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\begin{document}

\title{\textsubscript{}A Phase I study to evaluate the safety, tolerance and
pharmacokinetics of anti-Shiga toxin hyperimmune equine
F(ab')\textsubscript{2} fragments in healthy volunteers}



\author[1]{yanina Hiriart}%
\author[2]{Paula Scibona}%
\author[2]{Augusto Ferraris}%
\author[2]{Waldo Belloso}%
\author[2]{Valeria Beruto}%
\author[3]{Facundo Garcia-Bournissen}%
\author[1]{Vanesa Zylberman}%
\author[4]{Luciana Muñoz}%
\author[1]{Fernando Goldbaum}%
\author[4]{Linus Spatz}%
\author[4]{Mariana Colonna}%
\author[4]{Santiago Sanguineti}%
\author[2]{Ventura Simonovich}%
\affil[1]{CONICET}%
\affil[2]{Hospital Italiano de Buenos Aires}%
\affil[3]{London Health Sciences Centre}%
\affil[4]{Inmunova}%


\vspace{-1em}



  
  \date{May 18, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
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\endgroup





\selectlanguage{english}
\begin{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.
INM004, composed of F(ab')\textsubscript{2} fragments from equine
immunoglobulins, 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
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 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 (C\textsubscript{max}) of INM004 were 45.1 \selectlanguage{greek}µ\selectlanguage{english}g.mL-1 and
77.7 \selectlanguage{greek}µ\selectlanguage{english}g.mL-1 for different doses, within two hours after infusion. The
serum concentration declined in a biphasic manner (t\textsubscript{1/2}
range 30.7-52.9 hours). Systemic exposures showed accumulation in the
repeated dose regimen (C\textsubscript{max} Day1 85.7 vs.149 \selectlanguage{greek}µ\selectlanguage{english}g.mL-1
Day3). These results supporting progression into the phase 2 trial in
children with HUS%
\end{abstract}%



\sloppy


A Phase I study to evaluate the safety, tolerance and pharmacokinetics
of anti-Shiga toxin hyperimmune equine F(ab')\textsubscript{2} fragments
in healthy volunteers

Authors

Yanina Hiriart\textsuperscript{1-2\#}, Paula Scibona\textsuperscript{3},
Augusto Ferraris\textsuperscript{5}, Waldo H.
Belloso\textsuperscript{4}, Valeria Beruto\textsuperscript{3}, Facundo
Garcia Bournissen\textsuperscript{6}, Vanesa
Zylberman\textsuperscript{1-2}, Luciana Mu\selectlanguage{ngerman}ñoz\textsuperscript{2},
Fernando Goldbaum\textsuperscript{1,2,7}, Linus
Spatz\textsuperscript{2}, Mariana Colonna\textsuperscript{2}, Santiago
Sanguineti\textsuperscript{2}, Ventura A. Simonovich\textsuperscript{3}.

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')\textsubscript{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\textsuperscript{\#} Actually, Instituto de estudios Inmunológicos y
Fisiopatológicos, CONICET, UNLP, Boulevard 120 1489, CP 1900, La Plata,
Buenos Aires, Argentina

ABSTRACT

Shiga toxin-producing \emph{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')\textsubscript{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 \selectlanguage{greek}µ\selectlanguage{english}g.mL-1 and 77.7 \selectlanguage{greek}µ\selectlanguage{english}g.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
\selectlanguage{greek}µ\selectlanguage{english}g.mL-1 and 10300 \selectlanguage{greek}µ\selectlanguage{english}g.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\selectlanguage{ngerman}´)\textsubscript{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')\textsubscript{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 \emph{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 \emph{E.
coli} diarrhea with or without blood\textsuperscript{1}. 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 transplantation\textsuperscript{2}.
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
cytotoxins\textsuperscript{3}. 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
option\textsuperscript{4}. Their safety and effectiveness have been
widely demonstrated in rabies virus or botulinum toxin exposures, and in
the treatment of venomous snake and scorpion
bites\textsuperscript{5--8}. Furthermore, EpAbs present several
advantages over mAbs. First, they are composed of
F(ab)'\textsubscript{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')\textsubscript{2}fragments from equine immunoglobulins that
efficiently neutralizes eight variants of Stx1 and Stx2 \emph{in vitro}
and \emph{in vivo} in an animal model of STEC-HUS potentially preventing
the onset of HUS\textsuperscript{9}. No significant toxicity was
observed in preclinical studies, both in single-dose and repeated-dose
toxicity studies in mice and rabbits\textsuperscript{10}.

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 \emph{in vitro} .

Methods

\emph{Study design}

We conducted a single-center, randomized, Phase 1, single blind,
placebo-controlled clinical trial to evaluate the safety, tolerability,
and PK of INM004 in two stages. In stage I, a single dose was given (at
2 dose levels, 2 mg/kg and 4 mg/kg), and in stage II, 3 daily 4 mg/kg
doses were given. The study enrolled healthy adult volunteers between
December 2017 and September 2018 in a single site in Argentina.

Eligible participants included fourteen healthy volunteers 18-55 years
old, with a body mass index (BMI) of 19-27 kg.m-2, normal laboratory
values, chest ray, and electrocardiogram recordings. Exclusion criteria
included history of equine serum allergy or any history of allergy,
prior administration of equine serum for other indications (anti-tetanus
serum, anti-ophidic serum, anti-arachnid toxin serum, among others),
pregnancy, seropositivity for HIV, hepatitis B or C, mental condition,
drug or alcohol abuse, any history of cardiovascular, hepatic,
pulmonary, gastrointestinal, hematological, or neurological illness,
having taken any prescription drugs within the two weeks before the
inclusion date, infectious disease within 30 days before the inclusion,
or having a personal bond with any of the study personnel.

All study subjects provided written informed consent after receiving
comprehensive information of the objectives, methods, and potential
harms related to their participation in the study. The present study was
approved by the local ethics committee and the Argentine National Food
and Drug Regulatory Agency, Administración Nacional de Medicamentos,
Alimentos y Tecnología Médica (ANMAT). The study was prospectively
registered in clinicaltrials.gov (\emph{NCT03388216}).

\emph{Blinding and randomization}

Study subjects were masked to treatment allocation and were randomized
to either treatment or placebo group when admitted to the Hospital at
day 0. We established a system of opaque closed envelopes containing the
arm designation. The pharmacist of the study randomly selected the
envelope for each study subject in the presence of the principal
investigator. For safety reasons, the researchers responsible for the
clinical trial were aware of treatment allocation. INM004 was provided
to the nursing staff in anonymized bags with the preparation already
reconstituted and labeled with participant and treatment identification
number without the specific content (INM004 or placebo) so that the
subject could not infer the assigned treatment. The physician
responsible for safety monitoring during the infusion of INM004 or
placebo was blinded to treatment allocation.

\emph{Procedures}

INM004 was formulated by Inmunova and manufactured under GMP standards
and supplied in vials containing: Total proteins (equines) 125,0 mg;
Sodium chloride 45,0 mg; Hydrochloric Acid/Sodium Hydroxide as much as
suffices to keep pH between 6,0 and 7,0; Water for injection as much as
suffices to 5 mL.

Safety monitoring and tolerability of INM004 were assessed by monitoring
adverse events (AEs), laboratory test values, and vital signs (blood
pressure, heart rate and complete physical examination and 12 lead
electrocardiogram examinations).

The trial was divided into two stages. In Stage I, 8 study subjects were
divided in two cohorts of 4 patients to receive a single dose of INM004
or placebo at an INM004:placebo rate of 3:1 with dose escalation design.
Thus, after a screening period of two weeks, study participants in the
first and second cohort received 2 mg.kg-1 protein of INM004 or placebo
and 4 mg.kg-1 of INM004 or placebo, respectively. We selected an initial
dose of 2 mg.kg-1 based on the kinetics and effectiveness determined in
preclinical studies. This dose was determined after applying a
correction factor of 5 to the maximum dose of 116 mg.kg-1 administered
in mice following the allometric approach recommended by the US Food and
Drug Agency´s guidelines. We used this correction factor since the NOAEL
was not reached with the highest dose tested in mice, and similar equine
Fab products already available for human use have demonstrated safety in
humans at higher protein doses.

Four participants received sequentially an initial dose of 2 mg.kg-1
intravenously, with ClNa 0.9\% in a total volume of 100 ml administered
at 2 ml.min-1. Once we established that the drug was well tolerated,
four more volunteers were enrolled and received 4 mg.kg-1. Both cohorts
of stage I had a INM004:placebo rate of 3:1.

Stage II recruited and randomized sequentially 6 study subjects to
receive INM004 or placebo (INM004:placebo rate of 5:1). After a
screening period of two weeks, participants received three repeated
doses of 4 mg.kg-1 of INM004 or placebo every 24 hours.

Four visits for PK sampling and a final safety follow-up visit at 30
days after hospital discharge were pre-scheduled. In case of occurrence
of adverse events (AEs), unscheduled visits or hospitalizations were
performed until resolution of the event.

\emph{PK sampling}

PK parameters of INM004 levels were determined in all subjects receiving
at least one dose of the medication. In stage I the participants were
hospitalized for 24 hours after the infusion and PK samples were
obtained at the following times: 0, 15, 30, 45, 60, 90, 120, 150, 180,
210, 240, 270, 300, 360, and 480 min, and at 12, 24, 48, and 72 hours
after infusion. A baseline and final laboratory tests for immunogenicity
were obtained at day 0 and 30, respectively. Ambulatory PK samples,
clinical visits, and safety laboratories at days 3, 4, 5, and 30 after
infusion were scheduled. During stage II, the participants received
three repeated doses of 4 mg.kg-1 every 24 hours during 3 days. The
volunteers stayed hospitalized for 24 hours after the last infusion. PK
samples were obtained at the following times: 0, 30, 60, 120, 180, 240,
360, and 480 min, and at 12 and 24 hours after first infusion; at 60,
120, 360 min, and 12 and 24 hours after the second infusion; and at 60,
120, 360 min, and 12, 24, 48, 72, 96 and 120 hours, and at day 30 after
the third infusion. Ambulatory visits and safety laboratory tests were
identical to those scheduled in stage I.

Quantification of the F(ab´)\textsubscript{2} equine polyclonal antibody
concentrations in plasma was performed by Enzyme-Linked Immunosorbent
Assay (ELISA) developed for preclinical studies of INM004 and validated
for human plasma samples\textsuperscript{11}. Blood samples (5 ml) for
PK analyses were collected by venipuncture, and the results analyzed by
non-compartmental (NCA) PK methods. The fixed main effects treatment,
period and sequence were analyzed for the PK parameters of interest
{[}C\textsubscript{max}, AUC (0-t) and AUC (0-Inf){]}. The AUC was
obtained from the graph of the drug plasma levels over time for each
subject. The 90\% confidence interval limits for PK were calculated.
C\textsubscript{max} and T\textsubscript{max} were estimated directly
from the curve with interpolation. AUC (0-inf) and AUC (0-t) were
calculated using the trapezoidal rule based on the individual
concentrations observed. The elimination half-time (t½) was calculated
as Ln (2) / \selectlanguage{greek}λ\selectlanguage{english}z, where \selectlanguage{greek}λ\selectlanguage{english}z was estimated from a log-linear regression of
the last 3 to 5 points of the curve, after reaching plateau.
Coefficients of variation were calculated for Cmax and AUC(0-inf).

\emph{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
described\textsuperscript{3}. 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\selectlanguage{ngerman}´)\textsubscript{2}. The estimated concentrations of INM004
(determined by ELISA) in the samples at the evaluated times were 40, 15
and 7 \selectlanguage{greek}µ\selectlanguage{english}g.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 \textless{}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\selectlanguage{ngerman}® 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 C\textsubscript{max} values were 45.1 \selectlanguage{greek}µ\selectlanguage{english}g.mL-1 and 77.7 \selectlanguage{greek}µ\selectlanguage{english}g.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 t\selectlanguage{ngerman}\textsubscript{½} 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\textsubscript{(0-last)} values were 1180 \selectlanguage{greek}µ\selectlanguage{english}g.h.mL-1 and 2373
\selectlanguage{greek}µ\selectlanguage{english}g.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 t\textsubscript{1/2} 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 C\textsubscript{max} 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 C\textsubscript{max} and AUC,
respectively.

Geometric mean C\textsubscript{max} value was 149 \selectlanguage{greek}µ\selectlanguage{english}g.mL-1 with
corresponding geometric mean AUC(0-\selectlanguage{greek}τ\selectlanguage{english}), AUC(0-inf) and AUC(total) (i.e.
the total AUC for Days 1 to 3 combined) values of 2460 \selectlanguage{greek}µ\selectlanguage{english}g.h.mL-1, 10300
\selectlanguage{greek}µ\selectlanguage{english}g.h.mL-1 and 14600 \selectlanguage{greek}µ\selectlanguage{english}g.h.mL-1, respectively. The between-subject
variability, as assessed from the geometric percent CV, was low (CV\%
\textless{}25\%) for both C\textsubscript{max} 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 (\textless{}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 trials\textsuperscript{12--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\selectlanguage{ngerman}´)\textsubscript{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´)\textsubscript{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, respectively\textsuperscript{15}.
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-19\textsuperscript{16}.

This study proves that the production of equine
F(ab')\textsubscript{2}for 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 (\emph{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.

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Figure 1. Study flow chart.

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\textbf{Hosted file}

\verb`Figure 1 FIH SUH.pptx`  available at \url{https://authorea.com/users/468443/articles/654566-a-phase-i-study-to-evaluate-the-safety-tolerance-and-pharmacokinetics-of-anti-shiga-toxin-hyperimmune-equine-f-ab-2-fragments-in-healthy-volunteers}\selectlanguage{english}
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Figure 2. Combined individual PK serum concentrations (\selectlanguage{greek}µ\selectlanguage{english}g.mL-1) of INM
004 vs time (h) of study subjects. Panel A Stage I, Cohort I (2.00
mg.kg-1 single dose regime). Panel B Stage I, Cohort II (4.00 mg.kg-1
single dose). Panel C, Stage II (4.00 mg.kg-1 repeated dose regime).

\textbf{Hosted file}

\verb`Figure 2 FIH SUH.docx`  available at \url{https://authorea.com/users/468443/articles/654566-a-phase-i-study-to-evaluate-the-safety-tolerance-and-pharmacokinetics-of-anti-shiga-toxin-hyperimmune-equine-f-ab-2-fragments-in-healthy-volunteers}\selectlanguage{english}
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\includegraphics[width=0.70\columnwidth]{figures/Figure-3-Neutralization/Figure-3-Neutralization}

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Figure 3. Neutralizing capacity of INM 004 in 3 study subjects.\selectlanguage{english}
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\includegraphics[width=0.70\columnwidth]{figures/Table-2-FIH-SUH/Table-2-FIH-SUH}

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