Importance of bioimaging in assessing the preclinical safety and in vivo biodistribution of covifab, an RBD-specific F(AB′)2 fragment derived from equine polyclonal antibodies

SALINAS, F.; MARELLI, B. E.; SANGUINETI, S.; GOLDBAUM, F.; MUÑOZ, L.; ETCHEVERS, L.; SILVESTRINI, P.; NOTARO, U.; SALVETTI, N.; ZYLBERMAN, V.; ORTEGA, H. H.

Congreso; LXVII Reunión anual de la Sociedad Argentina de Investigación Clínica (SAIC); 2022

SARS-CoV-2 pandemic has required the urgent development and approval of treatments precluding the traditional drug discovery pathways, given their typically long times. In this sense, the need for tools that accelerates the evaluation process of new therapies, taking advantage of technological evolution in the sciences, has been highlighting. In vivo bioluminescent imaging systems are cutting-edge technologies in biomedicine due to their advantages of high sensitivity, non-invasiveness, non-radioactivity and low cost. The search for the more effective therapy against COVID-19 has prompted the investigation of different forms of treatment, including antivirals, corticosteroids, monoclonal and polyclonal antibodies, as well as vaccines1,2. Immunotherapies with hyperimmune serum have been applied as a therapeutic approach for numerous diseases. In particular, equine polyclonal antibodies (EpAbs) have been used for decades in the management of clinical emergencies, and are the technological base of the first treatment for hemolytic-uremic syndrome developed in Argentina3.In this context, a therapeutic hyperimmune serum named CoviFab® was developed for the treatment of COVID-19 patients. CoviFab® is based on EpAbs from which the F(ab´)2 fragments were obtained and purified, diminishing the risk of serum sickness, mainly due to the absence of Fc fragments4. CoviFab® recognizes numerous epitopes (limiting the risk of viral escape mutations) and has a greater avidity than monoclonal antibodies for their cognate antigens5. Understanding the urgent need for pharmacokinetics and preclinical data to comply with regulatory requirements for approval the beginning of clinical phases, our objective was to establish the biodistribution and safety of CoviFab® under good laboratory practice (GLP-OECD) standards, and thus validating the use of bioimaging as a useful tool in preclinical drug studies.CoviFab® labeled with IRDye® 800CW fluorescent dye was administered in male BALB/cCmedc mice, 6-7 weeks old, 21.1+/-1.5 g, and imaged in vivo and ex vivo with a Pearl® Trilogy Imager LICOR-Bioscences Imaging System. The treated group was administered with a dose of 4 mg/kg of Covifab® intravenously at 0 and 48 h. Then, in vivo NIR (near-infrared wavelength) images were obtained at 1, 12, 24, 36, 48, 49, 60, 60, 72, 84, 96, 108, 120, 132, and 144 h after first administration. At 96 and 144 h, mice (n=6) were sacrificed for ex vivo imaging, pathological-clinical and histopathological analyses (Figure 1). Control animals (n=5) were euthanized 144 h after the administration of saline solution. Hematologic parameters were analyzed, including red blood cells, hematocrit, hemoglobin, mean cell volume of red blood cells, mean cell hemoglobin, mean corpuscular hemoglobin concentration, platelet, white blood cell with differential count of granulocyte, lymphocyte, and monocyte. Clinical chemistry was determined with validated micro-methods for small volumes and blood urea nitrogen, creatinine, alanine aminotransferase, aspartate aminotransferase, total protein, albumin, globulins, and glucose were measured. A sandwich ELISA developed and validated according to FDA guidelines (Guidance for Industry: Bionalytical Method Validation) was used to determine the concentration of equine F(ab’)2 in serum samples with average intra-assay coefficient of variation of 5.67 % and inter-assay of 7.49 %.In the toxicological study, intravenous administration of CoviFab® revealed no significant changes in serum clinical chemistry and hematological biomarkers at 96 and 144 h. Also, no macroscopic changes were observed in the necropsy without microscopic lesions in the pivotal organs. The in vivo bioimaging study showed that CoviFab® was localized rapidly in all regions tested. In the kidney and bladder areas, labeled CoviFab® was visualized 24 h after each injection, with significantly increased (P < 0.05) fluorescence relative to basal. In addition, in the liver and ears (used to assess widespread distribution), fluorescence was higher (P < 0.05) than basal throughout the study period. In all organs, the profile was similar, with the highest fluorescence 1 h after each injection and gradually decreasing until the end of the study. A highest (P < 0.05) relative fluorescence was observed in kidney and bladder areas, indicating that this could be the main excretion pathway, which was later confirmed by the fluorescence values observed in urine, although this fluorescence could be partially due to fluorophore cleavage. The ex vivo imaging study supports the information about the biodistribution determined in vivo, allowing to confirm that, in most organs, CoviFab® remains for more than 144 h after the first administration (96 h after the second administration). The fluorescent signals detected in the liver, kidney, adrenal glands, stomach/intestine, testicles, spleen, lung, heart, brain, eyes, bladder, and blood showed significant differences compared to control (P < 0.05). This long persistence time circulation is consistent with that described for mono- and polyclonal antibodies6,7,8.The correlation analysis between ex vivo organ fluorescence, urine and feces fluorescence, and serum F(ab′)2 concentration shows that in vivo imaging is a strong predictor of biodistribution in pharmacokinetic studies (r > 0.95; P < 0.05).These preclinical data confirmed that CoviFab® was safe with no observable adverse effects in mice. In addition, in vivo and ex vivo NIR imaging data indicate its localization and permanence in the organs of interest for COVID-19 (as lung) demonstrating the usefulness of this bioimaging system as a strong predictor of biodistribution and obtaining relevant results for a rapid drug development process.Although progress with the application of vaccines and other therapeutic measures is being made, the COVID-19 widespread pandemic continues to spread throughout the globe, with high mortality rates, especially in high-risk patients, which currently remains a major public health concern. The new drug discovery process has been difficult due to very little knowledge about the molecular mechanism involved in SARS-CoV-2 infection. There are currently several approved vaccines for COVID-19 and few new approved therapies, while many potential treatments are being evaluated in ongoing clinical trials. In this sense, many possible therapies have been pre-clinically and clinically tested, and many more are in process. However, only a few were found to be effective, undergoing the clinical trials. Our results supported the development of CoviFab®, helping to rapidly move towards clinical phases in the pandemic circumstances. The product was approved by the Argentine health authorities in December 20209, with a rapid use during the emergency4. Recently a retrospective cohort study, CoviFab®, showed adequate safety and effectiveness in the treatment of hospitalized patients with severe SARS-CoV-2 disease10, confirming the impact of our preclinical development, in this context of translational medicine.