Vaughn Smider - US grants

DESCRIPTION (provided by applicant): (13) Smart Biomaterials - Theranostics 13-EB-101*Theranostics: Combined delivery of diagnostic and therapeutic agents. Multimeric polyspecific protein therapeutics are difficult to design and produce. The ability to link binding, effector, and imaging components together in a single molecule would be a significant advantage. We propose to develop unnatural amino acid- oligonucleotide technology (UAA-oligo) in order to precisely control formation of modular therapeutics and diagnostics. An unnatural amino acid engineered in a single position will allow site-specific coupling of an oligonucleotide. The oligonucleotide can then be hybridized to its complement which is conjugated to a second modular protein. The base-pairing rules of DNA will allow higher order heterodimers, trimers, tetramers, and other multimers to be produced. This strategy will allow diagnostic and therapeutic modules to be coupled in a single molecule to produce a "Theranostic". PUBLIC HEALTH RELEVANCE: The ability to create modular protein therapeutics, such that novel binding, effector, and diagnostic proteins can be combined in a single "theranostic" has considerable importance in medicine. Specifically, this proposal aims to create the platform for discovery of such molecules as well as for their facile construction. We will use oligonucleotides and unnatural amino acids to site specifically link two or more proteins together through the complementary base pairing of nucleic acids. Molecules that we create could simultaneously detect, bind, and kill tumor cells through imaging, binding, and toxin modules. This "lego" block assembly process will allow a vast array of combinatorial protein theranostic modules to be created and evaluated against a myriad of diseases.

DESCRIPTION (provided by applicant): Antibody molecules are enormously important as therapeutic and diagnostic molecules. More recently, unique scaffolds like the VHH of camelids, or even non-antibody frameworks like knottins have become important in biomedicine. Antibody diversity and antigen binding in mammals is often restricted to the CDR loops of the immunoglobulin fold. In experiments challenging this paradigm, we have recently solved the crystal structures of two bovine antibodies containing ultralong CDR H3s (56 and 61 amino acids) and also deep sequenced the ultralong repertoire. Our data reveal that these CDR H3s form a very unusual architecture composed of a long ?-strand stalk which supports a disulfide rich knob that protrudes far from the immunoglobulin surface. Interestingly, the two different antibodies contain different patterns of disulfides, which result in different knob structures. Dee sequencing reveals extensive diversity in the ultralong CDR H3s where a multitude of different disulfides could potentially form within the knob. Thus, the bovine antibody system can produce an unprecedented repertoire of mega CDR H3s that may result in an impressive diversity of minifolds containing combinations of somatically generated disulfides. Thus, antibody diversity is located in a new minifold supported by the immunoglobulin domain. We will perform structural, functional, and engineering studies to investigate the properties of this new antibody class, as well as to lead the way to developing this unique structure into therapeutics.

? DESCRIPTION (provided by applicant): Breast cancer is a highly heterogeneous disease with five major molecular subtypes distinguished by gene and protein expression. While treatments targeting endocrine functions of luminal A and luminal B breast cancers, and antibody therapy of Her2 positive tumors have reduced patient mortality in these groups, patients with triple negative breast cancers (TNBC) lacking estrogen and progesterone receptors as well as HER2, have no access to targeted therapies. The goal of this project is to identify novel targets in TNBC with potentially therapeutic and diagnostic antibodies. This will be accomplished by combining novel approaches to elucidate the antibody fingerprint of triple negative breast cancer cells through screening against spatially addressed human germline IgG and bovine antibody libraries. We generated a human germline antibody library with rationally designed structural diversity. To further enhance antibody diversity and enable unique discovery of otherwise hard-to-target antigens, we included cow antibody structures. While the majority of mammalian antibodies bind antigens with six CDR loops, arranged to form a flat binding surface, we recently identified ultra-long CDR H3 regions from cow antibodies that have a protruding stalk and knob architecture. This structure is highly diverse and allows specific recognition of antigen cavities, enzyme active sites, pores or channels, or unique conformational epitopes in complex targets. To elucidate the antibody fingerprint of TNBC cells, we defined two Specific Aims: Aim 1: Generate antibody fingerprints against breast cancer cell lines by high throughput flow cytometry and identify antigen-antibody pairs specific for TNBC cells. A panel of breast cancer cell lines from each of the 5 classes of breast cancer and normal breast epithelial cells will be screened against the human germline and cow antibody libraries. TNBC-specific antibodies will be identified, tested for binding specificity and affinity, and affinity matured if desired. We established feasibility and robustness of the approach for hard-to-target proteins such as GPCRs with multispanning transmembrane regions expressed on HEK293 cells. Aim 2: Use antibody fingerprints to identify targets and evaluate therapeutic and diagnostic potential of antibodies specific for triple negative breast cancer cells. Antibody targes will be determined by proteomic analyses. Reactivity and target specificity for TNBC will be assessed by antibody screening on clinical breast cancer samples. Functional properties of TNBC specific antibodies will be tested for endogenous cell growth inhibition. Antibody drug conjugates will be evaluated for direct target cell killing. Suitability of antibodies for T- cell directed therapy can be analyzed after generating bispecific antibody versions. Our study could identify clinically relevant targets on triple negative breast cancer cells, and simultaneously provide antibodies with therapeutic and diagnostic potential for future evaluation and development.

? DESCRIPTION (provided by applicant): A key feature of several anti-HIV neutralizing antibodies is the unusually long length of their CDR H3s. Cow antibodies are unique in having ultralong CDR3 regions that can be over 60 amino acids in length and are cysteine-rich. Crystal structures of two different antibody Fab fragments reveal that these CDR3s form a very unusual architecture composed of a long ?-strand stalk which supports a disulfide rich knob that protrudes far from the immunoglobulin surface. Interestingly, different antibodies contain different patterns of disulfides, which result in different knob structures. Deep sequencing reveals extensive diversity in the ultralong CDR3s where a multitude of different disulfides could potentially form within the knob. Analysis of clonally derived sequences suggests that this diversity results from somatic hypermutation of an ultralong germline D region that has a severe codon bias towards mutation to cysteine. Thus, the bovine antibody system may produce an unprecedented repertoire of mega CDR3s that fold into an impressive diversity of minifolds containing combinations of somatically generated disulfides. We have immunized cows using the BG505 gp140 trimer and found that they are capable of making a robust and broadly neutralizing serum antibody response. In this exploratory proposal, we will take advantage of the unusual ultralong cow antibody repertoire to generate new monoclonal antibodies against HIV gp120 with the goal of identifying new neutralizing epitopes on HIV.

Vaccines are the primary means by which to prevent, control, or eradicate infectious diseases. While many vaccines have been successfully developed and have resulted in enormous medical and veterinary benefit, there are certain viruses that have eluded effective vaccine development. Generally, viruses with multiple strains or that have high mutation rates can evade neutralizing antibodies, as their surface determinants are variable and result in the inability of neutralizing antibodies raised against one strain to bind and neutralize alternative strains. Certain rare epitopes, however, are required for viral infection and are conserved across strains. Interestingly, neutralizing antibodies against these rare epitopes tend to have long CDR H3 regions. In the case of HIV, long CDR H3s can pierce the viral glycan shield and reach into the conserved epitope on the gp120 spike protein. While long CDR H3 regions in human antibodies are infrequent in the repertoire, cattle routinely produce long (20-40 amino acids) and ultralong (40-70 amino acids) CDR H3 regions that have unique ?stalk? and ?knob? structural features that protrude far from the antibody surface. Therefore, cattle may be an excellent model organism to identify and define new and conserved neutralizing epitopes in these challenging viruses. Indeed, in preliminary experiments we have found that cattle make a robust and broadly neutralizing antibody response to the HIV gp120 antigen. Here we propose to use the unique cow antibody repertoire to define new conserved neutralizing epitopes on two viruses of great importance to human and animal health, HIV and BVDV. Effective vaccines against both of these viruses have been a major challenge to develop. We will immunize animals against these viruses, generate monoclonal antibodies that neutralize the virus as well as related strains, and molecularly map the antigen-antibody interaction using mutagenesis and structural biology techniques. Definition of new conserved epitopes could lead to engineered epitope-specific vaccines. Thus, the outcomes of this proposal could enable generation of next-generation vaccines for these two viruses, but could also have broad utility in vaccine development against other challenging viruses in the future.