Arun K. Kashyap

Combinatorial antibody libraries from survivors of the Turkish H5N1 avian influenza outbreak reveal virus neutralization strategies

The widespread incidence of H5N1 influenza viruses in bird populations poses risks to human health. Although the virus has not yet adapted for facile transmission between humans, it can cause severe disease and often death. Here we report the generation of combinatorial antibody libraries from the bone marrow of five survivors of the recent H5N1 avian influenza outbreak in Turkey. To date, these libraries have yielded >300 unique antibodies against H5N1 viral antigens. Among these antibodies, we have identified several broadly reactive neutralizing antibodies that could be used for passive immunization against H5N1 virus or as guides for vaccine design. The large number of antibodies obtained from these survivors provide a detailed immunochemical analysis of individual human solutions to virus neutralization in the setting of an actual virulent influenza outbreak. Remarkably, three of these antibodies neutralized both H1 and H5 subtype influenza viruses.

Cross-neutralization of influenza A viruses mediated by a single antibody loop

Immune recognition of protein antigens relies on the combined interaction of multiple antibody loops, which provide a fairly large footprint and constrain the size and shape of protein surfaces that can be targeted. Single protein loops can mediate extremely high-affinity binding, but it is unclear whether such a mechanism is available to antibodies. Here we report the isolation and characterization of an antibody called C05, which neutralizes strains from multiple subtypes of influenza A virus, including H1, H2 and H3. X-ray and electron microscopy structures show that C05 recognizes conserved elements of the receptor-binding site on the haemagglutinin surface glycoprotein. Recognition of the haemagglutinin receptor-binding site is dominated by a single heavy-chain complementarity-determining region 3 loop, with minor contacts from heavy-chain complementarity-determining region 1, and is sufficient to achieve nanomolar binding with a minimal footprint. Thus, binding predominantly with a single loop can allow antibodies to target small, conserved functional sites on otherwise hypervariable antigens.

Protection from the 2009 H1N1 Pandemic Influenza by an Antibody from Combinatorial Survivor-Based Libraries

Influenza viruses elude immune responses and antiviral chemotherapeutics through genetic drift and reassortment. As a result, the development of new strategies that attack a highly conserved viral function to prevent and/or treat influenza infection is being pursued. Such novel broadly acting antiviral therapies would be less susceptible to virus escape and provide a long lasting solution to the evolving virus challenge. Here we report the in vitro and in vivo activity of a human monoclonal antibody (A06) against two isolates of the 2009 H1N1 pandemic influenza virus. This antibody, which was obtained from a combinatorial library derived from a survivor of highly pathogenic H5N1 infection, neutralizes H5N1, seasonal H1N1 and 2009 “Swine” H1N1 pandemic influenza in vitro with similar potency and is capable of preventing and treating 2009 H1N1 influenza infection in murine models of disease. These results demonstrate broad activity of the A06 antibody and its utility as an anti-influenza treatment option, even against newly evolved influenza strains to which there is limited immunity in the general population.

Combinatorial surrobody libraries

A unique type of combinatorial protein libraries has been constructed. These libraries are based on the pre-B cell receptor (pre-BCR). The pre-BCR is a protein that is produced during normal development of the antibody repertoire. Unlike that of canonical antibodies, the pre-BCR subunit is a trimer that is composed of an antibody heavy chain paired with two surrogate light chain (SLC) components. Combinatorial libraries based on these pre-BCR proteins in which diverse heavy chains are paired with a fixed SLC were expressed in mammalian, Escherichia coli, and phagemid systems. These libraries contain members that have nanomolar affinity for antigen. We term this type of antigen-binding protein a “surrobody” to distinguish it from the canonical antibody molecule.

Surrobodies with functional tails.

Surrobodies(2) are a unique type of binding protein based on the pre-B-cell receptor (pre-BCR). The pre-BCR is transiently expressed during development of the antibody repertoire. Unlike heterotetrameric canonical antibodies that are composed of identical pairs of heavy and light chains, the pre-BCR is a heterohexameric complex composed of identical pairs of heavy chains that are each paired with a two-subunit surrogate light chain (SLC). The SLC contains nonimmunoglobulin-like peptide extensions on each of the two SLC components. This arrangement provides unique opportunities for protein engineering by functional derivatization of these nonimmunoglobulin-like tails. Here we report recombinant fusions to these tails with either a fully active cytokine or with single-chain variable fragment (scFv) domains to generate Surrobodies with unique functions or Surrobodies that are bispecific with respect to targeted binding.

Dual Agonist Surrobody Simultaneously Activates Death Receptors DR4 and DR5 to Induce Cancer Cell Death

Death receptors of the TNF family are found on the surface of most cancer cells and their activation typically kills cancer cells through the stimulation of the extrinsic apoptotic pathway. The endogenous ligand for death receptors 4 and 5 (DR4 and DR5) is TNF-related apoptosis-inducing ligand, TRAIL (Apo2L). As most untransformed cells are not susceptible to TRAIL-induced apoptosis, death receptor activators have emerged as promising cancer therapeutic agents. One strategy to stimulate death receptors in cancer patients is to use soluble human recombinant TRAIL protein, but this agent has limitations of a short half-life and decoy receptor sequestration. Another strategy that attempted to evade decoy receptor sequestration and to provide improved pharmacokinetic properties was to generate DR4 or DR5 agonist antibodies. The resulting monoclonal agonist antibodies overcame the limitations of short half-life and avoided decoy receptor sequestration, but are limited by activating only one of the two death receptors. Here, we describe a DR4 and DR5 dual agonist produced using Surrobody technology that activates both DR4 and DR5 to induce apoptotic death of cancer cells in vitro and in vivo and also avoids decoy receptor sequestration. This fully human anti-DR4/DR5 Surrobody displays superior potency to DR4- and DR5-specific antibodies, even when combined with TRAIL-sensitizing proapoptotic agents. Moreover, cancer cells were less likely to acquire resistance to Surrobody than either anti-DR4 or anti-DR5 monospecific antibodies. Taken together, Surrobody shows promising preclinical proapoptotic activity against cancer cells, meriting further exploration of its potential as a novel cancer therapeutic agent. Mol Cancer Ther; 15(1); 114–24. ©2015 AACR.

Abstract 4318: Development of a novel SurrobodyTM that simultaneously activates both death receptors DR4 and DR5 and induces cancer cell death with high potency.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Death receptors of the Tumor Necrosis Factor (TNF) family are found on surface of most cancer cells and their activation typically kills cancer cells through the stimulation of the extrinsic apoptotic pathway. The endogenous ligand for death receptors-4 and -5 (DR4 and DR5) is tumor necrosis factor-related apoptosis-inducing ligand, TRAIL (Apo2L). TRAIL is expressed by many cell types, including Natural Killer (NK) cells that are involved in the immune surveillance and elimination of cells in vivo. Given that most normal untransformed cells are not susceptible to TRAIL-induced apoptosis, activators of death receptors have emerged as promising cancer therapeutic agents. One strategy to stimulate death receptors in cancer patients has been to use soluble human recombinant TRAIL protein, but this agent has limitations of a short half-life and decoy receptor sequestration. An alternative strategy to evade decoy receptor sequestration and to have improved pharmacokinetic properties is to generate DR4 or DR5 agonist antibodies. To this end, several agonistic monoclonal antibodies have been reported that overcome the limitations of short half-life, but exhibited the limitation of activating only one of the two death receptors. The SurrobodyTM binding protein is a novel therapeutic protein structure based upon the pre-B cell antigen receptor common chain that is capable of being engineered to bind a diversity of targets with high affinity and specificity. We exploited a previously discovered SurrobodyTM that potently activates both DR4 and DR5 in vitro and in vivo. Here we show experimental results for a dual agonist SurrobodyTM that offers a powerful and likely more reliable strategy for cancer therapeutics based on simultaneous stimulation of both TRAIL receptors, DR4 and DR5. Citation Format: Snezana Milutinovic, Sihong Zhou, Carina Wimer, Paul W. Diaz, Arun K. Kashyap, Ramesh R. Bhatt, John C. Reed. Development of a novel SurrobodyTM that simultaneously activates both death receptors DR4 and DR5 and induces cancer cell death with high potency. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4318. doi:10.1158/1538-7445.AM2013-4318