Abstract
Background
Immune checkpoint blockade is minimally active in unselected castration-resistant prostate cancer (CRPC) and does not reproducibly yield durable decreases in prostate-specific antigen (PSA) levels.
Methods
The Quick Efficacy Seeking Trial was designed to employ a combination of agents to initiate an immune response (with BN-Brachyury vaccine), potentiate that response (with nogapendekin-alfa inbakicept (NAI), an interleukin (IL)-15 receptor superagonist), and reduce or eliminate immunosuppressive entities in the tumor microenvironment (with bintrafusp alfa, a dual inhibitor of programmed death-ligand 1 and transforming growth factor beta). Epacadostat (an indoleamine 2,3-dioxygenase (IDO) inhibitor) was also employed in one cohort to reduce immune suppression induced by IDO’s conversion of tryptophan to kynurenine.
Results
Patients with CRPC enrolled sequentially to receive vaccine + bintrafusp alfa (Arm 2.1), vaccine + bintrafusp alfa + NAI (Arm 2.2), and vaccine + bintrafusp alfa + NAI + epacadostat (Arm 2.3), with the primary objective to determine response rate. Adverse events in Arms 2.1 and 2.2 were manageable and consistent with the safety profiles of each agent individually, and notable for five individuals developing isolated adrenocorticotropic hormone deficiency. Arm 2.3 was closed early due to skin toxicity. Sustained declines in PSA were seen in 1/13 (8%) patients in Arm 2.1, 7/24 (29%) patients in Arm 2.2, including six with proficient mismatch repair/microsatellite stable tumors, and 0/6 (0%) patients in Arm 2.3.
Conclusions
Analyses of peripheral immune profiles provided evidence of a multifaceted antitumor immune response, including IL-15 receptor superagonist NAI-dependent expansion and activation of natural killer cells and CD8+ T cells, increased effector-to-suppressor immune cell ratios, and induction of cytotoxic immune gene programs.
Trial registration number
Keywords: Prostate Cancer, Cytokine, Combination therapy, Immunotherapy, Vaccine
WHAT IS ALREADY KNOWN ON THIS TOPIC
Immune checkpoint blockade monotherapy has failed to produce reproducible benefit for patients with proficient mismatch repair (pMMR) castration-resistant prostate cancer (CRPC).
WHAT THIS STUDY ADDS
The Quick Efficacy Seeking Trial is the first in-human evidence suggesting that combination immunotherapy targeting transforming growth factor beta, programmed death-ligand 1, and tumor antigen, with an interleukin-15 receptor superagonist induces durable responses in pMMR CRPC.
HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY
To our knowledge, this is the first report of an active, multimodal immuno-oncology regimen in this difficult-to-treat population. Our findings provide the rationale for continued investigation of this combination treatment regimen directed against different arms of the immune system, and with the ability to modify the tumor cell phenotype and microenvironment, in patients with CRPC.
Introduction
There is no defined role of immune checkpoint blockade (ICB) immunotherapy in unselected patients with prostate cancer.1 Several studies, conducted in various treatment contexts, have failed to demonstrate reproducible clinical benefit or declines in prostate-specific antigen (PSA) levels,2–8 with the exception of patients with metastatic solid tumors with microsatellite instability high (MSI-H)/mismatch repair (MMR) deficient (dMMR) and/or high tumor mutational burden (≥10 mutations per megabase).9 10 However, individuals with MSI-H/dMMR or high mutational burden make up a small proportion of patients with metastatic castration-resistant prostate cancer (mCRPC) (approximately 5%).11 A growing pipeline of existing therapies and therapies in development hold promise to harness the immune system and direct it to eliminate prostate cancer cells.
Given evidence that therapeutic vaccines can generate tumor-specific immunity leading to improved clinical outcomes,12–14 we sought to develop a combination regimen employing various agents to initiate an immune response (with a vaccine), potentiate that response (with an immune stimulating interleukin (IL)-15 cytokine), and reduce or eliminate immunosuppressive entities in the tumor microenvironment (with ICB/transforming growth factor beta (TGF-β) inhibition, and indoleamine 2,3-dioxygenase (IDO) inhibition). To achieve this, we used a replication deficient poxviral vaccine BN-Brachyury (vaccine) targeting the transcription factor brachyury, which is key to invasion and metastasis of carcinoma cells,15 and in prostate cancer associates with poor prognostic features.16 For this combination study we selected three companion therapeutic agents—bintrafusp alfa (a dual inhibitor of programmed death ligand-1 (PD-L1) and TGF-β), NAI (also known as nogapendekin alfa inbakicept, an IL-15 receptor superagonist) and epacadostat (an IDO inhibitor)—for their ability to provide complementary immune enhancements as described previously.17 NAI was included for its ability to activate and expand natural killer (NK) and CD8+ T cells, bintrafusp alfa was selected to eliminate immune suppressive entities including TGF-β and PD-L1 in the tumor microenvironment, and epacadostat was used to reduce potential immune suppression induced by IDO’s conversion of tryptophan to kynurenine. This combination of agents also has the potential to alter tumor cell phenotype, rendering tumor cells more susceptible to lysis. Bintrafusp alfa, for example, via inhibition of TGF-β, has been shown to reverse tumor cell plasticity and increase the sensitivity of cancer cells to chemotherapy.18 Promising preclinical antitumor activity with various combinations of these classes of agents has been shown and provided the rationale for the Quick Efficacy Seeking Trial (QuEST1).19–23 Results from this adaptive design trial (NCT03493945) are presented here.
Methods
Trial design
This was a single-center study conducted at the National Institutes of Health (NIH) Clinical Center, Bethesda, Maryland, USA. The trial was approved by the NIH Institutional Review Board and conducted in accordance with the Declaration of Helsinki and Good Clinical Practice standards. All patients provided written informed consent prior to study enrollment. Using a standard 3+3 design, patients with metastatic solid tumors and measurable disease enrolled to a dose-finding arm (Arm 1.1) for NAI in combination with bintrafusp alfa. Via an adaptive design described in figure 1, patients with prostate cancer sequentially enrolled to Arms 2.1A, 2.2A and 2.3A, where they received BN-Brachyury vaccine + bintrafusp alfa in Arm 2.1A, BN-Brachyury vaccine + bintrafusp alfa + NAI in Arm 2.2A, and BN-Brachyury vaccine + bintrafusp alfa + NAI + epacadostat in Arm 2.3A. Part “A” (Arms 2.1A, 2.2A, 2.3A) preceded Part “B”. Per protocol, enrollment to each Part “B” arm (ie, Arm 2.1B, 2.2B or 2.3B) began only if the respective “A” arm had ≥2 clinical responses among the 13 patients enrolled, as depicted in figure 1.
Figure 1.
Trial schema for patients with prostate cancer receiving combination immunotherapy. In Part A, 13 participants with CRPC enrolled to Arm 2.1A. Following establishment of the safety of the Arm 2.1A regimen, 13 new participants with CRPC enrolled to Arm 2.2A. Following establishment of safety with the Arm 2.2 regimen, up to 13 new participants were eligible to enroll to Arm 2.3A, and 6 participants enrolled before enrollment to Arm 2.3A was stopped due to skin toxicity. Part B began after completion of Arm 2.3A. In Part B, 12 participant expansion arms (ie, Arms 2.1B, 2.2B, and 2.3B) were opened for the corresponding treatment regimen if that regimen showed clinical activity (≥2 responses) in Part A. All participants remained on the study arm they were assigned to at enrollment and did not change treatment regimens. CRPC, castration-resistant prostate cancer; NAI, nogapendekin-alfa inbakicept; PSA, prostate-specific antigen; QuEST1, Quick Efficacy Seeking Trial.
For the prostate cancer Arms (2.1, 2.2, and 2.3), patients were eligible if ≥18 years old with an Eastern Cooperative Oncology Group performance status of ≤1, normal organ and bone marrow function, and histologically or cytologically proven prostate cancer that is castration-resistant, that is, testosterone levels <50 ng/dL or 1.7 nmol/L despite androgen-deprivation therapy (ADT). Eligible individuals had radiographically proven metastases or PSA progression defined as rising values separated by >1 week, that is, two separate increasing values over a minimum of 1 ng/mL (Prostate Cancer Working Group 3 PSA eligibility criteria). Patients with CRPC continued ADT (if not status post-bilateral orchiectomy) and per eligibility were minimally symptomatic/asymptomatic and did not require regular use of narcotic analgesics. Patients on chronic immunosuppression within 28 days of enrollment, positive for HIV, or with active autoimmune disease were excluded. Patients with type 1 diabetes mellitus, vitiligo, psoriasis, hypothyroid or hyperthyroid disease not requiring concurrent immunosuppression, or with other endocrine disorders on replacement hormones were not excluded if the condition(s) were well controlled. Patients with mCRPC with a history of brain/leptomeningeal metastases were excluded. Eligible patients included those who were ≥28 days post major surgery and in receipt of other investigative or chemotherapeutic oncologic agents or radiation treatment, excluding bone-directed palliative radiotherapy. Concurrent use of agents that can decrease PSA (eg, saw palmetto) was prohibited in all patients with CRPC. A protocol amendment approved in 2021 allowed individuals progressing on an androgen receptor pathway inhibitor (ARPI) to continue such agents (eg, abiraterone acetate plus prednisone, enzalutamide, and darolutamide) on study.
Bintrafusp alfa 1,200 mg was administered intravenously on day 1 and day 15 of each 28-day cycle. The BN-Brachyury vaccine platform was administered subcutaneously (sc) as a priming dose (modified vaccinia Ankara (MVA)-BN-Brachyury 2.0×108 international unit (IU)) on day 1 of both cycle 1 and cycle 2, followed by booster doses (fowlpox virus-brachyury 1.0×109 IU sc) started 2 weeks after the second dose of MVA-BN-Brachyury, then every 4 weeks until 6 months, then every 3 months until reaching 2 years from the first dose of MVA-BN-Brachyury. NAI was given sc on the abdomen on days 1 and 15 of each 28-day cycle. All patients with prostate cancer who received NAI (Arms 2.2 and 2.3) received 15 µg/kg sc on days 1 and 15 of each cycle. De-escalation of NAI dosing was allowed for NAI-attributed toxicities per investigator discretion (online supplemental table S1). Epacadostat was administered at a dose of 600 mg orally two times per day (Arm 2.3 only).
jitc-14-7-s001.pdf (1.9MB, pdf)
The primary objective was to determine the clinical benefit (ie, response rate) for each of the regimens given in a respective arm (figure 1). For patients with prostate cancer, response was defined as partial response (PR) or complete response (CR) by Response Evaluation Criteria in Solid Tumors (RECIST) V.1.1 (if RECIST V.1.1–measurable disease was present at baseline) and/or by PSA decrease of ≥30% from baseline sustained for >21 days. CT imaging (and technetium bone scan for prostate cancer patients only) was obtained at baseline and every 3 months for restaging. Secondary endpoints were progression-free survival (PFS) for each given treatment regimen and safety of each treatment regimen assessed by Common Terminology Criteria for Adverse Events V.5.0. Exploratory analyses focused on peripheral immune modulation as a mechanistic correlate of clinical activity. Some participants were followed for long-term outcomes on a separate clinical protocol (NCT00451022).
Peripheral immune analysis
Peripheral blood was collected at baseline and at serial time points during treatment. Blood collected in serum separator tubes was centrifuged, and the resulting serum was stored at −80°C. Blood was also collected in sodium heparin tubes and peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque density gradient separation. PBMCs were cryopreserved in 90% heat-inactivated human AB serum supplemented with 10% dimethyl sulfoxide and stored in liquid nitrogen until use. Peripheral immune analyses were performed in 13 patients in Arm 2.1A and 24 patients in Arm 2.2A&B before and after treatment with available research samples. Tumor-associated antigen (TAA)-specific T cells to the vaccine target antigen brachyury and the prostate cancer-associated “cascade” antigen mucin-1 (MUC-1) were measured and analyzed in PBMCs using methods previously described.24 Serum levels of IL-8, IL-6, interferon gamma (IFN-γ), tumor necrosis factor-alpha (TNFα), IL-10, programmed cell death protein-1 (PD-1), TGF-β, granzyme B, and CD27 were analyzed as previously described.25 Frequencies of 158 PBMC subsets were determined and calculated as percent of total PBMCs as previously described, with the gating strategy outlined in online supplemental figure S1.24 Complete blood counts with differential were performed at the National Cancer Institute’s Center for Cancer Research (CCR). Bulk RNA sequencing (RNA-seq) of PBMCs was performed in a subset of patients who were also enrolled on a biospecimen collection protocol (NCT00034216) (n=6 in Arm 2.1A and n=6 in Arm 2.2A&B) at baseline and 2 weeks on treatment by the CCR Sequencing Facility (Frederick, Maryland, USA). Details of this analysis are described in online supplemental methods.
Statistical analyses
The survival and survminer packages in R were used to plot Kaplan-Meier curves, and confidence intervals (CIs) for PFS and overall survival (OS) were calculated with the Brookmeyer-Crowley method. Statistical analyses of frequencies of peripheral immune cells and levels of serum analytes were performed using RStudio (RStudio V.2024.04.1+748 for Macintosh, Boston, Massachusetts, USA) and GraphPad Prism (GraphPad Prism V.0.4.0 for Macintosh, San Diego, California, USA). Differences in peripheral immune cells and serum analytes between two groups were analyzed for statistical significance using a Mann-Whitney U test, with p value <0.05 considered statistically significant. For paired analyses of RNA-seq data assessing the change in each arm from baseline to 2 weeks, differentially expressed genes with a p value <0.05 and a fold change threshold of >1.2 were used to select the gene list for pathway analysis, and p values for enriched pathways were calculated by Fisher’s exact test. For RNA-seq analyses assessing the difference between responding and non-responding patients at baseline and of the change from baseline to 2 weeks, differentially expressed genes with a p value <0.1 and a fold change threshold of >1.2 were used to select the gene list for pathway analysis, and p values for enriched pathways were calculated by Fisher’s exact test. All p values were two-tailed and reported without adjustment for multiple comparisons in this hypothesis-generating study.
Results
Patient characteristics
A total of 58 patients enrolled and received treatment from April 2018 through February 2024. Arm 1.1 was designed to define the recommended phase 2 dose (RP2D) of NAI in combination with bintrafusp alfa, and enrolled 14 individuals with solid tumors, irrespective of tumor type. There were no dose-limiting toxicities in Arm 1.1 and the RP2D of NAI for use in combination with bintrafusp alfa was 15 µg/kg sc. The total number of individuals treated on each prostate cancer arm was as follows: Arm 2.1A (n=13), Arm 2.2A (n=13), Arm 2.2B (n=12), Arm 2.3A (n=6) (figure 1). Due to lack of efficacy signal in Part A for both Arm 2.1A and Arm 2.3A, no patients enrolled to Arm 2.1B and Arm 2.3B. Data cut-off for survival analyses was April 2024. Median follow-up for patients with prostate cancer by reverse Kaplan-Meier method was 32.3, 42.6 and 43 months for Arm 2.1A, Arm 2.2A&B and Arm 2.3A, respectively. Baseline characteristics of patients with prostate cancer are listed by arm in table 1.
Table 1.
Patient characteristics in Arms 2.1, 2.2 and 2.3
| Characteristic* | Treatment | ||
| Doublet Arm 2.1A (n=13) | Triplet Arm 2.2A&B (n=25) | Quadruplet Arm 2.3A (n=6) |
|
| Median age in years (range) | 66 (47–82) | 66 (55–79) | 75 (61–82) |
| Sites of disease | |||
| Non-metastatic | 2 | 1 | 0 |
| Lymph node | 3 | 13 | 3 |
| Visceral | 1 | 2 | 1 |
| Bone | 8 | 23 | 6 |
| Bone only | 4 | 11 | 2 |
| Median Gleason score (range) | 8 (7–9) | 9 (7–10) | 9 (5–10) |
| 5 | 0 | 0 | 1 |
| 6 | 0 | 0 | 0 |
| 7 | 2 | 4 | 1 |
| 8 | 6 | 8 | 1 |
| 9 | 4 | 9 | 2 |
| 10 | 0 | 3 | 1 |
| Unknown | 1 | 1 | 0 |
| Median pretreatment PSA (ng/dL) (range) | 9 (1.97–306.3) | 18 (2.68–1000) | 189 (2.6–507) |
| Naïve to treatment for CRPC | 9 | 6 | 2 |
| Prior ARPI | 4 | 19 | 5 |
| Prior chemotherapy for CRPC | 1 | 4 | 1 |
| Prior sipuleucel-T | 0 | 4 | 0 |
| Prior, other experimental vaccine | 4 | 2 | 0 |
| Prior anti-PD-1/PD-L1 | 0 | 2 | 0 |
| Continued ARPI on trial | 0 | 6 | 0 |
| # with baseline LDH ≥ULN | 2 | 12 | 2 |
| # with baseline ALP ≥ULN | 1 | 6 | 1 |
| # with baseline albumin <35 g/L | 0 | 0 | 0 |
*Characteristics at baseline prior to treatment initiation.
ALP, alkaline phosphatase; ARPI, androgen receptor pathway inhibitor; CRPC, castration-resistant prostate cancer; LDH, lactate dehydrogenase; PD-1, programmed cell death protein 1; PD-L1, programmed death-ligand 1; PSA, prostate-specific antigen; ULN, upper limit of normal.
Safety
Safety results from Arm 1.1 (dose escalation of NAI for RP2D identification when used in combination with bintrafusp alfa) are depicted in online supplemental table S2. As four patients needed to be replaced on dose level (DL) 2 due to missed doses of medication unrelated to toxicity, a total of 10 individuals received DL2. A dose of 15 µg/kg was identified as RP2D for NAI in combination with bintrafusp alfa for evaluation in the prostate cancer arms.
Treatment-related adverse events (TRAEs) ≥ Grade 2 for patients with prostate cancer treated in Arms 2.1, 2.2A&B, and 2.3A are shown (table 2). TRAEs of all grades for these patients are listed in online supplemental tables S3–S5. Adverse events were consistent with the known safety profile of each individual agent, with the exception of five individuals who developed isolated adrenocorticotropic hormone deficiency (IAD) in Arm 2.1A (n=1), Arm 2.2A (n=3) and Arm 2.3A (n=1). This will be discussed below. All except one of these individuals who developed IAD (in Arm 2.3A) developed clinical responses, defined as PR or CR by RECIST V.1.1 (if measurable disease present at baseline) or PSA decrease of ≥30% from baseline sustained for >21 days. Four of the seven individuals with clinical responses in Arm 2.2A&B did not develop IAD on study. In all cases, IAD was medically manageable with oral hydrocortisone at physiologic replacement doses. In Arm 2.3A, 3/6 individuals experienced Grade 3 skin toxicity and accrual to Arm 2.3A was discontinued following an ad hoc interim analysis.
Table 2.
Treatment-related adverse events >Grade 2 in Arm 2.1A, Arm 2.2A/B, and Arm 2.3A
| TRAE | Doublet Arm 2.2A (n=13) |
Triplet Arm 2.2A&B (n=25) |
Quadruplet Arm 2.3A (n=6) |
|||
| Grade 3 | Grade 4 | Grade 3 | Grade 4 | Grade 3 | Grade 4 | |
| N (%) | N (%) | N (%) | N (%) | N (%) | N (%) | |
| Anemia | 0 | 0 | 1 (4) | 0 | 2 (33.3) | 0 |
| Blood and lymphatic system disorders - other, specify: immune-mediated neutropenia | 0 | 0 | 1 (4) | 1 (4) | 0 | 0 |
| Cystitis non-infective | 0 | 0 | 1 (4) | 0 | 0 | 0 |
| Endocrine disorders - other, specify: isolated ACTH deficiency | 1 (7.6) | 0 | 0 | 0 | 0 | 0 |
| Eosinophilia | 0 | 0 | 1 (4) | 0 | 0 | 0 |
| Hematuria | 0 | 0 | 1 (4) | 0 | 1 (16.7) | 0 |
| Lipase increased | 0 | 0 | 0 | 0 | 1 (16.7) | 0 |
| Metabolism and nutrition disorders - other, specify: immune-mediated diabetes | 0 | 0 | 1 (4) | 0 | 0 | 0 |
| Pancreatitis | 1 (7.6) | 0 | 0 | 0 | 0 | 0 |
| Pruritus | 0 | 0 | 0 | 0 | 1 (16.7) | 0 |
| Rash maculopapular | 0 | 0 | 0 | 0 | 2 (33.3) | 0 |
| Tumor hemorrhage | 1 (7.6) | 0 | 0 | 0 | 0 | 0 |
TRAEs in patients who received BN-Brachyury vaccine + bintrafusp alfa on Arm 2.1A, BN-Brachyury vaccine + bintrafusp alfa + NAI on Arm 2.2A or 2.2B, or BN-Brachyury vaccine + bintrafusp alfa + NAI + epacadostat on Arm 2.3A. If a patient experienced multiple grades of the same event, each grade of that event is represented. If a patient experienced the same grade of an event multiple times, that TRAE is represented only once. There were no Grade 5 TRAEs.
ACTH, adrenocorticotropic hormone; NAI, nogapendekin alfa inbakicept; TRAEs, treatment-related adverse events.
Clinical activity
In Arm 1.1, where patients with advanced solid tumors received dose escalation of NAI with bintrafusp alfa, one of seven microsatellite stable (MSS)/proficient mismatch repair (pMMR) individuals with metastatic colorectal cancer who enrolled on DL2 had a durable partial response by RECIST V.1.1. There were no other objective responses in this arm.
The rest of the patients enrolled in this study had prostate cancer and enrolled to Arms 2.1A, 2.2A, 2.3A, or to Arm 2.2B. Arm 2.2B was the only arm in Part “B” that opened per prespecified efficacy thresholds in Part “A”. PSA response and best overall response for patients with prostate cancer in Arms 2.1A, Arm 2.2A, Arm 2.3A, and Arm 2.2A&B by RECIST V.1.1 are summarized in table 3. Mean and median time on treatment by arm is summarized in online supplemental table S6. One individual with pMMR disease who received vaccine plus bintrafusp alfa in Arm 2.1A had a PSA response durable for 13 months (figure 2A,B, table 3). This did not satisfy the predefined threshold for efficacy in Arm 2.1 (ie, ≥2 clinical responses). Arm 2.1B did not open for enrollment and the regimen was not tested further. For Arm 2.1A, median PFS was 4.6 months (95% CI 1.70 months to not estimable), 6-month PFS was 35.4% (95% CI 9.7% to 63.1%) and 12-month OS was 92.3% (95% CI 56.6% to 98.9%) (online supplemental figure S2A,B).
Table 3.
Composite responses by PSA and RECIST V.1.1 response
| Treatment | Number of patients | Number with PSA response | Number with RECIST V.1.1 measurable disease at enrollment | BOR by RECIST V.1.1* | ||||
| PD | SD | PR | CR | Not evaluable for BOR by RECIST V.1.1 | ||||
| BN-Brachyury + bintrafusp alfa (Arm 2.1A) | 13 | 1 | 3 | 4 | 8 | 0 | 0 | 1 |
| BN-Brachyury + bintrafusp alfa + NAI (Arm 2.2A) | 13 | 6 | 2 | 4 | 7 | 2 | 0 | 0 |
| BN-Brachyury + bintrafusp alfa + NAI + epacadostat (Arm 2.3A) | 6 | 0 | 1 | 2 | 2 | 0 | 0 | 2 |
| BN-Brachyury + bintrafusp alfa + NAI (Arm 2.2A&B) |
24† | 7‡ | 6 | 13 | 9 | 2 | 0 | 1 |
The table depicts the number of patients with PSA declines ≥30% of baseline, sustained for >21 days and best overall response.
*Includes individuals with measurable and non-measurable disease at baseline.
†One individual on Arm 2.2B withdrew 1 week after enrolling to pursue a standard treatment newly available to him and is not evaluable for PSA response.
‡One individual was dMMR (PMS2 loss on IHC); all other responders had pMMR disease.
BOR, best overall response; CR, complete response; dMMR, mismatch repair deficient; IHC, immunohistochemistry; NAI, nogapendekin alfa inbakicept; PD, progressive disease; pMMR, proficient mismatch repair; PR, partial response; PSA, prostate-specific antigen; RECIST, Response Evaluation Criteria in Solid Tumors; SD, stable disease.
Figure 2.
Maximal PSA decreases from baseline and PSA spider plots in patients with prostate cancer receiving combination immunotherapy. Waterfall plots depict maximal PSA decline compared with baseline ordered from worst to best responder left to right for patients in Arm 2.1A treated with BN-Brachyury vaccine + bintrafusp alfa (A), in Arm 2.2A treated with BN-Brachyury vaccine + bintrafusp alfa + NAI (C), in Arm 2.3A treated with BN-Brachyury vaccine + bintrafusp alfa + NAI + epacadostat (E), and in Arm 2.2A&B treated with BN-Brachyury vaccine + bintrafusp alfa + NAI (G). Brown dotted line indicates a 30% reduction of PSA compared with baseline. For waterfall plots, green bars indicate individuals naïve to treatment for CRPC, black bars indicate individuals who previously received chemotherapy for CRPC, and red bars indicate individuals who previously received ARPI for CRPC. For individuals with known tumor MMR status, purple dots indicate dMMR disease and blue dots indicate pMMR disease on waterfall plots. On spider plots, coloring of the dots to purple and blue indicates dMMR and pMMR disease, respectively. The individual represented by the gray bordered bar (G) had a somatic CDK12 mutation. Duration of PSA response is indicated on waterfall plots. A black +sign indicates that a patient’s response was ongoing at the time of starting another treatment and was no longer evaluable for duration of response (C, G). A blue +sign indicates ongoing response at the time of data cut-off (G). The individual with an “x” on his bar (C, G) indicates that maximal PSA response occurred after receiving palliative radiation to bone metastases. The individual with an asterisk (*) experienced a PSA decline after receiving palliative radiation to bone metastases (E). Spider plots depict PSA at baseline and subsequent changes on treatment for patients in Arms 2.1A (B), 2.2A (D), 2.3A (F), and 2.2A&B (H). Y-axis represents percent change in PSA compared with baseline (% change=0 at month=0). X-axis represents time in weeks from the initiation of treatment, up to 200 weeks. Brown dotted line indicates a 30% reduction of PSA compared with baseline. Green lines on spider plots indicate individuals naïve to treatment for CRPC. Black lines on spider plots indicate individuals who previously received chemotherapy for CRPC. Red lines on spider plots indicate individuals who previously received ARPI for CRPC. ADT, androgen deprivation therapy; ARPI, androgen receptor pathway inhibitor; CRPC, castration-resistant prostate cancer; dMMR, mismatch repair deficient; MSI, microsatellite instability; NAI, nogapendekin alfa inbakicept; pMMR, proficient MMR; PSA, prostate-specific antigen.
Six out of 13 patients treated in Arm 2.2A with vaccine + bintrafusp alfa + NAI had a PSA response (figure 2C,D, table 3), including two individuals with RECIST V.1.1-measurable disease who developed PRs. Imaging and PSA curves from the two patients developing PRs are shown in online supplemental figure S3. For Arm 2.2A, median PFS was 4.24 months (95% CI 2.70 months to 10.9 months), 6-month PFS was 35.0% (95% CI 16.4% to 54.3%) and 12-month OS was 91.2% (95% CI 68.8% to 97.7%) (online supplemental figure S2C,D).
With the exception of one participant who experienced a sustained PSA decline after receiving palliative radiation to bone metastases, there were no PSA responses (figure 2E,F, table 3) or RECIST V.1.1 responses in the six individuals who received treatment in Arm 2.3A with vaccine + bintrafusp alfa + NAI + epacadostat before its closure due to toxicity. Six-month PFS was not estimable and 12-month OS was 83.3% (95% CI 27.3% to 97.5%) (online supplemental figure S2E,F).
Due to satisfaction of the threshold for efficacy in Arm 2.2A (ie, ≥2 clinical responses), an expansion arm (Arm 2.2B) opened to increase the number of patients treated with vaccine + bintrafusp alfa + NAI. In total, 25 patients received this triplet regimen and 24 were evaluable for clinical response. Seven of 24 (29%) had PSA responses (figure 2G,H, table 3), including two individuals with RECIST V.1.1-measurable disease who had PRs. Characteristics of the patients who had responses following treatment with vaccine + bintrafusp alfa + NAI are listed in online supplemental table S7. Apart from one individual with loss of the MMR gene PMS2 as measured by immunohistochemistry (IHC), all of these responding individuals were pMMR/MSS by either IHC or next generation sequencing. One of the responding individuals had a germline CDK12 mutation and had not previously received ICB therapy. For responders who were treated with vaccine + bintrafusp alfa + NAI (Arm 2.2A&B), median duration of response was 13.8 months (95% CI 2.5 months to not estimable). For patients who were treated with vaccine + bintrafusp alfa + NAI (Arm 2.2A&B), median PFS was 4.24 months (95% CI 2.7 months to 10.9 months), 6-month PFS was 35.0% (95% CI 16.4% to 54.3%) and 12-month OS was 91.2% (95% CI 70.0% to 97.8%) (online supplemental figure S2G,H).
Peripheral immune analyses
Antigen-specific CD4+ and CD8+ T-cell responses to the vaccine target antigen brachyury, and the “cascade” antigen MUC-1 to define antigen spreading, were evaluated in PBMCs for patients in Arm 2.1A and Arm 2.2A&B at baseline, 2 weeks and 10 weeks on treatment following in vitro stimulation of PBMCs with peptide pools of tumor-associated antigens followed by intracellular cytokine staining in CD4+ and CD8+ T cells. The absolute number of T cells at each time point producing IFN-γ, TNFα, IL-2, or positive for the degranulation marker CD107a, or positive for two or more of these markers per 1×106 PBMCs is shown in online supplemental tables S8 and S9. The majority of patients developed T-cell responses against brachyury (17/31, 55%) and MUC-1 (19/31, 61%) after therapy (online supplemental figure S4), with many of these responses being multifunctional (ie, positive for two or more of the functional markers evaluated, online supplemental figure S5). Example flow cytometry plots demonstrate a developed CD4+ T-cell response against brachyury peptides (online supplemental figure S6) and MUC-1 peptides (online supplemental figure S7) at 10 weeks of treatment, including multifunctional T cells producing both IFN-γ and TNFα after peptide stimulation. As discussed below, no major differences in the development of antigen-specific T-cell responses were noted in Arm 2.1 and Arm 2.2.
We next compared peripheral immune cell subsets, levels of soluble factors, and gene expression profiles of PBMCs in patients enrolled in Arms 2.1A and 2.2A&B to evaluate the contribution of NAI to the combination therapy. While no major differences were observed at baseline between patients in Arm 2.1 and Arm 2.2, multiple differences in the peripheral immune system were noted 2 weeks after treatment was initiated. Patients in Arm 2.2 (receiving NAI) had a greater increase in absolute lymphocyte count (ALC) after 2 weeks of treatment compared with patients in Arm 2.1 (p<0.0001) (figure 3A). Patients in Arm 2.2 also had a greater increase in total NK cells after 2 weeks with a median increase of 238%, compared with 30% for patients in Arm 2.1 (p<0.0001) (figure 3B). A greater increase in mature NK cells (p<0.0001) and NK cells expressing the activating receptors NKp46 (p<0.0001), CD226 (p=0.0003) and NKp30 (p=0.0010) was also measured in Arm 2.2 compared with Arm 2.1 (figure 3B). Representative flow cytometry plots showing quantification of total NK cells, mature NK cells, and NKp46+ NK cells at baseline and 2 weeks are shown for patients in both treatment arms (online supplemental figure S8). After 2 weeks, combination therapy that included NAI also resulted in some minor decreases in other classic cell types, including total CD4+ T cells, plasmacytoid dendritic cells, B cells, regulatory T cells (Treg), conventional dendritic cells, and myeloid-derived suppressor cells (MDSC), while total CD8+ T cells, NKT cells, and monocytes were not differentially changed (online supplemental figure S9A). Greater increases in the ratios of effector cell to suppressor cell subsets, including the ratio of CD8 to Treg (p=0.0400), NK to Treg (p=0.0001) and NK to MDSC (p<0.0001) were noted for patients in Arm 2.2 compared with Arm 2.1 (figure 3C). Patients in Arm 2.2 who received NAI also had a greater increase in serum granzyme B at 2 weeks than patients in Arm 2.1 (p=0.0014) (figure 3D), along with trending greater increases in soluble CD27, a measure of enhanced T-cell activation, and IFN-γ (online supplemental figure S9B). Quantification of paired changes in values prior to calculation of percent change showed similar findings (online supplemental figure S10). In gene expression analyses of PBMCs, patients in Arm 2.2 demonstrated increases at 2 weeks in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway “Natural killer cell mediated cytotoxicity” (p<0.0001) (figure 3E) and the Gene Ontology (GO) pathway “Regulation of T cell mediated cytotoxicity” (p=0.0289) (figure 3F), while neither of these pathways was enhanced after therapy for patients in Arm 2.1. Taken together, these results show statistical differences in both cellular and soluble immune responses between patients treated in Arm 2.1 and Arm 2.2.
Figure 3.
Differences in peripheral immune cells and serum analytes after 2 weeks of treatment between patients enrolled in Arms 2.1A and 2.2A&B. Differences in PBMC subsets and serum analytes after 2 weeks of treatment between patients enrolled in Arms 2.1A and 2.2A&B were compared to assess the contribution of nogapendekin-alfa inbakicept to the combination therapy. Differences in the change in complete blood counts (A), NK cell subsets (B), ratios of effector cells to suppressor cells (C), and serum analytes (D) at 2 weeks compared with baseline are shown between patients in Arms 2.1A and 2.2A&B. PBMC subsets were calculated as a percentage of PBMC before percent change was determined. Differences in gene expression profiles at baseline (Pre) and 2 weeks (W2) for patients on Arms 2.1A and 2.2A&B, contributing to the Kyoto Encyclopedia of Genes and Genomes pathway “Natural killer cell mediated cytotoxicity” (E) and the Gene Ontology pathway “Regulation of T cell mediated cytotoxicity” (F). The z-scores for gene expression levels were calculated by normalizing the gene expression values across samples. P values for box plots in A–D were calculated using a Mann-Whitney U test. Red dots indicate patients who responded to treatment. P values for RNA-sequencing data in E–F were calculated using a paired Fisher’s exact test. P value <0.05 was considered statistically significant. MDSC, myeloid-derived suppressor cells; NK, natural killer; NS, not significant; PBMC, peripheral blood mononuclear cells; Treg, regulatory T cells.
Differences in serum analytes, frequencies of immune cell subsets, and gene expression profiles of PBMCs at baseline and during treatment were also compared between clinical responders and non-responders who received vaccine + bintrafusp alfa + NAI (in Arm 2.2A&B) (figure 4). Responding patients had higher frequencies of CD4+ and CD8+ T cells expressing the costimulatory molecule 4–1BB at baseline (p=0.0106 and p=0.0054, respectively), with enhanced levels persisting at 2 weeks (p=0.0149 and p=0.0225, respectively) (figure 4A). Responding patients also had lower frequencies of immunosuppressive subsets, including MDSCs at baseline (p=0.0337), which persisted at 10 weeks (p=0.0420), and trending lower levels of Tregs at baseline and after 2 weeks of treatment (figure 4B). Responding patients who were treated with vaccine + bintrafusp alfa + NAI also had higher ratios of effector T cell to suppressor cell immune subsets at baseline, including CD4 to Treg (p=0.0106), CD8 to Treg (p=0.0195), CD4 to MDSC (p=0.0195) and CD8 to MDSC (p=0.0400), compared with non-responders, with differences in several of these ratios persisting throughout treatment (figure 4C). Although all patients in Arm 2.2 experienced increases in NK cell frequencies after 2 weeks of treatment, levels of immature NK cells expressing the activating receptors NKp30 (p=0.0240) and NKp46 (p=0.0225) were higher at 10 weeks in responders than non-responders (figure 4D). While no serum cytokines evaluated here were different between responders and non-responders at baseline, there were trending higher IFN-γ levels at 10 weeks in responding than non-responding patients (figure 4E), and a significantly greater increase in IFN-γ at 10 weeks compared with baseline for responders than non-responders (p=0.0030, figure 4F). Gene expression analyses of PBMCs supported the immune cell findings obtained through flow cytometry-based analyses, with patients responding to vaccine + bintrafusp alfa + NAI having enhancement in the GO pathway “Immune response” (p<0.0001) and the KEGG “T cell receptor signaling pathway” (p=0.0001) at baseline compared with non-responders (figure 4G). A greater increase in the KEGG pathway “Natural killer cell mediated cytotoxicity” (p=0.0257) was also seen at 2 weeks compared with baseline for responding patients than non-responding patients, further supporting NK-cell involvement in the therapeutic efficacy of this combination (figure 4H). Collectively, these findings show multiple differences in the cellular immune profile of clinical responders and non-responders at baseline who are treated with vaccine + bintrafusp alfa + NAI, with some of these differences persisting throughout treatment.
Figure 4.
Differences in PBMC subsets and serum analytes at baseline, 2 weeks, and 10 weeks between responding and non-responding patients enrolled in Arm 2.2A&B. Differences in frequencies of T-cell subsets (A), suppressive cell subsets (B), ratios of effector cells to suppressor immune cells (C), NK cell subsets (D), and IFN-γ serum levels (E) at baseline, 2 weeks, and 10 weeks in R and NR patients in Arm 2.2A&B. The percentage change in IFN-γ from baseline to 10 weeks between R and NR patients is shown (F). PBMC subsets were calculated as a percentage of PBMC. Differences in gene expression profiles at baseline, contributing to the Gene Ontology pathway “Immune response” and the KEGG pathway “T cell receptor signaling pathway” (G). Difference in the change in gene expression profiles at 2 weeks compared with baseline, contributing to the KEGG pathway “Natural Killer Cell Mediated Cytotoxicity” (H). The z-scores for gene expression levels were calculated by normalizing the gene expression values across samples. P values for box plots in A–F were calculated using a Mann-Whitney U test. P values for RNA-sequencing data in G–H were calculated using a Fisher’s exact test. P value <0.05 was considered statistically significant. IFN, interferon; KEGG, Kyoto Encyclopedia of Genes and Genomes; MDSC, myeloid-derived suppressor cells; NK, natural killer; NR, non-responding; PBMC, peripheral blood mononuclear cells; R, responding; Treg, regulatory T cells.
Discussion
ICB monotherapy has failed to produce reproducible benefit for patients with pMMR CRPC.26 27 We sought to interrogate a set of three rationally designed immunotherapy regimens that included ICB to generate immune-mediated antitumor activity, potentiate that response, and eliminate immunosuppressive entities in the tumor microenvironment of patients with CRPC. The doublet of vaccine + bintrafusp alfa had minimal clinical activity (1/13 responses). The addition of the IL-15 receptor superagonist NAI to vaccine and bintrafusp alfa resulted in an increased frequency of clinical responses (7/24) in individuals with CRPC receiving the triplet regimen. Six of these individuals notably had pMMR disease. A composite response rate of 26% (ie, in 6 patients with pMMR disease out of 23 unselected patients) is notably higher than previous reports of single-agent ICB in patients with unselected CRPC, where efficacy has been limited.2 3 6
To our knowledge, this is the first in-human evidence suggesting that while a vaccine and immune checkpoint inhibition alone may be insufficient to produce significant clinical activity, the addition of an immunocytokine—which increases the number and functional capacity of NK cells and T cells—may lead to meaningful clinical activity. The arm that added epacadostat to vaccine, bintrafusp alfa, and NAI closed early due to skin toxicity. This early closure and resulting small sample size limit the assessment of this combination’s efficacy.
The majority of patients receiving vaccine + bintrafusp alfa or vaccine + bintrafusp alfa + NAI developed antigen-specific T cells to the vaccine encoded antigen brachyury, as well as to a prostate TAA not encoded by the vaccine, MUC-1, indicating a cascade T-cell response. The development of TAA-specific T-cell responses, however, did not differ between treatment arms, and was not associated with clinical response. This is likely due to other pharmacodynamic effects on the immune system from the triplet combination therapy that may contribute to clinical responses, including a robust increase in NK cell number and activity with NAI and reduction of immune suppressive entities with bintrafusp alfa. Moreover, the potential induction of T-cell responses against neoepitopes, which may substantially contribute to the antitumor activity observed with this combination therapy, could not be evaluated in the current study due to lack of tumor biopsies.28 Deep interrogation of the peripheral immune system indeed revealed multiple differences between treatment arms when NAI was added to the regimen, and identified several immune parameters that differed among clinical responders and non-responders both before and throughout the course of treatment. Greater increases in ALC, frequencies of total and activated NK cells, ratios of effector (NK and CD8) to suppressor cells, serum granzyme B, and gene expression profiles related to NK and T-cell cytotoxicity were noted following patients receiving the triplet therapy that included NAI. It should be noted that changes in frequencies of a given population may be influenced by expansion or contractions of other cell types, and our experimental flow cytometry setup, performed in cryopreserved PBMCs and without inclusion of counting beads, limited our ability to assess absolute quantification of immune cell subset numbers, which may provide further biologically relevant findings. Our findings, however, are consistent with previous reports of NAI enhancing both the frequency and number of NK and CD8+ T cells, both as a monotherapy29–31 and in combination with ICB.32 These analyses revealed changes in the peripheral immune cell compartment with NAI not previously reported, including a robust activation of mature NK cells and NK cells expressing activating receptors, and a further adjustment of the immune system towards enhanced effector and lessened suppressor immunity. Overall, the data collectively show that the addition of NAI increases the number of activated T cells in the context of PD-L1 blockade and vaccination and induces a more robust activation of NK cells.
Baseline peripheral immune profiles, including different frequencies of T-cell subsets, have been associated with clinical response to ICB in multiple other studies (reviewed in33), indicating that easily accessible immune features from patients’ blood can give insights into the likelihood of who may benefit from treatment. In the current study, peripheral immune analyses linked to clinical response with the triplet therapy (Arm 2.2, figure 4) suggest that a more primed immune profile at baseline, supported by enhanced T-cell activation and reduced suppressor cells, provides a favorable environment for response. The robust increase in NK cells induced with NAI may also contribute to the antitumor activity of this agent in combination therapies by providing a means for immune-mediated killing that is functionally independent of intact antigen processing machinery. NKp30 and NKp46 are NK cell receptors that recognize ligands upregulated by tumors, allowing for NK cell activation and NK cell-mediated clearance of tumor cells.34 Immature NK cells can differentiate into cytotoxic NKs on activation, and enhanced levels at later on-treatment time points may represent an ongoing replenishment of the NK cell pool in responding patients.35 Furthermore, IFN-γ is a master regulator cytokine produced by both activated CD8+ T cells and NK cells that promotes tumor cell killing.36 The greater increase in this cytokine at later time points in responders than non-responders illustrates the continued engagement of this compartment of the immune system in patients responding to the triplet therapy. These findings provide evidence that manipulation of multiple components of the immune system via rationally designed combination immunotherapy regimens can yield promising antitumor activity in patients with CRPC via immunomodulation.
At the time of this trial’s inception, the investigational plan was that if the trial was successful, a subsequent study would be conducted to deconvolute any clinical activity observed with combination immunotherapy. Further clinical development of bintrafusp alfa and BN-Brachyury vaccine, however, has been discontinued, thus limiting the ability of a subsequent clinical study to be performed to determine the contribution of these specific individual agents, amounting to a major limitation. However, other agents targeting TGF-β in combination with PD-1/L1 blockade,37 38 as well as brachyury-directed therapies,39 are in active clinical development. Therefore, it would be feasible to replicate a similar triplet regimen as the one in Arm 2.2 by combining these agents with NAI in future studies. Additional limitations of this trial included small sample size and heterogeneous population spanning non-metastatic CRPC to mCRPC, including patients exposed to multiple lines of prior treatment. In 2021, a protocol amendment aimed to increase accrual allowed, in patients progressing on an ARPI, the continuation of that ARPI on study, which introduced an additional variable into this trial. Studies in a larger, more homogenous population will be needed to define efficacy of targeting TGF-β, PD-L1, and a tumor antigen along with a cytokine.
All clinical responders in the current study had MMR/MSI status assessed retrospectively. However, since this study was initiated prior to widespread use of next generation sequencing testing of MMR status, not all participants had known MMR status (or available tissue) and MMR was assessed by varying means in an exploratory fashion. Future studies should have MMR assessed centrally, in all participants at study entry.
In addition to increased antitumor activity, the addition of cytokine to the combination of vaccine and dual blockade of PD-L1/TGF-β was associated with an increase in the incidence of IAD between these two small datasets. Interestingly, IAD was not observed in any individuals not developing a clinical response, with the exception of one individual in Arm 2.3. This individual had a PSA decline, but it did not meet the prespecified criteria for response. The single patient who responded to vaccine + bintrafusp alfa in Arm 2.1 experienced IAD. Of the seven responders (n=1 with dMMR disease) who received vaccine + bintrafusp alfa + NAI in Arm 2.2, three also experienced IAD.
The co-occurrence of IAD and clinical response in this dataset is hypothesis generating. An assumption that may explain this unexpected finding is cross-reactivity between vaccine-induced brachyury-specific T cells and another T-box protein, TBX19 (T-pit), which shares 55% identical amino acids with brachyury mostly in the DNA binding domain and is highly expressed in the pituitary gland; however, no data are currently available at this time to support this hypothesis. Notably, one individual with IAD did not have a T-cell response to brachyury detected in peripheral blood (online supplemental figure S2), and the magnitude of brachyury-specific T cells induced in patients with and without IAD was similar. These observations do not support the hypothesis that brachyury-specific T cells target TBX19-derived epitopes in the pituitary gland. Future studies with human peripheral blood from individuals who receive this combination therapy would be needed to assess the ability of brachyury-specific T cells to lyse TBX19-expressing cells; however, due to the limited remaining research samples and the discontinuation of the development of bintrafusp alfa and BN-brachyury vaccine, these analyses are not possible at this time.
The combination of a tumor-directed vaccine with ICB and an immunocytokine produced a higher response rate (ie, 26%) than previously reported in patients with MSS/pMMR CRPC. To our knowledge, this is the first report of an active, multimodal immuno-oncology regimen in this difficult-to-treat population. Immune analyses revealed complex changes in peripheral immune profiles providing evidence of a multifaceted antitumor immune response and identified multiple immune correlates of clinical activity. These findings provide the rationale for continued investigation of this combination treatment regimen directed against different arms of the immune system, and with the ability to modify the tumor cell phenotype and microenvironment, in patients with CRPC.
Acknowledgments
Employees of Merck KGaA, Darmstadt, Germany, Bavarian Nordic, and Incyte reviewed this manuscript for medical accuracy only. The authors are fully responsible for the content of this manuscript, and the views and opinions described in the publication reflect solely those of the authors. The authors thank Seth J Steinberg for his assistance in statistics, the NCI CCR Sequencing Core Facility for their technical support, and Debra Weingarten for her editorial assistance in preparing this manuscript. The authors thank the patients, their families and the team at the Center for Immuno-Oncology, CCR, NCI.
Footnotes
Contributors: Concept and design: JMR, RND, JSc, and JLG. Acquisition, analysis, or interpretation of data: JRM, RAM, RND, NJT, FK, JSt, CP, LAH, JDR, JLM, LC, MTL, TJM, MC, JSc, and JLG. Involved in patient care and acquisition of research samples: JMR, RAM, FK, JSt, JDR, JLM, LC, and JLG. Performed experiments and interpreted data from peripheral blood of patients: RND, NJT, and JSc. Developed and/or applied methodology for gene expression analyses: NJT, TJM and MC. Involved in data curation: JRM, RND, NJT, CP, LAH, MTL, JSc, and JLG. Supervised the study: JMR, RND, JSc, and JLG. Drafted the manuscript: JRM, RND, NJT, and JLG. Acquired funding for this project: JSc and JLG. Critical review of the manuscript for important intellectual content: All authors. JLG is the guarantor.
Funding: This research was supported in part by the Intramural Research Program of the National Institutes of Health (NIH) (ZIA BC 010666) and through Cooperative Research and Development Agreements (CRADAs) between the National Cancer Institute (NCI) and Bavarian Nordic, Hellerup, Denmark (CRADA 02561); EMD Serono Research & Development Institute, Inc., Billerica, Massachusetts, USA (CRADA 02666) and affiliate of Merck KGaA, Darmstadt, Germany (CrossRef Funder ID 10.13039/100004755); ImmunityBio, Culver City, California, USA CRADA (02997); and Incyte, Wilmington, Delaware, USA (CRADA 03142). This study was sponsored by the National Cancer Institute (NCI), Center for Cancer Research (CCR), Office of Sponsor and Regulatory Oversight (OSRO).
Disclaimer: The sponsor reviewed and approved the study design but had no role in data collection, data analysis, data interpretation, manuscript preparation, or the decision to submit the manuscript for publication. The contributions of the NIH authors are considered Works of the US Government. The findings and conclusions presented in this paper are those of the authors and do not necessarily reflect the views of the NIH or the US Department of Health and Human Services.
Competing interests: PS-S is in management at ImmunityBio and is a stockholder of the company. JMR, RAM and JSt’s work on this manuscript was conducted while they were employed at the National Cancer Institute, NIH. Currently, JMR is an employee of K36 Therapeutics, Cambridge, Massachusetts, USA; RAM is an employee of AstraZeneca, Gaithersburg, Maryland, USA, and JSt is an employee of Bristol Myers Squibb, Lawrence Township, New Jersey, USA. NIH authors declare they have no competing interests.
Provenance and peer review: Not commissioned; externally peer reviewed.
Supplemental material: This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.
Data availability statement
Data are available upon reasonable request.
Ethics statements
Patient consent for publication
Not applicable.
Ethics approval
Ethics approval and consent to participate: This study was performed in accordance with all applicable regulatory requirements. All clinical protocols were approved by the National Institutes of Health Institutional Review Board and conducted in accordance with the Declaration of Helsinki and Good Clinical Practice standards. All patients provided written informed consent prior to study enrollment.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
jitc-14-7-s001.pdf (1.9MB, pdf)
Data Availability Statement
Data are available upon reasonable request.