Academic Appointments


  • Instructor, Medicine

All Publications


  • Cardiovascular Risks in Patients with COVID-19: Potential Mechanisms and Areas of Uncertainty. Current cardiology reports Cheng, P., Zhu, H., Witteles, R. M., Wu, J. C., Quertermous, T., Wu, S. M., Rhee, J. W. 2020; 22 (5): 34

    Abstract

    COronaVirus Disease 2019 (COVID-19) has spread at unprecedented speed and scale into a global pandemic with cardiovascular risk factors and complications emerging as important disease modifiers. We aim to review available clinical and biomedical literature on cardiovascular risks of COVID-19.SARS-CoV2, the virus responsible for COVID-19, enters the cell via ACE2 expressed in select organs. Emerging epidemiological evidence suggest cardiovascular risk factors are associated with increased disease severity and mortality in COVID-19 patients. Patients with a more severe form of COVID-19 are also more likely to develop cardiac complications such as myocardial injury and arrhythmia. The true incidence of and mechanism underlying these events remain elusive. Cardiovascular diseases appear intricately linked with COVID-19, with cardiac complications contributing to the elevated morbidity/mortality of COVID-19. Robust epidemiologic and biologic studies are urgently needed to better understand the mechanism underlying these associations to develop better therapies.

    View details for DOI 10.1007/s11886-020-01293-2

    View details for PubMedID 32350632

  • The Environment-Sensing Aryl-Hydrocarbon Receptor Inhibits the Chondrogenic Fate of Modulated Smooth Muscle Cells in Atherosclerotic Lesions. Circulation Kim, J. B., Zhao, Q., Nguyen, T., Pjanic, M., Cheng, P., Wirka, R., Travisano, S., Nagao, M., Kundu, R., Quertermous, T. 2020

    Abstract

    Background: Smooth muscle cells (SMC) play a critical role in atherosclerosis. The Aryl hydrocarbon receptor (AHR) is an environment-sensing transcription factor that contributes to vascular development, and has been implicated in coronary artery disease (CAD) risk. We hypothesized that AHR can affect atherosclerosis by regulating phenotypic modulation of SMC. Methods: We combined RNA-Seq, ChIP-Seq, ATAC-Seq and in-vitro assays in human coronary artery SMC (HCASMC), with single-cell RNA-Seq (scRNA-Seq), histology, and RNAscope in an SMC-specific lineage-tracing Ahr knockout mouse model of atherosclerosis to better understand the role of AHR in vascular disease. Results: Genomic studies coupled with functional assays in cultured HCASMC revealed that AHR modulates HCASMC phenotype and suppresses ossification in these cells. Lineage tracing and activity tracing studies in the mouse aortic sinus showed that the Ahr pathway is active in modulated SMC in the atherosclerotic lesion cap. Furthermore, scRNA-Seq studies of the SMC-specific Ahr knockout mice showed a significant increase in the proportion of modulated SMC expressing chondrocyte markers such as Col2a1 and Alpl, which localized to the lesion neointima. These cells, which we term "chondromyocytes" (CMC), were also identified in the neointima of human coronary arteries. In histological analyses, these changes manifested as larger lesion size, increased lineage-traced SMC participation in the lesion, decreased lineage-traced SMC in the lesion cap, and increased alkaline phosphatase activity in lesions in the Ahr knockout compared to wild-type mice. We propose that AHR is likely protective based on these data and inference from human genetic analyses. Conclusions: Overall, we conclude that AHR promotes maintenance of lesion cap integrity and diminishes the disease related SMC-to-CMC transition in atherosclerotic tissues.

    View details for DOI 10.1161/CIRCULATIONAHA.120.045981

    View details for PubMedID 32441123

  • Cardiovascular Complications in Patients with COVID-19: Consequences of Viral Toxicities and Host Immune Response Curr Cardiol Rep Zhu, H., Rhee, J., Cheng, P., Waliany, S., Chang, A., Witteles, R. M., Maecker, H., Davis, M. M., Nguyen, P. K., Wu, S. M. 2020; 22 (5)
  • Outcomes in Patients With Cardiac Amyloidosis Undergoing Heart Transplantation. JACC. Heart failure Barrett, C. D., Alexander, K. M., Zhao, H., Haddad, F., Cheng, P., Liao, R., Wheeler, M. T., Liedtke, M., Schrier, S., Arai, S., Weisshaar, D., Witteles, R. M. 2020

    Abstract

    The purpose of this study is to report outcomes after heart transplantation in patients with cardiac amyloidosis based on a large single-center experience.Cardiac amyloidosis causes significant morbidity and mortality, often leading to restrictive cardiomyopathy, progressive heart failure, and death. Historically, heart transplantation outcomes have been worse in patients with cardiac amyloidosis compared with other heart failure populations, in part due to the systemic nature of the disease. However, several case series have suggested that transplantation outcomes may be better in the contemporary era, likely in part due to the availability of more effective light chain suppressive therapies for light chain amyloidosis.This study examined all patients seen between 2004 and 2017, either at the Stanford University Medical Center or the Kaiser Permanente Santa Clara Medical Center, who were diagnosed with cardiac amyloidosis and ultimately underwent heart transplantation. This study examined pre-transplantation characteristics and post-transplantation outcomes in this group compared with the overall transplantation population at our center.During the study period, 31 patients (13 with light chain amyloidosis and 18 with transthyretin [ATTR] amyloidosis) underwent heart transplantation. Patients with ATTR amyloidosis were older, were more likely to be male, had worse baseline renal function, and had longer waitlist times compared with both patients with light chain amyloidosis and the overall transplantation population. Post-transplantation, there were no differences in post-operative bleeding, renal failure, infection, rejection, or malignancy. There was no significant difference in mortality between patients who underwent heart transplantation for amyloid cardiomyopathy and patients who underwent heart transplantation for all other indications.In carefully selected patients with cardiac amyloidosis, heart transplantation can be an effective therapeutic option with outcomes similar to those transplanted for other causes of heart failure.

    View details for DOI 10.1016/j.jchf.2019.12.013

    View details for PubMedID 32387068

  • Coronary Disease Associated Gene TCF21 Inhibits Smooth Muscle Cell Differentiation by Blocking the Myocardin-Serum Response Factor Pathway. Circulation research Nagao, M., Lyu, Q., Zhao, Q., Wirka, R. C., Bagga, J., Nguyen, T., Cheng, P., Kim, J. B., Pjanic, M., Miano, J. M., Quertermous, T. 2019

    Abstract

    Rationale: The gene encoding transcription factor TCF21 has been linked to coronary artery disease (CAD) risk by human genome wide association studies (GWAS) in multiple racial ethnic groups. In murine models, Tcf21 is required for phenotypic modulation of smooth muscle cells (SMC) in atherosclerotic tissues and promotes a fibroblast phenotype in these cells. In humans, TCF21 expression inhibits risk for CAD. The molecular mechanism by which TCF21 regulates SMC phenotype is not known. Objective: To better understand how TCF21 affects SMC phenotype, we sought to investigate the possible mechanisms by which it regulates the lineage determining myocardin (MYOCD)-serum response factor (SRF) pathway. Methods and Results: Modulation of TCF21 expression in HCASMC revealed that TCF21 suppresses a broad range of SMC markers, as well as key SMC transcription factors MYOCD and SRF, at the RNA and protein level. We conducted chromatin immunoprecipitation (ChIP)-sequencing to map SRF binding sites in HCASMC, showing that binding is colocalized in the genome with TCF21, including at a novel enhancer in the SRF gene, and at the MYOCD gene promoter. In vitro genome editing indicated that the SRF enhancer CArG box regulates transcription of the SRF gene, and mutation of this conserved motif in the orthologous mouse SRF enhancer revealed decreased SRF expression in aorta and heart tissues. Direct TCF21 binding and transcriptional inhibition at co-localized sites were established by reporter gene transfection assays. Chromatin immunoprecipitation and protein co-immunoprecipitation studies provided evidence that TCF21 blocks MYOCD and SRF association by direct TCF21-MYOCD interaction. Conclusions: These data indicate that TCF21 antagonizes the MYOCD-SRF pathway through multiple mechanisms, further establishing a role for this CAD associated gene in fundamental SMC processes and indicating the importance of smooth muscle response to vascular stress and phenotypic modulation of this cell type in CAD risk.

    View details for DOI 10.1161/CIRCRESAHA.119.315968

    View details for PubMedID 31815603

  • TCF21 and AP-1 interact through epigenetic modifications to regulate coronary artery disease gene expression GENOME MEDICINE Zhao, Q., Wirka, R., Trieu Nguyen, Nagao, M., Cheng, P., Miller, C. L., Kim, J., Pjanic, M., Quertermous, T. 2019; 11
  • Large-Scale Assessment of a Smartwatch to Identify Atrial Fibrillation. The New England journal of medicine Perez, M. V., Mahaffey, K. W., Hedlin, H., Rumsfeld, J. S., Garcia, A., Ferris, T., Balasubramanian, V., Russo, A. M., Rajmane, A., Cheung, L., Hung, G., Lee, J., Kowey, P., Talati, N., Nag, D., Gummidipundi, S. E., Beatty, A., Hills, M. T., Desai, S., Granger, C. B., Desai, M., Turakhia, M. P., Apple Heart Study Investigators, Perez, M. V., Turakhia, M. P., Lhamo, K., Smith, S., Berdichesky, M., Sharma, B., Mahaffey, K., Parizo, J., Olivier, C., Nguyen, M., Tallapalli, S., Kaur, R., Gardner, R., Hung, G., Mitchell, D., Olson, G., Datta, S., Gerenrot, D., Wang, X., McCoy, P., Satpathy, B., Jacobsen, H., Makovey, D., Martin, A., Perino, A., O'Brien, C., Gupta, A., Toruno, C., Waydo, S., Brouse, C., Dorfman, D., Stein, J., Huang, J., Patel, M., Fleischer, S., Doll, E., O'Reilly, M., Dedoshka, K., Chou, M., Daniel, H., Crowley, M., Martin, C., Kirby, T., Brumand, M., McCrystale, K., Haggerty, M., Newberger, J., Keen, D., Antall, P., Holbrook, K., Braly, A., Noone, G., Leathers, B., Montrose, A., Kosowsky, J., Lewis, D., Finkelmeier, J. R., Bemis, K., Mahaffey, K. W., Desai, M., Talati, N., Nag, D., Rajmane, A., Desai, S., Caldbeck, D., Cheung, L., Granger, C., Rumsfeld, J., Kowey, P. R., Hills, M. T., Russo, A., Rockhold, F., Albert, C., Alonso, A., Wruck, L., Friday, K., Wheeler, M., Brodt, C., Park, S., Rogers, A., Jones, R., Ouyang, D., Chang, L., Yen, A., Dong, J., Mamic, P., Cheng, P., Shah, R., Lorvidhaya, P. 2019; 381 (20): 1909–17

    Abstract

    BACKGROUND: Optical sensors on wearable devices can detect irregular pulses. The ability of a smartwatch application (app) to identify atrial fibrillation during typical use is unknown.METHODS: Participants without atrial fibrillation (as reported by the participants themselves) used a smartphone (Apple iPhone) app to consent to monitoring. If a smartwatch-based irregular pulse notification algorithm identified possible atrial fibrillation, a telemedicine visit was initiated and an electrocardiography (ECG) patch was mailed to the participant, to be worn for up to 7 days. Surveys were administered 90 days after notification of the irregular pulse and at the end of the study. The main objectives were to estimate the proportion of notified participants with atrial fibrillation shown on an ECG patch and the positive predictive value of irregular pulse intervals with a targeted confidence interval width of 0.10.RESULTS: We recruited 419,297 participants over 8 months. Over a median of 117 days of monitoring, 2161 participants (0.52%) received notifications of irregular pulse. Among the 450 participants who returned ECG patches containing data that could be analyzed - which had been applied, on average, 13 days after notification - atrial fibrillation was present in 34% (97.5% confidence interval [CI], 29 to 39) overall and in 35% (97.5% CI, 27 to 43) of participants 65 years of age or older. Among participants who were notified of an irregular pulse, the positive predictive value was 0.84 (95% CI, 0.76 to 0.92) for observing atrial fibrillation on the ECG simultaneously with a subsequent irregular pulse notification and 0.71 (97.5% CI, 0.69 to 0.74) for observing atrial fibrillation on the ECG simultaneously with a subsequent irregular tachogram. Of 1376 notified participants who returned a 90-day survey, 57% contacted health care providers outside the study. There were no reports of serious app-related adverse events.CONCLUSIONS: The probability of receiving an irregular pulse notification was low. Among participants who received notification of an irregular pulse, 34% had atrial fibrillation on subsequent ECG patch readings and 84% of notifications were concordant with atrial fibrillation. This siteless (no on-site visits were required for the participants), pragmatic study design provides a foundation for large-scale pragmatic studies in which outcomes or adherence can be reliably assessed with user-owned devices. (Funded by Apple; Apple Heart Study ClinicalTrials.gov number, NCT03335800.).

    View details for DOI 10.1056/NEJMoa1901183

    View details for PubMedID 31722151

  • Coronary artery disease genes SMAD3 and TCF21 promote opposing interactive genetic programs that regulate smooth muscle cell differentiation and disease risk PLOS GENETICS Iyer, D., Zhao, Q., Wirka, R., Naravane, A., Trieu Nguyen, Liu, B., Nagao, M., Cheng, P., Miller, C. L., Kim, J., Pjanic, M., Quertermous, T. 2018; 14 (10)
  • What's in a Name? Factors That Influence the Usage of Generic Versus Trade Names for Cardiac Medications Among Healthcare Providers CIRCULATION-CARDIOVASCULAR QUALITY AND OUTCOMES Ouyang, D., Tisdale, R., Cheng, P., Chi, J., Chen, J. H., Ashley, E. 2018; 11 (8)
  • Coronary artery disease genes SMAD3 and TCF21 promote opposing interactive genetic programs that regulate smooth muscle cell differentiation and disease risk. PLoS genetics Iyer, D., Zhao, Q., Wirka, R., Naravane, A., Nguyen, T., Liu, B., Nagao, M., Cheng, P., Miller, C. L., Kim, J. B., Pjanic, M., Quertermous, T. 2018; 14 (10): e1007681

    Abstract

    Although numerous genetic loci have been associated with coronary artery disease (CAD) with genome wide association studies, efforts are needed to identify the causal genes in these loci and link them into fundamental signaling pathways. Recent studies have investigated the disease mechanism of CAD associated gene SMAD3, a central transcription factor (TF) in the TGFβ pathway, investigating its role in smooth muscle biology. In vitro studies in human coronary artery smooth muscle cells (HCASMC) revealed that SMAD3 modulates cellular phenotype, promoting expression of differentiation marker genes while inhibiting proliferation. RNA sequencing and chromatin immunoprecipitation sequencing studies in HCASMC identified downstream genes that reside in pathways which mediate vascular development and atherosclerosis processes in this cell type. HCASMC phenotype, and gene expression patterns promoted by SMAD3 were noted to have opposing direction of effect compared to another CAD associated TF, TCF21. At sites of SMAD3 and TCF21 colocalization on DNA, SMAD3 binding was inversely correlated with TCF21 binding, due in part to TCF21 locally blocking chromatin accessibility at the SMAD3 binding site. Further, TCF21 was able to directly inhibit SMAD3 activation of gene expression in transfection reporter gene studies. In contrast to TCF21 which is protective toward CAD, SMAD3 expression in HCASMC was shown to be directly correlated with disease risk. We propose that the pro-differentiation action of SMAD3 inhibits dedifferentiation that is required for HCASMC to expand and stabilize disease plaque as they respond to vascular stresses, counteracting the protective dedifferentiating activity of TCF21 and promoting disease risk.

    View details for PubMedID 30307970

  • HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS ASSOCIATED WITH IMPLANTATION OF VENTRICULAR ASSIST DEVICES AND OUTCOMES FOLLOWING SUBSEQUENT ORTHOTROPIC HEART TRANSPLANTATION (OHT) Cheng, P., Davis, M., Ha, R., Martin, B., Banerjee, D. ELSEVIER SCIENCE INC. 2017: 864
  • Persistent Fever Can be Associated with Transient but Severe Hemophagocytic Lymphohistiocytosis (HLH) in Adult Recipients of Ventricular Assist Devices (VAD) for Treatment of Cardiomyopathy (CM) Martin, B., Cheng, P. A., Banerjee, D., Ha, R. AMER SOC HEMATOLOGY. 2016
  • Fibronectin mediates mesendodermal cell fate decisions. Development (Cambridge, England) Cheng, P., Andersen, P., Hassel, D., Kaynak, B. L., Limphong, P., Juergensen, L., Kwon, C., Srivastava, D. 2013; 140 (12): 2587–96

    Abstract

    Non-cell-autonomous signals often play crucial roles in cell fate decisions during animal development. Reciprocal signaling between endoderm and mesoderm is vital for embryonic development, yet the key signals and mechanisms remain unclear. Here, we show that endodermal cells efficiently promote the emergence of mesodermal cells in the neighboring population through signals containing an essential short-range component. The endoderm-mesoderm interaction promoted precardiac mesoderm formation in mouse embryonic stem cells and involved endodermal production of fibronectin. In vivo, fibronectin deficiency resulted in a dramatic reduction of mesoderm accompanied by endodermal expansion in zebrafish embryos. This event was mediated by regulation of Wnt signaling in mesodermal cells through activation of integrin-β1. Our findings highlight the importance of the extracellular matrix in mediating short-range signals and reveal a novel function of endoderm, involving fibronectin and its downstream signaling cascades, in promoting the emergence of mesoderm.

    View details for DOI 10.1242/dev.089052

    View details for PubMedID 23715551

    View details for PubMedCentralID PMC3666385

  • MicroRNA-10 regulates the angiogenic behavior of zebrafish and human endothelial cells by promoting vascular endothelial growth factor signaling. Circulation research Hassel, D., Cheng, P., White, M. P., Ivey, K. N., Kroll, J., Augustin, H. G., Katus, H. A., Stainier, D. Y., Srivastava, D. 2012; 111 (11): 1421–33

    Abstract

    Formation and remodeling of the vasculature during development and disease involve a highly conserved and precisely regulated network of attractants and repellants. Various signaling pathways control the behavior of endothelial cells, but their posttranscriptional dose titration by microRNAs is poorly understood.To identify microRNAs that regulate angiogenesis.We show that the highly conserved microRNA family encoding miR-10 regulates the behavior of endothelial cells during angiogenesis by positively titrating proangiogenic signaling. Knockdown of miR-10 led to premature truncation of intersegmental vessel growth in the trunk of zebrafish larvae, whereas overexpression of miR-10 promoted angiogenic behavior in zebrafish and cultured human umbilical venous endothelial cells. We found that miR-10 functions, in part, by directly regulating the level of fms-related tyrosine kinase 1 (FLT1), a cell-surface protein that sequesters vascular endothelial growth factor, and its soluble splice variant sFLT1. The increase in FLT1/sFLT1 protein levels upon miR-10 knockdown in zebrafish and in human umbilical venous endothelial cells inhibited the angiogenic behavior of endothelial cells largely by antagonizing vascular endothelial growth factor receptor 2 signaling.Our study provides insights into how FLT1 and vascular endothelial growth factor receptor 2 signaling is titrated in a microRNA-mediated manner and establishes miR-10 as a potential new target for the selective modulation of angiogenesis.

    View details for DOI 10.1161/CIRCRESAHA.112.279711

    View details for PubMedID 22955733

    View details for PubMedCentralID PMC3525481

  • Notch post-translationally regulates β-catenin protein in stem and progenitor cells. Nature cell biology Kwon, C., Cheng, P., King, I. N., Andersen, P., Shenje, L., Nigam, V., Srivastava, D. 2011; 13 (10): 1244–51

    Abstract

    Cellular decisions of self-renewal or differentiation arise from integration and reciprocal titration of numerous regulatory networks. Notch and Wnt/β-catenin signalling often intersect in stem and progenitor cells and regulate each other transcriptionally. The biological outcome of signalling through each pathway often depends on the context and timing as cells progress through stages of differentiation. Here, we show that membrane-bound Notch physically associates with unphosphorylated (active) β-catenin in stem and colon cancer cells and negatively regulates post-translational accumulation of active β-catenin protein. Notch-dependent regulation of β-catenin protein did not require ligand-dependent membrane cleavage of Notch or the glycogen synthase kinase-3β-dependent activity of the β-catenin destruction complex. It did, however, require the endocytic adaptor protein Numb and lysosomal activity. This study reveals a previously unrecognized function of Notch in negatively titrating active β-catenin protein levels in stem and progenitor cells.

    View details for DOI 10.1038/ncb2313

    View details for PubMedID 21841793

    View details for PubMedCentralID PMC3187850

  • Reporter-based isolation of induced pluripotent stem cell- and embryonic stem cell-derived cardiac progenitors reveals limited gene expression variance. Circulation research van Laake, L. W., Qian, L., Cheng, P., Huang, Y., Hsiao, E. C., Conklin, B. R., Srivastava, D. 2010; 107 (3): 340–47

    Abstract

    Induced pluripotent stem (iPS) cells can differentiate into multiple cell types, including cardiomyocytes and have tremendous potential for drug discovery and regenerative therapies. However, it is unknown how much variability exists between differentiated lineages from independent iPS cell lines and, specifically, how similar iPS cell-derived cardiomyocytes (iPS-CMs) are to embryonic stem (ES) cell-derived cardiomyocytes (ES-CMs).We investigated how much variability exists between differentiated lineages from independent iPS cell lines and how similar iPS-CMs are to ES-CMs.We generated mouse iPS cells in which expression of NKX2-5, an early cardiac transcription factor, was marked by transgenic green fluorescent protein (GFP). Isolation of iPS- and ES-derived NKX2-5-GFP(+) cardiac progenitor pools, marked by identical reporters, revealed unexpectedly high similarity in genome-wide mRNA expression levels. Furthermore, the variability between cardiac progenitors derived from independent iPS lines was minimal. The NKX2-5-GFP(+) iPS cells formed cardiomyocytes by numerous induction protocols and could survive upon transplantation into the infarcted mouse heart without formation of teratomas.Despite the line-to-line variability of gene expression in the undifferentiated state of ES and iPS cells, the variance narrows significantly in lineage-specific iPS-derived cardiac progenitors, and these progenitor cells can be isolated and used for transplantation without generation of unwanted cell types.

    View details for DOI 10.1161/CIRCRESAHA.109.215434

    View details for PubMedID 20558827

    View details for PubMedCentralID PMC2919280

  • A regulatory pathway involving Notch1/beta-catenin/Isl1 determines cardiac progenitor cell fate NATURE CELL BIOLOGY Kwon, C., Qian, L., Cheng, P., Nigam, V., Arnold, J., Srivastava, D. 2009; 11 (8): 951–U96

    Abstract

    Regulation of multipotent cardiac progenitor cell (CPC) expansion and subsequent differentiation into cardiomyocytes, smooth muscle or endothelial cells is a fundamental aspect of basic cardiovascular biology and cardiac regenerative medicine. However, the mechanisms governing these decisions remain unclear. Here, we show that Wnt/beta-catenin signalling, which promotes expansion of CPCs, is negatively regulated by Notch1-mediated control of phosphorylated beta-catenin accumulation within CPCs, and that Notch1 activity in CPCs is required for their differentiation. Notch1 positively, and beta-catenin negatively, regulated expression of the cardiac transcription factors, Isl1, Myocd and Smyd1. Surprisingly, disruption of Isl1, normally expressed transiently in CPCs before their differentiation, resulted in expansion of CPCs in vivo and in an embryonic stem (ES) cell system. Furthermore, Isl1 was required for CPC differentiation into cardiomyocyte and smooth muscle cells, but not endothelial cells. These findings reveal a regulatory network controlling CPC expansion and cell fate that involves unanticipated functions of beta-catenin, Notch1 and Isl1 that may be leveraged for regenerative approaches involving CPCs.

    View details for DOI 10.1038/ncb1906

    View details for Web of Science ID 000268593200008

    View details for PubMedID 19620969

    View details for PubMedCentralID PMC2748816

  • Evolution of an interloop disulfide bond in high-affinity antibody mimics based on fibronectin type III domain and selected by yeast surface display: Molecular convergence with single-domain camelid and shark antibodies JOURNAL OF MOLECULAR BIOLOGY Lipovsek, D., Lippow, S. M., Hackel, B. J., Gregson, M. W., Cheng, P., Kapila, A., Wittrup, K. D. 2007; 368 (4): 1024-1041

    Abstract

    The 10th human fibronectin type III domain ((10)Fn3) is one of several protein scaffolds used to design and select families of proteins that bind with high affinity and specificity to macromolecular targets. To date, the highest affinity (10)Fn3 variants have been selected by mRNA display of libraries generated by randomizing all three complementarity-determining region -like loops of the (10)Fn3 scaffold. The sub-nanomolar affinities of such antibody mimics have been attributed to the extremely large size of the library accessible by mRNA display (10(12) unique sequences). Here we describe the selection and affinity maturation of (10)Fn3-based antibody mimics with dissociation constants as low as 350 pM selected from significantly smaller libraries (10(7)-10(9) different sequences), which were constructed by randomizing only 14 (10)Fn3 residues. The finding that two adjacent loops in human (10)Fn3 provide a large enough variable surface area to select high-affinity antibody mimics is significant because a smaller deviation from wild-type (10)Fn3 sequence is associated with a higher stability of selected antibody mimics. Our results also demonstrate the utility of an affinity-maturation strategy that led to a 340-fold improvement in affinity by maximizing sampling of sequence space close to the original selected antibody mimic. A striking feature of the highest affinity antibody mimics selected against lysozyme is a pair of cysteines on adjacent loops, in positions 28 and 77, which are critical for the affinity of the (10)Fn3 variant for its target and are close enough to form a disulfide bond. The selection of this cysteine pair is structurally analogous to the natural evolution of disulfide bonds found in new antigen receptors of cartilaginous fish and in camelid heavy-chain variable domains. We propose that future library designs incorporating such an interloop disulfide will further facilitate the selection of high-affinity, highly stable antibody mimics from libraries accessible to phage and yeast surface display methods.

    View details for DOI 10.1016/j.jmb.2007.02.029

    View details for Web of Science ID 000246265200009

    View details for PubMedID 17382960