Neutralizing capacity
The neutralizing capacity of INM004 in a subset of study subjects’ sera obtained at 1, 24, and 72 hours post-administration was evaluated over the toxic effect of Stxs on Vero cells as previously described3. As a positive control we used the pool of baseline samples (before administration) of the three evaluated volunteers, spiked with an estimated average concentration of F(ab´)2. The estimated concentrations of INM004 (determined by ELISA) in the samples at the evaluated times were 40, 15 and 7 µg.mL-1, respectively.
Statistical analyses
Continuous variables were summarized as mean, median, interquartile range (IQR) minimum, maximum and standard deviation (SD) and categorical or ordinal variables as percentages. Analysis was based strictly on observed cases, i.e., no replacement of missing data was planned in general. Statistical significance threshold was set for two-sided probability values at <0.05.
After completing Stage I, an interim analysis of the PK data and safety data was performed to assess if the PK and safety profile were acceptable to initiate Stage II. The blinded analysis was performed by a Medical Monitor not related to the clinical research team or the Sponsor. The dose and interdose interval for Stage II was defined taking into consideration the results obtained in the interim analysis.
All statistical analyses, listing, tabulations, and figures were performed by the Department of Biostatistics of Linical S.L.U., using SAS® Version 9.4 or later. PK analyses were performed using validated Phoenix WinNonlin (v.8.1, Certara USA, Inc., USA).
Results
Twenty-two healthy volunteers were screened, and 14 fulfilled inclusion criteria without reasons for exclusion. These fourteen subjects were randomized to receive INM004 or placebo, without differences in baseline characteristics. Table 1 summarizes clinical and demographic data of study participants. Three (21.4%) subjects reported at least one past medical condition.
INM004 infusions were well tolerated in all Stages of the study. Eighteen treatment emergent AEs (TEAEs, i.e. after administration of the study medication) were reported by eight (57.1%) subjects: three in Stage I (one in the placebo group) and five in Stage II (one in the placebo group). All TEAEs lasted for 24 hours or less and resolved completely without need for changes in the treatment schedule or specific treatment. Half of the reported TEAEs were of mild intensity (9/18 TEAEs reported) and the remaining half of moderate intensity, without serious or severe AEs. Of the total reported TEAEs, four were possibly drug related. These related TEAEs included: rhinitis, headache and flushing experienced by three (60.0%) subjects in the INM004 group at Stage II (Supplementary appendix Table s1). For full details on the occurrence of AE, see Tables s2 and s3.
All study subjects remained asymptomatic without the need of any specific intervention. No clinically significant changes in laboratory values were observed from baseline to hospitalization discharge.
PK analyzes
Basic PK parameters of INM004 are summarized in Table 2. Following a single intravenous infusion dose of INM004 to healthy subjects at doses of 2.00 mg.kg-1 and 4.00 mg.kg-1, peak concentrations were observed within 2 hours after the end of the infusion (Figure 2, Panel A and B). Mean Cmax values were 45.1 µg.mL-1 and 77.7 µg.mL-1 following the 2.00 mg.kg-1 and 4.00 mg.kg-1 doses, respectively. The serum concentration versus time profiles of INM004 declined in a biphasic manner, with t½ values ranging from 30.7 h to 41.8 h following the 2.00 mg.kg-1 dose and from 33.2 h to 52.9 h following the 4.00 mg.kg-1 dose. The corresponding geometric mean AUC(0-last) values were 1180 µg.h.mL-1 and 2373 µg.h.mL-1, respectively.
In Stage II, following repeated 4.00 mg.kg-1 intravenous infusions of INM004 to healthy subjects, the serum concentrations of INM004 on Day 3 declined in a biphasic manner, with t1⁄2 values ranging from 60.3 h to 95.0 h (Figure 2, Panel C). Systemic INM004 exposures increased with each subsequent dose in an approximately dose-proportional manner, with a 2-fold increase in dose resulting in a 1.7 fold increase in Cmax and a 2.0-fold increase in AUC(0‑inf). Following intravenous administration, the Day 3 geometric mean clearance of INM-004 was 1.63 mL/h/kg and the corresponding Vz value was 172 mL.kg-1.
As expected, accumulation of INM004 was observed on Day 3 when compared to Day 1 in the repeated dose regime, with mean relative accumulation values (RA) of 1.27 and 1.41 for Cmax and AUC, respectively.
Geometric mean Cmax value was 149 µg.mL-1 with corresponding geometric mean AUC(0-τ), AUC(0-inf) and AUC(total) (i.e. the total AUC for Days 1 to 3 combined) values of 2460 µg.h.mL-1, 10300 µg.h.mL-1 and 14600 µg.h.mL-1, respectively. The between-subject variability, as assessed from the geometric percent CV, was low (CV% <25%) for both Cmax and AUC (0‑inf) for both Stage I and II measures.
The inter-subject variability, as assessed from the geometric coefficient of variation %, was low (<25%) for both Cmax and AUC(0‑inf) in both single and repeated dose regimens.
Neutralizing capacity
Serum samples of 3 study subjects at 1, 24 and 72 hours after INM004 infusion were analyzed to evaluate its Stx neutralizing capacity. All samples evaluated with the in vitro cytotoxic assay in Vero cells showed neutralizing activity (Figure 3), inhibiting the toxicity of the Stx under test conditions in a comparable way to the diluted product in the serum of the same subjects before administration.
Discussion
Our study describes the first in-human use of INM004, an anti-Stx product obtained from serum of equine animals that were previously immunized against epitopes of Stx2B and Stx1B, this is the first study of this kind in Argentina and a very important step regarding potential treatments for neglected diseases such as HUS.
HUS is an orphan pediatric disease for which no specific therapies have been developed yet. Several overlapping or complementary strategies for preventing and treating STEC-HUS have been proposed previously without success. Specific therapies with monoclonal antibodies (mAbs) that target Stx and prevent its internalization through its specific receptor Gb3 (globotriaosylceramide) were previously evaluated with promising preclinical results, but thus far there is no evidence of conclusive effectiveness from clinical trials12–14.
In consequence, clinical research and drug development for treatment of STEC-HUS is urgently needed and this study is an important step towards the development of novel targeted therapies for STEC-HUS.
We examined the safety, tolerability, and PK properties of INM004, with encouraging results. The infusion of a single dose and repeated doses was not associated with major side-effects. Laboratory parameters presented transient minor variations without clinical significance, and there was no evidence of immunogenicity against the product in any of the healthy volunteers. In addition, the neutralizing capacity of INM004 was preserved at the concentrations achieved, confirming that INM004 does not lose its neutralizing capacity in the bloodstream after infusion.
INM004 PK were similar to other equine F(ab´)2 products. A study by Lopardo et al. including adult patients with moderate to severe COVID-19 infection treated with RBD-specific polyclonal F(ab´)2 yielded similar PK parameter results among ill patients with COVID-19, with a median half-life of 58.9 h and Cmax1 of 84.6 mg.l-1 and 102.4 at 1 h and 49 h, respectively15. With repeated dose regimen, INM004 accumulated and its half-life increased in a dose-proportional manner with low inter-subject variability.
INM004 infusions were not associated with serious or severe AEs and only mild or moderate intensity infusion related TEAEs were reported, mainly headache, flushing and rhinitis, all of which were transient and resolved without sequelae. No hypersensitivity events were reported in our study. In keeping with the aforementioned study by Lopardo et al., rates of adverse events between treatment and placebo groups were similar, including severe and emergent treatment adverse events. Similarly, observational data following the widespread use of these EpAbs for the treatment of COVID-19 in Argentina revealed a low rate of severe AE, with a cumulative incidence of hypersensitivity reactions of 1-2% under real-world conditions among 10,728 patients with COVID-1916.
This study proves that the production of equine F(ab’)2for the treatment of STEC HUS is feasible, since INM004 manufacturing can be easily scaled up. A Phase II open label study, to evaluate the pharmacokinetics, safety, and exploratory efficacy of INM004 in the treatment of pediatric patients with STEC-HUS was started in October 2022 in Argentina (NCT05569746), where HUS is an endemic disease with approx. 400 cases annually. This trial is being conducted in 16 clinical sites, and includes children between 1 and 12 years of age, with a clinical diagnosis compatible with STEC-HUS. Should this clinical trial be successful, a Phase III, double blind, clinical trial will be executed to confirm INM004 efficacy in the treatment of STEC-HUS in pediatric patients with the view to become the first line therapy for an orphan disease with global impact.
In conclusion, in this first human study of INM004, the administration was safe and well tolerated, allowing the pharmacokinetic data an adequate allometric conversion for the aforementioned trial in children with HUS whose main objective is to evaluate safety as well as exploratory efficacy of INM004 as a valid new therapeutic approach for STEC-HUS in children.
*Acknowledgements.
Special thanks to Cintia Cruz from Clinical Pharmacology Section, Hospital Italiano de Buenos Aires and Rosario Aragone and María Emilia Murello from Clinical Trials Section, Hospital Italiano de Buenos Aires.
* Conflict of interest statement
This study was sponsored by Inmunova. Yanina Hiriart, Linus Spatz, Mariana Colonna and Santiago Sanguineti are employees of Inmunova. Fernando Goldbaum is the scientific director of Inmunova.
*Author contribution statement
All authors participated in the design, and data analysis of the study. VS, PS, VB and WHB participated in patient enrollment, study procedures and data collection. YH and LM developed and performed the ELISA assay for PK analysis. YH and MC collected and analyzed PK data. YH performed the neutralization assay. All authors participated in writing this manuscript, and read and approved its final version.
*Funding. The research leading to these results received funding from Inmunova.
References
1. Gianantonio, C. A., Vltacco, M., Mendilaharzu, F., Gallo, G. E. & Sojo, E. T. The Hemolytic-Uremic Syndrome. Nephron 11 , 174–192 (1973).
2. Caletti, M. G., Petetta, D., Jaitt, M., Casaliba, S. & Gimenez, A. [Hemolytic uremic syndrome (HUS): medical and social costs of treatment]. Medicina (B Aires) 66 Suppl 3 , 22–6 (2006).
3. Karmali, M. A., Petric, M., Lim, C., Fleming, P. C. & Steele, B. T. Escherichia coli cytotoxin, haemolytic-uraemic syndrome, and haemorrhagic colitis. Lancet 2 , 1299–1300 (1983).
4. Ascoli, C. A. & Aggeler, B. Overlooked benefits of using polyclonal antibodies. Biotechniques 65 , 127–136 (2018).
5. Chippaux, J. P. et al. Clinical safety of a polyvalent F(ab’)2 equine antivenom in 223 African snake envenomations: a field trial in Cameroon. VAO (Venin Afrique de l’Ouest) Investigators. Trans R Soc Trop Med Hyg 92 , 657–62.
6. Boyer, L. et al. Safety of intravenous equine F(ab’)2: Insights following clinical trials involving 1534 recipients of scorpion antivenom. Toxicon 76 , 386–393 (2013).
7. Quiambao, B. P. et al. Rabies Post-Exposure Prophylaxis in the Philippines: Health Status of Patients Having Received Purified Equine F(ab’)2 Fragment Rabies Immunoglobulin (Favirab). PLoS Negl Trop Dis 2 , e243 (2008).
8. Reveneau, E., Cottin, P. & Rasuli, A. Two decades of pharmacovigilance and clinical experience with highly purified rabies immunoglobulin F(ab’)2 fragments. Expert Rev Vaccines16 , 273–287 (2017).
9. Hiriart, Y. et al. [Development of a product anti-Shiga toxin for prevention of the hemolytic uremic syndrome]. Medicina (B Aires) 78 , 107–112 (2018).
10. Yanina, H. et al. Preclinical Studies of NEAST (Neutralizing Equine Anti-Shiga Toxin): A Potential Treatment for Prevention of Stec-Hus. International Journal of Drug Development and Research11 , (2019).
11. Santiago, G. et al. Development and Validation of an ELISA to Evaluate Neutralizing Equine Anti Shiga Toxin Antibodies in Preclinical Studies. Venoms and Toxins 2 , (2022).
12. López, E. L. et al. Safety and pharmacokinetics of urtoxazumab, a humanized monoclonal antibody, against Shiga-like toxin 2 in healthy adults and in pediatric patients infected with Shiga-like toxin-producing Escherichia coli. Antimicrob Agents Chemother54 , 239–243 (2010).
13. Bitzan, M. et al. Safety and pharmacokinetics of chimeric anti-shiga toxin 1 and anti-shiga toxin 2 monoclonal antibodies in healthy volunteers. Antimicrob Agents Chemother (2009) doi:10.1128/AAC.01661-08.
14. H., T. et al. Effect of an Oral Shiga Toxin-Binding Agent on Diarrhea-Associated Hemolytic Uremic Syndrome in Children: A Randomized Controlled Trial. J Am Med Assoc 290 , 1337–1344 (2003).
15. Lopardo, G. et al. RBD-specific polyclonal F(ab´)2 fragments of equine antibodies in patients with moderate to severe COVID-19 disease: A randomized, multicenter, double-blind, placebo-controlled, adaptive phase 2/3 clinical trial. EClinicalMedicine 34 , 100843 (2021).
16. DIRECCIÓN DE EVALUACIÓN Y REGISTRO DE MEDICAMENTOS. Disposición 2022-951-APN-ANMAT. https://www.argentina.gob.ar/sites/default/files/dispo_0951-22_covifab_reins.pdf (2022).