Abstract
The immunocytokine NHS-IL12 delivers IL-12 to the tumor microenvironment by targeting DNA/histones in necrotic areas. The first-in-human clinical trial administered NHS-IL12 subcutaneously in 59 patients treated every four weeks (Q4W), with a maximum tolerated dose of 16.8 mcg/kg. The phase I study was expanded to include a high-exposure cohort that received bi-weekly treatment (Q2W) with two dose levels of NHS-IL12: 12.0 mcg/kg and 16.8 mcg/kg.
Here, patients given NHS-IL12 were analyzed both prior to and early after treatment for effects on 10 serum soluble analytes, complete blood counts, and 158 peripheral immune subsets. Higher levels of immune activation were seen with a dose of 16.8 mcg/kg versus 12.0 mcg/kg in patients in the high-exposure cohort, as evidenced by greater increases in serum IFNγ, TNFα, and soluble PD-1, and greater increases in frequencies of peripheral ki67+ mature natural killer (NK), CD8+T, and NKT cells. Greater immune activation was also seen in the Q2W versus Q4W cohort, as demonstrated by greater increases in pro-inflammatory serum analytes, ki67+ CD8+ T, NK, and NKT cells, intermediate monocytes, and a greater decrease in CD73+ T cells. Specific immune analytes at baseline including lower levels of monocytes and plasmacytoid dendritic cells, and early changes after treatment such as an increase in refined NK cell subsets and total CD8+ T cells, associated with better clinical response.
These findings may help to guide future schedule and dosing regimens of clinical studies of NHS-IL12 as monotherapy and in combination therapies.
Keywords: NHS-IL12, interleukin 12, immunocytokine, immunotherapy, cancer, peripheral immunome
1. Introduction
Interleukin 12 (IL-12) is a pro-inflammatory cytokine produced by activated dendritic cells (DC), macrophages, and neutrophils [1] and acts mainly on natural killer (NK) cells, natural killer T (NKT) cells, and CD8+ T cells to enhance proliferation [2, 3] and cytotoxicity [4, 5]. IL-12 drives CD4+ T cells towards a T helper 1 (Th1) phenotype [6, 7] and leads to interferon gamma (IFNγ) production by T and NK cells [4, 8–10], thus promoting cell-mediated immunity. In addition, IL-12 can alter the function of immunosuppressive cells in the tumor microenvironment (TME) by inhibiting and reprogramming tumor-associated macrophages (TAM) and myeloid-derived suppressor cells (MDSC) to enhance pro-inflammatory function, antigen presentation and support of T cell responses [11–15].
IL-12 treatment in preclinical mouse models, alone or in combination with other therapies, yielded promising results with significant antitumor activity and improved survival [16–19]. However, recombinant human IL-12 (rhIL-12) in clinical studies resulted in serious adverse effects. Intravenous administration of rhIL-12 in the first-in-human phase I clinical trial was generally well tolerated [20], but a subsequent phase II study resulted in severe toxicities, with 12 out of 17 patients requiring hospitalization and two patient deaths [21]. Further studies tested subcutaneous administration of rhIL-12 in metastatic melanoma [22] and advanced renal cell carcinoma [23], which was relatively well-tolerated. While rhIL-12 treatment continued to be tested at varying treatment schedules, patients experienced toxicities without satisfactory therapeutic efficacy, partly explained by an inability of rhIL-12 to reach biologically relevant levels of IL-12 induced IFNy in the TME [24–26]. The efficacy of systemic IL-12 was possibly limited by tachyphylaxis, where patients had a diminished rise in IFNy and therefore less responsiveness upon subsequent treatments [20, 27].
NHS-IL12 is an immunocytokine fusion protein composed of two molecules of IL-12 fused to each of the heavy-chains of the human IgG1 NHS76 antibody [28, 29]. NHS76 targets DNA/histones exposed in necrotic areas of solid tumors, thus delivering IL-12 to the TME, sparing systemic accumulation [30]. Preclinical studies using a murine version of the immunocytokine, designated NHS-muIL12, resulted in lower levels of serum IFNγ induction, greater accumulation in tumors, and greater antitumor effects compared to recombinant murine IL-12 [28]. The antitumor response was dose-dependent, and resulted in immune changes, including enhanced maturation of splenic DC and activation of splenic and tumor-infiltrating NKs, and splenic CD8+ T cells. Antitumor activity, resulting in complete cures and long-term survival benefit, was also seen in murine models of bladder cancer, and was dependent on both NK and CD8+ T cells [31]. Additional studies in bladder cancer mouse models found that NHS-IL12 induced antitumor activity with reductions in intratumoral monocytic MDSC, macrophages, and transforming growth factor beta (TGFβ), resulting in CD4+ and CD8+ antitumor T cell responses [32]. In addition, NHS-IL12 was safely administered to dogs that developed malignant melanoma naturally, with 2 out of 7 developing a partial response [33].
Preclinical findings led to the first-in-human clinical trial of NHS-IL12 (NCT01417546) [34]. NHS-IL12 was initially administered subcutaneously in 59 patients in a single-ascending dose cohort in which 22 patients were treated on day one and evaluated on day 28, and multiple ascending dose and expansion cohorts in which 37 patients were treated on day one and then every four weeks. IFNγ and IL-10 showed a time-dependent increase after administration of NHS-IL12, which rose to a lesser degree after the second dose, and is possibly reflective of the tachyphylaxis phenomenon. The maximum tolerated dose (MTD) was determined to be 16.8 mcg/kg. Overall, among 59 patients enrolled, 11 experienced a grade 3 treatment-related adverse event (TRAE), 1 experienced a grade 4 TRAE, and toxicity was slightly higher for patients treated with the MTD compared to lower doses. Of 30 patients with evaluable disease, 15 had a best overall response (BOR) of stable disease.
To determine the optimal treatment schedule and dosing regimen to achieve immune activation, clinical response, and limit toxicities, the phase I study was expanded to include a high-exposure cohort of subjects (n=13) who received bi-weekly treatment at one of two dose levels of NHS-IL12: 12.0 mcg/kg and 16.8 mcg/kg [35]. Bi-weekly NHS-IL12 was found to be well tolerated, with 1 dose limiting toxicity (Grade 3 AST/ALT elevation) and 3 other grade 3 TRAE noted (flu-like symptoms, decreased absolute lymphocyte count, and decreased white blood cell count); however, most adverse events were low grade and self-limiting. With bi-weekly dosing of NHS-IL12, 50% of evaluable patients (6/12) experienced stable disease. In this study, the peripheral immune responses of patients in the high-exposure cohort (receiving 12.0 mcg/kg and 16.8 mcg/kg Q2W NHS-IL12) and original cohort (receiving 16.8 mcg/kg Q4W NHS-IL12) were evaluated to determine if differences in dose level and schedule of NHS-IL12 resulted in changes in both serum soluble factors reflective of immune changes and in 158 immune cell subsets. Immune changes were also evaluated in the high exposure cohort for association with clinical response.
2. Materials and methods
2.1. Description of patients
Immune parameters were evaluated in 23 patients with advanced cancers enrolled in a phase I, open label, dose-escalation study at the National Cancer Institute (NCI) (NCT01417546). Patients were selected for analysis based on blood availability at pre- and post-timepoints, and patient characteristics are summarized in Table 1. Of 13 patients enrolled who received NHS-IL12 Q2W in the high-exposure cohort, blood from pre- and post-timepoints (baseline, day 14, day 28) were available for analysis in 12 patients (6 given 12.0 mcg/kg, and 6 given 16.8 mcg/kg). Peripheral blood was also evaluated at comparable time points from 11 patients who received NHS-IL12 Q4W in the original dose escalation cohort, where 16.8 mcg/kg was determined to be the MTD. All patients provided signed, informed consent prior to study enrollment. Patients were classified as having stable disease or progressive disease based on CT measurement or tumor markers, if available, at 8 weeks. Complete blood counts (CBC) with differential were performed at the National Cancer Institute’s Center for Cancer Research.
Table 1. Description of patients given NHS-IL12.
Q2W refers to patients given NHS-IL12 at baseline and then every two weeks; Q4W refers to patients given NHS-IL12 at baseline and then every four weeks.
Schedule | Dose (mcg/kg) | BOR | Ca Type | Age | Sex |
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12.0 | NE | Rectal | 40 | M | |
SD | Cervical | 35 | F | ||
SD | Prostate | 72 | M | ||
SD | Prostate | 56 | M | ||
SD | Prostate | 79 | M | ||
PD | Colon | 65 | F | ||
Q2W |
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16.8 | SD | Vaginal | 54 | F | |
SD | Prostate | 69 | M | ||
PD | Cervical | 44 | F | ||
PD | Cervical | 64 | F | ||
PD | Colon | 51 | F | ||
PD | Prostate | 71 | M | ||
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Q4W | 16.8 | NE | Prostate Ca | 60 | M |
NE | Ovarian Ca | 53 | F | ||
SD | Breast Ca | 78 | F | ||
SD | Prostate Ca | 75 | M | ||
SD | Chordoma | 57 | M | ||
PD | Prostate Ca | 67 | M | ||
PD | Intestinal Ca | 54 | M | ||
PD | Ovarian Ca | 70 | F | ||
PD | Colorectal | 50 | F | ||
PD | Liver Ca | 85 | M | ||
PD | Colon Ca | 43 | F |
Best overall response, BOR; stable disease, SD; progressive disease, PD; non-evaluable, NE; cancer, ca; male, M; female, F. [refs. 34 and 35; NCT01417546.]
2.2. Serum analytes
Blood was collected in serum separator tubes, centrifuged, and serum stored at −80°C prior to analysis. Serum levels of cytokines and soluble factors were evaluated in 23 patients before and after treatment with NHS-IL12. Soluble (s) CD27 (sCD27), sCD40L, sPD-1, TGFβ1, and granzyme B were measured with commercially available kits according to the manufacture’s instructions. sCD27 and sCD40L were evaluated using Human Instant ELISA kits from Life Technologies (Carlsbad, California, USA), sPD-1 was measured using ELISA kits from Abcam (Cambridge, UK), and TGFβ1 and granzyme B were measured using ELISA kits from R&D Systems (Minneapolis, Minnesota, USA). IL-8, IL-6, IFNγ, TNFα, and IL-10 were measured by a V-PLEX Meso Scale Discovery platform for human samples per the manufacturer’s instructions (Meso Scale Diagnostics, Rockville, Maryland, USA).
2.3. 158 peripheral blood mononuclear cell (PBMC) subsets
Blood was collected in sodium heparin tubes and PBMCs were isolated after Ficoll-Hypaque density gradient separation. PBMCs were then cryopreserved in 90% heat-inactivated human AB serum and 10% dimethyl sulfoxide at a concentration of 1×107 cells/mL and then analyzed in a single assay. PBMCs were stained with 30 antibodies in four multi-color flow cytometry panels to identify 158 subsets, including 10 classic subsets (CD4+ and CD8+ T cells, T regulatory cells (Treg), B cells, NK cells, NKT cells, conventional dendritic cells (cDC), plasmacytoid dendritic cells (pDC), MDSC, and monocytes), and 148 refined subsets related to maturation and function of the parental cell types (Supplemental Table 1), using methods previously described [36–38]. Flow cytometry files were acquired on an LSR Fortessa equipped with five lasers and analyzed using FlowJo v.9.9.6 for Macintosh, with nonviable cells excluded and negative gates based on fluorescence-minus-one controls. The frequency of all subsets was calculated as a percentage of PBMC to eliminate any bias that might occur in the smaller populations with fluctuations in leukocyte subpopulations.
2.4. Statistical analysis
Statistical analyses were performed using RStudio (RStudio version 2022.2.3.492 for Macintosh, Boston, Massachusetts, USA) and GraphPad Prism (GraphPad Prism version 9.4.0 for Macintosh, San Diego, California USA). Changes in immune parameters in paired pre- and post-treatment samples were analyzed for statistical significance using a Wilcoxon signed-rank test. Differences between groups were assessed for the significance of the difference using a Mann-Whitney test. p values <0.05 were considered statistically significant. All p values were two-tailed and reported without adjustment for multiple comparisons in this hypothesis-generating study.
3. Results
3.1. Effect of NHS-IL12 dose on immune responses early after treatment
Changes in immune parameters were first analyzed at 14 and 28 days after treatment compared to baseline from patients in the high-exposure bi-weekly cohort (Q2W) treated with a dose level of 12.0 mcg/kg NHS-IL12. Among the absolute levels of serum cytokines (n=6) and other soluble factors related to immunity (n=4) evaluated, only sCD27 at day 14 (p=0.031) was increased with 12.0 mcg/kg of NHS-IL12 (Fig. 1A). In contrast, patients treated with 16.8 mcg/kg of NHS-IL12 had elevations in absolute serum levels of IFNγ (p=0.031), TNFα (p=0.031), IL-10 (p=0.031), and sPD-1 (p=0.031), both at days 14 and 28, while granzyme B (p=0.031) and sCD27 (p=0.031) were increased only at day 28 after therapy. There was no change in absolute serum levels of IL-8, IL-6, TGFβ1, or sCD40L after treatment with either dose level of NHS-IL12 administered Q2W (Supplemental Table 2).
Figure 1. Effect of dose of NHS-IL12 on serum cytokines and soluble factors.
Levels of serum cytokines and soluble factors at baseline, day 14 and day 28, in patients receiving Q2W NHS-IL12 at 12mcg/kg or 16.8 mcg/kg (A). The percent change was compared at day 28 vs baseline between patients receiving Q2W 12 mcg/kg and 16.8 mcg/kg NHS-IL12 (B). In A, dashed lines indicate when patients received NHS-IL12 and median fold change vs baseline (D0) is indicated in gray italics. Significant differences between groups include those with p < 0.05 (calculated by Wilcoxon signed-rank test in A and Mann-Whitney test in B). The Limit of Detection (LOD) is indicated for IL-10 (0.185 pg/ml); samples that fell below that value were set to the LOD for analyses. Bars in B indicate median with interquartile range. IFNγ, interferon gamma; TNFα, tumor necrosis factor alpha; IL-10, interleukin 10; sCD27, soluble CD27; sPD-1, soluble programmed cell death protein 1.
Differences in the percent change of serum analytes from baseline to days 14 and 28 after treatment were also compared between dose levels. No differences between dose levels were observed at day 14 versus baseline; however, at day 28 versus baseline, there was a greater increase in IFNγ (p=0.030), TNFα (p=0.017), and sPD-1 (p=0.030), and trends of greater increases in IL-10 and granzyme B in patients treated with 16.8 mcg/kg compared to 12.0 mcg/kg (Fig. 1B).
Among the CBC parameters assessed, patients treated with 12.0 mcg/kg of NHS-IL12 Q2W had a decrease in absolute neutrophil count (ANC, p=0.031) at day 14 and day 28, as well as a decrease in total white blood cell count (WBC, p=0.031) at day 28 (Table 2A and B). Changes in individual patients from Table 2 are displayed in Supplemental Fig. 1. For patients treated with 16.8 mcg/kg NHS-IL12 Q2W, ANC (p=0.031) and WBC (p=0.031) were decreased at day 14 but returned to baseline by day 28, while absolute lymphocyte count (ALC, p=0.031) was decreased at day 28 (Table 2A and B). There was no difference in the percent change in CBCs at day 14 or day 28 versus baseline between the dose levels of NHS-IL12.
Table 2. Effect of dose of NHS-IL12 on complete blood counts and immune subsets.
Changes in complete blood counts, classic PBMC types, and notable refined PBMC subsets reflective of maturation and function are shown in patients treated with Q2W 12mcg/kg or 16.8mcg/kg NHS-IL12 at 14 days vs baseline (A) or 28 days vs baseline (B). All data presented and comparisons made for the 158 subsets (Supplemental Table 1) were performed on subset frequencies calculated as a percent of PBMC. Tables display median values, p values calculated by the Wilcoxon signed-rank test, and the percentage of patients with a >25% change in each subset compared to baseline. Subsets with significant changes at post timepoints vs baseline are those with p < 0.05, difference in medians >0.05, and ≥50% of patients having a >25% change. Only subsets with significant changes are shown.
A. 14 days after 1st dose vs Baseline | |||||||||||||||
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Patients treated with 12 mcg/kg NHS-IL 12 Q2W | Patients treated with 16.8 mcg/kg NHS-IL 12 Q2W | ||||||||||||||
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Analyte | Direction of Change | Median Baseline | Median Week 2 | % with >25% Increase | % with >25% Decrease | p-value | Analyte | Direction Of Change | Median Baseline | Median Week 2 | % with >25% Increase | % with >25% Decrease | p-value | ||
CBCs (n=6) | WBC (K/ul) | = | 5.76 | 5.02 | 0 | 17 | 0.0625 | CBCs (n=6) | WBC (K/ul) | ↓ | 5.94 | 4.40 | 50 | 0 | 0.0313 |
ANC (K/ul) | ↓ | 3.66 | 3.09 | 0 | 67 | 0.0313 | ANC (K/ul) | ↓ | 3.99 | 2.57 | 83 | 0 | 0.0313 | ||
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Classic Subsets (n=6) | NKT | = | 3.19 | 4.61 | 83 | 0 | 0.0938 | Classic Subsets (n=6)* | NKT | ↑ | 1.55 | 2.39 | 0 | 83 | 0.0313 |
Monocytes | = | 20.41 | 24.05 | 33 | 17 | 0.3125 | Monocytes | ↑ | 23.66 | 28.34 | 0 | 50 | 0.0313 | ||
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Refined Subsets (n=6) | NK ki67+ | ↓ | 0.71 | 0.36 | 0 | 67 | 0.0313 | Refined Subsets (n=6)* | NK ki67+ | = | 0.39 | 0.38 | 50 | 50 | 0.8438 |
NK mature ki67+ | ↓ | 0.52 | 0.23 | 0 | 100 | 0.0313 | NK mature ki67+ | = | 0.27 | 0.23 | 17 | 50 | 0.4375 | ||
CD4 CD73+ | ↓ | 4.32 | 2.71 | 0 | 50 | 0.0313 | CD4 CD73+ | = | 2.80 | 1.68 | 0 | 60 | 0.0625 | ||
Treg HLA-DR+ | ↓ | 0.20 | 0.11 | 0 | 83 | 0.0313 | Treg HLA-DR+ | = | 0.08 | 0.09 | 20 | 20 | 0.4375 | ||
NK PD-1 + | ↑ | 0.04 | 0.12 | 100 | 0 | 0.0313 | NK PD-1 + | = | 0.01 | 0.01 | 67 | 0 | 0.0313 | ||
NK Tim3+ | ↑ | 0.06 | 0.15 | 100 | 0 | 0.0313 | NK Tim3+ | = | 0.07 | 0.29 | 83 | 0 | 0.0625 | ||
NKT PD-1 + | = | 0.59 | 1.01 | 83 | 0 | 0.0938 | NKT PD-1 + | ↑ | 0.22 | 0.39 | 83 | 0 | 0.0313 | ||
CD8 ki67+ | = | 0.27 | 0.78 | 50 | 17 | 0.4375 | CD8 ki67+ | ↑ | 0.13 | 0.49 | 100 | 0 | 0.0313 | ||
Int Monocytes | = | 0.29 | 0.64 | 83 | 17 | 0.4375 | Int Monocytes | ↑ | 0.43 | 1.01 | 83 | 0 | 0.0313 | ||
Monocytes PD-L1 + | = | 7.39 | 8.64 | 67 | 17 | 0.1563 | Monocytes PD-L1 | ↑ | 7.32 | 12.66 | 83 | 0 | 0.0313 | ||
Class Monocytes | = | 16.01 | 21.67 | 33 | 17 | 0.1563 | Class Monocytes | ↑ | 18.18 | 23.73 | 100 | 0 | 0.0313 | ||
B. 28 days (14 days after 2nd dose) vs Baseline | |||||||||||||||
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Patients treated with 12 mcg/kg NHS-IL 12 Q2W | Patients treated with 16.8 mcg/kg NHS-IL 12 Q2W | ||||||||||||||
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Analyte | Direction of Change | Median Baseline | Median Week 4 | % with >25% Increase | % with >25% Decrease | p-value | Analyte | Direction of Change | Median Baseline | Median Week 4 | % with >25% Increase | % with >25% Decrease | p-value | ||
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CBCs (n=6) | WBC (K/ul) | ↓ | 5.76 | 4.30 | 0 | 50 | 0.0313 | CBCs (n=6) | WBC (K/ul) | = | 5.94 | 4.15 | 67 | 0 | 0.0625 |
ALC (K/ul) | = | 1.46 | 1.14 | 17 | 67 | 0.1563 | ALC (K/ul) | ↓ | 1.16 | 0.98 | 50 | 0 | 0.0313 | ||
ANC (K/ul) | ↓ | 3.66 | 2.58 | 0 | 83 | 0.0313 | ANC (K/ul) | = | 3.99 | 2.59 | 83 | 0 | 0.0625 |
n = n-1 for CD4 and Treg (CD4 antibody failed to bind to CD4 epitope for one patient). PBMC, peripheral blood mononuclear cells; CBC, complete blood count; WBC, white blood cell count; ANC, absolute neutrophil count; NKT, natural killer T; NK, natural killer; Treg, regulatory T; HLA-DR, human leukocyte antigen DR isotype; PD-1, programmed cell death protein 1; Tim3, T-cell immunoglobulin and mucin-domain containing-3; Int, intermediate; PD-L1, programmed death-ligand 1; Class, classical; ALC, absolute lymphocyte count.
For patients treated with 16.8 mcg/kg NHS-IL12 Q2W, there was an increase in total NKT cells (p=0.031) and monocytes (p=0.031) 14 days after the first dose (Table 2A). There were no other changes in classic PBMC cell types after 14 or 28 days in patients receiving 12 mcg/kg or 16.8 mcg/kg NHS-IL12 at the Q2W schedule (Supplemental Table 2A and B); however, there were notable changes in specific refined PBMC subsets for patients given either dose level at day 14 versus baseline. For patients receiving 12.0 mcg/kg, there was an increase in NK cells expressing the activation markers PD-1 (p=0.031) and Tim3 (p=0.031), and a decrease in proliferative ki67-expressing total NK (p=0.031) and mature NK (p=0.031) 14 days after treatment (Table 2A). Although total CD4+ T cells were unchanged (Supplemental Table 2A) there were decreases at day 14 in CD4+ T cells expressing CD73 (p=0.031), a protein involved in adenosine metabolism, and suppressive HLA-DR+ Tregs (p=0.031). Different changes in refined PBMC subsets were seen at day 14 in patients given 16.8 mcg/kg NHS-IL12, including increases in proliferative ki67-expressing CD8+ T cells (p=0.031), PD-1-expressing NKT cells (p=0.031), and PD-L1-expressing monocytes (p=0.031), classical monocytes (p=0.031), and intermediate monocytes (p=0.031) (Table 2A).
Differences in the percent change of PBMC subsets from baseline to days 14 and 28 after treatment were also compared between dose levels. Here, there was a greater increase at day 14 in ki67-expressing NKT (p=0.026) and mature NK (p=0.041) cells (Fig. 2A), and a greater increase at day 28 in ki67-expressing NKT (p=0.008) and CD8+ T (p=0.032) cells for subjects given 16.8 mcg/kg compared to 12.0 mcg/kg NHS-IL12 (Fig. 2B). Alternatively, there was a greater decrease at day 28 in immature NK cells (p=0.032) in patients receiving the higher dose level of NHS-IL12 (Fig. 2B).
Figure 2. Effect of dose of NHS-IL12 on changes in peripheral blood mononuclear cell subsets after therapy.
The percent change of the frequency of peripheral blood mononuclear cell (PBMC) subsets (calculated as percent of PBMC) was compared at day 14 vs baseline (A) and day 28 vs baseline (B) in patients receiving 12 mcg/kg or 16.8mcg/kg NHS-IL12 at a Q2W schedule. Significant differences between groups include those with p < 0.05 (calculated by Mann-Whitney test). Bars indicate median with interquartile range. NKT, natural killer T; NK, natural killer.
3.2. Effect of NHS-IL12 schedule on immune responses early after treatment
Patients treated with 16.8 mcg/kg NHS-IL12 every 2 weeks (Q2W) or every 4 weeks (Q4W) were analyzed for changes in immune parameters at days 14 and 28. At the day 14 timepoint, where patients on both schedules had received a single dose, serum levels of IFNγ, TNFα, IL-10, and sPD-1 were increased for both the Q2W and Q4W patients (Fig. 3A). At this time, granzyme B and sCD27 were also increased in patients receiving the Q4W but not the Q2W treatment schedule. At day 28, IFNγ (p=0.031), TNFα (p=0.031), granzyme B (p=0.031), sCD27 (p=0.031), and sPD-1 (p=0.031) were increased only for patients on the Q2W schedule and not the Q4W schedule of NHS-IL12. IL-10, in contrast, was elevated at day 28 for patients on either treatment schedule (p=0.031 for Q2W and p=0.029 for Q4W). Serum levels of IL-8, IL-6, TGFβ1, and sCD40L were unchanged after 14 and 28 days with either treatment schedule of NHS-IL12 (Supplemental Table 3).
Figure 3. Effect of dose scheduling of NHS-IL12 on serum cytokines and soluble factors.
Levels of serum cytokines and soluble factors at baseline, day 14 and day 28 in patients receiving Q2W or Q4W NHS-IL12 at 16.8 mcg/kg (A). The percent change was compared at day 28 vs baseline between patients receiving 16.8 mcg/kg at Q2W vs Q4W (B). For the Q2W schedule, this corresponds to 14 days after the second dose, and for the Q4W schedule, this corresponds to 28 days following the first dose of NHS-IL12. In A, dashed lines indicate when patients received NHS-IL12 and median fold change vs baseline (D0) is indicated in gray italics. Significant differences between groups include those with p < 0.05 (calculated by Wilcoxon signed-rank test in A and Mann-Whitney test in B). The Limit of Detection (LOD) is indicated for IL-10 (0.185 pg/ml); samples that fell below that value were set to the LOD for analyses. Bars in B indicate median with interquartile range. IFNγ, interferon gamma; TNFα, tumor necrosis factor alpha; IL-10, interleukin 10; sCD27, soluble CD27; sPD-1, soluble programmed cell death protein 1.
Differences were noted in specific serum cytokines and soluble factors between the treatment schedules when comparing the percent change at day 28 versus baseline. Patients receiving the Q2W schedule had greater increases in IFNγ (p=0.0002), TNFα (p=0.0006), IL-10 (p=0.0002), sCD27 (p=0.007), and sPD-1 (p=0.0006), and a trending greater increase in granzyme B compared to patients receiving the immunocytokine at the Q4W schedule (Fig. 3B).
Aside from a reduction in ALC at day 28 in patients receiving Q2W NHS-IL12, no other changes in CBCs or classic PBMC cell types were observed in patients receiving either the Q2W or Q4W treatment schedule (Supplemental Table 3). There was, however, a decrease in ki67+ NK cells (p=0.002) noted at day 28 in patients receiving NHS-IL12 at the Q4W schedule (Supplemental Table 3). Several differences in the percent change of PBMC subsets from baseline to day 28 after treatment were also observed between the two treatment schedules. Greater increases in specific refined PBMC subsets reflective of enhanced immune activation were noted in patients receiving the Q2W schedule than the Q4W schedule, including ki67-expressing CD8+ T (p=0.0005), NK (p=0.009), and NKT (p=0.0005) cells, and intermediate monocytes (p=0.006) (Fig. 4). In addition, greater decreases in CD4+ and CD8+ T cells that express the immune inhibitory marker CD73 (p=0.018 and p=0.009, respectively) were observed after 28 days in those patients receiving Q2W NHS-IL12 than Q4W NHS-IL12 (Fig. 4).
Figure 4. Effect of dose scheduling of NHS-IL12 on changes in peripheral blood mononuclear cell subsets after therapy.
The percent change of the frequency of peripheral blood mononuclear cell (PBMC) subsets (calculated as percent of PBMC) were compared at day 28 vs baseline in patients receiving 16.8mcg/kg NHS-IL12 at Q2W and Q4W schedules. For the Q2W schedule, this corresponds to 14 days after the second dose, and for the Q4W schedule, this corresponds to 28 days following the first dose of NHS-IL12. Significant differences between groups include those with p < 0.05 (calculated by Mann-Whitney test). Bars indicate median with interquartile range. NK, natural killer; NKT, natural killer T.
3.3. Immune correlates of clinical response
The immune status of patients prior to therapy was next assessed for association with clinical response. Patients in the high-exposure cohort (given either dose level of NHS-IL12) who developed a BOR of stable disease were compared to those developing progressive disease. Prior to therapy, lower frequencies of monocytes (p=0.017) and pDCs (p=0.037) at baseline were associated with the development of stable disease following treatment with NHS-IL12 (Fig. 5A). We next interrogated whether early changes in any immune parameters were associated with BOR. Two refined NK subsets, double positive (DP; CD16+ CD56br) NK cells and immature (CD16− CD56br) NK cells, were increased after 14 days to a greater extent in patients who developed stable disease compared to progressive disease (Fig. 5B). When calculated as a percent of total PBMC, DP NK were increased in 6/6 patients with stable disease compared to 3/6 patients with progressive disease, while immature NK were increased in 5/6 patients with stable disease compared to 1/5 patients with progressive disease. Furthermore, a greater increase in DP (p=0.017) and immature NK cells (p=0.009) was seen over baseline in those patients with disease stabilization when these subsets were calculated as a percentage of parent (total NK cells). The greater increase in immature NK cells observed in those patients with stable disease compared to progressive disease persisted at the day 28 timepoint following initiation of therapy (p=0.019) (Fig. 5C). Total CD8+ T cells, calculated as a percent of total PBMC, were also increased at day 28 to a greater extent in patients with disease stabilization, with 5/6 patients with stable disease compared to 1/4 patients with progressive disease having an increase in the frequency of this subset (Fig. 5C). In addition, there was a greater increase in CD8+ T cells (p=0.019) calculated as a percentage of parent (total lymphocytes) in patients who developed stable compared to progressive disease at this timepoint (Fig. 5C).
Figure 5. Association between best overall response and immune parameters in patients treated with NHS-IL12.
Immune parameters were compared at baseline and as percent change from baseline to day 14 or day 28 post-treatment in patients treated with 12mcg/kg (open circles) or 16.8mcg/kg (closed circles) Q2W NHS-IL12 with a best overall response (BOR) of stable disease (SD) vs progressive disease (PD). Significant differences at baseline between patients with BOR of SD and PD are shown (A). Immune parameters were analyzed at pre- and post-timepoints for patients with BOR of SD and PD, and significant differences in the percent change of immune subsets as a percent of parental cell type from baseline to day 14 (B) and day 28 (C) are shown between patients with BOR of SD and PD. Analytes with differences between groups are those with p < 0.05 (calculated by the Mann-Whitney test). Bars indicate median with interquartile range. pDC, plasmacytoid dendritic cells; NK, natural killer; DP, double positive.
Discussion
In the current exploratory study, we assessed immune changes in relation to the dose level and schedule of NHS-IL12 in the first-in-human trial to determine the optimal treatment regimen for immune activation. Three groups of patients were evaluated: (1) Q4W, 16.8 mcg/kg, (2) Q2W, 12.0 mcg/kg, and (3) Q2W, 16.8 mcg/kg. In the high exposure cohort, we found that a higher dose of NHS-IL12 (16.8 mcg/kg) resulted in more robust immune activation compared to a lower dose (12.0 mcg/kg). A panel of serum cytokines and soluble factors reflective of immune activation and suppression, some of which have been previously shown to change with NHS-IL12 treatment and associate with response to other therapies, were analyzed at pre- and post-treatment timepoints [34, 36]. Greater increases were seen at the higher dose level in the serum pro-inflammatory cytokines IFNγ and TNFα, and sPD-1. Increases in sPD-1 post-therapy have been associated with improved survival in various cancers and may indicate re-activation of CD8+ T cells [39]. In addition, there was also a greater expansion of proliferative immune cells expressing ki67, including NK cells, CD8+ T cells, and NKT cells, in patients treated with the higher dose level. IL-12 is well known to promote the proliferation of immune effector cells [40], and in preclinical studies, the antitumor effects of NHS-IL12 have been shown to be dependent on both CD8+ T and NK cells [28, 31].
We similarly found evidence of greater immune activation in patients treated with 16.8 mcg/kg NHS-IL12 every two weeks as opposed to every four weeks. Here, there were greater increases in multiple serum analytes, including granzyme B, which is suggestive of increased cytotoxicity, and greater increases in proliferative CD8+ T, NK, and NKT cells in patients receiving NHS-IL12 at the more frequent schedule. IFNy was also increased to a greater extent for patients treated every two weeks compared to every four weeks. It is important to note that IFNy did not fully resolve to baseline levels by the time of the second dose for Q2W dosing, but did return to baseline at the second dose for Q4W dosing. A diminished IFNy response due to tachyphylaxis may occur with the Q2W treatment schedule following subsequent doses; however, blood availability at later time points limited our ability to perform analyses beyond day 28. The clinical activity and toxicity of both dose levels will need to be evaluated in subsequent clinical studies. Notably, patients receiving NHS-IL12 Q2W also had greater decreases in CD73-expressing CD4+ and CD8+ T cells, which may be suggestive of diminished immunosuppression. CD73 catalyzes the breakdown of adenosine monophosphate to adenosine, which is suppressive of T cell function, and has been associated with tumor progression in preclinical studies and as a negative prognostic marker in cancer patients [41].
This is the first study to report on the association of immune correlates with clinical response in patients treated with NHS-IL12. We found higher baseline frequencies of monocytes and pDCs to associate with progressive disease. Higher peripheral monocytes [42–46] and pDCs, an immune subset thought to be tolerogenic and capable of promoting tumor immune escape [47], have both been shown to correlate with poor clinical outcome in cancer patients. Early increases after therapy in specific PBMC subsets, including refined NK subsets and total CD8+ T cells, also significantly associated with clinical response. Consistent with these findings, the antitumor activity of NHS-IL12 in preclinical studies has been found to be dependent on both NK cells and CD8+ T cells [31]. While this is a relatively small exploratory study, our findings that the baseline immune status and early changes in specific immune parameters after treatment with NHS-IL12 associate with clinical outcome may help to predict which patients may benefit from treatment. Larger studies, however, will need to be conducted to confirm these findings.
Preclinical mouse studies have shown the deposition of NHS-IL12 in the tumor. This was shown with a fluorescently labeled murine version of NHS-IL12 (NHS-muIL12) accumulating in the tumors of mice to a greater extent than a structurally similar murine version without a tumor-targeting component (BC1-muIL12) [28]. In addition, NHS-muIL12 binding to exposed nuclei in necrotic tumor regions was demonstrated through immunohistochemistry. Unfortunately, no tumor biopsies were obtained in this clinical study, so it was impossible to replicate the preclinical findings in the clinical study.
Further clinical development of NHS-IL12 has continued and is being tested both preclinically and clinically in combination with other therapies. NHS-IL12 plus avelumab (anti-PD-L1) was investigated preclinically as a strategy to boost the immune response and antitumor activity of checkpoint inhibition, and showed enhancement of tumor-specific immune memory, CD8+ T and NK cell proliferation, and tumor-antigen-specific CD8+ T cells that correlated with antitumor activity [48, 49]. Based on these findings, NHS-IL12 was combined with avelumab in a phase 1b study (NCT02994953) in patients with metastatic solid tumors, where some patients achieved prolonged partial and complete responses [50]. Preclinically, NHS-IL12 has also been combined with a human papillomavirus (HPV)-16 E6/E7 targeting liposomal vaccine PDS0101 and bintrafusp alfa, an anti-PD-L1/TGFβ trap [51]. In these studies, the triple combination therapy led to maximum antitumor responses and greater increases in T cell infiltration and T cell clonality compared to any singlet or doublet therapies tested. A phase II trial (NCT04287868) evaluating this triple therapy is underway, and a manageable safety profile and preliminary evidence of clinical activity for patients with both checkpoint naïve and refractory advanced HPV-16+ cancers has been reported [52]. In checkpoint naïve disease, 7/8 patients (88%) had objective responses including 1 delayed response after initial progression. In patients with checkpoint refractory disease, 10/22 (45%) had disease reduction including 6/22 (27%) with objective responses. Of interest to the current study, in checkpoint refractory patients, NHS-IL12 dosing was reported to have affected response rates, with 5/8 patients (63%) receiving the 16.8 mcg/kg dose having an objective response compared to 1/14 (7%) who received a lower dose of NHS-IL12 (8 mcg/kg). Despite differences in response rates with higher versus lower doses of NHS-IL12, survival outcomes were similar irrespective of dose.
Preclinical studies have also evaluated the rationale of combining NHS-IL12 with tumor necrosis inducing agents to enhance the tumor targeting of this immunocytokine. The combination of NHS-IL12 with docetaxel, for example, led to the regression of large (>500 mm3), well-established murine tumors in a synergistic fashion [29]. Based on these preclinical findings, NHS-IL12 has been combined with docetaxel in an ongoing phase I/II trial (NCT04633252). Preclinical studies combining NHS-IL12 with the histone deacetylase (HDAC) inhibitor entinostat have also shown impressive findings in poorly immunogenic tumors and mouse models of checkpoint resistance [53, 54]. These studies have provided the rationale for an ongoing clinical study (NCT04708470) testing the combination of NHS-IL12, entinostat, and bintrafusp alfa in patients with checkpoint refractory tumors.
In summary, we found a higher dose of NHS-IL12 consisting of 16.8 mcg/kg compared to 12.0 mcg/kg and more frequent dosing of Q2W compared to Q4W resulted in enhanced immune activation for patients with advanced cancer. With enhanced immune activation is the potential for increased toxicities, which needs to be considered when determining the optimal dose and schedule of this agent. In addition, cytokine levels remained elevated for patients on a Q2W schedule at the time of re-treatment, suggesting the possibility of a lack of resolution from the previous dose, and the potential of a less robust immune response for subsequent doses. This was an exploratory study with, unfortunately, a small number of patients. It should be emphasized that the results showing statistical significance should be regarded as trends. The findings presented here, however, may help to guide future schedule and dosing regimens for NHS-IL12 as monotherapy or in combination with other therapies in the clinic.
Supplementary Material
HIGHLIGHTS.
The effect on multiple components of the peripheral immunome from the first-in-human trial of the tumor targeting immune-cytokine NHS-IL12
Greater immune activation is seen with both a higher dose and a more frequent dosing schedule of NHS-IL12
Immune analytes of patients at both baseline and early after treatment with NHS-IL12 associate with clinical response
These findings will help guide future schedule and dosing regimens of NHS-IL12
Acknowledgments
The authors thank Debra Weingarten for her editorial assistance in the preparation of this manuscript.
Funding Information
This research was sponsored and supported by the Intramural Research Program of the Center for Cancer Research, National Cancer Institute (NCI), NIH. EMD Serono Research & Development Institute, Inc., Billerica, MA, USA, an affiliate of Merck KGaA (CrossRef Funder ID: 10.13039/100004755) supplied NHS-IL12 under a Cooperative Research and Development Agreement (CRADA) with the NCI.
Footnotes
Author Statement
Conception/design: Nicole J Toney, James L Gulley, Jeffrey Schlom, Renee N Donahue. Provision of study materials or patients: Margaret E Gatti-Mays, Nicholas P Tschernia, Julius Strauss, James L Gulley, Jeffrey Schlom. Collection and/or assembly of data: Nicole J Toney, Renee N Donahue. Data analysis and interpretation: Nicole J Toney, Jeffrey Schlom, Renee N Donahue. Manuscript writing: Nicole J Toney, Jeffrey Schlom, Renee N Donahue. Final approval of manuscript: all authors. All authors reviewed and approved the manuscript prior to submission.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Appendix A. Supplementary material.
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Data Availability
Data will be made available on request.
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Data Availability Statement
Data will be made available on request.