| CPC C40B 40/10 (2013.01) [A61P 31/14 (2018.01); C07K 16/1003 (2023.08); C07K 16/2803 (2013.01); A61K 2039/505 (2013.01); C07K 2317/565 (2013.01); C07K 2317/92 (2013.01)] | 11 Claims |
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1. A method of treating a subject with coronavirus infection, comprising administering to the subject a pharmaceutical composition comprising:
a mixture of at least 100 unique human recombinant antibodies specifically binding to SARS-CoV-2 receptor binding domain (RBD) or SARS-CoV-2 spike S1, wherein the mixture has an EC50 lower than 0.001 mg/ml, and
a pharmaceutically acceptable excipient,
wherein the mixture of at least 100 unique human recombinant antibodies has been generated by the process of
a. isolating single cells from a blood sample from a human donor exposed to an antigen of SARS CoV-2;
b. amplifying polynucleotides, wherein each of the polynucleotides encodes a cognate pair of heavy chain and light chain variable regions from one of the single cells by overlap extension reverse transcriptase polymerase chain reaction (OE-RT-PCR);
c. cloning the polynucleotides obtained from the amplification into expression vectors, thereby obtaining constructs encoding antibody fragments;
d. expressing the antibody fragments from the constructs;
e. enriching a subset of the constructs based on the binding activities of the antibody fragments against SAR-CoV-2 RBD or SAR-CoV-2 spike S1;
f. generating antibody expression constructs using the subset of the constructs of step e wherein each of the antibody expression constructs encodes a light chain variable region, a kappa or lambda-type light chain constant region, a heavy chain variable region, and a heavy chain IgG constant region,
g. introducing the antibody expression constructs of step f into a cell line, and
h. expressing antibodies from the antibody expression constructs in the cell line, thereby obtaining the mixture of at least 100 unique human recombinant antibodies;
wherein each of the at least 100 unique human recombinant antibodies is an IgG antibody comprising a cognate pair of heavy chain and light chain variable regions; and
wherein each of the cognate pair of heavy chain and light chain variable regions comprises a heavy chain variable region and a light chain variable region from a single cell out of the blood sample.
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