WHAT IS ALREADY KNOWN ON THIS TOPIC

IL-7 is a cytokine that is essential in the role of T-cell development, proliferation and homeostasis. Administration of IL-7 to people with HIV (PWH) without significant immune reconstitution following antiretroviral therapy and in patients with idiopathic CD4+ lymphopenia was associated with circulatory T-cell increases. We hypothesize that IL-7 treatment may be beneficial in Kaposi sarcoma (KS), where CD4+ T cell lymphopenia is associated with KS onset. There are no studies evaluating IL-7 in KS.

WHAT THIS STUDY ADDS

In participants with KS (with and without HIV) who received IL-7 (efineptakin alfa (NT-I7)), there were increases with number of CD4+ and CD8+ T cells, in particular among PWH who had low CD4+ T cell counts at baseline. Of evaluable participants, 42% had partial response to IL-7.

HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY

NT-I7 administration demonstrated preliminary evidence of activity among PWH in whom CD4+ T cell counts are associated with KS onset and persistence. With up to four intramuscular injections, this treatment may be beneficial in limited-resource settings, such as sub-Saharan Africa, where KS is highly prevalent. However, more data are needed to identify an optimal dose of IL-7 in this population.

Introduction

Kaposi sarcoma (KS) is an angioproliferative tumor caused by KS herpesvirus (KSHV), also known as human herpesvirus 8.1 There are five major established epidemiological variants of KS: (1) a classic form that is typically observed in elderly men, (2) an endemic subtype that is prevalent in sub-Saharan Africa, (3) epidemic KS that is seen among individuals with HIV infection, (4) an iatrogenic form that is noted in transplant-recipients, and (5) among HIV-negative men who have sex with men.2 In the USA, epidemic KS is the most common epidemiological subtype and KSHV transmission is high among men who have sex with men.3 4 KSHV is endemic in sub-Saharan Africa, and due to the high prevalence of HIV, KS is the leading cause of morbidity and mortality among men in this region.5 In these epidemiological subtypes, there is a strong association between immune dysregulation of the host and KSHV infection that leads to the onset and persistence of KS. Several studies have shown significantly lower total absolute lymphocyte counts (ALCs) and CD4+ T-cell counts in patients with classic and HIV-associated KS as compared with age-matched and sex-matched controls.6 7 HIV-associated KS is also associated with increased frequencies of exhausted and immunosenescent T-cells, with lower frequencies of naïve T-cells.7

Among people with HIV (PWH), initiation of antiretroviral therapy (ART) leads to immune reconstitution and, in some cases, increases in T-cell responses against KSHV, which may lead to beneficial KS responses.8 9 ART is an essential component of treatment for HIV-associated KS and, in some cases, ART alone can lead to regression of mild KS with limited cutaneous involvement.10 For patients with extensive KS and those with other epidemiological KS subtypes requiring systemic treatment, the existing standard first-line treatment options include chemotherapy agents, such as liposomal doxorubicin or paclitaxel, used intermittently over many years.11 However, prolonged chemotherapy administration may contribute to immunosuppression, which may impair immune reconstitution and increase the risk of opportunistic infections and secondary malignancies.12 As KS pathogenesis is related to immune dysregulation, immunotherapy options that attenuate senescence and augment immune surveillance may be favorable treatment approaches in KS.13

Several immunotherapy and immunomodulatory options have been investigated in KS. Immunomodulatory agents, such as low-dose interferon-alpha 2b and pomalidomide have been approved for HIV-associated KS, and pomalidomide is also approved in patients with HIV-negative KS.14 15 In addition to decreasing T-cell senescence and increasing T-cell activation, pomalidomide has been shown to transiently increase CD4+ and CD8+ T-cell counts.16 17 Pembrolizumab, an anti-programmed death-1 (PD-1) inhibitor, has been evaluated in HIV-associated, classic and endemic KS. Pembrolizumab has been shown to have an acceptable safety profile along with evidence of activity against KS in several studies.18 19 In addition to enhancing CD8+ effector antitumor responses in cancer, anti-PD1 agents may contribute to antitumor activity by expanding CD4+ T-cell functions and T-cell receptor diversity.20 21 Given that these agents are noted to modulate and increase CD4+ and CD8+ T-cells and their functions as one of many mechanistic actions, identifying a therapeutic option that specifically increases and potentiates these T-cell populations may be beneficial for the treatment of KS.

Interleukin-7 (IL-7), which is produced by endothelial, epithelial, and stromal cells in the bone marrow, thymus, and lymph nodes plays an important role in T-cell development and homeostasis throughout life.22 23 This cytokine stimulates T-cell proliferation (including naïve T-cells) and prolongs survival of mature T-cells. Several studies have demonstrated increases in serum or plasma IL-7 levels in patients with HIV-associated, chemotherapy-associated or idiopathic CD4+ T lymphopenia.24 25 In HIV infection and other lymphopenic states, a significant inverse correlation has been observed between serum IL-7 levels and CD4+ T-cell counts (below 250 cells/µL) due to a compensatory homeostatic response.26 Administration of short-acting recombinant IL-7 in PWH has been shown to increase peripheral T-cells, broaden T-cell receptor diversity, and increase CD4+ and CD8+ T-cell counts.27–29 IL-7 was also administered to patients with cancer and resulted in a similar effect on T-cell profiles.30

Efineptakin alfa (NT-I7, also known as rhIL-7-hyFc) is a long-acting immunoglobulin fusion protein composed of a recombinant form of endogenous human IL-7 fused to a hybrid crystallizable fragment region that extends the half-life of this compound. NT-I7 was well-tolerated and increased ALC, CD4+, and CD8+ T-cell counts following a single dose as a monotherapy agent in healthy volunteers.31 Apart from regulatory T-cells, all the other T-cell subsets proliferated and expanded for 56 days in a dose-dependent manner as shown in a previous study.25 It is hypothesized that administration of NT-I7 may lead to enhanced T-cell mediated antitumor immune responses, specifically in individuals with low numbers of circulating T-cells.

Here, we report the results of Cancer Immunotherapy Trials Network 17 (CITN-17), a phase I dose-escalation multicenter study of NT-I7 in patients with KS. This study evaluated the safety of NT-I7 monotherapy, its impact on CD4+ and CD8+ T-cell kinetics, and the antitumor activity in participants with KS with and without HIV.

MethodsStudy design, safety monitoring and study participants

CITN-17 is a multicenter, phase I, single-arm study with a 3+3 dose escalation design of NT-I7 that was initiated in 2021 in the USA. Four doses of NT-I7 were administered intramuscularly every 9 weeks, and three dose levels (DL) were investigated (480 µg/kg, 960 µg/kg and 1200 µg/kg). Dose-limiting toxicities (DLTs), defined as any adverse event (AE) related to NT-I7 and met at least one of the hematologic or non-hematologic criteria as defined in the protocol, were evaluated in the first 28 days of treatment. Participants >18 years, Eastern Cooperative Oncology Group <2, with histologically confirmed KS and evidence of evaluable disease per the AIDS Clinical Trials Group (ACTG) criteria were eligible. Any stage of KS was permissible; however, participants with visceral disease were included if they did not require immediate intervention and had limited tumor-related symptoms. PWH were required to be on effective ART for at least 3 months with inadequate regression of KS or persistent KS affecting their quality of life. PWH were required to have an undetectable HIV viral load (VL) of <40 copies/mL.

Previous studies of IL-7 in PWH had demonstrated HIV viremia (“blips”) of >50 copies/mL of unclear clinical significance.29 Therefore, HIV VL was measured prior to every cycle and week 4 and 13. If there was an increase in HIV VL to over 400 copies/mL, the next cycle was held until HIV VL was <400 copies/mL with an evaluation of medication adherence, and HIV resistance testing was performed if HIV VL was >1000 copies.

KS response was evaluated using the modified ACTG KS response criteria15 32 prior to every cycle (weeks 0, 9, 18 and 27), week 13 and week 36 at the end of treatment (EOT). The primary objective was to determine the safety and AE profile of NT-I7 in three DL and estimate the maximum tolerated dose (MTD). Safety was evaluated using the National Cancer Institute Common Terminology Criteria for Adverse Events V.5.0. The secondary endpoints included an evaluation of the objective response rate of NT-I7 in participants with KS and the kinetics of CD4+ and CD8+ T-cells in the blood during the study.

Correlative studies

Clinical cell counts: Complete blood count testing to determine ALC, and CD4+ and CD8+ T-cell count testing were performed on longitudinally collected, locally processed blood samples at each clinical site using CLIA-certified assays.

Whole blood immunophenotyping: The T-cell subsets were defined using the following flow cytometry gating criteria: CD4 T cells (CD45+/CD3+/CD4+); CD8 T cells (CD45+/CD3+/CD8+) Central Memory (CD45RA−/CD197+(CCR7)); Effector Memory (CD45RA−/CD197−); Terminal Effector (CD45RA+/CD197−); and Tscm (CD45RA+/CD197+/CD27+CD28+/CD95+/CD127+).33 34 The absolute number of Naive (CD4+ or CD8+) T cells was determined by subtracting the number of Tscm from the absolute number of (CD45RA+/CD197+) CD4+ or CD8+ T cells, respectively.

The change in absolute numbers, frequencies and phenotypes of T cells and other PBMC subsets were evaluated at the Central Immune Monitoring Lab (CIML) at Fred Hutchinson Cancer Center by flow cytometry using whole blood received and processed within 24 hours of blood draw.

To assess the absolute numbers of lymphocyte subpopulations in the periphery, whole blood collected in ACD vacutainers was stained (100 µL, ~24 hours after collection) in Trucount tubes (BD 340334) with a validated 14-color antibody panel staining mix containing 1x Brilliant Stain Buffer (BD 563794), FACS Buffer (2% FBS in DPBS (Gibco 14190144)) and the following antibodies: CD3 BUV805 (BD 612895), CD4 BUV661 (BD 612962), CD8 BUV496 (BD 564804), CD28 BB700 (BD 745905), CD45 BUV395 (BD 563792), CD45RA vioGreen (Miltenyi Biotec 130-113-369), CD127 PE-Cy5 (BioLegend 351324), CD197 APC-Fire 750 (BioLegend 353246), CD279 PE-Cy7 (BD 561272), CD366 BV421 (BioLegend 345007), CD185 BB515 (BD 564624), CD95 PE (BioLegend 305608), CD27 BV650 (BD 563228), and CX3CR1 PE-Dazzle 594 (BioLegend 341624). The samples were incubated at room temperature for 15 min in the dark. Subsequently, 900 µL of 1x FACS Lysing Buffer (BD 349202) was added to stained samples for 15 min at room temperature before freezing at −80°C. Thawed samples were collected in batches on a BD Symphony A1 and analyzed using FlowJo V.10.8 software (BD Life Sciences).

Immunogenicity and serum NT-I7 concentration: Serum NT-I7 concentration and anti-drug antibodies (ADA) blood draw specimens were processed on site at local labs immediately after collection and shipped in batch on ice to CIML. CIML organized batch shipments of samples to BioAgilytix (Durham, NC) for analyses. ADA and neutralizing ADAs (NADA) to NT-I7 were evaluated before the first dose of NT-I7, prior to each cycle and at the EOT using a risk-based, tiered bridging electrochemiluminescence ELISA-based testing approach. The assay included a core assay comprising screening, confirmatory, and titer testing for detecting ADA to NT-I7 and an IL-7 epitope-specific assay. ADA-positive patient serum samples were further tested for the presence or absence of NADA using a cell-based assay.

Serum NT-I7 concentrations were quantified in samples obtained prior to administration of study therapy at each cycle, 3 hours post NT-I7 administration, at week 1, 9 weeks and at EOT. The assay to evaluate serum NT-I7 did not specifically differentiate between endogenous IL-7 and the study therapy. NT-I7 concentrations were quantified using an electrochemoluminiscent sandwich immunoassay in which anti-huIL-7 capture antibody was immobilized, analyte was bound to the capture antibody, and SULFO-TAG anti-huIL-7 detection antibody was used to generate an electroluminescent response that is directly proportional to the amount of analyte detected in the well. Back-calculated concentrations of NT-I7 were generated using a 5-parameter logistic curve fit with 1/y2 weighting.

Statistical analysis

The primary objective of this study was to determine the safety and the MTD of NT-I7 in patients with KS. The safety analyses included data from participants who received at least one dose of NT-I7. The KS antitumor effect was evaluated using the modified ACTG criteria.15 32 The best response obtained within 12 months of the first dose of NT-I7 was used in all evaluable participants across the study doses. ALC, CD4+ and CD8+ levels and flow-cytometry enumeration data were evaluated by non-parametric analyses using the Wilcoxon signed-rank test. Differences were evaluated between responders and non-responders (which included one participant who experienced a DLT and came off treatment at week 5, and therefore, was not evaluable for the first KS response assessment at week 9) using Mann-Whitney U test (Wilcoxon rank sum test) and Wilcoxon signed rank test, as appropriate. These p value analyses were not adjusted for multiple comparisons. CD4+ and CD8+ T-cell ratio kinetics over the course of the study were evaluated using a t-test assuming unequal variances; and the overall p value was based on a mixed model fitted with treatment interval and responder status fitted as fixed effects adjusted for baseline, and a random intercept allowed. ADA data were evaluated using the Student’s t-test (two tails, type equal variance).

ResultsStudy participant characteristics

Between May 2022 and December 2023, eight cisgender male participants were enrolled on the study; six participants were enrolled to DL1 and two participants were enrolled to DL2. Among all participants, the median age was 55 years and 75% of participants were white (table 1). 75%t of all participants had stage T0 KS, and all participants with T0 disease were enrolled in DL1. Both participants in DL2 had stage T1 disease; one of these participants had pulmonary KS involvement and recurrent pleural effusions. Half of all the participants had prior systemic therapy, which consisted of chemotherapy. Among participants enrolled in DL1, two participants had prior radiation, and one participant had prior surgical removal of KS. Five of the eight participants had an HIV diagnosis, including both participants in DL2. Overall, the HIV VL was undetectable (<40 copies/mL) for PWH at baseline. Among all enrolled participants, the median CD4+ T-cell count was 650 cells/µL, CD8+ T-cell count was 592 cells/µL and median CD4+/CD8+ ratio was 1.28. Participants in DL2 had a lower median CD4+ T-cell count of 119 cells/µL and a CD4+/CD8+ ratio of 0.22 compared with participants in DL1.

Table 1

Baseline participant and disease characteristics among all enrolled participants and by dose level of study therapy

Treatment administration and safety

Among all the participants, the median number of NT-I7 treatments administered was 3.5 doses. Four of the eight participants (three in DL1 and one in DL2) completed all four doses of the NT-I7. The reasons for discontinuation among the other four participants were a DLT, disease progression, withdrawal by a participant and one participant discontinued treatment per physician discretion.

All participants were evaluable for DLTs. All NT-I7-related toxicities were grade 1–2 AEs (table 2). Among all eight participants, the most common AE related to the NT-I7 was a grade 1–2 local injection site reaction that was observed in seven participants. The injection site reactions were managed with antihistamines and topical steroids on the study. One participant who experienced a grade 2 injection site reaction following the first dose opted to come off treatment 5 weeks after cycle 1, thus meeting the criteria for a DLT and serious AE (SAE). A second participant in DL2 had an SAE due to hospitalization as a result of grade 2 infection and grade 3 chylothorax that was not related to NT-I7 administration. This participant had pleural effusions at baseline related to KS and chose to discontinue treatment following the hospitalization. Other toxicities included grade 1–2 alanine aminotransferase, and aspartate aminotransferase increases noted in 67% and 50% of the participants in DL1 but not among participants in DL2. Grade 2 increases in ALC were noted in 50% of all participants, which is expected per the activity of the NT-I7.

Table 2

Treatment-emergent adverse events (TEAE) that were judged as possibly related, probably related, or definitely related to study drug

Among participants with HIV, two participants (one in each DL) had an increase in the HIV VL between 40 and 200 copies/mL at week C2W09, but the HIV VL returned to undetectable levels following C3W18.

KS activity and outcomes

One participant who experienced a DLT within the first cycle came off treatment at week 5 and, therefore, was not evaluable for the first KS response assessment at week 9 prior to the second cycle of treatment. Seven participants were evaluable for KS response: three participants experienced a partial KS response (PR), three participants had stable disease as a best response and one participant without HIV had progression of KS. All participants who had a response were PWH, and two of the three responders received DL2 doses (figure 1A,B). The overall objective response rate to NT-I7 was 42.9% (95% CI 9.9%, 81.6%). There were no deaths reported in the study.

Figure 1

Example of partial response to NT-I7 therapy on study. (A) Study participant with target KS lesion at C1W00 prior to NT-I7 administration. (B) Study participant with evidence of confirmed partial response to NT-I7 at C3W18 with evidence of lightening and flattening of target lesion 4. KS, Kaposi sarcoma.

ALC, CD4+ and CD8+ T-cell kinetics

The pharmacodynamic impact of NT-I7 on lymphocyte populations is summarized in figure 2 and online supplemental table 1, and individual data are shown in online supplemental figure 1. Following the first dose of NT-I7, there was a minimal increase in ALC after the first week (p=0.38). However, the ALC peaked significantly (~3.5-fold, figure 2A) from baseline at week 4 (C1W04) (p=0.02). There was a significant elevation in the ALC from baseline to C2W13 and C3W18. Missing data at C4W27, EOT and at safety follow-up in four participants limited further evaluation of these time points. CD4+ T-cell increases (~5-fold from baseline) and CD8+ T cell increases (~3.5-fold from baseline) (figure 2B) were noted at C1W04 and began to decrease thereafter but remained elevated compared with the baseline till C2W13 (p=0.03). Notably, the numbers of observed circulating T cells did not increase above the C1W04 peak, despite subsequent doses of NT-I7. To investigate more specifically which T cell subsets were affected by treatment, we performed high-dimensional flow cytometry on whole blood. The overall profile of these analyses paralleled the clinical counts. For both CD4+ and CD8+ T-cells, all measured subsets increased significantly, including the effector memory and central memory subsets, which increased proportionally from C1W04 (p=0.003(CD4+CM); p=0.0003 (CD8+CM); p=0.005 (CD4+EM); p=0.001 (CD8+EM) figure 2C,D). Importantly, within CD4+ T cell subsets, there was a significant expansion of the naïve T-cell subsets from baseline to C1W04 (p=0.02) which persisted through the course of treatment (figure 2C). In both the CD4+ and CD8+ compartments, there was rapid expansion of the memory stem T-cell (Tscm) subset by C1W01 (figure 2C,D) which appeared to be the most affected subset of CD8+ T cells (figure 2D). Importantly, for both CD4+ and CD8+ T-cells, terminal effector cells had also increased from baseline.

Figure 2

Impact of NT-I7 (IL-7) on ALC, T-cells and T-cell subsets across study. Red arrows above time points indicate NT-I7 administration. (A) ALC changes across the study in all participants, absolute counts (left panel) and fold-change from baseline (right panel). (B) Absolute clinical cell counts (left panel) and fold-change (right panel) from baseline of CD4 and CD8 T cells among all participants at time points on the study. *Indicates statistical significance where p<0.05 in paired analyses compared with baseline. Blue asterisk corresponds to CD4 T cells and red astrisk corresponds to CD8 T cells. (C) Changes in absolute CD4 T-cell differentiation subsets (left panel) and fold change (right panel) over the course of the study. (D) Changes in absolute CD8 T-cell differentiation subsets (left panel) and fold change (right panel) over the course of the study. ALC, absolute lymphocyte count; CM, central memory; EM, effector memory; EOT, end-of-treatment; N, Naïve; TE, terminal effector; TSCM, memory stem T-cell.

We evaluated whether changes in ALC and in the CD4+ and CD8+ T-cell numbers and/or fold-expansion with study treatment were related to NT-I7 clinical responses in all participants. Evaluable participants with objective KS responses to NT-I7 treatment in this study were PWH. The absolute numbers and fold-change in ALC, CD4+ and CD8+ T-cells did not change by treatment response (figure 3A–C, online supplemental table 1). Among the treatment responders, the median ALC (figure 3A) and CD4+ T-cell counts (figure 3B) were lower at baseline, although not statistically significantly different as compared with those who did not respond to NT-I7 (p=0.39 for ALC, p=0.22 for CD4+cells). There were no statistically significant differences in CD8+ T cell counts or fold-change from baseline between responders and non-responders over the course of the study (figure 3C). The median CD4+/CD8+ ratio at baseline for seven evaluable participants was 0.98 and at C2W13 was 1.18. Interestingly, when comparing the CD4+/CD8+ ratio by response, there were statistically significant differences in CD4+/CD8+ ratio between treatment responders and non-responders at both baseline (p=0.011) and throughout the treatment (figure 3D); with responders having a lower CD4/CD8 ratio. Despite persistently low CD4+/CD8+ ratios as compared with those who did not respond to NT-I7, participants with response reported decrease in KS-associated edema and had objective improvement in the KS lesions (figure 1A,B).

Figure 3

Changes in ALC, CD4 and CD8 T-cells relative to clinical response status to NT-I7. (A) ALC absolute (left panel) and fold-change (right panel) changes by response. (B) Absolute (left panel) and fold-change (right panel) CD4 T-cell clinical counts and changes by response. (C) Absolute (left panel) and fold-change (right panel) CD8 T-cell clinical counts and changes by response. (D) CD4/CD8 ratio by response to NT-I7. Statistically significant difference between responders and non-responders is noted. Non-responders include one participant who experienced a DLT and was not evaluable for response. ALC, absolute lymphocyte count; DLTs, dose-limiting toxicities.

Immunogenicity assessment

Treatment-emergent ADA and their effect on NT-I7 serum concentrations, pharmacodynamics and safety were also analyzed as part of the safety assessment. ADA was not detected at baseline in any of the seven participants tested (0/7, 0%). After two doses of NT-I7 (C2W09), six participants developed treatment-emergent ADA (6/7, 85.7%), even if titers were low (ADA titer range 30–2430). Interestingly, one participant who did not develop ADA was receiving the higher DLs (960 µg/kg) and had the lowest CD4 T cell counts at baseline (59 cells/µL). ADA was persistent and remained detectable over the follow-up period. Five ADA samples had neutralizing activity at the first time of detection (5/6, 83.3%) while the remaining sample showed neutralizing activity at a later time point (EOT, week 18).

NT-I7 serum concentrations (figure 4A) were easily detectable 3 hours after administration, significantly decreased by C1W01 and were back to baseline before the next administration. Each subsequent dose of NT-I7 resulted in an increase in NT-I7 concentration, and higher in the higher DL participants, but showing decreased peak concentrations with each subsequent dose of NT-I7 administration (figure 4B). However, development of ADA did not affect the pharmacodynamics of NT-I7 and could be a physiological mechanism to maintain ALC levels within physiological range. Despite the low sample number in this study, and consistent with this hypothesis, ADA was developed at peak increase of ALC and ALC levels remained stable thereafter (figure 4C). The participant with a treatment response who did not develop ADA had significantly lower baseline ALC (0.63 vs an average of 1.85 k/µL) and lower peak ALC (2.07 vs an average of 6.5 k/µL) (figure 4D). The ADA levels and serum NT-I7 levels by participant are noted in online supplemental figure 2.

Figure 4

NT-I7 serum concentrations, anti-drug antibodies (ADA) titers and absolute lymphocyte counts (ALC) measurements in study participants. (A) NT-I7 serum concentrations after a single dose of NT-I7, by dose level. C1W00: predose; C1W00H3: 3 hours postadministration; C1W01: 168 hours postadministration; C1W09: 1512 hours postadministration. (B) NT-I7 serum concentrations before and 3 hours postadministration for the first three doses, by dose level. (C) ALC measurements and development of ADA during study. (D) ALC levels over the course of study in participants with treatment-emergent ADA and one participant who did not develop ADA. Gray areas in (C, D) between 2.5 and 4.5 K/µL denote lymphocytosis.

Discussion

In this study evaluating IL-7 in participants with KS, doses of NT-I7 of 480 µg/kg and 960 µg/kg in eight participants were associated with grade 1 and 2 treatment-related AEs. Treatment was safe among PWH with no significant impact on HIV VL. However, the primary objective was not met due to early study closure following termination of funding. Despite early termination, we noted that among seven response-evaluable participants, the majority of whom had prior systemic KS therapy a response in a subset of participants. The overall response rate of NT-I7 was 42.9%. KS responses in this study were observed in three participants with HIV. These PWH with KS responses to NT-I7 had low CD4+/CD8+ T-cell ratios of less than 0.5 that were persistently low as compared with those whose KS did not respond to treatment. Among all participants who received NT-I7, there was an expansion of several T-cell subsets, including CD4+ naïve T-cells, CD8+ memory stem T-cells and effector T-cells in both CD4+ and CD8+ subsets that may have impacted KS activity in those with lower CD4+ and CD8+ T-cell counts.

In this small study of seven evaluable participants, the overall response rate of NT-I7 in KS is lower than response rates observed with other cytotoxic chemotherapy agents for KS which ranges from 50% to 70%,35 36 and immunomodulatory therapies such as pomalidomide or lenalidomide with response rates of 60%–70%.15 37 However, CITN-17 enrolled a heterogeneous population of participants with and without HIV as well as those with and without prior systemic KS therapy. Many of the seminal studies evaluating KS therapies have focused on participants with HIV. Three of the five PWH (60%) in this study experienced a KS response to NT-I7, which is similar to other aforementioned studies evaluating treatments in HIV-associated KS. With regard to safety, there were no grade three events related to the study therapy. The most common AEs were local injection site reactions that were managed with antihistamines and topical steroids and with oral steroids in one participant. An injection site reaction was a DLT in one participant at a dose of 480 µg/kg but this was not observed in other participants who were treated with NT-I7 at the higher DL.

KS is a malignancy that arises in the context of inadequate immunological reconstitution. Low CD4+/CD8+ ratios have been shown to increase the risk of KS incidence in a large cohort of PWH.38 Low CD4+/CD8+ ratios are also associated with increased markers of T-cell senescence, activation and exhaustion despite well-controlled HIV39 40 indicate the presence of immune dysregulation and inflammation; these characteristics are considered hallmarks of cancer.41 At baseline, participants with a KS response in this study had lower CD4+/CD8+ ratios as compared with non-responders. Lower CD4+/CD8+ ratios between responders and non-responders in the CD4+/CD8+ ratio persisted from baseline to C2W13. Moreover, we did not observe increases in the CD4+/CD8+ ratio during the study that may account for KS response. The reasons for this apparent incongruity are unclear but may be due expansion of T-cell subsets by IL-7. More specifically, there may be differential expansion and/or activation of minor T-cell subsets, changing the balance between regulatory and effector T cell mechanisms leading to this observation. Such minor subset ratio differences or balances may not be readily conspicuous in our assays and may not effect changes in overall CD4+/CD8+ ratios over the course of treatment.

IL-7 through its activation of the JAK/STAT pathway has been shown to be essential for B-cell and T-cell development, proliferation and survival.23 42 Among all study participants, there were increases following IL-7 administration in the fold-change of several CD4+ and CD8+ T cell subsets. The proportion of CD4+ naïve T cell populations appeared to be higher, and the proportion of memory stem T-cells were higher in the CD8+ T cell subsets, after dosing. Central memory T cells also increased in both CD4+ and CD8+ T cell subsets. These changes were seen within 1 week of treatment administration and persisted with the subsequent doses of NT-I7. The changes observed in the T-cell subsets were similar to other studies evaluating IL-7 administration in PWH,29 patients with metastatic melanoma,30 and those receiving treatment following allogenic hematopoietic stem cell transplantation, where naïve T cell subsets increased.43 Though this analysis did not specifically evaluate regulatory T-cell subsets, which have immunosuppressive properties, several studies investigating administration of IL-7 and NT-I7 have shown increase in naïve and central memory T-cell subsets without increases in regulatory T-cells in patients with lymphopenia.29–31 44 Other possible antitumor mechanisms of IL-7 in responders may be due to the observed increases in naïve T-cell and central memory T-cell subsets. Increases in these subsets may augment immune response to tumor antigens and improve adaptive immunity in KS. PWH with KS have been shown to have higher levels of immunosenescent T-cells and lower naïve T-cells as compared with PWH alone.7 Therefore, treatment responses observed in PWH within this study may be due to the greater degree of immune dysregulation among PWH and KS. There are ongoing studies of KSHV-specific T cell responses and T-cell receptor changes in the KS tissues that may help further our understanding of the mechanism of action of IL-7 and its impact on KS.

In patients with lymphopenia, previous analyses have shown that endogenous IL-7 levels are high due to homeostatic regulation of T-cells.26 Consistent with physiological homeostatic regulation of both IL-7 levels and T cell counts, we observed treatment-emergent ADA that seem to maintain ALC levels at high but physiological levels. Correspondingly, the only participant who did not develop ADA had the lowest baseline ALC and peak ALC and did not need homeostatic compensatory mechanisms. Due to the limited number of participants in this study, as well as the heterogeneity of the study population, it is challenging to draw definitive conclusions from these data. However, we did not observe any relationship between treatment-emergent ADA and efficacy or safety outcomes.

Previous studies have demonstrated increases in HIV VL or “blips” following IL-7 administration.28 45 All participants in the study were required to have an undetectable HIV VL at baseline and two participants had increases in HIV VL up to 200 copies/mL at C2W9 that subsequently resolved prior to the next cycle. An analysis of the HIV reservoir was not evaluated in this study. Vandergeeten et al demonstrated that IL-7 led to HIV persistence among participants receiving ART by enhancing residual levels of viral production and inducing proliferation of latently infected CD4+ T cells.46 Though IL-7 alone may not contribute to eradication of HIV, combination strategies may help advance the treatment of both KS and HIV. A recent study of pembrolizumab, an anti-PD-1 inhibitor, in participants with HIV-associated KS resulted in a response rate of 62%.47 The same anti-PD-1 inhibitor led to reversal of HIV latency in CD4+ T-cells after the first infusion in participants with HIV and cancer.48 Combining IL-7 with a PD-1 inhibitor would be a logical next treatment approach and may be beneficial as a “kick and kill” strategy for the HIV latency reservoir but also as two agents that independently have demonstrated activity in KS.

There were important limitations in this study that impact our findings. Most significantly, the limited study population impairs our ability to fully evaluate the primary objective of identifying an MTD of NT-I7. Moreover, the participants have different epidemiological subtypes of KS; therefore, it is unclear whether the responses observed in PWH were due to chance or a biological effect of the therapies. Despite these limitations, this study provides important insights on the use of IL-7 as a therapy for KS. Though IL-7 has been shown to be useful in CD4 T-cell restoration among PWH, clinical benefit in a specific disease state has not been clearly demonstrated in prior studies. Evidence of preliminary activity of IL-7 in a malignancy associated with CD4+ lymphopenia is clinically meaningful. Additionally, as IL-7 has a reasonable safety profile and does not require intravenous administration, this may be beneficial in limited-resource settings, such as certain areas of sub-Saharan Africa, where KS is highly prevalent and infusion-based therapies and management of chemotherapy-related toxicities may be challenging.

In summary, NT-I7 administration was safe among participants with KS and led to increases in overall lymphocyte counts and increases in naïve and central memory T-cell subsets. Administration was also associated with KS responses in 42% of participants. NT-I7 demonstrated a similar safety profile to prior studies of IL-7 administration with evidence of preliminary activity of KS in PWH. Future studies need to fully evaluate the appropriate dose of NT-I7 in this population to identify the MTD.

Data availability statement

All data relevant to the study are included in the article or uploaded as supplementary information.

Ethics statementsPatient consent for publication

Consent obtained directly from patient(s).

Ethics approval

This study involves human participants and was approved by National Cancer Institute Central Institutional Review Board and participating sites (reference/ID number: CITN-17). Participants gave informed consent to participate in the study before taking part.

Acknowledgments

We thank participants and their families for their participation in this study. We also thank the medical teams at each research site who cared for study participants. We thank Adam Rupert and Kyndal Cook at the AIDS Monitoring Laboratory for their analysis of serum IL-7 levels in participant samples. This work was supported in part by a grant from the National Cancer Institute (UM1CA154967) and funding from NIT.