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).