This aspect will be further clarified in future studies. == Fig. 3 days post infection, treated mice do not exhibit disease symptoms while 80% of control animals succumb to the infection. == Introduction == Human monoclonal antibodies (mAbs), specifically targeting surface viral proteins, have increasingly demonstrated prophylactic and therapeutic efficacy against various viruses, including HIV, Ebola, and the pathogenic beta-coronaviruses Middle East respiratory syndrome coronavirus and severe acute respiratory syndrome coronavirus (SARS-CoV)15. Neutralizing Abs constitute a highly Rabbit Polyclonal to SSTR1 promising approach for treating and preventing infection by the novel SARS-CoV-2 (ref.6). The viral surface spike glycoprotein is essential for viral attachment, fusion, and entry into human cells, and thus considered as the major target for therapeutic neutralizing Abs713. Specifically, highly potent neutralizing Abs target and block the binding of the receptor binding domain (RBD) located in the S1 subunit of the spike to the human angiotensin-converting enzyme 2 (hACE2)7,10,14. A reliable animal model for COVID-19 is essential for the development of anti-SARS-CoV-2 countermeasures and for deciphering the pathogenicity of the disease15. Accordingly, mouse models that exploit the recombinant hACE2 expression, either by transgenic or by viral-transduction approaches were developed1628. A transgenic mouse strain expressing hACE2 under the K18 promoter (K18-hACE2) was shown to be highly susceptible to SARS-CoV-2 infection, resulting in significant viral load in the lungs, heart, brain, and spleen as well as mortality26,27,29. In response to the urgent need for Ab-based therapy for COVID-19, several reports have demonstrated efficacy against SARS-CoV-2 infection by neutralizing Abs, primarily as prophylactic protection10,19,3032. These studies were based on non-lethal COVID-19 models of mice transduced to express hACE2, and did not demonstrate the efficacy of Ab-based passive therapy administered at a significant time post-infection. We have previously reported the isolation of human-neutralizing Abs selected against SARS-CoV-2 RBD by extensive screening of a phage-display library generated from lymphocytes collected from infected individuals. Among these Abs, MD65 exhibited the highest neutralization potency in vitro11. In this work, we evaluate the therapeutic efficacy of the MD65 Ab in a lethal COVID-19 animal model by assessing its prophylaxis and treatment abilities to protect K18-hACE2-infected mice. == Results and discussion == == Reformatting and characterization of the MD65 Ab == The initial binding and neutralization characterization of MD65 Ab LOXL2-IN-1 HCl were conducted following the expression of this Ab as a single-chain human-Fc recombinant form (scFv-Fc)11. Here, towards implementation of this Ab as a bona fide therapeutic product appropriate for human use, it was re-cloned and produced as a full recombinant IgG molecule of the IgG1/k isotype which includes the triple mutation M252Y/S254T/T256E (YTE) in the Fc region. These modifications increase the Ab affinity towards the human FcRn33at acidic pH and thereby prolong its serum half-life, a LOXL2-IN-1 HCl parameter that is essential for a high therapeutic value3436. Characterization of three versions of the MD65 Ab: scFv-Fc, IgG, and IgG-YTE, established that overall they are comparable with regard to their antigen-binding performance (Fig.1a) and that the IgG versions exhibit slightly favorable affinity when compared to the scFv-Fc one (apparentKDof 0.7 versus 1.0 nM, respectively). Similarly, the two MD65 IgG formats possess equivalent LOXL2-IN-1 HCl SARS-CoV-2 neutralization potency in vitro (NT50of 40 ng/ml), which is slightly better than that of the scFv-Fc Ab (NT50of 67 ng/ml; Fig.1b). In addition, all three Ab versions were LOXL2-IN-1 HCl shown to effectively prevent.