My research in the Garcia Lab is focused on understanding the molecular basis for Notch receptor-ligand interactions, a critical signaling event for mammalian cell fate determination and the pathogenesis of many cancers. Until recently, we had not been able to “see” how Notch receptors engage ligands Delta-like and Jagged because their nearly undetectable binding affinity prevents reconstitution of stable complexes for structural studies. To overcome this obstacle, we used in vitro evolution to engineer mutations in Delta-like 4 (DLL4) that enhanced affinity for Notch1 and facilitated co-crystallization of the complex. The Notch1-DLL4 structure revealed an antiparallel, two-site binding interface in which O-linked glycan modifications of Notch1 residues make specific and essential contacts with DLL4. Changes in Notch glycosylation state are known to bias recognition towards certain ligands, and the Notch1-DLL4 structure thus rationalizes a mechanism for glycan-mediated tuning of Notch-ligand selectivity. The elucidation of a direct chemical role for O-glycans in Notch1 ligand engagement demonstrates how, by relying on posttranslational modifications of their ligand binding sites, Notch proteins have linked their functional capacity to developmentally regulated biosynthetic pathways

Gaining structural access to the Notch-ligand interface was a critical first step toward improved therapeutic targeting of the pathway. With this structural information to guide us, we will now be able to design novel ligands with enhanced selectivity and affinity for a number of immunologically focused applications. For example, we are generating (1) receptor-specific ligands that inhibit tumor growth while minimizing off-target toxicity, (2) stromal cell lines that express high-affinity ligands to enhance T-cell maturation in vitro, and (3) bi-specific ligands that activate Notch on desired cell types. In the future, we hope to create a diverse “toolbox” of engineered ligands for use as diagnostics and therapeutics in a variety of contexts.

Honors & Awards

  • K99 Pathway to Independence Award, National Cancer Institute (NIH) (May 2016-May 2021)
  • Irvington Postdoctoral Fellowship, Cancer Research Institute (July 2013-present)
  • Stanford Immunology Training Grant (T32), NIH (April 2013-June 2013)
  • Woodrow Wilson Undergraduate Research Fellowship, Johns Hopkins University (2001-2005)

Professional Education

  • Bachelor of Arts, Johns Hopkins University (2005)
  • Doctor of Philosophy, Washington University (2012)

Stanford Advisors

Lab Affiliations

All Publications

  • Antibodies to Interleukin-2 Elicit Selective T Cell Subset Potentiation through Distinct Conformational Mechanisms IMMUNITY Spangler, J. B., Tomala, J., Luca, V. C., Jude, K. M., Dong, S., Ring, A. M., Votavova, P., Pepper, M., Kovar, M., Garcia, K. C. 2015; 42 (5): 815-825


    Interleukin-2 (IL-2) is a pleiotropic cytokine that regulates immune cell homeostasis and has been used to treat a range of disorders including cancer and autoimmune disease. IL-2 signals via interleukin-2 receptor-β (IL-2Rβ):IL-2Rγ heterodimers on cells expressing high (regulatory T cells, Treg) or low (effector cells) amounts of IL-2Rα (CD25). When complexed with IL-2, certain anti-cytokine antibodies preferentially stimulate expansion of Treg (JES6-1) or effector (S4B6) cells, offering a strategy for targeted disease therapy. We found that JES6-1 sterically blocked the IL-2:IL-2Rβ and IL-2:IL-2Rγ interactions, but also allosterically lowered the IL-2:IL-2Rα affinity through a "triggered exchange" mechanism favoring IL-2Rα(hi) Treg cells, creating a positive feedback loop for IL-2Rα(hi) cell activation. Conversely, S4B6 sterically blocked the IL-2:IL-2Rα interaction, while also conformationally stabilizing the IL-2:IL-2Rβ interaction, thus stimulating all IL-2-responsive immune cells, particularly IL-2Rβ(hi) effector cells. These insights provide a molecular blueprint for engineering selectively potentiating therapeutic antibodies.

    View details for DOI 10.1016/j.immuni.2015.04.015

    View details for Web of Science ID 000354827400009

    View details for PubMedID 25992858

  • Structural biology. Structural basis for Notch1 engagement of Delta-like 4. Science Luca, V. C., Jude, K. M., Pierce, N. W., Nachury, M. V., Fischer, S., Garcia, K. C. 2015; 347 (6224): 847-853


    Notch receptors guide mammalian cell fate decisions by engaging the proteins Jagged and Delta-like (DLL). The 2.3 angstrom resolution crystal structure of the interacting regions of the Notch1-DLL4 complex reveals a two-site, antiparallel binding orientation assisted by Notch1 O-linked glycosylation. Notch1 epidermal growth factor-like repeats 11 and 12 interact with the DLL4 Delta/Serrate/Lag-2 (DSL) domain and module at the N-terminus of Notch ligands (MNNL) domains, respectively. Threonine and serine residues on Notch1 are functionalized with O-fucose and O-glucose, which act as surrogate amino acids by making specific, and essential, contacts to residues on DLL4. The elucidation of a direct chemical role for O-glycans in Notch1 ligand engagement demonstrates how, by relying on posttranslational modifications of their ligand binding sites, Notch proteins have linked their functional capacity to developmentally regulated biosynthetic pathways.

    View details for DOI 10.1126/science.1261093

    View details for PubMedID 25700513

  • Structure of the St. Louis Encephalitis Virus Postfusion Envelope Trimer JOURNAL OF VIROLOGY Luca, V. C., Nelson, C. A., Fremont, D. H. 2013; 87 (2): 818-828


    St. Louis encephalitis virus (SLEV) is a mosquito-borne flavivirus responsible for several human encephalitis outbreaks over the last 80 years. Mature flavivirus virions are coated with dimeric envelope (E) proteins that mediate attachment and fusion with host cells. E is a class II fusion protein, the hallmark of which is a distinct dimer-to-trimer rearrangement that occurs upon endosomal acidification and insertion of hydrophobic fusion peptides into the endosomal membrane. Herein, we report the crystal structure of SLEV E in the posfusion trimer conformation. The structure revealed specific features that differentiate SLEV E from trimers of related flavi- and alphaviruses. SLEV E fusion loops have distinct intermediate spacing such that they are positioned further apart than previously observed in flaviviruses but closer together than Semliki Forest virus, an alphavirus. Domains II and III (DII and DIII) of SLEV E also adopt different angles relative to DI, which suggests that the DI-DII joint may accommodate spheroidal motions. However, trimer interfaces are well conserved among flaviviruses, so it is likely the differences observed represent structural features specific to SLEV function. Analysis of surface potentials revealed a basic platform underneath flavivirus fusion loops that may interact with the anionic lipid head groups found in membranes. Taken together, these results highlight variations in E structure and assembly that may direct virus-specific interactions with host determinants to influence pathogenesis.

    View details for DOI 10.1128/JVI.01950-12

    View details for Web of Science ID 000312934400012

    View details for PubMedID 23115296

  • Crystal Structure of the Japanese Encephalitis Virus Envelope Protein JOURNAL OF VIROLOGY Luca, V. C., Abimansour, J., Nelson, C. A., Fremont, D. H. 2012; 86 (4): 2337-2346


    Japanese encephalitis virus (JEV) is the leading global cause of viral encephalitis. The JEV envelope protein (E) facilitates cellular attachment and membrane fusion and is the primary target of neutralizing antibodies. We have determined the 2.1-Å resolution crystal structure of the JEV E ectodomain refolded from bacterial inclusion bodies. The E protein possesses the three domains characteristic of flavivirus envelopes and epitope mapping of neutralizing antibodies onto the structure reveals determinants that correspond to the domain I lateral ridge, fusion loop, domain III lateral ridge, and domain I-II hinge. While monomeric in solution, JEV E assembles as an antiparallel dimer in the crystal lattice organized in a highly similar fashion as seen in cryo-electron microscopy models of mature flavivirus virions. The dimer interface, however, is remarkably small and lacks many of the domain II contacts observed in other flavivirus E homodimers. In addition, uniquely conserved histidines within the JEV serocomplex suggest that pH-mediated structural transitions may be aided by lateral interactions outside the dimer interface in the icosahedral virion. Our results suggest that variation in dimer structure and stability may significantly influence the assembly, receptor interaction, and uncoating of virions.

    View details for DOI 10.1128/JVI.06072-11

    View details for Web of Science ID 000299862500041

    View details for PubMedID 22156523

  • Hepatitis C virus epitope exposure and neutralization by antibodies is affected by time and temperature VIROLOGY Sabo, M. C., Luca, V. C., Ray, S. C., Bukh, J., Fremont, D. H., Diamond, M. S. 2012; 422 (2): 174-184


    A recent study with flaviviruses suggested that structural dynamics of the virion impact antibody neutralization via exposure of ostensibly cryptic epitopes. To determine whether this holds true for the distantly related hepatitis C virus (HCV), whose neutralizing epitopes may be obscured by a glycan shield, apolipoprotein interactions, and the hypervariable region on the E2 envelope protein, we assessed how time and temperature of pre-incubation altered monoclonal antibody (MAb) neutralization of HCV. Notably, several MAbs showed increased inhibitory activity when pre-binding was performed at 37°C or after longer pre-incubation periods, and a corresponding loss-of-neutralization was observed when pre-binding was performed at 4°C. A similar profile of changes was observed with acute and chronic phase sera from HCV-infected patients. Our data suggest that time and temperature of incubation modulate epitope exposure on the conformational ensembles of HCV virions and thus, alter the potency of antibody neutralization.

    View details for DOI 10.1016/j.virol.2011.10.023

    View details for Web of Science ID 000299755900003

    View details for PubMedID 22078164

  • Neutralizing Monoclonal Antibodies against Hepatitis C Virus E2 Protein Bind Discontinuous Epitopes and Inhibit Infection at a Postattachment Step JOURNAL OF VIROLOGY Sabo, M. C., Luca, V. C., Prentoe, J., Hopcraft, S. E., Blight, K. J., Yi, M., Lemon, S. M., Ball, J. K., Bukh, J., Evans, M. J., Fremont, D. H., Diamond, M. S. 2011; 85 (14): 7005-7019


    The E2 glycoprotein of hepatitis C virus (HCV) mediates viral attachment and entry into target hepatocytes and elicits neutralizing antibodies in infected patients. To characterize the structural and functional basis of HCV neutralization, we generated a novel panel of 78 monoclonal antibodies (MAbs) against E2 proteins from genotype 1a and 2a HCV strains. Using high-throughput focus-forming reduction or luciferase-based neutralization assays with chimeric infectious HCV containing structural proteins from both genotypes, we defined eight MAbs that significantly inhibited infection of the homologous HCV strain in cell culture. Two of these bound E2 proteins from strains representative of HCV genotypes 1 to 6, and one of these MAbs, H77.39, neutralized infection of strains from five of these genotypes. The three most potent neutralizing MAbs in our panel, H77.16, H77.39, and J6.36, inhibited infection at an early postattachment step. Receptor binding studies demonstrated that H77.39 inhibited binding of soluble E2 protein to both CD81 and SR-B1, J6.36 blocked attachment to SR-B1 and modestly reduced binding to CD81, and H77.16 blocked attachment to SR-B1 only. Using yeast surface display, we localized epitopes for the neutralizing MAbs on the E2 protein. Two of the strongly inhibitory MAbs, H77.16 and J6.36, showed markedly reduced binding when amino acids within hypervariable region 1 (HVR1) and at sites ∼100 to 200 residues away were changed, suggesting binding to a discontinuous epitope. Collectively, these studies help to define the structural and functional complexity of antibodies against HCV E2 protein with neutralizing potential.

    View details for DOI 10.1128/JVI.00586-11

    View details for Web of Science ID 000291932400018

    View details for PubMedID 21543495