Interleukin-Based Treatments Find Footing in Ongoing Research

Targeted Therapies in Oncology, August 2022, Volume 11, Issue 11

Current- generation cytokines are being engineered to extend their half-life and improve tumor targeting using polyethylene glycol conjugation, fusion to tumor-targeting antibodies, alterations of cytokine/cell receptor–binding affinity, and other strategies.

The discovery of antitumor activities of pro-inflammatory cytokines in animal models led to subsequent approvals of recombinant interferon alpha (INF-α) and IL-2.1 Challenges of targeted cancer treatment with cytokines include the short half-life of cytokines in the circulation, significant toxicities at therapeutic doses, and initial lack of efficacy.2,3 Subsequent immunotherapies such as immune checkpoint inhibitors (ICIs) and chimeric antigen receptor T cells were found to be more effective, and their use superseded and overshadowed that of cytokine-based therapies.1,3 Advances in genomics, molecular engineering, and molecular analysis have helped renew interest in the antitumor properties of cytokines and their use as single agents or in combination with other immunotherapies such as ICIs.1,3 Current- generation cytokines are being engineered to extend their half-life and improve tumor targeting using polyethylene glycol conjugation, fusion to tumor-targeting antibodies, alterations of cytokine/cell receptor–binding affinity, and other strategies.1,2 A significant number of recent studies have evaluated the clinical use of these cancer therapeutics based on numerous cytokines that include, but are not limited to, IFN-α, IL-2, IL-15, IL-21, IL-12, and IL-10.1-3

Svetomir N. Markovic, MD, PhD, a professor of medicine and oncology and consultant in the Division of Medical Oncology at Mayo Clinic in Rochester, Minnesota, discussed cytokine-based cancer treatments in an interview with Targeted Therapies in Oncology™. Markovic noted that initial antitumor cytokines were used like most chemotherapeutic and cytotoxic drugs.

Cytokines are hormones of the immune system that were developed the way we develop most drugs in cancer, by giving people the maximally tolerated dose.” He pointed out that cytokine therapies, in contrast, should likely follow the “Goldilocks principle.” “There are optimal doses for hormones.

Think of diabetes and insulin; there can be too much, or too little, or just right.” This is likely the reason behind the initial poor performance of recombinant IFN-α and IL-2. “The IL-2 doses gave us approximately 30 seconds of benefit and then the body started fighting us at every additional treatment, trying to shut down all of the toxicity we were generating.”

Here we discuss some of the most recent trial results and upcoming trials designed to evaluate new cytokine-based cancer therapies.

Recent Trials


SOT101 is a fusion protein consisting of IL-15 linked to the Sushi domain of IL-15 receptor α that displayed a favorable safety profile and promising preliminary efficacy signals in a phase 1 study enrolling patients with advanced solid tumors (NCT04234113).4 This dose-escalation study is evaluating subcutaneous injections of SOT101 as monotherapy and in combination with pembrolizumab (Keytruda) administered in 3-week cycles.4

A total of 51 patients have been treated: 30 with monotherapy and 21 with combination therapy.4 Pyrexia, chills, lymphopenia, anemia, transaminase elevation, and vomiting were the most common treatment-emergent adverse events (TEAEs), and all were transient.4 The majority of TEAEs were mild (grade 2 or less), and no treatment-related deaths were observed. A recommended phase 2 dose of 12 μg/kg for SOT101 has been determined.4

The observed clinical benefit rate of SOT101 monotherapy was 38% among the 13 patients who received doses of 6 μg/kg to 12 μg/kg. Dose ranges for the monotherapy group were 0.25 μg/ kg to 15 μg/kg and 1.5 μg/kg to 12 μg/kg in the combination therapy group.4 A single patient with ICI-refractory squamous cell carcinoma of the skin experienced a partial response (PR; duration, 46 days), and 4 ICI-pretreated patients had stable disease (SD) ranging from 33 to 183 days.4 The median duration of clinical benefit was 190 days, with a median duration on treatment of 84 days.4

The observed clinical benefit rate in patients receiving the pembrolizumab combination was 63% across all SOT101 doses.4 A single ICInaïve patient with mesothelioma experienced a complete response (CR) that was ongoing in cycle 5 as of January 2022.4 Three patients (2 ICI-pretreated) experienced PR (range, 51-232 days; 2 ongoing), and 5 patients (3 ICI pretreated) experienced SD (range, 92-340 days; 2 ongoing).4 The preliminary median duration of clinical benefit for combination treatment was 131 days, with a median treatment duration of 113 days (range, 7-429).4

N-803 Plus BCG

Intravesical administration of N-803 (Anktiva), a mutant IL-15–based immunostimulatory fusion protein complex (IL15RαFc), in combination with BCG in BCG-naïve patients with non–muscle-invasive bladder cancer (NMIBC) resulted in CRs in all patients for the study duration of 24 months in a recent phase 1b trial.5 This open-label, 3-cohort, multicenter trial (QUILT-3.032; NCT03022825) evaluating intravesical injection of N-803 with BCG in BCG-unresponsive high-grade NMIBC resulted in significant response rates and clinical outcomes.5

Primary end points included CR at any time for patients with carcinoma in situ (CIS) and disease-free survival (DFS) rate at 12 months for patients with papillary NMIBC.5 A total of 160 patients 83 CIS, 77 papillary) have been enrolled to date, with a median of 12 prior BCG doses and a mean of 4 prior transurethral resections of bladder tumors.5 Patients with CIS had a CR rate of 71% (59/83), and the median duration of CR was 24.1 months; 91% avoided cystectomy and 96% achieved 24-month bladder cancer–specific progression-free survival (PFS; progression was defined as progression to MIBC).5 Patients with papillary disease had a 57% 12-month DFS rate and a 48% 24-month DFS rate, and 95% avoided cystectomy. 5 For responders requiring cystectomy (n = 4), median time to procedure was 12.9 months vs 7.8 months for nonresponders requiring cystectomy (n = 8).5

No grade 4 or 5 treatment-related AEs (TRAEs) were observed, and the most common grade 1 or 2 TRAEs were dysuria (22%), pollakiuria (19%), hematuria (18%), fatigue (16%), and urgency (12%).5 Bladder cancer–specific overall survival (OS) at 2 years was 99% in patients with BCG-unresponsive NMIBC, and the activity of N-803 was confined to the bladder, with no measurable systemic levels.5 The developer of N-803, ImmunityBio, Inc, submitted a biologics license application to the FDA on May 23, 2022, for the use of N-803 with BCG to treat BCG-unresponsive NMIBC CIS with or without Ta or T1 disease.6


SRF388 is a fully human IgG1 blocking antibody that targets IL-27, thereby preventing it from upregulating inhibitory immune checkpoint receptors and downregulating proinflammatory cytokines.7 A phase 1 dose-escalation study recruited patients with advanced treatment-refractory solid tumors to determine preliminary safety and recommended phase 2 doses of SRF388 as monotherapy and in combination with pembrolizumab (NCT04374877).7

A total of 29 patients were enrolled to receive SRF388 monotherapy doses of 0.003 to 20 mg/kg; 62% were female, 80% had prior PD-L1 blockade, and 48% had 4 or more prior therapies.7 The median age of patients was 64 years, with a median time on study of 9 weeks (range, 0-59).7 TRAEs were observed in 21% of patients, with only treatment-related fatigue reported in more than 10% (n = 3) and all AEs were considered low grade.7

A confirmed PR was observed in a patient with highly treatment-refractory NSCLC at 8 weeks; the PR was durable for 20 weeks.7 SD was observed in 9 patients (31%) at 8 weeks, with 6 of 9 maintaining durable disease control at 6 months.7 Durable disease control for more than 20 weeks was observed in 43% (n = 3/7) of patients with clear cell renal cell carcinoma (ccRCC). The pembrolizumab safety cohort (n = 10) and stage 1 of the ccRCC monotherapy expansion (n = 17) have fully enrolled.7 A phase 2 dose of 10 mg/kg was selected based on study results, with dosing every 3 to 4 weeks.7

TransCon IL-2 β/γ

TransCon IL-2 β/γ is a novel, longacting prodrug with sustained release of an IL-2Rβ/γ–selective IL-2 analogue designed to address the shortcomings of current IL-2 cancer immunotherapies such as aldesleukin (a recombinant form of IL-2; Proleukin). The severe AEs associated with these agents, such as cytokine release syndrome and vascular leak syndrome, likely result from the frequent high doses necessitated by their short half-life and high peak serum concentration (Cmax).8

In a preclinical study, cynomolgus monkeys received 2 to 3 doses of TransCon IL-2 β/γ intravenously every 2 weeks.8 TransCon IL-2 β/γ produced a robust increase in absolute lymphocyte count (ALC) driven by potent activation and expansion of CD8-positive (+) T cells, natural killer (NK) cells, and γδ T cells relative to CD4+ T cells and T-regulatory cells (Tregs).8 After administration, investigators observed the following increases compared with predose levels: greater than 20-fold in ALC, 19-foldin CD8+ T cells, 50-fold in effector memory CD8+ T cells, 18-fold in NK cells, and 400-fold in γδ T cells.8 TransCon IL-2 β/γ had a long effective half-life (> 30 hours) and a low Cmax. Levels of IL-5, IL-6, IFN-β, and tumor necrosis factor-α remained low, whereas an induction of soluble CD25 and the chemokine MCP-1 was seen.8 No signs of vascular damage, tissue necrosis, pulmonary edema, or eosinophilia were observed on histopathology.8

These results suggest the potential for substantial improvement in the therapeutic index beyond that of current recombinant IL-2 therapies. TransCon IL-2 β/γ is being evaluated in the phase 1/2 IL Believe trial (NCT05081609) as monotherapy and in combination with pembrolizumab (see page 35).8

Markovic explained the concept behind this new IL-2–based therapy. “One potential explanation for the failure of the original IL-2 treatment [aldesleukin] was that it engaged the high-affinity [α/β/γ] receptor, thereby preferentially stimulating [immunosuppressive Tregs]. If we remove the [part of IL-2 that] binds to the high-affinity receptor and [bind it to] the low-affinity [β/γ] receptor [instead], there is potential for greater benefit.”

However, Markovic has his doubts about this approach: “I think one of the reasons high-dose IL-2 failed is not just because it triggered Tregs but because it triggered such an explosion of immune activation that the entire immunoregulatory system [was stimulated] to shut it down.”

Modakafusp Alfa

Modakafusp alfa (TAK-573) is a unique immune-targeting cytokine that is also

undergoing evaluation. The agent targets CD38-expressing cells with attenuated IFNα2b moieties.9 Increased target selection is achieved by the specificity for CD38 and reduced IFN receptor–binding affinity of this agent’s 2 attenuated IFNα2b molecules, which are genetically fused to the Fc portion of a humanized, anti-CD38, IgG4 monoclonal antibody.9 Although modakafusp alfa has shown strong clinical responses and immune activation in refractory/relapsed multiple myeloma, a phase 1b/2 study (NCT04157517) was conducted to investigate its safety and preliminary efficacy in metastatic solid tumors.9

A total of 21 patients with a median age of 63 years and 3 median prior lines of therapy, 71.4% of whom had gastrointestinal malignancies, received a dose of modakafusp alfa 0.1 to 1.5 mg/kg every 3 weeks.9 TRAEs occurred in 81% of patients and included infusion-related reactions (52.4%), chills (47.6%), and nausea (33.3%); grade 3 or greater TRAEs were reported in 42.9% of patients and included neutropenia (14.3%) and hypertension (9.5%).9

A greater than dose-proportional exposure increase was observed across the dosing range, with no exposure accumulation associated with dosing every 3 weeks. All patients (100%) developed posttreatment antidrug antibodies; the investigators are currently characterizing this response and its impact.9 A best response of SD was observed in 7 of the 14 response-evaluable patients, including 1 patient with cutaneous melanoma who experienced 21% target lesion reduction.9 These results provided proof of mechanism and a recommended phase 2 dosage of 1.0 mg/kg every 3 weeks, which will be tested in combination with an ICI in selected tumor types.9

NT-I7 With Pembrolizumab

Phase 2a of the ongoing KEYNOTE A60 trial (NCT04332653) is evaluating NT-I7 (efineptakin alfa), recombinant human IL-7 protein fused to a hybrid Fc region of a human antibody, in combination with pembrolizumab. Patients with ICI-naïve relapsed/ refractory microsatellite-stable colorectal cancer (MSS-CRC) or pancreatic ductal adenocarcinoma (PDAC) are eligible, as are those with ICI-treated triple-negative breast cancer (TNBC), non–small cell lung cancer (NSCLC), and SCLC.10 The primary end point is objective response rate, with secondary end points of duration of response, disease control rate, PFS, and OS.10

Ninety-two patients with metastatic or locally advanced cancer who had received a median of 3 prior treatments were enrolled as of January 2022 (PDAC, n = 32; MSS-CRC, n = 28; NSCLC, n = 22; TNBC, n = 6; SCLC, n = 4).10 Results from the 71 evaluable patients are shown in the TABLE10.

A sustained peripheral lymphocyte increase of approximately 3 times the baseline level was observed in all arms.10 NT-I7– related AEs occurred in 67 (72.8%) of the 92 treated patients, including 56.5% grade 1 or 2, 14.1% grade 3, and 2.2% grade 4.10 These results suggest the addition of NT-I7 to ICI therapy can help overcome primary resistance to ICIs in ICI-naïve MSS-CRC and PDAC, and acquired ICI resistance in ICI-treated TNBC, NSLC, and SCLC.10

Trials in Progress


AU-007 is a computationally designed monoclonal antibody that binds to IL-2 on its CD25-binding epitope, resulting in redirection of IL-2 (either endogenously produced or exogenously administered, eg, as aldesleukin) toward activation of immune-stimulating T effector and NK cells while diminishing Treg activation and expansion.11 AU-007 also converts a Treg-mediated autoinhibitory loop into an immune-stimulating loop by binding and redirecting newly secreted endogenous IL-2.11 A recently initiated phase 1/2 study (CP-AU-007-01; NCT05267626) is evaluating the safety, tolerability, and initial efficacy of AU-007 alone or combined with aldesleukin in patients with advanced solid tumors, including melanoma, RCC, and 17 others.11 Phase 1 consists of 3 escalation arms. In arm A, patients receive escalating doses of AU-007 monotherapy every 2 weeks; in arm B, a single dose of aldesleukin is received with the initial AU-007 dose; and in arm C, patients receive combination therapy with both drugs every 2 weeks.11 Matching expansion cohorts of up to 20 patients each are planned for phase 2.11

IL Believe

IL Believe is a multicenter, first-inhuman, phase 1/2 study in 3 parts evaluating

TransCon IL-2 β/γ in adults with locally advanced or metastatic solid tumors.12 Patients who are included need 1 or more measurable lesions per RECIST 1.1 and an ECOG score of 2 or less.12

Evaluating safety and tolerability and defining the maximum tolerated dose along with the recommended phase 2 dose are primary objectives.12 Parts 1 and 2 (phase 1; n = 15 patients each) involve dose escalation of TransCon IL-2 β/γ alone or in combination with pembrolizumab.12 Part 3 is a phase 2 evaluation of the preliminary clinical efficacy of TransCon IL-2 β/γ combined with standard-of-care chemotherapy.12


RTX-224 is an off-the-shelf allogeneic therapy consisting of red blood cells engineered to express the costimulatory 4-1BB ligand and IL-12.13 RTX-224 distribution is restricted to the vasculature and spleen, which should limit toxicities previously observed with agonist 4-1BB monoclonal antibodies and recombinant IL-12, which are distributed systemically.13

RTX-224-01 (NCT05219578) is a phase 1/2, first-in-human, multicenter, doseescalation and -expansion study of RTX-224 in patients with relapsed/refractory urothelial cancer, squamous cell carcinoma of the head and neck, NSCLC, TNBC, and cutaneous melanoma.13

Approximately 28 patients will be enrolled to evaluate safety, tolerability, pharmacokinetics, pharmacodynamics, and antitumor activity.13


XTX202 is a masked, tumor-selective recombinant human IL-2 protein designed to be pharmacologically active (unmasked by matrix metalloproteases) only in tumor tissue and inactive in the systemic circulation.14 The agent was designed with the goal of producing a localized antitumor immune response and limiting exposure of the active form in nontumor tissue.14 XTX202-01 (NCT05052268) is a first-in-human phase 1/2 study of XTX202. Phase 1 will determine the recommended phase 2 dose, and phase 2 will evaluate the efficacy of XTX202 monotherapy in patients with metastatic RCC and unresectable or metastatic melanoma.14

Final Thoughts

Markovic compared the immune system to an orchestra, with 800 to 1000 different regulators working in concert. The interplay between these regulators driving acute or chronic inflammation remains to be elucidated. “I think cytokines have a role, but I also sense chemokines have a bigger role, though they are much more difficult to target,” he stated concerning ICIs and cytokine-based therapies.

He added that the first iteration of cytokine-based therapies taught investigators that increasing doses until toxicity occurs is not the answer but focusing on biological measurements is. Herein lies another issue for Markovic and colleagues: “We measure many parameters, but finding those most relevant remains a challenge.” He again equated this to diabetes and insulin; without assessing blood glucose levels, you cannot determine an adequate dose of insulin. Therefore, what should be measured to determine the adequate dose of cytokines?

Markovic remains proud of the advances made in immunotherapy cancer treatments and has significant hope for the future. “The more we understand and unravel the complexities of multicytokine regulation in blood—what happens in the lymph node downstream of cancer, what is happening in the tumor itself, why the immune system is allowing the tumor to grow instead of eliminating it—[the closer we will get to highly efficacious and safe cytokine-based cancer therapies]. We’ve learned more in the past decade than in the previous 5 decades,” summarized Markovic.

“I think working through those conceptual frames with novel agents will get us there, and science will win out.”


1. Berraondo P, Sanmamed MF, Ochoa MC, et al. Cytokines in clinical cancer immunotherapy. Br J Cancer. 2019;120(1):6-15. doi:10.1038/s41416-018-0328-y

2. Xue D, Hsu E, Fu YX, Peng H. Next-generation cytokines for cancer immunotherapy. Antib Ther. 2021;4(2):123-133. doi:10.1093/abt/tbab014

3. Rallis KS, Corrigan AE, Dadah H, et al. Cytokine-based cancer immunotherapy: challenges and opportunities for IL-10. Anticancer Res. 2021;41(7):3247-3252. doi:10.21873/anticanres.15110

4. Garralda E, Naing A, Galvao V, et al. Interim safety and efficacy results from AURELIO-03: a phase 1 dose escalation study of the IL-2/IL-15 receptor superagonist SOT101 as a single agent and in combination with pembrolizumab in patients with advanced solid tumors. J Clin Oncol. 2022;40(suppl 16):2502-2502. doi:10.1200/JCO.2022.40.16_suppl.2502

5. Chamie K, Chang SS, Gonzalgo M, et al. Final clinical results of pivotal trial of IL-15RαFc superagonist N-803 with BCG in BCG-unresponsive CIS and papillary nonmuscle-invasive bladder cancer (NMIBC). J Clin Oncol. 2022;40(suppl 16):4508-4508. doi: 10.1200/JCO.2022.40.16_suppl.4508

6. ImmunityBio submits biologics license application for N-803 Plus BCG for patients with BCG-unresponsive non-muscle invasive bladder cancer carcinoma in situ. ImmunityBio; May 23, 2022, 2022. Accessed July 11, 2022.

7. Naing A, Mantia C, Morgensztern D, et al. First-in-human study of SRF388, a first-in-class IL-27 targeting antibody, as monotherapy and in combination with pembrolizumab in patients with advanced solid tumors. J Clin Oncol. 2022;40(suppl 16):-2501. doi:10.1200/JCO.2022.40.16_suppl.2501

8. Rosen D, M.nsson Kvarnhammar A, Olling JD, et al. TransCon IL-2 β/γ, a novel long-acting prodrug with sustained release of an IL-2Rβ/γ-selective IL-2 analog, demonstrates improved pharmacokinetics and profound expansion of cytotoxic immune cells in non-human primates. J Clin Oncol. 2022;40(suppl 16l):2563-2563. doi:10.1200/JCO.2022.40.16_suppl.2563

9. Johnson ML, Abdul-Karim R, Sommerhalder D, et al. Dose escalation of a phase 1b/2 study of modakafusp alfa, an immune-targeting attenuated cytokine, in patients (pts) with metastatic solid tumors. J Clin Oncol. 2022;40(suppl 16):2503-2503. doi:10.1200/JCO.2022.40.16_suppl.2503

10. Naing A, Mamdani H, Barve MA, et al. Efficacy and safety of NT-I7, long-acting interleukin-7, plus pembrolizumab in patients with advanced solid tumors: Results from the phase 2a study. J Clin Oncol. 2022;40(suppl 16):2514-2514. doi:10.1200/JCO.2022.40.16_suppl.2514

11. Vasselli JR, Frentzas S, Weickhardt AJ, et al. Trial in progress: A phase 1-2, first-in-human, open label, dose escalation and expansion study of AU-007, a monoclonal antibody that binds to IL-2 and inhibits IL-2Rα binding, in patients with advanced solid tumors. J Clin Oncol. 2022;40(suppl 16):TPS2671-TPS2671. doi:10.1200/JCO.2022.40.16_suppl.TPS2671

12. Starodub A, Gabrail NY, Zhang Y, et al. IL believe: A phase 1/2, open-label, dose escalation and dose expansion study of TransCon IL-2 β/γ alone or in combination with pembrolizumab or standard-of-care chemotherapy in patients with locally advanced or metastatic solid tumors. J Clin Oncol. 2022;40(suppl 16):TPS2695-TPS2695. doi:10.1200/JCO.2022.40.16_suppl.


13. Spira AI, Johnson ML, Ren A, et al. A phase 1/2 study of RTX-224, an engineered red blood cell expressing 4-1BB ligand and membrane- bound IL-12, for the treatment of patients with select advanced solid tumors. J Clin Oncol. 2022;40(16_suppl):TPS2680-TPS2680. doi:10.1200/JCO.2022.40.16_suppl.TPS2680

14. McKean M, Powderly JD, Duncan M, et al. A first-in-human, multicenter, phase 1/2, open-label study of XTX202, a masked and tumor-selective recombinant human interleukin-2 (IL-2) protein, in patients with advanced solid tumors. J Clin Oncol. 2022;40(suppl 16):TPS2697-TPS2697. doi:10.1200/JCO.2022.40.16_suppl.TPS2697