Computational design of nanomolar-binding antibodies specific to multiple SARS-CoV-2 variants by engineering a specificity switch of antibody 80R using RosettaAntibodyDesign (RAbD) results in potential generalizable therapeutic antibodies for novel SARS-CoV-2 virus.

The human infectious disease COVID-19 caused by the SARS-CoV-2 virus has become a major threat to global public health. Developing a vaccine is the preferred prophylactic response to epidemics and pandemics. However, for individuals who have contracted the disease, the rapid design of antibodies that can target the SARS-CoV-2 virus fulfils a critical need. Further, discovering antibodies that bind multiple variants of SARS-CoV-2 can aid in the development of rapid antigen tests (RATs) which are critical for the identification and isolation of individuals currently carrying COVID-19. Here we provide a proof-of-concept study for the computational design of high-affinity antibodies that bind to multiple variants of the SARS-CoV-2 spike protein using RosettaAntibodyDesign (RAbD). Well characterized antibodies that bind with high affinity to the SARS-CoV-1 (but not SARS-CoV-2) spike protein were used as templates and re-designed to bind the SARS-CoV-2 spike protein with high affinity, resulting in a specificity switch. A panel of designed antibodies were experimentally validated. One design bound to a broad range of variants of concern including the Omicron, Delta, Wuhan, and South African spike protein variants.

The Landscape of Neutralizing Monoclonal Antibodies (nAbs) for Treatment and Prevention of COVID-19.

Purpose: After nearly 3 years of the COVID-19 pandemic, even though a vast body of knowledge and products (including vaccines and treatments) have been developed and disseminated, the virus is still evolving and new variants arising. Consequently, thousands of lives continue to be lost. Neutralizing monoclonal antibodies (nAbs) are promising drugs that emerged to treat SARS-CoV-2. In the uncertainty of the current situation, there is the question of whether organizations should continue to invest in this technology. To help decision-making in scientifical and pharmaceutical organizations, it is of major importance to monitor the development of products and technologies. Therefore, the aim of this study is analyze the landscape of nAbs for COVID-19. Methods: The scenario of 473 biotherapeutics focusing on nAbs was evaluated using foresight techniques and a review of literature. Data were obtained from structured and semi-structured databases and processed for treatment, cleaning, consistency, validation, and enrichment. Results: We identified 227 nAbs and performed an extensive literature review of 16 nAbs in late clinical development, including development technologies, responses to variants of concern (VOCs), manufacturing, and clinical aspects. Conclusions: Even though the emergence of new VOCs is a threat to the effectiveness of this treatment, demanding constant genomic surveillance, the use of nAbs to treat and prevent COVID-19 will probably continue to be relevant due to excellent safety profiles and the possibility of immediate immunity transfer, especially in patients showing inadequate immunological response to vaccination. Therefore, we suggest that organizations should keep investing in improvements in this technology.

Pre-Clinical Development of a Potent Neutralizing Antibody MW3321 With Extensive SARS-CoV-2 Variants Coverage.

Since the outbreak of the coronavirus disease 2019 (COVID-19) pandemic, several variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have emerged and have consistently replaced the previous dominant variant. Therapeutics against variants of SARS-CoV-2 are urgently needed. Ideal SARS-CoV-2 therapeutic antibodies would have high potency in viral neutralization against several emerging variants. Neutralization antibodies targeting SARS-CoV-2 could provide immediate protection after SARS-CoV-2 infection, especially for the most vulnerable populations. In this work, we comprehensively characterize the breadth and efficacy of SARS-CoV-2 RBD-targeting fully human monoclonal antibody (mAb) MW3321. MW3321 retains full neutralization activity to all tested 12 variants that have arisen in the human population, which are assigned as VOC (Variants of Concern) and VOI (Variants of Interest) due to their impacts on public health. Escape mutation experiments using replicating SARS-CoV-2 pseudovirus show that escape mutants were not generated until passage 6 for MW3321, which is much more resistant to escape mutation compared with another clinical staged SARS-CoV-2 neutralizing mAb MW3311. MW3321 could effectively reduce viral burden in hACE2-transgenic mice challenged with either wild-type or Delta SARS-CoV-2 strains through viral neutralization and Fc-mediated effector functions. Moreover, MW3321 exhibits a typical hIgG1 pharmacokinetic and safety profile in cynomolgus monkeys. These data support the development of MW3321 as a monotherapy or cocktail against SARS-CoV-2-related diseases.

Comparison of Monoclonal Antibody Cocktail (Casirivimab-Imdevimab) Treatment with Remdesivir and Favipiravir in Mild to Moderate COVID-19 Infection: A Retrospective Study

Introduction: Favipiravir and remdesivir are antiviral drugs being used in the present pandemic and were also used previously for other viral infections in the past. Monoclonal antibody (Mab) Casirivimab-Imdevimab is a Coronavirus Disease 2019 neutralising antibody approved in the last one year. Therefore, a clinical comparison with the existing treatment modalities is imperative. Aim: To compare Mab with remdesivir and favipiravir for mild to moderate COVID-19 disease. Materials and Methods: A retrospective, observational and single-centre study was conducted at a COVID-19 infection facility and private tertiary care hospital, Mumbai, Maharashtra, India. Data of patients admitted during the period of 1st June 2021 to 31st August 2021 was collected and analysed in the months of September 2021 and October 2021. Adults participants diagnosed to have COVID-19 infection, not requiring critical care or oxygen therapy were included in the study. Time to recovery from treatment onset and the need for treatment escalation were the primary outcome measures. Data was entered into Microsoft Excel spreadsheet version 16 and analysed. Statistical analysis was carried out using Chi-square test for the significance of association between tabulated values of data for qualitative and categorical data. Two-tailed unpaired t-test and Analysis of Variance (ANOVA) was used for quantitative tabulated data. Results: This study included 158 participants, grouped into remdesivir(n=63),favipiravir(n=30)andMab(n=65)treatmentgroups. Gender distribution was comparable in all groups (p-value=0.08). The three groups were compared for need of treatment escalation and time of recovery. The Mab treatment group (on comparing with other treatment arms) had earlier symptom recovery when given to patients with mild COVID-19 disease (p-value=0.006 for major symptoms) or when treatment was started within five days of symptom onset (p-value <0.001). Patients in Mab treatment group with mild illness required no treatment escalation compared to other groups (p-value=0.011). However, time to recovery patients in all treatment groups was comparable in case of patients with moderate COVID-19 illness (p-value=0.7381). In patients with moderate COVID-19 illness Mab treatment group required more frequent treatment escalation compared to remdesivir treatment group (p-value=0.044), when treatment was started within 5 days of symptom onset remdesivir and mab were comparable for treatment escalation (p=0.144). Vaccination status of the three groups differed significantly (p-value=0.033) hence a further subanalysis was done. On further analysis, non-vaccinated patients receiving Mab recovered from minor symptoms (p-value=0.0006) earlier than those receiving Remdesivir. Amongst the participants of the Mab treatment-group, vaccinated and non-vaccinated patients had comparable recovery time and need for treatment escalation (p-value=0.57 and p-value=0.76, respectively). Participants who received Mab-treatment within five days of symptom onset;recovered earlier compared to those who received Mab treatment after five days (p-value=0.019). Conclusion: Monoclonal antibody treatment group compared to the other treatment groups had earlier recovery in non vaccinated patients, mild COVID-19 disease, and when treatment was started before or on the 5th day of symptom onset.

Efficacy of Antibody Cocktail Drug in COVID-19 Positive Patients: A Retrospective Single-centered Study

Introduction: Neutralising monoclonal antibodies (mABs) have been proposed and developed for the treatment of Coronavirus Disease-2019 (COVID-19) patients with mild to moderate diseases and to prevent further progression. The combination of Casirivimab and Imdevimab blocks the entry of virus into cells by attaching to receptor binding domain of Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) spike glycoprotein. The mABs are utilised as a pre-emptive strategy in certain high-risk groups such as those suffering from chronic liver, kidney and respiratory disease, malignancies and other immunocompromised states where efficacy of vaccines may be suboptimal. Aim: To evaluate the clinical outcomes in COVID-19 patients who were treated with Antibody Cocktail drug (casirivimab and imdevimab). Materials and Methods: A retrospective observational study was conducted in patients confirmed positive for SARS-CoV-2 from June 2021 to January 2022 and subsequently, the collected data was analysed from May 2022 to June 2022. The study was conducted in a tertiary care referral hospital in eastern India. All eligible patient subsequently received casirivimab and imdevimab at COVID-19 facility. Monitoring of patients was done upto 12 hour postinfusion. Demographic parameters, routine investigations and clinical outcomes were assessed. Data entry was done using Microsoft Excel. Data was entered, coded and analysed using International Business Machines (IBM) Statistical Package for the Social Sciences (SPSS) version 21.0. All analysis was done at a preset alpha error of 5% and results expressed at confidence levels of 95%. Results: Total 104 eligible cases were taken in present study. Nearly, 93% of those patients who had not been vaccinated were at higher risk for having severely elevated levels of C-Reactive Protein (CRP) as compared to 48% of those with COVID-19 vaccination. Nearly, 9 out of 10 patients with moderate-severe CRP levels were at nine times more risk for longer duration of hospitalisation as compared to normal levels of CRP. All patients having moderate-severe CRP levels required mechanical ventilation in comparison to mild CRP levels. Patients with comorbidities were more likely to get severe COVID-19 infections (p-value ≤0.05). Unvaccinated subjects were more likely to have severe infections than vaccinated subjects. (p-value ≤0.05). Prolonged hospitalisation (>7 days) was statistically significant in severe COVID-19. Unvaccinated subjects had a statistically significant rise in CRP over vaccinated subjects. The majority of the patients receiving antibody cocktail did not require prolonged hospitalisation while a minor fraction required invasive ventilation. Antibody cocktail was safe, well tolerated and had good efficacy and low mortality rate as compared to other modalities of treatment in this study. Conclusion: The duration of hospitalisation and outcomes were superior in patients having mild to moderate COVID-19 who received antibody cocktail without any serious side-effects.

Antibodies to watch in 2023.

In this 14th installment of the annual Antibodies to Watch article series, we discuss key events in commercial monoclonal antibody therapeutics development that occurred in 2022 and forecast events that might occur in 2023. As of mid-November, 12 antibody therapeutics had been granted first approvals in either the United States or European Union (tebentafusp (Kimmtrak), faricimab (Vabysmo), sutimlimab (Enjaymo), relatlimab (Opdualag), tixagevimab/cilgavimab (Evusheld), mosunetuzumab (Lunsumio), teclistamab (TECVAYLI), spesolimab (SPEVIGO), tremelimumab (Imjudo; combo with durvalumab), nirsevimab (Beyfortus), mirvetuximab soravtansine (ELAHERE™), and teplizumab (TZIELD)), including 4 bispecific antibodies and 1 ADC. Based on FDA action dates, several additional product candidates could be approved by the end of 2022. An additional seven were first approved in China or Japan in 2022, including two bispecific antibodies (cadonilimab and ozoralizumab). Globally, at least 24 investigational antibody therapeutics are undergoing review by regulatory agencies as of mid-November 2022. Our data show that, with antibodies for COVID-19 excluded, the late-stage commercial clinical pipeline grew by ~20% in the past year to include nearly 140 investigational antibody therapeutics that were designed using a wide variety of formats and engineering techniques. Of those in late-stage development, marketing application submissions for at least 23 may occur by the end of 2023, of which 5 are bispecific (odronextamab, erfonrilimab, linvoseltamab, zanidatamab, and talquetamab) and 2 are ADCs (datopotamab deruxtecan, and tusamitamab ravtansine).

A combination of two human neutralizing antibodies prevents SARS-CoV-2 infection in cynomolgus macaques.

BACKGROUND: Human monoclonal antibody (mAb) treatments are promising for COVID-19 prevention or therapy. The pre-exposure prophylactic efficacy of neutralizing antibodies that are engineered with mutations to extend their persistence in human serum and the neutralizing antibody titer in serum required for protection against SARS-CoV-2 infection remain poorly characterized. METHODS: The Fc region of two neutralizing mAbs (COV2-2130 and COV2-2381) targeting non-overlapping epitopes on the receptor binding domain of SARS-CoV-2 spike protein was engineered to extend their persistence in humans and reduce interactions with Fc gamma receptors. We assessed protection by individual antibodies or a combination of the two antibodies (designated ADM03820) given prophylactically by an intravenous or intramuscular route in a non-human primate (NHP) model of SARS-CoV-2 infection. FINDINGS: Passive transfer of individual mAbs or ADM03820 conferred virological protection in the NHP respiratory tract in a dose-dependent manner, and ADM03820 potently neutralized SARS-CoV-2 variants of concern in vitro. We defined a protective serum-neutralizing antibody titer and concentration in NHPs for passively transferred human antibodies that acted by direct viral neutralization. CONCLUSIONS: In summary, we demonstrate that neutralizing antibodies with extended half-life and lacking Fc-mediated effector functions are efficient for pre-exposure prophylaxis of SARS-CoV-2 infection in NHPs. These results support clinical development of ADM03820 for COVID-19 prevention. FUNDING: This research was supported by a contract from the JPEO-CBRND (W911QY-20-9-003, 20-05); the Joint Sciences and Technology Office and Joint Program Executive Office (MCDC-16-01-002 JSTO, JPEO); a DARPA grant (HR0011-18-2-0001); an NIH grant (R01 AI157155); and the 2019 Future Insight Prize from Merck KGaA.

Antibodies to watch in 2022.

In this 13th annual installment of the annual 'Antibodies to Watch' article series, we discuss key events in commercial antibody therapeutics development that occurred in 2021 and forecast events that might occur in 2022. Regulatory review of antibody therapeutics that target the SARS-CoV-2 coronavirus proceeded at an unprecedented pace in 2021, resulting in both emergency use authorizations and full approvals for sotrovimab, regdanvimab, REGEN-COV2, as well as others, in numerous countries. As of November 1, a total of 11 antibody therapeutics had been granted first approvals in either the United States or European Union in 2021 (evinacumab, dostarlimab loncastuximab tesirine, amivantamab, aducanumab, tralokinumab, anifrolumab, bimekizumab, tisotumab vedotin, regdanvimab, REGEN-COV2). The first global approvals of seven products, however, were granted elsewhere, including Japan (pabinafusp alfa), China (disitamab vedotin, penpulimab, zimberelimab), Australia (sotrovimab, REGEN-COV2), or the Republic of Korea (regdanvimab). Globally, at least 27 novel antibody therapeutics are undergoing review by regulatory agencies. First actions by the Food and Drug Administration on the biologics license applications for faricimab, sutimlimab, tebentafusp, relatlimab, sintilimab, ublituximab and tezepelumab are expected in the first quarter of 2022. Finally, our data show that, with antibodies for COVID-19 excluded, the late-stage commercial clinical pipeline of antibody therapeutics grew by over 30% in the past year. Of those in late-stage development, marketing applications for at least 22 may occur by the end of 2022.

Llamanade: An open-source computational pipeline for robust nanobody humanization.

Nanobodies (Nbs) have emerged as a promising class of biologics. Despite having marked physicochemical properties, Nbs are derived from camelids and may require humanization to improve translational potentials. By systematically analyzing the sequence and structural properties of Nbs, we found substantial framework diversities and revealed the key differences between Nbs and human immunoglobulin G antibodies. We identified conserved residues that may contribute to enhanced solubility, structural stability, and antigen binding, providing insights into Nb humanization. Based on big data analysis, we developed "Llamanade," an open-source software to facilitate rational humanization of Nbs. Using sequence as input, Llamanade can rapidly extract sequence features, model structures, and optimize solutions to humanize Nbs. Finally, we used Llamanade to successfully humanize a cohort of structurally diverse and potent SARS-CoV-2 neutralizing Nbs. Llamanade is freely available and will be easily accessible on a server to support the development of therapeutic Nbs into safe and effective trials.

Whole blood stability evaluation of monoclonal antibody therapeutics using volumetric absorptive microsampling.

Volumetric absorptive microsampling (VAMS) is increasingly utilized for both nonclinical and clinical pharmacokinetic studies. Currently, VAMS is employed as the sampling method for the detection of antibodies for coronavirus disease 2019. Biotherapeutics whole blood stability on VAMS presents as a critical concern for the health and pharmaceutical industries. In this follow-up to our previous publication, we evaluated daclizumab and trastuzumab whole blood sample stability on VAMS. The drug recovery data we observed at room temperature for short term and -80°C for long term was very encouraging. The knowledge could help us better understand and plan important investigation timelines, especially pandemic situations where human whole blood samples from a large population are collected and in urgent need of data analysis.

Rapid exploration of the epitope coverage produced by an Ebola survivor to guide the discovery of therapeutic antibody cocktails.

BACKGROUND: Development of successful neutralizing antibodies is dependent upon broad epitope coverage to increase the likelihood of achieving therapeutic function. Recent advances in synthetic biology have allowed us to conduct an epitope binning study on a large panel of antibodies identified to bind to Ebola virus glycoprotein with only published sequences. METHODS AND RESULTS: A rapid, first-pass epitope binning experiment revealed seven distinct epitope families that overlapped with known structural epitopes from the literature. A focused set of antibodies was selected from representative clones per bin to guide a second-pass binning that revealed previously unassigned epitopes, confirmed epitopes known to be associated with neutralizing antibodies, and demonstrated asymmetric blocking of EBOV GP from allosteric effectors reported from literature. CONCLUSIONS: Critically, this workflow allows us to probe the epitope landscape of EBOV GP without any prior structural knowledge of the antigen or structural benchmark clones. Incorporating epitope binning on hundreds of antibodies during early stage antibody characterization ensures access to a library's full epitope coverage, aids in the identification of high quality reagents within the library that recapitulate this diversity for use in other studies, and ultimately enables the rational development of therapeutic cocktails that take advantage of multiple mechanisms of action such as cooperative synergistic effects to enhance neutralization function and minimize the risk of mutagenic escape. The use of high-throughput epitope binning during new outbreaks such as the current COVID-19 pandemic is particularly useful in accelerating timelines due to the large amount of information gained in a single experiment.

Antibodies to watch in 2021.

In this 12th annual installment of the Antibodies to Watch article series, we discuss key events in antibody therapeutics development that occurred in 2020 and forecast events that might occur in 2021. The coronavirus disease 2019 (COVID-19) pandemic posed an array of challenges and opportunities to the healthcare system in 2020, and it will continue to do so in 2021. Remarkably, by late November 2020, two anti-SARS-CoV antibody products, bamlanivimab and the casirivimab and imdevimab cocktail, were authorized for emergency use by the US Food and Drug Administration (FDA) and the repurposed antibodies levilimab and itolizumab had been registered for emergency use as treatments for COVID-19 in Russia and India, respectively. Despite the pandemic, 10 antibody therapeutics had been granted the first approval in the US or EU in 2020, as of November, and 2 more (tanezumab and margetuximab) may be granted approvals in December 2020.* In addition, prolgolimab and olokizumab had been granted first approvals in Russia and cetuximab saratolacan sodium was first approved in Japan. The number of approvals in 2021 may set a record, as marketing applications for 16 investigational antibody therapeutics are already undergoing regulatory review by either the FDA or the European Medicines Agency. Of these 16 mAbs, 11 are possible treatments for non-cancer indications and 5 are potential treatments for cancer. Based on the information publicly available as of November 2020, 44 antibody therapeutics are in late-stage clinical studies for non-cancer indications, including 6 for COVID-19, and marketing applications for at least 6 (leronlimab, tezepelumab, faricimab, ligelizumab, garetosmab, and fasinumab) are planned in 2021. In addition, 44 antibody therapeutics are in late-stage clinical studies for cancer indications. Of these 44, marketing application submissions for 13 may be submitted by the end of 2021. *Note added in proof on key events announced during December 1-21, 2020: margetuximab-cmkb and ansuvimab-zykl were approved by FDA on December 16 and 21, 2020, respectively; biologics license applications were submitted for ublituximab and amivantamab.