A Phase I study to evaluate the safety, tolerance and pharmacokinetics of anti-Shiga toxin hyperimmune equine F(ab’)2 fragments in healthy volunteers
Authors
Yanina Hiriart1-2#, Paula Scibona3, Augusto Ferraris5, Waldo H. Belloso4, Valeria Beruto3, Facundo Garcia Bournissen6, Vanesa Zylberman1-2, Luciana Muñoz2, Fernando Goldbaum1,2,7, Linus Spatz2, Mariana Colonna2, Santiago Sanguineti2, Ventura A. Simonovich3.
1-National Scientific and Technological Research Council, CONICET, Godoy Cruz 2290 (C1425FQB) CABA, Argentina
2-Inmunova S.A Av. 25 de Mayo 1021 (CP1650), San Martín, Buenos Aires, Argentina
3-Clinical Pharmacology Section, Hospital Italiano de Buenos Aires, Tte. Gral. Juan Domingo Perón 4190, CP (C1181ACH), CABA, Argentina
4-Terra Nova Innovation Unit. Hospital Italiano de Buenos Aires, Tte. Gral. Juan Domingo Perón 4190, CP (C1181ACH) CABA, Argentina
5-Internal Medicine Section, Hospital Italiano de Buenos Aires, Tte. Gral. Juan Domingo Perón 4190, CP (C1181ACH), CABA, Argentina
6-Division of Pediatric Clinical Pharmacology, Department of Pediatrics, Schulich School of Medicine & Dentistry, University of Western Ontario, London, Ontario, Canada
7-CRIP, Centro de rediseño e ingeniería de proteínas, 25 de Mayo y Francia (CP 1650) San Martín, Buenos Aires, Argentina
YH and PS equally contributed to this work
* Correspondence to:
Ventura.simonovich@hospitalitaliano.org.ar
*Cover title: Safety of anti-Stx (Fab’)2 fragments
The authors confirm that the Principal Investigator for this paper is Ventura Simonovich and that he had direct clinical responsibility for patients.
Word count: The main text has 2961 words, three figures, two tables and three supplementary tables as appendix
11# Actually, Instituto de estudios Inmunológicos y Fisiopatológicos, CONICET, UNLP, Boulevard 120 1489, CP 1900, La Plata, Buenos Aires, Argentina
ABSTRACT
Shiga toxin-producing Escherichia coli -associated hemolytic uremic syndrome STEC-HUS is considered a toxemic disorder in which early intervention with neutralizing antibodies may have therapeutic benefits. We developed a novel therapy, INM004, composed of F(ab’)2 fragments from equine immunoglobulins that neutralizes Stx1/Stx2, potentially preventing the onset of HUS.
A single-center, randomized, Phase 1, single-blind, placebo-controlled clinical trial to evaluate INM004 safety, tolerance, and pharmacokinetics (PK) in healthy adult volunteers, was conducted in two stages; In Stage I, eight subjects were divided in two cohorts (n=4) to receive a single INM004 dose of 2 or 4 mg.kg-1, or placebo (INM004:placebo rate 3:1). In Stage II six study subjects received either three INM004 doses of 4 mg.kg-1 repeated every 24 h, or placebo (INM004:placebo rate of 5:1). Hospital discharged was 24 hours after the last infusion. INM004 was quantified by ELISA in serum samples obtained at predefined times. Safety and tolerability were assessed in both Stages by monitoring adverse events (AEs), laboratory test values, and vital signs. Eight subjects (57.1%) experienced treatment-emergent AEs (TEAEs), that resolved within 24 hours without requiring changes in treatment or additional intervention. No serious AEs were reported. Most TEAEs were of mild or moderate intensity, and four were possibly drug-related. Peak concentrations of INM004 occurred within 2 hours after infusion, with median Cmax values of 45.1 µg.mL-1 and 77.7 µg.mL-1 for different doses. The serum concentration of INM004 declined in a biphasic manner (t1/2 range 30.7-52.9 hours). Systemic exposures increased with each subsequent dose in a dose-proportional manner, exhibiting accumulation. Geometric median Cmax and AUC values were 149 µg.mL-1 and 10300 µg.h.mL-1, respectively, in the repeated dose regimen. The results obtained in this First in Human Study supporting progression into the phase 2 trial in children with hemolytic uremic syndrome.
Keywords: HUS treatment, F(ab´)2 fragments, pharmacokinetics, anti-shiga toxins, INM004
What is already known about this subject:
STEC-HUS is a serious foodborne disease worldwide for which there are currently no specific therapeutic options availables.
STEC-HUS is considered a toxemic disorder more than a bacterial disease, suggesting that treatment with neutralizing antibodies may have a therapeutic benefit.
What this study adds
Equine Anti Shiga Toxin F(ab’)2 immunoglobulins fragments (INM004) is a novel therapy for STEC infections which have an excellent profile of safety.
In this phase I study, INM004 showed adequate tolerability and safety in healthy volunteers, providing information on the pharmacokinetics of this novel passive immunotherapy to support further clinical development, including clinical trials in children suffering HUS.
Introduction
Shiga toxin-producing Escherichia coli -associated hemolytic uremic syndrome (STEC-HUS) is a disease characterized by microangiopathic hemolytic anemia, thrombocytopenia and acute renal compromise of varying extent, occurring after an episode of E. coli diarrhea with or without blood1. STEC-HUS affects predominantly pediatric patients. STEC-HUS poses a high social and economic burden, particularly due to the frequent need for long-term renal replacement therapy and renal transplantation2. STEC-HUS was described for the first time in 1955 by Gasser et. al, and 30 years later Karmali et al. demonstrated the association between this syndrome and diarrhea caused by bacteria producing cytotoxins3. These cytotoxins, called Shiga-type toxins or shigatoxins (Stx) can cause renal, neurological, intestinal or heart complications leading in some cases to chronic renal impairment, and even death. The development of effective treatments for STEC-HUS remains a priority. Polyclonal antibodies (pAbs) and specifically equine polyclonal antibodies (EpAbs) present an interesting therapeutic option4. Their safety and effectiveness have been widely demonstrated in rabies virus or botulinum toxin exposures, and in the treatment of venomous snake and scorpion bites5–8. Furthermore, EpAbs present several advantages over mAbs. First, they are composed of F(ab)’2 fragments generated by pepsin digestion that retain the bivalent binding capacity of IgG immunoglobulins but lack the constant region (Fc) potentially responsible for Fc-triggered side effects. Second, EpAbs recognize a vast array of epitopes and tend to develop greater avidity than mAbs for their cognate antigens. Finally, EpAbs are considerably easier than mAbs to produce, allowing manufacturers to rapidly achieve large-scale production.
We developed a novel therapy composed of F(ab’)2fragments from equine immunoglobulins that efficiently neutralizes eight variants of Stx1 and Stx2 in vitro and in vivo in an animal model of STEC-HUS potentially preventing the onset of HUS9. No significant toxicity was observed in preclinical studies, both in single-dose and repeated-dose toxicity studies in mice and rabbits10.
The aim of this phase I study was to evaluate the safety, tolerance, and pharmacokinetics (PK) of INM004 in human volunteers. In addition, the Stx neutralizing capacity of INM004 in the plasma of volunteers was evaluated in vitro .
Methods