The SB102 study was a phase 1, randomized, double-blind, placebo-controlled, dose-escalation study designed to assess the safety, tolerability, PK, and PD of SON-1010 administered as a single subcutaneous (SC) injection in healthy volunteers ( Figure 2 ). A flow diagram for sentinel participants and the rest of each cohort is shown in Supplementary Figure S1 . Participants had to be 18-54 years old and healthy based on their medical history, physical examination, and clinical laboratory testing (see the full list of Inclusion/Exclusion Criteria in the Supplementary Material ). SB102 was conducted at a single site in Melbourne, Victoria, Australia; blinding included the participants, site staff, clinical research organization (CRO), sponsor, and medical monitors, as well as the Safety Review Committee (SRC). An exploratory objective was to evaluate the relationship between PK and PD in SON-1010 dosing. Safety was carefully tracked, along with the evaluation of acute inflammatory cytokine responses to help with dose escalation decisions. The study was performed in accordance with the Declaration of Helsinki, Council for International Organizations of Medical Sciences International Ethical Guidelines, and Good Clinical Practice, and was approved by the Alfred Human Research Ethics Committee (Alfred Health, Melbourne, VIC) as authorized by the Australian Government through the National Health and Medical Research Council (NHMRC) on 2Aug2022, once the safety of the first two dose levels had been reviewed in SB101.

A SAD approach was used in SB102, including the dosing of sentinel participants before putting larger numbers at risk, with placebo participants in each of five cohorts designated S1 to S5, with eight participants in each dose cohort randomized to receive either SON-1010 (n=6) or a placebo (n=2) ( Table 1 ). Blinding was maintained until the database was closed. One similar trial, a large phase 1 dose escalation study using rIL-12 that was conducted in healthy volunteers as a medical countermeasure for acute radiation syndrome (30), was used as the basis for initial dose selection in the SON-1010 clinical program (31). The first cohort in SB102 received 50 ng/kg SON-1010, which is the molar equivalent of the lowest rIL-12 dose that showed a measurable IFNγ response in that earlier study of healthy volunteers.

Participants were followed for 3 days in confinement at the Nucleus Clinical Research Unit and then as outpatients for a total of 28 days after dosing to assess safety, tolerability, and laboratory responses. Safety, including an assessment of adverse events and all available laboratory results, was reviewed at each SRC meeting after all participants in that cohort had completed day 22. If dose-limiting toxicities (DLTs) or a lack of tolerability were observed in at least one participant at a given dose level, the highest previously evaluated dose level without a DLT was defined as the MTD. The cytokine responses are likely to be a better early indicator of an inflammatory response related to SON-1010 than clinical AEs, so, the PD response was followed closely as the dose was escalated with a rapid assessment of ‘acute inflammation’ labs from day to 1-8 (IFNγ, IL1ß, IL-6, IL-8, IL-10, IL-12, and TNFα by a Luminex assay, Crux Biolabs, Melbourne, Australia). The SRC reviewed these results, then authorized the subsequent dose level for enrollment of the next sentinel participants.

Safety and tolerability were determined by assessing treatment-emergent adverse events (TEAEs), vital signs, laboratory test results (hematology, biochemistry, coagulation, thyroid function tests, and urinalysis), electrocardiograms, and physical examination findings. Adverse events were graded according to the current version of the Common Terminology Criteria for Adverse Events (CTCAE) and were categorized as mild (grade 1), moderate (grade 2), severe (grade 3), life-threatening (grade 4), or death (grade 5), or as serious AEs (SAEs) according to standard definitions. Causal relationships of TEAEs to SON-1010 administration were judged by the investigator as either “unrelated”; or “possibly”, “probably”, or “definitely” related (treatment-related TEAEs). All TEAEs were coded using the Medical Dictionary of Regulatory Activities (MedRA v24.0).

Serum SON-1010 concentrations were assessed using a validated electrochemiluminescence-based immunoassay (ECLIA) for IL-12 (Mesoscale Discovery [MSD] Cat# K151QVD) at Celerion (Omaha, NB, USA). Urine concentrations were evaluated at 360biolabs (Melbourne, Australia) with the same kit after qualification for its use with urine. Primary PK parameters were calculated from concentration versus time data using non-compartmental analysis (NCA), including maximum serum concentration (C_max); area under the serum concentration vs. time curve (AUC) from the first dose until 24, 48, or 168 h (AUC_0-x); AUC until the last quantifiable concentration (AUC_0-t); an estimate of the total AUC (AUC_inf); time to peak serum concentration (T_max); terminal elimination half-life (T_½), apparent clearance (CL/F); and apparent volume of distribution (V_z/F). The formal PD parameters included IFNγ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, and TNFα levels, which were determined using a qualified multiplex assay (MSD Cat# K15049G) (Celerion).

The analysis sets for this study included the enrolled set (participants who signed the informed consent form [ICF], met eligibility criteria, and were approved for randomized treatment), the safety set (all participants who received at least one dose of SON-1010 or placebo), and the PD set (participants with sufficient PD samples available). All analysis datasets and outputs were produced by the Biostatistics Department of Resolutum Global using SAS^® Version 9.4 (SAS Institute, Cary, North Carolina, USA). The sample size selected (6 active participants per cohort) was based on common practice in phase 1 dose escalation studies. Non-compartmental analysis to estimate PK parameters was performed using R version 4.3.0 (32) and the pkr package version 0.1.3 (R Foundation for Statistical Computing, Vienna, Austria).

Blinded dosing in each cohort started with two sentinel participants (one active and one placebo), followed approximately a week later by six participants in the rest of each cohort to assess the safety, tolerability, PK, and PD without the background of prior chemotherapy (29). SON-1010 administration was generally safe and well-tolerated at all doses in this population of healthy volunteers. There were no grade ≥ 3 treatment-emergent adverse events (TEAEs) that were considered related to treatment. There were no serious adverse events (SAEs) and no TEAEs leading to discontinuation of the study.

Participants receiving SON-1010 at doses of 100 ng/kg or higher tolerated the injection but generally experienced more TEAEs than participants receiving SON-1010 at 50 ng/kg ( Table 2 ). However, there was no clear evidence of a dose-related effect among the higher-dosing cohorts. Headache (10/23, 43.4%), myalgia, injection site pain or induration, and pyrexia (3/23, 13.0% each) were reported as related events more frequently among SON-1010 treated participants than among placebo group, who only included one with headache or injection site pain (1/8, 12.5%), with no clear relationship between the SON-1010 dose cohort and frequency. Most TEAEs were mild and were considered possibly, probably, or definitely related to SON-1010. One participant (12.5%) in Cohort S1 developed grade 2 neutropenia on day 5 that returned to normal by day 10. One participant each (12.5%) in Cohorts S2 and S3 reported a recurrent moderate/grade 2 headache requiring acetaminophen for control, and one participant (14.3%) in Cohort 4 reported grade 2 flu-like symptoms requiring acetaminophen for 5 days. One placebo participant (12.5%) reported a grade 2 headache requiring acetaminophen that was considered related as well. During the trial, the most frequently prescribed concomitant medication was acetaminophen (15/31, 48.4%), which was administered to one placebo vs. five of the six active participants in the SON-1010 100 ng/kg cohort. Other concomitant medications were administered to a single participant, with no apparent patterns across treatments or cohorts.

Dose-related decreases in neutrophils, lymphocytes, and platelets were observed 24-72 hours after the administration of SON-1010 ( Figure 3 ), with resolution by day 7 to 10, which is consistent with other studies that used rIL-12 (30) or rIFNγ (36). Alanine aminotransferase (ALT) levels increased over the first 5 to 10 days then returned to baseline, with lower increases in aspartate aminotransferase (AST) levels or other liver enzymes. All values returned to baseline within a short period of time. A dose-related increase in C-reactive protein concentration was also observed on day 2, which returned to baseline values by day 7. Acute inflammation was assessed in each cohort by Luminex assay, for review by the SRC ( Supplementary Figure S2 ). An increase in IFNγ was observed in all SON-1010 dose cohorts, as well as a much smaller dose-dependent increase in TNFα, IL-8, and IL-10 concentrations. These changes occurred within 24 to 48 h after administration of the study drug. There were minimal transient increases in IL-6 concentrations that were not dose related. All values returned to baseline within a few days. There were no notable changes in vital signs or electrocardiograms. No TEAEs or cytokine responses were observed that might suggest cytokine release syndrome (37).

Studying SON-1010 in healthy volunteers in SB102 was an important objective for this non-genotoxic therapeutic oncology drug candidate, as it provided an opportunity to evaluate PK and PD without interference from prior chemotherapy (29, 33). Mean serum concentration versus time profiles following the single SC injection of SON-1010 are presented for the first week (Figure 4). Between the SON-1010 lowest- (50 ng/kg) and highest- (300 ng/kg) dose cohorts (a 6x escalation in dose), the serum C_max increased by 4.5x (Momentum Metrix, Dublin, CA), and the time to reach that (T_max) was approximately 11 h ( Table 3 ). This was associated with a corresponding 4.5 × increase in the exposure area under the concentration time curve (AUC) from time zero to the time of last observable concentration (AUC_0-t), and the shape of the curves indicated typical two-compartment elimination kinetics ( Figure 4 ). The mean T_½ across all dose cohorts was 104 h, and the serum concentrations for the majority of the participants remained above the lower limit of quantitation (LLOQ) for 336 h. The mean C_max value increased in a less than proportional manner between dose cohorts, yielding nonlinear PK. The geometric mean C_max values of the low- and high-dose cohorts were 29 pg/mL and 131 pg/mL, respectively. The low dose cohort reported mean AUC_0-t and AUC_0-inf of 1340 (CV% 41.5) h•pg/mL and 1500 (CV% 8.5) h•pg/mL, respectively. The high-dose cohort reported 6030 (CV% 47.1) h•pg/mL and 9850 h•pg/mL, respectively. Urine SON-1010 concentrations were below the level of quantitation at all time points, so that route of elimination was not included in the analysis. The SON-1010 PK parameters C_max, AUC_0-24, and AUC_0-48, are shown graphically in Supplementary Figure S3A . There was relatively large variability in the mean SON-1010 PK parameters with poor linear fits (R² adj < 0.8), and nearly all parameters had a geometric mean CV% greater than 30%; therefore, N was too low to calculate accurate summary statistics for other PK parameters, such as CL/F and V_Z/F.

Interim data from repeat dosing in patients with advanced solid tumors in study SB101, including dose escalation up to the same maximum dose used in SB102, are now available ( Figure 5 ) (28). Interestingly, the SON-1010 concentration curves, using the same assay in cancer patients, compared with a single dose in healthy volunteers ( Figure 4 ) showed an atypically dissimilar contour. Single-compartment elimination kinetics were noted in patients with cancer, compared to the two-compartment elimination kinetics observed in the healthy volunteers. The C_max and AUC PK parameters in SB101 were similar after the second dose compared to the first dose in SB102, while the IFNγ PD parameters of C_max and AUC were suppressed in SB101 ( Supplementary Figures S3B, S4B ), presumably by the induction of SOCS proteins (34).

Endogenous biomarkers of interest included IFNγ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10 and TNFα. Of these, only IFNγ, TNFα, IL-6, IL-8, and IL-10 met the criteria for analysis, as fewer than 20% of the data were below the limit of quantitation. Mean serum concentration versus time profiles following the single SC injection of SON-1010 are presented for the first 2 weeks ( Figure 6 ). Apart from IL-6, the concentrations remained above the LLOQ for all study participants.

A summary of the NCA parameter values after a single SC dose showed that IFNγ was the most prominent cytokine responding ( Table 4 ). The mean C_max value disproportionately increased between the wide range of doses tested, peaking at 977 pg/mL in the highest dose cohort (300 ng/kg). The time taken to achieve maximal IFNγ blood concentrations varied greatly between cohorts and did not correlate with the dose, with the mean time required to peak ranging from 28.8 to 85.0 hours. The AUC_0-t also increased disproportionately following the cohort doses and rose to 106,000 h*pg/mL in the highest-dose cohort. However, the partial areas under the concentration-time curve from time zero to 24 h, 48 h, and 168 h increased in a dose-dependent manner. The SON-1010 PD parameters C_max, AUC_0-24, and AUC_0-48, are shown graphically in Supplementary Figure S4A .

Another consideration is to compare SON-1010 PK with the IFNγ PD response after a single dose in healthy volunteers. The greatest linear correlation was observed between C_max PK and AUC_0-24h IFNγ PD (Pearson correlation coefficient = 0.77, p<0.001) ( Supplementary Figure S5 ). Although it was much less responsive, the C_max value for IL-10 increased with each higher SON-1010 dose cohort, peaking at 2.75 pg/mL with the highest dose. The mean time taken to achieve C_max ranged from 36.4 to 67.7 h. Although the analyzed IL-10 AUC metrics appeared to suggest dose proportionality, this was less clear for the other cytokines studied. The mean maximum IL-6, IL-8, and TNFα concentrations achieved after a 300 ng/kg dose of SON-1010 were 5.4, 24.6, and 4.6 pg/mL, respectively. The mean times to achieve C_max were 27.6, 52.2, and 48.1 h, respectively.

Early efforts to advance rIL-12 into the clinic showed in the first phase 1 study that doses up to 500 ng/kg daily could be administered intravenously (IV) with acceptable levels of safety, starting two weeks after a test dose (4). Weekly SC dosing was also well-tolerated at that dose (38). However, in the subsequent phase 2 study of 17 patients who received daily rIL-12 IV, 12 patients were hospitalized and two patients died (2). A thorough scientific investigation to determine the cause of this unexpected toxicity failed to identify any difference in the drug products used or the patient populations enrolled in the two IV studies that could have accounted for the profound difference in toxicity. The schedule-dependent toxicity of rIL-12 and an abrupt increase in IFNγ levels were verified in mice and nonhuman primates to be a form of tachyphylaxis; therefore, the dosing level misdirection was thought to have been due to PD effects.

The single test dose injection of rIL-12 administered 2 weeks before consecutive dosing in that first phase 1 study, but not after daily administration in the phase 2 study, apparently had a profound abrogating effect on rIL-12-induced IFNγ production and toxicity with subsequent doses. This was likely a result of the induction of suppressors of cytokine signaling (SOCS) proteins ( Figure 7 ) that normally regulate inflammatory responses (34). Competitive binding of SOCS proteins to the phosphorylated tyrosine residues of cytokine receptors prevents the docking of STAT proteins and the transfer of the signals, thereby reducing toxicity. When the second rIL-12 dose was delayed in phase 1, the subsequent toxic effects may have been suppressed by lingering SOCs proteins. However, in the next study the mean serum IFNγ levels rose to over 25,000 pg/mL after starting with daily injection of rIL-12. This was attributed, at least in part, as the cause of the toxicity, compared to an IFNγ peak of 5,000 pg/mL in phase 1 after the test dose. Interestingly, the phase 2 IFNγ peak was about the same peak level that was measured after intramuscular administration of rIFNγ at its MTD of 5.0 mg/m² in patients with cancer in an early phase 1 trial (36), where the most notable toxicity was fatigue. Peak IFNγ levels appeared to correlate with maximum toxicity in that study. Perhaps the more severe toxicity observed in the phase 2 study of rIL-12 was related to the sustained level of IFNγ that was secondarily induced, which had peaked at approximately 10,000 pg/mL after IV administration of 500 ng/kg rIL-12 in phase 1 (4).

Since the initial phase 2 study of rIL-12 (2), numerous trials have been conducted to determine the optimal dosing schedule and potential utility of various forms of rIL-12, both in patients with cancer and in healthy volunteers. In the largest study to date of rIL-12 in healthy volunteers, designed to study its use as a medical countermeasure for humans exposed to lethal radiation, 32 subjects were initially enrolled in a SAD format starting at 2 μg (as a standardized dose) and the MTD was found to be 12 μg when given SC as a standardized dose (30). The maximum serum concentration (C_max) values generally increased with increasing dose levels, except for the 20 μg cohort, where only one participant was dosed. Sixty patients were enrolled in a placebo-controlled expansion study at the MTD of 12 μg. In both studies, the most common AEs related to rIL-12 were headache, dizziness, and chills during the first few days of treatment. No immunogenicity was observed. Two-compartment elimination of rIL-12 was noted, suggesting significant distribution into extravascular spaces; the initial T_½ was 8.7 ± 4.7 hours and the C_max was 57.7 ± 49.8 pg/mL. rIL-12 triggered transient reductions in neutrophils, platelets, reticulocytes, lymphocytes, NK cells, and CD34⁺ hematopoietic progenitor cells by day 2, and correlated with induced increases in IFNγ and CXCL10. The cell subsets returned to above normal by day 7, and all parameters normalized over 1–2 weeks. This suggests that splenic sequestration or margination may account for the cell count changes, rather than destruction, as the return to normal in the standard complete blood count was faster than would be expected from simple replacement.

Multiple studies in patients with various types of cancer have shown similar effects of rIL-12 dosing strategies, ranging from daily to 2 to 3 times weekly. Most studies reported an MTD of 500–1000 ng/kg after IV or SC administration. Several attempts have been made to extend T_½ for a superior PK profile; the most advanced is NHS-IL12, an immunocytokine composed of two rIL-12 heterodimers, each fused to the heavy chain of an antibody that binds to DNA (39). The C_max of NHS-IL12 was reached at 36 h, and time-dependent elevations of IFNγ and IL-10 were observed after SC NHS-IL12 administration, which returned to normal by day 7. The MTD of NHS-IL12 was determined to be 16.8 μg/kg, which is much higher than the MTD for rIL-12 of 0.5 to 1 μg/kg, suggesting the possibility of steric hindrance of the cytokine portion of NHS-IL12. Note that NHS-IL12 was originally administered every 4 weeks as monotherapy and is currently being developed in combination with a checkpoint inhibitor given every 2 weeks (40).

The preclinical testing of SON-1010 has been extensive (25, 41). An early proof-of-concept study of F_HAB in the TGFβ⁺ mouse 4T1 breast tumor model (42) showed the accumulation and prolonged retention of F_HAB in the tumor as well as when anti-TGFβ was linked to the scFv, whereas the anti-TGFβ antibody alone first accumulated in tumor tissue, then rapidly diffused out. In another study, murine (m) rIL-12 linked to F_HAB caused up to a 10-fold increase in serum half-life in mice, compared with the rIL-12 control (41).

Murine rIL-12 (mIL-12) causing a reduction in pulmonary metastases or SC growth of B16F10 melanoma in mice was demonstrated as early as 1993 (23). Dose-dependent increases in anti-tumor activity were also demonstrated with mIL12-F_HAB in that melanoma model, producing a corresponding increase in tumor-infiltrating activated NK and CD8⁺ T cells (41). Single doses of mIL12-F_HAB were up to 30-fold more effective (by molar equivalence) in reducing B16F10 tumor growth and extending survivability, compared with mIL-12 alone, in a dose-dependent manner in tumor-bearing mice compared to placebo. This resulted in a corresponding increase in the immune response, as reflected by the increased splenic weight and serum IFNγ levels, which was transient and had no effect on mouse body weight. Toxic inflammatory responses were only observed at high levels of mIL12-F_HAB (30 µg/mouse), including moderate increases in IL-6, C-reactive protein, and transaminase levels. Overall, a comparison of the PD and toxicological effects of mIL12-F_HAB in mice suggests that, while the model may be limited to lower doses, B16F10 tumors are well controlled in a dose range that is non-toxic by these measures. Biodistribution studies also suggest delivery to and retention of mIL12-F_HAB in tumor tissue (25).

An in vitro evaluation of SON-1010 using cells from Syrian hamsters, Sprague Dawley rats, beagles, cynomolgus macaques, or humans was tested for albumin binding, potency, and binding of the complex to FcRn; only macaque cells responded physiologically (26). Therefore, macaques have been used for single- and multiple-dose toxicological testing of products based on the F_HAB platform in vivo. After a single SC dose of SON-1010, drug-related changes in clinical observations, body weight, clinical pathology, cytokines, and immunophenotyping were tolerated up to 250 µg/kg in macaques and were consistent with the anticipated on-target effects of rIL-12 (20). Most parameters recovered to pre-study values by day 22, and SON-1010 displayed improved PK characteristics compared to those reported for rIL-12. In the GLP toxicology study, three SC injections of SON-1010 were tolerated in monkeys at up to 62.5 µg/kg/dose. Hematological changes in red blood cells, reticulocytes, platelets, and neutrophils were suggestive of accelerated maturation, along with transient suppression of monocyte, lymphocyte, eosinophil, and white blood cell counts. Minimal changes occurred in the clinical chemistries. Cytokine data showed SON-1010-related effects on IFNγ, with minimal or no changes in IL-6, IL-8, IL-10, IL-1β, or TNFα. The no adverse effect level (NOAEL) in Cynomolgus macaques following repeated SC administration of SON-1010 was defined as 62.5 µg/kg/dose.

The unusual PK results comparing these two clinical studies suggest the potential for an improved local immune response due to accumulation in the TME in patients, which could make SON-1010 more effective than prior efforts with systemic immunotherapy using rIL-12. The dose relationship also suggests TMDD, perhaps due to the retention of SON-1010 caused by albumin binding to SPARC (27) and its slow release from the tumor tissue. Based on the SON-1010 concentration curves, a dose interval of 3 weeks produces minimal accumulation of SON-1010 before the next dose; therefore, any accumulation of the drug is unlikely to be physiologically significant. The drug product used in SB101 was a liquid formulation manufactured in a fed-batch process, while that used in SB102 was lyophilized and had been produced using a perfusion process, which may have accounted for the distinctive PK profiles. However, both drug products passed GMP release and stability testing with nearly identical results, including potency testing using an IL-12 HEK-Blue bioassay that assesses IFNγ production (43). Although subtle differences in biomolecule manufacturing lots are common and can include minor differences caused by deamidation or glycosylation (44), both lots met manufacturing specifications and were considered to be physically and functionally identical. Thus, variations such as these would not be expected to cause the drug elimination profile differences that were observed. Further testing with subsequent doses is required to substantiate the safety of prolonged dosing, which is planned for the next study (SB221).

Overall, the IFNγ PD response with a single dose in SB102 was dose-related, controlled, and prolonged without the stimulation of a more toxic cytokine response ( Figure 6 ), which may be required to initiate tumor control in humans, as in mice (45). Neutropenia, lymphopenia, and thrombocytopenia have been reported as common AEs with rIL-12. In the large dose-escalation study of rIL-12 in healthy volunteers (30), dose-related neutropenia reached a nadir on day 5 after a single dose and the mean returned to baseline (or above) by 2 weeks. Neutropenia, lymphopenia, and thrombocytopenia were seen in both SB101 and SB102 with similar nadir and recovery times. The SON-1010 C_max can also be compared with the IFNγ response using AUC_0-48h in the SB101 cancer patients using a Pearson correlation coefficient ( Supplementary Material Figure S5 ). The Pearson correlation coefficient measures linear correlation between two sets of data and is the ratio between the covariance of two variables and the product of their standard deviations; thus, it is essentially a normalized measurement of the covariance, such that the result always has a value between -1 and 1. The Pearson coefficient using C_max vs AUC_0-24 in SB102 was also significant. The longer time to C_max may reflect retention in the TME in the cancer patients.

Drugs such as SON-1010, which induce IFNγ in the TME, upregulate the expression of PD-L1 on tumor cells and/or immune cells (46). While there is a reasonable chance that SON-1010 inhibits tumor growth at higher doses, owing to its improved targeting of the TME, SON-1010 may have its greatest effect in treating cancer in combination with an immune checkpoint inhibitor (47). The next development step is to determine the SON-1010 MTD when combined with an immune checkpoint inhibitor in patients with a tumor that is high in SPARC, such as platinum-resistant ovarian cancer, which continues to be a high unmet need indication. Proof-of-concept will be assessed in this population in study SB221, using the combination of SON-1010 with atezolizumab, compared with SON-1010 alone or standard-of-care therapy.