STATEMENT REGARDING PRIOR DISCLOSURES BY THE INVENTOR OR A JOINT INVENTOR UNDER 37 C.F.R. 1.77(B)(6)

Most of the subject matter of the invention described in the present application was published by the inventors, Ing-Chien Chen, Yi-Kai Chiu, Chung-Ming Yu, Cheng-Chung Lee, Chao-Ping Tung, Yueh-Liang Tsou, Yi-Jen Huang, Chia-Lung Lin, Hong-Sen Chen, Andrew H.-J. Wang, and An-Suei Yang in an article titled “High throughput discovery of influenza virus neutralizing antibodies from phage-displayed synthetic antibody libraries.” The article was published on Oct. 31, 2017 on Scientific Reports 7, Article number: 14455 (2017). The publication was made by and/or originated from all member of the inventive entity of the present invention, and the entirety of this article is incorporated herein by reference. A copy of the article is provided in a concurrently filed Information Disclosure Statement pursuant to the guidance of 78 Fed. Reg. 11076 (Feb. 14, 2013).

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present disclosure relates to a method of screening of neutralizing antibodies. More specifically, the present disclosure relates to a method of high-throughput screening of neutralizing antibodies against various viruses, including an influenza virus.

2. Description of Related Art

Yearly outbreaks of influenza virus infections lead to substantial social and economic burden worldwide. The economic burden due to influenza in the US alone in 2003 has been estimated to be about $87.1 billion. However, the loss of lives due to influenza dwarfs the economic burden: Global annual seasonal influenza attack rate, estimated by World Health Organization (WHO), has been 5-10% in adults and 20-30% in children, leading to 1 billion annual cases of influenza infection, 3-5 million annual cases of severe illness and 250,000-500,000 annual deaths, who are mostly high risk groups such as elderly, infants, and people with underlying chronical illness.

Pandemic influenza A virus (IAV) outbreaks pose much more severe challenges comparing with seasonal influenza epidemics. The pandemic influenza attack rates are much higher (over 50% attack rate in the 1918 flu pandemic) and young adult mortality rate between the ages of 20 and 40 years is particularly alarming, accounting for more than half of the deaths as in the 1918 flu pandemic. The pandemic outbreaks are particularly devastating because of the bourgeoning human population and interconnected traveling patterns. Three major influenza pandemics occur in the past century: 1918 H1N1 Spanish, 1957 H2N2 Asian, and 1968 H3N2 Hong Kong. The total deaths caused by these three major human influenza pandemics have been estimated to be 50-100 million worldwide, where the mortality rate for 1918 flu pandemic alone was 2%, causing 20-100 million deaths. The 21th century's first pandemic, unpredictedly caused by 2009 H1N1 influenza virus (Mexico) of swine origin, is reported by WHO of millions of cases and 16813 deaths (as of 2010 Mar. 19). While the next pandemic influenza outbreak has been unpredictable with our current knowledge, the concern over an avian strain of pandemic virus is certainly warranted, as revealed by the emergence of avian influenza strains, including H5N1 (Hong Kong) in 1997, H7N9 (China), H10N8 (China) and H6N1 (Taiwan) in 2013 and H5N6 (Hong Kong) in 2014, among which the highly pathogenic avian influenza H5N1 (Hong Kong) and H7N9 (China) are of particular concern. If virus genome reassortment or antigenic drift are to result in the new H7N9/H5N1 avian influenza virus transmissible among humans, a new pandemic could arise.

Countermeasure for IAV epidemics and pandemics have not provided enough protection to human population. Seasonal trivalent or quardrivalent influenza vaccines are conditionally effective, depending on the match of the predicted vaccine strains and the strains in circulation each year. More importantly, the majority of human population does not have protective immunity against pandemic influenza viruses and vaccines against the pandemic strains would be available with considerable delay of at least 5-6 months after the availability of the vaccine virus to vaccine manufacturers. By the time when the vaccine becomes available, substantial mortality, morbidity and economic loss could have already occurred. Universal vaccines with efficacy against a broad spectrum of influenza strains are still under development; even effective vaccines are available, there would be a delay of about 2 weeks before developing protective immunity. Stockpile of antiviral drugs (mostly neuraminidase inhibitors such as oseltamivir (Tamiflu)) as strategy for curbing influenza pandemics has been questioned. Moreover, influenza strains with pandemic threats could be resistant to current antivirals, as drug-resistant influenza virus strains have occurred due to antigenic shift and antigenic drift.

Passive immunotherapy with neutralizing antibodies to treat severe IAV infections can be a viable strategy in mitigating the threats of influenza epidemic and pandemic outbreaks. Human antibody discoveries based on culturing/cloning human single memory and plasmablast B cells and displaying human antibody repertoires on phage particles have resulted in potent human neutralizing antibodies, many of which are broadly neutralizing antibodies (bnAbs) and are being tested for passive immunotherapy in human trials. However, affinity matured human antibodies with neutralizing efficacy against pandemic strains harboring genetic makeup unknown to human immune systems could be difficult to attain. Moreover, neutralization escape variants could eventually emerge due to the selection pressure of the widespread treatments with the neutralizing antibodies.

In view of the foregoing, there exists a need in the art for providing a method for high-throughput screening of neutralizing antibodies for use to respond to immediate threats of the emerging viral infections.

SUMMARY

The following presents a simplified summary of the disclosure in order to provide a basic understanding to the reader. This summary is not an extensive overview of the disclosure and it does not identify key/critical elements of the present invention or delineate the scope of the present invention. Its sole purpose is to present some concepts disclosed herein in a simplified form as a prelude to the more detailed description that is presented later.

In one aspect, the present disclosure is directed to a method for high-throughput screening of a neutralizing antibody specific to a virus. The present method comprises the steps of,

(a) providing a phage-displayed single-chain variable fragment (scFv) library that comprises a plurality of phage-displayed scFvs, wherein the heavy chain variable (VH) domain of each phage-displayed scFvs has a binding affinity to protein A, and the light chain variable (VL) domain of each phage-displayed scFvs has a binding affinity to protein L;

(b) exposing the phage-displayed scFv library of the step (a) to a viral antigen derived from the virus:

(c) selecting, from the phage-displayed scFv library of the step (b), a plurality of phages that express scFvs exhibiting binding affinity to the viral antigen under an acidic condition;

(d) respectively enabling the plurality of phages selected in the step (c) to express a plurality of soluble scFvs;

(e) exposing the plurality of soluble scFvs of the step (d) to the virus;

(f) determining the respective neutralizing efficacy of the plurality of soluble scFvs in the step (e); and

(g) based on the results determined in the step (f), selecting one soluble scFv that exhibits superior efficacy over the other soluble scFvs of the plurality of soluble scFvs as the neutralizing antibody.

The virus may be an influenza virus, a measles virus, a coronavirus, a mumps virus, a marburg virus, an ebola virus, a rubella virus, a rhinovirus, a poliovirus, a hepatitis virus, a smallpox virus, a varicella-zoster virus, a severe acute respiratory syndrome virus, a retrovirus, a rotavirus, an adenoviruses, an adeno-associated virus or an alphavirus. According to some embodiments of the present disclosure, the virus is an influenza virus; in these embodiments, the viral antigen for selecting scFv in the steps (b) and (c) is preferably hemagglutinin (HA) or neuraminidase (NA).

According to some embodiments of the present disclosure, the step (c) is carried out by subjecting the product of the step (b) to a buffer having a pH value ranging between 2-6.8. According to one working example, the buffer has a pH value of 5.0.

Also disclosed herein is a neutralizing antibody produced by the present method. According to some embodiments, the VL domain of the neutralizing antibody has the amino acid sequence at least 85% identical to any of SEQ ID NOs: 1-28; and the VH domain of the neutralizing antibody has the amino acid sequence at least 85% identical to any of SEQ ID NOs: 29-56. According to specific example of the present disclosure, the VL domain of the neutralizing antibody has the amino acid sequence 100% identical to any of SEQ ID NOs: 1-28; and the VH domain of the neutralizing antibody has the amino acid sequence 100% identical to any of SEQ ID NOs: 29-56.

Another aspect of the present disclosure pertains to a method of producing a recombinant antibody. The method comprises the steps of,

(a) selecting a neutralizing antibody by use of the present high-throughput screening method;

(b) extracting a phagemid DNA corresponding to the phage that expresses the neutralizing antibody of the step (a);

(c) respectively amplifying a first nucleic acid sequence that encodes a VL domain, and a second nucleic acid sequence that encodes a VH domain by PCR using the phagemid DNA of the step (b) as a template;

(d) inserting the first and second nucleic acid sequences into an expression vector that comprises a third and a fourth nucleic acid sequences, wherein the third nucleic acid sequence encodes the light chain constant (CL) domain of an immunoglobulin, and the fourth nucleic acid sequence encodes the heavy chain constant (CH) domain of the immunoglobulin; and

(e) transfecting a host cell with the expression vector of the step (d) that comprises the first, second, third, and fourth nucleic acid sequences so as to produce the recombinant antibody.

According to the embodiments of the present disclosure, the first nucleic acid sequence is disposed at the upstream of the third nucleic acid sequence, and the second nucleic acid sequence is disposed at the upstream of the fourth nucleic acid sequence. Preferably, the immunoglobulin is immunoglobulin G (IgG).

According to certain embodiments, the host cell of the step (e) is a mammalian cell.

The thus-produced recombinant antibody comprises a VL domain, a CL domain, a VH domain and a CH domain. According to some embodiments, the VL domain of the recombinant antibody has the amino acid sequence at least 85% identical to any of SEQ ID NOs: 1-28; and the VH domain of the recombinant antibody has the amino acid sequence at least 85% identical to any of SEQ ID NOs: 29-56. According to specific example of the present disclosure, the VL domain of the recombinant antibody has the amino acid sequence 100% identical to any of SEQ ID NOs: 1-28; and the VH domain of the recombinant antibody has the amino acid sequence 100% identical to any of SEQ ID NOs: 29-56.

Another aspect of the present disclosure is directed to a method of treating or preventing a virus infection in a subject in need thereof. The method comprises administering to the subject an effective amount of the present neutralizing antibody or the present recombinant antibody.

The subject treatable with the present method is a mammal; preferably, the subject is a human.

Many of the attendant features and advantages of the present disclosure will becomes better understood with reference to the following detailed description considered in connection with the accompanying drawings.

