Evaluation of a panel of therapeutic antibody clinical candidates for efficacy against SARS-CoV-2 in Syrian hamsters

Abstract

The COVID-19 pandemic spurred the rapid development of a range of therapeutic antibody treatments. As part of the US government's COVID-19 therapeutic response, a research team was assembled to support assay and animal model development to assess activity for therapeutics candidates against SARS-CoV-2. Candidate treatments included monoclonal antibodies, antibody cocktails, and products derived from blood donated by convalescent patients. Sixteen candidate antibody products were obtained directly from manufacturers and evaluated for neutralization activity against the WA-01 isolate of SARS-CoV-2. Products were further tested in the Syrian hamster model using prophylactic (-24 h) or therapeutic (+8 h) treatment approaches relative to intranasal SARS-CoV-2 exposure. In vivo assessments included daily clinical scores and body weights. Viral RNA and viable virus titers were quantified in serum and lung tissue with histopathology performed at 3d and 7d post-virus-exposure. Sham-treated, virus-exposed hamsters showed consistent clinical signs with concomitant weight loss and had detectable viral RNA and viable virus in lung tissue. Histopathologically, interstitial pneumonia with consolidation was present. Therapeutic efficacy was identified in treated hamsters by the absence or diminution of clinical scores, body weight loss, viral loads, and improved semiquantitative lung histopathology scores. This work serves as a model for the rapid, systematic in vitro and in vivo assessment of the efficacy of candidate therapeutics at various stages of clinical development. These efforts provided preclinical efficacy data for therapeutic candidates. Furthermore, these studies were invaluable for the phenotypic characterization of SARS CoV-2 disease in hamsters and of utility to the broader scientific community.

Keywords: Animal model; Antibody cocktail; COVID-19; Monoclonal antibody; SARS-CoV-2; Therapeutic antibody.

Fig. 1

Hamster efficacy study overview: in vivo pre-exposure prophylaxis and/or post-exposure treatment Three candidate antibody therapeutics were evaluated in each experiment with 2 doses of single treatments administered intraperitoneally (IP) at a standardized dose (10 mg/kg) and a company-selected dose. Mock-IP-treated (sham, phosphate buffered saline [PBS]) and mock-intranasal (IN)-exposed hamsters were included as negative controls. In these studies, the hamsters were treated IP with antibody/sham 24 h prior to IN exposure to 5 log₁₀ PFU SARS-CoV-2, while in therapeutic studies, hamsters were treated 8 h following virus exposure. Following virus exposure, each hamster was weighed daily. Half of the hamsters in each group were euthanized on day 3 post-exposure and the other half of each group was euthanized on day 7. Following euthanasia, blood and lung tissues were collected at necropsies for further analysis.

Fig. 2

Neutralization of authentic SARS-CoV-2 (the progenitor WA-01 isolate) by a panel of 16 therapeutic antibody candidates The 50% neutralization titers were calculated referencing “virus only” controls for each run. A four-parameter logistical analysis was performed on the full dilution series using Prism (GraphPad Software, San Diego, CA, USA). Analysis was done in quadruplicate. The regression was performed using all four replicates per dilution and the precise titer was calculated from the regression curve. Most monoclonal therapeutics tested had similar neutralization curves with EC50 values around 10² ng/mL. Polyclonal products (AE, AG, AJ, Q) require higher concentrations with EC50 values around 10⁵ ng/mL.

Fig. 3

Change in body weight (%) from days post-exposure (Day 0) 3A. Most compounds protect against body weight loss when administered prophylactically; 3B. Many compounds protect against body weight loss when administered post-exposure, albeit with wider variance when compared to prophylaxis studies. Variation was noted between hamsters treated across labs, although mock exposure groups were comparable. Antibodies tested in each study were as follows: IRF-A (−24 h):Y, AL, M; IRF-B (−24 h): AM, AC, AB; IRF-C (−24 h): D, Z, convalescent plasma; RIID-1 (−24 h): I, AQ, S; RIID-2 (−24 h):AF, AE, convalescent plasma, normal plasma; IRF-D (+8 h): AM, AC, AB; IRF-E (+8 h): Y, AL, M; IRF-F (+8 h): D, Z, AE, convalescent plasma; RIID-3 (+8 h): I, AQ, S.

Fig. 4

Lung tissue virus titer and viral load detected by qPCR Virus titers (upper panel) and subgenomic viral RNA (lower panel) from lung tissues collected at day 3 post-exposure were measured after (A) prophylactic or (B) post-exposure administration of antibodies in SARS-CoV-2-exposed hamsters. There was significant viral shedding observed in mock-treated controls. Minimal viral shedding was observed on day 7. Samples with PFU/g (titer) or subgenomic RAN gene copies/mg (gene copies) calculated to be below the assays lower limit of quantification (LLOQ) are represented using unfilled circles, while values above the LLOQ are represented using filled circles.

Fig. 5

Interstitial pneumonia (IP) lung pathology scores Lung sections taken at days 3 and 7 post exposure were examined by light microscopy and semiquantitatively scored on a scale ranging from 0 to 4. Scoring based on the percentage of interstitial pneumonia: 0: Below lower limit of perception (LLOP, estimated by model); 1: LLOP - ≤25%; 2: 26–50%; 3: 51–80%; 4: ≥80%. Protection from SARS-CoV-2-induced pneumonia was determined following prophylactic (A) or post-exposure (B) antibody administration.

Fig. 6

Pseudovirus neutralization antibody measured in serum collected on day 3 The serum dilution required to achieve 50% neutralization of SARS-CoV-2 pseudovirus, an indication of the presence of a nAb therapeutic in the hamster serum, were measured in prophylactic (A) or post-exposure (B) experiments in SARS-CoV-2-exposed hamsters. In treated hamsters, most serum samples showed 50% neutralization of pseudovirus at high dilutions, indicating that antibodies were present in the serum at day 3.

Fig. 7

In vivo assay overview Colored boxes correspond to protective effects of therapeutics in study experiments compared with controls.

Fig. 8

Prophylactic (-24h) and post-virus-exposure (+8h) antibody treatment correlations across endpoints Numerals indicate Spearman rank correlations by endpoint. Comparison of endpoints across prophylactic and post-virus-exposure treatment studies. In vitro live virus neutralization assays did not correlate well with in vivo virology or pathology assays, but correlated well with pseudovirus neutralization serology. Pathology endpoints (including body weight loss AUC and interstitial pneumonia) showed some correlation to virology endpoints including reduction in viral shedding (PCR) and plaque formation using day 3 lung samples (−24h only). Correlations were less clear overall in the post virus-exposure treatment (+8h) study. Virology endpoints (viral plaque and PCR) were closely correlated to each other in the pre-exposure prophylaxis (−24h) studies but less correlative in the post-exposure treatment (+8h) studies. Serology endpoints (pseudovirus neutralization) were most clearly correlated to reduction in plaque formation (−24 and +8h), PCR, and body weight change (−24h only). Overall, endpoints were more highly correlated in the pre-exposure prophylaxis (−24h) studies when compared to the post-virus-exposure treatment (+8h) studies.