The growth and potential of human antiviral monoclonal antibody therapeutics

Abstract

Monoclonal antibodies (mAbs) have long provided powerful research tools for virologists to understand the mechanisms of virus entry into host cells and of antiviral immunity. Even so, commercial development of human (or humanized) mAbs for the prophylaxis, preemptive and acute treatment of viral infections has been slow. This is surprising, as new antibody discovery tools have increased the speed and precision with which potent neutralizing human antiviral mAbs can be identified. As longstanding barriers to antiviral mAb development, such as antigenic variability of circulating viral strains and the ability of viruses to undergo neutralization escape, are being overcome, deeper insight into the mechanisms of mAb action and engineering of effector functions are also improving the efficacy of antiviral mAbs. These successes, in both industrial and academic laboratories, coupled with ongoing changes in the biomedical and regulatory environments, herald an era when the commercial development of human antiviral mAb therapies will likely surge.

Figure 1. Human antibody techniques.

(a) Phage display exemplifies human antibody library display techniques (phage, bacteria, yeast, mammalian cell and ribosome). Three steps are included in this technique: antibody library construction and display onto the phage surface, selection by panning the library against antigen (Ag) targets, and screening for desired specificity. Diverse human immunoglobulin-variable-region gene segments (as scFv or Fab fragments) are amplified from human B cells of immune or non-immune sources to construct the antibody library. The library is then cloned for display on the surface of the phage. Selection against the desired target is then performed using the phage display library; antibodies that do not bind are washed away and the binders are eluted and amplified by infection of Escherichia coli. After several rounds of such selection, desired specificity can be screened using enzyme-linked immunosorbent assay (ELISA) or techniques such as fluorescent-activated cell sorting (FACS) if a cell-membrane bound protein is the target. Once the desired specificity is obtained, the genes of antibody variable regions can be cloned into whole human IgG expression vectors and transfected into cell lines to produce fully human mAbs (hmAbs). (b) Transgenic mouse. The mouse immunoglobulin genes have been genetically knocked out and replaced with human counterparts. The transgenic mouse will make human antibodies after foreign antigen immunization. The B cells harvested from immunized mice are immortalized by fusion with a myeloma cell line, as in traditional hybridoma technology. The hybridomas are then screened for desired specificity. (c) Memory B-cell immortalization. Memory B cells (CD22⁺ IgM⁻, IgD⁻, IgA⁻) are isolated from peripheral blood mononuclear cells (PBMCs). They are immortalized by EBV in the presence of a CpG oligodeoxynucleotide and irradiated allogeneic PBMCs. The culture supernatants are then screened directly for specific antibodies. Positive cultures are further cloned by limiting dilution and fully human mAbs can then be produced from the cloned B cells. (d) CDR grafting exemplifies humanization. CDR residues from variable region of a mouse mAb are transferred to human antibody frameworks that have high sequence homology with the mouse counterparts. Katie Ris-Vicari

Figure 2. Mechanisms of viral neutralization.

(a) Antibodies block receptor engagement by binding to spikes on an enveloped virus. (b) Antibody blocks virus entry by binding to a viral cellular receptor (or coreceptor) on cell surface. (c) Post-binding/pre-fusion neutralization occurring inside endosome. For some viruses, conformational changes in viral proteins required for fusion are triggered by the low pH in the endosome. MAbs that block the requisite interactions between viral and endosomal membrane proteins would delay or prevent the penetration of the viral core into the target-cell cytoplasm. (d) Post-binding/pre-fusion neutralization occurring at the cell membrane. Antibodies binding to non-receptor binding regions of the viral envelope can neutralize viral infection through interfering with conformational changes that are required for membrane fusion. (e) Inhibition of the release of progeny virus. For example, mAbs to influenza A virion surface neuraminidase prevent the release of virions from the infected cell surface. Not shown in the figure are neutralizing effects of antibodies on the virus before cell binding, which include antibody-mediated virus aggregation to reduce the number of infectious particles. Katie Ris-Vicari

Figure 3. Antiviral mechanisms mediated by antibody Fc.

Antibody-dependent cell-mediated cytotoxicity (ADCC) is shown on the left. Antibody opsonization and activation of the complement leads to antibody-dependent, complement-mediated virolysis, phagocytosis directed against free virions or infected cells (only the free virus is shown) are shown on the right. NK, natural killer. Katie Ris-Vicari

Figure 4. Human mAb combinational therapy.

Two separate neutralizing epitopes that are prevalent on the envelope glycoprotein of a circulating virus are shown. Neutralizing antibody nAb1 and nAb2 can recognize the neutralizing epitope EI (nEI) and EII (nEII) on the circulating strain (row 1). The combination of nAb1 and nAb2 will provide broad neutralization activity and can prevent neutralization escape. A natural variant of nEII or neutralization escape mutant of nAb2 is still recognized by nAb1 (row 2) and nAb4. Likewise, a natural variant of nEI or neutralization escape mutant of nAb1 is still recognized by nAb2 (row 3) and nAb3. Both nAb3 and nAb4 are available and can be used to maintain two nAbs against circulating variant viruses. Only rarely would a combination of nAb3 and nAb4 be required (row 4). Katie Ris-Vicari