Kevin Kramer

Development of antibody therapeutics for cancer immunology

Project Abstract

Recent advances in computational and bioengineering sciences have allowed us to better understand the interactions between antibodies and antigen, to design immunogens capable of eliciting target antibody specificities, and to engineer and optimize antibodies as clinical products. The field of antibody engineering is of particular interest. Antibodies are a $75+ billion industry, projected to increase to nearly$125 billion/year over the next several years. Antibodies have proven to be effective as preventive or therapeutic measures against viruses, cancers, autoimmune disorders, and other diseases, and even more targets are being studied in clinical trials. Specifically in cancer therapy, antibodies are associated with several different modes of action, such as direct tumor targeting or immunomodulation by checkpoint inhibition, with a number of antibodies against various types of cancers already FDA-approved or in various stages of clinical trials. The goal of the proposed project is to apply a variety of techniques from computational protein design, immunology, and structural biology, in order to develop novel antibody therapeutics for cancer immunology. Specifically, we will apply structure-based design techniques to develop improved antibody therapeutics against various targets – for example, Bevacizumab (Avastin, Genentech), a humanized monoclonal antibody that neutralizes the activity of human vascular endothelial growth factor. Overall, this project will provide a methodology for generating novel and improved cancer antibody therapeutics.

Project Update April 2018
In the past year, we have pursued multiple strategies to purify the protein nanoparticle, ferritin in a mammalian expression system. Before expression in HEK293 cells, we had to genetically link the antigens vascular endothelial growth factor (VEGF) and angiopoietin-2 (Ang2) to the N terminus of ferritin. This was accomplished by overlap extension PCR in which shared nucleotide sequences are cloned into each DNA fragment, facilitating the attachment of our proteins of interest and ferritin, separated by a flexible linker. After sequence confirmation, we devised numerous purification schemes to isolate protein nanoparticles, some of which have resulted in nanoparticle structures (Figure 1), albeit at low yield. Additional purification strategies are currently being explored. We are also exploring the opportunity of utilizing phage capsid particles for antigen display, which has the additional advantage of expression in bacteria, rather than mammalian cells. After successful purification and an appropriate yield is reached for our nanoparticle proteins, we will initiateIn the past year, we have pursued multiple strategies to purify the protein nanoparticle, ferritin in a mammalian expression system. Before expression in HEK293 cells, we had to genetically link the antigens vascular endothelial growth factor (VEGF) and angiopoietin-2 (Ang2) to the N terminus of ferritin. This was accomplished by overlap extension PCR in which shared nucleotide sequences are cloned into each DNA fragment, facilitating the attachment of our proteins of interest and ferritin, separated by a flexible linker. After sequence confirmation, we devised numerous purification schemes to isolate protein nanoparticles, some of which have resulted in nanoparticle structures (Figure 1), albeit at low yield. Additional purification strategies are currently being explored. We are also exploring the opportunity of utilizing phage capsid particles for antigen display, which has the additional advantage of expression in bacteria, rather than mammalian cells. After successful purification and an appropriate yield is reached for our nanoparticle proteins, we will initiate immunization studies. As an orthogonal direction within the goals of this project, we have also established a collaboration with the Massion lab, who have provided access to lung cancer samples. We are currently performing a pilot study of B-cell sorting followed by next-generation sequencing of one such sample. The initial B-cell sorting experiment resulted in ~45,000 B cells, which are currently being characterized on the 10X paired heavy-light antibody sequencing equipment from the VANTAGE sequencing core. These initial experiments will then be expanded to additional samples. This work will lead to novel insights into tumor-specific antibody responses.

Mentors

Primary: Ivelin Georgiev
Secondary: Jim Crowe (& formerly Melanie Ohi)
Type of Trainee
Graduate student

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