoculations as the primary vaccine series (PVS) and were randomized to receive either E39 or E39′ booster inoculations. Demographic, safety, immunologic, and disease‐free survival (DFS) data were collected and evaluated. Results Fifty‐one patients were enrolled; 29 in the VG and 22 in the CG. Fourteen patients received

Purpose: In phase I/II studies, nelipepimut-S (NP-S) plus GM-CSF vaccine was well tolerated and effectively raised HER2-specific immunity in patients with breast cancer. Results from a prespecified interim analysis of a phase III trial assessing NP-S + GM-CSF are reported. Patients and Methods: This multicenter, randomized, double-blind phase III study enrolled females ≥18 years with T1–T3, HER2 low–expressing (IHC 1+/2+), node-positive breast cancer in the adjuvant setting. Patients received 1,000 μg NP-S + 250 μg GM-CSF or placebo + GM-CSF monthly for 6 months, then every 6 months through 36 months. The primary objective was disease-free survival (DFS). Protocol-specified imaging occurred annually. New abnormalities were categorized as recurrence events; biopsy confirmation was not mandated. The interim analysis was conducted as specified in the protocol after 73 DFS events. Results: A total of 758 patients (mean age 51.8 years) were randomized. Adverse events were similar between groups; most common were injection-associated: erythema (84.3%), induration (55.8%), and pruritus (54.9%). There was no significant between-arms difference in DFS events at interim analysis at median follow-up (16.8 months). In the NP-S arm, imaging detected 54.1% of recurrence events in asymptomatic patients versus 29.2% in the placebo arm (P = 0.069). Conclusions: NP-S was well tolerated. There was no significant difference in DFS events between NP-S and placebo. Use of mandated annual scans and image-detected recurrence events hastened the interim analysis contributing to early trial termination.

Background: SV-BR-1-GM is a GM-CSF transfected breast cancer cell line which expresses HLA class I & II antigens. In a previous clinical trial, a partial response of widely metastatic breast cancer was seen in a patient who matched SV-BR-1-GM at HLA-DRB3*02:02. Here we report the safety and efficacy analysis with immunologic correlates of response in the initial patients in a phase I/IIa trial of SV-BR-1-GM in patients with advanced breast cancer Methods: This phase I/IIa trial enrolled patients with recurrent and/or metastatic breast cancer refractory to standard chemotherapy/targeted-therapy. Patients received low-dose cyclophosphamide 2-3d prior to intradermal injection of SV-BR-1-GM (20x106 cells divided into 4 sites) and interferon-α into the inoculation sites (10,000 IU/site) ˜2 & 4 days subsequently. Cycles were 2 weeks x3 then q mo x 3. Adverse events (AE) were evaluated after each inoculation and graded via CTCAE v4.03. Immunologic response was measured by delayed type hypersensitivity (DTH) after each inoculation. Disease response was evaluated radiographically q3 mo and as clinically indicated (clinical trial NCT03066947). Results: To date, twenty-two patients have been enrolled and 17 have been inoculated for a total of 39 SV-BR-1-GM inoculations given. Per inoculation, the maximum related AE was grade 1 in 64%, grade 2 in 7.7%, and grade 3 in 7.7%. There were no related grade >3 or unexpected AE. Efficacy data is available on the first six (Table). Tumor regression was seen in 2 patients. 01-002 presented with liver, bone and 20 classic miliary lung metastases (up to 9mm). This subject previously received 7 chemotherapy regimens. She matched SV-BR-1-GM at Class I & II HLA loci. Imaging at 3 mo showed virtually complete regression of all 20 identifiable lesions in the lungs. This response was maintained at 6 mo but the subject was taken off protocol because of disease progression (liver and bone). 01-005, matching HLA-A*24:02, had notable regression of cutaneous lesions, but progressed in pleural and pericardial effusions, had irreversible cardiac arrest (unlikely related). DTH increased in 01-002 from 4mm (first dose) to 47mm (8th dose). Three of 3 patients evaluated developed antibodies responses (as measured by flow cytometry with SV-BR-1) including 01-002. Interleukin 8 also increased in 01-002. Conclusions: SV-BR-1-GM in this regimen appears to be safe and well-tolerated. In this initial exploratory analysis, SV-BR-1-GM can produce regression of pre-treated metastatic breast cancer correlating with an immunologic response. HLA matching is being evaluated as a predictor of response. PatientAgeMetastatic Sites# Prior RegimensHLA Matches# of CyclesTumor Regression?01-00146Pleura, Lymph Nodes7 chemo/bio, 5 hormonalDRB3*02:021No01-00273Lung, Liver, Bone6 chemo, 1 hormonalA*24:02, DRB3*02:028Lungs01-00554Lymph nodes, Pleura, Skin3 chemo/bioA*24:022Skin02-00170Lymph nodes1 chemo/bioNone1No02-00361Bone, Brain3 chemoNone6No02-00474Lymph nodes, Cutaneous3 chemo/bio, 1 hormonalDRB3*02:022Lost to Follow-up Citation Format: Bhattacharya S, Holmes JP, Calfa C, Lukas J, Tan-Chiu E, Clifton GT, Peoples GE, Lacher M, Wiseman CL, Williams WV. Initial safety and efficacy of a phase I/IIa trial of a modified whole tumor cell targeted immunotherapy in patients with advanced breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-09-09.

Background:HER2 low-expressing (LE) (IHC 1-2+, FISH non-amplified) breast cancer (BC) patients (pts) have not benefited from HER2-directed therapy despite HER2 antigen availability. Triple negative BC (TNBC), in particular, is immunogenic and in need of additional therapeutic options. We have previously shown the HER2-derived nelipeptimut-S (E75) + GM-CSF (NeuVax) to be synergistic with trastuzumab (Tz) in pre-clinical and pilot clinical studies. In a planned interim analysis of a multi-center, prospective, randomized, single-blinded, placebo-controlled phase 2b trial of Tz + NeuVax vs Tz to reduce recurrence in HER2 LE, node-positive (NP) and/or triple negative BC (TNBC) pts, we previously reported that the NeuVax + Tz was safe without added cardiac toxicity and demonstrated a significant reduction of recurrences in TNBC pts. This analysis examines additional subsets in this trial. Methods:HER2 LE, NP and/or TNBC pts who were clinically disease-free after standard therapy were randomized to receive Tz+NeuVax (vaccine group; VG) or Tz+GM-CSF (control group; CG). All pts received 1 yr of Tz per label. NeuVax or GM-CSF was given every 3 weeks x 6 starting with the 3rdTz dose, and then boosted every 6 months x 4. This pre-specified interim analysis was triggered 6 months after last enrollment. The primary endpoint is intention-to-treat 24 month disease-free survival (DFS) evaluated by log rank. Results: Of 275 pts randomized in the study (VG n=136, CG n=139), 98 had TNBC (VG=53, CG=45). In the interim analysis, estimated disease-free survival (DFS) was assessed with a median follow up of 18.8 months. No significant clinicopathologic differences were seen between treatment groups. In the TNBC group, estimated DFS was higher overall in VG vs CG (91.9% v 69.9%, p=0.023; hazard ratio [HR] 0.29, 95% confidence interval [CI] 0.09-0.90). On TNBC subgroup analysis, estimated DFS was higher in VG vs CG among pts who received neoadjuvant chemotherapy (VG n=35, CG n=31; HR 0.26, CI 0.07-0.93; p=0.03), HER2 IHC 1+ BC (VG n=34, CG n=28; HR 0.20, CI 0.04-0.96; p=0.03), pts who were AJCC 7thedition stage I/II (VG n=37, CG n=27; HR incalculable, no recurrences in the VG, p=0.008), and pts 351yr of age (VG n=32 & CG n = 26; HR 0.26 CI 0.07,0.94; p=0.009). HRs did not appreciably vary based on the histologic grade or presence of lymphovascular invasion. Conclusion:Examining the subgroups from the pre-specified interim analysis demonstrates a highly significant clinical benefit in TNBC pts overall. Within the TNBC cohort, specific benefit was seen in pts who received chemotherapy neoadjuvantly, expressed lower HER2, were earlier stage, and were older in age. These factors may help enrich the TNBC population targeted in a definitive Phase 3 study in TNBC patients with residual disease after neoadjuvant chemotherapy. Citation Format: Clifton GT, Kemp Bohan PM, Hale DF, Myers JW, Brown TA, Holmes JP, Vreeland TJ, Litton JK, Murthy RK, Mittendorf EA, Peoples GE. Subgroups analysis of a multicenter, prospective, randomized, blinded phase 2b trial of trastuzumab + nelipeptimut-S (NeuVax) vs trastuzumab for prevention of recurrence in breast cancer patients [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-09-01.

Background Checkpoint inhibitors (CPI) in combination with cell‐based vaccines may produce synergistic antitumor immunity. The primary analysis of the randomized and blinded phase IIb trial in resected stage III/IV melanoma demonstrated TLPLDC is safe and improved 24‐month disease‐free survival (DFS) in the per treatment (PT) analysis. Here, we examine efficacy within pre‐specified and exploratory subgroups. Methods Stage III/IV patients rendered disease‐free by surgery were randomized 2:1 to TLPLDC vaccine versus placebo. The pre‐specified PT analysis included only patients completing the primary vaccine/placebo series at 6 months. Kaplan–Meier analysis was used to compare 24‐month DFS among subgroups. Results There were no clinicopathologic differences between subgroups except stage IV patients were more likely to receive CPI. In stage IV patients, 24‐month DFS was 43% for vaccine versus 0% for placebo (p = 0.098) in the ITT analysis and 73% versus 0% (p = 0.002) in the PT analysis. There was no significant difference in 24‐month DFS when stratified by use of immunotherapy or CPI. For patients with resected recurrent disease, 24‐month DFS was 88.9% versus 33.3% (p = 0.013) in the PT analysis. All benefit from vaccination was in the PT analysis; no benefit was found in patients receiving up to three doses. Conclusion The TLPLDC vaccine improved DFS in patients completing the primary vaccine series, particularly in the resected stage IV patients. The efficacy of the TLPLDC vaccine will be confirmed in a phase III study evaluating adjuvant TLPLDC + CPI versus Placebo + CPI in resected stage IV melanoma patients., TLPLDC is an autologous tumor lysate vaccine made with autologous dendritic cells to prevent recurrence in patients with high‐risk resected melanoma. In this analysis, the vaccine was found to have the most benefit in stage IV resected patients and may have a synergistic effect in combination with checkpoint inhibitors. ​

Introduction: Existing HER2-targeted therapies modulate the tumor microenvironment and the immunologic response cancer in a favorable way. While these therapies have made dramatic improvements in t...

Background We have completed a prospective, randomized, multi-center, double-blind, placebo-controlled phase IIb trial of the tumor lysate, particle loaded, dendritic cell (TLPLDC) vaccine given to prevent recurrences in patients with resected stage III/IV melanoma. During the trial, granulocyte colony stimulating factor (G-CSF) was administered to some patients to mobilize dendritic cells (DCs) precursors prior to harvest, allowing for similar DC yield with reduced blood draws. This study examines the impact of DC collection methods on vaccine effectiveness. Methods TLPLDC is produced by loading tumor lysate into pre-prepared yeast cell wall particles (YCWPs) and exposing them to autologous DCs. DC precursors were isolated either by collection of 50–70 mL of blood following pre-administration of 300µg of G-CSF 24–48 hrs prior, or collection of 120 mL of peripheral blood without G-CSF pretreatment based on patient and provider preference. Patients were randomized 2:1 to receive TLPLDC or placebo (DCs exposed to empty YCWPs). 1–1.5 × 106 cells/dose were injected intradermally at 0, 1, 2, 6, 12, and 18 months. Differences in disease free survival (DFS) and overall survival (OS) were analyzed by log rank. Results Of 144 patients randomized, 103 received TLPLDC and 41 received placebo. Within the TLPLDC group, 57 received pretreatment with G-CSF (TLPLDC+G-CSF) and 46 did not (TLPLDC–G-CSF). There were no significant clinicopathologic or treatment differences between the three treatment arms. 36-month DFS was significantly better in TLPLDC–G-CSF vs. TLPLDC+G-CSF or placebo (51.8% vs. 23.4% and 27.1% respectively, p=0.027) (figure 1). TLPLDC–G-CSF had correspondingly improved OS (92.9% vs. 62.8% and 72.3% respectively, p=0.022) (figure 2). Subgroup analysis revealed TLPLDC–G-CSF had increased DFS over TLPLDC+G-CSF or placebo in Stage IV (68.6% vs. 18.8% and 0.0% respectively, p=0.058). Similarly, the DFS survival benefit of TLPLDC–G-CSF was enhanced in patients who received prior immunotherapy (IO) (61.9% vs. 11.5% and 35.7% respectively, p=0.007) or checkpoint inhibitors (CPI) (48.5% vs. 10.6% and 37.5% respectively, p=0.039). Conclusions TLPLDC vaccine created without G-CSF pretreatment significantly improved 36-month DFS and OS compared to TLPLDC+G-CSF or placebo in stage III/IV (resected) melanoma patients. On further subgroup analysis, the increases in OS and DFS were more profound in patients who received additional immune therapies to include CPI. Ongoing evaluation will determine if G-CSF mobilization leads to collection of phenotypically different DCs. Based on these results, we are planning a phase III trial of TLPLDC–G-CSF + CPI vs. placebo + CPI in advanced melanoma post-resection. Trial Registration ClinicalTrials. gov Identifier: NCT02301611 Ethics Approval This study was reviewed and approved by the IRB or Independent Ethics Committee (IEC) of each participating center prior to study initiation.

Background The autologous tumor lysate, particle-loaded, dendritic cell (TLPLDC) vaccine is safe and effective in improving 24 and 36-month disease-free survival (DFS) in patients (pts) with resected stage III/IV melanoma who completed the primary vaccine series. The tumor lysate, particle only (TLPO) vaccine has been developed to accelerate production by omitting DC isolation and ex vivo loading in favor of in vivo phagocytosis of the TL-loaded particles. We are currently conducting a randomized and double-blind trial of the TLPO vs TLPLDC to improve DFS and overall survival (OS) in patients with resected late stage melanoma. Methods Patients with stage III/IV melanoma who were clinically disease-free after standard of care therapies were randomized to receive TLPO vs TLPLDC (2:1) as a continuation of the phase IIb trial comparing TLPLDC vs placebo (2:1). For the TLPLDC vaccine, autologous TL was loaded into yeast cell wall particles (YCWP) which were then phagocytized by isolated autologous DC ex vivo. For the placebo DC were loaded with empty YCWP. For TLPO, the autologous TL-loaded YCWP were coated with a chemoattractant and injected intradermally for in vivo phagocytosis. Some patients in the TLPLDC arm received G-CSF prior to DC harvest to minimize blood draw (60 mL instead of 120 mL without G-CSF). For all arms, six vaccine/placebo doses were administered intradermally at 0, 1, 2, 6, 12, and 18 mos. Data was analyzed by an intention-to-treat (ITT) analysis for DFS and OS by the Kaplan-Meier method and compared by log-rank test. Results 63 pts were randomized to TLPO (n=43) vs TLPLDC (n=20). The TLPO cohort contained more females and received less chemotherapy (0% vs 10%), but otherwise were comparable. There were no differences in DFS (p=0.948) or OS (p=0.779) between the two vaccines (figures 1&2). Comparing the TLPO pts to all other pts in the phase IIb trial [TLPLDC+G-CSF (n=57), TLPLDC-G-CSF (n=46), and placebo (n=41)] the TLPO arm had improved DFS compared to placebo (p=0.019) and TLPLDC+G-CSF (p=0.001), but roughly equivalent to the TLPLDC-G-CSF arm (p=0.276) (figure 3). A similar trend was seen in OS analysis, though differences were not statistically significant (figure 4). Conclusions TLPO and TLPLDC vaccines (without the use of G-CSF) improve DFS in patients with resected stage III/IV melanoma compared to placebo. The TLPO vaccine may offer advantages via reduced cost and vaccine production time. TLPO should be closely considered for further clinical trials. Trial Registration NCT02301611: Phase IIB TL + YWCP + DC in MelanomaTLPLDC IND#16101TLPO IND#17274 Ethics Approval This study was approved by WIRB; protocol #20141932

Background The tumor lysate, particle-loaded, dendritic cell (TLPLDC) vaccine is created ex vivo by loading autologous dendritic cells (DC) with yeast cell wall particles (YCWP) containing autologous tumor lysate, thus delivering tumor antigens to the DC cytoplasm via phagocytosis. TLPLDC then activates a robust T cell response against the unique antigens for each patient. The primary analysis of the prospective, randomized, multi-center, double-blind, placebo-controlled phase IIb trial in patients with resected stage III/IV melanoma showed TLPLDC improved 24-month disease-free survival (DFS) in the per-treatment (PT) analysis (patients completing the 6-month primary vaccine series). Here, we examine the secondary endpoint of 36-month DFS and overall survival (OS). Methods Patients with resected stage III/IV melanoma were randomized 2:1 to TLPLDC vaccine or placebo (autologous DC loaded with empty YCWP). Treatments were given at 0, 1, 2, 6, 12 and 18 months. The protocol was amended to include patients receiving concurrent checkpoint inhibitors (CPIs) to follow changes in standard of care. The co-primary endpoints were 24-month DFS by intention-to-treat (IT) analysis and per-treatment (PT) analysis, with secondary endpoints including 36-month DFS and OS by ITT and PT analysis, pre-specified analysis by stage, and safety as measured by CTCAE v4.03. Results Overall, 103 patients received TLPLDC and 41 placebo. In PT analysis, 65 patients received TLPLDC and 32 placebo. Total adverse events (AEs), grade 3+ AEs, and serious AEs (SAEs) were similar in placebo vs TLPLDC groups, with one related SAE per treatment arm. By ITT analysis, 36-month OS was 76.2% for TLPLDC vs 70.3% for placebo (HR 0.72, p=0.437) and 36-month DFS was 35.6% vs 27.1% (HR 0.95, p=0.841). By PT analysis, 36-month DFS was improved with TLPLDC (57.5% vs 35.0%; HR 0.50, p=0.025, figure 1). This effect was even more dramatic in resected stage IV patients (36-month DFS: 60.9% vs 0%; HR 0.12, p=0.001, figure 2). Conclusions This phase IIb trial again demonstrates the safety of the TLPLDC vaccine, and an improved 36-month DFS in patients with resected stage III/IV melanoma who complete the primary vaccine series, particularly in the stage IV subgroup. Next, a phase III trial will evaluate the efficacy of TLPLDC vaccine as adjuvant treatment for resected stage IV melanoma, with patients randomized to receive standard of care PD-1 inhibitors + TLPLDC versus PD-1 inhibitors + placebo. Trial Registration This is a phase IIb clinical trial registered under NCT02301611 Ethics Approval This study was approved by Western IRB, protocol 20141932.

HER2-targeted therapy has not benefited patients with low levels of HER2 expression; however, combination therapy may be effective. Primary analysis of a phase IIb trial investigating the HER2-derived vaccine nelipepimut-S (NPS) did not benefit the intention-to-treat population, but subset analysis showed a benefit in triple-negative breast cancer (TNBC) patients. The subset analysis of this multicenter, randomized, single-blind, phase IIb trial identified significant improvement in 36-month disease-free survival (DFS) between NPS (n = 55) and placebo (n = 44) in TNBC (HR 0.25, p = 0.01) and those who express HLA-A24 (HR 0.41, p = 0.05). The TNBC cohort demonstrated improved 36-month DFS in those with HER2 1+ expression (HR 0.17, p = 0.01), HLA-A24 positivity (HR 0.08, p 0.01), or in those who received neoadjuvant chemotherapy (HR 0.21, p 0.01). NPS vaccination with trastuzumab was associated with improved 36-month DFS among patients with TNBC. The observed benefit to this high-risk subgroup warrants confirmation in a phase III trial.

Cancer immunotherapy has largely been hindered by the ability of tumors to induce tolerance and escape the immune system. By targeting coinhibitory and costimulatory receptors in the immune system, it is possible to counteract the pathways that tumors utilize to avoid immune destruction. Ipilimumab, the first approved drug of this class, blocks the coinhibitory receptor CTLA-4 and has been shown to improve survival in advanced melanoma. Research into expanding the uses of CTLA-4 blockade as well as targeting alternative coinhibitory and costimulatory checkpoints is ongoing. Checkpoint blockade, particularly when combined with conventional therapies and cancer vaccines, has the potential to revolutionize cancer immunotherapy and provide new therapeutic options to a broad range of malignancies. This chapter will review the rationale for targeting immune checkpoints, the pathways of particular interest, and the research being conducted to bring checkpoint blockade to clinical use.